WO2009055788A1 - Pneumoreductive therapy and compositions useful therein - Google Patents

Abstract

One aspect of the invention relates to a composition comprising an amphiphile, wherein the composition upon administration to a mammalian lung modifies the surface tension of the pulmonary surfactant, thereby increasing lung recoil. Another aspect of the invention relates to a composition comprising an amphiphile and a spreading agent, wherein the composition upon administration to a mammalian lung modifies the surface tension of the pulmonary surfactant, thereby increasing lung recoil. Yet another aspect of the invention relates to an aforementioned composition, further comprising a phospholipid. Another aspect of the invention relates to a method of reducing lung volume or increasing lung recoil by administering to a lung of a mammal in need thereof a therapeutically effective amount of such a composition.

Description

Pneumoreductive Therapy and Compositions Useful Therein

RELATED APPLICATIONS This application claims the benefit of priority to United States Provisional Patent

Application serial number 60/982,934, filed October 26, 2007 and United States Provisional Patent Application serial number 61/077,209, filed July 1, 2008; which are hereby incorporated by reference in their entirety.

BACKGROUND Emphysema is a common, debilitating, and progressive form of chronic obstructive pulmonary disease (COPD) that affects between 2.0 and 3.0 million Americans, and 3 to 4 times that number of patients worldwide. [American Thoracic Society Consensus Committee "Standards for the diagnosis and care of patients with chronic obstructive pulmonary disease," Am. J. Resp. Crit. Care Med. 1995, 152, 78-83; and Pauwels, R., et al. "Global strategy for the diagnosis, management, and prevention of chronic obstructive pulmonary disease," Am. J. Resp. Crit. Care Med. 2001, 163, 1256-1271.] Cigarette smoking, the primary cause of this disease, remains common. Although emphysema is usually initiated by cigarette smoking, once the disease reaches an advanced stage, it tends to progress in an unrelenting fashion, even in the absence of continued smoking. [Rutgers, S. R., et al. "Ongoing airway inflammation inpatients with COPD who do not currently smoke," Thorax 2000, 55, 12-18.] Furthermore, following a plateau in the mid 1990's, the incidence of smoking is again increasing worldwide, particularly in developing countries among individuals less than 30 years of age. Estimates from the World Health Organization indicate that by 2020, emphysema, together with other forms of COPD, will be the fifth leading cause of death among individuals older than 45 years of age. Direct and indirect costs of caring for patients with advanced emphysema and COPD in the United States total 24 billion dollars annually, representing over 10% of the American health care budget. It is anticipated that emphysema will remain a major public health concern well into the future. The hallmark of emphysema is destruction of lung tissue caused by chronic exposure to noxious inhaled materials. Emphysema is characterized by destruction of the small airways and lung parenchyma due to the release of enzymes from inflammatory cells in response to inhaled toxins. [Stockley, R. "Neutrophils and protease/antiprotease imbalance," Am. J. Resp. Crit. Care Med. 1999, 160, S49-S52.] This process results in loss of lung elastic recoil, prolongation of the time required to complete exhalation, and progressive lung hyperinflation. [Ingenito, E. et al. "On the Role of Surface Tension in the Pathophysiology of Emphysema," Am. J. Respir. Crit. Care Med. 2005, 171, 300-304.] As the damaged lung increases in size, it eventually becomes too large to function effectively within the rigid, surrounding chest cavity. As a result, breathing is markedly compromised, and affected patients are left with limited respiratory reserve and reduced exercise capacity.

In contrast to other common forms of COPD, such as asthma and chronic bronchitis, conventional medical treatment is of limited value in patients with emphysema. Although each of these diseases causes chronic airflow obstruction, limited exercise capacity, and shortness of breath, the site and nature of the abnormalities in asthma and chronic bronchitis are fundamentally different from those of emphysema. In asthma and chronic bronchitis, smooth muscle constriction and mucus hyper-secretion result in airway narrowing, thereby causing airflow limitation. Thus, pharmacologic agents that relax airway smooth muscle and loosen accumulated secretions are effective at improving breathing function and relieving symptoms in these two diseases. Such agents include beta- agonist and anti-cholinergic inhalers, oral theophylline preparations, leukotriene antagonists, steroids, and mucolytic drugs. In contrast, airflow limitation in emphysema is, in large part, due to loss of lung elastic recoil as a consequence of tissue destruction, rather than airway narrowing or obstruction. Loss of recoil compromises the ability to fully exhale, leading to lung hyperinflation and gas trapping. Although bronchodilators, anti-inflammatory agents, and mucolytic agents are frequently prescribed for patients with emphysema, they are generally of limited utility since they are intended primarily for obstruction caused by airway disease. These classes of compounds do not address the loss of lung elastic recoil that is principally responsible for airflow limitation in emphysema. [Barnes, P. "Chronic Obstructive Pulmonary Disease," N. Engl. J. Med. 2000, 343(4), 269-280.]

Thus, existing therapies for patients with COPD, which relax constricted airways and reduce bronchial inflammation, address only minor aspects of the physiological defects responsible for the symptoms and functional limitation in emphysema. While these therapies reduce airway narrowing, this usually represents only a portion of the physiological abnormality in patients with emphysema, for whom destruction of lung tissue and loss of lung elastic recoil are usually important determinants of lung dysfunction.

Mammalian lung surfactant lines the surface of lung alveoli and modulates surface tension in the alveolar compartment at the air-liquid interface. In the absence of lung surfactant, the thin liquid film that lines the alveolar compartment would generate very high surface tension and result in lung collapse and respiratory failure. Surfactant protects the lung from collapse during exhalation by lowering surface tension at the air-liquid interface, maintaining patency of alveoli. During inhalation, lung surfactant increases the surface tension at the air-liquid interface imparting elastic recoil to the respiratory system. During the respiratory cycle, surfactant contributes approximately twice as much to lung elasticity as tissue elements.

Lung surfactant is comprised of a mixture of lipids and proteins, including phospholipids, such as phosphatidylcholine and phosphatidylglycerol, phosphatidylinositol, sphingomyelin, phosphatidylserine and phosphatidylethanolamine, of which the lipid dipalmitoylphosphatidylcholine (DPPC) makes up about 41% by weight. Changes in surfactant biochemical composition alter its interfacial properties which, in turn affect lung physiology. Certain human diseases, such as respiratory distress syndrome are caused by chemical alterations in surfactant composition which result in substantial increases in surface tension at air-liquid interface and stiffening of the lungs. In this and related diseases, surfactant replacements aimed at restoring normal surface tension properties are used therapeutically to reduce surface tension and improve lung function. [Veldhuizen et al. "The role of lipids in pulmonary surfactant," Biochim. Biophys. Acta 1998, 1408, 90- 108.}

Under normal physiological conditions, recoil pressure (P _tp) is determined by 2 factors: 1) the elastic forces of the collagen and elastin fibers (P_tls) that constitute the axial fiber network of the lung, and 2) the elastic surface tension forces associated with the lung surfactant film (P_γ) that lines the surface of the alveolar sacs. While both contribute to recoil, tissue forces and surface tension forces do not act in a simple additive fashion (i.e., mechanical parallel). Rather, their relationship is best described by Equation (a): P_tp = P_tis + P_γ + P_duct Eq. (a) where Pduct accounts for the "additional" recoil force caused by surface tension pf the lung surfactant film stretching alveolar duct fibers, effectively making the lung tissues "stiffer." In emphysema, the destruction of tissue fibers causes a decrease in tissue elastic recoil (P_t]S), since there are fewer fibers. P_γ is also decreased since the contribution of surface tension to lung recoil is proportional to the alveolar surface area on which lung surfactant acts, and surface area is substantially reduced in emphysema as a consequence of tissue destruction. In addition, P_duct, which is proportional to P_γ, is reduced. Thus, in emphysema, each determinant of lung recoil is reduced. Therefore, P_tp can theoretically be increased by any intervention that increases P_tls, P_γ, and/or P_duct,the individual factors of lung recoil. Practical considerations limit the range of therapeutic options, however. For example, despite the fact that emphysema is principally a disease of tissue destruction, modulating P_tis directly is not straightforward. Medical treatments designed to increase P_tIS by replacing or repairing damaged collagen and elastin are not currently available. Alternatively, using a pharmaceutical composition to increase recoil by modulating P₇ and Pd_uct is both rational and achievable. Equation (a), supra, P_tp = Ptis + Pγ + Pduct, suggests that any substance that increases surface tension (γ), is biocompatible, and can be delivered to the alveolar air-liquid interface is capable of increasing lung recoil (i.e., P^) and in doing so, functioning as pneumoreductive therapy (PRT) agent. A therapeutically effective PRT material must, when combined with native surfactant be capable of increasing the surface tension at the alveolar air-liquid interface during lung inflation so as to increase recoil, while still permitting low surface tensions to be reached during exhalation to prevent alveolar collapse. The overall consequence of such a biophysical effect in the emphysema lung would be to increase lung recoil and reduce hyperinflation without causing alveolar collapse.

This unique biophysical capability is not one imparted by conventional surfactant replacement therapies that are currently marketed and used to treatment such conditions as infant respiratory distress syndrome, congenital diaphragmatic hernia, and acute respiratory distress syndrome. Conventional surfactant replacements are designed to replicate the function of natural lung surfactant, and reduce surface tension uniformly. Administering these types of agents to the emphysema lung, while safe, would not produce physiological benefit. It has been discovered that it is possible to safely and effectively increase lung recoil using a composition that safely modulates surface tension of mammalian native lung surfactant. The composition can be administered to the lung to increase P_Yi and accordingly, P_duct, thereby improving lung recoil.

SUMMARY

One aspect of the invention relates to a composition for pneumoreductive therapy, comprising an amphiphile, where the composition upon administration to a mammalian lung modifies the surface tension of the pulmonary surfactant, thereby increasing lung recoil. Another aspect of the invention relates to a composition for pneumoreductive therapy, comprising an amphiphile and a spreading agent, where the composition upon administration to a mammalian lung modifies the surface tension of the pulmonary surfactant, thereby increasing lung recoil. Yet another aspect of the invention relates to an aforementioned composition, further comprising a phospholipid. Another aspect of the invention relates to a method of reducing lung volume or increasing lung recoil by administering to a lung of a mammal in need thereof a therapeutically effective amount of an aforementioned composition. Additional advantages and features of the present invention will become apparent from the following detailed description of various non- limiting embodiments of the invention.

BRIEF DESCRIPTION OF THE FIGURES

Figure l(a) is a graph showing the effect of systematically varying the DPPC:H ratio (at fixed tyloxapol) on γ_max and γ_min when mixed with calf surfactant (CS) at a ratio of 30:1.

Figure l(b) is a graph showing the effect of varying the ratio of D:H:T to CS. Increasing DHT produces a dose-dependent change in surface tension.

Figure l(c) is a graph show γ vs. area (A) profiles (in triplicate) for samples of DHT 532 mixed with CS in a ratio of 30:1. Figure 2 is a graph showing the effect of DHT 532 v. saline on lung volumes following intratracheal administration. DHT 532 caused reductions in three relevant physiological parameters: residual volume ("RV"), functional residual capacity ("FRC"), and total lung capacity ("TLC").

Figure 3(a) is a graph showing the effects of nebulized DHT 532 on static lung compliance ("Cstat") in healthy rats for administered doses ranging from 3 to 30 mg/kg. Figure 3(b) is a graph showing the effects of nebulized DHT 532 on dynamic elastance in healthy rats for administered doses ranging from 3 to 30 mg/kg.

Figure 3(c) is a graph showing the effects of nebulized DHT 532 on airway resistance in healthy rats for administered doses ranging from 3 to 30 mg/kg. Figure 4(a) is a graph showing static compliance absolute values over time.

Figure 4(b) is a graph showing static compliance changes from baseline. Error bars represent one standard deviation.

Figure 5 is a bar graph showing percentage changes from baseline in static compliance as a function of dose (mg/kg) of OS or OTs. Error bars represent SEM.

DETAILED DESCRIPTION

DEFINITIONS

For convenience, certain terms employed in the specification and appended claims are collected here. These definitions should be read in light of the entire disclosure and understood as by a person of skill in the art.

The indefinite articles "a" and "an," as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean "at least one."

The phrase "and/or," as used herein in the specification and in the claims, should be understood to mean "either or both" of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with "and/or" should be construed in the same fashion, i.e., "one or more" of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the "and/or" clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to "A and/or B", when used in conjunction with open-ended language, such as "comprising" can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc. As used herein in the specification and in the claims, the phrase "at least one," in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase "at least one" refers, whether related or unrelated to those elements specifically identified. Thus, as a non- limiting example, "at least one of A and B" (or, equivalently, "at least one of A or B," or, equivalently "at least one of A and/or B") can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.

It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited. In the claims, as well as in the specification above, all transitional phrases, such as

"comprising," "including," "carrying," "having," "containing," "involving," "holding," "composed of," and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases "consisting of and "consisting essentially of shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03.

The term "alkyl" is art-recognized, and includes saturated aliphatic groups, including straight-chain alkyl groups, branched-chain alkyl groups, cycloalkyl (alicyclic) groups, alkyl substituted cycloalkyl groups, and cycloalkyl substituted alkyl groups. In certain embodiments, a straight chain or branched chain alkyl has about 30 or fewer carbon atoms in its backbone (e.g., Ci-C₃₀ for straight chain, Ci-C_3O for branched chain), and alternatively, about 20 or fewer. Likewise, cycloalkyls have from about 3 to about 10 carbon atoms in their ring structure, and alternatively about 5, 6 or 7 carbons in the ring structure.

Unless the number of carbons is otherwise specified, "lower alkyl" refers to an alkyl group, as defined above, but having from one to about ten carbons, alternatively from one to about six carbon atoms in its backbone structure. Likewise, "lower alkenyl" and "lower alkynyl" have similar chain lengths.

The terms "alkenyl" and "alkynyl" are art-recognized and refer to unsaturated aliphatic and alicyclic groups analogous in length and substitution to the alkyls described above, but that contain at least one double or triple bond, respectively. The terms "amine" and "amino" are art-recognized and refer to both unsubstituted and substituted amines.

The definition of each expression, e.g., alkyl, m, n, and the like, when it occurs more than once in any structure, is intended to be independent of its definition elsewhere in the same structure. Certain compounds contained in compositions of the present invention may exist in particular geometric or stereoisomeric forms. The present invention contemplates all such compounds, including cis- and trans-isomers, R- and S-enantiomers, diastereomers, (D)- isomers, (L)-isomers, the racemic mixtures thereof, and other mixtures thereof, as falling within the scope of the invention. Additional asymmetric carbon atoms may be present in a substituent, such as an alkyl group. All such isomers, as well as mixtures thereof, are intended to be included in this invention.

It will be understood that "substitution" or "substituted with" includes the implicit proviso that such substitution is in accordance with permitted valence of the substituted atom and the substituent, and that the substitution results in a stable compound, e.g., which does not spontaneously undergo transformation, such as by rearrangement, cyclization, elimination, or other reaction.

The term "substituted" is also contemplated to include all permissible substituents of organic compounds. In a broad aspect, the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and nonaromatic substituents of organic compounds. Illustrative substituents include, for example, hydroxy, amino, amido, carboxy, ester, formyl, acetyl, alkylcarbonyl, alkoxy, cyano, halo, nitro, sulfhydryl, etc. The permissible substituents may be one or more and the same or different for appropriate organic compounds. For purposes of this invention, the heteroatoms, such as nitrogen may have hydrogen substituents and/or any permissible substituents of organic compounds described herein which satisfy the valences of the heteroatoms. This invention is not intended to be limited in any manner by the permissible substituents of organic compounds.

LUNG VOLUME REDUCTION THERAPY WITH SURFACTANT COMPOSITIONS

One aspect of the invention relates to compositions useful for treating patients with COPD, such as emphysema. Another aspect of the invention relates to methods for treating COPD using pneumoreductive therapy (PRT).

Another aspect of the invention relates to a composition comprising an amphiphile, wherein the composition upon administration to a mammalian lung modifies the surface tension of the pulmonary surfactant, thereby increasing overall lung recoil. In certain embodiments, the pulmonary surfactant has a maximum surface tension and a minimum surface tension; wherein the increase in overall lung recoil is substantially due to an increase in the maximum surface tension of the pulmonary surfactant; and the increase in the minimum surface tension of the pulmonary surfactant is less than about 10 dyn/cm.

Yet another aspect of the invention relates to a composition comprising an amphiphile and a spreading agent, wherein the composition upon administration to a mammalian lung modifies the surface tension of the pulmonary surfactant, thereby increasing overall lung recoil. In certain embodiments, the pulmonary surfactant has a maximum surface tension and a minimum surface tension; wherein the increase in overall lung recoil is substantially due to an increase in the maximum surface tension of the pulmonary surfactant; and the increase in the minimum surface tension of the pulmonary surfactant is less than about 10 dyn/cm.

COMPOSITIONS FOR PRT

Compositions that are appropriate for use as PRT agents include combinations of C)₆ and/or Ci₈ amphiphiles, such as Ci₆ and Ci₈ amphipathic alcohols including hexadecanol and octadecanol, Ci₆ and Ci₈ fatty acids, such as palmitic acid and stearic acid, and Ci₆ and Ci₈ diacylphospholipids including DPPC and DSPC. To facilitate the transport of Ci₆ and Ci₈ amphiphiles into the surface film interface and enable them to interact biophysically with other constituents of native surfactant, it has been determined that a small amount of spreading agent or detergent is desirable in certain embodiments. Thus, formulations that can be used as safe and effective PRT agents comprise a composition of the general formula:

X/Y wherein X is a phospholipid containing a choline headgroup and Ci₃-C₁₉ acyl chains, e.g., DPPC = dipalmitoylphosphatidylcholine, DSPC = distearylphosphatidylcholine, PSPC = palmitoylstearylphosphosphatidylcholine;

Y is a C 1₃-Ci ₉ amphiphile with the general structure of

R₁-OR₂; wherein Ri is a Ci₃-CiQ alkyl chain; and

R₂ is H, O=CR₃, or lower alkyl, and R₃ is CH₃ or lower alkyl to C₂₀ alkyl, e.g., hexadecanol, octadecanol, hexadecyl acetate; or Y is a C1₃-C ₁₉ amphiphile with the general structure of: R₅-COOR₄; wherein R₅ is a Ci₃-C ₁₉ alkyl chain; and R₄ is H or lower alkyl to Ci₈ alkyl, e.g., palmitic acid, stearic acid, ethyl palmitate, methyl stearate, hexadecyl palmitate.

Alternatively, formulations that can be used as safe and effective PRT agents comprise a composition of the general formula:

XIYIZ wherein X is a phospholipid containing a choline headgroup and Ci₃-C ₁₉ acyl chains, e.g., DPPC = dipalmitoylphosphatidylcholine, DSPC = distearylphosphatidylcholine, PSPC = palmitoylstearylphosphosphatidylcholine;

Y is a Ci₃-CiQ amphiphile with the general structure of

Ri-OR₂; wherein Ri is a C _{I 3}-Ci ₉ alkyl chain; and

R₂ is H, O=CR₃, or lower alkyl, and R₃ is CH₃ or lower alkyl to C₂₀ alkyl, e.g., hexadecanol, octadecanol, hexadecyl acetate; or Y is a Ci₃-C ₁₉ amphiphile with the general structure of: R₅-COOR₄; wherein R₅ is a C _{I 3}-Ci ₉ alkyl chain; and R₄ is H or lower alkyl to C₁₈ alkyl, e.g., palmitic acid, stearic acid, ethyl palmitate, methyl stearate, hexadecyl palmitate;

Z is a spreading agent, e.g., Triton, Tween®, Brij, cholesterol, cholesterol esters, lysophospholipids, sucrose esters. In certain embodiments, the spreading agent is Triton WRl 339 or tyloxapol. In certain embodiments, the spreading agent is a sucrose ester, sucrose palmitate or sucrose stearate (tradename Surfhope).

In some embodiments, X, Y and Z of the formula XIY /Z have a relative mass ratios ranging from: X = 0 - 10, Y = 2 - 10, Z = 0.01 - 4.

In certain embodiments, the compositions of the present invention, when in contact with mammalian lung surfactant in a ratio of 1 : 10, cause an increase in surface tension during film expansion (γ_max) of about 1 to 15 dyn/cm. In other embodiments, the compositions increase γ_max by about 1 to 10 dyn/cm, or about 1 to 5 dyn/cm. In certain embodiments, while increasing γ_max as explained above, the compositions do not cause significant surface tension effects during film compression (γ_mi_n)- In certain embodiments, γ_mm is varied by about 0 to 10 dyn/cm, about 0 to 5 dyn/cm, or about 0 to 3 dyn/cm.

The compositions of the present invention are capable of increasing lung recoil pressure in a mammalian lung. In certain embodiments, lung recoil pressure is increased by about 10 - 100%, about 10 - 50 %, about 10 - 30%.

In certain embodiments, the compositions are capable of increasing mammalian lung expiratory flows (V_max). In certain embodiments, V_max is increased by about 5 - 100%, about 10 - 50 %, or about 10 - 30%. In certain embodiments, the compositions are capable of increasing mammalian lung forced expiratory volume in the first second (FEVj) by about 5 - 100%, about 5 - 50 %, about 5 - 20 %. FEVj, the volume of air that can be forced out in one second, is an important measure of pulmonary function. In certain embodiments, the compositions of the invention are capable of reducing residual volume (RV) in a mammalian lung, the amount of gas that remains trapped in the lung and contributes to hyperinflation in emphysema. In certain embodiments, residual volume is reduced by about 5 to 30%, about 5 to 20%, about 5 - 10 %.

In certain embodiments, the compositions of the present invention are capable of decreasing static compliance in a mammalian lung. In certain embodiments, static compliance is decreased by about 5- 30 % or about 5 - 20 %.

In certain embodiments, the compositions of the present invention are capable of increasing dynamic elastance in a mammalian lung by about 5 to 50%.

In certain embodiments, the compositions useful for PRT comprise a surfactant; or a surfactant and a spreading agent. As used herein, the term "surfactant" refers to organic compounds that are amphiphilic, meaning they contain both hydrophobic groups and hydrophilic groups. In other words, the terms "surfactant" and "amphiphile" are used interchangeably herein. Therefore, they are generally soluble in both organic solvents and water. Surfactants reduce the surface tension of water by adsorbing at the liquid-gas interface. The surfactants used in the compositions of the present invention can be anionic, neutral and zwitterionic.

In certain embodiments, the composition comprises an amphiphile. As used herein, the term "amphiphile" refers to compounds comprising at least one Cn to Ci ₉ hydrocarbon chain, and at least one polar group, such as a carboxylic acid or an ester of a carboxylic acid, e.g., acetates. Exemplary amphiphiles include hexadecanol, octadecanol and hexadecyl acetate.

In certain embodiments, the aforementioned composition comprises a phospholipid. As used herein, the term "phospholipid" refers to lipids containing a phosphate group, and at least non-polar saturated or unsaturated hydrocarbon group, including saturated and unsaturated hydrocarbons. General types of phospholipids include phosphoglycerides, such as phosphatidylcholines (e.g., dipalmitoylphosphatidylcholine (DPPC) and disteroylphosphatidylcholine (DSPC)), phosphatidylinositol, phosphatidylethanolamines, phosphatidylserines, and diphosphatidylglycerals; and sphingomyelins.

In certain embodiments, the phospholipid is a phosphatidylcholine. In certain embodiments, the phospholipid has the general formula I:

wherein R¹, and R², are for each occurrence independently selected from the group consisting of alkyl, alkenyl, alkynyl, subject to the proviso that at at least one of Ri or R₂ is selected from the group consisting of Ci_3-19 alkyl, Ci_{3- 19} alkenyl, and C1₃-19 alkynyl; and

R₃, R₄, and R₅ are for each occurrence independently selected from the group consisting of hydrogen, lower alkyl, lower alkenyl, and lower alkynyl.

In certain embodiments, at least one of R¹ and R² of the phospholipid of Formula I is C₁₃-₁9 alkyl. In certain embodiments, at least one of R¹ and R² is Ci₅ or C π alkyl.

In other embodiments, the phospholipid is DPPC, DSPC, PSPC, SPPC.

In certain embodiments, the surfactant is a fatty acid, an ester of a fatty acid, a fatty alcohol. In certain embodiments, the surfactant is hexadecanol (cetyl alcohol), octadecanol, palmitic acid, an ester of palmitic acid, stearic acid or an ester of stearic acid.

In certain embodiments, the surfactant is selected from the group consisting of palmitic acid and hexadecanol.

In certain embodiments, the surfactant is selected from the group consisting of stearic acid and octadecanol.

Those skilled in the art will readily appreciate that where the aforementioned surfactant structures contain a negative charge, e.g., a carboxylate, respectively, the molecule also contains a corresponding pharmaceutically acceptable counterion. When the surfactant contains an overall negative charge, a suitable counterion can be, for example, cations based on alkali metals or alkaline earth metals, such as lithium, sodium, potassium, calcium, magnesium and aluminum salts and the like and nontoxic quaternary ammonia and amine cations including ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, diethylamine, ethylamine and the like.

The term "spreading agent" as used herein refers to a compound that facilitates the incorporation of the surfactant into the native lung surfactant. Examples of spreading agents include nonionic polymers of the alkyl aryl polyether alcohol type, such as Tyloxapol. Other spreading agents include Tween® 20 (polysorbate 20), Tween® 40 (polysorbate 40), and Tween® 80 (polysorbate 80), poloxamer, poloxamine, span, Brij, cholesterol, cholesterol esters, and sucrose esters.

In certain embodiments, the spreading agent is tyloxapol (4-(l, 1,3,3- tetramethylbutyl)phenol polymer, or Triton WR 1339). Tyloxapol is a non-ionic polymeric detergent that aids in dispersion of surfactants. Tyloxapol has been utilized clinically as a dispering agent in synthetic lung surfactant formulations, and as a mucolytic agent in patients with bronchiectasis and chronic bronchitis.

In certain embodiments, the spreading agent is a sucrose ester, such as sucrose palmitate or sucrose stearate. While not being bound by any particular theory, the proposed mechanism of action involves initial adsorption of the surfactant or the both the surfactant and the phospholipid (when the composition comprises a surfactant and a phospholipid) into the air-liquid interface in a mammalian lung, followed by incorporation into the native lung surfactant film. For example, hexadecanol (H), or its metabolite palmitic acid (P) and DPPC are adsorbed and incorporated into the native lung surfactant. This would lead to molecular ordering and formation of crystal H-DPPC lipid structures on the lung surfactant film surface. [Lee, K.Y.C. et al. "Influence of palmitic acid and hexadecanol on the phase transition temperature and molecular packing of dipalmitoylphosphatidyl-choline monolayers at the air water interface," J. Chem. Phys. 2002, 116;2:774-783.]. Upon compression, these rigid formations fold and stack at the surface, and break off and desorb as large aggregates rather than as individual molecules or small aggregates. Minimum surface area is therefore achieved with a substantial fraction of surface-active material folded and stacked upon itself at the interface, and sequestered in a subphase as large, slowly adsorbing aggregates. During film expansion, adsorption of lung surfactant containing added DPPC and H, from the subphase is slower than normal since the majority of surface-active material in the subphase is arranged in large aggregates. Expansion is also accompanied by unfolding of the rigid lipid structures at the interface, but the rate of unfolding, which varies with film rigidity, is slower than normal liquid phase adsorption. As a consequence, surface tension rises more rapidly and to higher values in the presence of H+DPPC than normal. By varying the amounts of H or P in the film, it should be possible to modulate the kinetics of unfolding to achieve a therapeutically effective increase in γ_maχ without substantially increasing γ_min, or reducing dγ/dA.

In certain embodiments, the ratio of the phospholipid X to the amphiphile Y ranges from about X(O-10:Y):Y(2-10). In other embodiments, the ratio is about 2:5 to about 1:2. In certain embodiments, the ratio is about 2:9 to about 1:2. hi certain embodiments, the ratio is about 3:9 to about 1 :2. hi certain embodiments, the ratio is about 16:36, about 4:9, or about 3:10.

In certain embodiments, the ratio of the phospholipid X to the amphiphile Y to the spreading agent Z ranges from about X(0-10:Y):Y(2-10):Z(0.01-4). In other embodiments, the ratio is about 6:2:2, 5:3:2, 4:4:2, or 3:5:2. In other embodiments, the ratio is about 5:3:2, or 5:3:1, or 5:3:0.5, or 4:9:1, or 4:9:0.5, or 4:9:0.25 or 0:9:1 or 0:3:1. ADDITIONAL THERAPEUTIC AGENTS

Additional therapeutic agents may be incorporated in the compositions of the present invention. In general, therapeutic agents which may be incorporated include, but not limited to: anticholinergics; bronchodilators; anti-inflammatory agents, including steroidal and non-steroidal anti-inflammatory agents; anti-infective, such as antibiotics and antiviral agents (as mentioned above); analgesics and analgesic combinations; antiasthmatic agents; antidiuretic agents; antihistamines; antineoplastics; sympathomimetics; cough and cold preparations, including decongestants; immunosuppressives; parasympatholytics; naturally derived or genetically engineered proteins, polysaccharides, glycoproteins, or lipoproteins. Suitable pharmaceuticals for parenteral administration are well known as is exemplified by the Handbook on Injectable Drugs, 6^th Edition, by Lawrence A. Trissel, American Society of HosLVRTal Pharmacists, Bethesda, Md., 1990 (hereby incorporated by reference). In certain embodiments, the aforementioned compositions of the present invention further comprise an additional therapeutic agent selected from bronchodilators, anticholinergics, non-steroidal antiinflammatories and steroidal anti-inflammatories. Bronchodilators that may be included in the compositions of the present invention include: theophylline; beta-agonists, such as albuterol, lev-albuterol, salbutamol, epinephrine, salmeterol, formoterol, pirbuterol, and the like. Anticholinergics, include ipratropium bromide and the like.

Steroidal anti-inflammatories that may be incorporated into the compositions of the invention include: fluticasone, ciclesonide, prednisone, prednisolone, methylprednisolne, dexamethasone and its derivatives, cortisone, hydrocortisone, fludrocortisone, betamethasone, budesonide, triamcinolone, beclometasone, and the like.

ADDITIONAL COMPONENTS

The composition of the present invention may further comprise pharmaceutically acceptable carriers and/or excipients. The compositions of the invention may be in the form of a powder, which may optionally be suspended into a sterile liquid, such as water, saline, aqueous buffer, alcohols, and polyols (such as glycerol, propylene glycol, and polyethylene glycol). Pharmaceutical compositions of the invention also may be in the form of a suspension in a liquid, for example pharmaceutically-acceptable sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions. Examples of suitable aqueous and nonaqueous carriers, diluents, solvents or vehicles include water, ethanol, and polyols (such as glycerol, propylene glycol, and polyethylene glycol).

These compositions may also contain adjuvants, such as preservative, wetting agents, emulsifying agents, and dispersing agents. Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like.

The surfactant, phospholipid or spreading agent used in the compositions of the present invention may be used in the form of pharmaceutically-acceptable salts derived from inorganic or organic acids. By "pharmaceutically-acceptable salt" is meant those salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit/risk ratio. Pharmaceutically- acceptable salts are well-known in the art. For example, S. M. Berge, et al. describe pharmaceutically-acceptable salts in detail in J. Pharmaceutical Sciences, 1977, 66: 1 et seq.

METHODS OF THE INVENTION

Aspects of the invention relate to methods of increasing lung recoil or decreasing lung volume. According to the invention, increased lung recoil or decreased lung volume can be accomplished non-surgically, rather than by procedures that disrupt the integrity of the chest wall [Ingenito et al, Am. J. Resp. Crit. Care Med. 2001, 164, 295-301; Ingenito et al, Am. J. Resp. Crit. Care Med. 2000, 161, A750; and Ingenito et al, Am. J. Resp. Crit. Care Med. 2001, 163, A957.] In one aspect of the invention, the method of lung recoil increase or lung volume decrease comprises administering by inhalation any of the aforementioned compositions of the invention to a mammalian lung, wherein the composition comprises a surfactant; or a surfactant and a spreading agent. In other embodiments, the composition further comprises an amphiphile. In still other embodiments, the composition comprises the formula X:Y or X:Y:Z, as described above. In certain embodiments, administration by inhalation is achieved using a nebulizer

(e.g., a jet nebulizer, ultrasonic nebulizer or a vibrating mesh nebulizer), a metered-dose inhaler, or a dry-powder inhaler.

In certain embodiments, the method comprises administering a composition for PRT via a nebulizer. A "nebulizer" is a device used to administer medication to people in forms of a liquid mist to the airways. Nebulizers often pump air or oxygen through a composition to turn it into a vapor, which is inhaled by a patient. The vaporized composition is inhaled through a tube-like mouthpiece, or in some instances, the composition is inhaled through a rubber face mask.

Some nebulizers/inhalers are manually activated, while others are breath activated, and do not require manual activation. These inhalers automatically sense the patient breathing in and deliver the medication.

In certain embodiments, the composition is administered via an asthma spacer, which is an enclosed plastic chamber that mixes the medication with air in a simple tube, making it easier for patients to receive a full dose of the drug. In certain embodiments, the composition is administered via the use of finely divided dry powder. Dry powder devices use a priming procedure to place a does of a powder ready for the patient to take. The operator puts the release end of the inhaler into his mouth and takes a deep inhalation, holding their breath for 10 seconds.

In certain embodiments, the composition is administered to the lung as a bolus. For example, the composition can be administered using a bronchoscope. The composition may be administered intratracheal bolus administration, intermittent repeat nebulized dosing, or continuous nebulized dosing (in ventilated patients).

In certain embodiments, the method of increasing lung recoil or decreasing lung volume comprises administering to a mammalian lung a composition that modulates the surface tension of native lung surfactant, wherein the composition comprises a surfactant; or a surfactant and a spreading agent.

In certain embodiments, the mammal has emphysema. In certain embodiments, the mammalian lung is hyperinflated.

In certain embodiments, the administration of the composition is repeated weekly, daily, two times a day or three times a day for a period of time.

EXEMPLIFICATION

The invention now being generally described, it will be more readily understood by reference to the following examples, which are included merely for purposes of illustration of certain aspects and embodiments of the present invention, and are not intended to limit the invention.

EXAMPLE 1

IN VITRO CALFLUNG SURFACTANT TEST

Suspensions of palmitic acid (P) or hexadecanol (H) mixed with dipalmitoyl- phosphatidylcholine (DPPC) were prepared in methanol, dried under vacuum, and resuspended in buffered saline (pH 7.1 - 7.4). Homogeneous suspensions were then mixed with freshly isolated calf lung surfactant (CLS) at various ratios. Surface tension v. surface area profiles during isothermal (37 ⁰C) cyclic film expansion at oscillation frequencies of 20 cycles/min and relative surface area changes of 70% (approximately equivalent to a vital capacity maneuver) were determined using a pulsating bubble surfactometer (Electronetics Corporation, Jacksonville, FL). Preliminary studies indicated that addition of a spreading agent, the nonionic surfactant tyloxapol, substantially improved spreading of the DPPC/H and DPPC/P mixtures, and reduced the time required to reach steady state surface tension values during sinusoidal changes in surface area. Formulations containing DPPC/H/tyloxapol in varying ratios or DPPC/P/tyloxapol in varying ratios were mixed with CLS (at fixed CLS concentration of 2 mg/mL) in saline containing 2.5 mM CaCl₂. Surface tension v. surface area plots were recorded at steady state after 15 minutes of oscillating at 20 cycles/min, 37⁰C. The effects of each surfactant modifier on native surfactant biophysical properties were expressed in terms of y_m3X, γ_mi_n, and γ_eqUii, and are summarized in Figure 5 for samples prepared with H. Results using P were similar. The results for DPPC/H/tyloxapol ("DHT") are summarized in Figure 4a. Based on this study, the formulation containing DHT in a 5:3:2 weight ratio ("DHT 532") caused an increase in Y_max, without substantially increasing γ_mj_n. This is the pattern of change in surface film recoil that would be required to increase lung recoil, and therefore correct the loss of tissue recoil that occurs in patients with emphysema. In additional studies, the ratio of CLS to DHT 532 was varied (1:1, 3:1, 10:1, 30:1, and 100:1) to assess the magnitude of LVRT. Biophysical changes associated with these effects are also summarized in Figure 4b, and illustrate the amount of DHT 532 that would be required to alter surfactant surface tension properties to produce the desired therapeutic effect.

EXAMPLE 2 Additional studies were performed to further assess the effects of PRT on dynamic surfactant biophysical behavior using DHT 532 as an example,. These studies were performed using a second surface balance system known as a pendant drop surfactometer (PDS). Similar to the PBS system, the PDS system gives detailed information about surface tension surface area properties but is a more precise measurement. The PDS measurements were used to obtain a second, independent assessment of the biophysical effects of C16 and Cl 8 amphiphiles on surfactant behavior. In these measurements, the drop, containing surfactant with varying amounts of DHT 532, was continuously expanded by volumetric injection of the DHT components from a syringe, at a constant rate of 0.25 μL / second. The drop was expanded until it either fell off the needle or was larger than the video frame. All test preparations were prepared by dissolving components in tert-butanol followed by freezing and freeze drying. Results are summarized in Table 1. The results demonstrate that DHT532 had a consistent effect on the biophysical properties of native lung surfactant to increase surface tension, as outlined in Table 1. The increase in surface tension observed would be expected to increase lung recoil in the emphysemic lung, thereby improving overall lung function.

Table 1.

These observations indicate that DHT 532 is expected to produce physiologically beneficial changes in lung elasticity without promoting a significant increase in γ_min.

EXAMPLE 3 IN VIVO EFFECTS OF DHT 532 ONLUNG PHYSIOLOGY ADMINISTERED ASAN INTRATRACHEAL BOLUS IN HEALTHY RATS

Studies testing the safety and effectiveness of single-dose PRT were performed in healthy rats to determine whether increases in lung elastance could be achieved in vivo without adverse effects on lung volumes or short-term survival using Cl 6 amphiphiles, as suggested by the in vitro PBS and PDS results referenced in Example 2. Initial studies compared physiological responses to intratracheal DHT 532 (5 mg/kg in 1 mL volume) or saline control (1 mL volume) in tracheostomized, lightly anesthetized (ketamine 50 mg/kg and xylazine 5 mg/kg i.p.), ventilated rats for 60 minutes following bolus dose administration. Measurements of lung volumes were recorded by whole body plethysmography at three baseline time points over 30 minutes prior to dosing, and at 15 minute intervals up to 1 hour following dosing during periods of spontaneous breathing while off ventilator support. Baseline values represented the average of the three pre- treatment values for each physiological parameter, and post-treatment responses represented the average of the four values recorded during the 60 minutes following dosing. The variable increases in lung volume observed following adminstration of saline alone were interpreted as reflecting gas trapping as a consequence of foaming caused by the dilutional effect of adding saline to native lung surfactant, as seen in Table 3. Physiological responses to DHT 532 are summarized in Table 4 and Figure 6, where "RV" is residual volume (equal to trapped gas), "FRC" is functional residual capacity (equal to gas volume at passive end exhalation), and "TLC" is total lung capacity (equal to gas volume at full lung inflation). Results indicate that relative to saline, LVRT therapy administered as a bolus produced consistent reductions in all relevant lung volume parameters. The magnitude of changes in physiological response observed would be expected to have beneficial physiological effects in a patient with advanced emphysema.

EXAMPLE 4

IN VIVO EFFECTS OF DHT 532 ON L UNG PHYSIOLOGY ADMINISTERED AS A NEBULIZED SUSPENSION TO HEALTHY RATS USING THE AEROGEN VIBRATING MESHNEBULIZER SYSTEM

The effect of single dose nebulized DHT 532 on lung physiology in healthy rats was then tested to confirm that delivery via nebulization produces favorable physiological effects simlar to those observed with intratracheal bolus dosing. An initial study examined the effects of nebulized DHT 532 on lung physiology administered at a single dose of 15 mg/kg (total material, 7.5 mg/kg DPPC). Eighteen healthy Sprague Dawley rats (300-400 g) were randomized to two groups: saline control (n=9) and DHT 532 treatment (n=9). Animals were anesthetized with tribromoethanol, tracheostomized, and stabilized on a mechanical ventilator.

Baseline meaurements of static lung elastance (Cstat = ΔVolume/ΔPressure over full lung inflation) and dynamic lung elastance (H) and airway resistance (R) were measured at 15 minute intervals starting 45 minutes prior to dose administration, and continuing for 45 minutes following dose administration. Baseline values represent the mean of the three pretreatment measurements, and post-treatment values the mean of the three post treatment values.

Responses to nebulized DHT 532 administered at a dose of 15 mg/kg and concentration of 5 mg/mL using the Aerogen vibrating mesh nebulizer are summarized in Table 4.

Statistically significant reductions in static elastance (Cstat) (p = 0.039, paired t test) and increases in dynamic elastance (H) (p = 0.0005, paired t test) from baseline were observed in the DHT 532 treatment group. Compared to changes observed in the saline control group, the reductions in Cstat in the DHT 532 treatment group were statistically significant (p = 0.045).

Results confirm that DHT 532 administered over 15 to 20 minutes as a single dose of 15 mg/kg at a concentration of 5 mg/mL in saline using an Aerogen vibrating mesh nebulizer produces statistically significant decreases in static compliance and dynamic elastance without detectable adverse effects. Changes of this type would be expected to produce physiological benefit in patients with emphysema.

EXAMPLE 5

CHARACTERIZATION OF THE EFFECT OF DIFFERENT DOSES OF DHT 532 ON L UNG PHYSIOLOGY ADMINISTERED AS A NEB ULIZED SUSPENSION TO HEALTHY RATS

The effect of single dose nebulized DHT 532 in healthy rats was then tested in a second study to further characterize the dose range over which beneficial changes in lung physiology can be safely produced. Sixteen healthy Sprague Dawley rats (300-400 g) were randomized to four groups: saline control (n=9), DHT 532 treatment at 3 mg/kg (n=3), DHT 532 treatment at 15 mg/kg (n=9), and DHT 532 treatment at 30 mg/kg (n=3). Animals were anesthetized with tribromoethanol, tracheostomized, and stabilized on mechanical ventilation

Baseline meaurements of static and dynamic lung elastance and airway resistance were measured at 15 minute intervals starting 45 minutes prior to dose administration, and continuing for 45 minutes following dose administration. Baseline values represent the mean of the three pretreatment measurements, and post-treatment values the mean of the three post treatment measurements.

Response to treatment for animals in the 3 mg/kg and 30 mg/kg group are summarized in Table 5 (saline control and 15 mg/kg group are summarized in Table 4, above). Responses across all groups are summarized in Figure 3. These results indicate a dose-response relationship between lung physiology and administered dose over the dosing range from 3 to 30 mg/kg. Physiolgical changes of this magnitude in emphysema are expected to be sufficient to produce physiological benefit. Table 5: Response to Single Dose Nebulized DHT 532 in Healthy Rats -Dose Response.

EXAMPLE 6

CHARACTERIZATIONOFDELIVERED DOSE OF DHT 532 USING VARIOUS COMMERCIALLY AVAILABLE NEBULIZER SYSTEMS

Studies were performed to characterize the efficiency of DHT532 delivery using different nebulizer systems, because an effective pairing of a lipid-based drug, such as DHT 532, with an effective nebulizer will be requried for clinical application. DHT 532 was suspended in saline by vortexing, nebulized through a particle sizer/impactor unit, and the amounts of D and H delivered in the form of respirable particles (defined as partciles < 4.7 microns in aerodynamic diameter) were measured. Testing was performed using six different nebulizers: 2 vibrating mesh (the Aero gen Aeroneb^® Go and Omron NE-U22V MicroAIR^®); 2 jet (the CIS Aerotech™ II nebulizer and Pari LC Star^®); and 2 ultrasonic (the Systam LS 290 and Sigma Neb 3060).

Studies were performed according to the following protocol. One hundred sixty (160) mg of DHT 532 was suspended in 4 mL saline and a uniform suspension generated by vortexing for 30 seconds. The drug was then drawn into a syringe and injected into the nebulizer chamber. Preparations were aerosolized for 20 minutes while operating each nebulizer unit in accordance with manufacturer's specifications. The outflow stream from the nebulizer was directed through a TSI Incorporated Single Stage Impactor, Model 3306 (particle size cut-off of 4.7 μm aerodynamic diameter) and TSI Incorporated Aerodynamic Particle Sizer, Model 3321 (TSI, St. Paul, MN). Non-respirable particles (defined as those > 4.7 μm aerodynamic diameter) were collected on an impaction plate, while respirable particles (defined as those < 4.7 μm aerodynamic diameter) were collected on a glass fiber filter. Nebulized material from the impactor plate and glass filter was extracted into organic solvent and analyzed by HPLC, providing quantitative assessment of fractional delivery of D and H.

Results for samples containing 40 mg/mL of DH 53 aerosolized for 20 minutes are presented in Table below.

Results in Table 6 show that vibrating mesh nebulizers delivered substantially more PRT active ingredient (D+H) in the form of respirable particles (ie, Filter H+D) than either ultrasonic or jet nebulizers. The Aerogen Aeroneb^® Go vibrating mesh nebulizer delivered the greatest mass of DHT 532 over 20 minutes.

Mass of nebulized material = (Weight of nebulizer + solution prior to initiating nebulization - Weight of nebulizer + remaining solution after nebulization).

Plate (H+D) = mass of H+D deposited on the impaction plate, which represents the amount of H+D in the form of particles > 4.7 microns aerodynamic diameter.

Filter (H+D) = mass of H+D deposited on the impaction plate, which represents the amount of H+D in the form of particles < 4.7 microns aerodynamic diameter.

Amount theoretically nebulized = Mass of nebulized material multiplied by the total percentage of solids m the starting suspension (which equals 5%). Efficiency of delivery = Total mass of H+D nebulized by the device, including material deposited on the plate and filter (Plate (H+D) + Filter (H+D)) divided by the amount theoretically nebulized.

EXAMPLE 7

MANUFACTURE OF DHT532

A compostion of DPPC, H and tyloxapol in a 5:3:2 ratio ("DHT 532") was prepared by dissolution of the constituents in warm t-butanol under stirring at a final concentration of 25 mg/mL. The warm solution was filtered through a 0.22 μm filter and 10 mL were filled into clear 10 mL serum vials and frozen. The frozen solution was lyophilized, sealed and capped.

EXAMPLE 8

PRT WITH DAPC

Diarachidoylphosphocholine (DAPC; a phospholipid with a C20 acyl chain) was dissolved in tert-butylalcohol and dried under vacuum to render a powder for resuspension in saline. Samples of DAPC, DAPC containing hexadecanol and tyloxapol in a ratio of 5:3:2 by mass, and DAPC containing hexadecanol in a ratio of 1:1 by mass were prepared, resuspended in saline by mixing followed by sonication, and administered via nebulizer to Tight skin (Tsk) mice, a strain with congenital emphysema as a consequence of a mutation in the fibrillin- 1 gene. Results are summarized in Table 7 below. Each test formulation was evaluated in a single test animal to explore whether DAPC prepared in this fashion showed any evidence of beneficial physiological effects.

Table 7: Effects of treatment with DAPC 532

Results indicate that compared to DHT 532, which produces 2-15% reductions in Cst and 10-25% increases in dynamic elastance, DAPC-containing formulations failed to produce physiological benefit. These data suggest that materials with acyl side chains 20 carbons in length are less effective for producing the types of changes in lung recoil expected to be beneficial in patients with emphysema.

EXAMPLE 9 IN VIVO EFFECTS OF DHT 49X ON LUNG PHYSIOLOGY ADMINISTERED AS A NEBULIZED SUSPENSION TO HEALTHY RATS

Based on pharmacokinetic modeling, a formulation of D, H, and T at a ratio of 4/9/1 when nebulized three times a day would establish a steady state concentration of DHT in the lung 5/3/2 after 5 days. In vitro testing confirmed that DHT4/9/1 has desirable effects on surfactant function. The purpose of this study was to test the acute in vivo effects of DHT4/9/1 in rats. Formulations containing less T (DHT4/9/.5 and 4/9A25) and their corresponding steady state formulations (5/3/1 and 5/3Λ5) were also tested.

The mean of the three baseline time points for each animal was compared with the mean of the three post-treatment time points for the same animal to compute the change from baseline. The mean changes from baseline were compared by one-way ANOVA. Tukey's post-hoc test was used for between groups comparisons with statistical significance defined as p≤.05. For static lung compliance, DHT 4/9A25 and 4/9A5 caused decreases of 4.8 and 5.8% respectively, while DHT4/9/1 increased static compliance 12%. The corresponding steady state formulations (5/3Λ5, 5/3/1, and 5/3/2) decreased static compliance 8.7, 3.3, and 3.1% respectively. (See Figure 4.)

Changes in dynamic elastance from baseline are described in Table 8 below. All three formulations appear to have desirable in vivo properties for lung recoil increases both at initial ratios and at steady state.

Table 8. Dynamic elastance,percentage change from baseline at various levels of positive end expiratory pressure (PEEP).

(Values represent mean percent changes from baseline for each group)

DHT4/9/.25 and 4/9A5 appear to be more effective at reducing static compliance than DHT4/9/1 at both initial and steady state ratios. All three formulations increase dynamic elastance at PEEP 3 and 6 at both initial and steady state ratios. Changes of this type would be expected to produce physiological benefit in patients with emphysema.

EXAMPLE 10

PREPARATION OF OCTADECANOL INHALATION SUSPENSIONS

The examples summarized above report favorable results obtained using Cl 6 amphiphile systems, while studies using DAPC (Example 8) suggest that C20 amphiphiles are less effective in altering the surface film propoertis of lung surfactant in a way that would produce physiological benefit in emphysema. Testing was undertaken using octadecanol, a Cl 8 amphiphile, to explore whether this class of agent would produce benefits similar to those obtained with C16 amphiphiles. A first step towards this assessment was development of methods for manufacturing C18-containing amphiphile suspensions with particle sizes that could be readily nebulized.

Manufacturing methods were assessed as follows. Aqueous phase components (listed in Table 9 below for each experiment) were mixed and warmed on a hot plate to 70 or 80 ⁰C with stirring. Oil phase components (listed in Table 9 below for each experiment) were melted together on a hot plate and mixed together. The oil phase was poured into the aqueous phase, and the mixture was homogenized using an IKA Ultra-Turrax homogenizer at its highest setting, about 24,000 RPM. Homogenization of the two component mixture was continued until the particle size, as measured by a Malvern Mastersizer 2000E, remained constant. The homogenization typically required 30 to 60 minutes. The mixture was pumped through a heat exchanger immersed in ice water, which rapidly cooled (residence time of about 1 minute) the mixture to 10 to 20 ⁰C. The final particle size was then determined. The diameter which is greater than 90 % of the particles, on a mass basis, is reported in Table 9.

Table 9. Diameter of octahedral inhalation suspensions prepared using various conditions.

Experiments 112-184, 112-186, and 112-190 demonstrate the effect of various Surfhopes in preparing octahedral inhalation suspensions.

Experiments 121-067, 112-068 demonstrate that glycerol can be used in the aqueous phase, to make the suspending medium isotonic.

Experiments 121-090, 121-120, and 121-097 demonstrate that suspensions having higher solids content can be prepared.

Experiments 121-101 and 121-121 demonstrates the use of Span 60 and Tyloxapol as excipients.

Experiments 121-103 and 121-174 demonstrate that other lipophilic materials, such as cholesterol and ascorbyl palmitate, can be added to the oil phase. EXAMPLE 11

TESTING OF SURFACTANTMODIFYINGAGENTS PREPARED WITH Cl 8 AMPHIPHILES (OTSPANAND OS) IN VIVO This set of experiments was conducted to evaluate the in vivo efficacy of two surfactant modifying formulations containing Cl 8 amphiphiles in altering surface tension properties in a way that would safely increase lung recoil and produce therapeutic benefit in the emphysematous lung. The two formulations are: octadecanol with sucrose ester as excipient (OS; lot#121-093), and octadecanol with tyloxapol and Span 60 (OTSpan; lot#121-094) as excipients.

MATERIALS AND METHODS Reagents and equipment:

1. OS 45 mg/mL solution 2. OTSpan 45 mg/mL solution

3. Scireq Flexivent pulmonary oscillatory mechanics system

4. 14 g tracheal cannulae

5. Aerogen nebulizer system Procedures: Anesthesia was induced with ketamine 90 mg/kg and xylazine 5 mg/kg IP, and maintained with ketamine 50-100 mg/kg IP Q30-60 min. Animals were tracheostomized with a 14g stainless steel cannula, calibrated for use with a commercially available computer-controlled small animal ventilator system. The animals were mechanically ventilated with RR 150, TV 10 mL/kg. Lung physiology, including quasi-static pressure volume curves (for determining static lung compliance) and dynamic impedance (including measures of dynamic lung elastance and lung resistance measured at 3, 6, and 9 cm H2O PEEP) were measured. Baseline measurements were made every 15 minutes following the dosing of test materials via nebulalizer. .Inhaled surfactant formulations were diluted in 2.5% glycerol and administered using the Aerogen Pro nebulizer system. During administration, ventilator settings were changed to RR 100 and TV 20 mg/kg to improve delivery.

Following the last physiological assessment, animals were euthanized with phenobarbital 100 mg/kg administered intraperitoneally. The abdominal and thoracic organs were inspected. The pulmonary vasculature was flushed with saline via a catheter inserted into the right ventricle. The lungs and heart were removed enbloc and inflated with 10% buffered formalin. Samples of lung, heart, liver, kidney, and spleen were collected and preserved in 10% formalin for histologic processing and microscopic examination.

RESULTS

Twenty-three animals were treated with varying doses of the two test formulations (OS and OTSpan) as summarized in Table 10.

As shown in Figure 5, OS reduced static compliance 8.5, 11.1 and 13.7% at 14, 28, and 56 mg/kg, respectively. OTSpan reduced static compliance 4.1, 9.9 and 40.5% at 14, 28, and 56 mg/kg, respectively. The reduction in static compliance with OTSpan at 56 mg/kg was statistically significantly larger than OS at the same dose and OTSpan at lower doses.

All animals had normal appearing abdominal and thoracic organs at necropsy. There was no evidence of focal pulmonary atelectasis.

Conclusions

These physiological results indicate that both OS and OTSpan decrease static compliance in a dose-dependent manner, and could both be effective as inhaled agents for improving lung physiology in emphysema. EQUIVALENTS

While several embodiments of the present invention are described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and/or structures for performing the functions and/or obtaining the results and/or one or more of the advantages described herein, and each of such variations and/or modifications is deemed to be within the scope of the present invention. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and/or configurations will depend upon the specific application or applications for which the teachings of the present invention is/are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, the invention may be practiced otherwise than as specifically described and claimed. The present invention is directed to each individual feature, system, article, material, kit, and/or method described herein. In addition, any combination of two or more such features, systems, articles, materials, kits, and/or methods, if such features, systems, articles, materials, kits, and/or methods are not mutually inconsistent, is included within the scope of the present invention.

Claims

We claim:

I . A composition comprising an amphiphile and a spreading agent, wherein said composition upon administration to a mammalian lung modifies the surface tension of the pulmonary surfactant, thereby increasing overall lung recoil.

2. The composition of claim 1, wherein said pulmonary surfactant has a maximum surface tension and a minimum surface tension; said increase in overall lung recoil is substantially due to an increase in the maximum surface tension of the pulmonary surfactant; and the increase in the minimum surface tension of the pulmonary surfactant is less than about 10 dyn/cm.

3. The composition of claim 1, wherein the amphiphile is a fatty acid, an ester of a fatty acid, a fatty alcohol, or an ether of a fatty alcohol.

4. The composition of claim 1, wherein the amphiphile is represented by:

Ri-OR₂, wherein Ri is a C]₄-C₂₂ alkyl; R₂ is H, -C(O)R₃, or lower alkyl; and R₃ is a Ci-C₂₂ alkyl; or R₅-C(O)OR₄, wherein R₄ is H or Ci-C₂₂ alkyl; and R₅ is a Ci₃-C₂] alkyl.

5. The composition of claim 1, wherein the amphiphile is selected from the group consisting of hexadecanol, octadecanol, palmitic acid, an ester of palmitic acid, stearic acid, and an ester of stearic acid.

6 The composition of claim 1, wherein the amphiphile is palmitic acid or hexadecanol.

7. The composition of claim 1, wherein the amphiphile is stearic acid or octadecanol.

8. The composition of any one of claims 1-7, wherein the spreading agent is selected from the group consisting of Tyloxapol, Triton WR 1339, Tween® 20 (polysorbate 20), Tween® 40 (polysorbate 40), Tween® 80 (polysorbate 80), poloxamer, poloxamine, span, Brij, cholesterol, cholesterol esters and sucrose esters.

9 The composition of any one of claims 1-7, wherein the spreading agent is Tyloxapol.

10. The composition of any one of claims 1-7, wherein the spreading agent is a sucrose ester.

I 1. The composition of any one of claims 1-7, wherein the spreading agent is sucrose palmitate or sucrose stearate.

12. The composition of any one of claims 1-11, wherein the mass ratio of the amphiphile to the spreading agent is about 1:1 to about 40:1.

13. The composition of claim 12, wherein the mass ratio is about 1 :1 to about 20:1.

14. The composition of claim 12 wherein the mass ration is about 1 :1 to about 10:1.

15. The composition of any one of claims 1-14, further comprising a phospholipid.

16. The composition of claim 15, wherein the phospholipid is a phosphoglyceride or a phosphatidylcholine

17. The composition of claim 15 , wherein the phospholipid is DPPC, DSPC, PSPC, SPPC, or a mixture thereof.

18. The composition of claim 15, wherein the phospholipid is DPPC.

19. The composition of any one of claims 15-18, wherein the mass ratio of the phospholipid to amphiphile to spreading agent is about 2:5:1 to about 20:40:1.

20. The composition of claim 19, wherein the mass ratio is about 2:9:1 to about 20:40:1.

21. The composition of claim 19, wherein the mass ratio is about 3:9:1 to about 20:40:1.

22. The composition of claim 19, wherein the mass ratio is about 16:36:1, about 4:9:1 or about 3:10:1.

23. The composition of claim 15, wherein the phospholipid is DPPC; the amphiphile is hexadecanol; and the spreading agent is Tyloxapol; and the mass ratio of DPPC to hexadecanol to Tyloxapol is about 16:36:1.

24. A composition comprising an amphiphile, wherein said composition upon administration to a mammalian lung modifies the surface tension of the pulmonary surfactant, thereby increasing overall lung recoil.

25. The composition of claim 24, wherein said pulmonary surfactant has a maximum surface tension and a minimum surface tension; said increase in overall lung recoil is substantially due to an increase in the maximum surface tension of the pulmonary surfactant; and the increase in the minimum surface tension of the pulmonary surfactant is less than about 10 dyn/cm.

26. The composition of claim 24, wherein the amphiphile is a fatty acid, an ester of a fatty acid, a fatty alcohol, or an ether of a fatty alcohol.

27. The composition of claim 24, wherein the amphiphile is represented by:

Ri-OR₂, wherein Ri is a Ci₄-C₂₂ alkyl; R₂ is H, -C(O)R₃, or lower alkyl; and R₃ is a Ci-C₂₂ alkyl; or R₅-C(O)OR₄, wherein R₄ is H or CpC₂₂ alkyl; and R₅ is a Ci₃-C₂I alkyl.

28. The composition of claim 1, wherein the amphiphile is selected from the group consisting of hexadecanol, octadecanol, palmitic acid, an ester of palmitic acid, stearic acid, and an ester of stearic acid.

29. The composition of claim 24, wherein the amphiphile is palmitic acid or hexadecanol.

30. The composition of claim 24, wherein the amphiphile is stearic acid or octadecanol.

31. The composition of any one of claims 24-30, further comprising a phospholipid.

32. The composition of claim 31, wherein the phospholipid is a phosphoglyceride or a phosphatidylcholine

33. The composition of claim 31, wherein the phospholipid is DPPC, DSPC, PSPC, SPPC, or a mixture thereof.

34. The composition of claim 31 , wherein the phospholipid is DPPC.

35. The composition of claim 31, wherein the phospholipid is DPPC and the amphiphile is hexadecanol.

36. A method for reducing lung volume in a mammal, comprising administering to a lung of a mammal in need thereof a therapeutically effective amount of a composition of any one of claims 1-35.

37. The method of claim 36, wherein the administration is via a nebulizer, an asthma spacer, or a bronchoscope.

38. The method of claim 36 or 37, wherein the composition is a powder.

39. The method of any one of claims 36-38, wherein the mammal has emphysema.

40. The method of any one of claims 36-39, wherein administration of the composition results in an increase in the lung recoil.

41. The method of any one of claims 36-40, wherein the interior of the lung comprises a film having a surface tension, and administration of the composition results in an increase in film surface tension during film expansion (γ_maχ) of about 1-15 dyn/cm, about 1-10 dyn/cm, or about 1-5 dyn/cm.

42. The method of claim 41, wherein the administration of the composition results in an increase in film surface tension during film compression (γ_mi_n) of about 0-10 dyn/cm, about 0-5 dyn/cm, or about 0-3 dyn/cm.

43. The method of any one of claims 36-42, wherein the administration of the composition increases lung recoil pressure by about 10-100%, about 10-50%, or about 10- 30%.

44. The method of any one of claims 36-42, wherein the administration of the composition increases lung expiratory flows (V_max) by about 5-100%, about 10-50%, or about 10-30%.

45. The method of any one of claims 36-42, wherein the administration of the composition increases lung FEVi by about 5-100%, about 5-50%, or about 5-20%.

46. The method of any one of claims 36-45, wherein the administration of the composition decreases residual volume (RV) in the lung by about 5-30%, about 5-20%, or about 5-10%.

47. The method of any one of claims 36-46, wherein the administration of the composition decreases static compliance in the lung by about 5-30% or about 5-20%.

48. The method of any one of claims 36-47, wherein the administration of the composition increases dynamic elastance in the lung by about 5-50%.

49. The method of any one of claims 36-48, wherein the administration is repeated weekly for a period of time.

50. The method of any one of claims 36-48, wherein the administration is repeated daily for a period of time.

51. The method of any one of claims 36-48, wherein the administration is repeated two times a day for a period of time.

52. The method of any one of claims 36-48, wherein the administration is repeated three times a day for a period of time.

53. The method of any one of claims 36-52, wherein said therapeutically effective amount of said composition is about 1-350 mg per administration.

54. The method of any one of claims 36-52, wherein said therapeutically effective amount of said composition is about 5-75 mg per administration.

55. The method of any one of claims 36-52, wherein said therapeutically effective amount of said composition is about 10-50 mg per administration.