Use of ghrelin splicing version for treating cachexia and/or anorexia, and/or anorexia-cachexia, and/or eating disorder, and/or lipodystrophy, and/or muscle atrophy, and/or appetite stimulation

FIELD: medicine.

SUBSTANCE: invention provides using a prepared compound of a ghrelin splicing version for preparing an effective drug preparation activating body weight and food intake gain and/or stimulating growth hormone release, as well as for treating or preventing cachexia, lipodystrophy and muscle atrophy.

EFFECT: higher clinical effectiveness.

6 cl, 6 dwg, 13 ex

 

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the priority of provisional patent application U.S. No. 60/781860, filed March 13, 2006, which is fully incorporated here by reference.

The technical FIELD

The present invention relates to compounds for the treatment or prevention of cachexia, and/or lipodystrophy, and/or muscle wasting and related state.

BACKGROUND of INVENTION

Ghrelin is a bioactive peptide that stimulates food intake, increased body weight and obesity in rodents (Tschop M. et al., Nature 407: 903-13 (2000); Wren, A.M. et al., Diabetes 50: 2540-47 (2001)). Emergency administration of ghrelin stimulates food intake in healthy men and women (Wren A.M. et al., J. Clin. Endocrinol. Metab. 86: 5992-95 (2001); Druce M.R. et al., Int. J. Obes. Relat. Metab. Disord. 29: 1130-36 (2005)), as well as in cancer patients with anorexia (Neary N.M. et al., J. Clin. Endocrinol. Metab. 89: 2832-36 (2004)). Repeated administration of ghrelin increases lean body mass, body weight and food intake in patients with cachexia in patients with chronic obstructive pulmonary disease (COPD) (Nagaya N. et al., Chest 128: 1187-93 (2005)) and reduces muscle wasting in patients with chronic heart failure (Nagaya N. et al., Circulation 110: 3674-79 (2004)). A similar effect was also demonstrated in the model in mice (T. Hanada et al., Biochem. Biophys. Res. Common. 301: 275-79 (2003)).

Tumor growth is associated with CA is one Central metabolic and neurochemical changes, which lead to the syndrome of anorexia-cachexia. Anorexia is defined as a loss of desire to eat, while cachexia is the result of a progressive decline in skeletal muscle mass and less fat tissue, occurring just before it becomes apparent weight loss. Syndrome cancer anorexia-cachexia highly prevalent among cancer patients, has a great impact on morbidity and mortality and affects the patient's quality of life. However, its clinical significance is often overlooked, and usually start treatment only during the running stages of the disease (Laviano A. et al., Nat. Clin. Pract. Oncol. 3: 158-65 (2005)).

Ghrelin is secreted in pre-meal situation, starting 1-2 hours before a meal, leading to sharp, short-term spike in ghrelin levels in plasma before a meal, and continued a short time after the beginning of the meal. Because ghrelin is the only known produced perifericheskie causing appetite (appetite stimulant) substance is believed that the increased levels of ghrelin in the plasma is critical for the beginning of the meal.

Playing the role of a key stimulator of appetite, ghrelin, released from cells of the endocrine system in the mucosa of the gastrointestinal tract, can operate the AK locally as a paracrine substance, and Central as a hormone, as described below in the section related to cancer cachexia.

GHRL gene (ghrelin) encodes multiple products resulting from alternative playerowner transcripts of various types of splitting reprobation and various post-translational modifications (M. Kojima &Kangawa, M., Physiol. Rev. 85: 495-522 (2005); Zhang J.V. et al., Science 310: 996-99 (2005)). In addition, various fabrics are produced by various degradation products (C. De Vriese et al., Endocrinology 145: 4997-5005 (2004)). Some of these products GHRL described here.

Ghrelin is a peptide of 28 amino acids with n-octanoyl side chain of the third serine, resulting from the cleavage of the signal peptide and propeptide from preproghrelin of 117 amino acids and acylation. Acylated N-end of ghrelin necessary for endocrine functions (Kojima, M. et al., Nature 402: 656-60 (1999); M.A. Bednarek et al., J. Med. Chem. 43: 4370-76 (2000)). It is shown that ghrelin without acyl, you do not have endocrine functions, has an antagonistic effect on the influence of ghrelin on glucose production in vitro (C. Gauna et al., J. Clin. Endocrinol. Metab. 89: 5035-42 (2004)). Alternative splanirovannaya ghrelin mRNA encodes preprepared of 116 amino acids, which is then processed in des-Gln14-ghrelin and processionary peptide of 27 amino acids (H. Hosoda et al., J. Biol. Chem. 275: 21995-22000 (2000)). Another peptide, obestatin, Tsaplya the Xia from preproghrelin and has no overlapping sequences with protestirovanny peptide ghrelin. It is shown that this peptide has some antagonistic effect on acylated ghrelin, inhibiting food intake and increased body weight (Zhang J.V. et al., Science 310: 996-99 (2005)). Can also be a functioning one peptide, With the end preproghrelin of 66 amino acids (C. Pemberton et al., Biochem. Biophys. Res. Comm. 310: 567-73 (2003)). Multiple isoforms, including isoforms encoded by different splicing variants, known to other proteins, for example, growth factor vascular endothelial (VEGF), and different isoforms have a common role in the development of blood vessels, while they differ in some other properties, such as the binding affinity of (Neufeld, G. et al., FASEB J. 13: 9-22 (1999)). So many products GHRL gene may reflect a similarly complex control of the endocrine and paracrine actions of ghrelin isoforms.

Previously it was shown that ghrelin administration by continuous infusion doses of 5 pmol/kg/min for a period of 270 minutes increases food intake in healthy humans (Wren A.M. et al., J. Clin. Endocrinol. Metab. 86: 5992-95 (2001)). It was also shown that infusion of ghrelin within 90 minutes can increase by 30% of the food intake of patients with cancer cachexia (Abstract P09, Digestive Hormones, Appetite and Energy Balance, Baylis and Starling meeting, London, June 2003). Recently it was shown that subcutaneous injection of 3.6 nmol/kg acylated ghrelin before a meal, providing, that is they way imitation is close to occurring situations prior to food intake, increased energy intake by 27%. Also, apparently, ghrelin increased the perceived palatability of the proposed food product (Druce M.R. et al., Int. J. Obes. Relat. Metab. Disord. 29: 1130-36 (2005)).

These studies demonstrate that parenteral administration of ghrelin can increase appetite as in normal subjects and in patients with loss of appetite. In addition, the applicant has found that it is possible to obtain a significant effect of increasing body weight and a significant increase in food intake with the help of a new splicing variant of ghrelin (see in the ownership and in the process of simultaneous consideration of the published patent application U.S. No. 2005/0059015 included here by reference) with the introduction of the subject, in particular, when introduced subcutaneously before meals, providing, thus, imitation is close to occurring situations, prior to the meal. The effect of the new variant splicing of the ghrelin of the applicant to increase the weight focused mainly on lean mass, while the effect of ghrelin on weight gain focused mainly on fat mass.

SUMMARY of the INVENTION

One aspect is similar to the variant splicing of the ghrelin compound having the formula Z1-(1)m-(X2)-(X3)n-Z2, where Z1 is an optionally present protective group; each X1 is independently selected from naturally occurring amino acid and a synthetic amino acid; x2 is selected from naturally occurring amino acids and synthetic amino acids, with the specified amino acid modified large hydrophobic group; each X3 is independently selected from naturally occurring amino acids and synthetic amino acids, and one or more of X1 and X3 can be optionally modified by a large hydrophobic group; Z2 is an optionally present protective group; m is an integer in the range of 1-10; n is an integer in the range 4-92; the condition that the length of the compound according to formula Z1-(X1)m-(X2)-(X3)n-Z2 is 15-94 amino acids, and this connection is homologous to at least 80% to SEQ ID NO: 1.

Another aspect is a pharmaceutical composition containing such variant splicing of the ghrelin compound having the formula Z1-(X1)m-(X2)-(X3)n-Z2, where Z1 is an optionally present protective group; each X1 is independently selected from naturally occurring amino acid and a synthetic amino acid; x2 is selected from naturally occurring amino acids and synthetic amino acids, with the specified aminocyclopropane large hydrophobic group; each X3 is independently selected from naturally occurring amino acids and synthetic amino acids, and one or more of X1 and X3 can be optionally modified by a large hydrophobic group; Z2 is an optionally present protective group; m is an integer in the range of 1-10; n is an integer in the range 4-92; provided that the length of the compound according to formula Z1-(X1)m-(X2)-(X3)n-Z2 is 15-94 amino acids, and this connection is homologous to at least 80% to SEQ ID NO: 1.

A further aspect is a method of treating cachexia, including introduction to the needy in the mammal pharmaceutically acceptable amount of (a) variant splicing of the ghrelin; (b) such option splicing of the ghrelin compounds having the formula Z1-(X1)m-(X2)-(X3)n-Z2, where Z1 is an optionally present protective group; each X1 is independently selected from naturally occurring amino acid and a synthetic amino acid; x2 is selected from naturally occurring amino acids and synthetic amino acids, with the specified amino acid modified large hydrophobic group; each X3 is independently chosen of the naturally occurring amino acids and synthetic amino acids, and one or more of X1 and X3 can be optional, modifica ofany large hydrophobic group; Z2 is an optionally present protective group; m is an integer in the range of 1-10; n is an integer in the range 4-92; provided that the length of the compound according to formula Z1-(X1)m-(X2)-(X3)n-Z2 is 15-94 amino acids, and this connection is homologous to at least 80% to SEQ ID NO: 1; or (C) mixtures thereof.

An additional aspect is a method of prevention of cachexia, including introduction to the needy in the mammal pharmaceutically acceptable amount of (a) variant splicing of the ghrelin; (b) such option splicing of the ghrelin compounds having the formula Z1-(X1)m-(X2)-(X3)n-Z2, where Z1 is an optionally present protective group; each X1 is independently selected from naturally occurring amino acid and a synthetic amino acid; x2 is selected from naturally occurring amino acids and synthetic amino acids, with the specified amino acid modified large hydrophobic group; each X3 is independently chosen of the naturally occurring amino acids and synthetic amino acids, and one or more of X1 and X3 can be optionally modified by a large hydrophobic group; Z2 is an optionally present protective group; m is an integer in the range of 1-10; n is a whole is th number in the range 4-92; provided that the length of the compound according to formula Z1-(X1)m-(X2)-(X3)n-Z2 is 15-94 amino acids, and this connection is homologous to at least 80% to SEQ ID NO: 1; or (C) mixtures thereof.

A further aspect is a method of stimulation of appetite, food intake and/or increasing the weight, including the introduction to the needy in the mammal pharmaceutically acceptable amount of (a) variant splicing of the ghrelin; (b) such option splicing of the ghrelin compounds having the formula Z1-(X1)m-(X2)-(X3)n-Z2, where Z1 is an optionally present protective group; each X1 is independently selected from naturally occurring amino acid and a synthetic amino acid; x2 is selected from naturally occurring amino acids and synthetic amino acids, with the specified amino acid modified large a hydrophobic group; each X3 is independently selected from naturally occurring amino acids and synthetic amino acids, and one or more of X1 and X3 can be optionally modified by a large hydrophobic group; Z2 is an optionally present protective group; m is an integer in the range of 1-10; n is an integer in the range 4-92; provided that the length of the compound according to formula Z1-(X1)m-(X2)-(X3)n-Z2 is 15-94 amino acids, and this connection is% homologous to at least 80% OF SEQ ID NO: 1; or (C) mixtures thereof.

Another aspect is a method for the treatment of lipodystrophy, including introduction to the needy in the mammal pharmaceutically acceptable amount of (a) variant splicing of the ghrelin; (b) such option splicing of the ghrelin compounds having the formula Z1-(X1)m-(X2)-(X3)n-Z2, where Z1 is an optionally present protective group; each X1 is independently selected from naturally occurring amino acid and a synthetic amino acid; x2 is selected from naturally occurring amino acids and synthetic amino acids, with the specified amino acid modified large hydrophobic group; each X3 is independently chosen of the naturally occurring amino acids and synthetic amino acids, and one or more of X1 and X3 can be optionally modified by a large hydrophobic group; Z2 is an optionally present protective group; m is an integer in the range of 1-10; n is an integer in the range 4-92; provided that the length of the compound according to formula Z1-(X1)m-(X2)-(X3)n-Z2 is 15-94 amino acids, and this connection is homologous to at least 80% to SEQ ID NO: 1; or ((C) mixtures thereof.

A further aspect is a way to prevent lipodystrophy, including introduction to the needy in the mammal, formats whitesky acceptable amount of (a) variant splicing of the ghrelin; (b) such option splicing of the ghrelin compounds having the formula Z1-(X1)m-(X2)-(X3)n-Z2, where Z1 is an optionally present protective group; each X1 is independently selected from naturally occurring amino acid and a synthetic amino acid; x2 is selected from naturally occurring amino acids and synthetic amino acids, with the specified amino acid modified large hydrophobic group; each X3 is independently selected from naturally occurring amino acids and synthetic amino acids, and one or more of X1 and X3 can be optionally modified large hydrophobic group; Z2 is an optionally present protective group; m is an integer in the range of 1-10; n is an integer in the range 4-92; provided that the length of the compound according to formula Z1-(X1)m-(X2)-(X3)n-Z2 is 15-94 amino acids, and this connection is homologous to at least 80% to SEQ ID NO: 1; or (C) mixtures thereof.

Another aspect is a method of treating muscle wasting, including introduction to the needy in the mammal pharmaceutically acceptable amount of (a) variant splicing of the ghrelin; (b) such option splicing of the ghrelin compounds having the formula Z1-(X1)m-(X2)-(X3)n-Z2, where Z1 is an optionally present protective the th group; each X1 is independently selected from naturally occurring amino acid and a synthetic amino acid; x2 is selected from naturally occurring amino acids and synthetic amino acids, with the specified amino acid modified large hydrophobic group; each X3 is independently selected from naturally occurring amino acids and synthetic amino acids, and one or more of X1 and X3 can be optionally modified by a large hydrophobic group; Z2 is an optionally present protective group; m is an integer in the range of 1-10; n is an integer in the range 4-92; provided that the length of the connection in accordance with the formula Z1-(X1)m-(X2)-(X3)n-Z2 is 15-94 amino acids, and this connection is homologous to at least 80% to SEQ ID NO: 1; or (C) mixtures thereof.

A further aspect is a method to prevent muscle wasting, including introduction to the needy in the mammal pharmaceutically acceptable amount of (a) variant splicing of the ghrelin; (b) such option splicing of the ghrelin compounds having the formula Z1-(X1)m-(X2)-(X3)n-Z2, where Z1 is an optionally present protective group; each X1 is independently selected from naturally occurring amino acid and a synthetic amino acid; x2 is chosen from occurring in the origin of amino acids and synthetic amino acids, while this amino acid is modified large hydrophobic group; each X3 is independently selected from naturally occurring amino acids and synthetic amino acids, and one or more of X1 and X3 can be optionally modified by a large hydrophobic group; Z2 is an optionally present protective group; m is an integer in the range of 1-10; n is an integer in the range 4-92; provided that the length of the compound according to formula Z1-(X1)m-(X2)-(X3)n-Z2 is 15-94 amino acids, and this connection is homologous on at least 80% to SEQ ID NO: 1; or (C) mixtures thereof.

An additional aspect is a method of treating cachexia and/or anorexia and/or anorexia-cachexia, and/or malnutrition and/or lipodystrophy and/or stimulate appetite, including introduction to the needy in the mammal pharmaceutically acceptable number of amplifying secretion of means, including (a) variant splicing of the ghrelin; (b) such option splicing of the ghrelin compound having the formula Z1-(X1)m-(X2)-(X3)n-Z2, where Z1 is an optionally present protective group; each X1 is independently selected from naturally occurring amino acid and a synthetic amino acid; x2 is selected from naturally occurring amino acids and synthetic aminoxy the lots, while this amino acid is modified large hydrophobic group; each X3 is independently selected from naturally occurring amino acids and synthetic amino acids, and one or more of X1 and X3 can be optionally modified by a large hydrophobic group; Z2 is an optionally present protective group; m is an integer in the range of 1-10; n is an integer in the range 4-92; provided that the length of the compound according to formula Z1-(X1)m-(X2)-(X3)n-Z2 is 15-94 amino acids, and this connection is homologous on at least 80% to SEQ ID NO: 1; or (C) mixtures thereof; and a treatment selected from the group consisting of prophylaxis or treatment of cachexia, prevention or treatment of lipodystrophy, stimulate appetite, stimulation of food intake, stimulation of weight gain, increased body fat mass, increase lean body mass, or combinations thereof.

A further aspect is a kit for introduction of variant splicing of the ghrelin or a similar variant splicing of the ghrelin compounds, comprising (a) a dosage form containing a pharmaceutically acceptable amount of (1) variant splicing of the ghrelin; (2) such option splicing of the ghrelin compounds having the formula Z1-(X1)m-(X2)-(X3)n-Z2, where Z1 is an optionally present protective is the Rupp; each X1 is independently selected from naturally occurring amino acid and a synthetic amino acid; x2 is selected from naturally occurring amino acids and synthetic amino acids, with the specified amino acid modified large hydrophobic group; each X3 is independently selected from naturally occurring amino acids and synthetic amino acids, and one or more of X1 and X3 can be optionally modified by a large hydrophobic group; Z2 is an optionally present protective group; m is an integer in the range of 1-10; n is an integer in the range 4-92; provided that the length of the connection in accordance with the formula Z1-(X1)m-(X2)-(X3)n-Z2 is 15-94 amino acids, and this connection is homologous to at least 80% to SEQ ID NO: 1; or (3) mixtures thereof; and (b) optional instructions in relation to the introduction of (a).

Another aspect is for a method of producing such variant splicing of the ghrelin compounds, comprising (a) providing a cDNA comprising a polynucleotide sequence encoding such variant splicing of the ghrelin connection described above; (b) embedding the specified expressing the cDNA in the vector so that cDNA was functionally linked to the promoter; (C) the introduction of the specified expressing vector into the cell-the household is in, whereby specified a host cell produces the specified similar variant splicing of the ghrelin connection; and (d) optional selection of such option splicing of the ghrelin compounds produced in stage (C).

Other objects and advantages will be apparent skilled in the art specialists after a reference to the detailed description which follows.

A BRIEF description of the SEQUENCES

SEQ ID NO: 1 is a variant splicing preproghrelin person after the signal sequence.

SEQ ID NO: 2 represents a variant of the splicing of the ghrelin man of 22 amino acids.

SEQ ID NO: 3 represents a variant of the splicing of the ghrelin man of 24 amino acids.

SEQ ID NO: 4 represents a modified version of the splicing of the ghrelin man of 24 amino acids.

SEQ ID NO: 5 represents a variant of the splicing of the ghrelin man of 29 amino acids.

SEQ ID NO: 6 is a fragment of a full-sized version of the splicing of the ghrelin person.

SEQ ID NO: 7 represents a variant of the splicing preproghrelin mouse after the signal sequence.

SEQ ID NO: 8 represents a variant of the splicing preproghrelin rats after the signal sequence.

BRIEF DESCRIPTION of FIGURES

Fig. 1A is a line graph, monsterhouse cumulative increase in body weight in mice 129Sv, the treated acidified by alternative splicing of the ghrelin (SEQ ID NO: 2 and 5), compared with controls, treated by the media. Acylated variant splicing of the ghrelin causes an increase in body weight in male wild-type mice (n=10 per group, P = 0.0001). For mice treated once a day for two weeks acylated by alternative splicing of the ghrelin (0.8 mg/kg, subcutaneously), cumulative weight gain in animals of group SEQ ID NO: 2 was 3.4 times greater than that of control animals, which were injected with the carrier. Cumulative weight gain in animals of group acylated SEQ ID NO: 5 was 2 times greater than that of control animals, which were injected with the carrier. Fig. 1B is a line graph showing the cumulative food intake 129Sv mice, treated acidified by alternative splicing of the ghrelin (SEQ ID NO: 2 and 5), compared with controls, treated by the media. Treatment acylated by alternative splicing of the ghrelin increased food intake in the wild-type mice. Mouse treated once a day for two weeks acylated by alternative splicing of the ghrelin (0.8 mg/kg, subcutaneously), spruce 13% more than the control animals, which were injected media (n=10 per group, P = 0,0004).

Fig. 2A is a line graph showing cumulative Uwe is ikenie body weight in 129Sv mice, the treated acidified by alternative splicing of the ghrelin (SEQ ID NO: 2 and 4) and palleroni SEQ ID NO: 5, compared with controls, treated by the media. Acylated variant splicing of the ghrelin causes an increase in body weight in male wild-type mice (n=8 per group, P = 0.0001). For mice treated once a day for seven days acylated or palleroni option splicing of the ghrelin (7.2 mg/kg, subcutaneously), cumulative weight gain in animals of group SEQ ID NO: 2 and SEQ ID NO: 4 was 2.2 times greater than that of control animals, which were injected with the carrier. Cumulative weight gain in animals of group palleroni SEQ ID NO: 5 was 25% less than the control animals, which were injected with the carrier. Fig. 2B is a line graph showing the cumulative food intake 129Sv mice, treated acidified by alternative splicing of the ghrelin (SEQ ID NO: 2 and 4) and palleroni SEQ ID NO: 5, compared with controls, treated by the media. Treatment acylated by alternative splicing of the ghrelin increased food intake in the wild-type mice. Mouse treated once a day for seven days acylated variants of splicing of the ghrelin (7.2 mg/kg, subcutaneously), ate 18% more than the control mice, which were injected media (n=8 per group). The mouse will expose the s treatment once a day for seven days, SEQ ID NO: 5 (7.2 mg/kg, subcutaneously), ate at 2% less than the control animals, which were injected media (n=8 per group).

Fig. 3 is a histogram showing the concentration of growth hormone in serum after injection of acylated variants of splicing of the ghrelin (SEQ ID NO: 2, 4, 5) and palleroni SEQ ID NO: 5, compared with controls, which was introduced to the media. Demonstrates the effect of saline or acylated variants of splicing of the ghrelin (0.8 mg/kg, subcutaneously) on growth hormone in plasma after 10 minutes and 20 minutes after the injection of the wild-type mice (n=5 per group).

Fig. 4 is a histogram showing the change in body composition in mice 129Sv, treated acidified by alternative splicing of the ghrelin (SEQ ID NO: 2 and 4), and mice treated palleroni SEQ ID NO: 5, compared with controls, treated by the media and treated with ghrelin. Demonstrates the effect of daily subcutaneous treatment for seven days with saline, the ghrelin or acylated by alternative splicing of the ghrelin (7.2 mg/kg, subcutaneously) on fat and lean body mass as measured by NMR.

DETAILED description of the INVENTION

Applicants specifically include the full content of all mentioned in this description links. In addition, when an amount, concentration, or other value or parameter p is iodide or in the form of a range, the preferred range or enumeration of upper preferable values and lower preferable values, this should be understood as a disclosure, in particular, all ranges formed by any pair of any upper range limit or preferred value and any lower range limit or preferred value, regardless of whether disclosed whether the ranges separately. At the mention here of the range of numerical values, unless stated otherwise, the range, as expected, includes its final value, and all integers and parts within range. Is not intended that the scope of the invention limited to the specific values that are referenced when defining a range.

In the context of this description will be used a number of terms.

Used herein, the term "affinity" refers to the strength of the connection between receptors and their ligands, for example between an antibody and its antigen.

Used herein, the term "amino acid residue" means an amino acid that is formed by chemical cleavage (hydrolysis) of the polypeptide on its peptide linkages. The described amino acid residue preferably is in the "L" isomeric form. However, the amino acid comprises any amino acid, such as L-amino acids, D-amino acid, alpha-amino acid, beta-amino acids which, gamma-amino acid, natural amino acid and a synthetic amino acid, etc. until the polypeptide retains the desired functional property. NH2refers to the free amino group present on aminocore polypeptide. COOH refers to the free carboxyl group present on the carboxyl end of the polypeptide. Standard abbreviations for amino acid residues of the polypeptide shown in table 1.

Table 1
Single-letter codeThree-letter codeAmino acid
AAlaAlanine
BAsx, Asn and/or AspAspartic acid
and/or asparagine
CCysCysteine
DAspAspartic acid
EGluGlutamic acid
FPhe Phenylalanine
GGlyGlycine
HHisHistidine
IIleIsoleucine
KLysLysine
LLeuLeucine
MMetMethionine
NAsnAsparagine
PProProline
QGlnGlutamine
RArgArginine
SSerSerine
TThrThreonine
VValValine
WTrpTryptophan
XXaaUnknown or other
YTyrTyrosine
ZGlx, Gln and/or GluGlutamic acid
and/or glutamine
-Dpr2,3-diaminopropionic
acid

It should be noted that all the presents here a formula for the sequence of amino acid residues are oriented from left to right in the conventional direction from aminobenzo to the carboxyl end. In addition, when a broad definition of the phrase "amino acid residue" includes the amino acids listed in table 1, and modified and not naturally occurring amino acid residues. In addition, it should be noted that a dash at the beginning or end of the sequence of amino acid residues indicates a peptide bond with an additional sequence of one or more amino acid residues or covalent bond with a group on aminocore, such as, NH2or acetyl, or a carboxyl group at the end, such as COOH.

Use is used here, the term "antineoplastic treatment" means treatment, aimed at stopping or reducing abnormal growth of tissue (such as neoplasma) of the individual. Examples of such treatment include anti-cancer therapy, such as radiotherapy or chemotherapy.

"Appetite" in respect of an individual is determined by measuring the amount of food consumed and by assessing the willingness of the individual features. Here appetite (i.e. hunger) is usually defined by means of a short questionnaire given to individuals on a random basis several times a week. Typically, subjects evaluated their hunger, preoccupation with thoughts about food or the desire to eat large quantities and different types of food, answering questions using analogue scale from 1 (not at all) to 5 (extremely).

"Body mass index" or "BMI" is the ratio of growth to the weight of the individual. BMI is determined by calculating the weight in kilograms divided by the square of height in meters. The "normal" BMI range is 18.5-25.

"Fat weight" can be defined, for example, using the method of the fat folds. In the method of the folds of fat used compasses type of tweezers to determine the subcutaneous fat by measuring the thickness of skin folds in representative locations on the body. These measurements of skin folds are then used to calculate body fat or by addition of various indicators measured the th, or use this value as an indication of the relative degree of obesity among individuals, or through the use of measurement in mathematical equations, which were developed for the prediction of percent body fat (Fogelholm M. & van Marken Lichtenbelt, W., Eur. J. Clin. Nutr. 51: 495-503 (1997)).

Used herein, the term "equivalent concentration" means the equivalent dose is similar to the variant splicing of the ghrelin compounds with in vitro and/or in vivo response, the same response calculated by curve dose-response variant splicing of the ghrelin.

"Dissociation constant" or "Kd" is a value that characterizes the strength of the connection (or affinity, or avidity) between receptors and their ligands, for example, an antibody and its antigen. The smaller the Kd, the stronger the binding.

"Fusion polypeptide" is a polypeptide comprising at least two polypeptide and a binding sequence for the functional binding of the two polypeptides into one continuous polypeptide. Two polypeptide bound in the fused polypeptide, usually come from two independent sources, and, therefore, fused polypeptide includes two related polypeptide not found, usually associated in nature.

As used here, "ghrelin man" is a polypeptide having aminokislot the second sequence, stated in No. of access GenBank® - NP_057446 or Swiss-Prot-ID GHRL_HUMAN. Preprotein ghrelin person has a 117 amino acids. This preprotein subject to the following posttranslational processing. The signal peptide (amino acids 1-23) is removed, and the remaining 94 amino acids are broken down by protease ensuring the Mature ghrelin of 28 amino acids (amino acids 24-51) or Mature ghrelin of the 27 amino acids (amino acids 24-50) and Mature obestatin of the 23 amino acids (amino acids 76-98). Mature peptides ghrelin of the 27 or 28 amino acids may further be modified in position 26 serine in preprotein with or On-octanone group, or O-technoloy group. The Mature peptide obestatin may further be modified at position 98 lysine in preprotein with amide groups. Known additional preprotein ghrelin, which has no glutamine in position 37 preprotein.

"Alternative splicing of the ghrelin" is a polypeptide having the amino acid sequence represented in SEQ ID NO: 1, or any peptide of 15 amino acids or more of SEQ ID NO: 1 with a post-translational modification or without it, or any homologue of SEQ ID NO: 1 set forth in SEQ ID NO: 7 or SEQ ID NO: 8, and/or any peptide of 15 amino acids or more of SEQ ID NO: 7 or SEQ ID NO: 8 with a post-translational modification or without it.

"Such variant is lisinge ghrelin connection", as used here, refers to any compound that plays the function of splicing variant of ghrelin in particular splicing variant of human ghrelin, leading, in particular, regarding the functions of splicing variant of ghrelin, the desired therapeutic effects described herein, such as stimulation of appetite and/or treatment and/or prevention of cachexia, and is defined by formula I: Z1-(X1)m-(X2)-(X3)n-Z2, where Z1 is an optionally present protective group; each X1 is independently selected from naturally occurring amino acids and a synthetic amino acid; x2 is selected from naturally occurring amino acids and synthetic amino acids, with the specified amino acid modified large hydrophobic group; each X3 is independently selected from naturally occurring amino acids and synthetic amino acids, and one or more of X1 and X3 can be optionally modified by a large hydrophobic group; Z2 is an optionally present protective group; m is an integer in the range of 1-10; n is an integer in the range 4-92; provided that the length of the compound according to formula Z1-(X1)m-(X2)-(X3)n-Z2 is 15-94 amino acids, and this connection is homologous to at least 80% (or in alternative embodiments implement the program of the present invention to 85%, 90%, 93%, 95%, 97%, 98%, 99%, 100%) SEQ ID NO: 1. In a preferred embodiment of the present invention, the length of such variant splicing of the ghrelin connection is 22-29 amino acids.

"Immunological excellent" refers to the ability to establish the difference between the two polypeptides for the ability of the antibodies specifically bind to one of the polypeptide and does not bind specifically to another polypeptide.

"Individual" is an animal or a human, susceptible to the condition, in particular kactionmenu condition defined here. In preferred embodiments, the implementation of the present invention the individual is a mammal, including human and non-human mammals, such as dogs, cats, pigs, cows, sheep, goats, horses, rats and mice. In the most preferred embodiment of the present invention the individual is the man.

"Dedicated" is used to describe a variety of similar variant splicing of the ghrelin compounds, i.e. polypeptides and nucleotides disclosed here that have been identified and separated and/or purified from a component of its natural environment. Components of the admixture of the natural environment are materials that normally would prevent diagnostic or therapeutic applications of the polypeptide, and could the t to include enzymes, hormones and other relcovaptan or nebulophone solute. In preferred embodiments, the implementation of the present invention is similar to the variant splicing of the ghrelin connection is subjected to cleaning.

As used here, "modified amino acid" is an amino acid, any group which is chemically modified.

As used here, "non-standard amino acid" is an amino acid that does not belong to the 20 standard amino acids. Non-standard amino acids are usually formed due to chemical modifications of the standard amino acids. They may also be formed in nature in the form of intermediate metabolic pathways or microorganisms and/or plants.

"Monoclonal antibody"in its various grammatical forms refers to a population of antibody molecules that contain only one kind of binding site antibody capable of immunologically to interact with a specific antigen.

"Neetilirovannogo such variant splicing of the ghrelin connection" is a similar variant splicing of the ghrelin connection, defined above, which does not contain acyl groups attached to any of its amino acids, the component parts.

"Palliative treatment" before the hat is treatment, which alleviated or attenuated symptoms or disorders, but no effect of treatment.

"Polyclonal antibodies are a mixture of molecules of antibody recognizes this specific antigen; therefore, polyclonal antibodies may recognize different epitopes in the specified antigen.

"Polypeptide" refers to a molecule comprising amino acid residues that do not contain links other than amide bonds between adjacent amino acid residues.

"Processionary ghrelin" means acylated ghrelin of the 27 or 28 amino acid residues (post-translational product of the cleavage and acylation preproghrelin length 116 or 117 amino acid residues, respectively (for example, SWISS-PROT Q9UBU3 GHRL_HUMAN).

"Receptor" is a molecule such as a protein, glycoprotein, etc. that can specifically (not randomly) to communicate with another molecule.

"Enhancing the secretion agent" is a substance that stimulates the release of growth hormone, such as ghrelin or similar to ghrelin connection. Enhancing the secretion of a tool in accordance with the present description can, for example, be selected from L-692-429 and L-692-585 (connection betalaktam; available from Merck & Co., Inc., Whitehouse Station, NJ), MK677 (Spirogyra, available from Merck), G-7203, G-7039, G-7502 (peptidomimetics in the form of isoniazid is cotinuous acid, available from Genentech, Inc., South San Fraisco, Calif.), NN703 (Novo Nordisk Inc., Princeton, NJ) or ipamorelin. In particular, amplifying secretion of the tool is similar to the variant splicing of the ghrelin connection, including the option of splicing of the ghrelin man of 29 amino acids, variant splicing of the ghrelin man of 24 amino acids or splicing of the ghrelin man of 22 amino acids (e.g., SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5). In one embodiment, amplifying secretion of growth hormone remedy may be palleroni, for example palleroni form of variant splicing of the ghrelin or palleroni similar variant splicing of the ghrelin connection.

"Molecule surfactant is a molecule comprising a hydrophobic part and a hydrophilic part, i.e. a molecule that is able to be present in the interphase between a lipophilic phase and a hydrophilic phase.

Indications

The present description relates to the application enhances the secretion of tools, such as such variant splicing of the ghrelin connection, for the treatment or prevention of conditions, for example, related to abnormal weight loss or loss of lean and/or fat mass, including (a) the prevention or treatment of cachexia, and/or (b) the prevention or treatment of lipodystrophy, and/or (C) stimulation of appetite, and/or (d) stimulation energy consumption the Oia food and/or (e) stimulation of weight gain, and/or (f) increase in body fat mass, and/or (g) increase lean body mass. In particular, the present description relates to the treatment or prevention of cachexia, and/or stimulate appetite, stimulation of appatichnosti, improve quality of life, most preferably the prevention or treatment of cachexia.

Cachexia

Cachexia is one of the most upset and pernicious symptom of several serious diseases, such as cancer, robbing individuals of their energy, sense of well-being, quality of life and increasing their dependence on others. Cachexia frequently accompanies malignant tumors of the pancreas, stomach, esophagus, lung and intestines.

The main feature of cachexia is weight loss, not only in adipose tissue, but also muscle tissue and even bone. This Nazirova fabric also known as "lean body mass". In addition, there is loss of appetite (anorexia), weakness (asthenia) and reduced hemoglobin (anemia).

Treatment of cachexia is not just something like a larger food intake. Even if an individual wants to eat, even if one tries, even if the individual is given nutrients through gastric tube or intravenously, the condition usually does not change.

A recent study showed that the present state is considered as part of the reaction to the presence of underlying disease (Laviano A. et al., Nat. Clin. Pract. Oncol. 2: 158-65 (2005)). A recent study also showed that in some cases the tumor produce substances that cause cachexia (Esper D.H. & Harb W.A., Nutr. Clin. Pract. 20: 369-76 (2005)).

Cachexia, or wasting, as it can also be called, is observed in several diseases, such as AIDS, cancer, post-hip fracture, chronic heart failure, chronic lung disease, such as COLD (chronic obstructive lung disease and COPD (chronic obstructive pulmonary disease), liver cirrhosis, renal failure, autoimmune diseases such as rheumatoid arthritis and systemic lupus, sepsis, tuberculosis, cystic fibrosis, Crohn's disease and severe infection. In addition, the depletion is also observed during aging.

Although cachexia is a complex metabolic syndrome, which is observed in these patients, it is usually recognized as a progressive loss of weight with the depletion of the owner reserves of adipose tissue and skeletal muscle.

Cancer cachexia

The essence of the syndrome cancer cachexia is associated with the problem of progressive tumor growth and catabolic side effects common antineoplastic therapy. These two phenomena cause changes in the neuroendocrine system, production is noreste of proinflammatory cytokines and the release of specific cancer kagechika factors. In turn, these mediators cause a decrease in food intake, abnormalities in metabolism, or a combination of these two effects.

It is reported that cancer cachexia occurs in approximately half of all cancer patients, and it is associated with more than 20 percent of deaths from cancer (M.J. Tisdale, Nat. Rev. Cancer 2: 862-71 (2002)). This condition often occurs during running of cancer, in particular, when in the body are metastatic tumors. Cachexia is also more frequent in children and elderly patients. Also consistently identified specific cancers, for which the frequency of cancer cachexia is particularly high: cancers of the upper section of the gastrointestinal tract (including the pancreas, stomach, esophagus and liver (Bruera E., Br. Med. J. 315: 1219-22 (1997); Palesty J.A. et al., Dig. Dis. 21: 198-213 (2003)), lung cancer, in particular small cell lung cancer; head and neck cancer; cancer of the colon and rectum, other solid tumors (Bruera E., Br. Med. J. 315: 1219-22 (1997)). IWL (involuntary weight loss) was associated with a reduction by approximately 50% survival rate and reduced tolerance to anticancer therapy (Laviano A. et al., Nat. Clin. Pract. Oncol. 2: 158-65 (2005)). Locations of cancer associated with the highest risk of weight loss, are location, affecting europamusicale tract (lung, th is low and the neck and the esophagus and the gastro-intestinal system, especially the pancreas, stomach and liver. In addition, at the time of diagnosis, 80% of all patients with cancer of the upper section of the gastrointestinal tract and 60% of all lung cancer patients have experienced significant weight loss (Bruera E., Br. Med. J. 315: 1219-22 (1997)). On average, the prevalence of cachexia increases from 50% to over 80% before his death, and for more than 20% of patients with cachexia is a major cause of death (Bruera E., Br. Med. J. 315: 1219-22 (1997)).

Detection of cancer cachexia

The nutritional status is assessed through a combination of clinical assessment, anthropometric tests (weight, thickness of skin folds and medium length arm circumference) and image acquisition (DEXA scans, MR scans, CT scans and measurement of the bioelectric impedance). Cachexia is usually suspect, if involuntary weight loss of greater than 5% preclinical weight is observed within the period of six months - especially if it is combined with muscle exhaustion.

The most commonly used laboratory value is serum albumin. However, it is a nonspecific indicator. Other markers are proteins with a short half-period of existence; it was also used transferrin and transthyretin.

Other markers of cachexia are IGF-1, IGFBP-3, ALP (alkaline phosphatase) and testosterone.

The link between cancer and cancer cachexia

Cancer can cause cachexia through a number of mechanisms, including the induction of anorexia and/or increase or change in metabolism, as described below.

Anorexia

It is shown that energy intake is significantly reduced in losing weight cancer patients. Cancer patients can often suffer from physical obstruction of the gastrointestinal tract, pain, depression, constipation, malabsorption, malnutrition or side effects of treatment (such as, for example, treatment with opiates, radiotherapy or chemotherapy), all of which can reduce food consumption (Barber M.D. et al., Surg. Oncol. 8: 133-41 (1999)). Accompanying cancer hypercalcemia may also cause nausea, vomiting and loss of appetite.

However, there is still a large number of cancer patients who have no obvious clinical cause reduced food intake.

The Central mechanism induced cancer anorexia and cachexia is complex and includes many different cytokines, hormones and other evidence produced by cancer cells.

Leptin

In normal physiological situations, leptin plays an important role in the initiation of the adaptive response to fasting as a weight loss causes a decrease in the levels of leptin in proportion to the loss of body fat. However, in cancer patients increased levels of cytokines (n is an example, IL-1, IL-6, TNF-α, IFN-γ)produced by cancer cells, can stimulate the expression and/or release of leptin. Another possible mechanism of cytokines is that they reproduce the hypothalamic effect of extensive negative feedback signal from leptin, leading to prevention of the normal compensatory mechanism related to food intake and body weight.

NPY (neuropeptide Y)

Hypothalamic NPY system is one of the key neural pathways violated in anorexia and cancer induced by IL-1 or other cytokines. Cytokines reduce sensitivity to NPY. It is shown that NPY is regulating growth factor for tumors of the neuroendocrine system, by acting as autocrine activation of proliferation of tumor cells or apoptosis, and through the development of blood vessels (Kitlinska, J. et al., Cancer Res. 65: 1719-28 (2005)). In addition, we discovered that the receptors for Y1 and Y2 are expressed in cancers of the breast, cancer of the adrenal gland and related tumors, pochernkletocny carcinomas and cancers of the ovary as in tumor cells and related tumors of the blood vessels. Their widespread expression in cancer cells allows them to mediate the effects of NPY on the proliferation of cancer cells and the blood supply to tumors (for review see Körner M & Reubi JC, Peptides 28: 419-25 (2007)).

Mesland the Cortina

Aberrant signaling from melanocortins may be a contributing factor as anorexia and cachexia. Despite the obvious weight loss, which, as would be normal to expect that suppresses the transmission system signal from anorectic of melanocortins as ways to save energy, the system melanocortin remains active during induced cancer cachexia. Central locking melanocortin with AgRP (agouti related peptide) or other antagonists reversible anorexia and cachexia in animal models, which indicates a pathogenic role of this system (Wisse B.E. et al., Ann. N.Y. Acad. Sci. 994: 275-81 (2003)). In addition, recent experiments have shown that blocking the transmission of signals from melanocortin using antagonists against receptor melanocortin MS(4) reduces accompanying the disease anorexia and emaciation in models of cancer and kidney failure in rodents (DeBoer MD & Marks DL, Nat. Clin. Pract. Endocrinol. Metab. 2: 459-66 (2006)).

Metabolism

The hypermetabolism is defined as the increased energy expenditure resting (REE) is the main symptom of cachexia. Total energy consumption includes REE (approximately 70%) and conscious consumption (approximately 25%), and the energy used in digestion (5%). Conscious consumption can be reduced cachexia, Kotor, which may clinically manifest as apathy, fatigue and depression.

Orexigen (appetizing) and anorectic signals, as is well known, respectively reduce and increase the activity of the sympathetic nervous system, which regulates REE by activation of thermogenesis in brown adipose tissue in rodents and possibly muscle men, through the induction of mitochondrial separating proteins (UCP) (R. Alvarez et al., J. Biol. Chem. 270: 5666-73 (1995)). It has been suggested that the activation of UCP in muscle and white adipose tissue cytokines could be one of the molecular mechanisms underlying the increased production of heat and muscle wasting (Inui A., CA Cancer J. Clin. 52: 72-91 (2002); Fearon K.C. & Moses A.G., Int. J. Cardiol. 85: 73-81 (2002)).

Cancer patients has also been described modified food metabolism. Solid tumors produce large amounts of lactate, which is converted back into glucose through a process that uses large amounts of ATP and is very inefficient in terms of energy that advanced, thus, increases energy consumption. In addition, it was shown that originating from tumors mobilizing lipids factor (LMF) acts directly on adipocytes and induces an increase in lipolysis, leading to release of free fatty acids and glycerol (Islam-Ali B. et al., Br. J. Cancer 85: 758-63 (2001)) and reducing the toxicity of free radicals in tumor is ledah (Sanders PM & Tisdale MJ, Br. J. Cancer 90: 1274-78 (2004)).

Also suggested that increased levels of cytokines may indirectly induce catabolism of muscle proteins by affecting the process of muscle recovery (Islam-Ali B. et al., Br. J. Cancer 85: 758-63 (2001)).

The rationale for the use of amplifying secretion of funds for the treatment of cancer cachexia

Not bound by theory, the rationale for treatment enhances the secretion of means, in particular, such a variant splicing of the ghrelin connection, based on the following: option splicing of the ghrelin released from endocrine cells in the mucosa of the gastrointestinal tract, can act as locally as paracrine substances, and Central as a hormone. Locally variant splicing of the ghrelin may act as an initiator of afferent activity, for example, afferent vagusnye neurons. Such neurons will transmit the stimulus from variant splicing of the ghrelin in the Central nervous system, such as the core of a single path (NTS), which then communicate with the centers that regulate appetite and energy homeostasis, such as paraventrikulyarnoe the nucleus and the arcuate nucleus in the hypothalamus. As hormone variant splicing of the ghrelin are believed to act on the Central regulating appetite O.G. ROMs- (proopiomelanocortin) and NY/AgRP-neurons, which Express receptors for splicing variants of ghrelin.

Recently described that ghrelin is moved through the barrier the blood - brain (Banks W.A. et al., J. Pharmacol. Exp. Ther. 302: 822-27 (2002)). It is important to note that in the Central regulating appetite center, for example, in NPY/AgRP-neurons, i.e. neurons of the first level in a stimulating branch control appetite, ghrelin, acting through stimulating the receptors, ghrelin is the only known stimulatory input from the periphery. All known hormones and neurotransmitters, such as leptin, insulin, PYY3-36, α-MSH, etc. that act as inhibitors of the NPY/AgRP neurons in this important controlling appetite centre. Because NPY system supression during induced cancer cachexia, stimulation of this system with the help of ghrelin may be able to normalize the condition. Similarly, melanocortin, which is active during induced cancer cachexia may be Engibarov by ghrelin and alternative splicing of the ghrelin through the stimulation of AgRP.

Also it is shown that increasing the concentration of ghrelin leads to increased ACTH (adrenocorticotropic hormone) from the resulting increased levels of cortisol. This action can have important beneficial effects for the treatment of cachexia, because cortisol reduces the levels of cytokines (such as IL-1, IL-6, TNFα, IFN-α). Introduction glucocorticoids are already widely used in the palliative management of symptoms related to cancer (Inui A., CA Cancer J. Clin. 52: 72-91 (2002)). In addition, it was shown that injection of the cardiovascular system ghrelin reduces the temperature of the body in rodents, indicating that the decrease in REE (Lawrence C.B. et al., Endocrinology 143: 155-62 (2002)). Again there is not bound by theory, the assumption that the alternative splicing of the ghrelin-like wild-type ghrelin, will reverse the increase in REE, which is an important feature of cachexia, described above.

Enhance the secretion of the tool, in particular, such a variant splicing of the ghrelin connection, you can enter using any suitable scheme, taking into account information about the expected progression of cancer, as well as the scheme antineoplastic therapy.

In one embodiment, the present invention provides that in accordance with the present description amplifying secretion means you can enter any individual suffering from any type of cancer, regardless of etiology, for successful treatment, mitigation or prevention of cancer cachexia.

Thus, in one preferred embodiment of the present invention the treatment of the individual amplifying secretion of means, such as a variant splicing of the ghrelin or under the Noah option splicing of the ghrelin connection, intended for the treatment or prevention of cancer cachexia caused, for example, one or more of the following types of cancer: acute lymphoblastic leukemia, acute myeloid leukemia, adrenocortical carcinoma, associated with AIDS, cancers, AIDS-related lymphoma, cancer of the anal canal, astrocytoma, astrocytoma of the cerebellum in children, basal cell carcinoma of the brain in children, cancer, extrahepatic bile duct, bladder cancer, bone cancer, osteosarcoma/malignant fibrous histiocytomas, glioma, brain stem, brain tumor, breast cancer, adenomas/carcinoids of the bronchi in men, Burkitt's lymphoma, carcinoid tumor primary carcinoma of unknown localization, lymphoma of the Central nervous system, primary astrocytoma/malignant glioma of the brain, cervical cancer, cancers in children, chronic lymphocytic leukemia, chronic myelogenous leukemia, chronic myeloproliferative disorders, colon cancer, T-cell lymphoma of the skin, cancer of the endometrium, the ependymal glioma, esophageal cancer in children, family of tumors Ewing, veneciano tumor of germ cells, a tumor of germ cells outside the reproductive gland in children, cancer, eye cancer, eye - intraocular melanoma, retinoblastoma - cancer of the bile HSS is OC, gastric cancer, carcinoid tumor of the gastrointestinal tract, trophoblastic tumor cancer, glioma, Volokolamsky leukemia, head and neck cancer, liver cancer, Hodgkin's lymphoma, cancer of the pharynx-esophagus, glioma hypothalamus and optic tracts, intraocular melanoma - insuloma (pancreatic internal secretion), kidney cancer - Kaposi's sarcoma, laryngeal cancer, cancer of the lip and oral cavity, lung cancer, non-small cell lung cancer, small cell lymphoma, AIDS-related lymphoma, T-cell lymphoma of the skin, non-macroglobulinemia, malignant fibrous histiocytoma bone/osteosarcoma, waldenstrom, medulloblastoma, melanoma cancer cells, Merkel, mesothelioma, malignant mesothelioma in adults, metastatic squamous neck cancer in patients with primary syndrome multiple endocrine neoplasia of unknown origin, multiple myeloma/plasmacytomas neoplasmas children fungoides granuloma, myelodysplastic syndromes, myelodysplastic/myeloproliferative diseases, myeloma, multiple chronic myeloproliferative disorders, cancer of the nasal cavity and paranasal sinus cancer, nasopharyngeal, neoplasmas in children, cancer of the oropharynx, osteosarcoma/malignant fibrous, histio what ICOMOS bones, ovarian cancer, epithelial cancer of the ovary in children, tumors of germ cell ovarian tumor ovarian low potential malignancy, pancreatic cancer, cancer of the paranasal sinus and nasal cavity cancer parathyroid cancer, senile cancer, pheochromocytoma, pinealoblastoma and above cerebellar Tentorium, primitive neuroectodermal tumors, pituitary tumor in children, plavalaguna blastoma, prostate cancer, cancer of the renal pelvis and ureter, transitional cell cancer, retinoblastoma, rhabdomyosarcoma, a cancer of the salivary gland in children, occurring in adults with soft tissue sarcoma, sarcoma, sarcoma cancer in children, the retikulez Cesari, skin cancer (non-melanoma), carcinoma skin cancer Merkel cells of the small intestine, located above the cerebellar Tentorium, primitive neuroectodermal tumors, T-cell lymphoma of the skin in children, testicular cancer, thymoma and carcinoma of the thymus, thyroid cancer, transitional cell cancer of the renal pelvis and ureter, trophoblastic tumor, cancer of the ureter and renal pelvis in pregnant women, transitional cell cancer, cancer of the urethra, cancer, endometrial uterine sarcoma of the uterus, cancer of the vagina, gliomas of the optic tract and hypothalamus, macroglobulinemia the th waldenstrom in children, the Wilms tumor.

As discussed above, cancer cachexia may be caused by catabolic disorder, for example hypermetabolism condition described above, resulting from or progressive tumor growth, or catabolic side effects of anticancer therapy. However, cancer cachexia may also be caused by such anorectics violation, as is the case when the individual is suffering from cancer, has no appetite, or location of the tumor reduces the consumption of food or hinders him.

Accordingly, one alternative implementation of the present invention is intended for the treatment or prevention through enhancing the secretion of tools, such as such variant splicing of the ghrelin connection, cancerous cachexia, due to catabolic violation. This is particularly applicable when the cancer is cancer of the gastrointestinal tract, especially cancer of the upper section of the gastrointestinal tract (it should be understood that the term "cancer of the upper section of the gastrointestinal tract also covers pancreatic cancer), lung cancer (especially small cell lung cancer) and/or liver cancer (it should be understood that the term "liver cancer" also includes metastatic cancer process in the liver).

Another variant of implementation of the present invented what I intended for the treatment or prevention through enhancing the secretion of funds such as such variant splicing of the ghrelin connection, cancerous cachexia caused anorectics violation.

Another variant implementation of the present invention is intended for the treatment or prevention through enhancing the secretion of tools, such as such variant splicing of the ghrelin connection, cancerous cachexia, regardless of how cancer is caused cachexia and cachexia caused by a combination of catabolic disorders and anorectics violations.

In a preferred embodiment, the present invention enhances the secretion of a tool, such as a variant splicing of the ghrelin or a similar variant splicing of the ghrelin connection, used for the treatment or prevention of cancer cachexia induced solid tumor.

Another subgroup of cancers are cancers with anorexia caused by dysregulation of the Central regulating the appetite center in the hypothalamus, when eliminating other possible causes of the decrease in food intake.

The specific individuals in the last stages of cancer, when no further treatment of cancer, the treatment option splicing of the ghrelin as a palliative treatment to increase food consumption, improve digestion and improve metabolism may be beneficial. Accordingly, the other is the aspect relates to palliative care to increase food consumption, improve digestion and metabolism of the individual in need thereof, for example, when the specified individual suffers fired cancer, in particular cancer last stages.

In accordance with the above disclosed here are applicable connection, in honesty, for treating or preventing cachexia in an individual suffering from the following forms of cancer europamusicale tract: cancer of the pancreas, cancer of the upper section of the gastrointestinal tract, such as stomach cancer and/or esophageal cancer, head and neck cancer, in particular thyroid cancer or cancer of the salivary iron, and lung cancer, in particular small cell lung cancer.

In another preferred embodiment, the present invention enhances the secretion of a tool, such as a variant splicing of the ghrelin or a similar variant splicing of the ghrelin connection, used for the treatment or prevention of cancer cachexia caused by cancer of the lower section of the gastrointestinal tract, such as cancer of the colon and rectum, particularly cancer of the colon.

In another preferred embodiment, the present invention enhances the secretion of a tool, such as a variant splicing of the ghrelin or a similar variant splicing of the ghrelin connection, used for treating or preventing a cancer to the hexie, caused by endocrine cancer, i.e. cancer endocrine organ of the body of the individual.

Disclosed here connections are also applicable to the treatment of individuals suffering from ovarian cancer or breast cancer.

In a further preferred embodiment of the present invention enhances the secretion of a tool, such as a variant splicing of the ghrelin or a similar variant splicing of the ghrelin connection, used for the treatment or prevention of cancer cachexia caused in whole or in part the anti-cancer treatment such as chemotherapy or radiotherapy, or a combination of both.

In one preferred embodiment of the present invention, the individual subjected to the treatment of cancer cachexia is older, for example, age 60 to 120 years, for example, age 70-120 years, for example, the age of 80-120 years, for example, age 90-120 years. Equally preferred are embodiments of the present invention, when the specified individual is a child, for example, age 0-20 years, for example, age 0-15 years, for example, age 0-10 years, for example, age 0-5 years, for example, age 0-1 year, for example, a newborn's age less than 2 months.

In one embodiment of the present invention preferably enhances the secretion among the STV, such as such variant splicing of the ghrelin connection, prophylactically to prevent kahetinskogo state. In this embodiment of the present invention, the treatment can begin before any antineoplastons treatment. Enhance the secretion of the tool can be entered continuously during antineoplastic treatment, or it may be administered at intervals, for example between periods antineoplastic therapy. With the introduction during antineoplastic therapy and, in particular, between periods antineoplastics therapy is a risk that the exposed treatment of an individual found to infection and/or other complications may be reduced due to better health conditions of the individual.

The treatment of cancer cachexia using amplifying secretion of means, such as a variant splicing of the ghrelin or a similar variant splicing of the ghrelin compound, can be accomplished using any known in the art, the method of administration. Preferably, the treatment can be accomplished using any of these techniques, preferably using intravenous or subcutaneous administration, most preferably using the methods subcutaneous injection.

Lipo

In another aspect of the present description refers to when is teniu enhance the secretion of funds such as a variant splicing of the ghrelin or a similar variant splicing of the ghrelin connection, described above, to ensure lipodystrophies syndrome or manufacture of a medicinal product for the treatment of lipodystrophies syndrome. Lipodystrophies syndromes comprise a heterogeneous group of rare disorders characterized by partial or generalized loss of fatty deposits (Garg A., Am. J. Med. 108: 143-52 (2002)). There are several different types of lipodystrophy, and the degree of fat loss can vary from very small areas with a decrease in adipose tissue to the almost complete absence of adipose tissue. Some patients may have only cosmetic problems, while others may have severe metabolic complications such as dyslipidemia, fatty liver degeneration and severe insulin resistance (M.L. Reitman et al., Trends Endocrinol. Metab. 11: 410-16 (2000)). These violations can be either hereditary (familial or hereditary lipodystrophies) or can arise secondarily on the various types of diseases or drugs (acquired lipodystrophy). Inherited lipodystrophy can be caused by mutations in the gene.

Identified several genes responsible for different types of hereditary lipodystrophy. They include AGPAT2 (1-acylglycerol the l-3-phosphate-O-acyltransferase 2), gene BSCL2 (inherited lipodystrophy 2 Berardinelli-Seip) in hereditary generalized lipodystrophy (CGL), gene Lamin A/C (LMNA) when many family partial lipodystrophy Dunnigan (familial partial lipodystrophy) and PPARG gene (activated proliferation peroxisome gamma receptor) family of partial lipodystrophy. Several other candidate genes investigated in the present time for other sets of hereditary lipodystrophy.

Acquired by lipodystrophies are, for example, HAART (induced by highly active antiretroviral therapy)/HIV lipodystrophy in HIV-infected patients (LD-HIV), acquired generalized lipodystrophy (AGL), acquired partial lipodystrophy (APL) and localized lipodystrophy. Acquired lipodystrophy does not have a direct genetic basis. More likely to be involved in many mechanisms. One such mechanism may be an autoimmune reaction that destroys the normal fat cells (A. Misra et al., Medicine (Baltimore) 83: 18-34 (2004)).

HAART and HIV-induced lipodystrophy has become the most common acquired form of generalized lipodystrophia. The General occurrence of these physical abnormalities is approximately 50% after 12-18 months of treatment with protease inhibitors. The difference between these messages is in the range from 18% to 83% of the and affecting factors such as the type and duration of antiretroviral therapy. It has been suggested that the syndrome of lipodystrophy associated with protease inhibitors, may be due to partial analogy between the regulatory lipids and adipocyte proteins and the catalytic site of the protease of HIV-1 is bound to the protease inhibitors (A. Carr et al., Lancet 351: 1881-83 (1998)).

Localized lipodystrophy defined as a localized loss of subcutaneous fat from small areas or parts of limbs. Can be one or more pathological changes characterized by the crisis-ridden regions corresponding to the loss of subcutaneous fat. In some cases it may be accompanied by painful hselkovymi bulges in the skin. It usually occurs in patients with diabetes in the injection of insulin. In some patients, fat loss takes place in areas which are often applied pressure (e.g., compression hip compensating bone plate).

The pathogenesis of lipodystrophy mostly not known. However, accumulating data indicate that the mitochondrial defect as one of the factors increased induction of apoptosis in adipocytes (A. Misra et al., Medicine (Baltimore) 83: 18-34 (2004)). Several proteins encoded by HIV-1 triggers apoptosis by inducing mitochondrial permeability of the membrane is wound. Several nucleoside analogues, is clinically used for the treatment of HIV-1, inhibit the replication of mitochondrial DNA and/or increase the frequency of mutations in mitochondrial DNA. Both of these factors can cause severe mitochondriopathy and to contribute to the lipo. The treatment, which could inhibit the apoptosis of adipocytes, could be a very useful treatment and especially prevention of the development of lipodystrophy, particularly in HIV/HAART-induced form.

Metabolic consequences of lipodystrophy is very important for overall health and survival. The fact that insulin resistance and the subsequent progression of diabetes may be the result or obesity or lipodystrophy, reflects the crucial role of adipose tissue in the metabolism of carbohydrates and lipids. In the absence of adequate functionality adipocyte excess calories can not be discharged in their normal depot store; instead, they accumulate in the form of increased stocks of triglycerides in the liver, skeletal and cardiac muscle and in β-cells of the pancreas. This Snegireva lipid accumulation, is still unknown manner, is associated with impaired insulin action and often diabetes.

In addition to their passive role as depot-store normal adipocytes secrete a number of peptides ("adipokines"), which may in order to influence insulin sensitivity and/or energy balance (B.B. Kahn & J.S. Flier, J. Clin. Invest. 106: 473-81 (2000); A.H. Berg et al., Trends Endocrinol. Metab. 13: 84-89 (2002)). They include the potential insulinsensitizing, such as leptin and Acrp30 (also known as adiponectin), and antagonists of insulin, including TNF-α, IL-6, and resistin. Insulin resistance when lipodystrophia can, therefore, be the result of disturbed lipid threads and/or disorders of secretion of adipokines.

It was reported that treatment with rhGH causes a reduction in the size of the "Buffalo hump" (the accumulation of fat on the neck and at the top of the back) and a fat body in a small number of patients. However, fat loss and lipid abnormalities not improve, and control blood glucose worsens (Lo J.C. et al., J. Clin. Endocrinol. Metab. 86: 3480-87 (2001)).

Treatment of lipodystrophy using amplifying secretion of means, such as a variant splicing of the ghrelin or a similar variant splicing of the ghrelin compound, can be accomplished using any known in the art, the method of administration. Preferably, the treatment can be accomplished using any of these techniques, preferably using intravenous or subcutaneous administration, most preferably using the methods subcutaneous injection.

Quality of life

In all variants of implementation it is preferable that the method of treatment and/or pharmaceuticas is their composition, and/or compounds of the present description have been able to provide the individual being thus treated, improved quality of life (QOL), for example, affirm improved appetite and/or body weight, and/or nutritional status, and/or physical function. Thus, one aspect relates to improvements in quality of life using the amplifying secretion of means, such as a variant splicing of the ghrelin or a similar variant splicing of the ghrelin connection described above.

In another embodiment, the present invention indicated improvement in QOL of the individual assessed using the questionnaire "Quality of life", which is known to the skilled in the art specialist.

Two of the approved research quality of life, preferred for use in evaluating the improvement of QOL caused by the introduction of disclosed here compounds represent the following studies: (i) a brief study of the health research health effects (SF-36), and (ii) the questionnaire EORTC QLQ-C30 (+3). SF-36 contains 36 questions with which evaluated eight aspects of QOL of the patient: physical functioning (PF), role-physical functioning (RP), bodily pain (BP), General health (GH), vitality (VT), social functioning (SF), role-emotional function is inromania (RE) and mental health (MH). In accordance with the guidelines and instructions in relation to the interpretation of the answers to the questions on scales summarize and linearly transform in terms of a scaling factor, which can range from 0 (representing poor health) to 100 (representing optimal health). Confirmed the correctness of the Swedish version, and were presented normative data for the General population of Swedes (M. Sullivan et al., “Hälsoenkät: Svensk Manual och Tolkningsquide” (SF-36 Health Survey, Swedish Manual and Interpretation Guide), Göteborg, Sahigrenska University Hospital, 1994).

EORTC QLQ-C30 (version 3.0) is the main questionnaire of 30 items designed to assess QOL among patients (developed by a group of EORTC Quality of Life Study), see the website of the National Institutes of Health and the see, for example, a sample of the EORTC QLQ-C30 (version 3.0, available on the EORTC website and is incorporated here by reference). The correctness of the first version was confirmed on cancer patients, and was published reference data from the General population. The questionnaire includes five functional scales: physical functioning (five questions), role functioning (two questions), emotional functioning (four items), cognitive functioning (two questions) and social functioning (two questions). There are three scales of symptoms: fatigue (three questions), nausea and vomiting (two questions) and pain (two in the dew). There are six separate positions in shortness of breath, insomnia, loss of appetite, constipation, diarrhea and financial difficulties. Ask two General questions about the patient's state of health and overall QOL. All scales and values of one position are within the range of indices from 0 to 100. A high indicator for scales of functioning and General health and QOL represents a high level of functioning/health status and QOL. A high indicator for scales of symptoms and positions represents a high level of symptoms/problems. Indicators of QOL can be calculated in accordance with the manual on the measurement of the EORTC QLQ-C30.

Preferred evaluation forms improve the quality of life of patients after treatment one or more compounds that enhance the secretion of means are presented in table 9 below.

In preferred embodiments, the implementation of the present invention the treatment of the patient with the condition(s)described herein leads to a significant improvement of QOL of the patient. Preferably, the treatment leads to a significant increase in QOL determined using any method validation QOL, including, but without limitation, the above-mentioned questionnaires, for example, the increase(s) QOL or composite measure of QOL that are suitable for the way an individual determination, or reduce pokazatelyey), related to symptoms and/or problems, respectively.

This increase or decrease, respectively, is preferably 1% over the rate obtained before treatment, more preferably 2%, even more preferably 5%, for example 10%, even more preferably 20%, 50% or 75% above the rate before treatment. In another embodiment of the present invention, the treatment leads to a detectable increase the QOL indicator so that the indicator after treatment is equal to the average found in a comparable pool of healthy subjects, or close to such a "normal" rate, i.e. more than 50%, even more preferably 60% and more preferably 75%. In addition, in another embodiment of the present invention, the treatment leads to a decrease of the indicator(s)associated with symptoms and/or problems, constituting at least 1%, more preferably 3%, even more preferably 5% or more, preferably 10%, 20%, 30% or 50% of the indicator(s) prior to treatment. These increase or decrease, respectively, can be one, several, or all aspects of the method of determining the QOL of an individual or of a composite indicator, if appropriate.

Stimulation of appetite, food intake, weight gain, increase lean body mass, the expansion of the body fat mass

As discussed above, the promotion of weight gain or promoting weight maintenance, in particular, individuals suffering from pathological weight loss is not just something like stimulation of appetite and/or food intake, but rather also the correction of the imbalance between energy intake and energy expenditure, i.e. the overall metabolism of the body. However, stimulation of appetite will still be beneficial for some individuals, particularly for those individuals who have the disease process has led to a decrease in appetite, which will naturally lead to unhealthy weight loss. Thus, one aspect relates to stimulate appetite by administering enhance the secretion of tools, such as such variant splicing of the ghrelin connection. Stimulation of appetite can be defined using, for example, visual analog scales for measurement of appetite, feelings of hunger or satiety. In a preferred embodiment of the present invention stimulation is the stimulation to at least 5% compared with appetite before treatment, for example, 10% higher, more preferably 20% higher, or even more preferably 30%, 40% or 50% higher.

Stimulation of appetite does not necessarily lead to an increase in food intake, and, therefore, present the Scripture, also refers to another aspect, stimulation of food intake by introducing enhance the secretion of tools, such as such variant splicing of the ghrelin connection.

Food consumption can be measured using a variety of methods, including the message itself using, for example, diaries or questionnaires, measurement of the absorption of calories food pantry, the use of weighing food before they are received or the weighting and analysis of dual quantities of food. Food consumption can be measured on a food basis, daily basis, weekly basis or monthly basis. In a preferred embodiment of the present invention, the treatment leads to an increase of 1% in food intake, for example, an increase of 2%, more preferably 3% or 5%or 7% and even more preferably 10% above average food consumption prior to treatment. In another embodiment of the present invention, the treatment leads to increased absorption of calories regardless of changes in food consumption, since the amount of consumed food may not be directly related to calories consumed, as various items of food, such as fat, carbohydrates and proteins contain different amounts of calories for the amount of food. In a preferred embodiment of the present invention the treatment with the result in the increase of 1% absorption of calories for example, an increase of 2%, more preferably 3% or 5%or 7% and even more preferably 10% increase absorption of calories.

Another aspect relates to the stimulation of weight gain or to maintain a stable body weight by administering such variant splicing of the ghrelin connection.

It is preferable to stimulate food intake and weight gain applicable amplifying secretion of a tool, such as a similar variant splicing of the ghrelin connection. More preferably enhances the secretion of a tool, such as a similar variant splicing of the ghrelin connection, used for stimulating weight gain or maintain a stable body weight.

As discussed below, it is preferable for enhancing the secretion of a tool, such as a similar variant splicing of the ghrelin connection was administered before a meal, for example, within 180 minutes before a meal, for example, within 150 minutes before a meal, for example, within 120 minutes before a meal, for example, within 100 minutes before a meal, for example, within 80 minutes before a meal, for example, within 60 minutes before a meal, for example, within 45 minutes before a meal, for example, within 30 minutes before a meal, for example, within 15 minutes before a meal. In addition, it is preferable that such variant splicing of the ghrelin of the giving was administered subcutaneously.

In addition, amplifying secretion of a tool, such as a similar variant splicing of the ghrelin connection, you can enter in order to contribute to the preservation of physical functioning and/or to facilitate the recovery of physical function, for example, individuals recovering from major surgery, such as the introduction of the prosthesis of the hip joint, amputation, broken bones, and open heart surgery.

In particular, amplifying secretion of a tool, such as a similar variant splicing of the ghrelin connection applicable for the treatment of a subject with low birth weight or to prevent weight loss to the state of low weight. Subjects with low birth weight include subjects with a body weight less than about 3%, 5% or less, 10% or less, 20% or less, or 30 or less, the lower limit of "normal" weight range, or BMI. Normal weight ranges are well known in the art and include such factors as the patient's age, height and body type. In addition, disclosed here, the compound suitable for the treatment of patients affected by involuntary weight loss before treatment, for example, weight loss of 1% per month, 2% or less per month, or 5% or less within 6 months.

The increase in weight or appetite may be useful for the patient, Meuse what about the disease or being treated, which affects the weight or appetite. In addition, for example, farm animals such as pigs, cows or chickens, can be subjected to a treatment for increasing the weight.

The increase in body fat mass of an individual can be easily assessed by a qualified technician using a number of known methods. One variant of implementation of the present invention refers to an increase in fat body mass without increasing the overall weight of the individual. The preferred implementation of the present invention leads to an increase of body fat by 2%, compared with fat before treatment, more preferably 4%, for example 5%, and 8%, and 10%, and even more preferably 20% or 40% above the values before treatment.

Additional state of the individual, which can be treated in accordance with the present description, include the neurogenic bulemia, anorexia, erectile dysfunction in men, sexual dysfunction in women, the decrease of intensity of ischemic damage to the nerve or muscle, and systemic lupus erythematosus.

Subcutaneous administration

It is important to note that ghrelin variant splicing of the ghrelin activates the receptor GHS and additional receptors, which are still identified. These receptors are found on producing GH cells in the hypothalamic centers controlling Appeti is a and the number of additional places in the body. In the Central nervous system, these receptors configured to receive signals from local, containing variant splicing of the ghrelin neurons. Perifericheskie-secreted or artificially introduced variants of splicing of the ghrelin will reach such places and will pass through the barrier the blood - brain, specifically activating the appropriate receptors and initiating a specific way. However, the currently available "so-called" enhancing the secretion of GH funds, which are small organic compounds such as MK-0677 (Merck), aimed, as a rule, the binding with the receptor GHS, will pass through the barrier the blood - brain and also to reach these places, activating various related receptor GHS path, and consequently the danger of calling unwanted side effects such as dizziness, nausea, fall, elevated levels of glucose in serum glucose and insulin and darkened vision. Thus, such compounds, which do have the advantage that they are, for example, orally active, will not be optimal for reproducing nature prior to food intake, inducing appetite jump variant splicing of the ghrelin because they activate non-specific all related receptor GHS way in the body in Front, using natural peptide, the variant splicing of the ghrelin or its homologues, and entering it perifericheskie, as in the preferred embodiment, the present invention ensures that only relevant regulating appetite receptors variant splicing of the ghrelin and path are reached and stimulated.

Part of the alternative implementation of the present description may be any form of parenteral administration, which will ensure that the receptor variant splicing ghrelin, which are usually the target for perifericheskie-produced variant splicing of the ghrelin in pre-meal situation, will be exposed to levels of bioactive forms of variant splicing of the ghrelin sufficient to ensure a strong and appropriate stimulation of appetite, without calling desensitization of the system. However, taking into account that being treated individuals may need to receive treatment during much of the period, such as weeks or months, preferably so that it fit in well with the form of administration. Accordingly, it is preferable for enhancing the secretion of a tool, such as a similar variant splicing of the ghrelin compound was administered subcutaneously in number, making it possible to achieve sufficient levels of bioactive forms of variant splicing GRE is in, i.e. acylated form to receptors to the upcoming meal.

In the present description, preferably discusses how the introduction of amplifying secretion of tools, such as splicing variant of ghrelin, through which plays a physiologically preceding the meal situation as close as possible and still provides patients the increase in food intake, for example, weak elderly patients, patients after surgery and/or patients with loss of appetite as part of cachexia caused, for example, cancer, heart disease, etc. sufficient extension of the stimulating signal to their regulating appetite receptors splicing variant, which is usually achieved by alternative splicing of the ghrelin in pre-meal situation.

A bolus

In addition, from the point of view of molecular pharmacology it is important to note that, as found on the ghrelin receptor and, therefore, the receptor variant splicing of the ghrelin effect of short-term jumps in the concentration of ghrelin. The receptor GHS-R 1a (1A receptor enhances the secretion of growth hormone funds) belongs to the class associated with G protein receptor or receptors TM that continuous exposure to agonist desensitized undergo internalization and supressents. These mechanics is what we which are inherent in the whole system of signal transduction include processes such as phosphorylation of the receptor (which itself reduces the affinity of the receptor to the agonist) binding of inhibitory proteins, such as arrestin, (which sterically block the binding molecules of signal transduction, such as G proteins). Another part mediated by agonist desensitization process is the internalization of the receptor (i.e. the physical movement of the receptor from the cell surface, where it could link agonist), and suppression of the receptor (i.e. reduce production/expression of the receptor). For the internalization of the receptor could, after a short-term impact on receptor agonist, to follow the process of desensibilization when the receptor is exposed to dephosphorylating and is restored to the cell surface in order to re-use. Hasn't bound by theory, the assumption that long-term stimulation, which would occur, for example, during prolonged continuous infusion of the agonist, the process of suppression of receptor provides that the system of signal transduction of target cells adapt to this situation.

The optimal introduction

The present description also provides a procedure for the optimal introduction patients splicing variant of ghrelin, h is ordinary to obtain maximum response and to avoid for example, mechanisms of desensitization.

Accordingly, one aspect relates to the introduction enhancing the secretion of tools, such as such variant splicing of the ghrelin connection, boles, preferably bolus before each main meal. It was found that in contrast to the continuous processes introduction in the prior art as a bolus leads not only to stimulate appetite, but also to stimulate food intake and more importantly to stimulate weight gain. Hasn't bound by theory, the assumption that subcutaneous injection, intravenous injection or intermittent infusion before a meal appropriate doses increase secretion of means, such as a variant splicing of the ghrelin or a similar variant splicing of the ghrelin connection will ensure that strong stimulation inducing appetite receptor variant splicing of the ghrelin will be obtained with minimal restriction, for example, the mobility of the patient. Thus, for example, patients with a hip fracture can, in the situation after the operation, be subjected to treatment prior to the meal period and, if required, during the meal, to move freely and to participate in important postoperative physiotherapy schemes. In one preferred embodiment, the implementation of the Oia, the present invention enhances the secretion of the tool, such as a variant splicing of the ghrelin or a similar variant splicing of the ghrelin connection, enter the bolus in the amount equal to 10 mcg per kg of body weight.

Such variant splicing of the ghrelin connection

In the disclosed here are methods you can use any enhancing the secretion of a tool, such as a variant splicing of the ghrelin or a similar variant splicing of the ghrelin connection. One of the preferred types similar variant splicing of the ghrelin compounds described herein is a compound comprising a structure defined by formula I: Z1-(X1)m-(X2)-(X3)n-Z2, where Z1 is an optionally present protective group; each X1 is independently selected from naturally occurring amino acid and a synthetic amino acid; x2 is selected from naturally occurring amino acids and synthetic amino acids, with the specified amino acid modified large hydrophobic group; each X3 is independently selected from naturally occurring amino acids and synthetic amino acids, and one or more of X1 and X3 can be optionally modified by a large hydrophobic group; Z2 is an optionally present protective group; m is an integer in the range of 1-10; n is an integer in the range 4-92; provided that the length with the Union in accordance with the formula Z1-(X1)m-(X2)-(X3)n-Z2 is 15-94 amino acids, and this connection is homologous to at least 80% (or in alternative embodiments, the implementation of 85%, 90%, 93%, 95%, 97%, 98%, 99%, 100%) SEQ ID NO: 1. In a preferred embodiment of the present invention, the length of such variant splicing of the ghrelin connection is 22-29 amino acids.

Accordingly, the term "amplifying secretion agent" includes naturally occurring variant splicing of the ghrelin man of 29 amino acids, amino acid sequence of which is presented in SEQ ID NO: 5, as well as naturally occurring variant splicing of the ghrelin man of 22 amino acids, amino acid sequence of which is presented in SEQ ID NO: 2, and naturally occurring variant splicing of the ghrelin man of 24 amino acids, amino acid sequence of which is presented in SEQ ID NO: 3.

The present description includes the diastereomers, and racemic and split enantiomerically pure form. Enhance the secretion of the tool may contain D-amino acids, L-amino acids, alpha-amino acid, beta-amino acid, gamma-amino acid, natural amino acid and/or synthetic amino acid or the like, or a combination of both. Preferably the amino acids present in this variant splicing of the ghrelin connection represent the L-enantiomers.

Such variant splicing of the ghrelin is Obedinenie preferably includes the amino acid modified large hydrophobic group. The number of amino acids that are N-terminal relative to the modified amino acid, preferably is within the range that makes 1-9. Accordingly, m is preferably an integer in the range 1-9, for example, 1-8, for example, 1-7, for example, 1-6, for example, 1-5, for example, 1-4, for example, 1-3, for example 1-2, e.g., 2.

More preferably, when the number of amino acids that are N-terminal relative to the modified amino acid, is low, for example, 1-3, for example, 1-2. Most preferably 2 amino acids are N-terminal position relative to the modified amino acid.

In a preferred embodiment of the present invention (X1)m has a Gly residue at the N-terminal part of the sequence. Accordingly, in the preferred embodiment of the present invention (X1)m is chosen from the sequences Gly, Gly-Ser, Gly-Cys, Gly-Lys, Gly-Asp, Gly-Glu, Gly-Arg, Gly-His, Gly-Asn, Gly-Gln, Gly-Thr and Gly-Tyr. More preferably, X1)m is selected from Gly-Ser and Gly-Cys, most preferably (X1)m is a Gly-Ser.

In other words, in the preferred embodiment of the present invention is similar to the variant splicing of the ghrelin compound selected from Z1-Gly-(X1)m-(X2)-(X3)n-Z2 (formula II), Z1-Gly-Ser-(X2)-(X3)n-Z2 (formula III) and Z1-Gly-(X2)-(X3)n-Z2 (formula IV). More pre is respectfully similar variant splicing of the ghrelin compound has the formula III.

As described above, and x2 can be any amino acid, modified large hydrophobic group. In particular, x2 are selected from the group consisting of modified Ser, modified Cys modified Asp, modified Lys modified Trp modified Phe modified Ile and modified Leu. More preferably, x2 is chosen from the group consisting of modified Ser, modified and modified Cys Lys; most preferably x2 is a modified Ser.

In addition, (X1)m-(X2) preferably represents Gly-Xaa-Ser∗ or Gly-Xaa-Cys∗, where Xaa represents any amino acid, more preferably (X1)m-(X2) represents Gly-Ser-Ser∗ or Gly-Ser-Cys∗, where ∗ indicates that the amino acid residue modified large hydrophobic group.

(X3)n preferably includes a sequence that is a fragment of variant splicing ghrelin, such as splicing variant of human ghrelin. Respectively (X3)n preferably includes a fragment of the following sequence (SEQ ID NO: 6): Phe Leu Ser Pro Glu His Gln Arg Val Gln Val Arg Pro Pro His Lys Ala Pro His Val Val Pro Ala Leu Pro Leu Ser Asn Gln Leu Cys Asp Leu Glu Gln Gln Arg His Leu Trp Ala Ser Val Phe Ser Gln Ser Thr Lys Asp Ser Gly Ser Asp Leu Thr Val Ser Gly Arg Thr Trp Gly Leu Arg Val Leu Asn Arg Leu Phe Pro Pro Ser Ser Arg Glu Arg Ser Arg Arg Ser His Gln Pro Ser Cys Ser Pro Glu Leu.

In the preferred embodiment, this is part II of the invention the length of such variant splicing of the ghrelin connection essentially similar to long processioning ghrelin person, i.e. is 29 amino acids. Accordingly, preferably n is an integer in the range of 4-25, for example, 4-24, for example, 4-22, for example, 4-15, for example, 4-10, for example, 10-25, for example, 10-24, for example, 15-25, for example, 15-24. Most preferably such variant splicing of the ghrelin connection is a variant splicing of the ghrelin man of 29 amino acids, amino acid sequence of which is presented in SEQ ID NO:5; option splicing of the ghrelin man of 22 amino acids, amino acid sequence of which is presented in SEQ ID NO:2; variant splicing of the ghrelin man of 24 amino acids, amino acid sequence of which is presented in SEQ ID NO:3; or a variant splicing of the ghrelin man of 24 amino acids having in the third position, the remainder of 2,3-diaminopropionic acid (Dpr), the amino acid sequence of which is presented in SEQ ID NO:4.

Functionality

Enhance the secretion of the tools described here, is active against receptor described here for GHS, i.e. GHS-R 1a. Connections can contact GHS-R 1a and preferably stimulate the activity of the receptor. In some embodiments, implementation of the present invention compounds can communicate with other receptors and do not necessarily stimulate their activity.

The activity of the receptor can be measured with use the of different methods, such as the detection of changes in intracellular conformation of the receptor that are associated with G-protein activity and/or intracellular carriers. One simple indicator of the ability of such variant splicing of the ghrelin connection to activate the receptor variant splicing of the ghrelin is the measurement of its ES, i.e. the dose at which the compound is able to activate the induction of the receptor with a half maximal effect connection. When measuring, for example IS, the receptor may be or downregulation of endogenous on the source cell cultures, such as the pituitary cells, or heterogeneously expressed on cells, travelbank the ghrelin receptor. Assays using intact cells or assays using membranes prepared from one or two of these types of cells can be used depending on the analysis type.

Because the receptor, as usually considered, is associated mainly transmitted signal Gq, you can use any suitable analysis, which allows you to monitor activity on the transmission path of the signal Gq/G11, for example,

1) Analysis, which is the activation of Gq/G11 performed, for example, by measuring binding GTPgS, combined, for example, by precipitation using antibodies against G-alpha-q or -11 to increase the signal to the mind. With this study it is possible to detect the binding of G-proteins other than Gq/11.

2) Analysis, which is the activity of phospholipase C (PLC), one of the first effector molecules in the direction of the way down, for example, by measuring the accumulation of intitolata, which is one of the products PLC.

3) later in the cascade signal is the mobilization of calcium from intracellular storage, which can be determined using any method known to the specialist with an average level of competence in this field of technology.

4) you Can also define another downstream molecules of signal transmission, for example, the activity of different types of kinases MAR (p38, jun, etc), translocation NKκB and managed CRE gene transcription.

5) the Binding of fluorescently labeled arrestin activated receptor ghrelin.

Examples suitable for use in determining the functionality enhances the secretion of funds protocols shown in example 4 below.

In one embodiment, the binding compound to the receptor GHS-R 1a can be determined using the analysis described here above.

Such variant splicing of the ghrelin compound preferably has at least about 50%, at least about 60%, at least arr is siteline 70%, at least about 80% or at least about 90% of the functional activity relative to ghrelin human wild-type of the 28 amino acids determined using the analysis described here above, and/or IS greater than about 1000, greater than about 100, or greater than about 50, or greater than about 10. Excess refers to the activity and, thus, indicates that a smaller amount is required to achieve inhibition of the binding.

In one embodiment of the present invention the compound has an activity (IES) in relation to the GHS-R 1a constituting less than 500 nm. In another embodiment of the present invention the compound has an activity (IES) in relation to the GHS-R 1a constituting less than 100 nm, for example, less than 80 nm, for example, less than 60 nm, for example, less than 40 nm, for example, less than 20 nm, for example less than 10 nm, for example, less than 5 nm, for example, less than 1 nm, for example, less than 0.5 nm, for example, less than 0.1 nm, for example, less than 0.05 nm, for example, less than 0.01 nm.

In a further embodiment of the present invention, the dissociation constant (Kd) connection is less than 50 nm. In a still further embodiment of the present invention, the dissociation constant (Kd) of the ligand is less than 100 nm, for example, less than 80 nm, for example, less than 60 nm, for example less than 40 nm, for example, less than 20 nm, for example less than 10 nm, for example, less than 5 nm, for example, less than 1 nm, for example, less than 0.5 nm, for example, less than 0.1 nm, for example, less than 0.05 nm, for example, less than 0.01 nm.

The analysis of binding can be performed using recombinante produced polypeptide receptors present in different environments. Such environments include, for example, cell extracts and purified cell extracts containing polypeptide receptor, expressed from recombinant nucleic acids or naturally occurring nucleic acids, and include, for example, using a purified polypeptide receptor GHS produced by recombinant means or from naturally occurring nucleic acid that is introduced into a different environment.

Use recombinante downregulation of the receptor GHS offers several advantages, such as the ability to Express the receptor described in cellular system, so that the connection response in the receptor can be easily differentiated from responses in other receptors. For example, the receptor can be Express in a cell line such as HEK 293, COS 7 and CHO, not expressing typically the receptor, with the help expressing vector, with the same cell line without expressing vector can act as a con is playing.

Identity and homology

The term "identity" and "homology", as it should be interpreted, means the percentage of amino acid residues in the candidate sequence that are identical with the residues corresponding to the sequence with which it is compared, after aligning the sequences and introducing gaps, if necessary to achieve the maximum percent identity for the entire sequence, and without considering any conservative substitutions as part of the identity of the sequences. Either N - or C-terminal extension or insertion will not be considered as reducing identity or homology. Methods and computer programs for alignment are well known in the art. The sequence identity can be determined using the software for sequence analysis (for example, a software package for sequence analysis, Genetics Computer Group, University of Wisconsin Biotechnology Center, Madison, Wis.). This software compares similar sequence by setting the degrees of homology to various substitutions, deletions and other modifications.

The homologue of variant splicing of the ghrelin with one or more precisely defined here sequences may differ by one or more amino acids compared the structure with defined sequences but can perform the same function, i.e., the homolog may be provided as a functional equivalent of a specified sequence.

Preferably a homologue of variant splicing of the ghrelin is similar variant splicing of the ghrelin connection described above.

As described above, the homologue of any of the sequences defined here can be defined as i) homologues comprising amino acid sequence that can be recognized by the antibody also recognizes a variant splicing of the ghrelin man of 22 amino acids and/or 24 amino acids, and/or splicing of the ghrelin man of 29 amino acids, preferably acylated variant splicing of the ghrelin man of 22 amino acids and/or 24 amino acids, and/or acylated variant splicing of the ghrelin man of 29 amino acids, and/or (ii) homologues comprising the amino acid sequence capable of selectively contact the GHS-R 1a, and/or (iii) homologues having affinity binding to GHS-R 1a, essentially similar or higher than option splicing of the ghrelin man of 22 amino acids and/or 24 amino acids, and/or splicing of the ghrelin man of 29 amino acids, preferably acylated variant splicing of the ghrelin man of 22 amino acids and/or 24 amino acids, and/or acylated variant splicing of the ghrelin man of 29 and is of inoculat. (Alternative splicing of the ghrelin man of 22 amino acids and/or 24 amino acids, and/or 29 amino acid may have a sequence represented in SEQ ID NO: 2, SEQ ID NO: 3; SEQ ID NO: 4 and SEQ ID NO: 5, and the acylation he alleroed in position 3).

Used here antibody can be an antibody that binds the N-terminal region variant splicing of the ghrelin or C-terminal region variant splicing ghrelin, preferably N-terminal region. Antibodies may be antibodies described Ariyasu, H. and others (Endocrinoly 143: 3341-50 (2002)).

Given as examples of homologues include one or more conservative amino acid substitutions include one or more conservative amino acid substitutions within the same group in advance of certain amino acids, or many conservative amino acid substitutions, each of which is a conservative substitution created by replacement within a great group of pre-defined amino acids. Homologues may, therefore, include conservative substitutions, independent from one another, and at least one glycine (Gly) specified homologue substituted amino acid selected from the group of amino acids consisting of Ala, Val, Leu and Ile; and regardless of this homologues, in which at least one of these alanine (Ala) specified homologue substituted amino acid selected from the group of amino acids, SOS is oasa from Gly, Val, Leu and Ile; and regardless of this homologues, in which at least one valine (Val) specified homologue substituted amino acid selected from the group of amino acids consisting of Gly, Ala, Leu and Ile; and regardless of this homologues, in which at least one of these Lachinov (Leu) specified homologue substituted amino acid selected from the group of amino acids consisting of Gly, Ala, Val, and Ile; and regardless of this homologues, in which at least one isoleucine (Ile) of these homologues substituted amino acid selected from the group of amino acids consisting of Gly, Ala, Val and Leu; and regardless of this homologues, in which at least one of these aspartic acid (Asp) specified homologue substituted amino acid selected from the group of amino acids consisting of Glu, Asn and Gln; and regardless of this homologues, in which at least one of these phenylalanine (Phe) of these homologues substituted amino acid selected from the group of amino acids consisting of Tyr, Trp, His, Pro, and preferably selected from the group of amino acids consisting of Tyr and Trp; and regardless of this homologues, in which at least one of these tyrosines (Tyr) of these homologues substituted amino acid selected from the group of amino acids consisting of Phe, Trp, His, Pro, preferably an amino acid selected from the group of amino acids consisting of Phe and Trp; and n is dependent on this homologues, in which at least one of these residues (Arg) indicated fragment is substituted by an amino acid selected from the group of amino acids consisting of Lys and His; and, irrespective of this homologues, in which at least one lysine (Lys) of these homologues substituted amino acid selected from the group of amino acids consisting of Arg and His; and, irrespective of this homologues, in which at least one of these asparagine (Asn) of these homologues substituted amino acid selected from the group of amino acids consisting of Asp, Glu and Gln; and independently homologues, in which at least one glutamine (Gln) of these homologues substituted amino acid selected from the group of amino acids consisting of Asp, Glu, and Asn; and regardless of this homologues, in which at least one Proline (Pro) of these homologues substituted amino acid selected from the group of amino acids consisting of Phe, Tyr, Trp and His; and, irrespective of this homologues, in which at least one of the specified cysteine (Cys) of these homologues substituted amino acid selected from the group of amino acids consisting of Asp, Glu, Lys, Arg, His, Asn, Gln, Ser, Thr and Tyr.

Conservative substitutions may be introduced at any position of a preferred predetermined sequence. However, it may be desirable introduction of nonconservative substitutions, in particular, but without limitation, the non-conservative replacement of the s in one or more positions.

Non-conservative substitution leading to the formation of a functionally equivalent homologue sequences here could, for example i) differ substantially in polarity, for example a residue with a polar side chain (Ala, Leu, Pro, Trp, Val, Ile, Gly, Leu, Phe or Met), is replaced by a residue with a polar side chain, such as Ser, Thr, Cys, Tyr, Asn or Gln, or a charged amino acid such as Asp, Glu, Arg, or Lys, or replacement of a charged or polar residue non-polar residue; and/or ii) differ substantially in its effect on the orientation of the polypeptide backbone, for example, replacing Pro or Gly by another residue; and/or iii) differ substantially in electric charge, for example substitution of a negatively charged residue such as Glu or Asp for a positively charged residue such as Lys, His or Arg (and Vice versa); and/or iv) differ substantially in steric bulk, for example substitution of a large volume of residue such as His, Trp, Phe or Tyr at residue having a small side chain, e.g. Ala, Gly or Ser (and Vice versa).

In one embodiment, the replacement of amino acids can be carried out on the basis of the values of their hydrophobicity and hydrophilicity, and the relative similarity of the substituents of the side chains of amino acids, including charge, size, etc. are Examples of amino acid substitutions that take into account time the ranks of the above characteristics, well-known skilled in the art specialists and include, for example, arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine.

In a preferred embodiment of the present invention binding domain comprises a homolog having the amino acid sequence homologous to at least 60% of SEQ ID NO: 1. More preferably, the homology is at least 65%, for example, homology SEQ ID NO: 1 in at least 70%, such as 75%, for example, 80%, such as 85%, for example 90%, such as 95%, for example 98%. In a more preferred embodiment of the present invention noted above percentages relate to the identity of the sequence of the homologue is compared with SEQ ID NO: 1. The homologues of SEQ ID NO: 1 may be an option splicing of the ghrelin man of 22 amino acids (SEQ ID NO: 2), variant splicing of the ghrelin man of 24 amino acids (SEQ ID NO: 3) or a variant splicing of the ghrelin man of 29 amino acids (SEQ ID NO: 5). Other homologues are the variants described in EP 1197496 (Kangawa), incorporated here by reference.

A large hydrophobic group

A large hydrophobic group enhances the secretion of the means disclosed here, is any large hydrophobic group capable of disallowances relin human wild type of 28 amino acids, or its counterpart, the affinity of binding to GHS-R 1a and/or GS-R-1b. Any suitable amino acid can be modified in any suitable large hydrophobic group. In a preferred embodiment of the present invention, the Ser residue (preferably amino acids with the number 3 in the amino acid chain variant splicing of the ghrelin) modify a large hydrophobic group. If the modified amino acid includes, for example, -OH, -SH, -NH or-NH2as a replacement group in its side chain is preferred replacement band formed by acylation. The communication method may, therefore, be selected from the group consisting of ether complex, a simple ester, tiefer, amide and urea. For example, if the modified amino acid is serine, threonine, tyrosine or hydroxyproline, an amino acid has a hydroxyl group in the side chain. If the modified amino acid is cysteine, the amino acid has mercaptopropyl in the side chain. If the modified amino acid is lysine, arginine, histidine, tryptophan, Proline or hydroxyproline, an amino acid has an amino group or aminogroup in the side chain.

Hydroxyl group, mercaptopropyl, amino group and aminogroup described above can, therefore, be chemically modified. I.e. hydroxyl group or marketgroup what have you, for example, subjected to etherification, esterification, diatrypaceae or diesterification. Aminogroup can, for example, be aminoethanethiol, iminodiacetonitrile or alkylation. The amino group can, for example, be subjected to amidation, miamidian or carbamimidoyl. In addition, mercaptopropyl can, for example, to turn into a disulfide, aminogroup can, for example, be subjected to amidation or miamidian, and the amino group may be, for example, alkylated or thiocarbamide.

In a preferred embodiment of the present invention the modified amino acid is Ser, linked through a complex essential connection with a hydrophobic group, or in another preferred embodiment of the present invention the modified amino acid is Dpr, connected through the amide bond with a hydrophobic group.

The hydrophobic group may be any group with a saturated or unsaturated alkyl or acyl group containing one or more carbon atoms. In one embodiment of the present invention a large hydrophobic group is the acyl group include groups formed by removing a hydroxyl group from an organic carboxylic acid, organic sulfonic acids, or organic phosphorus acids. Organicheskoi the carboxylic acid includes, for example, fatty acids, and the number of its carbon atoms is preferably leaves 1-35. In the organic acid or organic phosphoric acid number of carbon atoms preferably leaves 1-35.

Accordingly acyl group preferably selected from C1-C35acyl group, for example, With1-C20acyl group, for example, With1-C15acyl group, for example, With6-C15acyl group, for example, With6-C12acyl group, for example, With8-C12acyl group. More preferably acyl group selected from the group consisting of C7acyl group8acyl group9acyl group10acyl group11acyl group and12acyl group. Such acyl group can be formed from octanoic acid (preferably Caprylic acid), decanoas acid (preferably capric acid) or dodecanol acid (preferably lauric acid), and their monoenoic or polyene fatty acids.

In addition, the modified amino acid may be any amino acid, in which the group is modified, as described in EP 1197496 (Kangawa), incorporated here by reference.

Protective group

Such variant splicing of the ghrelin connection according to the present description may include a protective group on the N-end or From the end, or at both ends. Protective group, covalently linked to the N-terminal amino group reduces the reactivity of aminocore in vivo. Protecting aminocore groups include, for example, With1-C10alkyl, C1-C10substituted alkyl, C2-C10alkenyl,2-C10replaced alkenyl, aryl, C1-C6alkylaryl, C(O)-(CH2)-(C1-C6alkyl)-COOH, C(O)-(C1-C6alkyl), C(O)-aryl, C(O)-O-(C1-C6alkyl), or C(O)-O-aryl. Preferably protects aminocore group is acetyl, propyl, succinyl, benzyl, benzyloxycarbonyl or tert-butyloxycarbonyl.

Protective group, covalently linked to the C-terminal carboxyl group reduces the reactivity of the carboxyl end in vivo. Protecting the carboxyl end group is preferably attached to the α-carbonyl group of the last amino acid. Protecting the carboxyl end groups include, for example, amide, methylamide and ethylamide.

Conjugates

Enhancing the secretion of a tool, such as a similar variant splicing of the ghrelin connection may be provided in the form of a conjugate enhances the secretion of funds, i.e. molecules comprising amplifying secretion tool conjugated with another element. Another element may be any vases is in, which can give improved properties enhance the secretion of the tool, for example, in relation to the increased stability of the half-period of existence, etc. are Examples of suitable elements are described as follows. For example, amplifying secretion of the tool can be konjugierte with the peptide, for example, the peptide has the effect on the receptor nociceptin ORL1. In one embodiment of the present invention, the conjugate is a conjugate version of the splicing of the ghrelin or its derivative, or homologue peptide that effect on ORL1, i.e. the peptide Ac-RYY(RK)(WI)RK)-NH2where in brackets shows the allowable variation of amino acid residues. Examples of other suitable peptides are found in published patent application U.S. No. 2003/040472 and 2002/004483 and U.S. patent No. 5869046, all of which are included here by reference.

In another embodiment, the present invention enhances the secretion of a tool, such as a variant splicing of the ghrelin and similar variant splicing of the ghrelin connection, kongugiruut with polymer molecule. The polymer molecule can be any suitable polymer molecule, such as a natural or synthetic polymer, typically with a molecular weight in the range of about 1-100 kDa, for example, approximately 3-20 kDa, for example, approximately 5-10 kDa. The polymer connec the Ute to the reactive group, present to enhance the secretion of the vehicle, such as an amino group or Tilney group. Examples of suitable polymer molecules include polymer molecules selected from the group consisting of polyalkyleneglycol (RAO), including polyalkyleneglycol (PAG), for example, linear or branched polyethylene glycol (PEG) and polypropyleneglycol (PPG); polyvinyl alcohol (PVA); polycarboxylate; poly(vinylpyrrolidone), polyethylene-copolymer of maleic anhydride; polystyrene-copolymer of maleic anhydride; and dextran, including carboxymethylcysteine. Preferably the polymer molecule is a molecule of PEG, in particular monofunctional PEG, such as methoxypolyethyleneglycol (mPEG). Suitable activated PEG molecules are available from, for example, Nektar Therapeutics Inc. (Huntsville, Ala) or Valentis, Inc. (Burlingame, Cal). In the alternative case, the molecules of the polymers can be activated using conventional methods known in the art, for example, as described in the application WO 90/13540 included here by reference. Specific examples of activated PEG polymers include linear PEG: NHS-PEG (e.g., SPA-PEG, SSPA-PEG, SBA-PEG, SS-PEG, SSA-PEG, SC-PEG, SG-PEG and SCM-PEG), NOR-PEG, BTC-PEG, EPOX-PEG, NCO-PEG, NPC-PEG, CDI-PEG, ALD-PEG, TRES-PEG, VS-PEG, IODO-PEG and MAL-PEG, branched PEG, such as PEG2-NHS and those disclosed in U.S. patent No. 5932462 and 5643576, both of which included codeposition links.

Pegylation (i.e., the conjugation of the polypeptide enhances the secretion of funds and the activated polymer molecule) is carried out in accordance with recognized procedures, such as described in the following reference documents (which also describe suitable methods of activation of polymer molecules): R.F. Taylor (1991) “Protein Immobilization. Fundamentals and Applications”, Marcel Dekker, N.Y.; S.S. Wong (1992) “Chemistry of Protein Conjugation and Crosslinking”, CRC Press, Boca Raton; G.T. Hermanson et al. (1993) “Immobilized Affinity Ligand Techniques”, Academic Press, N.Y.).

Also provided according to the present description, the binding molecules of the polymer to enhance the secretion of the tool through the linker. Suitable linkers are well known to the skilled technician. A preferred example is cyanuric acid chloride (Abuchowski, A. et al., J. Biol. Chem. 252: 3578-81 (1977); U.S. patent No. 4179337; Shafer et al., J. Polym. Sci. Polym. Chem. Ed. 24: 375-78 (1986)).

In yet another embodiment, the present invention enhances the secretion of the tool kongugiruut with oligosaccharide molecule, such as dextran, glycan, transferrin, etc. Such conjugation can be accomplished in accordance with recognized methods, such as methods that are available from Neose Technologies, Inc. (Horsham, PA).

In yet another embodiment, the present invention enhances the secretion of the tool kongugiruut with the Fc fragment of the IgG molecule, usually in the form of a fused protein. E.g. the measures bind to the receptor epitope salvation of the Fc fragment of IgG (i.e. the Fc fragment of immunoglobulin isotype IgG) include in enhancing the secretion of the tool in order to increase its circulatory half-life existence, but without losing its biological activity. This can occur by any method, for example, by mutation of the appropriate region in enhancing the secretion of the tool to simulate the Fc fragment, or by incorporating the epitope into a peptide tag that is then drained from the amplifying secretion means on one of the two ends or include in the middle, or by synthesis of DNA or peptide.

Bind to the receptor epitope of salvation is any suitable epitope known to the skilled in the art specialist, and its nature will depend, for example, of the type subjected to modification enhances the secretion of funds. The epitope include enhancing the secretion of the tool, so that the biological activity of enhancing the secretion of funds saved, i.e. the epitope is not prejudicial conformation enhances the secretion of funds or does not affect its binding to ligands, which ensures its biological activity.

In the alternative case to ensure enhancing the secretion of funds in the form of a conjugate enhances the secretion of the tool can modify the synchronize with the inclusion of suitable reactive groups, whereby the modified thus enhancing the secretion of a tool capable of forming the conjugate in vivo (after the introduction of the individual) due to covalent binding with available reactive functionalities on blood components. The present description also relates to such modification enhances the secretion of tools and methods of use thereof. Also present description relates to conjugates formed in vitro between modified enhancing the secretion of the means described above, and a component of blood. It is envisaged that the conjugates formed in accordance with this embodiment of the present invention, have an increased half-life existence in vivo compared to the corresponding unmodified enhance the secretion of the tool.

In accordance with this embodiment of the present invention enhances the secretion of the tool is modified chemically active group reactive element). Reactive element can, for example, to choose from a wide variety of active carboxyl groups, particularly esters, where the hydroxyl component is physiologically acceptable. Such groups can be selected from the group consisting of N-hydroxysuccinimide (NHS), N-hydroxysultaine (sulfo-NHS), Malay is eventinstanceid (MBS), gamma maleimidophenylmethacrylates ester (GMBS) and multimediaphoto acid (MPA). The main targets of this group of elements are the primary amines on the component of blood. Another group of active elements is maleimido-containing group, such as MPA and gamma maleimidomethyl (GMBA). These groups react with thiol groups present on the component of blood (Lee V.H.L. in “Peptide and Protein Drug Delivery”, New York, N.Y., M. Dekker, 1990).

Component of blood, which modified the amplifying secretion tool developed for entering into the reaction, can be any component of blood that is available to the group is the target, for example, the amino group or Tilney group, and which is suitable as a carrier for binding of the modified enhancing the secretion of funds in vivo and, thus, extends its circulatory half-life existence. Examples of such blood components is serum albumin and IgG.

As noted above, the covalent linking of a modified reinforcing the secretion of funds from the blood component can be successfully carried out in vivo by administering a modified reinforcing the secretion of funds directly to the patient. The introduction can be performed in any suitable form, for example, in the form of a bolus, or be injected slowly over time t is by infusion using a metered supply or the like In the alternative case, the conjugate enhances the secretion of the tool/component of blood can also be prepared ex vivo Association of blood with modified amplifying secretion tool, enabling covalent binding of a modified reinforcing the secretion of funds with reactive functional groups on blood components and then returning or entering conjugate the blood of the individual.

In addition, the above can also be performed using the first cleaning component of blood of an individual or limited number of components, such as red blood cells, immunoglobulins, serum albumin, or the like, and combining the component or components ex vivo with chemically active amplifying secretion of means.

Getting such option splicing of the ghrelin connections

Such variant splicing of the ghrelin connection can be obtained using methods well known in the art. For example, a polypeptide region of this variant splicing of the ghrelin compounds can chemically or biochemically synthesize and modify. Methods of chemical synthesis of polypeptides are well known in the art (see, for example, Lee V.H.L in “Peptide and Protein Drug Delivery”, New York, N.Y., M. Dekker, 1990). Examples of methods of biochemical synthesis, involving the introduction of a nucleic acid which acid in a cell and expression of nucleic acids, provided F.M. Ausubel and others in “Current Protocols in Molecular Biology”, John Wiley, 1987-1998, and Sambrook and others in “Molecular Cloning, A Laboratory Manual, 2ndEdition, Cold Spring Harbor Laboratory Press, 1989, all of which are included here by reference. Other cited as an example by the method described in U.S. patent No. 5304489 included here by reference, is the use of transgenic mammals having aimed at the breast mutations that lead to the production and secretion of the synthesized similar variant splicing of the ghrelin compounds in the milk of the transgenic mammal.

Such variant splicing of the ghrelin compounds can also recombinante to obtain using conventional methods of expression known in the art. Polynucleotide encoding desired such variant splicing of the ghrelin connection, functionally associated with the promoter in expressing vector, suitable for any convenient host. Both eukaryotic and prokaryotic systems, the hosts used in the formation of such recombinant variant splicing of the ghrelin compounds. Such variant splicing of the ghrelin connection then isolated from lysed cells or from the culture medium and purified to the extent necessary for its intended use.

Accordingly, a further option implemented the I of the present invention is a method of producing such variant splicing of the ghrelin connection includes stage (a) ensure cDNA comprising a polynucleotide sequence encoding such variant splicing of the ghrelin connection; (b) embedding the specified expressing the cDNA in the vector so that cDNA was functionally linked to the promoter; and (C) the introduction of the specified expressing vector into the cell host, whereby specified a host cell produces the specified similar variant splicing of the ghrelin connection.

In one aspect of this variant implementation of the present invention the method also includes a step of selection variant splicing of the ghrelin compound obtained in stage C). Another embodiment of the present invention is similar to the variant splicing of the ghrelin compound obtained by the method described in the preceding paragraph. Expressing the vector represents expressing any system of mammalian, yeast, insect or bacteria, known in the art. Commercially available vectors and expressing system available from many vendors, including Genetics Institute (Cambridge, Mass), Stratagene (La Jolla, Calif.), Promega (Madison, Wis.) and Invitrogen (San Diego, Calif.). If it is desirable to increase the expression and facilitate proper installation of the protein, the percentage of codons and the codons pairing sequence optimize on what I'm expressing specific organism, brought expressing vector, as explained in U.S. patent No. 5082767, the full content of which is included, therefore, by reference.

In another embodiment, the present invention is often a useful addition to the recombinant polynucleotide additional nucleotide sequence(s)which encodes a secretory or leader sequences, proposedvalue, sequences that facilitate purification such as multiple histidine residues, or an additional sequence for stabilization during recombinantly products.

The introduction of polynucleotide encoding such variant splicing of the ghrelin connection in the cell-master can be done using transfection using calcium phosphate mediated by DEAE-dextran transfection mediated by cationic lipid transfection, electroporation, transduction, transfection or other means. Such methods are described in many standard laboratory manuals, such as Davis et al., (1986) Basic Methods in Molecular Biology, ed., Elsevier Press, NY, the full content of which is included, therefore, by reference. In particular, it is envisaged that such variant splicing of the ghrelin compounds disclosed herein may incidentally be expressed in a cell-master, to the Torah there is no recombinant vector, natural or produced by the cell.

Such variant splicing of the ghrelin compounds can be extracted and cleaned from cultures of recombinant cells using well known methods, including differential extraction, precipitation with ammonium sulfate or ethanol, acid extraction, anyone - or cation-exchange chromatography, chromatography on phosphocellulose, chromatography based on hydrophobic interaction, affinity chromatography, chromatography on hydroxylapatite and chromatography using lectin (see, for example, “Methods in Enzymology: Aqueous Two-Phase Systems”, Walter H. et al. (eds.), Academic Press (1993), incorporated here by reference, for a variety of purification methods for proteins). In one embodiment of the present invention for purification using liquid chromatography high-resolution ("HPLC"). Recombinante produced a variant of this variant splicing of the ghrelin connection can largely be cleaned using the methods described herein or otherwise known in the art, such as, for example, one-step method described by Smith & Johnson, Gene 67: 31 40 (1988), the complete contents of which is included, therefore, by reference. Such variant splicing of the ghrelin connection you can also clear from recombinant sources using what Finance antibodies against such variant splicing of the ghrelin compounds, for example, those described here, in ways that are well known in the field of protein purification.

In one embodiment, the present invention recombinante expressed similar variant splicing of the ghrelin connection purified using standard lateral flow methods. In such procedures, a solution containing of interest such variant splicing of the ghrelin connection, such as culture medium or cell extract, is applied on a column containing antibodies against such variant splicing of the ghrelin connection attached to a chromatographic matrix. Recombinant similar variant splicing of the ghrelin connection provide the opportunity to contact immunochromatography column. Subsequently, the column is washed to remove nonspecific proteins bound peroxidase. Specifically associated secreted similar variant splicing of the ghrelin connection is then removed from the column and was isolated using standard methods.

Depending on the host used in the procedure of recombinant products, such variant splicing of the ghrelin connections can be glycosylamine or can be deglycosylation. In addition, the polypeptides present asego of the invention may also include an initial modified methionine residue, in some cases, the result is mediated by host processes. So in the art it is well known that N-terminal methionine encoded by the codon to initiate translation, usually effectively manage any protein after translation in all eukaryotic cells. Although the N-terminal methionine on most proteins also effectively removed in most prokaryotes, for some proteins, this prokaryotic removal process is inefficient, depending on the nature of amino acids, which are covalently linked to the N-terminal methionine.

The pharmaceutical composition

Although the compounds or salts of the present description can be entered in the form of the raw chemical, it is preferable to present them in the form of pharmaceutical compositions. Accordingly, one aspect relates to pharmaceutical compositions containing such variant splicing of the ghrelin connection defined in formula I.

Another variant of implementation of the present invention relates to pharmaceutical compositions containing a mixture of at least two different such variant splicing of the ghrelin compounds such as a mixture of such variant splicing of the ghrelin compounds acylated With8the acyl, and such variant splicing of the ghrelin compounds acylated With10the acyl. Expresses who I am not bound by theory assumption, this mixture will have a greater half-life existence in the plasma.

In yet another embodiment of the present invention the pharmaceutical composition comprises acylated similar variant splicing of the ghrelin connection, optional with different length acyl chains, preferably selected from the group consisting of C7acyl group9acyl group and11acyl group, optionally in combination with disallowances such variant splicing of the ghrelin connection.

Another aspect relates to pharmaceutical compositions containing any amplifying secretion of a tool, such as any such variant splicing of the ghrelin connection described above, or its pharmaceutically acceptable salt and pharmaceutically acceptable carriers and/or excipients, with the specified composition further contains a transport molecule. Transport of molecules, mainly added in order to increase the half-period of existence ALLROUNDER connection, preventing premature disallowance because disallowances variant splicing of the ghrelin may be inactive in relation to the GHS-R 1a.

Transport molecules act through the inclusion in the connection, the open here, or attaching to him in this connection. You can use the any suitable transport molecule, known qualified. Examples of transport molecules are transport molecules described in the section "Conjugates" above. Other preferred examples include liposomes, micelles, and/or microspheres.

Standard liposomes are usually composed of phospholipids (neutral or negatively charged) and/or cholesterol. Liposomes are vesicular structures that are based on the lipid bilayer surrounding water departments. They can vary according to the physical-chemical properties, such as size, lipid composition, surface charge and number, and the fluidity of the phospholipid bilayer. The most commonly used lipids for the formation of liposomes are 1,2-Dilauroyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMP), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DP), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DP), 1,2-dimyristoyl-sn-glycero-3-phosphate (odnonatrieva salt) (DMPA), 1,2-dipalmitoyl-sn-glycero-3-phosphate (odnonatrieva salt (DPPA), 1,2-dioleoyl-sn-glycero-3-phosphate (odnonatrieva salt) (DPA), 1,2-dimyristoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (sodium salt) (DMPG), 1,2-dipalmitoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] sodium salt) (DPG), 1,2-dioleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (sodium salt) (DPG), 1,2-dimyristoyl-sn-glycero-3-[phospho-L-serine] (sodium salt) (DMPS), 1,2-dipalmitoyl-sn-glycero-3-[phospho-L-serine] (sodium salt) (DPS), 1,2-dioleoyl-sn-glycero-3-[phospho-L-serine](sodium salt) (DOPS), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(glutaryl) (sodium salt) and 1,1',2,2'-tetrameristaceae (ammonium salt). Preferred compositions are composed of DPPC in combination with another lipid or modifier of liposomes, for example, in combination with cholesterol and/or phosphatidylcholine.

Long-circulating liposomes are characterized by their ability to penetrate from the blood vessels into the tissue in those areas of the body where vascular permeability increased. The preferred method of obtaining such long-circulating liposomes is joining the hydrophilic polymer polyethylene glycol (PEG) covalently to the outer surface of the liposomes. Some of the preferred lipid is 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (ammonium salt), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-5000] (ammonium salt), 1,2-dioleoyl-3-ammonium-propane (chloride) (DOTAP).

Possible applicable for liposomes, the lipids are supplied Avanti Polar Lipids, Inc., (Alabaster, AL). In addition, the suspension of liposomes viewlocity protecting lipids funds which protect lipids from free radical damage and the resulting perechisleniya lipids during storage. Preferred are lipophilic free radical absorbers, such as alpha-tocopherol, and water-soluble iron-specific chelating agents, such as ferrioxamine.

For preparation of liposomes there are a number of ways, described, for example, Szoka F. & Papahadjopolous D., Ann. Rev. Biophys. Bioeng. 9: 467-508 (1990); U.S. patent No. 4235871, 4501728 and 4837028; all of which are included here by reference. Another way we obtain a multi-layered vesicles of heterogeneous sizes. In this way forming vesicles, the lipid is dissolved in a suitable organic solvent or solvent system and dried under vacuum or inert gas for formation of a thin lipid layer. If you want this layer can be re-dissolved in a suitable solvent, such as tertiary butanol, and then subjected to lyophilization to form a more homogeneous lipid mixture, which is more easily gidratirutmi powdered form. This layer is covered with an aqueous solution of a specialized drug and target connections, and give him the opportunity to hydrogenate itself usually for 15-60 minute period with stirring. Size distribution obtained as a result of multi-stakeholder who eunuch vesicles can be moved in the direction of a smaller size using the hydration of the lipids in a more intensive mixing or by adding a solvent detergent, such as dezoksiholatom.

Micelles are formed by surface-active substances (molecules that contain a hydrophobic portion and one or more ionic or otherwise strongly hydrophilic groups) in aqueous solution. Increasing the concentration of solid surfactants, his monolayers adsorbed at the interface air/water or glass/water, become so tightly Packed that in order to continue seeding requires excessive compression of the molecules of surface-active substances already in the two monolayers. Further increase in the number of dissolved surfactants beyond this concentration causes the aggregation of amounts equivalent to new molecules in micelles. This process begins at a characteristic concentration called critical micelle concentration.

The shape of the micelles formed in dilute solutions of surface-active substances, is roughly spherical. The polar group of the heads of the molecules of surface-active substances are located in the outer spherical shell, while their hydrocarbon chains are oriented toward the center, forming a spherical core of the micelles. The hydrocarbon chain is randomly rolled and twisted, and the interior of the micelle is non-polar, such fluid nature of the micelles polyoxyethylenic nonionic detergents polyoxyethylene components are oriented outwards and permeable to water. This accommodation is energetically favorable because gabriellae group heads in contact with water and hydrocarbon components are removed from the aquatic environment and is partially protected from contact with water, the polar groups of heads. The hydrocarbon tails of the molecules of surface-active substances located in the interior of the micelles interact with each other through weak forces van der Waals forces.

The size of the micelles or the number of aggregation is regulated mainly geometric factors. The radius of the hydrocarbon core cannot exceed the length of the long hydrocarbon chain of the molecule surfactants. Therefore, increasing the chain length or move one step up in the homologous series leads to an increase in the aggregation number of spherical micelles. If the concentration of surfactant increases beyond a small percentage, and if you add electrolytes (in the case of ionic surfactants), or to raise the temperature (in the case of non-ionic surfactants), the size of the micelles increases. Under these conditions, the micelles are too large to remain spherical, and become ellipsoidal, cylindrical or, ultimately, lamellar form.

In the micelles of the present description, you can use the SQL conventional surfactants, well known to the skilled in the art specialist. Suitable surfactants include sodium laureate, sodium oleate, sodium laurylsulfate, monododecyl ether ataxiatelangiectasia, octoxynol 9 and PLURONIC®F-127 (BASF Corp., Florham Park, NJ). Preferred surfactants are non-ionic polyoxyethylene and polyoxypropylene detergents that are compatible with intravenous injection, such as TWEEN®-80, PLURONIC®F-68, n-octyl-beta-D-glucopyranoside, etc. in Addition to the formation of micelles can also be used phospholipids, such as phospholipids, are described for use in obtaining liposomes.

In another preferred embodiment of the present invention, the compounds disclosed here, prepared as described in the literature for the route of administration selected from buccal delivery, sublingual delivery, transcutaneous delivery, inhalation and needleless injection, such as by using methods developed by Powderject.

Inhalation disclosed here, the compounds can be prepared using methods known to skilled in the art specialists, for example, aerosol, dry powder, or dissolved in, for example, microcaps, preferably in an appliance designed for such delivery (e.g., commercial is available from Aradigm Corp. (Hayward, Cal.), Alkermes, Inc. (Cambridge, Mass.) or Nektar Therapeutics (San Carlos, Cal.)).

Introduction

Appropriate schemes the introduction of various compounds and methods of the present description are preferably determined taking into account factors well known in the art, including, for example, the type of person who administered the drug; the age, weight, sex and medical condition of the subject; the route of administration; the functioning of the kidneys and liver of the subject; the desired effect and the specific connection. Preferably the composition will contain about 0.5%-75% by weight, enhance the secretion of the means disclosed here, while the remaining part will consist of suitable pharmaceutical excipients.

For optimal precision in achieving concentrations of drugs within the range that provides efficacy without toxicity requires a scheme based on the kinetics of availability of medicines for sites to target. This includes taking into account distribution, balance, and excretion of drugs.

As described above, in one aspect of enhancing the secretion of a tool, such as a variant splicing of the ghrelin or a similar variant splicing of the ghrelin connection, injected subcutaneously.

In another aspect, amplifying secretion of a tool, such as a variant of splice the ha-ghrelin or a similar variant splicing of the ghrelin connection, impose a pre-meal bolus, the form of the introduction may be any suitable parenteral form. In a preferred embodiment, the present invention enhances the secretion of a tool, such as a variant splicing of the ghrelin or a similar variant splicing of the ghrelin connection, injected subcutaneously in the form of pre-meal bolus.

Enhancing the secretion of a tool, such as a variant splicing of the ghrelin or a similar variant splicing of the ghrelin connection, you can also enter during the meal bolus. The method of introduction during the meal includes subcutaneous injection, such as subcutaneous injected bolus.

Pharmaceutical compositions for parenteral administration include sterile aqueous and non-aqueous injectable solutions, dispersions, suspensions or emulsions, and sterile powders recovered in a sterile injectable solutions or dispersions prior to use. Other suitable forms of introduction include suppositories, sprays, solutions or suspensions, ointments, creams, gels, inhalers, skin patches, implants, pills, tablets, pellets and capsules.

The typical dose is within a concentration equivalent to from 10 ng to 10 mg variant splicing of the ghrelin / kg of body weight. The concentration and quantity are here e is wavelenth number of variant splicing ghrelin, the option of splicing of the ghrelin is a variant splicing of the ghrelin man of 22 amino acids (SEQ ID NO: 2), and/or splicing of the ghrelin man of 29 amino acids (SEQ ID NO: 5), and/or splicing of the ghrelin man of 24 amino acids (SEQ ID NO: 3), and/or splicing of the ghrelin man of 24 amino acids, with the remainder of the Drp in the third position (SEQ ID NO: 4). Equivalents can be tested as described herein in the section entitled "Functionality".

In a preferred embodiment of the present invention, the drug is administered at a concentration equivalent to from 0.1 μg to 1 mg variant splicing of the ghrelin / kg body weight, for example, from 0.5 μg to 0.5 mg variant splicing of the ghrelin / kg body weight, for example, from 1.0 μg to 0.1 mg variant splicing of the ghrelin / kg body weight, for example, from 1.0 μg to 50 μg variant splicing of the ghrelin / kg body weight, for example, from 1.0 μg to 10 μg variant splicing of the ghrelin / kg body weight.

As noted above, amplifying secretion of a tool, such as a variant splicing of the ghrelin or a similar variant splicing of the ghrelin connection, is preferably introduced in the form of a bolus. Accordingly, in one embodiment of the present invention, the drug is injected bolus before a meal, with the specified bolus contains the number amplify the its secretion or its salt, equivalent to 0.3 ág 600 mg splicing variant of ghrelin. More preferably, the drug is injected bolus before a meal, with the specified bolus contains the number of amplifying secretion or its salt, equivalent to 2 mg - 200 mg splicing variant of ghrelin, for example, 5,0 μg - 100 mg splicing variant of ghrelin, for example, 10 μg - 50 mg splicing variant of ghrelin, for example, 10 μg - 5 mg splicing variant of ghrelin, for example, 10 μg - 1 mg splicing variant of ghrelin.

It should be noted that normal, possible in the case of variant splicing of the ghrelin response that takes place before a meal, is a short-term spike in concentrations in plasma splicing variant of ghrelin, and that because of the relatively short half-existence of peptide intravenous injection of variant splicing of the ghrelin will provide similar short-term peak concentrations of variant splicing of the ghrelin. The administration should ensure that the main form in the circulation will be non-degraded, the bioactive form of the peptide, which will reach the receptor variant splicing of the ghrelin and encourage them.

Thus, to obtain the maximum effect of the medicinal product it is preferable to enter from one to three times daily, while ka is due the introduction is carried out within 45 minutes of ingestion, for example, within 30 minutes of a meal, for example, within 25 minutes of a meal, for example, within 20 minutes of eating, for example, within 15 minutes of eating, for example, within 10 minutes of eating, for example, within 5 minutes of eating. More preferably the drug is administered before each main meal, for example, administered three times daily.

Disclosed here compounds can also be prepared for nasal administration. Solutions or suspensions used directly in the nasal cavity using a conventional method, for example, with a pipette or spray solution or suspension. The composition can be provided in the form of single or repeated administration. In the latter case, use of a pipette, the patient can be achieved by introduction of an appropriate, predetermined volume of solution or suspension. In the case of the spray solution or suspension this can be achieved by means of a metering and spray pump.

Disclosed here is to prepare for aerosol administration, particularly in the respiratory tract and including intranasal administration. Compounds will generally have a small particle size, for example, about 5 microns or less. This particle size can be obtained from paasusaba, known in the art, for example, using fine grinding. The active ingredient is placed in a pressurized pack with a suitable gas propellant, such as hydrofluroalkane (HFA), for example hydrofluroalkane-134a and hydrofluroalkane-227, carbon dioxide or other suitable gas. Aerosol can appropriately also contain a surfactant such as lecithin. The dose of the drug can be controlled by using a controlled flow valve. Alternatively the active ingredients can be provided in the form of a dry powder, for example, a powder mix of the compound in a suitable basis for powder, such as lactose, starch, derivatives of starch, such as hypromellose and polyvinylpyrrolidine (PVP). Powder media will form a gel in the nasal cavity. The powder composition may be presented in the form of a standard dose of, for example, capsules or cartridges, for example, from gelatin or blister packs from which the powder can be introduced through the inhaler.

Composition introduced aerosol can be prepared, for example, as solutions in saline, using benzyl alcohol or other suitable preservatives, enhancers of absorption to increase the bioavailability using a fluorocarbon and/or COI is lsua other solubilizing and dispersing agents.

Compounds disclosed here can also be prepared for injection using a pen for injection in a manner similar to that used for placed in the cartridge of growth hormone (GH) or insulin. The cartridge contains disclosed here in connection solvents. The handle, which is, in essence, a needle, a syringe and an ampoule in one General control by turning, and make possible the introduction of different doses. This device offers simplicity, convenience, and signs of increased security for the delivery of compounds. It provides a simple construction of the device, a small number of stages of the introduction and click single-stage automatic dosing. Such a handle for dispensing can be obtained by a method known in the art. For example, several manufacturers offer handles for administering drugs to developers for use with the compounds of the developers in the form of medicines (BD Medical-Pharmaceutical Systems, Inc.; Owen Mumford Inc. etc.).

Compositions for oral administration

These types of amplifying secretion of tools that can remain biologically active in an individual after oral administration (such as, for example, small molecules and short peptides) can be prepared in the range of dosage forms for oral administration. Pharmaceutical the e compositions and dosage forms may contain as the active compounds disclosed herein compounds or their pharmaceutically acceptable salts, or crystalline form.

Pharmaceutically acceptable carriers can be either solid or liquid. Drugs in solid dosage forms include powders, tablets, pills, capsules, starch wafers, suppositories, and dispersible granules. A solid carrier can be one or more substances which may also act as diluents, corrigentov, solubilization, lubricants, suspendida substances, binders, preservatives, moisturizers, dezintegriruetsja tablets agents or sealing material.

For oral administration such fillers include, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talc, cellulose, glucose, gelatin, sucrose, magnesium carbonate, etc.

In powders, the carrier is a finely dispersed solid substance which is a mixture with finely dispersed active component. In tablets, the active compound is mixed with carrier having the necessary binding capacity in suitable proportions and pressed into the desired shape and size. The powders and tablets preferably contain from one to about seventy percent of the active compounds. Suitable carriers are magnesium carbonate, magnesium stearate, talc, sugar, lactose, aspect is h, dextrin, starch, gelatin, tragacanth gum, methylcellulose, sodium carboxymethylcellulose, low melting wax, cocoa butter, etc. the Term "drug", as expected, includes a composition comprising a disclosed here and in communication with a sealing material as a carrier providing a capsule in which the active ingredient with the carriers, or without them, surrounded by a media with which it is associated. Also included starch wafers and cakes.

As solid forms suitable for oral administration may be tablets, powders, capsules, pills, starch wafers and cakes.

Drops may contain sterile or non-sterile aqueous or oily solutions or suspensions and may be prepared by dissolving the active ingredient in a suitable aqueous solution, optionally including bactericidal and/or fungicidal agent and/or any other suitable preservative, and optionally containing a surfactant. The resulting solution can then be subjected to clarification by filtration, transferred to a suitable container, which is then subjected to sealing and sterilization by autoclaving or content at 98-100οWith in half an hour. In the alternative case, the solution can be sterilized by filtration and transferred aseptically into the container the EP. Examples of bactericidal and fungicidal agents suitable for inclusion in the drops, are finalstate nitrate or acetate (0.002 per cent), benzalkonium chloride (0.01 per cent) and chlorhexidine acetate (0.01 per cent). Suitable solvents for preparing an oil solution include glycerol, diluted alcohol, and propylene glycol.

Also included preparations of solid forms, which are supposed to turn shortly before use in the preparations of liquid forms for oral administration. Such liquid forms include solutions, suspensions and emulsions. These preparations may contain, in addition to the active component, colorants, corrigentov, stabilizers, buffers, artificial and natural sweeteners, dispersants, thickeners, solubilizing agents, etc.

Other forms suitable for oral administration include liquid preparations of forms, including emulsions, syrups, elixirs, aqueous solutions, aqueous suspensions, toothpaste, gel for mouth care, chewing gum or drugs in solid dosage forms, which are supposed to turn shortly before use in the preparations of liquid forms. The emulsion can be prepared in solution in aqueous solutions of propylene glycol, or they may contain an emulsifier, such as lecithin, orbitonasal or Arabian gum. Aqueous solutions can be prepared by dissolving the active component is in water and adding suitable colorants, Corrientes, stabilizers and thickeners. Aqueous suspensions can be prepared by dispersing finely dispersed active component in water with viscous material such as natural or synthetic gums, polymers, methylcellulose, sodium carboxymethylcellulose, and other well-known suspendresume funds. Drugs in solid dosage forms include solutions, suspensions and emulsions can contain, in addition to the active component, colorants, corrigentov, stabilizers, buffers, artificial and natural sweeteners, dispersants, thickeners, solubilizing agents, etc.

Compositions for parenteral administration

Disclosed here compounds can be prepared for parenteral administration (e.g. by injection, for example bolus injection or continuous infusion) or can be presented in the form of a standard dose vials filled syringes, infusion of a small volume or in packages of medicines for multiple injections with the added conservative. The compositions may take such forms as suspensions, solutions or emulsions in oily IDN aqueous media, for example, solutions in an aqueous solution of polyethylene glycol. Examples of oil or non-aqueous carriers, diluents, solvents or carriers include propylene glycol, polyethylene glycol, who astically oil (for example, olive oil) and injectable organic esters (for example, etiloleat) and may contain included in the composition of agents, such as preservatives, humectants, emulsifiers or suspendresume tools, stabilizers and/or dispersants. Alternatively the active ingredient may be in powder form, obtained by aseptic allocation of sterile solid or by lyophilization from solution, for preparation before use with a suitable carrier, e.g. sterile pyrogen-free water. Aqueous solutions should be appropriate to tabularity if necessary and the liquid diluent first need to give isotonicity with a sufficient amount of salt or glucose. Aqueous solutions, in particular, suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. All used sterile water environment is readily achievable using standard methods known to skilled in the art specialists.

Solutions of variant splicing of the ghrelin or a similar variant splicing of the ghrelin compounds, or their pharmaceutically acceptable salts (e.g. antigenic epitopes and protease inhibitors) can be prepared in water or saline, and optionally mixed with a nontoxic surfactant substance. The composition is for intravenous or intraarterial administration may include sterile aqueous solutions, which may also contain buffers, liposomes, diluents and other suitable additives.

Oil, applicable to parenteral compositions include mineral, animal, vegetable or synthetic oils. Specific examples of oils that are applicable in such compositions include peanut, soybean, sesame, cottonseed, corn, olive, vaseline and mineral. Fatty acids suitable for use in parenteral compositions include oleic acid, stearic acid and isostearoyl acid. Etiloleat and isopropylmyristate are examples of suitable esters of fatty acids.

Suitable for use in parenteral compositions Soaps include salts of alkali metals, ammonium and triethanolamine fatty acids, and suitable detergents include (a) cationic detergents such as, for example, dimethyldiallylammonium halide and alkylpyridine halide; (b) anionic detergents such as, for example, alkyl-, aryl - and reincorporate, alkyl-, olefin-, monoglycerides and ethers, sulfates, and sulfosuccinates; (C) nonionic detergents such as, for example, oxides, fatty amines, alkanolamide fatty acids and copolymers of polyoxyethylenesorbitan; (d) amphoteric detergents such as, for example, alkyl-beta-aminopropionic and Quaternary ammonium salts of 2-alkyl-imidazo the ins; and (e) mixtures thereof.

Parenteral compositions generally will contain from about 0.5 to about 25% by weight of active ingredient in solution. Can be used preservatives and buffers. To minimize or avoid irritation at the site of injection, such compositions may contain one or more nonionic surfactants having a hydrophilic-lipophilic balance (HLB) of from about 12 to about 17. The amount of surfactant in the compositions will generally be in the range of from about 5 to about 15% by weight. Suitable surfactants include esters polyethylenimine and fatty acids, such as orbitonasal and high molecular weight addition products of ethylene oxide to hydrophobic base formed by condensation of propylene oxide with propylene glycol. Parenteral compositions may be presented in sealed containers for single or multiple injection, such as ampoules and vials, and can be stored in a lyophilized condition requiring only the addition of the sterile liquid excipient, for example, water for injection immediately before use. Solutions and suspensions for improvised injection can be prepared from sterile powders, granules and tablets earlier the written type.

Pharmaceutical dosage forms suitable for injection or infusion can include sterile aqueous solutions or suspensions containing the active ingredient which are adapted for the introduction of encapsulation in liposomes. In all cases, the final dosage form should be sterile, fluid and stable under conditions of manufacture and storage.

Sterile injectable solutions are prepared by incorporation of variant splicing of the ghrelin or a similar variant splicing of the ghrelin compounds or their pharmaceutically acceptable salts in the required amount in the appropriate solvent with various other ingredients enumerated above, if required, followed by, for example, sterilization by filtration.

Compositions for local injection

Compounds disclosed here can also be delivered topically. Area for local injection include the surface of the skin and tissue of the mucous membranes of the rectum, nose, mouth, and larynx. Compositions for local insertion through the skin and mucous membranes should not cause signs of irritation, such as swelling or redness.

Compositions for local injection may include a pharmaceutically acceptable carrier adapted for topical administration. Thus, the composition may take the form of, for example, suspensions, solution, ointment, lotion, cream, spray, spray substances, suppositories, implants, inhalers, tablets, capsules, dry powder, syrup, balm or cakes. Methods of preparing such compositions are well known in the pharmaceutical industry.

Disclosed here are the links you can prepare for the local introduction of the epidermis as ointments, creams or lotions, or as transdermal patches. Ointments and creams may, for example, be prepared using an aqueous or oily base with the addition of suitable thickeners and/or gelling agents. Lotions can be prepared using water or oil base, and they will generally also contain one or more emulsifiers, stabilizers, dispersants, suspendida agents, thickeners or dyes. Compositions suitable for local injection in the mouth include pellet, containing the active ingredients in a flavored basis, usually sucrose and the Arabian or tragacanth gum; mints containing the active ingredient in an inert basis such as gelatin and glycerin or sucrose and Arabian gum; and liquid mouth rinse containing the active ingredient in a suitable liquid carrier.

Creams, ointments or pastes according to the present description are semi-solid compositions for external application is, containing the active ingredient. They can be obtained by mixing the active ingredient in finely dispersed or powdered form, solely or in solution or suspension in aqueous or non-aqueous fluid, with the aid of suitable mechanical equipment, with oily or amelanistic basis. The core can include hydrocarbons such as hard, soft or liquid paraffin, glycerol, beeswax, a metallic soap; a mucilage; an oil of natural origin such as almond, corn, peanut, castor or olive oil; lanolin or its derivatives, or a fatty acid such as stearic or oleic acid together with an alcohol such as propylene glycol, or a macrogel. The composition may include any suitable surfactant such as anionic, cationic or nonionic surface-active substance, such as ether, sorbitol or its derivative polyoxyethylene. Suspendresume agents such as natural gums, cellulose derivatives or inorganic materials such as silica and siliceous rocks, and other ingredients such as lanolin may also be included.

Lotions in accordance with the present description include lotions, suitable for application to the skin or eyes. Lotion for the eyes may include sterile aqueous of RA is creative, optionally containing a bactericidal substance, and can be prepared by methods similar to the methods of preparation of drops. Lotions or liquid ointment for application to the skin may also include an agent that accelerates the drying and cooling the skin, such as alcohol or acetone, and/or humectant, such as glycerol or an oil such as castor oil or peanut oil.

Described here are the links you can enter percutaneous. Percutaneous introduction usually includes the delivery of a pharmaceutical agent for percutaneous passage of drugs into the systemic circulation of the patient. Place skin include anatomical areas for percutaneous introduction of the medicinal product and include the forearm, abdomen, chest, back, buttock, mastoid region, etc.

Percutaneous delivered by the influence of the source of the active component on the patient's skin over an extended period of time. Transdermal patches have the added advantage of providing controlled delivery of complex joints in the body (see the Transdermal Drug Delivery: Developmental Issues and Research Initiatives, Hadgraft and Guy (eds.), Marcel Dekker, Inc., (1989); Controlled Drug Delivery: Fundamentals and Applications, Robinson and Lee (eds.), Marcel Dekker Inc., (1987); and Transdermal Delivery of Drugs, Vols. 1-3, Kydonieus and Berner (eds), CRC Press, (1987)). Such dosage forms can be prepared by dissolving, dispel the management or enabling otherwise disclosed here are the links to the appropriate environment, such as an elastomeric matrix material. Amplifiers absorption can also be used to increase the movement of compounds through the skin. The speed of such movement can be controlled or using the software controlling the speed of the membrane or by dispersing the compound in a polymer matrix or gel.

In the here described methods will find application of various types trandermal patches. For example, a simple patch can be made from the substrate material and acrylic adhesive. Prepare the composition of the active component of any amplifier in the adhesive filling solution, and allow thorough mixing. The solution is poured directly on the substrate material, and the solvent of the casting solution is evaporated in a drying oven, leaving a sticky film. For picking system can attach adhesive protective material.

In the alternative case for delivery of the disclosed here connection you can use the patch with a polyurethane matrix. Layers of this patch include padoku, polyurethane matrix with drug/amplifier, membrane, adhesive and anti-adhesive protective material. Polyurethane matrix prepared with the use of hardening at room temperature of the polyurethane prepolymer. Add water, Speer is a and complex to the prepolymer leads to the formation of sticky resistant elastomer, which can be poured directly on the substrate material.

In a further embodiment, will be used the patch matrix in the form of a hydrogel. Typically, the matrix in the form of a hydrogel will include alcohol, water, medicine and several hydrophilic polymers. This matrix in the form of a hydrogel can be included in a transdermal patch between the substrate and the adhesive layer.

In the here described methods also find use patches with the reservoir fluid. This patch includes an impermeable or semi-permeable, heat-sealable substrate, heat-sealable membrane, pressure-sensitive, communicating with leather-based substance acrylate and siliconized adhesive protective material. The substrate is glued by heating with a membrane to form a reservoir, which can then be filled with a solution of the complex, enhancers, gelling agents and other fillers.

Patches with a porous matrix similar in design and components with systems with a reservoir of liquid except that the gelled solution of the pharmaceutical agent - chemical modifier is enclosed in a thin porous layer, usually polyurethane. This porous layer located between the substrate and the membrane, which gum up the heat on the periphery of the patch.

For passive systems DOS is where it is refuelled the rate of release is normally controlled by the membrane, placed between the reservoir and the skin, the diffusion of the semiconductor device or the skin, serving as controlling the speed barrier in the delivery system (see, U.S. patent№ 4816258; 4927408; 4904475; 4588580; 4788062 and so on, all of which are included here by reference). The delivery rate of the drug will depend partly on the nature of the membrane. For example, the delivery rate of the drug through the membrane inside the body, as a rule, the higher the speed of delivery through the skin barrier. Most predominantly the speed with which the active compound is delivered from the device in the membrane control through the use of limiting speed of the membranes, which are placed between the reservoir and the skin. Assuming that the skin is sufficiently permeable to the active component (i.e. skin absorption above the rate of passage through the membrane), the membrane will serve to control the intensity of the dose experienced by the patient.

Suitable materials for the permeable membranes can be selected on the basis of the desired degree of permeability, the nature of active connections and mechanical considerations associated with the design of the device. Examples of materials for the permeable membranes include a wide variety of natural and synthetic polymers, such as polydimethylsiloxane (with likoni-rubber), copolymers of ethylene vinyl acetate (EVA), polyurethanes, copolymers of polyurethane-polyester, polyethylene, polyamides, polyvinylchloride (PVC), polypropylene, polycarbonate, polytetrafluorethylene (PTFE), cellulose materials, such as cellulose triacetate and cellulose nitrate/acetate, and hydrogels, for example, 2-hydroxyethylmethacrylate (HEMA).

The device may include other elements, such as other conventional components of therapeutic products, depending on the desired characteristics of the device. For example, disclosed here, the composition may also include one or more preservatives or bacteriostatic agents, e.g., methylhydroxybenzoate, propylhydroxybenzoate, chlorocresol, benzalkonium chlorides, and the like, These pharmaceutical compositions can also contain other active ingredients such as antimicrobial agents, particularly antibiotics, anesthetics, analgesics and anti-itching tools.

Compositions for administration in the form of suppositories

Disclosed here compounds can be prepared for administration in the form of suppositories. Typical suppository get through the provision of low-melting paraffin, for example, a mixture of glycerides of fatty acids or cocoa butter, which is first melted, and it is homogeneous, for example, by mixing, distribute the active ingredient. Rasplavlennogo the mixture is then poured into molds with convenient dimensions, allow it to cool and harden.

The active ingredient can be prepared in suppositories containing, for example, approximately 0.5% to approximately 50% of the uncovered here are the links located in the carrier of polyethylene glycol (PEG) (e.g., PEG 1000 [96%] and PEG 4000 [4%]).

Composition

A preferred aspect provides pharmaceutical compositions suitable for implementation in practice of therapeutic methods described herein. The pharmaceutical compositions can contain physiologically acceptable carrier together with at least one kind of reinforcing the secretion of means, such as a variant splicing of the ghrelin or a similar variant splicing of the ghrelin compound described herein, dissolved or dispersed therein as an active ingredient. In a preferred embodiment of the present invention the pharmaceutical composition is not immunogenic with the introduction of which is the human individual with a therapeutic purpose, unless the purpose is not inducing an immune response.

One aspect relates to pharmaceutical compositions containing at least one amplifying secretion of a tool, such as a variant splicing of the ghrelin or a similar variant splicing of the ghrelin compound as defined above in formula I. In a preferred is sustained fashion the embodiment of the present invention the pharmaceutical composition contains at least two different such variant splicing of the ghrelin connection defined above in formula I, to increase the treatment effect. The difference may, for example, be a compound with different atsilirovaniye discussed above.

Used herein, the terms "pharmaceutically acceptable", "physiologically tolerable" and grammatical variations, in the case when they refer to compositions, carriers, diluents and reagents, are used interchangeably and represent that the materials you can enter the person or to inflict on him without the production of undesirable physiological effects such as nausea, dizziness, gastric upset and the like

Preparation of pharmaceutical compositions containing the active ingredients dissolved or dispersed therein, is well established in the art. Typically, such compositions are prepared in the form of sterile injectable drugs or in the form of liquid solutions or suspensions, aqueous or non-aqueous; however, you can also make solid forms suitable for solution or suspension in liquid prior to use. The composition can also be turned into an emulsion.

The active ingredient can be mixed with excipients that are pharmaceutically acceptable and compatible with the active ingredient and is present in quantities of K is included for use in therapeutic methods, as described here. Suitable fillers are, for example, water, saline, dextrose, glycerol, ethanol or the like, and combinations thereof. In addition, if desired, the composition may contain minor amounts of auxiliary substances such as moisturizers or emulsifying agents, pH buffering agents and the like, which increase the effectiveness of the active ingredient. Preferably, the composition had a pH within the range of 3.5-8, for example, in the range of 4.5 to 7.5, for example, in the range of 5.5 to 7, for example, in the range of 6-7,5, most preferably approximately 7,3. However, as is clear to the skilled in the art specialist, the pH range can be set in accordance with being treated the individual and the insertion procedure. For example, certain amplifying secretion of tools, such as a variant splicing of the ghrelin variants and homologues of the splicing of the ghrelin can easily be stabilized at low pH; thus, in another preferred embodiment of the present invention, the composition has a pH within the range of 3.5-7, for example, 4-6, for example, 5-6, for example, 5,3-5,7, for example, 5,5.

Disclosed here, the pharmaceutical compositions can include pharmaceutically acceptable salts of the compounds. These salts are salts that are acceptable in their application for formats whitesage use, that means that the salt will preserve the biological activity of the parent compound and the salt will not have an adverse or harmful effects in its application and use for the treatment of diseases.

Pharmaceutically acceptable salts are prepared in a standard way. If the original connection is the basis, it is treated with an excess of an organic or inorganic acid in a suitable solvent. If the original connection is acid, it is treated with an inorganic or organic base in a suitable solvent.

Disclosed here are the links you can enter in the form of its alkali metal salt or alkaline-earth metal at the same time or together with a pharmaceutically acceptable carrier or diluent, especially and preferably in the form of pharmaceutical compositions, or, for example, oral, rectal or parenteral (including subcutaneous), in an effective amount.

Examples of pharmaceutically acceptable acid additive salts for use in pharmaceutical compositions of the present invention include salts derived from mineral acids, such as, for example, hydrochloric, Hydrobromic, phosphoric, metaphosphoric, nitric and sulphuric acids, and organic acids, such as tartaric, acetic, citric, malic, the breast is Aya, fumaric, benzoic, glycolic, gluconic, succinic acid, para-toluensulfonate and arylsulfonate.

Other suitable pharmaceutically acceptable salts include the acid additive salts (formed with free amino groups of the polypeptide). Other examples of salts include pharmaceutically acceptable acid additive salts, pharmaceutically acceptable metal salts, ammonium salts and alkylated ammonium salts. Acid additive salts include salts of inorganic acids, and organic acids. Typical examples of suitable inorganic acids include hydrochloric, Hydrobromic, yodiewonderdog, phosphoric, sulfuric, and nitric acid, etc. are Typical examples of suitable organic acids include formic, acetic, trichloroacetic, triperoxonane, propionic, benzoic, cinnamic, citric, fumaric, glycolic, lactic, maleic, malic, malonic, almond, oxalic acid, picric acid, pyruvic acid, salicylic acid, succinic acid, methanesulfonate, econsultation, tartaric, ascorbic, pambou, biotranslation acid, ethicalfashion, gluconic, citraconate, aspartic, stearic, palmitic, ethylenediaminetetraacetic (EDTA), para-aminobenzoic, glutamic acid, benzosulfimide and a pair of Tolu is sulfoxylate etc. Further examples of pharmaceutically acceptable salts of the accession of inorganic or organic acids include pharmaceutically acceptable salts listed in S.M. Berge et al., J. Pharm. Sci. 66: 1-19 (1977), the source of which is included here by reference. Examples of metal salts include salts of lithium, sodium, potassium and magnesium, etc.

Examples of ammonium and alkyl ammonium salts include ammonium salts, methylamine, dimethylamine, trimethylammonium, ethylamine, hydroxyethylamine, diethylamine, butylamine and Tetramethylammonium etc.

Salts formed free carboxyl groups can also be obtained from inorganic bases, such as, for example, hydroxides of sodium, potassium, ammonium, calcium or iron, and such organic bases as Isopropylamine, trimethylamine, 2-ethylaminoethanol, histidine, procaine and the like

The compounds or their pharmaceutically acceptable acid additive salts in the context of the present description also includes any hydrates (hydrated form).

For parenteral administration, you can use the solutions of compounds of the present invention in a sterile aqueous solution, aqueous solution of propylene glycol or sesame or peanut oil. Such aqueous solutions should be suitably buffered if necessary and the liquid will dilute the Yu should first give isotonicity with a sufficient amount of salt or glucose. Aqueous solutions, in particular, suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. All used sterile water environment is readily achievable using standard methods known to skilled in the art specialists.

Liquid compositions may also contain a liquid phase and in addition with the exception of water. Examples of such additional liquid phases are glycerin, vegetable oils, such as cottonseed oil, organic esters, such as etiloleat, and the emulsion of the type oil in water.

Suitable pharmaceutical carriers include inert solid diluents or fillers, sterile aqueous solution and various organic solvents. Examples of solid carriers are lactose, kaolin, sucrose, cyclodextrin, talc, gelatin, agar, pectin, Arabic gum, magnesium stearate, stearic acid or esters of cellulose and lower Akilov. Examples of liquid carriers are syrup, peanut oil, olive oil, phospholipids, fatty acids, amines, fatty acids, polyoxyethylene or water. Aerosol for nasal or compositions for inhalation can be prepared, for example, in the form of solutions in volitional solution, using benzyl alcohol or other suitable preservatives, enhancers of absorption to increase the bioavailability using CFT is Rupert and/or other solubilizing or dispersing agents.

The pharmaceutical compositions formed by combining disclosed here, the compounds and pharmaceutically acceptable carriers are then easily administered in a variety of dosage forms suitable for the disclosed routes of administration. The composition can be appropriately presented in the form of a standard dose using methods known in the field of pharmacy.

In a preferred embodiment of the present invention, the product contains amplifying secretion of the tool or its salt in the form of freeze-dried, and the product further contains a solvent, with the specified freeze-dried and the solvent are in separate compartments prior to the introduction. In another embodiment of the present invention the product is a solution enhances the secretion of the drug or its salt. In either embodiments, the solvent may be any suitable solvent such as the solvents described herein, and preferably the solvent is saline.

Another aspect relates to a method for preparing a medicament or pharmaceutical compositions containing the disclosed here, the connection comprising mixing at least one such variant splicing of the ghrelin compounds defined above in formula I with a physiologically acceptable to the carrier. A further aspect relates to pharmaceutical compositions containing as active ingredient a compound as described in formula I, or its pharmaceutically acceptable salt together with a pharmaceutically acceptable carrier. Accordingly, the composition can also include, transport molecules described above.

Combinatorial treatment

In a further aspect, compounds of the present invention can be administered in combination with additional pharmacologically active substances or other pharmacologically active material and/or you can type in combination with another therapeutic method. The phrase "in combination with any other substance(s) and/or therapeutic method(s)" means here that the other substance(s) and/or therapeutic method(s) designate the individual, thus being treated, before, during (including simultaneously) and/or after treatment of the individual amplifying secretion tool. In all cases, the combinatorial treatment described here, the combination can be in the form of systems of sets of parts, and the combined active substances can be used for the simultaneous, sequential or separate administration. In all cases, preferably the appointment of any of the drug mentioned here means is in pharmaceutically effective amounts, e.g. prescribing, includes the total amount of each active component of the medicinal product or farmatsevticheskii composition or method that is sufficient to demonstrate a significant benefit for the patient.

In the following sections combinatorial therapy for use in preferred embodiments, the implementation of this group as follows.

1) a Combination in which all of the active ingredients are regulating appetite agents or otherwise applicable to the treatment of cachexia and/or lipodystrophy.

Enhancing the secretion of means(a) in accordance with the present description can be entered in combination with other regulating appetite agents, including more than one type amplifying secretion of growth hormone means, such as another similar variant splicing of the ghrelin connection, such as a similar variant splicing of the ghrelin connection comprising a structure defined by formula I, described above. Other amplifying secretion products suitable for combination with another compound enhances the secretion of the tool, are any of the compounds enhance the secretion of the tools described here. In one preferred embodiment of the present invention splicing variant of ghrelin (the most preferable option splice the ha-ghrelin human) is administered in combination with such a great option splicing of the ghrelin connection - this combination, as envisaged, enhance and/or prolong the effect, amplifying secretion of funds for the ghrelin receptor. In a similar way to increase the efficiency of the ghrelin receptor individual, you can enter several different amplifying secretion of funds, for example, more than 2 different types of amplifying secretion of funds, for example 3, example 4, example 5, example 6, example 7, for example, more than 8 different types of amplifying secretion of funds. Enhancing the secretion of a tool in accordance with the present description, such as a variant splicing of the ghrelin or a similar variant splicing of the ghrelin compound(I)can also type in combination with a pharmaceutically effective amount of a growth hormone, including hGH.

In one preferred embodiment, the present invention enhances the secretion of a tool, such as a variant splicing of the ghrelin or a similar variant splicing of the ghrelin connection, you can type in combination with IGF-1, IGFBP-3, or ALP, preferably with IGF-1. The rationale for this combinatorial treatment is to increase the levels of IGF-1, IGFBP-3 and/or ALP, which, as found, are low in patients with cachexia individuals.

In a further embodiment, the present invention enhances the secretion of tools, such as a variant splicing of the ghrelin or podobn the e variant splicing of the ghrelin connection, you can type in combination with compounds which are known to stimulate the appetite, such as ghrelin antagonists of the receptor melanocortin, receptor agonists of NPY, including agonists, selective in relation to individual subtypes of receptors, neuropeptide Y, leptin or agonists, leptin receptor, cannabinoids, including marijuana and derivatives marijuana, neuroleptic drugs, particularly atypical antipsychotic agents such as sertindole, supplied, clozapine, risperidone, quetiapine, amisulpride, ziprasidone, and olanzapine.

2) Combination enhances the secretion of means, such as a variant splicing of the ghrelin or a similar variant splicing of the ghrelin connection with the active ingredient or therapy against the disease, which causes or is associated with the disease or condition under treatment enhances the secretion of a tool, such as a variant splicing of the ghrelin or a similar variant splicing of the ghrelin connection.

In particular, in relation to cancer cachexia introduction enhancing the secretion of tools, such as such variant splicing of the ghrelin connection can be performed in combination with any anti-cancer therapy, including antineoplastics chemotherapy, radiotherapy and surgery. In particular, it is used in combination with chemioterapia and radiotherapy. Thus, one variant of implementation relates to a method of treating cancer, including the appointment of an effective amount of radiotherapy and effective amount enhances the secretion of tools, such as such variant splicing of the ghrelin connection in accordance with the present description. The treatment enhances the secretion of means, such as a similar variant splicing of the ghrelin connection, you can start before the start of radiotherapy treatment. It can be given continuously during radiation, or it can be administered with intervals, for example, between periods of radiotherapy treatment.

Another variant of implementation relates to a method of treating cancer, including the appointment of an effective amount of antineoplastic chemotherapy and an effective amount enhances the secretion of tools, such as such variant splicing of the ghrelin connection in accordance with the present description. The treatment enhances the secretion of means, such as a similar variant splicing of the ghrelin connection, you can start before the start of chemotherapy treatment. It can be given continuously during chemotherapy, or it can be administered with intervals, for example, between periods chemotherapeutic therapy.

In addition, combinatorial treatment may be joint reinforcing composition is th secretion funds such as such variant splicing of the ghrelin connection, and antineoplastic chemotherapy.

Enhancing the secretion of a tool in accordance with the present invention, such as a variant splicing of the ghrelin or a similar variant splicing of the ghrelin compound can also be administered in combination with a pharmaceutically effective amount of glucocorticoid steroids and prokinetics treatment, and other treatments used for cancer therapy. Thus, in another preferred embodiment, the present invention enhances the secretion of a tool in accordance with the present invention, such as a variant splicing of the ghrelin or a similar variant splicing of the ghrelin compound is administered in combination with a pharmaceutically effective amount of one or more drugs: contributing to the onset or continuation of the pregnancy medicines, such as megestrol and/or cyproheptadine (and/or other antagonists of the receptor 5-HT); and/or branched chain amino acids; and/or Oxandrolone; and/or anti-TNF-α agents, such as infliximab, etanercept or adalimumab; and/or testosterone; and/or a mixture including medicines immunopathy, antioxidants and inhibitors SUCH; and/or cannabinoids; and/or eicosapentaenoic acid; and/or melatonin is; and/or thalidomide; and/or β2-adrenergic drugs, most preferably for the treatment of cachexia, such as cancer cachexia.

In yet another embodiment, the present invention enhances the secretion of a tool, such as a similar variant splicing of the ghrelin compound is administered in combination with anti-inflammatory compounds, preferably NSAID such as indomethacin, and inhibitors SUCH or inhibitors SUCH; and/or anti-TNF-α agents such as infliximab, etanercept or adalimumab. Another combination can be with erythropoietin/EPO. Another combination can be lower angiotensin II agents such as Vitor. Another combination can be selective modulator(s) of the androgen receptor. Another combination may be one or more of leptin agonists system the renin-angiotensin, agonists of opioid receptors or agonist-activated proliferation peroxisome gamma receptor.

Regarding the treatment of lipodystrophy another variant implementation of the present invention relates to the treatment, which enhances the secretion of a tool, such as a splicing variant of ghrelin, more preferably such variant splicing of the ghrelin compound is administered in combination with treatment against lipodystrophy, such as one or more treatment is or compounds, described here is suitable for the treatment lipodystrophies syndrome.

Thus, other pharmacologically active substances that can be administered in combination with the specified amplifying secretion of a tool, such as a similar variant splicing of the ghrelin connection, in the methods of the present description, include:

(a) Leptin: the leptin has a positive effect on the metabolic abnormalities associated with lipodystrophy (Oral E.A. et al., J. Clin. Endocrinol. Metab. 91: 621-28 (2006)). It is proved that this therapy is useful for patients suffering from low level of plasma leptin, and for patients with normal levels.

(b) the Agonist-activated proliferation peroxisome gamma receptor (PPAR-γ): In several studies demonstrated that PPAR-γ is important for the metabolism in adipocytes and metabolic syndrome, and suggested that agonists of PPAR-γ will reduce the symptoms of lipodystrophy (R.K. Semple et al., J. Clin. Invest. 116: 581-89 (2006)).

(C) Agonists system the renin-angiotensin: it was Shown that treatment with HAART increases the activity of ACE in T-cells, which means that agonists system the renin-angiotensin can improve HAART induced lipodystrophy (R.A. Hegele & Leff, T., J. Clin. Invest. 114: 163-65 (2004)).

(d) Agonists of opioid receptors: it is Shown that agonists of opioid receptors, such as naloxone is naltrexone, lengthen the period of time from treatment with protease inhibitors to the development of the first symptoms of lipodystrophy (AIDS Patient Care STDS 14: 283 (2000)).

(e) a Variant splicing of the ghrelin without acyl, found that splicing variant of ghrelin in combination with alternative splicing of the ghrelin without acyl reduces insulin resistance, which is an important feature of lipodystrophies syndrome (Koutkia, P. et al., Am. J. Physiol. Endocrinol. Metab. 286: E296-303 (2004)).

(f) Adiponectin and antidiabetic treatment, including other compounds for the treatment and/or prevention of insulin resistance and diseases in which insulin resistance is the pathophysiological mechanism.

(g) it was Reported that rhGH therapy causes a reduction in the size of the "Buffalo hump", body fat and increases lean body mass by a small number of patients (Lo J.C. et al., J. Clin. Endocrinol. Metab. 86: 3480-87 (2001)). However, fat loss and abnormalities of lipids did not improve, and control blood glucose deteriorated. Examples of syndromes, being treated with hGH include HIV, AIDS, and cancer. Hasn't bound by theory, the assumption that the treatment alternative splicing of the ghrelin or its analogue is to maintain and/or increase body fat in patients being treated with hGH, thereby effectively counteracting lipodystrophy caused hGH, or at least reducing this lipo. That is they way one preferred implementation of the present invention relates to the use of variant splicing of the ghrelin or a similar variant splicing of the ghrelin compounds in combination with growth hormone, preferably for individuals suffering from HIV or AIDS and/or cancer cachexia. Said treatment option splicing of the ghrelin or its analogue may be before and/or during and/or after the individual is subjected to treatment with growth hormone. Preferably the specified growth hormone is hGH.

(h) Treatment with various combinations enhance the secretion of the means described above in group 1), above.

3) Combination enhances the secretion of means, such as a variant splicing of the ghrelin or a similar variant splicing of the ghrelin connection with the active ingredient or therapy against symptoms associated with the disease or condition under treatment enhances the secretion of a tool, such as a variant splicing of the ghrelin or a similar variant splicing of the ghrelin connection.

One aspect relates to a combinatorial treatment in which one of the ingredients in the combination is used to treat symptoms or conditions that can occur in individuals suffering from cachexia. Thus, applications and combinatorial treatment, which include the purpose of reinforcing with whom crecio means, such as such variant splicing of the ghrelin connection in accordance with the present description may also include the treatment in combination with one or more of the

a) preventing and/or alleviating and/or treating clinical depression, combinatorial treatment which also includes the appointment of the antidepressant, it proletarienne forms or pharmaceutically acceptable salts of the specified antidepressant or specified proletarienne form. When the above combinatorial antidepressant treatment is preferably the inhibitor reuptake noradrenaline (NERI), a selective inhibitor of serotonin reuptake (SSRI), monoamine oxidase inhibitor (MAO), combined NERI/SSRI or an atypical antidepressant, proletarienne the form of said antidepressant or a pharmaceutically acceptable salt of the specified antidepressant or specified proletarienne form. The preferred antidepressants are an SSRI, it proletarienne form or pharmaceutically acceptable salt of the specified SSRI or specified proletarienne form. Preferably an SSRI is citalopram, ESCITALOPRAM, femoxetine, fluoxetine, fluvoxamine, indalpine, indeloxazine, milnacipran, paroxetine, sertraline, sibutramine, or zimeldine, Palekastro the form of an SSRI or a pharmaceutically p is yemlemuyu Sol specified an SSRI or specified proletarienne form. In certain embodiments of the implementation of combinatorial treatments of the above are preferred citalopram and ESCITALOPRAM, proletarienne form or pharmaceutically acceptable salt.

b) preventing and/or alleviating and/or treating vomiting conditions, including nausea and vomiting, combinatorial treatment which, in addition, includes the appointment of an antiemetic, it proletarienne forms or pharmaceutically acceptable salts of the specified antiemetic or specified proletarienne form. The preferred antiemetic used in combinatorial treatments in accordance with the present description, include meclizine hydrochloride, prochlorperazine, promethazine, trimethobenzamide hydrochloride and ondansetron hydrochloride. In particular, vomiting can be caused by cancer or by cancer treatment, or due to the cancer itself.

(C) preventing and/or alleviating and/or treating psychotic state, combinatorial treatment which, in addition, includes the appointment of adjunct, it proletarienne forms or pharmaceutically acceptable salts of the specified neuroleptic or specified proletarienne form. Preferred doses used in combinatorial treatments in accordance with this description is, include chlorpromazine, haloperidol, clozapine, loxapine, molindone hydrochloride, thiothixene, lanzarin, ziprasidone, ziprasidone hydrochloride, prochlorperazine, perphenazine, trifluoperazine hydrochloride and risperidone.

d) preventing and/or alleviating and/or treating anxiety, combinatorial treatment which also includes the appointment of sedatives, it proletarienne forms or pharmaceutically acceptable salts of the specified sedatives or specified proletarienne form. Preferred sedatives used in combinatorial treatments in accordance with the present description, include alprazolam, clonazepam, lorazepam, oxazepam, chlordiazepoxide hydrochloride, diazepam, buspirone hydrochloride, doxepin hydrochloride, hydroxyzine, pamoate and clonazepam.

Of course, combinations of the abovementioned groups (1-3) are also within the scope of this description.

Medical packaging

Disclosed here, the connection can be entered separately or in combination with pharmaceutically acceptable carriers or excipients, or for single or repeated administration.

The composition can be appropriately presented in the form of a standard dose using methods known to skilled in the art specialists.

Preferably, the connection is placed in accordance with the present description have been provided in the set. This set usually contains the active compound in dosage forms for injection. Dosage form contains an amount of active compound sufficient to obtain the desired effect when administered to a subject, preferably to at least one meal a day, preferably before each main example of food, for example, three times a day, during the course of 1 or more days. Therefore, it is preferable that the medical packaging included a number of standard doses corresponding to the relevant scheme introduction. Accordingly, in one embodiment of the present invention medical packaging includes a pharmaceutical composition comprising a compound as defined above, or its pharmaceutically acceptable salt and pharmaceutically acceptable carriers and/or excipients, with this pack contains 7 to 21 standard dose or a lot of them, therefore, contains the standard dose for injection for one week or introduction for several weeks.

In one embodiment of the present invention, the medical package is intended for introduction once a day during the week and includes 7 standard doses, in another embodiment of the present invention medical packaging is designed to put in place the program twice a day and includes 14 standard doses. In yet another preferred embodiment of the present invention medical packaging is designed for administration three times a day and includes 21 standard dose.

Standard doses are as defined above, i.e. a standard dose preferably contains the amount of such variant splicing of the ghrelin compounds or salts thereof equivalent to from 0.3 μg to 600 mg splicing variant of ghrelin, for example, from 2.0 μg to 200 mg splicing variant of ghrelin, for example, from 5.0 μg to 100 mg splicing variant of ghrelin, such as from 10 μg to 50 mg splicing variant of ghrelin, such as from 10 μg to 5 mg splicing variant of ghrelin, such as from 10 μg to 1.0 mg splicing variant of ghrelin.

Medical packaging may be in any form suitable for parenteral, in particular, subcutaneous, administration. In a preferred embodiment of the present invention, the packaging has the form of a cartridge, such as cartridge pens for injection, such as a pen for injection, known from treatment with insulin or hGH treatment.

When medical packaging includes more than one standard dose, preferably medical packaging has been provided a mechanism for establishing each of the introduction of only one standard dose.

Preferably, the kit contains instructions indicating the label of the dosage form to achieve the desired effect on the number of dosage forms, which should make for a specified period of time. Accordingly, in one embodiment of the present invention medical packaging includes instructions regarding the introduction of the pharmaceutical composition. In particular, these instructions may include instructions, guides on the introduction of a specified pharmaceutical composition or during a meal, or preferably for at most 45 minutes before a meal, for example, at most 30 minutes before a meal, for example, at most 25 minutes before a meal, for example, at most 20 minutes before a meal, for example, for at most 15 minutes before a meal, for example, at most 10 minutes before a meal, for example, for at most 5 minutes before a meal.

Method of controlling the effect of the treatment option splicing of the ghrelin and/or similar variant splicing of the ghrelin connection

Another aspect relates to a method for controlling the effect of the introduction of amplifying secretion of tools, such as such variant splicing of the ghrelin compounds disclosed here, in the method of the present description, including the definition of one or more markers, in particular markers selected from GH, IGF-1, IGFBP-3, ALP (with acid labeling), thyroid hormones, sex hormones and albumin; p is edocfile selected from IGF-1, IGFBP-3, ALP (with acid labeling); even more preferably IGF-1. All these markers are low in patients with cachexia and is expected to increase after treatment with alternative splicing of the ghrelin. Other markers that are expected to increase after treatment with alternative splicing of the ghrelin are the level of GH in the blood and the body weight. In addition, the expected change in body composition, and is expected to increase lean body mass. Changes in body composition can be assessed using magnetic resonance imaging (MRI) or NMR.

Thus, one way of implementing the present invention relates to a method of controlling effect on any of the treatments of the individual amplifying secretion by the means described herein, including determining the level in blood of the specified individual of one or more markers: selected from (i) IGF-1 and/or (ii) IGFBP-3, and/or (iii) ALP; and/or (iv) one or more thyroid hormones, and/or (v) one or more sex hormones; and/or (vi) of albumin, or preferably one or more markers: (i) IGF-1 and/or (ii) IGFBP-3, and/or (iii) ALP; and/or (iv) GH, and/or (v) of body weight; and/or (vi) body composition.

Methods of determining the level of substances in the blood of the individual are well known in the art. As an example, the selected blood sample can be tested using techniques such as Western is letting or solid-phase enzyme-linked immunosorbent assay (ELISA).

EXAMPLES

The present description is described below in the following examples. It should be understood that these examples, while indicating preferred embodiments of the present invention, is shown only for illustrative purposes. From the above discussion and these examples, skilled in the art, the technician can set the preferred features of this description and without departing from its essence and scope, can make various changes and modifications to adapt to different applications and conditions.

Examples 2, 5, 6, 7, 8 and 9 are working examples. Examples 1, 3, 4, 10 and 11 are prognostic examples.

Example 1

Analysis of competitive binding

Transfetsirovannyh cells COS-7 transferred into cultural tablets a day after transfection at a density of 1×105cells per well, with namiranian get 5-8% of the binding of the radioactive ligand. Two days after drasticly conduct experiments on competitive binding for 3 hours at 4οWith using 25 PM [125I]-ghrelin (GE Healthcare, Piscataway, NJ, USA). Analyses linking is carried out in 0.5 ml 50 mm Hepes buffer, pH 7.4, supplemented with 1 mm CaCl2, 5 mm MgCl2and 0.1% (W/W) bovine serum albumin, 40 μg/ml bacitracin. Nonspecific binding is determined is expressed as binding in the presence of 1 micromole unlabeled variant splicing of the ghrelin. Cells are washed twice with 0.5 ml chilled on ice buffer, and add 0.5-1 ml of buffer for lysis (8 M urea, 2% NP40 in 3 M acetic acid), and podkidyvajut associated radioactivity. Determine the exercise twice.

Example 2

Synthetic products such variant splicing of the ghrelin connection

Derivatives of amino acids and reagents for the synthesis can be obtained from commercial sources. Elongation of the peptide chain can be performed using a synthesizer Applied Biosystem 433A, produced by Perkin Elmer, and resin-derived protected peptide can be constructed using Boc - or Fmoc-way. From resin with the protected peptide obtained by using Boc-way, remove protection using anhydrous hydrogen fluoride (HF) in the presence of para-cresol, thereby releasing peptide, which is then cleaned. From resin with the protected peptide obtained by using Fmoc-way, remove protection using triperoxonane acid (TFA) or diluted TFA containing various acceptors, and the released peptide cleanse. The cleaning is performed using HPLC with a reverse phase column C4 or C18. The purity of the purified product can be confirmed by HPLC reverse phase, and its structure can be confirmed by analysis of amino acid composition and mass spectrometry.

Disclosed here, the peptides can be obtained General the accepted method of peptide synthesis. In particular, the synthesis of acylated alkyl or peptides cited as an example below.

Abbreviations: "NMR-resin" means 4-hydroxymethylbenzene-resin"; "Fmoc amide-resin" means a 4-(2',4'-acid-Fmoc-aminomethyl)phenoxyacetamide-resin; "PAM-resin" means phenylacetamido-resin; "HBTU" refers to 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexaflurophosphate; "TBTU" means 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate; "HOBt" refers to 1-hydroxybenzotriazole; "DCC" means dicyclohexylcarbodiimide; "DIPCI" means diisopropylcarbodiimide; "TFA" means triperoxonane acid; "DIPEA" means diisopropylethylamine; "TIPS" means triisopropylsilane; "Fmoc" refers to fluorenylmethoxycarbonyl; "Boc" means tert-butyloxycarbonyl; "Trt" means trityl; "Bu" means tert-butyl; "PMC" means 2,2,5,7,8-pentamethylchroman-6-sulfonyl; "Prl" means propionyl; "PhPrl" means phenylpropionyl; "Bzl" means benzyl; "Bom" means benzyloxyethyl; "Tos" means toluensulfonyl; "Cl-Z" means 2-chloro-benzyloxycarbonyl; "Pis" means 2-phenylisopropyl; "Mtt" means 4-methyldecyl; "DMF" means N,N-dimethylformamide; "NMP" means N-organic; "DMPA" means 4-dimethylaminopyridine; "HOSu" means N-hydroxysuccinimide; "Adod" means 2-aminododecanoic acid; "Aib" means 2-amino butylevoy acid; "Ape" means 5-aminopentanoic acid; "Cha" means cyclohexylamine; "Dap" means 2,3-diaminopropionic acid; "Nal" means nafcillin; "Nie" means norleucine.

In Fmoc-method of synthesis you can use the following protected amino acids: Boc-Gly, Fmoc-Gly, Fmoc-Ser(Bu), Fmoc-Ser(Trt), Fmoc-Glu(OBu), Fmoc-His(Boc), Fmoc-Gln(Trt), Fmoc-Arg(Pmc), Fmoc-Lys(Boc), Fmoc-Pro, Fmoc-Leu, Fmoc-Ala, Fmoc-Val, Fmoc-Phe, Fmoc-Phe, Fmoc-Ser(n-C8H17), Fmoc-Ser(n-C8H17),Fmoc-Cys(n-C8H17), Fmoc-Asp(OPis), Fmoc-Ser(Bzl), Fmoc-Cys(Trt), Fmoc-Dap(octanoyl), Fmoc-2-Nal, Fmoc-2-Nal, Fmoc-Nle, Fmoc-Lys(Mtt), Fmoc-Aib-OH, Fmoc-Asp(O-C7H15). In Boc-way: Boc-Gly, Boc-Ser(Bzl), Boc-Ser(Ac), Boc-Ser(Prl), Boc-Glu(OBzl), Boc-His(Bom), Boc-Gln, Boc-Arg(Tos), Boc-Lys(Cl-Z), Boc-Pro, Boc-Leu, Boc-Ala, Boc-Val, Boc-Phe, Boc-Cys(n-C8H17), Boc-Ape, Boc-Ser(n-C8H17).

Used equipment:

(a) Unit analytical system HPLC system Shimadzu LC-10A; column: YMC PROTEIN-RP (4.6 mm × 150 mm); column temperature: 40 º C; eluent: a linear gradient from 0 to 50% acetonitrile for 20 minutes in 0.1% triperoxonane acid; flow rate: 1 ml/min; detection: UV (210 nm); volume of injection: 10-100 × 10-9L.

(b) Unit preparative HPLC system: system Waters 600 Multisolvent Delivery; column: YMC-Pack ODS-A (5×10-9m, 20 mm × 250 mm, YMC-Pack PROTEIN-RP (5×10-9m, C4, 10 mm × 250 mm, YMC-Pack PROTEIN-RP (5×10-9m, C4, 20 mm × 250 mm, YMC-PROTEIN-RP (4.6 mm × 150 mm); eluent: suitable linear concentration gradient of acetonitrile in 0.1% triperoxonane acids is; flow rate: 10 ml/min (for columns with an inner diameter of 20 mm), 3 ml/min (for columns with an inner diameter of 10 mm), 1 ml/min (for columns with an inner diameter of 4.6 mm); detection: 210 nm, 260 nm; injection: 10-2000 × 10-9l (2000×10-9l or more was introduced through the pump).

(C) a Unit of mass spectrometer: Finnigan MAT TSQ700; ion source: ESI; the method of detection of ions: positive dispersion; voltage: 4.5 kV; capillary temperature: 250οC; mobile phase: a mixture of 0.2% acetic acid and methanol (1:1); flow rate: 0.2 ml/min; scan range of m/z 300-1500.

(d) a Unit for the analysis of amino acid sequences: Applied Biosystem 477A, the sequencer model 492, produced by Perkin Elmer.

(e) a Unit for analysis of amino acids: amino acid analyzer model L-8500, manufactured by Hitachi, Co., Ltd.; sample: if not stated otherwise, the sample is subjected to hydrolysis with 6 M HCl at 110 º C for 24 hours in a sealed tube.

Example of synthesis with azilsartan derivative (Fmoc-way derivatives as C-terminal amides) variant splicing of the ghrelin

GSS(CO-C7H15)FLSPEHQRVQVRPPHKAPHFmoc-His(Pmc)-HMP-resin (403 mg, 0.25 mmol, ABI Co., Ltd.) treated with 20% piperazine for 20 minutes and then subjected to introduction of Fmoc-amino acid using HBTU/HOBt and removal of Fmoc by piperazine sequentially to construct Fmoc-Ser(Bu)-Ser(Trt)-Phe-Leu-Ser(tu)-Pro-Glu(OBu)-His(Boc)-Gln(Trt)-Arg(Pmc)-Val-Gln-Val(Trt)-Arg(Pmc)-Pro-Pro-His(Boc)-Lys(Boc)-Ala(Boc)-Pro(Boc)-Pro-His(Pmc)-resin. After the introduction, in the end, Boc-Gly using DCC/HOBt resulting resin-protected peptide (1.3 g) is treated with a solution of 1% TFA-5% TIPS-methylene chloride (15 ml) for 30 minutes.

The peptide-resin was filtered, washed several times with methylene chloride (30 ml) and washed with 5% DIEA (10 ml) and then with methylene chloride (30 ml). The resulting de-Trt-peptide-resin (about 1.3 g) is subjected to swelling under the action of NMP (10 ml)and to it was added octane acid (144,2 mg, 1.0 mmol) and DIPCI (126,2 mg, 1.0 mmol) in the presence of DMAP (61,1 mg, 0.5 mmol) and make possible the interaction within 8 hours. The resin is removed by filtration and washed with NMP and then with methylene chloride, and then dried under vacuum to obtain about 1.2 g of resin with a protected peptide, and a side chain of the third serine is anjaneyaswami. To this product add the reagent unprotect (10 ml)consisting of 88% TFA - 5% phenol, 2% TIPS - 5% N2Oh, and the mixture is stirred at room temperature for 2 hours. The resin is removed by filtration, and the filtrate is concentrated, followed by addition of diethyl ether to the resulting residues for the formation of precipitates. The precipitates are removed by filtration and dried to obtain approximately 550 mg of the crude peptide. 200 mg of this product was dissolved in 10 ml of water and applied to YMC-Pack PROTEIN-RP (C4, 20 mm × 250 mm) and will versaut elution linear gradient (flow rate: 10 ml/min) from 0 to 54% acetonitrile in 0.1% triperoxonane acid for 60 minutes. The desired fractions are collected and subjected to lyophilization to obtain approximately 120 mg of the desired product.

Example of synthesis with azilsartan derivative (Fmoc-way connection in the form of C-terminal amides) variant splicing of the ghrelin (1-22)-NH2

GSS(CO-C7H15)FLSPEHQRVQVRPPHKAPH-NH2Fmoc-amide-resin (403 mg, 0.25 mmol, ABI Co., Ltd.) treated with 20% piperazine for 20 minutes and then subjected to introduction of Fmoc-amino acid using HBTU/HOBt and removal of Fmoc by piperazine sequentially to construct Fmoc-Ser(Bu)-Ser(Trt)-Phe-Leu-Ser(Bu)-Pro-Glu(OBu)-His(Boc)-Gln(Trt)-Arg(Pmc)-Val-Gln-Val(Trt)-Arg(Pmc)-Pro-Pro-His(Boc)-Lys(Boc)-Ala(Boc)-Pro(Boc)-Pro-His(Boc)-resin. After the introduction, in the end, Boc-Gly using DCC/HOBt resulting resin-protected peptide (approximately 550 mg) is treated with a solution of 1% TFA-5% TIPS-methylene chloride (10 ml) for 30 minutes. The peptide-resin is removed by filtration, washed several times with methylene chloride (30 ml) and washed with 5% DIEA (10 ml) and then with methylene chloride (30 ml). The resulting de-Trt-peptide-resin (approximately 750 mg) is subjected to swelling with NMP (10 ml)and to it was added octane acid (144,2 mg, 1.0 mmol) and DIPCI (126,2 mg, 1.0 mmol) in the presence of DMAP (61,1 mg, 0.5 mmol) and make possible the interaction within 4 hours. The resin is removed by filtration and washed with NMP and then with methylene chloride, and then dried under vacuum with recip is of approximately 800 mg of resin with a protected peptide, moreover, the side chain of the third serine is anjaneyaswami. This product added TFA (10 ml) and stirred at room temperature for 30 minutes. The resin is removed by filtration, and the filtrate is then concentrated, followed by addition of diethyl ether to the resulting residues for the formation of precipitates. The precipitates are removed by filtration and dried to obtain approximately 250 mg of the crude peptide. Approximately 200 mg of this product was dissolved in 10 ml of 30% aqueous acetic acid and applied to YMC-Pack PROTEIN-RP (C4, 20 mm × 250 mm) and subjected to elution with a linear gradient (flow rate: 10 ml/min) from 0 to 54% acetonitrile in 0.1% triperoxonane acid for 60 minutes. The desired fractions are collected and subjected to lyophilization to obtain approximately 150 mg of the desired product.

Example of synthesis with azilsartan derivative (Boc-way) variant splicing [Ser3(propionyl)]-ghrelin (1-22)

The resin with the protected option of the splicing of the ghrelin (4-22) GSS(CO-CH2CH3)FLSPEHQRVQVRPPHKAPH design on Boc-His(Tos)-Pam-resin (0.75 g, 0.5 mmol) using Boc-chemistry and a half (1.4 g) resin condensation of Boc-Ser(CO-CH2CH3)-OH, Boc-Ser(Bzl)-OH and Boc-Gly-OH. The resulting resin, 1.5 g, then treated with a mixture of HF and para-cresol (8,5 ml: 1.5 ml) at 0οC for 1 hour, and HF is evaporated. To balances add diatrofi the ether, whereby receive 671 mg of the crude peptide. This sample is then dissolved in 50% acetic acid (AcOH) and applied to preparative column YMC-Pack ODS-A (5×10-9m, 20 mm × 250 mm) and subjected to elution at a speed of 10 ml/min gradient from 0 to 95% acetonitrile in 0.1% TFA for 75 minutes. Containing the desired product fraction is subjected to lyophilization to obtain approximately br135.8 mg of the crude peptide. Part (0.5 mg) of this product applied on the column YMC-A-302 (C18, 4.6 mm × 150 mm) and subjected to elution at a flow rate of 1 ml/min gradient from 15% to 19% acetonitrile. This cleaning process is then repeated, and the desired fractions are combined with the receiving approximately 0,41 mg of the desired product.

Other compounds in accordance with the present invention can be obtained in a similar manner.

The above method used for the synthesis of acylated and palleroni SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 5.

Example 3

A randomized, chetyrehballnoe cross-test using one of the center for the study of the absolute bioavailability of intravenously administered variant splicing of the ghrelin and subcutaneously injected variant splicing of the ghrelin in three different single doses to healthy subjects

Purpose

Primary: to investigate the absolute bioavailability of three different prior to the splicing variant of ghrelin, input in the form of single intravenous and subcutaneous doses.

Secondary:

1) to investigate the degree of dose proportionality (dose proportionality) of increasing doses,

2) to examine and compare the pharmacodynamic profiles between treatments,

3) to evaluate the safety and local tolerability.

The design of the test: a Randomized, chetyrehballnoe cross-sectional test of an unbalanced block design with one center for the study of the absolute bioavailability of intravenously administered variant splicing of the ghrelin and subcutaneously injected variant splicing of the ghrelin in three different single doses in healthy subjects. For each route of administration will be used three doses: low, medium and high. To reduce the number of dotirovanii each individual and, therefore, decrease the duration of the test, each subject will only receive four doses of the total number of six doses, i.e. two levels of dose, administered as an intravenous or subcutaneous, respectively. Unbalanced block design will ensure that the levels of all three doses will be provided thus, although not all subjects will receive all dose levels. Sufficient washout period will be included between the periods of dosing of the individual.

Final provisions the Oia

Pharmacokinetics variant splicing of the ghrelin: AUC0-tAUC, Cmax, tmax, t, Cl/fVz/fClVz, t1/2

MRT-pharmacodynamics: GH: AUC, Cmaxand tmax, cardiac output, assessment of hunger, food intake/energy intake, the degree of pleasure associated with food consumption, body weight, energy expenditure, DEXA.

Safety: the safety and local tolerability will be assessed throughout the study using clinical assessment (physical examination and signs of life), electrocardiography, and laboratory tests (Hematology and clinical biochemistry).

The population for testing and calculation of power: health, which is the male subjects aged 18-45 years with a body mass index (BMI), components 19-26 kg/m2(includes both).

The primary goal of this research is the investigation of the absolute bioavailability of variant splicing of the ghrelin injected intravenously and subcutaneously. Unbalanced block design will be used to reduce the period of testing time and reduce the number of dotirovanii on the subject. The number of subjects required for the static analysis of the absolute bioavailability of the dose levels, as well as analysis of the degree of dose proportionality between doses will be calculated on the basis of existing the x data of the literature.

Products tested: variant splicing of the ghrelin for intravenous and subcutaneous injection.

Example 4

Functional tests on the ghrelin receptor

Transfection and tissue culture cells COS-7 grown in the modified Dulbecco environment Needle 1855, supplemented with 10% fetal calf serum, 2 mm glutamine, and 0.01 mg/ml gentamicin. Cells transferout using the method of precipitation of calcium phosphate with the addition of chloroquine, as previously described (B. Holst et al., Mol. Pharmacol. 53: 166-175 (1998)). For the experiments on the gene dosage use different amounts of DNA. The amount of cDNA (20 µg/75 cm2), resulting in maximum signal transmission, used for curves dose-response. Cells of SOME 293 grown in D-MEM, modified, Dulbecco environment Needle 31966 with high glucose, supplemented with 10% fetal calf serum, 2 mm glutamine, and 0.01 mg/ml gentamicin. Cells travelroute LipofectamineTM2000 (Invitrogen Corp., Carlsbad, Cal.).

The cycle of phosphatidylinositol: one day after transfection cells COS-7 incubated for 24 hours with 5 µci [3H]-myoinositol (GE Healthcare, Piscataway, NJ) in 1 ml of medium, supplemented with 10% fetal calf serum, 2 mm glutamine, and 0.01 mg/ml gentamicin, per well. Cells are washed twice with buffer is 20 mm HEPES, pH of 7.4, supplemented with 140 mm NaCl, 5 mm KCl, 1 mm MgSO4, 1 mm CaCl2, 10 mm glucose, 0,05% (weight/volume) b is whose serum and incubated in 0.5 ml buffer supplemented with 10 mm LiCl, when 37º within 30 minutes. After stimulation of various concentrations of peptide for 45 minutes at 37º cells extracted with 10% chilled on ice with perchloric acid followed by incubation on ice for 30 minutes the resulting supernatant neutralize KOH in HEPES buffer, and formed [3H]-insiststhat purified on anion exchange resin Bio-Rad AG 1-X8 (Bio-Rad Laboratories, Hercules, Cal.) in accordance with the manufacturer's instructions. The determination is performed twice.

Analysis of reporters CRE, SRE and NF-κ-B: Cells NEC (30,000 cells/well)seeded in 96-well plates, temporarily travelroute. In the case of the analysis of the CRE reporter cell travelroute mixture pFA2-CREB and reporter plasmid pFR-Luc (PathDetect CREB tran-Reporting System, Stratagene, La Jolla, Cal.) or SRE-Luc (PathDetect SRE Cis-Reporting System, Stratagene, La Jolla, Cal.) and the indicated amounts of DNA receptor. After transfection cells remain in low serum (2.5 percent) during the experiments and are processed with the proper inhibitor of intracellular signal transduction pathways. One day after transfection the cells treated with the appropriate ligands in the bulk of the analysis of 100 μl of medium for 5 hours a Analysis complete by washing the cells twice in PBS and add 100 ál for analysis Luciferace® (LucLite®, PerkinElmer, Inc., Wellesley, Mass). The luminescence is measured in the table counter (To Count NETT, Packard Instrument Co., Meriden, Conn.) within 5 seconds. Luminescence values result in relative light units (RLU).

Analysis of kinase MAR: Cells COS 7 (sowing density of 150,000 cells/well) transferout tablets for analysis. Two days after transfection with the indicated concentration of ligand added to the medium without any serum and incubated for 10 min at 37º. The reaction is stopped by removing the medium and two stages of leaching chilled PBS on ice. Cells are lysed in the buffer for sample and separated by electrophoresis in 10% SDS-page according to Laemmli U.K., Nature 227: 680-85 (1970). Proteins transferred to nitrocellulose, and Western blotting performed using dilution 1:5000 mouse monoclonal antibodies against phospho-ERK1/2 (Santa Cruz Biotechnology, Inc., Santa Cruz, Cal.). Total ERK protein was determined using a dilution of 1:10000 antibodies against ERK (Santa Cruz Biotechnology, Inc., Santa Cruz, Cal.). The blots examined using artemisinin second antibodies conjugated with horseradish peroxidase, visualize using a chemiluminescent reagent (GE Healthcare, Piscataway, NJ) and subjected to quantitative evaluation using densitometric analysis. Phosphorylation of ERK1/2 normalize in accordance with loading of the protein by expressing the data as the ratio of phospho-ERK1/2 to total ERK1/2. The results are expressed in percent of the values obtained is not stimulated fictitiously travelbank cells.

Example 5

Efficacy of subcutaneous administration of acylated variants of splicing of the ghrelin on weight gain and food consumption

Acylated variant splicing of the ghrelin (20 μg (Fig. 1A and 1B) or 180 mcg (Fig. 2A and 2B)or the media (1,6% mannitol) was administered once daily for 14 consecutive days (Fig. 1A and 1B) or 7 consecutive days (Fig. 2A and 2B) subcutaneously to groups of n=10 (Fig. 1A and 1B) or n=8 (Fig. 2A and 2B) male 129Sv mice. During the whole period of the study, mortality was not met none of the animals. Throughout the study period was not observed clinical signs none of the animals. All animals were subjected to end krovoisliania, under anesthesia WITH2immediately prior to euthanasia. The final blood collection was performed sequentially according to the number of the animal, and not according to the group.

Biochemistry: biochemical analysis blood was collected in not covered pre-labeled tubes. The tubes were pre-labeled and contain the following information: number of studies, the group number, animal number and date. After clotting, the blood from each animal was centrifuged, and serum was collected in two pre-labeled tubes and provided for analysis as follows: serum, 250 ál, kept at 2-8 ºc until analysis. Samples of the was adversely the following listed tests, using system Hitachi 917 on: creatinine, total bilirubin, glucose, triglycerides, cholesterol, HDL, LDL, total protein, globulin, albumin, urea, potassium, phosphorus, calcium, sodium, chloride, sGOT, sGPT, ALP.

Urine analysis: Urine was collected in pre-labeled tubes (as above) from all animals (if possible) before and/or after euthanasia. For all surviving animals collection of urine was performed sequentially according to the number of the animal, and not according to the group. An effort was made to maximize the number for conducting the following tests. Urine analysis was performed using a commercial probe for testing (Bayer Multistix®, 10SG)applied to the urine sample, and evaluating the following parameters: glucose, ketone, pH, leukocytes, blood, density, nitrite, bilirubin, urobilinogen and protein.

Procedures necropsy and macroscopic examination: All animals were subjected to a full and detailed autopsy. For all surviving animals autopsy was performed sequentially according to the number of the animal, and not according to the group, directly after the scheduled end krovoisliania. At necropsy, conduct thorough research, and any abnormalities or large pathological changes in tissues and/or organs noticed and log.

Collection of organs/tissues: the Following organs and tissue: head MH is, liver, kidney, stomach, pancreas, lungs, spleen, heart, epididymal WAT, retroperitoneal WAT, intercapillary Wat was removed and determined the weight as soon as possible after excision and removal of the attached fat and other connective tissues. All organs from one animal were collected in one container, pre-packaged with the following information: number of studies, the group number, animal number and date.

Assessment of body composition: the first and last day of treatment used NMR to analyze changes or fat, or lean body mass (Fig. 4).

Results: the cumulative increase in body weight in 129Sv mice, treated acidified by alternative splicing of the ghrelin was significantly higher (p=6E-0,5)than in controls treated with medium (2.2 g and 0.7 g, respectively). See Fig. 1A. Cumulative food intake 129Sv mice, treated acidified by alternative splicing of the ghrelin was significantly higher (p=0.04)than controls treated with vehicle (53,2 g and 47,21 g, respectively). See Fig. 1B. Throughout the study period was not observed events in the data on mortality and obvious clinical signs in response to treatment among any verifiable animals. Based on the above research results acylated splicing variant of graminaceous significantly activates the increase in body weight and food consumption.

Example 6

Efficacy of subcutaneous administration of acylated variants of splicing of the ghrelin on GH release

Acylated variants of splicing of the ghrelin (20 μg) or vehicle (1,6% mannitol) was administered via subcutaneous bolus injection (corresponding to 0.3 µmol/kg) of each of 5 mice. Blood samples were collected after 10 and 20 minutes after injection. Serum samples were stored at-70 º C and analyzed using the ELISA kit of human growth hormone in rats/mice DSL-10-72100 ACTIVE® (Diagnostic Systems Laboratories, Inc., Webster, Texas).

Results: the Concentration of growth hormone in serum after 10 minutes after subcutaneous injection acylated variants of splicing of the ghrelin or media was 2-14-fold higher in the group treated by alternative splicing of the ghrelin compared with the group treated with the carrier (see Fig. 3).

Example 7

Pharmacokinetics locator acylated variants of splicing of the ghrelin in the rat

Subcutaneous administration of splicing variants of ghrelin was carried out at three dose levels, components of 0.5, 2.5 and 10 mg/kg, corresponding to concentrations of 0.1, 0.5 and 2 mg/ml, respectively, and at a constant volumetric dose of 0.5 ml/kg of Intravenous carried out on the same dose level, amounting to 0.5 mg/kg, corresponding to a concentration of 0.1 mg/ml, and at a constant volumetric dose of 0.5 ml/kg of the Study included 9 SAMC is in and 9 female rats, Sprague-Dawley TM(SDTMfor each level of dose and route of administration.

The plan of taking blood samples was limited to 9 times krovoisliania for each level of dose to dose, 5, 15, 30, 60 and 90 min, 3, 5 and 24 hours after dose. Each group was divided into 3 subgroups, with each subgroup was administered 3 specific point in time krovoisliania to obtain samples of 3 individuals/point in time/group (a total of 27 individual samples/group). The average values of body weight in the group in the beginning of the study were similar among all groups and did not exceed ±20% of the average weight for each anatomical sex. Whole blood specimens were kept on ice after collection of blood before centrifugation. The resulting plasma samples were rapidly frozen in liquid nitrogen and kept in dry ice to move on-70 º C.

The concentration of the detected element in the plasma were determined using LC/MS/MS (liquid chromatography/mass spectrometry/mass spectrometry). Pharmacokinetic analysis of variant splicing of the ghrelin was based on the profiles of the average plasma concentration - time for each group doses obtained using nerazgrablennoi pharmacokinetic analysis obtained through the use of computer software "PK Solutions 2.0" (Summit Research Services, CO., USA).

Pharmacokinetic analysis to establish the author of the path revealed the values of AUC0-∞were similar for males and females who were administered a low dose of 6.1 and 5.2 µg · min/ml, respectively) and high dose (18,8 and 20.8 μg · min/ml, respectively). At high dose AUC values in females were significantly lower (49,1 μg · min/ml)than in males (79,2 μg · min/ml). Tmaxcame in 5 minutes after administration of the dose for all groups doses. The values of T1/2ranged from 17.4 to 26.4 minutes for male rats and from 10.7 to 28.9 minutes for female rats.

Example 8

The effect of a single emergency subcutaneous injection acylated variants of splicing of the ghrelin toxicity in rats

Acylated variant splicing of the ghrelin (2,5; 15 and 75 μg) or vehicle (saline) was administered once subcutaneously to groups of n=6 rats Sprague-DawleyTM(SDTM). During the whole period of the study, mortality was not met none of the animals. Throughout the study period was not observed clinical signs none of the animals. All animals were subjected to end krovoisliania, under anesthesia WITH2immediately prior to euthanasia.

Clinical signs:

Animals were observed individually after a dose at least once during the first 30 minutes, periodically during the first 24 hours, while special attention was paid at the time the first 4 hours, and clinical signs were log. Subsequently, animals were examined, and clinical signs were log once a day during the whole 14 days. Observations included changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions (e.g., diarrhea) and the activity of the autonomic nervous system (for example, lacrimation, salivation, piloerection, pupil size, unusual respiration). Also included changes in gait, posture and response to manipulation, as well as the presence of abnormal behavior, tremors, convulsions, sleep, and coma.

Body weight:

Determination of body weight of individuals was carried out shortly before the introduction of variant splicing of the ghrelin (day 0), after 2, 7 and 14 days after administration of the dose. Weight measurement motionless body was carried out just before the autopsy.

Clinical pathology:

Hematological, biochemical parameters and indices of coagulation, listed below, were determined in all surviving animals prior to the scheduled euthanasia.

Hematology: blood Samples were obtained after being deprived of food during the night. Blood samples (at least 500 ál of whole blood) were collected in pre-labeled, covered EDTA tubes containing the following information: number of studies, the group number, animal number and date. The samples were kept until analysis at 2-8 ºc. Hematolo the practical parameters, which determine, using hematological system ADVIA 120 (Beyer), are the number of leukocytes (WBC), the number of erythrocytes (RBC), hemoglobin (HGB), hematocrite number (HCT), mean volume of erythrocytes (MCV), mean hemoglobin (MCH), the average concentration of corpuscular hemoglobin (MCHC), platelets, leukocyte formula. The reticulocytes were counted manually.

Biochemistry: biochemical analysis blood was collected in not covered pre-labeled tubes. The tubes were pre-labeled and contain the following information: number of studies, the group number, animal number and date. After clotting, the blood from each animal was centrifuged, and 300 µl serum was collected in two pre-labeled tubes and provided for analysis, all the while keeping at 2-8 ºc until analysis. The samples were subjected to the following listed tests, using a system of HITACHI MODULAR P-800: creatinine, calcium, glucose, cholesterol, total protein, globulin, LDH, potassium, aspartate, aminotransferase (AST), CPK (IBS), phosphorus, urea, albumin, total bilirubin, alanine, aminotransferase (ALT), sodium, γ-glutamyltranspeptidase (GGT), chloride, triglycerides, high-density lipoprotein (HDL), low-density lipoprotein (LDL), alkaline phosphatase (ALP).

The coagulation indexes: For the analysis of coagulation of blood were collected from the POM is using krovoisliania behind orbital cavities under anesthesia CO 2covered trisodium citrate tubes. After blood collection, all samples of blood and serum was kept at 2-8 ºc until further analysis. Using system Sysmex CA-1500, the samples were subjected to the following listed tests: prothrombin time (PT), ART.

Urine analysis:

Individual samples emptied of urine were collected from all animals (if possible) prior to the scheduled end of the study or until a killing in the case of removal from the study for reasons of state of the animal, or by pressure on the abdominal area above the bladder, or collect emptied of urine, otherwise, directly from the bladder using a puncture of the bladder. The analysis was carried out using a commercial test set (Bayer Multistix®, 10 SG)applied to the sample is emptied of urine, and evaluating the following parameters: glucose, ketone, pH, leukocytes, blood, density, nitrite, bilirubin, urobilinogen and protein.

Procedures necropsy and macroscopic examination: All animals were subjected to a full autopsy after the scheduled euthanasia. At necropsy, all animals were subjected to a thorough investigation, including the external surface of the body, all the passages, cranial, thoracic and abdominal cavities and their contents. Noted and log any abnormalities or large pathological the systematic changes in the tissues and/or organs. Noted, there is a large pathological deviation on the outer surface of the body, which is located at the injection site or about him. A 4% solution of formaldehyde kept following tissues and/or organs, including tissue and/or organs with macroscopic changes: adrenal glands, thoracic aorta, brain, blind intestine, the colon, duodenum, epididymis, eye, sebaceous glands, which added to the lacrimal glands, heart, hip and knee, ileum, skinny intestine, kidney, lacrimal gland, liver, lungs, lymph nodes, superficial cervical lymph nodes, mesenteric, mammary gland + skin, oesophagus, optic nerves ovaries, pancreas, pituitary, prostate, rectum, salivary gland, sciatic nerve, seminal vesicles, skeletal muscle (thigh), skin at the site of injection, spleen, spinal cord (cervical, thoracic, lumbar), sternum (bone marrow, stomach, testes, thymus, thyroid (with parathyroid gland, if applicable), tongue, trachea, urinary bladder, uterus, the cervix, the vagina.

Weighing of organs/tissues and fixation of organs/tissues:

All the animals were weighed and fixation of organs/tissues. The wet weight of the above organs/tissues was determined by the AK possible after dissection and removal of the attached fat and connective tissue (in the case of paired organs were determined individual weight, but it was represented as the average body weight). All organs/tissues then were fixed and preserved in 4% formaldehyde solution (except for the eyes, optic nerves and sebaceous glands, which added to the lacrimal glands, which were fixed in a solution of Davidson) for at least the period of fixation within 48 h before delivery to tissues. In addition, any other organs/tissues with large macroscopic changes were preserved in 4% formaldehyde solution. Organs/tissues of each animal were Packed in plastic containers, each of which was marked by a number of studies, group number, animal number and date of the autopsy. Bone marrow was obtained from one femur was moistened fetal bovine serum in order to make possible a corresponding smear, and did smear on a clean, labeled glass slide using a second slide. Subsequently, the slides were left in the open air to dry and immersed in methanol for about 5 minutes for proper fixation. Preparing at least 2 slides/animal.

Results: during the study period was not observed events in the data on mortality and obvious clinical signs in response to treatment among any verifiable animals.

Example 9

The effect of a single emergency subcutaneous injection acylated variants of splicing of the ghrelin toxicity, toxicokinetic mini-pigs

In order to establish the maximum tolerated dose (MTD), representing the highest dose, do not cause unacceptable toxicity, or the maximum permissible dose (MFP) 2 mini-pigs Siwine/HsdScr:Sinclair group pre-test is administered increasing doses up to a maximum of 75 mg/kg acylated variants of splicing of the ghrelin. On the basis of effects observed in the preliminary test, the maximal single dose appropriately selected and administered to animals in the main study subcutaneously groups, including n=2 minisini. 1 day (day dose) collect blood samples for bioassay analyses in total 9 times: "0" - baseline pre-test, after 5, 15, 30, 60, 90 min, 3, 5 and 24 hours after dose. Receive assessment data standard RK-parameters (Cmax, Tmax, T1/2AUC). The mini-pigs in further observed for an additional 14-day observation period.

Received all pharmacokinetic parameters, and during the whole period of the study, mortality was not met none of the animals. Throughout the period of study, the project was not observed clinical signs none of the animals. All animals were subjected to end krovoisliania, under anesthesia WITH2immediately prior to euthanasia.

Clinical signs:

Animals are observed individually after a dose at least once during the first 30 minutes, periodically during the first 24 hours, with special attention given during the first 4 hours, and clinical signs shall be minuted. Subsequently, the animals examined, and clinical signs should log in once a day on the course as a whole 14 days. Observations include changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions (e.g., diarrhea) and the activity of the autonomic nervous system (for example, lacrimation, salivation, piloerection, pupil size, unusual respiration). Also include changes in gait, posture and response to manipulation, as well as the presence of abnormal behavior, tremors, convulsions, sleep, and coma.

Body weight:

Determination of individual body weight of animals carried out shortly before the introduction of the scanned item (day 0), after 2, 7 and 14 days after administration of the dose. Weight measurement motionless body exercise just before the autopsy. In the case of dead animals body weight measured as close as possible to death.

Clinical pathology:

Hematological, biochemical parameters and show the whether coagulation, listed below identify all surviving animals prior to the scheduled euthanasia.

Hematology: blood Samples obtained after deprivation of food during the night. Blood samples (at least 500 ál of whole blood) is collected in pre-labeled, covered EDTA tubes containing the following information: number of studies, the group number, animal number and date. The samples are kept up to delivery and analysis at 2-8 ºc. Hematological parameters, which determine, using hematological system ADVIA 120 (Beyer), are the number of leukocytes (WBC), the number of erythrocytes (RBC), hemoglobin (HGB), hematocrite number (HCT), mean volume of erythrocytes (MCV), mean hemoglobin (MCH), the average concentration of corpuscular hemoglobin (MCHC), platelets, leukocyte formula. The reticulocytes were counted manually.

Biochemistry: biochemical analysis of blood collected in not covered pre-labeled tubes. Tube pre-labeled and contain the following information: number of studies, the group number, animal number and date. After clotting, the blood from each animal centrifuged, and at least 300 µl serum collected in two pre-labeled tubes and provide for the analysis, all the while keeping at 2-8 ºc until analysis. The samples subjected to the following listed tests, using the ICI is it HITACHI MODULAR P-800, on: creatinine, calcium, glucose, cholesterol, total protein, globulin, LDH, potassium, aspartate, aminotransferase (AST), CPK (IBS), phosphorus, urea, albumin, total bilirubin, alanine, aminotransferase (ALT), sodium, γ-glutamyltranspeptidase (GGT), chloride, triglycerides, high-density lipoprotein (HDL), low-density lipoprotein (LDL), alkaline phosphatase (ALP).

The coagulation indexes: For the analysis of coagulation of blood is collected by krovoisliania behind orbital cavities under anesthesia CO2covered trisodium citrate tubes. After blood collection, all samples of blood and serum kept at 2-8 ºc until further analysis. Using system Sysmex CA-1500, the samples are subjected to the following listed tests: prothrombin time (PT), ART.

Urine analysis: Individual samples emptied of urine collected from all animals (if possible) prior to the scheduled end of the study or until a killing in the case of removal from the study for reasons of state of the animal, or by pressure on the abdominal area above the bladder, or collect emptied of urine, otherwise, directly from the bladder using a puncture of the bladder. The analysis is performed using a commercial test set (Bayer Multistix®, 10 SG)applied to the sample is emptied of urine, and evaluating the I following parameters: glucose, ketone, pH, leukocytes, blood, density, nitrite, bilirubin, urobilinogen and protein.

Procedures necropsy and macroscopic study:

All animals subjected to a full autopsy after the scheduled euthanasia. At necropsy, all animals undergo a thorough investigation, including the external surface of the body, all the passages, cranial, thoracic and abdominal cavities and their contents. Mark and should log any abnormalities or large pathological changes in tissues and/or organs. Note, there is a large pathological deviation on the outer surface of the body, which is located at the injection site or about him. A 4% solution of formaldehyde retain the following tissues and/or organs, including tissue and/or organs with macroscopic changes: adrenal glands, thoracic aorta, brain, blind intestine, the colon, duodenum, epididymis, eye, sebaceous glands, which added to the lacrimal glands, heart, hip and knee, ileum, skinny intestine, kidney, lacrimal gland, liver, lungs, lymph nodes, superficial cervical lymph nodes, mesenteric, mammary gland + skin, oesophagus, optic nerves ovaries, pancreas, pituitary, prostate, rectum, salivary gland, sciatic nerve, seminal vesicles, skeletal muscle (thigh), skin at the site of injection, spleen, spinal cord (cervical, thoracic, lumbar), sternum (bone marrow, stomach, testes, thymus, thyroid (with parathyroid gland, if applicable), tongue, trachea, urinary bladder, uterus, the cervix, the vagina.

Weighing of organs/tissues and fixation of organs/tissues:

For all animals spend weighing and fixation of organs/tissues. The wet weight of the above organs/tissues determine as soon as possible after dissection and removal of the attached fat and connective tissue (in the case of paired organs determine the individual weight, but it is presented as the average body weight). All organs/tissues then fix and retain a 4% solution of formaldehyde (except for the eyes, optic nerves and sebaceous glands, which added to the lacrimal glands, which are fixed in a solution of Davidson) for at least the period of fixation within 48 h before delivery to tissues. In addition, any other organs/tissues with large macroscopic changes retain a 4% solution of formaldehyde. Organs/tissues of each animal is Packed in plastic containers, each of which is marked by a number of studies, group number, animal number and date of the autopsy. Bone marrow is obtained from one of the femur, HC is Arnaut fetal bovine serum, to make possible a corresponding smear, and make strokes on a clean, labeled glass slide using a second slide. Subsequently, the slides leave in open air to dry and immersed in methanol for about 5 minutes for proper fixation. Prepare at least 2 slides/animal.

Results: during the study period was not observed events in the data on mortality and obvious clinical signs in response to treatment among any verifiable animals.

Example 10

Example repeated for 7, 10, 28 days or 3, 6, 9 months subcutaneous dose acylated variants of splicing of the ghrelin to rats/dogs/mini-pigs

Acylated splicing variant of ghrelin in different doses (0.2, 1.0, and 5 mmol/kg, all in 100 μl of media) or media (in saline) was injected once a day for different number of consecutive days (7, 10, 28 days or 3, 6, 9 months) subcutaneously to groups =10 subjects, which rats or bigamy, or people with cachexia. For 20 h after injection to determine the food intake, weight gain and spontaneous motor activity.

Example 11

Examples of evaluation using diaries/questionnaires of quality of life to patients is now

EORTC QLQ-C30 (Aaronson et al., J. Natl. Cancer Inst. 85: 365-76 (1993)), see the website of the National Institutes of Health and the see, for example, a sample of the EORTC QLQ-C30 (version 3.0), available on the EORTC website and is incorporated here by reference.

The authors of the present invention are interested in information about You and Your health. Please answer the following questions, noting the number that best applies to You. There is no "right" or "wrong" answers. All information will be treated confidentially.

Questions: See sample EORTC QLQ-C30 (version 3.0), available on the EORTC website and is incorporated here by reference.

Example 12

Examples of suitable compounds for preparing pharmaceutical compositions used in the present description

4% mannitol solution is prepared by dissolving D-mannitol in water for injection to achieve a final concentration of component 40 mg/ml of the mother solution variant splicing of the ghrelin prepared by dissolving splicing variant of ghrelin in TFA salt or acetate (5 mg) in 10 ml of 4% solution of mannitol, divided into aliquots and stored frozen (-70 º C) until use.

Solution for dispensing variant splicing of the ghrelin: each day the dose required amount of each check item is thawed and diluted with saline to the concentration of that component of 0.2 mg/ml

<> Solution for dispensing control element: Prepare in your every day dose by breeding a 4% solution of mannitol, saline solution in the same proportion as the solution for dispensing variant splicing of the ghrelin.

Patients: Patients with documented cancer cachexia and documented cancer cachexia with significant weight loss during the previous period and reduced appetite. Cachexia can be caused by any type of cancer, including, for example, esophagus, lung, breast, stomach, pancreas, nervous system and urinary tract, bone, hematological cancer, cancer of the genital tract, glands of external secretion of the glands of internal secretion, multiple endocrinology neoplasma, testicular cancer, prostate cancer, neurological cancer, skin cancer, thyroid, liver and colon.

The efficiency of variant splicing of the ghrelin will be evaluated in accordance with clinical assessments:

(1) Emergency food intake: Assessed by a specialist in the field of diet food consumption during the infusion.

(2) the Constant consumption of food: a Daily message about the amount of food consumed during the day, and evaluating the pleasure associated with food consumption. The correctness of this will be confirmed by the excretion of nitrogen in urine, based on what newnice, regarding power for 4 days.

(3) body Weight: the Standard and the graduated scale will be used in the clinic.

(4) the energy Expenditure resting (REE) is a very important indicator, since it is also affected by the condition of the disease, and body size.

(5) Test on physical activity: the Registrar of activity used in accordance with the standard Protocol described on the website of the Registrar activity.

(6) is Associated with health status QOL using standard forms, as described above.

(7) Clinical evaluation:

(i) the Excretion of nitrogen in urine: Collect urine for 24 h should be used as a confirmation of the reported food consumption.

(ii) Glucose in plasma FFA in plasma triglycerides in plasma glycerol in plasma and amino acids in plasma: Substances in the plasma, measured to ensure that the reported food consumption is in accordance with the absorbed amount of the accepted food.

(iii) Lean body mass and fat mass, as measured by the thickness of the TSF and the average length of arm circumference as an indicator of body composition.

(iv) the Fat of the whole body (and fat unbalanced mass) will be assessed by DEXA scan using the software 1.31 for Lunar DPX-L (Scanexport Medical Helsingborg, Sweden).

(v) Leptin in plasma: Leptin is produced by fat cells and by the associated from it. The level of leptin in the plasma provides an estimate of total fat load cells.

(vi) Ghrelin in plasma: the Level of the main ghrelin tends to increase in patients with cachexia.

(vii) GH in plasma: In previous studies, GH was measured as a control effect injection of ghrelin (Enomoto M. et al., Clin. Sci. (Lond). 105: 431-35 (2003)).

(viii) IGF-1: One definition IGF-I summarizes GH secretion over 24 h In healthy volunteers demonstrated that the levels of circulating IGF-I correlated with spontaneous GH secretion (Rose S.R. et al., N. Engl. J. Med. 319: 201-07 (1988). IGF-I may also increase, regardless of the increase GH by improving nutritional status.

(ix) IGFBP-3: One of the protein carrier for IGF-I. It increases in parallel IGF-I, but at a slower speed of response.

(x) Albumin: is an indicator of nutritional status.

(xi) Prealbumin: power status indicator, with a more rapid response to changes than albumin.

(xii) Cortisol: it is Shown that ghrelin administration increases the level of cortisol in serum (F. Broglio et al., J. Clin. Endocrinol. Metab. 88: 1537-42 (2003)). It is shown that corticosteroids have significant antiemetic effect and reduce fatigue and increase the pain relief that may be beneficial for cancer patients with cachexia. However, it has never been shown that cortisol increases the weight in cancer patients kahexiei.

(xiii) CRP and ESR: acute phase Proteins and ESR are often good indicators of systemic inflammation associated with cancer process (Inui A., CA Cancer J. Clin. 52: 72-91 (2002)).

Example 13

Treatment of patients with associated with cancer anorexia/cachexia

Patients with advanced cancer, suffering from the syndrome of anorexia/cachexia (ACS), such as patients with any type of deterioration incurable cancer, it is believed, will help this discussion about improving the quality of life, increased appetite, increased food intake, retention or weight gain pleasure from food and or fat.

Patients will receive subcutaneous injections of 10 mg/kg dose of variant splicing of the ghrelin and placebo. The Protocol will start at 08.00 after an overnight fast. In front from the elbow vein is inserted catheter size 22 for sampling blood. After a period of equilibrium of 30 min, subcutaneously will be introduced variant splicing of the ghrelin (10 µg/kg) or placebo (0.9% saline solution).

Passing the clinical trial treatment: Option splicing of the ghrelin will be available GMP-quality prepared ampoules 10 mg/kg from BACHEM AG, Switzerland or NeoMPS Inc., USA. Placebo consists of normal saline (or a medium used to dissolve the analyte), the cat is who will be provided by the hospital pharmacy. Variant splicing of the ghrelin was dissolved in saline solution, and the dose of 10 µg/kg variant splicing of the ghrelin will enter the patient.

Effectiveness evaluation:

(1) Associated with the consumption of food symptoms: estimated using an adapted version of the questionnaire, the Functional assessment of therapy of appetite and cachexia" (FAACT); related anorexia/cachexia questionnaire EORTC-QLQ-30 (see, for example, a sample of the EORTC QLQ-C30 (version 3.0), available on the EORTC website and is incorporated here by reference); related anorexia/cachexia questionnaire NCCTG (see the website of the National Institutes of Health) and/or a rating scale symptom of Edmonton (see Bruera E. et al., J. Palliat. Care 7: 6-9 (1991)).

(2) Quality of life will be assessed using the questionnaire EORTC-QLQ-C30 (see example 9).

(3) food Consumption and preference food products: determination of food consumption will be carried out by calculating the percentage of food consumed by the patient at every meal, specialist clinics in the field of diet therapy is to estimate the preferences of foods as part of their normal ratings.

(4) the Pleasure of food: will be assessed after the second Breakfast using the visual analogue scale, following the established bindings.

(5) the Perceived appetite, hunger, nausea and saturation: will be evaluated in the morning, before infusion and before and after the second Breakfast use is of a visual analogue scale, following the established bindings. The applicant will also use the shorter form, specially selected for the sense of taste.

(6) the growth Hormone (GH): since GH directly reflect biological function of ghrelin after injection of ghrelin GH increases quickly, the applicant will also monitor the levels of GH in the same moments of time, and ghrelin. Will be a standard analysis of ghrelin.

(7) body Composition: body composition will be assessed using BMI, analysis isoprothiolane and measuring the absorption of two photons/measuring the absorption of x-rays with two energies (DEXA).

(8) the Levels of albumin and transferrin will be determined as parameters of nutritional status.

(9) cardiovascular autonomic function: for vegetative screening violations will be recorded a 20-minute electromyogram Holter and to determine the value of SDNN.

(10) the Mediators of the syndrome of primary anorexia/cachexia: mediators of Pro-inflammatory response (CRP, IL-6, TNF-α), activated metabolism (free fatty acids, triglycerides, insulin, glucose, leptin), the axis of the gut-brain (ghrelin) and somatotropic axis (IGF-1, free testosterone) will be determined as the baseline in the first week. The urine sample will be reserved for evaluation inducing proteolysis of factor (PIF), mediate is and paraneoplastic syndrome of anorexia/cachexia.

1. Such variant splicing of the ghrelin connection, activating the increase in body weight and food consumption and/or stimulating the release of growth hormone having the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4, and which may be optionally modified chemically active group, without altering the biological function of the specified connection.

2. Such variant splicing of the ghrelin compound according to claim 1, where the second or third amino acid compounds modified octanone group.

3. Such variant splicing of the ghrelin compound according to claim 1 or 2, where the connection is modified chemically active group.

4. The use of such variant splicing of the ghrelin compounds for the manufacture of a medicinal product for the treatment or prevention of cachexia, and this variant splicing of the ghrelin compound has amino acid sequence SEQ ID NO: 2 or SEQ ID NO: 4.

5. The use of such variant splicing of the ghrelin compounds for the manufacture of a medicinal product for the treatment or prevention of lipo, and this variant splicing of the ghrelin compound has amino acid sequence SEQ ID NO: 2 or SEQ ID NO: 4.

6. The use of such variant splicing of the ghrelin compounds for the manufacture of a medicinal product for the treatment or before the prevention of muscle wasting, moreover, this variant splicing of the ghrelin compound has amino acid sequence SEQ ID NO: 2 or SEQ ID NO: 4.



 

Same patents:

FIELD: chemistry.

SUBSTANCE: invention relates to a method of purifying a cyclic or an acyclic peptide selected from a group comprising eptifibatide, exenatide, atosiban and nesiritide or combination thereof, from a mixture containing at least one impurity, involving contacting said mixture with a reverse phase HPLC matrix and an ion-exchange chromatography matrix and obtaining a purified peptide product with purity of at least 96% and, preferably, at least about 99%.

EFFECT: obtaining a purified peptide product.

11 cl, 4 tbl, 18 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to medicine and chemical technology. What is offered is a method for producing the peptide Exenatide. The peptide Exenatide prepared by synthesis may be used for producing drugs for type 2 diabetes mellitus.

EFFECT: higher yield of the unpurified peptide (42%-60%), as well as higher purity (58%-75%) and content of the end product in a mixture (33%-42%); besides, a cheaper condensing agent (DIC) is used at all the condensation stages, while the synthesis of more than one short fragments simultaneously enables faster end peptide process.

3 cl, 2 tbl, 8 ex

FIELD: chemistry.

SUBSTANCE: invention relates to synthesis of insulinotropic peptides, which are synthesised using an approach based on solid-phase and liquid-phase (hybrid) synthesis. Overall, said approach involves synthesis of three different intermediate peptide fragments using solid-phase chemistry. Liquid-phase chemistry is then employed to add an amino acid product one of the fragments. The fragments are then linked with each other using solid and liquid phases. This invention can be used primarily to obtain insulinotropic peptides, such as GLP-1(7-36), and analogues thereof which may or many not occur under natural conditions.

EFFECT: use of pseudo-proline in one of the fragments makes solid-phase synthesis of said fragment easier and also makes easier subsequent linking of said fragment with other fragments in liquid phase.

20 cl, 1 dwg, 14 tbl, 15 ex

FIELD: chemistry.

SUBSTANCE: invention relates to novel GLP-1 compounds with prolonged effect and therapeutic application thereof.

EFFECT: improved method.

26 cl, 22 ex

FIELD: medicine.

SUBSTANCE: there are described target-specific compounds representing glucagon-like protein-1 (GLP-1R) receptor agonist conjugates with a linker covalently bound with an antigen-binding antibody site. The invention also refers to methods for preventing or treating diabetes or diabetes-associated conditions.

EFFECT: compounds maintain insulnotropic activity combined with manifestation of prolonged elimination half-life.

39 cl, 44 dwg, 4 tbl, 29 ex

FIELD: medicine.

SUBSTANCE: there are described peptide exendin-4 derivatives amine-modified at a C-terminus and having a N-terminus bound with GLP-1-receptors, and partially or completely contain a peptide amino acid sequence of exendin-4. There is disclosed an application of the peptide exendin-4 derivatives for preparing a diagnostic and therapeutic agent for diseases whereat the GLP-1-receptors expression matters, for determination of the density of insulin-producing tissue cells, for determination of the GLP-1-receptors expression or density.

EFFECT: invention can be used for preparing the diagnostic and therapeutic agent for benign and malignant diseases whereat the GLP-1-receptors expression matters.

25 cl

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

FIELD: chemistry.

SUBSTANCE: invention relates to novel glucagons-like peptide-1 (GLP-1) derivatives which have a prolonged activity profile, their pharmaceutical compositions and use of the compounds in preparing a medicinal agent for treating or preventing hyperglycemia, diabetes 2 or obesity. The compound is a therapeutic GLP-1 polypeptide bonded to a residue bonded with albumin through a hydrophilic separator.

EFFECT: higher effectiveness of derivatives and treatment method.

26 cl, 66 ex

FIELD: medicine.

SUBSTANCE: present invention concerns new, selectable hybrid polypeptides expressing at least two hormonal activities containing a first biologically active module of a peptide hormone covalently bonded with at least one additional biologically active module of the peptide hormone.

EFFECT: polypeptides can be used as agents for treatment and prevention of metabolic diseases and disorders associated with overweight.

19 cl, 6 dwg, 6 tbl, 4 ex

FIELD: chemistry, biochemistry.

SUBSTANCE: claimed invention relates to biologically active compounds and includes novel peptides containing to 15 amino acid residues, including sequence Thr-Ser-Asp-Xaa-Xaa, where Xaa represents optionally substituted biphenylalanine, and possessing activity of GLP-1 receptor.

EFFECT: obtaining peptides demonstrating higher resistance to proteolytic decomposition, which makes them candidates for therapy by oral or parenteral introduction.

6 cl, 7 dwg, 4 tbl, 14 ex

FIELD: chemistry.

SUBSTANCE: invention relates to peptidyl analogues of ghrelin having greater stability which are active with respect to the GHS receptor, having the formula given below: (R2)-A2-A3-A4-A5-A6-A7-A8-A9-A10-A11-A12-A13-A14-A15-A16-A17-A18-A19-A20-A21-A22-A23-A24-A25-A26-A27-A28-Rl, where values of A1-A28, R1 and R2 are given in the description, pharmaceutically acceptable salts thereof and pharmaceutical compositions containing an effective amount of said compound, as well as therapeutic and non-therapeutic applications thereof.

EFFECT: high stability.

22 cl, 3 tbl, 11 ex

FIELD: chemistry.

SUBSTANCE: invention relates to methods for synthesis of nonapeptide ethylamide, having strong LH-RH/FSH-RH activity, of formula pGlu-His-Trp-Ser-Tyr-D-Ser(But)-Leu-Arg-Pro-NH-C2H5·2AcOH (I), and intermediate compounds for synthesis thereof. The nonapeptide ethylamide is obtained via condensation of a C-terminal tetrapeptide of formula H-D-Ser(But)-Leu-Arg-Pro-NH-C2H5·HCl (II) with a dipeptide of formula: X-Ser-Tyr-OH (IV), where X is a protective group. The obtained N-substituted hexapeptide ethylamide of formula X-Ser-Tyr-D-Ser(But)-Leu-Arg-Pro-NH-C2H5·HCl (III) is treated with an unblocking agent to remove the N-protective group, and then condensed with a tripeptide of formula pGlu-His-Trp-OH·HCl (V) and the end product is purified through chromatography and extracted in form of a monoacetate salt.

EFFECT: high output.

4 cl, 1 ex

FIELD: pharmaceutical chemistry.

SUBSTANCE: invention refers to the field of pharmaceutical chemistry, and more precisely to a new way of buserelin production with the formula: pGlu-His-Trp-Ser-Tyr-D-Ser(Bu1)-Leu-Arg-Pro-NH-C2H5·2AcOH (I) consisting in the fact that the synthesis is performed by means of condensing of C-ended protected dipeptide with the formula: X-pGlu-His-OH (IIa), where X is a protective group, with N-ended protected heptapeptide with the formula: H-Trp-Ser-Tyr-D-Ser(Bu1)-Leu-Arg-Pro-NH-C2H5 (III) and obtained N-protected nonapeptide ethylamide with the formula: X-pGlu-His-Trp-Ser-Tyr-D-Ser(Bu1)-Leu-Arg-Pro-NH-C2H5 (IV) that is treated with an unblocking agent in order to remove the N-protective group, and the end product is extracted with the help of chromatography.

EFFECT: way of buserelin production.

4 cl, 9 ex

FIELD: medicine.

SUBSTANCE: invention refers to medicine, namely to obstetrics, and concerns treating placental insufficiency in trimester II-III of pregnancy. For this purpose, conventional therapy is complemented with oxygen therapy and foetal sex determination. Those pregnant women with a predicted female foetuses require therapeutic inhalations with mixed gas containing oxygen - 40%, atmospheric air - the rest at air flow 5-6 litres per minute, length of a session 30 min once a day, the therapeutic course 10 procedures, while those pregnant women carrying female foetuses require therapeutic inhalations with mixed gas containing oxygen - 60%, atmospheric air - the rest at air flow 5-6 litres per minute, length of a session 45 min once a day, the therapeutic course 15 procedures.

EFFECT: method provides effective treatment of placental insufficiency with reduced side effects ensured by dosed oxygen therapy shown by foetus sex determination test.

2 ex

Ghrelin analogues // 2427587

FIELD: medicine.

SUBSTANCE: invention relates to peptide analogues of formulae (I) and (II): (R2R3)- A1-A2-A3-A4-A5-A6-A7-A8-A9-A10-A11-A12-A13-A14-A15-A16-A17-A18-A19-A20-A21A22-A23-A24-A25-A26-A27-A28-R1 in which A1-A28 and R2-R3 are defined in the description for each of the formulae (I) and (II), pharmaceutically acceptable salts thereof and pharmaceutical compositions containing an effective amount of formula (I) compounds and which can be used in the method of suppressing growth hormone secretion and a method of screening a compound which is capable of binding with the GHS receptor.

EFFECT: high efficiency of using the composition.

31 cl, 5 tbl, 2 ex

FIELD: chemistry.

SUBSTANCE: invention relates to compounds of formula (I) or (II) or pharmaceutically acceptable salts thereof, in which X is or ; Y is H; Z is -C(O)-; R1 and R3 each independently denotes H or (C1-C4) alkyl; R2 and R4 each independently denotes , , or ; R5 denotes H or (C1-C6) alkyl; R8 and R9 each independently denote (C1-C6) alkyl; and Q is H.

EFFECT: possibility of use in stimulating the growth hormone in a subject based on the said compounds.

49 cl, 2 tbl, 57 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: present invention refers to administration of formula (I) described new triazole derivatives as greline-like ligands of growth hormone secretion stimulating receptors (GHSS receptors) that can be effective in treatment or prevention of GHSS receptors mediated physiological and/or pathophysiological conditions in mammals, preferentially in humans. Formula (I): where R1 is chosen from a group including hydrogen atom, (C1-C12)alkyl phenyl, naphthyl, (C5-C14)phenyl(C1-C12)alkyl, indolylalkyl which can contain up to 3 substitutes independently chosen from a group including halogen, -F, -CI, -Br, -I, -NO2, (C1-C12)alkyl, phenyl, naphthyl, -O-(C1-C12)alkyl; R2 is chosen from a group including (C1-C12)alkyl, phenyl, naphthyl, (C5-C14)phenyl (C1-C12)alkyl, indolylalkyl which can contain up to 3 substitutes independently chosen from a group including halogen, -F, -Cl, -Br, -I, -NO2, (C1-C12)alkyl, phenyl, naphthyl, -O-(C1-C12)alkyl; one of radicals R3 and R4 represents hydrogen atom, and other radical are chosen from a group including hydrogen atom, phenyl, naphthyl, indolylalkyl; R5 is chosen from a group including hydrogen atom, phenyl, naphthyl, -CO-(C3-C8)cycloalkyl, -CO-phenyl, -CO-(C5-C7)heteroaryl containing 1, 2 nitrogen atoms, -CO-(C3-C7)heteroaryl(C1-C4)alkyl containing 1, 2 nitrogen atoms, -CO-(C5-C6)heterocyclyl containing 1, 2 nitrogen or oxygen atoms, -CO-C*(R9R10)-NH2, -CO-CH2-C*(R9R10)-NH2, -CO-C*(R9R10)-CH2-NH2, phenylsulfonyl which can contain up to 3 substitutes independently chosen from a group including halogen, -F, -CI, -Br, -I, -N3, -CN, -NR7R8, -OH, -NO2, (C1-C4)alkyl; R6 represents hydrogen atom; R7 and R8 represent hydrogen atom; R9 and R10 are independently chosen from a group including hydrogen atom and (C1-C4)alkyl; m relates to 0, 1 or 2, and preferentially 0; and * means carbon atom in a R or S configurations, if it is chiralene.

EFFECT: invention refers to GHSS receptors antagonists and agonists which can be used for modulation of these receptors and are effective in treatment of said conditions, particularly growth impairment, cachexia, short-term, intermediate and/or long-term energy balance control; short-term, intermediate and/or long-term food intake control (stimulation and/or suppression); adipogenesis, adiposity and/or obesity; body weight growth and/or reduction in mammals.

22 cl, 6 tbl, 15 ex, 46 dwg

FIELD: chemistry; biochemistry.

SUBSTANCE: proposed are human growth hormone conjugates, obtained by removing a hydrogen atom from -NH2 in the Gln side chain which is formed from the human growth hormone or a human growth hormone compound.

EFFECT: design of an efficient method of producing human growth hormone conjugates.

6 cl, 14 ex

Ghrelin analogues // 2373220

FIELD: medicine.

SUBSTANCE: invention relates to a peptidyl analogue representing (Aib2, Glu3(NH-hexyl))ghrelin(1-28)-NH2 displaying agonistic activity with respect to ghrelin receptor.

EFFECT: invention can be used for treatment or prevention or decrease in probability or severity of disease or condition accompanied by weight loss.

8 cl, 1 tbl, 6 ex

FIELD: chemistry.

SUBSTANCE: invention relates to dihydropyridine derivatives with general formula , or their pharmaceutically acceptable salts, where R1 is C1-6alkyl or phenyl, C1-5heteroaryl, both optionally substituted with one or more substitutes, chosen from hydroxy, amino, halogen, nitro, cyano; R2, R3 are independently C1-4alkyl, C1-4alkoxy group, C2-4alkenyloxy group, C3-4alkynyloxy group, halogen; R4 is C1-6alkyl, C3-6cycloalkyl, C3-6cycloalkylC1-4alkyl, C6-10aryl, C6-10arylC1-4alkyl, C1-9heteroaryl, where the (hetero)aryl group is optionally substituted with one or more substitutes, chosen from hydroxy, amino, halogen, nitro, trifluoromethyl, cyano, C1-4alkyl, C2-4alkenyl, C2-4alkynyl, C1-4alkoxy group, C1-4(di)alkylamino group, and, if R4 is a phenyl, additionally from C1-4alkylthio group, C1-4alkylsulphonyl, R5-oxycarbonyl, R5-carbonyl or R5,R6-aminocarbonyl; X is SO2, CH2, C(O) or X is absent, where X is CH2, R4 can additionally represent R5-oxycarbonyl, or R5-carbonyl; R5, R6 are independently H, C1-4alkyl, C2-4alkenyl, C2-4alkynyl, C3-6cycloakyl, C3-6cycloalylC1-4alkyl, C2-6heterocycloalkyl, C2-6heterocycloalkylC1-4alkyl, C1-4alkoxycarbonylC1-4alkyl, C1-4(di)alkylaminocarbonylC1-4alkyl or C6-10arylaminocarbonylC1-4alkyl, C1-9heteroarylaminocarbonylC1-4alkyl, C6-10aryl, C1-9heteroaryl, C6-10arylC1-4alkyl, C1-9heteroarylC1-4alkyl, where the (hetero)aryl group is optionally substituted with one or more substitutes, chosen from hydroxy, amino, halogen, nitro, trifluoromethyl, cyano, C1-4alkyl, C2-4alkenyl, C2-4alkynyl, C1-4alkoxy group, C1-4(di)alkylamino group or R5, R6 in R5, R6- aminocarbonyl group can be bonded to a C2-6heterocycloalkyl ring, as well as to a pharmaceutical composition with antagonistic activity towards FSH receptor and to use of these compounds for making medicinal agents.

EFFECT: compounds which are suitable for treating fertility disorders are obtained and described.

11 cl, 33 ex

Treatment method // 2397778

FIELD: medicine.

SUBSTANCE: invention relates to medicine, and deals with treatment of pathologic states connected with increased level of growth hormone (GH) or insulin-like growth factor 1(IGF). For this purpose realised is complex treatment which includes introduction of growth hormone antagonist and somatostatin or somatostatin analogue in developed doses and regimens.

EFFECT: method ensures normalisation of GH and IGF-1 levels in patients resistant to monotherapy with somatostatin analogues during at least 6-month treatment course.

15 cl, 1 tbl, 1 dwg, 1 ex

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