Selective r-cadherin antagonists and methods

FIELD: chemistry.

SUBSTANCE: proposed here is an isolated cyclic peptide, with amino acid sequence Cys-Ile-Xaa-Ser-Cys (SEQ ID NO:7); where Xaa is an amino acid residue, chosen from a group comprising Asp, Asn, Glu and Gin, and containing a disulphide bond between two Cys residues, which can be used as a selective antagonist of R-cadherin of mammals.

EFFECT: invented selective peptide-antagonists of R-cadherin can be used for inhibiting targeting of hematopoietic stem cells (HSC) on a developing vascular tree, for inhibiting cytoadherence caused by R-cadherin and inhibiting retina angiogenesis.

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Cross-reference to related applications

The application claims the priority of provisional application U.S. serial number 60/467188, filed may 1, 2003, which are hereby incorporated by reference.

Statement of interest of the government

The invention is made with the support of the government of the United States, grants No. EY11254 and EY12598 the National institutes of Health. The United States government has certain rights in the present invention.

The technical field to which the invention relates

In General, the invention relates to antagonists of cell adhesion molecules mammals. More specifically, the invention relates to selective peptidyl, which are antagonists of R-cadherin (cadherin-4) in mammals and to methods of inhibiting cell adhesion and, consequently, angiogenesis of the retina.

The level of technology

The family of cadherin molecules consists of transmembrane glycoproteins that function in a calcium-dependent selective intercellular interactions. These molecules play an important role during embryonic development and tissue morphogenesis, participating in cell-cell recognition and sorting of cells. The subfamily of catherinew (classic cadherin, protocadherin, desmocollin and other proteins related to cadherin) are characterized by RA the various extracellular cadherin domains, a single transmembrane segment and a single cytoplasmic domain. Reportedly, the so-called classical cadherin (that is, E, P, N and R-cadherin) contain five tandemly repeated extracellular cadherin domains (EC1-EC5), are preferably in homophilic interactions, and a highly conserved cytoplasmic tail, which mediates specific signals adhesion in the cell.

Mediated Katherina intercellular adhesion occurs as the expression of many molecules of the cadherin in the interaction of adjacent cells, resulting in the formation of adhesive bonds. According to the model of the zipper for cadherin proposed by Shapiro et al. Nature 1995; 374 (6520): 327-37, cadherin molecules in the membrane of one cell to form a tight dimer with parallel chains (i.e. the so-called CIS-dimers). As shown in figure 1, these CIS-dimers are then contacted with dimers cadherin expressed on adjacent cells (that is TRANS-dimerization). When supported by sufficient interaction can occur clustering of cadherin, as an increasing number of molecules cadherin is involved in website interaction, which leads to the entanglement of the molecules of the two surfaces of the cells. Thus, relatively weak interaction can be combined with the formation of Avelino strong intercellular adhesion.

First, when the adhesion cadherin intracellular signals through interaction of the cytoplasmic tails of cadherin molecules α and β catenin lead to the transformation of the cytoskeleton. Although the Association with actin, as is, does not affect homophile binding them together helps to keep the molecules of the cadherin in places of interaction. In symbiotic type relationship clustering of cadherin causes the conversion of the cytoskeleton and provides attachment points on the membrane that are important for the cellular changes that occur after the formation of adhesive contacts. At this time, the Association with the cytoskeleton maintains cadherin in places of interaction and helps to involve new molecules cadherin, thus leading to a clustering of cadherin. During clustering of cadherin calcium plays an important role of the cofactor. In solutions with insufficient concentration of calcium ions (i.e. below approximately 2 mm) cadherin loses its function, and the molecules become more sensitive by degradation. Calcium is essential for stabilizing the structure of molecules cadherin and ensures the correct orientation of the adjacent surfaces cadherin, therefore, in his absence, there is a loss of cadherin function and the prot is knowing degradation.

Despite the fact that, reportedly, each of the five extracellular domains of the classical cadherin from EC1 to EC5, plays an important role in ensuring the dimerization of cadherin-based mutational analysis, it was assumed that most of the residues that form the surface of the dimerization was detected at the N end of the larger domain of cadherin (EC1) (Kitagawa, et al., Biochem. Biophys. Res. Commun., 2000; 271(2):358-63). However, the specific mechanisms of homodimerization between cadherin molecules known to relatively few.

Katherine play a significant role in the control of neurons and the development of the Central nervous system. Reportedly, the different parts of the brain are determined by the expression of different types of cadherin during differentiation. Catherine also play an important role in neural development of the retina due to the specific expression in various regions of the developing retina. For example, it is reported that during development of the retina of the chicken embryo, B-cadherin found only in Muller glia, while certain populations of bipolar cells Express R-cadherin (also known as cadherin-4). Amacrine cells and a subpopulation of ganglion cells Express cadherin 6B and 7. Internal plexiform layer of the retina the same cadherin are only expressed in the sublayers related synapsin-I-positive n is runumi endings, that gives grounds for assuming that the different expression profiles contribute to the formation of the synapse between specific subpopulations of neurons during development of the retina. In the embryonic optic nerve axon growth ganglion cells mediated adhesion to N-cadherin with glial cells expressing R-cadherin.

Adhesion of catherinew also plays a role in vascularization of the retina during development (Dorrell et al. Invest. Ophthalmol. Vis. Sci. 2002; 43 (11):3500-10). Violation of adhesion of R-cadherin in the formation of the superficial plexus of blood vessels leads to the loss of complex compounds of vessels observed in the normal formation of blood vessels. When blocking the adhesion of R-cadherin during subsequent formation of the deep vascular layer key guiding signals are lost, which leads to migration of the vessel beyond the normal depth plexus of vessels in the optic layer receptors.

The retina consists of distinct layers of neural, glial and vascular elements. Any disease or condition that is even slightly modifies the layers of the retina, can cause degeneration of neurons and a significant loss of vision. For more than 70 years as a model for many of the diseases that lead to cell death of the visual receptors investigated mice with retinal degeneration mouserd/rd ). The mouse isrd/rdthe degeneration of the optic receptors begins in the first three weeks after birth, since the rod-shaped cells undergo apoptosis, which occurs as a result of mutations in β subunit of cGMP-dependent phosphodiesterase, which leads to the death of cones, the visual receptors. Vascular atrophy in the retina is also associated in time with the death of the cells of the visual receptors in micerd/rd. Apparently, the normal formation of the vascular network occurs when the development of the three functional layers within the first three weeks. However, when the blood vessels in the deep vascular layer begin to degenerate in the second week, and by the end of the fourth week after birth see a significant decrease in the number of vessels, there is almost complete disappearance of deep and intermediate vascular plexus.

In normal blood and bone marrow of an adult organism present population of hematopoietic stem cells, which can differentiate different subpopulations of cells positive line (Lin+HSC) or negative line (Lin-HSC). In addition, the authors of the present invention found that subpopulations Lin-HSC are endothelial precursor cells (EPC)capable of forming blood vesselsin vitroandin vivo. EPC in which ulali Lin -HSC can influence and stabilize degenerating vasculature in micerd/rdwhen intraocular administration to mice. In the superficial vascular layer was introduced astrocytes, target Lin-HSC and watched endogenous developing vascular network after injection on day 2 after birth (P2). Upon reaching endogenous vascular network of the periphery of the retina, which were aimed Lin-HSC, cells were embedded in new blood vessels with formation of functional mosaic vessels with mixed populations of introduced Lin-HSC and endogenous endothelial cells. In addition, Lin-HSC focus on areas of deep and intermediate vascular layer of the retina pre-vascularization of these layers due to endogenous endothelial cells. The inclusion Lin-HSC restores deep vasculature of micerd/rdabout 2-3 times stronger compared to normal mice and the control mice, which were injected Lin+HSC. In addition, recovery of deep vascular network prevents degradation of the visual receptors in the outer nuclear layer of the retina. However, since there is no basis for the proposal that these stem cells can undergo differentiation into neurons of the retina or glial cells, the mechanism of protection of neurons remains unknown.

Targeting Lin-HSC on astrocytes and deep vascular area before natural vascularization suggests that Lin-HSC Express molecules on the cell surface that act when you are targeting a similar targeting of endogenous endothelial cells during development. Adhesion of R-cadherin plays an important role in targeting endothelial cells with astrocytes and vascular plexus during angiogenesis in the retina during development.

R-cadherin was identified and sequenced a large number of mammals. Figure 2 shows the amino acid sequence (SEQ ID NO: 1) option preprally R-cadherin person described by Kitagawa et al. in the database SWISS-PROT number NP 001785, version, NP 001785,2, GI: 14589893, the description of which is given here as a reference. SEQ ID NO: 1 includes an amino acid sequence IDSMSGR (SEQ ID NO: 2) in the provisions 222-228.

Figure 3 shows the amino acid sequence (SEQ ID NO: 3) yet another variant, prefrably R-cadherin person described Tanihara et al. in the database SWISS-PROT number NP P55283, version P55283, GI: 1705542, the description of which is given here as a reference. SEQ ID NO: 3 comprises the amino acid sequence INSMSGR (SEQ ID NO: 4) in the provisions 222-228.

Figure 4 shows the amino acid sequence (SEQ ID NO: 5) option preprally R-cadg the Rina mouse (mouse home), described by Hutton et al. in the database SWISS-PROT number NP 033997, version, NP 033997,1, GI:6753376, the description of which is given here as a reference. SEQ ID NO: 5 comprises the amino acid sequence IDSMSGR (SEQ ID NO: 2) in the provisions 219-225.

Non-selective peptide antagonists catherinew, including the amino acid sequence His-Ala-Val (HAV), described Blaschuket al. in U.S. patent No. 6465427, No. 63456512, No. 6169071, and No. 6031072. Blaschuket al. describe both linear and cyclic peptide antagonists catherinew, all of which can counteract many types of cadherin molecules without distinction.

Selective peptide antagonists of N-cadherin, which contain the amino acid sequence Ile-Asn-Pro (INP), were described by Williams et al., Mol. Cell Neurosci.,2000; 15(5): 456-64. While HAV peptides are non-specific antagonists catherinew, peptide antagonists INP described by Williams et al., are specific in relation to N-cadherin and do not show significant binding to other molecules cadherin, such as R-cadherin.

Due to the different distribution of cell adhesion molecules in various tissues of the body, there is a continuing need in the antagonists, selective to the specific cell adhesion molecules, particularly antagonists, selective to R-cadherin. Selective peptides-antagoni what you R-cadherin of the present invention satisfy this need.

The invention

The selected peptide, used as a selective antagonist of R-cadherin mammals, contains from 3 to 30 amino acid residues, three of the neighboring residue peptides have the amino acid sequence Ile-Xaa-Ser (IXS), where Xaa represents an amino acid residue selected from the group including Asp, Asn, Glu and Gln. Preferably, Xaa represents Asp or Asn. In one of the preferred embodiments, the peptide contains at least seven amino acid residues, and seven neighboring amino acid residues of the peptide have the amino acid sequence Ile-Xaa-Ser-Met-Ser-Gly-Arg (SEQ ID NO: 6), where Xaa represents the same residues as defined above. The present invention also relates to pharmaceutical compositions containing the peptide antagonist of R-cadherin in a pharmaceutically acceptable carrier.

In another preferred embodiment, the peptide is a cyclic peptide containing from 3 to 10 amino acid residues, arranged in a ring, three neighboring residue peptides have the amino acid sequence Ile-Xaa-Ser, where Xaa represents an amino acid residue selected from the group comprising from Asp, Asn, Glu and Gln. Preferably, Xaa represents Asp or Asn.

Preferred cyclic peptide has the formula:

where X1and X2independently represent an amino acid residue or set to 10 amino acid residues, connected by peptide bonds; Y1and Y2independently represent amino acid residues linked together by a disulfide bond, and Xaa is an amino acid residue selected from the group including Asp, Asn, Glu and Gln.

Particularly preferred cyclic peptide has the amino acid sequence: cyclic Cys-Ile-Xaa-Ser-Cys (SEQ ID NO: 7); where Xaa represents an amino acid residue selected from the group including Asp, Asn, Glu and Gln, and the peptide ring is formed via disulfide bond between two cysteine residues.

The method of inhibition mediated R-Katherina cell adhesion involves the introduction of cells expressing R-cadherin in contact with inhibiting the adhesion amount of a selective peptide antagonist of R-cadherin of the present invention. For example, retinal angiogenesis inhibited by injection to a patient suffering from a pathological vascular retinal angiogenesis, inhibiting angiogenesis amount of a peptide antagonist of R-cadherin of the present invention. Similarly, targeting negative lines of hematopoietic stem cells in the developing vascular network inhibited by BB is the origin of stem cells in contact with the inhibiting targeting the vascular network of a number of selective peptide antagonist of R-cadherin of the present invention. Inhibited targeting Lin-HSC, such as endothelial precursor cells, at the developing vascular network is useful for treatment of diseases associated with abnormal development of blood vessels, such as age-related macular degeneration and diabetic retinopathy.

Brief description of drawings

In the section of the drawings figure 1 shows the pattern of clustering of cadherin and cadherin-regulated cell adhesion;

figure 2 shows amino acid sequence variants of preprally R-cadherin of human (SEQ ID NO: 1), containing the sequence IDSMSGR (SEQ ID NO: 2) at residues 222-228;

figure 3 shows amino acid sequence variants of preprally R-cadherin of human (SEQ ID NO: 3)containing the sequence INSMSGR (SEQ ID NO: 4) at residues 222-228;

figure 4 shows amino acid sequence variants of preprally R-cadherin of mouse (SEQ ID NO: 5)containing the sequence IDSMSGR (SEQ ID NO: 2) at residues 229-225;

figure 5 (A) shows homology sequences at residues 24-92 N-cadherin mouse and various R-cadherine (conservative residues (blue) and nonconservative (red) residues); there is a homology between R-Katherine mammals from human, mouse and rat, each of which contains a sequence of IDS or INS at residues 53-55, unlike R-cadherin is urity and N-cadherin mouse which contain the sequence IDP and INP, respectively, in residues 53-55; (B) cyclic and linear peptides, in this field, the corresponding residues of R-cadherin of mouse and human and N-cadherin mouse, synthesized, along with the corresponding control peptides: cyclic CIDSC (SEQ ID NO: 8), cyclic CINPC (SEQ ID NO: 9), IDSMSGR (SEQ ID NO: 2), IDSASGR (SEQ ID NO: 10), INPASGQ (SEQ ID NO: 11), cyclic CSDIC (SEQ ID NO: 12) and cyclic CRADC (SEQ ID NO: 13); partial sequence cadherin, is shown in figure 5 (A)represent from top to bottom: N-cadherin mouse (SEQ ID NO: 14), R-cadherin mouse (SEQ ID NO: 15), R-cadherin rat (SEQ ID NO: 16), R-cadherin human (SEQ ID NO: 17), the other R-cadherin human (SEQ ID NO: 18) and R-cadherin chicken (gallus gallus) (SEQ ID NO: 19);

figure 6 (A) shows A micrograph showing the aggregation of L-cells expressing R-cadherin and N-cadherin; (B) presents a bar chart of the percent aggregation of L-cells, mediated by R and N-Katherine in the presence and in the absence of calcium;

7 demonstrated stable transfection of L cells R-Katherina and N-Katherina; (a) Western blot turns R-cadherin; (B) Western blot turns N-cadherin; (C-E) micrograph painted L-cells, demonstrating the expression of R-cadherin (C) and N-cadherin (D), compared with cells not expressing any R-cadherin or N-cadherin (E);

on Fig graphically presents select the inhibition of aggregation of L-cells, expressing cadherin, using peptides containing IDS that are associated with cells expressing R-cadherin, compared with peptides containing INP, which are associated with cells expressing N-cadherin;

figure 9 reflects the selective inhibition of vascularization of the mouse retina after intravitreal injection of cyclic CIDSC (SEQ ID NO: 8) compared with the cyclic CINPC (SEQ ID NO: 9); (A) show micrographs of retinas of micerd/rdat the stage of development of P2; (B) show micrographs of retinas of micerd/rdat the stage of development P7; (C) is a column chart superficial vascularization; (D) is a column chart deep vascularization;

figure 10 shows the results of flow cytometry analysis of the expression of R-cadherin in hematopoietic stem cells (HSC);

figure 11 displays the cross-sectional micrographs of retinas of micerd/rdtreated with various peptides of the invention and the control peptides; and

on Fig reflected blocking targeting Lin-HSC at the developing vascular network of the retina peptides antagonists R-cadherin of the invention.

A detailed description of the preferred embodiments

As used here and in the accompanying claims, the term "cyclic peptide" refers to what olecular, contains many amino acids connected in a chain by peptide bonds, the ends of the chain are connected with the formation of rings of amino acid residues. The cyclic peptide may be linked to the peptide bond, a disulfide bond between the two amino acid residues, such as cysteine residues, or by any other suitable linking group. Ones linking group can be any chemical compound capable of reacting with functional groups on each end of the peptide chain to the formation of ties between them. For example, the two ends of the peptide chain can be associated with non-protein amino acids, such as, for example, 3-aminobutyric acid, or disulfide formed ones thiol groups, such as amide thioglycolic at the amino end and an amide formed from 2-aminoethanethiol at the carboxy-end.

As used here and in the accompanying claims, the term "pharmaceutically acceptable" and its grammatical variants, when referring to the media and other inert fillers, means that such materials can be used to introduce the patient to the person without the formation of undesirable physiological side effects such as irritation of the retina or eyes, nausea, dizziness, unclear or limited vision, cytotec what was mentioned and the like.

The term "amino acid"as used here and in the accompanying claims, in General, refers to any alpha amino acid. Preferably, the peptides of the present invention contain 21 amino acids encoded by the genetic code, although it may also include modified amino acid residues. Amino acids can be in the L, D or D, L form. Preferably, the peptides of the present invention contain amino acids L-shape. To minimize the possibility proteinasesdegradationin vivoentered the peptides of the present invention can include one or more amino acid residues in D-form.

The selected peptide, which is a selective antagonist of R-cadherin mammal contains from 3 to 30 amino acid residues, three of the neighboring residue peptides have the amino acid sequence Ile-Xaa-Ser. Xaa represents an amino acid residue selected from the group consisting of Asp, Asn, Glu and Gln. Preferably, Xaa represents Asp or Asn. The peptide antagonist of R-cadherin of the present invention may be linear or cyclic.

Selective peptide antagonist of R-cadherin in the present invention plays the sequence Ile-Asp-Ser and Ile-Asn-Ser found in the EC1 domain of R-cadherin mammals, but not in other cadherin molecules. Peptides containing the sequence Ile-Xaa-Ser, m is able to communicate with molecules of R-cadherin mammals and to counteract them. Xaa preferably represents the residue of aspartic acid (Asp) or an asparagine residue (Asn), to match the natural sequence in the molecules of R-cadherin mammals. Residues of glutamic acid (Glu) and glutamine (Gln) are also suitable as Xaa because of its chemical similarity to Asp and Asn, respectively.

In one preferred embodiment, the peptide contains at least seven amino acid residues, and seven neighboring amino acid residues of the peptide have the amino acid sequence Ile-Xaa-Ser-Met-Ser-Gly-Arg (SEQ ID NO: 6). Xaa represents an amino acid residue selected from the group consisting of Asp, Asn, Glu and Gln. Preferably, Xaa represents Asp or Asn.

In another preferred embodiment, the peptide is a cyclic peptide containing from 3 to 10 amino acid residues, arranged in a ring, three neighboring residue peptides have the amino acid sequence Ile-Xaa-Ser, where Xaa represents an amino acid residue selected from the group including Asp, Asn, Glu and Gln. Preferably, Xaa represents Asp or Asn.

Preferred cyclic peptide with five amino acids, arranged in a ring has the formula:

where X1and X2independently represent an amino acid residue or mnozhestvennosti residues to 10, linked by peptide bonds; Y1and Y2independently represent amino acid residues linked together by a disulfide bond, and Xaa is an amino acid residue selected from the group including Asp, Asn, Glu and Gln. Preferably, Y1and Y2represent cysteine residues, connected by a disulfide bond (i.e. the rest of the cystine).

Particularly preferred cyclic peptide has the amino acid sequence: cyclo Cys-Ile-Xaa-Ser-Cys (SEQ ID NO: 7); where Xaa represents an amino acid residue selected from the group including Asp, Asn, Glu and Gln, and the ring formed by using a disulfide bond between two cysteine residues. Preferably, Xaa represents Asp or Asn.

The method of inhibition mediated R-Katherina cell adhesion involves the introduction of cells expressing R-cadherin in contact with inhibiting the adhesion amount of a selective peptide antagonist of R-cadherin of the present invention. Cells can enter into contact withwith peptideantagonistin vivoby introducing inhibition of cell adhesion amount of the antagonist to a mammal suffering from a disease or condition the treatment of which can be achieved by inhibition mediated R-Katherina cell adhesion (for example, diseases of sets the weave characterized by abnormal proliferation of blood vessels). For example, a person suffering from age-related macular degeneration or diabetic retinopathy can be treated with a selective peptide antagonist of R-cadherin of the present invention. Preferably, the antagonist is administered in the form of a pharmaceutical composition comprising the antagonist and a pharmaceutically acceptable carrier.

For selective targeting or antagonism R-cadherin peptides and compositions of the present invention can be introduced in a therapeutically effective amount of a parenteral, oral, by inhalation, or topically in a standard dosage form together with pharmaceutically acceptable carriers, means of delivery and adjuvants. The term "parenteral"as used here, includes intravenous, subcutaneous, intramuscular, epigastric, intraorbital (for example, in the vitreous body) and intraperitoneal, and an introduction by infusion.

You can use any suitable route of administration, and the pharmaceutical composition, including selective peptide antagonist of R-cadherin of the present invention, is administered in a dose effective for the intended treatment. The person skilled in the art will readily determine a therapeutically effective amount for treatment of a specific m the medical condition or inhibiting its development, using preclinical and clinical studies, known in the medical field.

The term "therapeutically effective amount", as used here, refers to the amount of active ingredient that has a biological or therapeutic effect on medical tissue, system, animal or human that is being hunted by a doctor or researcher.

The term "inhibit", as used here, refers to slow down, delay or termination of the medical condition or biochemical interaction, but does not necessarily indicate the complete cessation of status or complete elimination of interaction. Lasting effect in a patient or prolonged decline in the severity of symptoms indicates that a medical condition is positively controlled (i.e. ingibirovalo).

Dosage regimes for peptide antagonists of R-cadherin of the present invention and containing compositions based on several factors, such as age, weight, gender and type of disease in the patient, the severity of the condition, the route of administration and antagonistic activity of specific peptide antagonist. The dosage can be changed depending on the above factors. For example, for inhibiting angiogenesis of the retina used dosage levels with the order from about 0.01 mg to the roughly 1000 mg per kilogram of body weight. The preferred dosage levels are in the range of from about 0.01 mg to about 100 mg per kilogram of body weight.

For administration by injection a composition comprising the peptide of the present invention, constitute, together with pharmaceutically acceptable carrier, such as water, saline or an aqueous solution of dextrose. For injection, the typical daily dose is from about 0.01 mg to about 100 mg per kilogram of body weight, which is administered daily as a single dose or as multiple doses depending on the above factors.

The pharmaceutical compositions of the present invention, containing a selective peptide antagonist of R-cadherin of the invention and a pharmaceutically acceptable carrier may also contain pharmaceutically acceptable inert fillers. Pharmaceutically acceptable inert fillers which may be included in the pharmaceutical compositions of the present invention, include, for example, physiologically tolerated surfactants, solvents, buffered agents, preservatives and the like, are well known in the prior art.

For example, for inhibiting angiogenesis of the retina to a patient suffering from a pathological proliferation of blood vessels in the retina, is administered a therapeutically effective the number of peptide antagonist of R-cadherin of the present invention. Entered the peptide selectively binds to R-Katherina in the retina, thus interrupting and inhibiting angiogenesis in it. Preferably, the peptide antagonist is administered by injection into the vitreous body.

Targeting Lin-HSC at the developing vascular network is inhibited by injecting stem cells into contact with inhibitory targeting the vascular network of a number of selective peptide antagonist of R-cadherin of the present invention. Inhibited targeting Lin-HSC, such as endothelial precursor cells, at the developing vascular network can be used in the treatment of diseases associated with abnormal development of blood vessels, such as age-related macular degeneration and diabetic retinopathy. Preferably, Lin-HSC bring into contact thein vivoby introducing peptide antagonists of R-cadherin of the present invention to a mammal, such as man, suffering from a vascular proliferative disease or condition.

The following non-limiting examples are presented to further illustrate various aspects of the invention. To a person skilled in the art it is obvious that modifications of the examples and illustrated embodiments disclosed here, can be implemented without deviating from the essence and scope of izopet the deposits.

Example 1. Synthesis of peptide

The peptides of the present invention and various control peptides were synthesized in the Central institution of proteins and nucleic acids, The Scripps Research Institute with the use of solid-phase synthesis and purified to the highest possible degree of purity (purity more than 95%) in accordance with the data of HPLC analysis. The sequence of the peptides was analyzed using mass spectrometry to confirm the correct synthesis of peptides. All peptides blocked at the amino-end of the amide group and azetilirovanie at the carboxy-end. Cyclic peptides were prepared with cysteine residues at the amino - and carboxy-ends, to create a disulfide bond and the formation of a ring containing five amino acid residues. Figure 5 (B) shows the resulting peptides: cyclic CIDSC (SEQ ID NO: 9), cyclic CINPC (SEQ ID NO: 9), IDSMSGR (SEQ ID NO: 2), IDSASGR (SEQ ID NO: 10), INPASGQ (SEQ ID NO: 11), cyclic CSDIC (SEQ ID NO: 12) and cyclic CRADC (SEQ ID NO: 13).

Example 2. Transfection of L-cells

R-cadherin mouse and plasmids N-cadherin were kindly provided by Dr. Masatoshi Takeichi (Kyoto University, Japan). The plasmid was subcloned into the vector pDsRed2-N1 (Clontech) to encode an integral protein and red fluorescent protein (RFP), attached to the C-end of cadherin molecules. L-cells (cells fibroblasts L929 mouse, ATCC #CRL-2148) was stable transfusional either R or N-cadherin the pDsRed2-N1 using system transfection calcium phosphate (Life Technologies) according to the manufacturer's Protocol. After selection by growing in nutrient media with the addition of geneticin (700 ng/ml geneticin G418, Gibco BRL) was selected positive clones. The cells were examined for expression of RFP and tested on the expression of cadherin by immunofluorescence staining and Western blot turns. Figure 6 illustrates the aggregation of L-cells expressing R and N-cadherin. Figure 6 (A) shows the micrograph of L-cells expressing R-cadherin (left) and N-cadherin (in the middle), aggregation in media containing calcium, compared with nitrostilbene L-cells, which are not aggregated. 6 (B) is a bar chart illustrating the percentage of aggregation of cells, shown in Fig.6 (A). Cells transfetsirovannyh N-Katherina and R-Katherina, trypsinization in buffer containing approximately 5 mm of calcium chloride (denoted as TC), formed large clusters of cells, whereas endogenous L-cells showed little aggregation in buffer containing calcium. Cells trypsinization with EDTA in the buffer without calcium (denoted as TE), showed a slight aggregation, regardless, was transfusional cells Katherine or not.

Example 3. Cell culture and immunofluorescence

Transfetsirovannyh L cells or L-cells of the wild type were grown in modified CPE is e Eagles (MEM) with the addition of a basic salt solution Earl's, 2 mm Glutamax, 1 mm sodium pyruvate, 0.1 mm nonessential amino acids and 10% fetal bovine serum. Transfetsirovannyh cell line was grown in media with the addition of approximately 700 μg/ml G418 (all reagents culture media were from Gibco BRL). For immunofluorescence cells were grown to approximately 75% confluence on top of the glass strips. The cells were fixed in 4% paraformaldehyde for about half an hour, followed by blocking with 5% normal goat serum and 5% fetal production of serum in 1x phosphate buffer saline (PBS). Polyclonal antibodies goats against R-cadherin or N-cadherin (Santa Cruz) was used with a dilution 1:200, and the fluorescence was provided by incubation with a labeled secondary IgG anti-goat Alexa488 (Molecular Probes). Image made using fluorescent confocal microscope (Radiance 200 (BioRad). For immunoblot analysis cells were dissolved in buffer containing 1% Triton X-100. Approximately 50 μg of the full cell lysate was added to each track 8% polyacrylamide gel and proteins were separated by electrophoresis. To visualize the bands used monoclonal antibodies (1:1000, BD Biosciences), specific for N-cadherin or R-cadherin.

Figure 7 (A) shows the Western blot turns native L-cells and L-cells transfected with R-Katherina and N-catherinem the colored antibody R-cadherin. Only cells transfetsirovannyh R-Katherina, showed significant levels of expression of R-cadherin. Figure 7 (B) shows the Western blot turns native L-cells and L-cells transfected with R-Katherina and N-Katherina and stained with antibody to N-cadherin. Only cells transfetsirovannyh N-Katherina, showed significant levels of expression of N-cadherin. Fig.7 (C) is a fluorescence micrograph of cells expressing R-cadherin labeled with a fluorescent antibody cadherin, demonstrating expression on the cell surface only of molecules of R-cadherin. Fig.7 (D) is a fluorescence micrograph of cells expressing N-cadherin labeled with a fluorescent antibody cadherin, demonstrating expression on the cell surface only of molecules N-cadherin. 7 (E) is a fluorescent micrograph of native L-cells exposed to fluorescent antibody cadherin, but not showing the expression of cell surface molecules cadherin any type.

Example 4. Test for aggregation

L-cells were grown to a high level of merger and subsequent trypsinization with 0.01% trypsin + 5 mmCaCl2and without EDTA (TC) or with 0.01% trypsin with 0.1 mm EDTA and without calcium (TE). Cells were collected and washed with subsequent resuspending in vernom Hanks solution (HBSS) + 1% BSA (TC) or without (TE) 5 mm CaCl 2. Cells were cultured at 37°C in 0.5 ml of solution at 2 × 105cells in each well of a 24-hole plates with shaking at about 60-70 rpm with varying concentrations of peptide. All tests were performed three times. The degree of aggregation of the cells represented by the ratio of the total number of particles after 2 hours of incubation (N2hr) to the initial number of particles (N0). Particles were counted on hemocytometer using the sum of 8 separate 20 ml of accounts in the hole until (N0), and after (Nt) incubation. The results are illustrated in Fig.

Example 5. Treatment of mice by injection of the peptide into the vitreous body

Peptides were dissolved in PBS + 10% dimethyl sulfoxide to a concentration of 10 mm. Approximately 0.5 μl or 1.0 ál of 10 mm solution of peptide was injected into the vitreous cavity of 2-day or 7-day-old mice, respectively. In the P5 and P11 of the retina were prepared in accordance with the description, and blood vessels and astrocytes visualized using immunohistochemistry. Quantitative analysis of peripheral vascularization, the length of the vessels and the area of the vessel surface during formation of blood vessels is received by a display of retinas with injection under the same settings microscopy. Then got the number using LASERPIX software® (BioRad) using the control mice generate the same litter, used for the initial normalization of the degree of vascularization of the retina. Quantitative analysis of the effect on the formation of deep vessels obtained by focusing on the anterior chamber of the eye to the plexus of vessels normal depth using confocal microscopy and counting the number of vessels penetrating into the layer of visual receptors. The results are shown in Fig.9.

Example 6. The selection of stem cells and enrichment

Bone marrow cells were isolated from adult transgenic mice, in which reproduced GFP was merged with the promoter β-actin (ACTbEGFP, the Jackson Laboratory, Bar Harbor, Maine). Then gathered monocytes by separation using density gradient using Histopaque (Sigma) and marked panel antibodies conjugated with Biotin line (CD45, CD3, Ly-6G, CD11, TER-119, Pharmingen, San Diego, CA) for selection Lin-. Cells Lin+and Lin-were separated using column magnetic separation (MACS, Miltenyi Biotech, Auborn, CA). As it was determined that cells CD31-represent the best control defunct HSC subpopulations that are identified with the help of targeting vessels, cells CD31-separated from monocytes using MACS separation using antibodies to CD31 and used as a negative control for functioning Lin-HSC. HSC from wild-type mice were analyzed for exp is essay R-cadherin using labeling of cells with antibodies anti-R-cadherin (sc-6456, Santa Cruz Biotech) and labeled with Alexa-488 secondary antibodies anti-goat donkey (molecular probes) and using the flow cytometer FACS calibur (Beckton Dickinson, Franklin Lakes, NJ). The results are shown in figure 10.

Example 7. Cultivation of cells HSC, injections and quantitative analysis

Before injection Lin-HSC were cultured with 100 nm antibody, blocking R-cadherin (SC-6456, Santa Cruz Biotech) or preimmune goat IgG in saline solution with phosphate buffer for about 1 hour at approximately 37°C. Intravitreal injection into the eye P6 performed with the use of 0.5 μl of a solution HSC. Then the retina was investigated in P12 in the form of total drug or slices. Targeting Lin-HSC quantified by counting the total number of stem cells in the retina using eight different areas of the review of the retina: left, right, top and bottom quadrants (3/4 of the distance to the periphery of the retina), two intermediate quadrant (1/4-1/2 of the distance to the periphery), the injection and the area of the optic nerve. These cells are characterized by localization in shallow, intermediate or deep layers or absence of target cells, which lie in the back layer of the visual receptors). The number anaclaudia cells in the layer of visual receptors is presented as a percentage of total observed the stem cells. The results are presented in figure 11 and Fig.

DISCUSSION

Selective peptide antagonist of R-cadherin in the present invention act as peptides that mimic the key motives for the recognition of R-cadherin (i.e. the sequence IDS and INS found in the R-cadherin mammals). Without being bound to theory, the peptide antagonists of the present invention will likely block the function of adhesion molecules R-cadherin due to competitive interactions with the EC1 domains of molecules R-cadherin on the surface of cells. The peptide antagonists of the present invention can be used for studying the molecular functions and in the treatment of diseases associated with cell adhesion, and, in General, they are easier to diffuse into the tissue afterinjectionsin vivothan antagonists based on antibodies.

The tissue morphogenesis during the development of most tissues, including nervous tissue of the retina, involves the selective binding of adhesion molecules between cells. Such selectivity of binding allows you to group similar differentiated cells and prevents cell types to invade mismatched fabric structure. However, despite extensive study of the properties and functions of catherinew, in particular N - and E-cadherine, the General mechanism responsible for the specificity cadherin, not yet open. The peptide antagonist of R-cadherin p the present invention selectively interact with molecules of R-cadherin mammals without significant binding to other classes cadherin. These peptides contain the sequence IDS (or its homologues INS, IES and IQS), which corresponds to the region in the EC1 domain of cadherin, residues 53 to 55 of SEQ ID NO: 17 and 18, in which, reportedly, there are important interactions on the surface of the adhesion on the basis of structural, mutational analysis and analysis of homology sequences.

Without being bound to theory, it is believed that the motive IDS establishes direct contacts with sequence VDI adjacent molecules of the cadherin on the surface of adhesion. Because residues 53-55 of catherinew, apparently, needed to transversale, and because unlike other areas that are important for adhesion, this short amino acid sequence is not conserved classic family members of catherinew, this area acts as a determinant of specificity cadherin. In fact, cyclic IDS (CIDSC, SEQ ID NO: 8) selectively inhibits cell aggregation mediated R-Katherina, while the corresponding analogue of N-cadherin, cyclic INP (CINPC, SEQ ID NO: 9), selectively inhibits cell aggregation mediated N-Katherina. N-cadherin and R-cadherin are the most homologous family member of catherinew. In fact, although all cadherine, including R - and N-cadherin, mainly interact homophily way, R - and N-cadh the Rin are the only two classic family members of catherinew, among them was observed functional heterodimer. Thus, it is very unlikely that cyclic IDS and cyclic INP have different functional properties in relation to N and R-cadherin, but matching properties in respect of any other member of the family of catherinew. These studies demonstrate that the motive IXS (where X is a D, N, E, or Q, that is, corresponds to residues 53-55 R-cadherin and their homologues) plays an important role in ensuring homologous binding molecules of R-cadherin. Apparently, based on the specificity of IXS to R-cadherin and INP to N-cadherin, respectively, placed the remains of other members of the family of catherinew (for example, motif IER E-cadherin and motive IEK P-cadherin) also provide specificity to the other classic cadherins.

Previous studies have shown that antibodies against R-cadherin interrupt the vascularizationretinain vivo. Apparently, the vascularization of the superficial plexus is terminated by reason of violation of indirect R-Katherina guide signals transmitted by astrocytes located in front of endothelial cells. The expression of R-cadherin was also observed in areas that would later discover deep plexus of vessels, immediately before the invasion of blood vessels. It is believed that the molecules of R-cadg the Rina in these areas send endothelial cells on the right plexus of vessels, since the injection locking R-cadherin antibodies causes the vessel to bypass the normal vascularization layers.

Currently, a similar phenotypes vessels produced by injection of cyclic IDS (CIDSC, SEQ ID NO: 8) as in the course of the superficial and the deep vascularization of the retina. As cyclic IDS selectively substantially interrupted aggregation, mediated R-Katherina,in vitro(that is, without significant interruption aggregation, mediated by N-Katherina), it is likely thatin vivothe phenotype of vessels were also produced due to interactions with high affinity cyclic IDS with R-Katherina. In addition, injection of cyclic INP (CINPC, SEQ ID NO: 9), which was an effective inhibitor of aggregation, mediated by N-Katherina, but not the aggregation of R-cadherinin vitrodid not cause significant phenotype of retinal vessels. Collectively, these results confirm the specific role of R-cadherin in the control vessels.

Development of selective peptide antagonists of R-cadherin of the present invention was based on structural, biochemical and mutational analysis of the various members of the family classic catherinew. As you know, the tryptophan-2 is important for the function of catherinew along with a sequence of HAV at amino acid residues 79-81 N-cadherin and R-cadherin. F. chicosci, reportedly, linear and cyclic peptides containing the sequence HAV, block-mediated N-Katherinathe growth of neuritisin vitro. However, these sequences are completely conserved in all of cadherin molecules and therefore cannot give a binding specificity. Other residues should also establish important contacts on the surface of the dimerization, with some nonconservative residues are important for the recognition of catherinew. Focused on the residues in the amino-terminal repeat cadherin (EC1). The most important contact residues were localized in three areas, 35-45 amino acids, amino acids 53-59 and amino acids 79-86. Residues 53-59 contained in most data cadherin-specific residues that are potentially important in shaping the surface of the dimerization. Of these residues 53-55 had special significance. Thus, the peptide analogs were developed from this area to increase the probability of obtaining R-cadherin-specific peptides. Developed similar peptides against sequences from the N-cadherin mouse, the closest family member of catherinew, and other control peptides and used for comparative analysis.

Aggregation-mediated Katherina

Selected cells in mouse fibroblasts (line L929), usually called aimie L-cells, since they are known to contain no endogenous expression of cadherin. Created transfectants resistant to R-cadherin, and used them to verify the validity of the designed peptides on the aggregation-mediated R-Katherina. Also created transfectants resistant to N-cadherin, and used them to assess the peptides from the point of view of selectivity to Catherine, on the basis of their effect on aggregation, mediated by N-Katherina. The immunoblot analysis revealed high levels of R-cadherin and the absence of expression of N-cadherin in clone 8 R-cadherin (R-cad8), while in clone N-cadherin 3 (N-cad3) found high levels of expression of N-cadherin and the absence of expression of R-cadherin, as shown in Fig.7. Immunofluorescence confirmed the expression data in the selected clones corresponding cadherin. When testing in the aggregation analysis revealed morphological changes transfinitely lines of cells after transfection cadherin. While the parent (i.e. nitrostilbene) L-cells remained divided in separate cellular particles, mouse, transfetsirovannyh R-Katherina and N-Katherina, formed large, calcium-dependent (buffer TC) clusters of cells due to the tight intercellular communication, as shown in Fig.6 (A). Clusters mediated R-Katherina aggregation eliminated by using starting the second trypsinization cells with EDTA solution and aggregation in the buffer without calcium (buffer, as shown in Fig.6 (B).

The effect of the peptide on the aggregation of cells

Peptides were added with varying concentrations in wells aggregation to test their effectiveness in blocking mediated Katherina aggregation. Cyclic IDS inhibited mediated R-Katherina aggregation with IC50approximately 300 μm. Linear peptide EDSMSGR (SEQ ID NO: 2) also blocked mediated R-Katherina aggregation. However, its effectiveness (IC50˜ 900 μm) was approximately 3 times lower than for cyclic IDS. Since cyclic peptides also turned out to be much more soluble and simpler to use than linear peptides, further emphasis was made solely on the analysis of cyclic peptides (Fig (A)). The effect of cyclic IDS was specific to R-cadherin, because the aggregation of N-cadherin was observed mild action. In contrast, the corresponding sequence specific to N-cadherin, cyclic INP, inhibited mediated N-Katherina aggregation with IC50slightly below 300 microns (Fig (B)) is similar to the action of cyclic IDS on the aggregation of R-cadherin. Cyclic INP had a mild effect on mediated R-Katherina aggregation.

Other control peptides, cyclic RAD (CRADC, SEQ ID NO: 13) and cyclic SDI (CSDIC, SEQ ID NO: 12) had a minor effect on AG is agazio, indirect or R-Katherina or N-Katherina. As a comparison, the tested cyclic peptide HAV (CHAVC, SEQ ID NO: 20), already known as effective in blocking adhesion mediated by any classical cadherin molecules. In the analysis of cyclic HAV blocked the aggregation-mediated R-Katherina and N-Katherina with IC50in the range between 150 and 200 microns. Thus, the cyclic IDS and cyclic INP is selectively blocked the adhesion of R or N-cadherin, respectively, only with a slightly lower sredstvami than the non-specific peptide that blocks all Katherine. It was shown that in previous studies using antibodies against R-cadherin, normal vascularization of the development of the retina is interrupted. These antibodies were also effective when the interrupt aggregation, mediated by Katherina in the system analysis with IC50approximately 10 nm, as shown in Fig (C).

Effect of peptides on the vascularization of the retina

Peptides were injected into the vitreous cavity of the eye of newborn mice. When the cyclic peptides IDS or cyclic HAV was injected into the eyes of a two-day mice, and emerging as a result of this vascular network investigated three days later, on day 5 after birth (P5), the formation of blood vessels was interrupted, and the results were similar to that observed when the injection is of NITEL. These retina characterized as containing less extensive peripheral vascularization and fewer interconnected vessels in areas of vascularization compared with normal control mice of the same litter without injections. In General, the vascularization of the surface layer was reduced by half due to peptides that block R-cadherin, while the retina by injecting N-cadherin-specific cyclic INP were relatively normal (see Fig.9(A-C)).

Selective peptide antagonist of R-cadherin of the present invention is also interrupted the normal vascularization of the deep layers of the retina. With the introduction of the cyclic peptide IDS in P7 immediately before penetration into the depth of the vessels of the superficial vascular network and the beginning of the formation of the deep plexus of vessels formed vascular network P11 was characterized by numerous processes of vessels that have migrated beyond the normal depth plexus of vessels in the avascular layer of the visual receptors. In addition, it is similar to the effect observed before the introduction of antibodies R-cadherin. On the contrary, the deep plexus of blood vessels of the eye with the injection of a cyclic peptide INP formed normally, as shown in Fig.9 (B and D).

R-cadherin is expressed Lin-HSC

The expression of R-cadherin hematopoi the terms stem cell (HSC) were analyzed to determine expressibility whether the cell adhesion molecules R-cadherin on the cell surface of cells, which functionally readying. Using flow cytometry analysis, R-cadherin expressively on the cell surface of almost 80% of the subpopulation Lin-HSC, while only 30% of the cells Lin+Express R-cadherin (figure 10). On the basis of the relative intensities of fluorescence between the two populations of cells, it is likely that cells Lin-also Express higher concentrations of R-cadherin on the cell surface than a small part of the R-cadherin-positive cells Lin+. Thus, the majority of cells into subpopulations that are functionally target vascular network of the retina, Express R-cadherin, unlike most cells anaclaudia subpopulations. It is interesting to note that other HSC subpopulations, which presents CD31, CD34 and Mac1-negative and do not possess the function of targeting, contained even fewer cells expressing R-cadherin.

Antibodies and peptides that block R-cadherin, terminating targeting HSC

To study the extent to which intercellular adhesion R-cadherin functions in targeting HSC at different layers of the retinal vessels, Lin-HSC blocked using R-cadherin-specific blocking antic the l before injection. Six days after injection of normal Lin-HSC found localized in only three layers of blood vessels: 1) superficial plexus of vessels localized in the layer of ganglion cells, 2) deep plexus of vessels, localized near the outer plexiform layer and 3) the intermediate layer, localized in the front edge of the inner nuclear layer. Figure 11 (A) shows cross-sections of retinas after injection of normal Lin-HSC (left), Lin-HSC, cultured with antibodies R-cadherin blocking adhesion (middle), and Lin-HSC, cultivated with preimmune goat IgG (right).

With prior cultivation Lin-HSC with antibodies anti-R-cadherin before injection many of these cells have lost the ability to correct targeting, while cells pre-cultured with preimmune IgG, functioning as an unlocked HSC. Apparently, targeting the deep and intermediate layers of the vessels affected, in particular, blocking the adhesion of R-cadherin, because in these areas found localized relatively few HSC blocked R-Katherina. Cells, localized in the superficial plexus of blood vessels, also seemed less organized and were not localized in conjunction with endogenous network of vessels in the same step is no, as normal Lin-HSC or pre-cultured with preimmune IgG.

Many of Lin-HSC pre-cultured with the antibody R-cadherin, migrated through the retina, through all three layers of the vessel and joined to the outer edge of the visual receptors near the RPE layer. Almost half of HSC with blocked R-Katherina found on the outer edge of the layer of visual receptors (11(B)). For comparison, in the control retinas with HSC injection, pre-cultivated with preimmune IgG, only 15% of the HSC did not focus on this area. A large part of anacalypsis cells from control retinas found near the injection site, and they probably can be attributed to the cells, which were issued under the retina when removing the needle. With the exception of the injection site from the calculation of the number anacalypsis HSC, cultivated with preimmune IgG, decreased to 10%. Because usually Lin-HSC almost not observed in this "especially the deep" layer, this small percentage anacalypsis HSC, cultivated with preimmune IgG can probably be attributed to the fact that preimmune IgG could bind approximately 10% of the cells Lin-(figure 10). These related molecules of IgG can not specific to prevent proper adhesion just because of steric hindrance. However, the difference between the quantity of glue is OK, anacalypsis due to a specific block of R-cadherin and non-specific blocking IgG, is significant.

On Fig (A) shows the confocal images through the z-plane three plexuses of vessels and the outer edge of the layer of visual receptors. Normal targeting appropriate plexus of blood vessels and along the endogenous vessels (red) was observed in Lin-HSC (green), blocked preimmune IgG goat. Blocking adhesion of R-cadherin led to the localization of multiple Lin-HSC at the outer edge of the layer of the visual receptors, and cells nalivalasja normal plexus of vessels sought to the accumulation and were not localized along the endogenous vessels (red). Fig (B) is a column chart showing what percentage anacalypsis cells in relation to the entire population of HSC in the retina was significantly higher for populations Lin-HSC-locked R-Katherina values (P<0,01).

The foregoing description should be considered as illustrative but not as limitations. However, other options within the essence and scope of the present invention can be easily presented by experts in the field of technology.

1. The selected cyclic peptide having the amino acid sequence Cys-Ile-Xaa-Ser-Cys (SEQ ID NO:7);

2. Cyclic peptide according to claim 1, where XAA represents Asp or Asn.

3. Cyclic peptide according to claim 1, where XAA represents Asp.

4. Cyclic peptide according to claim 1, where XAA represents Asn.

5. Pharmaceutical composition for inhibiting angiogenesis of the retina containing the selected peptide according to claim 1 and a pharmaceutically acceptable filler.

6. The method of inhibition mediated R-Katherina cell adhesion, including the conversion of mammalian cells expressing the molecule R-cadherin on the surface, in contact with the inhibiting adhesion amount of a cyclic peptide according to claim 1.

7. Method of inhibiting angiogenesis of the retina, including the introduction of a patient suffering from a pathological vascular angiogenesis of the retina, the number of cyclic peptide according to claim 1, inhibiting angiogenesis.

8. Method of inhibiting targeting hematopoietic stem cell (HSC) at the developing vascular network, for inhibition mediated R-Katherina cell adhesion and inhibition of angiogenesis of the retina, including bringing the above HSC in contact with inhibitory targeting the vascular network of a number of selected cyclic peptide according to claim 1.



 

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