A PROCESS FOR THE PURIFICATION OF NATURAL PULMONARY SURFACTANT MATERIAL USING SUPERCRITICAL FLUIDS

Abstract: Natural pulmonary surfactant material, consisting of a mixture of phospholipids and a small percentage of specific proteins, is purified by means of supercritical fluids, particularly with CO2.

Description: A PROCESS FOR THE PURIFICATION OF NATURAL PULMONA SURFACTANT MATERIAL USING SUPERCRITICAL FLUIDS The present invention relates to a process for th purification of animal pulmonary surfactant by means o extraction and separation of undesired component consisting of lipidic substances of a low polar power. Pulmonary surfactant (PS) is a vital material coating the surface of pulmonary alveoli, which act reducing the surface tension at the air-liqui interface, thus stabilizing alveoli and preventing the from collapsing during respiration. Lack of PS causes a severe respiratory diseas named respiratory distress, which is the cause of hig morbidity and mortality, particularly in pre-ter infants and in adults affected by various pathologie involving a severe pulmonary insufficiency. The effectiveness of a substitutive therapy base on the instillation of exogenous pulmonary surfactan material in lungs has already been widely proved. Natural surfactants and pharmaceutica compositions for the use thereof in respirator distress have been disclosed in a number of patents: G 2,050,832, US 4,397,839 and EP 0,119,056 (Toky Tanabe); EP 0,145,005 (Veb Arzneimittel Dresden); E 0,286,011 (Chiesi). All of the processes for the preparation o surfactant material starting from animal lung disclosed in said patents are substantially based on a extraction step, characterized by a number of washing centrifugation and extraction operations, with suitabl organic solvents, and a subsequent purification step i order to separate the active fraction and remov impurities as well as undesired inactive components. The purification step is of paramount importanc in obtaining an active material having a well-defined reproducible composition. Natural PS is a complex lipoprotein mainl consisting of phospholipids and specific proteins. Phospholipids, particularly phosphatidylcholin 10 (PC), its disaturated form dipalmitoy phosphatidylcholine (DPPC) and phosphatidylglycero play an essential role in the physiological function o natural surfactant in reducing the surface tension i alveoli. 15 Specific apoproteins of surfactant, identified a SP-B and SP-C, are also vital for the biological actio of natural surfactant, since they promote surfactan adsorption and distribution on the lung surface. Surfactant disclosed in GB 2,050,832 and in US ?<-> 4,397,839 is subjected to a series of dialysis, centrifugation, washing, filtration, drying (or freeze- drying) procedures in order to remove impurities and the neutral lipids and cholesterol excesses. Nevertheless, the resulting active material has a I' widely variable composition, in fact the inventors themselves, in the subsequent EP Patent 0,119,056, make use of a semi-synthetic preparation consisting of a natural lipoprotein, extracted from mammal lung, mixed with phospholipids and synthetic fatty acids, in order ~ >< > to obtain a better composition. Surfactant disclosed in EP 0,145,005 is purified by extraction with a water-insoluble alcohol an subsequent separation from the fraction which i insoluble in said alcohol. However, an active materia is obtained having a very low content in phospholipids ι> which are present in a ratio of 40-70% by weight. On the contrary, surfactant disclosed in E 0,286,011, which is of a completely natural origin, ha a satisfactory composition characterized by a hig polar lipid content and substantially free from suc ιo inactive components as carbohydrates, neutral lipid and cholesterol. In this case, the purification of th crude surfactant material is carried out by elutio through a column of Lipidex R , a particularl hydrophobic resin which is specifically suitable fo 15 the separation of polar lipids. However thi purification method, although proving to be highl effective, suffers from a series of drawbacks o industrial scale, above all the use of large amounts o chlorinated solvents. .'•"' The extraction with supercritical fluid provide an effective alternative method for the purification of natural substances. Supercritical fluids are substances which i ambient conditions behave as gas, whereas at 2'ι temperatures and pressures higher than the critical Temperature (cT) and the critical Pressure (cP) are i the liquid state. The first examples of the use of the extractio with supercritical fluids relate to extraction of hop, f' the decaffeinization of coffee, the extraction of nicotine from tobacco, the fractioning of oils and fats, the extraction of essential oils used both perfumes and as flavours, the extraction of vegetab oils of various type. PCT Application 87/02697 discloses a process f 5 the extraction of animal material components by the u of supercritical fluids. The process consists contacting homogenates of animal tissues or organs wi the supercritical fluid, thereafter carrying out t extraction under specific operative conditions. in However, the use of said process in t purification of a complex lipoprotein such as PS, whi is physically organized in the form of lamellar bodi or tubular myelin, turned out to be ineffective. Now it has been found, and it is the object of t .1" present invention, that the procedure wi supercritical fluids can advantageously be used for t purification of PS if the extractive step is preced by dispersing the material on a high surface area ine solid, to increase as much as possible the surface ar -'" of the material to extract, therefore promoting t permeation of the material itself with the solven increasing its extractive power. Therefore, one of the objects of the invention provided by an improved process for the purification ? r j natural PS. A further object of the invention is the use supercritical fluids for the extraction of materia which are components of animal tissues or organs, whi process is characterised in that the starting materi Λ > is previously dispersed on an inert carrier material order to promote the extraction. The supercritical fluid is preferably carbo dioxide, since it is non toxic, non flammable, no corrosive, it has a low critical temperature (31.06 β C), a high volatility under low pressure and can b easily be recovered from the solute of the supercritical phase. Moreover, carbon dioxide is markedly apolar, which, in the case of the use with PS, allows to avoid the extraction of highly polar products such as sugars, glycosides, inorganic salts, amino i f' acids and aromatic substances. Furthermore, the low cost, the easiness of achievement and the lack of aggressivity against proteins make it an optimum extraction solvent. It should also be evidenced that, in the 15 extraction with carbon dioxide, mechanical energy is used to separate the solvent from the extract, instead of thermal energy, which is the case with conventional techniques, therefore improving the thermodynamic yield of the system. > ' ' The supercritical region is the area in which the extraction process takes place. In this area, the gas cannot be liquefied due to its pressure; moreover, in this state, carbon anhydride can be liquefied by cooling, thus avoiding condensation. ?' The use of the supercritical fluid technique in the process for the purification of PS involves remarkable advantages: 1. achievement of a product which is substantially free from residual solvents; 30 2. drastic reduction of chlorinated and non chlorinated solvents, which are present in the process in very low amounts only in the separatio of surfactant from the inert carrier, in fact, th amount of solvent used in the present invention i from about 30 to about 50 ml per gram of purifie 5 product, whereas the process disclosed in EP 286011 uses about 600 ml per gram of purifie product; 3. cut in the costs of the purification plant. As an inert carrier to disperse the material to b l' ~ j extracted, glass beads of a suitable size, Extrelut R , Celite (R) and the like can be used. Celite (R) is preferred for both the good yield and the low cost. The weight ratio of inert material to material t extract ranges between 0.5:1 to 3:1, 1:1 being 15 preferred. The extraction is carried out at temperatures from 35 β C to 55 β C, preferably at 40 β C, under pressures from 180 to 300 atmospheres, preferably 200, for a time varying from 3 to 9 hours, preferably 7 hours. 0 In a preferred embodiment of the invention, the extraction step is subdivided in two cycles, evaporating to dryness the material from the first extraction cycle and subjecting the resulting powder to a second extraction cycle. Said procedure involves a 5 better extraction of low polar substances. The following examples evidence the optimum operative conditions to obtain the best extraction performances. In the examples, only supercritical CO was used, < " ι however, other supercritical fluids or possible combinations of supercritical fluids can obviously be used optionally in the presence of a cosolvent, i understood by those skilled in the art. The extraction plant is not critical to th purposes of the present invention, since th conventional plants operating with supercritical fluid can be used. Any changes in the configuration and parameters o the used plant can be carried out by those skilled i the art, without departing from the spirit and scope o the invention. The crude paste used in the examples was obtaine according to the procedure disclosed in EP 0, 286,011. EXAMPLE 1 30 g of PS crude paste are dissolved in 200 ml o chloroform at room temperature, with stirring. When th dissolution is complete, 30 g of Celite 545 (Merc 2693) are added in a 1:1 w/w ratio. The solvent i evaporated to dryness under vacuum at 35 β C. A powder i obtained, partly conglomerated in granules, which i placed into the plant extractor. An extraction cycle i carried out under the following conditions: fluid carbon dioxide pressure 200 bars extractor temperature 40 β C separator temperature 34 β C The cycle lasts about 3 hours, then the mixture i removed from the extractor and suspended in 500 ml o chloroform. The suspension is stirred for 5 min. an the solvent is evaporated to dryness at 35 β C. Th obtained powder is loaded again into the plan extractor and an extraction cycle is repeated, unde the same conditions, for about 3 hours. Finally the mixture is removed from the extractor suspended in 500 ml of chloroform, stirring for 10 min at room temperature, and filtered with suction. Th 5 resulting clear chloroform solution is evaporated t dryness under vacuum at 35 β C, to obtain 24 g of surfactant purified paste. The separator is washed twice with 150 ml o chloroform, the two washings are combined an 10 evaporated under vacuum at 35 β C. 5.2 g of impuritie are obtained. EXAMPLE 2 Similar results can be obtained following th procedures described in example 1, without resuspendin 15 the material in chloroform half way through the cycle. In this case, the mixture from the extractor i mixed by hand and loaded again, to repeat th extraction cycle under the same operative conditions. EXAMPLE 3 20 Following the same procedure as in examples 1 an 2, but carrying out the extraction cycle under pressure of 250 bars, 23.5 g of purified surfactant paste were obtained from 30 g of the starting PS crude paste, separating 6.5 g of impurities. 2.. EXAMPLE 4 30 g of PS crude paste are dissolved in 400 ml of chloroform at room temperature, with stirring. When the dissolution is complete, 60 g of Celite 545 (Merck 2693) are added in a 2:1 w/w ratio. The solvent is 30 evaporated to dryness under vacuum at 35*C. A powder is obtained which is placed into the plant extractor. An operative cycle is carried out under th following conditions: fluid carbon dioxide pressure 200 bars extractor temperature 40°C separator temperature 34"C The cycle lasts about 3 hours 30 in. , then the mixture is removed from the extractor and suspended i 500 ml of chloroform. The suspension is stirred for 5 min. and the solvent is evaporated to dryness at 35 * C. The obtained powder is loaded again into the plant extractor and an extraction cycle is repeated, under the same conditions, for about another 3 hours 30 min. Finally the mixture is removed from the extractor, suspended in 500 ml of chloroform, stirring for 10 min. at room temperature, and filtered with suction. The filtered solid is resuspended in 500 ml of chloroform and filtered with suction again. The two chloroform solutions obtained are evaporated to dryness under vacuum at 35°C, to obtain 23.7 g of a surfactant purified paste. The separator is washed twice with 150 ml of chloroform, the two washings are combined and evaporated under vacuum at 35 β C. 5.0 g of impurities are obtained. To stress the importance of the dispersion step of the material to extract on an inert carrier, as well as that of the general operative conditions, an example of a preparation carried out according to the prior art teachings is also reported. EXAMPLE 5 30 g of PS crude paste are placed into the plan extractor. An extraction cycle is carried out under th 5 following conditions: fluid carbon dioxide pressure 200 bars extractor temperature 40 * C separator temperature 34 * C 10 The cycle lasts 7 hours, then the extractor i unloaded and washed with 600 ml of chloroform. The paste is combined with the chloroform and th suspension is stirred to complete dissolution. Th solution is evaporated to dryness at 35 β C under vacuum, 15 to obtain 28.5 g of purified paste. The separator is washed twice with 150 ml o chloroform, the two washings are combined an evaporated under vacuum at 35 * C, to obtain 1.2 g of impurities. i-o The PS preparations obtained according to the above examples were analyzed against a control sample, i.e. a preparation obtained according to the purification procedure disclosed in EP 0,286,011 (Lipidex R column). The preparation of examples 1-4 25 evidenced a cholesterol and triglycerides content meeting the quality requirements; moreover, compared with the control sample, said preparations had a content in residual solvents lower than that of the control sample. 0 On the contrary, the preparation of example 5 showed an analytical profile not meeting the required parameters. This proves the criticality of t predispersion of the material to be extracted on t inert carrier, which is characteristic of the proce of the invention. More particularly, in the following Table t analytical data of PS samples obtained according to t invention (examples 1 and 4), to example 5 (no pr dispersion) and EP 0,286,011 (control) are reported. TABLE 1 Determination of the percent content in tot phosphorous (as an index of the phospholipids content) proteins, cholesterol and triglycerides in PS samples. % Content Example 1 Example 4 Example 5 Control The PS purified paste obtained according t examples 1 and 4 of the invention was resuspended i physiological saline at a concentration of 80 g o phospholipids/ml and subjected to an evaluation of th effects on the pulmonary mechanics of foetuses o prematurely born rabbits, according to the procedur described by Robertson B. and Lachmann B. i Experimental Lung Research 14: 279-310, 1988, to verif that the novel purification procedure does not affec the biological properties in any way. The results obtained in the animal confirmed th effectiveness of said preparations, which proved t have the same activity degree as the one of the produc obtained by the column purification procedure.

Claims: CLAIMS 1. A process for the purification of components of animal organs, by use of supercritical fluids, optionally in the presence of a cosolvent in which process said materials are previously dispersed on an inert carrier material. 2. A process for the purification of natural pulmonary surfactant, in which process the crude paste of said surfactant, previously dispersed on an inert carrier material, is subjected to extraction with a supercritical fluid. 3. A process according to claim 1, in which process the supercritical fluid is carbon dioxide. 4. A process according to claims 1-2, in which process the ratio of crude paste to inert carrier material is from 0.5:1 to 3:1. 5. A process according to claim 1, in which process said inert material is selected from glass beads, Extrelut R , Celite< R) . 6. A process according to any one of the above claims, in which process the extraction is carried out at a temperature from 35 to 55 β C, under a pressure from 180 to 300 atmospheres and for a time from 3 to 9 hours. 7. A process according to any one of the above claims, in which process the extraction cycle can be repeated. 8. Natural pulmonary surfactant obtained by means of the purification process of claims 1-6.

Inventors: CHIESI PAOLO, PIGHI ROBERTO, RECCHIA WALTER, SERVADIO VITTORINO, VENTURA PAOLO, CHIESI, PAOLO, PIGHI, ROBERTO, RECCHIA, WALTER, SERVADIO, VITTORINO, VENTURA, PAOLO
Applicants: CHIESI FARMA SPA, CHIESI PAOLO, PIGHI ROBERTO, RECCHIA WALTER, SERVADIO VITTORINO, VENTURA PAOLO, CHIESI FARMACEUTICI S.P.A., CHIESI, PAOLO, PIGHI, ROBERTO, RECCHIA, WALTER, SERVADIO, VITTORINO, VENTURA, PAOLO
Application year: 1993
Main classification: C07K3
IPC codes: B01D11, C07K14