Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
  • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
  • A61K35/30—Nerves; Brain; Eyes; Corneal cells; Cerebrospinal fluid; Neuronal stem cells; Neuronal precursor cells; Glial cells; Oligodendrocytes; Schwann cells; Astroglia; Astrocytes; Choroid plexus; Spinal cord tissue
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
  • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
  • A61K35/42—Respiratory system, e.g. lungs, bronchi or lung cells

Definitions

• 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.
• 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.
• 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.
• SP surfactant associated peptides
• the presence of carbohydrates was detected electronically and biochemically, but has not yet been quantified.
• 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.
• 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.
• an insoluble adsorbent for example diatomaceous earth
• 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.
• 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.
• 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.
• 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.
• lung surfactant which contains a lipid component and a protein component
• respiratory distress syndrome for the therapeutic treatment of respiratory distress syndrome is suitable to provide.
• tissue residues which also arise from the aqueous extraction of the proteinase inhibitor aprotinin from lung tissue
• 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.
• 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.
• brain, liver, heart, spleen and / or lung tissue of cattle can be used as the starting material for the production of the preparation according to the invention.
• 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.
• 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.
• 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.
• dried organic tissue material for example brain powder (obtained by crushing, grinding and freeze-drying frozen beef brains)
• brain powder obtained by crushing, grinding and freeze-drying frozen beef brains
• 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.
• 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).
• 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 ⁇ m 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 ⁇ m are formed, and the micro-suspension obtained in this way by 0.2 ⁇ m 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.
• 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.
• 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.
• 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.
• 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.
• 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:
• Thorax-lung compliance is defined as the elastic volume expansion of the lungs in the thorax.
• 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.

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Citations (9)

• 1988
  • 1988-07-22 CH CH2803/88A patent/CH677879A5/de not_active IP Right Cessation
• 1989
  • 1989-07-18 WO PCT/CH1989/000135 patent/WO1990000898A1/de unknown

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