Polypeptides for lung surfactant compositions

Abstract

The present disclosure provides synthetic lung surfactant proteins, methods for manufacturing synthetic lung surfactant proteins, and therapeutic compositions comprising synthetic lung surfactant proteins. Also described are methods for delivering a therapeutic agent using the lung surfactant protein and compositions comprising the lung surfactant proteins. The compositions and methods find use in treating, for example, cancer, metabolic disorders, cardiovascular disorders, neurological disorders and lung and respiratory disorders.

Description

POLYPEPTIDES FOR LUNG SURFACTANT COMPOSITIONSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional application no. 63 / 528,796, filed July 25, 2023, which is incorporated by reference herein in its entirety.STATEMENT REGARDING GOVERNMENT INTEREST

[0002] This invention was made with Government support under contract 2210373 awarded by National Science Foundation. The Government has certain rights in the invention.REFERENCE TO SEQUENCE LISTING

[0003] This application contains a Sequence Listing which has been submitted electronically in XML format. The Sequence Listing XML is incorporated herein by reference. The XML file, created on July 23, 2024, is named 117313-0019_8003_WO00_SL and is 124,315 bytes.TECHNICAL FIELD

[0004] The subject matter described herein relates to lung surfactant proteins and compositions and their use in treatment.BACKGROUND

[0005] Lung surfactant is a mixture of protein and phospholipid that facilitates lung inhalation and exhalation at the gas / liquid interface of the interior of alveoli sacs within the deepest recesses of the lung where circulating blood cells carry oxygen from the lungs and return carbon dioxide for exhalation. There are four surfactant proteins identified in air-breathing mammals: surfactant protein A, surfactant protein B, surfactant protein C and surfactant protein D.Surfactant protein A (SP-A, SFTPA1, SFTP1) and surfactant protein D (SP-D, SFTPD, SFTP4) are water-soluble and have collagen-like domains. Both SP-A and SP-D are part of the innate immune system’s collection. Surfactant protein B (SP-B, SFTP3, SFTB3) is a lung surfactant needed for lung expansion and compression. It is lipid-associated and assists the transport of lipid molecules into and out of alveolar membrane tissue. Surfactant protein C is an integral membrane protein that is also found in lung surfactant and is regulated by SP-B.

[0006] Both hydrophilic and hydrophobic regions are found within surfactant protein B (SP-B) and lipids of the surfactant. Lung surfactant also contains SP-C, a strongly hydrophobic peptide component. Lung surfactant also contains dipalmitoylphosphatidylcholine (DPPC), other phospholipids, and cholesterol. The phospholipid components reduce surface tension by positioning hydrophilic head groups in the aqueous-based alveolar fluid and hydrophobic tailsfacing towards the air where gas exchanges occur by adsorbing oxygen at the air-water interface of alveoli.

[0007] Lung surfactants have application in treating respiratory diseases, which affect both premature infants and adults. In premature infants, as commonly seen in the NICU, the infant is diagnosed with Infant Respiratory Distress Syndrome (IRDS) also known as neonatal respiratory distress syndrome (NRDS) and respiratory distress syndrome of prematurity (RDS). This illness was initially known as hyaline membrane disorder. RDS is a consequence of lung development immaturity as the lungs are structurally unable to produce sufficient levels of pulmonary surfactant that support independent breathing until around 35 weeks gestational age. Further, injury resulting from the treatment of the premature infant with supplemental oxygen, ventilator-induced lung injury, and instillation of lung surfactant can result inflammation resulting in bronchopulmonary dysplasia (BPD) and bacterial infection leading to late onset sepsis. In the 1990s the use of animal-derived lung surfactant products and less aggressive oxygen supplementation shifted the primary cause of BPD from ventilation-induced lung injury to inflammation of immature lung tissue from exposure to oxygen. However, the use of animal- derived lung surfactants may also contribute to lung inflammation resulting in BPD. In adults, adult respiratory distress syndrome (ARDS) can occur due to inflammation and subsequent degradation of lung tissue. These tissues become permeable which facilitates increased plasma in the lung. The plasma inhibits the biological actions of lung surfactant. Additional illnesses, such as COPD and emphysema are likely due to chronic inflammation. The adult respiratory distress syndromes can be a result of acute lung injury, ventilator-induced lung injury, and / or systemic inflammatory response syndrome and / or from physical insults to the lung tissues including shock, bacterial, viral and nosocomial pneumonias, and inhalation of toxic gases, vapors, fumes and particles and injuries such as mechanical ventilation, administration of oxygen, aspiration, and intubation.

[0008] Current therapeutics to replace deficient mammalian lung surfactant in pre-term infants suffering from the respiratory diseases described above are derived from minced bovine and porcine lung and calf lung lavage. A synthetic lung surfactant, CHF5633, contains an SP-B fragment analog and an SP-C analog. The SP-B fragment analog included in this synthetic lung surfactant contains less than half of SP-B sequence may limit therapeutic utility. Animal derived lung surfactant has several concerns ranging from variable composition, risk of contamination by animal pathogens, poor heat stability, and mishandling resulting in failure to adequately resuspend the therapeutic during administration. These products also may induceimmunogenic responses as the amino acid sequence of pig, cow, and sheep SP-B differ from human SP-B by at least 12%.

[0009] Lung surfactants also find use as pulmonary drug delivery carriers. Pulmonary delivery of drug is attractive because the lung is capable of absorbing pharmaceuticals either for local deposition or for systemic delivery. The high permeability and large absorptive surface area of lungs and good blood supply are advantageous for drug absorption.

[0010] There remains an unmet need for a lung surfactant composition that more closely matches human lung surfactant, is free of the potential for animal pathogen contaminants and of a consistent, immune tolerant composition. Further, there remains a need for a composition and / or lung surfactant composition that can be used as a drug delivery platform to deliver a bioactive or therapeutic agent for the treatment of diseases, such as inflammatory disorders, cancer, fibrosis, lung leakage, cardiovascular disorders, neurological disorders, metabolic disorders, and hypertension. The present disclosure provides compositions comprising surfactant polypeptides for use as a drug delivery carrier for delivery of a therapeutic agent (bioactive compound) via the pulmonary route of administration.

[0011] The foregoing examples of the related art and limitations related therewith are intended to be illustrative and not exclusive. Other limitations of the related art will become apparent to those of skill in the art upon a reading of the specification and a study of the drawings.BRIEF SUMMARY

[0012] The following aspects and embodiments thereof described and illustrated below are meant to be exemplary and illustrative, not limiting in scope.

[0013] In one aspect, a polypeptide or a surfactant polypeptide is provided comprising the amino acid sequence of FPIPLPYX1WLX2RALIKRIQAX3IPKGALAVAVAQVCRVVPLVAGGICQCLAERYSVILL DTLLGRX4 LPQLVX5RLVLRX6S (SEQ ID NO: 1) or an amino acid sequence of at least about 90% sequence identity to SEQ ID NO: 1, where Xi, X2, X5 and X<> are, independently, selected from the group consisting of L-threonine, D- threonine, L-tyrosine, D-tyrosine, L-alanine, D-alanine, L-leucine, D-leucine, L-aspartic acid, D-aspartic acid, L-glutamic acid, D-glutamic acid, L-cysteine, D-cysteine, valine, serine, threonine, glycine, norvaline, and 2-aminoisobutyric acid, and X3 and X4 are, independently, an amino acid that is non-oxidizable. In an embodiment, the surfactant polypeptide is an SP-B lung surfactant polypeptide.

[0014] In another aspect, provided herein is a polypeptide or a surfactant polypeptide monomer or dimer comprising the amino acid sequence ofFPIPLPYX1WLX2RALIKRIQAX3IPKGALAVAVAQVCRVVPLVAGGICQCLAERYSVILL DTLLGRX4LPQLVX5RLVLRX6S (SEQ ID NO: 10) or an amino acid sequence of at least about 90% sequence identity to SEQ ID NO: 10, where Xi, X2, X5 and Xe are, independently, A or C, and X3 and X4are Nle. In an embodiment, the surfactant polypeptide is an SP-B lung surfactant polypeptide.

[0015] In an embodiment, a polypeptide comprising or consisting of an amino acid sequence of SEQ ID NO: 1, or an amino acid sequence of at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 1, is provided, wherein Xi and Xe are independently, selected from the group consisting of L- threonine, D-threonine, L-tyrosine, D-tyrosine, L-alanine, D-alanine, L-leucine, D-leucine, L- aspartic acid, D-aspartic acid, L-glutamic acid, D-glutamic acid, L-cysteine, D-cysteine, valine, serine, threonine, glycine, norvaline, and 2- aminoisobutyric acid, X2 and X5 are L-cysteine, and X3 and X4are, independently, an amino acid, natural or unnatural, standard or non-standard, that is non-oxidizable.

[0016] In an embodiment, a polypeptide comprising or consisting of an amino acid sequence of SEQ ID NO: 1, or an amino acid sequence of at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 1, is provided, wherein X2 and X5 are independently, selected from the group consisting of L- threonine, D-threonine, L-tyrosine, D-tyrosine, L-alanine, D-alanine, L-leucine, D-leucine, L- aspartic acid, D-aspartic acid, L-glutamic acid, D-glutamic acid, L-cysteine, D-cysteine, valine, serine, threonine, glycine, norvaline, and 2-aminoisobutyric acid, Xi and Xe are L-cysteine, and X3 and X4are, independently, an amino acid, natural or unnatural, standard or non-standard, that is non-oxidizable.

[0017] In one embodiment, a surfactant polypeptide dimer comprises the amino acid sequence of SEQ ID NO: 11.

[0018] In another embodiment, a surfactant polypeptide dimer comprises the amino acid sequence of SEQ ID NO: 12.

[0019] In another embodiment, a surfactant polypeptide dimer comprises the amino acid sequence of SEQ ID NO: 13.

[0020] In an embodiment, the polypeptide is a synthetic polypeptide. In an embodiment, the polypeptide is a recombinant polypeptide. In an embodiment, the polypeptide is a human SP-B polypeptide and / or an analog of a human SP-B polypeptide.

[0021] In another aspect, a lung surfactant composition comprising one or more surfactant polypeptides described herein in combination with a phospholipid, cholesterol, or a combinationthereof is provided. In certain embodiments, the lung surfactant composition comprises one or more of a surfactant polypeptide monomer or dimer, an SP-C polypeptide, an SP-C polypeptide analog, a phospholipid, and cholesterol.

[0022] In an embodiment, the surfactant polypeptide is not CWLCRALIKRIQALIPKGGRLLPQLVCRLVLRCS (SEQ ID NO: 30).

[0023] In an embodiment, the surfactant polypeptide is not CWLRALIKRIQAX1IPKGGRX2LPQLVCRLVLRCS, where Xi is M, I, L or Nle and where X2is M, I, L or Nle (SEQ ID NO: 31).

[0024] In an embodiment, the surfactant polypeptide is not FP XiPLPY C X2LCRALIKRIQA X3IPKGGR X4LPQLVCRLVL X5CS (SEQ ID NO: 32), where Xi is L, I, or C; X2is W, I, or L; X3 and X4 are, independently, M, I, L, or Nle; and X5 R or T.

[0025] In another aspect, a method for delivering a therapeutic agent to a subject in need thereof is provided, the method comprising providing a composition comprising a surfactant polypeptide described herein and a therapeutic agent. In an embodiment, the method further comprises administering or instructing to administer the composition to a subject in need.

[0026] In another aspect, a method for delivering a therapeutic agent to a subject in need thereof is provided, the method comprising providing a lung surfactant composition comprising a surfactant polypeptide described herein and a therapeutic agent. In an embodiment, the method further comprises administering or instructing to administer the lung surfactant composition to a subject in need.

[0027] In an embodiment, the administering or instructing to administer comprises administering or instructing to administer by pulmonary delivery. In an embodiment, pulmonary delivery is selected from intratracheal instillation, inhalation and intranasal delivery.

[0028] In an embodiment, the composition or lung surfactant composition is in liquid form or in dry form.

[0029] In an embodiment, the therapeutic agent is for treating a disorder selected from the group consisting of serious respiratory illness associated with preterm birth, acute respiratory distress syndrome (ARDS), asthma, chronic obstructive pulmonary disease (COPD), emphysema, idiopathic pulmonary fibrosis (IPF), pulmonary arterial hypertension (PAH), and adenocarcinoma.

[0030] In an embodiment, the therapeutic agent is for treating a disorder selected from acute respiratory distress syndrome (ARDS), respiratory distress syndrome (RDS), meconium aspiration syndrome (MAS) and bronchopulmonary dysplasia.

[0031] In an embodiment, the therapeutic agent is for treating a disorder selected cancer, a metabolic disorder, a cardiovascular disorder and a neurological disorder.

[0032] In another aspect, a method for treating a disorder is provided. In an embodiment, the disorder is a cardiovascular disorder, a neurological disorder, a metabolic disorder, a cancer, or a lung or respiratory disorder. The method comprises providing for administration or administering to a subject in need thereof a composition comprising a surfactant polypeptide, a therapeutic agent for treating the disorder, and optionally one or more excipients. In an embodiment, the composition is a lung surfactant composition.

[0033] In some embodiments, the composition or lung surfactant composition is administered by intratracheal administration. In some embodiments, the composition or lung surfactant composition is administered in liquid form via nebulization or aerosolization. In other embodiments, the composition or lung surfactant composition is administered via an inhaler. In other embodiments, the composition or lung surfactant composition is administered intranasally.

[0034] In some embodiments, the lung or respiratory disorder is acute respiratory distress syndrome (ARDS), respiratory distress syndrome (RDS), meconium aspiration syndrome (MAS) or bronchopulmonary dysplasia (BPD).

[0035] In some embodiments, the subject is additionally administered a therapeutic agent, such an anti-inflammatory agent, anti-microbial agent, cardiovascular agent, or anti-cancer agent.

[0036] In another aspect, a method for manufacture of a surfactant polypeptide with an amino acid sequence of SEQ ID NO: 1 or an amino acid sequence of at least about 90% sequence identity to SEQ ID NO: 1 is provided, wherein Xi, X2, X5 and Xe are, independently, selected from the group consisting of L-threonine, D-threonine, L-tyrosine, D-tyrosine, L-alanine, D- alanine, L-leucine, D-leucine, L-aspartic acid, D-aspartic acid, L-glutamic acid, D-glutamic acid, L-cysteine, D-cysteine, valine, serine, threonine, glycine, norvaline, and 2- aminoisobutyric acid, and where X3 and X4 are, independently, an amino acid that is non-oxidizable. The method comprises reacting the surfactant polypeptide under conditions suitable for forming one or more internal disulfide bridges in a monomer and / or one or more interchain disulfide bridges resulting in the formation of a dimer. In an embodiment, the surfactant polypeptide is synthetic.

[0037] In some embodiments, the surfactant polypeptide of SEQ ID NO: 1 comprises one or more protected and / or unprotected amino acid residues. In an embodiment, the method comprises reacting the surfactant polypeptide with protected and unprotected groups with one or more reagents under conditions suitable for forming a first internal disulfide bridge and reacting the surfactant polypeptide with one or more reagents under conditions suitable for forming asecond internal disulfide bridge, while retaining one or more protecting groups in the surfactant polypeptide.

[0038] In another embodiment, the method comprises providing a surfactant polypeptide with an amino acid sequence of SEQ ID NO: 10, where X], X2, X5 and Xr, are, independently, A or C, and X3 and X4 are, independently, A or C, and X3 and X4 are Nle, and reacting the surfactant polypeptide under conditions suitable for forming one or more internal disulfide bridges. In an embodiment, the surfactant polypeptide is synthetic.

[0039] In a further aspect, provided herein is a method for manufacture of a lung surfactant polypeptide dimer comprising reacting a lung surfactant polypeptide monomer comprising an amino acid sequence of SEQ ID NO: 10 and one or more internal disulfide bridges and reacting the lung surfactant polypeptide monomer with a second lung surfactant polypeptide monomer comprising an amino acid sequence of SEQ ID NO: 10 and one or more internal disulfide bridges under conditions suitable for forming a dimer. In an embodiment, the lung surfactant polypeptide is synthetic.

[0040] In addition to the exemplary aspects and embodiments described above, further aspects and embodiments will become apparent by reference to the drawings and by study of the following descriptions. Additional embodiments of the present disclosure will be apparent from the following description, drawings, examples, and claims. As can be appreciated from the foregoing and following description, each and every feature described herein, and each and every combination of two or more of such features, is included within the scope of the present disclosure provided that the features included in such a combination are not mutually inconsistent. In addition, any feature or combination of features may be specifically excluded from any embodiment of the present disclosure. Additional aspects and advantages of the present disclosure are set forth in the following description and claims, particularly when considered in conjunction with the accompanying examples and drawings.BRIEF DESCRIPTION OF THE DRAWINGS

[0041] FIG. 1 is a graph showing dynamic surface tension, in mN / M, of compositions with dipalmitoylphosphatidylcholine (DPPC) and palmitoyloleoylphosphatidylglycerol (POPG) (7:3) and an SP-B or an SP-C surfactant polypeptide, the SP-B surfactant polypeptide having the amino acid sequence of'SEQ ID NO: 11 (open squares), SEQ ID NO: 12 (inverted triangles) or SEQ ID NO: 13 (diamonds), the SP-C surfactant polypeptide having the sequence of SEQ ID NO: 15. As controls, a composition of DPPC and POPG (7:3) with no surfactant protein (polypeptide) (closed circles) and bovine lipid extract surfactant (BLES) (open circles) were measured.

[0042] FIGS. 2A-2D are graphs of minimum surface tension, in mN / m, as a function of repetitive cycles (as described in Example 4) of compositions comprised of an SP-B surfactant polypeptide of SEQ ID NO: 11 (FIG. 2A), a surfactant polypeptide of SEQ ID NO: 12, (FIG. 2B) or a surfactant polypeptide of SEQ ID NO: 13 (FIG. 2C), or an SP-C surfactant polypeptide (SEQ ID NO: 15), where each composition also comprised DPPC and POPG (7:3). As controls, a composition of DPPC and POPG (7 :3) with no surfactant protein (open circles) and BLES (squares) were prepared and tested (FIG. 2D).

[0043] FIGS. 3A-3C illustrate dimers of the polypeptides of SEQ ID NO: 12 and SEQ ID NO: 13, where a homodimer of the polypeptide of SEQ ID NO: 12 is shown in FIG. 3A, a homodimer of the polypeptide of SEQ ID NO: 13 is shown in FIG. 3B and a heterodimer of the polypeptides of SEQ ID NO: 12 and SEQ ID NO: 13 is shown in FIG. 3C.

[0044] FIGS. 4A-4H show a reaction scheme for preparing a synthetic surfactant polypeptide (FIG. 4A) and ultra performance liquid chromatography (UPLC) scans (FIGS. 4B-4H). FIG. 4A discloses SEQ ID NOS 33-35, respectively, in order of appearance.

[0045] FIGS. 5A-5C show a reaction scheme for preparing a synthetic surfactant polypeptide (FIG. 5 A) and UPLC scans (FIGS. 5B-5C). FIG. 5 A discloses SEQ ID NO: 36.BRIEF DESCRIPTION OF THE SEQUENCESDETAILED DESCRIPTIONI. Definitions

[0046] Various aspects now will be described more fully hereinafter. Such aspects may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey its scope to those skilled in the art.

[0047] For convenience, certain terms employed in the specification, examples and claims are collected here. Unless defined otherwise, all technical and scientific terms used in this disclosure have the same meanings as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0048] Where a range of values is provided, it is intended that each intervening value between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the disclosure. For example, if a range of 1 pm to 8 pm is stated, it is intended that 2 pm, 3 pm, 4 pm, 5 pm, 6 pm, and 7 pm are also explicitly disclosed, as well as the range of values greater than or equal to 1 pm and the range of values less than or equal to 8 pm.

[0049] The singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to a "lipid" includes a single lipid as well as two or more of the same or different lipids, reference to an "excipient" includes a single excipient as well as two or more of the same or different excipients, and the like.

[0050] The word "about" when immediately preceding a numerical value means a range of plus or minus 10% of that value, e.g., "about 50" means 45 to 55, "about 25,000" means 22,500 to 27,500, etc., unless the context of the disclosure indicates otherwise, or is inconsistent with such an interpretation. For example, in a list of numerical values such as "about 49, about 50, about 55, "about 50" means a range extending to less than half the interval(s) between the preceding and subsequent values, e.g., more than 49.5 to less than 52.5. Furthermore, the phrases "less than about" a value or "greater than about" a value should be understood in view of the definition of the term "about" provided herein.

[0051] The compositions of the present disclosure can comprise, consist essentially of, or consist of, the components disclosed.

[0052] All percentages, parts and ratios are based upon the total weight of the topical compositions and all measurements made are at about 25 °C., unless otherwise specified.

[0053] An “amino acid” intends an organic compound that typically contains both amino and carboxylic acid functional groups, including natural and unnatural, standard and non-standard amino acids. Examples of unnatural amino acids include D-amino acids, homo amino acids, betahomo amino acids, N-methyl amino acids, alpha-methyl amino acids, and non-natural side chain variant amino acids. Examples of non-standard amino acids include cystine, desmosine, isodesmosine, hydroxyproline, hydroxylysine, gamma-carboxyglutamate, phosphoserine, phosphothreonine, phosphotyrosine, N-acetyl lysine, and methyllysine. The amino acid sequences of the present disclosure are shown according to the single-letter or three-letter designation corresponding to a given amino acid. The amino acid sequence contains (unless otherwise specified) a free amino group at the left end (amino terminus) and a free carboxyl group (unless otherwise specified) at the right end (carboxy terminus). Unless otherwise specified, all the amino acid residues identified herein are in the natural L-configuration and the sequences identified herein are reported according to standard abbreviations for amino acid residues as shown in Table 1.Table 1. Correspondence between standard amino acids and their one letter and three letter designations.

[0054] The term “amorphous powder” is used with reference to a product formed from the transformation of a wet material, e.g., a liquid, solution, suspension, emulsion, liposome and the like to a dry, microparticulate material by subjecting the wet material to at least one drying method including, but not limited to, spray drying, supercritical fluid freezing, bubble drying, and lyophilization as is known to the skilled artisan. The amorphous powder can be any noncrystalline solid matrix. The solid matrix can be a powder, tablet powder within a cake, tablet, and dispersed w / in a capsule.

[0055] The terms “bioactive agent” and “therapeutic agent” refer to a natural or synthetic moiety, substance, or chemical and the like which can impart a biological function, activity, or property. Examples include but are not limited to anti-inflammatory drugs, anti-infective drugs, bronchodilation drugs, antihistamines, cyclooxygenase inhibitors, leukotriene antagonists, phospholipase A2 (PLA2) inhibitors, platelet activating factor antagonists, anti-allergics, bronchodilators, analgesics, antibiotics, antibacterials, antifungals, antivirals, antiprotozoans, antihistamines, decongestants and anti-tussive drug substances, anticholinergics, P-blockers, adrenergic, f -adrenoreceptor agonist, anesthetics, anti-tuberculars, cardiovascular agents, agents for treating a neurological disorder, agents for treating a metabolic disorder, anti-cancer agents, enzymes, steroids, genetic material, nucleic acid vectors, and antisense agents. In some embodiments, the bioactive agent or therapeutic agent can be fused, attached, or complexed to or with a surfactant protein.

[0056] The term “emulsion” refers to a mixture, multiphasic biphasic solution, of two or more liquids that are normally immiscible, one dispersed in the other.

[0057] The terms “infant,” “neonatal infant,” “neonate” and “newborn” are used with reference to a baby in the first 28 days after birth. The terms can apply to premature, full term and postmature infants.

[0058] The term “lipid” or “lipid carrier” refer to a natural or synthetic compound, including for example fats, waxes, sterols, fat-soluble vitamins, monoglycerides, diglycerides, phospholipids, phosphoglycerolipids, saturated fatty acids, unsaturated fatty acids, fatty acid sugars, sphingolipids and digalactosylglycerolipids.

[0059] The term “lung surfactant composition” refers to a composition comprising one or more surfactant polypeptides and a lipid, with optional further components such as a buffer and / or bioactive or therapeutic agent(s). The surfactant polypeptide, also referred to as a “lung surfactant polypeptide” or “surfactant protein”, can be naturally occurring, recombinant or synthetic, and / or can be a homolog, analog, or a mimic of a naturally -occurring or native lung surfactant protein. The lipid can be naturally occurring, homolog, analog, mimic, modified and / or synthetic. The composition can be in liquid or dry form, and when liquid, can be a suspension or an emulsion.

[0060] The term “non-oxidizable” refers to any amino acid, D or L, natural or unnatural, comprising a sidechain that does not oxidize in the presence of air or normal physiological conditions

[0061] The phrase "pharmaceutically acceptable" refers to those compounds, salts, compositions, dosage forms, etc., which are— within the scope of sound medical judgment- suitable for use in contact with the tissues of human beings and / or other mammals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio. In some aspects, "pharmaceutically acceptable" means approved by a regulatory agency of the federal or a state government, or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in mammals (e.g., animals), and more particularly, in humans.

[0062] The term “phospholipid” refers to a class of lipids containing a phosphate group in the molecule. An example is a molecule constituted of glycerol, a phosphate group, and a neutral moiety, a positively charged moiety and / or a zwitter-ionic moiety. Another example is sphingomyelin, which is derived from sphingosine instead of glycerol. The glycerol moiety or the sphingosine residue can be esterified with long chain fatty acids (C14-C22) which in turn can be saturated (e.g., myristic, palmitic, and stearic acid), monounsaturated (e.g., oleic acid) or polyunsaturated (e.g., linoleic and arachidonic acid).

[0063] The term “polypeptide analogs of the native surfactant protein SP-C” includes polypeptides having an amino acid sequence in which, compared to the native SP-C proteins, one or more amino acids are missing or have been replaced by other amino acids, so long as the polypeptides, in a mixture with a lipid carrier, show pulmonary surfactant activity.

[0064] The term “polypeptide analogs of the native surfactant protein SP-B” includes peptides having an amino acid sequence in which, compared to the native SP-B proteins, one or more amino acids are missing so long as the polypeptides, in a mixture with a lipid carrier, show pulmonary surfactant activity.

[0065] The terms “polypeptide” and “protein” are used interchangeably to refer to amino acid residues joined by peptide bonds.

[0066] The term “premature” refers to a human baby born before reaching 37 weeks’ gestational age verses normally 40 weeks of gestational age at birth.

[0067] The term “reconstituted surfactant” refers to a lipid carrier to which polypeptide analogs of the surfactant proteins, made through recombinant technology or synthetic methods, have been added.

[0068] As used herein, the term “surfactant activity” refers to the ability of a surfactant polypeptide or a lung surfactant composition to lower surface tension. In vitro efficacy of exogenous lung surfactant polypeptides or lung surfactant compositions is commonly tested by measuring their ability to lowering the surface tension using a suitable apparatus, such as Wilhelmy Balance and Captive Bubble Surfactometer according to methods known to those skilled in the art. The disclosed surfactant polypeptides may or may not have surfactant activity.

[0069] The term “suspension” refers to a solid including, but not limited to an amorphous powder, dispersed in a liquid, such as an aqueous solution, dilute organic solution or buffering medium. Suspension can also refer to the resulting mixture of one or more lung surfactant protein(s) with one or more lipid(s).

[0070] The term "treating" is used herein in reference to methods of treating any disease, disorder or condition, such as a respiratory disorder, metabolic disorder, cancerous disorder, neurological disorder, cardiovascular disorder and the like, and generally includes the administration of a surfactant polypeptide, a composition with a surfactant polypeptide, such as a lung surfactant composition, which is capable of reducing the frequency of, or delaying the onset of, symptoms of the disease, disorder, or condition in a subject relative to a subject not receiving the surfactant polypeptide or composition comprising a surfactant polypeptide. This can include reversing, reducing, or arresting the symptoms, clinical signs, and underlyingpathology of a disease, disorder or condition in a manner to improve or stabilize a subject's condition.

[0071] By reserving the right to proviso out or exclude any individual members of any such group, including any sub-ranges or combinations of sub-ranges within the group, which can be claimed according to a range or in any similar manner, less than the full measure of this disclosure can be claimed for any reason. Further, by reserving the right to proviso out or exclude any individual substituents, analogs, compounds, ligands, structures, or groups thereof, or any members of a claimed group, less than the full measure of this disclosure can be claimed for any reason.

[0072] Throughout this disclosure, various patents, patent applications and publications are referenced. The disclosures of these patents, patent applications and publications in their entireties are incorporated into this disclosure by reference in order to more fully describe the state of the art as known to those skilled therein as of the date of this disclosure. This disclosure will govern in the instance that there is any inconsistency between the patents, patent applications and publications cited and this disclosure.II. Surfactant Polypeptides

[0073] In one aspect, an analog of a native SP-B polypeptide is provided, where the analog has an amino acid sequence represented by the general formula (I) (SEQ ID NO: 1) of: FPIPLPYX1WLX2RALIKRIQAX3IPKGALAVAVAQVCRVVPLVAGGICQCLAERYS VILLDTLLGRX4 LPQLVX5RLVLRX6S (I) or an amino acid sequence of at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 1 whereinXi (position 8), X2 (position 11), X5 (position 71) and Xe (position 77) are, independently, selected from:L-threonine, D-threonine, L-tyrosine, D-tyrosine, L-alanine, D-alanine, L-leucine, D-leucine, L- aspartic acid, D-aspartic acid, L-glutamic acid, D-glutamic acid, L-cysteine, D-cysteine, valine, serine, threonine, glycine, norvaline, and 2-aminoisobutyric acid, andX3 (position 21) and X4 (position 65) are, independently, any amino acid that is non-oxidizable.

[0074] In some embodiments, Xi, X2, X5, and X& at positions 8, 11, 71, and 77, respectively, are independently selected from L-cysteine, D-cysteine, L-alanine, D-alanine, serine, threonine and 2-aminoisobutyric acid (SEQ ID NO: 2). In some embodiments, Xi, X2, X5, and Xe are independently selected from 2-aminoisobutyric acid, norvaline, serine, valine, L-cysteine, andL-alanine (SEQ ID NO: 3). In some embodiments, X3 and X4 are independently selected from alanine, valine, leucine, isoleucine, phenylalanine, norleucine, and norvaline (SEQ ID NO: 7).

[0075] In an embodiment, Xi and Xe are dependently selected L-cysteine, D-cysteine, L- alanine, D-alanine, serine, threonine and 2-aminoisobutyric acid. In an embodiment, X2 and X5 are dependently selected from L-cysteine, D-cysteine, L-alanine, D-alanine, serine, threonine and 2-aminoisobutyric acid. In an embodiment, Xi and Xe are dependently selected L-cysteine, D-cysteine, L-alanine, and D-alanine. In an embodiment, X2 and X5 are dependently selected from L-cysteine, D-cysteine, L-alanine, and D-alanine.

[0076] In an embodiment, X3 and X4 are an amino acid that is non-oxidizable. In an embodiment, an oxidizable amino acid is one that reacts with oxygen with a loss of electrons. In an embodiment, X3 and X4 are independently and / or each an amino acid comprising a side chain that does not oxidize during manufacture, product storage, and / or preparation for therapeutic administration. In an embodiment, the amino acid at positions X3 and X4 are not cysteine, cystine, histidine, methionine, tryptophan and / or tyrosine, in D or L configuration. In an embodiment, a non-oxidizable amino acid intends an amino acid comprising a side chain that does not oxidize during manufacture, product storage, and / or preparation for therapeutic administration, but which may oxidize due to enzyme activity. For example, proline can be oxidized to hydroxyproline in the presence of some enzymes, and proline is considered herein to be a non-oxidizable amino acid in some embodiments. Contemplated for amino acid positions X3 and X4 of SEQ ID NO: 1 are amino acids that are not oxidizable. Exemplary non-oxidizable amino acids include, but are not limited to, all naturally occurring amino acids with the exceptions of cysteine, methionine, histidine, and tryptophan: L-alanine, L-valine, L-leucine, L- isoleucine, L-proline, L-phenylalanine, glycine, L-serine, L-threonine, L-tyrosine, L-lysine, L- arginine, L-aspartic acid, L-glutamic acid, L-asparagine, L-glutamine, and the corresponding D- isomers of these, and any amino acid with an alkyl sidechain, such as L-norleucine, L-norvaline and L-2-amino-butyric acid, and their corresponding D-isomers.

[0077] In some embodiments, X3 and X4 at positions 21 and 65, respectively, are any amino acid, D or L, natural or unnatural, other than cysteine, cystine, histidine, methionine, tryptophan and tyrosine (SEQ ID NO: 5). In some embodiments, one or both of X3 and X4 is norleucine, such as in SEQ ID NO: 10.

[0078] In some embodiments, Xi, X2, X5, and X are independently, alanine or cysteine, and X3 and X4 are norleucine as represented by the general formula la (SEQ ID NO: 10): FPIPLPY(C / A)WL(C / A)RALIKRIQA(Nle)IPKGALAVAVAQVCRVVPL (la) VAGGICQCLAERYSVILLDTLLGR(Nle) LPQLV(C / A)RLVLR(C / A)S.

[0079] In one embodiment, each of Xi, X2, X5, and X is cysteine and each of X3 and X4 is norleucine as set forth in SEQ ID NO: 11.

[0080] In another embodiment, each of Xiand Xe is cysteine and each of X2 and X5 is alanine as set forth in SEQ ID NO: 12.

[0081] In another embodiment, each of X2 and X5 is cysteine and each of Xi and Xs is alanine as set forth in SEQ ID NO: 13.

[0082] In another aspect, a lung surfactant polypeptide (also referred to herein as a surfactant polypeptide or polypeptide) comprises an amino acid sequence set forth in any one of SEQ ID NOs: 1-13, where the lung surfactant polypeptide is in the form of a monomer or dimer. In some embodiments, the dimer is a homodimer. In other embodiments, the dimer is a heterodimer. Examples of dimer polypeptides are described below with reference to FIGS. 3A-3C.

[0083] In one embodiment, the lung surfactant polypeptide is a dimer of a synthetic or recombinant polypeptide with an amino acid sequence SEQ ID NO: 10 or an amino acid sequence of at least about 90% sequence identity to SEQ ID NO. 10.

[0084] In some embodiments, the lung surfactant polypeptide is a dimer of a synthetic or recombinant polypeptide with an amino acid sequence SEQ ID NO: 11 or an amino acid sequence of at least about 90% sequence identity to SEQ ID NO: 11.

[0085] In some embodiments, the lung surfactant polypeptide is a dimer of a synthetic or recombinant polypeptide with an amino acid sequence SEQ ID NO: 12 or an amino acid sequence of at least about 90% sequence identity to SEQ ID NO. 12.

[0086] In some embodiments, the lung surfactant polypeptide is a dimer of a synthetic or recombinant polypeptide with an amino acid sequence SEQ ID NO: 13 or an amino acid sequence of at least about 90% sequence identity to SEQ ID NO. 13.

[0087] In some embodiments, a monomer or dimer having an amino acid sequence set forth in any one or SEQ ID NOs: 1-13 has a C-terminal end cap, an N-terminal end cap, or both. In some embodiments, the N-terminal cap is NH2.

[0088] In an embodiment, the SP-B lung surfactant polypeptide is an analog of a human native SP-B polypeptide or a human native SP-C polypeptide.

[0089] In some embodiments, a monomer or dimer having an amino acid sequence set forth in SEQ ID NO: 11, 12, or 13 includes a protecting group. In an embodiment, a monomer of SEQ ID NO: 24, SEQ ID NO: 25 or SEQ ID NO: 26 is contemplated. In an embodiment, a homodimer or a heterodimer comprised of SEQ ID NO: 24, SEQ ID NO: 25 and / or SEQ ID NO: 26 is contemplated. In an embodiment, a composition comprising a homodimer or aheterodimer comprised of SEQ ID NO: 24, SEQ ID NO: 25 and / or SEQ ID NO: 26 is contemplated, where the composition comprises one or more of the ingredients described herein.

[0090] In some embodiments, a lung surfactant polypeptide monomer or lung surfactant polypeptide dimer comprises an amino acid sequence set forth in any one of SEQ ID NOs: 1-13 with one or more internal disulfide bridges. In some embodiments, internal disulfide bridges are between amino acid residues Cl l and C71, between amino acid residues C8 and C77 or between amino acid residues C35 and C46. In some of the polypeptides containing one or more of these internal disulfide bridges, the polypeptide includes a protective group.

[0091] In some embodiments, a lung surfactant polypeptide homodimer or lung surfactant polypeptide heterodimer comprises an amino acid sequence set forth in any one of SEQ ID NOs: 10-13 has a disulfide bridge between amino acid residues C48 in each monomer of the homodimer or heterodimer. In one embodiment, a homodimer or heterodimer comprising an amino acid sequence set forth SEQ ID NO: 13 or SEQ ID Nos: 24-26 includes a disulfide bridge between C48 in one monomer and C48 in the other monomer. In another embodiment, a homodimer or heterodimer comprising an amino acid sequence set forth SEQ ID NO: 11 includes a disulfide bridge between C48 in one monomer and C48 in the other monomer. In another embodiment, a homodimer or heterodimer comprising an amino acid sequence set forth SEQ ID NO: 12 includes a disulfide bridge between C48 in one monomer and C48 in the other monomer. In another embodiment, a homodimer or heterodimer comprising an amino acid sequence set forth SEQ ID NO: 13 includes a disulfide bridge between C48 in one monomer and C48 in the other monomer.

[0092] In another embodiment, the homodimer or heterodimer includes a disulfide bridge between C48 of a monomer having the amino acid sequence set forth in SEQ ID NO: 11 and C48 of a monomer having the amino acid sequence set forth in SEQ ID NO: 12. In another embodiment, the homodimer or heterodimer includes a disulfide bridge between C48 of a monomer having the amino acid sequence set forth in SEQ ID NO: 11 and C48 of a monomer having the amino acid sequence set forth in SEQ ID NO: 13. In another embodiment, the homodimer or heterodimer includes a disulfide bridge between C48 of a monomer having the amino acid sequence set forth in SEQ ID NO: 12 and C48 of a monomer having the amino acid sequence set forth in SEQ ID NO: 13.

[0093] In an embodiment, a pharmaceutically acceptable salt of the lung surfactant polypeptide and / or a blocked N- and / or C-terminus derivatives, e.g., via acetylation and amidation, is provided. Pharmaceutically acceptable salts include for example, salts of hydrochloric acid, acetic acid, and trifluoroacetic acid.

[0094] In an embodiment, a starting polypeptide or an intermediate polypeptide for synthesizing a lung surfactant polypeptide is provided, where the lung surfactant polymer may have internal disulfide bridges in monomers and interchain disulfide bridges in dimers. Exemplary starting and intermediate polypeptides utilized or produced are identified by their amino acid sequences in SEQ ID NOs: 23-29 and SEQ ID NOs: 33-36. In the amino acid sequences shown below, modified cysteines are bolded and / or identified by their respective protecting groups (i.e., acetamidomethyl (Acm), trityl (Trt) and tert-Butyl (tBu)) for selective, stepwise disulfide bridge formation:SEQ ID NO: 23 FPIPLPYC(Acm)WLCRALIKRIQA-Nle-IPKGALAVAV AQVC(Acm)RVVPLVAGGIC(Acm)QC(tBu)LAERYSVILLDTLLGR-Nle- LPQLVCRLVLRC(Acm)SSEQ ID NO: 24 FPIPLPYCWLCRALIKRIQA-Nle-IPKGALAVAV AQVCRVVPLVAGGICQC(tBu)LAERYSVILLDTLLGR-Nle-LPQLVCRLVLRCSSEQ ID NO: 27 FPIPLPYC(Trt)WLC(Acm)RALIKRIQA-Nle-IPKGALAVAV AQVC(Acm)RVVPLVAGGIC(Acm)QC(tBu)LAERYSVILLDTLLGR-Nle-LPQL VC(Acm)RLVLRC(Trt)SSEQ ID NO: 28: FPIPLPYC(Acm)WLC(Trt)RALIKRIQA-Nle- IPKGALAVAVAQVC(Acm)RVVPLVAGGIC(Acm)QC(tBu)LAERYSVILLDTLLGR-Nle- LPQLVC(Trt)RLVLRC(Acm)SSEQ ID NO: 29 FPIPLPYC(Acm)WLC(Acm)RALIKRIQA-Nle-IPKGALAVAVAQVC(Trt)RVVPLVAGGIC(Trt)QC(tBu)LAERYSVILLDTLLGR-Nle- LPQLVC(Acm)RLVLRC(Acm)S.II. Lung Surfactant Compositions

[0095] In another aspect, a composition comprising one or more surfactant polypeptides (also referred to as a lung surfactant polypeptide) described herein, one or more excipients, and / or one or more SP-C polypeptides or analogs is provided.

[0096] In some embodiments, the excipient(s) include a phospholipid. In an embodiment, the phospholipid is a lipid in which one fatty acid has been replaced by a phosphate group and a simple organic molecule. Exemplary phospholipids are phosphatidylcholines (PC), phosphatidylethanolamine (PE) phosphatidylglycerol (PG), phosphatidylinositol (PI), and phosphatidylserine (PS). The glycerol moieties of the phospholipids are mainly esterified with long chain fatty acids which in turn can be saturated (e.g., myristic, palmitic, and stearic acid),monounsaturated (e.g., oleic acid) or polyunsaturated (e.g., linoleic and arachidonic acid). Any phospholipids may be used as an excipient in the lung surfactant composition.

[0097] In some embodiments, the phospholipids for use as excipients include phospholipids that are contained in natural pulmonary surfactant preparations, for example phosphatidylcholines (PC) such as dipalmitoylphosphatidylcholine (DPPC) and palmitoyloleoylphosphatidylcholine (POPC), and phosphatidylglycerols (PG), such as palmitoyloleoylphosphatidylglycerol (POPG), dioleoylphosphatidylglycerol (DOPG) and dipalmitoylposphatidylglycerol (DPPG). Other phospholipids which may be employed include phosphatidylinositol (PI), lysophosphatidylinositol (LPI), dilauroylphosphatidylcholine (DLPC), disteroylphosphatidylcholine (DSPC), behenoylphosphatidyl-choline, arachidoylphosphatidylcholine (ADPC), lysophosphatidylethanolamine (LPE), dipalmitoyl-sn- glycero-3-phosphoethanolamine (DPPE), l-palmitoyl-2-oleoyl-sn-glycero-3- phosphoethanolamine (POPE), lysophosphatidylserine (LPS), diether phosphono- phosphatidylglycerol (PG- 1), sphingophospholipids such as sphingomyelin, and combinations thereof. Additional phospholipids are described in U.S. Patent No. 9,074,018, which is incorporated by reference in its entirety.

[0098] In some embodiments, the excipients include cholesterol.

[0099] In some embodiments, the excipients include one or more neutral lipids, such as triacylglycerols, ceramides, and sphingomyelins, and / or free fatty acids.In some embodiments, the excipients include one or more of polyols, proteins, and / or sugars. Polyols can be selected from the group consisting of one or more of erythritol, inulin, lactitol, maltitol, mannitol, myoinositol, sorbitol, xylitol, and hydrates thereof. Sugars can include, but are not limited to, natural and synthetic sugar(s) including, but not limited to, dextran, fructose, galactose, glucose, inulin, maltose, mannitol, raffinose, melezitose, sorbitol, stachyose, sucrose, trehalose, starch, and hydrates thereof. The amino acids may include but are not limited to proteins having amino acid polymers containing phenylalanine, cystine, glycine, arginine, histidine, lysine, and leucine. The amino acid excipients can be selected from the group consisting of one or more of L-phenylalanine, L-cystine, glycine, L-arginine, L-histidine, L- isoleucine, L-lysine, and L-leucine, L-proline, L-methionine, L-threonine, L-tryptophan, L- valine, L-glutamic acid, L-aspartic acid, L-asparagine, L-glutamine, L-tyrosine, L-serine, L- alanine, tri-leucine and salts thereof.

[0100] In some embodiments, the excipients include a buffer. Exemplary buffers include, but are not limited to sodium, potassium, calcium and / or lithium buffers selected from the group including acetic acid, phosphoric acid, citric acid, boric acid, histidine, lactic acid,tromethamine, gluconic acid, aspartic acid, glutamic acid, tartaric acid, succinic acid, malic acid, fumaric acid, and alpha-ketoglutaric acid. The sodium, potassium, calcium and / or lithium buffer can have the sodium, potassium, calcium and / or lithium salt with the conjugate base or the salt’s conjugate acid. The selected buffer would be known to the skilled artisan to be suitable in spraydrying applications of the disclosed compositions.

[0101] Other excipients include sphingomyelins, glycolipids, and gangliosides; ceramides, sphingosines, and other excipients disclosed in PCT Publication No. 2022 / 082082, incorporated by reference herein.

[0102] In some embodiments, the excipients include a phospholipid, a cholesterol, a buffer, and / or any combination thereof.

[0103] In some embodiments, the composition further includes an SP-C polypeptide analog represented by the amino acid sequences set forth in SEQ ID NOs: 14-22. In some embodiments, the composition further includes a human SP-C polypeptide as represented by the amino acid sequences set forth in SEQ ID NO: 22. In some embodiments, the human SP-C polypeptide is synthetic.

[0104] In embodiments, the lung surfactant composition further comprises an SP-C polypeptide, or SP-C-like polypeptide, having an amino acid sequence selected from the group consisting of: SEQ ID NO: 14 IPSSPVHXiKRX2K-(X3)n-I-(X4)n-IXsGA-(X6)n-G-X7, where Xi,X2, X3, X4, Xs,Xe, and X7 are, independently, selected from the group consisting of Ala, Arg, Met, Leu, Lys, Gin, Glu, Ser, Tyr, Vai and Phe, and n is 1, 2, 3, 4, 5, 6, 7, 8, or 9. In an embodiment, each of Xi, X2, X3, X4. X5. Xe, and X7 is not Vai or is not Leu;SEQ ID NO: 15 IPSSPVHLKR LKLLLLLLLL 1LLLILGALL LGL;SEQ ID NO: 16 FGIPSSPVHXiKR-(X2)n-IX3VVVVVX4I-(X5)n-VGA-(X6)n-NleGX7, where Xi, X2, X3, X4, X5, Xe, and X7 are, independently, selected from the group consisting of Ala, Arg, Met, Leu, Lys, Gin, Glu, Ser, Tyr, and Phe, and n is 1, 2, 3, 4, 5, 6, 7, 8, or 9. In an embodiment, each of X1.X2. X3. X4, X5, Xe, and X7 is not Vai or is not Leu;SEQ ID NO: 17 FGIPSSPVHLKR LLILVVVV VLILLLLVGALL NleGL;SEQ ID NO: 18 FGIPCCPVHLKR LLIVVVVV VLIWVIVGALL MGL;SEQ ID NO: 19 IPSSPVHLKR LKLLLLLLLL ILLLILGALL MGL;SEQ ID NO: 20 LRIPCCPVNLKR LLVVWWVL VVVVIVGALL MGL;SEQ ID NO: 21 LLIPCCPVNI KRLLIWWVVL VWVIVGALLM GL;SEQ ID NO: 22 FGIPCCPVHLKR LLIVVVVV VLIWVIVGALL MGL, andpolypeptides having an amino acid sequence with at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 14-22.

[0105] The SP-C polypeptide, in an embodiment, retains the ability to insert into lipid bilayers and form alpha helices, thereby lowering the surface tension, and supporting the formation of lipid microstructures such as liquid ordered and liquid disordered regions.

[0106] In some embodiments, a lung surfactant composition comprising a lung surfactant polypeptide as described herein is administered without the inclusion of an exogenous therapeutic agent. For example, the lung surfactant composition is administered for treating respiratory distress syndrome in preterm infants (RDS), genetic mutations of the SP-B gene, and acute respiratory distress syndrome (ARDS), wherein the composition does not comprise a therapeutic agent, such as a drug or bioactive protein, other than the lung surfactant proteins of the composition.

[0107] In other embodiments, a lung surfactant comprises one or more bioactive and / or therapeutic agents. The bioactive and / or therapeutic agents can be any compound used in the prevention, treatment, or cure of disease, for the relief of pain, or to control or improve any physiological or pathological disorder in humans or animals. Examples of agents include compounds in the following classes: anesthetics, anticonvulsants, antidepressants, antidiabetic agents, antidotes, antiemetics, antihistamines, anti-infective agents, antineoplastics, anti- parkisonian drugs, antirheumatic agents, antipsychotics, anxiolytics, appetite stimulants and suppressants, blood modifiers, cardiovascular agents, central nervous system stimulants, drugs for Alzheimer's disease management, drugs for cystic fibrosis management, drugs for erectile dysfunction, gastrointestinal agents, hormones, drugs for the treatment of alcoholism, drugs for the treatment of addiction, immunosuppressives, mast cell stabilizers, drugs for migraines, motion sickness drugs, drugs for multiple sclerosis management, muscle relaxants, nonsteroidal anti-inflammatories, opioids, analgesics, stimulants, osteoporosis drugs, prostaglandins, respiratory agents, sedatives and hypnotics, smoking cessation aids, Tourette's syndrome agents, urinary tract agents, drugs for metabolic disorders, and vertigo agents.

[0108] In some embodiments, the bioactive and / or therapeutic agent is selected from the group consisting of anti-inflammatory agents, anti-infective agents, biologies, bronchodilation drugs, antihistamines, cyclooxygenase inhibitors, leukotriene antagonists, PLA2 inhibitors, PAF antagonists, analgesics, leukotriene inhibitors or antagonists, decongestants and anti-tussive drug substances, anticholinergics, P-blockers, adrenergic agonists (e.g., ai, a.2, Pi, P2, and P3), anesthetics, anti-tuberculars, cardiovascular agents and combinations thereof. In certainembodiments, the bioactive agent(s) can include, but are not limited to, imaging agents, enzymes, steroids, genetic material, nucleic acid vectors, antisense agents, nucleic acid aptamers, mesenchymal stem cells, CAR-T cells, biologies, proteins, peptides, combinations thereof, and any of the therapeutic agents disclosed in US Patent No. 9,050,267.

[0109] In some embodiments, the bioactive and / or therapeutic agent is an anti-inflammatory agent. Exemplary anti-inflammatory agents for use as therapeutic agents can include, but are not limited to omega-3 poly-unsaturated fatty acids (PUFA) including eicocsapentaenoic (EP A), docosahexaenoic acid (DHA), flunisolide, budesonide, tripedane, cortisone, fluticasone (e.g. propionate), mometasone (e.g. furoate), dexamethasone, beclomethasone, betamethasone, and triamcinolone (e.g. acetonide), adrenaline (ephedrine), fenoterol, formoterol, isoprenaline, metaproterenol, mometasone, prednisone, prednisolone, methyl prednisolone, and triamcinolone. Anti-inflammatory agents can also include nonsteroidal anti-inflammatory drugs (NSAIDs) including, but not limited to, one or more of aspirin, naproxen, acetaminophen, diclofenac, celecoxib, ibuprofen, budesonide, butixocort (tixocortol butyrate), diflunisal, indomethacin, etodolac, ketoprofen, ketorolac, nabumetone, oxaprozin, piroxicam, salsalate, sulindac, and tolmetin.

[0110] In some embodiments, the anti-inflammatory agent is a P2-adrenoreceptor agonist (bronchodilator) including, but not limited to, one or more of: albuterol (aka salbutamol), bitolterol, fenoterol, iosprenaline, levosalbutamol, orciprenaline, pirbuterol, proaterol, ritodrine, salbutamol, terbutaline, arformoterol, bambuterol, clenbuterol, formoterol, salmeterol, abediterol, carmoterol, indacaterol, olodaterol, vilanterol, isoxsuprine, mabuterol, and zilpaterol.

[0111] In some embodiments, the anti-inflammatory agent is a peptide, such as CATH-1 , CATH-2, LL-37, and CRAMP. Peptides, used as small molecule therapeutic agents against disease having an inflammatory component can include, but are not limited to, one or more of CATH-1, CATH-2, LL-37, and CRAMP. A review of therapeutic peptides can be found in La Manna, Sara et al. “Peptides as Therapeutic Agents for Inflammatory-Related Diseases” Int. J. Mol. Sci. (2018) 19(9)2714.

[0112] In some embodiments, the bioactive and / or therapeutic agent is an anti-infective agent, such as an antibiotic, an antibacterial, an antifungal, an antiviral, anti-protozoan, and / or antimicrobial peptide. Antimicrobial peptides as anti-infective bioactive or therapeutic agents are known to have activity against bacteria, viruses, fungi, and unicellular protozoa and can be components of innate immunity to protect the host against infections and are active in pathogen clearance. Reviews of antimicrobial peptides can be found in Mahlaupuu, Margit et al., (2016) Front. Cell. Infect. Microbiol. 27 Dec. 2016.

[0113] In other embodiments, the bioactive and / or therapeutic agent is an anti-cancer agent. Non-limiting examples of anti-cancer drugs that can be used with specific embodiments of the invention include Acivicin; Aclarubicin; Acodazole Hydrochloride; Acronine; Adriamycin; Adozelesin; Aldesleukin; Altretamine; Ambomycin; Ametantrone Acetate; Aminoglutethimide; Amsacrine; Anastrozole; Anthramycin; Asparaginase; Asperlin; Azacitidine; Azetepa; Azotomycin; Batimastat; Benzodepa; Bicalutamide; Bisantrene Hydrochloride; Bisnafide Dimesylate; Bizelesin; Bleomycin Sulfate; Brequinar Sodium; Bropirimine; Busulfan;Cactinomycin; Calusterone; Caracemide; Carbetimer; Carboplatin; Carmustine; Carubicin Hydrochloride; Carzelesin; Cedefingol; Chlorambucil; Cirolemycin; Cisplatin; Cladribine; Crisnatol Mesylate; Cyclophosphamide; Cytarabine; Dacarbazine; Dactinomycin; Daunorubicin Hydrochloride; Decitabine; Dexormaplatin; Dezaguanine; Dezaguanine Mesylate; Diaziquone; Docetaxel; Doxorubicin; Doxorubicin Hydrochloride; Droloxifene; Droloxifene Citrate;Dromostanolone Propionate; Duazomycin; Edatrexate; Eflornithine Hydrochloride; Elsamitrucin; Enloplatin; Enpromate; Epipropidine; Epirubicin Hydrochloride; Erbulozole; Esorubicin Hydrochloride; Estramustine; Estramustine Phosphate Sodium; Etanidazole; Etoposide; Etoposide Phosphate; Etoprine; Fadrozole Hydrochloride; Fazarabine; Fenretinide; Floxuridine; Fludarabine Phosphate; Fluorouracil; Flurocitabine; Fosquidone; Fostriecin Sodium; Gemcitabine; Gemcitabine Hydrochloride; Hydroxyurea; Idarubicin Hydrochloride; Ifosfamide; Ilmofosine; Interferon Alfa-2a; Interferon Alfa-2b; Interferon Alfa-nl; Interferon Alfa-n3; Interferon Beta-I a; Interferon Gamma-I b; Iproplatin; Irinotecan Hydrochloride; Lanreotide Acetate; Letrozole; Leuprolide Acetate; Liarozole Hydrochloride; Lometrexol Sodium; Lomustine; Losoxantrone Hydrochloride; Masoprocol; Maytansine; Mechlorethamine Hydrochloride; Megestrol Acetate; Melengestrol Acetate; Melphalan; Menogaril;Mercaptopurine; Methotrexate; Methotrexate Sodium; Metoprine; Meturedepa; Mitindomide; Mitocarcin; Mitocromin; Mitogillin; Mitomalcin; Mitomycin; Mitosper; Mitotane; Mitoxantrone Hydrochloride; Mycophenolic Acid; Nocodazole; Nogalamycin; Ormaplatin; Oxisuran; Paclitaxel; Pegaspargase; Peliomycin; Pentamustine; Peplomycin Sulfate; Perfosfamide; Pipobroman; Piposulfan; Piroxantrone Hydrochloride; Plicamycin; Plomestane; Porfimer Sodium; Porfiromycin; Prednimustine; Procarbazine Hydrochloride; Puromycin; Puromycin Hydrochloride; Pyrazofurin; Riboprine; Rogletimide; Safingol; Safingol Hydrochloride;Semustine; Simtrazene; Sparfosate Sodium; Sparsomycin; Spirogermanium Hydrochloride; Spiromustine; Spiroplatin; Streptonigrin; Streptozocin; Sulofenur; Talisomycin; Taxol; Tecogalan Sodium; Tegafur; Teloxantrone Hydrochloride; Temoporfin; Teniposide; Teroxirone; Testolactone; Thiamiprine; Thioguanine; Thiotepa; Tiazofuirin; Tirapazamine; TopotecanHydrochloride; Toremifene Citrate; Trestolone Acetate; Triciribine Phosphate; Trimetrexate; Trimetrexate Glucuronate; Triptorelin; Tubulozole Hydrochloride; Uracil Mustard; Uredepa; Vapreotide; Verteporfin; Vinblastine Sulfate; Vincristine Sulfate; Vindesine; Vindesine Sulfate; Vinepidine Sulfate; Vinglycinate Sulfate; Vinleurosine Sulfate; Vinorelbine Tartrate; Vinrosidine Sulfate; Vinzolidine Sulfate; Vorozole; Zeniplatin; Zinostatin; Zorubicin Hydrochloride. Additional antineoplastic agents include those disclosed in Chapter 52, Antineoplastic Agents (Paul Calabresi and Bruce A. Chabner), and the introduction thereto, 1202-1263, of Goodman and Gilman's “The Pharmacological Basis of Therapeutics”, Eighth Edition, 1990, McGraw-Hill, Inc. (Health Professions Division). Non-limiting examples for anti-cancer approved drugs include: abarelix, aldesleukin, aldesleukin, alemtuzumab, alitretinoin, allopurinol, altretamine, amifostine, anastrozole, arsenic trioxide, asparaginase, azacitidine, AZD9291, AZD4547, AZD2281, bevacuzimab, bexarotene, bleomycin, bortezomib, busulfan, calusterone, capecitabine, carboplatin, carmustine, celecoxib, cetuximab, cisplatin, cladribine, clofarabine, cyclophosphamide, cytarabine, dabrafenib, dacarbazine, dactinomycin, actinomycin D, Darbepoetin alfa, Darbepoetin alfa, daunorubicin liposomal, daunorubicin, decitabine, Denileukin diftitox, dexrazoxane, dexrazoxane, docetaxel, doxorubicin, dromostanolone propionate, Elliott's B Solution, epirubicin, Epoetin alfa, erlotinib, estramustine, etoposide, exemestane, Filgrastim, floxuridine, fludarabine, fluorouracil (5-FU), fulvestrant, gefitinib, gemcitabine, gemtuzumab ozogamicin, goserelin acetate, histrelin acetate, hydroxyurea, Ibritumomab Tiuxetan, idarubicin, ifosfamide, imatinib mesylate, interferon alfa 2a, Interferon alfa-2b, irinotecan, lenalidomide, letrozole, leucovorin, Leuprolide Acetate, levamisole, lomustine, CC U, meclorethamine, nitrogen mustard, megestrol acetate, melphalan, L-PAM, mercaptopurine 6- MP, mesna, methotrexate, mitomycin C, mitotane, mitoxantrone, nandrolone phenpropionate, nelarabine, Nofetumomab, Oprelvekin, Oprelvekin, oxaliplatin, paclitaxel, palbociclib palifermin, pamidronate, pegademase, pegaspargase, Pegfilgrastim, pemetrexed disodium, pentostatin, pipobroman, plicamycin mithramycin, porfimer sodium, procarbazine, quinacrine, Rasburicase, Rituximab, sargramostim, sorafenib, streptozocin, sunitinib maleate, tamoxifen, temozolomide, teniposide VM-26, testolactone, thioguanine, thiotepa, thiotepa, topotecan, toremifene, Tositumomab, Trametinib, Trastuzumab, tretinoin (ATRA), Uracil Mustard, valrubicin, vinblastine, vinorelbine, zoledronate and zoledronic acid.

[0114] In other embodiments, the bioactive and / or therapeutic agent is a cardiovascular agent, such as benazepril, captopril, enalapril, quinapril, ramipril, doxazosin, prazosin, clonidine, labetolol, candesartan, irbesartan, losartan, telmisartan, valsartan, disopyramide, flecanide, mexiletine, procainamide, propafenone, quinidine, tocainide, amiodarone, dofetilide, ibutilide,adenosine, gemfibrozil, lovastatin, acebutalol, atenolol, bisoprolol, esmolol, metoprolol, nadolol, pindolol, propranolol, sotalol, diltiazem, nifedipine, verapamil, spironolactone, bumetanide, ethacrynic acid, furosemide, torsemide, amiloride, triamterene, and metolazone.

[0115] In other embodiments, the bioactive and / or therapeutic agent is for treating a metabolic disorder. Examples include diabetes, obesity, Gaucher’s disease, hemochromatosis, phenylketonuria, Niemann- Pick disease, tyrosinemia, etc. Exemplary agents include but are not limited to benzphetamine, bupropion, eliglustat, naltrexone, diethylpropion, imigluycerase, insulin, liraglutide, methamphetamine, miglustat, nitisinone, orlistat, pegvaliase, phendimetrazine, phentermine, sapropterin, semaglutide, taliglucerase alfa, tirzepatide, velaglucerase alfa, and the like.

[0116] In other embodiments, the bioactive and / or therapeutic agent is for treating a neurological disorder, such as Parkinson’s disease, epilepsy, and migraine. In embodiments, the agent is an anti-migraine, an anti-epileptic or an anti-parkinson drug. Exemplary agents include but are not limited to almotriptan, alperopride, amitriptyline, atenolol, carbidopa-levidopa, clonidine, codeine, cyproheptadine, donepezil, dihydroergotamine, diltiazem, doxepin, ergotamine, eletriptan, fluoxetine, frovatriptan, gabapentin, isometheptene, lamotrigine, levetiracetam, lisinopril, lidocaine, lisuride, memantine, methysergide, metoclopramide, metoprolol, naratriptan, nadolol, nortriptyline, oxycodone, oxycarbazepine, paroxetine, pimozide, pizotifen, pizotyline, propanolol, protriptyline, propoxyphene, rizatriptan, sertraline, sumatriptan, timolol, tolfenamic acid, topiramate, verapamil, and zolmitriptan.

[0117] Pulmonary defects, including injuries to the microvasculature when the integrity of the endothelial cell making up these tissues is loosened, may lead to leakage of white and red blood cells, platelets, clotting factors and other components in blood circulation. Therefore, in some embodiments, bioactive or therapeutic agents which upregulate VE-cadherein on the surface of these cells are delivered via a lung surfactant composition. Exemplary bioactive or therapeutic agents include but not limited to focal adhesion kinase (FAK) and compounds that have FAK biologic activity, a nucleic acid vector that is comprised of the gene that codes for FAK, Angiotensin- 1, Angiotensin-1 agonists, Angiotensin-2, Angiotensin-2 antagonists, p38 mitogen activated protein (MAP) Kinase inhibitors, antibodies to MASP-2, and combinations thereof.

[0118] Studies were performed to characterize exemplary lung surfactant compositions prepared with a surfactant polypeptide as described herein, which is an SP-B polypeptide analog. Test compositions were prepared as described in Example 3, with exemplary lipids DPPC and POPG. The compositions comprised the surfactant polypeptide of SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13, which are polypeptide analogs of the native surfactant protein SP-B. Acomparative composition of DPPC and POPG with SP-C lung surfactant polypeptide of SEQ ID NO: 15 was also prepared.

[0119] The compositions were characterized by measuring surface tension, as described in Example 4. Results are shown in FIGS. 1-2. FIG. 1 shows dynamic surface tension, in mN / M, of three compositions with DPPC:POPG and either the SP-B lung surfactant polypeptide analog having the amino acid sequence ofSEQ ID NO: 11 (open squares), the SP-B lung surfactant polypeptide analog having the amino acid sequence ofSEQ ID NO: 12 (inverted triangles), the SP-B lung surfactant polypeptide analog having the amino acid sequence ofSEQ ID NO: 13 (diamonds), or the SP-C lung surfactant polypeptide having the sequence of SEQ ID NO: 15 (closed squares). Also shown is the surface tension of bovine lipid extract surfactant (BLES) (open circles) and of the lipid mixture alone (DPPC:POPG (7:3)) with no lung surfactant protein (closed circles). The data shows the composition with an SP-B polypeptide analog had a lower surface tension relative to a composition of the lipid alone, and that the SP-B polypeptide analogs were essentially as effective to reduce the composition surface tension as the human SP- C polypeptide.

[0120] FIGS. 2A-2D are graphs of minimum surface tension, in mN / m, as a function of repetitive cycles during the constrained sessile drop surfactometry (CDS) testing (as described in Example 4). In the CDS test, a constrained droplet mounted on the stage of a constrained sessile drop surfactometer is repeatedly expanded and compressed. The minimal surface tension of the compressed droplet during each cycle is determined. The compositions in the study were as described above with respect to FIG. 1. FIG. 2A shows the data for the composition of DPPC, POPG and surfactant polypeptide of SEQ ID NO: 11 (an analog of an SP-B lung surfactant), FIG. 2B shows the results for the composition of DPPC, POPG and SP-B lung surfactant polypeptide analog of SEQ ID NO: 12, and FIG. 2C shows the results for the composition of DPPC, POPG and SP-B lung surfactant polypeptide analog of SEQ ID NO: 13. FIG. 2D shows the results for the composition of DPPC, POPG and SP-C lung surfactant polypeptide (SEQ ID NO: 15). Each of FIGS. 2A-2D also show data for the control compositions of DPPC and POPG (7:3) with no lung surfactant protein (open circles) and BLES (squares).III. Methods of Treatment

[0121] In another aspect, a method for delivering a bioactive agent or a therapeutic agent to a subject in need is provided. The method comprises providing (i) a lung surfactant polypeptide for use in preparing a composition, (ii) a composition comprising a surfactant polypeptide, and / or (iii) a lung surfactant composition, where the composition comprises a bioactive or therapeutic agent. For simplicity, the term therapeutic agent is used in this section III. Inembodiments, the method comprises administering and / or instructing to administer a composition comprising a surfactant polypeptide or a lung surfactant composition that comprise a therapeutic agent.

[0122] In one embodiment, a method for treating a lung or respiratory disorder is provided. In an embodiment, the method comprises administering to a subject in need thereof, a lung surfactant composition as described herein. In another embodiment, the method comprises providing (i) a lung surfactant polypeptide for use in preparing a composition, (ii) a composition comprising a surfactant polypeptide, and / or (iii) a lung surfactant composition, where the composition comprises a therapeutic agent for treating the lung or respiratory disorder. Respiratory diseases and disorders for treatment broadly refer to conditions which make gas exchange an obstacle in air-breathing animals. Often there is an underlying pathology within the respiratory system’s organs, e.g., lungs, bronchi, pharynx, larynx, and diaphragm. Conditions of the respiratory tract can include the trachea, bronchi, bronchioles, alveoli, pleurae, pleural cavity as well as the nerves and muscles involved in respiration- the inhalation and exhalation of air and carbon dioxide, respectively. The alveoli make up the respiratory surface where gas exchange occurs by adsorbing oxygen at the air-water interface of alveoli. They are composed of microscopic air sacs that exchange oxygen at the liquid / gas interface on the interior surface of the alveoli with circulating blood which cares inhaled oxygen throughout the body and returns and exchanges carbon dioxide at the water / air interface into the alveoli, back into the lungs for exhalation. Respiratory diseases can be a consequence of blockage within the airway, an obstructive lung disease. A restrictive lung disease results from incomplete lung expansion and lung stiffness as seen in infant respiratory distress syndrome (IRDS). This can be associated with or resulting from deficient and / or dysfunctional lung surfactant production by the patient in need of treatment.

[0123] Respiratory diseases for treatment include, but are not limited to, respiratory illnesses associated with preterm birth, acute respiratory distress syndrome (ARDS), respiratory distress syndrome (RDS), including neonatal, infant and pediatric RDS, mechonium aspiration syndrome (MAS), bronchopulmonary dysplasia (BPD), asthma, chronic obstructive pulmonary disease (COPD), emphysema, idiopathic pulmonary fibrosis (IPF), pulmonary arterial hypertension (PAH), adenocarcinoma, pulmonary injury, infections leading to late onset sepsis (e.g., caused by bacterial, viral, and nosocomial infection sources), cystic fibrosis, and lysosomal storage diseases (LSDs), such as Gaucher disease (GD), Niemann-Pick disease, also known as acid sphingomyelinase deficiency (ASMD), and Fabry disease (FD). Complications of SARS COVID-19 may also benefit from the disclosed composition for use within a combinationtreatment therapeutic composition or for delivery of therapeutics alone or in combination. A detailed description of respiratory infections and diseases can be found in Dasaraju and Liu, Infections of the Respiratory System. In: Medical Microbiology, 4th ed. S. Baron et al., CoEditor, 1996, U. Texas Med. Branch, Galveston, TX. ISBN 0963117211 and in Matthay, M.A. et al., “Acute respiratory distress syndrome, Nat. Rev. Disease Primers 5, Article No. 18 (2019), each of which are incorporated by reference herein.

[0124] Neonatal, infant, and adult respiratory distress syndromes can result from and include but are not limited to acute lung injury (ALI), ventilator-induced lung injury (VILI), and systemic inflammatory response syndrome (SIRS). Moreover, ARDS can result from one or more causes including, but not limited to, shock, bacterial, viral, and nosocomial pneumonias, and inhalation of toxic gases, vapors, fumes, and particles.

[0125] Pulmonary injury can be a consequence of bronco-biopsy, mechanical ventilation, bronchopulmonary dysplasia, late onset sepsis, administration of oxygen, aspiration, inhalation of toxic gases, vapors, fumes and particles, and intubation. Pulmonary injury can also occur due to pancreatitis, transfusion associated acute lung injury, drug overdose with various agents, near drowning (inhalation of fresh or salt water), hemorrhagic shock or reperfusion injury, smoke inhalation, as well as aspiration of gastric contents into the lungs, poor sanitation, an infected wound, and inadvertent and / or accidental inhalation of chemical vapors, particulates, smoke and fumes.

[0126] Injury of the microvasculature can lead to leakage of white and red blood cells, platelets, clotting factors and other components in blood circulation. Therefore, the microvasculature of any organ, such as a lungs, will be adversely affected when the integrity of the endothelial cells underlying these tissues is loosened. In some embodiments, this can be countered by compounds that lead to upregulation of VE-cadherin on the surface of these cells by compounds such as focal adhesion kinase (FAK), angiopoietin- 1 , small GTPases, intracellular modulators, catenins, plakoglobin and VE-protein tyrosine phosphatase. In addition to acute respiratory distress syndrome, the resulting morbidities include microvascular coronary disease (stroke and myocardial infarction), acute and ventilator-induced lung injury, sepsis, pancreatitis, cerebral small-vessel disease, preeclampsia, pulmonary arterial hypertension (PAH), endothelial dysfunction in diabetes, diabetic cardiomyopathy, rheumatoid arthritis, systemic lupus erythematosus, asthma, neoplasms, diabetic retinopathy or age-related macular degeneration, and systemic sclerosis, may have a common etiologic linkage related to microvascular disease.

[0127] In addition, in patient with systemic capillary leak syndrome, characterized by arterial hypotension, hemoconcentration and low albumin levels with hypotensive shock and anasarca,pro-inflammatory and endothelial mediators are significantly increased, thereby necessitating prompt treatment.

[0128] In another embodiment, a method for treating lung tissue is provided. The method comprises exposing or contacting the lung tissue with a composition comprising a polypeptide or surfactant polypeptide and / or a lung surfactant composition. In an embodiment, the lung surfactant composition comprises a therapeutic agent. In an embodiment, the lung tissue is a lung, such as a lung intended for transplantation. In an embodiment, the exposing or contacting is ex vivo. In an embodiment, the method comprises obtaining or providing the lung tissue and / or the polypeptide or surfactant polypeptide and / or the lung surfactant composition. In an embodiment, the method comprises transporting the lung tissue while in contact or while being exposed to the polypeptide or surfactant polypeptide and / or the lung surfactant composition. The therapeutic agent, if present, can be any of the therapeutic agents described herein or known in the art to be suitable for treating a such a subject.

[0129] In another embodiment, a method for treating cancer comprises providing (i) a lung surfactant polypeptide for use in preparing a composition, (ii) a composition comprising a surfactant polypeptide, and / or (iii) a lung surfactant composition, where the composition comprises a therapeutic agent for treating cancer. In an embodiment, the method comprises administering or instructing to administer the composition or lung surfactant composition to a subject in need. Cancer is a physiological condition in mammals that is typically characterized by unregulated cell growth. The cancer may be a solid tumor, a metastasis, a blood cancer, and / or a solid pre-cancer. Examples of cancer include but are not limited to, carcinoma, blastoma, sarcoma and lymphoma, with specific cancers exemplified by squamous cell cancer, lung cancer (including small-cell lung cancer, non-small-cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung), glioma, melanoma cancer, cancer of the peritoneum, hepatocellular cancer, gastric, gastro esophageal or stomach cancer (including gastrointestinal cancer), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, soft tissue sarcoma, kidney or renal cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, Kaposi's sarcoma carcinoid carcinoma, and various types of head and neck cancer. A composition or lung surfactant composition comprising a therapeutic agent for treating cancer may comprise, for example, an anti-cancer agent, including but not limited to the agents described herein, or other therapeutic agents used in the treatment of subjects with cancer.

[0130] In another embodiment, a method for treating a metabolic disorder comprises providing (i) a lung surfactant polypeptide for use in preparing a composition, (ii) a composition comprising a surfactant polypeptide, and / or (iii) a lung surfactant composition, where the composition comprises a therapeutic agent for treating the metabolic disorder. In an embodiment, the method comprises administering or instructing to administer the composition or lung surfactant composition to a subject in need. In an embodiment, the metabolic disorder is diabetes, obesity, Gaucher’s disease, hemochromatosis, phenylketonuria, Niemann-Pick disease, tyrosinemia, or the like. In an embodiment, the therapeutic agent for treating the metabolic disorder is any one or more of the agents identified herein or known to a skilled artisan.

[0131] In another embodiment, a method for treating a cardiovascular disorder, disease or condition comprises providing (i) a lung surfactant polypeptide for use in preparing a composition, (ii) a composition comprising a surfactant polypeptide, and / or (iii) a lung surfactant composition, where the composition comprises a therapeutic agent for treating the cardiovascular disorder, disease or condition. In an embodiment, the method comprises administering or instructing to administer the composition or lung surfactant composition to a subject in need. In an embodiment, the cardiovascular disorder, disease or condition is an abnormal heart rhythm, cardiomyopathy, heart valve disease, pericardial disease, coronary artery disease, heart failure, or the like. In an embodiment, the therapeutic agent for treating the cardiovascular disorder, disease or condition is any one or more of the agents identified herein or known to a skilled artisan.

[0132] In another embodiment, a method for treating a neurological disorder comprises providing (i) a lung surfactant polypeptide for use in preparing a composition, (ii) a composition comprising a surfactant polypeptide, and / or (iii) a lung surfactant composition, where the composition comprises a therapeutic agent for treating the neurological disorder. In an embodiment, the method comprises administering or instructing to administer the composition or lung surfactant composition to a subject in need. In an embodiment, the neurological disorder is, for example, depression, Parkinson’s disease, epilepsy, or migraine.

[0133] Subjects for treatment may include neonates, infants, children, and adults, human or nonhuman.

[0134] In various embodiments, lung surfactant compositions described herein can be made by forming lung surfactant protein-lipid complexes comprising one or more of the SP-B lung surfactant polypeptide analogs described herein (monomer or dimer) in combination with one or more excipients, optionally in further combination with SP-C lung surfactant polypeptide or an SP-C lung surfactant polypeptide analog. Exemplary SP-B polypeptides for use as monomers ordimers may include SP-B lung surfactant polypeptide analogs having an amino acid sequence set forth in any one of SEQ ID NOs: 1-10. Exemplary SP-C lung surfactant polypeptides for use in combination with the SP-B lung surfactant polypeptide analogs may include amino acid sequence set forth in any one of SEQ ID NOs: 14-22.

[0135] Lung surfactant compositions may be prepared by mixing a solution or a suspension of one or more lung surfactant polypeptides, lipids, excipients, and / or therapeutic agents; and forming an emulsion or suspension. The resulting emulsion or suspension may be subsequently dried, e.g., by lyophilization or spray-drying into an amorphous powder. As noted above, the lung surfactant compositions for treatment may further include cholesterol and / or a variety of excipients. In some embodiments, one or more therapeutic agents can be added either prior to or after formation of the emulsion and / or suspension. The amorphous powder may be reconstituted in a suitable liquids and solution described herein to facilitate appropriate dosing concentrations (in mg / kg) and dosage volumes of lung surfactant protein (and optionally SP-Cs and / or other therapeutic agents), can be adjusted based on the age, weight, and overall health of the subject (e.g., mammal, human) to be treated.

[0136] Lung surfactant compositions containing the aforementioned components may be prepared or reconstituted in fixed amounts or quantitative ratios relative to the overall composition. For example, the weight ratios of SPs and lipids may vary in composition, amounts, and chemical nature. For example, in some embodiments, the surfactant proteins may be present in in an amount of between about 0.01-5 wt.%, 0.01-3 wt.%, 0.01-2 wt.%, 0.1-5 wt.%, 0.1-3 wt.%, 0.1-2 wt.%, 0.1-1 wt.%, 0.2-5 wt.%, 0.2-3 wt.%, 0.2-2 wt.%, or 0.2-lwt %, 0.5-5 wt.%, 0.5-3 wt.%, 0.5-2 wt.%, 0.5-1 wt.%, 1-5 wt.%, 1-3 wt.%, or 1-2 wt. %, relative to the total weight of the composition or lipid complex. Further, in certain embodiments, the surfactant polypeptides may be present in oxidized form.

[0137] Similarly, the weight ratios of the SP-B analog(s) (or SP-Bs) to SP-C or SP-C analogs (hereinafter collectively as “SP-C analogs or SP-Cs”) can vary. In some embodiments, the SP- Bs may be present in the composition or lipid complex in an amount of between about 0.01 -5 wt.%, 0.01-3 wt.%, 0.01-2 wt.%, 0.1-5 wt.%, 0.1-3 wt.%, 0.1-2 wt.%, 0.1-1 wt.%, 0.2-5 wt.%, 0.2-3 wt.%, 0.2-2 wt.%, or 0.2-lwt %, 0.5-5 wt.%, 0.5-3 wt.%, 0.5-2 wt.%, 0.5-1 wt.%, 1-5 wt.%, 1-3 wt.%, or 1-2 wt. %, relative to the total weight of components in the composition and / or relative to the total weight of SP-C in the composition.

[0138] The weight ratio of surfactant proteins to lipids can be adjusted and to facilitate ease of spreading of the lung surfactant composition within the alveoli. Naturally occurring lung surfactant compositions contain about 90% lipid and 10% protein. However, lipid and surfactantprotein selection and amino acid sequence optimization can help to improve penetration of the lipid(s) into the alveoli’s lipid bi-layer and can facilitate gas exchanges by adsorbing oxygen at the air-water interface of alveoli. Further, the addition of an anti-inflammatory and / or anti- infective agents can preclude degradation of surfactant proteins.

[0139] In some embodiments, weight ratio of lipid to SP-B or SP-B / Cs in the lung surfactant composition can range from 8.0:0.10, 8.0:0.25, 8.0:05.0, 8.0:0.75, 8.0:1.0, 8.0:1.25, 8.0:1.50, 8.0:1.75, 9.0:0.10, 9.0:0.25, 9.0:05.0, 9.0:0.75, 9.0:1.0, 9.0:1.25, 9.0: 1.50, 9.0:1.75, 10.0:0.10, 10.0:0.25, 10.0:05.0, 10.0:0.75, 10.0: 1.0, 10.0: 1.25, 10.0: 1.50, 10.0:1.75, 11.0:0.10, 11.0:0.25, 11.0:05.0, 11.0:0.75, 11.0: 1.0, 11.0:1.25, 11.0: 1.50, 11.0: 1.75, 12.0: 0.10, 12.0:0.25, 12.0:05.0, 12.0:0.75, 12.0: 1.0, 12.0:1.25, 12.0: 1.50, 12.0:1.75, 13.0:0.10 and any weight ratios or weight ratio ranges derivable from the aforementioned values.

[0140] Phospholipids, such as DPPC, in conjunction with another lipid can improve uniformity of coverage as well as ability of the lung surfactant composition to penetrate into the deep recesses of the lung’s alveoli. Drying a lung surfactant composition into an amorphous powder that can be resuspended prior to use can restore the ability of the lung surfactant composition to spread over the lung and alveoli surfaces without the loss of the ability to alter surface tension and can facilitate transfer of lipid(s) between lipid bilayers and other lipid structures.

[0141] The lung surfactant compositions may be prepared according to conventional techniques known in the pharmaceutical industry. In some embodiments, these techniques include the step of e.g., admixing a lung surfactant polypeptide and a phospholipid(s) or other lipid with an organic solvent. The solvent is then removed by dialysis, evaporation , exposure to vacuum, and / or by other appropriate technique well known to the skilled person in the art, such as lyophilization and spray-drying. The obtained powder is then uniformly and intimately brought into association with liquid carriers or finely divided solid carriers or both. Methods for preparing dry powder formulations comprising spray-dried particles for inhalation are disclosed in U.S. Patent No. 9,050,267, the disclosure of which is incorporated by reference herein.

[0142] In certain embodiments, the viscosity of the lung surfactant composition is less than 20 centiPoises (cP), preferably less than 15 cP, at 25 °C. Viscosity measurements can be made using viscometer according to methods known in the art.

[0143] The lung surfactant compositions may be concentrated or dilute with respect to the concentration of lung surfactant polypeptide in the composition. Concentrated lung surfactant compositions are typically used for “bolus” type administrations. For “bolus” type administrations, the concentration of lung surfactant polypeptide in the lung surfactant composition, often in the form of a solution or suspension, is in the range of 5-200 mg / mL, 5-100 mg / mL, 25-200 mg / mL, 25-150 mg / mL, 20-125 mg / mL, 25-80 mg / mL, or 20-80 mg / mL. In one embodiment, where the lung surfactant composition is to be administered by intratracheal instillation as a suspension in physiological saline (0.9% w / v sodium chloride in water), the concentration of lung surfactant polypeptide can be about 80 mg / mL or between about 50-150 mg / mL.

[0144] Dilute lung surfactant compositions are typically used for “lavage” type administrations. When used for lavage administration, a typical lung surfactant polypeptide concentration is in a range of about 0.1-25 mg / mL, 0.1 to 20 mg / mL, 0.5-25 mg / mL, 0.5-20 mg / Ml or 0.5 to 10 mg / mL (in terms of mg lung surfactant polypeptide per mL of solution or suspension). Because of the more diluted nature, the viscosity of these compositions would be lower.

[0145] In certain embodiments, the lung surfactant composition is administered in the form of a solution, dispersion, suspension, or dry powder. In some embodiments, dried compositions comprising the lung surfactant polypeptides are dissolved or suspended in a suitable physiologically tolerable solvent or re-suspension carrier, such as water or a physiological saline aqueous solution (0.9% w / v NaCl) are generally sterile and may additionally include pH buffering agents, diluents, and other suitable additives. The mixture of lung surfactant polypeptides, phospholipids and other excipients can be sterilized before removing the solvent for example by sterile filtration. In certain other embodiments, the reconstituted surfactant composition is terminally sterilized according to methods well known in the art.

[0146] In certain embodiments, the lung surfactant composition is a suspension in a buffered physiological saline aqueous solution in a single-use vial. In certain embodiments, the lung surfactant polypeptide concentration in the lung surfactant composition may range from between about 0.1-250 mg / mL, 0.5-200 mg / mL, 1-200 mg / mL, 5-200 mg / mL, 10-150 mg / mL, 10-125 mg / mL, 10-100 mg / mL, 20-150 mg / mL, 20-125 mg / mL, 20-100 mg / mL, 20-90 mg / mL, 25-150 mg / mL, 25-125 mg / mL, 25-100 mg / mL, 25-90 mg / mL or 25-80 mg / mL. The lung surfactant composition may further comprise electrolytes, such as calcium, magnesium, and / or calcium salts, including e.g., calcium chloride.

[0147] A variety of methods for administering the lung surfactant compositions are available and known to those of skill in the art. Depending on the disease and the subject, e.g., an infant with respiratory distress syndrome or an adult with a neurological or cardiovascular condition, different treatment methods can be appropriate. In some embodiments, the lung surfactant composition is administered via intratracheal instillation (as an infusion or bolus). In some embodiments, the lung surfactant composition is administered via nebulization or aerosolization. In some embodiments, the lung surfactant composition is in dry form, which can beadministered with an inhaler (e.g. a dry powder inhaler). In some embodiments, the lung surfactant composition is administered intranasally.

[0148] Effective doses of the lung surfactant polypeptide and optionally of a therapeutic agent, in the lung surfactant compositions may vary depending on a range of factors, including type of the disease, means of administration, weight of the subject, and physiological state of the subject, and whether treatment is prophylactic or therapeutic. In general, a dose is from 0.01 mg to 10 g per kg of body weight, preferably from 0.1 to 1 g per kg of body weight and the frequency of administration can vary depending on whether the treatment is prophylactic or therapeutic. Typically, a dose of about 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, 10 mg / kg, 12 mg / kg, 15 mg / kg, 20 mg / kg, 25 mg / kg, 35 mg / kg, 50 mg / kg, 100 mg / kg, 200 mg / kg, or of a dose in a range between any two of these recited values (e.g. 5-200 mg / kg, 5-12 mg / kg, 8-35 mg / kg, 10-200 mg / kg, 10-100 mg / kg, etc), is administered in one dose. For use in newborns, one or two administrations of a dose of any of these recited values or of a dose in a range between any two of the recited values are generally sufficient.

[0149] In an embodiment, the lung surfactant composition is administered by endotracheal instillation to patients (e.g., pre-term infants) kept under continuous or intermittent positive pressure ventilation (IPPV). Alternatively, the lung surfactant composition may be administered using a thin catheter placed in the trachea and the patient respiration supported with specially designed nasal devices, such as masks, prongs, or tubes according to methodology known as nasal Continuous Positive Airway Pressure (nCPAP). Such an approach would be only possible with a lung surfactant composition having low viscosity, since a high viscosity would make the passage of the surfactant through the thin catheter more difficult.

[0150] In instances where the patient suffers from a respiratory distress condition associated with pulmonary inflammation, pulmonary infection or pulmonary contusion, particular treatment modalities can be recommended. In one such therapeutic method, lavage of the patient’s lungs with a lung surfactant composition is performed over a single or multiple treatments.

[0151] The lung surfactant compositions are suitable to prevent, delay, alleviate, arrest, or inhibit development of the symptoms or conditions associated with a respiratory disease or disorder.

[0152] In embodiments, the pharmaceutical lung surfactant compositions are useful for the prophylaxis and / or treatment of a variety of lung or respiratory disorders, including but not limited to respiratory distress syndrome (RDS) in prematurely born babies, RDS in adults (ARDS), meconium aspiration syndrome (MAS), bronchopulmonary dysplasia (BPD), otherdiseases related to a surfactant-deficiency or dysfunction, such acute lung injury (ALI), chronic obstructive pulmonary disease (COPD), asthma, respiratory infections (e.g. pneumonia, pneumocystis carinii, cystic fibrosis and respiratory syncytial virus) as well as for the treatment of serous otitis media (glue ear).

[0153] In a further aspect, provided is a kit comprising: a) a preparation comprising a lung surfactant polypeptide as described herein, optionally an SP-C polypeptide, in a powder form in a first unit dosage form; b) a pharmaceutically acceptable carrier, such as a sterile aqueous physiological saline solution, in a second unit dosage form; and c) container means for containing the first and second unit dosage forms.

[0154] It will be appreciated that the lung surfactant compositions may comprise a polypeptide or surfactant polypeptide in monomer form, in dimer form, or a mixture thereof. Exemplary dimers are illustrated in FIGS. 3A-3C where a homodimer of SEQ ID NO: 12 is shown in FIG. 3A, a homodimer of SEQ ID NO: 13 is shown in FIG. 3B and a heterodimer of SEQ ID NO: 12 and SEQ ID NO: 13 is shown in FIG. 3C. It will be appreciated that homodimers and heterodimers of any of the polypeptides or surfactant polypeptides are contemplated. In some embodiments, the dimers is formed of two monomers, each having, independently, an amino acid sequence set forth in any one or SEQ ID NOs: 1-13. One or both of the monomers can have C-terminal end cap, an N-terminal end cap, or both. In some embodiments, the N-terminal cap is NH2, as illustrated in the exemplary dimers of FIGS. 3A-3C. The dimers can also have one or more internal disulfide bridges, as illustrated in the exemplary dimers of FIGS. 3A-3C, in addition to one or more interchain disulfide bridges that form the dimer. Reaction schemes for dimer formation are described below.IV. Methods of Manufacture

[0155] In another aspect, a method for manufacture of a lung surfactant polypeptide comprising an amino acid sequence of SEQ ID NO: 1 or an amino acid sequence of at least about 90% sequence identity to SEQ ID NO: 1 is provided, where Xi, X2, X5 and X& are, independently, selected from the group consisting of L-threonine, D-threonine, L-tyrosine, D-tyrosine, L- alanine, D-alanine, L-leucine, D-leucine, L-aspartic acid, D-aspartic acid, L-glutamic acid, D- glutamic acid, L-cysteine, D-cysteine, valine, serine, threonine, glycine, norvaline, and 2- aminoisobutyric acid, and where X3 and X4 are, independently, an amino acid that is non- oxidizable. The method comprises reacting the lung surfactant polypeptide under conditions suitable for forming one or more internal disulfide bridges in a monomer and / or reacting a second lung surfactant polypeptide under conditions suitable for forming one or more interchaindisulfide bridges resulting in formation of a dimer. In embodiments, the lung surfactant polypeptide is synthetic and / or is synthetically or recombinantly produced.

[0156] In some embodiments, the lung surfactant polypeptide comprises one or more protected and / or unprotected amino acid residues where the method comprises the step of reacting the lung polypeptide with protected and unprotected groups one or more reagents under conditions suitable for forming a first internal disulfide bridge; and reacting the lung surfactant polypeptide with one or more reagents under conditions suitable for forming a second internal disulfide bridge and retaining one or more protecting groups in the lung surfactant polypeptide.

[0157] In one embodiment, the method comprises providing a composition that comprises a surfactant lung polypeptide with an amino acid sequence of SEQ ID NO: 10, where Xi, X2, X5 and X<, are, independently, A or C, and X3 and X4 are, independently, A or C, and X3 and X4 are Nle, and reacting the lung surfactant polypeptide with one or more reagents under conditions suitable for forming one or more internal disulfide bridges.

[0158] In a further aspect, provided herein is a method for manufacture of a lung surfactant polypeptide dimer comprises reacting a lung surfactant polypeptide monomer comprising an amino acid sequence of SEQ ID NO: 10 and one or more internal disulfide bridges and reacting the lung surfactant polypeptide monomer with a second lung surfactant polypeptide monomer comprising an amino acid sequence of SEQ ID NO: 10 and one or more internal disulfide bridges with one or more reagents under conditions suitable for forming a dimer. In embodiments, the lung surfactant polypeptide is synthetic and / or is synthetically or recombinantly produced.

[0159] In another embodiment, the method comprises providing a lung surfactant polypeptide monomer where amino acid residues Cl l and C71 are unprotected and amino acid residues C8, C35, C46, C48 and C77 are protected, and reacting the lung surfactant polypeptide monomer under conditions suitable for forming a C11-C71 disulfide bridge; and reacting the lung surfactant polypeptide monomer containing the Cl 1-C71 disulfide bridge with an oxidizing agent to form a C8-C77 disulfide bridge and a C35-C46 disulfide bridge, while retaining a protecting group at C48.

[0160] In another embodiment, the method comprises providing a lung surfactant polypeptide monomer where amino acid residues Cl 1 and C71 are unprotected and amino acid residues C8, C35, C46, C48 and C77 are protected, and reacting the lung surfactant polypeptide monomer with an oxidizing agent to form a C11-C71 disulfide bridge, a C8-C77 disulfide bridge, and a C35-C46 disulfide bridge, while retaining a protecting group at C48.

[0161] In some embodiments, the method further comprises the step of reacting two lung surfactant polypeptide monomers to remove the protecting groups at C48 to thereby form a lung surfactant polypeptide dimer. In an embodiment, one or both of the lung surfactant polypeptide monomers is synthetic and / or is synthetically produced and / or the dimer is synthetic.

[0162] In some embodiments, one or more steps is done under microwave irradiation to accelerate the reaction. In an embodiment, the step of removing the protecting group at C48 with trifluoroacetic acid (TFA) is done under microwave irradiation.

[0163] In some embodiments, the protecting group at amino acid residues C8, C35, C46 and C77 is different from the protecting group at C48.

[0164] In some embodiments, the protecting group at amino acid residues C8, C35, C46 and C77 is acetamidomethyl (Acm) or trityl (trt) or a combination thereof.

[0165] In some embodiments, the protecting group at amino acid residue C48 is tert-butyl (tBu).

[0166] In some embodiments, the oxidizing reagent is selected from the group consisting of iodine, Hg(II) HBr / AcOH, TFA / TIS (90:10), HBF4 / scavengers, CuSO4-cysteamine, and guanidinium (Gdm)-HCl / HEPPS buffer (pH 7.3).

[0167] In some embodiments, the reagents for forming the disulfide bridges include NH3.

[0168] In some embodiments, the method further comprises the step of drying or lyophilizing the polypeptide.

[0169] In an exemplary synthesis methodology described in the Example 1 and in FIGS. 4A- 4H, the polypeptide represented by SEQ ID NO: 23 and / or SEQ ID NO: 33 (FIG. 4A) serves as a starting material for preparing a lung surfactant polypeptide with internal disulfide bridges. In this method, a first internal disulfide bridge is formed at residue positions Cl 1-C71, then at C8- C77 and C35-C48, and then optionally followed by interchain C-C bridge formation at C48 to form a dimer.

[0170] The polypeptide represented by SEQ ID NO: 24 (or SEQ ID NO: 35 (FIG. 4A)) shows the polypeptide of SEQ ID NO: 23 (or SEQ ID NOs: 33 and 34, FIG. 4A) after oxidation (e.g., with I2), resulting in formation of 3 disulfide bridges, C 11 -C71 , C8-C77, and C35-C46.

[0171] The polypeptide represented by SEQ ID NO: 28 (or SEQ ID NO: 36 (FIG. 5A)) show an exemplary polypeptide starting material for an alternative protection strategy described in Example 2, FIGS. 5A-5C.V. Examples

[0172] The following examples are illustrative in nature and are in no way intended to be limiting.EXAMPLE 1PREPARATION OF SYNTHETIC LUNG SURFACTANT POLYPEPTIDE

[0173] With reference to FIG. 4A, 1 mg of starting material, purified protein (SEQ ID NO: 23 or SEQ ID NO: 33) was dissolved in a solution of khO / AcN 3:7 (1 mL) followed by addition of ammonia to pH~8. After 6 hours ultra performance liquid chromatography (UPLC) shows the completion of the reaction and formation of Cysl 1-Cys71 disulfide bridge (FIGS. 4B-4D).

[0174] To the reaction mixture, 10% of TFA was added, followed by the addition of 10 eq. of L dissolved in methanol. After 30 minutes the reaction was complete. UPLC (FIGS. 4E-4H) shows the presence of a peak with a mass consistent with a protein with 3 disulfide bridges (Cys8-Cys77 disulfide bridge, Cysl 1-Cys71 disulfide bridge, and a C35-C46 disulfide bridge), while retaining a protecting group at C48 (Cys48tBu).EXAMPLE 2PREPARATION OF SYNTHETIC LUNG SURFACTANT POLYPEPTIDE

[0175] With reference to FIG. 5A, a stepwise synthesis of a synthetic lung surfactant protein was prepared by starting with the polypeptide of SEQ ID NO: 28 or SEQ ID NO: 36. The starting material comprised a pseudoproline Fmoc-Asp(OtBu)-Thr(psiMe,Mepro)-OH as indicated to reduce aspartimide formation and aggregation. 400 micromolar of protide rink amide resin (loading 0.20 mmol / g) was used. Double couplings were on amino acid residues at positions 21Nle to A42 and at S54 to P66 and at C71 to R76 (underlined in FIG. 5A), and Pseudoproline Asp-Thr. Cleavage was effected by Mixture K (87,5% TFA, 5% Phenol, 5% Water, 5% Thioanisole, 2,5% EDT): Recovered 1.9 g Yield 60%

[0176] Crude material was stirred overnight in a solution of ILO / AcN + 0.1% Formic acid to remove TFA adduct. UPLC scans are shown in FIGS. 5B-5C.EXAMPLE 3PREPARATION OF A COMPOSITION COMPRISING A SYNTHETIC LUNG SURFACTANT POLYPEPTIDE

[0177] A composition comprising DPPC, POPG and an SP-B analog was prepared for in vitro studies. Stock solutions of DPPC (40 mg / mL), POPG (20 mg / mL), and cholesterol (5 mg / mL), each in chloroforrmmethanol (1:1) were prepared. A stock solution of an SP-B analog (1 mg / mL) in distilled water was prepared.

[0178] From the stock solutions, compounds soluble in chloroform methanol were first mixed together in a test tube to form a lipid mixture with 17.5 pL DPPC (7 mg), 15 pL POPG (3 mg), and 10 pL cholesterol (0.5 mg). Then, SP-B from the SP-B stock solution (20 pg; 20 pL) was added to the lipid mixture. The lipid / protein mixture was dried manually by flowing nitrogen gas through a glass pipet while rotating the test tube. This resulted in the formation of a shell onthe inner surface of the test tube. The dried material was then then placed in a vacuum desiccator for additional drying.

[0179] Prior to use, glass beads were added to the test tube and 0.5 mb of resupension buffer (2.5 mM Hepes, 1.5 mM CaCh, and 140 mM NaCl at pH of 7.4) was added. Samples were resuspended by vortexing.EXAMPLE 4ANALYSIS OF COMPOSITIONS COMPRISING A SYNTHETIC LUNG SURFACTANT POLYPEPTIDE

[0180] Compositions comprising dipalmitoylphosphatidylcholine (DPPC), palmitoyloleoylphosphatidylglycerol (POPG), and an SP-B analog or a SP-C protein were prepared as described in Example 3. Three different compositions with DPPC:POPG (7:3) and an SP-B analog were prepared, where the SP-B lung surfactant polypeptide analog was SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13. For comparison, a composition with DPPC:POPG (7:3) and the SP-C protein of SEQ ID NO: 15 was prepared. Each composition comprised 2 wt.% of the SP-B or SP-C protein in 2 mg / mL lipid. As a negative control, a composition with DPPC:POPG (7:3) was prepared. As a positive control bovine lipid extract surfactant (BLES) was used.

[0181] The surface tension behavior of the compositions was characterized using constrained sessile drop surfactometry (CDS). In this method, droplet surface tension was measured continuously during expansion and contraction. CDS measurements were conducted by loading a 10 pL of each test composition onto the drop pedestal and, using an external stepper motor which controls the motion of a capillary that adds or relieves compression on the droplet. The sharp edges of the pedestal allow for compression to low surface tension. Each droplet was subjected to 20 cycles of compression and expansion at a rate of 5 seconds / cycle and a compression ratio of approximately 27%. Surface tension was determined by shape-analysis of the droplets. Images of the droplets were recorded at a rate of one image per second and analyzed. The image with the lowest surface tension throughout each cycle was determined to be the minimum surface tension (MST) for that cycle. Analyses were repeated in triplicate.Results are shown in FIGS. 1-2.EXAMPLE 5METHOD OF TREATMENT

[0182] An infant presenting with respiratory distress is treated by administering to the nasal cavity a surfactant composition as described herein at a dose of about 100 mg / kg. In an embodiment, 15 pL is provided to the nasal cavity.EXAMPLE 6METHOD OF TREATMENT WITH A LUNG SURFACTANT COMPOSITION COMPRISING AN ANTI-INFLAMMATORY THERAPEUTIC AGENT

[0183] A lung surfactant composition was prepared as described in Example 3 with the SP-B lung surfactant polypeptide analog of SEQ ID NO: 11 and with albuterol. The lung surfactant composition is administered by inhalation to provide a therapeutic dose of albuterol to a subject.EXAMPLE 7METHOD OF TREATING A NEUROLOGICAL DISORDER

[0184] A lung surfactant composition was prepared as described in Example 3 with the SP-B lung surfactant polypeptide analog of SEQ ID NO: 11 and with sumatriptan. The lung surfactant composition is administered by inhalation to provide a therapeutic dose of sumatriptan to a subject.EXAMPLE 8METHOD OF TREATING A METABOLIC DISORDER

[0185] A lung surfactant composition was prepared as described in Example 3 with the SP-B lung surfactant polypeptide analog of SEQ ID NO: 11 and with semaglutide. The lung surfactant composition is administered by inhalation to provide a therapeutic dose of semaglutide to a subject.

[0186] While a number of exemplary aspects and embodiments have been discussed above, those of skill in the art will recognize certain modifications, permutations, additions, and subcombinations thereof. It is therefore intended that the following appended claims and claims hereafter introduced are interpreted to include all such modifications, permutations, additions, and sub-combinations as are within their true spirit and scope.

Claims

IT IS CLAIMED:

1. A polypeptide, comprising: a polypeptide with an amino acid sequence of SEQ ID NO: 1 or an amino acid sequence of at least about 90% sequence identity to SEQ ID NO: 1, whereinXi, X2, X5 and Xe are, independently, selected from the group consisting of L-threonine, D- threonine, L-tyrosine, D-tyrosine, L-alanine, D-alanine, L-leucine, D-leucine, L-aspartic acid, D-aspartic acid, L-glutamic acid, D-glutamic acid, L-cysteine, D-cysteine, valine, serine, threonine, glycine, norvaline, and 2-aminoisobutyric acid, andX3 and X4 are, independently, an amino acid, natural or unnatural, standard or non-standard, that is non-oxidizable.

2. The polypeptide of claim 1, wherein Xi, X2, X5 and Xe are, independently, selected from the group consisting of L-cysteine, D-cysteine, L-alanine, D-alanine, serine, threonine and 2- aminoisobutyric acid.

3. The polypeptide of claim 1, wherein Xi, X2, X5 and Xe are, independently, selected from the group consisting of 2-aminoisobutyric acid, norvaline, serine, valine, L-cysteine, and L- alanine.

4. The polypeptide of any one of claims 1-3, wherein X3 and X4 are, independently, any amino acid, D or L, natural or unnatural, other than cysteine, cystine, histidine, methionine, tryptophan and tyrosine.

5. The polypeptide of any one of claims 1-3, wherein X3 and X4 are, independently, selected from the group consisting of alanine, valine, leucine, isoleucine, phenylalanine, norleucine, norvaline.

6. The polypeptide of any one of claims 1-5, wherein the polypeptide is a dimer.

7. The polypeptide of claim 6, wherein the dimer is a homodimer or a heterodimer.

8. A polypeptide, comprising: a dimer of a synthetic polypeptide with an amino acid sequence of SEQ ID NO: 10 or an amino acid sequence of at least about 90% sequence identity to SEQ ID NO: 10, whereinXi, X2, X5 and Xe are, independently, A or C, andX3 and X4 are Nle.

9. A polypeptide, comprising: a dimer of a synthetic polypeptide with an amino acid sequence of SEQ ID NO: 11 or an amino acid sequence of at least about 90% sequence identity to SEQ ID NO: 11.

10. A polypeptide, comprising: a dimer of a synthetic polypeptide with an amino acid sequence of SEQ ID NO: 12 or an amino acid sequence of at least about 90% sequence identity to SEQ ID NO: 12.

11. A polypeptide, comprising: a dimer of a synthetic polypeptide with an amino acid sequence of SEQ ID NO: 13 or an amino acid sequence of at least about 90% sequence identity to SEQ ID NO: 13.

12. The polypeptide of any one of claims 8-11, wherein the dimer is a homodimer or a heterodimer.

13. The polypeptide of any one of claims 1-12, wherein the synthetic polypeptide has a C- terminal end cap, an N-terminal end cap, or both.

14. The polypeptide of claim 13, wherein the end cap is NEL.ta15. The polypeptide of any one of claims 1-14, wherein the polypeptide has one or more internal disulfide bridges.

16. The polypeptide of any one of claims 1 -11 , wherein the polypeptide has one or more internal disulfide bridges between amino acid residues Cll and C71, between amino acid residues C8-C77 or between amino acid residues C35-C46.

17. The polypeptide of claim 15 or claim 16, wherein amino acid residue C48 comprises a protecting group.

18. The polypeptide of any one of claims 6-12, wherein the dimer is via a disulfide bridge at amino acid residue C48.

19. A polypeptide, comprising: a synthetic or recombinant polypeptide with an amino acid sequence of SEQ ID NO: 11, SEQ ID NO: 12 or SEQ ID NO: 13.

20. The polypeptide of claim 19, wherein the polypeptide has a C-terminal end cap, an N- terminal end cap, or both.

21. A polypeptide, comprising: a polypeptide with an amino acid sequence of SEQ ID NO: 1 or an amino acid sequence of at least about 90% sequence identity to SEQ ID NO: 1 , whereinXi and Xe are independently, selected from the group consisting of L-threonine, D-threonine, L- tyrosine, D-tyrosine, L-alanine, D-alanine, L-leucine, D-leucine, L-aspartic acid, D-aspartic acid, L-glutamic acid, D-glutamic acid, L-cysteine, D-cysteine, valine, serine, threonine, glycine, norvaline, and 2-aminoisobutyric acid,X2 and X5 are L-cysteine, andX3 and X4 are, independently, an amino acid, natural or unnatural, standard or non-standard, that is non-oxidizable.

22. A polypeptide, comprising: a polypeptide with an amino acid sequence of SEQ ID NO: 1 or an amino acid sequence of at least about 90% sequence identity to SEQ ID NO: 1, whereinX2 and X5 are independently, selected from the group consisting of L-threonine, D-threonine, L- tyrosine, D-tyrosine, L-alanine, D-alanine, L-leucine, D-leucine, L-aspartic acid, D-aspartic acid, L-glutamic acid, D-glutamic acid, L-cysteine, D-cysteine, valine, serine, threonine, glycine, norvaline, and 2-aminoisobutyric acid,Xi and Xg are L-cysteine, andX3 and X4 are, independently, an amino acid, natural or unnatural, standard or non-standard, that is non-oxidizable.

23. A composition, comprising: a polypeptide according to any one of claims 1-22 and an excipient.

24. The composition of claim 23, wherein the excipient is a phospholipid.

25. The composition of claim 24, wherein the phospholipid is selected from dipalmitoylphosphatidylcholine (DPPC), dipalmitoylposphatidylglycerol (DPPG), dioleoylphosphatidylglycerol (DOPG), palmitoyloleoylphosphatidylglycerol (POPG), and palmitoyloleoylphosphatidylcholine (POPC).

26. The composition of any one of claims 22-25, wherein the excipient is cholesterol or a buffer.

27. The composition of any one of claims 22-26, further comprising an SP-C polypeptide or an SP-C polypeptide analog.

28. The composition of claim 27, where the SP-C polypeptide or SP-C polypeptide analog is a synthetic, human SP-C polypeptide.

29. The composition of claim 27 or claim 28, wherein the SP-C polypeptide is selected from the group consisting of SEQ ID NO: 14 where Xi. X2, X3, X4, X5, Xe, and X7 are, independently, selected from the group consisting of Ala, Arg, Met, Leu, Lys, Gin, Glu, Ser, Tyr, and Phe, and n is 1, 2, 3, 4, 5, 6, 7, 8, or 9, provided each of X1.X2. X3. X4, Xs. Xe, and X? is not Vai, SEQ ID NO: 15, SEQ ID NO: 16 where X1.X2. X3. X4, X.5.X6, and X7 are, independently, selected from the group consisting of Ala, Arg, Met, Leu, Lys, Gin, Glu, Ser, Tyr, Vai and Phe, and n is 1, 2, 3, 4, 5, 6, 7, 8, or 9; provided each of Xi,X2. X3,X4, X5, X6, and X7is not Vai, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, and apolypeptide having an amino acid sequence with at least about 90% sequence identity to any one of SEQ ID NOs: 14-22.

30. A composition, comprising: a polypeptide selected from the group consisting of SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 13 and an excipient.

31. The composition of claim 30, wherein the excipient is a phospholipid.

32. The composition of claim 31, wherein the phospholipid is selected from dipalmitoylphosphatidylcholine (DPPC), dipalmitoylposphatidylglycerol (DPPG), dioleoylphosphatidylglycerol (DOPG), palmitoyloleoylphosphatidylglycerol (POPG), and palmitoyloleoylphosphatidylcholine (POPC).

33. The composition of any one of claims 30-32, wherein the excipient is cholesterol.

34. The composition of any one of claims 30-33, further comprising an SP-C polypeptide or an SP-C polypeptide analog.

35. The composition of any one of claims 30-34, further comprising a therapeutic agent.

36. The composition of claim 35, wherein the therapeutic agent is selected from the group consisting of an anti-inflammatory agent, an anti-cancer agent, a cardiovascular agent, an agent for treating a metabolic disorder and an agent for treating a neurological disorder.

37. A method for administering a therapeutic agent to a subject in need, comprising: providing a composition according to any one of claims 22-34 comprising a therapeutic agent, and administering or instructing to administer the composition to a subject in need.

38. The method of claim 37, wherein the administering comprises administering by pulmonary delivery.

39. The method of claim 37, wherein pulmonary delivery is selected from intratracheal instillation, inhalation and intranasal delivery.

40. The method of claim 37 or claim 38, wherein the composition is in liquid form or in dry form.

41. The method of any one of claims 37-40, wherein the therapeutic agent is for treating a disorder selected from the group consisting of serious respiratory illness associated with preterm birth, acute respiratory distress syndrome (ARDS), asthma, chronic obstructive pulmonary disease (COPD), emphysema, idiopathic pulmonary fibrosis (IPF), pulmonary arterial hypertension (PAH), and adenocarcinoma.

42. The method of any one of claims 37-40, wherein the therapeutic agent is for treating a disorder selected from acute respiratory distress syndrome (ARDS), respiratory distress syndrome (RDS), meconium aspiration syndrome (MAS) and bronchopulmonary dysplasia.

43. The method of any one of claims 37-40, wherein the therapeutic agent is for treating a disorder selected cancer, a metabolic disorder, a cardiovascular disorder and a neurological disorder.

44. The method of any one of claims 37-42, wherein the subject is an adult or an infant.

45. A method for manufacture of a synthetic polypeptide, comprising: providing a synthetic polypeptide with an amino acid sequence of SEQ ID NO: 10, wherein Xi, X2, X5 and Xe are, independently, A or C, and X3 and X4 are, independently, A or C, and X3 and X4 are Nle, and reacting the polypeptide to form one or more internal disulfide bridges.

46. The method of claim 45, further comprising reacting the polypeptide with one or more internal disulfide bridges with a second polypeptide with an amino acid sequence of SEQ ID NO: 10, wherein Xi, X2, X5 and X<> are, independently, A or C, and X3 and X4 are Nle, to form a dimer.

47. The method of claim 46, wherein the second polypeptide has one or more internal disulfide bridges.

48. The method of any one of claims 45-47, wherein the dimer is formed by a disulfide bridge.

49. A method for manufacture of a synthetic polypeptide, comprising: providing a synthetic polypeptide with an amino acid sequence of SEQ ID NO: 1 or an amino acid sequence of at least about 90% sequence identity to SEQ ID NO: 1, whereinXi, X2, X5 and Xe are, independently, selected from the group consisting of L-threonine, D- threonine, L-tyrosine, D-tyrosine, L-alanine, D-alanine, L-leucine, D-leucine, L-aspartic acid, D-aspartic acid, L-glutamic acid, D-glutamic acid, L-cysteine, D-cysteine, valine, serine, threonine, glycine, norvaline, and 2-aminoisobutyric acid, andX3 and X4 are, independently, an amino acid that is non-oxidizable; and wherein the polypeptide comprises one or more protected and / or unprotected amino acid residues; reacting the polypeptide with one or more reagents under conditions suitable for forming a first internal disulfide bridge; andreacting the polypeptide with one or more reagents under conditions suitable for forming a second internal disulfide bridge while retaining one or more protecting groups in the polypeptide.

50. A method for manufacture of a synthetic polypeptide, comprising: providing a synthetic polypeptide with an amino acid sequence of SEQ ID NO: 10, wherein Xi, X2, X5 and Xe are, independently, A or C, and X3 and X4 are Nle, and wherein amino acid residues Cll and C71 are unprotected and amino acid residues C8, C35, C46, C48 and C77 are protected; reacting the polypeptide with one or more reagents under conditions suitable to form a disulfide bridge between amino acid residues Cl l and C71 ; and reacting the polypeptide with a C11-C71 disulfide bridge with an oxidizing agent to form a C8- C77 disulfide bridge and a C35-C46 disulfide bridge, while retaining a protecting group at C48.

51. A method for manufacture of a synthetic polypeptide, comprising: providing a synthetic polypeptide with an amino acid sequence of SEQ ID NO: 10, wherein Xi, X2, X5 and Xg are, independently, A or C, and X3 and X4 are Nle, and wherein amino acid residues Cll and C71 are unprotected and amino acid residues C8, C35, C46, C48 and C77 are protected; reacting the polypeptide with an oxidizing reagent to form a disulfide bridge between amino acid residues Cll and C71, a disulfide bridge between amino acid residues C8 and C77, and a disulfide bridge between amino acid residues C35 and C46, while retaining a protecting group at C48.

52. The method according to any one of claims 45-51, wherein the protecting group at amino acid residues C8, C35, C46 and C77 is different from the protecting group at C48.

53. The method according to any one of claims 45-51, wherein the protecting group at amino acid residues C8, C35, C46 and C77 is acetamidomethyl (Acm) or trityl (trt) or a combination thereof.

54. The method according to any one of claims 45-52, wherein the protecting group at amino acid residue C48 is tert-butyl (tBu).

55. The method of claim 54, further comprising reacting to remove the protecting group at C48 to thereby form a dimer polypeptide.

56. The method of any one of claims 45-55, wherein the oxidizing reagent is selected from the group consisting of iodine, Hg(II) HBr / AcOH, TFA / TIS (90 : 10), HBF4 / scavengers, CuSO4-cysteamine, and Gdm-HCl / HEPPS buffer (pH 7.3).

57. The method of any one of claims 45-56, wherein the reagent is NH3.

58. The method of any one of claims 45-57, further comprising drying or lyophilizing the synthetic polypeptide.

Images