HK1261654B - Microcrystalline diketopiperazine compositions and methods - Google Patents

Description

Microcrystalline diketopiperazine compositions and methods

This application is a divisional application of Chinese patent application 201480015837.1 (PCT/US2014/029491) filed on March 14, 2014.

Technical Field

Disclosed herein are microcrystalline diketopiperazine (DKP) particles, compositions, methods of preparing the particles, and methods of using the particles. In particular, the particles can be used as a delivery system for drugs or active agents in the treatment of diseases or conditions (e.g., of endocrine origin, including diabetes and obesity).

Background Art

The delivery of drugs has become an important problem for many years, especially when the compound to be delivered is administered orally to a subject, it is unstable under the conditions encountered in the gastrointestinal tract before reaching the target location. For example, oral administration is preferred in many cases, particularly considering the cost aspect of being easy to administer, patient compliance and reducing. However, during oral administration, many compounds do not work or show low or variable effectiveness. This may be because the drugs are unstable under the conditions of the digestive tract or because they are not effectively absorbed.

Because of these problems associated with oral drug delivery, drug delivery to the lungs has been studied. For example, drugs typically delivered to the lungs are designed to act on lung tissue, such as vasodilators, surfactants, chemotherapeutic agents, or vaccines for influenza or other respiratory diseases. Other drugs, including nucleotide drugs, have been delivered to the lungs because the lungs represent 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 lungs.

Also can be delivered to the lungs with agent medicine of systemic effect.The benefit of systemic agent delivery to the lungs comprises large surface area and is easy to be absorbed by the lung mucosal surface.There are many difficulties in pulmonary drug delivery system, for example the use of propellant, and the atomization of biological agent such as protein and peptide can cause the degeneration and excessive loss of agent to be delivered.Another problem relevant to all these forms of pulmonary drug delivery is: owing to make drug pass through all natural barriers (such as cilia along trachea) and attempt the problem of administration with uniform volume and weight, so be difficult to drug delivery to the lungs.

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

SUMMARY OF THE INVENTION

The present disclosure provides improved microcrystalline particles, compositions, methods for preparing the particles, and methods for improving drug delivery to the lungs to treat diseases and conditions in subjects. The embodiments disclosed herein achieve improved delivery by providing crystalline diketopiperazine compositions comprising microcrystalline diketopiperazine particles having high drug adsorption capacity, thereby generating powders of one or more active agents with high drug content. Powders prepared using the microcrystalline particles of the present invention can deliver increased drug content with smaller powder doses. Powders can be prepared by a variety of methods, including methods utilizing either surfactant-free solutions or surfactant-containing solutions, depending on the starting material.

Certain embodiments disclosed herein may include a powder comprising a plurality of substantially uniform microcrystalline particles, wherein the particles have a substantially hollow spherical structure and include an outer shell, which may be porous and comprises crystallites of diketopiperazine that are not self-assembled.

Certain embodiments disclosed herein include a powder comprising a plurality of substantially uniform microcrystalline particles, wherein the particles have a substantially hollow spherical structure and include an outer shell, which may be porous and includes crystallites of diketopiperazine that are not self-assembled, and wherein the particles have a volume median geometric diameter of less than 5 μm.

In a specific embodiment herein, up to about 92% of the microcrystalline particles have a volume median geometric diameter of ≤5.8 μm. In one embodiment, the shell of the particles is composed of interlocking diketopiperazine crystals having one or more drugs adsorbed on their surfaces. In some embodiments, the particles may entrap the drug in their internal void volume and/or a combination of drug adsorption on the crystal surface and drug entrapment in the internal void volume of the spheres.

In certain embodiments, a diketopiperazine composition comprising a plurality of substantially uniformly formed microcrystalline particles is provided, wherein the particles have a substantially hollow spherical structure and include an outer shell comprising crystallites of diketopiperazine that are not self-assembled; wherein the particles are formed by a process comprising the steps of combining a diketopiperazine having a trans isomer content in the range of about 45% to 65% and an acetic acid solution in the absence of a surfactant while homogenizing in a high shear mixer at a high pressure of up to 2,000 psi to form a precipitate; washing the precipitate in the suspension with deionized water; concentrating the suspension and drying the suspension 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) while mixing prior to the spray drying step, so that the active agent or active ingredient is adsorbed and/or embedded on or within the particles. The particles produced by this method may be in the submicron size range prior to spray drying.

In certain embodiments, a diketopiperazine composition is provided comprising a plurality of substantially uniformly formed microcrystalline particles, wherein the particles have a substantially hollow spherical structure and include an outer shell comprising crystallites of diketopiperazine that are not self-assembled, and the particles have a volume mean geometric diameter of 5 μm or less; wherein the particles are formed by a process comprising the steps of combining a diketopiperazine in solution and an acetic acid solution in the absence of a surfactant while homogenizing in a high shear mixer at a high pressure of up to 2,000 psi to form a precipitate; washing the precipitate in the suspension with deionized water; concentrating the suspension and drying the suspension 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) while mixing prior to the spray drying step, so that the active agent or active ingredient is adsorbed and/or embedded on or within the particles. The particles produced by this method may be in the submicron size range prior to spray drying.

In certain embodiments, a diketopiperazine composition is provided comprising a plurality of substantially uniformly formed microcrystalline particles, wherein the particles have a substantially hollow spherical structure and include an outer shell comprising crystallites of diketopiperazine that are not self-assembled, and the particles have a volume average geometric diameter of 5 μm or less; wherein the particles are formed by a process comprising the steps of combining a diketopiperazine in solution and an acetic acid solution in the absence of a surfactant and an active agent while homogenizing in a high shear mixer at a high pressure of up to 2,000 psi to form a precipitate; washing the precipitate in the suspension with deionized water; concentrating the suspension and drying the suspension 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) while mixing prior to the spray drying step, so that the active agent or active ingredient is adsorbed and/or embedded on or within the particles. The particles produced 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, protein, nucleic acid molecule, 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 may be an endocrine hormone, a neurotransmitter, a vasoactive peptide, a receptor peptide, a receptor agonist, or antagonist, etc. In some embodiments, the endocrine hormone is insulin, parathyroid hormone, calcitonin, glucagon, glucagon-like peptide 1, oxyntomodulin, peptide YY, leptin, or an analog of the 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 in which the composition comprises insulin as an active ingredient, the composition may contain an amount of up to, for example, 9 units or 10 units of insulin per milligram of powder to be delivered to the patient. In this embodiment, insulin can be delivered to the patient in an amount of up to, for example, 100 units in a single inhalation using a dry powder inhaler. The composition can be administered to a patient requiring 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 3 to 20 carbon atoms, including propyl, butyl, pentyl, hexyl, heptyl, etc.; and the formula 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 a salt thereof. In a specific embodiment, the diketopiperazine is (di-3,6-(N-fumaryl-4-aminobutyl)-2,5-diketo-diketopiperazine having the following formula:

In various embodiments, a method for preparing a dry powder comprising microcrystalline particles suitable for pulmonary administration is provided, wherein the method can be performed 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%.

Certain embodiments disclosed herein include methods for preparing dry powders comprising crystalline diketopiperazine microparticles from a starting material comprising the 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 aqueous ammonia to form a first solution;

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

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

atomizing the suspension under a stream of air or gas; and

The particles are reshaped into a dry powder by spray drying, which comprises microcrystalline particles having substantially hollow spheres.

In another embodiment, the method comprises:

dissolving a diketopiperazine in an 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 into a high shear mixer at an approximate pH of less than 6.0 under high pressure;

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

atomizing the suspension under a stream of air or gas; and

The particles are reshaped into a dry powder by spray drying, which comprises microcrystalline particles having substantially hollow spheres.

In one embodiment, the method comprises:

dissolving diketopiperazine in aqueous ammonia to form a first solution;

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

atomizing the suspension under a stream of air or gas; and

The particles are reshaped into a dry powder by spray drying, which comprises microcrystalline particles having substantially hollow spheres.

The method may further comprise the step of adding a third solution to the diketopiperazine crystal suspension prior to atomizing the suspension; wherein the solution comprises a drug or a pharmaceutically active ingredient, and the atomizing step may be performed under air or gas (including nitrogen) using an external mixing two-fluid nozzle entering a spray dryer equipped with a high efficiency cyclone separator.

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

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

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

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

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

When a disruption step is used, larger species of a bimodal distribution can be shifted to smaller sizes.

Certain embodiments include a method of forming microcrystalline particles of a diketopiperazine acid for use in preparing a dry powder that carries a large drug content, comprising using a diketopiperazine salt as a starting compound, including 2,5-diketo-3,6-di(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;

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

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

atomizing the suspension under a stream of air or gas; and

The particles are reshaped into a dry powder by spray drying, which comprises microcrystalline particles of diketopiperazine acid having substantially hollow spheres.

In one embodiment, the microcrystalline particles can be prepared by a method 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 so that the suspension comprises a bimodal particle size distribution, wherein the particles have a size ranging from about 0.2 μm to about 10 μm in diameter, wherein the average diameter of the first peak of particles is about 0.4 μm and the average diameter of the second peak of particles is about 2.4 μm, and spray drying the suspension to obtain a dry powder.

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

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

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, glucagon-like peptide 1, oxyntomodulin, peptide YY, leptin, or an analog of the endocrine hormone. The method may optionally comprise 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, the present method can be used to prepare a composition containing more than one active agent. The method for preparing such a composition comprises the following steps: preparing microcrystalline diketopiperazine particles containing more than one active agent, wherein each active agent/ingredient is treated separately in solution and added to a separate suspension of diketopiperazine particles, and the solution conditions are changed to promote adsorption of the active agent onto the surface of the particles, and then two or more separate suspensions containing the active agent are blended, and then the particles are dispersed and spray-dried. In a different process, the blend comprises a suspension containing diketopiperazine particles but without the active agent, for example, in order to achieve a lower total 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 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 uniform microcrystalline particles containing the drug; wherein the particles are shaped as substantially spheres having a substantially hollow core and the crystallites form the outer 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, exendin, parathyroid hormone, calcitonin, oxyntomodulin, and the like.

Other embodiments include a drug delivery system, which includes an inhaler with or without a cartridge, wherein the cartridge is a unit dose dry powder medicament container (e.g., cartridge), and a powder comprising particles disclosed herein and an active agent. In one embodiment, the delivery system used together with dry powder includes an inhalation system containing a high resistance inhaler, wherein the high resistance inhaler has an air duct, which can impart high resistance to the air flow through the duct so that the powder is deagglomerated and dispersed. In one embodiment, the resistance value of the inhalation system is, for example, about 0.065 to about 0.200 (√kPa)/liter per minute. In certain embodiments, dry powder can be effectively delivered by inhalation with an inhalation system, wherein the peak inhalation pressure difference can be in the range of about 2 to about 20kPa, and the resulting peak flow rate that it can produce is between about 7 and 70 liters/minute. In certain embodiments, the inhalation system is configured to provide a single dose by discharging powder from the inhaler as a continuous flow or one or more pulses of powder delivered to the patient. In some embodiments disclosed herein, the dry powder inhaler system includes a predetermined mass flow balance in the inhaler, wherein the inhaler conduit is designed to have different flow distributions during inhalation. In one embodiment, the inhaler can be used for the treatment of the patient's internal bleeding of the patient's internal bleeding.For example, account for and leave inhaler and enter about 10% to 70% of the flow balance of the total flow of patient and send by one or more distribution ends, wherein air-flow is by air duct, and this pipeline is designed to have the zone that comprises powder preparation, and wherein about 30% to 90% air-flow is to produce from other pipeline of inhaler during suction manipulation.In addition, side stream or the stream that does not enter and leave the powder container zone (for example by cartridge) can be recombined with the stream that leaves the powder distribution end in the inhaler, thereby fluidized powder is diluted, accelerated and finally deagglomerates before leaving mouthpiece.In one embodiment, the flow velocity in about 7 to 70 liters/minute scopes causes in the filling mass between 1 and 50mg, surpasses in 75% container or the cartridge content and is distributed.In certain embodiments, above-mentioned inhalation system can eject the powder dosage of respirable fraction/filling capacity higher than 40%, higher than 50%, higher than 60% or higher than 70% by percentage in single suction.

In certain embodiments, a drug delivery system comprising an inhaler can include an inhaler that is particularly suitable for use with a particulate form comprising a dry powder, such as a crystalline or amorphous form.

In some specific embodiments, an inhalation system is provided that includes a dry powder inhaler, a dry powder formulation comprising fumaryl diketopiperazine microcrystalline particles having a trans-isomer content of between 45% and 65% FDKP, 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 can be pre-filled in the inhaler. In this embodiment, the structural configuration of the inhalation system allows the inhaler's deagglomeration mechanism to produce a respirable fraction 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 greater than 85% of the powder drug contained in the container during administration. In certain embodiments, the inhaler can expel greater than 85% of the powder drug contained in a single inhalation. In one embodiment, the inhaler can expel greater than 90% of the cartridge or container contents in less than 3 seconds at a pressure differential between 2 kPa and 5 kPa with a fill mass of up to 30 mg.

The embodiments disclosed herein also include methods. In one embodiment, a method for treating an endocrine-related disease or condition comprises administering a dry powder formulation comprising FDKP microcrystalline particles (comprising FDKP having a trans isomer content of about 45% to about 65%) and a drug suitable for treating the disease or condition to a person in need thereof, wherein the microparticles are prepared by the method of the present invention. One embodiment includes a method for treating an insulin-related condition, comprising administering a dry powder comprising the above-mentioned FDKP microcrystalline particles to a person in need thereof. The method comprises administering a dry powder formulation containing fumaryl diketopiperazine microcrystalline particles having a trans isomer content in the range of about 45% to 65% to a subject, wherein the particles are hollow spheres and do not contain any surfactant. In various embodiments, insulin-related conditions may 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 insulin release, pancreatic β-cell function loss, pancreatic β-cell loss, and metabolic conditions. 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 thereof, or a combination thereof.

Another embodiment disclosed herein includes a method of delivering peptides (including GLP-1, oxyntomodulin, peptide YY, oxytocin, insulin) to a patient in need thereof, the method comprising administering a dry powder comprising diketopiperazine microcrystalline particles disclosed herein 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.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of this specification and are included to further illustrate certain aspects of the examples disclosed herein. The present disclosure may be better understood by referring to one or more of these drawings in conjunction with the detailed description of specific embodiments presented herein.

Figures 1A and 1B are scanning electron micrographs (SEM) of fumaryl diketopiperazine particles containing insulin and showing the solid composition of the lyophilized particles at low magnification (1A) and high magnification (1B).

2 depicts a graphical representation of the particle size distribution of the particles depicted in FIGs. 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 prepared from a suspension in which the microcrystalline particles are formed without the presence of a surfactant in any of the solutions used. The figure shows a typical bimodal distribution of microcrystalline particles as measured by the probability density function (pdf, left y-axis) and the cumulative distribution function (cdf, right y-axis).

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

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

FIG6 depicts a SEM (2500X) of one of the claimed embodiments showing microcrystalline particles made from a spray dried surfactant-free solution.

FIG7 depicts a graphical representation of the particle size distribution of spray-dried surfactant-free particles dispersed in water.

FIG8 depicts a graphical representation of the particle size distribution of spray-dried surfactant-free particles dispersed in 0.01 M HCl (pH 2).

9 depicts a graphical representation of the bimodal particle size distribution of a suspension formed by crystallization of Na ₂ FDKP with acetic acid in the presence of a surfactant.

10A and 10B depict two scanning electron micrographs at 2,500X (10A) and 10,000X (10B) magnification of particles prepared by spray drying a crystalline suspension made of _Na2FDKP .

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

12A and 12B are two scanning electron micrographs at 2,500X (12A) and 10,000X (12B) magnification of particles prepared by spray drying a crystalline suspension made of _Na2DKP .

Figure 13 depicts a graphical representation of the size distribution of particles formed by spray drying a suspension of FDKP formed by crystallization from a solution of _Na2FDKP and polysorbate 80. The particles were dispersed in water for measurement.

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

Detailed Description of the Invention

As described, drug delivery to the lungs provides many advantages. However, due to the problem of transmitting drugs through natural physical barriers, it is difficult to deliver drugs to the lungs with uniform volume and weight. Crystalline diketopiperazine compositions, dry powders, and methods for preparing particles are disclosed herein. Crystalline compositions and dry powders therefrom comprise diketopiperazine microcrystalline particles, which are substantially uniformly defined spheres comprising a shell and a core containing diketopiperazine crystals. In certain embodiments, the core can be hollow. In one embodiment, the diketopiperazine has a definite trans isomer content, which can be beneficial for particles as drug delivery agents, methods for preparing particles, and methods for treating with particles. Compared to standard prior art particles, the particles disclosed herein have a higher ability to carry and deliver drug content to patients in smaller doses.

As used herein, "analogs" include compounds that have structural similarity to another compound. Thus, compounds that have structural similarity to another (parent compound) that mimic the biological or chemical activity of a parent compound are analogs. As long as the analog can mimic the biological or chemical properties of the parent compound in some relevant manner, in the same way, complementarily, or competitively, there is no minimum or maximum number of elements or functional group substitutions required for a compound to be identified as an analog. In some cases, analogs comprise fragments of the parent compound that are independent or connected to another molecule and may also contain other variants. Analogs of the compounds disclosed herein may have activity equal to, less than, or greater than that of their parent compound.

As used in the present invention, the term "microparticle" refers to particles with a diameter of about 0.5 to about 1000 μm, regardless of exact external or internal structure. Microparticles with a diameter between about 0.5 and about 10 microns can successfully pass through most natural barriers to reach the lungs. The diameter must be less than about 10 microns in order to pass through the corners of the throat, and the diameter must be about 0.5 microns or larger to avoid being exhaled. In order to reach the deep lungs (or alveolar regions) where it is believed that the most effective absorption occurs, it is preferred to maximize the particle ratio contained in the "inhalable fraction" (RF), which is generally recognized as having an aerodynamic diameter of about 0.5 to about 5.7 microns, as measured using standard techniques (e.g., with an Anderson Cascade Impactor), although some references use slightly different ranges. Other impactors can also be used to measure aerodynamic particle size, such as NEXT GENERATION IMPACTOR ^™ (NGI ^™ , MSP Corporation), where the inhalable fraction is defined by similar aerodynamic size (e.g., <6.4 μm). In some embodiments, particle size is measured using a laser diffraction device, such as that disclosed in U.S. patent application Ser. No. 12/727,179, filed March 18, 2010, the relevant teachings of which are incorporated herein in their entirety, wherein the volume median geometric diameter (VMGD) of the particles is measured to evaluate the performance of the inhalation system. For example, in various embodiments, a cartridge emptying of ≥80%, 85%, or 90% and a VMGD of ejected particles ≤12.5 μm, ≤7.0 μm, ≤5.8 μm, or ≤4.8 μm can indicate increasingly better aerodynamic performance. The embodiments disclosed herein show that FDKP particles having a trans isomer content between about 45% and about 65% exhibit properties that are beneficial for drug delivery to the lungs, such as improved aerodynamic performance.

Inhalable fraction (RF/ filling amount) based on filling amount represents the powder dosage percentage ratio that is suitable for sucking that ejects from inhaler after the filling powder content as dosage is discharged, promptly ejects from filling dose and the particle percentage ratio that size is suitable for pulmonary delivery, and it is the measuring of particle aerodynamic performance.As described in the present invention, 40% or greater than 40% RF/ filling amount value reflects acceptable aerodynamic properties.In certain embodiments disclosed herein, inhalable fraction based on filling amount can be greater than 50%.In an exemplary embodiment, inhalable fraction based on filling amount can be up to about 80%, wherein use standard technique to measure, and about 80% filling amount ejects with particle size＜5.8 μ m.

As used herein, the term "dry powder" refers to a fine particle 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/filling amount value can depend on the inhaler used to deliver the powder. Powders are generally easy to agglomerate and some crystalline DKP particles form particularly sticky powders. One function of a dry powder inhaler is to deagglomerate the powder so that the resulting particles comprise an inhalable fraction suitable for delivering a dose by inhalation. However, the deagglomeration of sticky powders is not thorough usually, so when measuring the inhalable fraction delivered by the inhaler, the particle size distribution seen does not match that of the original particles, that is, the curve shifts to larger particles. The design of the inhaler is different in its deagglomeration efficiency, so the actual value of the RF/filling amount observed using different designs will also be different. However, the optimal RF/filling amount as a function of isomer content is the same between inhalers.

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

As used herein, the term "surfactant-free" is used to indicate that no surfactant is present in any reagent (including solution and/or suspension) used in the process for preparing microcrystalline particles.

As used herein, the term "grain" is used to refer to a complete crystalline unit of a diketopiperazine particle, which may be of varying sizes.

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

diketopiperazines

One class of drug delivery agents that has been used to overcome challenges in the pharmaceutical field, such as drug instability and/or poor absorption, is 2,5-diketopiperazine. 2,5-diketopiperazine is represented by a compound of Formula 1, shown below, wherein _E1 and _E2 are independently N or, more specifically, NH. In other embodiments, _E1 and/or _E2 are independently oxygen or nitrogen, such that one of the substituents of _E1 and _E2 is oxygen and the other is nitrogen, resulting in a substituted analogue, diketomorpholine; or when both _E1 and _E2 are oxygen, the formula results in a substituted analogue, diketodioxane.

These 2,5-diketopiperazines, particularly those with acidic _R and _R groups, have been shown to be useful for drug delivery, as described, for example, in U.S. Patent Nos. 5,352,461, entitled "Self-Assembling Diketopiperazine Drug Delivery System," 5,503,852, entitled "Method For Making Self-Assembling Diketopiperazine Drug Delivery System," 6,071,497, entitled "Microparticles For Lung Delivery Comprising Diketopiperazine," and 6,331,318, entitled "Carbon-Substituted Diketopiperazine Delivery System," each of which is incorporated herein by reference in its entirety for all of its teachings regarding diketopiperazines and diketopiperazine-mediated drug delivery. Diketopiperazines can be formed into microparticles that incorporate drugs or onto which drugs can be adsorbed. The combination of drugs and diketopiperazines can impart improved drug stability and/or absorption characteristics. These microparticles can be administered via a variety of routes. As dry powders, the microparticles can be delivered by inhalation to specific areas of the respiratory system, including the lungs.

Such prior art microparticles are typically obtained by pH-dependent precipitation of free acid (or base) to produce self-assembled microparticles with a rosette morphology composed of aggregated crystalline plates. The stability of the particles can be enhanced by a small amount of surfactant (such as polysorbate-80) in the DKP solution from which the particles are precipitated (see, for example, U.S. Patent No. 7,799,344, entitled "Method of drug formulation based on increasing the affinity of crystalline microparticle surfaces 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 dry powders thereof). Finally, the solvent can be removed to obtain a dry powder. Methods for removing the solvent include freeze drying and spray drying (see, for example, U.S. Patent No. 8,039,431 entitled "A method for improving the pharmaceutical properties of microparticles comprising diketopiperazine and an active agent" and U.S. Patent No. 6,444,226 entitled "Purification and stabilization of peptide and protein pharmaceutical agents," each of which is incorporated herein by reference in its entirety for all its teachings regarding the formation and loading of DKP microparticles and dry powders thereof). The particles disclosed herein differ from prior art particles in that they are physically and morphologically distinct entities and are made by an improved process. References herein to FDKP should be understood to refer to the free acid or dissolved anion.

Other prior art particles are obtained by spray drying a DKP solution to obtain amorphous DKP salt particles, which generally have a collapsed spherical morphology, such as those disclosed in US Patent Nos. 7,820,676 and 8,278,308, entitled "Diketopiperazine salts for drug delivery and related methods."

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

Fumaryldiketopiperazine (di-3,6-(N-fumaryl-4-aminobutyl)-2,5-diketopiperazine; FDKP) is a preferred diketopiperazine for pulmonary applications:

FDKP provides an advantageous microparticle matrix because it has low solubility in acid but readily dissolves at neutral or alkaline pH. These properties allow FDKP to crystallize and self-assemble into microparticles under acidic conditions. The particles readily dissolve under physiological conditions of neutral pH. As described, microparticles with diameters between approximately 0.5 and 10 μm can successfully cross most natural barriers to reach the lungs. Particles within this size range can be readily prepared from FDKP.

FDKP has two asymmetric centers on the diketopiperazine ring. FDKP is prepared as a mixture of geometric isomers, designated "cis-FDKP" and "trans-FDKP," depending on the arrangement of the side chains relative to the diketopiperazine "ring." The R,R and S,S enantiomers have propenyl (amidobutyl) "sidearms" extending from the same planar sides of the diketopiperazine ring (A and B below), and are therefore referred to as cis isomers; the R,S compound has "sidearms" extending from opposite planar sides of the diketopiperazine ring (C below), and is therefore referred to as the trans isomer.

FDKP microparticle powders having acceptable aerodynamic properties have been prepared using FDKP having a trans isomer content ranging from about 45 to about 65%, as tested using RF/fill volume in a moderate efficiency inhaler, such as that disclosed in U.S. Patent No. 7,464,706, entitled "Unit Dose Cartridge and Dry Powder Inhaler," which is incorporated herein by reference for its teachings in the same regard. Particles with isomer contents within this range also perform well in high-efficiency inhalers, such as those disclosed 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,” which are incorporated herein by reference for their teachings of the same. Powders containing microparticles containing greater than 65% trans-FDKP tend to have lower and more variable RF/fill levels. FDKP microparticles enriched in the trans isomer have an altered morphology and result in viscous suspensions that are difficult to handle.

Formulations of FDKP microparticles with a trans isomer content of about 45% to about 65% provide powders with acceptable aerodynamic properties, as disclosed in U.S. Patent No. 8,227,409, the disclosure of which is incorporated herein by reference for its teachings in the same regard. Formulations of FDKP particles with a defined specific surface area of less than 67 m ² /g also provide dry powders for inhalation with acceptable aerodynamic properties, as disclosed in U.S. Patent No. 8,551,528, filed June 11, 2010, entitled "Diketopiperazine Microparticles with Defined Specific Surface Areas," the disclosure of which is incorporated herein by reference for its teachings in the same regard. However, these FDKP powders tend to be sticky, and inhalers are designed to overcome this characteristic.

Therefore, there is a need to prepare diketopiperazine powders with less cohesive particle compositions, which would allow for more efficient drug delivery and less inhaler design around. The present disclosure establishes that the present method of preparing diketopiperazine microcrystalline particles, as exemplified by FDKP and FDKP disodium salt, provides microcrystalline dry powders with acceptable aerodynamic properties, wherein the powders are less cohesive, have varying densities, 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 was unexpected.

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

Active agent selection and integration

In exemplary embodiments comprising FDKP, at least as long as the microcrystalline particles described herein maintain the above-mentioned isomer content, they can adopt other additional properties that are conducive to delivery to the lungs and/or drug absorption. U.S. Patent No. 6,428,771, entitled "Method for Drug Delivery to the Pulmonary System", describes delivery of DKP particles to the lungs, which is incorporated herein by reference for teachings of the same aspects. U.S. Patent No. 6,444,226, entitled "Purification and Stabilization of Peptide and Protein Pharmaceutical Agents", describes methods that are conducive to adsorption of drugs to the surface of microparticles, which is also incorporated herein by reference for teachings of the same aspects. Microparticle surface properties can be manipulated to achieve desired properties 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", which is incorporated herein by reference for teachings of the same aspects. 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 adsorption of active agents onto microparticles. U.S. Patent No. 7,803,404 is also incorporated herein by reference for its teachings in the same regard. These teachings can be applied to adsorption of active agents onto crystals in suspension, for example, prior to spray drying.

Microcrystalline particles as described herein can include one or more activating agents. As used herein, "activating agent" that can be used interchangeably with "drug" refers to a pharmaceutical substance, including small molecule drugs, biological preparations and bioactive agents. Activating agent can be 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, and antibodies and fragments thereof, including but not limited to humanized antibodies or chimeric antibodies, F (ab), F (ab) ₂ , single-chain antibodies or single-chain antibodies fused with other polypeptides or therapeutic or diagnostic monoclonal antibodies against cancer antigens. Activating agent can belong to a variety of biological activities and species, such as vasoactive agents, neuroactive agents (including opioid agonists and antagonists), hormones, anticoagulants, immunomodulators, cytotoxic agents, antibiotics, antivirals, antigens, infectious substances, 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, lipokines, enkephalins, alkynes, cyclosporins, 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 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-phenylalanyl-L-propyl-L-arginine chloromethyl ketone), oxyntomodulin (O-3-hydroxy-1-oxaline), pyruvate, ... XM), 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, analgesics, nociceptin, nociceptin, orphanin FQ2, calcitonin, CGRP, angiotensin, substance P, neurokinin A, pancreatic polypeptide, neuropeptide Y, delta-somnia peptide and vasoactive intestinal peptide; and analogs of the active agents.

The drug content delivered on the microcrystalline particles formed from FDKP or FDKP disodium salt can typically be greater than 0.01% (w/w). In one embodiment, the drug content to be delivered using the microcrystalline particles can 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 contain 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 can vary depending on the form and size of the drug to be delivered. In one embodiment in which GLP-1 is used as the active agent, the GLP-1 content can be as high as 40% (w/w) of the powder content.

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

The method of preparing such a composition may include the steps of preparing microcrystalline diketopiperazine particles containing more than one active agent; wherein each active agent/ingredient is separately processed in solution and added to a separate diketopiperazine particle suspension, and then blending the two or more separate active agent-containing suspensions, and then dispersing and spray drying the particles.

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

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

Each of the multiple active agents can be adsorbed onto a separate aliquot or granule material. The aliquots of adsorbed granules can then be mixed together and spray dried. Alternatively, the aliquots can be free of active agent so that the total active agent content in the dry powder can be adjusted without changing the conditions used to adsorb the active agent onto the granules.

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

As will be apparent from the foregoing disclosure, the microparticles of the disclosed embodiments of the present invention can have a variety of different forms and can incorporate a variety of different drugs or active agents.

Example

The following examples are included to demonstrate the disclosed embodiments of the microcrystalline diketopiperazine particles. It will be understood by those skilled in the art that the techniques disclosed in the following examples represent techniques developed by the inventors to function well in practicing the present disclosure and, therefore, can be considered to constitute preferred modes for its practice. However, in light of the present disclosure, it will be understood by those skilled in the art that many changes may be made to the specific embodiments and still obtain the same or similar results without departing from the scope of the present invention.

Example 1

Preparation of Standard FDKP Microparticles - For comparison purposes, FDKP microparticles were prepared as standard particles using prior art methods such as those 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 their teachings on related subject matter. In summary, a typical FDKP particle formation process involves combining feed solutions of FDKP and acetic acid, each containing 0.05% (w/w) polysorbate 80 (PS80), in a high shear mixer. Table 1 below shows the composition of the FDKP and insulin stock solutions.

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

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

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

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

Can prepare concentrated insulin stock solution with 1 part of insulin and 9 parts of acetic acid of about 2 % by weight.Can add the load of about 11.4 % by weight in the suspension by gravimetric analysis of insulin stock solution.Can mix the suspension that contains insulin at least about 15 minutes, then use about 14 to about 15 % by weight ammoniacal liquor to titrate to about 4.5 pH from about 3.5 initial pH.Can use low temperature granulator to make suspension quick-frozen in liquid nitrogen to form particle, for example, as disclosed in U.S. Patent No. 8,590,320, its disclosure is incorporated into this paper in full by reference, and lyophilization produces the FDKP microparticle that is loaded with insulin loose, and it forms small crystal or the colony of FDKP particle that self-assembles into the open structure with as seen in Figure 1A and 1B.

The resulting particle samples were investigated to determine the size distribution of the particles in these suspensions, and the results are shown in FIG2 . The data in FIG2 shows a graphical representation of the particle size distribution measurements, which are 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 the suspension have a unimodal size distribution ranging from about 1.0 to about 10 μm in diameter and centered at or about 2 μm.

Preparation of Microcrystalline FDKP Particles – 2.5% (w/w) FDKP was dissolved in an alkaline aqueous ammonia solution (1.6% ammonia). A 10.5% (w/w) acetic acid stock solution was added to a high shear mixer (Sonolator) under high pressure at an approximate pH of 2.0 to prepare the particles. The resulting particles were washed in deionized water. It was found that the diketopiperazine microparticles were unstable in the absence of a surfactant in the solution; however, no surfactant was added to any of the solutions or reagents used in the preparation of the particles.

In these experiments, equal masses of approximately 10.5% by weight acetic acid and approximately 2.5% by weight FDKP solutions were fed through a 0.001 square inch nozzle at a temperature of approximately 16°C ± approximately 2°C and 2000 psi 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 was ultimately concentrated to approximately less than 5% solids, for example, approximately 2 to 3.5% based on the initial mass of FDKP. The solids content of the concentrated suspension can be determined by oven drying. For samples containing active ingredients (i.e., insulin and/or GLP-1), the above-described FDKP suspension was used, to which an insulin stock solution was added (insulin dissolved in 2% acetic acid was added to the suspension while mixing, and the pH of the suspension was then titrated to pH 4.5±0.3 with ammonium hydroxide. Similarly, GLP-1 dissolved in a 2% acetic acid stock solution was gravimetrically added to the FDKP-suspension under stirring. The GLP-1 FDKP suspension was titrated to pH 4.5±0.1. Each of the insulin-FDKP suspension and the GLP-1-FDKP suspension was independently dispersed into a Niro SD-Micro ^™ spray dryer equipped with a high-efficiency cyclone using an external mixing two-fluid nozzle. Nitrogen was used as the process gas (25 kg/h) and the atomizing fluid (2.8 kg/hr). The samples were processed in the spray dryer using the two processing conditions listed in Table 3.

Table 3.

For control samples, blank FDKP microcrystalline particles were prepared in the same manner (except for the insulin or GLP-1 loading step).

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

A sample of the suspension was freeze-dried without spray drying. Figure 4 is a SEM of the freeze-dried particles at 2,500x magnification. As seen in Figure 4, after freeze-drying a similar suspension, large, flake-like particles formed, and when resuspended in water, a much larger average size was achieved, as shown in Figure 5. Figure 5 shows the particle size distribution in a suspension of a freeze-dried sample of particles prepared without the use of a surfactant. In this study, the particle size of the resuspended particles increased from approximately 1 μm in diameter to approximately 90 μm or greater.

FIG6 shows a typical 2,500x magnification of a scanning electron micrograph of a powder sample of a surfactant-free formulation of microcrystalline FDKP particles formed using the method of the present invention and spray drying as described above. As seen in FIG6 , the structure of the particles is a uniform sphere including a crystal shell. When the suspension without surfactant is spray dried, particles with a physical diameter of about 4 μm are formed, as shown in FIG6 . Unlike standard FDKP particles, these particles dissociate into particles with a diameter of 0.2 μm when dispersed in water, as shown in FIG7 . Thus, it has been demonstrated that surfactants play a role in particle integrity. Dispersing the particles in 0.01 M hydrochloric acid inhibits particle dissociation, as shown in FIG8 . The dissolved FDKP can precipitate during spray drying and can be deposited along the boundaries between the primary particles and can act as a cement. The FDKP “cement” dissolves in water and the particles dissociate into 0.2 μm primary particles; the lower solubility of FDKP in acid prevents dissociation and protects particle integrity.

Example 2

Preparation of microcrystalline FDKP particles using diketopiperazine salts by an alternative method - Alternatively, FDKP crystals can be formed from a feed solution containing a surfactant. Without using ammonia as a reagent, a feed solution of FDKP was prepared by dissolving FDKP disodium salt ( _Na2FDKP ) in water containing polysorbate 80 (PS80) as a surfactant. 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 FEEDSONOLATOR crystallized FDKP and produced the bimodal particle size distribution shown in Figure 9. As shown in Figure 9, approximately 26% of the primary crystals formed had a diameter of approximately 0.4 μm and approximately 74% of the larger particles had a diameter of approximately 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 Figures 10A and 10B. Figures 10A and 10B show that the particles are similar and spherical in shape, but smaller than those shown in Figure 6 of Example 1, in which the particles were prepared using FDKP as the free acid. Table 4 below shows some physical properties measured for the powders prepared by lyophilization and the powders prepared by spray drying (SD) using the disodium salt of FDKP.

Table 4

The data showed that powders made from spray-dried particles exhibited a higher respirable fraction (62.8% vs. 28%), higher cartridge emptying (%CE, 88.2% vs. 83.8%), and higher bulk density (0.159 g/mL vs. 0.019 g/mL) and tap density (0.234 g/mL vs. 0.03 g/mL) compared to lyophilized powders.

Example 3

Preparation of Microcrystalline FDKP Particles Containing Active Agents - The active pharmaceutical ingredient (active agent) was introduced into the particles by adding a solution of the active agent to a suspension of surfactant-free FDKP crystals, followed by spray drying of 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 PS-80 in solution for the preparation of a powder for pulmonary inhalation. In this study, insulin was dissolved in dilute acetic acid and added to a suspension of surfactant-free FDKP crystals prepared as in Example 1 (Samples 1 and 2, Table 5). The suspension was spray dried to obtain a dry powder containing approximately 10% insulin by weight. Powder samples were subjected to various analyses, including delivery via a high-resistance inhaler and scanning electron microscopy, and the results are shown in Table 5. The particle size was approximately the same as that of the particles without insulin (Example 1), and the particle morphology (Figure 11) was the same as that shown in Figure 6. In addition, both Samples 1 and 2 were powders with lower compactness 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, and there is no obvious insulin deposition on the particle surface, suggesting that the insulin is 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 behaved differently from the standard granules. For example, at the same insulin content, the powder without surfactant released more efficiently from the inhaler (96.4%) than the standard granules (85%). The increase in the percentage of cartridge emptying (% CE) indicates that the powder has increased flowability. Since the inhaler used to test the powder was designed for the control powder, the respirable fraction (RF% based on the fill amount) was higher for the control granules.

Example 4

Preparation of Microcrystalline FDKP Particles Using Diketopiperazine Salts by an Alternative Method - In this study, a suspension of FDKP salt particles as described in Example 2 was prepared using FDKP disodium salt. An insulin solution was added to a suspension of FDKP microcrystals prepared as in Example 2 without surfactant. The suspension was spray dried to obtain a dry powder containing approximately 10% insulin by weight. The morphology of the particles formed is shown in the SEM of Figures 12A and 12B at 2,500x and 10,000x magnifications (respectively). As seen in Figures 12A and 12B, the morphology was identical to that of the particles without insulin, exhibiting a spherical structure with a median particle diameter of 2.6 μm as shown in Figure 13, and also exhibiting particles with diameters ranging from about 1.0 μm to about 10 μm.

Example 5

Preparation of Microcrystalline FDKP Particles Containing More Than One Active Agent – In another embodiment, the methods of the present invention can be used to prepare compositions containing more than one active agent. Methods for preparing such compositions include the steps disclosed 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 then dispersed and spray-dried as described above to prepare microcrystalline diketopiperazine particles containing more than one active agent. In one exemplary study, a combination powder of insulin and GLP-1 was prepared.

The FDKP crystalline suspension prepared as in Example 1 was mixed with solutions of various active agents (eg, ghrelin, low molecular weight heparin, oxyntomodulin) and spray dried to obtain particles with properties similar to those in Example 3.

Example 6

Preparation of microcrystalline FDKP particles containing two active agents -

A combination powder containing two active agents (GLP-1 and insulin) was prepared by first preparing a suspension of FDKP crystals containing insulin and a second suspension of crystals containing 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 crystal suspension was prepared as described in Example 1; after adding the active agent, the suspension was adjusted to pH 4.5 to promote adsorption onto the crystals. Figure 14 is a data graph showing the particle size distribution of the spray-dried combination powder (1) and the crystal suspension containing the individual active agents, FDKP-insulin (2) and FDKP-GLP-1.

As shown in Figure 14, the particle size distribution of the combined powder is concentrated between the particle size distributions of the two separate suspensions and is significantly narrower. The combined powder contains particles with a diameter of about 1 μm to about 10 μm. The grains containing insulin are smaller than the grains containing GLP-1, which have a diameter ranging from about 0.5 μm to about 50 μm (about 0.25 μm to about 10 μm). The atomization step in the spray drying can dissociate the initial grain colonies in the suspension and reform particles with a particle size distribution that depends on the conditions in the suspension and the spray drying conditions.

Example 7

A dry powder composition comprising crystalline diketopiperazine particles is administered to a subject.

A dry powder formulation containing microcrystalline diketopiperazine microparticles made with the disodium salt of FDKP ( _Na2FDKP ) was prepared as described above in Example 1, such that each milligram of the composition contained 9 units of insulin. A high-resistance inhaler (Dreamboat ^™ Inhaler, MannKind Corporation) containing a cartridge was prepared to contain 1 to 10 mg of insulin per dose for administration to subjects diagnosed with diabetes. The inhaler containing the insulin dose was provided to the patient to be treated, who inhaled the insulin dose in a single inhalation before, during, or after a meal.

Unless otherwise indicated, in all cases, all numerical values used in the specification and claims indicating the number of components, properties (such as molecular weight), reaction conditions, etc. are understood to be modified by the term "about". Therefore, unless otherwise indicated, the numerical parameters listed in the following specification and the appended claims are approximate values, which can be changed according to the desired properties that are desired to be obtained by the present invention. At the lowest level, each numerical parameter is not intended to limit the application of the doctrine of equivalents to the scope of claim protection, and at least each numerical parameter should be understood according to the significant digits of the reported numbers and by conventional rounding methods. Although the numerical range and parameters given by the broad scope of the present invention are set to approximate values, the numerical values shown in the specific examples can be reported as accurately as possible. However, any numerical value inherently contains certain errors, which are inevitably caused by the standard deviations present in its corresponding experimental determinations.

Unless otherwise specified in the present invention or clearly contradicted by the context, the description of the present invention (particularly in the context of the following claims) without the use of quantifiers should be understood to include both the singular and the plural. The present invention's enumeration of ranges of values is merely intended to serve as a shorthand method for individually referring to each individual numerical value falling within the range. Unless otherwise specified in the present invention, each individual value is included in this specification as if individually listed in the present invention. All methods described in the present invention can be performed in any suitable order, unless otherwise specified in the present invention or clearly contradicted by the context. The use of any and all examples or exemplary language provided by the present invention (e.g., "such as") is merely for the purpose of "better illustrating the present invention, rather than limiting the scope of the invention protected by the claims. Any language in the specification should not be understood to indicate that any unclaimed element is necessary for the implementation of the present invention.

Unless an alternative or alternatives are explicitly stated to be mutually exclusive, the term "or" as used in the claims means "and/or" even though the specification supports an alternative and a definition of "and/or".

The grouping of the alternative elements or embodiments of the present invention disclosed in the present invention should not be construed as limiting. Each group member can be referred to and claimed individually or in any combination with other elements found in the group or in the present invention. It is foreseeable that, for convenience and/or patentability reasons, one or more members in the group may be included in a group or deleted therefrom. When any such inclusion or deletion occurs, the present invention will regard the specification as including the group so modified, thereby meeting the written description of all Markush groups used in the appended claims.

Preferred embodiments of the present invention are described herein, including the best modes of carrying out the invention known to the inventors. Of course, variations of those preferred embodiments will become apparent to those of ordinary skill in the art after reading the foregoing description. The inventors anticipate that a skilled person may appropriately adopt such variations, and the inventors intend that the present invention be implemented in a manner different from that specifically described herein. Therefore, the present invention includes all modifications and equivalents of the subject matter recited in the appended claims as permitted by applicable law. In addition, the present invention encompasses any combination of the above-mentioned elements in all possible variations thereof, unless otherwise indicated herein or otherwise clearly contradicted by the context.

The specific embodiments disclosed herein may also be limited within the claims using the phrase "consisting essentially of" or "consisting essentially of" language. When used in a claim, whether filed by amendment or added, the transition term "consisting essentially of" excludes all elements, steps, or ingredients not specified in the claim. The transition term "consisting essentially of" limits the scope of the claim to the specified materials or steps and those that do not significantly affect the basic and novel characteristics. The embodiments of the invention protected by the claims are inherently or expressly described and enabled in the invention.

In addition, a number of references, including patents and printed publications, are cited in this specification. Each of the above-cited references and printed publications is hereby incorporated by reference in its entirety.

Furthermore, it should be understood that the embodiments of the present invention disclosed herein are intended to illustrate the principles of the present invention. Other modifications that may be employed are within the scope of the present invention. Therefore, the embodiments of the present invention that are available are provided by way of example, but not limitation, based on the present invention. Therefore, the present invention is not intended to be limited to the precise form shown and described.

Claims (16)

1. A crystalline dry powder composition comprising one or more active ingredients and microcrystalline particles of 3,6-di(N-fumaryl-4-aminobutyl)-2,5-diketopiperazine in a content of 45% to 65% of various trans isomers, said particles being uniform in size, having a hollow spherical structure and including a shell, said shell comprising 3,6-di(N-fumaryl-4-aminobutyl)-2,5-diketopiperazine crystals that do not self-assemble in a suspension, said particles being formed in the absence of a surfactant by a method comprising: forming 3,6-di(N-fumaryl-4-aminobutyl)-2,5-diketopiperazine particles in a suspension having a bimodal distribution in a particle size range of 0.05 μm to 10 μm; and atomizing said suspension under an air or gas stream using a spray dryer to form a dry powder. 2. The crystalline dry powder composition according to claim 1, wherein up to 92% of the microcrystalline particles have a median geometric diameter ≤ 5.8 μm. 3. The crystalline dry powder composition according to claim 1, wherein one or more active ingredients are peptides, proteins, nucleic acid molecules or small organic molecules. 4. The crystalline dry powder composition according to claim 1, wherein one or more active ingredients are vasoactive agents, neuroactive agents including opioid agonists and antagonists, hormones, anticoagulants, immunomodulators, cytotoxic agents, antibiotics, antiviral agents, antigens, infectious substances, inflammatory mediators, cell surface receptor agonists or antagonists. 5. The crystalline dry powder composition according to claim 1, wherein one or more active ingredients are cell surface antigens. 6. The crystalline dry powder composition according to claim 4, wherein one or more active ingredients are neuroactive agents. 7. The crystalline dry powder composition according to claim 3, wherein the one or more active ingredients are at least one of the following: insulin, parathyroid hormone (PTH), calcitonin, glucagon, glucagon-like peptide-1, gastrin, peptide YY, leptin, cytokines, lipid factors, enkephalin, alkynes, cyclosporine, anti-IL-8 antibody, IL-8 antagonist including ABX-IL-8; prostaglandins including PG-12, containing LY29311, BIIL LTB receptor blockers of 284 and CP105696; triptans and palmitate esters, growth hormone, parathyroid hormone-related peptide (PTHrP), auxin-releasing peptide, obesity-inhibiting peptide, enterostatin, granulocyte-macrophage colony-stimulating factor (GM-CSF), dextrin, clopidogrel, PPACK (D-phenylalanyl-L-propyl-L-arginine chloromethyl ketone), adiponectin, cholecystokinin (CCK), secretin, gastrin, motilin, somatostatin, brain natriuretic peptide (BNP), atrial natriuretic peptide (ANP), IGF-1, growth hormone-releasing factor (GHRF), integrin β-4 precursor (ITB4) receptor antagonists, analgesics, pain-sensitive peptides, pain stabilizers, orphanonephrine FQ2, CGRP, angiotensin, substance P, neurokinin A, pancreatic polypeptide, neuropeptide Y, delta-sleep-promoting peptide, and vasoactive intestinal peptide. 8. A dry powder comprising microcrystalline particles of 3,6-bis(N-fumaryl-4-aminobutyl)-2,5-diketopiramate suitable for pulmonary administration, said particles being prepared by a method comprising the following steps: a) Diketopiperazine particles according to claim 1 formed in a suspension having a bimodal distribution in a particle size range of 0.05 μm to 10 μm; b) Atomize the suspension using a spray dryer under an air or gas stream, and c) The particles are reshaped into dry powder by spray drying, the dry powder comprising microcrystalline diketopiperazine particles having hollow spheres. 9. The dry powder according to claim 8, wherein the method further comprises adding a solution containing one or more active agents to the suspension of step a). 10. The dry powder according to claim 8, wherein the method further comprises the step of adding a surfactant to the suspension. 11. The dry powder according to claim 10, wherein the surfactant comprises polysorbate 80. 12. The dry powder according to claim 9, wherein the one or more active agents are peptides, proteins, nucleic acid molecules or small organic molecules. 13. The dry powder according to claim 12, wherein the peptide is an endocrine hormone. 14. The dry powder according to claim 13, wherein the endocrine hormone is insulin, parathyroid hormone, calcitonin, glucagon, glucagon-like peptide-1, gastrin, peptide YY, or leptin. 15. Use of the crystalline dry powder composition according to any one of claims 1-7 and the dry powder according to any one of claims 8-14 in the preparation of a medicament for treating a disease or condition. 16. The use according to claim 15, wherein the crystalline dry powder composition or dry powder contains 0.01% (w/w) to 75% (w/w) of a pharmaceutical or active agent.