Biologically active derivatives of peptide vapeehptllteaplnpk

FIELD: biotechnologies.

SUBSTANCE: invention is related to biotechnology and represents substantially pure peptide for modulation of immune system, which is characterised with aminoacid sequence VAPEEHPTLLTEAPLNPK, and also pharmaceutical composition, which includes specified peptide.

EFFECT: possibility to modulate immune activity selected from the group that consists of suppression of T-lymphocytes transformation, suppression of activity of NK-cells and suppression of antibodies production.

7 cl, 5 dwg, 15 tbl, 12 ex

 

Background of the invention

The scope of the invention

The present invention relates to short peptides and their use. In particular, the present invention relates to short peptides with various kinds of biological activity.

Description of the prior art

Peptides known in the field of disease treatment and as pharmaceutical compositions. For example, in U.S. patent No. 6191113 described peptide with activity as a growth inhibitor of smooth muscle cells and, therefore, acceptable for the prevention and treatment associated with the growth of smooth muscle cells of pathological conditions such as atherosclerosis, restenosis after angioplasty, the narrowing of the lumen after the transplantation of blood vessels and smooth muscle sarcoma. In U.S. patent 6184208 described another peptide, which found that it modulates physiological processes, such as increased activity zone of epithelial growth and hair growth. In addition, in PCT publication no WO 03/006492 and in the patent application U.S. No. 10/237405 suggest that certain peptides and their pharmaceutical compositions are biologically active and capable of modulating immune responses.

Thus, the aim of the present invention is the provision of a short peptide or peptides with biological activity.

The invention

One aspect of the present invention relates to peptides derived from peptide CMS-010, containing 18 amino acids (VAPEEHPTLLTEAPLNPK) (SEQ ID No. 1), which revealed the presence of biological activity, where these peptides do not contain the sequence of the peptide CMS-010. To test samples of these peptides are chemically synthesized from L-amino acids. Additional aspects of the present invention relate to a selected or purified peptide that contains selected from SEQ ID No. 2-31 sequence, essentially consisting of or consisting of selected from SEQ ID No. 2-31 sequence, where the specified peptide does not contain the sequence of the peptide CMS-010 (SEQ ID No. 1). Another aspect relates to essentially pure peptides containing selected from SEQ ID No. 2-31 peptides, where these peptides do not contain the sequence of the peptide CMS-010.

Another aspect of the present invention provides an introduction peptide containing selected from SEQ ID No. 2-31 sequence, essentially consisting of or consisting of selected from SEQ ID No. 2-31 sequence, where the specified peptide does not contain the sequence of the peptide CMS-010, where the impact of the specified injection selected from the group consisting of suppressing transformation of immune cells, inhibit the activity of NK-cells and increased activity of N-cells, suppression of antibody formationin vivo, suppression of cell proliferation, suppression of tumor growth, weakening jade and reduce proteinuria. In some embodiments, the implementation of the suppression of the transformation of immune cells is the suppression of the transformation of T-lymphocytes by ConAin vitro. In some embodiments, the implementation of the suppression of the transformation of immune cells is the suppression of the transformation of T-lymphocytesin vivo. In some embodiments, the implementation of the suppression of cell proliferation is a suppression of the development of cell sarcomain vivo. In some embodiments, the implementation of the weakening of the jade is a weakening of nephritis caused by antibodies to renal epitopes.

Additional aspects of the present invention are peptide containing selected from SEQ ID No. 2-31 sequence, essentially consisting of or consisting of selected from SEQ ID No. 2-31 sequence, where the specified peptide does not contain the sequence of the peptide CMS-010 consisting of L-amino acids. In some embodiments, the implementation of the peptide that contains selected from SEQ ID No. 2-31 sequence, essentially consisting of or consisting of selected from SEQ ID No. 2-31 sequence, where the specified peptide does not contain the sequence of the peptide CMS-010, is p is the substance in its pure form.

Another aspect of the present invention relates to pharmaceutical compositions containing the peptide, which contains selected from SEQ ID No. 2-31 sequence, where the specified peptide does not contain the sequence CMS-010. In some embodiments, the implementation of the pharmaceutical composition containing peptide, which contains selected from SEQ ID No. 2-31 sequence, where the specified peptide does not contain the sequence of the peptide CMS-010 contain consisting of L-amino acid peptides.

Another aspect of the present invention are methods of obtaining pharmaceutical compositions that obtains a peptide that contains selected from SEQ ID No. 2-31 sequence, where the specified peptide does not contain the sequence of the peptide CMS-010, and mixing the peptide with a pharmaceutically acceptable carrier.

Another aspect of the present invention are ways to reduce the impacts of human diseases, including the introduction of a pharmaceutically effective dose of a peptide that contains selected from SEQ ID No. 2-31 sequence, where the specified peptide does not contain the sequence of the peptide CMS-010. In some embodiments, the implementation of the person suffering from a cellular proliferative and/or immunologic disorders. In some embodiments, the implementation of cell-proliferative the violations represent a malignant tumor, sarcoma and/or tumor.

An additional aspect of the present invention is a method of modulating the immune system of the individual, including the introduction of a pharmaceutically effective dose of a peptide that contains selected from SEQ ID No. 2-31 sequence, where the specified peptide does not contain the sequence of the peptide CMS-010.

Another aspect of the present invention is the use of a peptide containing selected from SEQ ID No. 2-31 sequence, where the specified peptide does not contain the sequence of the peptide CMS-010, as a pharmaceutical preparation. In some embodiments, the implementation of the peptide used for the treatment of painful conditions in the form of cell-proliferative disorders and/or immune disorders. In some embodiments, the implementation of the exposed treatment of cell-proliferative infringement is sarcoma.

Another aspect of the present invention is the use of a peptide containing selected from SEQ ID No. 2-31 sequence, where the specified peptide does not contain the sequence of the peptide CMS-010, as a modulator of the immune system. In some embodiments, the implementation of the modulation of the immune system represents an increase or suppressing the activity of NK cells.

An additional aspect of the present invention is the use of a peptide content of asego selected from SEQ ID No. 2-31 sequence, where specified, the peptide contains the sequence of the peptide CMS-010, as a nutritional Supplement.

Another aspect of the present invention is a molecule that contains superior-derived peptide containing selected from SEQ ID No. 2-31 sequence containing improves molecule, functionally associated with the specified peptide, where improving molecule increases therapeutic efficacy of the indicated peptide and where the specified peptide does not contain the sequence of the peptide CMS-010.

Brief description of drawings

Each of the five illustrative figures shows the chemical reaction of binding of peptides to steroid molecules.

The figure 1 presents a series of chemical reactions linking covalent coupling of the peptide with the estrone molecule.

The figure 2 presents a second alternative set of reactions to obtain the same relations as in figure 1.

Figure 3 includes a series of chemical reactions designed to link the covalent bond of the peptide molecule with estradiol.

Figure 4 includes a second series of chemical reactions to obtain the same relations as in figure 3.

The figure 5 presents the methods of linking covalent coupling of the peptide molecule with hydrocortisone.

A detailed description of the preferred option exercise

I. INTRODUCTION

About arozena, the peptide CMS-010 (SEQ ID No. 1) with the sequence VAPEEHPTLLTEAPLNPK has biological immunoregulatory activity (U.S. Patent Application No. 10/178684) and has therapeutic potential for use in humans. The present invention relates to fragments and derivatives CMS-010 with biological activity. In specific embodiments implementing the present invention relates to fragments and derivatives of the sequences presented as SEQ ID No. 2-31. In some embodiments, the implementation of the fragments may have substituents and/or additional molecular groups or can be functional derivatives VAPEEHPTLLTEAPLNPK (CMS-010). Use fragments and derivatives CMS-010 include the regulation of cells and tissues. Fragments and derivatives CMS-010 can be incorporated into pharmaceutical products and nutritional supplements.

It is clear that as another way of practical implementation of the present invention it is possible to add additional amino acids to the N-Termini or C-Termini of the peptide that contains selected from SEQ ID No. 2-31 sequence, essentially consisting of or consisting of selected from SEQ ID No. 2-31 sequence, where the specified peptide does not contain the sequence of the peptide CMS-010, and its functional derivatives. In such scenarios, the implementation of the peptide that contains selected from SEQ ID No. 2-31 posledovatel the face, essentially consisting or consisting of selected from SEQ ID No. 2-31 sequence, where the specified peptide does not contain the sequence CMS-010, and its functional derivatives retain one or more described here therapeutic or functional properties. For example, in some embodiments, the implementation of the described peptide may be added one or two amino acids without affecting its biological function. In some embodiments, the implementation of the molecules of smaller size, containing some VAPEEHPTLLTEAPLNPK (CMS-010), contain only a fragment of the sequence obtained from VAPEEHPTLLTEAPLNPK (CMS-010). In other embodiments, implementation of the molecules of smaller size, containing some VAPEEHPTLLTEAPLNPK (CMS-010), contain two or more fragments of the sequences obtained from a separate, non-contiguous fragments VAPEEHPTLLTEAPLNPK (CMS-010). For example, in some embodiments, the implementation of the molecules of smaller size, containing some VAPEEHPTLLTEAPLNPK (CMS-010), contain a sequence located near the N-Terminus VAPEEHPTLLTEAPLNPK (CMS-010), and a sequence located near the C-Terminus VAPEEHPTLLTEAPLNPK (CMS-010) without any intermediate sequence located between these sequences in VAPEEHPTLLTEAPLNPK (CMS-010). In additional embodiments, the implementation may add three or four amino acids of the saving function of the peptide, selected from the group consisting of fragments CMS-010 (VAPEEHPTLLTEAPLNPK) (where these fragments do not contain the sequence CMS-010) and its functional derivatives. They are all variants of the same peptide. In addition, for the practical implementation of another aspect of the present invention can be used derivative peptide, such as derived from a conservative substitution of one amino acid for another of the same functional class. For example, peptides with non-polar or hydrophobic side chains can be replaced one side of the group to another without compromising biological activity. In some embodiments, implementation of the sequence of the peptide fragment CMS-010 you can remove one, two or more amino acids, and, in spite of this, there is still the activity of the original peptide fragment. For example, a fragment of the peptide CMS-010 length of ten amino acids resynthesized without the 5th amino acids from N-Terminus sequence. Thus, the resulting variation in length of 9 amino acids, 4 amino acids from N-Terminus covalently linked to the 6-th amino acid from the N-end of the original series, and at the same time it has the same activity as the original fragment peptide of the ten amino acids. In some embodiments, the implementation, where two or more amino acids deleted from the sequence fragm the NTA peptide CMS-010, the deleted amino acids in the original sequence of the peptide fragment are adjacent to each other. In other embodiments, the implementation, where the sequence of the peptide fragment CMS-010 removed two or more amino acids, the amino acids are not in the original sequence of the peptide fragment adjacent to each other, but rather are separated from each other in the source sequence of amino acids that are stored in a truncated version of the peptide. In additional embodiments, the implementation of the fragment peptide CMS-010 removed three or more amino acids, some were removed from the original sequence of amino acids that are adjacent to each other, while one or more amino acids deleted from the original sequence that are not adjacent to any other remote from the initial sequence of amino acids. In additional embodiments of this invention for the formation of peptide variants can be integrated sequence of the linker/spacer, but the options are still active functional group, as in the original peptide used in this study. They are also considered variants of the peptides. As used here, the analog of peptide includes a peptide with the amino acid molecules that mimic the structure ol the native amino acids, for example, similar with different structure of the main chain, or substituted D-amino acids. As an additional example, although the amino acids, which are used for the synthesis of peptides, are in their optical isomer L-form peptides with one or more amino acids in the sequence, substituted D-forms may possess similar biological activity. As used in the claims, the term "functional derivative" includes fragments, variants, analogs, or chemical derivatives of the peptide.

"Essentially pure peptide" refers to peptides with the degree of purification, at least 10% wt./wt., more preferably 20%, more preferably 60% and much more preferably more than 90% cleared. In the preferred embodiment, the purification efficiency is more than 99%. Essentially pure peptide can be used for pharmaceutical and nutritional preparations, which, as described hereinafter, can represent a complex mixture.

"Modulation" refers to the effect on the cells caused by the introduction or exposure of the peptides of the present invention, where the introduction or the effects of peptides on cells leads to changes in cell activity. These changes may include an increase or suppression act and the activities of the cells. Increasing or suppressing the activity of cells may represent an increase or suppression of the rate of cell division and replication, increasing or suppressing the response of cells to other factors and/or increase or suppression of the level of production and/or secretion of cell proteins or substances.

"Cell proliferation" refers to the increase in the number of existing cells and may be a consequence of transformation or immortalization of the cell. Disorders of cell proliferation include as non-limiting examples of malignant tumors, benign tumors, tumors and sarcomas, and may include any number of cells. "Immunologic disorders" refers to the abnormal function or damaging the function of immune cells or other parts of the immune system. Such disturbances may be caused by suppression of activity of the cells or molecules or increased activity of cells or molecules.

Used in pharmaceutical preparations of the peptide that contains selected from SEQ ID No. 2-31 sequence, essentially consisting of or consisting of selected from SEQ ID No. 2-31 sequence, where the specified peptide does not contain the sequence of the peptide CMS-010, and its functional derivatives, may be used as a potential treatment of immunological disorders or diseases. Drugs can with erati peptide, contains selected from SEQ ID No. 2-31 sequence, essentially consisting of or consisting of selected from SEQ ID No. 2-31 sequence, where the specified peptide does not contain the sequence of the peptide CMS-010, and its functional derivatives, mixed with other active or inactive components, including other peptides, for example, to the same drug, you can add two to several (e.g., 3-5) of peptides with other ingredients or without other ingredients. Alternatively, the peptide contains selected from SEQ ID No. 2-31 sequence, essentially consisting of or consisting of selected from SEQ ID No. 2-31 sequence, where the specified peptide does not contain the sequence of the peptide CMS-010, and its functional derivatives can be used to obtain the drug together with is not listed here peptides. They can be administered intravenously, intramuscularly, intracutaneously, subcutaneously or intradermally. The method of introduction may also be an intra-arterial injection, which leads directly to the diseased organ. Other routes of administration is transdermal, inhaled in the form of a powder or aerosol and other forms of delivery, known to specialists in this field. Drugs can be taken orally, and they can contain media that you can use DL is preventing digestion of the peptide in the stomach after oral administration, or any other media known in the field (carriers for transdermal delivery, such as liposomes).

As used here, the term "hybrid peptide" is used in relation to peptides containing additional peptides embedded in the source of the biologically active peptide with the above sequence or its functional derivative, but still retains essentially the same activity. Additional peptides include leader peptides, for example, contains the amino acid sequence, which recognize one or more prokaryotic or eukaryotic cells as a signal for secretion of the hybrid protein into the external environment or in a cage. The secretion may be direct or indirect via secretory vesicles.

As used here, the terminology "essentially consisting of" refers to a peptide or polypeptide that contains selected from SEQ ID No. 2-31 sequence, essentially consisting of or consisting of selected from SEQ ID No. 2-31 sequence, where the specified peptide does not contain the sequence of the peptide CMS-010, and its functional derivatives with additional amino acids at the C-end and/or the N-end and which retain one or more activities of these peptides can be found here. Thus, as non-limiting is confident, where the activity of a peptide containing selected from SEQ ID No. 2-31 sequence, essentially consisting of or consisting of selected from SEQ ID No. 2-31 sequence, where the specified peptide does not contain the sequence of the peptide CMS-010, and its functional derivatives relates to the treatment and/or prevention of cell proliferative or immunologic disorders or diseases, peptide or polypeptide "consisting essentially" of the peptide that contains selected from SEQ ID No. 2-31 sequence, essentially consisting of or consisting of selected from SEQ ID No. 2-31 sequence, where the specified peptide does not contain the sequence of the peptide CMS-010, and its functional derivatives, has activity in the treatment and/or prevention of disorders or diseases, as presented here in respect of this peptide, and by itself does not possess any characteristics (i.e. prior to modification by joining one or more biologically active molecules, that can significantly reduce the ability of the peptide or polypeptide for the treatment or prevention of cell proliferative or immunologic disorders or which cause significant changes in the basic and novel characteristics of the peptide as a therapeutic and/or prophylactic agent for the above violations or Soboleva is th. Thus, in the previous example, natural full-sized polypeptide, which has the original activity, other than the treatment and/or prevention of cell proliferative or immunologic disorders, and that somewhere contains selected from SEQ ID No. 2-31 sequence, consists essentially of or consists of selected from SEQ ID No. 2-31 sequence (but does not contain the sequence CMS-010), and its functional derivatives are not a peptide or polypeptide "essentially consisting of" peptide, which contains selected from SEQ ID No. 2-31 sequence, essentially consists of or consists of selected from SEQ ID No. 2-31 sequence and its functional derivatives, the sequence of which contains a natural full-sized polypeptide. In the previous example obtained by means of genetic engineering a peptide or polypeptide which has the original activity, other than the treatment or prevention of cell proliferative or immunologic disorders, but includes the amino acid sequence of the peptide that somewhere contains selected from SEQ ID No. 2-31 sequence, essentially consists of or consists of selected from SEQ ID No. 2-31 sequence (but does not contain the sequence CMS-010), and its functional derivatives, may not be the peptide or polypeptide is m "essentially consisting of" peptide, which contains selected from SEQ ID No. 2-31 sequence, essentially consists of or consists of selected from SEQ ID No. 2-31 sequence (but does not contain the sequence CMS-010), and its functional derivatives, the sequence of which is obtained by means of genetic engineering peptide or polypeptide.

Specialists in this field can easily determine whether a peptide or polypeptide essentially of the peptide that contains selected from SEQ ID No. 2-31 sequence, essentially consisting of or consisting of selected from SEQ ID No. 2-31 sequence, where the specified peptide does not contain the sequence of the peptide CMS-010, and its functional derivatives by the above definitions by measuring the activity of the peptide or polypeptide using analyses of treatment or prevention of cell proliferative or immunologic disorders, which are provided here in relation to fragments and derivatives of the peptide VAPEEHPTLLTEAPLNPK (CMS-010).

In a preferred embodiment, the terminology "essentially consisting of" can also refer to peptides or polypeptides that have less than 5 amino acid residues in addition to the peptide that contains selected from SEQ ID No. 2-31 sequence, essentially consisting of or consisting of selected from SEQ ID No. 2-31 posledovatelno and, where specified, the peptide contains the sequence of the peptide CMS-010, and its functional derivatives. In a more preferred embodiment, the above terminology refers to peptides with 2 amino acid residues in addition to the peptide that contains selected from SEQ ID No. 2-31 sequence, essentially consisting of or consisting of selected from SEQ ID No. 2-31 sequence, where the specified peptide does not contain the sequence of the peptide CMS-010, and its functional derivatives. In an even more preferred embodiment, the above terminology refers to a peptide with a single amino acid residue in addition to the peptide that contains selected from SEQ ID No. 2-31 sequence, essentially consisting of or consisting of selected from SEQ ID No. 2-31 sequence, where the specified peptide does not contain the sequence of the peptide CMS-010, and its functional derivatives.

The pharmaceutical preparation can include any of the known pharmaceutical carriers. Examples of acceptable carriers include any of the well-known specialists in the field of the standard pharmaceutically acceptable carriers. They include as non-limiting examples of physiological saline solution, water, emulsions, including mixtures of water and oil or a triglyceride emulsion, and other types of means which, fillers, coated tablets and capsules. Acceptable carrier can be selected on the basis of route of administration of the pharmaceutical composition.

A peptide selected from the group consisting of selected from SEQ ID No. 2-31 sequence, essentially consisting of or consisting of selected from SEQ ID No. 2-31 sequence, where the specified peptide does not contain the sequence of the peptide CMS-010, and its functional derivatives can be entered via intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection, and subcutaneous implantation. The peptide can be entered in any form for oral administration such as tablet, capsule, suspension, solution, etc, in the usual form, without modification or in the form of delayed release, or with gastrointestinal protection or without it. In addition, the peptide can be used in any form for local use, such as ointment, cream, gel, etc. with any auxiliary device for transdermal application, or without it. The peptide can also be present in the form of its genetic sequence and clone in expressing the system alone or in combination with other sequences of the peptides to obtain molecules of the final peptide for use in the activity of the peptide, as described herein.

The dose of each peptide may leave 1 ng - 10 g per kg of body weight. Preferred injection method of administration dose is 10 ng - 10 mg / kg, and more preferably 1 μg to 1 mg per kg However, the effective dose may be lower than 1 ng per kg of body weight due to the fact that one or more of the peptides may act through receptors that induce a cascade of normal physiological response. Alternatively, one or more peptides can directly initiate a cascade of reactions. For oral administration, the number can be 1 ng to 10 g per kg of body weight per day, more preferably 0.1 mg to 1 g per kg of body weight per day and more preferably 1 μg to 10 mg per day.

II. GENE THERAPY AND TREATMENT

Gene therapy on the basis of the above sequences of peptides is implemented by constructing a nucleic acid sequence that encodes one of these peptides. Nucleic acid can be chemically synthesized and functionally ligitamate with the promoter and expressing cloned into the vector. Then for expression in human cells as a form of gene therapy expressing the vector is introduced into the human body. As used here, the term "genetic vectors" includes those expressing vectors. Vectors that can be used for gene therapy, VK is ucaut adeno-associated virus (Mizuno, M. et al. (1998). Jpn J Cancer Res 89, 76-80), the vector LNSX (Miller, A. D. et al. (1993) Methods Enzymol 217, 581-599) and lentivirus (Goldman, M. J. et al. (1997) Hum Gene Ther 8, 2261-2268).

Other media for the delivery of peptide include vectors encoding the desired peptide, which can be transferred into an organism capable of replication in a host organism, which want to introduce the peptide, without significant adverse effects on the health of the host organism. For example, expressing vectors can be transferred into the body, which is not pathogenic to the host organism, which want to introduce the peptide. In some embodiments, the implementation expressing vector produces the desired peptide in bacterial or fungal organism that does not have significant harmful impacts on the health of the host organism, which administered the peptide. For example, expressing a vector coding for the desired peptide may be expressing the vector that produces the desired peptide in the body, such as lactobacteria,E. Colior yeast. In one embodiment expressing vector produces the desired peptide in a microorganism, which is usually in the intestines of mammals or the microorganism to which a tolerant digestive tract of mammals. Some species of bacteria, in which the desired peptide can be expressed, including the indicate as non-limiting examples of species of Lactobacillussuch asL. acidophilus, L. amylovorus, L. casei, L. crispatus, L. gallinarum, L. gasseri, L. johnsonii, L. paracasei, L. plantarum, L. reuteri, L. rhamnosusor other; the speciesBifidobacteriumsuch asB. adolescentis, B. animalus, B. bifidum, B. breve, B. infantis, B. lactis, B. longumor other; Enterococcus faecalisorEnt. facium; Sporolactobacillus inulinus; Bacillus subtilisorBacillus cereus; Escherichia coli; Propionibacterium freudenreichii;orSaccharomyces cerevisiaeorSaccharomyces boulardii.

Chemically synthesized or obtained in other ways, including as non-limiting examples of reverse transcription of mRNA with obtaining cDNA molecules, nucleic acid sequences that encode any of the peptides of the present invention, well-known specialists in this field by means of genetic engineering embed in expressing vector for gene transfer in desirable organisms. Expressing vectors can represent DNA vectors or RNA vectors. For example, expressing vectors can be based on plasmid or viral genetic elements. Expressing vectors can be represented as vectors that replicate extrachromosomal, or vectors that integrate into the chromosome.

Expressing the vectors contain a promoter functionally linked to a nucleic acid that encodes a peptide of the present invention. The promoter can be a adjustable the th promoter, such as inducible promoter or a constitutive promoter. In some embodiments, the implementation to obtain the desired level of expression of the peptide promoter can be selected. In addition, if necessary, expressing vectors can contain other sequences to ensure the production, presentation and/or secretion of peptides. In some embodiments, the implementation of the nucleic acid encoding the peptide of the present invention, functionally linked to a nucleic acid sequence that directs the secretion of the peptide. For example, nucleic acid encoding the peptide of the present invention can be functionally linked to a nucleic acid that encodes a signal peptide.

In some embodiments, the implementation of expressing vectors, designed to encode the peptides of the present invention may constitute expressing vectors adapted for expression of the peptide of the present invention the components of the normal flora of the intestines of mammals bacterial species, such as species ofLactobacillusandBacillus subtilis. Examples of such expressing vectors can be found in U.S. patent No. 6100388 issued Casas, and No. 5728571 issued by Bellini, respectively. Thus, these documents are specifically incorporated here as reference in full. the learn to understand you can use any expressing the vector, which facilitates the expression of the peptide of the present invention in the body, which is not harmful to the health of the host organism, which is injected peptide.

In some embodiments, the implementation of expressing the vectors which are designed to encode the peptides of the present invention may constitute expressing vectors adapted for expression of the peptide of the present invention in a highly stable in the intestines of mammals, yeast species, such asSaccharomyces cerevisiae; or preferablySaccharomyces boulardiithat can colonize the human intestine and are used to treat certain forms of diarrhea. You can use yeast expressing vectors that constitutively Express heterologous proteins and peptides are highly stable, so well are transferred to daughter cells during mitosis and meiosis, and may include encoding the signal peptide or the peptide sequence, providing a high level secretion of recombinant protein. An example of such a yeast vector is shown in U.S. patent No. 6391585 issued by Jang et al., which therefore specifically incorporated here by reference in full.

Expressing the vectors encoding the peptides according to the present image is the shadow, you can enter in the body, designed for the expression of peptides by means known in the field of methods. These methods include traditional methods of transformation of bacteria, yeast or other microorganisms using, for example, competent due to the use of chemical methods bacterial cells transformed by electroporation or lithium acetate (for yeast), as well as the latest achievements in the field of transformation of bacterial species that are resistant to such procedures. In some embodiments, the implementation of expressing vectors injected into the lactobacilli, known as resistant to transformation, using the method described Leer et al. (WO 95/35389), the disclosure of which is incorporated here by reference in full. The input sequence can be integrated into the chromosomal DNA of the microorganism, or may remain as extrachromosomal DNA elements.

Then for ongoing immunotherapy obtained such ways of genetically engineered microorganisms containing expressing vector, it is possible to inoculate into the digestive tract, vagina, trachea, etc. In some embodiments, the implementation of organisms expressing the peptides of the present invention, taking into inactive form or preferably in a live form. In the gut e and microorganisms produce these peptides, release them into the lumen through the secretion or lysis of the microorganism or otherwise provide peptides master, after which the peptides carry out the intended action against the host organism. In other embodiments, implementation of the peptides provided to the owner of the mucous membranes of the nasal passages, vagina, or small intestine.

Another method of treatment is the application of liposomes as a delivery method of a specific nucleic acid in cells of the human body. Nucleic acid (such as expressing a vector containing a nucleic acid sequence encoding the peptide containing the selected from SEQ ID No. 2-31 sequence, essentially consisting of or consisting of selected from SEQ ID No. 2-31 sequence, where the specified peptide does not contain the sequence of the peptide CMS-010, and its functional derivatives) are placed in an environment that facilitates cellular recognition and integration into the chromosome, as described in Gao, X. and Huang, L. (1995) Gene Ther 2, 710-722 and U.S. patent 6207456. Alternatively, the peptide can be encapsulated in the liposome and deliver directly using the method described in U.S. patent 6245427. All of the above scientific publications and patents included here as reference in full.

The nucleic acid sequences are acceptable for gene therapy and treatment method, above, include sequences encoding these peptides and their functional derivatives. Any of numerous sequences of nucleic acids can be used for encoding these peptides and their derivatives on the basis of the degeneracy of the system codons.

The following links are included here as reference in full.

1. Principles of Pre-clinical Research of New Drugs, People's Republic of China. 1993, 7:134-135 Shuyun Xu, Rulian Bian, Chen Xiu. Methodology of pharmacological experiment. People's Health Publishing House. 1991, 1221-1234.

2. Principle of new drug research in pre-clinic issued by Ministry of Health, People's Republic of China. 1993, 7:140.

3. Jinsheng He, Ruizhu Li Tingyi Zong. The study on MTT reduction method of testing NK cell activity. China Immunology Journal. 1996, 1(6):356-358.

4. Quan Wang. Modern medical experiment method. People's Health Publishing House. 1998, 482-483.

5. Principle of new drug research in pre-clinic issued by Ministry of Health, People's Republic of China. 1993, 7:141.

6. Principle of new drug research in pre-clinic issued by Ministry of Health, People's Republic of China. 1993, 7:132-133.

7. Principle of new drug research in pre-clinic issued by Ministry of Health, People's Republic of China. 1993, 7:128-129.

8. Yuanpei Zhang, Huaide Su. Pharmacological experiment (second edition). People's Health Publishing House. 1998, 137-138.

9. Li Jiatai, clinical pharmacology (second edition). People's Health Publishing House. 1998, 1338-1339.

III. PEPTIDE CONJUGATES AND products WITH PEPTIDESCONTAINS SELECTED FROM SEQ ID No. 2-31 SEQUENCE, ESSENTIALLY CONSISTING of OR CONSISTING OF SELECTED FROM SEQ ID No. 2-31 SEQUENCE (WHERE THESE FRAGMENTS do NOT CONTAIN the SEQUENCE of the PEPTIDE CMS-010), AND FUNCTIONALNAME DERIVATIVES

To provide additional effect or use, or to increase therapeutic efficacy of biologically active peptides of the present invention can be konjugierte with other biologically effective or suitable molecules. In this area there are many possible molecules for conjugation, their biological effects and methods of conjugation of molecules with peptides. For other prospective partners by chemical conjugation reactions of conjugation with them these peptides can be derived by the person skilled in the art without undue experimentation. Effective molecules described below. Describe specific examples of how various peptides of the present invention can be konjugierte effective molecules, and biological properties of the product obtained by conjugation. It is clear that for the other peptides of the present invention can also be conjugation by similar reactions.

Peptide fragments containing selected from SEQ ID No. 2-31 sequence, essentially consisting of or consisting of selected from SEQ ID No. 2-31 sequence, where the specified peptide does not contain the sequence of the peptide CMS-010, and its functional derivatives may have different therapeutic effects on specific cell types or tissues. One is th of the essential objectives of the conjugation of molecules with peptide drug substance is the direction of the peptide in a specific area or Department of the organism of the individual, being treated. Thus, the peptide drug and its effects can be concentrated in the area of those types of cells or tissues that are being targeted therapeutic effect. This may increase the effect, which would have a similar molar amount of free, unconjugated peptide. On the contrary, the dosing of conjugated peptide drugs directed in the area of its therapeutic activity may be significantly smaller than necessary to achieve the same therapeutic effect dosing free unconjugated form of the drug.

Another favorable effect of the direction of peptide drugs in the region, where its activity is most desirable, is the reduction of unwanted side effects. Peptide drug, which is injected to cause a change in specific types of cells or tissues, can also act in other parts of the body of the individual, sometimes with adverse consequences. Through peptide in a desirable area of activity through conjugation with directing molecule, it is possible to reduce the concentration of peptide in other parts of the body of the individual and the corresponding side effects.

For directions to various wher is all over the body of the individual peptides, contains selected from SEQ ID No. 2-31 sequence, essentially consisting of or consisting of selected from SEQ ID No. 2-31 sequence, where the specified peptide does not contain the sequence of the peptide CMS-010, and its functional derivatives can be konjugierte with many molecules. For any peptide of the present invention to direct the peptide in a desirable area, you can use any of the methods of conjugation are described below, as well as the other way conjugation, well-known experts in this field. For example, it was shown selective delivery of drugs against hepatitis B in liver cells (Fiume et al., Ital J Gastroenterol Hepatol, 29(3): 275, 1997, included here as a reference in full). In this study, the researchers conjugatively adeninederivatives (ara-AMP), phosphorylated nucleoside analogue, active against hepatitis B virus, with lactosaminated human albumin, macromolecule, ending with galactogram. Hepatocytes Express a receptor protein that interacts with the terminal galactosyl balance with high affinity. By binding to the receptor conjugated drug selectively captured by hepatocytes. After the absorption of the conjugated drug is delivered in a complementary mechanism where it is broken down the ligature between the two components of conjugated drugs with the release of ara-AMP in its active form. In the research mentioned above, conjugated drug was as effective for the treatment of patients with chronic infection with hepatitis B, as free ara-AMP, but did not lead to clinical side effects, such as neurotoxicity, which resulted in the introduction of free ara-AMP. This approach can be applied to any of the peptides of the present invention.

In research related to the above study, the same research group (Di Stefano et al., Biochem. Pharmacol., 61 (4): 459, 2001), for directions compounds in the liver and the treatment of metastases in the liver chemotherapeutic agent against malignant tumors 5-fluoro-2-deoxyuridine (FUdR) conjugatively with lactosaminated poly-L-lysine. The drug selectively captured by the liver cells, which will assalut the relationship between FUdR and directing molecule. Then a portion of the free FUdR leave the liver cells and creates a local therapeutic concentrations of anti-malignant tumor. This concentration is sufficient for the pharmaceutical activity against cells of metastases, infiltrating the liver. Due to the selective concentration in the liver drug dosage conjugated drug may be significantly less than on the smaller pharmaceutically active dose of free unconjugated compounds. This strategy can be used against any of the peptides of the present invention. For example, the conjugation lactosaminated poly-L-lysine with a peptide that contains selected from SEQ ID No. 2-31 sequence, essentially consisting of or consisting of selected from SEQ ID No. 2-31 sequence (where specified peptide does not contain the sequence of the peptide CMS-010), and its functional derivatives can significantly reduce the dosage required for the treatment or prevention of cell proliferative disorders involving the liver tissue.

The direction of the connections in specific types of tissues or cells of an organism achieved for many different types of tissues or cells. For example, tumor cells often Express on their surface abnormally high levels of receptors for peptide hormones such as bombezin, LH, growth hormone-releasing hormone and somatostatin. In one study the connection against malignant tumor paclitaxel (Taxol) is selectively sent to the hormone secreting cells of the tumor, with a high density of expressing receptors for somatostatin, through conjugation of medicinal substance with octreotide, a somatostatin analogue. Conjugated with octreotide Taxol was as effective as free Taxol, but had a reduced toxicity is relative to normal cells. (Huang et al., Chem. Biol., 7(7): 453, 2000). By applying the methods of Huang et al. for conjugation of the peptides of the present invention with analogues agonists of the receptor peptide hormone, you can create a method of treatment directed specifically against cells expressing high levels of this particular receptor peptide hormone. This approach can be adapted to target cells with high expression of any number of receptors for peptide hormones. In another example, the direction of the drug in a specific tissue type, for the direction of the release specifically to the cells of the large intestine as the molecules of the carrier used poly-L-aspartic acid (Leopold et al., J. Pharmacokinet. Biopharm., 23(4): 397, 1995).

In addition to specific areas of a peptide drug to a particular cell type or tissue conjugation of peptides containing selected from SEQ ID No. 2-31 sequence, essentially consisting of or consisting of selected from SEQ ID No. 2-31 sequence (where these peptides do not contain the sequence of the peptide CMS-010), and their functional derivatives with molecules-carriers can provide other ways to improve the delivery of peptide drugs, thereby enhancing or otherwise improving their therapeutic effects. For any of the peptides of the present invention monobrominated any of the following technologies, as well as other techniques known to experts in this field. The effectiveness of any of the drugs decreases if the connection is not to deliver effectively to its target. The drug can be transported to the area of his actions actively or otherwise without significant reduction in activity due to metabolic conversion or degradation. Peptide drugs affected by the action of peptidases in the case of strongly charged molecules may be unable to transport through the lipid cell membrane and the membranes of endothelial cells, such as blood-brain barrier. Conjugation with other molecules relates to a method for protecting peptides from degradation and enhance the absorption of peptide drugs into cells or anatomical divisions, in which connection under normal conditions do not penetrate.

Making possible the access of peptides in the body, which they would not normally penetrate, methods of conjugation can make available new ways of introducing medicines. In the article by Patel et al., Bioconjugate Chem., 8 (3): 434, 1997, the chemistry which are described in detail below in example 5 and which is incorporated here by reference in full, researchers conjugatively peptide drug, known as strong boleutolyayushchikh, heptapeptide deltorphin with an organic molecule, which was developed specifically to give the ability of the peptide to penetrate the blood-brain barrier. It is possible to introduce the drug intravenously instead intracerebroventricular introduction.

In addition to giving the peptide's ability to penetrate the barrier molecule-carrier in Patel et al. was developed for specific areas in the endothelial cells that comprise the blood-brain barrier. Membrane of endothelial cells throughout the body, including the blood-brain barrier, are heterogeneous in relation to the specificity of the sequence and concentration of membrane-bound endopeptidase who are on their surfaces. When designing molecules use these characteristics to make possible the direction of the molecules of the carrier and its cargo. The molecule contains three chains of fatty acids, the free ends of which end up in the dipeptide Arg-Pro, which preferably communicates with endopeptidase blood-brain barrier. The implementation of the transport of charged molecules of peptide drugs can be achieved through lipophilic fatty acid chains. Thus, the triglyceride molecule, ending with the dipeptide, allows and direction, and tra the sport through the blood-brain barrier.

By way of conjugation improves the kinetics of the activity of peptide drugs. In respect of the peptide that contains selected from SEQ ID No. 2-31 sequence, essentially consisting of or consisting of selected from SEQ ID No. 2-31 sequence (where specified peptide does not contain the sequence of the peptide CMS-010), and its functional derivatives to improve the kinetics of activity of the peptide can use any of the following technologies conjugation, as well as other conjugation technology, well-known experts in this field. Patel et al. found that conjugated forms painkiller peptide not only able to penetrate from the bloodstream into the brain, but also have greater duration compared to the free peptide. Intravenous require more time to achieve a therapeutic effect, but the effect lasts longer and decreases slower than the effect of the free peptide, entered intracranial. The researchers found that conjugated peptide molecule largely stable in serum, however, has no effect when intracerebroventricular the introduction, which indicates that the molecule-media probably are degraded and removed during its transport from the bloodstream into the brain. They suggested that if the other changes in the kinetics is time, required for the transport of the conjugate and the degradation of the molecules of the carrier. Regardless of the mechanism of the delay in the clinic intravenous stability of the conjugated peptide molecules and longer to start and activity effects medicines may mean that it may be administered less frequently. Less frequent and thus more convenient dosing regimen increases the usefulness of the medicinal product as a means of treatment.

Specialists in this field it is clear that the methods and procedures Patel et al. are easily adaptable to the delivery of any of the peptides that fall within a limited range of sizes, including any of the peptides of the present invention. For example, for the treatment and/or prevention of cell proliferative or immunologic disorders peptide of the present invention, such as a movie VAPEEHPTLLTEAPLNPK (CMS-010)can be konjugierte with the above molecules, which were applied Patel et al. In the treatment of an individual with an infection that affects the brain, conjugium molecule may facilitate the penetration of fragments VAPEEHPTLLTEAPLNPK (CMS-010) from the bloodstream into the brain and contribute to the impact of fragments VAPEEHPTLLTEAPLNPK (CMS-010) on the cells or tissue of the brain. For such individuals modifications for changing the direction of action of the molecules of the carrier is also evident. Direction of the Commissioner, the property of the molecules of the carrier is a function of the characteristics of the two amino acids, which form a dipeptide mask at the end of the fatty acid chains. Dipeptide Arg-Pro preferably interacts with the membrane-bound region of endopeptidase detected on the surface of the endothelial membrane of the blood-brain barrier. In other endothelial cells and membranes potentially can be sent through other combinations of dipeptides.

The researchers also used conjugation to obtain peptide drugs that can effectively be absorbed in the digestive tract or skin. To improve absorption of the peptide that contains selected from SEQ ID No. 2-31 sequence, essentially consisting of or consisting of selected from SEQ ID No. 2-31 sequence (where specified peptide does not contain the sequence of the peptide CMS-010), and its functional derivatives can be applied to any of the following technologies conjugation to improve absorption, and other conjugation technology, well-known experts in this field. Kramer et al. describe the procedure for the conjugation of peptide drugs with bile acids. The absorption rate of the conjugated molecules after oral delivery of compounds is greatly increased compared with a single peptide (J. Biol. Chem., 269(14): 10621, 1994). Toth et al. (J. Med. Chem., 42(19): 4010, 1999), describe the conjugation of the peptide is cartonnage funds with antitumor properties with lipoaspiration (LAA) or liposaccharide (LS) in order to increase the rate of absorption and improve the delivery of antitumor peptide in the area of its activity. In their study derived somatostatin expressing strong antiproliferative properties, but has a reduced pharmacokinetics, conjugatively or LAA, or LS. The resulting conjugated drug had improved profile of absorption through the skin and intestinal epithelium and increased resistance to degradation and at the same time was active against tumor cells. These methods can be particularly useful for conjugation with any of the peptides of the present invention. By increasing the rate of absorption of molecules across the intestinal epithelium into the blood stream can be delivered and have an impact on being treated with the individual more peptide.

The conjugation can be used to provide slow release of the peptide drug. To provide sustained release of a peptide containing selected from SEQ ID No. 2-31 sequence, essentially consisting of or consisting of selected from SEQ ID No. 2-31 sequence (where specified peptide does not contain the sequence of the peptide CMS-010), and its functional derivatives can be applied to any of the following technologies conjugation to provide a slow-release, as well as other conjugation technology, well-known specialists in this area is. As can be seen from the work of Patel et al., slow release of the peptide drug can be achieved by means of conjugation. Another example is the work of Kim et al. (Biomaterials, 23: 2311, 2002), where recombinant human epidermal growth factor (rhEGF) before microencapsulation in biodegradable microspheres of a copolymer of lactic and glycolic acid (PLGA) conjugatively with polyethylene glycol (PEG). For delivery of various growth factors and morphogenetic protein microencapsulation in PLGA was used in several bands (Meinelet al., J. Controlled Rel., 70: 193, 2001). Due to conjugation with PEG rhEGF becomes resistant to the formation of insoluble aggregates and adsorbirovanny on the division surface water-organic phase in the formation of micelles with PLGA compared to unconjugated, free rhEGF. The pharmacokinetics of the drug conjugated hormone has been improved, showing a longer, more sustainable and, in General, the best activity of the drug in comparison with the free hormone, which was considered by the researchers, was the consequence of increasing the physical stability conjugated with PEG hormone. For any of the peptides of the present invention to create drugs with a slow release, you can use a similar strategy. For example, as long as the ANO below in example 1, fragments and derivatives VAPEEHPTLLTEAPLNPK (CMS-010) show a pronounced antiproliferative and immunomodulatory effects. By PEG conjugation with the peptide and making conjugated drug in microspheres of PLGA antiproliferative and immunomodulatory effects fragments and derivatives VAPEEHPTLLTEAPLNPK (CMS-010) can be longer and more stable, because the dosage of the medicinal product as it is released from the conjugate with PEG is more uniform and leads to more stable delivery of peptide drugs into the area of infection.

Slow release of peptide drugs can significantly increase its activity. To provide sustained release of a peptide containing selected from SEQ ID No. 2-31 sequence, essentially consisting of or consisting of selected from SEQ ID No. 2-31 sequence (where specified peptide does not contain the sequence of the peptide CMS-010), and its functional derivatives can be applied to any of the following technologies conjugation to provide prolonged release and other conjugation technology, well-known experts in this field. Oldham et al. (Int. J. Oncology, 16: 125, 2000) compared the remedy against malignant tumor paclitaxel with a new form Lek is stannage means by paclitaxel, conjugated with poly-L-glutamic acid (PG-TXL). It turned out that PG-TXL has a higher antitumor activity compared to free paclitaxel, suggesting that the drug has improved pharmacokinetic properties or probably even a better way of exposure. However, the researchers found that PG-TXL exerts its effects through the same mechanism of action to that of free drug inducyruya stop (blocking) of the cell cycle through a violation of polymerization of subparticles of microtubules. Experimental data suggest that the enhanced antitumor activity of conjugated drugs occurs due to prolonged and sustained release of free drug from the conjugate with maintaining therapeutic concentrations over a longer period of time compared with the introduction of the free peptide. By adding a tail of poly-L-glutamic acid to the peptide of the present invention with anti-infective properties can also enhance these properties.

Enzymatic degradation of peptides in some cases can reduce the effectiveness of peptides as drug sredstva reduce enzymatic degradation of the peptide, contains selected from SEQ ID No. 2-31 sequence, essentially consisting of or consisting of selected from SEQ ID No. 2-31 sequence (where specified peptide does not contain the sequence of the peptide CMS-010), and its functional derivatives can be applied to any of the following technologies conjugation to reduce enzymatic degradation, and other conjugation technology, well-known experts in this field. Researchers have developed many approaches to protect peptides from secreted into the lumen of the intestinal peptidases, as well as from membrane-bound peptidases. The latter are on the surface of the mucous tissue, the penetration of which is often a way of penetration of peptide drugs. Bernkop-Schurch et al. (J. Drug Target., 7: 55, 1999) reported on the establishment of peptide drugs drugs containing inhibitors of pepsin. Similar pepstatin covalently linked with mucoadhesive polymers; such a new inhibitor of pepsin included in the tablet formulation containing insulin. After incubation under laboratory conditions, stimulation of digestion all the insulin control pills was metabolized, while approximately 50% of insulin-containing inhibitor tablets were protected from degradation. In another study for inhibition of degradati the biologically active peptides mentioned above, the group applied the inhibitors at doses under normal conditions, leading to toxic side effects (Bernkop-Schnurch et al., Adv. Drug Del. Rev., 52: 127, 2001). This approach was used similar to the cellulose, the aminopolysaccharide coated chitosan, extracted from chitin, the major structural polysaccharide found in shellfish and other organisms. Through conjugation of protease inhibitors with chitosan and the inclusion of conjugated molecules is part of the preparation of peptide drugs found a significant inhibition of the proteases of the digestive tract with a bioavailability of peptide without side effects that can be expected from the introduction of free protease inhibitors. In this study, many of protease inhibitors separately and in combination were used for conjugation with chitosan carrier. Conjugate chitosan-EDTA well inhibited endogenous protease by binding mineral cofactors required for activity of specific proteases. Specialists in this field it is easy to understand that to impart useful properties to the peptide drug you can get a large number of possible combinations between molecules media and effector sites, any of which can be easily adapted for use of the peptide of the present invention. Upon receipt of the product for oral delivery of peptide using related the data with chitosan of protease inhibitors oral delivery of peptide according to the present invention can be applied instead of intramuscular injection. This approach does not exclude the use of more absorbable conjugated variant peptide that contains selected from SEQ ID No. 2-31 sequence, essentially consisting of or consisting of selected from SEQ ID No. 2-31 sequence (where specified peptide does not contain the sequence of the peptide CMS-010), and its functional derivatives (discussed in the paragraph above) to obtain an even higher level of bioavailability of the peptide and its derivatives.

In addition to the direction in the scope of another molecule peptides can serve as a guide molecule independently. For peptide that contains selected from SEQ ID No. 2-31 sequence, essentially consisting of or consisting of selected from SEQ ID No. 2-31 sequence (where specified peptide does not contain the sequence of the peptide CMS-010), and its functional derivatives for use of the peptide for directions molecules in a desirable area, you can apply any of the following technologies conjugation, as well as other conjugation technology, well-known experts in this field. For example, researchers took a drug against malignant tumor deformational (DFMO) and to guide actions conjugatively it with the peptide. DFMO is vysokotekhnologicheskoi substance that effectively you which indicates the death of many types of tumor cells. However, due to its rapid elimination from the body, its therapeutic value is limited. In this study, DFMO conjugatively with a separate fragment of α-melanotropin and analogues fragment containing two amino acid substitutions have been shown preferred binding to receptors on melanotropin cell line human melanoma (Suli-Vargha et al., J. Pharm. Sci., 86: 997, 1997). To facilitate the release of DFMO from peptide fragments by aminopeptidase drug conjugatively N-end of the peptides. The researchers found that conjugated drugs are more effective against cell death of melanoma than separate unconjugated drug.

The effects of the peptides of the present invention may partially be due to the inherent self-peptides directing abilities. For example, as in the case of the fragment of the α-melanotropin, individual peptide according to this invention may be contacted with a specific receptor located on the cell surface of various types. Through the use of this peptide as conjugate action of the medicinal product can be directed to the area of those cells of the individual, which should be treated.

Peptides as conjugates can perform functions other than the direction of the action is elsewhere. To increase the efficiency of peptide that contains selected from SEQ ID No. 2-31 sequence, essentially consisting of or consisting of selected from SEQ ID No. 2-31 sequence (where specified peptide does not contain the sequence of the peptide CMS-010), and its functional derivatives can be applied to any of the following technologies conjugation to increase therapeutic efficacy of the peptide, as well as other conjugation technology, well-known experts in this field. Fitzpatrick et al. improved conjugated remedy against malignant tumors, using a peptide spacer between the two molecules (Anticancer Drug Design, 10: 1, 1995). Previously to increase uptake by tumor cells and activity against tumor cells methotrexate conjugatively with serum albumin human (HSA). After absorption cell some of the methotrexate is released from the conjugate by lysosomal enzymes and can then exercise their cytotoxic effects. By embedding the linker peptide of the four amino acids between methotrexate and HSA, which is easily cleaved by lysosomal enzymes, increased the level derived from the conjugated molecules active methotrexate in the cells. The peptides of the present invention can exert its effects through specific interaction with the definitely enzymes. By embedding the peptide of the present invention in the conjugate molecule as a linker segment between the drug and its molecule carrier or add to another linker segment can alter pharmacokinetics. This allows you to get the predecessor of the medicinal product, which is more resistant or more susceptible to the action of proteases, which respectively reduce or increase the rate of release of the conjugate molecules of the drug. As can be seen from the above examples conjugated funds for chemotherapy, a change that determines the speed of the delivery molecule drugs, can significantly increase the effectiveness of the drug.

Effects of drugs on specific cell can vary depending on other factors such as the level of activation of the cells or the presence of other molecular signals near or inside the cell. In some cases, to ensure that the drug had the effect requires the presence of other molecules or signals. Damjancic et al. (Exp. Clin. Endocrin., 95: 315, 1990) studied the effects of human atrial natriuretic factor (hANP) in patients with inadequate synthesis of endogenous glucocorticoids. Peptide gave the patient the m syndrome glucocorticoid therapy or subsequent resumption of therapy with the use of dexamethasone. Patients answered hANP increased diuresis and excretion of sodium only when during concomitant treatment with dexamethasone gave them a peptide hormone. Treatment by hANP when the syndrome glucocorticoid therapy had no effect. Effect of concomitant steroid hormone can also be increased activity of the peptide. According to Zhu et al. (Acta Pharm. Sinica, 28: 166, 1993) activity painkiller peptide kyotorphin (KTP) in conjugation with hydrocortisone through a short linker segment was significantly increased compared with the impact of individual peptide. No effect was found after injection of hydrocortisone separately.

The results of these studies illustrate the ability of steroid hormones as molecules for conjugation or ingredients in the product to increase the activity of biologically active peptides. Any of the peptides of the present invention can also modulate or activate via conjugation or joint application to steroid hormones. Ways Zhu et al. can be easily adapted to the conjugation of steroid molecules with the peptide of the present invention. The figures from 1 to 5 also presents illustrative phase synthesis reaction for the binding of steroid hormones with any of the peptides according to the present image is the shadow.

The above examples provide illustrative methods of increasing the usefulness and activity of any of the peptides of the present invention. Additional development in this area will help to overcome obstacles when creating based on peptides effective methods of clinical treatment. Specialists in this field it is clear that the methods, reagents and protocols developed for applications in biochemistry, pharmaceutical research and clinical trials of peptide easy to apply for any of the peptides of the present invention.

Examples

PREREQUISITES

It can be expected that some amino acids of the sequence VAPEEHPTLLTEAPLNPK (CMS-010) can be more important for biological activity than others. In some embodiments, implementation of the present invention through the discovery of the active site/sites in VAPEEHPTLLTEAPLNPK (CMS-010) can be removed from the sequence of those amino acids that are not involved in the activity of the peptide, so that the biologically active molecule may be shorter. You can also recombination of various active parts of the peptide to obtain a new peptide molecules having a modified biological activity. The shortening of the biologically active molecule peptide may have biological and economic mn is an increase. Due to the shorter sequence biological properties of peptide modified, and such modifications may have potential therapeutic benefits such as modified half-life of the organism, the affinity towards the receptor or the profile of side effects. Shorter peptides cheaper to obtain and you can reduce the cost of production.

In order to detect active sites in VAPEEHPTLLTEAPLNPK (CMS-010), the authors conducted a series of experiments on truncated. CMS-010 was subjected to truncation for each of the peptide bonds with the N-Terminus and C-Terminus. The authors suggested that, if the active area will be cut off, the biological activity of the obtained pairs of peptides will decrease, disappear, or in some way modified (activation/inactivation of biological activity). After determining the position of the active site/sites in CMS-010 through a combination of different active sites can be constructed a number of new peptides.

In experiments, the inventors have identified a number of truncated or recombinant peptides with biological activity that have the potential for therapeutic application to the human or biological applications. This series of peptides listed in the following table 1. The results of the authors, see p is imarah after the table 1.

Table 1
Truncated and recombinant peptides based on the sequence CMS-010
PeptideSequenceSEQ ID No.
CMS-010.02APEEHPTLLTEAPLNPK2
CMS-010.03VAPEEHPTLLTEAPLNP3
CMS-010.04PEEHPTLLTEAPLNPK4
CMS-010.05VAPEEHPTLLTEAPLN5
CMS-010.07VAPEEHPTLLTEAPL6
CMS-010.08VAPEEHPTLLTEAP7
CMS-010.09EHPTLLTEAPLNPK8
CMS-010.11HPTLLTEAPLNPK9
CMS-010.12VAPEEHPTLLTE10
CMS-010.13PTLLTEAPLNPK 11
CMS-010.14VAPEEHPTLLT12
CMS-010.15TLLTEAPLNPK13
CMS-010.16VAPEEHPTLL14
CMS-010.17LLTEAPLNPK15
CMS-010.18VAPEEHPTL16
CMS-010.19LTEAPLNPK17
CMS-010.20VAPEEHPT18
CMS-010.21TEAPLNPK19
CMS-010.22VAPEEHP20
CMS-010.23EAPLNPK21
CMS-010.24APLNPK22
CMS-010.25VAPEEH23
CMS-010.26PLNPK24
CMS-010.27VAPEE25
CMS-010.28LNPK26
CMS-010.29VAPE27
CMS-010.31NPK28
CMS-010.32VA29
CMS-010.103VALLT30
CMS-010.105VANPK31

Example 1

Effect of peptides on the transformation of murine T lymphocytes induced by ConAin vitro

1.1 Substance

1.1.1 Peptides

All amino acids used were in the L-form: CS Bio Co., USA

1.1.2 Control and other reagents

Saline: OTSUKA Pharmaceutical Co., Ltd, PR China. Culture medium RPMI-1640 and fetal calf serum (ETS): Gibcol Co., USA. MTT and ConA: Sigma Co., USA

1.2 Animals

Mouse BALB/c (H-2d, SPF, age 6-8 weeks, weight 18-22 g): Military Medical Academy of Science, PR China.

1.3 Method[1]

Spleen of healthy mice under aseptic conditions were identified and manually dispersible in a solution of 10% ETS RPMI-1640 with the use of needles for injection. Next dispergirovannoyj cell suspension was sieved through a stainless steel sieve with a diameter of 150 μm caliber 100. Then the suspension of spleen cells were brought to a concentration of 4×106in ml and aliquots were distributed in 96-well culture plates. The peptides were dissolved in a simple environment RPMI-1640. The scheme of distribution groups below.

Peptide group: 100 µl of the working solution of peptide + 75 μl of the suspension of spleen cells + 25 µl of the working solution of ConA.

Control group with ConA: 100 µl RPMI-1640 + 75 μl of the suspension of spleen cells + 25 µl of the working solution of ConA.

Group negative control:125 μl of RPMI-1640 + 75 ál of cell suspension of the spleen.

The final concentration of ConA in the hole was 5 µg/ml Final concentration of the peptides in the wells was 80 μg/ml, 16 μg/ml, and 3.2 μl/ml of 0.64 μg/ml and 0,128 µg/ml of Each peptide group were placed in three parallel wells, and the control group were placed in eight or twelve holes. Cells were incubated for 68 hours at 37°C, 5% CO2. To obtain values ODNmfor each hole with control at 630 nm was used MTT method on the reader ELISA.

1.4 Results

Table 2
Effect of peptides on induced by ConA transform the human murine T-lymphocyte in vitro.
GroupConcentrationNOD
CMS-010.2680 mcg/ml30,353±0,016*
CMS-010.2616 mcg/ml30,356±0,006*
CMS-010.263,2 mg/ml3of 0.332±0,015*
CMS-010.26of 0.64 mg/ml30,348±0,025*
CMS-010.260,128 mg/ml30,354±0,017*
CMS-010.10580 mcg/ml30,407±0,019*
CMS-010.105of 0.64 mg/ml30,386±0,008*
Negative control-120,134±0,011*
Control is with ConA 5 mcg/ml120,467±0,043
*: compared with positive control group with ConA, P<0,05
Table 3
Effect of peptides on induced by ConA transformation of murine T-lymphocytein vitro.
GroupConcentrationNOD
CMS-010.3280 mcg/ml30,276±0,034*
CMS-010.3216 mcg/ml30,273±0,023*
CMS-010.323,2 mg/ml30,309±0,030*
CMS-010.32of 0.64 mg/ml30,321±0,048
CMS-010.320,128 mg/ml30,306±0,033*
Negative control5 mcg/ml 8to 0.108±0,012*
Control with ConA-80,358±0,028
*: compared with positive control group with ConA, P<0,05

1.5 Conclusion

Found that the CMS-010.26, CMS-010.32 and CMS-010.105 in acceptable concentrations compared with the positive control with ConA able with statistical significance to suppress induced by ConA transformation of murine T-lymphocytein vitro(P<0,05).

Example 2

Effect of peptides on the transformation of murine T-lymphocytes and the activity of NK-cellsin vivo

2.1 Substance

2.1.1 Peptides

All amino acids used were L-form: CS Bio Co., USA.

2.1.2 Control and other reagents

Cyclosporine A: Novartis Pharma AG., Switzerland. Saline: OTSUKA Pharmaceutical Co. Ltd, PR China. Culture medium RPMI-1640 and fetal calf serum (ETS): GIBCOL, USA. MTT and ConA: Sigma Co., USA

2.1.3 Animals

Mouse BALB/c (H-2d, SPF, aged 6-8 weeks, weighing 18-22 g, 50% females and 50% males): Military Medical Academy of Science, PR China

2.2 Method

2.2.1 the Division of animals into groups and introduction

Animals were randomly divided into two groups treated with the peptide (200 μg/kg / day and 50 mg/kg per day), the group is alocasia cyclosporine A (10 mg/kg per day) and the group treated with saline (0.5 ml / day). Each group consisted of 10 mice, in which half females and half males. All the test substance was dissolved in 0.5 ml of saline and administered intraperitoneally injected once a day for 20 days. The transformation of T-lymphocytes and the activity of NK-cells was determined in the day of the last injection, immediately after injection.

2.2.2 Transformation of T-lymphocytes[1-2]

On the day of the last injection of test substance after injection mice were killed by displacement of the cervical vertebrae. Spleens were isolated under aseptic conditions and was manually dispersible in a solution of 10% ETS RPMI-1640 with the use of needles for injection. Next dispergirovannoyj cell suspension was sieved through a stainless steel sieve with a diameter of 150 μm caliber 100 and brought to a concentration of 4×106/ml of cell Suspension was added to 96-well culture plates in 100 μl per well on the next following sample.

The analyzed wells:100 μl of the suspension of cells + 100 μl ConA

Control wells:100 μl of the suspension of cells + 100 μl of RPMI-1640

For cells of each animal was intended four analytical and four control wells. The plates were incubated at 37°C, 5% CO2for 68 hours. Then MTT was added and tablets to obtain the OD at 570 nm with control at 630 nm on the reader EISA. The stimulation index SI (%) = (OD analyzed in the hole/OD control wells) × 100%.

2.2.3 Effect of peptides on the activity of NK-cells[3-5]

Target cells YAC-1 were grown to log-phase and brought up to a quantity of 1×105/ml. Cells in the spleen of mice, obtained according to the above section 2.2.2, brought up to the number of 4×106/ml and used as effector cells. The cell suspension was added to 96-well tablets the following way:

Wells with effector cells:100 μl of the suspension of spleen cells + 100 μl of RPMI-1640

Analytical wells:100 μl of the suspension of spleen cells + 100 μl of the suspension of cells YAC-1

Wells with target cells:100 μl of the suspension of cells YAC-1 + RPMI-1640

Culture the tablet to cells, cells of each animal was intended three analytical and three control wells and was intended 12 holes to target cells. The plates were incubated at 37°C, 5% CO2within 4 hours, and then MTT was added and the received values of the OD at 570 nm with control at 630 nm on the reader ELISA. The activity of NK-cells (%) = 1-[(OD in the analyzed wells - OD in wells with effector cells)/ OD in wells with target cells]×100%.

2.3 Results

2.3.1 Experiment transformation of T-lymphocytes

Table 4
The effect of the peptide on the transformation of murine T-lymphocyte(1)
GroupDosingNSI
CMS-010.2450 mcg/day102,40±0,31**
CMS-010.26200 mcg/day72,19±0,59**
CMS-010.28200 mcg/day92,70±0,37**
Saline0.5 ml/day83,63±0,69
**: compared with the group treated with saline, P<0,01

Table 5
Effect of peptides on the transformation of murine T-lymphocyte(2)
GroupDosingNSI
CMS-010.25200 mcg/kg/su the key 102,43±0,69*
Saline0.5 ml/day103,15±0,83
*: compared with the group treated with saline, P<0,05

Table 6
Effect of peptides on the transformation of murine T-lymphocyte(3)
GroupDosingNSI
CMS-010.0450 µg/kg/day81,56±0,25**
Saline0.5 ml/day92,24±0,52
**: compared with the group treated with saline, P<0,01

Table 7
Effect of peptides on the transformation of murine T-lymphocyte(4)
Group DosingNSI
CMS-010.1250 µg/kg/day102,12±0,42**
CMS-010.1450 µg/kg/day92,12±0,51**
Saline0.5 ml/day102,96±0,61
**: compared with the group treated with saline, P<0,01

2.3.2 Experiment activity of NK-cells(1)

Table 8
Effect of peptides on the activity of murine NK cells(1)
GroupDosingNThe activity of NK-cells(%)
CMS-010.24200 mcg/kg/day1069,0±7,4*
CMS-010.2450 µg/kg/day1056,0±6,0**
CMS-010.26200 mcg/kg/day967,7±5,3**
CMS-010.2650 µg/kg/day1068,5±7,2*
CMS-010.28200 mcg/kg/day970,3±6,7*
Saline0.5 ml/day1076,0±4,3
*: compared with the group treated with saline, P<0,05: **: compared with the group treated with saline, P<0,01

Table 9
Effect of peptides on the activity of murine NK cells(2)
GroupDosingNThe activity of NK-cells (%)
CMS-010.1150 µg/kg/day963,5±4,7**
CMS-010.1350 µg/kg/who ducks 1070,9±17,5*
CMS-010.14200 mcg/kg/day843,1±13,7*
CMS-010.1450 µg/kg/day975,3±9,0**
Saline0.5 ml/day1055,3±6,1
*: compared with the group treated with saline, P<0,05: **: compared with the group treated with saline, P<0,01

2.4 Conclusion

It has been found that acceptable dosing CMS-010.04, CMS-010.12, CMS-010.14, CMS-010.24, CMS-010.25, CMS-010.26 and CMS-010.28 compared with controls treated with saline, suppress the statistical significance of the transformation of murine T-lymphocytein vivo(P<0,05).

It has been found that acceptable dosing CMS-010.24, CMS-010.26 and CMS-010.28 compared with controls treated with saline, suppress the statistical significance of the activity of NK-cellsin vivo(P<0,05).

It has been found that appropriate dosing CMS-010.11, CMS-010.13 and CMS-010.14 compared with controls treated with physiological shall actor, increase the statistical significance of the activity of NK-cellsin vivo(P<0,05).

Example 3

The effect of the peptide on the formation of antibodies in micein vivo

3.1 Substance

3.1.1 Peptide

All amino acids used were L-form: CS Bio Co., USA.

3.1.2 Control and other reagents

Cyclosporine A: Novartis Pharma AG., Switzerland. Saline: OTSUKA Pharmaceutical Co. Ltd, PR China

3.1.3 Animals

Mouse BALB/c (H-2d, SPF, aged 6-8 weeks, weighing 18-22 g, 50% females and 50% males): Military Medical Academy of Science, PR China

3.2 Method

3.2.1 the Division of animals into groups and introducing a test substance

Mice were randomly divided into three groups: treated with the peptide (200 μg/kg / day), cyclosporine A (10 mg/kg per day) and saline solution (0.5 ml). Each group consisted of 12 mice, 6 females and 6 males. The test substance was dissolved in 0.5 ml of saline and administered intraperitoneally injected once a day for 20 the next day.

3.2.2 Zoom in and count the number of antibodies[7]

The sheep erythrocytes (SRBC) resuspendable in physiological solution to 2% (vol./about.) and 0.2 ml resuspending cells were administered intraperitoneally injected to each mouse at 16 days after administration of the test substance. On the day of the last injection of test substance after poslenovogodnee blood was collected from the inner angle of the palpebral fissure and left at room temperature for one hour to separate the serum. After centrifugation at 200 g for 10 minutes, the serum was diluted 200 times with normal saline.

To obtain a working solution of the complement to one volume precipitated by centrifugation SRBC was added 10 volumes of fresh Guinea-pig serum. This mixture is gently stirred for 30 minutes at 4°C. By centrifugation SRBC was removed at 200 g for 10 minutes. To obtain a working solution of complement to the supernatant was added ten volumes of normal saline.

For analysis of titer mouse antibodies 0.2 ml of 1% SRBC suspension was added to 1 ml of diluted ice mouse serum from each mouse. Then added one ml of the working solution of complement and the mixture is incubated at 37°C for 20 minutes. The reaction was stopped by cooling each sample in ice for 10 minutes. Then, to obtain supernatant samples were centrifuged at 200 g for 10 minutes. To 1 ml of this supernatant was added to 3 ml Drabkina and left at room temperature for 10 minutes and then measured

OD540nm. The OD values540nmlysis-50 in the control was determined exactly according to the procedure for sample, except for the replacement of half of the SRBC saline and without removal by centrifugation deletirovannykh SRBC. The index is varodi sample (HC 50) = OD540nmsample/OD540nmlysis-50 × 200.

3.3 Results

Table 10
The effect of the peptide on the formation of mouse antibodies.
GroupDosingNHC50
CMS-010,26200 mcg/kg/day10141,3±29,3*
Cyclosporin a10 mg/kg/day12148,9±21,7*
Saline0.5 ml/day11167,6±21,5
*: compared with the group treated with saline, P<0,05

3.4 Conclusion

It has been found that acceptable dosing CMS 010.26 compared with the control group treated with saline solution suppresses the statistical significance of the formation of antibodies in mice (P<0,05).

Example 4

Effect of peptides on the growth of transplanted mouse KM cell sarcoma S180in vivo

4.1 Materials

4.1.1 Peptides

All amino acids used were L-form: CS Bio Co., USA.

4.1.2 Control and other reagents

Saline: OTSUKA Pharmaceutical Co. Ltd, PR China. Adriamycin: Zhejiang Haizheng Pharmaceutical Co., Ltd., PR China

4.1.3 Animals

Healthy mouse KM (SPF, age 6-8 weeks, weight 18-22 g): Military Medical Academy of Science, PR China

4.2 Method

4.2.1 the Division of animals into groups, the introduction of the test substance and the implantation of tumor cells[8]

Sarcoma cells S180transplanted administered intraperitoneally to mice KM for 6-8 days and aseptic conditions were selected ascitic fluid. The cell concentration was brought to 1 × 107in ml 10% ETS RPMI-1640 and 0.2 ml of cell suspension was injected into the axilla of each mouse KM to obtain mice with sarcoma. Mice transplanted with S180randomly divided into five groups: treated with peptide (two groups: 50 mg/kg / day and 10 mg/kg/day), adriamycin (2 mg/kg per day), cyclophosphamide (40 mg/kg/day) and saline solution (0.5 ml per day). Intraperitoneal test compounds was begun on the day of the transplantation of the tumor immediately after transplantation and continued to enter once per day for 20 consecutive days.

4.2.2 Determining the development of sarcoma

In the day of the last injection of test compounds after the introduction the of the mice were removed sarcoma and weighed. The diameters of each of the sarcoma was measured with calipers in three dimensions (A, B, C). The volume of sarcoma was calculated by the formula V = (1/6)πABC. The index of inhibition of tumor growth was determined by the formula: index of inhibition of tumor growth = (tumor weight in the control group - tumor weight in the group treated) / tumor weight in the control group × 100%.

4.3 Results

Table 11
Effect of peptides on the development of transplanted into a mouse KM cell sarcoma S180in vivo(1).
GroupDosingNWeight sarcoma
CMS-010.3150 µg/kg/day171,20±1,60*
CMS-010.3110 mcg/kg/day141,05±1,28*
CMS-010.10350 µg/kg/day151,48±1,44*
CMS-010.10310 mcg/kg/day151,72±1,53*
Adriamycin2 mg/kg/day171,52±1,75*
Saline0.5 ml/day125,07±5,46
*: compared with the group treated with normal saline, P<0,05

10
Table 12
Effect of peptides on the growth of transplanted mouse KM cell sarcoma S180in vivo(2)
GroupDosingNWeight sarcomaVolume sarcoma
CMS-010.02500 µg/kg/day100,97±0,85*0,65±0,67*
CMS-010.02250 mcg/kg/day100,68±0,72*0,36±0,40*
CMS-010.03500 µg/kg/day100,33±0,35*@ 0,27±0,33*@
CMS-010.03250 mcg/kg/day100,68±0,46*0,31±0,22*
CMS-010.04500 µg/kg/day100,62±0,44*0,40±0,28*
CMS-010.04250 mcg/kg/day100,27±0,19*@0,17±0,12*@
CMS-010.05500 µg/kg/day100,47±0,29*@0,34±0,22*
CMS-010.05250 mcg/kg/day100,56±0,33*0,23±0,19*@
CMS-010.07500 µg/kg/day100,52±0,25*0,37±0,20*
CMS-010.07250 mcg/kg/day100,32±0,14*@ 0,24±0,13*@
CMS-010.08500 µg/kg/day101,05±0,64*1,01±0,63
CMS-010.08250 mcg/kg/day100,38±0,27*@0,20±0,14*@
CMS-010.09500 µg/kg/day100,85±0,70*0,84±0,84
CMS-010.09250 mcg/kg/day100,45±0,38*@0,37±0,44*
CMS-010.11500 µg/kg/day101,14±0,74*0,95±0,54
CMS-010.11250 mcg/kg/day100,64±0,31*0,63±0,40*
CMS-010.13500 µg/kg/day100,73±0,43*0,38±0,23*
CMS-010.13250 mcg/kg/day100,92±0,56*0,91±0,59
CMS-010.14500 µg/kg/day90,67±0,70*0,56±0,53*
CMS-010.14250 mcg/kg/day100,44±0,30*@0,29±0,21*
CMS-010.15500 µg/kg/day100,68±0,36*0,63±0,35*
CMS-010.15250 mcg/kg/day90,45±0,35*@0,41±0,37
CMS-010.16500 µg/kg/day100,99±0,42*0,92±0,36
CMS-010.16250 mcg/kg/day90,63±0,47*0,61±0,44*
CMS-010.17500 µg/kg/day0,91±0,46*0,55±0,34*
CMS-010.17250 mcg/kg/day90,65±0,41*0,40±0,24*
CMS-010.18500 µg/kg/day90,59±0,48*0,58±0,42*
CMS-010.18250 mcg/kg/day90,44±0,31*@0,27±0,20*@
CMS-010.19500 µg/kg/day90,68±0,68*0,40±0,42*
CMS-010.19250 mcg/kg/day100,41±0,45*@0,46±0,58*
CMS-010.20500 µg/kg/day100,59±0,46*0,54±0,51*
CMS-010.20250 mcg/kg/day91,00±0,76* 0,88±0,77
CMS-010.21500 µg/kg/day100,44±0,22*@0,44±0,21*
CMS-010.21250 mcg/kg/day100,51±0,29*0,44±0,22*
CMS-010.22500 µg/kg/day100,85±0,73*0,73±0,87
CMS-010.22250 mcg/kg/day100,28±0,12*@0,24±0,08*@
CMS-010.23250 mcg/kg/day90,27±0,18*@0,21±0,15*@
CMS-010.24500 µg/kg/day101,20±0,79*0,62±0,47*
CMS-010.24250 mcg/kg/day100,61±0,39*0,36±0,30*
CMS-010.25500 µg/kg/day100,52±0,38*0,26±0,21*@
CMS-010.25250 mcg/kg/day90,65±0,53*0,48±0,37*
CMS-010.27500 µg/kg/day91,05±0,86*0,51±0,33*
CMS-010.27250 mcg/kg/day100,78±0,68*0,58±0,58*
CMS-010.29500 µg/kg/day100,55±0,41*0,40±0,11*
CMS-010.29250 mcg/kg/day101,24±0,72*1,02±0,66
CMS-010.31500 µg/kg/day100,78±0,89*0,43±0,50*
CMS-010.31250 mcg/kg/day 100,27±0,19*@0,25±0,20*@
CMS-010.32500 µg/kg/day100,41±0,35*@0,40±0,31*
CMS-010.32250 mcg/kg/day100,38±0,24*@0,25±0,14*@
Cyclophosphamide40 mg/kg/day101,07±0,80*0,76±0,66*
Saline0.5 ml/day101,87±0,521,20±0,28
*: compared with the group treated with normal saline, P<0,05
@compared with the group treated with cyclophosphamide P<0,05

4.4 Conclusion

It has been found that acceptable dosing CMS-010.103, CMS-010.02, CMS-010.03, CMS-010.04, CMS-010.05, CMS-010.07, CMS-010.08, CMS-010.09, CMS-010.11, CMS-010.13, CMS-010.14, CMS-010.15, CMS-010.16, CMS-010.17, CMS-010.18, CMS-010.19, CMS-010.20, CMS-010.21, CMS-010.22, CMS-010.23, CMS-010.24, CMS-010.25, CMS-010.27, CMS-010.29, CMS-010.31 and CMS-010.32 compared with group received the Shih normal saline suppress the statistical significance of the development of transplanted into a mouse KM cell sarcoma S 180in vivo(P<0,05).

Example 5

Effect of peptides on jade Masugi in rabbits

5.1. Substance

5.1.1 Peptides

All amino acids used were L-form: CS Bio Co., USA.

5.1.2 Control and other reagents

Dexamethasone sodium phosphate injection: Tianjin Jinyao Aminophenal Ltd., PR China. Saline: OTSUKA Pharmaceutical Co. Ltd., PR China. BCG vaccine: Beijing Institute of Biological Products, PR China. Lanolin: Tianjin sixth chemical product factory, PR China. Liquid paraffin: Tianjin sixth chemical product factory, PR China. Diagnostic reagent for serum BUN: BECKMAN 443350, USA. Diagnostic reagent for serum creatinine: BECKMAN 443340, USA.

5.1.3 Animals

One male sheep (aged 8 months)Department of Laboratory Animal, Tianjin Medical University, PR China. Rabbits (MDA, males, 2-2,5 kg): Beijing Fuhao Breed Farm, PR China.

5.2 Methods[9-10]

5.2.1 Getting the sheep antisera against the cortex of the kidney of the rabbit

5.2.1.1 Getting antigen against the cortex of the kidney of the rabbit

A healthy rabbit was anestesiologi 25% urethane in the amount of 4 ml/kg by intravenous injection into the ear vein and systemic heparinisation was intravenously injected heparin in the amount of 1250 U/kg Stomach rabbit dissected under aseptic conditions and expose the renal artery and vein. Renal artery was catheterizable and crossed renal veins. The kidneys were washed in saline solution until the kidney will not be gray. Then the kidneys were removed. The cortex of the kidney was separated and homogenized in 0.5 volume of ice-cold saline, and then kept at -20°C.

5.2.1.2 Obtaining antisera sheep

7.5 ml of the homogenate of kidney cortex were mixed with 2.5 ml complete adjuvant's adjuvant (lanolin in liquid paraffin in a ratio of 1:5, with 5 mg/ml of BCG vaccine). After complete emulsification antigen was administered to sheep in five different areas of the back, and 1 ml of the area once in two weeks, only three series of injections. Since the fourth immunization, homogenized 5 g of the cortex of the kidney with one volume of saline and was injected sheep intramuscularly into 5 areas, 1 ml of the area once in two weeks. The titer of sheep antibodies against rabbit was measured every two weeks by double immunodiffusion. When the titer reached 1:32, anticigarette sheep was collected from the carotid artery. Sheep anticigarette was mixed with an equal volume of rabbit erythrocytes and removal of antibodies to rabbit erythrocytes were kept at 4°C for 12 hours. Then for inactivation of complement and proteases anticigarette were collected by centrifugation and kept at 56°C. Anticigarette kept at -20°C.

5.2.2 the Division of animals into groups, the introduction of a test substance and a model jade Masugi

Twenty-two healthy rabbit randomly divided into 5 groups: alocasia peptide (107,3 µg/kg per day and 58.5 mg/kg per day, 4 rabbits per group), dexamethasone (0.1 mg/kg per day, 4 rabbit), saline (1 ml / day, 7 rabbits) and normal healthy (3 rabbits). 24 hours before creating a painful condition in rabbits was carried out by measurement of serum BUN, creatinine and protein in the urine. In the absence of abnormalities on these indicators in experimental animals has created a model jade Masugi by intravenous injection of sheep antisera against the cortex of the kidney rabbit ear vein, 0.5 ml per injection, one injection every 30 minutes, for only 4 injections in the rabbit. The group of normal healthy control rabbits were injected with saline in the same way. Intravenous administration of test compounds through the ear vein was started in the last day administration of antisera after injection, once a day, 1 ml injection; during the following 30 days.

5.2.3 Monitoring therapeutic effect

5.2.3.1 determination of the quantity of protein in the urine

The urine of each rabbit was collected over a 24-hour period once a week and by a method using sulfosalicylic acid was determined amount of protein.

5.2.3.2 Anatomo-pathological examination

To observe the effects of the peptides on the excretion of IgG antibodies sheep against the bark of rabbit kidneys of rabbits with nepriemyshi in the day of the last injection of the substance after the introduction of rabbits were killed by strangulation and removed the right kidney, froze, did a cut and then were labeled immunofluorescent label for the presence of IgG sheep. Thought fluorescently-positive region of each glomeruli. Researched thirty glomeruli on rabbit and calculated the average number of positive areas on glomeruli.

5.2.3.3 Statistics.

Statistical significance was determined by t-test software SPSS.

5.3 Results

Table 14
The effect of the peptide on the excretion of IgG antibodies sheep against the bark of rabbit kidneys of rabbits with jade Masugi
GroupThe dosage per dayNThe number of positive fields on glomerulo
CMS-010,26107,3 ág/kg55,6±1,2**
CMS-010,2658,5 ág/kg46,3±1,4**
Dexamethasone0.1 mg/kg47,5±1,0
Receiving fisiologicas the th solution 0.5 ml78,7±0,9
Normal, healthy-3-
**: compared with the group treated with normal saline, P<0,01

5.4 Conclusion

Found that the CMS-010.26 compared with the control group treated with saline solution capable with statistical significance reduce the severity of proteinuria and to encourage the breeding of sheep antibodies against the bark of rabbit kidneys in rabbits with jade Masugiin vivo, P<0,05.

Example 6

Effect of peptides on rats with jade Heimanin vivo

6.1 Substances

6.1.1 Peptide

All amino acids used were L-form: CS Bio Co., USA.

6.1.2 Control and other reagents

Injection dexamethasone sodium phosphate: Tianjin Jinyao Aminophenal Ltd., PR China. Saline: OTSUKA Pharmaceutical Co. Ltd., PR China. BCG vaccine: Beijing Institute of Biological Products. Lanolin: Tianjin sixth factory of chemical product, PR China. Liquid paraffin: Tianjin sixth factory of chemical product, PR China. Diagnostic reagent for serum BUN: BECKMAN 443350, USA. Diagnostic reagent for serum creatinine: BECKMAN 443340, USA.

6.1.3 Animals

Wistar rats (SPF, aged 6-8 weeks, weighing 150-200 g): Beijing Vital River Laboratory Animal Co., Ltd., PR China.

6.2 Methods[11-12]

6.2.1 Obtaining homogenate of the rat renal

The stomachs of healthy Wistar rats were dissected under aseptic conditions. Expose the portal vein and the inferior Vena cava. The portal vein was catheterizable and crossed the inferior Vena cava. The kidneys were washed in saline solution until such time as the cortex of the kidney was not grey. Kidneys were removed and separated the cortex of the kidneys. Then the cortex of the kidney homogenized in ice and kept at -20°C.

6.2.2 Obtaining antigen cortex of the kidney

Lanolin mixed with liquid paraffin in a ratio of 1:2 vol./about, was heated to 70°C under stirring and autoclaved. For complete adjuvant's adjuvant to a mixture of lanolin/paraffin was added sufficient to obtain a concentration of 3 mg/ml the number of BCG vaccine. The homogenate of the cortex of the kidneys full of beta-blockers and saline were mixed in the ratio 1:1:2 in a mortar until complete emulsification.

6.2.3 the Division of animals into groups and getting models

20 healthy Wistar rats were randomly divided into two groups: treated with the peptide (200 μg/kg per day) and saline (2 ml / day). Two ml of antigen was administered intraperitoneally administered to each rat once every two weeks, only 5 cycles introduction. The introduction of test compounds through intraperitoneally injection began in the Yan third immunization after immunization, once a day until the end of the experiment.

6.2.4 Monitoring effectiveness

6.2.4.1 determining the amount of protein in the urine

Determining the amount of protein in the urine began in the third week of the creation model. Urine samples were collected from each rat during a 24-hour period of time once every two weeks, and protein content was determined in urine by the method using sulfosalicylic acid.

6.2.5 Statistics

Values for each sample were compared by t-test using the software SPSS.

6.3 Results

Table 15
The effect of the introduction of the peptide at the concentration of protein in the urine (mg/DL) within 24 h in rats with jade Heiman
GroupNWeek 3Week 5Week 7Week 9
CMS-010.26106,2±2,33,1±1,43,9±1,6*3,8±2,2*
Receiving saline104,1±1,3 3,1±0,810,9±2,812,6±1,2
*: compared with the group treated with saline, P<0,05

6.4 Conclusion

It has been found that acceptable dosing CMS-010.26 compared with the group treated with physiological solution was reduced with statistical significance of the severity of proteinuria in rats with jade Heimanin vivo(P<0,05).

Links to examples 1-6

1. Shuyun Xu, Rulian Bian, Chen Xiu. Methodology of pharmacological experiment. People's Health Publishing House. 2002, 1:1426-1428.

2. Principles of Pre-clinical Research of New Drugs, People's Republic of China. 1993, 7:134-135.

3. Shuyun Xu, Rulian Bian, Chen Xiu. Methodology of pharmacological experiment. People's Health Publishing House. 2002, 1:1429.

4. Jinsheng He, Ruizhu Li Tingyi Zong. The study on MTT reduction method of testing NK cell activity. China Immunology Journal. 1996, 1(6):356-358.

5. Principles of Pre-clinical Research of New Drugs, People's Republic of China. 1993, 7:128-129.

6. Yuanpei Zhang, Huaide Su. Pharmacological experiment (second edition). People's Health Publishing House. 1998, 137-138.

7. Shuyun Xu, Rulian Bian, Chen Xiu. Methodology of pharmacological experiment. People's Health Publishing House. 2002, 1:1429.

8. Principles of Pre-clinical Research of New Drugs, People's Republic of China. 1993, 7:137-139.

9. Principles of Pre-clinical Research of New Drugs, People's Republic of China. 1993, 7:96.

10. Shuyun Xu, Rulian Bian, Chen Xiu. Methodology of pharmacological experiment. People's Health Publishing House. 2002, 1:1227-1228.

11. Principles of Pre-clinical Research of New Drugs, People's Republic of China. 1993, 7:97.

12. Shuyun Xu, Rulian Bian, Chen Xiu. Methodology of pharmacological experiment. People's Health Publishing House. 2002, 1:1227.

b> Example 7

Delivery of peptides obtained by means of genetic engineering of bacterial speciesLactobacillus

The following is an illustrative method of delivery of the peptides according to this invention to the owner, as described above. The DNA sequence encoding a peptide selected from the group consisting of fragments CMS-010 (VAPEEHPTLLTEAPLNPK) (where these fragments do not contain the sequence CMS-010) and their functional derivatives, synthesized by chemical methods and the DNA sequence was built in expressing vector using well-known specialists in the field of standard methods of genetic engineering. Selected expressing vector contains a constitutive functional inLactobacillithe promoter, polylinker for integration of DNA sequences in a particular orientation from 5' to 3', as well as the selective marker gene, which confers resistance to the antibiotic (to facilitate cloning procedures) and may contain other sequences, contributing to the generation and/or secretion of peptides, such as sequences of signal peptides. An example of such a vector are presented in U.S. patent No. 5592908 issued by Pavla, which is included here by reference in full. In summary, this patent describes several promoters, f is nctioning in the species Lactobacillusand how the development of new promoters in these bacteria, any of which expression of the peptide inLactobacillimay be functionally linked to a nucleic acid that encodes a peptide of the present invention. Nucleic acid encoding a signal peptide, such as peptides that are active in theLactobacillus lactiscontaining from 16 to 35 mostly hydrophobic amino acids described in the above U.S. patent No. 5529908, was built between the promoter and the nucleic acid that encodes a peptide of the present invention so that encodes the signal peptide nucleic acid were in the same reading frame with a nucleic acid that encodes a peptide of the present invention.

In addition to encoding the peptide sequence of synthesized DNA sequence may include sequences that facilitate legirovanie and cloning of a specified DNA expressing vector. For example, the recognition sites of the restriction enzyme, which correspond to the plots in polylinker vector, can be embedded into the synthesized DNA with 5' and 3' ends of the sequence, so the sequence can be cloned into the vector in the correct orientation. And vector, and the synthesized DNA was digested by specific enzymes, and setimagescale. After reactions ligation of vector and synthesized DNA was carried out by transformation into an acceptable strain ofE. Coli. Transformed bacteria were placed in a medium containing the antibiotic to which the vector confers resistance. The colony of transformed bacteria were selected for cultivation and procedures for obtaining plasmids; confirmed the presence of synthesized DNA in the correct orientation.

Then expressing this vector is transformed into a bacterial cell hostLactobacillussuch asL. acidophilus. Transformed cells were selected on the basis of in sequence vector of the selective marker, and secretion of the peptide can be confirmed by carrying out Western blot, conducting gel electrophoresis of peptides in the culture medium, or other standard methods. Chose transformed colony of bacteria and used to derive large-scale cultures, obtained by means of genetic engineering of bacteria. The culture obtained by means of genetic engineering of bacteria expressing the desired peptide, cultivated, and at least some of them were introduced into the digestive tract, the vagina, the trachea or another area of the host organism, in which bacteria are able to replicate. If desirable, a bacterial culture can about abacinate different ways to obtain an additive for enteral consumption by the owner. These treatments include lyophilization or other methods of preservation of bacteria, in addition to combining bacteria with media, such as solutions, solvents, dispersion media, moderators, emulsions and similar to them. The use of these funds for getting supplements well known in this field. For example, bacteria can be used to obtain dairy products or other foods for human consumption, so that the organism expressing the peptide, colonized the intestine of the host organism. A number of different ways to include a specific strains of lactic acid bacteria in the composition of foods, such as yogurt, kimchi, cheese and butter, are described in U.S. patent No. 6036952 issued by Oh, which is included here by reference in full. When the consumption of bacteria by any number of methods obtained by means of genetic engineering organisms can colonize the intestine and contribute to the provision and/or absorption of peptides according to this invention through the mucus layer of the intestine.

Example 8

Delivery of peptides obtained by means of genetic engineering formsBacillus subtilis

The following applies to another illustrative method of delivery specified above, the owner of the peptides according to this invention. The DNA sequence, codereuse the peptide, selected from the group consisting of fragments CMS-010 (VAPEEHPTLLTEAPLNPK) (where these fragments do not contain the sequence CMS-010), and their functional derivatives, synthesized by chemical methods, and the DNA sequence was built in expressing vector by methods of genetic engineering, where all the methods known in this field. Selected expressing a vector containing the Shuttle vector, such as pTZ18R (Pharmacia, Piscataway, NJ), are able to multiply andE. Coliand inB. Subtilisand containing the gene for resistance to the antibiotic for selection of transformed colonies of bacteria. This vector may contain a constitutive promoter, active in theB. subtilissuch as a promoter derived from a gene Sac BB. subtilisand the nucleotide sequence encoding a signal peptide that is active in theB. subtilisthat provides an effective export expressed heterologous protein from the bacterial cells. An example of such a vector is described in U.S. patent No. 6268169 issued Fahnestock, the description of which is included here by reference in full. In summary, as described above, DNA encoding the peptide according to this invention, was synthesized with sites for enzymes and/or other sequences to facilitate cloning DNA by methods known to experts in D. the authorized area. After transformation inE. Coli, sowing on a Petri dish, breeding and propagation of plasmids to obtain plasmid material plasmid transformed inB. Subtilisand transformed cells were selected on the basis of resistance to the antibiotic in the culture medium in a Petri dish.

The products of the peptide obtained by means of genetic engineeringB. subtilisand secretion obtained by means of genetic engineeringB. subtilisconfirmed using methods well known to specialists in this field, such as the incorporation of radioactive label into peptides for autoradiographical definition after the analysis of SDS-PAGE or Western blotting.

The culture obtained by means of genetic engineering of bacteria were grown and at least part of them were introduced into the digestive tract, the vagina, the trachea or other areas of the host organism, in which bacteria are able to replicate.

Example 9

Delivery of peptides obtained by means of genetic engineering of species of the yeastSaccharomyces

The following applies to another illustrative method of delivery of the peptides according to this invention described above master. The DNA sequence of such a peptide selected from the group consisting of fragments CMS-010 (VAPEEHPTLLTEAPLNPK) (where these fragments do not contain the sequence is CMS-010), and its functional derivatives synthesized by chemical methods and the DNA sequence was built in expressing vector by methods of genetic engineering, where all the methods known in this field. Selected expressing vector contains stably supported yeast expressing proteins a vector containing a constitutive yeast promoter, such as pADHl, sites for replication of the vector in yeast and inE. Coli, the gene or genes for breeding give the ability of auxotrophic yeast mutants to protocolname the type of food, polylinker (MCS) and, if desirable, encoding the signal peptide sequence. Vectors, such as this one, commercially available and well known in this field or can be easily constructed using standard methods. After embedding the synthesized DNA into a yeast vector, transformation inE. Coligrowing on a Petri dish transformedE. Colion the selective medium, selection of transformed bacterial colonies and obtain plasmid DNA grown from the selected colonies of bacteria cultures vector transformed inSaccharomyces cerevisiaeby well known methods, such as transformation with lithium acetate or electroporation. Selected for transformation strain ofSaccharomyces cerevisiae is an auxotrophic mutant strain, which for growth on plates with minimal medium requires the presence of a gene in the plasmid. Transformed yeast colonies were isolated by growing the yeast in the cups on the growing medium, devoid of the gene product located on the vector. Only those yeast that has received vector and the gene for breeding and Express the product of this gene are able to grow into colonies on minimal medium. Confirmation of the secretion of the peptide can be obtained by conducting a Western blot, after gel electrophoresis of peptides in the culture medium, or other standard methods.

A transformed colony of yeast was selected and used to obtain large-scale cultures. The culture obtained by means of genetic engineering of yeast expressing the desired peptide, cultivated, and at least part of it was introduced into the digestive tract, the vagina, the trachea or other areas of the host organism, in which bacteria are able to replicate. If desirable, yeast culture can be processed in various ways to obtain an additive for enteral consumption by the owner. These treatments include lyophilization or other methods of preservation of yeast, in addition to combining bacteria with media, such as Rast is ora, solvents, dispersion media, moderators, emulsions and similar to them. The use of these funds for getting supplements well known in this field. In another embodiment, the transformed yeast was used to create food products such as fermented milk products such as yogurt or kefir, by methods known to experts in this field. As in the case with live lactic acid bacterial cultures, such foods transformed yeast colonizing the intestine, at least temporarily, and serve for the presentation of peptides to the owner in the intestinal lumen.

Example 10

The direction of the peptide in a specific area

The following applies to an illustrative method for selective delivery of a peptide according to this invention in a specific Department, organ, cell type or organism. In this case, the cell-proliferative violation was treated by peptide selected from the group consisting of fragments CMS-010 (VAPEEHPTLLTEAPLNPK) (where these fragments do not contain the sequence CMS-010), and its functional derivatives in the kidneys of the individual. For example, fragments CMS-010 (VAPEEHPTLLTEAPLNPK) (where these fragments do not contain the sequence CMS-010) and their functional derivatives are covalently linked by means known in the Noah chemical reactions, with low molecular weight (LMW) lysozyme, commercially available protein site, focusing specifically in the renal tissue. Ways to achieve conjugation of LMW molecules with lysozyme as described in the documentation (Folgert et al., Br. J. Pharmcology, 136: 1107, 2002). The main methods of conjugation of proteins or peptides with each other is also indicated in the literature in this area (Fischer et al., Bioconj. Chem., 12: 825, 2001). The newly obtained conjugated sample of the peptide was then purified from the one used for the process of conjugation of chemicals by methods of chromatography, such as cation-exchange FPLC, and/or gradient centrifugation. After cleaning conjugated peptide was administered to an individual in need of treatment of cell-proliferative disorders of the kidneys. For their antiproliferative activity action fragments CMS-010 (VAPEEHPTLLTEAPLNPK) (where these fragments do not contain the sequence CMS-010) and their functional derivatives are preferably directed in the renal tissue by binding between them and LMW lysozyme, which is selectively concentrated in the renal tissue due to the affinity of LMW lysozyme to the cells of the proximal tubules of the kidney. Such preferred shipping can achieve greater antiproliferative effect compared with the effect of an equivalent number of moles of individual fragments CMS-010 (VAPEEHPTLLTEAPLNPK) (g is e these fragments contain a sequence CMS-010) and their functional derivatives. On the contrary, it can reduce the number of peptide drugs required to achieve a certain level of antiproliferative activity.

Example 11

The increase in the delivery of the peptide to the field of activity

The following are presented as illustrative of the method of increasing delivery of neuroactive peptide in the brain. The peptide of the present invention, which has its own impact on the neurons of the brain, synthesized well-known specialists in the field of chemical methods. Alternatively, it can be expressed in obtained by means of genetic engineering of microorganisms and isolated from the culture of these organisms, as detailed in the above examples. After getting in purified form of the peptide used in a number of organic chemical reactions to obtain a conjugate with triglyceride complex ether group attached to the peptide. Conjugated group consists of Quaternary substituted carbon center connected with the peptide of the present invention through an amide bond with the C-terminal carbon of the peptide. The other three groups, combined with a Quaternary carbon center, consist of carbon of ester groups with consisting of 16 carbon residue of the fatty acid chains. Themselves-chain fatty acids C is cancelaura terminal dipeptide group, known as a peptide mask, which makes the circuit more hydrophilic and direct them specifically to endothelial cells of the blood-brain barrier. The procedure of synthesis described in Patel et al., Bioconjugate Chem., 8(3): 434, 1997, and it uses the usual reagents and equipment, well-known experts in this field.

After the introduction of the individual in the peripheral region of the connection moves through the body via the circulatory system, interacting with endothelial membranes of the blood-brain barrier. Sequential degradation of the dipeptide of the mask and the lipid chains in the process of transport of molecules across the epithelial layer of the blood-brain barrier leads to the release of the peptide of the present invention in the area of the brain. There the peptide can interact with receptors on the surface of neurons to implement its impact on brain function. The time required to achieve drug brain with concomitant degradation of the media, changes the kinetics of activity of the medicinal product, creating a more stable and longer-lasting compared to intracerebroventricularly the introduction of the free peptide.

Example 12

Obtaining preparations of a peptide resistant to enzymatic degradation

The following applies to the sludge is istratively a method for preparation of biologically active peptide for oral administration, resistant to the activity of proteases and peptidases located throughout the surface of the digestive tract. In this example, the peptide selected from the group consisting of fragments CMS-010 (VAPEEHPTLLTEAPLNPK) (where these fragments do not contain the sequence CMS-010), and its functional derivative is used to produce a pharmaceutical preparation for oral administration to a patient. As described in Larionova et al. (Int. J. Pharma. , 189: 171, 1999), the peptide was used to obtain microparticles with soluble starch and protease inhibitor by Aprotinin, which is a strong inhibitor of many secreted into the lumen and membrane-bound brush edges of proteases. In summary, soluble starch, protease inhibitor Aprotinin and the peptide of the present invention was dissolved in aqueous buffer. The ratio of soluble starch, Aprotinin and peptide were determined by experimental methods, known to experts in the field; for example, to determine the ratio and get the most effective conditions for protein Larionova et al. in their study used the analysis to stimulate digestionin vitro. The aqueous solution was emulsiable under mechanical stirring in cyclohexane (1:3 ratio.vol.), containing 5% Span-80 and the nonionic surfactant. The solution of chloride of terephthaloyl in chloroform was added to the emulsion and others who have continued the stirring for 30 minutes, during which molecules of starch transversely sewn together with Aprotinin and peptide. Microparticles obtained in this process, washed successively with cyclohexane, 95% ethanol with 2% vol./about. the detergent Tween 85, 95% ethanol and water. Microparticles resuspendable in water and liofilizirovanny. Liofilizirovannoe connection can be placed in gelatin capsules for oral delivery to an individual in need of treatment.

After oral administration, the compound is released after the dissolution of the gelatin capsule. Microparticles are dissolved in the small intestine under the action of α-amylase on the starch molecules, which leads to a gradual release of Aprotinin and peptide according to this invention. Simultaneous strong release of protease inhibitor Aprotinin at the same time and in the same place as the peptide reduces the enzymatic degradation of peptide and increases the proportion of intact peptide available for absorption through the membrane of the intestine.

Although the present invention has been described with application in some cases, the above methods and data and specific examples of peptide fragments CMS-010 (VAPEEHPTLLTEAPLNPK) and its functional derivatives, it is clear that this is only an example and should not understand this as a limitation of the present invention. Also it should be clear that the fragments CMS-010 (VAPEEHPTLLTAPLNPK) (where these fragments do not contain the sequence CMS-010), and their functional derivatives represent specific embodiments of the present invention, and the above principles of the present invention can also be applied to other functionally equivalent peptides modified without disrupting the biological function of the fragments CMS-010 (VAPEEHPTLLTEAPLNPK) (where these fragments do not contain sequence CMS-010) and their functional derivatives. For example, the equivalents of the peptide fragments CMS-010 (VAPEEHPTLLTEAPLNPK) (where these fragments do not contain sequence CMS-010) and their functional derivatives include peptide fragments that have conservative amino acid substitutions (i.e. any of the V, A, P, E, H, L, A, N, K or T is substituted by another amino acid, with the remainder of the same biochemical type, such as hydrophobic, hydrophilic, positively or negatively charged groups). Another example of a peptide equivalent peptide fragments CMS-010 (VAPEEHPTLLTEAPLNPK) (where these fragments do not contain the sequence CMS-010) and their functional derivatives, is slightly longer peptide, such as peptide, extended by one or two amino acids, which stores the above-mentioned types of biological activity. In addition, although the above disease or disorder for therapeutic use of the fragm the tov CMS-010 (VAPEEHPTLLTEAPLNPK) (where these fragments do not contain sequence CMS-010) and their functional derivatives specifically related to cell-proliferative and immune disorders and/or diseases, these therapeutic applications use only as non-limiting examples, and they are not intended to limit the scope of the claims. It is clear that there are other potential/alleged use of fragments CMS-010 (VAPEEHPTLLTEAPLNPK) (where these fragments do not contain the sequence CMS-010) and their functional derivatives, such as use as additives for therapeutic feeding for modulating the immune system of a healthy individual or a patient with any of the immune and/or cellular proliferative disorders or diseases. Any of these applications is also in the scope of the present invention.

1. Essentially pure peptide to modulate the immune system, characterized by amino acid sequence SEQ ID NO:24.

2. The peptide according to claim 1, where the specified modulation of the immune system is an activity selected from the group consisting of suppressing transformation of T-lymphocytes, inhibit the activity of NK-cells and suppression of antibody formation.

3. Pharmaceutical composition for modulation of the immune system, containing essentially pure peptide consisting of the amino acid sequence of SEQ ID NO:24, and a pharmaceutically acceptable carrier.

4. The pharmaceutical composition according to claim 3, where the specified modulation of the immune system is the th modulation, selected from the group consisting of suppressing transformation of T-lymphocytes, inhibit the activity of NK-cells and suppression of antibody formation.

5. A method of obtaining a pharmaceutical composition for modulation of the immune system that obtains a peptide consisting of the amino acid sequence of SEQ ID NO:24, and mixing the indicated peptide with a pharmaceutically acceptable carrier.

6. The use of essentially pure peptide consisting of the amino acid sequence of SEQ ID NO:24 in the manufacture of drugs for the modulation of the immune system.

7. The use according to claim 6, where the specified modulation selected from the group consisting of suppressing transformation of T-lymphocytes, inhibit the activity of NK-cells and suppression of antibody formation.



 

Same patents:

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FIELD: medicine.

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FIELD: chemistry; biochemistry.

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FIELD: medicine.

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61 cl, 5 ex, 2 tbl, 12 dwg

FIELD: chemistry.

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9 cl, 14 dwg, 18 ex

FIELD: chemistry; medicine.

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14 cl, 18 dwg, 1 tbl, 10 ex

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12 cl, 4 tbl, 4 ex

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30 cl, 1 tbl, 14 dwg

Thrombopoietin // 2245365

FIELD: medicine, molecular biology, polypeptides.

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EFFECT: valuable medicinal properties of polypeptide.

21 cl, 92 dwg, 14 tbl, 24 ex

FIELD: biotechnology, molecular biology, medicine, genetic engineering, pharmacy.

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22 cl, 19 dwg, 18 tbl, 117 ex

FIELD: gene engineering, in particular purification and isolation of polynucleotides.

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52 cl, 14 dwg, 3 tbl

FIELD: chemistry of peptides, medicine.

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14 cl, 5 tbl, 5 dwg, 9 ex

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FIELD: organic chemistry, amino acids.

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EFFECT: valuable biological properties of compounds.

9 cl, 2 tbl, 1 ex

FIELD: food processing industry.

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16 cl, 8 ex

FIELD: biotechnology, veterinary science.

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EFFECT: valuable properties of factors.

35 cl, 9 dwg, 4 tbl, 4 ex

FIELD: biotechnology, molecular biology, medicine.

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EFFECT: valuable biological, medicinal and veterinary properties of polypeptide.

23 cl, 71 dwg, 12 tbl, 17 ex

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