Dimer "clover" peptide

 

(57) Abstract:

The invention relates to dimers "clover" peptides. "Clover" peptide contains a single domain in the form of a clover leaf and represents the ITF person or ITF rats or pS2 man or pS2 rats. The peptide is in the form of a dimer, in which two identical monomers linked by a disulfide bond between two cysteine residues. The molecular weight of the peptide is about 13,000. Culturing a suitable cell host transformed by DNA encoding the ITF human or rat or pS2 humans or rats and separate dimeric form from a Monomeric. The pharmaceutical composition includes the "clover" the peptide and a pharmaceutically acceptable diluent. The invention allows on the basis of dimeric forms of "clover" peptides to obtain medicines used for healing of damaged mucosa of the gastrointestinal tract such as peptic ulcers. 11 C. and 4 h.p. f-crystals, 9 Il., table 2.

The invention relates to dimers "clover" peptides, methods for producing dimers "clover" the peptides, pharmaceutical compositions comprising dimers "clover peptides, and their use for the treatment of gastrointestinal disorders.

"Clover" the peptides obrazek contain one or more common domains in the form of a clover leaf [1], each of which consists of amino acid sequences consisting of 38 or 39 amino acid residues, in which 6 residues polycystina connected in combination 1-5, 2-4 and 3-6, thus forming a characteristic structure in the form of a clover leaf.

Known to date, the "clover" the peptides mammals contain either one or two domains in the form of a clover leaf (see reviews [3-5]), while for Xenopus laevis described peptides and proteins containing one [6], [7], four [8] or six [9] domains in the form of a clover leaf. "Clover" peptides mammals, containing a single domain are associated with breast cancer pS2-peptide, known to humans [10, 11] and mouse [12], and intestinal "clover" factor (ITF) [13, 14] and rats [15, 16]. For humans [17], pigs [18] and mouse [17] described spasmolytic polypeptide (SP), containing two domains in the form of a clover leaf. People have all three "clover" peptide - hpS2, hITF and hSP - expressed in normal gastrointestinal tract: hSP and hpS2 - in the epithelial layer of the mucous membrane of the stomach [17, 19], a hITF - in the epithelial layer of the mucous membrane of the intestine and colon [13].

The physiological role of the "clover" the peptides are not very good Vij, related mucosal damage, such as inflammation of intestine [20-22], ulcer of stomach and duodenum [20, 23, 24]. Based on these data it was proposed reparative function "clover" peptides (e.g., in [22]). Recently the authors in [25-27] evidence has been obtained accelerate the recovery of damaged epithelial mucosa as a result of "clover" peptides. The mechanism by which the "clover" the peptides contribute to the implementation of their reparative functions, may lie in the formation of crosslinks between musinovym glycoproteins forming a viscoelastic gel layer, resistant to the action of digestive enzymes [3, 28].

Cloning of single-domain "clover" factor of the intestine in rats and humans and its use for the treatment of lesions of the gastrointestinal tract described [29].

Discovered the possibility of obtaining dimers "clover" factors that have only one domain in the form of a clover leaf and possess interesting pharmacological properties.

In accordance with this present invention relates to a "clover" peptide containing a single domain in the form of a clover leaf and existing in the form of di is x ulcers and other lesions, related mucosa, due to stabilization of the mucosal layer of the intestinal tract. The mechanism of this stabilization is not currently known. However, x-ray structural analysis (see [28]) pancreatic spasmolytic polypeptide (PSP) pigs, forming two domains in the form of a clover leaf, showed that most of the available amino acid residues participate in the formation of the slit width 8-10 detected in each domain. Preliminary experiments showed that this gap is able to accommodate part of the oligosaccharide chain, for example carbohydrate associated with mucinosis glycoprotein. If so, then the PSP with two such slots can sew molecules mucines with each other, helping them to form a protective gel that covers the epithelium of the mucosa. Now known form whether in vivo dimers to implement similar functions single-domain "clover" peptides (such as IFT and pS2) or they have a different mechanism of action. However, at present it is believed that the dimers such "clover" peptides can really sew molecules mucines, being so active form of these peptides.

Another aspect of the present invention relates to spouse cultivation of appropriate host cells, the transformed DNA sequence that encodes a "clover" peptide containing a single domain in the form of a clover leaf, under conditions that allow the accumulation of the peptide, and selection of culture formed dimer "clover" peptide.

Another aspect of the present invention relates to pharmaceutical compositions comprising dimer "clover" peptide containing a single domain in the form of a clover leaf, together with a pharmaceutically acceptable diluent or carrier.

Another aspect of the present invention relates to the application of dimer "clover" of a peptide containing a single domain in the form of a clover leaf as medicine and the use of dimer "clover" of a peptide containing a single domain in the form of a clover leaf, to obtain drugs for the prevention and treatment of gastrointestinal disorders.

Dimer "clover" factor can be, in particular, the dimer intestinal "clover" factor (ITF) or peptide associated with breast cancer (pS2).

In particular, the "clover" the human factor is the ITF, the amino acid sequence of the monomer which looks as follows:

ZEYVGLSANQCAVPAKDRVDCGYPHVTPKECNNRGCCFDSRIPGVPWCFKPLQEAECTF, where Z Preeti;

or human pS2, the amino acid sequence of the monomer which looks as follows:

ZAQTETCTVAPRERQNCGFPGVTPSQCANKGCCFDDTVRGVPWCFYPNTIDVPPEEECEF

where Z represents the residue Glu, Gln or pyrGlu or its homolog, can timeresults and exhibiting similar activity.

Homologues ITF and pS2 cover the peptides with the same character location of cysteine residues and disulfide bonds (Fig. 1) and a certain degree of homology (in the sense of either the identity of amino acid residues in the corresponding positions, or having conservative substitutions) in the loops 1, 2 and 3. Homology of amino acid sequence in the region of the loops can vary in the interval from 1 to 10 amino acid residues, and the number of amino acid residues in each loop (except cysteine residues) from 7 to 12, preferably from 9 to 10.

Homologues ITF and pS2 can contain one or more amino acid substitutions, deletions or insertions. The preemptive nature of these changes is that they do not impact significantly on coagulation and protein activity. The size of a small deletions, typically ranges from 1 to 3 amino acid residues in loops and from 1 to 10 amino acid residues in the N - and C-terminal regions; the peptide of up to 10 amino acid residues, or a small Supplement easy-to-clean, such as polyhistidine tract, an antigenic epitope or binding domain (General principles, see [30] ). Examples of conservative substitutions can be replaced within the group of basic amino acids (e.g. arginine, lysine, histidine), acidic amino acids (such as glutamic and aspartic amino acid), polar amino acids (such as glutamine and asparagine), hydrophobic amino acids (e.g. leucine, isoleucine, valine), aromatic amino acids (such as phenylalanine, tryptophan, tyrosine) and amino acids with small side of the radical (for example, glycine, alanine, serine, threonine, methionine).

For professionals in this field should be obvious that such replacement can be performed outside of the regions responsible for the function of the molecule, resulting in a still active polypeptide. Essential for the activity of these "clover" peptides amino acids can be identified in accordance with well-known specialists techniques, such as site-specific and alanine-scanning mutagenesis [31]. In the latter case, mutations introduced into each of the amino acid residues of the molecule and the resulting mutant molecules are tested for Biol the stomach) to identify those amino acid residues, who is responsible for the activity of the molecule.

Possible allelic variant, homolog, i.e., an alternative form of a gene that results from a mutation, or a modified peptide encoded by the mutant gene, but having essentially the same activity as the present peptide. Therefore, mutations can be silent (no change in the encoded polypeptide) or may encode a peptide with a modified amino acid sequence.

A homologue of this "clover" peptide addition may be a species homolog, i.e., the polypeptide with similar activity originating from other species, e.g. mouse, rat, rabbit, cow, pig or frog.

In the preferred embodiment dimer "clover" peptide according to the invention has a value of molecular weight approximately 13000. The dimer consists of two Monomeric "clover" peptides linked by a disulfide bond between two cysteine residues at position 57 for ITF-like monomers or in position 58 for S2-like monomers.

It is preferable to obtain dimer "clover" peptide using recombinant DNA technology. For this purpose, the DNA sequence encoding a "clover" peptide, mi techniques using hybridization with synthetic oligonucleotide probes [32] the DNA sequence, encoding the peptide in whole or in part. Preferred within the present invention is an encoding peptide DNA sequence of human origin, i.e., from a library of genomic DNA or cDNA of human rights.

The DNA sequence encoding the "clover" the peptide can also be obtained by chemical synthesis using conventional methods, for example phosphoamide () private method, described in [33], or the method described in [34]. In accordance with phosphoamide(and)Tim method, oligonucleotides are synthesized, for example, in an automatic DNA synthesizer, and then cleaned parts are annealed, with the subsequent ligation and cloned in suitable vectors.

The DNA sequence can also be obtained using the polymerase chain reaction using specific primers, as described in [35, 36] or above [32].

The DNA sequence encoding a "clover" peptide, usually introducing a recombinant vector that represents any of the vectors, suitable for carrying out procedures with recombinant DNA, and the choice of vector will often depend on the nature of the host cell into which it must be entered. So, this vector can be contained replica, i.e. to be a plasmid. On the other hand, the vector may be one of those vectors, which, falling into the cell host, integrate into the genome of the host cell and replicated together with the chromosome(s) into which they are integrated.

Preferably, the vector was expressed by a vector in which the DNA sequence encoding a "clover" peptide operatively connected to the additional areas required for transcription of the DNA. In General expressed by the vector originates from plasmid or viral DNA or contains elements of both. The term "operable connected" means that the parts so arranged that operate in cooperation with each other to perform their intended purposes, e.g. transcription starts at the promoter and passes on the entire stretch of DNA encoding a polypeptide.

The promoter may be any DNA sequence that demonstrates transcriptional activity in selected cell host originating from protein-coding genes, as homologous and heterologous cell-master.

Examples of suitable for carrying out the transcription of the DNA encoding the "clover" the peptides in mammalian cells promoters of irusa 2.

An example of a suitable promoter for use in insect cells is polyhedrin promoter [39, 40], P10-promoter [41], the main promoter of the protein polyhedrosis virus Autoqrapha californica [42] , the promoter of the "fast" early gene 1 baculovirus [43, 44] or the promoter of a delayed early gene of the baculovirus 39K [43, 44].

Appropriate promoters for use in yeast cells-the owners are the promoters of yeast glycolytic genes [45, 46], or alcohol dehydrogenase genes [47], or TRI1- [48], or ADH2-4C-promoters [49] .

Examples suitable for use in filamentous fungi as cell hosts promoters are, for example, ADH3-promoter [50] or tpiA-promoter. As examples of other useful promoters can result in the promoters of genes TAKA-amylase from A. oryzae, aspartic proteinase from Rhizomucor miehei, neutral-amylase from A. niger, colocataires-amylase from A. niger, glucoamylase (gluA) from A. niger or A. awamori, lipase from Rhizomucor miehei, alkaline protease from A. oryzae, triosephosphate from A. oryzae or acetamidate from A. nidulans. Preferred are promoters of genes TAKA-amylase and gluA. Appropriate promoters indicated, for example, in [51] and [52].

The DNA sequence encoding a "clover terminator gene growth hormone human [53], or, when the host cells are used mushrooms, the TPI1 terminator [46] or ADH3 [50]. The vector can also include items such as polyadenylation signals (for example, from Elb-region of SV40 or adenovirus 5), sequences that increase transcription (e.g., enhancer SV40), and sequences that enhance translation (such as encoding Rncy adenovirus VA).

The recombinant vector may also include a DNA sequence enabling the vector to replicate in this cell the owner. An example of such a sequence (when the host-cell is a cell of a mammal) is the origin of replication of SV40.

When the host-cell is a yeast cell, the corresponding sequences enabling the vector to replicate, are replication genes REP 1-3 and origin of replication of the yeast plasmid 2.

The vector may also contain a selective marker, e.g. a gene the product of which complements a defect in the host cell, such as a gene dehydropeptidase (DHFR) or TPI-gene of Schizosaccharomyces pombe (described in [54]), or a gene that gives resistance to drugs such as ampicillin, kanamycin, tetracycline, chloramphenicol, tx2">

In order to send the "clover" the peptide described in the present invention, in the secretory pathway of the host cell, the recombinant vector can be introduced secretory signal sequence (also known as a leader sequence, preprofessionals or predpolozhytelno). The secretory signal sequence is joined to a DNA sequence that encodes a "clover" peptide, in the correct reading frame. Typically, the signal sequence is located 5'-end DNA sequence that encodes the peptide. As a secretory signal sequence can be applied signal sequence normally associated with the peptide or the sequence of the gene encoding another secretory protein.

For secretion from yeast cells secretory signal sequence may encode any signal peptide, providing effective direction expressed "clover" peptide into the secretory pathway cells. The signal peptide can be from the nature of the available signal peptide (or its functional part) or a synthetic peptide. It was found that the relevant shirvany signal peptide of carboxypeptidase [56] , BAR1 signal peptide of yeast [57] or the signal peptide of yeast aspartic protease 3 (YAP3) [57].

In addition, for efficient secretion in yeast between the signal sequence and the DNA sequence that encodes a "clover" peptide, there may be a sequence encoding a leader peptide. The function of the leader peptide is that it allows the downregulation of the peptide to be directed from the endoplasmic reticulum to the Golgi complex and later in the secretory vesicula for secretion into the culture medium (i.e., directs the export of "clover" peptide across the cell wall or at least through the cellular membrane into periplasmatic space of the yeast cell). As a leader peptide may be a leader peptide of the yeast factor (its use is described, for example, in [54, 59-63]). On the other hand, a leader peptide may be artificial, in other words not natural. Synthetic leader peptide may, for example, be constructed as described in [64] or [65].

For use in filamentous fungi signal peptide can be conveniently obtained from the gene encoding the amylase or glucoamylase from Aspergillus sp., the gene encoding the lipase is the future TAKA-amylase from A. oryzae, neutral-amylase from A. niger, kislotostabilen-amylase from A. niger or the glucoamylase from A. niger. Appropriate signal peptides are described in [51] and [66].

For use in insect cells such a signal peptide can be obtained on the basis of the gene of the insect (see [67]), as, for example, the signal peptide of the precursor adipokinetic hormone lepidopteran Manduca sexta (see [68]).

Methods, used for ligating the DNA sequences encoding "clover" peptide, promoter and possibly a terminator and/or secretory signal sequence, respectively, and introduce them into suitable vectors containing the information necessary for replication, are well known to specialists in this field (cf. for example [32]).

A host cell, into which is injected the DNA sequence encoding the "clover" the peptide can be any cell capable of producing the polypeptide in dimeric form, including yeast, fungi and higher eukaryotes.

Examples of appropriate cell lines of mammalian cell lines are COS (ATSS CRL 1650), KSS (ATSS CRL 1632, ATSS CCL 10), CHL (ATSS CCL 39) or Cho (ATSS CCL 61). Methods of transfection of mammalian cells and each yeast cells include cells of Saccharomyces spp. or Schizosaccharomyces spp., in particular strains of Saccharomyces cerevisiae or Saccharomyces kluyveri. Methods of transformation of yeast cells of heterologous DNA and producing them in heterologous polypeptides are described, for example, in [38, 54, 76-78], each of which is thus given in the references. Transformed cells are selected on phenotype determined by a selective marker, usually in terms of resistance to drug drug or ability to grow in the absence of a particular component of the nutrient medium, such as leucine. Preferred for use in yeast is the vector ROT described in [76]. DNA sequences encoding "clover" peptide may be preceded by a signal sequence, and possibly the leader sequence, such as described above. Other examples of appropriate yeast cells are strains of Kluyveromyces, such as K. lactis, Hansenula, such as H. polymorpha, or Pichi, such as P. pastoris (cp. [79, 80]).

Examples of other fungal cells are cells of filamentous fungi, such as Aspergillus spp., Neurospora spp., Fusarium spp. or Trichoderma spp., in particular, A. oryzae, A. nidulans or A. niger. The use of Aspergillus spp. for protein expression are described, for example, in [51, 81, 82]. Transformation of F. oxysporum may, for example, be held the stage of the fungus, it can be transformed constructed according to the present invention DNA through the latter's integration into the chromosome of the host cell to obtain a recombinant host cell. Generally, it is believed that such integration has the advantage that the introduced DNA sequence would be more stably maintained in the cell. Integration of the designed DNA sequences in the chromosome of the host cell can be carried out in accordance with conventional ways, for example by homologous or heterologous recombination.

Transformation of insect cells and production of them in heterologous polypeptides can be carried out, as described in patents [39, 42-44, 85], included here in the list of references. As cell lines insects used as cell hosts, suitable cell line Lepidoptera, for example cells of Spodoptera frugiperda or cells of Trichoplusia ni (see [86]). For cultivation suit the conditions described, for example, in [87] and [88] or in any of the above links.

Subjected to transformation or transfection described above cell owners then cultured in an appropriate nutrient medium under conditions permitting the expression is in dimeric form. As cultivation media can be any conventional medium suitable for the growth of host cells, such as minimal or complex media containing appropriate supplements. Suitable media are commercially available or can be prepared according to published recipes (for example, in accordance with the catalogues of the American Type Culture Collection). Produced by cells of the "clover" the peptide can be isolated from the culture medium using standard techniques, including separation of host cells from the medium by centrifugation or filtration, deposition of proteinaceous components of the supernatant or filtrate by means of a salt, e.g. ammonium sulphate, purification by using a variety of chromatographic procedures, e.g. ion exchange chromatography, gel filtration, affinity chromatography or the like depending on the type of the considered polypeptide.

Dimer "clover" peptide can be entered in the pharmaceutical composition according to the invention according to any of the established methods of preparing pharmaceutical compositions, e.g. as described in [89]. Such a composition can be made suitable for the systematic injection or ipvy can be sterilized using standard methods of sterilization, well-known specialists in this field. The resulting aqueous solutions can immediately be packaged for use or filtered under aseptic conditions and dry lyophilized; liofilizovannye the drug before the introduction mixed with sterile water solution. The pharmaceutical composition may contain a pharmaceutically acceptable additive substances required to approximate physiological conditions, such as tabularasa agents that regulate the tone of the substance and the like, for example sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, etc.

The pharmaceutical composition of the present invention can also be adapted for nasal, transdermal, and rectal administration. Pharmaceutically acceptable excipient or diluent used in the composition may be any conventional solid filler. Examples of particulate fillers are lactose, Terra Alba, sucrose, talc, gelatin, agar, pectin, Arabic gum, magnesium stearate and stearic acid. Similarly, the carrier or diluent may include any known in the art helps to eliminate material such as glycerylmonostearate is th however, you will usually be in the range from 25 mg to 1 g

The concentration of the "clover" the peptide in such composition may vary within wide limits, for example from about 5% to about 100% by weight. The preferred concentration is in the area of 50-100% by weight. The standard dose for such a composition can typically contain from about 1 mg to about 200 mg, preferably from about 25 mg to about 75 mg, in particular about 50 mg of peptide.

According to the above is that we present in this invention dimer "clover" peptide is the active form of said peptide. In this regard, it is assumed that it will be useful when using it with the purpose of prevention or treatment of diseases of the gastrointestinal tract. More specifically it is supposed to use it for the treatment of gastric or peptic ulcers, inflammatory bowel disease, Crohn's disease or damage of the intestinal tract caused by radiation therapy, bacterial or other infections, and so on, the Dose of the polypeptide, administered to a patient will vary depending on the type and severity of the condition treated, but typically lies in the interval to the accompanying drawings, where

Fig. 1 - proposed structure of the intestinal "clover" factor (ITF) person. The primary amino acid sequence taken from [14], and disulfide bonds are placed on the basis of homology with the PSP and pS2 [2].

Fig. 2 - reversed-phase HPLC of the supernatant of the yeast strain HW756 expressing ITF rats, column (Vydac TR.

Fig. 3 - ion exchange chromatography of partially purified ITF person on the column with Fast Flow SP-Separate. The number of monomer and dimer ITF person was determined by analytical HPLC. Colored rectangles indicate the fractions collected for further purification of the Monomeric and dimeric forms. The dashed line corresponds to the NaCl concentration in the eluting solvent.

Fig. 4 - reverse-phase HPLC purified dimer ITF rats (A), monomer ITF () and dimer ITF person (s) for column (Vydac TR C4. The dotted line shows the concentration of acetonitrile in the eluting solvent.

Fig. 5 - mass spectra of purified ITF rats (dimer) (A), ITF person (monomer) (b) and ITF person (dimer) (C).

Fig. 6 - structure of the dimeric form ITF person.

Fig. 7 is a restriction map of plasmid KFN 1003.

Fig. 8 - design planirovanie ITF rats (rITF) and ITF person (hITF)

Cloning ITF rat and human were performed as described in [15, 16, 29] (rITF) and [13, 14] (hITF).

Construction of plasmids expressing rITF and hITF

According to the schemes shown in Fig.7 - 9, were constructed expressing plasmids pHW756 for secretion ITF rats and pHW1066 for secretion ITF person. Expressing yeast vector FN1003 (described in [90]) is a derivative plasmid of srot [91]. It contains as a selective marker TPI gene Schizosaccharomyces pombe (MOUTH) [53], the promoter three-sofastithertz S. cerevisiae (procedures defined in the TPI) and the terminator, which controls the expression [46].

Gene ITF rats initially cloned in Bluescript II KS(-) (Stratagene), from which it was replicated in accordance with Fig. 8. Auxiliary vector pSX54 providing useful cloning sites, composed of pUC18 and pDN1050 [92]. Synthetic DNA linker Ncol-PflM1 has the following nucleotide sequence:

1858: 5'-CATGGCTGAAAGATTGGAAAAGAGACAAGAGTTCGTTGGTTTGTCTCCATCCC AATGT-3' 58 N. p.

1862: 5'-TTGGGATGGAGACAAACCAACGAACTCTTGTCTCTTTTCCAATCTTTCAGC - 3' 51 N. p.

The linker encodes the 8 C-terminal amino acids of the leader sequence, as described in [93], with several modifications in the choice of codons, and the N-terminal part of the gene ITF rat has the following sequence: QEFVGLSPSQC. Signal Gene human ITF cloned in PUC19 and were propagated in accordance with Fig. 9. Synthetic DNA linker Ncol-BsaAl has the following sequence:

2292: 5'-CATGGCTGAAAGATTGGAAAAGAGAGAAGAATAC-3' 34 N. p.

2287: 5'-GTATTCTTCTCTCTTTTCCAATCTTTCAGC-3' 30 H. p.

The linker encodes the 8 C-terminal amino acids of the leader sequence, as described for construction rITF, and three N-terminal amino acids of the gene hITF are EEY. Signal and leader sequences are the same as before.

Expressing plasmids transformed strain S. cerevisiae MT 663 (E2-7B X E11-36-a/ tpi/tpi, pep 4-3/pep 4-3) with selection for growth on glucose as the sole carbon source.

Yeast transformants expressing the ITF rat and human, were named HW756 and HW1066 respectively.

Fermentation

The above transformants were cultured for 72 h at 30oC in yeast-peptone-dextrose (YPD) medium [94], supplemented with yeast extract (60 g/l). At the end of fermentati were achieved values of optical density at 660 nm, 153 and 232 for HW756 (rITF) and HW1066 (hITF), respectively. At the end of the fermentation the pH was brought to 2.5 with 1 M phosphoric acid and the yeast cells were removed by centrifugation at 3000 rpm for 15 minutes

Purification of recombinant rITF

Concentric is using analytical HPLC. Aliquots (typically 50-200 µl) was applied to a C4 HPLC column (0,46 x 25 cm) Vydac 214TP54 for reversed-phase chromatography equilibrated with 0.1% (v/v) TFA in 15% (v/v) acetonitrile, 30oC and a flow rate of 1.5 ml/min After 10 min isocratic elution concentration of acetonitrile in the eluting solvent was increased for 40 min to 55%. Measured the absorption at 214 nm. Were detected three peaks, eluruumina with retention times of 26.5 min, 27,3 min and 28.2 min (Fig. 2), which represents the dimeric form rITF. The number of peptides were determined using calibration hSP-standard [93].

The level of expression of recombinant ITF rats in this yeast system was 113 mg/l

Using centrifugation from the fermenter with a capacity of 10 l was allocated to 8.7 l of fermentation broth. To reduce the conductivity of the supernatant was diluted to 14.8 liters of distilled water. The sample was applied to a column (5 x 42 cm) with SP-Separate Fast Flow (Pharmacia) at a flow rate of 600 ml/h Before use, the column was balanced 50 mm formatnum buffer pH of 3.7. ITF rats were suirable column with 50 mm formic acid pH of 3.7, containing 50 mm NaCl. In process chromatography at a flow rate of 600 ml/h were collected fraction volume of 100 ml, which analyzer cm) with Amberchrome (G-71). Before use, the column was balanced 10 mm ammonium acetate buffer pH 4.8 at a flow rate of 0.5 l/h After application the column was washed with 0.5 l of equilibrating buffer and suirable 10 mm ammonium acetate buffer pH of 4.8, containing 60% (v/v) ethanol, at a flow rate of 0.1 l/h was Selected fractions containing rITF, a volume of 10 ml. of ethanol Concentration in the combined fractions was increased from 60% (v/v) to 87% (v/v) by adding 2 volumes of ethanol (99.9%, the v/v) and, cooling the resulting mixture to a temperature of minus 25oC for 16 h, besieged rITF.

The precipitate was collected by centrifugation for 1 h at 10000 g and -25oC and pererestorani at room temperature in 130 ml of 20 mm formic acid, pH 3.0. The sample was applied to a column (5 x 20 cm) with SP-Separate Fast Flow (Pharmacia) at a flow rate of 50 ml/h Before use, the column was balanced 20 mm formic acid, pH 3.0. Peptides were suirable from the column using a linear gradient composed of 1.5 liters of 50 mm formic acid, pH 3.0, and 1.5 l of formic acid, pH 3.0, containing 0.5 M NaCl. In process chromatography at a flow rate of 80 ml/h was selected fractions (10 ml), which was measured by optical density at a wavelength of 280 nm. In selected fractions were measured content rITF, then fractions, with the shares (900 ml) was applied to a preparative C4 HPLC column (2,2 x 25 cm) Vydac 214TP1022, equilibrated with 0.1% (v/v) TFA. Peptides were suirable at 25oC and a flow rate of 5 ml/min using a linear gradient (540 ml) composed of MeCN/H2O/TFA (10:89,9:0,1 v/v/v) and MeCN/H2O/TFA (65:34,9: 0,1 v/v/v). Recorded absorption in the ultraviolet region at a wavelength of 280 nm, were selected fractions of 10 ml and analyzed their content rITF. Containing rITF fractions were combined and the resulting solution was reduced in volume to 30% by centrifugation under vacuum. The final selection of rITF carried out by lyophilization. The total number rITF obtained from 8,7 l fermentation medium, was 236 mg, which corresponds to a total of 24% of the output at all stages of the cleanup.

Purification of recombinant hITF

The concentration of hITF in yeast fermentation nutrient medium and the fractions obtained during purification, was measured using HPLC system, identical to those described for rITF. Were found two peaks, eluruumina with retention times of 21.2 minutes and 27.1 min (Fig. 2), representing according to mass spectrometry and sequence analysis of dimeric and Monomeric forms of the hITF. The level of expression of recombinant ITF person in this yeast system was 90 mg/l

Using centrifugation from the fermenter VM is awn 24 h) 40 l of 10 mm formic acid pH of 2.5. The sample was applied (0.25 l/h) on a column (5 x 40 cm) with SP-Separate Fast Flow (Pharmacia). The column was washed with 5 l of 20 mm formic acid, pH 2.5 and 5 l of formic acid pH of 2.5, containing 1 M NaCl. Selected fractions with a volume of 100 ml and analyzed their content hITF (Fig. 3). With the columns were suirvey two forms hITF, one of which represented the Monomeric form (eluted at 0.5 M NaCl), and the other dimer hITF (eluted with 0,78 M NaCI). Fractions corresponding to each of the forms were combined separately.

Each of the two combined fractions were divided into three equal parts (approximately 700 ml) and was applied on the column, Vydac C4 214TP1022 (2,2 x 25 cm), equilibrated with 0.1% (v/v) TFA. Peptides were suirable at 25oC and a flow rate of 4 ml/min using a linear gradient (540 ml) composed of MeCN/H2O/TFA (10: 89,9: 0,1 v/v/v) and MeCN/H2O/TFA (65:34,9:0,1 v/v/v). Absorption in the ultraviolet region were recorded at a wavelength of 280 nm, were selected fractions of 10 ml and analyzed their content hITF.

The fractions containing hITF (monomer) and hITF (dimer), respectively, were pooled and precipitated peptides by adding ethanol to a concentration of 90% (v/v) and maintaining the mixture for 72 h at -25oC. the Precipitate was collected by centrifugation and was liofilizovane. The total PT total output in all stages of treatment, 50% and 65% for Monomeric and dimeric forms, respectively.

Characterization of recombinant rITF and hITF

After hydrolysis in 6 M HCl in evacuated sealed ampoule for 24, 48 and 96 h samples (50 μg) were analyzed on an automatic amino acid analyzer company Beckman Model 121 MB). The number of residues polycystina defined as S--(4-pyridylethyl)derived after you restore the disulfide bonds by tributylphosphine [95] with subsequent interaction with 4-vinylpyridine [96] . Hydrolysis of the samples treated with 4-vinylpyridine, conducted in 4 M methanesulfonic acid or 3 M mercaptoethanesulfonate acid at 110oC for 24 h, as described previously. Analysis of amino acid sequences was performed using the automated process of degradation on Adminu using gas-phase sequencing machine Model A company Applied Biosystems [97].

Mass spectrometric analysis was performed on the API III LC/MS/MS (Sciex, Thornhill, Ont., Canada). Triple quadrupole instrument, which allows to determine the value of the ratio of mass-to-charge (m/z) in the interval up to 2400 is connected to the interface for newmusictuesday (also called ion-spray) [98, 99] . The introduction of the sample was carried out using a syringe infusion pump (Sage Instruments, Cambridge, MA) through the plug capillary tube (EXT. D. 75 μm) is analogo adduct of polypropyleneglycol (PPG) at unit resolution. Precision measurements of the masses is usually greater than 0,02%.

In Fig. 4 shows the results of the analytical HPLC for treated rITF (Fig. 4A) and hITF (Fig. 4B and 4C). Recombinant rITF contains a mixture of three relatives of peptides for the separation of which there have been no attempts. As a result of mass spectrometric analysis were established three core values of molecular weights - 13112,2; 13096,6 and 13078,8 (Fig. 5A). The calculated value of molecular weight Monomeric form ITF rats, in which Cys-57 has a free-SH group is 6558,3. The calculated value of molecular weight dimeric form ITF rats (for example, S-S bridge between the two Cys-57) is 13114,6. From the values of molecular weights found for the recombinant ITF rats, it is clear that all three peptides represent a dimeric form rITF. For other "clover" peptides in which the N-terminal amino acid residue is Gin, for example PSP [17, 100], it is known that this residue has a tendency to cyclization with the formation of pyrrolidonecarboxylic acid (pyrGlu). For ITF rats with predicted N-terminal sequence Gln-Glu-Phe-Val-Gly, it is reasonable to assume that the N-terminal Gln residue can also cilitates education pyrGlu. Such a move will peterstone. The observed values of molecular weights in 13096,6 and 13078,8 (Fig.5A) correspond to the dimeric form ITF rats, in which one or two N-terminal Gln residue sallisbury. Calculated values of molecular weights for these forms are 13097,6 and 13080,6, which is in good accordance with the experimentally obtained values. Thus, data from HPLC (Fig.4A) and mass spectrometric analysis (Fig. 5A) implies that recombinant ITF rats includes 3 different dimeric forms: one containing 2 N-terminal Gln; one - 1 N-terminal Gln and 1 N-terminal pyrGlu; and one with 2 N-terminal pyrGlu. Table I shows the amino acid composition ITF rats, which is in good agreement with expected values.

Fig. 4B and 4C demonstrate the purity of the hITF (monomer) and hITF (dimer), respectively, according to analytical HPLC. Dimeric form (Fig. 4C) looks relatively clean, while for Monomeric (Fig. 4B) is visible contamination material, eluruumist before peptide. However, if rechromatography material of the main peak is quite similar to the appearance of the chromatogram (data not shown). Apparently, it mostly indicates atypical behavior hITF (monomer) with reversed-phase chromatography is as it was observed for recombinant hSP high degree of purification [93].

Using mass spectrometric analysis of the main peak hITF (monomer) was set to a molecular weight equal to 6694,0 (Fig. 5B). The value of the molecular weight calculated from the amino acid sequence (Fig. 1), is 6574,4 on the assumption that the Cys-57 is-SH form. Data sequencing (Table. II) show the presence of the expected N-terminal sequence: Glu-Glu-Tyr-Val-Gly-. The amino-acid analysis (Table. I) show the expected values except for the presence of 7.3 (8) cysteine residues. The presence of an additional cysteine residue, attached to Cys-57, may increase the value of molecular weight up to 6694,7, which is very close to the value found by mass spectrometry (6694,0). Because of this, you can put that in hITF (monomer) residue Cys-57 is connected by a disulfide bond with an additional cysteine residue. A minor peak (Fig. 5B) in the mass spectrum can be a another derivative of Cys-57 or may be due to the presence of drug impurities.

The calculated value of molecular weight hITF (dimer), in which two monomers are connected by a disulfide bond between two residues Cys-57, is 13146,8. This value is in good accordance with the value of Primera). The results of the analysis of amino acid sequences (Table. II) and composition (Table. I) are also in good accordance with the expected values.

Example 2

Assessment of biological effects of dimeric ITF using in vitro models of epithelial barrier (Heather Kindon et al., Trefoil Peptide Protection of Intestinal Epithelial Barrier Function; Gastroenterology 1995; 109 : 516-523)

ITF human or rat in dimeric form and/or glycoprotein was added to the Transwell monolayers cell line T84, obtained from cancer of the rectum of the person.

Intact monolayer missed less than 4% of the inert marker [3H]mannitol for 4 hours. As a result of his handling of toxic lecithin phytohemagglutinin (1 mg/ml), oleic acid (8 mmol/l) and touronline acid (12 mmol/l) or by toxin a from Clostridium difficile (0,7 µg/ml) barrier function is weakened and thus treated monolayer missed 36%, 62% and 45% [3H] mannitol, respectively.

Adding ITF person (1-5 µg/ml) led to the weakening of the damage the integrity of the monolayer on the value up to 52%. The defense was increased (up to 95%) with the simultaneous presence mucinosa glycoprotein rectum of man.

A similar effect was observed when dobavljaite these substances to provide some protection, when they added themselves, indicates that the protective properties inherent in each of them and are not the result of interaction between two components.

Concentration as ITF and mucinosa glycoprotein used in this study is equivalent to that estimated takes place on the surface of the gastrointestinal tract, therefore, the action of on this model of epithelial monolayer comparable with physiological, and we can conclude that the ITF both human and rat, in combination with musinovym glycoproteins protects the mucous membrane of the gastrointestinal tract from a variety of injuries.

Example 3

The influence of exogenous ITF on experimental damage of the stomach in rats in vivo (Mark W. Babyatsky, Gastroenterology 1996, 110; 489-497)

Damage to the stomach induced or intragastric introduction of absolute ethanol (1.0 ml), or subcutaneous administration of indomethacin (20 mg/kg). In different doses (0.5, 1, 5 and 10 mg, each dose dissolved in 1.0 ml of distilled water) was administered dimeric ITF rats at different time points both before and after damage. As control was used media or bovine serum albumin. Then rats were killed and examined the damage JY effect in relation to damage called as ethanol and indomethacinum (P < 0,005).

Example 4

The preparation of recombinant pS2 man

Recombinant pS2 man in Monomeric and dimeric form were obtained in Escherichia coli using recombinant plasmids for expresii pEZZ18, coding or serine (monomer), or cysteine (dimer) in regulation 58 of this protein (Chadwick MP, May FEB. Westley BR. Production and comparison of mature single domain "trefoil" peptides pNR-2/pS2 Cys58and pNR - 2/pS2 Ser58. Biochem. J. 1995, 308; 1001-1007). This expression system produces a composite protein that contains two artificial domain derived from Staphylococcus aureus protein a and protein pS2. Recombinant integral proteins were purified using affinity chromatography on the lgG sepharose and released pS2 protein by cleavage with factor XA. Then the monomer pS2 Ser58purified uninominal chromatography. Protein pS2 Cys58were treated with cysteine, was purified ionoobmennoi chromatography, and the dimer was purified by gel-filtration. The molecular weight of the monomer and the dimer, measured by mass spectrometry, laser desorption, was 6645 and 13378 and agree well with theoretical 6674 and 13340 Yes.

Example 5

Assessment of biological effects of dimeric or Monomeric pS2 man with ISOE is))

The in vitro model

The rate of migration of cells to the top edge of the damaged monolayer cell line NT rectal person increased by adding dimeric or Monomeric pS2 (0,65-325 μg/ml). Dimeric pS2 had a greater effect than Monomeric, all tested doses.

Model in vivo

Action dimeric and Monomeric pS2 (Monomeric Cys58replaced by Ser58were compared to models of wound healing in rats. Dimeric pS2 at a dose of 25 and 50 mg/kg per hour showed 50% and 70% recovery of damage of the stomach induced by indomethacin. Monomeric pS2 in the same dose was significantly less effective in restoring damage (about half).

According to the above, we can conclude that the pS2 restores damage to the stomach and has a protective and healing effect, and dimeric pS2 more effective than Monomeric.

Example 6

Obtaining dimer pS2 rats

Total RNA was extracted from various tissues of rats, including the stomach and intestines. Using reverse transcriptase PCR with different primers was obtained single-stranded cDNA.

Using the obtained cDNA and plasmids for mission pS2 rats in yeast was carried out in the same way, as described for ITF humans and rats in example 1.

Example 7

Due to the fact that the "clover" the peptides are not destroyed in the digestive tract, one of the ways of their administration is oral. These peptides can be introduced, for example, in the form of tablets, capsules or pills or suspended in the solution, such as a syrup, which is swallowed by the patient. In the alternative case, the solution containing the indicated peptide, can be entered as gastric lavage or in the form of a suppository. Pharmaceutical compositions containing a compound according to the invention, can be prepared by standard methods, for example as described in Remington: The Science and Practise of Pharmacy, 19thEd., 1995.

A typical tablet which is prepared according to the traditional technology, has the following recipe, mg;

Core:

Active connection (ITF dimer person) - 10

Colloidal silicon dioxide (Aerosil) and 1.5

Microcrystalline cellulose (Avicel) - 70

Modified cellulose resin (Ac-Di-Sol) - 7,5

Magnesium stearate - Ad.

Shell:

HPMC - 9

Mywacett 9-40 T - 0,9

First prepare the core material of the tablets, and then it is covered by the shell. Mywacett represents acylated monomer in the form of a clover leaf and representing ITF person, when this peptide is in the form of a dimer, in which two identical monomers linked by a disulfide bond between two cysteine residues and which has a molecular weight of about 13,000.

2. The peptide under item 1, in which the disulfide bond formed between two cysteine residues located at position 57 of each monomer.

3. "Clover" peptide containing a single domain in the form of a clover leaf and representing ITF rats, while the peptide is in the form of a dimer, in which two identical monomers linked by a disulfide bond between two cysteine residues and which has a molecular weight of about 13,000.

4. The peptide under item 3, in which a disulfide bond formed between two cysteine residues located at position 57 of each monomer.

5. "Clover" peptide containing a single domain in the form of a clover leaf and represents a pS2 man, when this peptide is in the form of a dimer, in which two identical monomers linked by a disulfide bond between two cysteine residues and which has a molecular weight of about 13,000.

6. The peptide under item 5, in which a disulfide bond formed between two cysteine residues, located in Polo the East, and represents a pS2 rats, when this peptide is in the form of a dimer, in which two identical monomers linked by a disulfide bond between two cysteine residues and which has a molecular weight of about 13,000.

8. The peptide under item 7, in which a disulfide bond formed between two cysteine residues located at position 58 of each monomer.

9. "Clover" peptide according to any one of paragraphs.1, 3, 5 and 7 for use as a medicine for healing of damaged mucosa.

10. "Clover" peptide according to any one of paragraphs.1, 3, 5 and 7 for use in the preparation of medicines for the healing of damaged mucosa.

11. A method of obtaining a peptide under item 1 or 2, wherein culturing a suitable cell host transformed by a DNA sequence that encodes ITF person, under conditions that allow the production of ITF, and separate dimeric form ITF man from Monomeric.

12. A method of obtaining a peptide under item 3 or 4, in which cultivated a suitable cell host transformed by a DNA sequence that encodes ITF rats, under conditions that allow the production of ITF, and separate dimeric form ITF rats from Monomeric.

13. A method of obtaining a peptide p is teruya pS2 man under conditions that allow the production of pS2, and separate dimeric form pS2 man from Monomeric.

14. A method of obtaining a peptide according to p. 7 or 8, wherein culturing a suitable cell host transformed by a DNA sequence that encodes a pS2 rats, under conditions that allow the production of pS2, and separate dimeric form pS2 rats from Monomeric.

15. Pharmaceutical composition for healing of damaged mucosa of the gastrointestinal tract such as peptic ulcers, which includes an active ingredient and a pharmaceutically acceptable diluent or carrier, wherein as the active ingredient it contains the "clover" the peptide according to any one of paragraphs.1 - 8.

 

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The invention relates to therapeutic peptides

New peptides // 2162855

The invention relates to medicine, in particular to a gastroenterologist, and can be used to stop bleeding from gastroduodenal ulcers

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The invention relates to medicine, in particular to the development and application of new anxiolytic drug

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The invention relates to new peptide exhibiting antitumor activity, more specifically to Pentapeptide, the methods of its production and peptide compounds exhibiting anti-tumor activity

FIELD: medicine, cardiology.

SUBSTANCE: the suggested method should be performed at the background of medicinal therapy with preparations out of statins group, tevetene, polyoxidonium and conducting seances of plasmapheresis by removing 800 ml plasma twice weekly with N 5 due to additional intramuscular injection of immunophan 0.005%-1.0 with N 10 and fluimucyl 300 mg intravenously daily with N 5-10, total course of therapy lasts for 2 mo. The method provides modulation of leukocytic functional activity, moreover, due to altered cytokine profile and, thus, through disintegration of protein-lipid complexes participating in the development of atherosclerotic platelets.

EFFECT: higher efficiency of therapy.

3 ex

FIELD: medicine, phthisiology, anesthesiology.

SUBSTANCE: during the day of operation one should perform autohemotransfusion, then introduce epocrine intravenously by drops at the dosage of 50-200 U/kg patient's body weight; next day after interference one should inject epocrine subcutaneously at the dosage of 25-100 U/kg; at hematocrit level being below 35% 48 h after operation it is necessary to repeat subcutaneous injection of the above-mentioned preparation at the dosage not exceeding 50 U/kg. The present innovation favors hemopoiesis stimulation in postoperational period, that, in its turn, accelerates postoperational rehabilitation in patients of this group and enables, also, to avoid allotransfusions being dangerous because of immunoconflicting reactions.

EFFECT: higher efficiency of compensation.

1 ex, 1 tbl

FIELD: pharmaceutical chemistry, medicine.

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EFFECT: higher efficiency of therapy.

2 cl, 6 dwg, 8 ex, 5 tbl

FIELD: genetic engineering, medicine.

SUBSTANCE: invention relates to T-cell receptor sequence being detected in patients with extended sclerosis and is useful in diagnosis and therapy. Oligonucleotide including sequence which represents or is derived from 5'-CTAGGGCGGGCGGGACTCACCTAC-3' or nucleotide sequence being fully complementary thereto. Oligonucleotide together with nuclear acid including nearly 15-30 oligonucleotides, which doesn't comprise oligonucleotide sequence and presents in region from Vβ to Jβ of Vβ13.1 gene in T-cell Vβ13.1-subgroup, wherein oligonucleotide and nuclear acid sequences don't present in the same chain of pair sequences of Vβ13.1 gene, is used in Vβ13.1 gene part amplification. In method for detection of LGRAGLTY motive, which is present in T-cell receptors of T-cell Vβ13.1-subgroup, oligonucleotide is used in combination with labeling particle. Once LGRAGLTY motive is detected, development monitoring and treatment are carried out by removing of LGRAGLTY motive-containing peptide.

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21 cl, 7 dwg, 3 tbl, 3 ex

FIELD: medicine.

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EFFECT: higher efficiency and prophylaxis.

4 dwg, 5 ex

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EFFECT: valuable medicinal properties of agent.

3 cl, 4 tbl, 2 ex

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EFFECT: higher efficiency of therapy.

1 ex

FIELD: medicine, first aid, anesthesiology, resuscitation, surgery.

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EFFECT: higher efficiency.

3 ex

FIELD: bioorganic chemistry.

SUBSTANCE: invention provides somatostatin agonists of general formula: A1-cyclo{Cys-A2-D-Trp-A3-A4-Cys}-A5-Y1 (I), wherein A1 represents aromatic D- or L-α-amino acid selected from Phe, D-Phe, Tyr, D-Tyr, β-Nal, D-β-Nal, Cha, or D-Cha; A2 aromatic α-amino acid selected from Phe, Tyr, β-Nal, and Cha; A3 Lys or Orn; A4 β-hydroxyvaline, Ser, hSer, or Thr; A5 β-hydroxyvaline, Ser, hSer, or Thr; and Y1 represents NH2; amide nitrogen atoms of peptide groups and amine group of A1 in compound I are optionally substituted by methyl group, provided that at least one methyl group is available and that compound I cannot have following formula: D-Phe-cyclo{Cys-Phe-D-Trp-Lys-(N-Me-Thr)-Cys}-Thr-NH-2. pharmaceutically acceptable salts of compound I are also claimed.

EFFECT: expanded synthetic possibilities in peptide synthesis.

24 cl, 2 tbl, 18 ex

Oligopeptides // 2260597

FIELD: organic chemistry, peptides, biochemistry.

SUBSTANCE: invention describes oligopeptide or its salt taken among the group consisting of oligopeptide (1) and (2): Lys-Ser-Ile-Glu-Gln-Ser-Cys-Asp-Gln-Asp-Glu (I), Ser-Ile-Glu-Gln-Ser-Cys-Asp-Gln-Asp-Glu (II); Ser-Ile-Glu-Gln-Ser-Cys-Asp-Gln-Asp (III); Ser-Ile-Glu-Gln-Ser-Cys-Asp-Gln (IV); Ser-Ile-Glu-Gln-Ser-Cys-Asp (V); Ser-Ile-Glu-Gln-Ser-Cys (VI); Ile-Glu-Gln-Ser-Cys-Asp-Gln-Asp-Glu (VII); Glu-Gln-Ser-Cys-Asp-Gln-Asp-Glu (VIII); Gln-Ser-Cys-Asp-Gln-Asp-Glu (IX); 2) oligopeptide with amino acid sequence obtained by deleting by C- or N-end of one or some amino acids in any amino acid sequence (I)-(IX), and the modified oligopeptide representing oligopeptide biotinylated or dimerized by sulfhydryl group of cysteine residue based on oligopeptide determined in (1) or (2). Oligopeptides elicit activity with respect to hair growth stimulation.

EFFECT: valuable properties of oligopeptides.

11 cl, 6 dwg, 4 ex

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