Marked peptides binding hepatocyte growth factor (hgf) for visualisation

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention concerns biochemistry and medicine area. What is presented is a visualisation agent representing a conjugate of structure I as described in the patent claim. The visualisation agent refers to marked cMet-binding peptides. These peptides include a mark with an optical reporter group applicable for visualisation in vivo with the use of light within the range of wave length in the spectrum of 600-1200 nm. There are also presented a pharmaceutical composition for optical visualisation, containing the visualisation agent, a kit for preparing it and a method for optical visualization of a mammalian body in vivo. What is also presented is a method for managing the patients suffering colorectal cancer, involving the stage of optical visualisation in vivo.

EFFECT: presented visualisation agent possess higher cMet-binding affinity and selective cell targeting in vivo.

28 cl, 2 tbl, 7 ex

 

The scope of the invention

The present invention relates to labeled cMet-binding peptides suitable for optical imaging in vivo. These peptides include the label of the optical reporter group suitable for imaging in the spectral range from red to near infrared. Also disclosed are methods of imaging in vivo, in particular for use in the diagnosis of colorectal cancer (CRC).

Prior art

In WO 2005/030266 revealed that there is a medical need for early diagnosis of colorectal cancer (CRC). In WO 2005/030266 disclosed optical contrast imaging agents having affinity to the biological target, abnormally expressed in CRC. This biological target is selected from the following: cyclooxygenase-2 (MOR-2), beta-catenin, E-cadherin, P-cadherin, various kinases, Her-2, matrix metalloproteinase (MMP), cycline, P53, thymidylate synthase, receptors, growth factor vascular endothelial (VEGF), receptors for epidermal growth factor, K-ras, protein adenomatous polyposis of the colon, cathepsin, receptor activator of plasminogen urokinase type (uPAR), a growth factor receptor of hepatocytes (cMet), mucines and gastric receptors. It is said that such preferred target (page 7, lines 11-12) are: cMet, MMP-14, SOH-2, beta-catenin and cathepsin Century, the Vector of WO 205/030266 can represent: peptide peptido group, oligonucleotide, oligosaccharide, related to lipid compound or organic small molecule, such medicines. Reporter group preferably is a dye that interacts with light in the wavelength range from the ultraviolet to the infrared region of the electromagnetic spectrum.

Growth factor hepatocyte (HGF), also known as the scattering factor (scatter factor, SF), is a growth factor that is involved in various physiological processes such as wound healing and angiogenesis. The interaction of HGF with its high-affinity receptor (cMet) involved in the growth, invasion and metastasis of tumors.

In Knudsen et al. the role of HGF and cMet in prostate cancer with possible use for imaging and therapy [Adv.Cancer Res., 91, 31-67 (2004)]. Labeled antibodies against the met for diagnosis and therapy are described in WO 03/057155.

In WO 2004/078778 disclosed polypeptides or multimeric peptide constructs that bind to cMet or a complex containing cMet and HGF. Described approximately 10 different structural classes of peptides. In WO 2004/078778 revealed that these peptides may be in the state of detectable label for in vitro and in vivo applications or medicine for therapeutic applications. Detected label can be a: an enzyme, Fluor is santoe connection, optical dye, ion of a paramagnetic metal, ultrasound contrast agent or a radionuclide. It is said that the preferred label according to WO 2004/078778 are radioactive or paramagnetic and, most preferably, include metal chelated metal-chelating agent.

The present invention

In the present invention the proposed imaging agents suitable for optical imaging in vivo that contain cMet-binding cyclic peptides and optical reporter rendered grouping suitable for rendering the body of a mammal in vivo using light in the range of wavelengths from green to near-infrared spectral range 500-1200 nm. cMet-binding cyclic peptides belong to one of the structural classes of peptide from WO 2004/078778 and have optimal binding affinity of against cMet. These peptides are derived from the phage display technique and selected based on their affinity against cMet and lack of competition with HGF as described in WO 2004/078778. cMet-binding peptides of the present invention preferably have at least one of their ends, protected groups, any abscopal metabolism (MIG). This is an important factor for use in vivo, where the endogenous enzymes and peptidases otherwise will be fast metabolismof the peptide with the subsequent loss of cMet-binding affinity and thereby loss of selective targeting in vivo.

In the present invention proposed the best way of using cMet-binding peptides in vivo, including the use of optical reporter, in contrast to other visualization techniques (e.g., nuclear, magnetic resonance imaging (MRI) or ultrasound), and also proposed preferred optical rendered reporters. Region of the spectrum from green to near-infrared light with a wavelength of 500-1200 nm) is preferred, as this area has minimal spectral overlap with endogenous tissues and substances such as hemoglobin, porphyrin, melanin and collagen [Licha, Topics Curr. Chem., 222, 1-29 (2002)]. Other important factors contributing to the autofluorescence, are restored nicotinamide-adenindinucleotide (NADH), flavin-adenindinucleotide (FAD) and elastin.

Detailed description of the invention

In the first aspect of the present invention is proposed agent imaging, comprising the conjugate of the formula I:

where:

Z1attached to the N-end smbr and represents N or MIG;

Z2attached to the end of cmvr and is a HE, OBcor MIGwhere Bcis a biocompatible cation;

smbr is a cMet-binding cyclic peptide of amino acids 17-30, which is cancel amino acid sequence (SEQ-1):

Cysa-X1-Cysc-X2-Gly-Pro-Pro-X3-Phe-Glu-Cysd-Trp-Cysb-Tyr-X4-X5-X6; where

X1represents Asn, His, or Tyr;

X2represents Gly, Ser, Thr or Asn;

X3represents Thr or Arg;

X4represents Ala, Asp, Glu, Gly or Ser;

X5represents Ser or Thr;

X6represents Asp or Glu;

and each of Cysa-drepresents a cysteine residue, so that the residues a and b, and c and d cyklinowanie with the formation of two separate disulfide bonds;

MIGis a group inhibitory metabolism, which is a biocompatible group, inhibitory or inhibiting the metabolism smbr-peptide in vivo;

L represents a synthetic linker group of formula(A)m-where each And independently represents-CR2-, -CR=CR-, -C≡C-, -CR2CO2-, -CO2CR2-, -NRCO-, -CONR-, -NR(C=O)NR-, -NR(C=S)NR-, -SO2NR-, -NRSO2-, -CR2OCR2-, -CR2SCR2-, -CR2NRCR2-With4-8cyclohexanoltramadol group4-8cycloalkenyl group5-12Allenova group or3-12heteroarenes group, an amino acid, a sugar or a monodisperse polietilenglikoli (PEG) is a structural unit;

each R is independently selected from H, C1-4of alkyl, C2-4alkene is a, With2-4the quinil,1-4alkoxyalkyl or1-4hydroxyalkyl;

m is an integer from 1 to 20;

n is an integer 0 or 1;

IM is an optical reporter rendered grouping suitable for rendering the body of a mammal in vivo using light in the wavelength range at wavelengths from green to near-infrared: 600-1200 nm.

The term "agent visualization" means a connection that is suitable for rendering the body of a mammal in vivo. Preferably, the mammal is a human. Visualization can be invasive (e.g., intra-operative or endoscopic) or non-invasive. The preferred method of visualization is an endoscopy. Although the conjugate of formula I suitable for imaging in vivo, it can also be used in vitro (for example, for analyses of quantitative determination of cMet in biological samples or imaging cMet in tissue samples). Preferably, the agent imaging is used to visualize in vivo.

The group Z1replaces the amino group of the last amino acid residue. Thus, when Z1represents H, amino-end smvr free ends of the group of NH2the last amino acid residue. The group Z2replaces the carbonyl group of the last Amin is an acid residue. Thus, when Z2is a HE, carboxy-end smvr free ends by a group of CO2H last amino acid residue, and when Z2represents OBcthis terminal carboxyl group is ionized in the form of a group of CO2Bc.

The term "group, inhibiting metabolism" (MIGmeans biocompatible group which inhibits or suppresses metabolism smbr-peptide in vivo or amino end (Z1or carboxy-late (Z2). Such groups are well known to experts in the art and appropriate selection for amino end of the peptide from the N-acylated groups,- NH(C=O)RGwhere the acyl group is -(C=O)RGhas RGselected from C1-6of alkyl, C3-10aryl groups, or contains polietilenglikoli (PEG) structural unit. Suitable PEG group described below for the linker group (L). Preferred such groups PEG are biomodification formula IA or IB. Preferred such aminobenzene group MIGrepresent acetyl, benzyloxycarbonyl or TRIFLUOROACETYL, most preferably acetyl.

Suitable groups, inhibiting metabolism, peptide carboxyl end include: carboxamid, tert-butyl methyl ether, benzyl ether, cyclohexyloxy ether, aminoplast or polite angelically (PEG) structural unit. A suitable group of MIGfor the carboxyl terminal amino acid residue smbr-peptide is where the terminal amino group of amino acid residue N-alkylated C1-4alkyl group, preferably methyl group. Preferred such groups MIGare carboxamid or PEG, the most preferred such groups are carboxamid.

Formula I means that the group -(L)n[IM] can be attached at any position Z1, Z2or SPSR. For Z1or Z2group -(L)n[IM] can be either attached to the group of MIGwhere one of the Z1/Z2is a MIG. When Z1represents N, or Z2is a HE, joining the group -(L)n[IM] the position of the Z1or Z2gives compounds of formula [IM]-(L)n-[cMBP]-Z2or Z1-[cMBP]-(L)n-[IM], respectively. Inhibition of metabolism smvr one of the peptide may also be achieved by joining the group -(L)n[IM] in this way, but -(L)n[IM] is not covered by the definition of MIGaccording to the present invention.

Group -(L)nthe formula I can be attached in any suitable position IM. Group -(L)nor takes the place of the existing will replace the La IM either covalently attached to the existing Deputy IM. Group -(L)n- preferably attached via carboxialkilnuyu Deputy IM.

The term cMet-binding cyclic peptide" (smbr) means a peptide that binds with high-affinity receptor growth factor hepatocyte (HGF), also known as cMet (c-Met or a growth factor receptor of hepatocytes). Suitable smbr-peptides of the present invention have an apparent dissociation constant (KDfor cMet complex cMet/HGF less than approximately 20 nm. These smbr-peptides containing Proline residues, and it is known that these residues may exhibit CIS/TRANS isomerization of the primary amide linkages. smbr-peptides of the present invention includes all such isomers.

The term "biocompatible cation" (Bcmeans positively charged counterion which forms a salt with ionized negatively charged group, where specified positively charged counterion is also non-toxic and therefore suitable for introduction into the body of a mammal, especially a human body. Examples of suitable biocompatible cations include alkali metals sodium and potassium; alkaline earth metals calcium and magnesium; ammonium ion. Preferred biocompatible cations are sodium and potassium, the most FAV is preferably sodium.

The term "amino acid" means L - or D-amino acid, similar amino acids (for example, nafcillin) or mimetic amino acids, which can be of natural origin or obtained thin synthesis, and can be optically pure, i.e. in the form of an individual enantiomer and therefore chiral, or in the form of a mixture of enantiomers. For labeling of amino acids in this description used common 3-letter or one-letter abbreviations. Preferably, the amino acids of the present invention are optically pure. The term "mimetic amino acids" means synthetic analogues of the natural amino acids, which are isostere, that is designed to mimic the steric and the electronic structure of the natural compound. Such isostere well known to experts in the art and include depsipeptide, retro inverso-peptides, thioamides, cycloalkanes or 1,5-disubstituted tetrazole, but not limited to [see M. Goodman, Biopolymers, 24, 137, (1985)].

The term "peptide" means a compound containing two or more amino acids, as defined above, linked by peptide bond (i.e. the amide bond attaching Amin one amino acid to carboxyl other amino acids). The term "mimetic peptide" or "mimetic" refers to biologically active compounds that what can be carried on the biological activity of the peptide or protein, but no longer peptide according to the chemical nature, i.e. they no longer contain any peptide bonds (i.e. amide bonds between amino acids). Here, the term "mimetic peptide" is used in a broader sense to include molecules that are no longer fully peptide in nature, such as pseudopeptide, polypeptide and peptide.

The term "optical reporter rendered grouping (IM) refers to a fluorescent dye or chromophore capable of detection either directly or indirectly in the procedure optical imaging using light in the wavelength range at wavelengths from green to near-infrared (500-1200 nm, preferably 600-1000 nm). Preferably IM has fluorescent properties.

Provided that one of the roles of the linker group -(A)mformula I is to alienate IM from the active site smbr-peptide. This is especially important when rendered grouping is relatively bulky, so as not to hinder the interaction with the enzyme. This can be achieved by a combination of flexibility (for example, simple alkyl chain), so this volume group has the freedom to samorazdrazheniya away from the active site, and/or stiffness, such as cycloalkenyl or aryl spacer, which orients IM away from the active site. P is the Herod of the linker group can also be used to modify bearsdley agent imaging. Thus, for example, the inclusion of ester groups in the linker will help minimize binding to plasma proteins. When -(A)m- contains polietilenglikoli (PEG) is a structural unit or a peptide chain of 1-10 amino acid residues, the linker group may be a function of the modification of pharmacokinetic parameters and rate of clearance from blood agent imaging in vivo. Such "biomodification" linker group can accelerate the clearance of the agent visualization of the background tissue, such as muscle or liver, and/or from the blood, thereby giving the best diagnostic image due to less background noise. Beatifically of the linker group can also be used to promote specific ways of excretion, for example, through the kidneys, in contrast to the path through the liver.

The term "sugar" means mono-, di - or trisaccharide. Suitable sugars include glucose, galactose, maltose, mannose and lactose. Perhaps sugar can be functionalized to allow for easy combination with amino acids. Thus, for example, glucosamine derived amino acid can be conjugated with other amino acids through peptide bonds. Glucosamine derived aspartic acid (commercially available from NovaBiochem) is one such example:

Preferred indications

Molecular weight agent imaging is appropriate, up to 8000 daltons. Preferably, the molecular weight is in the range from 2800 to 6000 daltons, most preferably from 3000 to 4500 daltons, particularly preferably from 3200 to 4000 daltons.

The preferred imaging agents of the present invention have both peptide end-protected groups MIGi.e. preferably both Z1and Z2represent MIGthat will, as a rule, different. As indicated above, one of the Z1/Z2could possibly be a -(L)n[IM]. The presence of both a substituted peptide ends in this case, it is important for applications of visualization in vivo, as otherwise expected a fast metabolism with subsequent loss of affinity selective binding in respect of cMet. When both Z1and Z2represent MIGpreferably Z1represents acetyl, and Z2is a primary amide. Most preferably, Z1represents acetyl, and Z2is a primary amide, and a group -(L)n[IM] attached to the ε-amino group of the side chain of the lysine residue in SPSR.

Preferred smbr-peptides of the present invention have KDin respect of which wyzwania cMet complex cMet/HGF less than approximately 10 nm (based on the analysis of measurements of the polarization of fluorescence), most preferably in the range from 1 to 5 nm, ideally less than 3 nm,

The peptide sequence (SEQ-1):

Cysa-X1-Cysc-X2-Gly-Pro-Pro-X3-Phe-Glu-Cysd-Trp-Cysb-Tyr-X4-X5-X6(SEQ-1)

smvr formula I is a sequence of 17-dimensional peptide, responsible mainly for the selective binding to cMet. When smbr-peptide of the present invention contains more than 17 amino acid residues, the remaining amino acids can be any amino acid except cysteine. Additionally, unprotected cysteine residues can cause unwanted scrambled specific disulfide bridges Cysa-Cysband Cysc-Cysd. Additional peptides preferably contain at least one amino acid residue with a side chain suitable for easy conjugation group -(L)n[IM]. Suitable such residues include residues Asp or Glu for conjugation with the group -(L)n[IM], functionalized with amino groups, or a Lys residue for conjugation with the group -(L)n[IM], functionalized by carboxyglutamic or active complex ether. Amino acid residues for conjugation -(L)n[IM] appropriately localized away from the 17-dimensional binding site smbr-peptide (SEQ-1) and the pre is respectfully localized at the C - or N-end. Preferably, the amino acid residue for conjugation is a Lys residue.

Substitution of the tryptophan residue SEQ-1 was evaluated with the known amino acid substituents phenylalanine and naphthylamines. However, it was discovered the loss of affinity in respect of cMet, confirming the fact that the tryptophan residue is important for activity.

Preferably, when smbr-peptide further comprises a N-terminal serine residue with getting 18-Mer (SEQ-2):

Ser-Cysa-X1-Cysc-X2-Gly-Pro-Pro-X3-Phe-Glu-Cysd-Trp-Cysb-Tyr-X4-X5-X6(SEQ-2)

In addition to the SEQ-1 or preferably SEQ-2, smvr most preferably additionally contains one of the following:

(1) the residue is Asp or Glu within 4 amino acid residues of the peptide C - or N-Terminus smbr-peptide, and -(L)nIM functionalized amino group which is conjugated with carboxyla side chain of the specified residue is Asp or Glu obtaining amide linkages;

(2) a Lys residue within 4 amino acid residues of the peptide C - or N-Terminus smbr-peptide, and -(L)nIM functionalized carboxyl group which is conjugated to the ε-amino group of the side chain of the specified residue Lys obtaining amide bond.

Preferred smbr-peptides contain a 22-dimensional amino acid sequence (SEQ-3):

Ala-Gly-Ser-Cysa-X 1-Cysc-X2-Gly-Pro-Pro-X3-Phe-Glu-Cysd-Trp-Cysb-Tyr-X4-X5-X6-Gly-Thr (SEQ-3)

smbr-peptides of the present invention preferably have X3representing Arg.

smbr-peptide preferably further comprises, in addition to the SEQ-1, SEQ-2 or SEQ-3, any N - or C-end of the linker peptide selected from:

-Gly-Gly-Gly-Lys- (SEQ-4), -Gly-Ser-Gly-Lys- (SEQ-5) or

-Gly-Ser-Gly-Ser-Lys- (SEQ-6).

The Lys residue of the linker peptide is the most preferred point localization for conjugation group -(L)n[IM]. Especially preferred smbr-peptides containing SEQ-3, together with the linker peptide SEQ-4, giving 26-dimensional amino acid sequence (SEQ-7):

Ala-Gly-Ser-Cysa-Tyr-Cysc-Ser-Gly-Pro-Pro-Arg-Phe-Glu-Cysd-Trp-Cysb-Tyr-Glu-Thr-Glu-Gly-Thr-Gly-Gly-Gly-Lys (SEQ-7)

smbr-peptides with SEQ-1, SEQ-2, SEQ-3 and SEQ-7 preferably have Z1and Z2both represent MIGand, most preferably, have Z1that represents acetyl, and Z2representing a primary amide.

Group -(L)n[IM] suitably attached to one of the groups Z1or Z2or aminokislotnogo residue smbr-peptide, which is different from the cMet-binding sequence SEQ-1. Preferred amino acid residues and sites of conjugation are as described above, When the group -(L) n[IM] attached to the Z1or Z2it can take the place of the Z1or Z2during conjugation at the N - or C-end and block the metabolism in vivo in this way.

A preferred group IM have spread the delocalized electron system, for example, cyanine, merocianine dyes, indocyanines, phthalocyanines, naphthalocyanines, triphenylmethane, porphyrins, prelievi dyes, thiapyrilium dyes, squarewave dyes, croconaw dyes, asplenietea dyes, ingoonline, benzobisoxazole dyes, benzothiadiazines dyes, anthraquinones, naftochinona, endocrine, phthaloylation, triperoxonane, azo dyes, dyes with intramolecular and intermolecular charge-transfer complexes and dyes, trophy, tetrazine, bis(dithiolene)new complexes, bis(benzene-dithiolate)ing complexes, iodoaniline dyes, bis(S,O-dithiolan)type complexes. Fluorescent proteins such as green fluorescent protein (GFP) and modification of GFP, which have different properties absorption/emission, are also useful. Some rare earth metals such as europium, samarium, terbium or dysprosium) are used in some contexts, because they are fluorescent nanocrystals (quantum dots).

Specific examples of chromophores that can be COI is used, include fluorescein, sulforhodamine 101 (Texas red), rhodamine B, rhodamine 6G, rhodamine 19, indocyanine green, 2nd SUV, So, So, So, So, So, Marina Blue, Pacific Blue, Oregon Green 488, Oregon Green 514, tetramethylrhodamine, Alexa Fluor 350, Alexa Fluor 430, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 555, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor 700 and Alexa Fluor 750. Cyanine dyes are particularly preferred. Licha et al. lead the review of dyes and conjugates with dyes for optical imaging in vivo [Topics Curr. Chem., 222, 1-29 (2002); Adv.Drug Deliv.Rev., 57, 1087-1108 (2005)].

Preferred cyanine dyes, fluorophores which are represented by formula II:

where each X' is independently selected from: -C(CH3)2, -S-, -O - or-C[(CH2)andCH3][(CH2)bM], where a is an integer from 0 to 5, b is an integer from 1 to 5, and M represents a group selected from G or SO3M1or N;

each Y' independently represents 1-4 groups selected from the group consisting of:

H, -CH2NH2, -SO3M1, -CH2The SOOMA1, -NCS, and F, and where these groups Y' are located on any position of the aromatic ring;

Q' is independently selected from the group consisting of: H, SO3M1, NH2, COOM1, ammonium, ester groups, benzyl and group (G;

M1the present is the focus of a N or B c;

I is an integer from 1 to 3;

and m is an integer from 1 to 5;

where at least one of X', Y' and Q' includes a group G;

G represents a reactive or functional group suitable for attaching to smvr-peptide.

Group G interacts with a complementary group smbr-peptide, forming a covalent bond between the fluorophore cyanine dye and smbr-peptide. G may be a reactive group that can interact with a complementary functional group of the peptide or, alternatively, may include a functional group which can interact with the reactive group smbr-peptide. Examples of reactive functional groups include active esters; isothiocyanates; maleimide; halogenoacetyl; gelegenheid; hydrazide; vinylsulfonic; dihlotiazid; phosphoramidite; hydroxyl; amino; sulfhydryl; carbonyl; carboxylic acid and thiophosphate. Preferably, G represents an active ester.

The term "activated ester" or "active ester" means an ester derivative of the associated carboxylic acid, which is a better leaving group and therefore provides an easier interaction with the nucleophile, such as amines. Examples of suitable activehistory esters are: N-hydroxysuccinimide (NHS), sulfo-Succinimidyl ether, pentafluorophenol, pentafluorothiophenol, para-NITROPHENOL, hydroxybenzotriazole and Rumor (i.e. hexaphosphate benzotriazol-1-yl-oxtriphylline). Preferred active esters are N-hydroxysuccinimide or pentafluorophenol esters, especially N-hydroxysuccinimide esters.

In a preferred embodiment of formula II:

each X' is selected from the group- (CH3)2- and- (CH3)[(CH2)4M]-,

where M is a group G or-SO3M1;

each Y' is a SO3M1N or 1-4 F atoms;

each Q' is selected from a group G and SO3M1;

I preferably equal to 2, and m is preferably equal to 3, 4 or 5;

where, when one of X' or Q' represents a group G, it is most preferably represents Succinimidyl ester.

Especially preferred cyanine dyes represented by formula III:

where R1and R2independently represent H or SO3M1and at least one of R1and R2represents the SO3M1where M1represents N or Bc;

R3and R4independently represent a1-4alkyl or C1-6carboxyethyl;

R5, R 6, R7and R8independently represent groups of Ra;

where Rarepresents a C1-4alkyl, C1-6carboxylic or -(CH2)kSO3M1where k is an integer 3 or 4;

provided that the cyanine dye has a total of 1-4 Deputy SO3M1in groups of R1, R2and Ra.

Preferred dyes of the formula III is selected so that was attended by at least one1-6carboxialkilnuyu group, to facilitate conjugation with cmvr.

Preferred individual dyes of the formula III are listed in Table 1.

Table 1
The chemical structure of the individual cyanine dyes
Table 1The title dye
Su(1)Su(2)Su**Alexa647
R1NSO3HSO3HSO3H
R2 SO3HSO3HSO3HSO3H
R3CH3CH3CH3Rf
R4CH3CH3CH3CH3
R5CH3CH3CH3CH3
R6CH3CH3-(CH2)4SO3HCH3
R7RfRfRf-(CH2)3SO3H
R8CH3Et-(CH2)4SO3H-(CH2)3SO 3H

where Rfrepresents -(CH2)5COOH.

Especially preferred dyes of formula II are So** and Alexa647, and A** is the most preferred.

When there is a synthetic linker group (L), it preferably contains a terminal functional groups that facilitate conjugation with [IM] and Z1-[cMBP]-Z2. When L contains a peptide chain of 1-10 amino acid residues, these amino acid residues preferably selected from glycine, lysine, arginine, aspartic acid, glutamic acid or serine. When L contains grouping PEG, it preferably contains a structural unit obtained by oligomerization of monodisperse PEG-like structures of formula IA or IB:

17-amino-5-oxo-6-Aza-3,9,12,15-tetrachloroplatinate acid of formula IA, where p is an integer from 1 to 10. Alternatively, it may be used PEG-like structure on the basis of the propionic acid derivative of the formula IB:

where p is as defined for formula IA, and q is an integer from 3 to 15.

In formula IB, preferably p is 1 or 2, and q is preferably equal to the number of from 5 to 12.

When the linker group does not contain a PEG or peptide chain, the preferred groups L are on the main chain of the United atoms, forming a group -(A)m- from 2 to 10 atoms, most preferably from 2 to 5 atoms, particularly preferably from 2 or 3 atoms. Minimum linker group with the main chain of 2 atoms gives the advantage that rendered the group well separated, so that any undesirable interaction is minimized.

In formula I, n is preferably 0 or 1, most preferably 0, i.e. there is no linker groups.

The preferred imaging agents of the present invention are represented by formula IV:

where the group (L)n[IM] attached to the ε-amino group of the Lys residue. The preferred imaging agents of formula IV have MIG(N-terminal Ala), which represents acetyl, and MIG(C-terminal Lys), which is a primary amide. In formula IV, n is preferably equal to zero, and IM is preferably a cyanine dye, most preferably cyanine dye of formula II, Particularly preferred imaging agents of formula IV have IM representing A** or Alexa647, most preferably Cu**.

Peptides of the formula Z1-[cMBP]-Z2according to the present invention can be obtained by the production method, including:

(1) solid-phase peptide synthesis of the linear peptide, which has takauji peptide sequence as desired smbr-peptide, and in which Cysaand Cysbnot protected, and Cys residuescand Cysdhave a protective group for thiol;

(2) processing of the peptide from step (1) water base solution with obtaining monocyclic peptide from the first by a disulfide bond linking Cysaand Cysb;

(3) remove with Cyscand Cysdprotective groups for thiol and cyclization with getting the second disulfide bond connecting Cyscand Cysd, to give the desired bicyclic peptide product Z1-[cMBP]-Z2.

The term "protective group" means a group which inhibits or suppresses undesired chemical reactions, but which is designed to be sufficiently reactive so that it can be removed with interesting functional groups in a fairly mild conditions that do not change the rest of the molecule. After deciphering get the desired product. Protective groups for amino is well known to specialists in this field of technology, and they are appropriately selected from: Boc (where Boc is a tert-butoxycarbonyl), Fmoc (where Fmoc is fluorenylmethoxycarbonyl), TRIFLUOROACETYL, allyloxycarbonyl, Dde [1-(4,4-dimethyl-2,6-dioxocyclohex)-ethyl] or Npys (i.e. 3-nitro-2-pyridylsulfonyl). Suitable protective groups for tio is and represent Trt (trityl), ACM (atsetamidometil), t-Bu (tert-butyl), tert-butylthio, methoxybenzyl, methylbenzyl or Npys (3-nitro-2-pyridylsulfonyl). The use of other protective groups described in "Protective Groups in Organic Synthesis", Theorodora W. Greene and Peter G.M.Wuts, (John Wiley & Sons, 1991). Preferred protective groups for amino represent the BOC and Fmoc, most preferably Boc. Preferred protective groups for amino represent Trt and Acm.

In Examples 1 and 2 proposed more specific description. For more details, solid-phase peptide synthesis are described in P. Lloyd-Williams, F. Albericio and E. Girald; Chemical Approaches to the Synthesis of Peptides and Proteins, CRC Press, 1997. smbr-peptides are best kept in an atmosphere of inert gas and kept in the freezer. When used in solution, it is better to avoid pH values above 7, as this increases the risk of scrambling of disulfide bridges.

The imaging agents can be obtained as described in the third aspect (below).

In the second aspect of the present invention proposed a pharmaceutical composition comprising the agent visualization according to the first aspect together with a biocompatible carrier in a form suitable for administration to a mammal.

"Biocompatible carrier" is a fluid, in particular liquid, in which the agent visualization can be suspended or dissolved, so that the composition was the physiologically tolerable, that is, could be introduced into the body of a mammal without toxicity or undue discomfort. Biocompatible carrier is an appropriate injectable carrier liquid such as sterile pyrogen-free water for injection; an aqueous solution, such as saline (which may be advantageously adjusted so that the final product for injection was isotonic); an aqueous solution of one or more than one substance, regulatory toychest (for example, salts of plasma cations with biocompatible counterions), sugars (for example glucose or sucrose), sugar alcohols (for example sorbitol or mannitol), glycols (for example glycerol) or other non-ionic polyol (e.g. polyethylene glycols, propylene glycols and the like). Preferably, the biocompatible carrier is a pyrogen-free water for injection or isotonic saline solution.

Agents visualization and biocompatible carrier, each offered in suitable vials or vessels, comprising a sealed container, which provides for the maintenance of sterile integrity and/or radioactive safety, and possibly inert gas in the free space above the product (e.g., nitrogen or argon), at the same time providing the ability to add and retrieve restaurantprices or cannula. Preferred such container is a sealed airtight membrane of the vial, where the gas-tight cover is pressed additional capping agent (usually aluminum). The specified cover is suitable for piercing the syringe or multiple puncture needle for subcutaneous injection (for example, a sealed cover with the pressed membrane), at the same time maintaining sterile integrity. Such containers have the added advantage that the cover can withstand the vacuum if necessary (for example, to change the gas in the free space above the product or degassing of solutions) and to withstand changes in pressure, such as pressure drop, preventing thus the penetration of external atmospheric gases, such as oxygen or water vapor.

Preferred mnogorazovye containers include a vial with a single content (for example, a volume of from 10 to 30 cm3), which contains many doses to the patient, and a single dose to the patient can thus be extracted into the syringe for clinical use in different time intervals within an acceptable shelf-life of the drug in accordance with this clinical situation. Pre-filled syringes designed to house a single dose for a person, or standard doses of the Sabbath." and, thus, preferably represent a disposable syringes or other, suitable for clinical use. The pharmaceutical compositions of the present invention preferably have a dosage that is appropriate for the individual patient, and proposed a suitable syringe or container, as described above.

The pharmaceutical composition may contain additional excipients, such as an antimicrobial preservative, pH-adjusting agent, a filler, a stabilizer or an agent of regulating osmollnosti. The term "antimicrobial preservative" means an agent which inhibits the growth of potentially harmful microorganisms, such as bacteria, yeast or fungi. Antimicrobial preservative may also be some bactericidal properties, depending on the dosage. The primary role of the antimicrobial(s) preservative(s) of the present invention is to inhibit the growth of any such microorganism in the pharmaceutical composition. However, antimicrobial preservative may also may be used to inhibit the growth of potentially harmful microorganisms in one or more than one component sets used for the preparation of specified composition prior to introduction. Suitable antimicrobial preservatives is clucalc: parabens, that is, methyl-, ethyl-, propyl - or butylparaben, or mixtures thereof; benzyl alcohol; phenol; cresol; cetrimide and thiomersal. Preferred antimicrobial preservatives are parabens.

The term "pH-regulating agent" means a compound or mixture of compounds used to ensure that the pH value of the composition was within acceptable limits (approximately pH from 4.0 to 10.5) for the introduction of a human or mammal. Suitable such pH-adjusting agents include pharmaceutically acceptable buffers, such as trichinosis, phosphate or TRIS [i.e. Tris(hydroxymethyl)-aminomethane], and pharmaceutically acceptable bases, such as sodium carbonate, sodium bicarbonate or mixtures thereof. When the composition is used in the form of a kit, pH-regulating agent may possibly be offered in a separate vial or container, so that the user of this set could regulate the pH value as part of multistage procedure.

The term "filler" means a pharmaceutically acceptable filling agent that can facilitate the handling of the material during production and lyophilization. Suitable fillers include inorganic salts such as sodium chloride, and water-soluble sugar or sugar alcohols such as sucrose, maltose, mannitol or trehalose.

Farmaceuticas is their composition according to the second aspect can be manufactured in aseptic production conditions (for example, sterile room) to give the desired sterile, pyrogen-free product. Preferably, when the key components, especially the associated reagents, as well as those pieces of equipment that come in contact with the agent visualization (e.g., vials)were sterile. Components and reagents can be sterilized by methods known in the art, including: sterile filtration, terminal sterilisation using e.g. gamma-irradiation, autoclaving, dry heat or chemical treatment (e.g. with ethylene oxide). It is preferable to sterilize some of the components in advance, so that it was required to perform a minimum number of manipulations. However, as a precaution, it is preferable to include at least the stage of sterile filtration as the final stage in the manufacture of pharmaceutical compositions.

The pharmaceutical composition according to the second aspect may possibly be made from set, as described in the fourth aspect below.

In the third aspect of the present invention, a method for obtaining agent visualization according to the first aspect, including one of the stages(1)-(4):

(1) the interaction smbr-peptide formula Z1-[cMBP]-Z2where Z1represents H, and Z2is a MIGwith a compound of formula Y 1-(L)n-[IM]the receiving agent visualization of the formula I, where [IM] anywhereman position Z1;

(2) the interaction smbr-peptide formula Z1-[cMBP]-Z2where Z1and Z2both are MIGand SPSR contains the residue is Asp or Glu within 4 amino acid residues from the C - or N-Terminus smbr-peptide, and all other residues Asp/Glu smbr-peptide is protected, with a compound of formula Y2-(L)n-[IM]the receiving agent visualization of the formula I, where [IM] anywhereman at the specified residue is Asp or Glu this smbr-peptide;

(3) the interaction smbr-peptide formula Z1-[cMBP]-Z3where Z1is a MIGand Z3represents a group Z2or activated ester, and all other residues Asp/Glu smbr-peptide is protected, with a compound of formula Y2-(L)n-[IM]the receiving agent visualization of the formula I, where [IM] anywhereman position Z2;

(4) the interaction smbr-peptide formula Z1-[cMBP]-Z2where Z1and Z2both are MIGand SPSR contains Lys within 4 amino acid residues from the C - or N-Terminus smbr-peptide, with the compound of the formula Y1-(L)n-[IM]the receiving agent visualization of the formula I, where [IM] anywhereman on residue Lys of this smbr-peptide;

where Z1, smvr, Z2MIG, L, n and IM are the one whom they as defined in the first aspect (above), and

Z3represents a group Z2or activated esters;

Y1is a carboxylic acid, activated ester, isothiocyanate or thiocyanato group;

Y2represents an amino group.

The term "activated ester" or "active ester" and their preferred embodiments are as described above. Y2preferably represents a primary or secondary amino group, most preferably a primary amino group.

Connection Z1-[cMBP]-Z2preferably has both Z1and Z2representing MIG. Preferred smbr-peptides and Z1/Z2are as described in the first aspect. In particular, preferably when smbr-peptide contains the residue is Asp, Glu or Lys to facilitate conjugation, as described for the preferred smbr-peptides according to the first aspect. Particularly preferably, when smbr-peptide contains a Lys residue, as described in stage (4).

Obtain Z1-[cMBP]-Z2described in the first embodiment (above). The peptide Z1-[cMBP]-Z3where Z3represents an active ester, can be obtained from the Z1-[cMBP]-Z2where Z2is a HE or a biocompatible cation (Bc), conventional methods.

Optical reporter dyes (IM), functionalityand appropriate for conjugation with peptides, commercially available from GE Healthcare Limited, Atto-Tec, Dyomics, Molecular Probes, and others. Most of these dyes are available as NHS esters.

Methods of conjugation of suitable optical reporters (IM), in particular dyes, amino acids and peptides described Licha (see above)and Flanagan et al. [Bioconj. Chem., 8, 751-756 (1997)]; Lin et al, [ibid, 13, 605-610 (2002)] and Zaheer [Mol. Imaging, 1(4), 354-364 (2002)]. In the methods of conjugation of the linker group (L) with cmvr-peptide used chemical technology, similar to those involving the dyes separately (see above), and they are known in this technical field.

In the fourth aspect of the present invention proposed a kit for the preparation of pharmaceutical compositions according to the second aspect contains the agent visualization according to the first aspect of sterile solid form, so that after dilution with sterile additive biocompatible carrier according to the second aspect of the ongoing dissolution gives the desired pharmaceutical composition.

In this respect, the agent visualization and other possible excipients, as described above, may be offered in the form of lyophilized powder in a suitable vial or container. In this case, the agent is suitable for cultivation as Emim biocompatible carrier with obtaining a pharmaceutical composition in a sterile pyrogen-free form, ready for administration to a mammal.

The preferred sterile solid form of the agent visualization is a liofilizirovannoe solid. Sterile solid form preferably proposed container, pharmaceutical grade, as described for the pharmaceutical composition (above). When the set is lyophilized, the drug may may contain cryoprotector selected from a saccharide, preferably mannitol, maltose or trizina.

In the fifth aspect of the present invention, a method of optical imaging of the body of a mammal in vivo, including the use of agent visualization according to the first aspect or the pharmaceutical composition according to the second aspect of obtaining images of sites overexpression or localization of cMet in vivo.

The term "optical imaging" refers to any way in which get the image for detection, staging of disease or diagnosis of disease, monitoring disease progression or to monitor the treatment of the disease, based on the interaction with light in the spectral range from red to near infrared (wavelength 600-1200 nm). Optical visualization advanced includes all the ways from direct visualization without the use of any device, and including the use of devices such as decomp is cnie videoslittle devices, catheters and equipment for optical imaging applications, such as automated equipment for the tomographic images. Means and methods of measurements include: fluorescent imaging; endoscopy; fluorescence endoscopy; optical coherence tomography; visualization of the transmittance; adjustable time visualization of the transmittance; confocal imaging; nonlinear microscopy; the photoacoustic imaging; acousto-optical visualization; spectroscopy; scattering spectroscopy; interferometry; coherence interferometry; diffuse optical tomography and indirect fluorescence diffuse optical tomography (system operating in a continuous mode, time and frequency system), and measurement of light scattering, absorption, polarization, luminescence, lifetime fluorescence, quantum yield and quenching of fluorescence, but not limited to. Additional details of these techniques are proposed: (Tuan Vo-Dinh (editor): "Biomedical Photonics Handbook" (2003), CRC Press LCC; Mycek & Pogue (editors): "Handbook of Biomedical Fluorescence" (2003), Marcel Dekker, Inc.; Splinter & Hopper: "An Introduction to Biomedical Optics" (2007), CRC Press LCC.

Region of the spectrum from green to near-infrared preferably represented by the wavelengths of 600-1000 nm. The method of optical imaging is preferable, not only is em a fluorescence endoscopy. The body of a mammal according to the fifth aspect preferably is a human body. The preferred embodiment of the agent visualization are as described for the first aspect (above). In particular, preferably, when using a fluorescent dye.

In the method according to the fifth aspect, the agent visualization or pharmaceutical composition preferably is pre-injected into a specified body of a mammal. The term "pre-enter" means that this stage involving a physician, which gives the agent visualization of the patient, for example, in the form of intravenous injection, has already been carried out prior to visualization. This embodiment includes the use of agent visualization according to the first embodiment for the production of a diagnostic agent for diagnostic imaging of painful conditions of the body of a mammal in vivo, involving cMet.

The preferred method for optical imaging according to the fifth aspect is a fluorescent reflective imaging (Fluorescence Reflectance Imaging (FRI). When FRI agent visualisation of the present invention is administered to a subject, the subject of the diagnosis, and then the surface tissue of the subject Shine exciting light, as a rule, with the initiation of continuous waves (CW). The light excites the reporter molecule (IM). is luorescence agent imaging which generates excitation light, detected using a fluorescence detector. The reflected light is preferably filtered to separate the fluorescent component (exclusively or partially). Based fluorescent glow form the image. As a rule, spend a minimal amount of processing (not using the processor to calculate the optical parameters, such as lifetime, quantum yield, and others), and this image shows the intensity of fluorescence. The agent visualization is intended to concentrate in the affected area, causing a higher fluorescence intensity. Thus, the painful region gives a positive contrast in the picture fluorescence intensity. The image is preferably obtained using a CCD camera or the chip, so that it is possible to visualize in real-time.

The wavelength of excitation varies depending on the type of dye. The equipment for generating the exciting light can be a traditional source of exciting light such as laser (for example ion laser, laser dyes or semiconductor laser); source halogen light or xenon light source. Various optical filters may be used to obtain optimum is Lina wave excitation.

The preferred method FRI comprises the following stages:

(1) the surface of the tissue of interest in the body of a mammal Shine exciting light;

(2) using a fluorescence detector that detects fluorescence from the agent visualization generated by excitation rendered grouping (IM);

(3) the light detected by the fluorescence detector, possibly filtered to separate the fluorescent component;

(4) fluorescent glow in the stages (2) or (3) form the image of the specified surface tissue of interest.

At the stage (1) stimulating light preferably is a continuous wave (CW) by nature. At stage (3) of the detected light is preferably filtered. Especially preferred method of FRI is a fluorescence endoscopy.

In an alternative visualization method according to the fifth aspect of the use of the migration of photons in the frequency domain (frequency-domain photon migration, FDPM). It has advantages over methods that use continuous wave (CW), where an important deeper level detection IM in the tissue [Sevick-Muraca et al., Curr. Opin. Chem. Biol., 6, 642-650 (2002)]. For such a frequency/time visualization is preferred if the IM has fluorescent properties that can be modulated depending on the depth of tissue damage being rendered, and ti is and the equipment used.

The way FDPM is as follows:

(a) light-scattering biological tissue of a specified body of a mammal having a heterogeneous composition is exposed to light from a light source with a predetermined variable time-intensity for excitation agent visualization, in which see the multiple scattering of the exciting light in a specified tissue;

(b) repeatedly detects scattered light from the tissue in response to the specified effect;

(C) conduct quantitative determination parameter fluorescence throughout the tissue on the basis of this radiation by establishing a series of indicators using a processor, where each of the indicators corresponds to the level parameter fluorescence in various positions within this tissue, where the level parameter fluorescence varies according to the heterogeneous composition of this fabric; and

(g) generate the image of this tissue by imaging heterogeneous tissue composition in accordance with the performance stage (b).

Parameter fluorescence stage (C) preferably corresponds to the capture agent visualization and preferably additionally includes a comparison of some quantitative indicators in accordance with the coefficients of absorption and scattering in the tissue to put in place the surveillance agent imaging. Parameter fluorescence stage (C) preferably corresponds to at least one of the following: life time fluorescence quantum yield of fluorescence, fluorescence yield and the capture agent imaging. Parameter fluorescence preferably not depends on the intensity of emission and does not depend on the concentration of the agent visualization.

Quantitative determination of stage (C) preferably includes: (1) establishing performance indicator, (2) determining the calculated emission as a function of this performance indicator, (3) comparison of the calculated emission with the emission at the specified detection for error detection, (4) offer a modified estimate of the fluorescence as a function of the error. Quantitative determination preferably includes the definition of indicators based on mathematical relationships that model the behavior of multiple scattering of light in this tissue. The method according to the first embodiment preferably includes monitoring the metabolic properties of tissue in vivo by detecting the variation of the specified parameter fluorescence.

Optical visualization of the fifth aspect is preferably used to facilitate the tactics of patients with colorectal cancer (CRC). The term "clinical management of patients with CRC" means the use of de is the design, determining the stage of the disease, diagnosis, monitoring of disease progression or treatment monitoring. Additional details of suitable optical imaging is considered Sevick-Muraca et al. [Curr. Opin. Chem. Biol., 6, 642-650 (2002)].

In the sixth aspect of the present invention, a method for detecting, for determining the stage of the disease, diagnosis of disease, monitoring disease progression or monitoring the treatment of colorectal cancer (CRC) is the body of a mammal, including a method of optical imaging in vivo according to the fifth aspect.

The invention is illustrated non-limiting Examples are described in detail below. In Example 1, the proposed synthesis smbr-peptide according to the invention (Compound 1). In example 2, the proposed synthesis related peptide as a negative control, in which the peptide sequence of Connection 1 is subjected to scrambling. In example 3, the proposed synthesis of cyanine dye So**preferred dyes according to the invention. In Example 4, the proposed active synthesis of ester A**. In Example 5, the proposed pairing of cyanine dyes according to the invention with peptides (smbr-peptide and control). Compounds 3-7 were compared in this way. In Example 6, a method for determining the affinity of the peptides against cMet in vitro. Result is you show, the binding is selective, even when attached optical reporter rendered grouping (cyanine dye). In Example 7 proposed data on in vivo testing of Compounds 5 and 7 on animal models of cancer. Shows excellent correlation of tumor:background for Compounds 5, while for Compound 7 (negative control) revealed no differences between tumor and background.

Reduction

Used commonly accepted single-letter or 3-letter abbreviations of amino acids.

AFM:atsetamidometil
ACN (or MeCN):acetonitrile
Boc:tert-butyloxycarbonyl
DCM:dichloromethane
DMF:dimethylformamide
DMSO:the sulfoxide
Fmoc:9-fluorenylmethoxycarbonyl
HBTU:hexaphosphate O-benzotriazol-1-yl-N,N,N',N'-tetramethylurea
HPLC:highly effective is liquid chromatography
HSPyUhexaphosphate O-(N-Succinimidyl)-N,N,N',N'-tetramethylurea
NHS:N-hydroxy-succinimide
NMM:N-methylmorpholin
NMP:1-methyl-2-pyrrolidinone
Pbf:2,2,4,6,7-pentamethylcyclopentadiene-5-sulfonyl
PBS:buffered phosphate saline
tBu:tert-butyl
TFA:triperoxonane acid
TIS:triisopropylsilane
Trt:trail

Table 2
The structure of the compounds according to the invention
No.The structure of connections
1AC-AGSCYCSGPPRFECWCYETEGTGGGK-NH2
2AC-TGECTCPYWEFRPCECGSSGAGGGK-NH 2(negative control)
3
Ac-AGSCYCSGPPRFECWCYETEGTGGGK(ε-Cy5)-NH2
4
(negative control)

5
Ac-AGSCYCSGPPRFECWCYETEGTGGGK(ε-Cy5**)-NH2
6Ac-AGSCYCSGPPRFECWCYETEGTGGGK(ε-Alexa647)-NH2
7Ac-TGECTCPYWEFRPCECGSYSGAGGGK(ε-Cy5**)-NH2(negative control)

Example 1: Synthesis of Compounds 1

Stage (a): synthesis of the protected linear peptide predecessor

The linear peptide, the precursor has the structure:

Ac-Ala-Gly-Ser-Cys-Tyr-Cys(Acm)-Ser-Gly-Pro-Pro-Arg-Phe-Glu-Cys(Acm)-Trp-Cys-Tyr-Glu-Thr-Glu-Gly-Thr-Gly-GIy-Gly-Lys-NH2

The peptidyl-resin H-Ala-Gly-Ser(tBu)-Cys(Trt)-Tyr(tBu)-Cys(Acm)-Ser(tBu)-Gly-Pro-Pro-Arg(Pbf)-Phe-Glu(OtBu)-Cys(Acm)-Trp(Boc)-Cys(Trt)-Tyr(tBu)-Glu(OtBu)-Thr(ψMe,Mepro)-Glu(OtBu)-Gly-Thr(tBu)-Gly-Gly-Gly-Lys(Boc)-polymer were synthesized on a peptide synthesizer, Applied Biosystems 433A using Fmoc-chemistry, from 0.1 IMO the ü resin Rink Amide Novagel. In further stages of the combination caused an excess of 1 mmol of pre-activated amino acids (using HBTU). In this sequence was built Glu-Thr-pseudoproline (Novabiochem 05-20-1122). The resin was transferred into a nitrogen purged apparatus and treated with a solution of acetic anhydride (1 mmol) and NMM (1 mmol)dissolved in DCM (5 ml) for 60 minutes. A solution of the anhydride was removed by filtration and the resin washed with DCM and dried in a stream of nitrogen.

Simultaneous removal of the protective groups of the side chains and cleavage of the peptide from the resin was carried out in TFA (10 ml)containing 2.5% TIS, 2.5% of 4-thiocresol and 2.5% water for 2 hours and 30 minutes. The resin was removed by filtration, TFA was removed in vacuo and to the residue was added diethyl ether. Formed precipitate was washed with diethyl ether and dried in air to obtain 264 mg of the crude peptide.

Purification by preparative HPLC (gradient: 20-30% B over 40 min where a is an H2O/0.1% of TFA, and represents ACN/0.1% of TFA, flow rate: 10 ml/min, column: Phenomenex Luna 5µ C18 (2) 250×21,20 mm, detection: UV 214 nm, retention time of product: 30 min) of the crude peptide gave 100 mg of pure linear precursor Compound 1. The pure product was analyzed by analytical HPLC (gradient: 10-40% B over 10 min, where a is an H2O/0.1% of TFA, and is particularly the ACN/0.1% of TFA, flow rate: 0.3 ml/min, column: Phenomenex Luna 3µ C18 (2) 50×2 mm, detection: UV 214 nm, retention time of product: 6,54 min). An additional feature of the product was performed using mass spectrometry with ionization by elektrorazpredelenie (calculated: 1464,6,found: 1465,1).

Stage (b): Education monocyclic Cys4-16 disulfide bridge

Cys4-16; Ac-Ala-Gly-Ser-Cys-Tyr-Cys(Acm)-Ser-Gly-Pro-Pro-Arg-Phe-Glu-Cys(Acm)-Trp-Cys-Tyr-Glu-Thr-Glu-Gly-Thr-Gly-Gly-Gly-Lys-NH2

The linear precursor from step (a) (100 mg) was dissolved in a mixture of 5% DMSO/water (200 ml) and the pH of this solution was brought to pH 6 using ammonia. The reaction mixture was stirred for 5 days. Then the pH of this solution was brought to pH 2 using TFA, and the largest part of the solvent was removed by evaporation in a vacuum. The remainder (40 ml) was injected portions in the column for preparative HPLC purification of the product.

Purification by preparative HPLC (gradient: 0% B for 10 min, then 0-40% B over 40 min where a is an H2O/0.1% of TFA, and represents ACN/0.1% of TFA, flow rate: 10 ml/min, column: Phenomenex Luna 5µ C18 (2) 250×21,20 mm, detection: UV 214 nm, retention time of product: 44 min) of the residue gave 72 mg of pure monocyclic precursor Compounds 1.

Pure product (as a mixture of isomers RR) were analyzed by analytical HPLC (gradient: 10-40% B over 10 min, where a is an H2O/0.1% of TFA, and represents TSA/0,1% TFA, flow rate: 0.3 ml/min, column: Phenomenex Luna 3µ C18 (2) 50×2 mm, detection: UV 214 nm, retention time of product: lower than the 5.37 min (P1); 5,61 min (P2); 6,05 min (P3)). An additional feature of the product was performed using mass spectrometry with ionization by elektrorazpredelenie (calculated: 1463,6,found: 1464,1 (P1); 1464,4 (P2); 1464,3 (P3)).

Stage: Formation of secondary Cys6-14 disulfide bridge (Connection 1)

Monocyclic precursor from step (b) (72 mg) was dissolved in a mixture of 75% Asón/water (72 ml) under a protective layer of nitrogen. Added 1 M HCl (7.2 ml) and 0.05 M I2in the Asón (4.8 ml) in that order and the mixture was stirred for 45 minutes was Added 1 M ascorbic acid (1 ml) to give colorless mixture. The largest part of the solvents evaporated in vacuum and the residue (18 ml) was diluted with a mixture of water/0.1% of TFA (4 ml) and the product was purified using preparative HPLC.

Purification by preparative HPLC (gradient: 0% B for 10 min, then 20-30% B over 40 min where a is an H2O/0.1% of TFA, and represents ACN/0.1% of TFA, flow rate: 10 ml/min, column: Phenomenex Luna 5µ C18 (2) 250×21,20 mm, detection: UV 214 nm, retention time of product: 43-53 min) of the residue gave 52 mg of pure Compound 1./p>

The pure product was analyzed by analytical HPLC (gradient: 10-40% B over 10 min, where a is an H2O/0.1% of TFA, and represents ACN/0.1% of TFA, flow rate: 0.3 ml/min, column: Phenomenex Luna 3µ C18 (2) 50×2 mm, detection: UV 214 nm, retention time of product: 6,54 min). Additional characterization was performed using mass spectrometry with ionization by elektrorazpredelenie (calculated: 1391,5,found: 1392,5).

Example 2: Synthesis of Compounds 2

Ac-Thr-Gly-Glu-Cys-Thr-Cys(Acm)-Pro-Tyr-Trp-Glu-Phe-Arg-Pro-Cys(Acm)-Glu-Cys-Gly-Ser-Tyr-Ser-Gly-Ala-Gly-Gly-Gly-Lys-NH2

Compound 2 represents the negative control, where the peptide sequence of Compound 1 was subjected to scrambling.

Stage (a): synthesis of the protected linear peptide predecessor

The peptidyl-resin H-Thr(tBu)-Gly-Glu(OtBu)-Cys(Trt)-Thr(tBu)-Cys(Acm)-Pro-Tyr(tBu)-Trp(Boc)-Glu(OtBu)-Phe-Arg(Pbf)-Pro-Cys(Acm)-Glu(OtBu)-Cys(Trt)-Gly-Ser(tBu)-Tyr(tBu)-Ser(ψMe,Mepro-Gly-Ala-Gly-Gly-Gly-Lys(Boc)-polymer were synthesized on a peptide synthesizer, Applied Biosystems 433A using Fmoc-chemistry, starting with 0.1 mmol of resin Rink Amide Novagel. On stage combination inflicted excess of 1 mmol of pre-activated amino acids (using HBTU). In this sequence was built Tyr-Ser-pseudoproline (Novabiochem 05-20-1014). The resin was transferred into a nitrogen purged apparatus and processed the Ali solution of acetic anhydride (1 mmol) and NMM (1 mmol), dissolved in DCM (5 ml) for 60 minutes. A solution of the anhydride was removed by filtration and the resin washed with DCM and dried in a stream of nitrogen.

Simultaneous removal of the protective groups of the side chains and cleavage of the peptide from the resin was carried out in TFA (10 ml)containing 2.5% TIS, 2.5% of 4-thiocresol and 2.5% water for 2 hours and 10 minutes. The resin was removed by filtration, TFA was removed in vacuo and to the residue was added diethyl ether. Formed precipitate was washed with diethyl ether and dried in air to obtain 216 mg of the crude peptide.

Purification by preparative HPLC (gradient: 20-30% B over 40 min where a is an H2O/0.1% of TFA, and represents ACN/0.1% of TFA, flow rate: 50 ml/min, column: Phenomenex Luna 5µ C18 (2) 250×50 mm, detection: UV 214 nm, retention time of product: 34,1 min) of the crude peptide gave pure DX-1662 negative control linear precursor, dissolved in 200 ml of a mixture of ACN/water. The pure product was analyzed by analytical HPLC (gradient: 10-40% B over 5 min, where a is an H2O/0.1% of TFA, and represents ACN/0.1% of TFA, flow rate: 0.6 ml/min, column: Phenomenex Luna 3µ C18 (2) 20×2 mm, detection: UV 214 nm, retention time of product: 3,52 min). An additional feature of the product was performed using mass spectrometry with ionization by elektrorazpredelenie ( calculated: 1464,6,detected; 1464,9).

Stage (b): Education monocyclic Cvs4-16 disulfide bridge

Cys4-16; Ac-Thr-Gly-Glu-Cys-Thr-Cys(Acm)-Pro-Tyr-Trp-Glu-Phe-Arg-Pro-Cys(Acm)-Glu-Cys-Gly-Ser-Tyr-Ser-Gly-Ala-Gly-Gly-Gly-Lys-NH2

To a solution of the negative control linear precursor from step (a) (200 ml, see 4.3.1) was added DMSO (10 ml) and the pH of this solution was brought to pH 7 using ammonia. The reaction mixture was heated at 40°C for 18 hours, then at 60°C for 60 minutes, the pH Value of this solution was brought to pH 2 using TFA and ACN was removed by evaporation in a vacuum. This residue was subjected to purification preparative HPLC.

Purification by preparative HPLC (gradient: 0% B for 5 min, then 20-30% B over 60 min, where a is an H2O/0.1% of TFA, and is an ACM/0,1% TFA, flow rate: 50 ml/min, column: Phenomenex Luna 5µ C18 (2) 250×50 mm, detection: UV 214 nm, retention time of product: 29,6 min) of the residue gave pure negative control monocyclic predecessor in 100 ml of a mixture of ACN/water. The pure product was analyzed by analytical HPLC (gradient: 10-40% B over 5 min, where a is an H2O/0.1% of TFA, and represents ACN/0.1% of TFA, flow rate: 0.6 ml/min, column: Phenomenex Luna 3µ C18 (2) 20×2 mm, detection: UV 214 nm, retention time of product: of 3.46 min). An additional is the distribution of the characteristics was performed using mass spectrometry with ionization by elektrorazpredelenie ( calculated: 1463,6,found: 1463,7).

Stage: Formation of secondary Cys6-14 disulfide bridge (Compound 2)

Cys4-16, 6-14; Ac-Thr-Gly-Glu-Cys-Thr-Cys-Pro-Tyr-Trp-Glu-Phe-Arg-Pro-Cys-Glu-Cys-Gly-Ser-Tyr-Ser-Gly-Ala-Gly-Gly-Gly-Lys-NH2

The solution of the negative control monocyclic precursor from step (b) (100 ml) was diluted Asón (100 ml). Added 1 M HCl (5 ml) and 0.05 M I2in the Asón (7 ml) in that order under a protective layer of argon and the mixture was stirred for 20 minutes was Added 1 M ascorbic acid (1 ml) to give colorless mixture. The largest part of the solvents evaporated in vacuum and the residue (30 ml) was diluted with a mixture of water/0.1% of TFA (100 ml) and the product was purified using preparative HPLC.

Purification by preparative HPLC (gradient: 0% B for 10 min, then 20-30% B over 60 min, where a is an H2O/0.1% of TFA, and represents ACN/0.1% of TFA, flow rate: 50 ml/min, column: Phenomenex Luna 5µ C18 (2) 250×50 mm, detection: UV 214 nm, retention time of product: 32,8 min) of the residue gave 30 mg of pure Compound 2. The pure product was analyzed by analytical HPLC (gradient: 10-40% B over 10 min, where a is an H2O/0.1% of TFA, and represents ACN/0.1% of TFA, flow rate: 0.3 ml/min, column: Phenomenex Luna 3µ C18 (2) 50×2 mm, detection: UV 214 nm, retention time of product: 6,54 min). To anitelea characterization was performed using mass spectrometry with ionization by elektrorazpredelenie ( calculated: 1391,5,found: 1392,5).

Example 3: Synthesis of cyanine dye 2-{(1E,3E,5E)-5-[1-(5-carboxypentyl)-3,3-dimethyl-5-sulfo-1,3-dihydro-2H-indol-2-ilidene]-Penta-1,3-dienyl}-3-methyl-1,3-bis(4-sulfobutyl)-3H-indole-5-sulfonate (Cy5**)

(3A) 5-Methyl-6-oxapentane-1-Sultonova acid

To a suspension of sodium hydride (12.0 g, 60% NaH in mineral oil) in DMF (100 ml) was added dropwise ethyl-2-methylacetoacetate (50 g) in DMF (25 ml) under cooling in an ice bath for 1 hour (internal temperature 0-4°C). This mixture was left to warm to ambient temperature for 45 minutes under stirring, and then cooled. Then added dropwise over 15 minutes a solution of 1,4-butanesultone (45 g) in DMF (25 ml). The final mixture was heated at 60°C for 18 hours. The solvent was removed by rotary evaporation and the residue was distributed between water and diethyl ether. The aqueous layer was collected, washed with a fresh portion of diethyl ether and subjected to rotary evaporation to obtain an adhesive foam. This is an intermediate substance was dissolved in water (100 ml) was added sodium hydroxide (17.8 g) for 15 minutes under stirring. This mixture was heated at 90°C for 18 hours. The pH of the cooled reaction mixture was brought to ~pH 2 by adding concentrated the Oh hydrochloric acid (~40 ml). The solution was subjected to evaporation on a rotary evaporator and dried in vacuum. The yellow solid was washed with ethanol containing 2% hydrochloric acid (3×150 ml). The ethanol solution was filtered, evaporated on a rotary evaporator and dried in vacuum to obtain a yellow solid. Exit 70,

(3b) Dikalova salt of 2,3-dimethyl-3-(4-sulfobutyl)-3H-indole-5-sulfonic acid

4-Hydrazinobenzothiazole acid (40 g), 5-methyl-6-oxalate-1-sulfonic acid (3A, 60 g) and acetic acid (500 ml) were mixed and boiled under reflux for 6 hours. The solvent was filtered and evaporated on a rotary evaporator and dried in vacuum. The solid was dissolved in methanol (1 l). To it was added 2 M methanolic potassium hydroxide (300 ml). This mixture was stirred for 3 hours and then the volume of solvent was reduced by 50%using rotary evaporation. The precipitate was filtered off, washed with methanol and dried in vacuum. Exit 60, MS (LCMS): MH+362. ACC. Mass: Found 362,0729. MN+=C14H20NO6S2requires m/z 362,0732 (-0,8 million-1).

(3V) Dikalova salt of 2,3-dimethyl-1,3-bis(4-sulfobutyl)-3H-indole-5-sulfonate

2,3-Dimethyl-3-(4-sulfobutyl)-3H-indole-5-sulfonic acid (3b; 60 g) was heated with 1,4-butanesultone (180 g and tetramethylsilane (146 ml) at 140°C for 16 hours. The obtained red solid was washed with diethyl ether, was ground to a powder and dried in vacuum. Exit 60,

(3G) Su** as a TFA salt

K+-salt of 1-(5'-carboxypentyl)-2,3,3-trimethyl-indolinone-5-sulfonic acid (2.7 g), monohydrochloride bis(phenylimine)malonic aldehyde (960 mg), acetic anhydride (36 ml) and acetic acid (18 ml) was heated at 120°C for 1 hour to obtain a dark brown-red solution. This reaction mixture was cooled to ambient temperature. To this mixture was added 2,3-dimethyl-1,3-bis(4-sulfobutyl)-3H-indole-5-sulfonate (from 3V; 8,1 g) and potassium acetate (4.5 g) and was stirred for 18 hours at ambient temperature. Received blue solution was besieged by using ethyl acetate and dried in vacuum. The crude dye was purified by liquid chromatography (RPC18.gradient water + 0.1% of TFA/MeCN + 0.1% of TFA). Fractions containing the main peak of the dye was collected, combined and evaporated in vacuum to obtain specified in the header of dye 2, UV/Vis (water + 0.1% of F): 650 nm. MS (time-of-Flight mass spectrometry with ionization by laser desorption from the matrix (MALDI-TOF)): MH+ 887,1. MH+=C38H50N2O14S4requires m/z 887,1.

Example 4: Synthesis diisopropylethylamine salt of 2-[(1E,3E,5E)-5-(1-{6-[(2,5-dioxopiperidin-1-yl)oxy]-6-oxohexyl}-3,3-dimethyl-5-with whom life-1,3-dihydro-2H-indol-2-ilidene)Penta-1,3-dienyl]-3-methyl-1,3-bis(4-sulfobutyl)-3H-indole-5-sulfonate (NHS ester Su**)

S** (Example 3; 10 mg) was dissolved in anhydrous DMSO (3 ml); it was added HSPyU (20 mg) and N,N'-diisopropylethylamine (80 ál). The resulting solution was stirred for 3 hours, after which TLC (RPC18 water/N) revealed complete reaction. The dye was isolated by precipitation in ethyl acetate/diethyl ether, was filtered, washed with ethyl acetate and dried in vacuum. UV/Vis (water) 650 nm. MS (MALDI-TOF) MH+ 983,5. MH+=C42H53N3O16S4requires m/z 984,16.

Example 5: the Conjugation of dyes, synthesis of Compounds 3-7

Cys4-16, 6-14; Ac-Ala-Gly-Ser-Cys-Tyr-Cys-Ser-Gly-Pro-Pro-Arg-Phe-Glu-Cys-Trp-Cys-Tyr-Glu-Thr-Glu-Gly-Thr-Gly-Gly-Gly-Lys(Cy5)-NH2(Compound 3)

Compound 1 (10 mg), NMM (4 ml) and NHS ester Su (5,7 mg; GE Healthcare PA15104) was dissolved in NMP (1 ml) and the reaction mixture was stirred for 7 hours. Then this reaction mixture was diluted with a mixture of 5% ACN/water (8 ml) and the product was purified using preparative HPLC.

Purification by preparative HPLC (gradient: 5-50% B over 40 min where a is an H2O/0.1% of HCOOH, and represents ACN/0.1% of HCOOH, flow rate: 10 ml/min, column: Phenomenex Luna 5µ C18 (2) 250×21,20 mm, detection: UV 214 nm, retention time of product: 35,5 min) of the crude peptide gave 8,1 mg of pure Compound 3. The pure product was analyzed by analytical HPLC (gradient: 5-50% B over 10 min, where An a H 2O/0.1% of HCOOH, and represents ACN/0.1% of HCOOH, flow rate: 0.3 ml/min, column: Phenomenex Luna 3µ C18 (2) 50×2 mm, detection: UV 214 nm, retention time of product: 8,15 min). Additional characterization was performed using mass spectrometry with ionization by elektrorazpredelenie (calculated: 1710,6,found: 1711,0).

Compound 4 was obtained in the same way; mass spectrometry with ionization by elektrorazpredelenie (calculated: 1710,6,found: 1710,9).

Other conjugates of the dye-peptide (Compounds 5-7) was obtained in a similar way. Alexa647 was provided by Molecular Probes (A):

Compound 5 (calculated: 1825,7,found: 1825,9),

Compound 6 (calculated: 1811,7,found: 1812,0),

Compound 7 (calculated: 1825,7,found: 1826,2).

Example 6: analysis of the polarization of fluorescence in vitro

The principle of the polarization of fluorescence can be briefly described as follows:

Monochromatic light passes through a horizontal polarizing filter and excites the fluorescent molecules in the sample. Only those molecules that PRA is ilen oriented vertically polarized plane of the light absorption, excited and then emit radiation. The emitted radiation is measured in both the horizontal and vertical planes. Index anisotropy (A) represents the ratio between the light intensities according to the following equation:

Measurement of the anisotropy of fluorescence was carried out in 384-well microplate in a volume of 10 μl in a buffer for binding (phosphate buffered saline (PBS), 0.01% of Tween-20, pH 7.5) using a tablet reader polarized fluorescence Tecan Safire (Tecan, USA) at wosb/ispos nm. The concentration of labeled dye peptide was kept constant (20 nm) and the concentration of human or mouse c-Met/Fc Chimera (R&D Systems) or Semaphorin 6A (R&D Systems) was varied in the range of 0-150 nm. Mixture to bind balanced in the microplate for 10 minutes at 30°C. the Observed change in anisotropy was tailored to the equation:

where robsrepresents the observed anisotropy, rfreerepresents the anisotropy of the free peptide, rboundrepresents the anisotropy of the bound peptide, KDrepresents the dissociation constant, cMet represents the total concentration of c-Met, and P represents the total concentration of labeled dye peptide. This equation assumes that the synthetic peptide and receptor form a reversible complex in solution in the stoichiometric ratio 1:1. Approximation of data was performed through a non-linear regression using the software GraphPad Prism, obtaining values of KD(linking one site).

Compounds 3 and 4 were tested in relation to the binding of human and mouse c-Met (Fc Chimera). The results showed thatDis 3+/-1 nm in relation to the binding of Compound 3 with a human C-Met. No binding was observed for Compounds 4 with a human C-Met. In addition, Compounds 3 and 4 showed no binding to mouse c-Met in the tested range.

Using this method it was found that Compound 5 has KD1,1 nm against human cMet.

Example 7: testing of Compounds 5 and 7 in vivo

(a) Animal model

This study used Nude mice 54 Female BALB C/a with a mutation in the gene nude (Bom). The use of these animals approved by the local Committee for ethical research. BALB/And nude is an inbred mouse line with compromised immunological reactivity with a high degree of development of human tumors compared to other mouse lines nude. The mice were 4 weeks of age upon arrival and had a body weight of about 20 grams at the beginning of the study. Animals were kept in individually ventilated cages (IVC, Sanbur BK) filtered through high-efficiency air filter (HEPA) air. The animals had unlimited access to feed Rat and Mouse nr. 3 Breeding" (Scanbur BK) and tap water, podstilaemoi adding HCl to the molar concentration of 1 mm (pH 3.0).

Cells of colorectal cancer HCT-15 are the origin of human carcinomas of the colon and Express c-Met, as reported according to Zeng et al. [Clin. Exp. Metastasis, 21, 409-417. (2004)]. It is proved that this cell line is tumorigenic in subcutaneous inoculation of nude mice [Flatmark et al., Eur. J. Cancer 40, 1593-1598 (2004)].

Cells HCT-15 was grown and prepared for subcutaneous inoculation in RPMI medium (Sigma, catalog number # R0883) with 10% serum and penicillin/streptomycin. The strains were subjected to four passages and passage number four (P4) were frozen for storage in liquid nitrogen at a concentration of 3×107cells/flask in culture medium containing 5% dimethyl sulfoxide (DMSO). On the day of transplantation, the cells were subjected to rapid thawing in a water bath at 37°C (approximately 2 min), washed and resuspendable in PBS/2% serum (centrifugation at 1200 rpm for 10 min). Thorough mixing of the cells in vials provided whenever their aspiration into the dosing syringe. Cell suspension volume of 0.1 ml were injected with subcutaneous (s.c.) in the scapular region and back region, using a needle with a hole of small size (25 G), while the animals were under dei is the influence of anesthesia light gas. Then, animals were returned to their cages and left for the growth of tumors 13-17 days. Animals were given an acclimatization period of at least 5 days before inoculation.

(b) Procedure

All test substances were diluted in PBS from a lyophilized powder. A small stack of white paper for printer subjected visualization with obtaining a planar image, which was used to correct inhomogeneities in the x-ray. The test substances were injected with intravenously in the lateral part of the caudal vein with physical locks. The injection volume was 0.1 ml, which corresponds to a dose of 1 nmol test substance to the animal. After injection, animals were returned to their cages. Animals were killed immediately prior to visualization by displacement of the cervical vertebrae. For each test substance were noted time optimal visualization, based on a comparison of rates of leaching in the skin and muscle tissue in a limited number of animals (n is 1-6). Time visualization for Compounds 3 and 4 was 120 minutes after injection. For each animal produced subcutaneous pathologic-anatomic slices grown tumors. A thin plate of a thickness of approximately 1.6 mm and 3-4 mm in diameter were separated from the edge of one of the tumors. This slice of the tumor and then machining the Gali visualization plot against the normal colon from the same animal.

(C) Visualization

Visualization was carried out through clinical laparoscope, adapted for use with a light source for excitation of the reporter and the filtration system to extract the fluorescent component. Laser 635 nm was used for excitation of the reporter molecule. The camera Hamamatsu or SA ERG CCD was used as detector. This camera uses Binning Mode 2×2 zero gain. The standard exposure time for imaging of the colon was 10 sec. Calibration of the measurement system shows that it is a 10-second exposure time using this visualization system of the animal corresponds to a 40-millisecond exposure using a clinically relevant source of light, field of view and distance to the surface of the fabric. The intensity distribution in the image is corrected in respect of inhomogeneities in the radiation through the system calibration data. The ratio of target:the background was calculated from the areas of interest placed over the tumor, and normal background of the colon. Images were assessed visually, using the standard evaluation system used by the recipient, performing characteristic analysis.

(g) the Results

Compound 5 showed the ratio of tumor:normal tissue 1,46:1, and the corresponding scrambled control the hydrated peptide with the same dye (Compound 7) showed the ratio was 1.04:1. With Compound 5 was easily identifiable tumor, while nothing could be distinguished against the background when using Compound 7.

1. The agent visualization that represents the conjugate of formula I:
,
where Z1attached to the N-end smbr and represents N or M1G;
Z2attached to the end of cmvr and is a HE, OBCor MIGwhere BCis a biocompatible cation;
smbr is a cMet-binding cyclic peptide of 17 to 30 amino acids, including amino acid sequence (SEQ-1): Cysa-X1-Cysc-X2-Gly-Pro-Pro-X3-Phe-Glu-Cysd-Trp-Cysb-Tyr-X4-X5-X6;
where X1represents Asn, His, or Tyr;
X2represents Gly, Ser, Thr or Asn;
X3represents Thr or Arg;
X4represents Ala, Asp, Glu, Gly or Ser;
X5represents Ser or Thr;
X6represents Asp or Glu;
and each of Cysa-drepresents a cysteine residue, so that the residues a and b, and c and d cyklinowanie with the formation of two separate disulfide bonds;
MIGis a group inhibitory metabolism, which is a biocompatible group, inhibitory or inhibiting the metabolism smbr-peptide in vivo;
L t is made by a synthetic linker group of formula(A) m-where each And independently represents-CR2-, -CR=CR-, -C≡C-, -CR2CO2-, -CO2CR2-, -NRCO-, -CONR-, -NR(C=O)NR-, -NR(C=S)NR-, -SO2NR-, -NRSO2-, -CR2OCR2-, -CR2SCR2-, -CR2NRCR2C4-8cyclohexanoltramadol group4-8cycloalkenyl group5-12Allenova group or3-12heteroarenes group, an amino acid, a sugar or a monodisperse polietilenglikoli (PEG) is a structural unit;
each R is independently selected from H, C1-4of alkyl, C2-4alkenyl,2-4the quinil,1-4alkoxyalkyl or1-4hydroxyalkyl;
m is an integer from 1 to 20;
n is an integer 0 or 1;
IM is an optical reporter rendered grouping suitable for rendering the body of a mammal in vivo using light in the wavelength range at wavelengths from green to near-infrared: 600-1200 nm.

2. Agent imaging according to claim 1, where, in addition to the SEQ-1, smvr further comprises residues Asp or Glu within 4 amino acid residues from the C - or N-Terminus smbr-peptide, and -(L)nIM functionalized amino group which is conjugated with carboxyla side chain of the specified residue is Asp or Glu with the formation of amide linkages.

3. Agent imaging according to claim 1 or 2, where, in addition to the SEQ-1, smvr additional content is t a Lys residue within 4 amino acid residues from the C - or N-Terminus smbr-peptide, and -(L)nIM functionalized carboxyl group which is conjugated to the ε-amino group of the side chain of the specified Lys residue with the formation of amide linkages.

4. Agent imaging according to claim 1, where smvr includes the amino acid sequence SEQ-2 or SEQ-3:
Ser-Cysa-X'-Cysc-Gly-Pro-Pro-X3-Phe-Glu-Cysd-Trp-Cysb-Tyr-X4-X5-X6(SEQ-2);
Ala-Gly-Ser-Cysa-X1-Cysc-X2-Gly-Pro-Pro-X3-Phe-Glu-Cysd-Trp-Cysb-Tyr-X4-X5-X6-Gly-Thr (SEQ-3).

5. Agent imaging according to claim 1, where X3represents Arg.

6. Agent imaging according to claim 4, where, in addition to the SEQ-1, SEQ-2 or SEQ-3, smvr additionally contains at the N - or C-end of the linker peptide selected from-Gly-Gly-Gly-Lys- (SEQ-4), -Gly-Ser-Gly-Lys- (SEQ-5) or-Gly-Ser-Gly-Ser-Lys- (SEQ-6).

7. Agent imaging according to claim 6, where smbr has the amino acid sequence (SEQ-7):
Ala-Gly-Ser-Cysa-Tyr-Cysc-Ser-Gly-Pro-Pro-Arg-Phe-Glu-Cysd-Trp-Cysd-Tyr-Glu-Thr-Glu-GIy-Thr-Gly-Gly-Gly-Lys.

8. Agent imaging according to claim 1, where both Z1and Z2independently represent MIG.

9. The agent visualization of claim 8, where Z1=MIG= acetyl, and Z2=MIG= primary amide.

10. Agent imaging according to claim 1, where n is equal to 0.

11. Agent imaging according to claim 1, where IM represents a dye having the maximum absorption in the range 600-1000 nm.

12. The agent visualization on the .11, where IM is a cyanine dye.

13. The agent visualization indicated in paragraph 12, where the cyanine dye represented by formula III:
,
where R1and R2independently represent H or SO3M1and at least one of R1and R2represents the SO3M1where M1represents N or Bc;
R3and R4independently represent a1-4alkyl or C1-6carboxyethyl;
R5, R6, R7and R8independently represent groups of Ra;
where Rarepresents a C1-4alkyl, C1-6carboxylic or -(CH2)kSO3M1,
where k is an integer 3 or 4;
provided that the cyanine dye has a total of from 1 to 4 substituents SO3M1in groups of R1, R2and Ra.

14. Agent imaging according to claim 1, where SPSR is such as defined in claim 7, Z1and Z2are as defined in claim 9, and IM is the same as defined in PP and 13.

15. Pharmaceutical composition for optical imaging in vivo, containing the agent imaging according to any one of claims 1 to 14, together with a biocompatible carrier in a form suitable for administration to a mammal.

16. The pharmaceutical composition according to § 15 in a dosage that is appropriate for individual PAC is enta, offered in a suitable syringe or container.

17. The method of receiving agent imaging according to any one of claims 1 to 14, comprising one of the steps (1)to(4):
(1) the interaction smbr-peptide formula Z1-[cMBP]-Z2where Z1represents H, and Z2is a MIGwith a compound of formula Y1-(L)n-[IM] the receiving agent visualization of the formula I, where [IM] anywhereman position Z1;
(2) the interaction smbr-peptide formula Z1-[cMBP]-Z2where Z1and Z2both are MIGand SPSR contains the residue is Asp or Glu within 4 amino acid residues with a C - or M-end smbr-peptide, and all other residues Asp/Glu smbr-peptide is protected, with a compound of formula Y2-(L)n-[IM] the receiving agent visualization of the formula I, where [IM] anywhereman at the specified residue is Asp or Glu this smbr-peptide;
(3) the interaction smbr-peptide formula Z1-[SPSR]-Z3where Z1is a MIGand Z3represents a group Z2or activated ester, and all other residues Asp/Glu smbr-peptide is protected, with a compound of formula Y2-(L)n-[IM] the receiving agent visualization of the formula I, where [IM] anywhereman position Z2;
(4) the interaction smbr-peptide formula Z1-[cMBP]-Z2where Z1and Z2both represent the OI M IGand SPSR contains a Lys residue within 4 amino acid residues from the C - or N-Terminus smbr-peptide, with the compound of the formula Y1-(L)n-[IM] the receiving agent visualization of the formula I, where [IM] anywhereman on residue Lys of this smbr-peptide;
where Z1, smvr, Z2MIG, L, n and IM are as defined in claim 1, and
Z3represents a group Z2or activated esters;
Y1is a carboxylic acid, activated ester, isothiocyanate or thiocyanato group;
Y2represents an amino group.

18. The method according to 17, in which the use stage of the interaction (4).

19. Kit for the preparation of a pharmaceutical composition according to item 15 or 16, containing the agent imaging according to any one of claims 1 to 14 in a sterile solid form and a biocompatible carrier, so that after dilution with sterile additive biocompatible carrier according to item 15 or 16 occurred dissolution of obtaining the desired pharmaceutical composition.

20. Set according to claim 19, where sterile solid form is a liofilizirovannoe solid.

21. The method of optical imaging of the body of a mammal in vivo comprising introducing agent imaging according to any one of claims 1 to 14 or a pharmaceutical composition according to item 15 or 16 with images of sites overexpression and the localization and cMet in vivo.

22. The method according to item 21, wherein the agent imaging according to any one of claims 1 to 14 or a pharmaceutical composition according to item 15 or 16 pre-injected into the body of the specified mammal.

23. The method according to item 22, including the stage at which:
(1) the surface of the tissue of interest in the body of a mammal Shine exciting light;
(2) using a fluorescence detector that detects fluorescence from the agent visualization generated by excitation rendered grouping (IM);
(3) the light detected by the fluorescence detector, possibly filtered to separate the fluorescent component;
(4) fluorescent glow in the stages (2) or (3) form the image of the specified surface tissue of interest.

24. The method according to item 23, where the exciting light on the stage (1) is a continuous wave (CW) by nature.

25. The method according to item 22, wherein:
(a) light-scattering biological tissue of a specified body of a mammal having a heterogeneous composition is exposed to light from a light source with a predetermined variable time-intensity for excitation agent visualization, in which see the multiple scattering of the exciting light in a specified tissue;
(b) repeatedly detects scattered light from the tissue in response to the specified effect; (C) conduct quantitative determination parameter fluorescence throughout the tissue on the basis of this radiation by establishing a series of indicators using a processor, where each of the indicators corresponds to the level parameter fluorescence in various positions within this tissue, and where the level parameter fluorescence varies according to the heterogeneous composition of this fabric; and
(g) generate the image of this tissue by imaging heterogeneous tissue composition in accordance with the performance stage (b).

26. The method according to item 21, where the means of optical imaging includes fluorescent endoscopy.

27. The method according to any of p-26, where optical imaging in vivo use when performing the detecting, determining the stage of the disease, diagnosis, monitoring of disease progression or monitoring the treatment of colorectal cancer (CRC).

28. Method of managing patients with colorectal cancer (CRC), including the detection, staging of disease, diagnosis, monitoring of disease progression or monitoring the treatment of colorectal cancer the body of a mammal, including a method of optical imaging in vivo according to any one of p-26.



 

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EFFECT: high output of ovomucoid and high degree of purity.

1 tbl, 5 ex

FIELD: medicine.

SUBSTANCE: invention relates to field of medicine, namely to diagnostics. In order to estimate uterine tube patency chromosalpingoscopy during laparoscopy and visual assessment of uterine tube state and patency are performed. During chromosalpingoscopy determined are: rate of liquid dye passage along uterine tube, type of outflowing liquid dye and degree of uterine tube patency. Uterine tube patency is determined by point system: 5 points - liquid enters pelvis minor by tube 1-3 seconds after introduction by free wide flow through free fimbriae; 4 points - liquid enters pelvis minor after 2-5 seconds in form of stream; 3 points - liquid enters pelvis minor with 5-10 second delay, and uterine tube considerably expands and liquid is released in fast large drops; 2 points - liquid is released in slow drops after considerable delay or dye reintroduction; 1 point - liquid soaks through fimbrial part of tube; 0 points - liquid does not enter pelvis minor at all, fimbrial part of tube is not formed, expressed adhesive process is observed.

EFFECT: method increases accuracy of diagnostics due to quantitative estimation of degree of uterine tube patency.

4 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to chemical-pharmaceutical industry and medicine and represents a contrast agent for T1 and/or T2 magnetic resonant scanning consisting of a nano-sized superparamagnetic powder of cubic cobalt ferrite spinel CoxFe3-xO4, wherein 0.1 ≤ x ≤ 0.99 of particle size 3÷20 nm.

EFFECT: invention provides preparing the contrast agent having a simultaneous effect on relative positive T1 and negative T2 contrasts in magnetic resonant scanning.

4 cl, 3 tbl, 7 dwg

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to an X-ray agent for X-ray diagnosing of various organs. The declared agent contains 2.0-9.0 wt % of tantalate in the form of nanoparticles of average size 5 nm of at least one element specified in a group consisting of yttrium, lanthanum, cerium, praseodymium, neodymium, samarium, europium, gadolinium, terbium, dysprosium, holmium, erbium, thulium, ytterbium, lutetium, or bismuth, 0.3-1.2 wt % of natural polysaccharide and water.

EFFECT: invention provides high contrast ratio within the whole range of X-ray powers applied in medical X-ray diagnosing (10-100 keV) and having high sedimentation stability.

2 cl

FIELD: medicine.

SUBSTANCE: invention relates to ophthalmology and is intended for volumetric echography of orbit in case of anophthalmia or subatrophy of eye. For this purpose claimed is immersion medium which represents cross-linked polymer hydrogel based on modified polyvinyl alcohol of general formula: where R - H, R1 - OH, R2 - unsaturated group, R3 - O-CO-R4, R4 - CH3 or residue of other acid, from polyvinyl ester of which polyvinyl alcohol was obtained, and m=80-99 mol %, n=0.5-15 mol %, k=0-12 mol %. Said emersion medium is used in echography method in form of 4-6 mm thick disc with size corresponding to opening into orbit. Disc is laid on anterior surface of closed eyelids.

EFFECT: invention ensures optimisation of orbit content image with absence of complications, trauma of eye tissues, as well as reduction of examination time and absence of psychological trauma.

2 cl, 5 ex

FIELD: chemistry.

SUBSTANCE: invention relates to metal chelates of formula I: where PEG-Pf denotes a perfluorinated PEG radical having 4-30 carbon atoms, including a) at least one perfluorinated PEG radical of formula XXI: CF3-(CF2)n"'[-O-(CF2)2]m"'-O-(CF2)-, where n"' denotes an integer between 0 and 6, m''' denotes an integer between 1 and 14; linker denotes a linker group which links the PEG-Pf radical with the skeleton; skeleton denotes a trivalent radical which is a nitrogen-containing radical selected from amino acids having a side functional chain, an alkylene diamine radical and derivatives thereof, nitrogen and 3,5-diaminobenzoic acid, K denotes a chelate radical consisting of a chelating radical and at least one equivalent metal ion with atomic number of 57-83. Also disclosed is use of metal chelates to produce contrast agents.

EFFECT: invention enables to obtain contrast agents for MRT, having good imaging properties and high transferability.

9 cl, 3 dwg, 4 tbl, 37 ex

FIELD: chemistry.

SUBSTANCE: invention relates to metal chelates of formula I: where PEG-Pf denotes a perfluorinated PEG radical having 4-30 carbon atoms, including a) at least one perfluorinated PEG radical of formula XXI: CF3-(CF2)n"'[-O-(CF2)2]m"'-O-(CF2)-, where n"' denotes an integer between 0 and 6, m''' denotes an integer between 1 and 14; linker denotes a linker group which links the PEG-Pf radical with the skeleton; skeleton denotes a trivalent radical which is a nitrogen-containing radical selected from amino acids having a side functional chain, an alkylene diamine radical and derivatives thereof, nitrogen and 3,5-diaminobenzoic acid, K denotes a chelate radical consisting of a chelating radical and at least one equivalent metal ion with atomic number of 57-83. Also disclosed is use of metal chelates to produce contrast agents.

EFFECT: invention enables to obtain contrast agents for MRT, having good imaging properties and high transferability.

9 cl, 3 dwg, 4 tbl, 37 ex

FIELD: medicine.

SUBSTANCE: spiral computed tomography of facial bones is performed by scanning at the section thickness min. 3 mm followed by reconstruction of the formed images in a planar mode in a front plane. Output hole areas of the trifacial nerve branches are measured from injured and healthy sides at the level of an expected injury. If observing decrease of the area from the injured side more than by 25% as compared with the healthy side, compression of peripheral branches of trifacial nerve in this zone.

EFFECT: technique enables higher diagnostic reliability that is ensured by detection of the disease within all the three zones of the branches of trifacial nerve.

2 ex

FIELD: medicine.

SUBSTANCE: for the purpose of contrast study of colonic residuals in virtual colonoscopy of constrictive colonic tumour, virtual colonoscopy is conducted. It involves an enema with a concentrated barium suspension 72 hours before virtual colonoscopy; it is followed by prescribing a laxative at 1 sachet of Fortrans per 30 kg of body weight and a liquid diet with limited fats and vegetable fibres between the contrast study of colonic residuals and virtual colonoscopy.

EFFECT: method enables conducting the contrast study of colonic residuals with the minimum barium suspension concentration.

1 dwg, 1 ex

Contrast agents // 2469021

FIELD: chemistry.

SUBSTANCE: present invention relates to novel compounds which contain two bonded iodinated phenyl group of general formula R-N(CHO)-X-N(R3)-R (I), where R, R3 and X assume values given in the claim, as well as use as contrast agents in diagnostic X-ray imaging, and X-ray diagnostic compositions containing such compounds.

EFFECT: obtaining novel contrast agents.

15 cl, 22 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to medicine and represents gel-forming mixed dextran esters containing phosphate and carbamate groups of general formula: {C6H7O2(OH)3-x-y{[(OP(O)ONa)mONa)]xl[(O2P(O)ONa)k]x2}x(OCONH2)y}n, wherein x=x1+x2 is a degree of substitution in phosphate groups (mono- and diesters), x=0.47-1.09; X1 is a degree of substitution in monoesters, X1=0.01-0.48; m is a number of phosphates in monoesters, m=1-2; x2 is a degree of substitution in diesters, x2=0.01-1.09; k is a number of phosphates in diesters, k=1-2; y is a degree of substitution in carbamate groups, y=0.39-1.23; n is a degree of polymerisation, 20≥n≤1000.

EFFECT: invention provides producing low-toxic low- and high-substituted dextran phosphates in the form of hydrogels containing additionally carbamate groups and possessing antiproliferative activity with respect to cancer cells.

2 cl, 3 dwg, 14 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to pharmaceutical industry and represents a composition for normalising body microflora containing at least 3 prebiotic substances taken of four groups of prebiotic with various molecular chain lengths: monomer prebiotics (number of chains 1); dimer prebiotics (number of chains 2); oligomer prebiotics with number of chains 3 to 10; polymer prebiotics with number of chains more than 10; with the ingredients of the composition found in certain proportions, wt %.

EFFECT: invention provides recovery, maintaining of the qualitative-quantitative formula and activation of bacteria of various geni of normal intestinal microflora for adequate implementation of the microbiocenosis function along the full length of the digestive tract starting from an oral cavity and an oesophagus to a lower large intestine, as well as recovery of microbiocenosis of the other intestines.

16 cl, 12 ex

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