NZ619041B2 - Inhalable Coenzyme Q10 formulations and methods of use thereof - Google Patents

Abstract

Inhalable pharmaceutical compositions can include an aqueous dispersion of particles including a hydrophobic bioactive agent in particular Coenzyme Q10 (ubiquinone) suitable for continuous aerosolization. Due to their chemical composition and methods of manufacture, the pharmaceutical compositions exhibit distinctive physicochemical properties that provide advantageous aerosol transmission and output. the compositions comprises: a dispersion of liposomal particles having an average diameter between about 30 and 500 nm, each liposomal particle comprising a hydrophobic bioactive agent, a phospholipid selected from dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), dimyristoylphosphatidylcholine (DMPC), or a combination thereof, and an aqueous dispersion vehicle, wherein the ratio of hydrophobic bioactive agent phospholipid is between about 5:1 and about 1:5, the hydrophobic bioactive agent is between about 0.1 and 30 % w/w of the composition, the phospholipid is between about 0.1 and 30 % w/w of the composition, and the liposomal particles are dispersed within the aqueous dispersion vehicle xhibit distinctive physicochemical properties that provide advantageous aerosol transmission and output. the compositions comprises: a dispersion of liposomal particles having an average diameter between about 30 and 500 nm, each liposomal particle comprising a hydrophobic bioactive agent, a phospholipid selected from dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), dimyristoylphosphatidylcholine (DMPC), or a combination thereof, and an aqueous dispersion vehicle, wherein the ratio of hydrophobic bioactive agent phospholipid is between about 5:1 and about 1:5, the hydrophobic bioactive agent is between about 0.1 and 30 % w/w of the composition, the phospholipid is between about 0.1 and 30 % w/w of the composition, and the liposomal particles are dispersed within the aqueous dispersion vehicle

Description

BLE COENZYME Q10 FORMULATIONS AND METHODS OF USE THEREOF AH26(11154837_1):LNB conventional techniques for delivery of agents to the lung can be ineffective, inefficient, and/or insufficient. For example, many known methods e aerosols that have ts that are two large to deliver a pharmaceutical to the lung, and/or that are too inconsistent to reliably deliver a specific dose. Particle formation technologies developed to address issues such as particle size, for example mechanical micronization processes and on-based phase separation processes, can have additional limitations.

Mechanical micronization s such as g can cause thermal and/or mechanical degredation of the pharmaceutical. Spray drying, r method used to micronize drug nces, can lead to difficulty in collecting small particles.

SUMMARY OF THE INVENTION The invention provides inhalable pharmaceutical compositions having an aqueous sion of particles including a hydrophobic bioactive agent. Due to their chemical composition and methods of manufacture, the ceutical compositions exhibit distinctive physicochemical properties that provide advantageous aerosol transmission and , including continuous aerosolization. Accordingly, the invention provide improved methods for the treatment of diseases, including cancer, and compositions capable of delivering bioactive agents to aid in the treatment of diseases and other conditions, including by inhalation to the lungs.

Since a large amount of the available surface area of the lung is located in the deep lung, drug delivery can be facilitated by aerosol delivery of particles to the peripheral alveoli of the deep lung. In contrast, particles deposited in the upper respiratory tract can be rapidly removed by the mucociliary escalator, uently transported to the throat, and swallowed or removed by coughing. The invention, in various aspects and embodiments provides for the delivery of hydrophobic bioactive agents (e. g., including drugs that are strictly hydrophobic, ilic, and/or poorly water soluble), which are generally difficult to adequately aerosolize, to the deep lung (as well as other regions of the respiratory . In particular, the invention can provides for the continuous zation of nanodispersions of hydrophobic drugs for therapeutic use.

Other advantages of the various aspects and embodiments of the invention include, but are not limited to, high aerosol output (e. g., as ed by total aerosol [0007a] According to a first aspect of the invention there is provided an inhalable pharmaceutical composition comprising a dispersion of particles suitable for continuous aerosolization, wherein the particle is a liposome and/or otherwise stabilized er with a olipid, the composition comprising: a sion of particles having an average diameter between about 30 and 500 nm, each particle sing a hydrophobic bioactive agent, a phospholipid, and an aqueous dispersion vehicle, wherein the ratio of hydrophobic bioactive phospholipid is between about 5:1 and about 1:5, the hydrophobic bioactive agent is between about 0.1 and 30 % w/w of the composition, the phospholipid is between about 0.1 and 30 % w/w of the composition, and the particles are dispersed within the aqueous dispersion vehicle, wherein the hydrophobic bioactive agent comprises coenzyme Q10 (CoQ10); wherein the olipid comprises dipalmitoylphosphatidylcholine , distearoylphosphatidylcholine (DSPC), dimyristoylphosphatidylcholine (DMPC), or a combination f; and wherein, upon administration to a subject, the composition is characterized by continuous aerosolization sufficient to provide a therapeutic dose of the hydrophobic bioactive agent to the subject. [0007b] According to a second aspect of the ion there is provided an inhalable pharmaceutical composition comprising a dispersion of particles suitable for continuous aerosolization, wherein the particle is a liposome and/or otherwise stabilized together with a phospholipid, the composition comprising: a dispersion of particles having an average diameter between about 30 and 300 nm, each particle comprising CoQ10, DPPC, and an aqueous sion vehicle, wherein the ratio of CoQ10:DPPC is between about 5:1 and about 1:5, the CoQ10 is between about 0.1 and 6 % w/w of the composition, and the particles are dispersed within the aqueous sion vehicle, and wherein, upon AH26(11154837_1):LNB administration to a subject, the composition is characterized by continuous aerosolization sufficient to provide a therapeutic dose of the hobic bioactive agent to the subject. [0007c] According to a third aspect of the invention there is provided an inhalable pharmaceutical composition comprising a sion of particles suitable for continuous aerosolization, wherein the particle is a liposome and/or otherwise stabilized together with a olipid, the composition comprising: a dispersion of particles having an average diameter between about 30 and 300 nm, each particle comprising CoQ10, DSPC, and an aqueous dispersion vehicle, wherein the ratio of CoQ10:DSPC is between about 5:1 and about 1:5, the CoQ10 is between about 0.1 and 6 % w/w of the composition, and the particles are dispersed within the aqueous dispersion e, and wherein, upon administration to a subject, the ition is characterized by continuous aerosolization sufficient to e a therapeutic dose of the hydrophobic bioactive agent to the subject. [0007d] According to a fourth aspect of the ion there is provided an inhalable pharmaceutical composition comprising a dispersion of particles suitable for continuous aerosolization, wherein the particle is a liposome and/or otherwise stabilized together with a phospholipid, the composition comprising: a dispersion of particles having an average er between about 30 and 300 nm, each particle comprising CoQ10, DMPC, and an aqueous dispersion vehicle, wherein the ratio of CoQ10:DMPC is between about 5:1 and about 1:5, the CoQ10 is between about 0.1 and 6 % w/w of the composition, and the particles are dispersed within the s dispersion vehicle, and wherein, upon administration to a subject, the composition is characterized by continuous aerosolization sufficient to provide a therapeutic dose of the hydrophobic bioactive agent to the subject. [0007e] According to a fifth aspect of the invention there is provided a method for ing an inhalable pharmaceutical composition comprising the steps of: hydrating a phospholipid, thereby forming a hydrated olipid; mixing the hydrated phospholipid, a hydrophobic bioactive agent, and an aqueous dispersion vehicle, y producing a e; and homogenizing the e, thereby producing a dispersion of particles comprising the phospholipid and hydrophobic bioactive agent dispersed within the aqueous dispersion vehicle and having an average diameter between about 30 and 500, wherein the ratio of hydrophobic bioactive agent:phospholipid is between about 5:1 and about 1:5, the hydrophobic bioactive agent is between about 0.1 and 30 % w/w of the composition, and the phospholipid is between about 0.1 and 30 % w/w of the composition, wherein the particle is a me and/or ise ized er with a AH26(11154837_1):LNB olipid; n the hobic bioactive agent comprises coenzyme Q10 (CoQ10); wherein the phospholipid comprises dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine , dimyristoylphosphatidylcholine , or a combination thereof; and wherein, upon administration to a subject, the composition is characterized by continuous aerosolization sufficient to e a therapeutic dose of the hobic bioactive agent to the subject. [0007f] According to a sixth aspect of the invention there is provided the use of an inhalable composition for the preparation of a medicament for the treatment of a lung disease wherein said composition is formulated for administration to a subject by: aerosolizing a dispersion of particles, thereby forming a respirable aerosol comprising a plurality of droplets having a mass median aerodynamic diameter (MMAD) between about 1 and 5 µm, wherein the sion of particles has an average diameter between about 30 and 500 nm, each particle comprising a hydrophobic bioactive agent and a phospholipid dispersed within an aqueous dispersion vehicle, wherein the ratio of hydrophobic bioactive agent:phospholipid is between about 5:1 and about 1:5, the hydrophobic bioactive agent is between about 0.1 and 30 % w/w of the composition, and the phospholipid is n about 0.1 and 30 % w/w of the composition, n the particle is a liposome and/or otherwise stabilized er with a phospholipid; wherein the hydrophobic bioactive agent comprises coenzyme Q10 (CoQ10); wherein the phospholipid comprises itoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), dimyristoylphosphatidylcholine (DMPC), or a combination thereof; and n the composition is formulated for continuous aerosolization sufficient to deliver a therapeutic dose of the hydrophobic bioactive agent to the subject.

In another aspect, the invention features an inhalable pharmaceutical composition comprising a dispersion of liposomal particles suitable for continuous aerosolization. The composition includes a dispersion of liposomal particles having an average diameter n about 30 and 500 nm, each liposomal particle comprising a hydrophobic bioactive agent, a phospholipid, and an aqueous dispersion vehicle. The ratio of hydrophobic bioactive agent:phospholipid is n about 5:1 and about 1:5, the hydrophobic bioactive agent is between about 0.1 and 30 % w/w of the composition, the phospholipid is n about 0.1 and 30 % w/w of the composition, and the liposomal particles are dispersed within the aqueous dispersion vehicle. And, upon administration to 11259033 a subject, the composition is characterized by continuous aerosolization sufficient to provide a therapeutic dose of the hydrophobic ive agent to the subject.

AH26(11154837_1):LNB In another aspect, the invention features, an inhalable pharmaceutical composition comprising a sion of liposomal particles suitable for continuous aerosolization. The composition includes a dispersion of liposomal les having an average diameter between about 30 and 500 nm, each liposomal particle comprising a hobic ive agent, a phospholipid, and an aqueous dispersion vehicle. The ratio of hydrophobic bioactive agent:phospholipid is between about 5:1 and about 1:5, the hobic bioactive agent is between about 0.1 and 30 % w/w of the composition, the phospholipid is n about 0.1 and 30 % w/w of the composition, and the liposomal particles are dispersed within the aqueous dispersion vehicle. And, upon continuous aerosolization, the composition is capable of achieving a total emitted dose (TED) of at least about 2,900 ug over 15 seconds.

In still another aspect, the invention features an inhalable pharmaceutical composition sing a dispersion of liposomal particles suitable for continuous lization. The composition includes a dispersion of liposomal particles having an average diameter between about 30 and 300 nm, each mal particle comprising CoQ10, dipalmitoyl phosphatidylcholine (DPPC), and an aqueous dispersion vehicle.

The ratio of CoQ10:DPPC is between about 5:1 and about 1:5, the CoQ10 is between about 0.1 and 6 % w/w of the composition, and the liposomal particles are dispersed within the aqueous dispersion vehicle. And, upon administration to a subject, the composition is characterized by continuous aerosolization ient to provide a therapeutic dose of the hydrophobic bioactive agent to the subject (or, alternatively, the composition can be characterized by another pharmacokinetic property such as being capable of achieving a ive agent concentration of at least about 500 ug/g wet lung tissue or a total emitted dose (TED) of at least about 2,900 ug over 15 seconds).

In yet another aspect, the invention features an inhalable pharmaceutical composition comprising a dispersion of liposomal particles suitable for continuous aerosolization. The ition includes a dispersion of mal les having an average diameter between about 30 and 300 nm, each liposomal particle comprising CoQ10, distearoyl phosphatidylcholine , and an aqueous dispersion vehicle. The ratio of CoQ10:DSPC is between about 5:1 and about 1:5, the CoQ10 is between about 0.1 and 6 % w/w of the composition, and the liposomal particles are dispersed within the aqueous dispersion vehicle. And, upon administration to a subject, the composition is characterized by continuous aerosolization sufficient to provide a therapeutic dose of the hydrophobic bioactive agent to the subject (or, alternatively, the composition can be characterized by another pharmacokinetic ty such as being capable of achieving a bioactive agent concentration of at least about 500 ug/g wet lung tissue or a total emitted dose (TED) of at least about 2,900 ug over 15 seconds).

In still yet another aspect, the invention features an inhalable pharmaceutical composition comprising a dispersion of liposomal particles suitable for continuous aerosolization. The ition includes a dispersion of liposomal particles having an average diameter between about 30 and 300 nm, each liposomal particle comprising CoQ10, dimyristoyl phosphatidylcholine (DMPC), and an aqueous dispersion vehicle.

The ratio of DMPC is between about 5:1 and about 1:5, the CoQ10 is between about 0.1 and 6 % w/w of the composition, and the liposomal particles are dispersed within the aqueous dispersion vehicle. And, upon administration to a subject, the composition is characterized by continuous aerosolization sufficient to provide a eutic dose of the hydrophobic ive agent to the subject (or, alternatively, the ition can be characterized by another pharmacokinetic property such as being capable of achieving a bioactive agent concentration of at least about 500 ug/g wet lung tissue or a total emitted dose (TED) of at least about 2,900 ug over 15 seconds).

In still another aspect, the invention features a method for preparing an inhalable pharmaceutical composition. The method includes the steps of: (i) hydrating a phospholipid, thereby forming a ed olipid; (ii) mixing the hydrated phospholipid, a hydrophobic ive agent, and an aqueous dispersion vehicle, thereby producing a mixture; and (iii) nizing the e, thereby ing a dispersion of liposomal particles comprising the phospholipid and hydrophobic bioactive agent dispersed within the aqueous dispersion vehicle and having an e er between about 30 and 500. The ratio of hydrophobic bioactive agent:phospholipid is between about 5:1 and about 1:5, the hydrophobic bioactive agent is between about 0.1 and 30 % w/w of the composition, and the phospholipid is between about 0.1 and 30 % w/w of the composition. And, upon administration to a subject, the composition is characterized by continuous lization sufficient to provide a therapeutic dose of the hydrophobic WO 74559 2012/043001 bioactive agent to the subject (or, alternatively, the composition can be characterized by another pharmacokinetic property such as being capable of achieving a bioactive agent concentration of at least about 500 ug/g wet lung tissue or a total emitted dose (TED) of at least about 2,900 ug over 15 seconds).

In yet another aspect, the invention features a method for administering an inhalable pharmaceutical composition. The method includes the steps of: (i) aerosolizing a dispersion of liposomal les, thereby forming a respirable aerosol comprising a plurality of droplets having a mass median aerodynamic diameter (MMAD) between about 1 and 5 um, and (ii) ring a therapeutically effective amount of the hydrophobic bioactive agent to a lung of a subject in need of treatment.

The dispersion of liposomal particles has an average diameter between about 30 and 500 nm, each liposomal particle comprising a hydrophobic bioactive agent and a phospholipid dispersed within an aqueous dispersion vehicle. The ratio of hydrophobic bioactive agent:phospholipid is between about 5:1 and about 1:5, the hydrophobic bioactive agent is between about 0.1 and 30 % w/w of the composition, and the olipid is between about 0.1 and 30 % w/w of the composition. And, upon administration to a subject, the composition is terized by continuous aerosolization sufficient to e a therapeutic dose of the hydrophobic bioactive agent to the subject (or, alternatively, the ition can be characterized by another pharmacokinetic ty such as being capable of achieving a bioactive agent concentration of at least about 500 ug/g wet lung tissue or a total emitted dose (TED) of at least about 2,900 ug over 15 seconds).

In still yet r aspect, the invention features an inhalable ceutical composition ed by a process including the steps of: (i) hydrating a phospholipid, thereby forming a hydrated phospholipid; (ii) mixing the hydrated olipid, a hydrophobic bioactive agent, and an aqueous dispersion vehicle, thereby ing a mixture; and homogenizing the mixture, thereby producing a dispersion of liposomal particles comprising the phospholipid and hydrophobic bioactive agent dispersed within the aqueous dispersion vehicle and having an average diameter between about 30 and 500, where the ratio of hydrophobic bioactive agent:phospholipid is between about 5:1 and about 1:5, the hydrophobic bioactive agent is between about 0.1 and 30 % w/w of the composition, and the phospholipid is n about 0.1 and 30 % w/w of the ition. And, upon stration to a subject, the composition is characterized by continuous aerosolization sufficient to provide a therapeutic dose of the hydrophobic bioactive agent to the subject (or, alternatively, the composition can be characterized by another pharmacokinetic property such as being capable of ing a bioactive agent concentration of at least about 500 ug/g wet lung tissue or a total emitted dose (TED) of at least about 2,900 ug over 15 seconds).

In still another aspect, the invention features a method for ng a laser diffraction particle size system for continuously measuring a continuous aerosol. The method includes the steps of: (i) providing a laser diffraction particle size system comprising a nebulizer reservoir, membrane, laser beam, lens, and air suction source; (ii) positioning the zer reservoir with the membrane above the upper edge of the laser beam and at a distance between the lens and the center of an l cloud chamber; and (iii) positioning the air suction source beneath the laser beam. The adapted system avoids fogging of the lens by continuously exhausting the aerosol cloud chamber while continuously measuring transmission of the aerosol during continuous aerosolization.

In yet another aspect, the invention features a laser diffraction particle size system for continuously measuring a continuous aerosol. The system includes (i) a nebulizer reservoir positioned with a membrane above an upper edge of a laser beam and at a distance between a lens and the center of an aerosol cloud chamber; and (ii) an air suction source positioned beneath the laser beam. The system avoids fogging of the lens by continuously exhausting the aerosol cloud while continuously measuring transmission of the aerosol during continuous aerosolization.

In still yet another aspect, the invention features a method for continuously measuring a continuous l. The method includes the steps of: (i) ing a continuous aerosol to a laser diffraction particle size system, the system sing a nebulizer reservoir positioned with a ne above an upper edge of a laser beam and at a distance between a lens and the center of an aerosol cloud chamber, and an air suction source positioned beneath the laser beam, and (ii) continuously measuring transmission of the aerosol while the system avoids fogging of the lens by continuously exhausting the l cloud chamber.

In still yet another aspect, the invention features a method for manufacturing and verifying the average percent transmission (APT) of an inhalable pharmaceutical composition. The method includes the steps of: (i) hydrating a phospholipid, thereby forming a hydrated phospholipid; (ii) mixing the hydrated phospholipid, a hobic bioactive agent, and an aqueous dispersion e, thereby producing a mixture; (iii) homogenizing the mixture, thereby producing a dispersion of liposomal particles comprising the phospholipid and hydrophobic bioactive agent dispersed within the s dispersion vehicle and having an average diameter between about 30 and 500, wherein the ratio of hydrophobic bioactive agent:phospholipid is between about 5:1 and about 1:5, the hydrophobic bioactive agent is between about 0.1 and 30 % w/w of the composition, and the phospholipid is between about 0.1 and 30 % w/w of the composition; (iv) aerosolizing the dispersion of liposomal particles, thereby forming a respirable aerosol comprising a plurality of droplets, each droplet sing a sion of liposomal particles and having a mass median aerodynamic diameter (MMAD) between about 1 and 5 pm; (v) providing the respirable l to a laser ction particle size system, the system comprising a nebulizer reservoir positioned with a membrane above an upper edge of a laser beam and at a distance between a lens and the center of an aerosol cloud chamber, and an air suction source positioned beneath the laser beam; and (vi) uously ing transmission of aerosol with the laser diffraction particle size system, thereby determining if the composition is characterized by a predetermined APT value.

In different embodiments, any of the above aspects can be combined with any one or more or the features below, as well as any one or more of the features in the ed description and examples.

In s embodiments, the aqueous dispersion vehicle comprises water or an aqueous salt solution. The aqueous dispersion vehicle can be a buffer such as phosphate buffered saline.

In some embodiments, the dispersion of mal particles is in the form of a continuous respirable aerosol comprising a ity of aqueous ts ning a dispersion of liposomal particles and having a mass median aerodynamic diameter (MMAD) between about 1 and 5 pm.

In certain embodiments, the composition is characterized by an APT between about 50 and 100 % over at least 15 minutes of continuous aerosolization. The composition can be characterized by an APT between about 50 and 100 %, between about 60 and 100 %, n about 70 and 100 %, between about 80 and 100 %, between about 90 and 100 %, between about 50 and 95 %, between about 60 and 95 %, between about 70 and 95 %, between about 80 and 95 %, n about 90 and 95 %, n about 50 and 90 %, between about 60 and 90 %, between about 70 and 90 %, between about 80 and 90 %, less than about 50 %, less than about 55 %, less than about 65 %, less than about 70 %, less than about 75 %, less than about 80 %, less than about 85 %, less than about 90 %, less than about 95 %, less than about 100 %, or any sub- range or value therebetween. The continuous aerosolization can have a duration of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13,14, 15, 20, 25, 30, 35, 40, 50, or 60 minutes.

The plurality of droplets can have a MMAD between about 1 and 5 pm over at least 15 minutes of continuous aerosolization.

In various embodiments, the composition is characterized by an APT between about 50 and 100 % and after at least seven days of storage. The liposomal particles have an average diameter between about 30 and 500 nm after at least seven days of storage. Storage can be at t ions or other controlled conditions (e. g., in a refrigerator).

In some embodiments, the composition can be characterized by one or more physicochemical property. The composition can have a flow index of about 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, or 1.3. The ition can have a viscosity of about 0.1, 0.15, 0.2, 1, 100, or 110 CR The ition can have a zeta potential of about 2.5, 1.5, -2.5, -10, -50, -55, or -60 mV. The composition can have a surface tension of about 25, , 35, 40, 45, or 50 mN/m. The composition can have a yield stress of about 11, 12, 13, 14, 15, 16, 17, or 18 mPa. The dispersion of liposomal particles can have an average diameter between about 30 and 100 nm, 50 and 150 nm, 30 and 300 nm, 100 and 400 nm, or 200 and 300 nm. The composition can have a polydispersivity index (PDI) of about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, or 0.7. The composition can have a TAO of at least about 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 %. The composition can have a TED of at least about 3,600, 3,900, 4,300, or 4,600 ug over 15 seconds (e.g., as measured by DUSA, see Example 2). The composition can be characterized by non- Newtonian fluid behavior.

In various embodiments, the ity of droplets can have a mass median aerodynamic diameter (MMAD) of about 1, 2, 3, 4, or 5 um. The plurality of droplets can have a geometric standard deviation (GSD) of less than about 2.5, 2.4, 2.3, 2.2, 2.1, 2.0, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1.0, 0.9, 0.8, 0.7, 0.6, or 0.5.

In some embodiment, the hydrophobic bioactive agent includes one or more analgesics, nflammatory agents, anthelmintics, rrhythmic agents, anti- bacterial agents, anti-viral agents, anti-coagulants, anti-depressants, anti-diabetics, anti- epileptics, anti-fungal , anti-gout agents, anti-hypertensive agents, anti-malarials, anti-migraine agents, anti-muscarinic agents, anti-neoplastic agents, erectile dysfunction improvement , immunosuppressants, anti-protozoal , anti-thyroid agents, anxiolytic agents, sedatives, hypnotics, neuroleptics, kers, cardiac inotropic agents, corticosteroids, diuretics, anti-parkinsonian agents, gastro-intestinal agents, histamine receptor antagonists, keratolytics, lipid regulating agents, anti-anginal agents, cox-2 inhibitors, leucotriene inhibitors, macrolides, muscle relaxants, nutritional agents, opioid analgesics, protease inhibitors, sex hormones, ants, muscle relaxants, anti- osteoporosis agents, anti-obesity agents, cognition enhancers, anti-urinary incontinence agents, nutritional oils, anti-benign prostate hypertrophy agents, ial fatty acids, non-essential fatty acids, and combinations thereof. The hydrophobic bioactive agent can include one or more hobic anti-inflammatory d, NSAID agent, antibacterial agent, antifungal agent, herapeutic agent, vasoldilator, or a combination thereof.

In certain embodiments, the hydrophobic bioactive agent includes one or more of acutretin, albendazole, rol, luthemide, amiodarone, amlodipine, amphetamine, ericin B, atorvastatin, atovaquone, azithromycin, baclofen, beclomethsone, benezepril, benzonatate, betamethasone, bicalutanide, budesonide, bupropion, busulphan, butenafine, calcifediol, calciprotiene, calcitriol, camptothecan, artan, capsaicin, carbamezepine, carotenes, celecoxib, cerivistatin, cetrizine, chlorpheniramine, cholecalciferol, cilostazol, cimetidine, cinnarizine, ciprofloxacin, cisapride, clarithromycin, clemastine, clomiphene, clomipramine, clopidrogel, codeine, coenzyme Q10, cyclobenzaprine, cyclosporine, danazol, dantrolene, dexchlopheniramine, diclofenac, dicoumarol, digoxin, dihydroepiandrosterone, dihydroergotamine, dihydrotachysterol, dirithromycin, donepezil, efavirenz, eposartan, ergocalciferol, ergotamine, essential fatty acid sources, etodolac, etoposide, famotidine, fenofibrate, fentanyl, fexofenadine, finasteride, flucanazole, flurbiprofen, fluvastatin, fosphenytion, frovatriptan, furazolidone, gabapentin, gemfibrozil, glibenclamide, ide, glyburide, glymepride, griseofulvin, halofantrine, ibuprofen, irbesartan, irinotecan, isosorbide dinitrate, isotreinoin, itraconazole, ivermectin, ketoconazole, ketorolac, lamotrigine, razole, leflunomide, lisinopril, loperamide, loratadine, lovastatin, roxine, lutein, lycopene, medroxyprogesterone, mefepristone, mefloquine, megesterol acetate, one, methoxsalen, metronidazole, zole, midazolam, miglitol, dil, ntrone, montelukast, nabumetone, nalbuphine, naratiptan, nelfinavir, pine, nilsolidipine, nilutanide, nitrofurantoin, nizatidine, omeprazole, oprevelkin, osteradiol, oxaprozin, paclitaxel, paricalcitol, paroxetine, pentazocine, pioglitazone, pizofetin, pravastatin, prednisolone, probucol, terone, ephedrine, pyridostigmine, rabeprazole, raloxifene, refocoxib, repaglinide, rifabutine, rifapentine, rimexolone, ritanovir, rizatriptan, rosigiltazone, saquinavir, sertraline, sibutramine, sildenafil citrate, simvastatin, mus, spironolactone, sumatriptan, tacrine, tacrolimus, tamoxifen, tamsulosin, targretin, tazarotene, telmisartan, teniposide, terbinafine, terzosin, tetrahydrocannabinol, tiagabine, ticlidopine, tirofibran, tizanidine, mate, topotecan, toremifene, tramadol, oin, troglitazone, oxacin, valsartan, venlafaXine, vertoporfin, vigabatrin, vitamin A, vitamin D, vitamin E, vitamin K, zafirlukast, zileuton, zolmitriptan, zolpidem, zopiclone, and combinations thereof.

In various embodiments, the hydrophobic bioactive agent also includes an additive selected from the group consisting of deoxyglucoses, deoxyglucose salts, dihydroxy acetone, succinates, pyruvates, es, fumarates, malates, malonates, lactates, glutarates, and combinations thereof. The additive can be 2-deoxyglucose, 2- lucose phosphate, 6-deoxyglucose, 6-deoxyglucose phosphate, dihydroxy acetone, and combinations thereof.

WO 74559 In some embodiments, the hydrophobic bioactive agent includes COQ10.

The CoQ10 can substituted by an additive at the 1 position, the 4 position, or combinations thereof.

In certain embodiments, the hydrophobic bioactive agent is about 4 % W/W or less of the composition. The hobic bioactive agent can be about 6, 5, 4, 3, 2, or 1 % W/W or less of the composition.

In various embodiments, the phospholipid includes one or more of lecithin, lysolecithin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylglycerol, phosphatidic acid, phosphatidylserine, lysophosphatidylcholine, lysophosphatidylethanolamine, lysophosphatidylglycerol, lysophosphatidic acid, osphatidylserine, PEG-phosphatidylethanolamine, PVP- phosphatidylethanolamine, and combinations thereof. The phospholipid can include DPPC, DSPC, DMPC, or a combination thereof. The phospholipid can be a substantially pure phospholipid. The phospholipid can be about 3 % W/W or less of the composition.

In some embodiments, the ratio of hydrophobic bioactive agent:phospholipid is about 1: 1, 4:3, or 4:2.5. The ratio of hydrophobic bioactive agent:phospholipid can be about 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, or any value therebetween.

In certain embodiments, the olipid is in combination with one or more ents, antifoaming agents, acidifiers, alkalizers, buffers, antimicrobial agents, antioxidants, binders, solubilizing agents, solvents, viscosity modifiers, humectants, thickening , and combinations thereof. Alternatively, the composition can consist essentially of the hydrophobic bioactive agent, olipid, and aqueous dispersion vehicle.

In s embodiments, the composition includes sodium chloride in an amount less than about 1.0% W/v of the composition. The composition can include a salt in an amount making the composition essentially isosmotic with the human lung.

In some embodiments, the dispersion is suspension, nano-suspension, emulsion, or microemulsion.

In certain ments, the method also includes aerosolizing the sion of mal particles, thereby forming a respirable aerosol comprising a plurality of droplets, each droplet sing a dispersion of liposomal particles and having a mass median aerodynamic diameter (MMAD) between about 1 and 5 pm.

In various embodiments, mixing includes high shear mixing for up to about 5 minutes at about 10,000 to 20,000 rpm and at about 50 to 65 0C. Mixing can last for up to about 1, 2, 3, 4, or 5 minutes. Mixing can be at about 10,000, 11,000, 12,000, 13,000, 14,000, 15,000, 16,000, 17,000, 18,000, 19,000, or 20,000 rpm. Mixing can take place at about 50, 55, 60, or 65 0C. Temperature can vary depending upon the melting point of the hydrophobic bioactive agent used.

In some embodiments, homogenizing includes microfluidization.

Homoginization can e ultrasonic homogenization. Homogenizing can include high pressure homogenization for about 1-50 passes at about 30,000 psi and at about 50 to 65 0C. Homoginization can be for about 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 passes. The pressure can be about 25,000, 26,000, 27,000, 28,000, 29,000, 30,000, 31,000, 32,000, 33,000, , or 35,000 psi. The temperature can be about 50, 55, 60, or 65 0C. Temperature can vary depending upon the melting point of the hydrophobic ive agent used.

In n embodiments, aerosolization includes vibrating mesh nebulization.

Any le method for continuous nebulization can be adapted for use with the present invention.

In various embodiments, ry achieves a mass fraction deposited of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 %.

In some embodiments, delivery achieves local delivery to the lung substantially without systemic delivery.

In n embodiments, delivery achieves an ed amount of the hydrophobic bioactive agent in the lung for at least 48 hours after administration.

In various embodiments, upon continuous aerosolization, the composition is capable of achieving a bioactive agent concentration of at least about 900, 800, 700, 600, 500, 400, 300, 200, or 100 ug/g wet lung tissue. It will be tood that the ed wet lung tissue concentration will be effected by the subject, method of administration, and formulation, among other things. Therefore, in various embodiments, the bioactive 2012/043001 agent concentration can be a eutically adequate or therapeutically desirable amount of the particular bioactive agent being used.

In some embodiments, delivering a therapeutically effective amount of the hydrophobic bioactive agent comprises metering a dose of the bioactive agent.

In certain embodiments, the subject has cancer. The cancer can be lung cancer. More generally, the subject can have any one or more afflictions affecting the atory tract including, but not d to, one or more of asthma, allergies, chronic obstructive pulmonary disease, chronic itis, acute bronchitis, emphysema, cystic fibrosis, pneumonia, ulosis, pulmonary edema, acute atory distress syndrome, coniosis, interstitial lung e, pulmonary edema, pulmonary embolism, ary ension, pleural effusion, pneumothorax, mesothelioma, amyotrophic lateral sclerosis, myasthenia gravis, and lung disease.

In various embodiments, the ition does not include an opsonization reducer (e. g., an opsonization reducer that interferes with aerosolization). For example, the composition can specifically e a polyoxyethylene polyoxypropylene block polymer such as a Poloxamer (e. g., poloxymer 188), Pluronic, Lutrol, and Superonic. In another example, the composition can specifically exclude polyethylene glycol (PEG) of various chain lengths, polysaccharides, other PEG-containing copolymers, poloxamines, and the like. Alternatively, formulations in accordance with the invention can include one or more opsonization enhancers in an amount that does not substantially interfere with aerosolizlation, for example, if the amount opsonization enhancer imparts an otherwise desirable property on the formulation. In one embodiment, the composition includes a polyoxypropylene-poloxyethylene block polymer at 0.001-5% by weight of the total composition.

The present invention is described in further detail by the figures and examples below, which are used only for illustration purposes and are not limiting.

DESCRIPTION OF THE DRAWINGS shows a schematic diagram of aerosolization of drug dispersions using a vibrating mesh nebulizer. shows a schematic of several manufacturing processes. shows an X-Ray diffraction pattern of bulk powdered CoQ10. shows a differential scanning calorimetry thermogram of bulk powdered CoQ10. shows a Scanning Electron Microscopy (SEM) picture of bulk ed CoQ10. shows particle size distributions of CoQ10 dispersions prepared using different manufacturing processes. shows particle size distributions, ed by Laser Diffraction (LD), of aqueous dispersions of CoQ10 following preparation in the microfluidizer and after 7 days (Formulation A, Table l). shows Z-average and PdI values of aqueous dispersions of CoQ10 following preparation in the microfluidizer and after 7 days (Formulation A, Table 1).

Statistical differences were not found for drug particle size bution characteristics (Z-average and PdI) neither in formulations prepared with different number of microfluidization passes and ed following preparation nor when the same ations were compared at days 0 and 7. shows hydrodynamic diameters and polydispersity of aqueous sions of CoQ10 (Formulation B, Table 1) following preparation in the microfluidizer using lecithin (top) or DPPC (bottom). (*P S 0.05 when compared to 10 passes; §Not statistically different when compared to the lecithin sion prepared with same number of microfluidization passes). shows a Malvern Spraytec® coupled with inhalation cell. shows a schematic diagram of n Spraytec® with inhalation cell in horizontal position. shows a schematic diagram of the “open bench” method discussed in connection with the Examples below (distances: between ne and upper edge of laser beam: 25 mm; n lens and center of aerosol cloud: 25 mm; air suction beneath laser beam: 10 cm). shows transmittograms of lecithin dispersions of CoQ10 (Formulation C, Table 1). Results are expressed as means (n = 3) of percentage transmission relative to nebulization of CoQ10 dispersions for 15 minutes. The slope values from the linear sion analysis of the curves are evaluated as measurement of steadiness in aerosol production. shows the slope of transmittograms (top) and Total Aerosol Output (TAO — bottom) for nebulization of lecithin dispersions of CoQ10 lation C, Table 1) during 15 minute nebulization events. (*P S 0.05 compared to other formulations). shows a particle size distributions analyses of aqueous dispersions of CoQ10 (Formulation C, Table 1) following preparation in the luidizer using laser diffraction (left) and dynamic light scattering (right). (*P S 0.05 compared to formulations analysed following preparation; §P S 0.05 ed to other formulations at day 7). shows Zeta potential and surface tension values related to formulations of CoQ10 processed at different number of microfluidization passes (Formulation C, Table 1). Columns and error bars represent means and standard errors, respectively (n = 10 for zeta potential and n = 5 for surface tension). The temperature during surface tension measurement was 25 0C. (*P S 0.05 when compared to 10 passes, §Not statistically different). shows elements of the Herschel-Bulldey model for aqueous dispersions of CoQ10 processed at different number of microfluidization passes (Formulation C, Table 1). No statistical differences were found. shows a tic m of Dose Uniformity Sampling Apparatus (DUSA) for Dry Powder Inhalers (DPIs) adapted for nebulizers. shows a particle size distributions from laser ction technique of aqueous dispersions of CoQ10 following 50 passes in the uidizer. Results are expressed as means i standard deviations (n = 3). Some rd deviations are too small to be e on the graphs. shows Z-average and PdI values of aqueous dispersions of CoQ10 following 50 passes in the microfluidizer. Results are expressed as means i rd deviations (n = 3). Some standard deviations are too small to be visible on the graph (n = 3). §Not statistically different. shows Zeta potential of COQ10 dispersions. Results are expressed as means i standard deviation (n = 3). >“P < 0.05 when compared to synthetic phospholipids. shows surface tension of COQ10 dispersions. Results are expressed as means i standard error (n 2 5). The ature values during measurement were 25 0C, 25 0C, 19 0C and 17 0C, respectively. §Not statistically different. shows elements of the Herschel-Bulldey model for aqueous dispersions of COQ10, expressed as means i standard deviations (n = 3). Yield stress of DSPC formulation is not presented because it s Power Law model. Some standard deviations are too small to be visible on the graph. >“P < 0.05. §Not statistically different. shows an example schematic of a general flow curve of aqueous dispersions. shows rheological or of COQ10 dispersions. Graphs ted in ent scales are sed as means i standard deviations (n = 3). shows transmittograms of saline (control) and lecithin, DMPC, DPPC and DSPC dispersions of COQ10. s are sed as means (n = 3) of percentage transmission relative to zation of COQ10 dispersions for 15 minutes.

The slope values from the linear regression analysis of the curves are evaluated as measurement of steadiness in aerosol production. shows slope of transmittograms (top) and Total Aerosol Output, TAO (bottom), expressed as means i standard deviations (n = 3) relative to nebulization of COQ10 dispersions for 15 minutes. §Not statistically ent. shows TED from NGI (top) and from DUSA for DPI adapted for nebulizers (bottom) of dispersions of COQ10. Results are sed as means i standard deviations (n = 3) of total drug deposited within a 15 second period at initial and final phases of a 15-minute nebulization event. TED: Total Emitted Dose; DUSA: Dose Uniformity Sampling Unit; DPI: Dry Powder Inhaler. >“P < 0.05 when compared to synthetic phospholipids. TP < 0.05 within nebulization event. §Not statistically different compared to each other. iNot statistically different compared to other synthetic phospholipids. shows in vitro deposition profiles of lecithin, DMPC, DPPC and DSPC dispersions of CoQ10 at a flow rate of 15 L/min using an Aeroneb Pro® nebulizer. Results are expressed as means i standard deviations (n = 3) of the percentage of total drug deposited within a 15-second period at initial and final phases of a ute nebulization event. shows in vitro deposition profiles of lecithin, DMPC, DPPC and DSPC dispersions of CoQ10 at a flow rate of 15 L/min using an Aeroneb Pro® nebulizer. s are expressed as means i standard deviations (n = 3) of the drug amount deposited within a 15-second period at initial and final phases of a 15-minute nebulization event. shows the aerodynamic properties of lecithin, DMPC, DPPC and DSPC dispersions of CoQ10 at a flow rate of 15 L/min using an Aeroneb Pro® nebulizer. Results are expressed as means i standard ions (n = 3) of MMAD or GSD within a 15-second period at initial and final phases of a 15-minute nebulization event. >“P < 0.05 within zation event. §P < 0.05 when compared to each other.

A shows the TED NGI and TED DUSA values for the studied formulations. B shows estimated total dose (FPDet) and fraction (FPF) of aerosolized fine particles from in, DMPC, DPPC and DSPC dispersions of CoQ10 at a flow rate of 15 L/min using an Aeroneb Pro® zer. Results are expressed as means i standard deviations (n = 3) related to a 15-second period at l and final phases of a 15-minute nebulization event. >“P < 0.05 when compared to synthetic phospholipids. [P < 0.05 within nebulization event. §Not statistically different compared to each other. 1P < 0.05 when compared to each other. shows average Dv(50) of CoQ10 dispersions aerosolized using b Pro® nebulizer for 15 minutes (n = 3). shows an example nose-only dosing tus used to aerosolize CoQ10 to mice. Six mice are individually restrained in a tube, exposing their noses to the chamber. The nebulizer is positioned between the chamber and the fan that will provide sufficient airflow to fill the chamber with the drug aerosol. The tubing system is open to avoid drug recirculation. shows estimated drug concentration-time profiles of CoQ10 inside the nose-only tion chamber. shows tive estimated doses of CoQ10 from synthetic phospholipid formulations aerosolized to mice into a nose-only inhalation chamber during 15 minutes. shows mean lung concentrations normalized to wet lung tissue of CoQ10 from synthetic olipid dispersions following aerosolization to mice into a nose-only inhalation chamber during 15 minutes. Error bars indicate standard deviation (n = 6). shows mean lung concentrations normalized to animal body weight of CoQ10 from synthetic phospholipid dispersions following aerosolization to mice into a nose-only inhalation chamber during 15 minutes. Error bars indicate standard deviation (n = 6). shows deposition of CoQ10 in the nasal cavity of mice 0.5 and 1 hour post 15-minute nebulizer . Results are sed as means i standard deviations (n = 6). >“P < 0.05 when compared to control group. [P < 0.05 when compared within the same group. shows transmittograms of aerosolization of DMPC-stabilized sions with different concentrations of CoQ10. shows transmittograms of aerosolization of DMPC- and DSPC— stabilized dispersions, as compared to an intravenous formulation that includes a particular opsonisation reducer. FIGS. 39-41 show further charachterization of the formulations studied in connection with . Other features and advantages of the invention will be apparent from the following detailed description, examples, and claims.

DETAILED DESCRIPTION OF THE INVENTION As discussed above, the ion provides inhalable pharmaceutical compositions having an aqueous dispersion of particles including a hydrophobic bioactive agent. Due to their al ition and methods of cture, the pharmaceutical compositions t distinctive physicochemical properties that provide advantageous l transmission and output, including stable and continuous aerosolization.

CoQ10 was used as an exemplary hydrophobic bioactive agent. Coenzyme Q10, also known as CoQ10, ubiquinone or ubidecarenone, occurs naturally in the body.

CoQ10 participates in electron transport and proton transfer in mitochondrial respiration.

Therefore, altering the levels of this antioxidant may have an impact on biological activities such as aging, neurodegenerative and cardiovascular diseases, and cancer.

CoQ10 is a -water soluble compound presented as a yellow or orange crystalline powder. The highest plasma concentration of CoQ10 reported in the literature is 10.7 umol/L (approximately 9 , which was obtained by administration of a solubilized oral formulations (e. g., commercially available dietary supplement or “nutraceutical”). Nevertheless, the maximum ted dose (MTD) has yet to be determined. The present invention provides ations of CoQ10 for pulmonary delivery with advantageous cokinetic es that will improve the pharmacodynamic responses for treating respiratory system malignancies. By delivering a high amount of drug to the disease site, a lower dose can be used (as compared to intravenous or oral administration).

The following description provides further detail regarding the inventive compositions (including the hobic ive agents, phospholipids, aqueous dispersion vehicles, and other components), methods of manufacture (including mixing, homogenization, and aerosolization), and methods of treatment (including pharmacokinetics, pharmacodynamics, and indications). Finally, the detailed description provides illustrative es of the invention, including Example 1: pment and Characterization of Phospholipid-Stabilized Submicron Aqueous Dispersions of CoQ10 Adapted for uous Nebulization; Example 2: Prediction of In Vitro Aerosolization Profiles Based on Rheological Behaviors of Aqueous Dispersions of CoQ10; Example 3: Pulmonary Deposition and Systemic Distribution in Mice of Inhalable ations of CoQ10; Example 4: Low Concentration Range Determination of Hydrophobic Drugs Using HPLC; e 5: Determination of Suitable Hydrophobic Drug Concentrations in Phospholipid Nanodispersions Suitable for Continuous Nebulization; and Example 6: Measuring tory Reponse to Pulmonary Administration of Dispersions of Phospholipid Encapsulated Hydrophobic ive Agents.

ITIONS In various ments, inhalable pharmaceutical compositions according to the ion include aqueous dispersion of particles le for continuous aerosolization. The particles each include a hydrophobic bioactive agent and a olipid, and are dispersed within an aqueous dispersion vehicle. In some embodiments, the particles are liposomal particles, or include a fraction of liposomal particles. In some embodiments, the composition can consist essentially of the hydrophobic bioactive agent, phospholipid, and aqueous dispersion vehicle. However, other embodiments including one or more additional components are possible. Various ents for inclusion in the inventive compositions are sed, in turn, below.

Hydrophobic ive Agents In various embodiments, one or more hydrophobic bioactive agents (also known as lipophilic bioactive agents) can be prepared in inhalable pharmaceutical compositions. Hydrophobic bioactive agents are vely insoluble in water. For example, a hydrophobic bioactive agent can have a solubility in water of less than about 1 part of bioactive agent in about 1000 parts of water.

Suitable lipophilic bioactive agents can include, but are not limited to, analgesics, anti-inflammatory agents, anthelmintics, anti-arrhythmic agents, anti- bacterial , anti-viral agents, anti-coagulants, anti-depressants, anti-diabetics, anti- epileptics, anti-fungal agents, anti-gout agents, anti-hypertensive agents, anti-malarials, anti-migraine agents, anti-muscarinic , anti-neoplastic agents, erectile dysfunction improvement agents, immunosuppressants, anti-protozoal agents, anti-thyroid agents, anxiolytic agents, sedatives, hypnotics, neuroleptics, B-Blockers, cardiac inotropic , corticosteroids, diuretics, arkinsonian agents, gastro-intestinal agents, histamine receptor antagonists, keratolytics, lipid regulating agents, anti-anginal agents, cox-2 inhibitors, leucotriene inhibitors, macrolides, muscle relaxants, nutritional agents, opioid analgesics, protease inhibitors, sex hormones, stimulants, muscle nts, anti- osteoporosis agents, besity agents, cognition enhancers, anti-urinary incontinence agents, nutritional oils, anti-benign prostate hypertrophy agents, essential fatty acids, non-essential fatty acids, combinations thereof, and the like.

Non-limiting es of suitable hydrophobic active agents e, but are not d to, acutretin, albendazole, albuterol, luthemide, amiodarone, amlodipine, amphetamine, amphotericin B, atorvastatin, atovaquone, azithromycin, baclofen, beclomethsone, benezepril, benzonatate, betamethasone, bicalutanide, budesonide, bupropion, han, butenafine, calcifediol, calciprotiene, calcitriol, camptothecan, candesartan, capsaicin, carbamezepine, carotenes, xib, ceriVistatin, cetrizine, chlorpheniramine, cholecalciferol, cilostazol, dine, cinnarizine, ciprofloxacin, cisapride, clarithromycin, clemastine, clomiphene, clomipramine, clopidrogel, codeine, coenzyme Q10, cyclobenzaprine, cyclosporine, danazol, dantrolene, dexchlopheniramine, diclofenac, dicoumarol, digoxin, dihydro epiandrosterone, dihydroergotamine, dihydrotachysterol, dirithromycin, donepezil, efaVirenz, eposartan, ergocalciferol, ergotamine, ial fatty acid s, etodolac, etoposide, famotidine, fenofibrate, fentanyl, fexofenadine, finasteride, flucanazole, flurbiprofen, fluvastatin, fosphenytion, frovatriptan, furazolidone, ntin, gemfibrozil, glibenclamide, glipizide, glyburide, glymcpride, griseofulVin, ntrine, ibuprofen, irbesartan, irinotecan, isosorbide ate, isotreinoin, nazole, ivermectin, ketoconazole, ketorolac, lamotrigine, lanosprazole, leflunomide, lisinopril, loperamide, loratadine, lovastatin, L-thryroxine, lutein, lycopene, medroxyprogesterone, mefepristone, mefloquine, megesterol acetate, methadone, methoxsalen, metronidazole, miconazole, midazolam, miglitol, minoxidil, mitoxantrone, ukast, nabumetone, nalbuphine, naratiptan, nelfinaVir, nifedipine, nilsolidipine, nilutanide, nitrofurantoin, nizatidine, omeprazole, oprevelkin, osteradiol, oxaprozin, paclitaxel, paricalcitol, paroxetine, pentazocine, pioglitazone, pizofetin, tatin, prednisolone, probucol, progesterone, pseudoephedrine, pyridostigmine, rabeprazole, raloxifene, refocoxib, repaglinide, rifabutine, rifapentine, rimexolone, ritanovir, rizatriptan, ltazone, saquinaVir, sertraline, sibutramine, sildenafil citrate, simvastatin, sirolimus, spironolactone, sumatriptan, e, tacrolimus, tamoxifen, osin, targretin, tazarotene, telmisartan, teniposide, terbinafine, terzosin, tetrahydrocannabinol, ine, ticlidopine, tirofibran, tizanidine, topiramate, can, toremifene, tramadol, tretinoin, troglitazone, trovatloxacin, valsartan, venlafaxine, vertoporfin, Vigabatrin, Vitamin A, vitamin D, n E, vitamin K, zafirlukast, zileuton, zolmitriptan, zolpidem, zopiclone, combinations thereof, and the like. Salts, isomers and/or other tives of the above- listed bioactive agents can also be used, as well as combinations thereof.

In various embodiments, CoQ10 can be the hydrophobic bioactive agent (e.g., alone, or in combination with one or more additional ive agents). CoQ10, sometimes referred to herein as CleO or ubidccarenone, is a popular nutritional supplement and can be found in capsule form in nutritional stores, health food stores, pharmacies, and the like as a vitamin-like supplement that is hypothesized to help protect the immune system through the antioxidant properties of ubiquinol, the reduced form of CleO (ubiquinone). As used herein, CoQ10 can also include derivatives thereof, including, for example, ubiquinol. Similarly CleO can also include analogues of ubiquinone and nol, and precursor compounds as well and combinations thereof.

In various embodiments, the lipophilic bioactive agent, such as coenzyme Q10, can be combined with other bioactive agents or compounds for administration in vivo. Likewise, any bioactive agent can be combined with additional additives and/or excipients. The other bioactive , additives, and/or ents can be hydrophobic or hydrophilic.

] Combinations of bioactive agents can be utilized in accordance with the present disclosure for the treatment of cancers ing, but not limited to, lung cancer.

For example, a lipophilic bioactive agent, such as CleO, can be combined with deoxyglucoses, including 2-deoxyglucose and/or 2-deoxyglucose salts, 6-deoxyglucose and/or 6-deoxyglucose salts, as a mixture or blend and administered to a patient in vivo.

Suitable salts can include phosphates, lactates, pyruvates, ybutyrates, combinations thereof, and the like. In some embodiments the salt can be a phosphate such as 2-deoxyglucose phosphate, 6-deoxyglucose phosphate, combinations thereof, and the like. In other embodiments, the e or quinol ring of ubiquinone or ubiquinol can be substituted at the 1 position, the 4 position, or both, by the deoxyglucose or salts thereof, such as 2-deoxyglucose or 6-deoxyglucose or salts thereof, including 2-deoxyglucose phosphate or 6-deoxyglucose phosphate, with the substituted none or ubiquinol then administered to a patient. 2012/043001 Similarly, dihydroxy acetone can be ed with COQ10 as a mixture or blend and administered to a patient in vivo. In such embodiments, the quinone or quinol ring of none or ubiquinol can be substituted at the 1 position, the 4 position, or both, with the dihydroxy acetone, with the substituted ubiquinone or ubiquinol then administered to a patient. In other embodiments, compounds which can be administered with the lipophilic bioactive agent, such as me Q10, include succinates, pyruvates, citrates, fumarates, malates, malonates, lactates, ates, combinations thereof, and the like, with ic examples including, but not limited to, sodium succinate, potassium succinate, combinations thereof, and the like.

Phospholipids In various embodiments, the bioactive agent is sed within a liposome and/or otherwise stabilized together with a phospholipid. Liposomes can be formed from one or more liposome-forming compounds such as phospholipids. Similarly, the bioactive agent and phospholipid can form other physical ement such as mixtures and dispersions. Compositions in accordance with the invention can include predominantly liposomal arrangement, a fraction of liposomes together with other arrangements, or can be ially devoid of liposomes. Although various compounds and combinations thereof, are possible, the final composition must ultimately exhibit the distinctive physicochemical properties of the invention, which provide advantageous aerosol transmission and output, pharmacokinetics, and/or pharmacodynamics.

Suitable phospholipids and/or phospholipid derivatives/analogs for forming mes include, but are not limited to, lecithin, lysolecithin, phosphatidylcholine (e.g. dipalmitoyl phosphatidylcholine (DPPC) or dimyristoyl phosphatidylcholine (DMPC), phosphatidylethanolamine, phosphatidylinositol, phosphatidylglycerol, phosphatidic acid, phosphatidylserine, lysophosphatidylcholine, lysophosphatidylethanolamine, lysophosphatidylglycerol, osphatidic acid, lysophosphatidylserine, PEG- atidylethanolamine, PVP-phosphatidylethanolamine, ations thereof, and the like.

In one embodiment, the phospholipid is a lecithin. Lecithin can be derived from egg or soybean. Such lecithins include those commercially ble as PHOSPHOLIPON® 8SG, PHOSPHOLIPON® 90G, and PHOSPHOLIPON® 90H (the fully hydrogenated version of PHOSPHOLIPON® 90G) from American Lecithin Company, , CT (part of Lipo Chemicals, Inc. — the Lipo phospholipid catalog lists other potentially suitable phospholipids, for example those le for parenteral use). Other suitable lecithins include LECINOL S-10® lecithin available, for e, from Nikko Chemicals, NOF (Japan), Lipo Chemicals, Inc., and Genzyme Corporation, as well as other commercial suppliers. Alternatively, in some ments it can be ageious to select one or more phospholipids that are less hydrophilic than lecithin. olipids can be selected to confer a negative e charge to the ing liposome vesicles, which can reduce processing time and process , and which can aid in the formation of stable liposomes and lization. For example, a high phosphatidylcholine content lecithin (e. g., dipalmitoyl phosphatidylcholine or dimyristoyl phosphatidylcholine) can be utilized to form a liposome. An example high phosphatidylcholine lecithin is PHOSPHOLIPON® 85G, which ns a minimum of 85% of alinoleic acid based-phosphatidylcholine. This lecithin is easy to use and is able to produce submicron liposomes at low process temperatures (from about 20 0C to about 55 0C) without the addition of any other special additives. PHOSPHOLIPON® 85G contains, in addition to phosphatidylcholine, approximately 5-7% phosphatidic acid.

Aqueous Dispersion Vehicles An s medium, for example water, is required in order to form an aqueous dispersion according to the present invention. Example aqueous dispersion vehicles include water, saline (e. g., iso-osmotic saline, a saline solution that will make the final formulation iso-osmotic with a subject’s lung), and aqueous buffers (e. g., phosphate buffered saline). Other suitable aqueous dispersion vehicles can include other aqueous solutions that are compatible with the desired chemical composition, cturing method, and/or l use.

Additional components Pharmaceutical compositions in accordance with the invention can include one or more additional components in addition to the one or more ive agent, one or more phospholipid, and one or more aqueous dispersion vehicle. Additional components can be used, for example, to enhance formulation of the liposomes possessing a lipophilic ive agent, to improve overall rheological and processing WO 74559 properties of the liposomes, and to insure microbiological integrity of the resulting liposomal concentrate during storage. Such components include, Without limitation, absorbents, antifoaming agents, acidifiers, alkalizers, buffers, antimicrobial agents, antioxidants (for example ascorbates, tocopherols, butylated hydroxytoluene (BHT), polyphenols, phytic acid), binders, biological additives, chelating agents (for example, disodium ethylenediamine tetra acetic acid , tetrasodium EDTA, sodium metasilicate, and the like), denaturants, external analgesics (for example aspirin, nonsteroidal anti-inflammatories and the like), steroidal anti-inflammatory drugs (such as hydrocortisone and the like), preservatives (for example imidazolidinyl urea, diazolidinyl urea, phenoxycthanol, methylparaben, araben, propylparaben, and the like), reducing agents, solubilizing agents, solvents, viscosity modifiers, humectants, thickening agents, surfactants, s, stabilizers, rs, se inhibitors, antioxidants, absorption enhancers, and ations thereof. Such additional components can be present in an amount from about 0.001% by weight to about 10% by weight of the dispersion.

] The excipients and adjuvants that can be used in the present sure, While potentially having some activity in their own right, for example, as antioxidants, generally include compounds that enhance the efficiency and/or efficacy of the active agents. It is also possible to have more than one excipient, adjuvant, or even active agents in a given respirable ate.

Excipients can be selected and added either before or after the drug or bioactive age particles are formed, in order to enable the drug or bioactive age particles to be homogeneously admixed for appropriate administration. Excipients can include those items described above as suitable for formation of liposomes. Other suitable excipients include rs, tion enhancers, solubility enhancing agents, dissolution rate ing agents, stability enhancing agents, bioadhesive agents, controlled release agents, flow aids and processing aids. In some ments, suitable excipients include cellulose ethers, acrylic acid polymers, bile salts, and combinations thereof. Other suitable ents include those described in detail in the Handbook of Pharmaceutical Excipients, published jointly by the an Pharmaceutical Association and The Pharmaceutical Society of Great Britain, the Pharmaceutical Press, 1986, relevant portions of which are incorporated by reference . Such excipients are commercially available and/or can be prepared by techniques within the purview of those skilled in the art.

Excipients can also be chosen alone or in combination to modify the intended function of the effective ingredients by improving flow, or bioavailability, or to control or delay the release of the active agent. Specific non-limiting examples of excipients include: SPAN 80, TWEEN 80, BRIJ 35, BRIJ 98, PLURONICS, SUCROESTER 7, SUCROESTER II, SUCROESTER 15, sodium lauryl sulfate, oleic acid, h-9, laureth-8, lauric acid, vitamin E, TPGS, GELUCIRE 50/13, GELUCIRE 53/1 0, LABRAFIL, dipalmitoyl adityl choline, glycolic acid and salts, holic acid and salts, sodium fusidate, cyclodextrins, polyethylene glycols, labrasol, polyvinyl alcohols, polyvinyl pyrrolidones, tyloxapol, cellulose derivatives, polyethoxylated castor oil tives, ations thereof, and the like.

Examples of le humectants include, but are not limited to, s and polyol derivatives, including ol, diglycerol, triglycerol, ethylene glycol, propylene glycol, butylene glycol, pentylene glycol (sometimes referred to herein as 1,2-pentane diol), isopreneglycol (1,4-pentane diol), 1,5-pentane diol, ne glycol, erythritol, 1,2,6-hexanetriol, polyethylene glycols (“PEG”) such as PEG-4, PEG-6, PEG-7, PEG-8, PEG-9, PEG-IO, PEG-12, PEG-14, PEG-I 6, PEG-18, PEG-20, and combinations thereof, sugars and sugar derivatives (including, inter alia, fructose, glucose, maltose, maltitol, mannitol, inositol, sorbitol, sorbityl silanediol, sucrose, trehalose, xylose, xylitol, glucuronic acid and salts thereof), ethoxylated sorbitol (Sorbeth-6, Sorbeth-20, Sorbeth-30, Sorbeth-40), combinations thereof, and the like. In other ments, glycols such as butylene , 1,2-pentane diol, glycerin, 1,5-pentane diol, combinations thereof, and the like, can be utilized as a humectant. Where utilized, any of the above ants, including combinations thereof, can be present in amounts from about 0.1 % by weight to about 20% by weight of the second dispersion, in ments from about 1% by weight to about 5% by weight of the second dispersion.

In some embodiments, a preservative such as phenoxycthanol and a humectant such as propylene glycol can both be included in the formulation. The propylene glycol can provide humectant activity and assist in the preservation of the concentrate when combined with phenoxyethanol. The phenoxyethanol and ene glycol mix can be water soluble and latile. This embodiment is in contrast with the use of ethanol for preservation, which is often utilized by suppliers of mal sions. Where present, such preservatives can be present in amounts from about 0.01% by weight to about 3% by weight of the formulation. n embodiments can include a dispersion stabilizing agent. Example dispersion stabilizing agents include Polyethoxylated (a/k/a pegylated) castor oil (Cremophor® EL), Polyethoxylated hydrogenated castor oil (Cremophor® RH 40), Tocopherol polyethylene glycol succinate (Pegylated n E, n E TPGS), Polysorbates (Tweens®), Sorbitan fatty acid esters ®), Bile acids and bile-acid salts and DMPC.

] Certain embodiments can exclude opsonization reducers (e.g., opsonization reducers that can interfere with aerosolization). For example, the composition can specifically exclude a polyoxyethylene polyoxypropylene block polymer such as a Poloxamer (e.g., poloxymer 188), Pluronic, Lutrol, and Superonic. In another example, the composition can specifically exclude polyethylene glycol (PEG) of various chain lengths, polysaccharides, other PEG-containing copolymers, poloxamines, and the like.

Alternatively, formulations in accordance with the invention can include one or more opsonization enhancers in an amount or kind (e. g., suitable HLB) that does not substantially interfere with lizlation, for e, if the amount opsonization enhancer imparts an otherwise desirable property on the formulation. In one embodiment, the ition includes a polyoxypropylene-poloxyethylene block polymer at 0.001-5% by weight of the total composition. In another embodiment, the formulation includes a vely small amount of one or more hydrophilic polymers, to improve stability and increase TAO while maintaining effective and continuous aerosolization.

Formulations can include pulmonary surfactants and/or mucolytic agents.

Suitable pulmonary surfactants e, but are not limited to, pulmonary surfactant preparations having the function of natural pulmonary surfactant. These can include both natural and tic pulmonary surfactants. In various embodiments, compositions which contain phospholipids and/or pulmonary surfactant proteins can be utilized.

Exemplary phospholipids that can be used as pulmonary surfactants include dipalmitoylphosphatidylcholine (DPPC), palmitoyloleylphosphatidylglycerol (POPG) and/or phosphatidylglycerol (PG). Other suitable phospholipids include mixtures of various phospholipids, for example, mixtures of dipalmitoylphosphatidyicholine (DPPC) and oyloleylphosphatidylglycerol (POPG) at a ratio of from about 7 to about 3 to from about 3 to about 7.

] Commercial products that can be used as pulmonary surfactants include CUROSURF® (INN: PORACTANT ALFA) (Serono, Pharma GmbH, Unterschleipheim), a natural surfactant from nized porcine lungs; SURVANTA® (INN: BERACTANT) (Abbott GmbH, Wiesbaden), extract of bovine lungs; ACT® (INN: BOVACTANT) (Boehringer Ingelheim), extract of bovine lungs; EXOSURF® (INN: COLFOSCERIL PALMITATE) (GlaxoSmithKline), a synthetic phospholipid containing excipients; SURFACTEN® (INN: SURFACTANT- TA) (Mitsubishi Pharma Corporation), a pulmonary surfactant extracted from bovine lungs; INFASURF® (INN: CALFACTANT) (Forest Pharmaceuticals), a surfactant extracted from calf lungs; ALEC® (INN: PUMACTANT) (Britannia Pharmaceuticals), an artificial surfactant of DPPC and PO; and BLES® (BLES mical Inc.), a bovine lipid t surfactant.

Suitable pulmonary surfactant proteins include both ns obtained from natural sources, such as pulmonary lavage or extraction from amniotic fluid, and ns prepared by genetic engineering or chemical synthesis. Pulmonary surfactant proteins designated by SP-B (Surfactant Protein-B) and SP-C (Surfactant Protein-C) and their modified derivatives, including recombinant forms of the ns, can be ed in some embodiments.

Suitable mucolytic agents include, but are not limited to, guaifenesin, iodinated glycerol, glyceryl guaiacolate, terpin hydrate, ammonium chloride, N- acetylcysteine, xine, ambroxol, iodide, their pharmaceutically acceptable salts, and combinations thereof.

In some embodiments, the amount of preservatives utilized in a ition of the t disclosure including a lipophilic bioactive agent in liposomes can also be reduced by the inclusion of additional additives. For example, the amount of preservatives can be reduced in a composition of the present disclosure by the addition of multifunctional diols including, but not limited to, l,2-pentane diol, l,4-pentane diol, hexylene glycol, propylene glycol, l,3-butylene glycol, glycerol or diglycerol, combinations thereof, and the like, and by lowering the water activity, AW, via the addition of humectants described above and through the addition of the soluble ingredients. Other examples include soluble ingredients such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents and the like, for example, sodium acetate, sodium chloride, ium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, and the like. Other buffers that can be added include sodium hydroxide, potassium hydroxide, ammonium hydroxide, hanolamine, diethanolamine, triethanolamine, diisopropanolamine, ethylpropanol, hamine, tetrahydroxypropyl ethylenediamine, citric acid, acetic acid, lactic acid, and salts of lactic acid including sodium lactate, potassium lactate, lithium e, calcium e, magnesium lactate, barium lactate, aluminum lactate, zinc lactate, sodium citrate, sodium acetate, silver lactate, copper lactate, iron e, manganese lactate, ammonium lactate, combinations thereof, and the like.

In some ments, solubilization of a lipophilic bioactive agent such as CleO in a al that has both lipophilic and hilic properties can assist in liposome formulation by forming water-dispersible CleO for encapsulation by a high phosphatidylcholine lecithin, such as OLIPON® 85G. le solubilizing agents for the lipophilic ive agent include, for example, polyoxyalkylene dextrans, fatty acid esters of saccharose, fatty alcohol ethers of oligoglucosides (e.g., the akylpolyglucosides such as TRITONTM), fatty acid esters of ol (e.g., glycerol mono/distearate or glycerol urate), and polyoxyethylene type compounds (e.g., polyoxyethylene, polyethylene glycol, polyethylene oxide, SOLUTOLTM CREOMOPHORTM, MACROGOLTM, CARBOWAXTM, POLYOXYLTM). le solubilizers also include polyethoxylated fatty acid esters of sorbitan (e.g., Polysorbates, such as TWEENTM, SPANTM, including Polysorbate 20 and Polysorbate 80), fatty acid esters of poly(ethylene oxide) (e.g., polyoxyethylene stearates), fatty alcohol ethers of poly(ethylene oxide) (e.g., polyoxyethylated lauryl ether, polyoxyethylene 20 oleyl ether (BRIJ 98)), alkylphenol ethers of poly(ethylene oxide) (e. g., polyethoxylated octylphenol), yethylene-polyoxypropylene block copolymers (also known as poloxamers, such as “PLURONICS”, including PLURONIC F-127, a poloxamer 407 stabilizer), and ethoxylated fats and oils (e. g., lated castor oil, or polyoxyethylated castor oil, also known as polyethylene glycol-glyceryl triricinoleate), as well as combinations f.

In some embodiments, suitable solubilizing agents include Polysorbates, e.g. those sold under the brand name TWEENTM. Examples of such rbates include Polysorbate 80 (TWEENTM 80), Polysorbate 20 (TWEENTM 20), Polysorbate 60 (TWEENTM 60), Polysorbate 65 (TWEENTM 65), Polysorbate 85 (TWEENTM 85), and the like, and combinations including these materials with other similar surfactants, including ARLACEL® tants, as long as the HLB (Hydrophile-Lipophile Balance) of the surfactant and surfactant mixture favors the formation of an O/W type emulsion system.

In some embodiments the active agent(s) can be in solution with one or more organic solvents, or a combination thereof. The organic solvents can be water miscible or water immiscible. Suitable organic solvents include, but are not limited to, ethanol, methanol, tetrahydrofuran, acetonitrile, acetone, tert-butyl alcohol, dimethyl sulfoxide, N,N-dimethyl formamide, diethyl ether, methylene chloride, ethyl acetate, isopropyl acetate, butyl acetate, propyl acetate, e, hexane, heptane, pentane, 1,3-dioxolane, isopropanol, n-propanol, propionaldehyde, combinations thereof, and the like.

S OF MANUFACTURE Methods for preparing inhalable pharmaceutical compositions in ance with the invention e (i) hydrating a phospholipid, thereby forming a hydrated phospholipid; (ii) mixing the ed phospholipid, a hydrophobic bioactive agent, and an aqueous dispersion e, thereby producing a mixture; and (iii) homogenizing the mixture, thereby producing a dispersion of liposomal particles comprising the olipid and hydrophobic ive agent dispersed within the aqueous sion vehicle and having an average diameter between about 30 and 500 nm. The ratio of hydrophobic bioactive agent:phospholipid is between about 5:1 and about 1:5, the hydrophobic bioactive agent is between about 0.1 and 30 % w/w of the composition, and the phospholipid is between about 0.1 and 30 % w/w of the composition. As a result of the specific formulation and method of manufacture, the composition is characterized by advantageous ties, for example, an average percent ission (APT) between about 50 and 100 % upon continuous aerosolization. Alternatively, the composition can be characterized by other pharmacokinetic properties, such as that, upon continuous aerosolization, the composition is capable of achieving a bioactive agent concentration of at least about 500 ug/g wet lung tissue or a total d dose (TED) of at least about 2,900 ug over 15 seconds.

Although specific embodiments are sed , the dispersions and aerosols of the invention can be ed using various techniques within the purview of those skilled in the art. Such methods include fast freezing methods, precipitation s, emulsion methods and high pressure homogenization methods, for example, as described in 669, the entire contents of which are hereby incorporated herein by reference. Aqueous dispersions according to the present invention can be prepared using any suitable method (e. g., microfluidization) such as those described in U.S. patent applications U.S. 61/313,605, U.S. 61/313,632, U.S. 61/385,194 and U.S. 61/3 85,107, the entire contents of each of which are hereby incorporated herein by reference.

Prior to mixing and nization, it can be helpful to use a solubilizer and/or heating, to help solubilize the ilic bioactive agent. The temperature of heating and time of heating can depend upon the specific lipophilic bioactive agent, the intrinsic thermal stability of the bioactive agent, and lizer utilized. For example, in some embodiments the lipophilic bioactive agent and solubilizer can be heated to a temperature of from about 40 0C to about 650 C, or from about 500 C to about 600 C, or from about 500 C to about 550 C, for a period of time from about 1 minute to about 60 minutes, or about 15 minutes to about 45 minutes, or about 20 minutes to about 30 s. The weight ratio of lipophilic bioactive agent to solubilizer may be about 1:1, in embodiments from about 1:1 to about 4:2, in other embodiments from about 1:2 to about 3 :2.

For example, a solubilizer such as Polysorbate 80 can be capable of dissolving a lipophilic bioactive agent, in embodiments CoQ10, at high levels, with the lipophilic bioactive agent completely soluble in the solubilizer at a ratio of from about 1:2 to about 3:2, when heated to a temperature of from about 50 0C to about 55 0C, a temperature which exceeds the melting point of CoQ10 (which is from about 47 0C to about 48 0C).

As noted above, the amount of solubilizer added to a lipophilic bioactive agent can depend upon the solubilizer, the lipophilic ive agent, and the phospholipids utilized to form the liposomes. In some embodiments, the solubilizer can be present in an amount from about 0.2% to 12% by weight, or about 1.5 % to 6.5% by weight.

The solution of lipophilic ive agent and solubilizer can then be combined with a phospholipid (e. g., to form liposomes) which are in turn formed into a dispersion with an aqueous dispersion vehicle. To prepare the dispersion, the phospholipids and aqueous dispersion vehicle can be mixed together and heated, to approximately 50 0C to 60 0C, e. g., 55 0C, for between about 1-24 hours or for between about 1-8 hours, e.g., about 1 hour.

Suitable fast ng methods for forming aerosolized particles include those ed to herein as spray ng into liquid (SFL), as described in US. Patent No. 6,862,890, the entire disclosure of which is incorporated by nce herein, and ultra- rapid freezing (URF), as described in US. Patent Application Publication No. 2004/0137070, the entire disclosure of which is incorporated by reference herein. In some embodiments, a suitable SFL method can include mixing an active agent with a solution agent, and spraying the effective ingredient-solution agent mixture through an insulating nozzle located at, or below, the level of a cryogenic liquid, so that the spray generates frozen particles. In some embodiments, a suitable URF method can include contacting a solution including an active agent and at least one freezable organic solvent with a cold e so as to freeze the solution, and removing the organic solvent. le precipitation methods for forming aerosolized les include those referred to herein as evaporative precipitation into s solution (EPAS), as bed in U. S. Patent No. 062, the entire disclosure of which is incorporated by reference , and controlled precipitation (CP), as described in US. Patent Application Publication No. 2003/0049323, the entire disclosure of which is incorporated by reference herein. In some ments, a suitable EPAS method can include dissolving a drug or other active agent in at least one organic solvent to form a drug/organic mixture, ng the rganic mixture into an aqueous solution, while concurrently evaporating the organic solvent in the presence of the aqueous solution to form an aqueous dispersion of the drug particles. In some embodiments, a le CP method can include recirculating an anti-solvent through a mixing zone, dissolving a drug or other active agent in a solvent to form a solution, adding the solution to the mixing zone to form a particle slurry in the anti-solvent, and recirculating at least a portion of the particle slurry back through the mixing zone.

Suitable emulsion methods for g lized particles include those referred to herein as HIPE (high internal phase emulsions), as described in U.S. Patent Nos. 5,539,021 and 5,688,842, the entire disclosures of each of which are incorporated by nce . In some embodiments, a le HIPE method can include continuously merging into a disperser, in the presence of an emulsifying and stabilizing amount of a surfactant, a continuous phase liquid stream having a flow rate RJ, and a disperse phase liquid stream having a flow rate R2, and mixing the merged streams with a sufficient amount of shear with R2:R1 sufficiently constant, to form a high internal phase ratio emulsion without phase inversion or stepwise distribution of an internal phase into an external phase.

Suitable high re homogenization methods for forming aerosolized particles include those using homogenizer and uidizer, for e, as described in U.S. patent applications U.S. 61/313,605, U.S. ,632, U.S. 61/385,194 and U.S. 61/3 85,107.

The above methods can produce particles and aerosolized particles that are crystalline or amorphous in morphology. Advantageously, none of these methods require mechanical milling or other similar unit operations that can cause thermal degradation of the active agent.

One or more of the formulations components (e.g., the hydrophobic ive agent, phospholipid, and/or aqueous dispersion vehicle) can be homogenized by mixing at high shear to form a liposomal concentrate utilizing homogenizers, mixers, blenders and similar apparatus within the purview of those skilled in the art. In some embodiments, commercially available homogenizers including an Ultra-Turrax TP 18/10 Homogenizer or similar types of stator/rotor homogenizers made by Gifford-Wood, Frain, IIQA and others as well as multi-stage nizers, colloid mills, sonolators or other types of homogenizers can be used to produce submicron liposomal dispersions of the lipophilic bioactive agent. The stator/rotor type homogenizers bed above have an operational range of from about 100 rpm to about 15,000 rpm and can be supplied with a range of low shear, standard shear, and high shear head screens.

Homogenization can be carried out by mixing the two phases at suitable speeds of, for example, from about 5,000 rpm to about 15,000 rpm, in some embodiments about 5,000, 7,500, 10,000, 12,500, or 15,000 rpm or and value or range therebetween. The shear rate of the homogenizer can also be increased or decreased independent of the speed of the homogenizing shaft by increasing or decreasing the size of the sing screen surrounding the homogenizer head.

In some embodiments, liposomes can be produced with both a standard emulsification screen and a high shear screen supplied for the Silverson L4RT homogenizer. Mixing can occur for a suitable period of time of less than about 90 s, in embodiments from about 2 minutes to about 60 minutes, in embodiments from about 5 minutes to about 45 minutes. In one embodiment, mixing may occur for up to almost 5 minutes. The resulting liposomes can have a particle size of from about nm to about 500 nm, 50 nm to about 200 nm, from about 50 nm to about 150 nm, from about 50 nm to about 100 nm, from about 50 nm to about 75 nm, from about 75 nm to about 100 nm, from about 100 nm to about 150 nm.

In embodiments, the components being mixed can be heated to a ature between about 45 0C to about 65 0C, in embodiments from about 50 0C to about 55 OC, and mixed with high shear homogenization at speeds and for periods of time described above to form ron mes of CoQ10. Where the lipophilic bioactive agent is CoQ10, the processing temperature for the CoQ10 phase, the water/phospholipid phase, and the combined phases should not exceed about 65 0C in order to avoid oxidative degradation of the CoQ10. However, processing the mixture at a temperature from about 50 0C to about 60 0C can be desirable to obtain a desired viscosity of the concentrate of from about 5,000 CF to about 0 CF, in embodiments from about ,000 CF to about 40,000 cP at from about 35 0C to about 45 0C. In some embodiments, processing for extended periods, e. g., for up to about 60 minutes at the speeds noted above within this ature range, should not adversely impact the integrity of the resulting liposomes.

The particle size of the lipophilic bioactive agent dispersion can be reduced by utilizing mechanical devices, such as, e.g., milling, application of ultrasonic energy, forming colloidal-sized ts in a spray system, or by shearing the particles in a liquid flowing at high velocity in a restricted passage. Significant energy can be ed to cleave bulk particles. The smaller particles increase the interfacial area of the active agent. In some embodiments, surfactants are used to reduce the interfacial energy, y stabilizing the dispersion. The le size ines the total interfacial area and, thus, the interfacial energy that must be accommodated to achieve a stable system.

As the particle size decreases, increasing energy is required to produce the particle, and since the total e area increases, the surfactant must accommodate a greater interfacial energy.

In a preferred embodiment, the particle size of the bioactive agent dispersion is reduced by using a Microfluidizer. In some embodiments, in reducing the dispersion particle size, it can be desirable for the CoQ10 mixture to pass through several cycles in a Microfluidizer to obtain the desired particle size. For e, a phospholipid dispersion of a bioactive agent (e.g., CoQ10) of the invention can be passed through at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 or more cycles in a Microfluidizer.

Preferably, the phospholipid dispersion of a bioactive agent (e. g., CoQ10) is passed through a sufficient number of cycles in a Microfluidizer to obtain a preferred le size, e. g., a particle size suitable for asal delivery, e. g., via a nebulizer.

Suitable Microfluidizers for use with the invention include, for example, the M110P which is available through Microfluidics, Inc. (MFI). The M110P has a 75-um passage and a F12Y interaction chamber. In processing M3, the Microfluidizer has an inlet pressure of 25,000 psi. us other uidizers are commonly known in the art and are contemplated as being suitable for use in the methods of the invention.

Microfluidizers using in the invention can have an inlet pressure of at least about 20,000 psi, at least about 25,000 psi, and preferably at least about 30,000 psi.

In the examples provided herein, after a minimum of 10 cycles through M110P Microfluidizer with an F12Y interaction chamber with 75-um passages, particles of less than 160 nm mean diameter were produced with lecithin and particles of about 110 nm were produced with DPPC. One of ordinary skill in the art will understand that the ve amounts of the liophilic bioactive agent (e. g., CoQ10), phospholipids (e.g., lecithin, DPPC or DMPC) and aqueous dispersion vehicle can be ed based upon desired properties such as the desired therapeutic use, aerosolization, cokinetics, and/or pharmacodynamics. In the examples provided herein, the Microfluidizer operated at a pressure of about 30,000 PSI, gh other pressures can be used in other embodiments.

Aerosolization Methods in ance with the invention can include aerosolizing the dispersion of mal particles, thereby forming a respirable aerosol comprising a plurality of droplets, each droplet comprising a dispersion of liposomal particles and having a mass median aerodynamic diameter (MMAD) between about 1 and 5 pm.

Though, in some embodiments, particles can have diameters less than 1 pm and/or greater than 5 pm.

Figure 1A shows a schematic of pulmonary delivery of an aqueous liposomal dispersion of a hydrophobic bioactive agent in accordance with the invention. The bulk drug is formulated into a phospholipid-stabilized s dispersion with small (drug) particle size that is lized using the vibrating-mesh nebulizer into droplets containing small drug particles. For definition purposes, “particle” is referring the internal phase of the aqueous dispersion and “droplet” is referring the result of becoming l generated. In various embodiments, each t contains a certain number of drug les. Figure 1B shows three different tested manufacturing processes for obtaining an aqueous dispersion with a small drug particle size. For the purposes of , a phospholipid dispersion containing 6% w/w of lecithin in water was added to the molten CoQ10 (1% w/w) at 55 OC. The formulation was then processed as follows (1) High Shear Mixing -Turrax® TP 18/10 Homogenizer with 8 mm rotor blade, IM- Werke, Staufen, Germany): 100 mL of formulation was d at 300 rpm and high shear mixed at 10-12 thousands rpm for 45 s; (2) Microfluidization (M-110Y High Pressure Pneumatic Microfluidizer®, Microfluidics, Newton, MA USA): This process works by having two jet streams in opposite directions. Each pass represents one chance that the drug particles have to collide against each other during this process, breaking apart and ng smaller. The formulation was predispersed using probe sonication for 2 minutes, followed by 30 passes at approximately 13 Kpsi; or (3) Ultrasonication (Omni Sonic Ruptor-250® Ultrasonic Homogenizer with 5/32" (3.9mm) Micro-Tip Probe, Omni International, Kennesaw, GA, USA): at 125W for 60 minutes.

A comparison of the results of these different manufacturing methodologies are shown in Figure 5 and discussed in further detail below.

Production and delivery of aerosols in accordance with the present invention can be achieved through any suitable delivery means for continuous nebulization or aqueous liposomal dispersions, including nebulizers. The most suitable ry means will depend upon the active agent to be delivered to the lung, the other components of the formulation, the d effective amount for that active agent, and characteristics specific to a given patient. Given the present disclosure, the details of selecting and operating such devices are within the purview of those skilled in the art.

In various embodiments, aerosols in accordance with the invention can be delivered by an ultrasonic wave nebulizer, a jet nebulizer, a soft mist inhaler, an onic vibrating mesh nebulizer or other nebulizer utilizing vibrating mesh technology. For example, suitable ultrasonic wave nebulizers include Omron NE-Ul7 ble from Omron Corporation of Japan and Beurer Nebulizer IH30 available from Beurer GmbH of Germany. Suitable jet nebulizers include, for example, AquaTower available from A&H Products, Inc. of Oklahoma. Suitable soft mist nebulizers include, for example, at Soft Mist ble from nger Ingelheim GmbH of Germany. Suitable vibrating mesh nebulizers e, for example, Pari eFlow ble from Pari Pharma GmbH of Germany, Respironics i-Neb available from onics Inc. of Pittsburg, Pennsylvania, Omron MicroAir available from Omron Corporation of Japan, Beurer Nebulizer IH50 available from Beurer GmbH of Germany, and Aerogen Aeroneb available from Aerogen Ltd. of Ireland. With t to the present invention, a nebulizer is selected for inhalation therapy over pressurized d Dose Inhalers (pMDIs) and Dry Powder Inhalers (DPIs) by virtue of their capability of delivering high amounts of drugs via e breathing. Therefore, patients with impaired pulmonary function (e. g. lung cancer patients) are not expected to experience ulty during administration of the drug.

While the instant disclosure has discussed inhalation formulations in some detail, depending on the specific conditions being treated, the lipophilic bioactive agents, described above can also be ated and administered by other systemic and/or local routes. For example, aerosols can be delivered selectively to one or more regions of the respiratory tract, mouth, trachea, lungs, nose, mucosa, sinuses, or a combination f.

Delivery can achieve one or more of topical, local, or systemic delivery, or a combination thereof. Alternatively, aerosols can also be used for non-inhalation delivery. Compositions of the present invention can also be administered in vitro to a cell (for example, to induce apoptosis in a cancer cell in an in vitro culture or for scientific, al, or pre-clinical mentation) by simply adding the composition to the fluid in which the cell is contained.

METHODS OF TREATMENT Compositions of the present disclosure can be ed to administer ilic bioactive agents for the treatment of any disease or condition which may t from the application of the lipophilic bioactive agent, ing those disclosed in International Publication No. , the entire disclosure of which is incorporated by reference herein.

Method for stering an inhalable pharmaceutical composition in accordance with the present invention can include the steps of: (i) aerosolizing a dispersion of mal particles, thereby forming a respirable aerosol comprising a plurality of droplets having a mass median aerodynamic diameter (MMAD) between about 1 and 5 um and (ii) delivering a therapeutically effective amount of the hydrophobic bioactive agent to a lung of a subject in need of treatment. Further, the dispersion of liposomal particles has an average er between about 30 and 500 nm, each liposomal particle comprising a hydrophobic bioactive agent and a phospholipid dispersed within an aqueous dispersion vehicle. Furthermore, the ratio of hydrophobic bioactive agent:phospholipid is between about 5:1 and about 1:5, the hydrophobic ive agent is between about 0.1 and 30 % w/w of the composition, and the phospholipid is between about 0.1 and 30 % w/w of the composition.

As a result of the specific formulation and method of manufacture, the composition is characterized by ageous properties, for example, an average percent transmission (APT) between about 50 and 100 % upon continuous aerosolization.

Alternatively, the composition can be characterized by other pharmacokinetic ties, such as that, upon continuous lization, the ition is capable of achieving a bioactive agent concentration of at least about 600 ug/g wet lung tissue or a total emitted dose (TED) of at least about 2,900 ug over 15 seconds.

Other cokinetic properties can include mass fraction deposited, amount of drug and/or formulation delivered to the target, and residence time at the target. In some embodiments, the invention can be used to deposit a mass fraction of at least about 1, 5, 10, 15, or 20 %. The invention can also be used to facilitate delivery of over 0.25 pg/g of an active agent to the deep lung. In certain embodiments delivery to the lung can be of at least about 1, 5, 10, 15, 20, 25, 30, 50, 100, 200, 300, 400, or 500 ug/g of bioactive agent in lung tissue. Furthermore, the formulations can remain in the lungs (e. g., “residence time”) for a period of at least about 2, 4, 6, 8, 10, 12, 24, or 48 hours.

The terms “pharmaceutically effective amount” and “therapeutically effective amount” as used herein include a quantity or a concentration of a bioactive agent or drug that produces a desired pharmacological or therapeutic effect when administered to an animal t, including a human. The amount of active agent or drug that es a pharmaceutically effective amount or a therapeutically effective amount can vary according to factors such as the type of drug utilized, the potency of the particular drug, the route of stration of the formulation, the system used to administer the formulation, combinations thereof, and the like.

The terms “treatment” or “treating” herein include any treatment of a disease in a mammal, including: (i) preventing the disease, that is, causing the clinical symptoms of the disease not to develop; (ii) inhibiting the disease, that is, arresting the pment of al symptoms; and/or (iii) relieving the disease, that is, causing regression of the clinical symptoms.

] In some embodiments, compositions of the present disclosure can be utilized in the treatment of cancer. As used herein, r” refers to all types of cancer or neoplasm or malignant tumors found in mammals, including, but not limited to: leukemias, lymphomas, melanomas, carcinomas and sarcomas. As used herein, the terms r,77 6‘neoplasm,” and “tumor,” are used interchangeably and in either the singular or plural form, refer to cells that have undergone a malignant transformation that makes them pathological to the host organism.

] Primary cancer cells (that is, cells obtained from near the site of malignant transformation) can be readily distinguished from non-cancerous cells by well- established techniques, including histological examination. The definition of a cancer cell, as used herein, es not only a primary cancer cell, but any cell derived from a cancer cell ancestor. This includes metastasized cancer cells, and in vitro cultures and cell lines derived from cancer cells.

] When referring to a type of cancer that normally manifests as a solid tumor, a “clinically detectable” tumor is one that is detectable on the basis of tumor mass, e. g., by procedures such as CAT scan, MR imaging, X-ray, ultrasound or palpation, and/or which is detectable because of the expression of one or more cancer-specific antigens in a sample obtainable from a patient. es of cancers include cancer of the brain, breast, pancreas, cervix, colon, head and neck, kidney, lung, non-small cell lung, melanoma, elioma, ovary, sarcoma, stomach, uterus and Medulloblastoma.

The term “sarcoma” generally refers to a tumor which is made up of a substance like the nic connective tissue and is generally composed of closely packed cells embedded in a fibrillar or homogeneous substance. Examples of sarcomas which can be d with compositions including aerosolized particles of the present disclosure, and optionally a potentiator and/or chemotherapeutic agent include, but not limited to, a chondrosarcoma, fibrosarcoma, lymphosarcoma, melanosarcoma, myxosarcoma, osteosarcoma, e sarcoma, liposarcoma, ar soft part sarcoma, ameloblastic sarcoma, botryoid sarcoma, chloroma sarcoma, chorine carcinoma, embryonal a, Wilms’ tumor sarcoma, trial sarcoma, stromal sarcoma, Ewing’s sarcoma, fascial a, fibroblastic sarcoma, giant cell sarcoma, granulocytic a, Hodgkin’s sarcoma, idiopathic multiple pigmented hemorrhagic sarcoma, immunoblastic sarcoma of B cells, lymphoma, blastic sarcoma of T-cells, Jensen’s sarcoma, Kaposi’s sarcoma, r cell sarcoma, arcoma, leukosarcoma, malignant mesenchymoma sarcoma, parosteal sarcoma, reticulocytic sarcoma, Rous sarcoma, serocystic sarcoma, synovial sarcoma, and telangiectatic sarcoma.

The term “melanoma” includes a tumor arising from the cytic system of the skin and other organs. Melanomas which can be treated with compositions including aerosolized particles of the present disclosure include, but are not limited to, acral-lentiginous melanoma, amelanotic ma, benign juvenile I melanoma, Cloudman’s melanoma, 891 melanoma, Harding-Passey melanoma, juvenile melanoma, lentigo maligna melanoma, malignant melanoma, nodular ma, subungual melanoma, and superficial spreading melanoma.

The term “carcinoma” refers to a malignant new growth made up of epithelial cells tending to infiltrate the surrounding tissues and give rise to metastases.

Carcinomas which can be treated with compositions including aerosolized particles of the disclosure include, but are not d to, acinar carcinoma, acinous carcinoma, adenocystic carcinoma, adenoid cystic carcinoma, carcinoma adenomatosum, carcinoma of adrenal , alveolar carcinoma, alveolar cell carcinoma, basal cell carcinoma, carcinoma basocellulare, basaloid carcinoma, basosquamous cell oma, bronchioalveolar carcinoma, bronchiolar carcinoma, bronchogenic carcinoma, cerebriform carcinoma, cholangiocellular carcinoma, chorionic carcinoma, colloid carcinoma, comedo carcinoma, corpus carcinoma, cribriform carcinoma, oma en cuirasse, carcinoma cutaneum, cylindrical carcinoma, cylindrical cell carcinoma, duct carcinoma, carcinoma durum, embryonal carcinoma, encephaloid carcinoma, epiermoid carcinoma, carcinoma epitheliale adenoides, exophytic oma, carcinoma ex ulcere, carcinoma um, gelatiniform carcinoma, gelatinous carcinoma, giant cell carcinoma, carcinoma ocellulare, glandular oma, granulosa cell carcinoma, hair-matrix carcinoma, hematoid oma, hepatocellular carcinoma, Hurthle cell carcinoma, hyaline carcinoma, hypemephroid carcinoma, infantile embryonal carcinoma, oma in situ, intraepidermal carcinoma, pithelial carcinoma, cher’s carcinoma, Kulchitzky-cell carcinoma, large-cell carcinoma, lenticular carcinoma, carcinoma lenticulare, lipomatous carcinoma, lyrnphoepithelial carcinoma, carcinoma medullare, medullary carcinoma, melanotic carcinoma, carcinoma moue, mucinous carcinoma, carcinoma muciparum, carcinoma mucocellulare, idermoid oma, carcinoma mucosum, mucous carcinoma, carcinoma myxomatodes, nasopharyngeal carcinoma, oat cell oma, carcinoma ossificans, osteoid carcinoma, papillary carcinoma, periportal carcinoma, preinvasive carcinoma, prickle cell carcinoma, pultaceous carcinoma, renal cell carcinoma of kidney, reserve cell carcinoma, oma sarcomatodes, schneiderian carcinoma, scirrhous oma, carcinoma scroti, signet- ring cell carcinoma, carcinoma simplex, cell carcinoma, solenoid carcinoma, spheroidal cell carcinoma, spindle cell carcinoma, carcinoma spongiosum, squamous carcinoma, squamous cell carcinoma, string carcinoma, carcinoma telangiectaticum, carcinoma telangiectodes, transitional cell carcinoma, carcinoma tuberosum, tuberous carcinoma, ous carcinoma, and the like.

Additional cancers which can be treated with compositions including aerosolized particles of the present disclosure include, for example, Hodgkin’s Disease, dgkin’s Lymphoma, le myeloma, neuroblastoma, breast cancer, ovarian , lung cancer, rhabdomyosarcoma, primary thrombocytosis, primary macroglobulinemia, small-cell lung tumors, y brain tumors, stomach cancer, colon cancer, malignant pancreatic insulanoma, ant carcinoid, urinary; bladder cancer, ignant skin lesions, testicular cancer, lymphomas, thyroid cancer, neuroblastoma, esophageal cancer, genitourinary tract cancer, malignant hypercalcemia, cervical cancer, endometrial cancer, adrenal cortical cancer, and prostate cancer.

Although various s have been discussed in detail, the compositions and methods of the invention are applicable to other respiratory, oral, nasal, sinus, and pulmonary pathologies including, but not limited to, asthma, allergies, c obstructive pulmonary disease, chronic bronchitis, acute bronchitis, emphysema, cystic fibrosis, pneumonia, tuberculosis, pulmonary edema, acute respiratory distress syndrome, coniosis, interstitial lung e, pulmonary edema, ary embolism, pulmonary hypertension, pleural effusion, pneumothorax, mesothelioma, amyotrophic lateral sclerosis, myasthenia gravis, and lung disease.

EXAMPLES WO 74559 The following Examples are intended to be illustrative only and are not intended to limit the scope of the invention.

Example 1: Development and Characterization of Phospholipid- Stabilized Submicron Aqueous Dispersions of CoQ10 d for Continuous Nebulization This example provides methods for developing suitable formulations for pulmonary delivery of hobic drugs using CoQ10 as a case study. Excipients (e.g., phospholipids) and an aerosolization device (e. g., b Pro® vibrating-mesh nebulizer) were selected after an initial study (data not shown). Initial characterization of the bulk drug using X-ray diffraction (XRD), Differential Scanning Calorimetry (DSC), Laser Diffractometry (LD) and Scanning Electron Microscopy (SEM) was performed. High shear mixing, high pressure homogenization or ultrasonication was then evaluated as feasible manufacturing processes to obtain small le size dispersions of CoQ10. Following selection of an appropriate process, parameters affecting drug particle size were studied. Using LD and gravimetrical analysis, zation was evaluated to assess the performance of the drug-excipients-device combination. CoQ10 powder studied was crystalline with a melting point imately at 51 0C with a particle size of 30 um. Therefore, particle downsizing was deemed necessary for ary delivery. Microfluidization was found to be a suitable method to prepare submicron drug particles in aqueous dispersions. The number of passes and type of phospholipids (lecithin or Dipalmitoyl Phosphatidylcholine — DPPC) used affected final drug particle size of the sions. Nebulization performance of lecithin- stabilized CoQ10 dispersions varied according to number of passes in the microfluidizer.

Furthermore, the rheology of these dispersions appeared to play a role in the aerosol generation from the active ing mesh nebulizer used. In conclusion, aqueous sions of CoQ10 were adequately produced using a microfluidizer with characteristics that were suitable for pulmonary delivery with a zer.

Materials and Methods Materials: Coenzyme Q10 was supplied by Asahi Kasei Corp. (Tokyo, Japan). Lecithin (granular, NF) was purchased from um Chemical Mfg. Corp.

(Gardena, CA, USA). Genzyme Pharmaceuticals (Liestal, Switzerland) provided 1,2- dipalmitoyl-sn-glycero-3phosphocholine (DPPC). Sodium chloride (crystalline, certified ACS) was acquired from Fisher al (Fisher Scientific, Fair lawn, NJ, USA) and the deionized water was obtained from a central reverse osmosis/demineralizer system commonly found in ch laboratories. The dispersant 1,3-propanediol (98%) was purchased from Sigma-Aldrich (St. Louis, MO, USA).

Ethanol 200 proof USP was purchased from Decon Laboratories (King of Prussia, PA, USA).

Bulk Characterization of CoQ10 X-ray diffraction (XRD): Testing was performed using a s Model 1710 X-ray diffractometer (Philips Electronic Instruments Inc., Mahwah, NJ, USA) with y monochromated radiation (CuKOtl, it = 1.54056 A) emitting at an accelerating e of 40 kV and 30 mA. The COQ10 powder was placed into a stage and the sample was d for diffraction patterns from 50 to 500 at 0.050 intervals of 20 angles, with dwell time of 3 seconds.

Difi‘erential ng Calorimetry (DSC): DSC testing was performed using a 2920 Modulated DSC (TA Instruments, New Castle, DE, USA) and analyzed using TA Universal Analysis 2000 Software. Powder of COQ10 was weighed (10.5 mg) into aluminum pan (kit 02190041, Perkin-Elmer Instruments, Norwalk, CT, USA) and crimped. At a heating rate of 10 OC/min, the thermal or of the sample was analyzed from 10 to 120 0C.

Laser Difi‘raction (LD): Bulk COQ10 powder was dispersed in 20% (V/V) 1,3-propanediol in deionized water for analysis of particle size distribution. This dispersed sample was then added to a small cell apparatus in a Malvem Mastersizer S® instrument (Malvern Instruments, Worcestershire, UK) equipped with a 300 mm lens until 5-10% ation was attained. The internal phase and dispersant refractive indexes were 1.45 and 1.33, respectively.

] Scanning Electron Microscopy (SEM): Analysis of physical appearance and estimation of particle size of bulk COQ10 were med using an SEM. An aluminum stage with adhesive carbon tape held the powder sample. g was carried out in a rotary-planetary-tilt stage with platinum-iridium using a Cressington Sputter Coater 208 HR (Cressington Scientific Instruments, Watford, England) under argon atmosphere. The SEM pictures were captured using SmartSEM® graphical user interface software in a Carl Zeiss Supra® 40VP Scanning Electron Microscope (Carl Zeiss AG, Oberkochen, Germany) operated at a working distance of 19 mm and at 5 kV of Electron High Tension (EHT).

] Development of a Manufacturing Methodology Three different manufacturing processes were tested in the present example in order to obtain an aqueous dispersion CoQ10 with a small drug particle size. Similar methods can be adapted to r optimize CoQ10 formulations and to provide formulations for other hydrophobic drugs. A olipid dispersion containing 6% w/w of lecithin (as the example olipid) in water was added to the molten CoQ10 (1% w/w) at 55 OC. The phospholipid concentration was above the critical micellar concentration (e. g., for in, depending on the source and processing method, CMC varies from 1.3 to 5.5 mg/mL). The formulation was then processed as follows.

High Shear : One hundred milliliters of formulation was stirred at 300 rpm and high shear mixed at 10,000-12,000 rpm for 45 minutes using an Ultra- Turrax® TP 18/10 Homogenizer with 8 mm rotor blade (IKA-Werke, Staufen, Germany).

High Pressure Homogenization: High pressure nization was achieved using a microfluidization process. Each pass represents an opportunity for the drug particles to collide against each other, thereby breaking apart and becoming smaller. The formulation was predispersed using probe sonication for 2 minutes, ed by 30 passes at imately 30,000 psi using an M-110Y High Pressure Pneumatic Microfluidizer® (Microfluidics, Newton, MA USA).

Ultrasonication: The formulation was ultrasonicated at 125 W for 60 minutes using an Omni Sonic Ruptor-250® Ultrasonic Homogenizer with 5/32 inch (3.9 mm) with a micro-tip probe (Omni International, Kennesaw, GA, USA).

] Formulation Development ] After selection of the manufacturing process, formulations were prepared with high pressure homogenization to determine the effect of the selected parameters and type of phospholipid on the particle size distribution of the drug dispersion. During preliminary studies, it was observed that the high solute concentration of formulations containing 6% w/w of lecithin did not produce aerosol from the Aeroneb Pro® vibrating- mesh micropump nebulizer. Further preliminary studies also showed that formulations containing a reduced concentration of lecithin (1% w/w, at 1:1 drug-to-lipid ratio) have presented sufficient stability for evaluation of nebulization performance following preparation. Therefore, reduction of phospholipid concentration was necessary while simultaneously g the concentration of CoQ10 nt at an adequate drug-tolipid ratio.

Following hydration, a phospholipid dispersion containing 1% w/w of phospholipid (lecithin or DPPC) in water was added to the molten CoQ10 (1% w/w) at 55 OC. The formulation was then persed using high shear mixing (Ultra-Turrax® TP 18/10 nizer with 8 mm rotor blade, IIQA-Werke, Staufen, Germany) for up to 5 s at 20,000 rpm. Subsequently, the formulation was passed through an M- 110P Bench-top Microfluidizer® (Microfluidics, Newton, MA USA) up to 100 times at approximately 30,000 psi while maintaining the temperature between 50 and 60 0C.

In testing the effects that the type of phospholipid and number of passes have on particle size distribution of the formulations, olipid dispersions were hydrated for approximately 1 hour without stirring (Table 1, ations A and B).

Formulations were then passed through a luidizer 10, 20, 30, 40 and 50 times when comparing different phospholipids; 20, 50, 70 and 100 times when evaluating the effect from number of passes. For nebulization performance tests, the phospholipid dispersions were hydrated ght with stirring and 0.9% w/v of sodium chloride was added to the final ation (Table 1, Formulation C).

The particle size distributions of the formulations were then analyzed using Laser Diffraction (LD) and/or Dynamic Light Scattering (DLS). The surface tension, zeta potential and rheology were also ted. For nebulization performance, aerosol output was performed using LD and gravimetrical is.

Characterization of Formulations Particle Size Distribution: Particle size distribution testing of the dispersed formulations was performed with LD using a wet sample dispersion unit stirring at 1,000 rpm coupled to a Malvem Spraytec® (Malvem Instruments, Worcestershire, UK) equipped with a 300 mm lens. The sed formulations were added to distilled water (dispersant) until approximately 5% laser obscuration was attained. The internal phase and dispersant refractive indexes were set as 1.45 and 1.33, respectively. A timed ement was performed for 45 seconds with 1 second sampling periods (a total of 45 measurements). Results are presented as Dv(X) and span, where X is the cumulative percentile of particles under the referred size (e. g. Dv(50) ponds to the median volume of the particles). Span is a measurement of particle size distribution calculated as [Dv(90) — Dv(10)]/Dv(50)]. A higher span indicates a more polydisperse particle size distribution.

] In addition, the nanoparticle hydrodynamic diameter of the sed formulations was characterized with DLS using a Malvem zer Nano S® rn Instruments, Worcestershire, UK) at 25 OC and uilibrated for 2 minutes. The intercept of the correlation function was n 0.5 and 1.0. The dispersion was diluted with led water.

Surface Tension: Surface tension testing was performed using a TA.XT.plus Texture Analyzer (Texture Technologies, Scarsdale, NY, USA) from the maximum pull on a disk as described in the previous chapter. Briefly, the container and glass disk probe were thoroughly degreased, cleaned with ethanol and allowed to dry.

The probe was attached to the texture analyzer arm, and lowered until the bottom surface of the probe contacted the surface of the liquid formulation contained in the reservoir.

The temperature of the liquid was ed and recorded. At the start of testing, the probe was raised from the surface of the liquid at a constant speed (0.05 mm/s) for 10 mm, while the texture analyzer registered at 5 points per second the force exerted as a function of either time or ce. Using the maximum (detachment) force the surface tension was calculated using Equation 1 below: X/k = 0.0408687 + 6.20312*(x’\2/v) — 0.0240752 (x’\2/v)’\2 (Equation 1) Where x is probe radius, v is volume and k is the meniscus coefficient. The density values used to calculate surface tension were assumed to be the same as the y of water at the measurement temperature.

Zeta Potential: Electrophoretic light scattering was used to m zeta potential testing with a ZetaPlus Zeta Potential Analyzer (Brookhaven Instruments Corp., Holtsville, NY, USA). The samples were analyzed at a constant temperature of OC and nt (neutral) pH. Samples were diluted with distilled water to conductance values of 300 to 550 uS. Each sample was subjected to 10 runs each, with a 5 second interval between measurements. gy: Rheological behavior of the dispersed formulations were tested using a AR-G2 rheometer (TA Instruments, New Castle, DE, USA) equipped with a cone-and-plate geometry (cone er: 40 mm; truncation: 54 um). Zero-gap and rotational mapping, respectively, were med prior to testing. All measurements were executed with fresh sample dispersion at a constant temperature of 25 0C with no pre-shear. Excess sample around the edge of the probe was trimmed and water added to the solvent trap compartment. The samples were measured at steady state flow step over a range of shear rates (300 to 10 s'l) decreasing logarithmically (10 points per decade).

The upper limit of shear rate was determined by hydrodynamic limitations (high probe speed will cause the liquid sample to spill away from the ement zone). The sample period was 10 seconds and considered in equilibrium after 2 utive analyses within 5% tolerance, not exceeding a maximum point time of 2 minutes. The s were evaluated using Rheology Advantage Data Analysis software (TA Instruments, New Castle, DE, USA).

] Nebulization mance: Based on previous experience, the performance of vibrating-mesh nebulizers can be ed by mesh clogging, resulting in variable l emission (e.g., intermittent mist), since this formulation is a dispersed system.

To analyze the nebulization performance of these formulations, we evaluate the changes in ission over time from LD technique measurements. The nebulization performance of the dispersions was evaluated using the “open bench” method with a Malvern Spraytec® instrument (Malvern Instruments, Worcestershire, UK) equipped with 300 mm lens. The nebulizer reservoir was positioned with the membrane at 25 mm above the upper edge of the laser beam and a ce of 25 mm between the lens and the center of the aerosol cloud. Air suction was positioned 10 cm beneath the laser beam. The device and air suction apparatus positions were maintained still throughout the whole measurement period. The internal phase and dispersant refractive indexes were 1.33 (water) and 1.00 (air), respectively. Formulation (10 mL) was added to the nebulizer reservoir. At the start of nebulization, aerosol characteristics were continuously measured every second for 15 minutes. The slope of the transmission-time curves mittograms) were considered when comparing the different phospholipid formulations.

In addition, the Total Aerosol Output (TAO) was gravimetrically measured for each of the formulations studied. Before aerosolization, the nebulizer was d after each formulation was dispensed into the reservoir. The remaining formulation in the nebulizer reservoir was re-weighed after oing 15 minutes of nebulization.

The difference in weight before and after nebulization s in the calculated TAO.

The weight of the nebulizer mouthpiece was not ered during the measurements.

Importantly, neither transmittogram nor TAO provide information regarding drug output from the nebulizer. Information is limited solely to total mass output ets emitted over time). In the aerosolization of these dispersions, droplets not containing drug particles (empty ts) are potentially generated. However, our purpose with this test is to igate the capability of a nebulizer such as the Aeroneb Pro® nebulizer to continuously and steadily aerosolize the aqueous dispersions of Coenzyme Q10 over time. Intermittent mist can be identified in the transmittograms while TAO elucidates the magnitude of total mass being aerosolized. Saline solution (12 mL of 0.9% w/v NaCl in water) was used as the control.

Statistical Analysis: The data is expressed as mean i standard deviation with the exception of surface tension and zeta potential s, which were expressed as mean i standard error. For gy studies, standard errors were provided by the software used to analyze the best fit of the results to the rheological models. Samples were analyzed at least in triplicate and evaluated for statistical differences with One- Way ANOVA for significance when p < 0.05 using NCSS/PASS software Dawson edition. Post hoc comparisons were performed to identify tically significant differences among groups using Kramer method. A paired t—test was performed to analyze statistical differences (p < 0.05) within the same formulation for stability of drug particle size over time and to analyze the effect of different phospholipids processed at the same uidization conditions.

Results and Discussion This e demonstrates the feasibility of the development of a suitable 2012/043001 ation of hydrophobic drugs (e.g., CoQ10) for ary delivery. In particular, the example trates how to different ochemical properties of drug dispersions can influence the nebulization performance. The example also trates how, transmission data from LD and gravimetrical analysis of zer output can be used to evaluate steady aerosolization as a function of time.

The XRD pattern of bulk CoQ10 shows two high intensity peaks (20) at approximately 18.65 and 22.80, indicating the crystalline structure of CoQ10 (Figure 2).

An endothermic peak at approximately 51 0C in the DSC thermogram indicates the low melting point of this compound (Figure 3). The CoQ10 drug particles are unsuitable for pulmonary delivery as bulk material, with Dv(50) of 29.87 pm and span value of 2.05 l.

The magnitude of the particle dimensions were also med by SEM pictures (Figure 4). The first ch to reduce particle size was performed with ball milling for 18 hours, which was unsuccessful because the CoQ10 turned into a cluster of drug mass.

This visual observation was confirmed by an increased particle size (Dv(50) = 29.87 pm, span = 2.282). Due to the low melting point of CoQ10, heat generated during the process and mechanical impact may have both contributed to this outcome. Similar results were found when bulk powder was cryomilled (data not shown).

Therefore, an ative approach to engineering CoQ10 particles for pulmonary delivery was required. High shear mixing, high pressure homogenization and ultrasonication were tested. The results shown in Figure 5 indicate that formulations prepared using shear force presented drug particles in dispersion with nearly a bimodal distribution, confirmed by a higher span value and Dv(50) around 1 pm (Table 2). Both microfluidization and ultrasonication presented a monodisperse, unimodal distribution with a Dv(50) value in the submicron range, so each method is capable of preparing a formulation with small drug particle size, to varying degrees and with varying size butions.

After selecting a process, Formulation A was processed to determine the ce relating the number of passes in the microfluidizer to drug particle size stability (Table l). The LD results show that, following preparation, all formulations presented particle size distribution in the submicron range (Figure 6). After 7 days, the formulations presented larger particles, as compared to the size immediately after preparation, regardless of the number of passes. The DLS results indicate that increasing the number of passes above 50 does not appear to e smaller hydrodynamic diameters or more monodisperse systems (Figure 7). A trough in particle size as function of number of passes has been previously reported and attributed to a ary particle growth due to fusion or Ostwald ripening during repeated homogenization. Nevertheless, no statistical difference was found for drug particle sizes between days 0 and 7 for any individual preparation with any different number of passes.

] Reduction in number of passes and evaluation of different phospholipids were investigated using Formulation B (Table l). DLS analysis shows that drug particle size decrease for increased number of microfluidization passes (e. g., up to 50 passes) for both lecithin and DPPC dispersions of CoQ10 (Figure 8). The DPPC formulation presented smaller particle sizes than the lecithin dispersions of CoQ10 at the same microfluidization conditions (e.g. number of discrete passes), with Z-averages in the ranges of 50-120 nm and 0 nm, respectively. Although the DPPC colloidal dispersion presented smaller PdI values than lecithin-stabilized formulations, both presented high polydispersity (PdI > 0.2). This result indicates that no more than 50 passes are needed to obtain ations with small particle sizes; the final colloidal system will depend on the olipid utilized.

After it was shown that small drug particle dispersion of CoQ10 can be prepared, ability to steadily nebulize these formulations was studied, along with the physicochemical properties influencing zation performance. ittent mist, which is undesirable, can occur when vibrating-mesh zers generate aerosols from suspended dosage forms. Therefore, formulations were evaluated for a lack of intermittent mist, indicating aerosolization continuity throughout the nebulization event.

In this example, a Malvern ec® was used to analyze transmission as a on of time, to select dispersed formulations that continuously aerosolize in an Aeroneb Pro® nebulizer. Alternative method for evaluating changes in nebulized droplet concentration over time are described in General Chapter <l601> of the United States Pharmacopoeia (USP) on the characterization of nebulizer products.

] Prior to setting up the Malvern Spraytec® with the “open bench” method, numerous attempts were made to perform tests using the Malvem-provide inhalation cell accessory (Figure 9). In this system, a laser beam is projected from the left side of the instrument s a detector positioned at the right side. The laser beam crosses the inhalation cell coupled to the Spraytec®. A nebulizer is positioned in front of the inhalation cell and a vacuum line is connected at the back of the cell. An air sheath provide by tubes in the middle of the cell helps direct aerosol droplets from the nebulizer towards the vacuum source. To evaluate nebulizer output, this setup was arranged with the inhalation cell in the horizontal position (900 angle) to measure aerosol generation as close as possible to the ing-mesh. The suction airflow rate was set to 30 L/min and the sheath airflow rate was set to 15 L/min (30 — 15 L/min = 15 L/min) to obtain a final airflow rate of 15 L/min. This airflow rate was selected to match that required to analyze nebulizer formulations in the Next Generation Impactor (NGI) for ison reasons.

An experimental artifact due to an inefficient air sheath in the Malvern Spraytec® was observed, causing the aerosol cloud to invade the detector lens compartment, causing continuously increasing obscuration and consequently reducing transmission. During operation of the inhalation cell a 0.45 pm HEPA membrane filter was positioned e with the vacuum source, to avoid damage to the vacuum source and to prevent exposure to the operator. r, the formulation lly clogged the filter pores, which created back pressure that overcomes the air sheath and directs the droplets towards the detection lens chamber. After the inhalation cell windows fog, transmission values do not return to 100% and inaccurate data provides the appearance of uninterrupted nebulizer operation. ore, a feasible measurement using this setup was not possible. Without wishing to be bound by any particular , it is believed that this was due to the fact that the amount of aerosol ed during each 15-minutes nebulization event was enormous compared to pMDI and DPI deVices, which the inhalation cell was primarily designed for. Therefore, while such known ories are useful in characterizing aerosol generation from those other deVices, they were not useful for continuous nebulizers ing to the present invention.

To overcome this artifact, an “open bench” method was developed. The position of the nebulizer reservoir was selected to avoid Vignetting (wide angle scattered light misses the detector field) while also avoiding recirculating droplets by positioning the air suction source properly for a continuous exhaustion of the generated droplets.

The transmittograms presented in Figure 10 show a nebulization event of 15 minutes for Formulation C (Table 1). At the end of this duration the transmission values go back up to 100% for all formulations, indicating that the measurement was properly performed with no fogging of the detector lens. The three formulations presented a steady nebulization for the l 5 minutes. After this time point, the ission related to the formulation of the 10 pass runs were sed at a different rate than formulations of the 30 and 50 pass runs. To evaluate the nebulization performance of these formulations, the transmittogram was fitted to a linear regression in order to analyze the slopes of the rate curves. By comparing their slopes, the stability of nebulization can be determined.

The slope values and TAO of Formulation C (Table 1) with different numbers of passes in the microfluidizer are presented in Figure 11. A lower slope value for formulations that were run at 10 passes was observed, as compared to 30 and 50 passes. This observation agrees with the relative TAO values. These data indicate that Formulation C (processed with 10 passes in the microfluidizer) presented er nebulization over time than the same ations prepared with increased processing.

Next, the physicochemical properties of Formulation C ed with 10, 30 and 50 passes were d to identify how processing influences nebulization performance. By analyzing hydrodynamic size in the dispersions (Figure 12), it was we observed from LD s that the particle size appeared to be increasing slightly over time with most particles remaining in the nanometer range. When comparing formulations analyzed at day 0 for LD and DLS, we conclude that LD is not a suitable technique for the same reasons described above, based on the Fraunhofer theory. The DLS results show that all ations presented a Z—average of approximately 260 nm.

After 7 days, Dv(50) is still below range of ement for LD technique whereas Z- average did not vary significantly for the 30 and 50 passes. From the particle size distribution results we can conclude that the formulations with the higher number of passes were stable for about 1 week. PdI was n 0.2 and 0.3 following preparation and showed some level of polydispersity after 7 days.

The s indicate that a greater hydrodynamic diameter was formed for these lecithin dispersions (approximately 260 nm) than was formed with the previous formulation analyzed (Formulation B: 120-170 nm). These differences can be explained, at least in part, by the difference in electrolyte concentrations of the formulations. Addition of 0.9% w/v of sodium de to Formulation C serves two purposes: to provide normal logical osmolarity and to reduce variability in aerosol generation from this active vibrating-mesh nebulizer. Solutions with such low ionic concentrations, have a reduced variability factor, increased aerosol output, and smaller droplet sizes. Without wishing to be bound by any particular , low electrolyte content is believed to help to overcome drop detachment resistance from the vibrating-mesh due to an improved electrical conductivity that suppresses the high electrostatic charge of water, which in turn favors aerosol generation.

However, the addition of sodium chloride can also cause colloid instability, according to the Derjaguin-Landau-Verwey-Overbeek (DLVO) theory of interactions of electrolytes on phospholipid es. In this case, a nonspecific adsorption based solely on electrostatic forces (no chemical interactions) can be caused by lent cations (e.g., Na+). A decrease in zeta potential caused by such cations can increase the flocculation rate (e.g., as analyzed by turbidimetry). The addition of the aforementioned salt following uidization was observed to change the dispersion color from dark orange to bright yellow. Despite extensive discussion concerning the mechanism of this colloid stability, t theories in d science are unable to fully explain this phenomenon. Drug particle size distribution of the aqueous dispersion alone does not appear to control zation performance because these dispersions had similar diameters (following preparation), but different aerosolization behavior.

] Increasing the number of microfluidization passes increases both the surface tension and the zeta ial (statistically significant when comparing formulations processed with 10 or 50 passes, see Figure 13). It has been hypothesized that a higher number of passes aids ulation. However, the role of surface n in aerosol generation from active vibrating-mesh nebulizers is not well understood. The present example did not identify a correlation between the Formulation C zeta potential and surface tension that correlates the ent number of microfluidizer passes and respective nebulization performance.

The rheology of the dispersions was studied by plotting the shear stress as a function of shear rate. The Herschel-Bulkley model, Equation 2, best ented the behavior of these three formulations: * yAn (Equation 2) a = 032 + K Where a is shear stress, 03) is yield stress, K is consistency index or viscosity, )1 is shear rate and n is flow index (n = 1: Newtonian fluid; n < 1: shear-thinning; n > 1: shear- ning). Standard errors are 32.74 i 3.58, 31.62 i 2.04, 35.92 i 3.57 for dispersions of CoQ10 prepared with 10, 30 and 50 microfluidization passes, respectively. The three elements of the Herschel-Bulkley model are presented in Figure 14. gh the values of each element are not statistically different by this metric, the similarity between the rheology results and the results of nebulization performance is evident.

Formulations of 30 and 50 passes presented a similar rheological behavior and nebulization performance, which were different from formulations of 10 passes.

Interestingly, all formulations presented thickening behavior (n > 1).

Characteristics like size, size distribution, shape, charge, and the interactions between particles and the surrounding fluid play significant roles in the rheological behavior of these systems. Therefore, it is not surprising that the rheological behavior of the formulations influence nebulization performance, which is a function of the interaction of all the physicochemical characteristics.

The invention provide the first known study investigating the capability of vibrating-mesh nebulizers to ly nebulize sions in which fluid rheology is analyzed as d to performing simpler kinematic viscosity measurements (e.g., the viscosity of the dispersion media per se, without considering the interactions between the sed particles with the surrounding fluid).

Example 2: Prediction of In Vitro Aerosolization Profiles Based on Rheological ors of Aqueous Dispersions of CoQ10 Aerosolization of dispersed formulations can generate droplets containing le drug concentration due to the heterogeneous nature of the dosage form.

Therefore, it can be important to characterize formulations for in vitro drug deposition, which can be med with cascade ors. Laser diffractometry (LD) can also be used for this purpose, but LD’s usefulness is generally limited to on dosage forms.

The nonhomogeneity of dispersions create droplets with geneous concentrations of drug particles, rendering LD unsuitable. The United States Pharmacopoeia (USP) recommends the Next Generation Impactor (NGI) be used for this testing.

Human alveolar surfactant es about 90% phospholipids and 10% neutral lipids. Among the phospholipids, phosphatidylcholine (PC) is predominant (76%), with DPPC being the main component (81% of PC) and dimyristoyl phosphatidylcholine (DMPC) and royl phosphatidylcholine (DSPC) each comprising 3% of PCs. DPPC and DSPC are also present in the mixture of phospholipids that comprise the excipient soybean lecithin, but their concentration varies widely depending on the lecithin source and extraction method.

] The present example provides methods and data for selecting phospholipids formulations in ance with the invention. The present example also provides, more particularly, methods and data for using synthetic phospholipids to prepare ations of CoQ10 having improved nebulization performance, and which have the ial to deliver a desirable Fine Particle Dose (FPD) of CoQ10. The example studied three synthetic phospholipids: DMPC, DPPC, and DSPC, which have l4, l6 and 18 carbons in their ted fatty acid chains and lar weights of 678, 734, and 790 g/mol, respectively.

In addition to the tests described in connection with Example 1, the synthetic phospholipid formulations were further terized for in vitro drug deposition using NGI and Total Emitted Dose (TED) using both NGI and a Dose Uniformity Sampling Apparatus (DUSA) for Dry Powder Inhalers (DPIs) adapted for nebulizers. The results were analyzed in conjunction with the nebulization performance tests for continuous aerosolization and for identifying the physicochemical properties governing the mechanism of aerosol generation of dispersed systems of CoQ10 from the ump nebulizer. The results of Example 1 were also further validated by demonstrating that the rheology of the dispersions plays a role in the hydrodynamics of aerosol production using active vibrating-mesh nebulizer.

Materials and Methods Materials: CoQ10 was supplied by Asahi Kasei Corp. (Tokyo, Japan).

Lecithin (granular, NF) was purchased from Spectrum Chemical Mfg. Corp. (Gardena, CA, USA). Genzyme Pharmaceuticals (Liestal, Switzerland) ed l,2-dimyristoyl- sn-glycero-3phosphocholine (DMPC), l,2-dipalmitoyl-sn-glycerophosphocholine , and l,2-distearoyl-sn-glycerophosphocholine (DSPC). DMPC was also purchased from Lipoid GmbH (Ludswighafen, Germany). Sodium chloride (crystalline, certified ACS) was acquired from Fisher Chemical r Scientific, Fair lawn, NJ, USA) and the deionized water was obtained from a central reverse osmosis/demineralizer system. Hexane and ethanol 200 proof were purchased from Sigma-Aldrich (St. Louis, MO, USA) and methanol from Fisher Chemical (Fisher Scientific, Fair lawn, NJ, USA), all of which were from HPLC grade. The external filter for NGI testing (glass fiber, GC50, 75 mm) and the filter for DUSA (glass fiber, AP40, 47 mm) testing were purchased from Advantec MFS Inc. (Dublin, CA, USA) and from Millipore (Billerica, MA, USA), tively. Syringes (1 mL) and syringe filters (hyperclean, 17 mm, 0.45 pm, PTFE) were obtained from Becton Dickinson (Franklin Lakes, NJ, USA) and Thermo Scientific (Bellefonte, PA, USA), respectively.

Formulation: Formulations (100 mL) were prepared using hot high pressure homogenization to determine the effect of the type of phospholipid on the aerosolization profile — nebulization performance and in vitro drug deposition of particles for ary delivery. 2.5% w/w was selected as the maximum phospholipid tration. During preliminary studies (see Example 5), it was found that the maximum nominal drug g that could be achieved for CoQ10 with ations not presenting intermittent mist within a 15-minute nebulization event using the Aeroneb Pro® zer was 4% w/w. Therefore, formulations with synthetic phospholipids were prepared at a drug-to-lipid ratio of 42.5.

Following overnight hydration while stirring, a phospholipid sion containing 2.5% w/w of phospholipid (DMPC, DPPC, or DSPC) in water was added to the molten CoQ10 (4% w/w) at 55 OC. The ation was then predispersed using high shear mixing with an Ultra-Turrax® TP 18/10 Homogenizer with 8 mm rotor blade (HQA-Werke GmbH, Staufen, Germany) for 5 minutes at 20,000 rpm. Subsequently, each formulation was passed 50 times through an M-l 10P Bench-top Microfluidizer® (Microfluidics, Newton, MA, USA) at approximately 30,000 psi while maintaining a temperature between 55 and 65 OC. Following microfluidization, 0.9% w/v of sodium chloride was added to the final formulation for reasons outlined in the previous example.

The particle size distributions of the formulations were then analyzed using Laser Diffraction (LD) and/or c Light Scattering (DLS). The surface tension, zeta potential and rheology were also evaluated. For nebulization performance, aerosol output generated from an Aeroneb Pro® nebulizer en, Galway, Ireland) was analyzed using LD and gravimetrical analysis. In vitro drug deposition was evaluated using a NGI while the TED was ed from both the NGI results and from measurement using a Dose mity Sampling Apparatus (DUSA). In addition to the characterization and nebulization performance presented in Example 1, the in vitro drug deposition of lecithin dispersion of CoQ10 (drug-to-lipid ratio: 1:1) passed 50 times through the Microfluidizer® was prepared and ed. This was evaluated against the synthetic phospholipid formulations (DMPC, DPPC, or DSPC dispersions of CoQ10).

Details of the preparation, characterization and evaluation of nebulization performance of the lecithin dispersion are presented in Example 1. Testing was performed immediately following preparation, except for stability of drug particle size in the dispersions in which the samples were tested 7 days after preparation.

Characterization le Size Distribution: Particle size distribution g of the dispersed formulations was performed with LD using a wet sample sion unit stirring at 1,000 rpm coupled to a Malvem Spraytec® (Malvem Instruments, Worcestershire, UK) equipped with a 300 mm lens. The dispersed formulations were added to led water (dispersant) until approximately 5% ation was ed. The internal phase and dispersant refractive indexes were 1.45 and 1.33, respectively. A timed measurement was performed for 45 seconds with 1 second sampling periods (a total of 45 data acquisition s). Results are ted as Dv(X) and span, where X is the cumulative percentile of particles under the referred size (e. g. Dv(50) corresponds to the median volume of the particles). Span is the measurement of particle size distribution calculated as [Dv(90) — Dv(10)]/Dv(50)]. A higher span indicates a more polydisperse particle size distribution.

The nanoparticle ynamic diameter of the dispersed formulations was also terized with DLS using a Malvern Zetasizer Nano ZS® (Malvern Instruments, tershire, UK) at 25 OC and uilibrated for 2 minutes. The intercept of the correlation on was between 0.5 and 1.0. Distilled water was used for dilution of the dispersions where needed.

Surface Tension: Surface tension testing was performed using a TA.XT.plus Texture Analyzer (Texture Technologies, Scarsdale, NY, USA). The container and glass disk probe were thoroughly degreased, d with ethanol and allowed to dry. The probe was ed to the texture analyzer arm, and lowered until the bottom surface of the probe contacted the surface of the liquid formulation contained in the reservoir. The temperature of the liquid was measured and recorded. At the start of testing, the probe was raised from the surface of the liquid at a constant speed (0.05 mm/s) for 10 mm, while the texture analyzer registered at 5 points per second the force exerted as a on of either time or ce. Using the maximum (detachment) force the surface tension was calculated using Equation 3 below: X/ k = 0.0408687 + 6.20312 * (XA2/V) * (XA2/V)’\2 (Equation 3) - 0.0240752 Where X is probe radius, V is volume and k is the meniscus coefficient. The density values used to calculate surface tension were assumed to be the same as the density of water at the measurement temperature.

Zeta Potential: Electrophoretic light scattering was used to perform zeta potential testing with a Malvern Zetasizer Nano ZS® (Malvern Instruments, Worcestershire, UK). The s were analyzed at a constant temperature of 25 OC and nt (neutral) pH. Samples were diluted with distilled water, obtaining conductivity values ranging from 400 to 1400 uS/cm. Each sample was analyzed in triplicate and subjected to 10 to 100 runs each measurement, with automatic optimization of attenuation and voltage selection.

Rheology: gical behavior of the dispersed formulations were tested using a AR-G2 rheometer (TA Instruments, New Castle, DE, USA) equipped with a cone-and-plate geometry (cone diameter: 40 mm; truncation: 54 um). ap and onal mapping were performed prior to g. All measurements were executed with fresh sample dispersion at a constant temperature of 25 0C with no pre-shear.

Excess sample around the edge of the probe was trimmed and water was added to the solvent trap compartment. The samples were measured at the steady state flow step over a range of shear rates (from 1000 to as low as 0.01 s'l) decreasing logarithmically (5 points per ). The lower and upper limits of shear rate were determined, respectively, by the instrument sensitivity and hydrodynamic limitations (high probe speed will cause the liquid sample to spill away from the measurement zone) for each formulation. The sample period was 20 seconds and considered in equilibrium after 2 consecutive analyses within 5% tolerance, not exceeding a maximum measurement time of 2 minutes. The results were evaluated using gy Advantage Data Analysis software (TA Instruments, New Castle, DE, USA).

Nebulization mance: The performance of ing-mesh nebulizers with dispersion ations can be affected by mesh clogging, ing in variable l emission (e.g., intermittent mist). To analyze the nebulization performance of the synthetic phospholipid formulations, the changes in transmission over time were evaluated from LD technique measurements. The nebulization performance of the dispersions was evaluated using the “open bench” method with a Malvem Spraytec® (Malvem Instruments, Worcestershire, UK) equipped with 300 mm lens. The nebulizer reservoir was positioned with the vibrating mesh d 25 mm above the upper edge of the laser beam at a distance of 25 mm n the lens and the center of the aerosol cloud. Air suction was positioned 10 cm beneath the laser beam. The device and air suction apparatus ons were not disturbed throughout the entire measurement period. The internal phase and dispersant refractive indexes were 1.33 (water) and 1.00 (air), respectively. Formulation (10 mL) was added to the nebulizer reservoir. At the start of nebulization, l characteristics were continuously measured every second for 15 minutes. The slope of the transmission-time curves (transmittograms) were considered when comparing the different olipid formulations.

In addition, the Total Aerosol Output (TAO) was gravimetrically measured for each of the formulations studied. Before aerosolization, the nebulizer was weighed after each formulation was dispensed into the reservoir. The remaining ation in the nebulizer reservoir was re-weighed after undergoing 15 minutes of nebulization.

The difference in weight before and after nebulization results in the calculated TAO.

The weight of the nebulizer mouthpiece was not considered during the measurements.

WO 74559 Importantly, neither ittogram nor TAO alone provide complete information regarding drug output from the nebulizer. Information is limited solely to total mass output ets emitted over time). In the aerosolization of these dispersions, droplets not containing drug particles (empty droplets) are potentially generated.

Intermittent mist can be identified in the transmittograms while TAO elucidates the magnitude of total mass being aerosolized. Saline on (12 mL of 0.9% w/v NaCl in water) was used as the control.

In vitro Aerodynamic Deposition: To evaluate in vitro aerosol deposition, within a 15-minute nebulization event, the first and last 15 s (herein called initial and final sections or phases) of l generation were collected using NGI or DUSA for DPI (both from Copley Scientific, Nottingham, UK). This design helps in determining whether the slope in transmission, previously observed for lecithin formulations and related to TAO (Chapter 4, Section 4.3), translates into similar drug mass output. [0023 9] To measure the aerodynamic properties of the formulations, the NGI was set up with airflow of 15 L/min and the drug collected from the induction port, the seven stages of the cascade impactor, the micro-orifice collector (MOC) and the external filter was analyzed using High Performance Liquid Chromatography (HPLC). The sum of the masses in each of the mentioned compartments of the NGI hardware setup provides the TED measured from the NGI. The mass deposited in each stage is also used to determine the tion n and to calculate the Mass Median Aerodynamic Diameter (MMAD) as bed in the General Chapter <601> of the USP. This parameter is the equivalent t size in which half (50%) of the droplets are smaller and the other half are larger than the specified cutoff diameter, based on the drug amount deposited in different stages of the NGI. The Geometric Standard Deviation (GSD) can be used to indicate the droplet size distribution around the MMAD. The FPD was calculated from the sum of drug mass deposited on impaction Stages 3 through 7, MOC and external filter (aerodynamic cutoff diameter below 5.39 um).

Losses can occur during the NGI analysis drug collection due to deposition in the nebulizer mouthpiece and/or inner compartments between stages of the cascade or. Mass balance can be performed to ascertain the extent of such losses. During preliminary studies, it was observed that a 15-minute aerosol generation from dispersions prepared with synthetic phospholipids caused high s of formulation to accumulate in the nebulizer mouthpiece. TED was evaluated from an adapted DUSA to confirm that able mass recovery was being achieved during the analysis (Figure ). During DUSA testing, the aerosol was deposited directly onto a glass fiber filter, positioned on one end of the DUSA, which was connected to a vacuum pump. The nebulizer mouthpiece was positioned on the opposite end, and directly connected to the DUSA using a silicone adapter. TED was determined from the drug amount collected in the glass fiber filter and from the internal walls of the DUSA, which was analyzed using HPLC generated data ing a timed nebulization.

To further analyze the dose, FPD results were extrapolated from ond measurements to calculate an estimated total delivered drug (estimated total FPD or FPDet) within a 15-minute period in accordance with Equation 4: vi . 'FE‘S‘ —FP.~::;;.. w :89;;- = E; ; +§——§“fin. -- ‘ “- - 55—1 (Equation 4) Where i is an integer number representing 15-second intervals (time duration of NGI and TED analyses). The j value is the subsequent integer number r than i, and n is the number of 15-second fractions within a 15-minute nebulization period (n = 60). Fine Particle Dose (FPDr) was also calculated based upon FPDet.

HPLC Analysis of CoQ10: This method was adapted from the previously developed method presented in e 4. A Waters HPLC and column system (Waters Co., Milford, MA, USA) ted to a UV detection utilized a 1525 binary pump, a 717 autosampler, a 2487 dual 7t absorbance or, set at 275 nm, and a Symmetry® RP-C8 column (3.9 X 150 mm, 5 pm) connected to Symmetry® C8 guard column (3.9 X 20 mm, 5 pm). A methanol:hexane mobile phase at 97:3 (v/v) and was eluted at a flow rate of 1.0 mL/min. Stock solution of CoQ10 was initially ved in hexane:ethanol at a ratio of 2:1 (v/v) and then diluted with the mobile phase to obtain the desired concentrations. The linearity range was determined by injecting 50 uL of samples at a controlled temperature of 40 0C. Chromatogram peaks were acquired within run time of 9 minutes and the peak areas were used to determine curve linearity.

All samples were collected from NGI and DUSA g with ethanol, with the exception of drug collection from the NGI plates (Stages 1 through 7 and MOC) for analysis of in dispersions. Due to the low solubility of the formulation in ethanol, a mixture of hexane:ethanol 2:1 v/v was utilized. The samples collected in glass fiber filters (external filter in NGI and filter from DUSA) were vortexed for 30 seconds prior to filtering with 0.45 pm syringe filters. Mobile phase was used for sample dilution. ] tical Analysis: The data is expressed as mean i standard deviation with the exception of surface tension, which was expressed as mean i rd error.

For rheology studies, standard errors were provided by the software used to analyze the best fit of the results to the rheological models. Samples were analyzed at least in triplicate and evaluated for statistical differences with One-Way ANOVA for significance when p < 0.05 using NCSS/PASS software Dawson edition. Post hoc comparisons were performed to identify statistically significant differences among groups using Tukey-Kramer method. A paired t—test was performed to analyze statistical differences (p < 0.05) within the same zation event for different formulations and to compare TED methods.

Results and Discussion tic phospholipids (DMPC, DPPC, and DSPC) were used to prepare CoQ10 formulations and compared the results with lecithin formulation analyzed in e 1. Because CoQ10 delivery is achieved via a sion, aerosolization can generate droplets containing differing amounts of drug. Therefore, the aerodynamic properties of the formulation were analyzed using a cascade impactor, based on the drug amount deposited in each stage of the NGI apparatus. Furthermore, TED was ed based on drug collected in a filter delivered directly from the nebulizer mouthpiece.

Nebulization performance ed with the aerodynamic ties of the dispersion can provide a basis for the comparison of the inhalable potential of the ations.

These characteristics also for the identification of physicochemical properties favoring effective drug emission of drug dispersions from a nebulizer.

The hydrodynamic size in the dispersions (Figure 16 and Table 3) show that the lecithin formulation drug particle size was predominantly in the submicron range.

Synthetic phospholipid formulations presented some larger particles, though is of Dv(X) and span does not present tical ences among formulations (excepting the Dv(10) of DMPC and DSPC dispersions). Further analysis of drug particle size distribution using DLS shows that in dispersions presented larger nanoparticles with a higher polydispersity than the synthetic olipid formulations (Figure 17).

Among synthetic phospholipids, the DSPC dispersion presented the largest drug nanoparticles while the DMPC formulation ted the most monodisperse profile.

Following processing, the synthetic phospholipids presented some microparticles, although the population of particles in the nanometric scale was primarily smaller than drug particles that were produced from lecithin dispersions of CleO.

The zeta potential of lecithin dispersion was significantly higher than that of the synthetic olipid dispersions (Figure 18). Without g to be bound by any particular theory, the mixture of different phospholipids at various trations depending on the source and extraction method for lecithin can lead to variable zeta potential values. The zeta potential values of synthetic olipids can be attributed to the presence of sodium chloride in the formulations e increases in ionic th at neutral pH can se the zeta potential of negatively charged olipids like DMPC, DPPC, and DSPC.

] Increasing the number of uidizer passes can cause a decrease in surface tension (e. g., possibly due to a more efficient ulation). For the synthetic phospholipid compositions, an increase in surface tension was observed which tracked the increase in the number of carbons in the acyl chains of the phospholipid. The formulations were designed to have the same amount of DMPC, DPPC, and DSPC at 2.5% w/w. However, the lar weight vary slightly due to the different number of carbons in each respective acyl chain. Accordingly, the molar concentrations of the phospholipids in the dispersions were 36.9, 34.1 and 31.6 mM, respectively. The structure of phospholipids in water dispersions depended directly on the number of phospholipid molecules. Therefore, without wishing to be bound by any particular theory, it is believed that the number of phospholipid molecules available in “solution” to cause a decrease in surface tension at a constant temperature can explain the differences in surface tension. It is noteworthy that the surface tension of the CleO dispersion prepared with lecithin, which is a mixture of phospholipids, falls between the values of DMPC and DSPC (Figure 19).

Particle characteristics such as size, size distribution, shape, charge, deformability, and the interactions between particles and the surrounding fluid can play a role in the rheological behavior of dispersed systems. To evaluate the gy of the dispersions, shear stress was d as a function of shear rate and the results were fit to the best rheological model. The Herschel-Bulldey model (See on 2 and corresponding text above) best represented most of the formulations.

The Power Law model is similar to Herschel-Bulldey, except that it does not present yield stress value. Standard errors are 35.92 i 3.57, 9.83 i 0.17, 10.27 i 0.35, 21.15 i 8.17 for lecithin, DMPC, DPPC and DSPC sions, respectively. The three elements of the Herschel-Bulkley model are presented in Figure 20. DSPC dispersion of CoQ10 was governed by Power Law and therefore did not t yield stress.

Interestingly, the yield stresses of the formulations are shown to be tically different but no trend was fied. DSPC formulation had a significantly higher Non- Newtonian viscosity than the other analyzed samples, possibly due to its t shear- thinning behavior (n < 1). Interestingly, the flow index results indicated that DPPC, DMPC, and lecithin dispersions respectively presented increasing thickening behavior (n > 1).

The rheology was further analyzed by holding shear rate and viscosity as the independent and dependent variables, tively, in order to fit the results to the general flow curve of aqueous dispersions (Figure 21). Graphical representations are presented in Figure 22, which clearly shows the accentuated DSPC formulation shear- thinning event. nt equations related to these models are shown in Table 4. By fitting these curves to the rheological models, it was found that the formulations presented different behavior (Table 5). Standard errors are 93.49 i 8.60, 43.27 i 10.55, 41.34 i 8.57, 16.00 i 4.74 for lecithin, DMPC, DPPC and DSPC dispersions, tively.

The lecithin formulation of CoQ10 fits to the Sisko model, indicating that the investigated shear rate range falls within the mid-to-high shear-rate range related to the general flow curve of dispersions. This is confirmed by the small characteristic time seen in Table 5 and the curve shape at higher shear rates shown in Figure 22. This result also confirms the shear-thickening behavior presented from the evaluation of the Herschel-Bulkley model e 20). Of the formulations studied, only the lecithin dispersion presented thixotropic behavior. This indicates a ependent change following interruption of shear stress (e. g., shear-thinning event) during structure recovery from the shear-thickening behavior presented by this dispersion in the shear rate range studied. Therefore, the synthetic phospholipid formulations promptly recover to their initial state at cessation of shear stress.

The DMPC and DPPC dispersions followed the Cross model, thus both zero- rate and infinite-rate viscosities are presented. However, the ations’ teristic times differ y, with the lowest value shown for the DMPC formulation. This indicates that, similarly to lecithin dispersion, the DMPC formulation falls towards the upper range of shear rate d to the general flow curve of dispersions (Table 5), explaining the second Newtonian plateau (3.66 cP) being greater than the first Newtonian zone (1.13 cP). Therefore, the gical behavior of the DMPC dispersion is closer to the Sisko than the Cross model. For this reason, both lecithin and DMPC dispersions present rate index (or Cross rate nt) values above unity, reflecting the absence of the power law region in the shear rate range investigated. When the viscosity within this specific range is appropriately ing from the first to the second Newtonian zone, 1 — m is close to the rate index n. The shear-thickening behavior is evident from the curve shape at higher shear rates (Figure 22). The larger characteristic time of the DPPC ation indicates that the curve falls more towards the lower range of shear rates and therefore supports the infinite-rate viscosity being smaller than the zero-rate viscosity. The Cross rate constant is close to unity, which indicates a degree of shear-thinning behavior in the power law region. Observation of the curve shape of DPPC dispersion in Figure 22 supports these findings and the relatively low degree of shear-thickening behavior presented in the Herschel-Bulldey model (Figure ). This relatively low degree of shear-thickening behavior, when compared to lecithin and DMPC formulations, can be attributed to differences in rheology at higher shear rates.

The rheological behavior of the DSPC followed the Williamson model. The statistically significant higher teristic time in conjunction with the flow curve shape of this dispersion indicate that the shear rate range investigated falls within the low-mid shear rate range of the l flow curve of dispersions (Figure 22). The rate index value reflects the shear-thinning behavior at the power law region (Table 5). [0025 6] As discussed in tion with Example 1, it can be important to investigate the capability of vibrating-mesh nebulizers to continuously and steadily aerosolize dispersions, with concomitant analysis of fluid gy as opposed to simpler tic viscosity measurements. Previous works have focused on the viscosity of the dispersion media per se, less of the interactions between the sed particles within the surrounding fluid. Because high frequency mechanical stress of the nebulizer is directly transferred to the formulation, analysis of rheology parameters at higher shear rates may better translate to what is actually occurring in the vicinity of the vibrating membrane.

Some standard error values ed from fitting the results to gical models can be considered relatively high. Without wishing to be bound by any particular theory, it is believed that these values can be attributed to a limited shear rate range studied using the mental design of the present examples. Although further and/or additional ments could be conducted to lower standard error, the understanding of formulation reaction to the stress applied nevertheless provides valuable information about what can be expected from the active membrane nebulization of such dispersions.

In order to compare the nebulization performance of the formulations, a Malvern Spraytec® was set up with the open bench method described in Example 1.

The transmittograms presented in Figure 23 show nebulization events with a 15 minute duration. At the end of this duration, the transmission values returns to 100%, indicating that the measurements were med properly and without detector lens fogging. To evaluate the nebulization performance of these formulations, the ittograms were fitted to a linear regression to analyze the slopes of the curves. The steadiness of a given nebulization event can be inferred from the slope. The slopes and the TAO results are presented in Figure 24. Aerosolization of the control (i.e., saline) was steadiest over time, as indicated by the slope of essentially zero and the highest TAO. The lecithin formulation exhibited steady nebulization for the initial 5 minutes (300 seconds), followed by an increase in ission. The DMPC sion exhibited a transmission profile with a pattern opposite to lecithin. At the start of nebulization, a slight slope was observed up to about 8 s (480 seconds), followed by steady nebulization. DPPC and DSPC dispersions presented a very shallow slope throughout nebulization.

The lecithin dispersion exhibited the highest slope and a low TAO (that was not statistically different from the DMPC formulation). Although the DPPC and DSPC formulations presented similar slopes (e.g., not statistically different), the TAO from DSPC showed a higher mass output than that the TAO from DPPC, despite both formulations being steadily zed. These results show the importance of analyzing the slope of the transmittograms in conjunction with the mass output (or TAO). The DSPC formulation presented the best results among the aqueous dispersions of CoQ10, ting a low slope value and the highest TAO among the phospholipid dispersions.

To ize, the order of sing nebulization performance in the studied formulations was: Lecithin < DMPC < DPPC < DSPC.

These gs can be evaluated concomitantly with the respective rheological behavior of the formulations at higher shear rates. Upon examination of the curves (Figure 22), at high shear rates lecithin and DMPC sions t the characteristic shear-thickening behavior following the second Newtonian plateau, which is confirmed by their low respective characteristic times. The occurrence of shear- thickening following the shear-thinning event can be attributed to an arrangement instability following the two-dimensional ng of the fluid. Being above a al shear stress can causes random arrangement of the dispersed particles, resulting in an increase in viscosity. The random arrangement can limit steady nebulization performance, as shown by these two formulations. On the other hand, the high characteristic times and shear-thinning behavior at the power law region presented by DSPC, and to a lesser extent DPPC, dispersions at high shear rates can explain their vely superior nebulization performance. These results t that a high characteristic time corresponding to a shear-thinning behavior at high shear rates may favor the nebulization performance, while shear-thickening (low characteristic time) may have the opposite effect. Therefore, these results suggest that the rheological behavior at high shear rates can be directly related to the nebulization performance of the dispersions.

] However, these data suggest that mass output may not be correlated (e. g., directly correlated) to drug on in the case of the nebulization of the dispersions described herein. Therefore, in order to measure drug aerosolization and to gain understanding of the namic ties of the formulations, the in vitro deposition of the phospholipid formulations of CleO was analyzed using NGI and adapted DUSA. Analysis of drug deposition at initial and final time fractions of the 15-minute nebulization period allowed for an evaluation of this data in conjunction with the nebulization mance.

The TED of in, DMPC, DPPC and DSPC formulations are presented in Figure 25. The lecithin dispersion of CoQ10 presented a statistically significant decrease in drug aerosolization comparing l and final phases of nebulization period, following both NGI and DUSA analysis. This difference in amount of drug emitted at the beginning and at the end of the nebulization confirms that the slope (25.99 X 10'3 i 2.80 X 10'3 %/s) observed in the results from nebulization performance using LD is not only related to decreased mass output, but also to the amount of drug being aerosolized.

Overall, the lecithin dispersion also ted a significantly smaller TED both at the initial and final phases when ed to the synthetic phospholipid formulations.

No statistical ence was found within the same nebulization event for the dispersions prepared with synthetic phospholipids under NGI analysis. However, the DMPC dispersion exhibited a smaller TED within the same nebulization event using the DUSA methodology. However, the SA results can be more relevant to the present analysis because the ts containing the drug are directly deposited in a filter whereas the TED/NGI results have potential losses associated with the NGI apparatus.

Regardless of the potential losses, a satisfactory mass balance was achieved because no statistical ence was identified in comparing the two methods’ determination of TED. The slope (16.06 X 10'3 i 2.88 X 10'3 %/s) from nebulization performance testing of DMPC dispersion is in agreement with the difference in drug amount being aerosolized within the 15-minute zation period. DPPC and DSPC dispersions of CoQ10 aerosolized in appr0Ximately equal. These results show that these formulations both eXhibit steady nebulization (e. g., as quantified in the relatively small linear sion slope values).

Aerodynamic properties that can affect pulmonary drug delivery are shown in Figures 26 and 27. The lecithin formulation exhibited a higher droplet size, as related to drug mass on deposited, at the initial stage of nebulization than at the final stage (Figure 26). The DMPC formulation, to a lesser extent, exhibited a similar pattern to the lecithin ation. The DPPC and DSPC formulations had a more ed droplet size throughout the 15 minute nebulization event. With respect to the drug amount deposited (as opposed to drug fraction), Figure 27 shows that the overall deposition of lecithin formulation was low both at the l and final phases (e. g., when compared to the other formulations). This result is in ent with the TED results. Among the three synthetic olipids studied, the DMPC formulation presented the lowest deposition, which is in agreement with the TAO and TED results. The DPPC and DSPC formulations had high drug amounts deposited and maintained consistent aerodynamic properties throughout the 15 minute nebulization event.

To further e the aerodynamic properties of the aerosolized dispersions, the MMADs and GSDs are presented in Figure 28. The MMAD and GSD values are initially similar for all four formulations. However, by the completion of the nebulization event, the values were different. This behavior indicates that the size of the emitted droplets containing drug nanoparticles is phospholipid dependent. Remarkably, the changes in transmittogram slope observed within the same nebulization event for lecithin and DMPC sions (Figure 24) are reflected not only in the amount of drug being aerosolized (TED results, Figure 25), but also on the aerodynamic properties shown in their in vitro NGI deposition profiles es 26 and 27). As the nebulization progresses, the droplets aerosolized became smaller and fewer.

A further understanding of the nebulization ’s potential for lung deposition can be obtained by analyzing fine particles (e. g., aerodynamic sizes below .39 pm). Figure 29A shows the TED NGI and TED DUSA values for the studied formulations. The TED NGI data suggests that only the lecithin formulation exhibited a significant ence in drug amount aerosolized when comparing the initial and final phases within a 15-minutes nebulization. The TED DUSA values show that the lecithin and DMPC formulations exhibited a difference in drug amount aerosolized when comparing the initial and final phases within a 15-minutes nebulization. The TED DUSA results can be considered more meaningful e droplets containing the drug are directly deposited in a filter for measurement, s the TED NGI results can have losses throughout the NGI equipment aerosol passageways. Figure 29B shows the FPDet and FPF values for the studied formulations. The FPF increased over time for all of the dispersions aerosolized with the Aeroneb Pro® nebulizer under the present experimental conditions, confirming that droplet sizes decreases during the course of nebulization. The FPD of the lecithin ation changes drastically during nebulization. The MMAD values of aerosolized DMPC dispersions decreases during zation, while FPD does not statistically change. The DPPC formulation exhibited steady nebulization performance and, consequently, consistent TED values throughout zation. Although the MMAD values are not statistically different, the FPD results show that the DPPC ation exhibit a higher amount of aerosolized drug by the end of nebulization. A similar behavior was observed for the DSPC formulation, but the results was not statistical significant (P = 0.08) based on this example alone.

Figure 30 shows that the geometric sizes of the droplets containing CoQ10 particles also decrease over time, especially in the lecithin and DMPC formulations.

Aerosols of the DPPC and DSPC formulations exhibited a relatively tent (e. g., similar to the saline l) droplet size during the 15 minute nebulization.

Discrepancies in aerodynamic and geometric sizes can be attributed to the ent experimental setups (see discussion in Example 1).

Table 6 show the edentedly high doses with the potential to reach the lungs (based on FPD) exhibited by the present invention, with the DPPC and DSPC formulations presenting the highest . These doses are approximately 10 to 40 times greater than itraconazole nanodispersions previously aerosolized using the same type of nebulizer (vibrating-mesh device, data not shown) and as much as 280 times greater than previous aerosolization of budesonide suspension (Pulmicort Respule®, AstraZeneca, UK) using a Sidestream® PortaNeb® jet zer -Aid Ltd., UK).

Of perhaps equivalent importance, the present invention allows for ing the quality and ty of nebulization (e. g., bolus vs. steady aerosol during nebulization event).

In some cases refinements can be necessary for effective drug loading. For example, water evaporation can occur during hot high pressure homogenization.

Similarly, the small volume of formulation prepared (e. g., 100 mL) can result in drug loss through deposition on the cturing equipment.

Finally, the observed changes in zation performance during nebulization events have been shown to correspond to differences in aerodynamic properties between the different formulations. Nevertheless, the rheological or of these formulations was shown to be compatible with active vibrating-mesh nebulizer for continuously nebulizing olipid-stabilized nanodispersions of hydrophobic bioactive agents. The concentration of the various elements of the dispersion (e.g., hydrophobic bioactive agent) has a significant role in determining the critical shear rate at which the shear thickening event post-second Newtonian plateau begins. Thus, knowledge of a dispersion’s rheology can be used to identify a maximum drug loading while still maintaining a desired nebulization performance. Nebulizer l generation occurs through application of a stress (e.g., air jet stream, ultrasonic force, vibrating- mesh) into or onto the bulk liquid formulation. Therefore, the methodology provided herein, including the ation of rheological studies of dispersions and analysis of nebulization performance using LD techniques, provides for the formulation development of hydrophobic drugs continuous nebulizer based inhalation therapy.

Example 3: Pulmonary Deposition and Systemic Distribution in Mice of Inhalable Formulations of CoQ10 Example 3 presents an tion of in vivo systemic distribution, lung, and nasal tions in mice following pulmonary delivery of CoQ10 formulations ed with synthetic phospholipids. Three synthetic phospholipids were selected to stabilize these sions based upon the experimental results presented above and because of the phospholipids physiological occurrence in the lungs: DMPC, DPPC, and DSPC. Lecithin was not selected as a results of its low in vitro deposition. The dosing apparatus includes a nose-only inhalation chamber receiving l generated by an Aeroneb Pro® vibrating-mesh nebulizer. The results showed the achievement of a high and sustained dose of CoQ10 to the mice’s lungs, which varied from 1.8 to 3.0% of the theoretical exposure.

Materials and Methods Materials: CoQ10 was supplied by Asahi Kasei Corp. , Japan).

Genzyme ceuticals (Liestal, Switzerland) provided l,2-dimyristoyl-sn-glycero phosphocholine (DMPC), 1,2-dipalmitoyl-sn-glycerophosphocholine (DPPC), and 1,2-distearoyl-sn-glycerophosphocholine (DSPC). DMPC was also obtained from Lipoid GmbH (Ludswighafen, Germany). Sodium chloride (crystalline, ied ACS) was acquired from Fisher Chemical (Fisher Scientific, Fair lawn, NJ, USA) and the deionized water was obtained from a l reverse osmosis/demineralizer system.

Mouse int tubes (item E2QY-PC), anterior nose inserts (item E2TE-N) and posterior holders (item E2TA-N) were purchased from le Toxicology Northwest (Richland, WA, USA). A fan (12V, 0.10A, model OD4020-12HB) was purchased from Knight Electronics (Dallas, TX, USA). HPLC grade hexane and ethanol 200 proof were purchased from Aldrich (St. Louis, MO, USA). Syringes (1 mL) and needles (gauges 21G1 and 23G1) were obtained from Becton Dickinson (Franklin Lakes, NJ, USA). Heparinized tubes (1.3 mL ubes Lithium Heparin (LH) with screw cap closure, product no. 41.1393.105) were purchased from Sarstedt AG & Co. (Numbrecht, Germany). Microcentrifuge tubes (1.5 mL, clear, RNase/DNase free, BL3152) were obtained from Bio-Link Scientific, LLC (Wimberley, TX, USA). ation: Formulations were prepared using high re homogenization as described in Example 2. To summarize, following overnight hydration while stirring, a olipid dispersion containing 2.5% w/w of phospholipids (DMPC, DPPC, or DSPC) in water was added to the molten CoQ10 (4% w/w) at 55 OC. The formulation was predispersed, using an Ultra-Turrax® TP 18/10 Homogenizer with 8 mm rotor blade, by high shear mixing (IKA-Werke, Staufen, Germany) for 5 s at 20,000 rpm. The formulation was then passed 50 times through a M-110P “Plug-and-Play” Bench-top Microfluidizer® (Microfluidics, Newton, MA USA) at imately 30,000 psi while maintaining a temperature n 55 and 65 OC. Following microfluidization, 0.9% w/V of sodium chloride was added to the final formulation. A formulation for the control group was similarly prepared using DPPC in absence of drug (CoQ10 was not added).

Pulmonary Delivery to Mice: Animals were caged in groups of 4 and maintained on a normal rodent chow diet with free access to water. A nose-only chamber apparatus capable of dosing six mice at a time was assembled as shown in Figure 31. Prior to dosing, CD-1® IGS ICR mice (Charles River Laboratories International, Inc., Wilmington, MA, USA) were individually acclimatized for approximately 10 minutes per day for 3 days into restraint tubes, cted by an anterior nose insert and a posterior holder. The dosing apparatus was placed inside a fume hood to collect escaping aerosol containing drug. To avoid influence from the airflow provided by the fume hood, an erlenmeyer container was placed at the end of the tubing system as a buffer. The airflow rate was set to 1 L/min to ensure proper drug aerosolization into the nose-only chamber (internal volume: 230 mL; diameter: 3.8 cm; length: 20.3 cm) using an Aeroneb Pro® vibrating-mesh nebulizer (Aerogen, Galway, Ireland). Following ation, all formulations (saline control, DMPC, DPPC, and DSPC) were dosed for 15 minutes to mice weighing from 23 to 33 g each, at time of dosing. Each single-dose studied group consisted of thirty-six male animals. At each time point (0.5, l, 3, 8, 24, and 48 hours after the end of the aerosolization event) six animals randomly selected from ent dosing events of the same formulation were sacrificed by narcosis with carbon dioxide. As part of the collection process, blood was awn by c puncture, lungs were harvested, and a nasal wash was performed.

The s were extracted for analysis with liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS).

Estimated Dose: To estimate the dose to which mice were d during this study, it was assumed that the nose-only chamber gradually fills with the aerosol containing CoQ10. Therefore, the drug concentration steadily increases until it reaches a plateau. At steady-state, it is also d that the rate of drug entering the chamber is equal to the rate of drug g the chamber (dC/dt=0). Therefore, Equation 5 can be used to measure the drug concentration inside the chamber at any given time: C = FPDr/F * (I — e"- k 1:) ion 5) Where C is the drug concentration, FPDr is the rate of delivery of the Fine le Dose (the amount of particles with aerodynamic cutoff diameter below 5.39 pm per minute) as determined in the us chapter, F is the airflow rate, k is the chamber air-change rate and t is any given time within the nebulization period. The chamber airchange rate, k, can be determined based on the airflow rate and on the chamber internal volume, V, based upon Equation 6: k = F/V (Equation 6) Based on these assumptions, the following on 7 describes the estimated dose delivered to mice: 1! ”2 Estimated {30:5- = RH22 ..-‘ 59:2» .2 t -335».m . ‘ .. {2% .1—:... {w 3' _ I}? g r ‘ " 2 (Equation 7) Where RMV is the species-specific Rate Minute Volume and t’ is the duration of the zation event. The estimated dose as calculated above can then be ized by the animal body weight, W (g). RMV is calculated in accordance with Equation 8: RMV = 4.19 * W"0.66 (Equation 8) Analysis of CoQ10 Levels in Lung Tissue, Blood Plasma, and Nasal Cavity: For each ment, CoQ10 levels were determined after liquid extraction using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS).

The methods were validated in the drug tration range of 0.1 to 600 ug/mL. The general sample preparation ols for lung tissue, blood plasma, and nasal cavity analysis are described below.

Following harvesting of the mice lungs, the tissue was weighed (wet weight), frozen in dry ice, and transferred to a -80 0C erator for storage until to analysis.

After samples were thawed for analysis, lung tissue (50 i 1.5 mg) was weighed subsequently homogenized with Dulbecco’s Phosphate Buffer Saline (dPBS).

Homogenate (100 uL) and internal standard were added to isopropanol (IPA) and vortexed. Following centrifugation, the supernatant (100 uL) was added to r tube containing IPA. The sample was vortexed again and transferred for LC-MS/MS analysis.

Following cardiac puncture, approximately 1 mL of mice blood was collected in heparinized tubes and kept in ice bath until centrifugation for 10 minutes at 7000 g.

The supernatant was then transferred to 1.5 mL microcentrifuge tubes and kept refrigerated at -80 0C until analysis (see lung tissue procedure bed above).

A solvent wash was performed to evaluate the amount of drug deposited into the nasal cavity. The murine nasal cavity was directly accessed from the posterior portion of the hard palate by ing a needle into the nasopharynx and flushing the nasal fossa with hexane:ethanol 2:1 (v/v). The t was collected in a scintillation vial from the anterior (frontal) portion of the nose and subsequently allowed to dry at room temperature. The sample was then re-suspended and injected into LC-MS/MS for quantification of CoQ10. tical Analysis: Samples were tested for normality using the Shapiro Wilk test (p < 0.05) and outliers were ed from data analysis. Pharmacokinetic parameters were determined using Microsoft Office Excel 2007 software (Redmond, WA) with the add-in program PKSolver. Statistical analysis was performed using NCSS/PASS software Dawson edition. At each time point, lung tissue samples were analyzed for statistical differences among different groups with One-Way ANOVA for significance (p < 0.05). The same analysis was med for nasal wash samples, with additional post hoc multiple comparison tests performed to identify statistically significant ences between treated and l groups using t’s method (p < 0.05). A paired t—test was performed to analyze statistical differences (p < 0.05) within the same treatment group for changes in drug deposition in the nasal cavity over time.

Results and Discussion A nebulizer was used to te aerosol for dosing mice for 15 minutes with control, DMPC, DPPC, and DSPC formulations. The dose delivered to the lungs was estimated based on the FPDr values, as ined during the in vitro characterization of drug deposition using the Next tion Impactor (NGI) bed in Example 2.

Figure 32 shows the calculated drug concentration-time profile within the dosing chamber. A plateau is reached at 3.0 minutes. The concentration at steady-state (CSS) is equal to FPDr since the airflow rate during this experiment was 1 L/min (Table 7). The chamber air-change rate was 4.35 min'l. The estimated doses delivered to mice of aerosolized DMPC, DPPC, and DSPC dispersions of CoQ10 for 15 minutes increases in this respective order (Figure 33). When normalized to the body weight of animals, similar estimated doses were delivered to mice receiving either DPPC or DSPC formulations. These doses of CoQ10 were found to be greater than when the mice were dosed with the DMPC formulation.

The drug concentration in plasma was below the quantitative level (0.1 ug/mL) for all d groups at every time point. The baseline concentration of CoQ10 in mice blood plasma is approximately 0.1 umol/L (86 ng/mL). In the lungs, the drug concentration was also below the quantitative level for the control group at every time point igated. However, Figure 34 shows that CoQ10 stays in the lungs at relatively high concentrations for up to 48 hours. The mechanism by which CoQ10 could be absorbed through the lung epithelium is unknown. Without wishing to be bound by any ular theory, it is believed that, despite the ilicity of CoQ10, passive diffusion is only part of a more complex absorption process involving an additional active and facilitated transport phenomena. It is possible that the relatively small amount of lungs to systemic translocation is, at least in part, part due to this low permeability. In addition, the dispersions are ated in the nano-size range, which are known (e. g., with respect particles below 0.2-0.5 um) to be stealth to alveolar macrophages. In addition to size, other ochemical properties of the drug can influence the translocation of nanoparticles across the air-blood r, for example: particle material, in vivo solubility, and binding affinity to cell membranes (e. g. through surface charge and structure). The presence of phospholipids in these formulations may have also caused a r lung peripheral distribution of the drug nanoparticles.

The translocation of insoluble nanoparticles across the air-blood barrier is known to be minimal compared to the long term clearance from the alveoli up to the mucociliary escalator and into the GI tract, which can take weeks. A significant spreading of drug towards the lung periphery due to the ce of phospholipids in the formulations investigated in this study is a possible contributing factor explaining why the clearance of CoQ10 from the lungs was not ed after 48 hours and similarly why the drug levels in the plasma were below the quantitative limit. Furthermore, because drug clearance from the lungs was not significant in the studied time period, the ation constants and half-lives could not be determined for the nebulized formulations.

Other pharmacokinetic parameters are presented in Table 8. The lung deposition profiles of aqueous dispersions of CoQ10 using different phospholipids presented relatively similar results. The Cmax ranged from 604.0 to 791.3 ug/g of wet lung tissue, and was observed 1 hour (tmax) post dosing for all treated groups. These values ate to approximately 4.0 to 5.0 mg/kg of mouse body weight and correspond to 1.8 to 3.0% of the tical exposure dose (Figure 35). The AUCO-48 results were surprisingly different; with the DMPC formulation of CoQ10 presenting the highest value regardless of whether the smallest estimated dose that the mice were exposed to was presented. Although DPPC and DSPC dispersions of CleO presented high estimated dose, their Cmax and 8 values varied widely. No statistical differences were found in drug tration at the same time point among the treated groups (Figures 34 and 35).

The drug deposition in the nasal cavity was lower than that which was measured in the lungs (Figure 36), not exceeding an average of 1.7 mg/kg of mouse body weight among the treated groups. Only the DPPC group demonstrated a statistically significant decreasing trend for the first two time points investigated. A small amount of CleO was observed in the control group, possibly from an endogenous source. Finally, all mice were alive and presenting healthy signs 48 hours after the end the nebulization event. This trates the safety of delivering high amounts of exogenous CleO to the lungs.

In Example 2, edentedly high doses with potential to reach the lungs based on FPDet results were predicted, with DPPC and DSPC formulations presenting the highest values. These doses are approximately 10 to 40 times greater than itraconazole nanodispersions previously aerosolized using the same type of nebulizer (vibrating-mesh device) and as much as 280 times greater than previous aerosolization of a budesonide suspension cort Respule®, AstraZeneca, UK) using a Sidestream® PortaNeb® jet nebulizer (Medic-Aid Ltd., UK). This Example verified that the high doses translated into an improved drug deposition in the lungs. Cmax values of CleO were as much as 75-fold and l65-fold higher than previous studies using the same zer to deliver dispersions of porine A and itraconazole, respectively (data not shown). These data present a significant improvement in delivery of high amounts of hydrophobic drug ly to the lungs. The in vitro methods of the invention for designing and screening formulations with optimized potential to deliver high drug amounts to the lungs were essential in achieving these results.

Example 4: Low Concentration Range Determination of Hydrophobic Drugs Using HPLC Preclinical and al studies require the determination of small amounts of compounds (e.g., hydrophobic drugs such as CleO) in different biological fluids and tissues. Currently, there are many analytical s of HPLC with ultraviolet (UV) detectors available. r, for high sensitivity is, more sophisticated and complex methods are required, for example: HPLC followed by chemical reactions, HPLC with electrochemical detectors (ECD) and liquid chromatography-triple quadrupole (tandem) mass spectrometry (LC — MS/MS). Among the ters for validation of HPLC methods are accuracy, precision, range, linearity and limits of detection (LOD) and quantification (LOQ). Signal-to-noise (S/N) ratio is a quick and simple method to determine LOD and LOQ, which are ial when ing low concentration of drugs.

Methods: A Waters HPLC and column system including a 1525 binary pump, a 717 autosampler, a 2487 dual 1 absorbance detector, set at 275 nm, and a Symmetry RP-C8 column 5 um (3.9 X 150 mm) connected to Symmetry C8 guard column 5 um (3.9 X 20 mm) was selected. The mobile phase (MP) includes Methanol:Hexane at 97:3 (v/v). Stock solution of pure CoQ10 was initially dissolved in Hexane:Ethanol (diluent) at a ratio of 2:1 (v/v) and subsequently diluted with the mobile phase to obtain the desired concentration. Limit of Detection (LOD), Limit of Quantification (LOQ) and linearity (3-interday curves) were determined by injecting 50 uL samples at a lled temperature of 30 0C. Chromatogram peaks were acquired within run time of 11 minutes at a flow rate of 1.0 mL/min. Area and height of peaks were used to determine curve linearity. LOD and LOQ were d by signal-to-noise (S/N) ratio calculations according to method from the European Pharmacopoeia, with minimum acceptable values of 3 and 10, respectively. Concentration points were 10, 25, 37.5 and 50 ng/mL (n = 6).

For mobile phase preparation, solvents were filtered prior to use h 0.45 um nylon membrane s and sparged for 10 minutes with helium gas. For preparation of stock and working standard solutions (500 ug/mL), 12.5 mg of CoQ10 was accurately weighed in a 25 mL amber volumetric flask and dissolved in hexane- ethanol (2:1 v/v). Subsequently, this stock standard solution was diluted with MP to 10 ug/mL. To avoid light degradation of the API, standard solutions were kept in amber containers during drug manipulation. g standard solutions were prepared by erring suitable aliquots of stock solution to transparent tubes and diluted to final 2012/043001 concentration with MP. Finally, the working standard solutions were transferred to polypropylene conical containers and placed them in amber HPLC vials for analyses.

Results: The retention time (RT) of CoQ10 was determined as imately 8 minutes and ion of blank sample nt) shown not to interfere in peak determination at 275 nm. Temperature control was observed to be essential to obtain ric peaks at lower concentrations. LOD and LOQ were defined as 10 ng/mL (n = 6; S/N ratio = 6.0; SD = 0.6; RSD = 10.5%) and 25 ng/mL (n = 6; S/N ratio = 12.6; SD = 1.3; RSD = 10.1%); respectively. The curve linearities were obtained using height or area of the chromatogram peaks in the range of 25 to 2500 ng/mL with r2 2 0.9999 (n=3 for each concentration).

Conclusion: The method can be used as an alternative to more complex and expensive methods for analysis of CoQ10 in small concentrations. The ease of sample preparation and small retention time allows for a quick analysis. The possibility of using either the area or the height of chromatogram peaks gives more lity to adapt this method to different applications. Further studies on extraction of CoQ10 from biological materials, stability, and internal standard ion are needed to define the role of this method. This study provides an alternative and suitably stable method to determine CoQ10 at very low concentrations using an economically viable RP-HPLC system.

Example 5: Determination of Suitable Hydrophobic Drug Concentrations in olipid Nanodispersions Suitable for Continuous Nebulization.

In developing hydrophobic drug formulations for continuous nebulization, it can be useful to establish a maximum nominal drug gs to phospholipid-stabilized dispersions that will sustain continuous vibrating-mesh nebulization. This is because, for example, vibrating-mesh nebulizer can exhibit problems such as variable aerosolization due to clogging of mesh pores that can be mitigated by appropriate formulation. s: ations were prepared based upon the general methods discussed in connection with Examples 1 and 2. For this study, specific dispersions were ed with 50 microfluidization discrete passes using 2.5% w/w of dimyristoyl phosphatidylcholine (DMPC) and 7.5%, 7.0%, 6.0%, 5.0%, or 4.0% w/w of COQ10.

The dispersions were then lized within 24 hour using an Aeroneb Pro® nebulizer for 15 minute lization event. The aerosolization profile was monitored via analysis of Total Aerosol Output (TAO) and using laser diffraction with a Malvem Spraytec® coupled with an inhalation cell as described above.

Results and Discussion: The nebulization performances of the DMPC- ized formulations are presented in Figure 37. As the hydrophobic drug concentration decreases, the aerosolization becomes more continuous. The TAO values for decreasing drug concentrations are, respectively, 1.25 g (12.4%), 1.62 g (16.1%) and 2.15 g )0 The TAO results are in agreement with the analysis of nebulization performance from laser diffraction, with increasing values as the drug concentrations decrease. The transmission values do not return to 100% at the end of nebulization, due to an experimental artifact. Although a formulation containing 5% w/w of CoQ10 was prepared, the analysis using laser diffraction could not be performed appropriately due to this artifact. Based on visual observation, it was determined that this drug concentration was not suitable for continuous aerosolization of the CoQ10 dispersion because of tion of intermittent mist during nebulization. For the 4.0% w/w CoQ10 formulation, this intermittence was only observed at the end phase of nebulization, therefore being chosen as a suitable l drug concentration. sion: The l concentration of 4% w/w of CoQ10 was determined to be the riate drug loading for uous aerosolization with the Aeroneb Pro® nebulizer as established using DMPC at 2.5% w/w to stabilize the dispersions. Nominal concentrations can vary depending upon the specific hydrophobic drug used, as well as other components of the formulation such as the phospholipid.

Example 6: Measuring Inflamatory e to Pulmonary Administration of Dispersions of Phospholipid Encapsulated Hydrophobic Bioactive Agents The inflammatory response to the administration of hydrophobic bioactive agents (e. g., as discussed in connection with Example 1-3 above) was measured.

Surgery is med on sacrificed mice to expose the pleural cavity and trachea at the throat. A small incision is cut into the a and a cannula possessing about a 23 gauge needle with a sheath of plastic tubing (about 0.037 inch e diameter (OD) and about 0.025 inch ID) is inserted through the incision to the base of the trachea and clamped to seal the opening. An aliquot (about 0.75 mL) of phosphate ed saline is instilled through the cannula into the lungs and removed to wash the bronchial and alveolar surfaces. This process is repeated for a total of three washes. The phosphate buffered saline containing cells is placed into centrifuge vials and centrifuged at about 3000 rpm (MiniSpin Plus, Eppendorf International, Hamburg, DE). The supernatant is removed leaving the collected cells in the pellet. The supernatant from the BAL hoalveolar Lavage) is analyzed by enzyme-linked immunosorbent assay (ELISA) for IL-12 elevation (n=2 per sample tested). Administering CoQ10 is not associated with a rise in IL-l2 levels and does not cause lung inflammation.

] Example 7: ison of nebulization performance between aqueous dispersions of CoQ10 and an intravenous formulation.

In order to more fully understand the effect of the inclusion and amount certain pharmaceutical formulation components on nebulization performance, the continuous aerosolization of several aqueous sions of CoQ10 and an intravenous formulation were studied. The results of this example are summarized in Figure 38, which shows transmittograms of aerosolization of DMPC- and DSPC-stabilized dispersions, as compared to an intravenous formulation that includes a ular opsonisation reducer. Additional data is presented in s 39-41.

Tested ations studied included (i) a saline control (0.9% w/w NaCl in water); (ii) Lecithin (50 passes, as presented in e 1); (iii) CQDPPC06 - formulation containing DPPC (4:2.5); (iv) CQDSPC01 - formulation containing DSPC (42.5); (v) 05 - formulation containing DMPC (4:2.5); (vi) CQDMPC06 - formulation containing DMPC (3:2.5); (vii) IV Cytotech - an intravenous ation ed by Cytotech Labs for analysis of nebulization performance, including CoQ10:DMPC:Poloxamer 188 (431.5). ations iii-vi were prepared by the method presented in Example 2. Formulation viii was prepared in accordance with the method presented in International Publication Number .

Saline, presented a slope close to zero and a high TAO, which indicates successful delivery of the solution using the nebulizer. Dispersion formulations prepared with DMPC (excepting the IV formulation), despite drug concentration differences, presented similar results both for slope and TAO, whereas lecithin (50 passes) presented the highest slope and a comparatively a low TAO. The ance of analyzing both TAO and slope are illustrated by these figures. Although formulations CQDPPC06 and CQDSPC01 presented similar slopes, the TAO from CQDSPC01 was higher than CQDPPCO6, g a higher output despite both being steady nebulized.

On the other hand, gh the IV formulation ted some zation, the aerosol output was the lowest among all formulations. ore, for all practical purposes, the IV formulation failed to continuously nebulize in that it could not be reasonably used for delivering a therapeutic dose of the bioactive agent. Formulation CQDSP01 presented the arguably best results among the aqueous dispersions of API 31510. The order of nebulization performance observed was (high to low): DSPC, DPPC, DMPC, lecithin, and IV Cytotech.

Figure 40 shows an is of drug particles dispersed in the ations studied in connection with Example 7. in, DMPC, and DSPC present predominantly submicron sizes, although only lecithin presented a low span.

Nevertheless, the lecithin formulation nanoparticles are relatively large (e. g., ~ 260 nm) and polydisperse (PdI > 0.2). The on of micron-size particles is largest in the DSPC formulation. The IV formulation presented a monodisperse distribution of ~ 60 nm particles. The DMPC and DPPC formulations present a mixture of small and large drug les.

Figure 41 shows another analysis of drug particles dispersed in the formulations studied in connection with Example 7. The surface charge of drug particles in dispersion was relatively low for the DMPC, DPPC, and DSPC formulations, as reflected by their zeta potential values. The lecithin formulation had the largest zeta potential, despite the lowest phospholipid concentration. The surface tension of the formulations increase with increasing hydrophobicity of synthetic phospholipids (increase in the number of carbons in lipid chain of phospholipids): DMPC < DPPC < DSPC. Interestingly, the e tension of lecithin, a mixture of phospholipids, falls within the DMPC and DSPC values. r, the mole fractions of the synthetic phospholipids are different because the formulations were prepared by weight (DMPC WO 74559 was the highest and DSPC was the lowest).

Without wishing to be bound by any ular , it is believed that the inclusion of poloxamer in the IV formulation was the predominant factor in the IV formulations weak nebulization performance. However, the differences in nebulization can also be potentially attributed to other factors including, but not limited to, the ion of PBS rather than saline in the IV formulation, ionic concentration and charge of the formulation (e. g., due to different s dispersion agents and/or the presence of the zation reducer), and/or differences in the manufacturing method.

LENTS The specification should be understood as disclosing and encompassing all possible permutations and combinations of the described aspects, embodiments, and examples unless the context indicates otherwise. One of ordinary skill in the art will appreciate that the invention can be practiced by other than the summarized and described aspect, embodiments, and examples, which are presented for purposes of illustration, and that the invention is limited only by the following claims.

Recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value g within the range. Unless otherwise indicated, each individual value is orated into the specification as if it were individually recited. Each of the documents cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, and instructions), are hereby incorporated by reference in their entirety.

] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims (61)

We Claim:

1. An inhalable pharmaceutical composition comprising a dispersion of particles suitable for continuous aerosolization, wherein the particle is a liposome and/or otherwise stabilized together with a phospholipid, the composition comprising: a dispersion of les having an average diameter between about 30 and 500 nm, each particle comprising a hydrophobic bioactive agent, a phospholipid, and an aqueous dispersion vehicle, wherein the ratio of hydrophobic bioactive agent:phospholipid is between about 5:1 and about 1:5, the hydrophobic bioactive agent is n about 0.1 and 30 % w/w of the composition, the phospholipid is between about 0.1 and 30 % w/w of the composition, and the particles are dispersed within the aqueous dispersion vehicle, wherein the hydrophobic bioactive agent comprises coenzyme Q10 ); wherein the phospholipid comprises dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), dimyristoylphosphatidylcholine (DMPC), or a combination thereof; and wherein, upon administration to a subject, the composition is characterized by continuous aerosolization ient to e a therapeutic dose of the hydrophobic bioactive agent to the subject.

2. The inhalable pharmaceutical composition of claim 1, wherein the aqueous dispersion vehicle comprises water or an aqueous salt solution.

3. The inhalable ceutical composition of claim 1, wherein the dispersion of particles is in the form of a continuous respirable l comprising a ity of aqueous droplets containing a dispersion of particles and having a mass median aerodynamic diameter (MMAD) between about 1 and 5 µm. 11154633

4. The inhalable pharmaceutical composition of claim 1, wherein the composition is terized by an average percent ission (APT) between about 50 and 100 % over at least 15 minutes of continuous aerosolization.

5. The inhalable pharmaceutical composition of claim 1, wherein the plurality of droplets has a MMAD between about 1 and 5 µm over at least 15 minutes of continuous aerosolization.

6. The inhalable pharmaceutical ition of claim 1, wherein the composition is characterized by an APT between about 50 and 100 % after at least seven days of storage.

7. The inhalable pharmaceutical ition of claim 1, n the composition is characterized by an APT between about 50 and 100 %, between about 60 and 100 %, between about 70 and 100 %, between about 80 and 100 %, between about 90 and 100 %, between about 50 and 95 %, between about 60 and 95 %, between about 70 and 95 %, between about 80 and 95 %, between about 90 and 95 %, between about 50 and 90 %, between about 60 and 90 %, between about 70 and 90 %, between about 80 and 90 %, less than about 50 %, less than about 55 %, less than about 65 %, less than about 70 %, less than about 75 %, less than about 80 %, less than about 85 %, less than about 90 %, less than about 95 %, less than about 100 %, or any nge or value therebetween.

8. The inhalable pharmaceutical composition of claim 1, wherein the particles have an average diameter between about 30 and 500 nm after at least seven days of storage.

9. The inhalable pharmaceutical composition of claim 1, wherein the composition is characterized by non-Newtonian fluid behavior.

10. The inhalable pharmaceutical composition of claim 1, wherein the composition has a flow index (n) of about 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, or 1.3. 11154633

11. The inhalable pharmaceutical composition of claim 1, wherein the composition has a viscosity () of about 0.1, 0.15, 0.2, 1, 100, or 110 cP.

12. The inhalable ceutical composition of claim 1, wherein the composition has a zeta potential of about 2.5, 1.5, -2.5, -10, -50, -55, or -60 mV.

13. The inhalable pharmaceutical composition of claim 1, wherein the ition has a surface tension of about 25, 30, 35, 40, 45, or 50 mN/m.

14. The inhalable pharmaceutical composition of claim 1, wherein the composition has a yield stress () of about 11, 12, 13, 14, 15, 16, 17, or 18 mPa.

15. The inhalable pharmaceutical composition of claim 1, wherein the dispersion of particles have an average diameter between about 30 and 100 nm, 50 and 150 nm, 30 and 300 nm, 100 and 400 nm, or 200 and 300 nm.

16. The inhalable pharmaceutical composition of claim 1, wherein the composition has a PDI of about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, or 0.7.

17. The inhalable ceutical composition of claim 1, wherein the composition does not comprise an opsonization reducer.

18. The ble ceutical composition of claim 1, wherein the composition has a total aerosol output (TAO) of at least about 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 %.

19. The inhalable pharmaceutical composition of claim 3, wherein the MMAD is about 1, 2, 3, 4, or 5 µm. 11154633

20. The inhalable pharmaceutical composition of claim 3, wherein the plurality of droplets have a geometric standard deviation (GSD) of less than about 2.5, 2.4, 2.3, 2.2, 2.1, 2.0, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1.0, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, or 0.1.

21. The ble pharmaceutical composition of claim 1, wherein the hydrophobic bioactive agent is about 4 % w/w or less of the ition.

22. The inhalable pharmaceutical composition of claim 1, n the hydrophobic bioactive agent is about 1 % w/w of the composition.

23. The inhalable pharmaceutical composition of claim 1, wherein the phospholipid is about 3 % w/w or less of the composition.

24. The inhalable pharmaceutical composition of claim 1, wherein the ratio of hydrophobic bioactive agent:phospholipid is about 1:1, 4:3, or 4:2.5.

25. The inhalable ceutical composition of claim 1, r comprising sodium chloride in an amount less than about 1.0% w/v of the composition.

26. The inhalable pharmaceutical composition of claim 1, further comprising a salt in an amount making the composition essentially tic with the human lung.

27. The inhalable pharmaceutical composition of claim 1, wherein the dispersion is suspension, nano-suspension, emulsion, or microemulsion.

28. The inhalable pharmaceutical composition of claim 1, wherein the hydrophobic bioactive agent comprises one or more hydrophobic nflammatory steroid, NSAID agent, antibacterial agent, antifungal agent, chemotherapeutic agent, vasoldilator, or a combination thereof. 11154633

29. An ble pharmaceutical ition comprising a dispersion of particles suitable for continuous aerosolization, wherein the particle is a liposome and/or otherwise ized together with a phospholipid, the composition comprising: a dispersion of particles having an average diameter between about 30 and 300 nm, each particle comprising CoQ10, DPPC, and an aqueous dispersion vehicle, wherein the ratio of CoQ10:DPPC is between about 5:1 and about 1:5, the CoQ10 is between about 0.1 and 6 % w/w of the composition, and the particles are dispersed within the s dispersion vehicle, and wherein, upon administration to a subject, the composition is characterized by continuous aerosolization ient to provide a therapeutic dose of the hydrophobic bioactive agent to the t.

30. An inhalable pharmaceutical composition comprising a dispersion of particles suitable for continuous aerosolization, wherein the particle is a liposome and/or otherwise stabilized together with a phospholipid, the composition comprising: a dispersion of particles having an average diameter between about 30 and 300 nm, each particle sing CoQ10, DSPC, and an aqueous dispersion vehicle, wherein the ratio of DSPC is between about 5:1 and about 1:5, the CoQ10 is between about 0.1 and 6 % w/w of the composition, and the particles are dispersed within the aqueous dispersion vehicle, and wherein, upon stration to a subject, the composition is characterized by continuous aerosolization sufficient to provide a therapeutic dose of the hydrophobic bioactive agent to the subject.

31. An inhalable pharmaceutical composition comprising a dispersion of particles suitable for continuous aerosolization, wherein the le is a liposome and/or otherwise stabilized together with a olipid, the ition comprising: a dispersion of particles having an average diameter between about 30 and 300 nm, each particle comprising CoQ10, DMPC, and an aqueous dispersion vehicle, 11154633 wherein the ratio of CoQ10:DMPC is between about 5:1 and about 1:5, the CoQ10 is between about 0.1 and 6 % w/w of the composition, and the particles are dispersed within the aqueous dispersion vehicle, and wherein, upon administration to a subject, the composition is characterized by continuous aerosolization sufficient to provide a therapeutic dose of the hydrophobic bioactive agent to the subject.

32. The inhalable pharmaceutical composition of claim 1, wherein, upon continuous lization, the composition is capable of achieving a bioactive agent concentration of at least about 500 µg/g wet lung tissue.

33. A method for preparing an inhalable pharmaceutical composition comprising the steps of: hydrating a phospholipid, y g a hydrated phospholipid; mixing the hydrated phospholipid, a hydrophobic bioactive agent, and an aqueous dispersion vehicle, thereby producing a mixture; and homogenizing the mixture, thereby producing a sion of particles comprising the phospholipid and hydrophobic bioactive agent dispersed within the aqueous dispersion vehicle and having an e er between about 30 and 500, wherein the ratio of hydrophobic bioactive agent:phospholipid is n about 5:1 and about 1:5, the hydrophobic bioactive agent is between about 0.1 and 30 % w/w of the composition, and the phospholipid is between about 0.1 and 30 % w/w of the composition, wherein the particle is a liposome and/or otherwise ized together with a phospholipid; wherein the hydrophobic bioactive agent comprises coenzyme Q10 (CoQ10); wherein the phospholipid comprises dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), dimyristoylphosphatidylcholine (DMPC), or a combination thereof; and n, upon administration to a subject, the composition is characterized by uous aerosolization sufficient to provide a therapeutic dose of the hydrophobic bioactive agent to the subject. 11154633

34. The method of claim 35, further comprising: aerosolizing the dispersion of particles, thereby g a respirable aerosol sing a ity of ts, each droplet sing a dispersion of particles and having a mass median aerodynamic diameter (MMAD) between about 1 and 5 µm.

35. The method of claim 33, wherein mixing comprises high shear mixing for up to about 5 minutes at about 10,000 to 20,000 rpm and at about 50 to 65 ºC.

36. The method of claim 33, wherein homogenizing comprises microfluidization.

37. The method of claim 33, wherein homogenizing comprises high pressure homogenization for about 1-50 passes at about 30,000 psi and at about 50 to 65 ºC.

38. The method of claim 33, wherein homogenizing ses ultrasonic homogenization.

39. The method of claim 33, wherein aerosolization comprises vibrating mesh nebulization.

40. The use of an inhalable composition for the preparation of a medicament for the treatment of a lung disease wherein said composition is formulated for administration to a subject by: aerosolizing a dispersion of particles, y forming a able aerosol comprising a plurality of droplets having a mass median aerodynamic diameter (MMAD) between about 1 and 5 µm, wherein the dispersion of particles has an average diameter between about 30 and 500 nm, each particle comprising a hydrophobic bioactive agent and a phospholipid dispersed within an aqueous dispersion vehicle, 11258975 wherein the ratio of hydrophobic bioactive agent:phospholipid is between about 5:1 and about 1:5, the hydrophobic ive agent is between about 0.1 and 30 % w/w of the composition, and the phospholipid is n about 0.1 and 30 % w/w of the composition, wherein the particle is a liposome and/or otherwise stabilized er with a phospholipid; wherein the hydrophobic bioactive agent comprises coenzyme Q10 (CoQ10); wherein the phospholipid comprises dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), dimyristoylphosphatidylcholine , or a combination thereof; and wherein the composition is formulated for continuous aerosolization sufficient to r a therapeutic dose of the hydrophobic ive agent to the subject.

41. The use of claim 40, wherein aerosolization comprises vibrating mesh nebulization.

42. The use of claim 40, n the aerosol is characterized by an APT between about 50 and 100 % over at least 15 minutes of continuous aerosolization.

43. The use of claim 40, wherein the aerosol is characterized by an APT between about 50 and 100 %, between about 60 and 100 %, between about 70 and 100 %, between about 80 and 100 %, between about 90 and 100 %, less than about 50 %, less than about 60 %, less than about 70 %, less than about 80 %, less than about 90 %, or less than about 100 %.

44. The use of claim 40, wherein the plurality of droplets has a MMAD between about 1 and 5 µm over at least 15 minutes of continuous aerosolization.

45. The use of claim 40, wherein the MMAD is about 1, 2, 3, 4, or 5 µm.

46. The use of claim 40, wherein the droplets have a ric standard deviation (GSD) of at least about 2.0. 11258975

47. The use of claim 40, wherein delivery achieves a mass fraction deposited of at least about 1, 5, 10, 15, or 20 %.

48. The use of claim 40, wherein delivery achieves local delivery to the lung substantially t systemic delivery.

49. The use of claim 40, n delivery achieves an elevated amount of the hydrophobic bioactive agent in the lung for at least 48 hours after administration.

50. The use of claim 40, wherein, upon continuous aerosolization, the composition is capable of achieving a ive agent concentration of at least about 500 µg/g wet lung tissue.

51. The use of claim 40, wherein the composition is formulated for administration of a therapeutically effective amount of the hydrophobic bioactive agent in a metered dose of the bioactive agent.

52. The use of claim 40, wherein the subject has lung cancer.

53. The use of claim 40, wherein the subject has one or more of , allergies, chronic obstructive pulmonary disease, chronic bronchitis, acute bronchitis, emphysema, cystic fibrosis, pneumonia, tuberculosis, pulmonary edema, acute respiratory ss syndrome, pneumoconiosis, interstitial lung disease, pulmonary edema, pulmonary embolism, pulmonary hypertension, pleural effusion, pneumothorax, elioma, amyotrophic lateral sis and myasthenia gravis. 11258975

54. The inhalable pharmaceutical ition of claim 1, wherein the composition is prepared by a process comprising the steps of: hydrating a phospholipid, thereby forming a hydrated phospholipid; mixing the ed phospholipid, a hydrophobic bioactive agent, and an aqueous dispersion vehicle, y producing a mixture; and homogenizing the e, thereby producing a dispersion of les and wherein, upon administration to a subject, the composition is characterized by continuous aerosolization sufficient to provide a therapeutic dose of the hydrophobic bioactive agent to the subject.

55. The inhalable pharmaceutical composition of claim 1, wherein, upon continuous aerosolization, the composition is capable of achieving a total emitted dose (TED) of at least about 2,900 µg over 15 seconds.

56. The inhalable pharmaceutical composition of claim 55, wherein the TED is at least about 3,600, 3,900, 4,300, or 4,600 µg over 15 seconds.

57. The inhalable pharmaceutical composition of claim 1, wherein the continuous aerosolization has a duration of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 50, or 60 minutes.

58. The inhalable pharmaceutical ition of claim 1, further comprising a polyoxypropylene-poloxyethylene block polymer at 0.001-5% by weight of the total ition.

59. Use of an inhalable pharmaceutical composition as described in any one of claims 1-32 in the preparation of a medicament for treating or preventing a lung disease.

60. The use of claim 59, wherein the disease is lung cancer. 11154633

61. The use of claim 59, n the disease is one or more of asthma, allergies, chronic obstructive ary disease, chronic itis, acute bronchitis, emphysema, cystic fibrosis, pneumonia, tuberculosis, pulmonary edema, acute respiratory distress syndrome, pneumoconiosis, interstitial lung disease, pulmonary edema, pulmonary embolism, pulmonary hypertension, pleural effusion, pneumothorax, mesothelioma, ophic lateral sclerosis, and myasthenia gravis. Berg LLC By the Attorneys for the Applicant SPRUSON & FERGUSON Per: 11154633 WO 74559