WO1990000898A1

WO1990000898A1 - A surface and biologically active preparation

Info

Publication number: WO1990000898A1
Application number: PCT/CH1989/000135
Authority: WO
Inventors: Roland Eichenberger; Peter Ries
Original assignee: Pentapharm Ag
Priority date: 1988-07-22
Filing date: 1989-07-18
Publication date: 1990-02-08
Also published as: CH677879A5

Abstract

A surface and biologically active preparation containing a lipid and a protein component which is suitable for treating the shortness of breath syndrome, obtainable: a) by extraction from organs of slaughtered animals or residues thereof remaining after the aqueous extraction from slaughtered animals' organs by means of an alkanol with 1 to 10 carbon atoms, separating the organ residue, concentrating the extract obtained to separate out the efffective substance, taking the latter up in an alkanol with 1 to 4 carbon atoms, separating the undissolved material and removing the alkanol, or b) by extraction from organs of slaughtered animals or residues thereof remaining after the aqueous extraction from slaughtered animals' organs by means of an aqueous electrolyte solution or an aqueous mono or disaccharide solution, separating the organ residue, concentrating the extract to separate out the effective substance, taking the latter up in an alkanol with 1 to 4 carbon atoms, separating the undissolved material and removing the alkanol.

Description

Surface active and biologically active preparation

The present invention relates to a surface-active and biologically active preparation which is obtained by extracting organs or organ residues from slaughtered animals and is suitable for the medical treatment of abnormal or pathological lung conditions in which the natural "lung surfactant system" is not present or is disturbed.

Since the investigations by Von Neergard and K. Wirz, which are described in Z. Ges. Exp. Med. 66, 371-381 (1929), it has been known that the presence of surface-active substances for the regulated gas exchange in the lungs Substances on the surface of the alveoli and bronchi are necessary. These physiological substances ensure the retraction of the alveoli in the expiration phase without a total collapse of the alveoli and the smooth expansion of the alveoli in the inspiration phase by changing the surface tension at the alveolus-air interface. The alveolar system shows one in the state of expiration

2 total surface area of approx. 80 m and in the condition of the Inspira

2 tion on a surface of approx. 120 m. The single ones

Alveoli are with a layer of type I pneumocytes and II lined. Type II pneumocytes are the cells responsible for the synthesis and removal of surface-active substances. The surface-active mixture of substances which is released by the pneumocytes is referred to in the specialist literature as "pulmonary surfactant" or only as "surfactant".

The natural surfactant of both humans and animals is not a pure, uniform substance, but a mixture in the form of an emulsion of lipids, primarily phospholipids, proteins and carbohydrates. According to K. Morgenroth in "The Surfactant System of the Lungs", Walter de Gruyter Verlag, Berlin, 1986, pp. 10-12, the surfactant is composed of approx. 90% lipids, thereof approx. 65% phosphatidylcholine and 10% phosphatidylglycerol, plus 10% proteins, plus a very small amount of carbohydrates. The most important functional components in the surfactant are both the dipalmitoylphosphatidylcholine, which makes up about 50% of the lipid portion, and the relatively low protein or polypeptide portion of about 10%. The protein content is described by R. King, J. Appl. Physiol .: Respirat. Environ. Exercise Physiol. 5_3, 1-8 (1982), as consisting of 2 groups of "surfactant associated peptides" (SP) with molecular weights of 28000-36000 and 5000-18000. The presence of carbohydrates was detected electronically and biochemically, but has not yet been quantified.

The neonatal respiratory distress syndrome ("infantile respiratory distress εyndrome" = IRDS) develops especially in the perinatal period of premature babies due to the lack of surfactant in the alveoli or due to insufficient surface activity of the surfactant. This prevents the alveolar system from developing in the lungs, which ultimately leads to asphyxiation. substitution with a biologically active, natural or artificial surfactant can be life-saving in these cases.

In adult respiratory distress syndrome (ARDS), there is also a disruption of the pulmonary surfactant system, which leads to an electectase with a often fatal outcome. Clinical and experimental studies by B. Lachmann et al. , "Adult respiratory distress syndrome", in: Robertson, Van Golde, Batenburg Eds., "Pulmonary Surfactant", Elsevier, Amsterdam-1984, pp. 505-548, have shown that the course of the ARDS through suractant substitution and artificial ventilation can be influenced favorably.

S. O'Neill et al. , At the. Ren. Respir. Dis. 130, 225-230 (1984), were able to show in experimental studies that the lung surfactant is of important importance in the non-specific defense against infection, since the surfactant increases the phagocytosis of bacteria through the macrophages of the lungs.

Through the activation of phagocytosis, the surfactant also has a significant influence on the defense reactions of the lungs against very fine dusts that are constantly taken up with the air we breathe (= pneumoconiosis).

The IRDS and the ARDS are treated with alternating success by administering natural surfactant from animal lungs, artificial lipid mixtures or combinations of natural surfactant and artificial lipid mixtures into the patient's lungs.

The natural surfactant is from C.N. Morley et al., The Lancet 81/1, 64-68 (1980), classified as unsuitable for therapeutic use because of insufficient or strongly changing biological activity.

USP 4,312,600 describes a synthetic, protein-free surfactant which is composed of dipalmitoyl lecithin and hexadecanol. European patent application No. 11 04 98 describes an artificial, protein-free surfactant which consists of phospholipids, neutral lipids and special fatty acids. European patent application No. 11 90 56 describes a combination of a synthetic surfactant, which contains choline phosphoglycerides, acid phospholipids and fatty acids, with a lipoprotein from mammalian lung with a molecular weight of approximately 30,000 to 38,000.

PCT patent application no. 087 02 37 describes pulmonary surfactant proteins which are suitable for increasing the activity of pulmonary surfactant and have molecular weights of 35 * 000 or from 5'500 to 9'000.

The previously mentioned artificial protein-free surfactant materials have not proven themselves as substitutes for natural lung surfactant. It has been found that a certain proportion of lipophilic proteins or polypeptides for the biological effectiveness of the

Surfactants is essential. For this reason, the last two patent applications also describe combinations of lipids with proteins isolated from animal lung, which are intended to increase the activity of artificial or natural surfactant. DE-0S No. 30 21 006 describes the production of a surfactant material for the treatment of respiratory distress syndrome, which is extracted from mammalian lungs with electrolyte solution and to achieve a phospholipid content of 75-95.5% with the required amount is enriched in synthetic phospholipids. The protein content is 0.5 - 5.0%.

European patent application No. 0145 005 describes an actant lung cure, a method for its Extraction and its pharmaceutical use. The extraction takes place either by extraction of lung tissue or an aqueous isotonic lung extract with a lipid solvent, removal of the undissolved components, evaporation of the lipid solvent, removal of the undissolved components, extraction of the active ingredient with alcohol, removal of the alcohol-insoluble components and removal of the alcohol, or by bringing an aqueous isotonic lung extract into contact with an insoluble adsorbent, for example diatomaceous earth, for the purpose of adsorbing the active ingredient, desorbing the active ingredient by means of organic solvents and removing the solvent. The great disadvantage of this process is that large amounts of toxic halogenated hydrocarbons or flammable petroleum ether have to be used as lipid solvents.

Furthermore, the scientific literature describes methods for the extraction of natural lung actant that are only suitable for the production of very small laboratory quantities, but not for large quantities. In these methods, the pulmonary surfactant is obtained by pervasal irrigation of individual lungs (S. Bondurant et al., J. Appl. Physiol. 1_7, 167 (1962)) or by bronchial irrigation of individual lungs (JA Clements, Proc. Soc. Exp. Biol. 95, 170 (1957)).

Scarpelli et al. , J. Appl. Physiol. 2_3, 880 - 886 (1967) obtain small amounts of surfactant by extracting dog or rabbit lung with isotonic NaCl solution and separating the components by gel filtration on Sephadex® G 200. However, gel filtration leads to material losses that are so high that the process for the production of commercial amounts of surfactant must be regarded as unusable.

All previously described processes for manufacturing Technical quantities of biologically active surfactants are characterized by the fact that for the isolation and purification of the surfactant, on the one hand, very complex, multi-step processes and, on the other hand, dangerous, organic solvents, such as, for example, highly toxic and highly polluting chlorinated hydrocarbons or the like highly flammable petroleum ether can be used.

The invention was based on the object of an industrially feasible, simple and harmless, environmentally friendly method for producing a surface-active and biologically active preparation (lung surfactant) which contains a lipid component and a protein component and for the therapeutic treatment of respiratory distress syndrome is suitable to provide. When the specified task was solved, it was surprisingly found that as raw material source for the production of the surface-active preparation, other organs of slaughter animals as well as tissue residues, which also arise from the aqueous extraction of the proteinase inhibitor aprotinin from lung tissue, are suitable for the extraction of the surface-active preparation are gnet. It has also surprisingly been found that the extraction of the surface-active preparation from ground animal carcasses or residues from already extracted lung tissue is simple and clean with alkanols, e.g. Ethanol.

According to the invention, the surface-active and biologically active preparation is obtainable a) by extraction of organs from slaughter animals or from organ residues which remain after aqueous extractions from slaughter animal organs, using an alkanol with 1 to 10 carbon atoms, separation of the organ residue, concentration of the extract obtained for the purpose of separating the active substance, inclusion of the latter in an alkanol with 1 to 4 carbon atoms, removal of undissolved material and removal of the alkanol, or b) by extraction of organs from slaughter animals or organ residues which remain after aqueous extractions from slaughter animal organs, using an aqueous electrolyte solution or an aqueous mono- or Disaccharide solution, separation of the organ residue, concentration of the extract for the purpose of separating the active substance, absorption of the latter in an alkanol with 1 to 4 carbon atoms, separation of undissolved material and removal of the alkanol.

In particular, brain, liver, heart, spleen and / or lung tissue of cattle (Bovidae), pigs, sheep, rabbits or poultry can be used as the starting material for the production of the preparation according to the invention. These organ tissues can be used in the fresh, frozen or dried state. Tissue residues that remain after aqueous extractions of the above-mentioned organ tissues can also be used, e.g. the tissue residue which is obtained in the aqueous extraction of bovine lung tissue for the purpose of obtaining aprotinin.

For the alcoholic extraction of the organ material, an alkanol with 1 to 10 carbon atoms, preferably an alkanol with 1 to 4 carbon atoms, e.g. Methanol, ethanol, n-propanol, isopropanol, n-butanol or isobutanol are used. The alkanol is expediently used in a quantitative ratio of 4 to 20 L per kg of dry substance of the organ material. If, as a starting material, an organ tissue residue that still contains water, e.g. A lung tissue residue originating from the extraction of aprotine can be used as follows:

The finely shredded organ tissue residue is together with the alkanol, for example »95% ethanol, in a ratio of about 8 L alkanol per kg of dry substance of the organic tissue residue, stirred for 30 minutes to 1 hour. The extraction mixture is filtered to remove tissue residues. The alkanol is removed from the aqueous-alcoholic extract by vacuum distillation. Two phases are obtained, namely an aqueous phase and a lipophilic phase. The phases are separated by filtration. The aqueous phase is discarded. The lipophilic phase is dissolved in an alkanol with 1 to 4 carbon atoms, for example methanol. The solution is filtered in order to remove remaining undissolved material. Evaporation of the filtrate in vacuo gives a crude extract which can be further purified. If dried organic tissue material, for example brain powder (obtained by crushing, grinding and freeze-drying frozen beef brains), is used as the starting material, the procedure can be as follows:

The finely shredded, dry organ tissue material is combined with the alkanol, e.g. 95% ethanol, in the ratio of 10 L alkanol per kg dry material, stirred for 20-30 minutes. The extraction mixture is filtered to remove tissue residues. The alkanol is removed from the alcoholic organ extract obtained by distillation in vacuo. The residue is mixed with an alkanol with 1 to 4 carbon atoms, e.g. Methanol, and the solution is filtered to remove residual undissolved material. Evaporation of the filtrate in vacuo gives a crude extract which can be further purified.

The extraction of organ material with aqueous electrolyte solutions can be carried out in the manner described below:

Finely chopped fresh, frozen or Dried organ material, for example finely comminuted fresh beef lung, or an organ residue obtained in an aqueous extraction of organ material, for example a lung tissue residue which was obtained after the aqueous extraction of beef lung for the purpose of obtaining aprotinin, is carried out together with an aqueous electrolyte solution at room temperature during Stirred for 30-60 minutes. A 0.5 to 5% aqueous solution of sodium chloride, potassium chloride or sodium acetate can be used as the aqueous electrolyte solution. A 0.5 to 2% aqueous sodium chloride solution is preferably used. The aqueous extraction mixture is centrifuged in order to separate undissolved tissue material. The aqueous extract is then concentrated in vacuo to effect the separation of the active substance. The active substance can be separated by two methods. One method consists in concentrating the aqueous extract until the extract residue is practically water-free. The second method consists of concentrating the aqueous extract to about 10 to 20% of its volume and adding an alkanol with 1 to 4 carbon atoms, for example methanol or ethanol, to the concentrate in order to effect the separation of the active substance. The alkanol is expediently used in a ratio of 2 volumes of alkanol to 1 volume of concentrate. The separated impure active substance or the extract residue is taken up in an alkanol with 1 to 4 carbon atoms, for example methanol or ethanol. The solution is freed from undissolved material by filtration. Evaporation of the filtrate gives a crude extract which can be further purified.

The crude extracts obtained from the alcoholic and aqueous extractions of organ material can be purified by chromatography on organic chromatography materials. materials, for example polydextranes, agarose, polyacrylamides or vinyl polymers, or on inorganic chromatography materials, for example silicon dioxide, silicates, aluminum oxide or phosphates. Alkanols with 1 to 4 carbon atoms, for example methanol or ethanol, or mixtures of these alkanols with other organic solvents, for example mixtures of methanol or ethanol with methylene chloride or ethyl acetate, can be used as the solvent for the chromatography. Aqueous electrolyte solutions or mixtures thereof with the alcohols mentioned can also be used as the solvent. The chromatography is expediently carried out in a fractional manner. The preliminary and secondary fractions obtained are discarded, while the cleaned surface-active and biologically active preparation is isolated from the main fraction by evaporating the solvent in vacuo.

The crude extracts can furthermore be separated into the lipid component and the protein component by chromatography. The two chromatographically cleaned components are combined again in order to obtain a finished surface-active and biologically active preparation (lung surfactant).

To produce a therapeutic agent for treating respiratory distress syndrome, the cleaned, surface-active and biologically active preparation is expediently converted into the galenical form of a sterile, aqueous-saline suspension, which can be applied as a bolus, spray or aerosol. Sterilization is carried out most gently by sterile filtration under aseptic conditions, in that the combined main fractions of the chromatography described above are filtered through a 0.2 μm sterile membrane and the subsequent process steps are carried out under aseptic conditions, or by cleaning and isolating them Active substance in a carrier liquid, for example isotonic sodium chloride solution, is so finely suspended by means of a microfluidizer or a homogenizer that particles with a maximum diameter of 0.2 μm are formed, and the micro-suspension obtained in this way by 0.2 μm sterile membrane is filtered.

The concentration of the surface-active and biologically active preparations in the carrier liquid is adjusted so that a concentration of 20-500 mg of purified preparations per ml of carrier liquid is obtained.

The invention is illustrated by the following examples.

Example 1 1.5 kg of beef lung residues with a dry residue of 25.9% by weight, which resulted from the aqueous extraction of aprotinin from frozen beef lung, were mixed with 3 L of 95% by volume ethanol and at room temperature for 30 Minutes stirred intensively. The alcoholic organ pulp was filtered on a coarse filter under vacuum and gave about 3 L of aqueous-alcoholic lung extract. The tissue residue was discarded. The alcohol was distilled off in vacuo, giving a two-phase mixture which consisted of an aqueous phase and a lipid phase. The lipid phase was separated from the aqueous phase by filtering the mixture through a paper filter. Extraction of the separated aqueous phase with methylene chloride and evaporation of the methylene chloride extract to dryness showed that no lipophilic substances remained in the aqueous phase.

When the phases were separated, 15.1 g of lipophilic substance remained on the filter paper. The received lipophilic substance was taken up in 150 ml of methanol and the solution was filtered clear. Thereafter, the methanol was evaporated in vacuo. 14.6 g of crude surface-active preparation were obtained. The crude preparation was dissolved in 120 ml of methanol. For purification, the solution was placed on a chromatography column which was filled with Sephadex® LH 20 (polydextran) and fractionated by means of methanol. The surface active chromatography fractions were combined and the methanol was evaporated in vacuo. 5.1 g of purified, surface-active preparation remained. A sample of this material was tested for biological activity in the rabbit fetus test according to the method described below and proved to be biologically active.

Example 2

1.0 kg of frozen bovine brain was chopped with a knife mixer into a fine paste which was freeze-dried. The 200 g of water-free lyophilizate obtained therefrom were crushed to a powder, placed in an extraction vessel and extracted with 2 L of 95% by volume ethanol with vigorous stirring. The mixture was then filtered through a G1 suction filter. The insoluble tissue residue was discarded, while the alcohol obtained was distilled off in vacuo from the 1.95 L alcoholic brain extract filtrate obtained. One received alε

Residue 57.4 g bovine brain lipids, which were taken up in 500 ml of methanol and stirred for 10 minutes. The insoluble fraction was then filtered off, whereupon the filtrate was evaporated to dryness in vacuo. 35 g of surface-active lipid material were obtained, which was taken up in 280 ml of ethanol for cleaning. The mixture was filtered and the filtrate was chromatographed on Sephadex® LH 60 with ethanol. With a faction Fractions of 20 ml eluate were collected. The surface-active fractions were combined and the alcohol was evaporated in vacuo at 30 ° C. The residue obtained was 8.8 g of surface-active preparation, which was found to be biologically active in rabbit fetus test.

Example 3

2.4 kg of fresh beef lung were ground in a cutter for 10 minutes to form a fine tissue pulp, transferred to an extractor, with the addition of 3.0 L 1, 5% NaCl solution for half an hour with vigorous stirring Extracted room temperature and then centrifuged. Centrifugation gave 3.1 L of cloudy supernatant and 2.3 kg of tissue residue, which was discarded. The supernatant was concentrated in vacuo to 400 ml, treated with 800 ml of 95% by volume ethanol and filtered. Filtration gave 8.1 g of lipophilic residue, which was taken up in 80 ml of methanol, stirred and filtered. The filtrate was evaporated in vacuo and gave 1.7 g of crude preparation, which in turn was taken up in 14 ml of methanol, stirred and filtered. The methanolic filtrate was further purified as described in Example 1 and gave 1 g of purified surface-active preparation which proved to be biologically active in the rabbit fetus test.

Example 4

800 kg of frozen beef lung were ground in 8 portions of 100 kg each with a knife cutter to a fine paste. The pulp of the lung was transferred to a 2000 L extractor and mixed with 1220 L water. The mixture was adjusted to a pH of 4 with hydrochloric acid, stirred for 1 hour, briefly heated to 80 ° C. and dried using a filter fitted with filter cloths. terpress filtered. The filtration gave the filtrate I and the tissue residue I. The tissue residue I was extracted again with 800 L of water for 30 minutes. The mixture was then filtered as before. The filtrate II and 515 kg of lung tissue residue II were obtained. By combining the filtrate II with the filtrate I, about 2000 l of solution were obtained which did not contain any surface-active substance. The solution was worked up further to obtain aprotinin. The 515 kg of moist lung tissue residue II were transferred to a 2000 L extractor, 1030 L of 95% by volume ethanol were added and the mixture was stirred intensively for 1 hour. The mixture was filtered using a filter press equipped with filter cloths. An aqueous-alcoholic lung extract and tissue residue were obtained, which was discarded. From the aqueous-alcoholic lung extract obtained, the alcohol was distilled off in vacuo, giving a two-phase mixture which consisted of an aqueous and a lipophilic phase. The phase separation was achieved by filtration through a nylon filter cloth and gave 5.1 kg of lipophilic substance. The lipophilic substance was taken up in 51 L of methanol and stirred for 30 minutes. Insoluble material was then removed by filtration through a cellulose filter layer. The methanolic filtrate was evaporated in vacuo and gave 4.3 kg of crude extract which, as described in Example 5, was further purified by chromatography.

Example 5

300 g of crude extract, which had been prepared according to Example 4, were dissolved in 2.4 L of methanol. The methanol solution was clarified and then applied to a chromatography column which was filled with 100 L Sephadex® LH (polydextran). After application the substance was chromatographed with 110 L of methanol. The eluate was collected in 5 L fractions and gave 22 fractions. The course of the chromatography was monitored by continuously recording the UV light absorption of the eluate. On the basis of the chromatographic curve obtained, the eluate fractions obtained were divided into 7 pre-fractions, 6 main fractions and 9 minor fractions. The preliminary and minor fractions were discarded. The 6 main fractions were combined, concentrated in vacuo and gave 104 g of purified preparation which proved to be biologically active in rabbit fetus test.

Example 6

8 g of crude extract, which had been prepared according to Example 4, were dissolved in 64 ml of methanol. The methanolic solution was filtered clear and then applied to a chromatography column which was filled with 10 L Sephadex® LH (polydextran). After the substance had been applied, it was chromatographed with 10.5 L of methanol. After an eluate flow of 3.5 L, the eluate was collected in 14 fractions of 640 ml each. The course of the chromatography was monitored by continuously recording the UV light absorption of the eluate and marking each change of fraction. On the basis of the chromatography curve obtained, as well as thin-layer chromatograms and protein determinations of the individual fractions, it was found that the protein component in fraction 1 and the lipid component in fractions 2-5, while the following 9 fractions only contained fiber. Fraction i was evaporated in vacuo and gave 0.5 g protein portion. Fractions 2-5 were combined, likewise evaporated in vacuo and gave 2.7 g of lipid fraction. The remaining 9 fractions were pooled, evaporated and gave 4.8 g of fiber. The lipid portion thus purified was mixed with the purified protein portion. The mixture proved to be biologically active in the rabbit fetus test.

Example 7

1.5 kg of moist lung tissue residue II with a dry residue of 28.7% by weight, which was obtained in the aproine recovery and had been obtained according to Example 4, was stirred with 3 L 1, 5% sodium chloride solution for 40 minutes. The mixture was then centrifuged and the supernatant was separated from the tissue residue. The 2.8 L excess obtained was practically evaporated to dryness in a vacuum rotary evaporator at 53 C. 72.8 g of residue were obtained, which was taken up in 360 ml of methanol. The mixture was filtered through a glass filter. The insoluble residue was washed with 180 ml of methanol and then discarded. The methanolic filtrate and the methanolic washing solution were combined and evaporated in vacuo. 17.53 g of crude extract were obtained, which, as described in Example 5, was further purified by chromatography. Finally, 4.3 g of purified surface-active preparation were obtained, which proved to be biologically active in the rabbit fetus test.

The biological activity of the surface-active preparations produced according to the examples was tested using the method of B. Lachmann on rabbit fetuses with immature lungs (see B. Lachmann, "New aspectε in = pathophysiology and therapy of respiratory disorders syndrome: criteria for charaeterization of exogenous" sur¬ factant deficient animal modeis "; in: Selected Topics in Pe _. rinatal Medicine, CIC Edizioni Internationali, Rome, 1986, p. 177).

Immature rabbit fetuses were born on the 27th day of pregnancy by hyterotomy. Immediately after birth, the living fetuses were tracheotomized, placed in a pressure-constant whole-body plethysmograph and ventilated with pure oxygen. The fetuses of a mother animal were divided into 2 groups I and II. Group I fetuses were injected into the trachea via a tracheal cannula each containing 300 mcl of a suspension of the surface-active specimen sample. The suspension was prepared by dissolving 500 mg of sample in 5 ml of physiological saline. Group II fetuses remained untreated and served as a control.

The device continuously registered the primary data, namely ventilation pressure and tidal volume, throughout the entire duration of the experiment. From the recorded primary data, 15 minutes after the first sample administration, the thorax-lung compliance was calculated and evaluated according to the following formula:

Lung volume in I mlJ compliance per kg fetus

Ventilation pressure in bar ^■ ]

Thorax-lung compliance is defined as the elastic volume expansion of the lungs in the thorax.

The results are summarized in the table below. Biological effectiveness

Compliance at

Sample from a ventilation assessment of Example No., pressure of 25 mbar V / iramεamkeit

1. Raw preparation 0.8 good

1. Cleaned preparation 1,2 good

2. Raw lipid material 0.5 insufficient 2. Purified lipophilic preparation 1.0 good

3. Rohsurfactant 1.5 very good

3. Purified surfactant 1.7 very good

7. Crude extract 0.8 good

7. Cleaned preparation 1.3 good

Control 0.1 insufficient

The untreated control animals had a compliance of about 0.1 ml / mbar per kg in rabbit fetus, i.e. that there was practically no lung volume extensibility. Samples which increase the compliance from 0.1 to 0.6 ml / mbar per kg in the test are considered unsatisfactory, samples which increase the compliance to 0.6-1.2 ml / mbar per kg are considered to be good and samples that increase compliance to 1.3-2.4 ml / mbar per kg or above are rated as very effective.

Claims

1. Surface-active and biologically active preparation which contains a lipid component and a protein component and is suitable for the treatment of respiratory distress syndrome, obtainable a) by extraction from organs from slaughter animals or from organ residues which remain after aqueous extractions from slaughter animal organs, by means of an alkali nols with 1 to 10 carbon atoms, separation of the organ residue, concentration of the extract obtained for the purpose of separating the active substance, absorption of the latter in an alkanol with 1 to 4 carbon atoms, separation of the undissolved material and removal of the alkanol, or b) by extraction of organs slaughter animals or organ residues that remain after aqueous extractions of slaughter animal organs, by means of an aqueous electrolyte solution or an aqueous mono- or di-saccharide solution, separation of the organ residue, concentration of the extract between ks separation of the active substance, absorption of the latter in an alkanol with 1 to 4 carbon atoms, separation of the undissolved material and ■ removal of the alkanol.

2. Preparation according to claim 1, obtainable from brain, liver, heart, spleen and / or lung tissue of cattle (Bovidae), pigs, sheep, rabbits or poultry.

3. Preparation according to patent claims 1 and 2, obtainable by extraction of a finely comminuted water-containing organ residue with the C -C alkanol, removal of undissolved constituents, evaporation of the alkanol, removal of the lipophilic phase of the extract obtained from the aqueous phase, absorption of the lipophilic phase in an alkanol with 1 to 4 carbon atoms, Separation of undissolved components and evaporation of the alkanol to obtain a crude extract.

4. Preparation according to claims 1-3, obtainable using a lung tissue residue which was obtained after the aqueous extraction of bovine lung for the purpose of obtaining aprotinin.

5. Preparation according to claims 1 and 2, obtainable by extraction of a finely comminuted dried organic material with the C.-C _ alkanol, separation of undissolved constituents, evaporation of the alkanol, absorption of the lipophilic residue in an alkanol with 1 to 4 carbon atoms, separation of undissolved constituents and evaporation of the alkanol to obtain a crude extract.

6. Preparation according to claims 1 and 2, obtainable by extraction of a finely comminuted fresh, frozen or dried organ material or an organ residue by means of a 0.5 to 5% aqueous electrolyte solution, preferably a 0.5 to 2 % aqueous sodium chloride solution, separation of undissolved tissue parts, concentration of the aqueous extract obtained to a practically anhydrous residue, absorption of the residue in an alkanol with 1 to 4 carbon atoms, removal of undissolved material and evaporation of the alkanol to obtain a crude extract ¬ tes.

7. Preparation according to claims 1 and 2, obtainable by extraction of a finely comminuted fresh, frozen or dried organ material or an organ residue by means of a 0.5 to 5% aqueous electrolyte solution, preferably a 0.5 to 2- % aqueous sodium chloride solution, separation of undissolved tissue parts, concentration of the aqueous extract obtained to 10-20% of its volume, addition of 2- times the volume of the concentrate of alkanol with 1 to 4 carbon atoms to the concentrate for the purpose of separating the active substance, separating and taking up the latter in an alkanol with 1 to 4 carbon atoms, separating undissolved material and evaporating the alkanol to obtain a crude extract .

8. Preparation according to claims 1-7, obtainable by purifying the crude extract by chromatographing a solution thereof in an alkanol with 1 to 4 carbon atoms or in a mixture of such an alkanol with another organic solvent.

9. Preparation according to claims 1-8, obtainable using methanol or ethanol, or a mixture of methanol or ethanol with ethyl acetate or methylene chloride as a solvent for chromatography.

10. Preparation according to claims 1-9, obtainable using a chromatography material based on polydextrane, agarose, polyacrylamide or vinyl polymer base.

11. Preparation according to claims 1-9, obtainable using a chromatography material based on silicon dioxide. Silicates, aluminum oxide or phosphates.

12. Preparation according to claims 1-11, obtainable by chromatographic separation of the crude extract into the lipid component and the protein component and mixing the purified components thus obtained.

13. A process for producing a surface-active and biologically active preparation which contains a lipid component and a protein component and is suitable for the therapeutic treatment of respiratory distress syndrome _. , characterized in that one a) organs from slaughter animals or organ residues which remain after aqueous extractions of slaughter animal organs, extracted by means of an alkanol with 1 to 10 carbon atoms, separating the organ residue, concentrating the extract obtained for the purpose of separating the active substance, the latter in an alkanol with 1 up to 4 carbon atoms, undissolved material is removed and the alkanol is removed, or that b) organs from slaughter animals or organ residues which remain after aqueous extractions of slaughter animal organs are extracted by means of an aqueous electrolyte solution or an aqueous mono- or disaccharide solution, the organ residue is separated off , the extract is concentrated for the purpose of separating the active substance, the latter is taken up in an alkanol with 1 to 4 carbon atoms, separated undissolved material and the alkanol is removed.

14. The method according to patent claim 13, characterized in that brain, liver, heart, spleen and / or lung tissue of cattle (Bovidae), pigs, sheep, rabbits or poultry is used.

15. The method according to claims 13 and 14, characterized in that a finely comminuted water-containing organ residue is extracted with the C -C _ alkanol, undissolved constituents are separated off, the alkanol evaporates, the lipophilic phase of the extract obtained from separating the aqueous phase, taking up the lipophilic phase in an alkanol having 1 to 4 carbon atoms, separating undissolved constituents and evaporating the alkanol to obtain a crude extract.

16. The method according to claims 13-15, characterized in that a lung tissue residue is used as the organ residue, which was obtained after the aqueous extraction of bovine lung for the purpose of obtaining aprotinin.

17. The method according to claims 13 and 14, characterized in that a finely comminuted, dried organ material is extracted with the C - C _ιn alkanol, separated undissolved constituents, that alkanol evaporates, the lipophilic residue in an alkanol with 1 to 4 carbon atoms, separates undissolved constituents and evaporates the alkanol to obtain a crude extract.

18. The method according to claims 13 and 14, characterized in that a finely shredded fresh, frozen or dried organ or an organ residue by means of a 0.5 to 5% aqueous electrolyte solution, preferably a 0 , 5- to 2% aqueous sodium chloride solution, extracted, undissolved tissue parts separated, the aqueous extract obtained is concentrated to a practically water-free residue, the residue is taken up in an alkanol with 1 to 4 carbon atoms, undissolved material is removed and that Alkanol evaporates to obtain a crude extract.

19. The method according to claims 13 and

14, characterized in that a finely comminuted fresh, frozen or dried organ material or an organ residue is extracted by means of a 0.5 to 5% aqueous electrolyte solution, preferably a 0.5 to 2% aqueous sodium chloride solution, separates undissolved tissue components, concentrates the aqueous extract obtained to 10-20% of its volume, adds 2 times its volume of alkanol with 1 to 4 carbon atoms to the concentrate in order to effect the separation of the active substances, separating the latter and takes up in an alkanol with 1 to 4 carbon atoms, separates undissolved constituents and evaporates the alkanol to obtain a crude extract.

20. The method according to claims 13-19, - 24 -

characterized in that the crude extract is purified by chromatography of a solution thereof in an alkanol having 1 to 4 carbon atoms or in a mixture of such an alkanol with another organic solvent.

21. The method according to claims 13-20, characterized in that methanol or ethanol, or a mixture of methanol or ethanol with ethyl acetate or methylene chloride is used as a solvent for chromatography.

22. The method according to claims 13-21, characterized in that one uses a Chromatographiemate¬ rial based on polydextran, agarose, polyacrylamide or vinyl polymer.

23. The method according to claims 13-21, characterized in that one uses a Chromatographiemate¬ rial based on silicon dioxide, silicates, aluminum oxide or phosphates.

24. Therapeutic agent for the treatment of respiratory distress syndrome, characterized by a content of surface-active and biologically active preparation according to claims 1-12.

25. A therapeutic agent according to claim 24, characterized in that it contains 20 to 500 mg of said preparation per ml of a carrier liquid.

26. Therapeutic agent according to claims 24 and 25, characterized in that the carrier liquid is an aqueous isotonic electrolyte solution.