WO2005073246A2

WO2005073246A2 - Method for preparing kl4 lung surfactant

Info

Publication number: WO2005073246A2
Application number: PCT/US2005/001567
Authority: WO
Inventors: Ralph Niven; Naina Patel
Original assignee: Discovery Laboratories, Inc.
Priority date: 2004-01-20
Filing date: 2005-01-21
Publication date: 2005-08-11
Also published as: WO2005073246A3

Abstract

Methods of making KL4 peptide and other LS mimetic peptide lung surfactants that do not employ the currently used laboratory and manufacturing processes which involve the use of organic solvents are provided.

Description

METHOD FOR PREPARING KL4 LUNG SURFACTANT

BACKGROUND OF THE INVENTION

Field of the Invention This invention relates to a non-film method of preparing LS mimetic peptide lung surfactant compositions without using organic solvents.

State of the Art Pulmonary surfactant (also referred to as "lung surfactant") is a complex mixture of lipids and proteins that promotes the formation of a monolayer at the alveolar air- water interface, and by reducing the surface tension, prevents the collapse of the alveolus during expiration. Lung surfactant (LS) lines the alveolar epithelium of mature mammalian lungs. Natural LS has been described as a "lipoprotein complex" because it contains both phospholipids and apoproteins that interact to reduce surface tension at the lung air-liquid interface. Four proteins have been found to be associated with lung surfactant, namely SP-A, SP-B, SP-C, and SP-D. Specifically, SP-B appears to be essential for the biophysical action of LS. It is accepted therapy for the treatment of a variety of respiratory disorders to administer LS to the patient's lungs. From a pharmacological point of view, the optimal exogenous LS to use in the treatment would be completely synthesized in the laboratory. In this regard, one mimetic of SP-B that has been found to be useful is KL4, which is a 21 amino acid cationic peptide.

KL4 is representative of a family of LS mimetic peptides which are described for example in U.S. Patent 5,260,273, which is hereby incorporated by reference. The current laboratory method for making developmental KL4 lung surfactant employs a film-forming process that is both time consuming and involves many procedural steps. The current laboratory method is a multi-step process as follows. The first step of the laboratory process involves solubilization of the active ingredients in ethanol. In this step dipalmitoyl phosphatidylcholine (DPPC), palmitoyloleoyl phosphatidylglycerol (POPG) and palmitic acid (PA) and KL4 are sequentially added to a large volume of organic solvent at 45-50°C with ultrasonication. This step yields a solution of the actives within the organic solvent. The second step is an organic solvent evaporation procedure. The evaporation is performed using a rotary evaporator at 55°C under vacuum leaving a partially dry thin film. The third step involves overnight or at least several hours of high vacuum extraction of organic solvent residue from the thin film evaporation product under desiccation. The fourth step involves the rehydration of the desiccated thin film evaporation product with an appropriate aqueous buffered solution. During the fifth step, the buffered mixture is sonicated until a uniform suspension is obtained. The final step involves addition of salt to achieve isotonicity. Similarly, the current large scale manufacturing process involves the use of thin film evaporation (TFE) and the introduction and subsequent removal of organic solvents and requires multiple time-consuming steps. The process consists of the following basic steps: 1) solubilizing the four primary formulation components, DPPC, POPG, PA and KL4 in ethanol; 2) adding aqueous buffer; 3) removing the ethanol utilizing TFE; and 4) vialing the final dispersion. The TFE unit operation itself is complex and has scaling limitations. Typically, a 1 ft TFE processes a 40-liter batch and the biggest comparable unit available is a 10 ft² TFE. In addition to the complications that could be introduced by the number of steps, there can also be complications using organic solvents. Such complications may include toxicity in humans, costs associated with effective and environmentally safe disposal, and the like. These complications are also introduced when other LS mimetic peptide lung surfactant compositions. Based upon the above, it is clear that a more simple and faster method of generating the surfactant is desirable. It is also desirable to produce the surfactant without involving organic solvents.

SUMMARY OF THE INVENTION This invention is directed to novel methods of making LS mimetic peptides, such as KL4, lung surfactants that avoid the use of organic solvents. Additional advantages and novel features of the invention will be set forth in part in the description which follows, and in part will become apparent to those skilled in the art upon examination of the following, or may be learned by practice of the invention. In a broad aspect, the present invention is directed to a method that avoids the use of organic solvents for preparing LS mimetic peptide lung surfactant compositions The method comprises: forming an alkaline aqueous dispersion comprising LS mimetic peptide and one or more lipids; adjusting the pH of the dispersion to a physiological pH of from about 6 to about 8 by addition of compatible acid; and adjusting the tonicity of the dispersion to be essentially isotonic with mammalian lung fluids by addition of salt. Also provided is a LS mimetic peptide lung surfactant composition prepared by the methods of this invention. This composition comprises an aqueous medium containing LS mimetic peptide, and one or more lipids, preferably, dipalmitoyl phosphatidylcholine (DPPC), palmitoyloleoyl phosphatidylglycerol (POPG) and palmitic acid (PA), a concentration of base suitable to set the pH of the composition to at least 8, a pH-adjusting concentration of compatible acid suitable to neutralize a portion of the base to adjust pH of the composition to be from about 6 to about 8, and a concentration of added salt to bring the dispersion into isotonicity with mammalian lung fluids. In a particular aspect, the present invention is directed to a method that avoids the use of organic solvents for preparing KL4 lung surfactant. The method comprises: forming an alkaline aqueous dispersion comprising KL4 peptide and one or more lipids; adjusting the pH of the dispersion to a physiological pH of from about 6 to about 8 by addition of compatible acid; and adjusting the tonicity of the dispersion to be essentially isotonic with mammalian lung fluids by addition of salt. Also provided is a KL4 lung surfactant composition prepared by the methods of this invention. This composition comprises an aqueous medium containing EX4 peptide, and one or more lipids, preferably, dipalmitoyl phosphatidylcholine (DPPC), palmitoyloleoyl phosphatidylglycerol (POPG) and palmitic acid (PA), a concentration of base suitable to set the pH of the composition to at least 8, a pH-adjusting concentration of compatible acid suitable to neutralize a portion of the base to adjust pH of the composition to be from about 6 to about 8, and a concentration of added salt to bring the dispersion into isotonicity with mammalian lung fluids.

BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 A illustrates the hysteresis behavior of a composition prepared by the method of this invention before the tonicity is adjusted. Fig. IB illustrates the hysteresis behavior of a composition prepared by a method of this invention after the tonicity is adjusted. Fig. 2A illustrates the thermal transition peaks of compositions prepared by the method of this invention. Fig. 2B illustrates the thermal transition peak of a composition prepared by conventional methods. Figs. 3 A, 3B, 4A and 4B illustrate the comparison of select waveforms and hysteresis behavior derived from the waveforms for compositions prepared by methods of this invention. Figs. 3 A and 4A illustrate a waveform and a hysteresis curve for a composition prior to the tonicity being adjusted while Figs. 3B and 4B illustrate a waveform and a hysteresis curve for a composition after the tonicity has been adjusted. Fig. 5 A illustrates the thermal transition peak of compositions prepared by the method of this invention. Fig. 5B illustrates the thermal transition peaks of two compositions, one with an equivalent ratio of POPG and the other with no POPG. Figs. 6A, 6B, 7A and 7B illustrate the comparison of select waveforms and hysteresis behavior derived from the waveforms for compositions prepared by methods of this invention. Figs. 6A and 7A illustrate a waveform and a hysteresis curve for a composition prior to the tonicity being adjusted while Figs. 6B and 7B illustrate a waveform and a hysteresis curve for a composition after the tonicity has been adjusted. Fig. 8A illustrates the thermal transition peak of a composition prepared by the method of this invention. Fig. 8B illustrates the thermal transition peaks of two compositions, one with an equivalent ratio of POPG and the other with no POPG. Fig. 9 illustrates a comparison of lung compliance of a composition prepared by the method of the invention with a control/vehicle. DETAILED DESCRIPTION OF THE INVENTION

A. Definitions And Overview As discussed above, the present invention is directed to a method of preparing LS mimetic peptide lung surfactant, such as KL4 lung surfactant, that avoids the use of organic solvents. Before the present invention is described in detail, it is to be understood that unless otherwise indicated this invention is not limited to any particular method, as such may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention. It must be noted that as used herein and in the claims, the singular forms "a,"

"and" and "the" include plural referents unless the context clearly dictates otherwise. In this specification and in the claims which follow, reference will be made to a number of terms which shall be defined to have the following meanings: All "amino acid" residues identified herein are in the natural L-configuration. In keeping with standard polypeptide nomenclature, J Biol. Chem. , 243 :3557-59, (1969), abbreviations for amino acid residues are as shown in the following Table of Correspondence:

Table of Correspondence

It should be noted that all amino acid residue sequences are represented herein by formulae whose left to right orientation is in the conventional direction of amino-terminus to carboxy-terminus. "LS mimetic peptides" as used herein refers to polypeptides with an amino acid residue sequence that has a composite hydrophobicity of less than zero, preferably less than or equal to -1, more preferably less than or equal to -2. The composite hydrophobicity value for a peptide is determined by assigning each amino acid residue in a peptide its corresponding hydrophobicity value as described in Hopp, et al. Proc. Natl. Acad. Sci., 78: 3824-3829 (1981), which disclosure is incorporated by reference. For a given peptide, the hydrophobicity values are summed, the sum representing the composite hydrophobicity value. These hydrophobic polypeptides perform the function of the hydrophobic region of the SP18, a known LS apoprotein. SP-18 is more thoroughly described in Glasser, et al., Proc. Natl. Acad. Sci., 84:4007-4001 (1987), which is hereby incorporated by reference. In a preferred embodiment, the amino acid sequence mimics the pattern of hydrophobic and hydrophilic residues of SP 18. A preferred LS mimetic peptide includes a polypeptide having alternating hydrophobic and hydrophilic amino acid residue regions and is characterized as having at least 10 amino acid residues represented by the formula: (Z_aU_b)_cZ_d Z and U are amino acid residues such that at each occurrence Z and U are independently selected. Z is a hydrophilic amino acid residue, preferably selected from the group consisting of R, D, E and K. U is a hydrophobic amino acid residue, preferably selected from the group consisting of N, I, L, C, Y, and F. The letters, "a," "b," "c" and "d" are numbers which indicate the number of hydrophilic or hydrophobic residues. The letter "a" has an average value of about 1 to about 5, preferably about 1 to about 3. The letter "b" has an average value of about 3 to about 20, preferably about 3 to about 12, most preferably, about 3 to about 10. The letter "c" is 1 to 10, preferably, 2 to 10, most preferably 3 to 6. The letter "d" is 1 to 3, preferably 1 to 2. By stating that the amino acid residue represented by Z and U is independently selected, it is meant that each occurrence, a residue from the specified group is selected. That is, when "a" is 2, for example, each of the hydrophilic residues represented by Z will be independently selected and thus can include RR, RD, RE, RK, DR, DD, DE, DK, etc. By stating that "a" and "b" have average values, it is meant that although the number of residues within the repeating sequence (Z_aUb) can vary somewhat within the peptide sequence, the average values of "a" and "b" would be about 1 to about 5 and about 3 to about 20, respectively. Exemplary preferred polypeptides of the above formula are shown in the Table of LS Mimetic Peptides. Table of LS Mimetic Peptides

¹ The designation is an abbreviation for the indicated amino acid residue sequence. A LS mimetic peptide of this invention can be synthesized by any techniques that are known to those skilled in the polypeptide art. An excellent summary of the many techniques available may be found in J.M. Steward and J.D. Young, "Solid Phase Peptide Synthesis," W.H. Freeman Co., (1969); J. Meienhofer, "Hormonal Proteins and Peptides," Vol. 2, p. 46, Academic Press (1983): E. Schroder and K. Kubke, "The Peptides," Vol. 1 Academic Press (1965); all of which are incorporated herein by reference. Particularly preferred LS mimetic peptides are RL4 and KL4. As just described, "KL4" or "KL4 peptide" is a cationic peptide, described in US Patent 5,407,914, which is hereby incorporated by reference, which contains repeating lysine and leucine residues. The amino acid chain for KL4 is as follows: KLLLLKLLLLKLLLLKLLLLK (SEQ. ID. NO. 1). This is described more thoroughly below. The term "LS mimetic peptide lung surfactant" or "LS mimetic peptide lung surfactant composition" refers to a composition which comprises an LS mimetic peptide and one or more lipids. In most preferred embodiments, the lipids include, dipalmitoyl phosphatidylcholine (DPPC), optionally palmitic acid (PA), and optionally palmitoyloleoyl phosphatidylglycerol (POPG). The term "KL4 lung surfactant" or "KL4 lung surfactant composition" refers to a composition which comprises KL4 peptide and one or more lipids. In most preferred embodiments, the lipids include, dipalmitoyl phosphatidylcholine (DPPC), optionally palmitic acid (PA), and optionally palmitoyloleoyl phosphatidylglycerol (POPG). The term "viscosity" refers to the internal resistance to flow exhibited by a fluid at a specified temperature; the ratio of shearing stress to rate of shear. A liquid has a viscosity of one Poise if a force of 1 dyne/square centimeter causes two parallel liquid surfaces one square centimeter in area and one square centimeter apart to move past one another at a velocity of 1 cm/second. One Poise equals one hundred centipoise. As used herein, the term "salt" incorporates both organic and inorganic salts comprising one or more cationic components and one or more anionic components wherein the charge of the cationic component(s) and anionic component(s) are balanced. Preferably the salt comprises a single cation which is monovalent, divalent or trivalent. More preferably, the salt or combinations of salts employed are selected to be biocompatible with mammalian lungs and to not impose a deleterious effect to the LS mimetic peptide lung surfactant composition or any of the individual components. Preferably, the cationic component of the salt is an alkali metal, such as lithium, sodium or potassium; or alkaline earth metal, such as calcium, magnesium or barium. Alternatively, other cations may be employed, such as ammonium (NFLt^"1") and ammonium derivatives. Preferable salts include sodium chloride, potassium chloride, ammonium chloride, sodium acetate, sodium cacodylate, sodium bicarbonate, lithium acetate, sodium pyruvate, sodium citrate, sodium phosphate, potassium phosphate, sodium borate, sodium maleate, sodium succinate, potassium phthalate, trimethamine chloride, magnesium chloride, calcium chloride, magnesium sulfate, calcium sulfate, and combinations thereof. The most preferable salts are sodium chloride, potassium chloride, and ammonium chloride. As can be seen in the lists of salts just provided, this invention contemplates the use of salts which may be described as buffers. It should be noted that the buffer salts are selected so as not to adversely affect the pH of the composition when used in the desired amount. It is well within the purview of one of skill in the art to determine the appropriate amount of buffer so as not to disturb the desired pH. Suitable buffers salts include, by way of example only, sodium TES (2-[(2-hydroxy-l,l- bis[hydroxymethyl]ethyl)amino]ethanesulfonic acid), glycine amide hydrochloride, Tris maleate, bicarbonate salts, imidazole hydrochloride, N-ethyl morpholine hydrochloride, hydroxylamine hydrochloride, ADA (N-(2-acetamido)-2-iminodiacetic acid), PIPES (piperazine-N,N'-bis(2-ethanesulfonic acid)), MOPS (3-(N-morpholino)butanesulfonic acid), HEPES (N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid)), HEPPSO (N-(2- hydroxyethyl)piperazine-N'-(2-hydroxypropanesulfonic acid)), tricine, bicine, CHES (2- (cyclohexylamino)ethanesulfonic acid), CAPS (3-(cyclohexylamino)-l-propanesulfonic acid) glycylglycine and combinations thereof. The term "biocompatible" as used herein means that the material so described is substantially nontoxic and nonimmunogenic. The term "alkaline" as used herein to describe solutions or dispersions means that the solution or dispersion has a pH of greater than 7. Preferably, the alkaline dispersions of this invention have a pH of above 8; more preferably, from about 9 to about 12; and still more preferably from about 9 to about 11. As used herein the term "physiological pH" refers to a pH that is characteristic of body fluids within the patient being treated. Typically, this refers to a pH that is from about 6 to about 8. More typically this refers to a pH of from about 7 to about 7.5. The term "pharmaceutically acceptable" or "physiologically acceptable" refers to molecular entities and compositions that can be delivered to the treated mammal with little or no adverse affect. "Tonicity" relates to the osmotic pressure of a solution. When comparing the tonicity of two solutions, they may be hypotonic, hypertonic or isotonic to one another. As used herein the term "isotonic" means that a solution has substantially the same osmotic pressure as another solution. For example, as used herein, the compositions of this invention are isotonic with mammalian fluids and more preferably mammalian lung fluid. The term "compatible acid" as used herein refers to an acid that does not significantly adversely affect the KL4 lung surfactant or LS mimetic peptide lung surfactant, or the components thereof. Suitable compatible acids include, by way of example, acetic acid, carbonic acid and the like. Often when the compatible acid is added to the compositions, the acid is added in an amount that is referred to as a "pH-adjusting concentration." As used herein, the term "pH-adjusting concentration" refers to an amount of acid that when added to the composition adjusts the pH of the composition to the desired physiological pH. As used herein, the term "base" refers to a substance that yields hydroxyl ions when dissolved in water and/or can act as a proton acceptor. Preferably, bases used in the methods of this invention are present as aqueous solutions. Representative bases employed include sodium hydroxide, potassium hydroxide, calcium hydroxide, and the like. As used herein, the term "organic solvent" refers to a liquid substance, that is not water, that can be used to dissolve or solubilize another substance, or in the context of the present invention, the components of the surfactant. Typical organic solvents include aliphatic hydrocarbons, aromatic hydrocarbons, esters, ethers, ketones, amines and nitrated and chlorinated hydrocarbons. Compositions of this invention are sometimes referred to as dispersions.

"Dispersions" refer to solutions, as well as suspensions and mixtures, preferably finely divided, of two or more phases, such as, for example, liquid-in-liquid, solid-in-liquid and the like which preferably can remain stable for extended periods of time. Preferably, the dispersions achieved with this invention are solutions or liquid-in-liquid dispersions. B. Compositions Pulmonary surfactant compositions that are prepared by the methods of this invention comprise KL4 or other LS mimetic peptides and are dispersed in one or more lipids. KL4 is a cationic peptide containing repeating lysine and leucine residues. The amino acid chain for KL4 is as follows: KLLLLKLLLLKLLLLKLLLLK (SEQ. ID. NO. 1). Methods of preparing the KL4 peptide may be found in U.S. Patents 5,789,381 and 5,164,369. As stated above, when present as KL4 lung surfactant, the KL4 or other LS mimetic peptides are dispersed in one or more lipids. The composition containing the KL4 or other LS mimetic peptides and the one or more lipids is a dispersion and is sometimes referred to herein as a "colloidal dispersion." To obtain the colloidal dispersion, the lipids are typically admixed with the peptide. Optionally, the one or more lipids and the peptide are admixed in a buffered aqueous medium. Typically, these lipids are commercially available materials. The term "lipid" as used herein refers to a naturally occurring, synthetic or semi-synthetic (i.e., modified natural) compound which is generally amphipathic. The lipids typically comprise a hydrophilic component and a hydrophobic component. Exemplary lipids include, for example, phospholipids, fatty acids, fatty alcohols, neutral fats, phospholipids, oils, glycolipids, surface-active agents (surfactants), aliphatic alcohols, waxes, terpenes and steroids. The phrase semi-synthetic (or modified natural) denotes a natural compound that has been chemically modified in some fashion. Preferably, the lipids of the invention are fatty acids, alcohols, esters and ethers thereof and fatty amines. Examples of phospholipids useful in the compositions of the invention include native and/or synthetic phospholipids. Phospholipids that can be used include phosphatidylcholines, phosphatidylglycerols, phosphatidylethanolamines, phosphatidylserines, phosphatidic acids, phosphatidylinositols, sphingolipids, diacylglycerides, cardiolipin, ceramides, cerebrosides and the like. Exemplary phospholipids include dipalmitoyl phosphatidylcholine (DPPC), dilauryl phosphatidylcholine (DLPC) (C12:0), dimyristoyl phosphatidylcholine (DMPC) (C14:0), distearoyl phosphatidylcholine (DSPC), diphytanoyl phosphatidylcholine, nonadecanoyl phosphatidylcholine, arachidoyl phosphatidylcholine, dioleoyl phosphatidylcholine (DOPC) (C18:l), dipalmitoleoyl phosphatidylcholine (C16:l), linoleoyl phosphatidylcholine (C18:2), dipalmitoyl phosphatidylethanolamine (DPPE), dioleoylphosphatidylethanolamine (DOPE), dioleoyl phosphatidylglycerol (DOPG), palmitoyloleoyl phosphatidylglycerol (POPG), dipalmitoyl phosphatidylglycerol (DPPG), distearoylphosphatidylserine (DSPS) soybean lecithin, egg yolk lecithin, sphingomyelin, phosphatidylinositols, diphosphatidylglycerol, phosphatidylethanolamine, and phosphatidic acids, Egg phosphatidylcholine (EPC). Examples of fatty acids and fatty alcohols useful in these mixtures include sterols, palmitic acid, cetyl alcohol, lauric acid, myristic acid, stearic acid, phytanic acid, dipalmitic acid, and the like. Preferably, the fatty acid is palmitic acid and preferably the fatty alcohol is cetyl alcohol. An example of a semi-synthetic or modified natural lipid is any one of the lipids described above which has been chemically modified. The chemical modification can include a number of modifications; however, a preferred modification is the conjugation of one or more polyethylene glycol (PEG) groups to desired portions of the lipid. Polyethylene glycol (PEG) has been widely used in biomaterials, biotechnology and medicine primarily because PEG is a biocompatible, nontoxic, nonimmunogenic and water-soluble polymer (Zhao and Harris, ACS Symposium Series 680: 458-72, 1997). In the area of drug delivery, PEG derivatives have been widely used in covalent attachment (i.e., "PEGylation") to proteins to reduce immunogenicity, proteolysis and kidney clearance and to enhance solubility (Zalipsky, Adv. Drug Del. Rev. 16:157-82, 1995). Lipids that have been conjugated with PEG are referred to herein as "PEG- lipids." Preferably, when PEG-lipids are used in methods and compositions of this invention, they are present in small amounts of alcohols and/or aldehydes. Preferably, the lipids contemplated for use in this invention are selected from the group consisting of phosphatidylcholines, phosphatidylserines, phosphatidylethanolamines, phosphatidylglycerols, diacylglycerides, phosphatidylinositols, sphingolipids, sterols, cardiolipin, fatty acids, ceramides, cerebrosides, PEG-lipids and combinations thereof. A particularly preferred colloidal dispersion comprises a dispersion of DPPC,

POPG, PA and KL4 or other LS mimetic peptide (weight ratio of approximately 7.5 : 2.5 : 1.5 : 0.267) in a physiologically acceptable aqueous buffer. The individual components may range from KL4 peptide or other LS mimetic peptide 1 part by weight; DPPC 20 to 100 parts by weight; POPG 0 to 50 parts by weight; and palmitic acid 0 to 25 parts by weight. The total concentration of phospholipid in the dispersion may range from 1 to over 80 mg/ml.

Preferred buffers are Tris acetate, Tris hydrochloride, Tris saline, sodium glycinate, sodium phosphate, potassium phosphate, and the like. The most preferred buffer is tris(hydroxymethyl)amino methane. The buffers are commercially available. The concentration of the KL4 lung surfactant composition or LS mimetic peptide lung surfactant composition so formed can be adjusted by conventional means including, for example, water addition or removal and the like. Optionally, the surfactant composition can be sterilized by conventional means including heat or e-beam sterilization and the like.

C. Methods This invention contemplates formulating KL4 or other LS mimetic peptide lung surfactant without using the thin film method, which involves the use of ethanol or other organic solvents. Specifically, the method includes the step of forming an alkaline aqueous dispersion comprising KL4 or another LS mimetic peptide and one ore more lipids. Preferably, the lipids are dipalmitoyl phosphatidylcholine (DPPC), optionally palmitic acid (PA), and optionally palmitoyloleoyl phosphatidylglycerol (POPG). The next step involves adjusting the pH of the dispersion to a physiological pH of from 6 to 8 by addition of compatible acid; and adjusting the tonicity of the dispersion to be essentially isotonic with mammalian lung fluids by addition of salt. In some specific embodiments of preparing KL4 lung surfactant, the method comprises: a) forming an alkaline aqueous solution comprising palmitic acid, KL4, DPPC and POPG; b) adjusting the solution to physiological pH by addition of a compatible acid; and c) adjusting the tonicity of the solution to be isotonic by addition of a salt. The alkaline aqueous solution comprises a physiological acceptable buffer, the buffer is tris(hydroxymethyl)amino methane, the pH of the alkaline aqueous solution is above 8.0, preferably from about 8.0 to about 12.0 and more preferably from about 9.0 to 11.0, the tonicity is adjusted by adding a solution of a multivalent salt or a monovalent salt, especially a solution of salt selected from the group consisting of sodium chloride, potassium chloride, ammonium chloride, sodium acetate, sodium citrate, sodium phosphate, potassium phosphate, sodium TES, sodium borate sodium cacodylate, glycine amide hydrochloride, sodium bicarbonate, lithium acetate, hydroxylamine hydrochloride, sodium maleate, sodium succinate, sodium pyruvate, potassium phthalate and salts of MES, ADA, PIPES, ALES, MOPS, HEPES, HEPPS, tricine, bicine, CHES, CAPS glycylglycine and combinations thereof. The first step involves forming the alkaline aqueous dispersion having a pH of at least 8. To a dispersion of a suitable buffer, such as Tris, KL4 or another LS mimetic peptide and one or more lipids such as, DPPC, optionally POPG, and optionally PA are added with stirring. Preferably, the components may be added one at a time and can be optionally sonicated or homogenized for approximately 1 to 10 minutes after their addition. Once all of the components have been added, a colloidal dispersion of the components is formed. Alternatively, all of the components may be added at once with stirring and followed by sonication or homogenization after all of the components have been added. The pH may be adjusted to form the alkaline dispersion after all of the components have been added or is preferably adjusted after each component has been added. The pH is adjusted to be alkaline by adding a sufficient amount of a base. Preferably, the pH is adjusted to be at least 8; more preferably between 9 and 12; and still more preferably between 9 and 11. The pH can be monitored by a pH meter. Optionally, the peptide may be dissolved or solubilized in a small amount of suitable acid prior to being combined with the lipids. The amount and concentration of acid is selected so the peptide, lipid(s) and buffer when combined remain alkaline and in the ranges just discussed. The appropriate amount and concentration of the acid can be readily determined by one of skill in the art. The base is selected so as to not significantly adversely affect any of the components individually or the dispersion that contains the components. Preferably, the base is aqueous. Suitable bases include, but are not limited to, sodium hydroxide, potassium hydroxide, calcium hydroxide, combinations thereof and the like. After the alkaline dispersion has been formed, the pH is then adjusted to physiological pH of from about 6 to 8 by addition of a compatible acid. More preferably, the pH is adjusted to between 7 and 8. The acid may be added drop-wise until the desired pH is reached. Suitable acids include acetic acid, carbonic acid and the like. The final step of the method involves adjusting the tonicity of the dispersion to be isotonic with mammalian lung fluids. It should be noted that the tonicity may have already been adjusted by the previous step of adding the compatible acid. However, a further adjustment may be made by adding a suitable concentration of salt to the dispersion. Alternatively, a salt of a suitable buffer may be added to adjust the tonicity or osmolality. When salts of buffers are employed, the amount and concentration of the salt is selected so as not to adjust the pH of the composition to which it is being added. As stated above, the salt may be inorganic or organic and may be monovalent or multivalent. Preferably, the salt is sodium chloride or potassium chloride. The salt is most commonly added as a concentrated aqueous solution. The osmolality may be monitored by a vapor pressure osmometer.

D. Characterization of the Compositions Without being limited to any one theory, it is believed that the direct aqueous suspension of the lipids and the LS mimetic peptide with the pH adjusted to be alkaline allows for similar interaction of the components as is demonstrated in the methods involving organic solvents. Due to the similar interaction, the compositions resulting from the methods of this invention have surprisingly and unexpectedly demonstrated comparable characteristics to that of the KL4 or other LS mimetic peptide lung surfactant formulated using organic solvents in laboratory as well as manufacturing methods.

1. Thermal Transition States To illustrate the similar characteristics of compositions prepared by the method described herein with compositions formed by methods of the art, thermal transition states were investigated. Representative thermal transition states of compositions prepared by methods of the invention are illustrated in Figures 2 A, 5 A, and 8 A. Presented in Figure 2B for comparison, is the thermal transition of Surfaxin® brand lung surfactant. This material is prepared using the organic solvent method and is commercially available from Discovery Labs (Doylestown, PA). The compositions of the current method exhibit very similar thermal transition states compared to compositions of the art. While it has been observed that there can be some POPG-excluded material present in the formulation (Figures 5B and 8B), it is contemplated that the reaction conditions can be optimized by the skilled artisan using routine procedures to suitably incorporate the POPG. Suitable optimization techniques include, by way of example only, simple homogenization, short-term storage to allow the components to reach equilibrium, and individual hydration of each component prior to mixing.

2. Surface Tension/Pressure The dynamic surface pressure of compositions prepared by the methods of this invention was measured using a pulsating bubble surfactometer (PBS). The dynamic surface pressure measurements when plotted against surface area present an isotherm showing expansion and compression. The surface pressure data of the compositions are presented in Figures 1A, IB, 4 A, 4B, 7 A, and 7B and demonstrate hysteresis behavior. The surface pressure data are consistent with dynamic surface pressures of KL4 lung surfactant compositions of the art. The hysteresis behavior just described is an indication of the composition's spreading properties. When administering an artificial lung surfactant, it is desirable that the lung surfactant have the ability to spread effectively across the alveolar epithelium. It has been discovered that the compositions made the method of this invention exhibit similar spreading properties compared to those compositions made by conventional means. E. Methods of Use and Administration The present invention is useful for generating KL4 lung surfactant compositions and other LS mimetic peptide lung surfactant compositions can be used in treatment of various respiratory disorders or in replacement therapy. Pulmonary surfactant finds particular utility in critical care settings, specifically in supplying lung surfactant to prematurely born infants, but also with patients with acute respiratory distress syndrome, acute lung injury, meconium aspiration syndrome and the like. When the surfactant is supplied to prematurely born infants, an aliquot of the surfactant composition is delivered, preferably by intratracheal instillation, to provide an effective dose of surfactant in the lungs of the treated patient. Preferably, a single surfactant dose ranges from about 100 to 250 mg/kg. It being understood, of course, that the exact dose of surfactant will depend upon factors such as the age and condition of the patient, the severity of the condition being treated, and other factors well within the skill of the attending clinician. Other methods of delivery include lavage, lung wash, aerosol and the like. When so employed, dose ranges are well within the skill of one in the art. When used as an aerosol preparation, the surfactant composition may be supplied in finely divided form in combination with a suitable propellant. Useful propellants are typically gases at ambient conditions and are condensed under pressure. Lower alkanes and fluorinated alkanes, such as Freon, may be used. The aerosol is packaged in a suitable container under pressure. Suitable dosage of the surfactant, whether aerosolized or delivered as a liquid bolus will be dependent on the patient's age and severity of the disorder and will be readily ascertainable by the attending clinician. The following examples are set forth to illustrate the claimed invention and are not to be construed as limitations thereof.

EXAMPLES Unless otherwise stated all temperatures are in degrees Celsius. It should be understood that when pH values are given they are approximate. In these examples and elsewhere, abbreviations have the following meanings:

μm m microns or micrometers ACN = acetonitrile cm = centimeter DPPC = 1 ,2-dipalmitoyl phosphatidylcholine FWDS = Fine White Dense Suspension Gly = glycine Gly-Na — glycine sodium sodium glycinate kJ = kilojoules L = Liter M = Molar MeOH = methanol mg = milligram ml = milliliter mm = millimeter mmoles = millimoles N = Normal nm = nanometers PA = palmitic acid PBS = Pulsating bubble surface tensionometer POPG = palmitoyl-oleoyl-phosphatidyl-glycerol sec = Seconds TFA = trifluoroacetic acid TPL = total phospholipid All of the starting materials for the formulations contemplated by this invention are commercially available from at least one of the following sources: Sigma- Aldrich (St. Louis, MO); Fisher Scientific (Pittsburgh, PA); Genzyme Corporation (Cambridge, MA); Bachem (King of Prussia, PA); and Avanti Polar Lipids (Alabaster, AL). In the examples presented below, various characterization and/or analytical tests were performed. Unless otherwise stated, the following protocols/equipment were utilized in the examples.

Rheology Viscosity of the compositions at 25°C was determined using a TA AR1000 Rheometer (TA Instruments, New Castle, DE) using a cone and plate geometry with a set temperature of 25°C. Aliquots were analyzed using a step flow program involving a linear ramp up at 1/sec (0-200 sec) and then down at 1/sec (0-200 sec) each with 15 data collection points. The viscosity value at approximately 157 sec^"1 from both the steps was recorded and the average of these two values was recorded. Surface Activity The compositions were diluted in matching buffer to various concentrations and analyzed using a pulsating bubble surfactometer (PBS) (Electronetics Corp., Seminole, FL) at an oscillation frequency of 20 cycles/minute and 5 minutes total run time at 37°C. Data were collected at 0, 1 and 5 minutes. Minimum (λ_mm) and maximum (λ_max) surface tension values were determined from the PBS waveforms. Assessment of the formulations' spreading properties at the indicated concentrations were made by plotting hysteresis activity curves generated from the PBS data. Particle Size The particle size analysis was performed on a Sympatec HELOS System (Sympatec Inc., Princeton, NJ). Approximately 50 mg of sample was diluted in 10 mL of 20 mM Tris- Ac, 130 mM NaCl buffer. The sample was vortexed and ultrasonicated for 10 seconds before the measurements. About 4 ml of this diluted sample was used for each measurement. The average median particle size obtained from 10 measurements was recorded.

Thermal Transition The compositions were analyzed on a multi-cell differential scanning calorimeter (MC-DSC) (Calorimetry Sciences Corporation, Lindon, UT)). Scan rates of

l°C/min were used, scanning over the 20°C to 80°C temperature range.

Lipid Recoveries RP-HPLC analysis was used to establish the concentration of DPPC, POPG and PA in the formulations. The analysis was carried out on a HP 1100 from Agilent Technologies (Palo Alto, CA). The samples were diluted to suitable concentrations in methanol with vortexing if necessary. The samples were injected onto a Zorbax C18 300SB column, 4.6 mm X 250 mm. With a mobile phase consisting of 90% v/v MeOH, 6% ACN, 4% H₂O, 0.2% TFA at a flow rate of 1.5 ml/min for 20 min. An evaporative light scattering detector (Sedex 75, Richard Scientific, CA) was used for detection of the components. Example 1

Formulation 1 The formulation was prepared on a 20 ml scale at 30-mg/ml TPL. First, 20.0 mmoles of Tris base (19.2 mL) was heated to 48-50°C in an ultrasonic water bath. To this PA (90 mg) was added and then the pH was adjusted to 10.0 with IN NaOH until a near clear solution was obtained. DPPC (450 mg), POPG (150 mg), and KL4 (18.7 mg) were then sequentially added. After the addition of each component, the composition was ultrasonicated for 5 minutes. A fine white homogenous dispersion was obtained. The pH was adjusted to 7.6 by adding sufficient IM acetic acid to achieve the final pH. Half of the dispersion was then treated with 0.26 ml of 5 M NaCl (130 mmoles) and then was ultrasonicated for 5 minutes at 48-50°C. This is referred to below as the "salt formulation." The half of the dispersion that was not treated is referred to as the "no salt formulation."

Analytical Characterization of Formulation 1 Table 1 tabulates the analytical summary for Formulation 1. The median particle size of the no-salt formulation was 10 μm (2.6 to 23.5 μm range) and the median particle size was 14.8 μm (3.2 to 38.8 μm range) for the salt formulation. Figure 1 A and Figure IB present the hysteresis behavior as measured by the PBS of the formulation in the presence and absence of salt. The formulation showed a single thermal transition both in the presence and absence of salt at 52.6°C and 53.1 °C respectively (refer to Figure 2A,

Form. A= no salt, Form. IB = salt). This behavior is similar to a "normal" formulation that is prepared by standard methods of the art that employ organic solvents (refer to Figure 2B). The apparent viscosity of the formulation was high both in the presence and absence of salt yielding values of 72.5 centipoise and 85.9 centipoise respectively for the no salt and salt formulations. The viscosity of the salt-containing formulation was similar to normally observed values. Again, "normally" observed values refers to values obtained from formulations that are prepared by methods of the art. Table 1: Analytical Summary

Example 2

Formulation 2 This composition was formulated on a 20 ml scale at 30-mg/ml TPL. First, 27.3 mmoles of Tris base (about 15 mL, pH approximately 9.3) was heated to 55-60°C in an ultrasonic water bath. To this PA (90 mg) was added and the pH was adjusted to approximately 11.0 with IN NaOH (13.2 mmoles) to yield a slightly hazy dispersion. To this DPPC (450 mg) and POPG (150 mg) were sequentially added with 5 min ultrasonication at each addition until a fine homogenous dispersion was obtained. The KL4 (18.7 mg) was first dissolved in 14 mM acetic acid (5 ml) to achieve a pH 3.3 and this was then added to the DPPC:POPG:PA mixture maintaining the lipids at 55-60°C in an ultrasonic water bath. A further amount of 1 M acetic acid was added drop- wise to give a pH of approximately 7.6. The pH of this dispersion was further adjusted to 6.4 using additional IM acetic acid. Half of the dispersion was then treated with 0.26 ml of 5 M NaCl (130 mmoles) with 5 min ultrasonication at 55-60°C. This yielded a fine white homogenous dense dispersion, which started to gel upon cooling to room temperature. This portion is referred to as the "salt formulation." The untreated half is referred to as the "no salt formulation."

Analytical Characterization of Formulation 2 Table 2 tabulates the analytical summary for the formulation. The median particle size of the no salt formulation was 22.6 μm (7.5 to 54.3 μm range) and 18.8 μm (4.4 to 79.8 μm range) for the salt formulation. The PBS data showed desirable surface tension properties for both the no salt and salt formulations. The minimum surface tension was 3.9 and 0.0 Dynes/cm respectively for the no salt and salt containing formulations. Figs. 3A, 3B, 4A, and 4B compares the waveforms at 5 min to the hysteresis behavior in the presence and absence of salt. The formulation presented two thermal transitions both in the presence and absence of salt at 51.1°C (no salt)/48.8°C (salt) and 56.0°C (no salt)/55.0°C (salt) respectively (refer to Figures 5A and 5B, Form. 2A = salt, Form. 2B = no salt). In the absence of the salt the ratio of Tm.i :Tm₂ was 1.9:1 and in the presence of salt was 1.4: 1.

Table 2: Analytical Summary for Formulation 2

Example 3

Formulation 3 In this example Gly buffer (pH 10) was used to control the amount of sodium ions introduced into the formulation. This composition was formulated on a 20 ml scale at 30-mg/ml TPL. First, 10 mM Gly-Na buffer, pH 10 (14 mL) was heated to 55 - 60°C in an ultrasonic water bath. To this PA (90 mg) was added with ultrasonication yielding a homogenous fine white dispersion. To this DPPC (450 mg) was added with ultrasonication to yield a dense white dispersion with a pH of 8.5. The pH was readjusted back to 10 with IN NaOH (18.6 mmoles). To this, POPG (150 mg) was added and ultrasonicated for 5 min to yield a homogenous fine white dispersion. To this, 20 mM Tris base was added. The KL4 (18.7 mg) was then dissolved in 80 mM acetic acid (5 ml) to give a pH of 3.3 and this was then added to the DPPC:POPG:PA mixture maintaining the lipids at 55-60°C in an ultrasonic water bath. The pH was adjusted to 6.5. Half of the dispersion was then treated with 0.26 ml of 5 M NaCl (130 mmoles) with 5 min ultrasonication at 55-60°C. This yielded a fine white dense homogenous dispersion, which started to gel on cooling to room temperature.

Analytical Characterization of Formulation 3 Table 3 tabulates the analytical summary for the formulation. The median particle size of the no salt formulation was 29.9 μm (9.7 to 73.0 μm range) and 34.0 μm (7.1 to 116.0 μm range) for the salt formulation. The apparent viscosity of the formulation with and without salt was 70.3 centipoise and 127.1 centipoise respectively. The minimum surface tension as measured by PBS was 3.7 and 0.9 Dynes/cm respectively for the no salt and salt-containing formulations. Figures 7 A and 7B compare the PBS waveforms at 5 min to the PBS hysteresis behavior in the presence and absence of salt. The formulation presented a single broad thermal transition in the absence of salt at 51.5°C with a poorly resolved right shoulder with apex at 55.3°C (refer to Figures 8A, 8B).

Table 3: Analytical Summary for Basic Process 3

Example 4

In vivo Activity of Formulations of the Invention The following example examined the in vivo activity of a KL4 surfactant formulation prepared by the methods of this invention. The KL4 surfactant was prepared to a concentration of 30 mg/ml according to the procedure in Example 1 with the following components DPPC 22.5 mg; POPG 7.5 mg; palmitic acid 4.05 mg and KL4 0.8 mg per ml of Tris-saline vehicle (final pH 7.7). The change in the volume of the respiratory system was measured by placing an animal, in this case a fetal-rabbit, in a plethysmograph, and measuring the change in volume of gas into and out of the plethysmograph with a flow meter, or pneumotachograph. The stiffness of the respiratory system was calculated by dividing the tidal volume change by the airway pressure change during a mechanical breath. The parameter for this stiffhess was the compliance of the respiratory system (Crs). In surfactant-deficient fetal-rabbits treated with surfactant, Crs was expected to increase during mechanical ventilation, and reach a plateau in approximately 10-30 minutes. This increase was due to the spreading of the surfactant and the establishment of a surface-active lining of the alveoli. In surfactant-deficient animals, there was still a small increase in the Crs with mechanical ventilation due to the release of minimal stores of surfactant, but their lungs remained stiff because there was insufficient surfactant to prevent alveolar collapse with each exhalation. The expected course of Crs is shown in Figure 9 for the mean Crs of six fetal rabbits treated with a 30 mg/ml formulation and six control animals treated with control/vehicle (all at 27 days gestation) prepared by the process essentially described in Example 1, during 30 minutes of mechanical ventilation. The raw data was an average of 20 seconds worth of compliance data, which was acquired every 2 minutes. Compliance values rose over time. The values for compliance were corrected for the weight of the pups. It can be seen that the novel process composition exhibits a significant trend (p < 0.05 at 30 min time point) toward improving compliance relative to a vehicle control. From the foregoing description, various modifications and changes in the composition and method will occur to those skilled in the art. All such modifications coming within the scope of the appended claims are intended to be included therein.

Claims

We Claim:

1. A method for preparing LS mimetic peptide lung surfactant which method comprises: a. forming an alkaline aqueous dispersion comprising LS mimetic peptide and one or more lipids; b. adjusting the pH of the dispersion to a physiological pH of from 6 to 8 by addition of compatible acid; and c. adjusting the tonicity of the pH-adjusted dispersion to be essentially isotonic with mammalian lung fluids by addition of salt.

2. A method for preparing KL4 lung surfactant which method comprises: a. forming an alkaline aqueous dispersion comprising KL4 peptide and one or more lipids; b. adjusting the pH of the dispersion to a physiological pH of from 6 to 8 by addition of compatible acid; and c. adjusting the tonicity of the pH-adjusted dispersion to be essentially isotonic with mammalian lung fluids by addition of salt.

3. The method of claim 1 or 2, wherein said alkaline aqueous dispersion comprises a physiologically acceptable buffer.

4. The method of claim 3, wherein said buffer is tris(hydroxymethyl)amino methane.

5. The method of claim 1 or 2, wherein the one or more lipids are individually selected from the group consisting of phosphatidylcholines, phosphatidylglycerols, phosphatidylserines, phosphatidylethanolamines, diacylglycerides, phosphatidylinositols, sphingolipids, sterols, cardiolipin, fatty acids, ceramides, cerebrosides, PEG-lipids and combinations thereof.

6. The method of claim 5, wherein the one or more lipids comprise dipalmitoyl phosphatidylcholine (DPPC), optionally palmitoyloleoyl phosphatidylglycerol (POPG) and optionally palmitic acid.

7. The method of claim 6, wherein the relative amounts of LS mimetic peptide, dipalmitoyl phosphatidylcholine (DPPC), palmitoyloleoyl phosphatidylglycerol (POPG) and palmitic acid and liquid phase are:

LS mimetic peptide 1 part by weight;

DPPC 20 to 100 parts by weight;

POPG 0 to 50 parts by weight; and palmitic acid 0 to 25 parts by weight.

8. The method of claim 6, wherein the relative amounts of KL4 peptide, dipalmitoyl phosphatidylcholine (DPPC), palmitoyloleoyl phosphatidylglycerol (POPG) and palmitic acid and liquid phase are:

KL4 peptide 1 part by weight;

DPPC 20 to 100 parts by weight;

POPG 0 to 50 parts by weight; and palmitic acid 0 to 25 parts by weight.

9. The method of claim 1 or 2, wherein said physiological pH is from about 6 to about 8.

10. The method of claim 1 or 2, wherein the alkaline aqueous dispersion has a pH of at least 8.

11. The method of claim 1 or 2, wherein said alkaline aqueous dispersion has a pH from about 9 to about 12.

12. The method of claim 1 or 2, wherein said alkaline aqueous dispersion has a pH from about 9 to about 11.

13. The method of claim 1, wherein the LS mimetic peptide is solubilized in a compatible acid prior to forming the alkaline aqueous dispersion.

14. The method of claim 2, wherein the KL4 peptide is solubilized in a compatible acid prior to forming the alkaline aqueous dispersion.

15. The method of claim 1 or 2, wherein said tonicity is adjusted by adding a solution of the salt.

16. The method of claim 1 or 2, wherein the salt is selected from the group consisting of sodium chloride, potassium chloride, ammonium chloride, sodium acetate, sodium cacodylate, sodium bicarbonate, lithium acetate, sodium pyruvate, sodium citrate, sodium phosphate, potassium phosphate, sodium borate, sodium maleate, sodium succinate, potassium phthalate, trimethamine chloride and combinations thereof.

17. The method of claim 16, wherein the salt is selected from the group consisting of sodium chloride, potassium chloride, ammonium chloride and combinations thereof.

18. The method of claim 16, wherein the salt is selected from the group consisting of magnesium chloride, calcium chloride, magnesium sulfate, calcium sulfate, and combinations thereof.

19. The method of claim 1 or 2, wherein the salt is selected from the group consisting of sodium TES (2-[(2-hydroxy-l,l-bis[hydroxymethyl]ethyl)amino]ethanesulfonic acid), glycine amide hydrochloride, Tris maleate, bicarbonate salts, imidazole hydrochloride, N-ethyl morpholine hydrochloride, hydroxylamine hydrochloride, , ADA (N-(2-acetamido)-2-iminodiacetic acid), PIPES (piperazine-N,N'-bis(2- ethanesulfonic acid)), MOPS (3-(N-morpholino)butanesulfonic acid), HEPES (N-(2- hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid)), HEPPSO (N-(2- hydroxyethyl)piperazine-N'-(2-hydroxypropanesulfonic acid)), tricine, bicine, CHES (2-(cyclohexylamino)ethanesulfonic acid), CAPS (3-(cyclohexylamino)-l- propanesulfonic acid) glycylglycine and combinations thereof.

20. A LS mimetic peptide lung surfactant composition comprising an aqueous medium containing an LS mimetic peptide, dipalmitoyl phosphatidylcholine (DPPC), palmitoyloleoyl phosphatidylglycerol (POPG) and palmitic acid, a concentration of base suitable to set the pH of the composition to at least 8, a pH-adjusting concentration of compatible acid suitable to neutralize a portion of the base to adjust pH of composition to be from about 6 to about 8, and a concentration of added salt to bring the dispersion into isotonicity with mammalian lung fluids.

21. A KL4 lung surfactant composition comprising an aqueous medium containing KL4 peptide, dipalmitoyl phosphatidylcholine (DPPC), palmitoyloleoyl phosphatidylglycerol (POPG) and palmitic acid, a concentration of base suitable to set the pH of the composition to at least 8, a pH-adjusting concentration of compatible acid suitable to neutralize a portion of the base to adjust pH of composition to be from about 6 to about 8, and a concentration of added salt to bring the dispersion into isotonicity with mammalian lung fluids.

22. A method for preparing KL4 lung surfactant which method comprises: a. forming an alkaline aqueous solution comprising palmitic acid, KL4, DPPC and POPG; b. adjusting the solution to physiological pH by addition of a compatible acid; and c. adjusting the tonicity of the solution to be isotonic by addition of salt.