DD225841C2 - METHOD FOR PRODUCING A MEANS FOR THE TREATMENT OF DISEASES INCREASED WITH CHANGES IN THE PULMONARY SURFACTANT SYSTEM - Google Patents

Description

For this 3 pages drawings

Field of application of the invention

The invention relates to a method for obtaining surfactant from the lungs of mammals. The active substance formed by type II alveolar cells can be used for the causal therapy of surfactant-deficient respiratory distress syndrome. The manufacturing process is applicable in the pharmaceutical industry.

Characteristic of the known technical solutions

The hyaline membrane disease (HMD) is based on a deficiency of alveolar surfactant in the lung and thereby causes the formation of atelectases. A drug and causal treatment option is seen in the administration of lung surfactant into the trachea. Animal experimental and initial neonatal clinical trials have been conducted with both natural surfactant and its major component, dipalmityllecithin, primarily in combination with phosphatidylglycerol. The natural surfactant is considered by some authors (MORLEY, CN, et al .: The Lancet 1 (1981), pp. 64-68) to be insufficient for therapeutic use because of insufficient and widely varying activity. T. FUJIWARA developed a semi-synthetic surfactant that consists of a mixture of naturally occurring surfactant from mammalian lungs and synthetic phospholipids. Good treatment success was achieved in 10 precocious newborns with HMD (FUJIWARA, T. et al.: The Lancet 1 (1980), p.55-59).

Recovery of lung surfactant may be obtained from lung homogenate (V. NEERGARD, K .: Verh. Dtsch. Ges. Inn. Med. 41 (1929), p.249; CLEMENTS, JA: Proc. Soc. Exp. Biol. (NY ) (1957) p.170), by pervasal lavage (BONDURANT, S. DAMILLER, J. Appl. Physiol 17 (1962), p 167) or bronchial lavage (CLEMENTS, JA: Proc. Soc. Biol. (NY) 95 (1957), p. 170). For a therapeutic use of the drug only isolation from severely minced lung tissue into consideration. However, the extracted extract contains not only the components of the surfactant system, but also, depending on the extraction conditions used, soluble and emulsifiable components of the lung and the blood contained in the lung.

One way to obtain this surface-active material for the treatment of HMD is described in DE-OS 3021006. By electrolytic solution, a raw material is obtained from mammalian lung to which the required amount of phospholipid is added to achieve a phospholipid content of 75-95.5%. The protein content is 0.5-5.0%. Test the separation of cellular debris at 500-1500 rpm the crude active ingredient mixture is obtained by centrifugation at 12000-16000UPM and 0-1O ⁰ C as a sediment. The further work-up is carried out by repeated resumption of the lipids in sodium chloride solution and separation by high-speed centrifugation. After dialysis, extraction with acetone and absorption of phospholipids in a chloroform-methanol mixture and evaporation of the solvent, a drug mixture is obtained, which is then added in relation to the phosphate content in the molar ratio of 1: 0.65: 0.12 dipalmityllecithin and phosphatidylglycerol ,

Other methods described in the literature (BROWN, ES, et al .: J. Appl. Physiol 14 (1959), p.717; JOBE, A .: Biochem Biophys Acta 489 (1977), p.440 KING, RJ; JACLEMENTS: Am. J. Physiol 223 (1972), pp. 707-714; BERGEN, P., et al.: IRCS Med. Sci. 9 (1981), p. 283 -284) for natural surfactant recovery are based on the extraction or rinsing of mammalian lung with electrolyte solutions and subsequent separation by multiple centrifugation at different g-numbers and temperatures, the separation of phospholipids requiring the use of high g-numbers and density gradients The scale-up brings high costs.

SCARPELLI, E.M., et al. (I. Appl. Physiol 23 (1967), pp. 380-386) obtained a crude surfactant by extraction of Huntf> and rabbit lung by means of isotonic sodium chloride solution and subsequent multi-stage centrifugation. For analytical purposes, the surfactant components were separated by gel filtration over Sephadex G 200. Due to the different particle sizes and the surface-active properties, gel filtration leads to high material losses and does not appear to be suitable for preparative purposes.

MORLEY, C.J., et. al. (The Lancet 1 (1981), pp. 64-68) administered to neonates with HMD a synthetic lung surfactant consisting of a mixture of dipalmityl lecithin and phosphatidylglycerol in the ratio 7: 3, the effect of which, however, is insufficient.

The preparation of a synthetic, protein-free surfactant consisting of dipalmitoyl lecithin and hexadecanol is described by Clements in U.S. Patent 4,312,860.

Object of the invention

The aim of the invention is to provide mammalian lung with a natural surfactant with high efficacy for the treatment of

Diseases associated with a change in the pulmonary surfactant system, in particular the

To find adult and newborn infectious diseases in a simple, technically scalable process with readily available excipients. This new therapeutic has extraordinary medical and humanitarian significance.

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Explanation of the essence of the invention

It was therefore the task of developing a technically simple, reproducible method, which leads to a new, natural surfactant of special composition and is suitable for the treatment of respiratory distress syndrome. In addition, a composition of the composition suitable for medical use should be indicated.

It has surprisingly been found that binding of the surfactant extracted from lung to an insoluble adsorbent, in particular silica gel or activated silicates and subsequent elution with phospholipid-suitable solvents in a simple manner a surfactant preparation can be obtained that a high pharmacological activity in the treatment of respiratory distress syndrome having.

In carrying out the process according to the invention, the extraction of the surfactant from shredded mammalian lung, preferably bovine or porcine lung, by solutions of mono- or disaccharides, preferably by isotonic glucose or sucrose solution - as known from the literature by means of electrolyte solution, e.g. isotonic sodium chloride solution. After separation of cell debris by centrifugation at low g-numbers in a known manner, the active ingredient mixture is adsorbed on silica gel or activated magnesium silicate. The reversible binding of active surfactant on silica gel was not to be expected since it is explicitly stated in the literature that the physical and chemical properties of this multicomponent mixture cause an irreversible binding to solid chromatography materials and thus an isolation of biologically active surfactant in this way not feasible is (KING, RJ: Federation Proceedings 33 [1974], pp. 2238-2247). After washing off non-adsorbed material by means of distilled water or acetone, the active ingredient is replaced by a solvent mixture, preferably by FOLCH with chloroform / methanol from the adsorbent and recovered by evaporation in vacuo or by drying by introducing nitrogen.

Obtained is a natural surfactant having a phospholipid content between 20 and 75%, preferably 40-50%. The preparation is characterized by a low protein content (s 0.5%). The low protein content appears to be important in view of the possible side effects of surfactant preparations, since the extraction process brings both serum proteins and organ-specific proteins into the extract, which may have antigenic properties when administered to humans (BERGMANN, K.-CH., LACHMANN. Z. Erkr. Atm.137 [1972], p.55-60; MORLEY, CN, et al.: The Lancet, 1 [1981], pp. 64-68).

From the obtained natural surfactant having a phospholipid content between 20 and 75%, a pharmaceutical agent is prepared by the methods described below.

The substance is precleared after solution in halogenated hydrocarbons through glass filters and filter paper and then sterile filtered through solvent-stable membranes. After evaporation of the solvent in vacuo, the residue obtained is emulsified with water for injection or sterile aqueous solution, filled into glass jars, frozen and lyophilized.

Isotonic sodium chloride or calcium chloride solutions or isotonic solutions of mono- and disaccharides can be used as aqueous solutions. For homogenization is advantageous an Ultra-Turrax. In addition, it is possible to add suitable agents for improving the emulsifying ability, increasing the stability and buffering to maintain certain pH ranges.

Another way to obtain a pharmaceutical agent is to emulsify the resulting natural surfactant - bypassing the sterile filtration in organic solvents - immediately in water or aqueous solutions, the above solutions and additives also apply here, and to autoclave the emulsion , After cooling, if necessary, emulsify again. The emulsion is then filled into glass jars, frozen and lyophilized

 The process according to the invention will be explained with reference to the following examples without thereby restricting them.

embodiments example 1

From 4.5 kg of fresh bovine lung larger blood vessels, trachea and adhering fatty tissue were removed and the lung was divided. In a meat grinder with a 4 mm perforated disc, after washing the lung pieces, which were freed from adhering blood, the reduction of the lung tissue took place. The pulp thus obtained was vigorously shaken in portions of 500 g each with 2% sucrose solution (114 g / l) for 10 minutes. Filtration through a plastic sieve and subsequent centrifugation of the filtrate at 500 g and 20 ° C gave 15.41 aqueous extract. 7.01 of this extract were stirred with 420 g of silica gel for column chromatography at 20 ⁰ C for 60 min. After 16 hours of standing at 4 ° C, the clear supernatant was siphoned off and discarded. The silica gel adsorbed with surfactant was washed after aspiration of the remaining supernatant with 11 distilled water on a G2 frit. The elution of the surfactant was carried out with 1.51 chloroform / methanol mixture (2: 1). The chloroform phase was separated and concentrated to dryness under nitrogen. As a result, 515 mg of the substance was obtained

 Example 2

8.41 of the aqueous extract described in Example 1 were stirred with Boog Florisil® 30min at 20 ⁰ C. After 16 hours of standing at 4 ° C, the clear supernatant was siphoned off and discarded. The further work-up was carried out as described in Example 1. 600 mg of substance could be obtained

 Example 3

2.5 kg of pulp, prepared as described in Example 1, was lyophilized and worked up after 3 weeks cold storage, as also described in Example 1, to an aqueous extract. The lung lyophilizate was used according to its original fresh weight. The extractant used was η deviations from Example 1 glucose solution (55 g / l).

7.11 of the aqueous extract was stirred with 420g of silica gel for the column chromatography 60min at 20 ⁰ C. After working up the adsorbate according to the variant shown in Example 1, 310 mg of substance could be obtained.

Example 4

36.0 kg of fresh bovine lung were processed into pulp as described in Example 1. 500 g of each of these minced lungs were each intensively stirred at 20 ° C. for 60 min with 2N of isotonic sodium chloride solution. After filtration through a Dederongaze and then centrifuging the filtrate at 500g and 20 ⁰ C 1201 aqueous extract could be recovered.

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Each 3Ol extract was stirred with 2.5 kg Florisil® 60min at 20 ⁰ C for. After the absorption of the adsorbent was the

Supernatant (each 26-281) decanted and stirred again with 2.0 kg Florisil® 60min at 20 ⁰ C. The individual Florisiladsorbate was washed with 51 distilled water and eluted with 6.41 chloroform / methanol mixture (2: 1).

The Ghloroformphasen were combined and after concentration under nitrogen, 12.2 g of substance were recovered.

Example 5

From 9.35 kg of fresh bovine lung were obtained in accordance with Example 1 in 500g portions, which were stirred vigorously with 2, oil of isotonic sodium chloride solution at 20 ⁰ C, 31.71 aqueous extract. 23.01 of this extract were stirred with 1.5 kg Florisil® 60 min at 2O ⁰ C. After separation of the supernatant, the surfactant-loaded Florisil® was divided into 2 equal portions. One half was washed with 4 l tap water and the other with 31 acetone. Each part was then eluted with 2: 1 chloroform-methanol mixture, the chloroform phases were separated and concentrated to dryness under nitrogen. The water-washed adsorbate gave 1, Og, the acetone-washed adsorbate 0.62 g of substance.

Example 6

8, Ol of the extract obtained in Example 5 were stirred with 1.2 kg of silica gel for 60 min at 20 ° C. After separating the

On the supernatant, the surfactant-loaded silica gel was washed with 4 l tap water and eluted with 21 dichloromethane-methanol mixture (2: 1). The concentrated to dryness dichloromethane phase provided 422 mg of substance.

Example 7

From 9, Ol aqueous extract, which was prepared as described in Example 5, after stirring with 2 kg of diatomaceous earth, washing the adsorbate with 101 tap water and elution of the surfactant material by 60 minutes stirring in 2,81 chloroform / methanol mixture ( 2: 1) 600 ml of chloroform phase are obtained. This was concentrated to dryness under nitrogen to yield 1.3 g of the substance.

Example 8

The aqueous extract prepared as described in Example 5, after stirring with 0.68 kg of silica gel, which had already been used in Example 5, could be regenerated by treatment with 2 N NaOH.

Washing of the adsorbate with 21% acetone and elution of the surfactant material by stirring for 60 minutes in 1000 ml of chloroform / methanol mixture 960 ml of chloroform phase. This was concentrated to dryness under vacuum to yield 0.6 g of the substance.

Example 9

9.0 kg of pulp, which was prepared as described in Example 1, was extracted in 18 portions of 0.5 kg each with 2, isotonic saline solution by vigorous shaking for 10 minutes and the Lungerückstände separated via plastic sieve and Dederongaze. The resulting extract was applied to a column filled with 2.0 kg Florisil® (diameter 15 cm, filling height 25 cm).

It was then washed with 10.01 of distilled water and the active ingredient with 7.51 chloroform-methanol mixture (2: 1) eluted. After settling overnight, the methanol phase of the eluate could be sucked off. The chloroform phase was concentrated to dryness in vacuo. 2.65 g of surfactant could be obtained.

Example 10

2.5 g of the active ingredient prepared according to the invention (38.2% phospholipid) were dissolved in 25.0 ml of chloroform (2nd AB-DDR), prefiltered over glass fiber filter paper (layer thickness 1.80 mm) and filter paper and then over Glasfaservorfilter (layer thickness 450μητι) on cellulose -Sterilfilter (0,2μηη pore diameter, 90μίτι layer thickness) sterile filtered. After concentration of the sterile solution on a vacuum rotary evaporator, the solvent residues were expelled by introducing sterile nitrogen while simultaneously evacuating the vessel. The substance thus obtained was emulsified with 20.0 ml of sterile water by means of an Ultra-Turrax and the emulsion was filled into 25 ml rolled rim bottles so that 4 bottles of 200 mg phospholipid and one residual bottle contained only 160 mg phospholipid. After freezing at -50 ⁰ C for 3 days ° were robes lyophilized, while the counter heater was set at a maximum of 2O ⁰ C. After lyophilization, the flasks were closed with puncture stopper B 180 and aluminum capsolut closure.

Example 11

1.5 g of the active ingredient prepared according to the invention (40.0% of phospholipid) were emulsified with 15.0 ml of distilled water by means of an Ultra-Turrax and autoclaved in an Erlenmeyer flask with external grounding stoppers for 15 minutes. After cooling, the emulsion was emulsified for a further 2 minutes under sterile conditions by means of an Ultra-Turrax to improve the quality and filled the emulsion so in 25ml roll edge bottles that contained 3 bottles of 200mg phospholipid. After freezing at -25 ⁰ C for 4 days, the samples were lyophilized, while the counter-heating was set to a maximum of 20 ⁰ C.

After lyophilization, the bottles were closed with piercing stopper B 180 and aluminum capsolut closure.

For the determination of the phospholipid content, the protein concentration, the cholesterol and neutral lipid content, the following analytical methods were used.

The phospholipid content was determined by a method published by BARTLETT (N.ZÖLLNER and D.EBERHAGEN: "Examination and determination of lipid in the blood", Springer-Verlag Berlin, Heidelberg, New York, 1965) (s = i 2%) The content of cholesterol was determined by a colorimetric method, whereby the cholesterol as well as the cholesterol ester is detected (s = ± 4%) .The neutral lipids were determined by the triglyceride determination method of the 2nd Pharmacopoeia of the GDR, Method 1 A.1.1 The protein content was determined according to a method described by HESSE and LINDNER (Zeitschrift für Allg. Mikrobiologie 11 [1971], pp. 585-594; ebenda 15 [1975], pp. 559-561), which It is particularly suitable for determining protein concentrations in aqueous emulsions (s = 3%). For the surfactant obtained in accordance with the examples according to the invention, the following composition was determined by the abovementioned method:

Table 1

composition

Phospholipid 20-75%

Cholesterol 10-40%

Neutral lipids 5-30%

Protein 0.2-0.5%

Water 0,5-5%

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The preparations obtained by the method according to the invention showed a high efficiency in the in vivo testing on animal models, without further additions were necessary. The proof was carried out on 2 internationally recognized models.

1. Influence of surfactant in adult respiratory distress syndrome (Figure 1, Table 2)

The influence of surfactant instillation in adult respiratory distress syndrome has been studied in guinea pigs. Before the acute respiratory inefficiency was produced, the animals were araised by general anesthesia, a catheter was introduced into the A. carotid for blood sampling and constantly ventilated with pure oxygen. The acute respiratory distress syndrome was achieved in the animals by 8 times bronchial leaching of the surfactant with warm physiological saline. The flush volumes corresponded to the vital capacity of the animals. At the end of the rinse procedure, the arterial oxygen tension in these animals was below 60mm Hg, despite ventilation with peak pressures of + 30cm H ₂ O, positive end-expiratory pressure (PEEP) of 8cm H ₂ O, and 10 and 40 minutes postoperatively half of the animals each received 1 ml of surfactant tracheal instilled in the last lung lavage. Blood gas analyzes were performed at 5.15 and 30 minutes after each surfactant dose. An optimal surfactant leads to an immediate increase in the arterial oxygen tension by an average of 200 mm Hg under absolutely the same ventilation conditions.

2. Influence of Surfactant in Immature Lungs (Fig. 2, Table 3)

To test the influence of surfactant on immature lungs, immature rabbit neonates were born by hysterotomy on the 27th pregnancy day (total pregnancy duration: 31 ± 1 day). The animals were tracheotomized immediately after birth and part received via the tracheostomy tube an average of 60μΙ surfactant instilliert in the trachea; untreated sister animals of the same litter served as controls. Subsequently, the animals were placed in a pressure-consistent whole-body plethysmograph (38 ^° C.) and ventilated with pure oxygen with continuous registration of respiratory pressure and respiratory volume. Primary data were used to calculate thorax-lung compliance. In some experiments, all animals were ventilated after birth for 10-15 minutes, and only then did a portion of the animals receive surfactant instillated via the trachea.

In both animal models good and excellent results could be achieved with the tested surfactant preparations.

Compatibility studies show that at concentrations of 4 mg / 0.5 ml / 20 g mouse and 8 mg / 10 ml / 20g mouse, i.p. applied, all animals are without findings in an observation period of 7 days.

When determining the microwave purity, no microorganisms can be detected in the preparation produced by the method according to the invention.

The application of the surfactant preparation according to the invention is carried out by tracheal instillation or droplet inhalation by means of nebulizer in all acute or chronic lung diseases that are associated with a surfactant deficiency. Therapy with the surfactant preparation is therefore indicated in adult respiratory distress syndrome caused by

- trauma (shock lung)

- Viral pneumonia

- Heart attack

- acute kidney failure

- gas poisoning

- burns

- over infusion ("overhydration")

- Drug poisoning

- Pulmonary contusion

- fat embolism

- sepsis

- Aspiration pneumonia

- Artificial respiration

- Oxygen poisoning

- Irradiation

- Post-perfusion lung

and all other internal and external pollutants leading to pulmonary edema as well as respiratory diseases

disturbed bronchial surfactant, such as. B. in all chronic obstructive pulmonary diseases (asthma, chronic

Bronchitis etc.). In addition, the surfactant preparation can be used to improve non-ciliary mucus transport as well

to support the local defense against infection of viral and bacterial infections (macrophage stimulation).

It goes without saying that a preparation which can be used in adult respiratory distress syndrome can also be used in neonatal Respiratory distress syndrome is applicable as a causal therapeutic. The administration of the surfactant preparation is based on the phospholipid in doses of 1-500mg / ml, wherein

for the treatment of adults adult 0.5g to? 0g, preferably 10 μl. 15g for the treatment of a newborn 0.1 to5.0g, preferably 0.2g, phospholipid may be needed.

The preparation according to the invention changes the surface tension of an aqueous phase.

Methodology:

The measurement of the surface tension and the recording of the hysteresis curves were carried out according to the known method

by means of a WILHELMY balance (type ATF 01, manufacturer: Fa.E.Biegler, Mauersbauch, Austria).

Substance use: 5Ml (60mgPhosphalipid / ml) Result: Fig. 3, substance of Example 4 Ordinate: surface tension Abscissa: relative surface

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Table 2

Arterial oxygen tension (mm Hg) in experimental respiratory distress syndrome due to lung flushing before and after surfactant instillation Guinea pig PaO ₂

Initial values 30 min after 30 min after

before lung after lung 1. instillation 2. instillation n

Rinse conditioner

example 1 not determined 55 ± 3 280 ± 33 305 ± 41 5 Example 2 375 ± 7 42 + 11 238 + 96 340 + 54 4 Example 3 355 + 14 51 ± 6 272 ± 52 315 ± 62 4 Example 4 335 ± 8 Example 5 46 ± 2 225 ± 81 300 ± 61 6 Washed water 315 ± 6 Washed acetone not determined 38 + 14 218 + 35 295 ± 38 3 Example 6 320 ± 11 49 ± 8 284 + 25 355 + 46 5 Example 7 370 ± 15 60 ± 17 252 ± 62 320 ± 48 4 Examples 350 ± 12 Example 9 not determined 61 ± 15 90 ± 51 113 ± 87 23 controls 330 ± 65 Table 3 Compliance (ml / cm H ₂ O · kg " ¹ ) at a ventilation pressure of phospholipid + 25 cm H ₂ O 30 minutes after birth η salary(%)

example 1 Example 2 Example 3 Example 4

Example 5

Washed water washed acetone

Examples Example 7 Examples Example 9

control animals

23.0 40.0 21.5 25.0

34.2 40.1 21.2 40.0 50.0 38.2

1,006 1,605 1,199 1.531

1,273

1,138

1,012

1,189

not determined

1,261

0.082

+0.727 ± 0.508 ± 0.24 ± 0.018

+0.33 ± 0.23 ± 0.023 +0.41

± 0.46 ± 0.056

 gene

Claims (5)

-ι- 257 641 6 Invention claims: 1. A process for the preparation of an agent for the treatment of diseases associated with a change in the pulmonary surfactant system by isotonic aqueous extraction of minced lungs of mammals and separation of the cell debris by centrifugation at low g-numbers, characterized in that the extract with contacting an insoluble adsorbent, washing the non-adsorbed materials, peeling the active ingredient with organic solvents or mixtures thereof, removing the organic solvent and bringing the resulting active ingredient into a pharmaceutical form suitable for application. 2. The method according to item 1, characterized in that for the replacement of the active substance of the adsorbent, a solvent mixture consisting of methylene chloride or chloroform and methanol in the ratio 2: 1 is used. 3. The method according to item 1, characterized in that the extraction of the minced lung is carried out with isotonic glucose or sucrose solution. 4. The method according to item 1, characterized in that one uses an adsorbent based on silica, preferably activated silica gel or magnesium silicate. 5. The method according to item 1 and 4, characterized in that the isotonic-aqueous extract is repeatedly brought into contact with fresh absorbent.