HK1261654A1 - Microcrystalline diketopiperazine compositions and methods - Google Patents

Description

Microcrystalline diketopiperazine compositions and methods

The application is a divisional application of Chinese patent application 201480015837.1(PCT/US2014/029491) with application date of 2014, 3, and 14.

Technical Field

Disclosed herein are microcrystalline Diketopiperazine (DKP) particles, compositions, methods of making the particles, and methods of using the particles. In particular, the particles are useful as delivery systems for drugs or active agents in the treatment of diseases or disorders (e.g. of endocrine origin, including diabetes and obesity).

Background

Drug delivery has been an important issue for many years, particularly when the compound to be delivered is administered orally to a subject, which is unstable under the conditions encountered in the gastrointestinal tract before reaching the target site. For example, oral administration is preferred in many cases, particularly in view of ease of administration, patient compliance and reduced cost. However, many compounds do not work or exhibit low or variable efficacy when administered orally. This may be because the drugs are not stable under the conditions of the digestive tract or because they are not efficiently absorbed.

Due to these problems associated with oral drug delivery, drug delivery to the lung was investigated. For example, drugs that are commonly delivered to the lung are designed to act on the lung tissue, such as vasodilators, surfactants, chemotherapeutic agents, or vaccines against influenza or other respiratory diseases. Other drugs, including nucleotide drugs, have been delivered to the lung because the lung represents a particularly suitable tissue for treatment, such as gene therapy for cystic fibrosis, where a retroviral vector expressing a defective adenosine deaminase is administered to the lung.

It is also possible to deliver agent drugs with systemic effects to the lung. Benefits of delivering systemic agents to the lung include large surface area and ease of uptake through the mucosal surfaces of the lung. Pulmonary drug delivery systems present many difficulties, such as the use of propellants, and nebulization of biological agents such as proteins and peptides can result in denaturation and excessive loss of the agent to be delivered. Another problem associated with all of these forms of pulmonary drug delivery is: it is difficult to deliver drugs to the lungs due to the problem of passing the drug through all natural barriers (such as cilia along the trachea) and trying to administer the drug in a uniform volume and weight.

Thus, there is room for improvement in pulmonary drug delivery.

Summary of The Invention

The present disclosure provides improved microcrystalline particles, compositions, methods of making particles, and methods of improving delivery of a drug to the lung for treating diseases and conditions in a subject. Embodiments disclosed herein achieve improved delivery by providing crystalline diketopiperazine compositions comprising microcrystalline diketopiperazine particles having high drug adsorption capacity, resulting in powders of one or more active agents with high drug content. Powders prepared with the microcrystalline particles of the present invention can deliver increased drug content in smaller powder doses. Powders can be prepared by a variety of methods, including methods that utilize surfactant-free solutions or surfactant-containing solutions depending on the starting materials.

Certain embodiments disclosed herein may include a powder comprising a plurality of substantially homogeneous microcrystalline particles, wherein the particles have a substantially hollow spherical structure and comprise a shell, which may be porous and comprise non-self-assembled diketopiperazine crystallites (crystallites).

Certain embodiments disclosed herein include powders comprising a plurality of substantially homogeneous microcrystalline particles, wherein the particles have a substantially hollow spherical structure and comprise an outer shell, which may be porous and comprise crystallites of a diketopiperazine that do not self-assemble, and the particles have a volume median geometric diameter (volumetrical) of less than 5 μm.

In one embodiment herein, up to about 92% of the crystallite particles have a volume median geometric diameter ≦ 5.8 μm. In one embodiment, the outer shell of the particle is composed of interlocking diketopiperazine crystals with one or more drugs adsorbed on their surface. In some embodiments, the particles can be a combination of embedding a drug in their internal void volume and/or adsorbing a drug to the surface of a crystallite and embedding a drug in the internal void volume of a sphere.

In certain embodiments, there is provided a diketopiperazine composition comprising a plurality of substantially uniformly formed microcrystalline particles, wherein the particles have a substantially hollow spherical structure and comprise a shell comprising grains of diketopiperazine that do not self-assemble; wherein the particles are formed by a process comprising the steps of: combining diketopiperazine and acetic acid solutions in solution having a trans isomer content in the range of about 45% to 65% in the absence of a surfactant while homogenizing in a high shear mixer at high pressures up to 2,000psi to form a precipitate; washing the precipitate in the suspension with deionized water; the suspension is concentrated and dried in a spray drying apparatus.

The method may further comprise the step of adding a solution containing an active agent or active ingredient (such as a drug or bioactive agent) with mixing prior to the spray drying step, such that the active agent or active ingredient is adsorbed and/or embedded onto or into the particles. The particles prepared by this method may be in the submicron size range prior to spray drying.

In certain embodiments, there is provided a diketopiperazine composition comprising a plurality of substantially uniformly formed microcrystalline particles, wherein the particles have a substantially hollow spherical structure and comprise an outer shell comprising crystallites of diketopiperazine that do not self-assemble, and the particles have a volume average geometric diameter (volumetric diameter) of less than or equal to 5 μm; wherein the particles are formed by a process comprising the steps of: combining the diketopiperazine and acetic acid solutions in solution in the absence of a surfactant while homogenizing in a high shear mixer at high pressures up to 2,000psi to form a precipitate; washing the precipitate in the suspension with deionized water; the suspension is concentrated and dried in a spray drying apparatus.

The method may further comprise the step of adding a solution containing an active agent or active ingredient (such as a drug or bioactive agent) with mixing prior to the spray drying step, such that the active agent or active ingredient is adsorbed and/or embedded onto or into the particles. The particles prepared by this method may be in the submicron size range prior to spray drying.

In certain embodiments, there is provided a diketopiperazine composition comprising a plurality of substantially uniformly formed microcrystalline particles, wherein the particles have a substantially hollow spherical structure and comprise a shell comprising grains of diketopiperazine that do not self-assemble, and the volume average geometric diameter of the particles is less than or equal to 5 μm; wherein the particles are formed by a process comprising the steps of: combining the diketopiperazine and acetic acid solutions in solution in the absence of a surfactant and in the absence of an active agent while homogenizing in a high shear mixer at high pressures up to 2,000psi to form a precipitate; washing the precipitate in the suspension with deionized water; the suspension is concentrated and dried in a spray drying apparatus.

The method may further comprise the step of adding a solution containing an active agent or active ingredient (such as a drug or bioactive agent) with mixing prior to the spray drying step, such that the active agent or active ingredient is adsorbed and/or embedded onto or into the particles. The particles prepared by this method may be in the submicron size range prior to spray drying.

In one embodiment, the composition may comprise microcrystalline particles containing one or more active ingredients; wherein the active ingredient is a peptide, a protein, a nucleic acid molecule, a small organic molecule, or a combination thereof. In embodiments where the active ingredient is a peptide, oligopeptide, polypeptide, or protein, the peptide, oligopeptide, polypeptide, or protein can be an endocrine hormone, neurotransmitter, vasoactive peptide, receptor agonist or antagonist, or the like. In some embodiments, the endocrine hormone is insulin, parathyroid hormone, calcitonin, glucagon-like peptide 1, oxyntomodulin, peptide YY, leptin, or an analog of said endocrine hormone. In some embodiments, the excipient may be incorporated into the particles by addition to one, another, or all of the raw materials used in the spray drying step.

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

In an exemplary embodiment, the crystalline diketopiperazine composition comprises a diketopiperazine of the formula 2, 5-diketo-3, 6-di (N-X-4-aminoalkyl) piperazine wherein alkyl represents an alkyl group containing from 3 to 20 carbon atoms including propyl, butyl, pentyl, hexyl, heptyl and the like; and is for example 2, 5-diketo-3, 6-di (N-X-4-aminobutyl) piperazine, wherein X is selected from fumaryl, succinyl, maleyl, malonyl and glutaryl or salts thereof. In one particular embodiment, the diketopiperazine is (di-3, 6- (N-fumaryl-4-aminobutyl) -2, 5-diketo-diketopiperazine having the formula:

in various embodiments, methods of preparing a dry powder comprising microcrystalline particles suitable for pulmonary administration are provided, wherein the methods can be performed using a surfactant-free solution or a surfactant-containing solution. In one aspect, the diketopiperazine contains a trans isomer content ranging from about 45% to 65%.

Certain embodiments disclosed herein include methods of preparing a dry powder comprising crystalline diketopiperazine microparticles from a starting material comprising a free acid diketopiperazine.

Certain embodiments disclosed herein include methods of preparing a dry powder comprising crystalline diketopiperazine microparticles from a starting material comprising a diketopiperazine salt.

In one embodiment, the method comprises:

dissolving diketopiperazine in ammonia water to form a first solution;

simultaneously feeding the first solution and a second solution comprising about 10.5% acetic acid at high pressure to a high shear mixer at an approximate pH of less than 6.0;

homogenizing the first solution and the second solution to form a suspension comprising diketopiperazine crystallites in the suspension, wherein the suspension has a bimodal distribution of crystallites having a particle size in the range of about 0.05 μm to about 10 μm diameter;

atomizing the suspension under air or gas flow; and

the particles are reshaped by spray drying into a dry powder comprising microcrystalline particles having substantially hollow spheres.

In another embodiment, the method comprises:

dissolving diketopiperazine in aqueous sodium hydroxide solution 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, at an approximate pH of less than 6.0 in a high shear mixer at high pressure;

homogenizing the first and second solutions to form a suspension comprising diketopiperazine crystallites in the suspension, wherein the suspension has a bimodal distribution of crystallites having a particle size in the range of about 0.05 μm to about 10 μm diameter and comprises a trans isomer content in the range of about 45% to 65%;

atomizing the suspension under air or gas flow; and

the particles are reshaped by spray drying into a dry powder comprising microcrystalline particles having substantially hollow spheres.

In one embodiment, the method comprises:

dissolving diketopiperazine in ammonia water to form a first solution;

simultaneously feeding a first solution and a second solution comprising about 10.5% acetic acid at high pressure at an approximate pH of less than 6.0 in a high shear mixer to form a suspension comprising diketopiperazine crystallites in the suspension, wherein the suspension has a bimodal distribution of crystallites having a particle size in the range of about 0.05 μ ι η to about 10 μ ι η in diameter;

atomizing the suspension under air or gas flow; and

the particles are reshaped by spray drying into a dry powder comprising microcrystalline particles having substantially hollow spheres.

The method may further comprise the step of adding a third solution to the diketopiperazine crystallite suspension prior to atomizing the suspension; wherein the solution contains a drug or pharmaceutically active ingredient and the atomization step can be carried out under air or gas (including nitrogen) using an external mixing two-fluid nozzle into a spray dryer equipped with a high efficiency cyclone.

In certain embodiments, the particles in the suspension have a particle size distribution as a bimodal curve, as determined by laser diffraction; wherein the first peak of the particles has an average particle size of about 0.2 μm to about 0.4 μm and the second peak of the particles has an average size of about 2.1 μm to about 2.4 μm diameter.

In some embodiments, the step of atomizing the suspension uses a nitrogen stream of about 700 liters of nitrogen per hour as the process gas, and the nozzle temperature may be maintained at about 25 ℃.

The microcrystalline particles formed by the above method do not self-assemble when suspended in a solution, such as water or other water-based solvent. In a particular embodiment, the process comprises a diketopiperazine of the formula 2, 5-diketo-3, 6-di (N-X-4-aminobutyl) piperazine, wherein X is selected from the group consisting of fumaryl, succinyl, maleyl, malonyl, and glutaryl. In one embodiment, the method comprises homogenizing a solution of a diketopiperazine in a high shear mixer, wherein the diketopiperazine is (di-3, 6- (N-fumaryl-4-aminobutyl) -2, 5-diketo-diketopiperazine or salts thereof, including disodium, dipotassium, magnesium, calcium and dilithium salts.

In one embodiment, a crystalline diketopiperazine composition comprising a plurality of microcrystalline particles that are substantially uniform in size is obtained as a product of a spray drying step.

In one embodiment, a crystalline diketopiperazine composition comprising a plurality of microcrystalline particles having a bimodal size distribution is obtained as a product of the grain formation step.

When a splitting (dispersion) step is used, the bimodal distribution of larger species can shift to smaller sizes.

Certain embodiments include a method of forming microcrystalline particles of diketopiperazine acid for the preparation of dry powders for carrying larger drug contents comprising using a diketopiperazine salt as a starting compound, including 2, 5-diketo-3, 6-bis (N-fumaryl-4-aminobutyl) piperazine disodium salt, the method comprising:

dissolving a diketopiperazine salt in water comprising a surfactant in an amount of about 0.2% to about 6% (w/w) to form a first solution;

simultaneously combining the first solution with a second solution comprising about 8% to about 12% (w/w) acetic acid in a high shear mixer at high pressure at an approximate pH of less than 6.0;

homogenizing the first solution and the second solution to form a suspension comprising diketopiperazine crystallites in the suspension, wherein the suspension has a bimodal distribution of crystallites having a particle size in the range of about 0.05 μm to about 10 μm in diameter;

atomizing the suspension under air or gas flow; and

the particles are reshaped by spray drying into a dry powder comprising microcrystalline particles of diketopiperazine acids having substantially hollow spheres.

In one embodiment, the microcrystalline particles may be prepared by a process comprising: preparing a first solution comprising a diketopiperazine (e.g., 2, 5-diketo-3, 6- (N-fumaryl-4-aminobutyl) piperazine disodium salt) and a surfactant (such as polysorbate 80) in water; preparing a second solution comprising acetic acid at a concentration of about 10.5% (w/w) and a surfactant at a concentration of about 0.5% (w/w); mixing the first solution and the second solution in a high shear mixer to form a suspension; optionally testing the suspension to determine the particle size distribution such that the suspension comprises a bimodal particle size distribution, wherein the particles have a size in the range of about 0.2 μm to about 10 μm diameter, wherein the first peak of the particles has an average diameter of about 0.4 μm and the second peak of the particles has an average diameter of about 2.4 μm, and spray drying the suspension to obtain a dry powder.

Certain embodiments may include a splitting step to reduce the size of the larger size population in the bimodal distribution, for example using ultrasound, agitation or homogenization. In some embodiments, the disruption step may be performed prior to nebulizing the suspension.

In some embodiments herein, the method for preparing microcrystalline diketopiperazine particles can further comprise a washing step with deionized water. In one embodiment, the atomization step may be performed, for example, using an external mixing two-fluid nozzle into a spray dryer equipped with a high efficiency cyclone.

The method may further comprise the step of adding a solution comprising one or more active agents to the suspension prior to dispersion and/or spray drying, wherein the active agent is a peptide, oligopeptide, polypeptide, protein, nucleic acid molecule, or small organic molecule. The peptide may be an endocrine hormone including insulin, parathyroid hormone, calcitonin, glucagon-like peptide 1, oxyntomodulin, peptide YY, leptin, or an analog of said endocrine hormone, and the like. The method may optionally include the step of adding a solution comprising a surfactant and/or a pharmaceutically acceptable carrier including amino acids (such as leucine, isoleucine) and/or monosaccharides, disaccharides or oligosaccharides (such as lactose, trehalose, etc.), or sugar alcohols (including mannitol, sorbitol, etc.).

In another embodiment, a composition comprising more than one active agent may be prepared using the method of the present invention. The process for preparing such compositions comprises the steps of: microcrystalline diketopiperazine particles comprising more than one active agent are prepared wherein each active agent/ingredient is treated separately in solution and separate diketopiperazine particle suspensions are added and the solution conditions are changed to promote adsorption of the active agent onto the surface of the crystallites, then two or more separate suspensions comprising the active agent are blended, then the particles are dispersed and spray dried. In a different procedure, the blend comprises a suspension containing diketopiperazine particles but no active agent, e.g., to achieve a lower total active agent content. In an alternative embodiment, one or more separate solutions containing a single active agent may be combined with a single suspension comprising diketopiperazine particles prior to dispersing and spray drying the particles. The resulting dry powder comprises a composition comprising two or more active ingredients. In these embodiments, the amount of each component in the composition can be controlled according to the needs of the patient population to be treated.

In another embodiment, the dry powder comprises a composition comprising 2, 5-diketo-3, 6-bis (N-X-4-aminobutyl) piperazine, wherein X is fumaryl, and the composition comprises substantially homogeneous drug-containing microcrystalline particles; wherein the particles are in the shape of substantially spheres having a substantially hollow core and the grains form the shell of the spheres. In another embodiment, the dry powder comprises a diketopiperazine of the formula 2, 5-diketo-3, 6-bis (N-X-4-aminobutyl) piperazine and a drug, wherein the drug is a peptide, wherein the peptide can have a variety of peptide lengths, molecular sizes, or masses, including: insulin, glucagon-like peptide-1, glucagon, toxin-specific exocrine peptide, parathyroid hormone, calcitonin, oxyntomodulin, etc.

Other embodiments include drug delivery systems including inhalers with or without a cartridge, wherein the cartridge is a unit dose dry powder medicament container (e.g., a cartridge), and a powder comprising the particles and active agent disclosed herein. In one embodiment, the delivery system for use with dry powders includes an inhalation system comprising a high resistance inhaler having an air conduit capable of imparting a high resistance to airflow through the conduit to deagglomerate and disperse the powder. In one embodiment, the resistance of the inhalation system has a value of, for example, about 0.065 to about 0.200(√ kPa)/liter per minute. In certain embodiments, the dry powder may be effectively delivered by inhalation with an inhalation system, wherein the peak inhalation pressure differential may be in the range of about 2 to about 20kPa, which may produce a resulting peak flow rate of between about 7 and 70 liters/minute. In certain embodiments, the inhalation system is configured to provide a single dose by causing the powder to be expelled from the inhaler as a continuous stream or one or more pulses of powder delivered to the patient. In some embodiments disclosed herein, the dry powder inhaler system comprises a predetermined mass flow balance within the inhaler, wherein the inhaler conduits are designed to have different flow profiles during inhalation. For example, a flow balance of about 10% to 70% of the total flow exiting the inhaler and into the patient is delivered through one or more dispensing tips, wherein the airflow is through an air conduit designed to have a region containing the powder formulation, and wherein about 30% to 90% of the airflow is generated from other conduits of the inhaler during inhalation maneuvers. In addition, the bypass flow or flow that does not enter and exit the powder container area (e.g., through the cartridge) may recombine with the flow exiting the powder dispensing end within the inhaler, causing the fluidized powder to be diluted, accelerated, and eventually deagglomerated before exiting the mouthpiece. In one embodiment, a flow rate in the range of about 7 to 70 liters/minute results in more than 75% of the container or cartridge contents being dispensed in a fill mass between 1 and 50 mg. In certain embodiments, the inhalation system described above may emit a powder dose in a single inhalation of inhalable fractions/loading of greater than 40%, greater than 50%, greater than 60% or greater than 70% in percentage.

In certain embodiments, a drug delivery system including an inhaler may include an inhaler that is particularly suitable for use with particulate forms (e.g., crystalline or amorphous forms) containing dry powders.

In some embodiments, provided inhalation systems include a dry powder inhaler, a dry powder formulation comprising fumaryl diketopiperazine microcrystalline particles 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 prefilled in the inhaler. In this embodiment, the structural configuration of the inhalation system allows the deaggregation mechanism of the inhaler to produce an inhalable fraction of greater than 50%; that is, more than half of the powder contained in the inhaler (cartridge) is ejected as particles smaller than 5.8 μm. The inhaler can expel more than 85% of the powdered medicament contained in the container during administration. In certain embodiments, the inhaler can discharge more than 85% of the powder medicament contained in a single inhalation. In one embodiment, the inhaler may discharge more than 90% of the cartridge content or container content in less than 3 seconds at a fill mass of up to 30mg at a pressure differential between 2kPa and 5 kPa.

in one embodiment, a method of treating an endocrine-related disease or condition comprises administering to a human in need thereof a dry powder formulation comprising FDKP microcrystalline particles (comprising FDKP having a trans isomer content that may be from about 45% to about 65%), wherein the microparticles are prepared by the method of the invention, and a medicament suitable for treating the disease or condition, one embodiment comprises a method of treating an insulin-related condition comprising administering to a human in need thereof a dry powder formulation comprising FDKP microcrystalline particles as described above, the method comprising administering to a subject a dry powder formulation comprising fumaryl diketopiperazine microcrystalline particles having a trans isomer content that is in the range of about 45% to 65%, wherein the particles are hollow spheres and do not comprise any surfactant.

Another embodiment disclosed herein includes a method of delivering a peptide (including GLP-1, oxyntomodulin, peptide YY, oxytocin, insulin) to a patient in need thereof, the method comprising: the dry powder comprising the diketopiperazine microcrystalline particles disclosed herein is administered to the deep lung by inhalation of the dry powder by the patient. In some aspects of this embodiment, specific features of the inhaler system are specified.

Drawings

The following drawings form part of the present specification and are included to further demonstrate certain aspects of the disclosed examples. The 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.

Fig. 1A and 1B are Scanning Electron Micrographs (SEM) of fumaryl diketopiperazine particles comprising insulin and showing the solid composition of the lyophilized particles at low magnification (1A) and high magnification (1B).

Fig. 2 depicts a graphical representation of the particle size distribution of the particles depicted in fig. 1A and 1B as measured by the probability density function (pdf, left y-axis) and cumulative distribution function (cdf, right y-axis) scales.

Figure 3 depicts a graphical representation of the particle size distribution of particles obtained from one embodiment of the preparation of a suspension in which microcrystalline particles are formed in the absence of surfactant in any solution used. The figure shows a typical bimodal distribution of crystallite particles as measured by the probability density function (pdf, left y-axis) and cumulative distribution function (cdf, right y-axis) scales.

Fig. 4 depicts an SEM at low magnification (2500X) of FDKP particles recovered from an embodiment herein in which a surfactant-free particle suspension was lyophilized.

Fig. 5 depicts a graphical representation of the lyophilized particle size distribution in the suspension as depicted in fig. 4 formed in the absence of surfactant and shows the increase in particle size as determined by the probability density function (pdf, left y-axis) scale and the cumulative distribution function (cdf, right y-axis) scale.

Fig. 6 depicts an SEM (2500X) of one embodiment of the claimed invention, showing microcrystalline particles made from spray dried surfactant free solution.

Figure 7 depicts a graphical representation of the particle size distribution of spray-dried surfactant-free particles dispersed in water.

Figure 8 depicts a graphical representation of the particle size distribution of spray-dried surfactant-free particles dispersed in 0.01M HCl (pH 2).

FIG. 9 depicts Na by acetic acid in the presence of a surfactant₂Graphical representation of the bimodal particle size distribution of the suspension formed by the crystallization of FDKP.

FIGS. 10A and 10B depict a cross-sectional view taken through a cross-section taken through Na₂Two scanning electron micrographs of particles prepared by spray drying a suspension of crystals made from FDKP at 2,500X (10A) and 10,000X (10B) magnification.

Fig. 11A and 11B depict scanning electron micrographs of spray-dried surfactant-free FDKP particles with about 10 wt% insulin at 2,500X (11A) and 5,000X (11B) magnifications.

FIGS. 12A and 12B are prepared by reacting Na₂Two scanning electron micrographs at 2,500X (12A) and 10,000X (12B) magnification of particles prepared by spray drying a suspension of crystals made of DKP.

FIG. 13 depicts a cross-sectional view of a₂Graphical representation of the size distribution of particles formed by spray drying a suspension of FDKP crystallized from a solution of FDKP and polysorbate 80. The particles were dispersed in water for assay.

Fig. 14 depicts a graphical representation of the particle size distribution of a spray-dried combined powder and a grain suspension with an individual active agent. 1 represents the particle size distribution of a combined microcrystalline powder composition comprising two different active agents; in separate suspensions of diketopiperazine-active agent particles, one composition containing FDKP-GLP-1 particles and the other containing a suspension of FDKP-insulin (3), which are combined prior to being spray dried.

Detailed Description

As mentioned, delivery of drugs to the lungs provides a number of advantages. But it is difficult to deliver the drug to the lungs in a uniform volume and weight due to the problem of transporting the drug across natural physical barriers. Disclosed herein are crystalline diketopiperazine compositions, dry powders, and methods of making particles. The crystalline composition and the dry powder therefrom comprise diketopiperazine microcrystalline particles that are substantially uniformly defined spheres comprising a shell comprising diketopiperazine crystallites and a core. In certain embodiments, the core may be hollow. In one embodiment, the diketopiperazines have a defined trans isomer content, which can facilitate particles as drug delivery agents, methods of making the particles, and methods of treatment using the particles. The particles disclosed herein have a higher capacity to carry and deliver drug content to a patient in smaller doses than standard prior art particles.

As used herein, "analog" includes a compound having structural similarity to another compound. Thus, a compound that mimics the biological or chemical activity of the parent compound with structural similarity to another (the parent compound) is an analog. So long as the analog is capable of modeling the biological or chemical properties of the parent compound in some relative manner, either identically, complementarily, or competitively, there is no minimum or maximum number of substitutions that would result in the compound being identified as the element or functional group required for the analog. In some cases, analogs comprise fragments of the parent compound, either alone or linked to another molecule, and may also contain other modifications. Analogs of the compounds disclosed herein may have equal, less, or greater activity than their parent compounds.

As used herein, the term "microparticle" refers to particles having a diameter of about 0.5 to about 1000 μm, regardless of the exact external or internal structure. Microparticles between about 0.5 and about 10 microns in diameter can successfully cross most natural barriers to the lungs. The diameter must be less than about 10 microns to pass through the corner of the throat, while the diameter must be about 0.5 microns or more to avoid exhalation. In order to reach the deep lung parts (or alveolar regions) where the most efficient absorption is believed to occur, it is preferred to maximize the proportion of particles contained in the "respirable fraction" (RF), which is generally accepted to use standard techniques (e.g. with Anderson multistage impact sampler (Anderson Cascade Imp)actor)) have an aerodynamic diameter of about 0.5 to about 5.7 microns, although some references use a slightly different range. Other IMPACTORs may also be used to determine aerodynamic particle size, such as NEXT genetic impact actuator^TM(NGI^TMMSP Corporation) in which the respirable fractions are of similar aerodynamic size (e.g.<6.4 μm). In some embodiments, the particle size is determined using a laser diffraction apparatus, such as the laser diffraction apparatus disclosed in U.S. patent application serial No. 12/727,179 filed 3/18/2010, the relevant teachings of which are incorporated herein in their entirety, wherein the Volume Median Geometric Diameter (VMGD) of the particles is determined to assess the performance of the inhalation system. For example, in various embodiments, VMGD with > 80%, 85%, or 90% cartridge empty and < 12.5 μm, < 7.0 μm, <5.8 μm, or < 4.8 μm ejected particles may exhibit increasingly better aerodynamic performance. Embodiments disclosed herein show that FDKP particles with trans isomer content between about 45% and about 65% exhibit properties that facilitate drug delivery to the lung, such as improved aerodynamic performance.

The respirable fraction based on the filling amount (RF/filling amount) represents the percentage of powder dose emitted from the inhaler suitable for inhalation after the discharge of the filling powder content used as a dose, i.e. the percentage of particles emitted from the filling dose and sized for pulmonary delivery, which is a measure of the aerodynamic properties of the particles. As described herein, RF/fill values of 40% or greater than 40% reflect acceptable aerodynamic properties. In certain embodiments disclosed herein, the respirable fraction based on the loading may be greater than 50%. In an exemplary embodiment, the respirable fraction may be up to about 80% based on the loading, where about 80% of the loading is emitted at a particle size <5.8 μm, as determined using standard techniques.

As used herein, the term "dry powder" refers to a fine particulate composition that is not suspended or dissolved in a propellant, carrier, or other liquid. This does not necessarily mean that all water molecules are completely absent.

The specific RF/fill value may depend on the inhaler used to deliver the powder. Powders are generally prone to agglomeration and certain crystalline DKP particles form particularly sticky powders. One function of a dry powder inhaler is to deagglomerate the powder so that the resulting particles contain an inhalable fraction suitable for delivering a dose by inhalation. However, deagglomeration of sticky powders is often not complete, so when determining the respirable fraction delivered by an inhaler, the particle size distribution seen does not match that of the original particles, i.e., the curve is shifted towards larger particles. The design of the inhaler will differ in its deagglomeration efficiency, and therefore the actual value of the RF/fill volume observed with different designs will also differ. However, the optimum RF/loading as a function of isomer content is the same between inhalers.

As used herein, the term "about" is used to indicate that a value includes the measured standard deviation of the equipment or method used to determine the value.

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

As used herein, the term "crystallites" is used to refer to the complete crystal units of diketopiperazine particles, which can be of varying sizes.

As used herein, "microcrystalline particles" comprise diketopiperazine grains having a particle size distribution of from 0.05 μm to about 100 μm, particle sizes of less than 50 μm, less than 20 μm, or less than 10 μm diameter, as determined by laser diffraction. In one embodiment, the size of the grains may be in the range of 0.01 to 1 μm.

Diketopiperazines

One class of drug delivery agents that has been used to overcome difficulties in the pharmaceutical field (e.g., drug instability and/or poor absorption) is 2, 5-diketopiperazines. The 2, 5-diketopiperazine is represented by a compound of the general formula 1 shown below, wherein E₁And E₂Independently N or more specifically NH. In thatIn other embodiments, E₁And/or E₂Independently oxygen or nitrogen, so that E₁And E₂Any one of the substituents of (a) is oxygen and the other is nitrogen, which formula gives the substituted analogue diketomorpholine; or when E₁And E₂When both are oxygen, this formula gives the substituted analog diketodioxane (diketodioxane).

These 2, 5-diketopiperazines have been shown to be useful for drug delivery, particularly those with acidic R₁And R₂Of groups such as described below: for example, U.S. Pat. No.5,352,461 entitled "Self Assembling Diketopiperidine Drug Delivery System"; U.S. Pat. No.5,503,852 entitled "Method For Making sectional device-assembling Diketopiprazine Drug Delivery System"; U.S. patent No.6,071,497 entitled "Microparticles For long Delivery Comprising Diketopiperazine" and U.S. patent No.6,331,318 entitled "Carbon-heated Diketopiperazine Delivery System," each of which is incorporated herein by reference in its entirety For all its teachings regarding Diketopiperazine and Diketopiperazine mediated drug Delivery. The diketopiperazine can form microparticles that incorporate the drug or microparticles to which the drug can be adsorbed. The combination of the drug and the diketopiperazine may confer improved drug stability and/or absorption characteristics. These microparticles can be administered by a variety of routes of administration. As a dry powder, the microparticles can be delivered by inhalation to specific regions of the respiratory system, including the lungs.

Such prior art microparticles are typically obtained by: the pH-based precipitation of the free acid (or base) produces self-assembled microparticles with a rose morphology, consisting of aggregated crystalline plates. The stability of the particles can be enhanced by a small amount of a surfactant, such as polysorbate-80, in the DKP solution from which the particles are precipitated (see, e.g., U.S. patent No.7,799,344 entitled "Method of drug formation based on the incubation of the affinity of crystalline particulate substrates for active agents," which is incorporated herein by reference in its entirety for all its teachings regarding the formation and loading of DKP microparticles and their dry powders). Finally, the solvent can be removed to obtain dry powder. Methods of solvent removal include lyophilization and spray drying (see, e.g., U.S. Pat. No.8,039,431 entitled "A method for improving the pharmaceutical properties of microorganisms comprising a liposome and an active agent" and U.S. Pat. No.6,444,226 entitled "Purification and stabilization of peptides and protein pharmaceuticals", each of which is incorporated herein by reference in its entirety for all teachings regarding the formation and loading of DKP microparticles and their dry powders). The particles disclosed herein differ from prior art particles in that they are both physically and morphologically distinct entities and are made by an improved process. Reference herein to FDKP is to be understood as a free acid or a dissolved anion.

Other prior art particles are obtained by spray drying a DKP solution to obtain amorphous DKP salt particles, which typically have a collapsed sphere morphology, such as those disclosed in U.S. patent nos. 7,820,676 and 8,278,308 entitled "diketopterazine salts for drug delivery related methods".

Methods of synthesizing diketopiperazines are described, for example, in katcallski 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 (Katchalski et al, known as lysine anhydride) can also be prepared by cyclodimerization of N- ε -P-L-lysine in molten phenol (this is analogous to the Kopple method), followed by removal of the blocked (P) -group by appropriate reagents and conditions. For example, the CBz protecting group can be removed using 4.3M HBr in acetic acid. This approach uses commercially available starting materials, which involve reaction conditions that reportedly preserve the stereochemistry of the starting materials in the product and all steps can be easily scaled up for production. Methods for synthesizing diketopiperazines are also described in U.S. patent No.7,709,639 entitled "Catalysis of Diketopiperazine Synthesis," the teachings of which in the same respect are also incorporated herein by reference.

Fumaryl diketopiperazines (di-3, 6- (N-fumaryl-4-aminobutyl) -2, 5-diketo-diketopiperazines; FDKP) is a preferred diketopiperazine for pulmonary use:

FDKP provides an advantageous microparticle matrix because it has low solubility in acids but dissolves readily in neutral or basic pH. These properties allow FDKP to crystallize and allow the crystals to self-assemble into microparticles under acidic conditions. The particles are readily soluble under physiological conditions where the pH is neutral. As noted, microparticles between about 0.5 and about 10 μm in diameter can successfully cross most natural barriers to the lungs. Particles in this size range can be readily prepared from FDKP.

FDKP has two asymmetric centers on the diketopiperazine ring. The FDKP prepared is a mixture of geometric isomers, which are designated as "cis-FDKP" and "trans-FDKP" according to the arrangement of the side chains relative to the central "ring" of the diketopiperazine. The R, R and S, S enantiomers have propenyl (amidobutyl) "side arms" that extend from the same planar side of the diketopiperazine ring (a and B below), and are therefore referred to as cis-isomers; and the R, S compounds have "side arms" extending from the opposite planar side of the diketopiperazine ring (C below) and are therefore referred to as trans isomers.

FDKP particulate powders having acceptable aerodynamic properties have been prepared with FDKP having a trans isomer content in the range of about 45 to about 65% by a moderately efficient Inhaler (such as U.S. Pat. No.' Unit DosecCarddge and Dry Powder InhalerDisclosed in No.7,464,706Inhaler, incorporated by reference herein for the same teachings) was tested with RF/fill volume. Particles having isomer contents within this range also perform well in high efficiency inhalers (such as those disclosed in U.S. patent No.8,499,757 entitled "a drive Powder inharaned System for Drug Delivery" filed on 12.6.2009, U.S. patent application No.8,424,518 entitled "drive Powder Inhaler and System for Drug Delivery" filed on 12.6.12.2009, U.S. patent application No.13/941,365 entitled "drive Powder Delivery System and Methods" filed on 12.7.4.2010, and U.S. patent application No.12/717,884 entitled "filed on 3.4.2010, which are incorporated herein by reference for the same teachings). Powders comprising microparticles containing greater than 65% trans FDKP tend to have lower and more variable RF/loading. Trans isomer-rich FDKP microparticles have altered morphology and can result in viscous suspensions that are difficult to handle.

The formulation of FDKP microparticles having a trans isomer content of about 45% to about 65% provides a powder with acceptable aerodynamic properties, as disclosed in U.S. patent No.8,227,409, the disclosure of which is incorporated herein by reference for the same teachings. The specific surface area is determined to be less than 67m²The formulation of FDKP granules/g also provides a dry powder for inhalation with acceptable aerodynamic properties, as disclosed in U.S. patent No.8,551,528 entitled "dikettopirazine Microparticles with Defined Specific Surface Areas", filed on 11.6.2010, the disclosure of which is incorporated herein by reference for the teachings of the same aspects. However, these FDKP powders tend to be sticky and inhalers are designed to overcome this property.

There is therefore a need to prepare diketopiperazine powders with less sticky particle compositions, which will allow for more efficient drug delivery and less inhaler bypass design (design around). The present disclosure identifies that the present method of preparing diketopiperazine microcrystalline particles as exemplified by FDKP and FDKP disodium salts provides microcrystalline dry powders with acceptable aerodynamic properties, where the powders are less viscous, vary in density, have alternative physical structures that do not self-assemble in suspension, and provide increased drug content capacity, including delivery of one or more active agents, which is unexpected.

It has been determined that by different methods of preparing diketopiperazine microparticles, uniformity of particle uniformity can be improved. The inventive method of making the composition and the compositions comprising the inventive microcrystalline diketopiperazine particles provide dry powders for pulmonary inhalation having advantageous physical and morphological aerodynamic properties.

Selection and integration of active agents

In exemplary embodiments comprising FDKP, the microcrystalline particles described herein may take on other additional properties that facilitate delivery to the lung and/or absorption of the drug, at least as long as they maintain the above-described isomer content. U.S. patent No.6,428,771 entitled "method for Drug Delivery to the Pulmonary System," which is incorporated herein by reference for the same teachings, describes the Delivery of DKP particles to the lung. U.S. Pat. No.6,444,226, entitled "Purification and stabilization of peptides and Protein Pharmaceutical Agents," describes a method for facilitating adsorption of a drug to the surface of microparticles, and is also incorporated herein by reference for its teachings in the same regard. The particulate surface properties can be manipulated to achieve the desired characteristics as described in U.S. patent No.7,799,344 entitled "Method of Drug formulated on incorporation of the affinity of Crystalline Microparticle Surfaces for active Agents" (which is incorporated herein by reference for the same teachings). U.S. Pat. No.7,803,404 entitled "Method of Drug Formation based on incorporation of the affinity of Active Agents for Crystalline particulate Surfaces" describes a Method of promoting the adsorption of an Active agent onto a particulate. Also, the teachings of U.S. patent No.7,803,404 in the same respect are incorporated herein by reference. These teachings can be applied to the adsorption of an active agent to a crystallite in suspension, for example, prior to spray drying.

The microcrystalline particles described herein may comprise one or more active agents. As used herein, "active agent" used interchangeably with "drug" refers to a pharmaceutical substance, including small molecule drugs, biologicals, and bioactive agents. The active agent may be of naturally occurring, recombinant, or synthetic origin, including proteins, polypeptides, peptides, nucleic acids, organic macromolecules, synthetic organic compounds, polysaccharides and other sugars, fatty acids, and lipids, as well as antibodies and fragments thereof, including but not limited to humanized or chimeric antibodies, F (ab)₂Single chain antibodies alone or fused to other polypeptides, or therapeutic or diagnostic monoclonal antibodies against cancer antigens. The active agent can belong to a variety of biological activities and classes, 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 particularly, the active agent may include, in a non-limiting manner, cytokines, lipid factors (lipokines), enkephalins, alkynes, cyclosporines, anti-IL-8 antibodies, IL-8 antagonists including ABX-IL-8; prostaglandins including PG-12, LTB receptor blockers comprising LY29311, BIIL 284, and CP 105696; 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, myostatin, enterostatin, granulocyte macrophage colony stimulating factor (GM-CSF), dextrin analogs, glucagon-like peptide 1(GLP-1), clopidogrel, PPACK (D-phenylpropanoid-L-propyl-L-arginine chloromethyl ketone), Oxyntomodulin (OXM), peptide YY (3-36) (PYY), adiponectin, cholecystokinin (CCK), secretin, gastrin, glucagon, motilin, growth hormoneinhibin, Brain Natriuretic Peptide (BNP), Atrial Natriuretic Peptide (ANP), IGF-1, Growth Hormone Releasing Factor (GHRF), integrin β -4 precursor (I T BETA 4) receptor antagonists, analgesics, nociceptin, orphyrin FQ2, calcitonin, CGRP, angiotensin, substance P, neurokinin A, pancreatic polypeptide, neuropeptide Y, delta-hypnotic peptide, and vasoactive intestinal peptide, as well as analogs of such active agents.

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

In one embodiment, compositions comprising more than one active agent may be prepared by adsorption using the methods of the invention, for example by binding the active agent to the crystallites prior to forming the dry powder.

In one embodiment, compositions comprising more than one active agent can be prepared using the methods of the invention by embedding the active agent between and within the crystallites, for example by spray drying the material without first adsorbing the active agent to the crystallites.

The process for preparing such compositions may comprise the steps of: preparing microcrystalline diketopiperazine particles comprising more than one active agent; wherein each active agent/ingredient is separately treated in solution and added to a separate diketopiperazine particle suspension, then two or more separate active agent-containing suspensions are blended, then the particles are dispersed and spray dried.

In certain embodiments, the grains may be mixed with a solution comprising one or more active agents.

In certain embodiments, the grains may be mixed with a solution comprising one or more active agents, wherein the solution conditions are altered to promote adsorption of the active agents onto the grain surfaces.

Each of the plurality of active agents may be adsorbed to a separate aliquot or particulate material. Aliquots of the adsorbed grains can then be mixed together and spray dried. Alternatively, the aliquot may be free of active agent, so that the total active agent content in the dry powder is adjusted without changing the conditions used to adsorb the active agent onto the grains.

In an alternative embodiment, one or more separate solutions containing a single active agent may be combined with the suspension comprising diketopiperazine particles prior to dispersion and spray drying to reshape the particles. The resulting dry powder composition comprises two or more active ingredients. In this embodiment, the amount of each component in the composition can be controlled according to the needs of the patient population to be treated.

It will be apparent from the foregoing disclosure that the microparticles of the disclosed embodiments of the present invention may take a variety of different forms and may incorporate a variety of different drugs or active agents.

Examples

The following examples are included to demonstrate embodiments of the disclosed microcrystalline diketopiperazine particles. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques developed by the inventors to function well in the practice of the present disclosure, and thus can be considered to constitute preferred modes for its practice. However, those of ordinary skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the scope of the invention.

Example 1

Preparation of standard FDKP microparticles-for comparison purposes FDKP microparticles were prepared as standard granules using prior art preparation methods such as disclosed in U.S. patent nos. 7,799,344, 7,803,404 and 8,227,409, the disclosures of which are incorporated herein by reference for the teachings of their related subject matter. In summary, a typical FDKP particle formation process: feed solutions each containing 0.05% (w/w) polysorbate 80(PS80) of FDKP and acetic acid were combined in a high shear mixer. Table 1 below shows the components of FDKP and insulin stock solutions.

Table 1 10.5% acetic acid solution filtered through 0.2 μm membrane

Components	By weight%
		Deionized water	89.00
GAA	10.50
		10% polysorbate 80	0.50

Table 2-2.5% FDKP solution filtered through 0.2 μm membrane

Components	By weight%
		Deionized water	95.40
FDKP	2.50
		NH4OH	1.60
10% polysorbate 80	0.50

A concentrated stock insulin solution may be prepared from 1 part insulin and 9 parts of about 2% by weight acetic acid. The insulin stock solution can be added gravimetrically to the suspension to achieve a loading of about 11.4% by weight. The insulin-containing suspension may be mixed for at least about 15 minutes and then titrated from an initial pH of about 3.5 to a pH of about 4.5 with about 14 to about 15 weight percent ammonia. The suspension may be snap frozen in liquid nitrogen using a cryogranulator to form granules, for example, as disclosed in U.S. patent No.8,590,320, the disclosure of which is incorporated herein by reference in its entirety, and lyophilized to produce discrete insulin-loaded FDKP microparticles that form small crystals or aggregates that self-assemble into FDKP granules having an open structure as seen in fig. 1A and 1B.

A sample of the particles formed was investigated to determine the size distribution of the particles in these suspensions, and the results are shown in figure 2. The data in fig. 2 shows a graphical representation of particle size distribution measurements, plotted on a logarithmic scale as a function of probability density (pdf, left y-axis) and cumulative distribution (cdf, right y-axis). The data shows that the particles in suspension have a monomodal size distribution ranging from about 1.0 to about 10 μm in diameter and centered at 2 μm or about 2 μm.

Microcrystalline FDKP granules were prepared-2.5% (w/w) FDKP was dissolved in basic aqueous ammonia solution (1.6% ammonia). A stock solution of 10.5% (w/w) acetic acid was added to a high shear mixer (Sonolater) under high pressure at an approximate pH of 2.0 to prepare granules. The formed particles were washed in deionized water. It was found that diketopiperazine microparticles were not stable in the absence of surfactant in the solution, however, no surfactant was added to any of the solutions or reagents in preparing the particles.

In these experiments, an equal mass of about 10.5 wt.% acetic acid and about 2.5 wt.% FDKP solution was fed through a 0.001 square inch nozzle at a temperature of about 16 ℃ ± about 2 ℃ and 2000psi using a dual feed high shear mixer to form a precipitate by homogenization. The precipitate was collected in a Deionized (DI) water reservoir of approximately equal mass and temperature. The precipitate was concentrated and washed with deionized water by tangential flow filtration. The suspension can be finally concentrated to a solids of about less than 5%, for example, about 2 to 3.5% based on the initial mass of FDKP. The solids content of the concentrated suspension can be determined by the drying method. For samples containing the active ingredient (i.e., insulin and/or GLP-1), the FDKP suspension described above was used, wherein the insulin stock solution was added to the suspension (insulin dissolved in 2% acetic acid was added to the suspension while mixing, then the suspension pH was titrated to pH4.5 + -0.3 with ammonium hydroxide. similarly, GLP-1 dissolved in 2% acetic acid stock solution was added gravimetrically to the FDKP-suspension with stirring, GLP-1FDKP suspension was titrated to pH4.5 + -0.1. each of the insulin-FDKP suspension and GLP-1-FDKP suspension was dispersed independently to a Niro SD-Micro equipped with a high efficiency cyclone using an external mixing two-fluid nozzle^TMIn a spray dryer. Nitrogen was used as the process gas (25kg/h) and atomizing fluid (2.8 kg/hr). The samples were treated in the spray dryer using the two treatment conditions listed in table 3.

Table 3.

For the control sample, blank FDKP crystallite particles were prepared in the same manner (with the exception of the insulin or GLP-1 loading step).

Figure 3 shows data from the above experiment in which the feed solution did not contain surfactant. Fig. 3 is a graph showing the particle size distribution of a suspension of FDKP particles, which shows a typical bimodal particle size distribution of the particles. The particle sizes herein range from about 0.1 to about 10 μm diameter, with one particle population centered at 0.2 μm diameter and another particle population centered at 2.1 μm diameter.

Samples of the suspension were lyophilized without spray drying. Fig. 4 is an SEM of the lyophilized particles at 2,500x magnification. As seen in figure 4, after lyophilization of the similar suspension, large flake-like particles were formed and when resuspended in water, much larger average sizes were obtained, as shown in figure 5. Figure 5 shows the particle size distribution in suspension of a sample freeze-dried from particles made without the use of a surfactant. In this study, the particle size diameter of the resuspended particles increased from about 1 to about 90 μm or more.

Fig. 6 shows a typical 2,500x magnification of a scanning electron micrograph of a powder sample from a surfactant-free formulation of microcrystalline FDKP particles formed using the method of the invention and spray drying as described above. As seen in fig. 6, the structure of the particles is a uniform sphere including a grain shell. When the suspension without surfactant was spray dried, particles with a physical diameter of about 4 μm were formed, as shown in fig. 6. Unlike standard FDKP particles, these particles dissociate into particles with a diameter of 0.2 μm when dispersed in water, as shown in fig. 7. Thus, surfactants have been shown to have a role in particle integrity. Dispersing the particles in 0.01M hydrochloric acid inhibited particle dissociation, as shown in fig. 8. The dissolved FDKP can precipitate during spray drying and can deposit along the boundaries between the primary particles and can act as a cement (cement). FDKP "cement" was dissolved in water and the particles dissociated into 0.2 μm primary particles; the lower solubility of FDKP in acid prevents dissociation and protects particle integrity.

Example 2

Microcrystalline FDKP particles were prepared by an alternative method using diketopiperazine salts-alternatively, FDKP crystallites can be formed from a surfactant-containing feed solution. By reacting FDKP disodium salt (Na) without using ammonia as a reagent₂FDKP) was dissolved in water containing polysorbate 80(PS80) as a surfactant to prepare a feed solution of FDKP. 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 DUAL feed sonolator crystallized FDKP and produced the bimodal particle size distribution shown in figure 9. As shown in fig. 9, about 26% of the formed primary crystals had a diameter of about 0.4 μm and about 74% of the larger particles had a diameter of about 2.4 μm. The suspension was treated and spray dried to obtain particles and observed under SEM. SEM micrographs were taken at 2,500x and 10,000x magnification and are shown in fig. 10A and 10B. Fig. 10A and 10B show that the particles are similar and spherical in shape, but smaller than that shown in fig. 6 of example 1, where FDKP was used as the free acid to prepare the particles. Table 4 below shows some of the physical properties determined for the powder prepared by lyophilization and the powder prepared by Spray Drying (SD) using FDKP disodium salt.

TABLE 4

The data show that the powder made from spray dried particles exhibited a higher respirable fraction (62.8% vs 28%), higher cartridge empty (% CE, 88.2% vs 83.8%), and higher bulk density (0.159g/mL vs 0.019g/mL) and tap density (0.234g/mL vs 0.03g/mL) than the lyophilized powder.

Example 3

Microcrystalline FDKP granules containing active agent-the active pharmaceutical ingredient (active agent) is incorporated into the granules by adding an active agent solution to a suspension of FDKP crystallites containing no surfactant, and then spray drying the mixture to remove the solvent as described in example 1. Control particles (FDKP-insulin) were also prepared for pulmonary inhalation powder by standard self-assembly methods using PS-80 in solution. In this study, insulin was dissolved in dilute acetic acid and a suspension of surfactant-free FDKP crystallites prepared as in example 1 was added (samples 1 and 2, table 5). The suspension was spray dried to obtain a dry powder containing about 10 wt% insulin. Powder samples were used for a variety of analyses, including delivery by high-resistance inhaler and scanning electron microscopy, and the results are shown in table 5. The size of the particles was approximately the same as the particles without insulin (example 1) and the particle morphology (fig. 11) was the same as shown in fig. 6. In addition, both samples 1 and 2 were powders with lower compactibility than the standard particles, and the sample 1 particles had a larger Specific Surface Area (SSA) than the control. The distribution of insulin is unknown, with no significant insulin deposition on the particle surface, indicating that insulin is either inside the particle or integrated into the particle wall.

TABLE 5

However, the data shown in table 5 show that at the same insulin content, the powder without surfactant behaves differently from the standard granules. For example, at the same insulin content, the powder without surfactant is released from the inhaler more efficiently (96.4%) than the standard particles (85%). An increase in the percentage of cartridge empty (% CE) indicates that the powder has increased flowability. Since the inhaler for the test powder was designed for the control powder, the respirable fraction (RF% based on loading) for the control particles was higher.

Example 4

Microcrystalline FDKP particles were prepared by an alternative method using diketopiperazine salts-in this study, FDKP disodium salt was used to prepare a suspension of FDKP salt particles as described in example 2. The insulin solution was added to the suspension of surfactant-free FDKP microcrystals as prepared in example 2. The suspension was spray dried to obtain a dry powder containing about 10 wt% insulin. The morphology of the formed particles (respectively) is shown in the SEM of fig. 12A and 12B at 2,500x and 10,000x magnification. As seen in fig. 12A and 12B, the morphology was the same as the particles without insulin, showing a spherical structure with a median particle diameter of 2.6 μm as shown in fig. 13, and also showing particles with diameters in the range of about 1.0 μm to about 10 μm.

Example 5

Preparation of microcrystalline FDKP granules containing more than one active agent-in another embodiment, a composition comprising more than one active agent may be prepared using the process of the present invention. The method of making such compositions includes the steps as disclosed above for each individual active agent to form an active agent-FDKP suspension of each of the active agents to be incorporated into the composition. The suspensions are then combined and blended to form a mixture. The blended mixture is then dispersed and spray dried as described above to produce microcrystalline diketopiperazine particles comprising more than one active agent. In one exemplary study, insulin and GLP-1 combination powders were prepared.

The suspension of FDKP crystallites prepared as in example 1 was mixed with a solution of various active agents (e.g. ghrelin, low molecular heparin, oxyntomodulin) and spray dried to obtain granules with properties similar to those in example 3.

Example 6

Preparation of microcrystalline FDKP granules containing two active agents

A combination powder containing two active agents (GLP-1 and insulin) is prepared by first preparing a suspension of FDKP crystallites containing insulin and a second suspension of crystallites containing GLP-1. The two suspensions are then mixed and the combined suspension is spray dried to obtain a dry powder containing the two active agents. A suspension of crystallites was prepared as described in example 1; after addition of the active agent, the suspension is adjusted to ph4.5 to facilitate adsorption onto the crystallites. Figure 14 is a graph of data showing the particle size distribution of spray dried combination powder (1) and a crystallite suspension containing the individual active agents, FDKP-insulin (2) and FDKP-GLP-1.

As shown in fig. 14, the particle size distribution of the combined powder was concentrated between the particle size distributions of the two separate suspensions and was significantly narrower. The composite powder comprises particles having a diameter of about 1 μm to about 10 μm. The insulin-containing crystallites are smaller (about 0.25 μm to about 10 μm) than the GLP-1 containing crystallites having a diameter in the range of about 0.5 μm to about 50 μm. The atomization step in spray drying can dissociate the initial crystallite colonies in the suspension and re-form particles with a particle size distribution that depends on the conditions in the suspension and the spray drying conditions.

Example 7

Administering a dry powder composition comprising crystalline diketopiperazine particles to a subject.

Preparation of a composition comprising the disodium salt of FDKP (Na) as described in example 1 above₂FDKP) to a powder formulation containing 9U of insulin per mg of composition. High resistance inhaler (Dreamboat) containing a cartridge^tmInhaler, mannikidd Corporation) was made to contain 1mg to 10mg per dose administered to subjects diagnosed with diabetes. An inhaler containing a dose of insulin is provided to the patient to be treated, who inhales the insulin dose in a single inhalation before, during or after a meal.

Unless otherwise indicated, all numbers expressing quantities of ingredients, properties (e.g., molecular weights), reaction conditions, and so forth, used in the specification and claims are to 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 following specification and attached 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. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

The use of quantitative terms in the context of describing the invention (especially in the context of the following claims) should be construed to include both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless the invention is otherwise indicated, each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

The term "or" as used in the claims means "and/or" unless it is expressly stated that an alternative or an alternative is mutually exclusive, although the description supports the definition of an alternative and "and/or".

Groupings of alternative elements or embodiments of the invention disclosed in the present application are not to be construed as limitations. Each group member may be referred to and claimed individually or in any combination with other members of the group or other elements found in the present invention. It is contemplated 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 is to be considered as including the group so modified as to satisfy the written description of all markush groups used in the appended claims.

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 those preferred embodiments will become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to 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 herein or otherwise clearly contradicted by context.

The specific embodiments disclosed herein are also to be limited in use by the claims as consisting of or consisting essentially of language. When used in a claim, the transitional term "consisting of … …" excludes all elements, steps or components not specified in the claims, whether filed in accordance with amendment or added. The transitional term "consisting essentially of … …" limits the scope of the claims to the specified materials or steps as well as those materials or steps that do not significantly affect the basic and novel characteristics. Embodiments of the claimed invention are described, either inherently or explicitly, and are practiced in the present invention.

In addition, a number of references, including patent and printed publications, are referred to in this specification. Each of the above-cited references and printed publications, respectively, is incorporated by reference herein in its entirety.

Furthermore, it is to be understood that the disclosed embodiments of the invention are merely illustrative of the principles of the invention. Other modifications that may be employed are within the scope of the invention. Thus, alternative models of the invention can be utilized in accordance with the teachings of the invention by way of example, but not by way of limitation. Accordingly, the invention is not to be limited exactly as shown and described.

Claims (15)

1. Use of a crystalline diketopiperazine composition comprising an active agent for treating endocrine-related disorders, wherein the composition comprises a plurality of microcrystalline particles that are substantially uniform in size, have a substantially hollow spherical structure, and comprise a shell comprising grains of non-self-assembled diketopiperazine.

2. Use according to claim 1, wherein the particles have a volume median geometric diameter of less than 5 μm.

3. The use of claim 1, wherein the composition further comprises a pharmaceutically acceptable carrier.

4. The use of claim 3, wherein the pharmaceutically acceptable carrier comprises at least one of an amino acid, a sugar, or a sugar alcohol.

5. The use of claim 4, wherein the amino acid comprises at least one of leucine and isoleucine.

6. The use of claim 4, wherein the sugar comprises at least one of a monosaccharide, a disaccharide, and an oligosaccharide.

7. The use of claim 5, wherein the amino acid is leucine.

8. The use of claim 3, wherein the active agent is a peptide, oligopeptide, polypeptide, protein, nucleic acid molecule or small organic molecule, wherein the peptide can be an endocrine hormone including insulin, parathyroid hormone, calcitonin, glucagon-like peptide 1, oxyntomodulin, peptide YY, leptin, or an analog of the endocrine hormone, or the like.

9. The use of claim 8, wherein the peptide is an endocrine hormone.

10. the use according to claim 3, wherein the active agent comprises at least one of vasoactive agents, neuroactive agents (including opioid agonists and antagonists), hormones, anticoagulants, immunomodulators, cytotoxic agents, antibiotics, antiviral agents, antigens, infectious agents, inflammatory mediators, hormones, cell surface receptor agonists and antagonists and cell surface antigens, more particularly, the active agents may include, without limitation, cytokines, lipid factors, enkephalins, alkynes, cyclosporines, anti-IL-8 antibodies, IL-8 antagonists including ABX-IL-8, prostaglandins including PG-12, LTB receptor blockers including LY29311, BIIL 284 and CP105696, triptans such as sumatriptan and palmitoleate, insulin and analogs thereof, growth hormone and analogs thereof, parathyroid hormone (PTH) and analogs thereof, parathyroid hormone-related peptides (PTHrP), ghrelin, leptin-releasing peptides, obesity inhibins, enterostatin, granulocyte colony stimulating factor (GM-CSF), GLP-1 and analogs thereof, ghrelin-1, ghrelin (PPG-gamma-.

11. The use of claim 3, wherein the composition further comprises a surfactant.

12. The use of claim 11, wherein the surfactant comprises polysorbate 80.

13. The use of claim 3, wherein the diketopiperazine is a diketopiperazine of the formula 3, 6-bis (N-X-4-aminobutyl) -2, 5-diketopiperazine, wherein X is selected from fumaryl, succinyl, maleyl, malonyl, and glutaryl.

14. The use according to claim 3, wherein the diketopiperazine is 3, 6-bis (N-fumaryl-4-aminobutyl) -2, 5-diketopiperazine.

15. The use of claim 10, wherein the active agent is PG-12.