Peptide compounds

FIELD: chemistry.

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

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

9 cl, 5 ex

 

This invention relates to new peptide compounds and their use in diagnostic methods optical imaging or treatment of the disease. More specifically, the invention relates to the use of such peptides as targeting vectors that bind to receptors associated with angiogenesis. The compounds can be used as contrast agents in the diagnosis or treatment of diseases associated with angiogenesis.

As a rule, new blood vessels can be formed through two different mechanisms: vasculogenesis or angiogenesis. Angiogenesis is the formation of new blood vessels through branching of existing vessels. The primary incentive for this process may be insufficient inflow of nutrients and oxygen (hypoxia) to the tissue cells. Cells can respond by secreting angiogenic factors, of which there are many; one of the frequently cited examples of vascular endothelial growth factor (VEGF). These factors initiate the secretion of proteolytic enzymes that destroy proteins of the basal membrane, as well as inhibitors that limit the effect of these potentially harmful enzymes. Another known effect of angiogenic factors is to cause migrate and division of endothelial cells. Endothelial cells, which are attached to the basal membrane and form a continuous layer around the blood vessels on the side opposite the lumen of the vessels, do not undergo mitosis. The combined effect of loss of attachment and signals from receptors for angiogenic factors causes endothelial cells to move, to share, to regroup and, finally, to synthesize basement membrane around new blood vessels.

Angiogenesis observed in the growth and remodeling of tissues, including wound healing and inflammatory processes. In addition, inhibition of angiogenesis is a promising strategy for cancer treatment. Transformations associated with angiogenesis, are also very promising for the diagnosis, a clear example of what is a malignant disease, but this concept is also promising for inflammation and many diseases associated with inflammation including atherosclerosis, and macrophages in early atherosclerotic lesions represent a potential source of angiogenic factors. These factors are also involved in the revascularization areas of infarction, suffered a heart attack, which occurs when the stenosis is eliminated in a short time.

Additional examples of undesirable conditions associated with NeoV what skolaryzacji or angiogenesis, the development or proliferation of new blood vessels, is presented below. In this regard, also made reference to WO 98/47541.

Diseases and indications associated with angiogenesis, are, for example, various forms of cancer and metastasis, such as cancer of the breast, skin, colorectal cancer, pancreatic cancer, prostate, lung or ovarian cancer.

Other diseases and indications are inflammation (e.g., chronic), atherosclerosis, rheumatoid arthritis and gingivitis.

Additional diseases and indications associated with angiogenesis are arteriovenous defects, astrocytomas, choriocarcinoma, glioblastomas, gliomas, hemangioma (children, capillary), hematoma, endometrial hyperplasia, myocardial ischemia, endometriosis, sarcoma, macular degeneration, melanoma, neuroblastoma, occlusal disease peripheral artery disease, osteoarthritis, psoriasis, retinopathy (diabetes, proliferative), scleroderma, seminoma and ulcerative colitis.

In angiogenesis involved receptors that are unique to endothelial cells and surrounding tissues. These markers include the receptors of growth factors such as VEGF and integranova family of receptors. Immunohistochemical studies demonstrated that sets the integrins, perhaps most significantly, the class αv, is expressed on the apical surface of blood vessels [Conforti, G., et al. (1992) Blood 80:37-446] and are available for targeting circulating in the circulatory system of the ligands [Pasqualini, R., et al. (1997) Nature Biotechnology 15:542-546]. α5β1 is also an important integrin facilitating the Assembly fibronectin matrix and initiation of attachment of cells to fibronectin. It also plays an important role in cell migration.

Integrin αvβ3is one of the receptors, which are known to be associated with angiogenesis. Stimulated endothelial cells seems to rely on this receptor in relation to survival during the critical period of the process of angiogenesis, as antagonists αvβ3integranova receptor interactions with ligand induce apoptosis and inhibit the growth of blood vessels.

Integrins are heterodimeric molecules, in which the α - and β-subunit penetrate the lipid bilayer of the cell membrane. the α-Subunit has four domains, binding of CA2+at its extracellular chain and β-subunit has a number of extracellular cysteine-rich domains.

Many ligands (e.g., fibronectin), involved in cell adhesion, contain Tripeptide sequence arginine-glycine-aspartic acid (RGD). The sequence is RGD, apparently, acts as the primary site of recognition for ligands, presenting this sequence, and receptors on the cell surface. It is usually assumed that the secondary interactions between the ligand and the receptor increase the specificity of the interaction. These secondary interactions can occur between groups of the ligand and the receptor, which is located directly adjacent to the RGD sequence, or in the websites which are located on the distance from the RGD sequence.

It is known that RGD-peptides are associated with a number of integranova receptors and can regulate a number of cellular phenomena that have important clinical applications. Perhaps the most widely studied effect of RGD-peptides and their imitators relates to their use as antithrombotic funds when they are targeted to integrin platelet Gpllbllla.

Inhibition of angiogenesis in tissues by injection of the antagonist of αvβ3 or αvβ5 antibody or peptides containing RGD described, for example, in WO 97/06791 and WO 95/25543. In EP 578083 described a number of monocyclic peptides containing RGD. Cyclic RGD-peptides containing multiple bridges, also described in WO 98/54347 and WO 95/14714.

Additional examples of RGD, including peptide compounds contained in WO 01/77145, WO 02/26776 and WO 03/006491. In WO 01/77145 disclosed bicyclic peptide is RGD-type conjugated with a reporter group. In WO 05/003466 also disclosed peptides RGD-type conjugated with fluorescein for use in optical imaging.

There is a clinical need to develop more specific non-invasive methods for imaging of diseases associated with angiogenesis, and treatment of such diseases. Such visualization techniques play a major role in the evaluation of new antiangiogenic therapies. The opportunity to assess the real level of angiogenesis has clinical significance in the diagnosis of diseases associated with angiogenesis, at an early stage. Optical imaging can be used to assess the level of angiogenesis, and in the proposed invention the new compounds are useful as contrast agents for optical imaging for this task.

Taking into account the needs in this area, in the proposed invention the peptide compounds labeled with cyanine dyes for use as contrast agents for optical imaging or therapeutic treatment. For efficient in vivo targeting and imaging integranova receptors associated with angiogenesis requires selective, high-affinity vector RGD-type, which is chemically stable. In addition, the path of excretion is important is the actor in the development of imaging agents with the aim of reducing problems emerging from the background noise. These conditions satisfy the peptide compounds labeled with cyanine dye described in this invention.

In accordance with one aspect of the invention proposed new peptide compounds as defined in the claims. These compounds have affinity against integranova receptors, for example by affinity against integrin αvβ3, and marked cyanine reporter dye.

Compounds or their physiologically acceptable salts, include the peptide vector and at least one cyanine dye, where the peptide vector comprises amino acid sequence X3G-D, and where the peptide vector and at least one cyanine dye is connected, preferably a covalent bond. X3represents arginine, N-methylarginine or arginine mimetic, G represents glycine and D represents aspartic acid. The peptide vector has affinity to integranova receptors, such as receptors αvβ3.

Cyanine dye (CyDye™) hereinafter designated by the letter Z. Cyanine dyes are compounds characterized by a polyene chain containing an odd number of carbon atoms linked by alternating simple and multiples, preferably double carbon-the low carbon bonds, terminated at each end by the amino group, one of which stereoselectivity. Cyanin and similar aryl-linker-modied chromophores may have suspended or condensed ring substituents. General description of cyanine dyes and their synthesis are described in US 6048982 and US 5268486. included in this description by reference. Cyanine dyes are particularly useful because of the wide range of spectral properties and structural variants. The range of cyanine dyes are well known and tested, they have low toxicity and are commercially available (GE Healthcare, formerly Amersham Biosciences). Cyanine dyes represent one family of high-intensity dyes with good solubility in water. They are not sensitive to pH in the range of pH 3-10, show low non-specific binding and are more photostability compared to fluorescein.

The preferred embodiment of the invention involves the conjugation of cyanine dyes with a peptide vectors, leading to a decrease of trapping in a pool of blood. In this embodiment of the invention the proposed connection, including cyanine dyes with two or one group of sulfonic acid or without it. Dyes with a reduced number of sulfonic acid groups, conjugated with PEP is igami, such as RGD-peptide, have a low binding to plasma and reduced nonspecific binding background cloth. Group, sulfonic acid does not give any hydrophilicity dyes, which is a necessary property for imaging in vivo. Cyanine dyes have been traditionally used in vitro, where polysulfonamide dyes was important to ensure that these dyes were very soluble in water. It has been unexpectedly discovered that by removing the sulfonic acid groups of the dye was obtained more optimal biodistribution of the compounds.

In this embodiment of the invention the inventors preferably used cyanine dyes, each of which contain 2 or 1 group sulfonic acid or does not contain sulfonic acid groups to reduce binding to blood plasma and nonspecific binding peptide compounds. It has been unexpectedly found that the compounds are sufficiently hydrophilic to dissolve in water.

Cyanine dye is preferably selected from the group consisting of carbocyanine, oxazinone, dialanine and aslanidou below General formula

In these structures the x groups R1 are identical or different and represent substituted or unsubstituted alkyl group, preferably C1-Sulkily, and can include a simple ester or a group-N-CO-N-. Alkyl group possibly substituted by carboxy groups, sulfonic acid, amine, ammonium, or a complex ester. The group R1 can form bridges with any of the carbon atoms of the unsaturated chains, for example the group-N-CO-N - or ether group. Groups R2 are identical or different and represent substituted or unsubstituted alkyl group. Alkyl group possibly substituted by carboxy groups or sulfonic acid, but preferably one of the groups R2 represent lower alkyl groups, for example C1-Sulkily, and most preferably a methyl group. Possible aromatic group denoted by dashed lines in order to cover the structure, including the condensed benzene rings and a condensed naftalie rings. Rings are substituted or unsubstituted. Rings can be substituted by sulfonic acid groups, carboxyl groups, hydroxyl groups, alkyl(sulfoalkyl)amino group, bis(sulfoalkyl)amino groups, sulfoalkyl, sulfoalkylation groups, alkyl or substituted alkyl or sulfoalkylation. Alkyl groups preferably represent lower alkali, for example containing from 1 to 6 carbon atoms. Y SEL is an hydrogen, halide group, an amino group or sulfonyl, and preferably represents hydrogen. Polyene chain cyanine dye may also contain one or more than one cyclic chemical group, forming bridges between two or more atoms of carbon polyene chain, for example by including the group-CO - between two carbon atoms of the chain, as in schwarenbach dye, or by adding an alkyl bridge. These bridges can serve to increase the chemical or the photostability of the dye.

In formulas I-IV, I represents a positive integer 1, 2, 3 or 4, which leads to trimethylamine, having a carbon bridge of three carbon atoms, pentamation, heptamethine or nonemotional cyanine dyes. Preferably cyanine dye is intimately or heptamethine dye carbon bridges, consisting of 5 and 7 carbon atoms, respectively.

In respect of formula I-IV preferred dyes contain a total of 2 or 1 sulfonic acid group or do not contain sulfonic acid groups attached to the indole rings or benzydamine rings.

Preferred dyes selected from the group of carbocyanines. And more preferred are carbocyanine dyes indole t the PA. Preferred dyes of this type are illustrated by the formula V:

where X represents a sulfonic acid group or is absent, the groups R1 are identical or different and represent substituted or unsubstituted lower alkyl group, for example substituted C1-Sulkily group. Alkyl group substituted, for example, carboxy groups, sulfonic acid, amino, ammonium or ester groups, for example, heterocyclic ether groups (for example NHS-ester). Groups R2 represent lower alkyl groups, such as C1-Sulkily, preferably a methyl group, possibly substituted, for example, carboxy groups or sulfonic acid. 1 represents 1, 2 or 3.

R1, R2 and X represent potential binding sites for binding of the dye with a peptide vector, and are preferred groups R1 and X. In the preferred embodiment, one group R1 is linked to a peptide vector, whereas the other group R1 represents a possibly substituted lower alkyl group.

The most preferred dyes are Cy5-NHS-ether-SO3and Cy7-NS-ether-SO3below:

Cy5-NHS-ether-SO3

Cy7-NHS-ether-SO3

Suitable cyanine dyes for use in the compounds according to the invention have a range of emission in the visible or near infrared region, preferably in the range of 500-900 nm, more preferably in the range of 650-850 nm.

Compounds according to the invention include the amino acid sequence X3-G-D that has affinity against integranova receptors. The connection preferably includes additional amino acids and possibly other groups, where the sequence X3-G-D represents a binding site peptide vector, functioning as a vector to bind to the receptor integranova type.

The connection according to the invention may be associated, for example, by formation of one or more ciclitira bridges in the area of peptide vector. Monocyclic peptide compound can be obtained by formation of a disulfide bond or thioester linkages between amino acids. Peptide compound comprising one cyclisme bridge, more specific αvβ3 and more preferably compared to the linear peptide. Compounds according to the invention preferably include two cyclessa bridge between different amino acids compounds or between amino acids and other groups. The term "cycle the existing bridges" refers to any combination of amino acids with functional groups, allowing you to enter a bridge or between amino acids and groups -(CH2)n-or -(CH2)n-C6H4-. n represents a positive integer from 1 to 10. Some preferred examples are disulfides, disulfide mimetic, such as -(CH2)4-carbamates, thioacetal, thioester bridges (citation or lanthionine) and bridges containing esters or ethers. Preferably one of the bridges forms a disulfide bond, and the second bridge contains a thioester (sulfide) connection.

Another embodiment of the compounds according to the invention are defined by formula (VIa)

where And are defined by the formula (VI)

and Z represents at least one cyanine dye associated with one or more than one of X1X6or X7And, perhaps via spacer elements group,

the connection involving two cyclessa bridge

where,

X3, G and D are as defined above;

Rarepresents a group -(CH2)n- or -(CH2)H-C6H4-which forms the land bridge that is associated with any of the X2, X4or X6where

n represents a positive integer from 1 to 10;

X1that is the link Il is 1, 2, 3, 4 or 5 amino acid residues, where at least one amino acid residue may functionalized spacer elements group, and the specified amino acid residue preferably has a functional side-chain such as an acid or amino group, and preferably selected from aspartic or glutamic acid, hemolysin or diaminoalkanes acid, such as lysine or diaminopropionic acid; and

X2and X4independently represent amino acid residues that can form cyclisme bridge, such as cysteine or homocysteine residues that form disulfide or thioester communication, or other amino acid residues capable of forming cyclisme bridge, such as aspartic acid and lysine, preferably X2and X4represent residues of cysteine or homocysteine, preferably X2and X4form cyclessa bridges between each other or with Raor X6; and

X5represents a hydrophobic amino acid or its derivative, preferably represents the tyrosine, phenylalanine, 3-itteratively or Neftyanoy residue, more preferably phenylalanine or 3-itteratively residue; and

X6represents an amino acid residue ways is hydrated to form cyclisme bridge, preferably tilligerry amino acid residue, preferably a cysteine or homocysteinemia residue, preferably X6forms cyclisme bridge with Ra, X2or X4; and

X7is a spacer elements or biomodification group or absent, preferably includes monodisperse polietilenglikolya (PEG) is a structural unit containing from 1 to 10 units of said structural unit, and the specified biomodification has a function to modify the pharmacokinetics and renal clearance of these agents. Additionally, X7also can represent from 1 to 10 amino acid residues, preferably including glycine, lysine, aspartic acid or serine. X7also may refer to a spacer or biomodification comprising amino acid residues, and PEG-like structure, preferably a combination of vitaminoterapiya and glycine. In the preferred embodiment X7is a unit consisting of monodisperse PEG-like structures, 17-amino-5-oxo-6-Aza-3,9,12,15-tetracosapentaenoic acid of formula (X)

where m is an integer from 1 to 10, and where the end is an amide or acid group. Found that biomodification X7option is ciruit the pharmacokinetics and the rate of clearance of blood from these compounds. Biomodification affect the decrease in absorption of the compounds in the tissue, i.e. muscle, liver and so on, thus providing better diagnostic visualization due to the smaller influence of the background. Secretion occurs mainly through the kidneys, and this is another advantage of this biomodification.

Peptide compound contains a peptide vector, a specific amino acid sequence formed by the X1, X2, X3, G, D, X4X5and X6formula VI, and this peptide is a targeting vector having affinity to integrin receptors associated with angiogenesis.

Depending on the location cyclessa bridges, connections include "discrete", "female" or "related" configuration. Preferably two of the bridge in each of the compounds are:

between Raand X6and between X2and X4(forming a nesting configuration);

between Raand X2and between X4and X6(discrete configuration);

between Raand X4and between X2and X6(forming an interconnected configuration).

In the preferred embodiment one bridge forms a thioester bond, and the second bridge forms a disulfide bond.

In the preferred embodiment of the compounds according to the invention are defined below forms the Lamy:

where Ra, X3, G, D, X5and X7are as defined for formula VI;

X'1contains amino acid residue with a functional side-chain such as an acid or amine group, and an amino acid, preferably selected from aspartic or glutamic acid, hemolysin or diaminoalkanes acid, such as lysine or diaminopropionic acid, preferably aspartic acid or lysine;

X'2X'4and X'6represent amino acid residues that form disulfide or thioester bond, such as cysteine or homocysteine, (shown disulfide and thioester bond);

W1is a spacer elements grouping or missing, and preferably is a derivative of glutaric and/or succinic acid, and/or based on polyethylene glycol units, binding cyanine reporter dye with the peptide. Other typical spacer elements (W1elements include polysaccharides structural polysaccharides cumulative type, polyaminoamide, and their methyl and ethyl esters, polypeptides, oligosaccharides and oligonucleotides, which may contain or not contain the sites of cleavage of f is rontani. The role of spacer elements grouping W1is to distantsiirovat relatively bulky dye from binding to the receptor domain of the peptide component;

h represents a positive integer 1 or 2;

and, when there is at least one group Z, Z is a cyanine dye.

Compounds preferably include only one z group.

Cyanine dye, denoted by Z, is associated with X'1, W1, X6or X7peptide vector, for example through the formation of amide linkages, sulfonamidnuyu communication or thioester linkages. Amide bond, for example, is formed by the reaction between an amine and a carboxyl group, sulfonamidnuyu communication, for example, is formed by the reaction between an amine and an activated sulfonic acid, and thioester bond, for example, is formed by the reaction between the thiol and halogen. X'1peptide vector containing at least one amino acid with functional side chain is preferred attachment point cyanine dye. Active esters of cyanine dyes, such as NHS-esters, are particularly useful in the synthesis of compounds that form amide bond with peptide vector.

In a preferred aspect of compounds of formula VII-IX is whether their physiologically acceptable salts have the following characteristics:

Rapreferably represents -(CH2)-.

In addition, X'1represents an amino acid residue with a functional side chain, such as acid or amino group, the amino acid is preferably selected from aspartic or glutamic acid, hemolysin or diaminoalkanes acid, such as lysine or diaminopropionic acid, preferably aspartic acid or lysine.

X'2X'4and X'6independently preferably represent a cysteine or homocysteinemia the rest.

X3preferably represents arginine.

X5preferably represents a tyrosine, phenylalanine, 3-ittrain or nafcillin, and more preferably phenylalanine or 3-ittrain.

X7and W1are as defined for formula VIb. Preferably X7contains from 1 to 10 units of monodisperse PEG block, or is absent, and W1preferably absent.

Z represents a cyanine dye or missing, so that the compound contains at least one grouping of cyanine dye.

In a preferred aspect the compound is a compound of formula VII (socket configuration) or their physiologically acceptable salts, and more prefer the LNO they have the above characteristics.

Any of the amino acid residues as defined in the formula VI, preferably may be a naturally occurring amino acid. In most cases, it is preferable that all amino acids in the peptide vector was in an L-shape. However, in some embodiments of the invention one, two, three or more than three amino acids in the peptide are preferably in the D-form. The inclusion of any amino acid in D-form can have a significant effect on increasing the stability of the compounds in the serum.

Some compounds according to the invention are high affinity RGD vectors-type. Used in this description, the term "high-affinity vector RGD-type" refers to compounds that have the Kiless than 10 nm and preferably less than 5 nm in the competitive analysis of binding against αvβ3 integrin and where a value of Kidetermined by competition with well-known high-affinity ligand echistatin. Methods of implementation of such competitive assays are well known in the art.

The connections defined in the present invention are unexpectedly stable in vivo and in conditions that are used when tagging using a cyanine dye.

Some examples of compounds according to the invention is illustrated below. Compounds a, B and C include pentamidine karbuz is anin respectively one, two or four groups, sulfonic acid, conjugated with RGD-containing peptide (Lys-Cys-Arg-Gly-Asp-Cys-Phe-Cys). The compounds a and B include dye Cy5, whereas the connection includes a dye Cy5.5:

Connection:

Connection B:

Connection:

Compound G:

The compound contains an RGD peptide-type (Lys-Cys-Arg-Gly-Asp-Cys-Phe-Cys)associated with the two groups of cyanine dye (Cy5).

Compound D: the Compound contains the peptide Lys-Cys-Arg-Gly-Asp-Cys-Phe-Cys associated with indocyanine green (ICG).

Connection: the Connection contains the peptide Lys-Cys-Arg-Gly-Asp-Cys-Phe-Cys associated with SUV.

Connection W: the Compound contains the peptide Lys-Cys-Arg-Gly-Asp-Cys-Phe-Cys associated with Cy5, where R1 is an alkyl, substituted ammonium group.

Connection 3: Peptide compound shown below, containing the peptide Asp-Cys-Arg-Gly-Asp-Cys-Phe-Cys may be associated with a cyanine dye through the formation of ties between aspartic acid (X1) with amino-functionalized cyanine dye, or by the reaction of NHS-ester cyanine dyes with amino-PEG, located in the X7.

Connection: the Connection contains the peptide Lys-Asp-Cys-Arg-Gly-Asp-Cys-Phe-Cys-Gly associated with Cy5.

Compound K: c[-Asp-D-Phe-Lys(Cy5.5)-Arg-Gly-]

The new compounds according to the invention can be used as contrast agents in optical imaging or treatment of diseases. The preferred embodiment of the invention are compounds described for use in optical imaging and preferably for the diagnosis of diseases associated with angiogenesis.

Compounds according to the invention are useful as imaging agents in the detection of angiogenesis in humans and animals. Products can also be useful in preclinical animal models and allow you to control therapeutic efficacy of new drugs in pharmaceutical research, for example, in Oncology.

The present invention also proposed a pharmaceutical composition comprising an effective amount of the compounds according to the invention or its salts, for example the amount, effective to enhance image contrast in in vivo imaging, together with one or more pharmaceutically acceptable adjuvants, excipients or diluents.

In addition, in the proposed invention the pharmaceutical composition for treating diseases, containing the th effective amount of the compound or its salts together with one or more pharmaceutically acceptable adjuvants, the excipients or diluents. The composition may be used in the treatment of diseases associated with angiogenesis, for example, by photodynamic therapy.

In accordance with another aspect of the invention the application of the compounds according to the invention for the manufacture of a contrast agent for optical imaging for use in the diagnostic method, in which specified a contrast agent injected into the human or animal and receive an image of at least part of the specified organism.

Thus it is assumed that the use of the compounds in the manufacture of therapeutic compositions (drug), and methods of therapeutic or prophylactic treatment of the human or animal, preferably for the treatment of diseases associated with angiogenesis, is an additional aspect of the invention.

In accordance with another aspect of the invention, a method for obtaining images of a human or animal by optical imaging, in which the specified organism, for example in the vascular system, the injected contrast agent and receive an image of at least part of the specified organism in which distributed the specified contrast agent, where as the specified contrast agent use is described connection.

the accordance with another aspect of the invention, a method for obtaining improved images of the human or animal by optical visualization preset songs with contrast agent, containing compound, as defined, which receive an image of at least part of the specified organism.

In accordance with another aspect of the invention, a method of monitoring the effect of treatment of the human or animal drug to combat a condition associated with angiogenesis, in which the specified organism is injected described connection and detects the specified capture agent cellular receptors, preferably receptors of endothelial cells, in particular receptors αvβ3, and specified the introduction and detection is possible, but is preferably carried out repeatedly, e.g. before, during, and after treatment with the indicated drug. This detection method includes an optical imaging.

Contrast agents according to the invention are intended for use in optical imaging. Any way to get the image for diagnosing disease, monitoring disease progression or monitoring the treatment of a disease, based on the interaction with the light of the electromagnetic spectrum in the range from ultrafilter to near infrared radiation, covered by the term optical imaging. Optical visualization includes all the ways, from direct visualization without application to the someone of the device and the application device, such as various indicators, catheters and equipment for optical imaging applications, such as computer equipment for the tomographic representation. Contrast agents useful in the methods of optical imaging and techniques change, including without limitation by them: fluorescent imaging; endoscopy; fluorescence endoscopy; optical coherence tomography; imaging based on light transmission; visualization on the basis of transmittance with time resolution; confocal imaging; nonlinear microscopy; the photoacoustic imaging; acoustic-optical imaging; spectroscopy; scattering spectroscopy; interferometry; coherence interferometry; diffuse optical tomography and fluorescently-mediated diffuse optical tomography (continuous-wave systems, with time intervals and frequency domains), and measurement of light scattering, absorption, polarization, luminescence, duration fluorescence quantum yield and quenching. Preferred are methods of optical imaging based on fluorescence identification or measurement.

Peptide vectors compounds of the present invention can be synthesized using known methods of chemical Sintesi especially useful is the solid-phase method of Merrifield, used in an automatic peptide synthesizer (J. Am. Chem. Soc, 85:2149 (1964)). In addition, the binding cyanine dye, such as an active ester cyanine dye, can also be performed automatically using an automatic peptide synthesizer with the formation of amide bond between the peptide and the group of cyanine dye. Other relationships between the cyanine dye and peptide, such as thioester or sulfonamidnuyu ligaments can also be obtained automatically, or the interaction of the dye and peptide can be accomplished by conventional manual chemical synthesis. Peptide synthesis using solid-phase methods based on sequential addition of protected amino acids, connected, possibly via a linker group to a solid-phase carrier. One of the commonly used methods, the α-amino group suitably protected colorability or labile with respect to the grounds of the protective groups. After adding and associating the first amino acid residue of the α-amino protective group is removed. The chain is lengthened by successively adding additional protected amino acid derivative or peptide fragments and/or appropriately derivatizing and protected derivatives of cyanine dye. Thus, the labeled dye the peptide compound according to the invention can be constructed by successive addition of amino acids or a derivative of cyanine dye.

Peptide vectors containing many bridges are synthesized with the use of various protective groups for cysteine, so that there is no uncertainty about the final stacked form of the vector. Can be used in the synthesis disclosed in WO 03/006491 describing how peptides are formed, comprising thioester and disulfide bridges. Thioester cyclization, for example, can be carried out as follows: Cys(t-Bu)-protected peptide is dissolved in a mixture of water/acetonitrile (1 mg/ml). The mixture is brought to pH 8 using dilute solution of ammonia, and stirred overnight.

Disulfide bridges can be formed by oxidation in DMSO/THF (dimethysulfoxide) as follows; the peptide is dissolved in 5% DMSO/triperoxonane acid (TFA) (1 mg/ml) and the mixture is stirred for 30 minutes.

Cyanine dyes are commercially available from GE Healthcare, formerly Amersham Biosciences, for example NHS-ester of Cy5, 1 mg, RA.

Peptide vectors and peptide compounds can be purified using high performance liquid chromatography (HPLC) and characterized by mass spectrometry and analytical HPLC before testing in vitro.

The present invention is additionally illustrated by the following, not limiting the scope of the invention examples.

Examples

Abbreviations:

TSTU: O-(N-Succinimidyl)-N,N,N',N'-tetramethylurea tetrafluoroborate

TFA: Triperoxonane acid

DMF: N,N-Dimethylformamide

NMM: N-Methylmorpholine

Example 1: Synthesis of Cys2-6: c[CH2CO-Lys(Cy5 mono-SO3)-Cys-Arg-Gly-Asp-Cys-Phe-Cys]-(PEG)n-NH2(n=1)

The above peptide was designed using standard methods of solid-phase peptide synthesis. Chloracetophenone peptide was tsalala from solid media and cyclically in solution with the formation of the first thioester of the bridge, and then a disulfide bridge. The NHS-Ester of Cy5-monocellate-mono-SO3(4.5 mg, 0,008 mmol) was obtained by processing TSTU (2.1 mg, 0,0076 mmol) and NMM (0,009 ml, 0.08 mmol) in DMF (2 ml) for 1 h Then the solution was added to the peptide (20 mg, to 0.016 mmol)and the reaction was continued throughout the night in the absence of light. DMF is evaporated under reduced pressure and the crude product was purified using reverse-phase preparative chromatography (column Vydac C18, TR; solvents: A=water/0.1% of TFA and B=CH3CN/0.1% of TFA; gradient 20-40% B over 60 min; flow 10 ml/min; detection at 254 nm) to give 4.9 mg (34%) of pure product (analytical HPLC: column Phenomenex Luna C18, 00G-4252-E0; solvents: A=water/0.1% of TFA and B=CH3CN/0.1% of TFA; gradient 25-45% B over 20 min; flow 1.0 ml/min; retention time of 15.2 min; detection at 214 and 254 nm). Additionally, the characterization was performed using mass spectrometry, to obtain a value of m/z 902,1 [MN2+].

Example 2: Synthesis of Cys2-6; C[CH2CO-Lys(Cy5 bis-SO3)-Cys-Ara-Gly-Asp-Cys-Phe-Cys]-(PEG)n-NH2(n=1)

The above peptide was designed using standard methods of solid-phase peptide synthesis. Chloracetophenone peptide was tsalala from solid media and cyclically in solution with the formation of the first thioester of the bridge, and then a disulfide bridge. Bicyclic peptide (24 mg, 0.02 mmol) were added as solids to a solution of Cy5 mono NHS-ester bis-SO3(7.5 mg, 0.01 mmol) in DMF (2 ml)and then was added NMM (0.01 ml, 0.09 mmol). The reaction was continued throughout the night in the absence of light. DMF is evaporated under reduced pressure and the crude product was purified using reverse-phase preparative chromatography (Vydac column 18, TR; solvents: A=water/0.1% of TFA and B=CH3CN/0.1% of TFA; gradient 10-30% B over 60 min; flow 10 ml/min; detection at 254 nm) to give 6.6 mg (37%) of pure product (analytical HPLC: column Phenomenex Luna C18, 00G-4252-E0; solvents: A=water/0.1% of TFA and B=CH3CN/0.1% of TFA; gradient 15-35% B over 20 min; flow 1.0 ml/min; retention time of 19.5 min; detection at 214 and 254 nm). Additional characterization was performed using mass spectrometry, getting m/z 949,1 [MN2+].

Example 3: Synthesis of Cys 2-6: c[CH2 CO-Lys(Cy7 bis-SO3)-Cys-Arg-Gly-Asp-Cys-Phe-Cys]-(PEG)n-NH2(n=1)

The above conjugate was designed using standard methods of solid-phase peptide synthesis. Chloracetophenone peptide was tsalala from solid media and cyclically in solution with the formation of the first thioester of the bridge, and then a disulfide bridge. NHS-Ester Cy7-monocellate-bis-SO3received by processing Cy7 (5.4 mg, 1 EQ.) TSTU (2.1 mg, of 0.95 EQ.) and NMM (5 EQ.) in DMF (2 ml) for 1 h Then the solution was added to the peptide (18,9 mg, 0,0156 mmol, 2 EQ.) in DMF (2 ml)and the reaction proceeded in the absence of light, usually during the night. DMF is evaporated under reduced pressure and the crude product was purified using reverse-phase preparative chromatography (column Vydac C18, TR; solvents: A=water/0.1% of TFA and B=CH3CN/0.1% of TFA; gradient 15-35% B over 60 min; flow 10 ml/min; detection at 254 nm) to give 5.2 mg (36%) of pure compound (analytical HPLC: column Phenomenex Luna C18, 00G-4252-E0; solvents: A=water/0.1% of TFA and B=CH3CN/0.1% of TFA; gradient 20-40% B over 20 min; flow 1.0 ml/min; retention time of 16.6 min; detection at 214 and 254 nm). Additional characterization was performed using mass spectrometry, getting m/z 961,9 [MH2+].

Example 4: Synthesis of Cys2-6: c[CH2CO-Lys(Cv7 mono-SO3)-Cys-Arg-Gly-Asp-Cys-Phe-Cy]-(PEG) n-NH2(n=1)

The peptide was designed using standard methods of solid-phase peptide synthesis. Chloracetophenone peptide was tsalala from solid media and cyclically in solution with the formation of the first thioester of the bridge, and then a disulfide bridge. NHS-Ester Cy7-monocellate-mono-SO3received by processing Cy7 (6 mg, 1 EQ.) TSTU (3 mg, of 0.95 EQ.) and NMM (5 EQ.) in DMF (1 ml) for 1 h Then the solution was added to the peptide (14,7 mg, 0.012 mmol, 1.2 EQ.) in DMF (2 ml)and the reaction continued overnight in the dark. DMF is evaporated under reduced pressure and the crude product was purified using reverse-phase preparative chromatography (column Vydac C18, TR; solvents: A=water/0.1% of TFA and B=CH3CN/0.1% of TFA; gradient of 25-35% B over 60 min; flow 10 ml/min; detection at 254 nm) to give 7.0 mg (38%) of pure compound (analytical HPLC: column Phenomenex Luna C18, 00G-4252-E0; solvents: A=water/0.1% of TFA and B=CH3CN/0.1% of TFA; gradient 30-40% B over 20 min; flow 1.0 ml/min; retention time of 17.4 min; detection at 214 and 254 nm). Additional characterization was performed using mass spectrometry, getting m/z 922,1 [MN2+].

Example 5: Analysis of protein-binding dye conjugates containing 1, 2 and 4 groups of sulfonic acid

Analysis of protein binding is painted to Nyugati according to the invention is carried out by means of equilibrium dialysis and speed dialysis as described in the book "Protein-Ligand Interactions: Hydrodynamics and calorimetry", edited by Stephen E. Harding and Babur Z. Chowdhry, Oxford University Press, published 2001, chapter 2 by Bent Honore, Department of Medical Biochemisty, University of Aarhus.

The results:

Connection:Protein binding (%)
No. of Sample 117
RGD-peptide Cy5 mono-SO3
No. b from Example 221
RGD-peptide Cy5-bis-SO3
No.
RGD-peptide Cy5.5-4-SO345

The examples demonstrate that cyanine dyes with a reduced number of sulfonic acid groups in conjugation with RGD-peptide have a smaller binding to blood plasma and reduced nonspecific binding background cloth.

1. The connection that represents the conjugate of the peptide vector and at least one cyanine dye, where the peptide vector containing the amino acid sequence X3G-D, and the connection is with the battle of the compound of formula VIIA:
,
where X3represents an arginine or N-methylarginine,
G represents glycine,
D represents aspartic acid,
Rarepresents a group -(CH2)n-which forms the land bridge that is associated with X'6where n represents a positive integer from 1 to 10,
h represents a positive integer 1 or 2,
X'1contains amino acid residue with a functional side chain, such as an amine group,
X'2X'4and X'6represent amino acid residues that form disulfide bonds or thioester bond,
X5represents phenylalanine,
X7is a spacer elements or biomodification group containing from 1 to 10 units of monodisperse polietilenglikoli (PEG) block,
there is one group Z, where Z represents a cyanine dye, and the specified cyanine dye each contains 0, 1 or 2 sulfonic acid group;
or its physiologically acceptable salt.

2. The compound according to claim 1, where the cyanine dye is selected from the group carbocyanines, oxazinone, dialanine and aslanidou.

3. The compound according to claim 1, where the cyanine dye is carbocyanine dye.

4. The compound according to claim 1, g is e R arepresents -(CH2)-.

5. The compound according to claim 1, where X'1selected from homolosine, lysine or diaminopropionic acid.

6. The compound according to claim 1, where X'2X'4and X'6independently represent a cysteine or homocysteinemia the rest.

7. The compound according to claim 1, where X3represents arginine.

8. The compound according to any one of claims 1 to 7, where cyanine dye Z associated with X'1or X7through amide sulfonamidnuyu or thioester linkages.

9. Pharmaceutical composition having an affinity for the αvβ3 integrin, and containing an effective amount of a compound according to any one of claims 1 to 8 together with one or more pharmaceutically acceptable adjuvants, excipients or diluents.



 

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FIELD: chemistry; biochemistry.

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

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FIELD: biotechnology.

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17 cl, 25 ex, 63 dwg, 2 tbl

FIELD: chemistry.

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EFFECT: invention enables to obtain a gadolinium complex with high relaxation efficiency.

1 cl, 1 tbl, 4 dwg

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