WO1999016421A1

WO1999016421A1 - Stabilized bioactive preparations and methods of use

Info

Publication number: WO1999016421A1
Application number: PCT/US1998/020613
Authority: WO
Inventors: Luis A. Dellamary; Thomas E. Tarara; Alexey Kabalnov; Jeffry G. Weers; Ernest G. Schutt
Original assignee: Inhale Therapeutic Systems, Inc.
Priority date: 1997-09-29
Filing date: 1998-09-29
Publication date: 1999-04-08
Also published as: MEP4108A; EA200000375A1; CA2304975A1; JP4526702B2; NO20001618D0; DK1019022T3; AU750567B2; IL135126A; ES2195415T3; JP2003525841A; JP2010047615A; KR20010030801A; CA2304820A1; JP6100114B2; JP2013199497A; DK1019022T4; KR100599634B1; AU9677098A; ES2205560T5; EP1019021A1

Abstract

Stabilized dispersions are provided for the delivery of a bioactive agent. The dispersions preferably comprise a plurality of perforated microstructures dispersed in a suspension medium that typically comprises a liquid fluorochemical. 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 directly administered to the lung of a patient using an endotracheal tube or bronchoscope.

Description

STABILIZED BIOACTIVE PREPARATIONS AND METHODS OF USE

Field of the Invention The present invention generally relates to formulations and methods for the administration of bioactive agents to a patient in need thereof. More particularly, the present invention relates to methods, systems and compositions comprising relatively stable dispersions of perforated microstructures in a suspension medium that are preferably administered via liquid dose instillation both for topical delivery to the lung, and for delivery via the lung to the systemic circulation.

Background of the Invention

The efficacy of many pharmaceutical agents is predicated on their ability to proceed to the selected target sites and remain there in effective concentrations for sufficient peπods of time to accomplish the desired therapeutic or diagnostic purpose Difficulty in achieving efficacy may be exacerbated by the location and environment of the target site as well as by the inherent physical characteristics of the compound administered For example, drug delivery via routes that are subject to repeated drainage or flushing as part of the body's natural physiological functions offer significant impediments to the effective administration of pharmaceutical agents In this respect, delivery and retention problems are often encountered when administering compounds through the respiratory or gastrointestinal tracts Repeated administration of fairiy large doses are often required to compensate for the amount of drug washed away and to maintain an effective dosing regimen when employing such routes. Moreover, the molecular properties of the pharmaceutical compound may impair the absorption through a given delivery route, thereby resulting in a substantial reduction in efficacy. For instance, insoluble particulates are known to be subject to phagocytosis and pinocytosis, resulting in the accelerated removal of the compound from the target site Such reductions in delivery and retention time complicate dosing regimes, waste pharmaceutical resources and generally reduce the overall efficacy of the administered drug.

In this respect, one class of delivery vehicles that has shown great promise when used for the administration of pharmaceutical agents is fluorochemicais. During recent years, fluorochemicais have found wide ranging application in the medical field as therapeutic and diagnostic agents. The use of fluorochemicais to treat medical conditions is based, to a large extent, on the unique physical and chemical properties of these substances. In particular, the relatively low reactivity of fluorochemicais allows them to be combined with a wide vaπety of compounds without altering the properties of the incorporated agent This relative inactivity, when coupled with other beneficial characteristics such as an ability to carry substantial amounts of oxygen, radioopaqueness for certain forms of radiation and low surface energies, have made fluorochemicais invaluable for a number of therapeutic and diagnostic applications. Among these applications is liquid ventilation. For all practical purposes, liquid ventilation became a viable technique when it was discovered that fluorochemicais could be used as the respiratory promoter. Liquid breathing using oxygenated fluorochemicais has been explored for some time For example, an animal submerged in an oxygenated fluorochemical liquid, may exchange oxygen and carbon dioxide normally when the lungs fill with the fluorochemical. In this regard it has been shown that mammals can derive adequate oxygen for survival when submerged by breathing the oxygenated fluorochemical liquid. In particular, it has been established that total liquid ventilation may keep mammals alive for extended periods prior to returning them to conventional gas breathing.

Those skilled in the art will appreciate that contemporary liquid ventilation is an alternative to standard mechanical ventilation which involves introducing an oxygeπatable liquid medium into the pulmonary air passages for the purposes of waste gas exchange and oxygenation Essentially, there are two separate techniques for performing liquid ventilation, total liquid ventilation and partial liquid ventilation. Total liquid ventilation or "TLV" is the pulmonary introduction of warmed, extracorporeally oxygenated liquid respiratory promoter (typically fluorochemicais) at a volume greater than the functional residual capacity of the subject. The subject is then connected to a liquid breathing system and tidal liquid volumes are delivered at a frequency depending on respiratory requirements while exhaled liquid is purged of CO, and oxygenated extracorporeally between the breaths. This often involves the use of specialized fluid handling equipment.

Conversely, partial liquid ventilation or "PLV" involves the use of conventional mechanical ventilation in combination with pulmonary administration of a respiratory promoter capable of oxygenation. In PLV a liquid, vaporous or gaseous respiratory promoter (ι.e. a fluorochemical) is introduced into the pulmonary air passages at volumes ranging from just enough to interact with or coat a portion of the pulmonary surface all the way up to the functional residual capacity of the subject. Respiratory gas exchange may then be maintained for the duration of the procedure by, for example, continuous positive pressure ventilation using a conventional open circuit gas ventilator Alternatively, gas exchange may be maintained through spontaneous respiration. When the procedure is over, the introduced respiratory promoter or fluorochemical may be allowed to evaporate from the lung rather than being physically removed as in TLV. For the purposes of the instant application the term "liquid ventilation" will be used in a generic sense and shall be defined as the introduction of any amount of respiratory promoter or fluorochemical into the lung, including the techniques of partial liquid ventilation, total liquid ventilation and liquid dose installation. Use of liquid ventilation may provide significant medical benefits that are not available through the use of conventional mechanical ventilators employing a breathable gas. For example, the weight of the respiratory promoter opens alveoli with much lower ventilator pressure than is possible with gas. Additionally, liquid ventilation using fluorochemicais as the respiratory promoter has been shown to be effective in πnsing out congestive materials associated with respiratory distress syndrome. Moreover, liquid ventilation has been shown to be a promising therapy for the treatment of respiratory distress syndromes involving surfactant deficiency or dysfunction Elevated alveolar surface tension plays a central role in the pathophysiology of the Respiratory Distress Syndrome (RDS) in premature infants and is thought to contribute to the dysfunction in children and adults Liquid ventilation, particularly using fluorochemicais, is effective in surfactant deficient disorders because it eliminates the air/fluid interfaces in the lung and thereby greatly reduces pulmonary surface tension Moreover, liquid ventilation can be accomplished without undue alveolar pressures or impairing cardiac output and provides excellent gas exchange even in premature infants. Finally, fluorochemicais have also been shown to have pulmonary and systemic anti inflammatory effects

In addition to liquid ventilation, it has been recognized that fluorochemicais may be effective in the pulmonary delivery of bioactive agents in the form of liquid or solid particulates. For example, pulmonary delivery of bioactive agents using fluorochemical suspensions is described in Sekms et al., U.S. Patent No.

5,562,608, Fuhrman, U S Patent No 5,437,272, Faithful) et al U S Patent No 5,490,498, Trevino et al U.S. Patent No. 5,667,809 and Schutt U.S. Patent No. 5,540,225 each of which is incorporated herein by reference The bioactive agents may preferably be delivered in conjunction with partial liquid ventilation or lavage Due to the physical characteristics of compatible respiratory promoters or fluorochemicais, the use of such techniques provides for improved dispersion of the incorporated agent in the lung thereby increasing uptake and increasing efficacy. Further, direct administration of the bioactive agent is particularly effective in the treatment of lung disease as poor vascular circulation of diseased portions of the lung reduces the efficacy of intravenous drug delivery. Besides treating pulmonary disorders, fluorochemical pharmaceutical formulations administered to the lung could also prove useful in the treatment and/or diagnosis of disorders such as RDS, impaired pulmonary circulation, cystic fibrosis and lung cancer. It will also be appreciated that, in addition to the pulmonary route of administration, fluorochemicais could advantageously be used for the administration of compounds via other routes such as topical, oral (e.g. for administration to the gastrointestinal tract), iπtrapeπtoneal, or ocular. Unfortunately, regardless of the administration route, the use of fluorochemical suspensions may result in unreliable and irreproducible drug delivery due to the admimstraion of a non homogeneous dispersion or instability of the particulates in the fluorochemical phase

More particularly, drug suspensions in liquid fluorochemicais comprise heterogeneous systems which usually require redispersioπ prior to use Yet, because of factors such a patient compliance obtaining a relatively homogeneous distribution of the pharmaceutical compound is not always easy or successful. In addition, prior art formulations comprising micronized particulates may be prone to aggregation of the particles which can result in inadequate delivery of the drug. Crystal growth of the suspensions via Ostwald ripening may also lead to particle size heterogeneity and can significantly reduce the shelf life of the formulation. Another problem with conventional dispersions is particle coarsening. Coarsening may occur via several mechanisms such as flocculation, fusion, molecular diffusion, and coalescence. Over a relatively short period of time these processes can coarsen the formulation to the point where it is no longer usable. As such, while such systems are certainly a substantial improvement over prior art non fluorochemical delivery vehicles, the drug suspensions may be improved upon to enable formulations with improved stability that also offer more efficient and accurate dosing at the desired site.

Accordingly, it is an object of the present invention to provide stabilized preparations for the administration of bioactive agents. It is another object of the present invention to provide methods, systems and compositions that advantageously allow for the efficient delivery of bioactive agents to the pulmonary air passages of a patient,

It is a further object of the present invention to provide for the delivery of bioactive agents to the systemic circulation of a patient.

It is yet another object of the present invention to provide stabilized preparations suitable for instillation to the pulmonary air passages 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 to selected physiological target sites using stabilized preparations. In preferred embodiments, the bioactive agents are in a form for administration to at least a portion of the pulmonary air passages of a patient via liquid dose instillation. More particularty, the present invention provides for the formation and use of stabilized dispersions and delivery systems comprising such dispersions, as well as individual components thereof. Unlike prior art suspensions or dispersions for drug delivery, 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 by flocculation, sedimentation or creaming. Moreover, the stabilized preparations of the present invention preferably comprise a suspension medium that further serves to reduce the rate of degradation with respect to the incorporated bioactive agent. In particularty preferred embodiments, the suspension medium will comprise a fluoriπated compound, fluorochemical or fluorocarbon. Those skilled in the art will appreciate that the disclosed stable preparations, and systems comprising those preparations, act to reduce dosing incongruities, retard degradation of incorporated bioactive agents and allow for more concentrated dispersions.

In a broad sense, the stabilized dispersions of the present invention comprise a continues phase suspension medium having a plurality of perforated microstructures dispersed or suspended therein wherein the stabilized dispersions are capable of being administered to the lung of a patient in need thereof. As discussed above, the disclosed preparations will preferably be administered at least a portion of the pulmonary air passages of a patient using liquid dose instillation (LDI). Those skilled in the art will appreciate that LDI comprises the direct instillation or administration of a liquid preparation to the lungs. Preferably, LDI comprises instillation of a bioactive preparation to the pulmonary air passages using a pulmonary delivery conduit. In this respect, the preparation may be delivered to an intubated patient through an eπdotracheal tube, or to a free-breathing patient via bronchoscope, or may even be administered using standard tubing and/or a syringe. It shouid be emphasized that the methods and systems disclosed herein may be used with both ventilated and nonventilated patients. Moreover, the present invention may be used in conjunction with liquid ventilation (e.g. both PLV and TLV). As the stabilized dispersions of the present invention may be administered by a variety of routes and methods, such as top-loading onto existing fluorochemical (i.e. in the lung), trickle-filling or lavage, dosages can be more effectively administered and controlled. Specifically, administration of bioactive agents in a fluorochemical, as is contemplated herein, provides a relatively anhydrous environment wherein the physiological uptake of the drug may be dramatically increased

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 and/or porous perforated microstructures that substantially reduce attractive molecular forces, such as van der Waals forces, which dominate pπor 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. reduce Hamaker constant differentials) and further stabilize the preparation The relatively homogeneous nature of these particulate dispersions or suspensions, inhibits detenoration thereby allowing for pharmaceutical preparations having enhanced stability.

With regard to the dispersed 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 compπse 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 material or configuration may be used to form the microstructure matrix With regard to the selected materials, it is desirable that the microstructure incorporates at least one surfactant. Preferably, this surfactant will compπse a phospho pid or other surfactant approved for pulmonary use. As to the configuration, particularly preferred embodiments of the invention incorporate spray dried, 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 stabilized dispersions for the delivery of a bioactive agent compnsing a biocompatible 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

It should further be appreciated that the suspension medium may be any liquid or compound that is in liquid form, under appropriate thermodynamic conditions, for formation of a compatible particulate dispersions. 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 liquid dose instillation, 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 compπse fluorochemicais (e.g. perfluorocarboπs or fluorocarbons) that are liquid at room temperature. As discussed above, It is well established that many fluorochemicais have a proven history of safety and bioco patibility in the lung. Further, in contrast to aqueous solutions, fluorochemicais do not negatively impact gas exchange. Moreover, because of their unique wettabihty characteπstics, fluorochemicais may be able to provide for the dispeπon of particles deeper into the lung, thereby improving systemic delivery. Finally, many fluorochemicais are also bacteπostatic thereby decreasing the potential for microbial growth in compatible preparations.

Accordingly, the present invention provides for the use of a liquid fluorochemical in the manufacture of a stabilized dispersion for the pulmonary delivery of a bioactive agent whereby the stabilized dispersion is directly administered to at least a portion of the pulmonary air passages of a patient in need thereof, said stabilized dispersion compnsing a fluorochemical suspension medium having dispersed therein a plurality of perforated microstructures compnsing at least one bioactive agent wherein the suspension medium substantially permeates said perforated microstructures.

It will further be appreciated that, in selected embodiments, the present invention comprises 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 comprise perforated microstructures that allow for the perfusion or permeation of the selected suspension medium

As such, preferred embodiments of the invention provide methods for forming a stabilized dispersion for direct pulmonary administration of a bioactive agent comprising the steps of: combining a plurality of perforated microstructures compnsing at least one bioactive agent with a predetermined volume of a biocompatible 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 stabilized 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 dispersion by reducing attractive van der Waals forces comprising the steps of: providing a plurality of perforated microstructures, combining the perforated microstructures with a biocompatible suspension medium comprising at least one liquid 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 comprise 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 systems for the direct pulmonary administration of a bioactive agent to a patient compnsing- a fluid reservoir, a stable dispersion in said fluid reservoir wherein said stabilized dispersion compnses a biocompatible suspension medium having a plurality of perforated microstructures dispersed therein, said perforated microstructures compnsing at least one bioactive agent; and a pulmonary delivery conduit operably associated with said fluid reservoir wherein the delivery conduit is capable of administering the stabilized dispersion to at least a portion of the pulmonary air passages of a patient in need thereof.

Those skilled in the art will appreciate the term "pulmonary delivery conduit", as used herein, shall be construed in a broad sense to comprise any device or apparatus, or component thereof, that provides for the instillation or administration of a liquid in the lungs In this respect a pulmonary delivery conduit or delivery conduit shall be held to mean any bore, lumen, catheter, tube, conduit, syringe, actuator, mouthpiece, endotracheal tube or hronchoscope that provides for the administration or instillation of the disclosed dispersions to at least a portion of the pulmonary air passages of a patient in need thereof. It will be appreciated that the delivery conduit may or may not be associated with a liquid ventilator or gas ventilator In particularly preferred embodiments the delivery conduit shall compπse an endotracheal tube or bronchoscope. Yet another associated advantage of the present invention is the effective 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 antiallergics, bronchodilators, bronchoconstπctors, pulmonary lung surfactants, analgesics, antibiotics, leukotnene inhibitors or antagonists, anticholinergics, mast cell inhibitors, aπtihistamines, antiiπflammatoπes, antmeoplastics, 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.

Accordingly, the present invention provides methods for the delivery of one or more bioactive agents compnsing the steps of: providing a stabilized dispersion compnsing a biocompatible suspension medium having dispersed therein a plurality of perforated microstructures wherein said perforated microstructures comprise a bioactive agent, and admimstenng a therapeutically effective amount of said stabilized dispersion to at least a portion of the pulmonary passages of a patient in need thereof

While the stabilized dispersions of the present invention are particularly suitable for the pulmonary administration of bioactive agents, they may also be used for the localized or systemic administration of compounds to any location of the body. Accordingly, it should be emphasized that, in preferred embodiments, the formulations may be administered using a number of different routes including, but not limited to, the gastrointestinal tract, the respiratory tract, topically, intramuscularly, intrapeπtoneally, nasally, vaginally, rectally, aurally, orally or ocularly. With respect to particulate dispersions the selected bioactive agent, or agents, may be used as the sole structural component of the perforated microstructures Conversely, the perforated microstructures may comprise one or more components (i e structural materials, surfactants, excipients, etc ) in addition to the incorporated bioactive agents In particularly preferred embodiments, the suspended perforated microstructures will compπse 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.

The stabilized dispersions of the invention may optionally compπse one or more additives to further enhance stability or increase biocompatibility For example, various 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 expenmentation 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 descnption of preferred exemplary embodiments thereof

Detailed Descπption 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 methods and compositions that allow for the formation of stabilized suspensions that may advantageously be used for the delivery of bioactive agents

The enhanced stability of the suspensions is primarily 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 regard, the perforated microstructures comprise pores, voids, and 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 hollow and porous, almost honeycombed or foam like in appearance In especially preferred embodiments the perforated microstructures comprise hollow, porous spray dried microspheres. With respect to the instant specification, the terms "perforated microstructures" and "perforated microparticles" are used to describe porous products, preferably comprising a bioactive agent, distributed throughout the suspension medium in accordance with the teachings herein Accordingly, the subject terms may be used interchangeably throughout the instant specification unless the contextual setting indicates otherwise When the perforated microstructures are placed in the suspension medium (i.e. propellant), the suspension medium is able to permeate the particles, thereby creating a "homodispersioπ", wherein both the continuous and dispersed phases are substantially indistinguishable. Since the defined or "virtual" particles (i e. comprising the volume circumscribed by the microparticulate matrix) are made up almost entirely of the medium in which they are suspended, the forces driving particle aggregation (flocculation) are minimized. Additionally, having the microstructures filled with the medium, thereby effectively slowing particle creaming or sedimentation minimizes the differences in density between the defined particles and the continuous phase.

Due to their stability and substantially homogenous nature, the stabilized suspensions of the present invention are compatible with inhalation therapies and may be used in conjunction with metered dose inhalers, dry powder inhalers and nebulizers In particularly preferred embodiments the disclosed perforated microstructures may be dispersed in a suitable suspension medium (e.g. a long chain liquid fluorochemical) and directly administered to the pulmonary air passages of a patient in need thereof For the purposes of the instant specification, methods comprising direct administration of a stabilized dispersion to the lungs such as through an endotracheal tube or a broπchoscope, will be termed liquid dose instillation While the compositions of the present invention are particularly effective for pulmonary drug delivery, it will be appreciated that they may also be used to drugs to a variety of physiological sites including body cavities and organs. Accordingly, the stabilized dispersions may be administered topically, subcutaneously intramuscularly, intrapeπtoneally, nasally, vaginally, rectally, orally or ocularly.

In contrast to many prior art suspensions, the dispersions of the present invention are designed not to increase repulsion between particles, but rather to decrease attractive forces. The principal forces driving flocculation in nonaqueous media are van der Waals (VDW) attractive forces. VDW forces are quantum mechanical in origin, and can be visualized as attractions between fluctuating dipoles (i.e. induced dipole induced 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 sums 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 VDW potential, V _A , can be approximated by. _{v =} ^{~ A} ,// ^ i -ft . where ^^ is the effective Hamaker constant which

6 -7 . ( /- , + Λ . ) accounts for 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 polaπzabilities of the dispersed particles and the suspension medium:

^A _ejr = (_SJΛ-_SM ~ •_s}A_PΛRT )Z where -4 and A_PART 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 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 Besides 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 provide a good indication as to the stability of the preparation. Alternatively, one could select relatively compatible perforated microstructure components and suspension mediums using characteristic physical values that coincide with measurable Hamaker constants but are more readily discernible.

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 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 expenmentation. 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-octaπe 1.397

DMPC 1 43

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 perforated particles or 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 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 excipients are particularly compatible without undue experimentation given the instant disclosure. The ultimate choice of preferred excipients 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 provides for stabilized dispersions, at least in part, by immobilizing the bioactive agent(s) and excipients (including surfactants) 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 alluded to above, the minimization of density differences between the particles and the continuous phase is largely dependent on the perforated and/or hollow nature of the 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/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%, and 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 a matrix or shell volumes typically contπbutes little to the virtual particle density which is overwhelmingly dictated by the suspension medium found therein. Of course, in selected embodiments the excipients 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 substantially eliminating the attractive van der

Waals forces. 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/cm³ 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 viscoelastic behavior. Rheological behavior of this type is counterproductive in the administration of bioactive agents. 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 denved 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 porous structures of the present invention do not exhibit undesirable viscoelastic 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 the disclosed suspensions is thought to be due, at least in large part, to the relatively low VDW 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, more preferably 0.001 0.01. Yet other preferred embodiments comprise stabilized suspensions having volume fractions from approximately 0.01 to approximately 0.1.

The perforated microstructures of the present invention may also be used to stabilize dilute suspensions of micronized bioactive agents. In such embodiments the perforated microstructures may be added to increase the volume fraction of particles in the suspension, thereby increasing suspension stability to creaming or sedimentation. Further, in these embodiments the incorporated microstructures may also act in preventing close approach (aggregation) of the micronized 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.

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, flocculation 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 suspension, a preferred method for the purposes of the present invention compnses 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 a particulate suspension is sealed glass vials The vials are agitated or shaken to provide relatively homogeneous dispersions which are then set aside and observed using appropriate instrumentation or by eye The time necessary for the suspended particulates to cream to 1 /2 the volume of the suspension medium (i.e to nse to the top half of the suspension medium) or to sediment within 1 /2 the volume (i e to settle in the bottom half of the medium) is then noted. Suspension formulations having a creaming time greater than 1 minute are preferred and indicates 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 peπods for sedimentation times are similarly indicative of compatible dispersions Regardless of the ultimate composition or precise creaming time, the stabilized respiratory dispersions of the present invention compπse a plurality of perforated microstructures or microparticulates that are dispersed or suspended in the suspension medium. Preferably the perforated microstructures comprise a structural matπx that exhibits, defines or comprises 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 characteπstics is contemplated as being within the scope of the invention Accordingly, while preferred embodiments can compπse approximately microsphencal shapes, collapsed, deformed or fractured particulates are also compatible. With this caveat, it will be appreciated that particularly preferred embodiments of the invention comprise spray dried hollow, porous microspheres In order to maximize dispersion stability and optimize bioavailabi ty upon administration, the mean geometnc particle size of the perforated microstructures is preferably about 0.5 50 m, more preferably 1 30 m Unlike aerosolization techniques, liquid dose instillation or administration of bioactive agents does not depend critically on the aerodynamic properties of the particle for efficient biodistπbution Rather, the unique wettability characteristics of the FC suspension medium and the homogeneous nature of the dispersion promotes efficient biodistπbution Thus, there may be some advantage to using larger particles (i e. 5 30 μm) for this application, since recent studies (Edwards et al, Science 1997, 276:1868 1871, which is incorporated herein by reference) have suggested that large porous particles may be able to provide a sustained release of bioactive agent Edwards et a/. claim that their large porous particles are effective sustained release agents upon inhalation because they are too large to be effectively cleared by pulmonary macrophages, yet light enough to penetrate deep into the lung, thereby avoiding clearance by the mucociliary escalator In this regard it will be appreciated that the compositions and methods of the present invention may provide for the deep lung deposition of the bioactive particulates thereby countering, at least in part, the mucociliary escalator Accordingly, larger perforated microstructures having a geometπc diameter of greater than approximately 5 m may prove to be particularly effective when administered (ι.e. by LDI) using the disclosed dispersions Besides the aforementioned advantages, there may be significant differences in local versus systemic bioavailability depending upon the size of the hollow porous particles delivered via liquid dose instillation For example it is easy to envision that smaller particles (ca. 1 μm) may be more efficiently delivered to the alveolus than large particles (ca 20 μm) The choice of particle size will ultimately be dependent on the nature of the bioactive agent and its intended site of action In especially preferred embodiments the perforated microstructures will comprise a powder of dry, hollow, porous microspheπcal shells of approximately 1 to 30 m in diameter, with shell thicknesses of approximately 0 1 to approximately 0.5 m. It is a particular advantage of the present invention that the particulate concentration of the dispersions and structural matnx components can be adjusted to optimize the delivery characteristics of the selected particle size.

As alluded to throughout the instant specification the porosity of the microstructures may play a significant part is 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.

Whatever configuration and/or size distπbution is ultimately selected for the perforated microstructure, the composition of the defining structural matnx may compnse any one of a number of biocompatible mateπals It will be appreciated that, as used herein, the terms "structural matrix" or "microstructure matnx" are equivalent and shall be held to mean any solid mateπal 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 matnx composes a spray dried hollow porous microsphere incorporating at least one surfactant. For other selected embodiments the particulate mateπal may be coated one or more times with polymers, surfactants or other compounds which aid suspension.

More generally, the perforated microstructures may be formed of any biocompatible mateπal that is relatively stable and preferably insoluble with respect to the selected suspension medium and can provide the necessary perforated configuration. While a wide variety of materials may be used to form the particles, in particularly preferred embodiments the structural matnx is associated with, or comprises, a surfactant such as phospho pid or fluoππated surfactant. Although not required, the incorporation of a compatible surfactant can improve the stability of the respiratory dispersions, increase pulmonary deposition and facilitate the preparation of the suspension. Moreover, bv altering the components, the density of the structural matrix 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, the perforated microstructures preferably comprise at least one bioactive agent.

As set forth above the 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. With respect to MDIs surfactants further serve to lubricate the metering valve, thereby ensuring consistent reproducibility of valve actuation and accuracy of dose dispersed 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 structural matrix 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 structural matrix and the suspension medium. The surfactant may comprise a single compound or any combination of compounds, such as in the case of co surfactants. Particularly preferred surfactants are substantially insoluble in the propellant, nonfluonnated, and selected from the group consisting of saturated and unsaturated lipids, nomonic 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) fluoπnated 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 structural matrix. 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. C₁₆ C₂₂) saturated lipids and more preferably comprise phospholipids. Exemplary phospholipids useful in the disclosed stabilized preparations compnse egg phosphatidylcholiπe, dilauroylphosphatidylcholine, dioleylphosphatidylchohne, dipalmitoylphosphatidyl-chohne, disteroylphosphatidylchohne, short chain phosphatidylcho nes, phosphatidylethanolamine, dioleylphosphatidylethaπolamine, phosphatidylseπne, phosphatidylglycerol, phosphatidylinositol, glycolipids, ganglioside GM1 , sphmgomyelin, phosphatidic acid, cardiolipm; lipids bearing polymer chains such as polyethylene glycol, chitin, hyaluronic acid, or polyvinylpyrrolidone; lipids bearing sulfonated mono , di , and polysacchandes; fatty acids such as palmitic acid, stearic acid, and oleic acid; cholesterol, cholesterol esters, and cholesterol hemisuccinate. Due to their excellent biocompatibility characteristics, phospholipids and combinations of phospholipids and poloxamers are particularly suitable for use in the stabilized dispersions disclosed herein Compatible nonionic detergents comprise, sorbitan esters including sorbitan tnoleate (Span" 85), sorbitan sesquioleate, sorbitan monooleate, sorbitan monolaurate, polyoxyethylene (20) sorbitan monolaurate, and polyoxyethylene (20) sorbitan monooleate, oleyl polyoxyethylene (2) ether, stearyl polyoxyethylene (2) ether, lauryl polyoxyethylene (4) ether, glycerol esters, and sucrose esters. Other suitable nonionic detergents can be easily identified using McCutcheon's Emulsifiers and Detergents (McPublishing Co , Glen Rock, New Jersey) which is incorporated herein in its entirety. Preferred block copolymers include diblock and tπblock copolymers of polyoxyethylene and polyoxypropylene, including poloxamer 188 (PluromZ F 68), poloxa er 407 (Pluronic " F 127), and poloxamer 338 Ionic surfactants such as sodium sulfosuccmate, and fatty acid soaps may also be utilized. In preferred embodiments the microstructures may comprise oleic acid or its alkali salt. In addition to the aforementioned surfactants, cationic surfactants or lipids are preferred especially in the case of delivery or RNA or DNA. Examples of suitable cationic lipids include. DOTMA, N [1 (2,3 dioleyloxylpropyll N,N,N tπmethylammonium chloride; DOTAP, 1 ,2 dioleyloxy 3 (trιmethylammonιo)propane; and DOTB, 1,2 dιoleyl-3 (4'-trιmethylammonιo)butanoyl-sn glycerol. Polycationic ammo acids such as polylysme, and polyargimne are also contemplated Those skilled in the art will further appreciate that a wide range of surfactants 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 empirical 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 duπng processing to form the structural matrix

On a weight to weight basis, the structural matrix of the perforated microstructures may compnse relatively high levels of surfactant In this regard, the perforated microstructures will preferably comprise greater than about 1 %, 5%, 10%, 15%, 18%, or even 20% w/w surfactant. More preferably, the perforated microstructures will compπse greater than about 25%, 30%, 35%, 40%, 45%, or 50% w/w surfactant Still other exemplary embodiments will compπse 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 compnse essentially 100% w/w of a surfactant such as a phospho pid. Those skilled in the art will appreciate that, in such cases, the balance of the structural matrix (where applicable) will preferably compπse a bioactive agent or non surface active excipients or additives.

While such surfactant levels are preferably employed in perforated microstructures, they may be used to provide stabilized systems compnsing relatively nonporous, or substantially solid, particulates. That is, while preferred embodiments will compnse perforated microstructures or microspheres associated with high levels of surfactant, acceptable dispersions may be formed using relatively low porosity particulates of the same surfactant concentration (i e. greater than about 10% or 20% w/w) In this respect such embodiments are specifically contemplated as being within the scope of the present invention.

In other preferred embodiments of the invention the structural matrix defining the perforated microstructure optionally compnses synthetic or natural polymers or combinations thereof. In this respect useful polymers compπse polylactides, polylactide glycolides, cyclodextnns, polyacrylates, methylcellulose, carboxymethylcellulose, polyvinyl alcohols, polyanhydπdes, polylactams, polyvinyl pyrrolidones, polysacchandes (dextrans, 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 the effectiveness of the bioactive agent.

Besides the aforementioned polymer materials and surfactants it may be desirable to add other excipients to an aerosol formulation to improve microsphεre 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, antioxidants, and chemical stabilizers Further, vaπous excipients may be incorporated in, or added to, the particulate matrix to provide structure and form to the perforated microstructures (i e. microspheres) These excipients may include, but are not limited to, carbohydrates including monosacchaπdes, disacchandes and polysacchandes. For example, monosacchandes such as dextrose (anhydrous and monohydrate), galactose, mannitol, D mannosε, sorbitol, sorbose and the like, disacchandes such as lactose, maltose, sucrose, trehalose, and the like, trisacchaπdes such as raffinose and the like; and other carbohydrates such as starches (hydroxyethylstarch), cyclodextnns and maltodextπns Ammo acids are also suitable excipients with glycine preferred. 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, trotnethamine hydrochlonde) and buffers is also contemplated. Yet other preferred embodiments include perforated microstructures that may comprise, or may be coated with, charged species that prolong residence time at the point of contact or enhance penetration through mucosae. For example, aniomc 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, polylysine, polylactic acid and chitosan.

In addition to, or instead of, the components discussed above, the perforated microstructures will preferably compπse 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 presence or absence of a disease in a patient andlor in methods for treating a disease 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 treatment 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 perforated microstructures may incorporate much less bioactive agent depending on the activity thereof.

Accordingly, for highly active matenals, 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 embodiments of the invention may comprise 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 particularly 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 matnx or particulate. The precise amount of bioactive agent incorporated in the stabilized dispersions of the present invention is dependent upon the agent of choice, the volume of suspension media required to effectively distnbute the drug, 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 are suitable for pulmonary administration in conjunction with the teachings herein include any drug that may be presented in a form which is relatively insoluble in the selected medium and subject to pulmonary uptake in physiologically effective amounts. Compatible bioactive agents may compπse hydrophilic and pophilic respiratory agents, bronchodilators, pulmonary lung surfactants, antibiotics, antivirais, anti inflammatoπes, steroids, antihistamintcs, histamine antagonists, leukotnene inhibitors or antagonists, anticholinergics, anti neoplasties, anesthetics, enzymes, lung surfactants, cardiovascular agents, genetic material including DNA and RNA, viral vectors, immunoactive agents, imaging agents, vaccines, immunosuppressive agents, peptides, proteins and combinations thereof. Particularly preferred bioactive agents, for localized administration 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, i.e cromoglycate (e.g. the sodium salt), and albuterol (e g. the sulfate salt). For systemic delivery (e.g for the treatment of autoimmune diseases such as diabetes or multiple sclerosis), peptides and proteins are particularly preferred.

Exemplary medicaments or bioactive agents may be selected from, for example, analgesics, e g codeine, dihydromorphine, ergotamine, fentaπyl, or morphine, anginal preparations, e.g. diltiazem, mast cell inhibitors, ε g cromolyn sodium; antiinfectives, e g. cephalosporins, macrolides, quinolines, penicillins, streptomycin, sulphoπamides, tetracycliπes and pentamidine, antihistammes, e.g. methapyπleπe, anti inflammatoπes, e g. fluticasone propionatε, beclomethasonε dipropionate, flunisolide, budesonide, tnpedane, cortisone, predπisone, predmsilone, dexamethasone, betamethasone, or tπarncinolone acetomde; antitussives, e.g. noscapine; bronchodilators, e g ephednne, adrenaline, fenoterol, formoterol, isoprenaline, metaproterenol, salbutamol, albuterol, salmeterol, terbutalme, diuretics, e.g. amilonde, anticholinergics, e.g. ipatropium, atropine, or oxitropium; lung surfactants e.g Surfaxin, Exosurf, Survanta; xanthines, e g aminophyllme, theophylline, caffeine, therapeutic proteins and peptides, e.g. DNAse, insulin, glucagon, T cell receptor agonists or antagonists, LHRH, nafare n, goserehn, leuprolide, interferon, rhu IL 1 receptor, macrophage activation factors such as lymphokines and muramyl dipeptides, opioid peptides and neuropeptides such as enkaphalins, eπdorphins, remn inhibitors, cholecystokimns, growth hormones, leukotnene 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, genetic tolenzation or antisense applications, may be incorporated in the disclosed dispersions as described herein. Representative DNA plasmids include, but are not limited to 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 the enzyme β galactosidase)

With respect to particulate dispersions, the selected bioactive agent(s) may be associated 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 the structural matrix, perforated microstructure, relativεly non porous particle or discontinuous phase may comprise, incorporate, adsorb, absorb, be coated with, or be formed by the bioactive agent. Where appropriate, the medicaments may be used in the form of salts (e 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 the medicament and/or, to minimize the solubility of the medicament in the suspension medium

It will further be appreciated that 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 individually in separate species that are combined in the suspension medium or continuous phase For example, two or more bioactive agents may be incorporated in a single feed stock preparation and spray dπed to provide a single microstructure species comprising a plurality of medicaments Conversely, 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 delivery systems as described below Further, as briefly alluded to above, the perforated microstructures (with or without an associated medicament) may be combined with one or more conventionally micronized bioactive agents to provide the desired dispersion stability

Based on the foregoing, it will be appreciated by those skilled in the art that a wide vaπety of bioactive agents may be incorporated in the disclosed stabilized dispersions Accordingly, the list of preferred bioactive agents above is exemplary only and not intended to be limiting. It will also be appreciated by those skilled in the art that, the proper amount of bioactive agent and the timing of the dosages may be determinεd for the formulations in accordance with already existing information and without undue expeπmentation

As seen from the passages above, various components may be associated with, or incorporated in the perforated microstructures of the present invention Similarly, several techniques may be used to provide particulates having the desired morphology (e g a perforated or hollow/porous configuration) and density Among other methods, perforated microstructures compatible with the instant invention may be formed by techniques including lyophilization, spray drying, multiple emulsion, micronization, or crystallization It will further be appreciated that the basic concepts of many of these techniques are well known in the prior art and would not, in view of the teachings herein, require undue experimentation to adapt them so as to provide the desired perforated microstructures.

While several procedures are generally compatible with the present invention, particularly preferred embodiments typically comprise 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 material 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 general, spray drying consists of bringing together 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 be 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 the dried 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, e.g. the simultaneous spraying of two solutions using a double nozzle technique. More specifically, a water-m-oil emulsion can be atomized from one nozzle and a solution containing an anti adherent 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 critical and would be apparent to the skilled artisan in view of the teachings herein.

While typical spray-dned particles are approximately spherical in shape, nearly uniform in size and frequently hollow, there may be some degree of irregularity in shape depending upon the incorporated medicament and the spray drying conditions. In many instances the dispersion stability of spray-dned microspheres appears to be more effective if an inflating agent (or blowing agent) is used in their production. Particularly preferred embodiments may comprise, an emulsion with the inflating agent as the disperse or continuous phase (the other phase being 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 5000 to 15,000 psi This process forms an emulsion, preferably stabilized by an incorporated surfactant, typically compnsing submicroπ droplets of water immiscible blowing agent dispersed in an aqueous continuous phase. The formation of such dispersions using this and other techniques are common and well known to those in the art. The blowing agent is preferably a fluonnated compound (e.g. perfluorohexaπe, perfluorooctyl bromide, perfluorodecalin, perfiuorobutyl ethane) which vaporizes during the spray drying process, leaving behind generally hollow, porous, aerodyπamically light microspheres. As will be discussed in more detail below, other suitable blowing agents include chloroform, Freons and hydrocarbons. Nitrogen gas and carbon dioxide are also contemplated as a suitable blowing agent.

Although the perforated microstructures are preferably formed using a blowing agent as described above, it will be appreciated that, in some instances, no blowing agent is required 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 (e.g., elevated temperatures) that generally lead to the formation of hollow, relatively porous microparttcles. Moreover, the medicament may possess special physicochemical properties such as, high crystallinity, elevated melting temperature, surface 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 the blowing agent, its concentration in the feed stock (i.e. 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 perforated microstructures having particularly desirable characteristics More particularly, in this novel and unexpected aspect of the present invention it has been found that the use of fluonnated 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 fluonnated 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 perflubron, perfluorodecalin) In addition, blowing agents with relatively low water solubility ( < 10⁶ M) are preferred since they enable the production of stable emulsion dispersions with mean weighted particle diameters less than 0 3 μm 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 compnse one or more bioactive agents, one or more surfactants, or one or more excipients. Of course, combinations of the aforementioned components 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 dunng 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 outgassing apparently leads to the formation of pores or other defects in the crust At the 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 resistance to mass transfer caused by an increased internal viscosity. Once 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 dependent 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 blowing agents may be retained in the resulting spray dried product That is, the spray dried perforated microstructures 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 caused by residual blowing agent 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 the stability of the respiratory dispersions. Conversely, the residual blowing agent can generally be removed relatively easily with a post-production evaporation step in a vacuum oven. Optionally, pores may be formed by spray drying a bioactive agent and an excipient that can be removed from the formed microspheres under a vacuum.

In any event, typical concentrations of blowing agent in the 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 preferably be greater than about 10%, 20%, 30%, 40% 50% or even 60% w/v. Yet other feed stock emulsions may comprise 70%, 80%, 90% or even 95% w/v of the selected high boiling point compound.

In preferred embodimεnts, another method of identifying the concentration of blowing agent used in the feed is to provide it as a ratio of the concentration of the blowing agent to that of the stabilizing surfactant (i e phospholipid) in the precursor emulsion For fluorocarbon blowing agents such as perfluorooctyl bromide and phosphatidylcho πe, the ratio may be termed a perfluorocarbon/phosphatidylcholine ratio (or PFC/PC ratio). While phosphotidylcholme is a preferred surfactant, those skilled in the art will appreciate that other surfactants may provide acceptable emulsions and may be substituted therefore In any event, 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, it will be appreciated that higher PFC/PC ratios typically lead to particulates exhibiting greater porosity. Accordingly, altering the PFC/PC ratio in the feed stock emulsion may advantageously control the morphology of the resulting microstructures In this regard, the use of higher PFC/PC ratios tends to provide 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 provide structures that are particularly compatible with the dispersions disclosed herein.

While relatively high boiling point blowing agents comprise one preferred aspect of the instant invention, it will be appreciated that more conventional blowing or inflating agents may also be used to provide compatible perforated microstructures. Generally, the inflating agent can be any matεπal that will turn to a gas at some point during the spray drying or post production process. Suitable agents include: 1 Dissolved low boiling (below 100 C) solvents with limited irascibility with aqueous solutions, such as methylene chloride, acetone and carbon disulfide used to saturate the solution at room temperature. 2. A gas, e.g. C0₂ or N₂, used to saturate the solution at room temperature and elevated pressure (e.g. 3 bar). The droplets are then supersaturated with the gas at 1 atmosphere and 100 C.

3 Emulsions of immiscible low boiling (below 100 C) liquids such as Freon 1 13, perfluoropentane, perfluorohexane, perfluorobutane, pentane, butane, FC 1 1, FC 11 B1, FC 11 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 1 112, FC 1121 and FC-1 131. With respect to these lower boiling point inflating agents, they are typically added to the feed stock in quantities of about 1 % to 80% w/v of the surfactant solution Approximately 30% w/v inflating agent has been found to produce a spray dried powder that may be used to form the stabilized dispersions of the present invention. Regardless of which blowing agent is ultimately selected, it has been found that compatible perforated microstructures may be produced particularly efficiently using a Buchi mini spray drier (model B

191 , Switzerland). As will be appreciated by those skilled in the art, the inlet temperature and the outlet temperature 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 the desired activity of the medicament. In this regard, the inlet and outlet temperatures are adjusted depending on the melting characteristics of the formulation components and the composition of the feed stock The inlet temperature may thus be between 60°C and 170°C, with the outlet temperatures of about 40°C to 120°C depending on the composition of the feed and the desired particulate characteristics Preferably these temperatures will be from 90°C to 120°C for the inlet and from 60°C to 90°C for the outlet. The flow rate that is used in the spray drying equipment will generally be about 3 ml per minute to about 15 ml per minute. The atomizer 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 empiπcal testing, with due reference to the examples that follow Of course, the conditions may be adjusted so as to preserve biological activity in larger molecules such as proteins or peptides.

Particularly preferred embodiments 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 compπse an excipient comprising a hydrophilic moiety such as, for example, a carbohydrate (i.e. glucose, lactose, or starch) in addition to any selected surfactant. In this regard various starches and denvatized starches suitable for use in the present 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 isotonicity, hyperosmolaπty, or hyposmolaπty) Examples of suitable salts include sodium phosphate (both monobasic and dibasic), sodium chloπde, calcium phosphate, calcium chloride and other physiologically acceptable 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. It will also be appreciated that the drug may be incorporated in the form of a solid particulate dispersion The concentration of the drug used is dependent on the dose of drug required in the final powder and the performance of the MDI drug suspension (e g , fine particle dose). As needed, cosurfactants 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 selected embodiments an oil in water emulsion is then formed in a separate vessel The oil employed is preferably a fluorocarbon (e.g., perfluorooctyl bromide, perfluorodecalin) 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 reduce the particle size Typically the emulsion is processed at 12,000 to 18,000 psi for 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 agent is solubi zed separately for the purposes of the instant discussion it will be appreciated that, in other embodiments, the bioactive agent may be solubilized (or dispersed) directly in the emulsion In such cases, the bioactive emulsion is simply spray dried without combining a separate drug preparation. In any event, operating conditions such as inlet and outlet temperature, feed 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 the 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 100% 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 view 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. Along with spray drying the perforated microstructures of 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 the 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 be dπed without elevated temperatures (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 the perforated microstructures without compromising physiological activity. Methods for providing iyophilized particulates are known to thosε 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 processes may be used to provide microstructures having the desired porosity and size they are conformance with the teachings herein and are expressly contemplated as being within the scope of the instant invention.

Besides the aforementioned techniques, the perforated microstructures of the present invention may also be formed using a double emulsion method. In the double emulsion method the medicament is first dispersed in a polymer dissolved in an organic solvent (e g methylene chloride) by sonication or homogenization. This primary emulsion is then stabilized by forming a multiple emulsion in a continuous aqueous phase containing an emulsifier such as polyvinylalcohol Evaporation or extraction using conventional techniques and apparatus then removes the organic solvent. The resulting microspheres are washed, filtered and dried pnor to combining them with an appropriate suspension medium in accordance with the present invention.

Regardless of how the microstructures or particles are formed, the selected suspension media used to provide the desired stabilized dispersion is preferably compatible with pulmonary administration. In general, the selected suspension medium should be biocompatible (i.e. relatively non toxic) and non reactive with respect to the suspended perforated microstructures comprising the bioactive agent Preferred embodiments compπse suspension media selected from the group consisting of fluorochemicais, fluorocarbons (including those substituted with other halogens), perfluorocarboπs, fluorocarbon/hydrocarbon diblocks, hydrocarbons, alcohols, ethers, or combinations thereof It will be appreciated that the suspension medium may comprise a mixture of various compounds selected to impart specific characteristics. It will also be appreciated that the perforated microstructures are preferably insoluble in the suspension medium, thereby 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 selectεd suspension medium is bactεnostatic.

As indicated above, the suspension media may comprise any one of a number of different compounds including hydrocarbons, fluorocarbons or hydrocarbon/fluorocarbon diblocks In general, the contemplated hydrocarbons or highly fluonnated or perfluonπated compounds may be linear, branched or cyclic, saturated or unsaturated compounds Conventional structural deπvatives of these fluorochemicais and hydrocarbons are also contemplated as being within the scope of the present invention. Selected embodiments comprising these totally or partially fluonnated compounds may contain one or more hetero atoms including bromine or chlorine. Preferably, these fluorochemicais comprise from 1 to 16 carbon atoms and include, but are not limited to, linear, cyclic or polycyclic perfluoroalkanes, bιs(perfluoroalkyl)alkenes, perfluoroethers, perfluoroamines, perfiuoroalkyl bromides and perfiuoroalkyl chlorides such as dichlorooctane. Particularly preferred fluonnated compounds for use in the suspension medium may comprise perfluorooctyl bromide, C_βF,₇Br (PFOB or perflubron), dichlorofluorooctane C_βF,₆CI₂, and the hydrofluoroalkane perfluorooctyl ethane C_eF,₇C₂H₅ (PFOE). In selected embodiments the suspension medium will compπse a compound (particularly a fluorochemical) having a positive spreading coefficient Other useful preparations may comprise perfluorohexane or perfluoropentane as suspension media. More generally, exemplary fluorochemicais which are contemplated for use in the present invention generally include halogenated fluorochemicais (i.e. C-F_2nttX, XC_nF₂.X, where n = 2 10, X = Br, CI or I) and, in particular, 1 bromo F butane n C₄F_gBr, 1 bromo F hexane (n C₆F,₃Br), 1 bromo F heptane (n C₇F,₅Br), 1 ,4 dibromo F butane and 1,6-dιbromo-F hexane. Other useful brominated fluorochemicais are disclosed in US Patent No. 3,975,512 to Long, which is incorporated herein by reference. Specific fluorochemicais having chloπde substituents, such as perfluorooctyl chloride (n-C_βF₎₇CI), 1,8-dιchloro-F-octane (n-CIC_βF,₆CI), 1,6-dιchloro F hexane (n-CIC₆F₁₂CI), and 1, 4 dichloro F butane (n CIC₄F₈CI) are also preferred.

Fluorocarbons, fluorocarbon-hydrocarboπ compounds and halogenated fluorochemicais containing other linkage groups, such as esters, thioethers and amines are also suitable for use as suspension media in the present invention. For instance, compounds having the general formula, C_nF_2ntl0C_mF_2m<.|, or C_n 2_n*|CH = CHC_mF_2m,|, (as for example C₄F₉CH= CHC₄F_g (F 44E), i C₃F_gCH= CHC₆F₁₃ |F ι36E), and C_BF,₃CH= CHC₆F,₃ (F 6BE)) where n and m are the same or different and n 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 C_nF_2l„, C_mH_2m<1 and C_nF_2n,,C_mH_2m), where n = 2 12; m = 2 16 or C_PH_2|)-, C_nF_2n C_mH_2m,„ where p = 1 12, m = 1 12 and n = 2-12. Preferred compounds of this type include C_eF,₇C₂H₆ C₆F₁₃C,₀H₂, C₈F,₇C_βH,₇ C₆F,₃CH= CHC_BH,₃ and

C₈F,₇CH= CHC,₀H₂|. Substituted ethers or polyethers (i.e. XC-F^OC/a-X, XCF0C_nF_2n0CF₂X, where n and m = 1-4, X = Br, CI or I) and fluorochemical hydrocarbon ether diblocks or tπblocks (i.e. C_nF_2n<, 0 C_mH_2m„,, where n = 2 10; m = 2 16 or C.H₂₍ 0 C.F₂. 0 CJ.^,, where p = 2-12, m = 1 12 and n = 2-12) may also used as well as C„F_2n,,0 C_mF_2m0C_pH_2p»,, wherein n, m and p are from 1 12. Furthermore, depending on the application, perfluoroalkylated ethers or polyethers may be compatible with the claimed dispersions.

Polycyclic and cyclic fluorochemicais, such as C,₀F,₈ (F deca n or perfluorodecalin), perfluoroperhydrophenanthrene, perfluorotetramethylcyclohexane (AP-144) and perfluoro n butyldecalin are also within the scope of the invention Additional useful fluorochemicais include perfluoπnated amines, such as F tπpropylamine ("FTPA") and F tπbutylamine ("FTBA"). F 4-methyloctahydroquιnolιzιne ("FMOQ"), F N-methyl- decahydroisoquinoline ("FMIQ"), F N methyldecahydroquinoline ("FHQ"), F N cyclohexylpyrrolidine ("FCHP") and F 2 butyltetrahydrofuran ("FC-75"or "FC-77"). Still other useful fluonnated compounds include perfluoropheπanthrene, pεrfluoromethyldecalin, perfluorodimethylethyicyclohexanε, perfluorodimethyldecalin, perfluorodiethyldecalin, perfluoromethyladamantane, perfluorodimethyiadamantane. Other contemplated fluorochemicais having πonfluoππe substituents, such as, perfluorooctyl hydride, and similar compounds having different numbers of carbon atoms are also useful. Those skilled in the art will further appreciate that other vaπously modified fluorochemicais 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 the stabilized dispersions of the present invention.

Yet other specific fluorocarbons, or classes of fluonnated compounds, that may be useful as suspension media include, but are not limited to, fluoroheptane, fluorocycloheptane fluoromethylcycloheptane, fluorohexane. fluorocyclohexane, fluoropentane, fluorocyclopentane, fluoromethylcyclopentane, fluorodimethylcyclopentanes, fluoromethylcyclobutane, fluorodimethylcyclobutane, fluorotπmethylcyclobutane, fluorobutane, fluorocyclobutane, fluoropropane, fluoroethers, fluoropolyethers and fluorotπethylamiπεs Such compounds are generally environmentally sound and are biologically non reactive. While any biocompatible fluid compound may be usεd in conjunction with the present invention, the selected suspension medium will preferably have a vapor pressure less than about 5 atmospheres and more preferably less than about 2 atmospheres 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 used in conjunction with compressed air nebulizers, ultrasonic nebulizers or with mechanical atomizers to provide effective ventilation therapy. Moreover, more volatile compounds may be mixed with lower vapor pressure components to provide suspension media having specified physical charactenstics selected to further improve stability or enhance the bioavailability of the dispersed bioactive agent.

Other embodiments of the present invention will comprise suspension media that boil at selected temperatures under ambient conditions d e. 1 atmosphere). For example, preferred embodiments will comprise suspension media compounds that boil above 0°C, above 5°C, above 10°C, above 15°, or above 20°C. In other embodiments, the suspension media compound may boil at or above 25°C or at or above 30°C In yet other embodiments, the selected suspension media compound may boil at or above human body temperature (i.e. 37°C), above 45°C, 55°C, 65°C,

75°C, 85°C or above 100°C.

The stabilized suspensions or dispersions of the present invention may be prepared by dispersal of the microstructures in the selected suspension medium, which may then be placed in a container or reservoir In this regard, the stabilized preparations of the present 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 energy to aid in dispersion (e.g. with the aid of sonication) is contemplated, particularly for the formation of stable emulsions or reverse emulsions Alternatively, the components may be mixed by simple shaking or other type of agitation The process is preferably earned out under anhydrous conditions to obviate any adverse effects of moisture on suspension stability. Once formed, the dispersion has a reduced susceptibility to flocculation and sedimentation.

It will also be understood that other components can be included in the pharmaceutical compositions of the present invention. For example, osmotic agents, stabilizers, chelators, buffers, 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 sterility, 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 types of agents can be determined empirically without undue expenmentation

The stabilized suspensions or dispersions of the present invention may be prepared by dispersal of the microstructures in the selected suspension medium that may then be placed in a container or reservoir. In this regard, the stabilized preparations of the present invention can be made by simply combining the components in sufficient quantity to produce the final desired dispersion concentration That is, the components of the preparations may be combined to provide a respiratory blend. Although the microstructures readily disperse without mechanical energy, the application of mechanical energy (e.g. sonication) to the respiratory blend to mix the components or aid in their dispersion is contemplated. Alternatively, the components may be mixed by simple shaking or other type of agitation The process is preferably carried out under anhydrous conditions to obviate any adverse effects of moisture on suspension stability. Once formed, the dispersion has a reduced susceptibility to flocculation and sedimentation

It will be appreciated that conventional pharmaceutical equipment and methodology may be used dunng production of the disclosed dispersions For example, commercially available spray drying and mixing equipment may be used to form the perforated microstructures and desired suspensions. Accordingly, it is submitted that the skilled artisan would have little trouble producing the pharmaceutical dispersions of the present invention on a commercial scale when in possession of the instant disclosure.

It will further be appreciated that the stabilized preparations 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, the formulations may be supplied as stable, preformed dispersions ready for administration or, as separate ready to mix components. When provided in a ready to use form, the dispersions may be packaged in single use containers or reservoirs (e.g. in glass vials comprising a few milliliters of the dispersion) or in multi use containers or reservoirs. When provided as individual components (e.g., as powdered microspheres and as neat suspension medium) the stabilized preparations may then be formed at any time prior to use by simply combining the contents of the containers as directed. For example, a small volume of concentrated dispersion could be diluted in a larger volume of neat fluorocarbon prior to its use in liquid ventilation. Additionally, due to the superior stability of the disclosed preparations, the kits may contain a number of ready to mix, or prepackaged dispersions in a single use form so that the user can readily select or modify the therapeutic regimen for the particular indication. In this regard, each of the containers may be fitted with a septum for direct removal of the dispersion or with appropriate tubing, cannulas, Luer fittings, etc. for association with a ventilator or endotracheal apparatus. It will also be appreciated that such kits may optionally include a bronchoscope or endotracheal apparatus (or components thereof) for administration of the preparations. 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 systemic conditions or disorders. In this regard, one particularly preferred embodiment comprises the systemic administration (e.g. delivery to the systemic circulation of a patient via the pulmonary air passages) of a bioactive agent It will further be appreciated that the precise dose administered will depend on the age and condition of the patient, the particular medicament used and the frequency of administration and will ultimately be at the discretion of the attendant physician When dispersions compnsing combinations of bioactive agents are administered, the dose of each agent will generally be that employed for each agent when used alone.

Direct administration of bioactive compounds is particularly effective in the treatment of pulmonary disorders especially where poor vascular circulation of diseased portions of a lung reduces the effectiveness of intravenous drug delivery Accordingly, stabilized dispersions administered to the lung may prove useful in the treatment and/or diagnosis of disorders such as respiratory distress syndrome, acute respiratory distress syndrome, lung contusions, divers lung, post traumatic respiratory distress, post surgical atelectasis, septic shock, multiple organ failure, Mendelssohn's disease, obstructive lung disease, pneumonia, pulmonary edema, impaired pulmonary circulation, cystic fibrosis and lung cancer In this regard, the stabilized dispersions are preferably used in conjunction with partial liquid ventilation or total liquid ventilation. Moreover, the present invention may further comprise introducing a therapeutically beneficial amount of a physiologically acceptable gas (such as nitric oxide or oxygen) into the pharmaceutical microdispεrsioπ pπor to, duπng or following administration.

As discussed throughout the instant specification, the compositions of the present invention may be administered to the lung using a pulmonary delivery conduit. Those skilled in the art will appreciate the term

"pulmonary delivery conduit", as used herein, shall be construed in a broad sense to comprise any device or apparatus, or component thereof, that provides for the instillation or administration of a liquid in the lungs. In this respect a pulmonary delivery conduit or delivery conduit shall be held to mean any bore, lumen, catheter, tube, conduit, syringe, actuator, mouthpiece, endotracheal tube or bronchoscope that provides for the administration or instillation of the disclosed dispersions to at least a portion of the pulmonary air passagεs of a patient in need thereof. It will be appreciated that the delivery conduit may or may not be associated with a liquid ventilator or gas ventilator. In particularly preferred embodiments the delivery conduit shall comprise an endotracheal tube or bronchoscope.

Accordingly, liquid dose instillation preferably involves the instillation of the perforated microstructures in a suitable suspension medium to an mtubated patient through an endotracheal tube, or to a free breathing patient via bronchoscope Other embodiments comprise the administration of the disclosed dispersions directly into the throat. That is, the formulations of the present invention may be "trickled" into the lungs of the patient as a bolus using standard tubing and/or a syπnge. Here it must be emphasized that the dispersions of the present invention may be administered to ventilated (e.g. those connected to a mechanical ventilator) or nonventilated, patients (e g those undergoing spontaneous respiration) Accordingly, in preferred embodiments the methods and systems of the present invention may comprise the use or inclusion of a mechanical ventilator. Further, the stabilized dispersions of the present invention may also be used as a lavage agent to remove debris in the lung, or for diagnostic lavage procedures. In any case the introduction of liquids, particularly fluorochemicais, into the lungs of a patient is well known and could be accomplished by a skilled artisan in possession of the instant specification without undue expeπmεntation.

It will be understood that, in connection with the present invention, the disclosed dispersions are preferably administered directly to at least a portion of the pulmonary air passages of a mammal. As used herein, the terms "direct instillation" or "direct administration" shall be held to mean the introduction of a stabilized dispersion into the lung cavity of a mammal. That is, the dispersion will preferably be administered through the trachea of a patient and into the lungs as a liquid. While the dispersions may be administered in the form of an aerosol or nebulized liquid, they will preferably be introduced as a volume of a relatively free flowing liquid passing through a delivery conduit and into the pulmonary air passages. In this regard, the flow of the dispersion may be gravity assisted or may be afforded by induced pressure such as through a pump or the compression of a syringe plunger. In any case, the amount of dispersion administered may be monitored by mechanical devices such as flow meters or by visual inspection

It will further be appreciated that, liquid ventilation (partial or total) involves the introduction of a respiratory promoter (typically a fluorochemical) to the lung for the promotion of physiological gas exchange. For partial liquid ventilation, the patient is preferably ventilated using a mechanical ventilator following pulmonary introduction of the liquid In accordance with the teachings herein the respiratory promoter may comprise a stabilized dispersion. For example, perforated microparticles comprising penicillin may be suspended in perfluorooctyl bromide to provide a stabilized dispersion that could be used for liquid ventilation. This dispersion could then be administered, at any volume up to functional residual capacity (FRC), to the lung of a patient as desenbed in U.S. Pat. Nos. 5,562,608, 5,437,272, 5,490,498, 5,667,809, 5,770,585 and 5,540,225 each of which is incorporated herein by reference. Alternatively, a concentrated, but relatively stable, dispersion could be packaged in a single dose configuration having a total volume on the order of a few milliliters or less. It will be appreciated that the relatively small volume could be administered directly to the lung However, in preferred embodiments this concentrated dispersion could be mixed with a larger volume of neat respiratory promoter (which may be the same or different as the suspension medium) pπor to introduction to the lung In still other embodiments the concentrated dispersion could be administered directly to the lung of a patient already containing respiratory promoter. That is, for mtubated patients undergoing partial liquid ventilation, the bioactive agent suspension may be top loaded onto an existing volume of a fluorochemical. In each of these cases, the respiratory promoter and/or suspension medium will provide for the efficient dispersal and deposition of the bioactive perforated microspheres on the lung membrane. More specifically, by providing for the administration of bioactive agents in what can be a relatively anhydrous environment, ι.e. in a fluorochemical, physiological uptake of the agent may be dramatically increased. This is particularly true of lung surfactants such as phospholipids As discussed more fully in Example XIV below the adsorption time for surfactant is exponentially decreased when it is brought into contact with a wetted surface (lung membrane) by a fluorochemical as opposed to an aqueous solution This is because adsorption of the surfactant from an anhydrous suspension medium into an aqueous environmεnt is thεrmodynamically very favorable. By way of contrast, there is no large dπving force when the surfactant is moving from one aqueous medium to another Accordingly, particularly preferred embodiments of the present invention compπse perforated microstructures associated with, or incorporating, natural or synthetic surfactants distributed in a fluorochemical suspension medium While the stabilized dispersions may be administered up to the functional residual capacity of the lungs of a patient, it will be appreciated that selected embodiments will compπse the pulmonary administration of much smaller volumes (e.g. on the order of a milliliter or less). For example, depending on the disorder to be treated, the volume administered may be on the order of 1, 3, 5, 10, 20, 50, 100, 200 or 500 milliliters. In preferred embodiments the liquid volume is less than 0.25 or 0.5 percent FRC For particularly preferred embodiments, the liquid volume is 0.1 percent FRC or less With respect to the administration of relatively low volumes of stabilized dispersions it will be appreciated that the wettabi ty and spreading characteristics of the suspension media (particularly fluorochemicais) will facilitate the even distribution of the bioactive agent in the lung However, in other embodiments it may be preferable to administer the suspensions a volumes of greater than 0.5, 0.75 or 0.9 percent FRC In any event, LDI treatment as disclosed herein represents a new alternative for critically ill patients on mechanical ventilators, and opens the door for treatment of less ill patients with bronchoscopic administration

While the stabilized dispersions of the present invention are particularly suitable for the pulmonary administration of bioactive agents, they may also be used for the localized or systemic administration of compounds to any location of the body. Accordingly, it should be emphasized that, in preferred embodiments, the formulations may be administered using a number of different routes including, but not limited to, the gastrointestinal tract, the respiratory tract, topically, intramuscularly, intrapeπtoneally, nasally, vaginally, rectally, aurally, orally or ocular. More generally, the stabilized dispersions of the present invention may be used to deliver agents topically or by administration to a non pulmonary body cavity. In preferred embodiments the body cavity is selected from the group consisting of the pentoneum, sinus cavity, rectum, urethra, gastrointestinal tract, nasal cavity, vagina, auditory meatus, oral cavity, buccal pouch and pleura Among other indications, stabilized dispersions compnsing the appropriate bioactive agent, (e g. an antibiotic or an anti inflammatory), may be used to treat infections of the eye, sinusitis, infections of the auditory tract and even infections or disorders of the gastrointestinal tract. With respect to the latter, the dispersions of the presεnt invention may be used to selectively deliver pharmaceutical compounds to the lining of the stomach for the treatment of H. pylon infections or other ulcer related disorders. The foregoing descπption will be more fully understood with reference to the following Examples. Such Examples, are, however, merely representative of preferred methods of practicing the present invention and should not be read as limiting the scope of the invention

I

Preparation of Hollow Porous Particles of Gentamicin Sulfate by Spray-Drying

40 to 60ml of the following solutions were prepared for spray drying-

50% w/w hydrogenated phosphatidylcho ne, E 100 3 (Lipoid KG, Ludwigshafen, Germany) 50% w/w gentamicin sulfate (Amresco, Solon, OH)

Perfluorooctylbromide, Perflubron (NMK, Japan) Deionized water

Perforated microstructures comprising gentamicin sulfate were prepared by a spray drying technique using a B 191 Mini Spray Drier (Buchi, Flawil, Switzerland) under the 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 m-water emulsions of perfluorooctyl bromide containing a 1.1 w/w ratio of phosphatidylchohne (PC), and gentamicin sulfate were prepared varying only the PFC/PC ratio 1 3 grams of hydrogenated egg phosphatidylchohne was dispersed in 25 mL deionized 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 during mixing (T = 60 70°C). After addition was complete, the fluorocarbon in water emulsion was mixed for an additional peπod of not less than 4 minutes The resulting coarse emulsions were then homogenized 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 deionized water and subsequently mixed with the perflubron emulsion immediately prior to the spray dry process. The gentamicin powders were then obtained by spray drying using the conditions described above. A free flowing pale yellow powder was obtained 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), of the samples obtained in Example I. In the 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 example, the formulation devoid of perfluorooctyl bromide produced microstructures that appeared to be highly agglomerated and readily adhered to the surface of the glass vial Similarly, smooth, spherically shaped microparticles were obtainεd whεn relatively little (PFC/PC ratio - 1 .1 or 2.2) blowing agent was used. However, as the PFC/PC ratio increased, the particles showed dramatic increases in porosity and surface roughness.

As revealed by transmission electron microscopy (TEM) cross sections of the particles revealed that the hollow nature of the microstructures was also enhanced by the incorporation of additional blowing agent. In this regard, both the hollow nature and wall thickness of the 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 walls appeared to decrease as the PFC/PC ratio increased. Substantially non-porous, relatively solid structures were obtained from formulations containing little or no fluorocarbon blowing agent. Conversely, the perforated microstructures produced using a relatively high PFC /PC ratio of approximately 45 proved to be extremely hollow with a relatively thin wall ranging from about 43.5 to 261 nm. In keeping with the teachings herein, both types of particles are compatible for use in the present invention.

III

Preparation of Hollow Porous Particles of Albuterol Sulfate bv Spray-Drying

Hollow porous albuterol sulfate 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 tεmpεraturε: 61 °C; fεed pump- 10%; N₂ flow: 2,800 L/hr The feed solution was prepared by mixing two solutions A and B immediately prior to spray drying.

Solution A: 20g of water was used to dissolve 1 g of albuterol sulfate (Accurate Chemical, Westbury, NY) 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 homogenized 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 (Atochem, Pans, France) was added dropwise during mixing. After the fluorocarbon was added, the emulsion was mixed for a penod of not less than 4 minutes. The resulting coarse emulsion was then passed through a high pressure homogenizer (Avestin, Ottawa, Canada) at

18,000 psi for 5 passes. Solutions A and B were combined and fed into the spray-dryer under the conditions described above.

A free flowing white powder 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, Amherst Process Instruments, Amherst, MA). Scanning electron microscopy

(SEM) analysis showed the powders to be spheπcal and highly porous. Thε tap density of the powder was determined to be less than 0.1 g/cm³. This foregoing example serves to illustrate the inherent diversity of the present 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 example.

IV

Formation of Porous Particulate Microstructures Comprising Mixture of Long-Chaiπ/Short-Chaiπ Phospholipids and Albuterol Sulfate

A dispersion for spray drying was prepared as described in Example III above, with the difference that 1 g of DSPC was dispersed with 100 mg of a short chain phospholipid, dioctylphosphatidylcholine (DOPC) (Avanti Polar Lipids, Alabaster, Alabama). The composition of the spray feed is shown in Table II immediatεly below. The resulting yield was 50%

Table II

Composition of the Spray Feed

V 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) undεr thε following spray conditions- aspiration

100%, inlet temperature: 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 immediately 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 homogenized 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). 27g of perfluorodecalin (Air Products, Allentown, PA) 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 (Avestin, Ottawa, Canada) at 18,000 psi for 5 passes. Solutions A and B were combined and fed into the spray dryer under the conditions described above A free flowing pale yellow powder was collected at the cyclone separator. The hollow porous cromolyn sodium particles had a volume weighted mean aerodynamic diameter of 1 23 ± 1 31 μm as determined by a tιme-of-flιght 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.

VI Preparation of Hollow Porous Particles of BDP by Spray-Drying Perforated microstructures comprising beclomεthasone dipropionate (BDP) 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, feed pump: 10%; N₂ flow 2,800 L/hr The feed stock was preparεd by mixing 0 11g of lactosε with a fluorocarbon in water emulsion immediately prior 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 dissolved in 2 ml of hot methanol The methanol was then evaporated to obtain a thin film of the phospholipid/steroid mixture. The phospholipid/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 minutes (T = 60 70°C) 8g of perflubron (Atochem, Pans, France) was added dropwise duπng mixing. After the addition was complete, the emulsion was mixed for an additional peπod of not less than 4 minutes. The resulting coarse emulsion was then passed through a high pressure homogenizer (Avestin, Ottawa, Canada) at 18,000 psi for 5 passes. This emulsion was then used to form the feed stock that was spray dned as descπbed 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 tπamcinolone acetomde (TAA) particles werε 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, feed pump. 10%; N₂ flow.

2,800 L/hr The feed stock was preparεd by mixing 0 57g of lactose with a fluorocarbon in water emulsion immediately 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 ml of hot methanol The methanol was then evaporated to obtain a thin film of the phospholipid/steroid mixture. The phospholipid/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 minutes (T = 60-70°C). 8g of perflubron (Atochem, Pans, France) was added dropwise duπng 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 (Avestin, Ottawa, Canada) at 18,000 psi for 5 passes. This emulsion was then used to form the feed stock that was spray dned as descπbed above. A free flowing white powder was collected at the cyclone separator. The 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 preparεd fay a spray drying technique with a B 191 Mini Spray Drier (Buchi, Flawil, Switzerland) under the following conditions: aspiration: 100%, inlet temperature. 80°C; outlet temperature: 61°C; feεd 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, Ludwigshafen, 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 (Avestin, Ottawa, Canada) at 18,000 psi for 5 passes.

Solutions A and B were combined and fed into the spray dryer under the conditions described above A free flowing pale yellow powder was collected at the cyclone separator. The hollow porous DNase I particles had a volume weighted mean aerodynamic diametεr of 1.29 ± 1 40 μm as dεtεrminεd 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 Thε tap density of the powder was determined to be less than 0.1 g/cm³.

The foregoing example 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 present invention may be foimulated to effectively incorporate larger, fragile moleculεs such as peptides, proteins and genetic mateπal.

IX Preparation of hollow porous powder bv spray drvino a αas-in-watar emulsion The following solutions werε prepared with water for injection: Solution 1 :

3.9% w/v m-HES hydroxyethylstarch (Ajinomoto, Tokyo, Japan)

3.25% w|v Sodium chloride (Mallinckrodt, St. Louis, MO)

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

0 42% w/v Sodium phosphate, monobasic (Mallinckrodt, St Louis, MO)

Solution 2:

0 45% w/v Poloxamer 188 (BASF, Mount Olive, NJ)

1.35% w/v Hydrogenated egg phosphatidylchohne, 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 dispersed in water using a high shear mixer. The solutions were combined following emulsiflcation and staurated with nitrogen pπor to spray drying.

The resulting dry, free flowing, hollow, spherical product had a mean particle diameter of 2 6 ± 1.5 μm. The particles, which may be used for the replacement or augmentation of lung surfactant, were spherical and porous as determined by SEM.

This example illustrates the point that a wide vaπεty of blowing agents (here nitrogen) may be used to provide microstructures exhibiting desired morphology. Indeed, 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 selected porosity.

X

Preparation of Perforated icrostructura Powder Containing Ampicillin

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

20% w/w Ampicillin, Biotech grade (Fisher Scientific, Pittsburgh, PA) 14.38% w/w Hydroxyethyl starch (Ajinomoto, Japan)

65.62% w/w Dipaimitoylphosphatidylcholinε (Gεnzyme, Cambridge, MA) Perfluorohexane (3M, St. Paul, MN) Deionized water

Hydroxyethyl starch, (HES; 0 9 g), and dipalmitoylphosphatidylcholme (DPPC; 4.11 g) were dispersed in 75 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 dispεrsion 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, 4.11 g) was then added dropwise during mixing (T = 5-10 C).

After thε addition was complete, the PFH in water emulsion was mixed on the Ultra Turrax for a total of not less than 4 minutes. 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 temperatures of thε spray dryer were 90 C and 55 C respectively The nebu zation air and aspiration flows were 1 ,800 L/hr and 100% respectively. A free flowing white powder comprising porous microspheres was obtained.

XI Preparation of Perforated Microstructure Powder Containing Insulin

The following materials were obtained and used to provide a feed stock.

0.0045% w/w Human Insulin, (Calbiochem, San Diego, CA) 17.96% w/w Hydroxyethyl starch (Ajinomoto, Japan) 82.04% w/w Dipalmitoylphosphatidylcholine (Gεnzyme, Cambridge, MA) Perfluorohexane (3M, St. Paul, MN)

Deionized water

Hydroxyεthyl starch, (HES; 1.35 g) and dipalmitoylphosphatidylcholine (DPPC; 6.16 g) were dispersed in

100 ml deionized water using an Ultra Turrax mixer (model T 25) at 10,000 rpm for approximately 2 mmutεs (T = 45-

50 C). Thε resulting DPPC/HES dispεrsion was thεn chilled in a ice bath Insulin (3 4 mg) was added and allowed to mix for 1 minute (T = 5 10 C). Perfluorohexane (PFH, 6.16 g) was then added dropwise 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 fed at a rate of 5 5 ml/mm. The inlet and outlet temperatures of the spray dryer were 80 C and 45 C respectively. The nebuhzation air and aspiration flows were 1,800 L/hr and 100% respectively. A free flowing, white powder comprising porous microspheres was obtained.

XII

Preparation of Fluorescent-Labeled

Perforated Microstructure Powder via Spray Drying The following materials were obtained and used to manufacture feed stock.

0.2% w/w Nitrobenzoyldiol Phosphatidylchohne (Avanti Polar Lipids, Alabaster, AL) 17.6% w/w Hydroxyethyl starch (Ajinomoto, Japan) 82.2% w/w Dipalmitoylphosphatidylcholine (Genzymε, Cambndgε, MA) Perfluorohexane (3M, St. Paul, MN) Deionized watεr

Dipalmitoylphosphatidylcholine (DPPC; 1 g) and nitrobenzoyldiol phosphatidylchohne (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 added to the DPPC/NBD PC thin film. The surfactants and starch were then dispersed in the aqueous phase using an Ultra Turrax mixer (model T 25) at 10,000 rpm for approximately 2 minutes (T = 45 50 C). The resulting

NBD PC/DPPC/HES dispersion 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 complete, the resulting PFH in water emulsion was mixed on the

Ultra-Turrax for an additional time of not less than 4 minutes. The fluoresceπtly 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 temperatures of the spray dryer were 100 C and 65 C respectively The nebuhzation air and aspiration flows were 1 ,800 L/hr and 100% respectively A free flowing, yellow powder compnsing perforated microstructures was obtained.

XIII 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 deπvεd lung surfactant, Alvεofact (Thomae, Biberach, Germany) and spray dned lung surfactant containing microshells were dissolved in normal saline at a concentration of 10 mg/ml and allowed to incubate for 15 minutes at 37 C. Prior to analysis, thε surfactant test solutions were vigorously shaken using a Vortex mixer for 30 seconds. The samples were analyzed for their surface properties using the Pulsating Bubble Surfactometεr at 37 C (model EC PBS B, Electronics, Amherst, NY) according to the manufacturers instructions Surfactant solutions were allowed to adsorb at minimum bubble diameter for 10 seconds, and bubble cycling was performed in the automatic mode (20 cyclεs/minutε) For each εxpeπmεnt, measurements were taken for approximately the first 10 cycles, then again at 2, 4, and 6 minutes

The main difference observεd bεtwεεn the neat and spray dried surfactant suspensions is the rate at which they adsorb to the bubble surface and thus lower the tension. The spray dried mateπals required 6 cycles to achieve low surface tension as compared with one cyclε for thε Alvεofact sample Howevεr, thε magnitude of thε tension at maximum, and minimum bubble diameter were found to be approximately the same.

For the Alveofact dispεrsion, thε tεnsion decreased from 32 mN/m at maximum diametεr to 4 mN/m at minimum in the first cycle With further pulsation, a steady state oscillation was reached with a maximum tension _mκ 33 mN/m and a minimum tension _mn 0 to 1 mN/m. For the spray dried lung microshεll dispεrsion, thε tension decreased from 36 mN/m at maximum diameter to 16 mN/m at minimum in the first cycle By the sixth pulsation, _m3< and _m„ werε respectively 36 and 2 mN/m Both the neat Alveofact and the spray dπed lung surfactant perforated microstructures satisfy the maximum and minimum surface 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 example illustrates that, the compositions and methods of the presεnt invention are particularly useful for the replacement or augmentation of lung surfactant in patients.

Example XIV Rapid Spreading of Spray-Dried Microshells in PFC's

Stabilized dispersions formed according to the present invention provide for enhanced surfactant spreading at the pulmonary air/water interface. In this regard, the equilibrium surface tension of dimyristoylphosphatidylcholine is ca. 22 mN/m. Aqueous based liposomes are adsorbed very slowly at the air/water interface as evidenced by the fact that, after 1800 seconds, the surface tension of an aqueous solution has not been significantly reduced. The slow adsorption for liposomes is due to the slow molecular diffusion of DMPC through the water phase. Surprisingly, adsorption of DMPC suspended in perflubron (PFOB) in the form of dry perforated microstructures is very fast, reducing the surface tension to equilibrium values within a fεw seconds. This rapid spreading and reduction of surface tension is indicative of what would occur upon contacting the perforated microstructures with a wettεd pulmonary membrane. More specifically, the prεsεnt example demonstrates that the disclosed stabilized dispersions provide for the effective delivery of lung surfactants, and drugs to the lung by liquid dose instillation.

XV

Pharmacokinetics for Insulin and Glucose

Following Administration via LDI vs. IM

The insulin formulation descπbed in Example XI was administered via liquid dose instillation (0.86 IU in 4.5 mi/kg of perflubron) and intramuscular (IM) to fasting rabbits. In the case of LDI administration, rabbits were anesthetized, intubated, placed on a respirator, and their lungs were instilled with ca. 4.5 ml/kg of perflubron. The hollow porous microsphere formulation of insulin was then top-loaded in a minimal perflubron volume onto the existing perflubron in the lung, at a dose of 0.86 lU/kg. Control animals were injected IM with a similar dose of insulin (Humulin R). Plasma levels of insulin were determined by a radioimmunoassay method, and thε dεcrεase in serum glucose levels were also determined. The results are shown in Tables HI and IV. Extremely fast uptake of insulin into the systεmic circulation was obsεrvεd following LDI administration. The relative bioavailability was found to be 53%. Little differences were noted in glucose modulation between the IM and LDI groups. These results show the utility of LDI administration in the systεmic delivery of bioactive agents.

Table III

Insulin Pharmacokinetics following LDI or IM administration to rabbits

Table IV

Serum glucose levels (mg/dl) following LDI or IM administration to rabbits

XVI Reduction in Rat Mortality Following Liguid Dose Instillation of Antibiotics

Male Wistar rats (ca. 500 g) were inoculated intratracheally with 10⁹ colony forming units of Streptococcus pneumomae The model is an acute pneumonia model with 100% of untreatεd control animals dying within 4 days of inoculation. Animals receiving 10 mg of ampicillin intramuscularly one day after inoculation exhibited improved survival with 27% of the animals surviving to 10 days. Animals receiving 10 mg of ampicillin (prepared according to Example X) in 10 ml of perflubron via LDI administration exhibited a survival of 87%. These results indicate that local antibiotic treatmεnt with the hollow porous microspheres of the present invention, is extremely efficient in reducing the mortality associated with life threatening bacterial infections.

XVII Ampicillin Concentrations in the Lung and Serum Following IM and LDI Administration

Ampicillin concentrations in lung tissue and serum were measured for the two treatmεnt groups in Examplε XV by a bioassay mεthod. In this method, 60μl of lung tissue homogenate, or serum obtained from the rats at various points after dosing is placed on a steπlε disk The disk is then placed on an agar plate covered with S pneumomae and incubated for 24 hr. Levels of antibiotic high enough to inhibit growth of S. pneumomae resulted in zones of growth inhibition around the disk The no growth zones were quantitated, and concentrations of antibiotic were calculated based on a standard curve.

The results for the IM and LDI groups are shown in Table V. Ampicillin has a short half life in serum as noted by the fact that ampicillin levels are undetectable following IM administration after only 2 hours. Following LDI administration, the serum levels persisted for at least 4 hours, indicating a sustained release of ampicillin into the blood. Similarly, the local lung concentrations were 250 times higher with LDI delivery and persisted for several days.

These results indicate that largε local antibiotic concentrations can be achieved at the site of the infection, without correspondingly high serum levels, following LDI administration. Moreover, unlike intramuscular administration, the higher concentrations provided by liquid dose instillation also persistεd for several days following administration. Such persistence could significantly reduce dosing requirements.

Table V

Ampicillin pharmacokinetics in rat lung: effect of mode of administration.

XVIII Gentamicin Biodistribution in Rabbit Lung

Comparison of biodistribution in New Zealand white rabbits at one hour post-administration of 5 mg/kg gentamicin by either IM or LDI methods was performεd. Thε gεntamicin was administered in an LDI volume of only 1.8 ml/kg. Individual lobes of the lungs were collected and analyzed quantitatively for gentamicin by an immunoassay method. The results are detailεd in Table VI. The lung gentamicin concentrations werε ca. 2 ordεrs of agnitudε higher following local administration (LDI) than for IM administration. Excellent biodistribution across the lung lobes was observεd following either IM or LDI administration.

Table VI

Biodistribution of Gentamicin (μg gentamiciπ/g tissue) in Rabbit Lungs Following LDI and IM Administration

Those skilled in the art will further appreciate that the present invention may be εmbodiεd in other specific forms without departing from the spint or central attributes thereof. In that the foregoing descπption of the present invention discloses only exemplary embodiments therεof, it is to bε undεrstood that, othεr vaπations are contemplated as being within the scope of the present invention. Accordingly, the present invention is not limited to the particular embodiments that have been descπbεd in detail herein. Rather, refεrence should be made to the appended claims as indicative of the scope and content of the invention.

Claims

WHAT IS CLAIMED-

1. Use of a liquid fluorochemical in the manufacture of a stabilized dispersion for the pulmonary delivery of a bioactive agent wh╬╡rein the stabilized dispersion is in a form for direct administeration to at least a portion of the pulmonary air passages of a patient in need thereof, said stabilized dispersion compnsing a fluorochemical suspension medium having dispers╬╡d therein a plurality of perforated microstructures compnsing at least one bioactive agent wherein the suspension medium substantially permeates said perforated microstructures.

2. The use of claim 1 wherein said pati╬╡nt is ventilated

3. The use of claim 1 wherein said patient is undergoing spontaneous respiration.

4 The use of any of claims 1 to 3 wherein said patient is undergoing partial liquid ventilation

5. The use of any of claims 1 to 4 wherein said perforated microstructures comp╧Çs╬╡ a surfactant.

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

7. The use of claims 5 or 6 wherein said surfactant is a phospholipid.

8. The use of claim 7 wherein said phospholipid is selected from the group consisting of dil auroyi phosphatt dylcholi ne, dioleylphosphatidylcholi╧Çe, dipalmitoyl phosphatidylchohne, disteroyl phosphatidylcholine, behenoylphosphatidyl cho ne, arachidoylphosphatidylcholme and combinations thereof

9. The use of any of claims 1 to 8 wherein said suspension medium and said perforated microstructures have a refractiv╬╡ index differential of less than about 0 5.

10. The use of any of claims 1 to 9 wherein said perforated microstructures comprise hollow porous microsph╬╡res

1 1. The use of any of claims 1 to 10 wherein said bioactive agent is selected from the group consisting of antiallergics, bronchodilators, pulmonary lung surfactants, analgesics, antibiotics, l╬╡ukotn╬╡n╬╡ inhibitors or antagonists, antihistamines, antiinflammato╧Çes, antineoplastics, anticholinergics, anesthetics, anti tuberculars, imaging agents, cardiovascular agents, enzym╬╡s, steroids, gen╬╡tic mate╧Çal, viral vectors, antisense agents, proteins, peptides and combinations thereof.

12. The use of any of claims 1 to 11 wherein said bioactive agent is delivered to the systemic circulation of said patient.

13. The use of any of claims 1 to 12 wh╬╡rein said liquid fluorochemical compnses perfluorooctyl bromide.

14. A method for forming a stabilized disp╬╡rsion for direct pulmonary administration of a bioactive agent comprising the steps of. combining a plurality of perforated microstructures compnsing at least one bioactive agent with a predetermined volume of a biocompatible 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 stabilized dispersion

15. The method of claim 14 wherein said perforated microstructures comprise a surfactant.

16 The method of claim 15 wherein said surfactant is sel╬╡cted from the group consisting of phospholipids, nonionic detergents, nonionic block copolymers, ionic surfactants, biocompatible fluonnated surfactants and combinations thereof.

17. The method of claim 15 or 16 wherein said surfactant is a phospholipid.

18 The m╬╡thod of claim 17 wherein said phospholipid is selected from the group consisting of dilauroylphosphatidylcholme, diole╬│lphosphatidylchohne, dipalmitoylphosphatidylcholine, dist╬╡roylphosphatidylchohn╬╡ beh╬╡noylphosphatidylcholm╬╡, arachidoylphosphatidylcholme and combinations thereof.

19. The method of claims 15 or 16 wh╬╡rein said perforated microstructures comprise a poloxamer selected from the group consisting of poloxamer 188, poloxamer 407, and poloxamer 338.

20 The method of any of claims 15 to 19 wherein said perforated microstructures comprise greater than about 10% w/w surfactant.

21 The method of any of claims 14 to 20 wherein said suspension medium and said perforat╬╡d microstructures have a refractive index differential of less than about 0.5.

22 The method of any of claims 14 to 21 wherein said perforat╬╡d microstructures compnse hollow porous microspheres.

23. The method of any of claims 14 to 22 wherein said stabilized dispersion compnses a homodispersion.

24 The method of any of claims 14 to 23 wherein said bioactive agent is selected from the group consisting of antiallergics, bronchodilators, pulmonary lung surfactants, analgesics, antibiotics, leukotnene inhibitors or antagonists, antihistamines, antiinflammatones, antineoplastics, anticholinergics, anesthetics, anti tuberculars, imaging agents, cardiovascular agents, enzymes, steroids, genetic mate╧Çal, viral vectors, antisense agents, proteins, peptides and combinations thereof.

25. The method of any of claims 14 to 24 wherein said suspension medium comprises a liquid fluorochemical

26. The method of claim 25 wherein said fluorochemical suspension medium comprises perfluorooctyl bromide.

27. A method for stabilizing a dispersion by reducing attractive van der Waals forces compnsing the steps of: providing a plurality of perforated microstructures; combining the perforat╬╡d microstructures with a biocompatible suspension medium comprising at least one liquid fluorochemical wherein the suspension medium and the perforated microstructures are selected to provide a refractive index differential valu╬╡ of less than about 0 5.

28 The method of claim 26 wherein said perforated microstructures comprise a surfactant

29. The method of claim 27 wherein said surfactant is selected from the group consisting of phospholipids, nonionic det╬╡rg╬╡nts, nonionic block copolymers, ionic surfactants, biocompatible fluonnated surfactants and combinations thereof.

30. The method of claims 27 or 28 wherein said surfactant is a phospholipid.

31. The method of claim 29 wherein said phospholipid is selected from the group consisting of dilauroylphosphatidylcholine, dioieylphosphatidylchohne, dipalmitoylphosphatidylcholine, disteroyl phosphatidylchohne behenoylphosphatidyl chohne, arachidoylphosphatidylcholme and combinations thereof.

32. The method of any of claims 26 to 31 wherein said perforat╬╡d microstructures compnse hollow porous microspheres.

33. The method of any of claims 26 to 32 wherein said bioactive agent is selected from the group consisting of antiallergics, bronchodilators, pulmonary lung surfactants, analgesics, antibiotics, leukotnene inhibitors or antagonists, antihistamines, antnnflammatones, antineoplastics, anticholinergics, anesthetics, anti tubercuiars, imaging agents, cardiovascular agents, enzymes, steroids, genetic matenal, viral vectors, antisense agents, proteins, peptid╬╡s and combinations thereof.

34. The method of any of claims 26 to 33 wherein said liquid fluorochemical exhibits a positive spreading coefficient.

35. The method of any of claims 26 to 34 wherein said liquid fluorochemical comprises perfluorooctyl bromide.

36. A stabilized dispersion for the delivery of a bioactive agent compnsing a biocompatible suspension medium having dispersed therein a plurality of perforated microstructures comprising at least one bioactive agent wherein said suspension medium substantially permeates said perforated microstructures.

37 Th╬╡ disp╬╡rsion of claim 36 wherein said perforated microstructures compnse a surfactant.

38. Th╬╡ dispersion of claim 37 wherein said surfactant is selected from the group consisting of phospholipids, nonionic detergents, nonionic block copolymers, ionic surfactants, biocompatible fluonnated surfactants and combinations thereof.

39. The dispersion of claims 37 or 38 wherein said surfactant is a phospholipid

40. The dispersion of claim 39 wherein said phospholipid is selected from the group consisting of dilauroylphosphatidylcholine, dioleylphosphatidylcholin╬╡, dipalmitoylphosphatidylcholine, disteroyl phosphatidylchohne behenoylphosphatidylchohne, arachidoylphosphatidylcholme and combinations thereof.

41 The dispersion of any of claims 36 to 40 wherein said perforated microstructures comprise hollow porous microspheres.

42. The dispersion of any of claims 36 to 41 wh╬╡r╬╡m said bioactive agent is selected from the group consisting of antiallergics, bronchodilators, pulmonary lung surfactants, analgesics, antibiotics, leukotnene inhibitors or antagonists, antihistamines, antnnflammatones, antineoplastics, anticholinergics, anesthetics, anti tubercuiars, imaging agents, cardiovascular agents, enzymes, steroids, genetic material, viral vectors, antisense agents, proteins, peptides and combinations thereof.

43 The dispersion of any of claims 36 to 42 wh╬╡rein said biocompatible suspension medium comprises a liquid fluorochemical.

44 The dispersion of claim 43 wherein said liquid fluorochemical comprises perfluorooctyl bromide.

45 A system for the direct pulmonary administration of a bioactive agent to a patient comprising: a fluid reservoir; a stable dispersion in said fluid reservoir wherein said stabilized dispersion compnses a biocompatible suspension medium having a plurality of perforat╬╡d microstructures dispersed therein, said perforated microstructures compnsing at least one bioactive agent; and a pulmonary dehv╬╡ry conduit operably associated with said fluid reservoir wherein the delivery conduit is capable of administering the stabilized dispersion to at least a portion of the pulmonary air passages of a patient in need ther╬╡of

46. The system of claim 45 wherein said pulmonary delivery conduit compnses a bronchoscope or endotracheal tube.

47. The system of claims 45 or 46 wherein said pulmonary delivery conduit is associated with a mechanical ventilator.

48. The system of any of claims 45 to 47 wherein said perforated microstructures comp╧Çse a surfactant.

49. The system of claim 48 wherein said surfactant is selected from the group consisting of phospholipids, nonionic deterg╬╡nts, nonionic block copolymers, ionic surfactants, biocompatible fluonnated surfactants and combinations thereof.

50. The system of claim 49 wherein said surfactant is a phospholipid.

51. The system of claim 50 wherein said phospholipid is selected from the group consisting of dilauroylphosphatidylcholine, dioieylphosphatidylchohne, dipalmitoylphosphatidylcholine, disteroyl phosphatidylchohne, behenoylphosphatidyl chohne, arachidoylphosphatidylcholme and combinations thereof.

52. The system of any of claims 45 to 51 wherein said bioactive agent is selected from the group consisting of antiallergics, bronchodilators, pulmonary lung surfactants, analgesics, antibiotics, leukotnene inhibitors or antagonists, antihistamines, antnnflammatones, antineoplastics, anticholinergics, anesthetics, anti tuberculars, imaging agents, cardiovascular agents, enzymes, steroids, genetic mate╧Çal, viral vectors, antisense agents, proteins, peptides and combinations thereof.

53. The system of any of claims 45 to 52 wherein said biocompatible suspension medium comprises a fluorochemical

54. The system of claim 53 wherein said fluorochemical comprises perfluorooctyl bromid╬╡.

55. A method for the delivery of one or more bioactive agents comprising the steps of: providing a stabilized dispersion comprising a biocompatible suspension medium having dispers╬╡d therein a plurality of perforated microstructures wherein said perforated microstructures comprise a bioactive agent; and administering a therapeutically effective amount of said stabilized dispersion to at least a portion of the pulmonary passages of a patient in ne╬╡d thereof.