WO2008013955A2 - Sustained release formulations for pulmonary delivery - Google Patents

Abstract

Compositions comprising at least one pharmacologically active protein, and at least one charged polymer; wherein the composition comprises insoluble complexes of the charged polymer and precipitated pharmacologically active protein; and wherein the composition is in dry powder form and the dry powder comprises particles in which at least an outermost portion is amorphous. Methods of making and using are also provided.

Description

SUSTAINED RELEASE FORMULATIONS FOR PULMONARY DELIVERY

FIELD OF THE INVENTION

[0001J The present invention relates to pharmaceutical formulations and related methods. For example, in one or more embodiments, the present invention relates to formulations for pulmonary delivery, including sustained release formulations. Formulations of the invention may include a peptide, such as insulin, and a precipitating agent, such as a polymer. In some embodiments, the present invention includes insulin and a poly-amino acid, such as polylysine. Such components can be formulated to form an insoluble complex, which acts to delay absorption of the drug.

BACKGROUND OF THE INVENTION

[0002] Administration of pharmaceutical formulations of peptides traditionally has been performed by injection, thereby avoiding drug degradation by the gastrointestinal tract. Injection, however, is generally regarded as being less than desirable, because of the immediate discomfort at the injection site, as well as because of long-term tissue damage that can be caused by repeated injections to a common area. Besides injection, other routes of administration include transdermal, intranasal, and pulmonary delivery.

[0003] Delivery of therapeutics through pulmonary routes is particularly advantageous. This approach eliminates the need for needles, limits irritation to the skin and body mucosa (common side effects of transdermally, iontophoretically, and intranasally delivered drugs), and eliminates the need for nasal and skin penetration enhancers (typical components of intranasal and transdermal systems that often cause skin or membrane irritations/dermatitis). Pulmonary administration is also economically attractive, amenable to patient self-administration, and is often preferred by patients over other alternative modes of administration. [0004] However, pulmonary administration poses a number of difficulties that are not encountered in other routes of administration. For example, whereas intranasal or transdermal administration involves placing the drug to be absorbed in immediate or very close proximity to the actual point of absorption, pulmonary administration requires administering the drug several feet away from the actual point of absorption. Thus, a pulmonary formulation must survive a relatively long journey through the mouth, down the trachea, and into the lungs. If not properly formulated and delivered, the drug will not reach the site of absorption in the distal lungs, and availability is compromised.

[0005] The problem becomes even more complicated when a controlled or delayed release of the pulmonarily delivered drug is desired. A number of methods have been employed to control the release rate of drugs from pulmonary pharmaceutical compositions (see, e.g., Zeng, X. M., et al., "The controlled delivery of drugs to the lung," Int. J. Pharmaceutics 124, 149-164 (1995)). Examples of these methods include, for example, the use of liposomes or biodegradable microspheres, and modification of the drug so that the active form of the drug is not readily released. Another method is to include the drug in an insoluble complex. For example, the injectable sustained release insulin formulations often contain insulin in a crystallized form, which releases insulin more slowly than compositions comprising free insulin. The insulin crystals that exhibit a satisfactory sustained release profile in injectable compositions, however, are not suitable for pulmonary delivery, because the crystals are too large and deposit prematurely before they reach the deep lung.

[0006] Crystallization procedures that result in microcrystals have also been attempted to manufacture small crystals of insulin that can be delivered to the lung, but these procedures can be complicated and tedious (WO 01/00674). Simple and effective methods of preparing sustained release compositions for pulmonary delivery are thus still desirable, and the need for improvement continues.

[0007] U.S. Published Application No. 2003/0068277, which is incorporated herein by reference in its entirety, discloses a method for pulmonary delivery of therapeutic, prophylactic, and diagnostic agents to a patient wherein the agent is released in a sustained fashion. The method involves administering to the respiratory tract of a patient in need of treatment, prophylaxis, or diagnosis, an effective amount of particles comprising a polycationic complexing agent which is complexed with a therapeutic, prophylactic, or diagnostic agent or any combination thereof having a charge capable of complexing with the polycationic complexing agent.

SUMMARY OF THE EWENTION

[0008] The present invention provides, in some embodiments, compositions comprising at least one pharmacologically active protein, and at least one charged polymer; wherein the composition comprises insoluble complexes of the charged polymer and precipitated pharmacologically active protein; and wherein the composition is in dry powder form and the dry powder comprises particles in which at least an outermost portion is amorphous.

[00091 The present invention also provides, in some embodiments, pharmaceutical formulations, comprising particles having an amorphous at least outermost portion, the particles having a mass median aerodynamic diameter of less than 10 μm, the particles comprising insulin and at least one positively charged biopolymer.

[0010] Also provided by the invention are, in some embodiments, methods of preparing a pharmaceutical composition comprising: combining, in a liquid composition, at least one pharmacologically active protein with at least one charged polymer, to form a protein-polymer mixture; precipitating the pharmacologically active protein from the protein-polymer mixture; and drying the liquid composition to form particles having an amorphous at least outermost portion, the particles comprising insoluble complexes of precipitated protein and charged polymer.

[0011] In some embodiments, the invention provides methods of preparing a pharmaceutical composition comprising: adding to a liquid composition at least one pharmacologically active protein and at least one charged polymer, wherein the addition of the charged polymer results in precipitation of the pharmacologically active protein; and drying the liquid composition to form particles comprising an amorphous at least outermost portion, the particles comprising insoluble complexes of precipitated protein and charged polymer,

{0012] In some embodiments, the invention provides, methods of preparing a pharmaceutical composition comprising: adding to a liquid composition comprising at least one charged polymer, at least one pharmacologically active protein, wherein the pharmacologically active protein precipitates upon addition to the liquid composition; and drying the liquid composition to form particles comprising an amorphous at least outermost portion, the particles comprising insoluble complexes of precipitated protein and charged polymer.

[0013] The present invention also provides, in some embodiments, pharmaceutical formulations, comprising particles comprising an amorphous at least outermost portion, the particles having a mass median aerodynamic diameter of less than 10 μm, the particles comprising insulin and polylysine; which particles exhibit a rate of insulin release of less than or equal to about 40% in 5 hours, less than or equal to about 50% in 10 hours, less than or equal to about 60% in about 15 hours, and less than or equal to about 70% in about 20 hours, as measured by diafiltration.

[0014] Still further, the invention provides, in some embodiments, methods of reducing blood glucose level in an animal, comprising pulmonarily administering to the animal a pharmaceutical formulation for inhalation, the formulation comprising particles comprising an amorphous at least outermost portion, the particles having a mass median aerodynamic diameter of less than 10 μm, the particles comprising insulin and at least one positively charged biopolymer, wherein the administration results in a reduction in blood glucose level for a period of at least about 6 hours.

[0015] The present invention provides, in some embodiments, compositions comprising at least one pharmacologically active protein, and at least one charged polymer; wherein the composition comprises insoluble complexes of the charged polymer and precipitated pharmacologically active protein; and wherein the composition is in dry powder form and the dry powder comprises particles having a mass median diameter of less than 5 μm.

[0016] The invention also provides, in some embodiments, compositions comprising at least one pharmacologically active protein, and at least one charged polymer; wherein the composition comprises insoluble complexes of the charged polymer and precipitated pharmacologically active protein^ and wherein the composition is in dry powder form and the composition comprises no lipid.

[0017] In some embodiments, the invention provides methods of preparing a pharmaceutical composition comprising: combining, in a liquid composition, at least one pharmacologically active protein with at least one charged polymer, to form a protein- polymer mixture, wherein the liquid composition comprises no ethanol; precipitating the pharmacologically active protein from the protein-polymer mixture; and drying the liquid composition to form particles comprising insoluble complexes of precipitated protein and charged polymer.

[0018] Other features and advantages of the present invention will be set forth in the description of invention that follows, and will be apparent, in part, from the description or may be learned by practice of the invention. The invention will be realized and attained by the compositions, devices, and methods particularly pointed out in the written description and claims hereof.

BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The present invention is further described in the description of invention that follows, in reference to the noted plurality of non-limiting drawings, wherein:

[0020] Figure 1 diagramrnatically illustrates formation of protein precipitates by cations. [0021] Figure 2 shows percent insulin complexed with poly-L-lysine and zinc in the presence and absence of sodium citrate.

[0022] Figure 3 shows percent insulin complexed with poly-L-lysine as a function of pH.

[0023] Figure 4 shows cumulative drug release as a function of time from insulin-Zn and insulin-poly-L-lysine formulations.

[0024] Figure 5 shows the effect of poly-L-lysine molecular weight on insulin binding.

[0025] Figure 6 shows scanning electron micrographs of polylysine-insulin powders: (a) PLl, (b) PL2, (c) PL3, and (d) non-PLL insulin powder.

[0026] Figure 7 shows the Dose Past ET tube before and after shipping of PLL-insulin powders (PLl, PL2 & PL3) relative to a non-PLL containing, immediate release powder (IR-I) run in duplicate (a & b).

[0027] Figure 8 shows blood glucose levels after administration of PLL formulations to beagle dogs.

DETAILED DESCRIPTION OF THE INVENTION

[0028] Unless otherwise stated, a reference to a compound or component includes the compound or component by itself, as well as in combination with other compounds or components, such as mixtures of compounds.

[0029] As used herein, the singular forms "a," "an," and "the" include the plural reference unless the context clearly dictates otherwise. [0030] Except where otherwise indicated, all numbers expressing quantities of ingredients, 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 to be considered as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should be construed in light of the number of significant digits and ordinary rounding conventions.

[0031] Additionally, the recitation of numerical ranges within this specification is considered to be a disclosure of all numerical values and ranges within that range. For example, if a range is from about 1 to about 50, it is deemed to include, for example, 1, 7, 34, 46.1, 23.7, or any other value or range within the range.

[0032] Before further discussion, a definition of the following terms will aid in the understanding of the present invention.

Definitions

[0033] The terms used in this disclosure are defined as follows unless otherwise indicated. Standard terms are to be given their ordinary and customary meaning as understood by those of ordinary skill in the art, unless expressly defined herein.

[0034] "Amino acid" refers to any compound containing both an amino group and a carboxylic acid group. Although the amino group most commonly occurs at the position adjacent to the carboxy function, the amino group may be positioned at any location within the molecule. The amino acid may also contain additional functional groups, such as amino, thio, carboxyl, carboxamide, imidazole, etc. An amino acid may be synthetic or naturally occurring, and may be used in either its racemic or optically active (D-, or L-) form. [0035] A "sustained release composition" is a composition that releases the active component slowly over a relatively longer period of time than an "immediate release" composition. In general, the active component is released over at least about 3 hours, or at least about 4 hours, or at least about 5 hours, or at least about 6 hours, or at least about 8 hours.

[0036] A substance is "amorphous" if particles of the substance possess no uniform shapes. For example, a crystal substance is not amorphous.

[0037] A "protein" is an organic compound comprising one or more chains of amino acid residues linked by peptide bonds. As used herein, the term "protein" encompasses proteins of any length and derivatives thereof, such as peptides, glycopeptides, lipopeptides, glycoproteins, and lipoproteins. These terms may be used interchangeably herein.

[0038] A "pharmaceutical protein" is a protein useful for pharmaceutical purposes.

[0039] A "sustained plasma level" of a protein for a specified period of time means that the protein can be detected in the plasma for a duration specified. A protein can be detected by any methods for detecting such protein, e.g., immunological, biochemical, or functional methods. For example, insulin can be detected by enzyme-linked immunosorbent assay (ELISA), mass spectrometry, or determination of blood glucose levels.

[0040] A "precipitating agent" is a chemical compound or mixture of chemical compounds that is capable of precipitating a protein of interest when the agent is added to an aqueous solution of the protein. A protein can be precipitated by a variety of mechanisms, including, but not limited to, affinity precipitation, salting out, and isoelectric precipitation. [0041] An "insoluble complex" is a complex that does not completely dissolve in an excess of water, or a designated solvent, where specified, in an hour at 37 ⁰C. Typically, an insoluble complex has a solubility of less than about 30%, i.e., less than about 30% of the complex is dissolved in an hour. The insoluble complex can have a solubility of less than about 20%, or less than about 10%, or less than about 5%.

[0042] "Insulin" as used herein includes proinsulin and encompasses any purified isolated polypeptide having part or all of the primary structural conformation (i.e., contiguous series of amino acid residues) and at least one of the biological properties of naturally occurring insulin. In general, the term "insulin" is meant to encompass natural and synthetically-derived insulin including glycoforms thereof as well as agonists and analogs thereof, including polypeptides having one or more amino acid modifications (deletions, insertions, or substitutions) to the extent that they substantially retain at least 80% or more of the therapeutic activity associated with full length insulin. Generally, the polypeptides with amino acid modifications will retain at least a 50% amino acid sequence identity with a native insulin. The insulins of the present invention may be produced by any manner, including, but not limited to, pancreatic extraction, recombinant expression, and in vitro polypeptide synthesis.

[0043] A composition that is "suitable for pulmonary delivery" refers to a composition that is capable of being aerosolized and inhaled by a subject so that a portion of the aerosolized particles reaches the lungs to permit penetration into the alveoli. Such a composition may be considered "respirable" or "inhaleable."

[0044] An "aerosolized" composition contains liquid or solid particles that are suspended in a gas (typically air), typically as a result of actuation (or firing) of an inhalation device such as a dry powder inhaler, an atomizer, a metered dose inhaler, or a nebulizer.

[0045] A "jet nebulizer" is a system, such as a device, that forces compressed air through a solution of a drug so that a fine spray can be delivered to a facemask and inhaled. Nebulizers often are used to administer drugs to those who lack the ability to use a metered-dose or breath-activated inhaler.

[0046] A "dry powder inhaler" is a device that is loaded with a unit dosage of the drug in powder form. Generally, the inhaler is activated by taking a breath. For example, a capsule or blister is punctured and the powder is dispersed so that it can be inhaled in, e.g., a "Spinhaler" or "Rotahaler." "Turbohalers" are fitted with canisters that deliver measured doses of the drug in powder form.

[0047J A "metered dose inhaler" or "MDI" is a device that delivers a measured dose of a drug in the form of a suspension of extremely small liquid or solid particles, which is dispensed from the inhaler by a propellant under pressure. Such inhalers are placed into the mouth and depressed (activated) to release the drug as the individual takes a breath.

[0048] As used herein, the term "emitted dose" or "ED" refers to an indication of the delivery of dry powder from an inhaler device after an actuation or dispersion event from a powder unit or reservoir. ED is defined as the ratio of the dose delivered by an inhaler device to the nominal dose (i.e., the mass of powder per unit dose placed into a suitable inhaler device prior to firing). The ED is an experimentally determined amount, and may be determined using an in vitro device set-up which mimics patient dosing. To determine an ED value, as used herein, dry powder is placed into a Pulmonary Delivery System (PDS) device (Nektar Therapeutics), described in U.S. Patent No. 6,257,233, which is incorporated herein by reference in its entirety. The PDS device is actuated, dispersing the powder. The resulting aerosol cloud is then drawn from the device by vacuum (30 L/min) for 2.5 seconds after actuation, where it is captured on a tared glass fiber filter (Gelman, 47 mm diameter) attached to the device mouthpiece. The amount of powder that reaches the filter constitutes the delivered dose. For example, for a capsule containing 5 mg of dry powder that is placed into an inhalation device, if dispersion of the powder results in the recovery of 4 mg of powder on a tared filter as described above, then the ED for the dry powder composition is 80% (= 4 mg (delivered dose)/5 mg (nominal dose)). [0049] A composition in "dry powder form" is a powder composition that typically contains less than about 20% moisture.

[0050] As used herein, "mass median diameter" or "MMD" refers to the median diameter of a plurality of particles, typically in a polydisperse particle population, i.e., consisting of a range of particle sizes. MMD values as reported herein are determined by laser diffraction (Sympatec Helos, Clausthal-Zellerfeld, Germany), unless the context indicates otherwise. Typically, powder samples are added directly to the feeder funnel of the Sympatec RODOS dry powder dispersion unit. This can be achieved manually or by agitating mechanically from the end of a VIBRI vibratory feeder element. Samples are dispersed to primary particles via application of pressurized air (2 to 3 bar), with vacuum depression (suction) maximized for a given dispersion pressure. Dispersed particles are probed with a 632.8 nm laser beam that intersects the dispersed particles' trajectory at right angles. Laser light scattered from the ensemble of particles is imaged onto a concentric array of photomultiplier detector elements using a reverse-Fourier lens assembly. Scattered light is acquired in time-slices of 5 ms. Particle size distributions are back-calculated from the scattered light spatial/intensity distribution using an algorithm.

[0051] "Mass median aerodynamic diameter," or "MMAD," is a measure of the aerodynamic size of a dispersed particle. The aerodynamic diameter is used to describe an aerosolized powder in terms of its settling behavior, and is the diameter of a unit density sphere having the same settling velocity, in air, as the particle. The aerodynamic diameter encompasses particle shape, density, and physical size of a particle. As used herein, MMAD refers to the midpoint or median of the aerodynamic particle size distribution of an aerosolized powder determined by cascade impaction at standard conditions using a Pulmonary Delivery System (PDS) device (Nektar Therapeutics), described in U.S. Patent No. 6,257,233, which is incorporated herein by reference in its entirety, unless otherwise indicated. [0052] "Fine particle fraction" is the fraction of particles with an aerodynamic diameter that is less than 5 microns. Where specified, the fine particle fraction may also refer to the fraction of particles with an aerodynamic diameter that is less than 3.3 microns.

[0053] A "pharmaceutically acceptable excipient or carrier" refers to an excipient that may optionally be included in the compositions of the present invention. Preferred are excipients that can be taken into the lungs with no significant adverse toxicological effects to the subject, particularly to the lungs of the subject.

[0054] "Glass transition temperature (Tg)", as used herein, is the onset of a temperature range at which a composition changes from a glassy or vitreous state to a syrup or rubbery state. Generally, Tg is determined using differential scanning calorimetry (DSC). The standard designation for Tg is the temperature at which onset of the change of heat capacity (Cp) of the composition occurs upon scanning through the transition. The definition of Tg, however, can be arbitrarily defined as the onset, midpoint or endpoint of the transition. For purposes of the present invention, we will use the onset of the changes in Cp when using DSC. See the article entitled "Formation of Glasses from Liquids and Biopolymers" by C. A. Angell: Science, 267, 1924-1935 (Mar. 31, 1995) and the article entitled "Differential Scanning Calorimetry Analysis of Glass Transitions" by Jan P. Wolanczyk: Cryo-Letters, 10, 73-76 (1989). For detailed mathematical treatment, see "Nature of the Glass Transition and the Glassy State" by Gibbs and DiMarzio: Journal of Chemical Physics, 28, NO. 3, 373-383 (March, 1958). These articles are incorporated herein by reference.

[0055] A "glass-forming excipient" is an excipient that, when added to a composition, promotes glassy state formation of the composition.

[0056] The term "glass" or "glassy state," as used herein, refers to a liquid that has lost its ability to flow, i.e. it is a liquid with a very high viscosity, wherein the viscosity ranges from 10¹⁰ to 10¹⁴ pascal-seconds. It can be viewed as a metastable amorphous system in which the molecules have vibrational motion and reduced rotational motion, but have very slow (almost immeasurable) translational motion when compared to the liquid state. As a metastable system, it is stable for long periods of time when stored well below the glass transition temperature. Because glasses are not in a state of thermodynamic equilibrium, glasses stored at temperatures at or near the glass transition temperature relax to equilibrium upon storage and lose their high viscosity. The resultant rubbery or syrupy flowing liquid can lead to physical instability of the product. A solvent evaporation technique (U.S. Pat. No. 6,309,671) can be used to achieve a glassy state, as well as other processes that can produce a glassy state with acceptable Tg, for example, freeze drying followed by milling for micronization.

[0057] "Treating or ameliorating" a disease or medical condition means reducing or eliminating the symptoms of the disease or medical condition. In some embodiments, "treating or ameliorating" a disease or medical condition will be directed at addressing the cause of the disease or medical condition. Treating a disease may result in cure of the disease.

10058] The term "diabetes and related conditions" refers to diseases or medical conditions caused by the lack or inaction of, or inability to utilize, insulin. Diabetes and related conditions include type I and type II diabetes, particularly type I diabetes.

[0059] A "pharmacologically effective amount" or "'physiologically effective amount" of a pharmaceutical composition is an amount sufficient for the intended purpose of the pharmaceutical composition. For example, an effective amount of a pharmaceutical composition to treat or ameliorate diabetes is an amount sufficient to reduce or eliminate the symptoms of diabetes, for example, an amount that is needed to provide a desired level of insulin in the bloodstream to result in reduced blood glucose. The pharmacologically effective amount of a given pharmaceutical composition will vary with factors such as the nature of the active component in the composition, the route of administration, the size and species of the animal to receive the composition, and the purpose of the administration. The suitable amount can be readily determined by one skilled in the art based upon available literature and the information provided herein. [0060] As used herein, "measured by diafiltration" means that the dissolution kinetics of the powders are evaluated in vitro in a flow-through experimental setup using diafiltration cells (Amicon). Each formulation is weighed (usually n=2) and dispersed in 35 mL of release medium consisting of 10 mM phosphate buffer, pH 6.9, with 150 mM sodium chloride and 0.1 mM sodium citrate (added to accelerate dissolution of the complexes and thus, drug release) in order to achieve a drug concentration of 0.2 mg/mL. The mixture is loaded into a 50-mL capacity diafiltration cell, stirred continuously at 150 rpm, and maintained at 37 ± 2 ⁰C. The use of a pressurized reservoir allows the diafiltration medium (same as release medium) to flow through each cell at approximately 30 mL/hr. Membrane filters with a molecular weight cut-off of, e.g., 1 kDa ensure that the drug released from the formulations is contained within the cell to allow quantitative measurement. Aliquots of 1 mL are withdrawn from each of the cells at predetermined intervals. A volume of 200 μL from each of the samples is centrifuged and the supernatant is analyzed by HPLC for free (released) insulin. Total drug content is also measured at selected sampling points to perform mass balance calculations. This may be achieved by reducing pH to 2.0-2.5 by adding IN HCl to dissolve all drug, followed by analysis using an HPLC assay.

Insoluble Complexes of the Present Invention

[0061J The present invention generally relates to pharmaceutical formulations and related methods. For example, in one or more embodiments, the present invention relates to formulations for pulmonary delivery, including sustained release formulations. Sustained release formulations of the present invention include, but are not limited to, formulations including an insoluble complex of a pharmacologically active protein and a precipitating agent, which can be used to deliver the protein in a sustained release manner.

(0062] Briefly, by way of background, insulin has been found to form a crystal structure with zinc at a ratio of two or four zinc molecules to each insulin hexamer. Reference is made to U.S. Patent Application Publication 2005/0203002, which describes insulin-zinc complexes containing higher levels of zinc, and which are amorphous rather than crystalline. Without wishing to be limited to any particular theory of operation, it appears that the high levels of zinc (or other precipitating agents) in the compositions of 2005/0203002, and that the charged polymers of the present invention, lead to the formation of "kinetically irreversible precipitates," which dissociate much more slowly than the insulin-zinc crystals described above. The term "kinetically irreversible" does not mean that the precipitation process is not reversible. Rather, it means that dissociation, with subsequent dissolution, is a slow process that is kinetically controlled.

10063] The formation of the "kinetically irreversible" protein precipitates may be induced by a variety of ways. Examples include, but are not limited to: (a) affinity precipitation via complexation and specific interactions with appropriate cations, such as divalent cation salts (e.g., zinc, magnesium, calcium salts, both organic and inorganic); (b) salting out with Hofmeister series salts; (c) volume exclusion induced by addition of appropriate quantities of large polymers, such as polyethylene glycol (different molecular weights), dextran, etc.; and (d) isoelectric precipitation by a pH adjuster (acid or base).

[0064] Charged polymers are the exemplified precipitating agent in the present invention. While the nature of the precipitating mechanism is not entirely understood, it is believed that proteins form insoluble complexes with the precipitating polymers primarily through charge-based interactions. Therefore, the present invention can be used to deliver proteins without limitation to the function, size, overall charge, or physical properties of the proteins.

[0065] It should be understood that the compositions of the present invention generally include a protein and a biopolymer. As discussed in that context, the "biopolymer" may be a poly-amino acid. Poly-amino acids may be homopolymers or heteropolymers. Homopolymers of amino acids are generally "non-naturally occurring," meaning that they do not exist in nature and are generally human-made. Heteropolymers may be either naturally occurring or non-naturally occurring. Naturally occurring heteropolymers can be used in accordance with the present invention to the extent that they produce the desired effect of precipitation of the pharmacologically active protein. In view of these points, compositions including a pharmacologically active protein but excluding any other naturally occurring biopolymer are expressly contemplated.

[0066J Charged polymers of the present invention include both biopolymers and non- biopolymers. Non-biopolymers are polymers formed from monomers not normally found in nature; examples include charged polymers, such as polyacrylic acid. Biopolymers include, but are not limited to, poly-amino acids, such as polylysine, polyhistidine, and polyarginine. The choices are not limited to these three, and others can be chosen, based upon the desired charge of the polymer.

[0067] The choice of the polymer can be determined through trial and error. In some instances, specific polymers will work particularly well with specific proteins, and this determination can be made by the person of skill in the art. For example, precipitation of compounds having a net negative charge can be induced by including positively charged polymers, and vice versa. Thus, proteins with isoelectric points less than 7 can be precipitated with polymers having positive charges under the same solution conditions; proteins with isoelectric points higher than 7 can be precipitated with polymers having negative charges.

[0068] Compositions and formulations of the present invention may comprise, in addition to a pharmaceutically relevant protein and polymer, one or more additional precipitating agents. Such agents include, but are not limited to, agents that act in a similar manner to the present polymers — such as by affinity complexation and specific interaction. Such agents include divalent metal cations, such as zinc.

|0069] Precipitation with divalent metal cations, such as zinc, is thought to occur through the formation of insoluble complexes with the cations through predominantly surface-exposed histidine residues and, to a lesser extent, cysteine, tryptophan, and glutamic acid residues. Zinc ions selectively precipitate proteins from solution by coordinating the lone pair electrons of heteroatoms on the side chains of these amino acids. The majority of the precipitation at low protein concentrations is thought to occur due to the interprotein crosslinking between metal-ion and free surface groups. Precipitations using zinc are very rapid and are found to be kinetically irreversible. Protons in solution also compete with zinc ions for protein-binding sites; during the course of a metal precipitation there is usually a change in pH as protons are displaced from their coordination sites by the stronger binding zinc ions. This competitive binding offers possibilities for control of the kinetics of the dissolution process.

10070] Divalent metal ions include the transition metal, alkaline metal, and alkaline earth metal ions. Transitional metal ions such as zinc, copper, cobalt and iron are particularly suitable.

[0071] It should be noted that the insulin-polymer complexes of the present invention are typically amorphous rather than crystalline. However, depending on the precipitation process, small amounts of crystals may exist in the compositions. Furthermore, the amorphous complexes of the present invention may be mixed with crystalline insulin complexes to make compositions with a mixed release feature. Accordingly, the compositions of the present invention may contain about one of the following dry weight percentages of amorphous complexes: 0%, 1%, 2%, 4%, 6%, 8%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80% and 90%.

[0072] Still further, it should be noted that the primary insoluble complexes of the present invention, which are generally amorphous and glassy, can be included in formulations that ultimately are not amorphous or glassy. For example, the present insoluble complexes may be included in perforated microstructures, which are neither amorphous nor glassy.

[0073] The Hofmeister series salts include, but are not limited to, for example, thiocyanide, nitrate, fluoride, chloride, bromine, iodine, citrate, acetate, phosphate, and sulfate. Cations of these salts include, for example, calcium, magnesium, sodium, potassium, ammonium, tetramethyl ammonium, cesium, and aluminum. The relative ability of the salts to precipitate a given protein depends on the nature of the protein, pH, and temperature, and can be experimentally determined.

10074] The compositions of the present invention typically comprise the precipitating agent at a solid weight percentage of about 0.01% to about 95%, or about 10% to about 85%, or about 30% to about 75%, or about 50% to about 70%. In some embodiments, the composition may comprise no precipitating agent, because when the pharmaceutically useful protein is precipitated by salting out, volume exclusion, or isoelectric precipitation, the precipitating agent only facilitates precipitation, rather than forming part of the insoluble complex. Once the precipitates form, the precipitating agent may optionally be removed from the suspension, leaving no or only trace amount of the precipitating agent in the final composition. The addition of excipient may also change the percentage of the precipitating agent. Thus, depending upon the amount of excipient, a composition of the present invention will comprise the precipitating agent at a solid weight percentage of at least about one of the following: 0%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or greater.

[0075] Any protein useful as a therapeutic agent can be delivered in an insoluble complex as described herein. The protein may also contain non-peptide moieties such as carbohydrate or lipid. Thus, these pharmaceutically useful proteins ("pharmaceutical proteins") of the present invention may include drugs which act on the peripheral nerves, adrenergic receptors, cholinergic receptors, the skeletal muscles, the cardiovascular system, smooth muscles, the blood circulatory system, synaptic sites, neuroeffector junctional sites, endocrine and hormone systems, the immunological system, the reproductive system, the skeletal system, autacoid systems, the alimentary and excretory systems, the histamine system, and the central nervous system. Suitable proteins may be selected from, for example, hypnotics and sedatives, psychic energizers, tranquilizers, respiratory drugs, anticonvulsants, muscle relaxants, antiparkinson agents (dopamine antagonists), analgesics, anti-inflammatories, antianxiety drugs (anxiolytics), appetite suppressants, antimigraine agents, muscle contractants, anti-infectives (antibiotics, antivirals, antifungals, vaccines) antiarthritics, antimalarials, antiemetics, anepileptics, bronchodilators, cytokines, growth factors, anti-cancer agents, antithrombotic agents, antihypertensives, cardiovascular drugs, antiarrhythmics, antioxidants, anti-asthma agents, hormonal agents including contraceptives, sympathomimetics, diuretics, lipid regulating agents, antiandrogenic agents, antiparasitics, anticoagulants, neoplasties, antineoplastics, hypoglycemics, nutritional agents and supplements, growth supplements, antienteritis agents, vaccines, antibodies, diagnostic agents, and contrasting agents. The pharmaceutical protein may act locally or systemically.

[0076] Examples of pharmaceutical proteins suitable for use in this invention include but are not limited to calcitonin, erythropoietin (EPO), Factor VIII, Factor IX, ceredase, cerezyme, cyclosporin, granulocyte colony stimulating factor (GCSF), thrombopoietin (TPO), alpha- 1 proteinase inhibitor, elcatonin, granulocyte macrophage colony stimulating factor (GMCSF), growth hormone, human growth hormone (HGH), growth hormone releasing hormone (GHRH), interferon alpha, interferon beta, interferon gamma, interleukin-1 receptor, interleukin-2, interleukin-1 receptor antagonist, interleukin-3, interleukin-4, interleukin-6, luteinizing hormone releasing hormone (LHRH), insulin, factor IX insulin, pro-insulin, insulin analogues (e.g., mono-acylated insulin as described in U.S. Pat. No. 5,922,675), C-peptide, somatostatin, somatostatin analogs including octreotide, vasopressin, follicle stimulating hormone (FSH), insulin- like growth factor (IGF), insulintropin, macrophage colony stimulating factor (M-CSF), nerve growth factor (NGF), tissue growth factors, keratinocyte growth factor (KGF), glial growth factor (GGF), tumor necrosis factor (TNF), endothelial growth factors, parathyroid hormone (PTH), Ilb/IIIa inhibitor, alpha- 1 antitrypsin, phosphodiesterase (PDE) compounds, respiratory syncytial virus antibody, deoxyribonuclease (DNase), bactericidal/permeability increasing protein (BPI), anti-CMV antibody, and where applicable, analogues, agonists, antagonists, and inhibitors of the above, including the synthetic, native, glycosylated, unglycosylated, pegylated forms, and biologically active fragments and analogs thereof. [0077] Compositions and corresponding doses of the pharmaceutical protein will vary with the bioactivity of the protein employed. For example, injectable insulin is measured in USP Insulin Units; one unit (U) of insulin is equal to the amount required to reduce the concentration of blood glucose in a fasting rabbit to 0.45 mg/ml (2.5 mM). Typical concentrations of insulin preparations for injection range from 30-100 Units/mL, which is about 3.6 mg of insulin per mL. The amount of insulin required to achieve the desired physiological effect in a patient will vary not only with the particulars of the patient and the disease (e.g., type I vs. type II diabetes) but also with the strength and particular type of insulin used. For instance, dosage ranges for regular insulin (rapid acting) are from about 0.3 to 2 U insulin per kilogram of body weight per day. The compositions of the present invention are, in one aspect, effective to achieve in patients undergoing therapy a fasting blood glucose concentration between about 90 and 140 mg/dl and a postprandial value below about 250 mg/dl. The precise dosages can be determined by one skilled in the art when coupled with the pharmacodynamics and pharmacokinetics of the precise pharmaceutical composition employed for a particular route of administration, and can readily be adjusted in response to periodic glucose monitoring.

[0078] Individual dosages (on a per inhalation basis) for inhaleable insulin compositions are typically in the range of from about 0.5 mg to 15 mg insulin, where the desired overall dosage is typically achieved in about 1-10 breaths, and preferably in about 1 to 4 breaths. On average, the overall dose of insulin administered by inhalation per dosing session will range from about 10 U to about 400 U, with each individual dosage or unit dosage form (corresponding to a single inhalation) containing from about 5 U to 400 U.

[0079] When the present invention is used to deliver insulin by inhalation to the lung, the amount of insulin in the composition will be that amount necessary to deliver a therapeutically effective amount of insulin per unit dose to achieve at least one of the therapeutic effects of native insulin, i.e., the ability to control blood glucose levels to near normoglycemia. In practice, this will vary widely depending upon the particular insulin, its activity, the severity of the diabetic condition to be treated, the patient population, the stability of the composition, and the like.

[0080] The composition will generally contain, in terms of solid weight, anywhere from about 1% to about 99%, typically from about 2% to about 95%, and more typically from about 5% to about 85% of the pharmaceutical protein. The percentage of the pharmaceutical protein in the composition will also depend upon the relative amounts of excipients/additives contained in the composition. More specifically, the composition will typically contain at least about one of the following solid weight percentages of the pharmaceutical protein: 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more. Powder compositions may contain at least about 60%, e.g., about 60-100% by weight of the pharmaceutical protein. It is to be understood that more than one pharmaceutical protein may be incorporated into the compositions described herein. Furthermore, the composition may also contain more than one form of the pharmaceutical protein, for example two or more insulins.

[0081] The molar ratio of the precipitating agent to the pharmaceutical protein in the compositions of the present invention may range from about 1 : 50 to about 500: 1. The ratio is more generally from about 1 :20 to about 100: 1 , or from about 1 : 10 to about 50: 1 , or from about 1 :5 to about 20: 1. The ideal molar ratio of the precipitating agent to the pharmaceutical protein may be determined by a person of ordinary skill in the art, and will generally be about one of the following: 1: 10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1 :2, 1:1, 2:1, 3:1, 4:1, 5: 1, 6: 1, 7:1, 8:1, 9:1, 10:1, 15:1, 20:1, 30:1, 40:1, 50:1, 60:1, 70:1, 80:1, 90:1, 100:1 or greater.

[0082] The formulations of the present invention, when measured by diafiltration, exhibit a rate of insulin release of less than or equal to about 40% in 5 hours, less than or equal to about 50% in 10 hours, less than or equal to about 60% in about 15 hours, and less than or equal to about 70% in about 20 hours. In some embodiments, the formulations of the invention, when measured by diafiltration, exhibit a rate of insulin release of less than or equal to about 30% in 5 hours, less than or equal to about 40% in 10 hours, less than or equal to about 50% in about 15 hours, and less than or equal to about 60% in about 20 hours. In some embodiments, the formulations of the invention, when measured by diafiltration, exhibit a rate of insulin release of less than or equal to about 20% in 5 hours, less than or equal to about 30% in 10 hours, less than or equal to about 40% in about 15 hours, and less than or equal to about 50% in about 20 hours.

[0083] The compositions of the present invention generally result in a detectable plasma level of the pharmaceutically useful protein that sustains for at least about 4 hours, or at least about 6 hours, or at least about 7, 8, 9, 10, or 12 hours, or more. When compared to the crystalline insulin-zinc complexes containing 2 or 4 zinc molecules per each insulin hexamer, in one or more embodiments, the compositions of the present invention result in a duration of plasma insulin that is at least about 1.5 times, or at least about 2 times, or at least about 2.5 times, or at least about 3 times, that of the crystalline insulin-zinc composition.

[0084J It should be noted that while insulin is exemplified herein, and that while pharmaceutical proteins are described generically, the present invention is not limited to protein pharmaceuticals. The invention extends to any pharmaceutical that can form an "insoluble complex" according to the invention, when combined with a charged polymer in an appropriate manner.

[0085] One embodiment of the present invention provides compositions that contain no protamine. Protamines are a group of proteins isolated from fish, and are commonly used in insulin formulations to prolong duration (see, e.g., Vanbever R. et al., "Sustained release of insulin from insoluble inhaled particles," Drug Dev. Res. 48, 178-185, 1999). However, protamines, as well as protamine-insulin complexes, have been shown to be potentially immunogenic (Samuel T. et al., "Studies on the immunogenicity of protamines in humans and experimental animals by means of a micro-complement fixation test," Clin. Exp. Immunol. 33(2), 252-260 (1978); Kurtz A. B. et al., "Circulating IgG antibody to protamine in patients treated with protamine-insulins," Diabetologia. 25(4), 322-324 (1983)). Since the compositions of the present invention are capable of sustained release in the absence of protamine, the present invention provides the option of including no protamine, thereby avoiding the adverse reactions that may be caused by protamine.

[0086] While the use of liposomes is also commonly employed to sustain duration of drug effect, the present invention does not require the use of liposomes. Accordingly, other embodiments of the present invention provide compositions that contain no lipid in addition to the pharmaceutical protein. However, having noted the possibility that the present compositions exclude lipids or the use of liposomes, it is also noted that the primary particles of the present invention can be included into liposomal formulations, described in more detail below.

Excipients

[.0087] The composition may further comprise excipients, solvents, stabilizers, membrane penetration enhancers, etc., depending upon the particular mode of administration and dosage form. Examples include carbohydrate excipients, either alone or in combination with other excipients or additives. Representative carbohydrates for use in the compositions of the present invention include sugars, derivatized sugars such as alditols, aldonic acids, esterified sugars, and sugar polymers. Exemplary carbohydrate excipients suitable for use in the present invention include, for example, monosaccharides such as fructose, maltose, galactose, glucose, D-mannose, sorbose, and the like; disaccharides, such as lactose, sucrose, trehalose, cellobiose, and the like; polysaccharides, such as raffinose, melezitose, maltodextrins, dextrans, starches, and the like; and alditols, such as mannitol, xylitol, maltitol, lactitol, xylitol sorbitol (glucitol), pyranosyl sorbitol, myoinositol and the like. Preferred are non-reducing sugars, sugars that can form a substantially dry amorphous or glassy phase when combined with the composition of the present invention, and sugars possessing relatively high glass transition temperatures, or Tgs (e.g., Tgs greater than 4O⁰C, or greater than 50⁰C, or greater than 60°C, or greater than 70⁰C, or having Tgs of 80⁰C and above). Such excipients may be considered glass-forming excipients. [0088] Additional excipients include amino acids, peptides and particularly oligomers comprising 2-9 amino acids, or 2-5 mers, and polypeptides, all of which may be homo or hetero species. Representative amino acids include glycine (gly), alanine (ala), valine (val), leucine (leu), isoleucine (ile), methionine (met), proline (pro), phenylalanine (phe), tryptophan (trp), serine (ser), threonine (thr), cysteine (cys), tyrosine (tyr), asparagine (asp), glutamic acid (glu), lysine (lys), arginine (arg), histidine (his), norleucine (nor), and modified forms thereof. These amino acids and amino acid polymers may be included in addition to the precipitating polymer of the present invention.

[0089) Also useful as excipients in inhaleable compositions are di- and tripeptides containing two or more leucyl residues, as described in Nektar Therapeutics' International patent application WO 01/32144, incorporated herein by reference in its entirety.

[00901 Also preferred are di- and tripeptides having a glass transition temperature greater than about 40⁰C, or greater than 50⁰C, or greater than 60⁰C, or greater than 70⁰C.

[0091] Although less preferred due to their limited solubility in water, additional stability and aerosol performance-enhancing peptides for use in the present invention include 4-mers and 5-mers containing any combination of amino acids as described above. The 4-mer or 5-mer may comprise two or more leucine residues. The leucine residues may occupy any position within the peptide, while the remaining (i.e., non- leucyl) amino acids positions are occupied by any amino acid as described above, provided that the resulting 4-mer or 5-mer has a solubility in water of at least about 1 mg/ml. In some embodiments, the non-leucyl amino acids in a 4-mer or 5-mer are hydrophilic amino acids such as lysine, to thereby increase the solubility of the peptide in water.

[0092] Exemplary protein excipients include albumins such as human serum albumin (HSA), recombinant human albumin (rHA), gelatin, casein, hemoglobin, and the like. The compositions may also include a buffer or a pH-adjusting agent, typically but not necessarily a salt prepared from an organic acid or base. Representative buffers include organic acid salts of citric acid, ascorbic acid, gluconic acid, carbonic acid, tartaric acid, succinic acid, acetic acid, or phthalic acid. Other suitable buffers include Tris, tromethamine hydrochloride, borate, glycerol phosphate, and phosphate. Amino acids such as glycine are also suitable.

[0093] The compositions of the present invention may also include one or more additional polymeric excipients/additives, e.g., polyvinylpyrrolidones, derivatized celluloses such as hydroxymethylcellulose, hydroxyethylcellulose, and hydroxypropylmethylcellulose, Ficolls (a polymeric sugar), hydroxyethylstarch (HES), dextrates (e.g., cyclodextrins, such as 2-hydroxypropyl-β-cyclodextrin and sulfobutylether-β-cyclodextrin), polyethylene glycols, and pectin.

[0094J The compositions may further include flavoring agents, taste-masking agents, inorganic salts (e.g., sodium chloride), antimicrobial agents (e.g., benzalkonium chloride), sweeteners, antioxidants, antistatic agents, surfactants (e.g., polysorbates such as 'TWEEN 20" and "TWEEN 80," and pluronics such as F68 and F88, available from BASF), sorbitan esters, lipids (e.g., phospholipids such as lecithin and other phosphatidylcholines, phosphatidylethanolamines, although preferably not in liposomal form), fatty acids and fatty esters, steroids (e.g., cholesterol), and chelating agents (e.g., zinc and other such suitable cations). The use of certain di-substituted phosphatidylcholines for producing perforated microstructures (i.e., hollow, porous microspheres) is described in greater detail below. Other pharmaceutical excipients and/or additives suitable for use in the compositions according to the present invention are listed in "Remington: The Science & Practice of Pharmacy," 21^st ed., Williams & Williams, (2005), and in the "Physician's Desk Reference," 60th ed., Medical Economics, Montvale, N.J. (2006).

[0095J Compositions in accordance with the present invention may exclude penetration enhancers, which can cause irritation and are toxic at the high levels often necessary to provide substantial enhancement of absorption. Specific enhancers, which are typically absent from the compositions of the present invention, are the detergent-like enhancers such as deoxycholate, laureth-9, DDPC, glycocholate, and the fusidates. Certain enhancers, however, such as those that protect the pharmaceutical protein from enzyme degradation, e.g., protease and peptidase inhibitors such as alpha- 1 antiprotease, captropril, thiorphan, and the HIV protease inhibitors, may, in certain embodiments of the present invention, be incorporated in the composition of the present invention.

[0096] Generally, the pharmaceutical formulations of the present invention will contain from about 1% to about 99% by weight excipient, or from about 5%-98% by weight excipient, or from about 15-95% by weight excipient. In some embodiments, spray-dried formulations will contain from about 0-50% by weight excipient, or from 0-40% by weight excipient. In general, a high concentration of the pharmaceutical protein is desired in the final pharmaceutical formulation.

Preparing Dry Powders Comprising Insoluble Complexes

[0097] Protein precipitation and recovery operations have been traditionally used for protein and peptide purification during downstream processing. The term "salting out" is used to describe an operation in which a reagent is added to a protein solution, causing the formation of insoluble protein particles. In most applications, the intention is to recover the protein in either the native form or one, which is readily returned to the native state upon reconstitution. In one or more embodiments, the present invention uses the salting-out phenomenon to create insoluble complexes of protein, which then act as depots for the drug.

[0098] hi some embodiments of the present invention, the formation of precipitates can be described by electrostatic interactions between the drug (insulin), and the positively charged cations, as a reversible process that follows the scheme illustrated in Figure 1. The first equation describes the protein equilibria in solution. The prevalent species in solution depends on the environmental conditions, such as pH, concentration, presence, and concentration of specific salts, etc. It is this species that then contributes in the subsequent reactions.

[0099] As indicated by equilibrium II in Figure 1, when a salting-out agent is added in the solution, the primary species' solubility decreases until supersaturation is achieved. At this point, insoluble "primary precipitates" are formed. In these precipitates, many primary particles are interacting to form a network of floes. Dependent of their density, these floes may remain suspended or precipitate. Using the appropriate processing conditions, which reflect the energy input in the flocculation system (e.g., mixing type, speed, aging), the floes may break to form smaller, secondary particles, composed of the primary particles.

[010OJ Generally, the conditions of the composition shown in the precipitation and complexation reactions, schematically illustrated in Figure 1, are not critical. The pH of the solution typically ranges from about 4 to about 9, such as from about 5 to about 8. The concentration of the protein is typically less than 20 mg/ml, such as about 3 mg/ml to about 12 mg/ml. The concentration of the polymer is typically less than 20 mg/ml, such as about 3 mg/ml to about 12 mg/ml. The temperature typically ranges from about 10 ⁰C to about 40 ⁰C. The ideal conditions for any particular protein/polymer combination can be determined through routine experimentation.

[0101] Similarly, the choice of charged polymer is not critical. Any of a number of charged polymers can be used successfully, and the particular choice may be based upon empirical observations and experimentation. There are a number of polymers and biopolymers that carry multiple positive or negative charges and that can be formulated to precipitate proteins and peptides. Most of these polymers have been used in downstream operations for protein purification, but have not been used as described herein. It is anticipated that, in following the present teachings, one of skill in the art could use such polymers to achieve the results described herein. The use of other charged polymers and biopolymers not described herein is expressly contemplated as falling within the scope of the present invention. [0102] In some embodiments, the choice of polymer is poly-L-lysine (PLL), which is the polymer exemplified herein. PLL is a biodegradable polymer that undergoes hydrolytic degradation to form lysine molecules. PLL is also commercially available in different molecular weight ranges. For instance, the molecular weight of the charged polymers of the present invention are typically less than 150 kDa, less than 70 kDa, less than 30 kDa, or less than 15 kDa. Upon mixing with drug molecules containing opposite charges, it will result in the formation of drug- PLL complexes, which will precipitate out of solution following the scheme shown in Figure 1.

[0103] In some embodiments, a suspension comprising the aforementioned precipitates can be dried to form a dry formulation. In some embodiments, such formulations are spray dried to form a dry powder. Such dry powder formulations comprise the precipitated protein complexed to the charged polymer.

[0104] One embodiment of the present invention provides dry powder compositions suitable for pulmonary delivery. Dry powder compositions of the present invention may be prepared by any of a number of drying techniques, including by spray drying. Spray drying of the compositions is carried out, for example, as described generally in the "Spray Drying Handbook," 5^th ed., K. Masters, John Wiley & Sons, Inc., NY, N.Y. (1991), and in Platz, R., et al., International Patent Publication Nos. WO 97/41833 (1997) and WO 96/32149 (1996), the contents of which are incorporated herein by reference.

[0105] Suspensions comprising the insoluble complexes of the present invention can be spray-dried in a conventional spray drier, such as those available from commercial suppliers such as Niro A/S (Denmark), Buchi (Switzerland) and the like, resulting in a dispersible, dry powder. Desirable conditions for spray drying will vary depending upon the composition components, and are generally determined experimentally. The gas used to spray dry the material is typically air, although inert gases such as nitrogen or argon are also suitable. Moreover, the temperature of both the inlet and outlet of the gas used to dry the sprayed material is such that it does not cause degradation of the pharmaceutical protein in the sprayed material. Such temperatures are typically determined experimentally, although in general the inlet temperature will range from about 50⁰C to about 200⁰C, while the outlet temperature will range from about 3O⁰C to about 15O⁰C. Parameters may include atomization pressures ranging from about 20-150 psi, or from about 30-100 psi. Typically the atomization pressure employed will be one of the following (psi): 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, or 120 or above.

[0106] Respirable compositions of the present invention having the features described herein may also be produced by drying certain composition components, which result in formation of a perforated microstructure powder as described in WO 99/16419, assigned to Alliance Pharmaceutical Corporation, the entire contents of which are incorporated by reference herein. The perforated microstructure powders typically comprise spray-dried, hollow microspheres having a relatively thin porous wall defining a large internal void. The perforated microstructure powders may be dispersed in a selected suspension media (such as a non-aqueous and/or fluorinated blowing agent) to provide stabilized dispersions prior to drying. The use of relatively low density perforated (or porous) microstructures or micropaiticulates significantly reduces attractive forces between the particles, thereby lowering the shear forces, increasing the flowability and dispersibility of the resulting powders, and reducing the degradation by flocculation, sedimentation or creaming of the stabilized dispersions thereof.

[0107] Alternatively, powders may be prepared by lyophilization, vacuum drying, spray freeze drying, super critical fluid processing (e.g., as described in Hanna, et al., U.S. Pat. No. 6,063,138), air drying, or other forms of evaporative drying.

[0108] In yet another approach, dry powders may be prepared by agglomerating the powder components, sieving the materials to obtain agglomerates, spheronizing to provide a more spherical agglomerate, and sizing to obtain a uniformly sized product, as described, e.g., in Ahlneck, C, et al., International PCT Publication No. WO 95/09616, 1995, incorporated herein by reference. [0109] Dry powders may also be prepared by blending, grinding, sieving, or jet milling composition components in dry powder form.

[0110] Once formed, the dry powder compositions are preferably maintained under dry (i.e., relatively low humidity) conditions during manufacture, processing, and storage. Irrespective of the drying process employed, the process will preferably result in ^• inhaleable, highly dispersible particles comprising the insoluble complexes of the present invention.

[0111] In one or more embodiments, powders of the present invention may be characterized by several features, most notably, (i) consistently high dispersibilities, which are maintained, even upon storage, (ii) small aerodynamic particles sizes (MMADs), (iii) improved fine particle dose values, i.e., powders having particles sized less than 10 microns, all of which contribute to the improved ability of the powder to penetrate to the tissues of the lower respiratory tract (i.e., the alveoli) for delivery to the systemic circulation. These physical characteristics of the inhaleable powders of the present invention, to be described more fully below, play a role in maximizing the efficiency of aerosolized delivery of such powders to the deep lung.

[0112] Dry powders of the present invention are typically composed of aerosolizable particles effective to penetrate into the lungs. As shown in Example 1 , the amorphous insulin-zinc complexes of the present invention have a much smaller diameter (about 1 μm) than the insulin-zinc crystals (about 20 μm), and are thus superior for pulmonary delivery. The particles of the present invention may generally have a mass median diameter (MMD), or volume median geometric diameter (VMGD), or mass median envelope diameter (MMED), or a mass median geometric diameter (MMGD), of less than about 20 μm, or less than about 10 μm, or less than about 7.5 μm, or less than about 4 μm, or less than about 3.3 μm, and usually are in the range of 0.1 μm to 5 μm in diameter. Preferred powders are composed of particles having an MMD, VMGD, MMED, or MMGD from about 1 to 5 μm. In some cases, the powder will also contain non-respirable carrier particles such as lactose, where the non-respirable particles are typically greater than about 40 microns in size.

[0113] The powders of the present invention may also be characterized by an aerosol particle size distribution — mass median aerodynamic diameter (MMAD) — typically having MMADs less than about 10 μm, such as less than 5 μm, less than 4.0 μm, less than 3.3 μm, or less than 3 μm. The mass median aerodynamic diameters of the powders will typically range from about 0.1-5.0 μm, or from about 0.2-5.0 μm MMAD, or from about 1.0-4.0 μm MMAD, or from about 1.5 to 3.0 μm. Small aerodynamic diameters may be achieved by a combination of optimized spray drying conditions and choice and concentration of excipients.

[0114] The powders of the present invention may also be characterized by their densities. The powder will generally possess a bulk density from about 0.1 to 10 g/cubic centimeter, or from about 0.1-2 g/cubic centimeter, or from about 0.15-1.5 g/cubic centimeter. In one embodiment of the present invention, the powders have big and fluffy particles with a density of less than about 0.4 g/cubic centimeter and an MMD between 5 and 30 microns. It is worth noting that the relationship of diameter, density and aerodynamic diameter can be determined by the following formula (Gonda, "Physico-chemical principles in aerosol delivery," in Topics in Pharmaceutical Sciences 1991, Crommelin, D.J. and K.K. Midha, Eds., Medpharm Scientific Publishers, Stuttgart, pp. 95-117, 1992).

[0115] The powders may have a moisture content below about 20% by weight, usually below about 10% by weight, or below about 5% by weight. Such low moisture- containing solids tend to exhibit a greater stability upon packaging and storage.

[0116] Additionally, the spray drying methods and stabilizers described herein are generally effective to provide highly dispersible compositions. Generally, the emitted dose (ED) of these powders is greater than 30%, and usually greater than 40%. In some 16939

embodiments, the ED of the powders of the present invention is greater than 50%, 60%, 70%, or higher.

[0117] A particular characteristic which usually relates to improved dispersibility and handling characteristics is the^' product rugosity. Rugosity is the ratio of the specific area (e.g., as measured by BET, molecular surface adsorption, or other conventional technique) and the surface area calculated from the particle size distribution (e.g., as measured by centrifugal sedimentary particle size analyzer, Horiba Capa 700) and particle density (e.g., as measured by pycnometry), assuming non-porous spherical particles. Rugosity may also be measured by air permeametry. If the particles are known to be generally nodular in shape, as is the case in spray drying, rugosity is a measure of the degree of convolution or folding of the surface. This may be verified for powders made by the present invention by SEM analysis. A rugosity of 1 indicates that the particle surface is spherical and non-porous. Rugosity values greater than 1 indicate that the particle surface is non-uniform and convoluted to at least some extent, with higher numbers indicating a higher degree of non-uniformity. For the powders of the present invention, it has been found that particles may have a rugosity of at least about 2, such as at least about 3, at least about 4, or at least about 5, and may range from 2 to 10, such as from 4 to 8, or from 4 to 6.

[0118] The drying operation may be controlled to provide dried particles having particular characteristics, such as a rugosity above 2, as discussed above. Rugosities above 2 may be obtained by controlling the drying rate so that a viscous layer of material is rapidly formed on the exterior of the droplet. Thereafter, the drying rate should be sufficiently rapid so that the moisture is removed through the exterior layer of material, resulting in collapse and convolution of the outer layer to provide a highly irregular outer surface. The drying should not be so rapid, however, that the outer layer of material is ruptured. The drying rate may be controlled based on a number of variables, including the droplet size distribution, the inlet temperature of the gas stream, the outlet temperature of the gas stream, the inlet temperature of the liquid droplets, and the manner in which the atomized spray and hot drying gas are mixed. [0119J In some embodiments of the invention, powder surface area, measured by nitrogen adsorption, typically ranges from about 6 m²/g to about 13 m²/g, such as from about 7 m²/g to about 10 m²/g. The particles often have a convoluted "raisin" structure rather than a smooth spherical surface.

[0120] A particularly preferred embodiment of the present invention is one where at least the outermost regions, including the outer surface, of the powder particles are in an amorphous glassy state. It is thought that when the particles have a high T_g material at their surfaces, the powder will be able to take up considerable amounts of moisture before lowering the T_g to the point of instability (T_g -T₅ of less than about 10⁰C).

[0121] The compositions described herein typically possess good stability with respect to both chemical stability and physical stability, i.e., aerosol performance over time. Generally, with respect to chemical stability, the pharmaceutical protein contained in the composition will degrade by no more than about 10% upon spray drying. That is to say, the powder will generally possess at least about 90%, or about 95%, or at least about 97% or greater of the intact pharmaceutical protein.

[0122] With respect to aerosol performance, compositions of the present invention are generally characterized by a drop in emitted dose of no more than about 20%, or no more than about 15%, or no more than about 10%, when stored under ambient conditions for a period of three months.

Perforated Microstructures Comprising Insoluble Complexes

[0123] In addition to formulating as the dry powders discussed above, the insoluble complexes formed according to the present invention can alternatively be formulated into perforated microstructures. Such microstructures, and methods of their manufacture, are described in U.S. Patent No. 6,565,885, the entire disclosure of which is incorporated herein by reference. [0124J Briefly, such perforated microstructures generally comprise a structural matrix that exhibits, defines, or comprises voids, pores, defects, hollows, spaces, interstitial spaces, apertures, perforations, or holes. The absolute shape (as opposed to the morphology) of the perforated microstructure is generally not critical and any overall configuration that provides the desired characteristics is contemplated as being within the scope of the invention. Accordingly, some embodiments comprise approximately microspherical shapes.

[0125] With respect to the composition of the structural matrix defining the perforated microstructures, they may be formed of any material that possesses physical and chemical characteristics that are compatible with any incorporated active agents. While a wide variety of materials may be used to form the particles, in some pharmaceutical embodiments, the structural matrix is associated with, or comprises, a surfactant such as phospholipid or fluorinated surfactant. Although not required, the incorporation of a compatible surfactant can improve powder flowability, increase aerosol efficiency, improve dispersion stability, and facilitate preparation of a suspension. It will be appreciated that, as used herein, the terms "structural matrix" or "microstructure matrix" are equivalent and shall be held to mean any solid material forming the perforated microstructures which define a plurality of voids, apertures, hollows, defects, pores, holes, fissures, etc. that provide the desired characteristics. In some embodiments, the perforated microstructure defined by the structural matrix comprises a spray dried hollow porous microsphere incorporating at least one surfactant. It will further be appreciated that, by altering the matrix components, the density of the structural matrix may be adjusted. Finally, as will be discussed in further detail below, the perforated microstructures would comprise at least one active or bioactive agent, which in the present invention, would take the form of an insoluble complex.

[0126] The perforated microstructures may include one or more pharmaceutically acceptable excipients. Examples of pharmaceutically acceptable excipients include, but US2007/016939

are not limited to, lipids, metal ions, surfactants, amino acids, carbohydrates, buffers, salts, polymers, and the like, and combinations thereof.

[0127] Examples of lipids include, but are not limited to, phospholipids, glycolipids, ganglioside GMl, sphingomyelin, phosphatidic acid, cardiolipin; lipids bearing polymer chains such as polyethylene glycol, chitin, hyaluronic acid, or polyvinylpyrrolidone; lipids bearing sulfonated mono-, di-, and polysaccharides; fatty acids such as palmitic acid, stearic acid, and oleic acid; cholesterol, cholesterol esters, and cholesterol hemisuccinate.

[0128] In one or more embodiments, the phospholipid comprises a saturated phospholipid, such as one or more phosphatidylcholines. Exemplary acyl chain lengths are 16:0 and 18:0 (i.e., palmitoyl and stearoyl). The phospholipid content may be determined by the active agent activity, the mode of delivery, and other factors. Phospholipids from both natural and synthetic sources may be used in varying amounts. When phospholipids are present, the amount is typically sufficient to coat the active agent(s) with at least a single molecular layer of phospholipid. In general, the phospholipid content ranges from about 5 wt% to about 99.9 wt%, such as about 20 wt% to about 80 wt%.

10129] Generally, compatible phospholipids comprise those that have a gel to liquid crystal phase transition greater than about 40⁰C, such as greater than about 60⁰C, or greater than about 80⁰C. The incorporated phospholipids may be relatively long chain (e.g., C^-C₂₂) saturated lipids. Exemplary phospholipids useful in the disclosed stabilized preparations include, but are not limited to, phosphoglycerides such as dipalmitoylphosphatidylcholine, distearoylphosphatidylcholine, diarachidoylphosphatidylcholine, dibehenoylphosphatidylcholine, diphosphatidyl glycerols, short-chain phosphatidylcholines, hydrogenated phosphatidylcholine, E- 100-3 (available from Lipoid KG, Ludwigshafen, Germany), long-chain saturated phosphatidylethanolamines, long-chain saturated phosphatidylserines, long-chain saturated phosphatidylglycerols, long-chain saturated phosphatidylinositols, phosphatidic acid, phosphatidylinositol, and sphingomyelin.

[0130] Examples of metal ions include, but are not limited to, divalent cations, including calcium, magnesium, zinc, iron, and the like. For instance, when phospholipids are used, the pharmaceutical composition may also comprise a polyvalent cation, as disclosed in WO 01/85136 and WO 01/85137, which are incorporated herein by reference in their entireties. The polyvalent cation may be present in an amount effective to increase the melting temperature (T_m) of the phospholipid such that the pharmaceutical composition exhibits a T_m that is greater than its storage temperature (T_s) by at least about 20⁰C, such as at least about 40⁰C. The molar ratio of polyvalent cation to phospholipid may be at least about 0.05:1, such as about 0.05:1 to about 2.0:1 or about 0.25:1 to about 1.0:1. An example of the molar ratio of polyvalent cation:phospholipid is about 0.50: 1. When the polyvalent cation is calcium, it may be in the form of calcium chloride. Although metal ion, such as calcium, is often included with phospholipid, none is required.

[0131] As noted above, the pharmaceutical composition may include one or more surfactants. For instance, one or more surfactants may be in the liquid phase with one or more being associated with insoluble complexes of the composition. By "associated with," it is meant that the pharmaceutical compositions may incorporate, adsorb, absorb, be coated with, or be formed by the surfactant. Surfactants include, but are not limited to, fiuorinated and nonfluorinated compounds, such as saturated and unsaturated lipids, nonionic detergents, nonionic block copolymers, ionic surfactants, and combinations thereof. It should be emphasized that, in addition to the aforementioned surfactants, suitable fiuorinated surfactants are compatible with the teachings herein and may be used to provide the desired preparations.

[0132] Examples of nonionic detergents include, but are not limited to, sorbitan esters including sorbitan trioleate (Span™ 85), sorbitan sesquioleate, sorbitan monooleate, sorbitan monolaurate, polyoxyethylene (20) sorbitan monolaurate, and polyoxyethylene (20) sorbitan monooleate, oleyl polyoxyethylene (2) ether, stearyl polyoxyethylene (2) ether, lauryl polyoxyethylene (4) ether, glycerol esters, and sucrose esters. Other suitable nonionic detergents can be easily identified using McCutcheon's Emulsifiers and Detergents (McPublishing Co., Glen Rock, New Jersey), which is incorporated by reference herein in its entirety.

[0133] Examples of block copolymers include, but are not limited to, diblock and triblock copolymers of polyoxyethylene and polyoxypropylene, including poloxamer 188 (Pluronic™ F-68), poloxamer 407 (Pluronic™ F- 127), and poloxamer 338. Examples of ionic surfactants include, but are not limited to, sodium sulfosuccinate, and fatty acid soaps.

[0134] Examples of amino acids include, but are not limited to, hydrophobic amino acids. Use of amino acids as pharmaceutically acceptable excipients is known in the art as disclosed in WO 95/31479, WO 96/32096, and WO 96/32149, which are incorporated herein by reference.

[0135J Examples of carbohydrates include, but are not limited to, monosaccharides, disaccharides, and polysaccharides. For example, monosaccharides such as dextrose (anhydrous and monohydrate), galactose, mannitol, D-mannose, sorbitol, sorbose, and the like; disaccharides such as lactose, maltose, sucrose, trehalose, and the like; trisaccharides such as raffinose and the like; and other carbohydrates such as starches (hydroxyethylstarch), cyclodextrins and maltodextrins.

[0136] Examples of buffers include, but are not limited to, tris or citrate.

[0137] Examples of acids include, but are not limited to, carboxylic acids.

[0138] Examples of salts include, but are not limited to, sodium chloride, salts of carboxylic acids, (e.g., sodium citrate, sodium ascorbate, magnesium gluconate, sodium gluconate, tromethamine hydrochloride, etc.), ammonium carbonate, ammonium acetate, ammonium chloride, and the like.

J0139J Examples of organic solids include, but are not limited to, camphor, and the like.

[0140] The pharmaceutical composition of one or more embodiments of the present invention may also include a biocompatible, such as biodegradable polymer, copolymer, or blend or other combination thereof. In this respect useful polymers comprise polylactides, polylactide-glycolides, cyclodextrins, polyacrylates, methylcellulose, carboxymethylcellulose, polyvinyl alcohols, polyanhydrides, polylactams, polyvinyl pyrrolidones, polysaccharides (dextrans, starches, chitin, chitosan, etc.), hyaluronic acid, proteins, (albumin, collagen, gelatin, etc.). Those skilled in the art will appreciate that, by selecting the appropriate polymers, the delivery efficiency of the composition and/or the stability of the dispersions may be tailored to optimize the effectiveness of the active agent(s).

[0141] Besides the above mentioned pharmaceutically acceptable excipients, it may be desirable to add other pharmaceutically acceptable excipients to the pharmaceutical composition to improve powder rigidity, production yield, emitted dose and deposition, shelf-life, and patient acceptance. Such optional pharmaceutically acceptable excipients include, but are not limited to: coloring agents, taste masking agents, buffers, hygroscopic agents, antioxidants, and chemical stabilizers. Further, various pharmaceutically acceptable excipients may be used to provide structure and form to the powder compositions (e.g., latex particles). In this regard, it will be appreciated that the rigidifying components can be removed using a post-production technique such as selective solvent extraction.

[0142] The pharmaceutical compositions may also include mixtures of pharmaceutically acceptable excipients. For instance, mixtures of carbohydrates and amino acids are within the scope of the present invention. [0143] The matrix material may comprise a hydrophobic or a partially hydrophobic material. For example, the matrix material may comprise a lipid, such as a phospholipid, and/or a hydrophobic amino acid, such as leucine or tri-leucine. Examples of phospholipid matrices are described in WO 99/16419, WO 99/16420, WO 99/16422, WO 01/85136, and WO 01/85137 and in U.S. Patent Nos. 5,874,064; 5,855,913; 5,985,309; and 6,503,480, and in copending and co-owned U.S. Application No. 10/750,934, filed on December 31, 2003, all of which are incorporated herein by reference in their entireties. Examples of hydrophobic amino acid matrices are described in U.S. Patent Nos. 6,372,258 and 6,358,530, and in U.S. Application Publication No. 2002/0177562, which are incorporated herein by reference in their entireties. When phospholipids are utilized as the matrix material, the pharmaceutical composition may also comprise a polyvalent cation, as disclosed in WO 01/85136 and WO 01/85137, which are incorporated herein by reference in their entireties.

[0144] According to another embodiment, release kinetics of the active agent(s) containing composition is controlled. According to one or more embodiments, the compositions of the present invention provide immediate release of the insoluble complexes. Alternatively, the compositions of other embodiments of the present invention may be provided as non-homogeneous mixtures of active agent incorporated into a matrix material and unincorporated active agent in order to provide desirable release rates of insoluble complex. According to this embodiment, active agents formulated using the emulsion-based manufacturing process of one or more embodiments of the present invention have utility in immediate release of the insoluble complex when administered to the respiratory tract. Rapid release is facilitated by: (a) the high specific surface area of the low density porous powders; (b) the small size of the insoluble complex that are incorporated therein, and; (c) the low surface energy of the powders.

10145] Alternatively, it may be desirable to engineer the powder matrix so that extended release of the insoluble complex is effected. This may be particularly desirable when sustained release is desired. For example, the nature of the phase behavior of phospholipid molecules is influenced by the nature of their chemical structure and/or preparation methods in spray-drying feedstock and drying conditions and other composition components utilized. In the case of spray-drying of active agent(s) solubilized within a small unilamellar vesicle (SUV) or multilamellar vesicle (MLV), the active agent(s) are encapsulated within multiple bilayers and are released over an extended time.

[0146] In contrast, spray-drying of a feedstock comprised of emulsion droplets and insoluble complex in accordance with the teachings herein leads to a phospholipid matrix with less long-range order, thereby facilitating rapid release of the insoluble complex. While not being bound to any particular theory, it is believed that this is due in part to the fact that the insoluble complexes are never formally encapsulated in the phospholipid, and the fact that the phospholipid is initially present on the surface of the emulsion droplets as a monolayer (not a bilayer as in the case of liposomes). The spray-dried powders prepared by the emulsion-based manufacturing process of one or more embodiments of the present invention often have a high degree of disorder. Also, the spray-dried powders typically have low surface energies, where values as low as 20 mN/m have been observed for spray-dried DSPC powders (determined by inverse gas chromatography). Small angle X-ray scattering (SAXS) studies conducted with spray- dried phospholipid powders have also shown a high degree of disorder for the lipid, with scattering peaks smeared out, and length scales extending in some instances only beyond a few nearest neighbors.

[0147] It should be noted that a matrix having a high gel to liquid crystal phase transition temperature may not be sufficient in itself to achieve sustained release of the insoluble complexes. Having sufficient order for the bilayer structures is also important for achieving sustained release. To facilitate rapid release, an emulsion-system of high porosity (high surface area), and minimal interaction between the insoluble complexes and phospholipid may be used. The pharmaceutical composition formation process may also include the additions of other composition components (e.g., small polymers such as Pluronic F-68; carbohydrates, salts, hydrotropes) to break the bilayer structure are also contemplated.

[0148] To achieve a sustained release, incorporation of the phospholipid in bilayer form may be used, especially if the insoluble complex is encapsulated therein. In this case increasing the T_m of the phospholipid may provide benefit via incorporation of divalent counterions or cholesterol. As well, increasing the interaction between the phospholipid and drug substance via the formation of ion-pairs (negatively charged active + stearylamine, positively charged active + phosphatidylglycerol) would tend to decrease the dissolution rate. If the active is amphiphilic, surfactant/surfactant interactions may also slow active dissolution.

[0149] The addition of divalent counterions (e.g., calcium or magnesium ions) to long- chain saturated phosphatidylcholines results in an interaction between the negatively charged phosphate portion of the zwitterionic headgroup and the positively charged metal ion. This results in a displacement of water of hydration and a condensation of the packing of the phospholipid lipid headgroup and acyl chains. Further, this results in an increase in the T_m of the phospholipid. The decrease in headgroup hydration can have profound effects on the spreading properties of spray-dried phospholipid powders on contact with water. A fully hydrated phosphatidylcholine molecule will diffuse very slowly to a dispersed crystal via molecular diffusion through the water phase. The process is exceedingly slow because the solubility of the phospholipid in water is very low (about 10^~10 mol/L for DPPC). The "dry" phospholipid powders according to one or more embodiments of this invention can spread rapidly when contacted with an aqueous phase, thereby coating dispersed insoluble complexes without the need to apply high energies.

[0150] For example, upon reconstitution, the surface tension of spray-dried DSPC/Ca mixtures at the air/water interface decreases to equilibrium values (about 20 mN/m) as fast as a measurement can be taken. In contrast, liposomes of DSPC decrease the surface tension (about 50 mN/m) very little over a period of hours, and it is likely that this reduction is due to the presence of hydrolysis degradation products such as free fatty acids in the phospholipid. Single-tailed fatty acids can diffuse much more rapidly to the air/water interface than can the hydrophobic parent compound. Hence, the addition of calcium ions to phosphatidylcholines can facilitate the rapid encapsulation of insoluble complexes more rapidly and with lower applied energy.

[0151] In another embodiment, the pharmaceutical composition comprises low density powders achieved by co-spray-drying insoluble complexes with a perfluorocarbon-in- water emulsion. The insoluble complexes may be formed by precipitation and may, e.g., range in size from about 45 μm to about 80 μm. Examples of perfluorocarbons include, but are not limited to, perfluorohexane, perfluorooctyl bromide, perfluorooctyl ethane, perfluorodecalin, and perfluorobutyl ethane.

[0152] In accordance with the teachings herein, the powder compositions may be provided in a "dry" state. That is, in one or more embodiments, the powders will possess a moisture content that allows the powder to remain chemically and physically stable during storage at ambient or reduced temperature and remain dispersible. In this regard, there is little or no change in primary powder size, content, purity, and aerodynamic powder size distribution.

[0153] In such embodiments, the moisture content of the powders is typically less than about 10 wt%, such as less than about 6 wt%, less than about 3 wt%, or less than about 1 wt%. The moisture content is, at least in part, dictated by the composition and is controlled by the process conditions employed, e.g., inlet temperature, feed concentration, pump rate, and blowing agent type, concentration, and post drying. Reduction in bound water typically leads to improvements in the dispersibility and flowability of phospholipid based powders, leading to the potential for highly efficient delivery of powdered lung surfactants or powder composition comprising insoluble complexes dispersed in the phospholipid. The improved dispersibility allows simple passive DPI devices to be used to effectively deliver these powders. [0154] Yet another version of the pharmaceutical composition includes powder compositions that may comprise, or may be partially or completely coated with, charged species that prolong residence time at the point of contact or enhance penetration through mucosae. For example, anionic charges are known to favor mucoadhesion while cationic charges may be used to associate the formed powder with negatively charged bioactive agents such as genetic material. The charges may be imparted through the association or incorporation of polyanionic or polycationic materials such as polyacrylic acids, polylysine, polylactic acid, and chitosan.

[0155] These unit dose pharmaceutical compositions may be contained in a container. Examples of containers include, but are not limited to, capsules, blisters, vials, ampoules, or container closure systems made of metal, polymer (e.g., plastic, elastomer), glass, or the like.

[0156] The container may be inserted into an aerosolization device. The container may be of a suitable shape, size, and material to contain the pharmaceutical composition and to provide the pharmaceutical composition in a usable condition. For example, the capsule or blister may comprise a wall which comprises a material that does not adversely react with the pharmaceutical composition. In addition, the wall may comprise a material that allows the capsule to be opened to allow the pharmaceutical composition to be aerosolized. In one version, the wall comprises one or more of gelatin, hydroxypropyl methylcellulose (HPMC), polyethyleneglycol-compounded HPMC, hydroxyproplycellulose, agar, aluminum foil, or the like. In one version, the capsule may comprise telescopically adjoining sections, as described for example in U.S. Patent No. 4,247,066, which is incorporated herein by reference in its entirety. The size of the capsule may be selected to adequately contain the dose of the pharmaceutical composition. The sizes generally range from size S to size 000 with the outer diameters ranging from about 4.91 mm to 9.97 mm, the heights ranging from about 11.10 mm to about 26.14 mm, and the volumes ranging from about 0.13 mL to about 1.37 mL, respectively. Examples of suitable capsules are available commercially from, for example, Shionogi Qualicaps Co. in Nara, Japan and Capsugel in Greenwood, South Carolina. After filling, a top portion may be placed over the bottom portion to form a capsule shape and to contain the powder within the capsule, as described in U.S. Patent Nos. 4,846,876 and 6,357,490, and in WO 00/07572, which are incorporated herein by reference in their entireties. After the top portion is placed over the bottom portion, the capsule can optionally be banded.

[0157J The powders and compositions of one or more embodiments of the present invention may be made by any of the various methods and techniques known and available to those skilled in the art. The pharmaceutical composition may be produced using various known techniques. For example, the composition may be formed by spray drying, lyophilization, milling (e.g., wet milling, dry milling), and the like.

[0158] In spray drying, the preparation to be spray dried or feedstock can be any solution, coarse suspension, slurry, colloidal dispersion, or paste that may be atomized using the selected spray drying apparatus. In the case of insoluble agents, the feedstock may comprise a suspension as described above. Alternatively, a dilute solution and/or one or more solvents may be utilized in the feedstock. In one or more embodiments, the feedstock will comprise a colloidal system such as an emulsion, reverse emulsion, microemulsion, multiple emulsion, particle dispersion, or slurry.

[0159] In one embodiment, the insoluble complex of the invention and the matrix material are added to an aqueous feedstock to form a feedstock solution, suspension, or emulsion. The feedstock is then spray dried to produce dried powders comprising the matrix material and the insoluble complex. Examples of suitable spray-drying processes are known in the art, for example as disclosed in WO 99/16419 and U.S. Patent Nos. 6,077,543; 6,051,256; 6,001,336; 5,985,248; and 5,976,574, which are incorporated herein by reference in their entireties.

[0160] Whatever components are selected, the first step in powder production typically comprises feedstock preparation. If a phospholipids-based powder is intended to act as a carrier for the insoluble complex, the selected active agent(s) may be introduced into a liquid, such as water, to produce a concentrated suspension. The concentration of insoluble complex and optional active agents typically depends on the amount of agent required in the final powder and the performance of the delivery device employed (e.g., the fine particle dose for a metered dose inhaler (MDI) or a dry powder inhaler (DPI)).

[0161] Any additional active agent(s) may be incorporated in a single feedstock preparation and spray dried to provide a single pharmaceutical composition species comprising a plurality of active agents. Conversely, individual active agents can be added to separate stocks and spray dried separately to provide a plurality of pharmaceutical composition species with different compositions. These individual species can be added to the suspension medium or dry powder dispensing compartment in any desired proportion and placed in the aerosol delivery system as described below. Polyvalent cations may be combined with the insoluble complex suspension, combined with the phospholipid emulsion, or combined with an oil-in-water emulsion formed in a separate vessel. The insoluble complex may also be dispersed directly in the emulsion.

[0162] For example, polyvalent cation and phospholipid may be homogenized in hot distilled water (e.g., 70⁰C) using a suitable high shear mechanical mixer (e.g., Ultra- Turrax model T-25 mixer) at 8000 rpm for 2 to 5 min. Typically, 5 to 25 g of fluorocarbon is added dropwise to the dispersed surfactant solution while mixing. The resulting polyvalent cation-containing perfluorocarbon in water emulsion may then be processed using a high pressure homogenizer to reduce the particle size. Typically, the emulsion is processed for five discrete passes at 12,000 to 18,000 PSI and kept at about 50⁰C to about 80⁰C.

[0163] When the polyvalent cation is combined with an oil-in-water emulsion, the dispersion stability and dispersibility of the spray dried pharmaceutical composition can be improved by using a blowing agent, as described in WO 99/16419, which is incorporated herein by reference in its entirety. This process forms an emulsion, optionally stabilized by an incorporated surfactant, typically comprising submicron droplets of water immiscible blowing agent dispersed in an aqueous continuous phase. The blowing agent may be a fluorinated compound (e.g. perfluorohexane, perfluorooctyl bromide, perfluorooctyl ethane, perfluorodecalin, perfluorobutyl ethane) which vaporizes during the spray-drying process, leaving behind generally hollow, porous aerodynamically light powders. Other suitable liquid blowing agents include non- fluorinated oils, chloroform, Freon® fluorocarbons, ethyl acetate, alcohols, hydrocarbons, nitrogen, and carbon dioxide gases. The blowing agent may be emulsified with a phospholipid.

[0164] Although the pharmaceutical compositions may be formed using a blowing agent as described above, it will be appreciated that, in some instances, no additional blowing agent is required and an aqueous dispersion of the insoluble complex and/or pharmaceutically acceptable excipients and surfactant(s) are spray dried directly. In such cases, the pharmaceutical composition may possess certain physicochemical properties (e.g., elevated melting temperature, surface activity, etc.) that make it particularly suitable for use in such techniques.

[0165] As needed, cosurfactants such as poloxamer 188 or span 80 may be dispersed into this annex solution. Additionally, pharmaceutically acceptable excipients such as sugars and starches can also be added.

[0166] The feedstock(s) may then be fed into a spray dryer. Typically, the feedstock is sprayed into a current of warm filtered air that evaporates the solvent and conveys the dried product to a collector. The spent air is then exhausted with the solvent.

[0167] Commercial spray dryers manufactured by Bϋchi Ltd. or Niro Corp. may be modified for use to produce the pharmaceutical composition. Examples of spray drying methods and systems suitable for making the dry powders of one or more embodiments of the present invention are disclosed in U.S. Patent Nos. 6,077,543; 6,051 ,256; 6,001,336; 5,985,248; and 5,976,574, which are incorporated herein by reference in their entireties. [0168] Operating conditions of the spray dryer such as inlet and outlet temperature, feed rate, atomization pressure, flow rate of the drying air, and nozzle configuration can be adjusted in order to produce the required powder size, and production yield of the resulting dry powders. The selection of appropriate apparatus and processing conditions are within the purview of a skilled artisan in view of the teachings herein and may be accomplished without undue experimentation. Exemplary settings are as follows: an air inlet temperature between about 60⁰C and about 170⁰C; an air outlet between about 40⁰C to about 120⁰C; a feed rate between about 3 mL/min to about 15 mL/min; an aspiration air flow of about 300 L/min; and an atomization air flow rate between about 25 L/min and about 50 L/min. The settings will, of course, vary depending on the type of equipment used. In any event, the use of these and similar methods allow formation of aerodynamically light powders with diameters appropriate for aerosol deposition into the lung.

[0169] Hollow and/or porous microstructures may be formed by spray drying, as disclosed in WO 99/16419, which is incorporated herein by reference. The spray-drying process can result in the formation of a pharmaceutical composition comprising powders having a relatively thin porous wall defining a large internal void. The spray-drying process is also often advantageous over other processes in that the powders formed are less likely to rupture during processing or during deagglomeration.

[0170] Pharmaceutical compositions useful in one or more embodiments of the present invention may alternatively be formed by lyophilization. Lyophilization is a freeze- drying process in which water, is sublimed from the composition after it is frozen. The lyophilization process is often used because biologicals and pharmaceuticals that are relatively unstable in an aqueous solution may be dried without exposure to elevated temperatures, and then stored in a dry state where there are fewer stability problems. With respect to one or more embodiments of the instant invention, such techniques are particularly compatible with the incorporation of peptides, proteins, genetic material and other natural and synthetic macromolecules in pharmaceutical compositions without compromising physiological activity. Lyophilized cake containing a fine foam-like structure can be micronized using techniques known in the art to provide powders of the desired size.

Administration of the Compositions

[0171 j The compositions of the present invention can be administered by any suitable route, such as pulmonary, intravascular, intramuscular, transdermal, subcutaneous, intraperitoneal, and oral. The compositions can be administered pulmonarily, particularly by inhalation, and most particularly by inhalation of a dry powder composition.

101721 The dry powder compositions as described herein may be delivered using any suitable dry powder inhaler (DPI), i.e., an inhaler device that utilizes the patient's inhaled breath as a vehicle to transport the dry powder drug to the lungs. Included are Nektar Therapeutics' dry powder inhalation devices as described in Patton, J. S., et al., U.S. Pat. No. 5,458,135, Oct. 17, 1995; Smith, A. E., et al., U.S. Pat. No. 5,740,794, Apr. 21, 1998; and in Smith, A. E., et al., U.S. Pat. No. 5,785,049, JuI. 28, 1998, incorporated herein by reference. When administered using a device of this type, the powdered medicament is contained in a receptacle having a puncturable Hd or other access surface, preferably a blister package or cartridge, where the receptacle may contain a single dosage unit or multiple dosage units. Convenient methods for filling large numbers of cavities (i.e., unit dose packages) with metered doses of dry powder medicament are described, e.g., in Parks, D. J., et al., International Patent Publication WO 97/41031, Nov. 6, 1997, incorporated herein by reference.

[0173] Other dry powder dispersion devices for pulmonary administration of dry powders include those described, for example, in Newell, R. E., et al, European Patent No. EP 129985, Sep. 7, 1988); in Hodson, P. D., et al., European Patent No. EP472598, JuI. 3, 1996; in Cocozza, S., et al., European Patent No. EP 467172, Apr. 6, 1994, and in Lloyd, L. J. et al., U.S. Pat. No. 5,522,385, Jun. 4, 1996, incorporated herein by reference. Also suitable for delivering the dry powders of the present invention are inhalation devices such as the Astra-Draco "TURBUH ALER." This type of device is described in detail in Virtanen, R., U.S. Pat. No. 4,668,218, May 26, 1987; in Wetterlin, K., et al., U.S. Pat. No. 4,667,668, May 26, 1987; and in Wetterlin, K., et al., U.S. Pat. No. 4,805,811, Feb. 21, 1989, all of which are incorporated herein by reference. Other suitable devices include dry powder inhalers such as Rotahaler™ (Glaxo), Discus™ (Glaxo), Spiros™ inhaler (Dura Pharmaceuticals), and the Spinhaler™ (Fisons). Also suitable are devices which employ the use of a piston to provide air for either entraining powdered medicament, lifting medicament from a carrier screen by passing air through the screen, or mixing air with powder medicament in a mixing chamber with subsequent introduction of the powder to the patient through the mouthpiece of the device, such as described in Mulhauser, P., et al, U.S. Pat. No. 5,388,572, Sep. 30, 1997, incorporated herein by reference.

[0174] The compositions of the present invention may also be delivered using a pressurized, metered dose inhaler (MDI), e.g., the Ventolin™ metered dose inhaler, containing a solution or suspension of drug in a pharmaceutically inert liquid propellant, e.g., a chlorofluorocarbon or fluorocarbon, as described in Laube, et al., U.S. Pat. No, 5,320,094, and in Rubsamen, R. M., et al, U.S. Pat. No. 5,672,581, both incorporated herein by reference.

[0175] Alternatively, the compositions described herein may be dissolved or suspended in a solvent, e.g., water or saline, and administered by nebulization. Nebulizers for delivering an aerosolized solution include the AERx™ (Aradigm), the Ultravent™ (Mallinkrodt), the Pari LC Plus™ or the Pari LC Star™ (Pari GmbH, Germany), the DeVilbiss Pulmo-Aide, and the Acorn II™ (Marquest Medical Products).

Utility

[0176] The compositions of the invention are useful, when administered pulmonarily in a therapeutically effective amount to a mammalian subject, for treating or preventing any condition responsive to the administration of the pharmacologically active compound in the formulation. For example, in cases in which the pharmacologically active compound is insulin, the condition being treated may be diabetes. Thus, for example, the present invention finds use in the treatment of diabetes.

[0177] The following examples are illustrative of the present invention, and are not to be construed as limiting the scope of the invention. Variations and equivalents of this example will be apparent to those of skill in the art in light of the present disclosure, the drawings, and the claims herein. Unless otherwise stated, all percentages are by weight of the total composition.

EXAMPLES

Example 1: Insulin-Poly-L-Lysine Formulations

[0178] This Example demonstrates dry powder formulations of insulin and polylysine, as well as their functional characteristics. More particularly, this example describes the preparation of particles containing precipitated insulin molecules with polylysine and their use in preparation of dry powder formulations that sustain and control the dissolution kinetics of the drug following pulmonary administration.

Experimental

[0179] Polylysine-insulin formulations are summarized in Table 1. Aqueous solutions of insulin were prepared by adding a weighed amount of insulin to an aqueous medium (usually a 2.6 mM sodium citrate buffer or DI water). The pH of the dispersion was reduced to 2.0-2.5 by addition of IN HCl solution to dissolve all insulin powder. The solution pH was then adjusted to 7.4 by addition of IN NaOH solution. Insulin- polylysine (PLL) complexes were prepared by adding a weighed amount of PLL to the insulin solution, which resulted in the formation of a cloudy suspension. The addition of PLL usually decreased the pH, which was re-adjusted to 7.4 by the drop-wise addition of a IN NaOH solution. The suspension was kept stirring at room temperature until spray- dried. The approximate Insulin and PLL compositions of the suspensions were: PLl: Insulin concentration, 10 mg/ml; PLL (MW 9.8 kDa) concentration, 5 mg/mL PL2: Insulin concentration, 5 mg/ml; PLL (MW 9.8 kDa) concentration, 10 mg/mL PL3: Insulin concentration, 5 mg/ml; PLL (MW 29.9 kDa) concentration, 10 mg/mL

[0180] The suspension containing Insulin-PLL complex was spray-dried using a Buchi 191 spray-dryer. The suspension was pumped through a specially designed tube using a Watson-Marlow pump. The typical spray drying conditions used for the preparation of the formulations are listed below:

Atomization pressure (psi): 60 Feed rate: 5 mL/min

Inlet temperature (⁰C): 125 + 3 Outlet temperature (⁰C): 68 + 4

Atomizer Jacket temperature (°C): 25 + 1

[0181] The formulations are summarized in Table I:

Table 1

Summary of PLL-Insulin Formulations

[0182] The effect of formulation conditions on the complexation behavior of PLL and insulin was evaluated. Effect of citrate: 6 mg/ml Insulin solutions were prepared in 2.6 mM sodium citrate (solution A) and in DI water (solution B). One of the following was added to 10 ml each of solutions A and B (n=2): 10 mg zinc chloride, 28 mg zinc chloride, and 100 mg PLL (MW 4-15 kDa), so as to achieve complexing-agent molar ratios with respect to insulin of 7, 20, and I₃ respectively. The resulting suspensions were stirred and centriftiged and the supernatant was analyzed for free insulin. Effect of pH: 5 ml of 20 mg/ml PLL (MW 4-15 kDa) was mixed with 5 ml of 12 mg/ml Insulin in DI water (pH 7.4) in 20 ml scintillation vials (n=8). Then pH of the resulting suspensions was adjusted to one of the following by addition IN NaOH solution (n=2): 7.4, 8.0, 9.0, and 10.0. The resulting suspensions were stirred and centrifuged. The supernatant was analyzed for free Insulin. The effect of pH and sodium citrate, a well-known glass stabilizer, was evaluated. The results are shown in Figures 2 and 3.

[0183] Sodium citrate is a chelating agent that has the potential to compete with insulin molecules to complex with PLL. This study was performed to determine whether the presence of low concentrations of sodium citrate in the formulation interferes with the ability of Insulin to complex with PLL. As can be seen from Figure 2, the presence of sodium citrate at concentrations of 2.6 mM had no adverse effect on the extent of insulin- PLL and insulin-Zn complexation.

[0184] Figure 3 shows the percent insulin bound to a fixed proportion of PLL as a function of pH. A change in formulation pH did not appear to have a significant effect on the extent of Insulin-PLL complexation.

[0185] The dissolution kinetics of the powders were evaluated in vitro in a flow through experimental setup using diafiltration cells (Amicon). Each formulation was weighed (usually n=2) and dispersed in 35 mL of release medium consisting of 10 mM phosphate buffer, pH 6.9, with 150 mM sodium chloride and 0.1 mM sodium citrate (added to accelerate dissolution of the insulin-PLL complexes and thus, drug release) in order to achieve an insulin concentration of 0.2 mg/mL. The mixture was loaded into a 50-mL capacity diafiltration cell, stirred continuously at 150 rpm, and maintained at 37 ± 2 ⁰C. The use of a pressurized reservoir allowed the diafiltration medium (same as release medium) to flow through each cell at approximately 30 mL/hr. Membrane filters with a molecular weight cut-off of 1 kDa ensured that the Insulin released from the formulations was contained within the cell to allow quantitative measurement. Aliquots of 1 mL were withdrawn from each of the cells at predetermined intervals. A volume of 39

200 μL from each of the samples was centrifuged and the supernatant was analyzed by HPLC for free (released) insulin. Total insulin content was also measured at selected sampling points to perform mass balance calculations. This was achieved by reducing pH to 2.0-2.5 by adding IN HCl to dissolve all insulin, followed by analysis using an HPLC assay.

[0186] As shown in Figure 4, the PLL complexes sustained the dissolution of insulin for more that 24 hours. All PLL formulations showed a small initial burst and insulin release kinetics even slower than the 20:1 zinc formulation.

[01871 Finally, the effect of PLL molecular weight on the extent of insulin binding was investigated. Briefly, 100 mg PLL of different molecular weights was added to 10 mL of 6 mg/mL insulin solution in DI water adjusted to pH 7.4. PLL molecular weight grades used in the study were 4-15, 15-30, 30-70, 70-150, and >300 kDa. The resulting suspensions were stirred and centrifuged and the supernatant was analyzed for free insulin. As illustrated in Figure 5, an increase in molecular weight decreases the ability of PLL to complex with insulin.

[0188] All three PLL powders were comprised primarily of spherical particles, as shown in Figure 6. Furthermore, most particles in PLl formulation seem to have rough surfaces. On the other hand, the particles of PL2 and PL3 formulations appear to have two types of surface characteristics: larger particles with a rough surface and some smaller particles with a smooth surface.

10189] Further, the PLL powders exhibited improved aerosol properties compared to non-PLL, glassy insulin powders, as illustrated in Table 2. ED, defined as the relative amount of powder loaded in the blister that leaves the device, was determined by gravimetric analysis of the powder collected on a glass fiber filter (Gellman Labs, Ann Arbor, Michigan). Mass median aerodynamic diameter (MMAD) was determined gravimetrically by inertial impaction with an eight-stage Andersen cascade impactor (Andersen Instruments, Smyrna, GA). MMAD was determined at flow rate of 28.3 L/min. The stage cut-offs were calculated using a modified Stokes' equation. Special adaptors were used to fit the mouthpiece and thus accommodate the different devices. Fine particle fraction (FPF), defined as the fraction of emitted drug mass in the respirable size range (either at <5 or <3.3 μm), was determined by interpolation of the Andersen deposition profiles. All aerosol tests were performed at room temperature and controlled relative humidity (RH) conditions of 35%-45%. The collection efficiency was calculated based on the total amount of collected powder relative to the total powder loaded in the blister and was determined gravirnetrically.

[0190] All PLL formulations demonstrated high Emitted Dose (ED) and average Fine Particle Fraction (FPF) less than 3.3 μm (39 - 42%). The Mass Median Aerodynamic Diameter (MMAD) ranged from 3.5-3.8 μm.

Table 2

Aerosol properties of PLL-insulin powders

[0191] All PLL-insulin powders exhibited excellent stability during shipping, as demonstrated in Figure 7. To determine powder stability during shipping, the insulin- PLL powders were packaged in unit-dose blisters and shipped in appropriate containers to the preclinical facility. The fraction of the powder emitted through the endotracheal (ET) tube before and after shipping was compared. [01921 The drug was administered to dogs using a pneumatically driven aerosol delivery system (PDADS). The PDADS involved a pulmonary delivery system (PDS), as disclosed in U.S. Pat. No. 6,257,233, which is incorporated by reference herein in its entirety, connected to a compressed air source. A known volume of compressed air was passed through the valve and the flow rate of this compressed air was monitored continuously by a flow meter. The compressed air was used to deliver aerosol to the dog through an endotracheal (ET) tube.

[0193] Figure 7 compares the dose past ET tube data on aerosol tests performed on both shipped and unshipped blisters. For the non-PLL, immediate release insulin formulation (IRl) a 10% drop in dose past ET tube was observed. In contrast, insulin-PLL formulations exhibited minimal to no reduction in the % dose past ET tube. This suggests that inclusion of PLL in the formulation reduces the susceptibility of the protein powder formulations to drop in ED upon shipping.

[0194] After aerosol administration to beagle dogs, the polylysine-insulin complexes significantly reduced the blood glucose levels and prolonged the reduced level to over 8 hours, as shown in Figure 8. This data provides a strong indication of prolonged insulin absorption by the animals' lungs.

[0195] Finally, due to the inclusion of glass formers, these PLL formulations are in the glassy state (data not shown).

[0196] In summary, this Example demonstrates that formulation of proteins with polylysines (or other positively or negatively charged polymers) results in formation of precipitates:

• at mass ratios greater than 30% w/w biodegradable polymer either positively or negatively charged,

• at polymer molecular weights of 3OK or less,

• containing glass forming excipients, and

• that complex at least 90% of the drug. [0197] The precipitates of the invention can be processed a spray drying process, as described above, to yield particles:

• highly dispersible over non-PLL containing counterparts,

• suitable for pulmonary delivery via active or passive DPI inhalers,

• that maintain their stability following powder shipping, in contrast to non-PLL containing powders, and

• that can prolong the dissolution kinetics and absorption kinetics of the active drug following administration in vivo.

[0198J Although the present invention has been described in considerable detail with regard to certain versions thereof, other versions are possible, and alterations, permutations and equivalents of the version shown will become apparent to those skilled in the art upon a reading of the specification and study of the drawings. Also, the various features of the versions herein can be combined in various ways to provide additional versions of the present invention. Furthermore, certain terminology has been used for the purposes of descriptive clarity, and not to limit the present invention. Therefore, any appended claims should not be limited to the description of the preferred versions contained herein and should include all such alterations, permutations, and equivalents as fall within the true spirit and scope of the present invention.

[0199J Having now fully described this invention, it will be understood to those of ordinary skill in the art that the methods of the present invention can be carried out with a wide and equivalent range of conditions, formulations, and other parameters without departing from the scope of the invention or any embodiments thereof.

|0200] All patents and publications cited herein are hereby fully incorporated by reference in their entirety. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that such publication is prior art or that the present invention is not entitled to antedate such publication by virtue of prior invention. Furthermore, it is noted that U.S. Patent Application entitled "INSULIN DERIVATIVE FORMULATIONS FOR PULMONARY DELIVERY" by Mei-Chang Kuo et al. and filed the same day as the present application is hereby fully incorporated by reference in its entirety.

Claims

What is claimed is:

1. A composition comprising at least one pharmacologically active protein, and at least one charged polymer; wherein the composition comprises insoluble complexes of the charged polymer and precipitated pharmacologically active protein; and wherein the composition is in dry powder form and the dry powder comprises particles in which at least an outermost portion is amorphous.

2. The composition according to claim 1, wherein the at least one pharmacologically active protein comprises insulin.

3. The composition according to claim 1, wherein the at least one charged polymer comprises a poly-amino acid.

4. The composition according to claim 3, wherein the poly-amino acid is chosen from polylysine, polyhistidine, and polyarginine.

5. The composition according to claim 4, wherein the poly-amino acid comprises polylysine.

6. The composition according to claim 5, wherein the poly-amino acid comprises poly-L-lysine.

7. The composition according to claim 1, further comprising at least one divalent cation.

8. The composition according to claim 7, wherein the molar ratio of protein: divalent cation is less than 1 : 1.

9. The composition according to claim 8, wherein the divalent cation is chosen from zinc, copper, cobalt, iron, and magnesium.

10. The composition according to claim 1, further comprising at least one glass-forming excipient.

11. The composition according to claim 10, wherein the glass-forming excipient is chosen from sucrose, raffinose, trehalose, sodium citrate, and human serum albumin.

12. The composition according to claim 1, wherein the at least outermost portion comprises a glassy state.

13. The composition according to claim 1, wherein the dry powder comprises particles having a mass median aerodynamic diameter of less than 10 μm.

14. The composition according to claim 13, wherein the dry powder comprises particles having a mass median aerodynamic diameter of less than 5 μm.

15. A pharmaceutical formulation, comprising particles having an amorphous at least outermost portion, the particles having a mass median aerodynamic diameter of less than 10 μm, the particles comprising insulin and at least one positively charged biopolymer.

16. The pharmaceutical formulation according to claim 15, wherein the at least one positively charged biopolymer comprises a poly-amino acid.

17. The pharmaceutical formulation according to claim 16, wherein the poly- amino acid is chosen from polylysine, polyhistidine, and polyarginine.

18. The pharmaceutical formulation according to claim 15, further comprising at least one glass-forming excipient.

19. The pharmaceutical formulation according to claim 18, wherein the glass- forming excipient is chosen from sucrose, raffinose, trehalose, sodium citrate, and human serum albumin.

20. The pharmaceutical formulation according to claim 15, further comprising at least one divalent cation.

21. The pharmaceutical formulation according to claim 20, wherein the molar ratio of protein:divalent cation is less than 1:1.

22. The pharmaceutical formulation according to claim 21, wherein the divalent cation is chosen from zinc, copper, cobalt, iron, and magnesium.

23. The pharmaceutical formulation according to claim 15, wherein the formulation comprises particles having a mass median aerodynamic diameter of less than 5 μm.

24. A method of preparing a pharmaceutical composition comprising: combining, in a liquid composition, at least one pharmacologically active protein with at least one charged polymer, to form a protein-polymer mixture; precipitating the pharmacologically active protein from the protein-polymer mixture; and drying the liquid composition to form particles having an amorphous at least outermost portion, the particles comprising insoluble complexes of precipitated protein and charged polymer.

25. The method according to claim 24, wherein the liquid composition comprises a glass-forming excipient chosen from sucrose, raffϊnose, trehalose, sodium citrate, and human serum albumin.

26. The method according to claim 24, wherein the liquid composition comprises at least one divalent cation.

27. The method according to claim 26, wherein the molar ratio of protein:divalent cation is less than 1: 1.

28. The method according to claim 27, wherein the divalent cation is chosen from zinc, cobalt, copper, iron, and magnesium.

29. The method according to claim 24, wherein the drying comprises spray- drying, freeze-drying, or spray-freeze drying.

30. A method of preparing a pharmaceutical composition comprising: adding to a liquid composition at least one pharmacologically active protein and at least one charged polymer, wherein the addition of the charged polymer results in precipitation of the pharmacologically active protein; and drying the liquid composition to form particles comprising an amorphous at least outermost portion, the particles comprising insoluble complexes of precipitated protein and charged polymer.

31. A method of preparing a pharmaceutical composition comprising: adding to a liquid composition comprising at least one charged polymer, at least one pharmacologically active protein, wherein the pharmacologically active protein precipitates upon addition to the liquid composition; and drying the liquid composition to form particles comprising an amorphous at least outermost portion, the particles comprising insoluble complexes of precipitated protein and charged polymer.

32. A pharmaceutical formulation, comprising particles comprising an amorphous at least outermost portion, the particles having a mass median aerodynamic diameter of less than 10 μm, the particles comprising insulin and polylysine; which particles exhibit a rate of insulin release of less than or equal to about 40% in 5 hours, less than or equal to about 50% in 10 hours, less than or equal to about 60% in about 15 hours, and less than or equal to about 70% in about 20 hours, as measured by diafiltration.

33. The pharmaceutical formulation according to claim 32, which, when administered to an animal in need of treatment, produces a reduction in blood glucose level for a period of at least about 7 hours.

34. The pharmaceutical formulation according to claim 33, which, when administered to an animal in need of treatment, produces a reduction in blood glucose level for a period of at least about 8 hours.

35. A method of reducing blood glucose level in an animal, comprising pulmonarily administering to the animal a pharmaceutical formulation for inhalation, the formulation comprising particles comprising an amorphous at least outermost portion, the particles having a mass median aerodynamic diameter of less than 10 μm, the particles comprising insulin and at least one positively charged biopolymer, wherein the administration results in a reduction in blood glucose level for a period of at least about 6 hours.

36. The method according to claim 35, wherein the administration results in a reduction in blood glucose level for a period of at least about 7 hours.

37. The method according to claim 36, wherein the administration results in a reduction in blood glucose level for a period of at least about 8 hours.

38. A composition comprising at least one pharmacologically active protein, and at least one charged polymer; wherein the composition comprises insoluble complexes of the charged polymer and precipitated pharmacologically active protein; and wherein the composition is in dry powder form and the dry powder comprises particles having a mass median diameter of less than 5 μm.

39. A composition comprising at least one pharmacologically active protein, and at least one charged polymer; wherein the composition comprises insoluble complexes of the charged polymer and precipitated pharmacologically active protein; and wherein the composition is in dry powder form and the composition comprises no lipid.

40. A method of preparing a pharmaceutical composition comprising: combining, in a liquid composition, at least one pharmacologically active protein with at least one charged polymer, to form a protein-polymer mixture, wherein the liquid composition comprises no ethanol; precipitating the pharmacologically active protein from the protein-polymer mixture; and drying the liquid composition to form particles comprising insoluble complexes of precipitated protein and charged polymer.

41. The method of claim 40, wherein the liquid composition comprises no organic solvent.