New biologically active peptides and their new application

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to the application of a biologically active peptide which represents the amino acid sequence SEQ ID No.1.

EFFECT: preparation of a drug for modulation of at least one of the following conditions: fatigue, liver glycogen level and blood lactic acid level.

30 tbl, 14 ex

 

Prior art

The technical field to which the invention relates

The present invention relates to short peptides and their application. In particular, the present invention relates to short peptides with biological activity.

A description of the associated level of technology

In the art known peptides for the treatment of diseases and in the form of pharmaceutical compositions. For example, in U.S. patent No. 6191113 disclosed peptide with inhibitory activity against the growth of smooth muscle cells and, therefore, applicable to the prevention and treatment of pathological conditions associated with the growth of smooth muscle cells, such as atherosclerosis, restenosis after angioplasty, stenosis of the lumen after transplantation, blood vessel and smooth muscle sarcoma. Patent No. USA 6184208 reveals another peptide as detected modulating physiological processes, such as the activity of the epithelial growth area in relation to weight gain and hair growth. Moreover, PCT publication WO 03/006492 and patent application U.S. No. 10/237405 suggested that certain peptides and their pharmaceutical compositions are biologically active and capable of modulating immune responses.

Therefore, the aim of the present invention is the provision of a short peptide Il the peptides, possessing biological activity.

A brief statement of the substance of the invention

Peptides in accordance with the present invention individually synthesized using standard chemical methods. Using various tests on animals and in vitro to analyze various biological functions of these peptides using methods described in the following references, as described below. The names of the peptides are given with the code “CMS”followed by a number. The peptide sequence and the corresponding ID numbers presented in table 1. In total, identified seven new peptides having biological activity in vivo, and they are indicated by an asterisk after the ID sequence numbers. Other peptides have been previously described in WIPO publication WO 03/006492, but new and inventive new signs were found in the present invention. For ease of reference sequences, which are found in the publication WO 03/006492, given the same ID number sequence.

Pro Thr Thr Lys Thr Tyr Phe Pro His Phe
Table 1
List ID No. sequencesThe title peptideThe peptide sequence
1CMS-001
2CMS-017Ile-Val-Thr-Asn-Thr-Thr
3CMS-008Lys Ala Val Gly His Leu Asp Asp Leu Pro Gly Ala Leu
4*CMS001.30Pro-Thr-Thr-Lys-Thr-Tyr
5*CMS001.31Pro-Thr-Thr-Lys-Thr-Tyr-phe-Pro
6No sequence
7CMS-014Ala Ala His His Pro Asp Asp Phe Asn Pro Ser Val
8*CMS024.04Tyr-Ser-Nle, where Nle=norleucine (2-aminocaproic acid)
9*CMS024.05Tyr-Thr-Val
10*CMS024.14(3,5-dibromo-Tyr-Ser-Leu
11*CMS024.16Leu-Tyr-Ser
12No sequence
No sequence
14No sequence
15CMS-023Ala Ala Phe
16No sequence
17No sequence
18No sequence
19No sequence
20No sequence
21CMS-030Phe Glu Glu Met

Accordingly in one aspect the present invention relates to essentially pure peptides having the sequence identified as SEQ ID No.4, 5, 8-11. Thus, the present invention also relates to essentially pure peptide comprising amino acid sequence selected from the group consisting of the C SEQ ID No.4, 5, 8-11. It also relates to essentially pure peptide consisting essentially of the amino acid sequence selected from the group consisting of SEQ ID No.4, 5, 8-11. The invention also relates to essentially pure peptide consisting of the amino acid sequence selected from the group consisting of SEQ ID No.4, 5, 8-11. In a specific implementation, the peptides can modulate, but are not limited to modulation, one or more of the following: fatigue, malnutrition, metabolic disorders, lipid metabolism disorders, the activity of the immune system, the effects of radiation, hepatitis, rejection of transplanted organ, the growth of the cancer and appetite.

In a more preferred implementation of the peptides can modulate, but are not limited to modulation of one or more of the following conditions: supply of glycogen in the liver, levels of lactic acid in the blood induced by irradiation of immunodeficiency, infectious hepatitis B, transplant rejection, skin, liver cancer, stomach cancer, inflammation, lipids, triglycerides and total blood cholesterol, hypersensitivity of the immune system, proliferation of T-lymphocytes, mixed lymphocyte proliferation, obesity and increased in comparison with normal body mass.

In another aspect, the present invention relates to essentially pure Pat the ladies, which are the functional derivatives of the peptides having the sequence ID No.4, 5, 8-11. Thus, the present invention also relates to essentially pure peptide comprising the amino acid sequence which is a functional derivative of a biologically active peptide, and this biologically active peptide has an amino acid sequence selected from the group consisting of SEQ ID No.4, 5, 8-11. It also relates to essentially pure peptide consisting essentially of the amino acid sequence which is a functional derivative of a biologically active peptide, and this biologically active peptide has an amino acid sequence selected from the group consisting of SEQ ID No.4, 5, 8-11. The invention also relates to essentially pure peptide consisting of the amino acid sequence, which is a functional derivative of a biologically active peptide, and this biologically active peptide has an amino acid sequence selected from the group consisting of SEQ ID No.4, 5, 8-11. In a particular implementation of the peptides, which is the functional derivative, can modulate, but are not limited to modulation, one or more of the following: fatigue, malnutrition, metabolic disorders, lie the stock exchange, acidosis, the activity of the immune system, the effects of radiation, hepatitis, rejection of transplanted organ, the growth of the cancer and appetite.

In another aspect, the present invention relates to a genetic vector comprising the nucleotide sequence encoding the peptide comprising the amino acid sequence selected from the group consisting of a peptide comprising one of SEQ ID No.4, 5, 8-11. It also refers to a genetic vector comprising a nucleotide sequence encoding a peptide consisting essentially of the amino acid sequence selected from the group consisting of a peptide comprising SEQ ID No.4, 5, 8-11. The invention also relates to a genetic vector comprising a nucleotide sequence encoding a peptide comprising a functional derivative of a biologically active amino acid sequence selected from the group consisting of SEQ ID No.4, 5, 8-11. It also refers to a genetic vector comprising a nucleotide sequence encoding a peptide consisting essentially of the amino acid sequence which is a functional derivative of a biologically active amino acid sequence selected from the group consisting of SEQ ID No.4, 5, 8-11.

In another aspect the present invention relates to gibr denim peptides, containing adjacent to the peptide leader peptide, and the peptide comprises, essentially consists of, or consists of an amino acid sequence selected from the group consisting of SEQ ID No.4, 5, 8-11. The present invention also relates to hybrid peptides containing adjacent to the peptide leader peptide, the peptide comprising a functional derivative of a biologically active peptide, a bioactive peptide comprises, essentially consists of, or consists of an amino acid sequence selected from the group consisting of SEQ ID No.4, 5, 8-11.

The present invention also relates to a genetic vector comprising the nucleotide sequence encoding the peptide comprising the leading amino acid sequence adjacent to the peptide comprising the amino acid sequence which is a functional derivative of a biologically active amino acid sequence selected from the group consisting of SEQ ID No.4, 5, 8-11. It also refers to a genetic vector comprising the nucleotide sequence encoding the peptide comprising the leading amino acid sequence adjacent to a nucleotide sequence that encodes a peptide consisting essentially of the amino acid sequence that is fun is inim derived biologically active amino acid sequence, selected from the group consisting of SEQ ID No.4, 5, 8-11.

In a particular implementation of the peptides obtained in any of the above-described genetic vectors, can modulate, but are not limited to modulation, one or more of the following: fatigue, malnutrition, metabolic disorders, lipid metabolism disorders, the activity of the immune system, the effects of radiation, hepatitis, rejection of transplanted organ, the growth of the cancer and appetite.

In another aspect the present invention relates to a micro-organism with a genome comprising a nucleotide sequence encoding the peptide comprising the amino acid sequence selected from the group consisting of SEQ ID No.4, 5, 8-11. It also refers to an organism with a genome comprising a nucleotide sequence encoding a peptide consisting essentially of the amino acid sequence selected from the group consisting of SEQ ID No.4, 5, 8-11.

In another aspect the present invention relates to a micro-organism with genetic material that includes the nucleotide sequence encoding the peptide, preferably an exogenous peptide comprising the amino acid sequence which is a functional derivative of a biologically active amino acid sequence selected from the group consisting of SEQID No.4, 5, 8-11. It also refers to an organism with a genetic composition that includes a nucleotide sequence encoding an exogenous peptide consisting essentially of the amino acid sequence which is a functional derivative of a biologically active amino acid sequence selected from the group consisting of SEQ ID No.4, 5, 8-11. When used in this description of exogenous peptide refers to a peptide having the amino acid sequence that differs from any other peptides, usually expressed by the organism in its natural, unmodified form.

In another aspect the present invention relates to a microorganism with a genetic composition that includes the nucleotide sequence encoding the peptide, preferably exogenous hybrid peptide comprising a leading amino acid sequence adjacent to the peptide, and the peptide includes an amino acid sequence selected from the group consisting of SEQ ID No.4, 5, 8-11. It also refers to an organism with a genome comprising a nucleotide sequence encoding a hybrid peptide comprising a leading amino acid sequence flanking the peptide consisting essentially of or consisting of the amino acid sequence, SEL is Anna from the group consisting of SEQ ID No.4, 5, 8-11.

In another aspect the present invention relates to a microorganism with a genetic composition that includes a nucleotide sequence encoding an exogenous hybrid peptide comprising a leading amino acid sequence adjacent to the peptide, and the peptide includes an amino acid sequence which is a functional derivative of a biologically active amino acid sequence selected from the group consisting of SEQ ID No.4, 5, 8-11. It also refers to an organism with a genetic composition that includes a nucleotide sequence encoding an exogenous hybrid peptide comprising a leading amino acid sequence flanking the peptide consisting essentially of or consisting of an amino acid sequence which is a functional derivative of a biologically active amino acid sequence selected from the group consisting of SEQ ID No.4, 5, 8-11.

In a particular implementation of the peptides obtained in any of the above microorganisms can modulate, but are not limited to modulation, one or more of the following conditions: fatigue, malnutrition, metabolic disorders, lipid metabolism disorders, the activity of the immune system, the effects of irradiation, heparin is it rejection of the transplanted organ, the growth of the cancer and appetite.

In another aspect the present invention relates to pharmaceutical compositions comprising essentially pure peptide comprising amino acid sequence selected from the group consisting of SEQ ID No.4, 5, 8-11. The invention also relates to pharmaceutical compositions comprising almost pure peptide consisting essentially of or consisting of an amino acid sequence selected from the group consisting of SEQ ID No.4, 5, 8-11.

The present invention also relates to pharmaceutical compositions comprising essentially pure peptide comprising a functional derivative of a biologically active peptide, a bioactive peptide includes an amino acid sequence selected from the group consisting of SEQ ID No.4, 5, 8-11. It also relates to pharmaceutical compositions comprising almost pure peptide consisting essentially of a functional derivative of a biologically active peptide, and this biologically active peptide has an amino acid sequence selected from the group consisting of SEQ ID No.4, 5, 8-11. It further relates to pharmaceutical compositions comprising almost pure peptide consisting of a functional derivative of a biologically Akti the nogo peptide, this biologically active peptide has an amino acid sequence selected from the group consisting of SEQ ID No.4, 5, 8-11.

In a particular implementation of the peptides presented in any of the above pharmaceutical compositions that can modulate, but are not limited to modulation, one or more of the following: fatigue, malnutrition, metabolic disorders, lipid metabolism disorders, the activity of the immune system, the effects of radiation, hepatitis, rejection of transplanted organ, the growth of the cancer and appetite.

In another aspect the present invention relates to a method for producing a pharmaceutical composition, comprising providing essentially pure peptide comprising amino acid sequence selected from the group consisting of SEQ ID No.4, 5, 8-11; and mixing the specified essentially pure peptide with a pharmaceutically acceptable carrier. It also relates to a method for producing a pharmaceutical composition, comprising providing almost pure peptide consisting essentially of or consisting of an amino acid sequence selected from the group consisting of SEQ ID No.4, 5, 8-11, and mixing the specified essentially pure peptide with a pharmaceutically acceptable carrier.

In another aspect, the present invention relates to a method according to the teachings of the pharmaceutical composition, including the provision of essentially pure peptide comprising the amino acid sequence which is a functional derivative of a biologically active peptide, and this biologically active peptide has an amino acid sequence selected from the group consisting of SEQ ID No.4, 5, 8-11; and mixing the specified essentially pure peptide with a pharmaceutically acceptable carrier.

The invention additionally relates to a method for producing a pharmaceutical composition, comprising providing essentially pure peptide consisting essentially of or consisting of an amino acid sequence which is a functional derivative of a biologically active peptide, and this biologically active peptide has an amino acid sequence selected from the group consisting of SEQ ID No.4, 5, 8-11; and mixing the specified essentially pure peptide with a pharmaceutically acceptable carrier.

In connection with any of the methods described above, the peptide can modulate, but are not limited to modulation, one or more of the following conditions: the activity of the immune system, fatigue, malnutrition, metabolic disorders, lipid metabolism disorders, the activity of the immune system, the effects of radiation, hepatitis, rejection of transplanted organ, the growth Raco is Oh tumors and appetite.

In another aspect the present invention relates to a method of treatment of a person, including the introduction of man pharmaceutically effective dose of essentially pure peptide comprising, essentially consisting of, or consisting of an amino acid sequence selected from the group consisting of SEQ ID No.4, 5, 8-11. It also relates to a method of treatment of a person, including the introduction of man pharmaceutically effective dose of essentially pure peptide comprising, essentially consisting of, or consisting of an amino acid sequence which is a functional derivative of a biologically active peptide, and this biologically active peptide has an amino acid sequence selected from the group consisting of SEQ ID No.4, 5, 8-11.

In the specific implementation described above, the peptides used for treatment of humans, can be used to modulate, but are not limited to it, one or more of the following human conditions: fatigue, eating disorders, metabolic disorders, disorders of lipid metabolism, acidosis, immune system activity, effects of exposure, hepatitis, rejection of transplanted organ, the growth of the cancer and appetite.

In connection with any of the above nucleotide sequences of the peptides and/or hybrid peptides, Express arausiaca with these sequences of nucleic acids, can modulate, but are not limited to modulation, the following conditions: fatigue, malnutrition, metabolic disorders, lipid metabolism disorders, the activity of the immune system, the effects of radiation, hepatitis, rejection of transplanted organ, the growth of the cancer and appetite.

In yet another implementation of the present invention relates to the use of biologically active peptide, and specified biologically active peptide has an amino acid sequence comprising, consisting essentially of or consisting of an amino acid sequence selected from the group consisting of SEQ ID No.1, 2, 15, or its functional derivatives, to obtain drugs to modulate at least one of the following conditions: hepatitis, fatigue, the inventory level of glycogen in the liver, levels of lactic acid in the blood and immunological reactions in the individual. It also relates to a method of modulation of hepatitis, the inventory level of glycogen in the liver, levels of lactic acid in the blood or immunological response in an individual, comprising the introduction of a pharmaceutically effective dose of a biologically active peptide, and specified biologically active peptide has an amino acid sequence consisting essentially of the amino acid sequence selected from the group, with Toyama of SEQ ID No.1, 2, 15, or its functional derivatives.

The present invention additionally relates to the use of biologically active peptide, and specified biologically active peptide has an amino acid sequence comprising, consisting essentially of or consisting of amino acids selected from the group consisting of SEQ ID No.3, 7, 21, or their functional derivatives, to obtain drugs to modulate at least one of the following conditions: fatigue, inflammation, appetite, body mass, fat body lipid levels in the blood level of triglycerides in the blood level of cholesterol in the blood, inflammation and immunity. The present invention also relates to a method of modulating at least one of the following conditions: fatigue, inflammation, appetite, body mass, fat body lipid levels in the blood level of triglycerides in the blood level of cholesterol in the blood, inflammation and immunity in an individual, comprising the introduction of a pharmaceutically effective dose of a biologically active peptide, and specified biologically active peptide has an amino acid sequence comprising, consisting essentially of or consisting of an amino acid sequence selected from the group consisting of SEQ ID No.3, 7, 21, or its functional derivatives.

To complement Inom the implementation of the present invention is applied to a food composition, containing a peptide comprising, consisting essentially of or consisting of amino acids selected from the group consisting of SEQ ID No.4, 5, 8-11, or its functional derivatives, and their use for the production of dietary supplements.

In an additional implementation of the present invention offers an improved derivative of the peptide SEQ ID No.4, 5, 8-11 and their functional derivatives. Reinforced derivatives of these peptides include molecule-amplifier, operatively associated with these peptides thus, to improve or to increase therapeutic efficacy of the peptide. The strengthening effect can be a prolonged effect, shortened the effect of delayed start effect, accelerated the effect, increased the intensity of the effect, reduced the intensity of the effect, the reduction of side effects, the creation of one or more effects of delayed-lowering effect, an accelerated decline effect and targeted delivery of the peptide to the specific location of the individual. Examples of such molecules amplifiers and amplified derivatives are described below. In some aspects of the invention molecules amplifiers can be treated or prevented, but are not limited to treatment and prevention, viral infections and immunologic disorders. Additional aspects of the present invention include methods of gain is of therapeutic effects of the peptide, comprising, consisting essentially of or consisting of a peptide selected from the group consisting of SEQ ID No.4, 5, 8-11, and its derivatives, including the specified peptide operatively linked to a molecule that enhances therapeutic effect. In some aspects of the invention specified operatively associated molecule, which enhances therapeutic effect is not a peptide, which is adjacent to the peptide, selected from the group consisting of SEQ ID No.4, 5, 8-11, and its derivatives, existing in the nature of the peptide.

In an additional aspect, the invention features a composition comprising essentially pure peptide comprising, consisting essentially of or consisting of an amino acid sequence selected from the group consisting of SEQ ID No.4, 5, 8-11, and its derivatives.

In an additional aspect, the invention features a method of reducing the effects of human disease by administering pharmaceutically effective dose of a biologically active peptide comprising, consisting essentially of or consisting of an amino acid sequence selected from the group consisting of SEQ ID No.4, 5, 8-11. In some implementations, the person suffers from at least the following conditions: fatigue, eating disorders, metabolic disorders, disorders of lipid metabolism, acidosis, immune system activity, effects of irradiation is to be placed, hepatitis, rejection of transplanted organ, the growth of the cancer and appetite.

In an additional aspect, the invention features a method of modulating or treating a condition involving the introduction of a biologically active peptide having an amino acid sequence comprising, consisting essentially of or consisting of SEQ ID No. 1 or its functional derivative, where the condition is selected from the group consisting of: fatigue, the inventory level of glycogen in the liver, levels of lactic acid and immune response in the individual.

In an additional aspect, the invention features a method of modulating the inventory level of glycogen in the liver, levels of lactic acid or immune response in an individual by administering a pharmacologically effective dose of a biologically active peptide comprising, consisting essentially of or consisting of the amino acid sequence of SEQ ID No. 1 or its functional derivatives.

In another aspect of the invention features a biologically active peptide having an amino acid sequence comprising, consisting essentially of or consisting of an amino acid sequence selected from the group consisting of SEQ ID No. 2 or 15 or its functional derivative to obtain drugs for the treatment of hepatitis.

In another aspect of the invention affords the tsya a method of treating hepatitis in an individual by administering a pharmacologically effective dose of a biologically active peptide, moreover, biologically active peptide has an amino acid sequence comprising, consisting essentially of or consisting of an amino acid sequence selected from the group consisting of SEQ ID No. 2 or 15 or its functional derivatives.

In an additional aspect, the invention features a biologically active peptide having an amino acid sequence comprising, consisting essentially of or consisting of SEQ ID No. 3 or its functional derivatives, to obtain medicines to alleviate fatigue.

In an additional aspect of the invention proposes a method of alleviating fatigue in an individual by administering a pharmacologically effective dose of a biologically active peptide having the amino acid sequence comprising, consisting essentially of or consisting of SEQ ID No. 3 or its functional derivatives.

In another aspect of the invention features the use of biologically active peptide having an amino acid sequence comprising, consisting essentially of or consisting of SEQ ID No. 21 or its functional derivatives, to obtain drugs to modulate at least one of the following conditions: inflammation, appetite, body mass, fat body lipid levels in the blood level of triglycerides in the blood, the level of Ho is esterina in the blood, inflammation and immunity.

In another aspect of the invention features a method of modulating at least one of the following conditions: inflammation, appetite, body mass, fat body lipid levels in the blood level of triglycerides in the blood level of cholesterol in the blood, inflammation and immunity, the individual, including the introduction of a pharmacologically effective dose of a biologically active peptide, and a biologically active peptide has an amino acid sequence comprising, consisting essentially of or consisting of SEQ ID No. 21 or its functional derivatives.

In an additional aspect of the invention features the use of biologically active peptide having an amino acid sequence comprising, consisting essentially of or consisting of SEQ ID No. 7 or its functional derivatives, to obtain drugs for the modulation of the immune system of the individual.

In another aspect of the invention features a method of modulating the immune system of an individual by administering a pharmacologically effective dose of a biologically active peptide having the amino acid sequence comprising, consisting essentially of or consisting of SEQ ID No. 7 or its functional derivatives.

A detailed description of the preferred option exercise

It is described in this description, the peptides was found to have significant biological activity. The peptides can be easily synthesized using standard methods of synthesis of L-amino acids, but they can also be synthesized using genetic engineering techniques using nucleic acid having a sequence encoding the individual peptides. All cited references are included in this description in its entirety.

As used herein, "essentially pure peptide" refers to peptides that are cleaned at least 40% and more preferably 60%, and more preferably more than 90%. In the most preferred implementation of the purity of approximately 99%-100%. Essentially pure peptide can be applied to obtain the pharmaceutical and food compositions which can be complex mixtures, as described below.

As used herein, the term "hybrid peptide" is used to refer to peptides that contain additional peptides included in described in this description of the original biologically active peptide having the sequence described above, or its functional derivative, but still retain essentially the same activity. Additional peptides include leading peptides, which contain, for example, amino acid serial is lnost, which is recognized by one or more prokaryotes or eukaryotes as a signal for secretion of the hybrid protein from the cell. The secretion may be the direct secretion or indirectly via secretory vesicles.

As described above, another implementation of the present invention is a peptide or polypeptide consisting essentially of the peptide of the present invention. As used herein, the term "consisting essentially of" refers to a peptide or polypeptide comprising the amino acid sequence of the peptides of the present invention together with additional amino acids at the carboxyl and/or aminobenzo and which retains the activity proposed in this description of the peptides of the present invention. So, as a non-limiting example, when the activity of the peptide of the present invention is the modulation of immune activity, peptide or polypeptide "consisting essentially of" peptide of the present invention must possess the ability to modulate immune activity, as suggested in this description, in the sense that the peptide should not have any characteristics that significantly reduce the ability of the peptide or polypeptide to modulate immune activity or which create real change compared with basic and new features PE the Chida as modulator of immune activity. Thus, in the example above, the full-size existing in the nature of the polypeptide having the original activity that is different from the modulation of immune activity, and contains somewhere within himself amino acid sequence of the peptide of the present invention, should not be a peptide or polypeptide "consisting essentially of" peptide of the present invention. Similarly, in the example above, the genetically engineered peptide or polypeptide which has the original activity that is different from the modulation of immune activity, but somewhere inside amino acid sequence of the peptide of the present invention, should not be a peptide or polypeptide "consisting essentially of" peptide of the present invention.

In a preferred implementation, the term "consisting essentially of" refers to peptides or polypeptides that have four or less amino acids in addition to one of the peptides of the present invention. In a more preferred implementation of the same term refers to peptides or polypeptides with two amino acids in addition to one of the peptides of the present invention. In the most preferred implementation of the same term refers to peptides or polypeptides with a single amino acid in addition to one is of the peptides of the present invention.

In addition to the example modulation of immune activity, used for the above illustration, the above definition also applies for all peptides of the present invention in respect of activity that provide such peptides. In particular, the above definition applies to the peptides of the invention having activity in the modulation of fatigue, eating disorders, metabolic disorders, disorders of lipid metabolism, acidosis, immune system activity, effects of exposure, hepatitis, rejection of transplanted organ, the growth of the cancer and appetite.

Specialists in the art can easily determine whether a peptide or polypeptide essentially the peptide of the present invention using the above definitions by measuring the activity of the peptide or polypeptide using the tests described below.

Specialists in the art can easily determine whether a peptide or polypeptide essentially the peptide of the present invention using the above definitions by measuring the activity of the peptide or polypeptide with the use of tests for modulation disorders, such as fatigue, malnutrition, metabolic disorders, lipid metabolism disorders, the activity of the immune system, the effects of radiation, hepatitis, is tworzenie transplanted organ, the growth of the cancer and appetite, which are offered in this description and in respect of a particular peptide of the present invention.

Used in this description and in the claims, the term "violation" includes any body States, which falls out of the normal range when measured by standard methods. For example, "lipid metabolism" refers to the condition in which the level of any of the lipids in the body exceeds the normal range for this specific subject by gender, height and age when determining the standard of medical diagnostic tests.

The peptide may be introduced by any suitable means. Examples of ways of doing are intravenous, intramuscular, intraperitoneal administration, subcutaneous administration, and subcutaneous implantation with the device to facilitate delivery, such as a liposome, or without protection with a constant release and the like. Thus, the present invention includes a device for injection of the peptide to a subject. In some implementations, the device may be a syringe. The peptide may be entered in any form of oral administration. Examples may include, but are not limited to, a tablet, capsule, suspension, solution, cake and the like, in the usual form of the be the modification or in the form of delayed release, or protection in the gastrointestinal tract, or without it. The peptide can be optionally applied to any form of local application, such as ointment, cream, gel, etc. with or without devices for facilitating transdermal delivery, or in the form of a powder inhaler, dissolved or in the form of liposomal protective form.

It is clear that it may be possible to add additional amino acids to the amino - or carboxyl ends are described and disclosed in this description of the peptides as another way to practice the present invention. For example, one or two amino acids can be added to opened the peptide without affecting its biological function. It may be also possible to add three or four amino acids at still retains features of the peptides. All this is referred to as variants of the same peptide. Alternative one or two amino acids may be deleted from the peptide without affecting its biological activity. Additionally it may be possible to remove three or four amino acids without affecting the biological function of the peptide. This is considered as fragments of the peptide. Moreover, in the practice of another aspect of the present invention can be applied derivative peptide, such as when a conservative substitution of one amino acid to another within the same is funkcionalnogo class. For example, peptides with non-polar or hydrophobic side chains may be possible to replace one side of the group to another without compromising biological activity. As an additional example, the peptide may be a linker/spacer education options, but options still retaining its active part as in the original peptide used in this study. It is also considered variants of the peptides. Similar peptide, as used herein, includes peptides that have amino acid molecules that mimic the structure of natural amino acids, for example similar with different structure of the skeleton, or replacement of a D-amino acid. As an additional example, although the amino acids used for peptide synthesis, are in their L optical isomer form, peptides with one or more amino acids in the sequence are replaced by D-form, can have similar biological activity. The term "functional derivative", as used in the claims is intended to include fragments, variants, analogs, or chemical derivatives of the peptide.

The application identified above peptides in the pharmaceutical compositions can be used as a possible treatment described in this description of violations, such as immunological is their violations, cancer, fatigue, graft rejection, and the like. The compositions can contain one of the identified peptides, mixed with other active or inactive components, including other peptides. Alternate one of the following peptides can be applied to obtain a composition together with not listed in this description and peptides. They can be inserted into the intravenous, intramuscular, intradermal, subcutaneous or vnutriaortalina form. The method of introduction may also be a intraarterial injection, which leads to the body in need of treatment. Other methods of introduction are transdermal introduction inhalation in the form of a powder or spray and other forms of delivery, known to the person skilled in the art. The composition can also be applied orally and it may contain media that can be used to prevent hydrolysis of the peptide in the stomach after oral administration, or any other media known in the art.

The pharmaceutical composition may include any of the known pharmaceutical carriers. Examples of suitable carriers include any of the standard pharmaceutically acceptable carrier well-known specialists in this field of technology. They include, but are not limited to, physiological sole is the second solution, water, emulsions, including mixtures of oil and water or emulsion of triglycerides, and other types of agents, fillers, coated tablets and capsules. Suitable carrier may be selected based on the method of administration of the pharmaceutical composition.

In an additional aspect of the present invention offers described in this description superior derived peptides and their functional derivatives. Biologically active peptides of the present invention can be conjugated with other biologically effective or useful molecules to provide additional effect or application, or to enhance their therapeutic efficacy. In the art there are many potential conjugating molecules, their biological effects and methods of conjugation of molecules with peptides. Examples of conjugating molecules include, but are not limited to, organic compound, a carbohydrate, a sugar, a polysaccharide, an amino acid, a polymer of amino acids, peptide, steroid, a protein isolated domain of a protein, a lipid molecule, fatty acid, bile acid, a polyamine, a protease inhibitor, and the like. Can also be used a combination of conjugating molecules.

Some of the peptides of the invention are individually therapeutic effect on a particular cell type or tissue. One important requirement to say kulam, conjugialis with peptide drugs, is targeting peptide to a specific location or area of the body of the individual being treated. In this way, the peptide drug and its effects can be concentrated in the localization of the cell or tissue type, which is intended therapeutic effect. This may enhance the effects compared with those who should have similar molecular amount of free, unconjugated peptide. Other requirements for conjugating molecules can include, for example, increasing the life of the peptide, the change in the solubility of the peptide, changes in the activity of the peptide and the change in the bioavailability of the peptide.

The chemical reaction for conjugation of these peptides with conjugating partners can be derived by a specialist in the art without undue experimentation. Different methods of conjugation are described, for example, Haas et al., Kidney Intl.,52(6):1693, 1997; Fiume et al., Ital J Gastroenterol Hepatol, 29(3):275, 1997; Di Stefano et al., Biochem. Pharmacol., 61(4):459, 2001; Huang et al., Chem. Biol., 7(7):453, 2000; Leopold et al., J. Pharmacokinet. Biopharm., 23(4):397, 1995; Patel et al., Bioconjugate Chem., 8(3):434, 1997; Kramer et al. J. Biol. Chem., 269(14): 10621, 1994; Toth et al. (J.Med.Chem., 42(19):4010, 1999; Kim et al., (Biomaterials, 23:2311, 2002; Oldham et al. (Int. J. Oncology, 16:125, 2000; Fitzpatrick et al. Anticancer Drug Design, 10:1, 1995, all of which are included in this description as a reference in all its full of the same.

Gene therapy and treatment method

Gene therapy based on the disclosed peptide sequences, can be done by creating a nucleic acid sequence that encodes one of these peptides. The nucleic acid may be synthesized chemically and operatively associated with the promoter and cloned in the expression vector. The expression vector is then introduced into the human body as a form of gene therapy for expression in human cells. Used in this description, the term "genetic vectors includes these expression vectors. Vectors which may be suitable for gene therapy include assosiated with adenovirus (Mizuno, M. et al. (1998), Jpn J Cancer Res 89, 76-80) vector LNSX (Miller, A.D. et al. (1993) Methods Enzymol 217, 581-599) and lentivirinae (Goldman, MJ. et al. (1997) Hum Gene Ther 8, 2261-2268), the disclosure of which is incorporated herein by reference in its entirety.

Other media for the delivery of peptides include expression vectors encoding the desired peptide, which can be moved in the body, which can be replicated in the body of the host, which it is desirable to introduce the peptide without significant adverse effects on the health of the host body. For example, expression vectors can be transferred into the body, which is not pathogenic organism x is zaina, which it is desirable to introduce the peptide. In some implementations expression vector produces the desired peptide in the body of bacteria or fungi that do not have a significant detrimental effects on the health of the host body, which is supposed to be a peptide. For example, the expression vector encoding the desired peptide can be an expression vector that produces the desired peptide in the body, such as lactobacilli, E. coli or yeast. In one implementation of the expression vector produces the desired peptide in the microbe, usually found in the gastrointestinal tract of mammals, or microbe, tolerant to the digestive tract of mammals. Some strains of microbes, which can be expressed desired peptide include, but are not limited to, strains of Lactobacillus such as L. acidophilus, L. amylovorus, L. casei, L. crispatus, L. gallinarum, L. gasseri, L. johnsonii, L. paracasei, L. plantarum, L. reuteri, L. rhamnosus or other; Bifidobacterium strains, such as B. adolescentis, B. animalus, B. bifldum, B. breve, B. infantis, B. lactis, B. longum or other; Enterococcus faecalis or Ent. facium; Sporolactobacillus inulinus; Bacillus subtilis or Bacillus cereus; Escherichia coli; Propionibacterium freudenreichii; or Saccharomyces cerevisiae or Saccharomyces boulardii.

Nucleic acid sequence that encode the peptides of the present invention, are synthesized chemically or produced using other methods, including, but neogranichivatsya this, reverse transcription of mRNA to obtain cDNA molecules that can be incorporated into expression vectors for gene transfer in the desired organisms using methods of genetic engineering, familiar to specialists in the field of technology. Expression vectors may represent a DNA vectors or RNA vectors. For example, expression vectors can be based on molecular or genetic elements of viruses. Expression vectors can represent vectors that are replicated outside the chromosomes, or vectors which integrate into the chromosome.

The expression vectors include a promoter operatively linked to nucleic acid encoding the peptide of the present invention. The promoter may be a regulated promoter, such as an inducible promoter or a constitutive promoter. In some implementations can be selected by the promoter to provide the desired level of expression of the peptide. In addition, if desired, expression vectors can include other sequences to stimulate production, presentation and/or secretion of peptides. In some implementations nucleic acid encoding a peptide of the present invention, operatively linked to a nucleic acid sequence, which controls the secretion of the peptide. For example, the nucleic acid is one encoding the peptide of the present invention may be operatively linked to a nucleic acid that encodes a signal peptide.

In some implementations expression vectors, which are constructed to encode the peptides of the present invention, can be represented as expression vectors adapted for expression of the peptide of the present invention in the strains of bacteria that make up the normal flora of the digestive tract of mammals, such as Lactobacillus and Bacillus subtilis. Examples of such expression vectors can be found in U.S. patent No. 6100388 owned Casas, and No. 5728571 owned by Bellini, respectively. It should be clear that can be used in any expression vector that facilitates the expression of the peptide of the present invention in the body, which is not harmful to the health of the host body, which must be entered peptide.

In some implementations expression vectors, which are created to encode the peptides of the present invention, can be represented as expression vectors adapted for expression of the peptide of the present invention in yeast strains that are well tolerant digestive tract of mammals, such as Saccharomyces cerevisiae; or, preferably, Saccharomyces boulardii, which can form colonies in adevaratele tract of man, and is used to treat certain forms of diarrhea. Can be used a yeast expression vectors that constitutively Express heterologous proteins and peptides, they are highly stable, thus, well transferred to progeny cells during mitosis and meiosis and may include the coding sequence for the signal peptide or peptides, which manages the high-level secretion of recombinant protein. An example of such a yeast vector is given in U.S. patent No. 6391585 owned by Jang et al., included in this description specifically by reference in its entirety.

Expression vectors encoding the peptides of the present invention, can be introduced into the body, which is planned for the expression of peptides using methods known in the art. These methods include traditional methods of transformation of bacteria, yeast or other microbes by application of chemically competent bacterial cells, electroporation or transformation with lithium acetate (for yeast), for example, and using the latest achievements in the transformation of bacterial strains that are not susceptible to these procedures. In some implementations expression vectors injected into the lactobacilli are known to be transformative, using the method disclosed Leer et al. (WO 95/35389), the disclosure of which is incorporated in this description by reference in its entirety. The input sequence can be incorporated into microbial chromosomal DNA or can be in the form of elements of extrachromosomal DNA.

This genetically engineered microbe containing the expression vector can then be used to inoculate into the digestive tract, vagina, trachea, etc. to achieve the supported immunotherapy. In some implementations organisms expressing the peptides of the present invention, are swallowed in an inactive form or, preferably, in a living form. In the digestive tract, these microorganisms produce these peptides, release them into the lumen through the secretion or lysis of the microorganism, or otherwise present the peptide to the owner, resulting in peptides produce intended them effect in the body of the host. In other implementations, the peptides are presented to the host on the mucous membrane of the nasal passages, vagina, or small intestine.

Another method of treatment is the use of liposomes as vehicles for the delivery of specific nucleic acids to cells in the human body. Nucleic acid (such as an expression vector containing a nucleic acid sequence that encodes described in this description of the PE the Chida) can be delivered in an environment that promote cell capture and integration into the chromosome, as described in Gao, X. and Huang, L. (1995) Gene Ther 2, 710-722 and U.S. patent No. 6207456, the disclosure of which is incorporated in this description by reference in its entirety. Alternative the peptide can be encapsulated in the liposome and delivered directly using the method described in U.S. patent No. 6245427, the disclosure of which is incorporated in this description by reference in its entirety. All scientific publications and these patents are included in this description by reference.

Nucleic acid sequence suitable for the above gene therapy, and the treatment method include sequences that encode these peptides and their functional derivatives. Any of the numbered sequences of nucleic acids may be used for encoding these peptides and their derivatives on the basis of the degeneracy of codons.

Example 1

The effect of CMS-001 and CMS-008 on caused by exercise fatigue in mice

Purpose: To explore relieves fatigue effect of CMS-001 and CMS-008 mice BALB/c

Methods: To monitor relieves fatigue actions of CMS-001 and CMS-008 model is used debilitating swimming BALB/c mice. Defined time exhausting voyage, liver glycogen, urea nitrogen in serum (BUN) and level Moloch the th acid in serum after administration of CMS-001 and CMS-008. Also determined the levels of malondialdehyde (MDA), superoxide dismutase (SOD), alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in serum to study the possible mechanism relieves fatigue actions of CMS-001 and CMS-008.

Results: It was found that the CMS-001 and CMS-008 in suitable concentrations can prolong the time of debilitating swimming mice, reduce BUN, to increase the level of liver glycogen and reduce the accumulation of lactic acid in the blood statistically significantly compared with the control saline (P<0,05). It was further discovered that the CMS-001 and CMS-008 is also able to suppress the increase of MDA levels, ALT and AST in the blood and increase the activity of SOD during exhausting exercise statistically significantly compared with the control saline (P<0,05).

Conclusion: It was found that the CMS-001 and CMS-008 have the ability to relieve fatigue and can be used for therapy-related fatigue States.

1.Materials and methods:

1.1.Medicine and chemicals

CMS-001 and CMS-008 synthesized on the order of Shenzhen Kangzhe Pharmaceutical Co. Ltd., Shenzhen, PR China.

Kit for determination of urea nitrogen: Beckman Coulter, Inc., Fullerton, California, USA.

Set anthracenemethanol reagent: Shanghai Union Fifth Laboratory, Shanghai, China, PRC.

Kits MDA and SOD: Nanjing Jiancheng on Bio-engineering Corporation, Nanjing, China, PRC./p>

Kits AST and ALT: Beijing Zhongsheng Bio-engineering High Technology Corporation, Beijing, PRC.

1.2.Animals

Male mice of BALB/c separate groups that do not contain pathogens, weighing 18-22 g were obtained from the Academy of military medicine and research Center of experimental animals, PR China.

Mice were randomly distributed into groups of CMS-001 (20 mg/kg/day, 5 mg/kg/day), CMS-008 (500 µg/kg/day, 125 mg/kg/day) and control with saline solution. The test substance was administered by intraperitoneal injection once a day for 30 days without a break. A week after the introduction of the test substance, the mice were trained to swim for 10 minutes at a water temperature of 25±1°C.

1.3.Experimental equipment

Swimming pool (50 cm×50 cm×40 cm).

Fully automated biochemistry analyzer RA-1000.

The automated analyzer of lactic acid 1500SPORT.

1.4.The effect of CMS-001 and CMS-008 on time exhausting swimming mice[1]

After 30 min after the last injection, the mice were weighed and the tails of mice at approximately 1 cm from the body attached pieces of lead corresponding to 5% of body weight. Mice were placed in a swimming pool with a water depth of 30 cm and a temperature of 25±1°C. the limbs of the mice were moved through the entire process. Recorded the voyage before the onset of death.

1.5.The effect is the development of CMS-001 and CMS-008 on the level of urea nitrogen in serum after a workout

After 30 min after the last injection mice without increasing the weight placed for swimming for 90 min in a swimming pool with a water depth of 30 cm and a temperature of 30±1°C. the Mice were then allowed to rest for 30 min and collected the blood from the eye of the sinus. Allocated serum and determined BUN by using a fully automated biochemical analyzer.

1.6.The effect of CMS-001 and CMS-008 on the level of glycogen in the liver of mice[2]

After 30 min after the last injection the mice were scored by decapitation, liver was separated, washed with saline and dried with filter paper. Carefully weighed 100 mg of liver and determined the level of liver glycogen by using a set anthracenemethanol reagent.

Mass glycogen (mg) per 100 g liver=DU/DS×0,5×about. fluid under homogenization/weight liver (g) ×100×0,9,

where DU = absorption sample

DS = absorption standard

About. liquid at homogenizing=8 ml.

1.7.The effect of CMS-001 and CMS-008 on the level of lactic acid in the blood of mice after exercise[3]

After 30 min after the last injection was initially selected 20 µl of blood from the ocular sinus. Mice were then weighed and the tails of mice at approximately 1 cm from the body attached pieces of lead, corresponding to 4% of body weight. Mice were placed for 10 min in a swimming pool with a water depth of 30 cm and te is the temperature of 30±1°C. The limbs of the mice were moved through the entire process. Immediately after a workout and after resting for 20 min again took 20 µl of blood from the eye of the sinus. The blood was added to 40 μl of hypotonic buffer and subjected to the action of ultrasound. The level of lactic acid in the blood was determined using the analyzer lactic acid.

The increase in the number of lactic acid after exercise calculated based on the level of lactic acid immediately after a workout, minus the level of lactic acid before workout.

The amount of lactic acid after relaxation was calculated based on the level of lactic acid immediately after a workout, minus the level of lactic acid after a rest.

1.8. The effect of CMS-001 and CMS-008 on the level of SOD, MDA, AST and ALT after exhausting workout

After 30 min after the last injection mice without increasing the weight placed for a swim in the swimming pool with a water depth of 30 cm and a temperature of 25±1°C[4]. As soon as the mouse began to sink, they were saved and immediately the blood was collected from the ocular sinus. Allocated serum and determined the levels of SOD, MDA, AST and ALT.

1.9. Statistical method

The difference between groups was analyzed using analysis ANOV dispersion.

2. Results

Salinetd align="center" namest="c2" nameend="c4"> 5
Table 11
The effect of CMS-001 and CMS-008 on time exhausting swimming mice
GroupDose (mg/kg/day)No.The sailing time (min)The increase in sailing time (%)
CMS-0012015and 99.8±30,6*48,0*
CMS-001518105,7±19,8*56,7*
CMS-00850015116,5±17,7*72,7*
CMS-00812518the 106.5±19,4*57,9*
-1667,5±28,0
*Compared with the control saline solution, P<0,05.
Table 1.2
The effect of CMS-001 and CMS-008 on the level of urea nitrogen in serum after a workout
GroupDose (mg/kg/day)No.The urea nitrogen (mmol/l)
CMS-00120177,3±2,1*
CMS-0015186,9±1,6*
CMS-008500177,1±2,0*
CMS-008 125187,2±2,3*
Saline-179,0±2,5
*Compared with the control saline solution, P<0,05.
Table 1.3
The effect of CMS-001 and CMS-008 on the level of glycogen in the liver of mice
GroupDose (mg/kg/day)No.Liver glycogen (mg/100 g liver tissue)
CMS-0012081567±604*
CMS-001510439±271*
CMS-0085008 1408±457*
CMS-008125101275±726*
Saline-10271±217
*Compared with the control saline solution, P<0,05.
Table 1.4
The effect of CMS-001 and CMS-008 on the level of lactic acid in the serum of mice after exercise
GroupDose (mg/kg/day)No.The increase in lactic acid after exercise (mol/l)The removal of lactic acid after a break (mol/l)
CMS-00120152,56±0,41*3,14±0,64*
CMS-001132,60±0,33*2,96±0,33*
CMS-008500152,23±0,30*3,71±0,22*
CMS-008125132,70±0,15*3,62±0,57*
Saline-144,60±0,162,00±0,44
*Compared with the control saline solution, P<0,05.
Table 1.5
The effect of CMS-001 and CMS-008 on the levels of SOD, MDA, AST and ALT in serum after exhausting workouts
GroupDose (µg/
kg/
day)
No. MDA (nmol/l)SOD (U/ml)AST (IU/l)ALT (IU/l)
CMS-0012094,4±0,2*400,6±45,6*117,0±19,2*32,7±3,0*
CMS-00850094,1±0,4*415,7±31,5*116,6±18,8*32,9±5,2*
Saline-96,9±0,3342±27,3145, 2mm±32,340,2±8,2

Conclusion

This study showed that the CMS-001 and CMS-008 have the following properties:

1. To prolong the voyage mice, which indicates the ability of CMS-001 and CMS-008 to increase the physical capacity of the animal[5].

2. To reduce BUN in mice, which indicates the ability of the CMS-001 and CMS-008 to reduce the need for the catabolism of protein for energy during physical activity [6].

3. To increase the reserves of glycogen in the liver of the animal during the holidays, which increases the physical features of animals[6].

4. To reduce the level of lactic acid in the blood after exercise to speed up the removal of lactic acid later, which indicates the ability of CMS-001 and CMS-008 reduce the rate of production of lactic acid or remove lactic acid during physical activity[7].

5. To reduce the level of MDA, which indicates the ability of CMS-001 and CMS-008 reduce the formation of free radicals during physical activity[8].

6. To increase the level of SOD, which indicates the ability of CMS-001 and CMS-008 to increase the removal of free radicals during physical activity[8].

7. To reduce the level of ALT and AST, which indicates the ability of CMS-001 and CMS-008 to protect the cells of the heart and liver from damage during exercise.

8. CMS-001 and CMS-008 can be suitable for treating disorders associated with fatigue during exercise.

Links:

The following links are included in this description by reference in its entirety.

1. Mizunoya W, Oyaizu S, Ishihara K5et al. Protocol for measuring the endurance capacity of mice in an adjustable-current swimming pool. Biosci Biotechnol Biochem. 2002 May; 66(5):1133-6.

2. HE Ling, WANG Ming, CHEN Run etc. The effect of blood lactic acid * blood serum carbamide nitrogen and liver hepatin affected by gen-seng. Prevent Medicine Literature Information, 2002, 8(3):293-294.

3. WANF Xiao-xue, QIU Juan, SONG Yu etc. Study on Effect of Theanine of Fatigue. China Commonality Sanitation Journal, 2002, 18(3):315-317.

4. Thomas D P > Marshall K I. Effects of repeated exhaustive exercise on myocardial subcellular membrane structure. Int J Sport Med > 1988, (9):257-260.

5. JIN Zong-lian. Evaluation principle and method of function food. Beijing: Beijing University Publishing Company, 1995.

6. Sanitation ministry, Evaluation of progress and test methods of health care food. Ministry of Public Health, PR China.

7. Westerblad, et al. Changes of intracellular pH due to repetitive stimulation of single fibers from mouse skeletal muscle. J Physiol; 1992 Apr; (499):49-71.

8. Groussard C, Rannou-Bekono F, Machefer G, et al. Changes in blood lipid peroxidation markers and antioxidants after a single sprint anaerobic exercise. Eur J Appl Physiol. 2003 Mar; 89(l):14-20.

Example 2

Immunoregulatory effect of CMS-001 in mice with induced radiation therapy immunodeficiency

Purpose: To investigate the immunoregulatory effect of CMS-001 in animal models of induced radiation therapy of immunodeficiency.

Ways: Murine model of immunodeficiency was originally based on the irradiation Cs137. Then typed CMS-001 and by using method MMT analyzed the change in the rate of proliferation of T-lymphocytes.

Results: It was found that at a dose of 20 mg/kg/day CMS-001 capable of statistically significant (P<0,05) increase the rate of proliferation of T-lymphocytes.

Materials and methods

Medicine and chemicals

CMS-001: Synthesized on the order of Shenzhen Kangzhe Pharmaceutical Co. Ltd., Shenzhen, PR China.

The Hanks solution, serum fruits calves and RPMI-1640: Hyclone, Logan, Utah.

ConA is MTT: Sigma Chemical Co., St. Louis, Missouri, USA.

1.2. Experimental animals

Male mice of BALB/c separate groups that do not contain pathogens (SPF), weighing 18-22 g were obtained from the Academy of military medicine and research Center of experimental animals, PR China.

Getting animal model, the introduction of CMS-001 and the measurement of the rate of proliferation of T-lymphocytes[1]

Male Balb/c mice were randomly divided into groups: CMS-001 (20 mg/kg/day, 5 mg/kg/day)control saline and normal control. All mice, except for the group of normal control, exhibited with 600 rad Cs137(82,83 rad/min for a period of 7.2 min). The test substance was dissolved in 0.5 ml of saline and injected intraperitoneally once a day for 15 days without a break after irradiation. The day after the last injection of test compound sterile seized the spleen in mice. The spleen was dispersively to single cells were washed and the cell concentration was brought to 4×106/ml with RPMI-1640. In wells of 96-well culture tablet made of 100 μl of the cells and 100 μl of Con A (to a final concentration of 5 μg/ml). Prepared audit blanki basal level without Con A. Cells were incubated at 37°C and 5% CO2for 68 hours. The proliferation of T-lymphocytes was determined by MTT method[2]. The stimulation index was calculated as follows clicks the zoom: the stimulation Index (SI)=average OD of wells with Con A/ average OD of the hole Blanca basal level.

2.Result

Table 2.1
The effect of CMS-001 on the proliferation of T-lymphocytes in mice with induced irradiation immunodeficiency
GroupDoseNo.SI
CMS-00120 mg/kg/day101,27±0,19*
Control saline-81,11±0,08
Normal control-103,42±0,93*
*Compared with the group with saline solution, P<0,05.

Conclusion

It was found that the CMS-001 capable statistically significantly stimulate the proliferation of T-lymphocytes in mice with irradiation induced immunodeficiency, which indicates the possibility of the use of CMS-001 as immunostimulant in immunocompromised patients after radiotherapy.

Links

The following links are included in this op is a description by reference in its entirety.

1. New drugs (Western medicine) research direction principle before clinic. Chinese Sanitation Ministry Drug Political Situation. 1993, 7:128-137.

2. Qiu Zhi-qiang. The influence of Fuzheng buxuegao to blood system and immunity function affected by radiotherapy. Lanzhou Medical Transaction, 2003, 3(28).

Example 3

The effect of CMS-014 on skin allograft survival in mice

Purpose: To investigate the immunosuppressive action of the CMS-014 model skin allograft.

Methods: Pieces of the skin of mice C57BL/6 were transplanted to BALB/c mice and determined the average time to engraftment (MST) skin allograft.

Results: It was found that the CMS-014 able statistically significantly (p<0,05) to prolong skin allograft viability compared with the control saline solution.

Conclusion: CMS-014 can be used as an immunosuppressive agent to control rejection reactions after organ transplantation.

Materials

Drugs and other chemicals

CMS-014: Synthesized on the order of Shenzhen Kangzhe Pharmaceutical Co. Ltd., Shenzhen, PR China.

Cyclosporine A (CsA): Novartis Pharmaceutical Co. Ltd., Basel, Switzerland.

Saline: China OTSUKA Pharmaceutical Co. Ltd., Tianjin, PR China.

Na2S: Tianjin Beilian Chemical Co. Ltd., Tianjin, PR China.

Animals

Recipients: mice Balb/c(H-2d) separate groups that do not contain pathogens (SPF), aged 6 weeks weighing 18-22 g: Academy of Sciences of military medicine, China.

Donor: mouse C57BL/6(H-2d), SPF, age 6 weeks, weight 18-22 g: Academy of Sciences of military medicine, China.

2. The formation of groups and treatment

2.1. The formation of groups and the introduction of the test substance

Balb/c mice were divided randomly into groups CMS-014 (500 µg/kg/day, 250 mg/kg/day, 125 mg/kg/day), cyclosporine A (10 mg/kg/day) and control saline (0.5 ml/day). Half of the mice were males and half females.

Test compounds were dissolved in 0.5 ml of saline and injected intraperitoneally for 5 days before transplantation of the skin, followed by daily injections until the final rejection of the skin graft.

2.2.Skin grafts

The flap of the coat from the back of Balb/c mice was removed with 8% solution of Na2S. the next day received the wound surface approximately 1 cm2surgical removal of the skin, after which the wound surface was placed a piece of skin of the tail full thickness size 1 cm2from the corresponding sex of the donor mouse C57BL/6J. The area of the surgery covered and protected by a layer impregnated with paraffin gauze with the floor covering adhesive. After 6 days after transplantation, the patch was removed[1]and mouse-recipients were observed daily for signs of allograft viability is. The final moment of rejection took appropriate to preserve the viability of allograft less than 10%[2].

Statistical analysis

Statistical analysis was performed using the test for survival Kaplan-Meier.

Results

Table 3.1
The effect of CMS-014 on MST allografts skin of mice
GroupDoseNo.MST (days)
CMS-014500 µg/kg/day99,8±0,4*
CMS-014250 mcg/kg/day109,8±0,5*
CMS-014125 mg/kg/day910,0±0,5*
CsA10 mg/kg/day911,6±0,8*
Saline0.5 ml/day88,1±0,4
*Compared with the group with saline solution, P<0,05.

Conclusion

It was found that the CMS-014 able statistically significantly (p<0,05) to prolong skin allograft survival compared with the control saline solution. CMS-014 can be used as an immunosuppressive agent to control rejection reactions after organ transplantation.

Links:

The following links are included in this description by reference in its entirety.

[1] Ming Jiankuo, Wang Xingbing, Huang Baojun, et al. Peptide Nucleic Acid Antisense Prolongs Skin Allograft Survival by Means of Blockade of CXCR3 Expression Directing T Cells into the Graft. The Journal of Immunology, 2003, 170:1556-1565.

[2] Steven H. Borenstein, Jeremy Graham, et al. CD8+ T Cells Are Necessary for Recognition of Allelic, But Not Locus-Mismatched or Xeno-, HLA Class I Transplantation Antigens. The Journal of Immunology, 2000; 165:2341-2353.

Example 4

Inhibitory effect of CMS-017 and CMS-023 on the hepatitis B virus in vitro

Example 4a. Hepatitis man

The tested peptides were added to the culture medium of the cell line 2.2.15. After incubation was determined by the concentration of the surface antigen of hepatitis B (HBsAg), e antigen hepatitis B (HBeAg) and DNA of hepatitis B virus (HBV-DNA) in culture medium and compared with control values. It was found that the peptides CMS-017 and CMS-023 in a suitable concentration have activity against hepatitis B virus in vitro, statistically significant compared to the control (P< 0,05). Is that these peptides can be used to treat infection of hepatitis B.

1. Materials

Peptides cms-017 and cms-023 were synthesized in order (from a source of l-amino acids) American Peptide Company, Inc., Sunnyvale, California, USA.

Cell line 2.2.2.15, transfected DNA of hepatitis B virus (HBV) human, was obtained from the National Center For Drug Screening (Shanghai, China) (test one) and Department Of Infectious Diseases Of The First Hospital affiliated to Beijing University (test two).

Environment for cell cultures MEM was from GIBCO®, Invitrogen, Carlsbad, California, USA.

The ELISA test kits for the analysis of HBsAg and HBeAg were from Shanghai Shiye Kehua Biotech. Company, Shanghai, PR China.

A set of fluorescent quantitative PCR for determination of HBV-DNA was from DA AN gene company of Zhongshan Medical University, Guangzhou, PR China.

Methods

Test 1: Inhibitory effect of peptides on HBsAg and HBeAg at the maximum non-toxic concentration

The 2.2.15 cells in log phase were collected and brought to a concentration of 2×106/ml MEM medium (containing 10% serum fruits calves, 100 mg/ml penicillin and 100 u/ml streptomycin). The suspension was inoculable on a 24-well plate for culturing 1.5 ml per well and incubated at 37°C, 5% CO2within 48 hours to attach. Added peptides to a final concentration of 400 μg/ml in 3 parallel wells per sample. Prepared a number of control Blanca (3 holes), in which the peptide was replaced by culture medium. Planches the t cells were then incubated at 37°C for 4 days, then the culture medium was replaced with freshly prepared medium (same composition as the original) and the plate incubated for 4 days. At the end of the incubation collected supernatant culture and determined the titers of HBsAg and HBeAg by using method ELISA according to the manufacturer's instructions of the kit for determination. The percent inhibition of drug was calculated as follows:

% inhibition=(mean concentrations of control-the Average concentration of sample)/Average concentration of control × 100%

Test 2: the Inhibitory effect of the peptides on HBV-DNA

Repeated the above test 1 obtaining and incubation of cell suspension, but when the concentration of the peptides 160 mcg/ml At the end of the incubation collected supernatant and determined the concentration of HBV DNA using fluorescent quantitative PCR according to the manufacturer's instructions of the kit for determination.

% inhibition=(Average concentration of DNA control-Average concentration of DNA sample)/Average concentration of DNA control × 100%

Statistics

For statistical analysis used t-test t-test. Statistically significant took P<0,05.

Results

Table 4.1
Inhibitory effect of peptides on HBsAg and HBeAg at 400 mcg/ml
PeptideThe ratio of inhibition to HBsAThe ratio of inhibition to Nwea
CMS-01768,6%*62,2%*
CMS-02354,4%*53,7%*
* Compared with the control blankom, P < 0,05.
Table 4.2
Inhibitory effect of peptides on HBV-DNA at 160 mg/ml
Peptide% inhibition of HBV-DNA
CMS-01790,8%*
CMS-02370,8%*
* Compared with the control blankom, P<0,05.

Conclusion

It was found that when a suitable concentration CMS-017 and CMS-023 able to inhibit the development of hepatitis B virus in vitro statistically significant compared with control (P<0,05). This indicates the possibility of using CMS-017 and CMS-023 for treatment is associated with hepatitis B viral infections.

Example 4b. The antiviral effect of CMS-017 hepatitis B ducks in vivo

PurposeTo investigate the antiviral effect of CMS-017 hepatitis B virus ducks (DHBV) in vivo.

Methods

CMS-017 was dissolved in saline and injected animal model of hepatitis B ducks Chongqing by intramuscular injection for 28 days. Were determined in the serum levels of DHBV DNA and DHBsAg (surface antigen of hepatitis B ducks) using the "dot blot" hybridization and ELISA, respectively, and compared with the control blankom.

Results

It was found that the CMS-017 able to reduce the levels of DHBV DNA and DHBsAg in serum during treatment (P<0,01). 7 days after discontinuation of treatment was observed return of symptoms.

Materials and methods

1. Animal model[2]

0.1 ml of DHBV DNA positive serum (5×107copies/ml) was inoculable into the abdominal cavity one-day ducks Chongqing for the formation of the animal model of hepatitis. 10 days after inoculation were collected blood samples from the jugular vein and confirmed the success of the infection using the "dot blot" hybridization with probe DHBV DNA labeled with digoxin[3]. Ducks were pokasivali up to 2 weeks of age for introduction into the study.

Divide into groups and introduction

Ducks with confirmed infection DHBV randomly distributed into the following groups:

1) control of the other group (n=12): Normal saline 1 ml / day, administered by intramuscular injection.

2) Group CMS-017 (n=12): 200 mcg/kg/day CMS-017 (dissolved in 1 ml normal saline) injected by intramuscular injection, 1 time per day.

Treatment continued for 4 weeks and observation continued for one week after stopping treatment. Selected 1 ml samples of blood from the external jugular vein at 0, 7, 14, 21, 28 and 35 days after the start of treatment. Serum was isolated[4]and kept at -20°C until analysis.

Monitored parameters

1) the Level of DHBV DNA in serum

Probe DHBV DNA was fluorescently labeled in accordance with the Protocol of the manufacturer of the kit prokachivanija (Roche Co.). Serum ducks were subjected to dot-blot (2 points on the sample) on nitrocellulose membrane and hybridized with a fluorescently labeled probe for the quantitative determination of DHBV DNA[3]. As internal standard used DHBV DNA in 40 ál+DHBsAg 100 μl. For amplification of fluorescence used fluorimetric reagent CDP-Star. Film scanning was used scanner Vuego Scan Brisa-620st), and for quantitative analysis of the blots used the Discovery Series Quantity One. The size of the blot was described as "volume" (volume=intensity×mm2).

2) the Level DHBsAg serum

Level DHBsAg was determined by the method of ELISA[5-8]and OP were obtained using reader for ELISA(Bio-TEK Co.) at 490 nm.

4. Statistical analysis

For each group used the paired t-test using SPSS software.

Results

1. Changes in the concentration of DHBV DNA in serum

Table 4.3
The titer of DHBV DNA in serum before, during and after the treatment
DHBV DNA (volume)
Before the treatmentWeek 1Week 2Week 3Week 4Week 5
Normal saline2055,9±521,81952,5±621,52048,6±692,32031,2±722,31886,0±641,32118,5±468,1
CMS-0172207,0±237,51991,8±378,9*1925,0±549,9*1743,9±555,0*1544,9±389,7*1742,7±437,0*
*Compared with the control saline solution, P<005.

1. Changes in the level DHBsAg serum

Table 4.4
OP DHBsAg serum before, during and after the treatment
Before the treatmentWeek 1Week 2Week 3Week 4Week 5
Normal saline0,949±
0,688
0,761±
0,540
0,892±
0,762
0,867±
0,802
0,701±
0,673
0,871±
0,634
CMS-0170,928±
0,402
0,761±
0,328
0,668±
0,310*
0,551±
0,268*
0,479±
0,279*
0,488±
of 0.182*
*Compared with the control saline solution, P<0,05.

Conclusion

It was found that in a suitable concentration CMS-017 has a statistically significant antihepatitis properties in vivo compared with the control saline (P< 0,05). CMS-017 can be used for treating disorders associated with viral infection.

Links:

The following links are included in this description by reference in its entirety.

1. Yaxi Chen, Guo shuhua Zhang Dingfeng, et al. Foundation and application of Chongqing duck hepatitis B model. Chinese Journal of Hepatology. 1993; 1(2):89-91.

2. Yaxi Chen, Guo shuhua, Chen Xuehua. Preparation and application of DHBV DNA probe labeled with digoxin. Journal of Chongqing University of Medical Sciences. 1994; 19(4):295-297.

3. Tang Ni, Huang Ailong, Guo shuhua, et al. Systemic foundation and application of serological parameters of humoral immunity to duck hepatitis B virus. Chinese Journal of Hepatology. 2001; 9(1):13-15.

4. Tang Ni, Huang Ailong, Guo shuhua, et al. Purification of Duck hepatitis B surface antigen and its applications. Journal of Chongqing University of Medical Sciences. 2001;26(l):14-16.

5. Tang Ni, Huang Ailong, Guo shuhua, et al. A comparison of specifically immune response in DHBV-infected ducks. Chinese Journal of Hepatology. 2001; 9(3): 166-168.

6. Tang Ni, Huang Ailong, Qi Zhenyuan, et al. Immune response of acutely infected ducks to duck hepatitis B virus. Chinese Journal of Microbiology and Immunology 2000; 2(4):24-29.

7. Yaxi Chen, Guo shuhua, Qi Zhenyuan, et al. An experimental study of lamivudine against duck hepatitis B virus in combination with famciclovir. Chinese Journal of Hepatology. 2001; 9(4):209-211.

8. Qiu Zhi-qiang. The influence of Fuzheng buxuegao to blood system and immunity function affected by radiotherapy. Lanzhou Medical Transaction, 2003,3 (28).

Example 5

The inhibitory activity of CMS024-16 on transplanted bare mice xenograft carcinoma of the stomach of a man BGC-823

1.Materials

1.1.Drugs and reagents

CMS024-16: Synthesized on the order of Shenzhen Kangzhe Pharmaceutical Co. Ltd., Shenzhen, PR China.

5-Fu: Tianjin Jinyao Amino Acid Co., Tianjin, PR China.

Siva is denied the fruits of calves: Hyclone, Logan, Utah.

The cell culture medium RPMI-1640: GIBCO®, Invitrogen, Carlsbad, California, USA.

1.2.Animals

Healthy females naked mice BALB/c (nu/nu), a separate group that does not contain pathogens (SPF), at 4-5 weeks of age were obtained from the Academy of military medicine, China.

1.3.Cell line

Cell line BGC-823 cell carcinomas of the human stomach: Cancer Research Department, China Medical Science Institute.

2.Methods

2.1.Getting the animal model and the determination of antitumor action[1]

Cell line BGC-823 carcinoma of the stomach of the man in log phase was brought to a concentration of 2×107/ml and then were inoculable subcutaneously in the back of hairless mice (0.1 ml per mouse). Inoculated animals were randomly distributed into groups CMS024-16 (160 µg/kg/day 320 mg/kg/day and 640 mg/kg/day, all at 0.2 ml/day), positive control (5-Fu, 20 mg/kg/day 0.2 ml/day) and negative control (normal saline, 0.2 ml/day). The introduction of the test substances by intraperitoneal injection was started the next day after implantation of the tumor, 1 time a day for 30 days without interruption. The day after the last injection of tumor mass was collected and determined their mass. The rate of inhibition=(Average weight of negative control-Average weight of test group)/(Average weight of negative control)×100%

2.2.Statistical analysis

The results were expressed as mean ± standads. For statistical analysis used ANOVA SPSS. Statistically significant took values of P <0,05.

3.Results

Table 5.1
The inhibitory activity of CMS024-16 on transplanted bare mice xenograft carcinoma of the stomach of a man BGC-823
GroupDoseNThe tumor weightThe rate of inhibition (%)
CMS024-16640 mcg/kg/day81,31±0,77*54,4*
CMS024-16320 mg/kg/day81,59±1,017*44,7*
CMS024-16160 µg/kg/day81,32±0,68*54,0*
5-Fu20 mg/kg/day91,70±0,70*40,8*
Normal saline0.2 ml/day82,87±1,05-
* Compared with a group of normal saline, p<0,05.

4.Conclusion

It was found that CMS024-16 dose 160-640 mg/kg/day is able to inhibit the growth of transplanted bare mice xenograft carcinoma of the stomach of a man BGC-823 statistically significant compared with the group of normal saline (P<0,05).

Link

The following link is included in this description by reference in its entirety.

1. Li XH, Zhang GY, Luo FJ, Xu MH, Li Q. The effect of Helicobacter pylori on the expression of metallo proteinases in gastric carcinoma cell lines. World J Chinese Digestion, 2003, ll(5):544-546.

Example 6

Inhibitory effect of peptides on the hepatocarcinoma H22 in vivo

Aim:To investigate the influence of peptides on the growth of hepatocarcinoma H22 in mice.

Methods: BALB/c mice were divided randomly into control group of saline, 5-Fu, normal control peptides. Cell carcinoma of the liver H22 transplanted by intraperitoneal injection, and the test substance was also introduced by intraperitoneal injection of 1 times in day. Recorded survival time of mice and against the Vali with the controls.

Results: It was found that at doses of 80 mcg/kg/day CMS-024.04, CMS-024.05, CMS-024.14, CMS-024.16 can prolong the life of mice with transplanted tumor cells H22 statistically significantly compared with the control group saline (P<0,05).

Conclusion: It was found that the CMS-024.04, CMS-024.05, CMS-024.14 and CMS-024.16 can prolong the life of mice with transplantirovannam liver cancer H22, which indicates the possibility of using these peptides for cancer treatment.

1.Materials and methods

1.1.Drugs and reagents

CMS-024.04, CMS-024.05, CMS-024.14 and CMS-024.16 cooked to order, Shenzhen Kangzhe Pharmaceutical Co. Ltd., Shenzhen, PR China.

5-FU (5-fluorouracil) was from Tianjin Jinyao Aminophenol Ltd., Tianjin, China.

Saline was from China OTSUKA Pharmaceutical Co. Ltd., Tianjin, China.

RPMI-1640 and serum fruits calves (FBS) were from GIBCO®, Invitrogen, Carlsbad, California, USA.

A solution of D-Hanks was from Sigma Chemical Co., St. Louis, Missouri, USA.

1.2.Animals

Healthy mice BALB/c (category CLA, 6-8 weeks, weight 18-22 g) were from the animal Center of Academy of military medicine, Beijing, China.

1.3.Cell line

Mouse-native cell line H22 carcinomas of the liver were from Tumor Department of Medical Institute of China Medical Academy of Science, Beijing, China.

1.4.The formation of groups of animals

Healthy BALB/c mice were randomly included in the CMS group-024.04, CMS-024.05, CMS-024.14 and CMS-024.16 (80 mg/kg/day), 5-Fu (20 mg/kg/day, once in two on the I), physiological saline (0.2 ml/day) and normal group (without inoculation of tumor cells).

1.5.Introduction murine models of carcinoma of the liver H22

Mice inoculated with hepatocarcinoma, scored after 6-8 days. Collected ascites and cell concentration was brought to 5×107/ml solution of D-Hanks. 0.2 ml content inoculable intraperitoneally to each mouse BALB/c mice, excluding normal mice control.

1.6.The introduction of the test substance

The introduction of the test substance was started the next day after inoculation of tumor cells. Peptides and saline were injected daily and 5-FU twice a day for 60 days without interruption until the death of the mice.

1.7.Record survival time

I recorded the time of death and expected lengthening survival time. Lengthening survival time was calculated as follows:

Lengthening survival (%)=(Average number of days of survival of the tested groups(Average number of days of survival of the control group saline solution)/(Average number of days of survival of the group of physiological solution)×100%.

Mice that survived for more than 60 days were considered as long-lived.

1.8.Statistical method

For statistical comparisons used the method of Kaplan-Meier, and values of P equal to or less than 0,05, take the Ali as statistically significant.

2.Results

The experiment was conducted in two different time periods with the results below.

Table 6.1
The effect of peptides on the survival of mice with transplanted hepatocarcinoma H22
GroupDoseNSurvival time (days)Survival time (days)Lengthening survival (%)
CMS-024.0480 ág/kg1619,0±0,920,2±4,4*15,8*
CMS-024.0580 ág/kg1619,0±1,019,3±2,4*10,4*
Control saline0.2 ml/day 18,0±0,317,4±2,1-
5-Fu20 mg/kg1630,0±2,032,5±6,3*84,5*
Normal-166060-
* Compared with control group of saline, P<0,05.
Table 6.2
The effect of peptides on the survival of mice with transplanted hepatocarcinoma H22
GroupDoseNSurvival time (days)Survival time (days)Lengthening survival (%)
CMS-024.1480 is kg/kg 1616,0±0,027,6±8,5*50,3
CMS-024.1680 ág/kg1618,0±1,029,0±7,9*57,8
Control saline-1616,0±0,118,4±3,3-
5-Fu20 mg/kg1618,0±0,424,5±11,1*33,3
Normal-166060-
* Compared with gr is ppoi control saline, P<0,05.

3.Conclusion

It was found that the CMS-024.04, CMS-024.05, CMS-024.14 and CMS-024.16 can prolong the life of mice with transplantirovannam liver cancer H22 statistically significant compared with control group of saline, which indicates the possibility of using these peptides for cancer treatment.

Links:

The following links are included in this description as a reference in their entirety.

[1] Zhai Y and ZJ Lu. Effect of thalidomide on tumor growth in mouse hepatoma H22 model. Ai Zheng, Dec 2003; 22(12):1301-6.

[2] Yang YX, Zhu L, X He, et al. Antitumor activity of mitoxantrone - nanosphere against murine liver tumor H22. Sichuan Da Xue Xue Bao Yi Xue Ban, Jan 2004; 35(l):68-70.

Example 7

The effect of CMS-030 on induced Karenina swelling of the limbs in rats

Purpose: To investigate the anti-inflammatory properties of CMS-030.

Methods: To study the anti-inflammatory properties of CMS-030 used animal model induced Karenina swelling of the extremities.

Results: It was found that at a dose of 2-20 µg/ml of CMS-030 able statistically significantly (P<0,01) to suppress the swelling of the limbs in the group with treatment compared with control groups.

Conclusion: it Was found that the CMS-030 has a statistically significant anti-inflammatory effect in animal models of induced Karenina swelling of the limbs in rats.

1.Materials and m is proposed

1.1.Drugs and reagents

CMS-030: synthesized on the order of Shenzhen Kangzhe Pharmaceutical Co. Ltd., Shenzhen, PR China.

Ceragenin: Sigma Chemical Co., St. Louis, Missouri, USA.

Dexamethasone (DXM): Tianjin JinYao Co. Ltd., Tianjin, PR China.

1.2.Animals

Female Wistar rats aged 6-8 weeks, weighing 180-220 g, Vitalriver Experiment Animal Co. Ltd., Beijing, PR China.

1.3.Methods[1]

The Wistar rats were randomly distributed in the DXM group (0.2 mg/kg/day), group control saline (1 ml/day) group three doses of CMS-030 (2, 10, 20 μg/ml/day). All drugs were diluted to 0.5 ml of saline and injected intraperitoneally (b) daily. After two weeks of drug administration on the right foot struck 0.15 ml of 1% Karenina in saline by subcutaneous injection. After 0.5 hour accurately measured the circumference of the right limbs and swollen limbs was calculated by subtracting the circumference to provocative tests of the value after provocative tests. The index of inhibition (%)=(swelling of a limb in the control group of saline-swelling of a limb in the group with medicine)/swollen limb in the control group saline solution×100%.

1.4.Statistical analysis

Statistical analysis was performed using one-way ANOVA analysis.

2.Results

Table 7.1
Anti-inflammatory effect of CMS-030 in relation induced Karenina swelling of the limbs in rats
GroupDoseNCircle up provocative tests (cm)Swelling (cm)The index of inhibition (%)
0.5 hour0.5 hour
CMS-0302 mcg/kg/d102,78±0,063,20±0,87**37,0**
CMS-03010 µg/kg/d10was 2.76±0,033,10±0,10**50,2**
CMS-03020 mg/kg/d102,74±0,043,12±0,05**43,8**
DXM0.2 mg/kg/d92,49±0,052,74±0,10** 63,0**
Fiziol. solution0.5 ml/d102,80±0,063,47±0,08-
** Compared with control group of saline, P<0,01.

Conclusion

Was established animal model of acute inflammation in vivo in the form of induced Karenina swelling of the limbs in rats and it was used to evaluate anti-inflammatory action of drugs[2]. Ceragenin able to induce excessive prostaglandin synthesis at the site of inflammation. Together with other vasoactive substances excessively produced prostaglandin usually induces local swelling. It was found that the CMS-030 able statistically significantly (P<0,01) to inhibit the swelling of the limbs, induced Karenina in rats. Thus it has been shown that the CMS-030 has anti-inflammatory properties.

Links:

The following links are included in this description as a reference in their entirety.

[1] Li Jinhua, Zhang Huiqing, Kezhi Zheng, et al. Inhibitory effects of Orgotein on the swelling of hind paw in rats induced by carrageenin. Suzhou University Journal of Medical Science, 2002, VoI, 22(4):386-388.

[2] Huang Zhili, Kagoshima Masatoyo, Kagawa Eiichiro, Anti-inflammatory and ulcerogenic effects of 3-(N,N-diethylamino) propylindometacin HCl. Acta Pharmacologica Sinica, 197, VoI, 18(4):306-308.

Example 8

The effect of CMS-030 on obesity

Purpose: To determine the effect of CMS-030 anti-obesity using animal models pericardium rats.

Methods: A model was developed obesity by feeding rats a highly nutritious diet for 6 weeks. Rats were then treated CMS-030 (subcutaneously at doses of 150, 300 and 600 mg/kg/day) or saline for 4 weeks. Rats were weighed once a week and scored to measure abdominal and semenkovich fat bodies and blood lipids at the end of the experiment.

Results: It was found that the CMS-030 able to reduce body weight, the performance of the fat bodies, serum triglycerides and total cholesterol in the serum of rats was statistically significant compared with the control group saline (P<0,05).

Conclusion: CMS-030 has anti-obesity properties and can be used for treating disorders diet-induced obesity.

Materials and methods

1.Materials

1.1.The test substance and animals

CMS-030 was synthesized on the order of Shenzhen Kangzhe Pharmaceutical Co. Ltd., Shenzhen, PR China.

Male rats Sprague-Dawley (SD), a separate group that does not contain pathogens, weight 135±15 g: The Experimental Animal Center of First Military Medical University.

1.2.Kits

Triglyceride set: Shanghai Rongcheng Biotechnology Laoratory.

Set for total serum cholesterol: Zhongsheng Beikong Biotechnology Holding Ltd.

2.Materials

Highly nutritious and normal food was prepared in accordance with the provisions of The Guideline for Pre-clinical Research of Anti-obesity Drug, issued by State Food and Drug Administration, PR China (SFDA)[1].

Model of obesity[2]was obtained by feeding rats a highly nutritious diet for 6 weeks. Rats with obesity then processed CMS-030 (150, 300 and 600 mg/kg/day, once a day, subcutaneously) or saline for 4 weeks. During the administration of the test substance all the rats were kept on a normal diet. Rats were weighed once a week and killed at the end of the experiment to measure abdominal and semenkovich fat bodies and mass of the spleen, liver, kidney and thymus. Took the blood of rats for analysis of blood lipids.

The index of the fat bodies was calculated as follows: weight of body fat (g)/body weight (g)×1000.

The index was calculated as follows: body weight (g)/ body weight (g) x 1000.

3.Statistical analysis

Data are presented as mean ± standard deviation. For comparison within each group or between groups used the paired t-test or one-way ANOVA analysis using the DAS program (Drug And Statistics Ver 1.0). P<0.05 is accepted as statistically significant.

4.Results

Table 8.1
The effect of CMS-030 on body weight of rats (unit:gram)
Groupn0 weeks1 week2 weeks3 weeks4 weeks
Normal control10224,6±
the 15.6*
252,0±
18,0*
277,9±
20,2*
305,0±
26,9*
332,0±22,7*
Control saline10342,6±
23,8
386,2±
26,9
386,1±
24,6
410,8±
25,7
408,8±24,5
CMS-030:
600 mg/kg/day
12339,0±
27,0
365,4±
25,7
355,1±
24,6*
361,7±
32,6*
369,9±30,0*
CMS-030:
300 mg/kg/day
12338,0±
22,8
369,7±
26,1
359,6±
23,7*
364,3±
22,6*
366,3±24,5*
CMS-030:
150 mg/kg/day
12327,0±
26,9
348,9±
23,9*
346,5±
22,7*
351,8±
25,0*
354,0±26,3*
*Compared with the control saline, *p<0,05.
Table 8.2
The effect of CMS-030 on the index of the fat bodies of rats
GroupnThe index of body fatThe index of body fat testes
Normal control104,3±1,2*4,6±0,9*
Control saline106,0±1,85,5±0,9
CMS-030: 600 mg/kg/day124,5±1,1*5,0±0,7*
CMS-030: 300 mg/kg/day123,8±2,1*4,0±1,1*
CMS-030: 150 mcg/kg/day123,9±1,6*3,6±0,9*
*Compared with the control saline, *p<0,05.
Table 8.3
The effect of CMS-030 on triglycerides (TG) and blood total cholesterol (TCH) in rats
Groupn TG (mg/DL)TCH (mg/DL)
Control saline8to 101.8±31,2to 333.3±24,5
Normal control836,4±9,1*225,7±55,0*
CMS-030: 600 mg/kg/day1240,6±7,9*to 228.1±39,8*
CMS-030: 300 mg/kg/day1132,7±6,7*234,9±39,1*
CMS-030: 150 mcg/kg/day1251,5±19,4*270,9±33,0*
*Compared with the control saline, *p<0,05.
Table 8.4
VL is of CMS-030 on food intake in rats (unit: g/day)
GroupOne weekTwo weeksThree weeksFour weeks
Normal control13,2±1,3*18,3±6,1*18,6±03517,6±2,7
Control saline11,7±2,315,5±3,018,21±0,4318,4±0,59
CMS-030: 600 mg/kg/day13,8±5,415,9±1,718,2±0,6918,5±0,61
CMS-030: 300 mg/kg/day13,2±4,816,9±1,618,1±0,7418,4±0,59
CMS-030: 150 mcg/kg/day 13,2±3,116,1±1,818,3±0,6717,8±0,53*
*Compared with the control saline, *p<0,05.
Table 8.5
The effect of CMS-030 on indexes organs in rats
GroupnThe index of thymusThe index liverIndex of spleen
Control saline121,1±0,325,5±2,32,0±0,3
Normal control81,3±0,325,5±1,22,4±0,2*
CMS-030: 600 mg/kg/day12 1,1±0,224,3±2,22,0±0,2
CMS-030: 300 mg/kg/day121,0±0,323,1±1,3*2,0±0,2
CMS-030: 150 mcg/kg/day120,9±0,225,9±3,62,0±0,2
*Compared with the control saline, *p<0,05.

Conclusion

It was found that the CMS-030 in a suitable dose can cause weight loss and reduce the levels of blood lipids in rats with dietary obesity was statistically significant in comparison with the control saline (P<0,05). During the introduction of substances appetite has not changed significantly. CMS-030 may be suitable for treatment related to overeating, obesity and hyperlipidemia.

Links:

The following links are included in this description as a reference in their entirety.

1. SFDA, PR China. The guideline for pre-clinical research of new drugs. 1993. 193-194.

2. Bays HE. Current and investigational anti-obesity agents and obesity terapeutic treatment targets. Obes Res. 2004; 12 (8):1197-1211.

Example 9

The effect of CMS-030 on the delayed hypersensitivity type mice

Purpose: To investigate the inhibitory effect of CMS-030 on the delayed hypersensitivity type (DTH) in mice.

Methods: To demonstrate the immunosuppressive actions of CMS-030 was used induced by 2,4-dinitrofluorobenzene (DNFB) swelling of the ear.

Results: It was found that at 10 mg/kg/day CMS-030 able to suppress induced by DNFB the ear swelling in mice was statistically significant compared with the control saline (P<0,01).

Conclusion: CMS-030 has immunosuppressive properties and may be suitable for the treatment of immune-related disorders.

1.Materials and methods

1.1.Drugs and reagents

CMS-030: Synthesized on the order of Shenzhen Kangzhe Pharmaceutical Co. Ltd., Shenzhen, PR China.

2,4-dinitrophenol (DNFB): Smack Co. Ltd.

Sodium sulfide (Na2S): Tianjin Beilian Chemical Co. Ltd., Tianjin, PR China.

1.2.Animals

Mouse Balb/c separate groups that do not contain pathogens (SPF), aged 6-8 weeks, weighing 18-22 g: Academy of military medicine, PR China.

1.3.Methods

BALB/c mice were randomly divided into control group saline (0.5 ml/day) and CMS-030 (10 µg/kg/day). The test substance was dissolved in 0.5 ml of saline and injected inside rushino 1 time a day for two weeks before sensitization.

In mice on the stomach was removed coat using an 8% solution of Na2S one day prior to sensitization. DNFB dissolved in a mixture of acetone/olive oil (4:1) to a final concentration of 1% and 50 µl was applied to the exposed area for sensitization. Four days after the initial sensitization 10 µl of 1% solution of DNFB was applied topically to the right ear to the call of inflammation of the delayed hypersensitivity type. The same volume of solvent without DNFB was applied to the left ear as a reference point. 24 hour used a 6-mm punch to get a piece of fabric ear the same localization of the left and right ear and the fabric is carefully weighed. The ear swelling was calculated by subtracting from the mass of tissue from the right ear tissue mass of the left ear of the same mouse[1]. The rate of inhibition (%)=(Swelling of the ear with the control saline-Swelling of the ear in the test group)/swelling in the ear with a control saline solution×100%.

1.4.Statistical analysis

Statistical analysis was performed one-way ANOVA analysis using SPSS.

2.Results

Table 9.1
Inhibitory effect of CMS-030 on DH in mice
GroupDose nSwelling of the ear (mg)The rate of inhibition (%)
CMS-03010 mcg/kg/day173,69±2,31*46,5*
Saline0.5 ml/day186,91±2,50-
* Compared with control group of saline, p<0,01.

3.Conclusion

It was found that the CMS-030 able to suppress the response of the delayed hypersensitivity type mice on DNFB statistically significantly compared with the control saline (P<0,05). This indicates that the CMS-030 can be used for the treatment of immune disorders associated with hypersensitivity.

Link:

The following link is included in this description by reference in its entirety.

[1] Li Weidong, Ren LianSheng, Lin Zhibin, et al. Preliminary study on immunomodulating actions of Actarit in mice. Journal of Beijing Medical University, 2000, VoI, 1(32): 1-3.

Example 10

Research immunosuppressive properties of CMS-030 in vivo

Purpose: To investigate the ability of CMS-030 to counteract the rejection of the allograft and the prob is mportant mechanisms of action.

Methods: Immunosuppressive properties of CMS-030 was observed using test proliferation of T-lymphocytes and the mixed reactions of lymphocytes (MLR) in vitro. The effect of CMS-030 on allograft survival in animal models of allograft skin and cardiac muscle of mice in vivo. We also observed the effect of CMS-030 on transformed T-cells and secretion of IL-2 by spleen cells of the allograft recipient.

Conclusion: Observed the ability of CMS-030 to suppress the reaction of rejection of the allograft. It can oposredovanie by suppressing the activity of T-lymphocytes and the secretion of IL-2 by lymphocytes.

1.Materials and methods

1.1.Animals

Five-week male and female Balb/c mice (H-2b) and C57BL/6J (H-2d) separate groups that do not contain pathogens: The Institute for Laboratory Animals of Military Medical Academy of Science (Beijing, China). Half males and half females.

Newborn mouse57BL/6J were their own cultivation.

1.2.Drugs and other chemicals

CMS-030: Synthesized on the order of Shenzhen Kangzhe Pharmaceutical Co. Ltd., Shenzhen, PR China.

Cyclosporine (CsA): Novartis Pharmaceutical Co. Ltd., Basel, Switzerland. Dilute to a final volume of 0.5 ml for all groups CsA.

Serum of cows, RPMI-1640, Hanks solution: GIBCO®, Invitrogen, Carlsbad, California, USA.

MTT, ConA: Sigma Chemical Co., St. Louis, Missouri, USA.

NaS: Tianjin Beilian Fine Chemicals Co., Ltd., Tianjin, PR China.

The ELISA kit for IL-2 mouse: R&D Systems Inc., Minneapolis, Minnesota, UA.

1.3.Groups of animals

To test in vivo mice were randomly distributed in groups

CMS-030 (10 µg/kg/day).

CMS-030 (2 mg/kg/day).

CsA (10 mg/kg/day).

Normal saline (0.5 ml/day).

The solutions were administered by intraperitoneal injection once a day for 5 days before transplantation and continued for 20 days after the operation.

1.4.The effect of CMS-030 on the proliferation of T-lymphocytes in vitro[1]

Spleen healthy Balb/c mice sterile were prepared and immersed in chilled on ice, a solution of D-Hank's. The spleen cells were obtained by disruption of the spleen frosted glass plates in RPMI-1640. Cells are washed twice in RPMI-1640 at 4°C and 1200 rpm for 10 min the Cells were then counted and brought to the concentration required for each analysis. Cell viability in the experiment was determined by the exception Trypanosoma blue and had to be more than 95%.

The spleen cells from normal mice were cultured in 96-well tablets with increase of concentration up to 4×105on the hole. To the wells were added CMS-030 at a final concentration of 40 μg/ml, 8 μg/ml, and 1.6 µg/ml and 0.32 µg/ml. the plates were incubated in humidified atmosphere at 37°C, 5% CO2for 68 hours in the presence of concanavalin A (ConA) at a final concentration of 5 µg/ml In the positive control group CMS-030 not doba is ranged. Lymphocyte proliferation was measured by colorimetric analysis with 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) at the end of incubation. To each well was added 20 μl of MTT and tablets additionally incubated for 4 hours. Then to each well was added 100 μl of 0.04 M HCl in isopropyl alcohol to dissolve formisano precipitate. The OD of each sample was measured spectrophotometrically at 570 nm correlated with 630 nm.

1.5.The effect of CMS-030 on mixed lymphocyte reaction (MLR) in vitro[2]

Spleen cells taken from healthy Balb/c mice were washed in chilled on ice with a solution of D-Hank's and resuspendable in RPMI-1640 up to 8×106/well for the formation of reactive cells. Suspension of spleen cells from normal mice C57BL/6J were incubated with mitomycin (25 μg/ml) for 45 minutes in RPMI-1640 at 37°C and 5% CO2, then washed with RPMI-1640 and resuspendable up to 8×106/well for the formation of stimulating cells. Using 96-well culture plate to test groups, add 100 mcg reactive cells, 100 μg stimulating cells and 20 μl of CMS-030 at a final concentration of 40 μg/ml, 8 μg/ml, and 1.6 µg/ml and 0.32 µg/ml In the positive control group was added 100 μl of reactive cells, 100 µl of stimulating cells and 20 μl of RPMI-1640. In the negative control group was added 200 μl of stimulating cells is 20 ál of RPMI-1640. For each combination used six parallel wells. After 5 days of incubation at 37°C and 5% CO2lymphocyte proliferation was measured by colorimetric analysis MMT.

1.6.The effect of CMS-030 on survival of heart transplant in vivo[3]

Heart donors were prepared from mice C57BL/6J 24 hours after birth. The heart was immersed in a solution of D-Hank's and removed the blood from the cavity of the heart. Hearts transplanted subcutaneously into the passages of the ear healthy adult recipients of Balb/c mice. The air from the heart and pass said light pressure. Starting from the sixth day after the transplantation, daily rented an electrocardiogram (ECG) transplanted heart. The absence of ECG signal for 3 days in a row pointed to a failed transplant surgery, and the mice were excluded from statistical analysis. ECG was monitored daily, and the rejection time was considered the day when the ECG signal was gone. The test substance was administered intraperitoneally, beginning 6 days after surgery. The group had 10 mice. Cyclosporine A was administered at a dose of 10 mg/kg/day, CMS-030 - the dose of 10 mg/kg/day or 2 mg/kg/day, and saline 0.5 ml/kg/day. Statistical analysis was performed using log-rank criterion Kaplan-Meier estimates for comparison with the group treated with saline solution.

1.7.The effect of CMS-030 on the lid skin allograft in vivo [4]

The area of hair on the back of Balb/c mice was removed with 8% solution of Na2S. the next day was created by the wound surface approximately 1 cm2surgical removal of the skin, and then place the wound surface was placed a piece of skin 1 cm2the full thickness of the tail of the appropriate gender of the mouse donor C57BL/6J. The operative field was closed and protected by a layer of paraffin gauze and covered with plaster. 8 days after transplantation, the patch was removed, and the mouse-recipient controlled daily for signs of allograft viability. For the end point of exclusion took when only less than 10% of allograft remained viable. Intraperitoneal treatment of the tested substances was started 6 days prior to surgery. The group had 10 mice. Cyclosporine A was administered at a dose of 10 mg/kg/day, CMS-030 - the dose of 10 mg/kg/day or 2 mg/kg/day, and saline 0.5 ml/kg/day. The treatment continued for another 20 days after the operation.

1.8.The effect of CMS-030 on the proliferation of T-lymphocytes in vivo[5]

Spleen cells taken from mice recipient graft skin were isolated and resuspendable in RPMI-1640 to 4×106/ml In 96-well plate was made in 100 μl of cells/well. In the wells for testing was added 100 μl of ConA to a final concentration of 5 µg/ml At entry checkpoints for important locations the e wells instead was added 100 μl of RPMI-1640. Four parallel wells for each combination. Cells were incubated for 68 hours at 37°C and 5% CO2. Lymphocyte proliferation was measured by the colorimetric MTT analysis. The OD of each sample was measured spectrophotometrically at 570 nm correlated with 630 nm. Data are presented as stimulation index, which was a OP test group divided by the OD of the control group.

1.9.The effect of CMS-030 on the level of IL-2 in vivo[6]

Spleen cells taken from mice recipient graft skin were isolated and resuspendable in RPMI-1640 to 2×106/ml 1.5 ml of cells per well was added to 24-well plate and incubated for 24 h for attachment. Added 100 μl of ConA to a final concentration of 10 µg/ml Cells were incubated for another 48 hours, after which the collected supernatant by centrifugation. The level of IL-2 in the culture supernatant were determined using ELISA.

1.10.Statistical analysis

For analysis of survival times allograft used the log-rank criterion Kaplan-Meier comparison with the control group. For other experiments used the comparison of the average with variance 2-sided t-student test.

2.Results

Table 10.1
The effect of CMS-030 on the proliferation of T-lymphocytes in vitro
GroupDose medicationsNo.OP
CMS-03040 mg/ml6of 0.332±0,062*
CMS-0308 mcg/ml60,281±0,041*
CMS-0301.6 ĩg/ml60,311±0,027*
CMS-0300,32 mg/ml60,315±0,043*
The positive control-60,421±0,055
Negative control-60,109±0,003*
* Compared with control group fisiologicas the solution, p<0,01.
Table 10.2
The effect of CMS-030 on mixed lymphocyte reaction (MLR) in vitro
GroupDose medicationsNo.OP
CMS-03040 mg/ml60,192±0,019*
CMS-0308 mcg/ml60,285±0,004*
CMS-0301.6 ĩg/ml60,361±being 0.036*
The positive control-60,440±0,043
Negative control-60,145±0,019
* Compared to g is uppoi control saline, p<0,01.
Table 10.3
The effect of CMS-030 on survival of heart transplant in vivo
GroupDose medicationsNo.The average survival time (days)
CMS-03010 µg/kg/d912,0±2,2*
CMS-0302 mcg/kg/d911,5±1,9*
CsA10 mg/kg/d813,8±1,3*
Saline0.5 ml/d109,1±1,4
* Compared with control group of saline, p<0,05.
Table 10.4
The effect is the development of CMS-030 on the survival of allograft skin in vivo
GroupDose medicationsNo.The average survival time (days)
CMS-03010 µg/kg/d914,1±1,2*
CMS-0302 mcg/kg/d1013,4±1,5
CsA10 mg/kg/d915,0±1,4*
Saline0.5 ml/d911,0±1,3
* Compared with control group of saline, p<0,01.
Table 10.5
The effect of CMS-030 on the proliferation of T-lymphocytes in vivo
GroupDose medications No.The stimulation index
CMS-03010 µg/kg/d91,6±0,3*
CMS-0302 mcg/kg/d101,9±0,5*
CsA10 mg/kg/d91,8±0,3*
Saline0.5 ml/d92,3±0,5
* Compared with control group of saline, p<0,01.
Table 10.6
The effect of CMS-030 on the level of IL-2 in vivo
GroupDose medicationsNo.IL-2 (PG/ml)
CMS-030 10 µg/kg/d9599,0±level of 121.8*
CMS-0302 mcg/kg/d10577,4±to 163.1*
CsA10 mg/kg/d9595,2±162,8*
Saline0.5 ml/d9787,4±227,8
* Compared with control group of saline, p<0,01.

Conclusion

It was found that at a concentration of from 0.32 to 40 μg/ml of CMS-030 capable of statistically significant inhibition of ConA-induced proliferation of T-lymphocytes in vitro, which indicates the ability of CMS-030 to inhibit the proliferation of T-lymphocytes in vitro. MLR is an in vitro experiment to determine the response of lymphocytes to different molecules HLA-II and is a model for prediction of potential rejection after organ transplantation[7]. It was found that at a concentration of from 1.6 to 40 μg/ml of CMS-030 capable of statistically inhib the encoded mixed lymphocyte reaction, that indicates the ability of CMS-030 reduce the potential rejection after organ transplantation.

Experiments with transplant hearts and allograft skin was an animal model to study the suppression of rejection after transplantation[7]. It was found that the CMS-030 at a dose of 2 mg/kg/day and 10 mg/kg/day is capable of statistically significantly prolong graft survival, which indicates the ability of CMS-030 to suppress graft rejection under the action of the host immune response. Analysis of spleen cells isolated from mice recipient of skin graft, showed that the CMS-030 able statistically to suppress and activation of lymphocytes and the secretion of IL-2 by T-lymphocytes, which indicates that the prolongation of allograft survival was achieved by suppressing the immune response of the animal recipient.

Links:

The following links are included in this description as a reference in their entirety.

[1] Roma Kalra, Shashi P. Singh, Juan C, et al. Immunosuppressive and Anti-Inflammatory Effects of Nicotine Administered by Patch in an Animal Model. Clinical and Diagnostic Laborarory Immunology, May 2004, 563-568.

[2] Dubey DP, Yunis I, Yunis EJ, et al. Cellar typing: mixed lymphocyte response and cell mediated lympholysis. American Society for Microbiology, 1986, 847-848.

[3] Vakeva A, Laurila P,Meri S, et al. Regulation of complement membrane attack complex formation in myocardial infarction. Am J Pathol, 1993,143:65.

[4] Ming Jiankuo, Wang Xingbing, Huang Baojun, et al. Peptide Nucleic Acid Antisense Prolongs Skin Allograft Survival by eans of Blockade of CXCR3 Expression Directing T Cells into the Graft. The Journal of Immunology, 2003,170:1556-65.

[5] Maria A. Puertollano, Manuel A. de Pablo, et al. Relevance of Dietary Lipids as Modulators of Immune Functions in Cells Infected with Listeria monocytogenes. Clinical and Diagnostic Laboratory Immunology. 2002, 9:352-357.

[6] Mayumi H,Himeno K,Shin T, et al. Drug-induced tolerance to allografts in mice. Immunobiology, 1985, 169(2):147-161.

[7] Rene J.Duquesnoy Li YP. Transplantation immunobiology. 2002, 10:5-7.

Example 11

The effect of peptides on exercise-induced fatigue in mice

Purpose: To investigate the effect of the peptides against fatigue in mice Balb/c mice.

Ways: The voyage of male Balb/c mice were used as animal model for studying the actions of the peptides against fatigue.

Results: It was found that the CMS-001.30 and CMS-001.31 can prolong the voyage mice was statistically significant compared with control (P<0,01).

Conclusion: CMS-001.30 and CMS-001.31 have the ability to reduce fatigue and can be used for treating disorders associated with fatigue.

1.Materials and methods

1.1.Medicine and chemicals

CMS-001.30 and CMS-001.31 were synthesized in order Shenzhen Kangzhe Pharmaceutical Co. Ltd., Shenzhen, PR China.

Erythropoietin (EPO): Japan Kunpeng Medical Corporation.

1.2.Animals

Male Balb/c mice separate groups that do not contain pathogens (SPF), weighing 18-22 g: Academy of Military Medicines and Sciences Experimental Animal Center, PR China.

1.3.The formation of groups and the method[1]

Male Balb/c mice were randomly divided into group the s CMS-001.30 (20 mg/kg/day), CMS-001.31 (20 mg/kg/day), EPO (1000 U/kg/day, three times a week) and a control saline solution. The test substance was dissolved in 0.5 ml of saline and injected intraperitoneally once a day for 30 days without interruption (EPO replaced with saline, if the EPO was not introduced). On the tenth day, the mice were trained to swim for 10 minutes at a water temperature of 25±1°C. After 30 min after the last injection of test substances mice were placed in a pool for swimming (50 cm×50 cm×40 cm). Water depth was 30 cm and water temperature 25±1°C. the limbs of the mice were moved through the entire process. Recorded time (min) swimming mice to death.

The increase of time exhausting voyage (ESTR) (%)=(Average time exhaustive swimming test group-Average time exhausting swimming group control saline solution)/(Average time exhausting swimming group control saline solution) ×100%.

1.4.Statistics

Differences between groups were analyzed using analysis of variance ANOVA

2.Results

Table 11.1
The effect of peptides on time exhausting swimming mice
Group DoseNo.The sailing time (min)ESTR (%)
CMS-001.3020 mg/kg/day20186,4±15,2*88,3*
CMS-001.3120 mg/kg/day20174,3±29,2*76,0*
EPO1000 U/kg/day, 3 times a week20126,1±20,4*31,3*
Saline0.5 ml/day20a 99.0±11,2-
* Compared with the group of saline, P<0,01.

3.Conclusion

It was found that the CMS-001.30 and CMS-001.31 have the ability to reduce fatigue and can be used for treating disorders associated with fatigue.

Link:

The following link is included in this description by reference in its entirety.

1. Mizunoya W, Oyaizu S, Ishihara K, et al.Protocol for measuring the endurance capacity of mice in an adjustable-current swimming pool. Biosci Biotechnol Biochm. 2002 May; 66(5):1133-1136.

On the basis of the above information from the disclosed peptides can be obtained by different pharmaceutical compositions. The pharmaceutical composition may include any of the known pharmaceutical carriers. Examples of suitable carriers include any of the standard pharmaceutically acceptable carriers known to specialists in this field of technology. They include, but are not limited to, physiological saline solution, water, emulsions, comprising a mixture of oil and water or emulsion of triglycerides, and other types of agents, fillers, coated tablets and capsules. Suitable carrier may be selected based on the method of administration of the pharmaceutical composition.

The pharmaceutical composition can be introduced through intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection, and subcutaneous implantation. The peptide can also type in any form of oral administration, such as, for example, tablet, capsule, suspension, solution and the like, in the usual form, without modification or in the form of slow release, or the presence or absence of gastrointestinal protection. The peptide can also type in any form of local application, such as ointment, cream, gel, etc. to facilitate transcutaneous device, or without it. The peptide can also be re which can be found in its genetic sequence and cloned in the expression system as such or in combination with other peptide sequences to obtain the resulting peptide molecules for use activity of the peptide, as described in this specification.

The dose of each peptide can be 1 ng to 10 g per kg of body weight. The preferred dose is 10 ng-10 mg / kg and more preferably 1 μg to 1 mg per kg for injection route of administration. However, the effective dose can be as low as 1 ng per kg of body weight, because one or more of the peptides may act through receptors that normally induce a cascade of normal physiological responses. Alternative one or more of the peptides can be simply initiated a cascade of reactions. For oral administration, the number can be 1 ng to 10 g per day per kg of body weight, more preferably 0.1 mg to 1 g per day per kg of body weight and even more preferably mcg 10 mg per day.

Based on the above peptide sequences, gene therapy can be carried out on the basis of methods known in the art, as well as on the basis of the patent publication WO 03/006492A2, which is incorporated in this description by reference in its entirety. The peptides may also be conjugated with other reinforcing molecules on the basis of indications disclosed in patent publication WO 2004/055042A1, which is incorporated in this description by reference in its entirety.

Links:

The following links are included in this description is tion as references in their entirety.

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

2. Shuyun Xu, Rulian Bian, Chen Xiu. Methodology of pharmacological experiment. People's Health Publishing House. 1991, 1221-1234.

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

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

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

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

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

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

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

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

Example 12

Delivery of peptides using genetically engineered bacterial strains Lactobacillus

Subsequent offered as one example of the method of delivery of the peptides of this invention to the owner, as described above. The DNA sequence that encodes one of the peptides listed in table 1 above, are synthesized by chemical methods and the sequence of the DNA insert in the expression vector using standard methods of genetic engineering, familiar to specialists in the field of technology. The selected expression vector contains a constitutive promoter, functionyou the th in Lactobacilli, a multiple cloning site for insertion of DNA sequences in a particular orientation from 5' to 3', as well as the selective marker gene, which confers resistance to the antibiotic (for assistance in cloning procedures), and may include other sequences to aid in the production and/or secretion of peptides, such as sequences of signal peptides. An example of such a vector is proposed in U.S. patent No. 5592908 owned by Pavla, which is included in this description by reference in its entirety. Briefly, this patent discusses several well-known promoters that work in strains of Lactobacillus and how disclosing new promoters in these bacteria, any of which may be operatively associated with a nucleic acid that encodes a peptide of the present invention, for expression of the peptide in Lactobacilli. Nucleic acid encoding a signal peptide, such as peptides comprising from 16 to 35 mostly hydrophobic amino acids, which are active in Lactobacillus lactis, described in U.S. patent No. 5529908, quoted above, is inserted between the promoter and the nucleic acid that encodes a peptide of the present invention, so that the nucleic acid encoding a signal peptide, is in reading frame with a nucleic acid that encodes a peptide of the present invention.

In addition the giving to the sequence, encodes a peptide synthesized DNA sequence may include sequences that facilitate legirovanie and cloning of a specified DNA in the expression vector. For example, the recognition sites of the enzymes, which are similar to those found in the multiple cloning site of the vector, can be incorporated into the synthesized DNA on the 5' and 3' ends of the sequence, so the sequence can be cloned in the vector in the correct orientation. As a vector, and the synthesized DNA hydrolyzing specific enzymes and then purified. For reactions ligation with the vector and synthesized DNA should be transformed into a suitable E. coli strain. Transformed bacteria are plated on medium containing the antibiotic to which the given vector resistance. Choose a colony of transformed bacteria for the growth of crops and procedures for obtaining plasmids; confirm the presence of the synthesized DNA in the correct orientation.

This expression vector is then transformed into the bacterial cell host strains of Lactobacillus, such as Lactobacillus acidophilus. Transformed cells are selected based on the property of selective marker found in the sequence of the vector, and the secretion of the peptide may be confirmed by performing Western blotting, run the gel-electropho is ESA peptides present in the growth environment, or other standard methods. A transformed colony of bacteria is chosen and used to derive large-scale cultures of genetically engineered bacteria. The culture of genetically engineered bacteria expressing the desired peptide, increasing and at least part thereof is introduced into the digestive tract, the vagina, the trachea or other areas of the host organism, in which bacteria are able to multiply. If this is desirable, bacterial culture can be treated in different ways to obtain supplements for consumption in the intestine of the host. This processing includes lyophilization or other methods of preservation of bacteria in addition to the combination of bacteria with agents-carriers, such as solutions, solvents, dispersion media, delay agents, emulsions and the like. The application of these agents to obtain additives well known in the art. For example, bacteria can be used to create a culture of dairy products or other foods for human consumption, so that the body expressing the peptide, colonize the digestive tract of the host body. A number of different ways including various strains of lactic acid bacteria in foods such as yogurt, Korean sauerkraut, the yr and oil, disclosed in U.S. patent No. 6036952 belonging to Oh, which is included in this description by reference in its entirety. When the consumption of bacteria through one of any number of paths constructed organisms may colonize the digestive tract and to enable the presentation and/or absorption of the peptides of this invention through the mucous lining of the digestive tract.

Example 13

Delivery of peptides using a genetically engineered form of Bacillus subtilis

Subsequent serves as another example of the method of delivery of the peptides of this invention to the owner, as described above. The DNA sequence that encodes one of the peptides listed in table 1 above, are synthesized by chemical methods and the sequence of the DNA insert in the expression vector using genetic engineering methods, and all methods known in the art. The selected expression vector includes a Shuttle vector, such as pTZ18R (Pharmacia, Piscataway, NJ), is able to replicate both in E. Coli and B. Subtilis, and containing the gene for resistance to the antibiotic for selection of transformed colonies of bacteria. This vector may contain a constitutive promoter, active in B. subtilis, such as a promoter derived from a gene Sac B B. subtilis, as well as nucleotide sequence, coderush the th signal peptide, active in B. subtilis, which manages the efficient export of expressed heterologous protein from the bacterial cells. An example of such a vector is disclosed in U.S. patent No. 6268169 owned Fahnestock, the disclosure of which is incorporated in this description by reference in its entirety. Briefly, as described above, DNA encoding the peptide of this invention, are synthesized with sites for enzymes and/or other sequences to facilitate cloning DNA using methods familiar to specialists in the field of technology. After transformation in E. coli, sowing, selection and replication of the plasmid to create a pool of plasmids, the plasmids are then transformed into B. subtilis and transformants are selected based on the property of resistance to the antibiotic in environments sowing.

Production of the peptide in genetically engineered B. subtilis and its secretion from it confirmed using methods well known to experts in the art, such as radioactive prokachivanie peptides for autoradiographical determine after analysis in DDS-Na, PAGE or Western blotting.

The culture of genetically engineered bacteria are increasing and at least part thereof is introduced into the digestive tract, the vagina, the trachea or other areas of the host organism, in which bacteria are able to multiply.

Note the R 14

Delivery of peptides using genetically engineered strains of Saccharomyces

Subsequent serves as another example of the method of delivery of the peptides of this invention to the owner, as described above. The DNA sequence that encodes one of the peptides listed in table 1 above, are synthesized by chemical methods and the sequence of the DNA insert in the expression vector using genetic engineering methods, and all methods known in the art. The selected expression vector includes a vector that stably supports the expression of yeast proteins, including constitutive yeast promoter, such as pADHl, sites, replication of the vector in yeast and in E. coli, the gene or genes that give prototropic auxotrophic yeast mutant for selection purposes, a multiple cloning site (MCS) and, if desired, sequences that encode a signal peptide. Vectors, such as this one, commercially available and well known in the art or can be easily constructed using standard methods. After inserting the synthesized DNA into a yeast vector, transformation in E. coli, sowing transformed E. coli on the selective environment, selection of transformed bacterial colonies and obtain plasmid DNA from rostov.anatoly bacteria from the selected colonies vector transform in Saccharomyces cerevisiae using well-known methods, such as transformation with lithium acetate or electroporation. The Saccharomyces cerevisiae strain selected for transformation, is an auxotrophic mutant strain, which will require the gene in the plasmid for growth on minimal media sowing. Transformed colonies of yeast produce with sowing the yeast growth medium with the absence of the gene provided by the vector. Only those yeast that has received vector and its selective gene and Express its gene product, will be able to grow in colonies on minimal media. Confirmation of the secretion of the peptide can be by performing Western blotting, perform gel electrophoresis of peptides present in the growth environment or other standard methods.

A transformed colony of yeast selected and used to obtain large-scale cultures. The culture of genetically engineered yeast expressing the desired peptide, increasing and at least part thereof is introduced into the digestive tract, the vagina, the trachea or other areas of the host organism, in which bacteria are able to multiply. If this is desirable, yeast culture can be processed using a variety of methods for obtaining supplements for consumption in the intestine of the host. This processing includes lyophilization or other methods of preservation of driven is in addition to the combination of bacteria with agents, carriers, such as solutions, solvents, dispersion media, delay agents, emulsions and the like. The application of these agents to obtain additives well known in the art. In another implementation of the transformed yeast is used to make food products such as fermented dairy products such as yogurt or kefir, using methods known to experts in this field of technology. As in the case of live cultures of lactobacilli in these foods, transformed yeast colonizing the digestive tract, at least temporarily, and serve for the presentation of peptides to the owner through the lumen of the intestine.

Although the present invention has been described using the above methods and data, as well as the specific examples described in the present description of the peptides in many cases, it is clear that this is only an example and should not be interpreted as limiting the present invention. It should also be understood that these described in this description of peptides represent an implementation of the present invention, and the same principle of the present invention can also be applied to other functionally equivalent peptides which are modified without affecting the biological function of these peptides. Moreover, although opisaneiye the disease or disorder for medical use of these peptides are listed to support their suitability, these medical applications are used only as non-limiting examples and should not be used to limit the scope of the claims. It is clear that there is another possible/intended use of these peptides and their functional derivatives, such as, but not limited to, use as a food additive to enhance or stimulate the immune system, relieve fatigue, reduce lactic acid in the blood of a normal subject or a patient with any infections. Any of these applications also falls in the scope of the present invention.

As for peptides with sequences that have been published previously, in the present invention proposes a new, unexpected, their use, and it is believed that this new evidence supports the use of some of these known peptides for industrial use, which has not previously been provided. In addition to new medical applications, as described above and in the claims, they can also be used as a dietary or nutritional supplements to improve the condition of the normal individual on the basis of the presented in this guidance.

The use of biologically active peptide, which represents the amino acid sequence of SEQ ID No.l, to obtain medicinal cf is DSTV for modulation, at least one of the following conditions: fatigue, the inventory level of glycogen in the liver and levels of lactic acid in the blood.



 

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31 cl, 3 tbl, 25 ex

FIELD: chemistry.

SUBSTANCE: method of obtaining dodecapeptide of the formula I: H-Asp-His-Leu-Asp-Lys-Gln-Thr-Gln-Thr-Pro-Lys-Thr-OH and tripeptide of the formula II: X-Asp(Y)-His-Leu-OH is the intermediate compound in its synthesis. Solid-phase synthesis of dodecapeptide I is realised by sequential growth of the peptide chain, beginning with the C-end dipeptidilpolymertill the obtaining of C-end nonapeptidilpolimer, which is condensed with the protected N-end tripeptide of the formula II: X-Asp (Y)-His-Leu-OH where X, Y are protected groups and the obtained dodecapeptidilpolymer is processed with an unblocking agent for removing the protective groups and the polymeric matrix and in 1 stage the end product is given out by means of HELC.

EFFECT: increasing the output of the end product and simplification of the process.

3 cl, 2 ex

FIELD: chemistry.

SUBSTANCE: invention relates to methods and reagents of labelling a vector such as a peptide, involving reaction of a compound of formula

with a compound of formula R*-L2-N3 (II) in the presence of a Cu (I) catalyst.

EFFECT: obtained labelled conjugates are useful as diagnostic agents, for example, as radiopharmaceutical preparations, more specifically for use in positron emission tomography (PET) or single-photon emission computed tomography (SPECT) or for radiotherapy.

7 cl, 13 ex

FIELD: chemistry.

SUBSTANCE: invention discloses use of peptide chemical molecules and also describes a pharmaceutical composition for stimulating growth hormone secretion and a composition used in veterinary to stimulate growth and/or exhibiting resistance to diseases in aquaculture or some other animal. The peptide chemical compounds are obtained via molecular simulation in silico, the structure of which enables the compounds to perform the functions as growth hormone peptide secretion stimulants.

EFFECT: invention enables to obtain peptide chemical compounds for stimulating growth and/or exhibiting resistance to diseases in aquaculture or some other animal.

8 cl, 12 dwg, 12 tbl, 11 ex

Peptide compounds // 2415149

FIELD: medicine, pharmaceutics.

SUBSTANCE: present invention refers to new peptide compounds, pharmaceutical compositions containing them, application of said compound for making a drug exhibiting activity with respect to hV1a receptor for treating related conditions, particularly depressed cases. The compounds are presented by general formula (I) which is specified in the description of the invention.

EFFECT: preparation of new peptide compounds.

12 cl, 1 tbl, 1 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to pharmacology and medicine. An agent represents peptide of general formula A-Pro-Tyr-B-X (I) where Pro can represent DPro, A is chosen from Ala, Gly, IIe, Leu, Met, Phe, Ser, Thr, Trp, Tyr, Val; B is chosen from Ala, Arg, Cly, IIe, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, Val; X-OH; -OCH3; -NH2; -Cly-Pro, and is described as perspective for producing new drugs able to reduce positive and negative symptomatology of schizophrenia and other psychoses.

EFFECT: offered is the agent exhibiting antipsychotic activity.

4 dwg, 9 tbl, 15 ex

FIELD: chemistry.

SUBSTANCE: invention relates to molecular pharmacology and specifically to a peptide which is part of an interleukine-15 (IL-15) sequence which can inhibit biological activity of the said molecule.

EFFECT: obtaining a peptide which inhibits T cell proliferation induced by IL-15, and apoptosis caused by tumour necrosis factor when bonding with the alpha subunit of the (IL-15R) receptor.

8 cl, 4 dwg, 5 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: present invention refers to application of peptide of formula (I): ((X)1(Y)m)n where peptide contains 3 to 200 amino acids and where 1, m and n represent integers within 0 to 10; X and Y which can be identical or different, represent cationic amino acids chosen from arginine and lysine in preparing drugs for treating fungal infection.

EFFECT: ensured applicability of peptide for preparing a drug for treating fungal infection.

26 cl, 53 dwg, 2 tbl, 19 ex

Rgd-like peptides // 2396271

FIELD: chemistry.

SUBSTANCE: invention discloses novel synthetic RGD-like peptides capable of dose-dependant inhibition of thrombocyte aggregation.

EFFECT: obtaining novel compounds capable of dose-dependant inhibition of thrombocyte aggregation.

2 tbl, 1 ex

FIELD: chemistry.

SUBSTANCE: invention relates to biotechnology and is a peptide which induces killer T cells ex vivo and which has an amino acid sequence as shown in one SEQ ID NOS: from 1 to 3. The disclosed peptide is used in an ex vivo agent for inducing anti-tumour immunity, in an ex vivo agent for inducing antigen-presenting cells, in an ex vivo agent which induces tumour-reactive T cells, as well as in an ex vivo pharmaceutical agent when treating or preventing tumours. The invention also relates to an antibody against the said peptide.

EFFECT: disclosed agents enable identification of glypican-3-derivative peptide, which can bond with HLA-A2, and activation of human killer T cells in order to provide an immunotherapy agent which may be effective in approximately 40% Japanese patients suffering from certain types of malignant tumours, accompanied by high level of GPC3 expression.

7 cl, 4 dwg, 1 tbl, 2 ex

FIELD: chemistry.

SUBSTANCE: invention relates to contrast agents which contain a peptide vector linked with uPAR, marked with a visualising group.

EFFECT: obtaining a contrast agent for detecting urokinase plasminogen activator receptor.

6 cl, 6 ex

Peptide compounds // 2393167

FIELD: chemistry.

SUBSTANCE: invention relates to novel peptide compounds and their use in diagnostic optical visualisation techniques. More specifically, the present invention pertains to use of such peptide compounds as targeted vectors which are related to receptors associated with angiogenesis. The compounds are labelled using at least one cyanine reporter dye.

EFFECT: obtaining compounds which can be used as contrast agents in optical visualisation when diagnosing diseases related to angiogenesis.

9 cl, 5 ex

FIELD: chemistry; biochemistry.

SUBSTANCE: invention relates to bioengineering and specifically to obtaining biologically active substances of peptide nature, which have growth factor activity towards fibroblast proliferation and can be used in medicine. An oligopeptide of formula A-X1-X2-X3-X4-X5-B is obtained through in silico construction, where A is F; X1 is E, or Q, or S; X2 is N, or Q, or A, or G; X3 is K, or R, or T; X4 is K, or E, or is absent, X5 is K, or L, or is absent and B is OMe - methyl.

EFFECT: invention enables obtaining an oligopeptide with transformation growth factor (TGF-β) and oncostatin M (OSM) towards fibroblast proliferation, and expansion of the range of effective therapeutic agents with wound-healing effect, which take part in closing wounds during inflammation and cicatrisation.

4 dwg, 2 ex

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