WO1999016420A9

WO1999016420A9 - Stabilized preparations for use in nebulizers

Info

Publication number: WO1999016420A9
Application number: PCT/US1998/020603
Authority: WO
Inventors: Ernest G Schutt; Thomas E Tarara; Luis A Dellamary; Alexey Kabalnov; Jeffry G Weers
Original assignee: Alliance Pharma; Ernest G Schutt; Thomas E Tarara; Luis A Dellamary; Alexey Kabalnov; Jeffry G Weers
Priority date: 1997-09-29
Filing date: 1998-09-29
Publication date: 1999-06-17
Also published as: WO1999016420A1

Abstract

Stabilized dispersions are provided for the delivery of a bioactive agent to the respiratory tract of a patient. The dispersions preferably comprise a stabilized colloidal system which may comprise a fluorochemical component. In particularly preferred embodiments, the stabilized dispersions comprises perforated microstructures dispersed in a fluorochemical suspension medium. As density variations between the suspended particles and suspension medium are minimized and attractive forces between microstructures are attenuated, the disclosed dispersions are particularly resistant to degradation, such as by settling or flocculation. In particularly preferred embodiments, the stabilized dispersions may be administered to the lung of a patient using a nebulizer.

Description

STABILIZED PREPARATIONS FOR USE IN NEBULIZERS

Field of the Invention

The present invention generally relates to formulations and methods for the administration of bioactive agents to a patient via the respiratory tract More particularly, the present invention relates to methods, systems and compositions comprising relatively stable dispersions that are preferably administered via nebulization both for topical delivery to the lung, and for delivery via the lung to the systemic circulation.

Background of the Invention

Targeted drug delivery means are particularly desirable where toxicity or bioavailability of the pharmaceutical compound is an issue. Specific drug delivery methods and compositions that effectively deposit the compound at the site of action potentially serves to minimize toxic side effects lower dosing requirements and decrease therapeutic costs. In this regard, the development of such systems for pulmonary drug delivery has long been a goal of the pharmaceutical industry.

The three most common systems presently used to deliver drugs locally to the pulmonary air passages are dry powder inhalers (DPIs), metered dose inhalers (MDIs) and nebulizers. MDIs, the most popular method of inhalation administration, may be used to deliver medicaments in a solubilized form or as a dispersion Typically, MDIs compπse a Freon or other relatively high vapor pressure propellant that forces aerosolized medication into the respiratory tract upon activation of the device Unlike MDIs, DPIs generally rely on the patient's inspiratory efforts to introduce a medicament in a dry powder form to the lungs. Finally, nebulizers form a medicament aerosol to be inhaled by imparting energy to a liquid solution More recently, direct pulmonary delivery of drugs dunng liquid ventilation or pulmonary lavage using a fluorochemical medium has also been explored. While each of these methods and associated systems may prove effective in selected situations, inherent drawbacks, including formulation limitations, can limit their use.

A key development, which has elevated the importance of pulmonary drug delivery systems, has been the emergence of new drugs deπved from biotechnology (e g. peptides, proteins, oligonucleotides and plasmids) The systemic delivery of these biopolymers has proven difficult, owing to their large molecular size, high surface charge, poor chemical and enzymatic stability, and low permeability across vaπous absorption barπers of the body Because of their low bioavailability by oral and transdermal routes of administration, drugs such as peptides are currently administered primarily by infusions or frequent injections The development of less invasive methods for deliveπng peptides and other biopolymers represents a large focus of current drug delivery research, and a number of sites of administration are being explored, including enhanced oral, nasal, and pulmonary delivery As indicated above, nebulizers are frequently used for drug delivery to the human lung and are particularly useful for the treatment of hospitalized or πonambulatory patients. There are two mam classes of devices, air jet nebulizers and ultrasonic nebulizers In air jet nebulizers compressed air is forced through an orifice. A liquid may then be withdrawn from a perpendicular nozzle (the Bernoulli effect) to mix with the air jet to form droplets. A baffle (or series of baffles) within the nebulizer is used to facilitate formation of the aerosol cloud. In contrast, ultrasonic nebulizers rely on the generation of ultrasound waves in an ultrasonic nebulizer chamber by a ceramic piezoelectnc crystal that vibrates at a precise frequency when electrically excited. The ultrasonic energy sets up high energy waves in the nebulizer solution, facilitating generation of an aerosol cloud.

Formulations for nebulizatioπ typically comprise aqueous based solutions Assuming that the solubility and stability of the active drug are adequate, an aqueous based formulation administered by πebulization is reasonable when the estimated minimal effective dose exceeds about 200 μ_. Continuous nebulization has long been an option for the delivery of topical lung therapy for the treatment of various lung diseases such as asthma, chronic obstructive pulmonary disease, emphysema, and bronchitis More recently, proteins such as DMase have been delivered by conventional jet nebulizers for their local effect on the lung. Unfortunately, continuous nebulization is an intπnsically inefficient way to deliver aerosolized medication. This fact is underscored by the observation that doses of bronchodilators delivered using nebulizers are three orders of magnitude greater than a bioequivalent dose delivered by MDI or dry powder generator. In addition to concerns with respect to device efficiency, concerns also exist with regards to changes in the formulation dunng the nebulization process. For example, drug concentration in the reservoir solution of an air jet nebulizer often increase over time Moreover, a change in drug concentration may imply a change in os olality of the aqueous solution, and hyperosmolar nebulizer solutions have been shown to cause broπchoconstπctioπ.

In terms of pulmonary delivery of bioactive agents to the systemic circulation via nebulization, most of the research has focused on the use of portable hand held ultrasonic nebulizers, also referred to as metered solution nebulizers These devices should not be confused with hand held nebulizers which require several minutes per treatment These devices, generally known as single-bolus nebulizers, aerosolize a single bolus of medication in an aqueous solution with a particle size efficient for deep lung delivery in one or two breaths. These devices fall into three broad categories.

The first category compπses pure piezoelectnc single-bolus nebulizers such as those described by Mutterlein, et. al., (J. Aerosol Med. 1988; 1 :231 ). In another category, the desired aerosol cloud may be generated by icrochannel extrusion single-bolus nebulizers such as those described in U.S. Pat No. 3,812,854. Finally, a third category comprises devices exemplified by Robertson, et. al , (WO 9211 1050) which descπbes cyclic pressunzation single bolus nebulizers. Each of the aforementioned references is incorporated herein in their entirety.

While such devices are an improvement over conventional hand held nebulizers that require treatment times of several minutes, they are somewhat limited by the fact that they employ multidose reservoirs. This is problematic for protein delivery applications where the product must remain sterile throughout the therapy program. At the very least

.0. use of these multidose reservoirs would require the use of preservatives, and even this approach is unlikely to be satisfactory under all product usage sceπaπos. In order to overcome some of these limitations, a unit dose system has recently been dεscπbed by Schuster, et, al , (Pharm. Res. 1997; 14-354 which is incorporated herein). However, problems remain even with such unit dose systems. For example, a pitfall with devices for the delivery of bioactive agents to the systemic circulation is that the bioactive agent must have long term stability in an aqueous phase. This is possible only for a select few peptides and proteins.

Accordingly, it is an object of the present invention to provide methods, compositions and systems for the effective pulmonary delivery of bioactive agents using nebulizers.

It is a further object of the present invention to provide methods and compositions for the stabilization of bioactive agents to be delivered using a nebulizer.

It is yet another object of the present invention to provide methods and preparations that advantageously allow for the efficient delivery of bioactive agents to the systemic circulation of a patient in need thereof.

Summary of the Invention These and other objects are provided for by the invention disclosed and claimed herein. To that end, the methods and associated compositions of the present invention provide, in a broad aspect, for the improved delivery of bioactive agents using stabilized preparations. Preferably, the bioactive agents are delivered to a patient via the respiratory tract. More particularly, the present invention provides for the formation and use of stabilized dispersions (also referred to as stabilized respiratory dispersions) and inhalation systems, including nebulizers comprising such dispersions, as well as individual components thereof. Unlike prior art formulations in a form for use in nebulizers, the present invention preferably employs novel techniques to reduce attractive forces between the dispersed constituents and to reduce density fluctuations in the stabilized dispersion thereby retarding degradation of the disclosed preparations by fiocculation, sedimentation or creaming. Moreover, the stabilized preparations of the present invention preferably compπse a suspension medium that further serves to reduce the rate of degradation with respect to the incorporated bioactive agent. In particularly preferred embodiments, the suspension medium will compπse a fluoπnated compound or fluorocarboπ. Those skilled in the art will appreciate that the disclosed stable preparations, and systems comprising those preparations, act to reduce dosing incongruities, thereby facilitating uniform drug delivery, allowing for more concentrated dispersions and, retarding the degradation of any labile biopolymers incorporated therein.

In a broad sense, the stabilized dispersions of the present invention incorporate colloidal preparations compnsing a nonaqueous continuous phase wherein the stabilized dispersions are capable of being nebulized or aerosolized to provide effective dosing to a patient in need thereof. For example, the stabilized dispersions may comprise any reverse emulsion or particulate dispersion that allows for the effective delivery of a bioactive agent to the pulmonary air passages of a mammal. Those skilled in the art will appreciate that, the disperse phase of such preparations may comprise liquid particulates in the case of reverse emulsions or non liquid particulates in the case of stabilized suspensions Accordingly, for the purposes of the present application the term "stabilized dispersion" shall be held to compπse colloidal systems compnsing reverse emulsions and particulate suspensions unless otherwise dictated by contextual constraints With respect to each of these cases, the stabilized dispersion may be used with a nebulizer to provide the desired aerosolized medicament for pulmonary administration

With regard to particularly preferred embodiments, the stabilized preparations of the present invention provide these and other advantages through the use of particulate suspensions compnsing hollow andlor porous perforated microstructures that substantially reduce attractive molecular forces, such as van der Waals forces, which dominate prior art dispersion preparations. More particularly, the use of perforated (or porous) microstructures or microparticulates that are permeated or filled by the surrounding fluid medium, or suspension medium, significantly reduces disruptive attractive forces between the particles. Additionally, the components of the dispersions may be selected to minimize differences in polaπzabilities (i.e. reduced Hamaker constant differentials) and further stabilize the preparation. The relatively homogeneous nature of these particulate dispersions or suspensions, inhibits deterioration thereby allowing for pharmaceutical preparations having enhanced stability. In addition to the heretofore unappreciated advantages associated with the formation of stabilized particulate dispersions, the perforated configuration and corresponding large surface area enables the microstructures to be more easily carried by the flow of gases during inhalation than non perforated particles of comparable size. This, in turn, enables the perforated microstructures or microparticles of the present invention to be carried more efficiently into the lungs of a patient than non perforated structures such as micronizεd particles or relatively nonporous microspheres In view of these advantages, dispersions comprising perforated microstructures are particularly compatible with inhalation therapies compnsing administration of the bioactive preparation to at least a portion of the pulmonary air passages. For the purposes of the present application, these stabilized dispersions intended for pulmonary drug delivery may be termed respiratory dispersions In particularly preferred embodiments, such respiratory dispersions are used in conjunction with nebulizers to effectively deliver a bioactive agent to the pulmonary air passages or nasal passages of a patient. For those embodiments comprising perforated microstructures, those skilled in the art will appreciate that they may be formed of any biocompatible mateπal providing the desired physical characteristics or morphology that allows for the preparation of stabilized dispersions In this respect, the perforated microstructures comprise pores, voids, defects or other interstitial spaces that allow the fluid suspension medium to freely permeate, or perfuse, the particulate boundary, thus reducing or minimizing density differences between the dispersion components Yet, given these constraints, it will be appreciated that any mateπal or configuration may be used to form the microstructure matπx. With regard to the selected materials, it is desirable that the microstructure incorporates at least one surfactant Preferably, this surfactant will compπse a phospholipid or other surfactant approved for pulmonary use As to the configuration, particularly preferred embodiments of the invention incorporate spray dπed, hollow microspheres having a relatively thin porous wall defining a large internal void, although, other void containing or perforated structures are contemplated as well.

Accordingly, select embodiments of the invention provide for stable respiratory dispersions for use in a nebulizer compnsing a suspension medium having dispersed therein a plurality of perforated microstructures compnsing at least one bioactive agent wherein said suspension medium substantially permeates said perforated microstructures.

While preferred embodiments of the invention compπse perforated microstructures, relatively nonporous or solid particulates may also be used to prepare dispersions that are compatible with the teachings herein That is, respiratory dispersions compnsing suspensions of relatively nonporous or solid particulates in a nonaqeous suspension medium are also contemplated as being within the scope of the present invention In this respect, such relatively nonporous particulates may comprise micromzed particles or nanocrystals. Accordingly, as used herein the term "particulate" shall be interpreted broadly to mean any non liquid particle comprising the discontinuous phase of a dispersion or suspension More specifically, it will be appreciated that the term "particulate" shall be held to comprise particles of any porosity, including both perforated microstructures and relatively nonporous particles

It should further be appreciated that the nonaqueous continuous phase or suspension medium, may be any liquid or compound that is in liquid form, under appropriate thermodynamic conditions, for formation of a compatible particulate dispersion or reverse emulsion. Unless otherwise dictated by contextual restraints, the terms "suspension medium," "suspension media" and "nonaqueous continuous phase" are held to be equivalent for the purposes of the instant application and may be used interchangeably. For embodiments wherein the stabilized dispersion is to be used in conjunction with a nebulizer, the suspension medium preferably comprises hydrocarbons or fluorocarbons having a vapor pressure less than about one atmosphere. That is, it will preferably be a liquid under standard conditions of one atmosphere and 25° C

In accordance with the teachings herein, particularly preferred suspension mediums or nonaqueous continuous phases compnse fluorochemicals |e g perfluorocarbons or fluorocarbons) that are liquid at room temperature It is well established that many fluorochemicals have a proven history of safety and bioco patibility in the lung. Further, in contrast to aqueous solutions, fluorochemicals do not negatively impact gas exchange Moreover, because of their unique wettability characteπstics, fluorochemicals may be able to carry an aerosolized stream of particles deeper into the lung, thereby improving systemic delivery Finally, many fluorochemicals are also bactenostatic thereby decreasing the potential for microbial growth in compatible nebulizer devices

As such, the present invention provides for the use of a liquid fluorochemical in the manufacture of a medicament for the pulmonary delivery of a bioactive agent whereby the medicament comprises a stabilized dispersion having a fluorochemical continuous phase which is nebulized using a nebulizer to form an aerosolized medicament compnsing said bioactive agent wherein said aerosolized medicament is administered to at least a portion of the pulmonary air passages of a patient in need thereof It will further be appreciated that, in selected embodiments, the present invention compπses methods for forming dispersions which comprise combining a plurality of particulates compnsing at least one bioactive agent with a predetermined volume of suspension medium, to provide a respiratory blend. The respiratory blend may then be mixed or otherwise agitated to provide a substantially homogeneous dispersion. Again, in preferred embodiments, the particulates will compπse perforated microstructures which allow for the perfusion or permeation of the selected suspension medium

Of course, in other embodiments the dispersion may compπse a reverse emulsion.

As such, preferred embodiments of the invention provide for the formation of stabilized respiratory dispersions compnsing the steps of. combining a plurality of perforated microstructures compnsing at least one bioactive agent with a predetermined volume of a nonaqueous suspension medium to provide a respiratory blend wherein said suspension medium permeates said perforated microstructures; and mixing said respiratory blend to provide a substantially homogeneous respiratory dispersion.

Along with the aforementioned advantages, the stability of the formed particulate dispersions may be further increased by reducing, or minimizing, the Hamaker constant differential between incorporated particulates, or perforated microstructures, and the suspension medium Those skilled in the art will appreciate that Hamaker constants tend to scale with refractive indices. In this regard, the present invention further provides methods for stabilizing a respiratory dispersion by reducing attractive van der Waals forces compnsing the steps of: providing a plurality of perforated microstructures; combining the perforated microstructures with a suspension medium comprising at least one fluorochemical wherein the suspension medium and the perforated microstructures are selected to provide a refractive index differential value of less than about 0 5 In accordance with the teachings herein, the particulates preferably compπse perforated microstructures and, in particularly preferred embodiments, the particulates will compπse hollow, porous microspheres

With regard to delivery of the stabilized preparations, another aspect of the present invention is directed to liquid inhalation systems for the administration of one or more bioactive agents to a patient As such, the present invention provides for inhalation systems for the pulmonary administration of a bioactive agent to a patient comprising- a fluid reservoir, a stable respiratory dispersion in said fluid reservoir wherein said stabilized dispersion composes a fluorochemical continuous phase and at least one bioactive agent; and a nebulizer operably associated with said fluid reservoir wherein the nebulizer is capable of aerosolizing and discharging the stable respiratory dispersion.

The respiratory dispersion may compπse a reverse emulsion, microemulsion or particulate suspension Preferably, the dispersion compnses a suspension medium having dispersed therein a plurality of perforated microstructures, which comprise at least one bioactive agent and are substantially permeated by the suspension medium Those skilled in the art will appreciate that the nebulizer may compπse an ultrasonic nebulizer, an air jet nebulizer and, most preferably, a single-bolus nebulizer. In any event, the disclosed systems of the present invention allow for the reproducible administration of bioactive agents having aerosolized particle size small enough to travel deep within the lung More specifically, the aerosolized medicament will preferably exhibit a fine particle fraction of greater than approximately 20% w/w.

Yet another associated advantage of the present invention is the effective pulmonary delivery of bioactive agents As used herein, the terms "bioactive agent" refers to a substance which is used in connection with an application that is therapeutic or diagnostic in nature, such as methods for diagnosing the presence or absence of a disease in a patient and/or methods for treating disease in a patient As to compatible bioactive agents, those skilled in the art will appreciate that any therapeutic or diagnostic agent may be incorporated in the stabilized dispersions of the present invention For example, the bioactive agent may be selected from the group consisting of aπtialiergics, bronchodilators, broπchoconstnctors, pulmonary lung surfactants, analgesics, antibiotics, leukotnene inhibitors or antagonists, anticholinergics, mast cell inhibitors, antihistamines, antnnfiammatoπes, antineoplastics, anesthetics, anti tuberculars, imaging agents, cardiovascular agents, enzymes, steroids, genetic mateπal, viral vectors, antisense agents, proteins, peptides and combinations thereof Particularly preferred bioactive agents comprise compounds which are to be administered systemically (i.e. to the systemic circulation of a patient) such as peptides, proteins or polynucleotides. As will be disclosed in more detail below, the bioactive agent may be incorporated, blended in, coated on or otherwise associated with the perforated microstructure. In other embodiments, the bioactive agent may be associated with the disperse phase (e.g , aqueous phase) of a reverse emulsion. Whatever form of stabilized dispersion is employed, the present invention provides methods for the pulmonary delivery of one or more bioactive agents compnsing the steps of- providing a stabilized respiratory dispersion compnsing one or more bioactive agents wherein the respiratory dispersion compπses a fluorochemical continuous phase; nebulizing said respiratory dispersion with a nebulizer to provide an aerosolized medicament; and ad inisteπng a therapeutically effective amount of said aerosolized medicament to at least a portion of the pulmonary passages of a patient in need thereof.

When the stabilized dispersion compπsεs a reverse emulsion, the bioactive agent preferably will be substantially associated with the dispersed droplets. With respect to particulate dispersions, the selected bioactive agent, or agents, may be used as the sole structural component of the particulates or perforated microstructures Conversely, the particulates, or perforated microstructures, may comprise one or more components (i e structural matenals, surfactants, excipients, etc ) in addition to the incorporated bioactive agents In particularly preferred embodiments, the suspended particulates or perforated microstructures will comprise relatively high concentrations of surfactant (greater than about 10% w/w) along with the incorporated bioactive agent(s) Finally, it should be appreciated that the particulate or perforated microstructure may be coated, linked or otherwise associated with the bioactive agent in a non integral manner. Whatever configuration is selected, it will be appreciated that the associated bioactive agent may be used in its natural form, or as one or more salts known in the art

In addition to reverse emulsions and suspensions of perforated microstructures, it must be emphasized that the present invention provides for the nebulization and pulmonary delivery of relatively stable particulate dispersions Those skilled in the art will appreciate that, due to other physiochemical characteristics, the morphology of incorporated particulates may vary without destabilizing the dispersion As such, stabilized dispersions may be formed with compatible particulates even if they exhibit relatively low porosity, or are substantially solid. That is, while particularly preferred embodiments of the present invention will compπse perforated microstructures or microspheres, acceptable dispersions may be formed using relatively low porosity particulates such as πaπocrystals, or microπized drugs. In this respect, such embodiments are specifically contemplated as being within the scope of the present invention

The stabilized dispersions of the invention may optionally compπse one or more additives to further enhance stability or increase biocompatibility For example, vanous surfactants, co solvents, osmotic agents, stabilizers, chelators, buffers, viscosity modulators, solubility modifiers and salts can be associated with the perforated microstructure, suspension medium, or both. The use of such additives will be understood to those of ordinary skill in the art and, the specific quantities, ratios, and types of agents can be determined empirically without undue expeπmentatioπ.

Other objects, features and advantages of the present invention will be apparent to those skilled in the art from a consideration of the following detailed descπptioπ of preferred exemplary embodiments thereof

Brief Descπption of the Drawings

Figs. 1 A1 to 1 F2 illustrate changes in particle morphology as a function of variation in the ratio of fluorocarbon blowing agent to phospholipid (PFC/PC) present in the spray dry feed The micrographs, produced using scanning electron microscopy and transmission electron microscopy techniques, show that in the absence of FCs, or at low PFC/PC ratios, the resulting spray dried microstructures comprising gentamicin sulfate are neither particularly hollow nor porous Conversely, at high PFC/PC ratios, the particles contain numerous pores and are substantially hollow with thin walls.

Fig. 2 is a scanning electron microscopy image of perforated microstructures comprising cromolyn sodium illustrating a preferred hollow/porous morphology

Fig 3 presents results of in vitro Andersen cascade impactor studies comparing the same hollow porous cromolyn sodium formulation delivered via MDI in HFA 134a, or from a long chain fluorocarbon (perfluorooctyl ethane) via nebulization Nebulized particles are observed to deposit onto later stages in the impactor, corresponding to improved systemic delivery in vivo. Detailed Descnption Preferred Embodiments

While the present invention may be embodied in many different forms, disclosed herein are specific illustrative embodiments thereof that exemplify the principles of the invention It should be emphasized that, the present invention is not limited to the specific embodiments illustrated. As set forth above, the present invention provides systems, methods and compositions that allow for the formation and administration of stabilized suspensions or dispersions, having a πonaqeous continuous phase, that may advantageously be used for the pulmonary delivery of bioactive agents in conjunction with a nebulizer In this regard, it will be appreciated that the stabilized dispersions may comprise any colloidal system, including, reverse emulsions, microemulsions or particulate (i e non liquid particles) dispersions that may be nebulized to effectively deliver a bioactive agent to the pulmonary air passages of a patient. Particularly preferred embodiments comprise stabilized dispersions incorporating a liquid fluorochemical continuous phase or suspension medium In any event, the stabilized dispersion will preferably be administered to the pulmonary air passages of a patient using a nebulizer (e g a single bolus type nebulizer)

Traditional pπor art nebulizer preparations typically compπse aqueous solutions of the selected pharmaceutical compound With such pπor art nebulizer preparations, it has long been established that corruption of the incorporated therapeutic compound can severely reduce efficacy For example, with conventional aqueous multi dose nebulizer preparations, bacterial contamination is a constant problem In addition, the solubilized medicament may precipitate out, or degrade over time, adversely affecting the delivery profile. This is particulariy true of larger, more labile biopolymers such as enzymes or other types of proteins. Precipitation of the incorporated bioactive agent may lead to particle growth that results in a substantial reduction in lung penetration and a corresponding decrease in bioavailability Such dosing incongruities markedly decrease the effectiveness of any treatment

The present invention overcomes these and other difficulties by providing stabilized dispersions with a nonaqueous continuous phase that preferably comprises a fluoπnated compound (i e a fluorochemical, fluorocarbon or perfluorocarbon). Particulariy preferred embodiments of the present invention comprise fluorochemicals that are liquid at room temperature As indicated above, the use of such compounds, whether as a continuous phase or, as a suspension medium, provides several advantages over prior art liquid inhalation preparations In this regard, it is well established that many fluorochemicals have a proven history of safety and biocompatibility in the lung Further, in contrast to aqueous solutions, fluorochemicals do not negatively impact gas exchange following pulmonary administration To the contrary, they may actually be able to improve gas exchange and, due to their unique wettability characteristics, are able to carry an aerosolized stream of particles deeper into the lung, thereby improving systemic delivery of the desired pharmaceutical compound In addition, the relatively non reactive nature of fluorochemicals acts to retard any degradation of an incorporated bioactive agent Finally, many fluorochemicals are also bactenostatic thereby decreasing the potential for microbial growth in compatible nebulizer devices As previously indicated, the present invention may comprise any one of a number of colloidal systems including, but not limited to, reverse emulsions, microemulsions and particulate dispersions. For the purposes of the instant application the terms shall be used in accordance with their common meanings unless otherwise dictated by contextual constraints. Thus, those skilled in the art will appreciate that emulsions (whether micro, or reverse [water-in oil]) will compπse a dispersion of liquid particulates in a liquid continuous phase. Conversely, a particulate suspension or dispersion shall, as used herein, be held to compπse a distribution of non liquid particles in a liquid continuous phase or suspension medium

While inhalation preparations compatible with the present invention may comprise any colloidal system that is capable of nebulization or aerosolization the following discussion, for the purpose of explanation, will largely be directed to particularly preferred embodiments of the present invention comprising stabilized particulate dispersions. It should be emphasized that, the scope and content of the present invention is not limited to these specific illustrative embodiments and, in particular, is not limited to those embodiments comprising particulate dispersions. While such dispersions are particularly effective in terms of stability and pulmonary distribution, nebulized reverse emulsions may also provide for the efficient pulmonary delivery of bioactive compounds As such, their use is specifically contemplated as being within the scope of the present invention

With regard to particulate dispersions, the enhanced stability provided by the suspensions of the present invention may be achieved by lowering the van der Waals attractive forces between the suspended particles, and by reducing the differences in density between the suspension medium and the particles. In accordance with the teachings herein, the increases in suspension stability may be imparted by engineering perforated microstructures that are then dispersed in a compatible suspension medium. In this respect, the perforated microstructures comprise pores, voids, hollows, defects or other interstitial spaces that allow the fluid suspension medium to freely permeate or perfuse the particulate boundary Particularly preferred embodiments comprise perforated microstructures that are both hollow and porous, almost honeycombed or foam like in appearance In especially preferred embodiments the perforated microstructures comprise hollow, porous spray dried microspheres. When perforated microstructures are placed in the suspension medium, the suspension medium is able to permeate the particles, thereby creating a "homodispersioπ", wherein both the continuous and dispersed phases are essentially indistinguishable Since the defined or "virtual" particles (i.e comprising the volume circumscribed by the microstructure matrix) are made up almost entirely of the medium in which they are suspended, the forces driving particle aggregation (fiocculation) are minimized Additionally, the differences in density between the defined or virtual particles and the continuous phase are minimized by having the microstructures filled with the medium, thereby effectively slowing particle creaming or sedimentation As such, the stabilized suspensions of the present invention are particularly compatible with inhalation therapies and may be used in conjunction with metered dose inhalers (MDIs), dry powder inhalers, and nebulizers More specifically, the particulate suspensions of the present invention may be designed to decrease the attractive forces between particles. The principal forces driving fiocculation in nonaqueous media are van der Waals attractive forces. Van der Waals forces are quantum mechanical in origin, and can be visualized as attractions between fluctuating dipoles (i.e. induced dipole-iπduced dipole interactions). Dispersion forces are extremely short range and scale as the sixth power of the distance between atoms. When two macroscopic bodies approach one another, the dispersion attractions between the atoms sum up. The resulting force is of considerably longer range, and depends on the geometry of the interacting bodies.

More specifically, for two spherical particles, the magnitude of the van der Waals potential, V _A , can be approximated by _{v =} - ^A ,JJ R _t R _ where A_eff \s the effective Hamaker constant which accounts for 6 H _β ( Λ , + Λ . ) the nature of the particles and the medium, H₀ is the distance between particles, and R, and R₂ are the radii of spherical particles 1 and 2. The effective Hamaker constant is proportional to the difference in the polanzabilities of the dispersed particles and the suspension medium, l = (^jA_Sil - /A _PΛRT ) ² , where

A and A_P4RT are the Hamaker constants for the suspension medium and the particles, respectively As the suspended particles and the dispersion medium become similar in nature, A_M and A_PART become closer in magnitude, and A_eff and V_A become smaller. That is, by reducing the differences between the Hamaker constant associated with suspension medium and the Hamaker constant associated with the dispersed particles, the effective Hamaker constant (and corresponding van der Waals attractive forces) may be reduced.

One way to minimize the differences in the Hamaker constants is to create a "homodispersion", that is make both the continuous and dispersed phases essentially indistinguishable as discussed above In addition to exploiting the morphology of the particles to reduce the effective Hamaker constant, the components of the structural matrix (defining the perforated microstructures) will preferably be chosen so as to exhibit a Hamaker constant relatively close to that of the selected suspension medium. In this respect, one may use the actual values of the Hamaker constants of the suspension medium and the particulate components to determine the compatibility of the dispersion ingredients and to provide a good indication as to the stability of the preparation. Alternatively, one could select relatively compatible perforated microstructure components and suspension mediums using readily discernible characteristic physical values that coincide with measurable Hamaker constants.

In this respect, it has been found that the refractive index values of many compounds tend to scale with the corresponding Hamaker constant Accordingly, easily measurable refractive index values may be used to provide a fairly good indication as to which combination of suspension medium and particle excipients will provide a dispersion having a relatively low effective Hamaker constant and associated stability It will be appreciated that, since refractive indices of compounds are widely available or easily derived, the use of such values allows for the formation of stabilized dispersions in accordance with the present invention without undue experimentation. For the purpose of illustration only, the refractive indices of several compounds compatible with the disclosed dispersions are provided in Table I immediately below.

Table I

Compound Refractive Index

HFA 134a 1.172

HFA-227 1.223

CFC 12 1.287

CFC-114 1.288

PFOB 1.305

Mannitol 1.333

Ethanol 1 361 n octane 1.397

DMPC 1 3

Pluronic F 68 1.43

Sucrose 1 538

Hydroxyethylstarch 1.54

Sodium chloride 1.544

Consistent with the compatible dispersion components set forth above, those skilled in the art will appreciate that the formation of dispersions wherein the components have a refractive index differential of less than about 0.5 is preferred. That is, the refractive index of the suspension medium will preferably be within about

0.5 of the refractive index associated with the suspended particles or perforated microstructures. It will further be appreciated that, the refractive index of the suspension medium and the particles may be measured directly or approximated using the refractive indices of the major component in each respective phase. For the particles or perforated microstructures, the major component may be determined on a weight percent basis. For the suspension medium, the major component will typically be derived on a volume percentage basis. In selected embodiments of the present invention, the refractive index differential value will preferably be less than about 0.45, about 0 4, about 0.35 or even less than about 0.3. Given that lower refractive index differentials imply greater dispersion stability, particularly preferred embodiments comprise index differentials of less than about 0.28, about 0.25, about 0.2, about 0.15 or even less than about 0.1. It is submitted that a skilled artisan will be able to determine which dispersion components are particularly compatible without undue experimentation given the instant disclosure. The ultimate choice of preferred components will also be influenced by other factors, including biocompatibility, regulatory status, ease of manufacture and cost In contrast to prior art attempts to provide stabilized suspensions which require surfactants that are soluble in the suspension medium, the present invention may provide stabilized dispersions, at least in part, by immobilizing the bioactive agent(s) within the structural matrix of the hollow, porous microstructures. Accordingly, preferred excipients useful in the present invention are substantially insoluble in the suspension medium. Under such conditions, even surfactants like, for example, lecithin cannot be considered to have surfactant properties in the present invention since surfactant performance requires the amphiphile to be reasonably soluble in the suspension medium The use of insoluble excipients also reduces the potential for particle growth by Ostwald ripening.

As discussed above, the minimization of density differences between the particles and the continuous phase may be improved by the perforated and/or hollow nature of incorporated microstructures, such that the suspension medium constitutes most of the particle volume. As used herein, the term "particle volume" corresponds to the volume of suspension medium that would be displaced by the incorporated hollow/porous particles if they were solid, i e. the volume defined by the particle boundary For the purposes of explanation these fluid filled particulate volumes may be referred to as "virtual particles." Preferably, the average volume of the bioactive agent and/or excipient shell or matrix (i.e. the volume of medium actually displaced by the perforated microstructure) comprises less than 70% of the average particle volume (or less than 70% of the virtual particle). More preferably, the volume of the microparticulate matrix comprises less than about 50%, 40%, 30% or even 20% of the average particle volume. Even more preferably, the average volume of the shell/matrix comprises less than about 10%, 5% or 3% of the average particle volume Those skilled in the art will appreciate that such matrix, or shell volumes typically contribute little to the virtual particle density that is overwhelmingly dictated by the suspension medium found therein. Of course, in selected embodiments the excipients or bioactive agents used to form the perforated microstructure may be chosen so the density of the resulting matrix or shell approximates the density of the surrounding suspension medium.

It will be appreciated that, the use of such microstructures will allow the apparent density of the virtual particles to approach that of the suspension medium. Moreover, as previously discussed, the components of the microparticulate matrix are preferably selected, as much as possible given other considerations, to approximate the density of suspension medium. Accordingly, in preferred embodiments of the present invention the virtual particles and the suspension medium will have a density differential of less than about 0.6 g/cm³. That is, the mean density of the virtual particles (as defined by the matrix boundary) will be within approximately 0.6 g/cm³ of the suspension medium. More preferably, the mean density of the virtual particles will be within 0.5, 0.4, 0.3 or

0.2 g/cm of the selected suspension medium In even more preferable embodiments the density differential will be less than about 0.1 , 0.05, 0.01 , or even less than 0.005 g/cm³. In addition to the aforementioned advantages, the use of hollow, porous particles allows for the formation of free-flowing dispersions comprising much higher volume fractions of particles in suspension. It should be appreciated that, the formulation of prior art dispersions at volume fractions approaching close packing generally results in dramatic increases in dispersion viscoeiastic behavior. Rheological behavior of this type is not appropriate for inhalation applications Those skilled in the art will appreciate that, the volume fraction of the particles may be defined as the ratio of the apparent volume of the particles (i.e. the particle volume) to the total volume of the system. Each system has a maximum volume fraction or packing fraction. For example, particles in a simple cubic arrangement reach a maximum packing fraction of 0.52, while those in a face centered cubic/hexagonal close packed configuration reach a maximum packing fraction of approximately 0 74 For non spherical particles or polydisperse systems, the derived values are different. Accordingly, the maximum packing fraction is often considered to be an empirical parameter for a given system.

Here, it was surprisingly found that, the use of porous structures in the present invention did not introduce undesirable viscoeiastic behavior even at high volume fractions approaching close packing To the contrary, they remain as free flowing, low viscosity suspensions having little or no yield stress when compared with analogous suspensions comprising solid particulates The low viscosity of disclosed preferred suspensions is thought to be due, at least in large part, to the relatively low van der Waals attraction between the fluid filled hollow, porous particles. As such, in selected embodiments the volume fraction of the disclosed dispersions is greater than approximately 0.3. Other embodiments may have packing values on the order of 0.3 to about 0.5 or, on the order of 0 5 to about 0.8, with the higher values approaching a close packing condition Moreover, as particle sedimentation tends to naturally decrease when the volume fraction approaches close packing, the formation of relatively concentrated dispersions may further increase formulation stability

Although the methods and compositions of the present invention may be used to form relatively concentrated suspensions, the stabilizing factors work equally well at much lower packing volumes and, such dispersions are contemplated as being within the scope of the instant disclosure. In this regard, it will be appreciated that dispersions comprising low volume fractions are extremely difficult to stabilize using prior art techniques. Conversely, dispersions incorporating perforated microstructures comprising a bioactive agent as described herein are particularly stable even at low volume fractions Accordingly, the present invention allows for stabilized dispersions, and particularly respiratory dispersions, to be formed and used, at volume fractions less than 0 3. In some preferred embodiments, the volume fraction is approximately 0 0001 0.3, or more preferably 0.001 - 0 01. Yet other preferred embodiments comprise stabilized suspensions having volume fractions from approximately 0 01 to approximately 0.1

In other preferred embodiments, perforated microstructures may be used to stabilize dilute suspensions of micromzed bioactive agents. In such embodiments the perforated microstructures mav be added to increase the volume fraction of particles in the suspension, thereby increasing suspension stability with respect to creaming or sedimentation. Further, in these embodiments, the incorporated microstructures may also act in preventing close approach (aggregation) of micromzed drug particles. It should be appreciated that, the perforated microstructures incorporated in such embodiments do not necessarily comprise a bioactive agent. Rather, they may be formed exclusively of various excipients, including surfactants.

Of course, it will also be appreciated that the stabilized dispersions of the present invention may comprise relatively solid or non perforated particulates without the addition of perforated microstructures That is, depending on the size, composition and density of the suspended microparticulates, as well as the selection of suspension medium, effective particulate dispersions for nebulization may be formed using relatively non porous or micromzed particulates. In a preferred embodiment, the suspended particulates may comprise πanocrystals such as those disclosed in U.S. Pat. No 5,667,809 which is incorporated herein by reference. As with embodiments comprising perforated microstructures, such preparations will preferably comprise a fluorochemical suspension medium. Accordingly, in a broad sense, the present invention provides for the formation and pulmonary administration of stabilized dispersions comprising relatively non porous particulates (e.g. micromzed particles), porous particulates (i e. hollow porous microspheres or perforated microstructures) and combinations thereof

While the stabilized dispersions may comprise particulates exhibiting various morphologies, particularly preferred embodiments of the present invention comprise a plurality of perforated microstructures or microparticulates that are dispersed, or suspended in the suspension medium. In such embodiments, the perforated microstructures compπse a structural matrix that exhibits, defines or composes voids, pores, defects, hollows, spaces, interstitial spaces, apertures, perforations or holes that allows the surrounding suspension medium to freely permeate, fill or pervade the microstructure The absolute shape (as opposed to the morphology) of the perforated microstructure is generally not cπtical and. any overall configuration that provides the desired stabilization characteristics is contemplated as being within the scope of the invention Accordingly, while preferred embodiments incorporating perforated microstructures can comprise approximately microspherical shapes, collapsed, deformed or fractured particulates are also compatible. With that caveat, it will be appreciated that particularly preferred embodiments of the invention compπse spray dπed hollow, porous microspheres.

In order to maximize dispersion stability and optimize distπbution upon administration, the mean geometric particle size of the perforated microstructures is preferably about 0.5 50 m, more preferably 1 30 m. It will be appreciated that, large particles (i.e. greater than 50 m) should not be used as large particles may tend to aggregate or, separate from the suspension and not be effectively nebulized. In especially preferred embodiments, the mean geometπc particle size (or diameter) of the perforated microstructures is less than 20 m or less than 10 m. More preferably, the mean geometπc diameter is less than about 5 m. In especially preferred embodiments, the perforated microstructures will compπse a powder of dry, hollow, porous microspherical shells of approximately 1 to 10 m m diameter, with shell thicknesses of approximately 0.1 m to approximately 0 5 m. It is a particular advantage of the present invention that, the particulate concentration of the dispersions and structural matπx components can be adjusted to optimize the delivery charactenstics of the selected particle size

As indicated throughout the instant specification, the dispersions of the present invention are preferably stabilized. In a broad sense, the term "stabilized dispersion" will be held to mean any dispersion that resists aggregation, fiocculation or creaming to the extent required to provide for the effective delivery of a bioactive agent. While those skilled in the art will appreciate that there are several methods that may be used to assess the stability of a given dispersion, a preferred method for the purposes of the present invention comprises determination of creaming or sedimentation time. In this regard, the creaming time shall be defined as the time for the suspended drug particulates to cream to 1/2 the volume of the suspension medium. Similarly, the sedimentation time may be defined as the time it takes for the particulates to sediment in 1 /2 the volume of the liquid medium One relatively simple way to determine the creaming time of a preparation is to provide the particulate suspension in a sealed glass vial. The vials are agitated or shaken to provide relatively homogeneous dispersions which are then set aside and observed using appropπate instrumentation or by visual inspection. The time necessary for the suspended particulates to cream to 1 /2 the volume of the suspension medium (i.e , to rise to the top half of the suspension medium), or to sediment within 1/2 the volume (i.e., to settle in the bottom 1 /2 of the medium), is then noted. Suspension formulations having a creaming time greater than 1 minute are preferred and indicate suitable stability. More preferably, the stabilized dispersions compπse creaming times of greater than about 2, 5, 10, 15, 20 or 30 minutes. In particularly preferred embodiments, the stabilized dispersions exhibit creaming times of greater than about 1, 1 5, 2, 2.5, 3, 4 or even 5 hours Substantially equivalent periods for sedimentation times are indicative of compatible dispersions.

With respect to the preparations of the present invention, the porosity of incorporated microstructures may contribute significantly to establishing dispersion stability. In this respect, the mean porosity of the perforated microstructures may be determined through electron microscopy coupled with modern imaging techniques More specifically, electron micrographs of representative samples of the perforated microstructures may be obtained and digitally analyzed to quantify the porosity of the preparation. Such methodology is well known in the art and, may be undertaken without undue experimentation.

For the purposes of the present invention, the mean porosity (i.e the percentage of the particle surface area that is open to the interior and/or a central void) of the perforated microstructures may range from approximately 0.5% to approximately 80% In more preferred embodiments, the mean porosity will range from approximately 2% to approximately 40%. Based on selected production parameters, the mean porosity may be greater than approximately,

2%, 5%, 10%, 15%, 20%, 25% or 30% of the microstructure surface area In other embodiments, the mean porosity of the microstructures may be greater than about 40%, 50%, 60%, 70% or even 80%. As to the pores themselves, they typically range in size from about 5 nm to about 400 nm, with mean pore sizes preferably in the range of from about 20 nm to about 200 nm. In particularly preferred embodiments the mean pore size will be in the range of from about 50 nm to about 100 nm. As may be seen in Figs. 1 A1 to 1 F2, and discussed in more detail below, it is a significant advantage of the present invention that the pore size, and porosity, may be closely controlled by careful selection of the incorporated components and production parameters. Along with the geometric configuration, the perforated or porous and/or hollow design of microstructures can also play an important role in the resulting aerosol properties during nebulization. In this respect, the perforated structure, and relatively high surface area of the dispersed microparticles, enables them to be earned along in the aerosol cloud during inhalation with greater ease and, for longer distances, than non perforated particles of comparable size. Because of their high porosity, the density of the particles is significantly less than 1.0 g/cm³, typically less than 0.5 g/cm³, more often on the order of 0.1 g/cm³, and as low as 0.01 g/cm³. Unlike the geometric particle size, the aerodynamic particle size, d_aer , of the perforated microstructures depends substantially on the particle density, p : d_aer = d_geop , where d_geo is the geometric diameter. For a particle density of 0 1 g/cm³, d_aer will be roughly three times smaller than d leading to increased particle deposition into the peripheral regions of the lung and correspondingly less deposition in the throat. In this regard, the mean aerodynamic diameter of the perforated microstructures is preferably less than about 5 μm, more preferably less than about 3 μm, and, in particularly preferred embodiments, less than about 2 μm. Such particle distributions will act to increase the deep lung deposition of the administered agent.

As will be shown subsequently in the Examples, the particle size distribution of the aerosol formulations of the present invention are measurable by conventional techniques such as cascade i paction, or by time of flight analytical methods. Determination of the emitted dose in nebulized inhalations was done according to the proposed U S Pharmacopeia method [Pharmacopeia! Previews, 22( 1996) 3065) which is incorporated herein by reference These and related techniques enable the "fine particle fraction ' of the nebulized aerosol, which corresponds to those particulates that are likely to effectively deposited in the lung, to be calculated As used herein, the phrase "fine particle fraction" refers to the percentage of the total amount of active medicament delivered per actuation from the mouthpiece onto plates 2 7 of an 8 stage Andersen cascade impactor Based on such measurements, the formulations of the present invention will preferably have a fine particle fraction for local airway delivery of approximately 20% or more, by weight of the perforated microstructures (w/w) More preferably, they will exhibit a fine particle fraction of from about 25% to 80% w/w, and even more preferably from about 30 to 70% wlw. In selected embodiments the present invention will preferably comprise a fine particle fraction of greater than about 30%, 40%, 50%, 60%, 70% or 80% by weight. For systemic delivery, the fine particle fraction will preferably be greater than 80% by weight, more preferably, greater than 90% by weight Whatever configuration and/or size distπbution is ultimately selected for the incorporated particulate (whether a perforated microstructure or relatively solid non porous particulate), the composition thereof may compπse any one of a number of biocompatible materials. With regard to perforated microstructures, it will be appreciated that, as used herein, the terms "structural matπx" 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 promote the formation of stabilized dispersions as explained above. The structural matnx may be soluble or insoluble in an aqueous environment. In preferred embodiments the perforated microstructure defined by the structural matπx compπses a spray dried hollow porous icrosphere incorporating at least one surfactant. For other selected embodiments, the particulate material may be coated one or more times with polymers, surfactants or other compounds which aid suspension.

More generally, particulates useful in the stabilized dispersions of the present invention may be formed of any biocompatible mateπal that is relatively stable and preferably insoluble with respect to the selected suspension medium. While a wide variety of materials may be used to form the particles, in particularly preferred embodiments, the particles (or structural matrix) is associated with, or comprises, a surfactant such as phospholipid or fluoπnated surfactant. Although not required, incorporation of a compatible surfactant can improve the stability of the respiratory dispersions, increase pulmonary deposition and facilitate the preparation of the suspension. Moreover, by alteπng the components, the density of the particle or structural matπx may be adjusted to approximate the density of the surrounding medium and further stabilize the dispersion, finally, as will be discussed in further detail below, perforated microstructures preferably compπse at least one bioactive agent. As set forth above, the relatively non porous particles or perforated microstructures of the present invention, may optionally be associated with, or compπse, one or more surfactants. Moreover, miscible surfactants may optionally be combined with the suspension medium liquid phase. It will be appreciated by those skilled in the art that, the use of surfactants, while not necessary to practice the instant invention, may further increase dispersion stability, simplify formulation procedures or increase bioavailability upon administration. Of course combinations of surfactants, including the use of one or more in the liquid phase and one or more associated with the perforated microstructures are contemplated as being within the scope of the invention. By "associated with or comprise" it is meant that the particle or perforated microstructure may incorporate, adsorb absorb, be coated with or be formed by the surfactant

In a broad sense, surfactants suitable for use in the present invention include any compound or composition that aids in the formation and maintenance of the stabilized respiratory dispersions by forming a layer at the interface between the particle and the suspension medium. The surfactant may compπse a single compound or any combination of compounds, such as in the case of co surfactants. Particularly preferred surfactants are substantially insoluble in the medium, noπfluoriπated, and selected from the group consisting of saturated and unsaturated lipids, nonioπic detergents, nonionic block copolymers, ionic surfactants, and combinations of such agents It should be emphasized that, in addition to the aforementioned surfactants, suitable (i.e. biocompatible) fluαπnated surfactants are compatible with the teachings herein and may be used to provide the desired stabilized preparations.

Lipids, including phospholipids, from both natural and synthetic sources are particularly compatible with the present invention and may be used in varying concentrations to form the particle or structural matπx.

Generally compatible lipids comprise those that have a gel to liquid crystal phase transition greater than about 40°C. Preferably, the incorporated lipids are relatively long chain (i.e. C_l8 Cy saturated lipids and more preferably compπse phospholipids. Exemplary phospholipids useful in the disclosed stabilized preparations comprise egg phosphatidylcholine, dilauroγlphosphatidylcholine, dioleylphosphatidylcholine, dipalmitoylphosphatidyl c olme, disteroγlphosphatidylchohne, short-chain phosphatidylcholines, phosphatidylethaπolamine, dioleylphosphatidylethanolamine, phosphatidylseπne, phosphatidylglycerol, phosphatidylinositol, glyco pids, gaπglioside GM1, sphingomγehπ, phosp atidic acid, cardiolipin; lipids beaπng polymer chains such as polyethylene glγcol, chitin, hyaluronic acid, or poiyvinylpγrrolidone; lipids bearing sulfonated mono , di , and polysacchaπdes; fatty acids such as palmitic acid, steaπc acid, and oleic acid; cholesterol, cholesterol esters, and cholesterol hemisuccinate. Due to their excellent biocompatibility characteπstics, phospholipids and combinations of phospholipids and poloxamers are particularly suitable for use in the stabilized dispersions disclosed herein.

Compatible πomomc detergents comprise: sorbitan esters including sorbitaπ tπoleate (Span⁰¹ 85), sorbitan sesquiolεate, sorbitaπ monooleate, sorbitaπ moπolaurate, polyoxyethylene (20) sorbitaπ 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 (McPub shing Co., Glen Rock, New Jersey) which is incorporated herein in its entirety. Preferred block copolymers include diblock and tπblock copolymers of polyoxyethylene and polyoxypropylene, including poloxamer 188 (Pluromc" F 68), poloxamer 407 (Pluromc " F 127), and poloxamer 338. Ionic surfactants such as sodium sulf osuccmate, and fatty acid soaps may also be utilized. In preferred embodiments the microstructures may compnse oleic acid or its alkali salt.

In addition to the aforementioned surfactants, catioπ'c surfactants or lipids are preferred especially in the case of delivery or RNA or DNA Examples of suitable cationr lipids include- cetylpyπdinium chloride, DOTMA, N [1-12,3 dιoleyloxy)propyl]-N,I\l,ιM tπmethylammonιum chloπde; DOTAP, 1,2 dioleyioxy 3

(tπmethylammonιo)propane; and DOTB, 1,2 dtoleyl 3 (4' tπmεthylammonio) butanoyi sn glycerol Polycationic ammo acids such as polylysiπe, and polyarginme are also contemplated.

Those skilled in the art will further appreciate that, a wide range of surfactants, including those not listed above, may optionally be used in conjunction with the present invention. Moreover, the optimum surfactant, or combination thereof, for a given application can readily be determined by empiπcal studies that do not require undue experimentation. It will further be appreciated that, the preferred insolubility of any incorporated surfactant in the suspension medium will dramatically decrease the associated surface activity As such, it is arguable as to whether these materials have surfactant like character prior to contracting an aqueous bioactive surface (e g the aqueous hypophase in the lung) Finally, as discussed in more detail below, surfactants comprising the porous particles may also be useful in the formation of precursor oil in water emulsions (i.e. spray drying feed stock) used during processing to form the structural matrix or bioactive particulate

Unlike pπor art formulations, it has surpπs gly been found that the incorporation of relatively high levels of surfactants (i e. phospholipids) may be used to increase the stability of the disclosed dispersions. That is, on a weight to weight basis, the structural matnx of the perforated microstructures may comprise relatively high levels of surfactant. In this regard, the perforated microstructures will preferably compπse greater than about 1 %, 5%, 10%, 15%, 18%, or even 20% w/w surfactant. More preferably, the perforated microstructures will comprise greater than about 25%, 30%, 35%, 40%, 45%, or 50% w/w surfactant Still other exemplary embodiments will comprise perforated microstructures wherein the surfactant or surfactants are present at greater than about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or even 95% w/w In selected embodiments the perforated microstructures will compπse essentially 100% w/w of a surfactant such as a phospholipid. Those skilled in the art will appreciate that, in such cases, the balance of the structural matπx (where applicable) will likely compπse a bioactive ageπt(s) or non surface active excιpιent(s) or addιtιve(s)

As previously indicated, stabilized dispersions comprising perforated microstructures merely represent a preferred embodiment of thε present invention Accordingly, while such surfactant levels are preferably employed in perforated microstructures, equivalent surfactant levels may also be used to provide stabilized systems compnsing relatively nonporous, or substantially solid, particulates That is, while preferred embodiments will compπse perforated microstructures or microspheres associated with high levels of surfactant, acceptable dispersions may be formed using relatively low or non porous particulates (e g micro zed particulates) of the same surfactant concentration In this respect such embodiments are specifically contemplated as being within the scope of the present invention. In other preferred embodimεnts, relatively non porous particles or the structural matπx defining the perforated microstructures optionally compπses synthetic or natural polymers or combinations thereof. In this respect useful polymers compπse polylactides, polylactide glycolides, cyclodextπns, polyacrylates, methylcellulose carboxymethylcellulose, polyvinyl alcohols, polyanhydπdes, polylactons, polyviπyl pyrrolidoπes, polysacchandes (dextraπs, starches, chitin, chitosan, etc ), hyaluronic acid, proteins, (albumin, collagen, gelatin, etc ) Those skilled in the art will appreciate that, by selecting the appropπate polymers, the delivery profile of the respiratory dispersion may be tailored to optimize thε effectiveness of the bioactive agent

In addition to the aforementioned polymer materials and surfactants, it may be desirable to add other excipients to an inhalation formulation to improve microsphere (or non porous particulate) rigidity, drug delivery and deposition, shelf life and patient acceptance. Such optional excipients include, but are not limited to: coloring agents, taste masking agents, buffers, hygroscopic agents, antioxidaπts, and chemical stabilizers. Further, excipients may be incorporated in, or added to, the particles or particulate matrix to provide structure and form to the perforated microstructures (i.e. microspheres). Such excipients may include, but are not limited to, carbohydrates including monosacchaπdes, disacchaπdes and polysacchandes. For example, monosacchandes such as dextrose (anhydrous and moπohydrate), galactose, mannitol, D maππose, sorbitol, sorbose and the like, disacchaπdes such as lactose, maltosε, sucrose, trehalose, and the like; tπsacchaπdes such as raffmose and the like; and other carbohydrates such as starches (hydroxyethylstarch), cyclodextnπs and maltodextnns. Ammo acids are also suitable excipients with glycine prefεrred. Mixtures of carbohydrates and ammo acids are further held to be within the scope of the present invention. The inclusion of both inorganic (e.g. sodium chloride, calcium chloride), organic salts (e.g. sodium citrate, sodium ascorbate, magnesium gluconate, sodium gluconate, tromethamine hydrochloπde) and buffers is also contemplated. Of course, it will be appreciated that, the selected excipients may be added to the dispersion as separate particles or perforatεd microstructures

Yet other preferred embodiments include non porous particles or perforated microstructures that may comprise, or may be coated with, charged speciεs that prolong residence time at thε 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 microparticulate with negatively charged bioactive agents such as genetic mateπal. The charges may be imparted through the association or incorporation of polyanionic or polycationic mateπals such as polyacrylic acids, polylysinε, polylactic acid and chitosan In addition to, or instead of, the components discussed above, the particles, perforated microstructures or aqueous emulsion droplets will preferably comprise at least one bioactive agent. As used herein, "bioactive agent" refers to a substance which is used in connection with an application that is therapeutic or diagnostic in nature, such as in methods for diagnosing the presεnce or absence of a disease in a patient and/or in methods for treating a disεasε in a patient. Particularly preferred bioactive agents for use in accordance with the invention include anti allergies, peptides and proteins, bronchodilators and anti inflammatory steroids for use in the treatmεnt of respiratory disorders such as asthma by inhalation therapy.

It will be appreciated that, the distributed particles or perforated microstructures of the present invention may exclusively compπse one or more bioactive agents (i.e. 100% w/w). However, in selected embodiments the particles or perforatεd microstructures may incorporatε much less bioactive agent depending on thε activity thereof. Accordingly, for highly active mateπals, the particles may incorporate as little as 0.001 % by weight, although a concentration of greater than about 0 1 % w/w is preferred Other εmbodimεnts of thε invention may compπse greater than about 5%, 10%, 15%, 20%, 25%, 30% or, even 40% w/w bioactive agent. Still more preferably, the particles or perforated microstructures may compπse greater than about 50%, 60%, 70%, 75%, 80% or, even 90% w/w bioactive agent. In particulariy preferred embodiments, the final stabilized respiratory dispersion desirably contains from about 40% 60% w/w, more preferably 50% - 70% w/w, and even more preferably, 60% 90% w/w of bioactive agent relative to the weight of the microparticulate matrix or particulate The precise amount of bioactive agent incorporated in the stabilized dispersions of the present invention is dependent upon thε agent of choice, the required dose, and the form of the drug actually used for incorporation Those skilled in the art will appreciate that, such determinations may be made by using well-known pharmacological techniques, in combination with the teachings of the present invention.

Accordingly, bioactive agents that may be administered in thε form of aerosolized medicaments in conjunction with the teachings herein include any drug that may be presented in a form which is subject to pulmonary uptake in physiologically effective amounts. In selected εmbodimεnts (ε g particulate dispersions), the incorporated agent will preferably be relatively insoluble in the suspension medium. In other embodiments, such as reverse emulsions, thε sεlεcted agεnt may bε substantially soluble in the disperse phase Particularly preferred embodiments compnsing a reverse emulsion will preferably comprise a hydrophilic bioactive agent.

In any case, compatible bioactive agents may compπse hydrophilic and lipophilic respiratory agents, bronchodilators, antibiotics, antivirals, anti inflammatoπes, steroids, antihistaminics, histamine antagonists, leukotnene inhibitors or antagonists, anticholinergics, antineoplastics, anesthetics, enzymεs, lung surfactants, cardiovascular agents, genetic material including DNA and RNA, viral vectors, immuπoactive agents, imaging agents, vaccines, immunosuppressive agents, peptides, proteins and combinations thereof Particularly prefεrred bioactive agents, for local administration using aerosolized medicaments in accordance with the present invention include, mast cell inhibitors (anti allergies), bronchodilators, and anti inflammatory steroids for use in the treatment of respiratory disorders such as asthma by inhalation therapy, for example cromoglycate (e g. the sodium salt), and albuterol

(e g thε sulfate salt). For systemic delivery (e.g for the treatment of autoimmune diseases such as diabetes or multiple sclerosis), peptides and proteins are particularly prefεrred.

Exemplary medicaments or bioactive agents may bε selεcted from for example, analgesics, e g codeinε, dihydromorphine, ergotamine, fentaπyl, or morphine, anginal preparations, e.g. diltiazem, mast cell inhibitors, e.g. cromolyn sodium; anti fectives, e g cephalospoπns, macrolides, qumolines, penicillins, streptomycin, sulphonarnides, tetracychnes and pentamidiπe, antihistamines, e.g methapyπlene, anti-iπflammatoπes, e.g. fluticasone propionate, heclomεthasonε dipropionate, flumsolide budesonide, tnpedane, cortisone, prednisone, prednisilone, dexamethasoπe, betamethasone, or tπamcinolone acetonide, antitussives, e g. noscapiπe, bronchodilators, e g ephedπnε, adrenaline, fenotεrol, formotεrol, isoprεnahne, metaproterεnol, salbutamol, albuterol, saimeterol, terbutaliπe; diuretics, e.g. amiloπde, anticholinergics, e.g. ipatropium, atropine, or oxitropium, lung surfactants e.g Surfaxin, Exosurf, Survanta; xanthines, e g ammophylline, theophylline, caffeine; therapeutic proteins and peptides, e.g. DNAse, insulin, glucagon, T cell receptor agonists or antagonists, LHRH, πafarelm, goserelin, leuprohdε, interferon, rhu IL 1 receptor, macrophage activation factors such as ly phokinεs and muramyl dipeptidεs, opioid peptides and neuropeptidεs such as εnkaphalms, εndorphins, rεnm inhibitors, cholecystokimns, growth hormones, leukotπεne inhibitors, α antitrypsin, and the like In addition, bioactive agents that comprise an RNA or DNA sequence, particularly those useful for gene therapy, genetic vaccination or tolenzation or antisense applications, may be incorporated in the disclosed dispersions as descπbed herein. Representative DNA plasmids include pCMVβ (available from Genzyme Corp, Framington, MA) and pCMV β gal (a

CMV promotor linked to the E coli Lac Z gene, which codes for thε εnzymε β galactosidasε)

With respect to particulate dispersions, thε sεlεctεd bioactive agent(s) may bε associatεd with, or incorporated in, the particles or perforated microstructures in any form that provides the desired efficacy and is compatible with the chosen production techniques. Similarly, the incorporated bioactive agent may be associated with the discontinuous phase of a reverse emulsion. As used herein, the terms "associate" or "associating" mean that thε structural matrix, perforated microstructure, relatively non porous particle or discontinuous phase may compπsε, incorporate, adsorb, absorb, be coated with or be formed by the bioactive agent. Wherε appropriate, the medicaments may be used in the form of salts (ε g. alkali metal or amine salts or as acid addition salts), or as esters, or as solvates (hydrates). In this regard, the form of the bioactive agents may be selected to optimize the activity and/or stability of thε medicament and/or, to minimize the solubility of the mεdicamεnt in the suspension medium.

It will further be appreciated that the aerosolized formulations according to the invention may, if desired, contain a combination of two or more active ingredients The agents may be provided in combination in a single species of perforated microstructure or particle or individually in separate species that are combined in the suspension medium or continuous phase For example, two or morε bioactive agents may bε incorporated in a single fεed stock preparation and spray dried to provide a single microstructure species comprising a plurality of medicaments Convεrsεly, the individual medicaments could be added to separate stocks and spray dried separately to provide a plurality of microstructure species with different compositions. These individual species could be added to the medium in any desired proportion and placed in inhalation delivery systems as described below. Further, as briefly mentioned above, the perforated microstructures (with or without an associated medicament) may be combined with onε or morε conventionally micromzed bioactive agents to provide the desired dispersion stability.

Based on the foregoing, it will be appreciated by those skilled in the art that a widε vaπεty of bioactive agents may be incorporated in thε disclosed stabilized dispersions. Accordingly, the list of preferred bioactive agents above is exemplary only and not intendεd to be limiting It will also bε apprεciatεd by those skilled in the art that, the proper amount of bioactive agent and the timing of the dosages may be determined for the formulations in accordance with already existing information and without undue expeπmentation As seen from the passages above, various components may be associatεd with, or incorporated in the discontinuous phase, perforated microstructures or particles of the present invention. Similarly, several techniques may be used to provide particulates having compatible physiochemical properties, morphology (i e. a perforatεd configuration) and density. Among other methods, perforated microstructures or particles compatible with the instant invention may be formed by techniques including lyophilization, spray drying, multiple emulsion, micrαnization, or crystallization In prefεrred embodiments, relatively non porous particles may be produced using techniques such as microπization, crystallization or milling. It will further bε appreciated that, the basic concepts of many of these techniques are well known in thε pπor art and would not, in view of the teachings herein, require undue experimentation to adapt them so as to provide the desired particulates While several procedures are generally compatible with the present invention, particularly preferred embodimεnts typically comprise particulates or perforated microstructures formed by spray drying. As is well known, spray drying is a one-step process that converts a liquid feed to a dried particulate form. With respect to pharmaceutical applications, it will be appreciated that spray drying has been used to provide powdered mateπal for various administrative routes including inhalation. See, for example, M Sacchetti and M M. Van Oort in- Inhalation Aerosols- Physical and Biological Basis for Therapy, A J Hickey, ed Marcel Dekkar, New York, 1996, which is incorporated herein by reference.

In gεπεral, spray drying consists of bringing togεthεr a highly dispersed liquid, and a sufficient volume of hot air to produce evaporation and drying of the liquid droplets. The preparation to be spray dried or feed (or feed stock) can bε any solution, course suspension, slurry, colloidal dispersion or paste that may be atomized using the selected spray drying apparatus. Typically, the feed is sprayed into a current of warm filtered air that evaporates the solvent and conveys thε dπεd product to a collector The spent air is then exhausted with the solvent Those skilled in the art will appreciate that several different types of apparatus may be used to provide the desired product For example, commercial spray dryers manufactured by Buchi Ltd or Niro Corp will effectively produce particles of desired size. It will further be appreciated that these spray dryers, and specifically their atomizers, may be modified or customized for specialized applications, i.e. the simultaneous spraying of two solutions using a double nozzle technique. More specifically, a water in oil emulsion can be atomized from one nozzle and, a solution containing an anti adherεnt such as mannitol can be co atomized from a second nozzle In other cases it may be desirable to push the feed solution though a custom designed nozzle using a high pressure liquid chromatography (HPLC) pump Provided that microstructures compnsing the correct morphology and/or composition are produced, the choice of apparatus is not cπtical and would be apparent to the skilled artisan in view of the teachings herein.

While thε resulting spray dried powdered particles typically are approximately spherical in shapε, nearly uniform in size and frequently are hollow, there may be some degree of irregularity in shape depending upon the incorporated medicament and the spray drying conditions In many instances, thε dispersion stability of spray dried microspheres or particles appears to be more effective if an inflating agent (or blowing agent) is used in their production. Particularly preferred embodiments may compπse an emulsion with the inflating agent as the disperse or continuous phase (the othεr phasε bεing aqueous in nature) The inflating agent is preferably dispersed with a surfactant solution, using, for instance, a commercially available microfluidizer at a pressure of about 5,000 to 15,000 psi. This process forms an emulsion, preferably stabilized by an incorporated surfactant, typically compnsing submicron droplets of water immiscible blowing agent dispersed in an aqueous continuous phase. The formation of such dispersions using this, and othεr tεchniquεs, are common and wεll known to those in the art The blowing agent is preferably a fluoπnated compound (e.g. perfluorohexane, perfluorooctyl bromide, perfluorodecalm, perfluorobutyl ethane) which vaporizes during thε spray drying procεss, leaving behind generally hollow, porous aerodynamically light microspheres. As will be discussed in more detail below, other suitable blowing agents include chloroform, Frεons, and hydrocarbons Nitrogen gas and carbon dioxide are also coπtεmplatεd as suitable blowing agents.

Although perforated microstructures are preferably formed using a blowing agent as described above, it will bε appreciated that, in some instances, no additional blowing agent is requirεd and an aqueous dispersion of the medicament and surfactant(s) are spray dried directly. In such cases, the formulation may be amenable to process conditions (ε g , elevated temperatures) that generally lead to the formation of hollow, relatively porous microparticles. M oreover, the medicament may possess special physicochemical properties (e.g., high crystallinity, elevated melting tempεraturε, surfacε activity, etc.) that make it particularly suitable for use in such techniques.

When a blowing agent is employed, the degree of porosity of the perforated microstructure appears to depend, at least in part, on the nature of thε blowing agεnt, its concentration in the feεd stock (i.ε as an emulsion), and the spray drying conditions. With respect to controlling porosity, it has surprisingly been found that the use of compounds, heretofore unappreciated as blowing agents, may provide particulates or perforatεd microstructures having particularly desirable characteristics. More particularly, in this novel and unexpected aspect of the presεnt invention, it has been found that the use of fluoπnated compounds having relatively high boiling points (i.e. greater than about 60°C) may be used to produce particulates that are especially suitable for inhalation therapies. In this regard, it is possible to use fluonπated blowing agents having boiling points of greater than about 70°C, 80°C, 90°C or even 95°C. Particularly preferred blowing agents have boiling points greater than the boiling point of water, i.e. greater than 100°C (e.g. perflubroπ, perfluorodecalm) In addition, blowing agents with relatively low water solubility ( < 10 ° M) are preferred since they enable the production of stable emulsion dispersions with mean weighted particle diameters less than 0.3 um. As indicated above, these blowing agents will preferably be incorporated in an emulsified feed stock prior to spray drying For the purposes of the present invention this feed stock will also preferably comprise one or more bioactive agents, one or more surfactants, or one or more excipients. Of course, combinations of the aforemεntionεd componεnts are also within the scope of the invention.

While not limiting the invention in any way, it is hypothesized that, as the aqueous feed component evaporates during spray drying it leaves a thin crust at the surface of the particle. The resulting particle wall or crust, formed during the initial moments of spray drying, appears to trap any high boiling blowing agents as hundreds of emulsion droplets (ca. 200 300 nm). As the drying process continues, the pressure inside the particulate increases, thereby vaporizing at least part of the incorporated blowing agent and, forcing it through the relatively thin crust. This venting or outgassmg, apparently leads to the formation of pores or other defects in the crust. At thε same time, remaining particulate components (possibly including some blowing agent) migrate from the interior to the surface as the particle solidifies. This migration apparently slows during the drying process as a result of increased resistancε to mass transfεr causεd by an incrεasεd intεrnal viscosity. Oncε the migration ceases, the particle solidifies, leaving vesicles, vacuoles or voids where the emulsifying agent resided. The number of pores, their size, and the resulting wall thickness is largely depεndεnt on the nature of the selected blowing agent (i.e. boiling point), its concentration in the emulsion, total solids concentration, and the spray-drying conditions.

It has been surprisingly found that substantial amounts of these relatively high boiling point blowing agents may be retained in the resulting spray dπεd product. That is, thε spray dπεd pεrforated microstructurεs may comprise as much as 5%, 10%, 20%, 30% or even 40% w/w of the blowing agent. In such cases, higher production yields were obtained as a result an increased particle density causεd by residual blowing agεnt It will be appreciated by those skilled in the art that, this retained fluonnated blowing agent may alter the surface characteristics of the perforated microstructures and further increase thε stability of the respiratory dispersions Conversely, the residual blowing agent can easily be removed with a post production evaporation step in a vacuum oven. Optionally, pores may bε formεd by spray drying a bioactive agεnt and an excipiεnt that can bε removed from the formed microspheres under a vacuum. In any evεnt, typical coπcεntrations of blowing agεnt in thε feed stock are between 5% and 100% w/v, and more preferably, between about 20% to 90% w/v. In other embodiments, blowing agent concentrations will prefεrably bε greater than about 10%, 20%, 30%, 40% 50% or even 60% w/v Yet other feεd stock emulsions may comprise 70%, 80%, 90% or even 95% w/v of the selected high boiling point compound.

In preferred embodiments, another method of identifying thε concentration of blowing agent usεd in the fεed is, to provide it as a ratio of the concentration of the blowing agεnt to that of the stabilizing surfactant (i.e. phospholipid) in thε precursor emulsion For fluorocarbon blowing agents such as perfluorooctyl bromide and phosphatidylcholme, the ratio may be termed a perfluorocarbon/phosphatidylcholine ratio (or PFC/PC ratio). Of course, it will be apprεciatεd that othεr compatible surfactants may also be usεd to providε compatible particulates. In any evεnt, the PFC/PC ratio will typically range from about 1 to about 60 and more preferably, from about 10 to about 50. For preferred embodiments the ratio will generally be greater than about 5, 10, 20, 25, 30, 40 or even 50 In this respect, Fig. 1 shows a series of pictures taken of perforatεd microstructures formed of phosphatidylcholme (PC) using various amounts of perfluorooctyl bromide (PFC), a relatively high boiling point fluorocarbon as the blowing agent. The PFC/PC ratios are providεd undεr εach subsεt of pictures, i.e. from

1 A to 1 F. Formation and imaging conditions are discussed in greatεr detail in Examples I and II below. With regard to thε micrographs, thε column on thε left shows the intact microstructures while the column on the right illustrates cross-sections of fractured microstructures from the same preparations.

As may easily be seεn in the Fig 1 , the use of higher PFC/PC ratios provides structures of a more hollow and porous nature. More particularly, those methods employing a PFC/PC ratio of greater than about 4.8 tended to providε structures that arε particularly compatible with the dispersions disclosed herein Similarly, Fig. 2, a micrograph which will be discussed in more detail in Example II below, illustrates a preferably porous morphology obtained by using higher boiling point blowing agents (in this case perfluorodecalm)

While relatively high boiling point blowing agents comprise one preferred aspect of the instant invention, it will bε appreciated that more conventional blowing or inflating agεnts may also bε usεd to providε compatible perforated microstructures. Generally, the inflating agent can be any mateπal that will turn to a gas at some point dunng the spray drying or post production process. Suitable agents include: 1. Dissolved low boiling (below 100 C) solvents with limited miscibility with aqueous solutions, such as methyleπε chloπdε, acetone and carbon disulfidε used to saturate thε solution at room tεmpεraturε. 2. A gas, e.g. C0₂ or N->, used to saturate the solution at room temperature and elevated pressure (e.g. 3 bar). The droplets arε then supersaturated with the gas at 1 atmosphere and 100 C 3. Emulsions of immiscible low boiling (below 100 C) liquids such as Freoπ 113, perfluoropentane. perfluorohexane, perfluorobutane, pentanε, butane, FC 1 1 , FC 11 B1 , FC 1 1 B2, FC 12B2, FC 21, FC 21 B1, FC

21 B2, FC 31 B1, FC 113A, FC 122, FC 123, FC-132, FC 133, FC-141, FC 141 B, FC 142, FC 151 , FC-152, FC 1112, FC 1121 and FC 1131.

With respect to these lower boiling point inflating agents, they are typically added to the feed stock in quantities of about 1 % to 40% v/v of the surfactant solution Approximately 15% v/v inflating agent has been found to produce a spray dπed powder that may be used to form the stabilized dispersions of the present invention.

Regardless of which blowing agent is ultimately selectεd, it has been found that compatible perforated microstructures or particles may be produced particularly efficiently using a Buchi mini spray drier (model B 191 ,

Switzerland) As will be apprεciatεd by thosε skillεd in thε art, thε inlet tεmpεraturε and thε outlεt tεmpεrature of the spray drier are not critical but will be of such a level to provide the desired particle size and to result in a product that has thε dεsirεd activity of thε medicament In this regard, the inlet and outlet temperatures are adjusted depending on the melting characteristics of thε formulation components and the composition of thε feed stock. The inlet temperature may thus be between 60°C and 170°C, with the outlet tεmperatures of about 40°C to 120°C depending on the composition of the feed and the desired particulate characteristics. Preferably, these temperaturεs will be from 90°C to 120°C for the inlet and from 60°C to 90°C for thε outlet. The flow rate which is used in the spray drying equipment will generally be about 3 ml per minute to about 5 ml per minute. Thε atomizεr air flow rate may vary between values of 1,200 liters per hour to about 3,900 liters per hour. Commercially available spray dryers are well known to those in the art, and suitable settings for any particular dispersion can be readily determined through standard empirical testing, with due reference to the examples that follow. Of course, the conditions may be adjusted so as to preserve biological activity in larger moleculεs such as protεins or peptides.

Particularly preferred embodimεnts of the present invention compπse spray drying preparations comprising a surfactant such as a phospholipid and at least one bioactive agent. In other embodiments, the spray drying preparation may further comprise an excipient compnsing a hydrophilic moiεty such as, for example, a carbohydrate |ι e glucose, lactose, or starch) in addition to any selected surfactant. In this regard, various starches and deπvatized starches are suitable for use in the presεnt invention. Other optional components may include conventional viscosity modifiers, buffers such as phosphate buffers or, other conventional biocompatible buffers or pH adjusting agents such as acids or bases, and osmotic agents (to provide isotomcity, hyperosmolaπty, or hyposmolaπty). Examples of suitable salts include sodium phosphate (both monobasic and dibasic), sodium chloride, calcium phosphate, calcium chloride and other physiologically acceptablε salts. Whatever components are selected, the first step in particulate production typically comprises feed stock preparation Preferably, the selected drug is dissolved in water to produce a concentrated solution The drug may also be dispersed directly in the emulsion, particularly in the case of water insoluble agents Alternatively, the drug may be incorporated in the form of a solid particulate dispersion. The concentration of thε drug used is dependent on the dose of drug required in thε final powder and the performance or efficiency of the nebulization dεvicε As nεeded, co surfactants such as poloxamer 188 or span 80 may be added to this annex solution. Additionally, excipients such as sugars and starches can also be added.

In selεctεd embodimεnts, an oil in water emulsion is then formed in a separate vessel The oil εmployed is preferably a fluorocarbon (e.g., perfluorooctyl bromide, perfluorodecalm), which is emulsified using a surfactant such as a long chain saturated phospholipid For example, one gram of phospholipid may be homogenized in 150 g hot distilled water (e.g., 60°C) using a suitable high shear mechanical mixer (e.g., Ultra Turrax model T 25 mixer) at 8000 rpm for 2 to 5 minutes Typically, 5 to 25 g of fluorocarbon is added dropwise to the dispersed surfactant solution while mixing. The resulting perfluorocarbon in-water emulsion is then processed using a high pressure homogemzer to reducε thε particle size. Typically, the emulsion is processεd at 12,000 to 18,000 psi, 5 discrete passes and kept at 50 to 80°C.

The drug solution and perfluorocarbon emulsion are then combined and fed into the spray dryer. Typically, the two preparations will be miscible as the emulsion will preferably comprise an aqueous continuous phase. While the bioactive agεnt is solubilized separately for the purposes of the instant discussion, it will be appreciated that, in other embodimεnts, thε bioactive agent may be solubilized (or dispersed) directly in the emulsion in such cases, thε bioactive emulsion is simply spray dried without combining a separate drug preparation.

In any event, operating conditions such as inlet and outlet temperature, feεd rate, atomization pressure, flow rate of the drying air, and nozzle configuration can be adjusted in accordance with the manufacturer's guidelines in order to produce thε required particle size and production yield of the resulting dry microstructures. Exemplary settings are as follows: an air inlet temperature between 60°C and 170°C; an air outlet between 40°C to 120°C, a feed rate between 3 ml to about 15 ml per minute, and an aspiration setting of 300 L/min and an atomization air flow rate between 1 ,200 to 2,800 L/hr. The selection of appropriate apparatus and processing conditions are well within the purview of a skilled artisan in viεw of the teachings herein, and may be accomplished without undue experimentation. In any event, the use of these and substantially equivalent methods provide for the formation of hollow, porous, aerodynamically light microspheres, with particle diameters appropriate for aerosol deposition into the lung. As described above, such particles are particularly effective in the formation of stabilized dispersions that are extremely compatible with the inhalation systems and nebulization techniques described more fully below.

Along with spray drying, particulates or perforated microstructures useful in the present invention may be formed by lyophilization. Those skilled in the art will appreciate that lyophilization is a freeze drying process in which water is sublimed from thε composition after it is frozen The particular advantage associated with the lyophilization process is that, biologicals and pharmaceuticals that are relatively unstable in an aqueous solution can bε dπεd without εlεvatεd tεmperatures (thereby eliminating the adverse thermal effects), and then stored in a dry state where there are few stability problems. With respect to the instant invention, such techniques are particularly compatible with the incorporation of peptides, proteins, genetic material and other natural and synthetic macromolecules in particulates or perforated microstructures without compromising physiological activity. Methods for providing lyophilized particulates are known to those of skill in the art and, it would clearly not require undue experimentation to provide dispersion compatible microstructures in accordance with the teachings herein. Accordingly, to the extent that lyophilization processεs may bε used to provide microstructures having the desired porosity and size, they are in conformance with the teachings herein and are expressly contemplated as being within the scope of the instant invention.

In addition to the aforementionεd techniques, perforated microstructures or particles of the present invention may also be formed using a double emulsion method. In the double emulsion method, the medicament is first dispεrsεd in a polymεr dissolved in an organic solvent (e g methylene chloride) by so cation or homogemzatioπ This primary εmulsioπ is then stabilized by forming a multiple emulsion in a continuous aqueous phase containing an emulsifiεr such as polyvinylalcohol The organic solvent is thεn removed by evaporation or extraction using conventional techniques and apparatus. The resulting microspheres are washed, filtered and lyophilized pπor to dispersion into suspension medium in accordance with the prεsεnt mvεntion While particulate suspensions comprising a non liquid dispersed phase are particularly compatible with the presεnt invention, it will be appreciatεd that, as discussed above, the stabilized dispersions may also comprise liquid in liquid colloidal systems, e.g. reverse emulsions and microemulsions. Those skilled in the art will appreciate that such systems are known in the art and stabilized dispersions compatible with the teachings herein may be provided without undue experimentation. In this regard, any reverse emulsion or microemulsioπ that is capable of being nebulized to provide a therapeutically effective aerosol for pulmonary administration is contemplatεd as being within the scope of the present invention. Preferably, the emulsions will be water in fluorochemical emulsions That is, the sεlεcted reverse emulsion or microemulsioπ will prefεrably compπsε a fluorochemical disperse phase with the other phase being aqueous in nature. Exemplary reverse emulsions useful with the present invention are disclosed in U S Pat No 5,770,585, pending U S S 08/487,612 and pending U S S N. 08/478,824 with each of the foregoing references incorporated herein by reference Such preparations may be stabilized by fluoππated or non fluoπnated surfactants With respect to this aspect of the invention, many of the fluorochemicals useful in the disclosed liquid in liquid preparations are the same as those that are useful as suspension mediums in the disclosed particulate dispersions Accordingly, while the following discussion is primarily directed to compatible suspension mediums for the distribution of non liquid particles, it will be apprεciatεd that thε samε compounds (ε g fluorochemicals) are useful in liquid in liquid dispersions that are compatible with the instant invention. Thus, while the term "suspension medium" or media will be used below, it should be understood that thesε same compounds may comprise emulsion phases in accordance with thε teachings herein.

Rεgardless of the sεlεctεd colloidal system, it is an advantage of the presεnt invention that biocompatible nonaqueous compounds may be used as suspension mediums or as a continuous phase. Particularly prefεrred suspension media are compatiblε with usε in nebulizers That is, they will be able to form aerosols upon the application of energy thereto. In general, the selectεd suspension medium should be biocompatible d e relatively non toxic) and non reactive with respεct to the suspendεd pεrforatεd microstructures compnsing the bioactive agent Prefεrred embodiments compπse suspension media selεctεd from the group consisting of fluorochemicals, fluorocarbons (including those substituted with other halogens), perfluorocarbons, fluorocarboπ/hydrocarbon diblocks, hydrocarbons, alcohols, ethers, or combinations thereof It will be appreciatεd that, thε suspension medium may comprise a mixture of vaπous compounds selected to impart specific characteπstics. It will also be appreciated that the perforated microstructures are prefεrably insoluble in thε suspεπsion mεdium, thεrεby providing for stabilized medicament particles, and effectively protecting a selected bioactive agent from degradation, as might occur dunng prolonged storage in an aqueous solution. In preferred embodiments, the selεcted suspension medium is bacteπostatic The suspεnsion formulation also protects the bioactive agent from degradation dunng the nebulization process.

As indicated above the suspension media may comprise any one of a number of different compounds including hydrocarbons, fluorocarbons or hydrocarbon/fluorocarboπ diblocks. In general, the contemplated hydrocarbons or highly fluoπnated or pεrfluoπnatεd compounds may bε linear, branched or cyclic, saturated or unsaturated compounds Conventional structural derivatives of these fluorochemicals and hydrocarbons are also contemplated as being within the scope of the present invention as well Selectεd embodiments compnsing thesε totally or partially fluonnated compounds may contain one or more hetero atoms and/or atoms of bromine or chloπne. Preferably, thesε fluorochemicals compnse from 1 to 16 carbon atoms and include, but are not limited to, linear, cyclic or polycyclic perfluoroalkanes, bιs(perfluoroalkyl)alkeπes, perfluoroethers, perfluoroammes, perfluoroalkyl bromides and perfluoroaikyl chlorides such as dichlorooctane Particularly preferred fluoπnatεd compounds for use in the suspension medium may comprise perfluorooctyl bromide C₈F,₇Br (PFOB or perflubron), dichlorofluorooctane C₉F,_sCl2 and the hydrofluoroalkane perfluorooctyl ethane C₈F, ₇C₂H₅ (PFOE). With respect to other embodiments, the use of perfluorohexane or perfluoropeπtane as the suspension medium is especially preferred.

More generally, exemplary fluorochemicals which are contemplatεd for usε in the present invention generally include halogεπated fluorochemicals (i e C,F,„,,X, XC„F₂.X, where n = 2 10, X = Br, Cl or I) and, in particular, 1 bromo F butanε n C.F₉Br, 1 bromo F hexane (n C₆F,₃Br), 1 bromo F hεptane (π C₇F,₅Br), 1 4 dibromo F butane and 1 6 dibromo F hexane. Other useful brominated fluorochemicals are disclosed in US Patent No. 3,975,512 to Long and are incorporated herein by refεrεncε Specific fluorochemicals having chlondε substituεnts, such as perfluorooctyl chloride (n C₈F, ,CI), 1,8 dichloro F octanε (n CIC_βF,₈CI), 1 ,6 dichloro F hexane (π CIC₆F_PCI), and 1 , 4 dichloro F butane (n CIC₄F_eCI) are also preferred.

Fluorocarbons, fluorocarbon hydrocarbon compounds and halogeπated fluorochemicals containing other linkage groups, such as εstεrs, thioεthers and amines are also suitable for use as suspension media in the present invention For instance, compounds having the general formula, C-,F₂-.,₁ OC_mF_2m<.„ or CJ^CH ' CHCJJ,.,, (as for example

C₄F₉CH= CHC₄F₉ (F 44E), ι C₃F₉CH = CHC₆F,₃ (F ι36E), and C₆F₁₃CH = CHC₆F,₃ (F 66E)) where n and m are the same or different and π and m are integers from about 2 to about 12 are compatible with teachings herein Useful fluorochemical hydrocarbon diblock and tnblock compounds include those with the general formulas CJ_2n., C_mH^, and CJ_2n,,C-,H_2m ,, where n - 2 12; m = 2 16 or C-H_2p., C„F₂„ C_mH₂-,.,, where p = 1 12, m = 1 12 and n - 2 12. Preferred compounds of this type include CJ, ,C₂H₅ C₆F,₃C₁₀H_2t C₈F, ₇C_eH,₇ C₆F₁₃CH =CHC₆H,₃ and CJ,₇CH =CHC₎₀H₂₎. Substituted ethers or polyethers (i.e XC-F₂.0CJ_2mX, XCF0CJ_2n0CF₂X, where n and m = 1 4, X = Br, Cl or I) and fluorochemical hydrocarbon ether diblocks or tnblocks (i.e. C-F₂-.. 0-C_rH_2m.,, where n - 2 10; m = 2-16 or C₅H_2p.|-0-C_nF_2n-0-C_mH_2ffl,ι, where p = 2 12, m = 1 12 and n = 2 12) may also used as well as C„F₂„,0 CJ₂-.0C_pH_2p.,, wherein n, m and p are from 1 12.

Furthermore, depending on the application, perfluoroalkylatεd ethers or polyethers may be compatible with the claimed dispersions.

Poiycyclic and cyclic fluorochemicals, such as C,₀F,₈ (F decalin or perfluorodecalm), perfluoropεrhydrophεnanthrεnε, pεrfluorotεtramethylcyclohexane (AP 144) and perfluoro π butyldecalm arε also within thε scope of the invention. Additional useful fluorochemicals include perfluoππated ammεs, such as F tripropylamine

("FTPA") and F tπbutylamiπe ("FTBA"). F 4 mεthyloctahydroquinolizine ("FMOQ"), F N methyl decahydroisoquinoline ("FMIQ"), F N-mεthyldecahydroquinoline ("FHQ"), F-N-cyclohεxylpyrrolidme ("FCHP") and F-2 butyltetrahydrofuraπ ("FC 75"or "FC 77"). Still other useful fluoπnated compounds include perfluorophεnanthrene, perfluoromethyldecalin, perfluorodimethylethylcyclohexaπe, perfluorodimethyldecaliπ, perfluorodiethyldecalin, perfluoromethyladamaπtane, perfluorodimethyladamantanε. Other contεmplatεd fluorochemicals having nonfiuoπne substituents, such as, perfluorooctyl hydnde, and similar compounds having different numbers of carbon atoms are also useful. Those skilled in thε art will further appreciatε that othεr variously modified fluorochemicals are encompassed within the broad definition of fluorochemical as used in the instant application and suitable for use in the present invention. As such, each of the foregoing compounds may be used, alone or in combination with other compounds to form thε stabilized dispersions of the present invention.

Specific fluorocarbons, or classes of fluoπnated compounds, that may be useful as suspension media include, but are not limited to. fluoroheptane, fluorocycloheptane fluoromethylcycloheptane, fluorohexaπe, fluorocyclohexane, fluoropentane, fluorocyclαpεntanε, fluoromεthylcyclopentane, fluorodimεthyicyclopentanεs, fluoromethylcyclobutane, fluorodimethylcyclobutane, fluorotπmethylcyclobutane, fluorobutane, fluorocyclobutane, fluoropropane, fluoroethers, fluoropolyethers and fluorotπethylaminεs. Such compounds are generally environmentally sound and are biologically non reactive.

While any fluid compound capable of producing an aerosol upon the application of energy may bε usεd in conjunction with the present invention, the selected suspension medium will preferably have a vapor pressure less than about 5 atmospheres and more prefεrably less than about 2 atmosphεres Unless otherwise specified, all vapor pressures recited herein are measured at 25°C. In other embodiments, preferred suspension media compounds will have vapor pressures on the order of about 5 torr to about 760 torr, with more preferable compounds having vapor pressures on the order of from about 8 torr to about 600 torr, while still more preferable compounds will have vapor pressures on the order of from about 10 torr to about 350 torr. Such suspension media may be usεd in conjunction with compressed air nebulizers, ultrasonic nebulizers or with mechanical atomizers to provide effective ventilation therapy. Moreovεr, more volatile compounds may be mixed with lower vapor pressure components to provide suspension media having specified physical characteπstics selεctεd to further improve stability or enhance the bioavailability of thε dispersed bioactive agent. Other embodimεnts of thε present invention will compπse suspension mεdia that boil at selected temperatures under ambient conditions (i.e. 1 atm). For example, preferred embodimεnts will compπse suspension media compounds that boil above 0°C, above 5°C, above 10°C, above 15°, or above 20°C In othεr embodiments, the suspension media compound may boil at or above 25°C or at or above 30°C. In yet other embodiments, the selectεd suspension media compound may boil at or above human body tempεrature (i.e. 37°C), above 45°C, 55°C, 65°C, 75°C, 85°C or above 100°C.

It will further be apprεciatεd that onε of ordinary skill in the art can readily detεrminε other compounds that would perform suitably in the presεnt invention which apparently do not exhibit a desirable vapor pressure and/or viscosity Rather, it will be understood that, certain compounds outside the prefεrrεd ranges of vapor pressure or viscosity can be used if they provide the desired aerosolized medicament. The stabilized suspεnsions or dispersions of the presεnt invention may be prepared by dispersal of the microstructures in the selεctεd suspension medium which may then be placed in a container or reservoir. In this regard, thε stabilized preparations of the prεsεnt invention can be made by simply combining the components in sufficient quantity to produce the final desired dispersion concentration. Although the microstructures readily disperse without mechanical energy, the application of mechanical enεrgy to aid in dispεrsion (e.g with the aid of sonication) is contemplated, particularly for the formation of stable emulsions or reverse emulsions. Alternatively, the components may be mixεd by simple shaking or othεr type of agitation The process is prefεrably carried out under anhydrous conditions to obviate any adverse effects of moisture on suspension stability Once formed, the dispersion has a reduced susceptibility to fiocculation and sedimentation

It will also be understood that, other components can be included in the pharmaceutical compositions of the present invention. For examplε, osmotic agεnts, stabilizers, chelators, buffεrs, viscosity modulators, salts, and sugars can be added to fine tune the stabilized dispersions for maximum life and ease of administration. Such components may be added directly to the suspension medium, ether phase of an emulsion or associated with, or incorporated in, dispersed particles or perforated microstructures. Considerations such as steπlity, isotonicity, and biocompatibility may govern the use of conventional additives to the disclosed compositions The use of such agents will be understood to those of ordinary skill in the art and, the specific quantities, ratios, and typεs of agεπts can bε dεtεrminεd empirically without undue expεπmεntation

Administration of bioactive agent may be indicated for the treatment of mild, moderate or severe, acute or chronic symptoms or for prophylactic treatment Moreover, the bioactive agent may be administered to treat local or systεmic conditions or disorders. It will be appreciated that, thε prεcisε dosε admimstεrεd will dεpεnd on thε agε and condition of the patient, the particular medicamεπt usεd and thε frequency of administration, and will ultimately be at the discretion of the attendant physician. When combinations of bioactive agents are employεd, thε dosε of εach componεnt of thε combination will generally be that employed for each component when used alone. As discussed throughout the specification, the stabilized dispersions disclosed herein, are preferably administered to the lung or pulmonary air passages of a patient via aerosolizatioπ, such as with a nebulizer. Nebulizers are well known in thε art and could easily bε employed for administration of the claimed dispersions without undue expεπmεntatioπ. Breath activated nebulizers, as well as those compnsing other types of improvements which have been, or will be, dεvεlopεd are also compatible with thε stabilized dispersions and prεsεnt invention and are contemplated as being with in the scope thereof.

While compatible bioactive agents may be administered using various systems, it will be appreciated that, in particularly preferred embodiments, the stabilized dispersions disclosed herein will be administered to the lung or pulmonary air passagεs of a patiεnt via nebulization Nebulizers are well known in the art and could easily be employεd for administration of the claimed dispersions without undue expεπmεntation. Nebulizers work by forming aerosols, that is converting a bulk liquid into small droplets suspεndεd in a breathable gas. Here, the aerosolized medicament to be administered (preferably to the pulmonary air passages) will compπse small droplets of suspension medium associated with relatively non porous particles, perforatεd microstructures, or disperse liquid phase compnsing a bioactive agent. In such embodiments, the stabilized dispersions of the present invention will typically be placed in a fluid reservoir operably associated with a nebulizer The specific volumes of preparation provided, means of filling the reservoir, etc., will largely be depeπdεnt on thε selection of the individual nebulizer and is well within the purviεw of thε skilled artisan Of course, the prεsεnt invention is entirely compatible with single-dose nebulizers and multiple dose nebulizers.

In any event, nebulizer εdiatεd aεrosolization typically requires an input of enεrgy in order to produce the increased surface area of the droplets and, in some cases, to provide transportation of the atomized or aerosolized medicament. One common modε of aerosolizatioπ is forcing a stream of fluid to be ejected from a nozzle, whereby droplets are formed. With respect to nebulized administration, additional energy is usually imparted to provide droplets that will be sufficiently small to be transportεd dεεp into thε lungs. Thus, additional εnergy is needed, such as that provided by a high velocity gas stream or a piezoelectnc crystal. Two popular types of nebulizers, jet nebulizers and ultrasonic nebulizers, rely on the aforεmentionεd methods of applying additional enεrgy to the fluid during atomization The jet nebulizer is well known and in widεspread use. In a jet nebulizer, compressed air is forced into a device containing a liquid to bε aerosolized, such as one of the suspεnsions of thε present invention. Thε compressed air draws the liquid through one or more small openings, thus generating the aerosol. The high velocity of the compressεd air providεs sufficient εnergy to enable the foπnation of droplets small enough for inhalation To aid in formation of uniformly smaller droplets, thε droplεts initially impact a baffle. There may bε other impaction sites onto which thε droplεts may be directed before the aerosol is earned out of the nebulizer by the flow of the compressed air. In preferred embodiments the compressed air may bε saturated with the suspension medium. This would allow the aerosolized droplets to bε dεpositεd in the lung, possibly facilitating enhaπcεd spreading of bioactive agent after initial deposition. Ultrasonic nebulizers do not require the usε of compressed air, and thus, may be similar to MDIs as to compactness and portability, though they operate under different physical pπnciples. Preferred ultrasonic nebulizers are those which are fairly small, portable, battery powered and capable of delivεnng sεvεral dosεs, εach of which comprises a single bolus of aerosolized solution. Such nebulizers may be termεd single-bolus nebulizers. Most devices are manually actuated, but some devices exist which are breath actuated. Breath actuated devices work by releasing aerosol when the device senses the patient inhaling through a circuit. Breath actuated nebulizers may also be placed in line on a ventilator circuit to release aerosol into thε air flow which compπsεs thε inspiration gasεs for a patiεnt

The heart of most species of ultrasonic nebulizer is a transducer made from a piezoεlectπc crystal. When oscillating energy is applied to the piezoelectric crystal, it will vibrate at the same frequency as the applied energy which is preferably in the ultrasonic range. This motion, when transmitted into a liquid, providεs the energy needed to aerosolize the liquid. The droplet size (count median diametεr) formεd by this εthod is a function of thε excitation frequency, the density of the liquid, and the surface tension of the liquid, whereas the rate of atomization is a function of the viscosity, surface tension, and vapor pressure.

One type of nebulizer is the Respimat (Boehπnger Iπgεlhεim, Gεrmaπy) which is manually actuated, hand-held and battery operatεd Whεn thε patient squeezes a tπgger on thε device, a droplet of solution (about 100 I) is metεred into a piεzoelectric plate about 1 cm in diameter. When eπεrgy is applied, the plate vibrates at about 10 MHz, resulting in the aerosolization of the solution which may then be inhaled by a patient.

Another type of ultrasonic nebulizer is the AeroDosε (AeroGeπ, Sunnyvale, CA) (DeYoung, "The AeroDose Multidose Inhaler Device Design and Delivery Characteπstics," Respiratory Drug Delivery VI, 1998, p 91 ) The battery powered AeroDose operates by means of a plate containing several hundred holes which vibrates at ultrasonic frequεnciεs. Whεn the top of the device is pressεd down, a mεtεnng pump delivers a dose of liquid from a multidose canister to the plate. The device is breath actuated, with aerosolization beginning when the device senses the inspiration of the patient The investigators for the AeroDosε report that they are able to achieve a median mass aerodynamic diametεr of 1.9 to 2.0 m using this device.

Yet another typε of ultrasonic nεbulizεπs that in PCT Publication No. W092/1 1050 to Robertson, et. al., In the Robertson device, the solution or other mateπal to be nebulized is drawn through numerous tiny holes in a metal plate which vibrates through the use of a piεzoelectnc device. When εnergy is applied, the aerosol is formed and will continue to form as long as energy is delivered to the piezoelectnc crystal. Thus, depending on the amount of time that the device is left on, it may serve as εithεr a single bolus device or a continuous nebulizer. As seεn abovε, ultrasonic nebulization dewcεs may act by ultrasonic εnεrgy alone, or may use ultrasonic energy in combination with other methods of aerosolization such as forcing or drawing a liquid or suspension through a mateπal with very small openings. Yet, regardless of the type of nebulizer selected, thε stabilized dispersions of the present invention provide a significant advantage due to their relatively homogenεous dispεrsion of the incorporated bioactive agent over a period of time. That is, the homogenεous dispεrsion of the incorporated particulates ensures that the amount of bioactive agent administered will be consistent no matter which fraction of the preparation in thε fluid reservoir is actually nebulized in εach individual actuation of thε nebulizer Similarly, whεn used for continuous administration over an exteπdεd period the stable, homogeneous dispersions of the present invention ensure that relatively constant levels of bioactive agent are delivered during each incremental period of time. In any event, it should be noted that the preceding examples of nebulizers are only for exεmplary purposes. As will be recogmzεd by oπε skilled in the art, other types of nebulizers, whether currently known or later invεntεd, may also be used for administration of the stabilized dispersions of thε present invention.

It will bε apprεciatεd that, thε stabilized preparations for use in nebulizers of the present invention may be advantageously supplied to the physician or other health care professional, in a sterile, prepackaged or kit form More particularly, thε formulations may be supplied as stable, preformed dispersions ready for administration or, as separata ready to mix components. When provided in a ready to use form, the dispersions may be packaged in single use containers or reservoirs, as well as in multi use containers or reservoirs. In either case, the container or reservoir may be associated with the selected nebulizer and used as described herein. When provided as individual components (e g., as powdεrεd microsphεrεs and as nεat suspεnsion mεdium) thε stabilized preparations may then be formed at any time prior to use by simply combining the contents of the containers as directεd Additionally, such kits may contain a number of ready to mix, or prepackaged components that may be packaged individually so that the user can then select the desired compoπeπt(s) for the particular indication or use In this regard, the user may then substitute selεcted components at will, or as indicated, during a particular course of treatment. It will also be appreciated that such kits may optionally include a nebulizer or that the preparation may be supplied in a disposable nebulizer

The foregoing description will be more fully understood with reference to the following Examples. Such Examples, are, howevεr, mεrεly represεntative of preferred methods of practicing the presεnt invention and should not be read as limiting the scope of the invention.

I

Preparation of Hollow Porous Pamelas of Gεntamicin Sulfate by Spray Drying 40 to 60ml of the following solutions were prepared for spray drying. 50% w/w hydrogenatεd phosphatidylcholme, E 100 3 (Lipoid KG, Ludwigshafen, Gεrmany) 50% w/w gentamicin sulfate (Amresco, Solon, OH) Perfluorooctylbromidε, Perflubron (NMK, Japan) Deioπized water

Perforated microstructures comprising gentamicin sulfate were prepared by a spray drying technique using a B 191 Mini Spray Drier (Buchi, Flawil, Switzerland) under thε following conditions- aspiration- 100%, inlet temperature. 85° C; outlet temperature: 61 °C, feed pump: 10%, N₂ flow. 2,800 L/hr Variations in powder porosity were examined as a function of the blowing agent concentration Fluorocarbon in-watεr emulsions of perfluorooctyl bromide containing a 1.1 w/w ratio of phosphatidylcholme (PC), and gentamicin sulfate wεrε prepared varying only the PFC/PC ratio 1 3 grams of hydrogenated egg phosphatidylcholme was dispersed in 25 mL deiomzed water using an Ultra Turrax mixer (model T 25) at 8000 rpm for 2 to 5 minutes (T = 60 70°C) A range from 0 to 40 grams of perflubron was added dropwise dunng mixing (T = 60 70°C) After addition was complete, the fluorocarbon in water emulsion was mixed for an additional period of not less than 4 minutes The resulting coarse emulsions were then homogεmzεd under high pressure with an Avestin (Ottawa, Canada) homogenizer at 15,000 psi for 5 passes Gentamicin sulfate was dissolved in approximately 4 to 5 mL dεiomzεd water and subsequently mixed with the perflubron emulsion immediately prior to thε spray dry process. The gentamicin powders were then obtained by spray drying using the conditions described above A free flowing pale yεllow powdεr was obtainεd for all perflubron containing formulations The yield for each of the various formulations ranged from 35% to 60%.

II Morphology of Gentamicin Sulfate Spray Dried Powders A strong dependence of the powder morphology, degree of porosity, and production yield was observed as a function of the PFC/PC ratio by scanning electron microscopy (SEM). A series of six SEM micrographs illustrating these observations, labeled 1 A1 to 1 F1 , are shown in the left hand column of Fig. 1. As seen in these micrographs, the porosity and surface roughness was found to be highly dependent on the concentration of the blowing agent, where the surface roughness, number and size of the pores increased with increasing PFC/PC ratios. For examplε, thε formulation dεvoid of perfluorooctyl bromide produced microstructures that appeared to be highly agglomeratεd and readily adhered to the surface of the glass vial. Similarly, smooth, spherically shaped microparticles were obtained when relatively little (PFC/PC ratio = 1 1 or 2 2) blowing agent was used As the PFC/PC ratio was increased the porosity and surface roughness increased dramatically

As shown in the right hand column of Fig. 1 , the hollow nature of the microstructures was also enhanced by the incorporation of additional blowing agent. More particularly, the series of six micrographs labeled 1 A2 to 1 F2 show cross sections of fractured microstructures as revεalεd by transmission electron microscopy (TEM). Each of these images was produced using the same microstructure preparation as was used to produce the corresponding SEM micrograph in thε left hand column. Both the hollow nature and wail thickness of thε resulting perforated microstructures appeared to be largely dependent on the concentration of the selected blowing agent. That is, the hollow nature of the preparation appeared to increase and the thickness of the particle wails appeared to decrease as the PFC/PC ratio mcreasεd. As may be seen in Figs. 1 A2 to 1 C2 substantially solid structures were obtained from formulations containing little or no fluorocarbon blowing agεnt Convεrsεly, thε perforated microstructures produced using a relatively high PFC /PC ratio of approximately 45 (shown in Fig. 1 F2 proved to be εxtrεmεly hollow with a relatively thin wall ranging from about 43.5 to 261 nm Both types of particles are compatible for use in the present invention.

Preparation of Hollow Porous Particles of Albuterol Sulfate by Spray Drying Hollow porous albuterol sulfate particles were prepared by a spray drying technique with a B 191 Mini Spray Drier (Buchi, Flawil, Switzerland) undεr thε following spray conditions- aspiration: 100%, inlet tempεraturε-

85°C; outlet temperature: 61 ° C; feed pump: 10%; N, flow: 2,800 L/hr. The feed solution was prepared by mixing two solutions A and B immediatεly prior to spray drying.

Solution A. 20g of watεr was used to dissolve 1 g of albuterol sulfate (Accurate Chemical, Westbury, NY) and 0 021 g of poloxamεr 188 NF grade (BASF, Mount Olive, NJ). Solution B A fluorocarbon in water emulsion stabilized by phospholipid was prepared in the following manner Thε phospholipid, 1 g EPC 100 3 (Lipoid KG, Ludwigshafen, Germany), was homogenizεd in 150g of hot deionized water (T = 50 to 60°C) using an Ultra Turrax mixer (model T 25) at 8000 rpm for 2 to 5 minutes (T = 60 70°C). 25g of perfluorooctyl bromide (Atochεm, Pans, France) was added dropwisε during mixing After the fluorocarbon was added, the emulsion was mixed for a period of not less than 4 minutes. The resulting coarse emulsion was then passed through a high pressure homogenizer (Avεstin, Ottawa, Canada) at 18,000 psi for 5 passεs.

Solutions A and B were combined and fed into the spray-dryer under the conditions described above. A free flowing whitε powdεr was collected at the cyclone separator The hollow porous albuterol sulfate particles had a volume-weighted mean aerodynamic diameter of 1.18 ± 1 42 μm as determined by a time-of flight analytical method (Aerosizer, Amhεrst Procεss Instruments, Amherst, MA) Scanning electron microscopy (SEM) analysis showed the powdεrs to be spheπcal and highly porous. The tap density of the powder was detεrminεd to be less than 0 1 g/cm³ This foregoing εxamplε sεrvεs to illustrate the inherent diversity of thε prεsεnt invention as a drug delivery platform capable of effectively incorporating any one of a number of pharmaceutical agents. The pπnciple is further illustrated in the next εxamplε.

IV Formation of Porous Particulate Microstructures Compnsing Mixtures of Long Chain/Short Cham Phospholipids and Albuterol Sulfate A dispersion for spray drying was prepared as described above, with the difference that 1 g of DSPC was dispersed with 100 mg of a short chain phospholipid, dioctylphosphatidylcholinε (DOPC) (Avanti Polar Lipids, Alabaster, Alabama). The composition of the spray feed is shown in Table II immediately below. Thε resulting yield was 50%.

Table II

Composition of the Spray Feεd

Preparation of Hollow Porous Particles of Cromolyn Sodium by Spray Drying Perforated microstructures comprising cromolyn sodium were prepared by a spray drying technique with a B 191 Mini Spray Drier (Buchi, Flawil, Switzerland) under the following spray conditions- aspiration- 100% inlet tempεraturε. 85°C, outlεt temperature: 61 °C, feed pump: 10%, N, flow. 2,800 L/hr. The feed solution was prepared by mixing two solutions A and B immediatεly prior to spray drying.

Solution A 20g of water was used to dissolve 1 g of cromolyn sodium (Sigma Chemical Co, St. Louis, MO) and 0 021 g of poloxamer 188 NF grade (BASF, Mount Olive, NJ)

Solution B. A fluorocarbon in water emulsion stabilized by phospholipid was prepared in the following manner The phospholipid, 1 g EPC 100 3 (Lipoid KG, Ludwigshafen, Germany), was homogεnizεd in 150g of hot dεionized water (T - 50 to 60°C) using an Ultra-Turrax mixer (model T 25) at 8000 rpm for 2 to 5 minutes (T = 60 70°C). 27g of perfluorodεcahn (Air Products, Allεntown, PA) was addεd dropwisε during mixing Aftεr the fluorocarbon was addεd, thε emulsion was mixed for at least 4 minutes The resulting coarse emulsion was then passed through a high pressure homogenizεr (Avεstin, Ottawa, Canada) at 18,000 psi for 5 passεs Solutions A and B were combined and fed into the spray dryer under thε conditions described above. A free flowing pale yellow powder was collected at the cyclone separator. The hollow porous cromolyn sodium particles had a volume weightεd mεan aεrodynamic diamεtεr of 1 23 ± 1 31 μm as determinεd by a timε-of flight analytical method (Aerosizer, Amherst Process Instruments, Amherst, MA). As shown in Fig. 2, scanning electron microscopy (SEM) analysis showed thε powdεrs to be both hollow and porous. The tap density of the powder was determined to be less than 0.1 g/cm³.

VI Preparation of Hollow Porous Particles of BDP by Spray Drying Perforated microstructures comprising beclomethasoπe dipropioπate (BDP) particles were prepared by a spray drying technique with a B 191 Mini Spray Drier (Bϋchi, Flawil, Switzerland) undεr thε following spray conditions: aspiration: 100%, inlet temperature: 85°C, outlet temperature: 61 °C; feed pump: 10%; N₂ flow: 2,800 L/hr The feεd stock was prepared by mixing 0 1 1 g of lactose with a fluorocarbon in water emulsion immεdiatεly pπor to spray drying. The emulsion was prepared by the technique described below. 74 mg of BDP (Sigma, Chemical Co , St Louis, MO), 0 5g of EPC 100 3 (Lipoid KG, Ludwigshafen,

Germany), 15mg sodium oleate (Sigma), and 7mg of poloxamer 188 (BASF, Mount Olive, NJ) were dissolvεd in 2 ml of hot mεthanol. The methanol was thεn εvaporatεd to obtain a thin film of thε phospholipid/steroid mixture. Thε phosphohpid/stεroid mixture was then dispersed in 64g of hot deionized water (T = 50 to 60°C) using an Ultra-Turrax mixer (model T 25) at 8000 rpm for 2 to 5 minutεs (T = 60 70°C). 8g of perflubron (Atochem, Pans, Francε) was added dropwise during mixing. After the addition was complete, the emulsion was mixed for an additional pεnod of not less than 4 minutes The resulting coarse emulsion was thεn passed through a high pressure homogemzer (Avestin, Ottawa, Canada) at 18,000 psi for 5 passes This εmuision was then usεd to form the feed stock which was spray dried as descπbεd above A free flowing white powder was collected at the cyclone separator. The hollow porous BDP particles had a tap density of less than 0.1 g/cm³.

VII Preparation of Hollow Porous Particles of TAA by Spray Drying Perforated microstructures comprising tnamciπolone acetoπide (TAA) particles were prepared by a spray drying technique with a B 191 Mini Spray Drier (Buchi, Flawil, Switzerland) under the following spray conditions- aspiration. 100%, inlet temperature: 85°C, outlet temperature. 61 °C, feεd pump. 10%, N, flow. 2,800 L/hr. The fεεd stock was prepared by mixing 0.57g of lactose with a fluorocarbon in water emulsion immediatεly prior to spray drying. The emulsion was prepared by the technique described below. 100mg of TAA (Sigma, Chemical Co., St. Louis, MO), 0.56g of EPC 100 3 (Lipoid KG, Ludwigshafen, Germany), 25mg sodium oleate (Sigma), and 13mg of poloxamer 188 (BASF, Mount Olive, NJ) were dissolved in 2 mi of hot methanol Thε methanol was thεn evaporated to obtain a thin film of thε phospholipid/stεroid mixture. The phosphohpid/steroid mixture was then dispersed in 64g of hot deionized water (T = 50 to 60°C) using an Ultra Turrax mixer (model T 25) at 8000 rpm for 2 to 5 minutεs (T = 60 70° C). 8g of perflubron (Atochem, Pans,

France) was added dropwise during mixing. After the fluorocarbon was added, the emulsion was mixed for at least 4 minutes The resulting coarse εmulsion was thεn passεd through a high pressure homogεnizεr (Avεstin, Ottawa, Canada) at 18,000 psi for 5 passes. This emulsion was than usεd to form thε feed stock which was spray dπed as descπbed above. A free flowing white powdεr was collected at the cyclone separator Thε hollow porous TAA particles had a tap density of less than 0.1 g/cm³.

VIII Preparation of Hollow Porous Particles of DNase I by Spray Drying Hollow porous DNase I particles were prepared by a spray drying technique with a B 191 Mini Spray- Drier (Buchi, Flawil, Switzεrland) undεr the following conditions- aspiration- 100%, inlet tempεraturε- 80°C; outlεt temperature: 61 °C, feed pump: 10%; N₂ flow: 2,800 L/hr The feed was prepared by mixing two solutions A and B immediately prior to spray drying.

Solution A. 20g of water was used to dissolve 0.5gr of human pancreas DNase I (Calbiochem, San Diego CA) and 0 012g of poloxamer 188 NF grade (BASF, Mount Olive, NJ). Solution B. A fluorocarbon-in-water emulsion stabilized by phospholipid was prepared in thε following way. The phospholipid, 0.52g EPC 100 3 (Lipoid KG, Ludwigshafεn, Germany), was homogenized in 87g of hot deionized water (T = 50 to 60°C) using an Ultra Turrax mixer (model T 25) at 8000 rpm for 2 to 5 minutes (T = 60 70° C) 13g of perflubron (Atochem, Pans, France) was added dropwise during mixing After the fluorocarbon was added, the emulsion was mixed for at least 4 minutes. The resulting coarse emulsion was then passed through a high pressure homogenizer (Avεstin, Ottawa, Canada) at 18,000 psi for 5 passεs.

Solutions A and B wore combinεd and fed into the spray dryer under the conditions described above. A freε flowing pale yellow powder was collected at thε cyclone separator The hollow porous DNase I particles had a volume-weighted mean aerodynamic diameter of 1.29 ± 1.40 μm as detεrminεd by a time of flight analytical method (Aerosizer, Amherst Process Instruments, Amherst, MA) Scanning electron microscopy (SEM) analysis showed the powders to be both hollow and porous. The tap density of the powder was determined to be less than 0.1 g/cm³.

The foregoing examplε further illustrates the extraordinary compatibility of the present invention with a variety of bioactive agents That is, in addition to relatively small hardy compounds such as steroids, the preparations of the prεsεnt invention may be formulated to effectively incorporate larger, fragile molecules such as peptides, proteins and genetic mateπal.

IX Preparation of hollow porous powder by spray drying a gas in water emulsion

The following solutions were prepared with water for injection: Solution 1 -

3.9% w/v m HES hydroxyethylstarch (Ajinomoto, Tokyo, Japan) 3.25% w/v Sodium chloride (Mallmckrodt, St. Louis, MO)

2.83% w/v Sodium phosphate, dibasic (Mallmckrodt, St Louis, MO)

0.42% w/v Sodium phosphate, monobasic (Mallmckrodt, St. Louis, MO)

Solution 2.

0.45% w/v Poloxamer 188 (BASF, Mount Olive, NJ)

1.35% wlv Hydrogenatεd egg phosphatidylcholme, EPC 3

(Lipoid KG, Ludwigshafen, Germany) The ingredients of solution 1 were dissolved in warm water using a stir plate. The surfactants in solution 2 were dispersεd in water using a high shear mixer. The solutions were combined following emulsification and saturated with nitrogen prior to spray drying.

The resulting dry, freε flowing, hollow, sphεπcal product had a mεaπ particle diametεr of 2.6 ± 1 5 μm. The particles, which may be used for the replacεment or augmentation of lung surfactant, were spherical and porous as determined by SEM.

The previous example illustrates the point that, a wide variety of blowing agents (here nitrogen) may be used to providε microstructures exhibiting desirεd morphology Indεεd, one of the primary advantages of the present invention is the ability to alter formation conditions so as to preserve biological activity (i.e. with proteins or lung surfactant) or produce microstructures having sεlεctεd porosity.

X

Preparation of Perforated Microstructure Powder Containing Ampicillin

The following materials were obtained and used to provide a feεd stock:

20% w/w Ampicillin, Biotech grade (Fisher Scientific, Pittsburgh, PA)

14.38% w/w Hydroxyεthyl starch (Ajinomoto Japan)

65.62% w/w Dipalmitoylphosphatidylcholine (Genzyme, Cambridge, MA)

Perfluorohexane (3M, St. Paul, MN)

Deionized water Hydroxyεthyl starch, (HES; 0.9 g), and dipalmitoylphosphatidylcholinε (DPPC; 4 1 1 g) werε dispersed in 75 ml deionized water usmq an Ultra-Turrax mixer (model T-25) at 10,000 rpm for approximately 2 minutes (T =^■ 45-50 C). The resulting DPPC/HES dispersion was chilled in an ice bath Ampicillin (1.25 g) was added and allowed to mix for 1 minute (T

= 5-10 C). Perfluorohexane (PFH, 4.1 1 g ) was then added dropwise during mixing (T = 5-10 C). After the addition was complete, thε PFH in water emulsion was mixed on the Ultra Turrax for a total of not less than 4 minutεs.

A perforated microstructure powder comprising ampicillin was obtained by spray-drying (Buchi, 191 Mini Spray Dryer, Switzerland) the ampicillin containing emulsion at a rate of 5 5 ml/mm. The inlet and outlet tempεraturεs of thε spray dryer were 90 C and 55 C respectively. The nebulization air and aspiration flows were 1 ,800 L/hr and 100% respectively. A free flowing white powder compnsing porous microspheres was obtained.

XI Effect of Spray Drying on the In-Vitro Activity of Lung Surfactant The activity of a spray dried lung surfactant preparation to lower the surface tension of a pulsating bubble was compared with the neat lung surfactant preparation Bovine derived lung surfactant, Alveofact (Thomae, Biberach, Germany) and spray dried lung surfactant containing microshells were dissolvεd in normal saline at a concentration of 10 mg/ml and allowed to incubate for 15 minutes at 37 C. Prior to analysis, the surfactant test solutions were vigorously shaken using a Vortex mixer for 30 seconds. The samplεs were analyzed for their surface properties using the Pulsating Bubble Surfactometer at 37 C (model EC PBS-B, Electronics, Amherst, NY) according to the manufacturers instructions. Surfactant solutions were allowed to adsorb at minimum bubble diametεr for 10 sεcoπds, and bubble cycling was performεd in thε automatic mode (20 cycles/minute). For each expeπmεnt, measurements were taken for approximately the first 10 cycles, then again at t= 2, 4, and 6 minutes.

The main difference observed between thε neat and spray dried surfactant suspensions is the rate at which they adsorb to the bubble surface and thus lower thε tension. Thε spray dnεd materials required 6 cycles to achievε low surface tension as compared with oπε cycle for the Alveofact sample. Howevεr, the magnitude of thε tεπsion at maximum, and minimum bubble diametεr wεrε found to bε approximately the same.

For the Alveofact dispersion, the tension decreasεd from 32 mN/m at maximum diameter to 4 mN/m at minimum in the first cycle With further pulsation, a stεady statε oscillation was reached with a maximum tεnsion _max 33 mN/m and a minimum tεnsion _nn 0 to 1 mN/m. For the spray-dried lung microshell dispersion, the tension decreased from 36 mN/m at maximum diameter to 16 mN/m at minimum in the first cycle. By the sixth pulsation, _m„ and _mιn werε respectively 36 and 2 mN/m. Both the neat Alveofact and thε spray dπεd lung surfactant perforated microstructures satisfy thε maximum and minimum surfacε tension requiremεnts for physiologically effective lung surfactants as outlined by Notter; [R.H. Notter, in Surfactant Replacement Therapy, (Eds: D.H. Shapiro, and R.H. Notter) Alan R. Liss, New York, 19891 these values should range from 35 to about 5 mN/m, respectively. This examplε illustrates that the compositions and methods of the presεnt invention arε particularly useful for the replacεmεnt or augmentation of lung surfactant in patients in need thereof.

XII Preparation of Perforated Microstructure Powder Containing Insulin

The following materials werε obtained and used to provide a feεd stock.

0 0045% w/w Human Insulin, (CalBiochεm, San Diego, CA) 17.96% wlw Hydroxyethyl starch (Ajinomoto, J apan) 82.04% wlw Dipalmitoylphosphatidylcholinε (Genzymε, Cambπdgε, MA) Pεrfluorohexane (3M, St. Paul, MN)

Dεionizεd watεr

Hydroxyεthyl starch, (HES; 1 35 g) and dipalmitoylphosphatidylcholiπε (DPPC, 6.16 g) wεrε dispersed in 100 ml deionized water using an Ultra-Turrax mixer (model T 25) at 10,000 rpm for approximately 2 minutes (T = 45 50 C). The resulting DPPC/HES dispersion was thεn chilled in a ice bath Insulin (3 4 mg) was added and allowed to mix for 1 minute (T = 5 10 C). Perfluorohexanε (PFH, 6.16 g) was then addεd dropwisε during mixing (T = 5 10 C). After the addition was complete, the resulting PFH in water emulsion was mixed with the Ultra Turrax) for a total of not less than 4 minutes. The insulin microstructure powder was obtained using a Buchi model 191 mini spray dryer (Buchi, Switzerland). The insulin containing emulsion was fεd at a ratε of 5 5 ml/mm. The inlet and outlεt temperatures of thε spray dryer were 80 C and 45 C respectively. The nebulization air and aspiration flows were 1,800 L/hr and 100% respectively. A free flowing, white powdεr compnsing porous microsphεrεs was obtained

XIII

Effect of Perflubron on the In Vitro Activity of DNAse I Bovine pancreas deoxyπbonuclease I, (DNAse I, Calbiochεm, San Diego, CA) was dispεrsεd in pεrflubron ( 1 mg/ml) and allowεd to incubate for 1 hour. The perflubron was then evaporated using a Savant Speed Vac (Famiiπgdale,

NY). Thε activity of thε perflubron treated DNAse I to cleave thε phosphodiεstεr linkages of DNA was compared with an uπtreatεd DNAse preparation. Seπal dilutions of a DNAse solution (1 mg/ml) was combined with 50 g DNA and dissolved in 500 L of a 10mM Tπs HCI buffer (6 3 pH) which contained 0 15 mg/ml CaCl, and 8 77 mg/ml NaCI The samples werε placed on an orbital shaker and incubated at 37 C for 30 minutes. The condition of the DNA in each sample after incubation was then exam εd elεctrophoretically over a 1 % agarose gεl which contained ethidium bromide for visualization. No difference in DNA cleavage was observεd bεtwεen the untreated and pεrflubron treated DNAse I samples

XIV The Preparation of DNAsε Microdispεrsion in Perflubron One milliliter of the following solution was prepared: 0.00001 %, w/v, Bovine pancreas deoxyπbonuclease I, (DNAse I) (Calbiochem, San Diego, CA) and 0.001 % polyvinyl pyrrolidonε (PVP) (Sigma, St. Louis, MO), was dissolved into a solution composed of 0.121 %, w/v, tπslhydrαxymethyD-aminomethane (Sigma), 0.0000015 %, w/v, CaCI₂-2H20 (Sigma) and 0.0000877% w/v, NaCI (Sigma). Thε pH of thε solution was adjustεd to 6.3 pπor to adding thε DNAsε or PVP

Onε hundred micrαliters of the DNAse/PVP solution was added to a 12x100 mm test tube containing 5 ml perfluorooctylεthanε (F Tεch, Japan). Thε tube was capped and submergεd in a sonicator bath (Branson Model 3200, Danbury, CT) for 5 seconds to obtain a milky dispersion in the perflubron. The suspension was then evaporatεd to drynεss using a Savant Spεed Vac (Model SC 200). The resulting dπεd microsphεrεs were resuspεndεd with 7 ml Perflubron. A milky DNAse/PVP m-perflubroπ suspension was obtained. Particle size analysis was done by laser diffraction (Hoπba LA 700, Irvine, CA) in the volume weighted modε. Approximately a 20 to 50 L aliquot of each sample was diluted in 9 to 10 ml of π-dodecane. The distribution shape "3", refractive indεx ratio of 1.1 and the fraction cell was used. The resulting rnicrodispersion had a mean droplεt diameter of 2.83 m. Examplεs XIII and XIV clearly demonstrate the feasibility of preparing enzymatically active stabilized dispersions in accordance with the present invention. This Example further illustrates that a number of techmquεs may be used to form compatible particulates useful in the disclosed dispersions.

XV

Preparation of Fluorescent Labeled Perforated Microstructure Powder via Spray Drying

The following matεπals wεrε obtainεd and usεd to manufacture fεεd stock:

0.2% w/w Nitrobenzoyldiol Phosphatidylcholme (Avaπti Polar Lipids, Alabaster, AL)

17.6% w/w Hydroxyεthyl starch (Ajinomoto, Japan) 82.2% w/w Dipalmitoylphosphatidylcholine (Genzyme, Cambridge, MA) Perfluorohεxanε (3M, St. Paul, MN)

Deiomzεd water

Dipalmitoylphosphatidylcholine (DPPC; 1 g) and nitrobenzoyldiol phosphatidylcholme (NBD-PC, 10 mg) were dissolved in 4 ml chloroform. The chloroform was then removed using a Savant Speed Vac (Model SC 200).

Hydroxyethyl starch, (HES; 0.9 g), dipalmitoylphosphatidyl choline (DPPC; 3.19 g) and 75 ml deionized water were then addεd to the DPPC/NBD PC thin film. The surfactants and starch were then dispεrsεd in thε aquεous phasε using an

Ultra-Turrax mixer (model T-25) at 10,000 rpm for approximately 2 minutes (T = 45-50 C). Thε resulting NBD

PC/DPPC/HES dispεrsion was chilled in an ice bath. Perfluorohexane (PFH, 4 11 g ) was then added dropwise during mixing (T = 5 10 C). After the addition was complεte, the resulting PFH in-water emulsion was mixed on the Ultra-Turrax for an additional time of not less than 4 mmutεs. Thε fluorεscεntly labeled microshell powder was obtained by spray drying (Buchi, 191 Mini Spray Dryer, Switzerland). The NBD PC/DPPC/HES containing emulsion was fed at a rate of 5.5 ml/mm. The inlet and outlet tempεratures of the spray dryer were 100 C and 65 C respectively. The nebulization air and aspiration flows were 1,800 L/hr and 100% respectively A free flowing, yellow powder comprising perforated microstructures was obtained.

XVI Inhalation Behavior of a Perforatεd Microstructure In Fluorocarbon Dispersion vs. Agueous Liposomes The nebulization profile as a function of the aerodynamic diametεr of a spray dπεd microshell in perflubron dispersion vs. an aqueous-based posomal dispersion was evaluated using an Andersen Cascade Impactor. For the experiments, compressed air servεd as thε earner and aεrosol generating gas. An air flow rate of 7 5 liters/mm. was established at a pressure of 20 p.s.i. Aerosols were generated w th a DeVilbiss air jet nebulizer (DeVilbiss Co., Somerset, PA). The nebulizer was connected to an Andersen cascade impactor (Sierra Andεrsεn 1 ACFM Nonviable Ambient Particle Sizing Sampler). The aqueous hposomal dispersion was prepared by dispersing fluorescent labeled microshells prepared as set forth in Example XIV in water, followed by somfication with a Vibracell sonicator (Somes Mateπals, 30 mm o.d titanium probe) at a power of 100 watts for approximately 2 minutes (T= 22-25 C). The same perforated microstructures were suspeπdεd in PFOB to providε a stabilized dispεrsion. 5 ml of either a 20 mg/ml fluorescεntly labeled microshell in PFOB dispersion or the aqueous fluorescently-labeled liposomes were nebulized for 4 minutes. The 8 stages of the impactor were then washed with chlorofomrmethaπol (2:1 v/v) Each stage extract was then transferred to a 2 milliliter volumetric flask and q.s. to the mark with chloroform:methaπol (2:1 v/v).

Thε extracts were mεasurεd for fluorεscεncε content using thε following conditions: „ = 481 nm ; _m = 528 nm and quantified by comparison to an external standard curve. Table III lists the characteristics of each cascade impactor stage, the inhalation behavior of the nebulized microshells and liposomεs The NBD PC mass distribution as a function of aεrodyπamic diameter was calculated using calibration curves described by Gonda, et. al„ [Gonda, I., Kayes, J.B., Groom, C.V., and Fildεs, F.J.T.; Characterization of hydroscopic inhalation aerosols. In: Particle Size Analysis 1981

(Eds. N.G. Stanlet-Wood, and T. Allen), pp. 31 43, Wiley Heydeπ Ltd, New York] and incorporated herein by reference.

Compaπsoπ of the two delivery vehicles rεvealεd that thε efficiency of nebulization was greatεr for thε liposomes. On the other hand, a higher percεntagε of the nebulized dose to smaller airway diameters could be achievεd with thε fluorocarbon delivered microstructures, which is reflection of it's smaller median mass aerodynamic diameter (MMAD), achieved due its hollow, porous nature. This Example and the results shown in Table III immediately below clearly illustrate that a number of different colloidal systems, including both particulate dispersions and hposomal preparations, are compatible with the present invention. Table III

Median Mass Aerodynamic Diameters of Hollow Microspheres vs. Liposomes

XVII

Andersεn Impactor Tεst for Assεssing Aεrosol Pεrformancε

Formulations described in Examples XVIII, XIX, XX and XXI comprising Cromolyn sodium were tested using commonly accεptεd pharmaceutical procedures. The mεthod utilized was compliant with thε United State

Pharmacopeia (USP) procedure (Pharmacopeial Previews (1996) 22.3065-3098) incorporated herein by reference. The Andersεn Impactor was associatεd with thε rεspεctivε nebulizer or metered dose inhaler as set forth in the following examplεs and collected aerosolized sample for a specified period.

Extraction procedure. Thε extraction from all the plates, induction port, and actuator were pεrf ormed in closed vials with 10 mL of a suitable solvent. The filter was installed but not assayed, because the polyacrylic binder interfered with the analysis The mass balance and particle size distribution trends indicated that thε deposition on the filter was negligibly small. Thε plates were extractεd with deionized water.

Quantitation procedure Cromolyn sodium was quantitated by absorption spectroscopy (Beckman DU640 spectrophotomεtεr) relative to an external standard curve with the extraction solvent as the blank. Cromolyn sodium was quantitated using the absorption peak at 326 nm

Calculation procedure. For each formulation, thε mass of the drug in the devicε as well as on thε induction port ( 1 ) and platεs (0 7) wεrε quantifiεd as described above. The Fine Particle Dosε and Fine Particle

Fraction was calculated according to the USP method referenced above. Throat deposition was defined as the mass of drug found in the induction port and on plates 0 and 1. The mean mass aerodynamic diameters (MMAD) and geometric standard diameters (GSD) were evaluated by fitting the experimental cumulative function with log normal distribution by using two paramεtεr fitting routine. Thε results of such mεasurεments arε prεsεntεd in subsequent examplεs.

XVlll Nεbuhzation of Porous Particulate Structures Compnsing Phospholipids and Cromolyn sodium in Perfluorooctylethane using a MicroMist Nebulizer Forty milligrams of the lipid basεd microsphεrεs containing 50% cromolyn sodium by weight (as from Example V) were dispersed in 10 ml perfluorooctylethane (PFOE) by shaking, forming a suspension. The suspension was nebulized until the fluorocarbon liquid was delivered or had evaporated using a Micro Mist (DeVilbiss) disposable nebulizer using a PulmoAide^' air compressor (DeVilbiss). As described above an Andersen Cascade Impactor was used to measure the resulting particle size distπbution. Thε impactor was disassεmblεd and the platεs of the impactor were extracted with water. Cromolyn sodium content was mεasured by UV adsorption at 326nm. The fine particle fraction is the ratio of particles deposited in stages 2 through 7 to those deposited in all stages of the impactor The fine particle mass is the weight of mateπal deposited in stages 2 through 7. The deep lung fraction is thε ratio of particles deposited in stages 5 through 7 of the impactor (which correlate to the alveoli) to thosε depositεd in all stagεs. The deεp lung mass is thε weight of matenal deposited in stages 5 through 7. Table IV immediatεly below provides a summary of the results.

Table IV

XIX Nebulization of Porous Particulate Structures Comprising Phospholipids and Cromolyn Sodium in Perfluorooctylethane using a Raindrop Nebulizer A quantity of lipid based microspheres containing 50% cromolyn sodium, as from Example V, wεighing 40 mg was dispersed in 10 ml perfluorooctylεthaπe (PFOE) by shaking, thereby forming a suspension. The suspension was nebulized until the fluorocarbon liquid was delivered or had evaporated using a Raindrop disposable nebulizer (Nellεor Puπtan Bennεt) coππectεd to a PulmoAide air compressor (DeVilbiss). An Andersen Cascade Impactor was used to measure the rεsulting particle size distπbution in thε manner described in Examples XVII and XVlll Table V immediately below providεs a summary of thε results. Table V

XX Nebulization of Agueous Cromolyn Sodium Solution The contεnts of plastic vial containing a unit dosε inhalation solution of 20 mg of cromolyn sodium in 2 ml puπfied water (Dey Laboratories) was nebulized using a Micro Mist disposable nebulizer (DeVilbiss) using a PulmoAide® air compressor (DeVilbiss) The cromolyn sodium solution was nebulized for 30 minutes An Andersen Cascadε Impactor was usεd to measure the resulting particle size distribution, by the method descπbεd above in Example XVII. Table VI immediatεly below providεs a summary of the results. In this regard, it will bε appreciatεd that, thε formulations nebulized from fluorocarbon suspension mediums in Examples XVlll and XIX provided a greater percentage of deep lung deposition than the aqueous solution

Table VI

XXI Preparation of a Metered Dose Inhaler of Cromolyn Sodium A pre weighεd amount of hollow porous cromolyn sodium particles prepared in Example V was placεd into a 10 ml aluminum can and dπεd in a vacuum ovεn undεr thε flow of nitrogεn for 3 4 hours at 40°C. The amount of powder filled into the can was determined by the amount of drug required to providε a desired thεrapεutic effect After this the can was crimp sεalεd using a DF31/50act 50ul valvε (Valois of Amεrica Grεεnwich, CT) and filled with HFA 134a propellant (DuPont, Wilmington DE) by overpressure through the stem Thε amount of propellant in the can was determined by weighing the can before and after the fill.

The filled MDI was then used to compare the administration of cromolyn sodium using a metεrεd dosε inhaler and a nεubulizer. More specifically, a cromolyn sodium preparation was nebulized and quantitated as descnbed in Example XVlll. The MDI was then associated with thε Andεrsεn impactor and discharged For the test 5 shots wεre sent to waste and, 20 shots were made into the Andersen impactor. A compaπson of the Andersεn cascadε impactor results for thε nebulized cromolyn sodium and the cromolyn sodium administered by the MDI is shown in Fig. 3. As seen in the Figure, a significantly greater percentage of the nebulized drug is found on plates 5 7 showing the enhanced potential for systemic delivery via nebulization

XXII

Nebulization of Porous Particulate Structures Comprising Mixtures of Long Chain/Short Chain Phospholipids and Albuterol Sulfate in Perflubron To further demonstrate the diversity of the present invention the spray dried powder from Example IV was dispersed in perflubron (Atochem, France) at 0.2 wt% concentration. The resulting stabilized dispεrsion did not show any visible sedimentation over 30 minutes and could be easily nebulized with a Pulmo-Neb Disposable Nebulizer (DeVilbiss,

Somerset, PA). A significant deposition of the powder was found on plates 4 and 5 of an Andersεn cascadε impactor, as judged by visual inspection, indicating that significant deposition is likely in human secondary and terminal bronchi.

Those skilled in the art will further appreciate that the present invention may be embodied in other specific forms without departing from the spmt or central attributes thereof In that the foregoing dεscπption of thε prεsεnt invention discloses only exemplary embodiments thereof, it is to be understood that, other vaπations are contemplated as being within the scopε of the presεnt invention. Accordingly, the presεnt invεntion is not limited to the particular embodiments which have been descnbed in detail herein. Rather, reference should be made to thε appended claims as indicative of the scopε and contεπt of the invention.

Claims

WHAT IS CLAIMED:

I Use of a liquid fluorochemical in the manufacture of a medicament for the pulmonary delivery of a bioactive agent whereby the medicamεnt compπses a stabilized dispersion having a fluorochemical continuous phase which is nebulized using a nebulizer to form an aerosolized medicamεnt comprising said bioactivε agεnt wherεin said aerosolized medicament is in a form for administration to at least a portion of the pulmonary air passages of a patient in need thereof

2. The use of claim 1 wherein said stabilized dispersion compπses a reverse emulsion, microemulsion or a particulate dispersion 3. The use of claim 1 wherein said stabilized dispersion compπses a plurality of particulates suspended in said fluorochemical continuous phase wherein said particulates are selected from the group consisting of micromzed particles, naπocrystals, spray dπed microspheres, perforated microstructures and combinations thereof.

4 The usε of claim 1 whεrem said stabilized dispersion compπses a plurality of perforatεd microstructures suspended in said fluorochemical continuous phase 5 The usε of claim 4 whεrεin said perforated microstructures compπsε a surfactant

6. The use of claim 5 wherein said surfactant is selected from the group consisting of phospholipids, nonionic detεrgents, nonionic block copoiymεrs, ionic surfactants, biocompatible fluoπnated surfactants and combinations thereof.

7 The usε of claims 5 or 6 wherein said surfactant is a phospholipid 8. The use of claim 7 wherein said phospholipid is selectεd from thε group consisting of dilauroylphosphatidylcho nε, diolεylphosphatidylchohnε, dipalmitoylphosphatidylcholine, disteroylphosphatidylcholine, behenoylphosphatidyl choline, arachidoylphosphatidylcholine and combinations thereof

9. The use of claims 4 to 8 wherein the mean aerodynamic diameter of the perforated microstructures is between 0 5 and 5 μm

10. The use of any of claims 1 to 9 wherein said bioactive agent is selected from the group consisting of antiallergics, bronchodilators, pulmonary lung surfactants, analgesics antibiotics, leukotπenε inhibitors or antagonists antihistamiπes, antimflammatoπes, antmeoplastics, anticholinergics, anesthetics, anti tuberculars, imaging agents, cardiovascular agents, enzymεs, stεroids, gεnεtic mateπal, viral vectors, antisensε agεnts, protεins, pεptides and combinations thereof.

I I The use of any of claims 1 to 10 wherεin said bioactivε agεnt is delivered to the systemic circulation of said patient.

12 A method for forming a stabilized respiratory dispεrsion compnsing thε stεps of combining a plurality of perforatεd microstructures compnsing at least one bioactive agent with a predetermined volume of a nonaqueous suspension medium to provide a respiratory blend wherein said suspension medium pεrmeatεs said perforatεd microstructures; and mixing said respiratory blend to provide a substantially homogeneous respiratory dispersion. 13. The method of claim 12 wherein said perforatεd microstructures comprise a surfactant

14. The method of claim 13 wherein said surfactant is selected from the group consisting of phospholipids, nonionic detergents, nonionic block copolymers, ionic surfactants, biocompatible fluoππatεd surfactants and combinations thereof.

15. The method of claim 13 or 14 wherein said surfactant is a phospholipid. 16. The method of claim 15 wherein said phospholipid is selected from the group consisting of diiauroylphosphatidylcholine, dioleylphosphatidylcholine, dipalmitoylphosphatidylcholine, disteroylphosphatidylcholine behenoylphosphatidylcholine, arachidoylphosphatidylcholine and combinations thereof

17. Thε mεthod of any of claims 12 to 16 wherein said suspension medium and said perforated microstructures havε a refractive index differential of lεss than about 0.5.

18. The method of any of claims 12 to 17 wherein said perforated microstructures compπse hollow porous microspheres.

19. The method of any of claims 12 to 18 wherein the mean aεrodynamic diameter of said perforatεd microstructurεs is bεtwεεn 0 5 and 5 μm 20. The method of any of claims 12 to 19 wherein said bioactive agent is selected from the group consisting of antiallergics, bronchodilators, pulmonary lung surfactants, analgesics, antibiotics, leukotnene inhibitors or antagonists, antihistamiπεs, antiiπflammatoπεs, antmeoplastics, anticholinergics, anεsthetics, anti tuberculars, imaging agεnts, cardiovascular agεnts, enzymεs, stεroids, gεnεtic matεπal, viral vεctors, antisεnsε agεnts, protεins, peptides and combinations thereof. 21. A method for stabilizing a respiratory dispεrsion by reducing attractive van der Waals forces compnsing the steps of. providing a plurality of perforated microstructures; combining the perforatεd microstructures with a suspension medium comprising at least one fluorochemical wherein the suspεnsion medium and the perforatεd microstructures are selected to provide a refractive index differential value of less than about 0.5.

22. The method of claim 21 whεrεin said perforated microstructures comprise a surfactant

23. Thε mεthod of claim 22 whεrεin said surfactant is sεlεctεd from the group consisting of phospholipids, nonionic detεrgents, nonionic block copolymers, ionic surfactants, biocompatible fluoπnated surfactants and combinations thereof.

24. The method of claim 22 or 23 wherein said surfactant is a phospholipid. 25 The method of claim 24 wherεin said phospholipid is sεlεcted from thε group consisting of dilauroylphosphatidylcholinε, dioleylphosphatidylchohπe, dipalmitoylphosphatidylcholine, disteroylphosphatidylcholine behenoylphosphatidylcholine, arachidoylphosphatidylcholine and combinations thereof.

26. The method of any of claims 21 to 25 wherein said perforatεd microstructures compπsε hollow porous microspheres.

27. The method of any of claims 21 to 26 whεrεin said bioactivε agent is selectεd from the group consisting of antiallergics, bronchodilators, pulmonary lung surfactants, analgesics, antibiotics, leukotnene inhibitors or antagonists, antihistaminεs, antiinflammatoπεs, antmeoplastics, anticholinergics, anεsthεtics, anti tuberculars, imaging agents, cardiovascular agents, enzymes, steroids, genetic matenal, viral vectors, antisense agents, protεins, peptides and combinations thereof

28. A stable respiratory dispersion for use in a nebulizer compnsing a suspension medium having dispersεd therein a plurality of perforatεd microstructures compnsing at lεast onε bioactivε agεπt whεrεin said suspension mεdium substantially permeates said perforatεd microstructures.

29 The dispersion of claim 28 wherein said perforatεd microstructurεs compπsε a surfactant 30. The dispersion of claim 29 wherein said surfactant is selεcted from the group consisting of phospholipids, nonionic detergεnts, nomomc block copolymεrs, ionic surfactants, biocompatible fluoπnatεd surfactants and combinations thereof.

31. The dispersion of claim 29 or 30 wherεin said surfactant is a phospholipid

32. The dispersion of claim 31 wherein said phospholipid is selectεd from the group consisting of dilauroylphosphatidylcholine, diolεylphosphatidylchohnε, dipalmitoylphosphatidylcholine, disteroylphosphatidylcholine behenoylphosphatidylcholine, arachidoylphosphatidylcholine and combinations thereof.

33. The dispersion of any of claims 28 to 32 wherein said suspension medium and said perforated microstructures have a refractive index differential of less than about 04 34 The dispersion of any of claims 28 to 33 wherein said perforated microstructures comprise hollow porous microspheres

35. The dispersion of any of claims 28 to 34 wherein the mean aerodynamic diameter of said perforatεd microstructures is between 0 5 and b μ

36. Thε method of any of claims 28 to 35 wherein said bioactive agent is selected from the group consisting of antiallergics, bronchodilators, pulmonary lung surfactants, analgesics, antibiotics, leukotnene inhibitors or antagonists, antihistamines, antiinflammatoπes, antmeoplastics, anticholinergics, anεsthetics, anti tuberculars, imaging agents, cardiovascular agents, enzymes, steroids, genetic matenal, viral vectors, antisense agents, proteins, peptides and combinations thereof

37. An inhalation system for the pulmonary administration of a bioactive agent to a patient compnsing. a fluid reservoir; a stable respiratory dispersion in said fluid reservoir wherein said stabilized dispersion compπses a fluorochemical continuous phase and at least one bioactive agεnt; and a nebulizer operably associated with said fluid reservoir wherein the nebulizer is capable of aerosolizing and discharging the stable respiratory dispersion.

38. The system of claim 37 wherein said stabilized dispersion compπses a reverse emulsion, microemulsion or a particulate dispersion.

39. The system of claim 37 wherein said stabilized dispersion comprises a plurality of particulates suspendεd in said fluorochemical continuous phase wherεin said particulates are selected from the group consisting of micromzed particles, naπocrystals, spray dned microspheres, perforated microstructures and combinations thereof.

40. The system of claim 37 wherein said stabilized dispersion comprises a plurality of perforated microstructures suspended in said fluorochemical continuous phase

41. The systεm of claim 40 wherein said perforated microstructures compπsε a surfactant 42. The system of claim 41 wherεin said surfactant is selected from the group consisting of phospholipids, nonionic detεrgents, nonionic block copolymers, ionic surfactants, biocompatible fluoπnated surfactants and combinations thereof.

43 The system of claims 41 or 42 whεrein said surfactant is a phospholipid.

44. The system of claim 43 wherein said phospholipid is selected from the group consisting of dilauroylphosphatidylcholme, dioleylphosphatidylchohne, dipalmitoylphosphatidylcholine, disteroylphosphatidylcholine, behenoylphosphatidyl choline, arachidoylphosphatidylcholine and combinations thereof.

45. The system of any of claims 40 to 44 wherein the mean aerodynamic diameter of the perforated microstructures is betwεεn 0 5 and b μ . 46. Thε system of any of claims 37 to 45 wherein said bioactive agent is selectεd from thε group consisting of antiallergics, bronchodilators, pulmonary lung surfactants, analgesics, antibiotics, leukotπεnε inhibitors or antagonists, antihistamines, antiinflammatoπes, antmeoplastics, anticholinergics, anesthetics, anti tuberculars, imaging agents, cardiovascular agents, enzymεs, stεroids, genetic matεπal, viral vεctors, antisense agents, protεins, pεptidεs and combinations thereof.

47. Thε systεm of any of claims 37 to 46 wherein said bioactive agent compπses a compound selectεd from thε group consisting of protεins, peptides and genetic material. 48. The system of any of claims 37 to 47 wherein said fluid resεrvoir is a multi dose resεrvoir or a single dose reservoir.

49. The system of any of claims 37 to 48 wherεin said nebulizer is a jet nebulizer, an ultrasonic nebulizer or a single-bolus nebulizer.

50. A method for thε pulmonary delivery of one or more bioactive agents compnsing the steps of: providing a stabilized respiratory dispersion compnsing one or more bioactive agents wherein the respiratory dispersion compπses a fluorochemical continuous phase; nebulizing said respiratory dispersion with a nebulizer to provide an aerosolized medicament; and administeπng a thεrapεutically effective amount of said aerosolized medicamεnt to at lεast a portion of the pulmonary passages of a patient in need thereof.