Polypeptides having the activity of pulmonary surfactant, and farmcampsite


(57) Abstract:

Describes the polypeptides of formula (I)

< / BR>
where a Is H or Phe; B is Phe or Trp and Ile, Leu or Ser, which have activity of pulmonary surfactant, which receive high yield and high purity and that its effectiveness is not inferior to natural pulmonary surfactant. They are suitable for the production of pharmaceutical compositions for the treatment of syndrome asphyxia in newborns and adults. 2 C. and 7 C.p. f-crystals, 1 table.

The invention relates to pulmonary surfactant polypeptides, methods for their production and to their containing compositions.

Light of all vertebrates contain a mixture of substances, known as "pulmonary surfactant". It has surface-active properties and so lowers the surface tension in the alveolar region of the lungs that prevents the collapse of the end sections of the Airways during exhalation. This mixture of substances regulates surface tension dynamic way, so that expected according to the law of Laplace collapse of small alveoli in favor of larger due adapt poverhnostnoaguoe stable structure of the lungs.

Pulmonary surfactant is secreted alveolar pneumocytes type II in the form of lamellar cells. They are compact formation of the phospholipid double layers with a high content of dipalmitoylphosphatidylcholine (DPPC) and phosphatidylglycerol (FG). Other major components contained in the pulmonary surface-active substances are proteins, which are referred to as SP-A, SP-B and SP-C SP-A is a high molecular weight glycoprotein that plays a major role in the regulation of secretion.

Protein SP-C and to a lesser extent SP-B function "thermodynamic catalysts in the formation of monomolecular surface film (in a more narrow sense - surfactants). Due to the presence of these proteins kinetics of expansion is greatly accelerated. Only as a consequence without delay it is possible to adapt the composition of the surfactant to the relevant requirements of the surface tension. These properties are reflected in the extremely hydrophobic nature of proteins, in particular SP-C.

By extraction of the lung tissue or washing the lungs of animals you can get drugs surfactants, to the application demonstrate ability to compensate for the lack of surface-active substances and thus suitable, for example, for the treatment of child syndrome dyspnea (respiratory distress syndrome of newborns (RDTS)). However, these drugs derived from animals, inherent severe disadvantages.

The composition of phospholipids strongly depends on the type of animal, its health and nutritional status and may be only to a limited extent balanced by the adulteration of certain components. The content of surface-active proteins, as well as the ratio of SP-B/SP-C is subject to the same uncertainties. In addition, in therapeutically applied mixture also contains possible products of proteolytic degradation of proteins or modified derivatives (e.g., oxidation of methionine). With continued use or application of large quantities of surfactants that may be necessary, for example, the syndrome of asthma in adults (light shock, respiratory distress syndrome of adults (rdsw)) or in other applications, for example, when using a surfactant as a "tug" to other substances in pulmonary applications, the issue of replenishment of the matter remains open.

Because of these problems it is proposed to solve the shackles and bacterial expression systems, can be produced in virtually unlimited quantities, and there is the possibility of using modern methods of analysis and quality control using synthetic phospholipids, can be obtained surfactant exactly defined composition. This substance can be optimally meet therapeutic requirements.

The Central part of the human protein SP-C (see formula I, where A is H or Phe; B - Cys; (C - Met), which is particularly important for the kinetics of expansion, consists exclusively of aliphatic, very hydrophobic amino acids such as valine, leucine and isoleucine. The length of this Central part (amino acids 12-34) allows you to integrate the peptide in monomolecular phospholipid film. In the sequence Pro-Cys-Cys-Pro (item 3-6) both Cys-residue theatrevision palmitic acid on SH-groups. Palmitic acid also improves the hydrophobic character of the entire protein and simultaneously closes both SH groups cysteine and protects them from oxidation and formation of a disulfide bridge. The Central region (amino acids 13-34) forms a transmembrane spatial spiral. This area is adjacent to the N-end using polar sequence, soderzhashchej recombinant SP-C and mutant SP-C. In this application, among other things, it is proposed to replace both cysteines at positions 4 and 5 two Suriname. The advantage of this when getting is that after selecting a very hydrophobic protein eliminates the need for technically difficult palmitoylation both cysteines.

Description of the invention

Unexpectedly, it was found that SP-C mutants that differ from the human protein SP-C replacement of both cysteines in positions 4 and 5, phenylalanine, or tryptophan, and the replacement of the methionine at position 32 isoleucine, leucine or serine, have no functional loss in comparison with the natural SP-C, and stability even surpass it. This greatly simplifies the preparation using genetic engineering techniques to achieve higher output. New polypeptides with pulmonary surface activity can be obtained with high purity.

The subject invention are polypeptides with pulmonary surface activity having the amino acid sequence of General formula I

< / BR>
where A is H or Phe;

B - Phe or Trp, and

C - Ile, Leu or Ser.

A preferred subject of the invention are polypeptides total Faure H, B denotes Phe and C denotes Ile.

Another subject of the invention are pharmaceutical compositions which are characterized in that they contain one or more polypeptides according to the invention, and optionally contain one or more pulmonary surfactant polypeptide from the group of SP-A and SP-B, preferably SP-B.

The polypeptides according to the invention can be obtained by known methods of solid-phase peptide synthesis, or by using the appropriate recombinant vectors in the cells of the host. Methods of constructing vectors, methods of transforming cells, methods of inducing expression of the protein in transformed cells and methods of isolation and purification of expressed proteins known to experts in the art (see , for example, WO 86/03408, WO 87/06588 and WO 91/18015). Upon receipt of vectors for the expression of SP-C in bacterial systems use conventional methods of recombinant DNA.

Expression of hydrophobic SP-C protein in bacteria is possible in large quantities and without prejudice to cell owners only in the form of suitable fused proteins, such as, for example, together with chloramphenicolchloramphenicol (CAT). For example, the vector pTrpAmpCAT152 to the patients DNA, coding for SP-C. CAT and SP-C are associated at the protein level with each other through sensitive to hydroxylamine the confluence (AsN Cly). Such vectors as pTrpAmpCAT152::SPC, allow controlled expression of corresponding fused proteins up to production scale (fermentation). The expression of the fused proteins causes the formation of Taurus inclusion in the cell host. The length of the lobe CAT in the fused protein may be modified in such a way that will achieve high outputs Taurus turn on when a high share of SP-C.

In addition to bacteria expression can occur in a large number of systems hosts, for example in mammalian cells, yeast and insects. Suitable for various host cells design DNA synthesized by known methods and is built in the usual way with the appropriate control sequences into the genome of host cells.

On the DNA synthesizer MilliGen/Biosearch Cyclone normal fostamatinib method can be synthesized two oligonucleotide DNA.

The first oligonucleotide DNA encoding forms (retranscribing) DNA strand length 118 nucleotides. This thread encodes (in the direction of 5'--->3') specific against EcoRI 5'-end d is na Leu-58 SP-C sequence-predshestvennika, i.e., Gly-1, respectively Leu-34 in the formula I. this is known amino acid sequence of the human SP-C protein according to the rules of the genetic code is translated into DNA. However, the sequence is modified in such a way that both cysteine at positions 28 and 29 SP-C sequences predecessor is replaced with phenylalanine, or tryptophan, and methionine at position 56 sequence predecessor is replaced by isoleucine, leucine or serine. In the same way can additionally be taken into account in the frequency of codon usage of the host cell. Modified SP-C sequence containing at positions 4 and 5 of phenylalanine and at position 32 isoleucine (numbering according to the formula (I), denoted in accordance with the conventional one-letter code for both of these amino acids as SPC34(FF/I). Next Smelova strand of DNA contains a TAA termination codon to terminate ribosomal translation, as well as specific in relation to > PST 3'-end. The second oligonucleotide DNA is a complementary, non-coding (antisense) strand, consisting of 110 nucleotides.

Synthetically derived SP-C-fragment DNA clone into suitable expression vector, such as pTrpAmpCAT152. This vector is composed of pKK233 (firm Ph the n on Trc-promoter, as in pTrpAmpCFT152. Can also be used and other inducible promoters.

For sublimirovanny SP-C-fragment complementary oligonucleotides DNA first hybridizing with each other. The resulting double strand of DNA has a protruding single-stranded ends (EcoRI/ > PST).

Embedding in vector DNA is carried out in the usual way after splitting vector DNA using EcoRI/ > PST, purification of the desired fragments of the vector DNA by electrophorese agarose gel and hybridization of SP-C-DNA and vector fragments by being coupled to ends. Then both pieces by known methods connected with each other by ligating.

For DNA amplification and plasmid isolation produce transformation in conventional protocols, for example, in calcifolic-competent cells of E. coli MM294 and for selection of bearing plasmid cells cover on LB-agar plates with ampicillin. From the obtained resistant to ampicillin (Amp-resistant) colonies produce plasmid DNA and analyze it using the appropriate combinations of restriction enzymes. Choose the clone with the expected restriction map of the DNA. By sequencing the plasmid sequence is confirmed by the rules of the ora allow the expression of the fused protein CAT::SPC under the control of the Trp promoter (or other promoters). Recombinant fused protein falls in the cells of the host after induction in the form of Taurus enable.

Fused protein CAT152: : SPC34(FF/I) has the following, presents the conventional one-letter code amino acid sequence:

< / BR>
34 amino acids SP-C(FF/I) provisions 153-186 fused protein are underlined and indication of provisions 1-34.

Subsequent cleavage with hydroxylamine to separate CAT and SP-C is between Asn-152 and Gly-153 (corresponds to 1-th amino acid in the SP-C peptide). Isolation and purification of SP-C peptide is carried out by normal in protein chemistry methods.

The polypeptides according to the invention can be applied separately or in combination in pharmaceutical compositions that meet the requirements of the treatment of the respiratory tract. These compositions are suitable not only for the treatment of syndrome asphyxia in preterm infants and in adults, but also for the treatment of pneumonia and bronchitis. In addition, the polypeptides according to the invention is suitable as a "tug" to drugs, administered by inhalation.

Along with polypeptide compositions contain phospholipids, preferably such phospholipids that are found in natural compounds possessing lagodellemeraviglia (FOPH) and/or phosphatidylglycerol (FG). To achieve optimal viscosity of the composition containing calcium ions or magnesium, and sodium chloride. To determine the type and quantity of individual components of the compositions of the specialist is able to focus, first, on the known composition of natural lung surfactant, and secondly, to the many proposals, known from the prior art, for example from the European patent applications EP-A 0119056 and EP-A 0406732.

Preferred compositions according to the invention contain 80 to 95 wt.% phospholipids, 0.5 to 3.0 wt.% polypeptides, 4 to 7 wt.% fatty acid, preferably palmitic acid, and 1 to 3 wt.% calcium chloride.

Example of getting

1. Producing strains of

Used producing strains of E. coli 199 is the origin of the strain MM294 E. coli K12, which is deposited under 5208 N in the German collection of microorganisms and cell cultures GmbH (DSM, Braunschweig).

The expression vector pTrpAmpCAT152: : SPC34(FF/I), containing the gene fused protein CAT152::SPC34(FF/I), was obtained from the DNA sequence of pBR322, i.e. ColEI-derived [E. Weber (ed), (1988), Biologische Sicherheit, Bundesministerium fur Forschung und Technologie, Bonn]. Plasmid pKK233-2, supplied by the company Pharmacia, using EcoRI/HindIII was cut Trc-promoter and replaced by a synthetic Trp-is Blasta (the binding site Trp-repressor), sequence Shine-Dalgarno (S/D sequence), and restriction sites for cloning. For Trp-promoter was inserted gene (CAT152), 152 encoding amino acids 5'-part of the bacterial chloramphenicol-acetyl-transferase. Part CAT152 DNA sequence was fused synthetic gene fragment that encodes a 34 amino acids similar to the human protein SP-C(FF/I). Functional transcription CAT: : SPC(FF/I) ends bacterial rrnB sequence T1T2, terminating transcription. The resulting design is referred to as pTrpAmpCAT152::SPC34(FF/I).

Vector pTrpAmpCAT::SPC(FF/I) has the following functional elements:

gene CAT152::SPC34(FF/I), managed Trp-promoter and T1T2 - terminator of transcription;

- ori-region and the neighbouring regions, which control the number of copies of plasmids;

gene Amp.

After placing the plasmid in the cell-master latter has a high number of copies of the gene CAT::SPC(FF/I) controlled Trp-promoter. Trp-repressor itself produces a host cell.

Enzymatic getting rCAT::SPC is regulated by the concentration of tryptophan in the environment, respectively the add - IAA ( - intolerability acid).

2. Periodic fermentation

Using PR is or initial culture (1 l) and at 37oC incubated with shaking and strong ampicillinampicillin selection. Growth is monitored by optical density at 578 nm. Upon reaching the initial culture of E. coli 199 optical density of more than 3 culture were seeded in 10-liter fermenter and continue growing bacteria at reduced ampicillinampicillin selection. After reaching the values of optical density between 5 and 6 10-liter culture is transferred into a 100-liter fermenter and continue to incubate under the same conditions. After sufficient growth by adding, for example, 40 mg/l IAA induce controlled Trp-promoter transcription CAT: : SPC(FF/I). After induction for collecting cells fermentation additionally continue for 4-5 hours

During fermentation directly supervise and regulate the partial pressure of oxygen (pO2), pH value and temperature of the broth in the fermenter. Value to maintain a constant pH using sodium hydroxide solution, the partial pressure of oxygen (pO2) regulate by introducing oxygen and using a speed mixer. The optical density at 578 nm and the concentration of source C in the medium determined at regular intervals. Foaming is controlled using a foam sensor, psovaya.

At different points in time are selected aliquots of the culture broth. After lysis of the bacteria E. coli proteins to control expression separated on polyacrylamide gel and stained. The percentage (dominant) proteins in the whole protein of E. coli was determined by densitometer. Shortly after induction of recombinant gene there is a new dominant protein band (rCAT::SPC).

The culture medium has the following composition.

Soy peptone of 27.0 g/l, yeast autolysate KAV 14 g/l, NaCl 5.0 g/l, K2HPO43H2O 6.0 g/l, KH2PO43.0 g/l, MgSO47H2O 0.5 g/l, glycerol (99,5%) 30.0 g/l, non J673 (firm Structol Comp.) 0.2 ml/l, L-tryptophan 80.0 mg/l and ampicillin 20 mg/l for the 1st pre-culture and 5 mg/l for the 2nd pre-culture and the 100-liter fermenter.

Prior to heating in an autoclave and sterilization of complex nutrient medium pH with 2N NaOH establish 6.8. For pre-culture in a 10-liter fermenter speed stirrer at 750 rpm, and the oxygen flow rate is 10 l/min at 37oC, for the main culture in a 100-liter fermenter speed stirrer is 400 rpm and the flow rate of oxygen is 70 l/min at 37ooC). Mixed cell suspension process (at room temperature) in a homogenizer high pressure (700 bar) and again collected in a sterile container made of stainless steel. Then immediately by filtration and/or centrifugation (centrifuge Sorvall RC2-B, 27000g) at 4oC collect bullock inclusion, resuspended in the buffer (about 1 l) and portions, for example, approximately 350 ml, transferred into a 1-liter round bottom flask and lyophilized within 96 hours Of 100-liter fermentation download get about 200 grams of dry Taurus include with the contents of the fused protein of more than 20 wt.%. Liofilizovannye bullock inclusion can be stored at -20oC for months.

3. The cleavage of the fused protein and purification of lipophilic peptide SP-C(FF/I)

100 g of Dry Taurus enable dissolve with light heating 1.6 l 8-molar solution of guanidine hydrochloride (917,1 g). Nerastvorim the residue is filtered through a folded filter. For cleavage of the fused protein on the binding site of Asn-Gly to the solution is added 167 g hydroxyammonium is the temperature under stirring. Upon completion of the reaction by the addition of 6.4 l Tris buffer (pH 8.0) precipitated SP-C(FF/I) and it is separated using centrifugation (centrifuge Sorvall RC2-B, 20000g). The supernatant decanted, precipitated by centrifugation SP-C, re-suspended in 400-500 ml of Tris-buffer and again centrifuged under the same conditions for 30 minutes

Precipitated by centrifugation SP-C dissolved in 3.5 l of a mixture of chloroform, methanol and hydrochloric acid (1,75 l CHCl3+ 1,75 l CH3OH + approximately 30 ml of 2N HCl). This crude solution of SP-C is further purified using preparative liquid chromatography high pressure (ghvd) on C8 as the material of inverted phases. The chloroform/methanol extract before loading on the column for preparative Ehud diluted with 90% methanol in a ratio of about 1: 2. From this solution in a column (diameter 5 cm) can be downloaded, for example, approximately 400 mg of SP-C(FF/I) (for example, diluted with 2 l of crude extract). SP-C(FF/I) elute in acidic conditions (pH 2-3) with a gradient of water/isopropanol (see table). After about 30 min chromatography was carried out in the area in which blueraven SP-C (UV-detection at 220 nm), collected 4-6 fractions of 200 ml Fractions checked with analytical GHW and stored in a freezer at -80oC. Purity SP-C(FF/F) is 98.5-99.5% pure.

Conditions of the separation are presented in the table.

4. Embedding of SP-C(FF/I) in the phospholipid matrix

Lipophilic peptide SP-C(FF/I) are mixed in isopropanole solution with components of the phospholipid matrix and precipitated by injection of dilute salt solution (0,065% weight/weight of NaCl) at room temperature in a homogeneous mixture with the other components of the phospholipid matrix. From the suspension of pulmonary surfactant on stakanchikov the centrifuge separates the pulmonary surfactant (LAW), resuspended in the electrolyte solution (NaCl, CaCl2) and using 0.1 N NaOH pH was adjusted to 6.5. This aqueous suspension is poured into 20 ml of the vials and lyophilized. Following the example of obtaining the weight and volume data refer to 10-gram of the drug pulmonary surfactant.

7,00 g Dipalmitoylphosphatidylcholine (DRPH), is 3.08 g of ammonium salt of palmitoyloleoylphosphatidylglycerol (POPG x NH4) and 0.25 g of palmitic acid are dissolved at 40oC in 200 ml of 90% isopropanol and then cooled to room temperature. The resulting solution of phospholipid volume of the CSO "spray solution" brought under stirring with a solution of bicarbonate (approximately 5 ml of 5% aqueous solution of NaHCO3) to 4.5.

"Spray solution" under vigorous stirring injected at room temperature with a speed of injection of 25 ml/min through a single nozzle 9.6 l of dilute NaCl solution (0,065% weight/weight). Formed opalescent solution, which after two hours exposure at 4-8oC when the injection of the electrolyte solution (3.0 g CaCl22H2O and 61.3 g of NaCl in 300 ml of H2O) in the sediment falls drug pulmonary surfactant. Suspension of pulmonary surfactant (total 10,8-11,0 l) was incubated overnight at 4oC, and then centrifuged for 30 min on stakanchikov the Sorvall centrifuge (RC2-B) at 16000g. To remove residual isopropanol formed during the centrifugation pellet resuspended in half the volume of 0.65% sodium chloride solution and again centrifuged. This stage is repeated 3-4 times. The cake obtained at the final stage of centrifugation, dissolved in 400 ml of 0.65% NaCI solution, pH using 0.1 N NaOH was adjusted to 6.5 and distribute portions at 6.2 g in 20-ml ampoule. The contents of the ampoules lyophilized as follows: freeze for 6 h at -45oC and normal pressure, dried wymiarow oC and 0.02 mbar.

In this way receive 65-66 vials containing 0,150 g pulmonary surfactant (calculated without NaCl).

Dry samples of pulmonary surfactant is to be stored in the refrigerator at 4oC and before use they must be resuspendable water or physiological NaCl solution (slurry concentration of 25 mg/ml).

Each ampoule contains: 95,6 mg dipalmitoylphosphatidylcholine, 42,1 mg palmitoyloleoylphosphatidylglycerol (ammonium salt), 2.7 mg of SP-C(FF/I), 6,8 mg palmitic acid, 2,9 mg of calcium chloride (anhydrous).

1. Polypeptides having the activity of pulmonary surfactant, a General formula I

< / BR>
where A is H or Phe;

B - Phe or Trp;

C - IIe, Leu or Ser.

2. Polypeptides under item 1, wherein A is H or Phe, B is Phe and C - Ile.

3. The polypeptide under item 1, characterized in that A - Phe, B is Phe and C - Ile.

4. The polypeptide under item 1, characterized in that A - H, B - Phe and C - Ile.

5. Pharmaceutical composition for the treatment of respiratory distress syndrome (RDS) in mammals, containing pulmonary surfactant polypeptide according to one of paragraphs.1 to 4, phospholipids, fatty acids and electrolytes is
Electrolytes - 1 - 3

6. The pharmaceutical composition according to p. 5, characterized in that as pulmonary surfactant contains at least one pulmonary surfactant polypeptide from the group of SP-A and SP-B.

7. The pharmaceutical composition according to p. 6, characterized in that as pulmonary surface-active substances it contains SP-B.

8. The pharmaceutical composition according to p. 5, characterized in that it contains phospholipids dipalmitoylphosphatidylcholine, palmitoyloleoylphosphatidylglycerol and/or phosphatidylglycerol.

9. The pharmaceutical composition according to p. 5, characterized in that as electrolytes it contains salts of calcium and/or sodium.


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