WO2009120619A2

WO2009120619A2 - Nuclease compositions, methods of making and using such compositions, and systems for pulmonary delivery of such compositions

Info

Publication number: WO2009120619A2
Application number: PCT/US2009/037930
Authority: WO
Inventors: Jeffry G. Weers; Mei-Chang Kuo; Thomas Tarara
Original assignee: Novartis Ag
Priority date: 2008-03-24
Filing date: 2009-03-23
Publication date: 2009-10-01
Also published as: WO2009120619A3

Abstract

A powder composition comprises particles including a nuclease, wherein the particles have a mass median aerodynamic diameter ranging from about 1 µm to about 5 µm. A pharmaceutical composition comprises a powder including an effective amount of nuclease and pharmaceutically acceptable excipient, wherein the powder comprises particles comprising less than 40 wt% of nuclease and having a mass median aerodynamic diameter ranging from about 1 µm to about 5 µm. Unit dosage forms, spray drying methods, and methods of treatment are also included.

Description

NUCLEASE COMPOSITIONS, METHODS OF MAKING AND USING SUCH COMPOSITIONS, AND SYSTEMS FOR PULMONARY DELIVERY OF SUCH COMPOSITIONS

RELATED APPLICATIONS

[001] This application relates to U.S. Provisional Application No. 61/070,693, filed

March 24, 2008, from which priority is claimed under 35 USC §119(e), the disclosure of which is incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

[002] One or more embodiments of the present invention include nuclease compositions, such as dry powders comprising nucleases, methods of making and using such compositions, and systems for pulmonary delivery of such compositions.

[003] A deoxyribonuclease (DNase, for short) is any enzyme that catalyzes the hydrolytic cleavage of phosphodiester linkages in the DNA backbone. Deoxyribonucl eases are thus one type of nuclease.

[004] DNase is a phosphodiesterase capable of hydrolyzing polydeoxyribonucleic acid.

DNase has been purified from various species to various degrees. The complete amino acid sequence for a mammalian DNase was first made available in 1973. See, e.g., Liao, et al., J. Bio.

Chem, 248, 1489 (1973).

[005] DNase has a number of known utilities and has been used for therapeutic purposes. Its principal therapeutic use has been to reduce the viscoelasticity of pulmonary secretions in such diseases as pneumonia and cystic fibrosis, thereby aiding in the clearing of respiratory airways. See, e.g., Lourenco, et al., Arch. Intern. Med. 142, 2299 (1982); Shak, et al.,

Proc. Nat. Acad. Sci. 87, 9188 (1990); and Hubbard, et al., New England Journal of Medicine

326, 812 (1992).

[006] DNA encoding human DNase has been isolated and sequenced and that DNA has been expressed in recombinant mammalian host cells, thereby enabling the production of human

DNase in commercially useful quantities. See, e.g., WO 90/07572 or Shak, et al., Proc. Nat.

Acad. Sci. 87, 9188 (1990). Recombinant human DNase (rhDNase) has been found to be useful clinically, especially in purified form such that the DNase is free from proteases and other proteins with which it is ordinarily associated in nature.

[007] The means and methods by which human DNase can be obtained in pharmaceutically effective form is described in WO 90/07572, cited above. Various specific methods for the purification of DNase are known in the art. See, e.g., Khouw, et al., U.S. Patent

No. 4,065,355, issued 27 December 1977; Markey, FEBS Letters 167, 155 (1984); and Nefsky, et al., Euro. Journ. Biochem. 179, 215 (1989).

[008] DNase can be employed as a mixture of deamidated and non-deamidated forms, or in isolated deamidated and non-deamidated forms, non-deamidated human DNase being regarded as the more active species. The preparation and separation of such forms are the subject matter of WO 90/07572, cited above.

[009] Dornase alfa (proprietary name Pulmozyme® from Genentech) is a highly purified solution of recombinant human deoxyribonuclease I (rhDNase), an enzyme which selectively cleaves DNA. Dornase alfa hydrolyzes the DNA present in sputum/mucus of cystic fibrosis patients and reduces viscosity in the lungs, promoting improved clearance of secretions.

This protein therapeutic agent is produced in Chinese hamster ovary cells. Dornase alfa is the most recent therapeutic agent developed with this basic mechanism of action. Prior to the cloning of the human enzyme, bovine DNase I was on the market for many years, though its utility was limited by the inherent antigenic response to a cow protein in the lungs of patients. Other

DNases, such as DNase II, have therapeutic potential as well, but as of yet no further DNases have been brought to market for cystic fibrosis.

[010] The current nominal dose for Pulmozyme in CF patients is 2.0 mg. The jet nebulizers used to administer Pulmozyme deliver about 10% of the nominal dose into patient's lungs, meaning that the required lung dose is about 200 μg. Pulmozyme requires 10-15 min administration time and also requires additional time to disassemble, clean, and sterilize the aerosol delivery equipment.

[01 1] EP 0 749 225, which is incorporated herein by reference, discloses the preparation of pharmaceutically acceptable formulations comprising spray-dried DNase in therapeutically effective form for administration into the lung of an individual.

[012] U.S. Patent No. 6,309,623, which is incorporated herein by reference, discloses preparation of hollow porous particles of DNase I by spray drying. [013] Cystic fibrosis (CF) is the most common life-shortening genetic disease in the

United States and Northern Europe, affecting approximately 30,000 individuals in the United States (Cunningham, J.C. et al., "An Introduction to Cystic Fibrosis for Patients and Families " 5th ed., Bethesda: Cystic Fibrosis Foundation (2003)) and a similar number of individuals in Western Europe. The genetic defect in this autosomal recessive disease is a mutation in the CF transmembrane conductance regulator (CFTR) gene, which codes for a chloride-channel protein (Collins, F. S., "Cystic Fibrosis Molecular Biology and Therapeutic Implications," Science 256:774-779 (1992)). Persons with CF typically suffer from chronic endobronchial infections, sinusitis, and malabsorption due to pancreatic insufficiency, increased salt loss in sweat, obstructive hepatobiliary disease, and reduced fertility (FitzSimmons, S. C, "The Changing Epidemiology of Cystic Fibrosis," J Pediatr 122:1-9 (1993)). Respiratory disease is a major cause of morbidity and accounts for 90% of mortality in persons with CF (Cystic Fibrosis Foundation, Cystic Fibrosis Foundation Patient Registry 2003 Annual Data Report, Bethesda, MD: Cystic Fibrosis Foundation, (2004); Davis, P.B. et al., "Cystic fibrosis," Amer J. Respir Crit Care Med 154(5): 1229-56 (1996)). Lung function (measured as forced expiratory volume at 1 second (FEVl % predicted) is a significant predictor of survival in CF. Two- year survival for a given population of persons with CF is reduced 2-fold with each 10% reduction in FEVl % predicted, and persons with FEVl below 30% of predicted have a 2-year survival below 50% (Kerem, E. et al., "Prediction of Mortality in Patients with Cystic Fibrosis," N Engl J Med 326:1 187-1191 (1992)). Rates of lung function loss vary both between individuals and over time for a given individual. Retrospective longitudinal analyses show rates of decline ranging from less than 2% Of FEVl % predicted per year to greater than 9% FEVl % predicted per year, with overall rate of decline strongly associated with age of death (Corey, M. et al., "Longitudinal Analysis of Pulmonary Function Decline in Patients with Cystic Fibrosis," J Pediatr 131(6):809-l (1997)). [014] CF patients suffer from thickened mucus caused by perturbed epithelial ion transport that impairs lung host defenses, resulting in increased susceptibility to early endobronchial infections with Staphylococcus aureus, Haemophilus influenzae, and P. aeruginosa. By adolescence, a majority of persons with CF have P. aeruginosa present in their sputum (Cystic Fibrosis Foundation Patient Registry 2003 Annual Data Report (2004)). Chronic endobronchial infections, particularly with P. aeruginosa, provoke a persistent inflammatory response in the airway that accelerates progressive obstructive disease characterized by diffuse bronchiectasis (Davis, P.B. et al. (1996), supra; Winnie, G.B. et al., "Respiratory Tract Colonization with Pseudomonas aeruginosa in Cystic Fibrosis: Correlations Between AxAi-P seudomonas aeruginosa Antibody Levels And Pulmonary Function," Pediatr Pulmonol 10:92- 100 (1991); Ballman, M. et al. "Long Term Follow Up of Changes in FEVl and Treatment Intensity During Pseudomonas Aeruginosa Colonisation in Patients with Cystic Fibrosis," Thorax 53:732-737 (1998); Pamukcu, A. et al., "Effects of Pseudomonas aeruginosa Colonization on Lung Function and Anthropometric Variables in Children with Cystic Fibrosis," Pediatr Pulmonol 19: 10-15 (1995)). A link between acquisition of chronic endobronchial P. aeruginosa infection, lung inflammation, loss of lung function, and ultimate death is suggested by significantly decreased survival associated with chronic P. aeruginosa infection (Henry, R.L. et al., "Mucoid Pseudomonas aeruginosa is a Marker of Poor Survival in Cystic Fibrosis," Pediatr Pulmonol 12(3):158-61 (1992)), and by the significant association of early acquisition of chronic P. aeruginosa infection and childhood mortality (Demko, CA. et al., "Gender Differences in Cystic Fibrosis: Pseudomonas aeruginosa Infection," J Clin Epidemiol 48: 1041-1049 (1995)). Chronic Therapies that either suppress bacterial loads in the lung (MacLusky, LB. et al, "Long- term Effects of Inhaled Tobramycin in Patients with Cystic Fibrosis Colonized with Pseudomonas aeruginosa " Pediatr Pulmonol 7(l):42-8 (1989)) or suppress resulting inflammation (Konstan, M. W. et al., "Effect of high-dose Ibuprofen in Patients with Cystic Fibrosis," N Engl J Med 332(13): 848-54 (1995)) have been shown to reduce rates of lung function decline in infected patients.

[015] Historically, the standard therapy for P. aeruginosa endobronchial infections was

14 to 21 days of parenteral antipseudomonal antibiotics, typically including an aminoglycoside. However, parenteral aminoglycosides, as highly polar agents, penetrate poorly into the endobronchial space. To obtain adequate drug concentrations at the site of infection with parenteral administration, serum levels approaching those associated with nephro-, vestibule-, and oto-toxicity are required ("American Academy of Otolaryngology. Guide for the evaluation of hearing handicap," JAMA 241(19):2055-9 (1979); Brummett, R.E., "Drug-induced ototoxicity," Drugs 19:412-28 (1980)). Inhalation administration of aminoglycosides offers an attractive alternative, delivering high concentrations of antibiotic directly to the site of infection in the endobronchial space while minimizing systemic bioavailability (Touw, DJ. et al., "Inhalation of Antibiotics in' Cystic Fibrosis," Eur Respir J 8:1594-604 (1995); Rosenfeld, M. et al., "Aerosolized Antibiotics for Bacterial Lower Airway Infections: Principles, Efficacy, and Pitfalls," Clinical Pulmonary Medicine 4(2):101-12 (1997)).

[016] One leading current treatment of P. aeruginosa infections in CF patients is

TOBI® tobramycin solution for inhalation, a preservative-free, stable, and convenient formulation of tobramycin (60 mg/mL tobramycin in 5 mL of 1/4 normal saline) for administration via jet nebulizer, developed by PathoGenesis Corporation, Seattle, Wash, (now Novartis AG, having a place of business in Emeryville, Calif). The combination of a 5 mL BID TOBI dose (300 mg tobramycin) and the PARI LC PLUS/PulmoAide compressor delivery system was approved by the FDA under NDA 50-753, December 1997, as a chronic intermittent therapy for the management of P. aeruginosa in CF patients, and remains the industry standard for this purpose. The process of inhalation of the commercially available 300 mg TOBI dose can take 20 minutes per dose with additional time required for set-up and nebulizer cleaning. The aerosol administration of a 5 ml dose of a formulation containing 300 mg of tobramycin in quarter normal saline for the suppression of P. aeruginosa in the endobronchial space of a patient is also disclosed in U.S. Pat. No. 5,508,269, the disclosure of which is incorporated herein in its entirety by this reference.

[017] In addition to inhaled antibiotics such as the commercially available TOBI® product, a variety of other chronic therapies are routinely prescribed to reduce the destructive cycles of obstruction, infection, and inflammation in the CF lung. Aggressive Airway Clearance Therapy (Reisman, JJ. et al.5 "Role of conventional physiotherapy in cystic fibrosis," J Pediatr 113(4):632-6 (1988)), inhaled bronchodilators (Konig P et al., "Short-term and Long-term Effects of Albuterol Aerosol Therapy in Cystic Fibrosis: A Preliminary Report," Pediatr Pulmonol 20(4):205-14 (1995)), and mucolytics such as recombinant human dornase alpha (rhDNase; Fuchs, HJ. et al., "Effect of Aerosolized Recombinant Human DNase on Exacerbations of Respiratory Symptoms and on Pulmonary Function in Patients with Cystic Fibrosis. The Pulmozyme Study Group," N Engl J Med 331(10):637-42 (1994)) are all prescribed chronically, creating a potential for significant treatment burden for persons with CF. It has been shown that adherence to therapies is a significant problem for persons with CF (Conway, S.P. et al., "Compliance with treatment in adult patients with cystic fibrosis," Thorax 51(l):29-33 (1996)) and that lack of compliance can vary by specific treatment (Abbott J et al., "Treatment Compliance in Adults with Cystic Fibrosis," Thorax 49(2): 115-20 (1994)). [018] Lower respiratory tract infections with pseudomonas aeruginosa (Psa) are a major cause of morbidity and mortality among patients with cystic fibrosis (CF) and non-CF bronchiectasis. Once an infection is established, even aggressive antibiotic treatments may only temporarily reduce the number of Psa organisms in the respiratory tract. As a result, many CF patients have persistent Psa infections requiring frequent hospital admissions for intravenous chemotherapy.

[019] Bronchiectasis is a condition characterized by progressive destruction and dilatation of airway walls due to infected retained secretions that result from a failure of airway defenses to maintain the sterile environment of the lower respiratory tract airways and lung parenchyma. The large volumes of infected secretions requiring aggressive antibiotic treatment at the onset of the infection and the presence of marked bacterial resistance to common and often used antibiotics represent significant barriers to effective therapy. The most effective treatment of bronchiectasis remains antibiotic therapy, usually administered systemically orally or by intravenous injection.

[020] Aminoglycosides are considered one of the most useful classes of antibiotics for treating Psa infections. However, antibiotic therapy of a variety of respiratory infections, in particular bronchiectasis, continues to represent a major medical challenge. [021] One of the major disadvantages of aminoglycosides is that they can induce fairly severe side effects. Aminoglycosides are generally poorly absorbed orally and, for this reason, are given intravenously or intramuscularly. Aminoglycosides active against Psa penetrate into sputum poorly, making it necessary to administer large systemic doses intravenously in order to optimize sputum penetration at the site of infection in the lung. Such high doses can produce both nephrotic and ototoxic effects, often causing permanent renal insufficiency and auditory nerve damage, with deafness, dizziness, and unsteadiness.

[022] At the same time, underdosing and incomplete courses of antibiotics are part of the problem of ineffective therapy. Potential consequences of underdosing respiratory tract infections include inadequate pathogen eradication, development of antibiotic resistance and lengthened eradication times, as well as potential for persistent clinical symptoms due to increasing lung injury, bronchiectasis, scarring, and premature death. [023] The overuse of antibiotics in the treatment of respiratory infections is a major problem and is increasingly regarded as such by both the medical community and the pharmaceutical industry. The Center for Disease Control (CDC) considers the growing problem of antibiotic resistance to be one of the most important public health challenges of our time. The CDC views overprescription of antibiotics as one of the prime culprits for the growing antibiotic resistance problem.

[024] In view of the above problems in antibiotic therapies, research has primarily focused on the discovery of new molecules to provide possible solutions. Alternatively, the potential effectiveness of treating infections of the respiratory tract with aminoglycosides administered by new drug delivery technologies such as inhalation aerosols has been investigated. In particular, aerosolized antibiotics have been administered by small volume nebulizers (SVN) driven ultrasonically or by air compressors.

[025]' For two decades, inhaled antibiotics have been used effectively for ameliorating chronic pulmonary infections in conditions such as cystic fibrosis and non-CF bronchiectasis. To date, the U.S. Food and Drug Administration (FDA) has approved only one aerosolized antiinfective: TOBI® (Novartis Vaccines and Diagnostics, Inc., Emeryville CA). TOBI is a tobramycin solution for inhalation by nebulization. Tobramycin (O-3-amino-3-deoxy-α-D- glucopyranosyl-(l-4)-O-[2,6-diarnino-2,3,6-trideoxy-a-D-ribo-hexopyranosyl-(l-6)]-2-deoxy-L- streptamine) is a water soluble, aminoglycoside antibiotic having a molceular weight of 467.52 g/mol. Tobramycin is effective against gram negative pathogens, in particular Pseudomonas aeruginosa, the key infective agent in CF patients.

[026] The formulated TOBI product is an aqueous solution, which is sterile, clear, slightly yellow, non-pyrogenic, and is pH and salinity adjusted. It comprises 300 mg of tobramycin free base in 5 ml of sodium chloride (2.25 mg/ml) at pH 6.0 and is stable at 2-8 C. for two years, or 28 days at room temp. The solution darkens in intense light. At pH 6.0, approximately 2.2 of the 5 tobramycin amino groups have been converted to sulfate salts. A dose is a single 300 mg ampoule bid (12 hours apart). Patients receive a 28 day "on" therapy followed by a 28 day "off period, to reduce the potential for development of resistant bacterial strains. Of the 300 mg inhaled, only approximately 10% or 30 mg is delivered to the lung. Systemic tobramycin given by IV injection has serious adverse effects including renal and ototoxicity. High IV doses are typically given due to poor penetration of the drug across the lung endothelium and into sputum. Clinical studies with TOBl have shown that inhaled tobramycin may lead to tinitus and voice alteration.

[027] Nebulization has many well documented disadvantages, including extended administration time, high cost, poor efficiency and reproducibility, risk of bacterial contamination, and the need for bulky compressors or gas cylinders. These disadvantages likely have an impact on patient compliance.

[028] Pulmonary delivery by aerosol inhalation has received much attention as an attractive alternative to intravenous, intramuscular, and subcutaneous injection, since this approach eliminates the necessity for injection syringes and needles. Pulmonary delivery also limits irritation to the skin and body mucosa which are common side effects of transdermal Iy, iontophoretically, and intranasally delivered drugs, eliminates the need for nasal and skin penetration enhancers (typical components of intranasal and transdermal systems that often cause skin irritation/dermatitis), is economically attractive, is amenable to patient self-administration, and is often preferred by patients over other alternative modes of administration. Administration of aminoglycoside dry powder aerosols to the lung has been attempted, but inefficient delivery devices and/or poorly dispersible lactose formulations limited these studies. [029] Dry powder inhalers are known in the art as disclosed, for example, in U.S. Pat.

Nos. 5,458,135; 5,740,794; 5,775,320; 5,785,049; 6,089,228; 6,257,233 and in copending U.S. application Ser. Nos., 10/601,127, filed 6/19/2003; 10/298,177, and 10/295,783, both filed 11/14/2002; and 60/136,518 filed May 28, 1999, all of which are hereby incorporated in their entirety by reference. In addition, U.S. Pat. No. 5,875,776 discloses a dry powder inhaler and discloses antibiotics such as gentamicin sulfate, amikacin sulfate, and tobramycin sulfate, among an extensive list of agents suitable for administration by the devices disclosed therein. No examples of formulations are disclosed. WO 00/35461 further discloses a method for treating bronchiectasis comprising the administration of an aminoglycoside aerosol. [030] In view of the known systems for administering nuclease aerosols, there remains a need for higher efficiency and more convenient systems. SUMMARY OF THE INVENTION

[031] Accordingly, one or more embodiments of the present invention include nuclease compositions, such as dry powders comprising nucleases, methods of making and using such compositions, and systems for pulmonary delivery of such compositions. [032] Other features and advantages of embodiments of the present invention will be set forth in the description of invention that follows, and in part will be apparent from the description or may be learned by practice of the invention. Embodiments of the invention will be realized and attained by the compositions and methods particularly pointed out in the written description and claims hereof,

[033] In one aspect, the present invention is directed to a powder composition, comprising particles comprising a nuclease, such as a DNase, wherein the particles have a mass median aerodynamic diameter ranging from about 1 μm to about 5 μm. [034] In one aspect, the present invention is directed to a powder composition, comprising particles comprising about 10-90 wt% of nuclease, such as a DNase, wherein the particles have a mass median aerodynamic diameter ranging from about 1 μm to about 5 μm. [035] In one aspect, the present invention is directed to a powder composition, comprising particles comprising about 20-80 wt% of nuclease, such as a DNase, wherein the particles have a mass median aerodynamic diameter ranging from about 1 μm to about 5 μm. [036] In one aspect, the present invention is directed to a powder composition, comprising particles comprising less than about 40 wt% of nuclease, wherein the particles have a mass median aerodynamic diameter ranging from about 1 μm to about 5 μm. [037] In another aspect, the present invention is directed to a pharmaceutical composition, comprising a powder comprising an effective amount of nuclease and pharmaceutically acceptable excipient, wherein the powder comprises particles comprising less than about 40 wt% of nuclease and having a mass median aerodynamic diameter ranging from about 1 μm to about 5 μm.

[038] In still another aspect, the present invention is directed to a unit dosage form, comprising a container containing a pharmaceutical composition comprising an effective amount of nuclease and pharmaceutically acceptable excipient, wherein the pharmaceutical composition comprises particles having a mass median aerodynamic diameter ranging from about 1 μm to about 5 μm.

[039] In a further aspect, the present invention is directed to a delivery system, comprising an inhaler and a pharmaceutical composition comprising particles comprising nuclease and pharmaceutically acceptable excipient, wherein the particles comprise less than 40 wt% of nuclease.

[040] In yet another aspect, the present invention is directed to a method of making spray-dried particles, comprising suspending nuclease in a liquid to form a feedstock and spray drying the feedstock to produce the spray-dried particles, wherein the particles comprise less than 40 wt% of nuclease and have a mass median aerodynamic diameter ranging from about 1 μm to about 5 μm.

[041] In still another aspect, the present invention is directed to a method of treating a condition associated with increased viscosity of pulmonary and/or nasal secretions, comprising administering by inhalation an effective amount of a composition comprising nuclease to a patient in need thereof, wherein the composition comprises a powder comprising particles comprising less than 40 wt% of nuclease and having a mass median aerodynamic diameter ranging from about 1 μm to about 5 μm.

[042] Further embodiments and aspects comprise any two or more of any of the foregoing features, aspects, versions or embodiments.

DRAWINGS

[043] Embodiments of the present invention are further described in the description of invention that follows, in reference to the noted plurality of non-limiting drawings, wherein:

[044] Figs. IA-IE show a passive inhaler device.

[045] Fig. 2 shows emptying profiles of hollow porous particles of DNase.

[046] Fig. 3 shows emptying profiles of amorphous drug particles of DNase.

[047] Figs 4A-4B are SEM images of particles of the present invention.

DESCRIPTION

[048] It is to be understood that unless otherwise indicated the present invention is not limited to specific formulation components, drug delivery systems, manufacturing techniques, administration steps, or the like, as such may vary. Unless otherwise stated, a reference to a compound or component includes the compound or component by itself, as well as the compound in combination with other compounds or components, such as mixtures of compounds.

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

[050] As used herein, the singular forms "a," "an," and "the" include the plural reference unless the context clearly dictates otherwise. Thus, for example, reference to "a phospholipid" includes a single phospholipid as well as two or more phospholipids in combination or admixture unless the context clearly dictates otherwise.

[051 ] When referring to an active agent, the term encompasses not only the specified molecular entity, but also its pharmaceutically acceptable, pharmacologically active analogs, including, but not limited to, salts, esters, amides, hydrazides, N-alkyl derivatives, N-acyl derivatives, prodrugs, conjugates, active metabolites, and other such derivatives, analogs, and related compounds. Therefore, as used herein, the term "nuclease" refers to nucleases per se or derivatives, analogs, or related compounds noted above, as long as such nucleases derivatives, analogs, or related compounds exhibit nuclease activity.

[052] As used herein, the terms "treating" and "treatment" refer to reduction in severity and/or frequency of symptoms, elimination of symptoms and/or underlying cause, reduction in likelihood of the occurrence of symptoms and/or underlying cause, and improvement or remediation of damage. Thus, "treating" a patient with an active agent as provided herein includes prevention of a particular condition, disease or disorder in a susceptible individual as well as treatment of a clinically symptomatic individual.

[053] As used herein, "effective amount" refers to an amount covering both therapeutically effective amounts and prophylactically effective amounts.

[054] As used herein, "therapeutically effective amount" refers to an amount that is effective to achieve the desired therapeutic result. A therapeutically effective amount of a given active agent will typically vary with respect to factors such as the type and severity of the disorder or disease being treated and the age, gender, and weight of the patient.

[055] As used herein, "prophylactically effective amount" refers to an amount that is effective to achieve the desired prophylactic result. Because a prophylactic dose is administered in patients prior to onset of disease, the prophylactically effective amount typically is less than the therapeutically effective amount.

[056] As used herein, the term "respiratory infections" includes, but is not limited to upper respiratory tract infections such as sinusitis, pharyngitis, and influenza, and lower respiratory tract infections such as tuberculosis, bronchiectasis (both the cystic fibrosis and non- cystic fibrosis indications), bronchitis (both acute bronchitis and acute exacerbation of chronic bronchitis), and pneumonia (including various types of complications that arise from viral and bacterial infections including hospital-acquired and community-acquired infections). [057] As used herein, "mass median diameter" or "MMD" refers to the median diameter of a plurality of particles, typically in a polydisperse particle population, i.e., consisting of a range of particle sizes. MMD values as reported herein are determined by laser diffraction (Sympatec Helos, Clausthal-Zellerfeld, Germany), unless the context indicates otherwise. Typically, powder samples are added directly to the feeder funnel of the Sympatec RODOS dry powder dispersion unit. This can be achieved manually or by agitating mechanically from the end of a VIBRI vibratory feeder element. Samples are dispersed to primary particles via application of pressurized air (2 to 3 bar), with vacuum depression (suction) maximized for a given dispersion pressure. Dispersed particles are probed with a 632.8 nm laser beam that intersects the dispersed particles' trajectory at right angles. Laser light scattered from the ensemble of particles is imaged onto a concentric array of photomultiplier detector elements using a reverse-Fourier lens assembly. Scattered light is acquired in time-slices of 5 ms. Particle size distributions are back-calculated from the scattered light spatial/intensity distribution using a proprietary algorithm.

[058] As used herein, "geometric diameter" refers to the diameter of a single particle, as determined by microscopy, unless the context indicates otherwise.

[059] As used herein, "mass median aerodynamic diameter" or "MMAD" refers to the median aerodynamic size of a plurality of particles or particles, typically in a polydisperse population. The "aerodynamic diameter" is the diameter of a unit density sphere having the same settling velocity, generally in air, as a powder and is therefore a useful way to characterize an aerosolized powder or other dispersed particle or particle formulation in terms of its settling behavior. The aerodynamic diameter encompasses particle or particle shape, density, and physical size of the particle or particle. As used herein, MMAD refers to the median of the aerodynamic particle or particle size distribution of an aerosolized powder determined by cascade impaction, unless the context indicates otherwise.

[060] As used herein, the term "emitted dose" or "ED" refers to an indication of the delivery of dry powder from an inhaler device after an actuation or dispersion event from a powder unit or reservoir. ED is defined as the ratio of the dose delivered by an inhaler device to the nominal dose (i.e., the mass of powder per unit dose placed into a suitable inhaler device prior to firing). The ED is an experimentally determined amount, and may be determined using an in vitro device set up which mimics patient dosing. To determine an ED value, as used herein, a nominal dose of dry powder (as defined herein) is placed into a a suitable inhaler device, for example, a Turbospin® DPI device (PH&T, Italy), described in U.S. Patent Nos. 4,069,819 and 4,995,385, which are incorporated herein by reference in their entireties. The a inhaler device is actuated, dispersing the powder. The resulting aerosol cJoud is then drawn from the device by vacuum (30 L/min) for 2.5 seconds after actuation, where it is captured on a tared glass fiber filter (Gelman, 47 mm diameter) attached to the device mouthpiece. The amount of powder that reaches the filter constitutes the delivered dose. For example, for a capsule containing 5 mg of dry powder that is placed into an inhalation device, if dispersion of the powder results in the recovery of 4 mg of powder on a tared filter as described above, then the ED for the dry powder composition is 80% [= 4 mg (delivered dose)/5 mg (nominal dose)]. [061] As used herein, "passive dry powder inhaler" refers to an inhalation device that relies upon a patient's inspiratory effort to disperse and aerosolize a pharmaceutical composition contained within the device in a reservoir or in a unit dose form and does not include inhaler devices which comprise a means for providing energy, such as pressurized gas and vibrating or rotating elements, to disperse and aerosolize the drug composition.

[062] As used herein, "active dry powder inhaler" refers to an inhalation device that does not rely solely on a patient's inspiratory effort to disperse and aerosolize a pharmaceutical composition contained within the device in a reservoir or in a unit dose form and does include inhaler devices that comprise a means for providing energy to disperse and aerosolize the drug composition, such as pressurized gas and vibrating or rotating elements. [063] Compositions including nucleases may include various forms and amounts of nucleases. For example, the amount of nuclease may range from at least about 0.01 wt%, such as at least about 1 wt%, at least about 10 wt%, at least about 50 wt%, at least about 90 wt%, at least about 95 wt%, or at least about 98 wt%.

[064] The pharmaceutical composition according to one or more embodiments of the invention may comprise one or more nuclease and, optionally, one or more other active ingredients and/or pharmaceutically acceptable excipients. For example, the pharmaceutical composition may comprise neat particles of nuclease, may comprise neat particles of nuclease together with other particles, and/or may comprise particles comprising nuclease and one or more active ingredients and/or one or more pharmaceutically acceptable excipients. [065] Thus, the pharmaceutical composition according to one or more embodiments of the invention may, if desired, contain a combination of nuclease and one or more other active ingredients. Examples of other active agents include, but are not limited to, agents that may be delivered through the lungs or nasal passages. For example, the other active agent(s) may be long-acting agents and/or active agents that are active against pulmonary and/or nasal infections such as antivirals, antifungals, and/or antibiotics, such as active agents that are effective against cystic fibrosis.

[066] Examples of antivirals include, but are not limited to, acyclovir, gangcyclovir, azidothymidine, cytidine arabinoside, ribavirin, rifampacin, amantadine, iododeoxyuridine, poscarnet, and trifluridine, as well as neuraminidase inhibitors, such as Zanamivir. [067] Examples of antifungals include, but are not limited to, azoles (e.g., imidazoles, itraconazole, pozaconazole), micafungin, caspafungin, salicylic acid, oxiconazole nitrate, ciclopirox olamine, ketoconazole, miconazole nitrate, and butoconazole nitrate. [068] Examples of antibiotics include, but are not limited to, penicillin and drugs of the penicillin family of antimicrobial drugs, including but not limited to penicillin-G, penicillin- V, phenethicillin, ampicillin, amoxacillin, cyclacillin, bacampicillin, hetacillin, cloxacillin, dicloxacillin, methicillin, nafcillin, oxacillin, azlocillin, carbenicillin, mezlocillin, piperacillin, ticaricillin, and imipenim; cephalosporin and drugs of the cephalosporin family, including but not limited to cefadroxil, cefazolin, caphalexn, cephalothin, cephapirin, cephradine, cefaclor, cefamandole, cefonicid, cefoxin, cefuroxime, ceforanide, cefotetan, cefinetazole, cefoperazone, cefotaxime, ceftizoxime, ceftizone, moxalactam, ceftazidime, and cefixime; aminoglycoside drugs and drugs of the aminoglycoside family, including but not limited to streptomycin, neomycin, kanamycin, gentamycin, tobramycin, amikacin, and netilmicin; macrolide and drugs of the macrolide family, exemplified by amphotericin B, azithromycin, clarithromycin, roxithromycin, erythromycin, lincomycin, and clindamycin; tetracyclin and drugs of the tetracyclin family, for example, tetracyclin, oxytetracyclin, democlocyclin, methacyclin, doxycyclin, and minocyclin; quinoline and quinoline-like drugs, such as, for example, naladixic acid, cinoxacin, norfloxacin, ciprofloxacin, ofloxicin, enoxacin, and pefloxacin; antimicrobial peptides, including but not limited to polymixin B, colistin, and bacatracin, as well as other antimicrobial peptides such as defensins, magainins, cecropins, and others, provided as naturally- occurring or as the result of engineering to make such peptides resistant to the action of pathogen-specific proteases and other deactivating enzymes; other antimicrobial drugs, including chloramphenicol, vancomycin, rifampicin, metronidazole, voriconazole, fluconazole, ethambutol, pyrazinamide, sulfonamides, isoniazid, and erythromycin. [069] When a combination of active agents is used, the agents may be provided in combination in a single species of pharmaceutical composition or individually in separate species of pharmaceutical compositions. Further, the pharmaceutical composition may be combined with one or more other active or bioactive agents that provide the desired dispersion stability or powder dispersibility.

[070] The amount of active agent(s), e.g., nuclease, in the pharmaceutical composition may vary. The amount of active agent(s) is typically at least about 0.5 wt%, such as at least about 1 wt%, at least about 2 wt%, at least about 5 wt%, at least about 10 wt%, at least about 20 wt%, at least about 30 wt%, at least about 40 wt%, at least about 50 wt%, at least about 60 wt%, at least about 70 wt%, or at least about 80 wt%, of the total amount of the pharmaceutical composition. The amount of active agent(s) generally varies between about 0.1 wt% to 100 wt%, such as about 1 wt% to about 95 wt%, about 2 wt% to about 90 wt%, about 30 wt% to about 80 wt%, about 40 wt% to about 70 wt%, about 50 wt% to about 60 wt%, about 1 wt% to about 20 wt%, about 2 wt% to about 10 wt%, about 5 wt% to about 50 wt%, or about 4 wt% to about 20 wt%.

[071] As noted above, the pharmaceutical composition may include one or more pharmaceutically acceptable excipient. Examples of pharmaceutically acceptable excipients include, but are not limited to, lipids, metal ions, surfactants, amino acids, carbohydrates, buffers, salts, polymers, and the like, and combinations thereof. [072] Examples of lipids include, but are not limited to, phospholipids, glycolipids, gangliostde GMl, sphingomyelin, phosphatidic acid, cardiolipin; lipids bearing polymer chains such as polyethylene glycol, chitin, hyaluronic acid, or polyvinylpyrrolidone; lipids bearing sulfonated mono-, di-, and polysaccharides; fatty acids such as palmitic acid, stearic acid, and oleic acid; cholesterol, cholesterol esters, and cholesterol hemisuccinate. [073] In one or more embodiments, the phospholipid comprises a saturated phospholipid, such as one or more phosphatidylcholines. Exemplary acyl chain lengths are 16:0 and 18:0 (i.e., palmitoyl and stearoyl). The phospholipid content may be determined by the active agent activity, the mode of delivery, and other factors.

[074] Phospholipids from both natural and synthetic sources may be used in varying amounts. When phospholipids are present, the amount is typically sufficient to coat the active agent(s) with at least a single molecular layer of phospholipid. In general, the phospholipid content ranges from about 5 wt% to about 99.9 wt%, such as about 20 wt% to about 80 wt%. [075] Generally, compatible phospholipids comprise those that have a gel to liquid crystal phase transition greater than about 40 °C, such as greater than about 60 ⁰C, or greater than about 80 °C. The incorporated phospholipids may be relatively long chain (e.g., Cj₆-C₂₂) saturated lipids. Exemplary phospholipids useful in the disclosed stabilized preparations include, but are not limited to, phosphoglycerides such as dipalmitoylphosphatidylcholine, distearoylphosphatidylcholine, diarachidoylphosphatidylcholine, dibehenoylphosphatidylcholine, dimyristoylphosphatidylcholine, diphosphatidyl_. glycerols, short-chain phosphatidylcholines, hydrogenated phosphatidylcholine, E-100-3 (available from Lipoid KG, Ludwigshafen, Germany), long-chain saturated phosphatidylethanol amines, long-chain saturated phosphatidylserines, long-chain saturated phosphatidylglycerols, long-chain saturated phosphatidylinositols, phosphatidic acid, phosphatidylinositol, and sphingomyelin. [076] Examples of metal ions include, but are not limited to, divalent cations, including calcium, magnesium, zinc, iron, and the like. For instance, when phospholipids are used, the pharmaceutical composition may also comprise a polyvalent cation, as disclosed in WO 01/85136 and WO 01/85137, which are incorporated herein by reference in their entireties. The polyvalent cation may be present in an amount effective to increase the melting temperature (T_m) of the phospholipid such that the pharmaceutical composition exhibits a T_n, which is greater than its storage temperature (T_s) by at least about 20 ⁰C, such as at least about 40 ⁰C. The molar ratio of polyvalent cation to phospholipid may be at least about 0.05:1, such as about 0.05:1 to about 2.0:1 or about 0.25: 1 to about 1.0:1. An example of the molar ratio of polyvalent cation:phospholipid is about 0.50:1. When the polyvalent cation is calcium, it may be in the form of calcium chloride. Although metal ion, such as calcium, is often included with phospholipid, none is required.

[077] As noted above, the pharmaceutical composition may include one or more surfactants. For instance, one or more surfactants may be in the liquid phase with one or more being associated with solid particles or particles of the composition. By "associated with" it is meant that the pharmaceutical compositions may incorporate, adsorb, absorb, be coated with, or be formed by the surfactant. Surfactants include, but are not limited to, fluorinated and nonfluorinated compounds, such as saturated and unsaturated lipids, nonionic detergents, nonionic block copolymers, ionic surfactants, and combinations thereof. It should be emphasized that, in addition to the aforementioned surfactants, suitable fluorinated surfactants are compatible with the teachings herein and may be used to provide the desired preparations. [078] Examples of nonionic detergents include, but are not limited to, sorbitan esters including sorbitan trioleate (Span™ 85), sorbitan sesquioleate, sorbitan monooleate, sorbitan monolaurate, polyoxyethylene (20) sorbitan monolaurate, and polyoxyethylene (20) sorbitan monooleate, oleyl polyoxyethylene (2) ether, stearyl polyoxyethylene (2) ether, lauryl polyoxyethylene (4) ether, glycerol esters, and sucrose esters. Other suitable nonionic detergents can be easily identified using McCutcheon's Emulsifiers and Detergents (McPublishing Co., Glen Rock, New Jersey), which is incorporated by reference herein in its entirety. [079] Examples of block copolymers include, but are not limited to, diblock and triblock copolymers of polyoxyethylene and polyoxypropylene, including poloxamer 188 (Pluronic™ F-68), poloxamer 407 (Pluronic™ F- 127), and poloxamer 338. [080] Examples of ionic surfactants include, but are not limited to, sodium sulfosuccinate, and fatty acid soaps.

[081] Examples of amino acids include, but are not limited to, hydrophobic amino acids. Use of amino acids as pharmaceutically acceptable excipients is known in the art as disclosed in WO 95/31479, WO 96/32096, and WO 96/32149, which are incorporated herein by reference. [082] Hydrophobic amino acids and lipids are capable of providing a particle surface of low surface energy. Magnesium stearate may also be used as an excipient to reduce surface energy.

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

[084] Examples of buffers include, but are not limited to, trϊs or citrate.

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

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

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

[088] The excipients may be glass forming excipients providing an amorphous glass, e.g., with a glass transition temperature that is at least 20 ⁰C greater than the storage temperature.

Glass forming systems are disclosed in U.S. Patent Nos. 6,258,341; 5,098,893; 5,928,469; and

6,071,428, which are incorporated herein by reference.

[089] The pharmaceutical composition of one or more embodiments of the present invention may also include a biocompatible, such as biodegradable polymer, copolymer, or blend or other combination thereof. In this respect useful polymers comprise polylactides, polylactide- glycolides, cyclodextrins, polyacrylates, methylcellulose, carboxymethylcellulose, polyvinyl alcohols, polyanhydrides, polylactams, polyvinyl pyrrolidones, polysaccharides (dextrans, starches, chitin, chitosan, etc.), hyaluronic acid, proteins, (albumin, collagen, gelatin, etc.).

Those skilled in the art will appreciate that, by selecting the appropriate polymers, the delivery efficiency of the composition and/or the stability of the dispersions may be tailored to optimize the effectiveness of the active agent(s).

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

[091] The pharmaceutical compositions may also include mixtures of pharmaceutically acceptable excipients. For instance, mixtures of carbohydrates and amino acids are within the scope of the present invention. Other combinations of excipients include, but are not limited to, (a) distearoylphosphatidylcholine to calcium chloride (e.g., in a 2:1 molar ratio); (b) core-shell particles comprised of a shell of trileucine, and a core comprised of glass forming excipients, including sodium citrate and trehalose.

[092] The compositions of one or more embodiments of the present invention may take various forms, such as dry powders, capsules, tablets, reconstituted powders, suspensions, or dispersions comprising a non-aqueous phase, such as propellants (e.g., chlorofluorocarbon, hydrofluoroalkane). The moisture content of dry powder may be less than about 15 wt%, such as less than about 10 wt%, less than about 5 wt%, less than about 2 wt%, less than about 1 wt%, or less than about 0.5 wt%. Such powders are described in WO 95/24183, WO 96/32149, WO 99/16419, WO 99/16420, and WO 99/16422, which are incorporated herein by reference in their entireties.

[093] One or more embodiments of the invention involve homogeneous compositions of nuclease incorporated in a matrix material with little, if any, unincorporated nuclease. For instance, at least about 40 wt%, at least about 50 wt%, at least about 60 wt%, at least about 70%, at least about 80%, at least about 90 wt%, at least about 95 wt%, or at least about 99 wt%, of the composition may comprise particles including both nuclease and matrix material. [094] In some cases, however, a heterogeneous composition may be desirable in order to provide a desired pharmacokinetic profile of the nuclease to be administered, and in these cases, a large nuclease particle (e.g., mass median diameter of about 3 μm to about 10 μm, or larger) may be used. [095] In one version, the pharmaceutical composition comprises nuclease incorporated into a phospholipid matrix. The pharmaceutical composition may comprise phospholipid matrices that incorporate the active agent and that are in the form of particles that are hollow and/or porous microstructures, as described in the aforementioned WO 99/16419, WO 99/16420, WO 99/16422, WO 01/85136, and WO 01/85137, which are incorporated herein by reference in their entireties. The hollow and/or porous microstructures are useful in delivering the nuclease to the lungs because the density, size, and aerodynamic qualities of the hollow and/or porous microstructures facilitate transport into the deep lungs during a user's inhalation. In addition, the phospholipid-based hollow and/or porous microstructures reduce the attraction forces between particles, making the pharmaceutical composition easier to deagglomerate during aerosolization and improving the flow properties of the pharmaceutical composition making it easier to process. [096] In one version, the pharmaceutical composition is composed of hollow and/or porous microstructures having a bulk density less than about 1.0 g/cm³, less than about 0.5 g/cm³, less than about 0.3 g/cm³, less than about 0.2 g/cm³, or less than about 0.1 g/cm³. For example, small porous particles of the present invention may have a bulk density ranging from 0.01 g/cm³ to 0.4 g/cm³, such as from 0.03 g/cm³ to 0.25 g/cm³. Particle density can be controlled by controlling the drying rate and surface composition of spray-dried particles, or by inclusion of a specific pore forming agent in the formulation. Preferred pore-forming agents are medium chain fluorocarbons such as perfluorooctyl bromide (PFOB), perfluorodecalin (PFD), and perfluorooctyl ethane (PFOE). Bulk densities are determined by simply weighing a no. 2 capsule (0.37 ml fill volume) with powder, and weighing the filled capsule. [097] By providing low bulk density particles or particles, the minimum powder mass that can be filled into a unit dose container is reduced, which eliminates the need for carrier particles. That is, the relatively low density of the powders of one or more embodiments of the present invention provides for the reproducible administration of relatively low dose pharmaceutical compounds. Moreover, the elimination of carrier particles will potentially reduce throat deposition and any "gag" effect or coughing, since large carrier particles, e.g., lactose particles, will impact the throat and upper airways due to their size.

[098] In one version, the pharmaceutical composition is in dry powder form and is contained within a unit dose receptacle which may be inserted into or near the aerosolization apparatus to aerosolize the unit dose of the pharmaceutical composition. This version is useful in that the dry powder form may be stably stored in its unit dose receptacle for a long period of time. The pharmaceutical compositions of one or more embodiments of the present invention may be stable for at least about 2 years. In some versions, no refrigeration may be required to obtain stability. In other versions, reduced temperatures, e.g., at 2-8 ⁰C, may be used to prolong stable storage. In many versions, the storage stability allows aerosolization with an external power source.

[099] It will be appreciated that the pharmaceutical compositions disclosed herein may comprise a structural matrix that exhibits, defines or comprises voids, pores, defects, hollows, spaces, interstitial spaces, apertures, perforations or holes. The absolute shape (as opposed to the morphology) of the perforated microstructure is generally not critical and any overall configuration that provides the desired characteristics is contemplated as being within the scope of the invention. Accordingly, some embodiments comprise approximately spherical shapes. However, collapsed, deformed or fractured particles are also compatible. [0100] In one version, the nuclease is incorporated in a matrix that forms a discrete particle, and the pharmaceutical composition comprises a plurality of the discrete particles. The discrete particles may be sized so that they are effectively administered and/or so that they are available where needed. For example, for an aerosolizable pharmaceutical composition, the particles are of a size that allows the particles to be aerosolized and delivered to a user's respiratory tract during the user's inhalation.

[0101] In some versions, the pharmaceutical composition comprises particles having a mass median diameter less than about 20 μm, such as less than about 10 μm, less than about 7 μm, or less than about 5 μm, and may, e.g., range from 1 μm to 10 μm, such as from 1 μm to 5 μm. The particles may have a mass median aerodynamic diameter ranging from about 1 μm to about 6 μm, such as about 1.5 μm to about 5 μm, or about 2 μm to about 4 μm. If the particles are too large, fewer particles will reach the deep lung. If the particles are too small, a larger percentage of the particles may be exhaled.

[0102] The matrix material may comprise a hydrophobic or a partially hydrophobic material. For example, the matrix material may comprise a lipid, such as a phospholipid, and/or a hydrophobic amino acid, such as leucine or tri-leucine. Examples of phospholipid matrices are described in WO 99/16419, WO 99/16420, WO 99/16422, WO 01/85136, and WO 01/85137 and in U.S. Patent Nos. 5,874,064; 5,855,913; 5,985,309; and 6,503,480, and in copending and co- owned U.S. Application No. 10/750,934, filed on December 31, 2003, all of which are incorporated herein by reference in their entireties. Examples of hydrophobic amino acid matrices are. described in U.S. Patent Nos. 6,372,258 and 6,358,530, and in U.S. Application No. 10/032,239, filed on December 21, 2001, which are incorporated herein by reference in their entireties.

[0103] When phospholipids are utilized as the matrix material, the pharmaceutical composition may also comprise a polyvalent cation, as disclosed in WO 01/85136 and WO 01/85137, which are incorporated herein by reference in their entireties. [0104] According to another embodiment, release kinetics of the active agent(s) containing composition is controlled. According to one or more embodiments, the compositions of the present invention provide immediate release of the active agent(s). Alternatively, the compositions of other embodiments of the present invention may be provided as non- homogeneous mixtures of active agent incorporated into a matrix material and unincorporated active agent in order to provide desirable release rates of antifungal agent. According to this embodiment, active agents formulated using the emulsion-based manufacturing process of one or more embodiments of the present invention have utility in immediate release applications when administered to the respiratory tract. Rapid release is facilitated by: (a) the high specific surface area of the low density porous powders and; (c) the low surface energy of the particles. [0105] Alternatively, it may be desirable to engineer the particle matrix so that extended release of the active agent(s) is effected. This may be particularly desirable when the active agent(s) is rapidly cleared from the lungs or when sustained release is desired. For example, the nature of the phase behavior of phospholipid molecules is influenced by the nature of their chemical structure and/or preparation methods in spray-drying feedstock and drying conditions and other composition components utilized. In the case of spray-drying of active agent(s) solubilized within a small unilamellar vesicle (SUV) or multilamellar vesicle (MLV), the active agent(s) are encapsulated within multiple bilayers and are released over an extended time. [0106] In contrast, spray-drying of a feedstock comprised of emulsion droplets and dispersed or dissolved active agent(s) in accordance with the teachings herein leads to a phospholipid matrix with less long-range order, thereby facilitating rapid release. While not being bound to any particular theory, it is believed that this is due in part to the fact that the active agent(s) are never formally encapsulated in the phospholipid, and the fact that the phospholipid is initially present on the surface of the emulsion droplets as a monolayer (not a bilayer as in the case of liposomes). The spray-dried particles prepared by the emulsion-based manufacturing process of one or more embodiments of the present invention often have a high degree of disorder. Also, the spray-dried particles typically have low surface energies, where values as low as 20 mN/m have been observed for spray-dried DSPC particles (determined by inverse gas chromatography). Small angle X-ray scattering (SAXS) studies conducted with spray-dried phospholipid particles have also shown a high degree of disorder for the lipid, with scattering peaks smeared out, and length scales extending in some instances only beyond a few nearest neighbors.

[0107] It should be noted that a matrix having a high gel to liquid crystal phase transition temperature is not sufficient in itself to achieve sustained release of the active agent(s). Having sufficient order for the bilayer structures is also important for achieving sustained release. To facilitate rapid release, an emulsion-system of high porosity (high surface area), and minimal interaction between the drug substance and phospholipid may be used. The pharmaceutical composition formation process may also include the additions of other composition components (e.g., small polymers such as Pluronic F-68; carbohydrates, salts, hydrotropes) to break the bilayer structure are also contemplated.

[0108] To achieve a sustained release, incorporation of the phospholipid in bilayer form may be used, especially if the active agent is encapsulated therein. In this case increasing the T_m of the phospholipid may provide benefit via incorporation of divalent counterions or cholesterol. As well, increasing the interaction between the phospholipid and drug substance via the _^ formation of ion-pairs (negatively charged active + steaylamine, positively charged active + phosphatidylglycerol) would tend to decrease the dissolution rate. If the active is amphiphilic, surfactant/surfactant interactions may also slow active dissolution.

[0109] The addition of divalent counterions (e.g., calcium or magnesium ions) to long- chain saturated phosphatidylcholines results in an interaction between the negatively charged phosphate portion of the zwitterionic headgroup and the positively charged metal ion. This results in a displacement of water of hydration and a condensation of the packing of the phospholipid lipid headgroup and acyl chains. Further, this results in an increase in the Tm of the phospholipid. The decrease in headgroup hydration can have profound effects on the spreading properties of spray-dried phospholipid particles on contact with water. [01 10] For example, upon reconstitution, the surface tension of spray-dried DSPC/Ca mixtures at the air/water interface decreases to equilibrium values (about 20 mN/m) as fast as a measurement can be taken. In contrast, liposomes of DSPC decrease the surface tension (about 50 mN/m) very little over a period of hours, and it is likely that this reduction is due to the presence of hydrolysis degradation products such as free fatty acids in the phospholipid. Single- tailed fatty acids can diffuse much more rapidly to the air/water interface than can the hydrophobic parent compound.

[01 11] In another version, the pharmaceutical composition comprises low density particles achieved by co-spray-drying with a perfluorocarbon-in-water emulsion. Examples of perfluorocarbons include, but are not limited to, perfluorohexane, perfluorooctyl bromide, perfluorooctyl ethane, perfluorodecalin, perfluorobutyl ethane.

[01 12] In accordance with the teachings herein the particle compositions will preferably be provided in a "dry" state. That is, in one or more embodiments, the particles will possess a moisture content that allows the powder to remain chemically and physically stable during storage at ambient or reduced temperature and remain dispersible. In this¹ regard, there is little or no change in primary particle size, content, purity, and aerodynamic particle size distribution. [0113] As such, the moisture content of the particles is typically less than about 10 wt%, such as less than about 6 wt%, less than about 3 wt%, or less than about 1 wt%. The moisture content is, at least in part, dictated by the composition and is controlled by the process conditions employed, e.g., inlet temperature, feed concentration, pump rate, and blowing agent type, concentration and post drying. Reduction in bound water leads to significant improvements in the dispersibiljty and flowability of phospholipid based powders, leading to the potential for highly efficient delivery of powdered lung surfactants or particle composition comprising active agent dispersed in the phospholipid. The improved dispersibility allows simple passive DPI devices to be used to effectively deliver these powders.

[0114] Yet another version of the pharmaceutical composition includes particle compositions that may comprise, or may be partially or completely coated with, charged species that prolong residence time at the point of contact or enhance penetration through mucosae. For example, anionic charges are known to favor mucoadhesion while cationic charges may be used to associate the formed particle with negatively charged bioactive agents such as genetic material. The charges may be imparted through the association or incorporation of polyanionic or polycationic materials such as polyacrylic acids, polylysine, polylactic acid, and chitosan. [01 15] These unit dose pharmaceutical compositions may be contained in a container.

Examples of containers include, but are not limited to, capsules, blisters, vials, ampoules, or container closure systems made of metal, polymer (e.g., plastic, elastomer), glass, or the like. [0116] The container may be inserted into an aerosolization device. The container may be of a suitable shape, size, and material to contain the pharmaceutical composition and to provide the pharmaceutical composition in a usable condition. For example, the capsule or blister may comprise a wall which comprises a material that does not adversely react with the pharmaceutical composition. In addition, the wall may comprise a material that allows the capsule to be opened to allow the pharmaceutical composition to be aerosolized. In one version, the wall comprises one or more of gelatin, hydroxypropyl methylcellulose (HPMC), polyethyleneglycol-compounded HPMC, hydroxyproplycellulose, agar, aluminum foil, or the like. In one version, the capsule may comprise telescopically adjoining sections, as described for example in U.S. Patent No. 4,247,066 which is incorporated herein by reference in its entirety. The size of the capsule may be selected to adequately contain the dose of the pharmaceutical composition. The sizes generally range from size 5 to size 000 with the outer diameters ranging from about 4.91 mm to 9.97 mm, the heights ranging from about 1 1.10 mm to about 26.14 mm, and the volumes ranging from about 0.13 mL to about 1.37 niL, respectively. Suitable capsules are available commercially from, for example, Shionogi Qualicaps Co. in Nara, Japan and Capsugel in Greenwood, South Carolina. After filling, a top portion may be placed over the bottom portion to form a capsule shape and to contain the powder within the capsule, as described in U.S. Patent Nos. 4,846,876 and 6,357,490, and in WO 00/07572, which are incorporated herein by reference in their entireties. After the top portion is placed over the bottom portion, the capsule can optionally be banded.

[0117] In one version, the pharmaceutical composition comprising nuclease is aerosolizable so that it may be delivered to the lungs of a patient during the patient's inhalation. In this way the nuclease in the pharmaceutical composition is delivered directly to the site of infection. This is advantageous over systemic administration. Because the active agent(s) often have renal or other toxicity, minimizing systemic exposure is typically preferred. Therefore, the amount of active agent(s) that may be delivered to the lungs is preferably limited to the minimum pharmacologically effective dose. By administering the active agent(s) directly to the lungs, a greater amount may be delivered to the site in need of the therapy while significantly reducing systemic exposure.

[01 18] The pharmaceutical compositions of one or more embodiments of the present invention lack taste. In this regard, although taste masking agents are optionally included within the composition, the compositions often lack taste even without a taste masking agent. [0119] The particles, particles, and compositions of one or more embodiments of the present invention may be made by any of the various methods and techniques known and available to those skilled in the art. The pharmaceutical composition may be produced using various known techniques. For example, the composition may be formed by spray drying, lyophilization, milling (e.g., wet milling, dry milling), and the like.

[0120] In spray drying, the preparation to be spray dried or feedstock can be any solution, coarse suspension, slurry, colloidal dispersion, or paste that may be atomized using the selected spray drying apparatus. In the case of insoluble agents, the feedstock may comprise a suspension as described above. Alternatively, a dilute solution and/or one or more solvents may be utilized in the feedstock. In one or more embodiments, the feed stock will comprise a colloidal system such as an emulsion, reverse emulsion, microemulsion, multiple emulsion, particle dispersion, or slurry.

[0121] In one version, the nuclease and the matrix material are added to an aqueous feedstock to form a feedstock solution, suspension, or emulsion. The feedstock is then spray dried to produce dried particles comprising the matrix material and the nuclease. Suitable spray- drying processes are known in the art, for example as disclosed in WO 99/16419 and U.S. Patent Nos. 6,077,543; 6,051,256; 6,001,336; 5,985,248; and 5,976,574, which are incorporated herein by reference in their entireties.

[0122] Whatever components are selected, the first step in particle production typically comprises feedstock preparation. If a phospholipid-based particle is intended to act as a carrier for the nuclease, the selected active agent(s) may be introduced into a liquid, such as water, to produce a concentrated suspension. The concentration of nuclease and optional active agents typically depends on the amount of agent required in the final powder and the performance of the delivery device employed (e.g., the fine particle dose for a metered dose inhaler (MDI) or a dry powder inhaler (DPI)). [0123] Any additional active agent(s) may be incorporated in a single feedstock preparation and spray dried to provide a single pharmaceutical composition species comprising a plurality of active agents. Conversely, individual active agents could be added to separate stocks and spray dried separately to provide a plurality of pharmaceutical composition species with different compositions. These individual species could be added to the suspension medium or dry powder dispensing compartment in any desired proportion and placed in the aerosol delivery system as described below.

[0124] Polyvalent cation may be combined with the nuclease suspension, combined with the phospholipid emulsion, or combined with an oil-in-water emulsion formed in a separate vessel. The nuclease may also be dispersed directly in the emulsion. [0125] For example, polyvalent cation and phospholipid may be homogenized in hot distilled water (e.g., 70 ⁰C) using a suitable high shear mechanical mixer (e.g., Ultra-Turrax model T-25 mixer) at 8000 rpm for 2 to 5 min. Typically, 5 to 25 g of fluorocarbon is added dropwise to the dispersed surfactant solution while mixing. The resulting polyvalent cation- containing perfluorocarbon in water emulsion may then be processed using a high pressure homogenizer to reduce the particle size. Typically, the emulsion is processed for five discrete passes at 12,000 to 18,000 PSl and kept at about 50 ⁰C to about 80 ⁰C. [0126] When the polyvalent cation is combined with an oil-in-water emulsion, the dispersion stability and dispersibility of the spray dried pharmaceutical composition can be improved by using a blowing agent, as described in WO 99/16419, which is incorporated herein by reference in its entirety. This process forms an emulsion, optionally stabilized by an incorporated surfactant, typically comprising submicron droplets of water immiscible blowing agent dispersed in an aqueous continuous phase. The blowing agent may be a fluorinated compound (e.g. perfluorohexane, perfluorooctyl bromide, perfluorooctyl ethane, perfluorodecalin, perfluorobutyl ethane) which vaporizes during the spray-drying process, leaving behind generally hollow, porous aerodynamically light particles. Other suitable liquid blowing agents include non-fluorinated-oils, chloroform, Freon® fluorocarbons, ethyl acetate, alcohols, hydrocarbons, nitrogen, and carbon dioxide gases. The blowing agent may be emulsified with a phospholipid. [0127] Although the pharmaceutical compositions may be formed using a blowing agent as described above, it will be appreciated that, in some instances, no additional blowing agent is required and an aqueous dispersion of the nuclease and/or pharmaceutically acceptable excipients and surfactant(s) are spray dried directly. In such cases, the pharmaceutical composition may possess certain physicochemical properties (e.g., elevated melting temperature, surface activity, etc.) that make it particularly suitable for use in such techniques. [0128] As needed, cosurfactants such as poloxamer 188 or span 80 may be dispersed into this annex solution. Additionally, pharmaceutically acceptable excipients such as sugars and starches can also be added.

[0129] The feedstock(s) may then be fed into a spray dryer. Typically, the feedstock is sprayed into a current of warm filtered air that evaporates the solvent and conveys the dried product to a collector. The spent air is then exhausted with the solvent. Commercial spray dryers manufactured by Bϋchi Ltd. or Niro Corp. may be modified for use to produce the pharmaceutical composition. Examples of spray drying methods and systems suitable for making the dry powders of one or more embodiments of the present invention are disclosed in U.S. Patent Nos. 6,077,543; 6,051,256; 6,001,336; 5,985,248; and 5,976,574, which are incorporated herein by reference in their entireties.

[0130] Operating conditions of the spray dryer such as inlet and outlet temperature, feed rate, atomization pressure, flow rate of the drying air, and nozzle configuration can be adjusted in order to produce the required particle size, and production yield of the resulting dry particles. The selection of appropriate apparatus and processing conditions are within the purview of a skilled artisan in view of the teachings herein and may be accomplished without undue experimentation. Exemplary settings are as follows: an air inlet temperature between about 60⁰C and about 170°C, such as between 80⁰C and 120°C; an air outlet between about 4O⁰C to about 120⁰C, such as about 50⁰C and 70⁰C; a feed rate between about 3 mL/min to about 15 mL/min; an aspiration air flow of about 300 L/min; and an atomization air flow rate between about 25 L/min and about 50 L/min. The solids content in the spray-drying feedstock will typically be in the range from 0.5 wt% to 10 wt%, such as 1.0 wt% to 5.0 wt%. The settings will, of course, vary depending on the type of equipment used. In any event, the use of these and similar methods allow formation of aerodynamical Iy light particles with diameters appropriate for aerosol deposition into the lung. [0131] Hollow and/or porous microstructures may be formed by spray drying, as disclosed in WO 99/16419, which is incorporated herein by reference. The spray-drying process can result in the formation of a pharmaceutical composition comprising particles having a relatively thin porous wall defining a large internal void. The spray-drying process is also often advantageous over other processes in that the particles formed are less likely to rupture during processing or during deagglomeration.

[0132] Pharmaceutical compositions useful in one or more embodiments of the present invention may alternatively be formed by lyophilization. Lyophilization is a freeze-drying process in which water is sublimed from the composition after it is frozen. The lyophilization process is often used because biologicals and pharmaceuticals that are relatively unstable in an aqueous solution may be dried without exposure to elevated temperatures, and then stored in a dry state where there are fewer stability problems. With respect to one or more embodiments of the instant invention, such techniques are particularly compatible with the incorporation of peptides, proteins, genetic material and other natural and synthetic macromolecules in pharmaceutical compositions without compromising physiological activity. Lyophiiized cake containing a fine foam-like structure can be micronized using techniques known in the art to provide particles of the desired size.

[0133] The compositions of one or more embodiments of the present invention may be administered by known techniques, such as inhalation, oral, intramuscular, intravenous, intratracheal, intraperitoneal, subcutaneous, and transdermal.

[0134] For example, the pharmaceutical compositions of one or more embodiments of the invention are effective in the treatment, including adjunctive treatment, of cystic fibrosis. [0135] In one version, the compositions, when inhaled, penetrate into the nasal cavities and/or airways of the lungs to achieve effective nuclease concentrations. [0136] In one or more embodiments of the invention, a pharmaceutical composition comprising nuclease is administered to the lungs of a patient in need thereof. For example, the patient may have been diagnosed with cystic fibrosis or the patient may be determined to be susceptible to cystic fibrosis.

[0137] Thus, the pharmaceutical compositions of one or more embodiments of the present invention can be used to treat and/or provide prophylaxis for a broad range of patients. A, suitable patient for receiving treatment and/or prophylaxis as described herein is any mammalian patient in need thereof, preferably such mammal is a human. Examples of patients include, but are not limited to, pediatric patients, adult patients, and geriatric patients [0138] In one version, an aerosolizeable pharmaceutical composition comprising nuclease is administered to the lungs and/or nasal cavity of a patient in a manner that results in an effective nuclease concentration.

[0139] In one version, the pharmaceutical composition comprising nuclease is administered so that a target concentration is maintained over a desired period of time. For example, it has been determined that an administration routine that maintains a target concentration of nuclease is effective in treating and/or providing prophylaxis. It has been further determined that by maintaining the nuclease concentration at the target lung concentration for a period of at least about 1 week, such as at least about 2 weeks, or at least about 3 weeks, a pulmonary and/or nasal mucus load can be effectively treated in some patients. [0140] The dosage necessary and the frequency of dosing for maintaining the nuclease concentration within the target concentration depends on the composition and concentration of the nuclease within the composition. In each of the administration regimens, the dosages and frequencies are determined to give a lung nuclease concentration that is maintained within a certain target range. In one version, the nuclease may be administered daily. In this version, the daily dosage of nuclease ranges from about 2 mg to about 75 mg, such as about 3 mg to about 50 mg, about 4 mg to about 25 mg, about 5 mg to about 20 mg, and about 7 mg to about 10 mg. [0141] The drug loading in the small porous particles of the present invention depends on a number of factors including: (a) the volume of the unit dose (blister or capsule); (b) the lung delivery efficiency achieved with the device; (c) factors related to the mechanism of device emptying. It is anticipated that the pulmonary delivery efficiency for the powder formulations of the present invention with portable, passive dry powder inhalers will be 40%-60%, suggesting that the required nominal dose will be about 500μg. Optimal performance of capsule-based devices (e.g., the inhaler shown in Figs. 1A-1E) depends on having sufficient mass in the capsule to facilitate proper capsule spinning and emptying characteristics. Based on these factors it is anticipated that drug loadings will typically range from 1 wt% to 20 wt%, such as 2 wt% to 10 wt%. The required drug loading will be greater for a blister-based inhaler. An example of such an inhaler is disclosed in PCT Application No. PCT/US2007/022830, filed October 25, 2007, and assigned to the same assignee as the invention herein,, and which is fully incorporated herein by reference. This device comprises a smaller volume for loading powder. In this case, the loading will typically range from 5 wt% to 50 wt%, such as 4 wt% to 20 wt%. The drug loading will provide for delivery of DNase in a single puff from a dry powder inhaler. The reduction in administration time is anticipated to improve patient compliance.

[0142] The dose may be administered during a single inhalation or may be administered during several inhalations. The fluctuations of lung nuclease concentration can be reduced by administering the pharmaceutical composition more often or may be increased by administering the pharmaceutical composition less often. Therefore, the pharmaceutical composition of one or more embodiments of the present invention may be administered from about three times daily to about once every two days.

[0143] The amount per dose of nuclease may be an amount that is effective generally ranges from about 0.01 mg/kg to about 5.0 mg/kg, such as about 0.4 mg/kg to about 4.0 mg/kg, or about 0.7 mg/kg to about 3.0 mg/kg.

[0144] Thus, in one version, the pharmaceutical composition may be delivered to the lungs of a patient in the form of a dry powder. Accordingly, the pharmaceutical composition ^• comprises a dry powder that may be effectively delivered to the deep lungs or to another target site. This pharmaceutical composition is in the form of a dry powder comprising particles or particles having a size selected to permit penetration into the alveoli of the lungs. [0145] In some instances, it is desirable to deliver a unit dose, such as powder doses of 5 mg or 10 mg or greater of nuclease to the lung in a single inhalation. The above described phospholipid hollow and/or porous dry powder particles allow for powder doses of about 5 mg or greater, often greater than about 10 mg, and sometimes greater than about 25 mg, to be delivered in a single inhalation and in an advantageous manner. Alternatively, a dosage may be delivered over two or more inhalations. For example, a 10 mg powder dosage may be delivered by providing two unit doses of 5 mg each, and the two unit doses may be separately inhaled. [0146] The nuclease treatment regimen of the present invention may be used alone or in combination with an agent for the treatment of endobronchial infections, particularly infections by P. aeruginosa. In this aspect of the invention, the one or more agent for the treatment of endobronchial infections may be an antibiotic, and may be administered during the first treatment period of nuclease treatment, during the second non-treatment period wherein no nuclease is administered to the endobronchial system of the patient, or during both the first and second treatment periods. In one embodiment of this aspect of the invention, the one or more agent for the treatment of endobronchial infections is administered during the second non- treatment period wherein no nuclease is administered to the endobronchial system of the patient. Suitable agents for the treatment of endobronchial infections include, for example, aminoclycosides such a tobramycin, non-aminoglycoside antiinfective agents, such as monobactam, β-lactam, macrolide, fluoroquinolone and/or glycopeptide antibiotic compounds. For example, the non-aminoglycoside antiinfective agent may be aztreonam. [0147] The dispersions or powder pharmaceutical compositions may be administered using an aerosolization device. The aerosolization device may be a nebulizer, a metered dose inhaler, a liquid dose instillation device, or a dry powder inhaler. The powder pharmaceutical composition may be delivered by a nebulizer as described in WO 99/16420, by a metered dose inhaler as described in WO 99/16422, by a liquid dose instillation apparatus as described in WO 99/16421 , and by a dry powder inhaler as described in U.S. Patent Application No. 09/888,311 filed on June 22, 2001, in WO 99/16419, in WO 02/83220, in U.S. Patent No. 6,546,929, and in U.S. Patent Application No. 10/616,448, filed on July 8, 2003, which are incorporated herein by reference in their entireties. As such, an inhaler may comprise a canister containing the particles or particles and propellant, and wherein the inhaler, comprises a metering valve in communication with an interior of the canister. The propellant may be a hydrofluoroalkane. [0148] Suitable passive dry powder inhalers include both capsule-based inhalers and blister-based inhalers. Suitable capsule-based inhalers include; devices by Nektar Therapeutics disclosed in U.S. Application Nos. 10/298,177; 10/295,783; 10/821,652; 10/821,624; 10/822,850; 10/704,160; 10/714,51 1 ; and 10/313,419, and US Patent Application Publication No. 2005-0150492, each of which are incorporated herein by reference. Devices sold or marketed under the following tradenames an/or trademarks may also be suitable: Handihaler (Boehringer Ingelheim), Eclipse (Aventis), AIR inhaler (Alkermes), Cyclohaler (Plastiape), Flowcaps (Hovione), Turbospin (PH&T), Monohaler (Pfizer), Spinhaler (Aventis), Rotahaler (GSK). Suitable blister-based inhalers include: the Diskus (GSK), the device of Nektar Therapeutics disclosed in PCT Application No. US2007/022830, which is incorporated herein by reference, Gyrohaler (Vectura), E-Flex, Microdrug, Diskhaler (GSK). Also contemplated are active dry powder inhalers including: the Exubera inhalation device, which is described in U.S. Patent No. 6,257,233, which is incorporated herein by reference, Aspirair (Vectura), and Microdose inhaler (Microdose).

[0149] The pharmaceutical composition of one or more embodiments of the present invention typically has improved emitted dose efficiency. Accordingly, high doses of the pharmaceutical composition may be delivered using a variety of aerosolization devices and techniques.

[0150] The emitted dose (ED) of these powders may be greater than about 30%, such as greater than about 40%, greater than about 50%, greater than about 60%, or greater than about 70%.

[0151] An example of a dry powder aerosolization apparatus particularly useful in aerosolizing a pharmaceutical composition 100 according to one or more embodiments of the present invention is shown schematically in Fig. IA. The aerosolization apparatus 200 comprises a housing 205 defining a chamber 210 having one or more air inlets 215 and one or more air outlets 220. The chamber 210 is sized to receive a capsule 225 which contains an aerosolizable pharmaceutical composition comprising nuclease. A puncturing mechanism 230 comprises a puncture member 235 that is moveable within the chamber 210. Near or adjacent the outlet 220 is an end section 240 that may be sized and shaped to be received in a user's mouth or nose so that the user may inhale through an opening 245 in the end section 240 that is in communication with the outlet 220.

[0152] The dry powder aerosolization apparatus 200 utilizes air flowing through the chamber 210 to aerosolize the pharmaceutical composition in the capsule 225. For example, Figs. 1 A-IE illustrate the operation of a version of an aerosolization apparatus 200 where air flowing through the inlet 215 is used to aerosolize the pharmaceutical composition and the aerosolized pharmaceutical composition flows through the outlet 220 so that it may be delivered to the user through the opening 245 in the end section 240. The dry powder aerosolization apparatus 200 is shown in its initial condition in Fig. IA. The capsule 225 is positioned within the chamber 210 and the pharmaceutical composition is contained within the capsule 225. [0153] To use the aerosolization apparatus 200, the pharmaceutical composition in the capsule 225 is exposed to allow it to be aerosolized. In the version of Figs. 1A-1E, the puncture mechanism 230 is advanced within the chamber 210 by applying a force 250 to the puncture mechanism 230. For example, a user may press against a surface 255 of the puncturing mechanism 230 to cause the puncturing mechanism 230 to slide within the housing 205 so that the puncture member 235 contacts the capsule 225 in the chamber 210, as shown in Fig. IB. By continuing to apply the force 250, the puncture member 235 is advanced into and through the wall of the capsule 225, as shown in Fig, IC. The puncture member may comprise one or more sharpened tips 252 to facilitate the advancement through the wall of the capsule 225. The puncturing mechanism 230 is then retracted to the position shown in Fig. ID, leaving an opening 260 through the wall of the capsule 225 to expose the pharmaceutical composition in the capsule 225.

[0154] Air or other gas then flows through an inlet 215, as shown by arrows 265 in Fig.

IE. The flow of air causes the pharmaceutical composition to be aerosolized. When the user inhales 270 through the end section 240 the aerosolized pharmaceutical composition is delivered to the user's respiratory tract. In one version, the air flow 265 may be caused by the user's inhalation 270. In another version, compressed air or other gas may be ejected into the inlet 215 to cause the aerosolizing air flow 265.

[0155] A specific version of a dry powder aerosolization apparatus 200 is described in

U.S. Patent Nos. 4,069,819 and 4,995,385, which are incorporated herein by reference in their entireties. In such an arrangement, the chamber 210 comprises a longitudinal axis that lies generally in the inhalation direction, and the capsule 225 is insertable lengthwise into the chamber 210 so that the capsule's longitudinal axis may be parallel to the longitudinal axis of the chamber 210. The chamber 210 is sized to receive a capsule 225 containing a pharmaceutical composition in a manner which allows the capsule to move within the chamber 210. The inlets 215 comprise a plurality of tangentially oriented slots. When a user inhales through the endpiece, outside air is caused to flow through the tangential slots. This airflow creates a swirling airflow within the chamber 210. The swirling airflow causes the capsule 225 to contact a partition and then to move within the chamber 210 in a manner that causes the pharmaceutical composition to exit the capsule 225 and become entrained within the swirling airflow. This version is particularly effective in consistently aerosolizing high doses of the pharmaceutical composition. In one version, the capsule 225 rotates within the chamber 210 in a manner where the longitudinal axis of the capsule is remains at an angle less than 80 degrees, and preferably less than 45 degrees from the longitudinal axis of the chamber. The movement of the capsule 225 in the chamber 210 may be caused by the width of the chamber 210 being less than the length of the capsule 225. In one specific version, the chamber 210 comprises a tapered section that terminates at an edge. During the flow of swirling air in the chamber 210, the forward end of the capsule 225 contacts and rests on the partition and a sidewall of the capsule 225 contacts the edge and slides and/or rotates along the edge. This motion of the capsule is particularly effective in forcing a large amount of the pharmaceutical composition through one or more openings 260 in the rear of the capsule 225,

[0156] In another passive dry powder inhaler version, the dry powder aerosolization apparatus 200 may be configured differently than as shown in Figs. IA- IE. For example, the chamber 210 may be sized and shaped to receive the capsule 225 so that the capsule 225 is orthogonal to the inhalation direction, as described in U.S. Patent No. 3,991 ,761; which is incorporated herein by reference in its entirety. As also described in U.S. Patent 3,991,761, the puncturing mechanism 230 may puncture both ends of the capsule 225. In another version, the chamber may receive the capsule 225 in a manner where air flows through the capsule 225 as described for example in U.S. Patent Nos. 4,338,931 and 5,619,985. In another version, the aerosolization of the pharmaceutical composition may be accomplished by pressurized gas flowing through the inlets, as described for example in U.S. Patent Nos. 5,458,135; 5,785,049; and 6,257,233, or propellant, as described in WO 00/72904 and U.S. Patent No. 4,1 14,615, which are incorporated herein by reference. These types of dry powder inhalers are generally referred to as active dry powder inhalers.

[0157] In one or more embodiments, a blister-based inhaler device can achieve a high drug loading loading. A specific example of such a device is that disclosed in the previously- referenced PCT Application No. PCT/US2007/022830. This device typically operates with a smaller volume for loading powder. In one or more embodiments with such device, the loading will typically range from 5 wt% to 50 wt%, such as 4 wt% to 20 wt%. In one or more embodiments with such device, the loading will typically range from about 0.7 to 8 mg per blister, such as from about 4 to 6 mg per blister. In one or more embodiments, such drug loading and device will provide for delivery of DNase (or nuclease) in a single puff from a dry powder inhaler,

[0158] The pharmaceutical composition disclosed herein may also be administered to the pulmonary and/or nasal air passages of a patient via aerosolization, such as with a metered dose inhaler. The use of such stabilized preparations provides for superior dose reproducibility and improved lung deposition as disclosed in WO 99/16422, which is incorporated herein by reference in its entirety. MDIs are well known in the art and could be employed for administration of the nuclease. Breath activated MDIs, as well as those comprising other types of improvements which have been, or will be, developed are also compatible with the pharmaceutical composition of one or more embodiments of the present invention. [0159] Nebulizers are known in the art and could easily be employed for administration of the claimed dispersions without undue experimentation. Breath activated nebulizers, as well as those comprising other types of improvements which have been, or will be, developed are also compatible with the stabilized dispersions, which are contemplated as being with in the scope of one or more embodiments of the present invention. Along with the aforementioned embodiments, the stabilized dispersions of one or more embodiments of the present invention may also be used in conjunction with nebulizers as disclosed in WO 99/16420, which is incorporated herein by reference in its entirety, in order to provide an aerosolized medicament that may be administered to the pulmonary and/or nasal air passages of a patient in need thereof. [0160] Along with DPIs, MDIs and nebulizers, it will be appreciated that the stabilized dispersions of one or more embodiments of the present invention may be used in conjunction with liquid dose instillation or LDI techniques as disclosed in, for example, WO 99/16421, which is incorporated herein by reference in its entirety. Liquid dose instillation involves the direct administration of a stabilized dispersion to the lung. In this regard, direct pulmonary and/or nasal administration of bioactive compounds is particularly effective in the treatment of disorders especially where poor vascular circulation of diseased portions of a lung reduces the effectiveness of intravenous drug delivery. With respect to LDI the stabilized dispersions are preferably used in conjunction with partial liquid ventilation or total liquid ventilation. Moreover, one or more embodiments of the present invention may further comprise introducing a therapeutically beneficial amount of a physiologically acceptable gas (such as nitric oxide or oxygen) into the pharmaceutical microdispersion prior to, during or following administration. [0161] The time for dosing is typically short. For a single capsule (e.g., 5 mg powder dose), the total dosing time is normally less than about 1 minute. A 2 capsule dose (e.g., 10 mg powder) usually takes about 1 min. A 5 capsule dose (e.g., 25 mg powder) may take about 3.5 min to administer. Thus, the time for dosing may be less than about 5 min, such as less than about 4 min, less than about 3 min, less than about 2 min, or less than about I min. [0162] A wide variety of nucleases may form a part of the present invention, and include both DNases and ribonucleases (RNases).

[0163] Numerous types of DNase and RNase. have been isolated, and may be characterized by factors such as substrate specificity, cofactor requirements, and whether they cleave nucleic acids internally (endonucleases), from the ends (exonucleases) or attack in both of these modes.

[0164] Specific nonlimiting examples include DNase 1 and RNase A. Other types include: Exonuclease III (E. coli) which removes mononucleotides from the 3^$ termini of duplex DNA; Mung Bean Nuclease (Mung bean sprouts), which digests single-stranded DNA to 5'- phosphorylated mono or oligonucleotides; Nuclease BAL 31 (Alteromonas) which functions as an exonuclease to digest both 5' and 3' ends of double-stranded DNA; Nuclease Sl (Aspergillus) which, depending upon the amount of enzyme used, digests single-stranded DNAs or RNAs (low concentrations), or double-stranded nucleic acids e.g. DNA:DNA, DNA:RNA and RNArRNA (large concentrations); and Ribonuclease Tl (Aspergillus) which cleaves RNA at 3' phosphates of guanine residues, producing oligonucleotides terminal guanosine 3' phosphates. [0165] The foregoing description will be more fully understood with reference to the following Examples. Such Examples, are, however, merely representative of methods of practicing one or more embodiments of the present invention and should not be read as limiting the scope of the invention.

EXAMPLES

Example I Preparation of Hollow Porous Particles of DNase by Spray-Drying

[0166] Phospholipid-based hollow porous DNase I particles were prepared by spray- drying an emulsion-based feedstock with a B-191 spray-drier (Buchi, Flawil, Switzerland). Three powders were prepared, in which the volume fraction of the dispersed phase was increased from 0% (liposomal feedstock) to 10% v/v to 20% v/v (emulsion-based feedstocks). The feedstock was prepared by combining a submicron PFOB-in-water emulsion prepared by high pressure homogenization with an annex solution comprised of DNase 1 and calcium chloride. The details of the emulsion and annex solution are captured below. Composition of the emulsion feedstock

[0167] The emulsion was prepared by first dispersing distearoylphosphatidylcholine,

DSPC (Avanti Polar Lipids, Alabama) in warm sterile water for irrigation (WFI) with a model T- 25 Ultraturrax high shear mixer operated at 1 1,500 rpm for 2 min. The speed of the mixer was increased to 13,000 rpm and the peril uorooctyl bromide, PFOB (Atofina, Paris, France) was added dropwise. After the addition was complete the resulting crude emulsion was allowed to mix for an additional 5 min. The emulsion was then transferred to an Emulsiflex C-5 high pressure homogenizer, for 5 discrete passes at increasing pressures ranging from 10 kpsi to 22 kpsi.

[0168] The resulting submicron emulsion was then combined with an annex solution comprising DNase I dissolved in an aqueous solution containing calcium chloride.

Composition of Annex Solution 0.163 g Calcium chloride 0.05 g DNase 1 14.0 g WFI

[0169] The resulting emulsion based feedstock (2% w/w total solids) containing DNase I dissolved in the continuous phase was spray-dried on a B-191 spray-drier under the following conditions:

Inlet temperature: 85 ⁰C Outlet temperature: 59 ⁰C Aspirator: 81% Pump %: 15% [0 J 70] The spray-drying process produced white powders comprising 2.5% DNase 1 with the balance of solid ingredients comprised of a 2: 1 molar ratio of DSPC: CaCl₂.

[0171] The powder prepared with 20% v/v PFOB was free-flowing with a low bulk density. The powder was observed to easily aerosolize during transfer into the storage receptacle. The volume of powder in the storage receptacle decreased as the volume fraction of PFOB was decreased due to increases in bulk density of the powder. The powder prepared in the absence of PFOB did not appear to flow or aerosolize to the same extent as the powders prepared by the emulsion-based process.

Example II Preparation of Core-Shell Amorphous Glass Particles of DNase by Spray-Drying

[0172] Spray-drying was used to prepare two lots of core-shell particles comprising a shell of the hydrophobic oligopeptide trileucine encapsulating an amorphous glass core comprising DNase and the glass stabilizing agents trehalose and sodium citrate/citric acid. The combination of sodium citrate/citric acid serves as a buffer to control pH, and limit deamidation in the protein. The preferred pH is in the range from pH 4.5 to 6.5. The optimal pH likely differs for formulations in solution versus in the solid state, and will require further investigation. Calcium ions are added to aid in the stabilization of DNase against aggregation in solution. [0173] The nominal compositions of the spray-dried particles are captured below:

[0174] The powders are manufactured by first preparing a solution comprising the components listed above at a total solids concentration of 1% w/w. The pH of the feedstock was ca., 5.

[0175] The resulting solution was then spray-dried with a B-191 spray-dryer (Buchi,

Flawil, Switzerland) under the following conditions: inlet temperature = 100 ⁰C; outlet temperature = 58 ⁰C; pump = 38-41% (7.1-7.6 ml/rnin), aspirator = 100%.

Example III Physicochemical Properties of Hollow Porous Particles of DNase I

[0176] The geometric particle size distributions of the spray-dried hollow porous particles as determined by laser diffraction (Sympatec, HELOS H1006 equipped with a RODOS T4.1 vibrating trough and disperser, Clausthal-Zellerfeld, Germany) at a driving pressure of 2 bar are detailed below.

[0177] The median diameter (x50) for the 20% v/v PFOB formulation is 3.4μm, decreasing to 3.0μm for the 10% v/v PFOB formulation. This is expected as the greater the volume fraction of PFOB in the formulation, the greater the degree of inflation and the larger the final diameter of the spray-dried particle. The median diameter (3.6 μm) of the particle prepared without blowing agent is likely the result of extensive particle agglomeration. This is reflected in the broad particle size distribution noted for this sample, where a geometric standard deviation (GSD) equals 2.6 versus 1.6 for the two formulations containing blowing agent. This formulation also exhibited significant variability in aerosol measurements, as discussed in Example V. [0178] The bulk density of the hollow porous powders decreased with increasing amounts of blowing agent, viz: 0.16 g/cm³ (0% PFOB), 0.041 g/cm³ (10% PFOB), 0.017 g/cm³ (20% PFOB).

Example IV Physicochemical Properties of Amorphous Glass Particles of DNase I

[0179] The geometric particle size distributions of the spray-dried amorphous particles comprising 2.5% w/w DNase as determined by laser diffraction (Sympatec, HELOS H1006 equipped with a RODOS T4.1 vibrating trough and disperser, Clausthal-Zellerfeld, Germany) at a driving pressure of 2 bar are detailed below.

[0180] The median diameters (x50 ~ 1.3 μm) are significantly less than observed for the formulations containing blowing agent, with a narrow GSD (~1.6 μm), [0181] The bulk density of the amorphous glass powders were 0.178 (lot # - 6093-03-

01), 0.237 (lot # - 6093-05-01), respectively. As expected the bulk densities of these powders are greater than those prepared using PFOB. The density can be decreased by the addition of greater percentages of trileucine in the formulation.

Example V Aerosol Properties of Hollow Porous Particles of DNase I

[0182] The aerosol particle size distributions of the hollow porous particle formulations of DNase I prepared according to Example I were determined by Andersen cascade impaction. The fill mass was 17 mg (3/4 capsule volume) for the 10% PFOB formulation and 20 mg for the 0% PFOB formulation. The inhaler shown in U.S. Application No. 10/822,850, which is incorporated by reference herein, a medium resistance device, was operated at a flow rate of 56 L/min. The mass of particles less than 3.3μm is thought to provide an approximation of total lung deposition, when operated at a flow rate of 28.3 L/min. At the 56 L/min flow rate utilized with the device shown in U.S. Application No. 10/822,850, which is incorporated by reference herein, the cutoff diameter needs to be corrected for the increases in inertial impaction which occur as a result of the increased flow rate. Hence a cutoff diameter of 2.3 μm is appropriate. Based on the current nebulized Pulmozyme product, the target FPD₂ i_μm is -200 μg (10% lung deposition). The results are captured below. FPD = fine particle dose.

[0183] The aerodynamic particle size distribution for the formulation without blowing agent is in the range from 1 to 5 μm, but outside the preferred range from 1 to 3 μm. Moreover there is significant variability in a PSD from run to run, owing to the poor powder flow noted with the powders prepared without blowing agent. The FPD_{2 3μm} is also significantly less than the target of 200 μg, indicating that the drug loading would need to be increased for powders of this type. The preferred embodiment with the lipid-based formulations is the inclusion of blowing agent in the formulation to drive the development of the preferred hollow porous particle morphology. In the case of the formulation comprising 10% PFOB, the MMAD was 2.2 μm, and the FPD_{2 3μm} was 195 μg. The FPD_23μm values suggest that the 200 μg target dose can be achieved with the compositions and targeted fill masses detailed in Example I.

Example VI Emptying Profiles of Hollow Porous Particles

[0184] The emptying profiles of the hollow porous particles of Example I from the device shown in U.S. Application No. 10/822,850, which is incorporated by reference herein, were examined by laser photometry at a flow rate of 56 L/min. AU three powders exhibit near complete emptying from the capsule within a volume of about 1 L or less, such as about 0.8 L or 0.7 L, or 0.6 L, as shown by the two lower plots of Fig. 2. The average flow profile for 12 pediatric patients with cystic fibrosis through the device shown in U.S. Patent Application Publication No. 2005-0150492 (USSNl 0/822,850), which is incorporated by reference herein, was explored previously (Standaert et al: Young cystic fibrosis patients can effectively use a novel high-payload capsule-based dry powder inhaler with tobramycin inhalation powder for inhalation. Pediatr Pulmonol (2004) 27:284). The mean peak flow rate was about 70 L/min, with a mean inhaled volume of 1.0 L. It is expected that the profiles shown in Figure 1 will shift to even faster emptying times at a flow rate of 70 L/min. Nonetheless, even at a flow rate of 56 L/min, it is likely that pediatric cystic fibrosis patients can effectively empty therapeutic doses of DNase aerosol from the portable, passive inhaler shown in U.S. Patent Application Publication No. 2005-0150492.

Example VII Aerosol Properties of Amorphous Glass Particles of DNase I

[0185] The aerodynamic particle size distributions of the amorphous glass formulations of DNase I prepared according to Example II were determined by Andersen cascade impaction at a capsule fill mass of 20 mg (500 μg DNase I). The inhaler shown in U.S. Patent Application Publication No. 2005-0150492, was operated at a flow rate of 56 L/min. The results are captured below.

[0186] The measured aerodynamic diameters (MMAD) are in the preferred range from 1 to 3 μm. The FPD₂3_μm values suggest that the 200 μg target dose can be achieved with small changes to the compositions and targeted fill masses detailed in Example II.

Example VIII Emptying Profiles of Amorphous Glass Particles

[0187] The emptying profiles of the hollow porous particles of Example I from the device shown in U.S. Patent Application Publication No. 2005-0150492, were examined by laser photometry at a flow rate of 56 L/min. The two powders (both 20 mg mass) exhibit near complete emptying from the capsule within a volume of about 1 L or less (Fig. 3). Hence, it is likely that pediatric cystic fibrosis patients can effectively empty therapeutic doses of DNase aerosol from the portable, passive inhaler shown in U.S. Patent Application Publication No. 2005-0150492. Such a device, in one or more exemplary forms or embodiments, comprises a handheld aerosol ization apparatus which comprises a housing defining a chamber having a plurality of air inlets, the chamber being sized to receive a receptacle which contains an aerosolizable pharmaceutical formulation; and an end section associated with the housing, the end section sized and shaped to be received in a user's mouth or nose so that the user may inhale through the end section to inhale aerosolized pharmaceutical formulation that has exited the receptacle. In one or more embodiments, the device further comprises a shield which covers at least one but not all of the air inlets, whereby the shield prevents blockage of the at least one air inlet by a user grasping the apparatus. In one or more embodiments the device further comprises a plurality of tangentially oriented slots within the chamber, such that when a user inhales, outside air is caused to flow through the tangential slots to create a swirling airflow within the chamber, which then causes the receptacle to move within the chamber in a manner that causes the pharmaceutical formulation to exit the receptacle and become entrained within the swirling airflow, thus assuring full and complete aerosolization.

Example IX

Preparation of High Load DNase Core-Shell Amorphous Glass Particles by Spray Drying [0188] Aerosol dry powders were prepared by spray drying bovine DNase I from aqueous solutions containing trileucine and sodium citrate as excipients using a Bϋchi 190 spray dryer.

[0189] More specifically DNase I solutions formed by dissolving bovine DNase I in water along with sodium citrate and trileucine, as shown below. The pH was adjusted to 4.3 by adding 2 M NaOH.

[0190] The bovine DNase I solutions was spray dried using a laboratory scale Biichi 190 spray dryer (Bϋchi Labortechnik, Ag., Meierseggstrasse, Switzerland) fitted with a modified cyclone, an atomizer nozzle, and a powder collection vessel. The atomizer of the spray dryer was operated with clean dry air. The liquid flow rate into the spray dryer was 5 ml/min. The inlet temperature was adjusted to achieve the target particle size and morphology (80 ⁰C to 150 ⁰C, e.g., 130 ⁰C). The outlet temperature ranged from about 30 ⁰C to 100 ⁰C, e.g., 75 ⁰C. The atomizer air pressure was set at 65 psi. The yield was 58.9%. [0191] The powders were transferred into a glovebox with a relative humidity less than

5% and placed into unit dosage forms (blister packs, BP's) suitable for use in a dry powder inhaler device as described in PCT Application No. US2007/022830, which is incorporated herein by reference. In other words, dry powders were packaged as premetered doses in individually manufactured blisters to fit a passive inhalation device. A nominal dose of 2 mg DNase 1 per blister could be achieved with a 2.3 mg dry powder fill-weight. Lower nominal doses could be achieved by reducing the blister fill-weight or by adjusting the DNase I to lower percentage in the formulation. In the latter case, a 40% DNase I formulation with 30% of trileucine and 30% sodium citrate would reduce the nominal dose of 0.92 mg with the same blister fill-weight.

Example X

SEM Images of DNAse Particles

[0192] Particles prepared in accordance with Example 1 (lot No. 6091-4) were imaged using SEM, and results are shown in Figs 4A (at 1000 magnification and 4B (20000 magnification).

[0193] While the invention has been described in connection with certain embodiments so that aspects thereof may be more fully understood and appreciated, it is not intended to limit the invention to these particular embodiments. On the contrary, it is intended to cover all alternatives, modifications and equivalents as may be included within the scope of the invention as defined by the appended claims.

Claims

WHAT IS CLAIMED IS:

1. A powder composition, comprising: particles comprising less than 40 wt% of nuclease, wherein the particles have a mass median aerodynamic diameter from about 1 μm to about 5 μm.

2. A pharmaceutical composition, comprising: a powder comprising: an effective amount of nuclease; and pharmaceutically acceptable excipient, wherein the powder comprises nuclease particles and having a mass median aerodynamic diameter from about 1 μm to about 5 μm.

3. The pharmaceutical composition of claim 2, wherein the pharmaceutically acceptable excipient comprises at least one member selected from carbohydrate, lipid, amino acid, buffer, and salt.

4. The pharmaceutical composition of claim 2, wherein the pharmaceutically acceptable excipient comprises phospholipid.

5. The pharmaceutical composition of claim 2, wherein the pharmaceutically acceptable excipient comprises metal ion.

6. The pharmaceutical composition of claim 2, wherein the pharmaceutically acceptable excipient comprises phospholipid and metal ion.

7. The pharmaceutical composition of claim 2, wherein the pharmaceutically acceptable excipient comprises trileucine.

8. The pharmaceutical composition of claim 2, wherein the pharmaceutical composition comprises hollow and/or porous particles.

9. The pharmaceutical composition of claim 2, wherein the pharmaceutical composition comprises particles comprising nuclease in a matrix formed by the pharmaceutically acceptable excipient.

10. The pharmaceutical composition of claim 9, wherein the pharmaceutically acceptable excipient comprises at least one phospholipid.

11. The pharmaceutical composition of claim 2, wherein the pharmaceutical composition comprises dry powder.

12. The pharmaceutical composition of claim 2, wherein the pharmaceutical composition further comprises a propellant.

13. The pharmaceutical composition of claim 12, wherein the propellant comprises at least one hydrofluoroalkane.

14. A unit dosage form, comprising: a container containing a pharmaceutical composition comprising: an effective amount of nuclease, and pharmaceutically acceptable excipient, wherein the pharmaceutical composition comprises particles having a mass median aerodynamic diameter from about 1 μm to about 5 μm.

15. The unit dosage form of claim 14, wherein the container comprises a capsule.

16. The unit dosage form of claim 14, wherein the particles comprise less than 40 wt% of nuclease.

17. The unit dosage form of claim 14, wherein the pharmaceutical composition comprises dry powder.

18. The unit dosage form of claim 14, wherein the pharmaceutically acceptable excipient comprises at least one member selected from carbohydrate, lipid, amino acid, buffer, and salt.

19. The unit dosage form of claim 14, wherein the pharmaceutically acceptable excipient comprises phospholipid.

20. The unit dosage form of claim 14, wherein the pharmaceutically acceptable excipient comprises metal ion.

21. The unit dosage form of claim 14, wherein the pharmaceutically acceptable excipient comprises phospholipid and metal ion.

22. The unit dosage form of claim 14, wherein the pharmaceutically acceptable excipient comprises trileucine.

23. The unit dosage form of claim 14, wherein the pharmaceutical composition comprises hollow and/or porous particles.

24. The unit dosage form of claim 14, wherein the pharmaceutical composition comprises particles comprising nuclease in a matrix formed by the pharmaceutically acceptable excipient.

25. The unit dosage form of claim 24, wherein the pharmaceutically acceptable excipient comprises at least one phospholipid.

26. A delivery system, comprising: an inhaler; and a pharmaceutical composition comprising particles comprising; nuclease, and pharmaceutically acceptable excipient, wherein the particles comprise less than 40 wt% of nuclease.

27. The delivery system of claim 26, wherein the inhaler comprises a dry powder inhaler.

28. The delivery system of claim 26, wherein the inhaler comprises a canister containing the particles and propellant, and wherein the inhaler comprises a metering valve in communication with an interior of the canister.

29. The delivery system of claim 26, wherein the inhaler comprises a nebulizer, and wherein the particles are suspended in a liquid.

30. The delivery system of claim 26, wherein the pharmaceutical composition comprises particles having a mass median aerodynamic diameter ranging from about 1 μm to about 5 μm.

31. The delivery system of claim 26, wherein the pharmaceutically acceptable excipient comprises at least one member selected from carbohydrate, lipid, amino acid, buffer, and salt.

32. The delivery system of claim 26, wherein the pharmaceutically acceptable excipient comprises phospholipid.

33. The delivery system of claim 26, wherein the pharmaceutically acceptable excipient comprises metal ion.

34. The delivery system of claim 26, wherein the pharmaceutically acceptable excipient comprises phospholipid and metal ion.

35. The delivery system of claim 26, wherein the pharmaceutically acceptable excipient comprises trileucine.

36. The delivery system of claim 26, wherein the pharmaceutical composition comprises hollow and/or porous particles.

37. The delivery system of claim 26, wherein the pharmaceutical composition comprises particles comprising nuclease in a matrix formed by the pharmaceutically acceptable excipient.

38. The delivery system of claim 37, wherein the pharmaceutically acceptable excipient comprises at least one phospholipid.

39. The delivery system of claim 26, wherein the pharmaceutical composition comprises dry powder.

40. The delivery system of claim 26, wherein the pharmaceutical composition further comprises a propel lant.

41. The delivery system of claim 40, wherein the propellant comprises at least one hydro fluoroalkane.

42. A method of making spray-dried particles, comprising: suspending nuςlease in a liquid to form a feedstock; and spray drying the feedstock to produce the spray-dried particles, wherein the particles comprise less than 40 wt% of nuclease and have a mass median aerodynamic diameter from about 1 μm to about 5 μm.

43. The method of claim 42, wherein the feedstock further comprises a pharmaceutically acceptable excipient.

44. The method of claim 42, wherein the spray-dried particles further comprise a pharmaceutically acceptable excipient.

45. The method of claim 42, further comprising collecting the spray-dried particles.

46. The method of claim 42, wherein the feedstock further comprises an emulsifying agent.

47. The method of claim 46, wherein the emulsifying agent comprises phosphatidylcholine.

48. The method of claim 47, wherein the phosphatidylcholine comprises distearoyl phosphatidylcholine.

49. The method of claim 42, wherein the feedstock further comprises a blowing agent.

50. The method of claim 42, wherein the spray-dried particles comprise nuclease in a phospholipid matrix.

51. The method of claim 42, wherein the spray-dried particles are hollow and/or, porous.

52. The method of claim 51, wherein the feedstock further comprises a hydrophobic amino acid.

53. The method of claim 42, wherein the feedstock further comprises trileucine.

54. A method of treating a condition associated with increased viscosity of pulmonary and/or nasal secretions, comprising: administering by inhalation an effective amount of a composition comprising nuclease to a patient in need thereof, wherein the composition comprises a powder comprising particles comprising less than 40 wt% of nuclease and having a mass median aerodynamic diameter from about 1 μm to about 5 μm.

55. The method of claim 54, wherein the administration comprises delivering 1 dose of the composition each day.

56. The method of claim 54, wherein the particles further comprise a pharmaceutically acceptable excipient.

57. The method of claim 56, wherein the pharmaceutically acceptable excipient comprises at least one member selected from carbohydrate, lipid, amino acid, buffer, and salt.

58. The method of claim 56, wherein the pharmaceutically acceptable excipient comprises phospholipid.

59. The method of claim 56, wherein the pharmaceutically acceptable excipient comprises metal ion.

60. The method of claim 56, wherein the pharmaceutically acceptable excipient comprises phospholipid and metal ion.

61. The method of claim 56, wherein the pharmaceutically acceptable excipient comprises trileucine.

62. The method of claim 56, wherein the pharmaceutical composition comprises hollow and/or porous particles.

63. The method of claim 56, wherein the pharmaceutical composition comprises particles comprising nuclease in a matrix formed by the pharmaceutically acceptable excipient.

64. The method of claim 63, wherein the pharmaceutically acceptable excipient comprises at least one phospholipid.

65. The method of claim 56, wherein the pharmaceutical composition comprises dry powder.

66. The method of claim 56, wherein the pharmaceutical composition further comprises a propellant.

67. The method of claim 66, wherein the propellant comprises at least one hydro fluoroalkane.

68. The method of claim 56, wherein the administration comprises delivering the composition in dry powder form using a dry powder inhaler.

69. The method of claim 56, wherein the composition comprises a propellant and wherein the administration comprises aerosolizing the composition by opening a valve to release the composition.

70. The method of claim 69, wherein the propellant comprises at least one hydrofluoroalkane.

71. The method of claim 56, wherein the administering comprises pulmonary administration.

72. The method of claim 56, wherein the administering comprises nasal administration.