Method for preparing pulmonary surfactant

Abstract

Method for preparing pulmonary surfactant The present invention relates to a method for preparing exogenous pulmonary surfactants, in particular modified native or reconstituted surfactants, in which the final filtration step is carried out without the use of chlorinated solvents. The present invention also relates to the products obtained by said method and to the corresponding pharmaceutical compositions.

Description

[Technical field]

[0001] The present invention relates to a method for preparing modified natural surfactants. The present invention also relates to the products obtained by said method, the corresponding pharmaceutical compositions and their uses. [Background technology]

[0002] Pulmonary surfactant is a lipid-protein mixture that lines the alveoli of the lungs. The presence of lipids as a monolayer at the air-liquid interface within the alveoli reduces surface tension, thereby reducing the tendency of the alveoli to collapse during expiration and possibly reducing the seepage of liquid into the air spaces.

[0003] Endogenous pulmonary surfactant comprises approximately 80% by weight phospholipids, 10% by weight protein and 10% by weight neutral lipids such as triglycerides, as well as additional minor components.

[0004] Among phospholipids, dipalmitoylphosphatidylcholine (DPPC) plays the most important role since it stabilizes the alveolar system by forming a monolayer at the air-liquid interface during the inspiration phase.

[0005] Natural pulmonary surfactants usually contain at least four proteins: SP-A, SP-B, SP-C and SP-D. Of these four, SP-B and SP-C are distinct low molecular weight hydrophobic proteins that have been shown to enhance the surface active properties of surfactant phospholipid mixtures, possibly by promoting lipid migration from the bulk phase lamellar organization to the air-water interface and by stabilizing lipid monolayers in exhaled air (see Hawgood S et al Biochim Biophys Acta. 1998 Nov 19;1408(2-3) pp 150-160; Johansson J Biochim Biophys Acta. 1998 Nov 19;1408(2-3) pp 161-72).

[0006] Pulmonary surfactant deficiency or dysfunction leads to a severe respiratory disease termed dyspnea, which is responsible for high morbidity and mortality, especially in preterm infants and adults suffering from various conditions associated with severe pulmonary regurgitation.

[0007] Replacement therapy with a variety of exogenous surfactants has proven beneficial in both experimental and clinical studies.

[0008] In particular, modified natural surfactants extracted from mammalian lungs are widely used as replacement therapies.

[0009] A widely used modified natural surfactant is Curosurf (登録商標) poractant alfa, derived from porcine lung, sold under the trademark poractant alfa (Chiesi Farmaceutici SpA, Italy); beractant (Survanta), derived from bovine lung (登録商標) , AbbVie Inc, USA) and bovactant (Alveofact (登録商標) Lyomark Pharma GmbH, Germany), and calfactant from calf lung (Infasurf (登録商標) , Ony Biotech, USA).

[0010] Typically, surfactant is removed from the airways of mammalian lungs by various methods, then extracted with an organic solvent and dried.

[0011] Hereinafter, the raw material obtained by drying is defined as a paste.

[0012] For example, US 6,129,934 discloses a method for obtaining calfactant, which involves removing surfactant from the airways of the lungs of calves by a process of bronchoalveolar lavage. The liquid collected by bronchoalveolar lavage is centrifuged to collect a concentrated surfactant precipitate. The surfactant precipitate is then brought to a desired volume by adding an aqueous solution such as saline.

[0013] Extraction of the surfactant from the aqueous suspension can be carried out using a variety of organic solvents, including, but not limited to, chloroform, benzene, a mixture of chloroform and methanol, a mixture of ether and ethanol, and a mixture of hexane and ethanol. In a preferred embodiment, a mixture of chloroform and methanol is used.

[0014] Alternatively, a method for obtaining beractant is disclosed in US 4,397,839, and includes the steps of: (a) contacting minced mammalian lung tissue with an electrolyte solution to obtain an extract; (b) centrifuging the extract to collect a crude precipitate; (c) adjusting the specific gravity of an aqueous suspension of the crude precipitate by adding sodium chloride and centrifuging the adjusted suspension to isolate a top layer comprising an emulsified scum layer; (d) dialyzing the aqueous suspension of the top layer and freeze-drying the dialyzed suspension to obtain a crude dry product; (e) contacting the crude dry product with an acetate ester to collect materials insoluble in the acetate ester, and then contacting the insoluble materials with an organic solvent mixture to obtain a refined filtrate; and (f) concentrating the refined filtrate to obtain a solid residue.

[0015] A method for preparing poractant alfa is disclosed in EP 286,011 and comprises the following steps: i) mincing the lungs of the animal and washing them with saline solution; ii) isolating the surfactant by subsequent filtration, centrifugation and extraction with a mixture of chloroform and methanol 2:1 (v / v); iii) recovering the raw lipid fraction via evaporation of the solvent to dryness; iv) eluting the latter with the resin Lipidex using 1,2-dichloroethane:methanol = 1:4 (v / v) as eluent. (登録商標) and purifying the product by reverse phase chromatography using -5000.

[0016] The purification step by chromatography columns is considered to be crucial to ensure modified natural surfactants with a well-defined and reproducible composition and has been optimized to obtain a surfactant enriched in the components believed to be responsible for the biological activity, i.e. polar lipids, mainly phospholipids, the hydrophobic proteins SP-C and SP-B, and substantially free of non-essential components, i.e. carbohydrates, neutral lipids, e.g. triglycerides, cholesterol and free fatty acids.

[0017] The need for improved surfactant preparations has led to the development of synthetic surfactants that mimic the composition of modified natural surfactants, known as reconstituted surfactants.

[0018] An example of a reconstituted surfactant is, but is not limited to, rutinactant (Surfaxin), a product known in the art with experimental code CHF 5633, having the composition disclosed in the abstract paragraphs on pages 9 and 10 of WO2010 / 139442. TM , Windtree Therapeutics, Inc., Warrington, Pa.) and elifactant.

[0019] Regardless of the type of surfactant, the components of the modified native surfactant paste or reconstituted surfactant are generally dissolved and undergo a further purification step by sterile filtration.

[0020] For example, according to EP 286,011, the pre-filtered paste can be dissolved in 98:2 chloroform / methyl alcohol (V / V).

[0021] However, the use of certain solvents, such as chloroform, can potentially affect both the remaining steps in the manufacturing process and the quality of the product. For example: i) Both pipes and filters are usually exposed to chemical stresses, which may reduce the range of usable materials; ii) As a result, the levels of extractable components in the product may require further processing; iii) The solvent evaporation process may take some time or multiple repetitions to remove residual chemical components.

[0022] Therefore, to reduce or mitigate the above risks and at the same time ensure all the quality attributes of the product, alternative processes involving different solvents are desirable.

[0023] On the other hand, due to the hydrophobic structure of phospholipids, it is very difficult to validate the solubilization phase in aqueous media. Therefore, new candidates must be organic solvents that meet several defined criteria, such as: i) It can be substituted for chloroform; ii) The presence of temperature-sensitive components such as proteins and the ability to dissolve surfactants at around body temperature. iii) allow a similar proportion of the problematic volume in the dissolution step; iv) being suitable for the evaporation step, i.e. having the right physical properties (boiling point, vapour pressure, etc.); v) Readily available in the market with reduced or no risk of phase-out.

[0024] This problem is solved by the present invention. Summary of the Invention

[0025] In a first embodiment, the present invention provides a method for preparing an exogenous pulmonary surfactant selected from a modified natural surfactant or a reconstituted synthetic surfactant, comprising the steps of: i) extracting said modified natural surfactant from the lungs of a mammal in the form of a dry paste or mixing the dry components of said reconstituted synthetic surfactant; ii) purifying the resulting paste or mixture; wherein the purification in step ii) is carried out by the following steps: iii) dissolving the modified native surfactant paste or the components of the reconstituted surfactant in an organic solvent comprising 2-methyl-2-propanol; iv) subjecting the resulting solution to sterilization by filtration; and v) drying process; The method includes:

[0026] Advantageously, a dry paste of the modified natural surfactant can be obtained from the mammalian lungs by lavage or column elution.

[0027] Preferably, the modified natural surfactant is poractant alfa.

[0028] Thus, in a preferred embodiment, the present invention provides a method for preparing poractant alfa, comprising the steps of: i) chopping the pig lungs in a blender; ii) extracting the minced lungs with a physiological solution, followed by filtering and centrifuging the mixture; iii) extracting the supernatant with an organic solvent or a mixture thereof, followed by evaporating the organic phase to dryness; iv) isolating the fraction containing polar lipids and hydrophobic proteins SP-C and SP-B; v) collecting and pooling said fractions and then evaporating the organic solution to dryness to obtain the surfactant in the form of a dry paste; vi) dissolving the resulting paste in an organic solvent comprising 2-methyl-2-propanol; vii) subjecting the resulting solution to sterilization; and viii) Drying process.

[0029] The present invention also relates to an exogenous pulmonary surfactant obtainable by the method of the present invention.

[0030] In another aspect, the present invention relates to a pharmaceutical formulation comprising as an active ingredient the exogenous pulmonary surfactant obtainable by the method of the present invention.

[0031] The present invention also relates to the exogenous pulmonary surfactant obtained by the method of the present invention for use in the prevention or treatment of various pulmonary disorders, abnormal conditions and diseases caused by or associated with pulmonary surfactant deficiency or dysfunction.

[0032] In a further aspect, the present invention relates to the use of the exogenous pulmonary surfactant obtained by the method of the present invention in the manufacture of a medicament for preventing or treating various pulmonary disorders, abnormal conditions and diseases caused by or associated with pulmonary surfactant deficiency or dysfunction.

[0033] Furthermore, the present invention provides a method for preventing or treating various pulmonary disorders, abnormal conditions and diseases caused by or associated with pulmonary surfactant deficiency or dysfunction, comprising administering to a patient in need of such treatment a therapeutically effective amount of exogenous pulmonary surfactant obtained by the method of the present invention. [Brief description of the drawings]

[0034] [Figure 1]Figure 2 shows the results of tidal volume (ml / kg) as a function of time / pressure for Curosurf® obtained by the method of the present invention compared to Curosurf® control and untreated animals.

[0035] definition The terms "surfactant" and "surface-active substance" are used synonymously.

[0036] For a comprehensive definition of the various types of exogenous pulmonary surfactant, see Wilson D. Expert Opin Pharmacother 2001, 2, 1479-1493.

[0037] The term "modified native surfactant" refers to a lipid extract of minced mammalian lung from which the hydrophilic proteins SP-A and SP-D have been removed and which contains certain amounts of the hydrophobic proteins SP-B and SP-C due to the lipid extraction process used in the preparation process. Depending on the extraction method, different amounts of phospholipids, non-surfactant lipids, and other minor components may be included.

[0038] The term "artificial surfactant" refers to synthetic compounds, primarily simple mixtures of phospholipids and other lipids, formulated to mimic the lipid composition and properties of natural surfactants, but which do not contain surfactant proteins.

[0039] The term "reconstituted" pulmonary surfactant refers to an artificial pulmonary surfactant to which has been added pulmonary surfactant proteins / peptides isolated from an animal or proteins / peptides produced by recombinant technology.

[0040] The term "dry" refers to the surfactant material obtained after evaporation of the organic solvent. The amount of residual solvent, including water, is limited.

[0041] The alcohol 2-methyl-2-propanol is also known as tert-butanol.

[0042] The term "polar lipids" includes primarily phospholipids as well as minor components such as plasmalogens, glycolipids, cardiolipin and lysophospholipids.

[0043] The term "phospholipid" refers to a lipid consisting of a non-polar hydrophobic tail, a glycerol or sphingosine moiety, and a polar head group. The non-polar hydrophobic tail is usually a long chain fatty acid that is saturated (e.g., myristic acid, palmitic acid, and stearic acid), monounsaturated (e.g., oleic acid), or polyunsaturated (e.g., linoleic acid and arachidonic acid).

[0044] The polar head has a phosphate group attached to a nitrogenous base.

[0045] The phospholipid fraction in surfactant mainly contains phosphatidylcholine (PC), phosphatidylinositol (PI), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylglycerol (PG) and sphingomyelin (SM).

[0046] The term "neutral fats" includes triglycerides, diglycerides and monoglycerides.

[0047] The term "size-exclusion liquid-gel chromatography" refers to a chromatography system in which the stationary phase is a gel that retains substances to be separated according to molecular size.

[0048] The term "biosimilar of poractant alfa" means a modified natural pulmonary surfactant that has an identical safety profile, is therapeutically equivalent, has a qualitative-quantitative compositional similarity of at least 80% (especially with regard to phospholipids and surfactant proteins SP-B and SP-C) and has a viscosity of 15 mPas (cP) or less at room temperature when suspended in aqueous solution at a concentration of 80 / mg / ml. Viscosity can be determined according to known methods.

[0049] The term "blender" refers to a device, typically of stainless steel, used to mix, grind, and chop organic materials. It generally consists of a container with rotating metal blades at the bottom, driven by an electric motor.

[0050] The term "sterile" refers to a product that meets the criteria for sterility according to the European Pharmacopoeia (Ph. Eur. 1998, Chapters 2.6.1 and 5.1.1). Further regulations regarding sterility of final products include the United States Pharmacopoeia 23 / NF18, 1995, pp. 1686-1690 and 1963-1975.

[0051] The "surfactant activity" of a surfactant preparation is defined as its ability to reduce surface tension.

[0052] The in vitro efficacy of exogenous surfactant preparations is typically tested by measuring their ability to reduce surface tension using a suitable device such as a Wilhelmy Balance or a Captive Bubble Surfactometer.

[0053] The in vivo effectiveness of exogenous surfactant preparations is generally tested by measuring two parameters: i) tidal volume, which is a measure of lung compliance; and ii) Lung gas volume, which is a measure of alveolar expansion or patency at end-expiration and hence the ability to form a stable phospholipid membrane within the alveoli at end-expiration.

[0054] Detailed Description of the Invention The characteristics of the present process, the resulting products and the corresponding pharmaceutical compositions are described in the detailed description below.

[0055] In a first embodiment, the present invention provides a method for preparing an exogenous pulmonary surfactant selected from a modified natural surfactant or a reconstituted synthetic surfactant, comprising the steps of: i) extracting said modified natural surfactant from the lungs of a mammal in the form of a dry paste or mixing the dry components of said reconstituted synthetic surfactant; ii) purifying the resulting paste or mixture; iii) wherein the purification of step ii) is carried out by the following steps: iv) dissolving the modified native surfactant paste or the components of the reconstituted surfactant in an organic solvent comprising 2-methyl-2-propanol; v) subjecting the resulting solution to sterilization by filtration; and vi) drying process; The present invention provides a method comprising:

[0056] Advantageously, pastes of modified natural surfactants can be prepared according to methods reported in the art, for example methods based on elution on chromatographic columns or bronchoalveolar lavage.

[0057] Preferably, the organic solvent comprises 2-methyl-2-propanol, more preferably the solvent consists solely of 2-methyl-2-propanol, in a further preferred embodiment, the solvent is anhydrous 2-methyl-2-propanol having a purity of greater than 99.5%.

[0058] The dissolution in step iii) of the method of the present invention is usually carried out with gentle heating, ie at a temperature of 30° C. to 40° C., preferably at 35° C.±1° C., with stirring.

[0059] The speed and duration of stirring must be determined by one skilled in the art, but this is typically 300-400 rpm for 15-30 minutes.

[0060] The person skilled in the art must determine the ratio between surfactant and organic solvent. In a preferred embodiment of the invention, said ratio is higher than 5% w / w. It has been found in practice that lower ratios do not result in complete dissolution.

[0061] It preferably contains 6 to 20% w / v, more preferably 8 to 12% w / v.

[0062] The sterilization by filtration in step iv) of the method of the present invention can be carried out according to the knowledge of the person skilled in the art.

[0063] Optionally, the solution is subjected to a clarification step through a membrane filter with a pore size of 0.45 microns; it is then sterilized by filtration, for example, using filters with pore sizes of 0.22 and 0.1 microns.

[0064] In step v) of the method of the invention, the solvent can be removed by methods known to those skilled in the art, for example by dialysis or evaporation under nitrogen and / or exposure to vacuum, or other suitable techniques such as freeze-drying.

[0065] The chemical and physical properties of 2-methyl-2-propanol as a solvent in the present invention allow for the advantageous application of more rapid drying techniques such as tangential flow filtration.

[0066] Notably, according to this process, the amount of residual 2-methyl-2-propanol at the end of the drying process is less than 3000 ppm, preferably less than 1500 ppm.

[0067] Any exogenous modified natural or reconstituted pulmonary surfactant may be used. Advantageously, the reconstituted surfactant is Surfaxinactant. TM , Windtree Therapeutics, Inc., Warrington, Pa.) and the surfactant referred to in the art as CHF 5633 (Chiesi Farmaceutici SpA, Italy); the modified natural surfactant is selected from poractant alfa (Curosurf (登録商標) ,Chiesi Farmaceutici SpA, Italy) and its biosimilar, Beractant (Survanta (登録商標), AbbVie Inc, USA) and bovacant (Alveofact (登録商標) , Lyomark Pharma GmbH, Germany), Calfactant (Infasurf (登録商標) , Ony Biotech, USA), Bles (登録商標) (Bles Biochemicals Inc, Canada), KeLiSu (Double Crane, China), Surfactant TA (Surfaten (登録商標) , Mitsubishi Tanabe Pharma Corporation, Tokyo) and other Surfacen (登録商標) , Beraksurf (登録商標) , Newfactan (登録商標) , Surfactant Gray (登録商標) and surfactant BL (登録商標) A preferred modified natural surfactant is poractant alfa.

[0068] Thus, in a preferred embodiment, the present invention relates to a method for preparing poractant alfa, comprising the steps of: i) mincing the pig lungs in a suitable blender; ii) extracting the minced lungs with a physiological solution, followed by filtering and centrifuging the mixture; iii) extracting the supernatant with an organic solvent and then evaporating the organic phase to dryness; iv) isolating the fraction containing polar lipids and hydrophobic proteins SP-C and SP-B; v) collecting and pooling said fractions and then evaporating the organic solution to dryness to obtain the surfactant in the form of a dry paste; vi) dissolving the modified natural surfactant paste in an organic solvent comprising 2-methyl-2-propanol; vii) subjecting the resulting solution to sterilization by filtration; and viii) Drying process.

[0069] Preferably, the organic solvent consists solely of 2-methyl-2-propanol.

[0070] As explained in more detail in the experimental section below with reference to Examples 3 and 4, the use of 2-methyl-2-propanol allows for the production of a product with the same qualitative and quantitative composition and similar tidal volumes as the Poractant Alpha control batch.

[0071] Step i) can be carried out according to the knowledge of the person skilled in the art.

[0072] In step ii) above, the minced lungs are extracted with a physiological solution, the mixture is filtered through a strainer and advantageously centrifuged at 1,000×g at 20° C. for less than 30 minutes, preferably 15 minutes, to remove cellular debris.

[0073] After cooling to 4° C. for 2 hours, the supernatant is further recentrifuged at 3,000×g, advantageously within 2 hours, preferably within 1 hour, more preferably for 15 minutes.

[0074] In step iii) above, the pelletized raw solid surfactant is removed and extracted with a mixture of halogenated hydrocarbons and short chain aliphatic alcohols as disclosed in EP 286,011.

[0075] The resulting solution is filtered, washed with water, and then evaporated to dryness under vacuum using equipment well known to those skilled in the art.

[0076] According to step iv), the separation of the fractions containing the components responsible for the biological activity (polar lipids, mainly phospholipids, hydrophobic proteins SP-C, SP-B) from the other fractions by extraction can be carried out according to methods known in the art (Saini RK et al Int J Molecular Sci 2021, 22, 1-19).

[0077] In the case of poractant alfa, the separation of the fraction containing the components responsible for the biological activity (polar lipids, mainly phospholipids, hydrophobic proteins SP-C, SP-B) from the other fractions (step iv) is advantageously carried out by size-exclusion gel chromatography using a lipophilic Sephadex derivative as stationary phase and a mixture of halogenated hydrocarbons and short-chain aliphatic alcohols as eluent, as reported in EP 286,011.

[0078] More advantageously, the stationary phase used to pack the chromatographic column is available from different suppliers such as Lipidex. (登録商標) -Sephadex, sold under the trademark 5000 (登録商標) It is composed of:

[0079] Alternatively, step iv) can be carried out using a method that does not use chlorinated solvents such as chloroform.

[0080] For example, step iv) can be carried out using a supercritical fluid, as described in EP 670846. Further adjustments of the inert support, the pressure and temperature of the supercritical fluid, and the type and amount of co-solvent can be made by the skilled person according to their knowledge.

[0081] Otherwise, said process can be carried out by first removing components such as cholesterol and mono-, di- and triglycerides by nanofiltration and ultrafiltration according to methods known in the art (C. Allegre et al. / Journal of Membrane Science 269 (2006) 109-117), and then obtaining the components responsible for the biological activity by ion exchange chromatography and a mixture of t-butanol and water as eluent (Separation of lipid mixtures, 1972. Lab. Tech. Biochem. Mol. Biol. 3, 393-469).

[0082] In said step v), the fractions containing polar lipids and hydrophobic proteins SP-B and SP-C are collected and pooled, and then the organic solution is evaporated to dryness at a temperature below 50°C, preferably below 40°C.

[0083] The above steps vi) to viii), carried out as disclosed above, are of particular importance for poractant alfa.

[0084] Said modified natural surfactants certainly have unique properties in terms of viscosity compared to others currently available.

[0085] According to the prior art, this is due to the fact that poractant alfa is essentially composed of components believed to be responsible for its biological activity, namely polar lipids and the hydrophobic proteins SP-C and SP-B, and that the phospholipid fraction is rich in polyunsaturated fatty acids, including two components, plasmalogens and PL (see Rudiger M et al. Am J Physiol Lung Cell Mol Physiol 2005, 288, 379-383).

[0086] Therefore, the solvent and conditions selected should ensure that the composition of the surfactant in the following steps vi) and vii) is not altered.

[0087] The paste of poractant alfa at the end of step viii) has the following composition (all values ​​given as a percentage of the total weight of the dry mass of the surfactant): Total phosphorus content (P): 3.6-4.2%; hydrophobic proteins SP-C and SP-B: 0.5–2.2%; Neutral lipid: 0.5% or less; Cholesterol: 0.5% or less; Free fatty acids (FFA): 1.5% or less, preferably less than 1.0%; Residual solvents: 6.0% or less;

[0088] As far as the phosphatidylcholine species are concerned, the surfactant has the following percent composition based on the total phosphorus content: Phosphatidylcholine (PC) content: 66-77%; Dipalmitoylphosphatidylcholine (DPPC) content: more than 28%, preferably 33-45%; Lysophspatidylcholine (LPC) content: 2.0% or less

[0089] Optionally, the dry paste may contain small amounts of components that are not essential for biological activity, such as neutral lipids, cholesterol, free fatty acids, carbohydrates and residual solvents.

[0090] Advantageously, the total amount of said non-essential components is less than 5%, preferably less than 3.5%, more preferably less than 2.5% and even more preferably less than 1% of the dry mass of the surfactant, calculated relative to the total weight of the surfactant.

[0091] The various components of the pulmonary surfactant can be determined according to methods reported in the art.

[0092] For example, the total phosphorus content can be estimated according to a colorimetric procedure based on the method of Barlett GR J Biol Chem, 1959, 234, 406-408.

[0093] The contents of PC and LPC can be estimated by the following procedure.

[0094] First, the phospholipid classes of surfactants are first separated by two-dimensional thin layer chromatography (TLC) according to Poorthius BJH et al. J Lipid Res 1976, 17, 433-436.

[0095] Silica corresponding to the PC and LPC spots (identified by comparison with reference standards) are removed from the TLC plate with an appropriate blade and transferred to a test tube.

[0096] Mineralization of the samples is carried out in each test tube according to the procedure reported for the determination of total phosphorus content (mentioned above).

[0097] The DDPC content can be estimated with OsO4 according to the method reported in Mason RJ J Lipid Res 1976, 17, 281-284.

[0098] The phosphorus content associated with DPPC is determined relative to the total phosphorus content.

[0099] The free fatty acids, neutral lipids (triglycerides, diglycerides and monoglycerides) and cholesterol (fractional free cholesterol and cholesterol esters) contents can be estimated by HPLC analysis by methods known in the art.

[0100] The content of hydrophobic proteins SP-C and SP-B can be measured using the commercially available kit "BCA Protein Quantitation Reagent" according to the following procedure. Otherwise, it can be measured according to HPLC or capillary electrophoresis methods reported in the art.

[0101] Carbohydrates can be quantified according to the method described by Dubois et al Anal Chem 1956, 28, 350-356 and expressed as glucose.

[0102] Residual solvent and residual water content are estimated by gas chromatography and the Karl Fischer method, respectively, according to methods well known to those skilled in the art.

[0103] The present invention also relates to a modified native surfactant extracted in the form of a paste from mammalian lungs, or a reconstituted surfactant, obtained or obtainable by the method of the present invention.

[0104] The present invention also relates to a pharmaceutical composition comprising a modified native surfactant extracted in the form of a paste from a mammalian lung, or a reconstituted surfactant, obtained or obtainable by the method of the present invention.

[0105] The composition is advantageously administered in the form of a solution, dispersion, suspension or dry powder. Preferably, the composition is in the form of a sterile formulation in which the pulmonary surfactant is suspended in a suitable solvent or resuspension medium, typically physiological aqueous sodium chloride solution.

[0106] Thus, to prepare a sterile formulation in the form of a suspension, the method of the invention may optionally comprise the following steps: -ix) resuspending the dried product in a sterile physiological aqueous sodium chloride solution at room temperature with stirring: -x) optionally adjusting the pH of the suspension to a desired value, and -xi) filling the suspension under sterile conditions into suitable single-use vials.

[0107] Step ix) is known in the art as thin film hydration and can be achieved by mechanical agitation or ultrasonic treatment, optionally followed by extrusion.

[0108] Advantageously, the concentration of the pulmonary surfactant ranges from about 2 mg to about 160 mg of surfactant per ml, preferably from 10 mg / ml to 100 mg / ml, more preferably from 20 mg / ml to 80 mg / ml. When using poractant alfa, the preferred concentration is 80 mg / ml.

[0109] The sterile preparation containing pulmonary surfactant obtained by the method of the present invention is administered by methods known to those skilled in the art, preferably by intratracheal placement (infusion or bolus) or nebulizer, and is useful for treating or preventing various pulmonary disorders, abnormal conditions and diseases caused by or associated with pulmonary surfactant deficiency or dysfunction. Examples include hyaline membrane disease, infantile and adult respiratory distress syndrome (IRDS and ARDS), acute lung injury (such as caused by inhalation of ozone, inhalation or close to inhalation of smoke), surfactant inactivation conditions induced by volume trauma and barotrauma, meconium aspiration syndrome, capillary leak syndrome, bacterial and viral pneumonia and bronchopulmonary dysplasia.

[0110] They are also useful in the treatment or prevention of other respiratory disorders such as pneumonia, bronchitis, COPD (chronic obstructive pulmonary disease), asthma and cystic fibrosis, and in the treatment of serous otitis media (glue ear).

[0111] The following examples illustrate the invention in detail. EXAMPLES

[0112] Example 1 - Preparation of a dry paste of Poractant Alpha Approximately 180 kg of porcine lungs are ground in a blender and the tissue fragments are washed with physiological solution. The mixture is filtered through a strainer and pre-centrifuged at 1000 × g and 20 °C for 15 min to remove cellular debris. The supernatant is then recentrifuged at 3000 × g and 4 °C for 2 h. The raw surfactant is removed, extracted with chloroform:methanol 2:1 (v / v), filtered, washed with water, and the organic phase is evaporated to obtain the raw lipid extract. The lipid fraction extract (approximately 500 g) is recovered in approximately 4 liters of chloroform:methanol 1:4 (v / v), filtered through a 2.5 micron membrane filter flooded with nitrogen at 0.2-0.4 atm, and then eluted with Lipidex with chloroform:methanol 1:4 (v / v) as the eluent. (登録商標) The mixture is separated by reverse phase chromatography using a column packed with -5000 packaging material 2.

[0113] Approximately 200 g of purified poractant alfa was obtained as dry material and tested using the analytical methods reported above.

[0114] The purified poractant alpha dry matter has the following composition (all values ​​are given as percentages of the total dry mass): Total phosphorus content: 3.8%; Phosphatidylcholine (PC) content: 68%; Dipalmitoylphosphatidylcholine (DPPC) content: 40%; Lysofuspatidylcholine (LPC) content: 1.0% hydrophobic proteins SP-C and SP-B: 1.6%; Free fatty acids: 0.9%; Neutral lipids: undetectable; Cholesterol: undetectable; Carbohydrates: Undetectable.

[0115] Example 2 - Determination of a Solvent Suitable for Sterilization by Filtration Evaluations are included for the alcohol chemical groups: ethanol, 1-propanol, 2-propanol, and 2-methyl-2-propanol (tert-butanol).

[0116] As an initial screening test, equal amounts of Poractan Alpha Paste (approximately 10 g) were diluted and mixed with 100 mL of each alcohol, in each case with slight heating (35°C) to promote dissolution and avoid precipitation.

[0117] The associated appearance was then visually assessed and compared to a reference solution of poractant alpha in chloroform. Of the alcohols tested, only tert-butanol provided a clear solution similar to the chloroform reference. The other alcohols yielded milky or very turbid, heterogeneous solutions.

[0118] After defining tert-butanol as an alternative solvent, it was decided to move to a filtration process to understand the product behavior during unit operations.

[0119] The following filtration system was used: -Pore diameter 0.45μm, filtration surface approximately 17cm 2 Fiberglass prefilter. -Pore diameter 0.22μm, filtration surface approximately 17cm 2 PVDF bioburden reduction filter. -Pore diameter 0.1μm, filtration surface approximately 17cm 2 PVDF sterilizing filter.

[0120] Filtration was performed by peristalsis and constant pressure monitoring, and the ΔP between the upstream and downstream sides of the membrane was calculated.

[0121] For comparison, the same test was performed on both t-butanol and chloroform solutions.

[0122] No criticality was observed in the process.

[0123] The resulting solution was evaporated in a rotary evaporator according to the following parameters: (登録商標) ) was used to dry the mixture: -Bath temperature: 35℃ -Cooler temperature: 15~17℃ -Vacuum: 45~50mbar - Rotation: 100 rpm

[0124] The solid paste was collected and resuspended in a sterile solution of 0.9% w / v sodium chloride in water. The final suspension was filled into vials and stored at 2-8°C.

[0125] Example 3 - Product characterization Based on the above process, the feasibility of the general product and the Curosurf (登録商標) Pilot batches were prepared to evaluate the effect of key critical quality attributes of

[0126] The dried paste was resuspended in sterile physiological aqueous sodium chloride solution and then transferred to Rotavapor (登録商標) Mixed in.

[0127] The suspension thus obtained was distributed into vials for use at a concentration of 80 mg of surfactant per ml of physiological aqueous sodium chloride solution.

[0128] Analytical testing was performed on the vials to determine whether the current specifications for the product were still being met despite the modifications applied to the manufacturing process.

[0129] The results met all specification criteria.

[0130] This batch was also tested for morphological imaging for particle characterization and compared to a reference batch produced under standard conditions, i.e., the current process using chloroform as the process solvent.

[0131] Curosurf obtained by the method of the present invention (登録商標) However, in terms of micelle size and micelle shape, (登録商標) It was found to be very similar to the control batch.

[0132] Example 4 - In vivo characterization To determine surfactant activity, the effect on tidal volume when administered intratracheally with the batches obtained according to the method of the invention was evaluated in an in vivo test using premature newborn rabbits.

[0133] Curosurf (登録商標) A control batch was used.

[0134] The surfactant preparation was administered at a standard dose of 2.5 ml / kg.

[0135] Immature newborn rabbits were artificially ventilated in parallel with a standardized series of peak insufflation pressures. The pressure was first set at 35 cmH2O for 1 min to open the lungs. After this recruitment manoeuvre, the pressure was reduced to 25 cmH2O for 15 min, then to 20 cmH2O and 15 cmH2O.

[0136] Finally, the pressure is increased again to 25 cmH2O over a period of 5 minutes, after which the lungs are ventilated with nitrogen for a further 5 minutes.

[0137] Tidal volumes expressed in ml / kg were measured and the results, presented as median values, are reported in FIG.

[0138] Curosurf obtained by the method of the present invention (登録商標) Animals treated with Curosurf (登録商標) It can be seen that the tidal volumes are similar to those obtained with the control batch.

Claims

1. A method for preparing an exogenous pulmonary surfactant selected from a modified natural surfactant or a reconstituted synthetic surfactant, comprising: i) extracting the modified natural surfactant from the lung of a mammal in the form of a dry paste or mixing the dry components of the reconstituted synthetic surfactant; ii) purifying the resulting paste or mixture; iii) the purification in step ii) is carried out by the following steps: iv) dissolving the paste of the modified natural surfactant or the components of the reconstituted surfactant in an organic solvent containing 2-methyl-2-propanol; v) subjecting the resulting solution to sterilization by filtration; and vi) a drying step, A method comprising the above steps.

2. The method according to claim 1, wherein the organic solvent consists only of 2-methyl-2-propanol.

3. The method according to claim 1, wherein the dissolving step i) is carried out with stirring at a temperature of 30°C to 40°C.

4. The method according to claim 3, wherein the stirring is carried out at a speed of 300 to 400 rpm for 15 to 30 minutes.

5. The method according to claim 1, wherein the ratio of the surfactant to the organic solvent in step 1 is 6 to 20% w / v.

6. The method according to claim 1, wherein the reconstituted surfactant is lucinactant or elifactant.

7. The method according to claim 1, wherein the modified natural surfactant is selected from the group consisting of poractant alfa and its biosimilars, beractant, calfactant, and bovactant.

8. The method according to claim 7, wherein the modified natural surfactant is poractant alfa.

9. The method according to claim 6, comprising the following steps: i) a step of mincing porcine lungs with a suitable blender; ii) a step of extracting the minced lungs with a physiological solution, then filtering and subjecting the mixture to centrifugation; iii) a step of extracting the supernatant with an organic solvent or a mixture thereof, and then evaporating the organic phase to dryness; iv) a step of separating the fraction containing polar lipids and hydrophobic proteins SP-C and SP-B; v) a step of collecting and pooling the said fractions, and then evaporating the organic solution to dryness to obtain the surfactant in the form of a dry paste; vi) a step of dissolving the paste of the modified natural surfactant in an organic solvent containing 2-methyl-2-propanol; vii) a step of subjecting the obtained solution to sterilization by filtration; and viii) a drying step.

10. The method according to claim 9, wherein the separation in step iv) is carried out by size exclusion liquid-gel chromatography.

11. The method according to claim 9, wherein the separation in step iv) is carried out without using a chlorine-based solvent.

12. The method according to claim 11, wherein the separation in step iv) is carried out by using a supercritical fluid.

13. The method according to claim 1, further comprising the following steps: ix) a step of resuspending the dried product in a sterile aqueous sodium chloride solution while stirring at room temperature; x) optionally, a step of adjusting the pH of the suspension to a desired value; and xi) a step of filling the suspension into a suitable single-use vial under sterile conditions.

14. An exogenous pulmonary surfactant obtained by the method according to any one of claims 1 to 13.

15. A pharmaceutical preparation containing an exogenous pulmonary surfactant obtained by the method according to any one of Claims 1 to 13.

16. An exogenous pulmonary surfactant obtained by the method according to any one of Claims 1 to 13 for use in the prevention or treatment of various pulmonary disorders, abnormalities and diseases caused by or associated with a deficiency or dysfunction of pulmonary surfactant.

17. The exogenous pulmonary surfactant according to Claim 16 for use in the treatment of neonatal respiratory distress syndrome (RDS).

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