Dry powder formulation

Abstract

To provide DKP microcrystals produced by an improved method which are not irreversibly self-assembled into fine particles.SOLUTION: Microcrystals are dispersed by spray, and can be re-formed into particles having a spherical shell form by spray drying. An activator and a filler, by spraying and drying solution containing components incorporated into microcrystalline diketopiperazine particles, can be incorporated into particles. Particularly, a microcrystalline particle composition is suitable for pulmonary drug delivery of one or a plurality of peptide, protein, nucleic acid molecule and / or organic small molecules.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Disclosed herein are microcrystalline diketopiperazine (DKP) particles, compositions, methods for making the particles, and methods for using the particles. In particular, the particles can be used as delivery systems for drugs or active agents in the treatment of endocrine-derived diseases or disorders, including, for example, diabetes and obesity. [Background technology]

[0002] Drug delivery has been a major problem for many years, especially when the compound that is orally administered to a subject and delivered is unstable under the conditions encountered in the gastrointestinal tract before reaching its target location.For example, in many cases, oral administration of drugs is preferred, especially in terms of ease of administration, patient compliance and cost reduction.However, many compounds are ineffective, or show low or variable efficacy when administered orally.This may be because the drug is unstable to the conditions in the gastrointestinal tract or is absorbed inefficiently.

[0003] Due to the problems associated with oral drug delivery, pulmonary drug delivery has been explored. For example, typical pulmonary drug delivery systems are designed to have an effect on lung tissue, such as vasodilators, surfactants, chemotherapy drugs, or vaccines for influenza or other respiratory diseases. Other drugs, including basic drugs, are delivered to the lung because it is a particularly suitable tissue for gene therapy treatment, such as in cystic fibrosis. In this case, a retroviral vector expressing a defective adenosine deaminase is administered to the lung.

[0004] Pulmonary drug delivery can also be carried out for drugs with systemic effects.The advantages of the lung in systemic drug delivery include a large surface area and the ease of uptake by the mucous membrane surface of the lung.Pulmonary drug delivery systems present many difficulties, for example, the use of propellants and the aerosolization of biological agents, which can denature proteins and peptides and lead to excessive loss of delivered drugs.Another problem associated with all of these forms of pulmonary drug delivery is that drug delivery to the lung is difficult due to the problems of getting the drug through all natural barriers, such as the cilia lining the trachea, and trying to administer a uniform drug volume and weight.

[0005] Thus, there is room for improvement in pulmonary delivery of drugs. Summary of the Invention [Means for solving the problem]

[0006] The present disclosure provides improved microcrystalline particles, compositions, particle manufacturing methods, and methods that enable improved pulmonary drug delivery for treating diseases and disorders in subjects. The embodiments disclosed herein achieve improved delivery by providing crystalline diketopiperazine compositions comprising microcrystalline diketopiperazine particles with a high capacity for drug adsorption, resulting in powders with a high drug load of one or more active agents. Powders produced using the microcrystalline particles of the present disclosure can deliver increased drug loads in smaller powder doses, facilitating drug delivery to patients. Powders can be produced by a variety of methods, including using surfactant-free or surfactant-containing solutions, depending on the starting material.

[0007] Certain embodiments described herein may include a powder comprising a plurality of substantially uniform microcrystalline particles, the particles having a substantially hollow spherical structure and may be porous, with a shell comprising non-self-assembled crystallites of diketopiperazine.

[0008] Certain embodiments described herein may include a powder comprising a plurality of substantially uniform microcrystalline particles, the particles having a substantially hollow spherical structure and may be porous, with a shell comprising non-self-assembled crystallites of diketopiperazine, the particles having a volume average geometric diameter of less than 5 μm.

[0009] In certain embodiments herein, up to about 92% of the microcrystalline particles have a volume average geometric diameter of 5.8 μm or less. In one embodiment, the particle shell is composed of connected diketopiperazine crystals with one or more drugs adsorbed on their surfaces. In certain embodiments, the particles may entrap drugs within their interior void volume and / or a combination of drugs adsorbed on the surface of the microcrystals and entrapped within the void volume of the interior of the spheres.

[0010] In certain embodiments, a diketopiperazine composition is provided comprising a plurality of substantially uniformly formed microcrystalline particles, the particles having a substantially hollow spherical structure and a shell comprising non-self-assembled diketopiperazine crystallites, the particles being formed by a process comprising combining a diketopiperazine having a trans-isomer content ranging from about 45% to about 65% in solution with an acetic acid solution in the absence of a surfactant, simultaneously homogenizing the diketopiperazine in a high-shear mixer under high pressure up to 2000 psi to form a precipitate, washing the precipitate in suspension with deionized water, concentrating the suspension, and drying the suspension in a spray dryer.

[0011] The method can further include adding, with mixing, a solution containing an active agent or ingredient, such as a drug or bioactive agent, prior to the spray drying step, such that the active agent or ingredient is adsorbed or entrapped on or within the particles. The particles produced by this method can be in the submicron size range prior to spray drying.

[0012] In certain embodiments, a diketopiperazine composition is provided comprising a plurality of substantially uniformly formed microcrystalline particles. The particles have a substantially hollow spherical structure and a shell comprising non-self-assembled diketopiperazine crystallites, the particles having a volume average geometric diameter of 5 μm or less. The particles are formed by a process comprising combining diketopiperazine in solution with an acetic acid solution in the absence of a surfactant and simultaneously homogenizing the mixture in a high-shear mixer under high pressure up to 2000 psi to form a precipitate, washing the precipitate in suspension with deionized water, concentrating the suspension, and drying the suspension in a spray dryer.

[0013] The method can further include adding, with mixing, a solution containing an active agent or ingredient, such as a drug or bioactive agent, prior to the spray drying step, such that the active agent or ingredient is adsorbed or entrapped on or within the particles. The particles produced by this method can be in the submicron size range prior to spray drying.

[0014] In certain embodiments, a diketopiperazine composition is provided comprising a plurality of substantially uniformly formed microcrystalline particles. The particles have a substantially hollow spherical structure and a shell comprising non-self-assembled diketopiperazine crystallites, the particles having a volume average geometric diameter of 5 μm or less. The particles are formed by a process comprising combining diketopiperazine in solution with an acetic acid solution in the absence of a surfactant and an active agent, simultaneously homogenizing the mixture in a high-shear mixer under high pressure up to 2000 psi to form a precipitate, washing the precipitate in suspension with deionized water, concentrating the suspension, and drying the suspension in a spray dryer.

[0015] The method can further include the step of adding a solution containing an active agent or ingredient, such as a drug or bioactive agent, by mixing prior to the spray drying step, such that the active agent or ingredient is adsorbed or entrapped on or within the particles. The particles produced by this method can be in the submicron size range prior to spray drying.

[0016] In one embodiment, the composition may comprise microcrystalline particles containing one or more active ingredients. The active ingredient may be a peptide, a protein, a nucleic acid molecule, a small organic molecule, or a combination thereof. In embodiments in which the active ingredient is a peptide, oligopeptide, polypeptide, or protein, the peptide, oligopeptide, polypeptide, or protein may include an endocrine hormone, a neurotransmitter, a vasoactive peptide, a receptor peptide, a receptor agonist, or a receptor antagonist. In certain embodiments, the endocrine hormone is insulin, parathyroid hormone, calcitonin, glucagon, glucagon-like peptide-1, oxyntomodulin, peptide YY, leptin, or an analog of these endocrine hormones. In embodiments, excipients may be incorporated into the particles by addition to one, another, or all of the feedstocks used in the spray-drying process.

[0017] In one embodiment in which the composition includes insulin as an active ingredient, the composition can contain insulin in an amount, for example, up to 9 or 10 units per milligram of powder delivered to the patient. In this embodiment, insulin can be delivered to the patient in an amount, for example, up to 100 units, in a single inhalation using a dry powder inhaler. The composition can be administered to a patient in need of insulin for the treatment of diabetes and / or hyperglycemia.

[0018] In exemplary embodiments, the crystalline diketopiperazine composition comprises a diketopiperazine of the formula 2,5-diketo-3,6-bis(N-X-4-aminoalkyl)piperazine, where the alkyl represents an alkyl containing 3 to 20 carbon atoms, including propyl, butyl, pentyl, hexyl, heptyl, and the like. The formula is, for example, 2,5-diketo-3,6-bis(N-X-4-aminobutyl)piperazine, where X is selected from the group consisting of fumaryl, succinyl, maleyl, malonyl, and glutaryl, or a salt thereof. In certain embodiments, the diketopiperazine is (bis-3,6-(N-fumaryl-4-aminobutyl)-2,5-diketo-diketopiperazine, having the formula: [ka]

[0019] In various embodiments, a method for producing a dry powder comprising microcrystalline particles suitable for pulmonary administration is provided. The method can be carried out using a surfactant-free solution or a surfactant-containing solution. In one aspect, the diketopiperazine comprises a trans-isomer content ranging from about 45% to 65%.

[0020] Certain embodiments described herein include methods for producing dry powders comprising crystalline diketopiperazine microparticles from starting materials comprising the free acid diketopiperazine.

[0021] Certain embodiments described herein include methods for producing dry powders comprising crystalline diketopiperazine microparticles from starting materials comprising diketopiperazine salts.

[0022] In one embodiment, the method comprises: dissolving diketopiperazine in aqueous ammonia to form a first solution; simultaneously feeding the first solution and a second solution containing about 10.5% acetic acid into a high shear mixer under high pressure and at a pH of less than about 6.0; homogenizing the first solution and the second solution to form a suspension comprising microcrystals of diketopiperazine in suspension, the suspension having a bimodal distribution of microcrystals having a particle size ranging from about 0.05 μm to about 10 μm in diameter; atomizing the suspension under an air or gas stream; and reforming the particles by spray drying to produce a dry powder comprising microcrystalline particles having substantially hollow spheres.

[0023] In another embodiment, the method comprises: dissolving a diketopiperazine in aqueous sodium hydroxide and optionally a surfactant to form a first solution; simultaneously feeding the first solution and a second solution comprising about 10.5% acetic acid, and optionally a surfactant, into a high shear mixer under high pressure and at a pH of less than about 6.0; homogenizing the first solution and the second solution to form a suspension comprising microcrystals of diketopiperazine in suspension, the suspension having a bimodal distribution of microcrystals having a particle size ranging from about 0.05 μm to about 10 μm in diameter and a trans isomer content ranging from about 45% to 65%; atomizing the suspension under an air or gas stream; and reforming the particles by spray drying to produce a dry powder comprising microcrystalline particles having substantially hollow spheres.

[0024] In one embodiment, the method comprises: dissolving diketopiperazine in aqueous ammonia to form a first solution; simultaneously feeding the first solution and a second solution comprising about 10.5% acetic acid into a high shear mixer under high pressure and at a pH of less than about 6.0 to form a suspension comprising microcrystals of diketopiperazine in suspension, the suspension having a bimodal distribution of microcrystals having a particle size ranging from about 0.05 μm to about 10 μm in diameter; atomizing the suspension under an air or gas stream; and reforming the particles by spray drying to produce a dry powder comprising microcrystalline particles having substantially hollow spheres.

[0025] The method can further include adding a third solution to the diketopiperazine microcrystalline suspension before spraying the suspension, the solution containing a drug or pharmaceutically active ingredient, and the spraying can be carried out using an externally mixed two-fluid nozzle inserted into a spray dryer equipped with a high-efficiency cyclone separator under air or gas, including nitrogen gas.

[0026] In certain embodiments, the particles in the suspension have a bimodal particle size distribution as measured by laser diffraction, with a first peak of particles having an average particle size of about 0.2 μm to about 0.4 μm in diameter and a second peak of particles having an average size of about 2.1 μm to about 2.4 μm in diameter.

[0027] In one embodiment, the suspension atomization process may use a nitrogen flow of about 700 liters per hour as the process gas, and the nozzle temperature may be maintained at about 25°C.

[0028] The microcrystalline particles formed by the above method do not self-assemble when suspended in a solution, such as water or other aqueous solvent. In certain embodiments, the method includes a diketopiperazine of formula 2,5-diketo-3,6-bis(N-X-4-aminobutyl)piperazine, where X is selected from the group consisting of fumaryl, succinyl, maleyl, malonyl, and glutaryl. In a specific embodiment, the method includes homogenizing a solution of the diketopiperazine in a high shear mixer, the diketopiperazine being (bis-3,6-(N-fumaryl-4-aminobutyl)-2,5-diketo-diketopiperazine or a salt thereof, including the disodium, dipotassium, magnesium, calcium, and dilithium salts.

[0029] In embodiments, a crystalline diketopiperazine composition comprising a plurality of microcrystalline particles of substantially uniform size is obtained as the product of the spray drying process.

[0030] In embodiments, a crystalline diketopiperazine composition comprising a plurality of microcrystalline particles having a bimodal size distribution is obtained as a product of the crystallization process.

[0031] When a crushing process is used, the larger species of the bimodal distribution can be shifted to smaller sizes.

[0032] Certain embodiments include a method for forming microcrystalline particles of diketopiperazine acid to produce a dry powder with high drug loading, the method comprising the use of a diketopiperazine salt, including 2,5-diketo-3,6-bis(N-fumaryl-4-aminobutyl)piperazine disodium salt, as a starting compound, the method comprising: dissolving a diketopiperazine salt in water containing a surfactant in an amount of about 0.2% (w / w) to about 6% (w / w) to form a first solution; simultaneously combining the first solution with a second solution comprising about 8% (w / w) to about 12% (w / w) acetic acid in a high shear mixer under high pressure and at a pH of less than about 6.0; homogenizing the first solution and the second solution to form a suspension comprising microcrystals of diketopiperazine in suspension, the suspension having a bimodal distribution of microcrystals having a particle size ranging from about 0.05 μm to about 10 μm in diameter; atomizing the suspension under an air or gas stream; and reforming the particles by spray drying to produce a dry powder comprising microcrystalline particles of diketopiperazic acid having substantially hollow spheres.

[0033] In a specific embodiment, the microcrystalline particles can be produced by a process comprising preparing a first solution containing a diketopiperazine, such as 2,5-diketo-3,6-(N-fumaryl-4-aminobutyl)piperazine disodium salt, in water and a surfactant, such as polysorbate 80; preparing a second solution containing acetic acid at a concentration of about 10.5% (w / w) and the surfactant at a concentration of about 0.5% (w / w); mixing the first and second solutions in a high shear mixer to form a suspension; optionally testing the suspension to determine a bimodal particle size distribution, with particles ranging in size from about 0.2 μm to about 10 μm in diameter, wherein a first peak of particles has an average diameter of about 0.4 μm and a second peak of particles has an average diameter of about 2.4 μm; and spray drying the suspension to obtain a dry powder.

[0034] Certain embodiments may include a disruption step, e.g., using sonication, agitation, or homogenization, to reduce the size of the larger sized populations in the bimodal distribution. In embodiments, a disruption step may be performed prior to spraying the suspension.

[0035] In embodiments herein, the method for producing microcrystalline diketopiperazine particles can further include a washing step using deionized water. In one embodiment, the spraying step can be carried out using, for example, an externally mixed two-fluid nozzle inserted into a spray dryer equipped with a high-efficiency cyclone separator.

[0036] The method can further include adding a solution containing one or more active agents to the suspension prior to dispersion and / or spray drying, where the active agents are peptides, oligopeptides, polypeptides, proteins, nucleic acid molecules, or small organic molecules. Peptides can include endocrine hormones, including insulin, parathyroid hormone, calcitonin, glucagon, glucagon-like peptide-1, oxyntomodulin, peptide YY, leptin, and analogs of these endocrine hormones. The method can optionally include adding a solution containing a surfactant and / or a pharmaceutically acceptable carrier, where the pharmaceutically acceptable carrier includes amino acids such as leucine and isoleucine, and / or monosaccharides, disaccharides, oligosaccharides such as lactose and trehalose, and / or sugar alcohols, including mannitol, sorbitol, and the like.

[0037] In another embodiment, compositions containing two or more active agents can be produced using the methods of the present disclosure. The method for producing such compositions includes producing microcrystalline diketopiperazine particles containing two or more active agents, where each active agent / active ingredient is processed separately in solution and added to a separate suspension of diketopiperazine particles, the solution conditions are modified to promote adsorption of the active agent onto the surface of the microcrystals, and then the two or more separate suspensions containing the active agents are blended before dispersing and spray-drying the particles. In a modified procedure, the blend includes a suspension containing diketopiperazine particles without the active agent, for example, to achieve a lower overall active agent content. In an alternative embodiment, one or more separate solutions containing a single active agent can be combined with a single suspension containing diketopiperazine particles before dispersing and spray-drying the particles. The resulting dry powder comprises a composition containing two or more active ingredients. In these embodiments, the amount of each ingredient in the composition can be controlled according to the needs of the patient population being treated.

[0038] In another embodiment, the dry powder comprises a composition comprising 2,5-diketo-3,6-bis(NX-4-aminobutyl)piperazine, where X is fumaryl, and the composition comprises substantially uniform microcrystalline particles comprising a drug. The particles are substantially spherical in shape with a substantially hollow core, and the microcrystals form the shell of the sphere. In another embodiment, the dry powder comprises a diketopiperazine of formula 2,5-diketo-3,6-bis(NX-4-aminobutyl)piperazine and a drug, where the drug is a peptide, and the peptide can be of various peptide lengths, molecular sizes, or molecular weights, including insulin, glucagon-like peptide-1, glucagon, exendin, parathyroid hormone, calcitonin, and oxyntomodulin.

[0039] Further embodiments include drug delivery systems comprising an inhaler with or without a cartridge, the cartridge containing a unit-dose dry powder drug container, e.g., a cartridge, and a powder containing the particles and active agent described herein. In one embodiment, a delivery system for use with dry powders includes an inhalation system comprising a high-resistance inhaler with 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 of, for example, about 0.065 (√kPa) / L to about 0.200 (√kPa) / L per minute. In certain embodiments, the dry powder can be effectively delivered by inhalation with an inhalation system that can have a peak inhalation pressure differential ranging from about 2 kPa to about 20 kPa, which can produce a peak flow rate of between about 7 and 70 L per minute. In certain embodiments, 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 waves (pulses) of powder delivered to the patient. In certain embodiments described herein, the dry powder inhalation system has a predetermined mass flow balance within the inhaler, and the inhalation conduit is designed to have a variable flow distribution during inhalation. For example, approximately 10% to 70% of the total flow leaving the inhaler and entering the patient is delivered by one or more dispensing ports, with the air flow passing through an air conduit designed with a region containing the powder formulation. Approximately 30% to 90% of the air flow occurs through other conduits of the inhaler during the inhalation maneuver. Additionally, bypass flow, or flow that does not enter or exit the powder-containing region, such as through the cartridge, can be recombined within the inhaler with the flow exiting the powder dispensing port to dilute, accelerate, and ultimately deagglomerate the fluidized powder before it exits the mouthpiece. In one embodiment, a flow rate ranging from approximately 7 L to 70 L per minute results in greater than 75% of the dispensed container or cartridge content at a fill mass between 1 mg and 50 mg. In certain embodiments, the inhalation system is capable of delivering greater than 40%, 50%, 60% or 70% of the respirable fraction of the powder dose per charge in a single inhalation.

[0040] In certain embodiments, the drug delivery system comprising an inhaler may comprise an inhaler particularly suited for use with particulate forms, including dry powders, such as crystalline or amorphous forms.

[0041] In certain embodiments, an inhalation system is provided that includes a dry powder inhaler, a dry powder formulation comprising microcrystalline particles of fumaryl diketopiperazine having an FDKP trans-isomer content of between 45% and 65%, and one or more active agents. 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 may be pre-loaded into the inhaler. In this embodiment, the structural configuration of the inhalation system enables the deagglomeration mechanism of the inhaler to achieve a respirable fraction of greater than 50%. That is, more than half of the powder contained in the inhaler (cartridge) is released as particles less than 5.8 μm. The inhaler is capable of releasing more than 85% of the powdered formulation contained in the container during dosing. In certain embodiments, the inhaler is capable of releasing more than 85% of the powdered formulation contained in a single inhalation. In one embodiment, the inhaler is capable of expelling more than 90% of the cartridge or container contents in less than 3 seconds at a pressure difference between 2 kPa and 5 kPa for fill masses ranging up to 30 mg.

[0042] The embodiments described herein also include methods. In one embodiment, a method for treating an endocrine-related disease or disorder comprises administering to a subject in need thereof a dry powder formulation comprising FDKP microcrystalline particles, the FDKP containing FDKP, which may have a trans-isomer content of about 45% to about 65%, and a drug suitable for treating the disease or disorder, wherein the microparticles are produced by the disclosed method. One embodiment includes a method for treating an insulin-related disorder comprises administering to a subject in need thereof a dry powder comprising the above-described FDKP microcrystalline particles. The method comprises administering to a subject a dry powder formulation comprising microcrystalline particles of fumaryl diketopiperazine having a trans-isomer content ranging from about 45% to 65%, the particles being hollow spheres and free of surfactant. In various embodiments, insulin-related disorders can specifically include or exclude any or all of the following: prediabetes, type 1 diabetes (honeymoon phase, post-honeymoon phase, or both), type 2 diabetes, gestational diabetes, hypoglycemia, hyperglycemia, insulin resistance, secretory dysfunction, impaired early release of insulin, loss of pancreatic beta cell function, loss of pancreatic beta cells, and metabolic disorders. In one embodiment, the dry powder comprises insulin. In other embodiments, the dry powder comprises oxyntomodulin, peptide YY, leptin, oxytocin, glucagon, exendin, a GLP-1 analog, or a combination thereof.

[0043] Another embodiment described herein includes a method of delivering peptides, including GLP-1, oxyntomodulin, peptide YY, oxytocin, and insulin, to a patient in need thereof, comprising administering a dry powder comprising diketopiperazine microcrystalline particles described herein to the deep lung via inhalation of the dry powder by the patient. Aspects of this embodiment embody certain features of the inhalation system.

[0044] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the examples described 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. [Brief explanation of the drawings]

[0045] [Figure 1A] FIG. 1 is a scanning electron microscope (SEM) image of insulin-containing fumaryl diketopiperazine particles, showing the solid composition of the freeze-dried particles at low magnification. [Figure 1B] FIG. 1 is a scanning electron microscope (SEM) image of insulin-containing fumaryl diketopiperazine particles, showing the solid composition of the freeze-dried particles at high magnification. [Figure 2] FIG. 1C shows a graph of particle size distribution of the particles shown in FIGS. 1A and 1B as measured by probability density function (pdf, left y-axis) and cumulative distribution function (cdf, right y-axis) scales. [Figure 3] 1 shows a graph of particle size distribution for particles obtained from an embodiment prepared with a suspension in which microcrystalline particles were formed without a surfactant in the use solution, showing a typical bimodal distribution of microcrystalline particles as measured by the probability density function (pdf, left y-axis) and cumulative distribution function (cdf, right y-axis) scales. [Figure 4] FIG. 1 is an SEM at low magnification (2500×) of FDKP particles recovered from an embodiment in which a surfactant-free particle suspension was freeze-dried. [Figure 5] FIG. 5 shows a graph of freeze-dried particle size distribution in the suspension shown in FIG. 4 formed without surfactant, showing the increase in particle size as measured by the probability density function (pdf, left y-axis) and cumulative distribution function (cdf, right y-axis) scales. [Figure 6] FIG. 1 is an SEM (2500×) of a claimed embodiment showing microcrystalline particles produced from a spray-dried surfactant-free solution. [Figure 7] FIG. 1 shows a graph of particle size distribution of spray-dried surfactant-free particles dispersed in water. [Figure 8]FIG. 1 shows a graph of particle size distribution of spray-dried surfactant-free particles dispersed in 0.01 M HCl (pH 2). [Figure 9] FIG. 1 shows a graph of bimodal particle size distribution of a suspension formed by crystallizing Na2FDKP with acetic acid in the presence of a surfactant. [Figure 10A] FIG. 1 is a scanning electron microscope image at 2500× magnification of particles prepared by spray drying a suspension of crystals prepared from Na2FDKP. [Figure 10B] FIG. 1 is a scanning electron microscope image at 10,000× magnification of particles prepared by spray drying a suspension of crystals prepared from Na2FDKP. [Figure 11A] FIG. 1 is a scanning electron microscope image at 2500× magnification of spray-dried surfactant-free FDKP particles with approximately 10 wt % insulin. [Figure 11B] FIG. 1 is a scanning electron microscope image at 5000× magnification of spray-dried surfactant-free FDKP particles with approximately 10 wt% insulin. [Figure 12A] FIG. 1 is a scanning electron microscope image at 2500× magnification of particles prepared by spray drying a suspension of crystals prepared from Na2DKP. [Figure 12B] FIG. 1 is a scanning electron microscope image at 10,000× magnification of particles prepared by spray drying a suspension of crystals prepared from Na2DKP. [Figure 13] 1 shows a graph of the size distribution of particles formed by spray drying a suspension of FDKP crystallized from a solution of Na2FDKP and polysorbate 80. The particles were dispersed in water for the measurement. [Figure 14]Figure 1 shows graphs of particle size distributions of spray-dried combination powders and crystalline suspensions with individual active agents. Figure 1 shows the particle size distributions of combination microcrystalline powder compositions containing two different active agents in separate diketopiperazine-active agent particle suspensions, where one composition containing particles of FDKP-GLP-1 and the other composition containing FDKP-insulin (3) in suspension were combined before being spray-dried. DETAILED DESCRIPTION OF THE INVENTION

[0046] As discussed above, pulmonary drug delivery offers many advantages. However, delivering drugs into the lungs is difficult due to problems with drug transport across natural physical barriers at uniform volumes and weights. Methods for producing crystalline diketopiperazine compositions, dry powders, and particles are described herein. The crystalline compositions and dry powders produced therefrom comprise diketopiperazine microcrystalline particles that are substantially uniformly defined spheres with a core and a shell containing microcrystals of diketopiperazine. In certain embodiments, the core can be hollow. In one embodiment, the diketopiperazine has a defined trans-isomer content, which can be beneficial for the particles, particle manufacturing methods, and therapeutic methods using the particles as drug delivery agents. The particles described herein have a higher capacity to load and deliver drug contents to patients at lower doses than standard prior art particles.

[0047] As used herein, "analog" includes compounds that share structural similarity with another compound. Thus, a compound that shares structural similarity with another compound (parent compound) that mimics the biological or chemical activity of the parent compound is an analog. There is no minimum or maximum number of element or functional group substitutions required for a compound to be considered an analog, so long as the provided analog can mimic the biological or chemical properties of the parent compound in some related manner—either identically, complementary, or competitively. In some instances, analogs include fragments of the parent compound, either isolated or attached to another molecule, and may contain other substitutions as well. Analogs of the compounds described herein may have activity comparable to, less than, or greater than their parent compounds.

[0048] As used herein, the term "fine particle" refers to particles having a diameter (particle size) of about 0.5 μm to about 1000 μm, regardless of their exact external or internal structure. Fine particles with diameters between about 0.5 and about 10 microns can reach the lungs and successfully pass through most natural barriers. A diameter less than about 10 microns is necessary to pass through the curvature of the throat, while a diameter greater than about 0.5 microns 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 effective absorption is believed to occur. While some references use somewhat different ranges, measured using standard techniques, such as an Andersen cascade impactor, particles with an aerodynamic diameter of about 0.5 microns to about 5.7 microns are generally acceptable. Other impactors, such as the NEXT GENERATION IMPACTOR™ (NGI™, MSP Corporation), can be used to measure aerodynamic particle size, with the respirable fraction defined by a similar aerodynamic size, e.g., less than 6.4 μm. In certain embodiments, a laser diffraction device is used to measure particle size, such as that described in U.S. Patent Application No. 12 / 727,179, filed March 18, 2010, which is incorporated herein in its entirety for its relevant teachings. Here, the volume mean geometric diameter (VMGD) of particles is measured to evaluate the performance of an inhalation system. For example, in various embodiments, cartridge ejection of 80%, 85%, or 90% or more and a VMGD of emitted particles of 12.5 μm, 7.0 μm, 5.8 μm, or 4.8 μm or less may indicate progressively better aerodynamic performance. The embodiments described herein demonstrate that FDKP particles having a trans isomer content between about 45% and about 65% exhibit beneficial characteristics for pulmonary drug delivery, such as improved aerodynamic performance.

[0049] Respirable fraction per fill (RF / Fill) represents the percentage of powder emitted from an inhaler in a dose that is suitable for respirability upon expulsion of a powder content filled for use in a medication. It is the percentage of particles from the filled dose that are emitted with an appropriate size for pulmonary delivery, and is a measure of particle dynamic performance. As described herein, an RF / Fill value of 40% or greater indicates acceptable aerodynamic performance characteristics. In certain embodiments described herein, the respirable fraction per fill can be greater than 50%. In exemplary embodiments, the respirable fraction per fill can be up to about 80%, with about 80% of the filled dose emitted with a particle size of less than 5.8 μm, as measured using standard techniques.

[0050] The term "dry powder" as used herein refers to a finely particulate composition that is not suspended or dissolved in a propellant, carrier, or other liquid, and is not necessarily meant to refer to the complete absence of all water molecules.

[0051] The specific RF / Fill value may depend on the inhaler used to deliver the powder. Powders in general tend to agglomerate, and certain crystalline DKP particles form particularly cohesive powders. One of the functions of a dry powder inhaler is to deagglomerate the powder so that the resulting particles contain a respirable fraction suitable for delivering a dose via inhalation. However, deagglomeration of cohesive powders is typically incomplete, and the particle size distribution seen when measuring the respirable fraction delivered by the inhaler will not match the size distribution of the initial particles; i.e., the profile will be shifted toward larger particles. Because inhaler designs vary in their deagglomeration efficiency, the absolute values ​​of RF / Fill observed using different designs will also vary. However, the optimal RF / Fill as a function of isomer content will be similar across inhalers.

[0052] As used herein, the term "about" is used to indicate a value that includes the standard deviation of measurement for the device or method being employed to determine the value.

[0053] As used herein, the term "surfactant-free (in the absence of surfactant)" is used to indicate that no surfactant is present in any of the reagents, including the solutions and / or suspensions, used in the method of making microcrystalline particles.

[0054] As used herein, the term "crystallite" is used to refer to an integral crystalline unit of a diketopiperazine particle, which may have varying sizes.

[0055] As used herein, "microcrystalline particles" include microcrystals of diketopiperazine having a particle size distribution of about 0.05 μm to about 100 μm, with particle sizes of less than 50 μm, less than 20 μm, or less than 10 μm in diameter as measured by laser diffraction. In embodiments, the microcrystals can range in size from 0.01 μm to 1 μm.

[0056] Diketopiperazine One class of drug delivery agents used to overcome pharmaceutical problems such as drug instability and / or poor absorption are 2,5-diketodiketopiperazines. 2,5-diketodiketopiperazines are represented by compounds of general formula 1, shown below, where E1 and E2 are independently N or, specifically, NH. In other embodiments, E1 and E2 are independently oxygen or nitrogen; when one of the E1 and E2 substituents is oxygen and the other is nitrogen, the formula is a substituted analog diketomorpholine. Alternatively, when both E1 and E2 are oxygen, the formula is a substituted analog diketodioxane. [ka]

[0057] These 2,5-diketodiketopiperazines have been shown to be useful in drug delivery, particularly those with acidic R1 and R2 groups, as described, for example, in U.S. Patent No. 5,352,461, entitled "Self-Assembling Diketopiperazine Drug Delivery System," U.S. Patent No. 5,503,852, entitled "Method For Making Self-Assembling Diketopiperazine Drug Delivery System," U.S. Patent No. 6,071,497, entitled "Microparticles For Lung Delivery Comprising Diketopiperazine," and U.S. Patent No. 6,331,318, entitled "Carbon-Substituted Diketopiperazine Delivery System." Each of these is incorporated herein by reference in its entirety for all that it teaches regarding diketopiperazines and diketopiperazine-mediated drug delivery. Diketopiperazines can be formed into microparticles that incorporate drugs or onto which drugs can be adsorbed. Combining a drug with a diketopiperazine can improve the stability and / or absorption characteristics of the drug. These microparticles can be administered by a variety of routes. As a dry powder, the microparticles can be delivered by inhalation to specific regions of the respiratory system, including the lungs.

[0058] Such prior art microparticles are typically obtained by precipitation based on the pH of free acid (or base), resulting in self-assembled microparticles containing aggregated crystalline plates with a reddish morphology. Particle stability can be improved by the addition of a small amount of surfactant, such as polysorbate 80, in the DKP solution from which the particles are precipitated (see, e.g., U.S. Pat. No. 7,799,344, entitled "Method of drug formulation based on increasing the affinity of crystalline microparticle surfaces for active agents," the disclosure of which is incorporated herein by reference in its entirety for all it teaches regarding the formulation and packing of DKP microparticles and their dry powders). Finally, the solvent can be removed to obtain a dry powder. Methods for removing the solvent include freeze-drying and spray-drying (see, e.g., U.S. Pat. No. 8,039,431, entitled "A method for improving the pharmaceutic properties of microparticles comprising diketopiperazine and an active agent," and U.S. Pat. No. 6,444,226, entitled "Purification and stabilization of peptide and protein pharmaceutical agents," each of which is incorporated by reference in its entirety for all they teach regarding DKP microparticles and the formulation and packing of dry powders thereof.) The particles described herein are physically distinct from prior art particles and are morphologically distinct entities, produced by improved methods. This disclosure describes FDKP, understood as the free acid or dissolved anion.

[0059] Other conventional particles are obtained by spray drying a DKP solution, as described in U.S. Pat. Nos. 7,820,676 and 8,278,308, entitled "Diketopiperazine salts for drug delivery and related methods," to yield amorphous DKP salt particles that typically have a collapsed spherical morphology.

[0060] Methods for synthesizing diketopiperazines are described, for example, in Katschalski, et al., J. Amer. Chem. Soc. 68, 879-880 (1946) and Kopple, et al., J. Org. Chem. 33(2), 862-864 (1968), the teachings of which are incorporated herein by reference in their entirety. 2,5-Diketo-3,6-di(aminobutyl)piperazine (referred to by Katschalski et al. as lysine anhydride) can also be prepared via cyclodimerization of N-ε-PL-lysine in dissolved phenol, similar to the Kopple method, followed by removal of the blocking (P) group with appropriate reagents and conditions. For example, the CBz protecting group can be removed using 4.3 M HBr in acetic acid. This route uses commercially available starting materials, involves reaction conditions that have been reported to maintain the stereochemistry of the starting material in the product, and all steps can be easily scaled up for manufacturing. Methods for synthesizing diketopiperazines are also described in U.S. Pat. No. 7,709,639, entitled "Catalysis of Diketopiperazine Synthesis," the disclosure of which is incorporated herein by reference for its teachings regarding the same.

[0061] Fumaryl diketopiperazine (bis-3,6-(N-fumaryl-4-aminobutyl)-2,5-diketo-diketopiperazine; FDKP) is one preferred diketopiperazine for pulmonary applications. [ka]

[0062] FDKP provides a useful microparticle matrix because it has low solubility in acidic conditions but easily dissolves at neutral or basic pH. These properties allow FDKP to crystallize and the crystals to self-assemble into microparticles under acidic conditions. The particles are easily dissolved under physiological conditions with a neutral pH. As mentioned above, microparticles with a diameter between about 0.5 μm and about 10 μm can reach the lungs and successfully pass through most natural barriers. Particles in this size range can be easily prepared from FDKP.

[0063] FDKP has two asymmetric centers in the diketopiperazine ring. FDKP is produced as a mixture of geometric isomers, identified as "cis-FDKP" and "trans-FDKP," depending on the configuration of the side chains relative to the central "ring" of the diketopiperazine. The R,R and S,S enantiomers have propenyl (aminobutyl) "side arms" projecting from the same planar side of the diketopiperazine ring (A and B below) and are therefore designated as cis isomers. Meanwhile, the R,S compound has a "side arm" projecting from the opposite planar side of the diketopiperazine ring (C below) and is designated as the trans isomer. [ka]

[0064] FDKP fine particle powders with acceptable aerodynamic performance, as measured by RF / Fill in a reasonably efficient inhaler, such as the MEDTONE® inhaler, described in U.S. Patent No. 7,464,706, entitled "Unit Dose Cartridge and Dry Powder Inhaler," have been produced from FDKP having a trans-isomer content ranging from about 45% to about 65%, the disclosure of which is incorporated herein by reference for its teachings regarding the same. Particles having an isomer content in this range also perform well with highly efficient inhalers, such as those described in U.S. Patent No. 8,499,757, filed June 12, 2009, entitled "A Dry Powder Inhaler and System for Drug Delivery," U.S. Patent No. 8,424,518, filed June 12, 2009, entitled "Dry Powder Inhaler and System for Drug Delivery," U.S. Patent Application No. 13 / 941,365, filed July 12, 2013, entitled "Dry Powder Drug Delivery System and Methods," and U.S. Patent Application No. 12 / 717,884, filed March 4, 2010, entitled "Improved Dry Powder Drug Delivery System." These disclosures are incorporated herein by reference for their teachings regarding the same. Powders comprising fine particles containing greater than 65% trans-FDKP tend to have lower and more variable RF / Fill. The trans-isomer-enriched microparticles of FDKP have altered morphology and also lead to viscous suspensions that are difficult to process.

[0065] Formulations of FDKP microparticles having a trans-isomer content of about 45% to about 65% provide powders with acceptable aerodynamic properties, as described in U.S. Patent No. 8,227,409, the disclosure of which is incorporated herein by reference for its teachings regarding the same. 2Formulations of FDKP particles having a defined specific surface area of ​​less than 1 / g also provide dry powders for inhalation with acceptable aerodynamic properties, as described in U.S. Patent No. 8,551,528, entitled "Diketopiperazine Microparticles with Defined Specific Surface Areas," filed June 11, 2010, the disclosure of which is incorporated herein by reference for its teachings regarding the same. However, these FDKP powders tend to be cohesive, and inhalers are designed to overcome this characteristic.

[0066] Therefore, it is desirable to produce diketopiperazine powders with less cohesive particle compositions, which allows for more effective drug delivery and less inhaler design overhead. In the present disclosure, it has been confirmed that a method for producing microcrystalline particles of diketopiperazine, exemplified by FDKP and FDKP disodium salt, provides microcrystalline dry powders with acceptable aerodynamic performance. The powders are less cohesive, have different densities, and have different physical structures that do not self-assemble in suspension, and provide unexpectedly improved drug loading capabilities, including delivery of one or more active agents.

[0067] It has been found that different methods for producing diketopiperazine microparticles can result in improved consistency in particle homogeneity. The disclosed methods for producing compositions and compositions comprising the disclosed microcrystalline diketopiperazine particles provide dry powders for pulmonary inhalation with advantageous physical and morphological aerodynamic properties.

[0068] Active Agent Selection and Incorporation

[0069] In exemplary embodiments including FDKP, other additional features beneficial for pulmonary delivery and / or drug absorption can be employed, at least so long as the microcrystalline particles described herein retain the isomer content described above. U.S. Patent No. 6,428,771, entitled "Method for Drug Delivery to the Pulmonary System," describes pulmonary delivery of FDKP particles, the disclosure of which is incorporated herein by reference for its teachings regarding same. U.S. Patent No. 6,444,226, entitled "Purification and Stabilization of Peptide and Protein Pharmaceutical Agents," describes a beneficial method for drug adsorption onto microparticle surfaces, the disclosure of which is incorporated herein by reference for its teachings regarding same. As described in U.S. Patent No. 7,799,344, entitled "Method of Drug Formulation based on Increasing the Affinity of Crystalline Microparticle Surfaces for Active Agents," the properties of the microparticle surface can be manipulated to achieve desired characteristics, the disclosure of which is incorporated herein by reference for its teachings regarding same. U.S. Patent No. 7,803,404, entitled "Method of Drug Formation based on Increasing the Affinity of Active Agents for Crystalline Microparticle Surfaces," describes a method for promoting the adsorption of active agents onto microparticles. U.S. Patent No. 7,803,404 is also incorporated herein by reference for its teachings regarding the same. These teachings can be applied to the adsorption of active agents onto microcrystals in suspension, for example, prior to spray drying.

[0070] The microcrystalline particles described herein can contain one or more active agents. As used herein, "active agent" is used interchangeably with "drug" and refers to pharmaceutical substances, including small molecule pharmaceuticals, biologics, and bioactive agents. Active agents can be of natural, recombinant, or synthetic origin and include proteins, polypeptides, peptides, nucleic acids, organic polymers, synthetic organic compounds, polysaccharides and other sugars, fatty acids, lipids, and antibodies and fragments thereof. This includes, but is not limited to, humanized or chimeric antibodies, F(ab)s, F(ab)2s, single-chain antibodies alone or fused to other polypeptides, or therapeutic or diagnostic monoclonal antibodies against cancer antigens. Active agents can be classified into various biological activities and types, such as vasoactive agents, neuroactive agents including opioid agonists and antagonists, hormones, anticoagulants, immunomodulators, cytotoxic agents, antibiotics, antivirals, antigens, infectious agents, inflammatory mediators, hormones, cell surface receptor agonists and antagonists, and cell surface antigens.More specifically, the active agent may be, but is not limited to, cytokines, lipokines, enkephalins, alkynes, cyclosporines, anti-IL-8 antibodies, IL-8 antagonists including ABX-IL-8, prostaglandins including PG-I2, LTB receptor blockers including LY29311, BIIL284 and CP105696, triptans such as sumatriptan and palmitoleate, insulin and its analogs, growth hormone and its analogs, parathyroid hormone (PTH) and its analogs, parathyroid hormone-related peptide (PTHrP), ghrelin, obestatin, enterostatin, granulocyte-macrophage colony-stimulating factor (GM-CSF), amylin, amylin analogs, glucagon-like peptide-1 (GLP-1), clopidogrel, PPACK (D-phenylalanine), riboflavin, riboflavin, riboflavin-like peptide-1 (RI-1), ... nitrile-L-prolyl-L-arginine chloromethyl ketone), oxyntomodulin (OXM), peptide YY(3-36) (PYY), adiponectin, cholecystokinin (CCK), secretin, gastrin, glucagon, motilin, somatostatin, brain natriuretic peptide (BNP), atrial natriuretic peptide (ANP), IGF-1, growth hormone-releasing factor (GHRF), integrin beta-4 precursor (ITB4) receptor antagonist, analgesic, nociceptin, nocistatin, orphanin FQ2, calcitonin, CGRP, angiotensin, substance P, neurokinin A, pancreatic polypeptide, neuropeptide Y, delta sleep-inducing peptide, vasoactive intestinal peptide, and analogs of such active agents.

[0071] The drug content delivered by microcrystalline particles formed from FDKP or FDKP disodium salt can typically be greater than 0.01% (w / w). In one embodiment, the drug content delivered by the microcrystalline particles can be about 0.01% (w / w) to about 75% (w / w), about 1% (w / w) to about 50% (w / w), about 10% (w / w) to about 30% (w / w), or about 10% (w / w) to about 20% (w / w). In one embodiment, for example, when the drug is insulin, the microparticles typically contain about 10% to 45% (w / w) or about 10% to about 20% (w / w) insulin. In certain embodiments, the drug content of the particles can vary depending on the form and size of the drug being delivered. In embodiments where GLP-1 is used as the active agent, the GLP-1 content may be up to 40% (w / w) of the powder content.

[0072] In embodiments, compositions containing two or more active agents can be prepared using the methods of the present disclosure by adsorption, for example, by binding the active agents to microcrystals prior to forming a dry powder.

[0073] In embodiments, compositions containing two or more active agents can be produced using the methods of the present disclosure by entrapping the active agents between and within the microcrystals, for example, by spray drying the material, without first adsorbing the active agents onto the microcrystals.

[0074] A method for producing such a composition can include producing microcrystalline diketopiperazine particles containing two or more active agents, where each active agent / component is processed in a separate solution and added to a separate suspension of diketopiperazine particles, and then the two or more separate suspensions containing the active agents are combined before dispersing and spray drying the particles.

[0075] In certain embodiments, the microcrystals may be mixed with a solution containing one or more active agents.

[0076] In certain embodiments, the microcrystals may be mixed with a solution containing one or more active agents, with the solution conditions altered to promote adsorption of the active agents onto the microcrystal surface.

[0077] Each of the multiple active agents can be adsorbed onto a separate aliquot or seed of microcrystals. The aliquot-adsorbed microcrystals can then be mixed together and spray-dried. Alternatively, the aliquots can contain no active agent at all, so as to adjust the overall content of active agent in the dry powder without changing the conditions used to adsorb the active agent onto the microcrystals.

[0078] In an alternative embodiment, one or more separate solutions containing a single active agent can be combined with the suspension containing the diketopiperazine particles before dispersion and spray drying to reform the particles. The resulting dry powder composition contains two or more active ingredients. In this embodiment, the amount of each ingredient can be controlled in the composition according to the needs of the patient population being treated.

[0079] As is evident from the foregoing disclosure, the microparticles of the embodiments described herein can take many different forms and can incorporate many different drugs or active agents. [Example]

[0080] The following examples are included to demonstrate embodiments of the microcrystalline diketopiperazine particles described herein. It should be understood by those skilled in the art that the techniques disclosed in the following examples represent techniques developed by the inventors that function well in the practice of the present disclosure and can therefore be considered to constitute preferred modes for its practice. However, those skilled in the art should understand, in light of the present disclosure, that many variations in the specific embodiments disclosed are possible and can still achieve the same or similar results without departing from the scope of the present invention.

[0081] Example 1 Production of standard FDKP microparticles

[0082] Use conventional manufacturing method to prepare FDKP microparticles for comparison.This is the standard particle as described in US Patent No. 7,799,344, US Patent No. 7,803,404 and US Patent No. 8,227,409, and these disclosures are incorporated herein by reference for the teaching of related subject matter.In summary, in the typical FDKP particle preparation method, the feed solution of FDKP and acetic acid, each containing 0.05% (w / w) polysorbate 80 (PS80), is combined in high-shear mixer.The following table 1 shows the components of FDKP and insulin stock solution.

[0083] [Table 1]

[0084] [Table 2]

[0085] A concentrated insulin stock solution can be prepared with 1 part insulin and 9 parts about 2% wt acetic acid. Insulin can be added gravimetrically to the suspension to obtain a loading of about 11.4% wt. The insulin-containing suspension can be mixed for at least about 15 minutes and then titrated to a pH of about 4.5 from an initial pH of about 3.5 with about 14 wt.% to about 15 wt.% aqueous ammonia. The suspension can be flash-frozen in liquid nitrogen to form pellets using a cryogranulator, as described, for example, in U.S. Pat. No. 8,590,320, the disclosure of which is incorporated herein by reference in its entirety. The bulk insulin-loaded FDKP microparticles can then be freeze-dried to form small crystals or clusters that self-assemble into FDKP particles with an open structure, as seen in FIGS. 1A and 1B.

[0086] A sample of the formed particles was examined to determine the size distribution of these particles in suspension. The results are shown in Figure 2. The data in Figure 2 shows a graph of the particle size distribution measurement, plotted on a logarithmic scale as a probability density function (pdf, left y-axis) and a cumulative distribution function (cdf, right y-axis). The data show that the particles in suspension have a single-peak size distribution ranging from about 1.0 μm to about 10 μm in diameter, centered at or near 2 μm.

[0087] Preparation of microcrystalline FDKP particles 2.5% (w / w) FDKP was dissolved in a basic solution of aqueous ammonia (1.6% ammonia). A 10.5% (w / w) acetic acid stock solution was added to a high-shear mixer (Snolator) under high pressure at a pH of approximately 2.0 to produce particles. The formed particles were washed in deionized water. While diketopiperazine microparticles have been found to be unstable in solution without a surfactant, no surfactant was added to either the solution or the reagents used to produce the particles in this example.

[0088] In these examples, a dual feed high shear mixer was used to mix equal masses of about 10.5 wt % acetic acid solution and about 2.5 wt % FDKP solution at about 16° C.±about 2° C., with a 0.001-in 2The suspension was fed at 2000 psi through an orifice and homogenized to form a precipitate. The precipitate was collected in a deionized (DI) water reservoir of approximately equal mass and temperature. The precipitate was concentrated and washed by tangential flow filtration with DI water. The suspension can be finally concentrated to a solids content of less than about 5%, for example, about 2% to 3.5% based on the initial mass of FDKP. The concentrated suspension can be analyzed for solids content by oven drying. For samples containing the active ingredients, i.e., insulin and / or GLP-1, the FDKP suspension described above was used by adding insulin stock solution (insulin dissolved in 2% acetic acid) to the suspension with mixing, and then titrating the pH of the suspension to pH 4.5 ± 0.3 with ammonium hydroxide. Similarly, GLP-1 stock solution dissolved in 2% acetic acid was gravimetrically added to the FDKP suspension with stirring. The GLP-1-FDKP suspension was then titrated to pH 4.5 ± 0.1. The insulin-FDKP suspension and the GLP-1-FDKP suspension were each dispersed independently using an externally mixed two-fluid nozzle inserted into a Niro SD-Micro™ spray dryer equipped with a high-efficiency cyclone. Nitrogen was used as the process gas (25 kg / h) and the atomizing fluid (2.8 kg / hr). The samples were processed using two process conditions for spray drying, as listed in Table 3.

[0089] [Table 3]

[0090] For control samples, blank FDKP microcrystalline particles were prepared similarly, omitting the insulin or GLP-1 loading step.

[0091] Figure 3 shows data from the above experiment without surfactant in the feed solution. Figure 3 is a graph showing the particle size distribution of a particle suspension of FDKP, showing a typical bimodal particle size distribution. The particle sizes range from about 0.1 μm to about 10 μm in diameter, with one particle population centered at 0.2 μm in diameter and another particle population centered at 2.1 μm in diameter.

[0092] A sample of the suspension was freeze-dried and not spray-dried. Figure 4 is an SEM at 2500x magnification of the freeze-dried particles. As seen in Figure 4, large flake-like particles were formed upon freeze-drying of a similar suspension, which reached a much larger average size when resuspended in water, as seen in Figure 5. Figure 5 shows the particle size distribution in the suspension of a freeze-dried sample from particles produced without the use of surfactant. In this study, the particle size diameter of the resuspended particles increased from about 1 μm to over about 90 μm.

[0093] Figure 6 shows a typical 2500x magnification scanning electron microscope image of a powder sample obtained from a surfactant-free preparation of microcrystalline FDKP particles formed using the method of the present disclosure and spray drying as described above. As seen in Figure 6, the particles are homogeneously spherical in structure with a shell of microcrystals. When the surfactant-free suspension was spray dried, particles with a physical diameter of approximately 4 μm were formed, as shown in Figure 6. Unlike standard FDKP particles, these particles dissociated into particles 0.2 μm in diameter when dispersed in water, as shown in Figure 7. This demonstrates that the surfactant plays a role in particle integrity. As demonstrated in Figure 8, dispersion of the particles in 0.01 M hydrochloric acid inhibited particle dissociation. It is believed that dissolved FDKP precipitates during spray drying, depositing along the boundaries between primary particles and acting like cement. The FDKP "cement" dissolves in water, causing the particles to dissociate into 0.2 μm primary particles. The lower solubility of FDKP in acid prevents dissolution and maintains particle integrity.

[0094] Example 2 Preparation of microcrystalline FDKP particles by an alternative method using diketopiperazine salts

[0095] Alternatively, FDKP microcrystals can be formed from a surfactant-containing feed solution. The FDKP feed solution was prepared by dissolving the disodium salt of FDKP (NaFDKP) in water containing polysorbate 80 (PS80) as a surfactant, without using ammonia as a reagent. A feed solution containing acetic acid (10.5% w / w) and PS80 (0.5% w / w) was also prepared. Mixing the two feed solutions in a dual-feed sonolater and crystallizing the FDKP resulted in the bimodal particle size distribution shown in Figure 9. As shown in Figure 9, approximately 26% of the primary crystals formed were approximately 0.4 μm in diameter, and approximately 74% of the larger particles had a diameter of approximately 2.4 μm. This suspension was processed and spray-dried to obtain particles, which were then observed by SEM. SEM micrographs were taken at 2500× and 10,000× magnification and are shown in Figures 10A and 10B. Figures 10A and 10B show that the particles are similar in shape to those shown in Figure 6 of Example 1, where particles were produced using FDKP as the free acid, but are smaller. Table 4 below shows some physical properties measured for powders produced by freeze drying and spray drying (SD) using FDKP disodium salt.

[0096] [Table 4]

[0097] The data show that powders produced from spray-dried particles exhibited a higher respirable fraction (62.8% vs. 28%), higher cartridge excretion (%CE, 88.2% vs. 83.8%), and higher bulk and tapped densities (0.159 g / mL and 0.234 g / mL) than lyophilized powders (bulk and tapped densities of 0.019 g / mL and 0.03 g / mL, respectively).

[0098] Example 3 Preparation of microcrystalline FDKP particles containing an active agent

[0099] Active pharmaceutical ingredients (active agents) were incorporated into particles by adding a solution of the active agent to a suspension of surfactant-free FDKP microcrystals, followed by spray drying the mixture to remove the solvent, as described in Example 1. Control particles (FDKP-insulin) were also prepared by a standard self-assembly method using PS80 in solution to produce a powder for pulmonary inhalation. In this study, insulin was dissolved in dilute acetic acid and added to a suspension of surfactant-free FDKP microcrystals prepared as in Example 1 (Samples 1 and 2, Table 5). The suspension was spray-dried to yield a dry powder containing approximately 10 wt% insulin. Various analyses were performed on the powder samples, including delivery through a high-resistance inhaler and scanning electron microscopy. The results are shown in Table 5. The particles were approximately the same size as the insulin-free particles (Example 1), and the particle morphology (Figure 11) was similar to that in Figure 6. Furthermore, both Sample 1 and Sample 2 were less dense than the standard particles, and the particles of Sample 1 had a larger specific surface area (SSA) than the control. Although the distribution of insulin is unknown, there is no obvious deposition of insulin on the particle surface, suggesting that insulin is present inside the particle or incorporated into the particle wall.

[0100] [Table 5]

[0101] However, the data presented in Table 5 show that the surfactant-free powder behaved differently from standard particles at the same insulin content. For example, at the same insulin content, the surfactant-free powder was released from the inhaler more efficiently (96.4%) than the standard particles (85%). The increase in cartridge exhaustion percentage (%CE) indicates increased powder flowability. The respirable fraction (%RF / Fill) was higher for the control particles because the inhaler used to test the powders was designed for the control powder.

[0102] Example 4 Preparation of microcrystalline FDKP particles by an alternative method using diketopiperazine salts

[0103] In this study, disodium salt of FDKP was used to prepare an FDKP salt particle suspension as described in Example 2. An insulin solution was added to a surfactant-free suspension of FDKP microcrystals prepared as in Example 2. The suspension was spray-dried to obtain a dry powder containing approximately 10 wt% insulin. The morphology of the formed particles is shown in Figures 12A and 12B by SEM at 2500x and 10000x magnification, respectively. As seen in Figures 12A and 12B, the morphology is similar to that of particles without insulin, exhibiting spherical structures with an average particle size of 2.6 μm, as shown in Figure 13, and also exhibiting particles with diameters ranging from approximately 1.0 μm to approximately 10 μm.

[0104] Example 5 Preparation of microcrystalline FDKP particles containing two or more active agents

[0105] In another embodiment, the methods of the present disclosure can be used to prepare compositions containing two or more active agents. The method for preparing such compositions includes the steps described above for each individual active agent to form an active agent-FDKP suspension for each active agent to be incorporated into the composition. The suspensions are then combined and blended to form a mixture. The blended mixture is dispersed and spray-dried as described above to produce microcrystalline diketopiperazine particles containing two or more active agents. In one exemplary study, a combination powder of insulin and GLP-1 was prepared.

[0106] A suspension of FDKP microcrystals prepared as in Example 1 was mixed with solutions of various active agents (e.g., ghrelin, low molecular weight heparin, oxyntomodulin) and spray-dried to obtain particles with properties similar to those in Example 3.

[0107] Example 6 Preparation of microcrystalline FDKP particles containing two active agents.

[0108] A combination powder with two active agents (GLP-1 and insulin) was produced by preparing a first suspension of FDKP microcrystals with insulin and a second suspension of microcrystals with GLP-1. The two suspensions were then mixed, and the combined suspension was spray-dried to obtain a dry powder containing both active agents. The microcrystal suspension was prepared as in Example 1, and after adding the active agents, the suspension was adjusted to pH 4.5 to promote adsorption onto the microcrystals. Figure 14 is a plot of data showing the particle size distribution of the spray-dried combination powder (1) and the microcrystal suspensions with the individual active agents, FDKP-insulin (2) and FDKP-GLP-1.

[0109] As can be seen in Figure 14, the particle size distribution of the combined powder was significantly narrower, intermediate between that of the two individual suspensions. The combined powder contained particles with diameters ranging from about 1 μm to about 10 μm. The insulin-containing microcrystals were smaller (about 0.25 μm to about 10 μm) than the GLP-1-containing microcrystals, which had diameters ranging from about 0.5 μm to about 50 μm. The atomization step in spray drying likely breaks up the original clusters of microcrystals in suspension and reforms particles with a size distribution that depends on the conditions in the suspension and the spray-drying conditions.

[0110] Example 7 Administering a dry powder composition comprising crystalline diketopiperazine particles to a subject

[0111] A dry powder formulation containing microcrystalline diketopiperazine microparticles made with the disodium salt of FDKP (NaFDKP) was prepared as in Example 1 above, containing 9 U of insulin per milligram of composition. High-resistance inhalers (Dreamboat™ inhalers, MannKind Corporation) containing cartridges were calibrated to contain 1 mg to 10 mg per dose and prepared for administration to subjects diagnosed with diabetes. The inhaler containing the insulin dose was provided to the patient to be treated, and the patient inhaled the insulin dose in a single inhalation at the start of, during, or after a meal.

[0112] Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and the like used in the specification and claims should be understood as being modified in all instances by the term "about." Accordingly, unless indicated to the contrary, the numerical parameters set forth in the specification and appended claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values ​​set forth in the specific examples are reported as precisely as possible. However, any numerical value inherently contains certain errors necessarily resulting from the standard deviation found in each testing measurement.

[0113] The terms "a," "an," "the," and similar referents in the context of describing the present invention (particularly in the context of the claims that follow) shall be construed to include both the singular and the plural unless otherwise stated or clearly contradicted by context. The designation of ranges of values ​​herein is merely intended to serve as a shorthand method of mentioning each separate value falling within the range individually. Unless otherwise stated, each separate value is incorporated herein as if set forth individually. All methods described herein can be performed in any suitable order unless otherwise stated or clearly contradicted by context. The use of any and all examples or exemplary language (e.g., "etc.") provided herein is merely to further clarify the invention and does not limit the scope of the invention or otherwise the claims. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

[0114] Use of the term "or" in the claims is used to mean "and / or," although the present disclosure supports a definition that refers to alternatives only and "and / or," unless expressly stated to refer to alternatives only, or unless the alternatives are mutually exclusive.

[0115] Groups of alternative elements or embodiments of the invention disclosed herein should not be construed as limiting. Each group member may be referenced and claimed individually or in any combination with other members of the group or other elements described herein. It is anticipated that one or more members of a group may be included in or deleted from a group for reasons of convenience and / or patentability. When any such inclusion or deletion occurs, the specification shall be deemed to include the group as modified to satisfy all Markush group descriptions used in the appended claims.

[0116] Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Of course, variations of these preferred embodiments will become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors anticipate that skilled artisans will employ such variations as appropriate, and the inventors intend that the invention may be practiced otherwise 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 invention unless otherwise indicated or clearly contradicted by context.

[0117] Specific embodiments described herein may be further limited in the claims using the terms "consisting of" or "consisting essentially of." Whether added by application or amendment, the transitional term "consisting of," when used in a claim, excludes any element, step, or ingredient not specified in the claim. The transitional term "consisting essentially of" limits the claim to the specified materials or steps and those that do not materially affect the claim's basic and novel characteristic(s). Such claimed embodiments of the invention are essentially or explicitly described and enabled herein.

[0118] Additionally, throughout this specification, numerous references are made to patents and printed publications. Each of the above-cited references and printed publications is individually incorporated herein by reference in its entirety.

[0119] Furthermore, it is to be understood that the embodiments of the invention disclosed herein are illustrative of the principles of the invention. Other variations that may be employed 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. Accordingly, the invention is not limited to that specifically shown and described.

[0120] (Addendum) (Appendix 1) A crystalline diketopiperazine composition comprising a plurality of substantially uniformly sized microcrystalline particles having a substantially hollow spherical structure and a shell comprising non-self-assembled diketopiperazine crystallites, said particles having a volume average geometric diameter of less than 5 μm.

[0121] (Appendix 2) 2. The crystalline diketopiperazine composition of claim 1, wherein up to 92% of the microcrystalline particles have a volume average geometric diameter of 5.8 μm or less.

[0122] (Appendix 3) 2. The crystalline diketopiperazine composition of claim 1, wherein the microcrystalline particles further comprise one or more active ingredients.

[0123] (Appendix 4) 4. The crystalline diketopiperazine composition of claim 3, wherein the one or more active ingredients are a peptide, a protein, a nucleic acid molecule, or an organic small molecule.

[0124] (Appendix 5) 5. The crystalline diketopiperazine composition of claim 4, wherein the peptide is an endocrine hormone.

[0125] (Appendix 6) 6. The crystalline diketopiperazine composition of claim 5, wherein the endocrine hormone is insulin, parathyroid hormone, calcitonin, glucagon, glucagon-like peptide-1, oxyntomodulin, peptide YY, leptin, or an analog of these endocrine hormones.

[0126] (Appendix 7) 2. The crystalline diketopiperazine composition of claim 1, wherein the diketopiperazine has the formula 2,5-diketo-3,6-bis(NX-4-aminobutyl)piperazine, where X is selected from the group consisting of fumaryl, succinyl, maleyl, malonyl, and glutaryl, or a salt thereof.

[0127] (Appendix 8) 8. The crystalline diketopiperazine composition of claim 7, wherein the diketopiperazine is (bis-3,6-(N-fumaryl-4-aminobutyl)-2,5-diketopiperazine.

[0128] (Appendix 9) a) forming diketopiperazine particles in a suspension having a bimodal distribution of particle sizes ranging from about 0.05 μm to about 10 μm; b) atomizing the suspension using a spray dryer under an air or gas stream; and c) reforming the particles by spray drying to a dry powder comprising microcrystalline diketopiperazine particles having substantially hollow spheres; 1. A method for producing microcrystalline diketopiperazine particles suitable for pulmonary administration as a dry powder, comprising:

[0129] (Appendix 10) 10. The method of claim 9, wherein a first peak of particles in the bimodal particle size distribution has an average size of about 0.2 μm to about 2.4 μm, and a second peak of particles has an average size of about 2.1 μm to about 2.4 μm.

[0130] (Appendix 11) 10. The method of claim 9, wherein the diketopiperazine has the formula 2,5-diketo-3,6-bis(NX-4-aminobutyl)piperazine, where X is selected from the group consisting of fumaryl, succinyl, maleyl, malonyl, and glutaryl.

[0131] (Appendix 12) 12. The method of claim 11, wherein the diketopiperazine is (bis-3,6-(N-fumaryl-4-aminobutyl)-2,5-diketopiperazine.

[0132] (Appendix 13) 10. The method of claim 9, further comprising adding a solution containing one or more active agents to the suspension in step a).

[0133] (Appendix 14) 10. The method of claim 9, further comprising adding a surfactant to the solution or suspension.

[0134] (Appendix 15) 15. The method of claim 14, wherein the surfactant is polysorbate 80.

[0135] (Appendix 16) 14. The method of claim 13, wherein the one or more active agents are peptides, proteins, nucleic acid molecules, or small organic molecules.

[0136] (Appendix 17) 17. The method of claim 16, wherein the peptide is an endocrine hormone.

[0137] (Appendix 18) 18. The method of claim 17, wherein the endocrine hormone is insulin, parathyroid hormone, calcitonin, glucagon, glucagon-like peptide-1, oxyntomodulin, peptide YY, leptin, or an analog of these endocrine hormones.

[0138] (Appendix 19) 12. The method of claim 9 or 11, wherein the diketopiperazine is a disodium, magnesium, lithium, calcium, or potassium salt of diketopiperazine.

[0139] (Appendix 20) dissolving a diketopiperazine salt in water containing a surfactant in an amount of about 0.2% (w / w) to about 6% (w / w) to form a first solution; simultaneously combining the first solution with a second solution comprising about 8% (w / w) to about 12% (w / w) acetic acid in a high shear mixer under high pressure and at a pH of less than about 6.0; homogenizing the first solution and the second solution to form a suspension comprising microcrystals of diketopiperazine in suspension, the suspension having a bimodal distribution of microcrystals having a particle size ranging from about 0.05 μm to about 10 μm in diameter; atomizing the suspension under an air or gas stream; and reforming the particles by spray drying to a dry powder comprising microcrystalline particles having substantially hollow spheres; 1. A method for producing a dry powder composition, comprising:

[0140] (Appendix 21) 20. The method of claim 19, wherein atomizing the suspension comprises using an externally mixed two-fluid nozzle inserted into a spray dryer equipped with a high-efficiency cyclone separator under nitrogen gas.

[0141] (Appendix 22) A method for treating a disease or disorder, comprising administering to a subject in need thereof the crystalline diketopiperazine composition of claim 4.

[0142] (Appendix 23) A method for treating hyperglycemia and / or diabetes, comprising administering to a patient in need thereof the crystalline diketopiperazine composition of claim 6.

[0143] (Appendix 24) 10. A dry powder comprising the crystalline diketopiperazine composition of claim 1.

[0144] (Appendix 25) 25. The dry powder of claim 24, further comprising one or more active agents, wherein the one or more active agents are peptides, proteins, nucleic acid molecules, small organic molecules, or analogs thereof.

Claims

1. a) forming diketopiperazine particles in a suspension having a bimodal distribution of particle sizes ranging from about 0.05 μm to about 10 μm and including one or more active agents; b) atomizing the suspension using a spray dryer under an air or gas stream; and c) reforming the particles by spray drying to a dry powder comprising microcrystalline diketopiperazine particles having substantially hollow spheres; and a method for administering the compound of formula (I) to a subject in need of administration by a method comprising the steps of: Dry powder formulation.

2. A first peak of particles in the bimodal particle size distribution has an average size of about 0.2 μm to about 2.4 μm, and a second peak of particles has an average size of about 2.1 μm to about 2.4 μm.

10. The dry powder formulation of claim 1.

3. The diketopiperazine of claim 2, wherein X is selected from the group consisting of fumaryl, succinyl, maleyl, malonyl, and glutaryl.

10. The dry powder formulation of claim 1.

4. The diketopiperazine is 3,6-bis(N-fumaryl-4-aminobutyl)-2,5-diketopiperazine. The dry powder formulation of claim 3.

5. The active agent is at least one endocrine hormone or an agonist of an endocrine hormone.

10. The dry powder formulation of claim 1.

6. The active agent is selected from the group consisting of cytokines, lipokines, enkephalins, alkynes, cyclosporines, anti-IL-8 antibodies, IL-8 antagonists including ABX-IL-8, PG-I2, LTB receptor blockers, triptans, insulin and analogs thereof, growth hormone and analogs thereof, parathyroid hormone (PTH) and analogs thereof, parathyroid hormone-related peptide (PTHrP), ghrelin, obestatin, enterostatin, granulocyte-macrophage colony-stimulating factor (GM-CSF), amylin, amylin analogs, glucagon-like peptide-1 (GLP-1), clopidogrel, PPACK (D-phenylalanyl-L-prolyl-L-arginine chloromethyl ketone), oxyntomo oxalin (OXM), peptide YY(3-36) (PYY), adiponectin, cholecystokinin (CCK), secretin, gastrin, glucagon, motilin, somatostatin, brain natriuretic peptide (BNP), atrial natriuretic peptide (ANP), IGF-1, growth hormone releasing factor (GHRF), integrin beta-4 precursor (ITB4) receptor antagonist, analgesic, nociceptin, nocistatin, orphanin FQ2, calcitonin, CGRP, angiotensin, substance P, neurokinin A, pancreatic polypeptide, neuropeptide Y, delta sleep-inducing peptide, vasoactive intestinal peptide, and analogs of said active agents.

10. The dry powder formulation of claim 1.

7. The active agent comprises at least one of proteins, polypeptides, peptides, nucleic acids, organic polymers, synthetic organic compounds, polysaccharides and other sugars, fatty acids, lipids, antibodies, and fragments thereof.

10. The dry powder formulation of claim 1.

8. The active agent comprises at least one of a humanized or chimeric antibody, F(ab), F(ab)2, single chain antibody alone or fused to other polypeptides, or a therapeutic or diagnostic monoclonal antibody against a cancer antigen, a vasoactive agent, a neuroactive agent, a hormone, an anticoagulant, an immunomodulator, a cytotoxic agent, an antibiotic, an antiviral, an antigen, an infectious agent, an inflammatory mediator, a cell surface receptor agonist and antagonist, and a cell surface antigen.

10. The dry powder formulation of claim 1.

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