Pharmaceutical Compositions and Methods

Abstract

Disclosed herein are stable liquid, suspension and dry powder compositions and methods for the delivery of stabilized biodegradable materials, including peptides and proteins, small molecules and methods for delivery in the treatment of pulmonary diseases. Specifically, the compositions include vasoactive intestinal peptides for pulmonary inhalation to treat respiratory and / or pulmonary diseases, including inflammation, acute and chronic lung injury and pulmonary edema.

Description

[Technical field]

[0001] Disclosed herein are stable pharmaceutical compositions and methods for the delivery of biodegradable substances, including peptides and proteins, as well as methods for the delivery of dry powders in the treatment of pulmonary diseases.Specifically, contemplated are solutions, suspensions and dry powders for pulmonary inhalation to treat respiratory disorders and / or pulmonary diseases, including inflammation, edema, acute and chronic lung injury. [Background technology]

[0002] Delivery of unstable drugs to treat diseases has been a major problem for many years. Many compounds are ineffective or have low or variable potency when administered orally. Oral administration of unstable compounds can lead to poor absorption of the compounds under conditions encountered in the gastrointestinal tract, resulting in reduced activity before reaching the target site. Oral administration of drugs is often desirable, especially in terms of ease of administration, patient compliance, and reduced costs, but increased drug content can lead to side effects, so alternative methods are needed to reduce the drug content used per tablet or capsule. For biologics, especially peptides and proteins, the acidic environment in the stomach is detrimental to maintaining their function, as most proteins are easily degraded.

[0003] Drug formulations containing labile drugs, including isolated biological materials including proteins and peptides, are most commonly formulated as injections. However, in injections, by entering the venous circulation and passing through the liver where these unstable compounds are metabolized, these unstable compounds can easily undergo denaturation and complete loss of functional activity. For example, some biological products lose their functional activity when removed from -20°C storage and placed at room temperature for a short period of time. Other isolated proteins and peptides undergo significant degradation, either by oxidation or ubiquitous proteases, when stored at 4°C without the addition of protease inhibitors. Most mammalian proteins and peptides degrade above 43°C. It is well known that at 55°C, complete denaturation of most proteins occurs in about 1-2 hours. In some cases, complete denaturation and destabilization of isolated proteins occurs even at room temperature.

[0004] Currently, primarily injectable compositions, as well as solutions and suspensions for nebulization, are available for delivering formulations containing unstable drugs as active agents to the lungs for treating local and systemic diseases. The preparation of injectable formulations and the special storage required creates challenges that prohibit their use, especially in subtropical and tropical climates where there is a great need and refrigeration and sterilization are not always readily available. Alternative methods and formulations are needed to stabilize biologically derived formulations for the treatment of subjects, including during epidemic outbreaks of diseases such as coronavirus diseases, including Covid-19.

[0005] Dry powder compositions for pulmonary inhalation and systemic administration of peptides, such as insulin (AFREZZA®), require cold storage prior to use. Although AFREZZA can be stored at ambient temperature for short periods of time, e.g., weeks to months, there remains a need to improve the stability of peptides in dry powder products. Similarly, there is a need to improve the stability of dry powder compositions intended for pulmonary delivery, particularly those containing biological molecules, to further extend the shelf life and facilitate storage and delivery prior to patient use, especially when refrigeration is not available.

[0006] Thus, there is room for improvement in the development of pharmaceutical formulations containing biological molecules for delivery to the lungs, particularly in the treatment of disease. Summary of the Invention [Means for solving the problem]

[0007] The present disclosure provides methods for treating acute or chronic pulmonary diseases or disorders, including inflammation, pulmonary fibrosis, and viral infections, including Covid-19 disease. The methods include administering to a subject pharmaceutical compositions, including suspension and dry powder compositions for inhalation, that include a labile active agent, which compositions provide improved stability of the active agent in solution or suspension at or above room temperature for extended periods of time without substantial loss of biological activity due to degradation of the active agent.

[0008] In one embodiment, an inhalable pharmaceutical formulation is provided, comprising a dry powder for inhalation, comprising unstable, rapidly degrading small or macromolecular molecules, where biological molecules, including peptides, proteins, particularly synthetically derived peptides, proteins, are easily destabilized in aqueous solution or suspension. In an exemplary embodiment, the pharmaceutical formulation is manufactured for inhalation into local lung tissue. The formulation is provided for delivery to the lung using a dry powder inhalation system, comprising a multi-use inhaler that can be used with replaceable unit dose cartridges or capsules. Alternatively, a single-use inhaler can be provided with an integrally built-in container that contains the formulation for a single use, or a multi-use inhaler can be provided with multiple doses that are integrated with the inhaler for multiple uses.

[0009] In another embodiment, a stable inhalable pharmaceutical formulation is provided comprising a dry powder comprising a protein or peptide and one or more pharma- ceutically acceptable carriers and / or excipients, said formulation being stable at room temperature, elevated temperature and / or elevated humidity. In one embodiment, the pharmaceutical formulation is stable for extended periods of time, for example, at temperatures above 4° C., above 10° C., above 20° C., or above 35° C. In one embodiment, the formulation is also stable in environments with relative humidity, such as environments with relative humidity greater than 5%, greater than 10%, greater than 30%, greater than 50%, greater than 60%, greater than 70%.

[0010] In exemplary embodiments, the pharmaceutical formulations for use in the methods of treating pulmonary disease are stabilized by one or more pharma- ceutically acceptable carriers and / or excipients, or combinations thereof, including, for example, buffers, salts, minerals, vitamins, antioxidants, polymers, sugars, including mannitol, xylitol, diketopiperazines and / or salts thereof, etc. In one embodiment, the dry powder composition can optionally include a surfactant, such as a polysorbate, e.g., polysorbate 80 and Tween.

[0011] In certain embodiments, the formulation comprises a dry powder comprising a therapeutic peptide for treating inflammatory diseases such as pulmonary edema, including vasoactive intestinal peptide (VIP) (including aviptadil acetate), its derivatives, its analogs, its pharma- ceutically acceptable salts, or combinations thereof, and one or more pharmaceutical excipients and / or carriers including a peptide stabilizer. In an exemplary embodiment, the pharma- ceutically acceptable carrier and / or excipient can be formulated for oral inhalation, which can form particles suitable for inhalation, such as diketopiperazines, including fumaryl diketopiperazine, where the particles form an amorphous powder, a crystalline powder, or a composite crystalline powder.

[0012] In one embodiment, the pharma- ceutically acceptable carrier or excipient comprises one or more sugars including mannitol, xylitol, sorbitol, and trehalose; one or more amino acids including arginine, glycine, leucine, isoleucine, trileucine, cysteine, lysine, methionine, and histidine; a surfactant including polysorbate 80; a cationic salt including monovalent, divalent, and trivalent salts including sodium chloride, potassium chloride, magnesium chloride, zinc chloride; a buffer including citrate and tartrate; vitamins including vitamin A, vitamin C, vitamin E, or a combination of one or more carriers and / or excipients.

[0013] In certain embodiments, the pharmaceutical dry powder composition comprises one or more pharmaceutical excipients and / or carriers including antioxidants, such as ascorbic acid or vitamin C, vitamin E, selenium, carotenoids including beta-carotene, lycopene, lutein, and zeaxanthin, glutathione, phenols, polyphenols, and flavonoids.

[0014] In certain embodiments, the pharmaceutical dry powder composition comprises vasoactive intestinal peptide (VIP), its derivatives, its analogs, its pharma- ceutically acceptable salts, and / or combinations thereof, including aviptadil, aviptadil acetate, a sugar, an amino acid, and / or an antioxidant, wherein the sugar is mannitol or trehalose, and the amino acid is leucine, isoleucine, methionine, or combinations thereof. In certain embodiments of the invention, the pharmaceutical dry powder composition further comprises an antioxidant selected from the group consisting of vitamin C, vitamin E, selenium, and glutathione.

[0015] In an exemplary embodiment, the pharmaceutical dry powder composition comprises vasoactive intestinal peptide (VIP), including aviptadil, aviptadil acetate, its derivatives, its analogs, its pharmaceutically acceptable salts, and / or combinations thereof, and said one or more pharmaceutically acceptable carriers and / or excipients include mannitol, methionine, leucine, and ascorbic acid.

[0016] In other embodiments, the inhalable pharmaceutical dry powder may further comprise an inorganic or organic salt or combination thereof, including sodium chloride, potassium chloride, magnesium chloride, zinc chloride, sodium citrate, sodium tartrate, or combinations thereof.

[0017] In one embodiment, the inhalable dry powder composition can include a buffering agent and a monovalent or divalent cation salt. In a particular embodiment, the formulation comprises a dry powder comprising VIP, a VIP derivative, a VIP analog, or a salt thereof, a buffering agent, and / or a divalent or monovalent cation provided by a salt including zinc citrate, zinc acetate, disodium tartrate, monosodium tartrate, sodium citrate, disodium citrate, trisodium citrate, zinc chloride, calcium chloride, magnesium chloride, sodium hydroxide, and the like. In one embodiment, the monovalent cations in the composition include sodium, potassium, and lithium. In an alternative embodiment, the formulation can include citric acid as an excipient.

[0018] In certain embodiments, dry powder compositions are provided that include an active agent, such as vasoactive intestinal peptide, in an amount of less than 40 wt%, less than 30 wt%, less than 20 wt%, or less than 10 wt% of the composition, and sugar in an amount of less than 90 wt%, less than 85 wt%, or less than 50 wt%. In certain embodiments, the amino acid component in the composition is in an amount of up to about 20 wt%, up to about 15 wt%, or up to about 10 wt% of the composition. In one embodiment, the amount of antioxidant in the composition can be up to about 10%, up to about 5%, or up to about 3% of the composition.

[0019] In certain embodiments, an inhalable dry powder composition for the treatment of pulmonary diseases, including acute or chronic infections of the lungs, comprises vasoactive intestinal peptide in an amount of up to 50 wt %, wherein the pharmacologic acceptable excipients or carriers are mannitol, leucine, and ascorbic acid.

[0020] 1. A method for producing a dry powder formulation comprising mixing or homogenizing a solution comprising a peptide or protein or analog thereof, and spray drying the solution to form a dry powder, the solution further comprising a sugar comprising mannitol, one or more amino acids comprising leucine or isoleucine, and an antioxidant comprising ascorbic acid or glutathione.

[0021] A method of treating a pulmonary disease comprising administering to a subject in need of treatment one or more doses of a dry powder composition by inhalation using a dry powder inhaler, the composition comprising vasoactive intestinal peptide, an analogue or derivative thereof, an amino acid, a sugar and an antioxidant. In one embodiment, the treatment comprises administering one or more doses of the dry powder composition per session. In one embodiment of the invention, the dose of the composition comprises up to about 500 μg, 300 μg, 200 μg, or 100 μg of vasoactive intestinal peptide per session.

[0022] In an exemplary embodiment, a method for treating a pulmonary disease comprises administering to a subject in need of treatment, including pulmonary edema, an inhalable pharmaceutical formulation comprising mannitol, leucine, methionine and vasoactive intestinal peptide, an analogue or derivative thereof, at least once daily, at a dose of up to 200 μg per administration. In one embodiment, the pharmaceutical formulation can be a solution, suspension or dry powder for inhalation and can be administered by nebulization, a metered dose inhaler or a dry powder inhaler.

[0023] In other embodiments described herein, methods of making stable compositions comprising vasoactive intestinal peptide, its derivatives, its analogs, or combinations thereof, and other rapidly degraded bioactive agents, or methods of using the compositions in the treatment of diseases and / or disorders of pulmonary or systemic origin are disclosed. In exemplary embodiments, an inhalation system is provided comprising a high resistance inhaler for single dose use for the treatment of pulmonary diseases comprising the compositions.

[0024] In another embodiment, the method of treatment comprises administering to a subject in need of treatment an inhalable pharmaceutical formulation comprising mannitol, leucine, methionine and vasoactive intestinal peptide, an analogue or derivative thereof, at least once a day, at a dose of up to 200 μg per administration. In one embodiment, the pharmaceutical formulation can be a solution, suspension or dry powder for inhalation, and can be administered by nebulization, metered dose inhaler or dry powder inhaler. In this and other embodiments, the disease to be treated includes, for example, pulmonary edema, Crohn's disease, heart failure, neurodegenerative diseases, etc. [Brief description of the drawings]

[0025] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the embodiments disclosed herein. The present disclosure may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0026] [Figure 1A] Scanning electron microscope images (SEM) of dry powders comprising crystalline particles of fumaryl diketopiperazine and vasoactive intestinal peptide (VIP), containing different amounts of VIP peptide. [Figure 1B] Scanning electron microscope images (SEM) of dry powders comprising crystalline particles of fumaryl diketopiperazine and vasoactive intestinal peptide (VIP), containing different amounts of VIP peptide. [Figure 1C] Scanning electron microscope images (SEM) of dry powders comprising crystalline particles of fumaryl diketopiperazine and vasoactive intestinal peptide (VIP), containing different amounts of VIP peptide. [Diagram 2] Graph representing standard curve of VIP in dry powder of data from an experiment. [Diagram 3] HPLC chromatogram of the VIP peptide used in the experiment. [Figure 4] HPLC chromatogram of the content of a dry powder containing crystalline FDKP and VIP from a sample produced by the method described in Example 1 herein. [Diagram 5] Graph of formulation experiment conducted to test solutions or suspensions containing various types of vasoactive intestinal peptide (VIP) as described in Example 2, showing the stability of VIP in compositions with various methionine contents relative to a control sample. [Figure 6] Graph of formulation experiments conducted to test various types of vasoactive intestinal peptide (VIP)-containing solutions or suspensions (see Table 2 below), showing stability in compositions with various pharmacologic acceptable excipients. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0027] Detailed Description of the Invention Disclosed herein are pharmaceutical dry powders for inhalation comprising vasoactive intestinal peptide, and methods of making powder compositions for the treatment of pulmonary diseases. The embodiments disclosed herein demonstrate that rapidly biodegradable active agents, including vasoactive intestinal peptide, can be formulated to be stable in compositions for the treatment of pulmonary diseases, including pulmonary edema.

[0028] Methods for producing dry powder formulations containing stabilized vasoactive intestinal peptide are provided, which facilitate the use of VIP in the treatment of disease, particularly pulmonary disease. Methods for producing stabilized VIP and dry powder compositions obtained therefrom are also provided.

[0029] The term "fine particles" as used herein refers to particles with a diameter of about 0.5 to about 1000 μm, regardless of the exact external or internal structure. Fine particles with a diameter of about 0.5 to about 10 microns are able to successfully pass through most of the natural barriers to reach the lungs. A diameter of less than about 10 microns is necessary to pass through the turns of the throat, and a diameter of about 0.5 microns or more is necessary to avoid exhalation. It is preferable to maximize the proportion of particles contained in the "respirable fraction" (RF) to reach the deep lung (or alveolar region), where most efficient absorption is believed to occur. This fraction is generally accepted to be particles with an aerodynamic diameter of about 0.5 to about 6 microns, although some sources use somewhat different ranges, measured by standard techniques, for example, using an Anderson Cascade Impactor. Other impactors can also be used to measure aerodynamic particle size, such as the NEXT GENERATION IMPACTOR™ (NGI™, MSP Corporation), where the respirable fraction is defined as a similar aerodynamic size, e.g., <6.4 μm. In some embodiments, a laser diffraction device is used to determine particle size, such as the laser diffraction device disclosed in U.S. Patent Application No. 12 / 727,179, filed March 18, 2010 (incorporated herein in its entirety for relevant teachings), and the volume median geometric diameter (VMGD) of the particles is measured to evaluate the performance of the inhalation system. For example, in various embodiments, a cartridge emptying of ≧80%, ≧85%, or ≧90%, and a VMGD of emitted particles of ≦12.5 μm, ≦7.0 μm, or ≦4.8 μm can indicate progressively better aerodynamic performance.

[0030] As used herein, the term "about" is used to indicate that a value includes the standard deviation of the measurement for the device or method employed to determine the value.

[0031] Respirable fraction at fill (RF / fill) represents the percentage of powder in a dose that is released from an inhaler upon expulsion of the powder content filled for use as a dose and is suitable for respirability, i.e., the percentage of particles released from a filled dose that are suitable for pulmonary delivery, and is a measure of the aerodynamic performance of microparticles. As described herein, RF / fill values ​​of 40% or greater reflect acceptable aerodynamic performance characteristics. In certain embodiments disclosed herein, the respirable fraction at fill can be greater than 50%. In exemplary embodiments, the respirable fraction at fill can be up to about 80%, where about 80% of the fill is released with a particle size of <5.8 μm as measured using standard techniques.

[0032] As used herein, the term "dry powder" refers to a particulate composition that is not suspended or dissolved in a propellant or other liquid, and does not necessarily imply a complete absence of water molecules.

[0033] As used herein, "amorphous powder" refers to a dry powder that does not have a distinct repeating form, shape or structure, and includes all non-crystalline powders.

[0034] In one embodiment, the dry powder is a relatively highly cohesive powder that requires optimal deagglomeration conditions. In one embodiment, the inhalation system provides a compact, reusable, breath-powered inhaler in combination with a single-use cartridge containing a pre-metered dose of the dry powder formulation.

[0035] The term "unit dose inhaler" as used herein refers to an inhaler that houses or is adapted to contain a single container containing a dry powder formulation and delivers a single dose of the dry powder formulation to a user by inhalation from the container. In some cases, multiple unit doses will be required to provide a specified dose to the user. In one embodiment, the inhaler is a dry powder inhaler, which can be single-use disposable or multiple-use reusable with a single unit dose container.

[0036] As used herein, the term "multi-dose inhaler" refers to an inhaler that includes multiple containers, each container containing a pre-metered dose of dry powder medicinal product, and the inhaler delivers a single dose of the medicinal powder at any one time via inhalation.

[0037] As used herein, a "container" is an enclosure configured to hold or contain a dry powder formulation, a powder-containing enclosure, and can be a capped or uncapped structure. The container can be provided separately from the inhaler or can be structurally integrated (e.g., non-removable) within the inhaler. Additionally, the container can be filled with dry powder. Cartridges can also include a container.

[0038] As used herein, "powder mass" refers to an aggregate or agglomerate of powder particles that are irregular in shape, such as width, diameter, and length.

[0039] As used herein, the term "microparticle" refers to a particle having a diameter of about 0.5 to about 1000 μm, regardless of the exact external or internal structure, however, microparticles for pulmonary delivery less than 4 10 μm are generally desired, and particularly microparticles for pulmonary delivery having a mean particle size of less than about 5.8 μm.

[0040] In one embodiment, a pharmaceutical composition is provided that includes a vasoactive intestinal peptide and a diketopiperazine, including 3,6-bis(4-fumaryl-4-aminobutyl)-2,5-diketopiperazine or a salt thereof.

[0041] In an alternative embodiment, a method for treating a pulmonary disease, including interstitial lung disease, e.g., idiopathic pulmonary fibrosis, inflammation, acute or chronic respiratory distress syndrome, is provided comprising administering to a subject in need of treatment a vasoactive intestinal peptide and formula: [ka] and optionally one or more pharmaceutical excipients and / or carriers as defined above for the formulation. In one embodiment of the invention, the formulation comprises a vasoactive intestinal peptide and a diketopiperazine, such as 3,6-bis(4-fumaryl-4-aminobutyl)-2,5-diketopiperazine, or a salt thereof, and optionally one or more amino acids, which are isoleucine, leucine, trileucine, cystine, cysteine, glycine, lysine, arginine, histidine, or methionine, and one or more sugars, including lactose, mannitol, mannose, sorbitol, trehalose, and the like. In this and other embodiments, the diketopiperazine is in the form of a dry crystalline carrier prior to making a suspension in water for use in the formulation, although in other embodiments other carriers can be used including sugars, oligosaccharides, or polysaccharides including lactose, trehalose, mannose, mannitol, or sorbitol, zinc citrate, and ascorbic acid, where the formulation is made by solution and spray drying methods, where the peptide can be in a buffer solution having a pH ranging from pH 3.5 to about pH 7, or from pH 4.5 to pH 6.5.

[0042] In a further embodiment, the formulation is an amorphous dry powder comprising microparticles of fumaryl diketopiperazine disodium containing vasoactive intestinal peptide, a surfactant, and an amino acid as disclosed above. In an embodiment, the formulation comprises a crystalline dry powder comprising a peptide, including a thermosensitive peptide, containing vasoactive intestinal peptide, wherein the dry powder is formed by mixing vasoactive intestinal peptide in a solution comprising a diketopiperazine suspension of crystalline particles at an adjusted pH ranging from pH 3.5 to about pH 5.0, and spray drying the suspension.

[0043] In an exemplary embodiment, a dry powder formulation is provided comprising a peptide or protein, wherein the peptide or protein is susceptible to oxidation or thermal degradation. In a particular embodiment, the dry powder formulation comprises a peptide comprising a vasoactive intestinal peptide, a derivative or analog thereof, one or more amino acids comprising leucine, isoleucine and methionine, one or more sugars comprising mannitol, xylitol and trehalose, and optionally one or more antioxidants comprising ascorbic acid, selenium, carotenoids and vitamin E. In one embodiment of the invention, the dry powder formulation comprises a vasoactive intestinal peptide, a sugar, at least one amino acid and optionally an antioxidant, wherein the amino acid is methionine and / or leucine, the antioxidant is ascorbic acid and the sugar is mannitol.

[0044] In alternative embodiments, the one or more pharma- ceutically acceptable carriers are selected from sugars, e.g., saccharides, disaccharides, oligosaccharides, amino acids, where the sugars are, e.g., trehalose, mannose, mannitol or sorbitol, polyethylene glycols, polyvinylpyrrolidone, and diketopiperazines capable of forming microparticles, including fumaryl diketopiperazine, succinyl diketopiperazine, maleyl diketopiperazine, malonyl diketopiperazine and oxalyl diketopiperazine, or disodium or magnesium salts thereof, and derivatives thereof.

[0045] In one embodiment, a stable formulation is provided for use as a solution or suspension for nebulization or as a dry powder for inhalation. In an embodiment, the formulation comprises one or more labile peptides for the treatment of pulmonary inflammation, including vasoactive intestinal peptide and / or prostaglandin PG I2, including derivatives or analogs such as treprostinil, one or more amino acids that are isoleucine, leucine, trileucine, cystine, cysteine, glycine, lysine, arginine, histidine, or methionine, and one or more sugars, including lactose, mannitol, mannose, sorbitol, trehalose, and the like. In this and other embodiments, the carrier may be a saccharide, oligosaccharide, or polysaccharide, including lactose, trehalose, mannose, mannitol, or sorbitol, zinc citrate, and ascorbic acid, where the formulation is produced in solution and by spray drying methods, where the peptide is in a buffer solution having a pH ranging from about pH 3.5 to about pH 7, or from pH 4.5 to pH 6.5. In this embodiment, the amount of vasoactive intestinal peptide is up to about 300 μg and the amount of treprostinil is up to about 200 μg in the composition per dosage form.

[0046] In this embodiment, for example, the peptide, including vasoactive intestinal peptide, or protein content may be provided in the formulation in an amount ranging from about 0.1% (w / w) to about 50% (w / w), from about 0.5% (w / w) to about 40% (w / w), from about 0.5% (w / w) to about 20% (w / w), or from about 1% (w / w) to about 10% (w / w).

[0047] In one embodiment, a method is provided for effectively delivering a formulation to the lungs of a subject, comprising providing to a subject in need of treatment an inhalation system comprising an inhaler including a cartridge containing a formulation comprising a dry powder comprising up to 200 μg or 10 wt% vasoactive intestinal peptide, up to about 90 wt% mannitol, up to about 15 wt% leucine, and up to about 5 wt% ascorbic acid. In this and other embodiments, the inhalation system delivers a powder plume comprising particles having a volume median geometric diameter (VMGD) of less than 8 μm. In an exemplary embodiment, the VMGD of the fine particles may range from about 4 μm to about 6 μm. In an exemplary embodiment, the VMGD of the powder particles may range from 3 μm to about 6 μm for a single inhalation of a formulation with a dry powder fill mass ranging from 1 mg and 10 mg. In this and other embodiments, the inhalation system delivers more than 40% or more than 60% of the dry powder formulation from the cartridge. In certain embodiments herein, the dry powder formulations can include from 10 μg to about 20 μg, from about 20 μg to about 50 μg, from about 50 μg to about 100 μg, from about 100 μg to about 150 μg, or from about 150 μg to about 200 μg of active agent, including VIP, in the composition. In some embodiments, the dry powder formulations can include more than 200 μg depending on the needs of the patient.

[0048] A further embodiment relates to a drug delivery system comprising an inhaler, a unit dose dry powder drug container, and a dry powder comprising a thermosensitive peptide as disclosed herein, comprising a formulation comprising vasoactive intestinal peptide, methionine, and ascorbic acid. In one embodiment, the formulation herein comprises vasoactive intestinal peptide, leucine, methionine, and ascorbic acid.

[0049] In one embodiment, a formulation comprising vasoactive intestinal peptide, a derivative, or an analog thereof, wherein the formulation is at least about 67 mg / mL. 2 In another embodiment, the formulation further comprises a diketopiperazine microparticle, the diketopiperazine microparticle comprising a microparticle of fumaryl diketopiperazine having a specific surface area of ​​less than about 35 m / g. 2 / g to about 67m 2In another embodiment, the specific surface area of ​​the diketopiperazine microparticles is about 35 m / g. 2 / g to about 62m 2 In another embodiment, the specific surface area of ​​the diketopiperazine microparticles is about 40 m / g. 2 / g to about 62m 2 / g of diketopiperazine microparticles. In one embodiment, the diketopiperazine has a trans isomer content of from 45% to about 59%, or from about 52% to about 57%.

[0050] In alternative embodiments, the FDKP microparticles contain a drug or active agent that is unstable in solution or suspension. In various alternative embodiments of the FDKP microparticles, the drug can be, for example, glucagon-like peptide-1 (GLP-1), glucagon, exendin, parathyroid hormone, calcitonin, oxyntomodulin, derivatives and / or analogs thereof, and peptides including the same. In one embodiment using FDKP microparticles, the peptide content can be varied depending on downstream processing conditions. In certain examples, the FDKP microparticles or other carrier particles can be prepared to have a drug / peptide content that can vary depending on the targeted or delivered dose. For example, if the drug is a vasoactive intestinal peptide, the peptide content component can be about 1 μg to about 300 μg, 10 μg to about 200 μg, 30 μg to 150 μg, or 50 μg to 100 μg per dose in the powder formulation. In certain embodiments, microparticles in suspension with pharma- ceutically acceptable carriers and / or excipients can be spray dried to produce a stable formulation.

[0051] Further embodiments relate to a drug delivery system comprising a combination of an inhaler, a unit dose dry powder pharmaceutical container, e.g., a cartridge or capsule, and comprising a dry powder formulation disclosed herein with an active agent. In one embodiment, a delivery system for use with dry powders includes an inhalation system comprising a high resistance inhaler having an air conduit that provides high resistance to airflow through the conduit for deagglomerating and dispensing the powder. In one embodiment, the inhalation system has a resistance value of, for example, about 0.065 (√kPa) / liter to about 0.200 (√kPa) / liter per minute. In certain embodiments, the dry powder can be effectively delivered by inhalation using an inhalation system in which the peak inhalation pressure differential can range from about 2 Pa to about 20 kPa, which can result in a resulting peak flow rate of between about 7 liters and about 70 liters per minute. In an embodiment, the inhalation system is configured to provide a single dose by expelling the powder from the inhaler as a continuous stream or as one or more pulses of powder delivered to the patient. In some embodiments disclosed herein, the dry powder inhaler system includes a predetermined mass flow balance within the inhaler. For example, a flow balance of about 10% to 70% of the total flow rate leaving the inhaler to the patient is delivered by one or more dispensing ports, whose airflow passes through the area containing the powder formulation, and about 30% to 90% of the airflow is generated from other conduits of the inhaler. In addition, bypass flow, or flow that does not enter or leave the powder containment area, such as through the cartridge, can be recombined with the flow exiting the powder dispensing port within the inhaler to dilute, accelerate, and ultimately deagglomerate the fluidized powder before it leaves the mouthpiece. In one embodiment, a flow rate of about 7 to about 70 liters per minute dispenses more than 75% of the container or cartridge contents at a fill mass between 1 mg and 50 mg, or 1 mg and 30 mg. In certain embodiments, the inhalation systems described above are capable of releasing a percentage of the inhalable fraction / load of a powder dose in a single inhalation that is greater than 40%, greater than 50%, greater than 60%, or greater than 70%.

[0052] In certain embodiments, an inhalation system is provided that includes a dry powder inhaler and a dry powder formulation. In some aspects of this embodiment of the inhalation system, the dry powder formulation is provided in a unit dose cartridge. Alternatively, the dry powder formulation can be preloaded into the inhaler. In this embodiment, the configuration of the structure of the inhalation system allows the deagglomeration mechanism of the inhaler to generate a respirable fraction of more than 50%, i.e., more than half of the powder contained in the inhaler (cartridge) is discharged as particles smaller than 5.8 μm. The inhaler can discharge 85% or more of the powdered medicament contained in the container during administration. In certain embodiments, the inhaler can discharge 85% or more of the powdered medicament contained in one inhalation. In one embodiment, the inhaler can expel 90% or more of the cartridge or container contents in less than 3 seconds with a pressure difference between 2 and 5 kPa with a filling mass in the range of up to 30 mg.

[0053] Another embodiment disclosed herein includes a method for producing microparticles suitable for pulmonary administration as a dry powder formulation, comprising carrier particles including diketopiperazine microparticles. In this and other embodiments, the dry powder formulation is obtained by spray drying a solution comprising a labile peptide, where one or more excipients are dissolved and mixed in an aqueous solution comprising a zinc salt and a citrate salt, then adding an amount of peptide with mixing to form a feed solution, and spraying the solution stream into a dry nitrogen gas stream at an inlet temperature of about 120°C to 150°C and an outlet temperature of about 60°C to 65°C, or 50°C to 75°C.

[0054] In some embodiments, about 67 m 2A method for producing diketopiperazine microparticles having a specific surface area of ​​less than 1 / g and / or a trans isomer ratio of about 45% to 65%, utilizing a diketopiperazine having the formula 2,5-diketo-3,6-bis(NX-4-aminobutyl)piperazine disodium salt or magnesium salt, where X is selected from the group consisting of fumaryl, succinyl, maleyl, and glutaryl. In an exemplary embodiment, the diketopiperazine has the formula (bis-3,6-(N-fumaryl-4-aminobutyl)-2,5-diketopiperazine or 2,5-diketo-3,6-bis(N-fumaryl-4-aminobutyl)piperazine.

[0055] Another embodiment disclosed herein includes a method of delivering a drug, e.g., a peptide such as vasoactive intestinal peptide, to a patient in need of drug delivery, comprising administering a dry powder to the deep lung by inhalation using a dry powder inhaler in which the patient inhales the dry powder, wherein the dry powder comprises diketopiperazine microparticles comprising vasoactive intestinal peptide, methionine and a citrate buffer, wherein the microparticles are formed of diketopiperazine and have a concentration of about 35 μg / mL. 2 / g to about 67m 2 / g or about 40m 2 / g to about 67m 2 / g and / or in microparticles having a trans isomer content raging from about 45% to about 65%.

[0056] Further embodiments include a method of treating pulmonary edema comprising administering a dry powder to a patient in need of treatment, the method comprising having the patient inhale the dry powder from a breath-powered inhaler containing the dry powder, such that the dry powder in aerosol form reaches the lungs. In some embodiments, the dry powder is delivered in one or more inhalations per treatment session. In certain embodiments, the treatment comprises delivering the dry powder in one inhalation per dose.

[0057] In embodiments in which a diketopiperazine is used as a carrier or excipient, fumaryl diketopiperazine 3,6-bis(N-fumaryl-4-aminobutyl)-2,5-diketo-diketopiperazine, FDKP) is a preferred diketopiperazine for pulmonary use, which has the formula [ka] (E)-3,6-bis[4-(N-carbonyl-2-propenyl)amidobutyl]-2,5-diketopiperazine or a pharma- ceutically acceptable salt thereof, including the disodium, magnesium, lithium and potassium salts.

[0058] FDKP microparticles with diameters between about 0.5 microns and about 10 microns can reach the lungs and successfully pass through most of the natural barriers. Particles of this size can be easily prepared from diketopiperazines with acidic groups such as carboxylate groups of FDKP (including related molecules such as 2,5-diketo-3,6-di(4-X-aminobutyl)piperazine, where X is succinyl, glutaryl or maleyl). Acid precipitation results in self-assembled particles composed of aggregates of crystalline plates. The size of these plates is related to the specific surface area of ​​the particle, which affects the structure, loading capacity, and aerodynamic performance of the particle. Figure 1 shows a scanning electron microscope image of dry powder particles containing crystalline plates of (E)-3,6-bis[4-(N-carbonyl-2-propenyl)amidobutyl]-2,5-diketopiperazine and vasoactive intestinal peptide. As can be seen in the microscope image, the dry powder particles appear substantially homogeneous.

[0059] The embodiment disclosed herein is approximately 67 m 2 This indicates that microparticles with a specific surface area of ​​less than 62 m / g exhibit beneficial properties for the delivery of drugs to the lungs, such as improved aerodynamic performance. 2 Alternative embodiments having a specific surface area of ​​less than 100 μm / g provide greater assurance that batches of particles will meet minimum aerodynamic performance standards. Since the SSA also impacts drug loading / loading capacity, in various embodiments, an SSA of ≥ 35, 40, or 45 m is used to improve drug adsorption capacity. 2In some embodiments, the SSA requires 35 ml / g depending on the activator used. 2 / g.

[0060] The microparticles described herein can include one or more active agents. As used herein, "active agent" is used interchangeably with "drug" and refers to pharmaceutical agents including small molecule pharmaceuticals, biological agents, and bioactive agents. Active agents can be of natural, recombinant, or synthetic origin, including proteins, polypeptides, peptides, nucleic acids, organic polymers, synthetic organic compounds, polysaccharides and other sugars, fatty acids, and lipids. Labile active agents can be categorized into a variety of biological activities and classes, including vasoactive agents, neuroactive agents, hormones, anticoagulants, immunomodulatory agents, cytotoxic agents, antibiotics, antivirals, antigens, infectious agents, inflammatory mediators, hormones, and cell surface antigens, and can be used to treat a variety of diseases or disorders. Certain embodiments herein exemplify compositions using vasoactive intestinal peptides for treating pulmonary inflammatory diseases, including pulmonary edema.

[0061] The drug content delivered depends on the need of the subject and the potency of the drug. In certain embodiments, microparticles are used that are made of FDKP with a trans isomer content between 45% and 65%, typically greater than 0.01%. In one embodiment, the drug content delivered in microparticles with the trans isomer content can range from about 0.01% to about 50%, which is typical for peptides such as vasoactive intestinal peptide.

[0062] The range of drug content delivered depends on the disease to be treated and the severity of the disease in the patient. For example, in the case of pulmonary delivery, the drug content in the formulation used is typically between about 0.01% and about 90%, depending on the form and size of the drug to be delivered and the required dose potency. In the case of vasoactive intestinal peptide, the preferred loading amount is about 0.5wt% to about 50wt%, or about 1wt% to about 20wt%. In some embodiments, the formulation can be delivered by alternative inhaler embodiments.

[0063] [Example] The following examples are included to illustrate the disclosed formulations and methods. Those skilled in the art should understand that the techniques disclosed in the following examples represent techniques that the inventors have found to work well in implementing the present disclosure, and therefore can be considered to constitute preferred modes for its implementation. However, those skilled in the art should understand in light of the present disclosure that many changes can be made in one disclosed embodiment and still obtain the same or similar results.

[0064] Example 1 Preparation, characterization and stability of vasoactive intestinal peptide dry powder: Vasoactive intestinal peptide (VIP) is a 28 amino acid peptide purchased from American Peptide Company, Sunnyvale, CA. Crystalline particles of fumaryl diketopiperazine (FDKP) were prepared, suspended in water, mixed with VIP solution, flash frozen as pellets in liquid nitrogen, and lyophilized to produce sample powders. Three powders with target peptide loadings or contents of 5%, 10%, and 20% were prepared and measured, resulting in powders with VIP contents of 5.6%, 10.7%, and 20.8% (Table 1 below). RF / filling ratios (free filling ratio of cartridge) were 56% (88%), 47% (76%), and 22% (95%) for the target loadings of 5%, 10%, and 20%, respectively, indicating that the powders could be aerosolized to form particles suitable for pulmonary delivery. Samples of the prepared powders were also used for scanning electron microscope (SEM) analysis (RJ Lee Group, Inc.). The results of these experiments are discussed below and with respect to Figure 1. Figures 1A, 1B, and 1C show the physical properties of crystalline FDKP powder forms containing various amounts of VIP, 5.6 wt%, 10.7 wt%, and 20.8 wt%, respectively. From the SEM, it appears that the particles remain identical with different amounts of VIP peptide, with no discernible change in their morphology.

[0065] Preparation of VIP powder: All powders made in the following examples were made in the same manner up to a target yield of 1000 mg. For example, for a target content of 10%, FDKP particle suspension (9012 mg 10.00% solids = 901.2 mg FDKP crystalline particles) was weighed into a 20 mL clear glass vial. The suspension was stoppered and mixed using a magnetic stirrer to prevent settling of precipitate. VIP solution (1001 mg 10% peptide in 3 wt% acetic acid) was added to the vial and mixed. The final composition ratio was approximately 10:90 VIP particles:FDKP particles. The VIP-FDKP suspension was adjusted to pH 4.49 by adding an aliquot of 15% aqueous ammonia. The suspension was pelleted in a small crystallization dish containing liquid nitrogen. The dish was placed in a freeze dryer and lyophilized. The shelf temperature was increased from -45°C to 25°C and held at 25°C for approximately 10 hours. The material was transferred to a clear glass vial. The total yield of powder after transfer to vials was 993 mg. The actual yield of all powders was between 985 mg and 1000 mg (yields 98.5-100%). Samples of the different powders were taken and analyzed by different methods.

[0066] Powder analysis: Determination of the percentage of peptide content was performed by high pressure liquid chromatography (HPLC) using a Phenomenex Gemini C18 column and mobile phase A of water containing 0.1% trifluoroacetic acid (TFA) and acetonitrile containing 0.1% (TFA), and the resulting data was analyzed using WatersEmpower™ software.

[0067] Sample powders were prepared at 1 mg / mL in 200 mM sodium phosphate, pH 7.5. Four VIP standards were prepared in 200 mM sodium phosphate, pH 7.5, independent of the VIP API. Standard curves are shown in Figure 2 and show that the VIP content in the formulation is linear without reaching saturation of the FDKP particles at the tested amounts. Figure 3 shows data from HPLC chromatograms of the VIP standards, showing a single peak for each standard tested. Figure 4 shows sample dry powders with various crystalline FDKP-VIP target contents, i.e., 5% (1=______), 10% (2= __ __ __ ) and 20% (3= - - - ), and their corresponding HPLC chromatograms. Table 1 shows some of the characteristics of the powders produced and their performance in tests using a dry powder inhaler. [Table 1]

[0068] The data show that powders from dry powder inhalers using VIP contents ranging from 5.6% to 20% were delivered at 76% to 95% loading, meaning that the powders were aerosolizable. RF / loading fraction indicates the percentage of particles that can reach the lungs due to their particle size. The data demonstrate that the 5.6% sample contains the highest percentage of particles that reach the lungs at 56%. The data also demonstrate that the dry powders could be loaded accurately, and that aerodynamic performance decreases as peptide content increases.

[0069] Example 2 VIP FDKP-suspension containing methionine: Methionine (added at levels ranging from 0.0 g to 0.0358 g) (methionine was added to give molar ratios of vasoactive intestinal peptide (VIP):methionine of 1:0, 1:1, 1:4, 1:8, 1:16, and 1:32) was dissolved in 3% aqueous acetic acid (acetic acid solutions were added at levels ranging from 0.54 g to 1.08 g depending on the VIP loading used to prepare 5% or 10% VIP solutions in acetic acid). 10% solutions of VIP were prepared by adding VIP (VIP was added at levels ranging from 0.05 g to 0.25 g) to methionine in 3% aqueous acetic acid. Crystalline (T)FDKP suspension (amount of T suspension used ranged from 9.8 g to 23.46 g, varying based on the amount of methionine and VIP added to the suspension) (T suspension used contained 8.61% solids) was added to the VIP / methionine / acetic acid solution. These mixtures were stirred until a homogenous suspension was obtained. Samples of the suspension were assayed for stability of VIP content by reverse phase (RP) HPLC to monitor the level of Met(O)-VIP formation. The remaining suspension was spray-dried or freeze-dried to a powder. Stability samples were analyzed and the results are shown in Figure 5 for 5 wt% and 10 wt% VIP spiked with various concentrations of methionine compared to the control. Figure 5 demonstrates that increasing the methionine content in solution increases the stability of the VIP peptide in solution or suspension and therefore will also increase the stability of the dry powder.

[0070] Example 3 Preparation of 10% VIP-Methionine FDKP-Powder: Methionine (methionine loading was varied between 0.18 g and 0.36 g, with VIP:methionine molar ratios of 1:16 and 1:32, respectively) was dissolved in 3% aqueous acetic acid (4.05 g of 3% aqueous acetic acid was used to dissolve the low level of methionine and 6.75 g was used to dissolve the high level of methionine). VIP (0.25 g) was added to the methionine / acetic acid solution and stirred until completely dissolved. T-suspension (8.61% solids) (T-suspension charge was 23.46 g for formulations containing low methionine amount and 21.37 g for formulations containing high methionine charge) was added to the VIP / methionine / acetic acid solution. The resulting suspension was stirred until homogenous and then pelleted in liquid nitrogen. The pelleted suspensions were dried by lyophilization using a program that started at -40°C under full vacuum, ramped the temperature at 0.2°C / min to 25°C, and held the temperature until each powder was completely dry.

[0071] Example 4 Preparation of VIP powder containing mannitol: Several VIP-containing solutions simulating a 10% VIP dry powder formulation were prepared and periodically analyzed by reverse-phase (RP) HPLC to assess the chemical stability of these solutions. Solutions containing mannitol alone, leucine alone, and their combinations were evaluated. Two solutions containing two antioxidants, methionine and ascorbic acid, were also evaluated.

[0072] Preparation of solutions: In the experiment, all solutions were prepared targeting a 10% VIP powder formulation, as shown in Table 2 below. For example, a 10% mannitol solution was prepared by adding 1 g mannitol to 9 g water, and a 1% leucine solution was prepared by adding 0.1 g leucine to 9.9 g water. The solids content of the XC suspension was 1.97%. For each solution, 5 mg of VIP was weighed into a small vial and other reagents were added according to Table 2 below. The solutions were stirred continuously at room temperature. Small aliquots were taken at each time point, diluted in diluent (0.2 M sodium phosphate, 3 mM methionine, pH 7), and analyzed by RP-HPLC. FDKP-VIP crystalline complex (XC) particle suspensions were also prepared as above and analyzed as described herein. [Table 2]

[0073] Solution stability experiments were performed as described above and the results are shown in Figure 6 with mannitol, leucine, methionine and XC suspensions. As shown in Figure 6, samples containing mannitol (Sample A), leucine (Sample B) and the mannitol + leucine sample (Sample D) showed little degradation over the course of 2 hours. The sample containing the antioxidant methionine (Sample E) also showed little degradation over the course of 2 hours. The XC suspension of Sample C showed less degradation than the suspension with added ascorbic acid (Sample F). Surprisingly, the results for ascorbic acid (Sample F) showed more degradation of the oxygen scavenger VIP. Presumably this is due to the concentrations used in the examples.

[0074] The terms "a" and "an" and "the" and similar referents used in the context of the description of the invention (particularly in the context of the claims below) are to be construed as covering both the singular and the plural, unless otherwise stated herein or otherwise clearly contradicted by context. The recitation of ranges of values ​​herein is merely intended to serve as a shorthand method for individually referring to each individual value falling within the range. Unless otherwise indicated herein, each individual value is incorporated herein as if it were individually set forth herein. All methods described herein may be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. Any examples or exemplary language (e.g., "such as") used herein are merely intended to better illustrate the invention and do not limit the scope of the claimed invention. No language in this specification should be construed as indicating any non-claimed element essential to the practice of the invention.

[0075] Use of the term "or" in the claims is used to mean "and / or" unless expressly indicated to refer to alternatives only or unless the alternatives are mutually exclusive, however, the present disclosure supports a definition that refers only to alternatives and "and / or."

[0076] Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member may be referenced and claimed individually or in any combination with other members of the group or other elements found herein. It is anticipated that for reasons of convenience and / or patentability, one or more members of a group may be included in or deleted from a group. When such inclusion or deletion is made, the specification shall be deemed to include the group as modified to satisfy all Marksch group descriptions used in the appended claims.

[0077] Preferred embodiments of the invention are described herein, including the best mode known to the inventors for carrying out the invention. Of course, variations on those preferred embodiments will become apparent to those skilled in the art upon reading the foregoing description. The inventors expect that such variations will be adopted by those skilled in the art as appropriate, and the inventors intend that the invention be practiced other than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the present invention unless otherwise stated herein or clearly contradicted by context.

[0078] Certain embodiments disclosed herein may be further limited in the claims using the language "consisting of" or "consisting essentially of." When used in the claims, whether as filed or added by amendment, the transitional term "consisting of" excludes any component, step, or ingredient not specified in the claim. The transitional term "consisting essentially of" limits the claim to those materials or steps specified and that do not materially affect the basic and novel characteristics. Thus, the claimed embodiments of the invention are essentially or explicitly described and enabled herein.

[0079] Additionally, throughout this specification, numerous patent and printed publications are cited, each of which is herein individually incorporated by reference in its entirety.

[0080] Moreover, it should be understood that the embodiments of the invention disclosed herein are illustrative of the principles of the invention. Other modifications that may be adopted are within the scope of the invention. Thus, by way of example, and not of limitation, alternative configurations of the invention may be utilized in accordance with the teachings herein. Thus, the invention is not limited to that precisely as shown and described.

[0081] (Additional Note) (Appendix 1) Dry powders for treating pulmonary diseases, comprising an inhalable dry powder pharmaceutical formulation comprising vasoactive intestinal peptide, a derivative thereof, an analog thereof, or a salt thereof, one or more amino acids, one or more antioxidants, and / or a pharma- ceutically acceptable carrier or excipient.

[0082] (Appendix 2) The dry powder described in Appendix 1, wherein the vasoactive intestinal peptide is up to 300 μg per treatment session.

[0083] (Appendix 3) 2. The dry powder of claim 1, wherein the pharma- ceutically acceptable carrier is a sugar selected from the group consisting of mannose, mannitol, trehalose, and sorbitol.

[0084] (Appendix 4) 2. The dry powder described in Appendix 1, wherein the pharma- ceutical acceptable carrier is mannitol.

[0085] (Appendix 5) The dry powder described in Appendix 1, wherein the pharma- ceutical acceptable carrier is a diketopiperazine.

[0086] (Appendix 6) 5. The dry powder of claim 4, wherein the diketopiperazine is fumaryl diketopiperazine or succinyl diketopiperazine.

[0087] (Appendix 7) 2. The dry powder of claim 1, wherein the one or more amino acids are selected from the group consisting of lysine, glycine, leucine, isoleucine, trileucine, histidine and methionine.

[0088] (Appendix 8) 8. The dry powder of claim 7, wherein the one or more amino acids are leucine and methionine.

[0089] (Appendix 9) 2. The dry powder of claim 1, wherein the one or more amino acids or antioxidants is methionine.

[0090] (Appendix 10) 2. The dry powder of claim 1, wherein the one or more antioxidants are selected from the group consisting of ascorbic acid, vitamin E, selenium, methionine and carotenoids.

[0091] (Appendix 11) 2. The dry powder of claim 1, wherein the dosage of the inhalable dry powder pharmaceutical formulation is up to 200 μg of vasoactive intestinal peptide, a derivative, analogue, or salt thereof, and up to 90% by weight of mannitol.

[0092] (Appendix 12) 1. An inhalable pharmaceutical formulation comprising mannitol, leucine and methionine, and a vasoactive intestinal peptide, a derivative, or an analog thereof, in a dosage of up to 200 μg for the treatment of edema in a subject.

[0093] (Appendix 13) 13. The inhalable pharmaceutical formulation of claim 12, manufactured in a dosage to be administered at least once daily.

Claims

1. Dry powders for treating pulmonary diseases, including inhalable dry powder pharmaceutical formulations comprising vasoactive intestinal peptide, its derivatives, analogs, or salts thereof, one or more amino acids, one or more antioxidants, and / or a pharma- ceutically acceptable carrier or excipient.

2. 2. The dry powder of claim 1, wherein the vasoactive intestinal peptide is up to 300 μg per treatment session.

3. 2. The dry powder of claim 1, wherein the pharma- ceutically acceptable carrier is a sugar selected from the group consisting of mannose, mannitol, trehalose and sorbitol.

4. 2. The dry powder of claim 1, wherein the pharma- ceutical acceptable carrier is mannitol.

5. 2. The dry powder of claim 1, wherein the pharma- ceutical acceptable carrier is a diketopiperazine.

6. 5. The dry powder of claim 4, wherein the diketopiperazine is fumaryl diketopiperazine or succinyl diketopiperazine.

7. 2. The dry powder of claim 1, wherein the one or more amino acids are selected from the group consisting of lysine, glycine, leucine, isoleucine, trileucine, histidine and methionine.

8. 8. The dry powder of claim 7, wherein the one or more amino acids are leucine and methionine.

9. 2. The dry powder of claim 1, wherein the one or more amino acids or antioxidants is methionine.

10. 2. The dry powder of claim 1, wherein the one or more antioxidants are selected from the group consisting of ascorbic acid, vitamin E, selenium, methionine and carotenoids.

11. 2. The dry powder of claim 1, wherein the dosage of the inhalable dry powder pharmaceutical formulation is up to 200 μg of vasoactive intestinal peptide, a derivative, analogue, or salt thereof, and up to 90% by weight of mannitol.

12. 1. An inhalable pharmaceutical formulation comprising mannitol, leucine and methionine, and a vasoactive intestinal peptide, a derivative, or an analog thereof, in a dosage of up to 200 μg for the treatment of edema in a subject.

13. 13. An inhalable pharmaceutical formulation according to claim 12, manufactured in a dosage to be administered at least once daily.

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