Phosopholipid-based powders for drug delivery

COMPLETE SPECIFICATION

STANDARD PATENT

Applicant

Invention

ALLIANCE PHARMACEUTICAL CORP.

Title :

PHOSOPHOLIPID-BASED POWDERS FOR DRUG DELIVERY

The following statement is a full description of this invention, including the best method of performing it known to me/us :

2006200768 24 Feb 2006

PHOSPHOLIPID-BASED POWDERS FOR DRUG DELIVERY

Field of the Invention

The present invention relates to particulate compositions suitable for drag delivery, preferably via inhalation. In particular, the present invention provides phospholipidcontaining particulate compositions comprising a polyvalent cation. The particulate compositions of the present invention exhibit an increased gel-to-liqitid crystal transition temperatures resulting in improved dispersibility and storage stability.

Background of the Invention

Phospholipids are major components of cell and «TE^ncUe membranes, blood lipoproteins, and lung surfactant. In terms of pulmonary drug delivery, phospholipids have 20 been investigated as therapeutic agents for the treatment of respiratory distress syndrome (i-e. exogenous lung surfactants), and as suitable excipients for the delivery of actives. The interaction of phospholipids with water is critical to the formation, maintenance, and function of each of these important biological complexes (McIntosh and Magid)- At low temperatures in the gel phase, the acyl chains are in a ctnribtinarionally well-ordered gtntp., 25 essentially in the aU-trans configuration. At higher temperatures, above the chain melting temperature, this chain order is lost, owing to an increase in gauche conformer content (Seddon and Cevc).

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derived finm bovine lungs (Survanta ®, Abbott Laboratories), porcine Jungs (CuroStrrf®,

Dey Laboratories), ar completely synthetic surfactants until no apoproteins (e.g. ALEC®, ExoSuri® Glaxo Wellcome)- To date, these products have been utilized for the treatment of infant respiratory distress syndrome (IRDS). None have been successful in recraving FDA approval for the treatment of adult respiratory distress syndrome (ARDS). The current ikifhut dose is 100 mgticg. For a 50 kg adult, this would translate into a dose of 5g. A dose of this amount can only he adinmlstem^ tn ARDS patiftnta hy djpret· wntffflatirm irrtn la

2006200768 24 Feb 2006 patient’s endotracheal tube, ar possibly via nabulization of aqueous dispersions of the surfactant material.

Instillation of surfactants leads to deposition primarily in the central airways, and little of the drug makes it to the alveoli, where it is needed to improve gas exchange in 5 these critically ill patients. Nebulizatitm of surfactant may allow for greater peripheral delivery, but is plagued by the fact that (a) current nebulizers are inefficient devices and only ca. 10% of the drug actually reaches the patients lungs; (b) the surfactant solutions foam during the nebuli2ation process, leading to complications and further loss of drug. It is believed that as much as 99% of the administered surfactant may be wasted due to poor

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decreased.

Further, lung surfactant has been shown to modulate mucous transport in airways. In this regard, die chronic administrating of surfactant for the treatment of patients with 15 chronic obstructive pulmonary disease (COPD) has been suggested. Still other indications with significantly lower doses may be open to treatment if a dry powder fonn of a lung surfactant were available. The powdered surfactant formulation may be purely synthetic (ie. with no added apoproteins). Alternatively, the powder formulation could contain the hydrophobic apoproteins SP-B or SP-C or alternative recombinant or synthetic peptide 20 mimetics (e^. KU).

Due to its spreading characteristics an lung epithelia, surfactant has been proposed as the ideal carrier for delivery of drugs to the lung, and via the lung to the systonic circulation. Once again, achieving efficient delivery to the lung is important, espetially in light of the potential high cost nf many of rhp mrrwrrf pnwincta, One potential Way to 25 deliver drugs in phospholipids is as a dry powder aerosolized to tile lung. Most fine powders (&lt; 5 pm) exhibit poor dispersibility. This can be problematic when attempting to

The major forces that control particle-particle interactions can be divided into short and long range forces. Long-range forces inoinde gravitational attractive forces and 30 electrostatics, where the interaction varies as the square of the separation distance. Shortrange attractive forces dominate for dry powders and include van der Waals interactions, hydrogen bonding, and liquid bridging. Liquid bridging occurs when water molecules axe ahle to irreversibly bind particles together.

Phospholipids are especially diffionlt to fonnnlate as dry powders as their low gel 35 to liquid crystal transition temperature (Tm) values and amorphous nature lead to powders which are very sticky and difficult to deaggregate and aerosolize. Phospholipids with Tm

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2006200768 24 Feb 2006 values less than 10®C (e.g- egg PC or any unsaturated lipids) form highly cohesive powders following Spray-drying. Inspection of the powders via scanning electron microscopy reveals highly agglomerated particles with surfaces that appear to have been melied/amiealed. Formulating phospholipid powders which have low Tm are problematic, especially if one hopes to achieve a certain particle morphology, as in the case of aerosol delivery. Thus, it would be advantageous to find ways to elevate the Tm of these lipids. Examples of particulate compositions incorporating a surfactant are disclosed in PCT publications WO 99/16419, WO 99/38493, WO 99/66903, WO 00/10541, and U.S. Patent Nos. 5,855,913. which are hereby incorporated in their entirety by reference.

Currently, lung surfactant is given to patients by intubating them and instilling a suspension of lung surfactant directly into the lungs- This is a highly invasive procedure which generally is nor performed on conscious patients, and as do most procedures, carries its own risks. Potential applications for lung surfactant beyond the current indication of respiratory distress syndrome in neonates are greatly limited by this method of artmtnisttation. For example, lung surfactant may be useful in a variety of disease stales that are, in part, due to decreased lung surfactant being present in the lungs. U.S. Pate*» Nos. 5,451,569.5,698,537, and 5,925337. and PCT publications WO 97/26863 and WO 00/27360, for example, disclose the pulmonary admmistration of lung surfactant to treat various conditions, the disclosures of which are hereby incorporated in their entirety by reference. Diseases that are thought to be possibly aggravated by lung surfactant deficiency include cystic fibrosis, chronic obstructive pulmonary disease, and asthma, just to name a few. The delivery of exogenous Jung surfactant, in a topical fashion, to patients suffering from these diseases may ameliorate certain signs and symptoms of the diseases. For chronic conditions, the regular (once or more times per day on a prolonged basis) 2$ delivery of lung surfactant via. intubation and instillation to ambulatory patients is impractical. Further, because of their high surface activity, lung surfactant suspensions are not amenable to nebulizntion due to foaming. The current delivery of phospholipid -based preparations by instillation or nebulizaticn are highly inefficient in delivering material to the peripheral lung. Therefore, the ability to deliver lung surfactant to patients via dry powder inhalation would be a tremendous advantage over the cunent method, siner. it would avoid the need for intubation, thereby expanding the potential uses of lung surfactant in the clinical setting

2006200768 24 Feb 2006

Summary of the Invention

The present invention provides far dry powder compositions of phospholipid suitable for drug delivery. According to a preferred embodiment· the phospholipid compositions are efficiently delivered to the deep lung. The phospholipid may be delivered alone, as in the case of lung surfactant or in c nm h; nation with another active agent and/or excipient The use of dry powder compositions may also open new indications for use since the patient need not be intubated. According to one the compositions of the present invention may be delivered from a simple passive DPI device. The present compositions allow for greater stability on storage, and for more efficient delivery to the

Jjt has been found in the present work that the gel to liquid crystal phase transition of the phospholipid, Tm, is critical ία obtaining phospholipid -based dry powders that both flow welt and are readily dispersible from a dry powder inhaler device. The present invention is related to the use of polyvalent cations, preferably divalent cations to dramatically increase the Tm of phospholipids. As used herein, “polyvalent cations” refers to polyvalent salts or their ionic components. fectemdnE the Tm. of the phospholipid leads to the following formulation improvements: (a) Increases in Tm allows the fonnulatar to increase the inlet and outlet temperatures on the spray-drier, or on a vacuum oven during a 20 secondary drying step, Higher temperatures allow the drying phase of the spray-drying to be controllable over a wider temperature range, thereby facilitating removal of trapped Wowing agent used in the manufacture of powders according to one aspect of the present invention; (b) Increases in Tin allow for a large difference between Tm and the storage temperature, thereby inipwving powder stability; (c) Increases in Tin yield phospholipids 25 in the gel state, where they are less prone to taking up water and water bridging phenomena (d) Increases in Tm yield phospholipids which are able to spread more effectively upon contact with lung epithelia than hydrated phospholipids, thereby allowing drugs to be more effectively distributed to the lung periphery; (e) Increases in Tm dramatienny improves the dispersibility of the resulting powders, (hereby improving ths emitted doss and particle _ fraction fallowing pulmonary delivery.

According to a preferred embodiment, the present invention relates to highly dispersible dry powder ertrnpratitinn^ of phospholipids suitable for pulmonary delivery. The compositions according to the present inventing are useful as syntheHe inng surfactants for the treatment of local lung conditions (e.g. asthma, COPD), ar as carriers for the pulmonary

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2006200768 24 Feb 2006

One aspect of the present invention is to provide powdered, dispersible compositions having stable dispersibility over time. The compositions exhibit a characteristic gel to liquid crystal phase transition temperature, Tm, winch is greater than a recommended storage temperature, Ts, typically room temperature, by at least 20 °C.

Preferably Tm is at least 40 °C greater than Ts.

It is a further aspect of foe present invention that the increases in Tm afforded by addition of divalent cations leads to foe ability to dry the powders in a secondary drying step at temperatures (Td) up to the Tm of the lipid. As well, it is possible to increase foe inlet and outlet temperatures on a spray-drier should a spray-dry process be employed (Td 10 =*Tm).

It is a farther aspect of the present invention to provide a powdered, dispersible farm of a lung surfactant having stable dispersibility over time and excellent spreading characteristics on an aqueous subphase.

It is a further aspect of foe present invention that foe improvements in dispersibility 15 obtained by foe present compositions allow far a simple, passive inhaler device to be utilized, in spite of the fact that particles less than 5 gm are contemplated and generally preferred. Present state-of-the-art formulations far fine particles utilize blends with large lactose particles to improve dispersibility. When placed in a passive DPI device such fammlatitms exhibit a strong dependence of emitted dose and lung deposition on foe patient’s inspiratory flowrate. The present compositions exhibit little flowrate dependence chi the emitted dose and lung deposition.

Brief Description of foe Drawings

Figure 1 is a graph depicting the physical stability of budesomde in pMDL

Figure 2 are SEM photographs the effect of calcium ion concentration on foe moiphology of spray-dried particles according to the invention.

Figure 3 is a graph depicting the spreading characteristics of powders of foe instant invention.

Definitions “Active agent” as described herein includes an agent, drug, compound, annpositxnn of matter ar mixture thereof which provides same diagnostic, prophylactic, ar pharmacologic, often beneficial, effect This includes foods, food supplements, nutrients, drugs, vaccines, vitamins, and other beneficial agents. As used herein, the terms farther 35 ar systemic effect in a patient The active agent that be delivered includes antihigtieg,

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2006200768 24 Feb 2006 antibodies, antiviral agents, anepileptics, analgesics, anti-inflammatory agents and bronchodilators, and viruses and may be inorganic and organic compounds, including, without limitation, drugs which act on the peripheral nerves, adrenergic receptors, cholinergic receptors, the skeletal muscles, the cardiovascular system, smooth muscles, the 5 blood circulatory system, synaptic sites, neuroeffector junctional sites, endocrine and hormone systems, the immunological system, the reproductive system, the skeletal system, antacoid systems, the alimentary and excretory systems, the histamine system and the central nervous system. Suitable agents may be selected from, far example, polysaccharides, steroids, hypnotics and sedatives, psychic energizers, tranquilizers, anticonvulsants, muscle relaxants, antiparidnson agents, analgesics, antitiuflamiiiatDries, muscle contractants, antimicrobials, antimalarials, hormonal agents including contraceptives, sympaihomimetics, polypeptides, and proteins capable of eliciting physiological effects, diuretics, lipid regulating agents, antiandrogenic agents, antiparusirfes, neoplasties, antineopiastics, hypoglycemics, nutritional agents and supplements, growth supplements, fats, aatienteritis agents, electrolytes, vaccines and diagnostic agents.

Examples of active agents useful in this invention include but are not limited to insulin, calcitonin, erythropoietin (EPO), Factor VTH, Factor K, ceredase, cerezyme, cyclosporine, granulocyte colony stimulating factor (GCSF), alpha-1 proteinase inhibitor, 20 elcatonin, granulocyte macrophage colony stimulating factor (GMCSF), growth hormone, human growth hormone (hGH), growth hormone releasing hormone (GHRH), heparin, low molecular weight heparin (LMWH), interferon alpha, interferon beta, interferon gamma, mterieultin-2, luteinizing hormone releasing hormone (LHRH), leuprolide, somatostatin,

2006200768 24 Feb 2006 present as bare nucleic add molecules, viral vectors, associated viral particles, nucleic acids associated ar incorporated within lipids or a lipid-coutaining material, plasmid DNA or RNA or other nucleic add construction of a type suitable for transfection or transformation of cells, particularly cells of the alveolar regions of the lungs. The active agents may be in various farms, such as soluble and insoluble charged or uncharged molecules, components of molecular complexes or pharmacologically acceptable salts. The active agents may be naturally occurring molecules or they may be reconihmantly produced, ar they may be analogs of the naturally occurring or recombinantiy produced active agents with one or more ammo acids added or deleted. Further. the active agent may 10 comprise live attenuated or killed viruses suitable for use as vaccines.

As used herein, the term “emitted dose” or “ED” refers to an indication of the delivery of dry powder from a suitable inhaler device after a firing or dispersion event from a powder unir or reservoir. ED is defined as the ratio, of the dose delivered by an inhaler device (described in detail below) to the nominal dose (Le., the mass of powder per 15 unit dose placed into a suitable inhaler device prior to firing). The ED is an experimentally-determined amount, and is typically determined using an tn-vftn> device set up which mimics patient dosing. To determine an ED value, a nominal dose of dty powder (as defined above) is placed into a suitable dry powder inhaler, which is than actuated, dispersing the powder. The resulting aerosol cloud is then, drawn by vacuum from the device, where it is captured on a tared filter attached to the device mouthpiece. The amount of powder that reaches the filter constitutes the delivered dose. For example, for a 5 mg, dry powder-containing blister pack placed into an inhalation device, if dispersion of the powder results in the recovery of 4 mg of powder on a taxed filter as described above, then the ED for the dry powder composition is: 4 mg (delivered dose)/5 mg (nominal dose) x

100-80%.

“Mass median diameter” or “MMD” is a measure of mean particle size, since the powders of the invention axe generally palydisperae (i.e_, consist of a range of particle sizes). MMD values as reported herein are determined by laser diffraction, nlthongh any number of commonly employed techniques can be used for measuring menu particle size.

“Mass median aerodynamic diameter” or “MMAD is a measure of the aerodynamic size of a disposed particle. The aerodynamic diamcterls used to describe an aerosolized powder in terms of its settling hahaviar, and is the diamehy of a mrit rlwisity sphere having the same settling velocity, generally in air, as ths particle. The aerodynamic

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2006200768 24 Feb 2006

Detailed Description of the Invention

The present invention is directed to the formulation of dry phospholipid 5 polyvalent cation based particulate composition. In particular, the present invention is directed to the use of polyvalent cations in the manufacture of phospholipid -containing, dispersible particulate compositions for pulmonary administration to the respiratory tract far local or systemic therapy via aerosolization, and to the particulate compositions martp.

thereby, The invention is based, at least in part, on the surprising discovery of the beneficial aerasolization and stabilization properties of phospholipid -containing particulate compositions comprising a polyvalent cation. These unexpected benefits include a dramatic increase in the gel-to-liquid crystal phase transition temperature (Tm) of the particulate composition, improved dispersibility of such particulate compositions, improved spreadability of the particulate compositions upon content with lung epithelia thsrehy allowing drugs to be more effectively distributed to the lung periphery, and improved

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It is surprisingly unexpected that the addition of a very hygroscopic salt such as calcium chloride would stabilize a dry powder prone to moisture induced destabilization, as one would expect that the calcium chloride would readily pick up water leading to particle 20 aggregation. However, this is not what is observed. In contrast. addition of calcium ions leads to a dramatic improvement in the stability of the dry phospholipid-based powder to humidity. While not being bound to any theory, it is believed that calcium ions are believed to intercalate the phospholipid membrane, thereby interacting directly with the negatively charged portion of the zwitterionic headgroup. The result of this interaction is increased dehydration of the headgroup area and comfcsisatian of the acyi-chaln packing, all of which leads to increaaed thermodynamic stability of the phospholipids.

The polyvalent cation far use in the present invention is preferably a divalent cation including calcram, magnesium, zinc, iron, and the ilk»- According to the invention, the polyvalent cation is present in an amoum effective to increase the Tm of the phospholipid such that the particulate composition exhibits a Tm which is greater than its storage temperature Ts by at least 20 °C. preferably at least 40°C. The molar ratio of polyvalent cation to phospholipid should be at least 0.05, preferably 0,05 - 2.0, and most preferahly 0.25 - 1.0. A molar ratio of polyvalent catiomphospholipid of about 0.50 is particularly preferred according to the present invention. Calcium, is the partirnlmty 35 preferred polyvalent cation of thepresent invention and is provided as calcium chloride.

hi a broad sense, phosphoEpid suitable for use in the present invention include any of those known in the art

According to a preferred embodiment, the phospholipid is most preferably a saturated phospholipid. According to a particularly preferred embodiment saturated phosphatidylcholines are used as the phospholipid of the present invention. Preferred acyl chain lengths are 16:0 and 18:0 (i.e. palmitoyl and stearoyl)- According to one embodiment directed to lung surfactant compositions, the phospholipid can make up to 90 to 99.9% w/w of the composition. Suitable phospholipids according to this aspect of the invention include natural or synthetic lung surfactants such as those commercially available under the trademarks ExoSurf. InfaSurf® (Ony, Inc.), Survanta, CuroSurf, and ALEC. Far drag delivery purposes wherein an active agent is included with the particulate composition, the phospholipid content will be dAtarmined by the drug activity, the mode of delivery, and other factors and will likely be in the range from about 20% to up to 99.9% w/w. Thus, drug loading can vary between about 0.1% and 80% w/w, preferably 5 - 70% w/w.

According to a preferred embodiment, it has been found in the present work that the Tm of the phospholipid is critical in ohtniring pbospholipid-based dry powders that both flow well and are readily dispersible from a dry powder inhaler (DPI). The Tm of the modified lipid microparticles can be manipulated by varying the amount of polyvalent cations in the formulation.

Phospholipids from both natural and synthetic sources are compatible with the present invention and may be.used in varying concentrations to farm the structural matrix. Generally compatible phospholipids comprise those that have a gel to liquid crystal phase transition greater than about 40°C_ Preferably the incorporated phospholipids are relatively long chain (i.e. C_ie-C2z) saturated lipids and more preferably comprise satmared phospholipids, most preferably saturated phosphatidylcholines having acyl chain lengths of 16:0 ar 18:0 (palmitoyl and stearoyl). Exemplary phospholipids useful in the disclosed stabilized preparations comprise, phrapbogly'y’r'r^¹? such as dipahnitoylphosphatidylcliQline, distemylphosphatidylcholine, diarachidoylphosphatidylchoiine dibehcnoylphosphatidylcholine, diphosphatidyl glycerol, short-chain phosphatidylcholines, lang-chain saturated phosphatidylethanolamines, longchain saturated phosphatidylserines, long-chain saturated phosphatidylglycerols, long-chain saturated phosphatidylinositols.

In addition to the phospholipid, a «v^rtrfaetant or combinations of surfactants, including die use of one ar more in the Equid phase and one or more associated with the particulate compositions are contemplated as being within fits scope of the inventian. By * associated with or comprise it is meant that the particulate compositions may incorporate.

2006200768 24 Feb 2006 adsorb, absorb, be coated with or be formed by the surfactant. Surfactants include fluorinated and nonfluorinated compounds and are selected from the group consisting of saturated and unsaturated lipids, norrionic detergents, nonionic block copolymers, ionic surfactants and combinations thereof. In those Embodiments comprising stabilized dispersions, such nonfluorinated surfactants will preferably be relatively insoluble in the suspension medium. It should be emphasized that, in addition to the aforementioned surfactants, suitable fluorinated surfactants ate compatible with the teachip gs herein and may be used to provide the desired preparations.

Compatible nomonic detergents suitable as co-surfactants comprise: sorbitan esters including sorbitan trioleate (Span™ 85), sorbitan sesquioleate, sorbitan monooleate, sorbitan mon olaurate, polyoxyethylene (20) sorbitan mondaurute, 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 entirely. Preferred block copolymers include diblock and triblcck copolymers of polyoxyethylene and polyoxypropylene, including poloxamer 188 (Plnrouic™ F-68), poloxamer 407 (Pluronic™ F-127), and poloxamer 338. Ionic surfactants such as sodium sulfosuccinate, and fatty acid soaps may also be utilized.

Other lipids including glycolipids, ganglioside GM1, sphingomyelin, phosphatidic acid, cardiolipin; lipids bearing polymer chains such as polyethylene glycol, chitin, hyaluronic acid, or polyvinylpyrrolidone; lipids bearing sulfonated mono-, di-, and polysaccharides; fatty acids such as palmitic acid, stearic acid, and oleic add; cholesterol, cholesterol esters, and cholesterol hennsuccmate may also be used in accordance with the 25 teachings of this invention.

It will further be appreciated that the particulate compositions according to the invention may, if desired, contain a comhinatiaD of two ar more active ingredients. The agents may be provided in combtnation in a single species of particulate composition or individually in separate species of particulate canpositions. For example, two or more 30 active agents may 1» incorporated in a single feed stock preparation and spray dried to provide a single particulate composition species comprising a plurality of active agents. Conversely, the individual actives could be added to separate stocks and spray dried separately to provide a plurality of particulate composition species with different coznpositicns. These individual species could be added to the suspension medium or dry 35 powder dispensing compartment in any desired proportion and placed in the aerosol dehvoy system as described below. Further, as alluded to above, the particulate compositions (with or without an associated agent) may be combined with one or mare conventional (e.g. a xmcranized drug) active or bioactivc agents to provide the desm-d dispersion stability or powder dispersibility.

Based on the foregoing, it will be appreciated by those skilled in the an that a wide variety of active agents may be incorporated in the disclosed particulate compositions.

Accordingly, the list of preferred active 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 agent and the timing of the dosages may be determined far the particulate compositions in accordance with already existing information and without undue experirpeti rating.

In addition to the phospholipid and polyvalent cation, the microparticles of the present invention may also include a biocompatible, preferably biodegradable polymer, copolymer, or blend ar other combination thereof. In this respect useful polymers comprise polylactides, polylactide-glycolides, cyclodextrins, polyacrylates, methylcellulose, carboxymethylcellulose, polyvinyl alcohols, polyanhydrides, polylactams. polyvinyl pyrrolidones, polysaccharides (dextrans, starches, chitin, chitosan, etc.), hyaluronic acid, proteins, (albumin, collagen, gelatin, etc.). Examples of polymeric resins that would be useful for the preparation of perforated ink microparticles include: styrene-butadiene, styrene-isoprene, styrene-acrylonitnle, ethylene-vinyl acetate, ethylene-asrylate, ethyleneacrylic acid, ethylene-methylacrylatale, ethylene-ethyl acrylate, vinyi-methyl methacrylate, acrylic acid-methyl methacrylate, and vinyl chloride-vinyl acetate. Those skilled in the art will appreciate that, by selecting the appropriate polymers, the delivery efficiency of the particulate compositions and/or the stability of the dispersions may be tailored to optimize the effectiveness of the active or agent

Besides the aforementioned polymer materials and surfactants, it may be desirable to add other excipients to a particulate composition to improve particle rigidity, production yield, emitted dose and deposition, shelf-life and patient acceptance. Such optional excipients inchide, but axe not limited to: coloring agents, taste masking agents, buffets, hygroscopic agents, antioxidants, and chemical stabilizers. Further, various excipients may be incorporated in, or added to, the particulate matrix to provide structure and form to the particulate compositions (Le. microspheres such as latex particles). In this regard it will be appreciated that the rigidifymg components can be removed using a post-production technique such as selective solvent extraetrnn.

Other excipients may include, but are not limited to, carbohydrates indudhrg monosaccharides, disaccharides and polysaccharides. For example, mouosaccharides such as dextrose (anhydrous and monohydraieX galactose, mannitol, D-jnatmose, sorbitol* sorbose and the like; disaccharides such as lactose, maltose, sucrose, trehalose, and the tike',

2006200768 24 Feb 2006 trisaccharides such as raffinose and the like; and other carbohydrates such as starches (hydroxyethylstaruh), cydodextrins and maltodextrins. Other excipients suitable far use with the present invention, including amino acids, are known in the art such as those disclosed in WO 95/31479, WO 96/32096, and WO 96/32149, Mixtures of carbohydrates 5 and ammo acids are further held to be within the scope of the present invention. The inclusion of both inorganic (e.g. sodium chloride, etc.), organic adds and their salts (e.g. carboxylic acids and their salts such as sodium citrate, sodium ascorbate, magnesium gluconate, sodium gluconate, tromethamine hydrochloride, etc.) and buffers is also contemplated. The inclusion of salts and organic solids such as ammamum carbonate, awnnoninm ager^tf·, ammonium chloride- or camphor are also ctmftgnpiatedYet other preferred embodiments include particulate compositions 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, anionic charges are known to favor mucoadhesion while cationic charges may be used to associate the fanned mfcraparticulate with negatively charged bioactive agents such as genetic material. The charges may be imparted through the association or incorporation of polyanionic or polycationic materials such as polyacrylic adds, polylysine, potyl&amp;ctic acid and chitosan.

According to a preferred embodiment, the particulate compositions may be used in the form Of dry powders or in the form of stabilized dispersions comprising a nan-aqueous phase. Accordingly, the dispersions or powders of the present invention may be used in conjunction with metered dose inhalers (MDIs), dry powder inhalers (DPIs), atomizers, nebulizers or liquid dose instillatinn fLDI) techniques to provide far effective drug delivery. With respect to inhalation therapies, those skilled in the art will appreciate that the hollow and porous microparticles of die present invention are particularly useful in DPIs. Conventional DPIs comprise powdered fornmlations and devices where a predetermined dose of medicament, either alone or in a blend with lactose carrier particles, is delivered as

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The medicament is formulated in a way such that it readily dispenses into discrete particles with an MMD between 0.5 to 20 gm, preferably 0.5-5gm, and arc further characterized by an aerosol particle size distribution less than about 10 gm mass median aondynamic diameter (MMAD), and preferably less than 5.0 pm- The mass median aerodynamic diameters of the powders will characteristically range from about 05 -10 pm, preferably from about 05 - 5.0 gm MMAD, mare preferably from about 1.0 - 4.0 gm MMAD.

He powder is actuated either by inspiration ar by some external delivery force, such as pressurized air. Examples of DPIs suitable for administration of the particulate

2006200768 24 Feb 2006 compositions of the present invention are disclosed in U.S. Patent Nos. 5,740,794, 5,785,049, 5,673,686, and 4,995,385 and PCT application nos. 00/72904,00/21594, and 01/00263, hereby incorporated in their entirety by reference. DPI fonnolations are typically packaged in single dose units such as those disclosed in the above mentioned patents or they employ reservoir systems capable of metering multiple doses with manual transfer of the dose to the device.

As discussed above, the stabilized dispersions disclosed herein may also be administered to the nasal or pulmonary air passages of a patient via aerosolization, such as with a metered dose inhaler, The use of such stabilized preparations provides far superior dose reproducibility and improved lung deposition aa disclosed in WO 99/16422, hereby incorporared in its entirety by reference, MDIs are well known in the art and could easily be employed for administration of the claimed dispersions without undue experimentation.

Breath activated MDIs, as well as those comprising other types of improvements which, have been, or will be, developed are also compatible with the Stabilized dispersions and present invention and, as such, are contemplated as being witbin the scope thereof. However, it should be emphasized that, in prefeued Embodiments, the stabilized dispersions may be administered with an MDI using a number of different routes including, but not limited to, topical, nasal, pulmonary ar oraL Those skilled in the ait will appreciate that, such routes are well known and that the dosing and administration procedures may be easily derived for the stabilized dispersions of the present invention.

Along with the aforementioned embodiments, the stabilized dispersions of the present invention may also be used in conjunction with nebulizers as disclosed in PCT WO 99/16420, the disclosure of which is hereby inctnporated in its entirety by reference, in order to provide an aerosolized medicament that may be administered to the pulmonary air passages of a patient in need thereof Nebulizers are well known in the art and could easily

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Breath activated nebulizers, as well as those comprising other types of improvements which have been, or will be, developed are also compatible with the stabilized dispersions and present invention and are contemplated as being with in the scope thereof.

Along with DPIs, MDIs and nehnlieMs. it will be appreciated that the stabilized dispersions of the present invention may be used in conjunction with liquid dose instillation or LDI techniques as disclosed in, for example, WO 99/16421 hereby incorporated in its entirety by reference. Liquid dose instillation involves the direct administration of a stabilized dispersion to the lung. In this regard, direct pulmonary administration of bioactive compounds is particularly effective in the treatment of rfisorrier*: especially where poor vascular circulation of diseased portions of a lung reduces the effectiveness of

2006200768 24 Feb 2006 intravenous drug delivery. With respect to LDI 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 microdispersion prior to, during or following administration.

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hollow and porous particulate compositions as disclosed in WO 99/16419, hereby incorporated in its entirety by reference. Such particulate compositions comprise particles having a relatively thin parous wall defining a large internal void, although, other void containing or perforated structures are contemplated as well· In preferred embodiments the particulate compositions will further comprise an active agent

Compositions according to the present invention typically yield powders with bulk densities less than 0.5 g/cm³ or 0.3 g/cm³, preferably less o.i g/cm³ and most preferably less than. 0.05 g/cm³. By providing particles with very low bulk density, the trnnimum .15 powder mass that can be filled into a unit dose container is reduced, which eliminates the need for carrier particles. That is, the relatively low density of the powders of the present invention provides for the reproducible administration of relatively low dose pharmaceutical compounds. Moreover, the elimination of carrier particles will potentially minimi ze throat deposition and any gag effect, since the large lactose particles will impact the throat and upper airways due to their size.

It will be appreciated that the particulate compositions disclosed herein comprise a structural matrix that exhibits, defines or comprises voids, pates, defects, hollows, spaces, interstitial spaces, apertures, perforations or holes. The absolute shape (as opposed to tire morphology) of the perforated microstructure is generally not critical and any overall configuration that provides the desired characteristics is conrexuplated as being within the scope of the invention. Accordingly, preferred embodiments can comprise approximately microspherical shapes. However, collapsed, deformed or fractured particulates are also compatible.

In accordance with the teachings herein the particulate compositions will preferably be provided in a dry” state. That is the microparticles will possess a moisture content that allows the powder to remain chemically and physically stable during storage at ambient temperaand easily dispersible- As evw*, for* mcistnre content of tine microparticles is typically less than 6% by weight, and preferably less 3% by weight In some instances the moisture content will be as low as 1% by weight. Of course it will be appreciated that the moisture content is, at least in part, dictated by the formulation and is

2006200768 24 Feb 2006 controlled by the process conditions employed, e.g., inlet temperature, feed concentration, pump rate, and blowing agent type, concentration and post drying.

Reduction in bound water leads to significant improvements in the dispersibility and flowability of phospholipid based powders, leading to the potential for highly efficient 5 delivery of powdered lung surfactants or particulate composition comprising active agent dispersed in ths phospholipid. The improved dispersibility allows simple passive DPI devices to be used to effectively deliver these powders.

Although the powder compositions ate preferably used for inhalation therapies, the powders of the present invention can also be administered by other techniques known in the 10 art, including, bitt not limited to intramuscular, intravenous, intratracheal, intraperitoneal. Subcutaneous, and transdermal, either as dry powders, reconstituted powders, or suspensions.

As seen from the passages above, various components may be associated with, or incorporated in the paniculate compositions 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), dispersibility and density. Among other methods, particulate compositions compatible with the instant¹ invention may be formed by techniques including spray drying, vacuum drying, solvent extraction, emulsification or lyophilization, and combinations thereof. It will further be appreciated that the basic concepts of many of these techniques an 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 particulate compositions.

While several procedures are generally compatible with the present invention, particularly preferred embodiments typically comprise particulate canq»sitians fanned by 25 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 far various administrative routes including inhalation. See, for example, M. Sacchetti and MM Van Oort itu inhalation Aerosols: Ptrysical 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 evaporatiem and drying of the liquid droplets. The preparation to be spray dried or feed (ar feed stock) can be any solution, course suspensitm, slnoy, colloidal dispersion, or paste that may be afawntwid using the selected spray drying 35 apparatus, hi preferred embodiments the feed stock will comprice a colloidal system such as an emulsion, reverse emulsion, nricroenmlsian, multiple emulsion,

2006200768 24 Feb 2006 dispersion, ar slurry. Typically the feed is sprayed into a current of wannfiltoed air that evaporates the solvent and conveys the dried product to a collector Ihe spent air is th**» 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 5 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 instantized for specialized applications, i.e. the simultaneous spraying of two solutions using a double nozzle technique. More specifically, IO a water-in-oil emulsion can be atomized from one nozzle and a solution con turning an antiadherent such as mannitol can be co-atomized from a second nozzle, hi other cases it may be desirable to push the feed solution though a custom designed nozzle using a high pressure Equid chromatography (HPLC) pump. Provided that microstructures comprising the correct morphology and/or composition are produced the choice of apparatus is not 15 - critical and would be apparent to the skilled artisan in view of the teachings herein.

Examples of spray drying methods and systems suitable for making the dry powders of the present invention are disclosed in U.S. Patent Nos. 6,077.543,6,051,256,6,001,336,

5,985,248, and 5,976,574, hereby incorporated in their entirety by reference.

While the resulting spray-dried powdered particles typically are approximately spherical in shape, nearly irttifa ja size and frequently are hollow, there may be degree of irregularity in shape depending upon the incorporated medicament and the spray drying conditions. In many instances dispersion stability and dispersibility of the particulate compositions appears to be improved if an inflating agent (or blowing agent) is used in their production as disclosed in WO 99/16419 cited above. Particularly preferred embodiments comprise an emulsion with the inflating agent as the disperse ar continuous phase. The inflating agent is preferably dispersed with a surfactant solution, using, far instance, a commercially available tmcroflnidizer at a pressure of about 5000 to 15,000 psi. This process farms an emulsian, preferably stabilized by an rnempomt^ gnrfaetant, typically camprisiiig submicron droplets of water immiscible blowing agent dispersed in an aqueous continuous phase. The formation of such emulszans using thia «nd other techniques are common and well known to those in the art. The blowing agent is preferably a fluorinated compound (e.g. perfluorohexane. perfluorooctyl bromide.

perfluorooctyl ethane, petfluarodecalin, perfluorobutyl ethane) which vaporizes during the spray-drying process, leaving behind generally hollow, porous aesodynamically light nticrospheres. Other suitable liquid binwing agents mdude nanfluorinated nite, chlorofonn. Freons, ethyl acetate, alcohols and hydrocarbons. Nitrogen and carbon dioxide

2006200768 24 Feb 2006 gases are also contemplated as a suitable blowing agent Ferfiuorpoctyl ethane is particularly preferred according to the invention.

Besides the aforementioned compounds, inorganic and organic substances which can be removed under reduced pressure by sublimation in a post-production step are also 5 compatible with the instant invention. These sublimating compounds can be dissolved nr dispersed as micronized crystals in the spray drying feed solution and include ammonium carbonate and camphor. Other compounds compatible with the present invention comprise rigidifying solid structures which can he dispersed in the feed solution or prepared in-situ. These structures are then extracted after the initial particle generation using a post10 production solvent extraction step. For example, latex particles can be dispersed and subsequently dried with other wall forming compounds, followed by extraction with a suitable solvent.

Although the particulate compositions are preferably formed using a blowing agent as described above, it will be appreciated that, m some instances, no additional blowing 15 agent is required and an aqueous dispersion of the medicament and/or excipients and surfectaijt(s) are spray dried directly, In such cases, the formulation may be amenable to process conditions (e.g., elevated temperatures) that may lead to the formation of hollow, relatively porous microparticles. Moreover, the medicament may possess special physicochemical properties (e.g., high crystallinity, elevated melting temperature, surface 20 activity, etc.) that makes it particularly suitable for use in such techniques.

Regardless of which blowing agent is ultimately selected, it has been found that compatible particulate compositions may be produced particularly efficiently using a Btlchi mini spray drier (model B-191. Switzerland). As will be appreciated by those skilled in the ait, the inlet temperature and the outlet temperature of the spray drier are not critical but

<img file="AU2006200768B2_D0012.tif" />

will be of such a level to provide th® desired particle ti»¹ 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 meTfing characteristics of the formulation components and the composition of the feed stock. The inlet temperature may Arcs 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 far the outlet The flow rate winch is used in the spray drying equipment will generally be about 3 ml per minute to about 15 ml per minuta. The atomizer air flow rate will vary between values of 25 liters per rrnnnte to about 50 liters per minute. Commercially available spray dryers are well known to those in the art, and suitable settings far any particular dispersion can be readily &lt;Wnrini tied through standard empirical testing, with due reference to the examples that

2006200768 24 Feb 2006 follow. Of course, the conditions may be adjusted so as to preserve biological activity in larger molecules such as proteins ar peptides.

Whatever components are selected, the first step in particulate production typically comprises feed, stock preparation. If the phospholipid based particle is intended to act as a 5 carrier for another active agent, the selected active agent is dissolved in a solvent, preferably water, to prodace a concentrated solution. The polyvalent cation may be added to the active agent solution ar may be added to die phospholipid emulsion as discussed below. The active agent may also be dispersed directly in the emulsion, particularly in the case of water insoluble agents. Alternatively, the active agent may be incarporared in the 10 fixcm of a solid particulate dispersion. The concentration of the active agent used is dependent cm the amount of agent required in the final powder and the performance of the delivery device employed (e.g., the fine particle dose for a MDI or DPI). As needed, cosurfactants such as poloxamer 188 or span 80 may be dispersed into this annex solution. Additionally, excipients such as sugars and starches can also be added.

hi selected embodiments a polyvalent cation-ccntaining oil-in-water emulsion is then farmed in a separate vessel. The oil employed is preferably a fluorocarbon (e.g., perfluorooctyl bromide, perfluorooctyl ethane, perfluorodecalin) which is emulsified with a phospholipid. For example, polyvalent cation and phospholipid may be homogenized in hot distilled water (e.g., 6D°C) using a suitable high shear mechanical mixer (e.g-, Ultra20 Tnrrax model T-25 mixer) at 8000 rpm for 2 to 5 mmutes. Typically 5 to 25 g of fluorocarbon is added dropwise to the dispersed surfactant solution while mixing. The resulting polyvalent caticn-containiug perfluorocarbon in water emulsion is then processed using a high pressure homogenizer to reduce the particle size. Typically the emulsion is processed at 12,000 to 18,000 psi, 5 discrete passes and kept at 50 to 80°C

The active agent solution and perfluorocarbon emulsion are then comhined 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 bioactrve ageut is solubilized separately for the purposes of the instant discussion it will be appreciated that, in other embodiments, the active agent may be solubilized (or dispersed) directly in the emulsion.

In such cases, the active emulsion is simply spray dried without combining a separate active agent preparation.

In any event, operating conditions such as mint and outlet temperature, feed rate, atonuzaticai pressure, flow rate of the drying air, and nosrie configuration can be adjusted in accordance with the manufecQuer¹ s grtirfrimea in order to produce th**, required particle 35 size, and production yield of the resulting dry particles. 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

2006200768 24 Feb 2006 feed rare between 3 ml to about 15 mJ per mmote; and an aspiration air flow of 300 L/mm. and an atomization air flow rate between 25 to 50 L/min. The selection of appropriate apparatus and processing conditions axe 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 far the formation of hollow porous aerodynamically light microparticles with particle diameters appropriate far aerosol deposition into the lung, microstructures that are both hollow and porous, almost honeycombed or foam-like in appearance. In especially preferred embodiments the particulate compositions comprise hollow, porous spray dried microparticles.

Along with spray drying, particulate compositions useful in the present invention may be formed by lyophilization. Those skilled in the art will appreciate that lyophilization is a freeze-drying process in which water is sublimed from 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 dried 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 particulate compositions without cnntp-mmising physiological activity. Methods for providing lyophilized particulates are known to those of skill in the art and it would clearly not require undue experimentation to provide dispersion compatible microparticles in accordance with the teachings heuein- The lyophilized cake containing a fine foam4ike structure can be micronized using techniques known in the art to provide 3 to 10/un sized particles. Accordingly, to the extent that lyophilization processes may be used to provide micropnrticles having the desired porosity and size they are in conformance with the teachings herein and are expressly contemplated as being within the scope of the ferfrot invention.

Besides the aforementioned techniques, the particulate compositions ar particles of the present invention may also be fanned Bring a math nd where a feed solution (either 30 emulsion or aqueous) containing wall fomring agents is rapidly added to a reservoir of heated oil (e.g. perflubrcn ar other high boiling FCs) under reduced pressure. The water and volatile solvents of the feed solution rapidly boils and are evaporated. This process provides a perforated structure from the wall forming agents similar to puffed rice or popcorn. Preferably the wall forming agents are insoluble in the heated oiL The resulting 35 particles can then separated from the heated oil using a filtering technique and subsequently dried under vacuum.

2006200768 24 Feb 2006

Additionally, rhe particulate compositions of the present invention may also be foamed 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, ethyl acBtate) 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 prior to combining them with an appropriate suspension medium in accordance with the present invention

Whatever production method is ultimately selected for production of the particulate compositions, the resulting powders have a number of advantageous properties that make them particularly compatible for use in devices for inhalation therapies. In particular, the physical characteristics of the particulate compositions malce them extremely effective for use in dry powder inhalers and in the formation of stabilized dispersions that may be used in conjunction with metered dose inhalers, nebulizers and liquid dose instillation, As such.

<img file="AU2006200768B2_D0013.tif" />

agents.

la order to maximize dispersibility, dispersion stability and optimize distribution upon administration, rhe mean geometric particle size of the particulate compositions is preferably about 0.5-50 pm, more preferably 1-20 pm and most preferably .5*5 pin. It will be appreciated that large particles (ie. greater than 50 pm) may not be preferred in applications where a valve or small orifice is employed, sines large particles tend to aggregate or separate from a suspension which could potentially clog the device. In especially preferred embodiments the mean geometric particle size (or diameter) of the particulate compositions is less than 20 pm or less than 10 pm. More preferably foe mean geometric diameter is leas than about 7 pm or 5 pm, even more preferably less than about 2-5 pm- Other preferred embodiments will comprise preparations wherein foe mean geometric diameter of foe particulate compositiDns is between about 1 pm and 5 pm hi especially preferred embodnnems the particulate compositions will comprise a powder of dry, hollow, parous mlctospherical shells of approximately 1 to 10 pm or 1 to 5 pm in diameter, with shell thictoasses of apptoxnnately 0.1 pm to approximately 0.5 pm It is a particular advantage of the present invention that the particulate concentration of the dispersions and structural matrix components can be adjusted to optimize the delivery characteristics of the selected particle «fee.

<img file="AU2006200768B2_D0014.tif" />

stabilized dispersiciss for use in pharma nemfoaj applications, it will be appreciated that the 20

2006200768 24 Feb 2006 particulate compositions and disclosed dispersions may be used for a number of nan pharmaceutical applications. That is, the present invention provides particulate compositions which have a broad range of applications where a powder is suspended and/or aerosolized. In particular, the present invention is especially effective where an 5 active or bioactive ingredient must be dissolved, suspended or solubilized as fast as possible. By increasing the surface area of the porous microparticles or by incorporation With suitable excipients as described herein, will result in an improvement in dispersibility, and/or suspension stability. fa this regard, rapid dispersement applications include, but are not limited to: detergents, dishwasher detergents, food sweeteners, condiments, spices, 10 mineral flotation detergents, thickening agents, foliar fertilizers, phytohormones, insect pheromones, insect repellents, pet repellents, pesticides, fungicides, disinfectants, perfumes, deodorants, etc.

The foregoing description will be more fully understood with reference to the following Examples. Such Examples, are, however, merely representative of preferred 15 methods of practicing the present invention and should not be read as limiting the scope of the invention. Example I

Effect of added calcium ions on the Tm of snrav-dried phospholipids

The effect of calcium ions on the gel-to-liquid crystal transition temperature (Tm) of spray-dried phospholipids was investigated. The resulting powders were examined visually for powder flow characteristics, characterized for Tm using a differential scanning calorimeter (DSC).

<img file="AU2006200768B2_D0015.tif" />

phosphatidylcholines, PCs (e.g., dipalmitoylphosphatidylcholine, DPPC or distearaylphosphatidylchnlme, DSPC) and varying amounts of calcium chloride were manufactured by an emulsion-based spray-drying process. Calcium levels were adjusted as mole ratio equivalents relative to the PC present, wiihCa/PC (mol/mol)=0to 1. Accordingly, g of saturated phosphatidylcholine (Genzyme Corp, Cambridge, MA) and 0 to 0.18 g of 30 calcium chloride dihydrate (Fisher Scientific Corp-, Pittsburgh, PA) were dispersed in approximately 40 mL of hot deionized water (T= 60-70° C) using an Ultta-Tunax T-25 mixer at 8,000-10.000ipm far 2 to 5 minutes. 18 g af perfhtorooctyl ethane, PFOE (FTech, Tokyo, Japan) was then added dropwise during mixing at a rate of 2-5 ml/mm. After the addition was complete, the emulsion was mixed for an addition period of not less than 4 35 minutes at 10,000 — 12JXX) tpm. The resulting coarse emulsion was then homogenized under

2006200768 24 Feb 2006 high pressure with an Avestm C-5 homogenizer (Ottawa, Canada) at 8,000-10,000 psi for 4 passes, and at 18,000-20,000 psi for a final pass.

The submicron fluorocarban-in-water emulsion was then spray-dried with a Bnchi B-191 Mini Spray-Drier (Flawil, Switzerland), equipped with a modified 2-fluid atomizer 5 under the fallowing conditions: inlet temperature = 85 °C; outlet temperature = 58°-61 ^eC; pump = 1.9 ml min'¹; atomizer pressure = 60-65 psig; atomizer flow rate = 30-35 cm. The aspiration flow (69-75%) was adjusted to maintain an exhaust bag pressure = 20-21 mbar.

The spray-dried phospholipid particles were collected using the standard Buchi cyclone separator. The volume-weighted mean geometric diameter (VMD) of tte dry 10 phospholipid particles was confirmed by laser diffraction (Sympatech Helos H1006,

Qavsthal-Zellerfeid, Germany), and ranged from 25 μπχ to 3.8 μιη depending on the formulation.

The resulting dry phospholipid particles were also characterized using a model 2920 DSC (TA Instruments) and by a Kad Fisher moisture analyzer. Approximately 05 to 2 mg 15 dry powder was weighed into aluminum sample pans and hermetically sealed. sample was analyzed using a modulated DSC mode under the following conditions: equilibration at 20 °C> and 2 °C/tamramp to 150 °C modulated +/-1 ®C every 60 sec. The phospholipid Tm was defined as the peak mavima of the first endothermic transition from reversing heat flow thermogram. For moisture analysis, approximately 50 mg powder was suspended in I 20 inLof anhydrous dimethylforanride (DMF). The suspension was teen injected directly into the titration cell and the moisture content was derived. The residual moisture content in the spray-dried DSPC particles is shown in Table la, and was found to decrease as a function of Ca/PC nude ratio. Tables lb and Ic present the Tm values for the various spray-dried FC particles as a function of the Ca/PC ratio. Hydrated DSPC and DPPC liposomes exhibit Tm values of 58 and 42 °C, respectively. Dramatic increases in Tm were observed following spray-drying, and with increases in calcium cantent. The powder formulations devoid of caldnm ions were highly cohesive, while the fonnulations incorporating added calcium were free-fiowing powders.

The present example illustrates that the hydration status of powdered phospholipid preparations greatly influences their inhoent thermodynamic and physicochemical characteristics, £.&amp;, Tmand flow properties. Increases in phospholipid Tm are believed to directly correlate with increases in thermal Stability, which could lead to an enhancement in long-term storage stability. In addition, moisture content may also lead to greater chemical stability.

2006200768 24 Feb 2006

Table la. Effect of Added Calcium on the Residual Moisture Content of SprayDried DSPC

<td> Ca/DSPC (mol/mol)</td><td> Water Content (%)</td>

<td> 0</td><td> 29</td>

<td> 025</td><td> 1.9</td>

<td> 050</td><td> 14</td>

Table lb. Effect of Added Calcium on the Tm of Spray-dried DSPC

<td> Ca/DSPC (mol/nrol)</td><td> TmCO</td>

<td> 0 (hydrated)</td><td> 58</td>

<td> 0</td><td> 79</td>

<td> 025</td><td> 85</td>

<td> 03</td><td> 98</td>

<td> 1.0</td><td> 126</td>

Table Ic. Effect of Added Calcium on the Tm of Spray-dried DPPC

<td> Ca/DPPC (moi/nwO</td><td> Tm(^eO</td>

<td> 0 (hydrated)</td><td> 42</td>

<td> 0</td><td> 63</td>

<td> 025</td><td> 69</td>

<td> 05</td><td> 89</td>

Example H

Effect of Added Magnesium Ions on Tm of Spray-dried Phospholipids

Phospholipid particles stabilized with magnesium ions were prepared by an emulsicm-based spray-drying rririmique. The emulsion feedstock was prepared according to the procedure described below, hi the first step, 045g of distearoyiphosphaddylcholine, 20 DSPC, and 0.126g magnesium chloride hexahydraxe (Fisher Scientific, Pittsburgh, PA) were dispersed in 41g of hot deionized water (T - 60 to 70°C) using an Ultra-Tunax mixer (model T-25) at 10,000 rpm far 2 mtn. 17g af perfluarooctyl ethane was then added drop wise at a rate of approximately 1-2 ml/min during mfring After the fluorocarbon addition was complete, the emulsion was mixed far an additional period of not less than 4 minutes.

The resulting coarse emulsion was then processed through a high pressure homogenizBr (Avestin, Ottawa, Canada) at 18,000 psi for 5 passes, to yield a suhnucron fluorocarbon-inwater emulsion stabilized by a n^cn'May’T of DSPC. The ounlsion was then spray-dried with a Buchi model B-I91 Mini Spray-Drier under the following spray conditions; aspiratioir^>99&, inlet temperainre=85^DC, outlet teraperamre=58°C, feed pump=1.9 mT-min^-1, and flow ra»s33 cmDiffei untial snanurtig calorimetric analysis of the dry particles revealed the Tm far the DSPC in the powder was 88°C as conqjared with 79“CfOT neat DSPC (Tahlelb). This

2006200768 24 Feb 2006 foregoing example illustrates the effect ions such as magnesium have upon the thermodynamic properties of dry phospholipid particles.

Example 411

Preparation of Snray.Dried Iaing Surfactant (ExoSurf⁸) Partides

Dry lung surfactant, particles having the ^1* components as ExoSurf® (GlaxoWellcome, Research Triangle Park. NC) were manufactured using a spray-drying process.

To achieve this end, the osmotic NaCl component of Exosurf was replaced m one fammlation by CaCl₂. Accordingly, 1.55 g of dipalnritoylpliDspbatidylcholine and 0.144g of calcium chloride dihydrate or sodium chloride were dispersed in 50 mL of hot deionized water (T=60-70 °C) using an Ultra-Tunax T-25 mixer at 8,000-10,000 rpm for 2 min, 185g of perfluorooctyl ethane was then added dropwise during mixing at a rate of 2-5 ml/nrin. After tije addition was complete, the emulsion was mixed for an additional period of not less than 4 minutes at 10,000-12^100 rpm. The resulting coarse emulsion was then homogenized under high pressure withan Avestin C-5 homogenizer (Ottawa, Canada) at 8,000-10,000 psi for 4 passes, and at 18,000 -20,000 psi for a final pass. In a separate flask, 0.12g of Tyloxapol® was dispersed in 10 g of hot deionized water (T=60-70 °C). The

Tyloxapol dispersion was then decanted into a vial that contained 0.174 g of cetyl alcohoL Hie vial was sealed and the cetyl alcohol was dispersed by placing it in a sonication bath for 15 minutes. The Tyloxapol/cetyl alcohol dispersion was added to the fluorocarbon emulsion and mixed for 5 min. The feed solution was then spray-dried with a Bucchi-191 Mini SprayDrier, equipped with a modified 2-fhtid atomizer under the following conditions? inlet temperature = 85 °C, outlet temperature = 58°-61 °C, pump = 1.9 ml mm*¹, atomizer pressure = 60-65 prig, atomizer flow rate = 30-35 cm. The aspiration flow (69-75%) was adjusted to maintain an exhaust bag pressure = 20-21 mbar. A free flowing white powder was collected using the standard Buchi cyclone separator.

The spray-dried powders were manually filled into a proprietary blister package and heat-sealed. The filling procedure was performed in a humidity controlled glove box (RH &lt; 2%). All blister packages were numbered, then weighed before and after filling to (feteuiiiiiB the amount of powder loaded. The filled blister packages were stared in a desiccating box operated at &lt; 2% RH until use. Tha powders were then tested for dispersibility from a DPI described in U.S. 5,740,794.

Emitted Dose testing of the formulations was assessed fallowing USP guidelines far inhalation products. The actuated dose was collected using a 1 - mtn*¹ flow rat* held far 2 seconds onto a type A/E glass filter (Gehnan, Aim Arbor, Ml)- The emitted dose was

2006200768 24 Feb 2006 calculated gravimerrically knowing the blister weight, total blister fin weight, and net change in filter weight.

Dry powder containing sodium chloride exhibited poor powder flow, and did net aerosolize well. In contrast, the formulation in which calcium chloride was substituted for

<img file="AU2006200768B2_D0016.tif" />

The differences in dispersibility between the two formulations is further reflected in the standard deviations of the emitted dose. Die foregoing example illustrates the ability of the present invention to alter and modulate the flow and emission properties of dry lipid particles through the inclusion of calcium ions.

Table XL Formulation of Highly Dispersible Dry Powder Lung Snrfa&lt;»tnn* Preparations

<td> Dry Powder Formulation</td><td> Ca/DSPC (mol/moi)</td><td> Emitted Dose (%)</td>

<td> Exosurf”</td><td> 0</td><td> 10 ±33</td>

<td> “Exosurf” + Calcium</td><td> 0.5</td><td> S7±3</td>

Example IV

Thermal Stability of Snrav-Dried Phospholipid Particles.

Ια the current example, the thermal stability of the spray-dried phospholipid particles prepared in example I were assessed. Accordingly 50 mg of powder was transferred into 20 mL glass vials and stored in a vacuum oven at 10Q°C for 1 hour. The volume-weighted mass median diameters (MMD) for the powders were determined using a SympaTech laser diffraction analyzer (HELOS HL006, Clansthal-Zeilerfeld, Germany) equipped with a RODOS type T4-1 vibrating trough. Approximately 1-3 mg of powder was placed in the powder feeder, which was subsequently atomized through a laser beam using bar of air pressure, 60 mbar of vacuum, 70% feed rate and 1.30 mm funnel gap. Dara was collected over an interval of 0,4 s, with a 175/im focal length, triggered at 1% obscuration. Particle size distributions were determined using a Fraflnhfifar model The volume-weighted mean aerodynamic diameters (VMAD) for the powders were with a model S050 Aerosizer*LD particle size analysis system (Amherst Process Instruments, Hadley, MA) equipped with an Aero-Sampler* chamber. Approximately 0-2 mg of powder was loaded into a specially designed DPI testing apparatus, fit this test, the powder was aerosolized by actuating a propellant can containing HFA-134a through the loaded sample chamber. The design of this apparatus is such to mumc artnatirm ftm an active DPI device and to offer some insight mtn powder flowability or its ability tn

2006200768 24 Feb 2006 deaggregaie. Narrow particle size distributions are preferred and are believed to be an indication of the powder's ability to deaggregate.

Table HI depicts the thermal stability and changes in particle size (MMD and VMAD) for the various spray-dried DSPC particles as a function of Ca/DSPC (mol/mol) 5 ratio. The thermal stability of the powders was found to increase with increasing calcium content. Significant structural and particle size changes were observed for the formulation devoid of calcium ions, as evidenced by particle sintering and large increases in MMD and VMAD. The addition of small amounts of calcium ions (Ca/DSPC = 0.25) resulting in a significant improvement in thermal stability erf the phospholipid particles. More surprising, 10 the spray-dried phospholipid formulation enriched at Ca/DSPC ratio of 0.5 completely tolerated the accelerated storage conditions, as no significant changes had occurred as a result of storage at 100’C for 1 hour. The above example further illustrates the enhanced thermal stability of spray-dried phospholipid particles afforded by the inclusion of calcium ions.

Table ΠΙ. Aerosol characteristics of Spray-Dried DSPC Powders following Storage at 100 °C fori hour

<td> Ca/DSPC (nuri/mol)</td><td> Tm (°C)</td><td> Thermal Stability</td><td> MMD₀(gm)</td><td> VMADo (gm)</td><td> MMD (gm)</td><td> VMAD (gm)</td>

<td> 0</td><td> 79</td><td> Sintering at5 min.</td><td> 33</td><td> 2.1</td><td> 5.7</td><td> Al</td>

<td> 0.25</td><td> 85</td><td> Sintering at 45 mm</td><td> 3.4</td><td> 1.B</td><td> 4.5</td><td> 2.1</td>

<td> 03</td><td> 98</td><td> No Change</td><td> 3.6</td><td> 1.7</td><td> 3.5</td><td> 1.8</td>

Example V

The Effect of Added Calcium Ions on pMDI Stability·

The objective of this study was to examine the effect added calcium had on the physical stability of lipid-based pMDI suspensions to moisture- Budesonide powders were prepared by spray-drying a feed solution comprised of micronized drug particles suspended in the aqueous phase of a fluarucarbcm-in-water emulsion. Accordingly, 0.8 g saturated egg 30 phosphatidylcholine (EPC-3, Lipoid KG, Ludwigshafen, Germany) was dispersed in approximately 80 mL hot deionized water (T=80° O using an Ultra-Turrax mixer at 8QQ0 rpm for 2 to 5 minutes 20g of perfluhran (φ = 0.09) was then added drop wise during muting. After the addition was complete, the errnjLsian was mixed far an additional period of not leSS than 4 mimttee The resulting cn^nat» mmlrion was hornngmim-d rmrW high prmuarrn 35 with an Avestin.C-5 homogenizer (Ottawa, Canada) at 18,000 psi for 5 passes. The resulting submicrun emnlsian was then combmed with a second aqueous phase contenting l-33g

2006200768 24 Feb 2006 budesonide suspended in a solution comprising 0 .4g d-Iactose monohydraie, and 0-0.134g calcium, chloride dissolved in approximately 30g of deionized water. The combined solution was then mixed using an Ultra-Turrax mixer ax 8000 rpm for 2 minutes to ensure dispersion of the budesonide particles. Hollow porous budesonide particles were prepared by spray5 drying the dispersion with a B-191 Mini Spray-Drier (Biichi, Flawil, Switzerland) under the following spray conditions: aspiration® 80%, inlet teurperatnre=85°C, outlet temperature=57°C, feed ptnnp^23 τηΤΛτπη, total air flow® 22.4 SCFM. Free flowing white powders were collected at the cyclone separator, Scanning electron microscopic (SEM) analysis showed the powders to be spherical and highly porous.

Approximately 40mg of spray-dried budesonide particles were weighed into 10 ml aluminum cans, and ciimp sealed (Pamasol 2005/10, Pfaffikon, Switzerland) with a DF30/50 ACT 50μ1 metering valve (Valois of Ammica- Greenwich, CT). The canisters were charged with 5 g HFA-134a (DuPont, Wilmington- DE) propellant by overpressure through the valve stem (Pamasol 8808). To elucidate differences between the budesonide formulations, propellant preparations that were spiked with varying amounts of water (0 to 1100 ppm) were utilized. The amount of the propellant in the can was determined by weighing the can before and after the fill. The final powder concentration in propellant was -0.8% w/w and formulated to provide a theoretical ex-valve dose of lOOgg budesonide per actuation. Powder dispersion was achieved by placing the canisters in a sonication bath for

15 min. The charged pMDIs were placed in quarantine for a period of 7 days at ambient conditions co allow the valve seals to seat

For the purpose of this study the aerosol fine particle fraction, FPF (% &lt;5.8 μιη) was used to assess changes in suspension physical stability that had occurred as a result of the water activity. The budesonide pMDIs were tested using commonly accepted pharmaceutical procedures. The method utilized was compliant with the United State Pharmacopeia (USP) procedure (Phaunacopeial Previews (1996) 22:3065-3098). After 5 waste shots, 20 doses from the test pMDIs were actuated into an Andersen Impactor. The extraction from all the plates, induction port, and actuator were performed in closed containers with an appropriate amount of mAthanobwater (1:1, v/v), The filter was

Budesonide was quantified by measuring the absorption at 245nm (Beckman DU640 spectrophotometer) and compared to an external standard curve with tbe extraction solvent as tbe blank. The FPF was calculated according to the USP method referenced above.

The effect of added calcium ions on tbe physical stability of the budesonide pMDIs 35 is depicted in Figure L The physical stability of the budesonide pMDIs was found to increase with increasing calcinm concentration. Smpxisingiy the tolerance of the budesonide pMDI

<img file="AU2006200768B2_D0017.tif" />

2006200768 24 Feb 2006 suspension to moisture increased from approximately 400 ppm to nearly 700 ppm by the inclusion of 4% calcium chloride into the formulation·

This example illustrates the enhanced stability of phospholipid-based pMDI particles afforded by the presence of calcium ions. The ability of a pMDI formulation to 5 tolerate increased levels of moisture will lead to an enhancement in their long-term storage stability. The presence of water fuels structural changes, which can lead to formation of liquid bridges between particles and/or recrystallization of components and changes in surface characteristics. The overall effect of moisture ingress for suspension pMDIs leads to particle coarsening and suspension instability, all of which can lead to product failure.

Example VI

The Effect of Added Calcium Ions cn Particle Morphology.

The objective of this Study was to examine the effect added calcium has upon the

<img file="AU2006200768B2_D0018.tif" />

micrographic (SEM) images of the spray-dried distearoylphosphatidylcholine particles prepared in example I were taken. The powders were placed on double sticky carbon graphite that was affixed on labeled aluminum stubs. The samples were then sputter-coated with a 250-300 A layer of gold/pafladimn. Samples were examined on a scanning electron microscope operated at an accelerating voltage of 20 Kev, and a probe current of 250 pAmps. Photomicrographs were digitally captured at a 20.000X magntfipatirm

The effect of calcium ion concentration tm the morphology of spray-dried DSPC partielea is iilustratpji in Figure IL Fortmtlatfims containing calcium ions had a highly ^e porous sponge-like inflated morphology, whereas the treat DSPC particles appeared melted and collapsed. The hollow porous morphology is characterized by powders that flow and aerosolize well, whereas the collapsed morphology results ία powders with poor flowability and dispersibility. No significant difference in morphology was observed as a result of calcium ion concentration, although the CaZDSPG? 0.25 formulation exhibited some degree of melted character as welL The decreased sensitivity of the powders with higher calcium content to melting and particle fusion is likely the result of the increased Tm values that allow far the powders to expert uryy a higher drying temj^rgtroT· while mmutnirnng the lipids in the gel state. The significant increases in Tm observed (Frampta j) lead to greater flexibility in spray-drying manufacture of these particles, and a significantly greater likelihood of achieving desired particle morphologies which are dependent on drying rates.

2006200768 24 Feb 2006

Example VII

Preparation of Snrav-PriedBudesoinde Partides.

Hollow porous budesonide particles were prepared by a two-step process. In the first step, 54mg of budesonide (Vmchem, Chatham, NJ.), and 0.775g of DSPC were dissolved in 2 ml of chlorofonn:niethaiiol (2:1). The chlorofornL'methaxml was then evaporated to obtain a thin film of the phospholipid/steroid mixture. The phospholipid/steroid mixture was then dispersed in 3O.5g of hot deionized water (T = 60 to

70°C) using an Ufaa-Turrax mixer (model T-25) at 8000 rpmfor 2 to 5 minutes. 12.8g of perfluoxwctyl ethane was then added dropwise during mixing. After the addition was complete, the emulsion was mixed for an additional period of not less than 4 minutes. The coarse emulsion was then passed through a high pressure homogenizer (Avestm, Ottawa,

Canada) at 18,000 psi for 5 passes. The resulting subnricrra fluorocarbon-in-water with steroid solubilized in the lipid monolayer surrounding the droplets was utilized as the feedstock in for the second step, Le. spray-drying on a B-I91 Mini Spray-Drier (Bilchi, Flawil, Switzerland). Calcium chloride (0 ar 0.65 mg) was added in 2^5g of water to the fluorocarbon-in-water emulsion immediately prior to spray drying. The following spray conditions were employed: aspiration^:100%, inlet cemperaturc=85°C, outlet temperaime=60^eC, feed pump=1.9 mL min'¹, Atomizer pressiirFj^0-65 psig, atomizer flow rate=30-35 cm. The aspiration flow (69-75%) was adjusted to maintain an exhaust bag . pressure of 30—31 inbar. Free flowing white powders were collected using a standard cyclone separator.

The resulting dry budesonide particles were characterized using DSC. Each sample was analyzed in a modulated DSC mode under the following conditions: eqmtihration at— 20°C. and 2 °C/min ranq> to 150“C modulated +1- 1°C every 60 sec. The phospholipid Tm was defined as the peak maxim» of the first endothermic tranritirm from each reversing heat flow thermogram. The phospholipid Tm for DSPC particles without added calcium is 79°C30 The addition of calcium ions in the budesonide foanulatian increased the Tm to 98’CL In addition, the powder formulations devoid of calcium had a cohesive flow character as compared to the caltriuntenriched fonnulation.

Ute aerosol characteristics of the calcium, containing formulation was examined in several passive dry pawdo^- inhaler devices (Eclipse®, Turbospin®, Cipla Rotahaler®, Glaxo 35 Rotahaler®®, and Hbvione FlowCaps® )- The emitted dose was determined gravimetrically at comfortable inhalation flow rate (peak flow rate=20-62L/min depending on tha resistance of the device), and at a forced inhalation flow rate (peak flow rate37-90Vmin). Under

2006200768 24 Feb 2006 comfortable inhalation flow conditions the range of emitted doses was between 89 tod 96% with a mean emitted dose of 94%. Under forced inhalation flow, the emitted dose varied between 94 and 103%, with a mean emitted dose of 99%· The feet that multiple devices with high and low resistance are able to effectively disperse the powders mure or less independent 5 of inspiratory flow rate speaks volumes to the dispersibility of the calcium contanring budesemde powder tested.

The above example further illustrates the ability of the present powder engineering technology to effectively modulate the Tmthrough fcrmnhtion changes. Increased (Tm’s) are desired as they often indicate increased physical stability and improved powder dispersibility.

Example VUI

Rapid Spreading of Sorav-Dcied DSPC Particlcs on an Air-Water Interface

The rapid spreading eharaetAnsties of the disclosed spray-dried phospholipid-based particles at the ak/warer interface are illustrated inFtg. HL Surface tension measurements were marte on a Knrg$ K12 tensiometer at 25^aC using the Wflhemey plate technique. To 20 measure surface tension, 20 mL of DI water ar DSPC liposome dispersion was placed in the thermostatic beaker. The platinum plate was taxed in the air and then dipped into the liquid and moved into the interface, after which measurements were taken. Far spray-dried DSPC particle analysis, measurements for DI water were made and confirmed to be 72 ± 1 mN/m. The glassware and plate were re-cleaned if the surface tension was not within 25 expectation. Approxinaiely 0.5 mg of dry DSPC crystal was sprinkled carefully onto the surface while the plate was dipped into the DI water. Measurements were started immediately after the powder was added. Care was taken to ensure dry powder did not adsorb to the plate. Measurements were ceased if any powder had contacted the plate surface. The equflibriumsurface tension of distearoylphosphatidylcholine (DSPC) is ca. 22 30 mN/m. Aqueous based DSPC liposomes adsorbed very slowly at the air/water interface as evidenced by the feet that after240 sec., the surface tension has not been significantly reduced. The alow adsorption far liposomes is due to the slow molecular diffusion of DSPC through the water phase, resulting from its extremely lew solubility in water. Surprisingly, the adsorption of DSPC in ths fonn of spray-dried DSPC particles is very fast, reducing the surface tension to equilibrium values within a few seconds. Moreover the inclusion of calcium ions had no effect cm the spreading of surfactant properties of the DSPC particles, This rapid spreading and reduction of surface tension is indicative of what would likely occur upon contacting the spray-dried phospholipid particles with a wetted

2006200768 24 Feb 2006 pulmonary membrane. Specifically, the present example provides a model for the effective deEvery of synthetic lung surfactants and drugs to the lung.

Example IX

Preparation of Nicotine Bitartrale Particles for pMDIsby Sorav-Drving

Hollow porous nicotine bitartnite particles were prepared by a spray-drying technique with a E-191 Mini Spray-Drier (Btichi, Hawil, Switzerland) under the following Spray conditions: aspiration: 80%, inlet temperature: 85°C; outlet temperature: 56°C; feed 10 pump: 2.3 mUuiin; air flow: 28 SCFM. The feed solution was prepared by mixing two solutions A and B immediately prior to spray drying.

Solution A: 5.2g of hot water (T= 50-60’C) was used to dissolve 0.60g of nicotine bitartrate (Sigma Chemicals, St Louis MO), 0.127g d-1 lactose (Sigma Chemicals, St Louis MO), and 90 mg calcium chloride dihydrate (Fisher Scientific, Fair Lawn, NJ).

Solution B: A fluorccarbon-in-water emulsion stabilized by phospholipid was prepared in the following manner The phospholipid, D.69g SPC-3 (Lipoid KG, Ludwigshafen, Germany) was dispersed in 29g of hot deionized water (T = 60 to 70°C) using an Ultra-Turrax mixer (model T-25) at 8000 ipm for 2 minutes (T = 6O-70°C). 30.2g of perfluorooctyl ethane (F-Tech, Japan) was added dropwise during mixing- After the fluorocarbon was added, the emulsion was mixed for a period of not less than 5 minutes ar 10000 rpm. The resulting coarse emulsion was then passed through a high pressure homogenizer (Avesdn, 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 geometric diameter of the nicotine bitarhate particles was confirmed by laser diffraction (Sympatnrh Helos H1006, Qansthal-Zellerfeld, Germany), where a volume weighted mean diameter (VMD) of 2.60^tm was found. Scarmmg electron microscopy (StM) analysis showed the powders to be spherical and parous. Differential scanning calorimetry analysis of the dry parties (TA Instruments) revealed the Tm for the . nicotine bitartrate in the powder to be 62°C, which is similar tn what is observed for spraydried neat materiaL

2006200768 24 Feb 2006

Eimnnie X

Prenararion of Phospholipid-Based Particles Containing Nicotine Bitertrate bv SpravDrying.

Hollow porous nicotine bitartrate panicles were prepared by a spray-drying technique with a B-191 Mini Spray-Drier (Biichi, Fiawii. Switzerland) under the following spray conditions: aspiration: 80%, inlet ternpBrntnre: 85°C; outlet temperature; ST’C; feed pump: 2.3 mL/min; total air flow: 22.4 SCFM.

A fluorocarbon-in-water emulsion stabilized by phospholipid was first prepared.

The phospholipid, 0.45g SPC-3 (Lipoid KG, Ludwigshafen, Germany), was homogenized in 30g of hot deionized water (T - 60 to 70®C) using an Ultra-Turrax mixer (model T-25) at 8000 rpm for 2 (T «= 60-70’C). 15g of perfluorooctyl ethane (F-Tecfa, Japan) was added (hopwise at a rate of approximately 1-2 ml/mm during mixing. After the fluorocarbon was added, the emulsion was mixed far a period of not less than 4 minutes. The resulting coarse emulsion was then processed through a high pressure homogenizer (A vestin, Ottawa, Canada) at 18,000 psi for 5 passes.

The emulsion was decanted into a beaker containing 8 mg sodium phosphate monobasic (Spectrum Chemicals, Gardena. CA) and 90 mg calcium chloride dihydrate (Fisher Scientific, Fair Lawn, NJ). The emulsion was allowed to stir for approximately 5 min. The emulsion was then dacantad into a haaker containing 0.225g nicotine bitartrate (Sigma Chemicals, St Louis MO) and was stirred for 5 minntas. The feed solution was fed into the spray-dryer under the conditions described above. A free flowing white powder was collected at the cyclone separator. The nicotine bitartrate particles had a volume-weighted mean aerodynamic diameter of 1.47 gm as determined by 25 a time-of-flight analytical method (Aerosizer, Amherst Process Instruments, Amherst,

MA). The geometric diameter of the nicotine brtartrate particles was detennmed by laser diffraction (Sympaiech Helos H1006, CUusthal-ZeUcafeld, Germany), where a volume weighted mean diameter (VMD) of 2-95/rm was found. Scanning electron microscopy

<img file="AU2006200768B2_D0019.tif" />

calorimetry analysis of the dry particles (TA Instruments) revealed the Tm for the nicotine

<img file="AU2006200768B2_D0020.tif" />

<img file="AU2006200768B2_D0021.tif" />

technology to effectively modulate the Tmthrough formulation ^hang^c

2006200768 24 Feb 2006

Example XI

The preparation of lung surfactant powders with and without the use of blowing agents was investigated. The resultant powders were characterized as to aerosol properties.

Preparation of powders

The annex solutions were prepared by wiring calcium or sodium chloride, cetyl alcohol, tyloxapol (Sigma), and Infasurf (ONY Inc.) as described in Table IV in a 20ml 10 glass vial to which was added an amount of hot deionized water (70° C) (approximately

0.54 g of sodium chloride were used instead of calcium chloride in lots 1S43-HSO3 and 04). The mixture was vortexed until all solids were fully dissolved. One lot, 1843-HS-04 used 200 ml ethanol as a solvent

The emulsions were prepared by adding DPPC into a beaker to which was added an amount of 70° C deionized water. The mixture was mixed in a mixer on low speed for approximately 2-3 minutes. When a blowing agent was used, PFOE was weighed out into a small flask and added dropwise into the DPPC/water mixture. The PFOE was added slowly, over the course of 1-2 minutes and the mixture was then allowed to continue mixing for an additional 1-2 minutes. Emulsion detail; are listed in Table IV.

When a blowing agent was used, the DPPC/water/PFOE mixture was then immediately removed from the mixer and run thmpgh a homogenizer four time» at 10,000 —13,000 psi. The sample was then run through a homogenizer a fifth time at 13,000 — 17,000 psi.

The annex solution was then added to the DPPC/water ar DPPC/water/PFOE emulsion with continued stirring an a hot plate set to the lowest temperature. The mixtures were kept at approximately 50° C during spray drying, whirf, was done at the conditions listed in Table V.

2006200768 24 Feb 2006

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2006200768 24 Fe

Table V Aerosol GfflraHsisws

<td> Lots</td><td> VfeH</td><td> Uftt Caa</td><td> ffl Weight</td><td> % Molduro</td><td></td><td> so</td><td> 'ifEC</td><td> %bBft</td><td> % Colh*t«i</td><td> MMAD</td><td> in</td><td> earn· steer</td><td> MND fSVM=&gt;A)</td>

<td> 1M3+&amp;O1</td><td> 430</td><td> 2</td><td> 22</td><td> 4Λ83</td><td> 837</td><td> 215</td><td> 25</td><td> zm</td><td> 90.B1</td><td> 343</td><td> 46</td><td> 1287</td><td> 2^3</td>

<td> 1843H&amp;O3</td><td> aao</td><td> No CaQ</td><td> 22</td><td> 2205</td><td> 10</td><td> 328</td><td> 327</td><td> 17.98</td><td> 1380</td><td> £5</td><td> 14</td><td></td><td></td>

<td> 1843+&amp;04</td><td> iao</td><td> No Caa</td><td> ao</td><td> 153S</td><td> 72</td><td> 218</td><td> 30.0</td><td> 3054</td><td> 1089</td><td></td><td></td><td></td><td></td>

<td> 164346®</td><td> 354</td><td> 2</td><td> 6Λ</td><td> 5503</td><td> Θ62Ε</td><td> 7.15</td><td> 106</td><td> 4.05</td><td> 69.15</td><td> 365</td><td> 44</td><td></td><td></td>

<td> 184SH&amp;2G</td><td> SS4</td><td> 2</td><td> 22</td><td></td><td> 8244</td><td> 4.11</td><td> so</td><td> 11-07</td><td> ffl.70</td><td> 2»</td><td> 57</td><td></td><td></td>

<td> 1843H&amp;3S</td><td> 335</td><td> 578</td><td> 22</td><td> 3722 2ffiKF</td><td> 81.63</td><td> 304</td><td> 3.7</td><td> 39</td><td> 8496</td><td> 4®</td><td> 29</td><td> 2683/ 2950</td><td> W</td>

<td> 1B43+&amp;38</td><td> 290</td><td> asr</td><td> 22</td><td> aoi</td><td> tn®</td><td> 2.93</td><td> 35</td><td> 325</td><td> 68,16</td><td> 4.14</td><td> 33</td><td> 2799</td><td> 393</td>

<td> 1843H&amp;BD</td><td> 27.3</td><td> 2</td><td> rad «Bat</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td> 1843HM1</td><td> 407</td><td> 2</td><td> 22</td><td> 33M</td><td> 8177</td><td> 283</td><td> 33</td><td> am</td><td> 9437</td><td> 3S2</td><td> 51</td><td></td><td></td>

<td> 1843FS51</td><td> 40.7</td><td> 2</td><td> 80</td><td></td><td> 7&amp;09</td><td> £33</td><td> 7.0</td><td> 4.19</td><td> 79.41</td><td> 385</td><td> 42</td><td></td><td></td>

<td> 184&amp;H&amp;85</td><td> 58.0</td><td> 5.70</td><td> 22</td><td> 209</td><td> 6787</td><td> 10.57</td><td> 137</td><td> £66</td><td> 71.85</td><td> 345</td><td> 46</td><td></td><td></td>

<td> 1848H&amp;S4</td><td> 11.5</td><td> 1.78</td><td> not dad</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td> 1B4344&amp;67</td><td> 57.8</td><td> 1</td><td> not ffled</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td> 18434/369</td><td> 11.6</td><td> 1</td><td> not filed</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td> 1S4344S-7O</td><td> 40.4</td><td> 1</td><td> 22</td><td> 1S4</td><td> 5455</td><td> 505</td><td> 93</td><td> &amp;a</td><td> 5354</td><td> 351</td><td> 45</td><td></td><td></td>

<td> 1848445-73</td><td> &amp;o</td><td> 340</td><td> 22</td><td> 2237, 2B9KF</td><td> 6937</td><td> 11.72</td><td> 188</td><td> 273</td><td> 71.77</td><td> 374</td><td> 39</td><td> 307</td><td> a®</td>

<td> ΙβΙΒΗ&amp;βΒ</td><td> 61Λ</td><td> 3J57</td><td> 22</td><td> BIOS)</td><td> B3.71</td><td> 889</td><td> 104</td><td> 7ΛΤ</td><td> 9045</td><td> asr</td><td> 45</td><td></td><td> 27S28Q /274</td>

<td> ιπ&amp;Η&amp;βο</td><td> ss</td><td> 357</td><td> 22</td><td> 4871</td><td> SUB</td><td> 4.16</td><td> 45</td><td> 367</td><td> 9367</td><td> 294</td><td> 58</td><td></td><td> 21® 210</td>

<td></td><td> 47.3</td><td> 357</td><td> 22</td><td> 5066</td><td> east</td><td> 29</td><td> 28</td><td> £13</td><td> 9340</td><td> 328</td><td> 9</td><td></td><td> 2Ή</td>

<td> 19S9+&amp;38</td><td> 394</td><td> 357</td><td> 22</td><td> 285B</td><td> ease</td><td> 10.77</td><td> 155</td><td> 269</td><td> 71®</td><td> 373</td><td> 41</td><td></td><td> 2£4/ am</td>

<td> 19594S33</td><td> 728</td><td> 2</td><td> 2?</td><td></td><td></td><td> 30B</td><td> 37</td><td> aaa</td><td> 8646</td><td> 363</td><td> 43</td><td></td><td> 2gaf 267</td>

<td> TO5M&amp;S0</td><td> 238</td><td> 2</td><td> rot filled</td><td> 3.190</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td> 166WB5I</td><td> 727</td><td> 2</td><td> 22</td><td> 4.964</td><td> flam</td><td> 18B</td><td> 2</td><td> 211</td><td> 835</td><td> 371</td><td> 43</td><td></td><td> 244</td>

2006200768 24 Feb 2006

Example ΧΤϊ

<img file="AU2006200768B2_D0024.tif" />

<img file="AU2006200768B2_D0025.tif" />

A single feed solution is prepared Under defined conditions. The feed solution is comprised of leuprolide acetate in the aqueous phase of a fhiorocarban-in-watBT emulsion. The emulsion composition is listed in Table VI below. Accordingly, DSPC and calcium chloride dihydrate are dispersed in approximately 400 mL SWFI (T=60 - 70 C) using an Ultm-Tunax T10 50 mixer at SOOQtpm for 2 to 5 minutes. The perfhihron is then added drop wise during mixing·

After the addition is complete, the emulsion is mixed for an additional period of not less than 5 minutes at 10,000 rpm. The resulting coarse emulsion is then homogenized under high pressure with an Avestin. C-5 homogenizer (Ottawa, Canada) at 19,000 psi for 5 discrete passes. The emulsion is transferred to the Potent Molecule Laboratory for Leuprolide Acetate addition and 15 spray drying.

Table VI.

Leuprolide Acetate Emnlskm Composition

<td> Emulsion Comuonents</td><td> Amount (grams)</td><td> % solids</td>

<td> DSPC</td><td> 7 33</td><td> 73%</td>

<td> Calcium Chloride</td><td> 0.67</td><td> 7%</td>

<td> Btrflnbrou</td><td> 200</td><td> NA</td>

<td> SWFI</td><td> 400</td><td> NA</td>

<td> Leuprolide Acetate</td><td> 2.00</td><td> 20%</td>

Aerosol Data:

Deposition analysis is performed using a multi-stage liquid impinger (MSU). The apparatus consists of four concurrent stages and a terminal filter, containing an aliquot at appropriate solvent for Leuprolide Acetate analysis. Deposition and emission dap* is reported in Table VHbelow.

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Tableau.

Leonrolide Acetate Aerosol Data

<td></td><td></td>

<td> Lot#</td><td> XB2316</td>

<td> Device</td><td> Tnrbospin</td>

<td> Flow Rate</td><td> 60 Lum</td>

<td> Emitted Dose</td><td> 96%</td>

<td> n=</td><td> 20</td>

<td> MM AD</td><td> 2.40</td>

<td> S4-Filter</td><td> 70%</td>

<td> n™</td><td> 4</td>

Example VUE

PTH Feed Solution Preparation

A single feed solution is prepared under defined conditions. The feed solution is comprised of parathyroid hormone in the aqueous phase of a fluorocarban-in-water emnimon The emulsion composition is listed in Table VIE below. Accordingly, DSPC and calcium chloride dihydrate are dispersed in approximately 40mL SWFI (T=60 - 70 C) using an UltraTunax T-50 mixer at SOOOrpm for 2 to 5 reroutes. The perfhtorooctylelhane is then added drop wise during mixing. After the addition is complete, the emulsion is mixed for an additional period of not less than 5 urinates at 10,000 rpm. The resulting coarse emulsion is then homogenized under high pressure with an Avestin C-5 homogenizer (Ottawa, Canada) at 19,000 psi for 5 discrete passes. The active drag is added to the emulsion and subsequently spray dried after mixing for a period of not less than 10 minntas.

<img file="AU2006200768B2_D0026.tif" />

<td> Brnnlsion Components</td><td> Amount (grams)</td><td> % solids</td>

<td> DSPC</td><td> 0.825</td><td> 82.5%</td>

<td> Calcium Chloride</td><td> 0.075</td><td> 73%</td>

<td> FerfiuaraoctytetiianetPFOK)</td><td> . ............2«</td><td> NA</td>

<td> SWFI</td><td> 40</td><td> NA</td>

<td> Parathyroid Honnone</td><td> 0.100</td><td> 10%</td>

Aerosol Data:

Deposition analysis is performed using an Anderson Cascade Impactor. The apparatus consists of seven concurrent stages and &amp; terminal filter. Aerosol deposition is measured gravimetrically and is repotted in Table IX below.

2006200768 24 Feb 2006

Table ΙΧ.

Parathyroid Hormone Aerosol Data

Inhalers

<td></td><td></td>

<td> Lots</td><td> 2193-1</td>

<td> Device</td><td> Turbospin</td>

<td> Flow Rate</td><td> 30Lpm</td>

<td> MMAD</td><td> 2.67</td>

<td> S44Filter</td><td> 59%</td>

<td> o=</td><td> 2</td>

Example XIV

Preparation of Metered Dose

Containing Nicotine Bitartrate Particles

50mg of nicotine bitartrate particles prepared in Examples IX, and X were weighed, into 10 ml aluminum cans, crimp sealed a DF30/50 RCU-20cs 50(41 valve (Valois of America, Greenwich, CT) and charged with HFA-134a (DuPont, Wilmington, DE) propellant by overpressure through the stem. A Pamasol (Pfaffikou, Switzerland) model 2005 small scale production plant complete with a model 2008 propellant pump was used for this purpose. The amount of the propellant in the can was dptw mined by weighing the can before and after the fin. The final powder concentration in propellant was 0.5 9b w/w and formulated to provide an approximate emitted dose of 110 pg nicotine bitartrate.

FbramnleXV

AndetserilniDactor Test for Assessing Nicotine Bit

a.pMDl Performance

The MDIs were tested using commonly accepted pharmaceutical procedures- The 25 method utilized was compliant with tbs United State Pharmaanpeta (usp) procedure (Rjaimacopeial Previews (1996) 223065-3098) incorporated herein by reference. After 5 waste shots, 20 doses tan the test pMDIs were acm atari into an Andersen Impactor.

Extraction procedure. The extraction from all the plates, induction part, and actuatar were performed in closed container with an appropriate amount of mstfaanokwater (1:1, v/v). 30 The filter was installed but not assayed, because the polyacryiic binder interfered with ths analysis. The mass balance and particle dy. digtrifrutfon trends radicated that the deposition on the filter was negligibly «mall.

Ommtitntian -procedure. Nicotine bitanrate was quantitated by measuring the absorption at 258nm (Beckman DU640 spectrophotometer) and compared to an external 35 standard curve with the extraction solvent as the blank.

Calculation procedure. Far each MD1 the mass of the drug in the stem (component 3), actuator (-2), induction port (-1) and plates (0-7) were quantified as described above. The

2006200768 24 Feb 2006

Fine Particle Dose and Hue Particle Fraction was calculated according to the USP method referenced above. Throat deposition was defined as the mass of drug found in the induction part and on plates 0 and 1. The mean mass aerodynamic diameters (MMAD) and geometric standard diameters (GSD) were evaluated by fitting the experimental cumulative function with 5 log-normal distribution by using two-parameter fitting routine. The results of these experiments are presented in subsequent examples.

ExamuleXVI

Andersen Cascade Impactor Results for Nicotine Bitartrate uMDI Formulations

The results of the cascade impactor tests for the nicotine bitartrate pMDIs prepared according to Example Xiv are shown below in Table X.

Table X...

<td colspan="4"> Nicotine Bitartrate -pMDIs</td>

<td></td><td> MMAD (GSD) pm</td><td> Fine particle fraction. Φ</td><td> Fine Particle Duse, its</td>

<td> Nfcotine/SPC-3/CaCy Lactose</td><td> 3.6 (2D)</td><td> 70</td><td> 74</td>

<td> Nicotine/SPC-i/CaC]?/ NaPbosphate</td><td> 3.0 (l-9&gt;</td><td> 73</td><td> 80</td>

Bath pMDI preparations were observed by visual inspection to have excellent suspension stability, where little or no creaming or sedimentation occurred over 1 hour. The lactose containnig formulations had a slightly larger MMAD and lower FPF and HD as 20 compared with the sodium phosphate formulation. The reduction in aerosol far the lactose fannnlaiion could be due to increased water content as evidenced in the reduced Tm.

2S

The invention has now been rtMnrihwi rn detail far purposes of clarity and understanding. However, it win be appreciated that certain changes and modifications may be practiced within die scope of ths appended claims.

2006200768 24 Feb 2006

In the claims which follow and in the preceding description of the invention, except where the context requires otherwise due to express language or necessary implication, the word “comprise” or variations such as “comprises” or “comprising” is used in an inclusive sense, i.e. to specify the presence of the stated features blit not to preclude the presence or addition of further features in various embodiments of the invention.

It will be clearly understood that, although a number of prior art publications are referred to herein, this reference does not constitute an admission that any of these documents forms part of the common general knowledge in the art, in Australia or in any other country.

H: \HaraF\Keep\Spec 03? . doc 23/02/06

2006200768 24 Feb 2006