New synthetic peptides, pulmonary surfactant composition, a drug for the treatment of respiratory distress syndrome

 

(57) Abstract:

Describes a new synthetic peptides having the following specific sequence of General formula I

Xaa-Pro-Val-Xbb-Xcc-Lys-Arg-W,

Xaa may be present or may present a Cys or Ser; bb represents His or Asn; CSU represents Leu or Ile; W represents the hydrophobic part of the peptide containing 12-20 molecules Leu and/or Nle, and Tolna or hydroxyl group can be allerban fatty acid C14-C18or subjected to reaction acetamidomalonate. Also describes compositions and drug-based compounds of the formula I for the treatment of respiratory distress syndrome. 5 S. and 2 C.p. f-crystals, 2 tab.

The present invention relates to new synthetic peptides. More specifically, it relates to synthetic peptide in combination with a mixture of peptides exhibits strong surface activity. The present invention also relates to intermediate compounds used to generate the synthetic peptides, the method of obtaining these synthetic peptides, pulmonary surface-active substances (surfactants) containing the peptides and see what Azania pulmonary surfactant as an active ingredient.

Respiratory distress syndrome is a condition in which the surface activity of the alveoli is reduced due to lack of pulmonary surfactant. This leads to collapse of the alveoli, which in turn leads to severe breathing disorders. This syndrome is common in premature babies and is a cause of high mortality. It is known that the composition of pulmonary surfactant is highly effective for the treatment of neonatal respiratory distress syndrome.

Adults are also prone to hypoxia, caused by various reasons, and there are many examples of diffuse darkening similar to frosted glass, observed in both lungs on x-ray images of the chest and breathing problems, despite the use of artificial respirator, etc. In the work of the author Ueda and others (see Hiromoto Yasuda, "Biosurfactants. Chapter 3. Medical Practices Using Surfactants. Section 1. Clinical Applications of Surfactants. V. Aspiration Pneumonia and Surfactants", page 184, 1990, Science Forum. Co. Ltd.) described 2 cases of pneumonia in adults (i.e. 1 case: pneumonia through inhalation of gaseous nitrate, and 2 case: recurrent aspiration pneumonia as a result of brain tumors, both of these cases were caused hypoxia and led to hudsen the Asti life of patients by injection of pulmonary surfactant in the respiratory tract. Postoperative respiratory failure can occur after heart surgery, because in the process of operation, the breathing stops. The effect of pulmonary surfactant on such respiratory disorders are also described in the literature (see, Shuichi Nosaka et al., Journal of Japanese Medical Society for Biological Interface, Vol. 22, p. 66, 1991).

Thus, substitution therapy, which consists in the introduction pulmonary surfactant from the outside and through the respiratory tract, provides a significant therapeutic effect in the treatment of respiratory distress syndrome.

Recently it was found 4 types of apoproteins that are unique pulmonary surfactant for mammals. This - SAC-apoprotein and surfactant-apoprotein D, which are hydrophilic, surfactant-apoprotein B (hereinafter referred to SP-B) and surfactant-apoprotein C (hereinafter referred to SP-C), which are hydrophobic (see Toyoaki Akino and Yoshio Kuroki, Respiration and Circulation, Vol. 38, N 18, p. 722, 1990; Hiromoto Yasuda et al., Biosurfactants: Chapter 2. The Biochemistry of Surfactants-Surfactants and Apoprotens, p. 131, 1990, Science Forum, Co. Ltd.).

SP-C (sequence No. 1), derived from the human lung consists of 35 amino acids and is vysokoriskovannym apoproteins, which is rich in valine and has phenylalanine as N-terminal amino acids, SP-C, isolated from lungs bulls (sequence is five amino acids in N-terminal position vary depending on animal species, they are almost fully comply with human SP-C

In Japanese patent N Hei-3-502095 also indicated that a synthetic peptide (sequence No. 4) with the following 32 amino acids, which are part of the structure of SP-C is a minimal fragment having a high surface activity, which is a mixture of the peptide with the lipid effectively used for the treatment of respiratory distress syndrome, and that the comparison of the surface activity of this minimal fragment of the peptide with the surface activity of the other fragments, synthetic peptides, which have a shorter amino acid sequence shows the decrease in surface activity is not due to the absence of a particular amino acid, but by reducing the length of the peptide chain.

Previously, some of the authors of the present invention conducted the work, the results of which showed that a mixture of synthetic peptide (hereinafter denoted TP-C), the structure of which is part of the structure of SP-C with lipids is effective in the treatment of respiratory distress syndrome and these compounds are used in the treatment of several patients (see Japanese patent N Hei-5-518188).

What casaldaliga, increasingly formed defective peptides, difficult isolation and purification of peptides, increases the time required to obtain peptides, it is increasingly difficult to arrange the receipt of peptides in large quantities, etc.

Also for reasons of safety, the composition of pulmonary surfactant are often supplied in the form of dry powders, which are diluted before use saline solution and injected into the suspension. In order to improve the dispersibility of the suspension containing pulmonary surfactant additive used suspendida agents, such as mannitol (see published Japanese patent N Hei-1-60451) and liophilization at a temperature of primary freezing from -1 to -10oC. However, these methods are difficult to implement in practice, and it was desirable to develop a more simple ways to get the drugs.

The composition of pulmonary surfactant (hereinafter referred to as S-35), obtained by combining SP-C with a mixture of lipids, which includes cholinesterase, acidic phospholipid and imokilly similar, has a very poor dispersibility in physiological solution, which complicates the preparation of the suspension, which must be sufficiently homogeneous, so that it can be used as it is moved cohesive ability of the peptide and the hydrophobic nature of the pulmonary surfactant.

Since TP-C has low solubility in most solvents, it was necessary to use triperoxonane acid (TFA) in the preparation of the composition of pulmonary surfactant. Problems with TP-C was that it took a long time to concentration and drying to remove as many of TPA, as well as that due to residual TFA suspension of pulmonary surfactant was acidified in the preparation of the composition of pulmonary surfactant.

The authors of the present invention have carefully studied synthetic peptides from the point of view of the above problems and have developed the present invention, revealing that new synthetic peptides (hereinafter referred to as "synthetic peptides according to the invention"), which contain the following amino acid sequence and hydrophilic peptide part of a specific sequence from the N-terminal part and a hydrophobic peptide portion, formed mainly by the sequence LEU and/or NLE in the C-terminal part, to easily select and clear, they can be produced in large quantities, they are well soluble in formic acid, TFA, triptoreline, dimethyl sulfoxide (DMSO), chloroform, mixtures of chloroform with methanol, methane is methanol compared to synthetic SP-C and TP-C. The authors present invention also found that pulmonary surfactant prepared from synthetic peptides according to the invention and mixtures of lipids, even in cases when they receive conventional methods with the use of freeze-drying at a temperature of -20oC or lower and without additives suspendida agents, form a fairly homogeneous suspension is more uniform in comparison with S-35 or synthetic pulmonary surfactant (hereinafter referred to as "SF-3"; see published Japanese patent N Hei-2-87685), which includes only the mixture of lipids consisting of cholinesterase, acidic phospholipid and gynecologi analog or compared with a substance (hereinafter called "S-TA; see published Japanese patent # Sho-61-9924), in addition to containing fatty acid, a substance composed of phospholipid, neutral lipid, cholesterol, carbohydrates, and minimal amounts of protein present in the lungs of mammals, and together with the synthetic peptides according to the invention have high surface activity, which is equivalent to surface activity of S-35, S-TA or pulmonary surfactant consisting of TP-C and a mixture of lipids.

Xaa-Pro-Val-Xbb-Xcc-Lys-Arg-W

(Xaa may be present or may present a Cys or Ser, Xbb PR="ptx2">

Synthetic peptide according to the invention contains a hydrophilic peptide part, described below in a specific sequence in the N-terminal part, and a peptide portion having a hydrophobic peptide portion containing mainly Lei and/or NLE in the C-terminal part, and is a synthetic peptide having a strong surface activity mixed with lipids.

Xaa-Pro-Val-Xbb-Xcc-Lys-Arg

(Xaa may be present or may present a Cys or Ser, Xbb is a GIS or ASN, and XCC represents LEU or Ile).

Although hydrophobic peptide portion consists of hydrophobic amino acids, as Lei, NLE, Ile, Val, Hairdryer, NCA and Three, it usually will include 12 or more, preferably 12 to 20 molecules Lei and/or NLE. Despite the fact that from the point of view of ease of synthesis, and so forth, it is preferable that this hydrophobic peptide part consisted of the same hydrophobic amino acids, it may consist of a suitable sequence of molecules Lei, NLE or may contain in its sequence from 1 to 5 molecules of Ile, Val, NVA, and Three other hydrophobic amino acids.

Synthetic peptides according to the invention include synthetic pepticheskogo peptide. Amino acids added to the N-terminal part can be a Cys or Ser. Further, to the N-terminal part can be added to a peptide with the sequence of hair-Gli-Ile-Pro. Tilney group or hydroxyl group present in the above-mentioned synthetic peptide, can be allievate fatty acid with 14 to 18 carbon atoms (preferably palmitic acid), or you can spend the reaction acetamidomalonate. Peptide added to the C-end, a sequence can have Gli-Ala-LEU-LEU or Gli-Ala-LEU-LEU-Met-Gli-Lei.

Further, the synthetic peptides according to the invention also include such synthetic peptides (with the exception of peptides, partially having the structure of natural SP-C), which contain the peptide group, with good hydrophilicity, and who show strong surface activity, if you put them with a mixture of lipids, even after the addition, deletion and substitution of one or more amino acids included in the composition of these peptides.

Synthetic peptides according to the invention can be obtained by chemical methods or by genetic engineering methods, although chemical methods are preferred from the viewpoint of separation and purification.

Hee the glasses and condensation of fragments with the use of liquid-phase synthesis or solid phase synthesis, such as methods based on the use of azides, acid anhydrides, acid anhydrides, mixed acid anhydrides, N-N'-dicyclohexylcarbodiimide (the way DCC), active ester (p-nitroanaline ether, p-hydroxysuccinimidyl, carbimazole and so on), redox activation methods N-N'-dicyclohexylcarbodiimide.

The invention also provides a method of condensation of the fragment to obtain the synthetic peptides according to the invention, in which the hydrophilic peptide group containing a protected N-end and protected functional side chains, serves as an intermediate connection.

Compared with the method of gradual lengthening of the chain method of condensation of the fragment makes it easier to clean the target substance, it is better suited for the synthesis of large quantities of peptides and prevents losses due to unexpected errors. Synthetic peptides according to the invention can be obtained by condensation of hydrophobic parts with pre-obtained hydrophilic peptide part, with the protected N-end and protected functional side chain, by liquid-phase or solid-phase synthesis. The choice of protective groups for the N-con protective group, used in the conventional synthesis of peptides. 9-Fluorenylmethoxycarbonyl (Fmoc), 2-chlorobenzenesulfonyl (2-ClZ) or tert-butyloxycarbonyl (Boc) can be used as a protective group for the terminal amino acid group, 9-fluorenylmethoxycarbonyl (Fmoc), tert-butyloxycarbonyl (Boc) or carbobenzoxy (Z) or tosyl (Tos) can be used as a protective group for lysine, TRT, Fmoc, Boc, dinitrophenol (D)Bom, Bz1 or tosyl can be used as a protective group for histidine and 4-methoxy-2,3,6-trimethylbenzenesulfonyl (Mtr), Pmc, Mts or tosyl can be used as a protective group for arginine. Thus, peptides that can be used as intermediates for the production of synthetic peptides according to the invention include Fmoc-Pro-Val-GIS(TRT)-LEU-Lys(Boc)-Arg(Mtr)

Fmoc-Pro-Val-ASN-LEU-Lys(BOC)-Arg(Mtr) and

Fmoc-Pro-Val-ASN-Ile-Lys(BOC)-Arg(Mtr).

Pulmonary surfactant (hereinafter referred to as "surfactants according to the invention can be obtained by connecting peptide according to the invention with a mixture of lipids comprising cholinesterase, acidic lipid and gynecologi analog

The ratio of the components of the composition should be selected so that the weight ratio of each of these components Popolare - 50,6 - 80,5 wt.%, acidic phospholipid - 4,5 - of 37.6 wt. %, similar to fatty acid - 4,6 - 24.6 wt.%.

Examples of cholinesterases, which can be used in surfactant according to the invention include 1,2-diacylglycerol-(3)-phosphocholine, such as 1,2-dipalmitoyl-(3)-phosphocholine (dipalmitoylphosphatidylcholine), 1,2-distroller-(3)-phosphocholine, 1-Palmitoyl-2-steamroller-(3)-phosphocholine and 1 stearoyl-2-palmitoylated-(3)-phosphocholine etc.; 1-alkyl-2-acylglycerol-(3)-phosphocholine, such as 1-hexadecyl-2-palmitoylated-(3)-phosphocholine and 1-octadecyl-2-palmitoylated-(3)-phosphocholine and so on; as well as 1-2-dialkylglycerol-(3)-phosphocholine, such as 1,2-dihexadecyl-(3)-phosphocholine, etc. Despite the fact that the above compounds have optical isomers based on the second carbon atom of glycerol residue, any isomer (D-, L - and DL-) can be used as surfactants according to the invention. In addition to single compounds among the above jointstereo in the present invention can be used in a mixture composed of two or more different 1,2-diacylglycerol-(3)-phosphocholine having acyl groups, preferably two saturated acyl groups of 12 to 24 carbon atoms, or a mixture consisting of videocase the EP: 1,2-diacyl-sn-glycero-(3)-phosphoric acid (L-phosphatidic acid), 1,2-diacyl-sn-glycero-(3)phospho-L-serine (phosphatidylserine), 1,2-diacyl-sn-glycero-(3)-phospho-sn-glycerol (phosphatidylglycerol) and 1,2-diacyl-sn-glycero-(3)-phospho-(1)-L-myo-Inositol (phosphatidylinositol). The first and second positions in these compounds can be substituted by the same or different acyl groups. Preferably, the indicated acyl group had 12 to 24 carbon atoms.

Examples of suitable imokilly analogues include free fatty acids, alkali metal salts of fatty acids, complex imokalee alkylether, imokalee glycerinated and amides of fatty acids as well as mixtures comprising two or more of these compounds, and alcohols, fatty acid amines and fatty acids.

In the present description, the term "analogs of fatty acids" encompasses the above-mentioned alcohols, fatty acids and aliphatic amines.

As the free fatty acid can be used myristic acid, palmitic acid or stearic acid, although it is preferable palmitic acid.

Sodium, potassium, magnesium and calcium salts of the above fatty acids can be used as a salt of an alkali metal fatty acid, although predpochtitelney of alkylation, although it is preferable to Etisalat. Esters of monoglyceride can be used as a complex ester of glycerin fatty acid, although it is preferable to monopolisation.

Alcohols with 14 to 18 carbon atoms can be used as alcohol fatty acid, although it is preferable hexadecylamine alcohol. Amines with 14 to 18 carbon atoms can be used as the aliphatic amine, although it is preferable to hexadecylamine.

The above cholinesterase, acidic phospholipids and imokalee analogues may represent the products isolated from plants or animals, semi-products or synthetic products, you can apply these types of products manufactured by the industry.

Surfactants according to the invention can be obtained by drying and curing at reduced pressure a mixture of solutions of peptide according to the invention with the above mixture of lipids, followed by suspendirovanie the obtained residue in a suitable suspendida liquid, followed by lyophilization.

Examples of solvents that can be used to obtain a solution of the peptide according to the invention include formic acid, TFA,Examples of solvents which can be used to obtain solutions of a mixture of lipids include chloroform and chloroform-methanol /1:2 to 5:1 (o/o)/.

Examples suspendida fluids include water and mixtures of water with ethanol /4: 1 - 20:1 (o/o)/, although preferred are mixtures of water with ethanol. The suspension is conducted for 5 to 60 minutes, preferably for 15 to 30 minutes, with 30 - 60oC, preferably at 40 - 50oC.

Surfactants according to the invention obtained in this way, inevitably contains a small amount of residual water. However, it is desirable to dry surfactant to the water content of 5.0 wt.% or lower relative to the total weight of the product. If surfactant is dried to such an extent, then the remainder of the ethanol becomes invisible in those cases, when using a mixture of water with ethanol.

Dry powder surfactant composition according to the invention can be suspended and dispersing until smooth in a solution containing fiziologicheskii is suitable for the concentration of salts of monovalent or divalent metal, such as 0.9% sodium chloride and 1.5 mm calcium chloride, or in solution in physiological buffer containing such salts. To prepare the solution using hand mixers or mixers with variable skorton suspension and pharmacological properties of the surfactants according to the invention, obtained by the above methods.

(1) Surface activity

1) Reduction of surface tension

The degree of decrease in surface tension was measured by the method described in the work of Tanaka et al. (Journal of Japanese Medical Society for Biological Interface, Vol. 13, N 2, p. 87, 1982).

Suspension surfactants according to the invention was administered in saline solution (surface tension 54,0 cm2) so that 1 cm2square had 1,0 - 2,0 g surfactants according to the invention. The specified surface area squeezed and expanded within 54,0 - 21,6 cm2within 2 - 5 minutes and continuously measured the surface tension at 37oC using the measuring device Wilhelmy (firm Kyowa Interface Science Co. Ltd.). The maximum surface tension was 24.7 - 34,1 Dyne/cm, and the minimum surface tension was 0.2 - 8,7 Dyne/cm, suggests that due to the action of surfactants according to the invention, the surface tension of physiological saline decreased.

Using the same method to measure the effect of SF-3 to reduce surface tension and got the following results: the maximum value of the surface tension - 26,8 is 50.3 Dyne/cm and minimum values of the surface tension - 1,0 - 13,5 DIN/see

Suspension surfactants according to the invention is dripped on the surface of the saline solution so that had to 0.8 - 1.5 mcg surfactant on 1 cm2the surface of the saline solution, and using vertical plate was measured by the change in surface tension, starting from the moment when a drop of suspension was hit on the solution. During the measurement, the temperature was 37oC.

The period balance - this is the time during which the surface tension reaches a constant value, starting from the moment when a drop of the suspension falls on a physiological solution, and the value after reaching this equilibrium is called the equilibrium surface tension.

Surfactants according to the invention was formed into a film on the surface section of gas-liquid within a short period of time (30 - 60 seconds) and reduced the surface tension to 26.7 - to 34.3 Dyne/see

The same method was measured by the ability of the SF-3 distributed on the surface, these measurements showed that the surface tension after 120 seconds decreased to 38.1 - to 52.9 Dyne/see

This fact indicates that the surfactants according to the invention is capable of rapidly p 3) adsorption on the surface of a section of gas-liquid

Prepared suspension of physiological solution containing 0.2 - 1.0 mg surfactant according to the invention of 1 ml at 37oC and measured the speed of adsorption of suspended surfactant according to the invention on the surface of a section of gas-liquid.

The adsorption was measured by the method described by King et al. (American Journal of discrimination, N 223, p. 715, 1972).

I.e., the suspension was injected into the bottom of a vessel made of Teflon with a diameter of 5 cm containing saline solution, and then stirred with a magnetic stirrer. The adsorption was determined from the change in surface tension after cessation of mixing.

Surfactants according to the invention has reduced the surface tension to values between 28.3 to 36.8 Dyne/cm for the period from 30 to 100 seconds after the termination of stirring, after which the surface tension remained unchanged.

This fact indicates that the surfactants according to the invention in suspended condition swam and adsorbiroval on the surface of a section of gas-liquid within 30-100 seconds and formed a film having a strong surface activity.

The same measurement is carried out for SF-3, showed that the constant surface tension in the range from 42,2 to 58.3 Dyne/cm was postignucem surfactants according to the invention, and that surfactants according to the invention have a pronounced ability to stimulate the adsorption on the surface.

(2) the Ability to move in suspension

Tests on the ability of pulmonary surfactant move in suspension was performed by the method described in Japanese patent Hei-4-76965.

The ability to move in the suspension was determined based on the dispersive ability of the pigment in specific points in time after the start of the suspension, and the maximum size of the dispersed particles after 2 minutes after the start of the suspension.

Tests dispersibility was carried out as follows. 60 mg each of pulmonary surfactant was poured into 20 ml vials. Then in each of these vials was added 2 ml of physiological solution and put the bottles in a shaker type Iwaki KM Shaker-V-S (produced by Iwaki Sangyo Co., Ltd. and shook with the intensity of 270 movements per minute. The degree of dispersion of each sample was visually observed using a magnifying glass every 30 seconds during the first 2 minutes after the start of shaking, every minute for 2 to 4 minutes after the start of shaking, and every 10 minutes after the first 4 minutes from start shaking.

The degree of transition in the suspension was determined through obrasovannii, if the vessel is not observed aggregations of matter, and if the composition is uniformly dispersed in saline solution before formation of a thick white suspension.

The efficiency of dispersion of each employee was determined at set points in time as the percentage of samples where the suspension ended, to the total number of samples (10 bottles), and then output the average for both employees.

The maximum size of dispersed particles was measured by the following method. 60 mg each pulmonary surfactant was poured into 20 ml vials. Then in each vial was added 2 ml of physiological solution and the vial was shaken for 2 minutes as described above. After that, under a microscope, they found that the largest particle and measure its diameter with a micrometer. Thus, it was found that surfactants according to the invention have mainly been suspended for 2 minutes, and the maximum particle size of these surfactants was 0.8 mm or less, which indicates a high ability to suspendirovanie.

(3) Pharmacological properties

1) Acute toxicity

Acute toxicity of surfactants according to the invention was tested on male animals mice 1CR is the situation for mice was 2.4 - 10.0 g/kg and 1.0 - 5.0 g/kg, respectively, for rats, the doses were 1.5 - 5.0 g/kg and 1.5 - 2.5 g/kg, respectively.

2) Subacute toxicity.

Surfactants according to the invention was administered intraperitoneally to adult Nesterovsky rats at daily doses of 300 - 600 mg/kg for 1 month. In relation to the mass of rats deviations were observed, not identified as abnormal histological observations with the naked eye.

3) Effect, which consists in maintaining the volume of the alveoli

Premature baby rabbit in the gestational period of 27 days practically do not produce pulmonary surfactant and are in a state of lack of pulmonary surfactant. Therefore, they were used as models for the study of neonatal respiratory distress syndrome.

5 young rabbit in the gestational period of 27 days was measured by the volume of the alveoli (hereinafter called lung volume) under various airway pressure at 37oC.

On the neck of the calf did an incision and using a water manometer attached to the trachea, conducted continuous measurements, starting at 5 minutes after injection of the surfactant according to the invention through the respiratory tract. Using a dual syringe pump with independent PR is to put the alveoli to expand. Then the airway pressure was lowered to 0 cm H2O in order to cause the collapse of the alveoli, and measured the amount of light at different pressures H2O. Then the volume of the lungs indicated in milliliters per 1 kg of body weight (ml/kg).

Surfactants according to the invention (60 mg/kg) was administered by direct injection into the respiratory tract of 0.05 - 0.5 ml of suspension in physiological solution with the concentration of the surfactant in 1,0 - 6,0 (V/o).

The volume of the lungs at the moment when the pressure is reduced to 5 cm H2O indicates the functional residual capacity and the higher the volume, the higher the activity of pulmonary surfactant.

As a control were injected saline solution without surfactants according to the invention. The volume of the lungs (at 5 cm H2O) in premature babies rabbit in the control group was 1 to 5 ml/kg, indicating that the alveoli barely expanded.

Full-term babies in the gestational period of 30 days have normal levels of pulmonary surfactant. The amounts of light such calves (at 5 cm H2O) make up 35 to 53 ml/kg, indicating that the alveoli expand enough and what you can call normal breathing.

In those cases, when introduced SF-3, the amounts of light (at 5 cm H2O) the premature deanesly surfactants according to the invention, the volume of the lungs (at 5 cm H2O) was 39 - 55 ml/kg, indicating that the surfactants according to the invention increase the volume of the lungs in premature babies up to normal levels.

As described above, the synthetic peptides according to the invention have the ability to greatly improve the surface activity of lipid mixtures. This allows the manufacture of medicinal preparations for the treatment of respiratory distress syndrome, which is effective against surface activity, the ability to suspendirovanie and pharmacological properties using surfactants according to the invention, which consist of synthetic peptides according to the invention and mixtures of lipids.

Compositions containing the surfactants according to the invention as an active ingredient, can also be used to treat other diseases for which treatment is applied pulmonary surfactant, including postoperative respiratory disorders, asthma, bronchitis, neonatal necrotizing enteritis, gastric ulcer and duodenal ulcer, respiratory diseases caused by viruses and tube obstruction, to prevent adhesions of fallopian tubes and postoperative SPECT, as well as extraordinary.

Pharmaceutical preparations according to the invention DL the mg surfactant per dose for adults. These doses are obtained by suspension in water, saline or buffers, etc. that are physiologically tolerable, at concentrations of 1.0 to 10.0% (V/o). This suspension is administered within 72 hours after the onset of respiratory failure by injection or spraying in the respiratory tract, produce from 1 to 10 times. The composition can breathe and resuspendirovania form, i.e. in the form of a powder. Dosage, method of application and the frequency of injection can be changed depending on the symptoms and the patient, as well as a companion treatment.

Pharmaceutical preparations according to the invention may contain, if necessary, such actual auxiliary substances, such as stabilizers, preservatives, isotherwise agents, buffers, suspendresume agents, antioxidants and surfactants, or such medications like bronchodilators, antiallergic drugs, carcinostatic tools, antiviral agents, anti-inflammatory drugs and antifungal drugs.

The medicinal drug of the invention may be in the form of liquid or powder. Pharmaceutical preparations according to the invention can be packaged in sealed vessels, such is the description of the present invention using examples below.

(1) Obtaining peptides

In the following examples, the molecular weight of the synthesized peptides was measured by the method of mass spectrometry with fast atom bombardment (MSBA). As the mass spectrometer used JMS-S102A (JEOL. Ltd.), and as the ion source is cesium gun (10 Kee).

Example 1

The peptide (Peptide A) with the sequence 5 was synthesized on the surface phenylacetylamino polymer (FEMME) method of solid-phase synthesis according to the method described in "The Peptides" (E. Gross and Meinenhofe J. publishers., Barany, G. and Merrifield, R. authors, vol. 2, page 1 - 284, Academie Press, New York, 1980).

Lazenby residue on the C-end transformed in tert-butyloxycarbonyl (Re-S) and associated it with a polymer FEMME through oxyethylenenitrilo. After binding C-Terminus, BOC-LEU-FEMME-polymer (0.70 mol/g, 0.35 g) was transferred into the reaction container peptide synthesizer (model AE, manufacturer Beekman Instruments, Inc.). Then towards the N-Terminus on the surface of the polymer introduced amino acids (which have been attached with suitable protective group) with symmetric anhydrides, in order to synthesize the fully protected peptide-O-polymer. However, by condensation of arginine produced a double N-terminal amino group of all amino acids were protected with BOC groups, and functional side chains were protected by the following groups (which were introduced before the amino acids used in the reaction):

Arg-TOZ : (tosyl)

Liz-2-ClZ : (2-chlorobenzenesulfonyl)

CIS-4MeBz1 : (4-methylbenzyl)

GIS-TOZ : (tosyl)

Completion of the condensation reaction of these compounds was confirmed using the Kaiser test using ninhydrin. The fully protected peptide-O-polymer (155 mg) for 5 minutes swollen in dichloromethane. Then the protective group of N-- BOC was removed with TFA containing 1% (V/o) indole and 0.1% (V/o) identicial. After this peptide was tsalala from polymer processing unprotected peptide-O-polymer anhydrous hydrogen fluoride (11 ml) to which was added p-cresol (1 ml), p-thiocresol (0.2 g) and DMSO (1 ml); the treatment was performed within a 60 minutes at 0oC.

Hydrogen fluoride and DMSO drove under vacuum at 0oC. the Separated peptide and the polymer was washed three times with cold diethyl ether (15 ml) were extracted and separated peptide by washing four times in cold TFA (5 ml). Extracted liquid was immediately filtered and added to ice water (150 ml) to fall into the residue of the crude peptide. The crude peptide was then centrifuged at the e repeated washing using diethyl ether, ethyl acetate and distilled water, received 84 mg of peptide A.

Then, this crude peptide was dissolved in 50% aqueous solution of DMSO and purified by high-performance liquid chromatography with reversed phase (HPLC) using "Bondasheres" and column C8-300 for the selection of the pure peptide A.

Elution was performed within a 5 minutes using 50% aqueous acetonitrile containing 0.1% TFA as eluent. Thereafter, the elution was made for 30 minutes at a linear concentration gradient formed by the above-mentioned eluent, and using 80% aqueous acetonitrile containing 0.1% TFA.

The presence of the peptide in the eluate was observed at 245 nm (spectrophotometer firm Japan Spectroscopic Co. Ltd., model 870-UV) and using a differential Refractometer (Shimadzu Manufacturing corporation, model RID-6A).

MSBA (M+H+) : 3837,1 (calculated molecular mass; 3835,9)

Example 2

The peptide (peptide) sequence 6 was synthesized by the method of solid-phase synthesis using mnogovershinnoe solid-phase synthesis system "Kokku - San" (trade name; manufacturer Kokusan Chemical Works Co. Co., Ltd.) using the methods described in "Solid Phase Peptide Synthesis-A PracCLASS="ptx2">

As the original polymer used N--9 - fluorenylmethoxycarbonyl-leucine-O-polymer (Fmoc-LEU-O-polymer) (0.20 mmol/0.5 g) in which N--9 - fluorenylmethoxycarbonyl-leucine (Fmoc-LEU) is associated with a copolymer of 4-(hydroxymethyl) and phenoxymethyl (copolymer 1% styrene with divinylbenzene). The specified polymer swollen in 20 minutes in N,N-dimethylformamide (DMF), then washed four times with DMF. Then added 20% piperidine in DMF and the mixture was stirred by shaking before removal of the protective groups. This operation was repeated three times to completely remove the protective group. Then thrice washed with DMF, three times washed with N-methyl-2-pyrrolidone and three times again DMF to remove excess piperidine in the polymer. The presence of piperidine was tested using litmus paper.

After this was added DMF (6 ml), Fm-Lei (0.5 mmol), N-hydroxybenzotriazole (0.5 mmol) and N,N'-diisopropylcarbodiimide (0.5 mmol) and the mixture was stirred by shaking for 90 minutes that was the reaction of condensation. The polymer was then washed four times with DMF to remove excess reagents. Completion of the condensation reaction using the Kaiser test using ninhydrin.

According to this scheme, carried out the synthesis and gradually debasement N-end and functional groups.

The condensation reaction for the introduction of arginine, lysine, histidine, Proline and cysteine was performed twice, each time for 120 minutes.

After that, the protected peptide-O-polymer was added 20% pyridine in DMF in order to remove the N-terminal protective group is Fmoc. Then the peptide-O-polymer was washed six times DMF and six times with methanol and dried under reduced pressure. Then to the dried peptide-O-polymer (100 mg) was added m-cresol (0.2 ml), 1,2-acondition (0.5 ml), thioanisole (1.2 ml), TFA (7.5 ml) and trimethylsilylacetamide (1,4 ml), additive produced under stirring in an ice bath. After that, the mixture was stirred for 120 minutes in an ice bath prior to the removal of the protective groups with functional side chains and to remove the peptide from the polymer and then filtered through a glass filter (C3). The filtrate was concentrated under reduced pressure to approximately 5 ml using an evaporator. Then added diethyl ether to peptide knocked out. The precipitate was collected on a glass filter (C3), five times washed with diethyl ether and, after drying under reduced pressure received 60 mg of peptide B.

N-terminal amino group of all amino acids were protected with Fmoc groups, and functional side chain b is XI-2,3,6-trimethylbenzenesulfonyl)

Liz-Sun : (tert-butyloxycarbonyl)

CIS-TRT : (trityl)

GIS TRT : (trityl)

Approximately 100 mg of the crude peptide was dissolved in TFA (1 ml) and added four times as much solvent in the mobile phase, i.e., 10 mm - mercaptoethanol in TFA-dichloromethane (5:95, o/o) with a solution of 20 mg/ml, which was purified HPLC using a column Asahipak GS-510 (diameter 7.5 x 500 mm) (trade name; manufacturer Asahi Chemical Industry Co., Ltd.), having pure peptide B.

As eluent used 10 mm - mercaptoethanol in TFA-dichloromethane (5: 95, o/o), elution was produced at a flow rate of 0.8 ml/min for 80 minutes. The presence of the peptide in the eluate was observed at 245 nm (spectrophotometer firm Japan Spectroscopic Co., Ltd., model 870-UV) and using the differential Refractometer (Shimadzu Manufacturing orporation, model RID-6A).

MSBA (M+H+): 3017,9 (calculated molecular mass; 3016,9).

Example 3

The peptide (peptide C) with a sequence of 7 was obtained by the method described in Example 2.

MSBA (M+H+): 3116,0 (calculated molecular weight: 3115,1).

Example 4

The peptide (peptide D) with a sequence of 8 was obtained by the method described in Example 2.

MSBA (M+H+): 2663,7 (calculated saying is that described in Example 2.

MSBA (M+H+): 2211,2 (calculated molecular mass; 2209,9).

Example 6

The peptide (peptide F) with a sequence of 10 was obtained by the method described in Example 2.

MSBA (M+H+): 2647,5 (calculated molecular mass; 2646,4).

Example 7

The peptide (peptide G) with a sequence of 11 was obtained by the method described in Example 2.

MSBA (M+H+): 3018,1 (calculated molecular mass; 3016,9).

Example 8

The peptide (peptide H) with a sequence of 12 was synthesized by the method of solid-phase synthesis using mnogovershinnoe solid-phase synthesis system described in Example 2. As the original polymer used N-N--9-fluorenylmethoxycarbonyl-norleucine-O-polymer (Fmoc-Nle-O-polymer) (0.20 mmol/0.5 g). The specified polymer swollen in 20 minutes in N,N-dimethylformamide (DMF), then washed four times with DMF. After this was added 20% piperidine in DMF and the mixture was stirred by shaking before removal of the protective groups. This operation was repeated three times to completely remove the protective group. After that, nine times washed with DMF to remove excess piperidine in the polymer. The presence of residual piperidine controlled using litmus paper.

Posmol), and the mixture was stirred by shaking for 90 minutes that was the reaction of condensation. The polymer was then washed four times with DMF to remove excess reagents. Completion of the condensation reaction was checked by Kaiser test using ninhydrin.

According to this scheme, carried out the synthesis and gradually added amino acids towards the N-Terminus on the surface of the polymer to obtain the peptide-O-polymer with a fully protected N-end and functional groups.

The condensation reaction for the introduction of arginine, lysine, histidine, Proline and cysteine was performed twice, each time for 120 minutes.

After that, the protected peptide-O-polymer was added 20% pyridine in DMF in order to remove the N-terminal protective group is Fmoc. Then the peptide-O-polymer was washed six times DMF and six times with methanol and dried under reduced pressure. Then to the dried peptide-O-polymer (100 mg) was added m-cresol (0.2 ml), 1,2-acondition (0.5 ml), thioanisole (1.2 ml), TFA (7.5 ml) and trimethylsilylpropyne (1,4 ml), additive produced under stirring in an ice bath. After that, the mixture was stirred for 120 minutes in an ice bath prior to the removal of the protective groups with functional side chains and to remove the peptide is approximately 5 ml using an evaporator. Then added diethyl ether to peptide knocked out. The precipitate was collected on a glass filter (C3), five times washed with diethyl ether and after drying under reduced pressure was obtained 65 mg of peptide h

N-terminal amino group of all amino acids were protected with Fmoc groups, and functional side chains were protected by the following groups (which were introduced before the amino acids used in the reaction):

Arg-Mtr : (4-methoxy-2,3,6-trimethylbenzenesulfonyl)

Liz-Sun : (tert-butyloxycarbonyl)

CIS-TRT : (trityl)

GIS-Sun : (tert-butyloxycarbonyl)

Approximately 10 mg of the crude peptide was dissolved in 3.0 ml of mixed solvent (chloroform-methanol, 2:1, o/o). Then the sample was purified on a column of Sephadex 1H-60 (diameter 2.5 cm x 90 cm), equilibrated mixed solvent (chloroform-methanol, 2:1, o/o) to obtain the pure peptide H.

The presence of the peptide in the eluate was observed at 245 nm (spectrophotometer firm Japan Spectroscopic Co., Ltd., model 870-UV) and using a differential Refractometer (Shimadzu Manufacturing corporation, model RID-6A).

MSBA (M+N1): 2663,6 (calculated molecular mass; 2662,5).

Example 9

The peptide (peptide I) sequence 13 received from the

Example 10

The peptide (peptide Q) with a sequence of 14 was obtained by the method described in Example 8.

MSBA (M+H+): 2663,8 (calculated molecular mass; 2662,5).

Example 11

The peptide (peptide K) with a sequence of 15 was obtained by the method described in Example 2.

MSBA (M+H+): 2663,5 (calculated molecular mass; 2662,5).

Example 12

The peptide (peptide L) with a sequence of 16 was obtained by the method described in Example 2.

MSBA (M+H+): 2503,6 (calculated molecular mass; 2502,4).

Example 13

The peptide (peptide M) with a sequence of 17 was obtained by the method described in Example 2.

MSBA (M+H+): 2736,7 (calculated molecular mass; 2735,5).

Example 14

The peptide (peptide N) with a sequence of 18 was obtained by the method described in Example 2.

MSBA (M+H+): 2640,4 (calculated molecular mass; 2639,4).

Example 15

The peptide (peptide O) with a sequence of 19 was obtained by the method described in Example 8.

MSBA (M+H+): 2640,3 (calculated molecular mass; 2639,4).

Example 16

Fmoc-Pro-Val-GIS(TRT)-LEU-Lys(BOC)-Arg(Mtr)

Named peptide (peptide P) was synthesized by the method of solid-phase Sint is Ali N--9 - fluorenylmethoxycarbonyl N--4 - methoxy-2,3,6 - trimethylbenzenesulfonyl-arginine-O-polymer (Fmoc-Arg(Mtr)-O-polymer) (0.20 mmol), in which N--9 - fluorenylmethoxycarbonyl N--4 - methoxy-2,3,6-trimethylbenzenesulfonyl-arginine is associated with 2-methoxy-4-alkoxybenzyl-polymer (polymer Sasrin" trade name of the company Bachem Co., Ltd). This polymer was gradually added amino acids towards the N-Terminus on the surface of the polymer in accordance with the Protocol Peptide Synthesizer System 9050" with the aim of synthesizing peptide-O-polymer with a fully protected N-end and functional groups.

Then the fully protected peptide-O-polymer washed five times with methanol and dried under reduced pressure. After that, the dried peptide-O-polymer (330 mg) was added a solution of TFA-dichloromethane (1:9, o/o); the additive is produced under stirring in an ice bath. Then the mixture was stirred in an ice bath for 30 minutes, then was stirred for 90 minutes at room temperature before removal of the peptide from the polymer, and the protective groups were still attached to the polymer. After that, the mixture was filtered through a glass filter (C3) and the filtrate was concentrated under reduced pressure to about 5 ml using an evaporator. Then added diethyl ether to peptide knocked out. The precipitate was collected on a glass filter (C3), washed toe

N-terminal amino group of all amino acids were protected with Fmoc groups, and functional side chains were protected by the following groups (which were introduced before the amino acids used in the reaction):

Arg-Mtr: (4-methoxy-2,3-trimethylbenzenesulfonyl)

Liz-Sun: (tert-butyloxycarbonyl)

GIS TRT: (trityl)

Then a solution of TGF-dichloromethane (1:99, o/o) was added to the crude peptide to obtain a sample solution 10 mg/ml, which was purified HPLC using a column Asahipak CS-510 (diameter: 21.5 x 500 mm) (trade name; company Asahi Chemical Industry Co., Ltd.), getting pure peptide P.

A solution of TFA-dichloromethane (1:99, o/o) was used as the eluate, the elution was performed at a flow rate of 8.1 ml/min for 120 minutes. The presence of the peptide in the eluate was observed at 245 nm (spectrophotometer firm Japan Spectroscopic Co., Ltd, model 870-UV) and using a differential Refractometer (Shimadzu Manufacturing orporation model RID-6A).

MSBA (M+H+): 1405,0 (calculated molecular mass; 1403,8).

Example 17

H-NLE(NLE)14 NLU-O-polymer was synthesized on solid-phase mnogovershinnoe system as described in Example 8.

Next, after the addition DMF) in the synthesized H-NLE(NLE)14 NLU-O-polymer instead of Fmoc-Arg(Mtr) Ali shaking for 8 hours. This condensation reaction was conducted twice. Completion of the condensation reaction was checked by Kaiser test using ninhydrin.

After that, the method described in Example 8, the protective group was removed with functional groups, and the peptide was separated from the resin and purified HPLC to obtain peptide sequence 13 (peptide I).

MSBA (M+H+): 2560,2 (calculated molecular mass; 2559,3).

Example 18

The peptide (peptide O), in which tirinya group in the peptide were acetamidomalonate (AFM) was obtained by the method described in Example 4, except that instead of Fmoc-Cys-(TRT) was used with Fmoc-Cys-(ACM).

MSBA (M+H+): 2734,7 (calculated molecular mass; 2733,6).

Example 19

Peptide R was obtained by the esterification of tylnej groups of peptide E palmitic acid by the method described in patent EP 0458167 A1 on names

MSBA (M+H+): 2448,5 (calculated molecular mass; 2448,3).

Comparative example 1

Peptide with sequence 20 was obtained by the method described in Example 2.

MSBA (M+H+): 2793,8 (calculated molecular mass; 2792,6).

Comparative example 2

Peptide T with a sequence of 21 was obtained by way of inoculating composition of the synthetic peptides according to the invention

Synthetic peptides according to the invention were subjected to acid hydrolysis using a solution of 12 N. HCl-TFA (2:1, o/o) containing 5% (m/o) phenol; the reaction was carried out under vacuum at 150oC for 1, 2, 4, 6, 12, 24, 48 and 72 hours, and the products of hydrolysis after removing them from the acid was subjected to analysis using "automatic analysis of amino acids Shimadzu" (1C-9A). Tryptophan (Three) peptide M was subjected to alkaline hydrolysis using 4,2 N. aqueous sodium hydroxide; the reaction was carried out for 16, 24 and 32 hours, after which the mixture was neutralized with HCl, and then analyzed using the "automatic analysis of amino acids". The amino acid composition, calculated on the basis of the coefficients of the acidity of the amino acids, the output of which was greatest during the hydrolysis for 1 to 72 hours, largely coincided with the indices calculated from the chemical formula.

The results are shown in Table 1.

(2) Obtaining surfactants according to the invention

Surfactants according to the invention was obtained by mixing the peptides according to the invention with three lipid components jointstereo, acidic phospholipid and imokilly analogue.

Example 20

Sterilized 1,2-dipalmitoyl; manufactured by Sigma Chemical Co., Ltd.) (450 mg) and myristic acid (200 mg) was dissolved at room temperature in a mixture of chloroform-methanol (2: 1, o/o) (100 ml), and 25 mg of peptide A dissolved in TFA (1.0 ml). The resulting solutions were mixed together, and then was dried and utverjdali under reduced pressure. The resulting residue is suspended in water-ethanol mixture (9: 1, o/o) (100 ml) for 15 minutes at 40oC. After freezing the resulting suspension at -50oC and subsequent drying for 36 hours under vacuum at 85 - 100 mgno received surfactant (2070 mg) as a white powder.

The resulting powder did not contain detectable quantities of ethanol, and the content of each component relative to the total weight of the surfactant was: 65.2 wt.% 1,2-dipalmitoyl-(3)-phosphocholine, and 21.7 wt.% 1,2-diacyl-sn-glycero-(3)-phospho-sn-glycerol, 9.7 wt. % myristic acid, 1.2 wt.% peptide A and 2.2 wt.% water.

Example 21

1,2-Dipalmitoyl-(3)-phosphocholine (300 mg), 1,2-diacyl-sn-glycero-(3)-phospho-sn-glycerol (with acyl group having 14-24 carbon atoms; manufactured by Sigma Chemical Co. Co., Ltd.) (100.0 mg) and palmitic acid (40,0 mg) was dissolved in a mixture of chloroform-methanol (2:1, o/o) (300 ml), and 10.0 mg of peptide B was dissolved in a mixture of chlorof the second pressure. The resulting residue is suspended in water-ethanol mixture (9:1, o/o) (100 ml) for 20 minutes at 45oC. After freezing the resulting suspension at -60oC and subsequent drying for 40 hours under vacuum at 60 - 110 mkno, got 459,1 mg surfactant in the form of a white powder.

The resulting powder did not contain detectable quantities of ethanol, and the content of each component relative to the total weight of the surfactant was : 65,3 wt.% 1,2 - dipalmitoyl-(3)-phosphocholine, 21.8 wt. % 1,2-diacyl-sn-glycero-(3)-phospho-sn-glycerol, to 8.7 wt.% palmitic acid, 2.2 wt.% peptide B and 2.0 wt.% water.

Example 22

1,2-Dipalmitoyl-(3)-phosphocholine (280,0 mg), 1,2-Dilauroyl-sn-glycero-(3)-phospho-sn-glycerol (120,0 mg) and palmitic acid (27.0 mg) was dissolved in a mixture of chloroform-methanol (2:1, o/o) (150 ml), and 2.8 mg of peptide C was dissolved in a mixture of chloroform-methanol (1:2, o/o) (0.5 ml). The resulting solutions were mixed together, and then was dried and utverjdali under reduced pressure. The resulting residue suspended in a mixture of water-ethanol (8:2, o/o) (100 ml) for 45 minutes at 40oC. After freezing the resulting suspension at -65oC and subsequent drying for 36 hours under vacuum at 50 - 80 mcno received 437,ethanol, and the content of each component relative to the total weight of the surfactant was: 64,0 wt.% 1,2-dipalmitoyl-(3)-phosphocholine, a 27.4 wt.% 1,2-Dilauroyl-sn-glycero-(3)-phospho-sn-glycerol, 6.2 wt.% palmitic acid, 0.6 wt.% peptide With and 1.8 wt.% water.

Example 23

Following the same procedure described in Example 21, but using peptide D instead of peptide B, and 1-Palmitoyl-2-oleyl-sn-glycero-(3)-phospho-sn-glycerol instead of 1,2-diacyl-sn-glycero-(3)-phospho-sn-glycerol (with acyl group having 14 to 24 carbon atoms; manufactured by Sigma Chemical Co., Ltd.), received 451,9 mg surfactant in the form of a white powder.

The resulting powder did not contain detectable quantities of ethanol, and the content of each component relative to the total weight of the surfactant was: 66,4 wt.% 1,2-dipalmitoyl-(3)-phosphocholine, 22,1 wt.% 1-Palmitoyl-2-oleoyl-sn-glycero-(3)-phospho-sn-glycerol, a 8.9 wt.% palmitic acid, 2.2 wt.% peptide and 0.4 wt.% water.

Example 24

1,2-Dipalmitoyl-(3)-phosphocholine (320,0 mg), 1,2-dimyristoyl-sn-glycero-(3)-phospho-sn-glycerol (80.0 mg) palmitic acid (60,0 mg) was dissolved in a mixture of chloroform-methanol (1:1, o/o) (200 ml), and of 14.0 mg of peptide E was dissolved in TFA (0.3 ml). The resulting solutions were mixed ode-ethanol (10: 1, o/a) (50 ml) for 60 minutes at 45oC. After freezing the resulting suspension at -45oC and subsequent drying for 24 hours under vacuum at 50 - 110 mkno, got 479,2 mg surfactant in the form of a white powder.

The resulting powder did not contain detectable quantities of ethanol, and the content of each component relative to the total weight of the surfactant was: 66,8 wt.% 1,2-dialettologia-(3)-phosphocholine, 16.7 wt.% 1,2-dimyristoyl-sn-glycero-(3)-phospho-sn-glycerol, 12.5 wt.% palmitic acid, 2.9 wt.% peptide E and 1.1 wt.% water.

Example 25

Following the same procedure described in Example 21, but using peptide F (22,0 mg) instead of peptide B (10.0 mg) and 1,2-distearoyl-sn-glycero-(3)-phospho-sn-glycerol instead of 1,2-diacyl-sn-glycero-(3)-phospho-sn-glycerol (with acyl group having 14 to 24 amoma carbon; manufactured by Sigma Chemical Co., Ltd.) received 463,9 mg surfactant in the form of a white powder.

The resulting powder did not contain detectable quantities of ethanol, and the content of each component relative to the total weight of the surfactant was: 64,7 wt. % 1-2 palmitoylated-(3)-phosphocholine, of 21.6 wt.% 1,2-distearoyl-sn-glycero-(3)-phospho-sn-glycerol, 8.6 wt. % palmitic acid, 4,7 mA is when using peptide C instead of peptide B, received 454,1 mg surfactant in the form of a white powder.

The resulting powder did not contain detectable quantities of ethanol, and the content of each component relative to the total weight of the surfactant was: 66,1 wt.% 1,2-dipalmitoyl-(3)-phosphocholine, to 22.0 wt.% 1,2-acyl-glycero-(3)-phospho-sn-glycerol (with acyl group having 14 to 24 carbon atoms; manufactured by Sigma Chemical Co., Ltd.), 8.8 wt.% palmitic acid, 2.2 wt.% peptide C and 0.9 wt.% water.

Example 27

1,2-Dipalmitoyl-(3)-phosphocholine (210 mg), 1,2-diacyl-sn-glycero-(3)-phospho-sn-glycerol (with acyl group having 14 to 24 carbon atoms; manufactured by Sigma Chemical Co., Ltd.) (90,0 mg) and stearic acid (33,0 mg) was dissolved in a mixture of chloroform-methanol (3:1, o/o) (100 ml), and 1.9 mg of peptide H was dissolved in methanol (0.5 ml). The resulting solutions were mixed together, and then was dried and utverjdali under reduced pressure. The resulting residue is suspended in water-ethanol mixture (9:1, o/o) (90 ml) for 15 minutes at 50oC. After freezing the resulting suspension at -55oC and subsequent drying for 28 hours under vacuum at 100 - 120 mcno received 340,2 mg surfactant in the form of a white powder.

The resulting powder was not contained measurable, obnarujeni.% 1,2-dipalmitoyl-(3)-phosphocholine, 26.5 wt.% 1,2-diacyl-sn-glycero-(3)-phospho-sn-glycerol, 9.7 wt.% stearic acid, 0.5 wt.% peptide H and 1.6 wt.% water.

Example 28

1,2-Dipalmitoyl-(3)-phosphocholine (210,0 mg), 1-Palmitoyl-2-oleoyl-sn-glycero-(3)-phospho-L-serine (90,0 mg) and palmitic acid (33,0 mg) was dissolved in a mixture of chloroform-methanol (4:1, o/o) (100 ml), and 11.0 mg of peptide I was dissolved in TFA (0.5 ml). The resulting solutions were mixed together, and then was dried and utverjdali under reduced pressure. The resulting residue is suspended in water-ethanol mixture (9:1, o/o) (110 ml) for 25 minutes at 45oC. After freezing the resulting suspension at -55oC and subsequent drying for 28 hours under vacuum at 100 - 120 mcno received Russia for USD 348.7 mg surfactant in the form of a white powder.

The resulting powder did not contain detectable quantities of ethanol, and the content of each component relative to the total weight of the surfactant was: 60,2 wt.% 1,2-dipalmitoyl-(3)-phosphocholine, and 25.8 wt.% 1-Palmitoyl-2-oleoyl-sn-glycero-(3)-phospho-L-serine, and 9.5 wt.% palmitic acid, 3.2 wt.% peptide I and 1.3 wt.% water.

Example 29

Following the same procedure described in Example 21, but using peptide Q instead of peptide B, received 459,3 mg drug victorii peptide K instead of peptide B, received 452,5 mg surfactant in the form of a white powder.

Example 31

Following the same procedure described in Example 21, but using peptide L instead of peptide B, received 456,6 mg surfactant in the form of a white powder.

Example 32

Following the same procedure described in Example 21, but using peptide M instead of peptide B, received 453,9 mg surfactant in the form of a white powder.

Example 33

Following the same procedure described in Example 21, but using peptide N instead of peptide B, received 452,5 mg surfactant in the form of a white powder.

Example 34

Following the same procedure described in Example 21, but using the peptide O instead of peptide B, received 458,1 mg surfactant in the form of a white powder.

Example 35

1,2-Dipalmitoyl-(3)-phosphocholine (30.0 mg), 1,2-diacyl-sn-glycero-(3)-phospho-sn-glycerol (with acyl group having 14 to 24 carbon atoms; manufactured by Sigma Chemical Co., Ltd.) (10.0 mg) and palmitic acid (4.0 mg) was dissolved in a mixture of chloroform-methanol (2:1. o/a) (30 ml), and 1.0 mg of peptide Q was dissolved in a mixture of chloroform-methanol (2:1, o/o) (2.0 ml). The resulting solutions were mixed together, and then was dried and utverjdali under reduced pressure. The resulting residue suspended in a mixture isC and subsequent drying for 36 hours under vacuum at 60 - 120 mcno got to 45.4 mg surfactant in the form of a white powder.

The resulting powder did not contain detectable quantities of ethanol, and the content of each component relative to the total weight of the surfactant was: 66,1 wt.% 1,2-dipalmitoyl-(3)-phosphocholine, to 22.0 wt.% 1,2-diacyl-sn-glycero-(3)-phospho-sn-glycerol, 8.8 wt.% palmitic acid, 2.2 wt.% peptide Q and 0.9 wt.% water.

Example 36

Following the same procedure described in Example 35, but using peptide R instead peptide Q, received of 45.7 mg surfactant in the form of a white powder.

Comparative example 3

Following the same procedure described in Example 21, except that instead of the solution of peptide B (10.0 mg) in a mixture of chloroform-methanol (2: 1, o/o) (2.0 ml), the solution was used peptide S (10.0 mg) in TFA (0.3 ml) and got to 455.2 mg surfactant in the form of a white powder.

The resulting powder did not contain detectable quantities of ethanol, and the content of each component relative to the total weight of the surfactant was: 65,9 wt.% 1,2-dipalmitoyl-(3)-phosphocholine, to 22.0 wt.% 1,2-acyl-sn-glycero-(3)-phospho-sn-glycerol (with acyl group having 14-24 carbon atoms; prvately example 4

Following the same procedure described in Example 21, except that instead of the solution of peptide B (10.0 mg) in a mixture of chloroform-methanol (2: 1, o/o) (2.0 ml) was used a solution of peptide T (10.0 mg) in TFA (0.3 ml) and received 456,0 mg surfactant in the form of a white powder.

The resulting powder did not contain detectable quantities of ethanol, and the content of each component relative to the total weight of the surfactant was: 65,8 wt.% 1,2-dipalmitoyl-(3)-phosphocholine, to 21.9 wt.% 1,2-acyl-sn-glycero-(3)-phospho-sn-glycero (with acyl group having 14 to 24 carbon atoms; manufactured by Sigma Chemical Co., Ltd.), 8.8 wt.% palmitic acid, 2.2 wt.% peptide T and 1.3 wt.% water.

Table 2 gives the results of tests on surface activity of the surfactants according to the invention and their ability to maintain alveolar volume.

Possible applications

As noted above, new synthetic peptides according to the invention can easily be distinguished and clean, you can get them in ways that are acceptable for mass production, these peptides have a high solubility in the usual solvents and easier suspended until a homogeneous suspension; these peptides possess surface activity equivalent surface and the Arata for the treatment of respiratory distress syndrome, which is a disease that gives heavy breathing.

1. Synthetic peptides having the following specific sequence of General formula I

Xaa-Pro-Val-Xbb-CSU-Lys-Arg-W

Xaa may be present or may present a Cys or Ser;

Xbb represents His or Asn;

CSU = Leu, Ile;

W represents the hydrophobic part of the peptide containing 12-20 molecules Leu and/or Nle,

and Tolna or hydroxyl group can be allerban fatty acid C14-C18or subjected to reaction acetamidomalonate.

2. Synthetic peptides, in which 12-20 hydrophobic amino acids that form the hydrophobic part of the synthetic peptides (p. 1) contain 1-5 molecules Ile, Val, Nva, Trp.

3. Synthetic peptide under item 1, which is S - acetamidomalonate form of the peptide of General formula I, where Xaa = Cys, Xbb = His, Xcc = Leu, W = (Leu)16.

4. Synthetic peptide under item 1, which is S - palmitoylation form of the peptide of General formula I, where Xaa = Cys, Xbb = His, Xcc = Leu, W = (Leu) 12.

5. The method of production of synthetic peptides under item 1 or 2 of General formula I, characterized in that the hydrophobic part of the peptide under item 1 or 2, W is subjected to condensation with the rest of frequent the P> 6. Pulmonary surfactant composition comprising the following components, percent by weight:

Synthetic peptides under item 1 or 2 - 0,1-5,0

Cholinesterase - 50,6-85,0

Acidic phospholipid - 4,5-37,6

Similar fatty acid - 4,6-24,6

7. Drug for the treatment of respiratory distress syndrome, containing as the active ingredient surfactant comprising a synthetic peptide under item 1 or 2, cholinesterase, acidic phospholipid and similar fatty acids.

 

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