A method for improving the pharmaceutic properties of microparticles comprising diketopiperazine and an active agent

<u>CROSS REFERENCE TO RELATED APPLICATIONS</u>

The present application claims the benefit under 37 CFR §119(e) of United States Provisional Application No. <patcit id="pcit0001" dnum="US60776605B"><text>60/776,605 filed February 22, 2006</text></patcit>, the contents of which is incorporated by reference herein in its entirety.

<u>BACKGROUND OF THE INVENTION</u>

<u>Field of the Invention</u>

The invention relates to the field of dry powder pharmaceuticals. The invention discloses methods of obtaining particles with improved aerodynamic performance or in which the active agent is more stable and efficiently delivered. More particularly, the present invention concerns methods for drying, particularly spray drying diketopiperazine-insulin (DKP-insulin) particles. The dry powders of the invention have utility as pharmaceutical formulations for pulmonary delivery.

<u>Description of the Related Art</u>

A number of different methodologies are employed in the art for preparing particles as a dry powder composition. These methodologies include, for example, lyophilization, evaporation, phase separation, and spray drying (see <patcit id="pcit0002" dnum="WO9116038A"><text>PCT Patent Application: WO 91/16038</text></patcit>). In the manufacture of dry powder pharmaceuticals some methods start with the components in solution and form the particles of the powder by removing solvent. Other methods form particles in a separate, earlier step, such as by precipitation, and can result in a particle in suspension, which must then be dried. Methods such as lyophilization and evaporation are often used particularly for drying or removing a solvent from particles in suspension, whereas spray drying has more typically been used for particle formation from solution. For example, see <patcit id="pcit0003" dnum="US5976574A"><text>U.S. Patent Nos.: 5,976,574</text></patcit>; <patcit id="pcit0004" dnum="US5985248A"><text>5,985,248</text></patcit>; <patcit id="pcit0005" dnum="US6001336A"><text>6,001,336</text></patcit>; <patcit id="pcit0006" dnum="US6051256A"><text>6,051,256</text></patcit>; <patcit id="pcit0007" dnum="US6077543A"><text>6,077,543</text></patcit>; <patcit id="pcit0008" dnum="US6365190B"><text>6,365,190</text></patcit>; <patcit id="pcit0009" dnum="US6372258B"><text>6,372,258</text></patcit>; <patcit id="pcit0010" dnum="US6423344B"><text>6,423,344</text></patcit>; <patcit id="pcit0011" dnum="US6479049B"><text>6,479,049</text></patcit>; <patcit id="pcit0012" dnum="US6509006B"><text>6,509,006</text></patcit>; <patcit id="pcit0013" dnum="US6569406B"><text>6,569406</text></patcit>; <patcit id="pcit0014" dnum="US6572893B"><text>6,572,893</text></patcit>; <patcit id="pcit0015" dnum="US6582728B"><text>6,582,728</text></patcit>; <patcit id="pcit0016" dnum="US6838076B"><text>6,838,076</text></patcit>; and <patcit id="pcit0017" dnum="US6896906B"><text>6,896,906</text></patcit>.

Lyophilization, or freeze drying, involves a process in which solvent, typically water, is removed from a product after it is frozen and placed under a vacuum, allowing the ice to change directly from solid to vapor without passing through a liquid phase. The process consists of three separate, unique, and interdependent processes; freezing, primary drying (sublimation), and secondary drying (desorption). During spray drying, a (generally aqueous) solution is introduced via a nozzle (e.g., a two fluid nozzle), spinning disc, or an equivalent device into a hot gas stream. Passage through the nozzle atomizes the solution into fine droplets. The heat energy supplied by the gas stream causes the evaporation of water or other solvents, thereby producing fine particles.

Drug delivery using substituted diketopiperazine microparticles has been described in <patcit id="pcit0018" dnum="US5352461A"><text>U.S. Patent Nos.: 5,352,461</text></patcit>; <patcit id="pcit0019" dnum="US5503852A"><text>5,503,852</text></patcit>; <patcit id="pcit0020" dnum="US6331318A"><text>6,331,318</text></patcit>; <patcit id="pcit0021" dnum="US6395774A"><text>6,395,774</text></patcit> and <patcit id="pcit0022" dnum="US6663898A"><text>6,663,898</text></patcit>. Pulmonary delivery of diketopiperazine microparticles as dry powders is described in <patcit id="pcit0023" dnum="US5503852A"><text>U.S. Patent Nos.: 5,503,852</text></patcit>; <patcit id="pcit0024" dnum="US6428771A"><text>6,428,771</text></patcit>; <patcit id="pcit0025" dnum="US6444226A"><text>6,444,226</text></patcit> and <patcit id="pcit0026" dnum="US6652885A"><text>6,652,885</text></patcit>. Various methods for forming and loading diketopiperazine particles for drug delivery are disclosed in <patcit id="pcit0027" dnum="US6444226B"><text>U.S. Patent No. 6,444,226</text></patcit>, <patcit id="pcit0028" dnum="US532063A"><text>U.S. Patent Application Nos. 11/532,063</text></patcit> and <patcit id="pcit0029" dnum="US11532065A"><text>11/532,065 both filed on September 14, 2006</text></patcit>, and <patcit id="pcit0030" dnum="US71752405P"><text>U.S. Provisional Patent Application Serial No: 60/717,524, filed on September 14, 2005</text></patcit>. Each of these documents is incorporated herein by reference for all they contain regarding diketopiperazines, diketopiperazine microparticles and their use in drug delivery. Dry powders made according to these teachings work well for pulmonary delivery; however there remains room for improvement of various pharmaceutic properties. The present invention serves to overcome the need in the art for obtaining improved particles having superior aerodynamics and providing more efficient delivery and greater stability of the active agent.

<u>SUMMARY OF THE INVENTION</u>

The present invention is directed to methods of obtaining an improved particle and/or an improved dry powder. The particles and powders contemplated by the present invention are comprised of a diketopiperazine derivative combined with an active agent. In particular embodiments of the present invention, the particle is a diketopiperazine-insulin particle formulation having improved stability, aerodynamic properties, and pharmacodynamic properties when dried by the process of spray drying as compared to that of freeze drying. In other embodiments, there is provided a spray-dried diketopiperazine-insulin particle formulation or dry powder.

In a particular embodiment of the present invention, the particle comprising a diketopiperazine is prepared and provided in a suspension, typically an aqueous suspension, to which a solution of the active agent is added. Active agents of the present invention may include one or more of the following: insulin, calcitonin, parathyroid hormone 1-34, or other bioactive fragment of parathyroid hormone, octreotide, leuprolide, and RSV peptide, felbamate, cannabinoid antagonists and/or agonists, muscurinic antagonists and/or agonists, heparin, low molecular weight heparin, cromolyn, sildenafil, vardenafil, tadalafil growth hormone, zidovudine (AZT), didanosine (DDI), granulocyte-colony stimulating factor (GCSF), lamotrigine, chorionic gonadotropin releasing factor, luteinizing release hormone, β-galactosidase, GLP-1, exendins 1-4, ghrelin, and fragments thereof, but are not limited to such. In another embodiment, the active agent is a peptide or protein such as insulin or an analogue thereof.

In a particular embodiment, the active agent is insulin or an analogue thereof.

The present invention discloses methods of obtaining particles with improved aerodynamic performance and in which the active agent is more stable and efficiently delivered. More particularly, the present invention concerns methods for drying, particularly spray drying, diketopiperazine-insulin particles. The dry powders have utility as pharmaceutical formulations for pulmonary delivery. In other embodiments, the diketopiperazine-insulin dry powders may be utilized for nasal delivery.

Thus, in particular embodiments the present invention provides a method of preparing a dry powder medicament with an improved pharmaceutic property, comprising the steps of: (a) providing a solution of a diketopiperazine; (b) providing a solution of an active agent; (c) forming particles; and (d) combining the diketopiperazine and the active agent; and thereafter (e) removing solvent by spray drying to obtain a dry powder, wherein the dry powder has an improved pharmaceutic property as compared to a dry powder obtained by removing solvent by lyophilization.

In another embodiment, the improved pharmaceutic property is selected from the group consisting of improved stability of the active agent, increased density of the dry powder, and improved aerodynamic performance of the dry powder. In still yet another embodiment, an improved aerodynamic performance of the dry powder is measured by the percentage of particles in the respirable range (respirable fraction) delivered from the inhaler. The respirable fraction, as contemplated in the present invention, may be greater than about 40% or greater than about 50%, or greater than about 60%, but is not limited to such.

In other embodiments of the present invention, it is contemplated that the insulin content of the microparticles is within the range of about 3% to about 50% by weight of the dry powder formulation. In other instances, the insulin concentration is within the range of about 7% to about 25% by weight. In preferred embodiments insulin content is about 19.0, 19.1, 19.2 19.3, 19.4, 19.5, 19.6, 19.7, 19.8, or 19.9% by weight. In another preferred embodiment, insulin concentration is at about 11% by weight. In still other preferred embodiments the insulin content is about 10, 12, 13, 14, 15, 16, 17, or 18 % by weight. In various embodiments, about is defined as ± 0.1, 0.2, 0.5, 1, or 2 %, so long as the uncertainty does not exceed 10% of the insulin content.

In still yet another embodiment, there is provided in the present invention a diketopiperazine having the formula 2,5-diketo-3,6-di(4-X-aminobutyl)piperazine, wherein X is selected from the group consisting of succinyl, glutaryl, maleyl, and fumaryl. In a preferred embodiment, the diketopiperazine is fumaryl diketopiperazine.

In yet another particular embodiment of the present invention there is provided a dry powder prepared according to the method of preparing a dry powder medicament with an improved pharmaceutic property, comprising the steps of: (a) providing a solution of a diketopiperazine; (b) providing a solution of an active agent; (c) forming particles; and (d) combining the diketopiperazine and the active agent; and thereafter (e) removing solvent by spray drying to obtain a dry powder, wherein the dry powder has an improved pharmaceutic property as compared to a dry powder obtained by removing solvent by lyophilization. In a further embodiment, the dry powder comprises an active agent such as insulin or an analogue thereof, but is not limited to such.

In still yet another particular embodiment, the present invention provides a method for delivering insulin to a patient in need thereof, comprising administering to the patient an effective amount of the dry powder.

The present invention also provides a dry powder having an improved pharmaceutic property wherein the improved property is improved delivery of the active agent whereby greater glucose disposal is achieved.

In still yet another particular embodiment of the present invention, there is provided a method of preparing a dry powder medicament with an improved pharmaceutic property, comprising: (a) providing a diketopiperazine in solution; (b) a step for forming particles comprising the diketopiperazine; (c) and removing solvent by spray drying to obtain a dry powder, wherein the dry powder has an improved pharmaceutic property as compared to a dry powder obtained by removing solvent by lyophilization. A further step comprising loading the particle with an active agent prior to the solvent removal step is also provided.

Another particular embodiment of the present invention provides a method of optimizing the aerodynamic performance of a diketopiperazine dry powder comprising the steps of: (a) precipitating a diketopiperazine from solution under a controlled temperature to form particles; (b) selecting a drying method based on said temperature; and (c) drying the particles. A further step comprising loading the particles with an active agent is also contemplated.

In particular embodiments the inlet temperature during spray drying is 105°C, 110°C, 120°C, 130°C, 140°C, or a range bounded by any pair of these values. In other particular embodiments the atomization pressure is 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1 bar or a range bounded by any pair of these values. In further particular embodiments the spray rate is 4.4, 7.6, 12.2 g/min, or a range bounded by any pair of these values. In still another particular embodiment of the present invention the outlet temperature is 75°C.

In a further embodiment, the diketopiperazine is fumaryl diketopiperazine, wherein the controlled temperature is between about 15°C to about 18°C and the selected drying method is spray drying. In other embodiments the controlled temperature is about 17°C. In still other embodiments the controlled temperature is less than or equal to about 13°C or greater than or equal to about 19°C.

In a further particular embodiments there is contemplated a particle containing about 11.0%, 11.1%, 11.2%, 11.3%, 11.4%, 11.5%, 11.6%, 11.7%, 11.8%, 11.9%, 12.0%, 12.1%, 12.2%, 12.3%, 12.4%, 12.5%, 12.6%, 12.7%, 12.8%, 12.9%, 13.0%, 13.1%, 13.2%, 13.3%, 13.4%, 13.5%, 13.6%, 13.7%, 13.8%, 13.9%, 14.0%, 14.1%, 14.2%, 14.3%, 14.4%, 14.5%, 14.6%, 14.7%, 14.8%, 14.9%, 15.0%, 15.1%, 15.2%, 15.3%, 15.4%, 15.5%, 15.6%, 15.7%, 15.8%, 15.9%, 16.0% or greater, insulin by weight. In a particular embodiment of the present invention there is provided a particle containing about 11.4% insulin by weight. In another particular embodiment there is contemplated a particle comprising up to 50% insulin by weight.

The active agent such as, but not limited to, insulin, comprised in a solution or suspension, is mixed with a suspension of a diketopiperazine wherein, the solution or suspension is in a suitable solvent for both the active agent and the diketopiperazine

In some embodiments, the present invention provides a method of obtaining a dry powder comprising a diketopiperazine and an active agent such as insulin, having improved pharmaceutic properties by precipitating the particles from a solution at a controlled temperature between about 15°C to about 18°C. In other embodiments the controlled temperature is about 17°C. In still other embodiments the controlled temperature is less than or equal to about 13°C or greater than or equal to about 19°C.

In other embodiments of the present invention the term 'Cartridge Fill Weight' as used herein refers to the quantity of drug product contained in a cartridge for an inhaler, typically 5-10 mg or more. In other embodiments the cartridge fill weight can vary from about 2.5 to 15 mg, 10 to 20 mg, or 5 to 30 mg.

In further embodiments the bulk or tapped density of the powder dried by spray drying is increased compared to a similar powder dried by lyophilization. In one such embodiment the density is greater by a factor of about 2 (1.7-2.3). Particular further embodiments include those limited to values disclosed in the examples or a range bounded by any pair of those values. In various embodiments the bulk density of the spray-dried powder is 0.150-0.200 g/cc. Particular embodiments include those limited to values disclosed in the examples or a range bounded by any pair of those values. In various embodiments the tapped density of the spray-dried powder is 0.250-0.300 g/cc. Particular embodiments include those limited to values disclosed in the examples or a range bounded by any pair of those values.

In yet another embodiment of the present invention the term 'Cartridge Emptying' as used herein refers to the percentage (%) of powder that is discharged from the inhaler upon activation (or discharge). This value is typically obtained by weighing the cartridge before and after discharge. Particular embodiments include those limited to values disclosed in the examples or a range bounded by any pair of those values.

In still yet another embodiment of the present invention the term 'Respirable Fraction (RF)' as used herein refers to the percentage (%) of particles in the respirable range (0.5-5.8 µm). The 'Respirable Fraction (RF) delivered' refers to the percentage of active ingredient able to reach the airways of the lung where the pharmaceutical effect is exerted. Particular embodiments include those limited to values disclosed in the examples or a range bounded by any pair of those values.

In another embodiment of the present invention the term 'Respirable Fraction Based on Fill' ('RF Based on Fill', '%RF on Fill' or '%RF/fill') as used herein refers to the percentage (%) of powder in the respirable range normalized by the quantity of powder in the inhaler. Particular embodiments include those limited to values disclosed in the examples or a range bounded by any pair of those values.

<u>BRIEF DESCRIPTION OF THE DRAWINGS</u>

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

<figref idref="f0001"><b>FIG. 1</b></figref><b>.</b> Demonstration that increased atomization pressure had a positive effect on the aerodynamics of the diketopiperazine-insulin formulations. The inlet temperature ranged from 110°C to 140°C and the outlet temperature was held constant at 75°C.

<figref idref="f0002 f0003"><b>FIGs. 2A-2E</b></figref><b>.</b> Evaluation of the accelerated stability of diketopiperazine-insulin formulations. The accelerated stability conditions were 40°C and 75% RH (relative humidity) for 10 days. A reduction in insulin loss in the spray-dried formulations is depicted in <figref idref="f0002"><b>FIG. 2A. FIG. 2B</b></figref> depicts a corresponding decrease in the formation of A-21, the primary degradation product of insulin under these conditions. <figref idref="f0003"><b>FIGs. 2C-2E</b></figref> demonstrate that the primary particles exhibit a decreased tendency to aggregate as the atomization pressure is increased from 0.4 bar (<figref idref="f0003"><b>FIG. 2C</b></figref>) to 0.6 bar (<figref idref="f0003"><b>FIG. 2D</b></figref>) to 0.7 bar <b>(</b><figref idref="f0003"><b>FIG. 2E</b></figref><b>)</b>. The measurements were obtained using laser diffraction.

<figref idref="f0004"><b>FIG. 3</b></figref><b>.</b> Demonstration of the effect of temperature on the aerodynamics of the diketopiperazine-insulin formulations. The outlet temperature was held at 75°C and the atomization pressure was held at 0.6 bar. The % RF on Fill (percent respirable fraction on a cartridge fill) remained relatively consistent over the temperature range.

<figref idref="f0005 f0006 f0007"><b>FIGs. 4A-4F</b></figref>. Demonstration that increased inlet temperature (drying rate) did not negatively impact the stability of the formulations. The accelerated stability conditions were 40°C and 75% RH for 10 days. <figref idref="f0005"><b>FIG. 4A</b></figref> depicts the percent loss of insulin. <figref idref="f0005"><b>FIG. 4B</b></figref> depicts formation of A-21, the most prevalent degradation product. <figref idref="f0006 f0007"><b>FIG. 4C-4F</b></figref> depicts a trend towards increased aggregation of primary particles (as shown by the particles size distribution obtained from laser diffraction) as the inlet temperature is increased from 105°C (<figref idref="f0006"><b>FIG. 4C</b></figref>) to 120°C (<figref idref="f0006"><b>FIGs. 4D</b></figref><b>and</b><figref idref="f0007"><b>4E</b></figref>) to 140°C (<figref idref="f0007"><b>FIG. 4F</b></figref>).

<figref idref="f0008 f0009"><b>FIGs. 5A-5E</b></figref>. Insulin distribution and particle morphology. <figref idref="f0008"><b>FIG. 5A</b></figref> shows that insulin is evenly distributed throughout the formulation independent of particle size. <figref idref="f0009"><b>FIGs. 5B-5E</b></figref> shows that the morphology of the spray-dried particles (<figref idref="f0009"><b>FIGs. 5C and 5E</b></figref>) and lyophilized particles <b>(</b><figref idref="f0009"><b>FIGs. 5B and 5D</b></figref>) is the same.

<figref idref="f0010"><b>FIGs. 6A-6B</b></figref>. Improvement in particle aerodynamics and insulin stability. <figref idref="f0010"><b>FIG</b>. <b>6A</b></figref> shows that %RF on Fill increases with atomization pressure at 0.7, 0.9 and 1.1 bar respectively. <figref idref="f0010"><b>FIG. 6B</b></figref> shows that %RF on Fill does not change with inlet temperature at 110°C, 120°C and 130°C respectively.

<figref idref="f0011 f0012 f0013 f0014"><b>FIGs. 7A-7K</b></figref>. Demonstration that insulin stability increases at higher inlet temperatures and atomization pressures. <figref idref="f0011"><b>FIG. 7A</b></figref> depicts measurement of the accelerated stability as percentage of insulin loss for powders spray dried at a pressure of 0.7 bar and inlet temperatures of 110°C, 120°C, and 130°C respectively. <figref idref="f0011"><b>FIG. 7B</b></figref> depicts measurement of the accelerated stability as percentage of insulin loss for powders spray dried at a pressure of 1.1 bar and inlet temperatures of 110°C, 120°C, and 130°C respectively. <figref idref="f0012 f0013 f0014"><b>FIGs. 7C-7K</b></figref> depicts minimal aggregation of primary particles (as shown by the particles size distribution obtained from laser diffraction) as the atomization pressure was varied from 0.7-1.1 bar and the inlet temperature was varied from 110°C, 120°C, and 130°C respectively.

<figref idref="f0015"><b>FIG. 8</b></figref>. Comparison of pharmacodynamic profiles (blood glucose reduction) following insufflation of 11.4% lyophilized FDKP/Insulin and 11.4% spray dried FDKP/Insulin in rats. Each animal received 3 mg of powder containing 11.4% insulin by weight. Each group contained 4 animals.

<figref idref="f0016"><b>FIG. 9</b></figref><b>.</b> Aerodynamic performance of FDKP/Insulin powders dried by spray drying or lyophilization. Two sets of suspensions (represented by squares and circles) were tested. Opened symbols represent spray-dried powders; filled symbols represent the lyophilized powders.

<figref idref="f0017"><b>FIGs. 10A-10B</b></figref><b>.</b> Stability data indicate that insulin loss (<figref idref="f0017"><b>FIG</b>. <b>10A</b></figref><b>)</b> and A-21 formation (<figref idref="f0017"><b>FIG. 10B</b></figref>) are reduced in the spray dried powder compared to the lyophilized powder. Both powders were adjusted to pH 4.5 prior to drying.

<u>DETAILED DESCRIPTION OF THE EMBODIMENTS OF THE INVENTION</u>

The success of any pharmaceutic particle depends not only on its efficacy in treating a disease or condition, but also having superior pharmaceutic properties over other known therapeutics. Desirable pharmaceutic properties sought in a dry powder particle include improved aerodynamics, pharmacodynamics and stability. However, producing particles with such properties is an ongoing challenge in the art. One approach to achieving this aim in the art, lies in the methodology used to manufacture particles.

Thus, the present invention provides the novel and unexpected discovery that the pharmaceutic properties of the dry powder can be generally improved by using spray drying in preference to lyophilization to remove solvent from the particles.

The present invention serves to overcome the shortcomings in the art by providing particles of diketopiperazine (DKP) combined with an active agent that are loaded and/or dried by a process to provide a dry powder having improved pharmaceutic properties. In particular embodiments, the present invention provides a particle, comprising a diketopiperazine combined with insulin, dried by spray drying. The invention further provides a spray-dried powder that demonstrates improved stability, aerodynamics or greater density, while maintaining at least similar pharmacodynamics as compared to the freeze-dried powder previously disclosed (see <patcit id="pcit0031" dnum="US6444226B"><text>U.S. Patent 6,444,226</text></patcit> entitled "Purification and Stabilization of Peptide and Protein Pharmaceutical Agents" and <patcit id="pcit0032" dnum="US71752405P"><text>U.S. Patent Application Serial Nos: 60/717,524, filed on September 14, 2005</text></patcit> and <patcit id="pcit0033" dnum="US11532063P"><text>11/532,063 filed September 14, 2006</text></patcit>, both entitled "Method of Drug Formulation Based on Increasing the Affinity of Active Agents for Crystalline Microparticle Surfaces"), each incorporated herein by reference for all they contain regarding diketopiperazine microparticle compositions.

Diketopiperazine particles for drug delivery can be formed and loaded with active agent by a variety of methods. Diketopiperazine solutions can be mixed with solutions or suspensions of an active agent and then precipitated to form particles comprising the active agent. Alternatively the DKP can be precipitated to form particles and subsequently mixed with a solution of the active agent. Association between the particle and the active agent can occur spontaneously, be driven by solvent removal, a specific step can be included prior to drying, or any combinations of these mechanisms applied to promote the association. Further variations along these lines will be apparent to one of skill in the art.

In one particular protocol the precipitated diketopiperazine particles are washed, a solution of insulin is added, the mixture frozen by dropwise addition to liquid nitrogen and the resulting frozen droplets (pellets) lyophilized (freeze-dried) to obtain a diketopiperazine-insulin dry powder. In other embodiments, the mixture can be dispersed into the liquid nitrogen by other means, for example, by spraying. In other protocols the precipitated diketopiperazine particles of the invention are washed, a solution of insulin added, the pH of the solution adjusted to promote insulin adsorption onto the particles, and solvent removed either by spray drying or freeze drying to obtain a diketopiperazine-insulin dry powder. Previously, lyophilization had been used for solvent removal and it had been expected that the use of spray drying for this purpose would produce similar results. As disclosed herein, it was surprisingly discovered that spray-dried dry powder possessed improved pharmaceutic characteristics. In particular the spray-dried powder had an improved respirable fraction (%RF), the insulin contained in the particles had greater stability against degradation and the particles had a greater density allowing higher doses to be loaded into any particular volume. Upon pulmonary administration, at least comparable amounts of insulin were delivered into the bloodstream as evidenced by at least comparable reductions in blood glucose. The performance of the spray-dried powders was superior to the lyophilized powders whether or not the preparation of the lyophilized samples included a pH-adjustment to promote association of the drug with the particle.

In a further refinement of the methodology, the temperature of the solution from which the DKP was precipitated was controlled. Surprisingly, FDKP particles precipitated from solutions at temperatures less than about 13°C or greater than about 19°C, dry powders with greater %RF were obtained using lyophilization for solvent removal. For FDKP particles precipitated from solutions at temperatures at about 17°C, dry powders with greater %RF were obtained using spray drying for solvent removal. In the remaining portions of the tested range, aerodynamic performance was similar with either drying method. Thus aerodynamic performance of DKP particles can be optimized by selecting a solvent removal procedure on the basis of the temperature of the solution from which the particles are precipitated. The dry powders obtained were characterized for aerodynamic properties (%RF, cartridge emptying, %RF/fill, mass median aerodynamic diameter [MMAD], geometric standard deviation [GSD]) and physicochemical properties (insulin content [% load], yield, density) as described in examples provided herein.

Surprisingly, the density of the spray-dried particles was roughly twice that of freeze-dried particles. This can be advantageous in providing higher doses. Dry powder inhalers generally impose a limit on the volume of powder, and thus the dosage of active agent, that can be delivered in a single operation. A powder of higher density, but at least similar respirable fraction, allows larger doses to be administered in a single operation, rather than requiring more operations per dose, formulations with higher % loading of active agent, or alternate inhaler or inhaler cartridge designs to accommodate various volumes of powder. Any of these alternatives entail greater development and/or production costs and also introduce issues of product complexity. Product complexity and requirements for multiple operations per dose additionally create issues with product acceptance and patient compliance. Thus this unexpected increase in powder density offers multiple advantages for the use of spray-dried powders as pharmaceutical products.

<b>1. Preparing Preformed Particles by Spray Drying</b>

Spray drying, as employed in the present invention, is a thermal processing method used to load and/or dry particles in a suspension in a liquid medium (solvent). As disclosed in the examples herein, a suspension of diketopiperazine particles and an insulin solution are mixed. Some or all of the insulin molecules then bind to the diketopiperazine particles. In various embodiments the diketopiperazine-insulin particles are then loaded and/or dried by spray drying and a dry powder is obtained. In an alternative embodiment, the active agent is added to a diketopiperazine solution prior to precipitation of the particles.

During spray drying, the aqueous mixture of diketopiperazine particles or diketopiperazine-insulin particles, are introduced via a nozzle (e.g., a two fluid nozzle or high pressure nozzle), spinning disc, or an equivalent device into a heated gas stream. Prior to being passed through the heated gas stream, the solution or suspension is atomized into fine droplets. The heat energy supplied by the gas stream causes the evaporation of water and other solvents in which the particles are suspended, thereby producing dry powder compositions.

In obtaining a dry powder comprising a diketopiperazine combined with insulin, as in embodiments of the present invention, the inventors found that the spray drying method generally provided particles with superior pharmaceutic properties compared to similar particles obtained by freeze drying. In obtaining the particles, the inventors took into consideration a number of parameters. These parameters included temperature, atomization pressure, solids content of the suspensions, percent of insulin loss, formation of A-21, aggregation of particles, and aerodynamic and biological performance.

The inlet temperature is the temperature of the gas stream leaving its source. The outlet temperature is a measure of the final temperature of the powder formulation and an indication of the utilization of the energy in the inlet air for drying and is a function of the inlet temperature and the heat load required to dry the product, along with other factors. The outlet temperature is selected based upon the lability of the macromolecule being treated.

The diketopiperazine/active agent mixture may be a suspension. The solvent, generally water, rapidly evaporates from the droplets producing a fine dry powder.

Spray drying is performed under conditions that result in a powder of homogeneous constitution having a particle size that is respirable, with low moisture content and other characteristics that allow for aerosolization. Preferably the particle size of the resulting powder is such that more than about 98% of the particles (by mass) have a diameter of about 10 µm or less with about 90% of the particles (by mass) have a diameter less than 5 µm. Alternatively, about 95% of the particles (by mass) have a diameter of less than 10 µm with about 80% of the particles (by mass) have a diameter of less than 5 µm. In certain embodiments, the dry powder has a mean particle size of 1 to 5 µm in diameter. The preceding embodiments relate especially to use of the powder in pulmonary delivery. Mean particle size can effect where in the respiratory tract particles are deposited and can also effect their bulk handling properties. For example nasal deposition is favored for particles with mean diameters greater than 20 µm. In other embodiments, the powder may be used to form tablets, packaged in capsules, or resuspended for oral administration or injection. Thus in various embodiments, the dry powder may comprise particles having a mean particle size of greater than about 10 µm, 20 µm, 30 µm, 40 µm, 50 µm, 60 µm, 70 µm, 80 µm, 90 µm, 100 µm. In another embodiment, the dry powder may comprise particles having a mean particle size of about 100 µm to about 500 µm. In other embodiments, the dry powder may comprise particles having a mean particle size of less than about 1 mm.

Suspensions of the present invention, comprising an active agent and a diketopiperazine may be spray-dried in conventional spray drying equipment such as the PHARMASD™ PSD-1 Spray Dryer or the SD-Micro™ Spray Dryer, as are well known in the art and obtainable from a commercial supplier (Niro Inc., Columbia, MD), thereby resulting in a dry powder comprised of such particles. It is noted that other conventional spray drying equipment may be used.

In conducting spray drying experimentation, methods such as rotary atomization, pressure atomization, and two-fluid atomization (for example, co-current two-fluid nozzle and/or fountain two-fluid nozzle) may be employed. Devices used in spray drying methodology are well known to one of ordinary skill in the art.

Although no special restrictions are placed on the nozzle of the atomizer used in the process of spraying, for a nozzle which can produce a spray-dry composition with a grain diameter suitable for nasal pharyngeal or pulmonary administration it is recommended in the art to use nozzles such as those in the following examples. For example, nozzle types "1A," "1," "2A," "2, " "3" and the like, (manufactured by Yamato Chemical Co.), or the SB Series SprayDry^® Nozzles (manufactured by Spraying Systems Co.), can be used with the spray-dryer. In addition, disks type "MC-50," "MC-65" or "MC-85," (manufactured by Okawara Kakoki Co.), can be used as rotary disks of the spray-drier atomizer.

In other embodiments, the inlet gas temperature used to dry the sprayed material is such that it does not cause heat deactivation of the active agent. The range of inlet temperatures may vary between about 50°C to about 200°C, preferably between about 110°C and 160°C. With well-stabilized agents, the inlet temperature can exceed 200°C. The temperature of the outlet gas used to dry the sprayed material may vary between about 35°C and about 100°C, preferably between 55°C and 85°C. In other embodiments, the outlet temperature may be preferably at 75°C. In another embodiment of the present invention, the inlet and outlet temperatures may be held at 120°C and 75°C respectively.

As disclosed above and elsewhere herein, terminology useful and applicable to the methods and compositions of the present invention are as follows:

The term "powder" means a composition that consists of fine solid particles that are capable of being dispersed in an inhalation device and inhaled by a subject. In preferred embodiments the particles reach the lungs or alveoli. Such a powder is said to be "respirable." Preferably the average particle size is less than about 10 microns (µm) in diameter with a relatively uniform spheroidal shape distribution. More preferably the diameter is less than about 7.5 µm and most preferably less than about 5.0 µm. Usually, the particle size distribution is between about 0.1 µm and about 8 µm in diameter, particularly about 0.3 µm to about 5 µm.

The term "dry" means that the powder composition is not suspended or dissolved in a propellant, carrier, or other liquid. It is not meant to imply a complete absence of water. The composition can have a moisture content such that the particles are readily dispersible in an inhalation device to form an aerosol. This moisture content is generally below about 10% by weight (% w) water, usually below about 5% weight and preferably less than about 3% weight.

The term "effective amount" is the amount that is needed to provide a desired response in the subject to be treated. The precise dosage will vary according to a variety of factors including, but not limited to, the age and size of the subject, the disease and the treatment being effected. The "effective amount" will also be determined based on the anticipated pharmacodynamic response or bioavailability.

<b>2. Diketopiperazines</b>

Diketopiperazines can be formed into particles that incorporate an active agent or particles onto which an active agent can be adsorbed. Diketopiperazines of the present invention include but are not limited 3,6-di(fumaryl-4 aminobutyl)-2,5-diketopiperazine also known as (E)-3,6-bis[4-(<i>N</i>-carboxyl-2-propenyl)amidobutyl]-2,5-diketopiperazine (which may also be referred to as fumaryl diketopiperazine or FDKP).

Other diketopiperazines that are contemplated in the present invention include 3,6-di(4- aminobutyl)-2,5-diketopiperazine; 3,6-di(succinyl-4-aminobutyl)-2,5-diketopiperazine (succinyl diketopiperazine or SDKP); 3,6-di(maleyl-4-aminobutyl)-2,5-diketopiperazine; 3,6-di(citraconyl-4-aminobutyl)-2-5-diketopiperazine; 3,6-di(glutaryl-4-aminobutyl)-2,5-diketopiperazine; 3,6-di(malonyl-4-aminobutyl)-2,5-diketopiperazine; 3,6-di(oxalyl-4-aminobutyl)-2,5-diketopiperazine and derivatives therefrom.

In brevity, diketopiperazines can be formed by cyclodimerization of amino acid ester derivatives, as described by <nplcit id="ncit0001" npl-type="s"><text>Katchalski, et al., (J. Amer. Chem. Soc. 68:879-80; 1946</text></nplcit>), by cyclization of dipeptide ester derivatives, or by thermal dehydration of amino acid derivatives in high-boiling solvents, as described by <nplcit id="ncit0002" npl-type="s"><text>Kopple, et al., (J. Org. Chem. 33(2):862-64;1968</text></nplcit>), the teachings of which are incorporated herein.

Methods for synthesis and preparation of diketopiperazines are well known to one of ordinary skill in the art and are disclosed in <patcit id="pcit0034" dnum="US5352461A"><text>U.S. Patents 5,352,461</text></patcit>; <patcit id="pcit0035" dnum="US5503852A"><text>5,503,852</text></patcit>; <patcit id="pcit0036" dnum="US6071497A"><text>6,071,497</text></patcit>; <patcit id="pcit0037" dnum="US6331318A"><text>6,331,318</text></patcit>; and <patcit id="pcit0038" dnum="US6428771A"><text>6,428,771</text></patcit>; and <patcit id="pcit0039" dnum="US208087A"><text>U.S. Patent Application No. 11/208,087</text></patcit> each of which is incorporated herein by reference for all they teach regarding diketopiperazines. United States Patent No. <patcit id="pcit0040" dnum="US6444226B"><text>6,444,226</text></patcit>, herein incorporated by reference for all it contains regarding diketopiperazine microparticles, describes preparing and providing microparticles of diketopiperazines in aqueous suspension to which a solution of active agent is added. This patent further describes a method of removing a liquid medium by lyophilization to yield microparticles comprising an active agent. See also United States Patent No. <patcit id="pcit0041" dnum="US6440463B"><text>6,440,463</text></patcit> and <patcit id="pcit0042" dnum="US532063A"><text>U.S. Patent Application Serial Nos: 11/532,063</text></patcit> and <patcit id="pcit0043" dnum="US11532025A"><text>11/532,025 both filed on September 14, 2006</text></patcit>, and <patcit id="pcit0044" dnum="US71752405P"><text>U.S. Provisional Patent Application Serial No: 60/717,524, filed on September 14, 2005</text></patcit>; each of which is incorporated herein by reference for all they teach regarding diketopiperazine microparticles.

It is further contemplated that the diketopiperazine-insulin particle formulations of the present invention can be administered by various routes of administration. As dry powders these particles can be delivered by inhalation to specific areas of the respiratory system, depending on particle size. Additionally, the particles can be made small enough for incorporation into an intravenous suspension dosage form. Oral delivery is also possible with the particles incorporated into a suspension, tablets, or capsules.

<b>3. Active Agents</b>

Embodiments of the present invention employ particles combining an active agent with a diketopiperazine. The term 'active agent' is referred to herein as the therapeutic agent, or molecule (such as protein or peptide or biological molecule), to be encapsulated, associated, joined, complexed or entrapped in or to the diketopiperazine of the present invention. Generally speaking, any form of an active agent can be combined with a diketopiperazine of the present invention. Active agents, as contemplated in the present invention, may or may not be charged.

Active agents contemplated for use in the compositions and methods described herein may include any polymer or large organic molecules, most preferably peptides and proteins. Examples include synthetic organic compounds, proteins and peptides, polysaccharides and other sugars, lipids, and nucleic acid sequences, having therapeutic, prophylactic, or diagnostic activities. Active agents may also include small molecules and vitamins. An active agent of the present invention may also be a vasoactive agent, a neuroactive agent, a hormone, an agent regulating metabolism, weight, or blood glucose levels, an anticoagulant, an immunomodulating agent, a cytotoxic agent, an antibiotic, an antiviral, an antisense molecule, or an antibody.

Examples of specific exemplary active agents have been listed above. In particular embodiments of the invention the active agent is insulin or an analogue thereof. Analogues with faster, slower, shorter, or longer action profiles are known in the art. Such analogues include those with altered amino acid sequences and those that have been covalently modified with other moieties, such as polyethylene glycol, or additional amino acids, such as in a fusion protein. Ultimately any molecule with a substantial portion of a wild type insulin molecule and physiologically relevant insulin activity is comprehended by this term.

Proteins as contemplated by the present invention are defined as consisting of 100 amino acid residues or more; in addition, peptides contemplated by the invention are less than 100 amino acid residues.

<b>4. Stabilizing Agents Contemplated in the Present Invention</b>

In further embodiments, there is contemplated by the present invention the use of stabilizing agents that may be contained in a suspension or solution comprising a diketopiperazine and an active agent which may be incorporated into the particle formulation.

Stabilizing agents may be included for conformational stability during the drying process. In addition, these stabilizing agents may further improve the aerodynamics or bioavailability of the dry powder diketopiperazine-insulin particle formulations of the present invention. Such stabilizing agents may comprise, but are not limited to, sugars, surface modifying agents, surfactants, hydrophobic amino acids such as tryptophan, tyrosine, leucine, phenylalanine, pharmaceutical carriers or excipients, and the like.

Stabilizing agents contemplated by the present invention are those preferably suitable for respiratory and pulmonary administration. In certain embodiments, it is preferred that the stabilizing agent be incorporated simultaneously into the diketopiperazine-insulin particle to produce a homogeneous powder. Alternatively, the stabilizing agent may be separately prepared in a dry powder form and combined with the spray dried diketopiperazine-insulin particle by blending.

In other instances, powder carriers may be employed such as, but not limited to carbohydrates, e.g., monosaccharides such as fructose, galactose, glucose, D-mannose, sorbose, and the like; disaccharides, such as lactose, trehalose, cellobiose, and the like; cyclodextrins, 2-hydroxypropyl-β-cyclodextrin; and polysaccharides, such as raffinose, maltodextrins, dextrans, and the like; amino acids, such as glycine, arginine, aspartic acid, glutamic acid, cysteine, lysine, and the like; organic salts prepared from organic acids and bases, such as sodium citrate, sodium ascorbate, magnesium gluconate, sodium gluconate, tromethamine hydrochloride, and the like; peptides and proteins, such as aspartame, human serum albumin, gelatin, and the like; alditols, such as xylitol, and the like. A preferred group of carriers may include trehalose, raffinose, maltodextrins, glycine, sodium citrate, tromethamine hydrochloride, human serum albumin, and mannitol. Such powder carriers will usually be crystalline (to avoid water absorption), but might in some cases be amorphous or mixtures of crystalline and amorphous forms. The size of the stabilizing agent particles may be selected to improve the flowability of the spray dried powder product.

Sugars as contemplated by the present invention include, but are not limited to, dextrose, lactose, and mannitol.

Surfactants as contemplated by the present invention include, but are not limited to, polysorbate 80 (PS80), lecithin, phosphatidylcholine, DPPC, sodium dodecylsulfate, and ionic detergents.

<u>EXAMPLES</u>

The following examples are included to demonstrate preferred embodiments of the present invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of 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 spirit and scope of the invention.

<u>Example 1</u>

<u>Effect of Atomization Pressure on Aerodynamics, Stability, and Aggregation</u>

Diketopiperazine derivative, 3,6-bis[<i>N</i>-fumaryl-<i>N</i>-(n-butyl)amino]-2,5-diketopiperazine (also referred to as 3,6-di(fumaryl-4 aminobutyl)-2,5-diketopiperazine, fumaryl diketopiperazine or FDKP; also termed (E)-3,6-bis[4-(N-carboxy-2-propenyl)amidobutyl]-2,5-diketopiperazine) were precipitated and washed. Insulin was loaded onto the FDKP particles by adjustment to a pH of approximately 4.45, and the FDKP-insulin particles were spray dried to obtain a FDKP-insulin dry powder. A pH of about 4.45 was found to increase the binding of insulin to FDKP particles as disclosed in <patcit id="pcit0045" dnum="US532063A"><text>U.S. Patent Application Serial Nos: 11/532,063</text></patcit> and <patcit id="pcit0046" dnum="US11532025A"><text>11/532,025 both filed on September 14, 2006</text></patcit>, and <patcit id="pcit0047" dnum="US71752405P"><text>U.S. Provisional Patent Application Serial No: 60/717,524, filed on September 14, 2005</text></patcit>.

The dry powders were characterized for various aerodynamic properties (%RF, cartridge emptying, %RF/fill, mass median aerodynamic diameter [MMAD], and geometric standard deviation [GSD]).

Table 1 and <figref idref="f0001">FIG. 1</figref> demonstrate the effect of the atomization (nozzle) pressure on the aerodynamic performance of the particles. The nozzle pressures ranged from 0.4 bar to 1.1 bar (Table 1). The respirable fraction on fill (% RF on Fill) improved as the atomization pressure was increased from 0.4 bar to 1.1 bar. <tables id="tabl0001" num="0001"><table frame="all"><title>Table 1. Effect of atomization pressure on aerodynamic properties. Outlet temperature was 75°C.</title><tgroup cols="7"><colspec colnum="1" colname="col1" colwidth="33mm" /><colspec colnum="2" colname="col2" colwidth="19mm" /><colspec colnum="3" colname="col3" colwidth="26mm" /><colspec colnum="4" colname="col4" colwidth="34mm" /><colspec colnum="5" colname="col5" colwidth="17mm" /><colspec colnum="6" colname="col6" colwidth="23mm" /><colspec colnum="7" colname="col7" colwidth="17mm" /><thead><row><entry align="center">Atomization Pressure (bar)</entry><entry align="center">Inlet T (°C)</entry><entry align="center">%RF delivered</entry><entry align="center">% Cartridge Emptying</entry><entry align="center">%RF fill</entry><entry align="center">MMAD (µm)</entry><entry align="center">GSD</entry></row></thead><tbody><row><entry valign="bottom" align="char" char=".">0.4</entry><entry align="center" valign="bottom">105</entry><entry valign="bottom" align="char" char="." charoff="14">34.7</entry><entry valign="bottom" align="char" char="." charoff="10">95.5</entry><entry valign="bottom" align="char" char="." charoff="22">33.1</entry><entry valign="bottom" align="char" char="." charoff="8">2.7</entry><entry valign="bottom" align="char" char="." charoff="11">2.2</entry></row><row><entry valign="bottom" align="char" char=".">0.5</entry><entry align="center" valign="bottom">105</entry><entry valign="bottom" align="char" char="." charoff="14">30.3</entry><entry valign="bottom" align="char" char="." charoff="10">92.1</entry><entry valign="bottom" align="char" char="." charoff="22">27.9</entry><entry valign="bottom" align="char" char="." charoff="8">3.3</entry><entry valign="bottom" align="char" char="." charoff="11">2.3</entry></row><row><entry morerows="3" valign="middle" align="char" char=".">0.6</entry><entry align="center" valign="bottom">105</entry><entry valign="bottom" align="char" char="." charoff="14">39.4</entry><entry valign="bottom" align="char" char="." charoff="10">95.6</entry><entry valign="bottom" align="char" char="." charoff="22">37.7</entry><entry valign="bottom" align="char" char="." charoff="8">2.5</entry><entry valign="bottom" align="char" char="." charoff="11">2.3</entry></row><row><entry align="center" valign="bottom">120</entry><entry valign="bottom" align="char" char="." charoff="14">45.5</entry><entry valign="bottom" align="char" char="." charoff="10">91.9</entry><entry valign="bottom" align="char" char="." charoff="22">41.8</entry><entry valign="bottom" align="char" char="." charoff="8">2.7</entry><entry valign="bottom" align="char" char="." charoff="11">2.2</entry></row><row><entry align="center" valign="bottom">120</entry><entry valign="bottom" align="char" char="." charoff="14">45.4</entry><entry valign="bottom" align="char" char="." charoff="10">92.2</entry><entry valign="bottom" align="char" char="." charoff="22">41.9</entry><entry valign="bottom" align="char" char="." charoff="8">2.5</entry><entry valign="bottom" align="char" char="." charoff="11">2.2</entry></row><row><entry align="center" valign="bottom">140</entry><entry valign="bottom" align="char" char="." charoff="14">42.4</entry><entry valign="bottom" align="char" char="." charoff="10">91.4</entry><entry valign="bottom" align="char" char="." charoff="22">38.8</entry><entry valign="bottom" align="char" char="." charoff="8">2.5</entry><entry valign="bottom" align="char" char="." charoff="11">2.2</entry></row><row><entry morerows="2" valign="middle" align="char" char=".">0.7</entry><entry align="center" valign="bottom">105</entry><entry valign="bottom" align="char" char="." charoff="14">48.2</entry><entry valign="bottom" align="char" char="." charoff="10">92.7</entry><entry valign="bottom" align="char" char="." charoff="22">44.7</entry><entry valign="bottom" align="char" char="." charoff="8">2.7</entry><entry valign="bottom" align="char" char="." charoff="11">2.2</entry></row><row><entry align="center" valign="bottom">110</entry><entry valign="bottom" align="char" char="." charoff="14">71.9</entry><entry valign="bottom" align="char" char="." charoff="10">68.9</entry><entry valign="bottom" align="char" char="." charoff="22">49.5</entry><entry valign="bottom" align="char" char="." charoff="8">2.3</entry><entry valign="bottom" align="char" char="." charoff="11">2.0</entry></row><row><entry align="center" valign="bottom">120</entry><entry valign="bottom" align="char" char="." charoff="14">57.7</entry><entry valign="bottom" align="char" char="." charoff="10">77.6</entry><entry valign="bottom" align="char" char="." charoff="22">44.8</entry><entry valign="bottom" align="char" char="." charoff="8">2.5</entry><entry valign="bottom" align="char" char="." charoff="11">2.0</entry></row><row><entry valign="bottom" /><entry align="center" valign="bottom">130</entry><entry valign="bottom" align="char" char="." charoff="14">63.5</entry><entry valign="bottom" align="char" char="." charoff="10">71.6</entry><entry valign="bottom" align="char" char="." charoff="22">45.5</entry><entry valign="bottom" align="char" char="." charoff="8">1.9</entry><entry valign="bottom" align="char" char="." charoff="11">2.0</entry></row><row><entry morerows="2" valign="middle" align="char" char=".">0.9</entry><entry align="center" valign="bottom">110</entry><entry valign="bottom" align="char" char="." charoff="14">68.4</entry><entry valign="bottom" align="char" char="." charoff="10">70.2</entry><entry valign="bottom" align="char" char="." charoff="22">48.0</entry><entry valign="bottom" align="char" char="." charoff="8">2.3</entry><entry valign="bottom" align="char" char="." charoff="11">2.0</entry></row><row><entry align="center" valign="bottom">120</entry><entry valign="bottom" align="char" char="." charoff="14">68.3</entry><entry valign="bottom" align="char" char="." charoff="10">74.9</entry><entry valign="bottom" align="char" char="." charoff="22">51.2</entry><entry valign="bottom" align="char" char="." charoff="8">2.1</entry><entry valign="bottom" align="char" char="." charoff="11">2.0</entry></row><row><entry align="center" valign="bottom">130</entry><entry valign="bottom" align="char" char="." charoff="14">55.4</entry><entry valign="bottom" align="char" char="." charoff="10">90.2</entry><entry valign="bottom" align="char" char="." charoff="22">49.9</entry><entry valign="bottom" align="char" char="." charoff="8">2.7</entry><entry valign="bottom" align="char" char="." charoff="11">2.0</entry></row><row><entry morerows="2" valign="middle" align="char" char=".">1.1</entry><entry align="center" valign="bottom">110</entry><entry valign="bottom" align="char" char="." charoff="14">64.2</entry><entry valign="bottom" align="char" char="." charoff="10">84.0</entry><entry valign="bottom" align="char" char="." charoff="22">54.0</entry><entry valign="bottom" align="char" char="." charoff="8">2.5</entry><entry valign="bottom" align="char" char="." charoff="11">1.9</entry></row><row><entry align="center" valign="bottom">120</entry><entry valign="bottom" align="char" char="." charoff="14">70.4</entry><entry valign="bottom" align="char" char="." charoff="10">70.8</entry><entry valign="bottom" align="char" char="." charoff="22">49.8</entry><entry valign="bottom" align="char" char="." charoff="8">2.0</entry><entry valign="bottom" align="char" char="." charoff="11">2.0</entry></row><row><entry align="center" valign="bottom">130</entry><entry valign="bottom" align="char" char="." charoff="14">71.7</entry><entry valign="bottom" align="char" char="." charoff="10">74.9</entry><entry valign="bottom" align="char" char="." charoff="22">53.7</entry><entry valign="bottom" align="char" char="." charoff="8">2.2</entry><entry valign="bottom" align="char" char="." charoff="11">2.0</entry></row></tbody></tgroup></table></tables>

The stability of the insulin was assessed as the percent loss of insulin (<figref idref="f0002">FIG. 2A</figref>) and the percent conversion to insulin degradation product A21-desamido insulin (% A21) under stress conditions (10 days at 40°C, 75% RH) (see <figref idref="f0002">FIG. 2B</figref>). For comparison, the bottom bar in each figure represents data obtained with freeze dried particles. The data demonstrate that, as the atomization pressure was increased, there was a general trend toward increased stability of the insulin in the diketopiperazine-insulin particles. Less formation of the A21 insulin degradation product was observed in all of the spray-dried particles as compared to freeze-dried particles (<figref idref="f0002">FIG. 2B</figref>).

To assess aggregation of the primary particles, the particle size distribution was determined from laser diffraction of a suspension of spray-dried particles using a Malvern Mastersizer 2000. Under the above trial conditions, a trend towards decreased aggregation of the primary diketopiperazine-insulin particles was observed with increased atomization pressure (<figref idref="f0003">FIGs. 2C-2E</figref>). It is observed that the size of the peak to the right, representing aggregated particles, decreases as the atomization pressure increases from 0.4 bar (<figref idref="f0003">FIG. 2C</figref>) to 0.6 bar (<figref idref="f0003">FIG. 2D</figref>), to 0.7 bar (<figref idref="f0003">FIG. 2E</figref>).

<u>Example 2</u>

<u>Effect of Inlet Temperatures on Aerodynamics, Stability, and Particle Aggregation</u>

Using particles prepared as above, spray dryer inlet temperature and process scalability were evaluated as shown in Table 2 below. In these experiments, the inlet temperature was varied from 105°C to 140°C and the outlet temperature was held constant at 75°C. The nozzle pressure was held constant at 0.6 bar.

It was observed that the increased inlet temperatures required an increase in the spray rate to maintain a consistent outlet temperature (Table 2). The increased spray rates produced dried particles at a greater production rate. The aerodynamics of the spray dried particles were assessed (Table 2). The % RF on Fill remained consistent over the temperature range studied (<figref idref="f0004">FIG. 3</figref>). <tables id="tabl0002" num="0002"><table frame="all"><title>Table 2. Effect of inlet temperature on particle aerodynamics. Nozzle pressure was maintained at 0.6 bar and the outlet temperature was 75 °C.</title><tgroup cols="8"><colspec colnum="1" colname="col1" colwidth="21mm" colsep="0" /><colspec colnum="2" colname="col2" colwidth="21mm" colsep="0" /><colspec colnum="3" colname="col3" colwidth="22mm" colsep="0" /><colspec colnum="4" colname="col4" colwidth="22mm" /><colspec colnum="5" colname="col5" colwidth="20mm" /><colspec colnum="6" colname="col6" colwidth="22mm" /><colspec colnum="7" colname="col7" colwidth="21mm" /><colspec colnum="8" colname="col8" colwidth="22mm" /><thead><row><entry align="center">Scale (g)</entry><entry align="center">Spray Rate (g/min)</entry><entry align="center">Inlet Temp. (°C)</entry><entry align="center">%RF</entry><entry align="center">% cartridge emptying</entry><entry align="center">%RF on Fill</entry><entry align="center">MMAD (µm)</entry><entry align="center">GSD</entry></row></thead><tbody><row><entry valign="bottom" align="char" char="." charoff="18">11.3</entry><entry align="center" valign="bottom">4.4</entry><entry align="center" valign="bottom">105</entry><entry valign="bottom" align="char" char="." charoff="17">39.4</entry><entry valign="bottom" align="char" char=".">95.6</entry><entry valign="bottom" align="char" char="." charoff="17">37.7</entry><entry valign="bottom" align="char" char="." charoff="9">2.5</entry><entry valign="bottom" align="char" char="." charoff="9">2.3</entry></row><row><entry valign="bottom" align="char" char="." charoff="18">11.3</entry><entry align="center" valign="bottom">7.6</entry><entry align="center" valign="bottom">120</entry><entry valign="bottom" align="char" char="." charoff="17">45.5</entry><entry valign="bottom" align="char" char=".">91.9</entry><entry valign="bottom" align="char" char="." charoff="17">41.8</entry><entry valign="bottom" align="char" char="." charoff="9">2.7</entry><entry valign="bottom" align="char" char="." charoff="9">2.2</entry></row><row><entry valign="bottom" align="char" char="." charoff="18">45.2</entry><entry align="center" valign="bottom">7.6</entry><entry align="center" valign="bottom">120</entry><entry valign="bottom" align="char" char="." charoff="17">42.4</entry><entry valign="bottom" align="char" char=".">91.4</entry><entry valign="bottom" align="char" char="." charoff="17">38.8</entry><entry valign="bottom" align="char" char="." charoff="9">2.5</entry><entry valign="bottom" align="char" char="." charoff="9">2.2</entry></row><row><entry valign="bottom" align="char" char="." charoff="18">11.3</entry><entry align="center" valign="bottom">12.2</entry><entry align="center" valign="bottom">140</entry><entry valign="bottom" align="char" char="." charoff="17">45.4</entry><entry valign="bottom" align="char" char=".">92.2</entry><entry valign="bottom" align="char" char="." charoff="17">41.9</entry><entry valign="bottom" align="char" char="." charoff="9">2.5</entry><entry valign="bottom" align="char" char="." charoff="9">2.2</entry></row></tbody></tgroup></table></tables>

Further, the data demonstrated that increasing the inlet temperature (drying rate) did not negatively impact the stability of the insulin on the particles. There was a trend toward increased insulin stability with increasing inlet temperature. Stability was measured as insulin lost and A21 formed (<figref idref="f0005">FIGS. 4A and 4B</figref>) after 10 days at 40°C/75%RH. However, under the above trial conditions, a trend toward increase aggregation of the primary diketopiperazine-insulin particles was observed with an increase in the inlet temperature (<figref idref="f0006 f0007">FIGs.4C-4F</figref>).

<u>Example 3</u>

<u>Insulin Recovery and Distribution</u>

In these experiments, a known mass of diketopiperazine particles was suspended in water. Enough insulin solution of known concentration was added to the suspension to give a theoretical composition of 11.4% insulin. The fumaryl diketopiperazine-insulin slurry was titrated to a pH of approximately 4.45 prior to spray drying.

Insulin distribution across particles was assessed as shown in <figref idref="f0008">FIG. 5A</figref>. These experiments were conducted using an Andersen Cascade Impactor. The powder was filled into cartridges and discharged through a MedTone^® inhaler into the Andersen cascade impactor. (The MedTone^® inhaler is described in <patcit id="pcit0048" dnum="US655153A"><text>U.S. Patent Application No. 10/655,153</text></patcit> entitled "Unit Dose Cartridge and Dry Powder Inhaler" which is incorporated herein by reference for all it contains regarding the inhaler device). The impactor classifies the particles by aerodynamic size. After discharge, the powder was recovered from each stage and assayed for insulin content (load). Insulin is shown to be evenly distributed throughout the formulation. Increasing the scale (grams of the powder), as shown in Table 2 above, by a factor of 4 was also found to be acceptable.

Particle morphology of the spray-dried and lyophilized particles was compared by scanning electron microscopy (SEM). <figref idref="f0009">FIG. 5B-5E</figref> shows the particle morphologies for the lyophilized formulation (<figref idref="f0009">FIGs. 5B and 5D</figref>) are comparable to those for the spray-dried formulation (<figref idref="f0009">FIGs. 5C and 5E</figref>).

<b><u>Summary of Examples 1-3</u></b>

The above data, show that: 1) increasing the atomization pressure decreased the aggregation of the primary particles; 2) increasing the inlet temperature had little impact on the particles aerodynamics; 3) increasing the inlet temperature was not observed to have a negative impact on the stability of the insulin; 4) increasing the inlet temperature resulted in greater aggregation of the primary particles; 5) spray-dried particles had increased insulin stability when compared to lyophilized particles of identical composition; and 6) spray-dried particles had similar morphology as lyophilized particles.

<u>Example 4</u>

<u>Determination of Spray-drying Parameters to Maximize Aerodynamic Performance</u>

Inlet temperature and atomization pressure were further evaluated using inlet temperatures of 110, 120 and 130°C and atomization pressures of 0.7, 0.9, and 1.1 bar (Table 3). <tables id="tabl0003" num="0003"><table frame="all"><title>Table 3. Effect of spray-drying parameters on particle aerodynamics</title><tgroup cols="7"><colspec colnum="1" colname="col1" colwidth="33mm" /><colspec colnum="2" colname="col2" colwidth="35mm" /><colspec colnum="3" colname="col3" colwidth="12mm" /><colspec colnum="4" colname="col4" colwidth="34mm" /><colspec colnum="5" colname="col5" colwidth="20mm" /><colspec colnum="6" colname="col6" colwidth="22mm" /><colspec colnum="7" colname="col7" colwidth="12mm" /><thead><row><entry colsep="0" align="center">Atomization pressure (bar)</entry><entry colsep="0" align="center">Inlet Temperature (°C)</entry><entry align="center">%RF</entry><entry align="center">% Cartridge emptying</entry><entry align="center">%RF on fill</entry><entry align="center">MMAD (µm)</entry><entry align="center">GSD</entry></row></thead><tbody><row><entry valign="bottom" align="char" char=".">0.7</entry><entry align="center" valign="bottom">110</entry><entry valign="bottom" align="char" char="." charoff="30">71.9</entry><entry valign="bottom" align="char" char="." charoff="11">68.9</entry><entry valign="bottom" align="char" char="." charoff="19">49.5</entry><entry valign="bottom" align="char" char="." charoff="9">2.3</entry><entry valign="bottom" align="char" char="." charoff="15">2.0</entry></row><row><entry valign="bottom" align="char" char=".">0.7</entry><entry align="center" valign="bottom">120</entry><entry valign="bottom" align="char" char="." charoff="30">57.7</entry><entry valign="bottom" align="char" char="." charoff="11">77.6</entry><entry valign="bottom" align="char" char="." charoff="19">44.8</entry><entry valign="bottom" align="char" char="." charoff="9">2.5</entry><entry valign="bottom" align="char" char="." charoff="15">2.0</entry></row><row><entry valign="bottom" align="char" char=".">0.7</entry><entry align="center" valign="bottom">130</entry><entry valign="bottom" align="char" char="." charoff="30">63.5</entry><entry valign="bottom" align="char" char="." charoff="11">71.6</entry><entry valign="bottom" align="char" char="." charoff="19">45.5</entry><entry valign="bottom" align="char" char="." charoff="9">1.9</entry><entry valign="bottom" align="char" char="." charoff="15">2.0</entry></row><row><entry valign="bottom" align="char" char=".">0.9</entry><entry align="center" valign="bottom">110</entry><entry valign="bottom" align="char" char="." charoff="30">68.4</entry><entry valign="bottom" align="char" char="." charoff="11">70.2</entry><entry valign="bottom" align="char" char="." charoff="19">48.0</entry><entry valign="bottom" align="char" char="." charoff="9">2.3</entry><entry valign="bottom" align="char" char="." charoff="15">2.0</entry></row><row><entry valign="bottom" align="char" char=".">0.9</entry><entry align="center" valign="bottom">120</entry><entry valign="bottom" align="char" char="." charoff="30">68.3</entry><entry valign="bottom" align="char" char="." charoff="11">74.9</entry><entry valign="bottom" align="char" char="." charoff="19">51.2</entry><entry valign="bottom" align="char" char="." charoff="9">2.1</entry><entry valign="bottom" align="char" char="." charoff="15">2.0</entry></row><row><entry valign="bottom" align="char" char=".">0.9</entry><entry align="center" valign="bottom">130</entry><entry valign="bottom" align="char" char="." charoff="30">55.4</entry><entry valign="bottom" align="char" char="." charoff="11">90.2</entry><entry valign="bottom" align="char" char="." charoff="19">49.9</entry><entry valign="bottom" align="char" char="." charoff="9">2.7</entry><entry valign="bottom" align="char" char="." charoff="15">2.0</entry></row><row><entry valign="bottom" align="char" char=".">1.1</entry><entry align="center" valign="bottom">110</entry><entry valign="bottom" align="char" char="." charoff="30">64.2</entry><entry valign="bottom" align="char" char="." charoff="11">84.0</entry><entry valign="bottom" align="char" char="." charoff="19">54.0</entry><entry valign="bottom" align="char" char="." charoff="9">2.5</entry><entry valign="bottom" align="char" char="." charoff="15">1.9</entry></row><row><entry valign="bottom" align="char" char=".">1.1</entry><entry align="center" valign="bottom">120</entry><entry valign="bottom" align="char" char="." charoff="30">70.4</entry><entry valign="bottom" align="char" char="." charoff="11">70.8</entry><entry valign="bottom" align="char" char="." charoff="19">49.8</entry><entry valign="bottom" align="char" char="." charoff="9">2.0</entry><entry valign="bottom" align="char" char="." charoff="15">2.0</entry></row><row><entry valign="bottom" align="char" char=".">1.1</entry><entry align="center" valign="bottom">130</entry><entry valign="bottom" align="char" char="." charoff="30">71.7</entry><entry valign="bottom" align="char" char="." charoff="11">74.9</entry><entry valign="bottom" align="char" char="." charoff="19">53.7</entry><entry valign="bottom" align="char" char="." charoff="9">2.2</entry><entry valign="bottom" align="char" char="." charoff="15">2.0</entry></row></tbody></tgroup></table></tables>

<figref idref="f0010">FIG. 6A</figref> summarizes the results of Table 3 as the %RF on fill versus the atomization pressure; <figref idref="f0010">FIG. 6B</figref> summarizes the results as %RF on fill versus the inlet temperature. Thus, the data show that increasing atomization pressure leads to improved aerodynamic performance and inlet temperature does not affect this parameter.

<u>Example 5</u>

<u>Effect of Inlet Temperature and Atomization Pressure on Stability and Aggregation</u>

The samples listed in Table 3 were analyzed for insulin stability and particle aggregation. As shown in <figref idref="f0011">FIGs. 7A and 7B</figref>, the results were consistent with those of Examples 1-3 in that the spray-dried samples showed less insulin loss than comparable lyophilized powders (the bottom bar in <figref idref="f0011">FIGs. 7A and 7B</figref>; loading of the particles used in the lyophilized samples included adjustment to pH 4.5, which as discussed in Example 1 above, increases the binding of insulin to FDKP particles).

The aggregation of the primary diketopiperazine-insulin particle was assessed under the conditions of increased inlet temperature and increased atomization pressure (<figref idref="f0012 f0013 f0014">FIGs. 7C-7K</figref>). The particle size distributions by laser diffraction were generally insensitive to atomization pressure and temperature over the ranges covered in this example. A small degree of aggregation was observed at 0.7 bar and inlet temperatures of 110°C and 120°C, but a unimodal distribution was obtained at all other conditions.

The results for the spray-dried samples as compared to the lyophilized samples show: 1) the atomization pressure can be increased to improve aerodynamics; 2) the inlet temperature has negligible effect on % RF on Fill; 3) insulin stability increases with increased inlet temperature; and 4) the increased inlet temperature and atomization pressure reduced aggregation of the primary insulin particles.

<u>Example 6</u>

<u>Insulin Pharmacodynamics with Spray-dried Particles</u>

Data from a rat insufflation study indicated that spray-dried FDKP-insulin powder provides at least comaprable glucose disposal as provided by lyophilized material. <figref idref="f0015">FIG. 8</figref> shows a comparison of pharmacodynamic profiles (blood glucose reduction) following insufflation of lyophilized and spray-dried 11.4% FDKP-insulin particles in rats. The glucose lowering capacity of spray-dried FDKP-insulin powder was found to be equivalent to that of lyophilized FDKP-insulin powder.

<u>Example 7</u>

<u>Aerodynamics and Stability of Spray-dried FDKP-insulin Powder</u>

Fumaryl diketopiperazine (FDKP)-insulin particles were prepared in a manner similar to that described above. That is, particles were mixed with an insulin solution to give particles containing 11.4% insulin by weight, and then the pH adjusted to promote insulin adsorption onto the particles. The resulting particle suspensions were dried by either spray drying or lyophilization. Table 4 shows the comparison of two 200g lots prepared using a commercial scale spray dryer with similar lyophilized samples. The bulk powders were tested for aerodynamic performance. Additional samples of bulk powders were stored at 40°C/75% RH for 15 days prior to evaluation for insulin loss and formation of A21-desamido insulin. The spray dried powder displayed an average respirable fraction on fill (%RF/fill) of 62%; compared to an average value of 54% for the lyophilized powder. The spray-dried powder also demonstrated superior stability. Insulin loss and A-21 formation of the spray-dried powder were about half that of the lyophilized powder. <tables id="tabl0004" num="0004"><table frame="all"><title>Table 4. Aerodynamics and stability of spray dried FDKP-insulin powder</title><tgroup cols="6"><colspec colnum="1" colname="col1" colwidth="51mm" /><colspec colnum="2" colname="col2" colwidth="13mm" /><colspec colnum="3" colname="col3" colwidth="35mm" /><colspec colnum="4" colname="col4" colwidth="16mm" /><colspec colnum="5" colname="col5" colwidth="25mm" /><colspec colnum="6" colname="col6" colwidth="27mm" /><thead><row><entry morerows="1" valign="middle">Manufacturing Process</entry><entry namest="col2" nameend="col4" align="center" valign="middle">Andersen cascade impactor</entry><entry namest="col5" nameend="col6" align="center" valign="middle">Accelerated stability</entry></row><row><entry align="center" valign="middle">%RF</entry><entry align="center" valign="middle">% Cartridge Emptying</entry><entry align="center" valign="middle">%RF/fill</entry><entry align="center" valign="middle">% Insulin Lost</entry><entry align="center" valign="middle">% A-21 Formed</entry></row></thead><tbody><row><entry align="center" valign="middle">Lyophilized (average of two lots*)</entry><entry align="center" valign="middle">55</entry><entry align="center" valign="middle">98</entry><entry align="center" valign="middle">54</entry><entry align="center" valign="middle">16.98</entry><entry valign="middle" align="char" char="." charoff="7">6.32</entry></row><row><entry align="center" valign="middle">Spray dried (average of two lots*)</entry><entry align="center" valign="middle">66</entry><entry align="center" valign="middle">94</entry><entry align="center" valign="middle">62</entry><entry align="center" valign="middle">8.83</entry><entry valign="middle" align="char" char="." charoff="7">2.63</entry></row></tbody></tgroup><tgroup cols="6" rowsep="0"><colspec colnum="1" colname="col1" colwidth="51mm" /><colspec colnum="2" colname="col2" colwidth="13mm" /><colspec colnum="3" colname="col3" colwidth="35mm" /><colspec colnum="4" colname="col4" colwidth="16mm" /><colspec colnum="5" colname="col5" colwidth="25mm" /><colspec colnum="6" colname="col6" colwidth="27mm" /><tbody><row><entry namest="col1" nameend="col6" align="justify">*-Lots were prepared in a similar manner.</entry></row></tbody></tgroup></table></tables>

<u>Example 8</u>

<u>Characterization of Spray-dried vs Lyophilized FDKP-insulin Powders</u>

In a further refinement of the process, the feed temperature of the FDKP solution was controlled. Stock solutions of fumaryl diketopiperazine (FDKP) were prepared and cooled to 11°C, 13°C, 15°C, 17°C, or 19°C and the FDKP particles were precipitated. Two different strategies were employed for loading and drying particles. In one strategy, the precipitated diketopiperazine particles were washed, an insulin solution was added and the pH adjusted to promote adsorption of insulin onto the particle, the mixture was frozen by dropwise addition to liquid nitrogen, and the resulting pellets were lyophilized (freeze-dried) to obtain a diketopiperazine-insulin dry powder. In another parallel protocol the precipitated diketopiperazine particles were washed, an insulin solution was added, the pH adjusted, and the diketopiperazine-insulin particle suspension was spray-dried to obtain a diketopiperazine-insulin dry powder.

Two sets of replicates were prepared and the dry powders were characterized for aerodynamic performance (%RF/fill, cartridge emptying, mass median aerodynamic diameter [MMAD] and geometric standard deviation [GSD]). These data are summarized in Table 5. The %RF/fill for these samples is shown in <figref idref="f0016">FIG. 9</figref>. The stability of the powders is compared in <figref idref="f0017">FIGs. 10A and 10B</figref>. As noted above, the spray-dried powders showed less insulin loss and less formation of A21-desamido insulin than the lyophilized samples.

The bulk density and tapped density of the spray-dried versus the lyophilized FDKP-insulin powder were assessed. The two sets of replicates were characterized for bulk and tapped density. Table 5 shows that the spray-dried powder is more dense (by about a factor of 2) than the lyophilized powder. The bulk and tapped density for the spray-dried materials averaged 0.2 g/cc and 0.29 g/cc respectively. The bulk and tapped densities for lyophilized FDKP-insulin averaged 0.09 g/cc and 0.13 g/cc respectively. These results were unexpected and surprising. This increase in density allows more powder to be placed in a single cartridge, thereby providing for higher dosages. <tables id="tabl0005" num="0005"><table frame="all"><title>Table 5. Effect of solution temperature on spray-dried and lyophilized FDKP-insulin particles</title><tgroup cols="8"><colspec colnum="1" colname="col1" colwidth="23mm" /><colspec colnum="2" colname="col2" colwidth="24mm" /><colspec colnum="3" colname="col3" colwidth="17mm" /><colspec colnum="4" colname="col4" colwidth="24mm" /><colspec colnum="5" colname="col5" colwidth="22mm" /><colspec colnum="6" colname="col6" colwidth="12mm" /><colspec colnum="7" colname="col7" colwidth="23mm" /><colspec colnum="8" colname="col8" colwidth="24mm" /><thead><row><entry align="center" valign="middle">Solution temperature (°C)</entry><entry align="center" valign="middle">Drying method</entry><entry align="center" valign="middle">%RF/fill</entry><entry align="center" valign="middle">% Cartridge Emptying</entry><entry align="center" valign="middle">MMAD (µm)</entry><entry align="center" valign="middle">GSD</entry><entry align="center" valign="middle">Bulk Density (g/cc)</entry><entry align="center" valign="middle">Tapped Density (g/cc)</entry></row></thead><tbody><row rowsep="0"><entry morerows="3" rowsep="1" align="center" valign="middle">11</entry><entry morerows="1" rowsep="1" valign="middle">spray-dried</entry><entry align="center" valign="middle">46.0</entry><entry align="center" valign="middle">87.0</entry><entry align="center" valign="middle">2.8</entry><entry align="center" valign="middle">1.9</entry><entry align="center" valign="middle">0.171</entry><entry align="center" valign="middle">0.260</entry></row><row><entry align="center" valign="middle">43.8</entry><entry align="center" valign="middle">92.1</entry><entry align="center" valign="middle">3.2</entry><entry align="center" valign="middle">1.8</entry><entry align="center" valign="middle">0.182</entry><entry align="center" valign="middle">0.267</entry></row><row rowsep="0"><entry valign="middle">lyophilized</entry><entry align="center" valign="middle">48.4</entry><entry align="center" valign="middle">98.4</entry><entry align="center" valign="middle">2.7</entry><entry align="center" valign="middle">2.1</entry><entry align="center" valign="middle">0.077</entry><entry align="center" valign="middle">0.121</entry></row><row><entry valign="middle" /><entry align="center" valign="middle">50.2</entry><entry align="center" valign="middle">96.1</entry><entry align="center" valign="middle">2.4</entry><entry align="center" valign="middle">2.1</entry><entry align="center" valign="middle">0.080</entry><entry align="center" valign="middle">0.122</entry></row><row rowsep="0"><entry morerows="3" rowsep="1" align="center" valign="middle">13</entry><entry morerows="1" rowsep="1" valign="middle">spray-dried</entry><entry align="center" valign="middle">57.4</entry><entry align="center" valign="middle">93.6</entry><entry align="center" valign="middle">2.4</entry><entry align="center" valign="middle">2.0</entry><entry align="center" valign="middle">0.157</entry><entry align="center" valign="middle">0.261</entry></row><row><entry align="center" valign="middle">54.9</entry><entry align="center" valign="middle">93.2</entry><entry align="center" valign="middle">2.2</entry><entry align="center" valign="middle">2.0</entry><entry align="center" valign="middle">0.156</entry><entry align="center" valign="middle">0.260</entry></row><row rowsep="0"><entry valign="middle">lyophilized</entry><entry align="center" valign="middle">55.4</entry><entry align="center" valign="middle">94.7</entry><entry align="center" valign="middle">2.6</entry><entry align="center" valign="middle">1.9</entry><entry align="center" valign="middle">0.089</entry><entry align="center" valign="middle">0.159</entry></row><row><entry valign="middle" /><entry align="center" valign="middle">54.8</entry><entry align="center" valign="middle">95.3</entry><entry align="center" valign="middle">2.3</entry><entry align="center" valign="middle">2.1</entry><entry align="center" valign="middle">0.077</entry><entry align="center" valign="middle">0.143</entry></row><row><entry morerows="3" align="center" valign="middle">15</entry><entry rowsep="0" valign="middle">spray-</entry><entry rowsep="0" align="center" valign="middle">60.2</entry><entry rowsep="0" align="center" valign="middle">93.9</entry><entry rowsep="0" align="center" valign="middle">2.2</entry><entry rowsep="0" align="center" valign="middle">2.1</entry><entry rowsep="0" align="center" valign="middle">0.153</entry><entry rowsep="0" align="center" valign="middle">0.254</entry></row><row><entry valign="middle">dried</entry><entry align="center" valign="middle">58.3</entry><entry align="center" valign="middle">94.2</entry><entry align="center" valign="middle">2.3</entry><entry align="center" valign="middle">2.0</entry><entry align="center" valign="middle">0.181</entry><entry align="center" valign="middle">0.274</entry></row><row rowsep="0"><entry valign="middle">lyophilized</entry><entry align="center" valign="middle">NM</entry><entry align="center" valign="middle">NM</entry><entry align="center" valign="middle">NM</entry><entry align="center" valign="middle">NM</entry><entry align="center" valign="middle">0.078</entry><entry align="center" valign="middle">0.127</entry></row><row><entry valign="middle" /><entry align="center" valign="middle">59.8</entry><entry align="center" valign="middle">94.5</entry><entry align="center" valign="middle">2.3</entry><entry align="center" valign="middle">2.1</entry><entry align="center" valign="middle">0.087</entry><entry align="center" valign="middle">0.141</entry></row><row rowsep="0"><entry morerows="3" rowsep="1" align="center" valign="middle">17</entry><entry morerows="1" rowsep="1" valign="middle">spray-dried</entry><entry align="center" valign="middle">62.4</entry><entry align="center" valign="middle">94.0</entry><entry align="center" valign="middle">2.4</entry><entry align="center" valign="middle">2.0</entry><entry align="center" valign="middle">0.177</entry><entry align="center" valign="middle">0.269</entry></row><row><entry align="center" valign="middle">61.5</entry><entry align="center" valign="middle">93.4</entry><entry align="center" valign="middle">2.2</entry><entry align="center" valign="middle">2.0</entry><entry align="center" valign="middle">0.186</entry><entry align="center" valign="middle">0.291</entry></row><row rowsep="0"><entry valign="middle">lyophilized</entry><entry align="center" valign="middle">52.3</entry><entry align="center" valign="middle">96.1</entry><entry align="center" valign="middle">2.4</entry><entry align="center" valign="middle">2.1</entry><entry align="center" valign="middle">0.087</entry><entry align="center" valign="middle">0.141</entry></row><row><entry valign="middle" /><entry align="center" valign="middle">58.6</entry><entry align="center" valign="middle">91.8</entry><entry align="center" valign="middle">2.2</entry><entry align="center" valign="middle">2.1</entry><entry align="center" valign="middle">0.083</entry><entry align="center" valign="middle">0.134</entry></row><row rowsep="0"><entry morerows="3" rowsep="1" align="center" valign="middle">19</entry><entry morerows="1" rowsep="1" valign="middle">spray-dried</entry><entry align="center" valign="middle">51.6</entry><entry align="center" valign="middle">81.8</entry><entry align="center" valign="middle">2.4</entry><entry align="center" valign="middle">1.8</entry><entry align="center" valign="middle">0.179</entry><entry align="center" valign="middle">0.279</entry></row><row><entry align="center" valign="middle">53.8</entry><entry align="center" valign="middle">86.8</entry><entry align="center" valign="middle">2.2</entry><entry align="center" valign="middle">2.0</entry><entry align="center" valign="middle">0.198</entry><entry align="center" valign="middle">0.300</entry></row><row rowsep="0"><entry valign="middle">lyophilized</entry><entry align="center" valign="middle">52.7</entry><entry align="center" valign="middle">97.2</entry><entry align="center" valign="middle">2.3</entry><entry align="center" valign="middle">2.0</entry><entry align="center" valign="middle">0.083</entry><entry align="center" valign="middle">0.126</entry></row><row><entry valign="middle" /><entry align="center" valign="middle">60.4</entry><entry align="center" valign="middle">94.7</entry><entry align="center" valign="middle">2.4</entry><entry align="center" valign="middle">2.1</entry><entry align="center" valign="middle">0.100</entry><entry align="center" valign="middle">0.142</entry></row></tbody></tgroup><tgroup cols="8" rowsep="0"><colspec colnum="1" colname="col1" colwidth="23mm" /><colspec colnum="2" colname="col2" colwidth="24mm" /><colspec colnum="3" colname="col3" colwidth="17mm" /><colspec colnum="4" colname="col4" colwidth="24mm" /><colspec colnum="5" colname="col5" colwidth="22mm" /><colspec colnum="6" colname="col6" colwidth="12mm" /><colspec colnum="7" colname="col7" colwidth="23mm" /><colspec colnum="8" colname="col8" colwidth="24mm" /><tbody><row><entry namest="col1" nameend="col8" align="justify">NM - not measured</entry></row></tbody></tgroup></table></tables>

Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as molecular weight, 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.

It is readily apparent to one skilled in the art that various embodiments and modifications can be made to the invention disclosed herein, without departing from the scope and spirit of the invention.

Following long-standing patent law, and as used herein, the use of the word "a" or "an" when used in conjunction with the term "comprising" in the claims and/or the specification may mean "one," but it is also consistent with the meaning of "one or more," "at least one," and "one or more than one" unless specifically noted.

Recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein.

It is contemplated that any method or composition described herein can be implemented with respect to any other method or composition described herein.

The use of the term "or" in the claims is used to mean "and/or" unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and "and/or."

Throughout this application, the term "about" is used to indicate that a value includes the standard deviation of error for the device or method being employed to determine the value. Further embodiments of the invention are also disclosed herein in the following numbered paragraphs: <ol id="ol0001"><li>1. A method of preparing a dry powder medicament with an improved pharmaceutic property, comprising the steps of: <ul id="ul0001" list-style="none" compact="compact"><li>providing a solution of a diketopiperazine;</li><li>providing a solution of an active agent;</li><li>forming particles of diketopiperazine; and</li><li>combining the diketopiperazine and the active agent; and thereafter</li><li>removing solvent by spray drying to obtain a dry powder, wherein the dry powder has an improved pharmaceutic property as compared to a dry powder obtained by removing solvent by lyophilization.</li></ul></li><li>2. The method of paragraph 1, wherein the improved pharmaceutic property is selected from the group consisting of improved stability of the active agent, increased density of the powder, and improved aerodynamic performance of the dry powder.</li><li>3. The method of paragraph 1, wherein the active agent is selected from the group consisting of insulin, calcitonin, parathyroid hormone 1-34, bioactive fragment of parathyroid hormone, octreotide, leuprolide, and RSV peptide, felbamate, cannabinoid antagonists and/or agonists, muscurinic antagonsits and/or agonistics, heparin, low molecular weight heparin, cromolyn, sildenafil, vardenafil, tadalafil, growth hormone, AZT, DDI, GCSF, lamotrigine, chorionic gonadotropin releasing factor, luteinizing release hormone, β-galactosidase, GLP-1, exendins 1-4, ghrelin, and fragments thereof.</li><li>4. The method of paragraph 1, wherein the active agent is a peptide or protein.</li><li>5. The method of paragraph 3, wherein the active agent is insulin or an analogue thereof.</li><li>6. The method of paragraph 5, wherein said insulin is within the range of about 3% to about 50% by weight of the dry powder formulation.</li><li>7. The method of paragraph 6, wherein said insulin is within the range of about 7% to about 19% by weight of the dry powder formulation.</li><li>8. The method of paragraph 7, wherein said insulin is at about 11 % by weight of the dry powder formulation.</li><li>9. The method of paragraph 7, wherein said insulin is at about 15% by weight of the dry powder formulation.</li><li>10. The method of paragraph 1, wherein said diketopiperazine is a diketopiperazine having the formula 2,5-diketo-3,6-di(4-X-aminobutyl)piperazine, wherein X is selected from the group consisting of succinyl, glutaryl, maleyl, and fumaryl.</li><li>11. The method of paragraph 10, wherein the diketopiperazine is fumaryl diketopiperazine.</li><li>12. The method of paragraph 1, wherein said improved pharmaceutic property is improved stability of the active agent of the particle.</li><li>13. The method of paragraph 1, wherein said improved pharmaceutic property is improved aerodynamic performance of the particle.</li><li>14. The method of paragraph 1, wherein said improved pharmaceutic property is increased density of the powder.</li><li>15. The method of paragraph 13, wherein said aerodynamic performance is measured by the percent respirable fraction on a cartridge fill.</li><li>16. The method of paragraph 15, wherein the percent respirable fraction is greater than about 40%.</li><li>17. The method of paragraph 15, wherein the percent respirable fraction is greater than about 50%.</li><li>18. The method of paragraph 15, wherein the percent respirable fraction is greater than about 60%.</li><li>19. A dry powder prepared according to the method of paragraph 1.</li><li>20. The dry powder of paragraph 19, wherein the active agent is insulin or an analogue thereof.</li><li>21. A method for delivering insulin to a patient in need thereof, comprising administering to the patient an effective amount of the dry powder of paragraph 19.</li><li>22. A method of preparing a dry powder medicament with an improved pharmaceutic property, comprising providing a diketopiperazine in solution; a step for forming particles comprising the diketopiperazine; and removing solvent by spray drying to obtain a dry powder, wherein the dry powder has an improved pharmaceutic property as compared to a dry powder obtained by removing solvent by lyophilization.</li><li>23. The method of paragraph 22, further comprising the step of loading the particle with an active agent prior to the solvent removal step.</li><li>24. A method of optimizing the aerodynamic performance of a diketopiperazine dry powder comprising the steps of: <ul id="ul0002" list-style="none" compact="compact"><li>precipitating a diketopiperazine from solution under a controlled temperature to form particles;</li><li>selecting a drying method based on said temperature; and</li><li>drying the particles.</li></ul></li><li>25. The method of paragraph 24, further comprising the step of loading the particles with an active agent.</li><li>26. The method of paragraph 24, wherein the diketopiperazine is fumaryl diketopiperazine, said controlled temperature is between about 15°C and about 18°C, and the selected drying method is spray drying.</li><li>27. The method of paragraph 26, wherein said controlled temperature is about 17°C.</li><li>28. The method of paragraph 24, wherein the diketopiperazine is fumaryl diketopiperazine, said controlled temperature is less than or equal to 13°C or greater than or equal to 19°C and the selected drying method is freeze drying.</li><li>29. The dry powder made by the method of paragraph 24.</li><li>30. The dry powder of paragraph 29, further comprising an active agent.</li><li>31. The dry powder of paragraph 30, wherein the active agent is insulin or an analogue thereof.</li></ol> Other objects, features and advantages of the present invention will become apparent from the detailed description provided herein. It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.