Surfactant preparations for pulmonary delivery, and methods for the preparation and use thereof

Abstract

A surfactant reagent, comprising about 1 to about 10 wt% of surfactant polypeptide and about 90 to 99 wt% of a lipid mixture and methods for the preparation and use thereof. The surfactant polypeptide is cationic and the lipid mixture consists of 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC), and one or both of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol sodium salt (POPG-Na), and one or both of 1-palmitoyl-2-oleoyl-glycero-3-phosphatidylcholine (POPC) or 2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC); or the surfactant polypeptide is anionic and the lipid mixture consists of POPC, DPPC, and 1,2-dioleoyl-3-trimethylammonium propane (DOTAP).

Description

SURFACTANT PREPARATIONS FOR PULMONARY DELIVERY, AND METHODSFOR THE PREPARATION AND USE THEREOF

[0001] The present application claims the benefit of United States Provisional Application No. 63 / 650,366, filed on May 21, 2024, from which priority is claimed and which is hereby incorporated by reference in its entirety.FIELD OF THE INVENTION

[0002] The present invention relates to the preparation of surfactant formulations for pulmonary administration and their use as vehicles for therapeutics.BACKGROUND OF THE INVENTION

[0003] The pulmonary surfactant system is essential to avoid alveolar collapse in mammalian lungs. It comprises a surface-active material composed of a major fraction of lipids and a small fraction of four specific proteins: the hydrophobic SP-B and SP-C, essential for the biophysical function of surfactant at the interface, and the two hydrophilic proteins SP-A and SP-D, involved in lung immune defense.

[0004] CUROSURF® (Chiesi Farmaceutici, S.p.A.) is a natural surfactant preparation used to treat premature infants for Respiratory Distress Syndrome. It is prepared from porcine lungs, and comprises almost exclusively phospholipids, in particular phosphatidylcholine (about 70% of the total phospholipid content) and about 1% of surfactant-specific low molecular weight hydrophobic proteins SP-B and SP-C. Similar animal-derived surfactant preparations include INFASURF® (ONY Biotech Inc.) and SURVANTA® (Abb Vie Inc.). Most commercially used naturally-derived surfactants are complex mixtures of phospholipids, neutral lipids, SP-B and SP- C. Although variations exist among different products, the main lipid components of these exogenous surfactants are dipalmitoylphosphatidylcholine (DPPC), other phosphatidylcholines with lower phase transition temperatures (PC), phosphatidylglycerols (PG), and neutral lipids like cholesterol. These lipids, together with SP-B and SP-C are ultimately responsible for the ability of exogenous surfactant to rapidly adsorb to the air-liquid interface and spread throughout the airway.

[0005] The goal of using exogenous surfactant for drug delivery is to open collapsed airways and areas with edema such that the therapeutic is able to reach the areas of the lung in need of treatment. Inhalation therapy for the delivery of drug substances to treat pulmonary disease haveseveral potential advantages in comparison to parenteral and oral delivery routes. Among these is the ability to bypass plasma drug metabolism systems, to release therapeutic ingredients directly at the target site for management of pulmonary disease, and to take advantage of the large surface area within the lungs for drug uptake. Formulations used for pulmonary delivery frequently involve surfactants and co-solvents to prepare stable formulations utilized for the pulmonary route of administration. Many studies have reported the efficiency and advantages of using pulmonary surfactant to transport different therapeutic molecules along the respiratory surface, such as hydrophobic drugs, antibiotics, proteins, and small-interfering RNA.

[0006] Although effective, animal -derived surfactant products have several issues with them including: 1) high manufacturing costs associated with the harvesting and processing of lung tissues from animals which results in batch-to-batch variability that requires complex compositional, biophysical, and bioactivity quality control testing; 2) the risk of exposure to prion-transmitted diseases, such as bovine spongiform encephalopathy; and 3) cultural and religious issues that arise due to the animal source being from cows or pigs. Recently, a peptide mimetic of SP-B had been developed. Known as Super Mini-B (SMB), the mimetic consists of the N-terminal (-residues 1-25) and C-terminal a-helices (-residues 63-78) of native SP-B connected with a -PKGG- turn to form a a-helix hairpin. Other surfactant protein mimetics are disclosed in US20180037610 and US20230107337, each of which is hereby incorporated by reference.SUMMARY OF THE INVENTION

[0007] It is an object of the invention to provide a surfactant reagent that is simple to manufacture, exhibits clinically relevant activity as a lung surfactant, is stable in room temperature storage, provides a hydration process that can be performed without specialized equipment, and can serve as a drug delivery vehicle.

[0008] In a first aspect, the present invention provides surfactant reagent, comprising: about 1 to about 10 wt% of surfactant polypeptide and about 90 to 99 wt% of a lipid mixture, wherein the surfactant polypeptide is cationic, and the lipid mixture consists ofone or more lipids selected from a first group consisting of l,2-dipalmitoyl-sn-glycero-3- phosphatidylcholine (DPPC), l,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC), or 1- palmitoyl-2-oleoyl-glycero-3 -phosphatidylcholine (POPC) or salts thereof, and one or more lipids selected from a second group consisting of l-palmitoyl-2-oleoyl-sn- glycero-3 -phosphoglycerol (POPG), or l,2-dioleoyl-sn-glycero-3-phosphoglycerol (DOPG) or salts thereof; or the surfactant polypeptide is anionic, and the lipid mixture consists of one or more lipids selected from a first group consisting of DPPC, DOPC, POPC, and salts thereof, and one or more lipids selected from a second group consisting of l,2-di-O-octadecenyl-3- trimethylammonium propane (DOTMA),l,2-dioleoyl-3 -trimethylammonium propane (DOTAP), and salts thereof, wherein the surfactant reagent is in the form of a dried cake or powder.

[0009] As described hereinafter, the formulations and methods of the present invention can advantageously create a dried cake or powder formulation of surfactant without the need to use standard lyophilization protocols and equipment. Lyophilization is a low temperature dehydration process that involves cooling the product below the triple point of water and lowering the air pressure, thereby removing the ice by sublimation, followed by a secondary adsorption stage during which the ionically-bound water molecules are removed. Agitation of the container containing a cake may create a loose powder, no longer recognizable as a lyophilized cake. The term “cake” as used herein refers to a stable solid material formed from lyophilization or sublimation of a formulation to remove solvents. See, e.g., Patel et al., Lyophilized Drug Product Cake Appearance: What Is Acceptable? Journal of Pharmaceutical Sciences 106 (2017) 1706-1721; Hedberg et al., Mechanical Behavior and Structure of Freeze-Dried Cakes, In: Ward, K., Matejtschuk, P. (eds) Lyophilization of Pharmaceuticals and Biologicals. Methods in Pharmacology and Toxicology. Humana Press, New York, NY. doi.org / 10.1007 / 978-1-4939- 8928-7 13.

[0010] The term “about” as used herein for a numeric value refers to + / - 10% or less of the recited numeric value. By way of example, a value of “about 1” refers to 0.9 to 1.1. “About X” also includes a range of values that are ±5%, ±2%, ±1%, ±0.5%, ±0.2%, or ±0.1% of X, where X is a numerical value.

[0011] The term “polypeptide” as used herein refers to a series of amino acids connected one to the other typically by peptide bonds between the a-amino and carboxyl groups of adjacent amino acids. The term “polypeptide” refers to any chain or chains of two or more amino acids and does not refer to a specific length of the product. Thus, peptides, dipeptides, tripeptides, oligopeptides, “protein”, “amino acid chain”, or any other term used to refer to a chain or chains of two or more amino acids, are included within the definition of “polypeptide” and the term “polypeptide” may be used instead of or interchangeably with any of these terms. The term “polypeptide” is also intended to refer to products of post-expression modifications of the polypeptide including without limitation glycosylation, acetylation, phosphorylation, amidation, derivatization by known blocking / protecting groups, proteolytic cleavage or naturally occurring amino acid modification. A polypeptide may be derived from a natural biological source or produced by recombinant technology, but is not necessarily translated from a designated nucleic acid sequence. It can be generated in any way, including by chemical synthesis.

[0012] In certain embodiments, the surfactant polypeptide is cationic. Examples of such cationic surfactant polypeptides include, but are not limited to, SP-B, SP-C, Mini-B, Super Mini- B, B-YL, KL4, SP-C33, SP-C30, and KALA. Most preferred are Super Mini-B. and Mini-B.

[0013] For cationic polypeptides, the lipid mixture preferably contains a weight ratio of the first group lipids to the second group lipids of between 1 :7 to 7: 1. In certain embodiments, the weight ratio is between 1 :2 and 2:1, and in preferred embodiments the weight ratio is between 1 : 1.5 and 1.5: 1.

[0014] In other embodiments, the surfactant polypeptide is anionic. Examples of such anionic surfactant polypeptides include, but are not limited to, GALA, E-Mini-B, Super E-Mini-B, and EL4.

[0015] In certain embodiments, a surfactant reagent as described herein comprises from about 1 wt% to about 6 wt% of surfactant polypeptide. In preferred embodiments, a surfactant reagent comprises about 2 to 4 wt% of a surfactant protein or peptide thereof, or combinations thereof. By way of example only, a dried surfactant reagent may comprise about 3 wt% of thecationic surfactant protein and the lipid mixture may consist of about 20 wt% POPG-Na, about 29.4 wt% POPC, and about 47.6 wt% DPPC. Similarly, a dried surfactant reagent may comprise about 3 wt% of an anionic surfactant protein and the lipid mixture may consist of about 20 wt% DOTAP, about 29.4 wt% POPC, and about 47.6 wt% DPPC.

[0016] In another aspect, the present invention provides a method of preparing an aqueous surfactant reagent, comprising:

[0017] rehydrating the surfactant reagent described herein in an aqueous buffer.

[0018] In a related aspect, the invention provides a therapeutic composition for pulmonary delivery, comprising: about 0.1 to about 1 wt% of a surfactant protein; about 0.9 to about 1 wt% lipid; and about 98 to about 99 wt% of one or more aqueous pharmaceutically acceptable excipients, wherein the surfactant polypeptide is cationic, and the lipid mixture consists of one or more lipids selected from a first group consisting of l,2-dipalmitoyl-sn-glycero-3- phosphatidylcholine (DPPC), l,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC), or 1- palmitoyl-2-oleoyl-glycero-3 -phosphatidylcholine (POPC) or salts thereof, and one or more lipids selected from a second group consisting of l-palmitoyl-2-oleoyl-sn- glycero-3 -phosphoglycerol (POPG), or l,2-dioleoyl-sn-glycero-3-phosphoglycerol (DOPG) or salts thereof; or the surfactant polypeptide is anionic, and the lipid mixture consists of one or more lipids selected from a first group consisting of DPPC, DOPC, POPC, and salts thereof, andone or more lipids selected from a second group consisting of l,2-di-O-octadecenyl-3- trimethylammonium propane (DOTMA),l,2-dioleoyl-3 -trimethylammonium propane (DOTAP), and salts thereof.

[0019] The aqueous buffer (also referred to as an aqueous excipient) can be any pharmaceutically acceptable buffer. Examples include, but are not limited to, bacteriostatic water for injection (BWFI), phosphate buffered saline (PBS), Ringer's solution and dextrose solution. Additionally, other materials desirable from a commercial and user standpoint may be included, such as excipients and diluents. Preferred aqueous buffers are PBS or 0.9% saline in 10-50 mM carbonate buffer, pH 6.4-7.4.

[0020] In certain embodiments, the aqueous surfactant reagent can comprise other pharmaceutical substances, excipients, etc. In various embodiments, these pharmaceutical substances may comprise lipid nanoparticles, small molecule therapeutics, biologies, antibiotics, antivirals, antibodies, anti-inflammatory compounds, chemotherapeutics, etc. Particularly preferred are pharmaceutical substances selected for pulmonary delivery. These may be intended for either local or systemic purposes.

[0021] In yet another aspect, the present invention provides a method for delivering a surfactant reagent to an individual in need thereof. This can comprise administering the surfactant as an aqueous reagent produced according to the methods described herein or as a dry powder.

[0022] The aqueous surfactant reagent may be administered by any conventional route including, but not limited to, mucosal, pulmonary, genitourinary, intrathecal, oral, rectal, topical, ocular, etc. routes. In certain preferred embodiments, the pulmonary route of administration is by intranasal or intratracheal installation or by inhalation.

[0023] In still another aspect, the present invention provides a method of producing a surfactant reagent, comprising mixing surfactant polypeptides and a lipid mixture in an organic:aqueous solvent system, followed by removing the organic:aqueous solvent system by sublimation or spray drying to produce the surfactant reagent in the form of a dried cake or powder, wherein(i) the surfactant polypeptide is at about 1 to about 10 wt% of the mixture,(ii) the organic: aqueous solvent system consists of one or more organic solvents selected from the group consisting of Cl -6 alcohols, acetone, and chloroform, and water, wherein the ratio of organic solvents: water in the organic:aqueous solvent system is 9: 1 or greater; and(iii)(a) the surfactant polypeptide is cationic, and the lipid mixture consists of one or more lipids selected from a first group consisting of l,2-dipalmitoyl-sn-glycero-3- phosphatidylcholine (DPPC), l,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC), or 1- palmitoyl-2-oleoyl-glycero-3 -phosphatidylcholine (POPC) or salts thereof, and one or more lipids selected from a second group consisting of l-palmitoyl-2-oleoyl-sn- glycero-3 -phosphoglycerol (POPG), or l,2-dioleoyl-sn-glycero-3-phosphoglycerol (DOPG) or salts thereof; or(iii)(b) the surfactant polypeptide is anionic, and the lipid mixture consists of one or more lipids selected from a first group consisting of DPPC, DOPC, POPC, and salts thereof, and one or more lipids selected from a second group consisting of l,2-di-O-octadecenyl-3- trimethylammonium propane (DOTMA),l,2-dioleoyl-3 -trimethylammonium propane (DOTAP), and salts thereof.

[0024] In certain embodiments, the one or more organic solvents are selected from the group consisting of tert-butanol, tert-amyl alcohol, methanol, ethanol, isopropanol, acetone, and 1- butanol, or combinations thereof. By way of example only, the organic:aqueous solvent system may be selected from tert-butanol: water, tert-butanol: water, methanol : tert-butanol: water, ethanol: tert-butanol: water, isopropanol : tert-butanol: water, acetone: tert-butanol: water, and tertamyl alcohol: water.

[0025] Sublimation to remove the solvent system may be carried out according to a standard lyophilization protocol between -80°C and 0°C. In preferred embodiments, the mixture of surfactant polypeptides, a lipid mixture, and an organic:aqueous solvent system is sublimated at between -20°C and 25°C under vacuum. By way of example, the sublimation may occur at 0°C to 4°C under vacuum.

[0026] In addition to sublimation, the dried cake or powder formulation of surfactant as described herein may be formed by spray drying. Spray drying is a fast, scalable, and continuous process to produce a fine powder suitable for inhalation, and the physical properties of the powder can be controlled via the spray drying process parameters. In preferred embodiments, the mixture of surfactant polypeptides, a lipid mixture, and an organic:aqueous solvent system.

[0027] Thus, in still another aspect, the present invention provides a method of producing a surfactant reagent, comprising mixing surfactant polypeptides and a lipid mixture in an organic:aqueous solvent system, followed by removing the organic:aqueous solvent system by spray drying to produce the surfactant reagent in the form of a dried powder, wherein(i) the surfactant polypeptide is at about 1 to about 10 wt% of the mixture,(ii) the organic: aqueous solvent system consists of one or more organic solvents selected from the group consisting of Cl -6 alcohols, acetone, and chloroform, and water, wherein the ratio of organic solvents: water in the organic:aqueous solvent system is 9: 1 or greater; and(iii)(a) the surfactant polypeptide is cationic, and the lipid mixture consists of one or more lipids selected from a first group consisting of l,2-dipalmitoyl-sn-glycero-3- phosphatidylcholine (DPPC), l,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC), or 1- palmitoyl-2-oleoyl-glycero-3 -phosphatidylcholine (POPC) or salts thereof, and one or more lipids selected from a second group consisting of l-palmitoyl-2-oleoyl-sn- glycero-3 -phosphoglycerol (POPG), or l,2-dioleoyl-sn-glycero-3-phosphoglycerol (DOPG) or salts thereof; or the surfactant polypeptide is anionic, and the lipid mixture consists of one or more lipids selected from a first group consisting of DPPC, DOPC, POPC, and salts thereof, and one or more lipids selected from a second group consisting of l,2-di-O-octadecenyl-3- trimethylammonium propane (DOTMA),l,2-dioleoyl-3 -trimethylammonium propane (DOTAP), and salts thereof.

[0028] In certain embodiments, the one or more organic solvents are selected from the group consisting of tert-butanol, tert-amyl alcohol, methanol, ethanol, isopropanol, acetone, and 1- butanol, or combinations thereof. By way of example only, the organic:aqueous solvent system may be selected from tert-butanol: water, tert-butanol: water, methanol : tert-butanol: water, ethanol: tert-butanol: water, isopropanol : tert-butanol: water, acetone: tert-butanol: water, and tertamyl alcohol: water.

[0029] Spray drying to remove the solvent system may be carried out at temperatures sufficient to evaporate the solvents quickly without damaging the lipid and surfactant polypeptide components. In preferred embodiments, the mixture of surfactant polypeptides, a lipid mixture, and an organic: aqueous solvent system is spray dried above the triple point of water, e.g., is carried out at between 4°C and 60°C. By way of example, the spray drying may occur at 25°C. In various embodiments, spray drying is performed with a pump speed of 2.9-3.1, atomizing air at 0.04-0.05, nitrogen gas blower at 5.5-6.0, inlet temperature at -10 to 25 °C and an outlet temperature 0- 55 °C. This is exemplary only and not meant to be limiting.

[0030] An understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention may be utilized, and the accompanying drawings.BRIEF DESCRIPTION OF THE FIGURES

[0031] Fig. 1 depicts a captive bubble surfactometry device test. 20 pL of hydrated surfactant samples were diluted with 180 pL of hydration buffer. A 50 pL bubble was then introduced into the 2.5 ml syringe and increments of 5 pL of the diluted surfactants or hydration buffer were injected near the interface of the air bubble and 10% sucrose solution, and pressure was applied by hand on the plunger several times. Changes to the shape of the bubble were monitored Top: Atmospheric pressure. Bottom: Compressed.

[0032] Fig 2. depicts a flattened bubble in the captive bubble surfactometry device after addition of surfactant and compressed.

[0033] Fig. 3 depicts efficacy test results of surfactant of the present invention compared to Survanta'^ in a pre-term rabbit model.

[0034] Fig. 4 depicts intranasal tolerability of surfactant hydrated with a niclosamide liposome. (n=6 BALB / c female mice / group). Each mouse was treated once daily for five days via intranasal (i.n.) administration of 50 pL per dose. Prior to each intranasal treatment, mice were anesthetized by intraperitoneal (i.p.) injection of ketamine / xylazine (50mg / kg / / 5mg / kg). Mice were monitored for body weight, morbidity and mortality through day 14. No mortality or significant weight loss was observed in mice from all groups.

[0035] Fig. 5 depicts niclosamide liposome and surfactant distribution of in mouse lungs. BALB / c mice were treated intranasally with surfactant rehydrated with a niclosamide liposome. The niclosamide liposome was formulated with DiR and the surfactant was labeled with Rhodamine. Lungs collected at 60 mins post treatment showed efficient distribution of surfactant (top) and the niclosamide liposome (bottom). Image taken at 4X magnification, Rhodamine detected at 530nm ex / 605nm em, and DiR detected at 630nm ex / 700nm em.

[0036] Fig. 6 depicts proteases retain their activities when the protease solutions were used to rehydrate a surfactant formulation. Lane l is a protein ladder; lane 2 is a protein with a Tobacco Etch Virus (TEV) protease site; lane 3 is the protein from lane 2 cleaved by TEV protease; lane 4 is the protein from lane 2 cleaved by a surfactant rehydrated with a TEV protease solution; lane 5 is empty; lane 6 is a protein with a thrombin protease site; lane 7 is the protein from lane 6 cleaved by thrombin; lane 8 is the protein from lane 6 cleaved by a surfactant rehydrated with a thrombin protease solution.

[0037] Fig. 7 depicts that the binding activity and specificity remains when an antibody solution was used to rehydrate a surfactant formulation. A protein with a 6-Histidine tag and a protein without a 6-Histidine tag (negative control) were coated onto a 96-well plate. A mouse monoclonal anti-poly-Histidine IgG2a antibody (Clone HIS-1; Sigma-Aldrich, St. Louis, MO) and a surfactant rehydrated with the same antibody was allowed to react with the coated proteins. Then, a horseradish peroxidase-conjugated anti-IgG2a antibody (goat anti-mouse;SouthemB iotech, Birmingham, AL) was incubated in the wells. Lastly, a substrate solution (ABTS; Life Technologies, Carlsbad, CA) was added to the wells, and after adding a stop solution, the absorbance of each well at 405 nm was recorded.

[0038] Fig. 8 depicts a mixture of the surfactant formulation with a liposomal sunscreen formulation that is effective at blocking UVB (290 nm - 320 nm) and UVA (320 nm - 400 nm) light.DETAILED DESCRIPTION OF THE INVENTION

[0039] The term “a” and “an” refers to one or more (i.e., at least one) of the grammatical object of the article. By way of example, “a cell” encompasses one or more cells.

[0040] As used herein, the terms “about” and “approximately,” when used to modify an amount specified in a numeric value or range, indicate that the numeric value includes ± 10%, and preferably ± 5% or ± 1% of the stated value.

[0041] In understanding the scope of the present disclosure, the terms “including” or “comprising” and their derivatives, as used herein, are intended to be open ended terms that specify the presence of the stated features, elements, components, groups, integers, and / or steps, but do not exclude the presence of other unstated features, elements, components, groups, integers and / or steps. The foregoing also applies to words having similar meanings such as the terms “including”, “having” and their derivatives. The term “consisting” and its derivatives, as used herein, are intended to be closed terms that specify the presence of the stated features, elements, components, groups, integers, and / or steps, but exclude the presence of other unstated features, elements, components, groups, integers and / or steps. The term “consisting essentially of,” as used herein, is intended to specify the presence of the stated features, elements, components, groups, integers, and / or steps as well as those that do not materially affect the basic and novel characteristic(s) of features, elements, components, groups, integers, and / or steps. It is understood that reference to any one of these transition terms (i.e. “comprising,” “consisting,” or “consisting essentially”) provides direct support for replacement to any of the other transition term not specifically used. For example, amending a term from “comprising” to “consisting essentially of or “consisting of would find direct support due to this definition for any elements disclosed throughout this disclosure. Based on this definition, any element disclosed herein or incorporated by reference may be included in or excluded from the claimed invention.

[0042] As used herein, a plurality of compounds, elements, or steps may be presented in a common list for convenience. However, these lists should be construed as though each member of the list is individually identified as a separate and unique member. Thus, no individual member of such list should be construed as a de facto equivalent of any other member of the same list solely based on their presentation in a common group without indications to the contrary.

[0043] The term “exemplary” is used herein to mean serving as an example, instance, or illustration. Any aspect or design described herein as “exemplary” is not necessarily to be construed as preferred or advantageous over other aspects or designs. Rather, use of the word exemplary is intended to present concepts in a more concrete fashion.

[0044] Certain molecules, constructs, compositions, elements, moieties, excipients, disorders, conditions, properties, steps, or the like may be discussed in the context of one specific embodiment or aspect or in a separate paragraph or section of this disclosure. It is understood that this is merely for convenience and brevity, and any such disclosure is equally applicable to and intended to be combined with any other embodiments or aspects found anywhere in the present disclosure and claims, which all form the application and claimed invention at the filing date. For example, a list of constructs, molecules, method steps, kits, or compositions described with respect to a construct, composition, or method is intended to and does find direct support for embodiments related to constructs, compositions, formulations, and methods described in any other part of this disclosure, even if those method steps, active agents, kits, or compositions are not re-listed in the context or section of that embodiment or aspect.

[0045] Standard methods in molecular biology are described in Sambrook, Fritsch and Maniatis (1982 & 1989 2ndEdition, 2001 3rdEdition) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Sambrook and Russell (2001) Molecular Cloning, 3rded., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Wu (1993) Recombinant DNA, Vol. 217, Academic Press, San Diego, CA). Standard methods also appear in Ausbel, et al. (2001) Current Protocols in Molecular Biology, Vols.1-4, John Wiley and Sons, Inc. New York, NY, which describes cloning in bacterial cells and DNA mutagenesis (Vol. 1), cloning in mammalian cells and yeast (Vol. 2), glycoconjugates and protein expression (Vol. 3), and bioinformatics (Vol. 4).

[0046] Methods for protein purification including immunoprecipitation, chromatography, electrophoresis, centrifugation, and crystallization are described (Coligan, et al. (2000) Current Protocols in Protein Science, Vol. 1, John Wiley and Sons, Inc., New York). Chemical analysis, chemical modification, post-translational modification, production of fusion proteins, glycosylation of proteins are described (see, e.g., Coligan, et al. (2000) Current Protocols in Protein Science, Vol. 2, John Wiley and Sons, Inc., New York; Ausubel, et al. (2001) Current Protocols in Molecular Biology, Vol. 3, John Wiley and Sons, Inc., NY, NY, pp. 16.0.5-16.22.17; Sigma-Aldrich, Co. (2001) Products for Life Science Research, St. Louis, MO; pp. 45-89;Amersham Pharmacia Biotech (2001) BioDirectory, Piscataway, N.J., pp. 384-391). Production, purification, and fragmentation of polyclonal and monoclonal antibodies are described (Coligan, et al. (2001) Current Protocols in Immunology, Vol. 1, John Wiley and Sons, Inc., New York; Harlow and Lane (1999) Using Antibodies, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Harlow and Lane, supra). Standard techniques for characterizing ligand / receptor interactions are available (see, e.g., Coligan, et al. (2001) Current Protocols in Immunology, Vol. 4, John Wiley, Inc., New York).

[0047] As used herein, the term “antibody” refers to any form of antibody that exhibits the desired biological activity. The term antibody includes antigen-binding portions, i.e., “antigen binding sites,” (e.g., fragments, subsequences, complementarity determining regions (CDRs)) that retain capacity to bind antigen, including (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHI domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CHI domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., (1989) Nature 341 :544-546), which consists of a VH domain; and (vi) an isolated complementarity determining region (CDR). Single chain antibodies are also included by reference in the term “antibody.” Preferred therapeutic antibodies are intact IgG antibodies. The term “intact IgG” as used herein is meant as a polypeptide belonging to the class of antibodies that are substantially encoded by a recognized immunoglobulin gamma gene. In humans this class comprises IgGl, IgG2, IgG3, and IgG4. In mice this class comprises IgGl, IgG2a, IgG2b, IgG3. The known Ig domains in the IgG class of antibodies are VH, Cyl, Cy2, Cy3, VL, and CL. AS used herein, unless otherwise indicated, “antibody fragment” or “antigen-binding fragment” refers to antigen-binding fragments of antibodies, i.e. antibody fragments that retain the ability to bind specifically to the antigen bound by the full-length antibody, e.g. fragments that retain one or more CDR regions. Examples of antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules, e.g., sc-Fv; nanobodies and multispecific antibodies formed from antibody fragments.

[0048] Monoclonal, polyclonal, and humanized antibodies can be prepared (see, e.g., Sheperd and Dean (eds.) (2000) Monoclonal Antibodies, Oxford Univ. Press, New York, NY; Kontermann and Dubel (eds.) (2001) Antibody Engineering, Springer-Verlag, New York; Harlow and Lane (1988) Antibodies A Laboratory Manual, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, NY, pp. 139-243; Carpenter, et al. (2000) J. Immunol. 165:6205; He, et al. (1998) J. Immunol. 160: 1029; Tang et al. (1999) J. Biol. Chem. 274:27371-27378; Baca et al. (1997) J. Biol. Chem. 272: 10678-10684; Chothia c / a / . (1989) Nature 342:877-883; Foote and Winter (1992) J. Mol. Biol. 224:487-499; U.S. Pat. No. 6,329,511).

[0049] An “isolated nucleic acid molecule” or “isolated polynucleotide” means a DNA or RNA of genomic, mRNA, cDNA, or synthetic origin or some combination thereof which is not associated with all or a portion of a polynucleotide in which the isolated polynucleotide is found in nature, or is linked to a polynucleotide to which it is not linked in nature. For purposes of this disclosure, it should be understood that “a nucleic acid molecule comprising” a particular nucleotide sequence does not encompass intact chromosomes. Isolated nucleic acid molecules “comprising” specified nucleic acid sequences may include, in addition to the specified sequences, coding sequences for up to ten or even up to twenty or more other proteins or portions or fragments thereof, or may include operably linked regulatory sequences that control expression of the coding region of the recited nucleic acid sequences, and / or may include vector sequences.

[0050] Pharmaceutical Compositions and Administration

[0051] To prepare pharmaceutical or sterile compositions of the invention (e.g., surfactant formulations) of the invention, a therapeutic (e.g., a surfactant polypeptide) is admixed with a pharmaceutically acceptable carrier or excipient. See, e.g., Remington's Pharmaceutical Sciences and U.S. Pharmacopeia: National Formulary, Mack Publishing Company, Easton, PA (1984).

[0052] Formulations of therapeutic agents may be prepared by mixing with acceptable carriers, excipients, or stabilizers in the form of, e.g., lyophilized powders, slurries, aqueous solutions or suspensions (see, e.g., Hardman, et al. (2001) Goodman and Gilman ’s The Pharmacological Basis of Therapeutics, McGraw-Hill, New York, NY; Gennaro (2000) Remington: The Science and Practice of Pharmacy, Lippincott, Williams, and Wilkins, New York, NY; Avis, et al. (eds.) (1993) Pharmaceutical Dosage Forms: Parenteral Medications, Marcel Dekker, NY; Lieberman, et al. (eds.) (1990) Pharmaceutical Dosage Forms: Tablets, Marcel Dekker, NY; Lieberman, et al. (eds.) (1990) Pharmaceutical Dosage Forms: Disperse Systems, Marcel Dekker, NY; Weiner and Kotkoskie (2000) Excipient Toxicity and Safety, Marcel Dekker, Inc., New York, NY).

[0053] Toxicity and therapeutic efficacy of pharmaceutical or sterile compositions of the invention, administered alone or in combination with another therapeutic agent, can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index (LD50 / ED50). The data obtained from these cell culture assays and animal studies can be used in formulating a range of dosages for use in humans. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration.

[0054] Suitable routes of administration include parenteral administration, such as intramuscular, intravenous, or subcutaneous administration and oral administration. Administration of pharmaceutical or sterile compositions of the invention, used to practice the method of the present invention, can be carried out in a variety of conventional ways, such as oral ingestion, inhalation, topical application or cutaneous, subcutaneous, intraperitoneal, parenteral, intraarterial or intravenous injection. In one embodiment, the pharmaceutical or sterile composition of the invention is administered intravenously. In another embodiment, the pharmaceutical or sterile composition of the invention is administered subcutaneously.

[0055] Alternatively, one may administer the surfactant reagent of the invention in a local rather than systemic manner, for example, directly into the airways as an aerosol or dry powder. An “aerosol” as used herein refers to a suspension of liquid or solid in the form of fine particles dispersed in a gas. As used herein, the term “particle” thus refers to liquids, e.g., droplets, and solids, e.g., powders. Pharmaceutical aerosols for the systemic delivery of conjugates of the invention to the lungs are, in one embodiment, inhaled via the mouth, and not via the nose. Alternatively, pharmaceutical aerosols for the delivery of conjugates of the invention to the lungs are, in one embodiment, introduced through direct delivery to a central airway, for example via an endotracheal tube or tracheotomy tube. In like manner, pharmaceutical aerosols for the systemic delivery of antibodies to the lungs are, in one embodiment, inhaled via the mouth, and not via the nose. Alternatively, pharmaceutical aerosols for the delivery of antibodies to the lungs are, in one embodiment, introduced through direct delivery to a central airway. In another embodiment, pharmaceutical aerosols for the systemic delivery of antibodies to the lungs areinhaled via the nose, and not via the mouth. In another embodiment, pharmaceutical aerosols for the systemic delivery of antibodies to the lungs are inhaled via the mouth and via the nose.

[0056] In one embodiment an aerosol delivery system includes a vibrational element constructed and arranged to vibrate an aperture plate having a plurality of apertures of defined geometry, wherein one side or surface of the aperture plate is in fluid connection with a solution or suspension of the conjugate. See, e.g., U.S. Pat. No. 5,758,637, U.S. Pat. No. 5,938,117, U.S. Pat. No. 6,014,970, U.S. Pat. No. 6,085,740, and U.S. Pat. No. 6,205,999, the entire contents of which are incorporated herein by reference. Activation of the vibrational element to vibrate the aperture plate causes liquid containing the conjugate in solution or suspension to be drawn through the plurality of apertures to create a low-velocity aerosol with a defined range of droplet (i.e., particle) sizes. Examples of this type of aerosol generator are commercially available from Aerogen, Inc., Sunnyvale, Calif.

[0057] In another embodiment an aerosol delivery system includes a pressurized container containing the conjugate in solution or suspension. The pressurized container typically has an actuator connected to a metering valve so that activation of the actuator causes a predetermined amount of the conjugate in solution or suspension within the container to be dispensed from the container in the form of an aerosol. Pressurized containers of this type are well known in the art as propellant-driven metered-dose inhalers (pMDIs or simply MDIs). MDIs typically include an actuator, a metering valve, and a pressurized container that holds a micronized drug suspension or solution, liquefied propellant, and surfactant. Historically, these MDIs typically used chlorofluorocarbons (CFCs) as propellants, including tri chlorofluoromethane, dichlorodifluoromethane, and dichlorotetrafluoromethane. Cosolvents such as ethanol may be present when the propellant alone is a relatively poor solvent. Newer propellants may include 1,1,1,2-tetrafhroroethane and 1,1,1,2,3,3,3-heptafluoropropane. Actuation of MDIs typically causes dose amounts of 50 pg-5 mg of active agent in volumes of 20-100 pL to be delivered at high velocity (30 m / sec) over 100-200 msec.

[0058] In other embodiments, an aerosol delivery system includes an air-jet nebulizer or ultrasonic nebulizer in fluid connection with a reservoir containing the conjugate in solution or suspension. Nebulizers (air-jet or ultrasonic) are used primarily for acute care of non-ambulatory patients and in infants and children. Air-jet nebulizers for atomization are considered portable because of the availability of small compressed air pumps, but they are relatively large and inconvenient systems. Ultrasonic nebulizers have the advantage of being more portable becausethey generally do not require a source of compressed air. Nebulizers provide very small droplets and high mass output. Doses administered by nebulization are much larger than doses in MDIs and the liquid reservoir is limited in size, resulting in short, single-duration therapy.

[0059] To generate an aerosol from an air-jet nebulizer, compressed air is forced through an orifice over the open end of a capillary tube, creating a region of low pressure. The liquid formulation is drawn through the tube to mix with the air jet and form the droplets. Baffles within the nebulizer remove larger droplets. The droplet size in the airstream is influenced by the compressed air pressure. Mass median diameters normally range from 2 to 5 pm with air pressures of 20 to 30 psig. The various commercially available air-jet nebulizers do not perform equally. This will affect the clinical efficacy of nebulized aerosol, which depends on the droplet size, total output from the nebulizer, and patient determinants.

[0060] Ultrasonic nebulizers generate aerosols using high-frequency ultrasonic waves (i.e., 100 kHz and higher) focused in the liquid chamber by a ceramic piezoelectric crystal that mechanically vibrates upon stimulation. Dennis J H et al. (1992) J Med Eng Tech 16:63-68; O'Doherty M J et al. (1992) Am Rev Respir Dis 146:383-88. In some instances, an impeller blows the particles out of the nebulizer or the aerosol is inhaled directly by the patient. The ultrasonic nebulizer is capable of greater output than the air-jet nebulizer and for this reason is used frequently in aerosol drug therapy. The droplets formed using ultrasonic nebulizers, which depend upon the frequency, are coarser (i.e., higher MMAD) than those delivered by air-jet nebulizers. The energy introduced into the liquid can result in an increase in temperature, which results in vaporization and variations in concentrations over time. This concentration variation over time is also encountered in jet nebulizers but is due to water loss through evaporation.

[0061] The choice between solution or suspension formulations in nebulizers is similar to that for the MDI. The formulation chosen will affect total mass output and particle size. Nebulizer formulations typically contain water with co-solvents (ethanol, glycerin, propylene glycol) and surfactants added to improve solubility and stability. Commonly, an osmotic agent is also added to prevent bronchoconstriction from hypoosmotic or hyperosmotic solutions. Witeck T J et al. (1984) Chest 86:592-94; Desager K N et al. (1990) Agents Actions 31 :225-28.

[0062] In yet other embodiments, the aerosol delivery system includes a dry powder inhaler in fluid connection with a reservoir containing the conjugate in powder form. The dry powder inhaler device may eventually replace MDIs for some indications in response to the internationalcontrol of chlorofluorocarbons in these latter products. Notably, this device can only deliver a fraction of its load in a respirable size range. Powder inhalers will usually disperse only about 10 to 20% of the contained drug into respirable particles. The typical dry powder inhaler device consists of two elements: the inhalation appliance to disperse unit doses of the powder formulation into the inspired airstream, and a reservoir of the powder formulation to dispense these doses. The reservoir typically can be of two different types. A bulk reservoir allows a precise quantity of powder to be dispensed upon individual dose delivery up to approximately 200 doses. A unit dose reservoir provides individual doses (e.g., provided in blister packaging or in gelatin capsule form) for inhalation as required. The hand-held device is designed to be manipulated to break open the capsule / blister package or to load bulk powder followed by dispersion from the patient's inspiration. Airflow will deaggregate and aerosolize the powder. In most cases, the patient's inspiratory airflow activates the device, provides the energy to disperse and deagglomerate the dry powder, and determines the amount of medicament that will reach the lungs.

[0063] Dry powder generators are subject to variability because of the physical and chemical properties of the powder. These inhalers are designed to meter doses ranging from 200 pg to 20 mg. The preparation of drug powder in these devices is very important. The powder in these inhalers requires efficient size reduction that is also needed for suspensions in MDIs. Micronized particles flow and are dispersed more unevenly than coarse particles. Therefore, the micronized drug powder may be mixed with an inert carrier. This carrier is usually a-lactose monohydrate, because lactose comes in a variety of particle size ranges and is well characterized. Byron P R et al. (1990) Pharm Res 7(suppl):S81. The carrier particles have a larger particle size than the therapeutic agent to prevent the excipient from entering the airways. Segregation of the two particles will occur when turbulent airflow is created upon patient inhalation through the mouthpiece. This turbulence of inspiration will provide a certain amount of energy to overcome the interparticulate cohesive and particle surface adhesive forces for the micronized particles to become airborne. High concentrations of drug particles in air are easily attained using dry powder generation, but stability of the output and the presence of agglomerated and charged particles are common problems. With very small particles, dispersion is difficult because of electrostatic, van der Waals, capillary, and mechanical forces that increase their energy of association. An example of a dry powder inhaler aerosol generator suitable for use with the present invention is the Spinhaler powder inhaler available from Fisons Corp., Bedford, Mass.

[0064] In some embodiments, the surfactant reagent of the invention is formulated in combination with, or administered in conjunction with, one or more therapeutic or diagnostic agents. Methods for co-administration or treatment with a second therapeutic agent are well known in the art, see, e.g., Hardman, el al. (eds.), 2001, Goodman and Gilman ’s The Pharmacological Basis of Therapeutics, 10th ed., McGraw-Hill, New York, NY; Poole and Peterson (eds.), 2001, Pharmacotherapeutics for Advanced Practice: A Practical Approach, Lippincott, Williams & Wilkins, Phila., PA; Chabner and Longo (eds.), 2001, Cancer Chemotherapy and Biotherapy, Lippincott, Williams & Wilkins, Phila., PA. The other agents include, but are not limited to, a cytotoxic, chemotherapeutic, cytostatic, anti -angiogenic or antimetabolite agents, a tumor targeted agent, an immune stimulating or immune modulating agent or an antibody conjugated to a cytotoxic, cytostatic, or otherwise toxic agent. The pharmaceutical composition can also be employed with other therapeutic modalities such as surgery, chemotherapy and radiation. Representative therapeutic agents (including prodrugs), prophylactic or diagnostic agents can be peptides, proteins, carbohydrates, nucleotides or oligonucleotides (e.g., siRNA, microRNA, DNA, and RNA), small molecules, or combinations thereof. Representative examples include:

[0065] 1. Bronchodilators

[0066] A bronchodilator is a type of medication that opens the airways to help you breathe easier. Short-acting bronchodilators are used in emergency situations or when needed for rapid removal. Some exemplary short-acting bronchodilators include anticholinergics (e.g., ipratropium (e.g., ATROVENT® in DUONEB®, in COMBIVENT®), P2- Agonists such as albuterol (e.g, VOSPIRE ER® in DUONEB® in COMBIVENT®) and levalbuterol (e.g.,XOPENEX®). Long-acting bronchodilators are used to treat COPD over the long term. They are usually taken once or twice daily for an extended period of time and they are formulated for inhalers or nebulizers. Some exemplary long acting bronchodilators include anticholinergics such as acridinium (e.g., TUDORZA®), tiotropium (e.g., SPIRIVA®), or umeclidinium (e.g., INCRUSE ELLIPTA®)), p2-agonists (e.g., alformoterol (e.g., BROVANA®), formoterol (e.g, FORADIL®, PERFOROMIST®), indacaterol (e.g, ARCAPTA®), salmeterol (e.g, SEREVENT®), and olodaterol (e.g, STRIVERDI RESPIMAT®).

[0067] 2. Corticosteroids

[0068] Corticosteroids help reduce inflammation in the body and make it easier for air to flow to the lungs. There are several corticosteroids. Some are prescribed with bronchodilators. This is because these two medications can work together to make breathing more effective. Fluticasone (e.g., FLOVENT®), budesonide (e.g., PULMICORT®), and prednisolone are medications commonly prescribed by physicians for chronic obstructive pulmonary disease (COPD).

[0069] 3. Methylxanthines

[0070] Some people have severe difficulties with COPD, and conventional or first-line treatment alone does not seem to help. In these cases, theophylline (e.g., THEO-24®, THEOLAIR®, ELIXOPHYLLINE®, QUIBRON-T®, UNIPHYL®, and ELIXOPHYLLIN® ) can be used to be taken with bronchodilators, which act as anti-inflammatory and relax airway muscles. Theophylline comes in pills or liquids to be taken on a once daily basis and / or combined with other medications.

[0071] 4. Phosphodiesterase-4 Inhibitors

[0072] This medication can help clear inflammation and improve airflow to the lungs. Roflumilast (e.g, DALIRESP®) is a phosphodiesterase-4 inhibitor that comes in a pill that can be taken once per day. It is usually prescribed with long-acting bronchodilators.

[0073] 5. Antimicrobial Agents

[0074] Antimicrobial agents are substances that kill or inhibit the growth of microorganisms (e.g., bacteria, fungi, viruses, or parasites). Antimicrobial agents include antiviral agents, antibacterial agents, antiparasitic agents, and antifungal agents. Representative antiviral agents include ganciclovir and acyclovir. Representative antibiotics include: aminoglycosides (e.g., streptomycin, amikacin, gentamicin, and tobramycin), ansamycins (e.g., geldanamycin and herbimycin), carbacephems, carbapenems, cephalosporins, sugars. Peptides (e.g. vancomycin, teicoplanin, and telavancin), lincosamides, lipopeptides (e.g., daptomycin), macrolides (e.g., azithromycin, clarithromycin, dirithromycin, and erythromycin), monobactam, nitrofurans, penicillins, polypeptides (e.g., bacitracin, colistin and polymyxin B), quinolones, sulfonamides, and tetracyclines.

[0075] Other exemplary antimicrobial agents include iodine, silver compounds, moxifloxacin, ciprofloxacin, levofloxacin, cefazolin, tigecycline, gentamicin, ceftazidime, ofloxacin, gatifloxacin, polyenes such as amphotericin B and nystatin, voriconazole, natamycin.

[0076] 6. Local Anesthetics

[0077] Local anesthetics are substances that have the effect of causing reversible local anesthesia and loss of pain sensation. Non-limiting examples of local anesthetics include: ambucaine, amolanone, amylocaine, benoxinate, benzocaine, betoxycaine, biphenamine, bupivacaine, butacaine, butamben. , butanilicaine, butetamine, butoxycaine, carticaine, chloroprocaine, cocaethylene, cyclomethicaine, dibucaine, dimethysoquin, dimethocaine, diperodone, dycyclonine, Ecgonidine, Ecgonine, Ethyl chloride , etidocaine, P-eucaine, euprocin, fenalcomine, formocaine, hexylcaine, hydroxytetracainetetracaine, isobutyl p-aminobenzoate, leucinocaine mesylate, levoxadrol ( levoxadrol), lidocaine, mepivacaine, meprilcaine, metabutoxycaine, methyl chloride, myrtecaine, naepaine, octacaine, orthocaine, oxethazaine, paretoxycaine, phenacaine, Phenol, Pipelocaine, Pyridocaine (piridocaine), polidocanol, pramoxine, prilocaine, procaine, propanocaine, proparacaine, propipocaine, propoxycaine, pseudococaine, pyrrocaine, ropivacaine, salicyl alcohol, tetracaine, tolycaine ), trimecaine, Zolamine, and combinations of any of these.

[0078] 7. Anti-Inflammatory and Immunosuppressive Agents

[0079] Anti-inflammatory agents reduce inflammation and include steroidal and nonsteroidal agents. Suitable steroidal active agents include glucocorticoids, progestins, mineralocorticoids and corticosteroids. Other exemplary anti-inflammatory agents include triamcinolone acetonide, fluocinolone acetonide, prednisolone, dexamethasone, loteprendol, fluoromethoIone, ibuprofen, aspirin, and naproxen. Exemplary immunomodulatory agents include cyclosporine, tacrolimus and rapamycin. Exemplary non-steroidal anti-inflammatory drugs (NSAIDs) include mefenamic acid, aspirin, diflunisal, salycilate, ibuprofen, naproxen, fenoprofen, ketoprofen, dexketoprofen, flurbiprofen, oxaprozin, loxoprofen, Indometacin, Slindak, Etodorak, Ketrolak, Diclofenac, Nabmeton, Pyroxi cum, Mel oxi cum, Tenoxicum, Doroxicum, Lomoxicum, Lornoxicum, Lornoxicum, Isoxicum, Mecrophenamic acid, Furphenamic acid, Turfenamic acid, Selecoxib (ELECOX) IB), Rofe Coxib, Valdecoxy, Palecoxy, Lumiracoxy, Etricoxy, Philocoxy, Sulfonanilide, nimesulide, niflumic acid, and Licoferone.

[0080] In some embodiments, the anti-inflammatory agent is an anti-inflammatory cytokine. Exemplary cytokines are IL- 10, TGF-P and IL-35. Anti-inflammatory cytokines in the context of biomaterial grafts, skin grafts, and hair grafts are cytokines that either induce an antiinflammatory immune environment or suppress an inflammatory immune environment. Activation of regulatory T cells (Treg) is involved in preventing rejection, inducing and maintaining peripheral tolerance in allografts. Thl7 cells are a subset of T helper cells characterized by the production of IL-17. Thl7 cells have been suggested to play a role in allograft rejection. In some embodiments, cytokines to be added to surfactant reagent of the inventions are those that induce Treg activation (e.g., IL-25) and those that suppress Thl7 activation (e.g., IL-25) to minimize rejection , IL- 10).

[0081] 8. Diagnostic agents

[0082] Exemplary diagnostic agents include paramagnetic molecules, fluorescent compounds, magnetic molecules, and radionuclides, x-ray imaging agents, and contrast agents. They may also be detectably labeled ligands or antibodies.

[0083] 9. Growth Factors and Cytokines

[0084] These are proteins that can stimulate cell growth, proliferation and / or cell differentiation. Non-limiting examples of growth factors include: transforming growth factor beta (TGF-beta), transforming growth factor alpha (TGF-alpha), granulocyte colony stimulating factor (GCSF), granulocyte macrophage colony, stimulatory factor (GM-CSF), nerve growth factor (NGF), neurotrophin, platelet-derived growth factor (PDGF), erythropoietin (EPO), thrombopoietin (TPO), myostatin (GDF8), growth differentiation factor-9 (GDF9) , acidic fibroblast growth factor (aFGF or FGF-1), basic fibroblast growth factor (bFGF or FGF-2), epidermal growth factor (EGF), vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF).

[0085] 10. Antibodies

[0086] In some embodiments, the formulation contains antibodies such as daclizumab, bevacizumab (Avastin®), ranibizumab (Lucentis®), basiliximab, ranibizumab, and pegaptanib sodium or peptide-like SN50, etc.

[0087] 11. Cofactors, Vitamins and Nutrients

[0088] Exemplary cofactors include vitamin A, vitamin C, vitamin D, biotin, vitamin E, and vitamin K. Exemplary essential nutrients are amino acids, fatty acids, and the like.

[0089] 12. Antiproliferative Agents

[0090] Representative anti -proliferative agents include, but are not limited to, the following small molecules (amsacrine, bleomycin, busulfan, capecitabine, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, clofarabine, crisantaspase, Cyclophosphamide, cytarabine, dacarbazine, dactinomycin, daunorubicin, docetaxel, doxorubicin, epipodophyllotoxin, epirubicin, etoposide, etoposide phosphate, fludarabine, fluorouracil, gemcitabine, hydroxycarbamide, idarubicin, ifosfamide, irinotecan , leucovorin, liposomal doxorubicin, lomustine, mechlorethamine, melphalan, mercaptopurine, mesna, methotrexate, mitomycin, mitoxantrone, oxaliplatin, paclitaxel, pemetrexed, pentostatin, procarbazine, raltitrexed, satraplatin, streptozocin, teniposide, tegafur- Uracil, temozolomide, teniposide, thiotepa, thioguanine, topotecan, treosulfan, vinblastine, vincristine, vindesine, vinorelbine, taxol and their derivatives, and antibodies such as trastuzumab (HERCEPTIN®), cetuximab, and rituximab (RITUXAN) ® or MABTHERA®), bevacizumab (AVASTIN®), and combinations thereof Representative proapoptotic agents include fludarabinetaurosporine, cycloheximide, actinomycin D, Examples include, but are not limited to, lactosylceramide, 15d-PGJ(2)5, and combinations thereof.

[0091] In particular embodiments, administration is accomplished intravenously, subcutaneously, intramuscularly, intraarterially, intratumorally, or by inhalation, aerosol delivery. As appropriate, administration by non-invasive routes (e.g., orally; for example, in a pill, capsule or tablet) is also within the scope of the present invention. Preferred routes include mucosal, pulmonary, genitourinary, intrathecal, oral, rectal, topical, ocular, etc. routes. In certain preferred embodiments, the pulmonary route of administration is by intranasal or intratracheal installation or by inhalation.

[0092] Determination of the appropriate dose is made by the clinician, e.g., using parameters or factors known or suspected in the art to affect treatment. Generally, the dose begins with an amount somewhat less than the optimum dose and it is increased by small increments thereafter until the desired or optimum effect is achieved relative to any negative side effects. Important diagnostic measures include those of symptoms of, e.g., the inflammation or level of inflammatory cytokines produced. In general, it is desirable that a biologic that will be used isderived from the same species as the animal targeted for treatment, thereby minimizing any immune response to the reagent. In the case of human subjects, for example, humanized and fully human antibodies may be desirable.

[0093] The pharmaceutical or sterile compositions disclosed herein may be provided by continuous infusion, or by doses administered, e.g., daily, 1-7 times per week, weekly, bi-weekly, monthly, bimonthly, quarterly, semiannually, annually etc. Doses may be provided, e.g., intravenously, subcutaneously, topically, orally, nasally, rectally, intramuscular, intracerebrally, intraspinally, or by inhalation. A total weekly dose is generally at least 0.05 pg / kg body weight, more generally at least 0.2 pg / kg, 0.5 pg / kg, 1 pg / kg, 10 pg / kg, 100 pg / kg, 0.25 mg / kg, 1.0 mg / kg, 2.0 mg / kg, 5.0 mg / mL, 10 mg / kg, 25 mg / kg, 50 mg / kg or more (see, e.g., Yang, et al. (IQQ^ New Engl. J. Med. 349:427-434; Herold, et al. (2002) New Engl. J. Med. 346: 1692-1698; Liu, et al. (1999) J. Neurol. Neurosurg. Psych. 67: 451-456; Portielji, et al. (20003) Cancer Immunol. Immunother. 52: 151-144). Doses may also be provided to achieve a pre-determined target concentration of pharmaceutical or sterile compositions in the subject’s serum, such as 0.1, 0.3, 1, 3, 10, 30, 100, 300 pg / mL or more. In other embodiments, a pharmaceutical or sterile composition of the present invention is administered, e.g., subcutaneously or intravenously, on a weekly, biweekly, "every 4 weeks," monthly, bimonthly, or quarterly basis at 10, 20, 50, 80, 100, 200, 500, 1000 or 2500 mg / subject.

[0094] The pharmaceutical or sterile compositions can be administered over a period of at least about 1 week, 2 weeks, 1 month (4 weeks), 6 weeks, 2 months, 3 months, 6 months, 1 year, 2 years, 3 years or longer, or as deemed appropriate by the treating physician. A chronic condition can exist for, e.g., at least about 6 weeks, 2 months, a year, or longer. The pharmaceutical or sterile compositions can be administered over a period of at least about 6 weeks, 2 months, 3 months or 6 months, a year, or even multiple years as required for medical care of an individual.

[0095] The pharmaceutical or sterile compositions can also be administered in an irregular manner. Early achievement of an effective target concentration (a therapeutic dose level) with a loading dose followed by maintenance dosing with the therapeutic (frontloading) may be more effective than conventional therapy in terms of requiring a lower total therapeutic dose and faster time to maximum target engagement. As used herein, such an administration protocol is referred to as a “loading / maintenance administration protocol.” An effective target therapeutic concentration may be reached in 4 weeks or less, preferably 3 weeks or less, more preferably 2weeks or less, most preferably 1 week or less, including 1 day or less using a loading dose. The target serum concentration is then maintained by administration of an equal or smaller (or less frequent) maintenance dose during the remainder of the treatment regimen or until suppression of disease symptoms is achieved.

[0096] The term “frontloading” when referring to drug administration refers to the initial loading dose, followed by the maintenance dose. The initial loading dose (single or multiple) is intended to more quickly increase the drug concentration of an animal or human patient to an effective target serum concentration. In various embodiments, frontloading is accomplished by initial dosing delivered over 3 weeks or less so that the therapeutic reaches the target concentration. Preferably, the loading dose or series of doses is administered for 2 weeks or less, more preferably 1 week or less, e.g., 1 day or less. Most preferably, the loading dosing is a single dosing, with no maintenance dosing thereafter for at least one week, and the loading dosing is administered in 1 day or less. The present invention includes loading and maintenance doses of frontloading drug delivery by mucosal, pulmonary, genitourinary, intrathecal, oral, rectal, topical, ocular, etc. routes. In certain preferred embodiments, the pulmonary route of administration is by intranasal or intratracheal installation or by inhalation.

[0097] Administration of the loading dose can be, for example, one or more doses at a time interval of at least about 1, 2, 3, 4, 5, 6, 7 or 8 weeks apart. In some embodiments, the at least one loading dose is administered by one or more intravenous injections and then at least one maintenance dose by one or more intravenous or subcutaneous administrations. In other embodiments, the instructions can be for administering at least one loading dose by, for example, one or more intravenous or subcutaneous administrations and at least one maintenance dose by one or more intravenous or subcutaneous administrations. In certain embodiments, both the at least one loading dose as well as the at least one maintenance dose is administered subcutaneously. In other embodiments, the at least one loading dose is administered by intravenous infusion followed by at least one maintenance dose administered subcutaneously. The choice of loading and maintenance dosages and intervals can be made according to the ability of the animal or human patient to tolerate administration of the therapeutic to the body and according to a desired level of drug to achieve.

[0098] In other embodiments, the at least one loading dose is administered by a pulmonary route followed by at least one maintenance dose administered by a pulmonary route which maybe the same or different. By way of example only, the loading administration is by intranasal or intratracheal installation and the maintenance administration is by inhalation.

[0099] A loading dose of a drug can be larger e.g., about 1.5, 2, 3, 4 or 5 times larger) than a subsequent maintenance dose. The one or more therapeutically effective maintenance doses can be any therapeutically effective amount described herein. The loading dose can be about 2 or 3 times larger than the maintenance dose. A therapeutic can be administered in two (or more) loading doses prior to the maintenance dose. A first loading dose of the therapeutic can be administered on day 1, a second loading dose can be administered, e.g., about 1 or 2 weeks later, and a maintenance dose can be administered, e.g., once weekly or once every 2 weeks thereafter for the duration of treatment. The first loading dose can be about 3 or 4 times larger than the maintenance dose, and the second loading dose can be about 2, 3, 4, 5, or more times larger than the maintenance dose.

[0100] As used herein, “inhibit” or “treat” or “treatment” includes a postponement of development of the symptoms associated with disease and / or a reduction in the severity of such symptoms that will or are expected to develop with said disease. The terms further include ameliorating existing symptoms, preventing additional symptoms, and ameliorating or preventing the underlying causes of such symptoms. Thus, the terms denote that a beneficial result has been conferred on a vertebrate subject with a disease.

[0101] As used herein, the term "effective amount" refers to an amount of a pharmaceutical or sterile compositions of the invention that, when administered alone or in combination with an additional therapeutic agent to a cell, tissue, or subject, is effective to cause a measurable improvement in one or more symptoms of disease, for example cancer or the progression of cancer. An effective dose further refers to that amount of the therapeutic sufficient to result in at least partial amelioration of symptoms. When applied to an individual active ingredient administered alone, an effective dose refers to that ingredient alone. When applied to a combination, an effective dose refers to combined amounts of the active ingredients that result in the therapeutic effect, whether administered in combination, serially or simultaneously. An effective amount of a therapeutic will result in an improvement of a diagnostic measure or parameter by at least 10%; usually by at least 20%; preferably at least about 30%; more preferably at least 40%, and most preferably by at least 50%. An effective amount can also result in an improvement in a subjective measure in cases where subjective measures are used to assess disease severity.

[0102] Preferred embodiments

[0103] The following are preferred embodiments of the invention.1. A surfactant reagent, comprising: about 1 to about 10 wt% of surfactant polypeptide and about 90 to 99 wt% of a lipid mixture, wherein the surfactant polypeptide is cationic, and the lipid mixture consists of one or more lipids selected from a first group consisting of l,2-dipalmitoyl-sn-glycero-3- phosphatidylcholine (DPPC), l,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC), or 1- palmitoyl-2-oleoyl-glycero-3 -phosphatidylcholine (POPC) or salts thereof, and one or more lipids selected from a second group consisting of l-palmitoyl-2-oleoyl-sn- glycero-3 -phosphoglycerol (POPG), or l,2-dioleoyl-sn-glycero-3-phosphoglycerol (DOPG) or salts thereof; or the surfactant polypeptide is anionic, and the lipid mixture consists of one or more lipids selected from a first group consisting of DPPC, DOPC, POPC, and salts thereof, and one or more lipids selected from a second group consisting of l,2-di-O-octadecenyl-3- trimethylammonium propane (DOTMA),l,2-dioleoyl-3 -trimethylammonium propane (DOTAP), and salts thereof, wherein the surfactant reagent is in the form of a dried cake or powder.2. A dried surfactant reagent according to embodiment 1, wherein the surfactant polypeptide is cationic and the lipid mixture contains a weight ratio of POPG to POPC, DOPC, or a combination POPC and DOPC, or their respective salts, of between 1 :7 to 7: 1.3. A dried surfactant reagent according to embodiment 1, wherein the surfactant polypeptide is cationic and the lipid mixture contains a weight ratio of POPG to POPC, DOPC, or a combination POPC and DOPC, or their respective salts, of between 1 :2 to 2: 14. A dried surfactant reagent according to embodiment 1, wherein the surfactant polypeptide is cationic and the lipid mixture contains a weight ratio of POPG to POPC, DOPC, or a combination POPC and DOPC, or their respective salts, is between 1 : 1.5 and 1.5: 15. A dried surfactant reagent according to one of embodiments 1-4, wherein the surfactant polypeptide is cationic and is selected from the group consisting of SP-B, SP-C, Mini-B, Super Mini-B, B-YL, KL4, SP-C33, SP-C30, KALA, and combinations thereof.6. A dried surfactant reagent according to embodiment 1, wherein surfactant polypeptide is anionic and is selected from the group consisting of GALA, E-Mini-B, Super E-Mini-B, EL4, and combinations thereof.7. A dried surfactant reagent according to one of embodiments 1-6, comprising about 2 to 4 wt% of surfactant polypeptide.8. A dried surfactant reagent according to one of embodiments 1-5 or 7, wherein the surfactant polypeptide is cationic and wherein the dried surfactant reagent comprises about 3 wt% of the cationic surfactant protein and wherein the lipid mixture consists of about 20 wt% POPG-Na, about 29.4 wt% POPC, and about 47.6 wt% DPPC.9. A dried surfactant reagent according to embodiment 1, 6 or 7, wherein the dried surfactant reagent comprises about 3 wt% of an anionic surfactant protein, and wherein the lipid mixture consists of about 20 wt% DOTAP, about 29.4 wt% POPC, and about 47.6 wt% DPPC.10. A dried surfactant reagent according to one of embodiments 1-5, 7, or 8, wherein the cationic surfactant protein, peptide, or combinations thereof is Super Mini-B.11. A dried surfactant reagent according to one of embodiments 1-5, 7, or 8, wherein the cationic surfactant protein, peptide analog thereof, or combinations thereof is Mini-B.12. A method of preparing an aqueous surfactant reagent, comprising: rehydrating the surfactant reagent according to one of embodiments 1-11 in an aqueous buffer.13. A method according to embodiment 12, wherein the aqueous buffer is phosphate buffered saline or 0.9% saline in 10 mM carbonate buffer or phosphate buffer, pH 6.5-7.4.14. A method according to one of embodiments 12 or 13, wherein the aqueous buffer further comprises lipid nanoparticles.15. A method according to one of embodiments 12-14, wherein the buffer or the lipid nanoparticles comprise a therapeutic molecule.16. A method according to embodiment 15, wherein the therapeutic molecule is hydrophobic.17. A method according to one of embodiments 12-16, wherein the aqueous buffer comprises a therapeutic polypeptide.18. A method according to embodiment 17, wherein the therapeutic polypeptide is an antibody.19. A method for delivering a surfactant reagent to an individual in need thereof, comprising: administering the surfactant as an aqueous reagent produced according to the method of one of embodiments 12-18.20. A method according to embodiment 19, comprising administering the aqueous surfactant reagent via a mucosal route.21. A method according to embodiment 19, comprising administering the aqueous surfactant reagent via a pulmonary route.22. A method according to embodiment 19, comprising administering the aqueous surfactant reagent via a genital route.23. A method according to embodiment 19, comprising administering the aqueous surfactant reagent via an oral route.24. A method according to embodiment 19, comprising administering the aqueous surfactant reagent via a rectal route.25. A method according to embodiment 19, comprising administering the aqueous surfactant reagent via a topical route.26. A method according to embodiment 19, comprising administering the aqueous surfactant reagent via an ocular route.27. A method according to embodiment 21, wherein the pulmonary route of administration is intranasal or intratracheal installation or by inhalation.28. A method for delivering a surfactant reagent to an individual in need thereof, comprising: administering the surfactant reagent according to one of embodiments 1-11 as a dried powder via inhalation.29. A method of producing a surfactant reagent, comprising: mixing a surfactant polypeptide and a lipid mixture in an organic:aqueous solvent system, wherein the surfactant polypeptide is at about 1 to about 10 wt% of the mixture, the organic:aqueous solvent system consists of one or more organic solvents selected from the group consisting of Ci-6 alcohols, acetone, and chloroform, and water, wherein the ratio of organic solvents: water in the organic:aqueous solvent system is 9: 1 or greater; and the surfactant polypeptide is cationic, and the lipid mixture consists of one or more lipids selected from a first group consisting of l,2-dipalmitoyl-sn-glycero-3- phosphatidylcholine (DPPC), l,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC), or 1- palmitoyl-2-oleoyl-glycero-3 -phosphatidylcholine (POPC) or salts thereof, and one or more lipids selected from a second group consisting of l-palmitoyl-2-oleoyl-sn- glycero-3 -phosphoglycerol (POPG), or l,2-dioleoyl-sn-glycero-3-phosphoglycerol (DOPG) or salts thereof; or the surfactant polypeptide is anionic, and the lipid mixture consists of one or more lipids selected from a first group consisting of DPPC, DOPC, POPC, and salts thereof, and one or more lipids selected from a second group consisting of l,2-di-O-octadecenyl-3- trimethylammonium propane (DOTMA),l,2-dioleoyl-3 -trimethylammonium propane (DOTAP), and salts thereof; andremoving the organic:aqueous solvent system by sublimation or spray drying to produce the surfactant reagent in the form of a dried cake or powder.30. A method according to embodiment 29, wherein the one or more organic solvents are selected from the group consisting of tert-butanol, tert-amyl alcohol, methanol, ethanol, isopropanol, acetone, and 1 -butanol, or combinations thereof.31. A method according to embodiment 30, wherein the organic:aqueous solvent system is selected from tert-butanol: water, tert-butanol: water, methanol : tert-butanol: water, ethanol :tert- butanol: water, isopropanol : tert-butanol: water, acetone: tert-butanol: water, and tert-amyl alcohol: water.32. A method according to one of embodiments 29-31, wherein the sublimation is carried out at between -20°C and 25°C under vacuum.33. A method according to one of embodiments 29-32, wherein the organic: aqueous solvent system is tert-butanol: water.34. A method according to one of embodiments 29-33, wherein the surfactant polypeptide is cationic and the lipid mixture contains a weight ratio of POPG-Na to POPC, DOPC, or a combination POPC and DOPC of between 1 :7 to 7: 1.35. A method according to one of embodiments 29-33, wherein the surfactant polypeptide is cationic and the lipid mixture contains a weight ratio of POPG to POPC, DOPC, or a combination POPC and DOPC, or their respective salts, of between 1 :2 to 2: 136. A method according to one of embodiments 29-33, wherein the surfactant polypeptide is cationic and the lipid mixture contains a weight ratio of POPG to POPC, DOPC, or a combination POPC and DOPC, or their respective salts, is between 1 : 1.5 and 1.5: 137. A method according to one of embodiments 29-36, wherein the surfactant polypeptide is cationic and is selected from the group consisting of SP-B, SP-C, Mini-B, Super Mini-B, B-YL, KL4, SP-C33, SP-C30, KALA, and combinations thereof.38. A method according to one of embodiments 29-36, wherein the surfactant polypeptide is anionic and is selected from the group consisting of GALA, E-Mini-B, Super E-Mini-B, EL4, and combinations thereof.39. A method according to one of embodiments 29-38, the surfactant polypeptide is at about 2 to 4 wt% of the mixture.40. A method according to one of embodiments 29-37 or 39, wherein the surfactant polypeptide is cationic and wherein the dried surfactant reagent comprises about 3 wt% of the cationic surfactant protein and wherein the lipid mixture consists of about 20 wt% POPG-Na, about 29.4 wt% POPC, and about 47.6 wt% DPPC.41. A method according to one of embodiments 29, 38 or 39, wherein the dried surfactant reagent comprises about 3 wt% of an anionic surfactant protein, and wherein the lipid mixture consists of about 20 wt% DOTAP, about 29.4 wt% POPC, and about 47.6 wt% DPPC.42. A method according to one of embodiments 29-37, 39, or 40, wherein the cationic surfactant protein, peptide, or combinations thereof is Super Mini-B.43. A method according to one of embodiments 29-37, 39, or 40, wherein the cationic surfactant protein, peptide analog thereof, or combinations thereof is Mini-B.44. A therapeutic composition for pulmonary delivery, comprising: about 0.1 to about 1 wt% of a surfactant protein; about 0.9 to about 1 wt% lipid; and about 98 to about 99 wt% of one or more aqueous pharmaceutically acceptable excipients, wherein the surfactant polypeptide is cationic, and the lipid mixture consists of one or more lipids selected from a first group consisting of l,2-dipalmitoyl-sn-glycero-3- phosphatidylcholine (DPPC), l,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC), or 1- palmitoyl-2-oleoyl-glycero-3 -phosphatidylcholine (POPC) or salts thereof, and one or more lipids selected from a second group consisting of l-palmitoyl-2-oleoyl-sn- glycero-3 -phosphoglycerol (POPG), or l,2-dioleoyl-sn-glycero-3-phosphoglycerol (DOPG) or salts thereof; orthe surfactant polypeptide is anionic, and the lipid mixture consists of one or more lipids selected from a first group consisting of DPPC, DOPC, POPC, and salts thereof, and one or more lipids selected from a second group consisting of l,2-di-O-octadecenyl-3- trimethylammonium propane (DOTMA),l,2-dioleoyl-3 -trimethylammonium propane (DOTAP), and salts thereof.45. A therapeutic reagent according to embodiment 44, wherein the aqueous buffer is phosphate buffered saline or 0.9% saline in 10 mM carbonate buffer, pH 6.45-7.4.46. A method according to one of embodiments 44 or 45, wherein the aqueous buffer further comprises lipid nanoparticles.47. A method according to one of embodiments 44-46, wherein the aqueous buffer or the lipid nanoparticles comprise a therapeutic molecule.48. A method according to embodiment 47, wherein the therapeutic molecule is hydrophobic.49. A method according to one of embodiments 44-48, wherein the aqueous buffer comprises a therapeutic polypeptide.50. A method according to embodiment 49, wherein the therapeutic polypeptide is an antibody.51. A method according to one of embodiments 44-50, wherein the surfactant polypeptide is cationic and the lipid mixture contains a weight ratio of POPG-Na to POPC, DOPC, or a combination POPC and DOPC of between 1 :7 to 7: 1.52. A dried surfactant reagent according to one of embodiments 44-50, wherein the surfactant polypeptide is cationic and the lipid mixture contains a weight ratio of POPG to POPC, DOPC, or a combination POPC and DOPC, or their respective salts, of between 1 :2 to 2: 153. A dried surfactant reagent according to one of embodiments 44-50, wherein the surfactant polypeptide is cationic and the lipid mixture contains a weight ratio of POPG to POPC, DOPC, or a combination POPC and DOPC, or their respective salts, is between 1 : 1.5 and 1.5: 154. A method according to one of embodiments 44-53, wherein the surfactant polypeptide is cationic and is selected from the group consisting of SP-B, SP-C, Mini-B, Super Mini-B, B-YL, KL4, SP-C33, SP-C30, KALA, and combinations thereof.55. A method according to one of embodiments 44-50, wherein the surfactant polypeptide is anionic and is selected from the group consisting of GALA, E-Mini-B, Super E-Mini-B, EL4, and combinations thereof.56. A method according to one of embodiments 44-55, the surfactant polypeptide is at about 2 to 4 wt% of the mixture.57. A method according to one of embodiments 44-56, wherein the dried surfactant reagent comprises about 3 wt% of the cationic surfactant protein and wherein the lipid mixture consists of about 20 wt% POPG-Na, about 29.4 wt% POPC, and about 47.6 wt% DPPC.58. A method according to one of embodiments 44-57, wherein the dried surfactant reagent comprises about 3 wt% of an anionic surfactant protein, and wherein the lipid mixture consists of about 20 wt% DOTAP, about 29.4 wt% POPC, and about 47.6 wt% DPPC.59. A method according to one of embodiments 44-54, 56 or 57, wherein the cationic surfactant protein, peptide, or combinations thereof is Super Mini-B.60. A method according to one of embodiments 44-54, 56 or 57, wherein the cationic surfactant protein, peptide analog thereof, or combinations thereof is Mini-B.

[0104] Examples

[0105] The following examples are presented in order to more fully illustrate certain embodiments of the invention. They should in no way, however, be construed as limiting the broad scope of the invention. One skilled in the art can readily devise many variations and modifications of the principles disclosed herein without departing from the scope of the invention.

[0106] EXAMPLE 1 : Formulation method and a precision captive bubble surfactometry assay for assessing surfactant activity

[0107] For a 1 mL final surfactant volume, 19.8 mg of l-palmitoyl-2-oleoyl-glycero-3- phosphatidylcholine (POPC), 32.0 mg of l,2-dipalmitoyl-sn-glycero-3 -phosphatidylcholine(DPPC), 13.4 mg of l-palmitoyl-2-oleoyl-sn-glycero-3 -phosphoglycerol sodium salt (POPG-Na; Avanti Polar Lipids), and 2 mg of a cationic, hydrophobic peptide (z.e., Super Mini-B or Mini-B; see Table EX4-1) were weighed and combined into a 4 mL borosilicate glass vial. A sample of the same amount of POPC, DPPC and POPG-Na, without polypeptide, were weighed into another 4 mL borosilicate glass vial. Each lipid and polypeptide powder mixture was dissolved in 3 mL of a solvent mixture composed of tert-butanol and distilled water at a 9: 1 volume ratio. The mixture was warmed to 44°C (in a water bath) and mixed gently until all the lipids and peptides had completely dissolved. The dissolved solution was solidified at -30°C. Vacuum was applied in order to speed up the sublimation process, and the vials were capped under vacuum. The vials were stored at 4°C or ambient temperature until use.

[0108] To hydrate the dried surfactant powder, the vacuum is released prior to the addition of 1 mL of sterile hydration buffer (10 mM sodium carbonate in 0.9% saline at pH 6.8) to each vial via a syringe by puncturing through the septum. The solution is gently shaken and can be warmed by hand until the surfactant powder has been suspended to form a milky solution.

[0109] The hydrated surfactant samples were analyzed by captive bubble surfactometry. A proprietary method was used to demonstrate surfactant activity. The method comprises the surfactant to be tested, a captive bubble device constructed in-house, and a well-characterized assay procedure to analyze the ability of the surfactant formulations to reduce the air pressure inside the bubble by expanding the surface area at the air-water interface. The standard procedure for preparing candidate surfactants for testing consists of diluting hydrated surfactant to a 1 :9 ratio of surfactant to hydration buffer, for example, 20 pL of surfactant and 180 pL of hydration buffer. The device consists of a 2.5 mL syringe (Hamilton Syringes) with a Luer Lock tip to lock a septum cap adapter in place which ensures that the solution and bubble do not leak out of the syringe when pressure is applied. The liquid that the bubble is suspended in is a 10% sucrose solution in water. Since the syringes have a Teflon plunger, we added an agarose plug (10% in water) to coat the plunger where the bubble is suspended since the Teflon disrupts the bubble at the interface between the air and Teflon, causing the bubble to collapse without surfactant. A 50 pL bubble at atmospheric pressure is injected with a 100 pL syringe before adding surfactant (see Figure 1, top). After capping the syringe with the septum adaptor and applying pressure to the syringe by pushing the plunger, the bubble shrinks according to the Ideal Gas Law, but maintains a relatively spherical shape (see Figure 1, bottom). After the bubble is injected and suspended in the 2.5 mL syringe with the agarose plug, the 1 :9 dilution of surfactantshould be added in via another syringe (e.g., 50-100 pL) or pipette (pipetting is less accurate). When the surfactant is injected, the septum adapter should be screwed on to the Luer Lock tip so no liquid escapes. The plunger should be oscillated 2-3 times to facilitate spreading of the surfactant across the bubble, enabling its surface activity at the air-water interface to reach the maximum potential. Afterwards, the plunger should be pressed down with enough pressure so that the bubble should flatten if: 1) the measurements were correctly done; 2) the surfactant works and; 3) the device was handled with caution and the assay was conducted correctly by the steps stated above for the best chances at success and minimal chances of error.

[0110] Following the procedure described above, 20 pL of the hydrated surfactant samples were diluted with 180 pL of the hydration buffer. A 50 pL bubble was then introduced into the 2.5 mL syringe with the agarose plug and 10% sucrose solution. Increments of 5 pL of the diluted surfactants or hydration buffer were injected near the interface of the air bubble and 10% sucrose solution. Between each 5 pL injection, the septum cap was placed on the Luer Lock of the 2.5 mL syringe and pressure was applied by hand on the plunger several times. Changes to the shape of the bubble were monitored (see Figure 2) and the spherical bubble typically flattens to a disk-like shape with an increase in surface area at the air-water interface and concomitant increase in volume. Additional 5 pL aliquots of each sample were added until flattening of the bubble was observed. The results are shown in Table EX1-1.

[0117] The results of this study demonstrate the basic formulation composition and process for producing the surfactant.

[0118] Table EX1-1. Surfactant composition examples and minimum weight of components in formulation required to flatten a bubble of air as measured by captive bubble surfactometryComposition by weight required to flatten air bubblePolypeptide POPC DPPC POPG-NaFormulation 1 Super Mini-B 9.9 pg 16 pg 6.7 pg(SMB); 1 pgFormulation 2 Mini-B; 1 pg 9.9 pg 16 pg 6.7 pgFormulation 3 None 39.6 pg 64 pg 26.8 pgBuffer only control Did not flatten bubble after six 5 pL injections

[0119] EXAMPLE 2. Determination of solvent compositions for surfactant formulations

[0120] A range of organic solvents were examined as key components of the preferred composition of the surfactant formulations. Different organic solvents that are miscible with water were tested in these studies. Examples of organic:aqueous solvents tested with the lipids and peptides are shown in Table EX2-1. As described in EXAMPLE 1, for a 1 mL final surfactant volume, 19.8 mg POPC, 32 mg DPPC, 13.4 mg POPG-Na, and 2 mg of SMB were weighed and combined into a 4 mL borosilicate glass vial. The lipids and peptide were dissolved in 3 mL of the solvent mixtures shown in Table EX2-1. The mixtures were warmed to 44°C (in a water bath) and mixed gently until all the lipids and peptide had completely dissolved. The dissolved solution was solidified and sublimed as described in EXAMPLE 1. The powder formulations were hydrated and the surfactant properties were measured by the captive bubble surfactometry assay as described in EXAMPLE 1.

[0121] Table EX2-1. Surfactant composition examples and minimum weight of components in formulation required to flatten a bubble of air as measured by captive bubble surfactometry.Composition by weight required to flatten air bubblePOPG-Peptide POPC DPPCNa te / 7-butanol: water 9: 1 v:v SMB; 1 pg 9.9 pg 16 pg 6.7 pg / c / 7-but anol: water 96:4 v:v SMB; 1 pg 9.9 pg 16 pg 6.7 pg methanol : / c77-but anol: water 4:5: 1 SMB; 1 pg 9.9 pg 16 pg 6.7 pg v:v:v ethanol : / c77-butanol: water 4:5: 1 SMB; 1 pg 9.9 pg 16 pg 6.7 pg v:v:v isopropanol : tert-butanol: water SMB; 2 pg 19.8 pg 32 pg 13.4 pg4:5:1 v:v:v acet on e: / c77-but anol: water 4:5: 1 SMB; 1 pg 9.9 pg 16 pg 6.7 pg v:v:v / c77-amyl alcohol : water 9: 1 v:v SMB; 3 pg 29.7 pg 48 pg 20.1pg

[0122] Combinations of lower melting point solvents with higher melting point solvents showed good activity upon hydration of the dry cake that had been sublimated. The mostpreferred solvents are those with higher melting points so that the sublimation process does not require cooling below standard refrigerator / freezer temperatures, re., >-20°C as opposed to typical lyophilization cooling temperatures (e.g., ranging from -20°C to -80°C.

[0123] EXAMPLE 3. Sublimation rather than lyophilization can be used to prepare the surfactant formulations

[0124] The identification of suitable organic: aqueous solvent compositions that facilitate sublimation processing for simplifying the surfactant production process allowed testing of different sublimation conditions. For example, for a 1 mL final surfactant volume, where 19.8 mg POPC, 32 mg DPPC, 13.4 mg POPG-Na, and 2 mg of SMB were dissolved in 3 mL of a solvent mixture composed of tert-butanol and distilled water at a 9: 1 volume ratio; the dissolved mixture was frozen at <-20°C overnight (or at -80°C for 30 minutes), then the solid was sublimed under vacuum at ambient temperature in a desiccator. Other solidification and sublimation temperatures can produce active dried surfactant and examples of conditions are shown in Table EX3-1. The results demonstrate that as long as the composition of the peptide, lipids and solvents will form a solid at any particular temperature, subsequent sublimation can be performed which generates dry surfactant that is active. Suitable temperatures are between about -20°C and about 4°C.

[0125] Table EX3-1. Solidification and sublimation conditions with different organic:aqueous solvent examples.Solidification Sublimation Temperatures* tert-butanol: waterTemperatures (°C) volume ratio(°C)98:2 4°C, on ice or -20°C -15, -10, -5, 0, 5, 10, 15°C96:4 on ice or -20°C -15, -10, -5, 0, 5, 10, 15°C90: 10 -20°C -15, -10, -5, 0, 5, 10, 15°C80:20 -20°C -15, -10, -5, 0, 5, 10, 15°C70:30 -20°C -15, -10, -5, 0, 5, 10, 15°C60:40 -20°C -15, -10, -5, 0, 5, 10, 15°C50:50 -20°C -15, -10, -5, 0, 5, 10, 15°C* Shelf temperature in a vacuum desiccator

[0126] EXAMPLE 4. Demonstration that combinations of charged, yet hydrophobic peptides in combination with lipids dissolved in the preferred solvent compositions and subjected to the sublimation process result in active surfactants.

[0127] The surfactant formulas with the lipid composition described in EXAMPLE 1 can be produced with other charged, hydrophobic peptides, or mixtures of peptides. Examples of cationic, hydrophobic peptides that can be formulated with lipids such as the ones described in EXAMPLE 1 are listed in Table EX4-1 [1-6], Their amino acid sequences, net charge and mean hydrophobicity per amino acid (<H>) based upon the Eisenberg consensus scale [7] are shown. Anionic, hydrophobic peptides can also be used for surfactant formulations. Examples of anionic, hydrophobic peptides that can be formulated with lipids are listed in Table EX4-2.

[0128] Table EX4-1. Amino acid sequences, net charge and mean hydrophobicity (<H>) of cationic, hydrophobic peptides.NetPeptide Amino acid sequence <H>ChargeSMB FPIPLPYCWLCRALIKRIQAMIPKGGRMLPQLVCRLVLRCS +7 0.12Mini-B CWLCRALIKRIQAMIPKGGRMLPQLVCRLVLRCS +7 0.02KL4 KLLLLKLLLLKLLLLKLLLLK +5 0.45SP-C33 IPSSPVHLKRLKLLLLLLLLILLLILGALLMGL +3 0.460SP-C33AIPSSPVHLKRLKLLLLLLLEILLKILGALLMGL +3 0.47B-YL FPIPLPYYWLYRALIKRIQALIPKGGRLLPQLVYRLVLRYS +7 0.12KALA WI<AALAI<ALAI<ALAI<HLAI<ALAI<ALI<ALAA +7 0.20SMB dIP FPLPYCWLCRALIKRIQAMIPKGGRMLPQLVCRLVLRCS +7 0.09RALA WRAALARALARALARHLARALARALRALAA +7 -0.01- “ion lock” salt bridge predicted to form between E and K

[0129] Table EX4-2. Amino acid sequences, net charge and mean hydrophobicity (<H>) of anionic, hydrophobic peptides.NetPeptide Amino acid sequence <H>ChargeSMB-E FPIPLPYCWLCEALIEEIQAMIPEGGEMLPQLVCELVLECS -7 0.55EL4 ELLLLELLLLELLLLELLLLE -5 0.63SP-C33-E IPSSPVHLEELELLLLLLLLILLLILGALLMGL -3 0.70GALA WEAALAEALAEALAEHLAEALAEALEALAA -7 0.38DALA WDAALADALADALADHLADALADALDALAA -7 0.34

[0130] In addition, the lipid components can also be varied in order to achieve optimal effectiveness at the air-water interface. In one embodiment, surfactant formulas with varying weight ratios of DPPC:POPC were tested (Table EX4-3). In another embodiment, POPC was substituted with 2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC; Lipoid GmbH), and POPG-Na was substituted with 2-dioleoyl-sn-glycero-3-phosphoglycerol sodium salt (DOPG- Na; Avanti Polar Lipids) (Table EX4-3). In another embodiment, cationic lipids were substituted for the anionic lipids (Table EX4-4). For example, l,2-dioleoyl-3-trimethylammonium-propane (DOTAP) or 1,2-di-O-octadecenyl -3 -trimethylammonium propane (DOTMA) replaced the POPG-Na.

[0131] Each sample in Tables EX4-3 and EX4-4 was prepared at the 1 mL scale, following procedures described in EXAMPLE 1. The powder formulations were hydrated as described and the surfactant properties were measured by captive bubble surfactometry. The results show that, for the cationic hydrophobic peptides, different unsaturated lipids can replace the POPC lipid and that the molar ratios with the saturated lipid (DPPC) can also be used. Similarly, different negatively charged lipids such as DOPG-Na can replace the POPG-Na and show good surfactant activity. Interestingly, replacement of the cationic hydrophobic peptide SMB, with other cationic hydrophobic peptides while generally active, displayed variable amounts required in order to observe flattening of the bubble. Surprisingly, some of the negatively charged hydrophobic peptides combined with the negatively charged lipids also were found to have reasonably good surfactant activity (e.g., the GALA peptide). When the peptides were formulated with cationic lipid mixtures, similar results were observed; good surfactant activity for cationic lipids and either cationic or anionic peptides had reasonable activity. In summary, these results demonstrate that, while positively and negatively charged peptides and lipids play role indefining surfactant formulations with good activity, other factors such as membrane fluidity and hydrophobicity of the peptides also are important factors to consider.

[0132] Table EX4-3. Formulations examples with varying anionic lipid and peptide compositions.Weight (in mg per mL of final surfactant formulation)DOPC DOPG- Peptide* CBS ResultADPPC POPC POPG-NaNa12.8 38.3 13.4 0 0 SMB 33.638.3 12.8 13.4 0 0 SMB 33.632 0 13.4 19.8 0 SMB 33.6-33632 19.8 0 0 13.4 SMB 33.632 0 0 19.8 13.4 SMB 33.632 19.8 13.4 0 0 Mini-B 134.4-16832 19.8 13.4 0 0 KL4 33.6-134.432 19.8 13.4 0 0 SP-C33 ion- 134.4 lock 132 19.8 13.4 0 0 SP-C33 134.432 19.8 13.4 0 0 B-YL 33.6-100.832 19.8 13.4 0 0 SMB-E 100.832 19.8 13.4 0 0 SMB dIP NC32 19.8 13.4 0 0 GALA 33.632 19.8 13.4 0 0 DALA 33.6-100.832 19.8 13.4 0 0 RALA 100.832 19.8 13.4 0 0 KALA 33.6-100.8*2 mg of each peptide per formulation;Atotal lipid and peptide in pg needed to flatten the bubble NC - no collapse or flattening of the bubble observed

[0133] Table EX4-4. Formulations examples with varying cationic lipid and peptide compositions.Weight (in mg per mL of final surfactant formulation) CBSDPPC POPC DOTAP DOTMA Peptide* ResultA32 19.8 12.1 0 SMB 33.632 19.8 12.1 0 KL4 67.2-100.832 19.8 12.1 0 SMB-E 33.632 19.8 12.1 0 GALA 67.232 19.1 0 12.1 DALA NC32 19.1 0 12.1 RALA NC32 19.1 0 12.1 KALA 33.632 19.1 0 12.1 B-YL 33.6*2 mg of each peptide per formulation;Atotal lipid and peptide in pg needed to flatten the bubbleNC - no collapse or flattening of the bubble observed

[0134] EXAMPLE 5 - Demonstration that the preferred compositions and method of preparation is easily scaleable

[0135] To prepare a 50 mL final surfactant volume, 990 mg of POPC, 1600 mg of 1, DPPC, 670 mg of POPG-Na, and 100 mg of SMB were weighed and combined into a 250 mL sterile borosilicate glass bottle. The lipids and peptide were then dissolved in a solvent mixture of 108 mL tert-butanol and 12 mL water; the mixture warmed to 44°C (in a water bath) and stirred until all the lipids and SMB had completely dissolved. The final volume was quantum satis to 150 mL by adding a 9: 1 (volume ratio) solvent of tert-butanol: water (prewarmed at 44°C). The dissolved solution was sterile filtered under a weak vacuum through a 0.22 pm polyethersulfone bottletop filter into a sterile 250 mL borosilicate glass bottle. The sterile-filtered solution was filled into 10 mL serum vials (6 mL of sterile solution per vial) and the solution solidified at <-30°C overnight. The solvent was then sublimated under vacuum at 0°C. When the samples dried, the vials were capped under vacuum, then sealed with aluminum seals. The vials were stored at 4°C or ambient temperature until use.

[0136] The same procedure was repeated at a 250 mL scale of final surfactant volume by increasing the amounts of all the components by a factor of five. Each 50 mL and 250 mL scale processing was repeated four times. The powder formulations from each batch were hydratedand the surfactant properties were measured by captive bubble surfactometry. All the surfactant formulations described in this EXAMPLE were able to flatten an air bubble in the captive bubble surfactometry assay, thus demonstrating that the process is robust and results in a surfactant composition that is reproducible.

[0137] The surfactant formulations produced in this example were also tested in a pre-term rabbit model of surfactant insufficiency. Pre-term rabbit pups (28 days gestation; n=12 / group) that lacked surfactant were instilled intratracheally with a clinically relevant volume of surfactant (3.25 mL per kg body weight) or Survanta® at 4.0 mL per kg body weight and the CRS / kg for each rabbit pup was measured. Comparison to the CRS / kg measured from rabbit pups not given surfactant (air control) was conducted and reported as the % increase in compliance. The results are displayed in Figure 3 and show that the captive bubble surfactometry assay reliably assesses the biological activity in an animal model of surfactant insufficiency.

[0138] EXAMPLE 6. Demonstration that small molecule drugs such as antibiotics, anticancer agents or respiratory medicines can be put in the hydration buffer without affecting the surfactant activity after hydration of the powder

[0139] The surfactant formulas described in the examples above are compatible with biologicals, pharmaceuticals, nutraceuticals and therapeutics, including antivirals, antibiotics, antifungals, anthelminthics, proteins such as enzymes and monoclonal antibodies, carbohydrates or nanoparticles. For example, a surfactant powder formulation containing SMB were hydrated with hydration buffer (10 mM sodium carbonate in 0.9% saline, pH 6.8) in which various small molecules have been dissolved. Examples of small molecule drugs solubilized in the hydration buffer are listed in Table EX6-1. All the surfactant formulations with therapeutics described in this EXAMPLE were able to still flatten the bubble in the captive bubble surfactometry assay.

[0140] The surfactant formulations hydrated with antibiotic solutions retained their antibiotic properties (see Table EX6-2). The surfactant formulation rehydrated with oseltamivir increased the mean day of death of mice challenged by influenza, compared to oseltamivir alone or surfactant alone (see Table EX6-3).

[0141] Table EX6-1. Small molecule drugs tested with surfactant formulation*.Concentration ofTherapeutic Type small molecule in CBS ResultAformulationOseltamivir Antiviral 10 mg / mL 33.6Penicillin Antibacterial 10 mg / mL 33.6Cephalexin Antibacterial 10 mg / mL 100.8Ceftriaxone Antibacterial 1 mg / mL 33.6Amikacin Antibacterial 5 mg / mL 33.6Amphotericin B Antifungal 1 mg / mL 100.8* Surfactant formulation containing POPC, DPPC, POPG-Na and SMB;Atotal lipid and peptide in pg needed to flatten the bubble

[0142] Table EX6-2. Antimicrobial activity of antibiotics with or without surfactant formulation.Amikacin Pseudomonas* Measured at 37°C

[0143] Table EX6-3. Mean day of death of mice challenged with 3 x LD50 influenzaA / Califomia / 04 / 2009 (H1N1) and treated with various formulations.

[0144] EXAMPLE 7 - Demonstration that lipid nanoparticles can be put in the hydration buffer without affecting the surfactant activity after hydration of the powder

[0145] The surfactant formulas can also be rehydrated with lipid nanoparticles. Some of the lipid nanoparticles used to rehydrate the surfactant powder are listed in Table EX7-1. For example, a 2 mL niclosamide lipid nanoparticle formulation composed of HSPC and DSPE- PEG2000 was used to rehydrate a surfactant powder formulation composed of 32 mg DPPC, 19.8 mg POPC, 13.4 mg POPG-Na and 2 mg SMB. All the surfactant formulations rehydrated with lipid nanoparticles described in this example were able to still flatten the bubble in the captive bubble surfactometry assay.

[0146] The surfactant formulation rehydrated with a liposomal niclosamide formula was well tolerated in mice (Figure 4), and the samples were effectively distributed to the lungs (Figure 5). The surfactant formulations rehydrated with a liposomal amphotericin B was able to inhibit and terminate growth Candida albicans (ATCC 10231).

[0147] Table EX7-1. Lipid nanoparicle tested with surfactant formulation*.Lipid nanoparticle Nanoparticle composition CBS ResultANiclosamide nanoparticle HSPC / DSPE-PEG2000 33.634.2 / 1.7 mg / mLAmphotericin B DPPC / Chol / DSPE-PEG2000 67.2 nanoparticle 41.3 / 7.0 / 2.1 mg / mLStearoyl-telmisartan HSPC / Chol / DSPE-PEG2000 33.6 nanoparticle 34.2 / 22.8 / 1.7 mg / mL* Surfactant formulation containing POPC, DPPC, POPG-Na and SMB;Atotal pg of lipid and peptide from the surfactant formulation needed to flatten the bubble

[0148] EXAMPLE 8 - Demonstration that biological molecules such as proteins including antibodies or enzymes, or polysaccharides can be put in the hydration buffer without affecting the surfactant activity after hydration of the powder

[0149] A surfactant formulation (composed of composed of 32 mg DPPC, 19.8 mg POPC, 13.4 mg POPG-Na and 2 mg SMB) rehydrated with palivizumab at 2 mg / mL was able to reduce RSV (respiratory syncytial virus) viral load in RSV challenged cotton rats (Table EX8-1).

[0150] Surfactant formulations (composed of 32 mg DPPC, 19.8 mg POPC, 13.4 mg POPG- Na and 2 mg SMB) rehydrated with protease solutions, such as thrombin, Tobacco Etch Virus (TEV) protease and trypsin, was able to retain the protease activities (Figure 6). The same surfactant formulation rehydrated with an antibody solution was able to retain its binding activity and specificity (Figure 7).

[0151] These surfactant formulations rehydrated with biological molecules, such as antibodies, enzymes, polysaccharides (e.g., sucrose) or double-stranded RNA, were able to flatten the bubble in the captive bubble surfactometry assay (Table EX8-2).

[0152] Collectively, Examples 6-8 show that the surfactant compositions can be re-hydrated with solutions containing small molecule drugs, nanoparticles and biologicals such as antibodies, polysaccharides, nucleic acids or enzymes without compromising the activity as a surfactant.

[0153] Table EX8-1. Lung viral titers. Each group of Sigmodon hispidus (n= 5 female / group) was challenged with IxlO5PFU of RSV / A2 intranasally (day 0), then treated by different formulations (intranasally) one day post-challenge (day 1). Lungs were harvested on day 5 for viral titer analysis. *p<0.5 to Group 1.

[0154] Table EX8-2. Biological molecules tested with surfactant formulation*.Biological molecule Type CBS ResultAMonoclonal Anti -polyHistidine Antibody 33.6Thrombin Protease 33.6Tobacco Etch Virus protease Protease 33.6Trypsin Protease 33.6Sucrose Polysaccharide 33.6Polyinosinic-polycytidylic acid Double-stranded 33.6(Poly EC) RNA; Toll-like receptor 3 agonist* Surfactant formulation containing POPC, DPPC, POPG-Na and SMB;Atotal lipid and peptide in pg needed to flatten the bubble

[0155] EXAMPLE 9 - Demonstration that the compositions and process results in a surfactant that is stable at a broad range of temperatures for long periods of time

[0156] Scaled batches of surfactant formulations prepared as described in EXAMPLE 5 were tested for stability under different temperatures. The dried surfactant formulations were stored at the temperatures indicated, then rehydrated with buffer and tested for surfactant activity by the captive bubble surfactometry assay described in EXAMPLE 1. Results showed that the driedsurfactant formulations can be stored at 4°C, ambient condition, 37°C or 45°C, for up to 2 years. (Table EX9-1).

[0157] Table EX9-1. Stability of three batches of surfactant formulation stored at different temperatures.StoraBatchTime 0 5 months 8 months 13 months4°C Pass Pass Pass PassAmbient temperature Pass Pass Pass Pass37°C Pass Pass Pass PassBatchTime 0 3 months 9 months 19 months 23 months4°C Pass Pass Pass Pass PassAmbient temperature Pass Pass Pass Pass Pass37°C Pass Pass Pass Fail PassBatch #3ATime 0 6 months 15 months4°C Pass Pass NTPass = diluted rehydrated formulations able to flatten air bubble in captive bubble surfactometry measurement; Fail = diluted rehydrated formulations unable to flatten air bubble in captive bubble surfactometry measurement; NT = not tested; * 1 vial tested per timepoint;A3 vials tested per timepoint; * all samples rehydrated readily except for this sample.

[0158] EXAMPLE 10 - Demonstration of the utility of the invention for treatment of diseases by application to various tissues (e.g., mucosal, topical, oral, ocular, auditory, vaginal, etc.)

[0159] To demonstrate the mucosal delivery of a vaccine, a surfactant formulation composed of 32 mg / mL DPPC, 19.8 mg / mL POPC, 13.4 mg / mL POPG-Na and 2 / mL mg SMB was rehydrated with an influenza vaccine formulation solution VesiVax®-M2e that contained 5 mg / mL influenza matrix protein 2 ectodomain (M2e) peptide and 53.2 mg / mL VesiVax® lipids[9-12], The final VesiVax®-M2e vaccine / surfactant mixture was able to flatten the bubble in the captive bubble surfactometry assay. A typical intranasal VesiVax®-M2e vaccine dose for mice is 40 to 100 pL, where two 20 to 50 pL doses were administered per nare at 36 hours apart [9], The surfactant properties of this VesiVax®-M2e composition is expected to increase the spreading of the vaccine throughout the surface area of the intranasal tract thus, enhancing absorptivity of the vaccine to the respiratory tract using much smaller volumes, e.g., down to 10 pL. Therefore, the effective dose or volume of vaccine administered via mucosal surfaces (e.g., intranasal) can be dramatically decreased.

[0160] To demonstrate the utility of the invention for topical applications, one volume of a surfactant formulation composed of 32 mg / mL DPPC, 19.8 mg / mL POPC, 13.4 mg / mL POPG- Na and 2 / mL mg SMB was mixed with one volume of a liposome-based sunscreen formulation (166 mg / mL Hydrogenated Soybean Phosphatidylcholine [HSPC], 12 mg / mL 1,2-Distearoyl-sn- glycero-3 -phosphoglycerol [DSPG], 35 mg / mL Cholesterol, 4 mg / mL tryptophan and 2 mg / mL octyldimethyl PABA; with particle size diameters ranging from 300-1,000 nm). The sunscreen formulation / surfactant mixture was able to flatten the bubble in the captive bubble surfactometry assay, and was adsorbed to human skin effectively, with moisturizing properties. Figure 8 shows that the sunscreen formulation / surfactant mixture is effective at blocking UVB (290 nm - 320 nm) and UVA (320 nm - 400 nm) light.

[0161] EXAMPLE 11 : References

[0162] 1. Walther, F.J., L.M. Gordon, and A. J. Waring, Design of Surfactant Protein BPeptide Mimics Based on the Saposin Fold for Synthetic Lung Surfactants. Biomedicine Hub, 2016. 1(3): p. 1-21.

[0163] 2 Walther, F. J., et al., Surfactant protein C peptides with salt-bridges ("ion-locks") promote high surfactant activities by mimicking the a-helix and membrane topography of the native protein. PeerJ, 2014. 2: p. e485.

[0164] 3 Walther, F. J., et al., A sulfur-free peptide mimic of surfactant protein B (B-YL) exhibits high in vitro and in vivo surface activities. Gates Open Res, 2018. 2: p. 13.

[0165] 4. Li, W ., F. Nicol, and F.C. Szoka, GALA: a designed synthetic pH-responsive amphipathic peptide with applications in drug and gene delivery. Adv Drug Deliv Rev, 2004. 56(7): p. 967-85.

[0166] 5 Sweet, D.G., et al., A first-in-human clinical study of a new SP-B and SP-C enriched synthetic surfactant (CHF5633) in preterm babies with respiratory distress syndrome. Arch Dis Child Fetal Neonatal Ed, 2017. 102(6): p. F497-F503.

[0167] 6. Cochrane, C.G., et al., The efficacy and safety of KL4-surfactant in preterm infants with respiratory distress syndrome. Am J Respir Crit Care Med, 1996. 153(1): p. 404-10.

[0168] 7. Eisenberg, D., R.M. Weiss, and T.C. Terwilliger, The helical hydrophobic moment: a measure of the amphiphilicity of a helix. Nature, 1982. 299(5881): p. 371-4.

[0169] 8. Connolly, K.L., et al., Pharmacokinetic Data Are Predictive of In Vivo Efficacy for Cefixime and Ceftriaxone against Susceptible and Resistant Neisseria gonorrhoeae Strains in the Gonorrhea Mouse Model. Antimicrob Agents Chemother, 2019. 63(3).

[0170] 9. Adler-Moore, J., et al., Characterization of the murine Th2 response to immunization with liposomal M2e influenza vaccine. Vaccine, 2011. 29(27): p. 4460-8.

[0171] 10. Ernst, W.A., et al., Protection against Hl, H5, H6 andH9 influenza A infection with liposomal matrix 2 epitope vaccines. Vaccine, 2006. 24(24): p. 5158-68.

[0172] 11. Fujii, G., W. Ernst, and J. Adler-Moore, The VesiVax system: a method for rapid vaccine development. Front Biosci, 2008. 13: p. 1968-80.

[0173] 12. Fujii, G., F.C. Szoka, and D.S. Watson, Methods and Compositions for LiposomalFormulations of Antigens and Uses Thereof, US #8,765,171 (July 1, 2014), #9,364,563 (June 14, 2016), #9,931,419 (April 3, 2018); EP 2,405,758 (April 27, 2016) CA. Notice of Allowance received May 17, 2017.

[0174] One skilled in the art readily appreciates that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. The examples provided herein are representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the invention.

[0175] It is to be understood that the invention is not limited in its application to the details of construction and to the arrangements of the components set forth in the following description or illustrated in the drawings. The invention is capable of embodiments in addition to those described and of being practiced and carried out in various ways. Also, it is to be understood thatthe phraseology and terminology employed herein, as well as the abstract, are for the purpose of description and should not be regarded as limiting.

[0176] As such, those skilled in the art will appreciate that the conception upon which this disclosure is based may readily be utilized as a basis for the designing of other structures, methods and systems for carrying out the several purposes of the present invention. It is important, therefore, that the claims be regarded as including such equivalent constructions insofar as they do not depart from the spirit and scope of the present invention.

[0177] While the invention has been described and exemplified in sufficient detail for those skilled in this art to make and use it, various alternatives, modifications, and improvements should be apparent without departing from the spirit and scope of the invention. The examples provided herein are representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the invention. Modifications therein and other uses will occur to those skilled in the art. These modifications are encompassed within the spirit of the invention and are defined by the scope of the claims.

[0178] It will be readily apparent to a person skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention.

[0179] All patent applications, patents, publications and other references mentioned in the specification are indicative of the levels of those of ordinary skill in the art to which the invention pertains and are each incorporated herein by reference. The references cited herein are not admitted to be prior art to the claimed invention.

[0180] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In the case of conflict, the present specification, including definitions, will control.

[0181] The terms “comprising”, “having”, “being of’ as in “being of a chemical formula”, “including”, and “containing” are to be construed as open terms (z.e., meaning “including but not limited to”) unless otherwise noted. Additionally, whenever “comprising” or another open-ended term is used in an embodiment, it is to be understood that the same embodiment can be more narrowly claimed using the intermediate term “consisting essentially of’ or the closed term “consisting of’.

[0182] Recitation of ranges of values are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. A range used herein, unless otherwise specified, includes the two limits of the range. For example, the terms “between X and Y” and “range from X to Y, are inclusive of X and Y and the integers there between. On the other hand, when a series of individual values are referred to in the disclosure, any range including any of the two individual values as the two end points is also conceived in this disclosure. For example, the expression “a dose of about 100 mg, 200 mg, or 400 mg” can also mean “a dose ranging from 100 to 200 mg”, “a dose ranging from 200 to 400 mg”, or “a dose ranging from 100 to 400 mg”.

[0183] The invention illustratively described herein suitably may be practiced in the absence of any element or elements, limitation or limitations which is not specifically disclosed herein. Thus, for example, in each instance herein any of the terms “comprising”, “consisting essentially of’ and “consisting of’ may be replaced with either of the other two terms. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the appended claims.

[0184] Other embodiments are set forth within the following claims.

Claims

We claim:

1. A surfactant reagent, comprising: about 1 to about 10 wt% of surfactant polypeptide and about 90 to 99 wt% of a lipid mixture, wherein the surfactant polypeptide is cationic, and the lipid mixture consists of one or more lipids selected from a first group consisting of l,2-dipalmitoyl-sn-glycero-3- phosphatidylcholine (DPPC), l,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC), or 1- palmitoyl-2-oleoyl-glycero-3 -phosphatidylcholine (POPC) or salts thereof, and one or more lipids selected from a second group consisting of l-palmitoyl-2-oleoyl-sn- glycero-3 -phosphoglycerol (POPG), or l,2-dioleoyl-sn-glycero-3-phosphoglycerol (DOPG) or salts thereof; or the surfactant polypeptide is anionic, and the lipid mixture consists of one or more lipids selected from a first group consisting of DPPC, DOPC, POPC, and salts thereof, and one or more lipids selected from a second group consisting of l,2-di-O-octadecenyl-3- trimethylammonium propane (DOTMA),l,2-dioleoyl-3 -trimethylammonium propane(DOTAP), and salts thereof, wherein the surfactant reagent is in the form of a dried cake or powder.

2. A dried surfactant reagent according to claim 1, wherein the surfactant polypeptide is cationic and the lipid mixture contains a weight ratio of POPG to POPC, DOPC, or a combination POPC and DOPC, or their respective salts, of between 1 :7 to 7: 1.

3. A dried surfactant reagent according to claim 1, wherein the surfactant polypeptide is cationic and the lipid mixture contains a weight ratio of POPG to POPC, DOPC, or a combination POPC and DOPC, or their respective salts, of between 1 :2 to 2: 1.

4. A dried surfactant reagent according to claim 1, wherein the surfactant polypeptide is cationic and the lipid mixture contains a weight ratio of POPG to POPC, DOPC, or a combination POPC and DOPC, or their respective salts, is between 1 : 1.5 and 1.5: 1.

5. A dried surfactant reagent according to one of claims 1-4, wherein the surfactant polypeptide is cationic and is selected from the group consisting of SP-B, SP-C, Mini-B, Super Mini-B, B-YL, KL4, SP-C33, SP-C30, KALA, and combinations thereof.

6. A dried surfactant reagent according to claim 1, wherein surfactant polypeptide is anionic and is selected from the group consisting of GALA, E-Mini-B, Super E-Mini-B, EL4, and combinations thereof.

7. A dried surfactant reagent according to one of claims 1-6, comprising about 2 to 4 wt% of surfactant polypeptide.

8. A dried surfactant reagent according to one of claims 1-5 or 7, wherein the surfactant polypeptide is cationic and wherein the dried surfactant reagent comprises about 3 wt% of the cationic surfactant protein and wherein the lipid mixture consists of about 20 wt% POPG-Na, about 29.4 wt% POPC, and about 47.6 wt% DPPC.

9. A dried surfactant reagent according to claim 1, 6 or 7, wherein the dried surfactant reagent comprises about 3 wt% of an anionic surfactant protein, and wherein the lipid mixture consists of about 20 wt% DOTAP, about 29.4 wt% POPC, and about 47.6 wt% DPPC.

10. A dried surfactant reagent according to one of claims 1-5, 7, or 8, wherein the cationic surfactant protein, peptide, or combinations thereof is Super Mini-B.

11. A dried surfactant reagent according to one of claims 1-5, 7, or 8, wherein the cationic surfactant protein, peptide analog thereof, or combinations thereof is Mini-B.

12. A method of preparing an aqueous surfactant reagent, comprising: rehydrating the surfactant reagent according to one of claims 1-11 in an aqueous buffer.

13. A method according to claim 12, wherein the aqueous buffer is phosphate buffered saline or 0.9% saline in 10 mM carbonate buffer or phosphate buffer, pH 6.5-7.4.

14. A method according to one of claims 12 or 13, wherein the aqueous buffer further comprises lipid nanoparticles.

15. A method according to one of claims 12-14, wherein the buffer or the lipid nanoparticles comprise a therapeutic molecule.

16. A method according to claim 15, wherein the therapeutic molecule is hydrophobic.

17. A method according to one of claims 12-16, wherein the aqueous buffer comprises a therapeutic polypeptide.

18. A method according to claim 17, wherein the therapeutic polypeptide is an antibody.

19. A method for delivering a surfactant reagent to an individual in need thereof, comprising: administering the surfactant as an aqueous reagent produced according to the method of one of claims 12-18.

20. A method according to claim 19, comprising administering the aqueous surfactant reagent via a mucosal route.

21. A method according to claim 19, comprising administering the aqueous surfactant reagent via a pulmonary route.

22. A method according to claim 19, comprising administering the aqueous surfactant reagent via a genital route.

23. A method according to claim 19, comprising administering the aqueous surfactant reagent via an oral route.

24. A method according to claim 19, comprising administering the aqueous surfactant reagent via a rectal route.

25. A method according to claim 19, comprising administering the aqueous surfactant reagent via a topical route.

26. A method according to claim 19, comprising administering the aqueous surfactant reagent via an ocular route.

27. A method according to claim 21, wherein the pulmonary route of administration is intranasal or intratracheal installation or by inhalation.

28. A method for delivering a surfactant reagent to an individual in need thereof, comprising: administering the surfactant reagent according to one of claims 1-11 as a dried powder via inhalation.

29. A method of producing a surfactant reagent, comprising: mixing a surfactant polypeptide and a lipid mixture in an organic:aqueous solvent system, wherein the surfactant polypeptide is at about 1 to about 10 wt% of the mixture, the organic:aqueous solvent system consists of one or more organic solvents selected from the group consisting of Ci-6 alcohols, acetone, and chloroform, and water, wherein the ratio of organic solvents: water in the organic:aqueous solvent system is 9: 1 or greater; and the surfactant polypeptide is cationic, and the lipid mixture consists of one or more lipids selected from a first group consisting of l,2-dipalmitoyl-sn-glycero-3- phosphatidylcholine (DPPC), l,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC), or 1- palmitoyl-2-oleoyl-glycero-3 -phosphatidylcholine (POPC) or salts thereof, and one or more lipids selected from a second group consisting of l-palmitoyl-2-oleoyl-sn- glycero-3 -phosphoglycerol (POPG), or l,2-dioleoyl-sn-glycero-3-phosphoglycerol (DOPG) or salts thereof; or the surfactant polypeptide is anionic, and the lipid mixture consists of one or more lipids selected from a first group consisting of DPPC, DOPC, POPC, and salts thereof, and one or more lipids selected from a second group consisting of l,2-di-O-octadecenyl-3- trimethylammonium propane (DOTMA),l,2-dioleoyl-3 -trimethylammonium propane (DOTAP), and salts thereof; andremoving the organic:aqueous solvent system by sublimation or spray drying to produce the surfactant reagent in the form of a dried cake or powder.

30. A method according to claim 29, wherein the one or more organic solvents are selected from the group consisting of tert-butanol, tert-amyl alcohol, methanol, ethanol, isopropanol, acetone, and 1 -butanol, or combinations thereof.

31. A method according to claim 30, wherein the organic:aqueous solvent system is selected from tert-butanol: water, tert-butanol: water, methanol : tert-butanol: water, ethanol :tert- butanol: water, isopropanol : tert-butanol: water, acetone: tert-butanol: water, and tert-amyl alcohol: water.

32. A method according to one of claims 29-31, wherein the sublimation is carried out at between -20°C and 25°C under vacuum.

33. A method according to one of claims 29-32, wherein the organic: aqueous solvent system is tert-butanol: water.

34. A method according to one of claims 29-33, wherein the surfactant polypeptide is cationic and the lipid mixture contains a weight ratio of POPG-Na to POPC, DOPC, or a combination POPC and DOPC of between 1 :7 to 7: 1.

35. A method according to one of claims 29-33, wherein the surfactant polypeptide is cationic and the lipid mixture contains a weight ratio of POPG to POPC, DOPC, or a combination POPC and DOPC, or their respective salts, of between 1 :2 to 2: 1.

36. A method according to one of claims 29-33, wherein the surfactant polypeptide is cationic and the lipid mixture contains a weight ratio of POPG to POPC, DOPC, or a combination POPC and DOPC, or their respective salts, is between 1 : 1.5 and 1.5: 1.

37. A method according to one of claims 29-36, wherein the surfactant polypeptide is cationic and is selected from the group consisting of SP-B, SP-C, Mini-B, Super Mini-B, B-YL, KL4, SP- C33, SP-C30, KALA, and combinations thereof.

38. A method according to one of claims 29-36, wherein the surfactant polypeptide is anionic and is selected from the group consisting of GALA, E-Mini-B, Super E-Mini-B, EL4, and combinations thereof.

39. A method according to one of claims 29-38, the surfactant polypeptide is at about 2 to 4 wt% of the mixture.

40. A method according to one of claims 29-37 or 39, wherein the surfactant polypeptide is cationic and wherein the dried surfactant reagent comprises about 3 wt% of the cationic surfactant protein and wherein the lipid mixture consists of about 20 wt% POPG-Na, about 29.4 wt% POPC, and about 47.6 wt% DPPC.

41. A method according to one of claims 29, 38 or 39, wherein the dried surfactant reagent comprises about 3 wt% of an anionic surfactant protein, and wherein the lipid mixture consists of about 20 wt% DOTAP, about 29.4 wt% POPC, and about 47.6 wt% DPPC.

42. A method according to one of claims 29-37, 39, or 40, wherein the cationic surfactant protein, peptide, or combinations thereof is Super Mini-B.

43. A method according to one of claims 29-37, 39, or 40, wherein the cationic surfactant protein, peptide analog thereof, or combinations thereof is Mini-B.

44. A therapeutic composition for pulmonary delivery, comprising: about 0.1 to about 1 wt% of a surfactant protein; about 0.9 to about 1 wt% lipid; and about 98 to about 99 wt% of one or more aqueous pharmaceutically acceptable excipients, wherein the surfactant polypeptide is cationic, and the lipid mixture consists of one or more lipids selected from a first group consisting of l,2-dipalmitoyl-sn-glycero-3- phosphatidylcholine (DPPC), l,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC), or 1- palmitoyl-2-oleoyl-glycero-3 -phosphatidylcholine (POPC) or salts thereof, and one or more lipids selected from a second group consisting of l-palmitoyl-2-oleoyl-sn- glycero-3 -phosphoglycerol (POPG), or l,2-dioleoyl-sn-glycero-3-phosphoglycerol (DOPG) or salts thereof; orthe surfactant polypeptide is anionic, and the lipid mixture consists of one or more lipids selected from a first group consisting of DPPC, DOPC, POPC, and salts thereof, and one or more lipids selected from a second group consisting of l,2-di-O-octadecenyl-3- trimethylammonium propane (DOTMA),l,2-dioleoyl-3 -trimethylammonium propane (DOTAP), and salts thereof.

45. A therapeutic reagent according to claim 44, wherein the aqueous buffer is phosphate buffered saline or 0.9% saline in 10 mM carbonate buffer, pH 6.45-7.4.

46. A method according to one of claims 44 or 45, wherein the aqueous buffer further comprises lipid nanoparticles.

47. A method according to one of claims 44-46, wherein the aqueous buffer or the lipid nanoparticles comprise a therapeutic molecule.

48. A method according to claim 47, wherein the therapeutic molecule is hydrophobic.

49. A method according to one of claims 44-48, wherein the aqueous buffer comprises a therapeutic polypeptide.

50. A method according to claim 49, wherein the therapeutic polypeptide is an antibody.

51. A method according to one of claims 44-50, wherein the surfactant polypeptide is cationic and the lipid mixture contains a weight ratio of POPG-Na to POPC, DOPC, or a combination POPC and DOPC of between 1 :7 to 7: 1.

52. A method according to one of claims 44-50, wherein the surfactant polypeptide is cationic and the lipid mixture contains a weight ratio of POPG to POPC, DOPC, or a combination POPC and DOPC, or their respective salts, of between 1 :2 to 2: 1.

53. A method according to one of claims 44-50, wherein the surfactant polypeptide is cationic and the lipid mixture contains a weight ratio of POPG to POPC, DOPC, or a combination POPC and DOPC, or their respective salts, is between 1 : 1.5 and 1.5: 1.

54. A method according to one of claims 44-53, wherein the surfactant polypeptide is cationic and is selected from the group consisting of SP-B, SP-C, Mini-B, Super Mini-B, B-YL, KL4, SP- C33, SP-C30, KALA, and combinations thereof.

55. A method according to one of claims 44-50, wherein the surfactant polypeptide is anionic and is selected from the group consisting of GALA, E-Mini-B, Super E-Mini-B, EL4, and combinations thereof.

56. A method according to one of claims 44-55, the surfactant polypeptide is at about 2 to 4 wt% of the mixture.

57. A method according to one of claims 44-54 or 55, wherein the dried surfactant reagent comprises about 3 wt% of the cationic surfactant protein and wherein the lipid mixture consists of about 20 wt% POPG-Na, about 29.4 wt% POPC, and about 47.6 wt% DPPC.

58. A method according to one of claims 44-50, 55 or 56, wherein the dried surfactant reagent comprises about 3 wt% of an anionic surfactant protein, and wherein the lipid mixture consists of about 20 wt% DOTAP, about 29.4 wt% POPC, and about 47.6 wt% DPPC.

59. A method according to one of claims 44-54, 56, or 57, wherein the cationic surfactant protein, peptide, or combinations thereof is Super Mini-B.

60. A method according to one of claims 44-54, 56, or 57, wherein the cationic surfactant protein, peptide analog thereof, or combinations thereof is Mini-B.

Images