Antibodies and pharmaceutical compositions containing them, which are suitable for inhibition of activity of metalloproteins

FIELD: biotechnologies.

SUBSTANCE: invention refers to a compound having general formula (I),

where: m and n are independent integer numbers of 1 to 6; each of X1-X3 and Y1-Y3 is O; each of R1-R3 is independently chosen from a group consisting of hydrogen or alkyl; and R is O-(CH2)x-C(-O)NR'-(CH2)y-NHR'. Besides, x and y are independent integer numbers of 1 to 6; and R' is chosen from the group consisting of hydrogen and alkyl. In addition, an antibody containing an antigen recognition area, use of the compound, pharmaceutical composition, use of the antibody, method for MMP-2 or MMP-9 activity inhibition in a sample and a composition for determining MMP-2 or MMP-9 are proposed.

EFFECT: invention allows obtaining compounds that are able to inhibit MMP-2 or MMP-9 activity.

11 cl, 16 dwg, 4 tbl, 11 ex

 

The SCOPE AND LEVEL of TECHNOLOGY

The present invention relates to molecules of hapten and the antibody directed against them, which can be used for inhibiting the activity of metalloproteins, such as metalloprotease, and to methods of using these antibodies for the treatment of diseases such as metastatic cancer, which are associated with abnormal activity of metalloproteins.

Matrix metalloprotease (MMPs) are key enzymes involved in the remodeling of extracellular matrix (ACM). These enzymes are able to break down the various components of the connective tissue of the articular cartilage and basement membranes.

Family genes MMP person consists of at least 28 structurally related proteins (see FIGURE 1), which have similar overall spherical topology (FIGURE 2 and Borkakoti, 1998). Each MMP secreted as an inactive latent proferment. Domain catalytic zinc consists of approximately 180 amino acids, where highly conservative sequence HE-GH-LGL-H gives three his-tag (i.e., N) residues that are associated with the metal ion zinc Zn(2+). The fourth ion binding site of the catalytic zinc in proferment associated with a cysteine residue (Morgunova and others, 1999), in which the enzyme activation is detached from the active site (van Warth and B is redal-Hansen, 1990). As a result, the fourth binding site in the activated MMP is the water molecule, which also has a hydrogen bond with the conservative glutamic residue. This process facilitates the hydrolysis of the peptide bond of the target substrate with an activated water molecule.

Uncontrolled destruction of the connective tissue by metalloprotease is a feature of many pathological conditions, probably resulting from excessive activity of MMP or unbalanced ratio between the natural tissue inhibitors of MMP (TIMP) and MSE. TIMP inhibit MMPs by forming stoichiometric complexes with the active binding site of zinc (Gomez and others, 1997; Henriet and others, 1999; Bode and others, 1999; will and others, 1996). When the levels of TIMP insufficient, slow progressive degradation of the ACM may result in the loss matrix of the cartilage in rheumatoid arthritis (Valachovic etc., Arthritis Rheum, 35:35-42, 1992) and osteoarthritis (Dean and others, J. Clin. Invest. 84:678-685, 1989) or degradation of the matrix of bone tissue in osteoporosis (hill and others, Biochem. J. 308:167-175, 1995). In other situations, such as congestive heart failure, may occur rapid degradation VKM heart (Armstrong and others, Canadian J. Cardiol. 10:214-220, 1994).

In addition, as you know, MMP plays a certain role in meiosis cytokines and chemokines, such as galectin-3 (About the LEng J., Biochemistry, 1994 33(47):14109-14), plasminogen (Patterson, B.K., JBC, 1997 272(46):28823-5), interleukin-8, connective tissue activating peptide III, platelet factor-4 (van den Steen, 2000 Blood. 2000 Oct 15; 96(8):2673-81.), prointernational-1β (Senbet, 1998), the α chain of the receptor for interleukin-2 [Su, B.K., Hsu, S.M., Ho, H., lien, H.K.RAKISHEV, Huang, S. Kaliev, Lin, A.D. "the New role of metalloproteinases in mediated cancer suppressing the immune response", Cancer Research (2001) 61, 237-242] and retransforming factor-β growth [TGF-β, Yu, K., Stamenkovic, I. Localized on the cell surface matrix metalloproteinase-9 proteoliticeski activates TGF-beta and promotes invasion and tumor angiogenesis", Genes Dev (2000) 14, 163-176].

Other pathological conditions that are also associated with the unregulated activity of MMP include rapid remodeling VKM metastatic tumor cells. In such States activated MMP or expressed by cancer cells or surrounding tissues. There is strong evidence that MMPs are involved in the growth and spread of tumors (see, for example, Davidson and others, Chemistry & Industry, 258-261, 1997, and referred to in article reference materials). In the process of metastasis of the tumor MMP are used for the destruction of the ACM, allowing the cancer cells of the primary tumor to invade neighboring blood vessels, where they are transported in different PRS is Ana and create secondary tumors. Invasive growth in these secondary areas mediated MMP, which destroys tissue. In addition, the activity of MMP promotes invasive growth of new blood vessels, called angiogenesis, which tumors grow beyond a certain size. It was demonstrated that among the members of a family of secreted MMP MMP-9 man, also known as gelatinase In, plays a major role not only in the catabolism of the extracellular matrix (ACM), but in the processing of proteins that are relevant to neurological diseases such as multiple sclerosis (PC) (Opdenakker, 2003). Recent studies have shown that MMP-9 plays a critical role in promoting autoimmune diseases by splitting pre-treated collagen type II (van den Steen, 2004). Products are fragments of collagen type II, which are the remaining epitopes, which are believed to generate autoimmune diseases.

Given the important role of MMPs in the physiology and pathology of man, not surprisingly, made numerous attempts to create drugs that inhibit excessive MMP activity.

Attempts drug development focus on those classes of inhibitors that contain a functional group, coordination of the zinc ion and this deactivates the target MSE. One is m this class of inhibitors are hydroxamate inhibitors, small peptide analogues of fibrillar collagens, which specifically interact in a bidirectional manner via the hydroxyl and carbonyl oxygen hydroxamic group with the zinc ion in the catalytic site [Grams and others, (1995), Biochem. 34:14012-14020; Bode and others, (1994), EMBO J., 13:1263-1269].

The MMP inhibitors on the basis of hydroxamate usually consist or of the carbon skeleton of the (WO 95/29892, WO 97/24117, WO 97/49679 and EP 0780386), peptidnogo skeleton (WO 90/05719, WO 93/20047, WO 95/09841 and WO 96/06074) or coworkers peptide backbone [Schwartz and others, Progr. Med. Chem., 29:271-334(1992); Rasmussen and others, Pharmacol. Ther., 75:69-75 (1997); Dennis and others, Invest. New Drugs, 15:175-185 (1997)]. Alternatively, they contain sulfamidoethanols group, which is linked on one side with a phenyl ring, and sulfamidate that is associated with hydroxamate group through a chain of one to four carbon atoms (EP 0757984 A1).

Other MMP inhibitors on the basis of thiol peptides are amides, which have activity, collagenase inhibitory (U.S. patent 4,595,700), N-carboxyquinolone containing beenrecognized that inhibit MMP-3, MMP-2 and collagenase (Duret and others, WO-9529689), lactam derivatives which inhibit MMP, TNF-alpha and aggrecanases (see U.S. patent 6,495,699), and tricyclic sulfonamidnuyu compounds (see U.S. patent 6,492,422).

Although inhibitors of MMP-based peptides have obvious therapeutic p. the potential their use in clinical therapy is limited. Hydroxamate-based peptide expensive to produce and have a low metabolic stability and oral bioavailability [e.g., batimastat (BB-94)]. These compounds quickly glucuronoside, are oxidized to carboxylic acid and excreted in the bile [Singh and others, Bioorg. Med. Chem. Lett. 5: 337-342, 1995; Hodgson, "Remodeling IDRM", Biotechnology 13: 554-557, 1995)]. In addition, inhibitors of MMP-based peptides often have the same or similar for each of the enzymes MMP. For example, BA timestat reportedly has IC50 values of approximately from 1 to 20 nmol against each of MMP-1, MMP-2, MMP-3, MMP-7 and MMP-9 [Rasmussen and others, Pharmacol. Ther., 75(1): 69-75 (1997)]. In addition, the use of multiple hydroxamate inhibitors was associated with serious side effects, such as mystiske-flight problems from marimastat (BB-2516), widespread ulcerative rash from CGS27023A (Novartis) [Levitt and others, 2001, Clin. Cancer Res. 7: 1912-1922] and liver, anemia, pain in shoulders and back, thrombocytopenia, nausea, fatigue, diarrhea and pulmonary veins from BAY12-9566 (Bayer) [Heath and others, 2001, Cancer Chemother. Pharmacol. 48: 269-274]. Moreover, clinical phase III trials in patients with advanced cancer who were prescribed marimastat, prinomastat (AG 3340, Agouron) and Bay 12-9566, not demonstrated clinical efficacy in the inhibition of metastasis (Zucker and the RV, 2000, Oncogene 19: 6642-50).

Other MMP inhibitors are chemically modified nemikrobnoy tetracyclines (HMT)that block the expression of several MMPsin vitro.However, it was found that the effectiveness of these compoundsin vivois limited, for example, the inhibitor HMT, doxycycline, reduced tissue levels of MMP-1 but not MMP-2, 3, or 9 in atherosclerotic plaques in the carotid artery in humans (axis ' and others, 2002, Stroke 33: 2858-2864).

A recently developed inhibitor mechanism of MMP, SB-3CT, which is based on x-ray crystallographic information about the active site of MMP (brown and others, 2000). X-ray absorption studies have shown that binding of this molecule with catalytic zinc reconstructs conformational environment around the metal ion in the active site back to the environment proferment [Kleifeld and others, 2001, J Biol. Chem. 276: 17125-31]. However, therapeutic efficacy of this substance remains to be determined.

Another class of natural inhibitors are monoclonal antibodies. Several antibodies have been selected against specific peptide sequences in the catalytic domain of MMP-1 (Galvez and others, 2001, J. Biol. Chem., 276: 37491-37500). However, although these antibodies were able to inhibit the activity of MMPin vitroresults demonstrating the effectiveness of such bodiesin vivohas not yet been received.

As is shown above, the catalytic site of MMP includes coordinated metal ion, which becomes available for binding of the substrate after enzyme activation (see Figa-C). Thus, the idea is that traditional antibodies directed at the primary amino acid sequence of the enzyme will not distinguish active from inactive forms of the enzyme and, therefore, will not be powerful inhibitors of these enzymes.

The authors of the present invention previously demonstrated that antibodies that recognize electronic and structural determinants of the catalytic site of MMP, are powerful inhibitors and, as such, can be used for the treatment of diseases associated with an unbalanced activity of MMP (see PCT publication WO 2004/087042).

Thus, there is a recognized need and the desire to get specific heptanone compounds that mimic the electronic and structural determinants of the catalytic site of metalloproteins as specific antibodies against them.

BRIEF description of the INVENTION

According to one aspect of the present invention proposed a compound having General formula (I):

where:

m and n are independently integers from 1 to 6;

X1-X3 and Y1-Y3 are independently O or S;

2)x-C(=O)NR'-(CH2)y-NR'r R"

whereas:

x and y are independently integers from 1 to 6; and

R' and R" independently are selected from the group consisting of hydrogen, alkyl and cycloalkyl.

According to other features of the preferred embodiments described below, the compound has the formula (II):

where R=-CH2-C(O)NH-CH2-CH2-NH2

According to another aspect of the present invention proposed a compound having the formula (II):

where R=-CH2-C(=O)NH-CH2-CH2-NH2

According to another aspect of the present invention proposed an antibody that contains a plot of the recognition of antigens that are able to specifically bind the above connection.

According to other characteristics of these preferred embodiments the area of recognition of antigens contains the amino acid sequence of a CDR selected from the group consisting of SEQ ID NO:7, 8, 9, 10, 11 and 12.

According to other characteristics of these preferred embodiments, the amino acid sequence of a CDR encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NO:13, 14, 15, 16, 17 and 18.

According to other characteristics of these preferred options OS is supervising the antibody is able to inhibit the activity of metalloproteins.

According to other characteristics of these preferred embodiments metalloproteins is the matrix metalloprotease.

According to other characteristics of these preferred embodiments the matrix metalloprotease is gelatinase.

According to other characteristics of these preferred embodiments gelatinase choose from a group of MMP-2 and MMP-9.

According to another aspect of the present invention, a method for production of an inhibitor of metalloproteinase, and this method includes the creation of antibodies directed to the above connection, creating this inhibitor metalloproteinase.

According to other characteristics of these preferred embodiments the antibodies are polyclonal antibodies.

According to other characteristics of these preferred embodiments the antibodies are monoclonal antibodies.

According to another aspect of the present invention proposed a pharmaceutical composition comprising the above-mentioned antibody and a pharmaceutically acceptable carrier.

According to another aspect of the present invention, a method of treatment of a disease associated with imbalanced or abnormal activity of metalloproteins the patient, and this method includes assigning patients who NTU therapeutically effective amount of any one of the antibodies on paragraphs 4-10, thereby treat the disease associated with imbalanced or abnormal activity of metalloproteins the patient.

According to other characteristics of these preferred embodiments the disease is an inflammatory disease of the internal organs.

According to another aspect of the present invention, a method for inhibiting the activity matrix metalloprotease in the cell, and the method includes the contact of cells with any one of the antibodies on paragraphs 4-10, this inhibiting the activity of matrix metalloprotease in the cell.

The present invention successfully overcomes the disadvantages of known configurations by providing new heptenophos compositions that can be used to generate antibodies that recognize the electronic and structural determinants of the catalytic site of metalloproteins.

If not stated otherwise, all technical and scientific terms in the present description have the same meaning that is commonly understood by the average expert in the field of the present invention. Although methods and materials similar or equivalent to the one described here, can be used in practice or testing of the present invention, suitable methods and materials are described below. In case of conflict, the main thing is the description of the invention,including definitions. In addition, the materials, methods and examples are only illustrative, but not restrictive.

BRIEF DESCRIPTION of DRAWINGS

Hereinafter the invention is described, with an example only, with reference to the accompanying drawings. Now with particular reference to the detailed drawings emphasizes that shows details are given for example only and for purposes of illustrative description of the preferred embodiments of the present invention and are presented for suggestions of what is considered the most appropriate and easily understood description of the principles and conceptual aspects of the invention. In this regard, no attempt is made to show structural details of the invention in more detail than is necessary for the fundamental understanding, the description taken with the drawings making apparent to experts in the field, as several forms of the invention can be implemented in practice. In the drawings:

FIGA-D diagram of the molecular structure of Co/ZnTCPP - [meso-Tetrakis (4-carboxyphenyl)-porfiriato] cobalt/zinc (II) (FIGA, Imisdp - [2-(2-aminoacylchromones)-ethoxymethyl]-Tris-[2-(N-(3-imidazol-1-yl-propyl))ethoxymethyl]methane, and conservative binding zinc-protein in the website of the catalytic zinc of MMP.

FILE-N - three spatial diagrams of the structures shown in FIGA-D. Note that ZnTCPP plan remains the major conformation, and Satsr has distorted conformation of the microcycle. It is noticeable that the structure Imisdp highly similar nearest environment of the ion catalytic zinc of MMP-9, as shown in .1G.

FIGA - structural overlay three-dimensional computed structures Imisdp (green carbon atoms) and three conserved histidine in the active site of MMP-9 (PDB code 1GKC, grey carbon atoms). Ion catalytic zinc is shown as the orange bowl, the water molecule is shown as a blue ball, nitrogen colored blue, oxygen is red-colored.

FIGW - structural overlay porphyrin ring ZnTCPP (CSD code AKICOM) (green carbon atoms) and three conservative histidines in the active site of MMP-9 (green carbon atoms PDB code 1GKC), ion catalytic zinc is shown as the orange ball, nitrogen painted blue.

FIGA-WITH - images of Western blotting showing the ability of mouse IgG - immobilized agarose monoclonal antibodies to pull the recombinant catalytic domain of MMP-2 (MMP-2cat) or Pro-MMP-2 and Pro-MMP-9 from the solution. In each experiment used antibodies S, E and E. FIGA - MMP-2cat (2 μg) were incubated with IgG-intimissi - agarose (control, lane 1) or a monoclonal antibody against Satsr, ZnTCPP and Imisdp (10 μg) - IgG-intimissi - agarose for 2 hours at 20°C, immunoprecipitate (tracks 2, 3, 5) were centrifuged and washed three times,separated on the gel SDS/PAGE and visualized by staining Kumasi. FIGW - Pro-MMP-2, Pro-MMP-9 were incubated with monoclonal antibodies-intimissi IgG - agarose in the same order as in A. Immunoprecipitate (tracks 2, 4, 6 on the left and 1, 3, 5 right) and the unbound fraction (tracks 1, 3, 5 left and 2, 4, 6 right) were separated on the gel SDS/PAGE and visualized by staining Kumasi. FIGS - air-conditioned environment of the cells NT that through the activation of ARMA (left) or not passed (right), was immunoassay using monoclonal antibodies against Atsr and analyzed by Western-blotting using antibodies specific against MMP-2.

FIGA-IN - graphics of Leinweber-Burke inhibition of MMP-2 and MMP-9 (V) using monoclonal antibodies against Satsr. Unit speed is µmol/s-1and the unit substrate is ámol-1. FIGA - concentration monoclonal antibodies were: 6 (black triangles), 18 (black squares), 24 (white circles), and 0 µmol (white squares). The concentration of MMP-2cat was 200 nmol. FIGW - Inhibition of full-length MMP-9, activated ARMA, the concentration of monoclonal antibodies was 6 (white squares), 12 (black triangles), 24 (white squares) and 0 µmol (black squares). The concentration of MMP-9 was 20 nmol. The model of inhibition shows that monoclonal antibody against Atsr behaves as a competitive inhibitor of MMP-2 and MM is-9.

5 is a graph showing the inhibition of MMP-2 and MMP-9 using monoclonal antibodies against Imisdp. The catalytic domain of MMP-9 (20 nmol) (black circles) or full length MMP-2 activated ARMA (black triangles, 5 nmol)was added to a mixture of fluorogenic substrate OCAcPLGLA2pr(Dnp)-AR-NH2 (10 µmol) in buffer solution R containing increasing concentrations of monoclonal antibodies. The lines represent the alignment according to the method of least squares to the equation:

vi/vo=(Km+[S])/(Km(1+[I]/Ki)+[S]), using the program Origin.

On FIGA shows the spectra of K-edge active and inhibited by monoclonal antibody against Atsr forms of MMP-2cat for zinc. Shows the normalized raw data XAS in the field of K-edge of the zinc active (points) and the complex of MMP-2cat - monoclonal antibody (solid line).

On FIGU shown marginal position in which the complex (solid line) MMP-2cat - monoclonal antibody is shifted towards higher energy with respect to active MMP-2cat (points).

On FIGS shows the results of EXAFS to active (black) and inhibited (green) forms of MMP-2cat. These results are presented in R-space and transformed back to k-space.

FIGA-IN - pictures showing the ability of monoclonal antibodies against Satsr to inhibit the activity gelatinase on the surface the displacement of the cells. Representative fluorescent micrographs of cells NT deposited on a cover glass coated with DQ-gelatin in the presence or absence of 1 µmol monoclonal antibodies E. Gelatinolytic activity of the cell surface was evaluated as a measure of fluorescence emitted from biodegradable gelatin. Untreated cells showed significant activity gelatinase on the cell surface, which was significantly ingibirovany in the presence of 1 µmol of monoclonal antibodies against Satsr. Staining with 4'-6-diamidino-2-phenylindole (DAPI), blue color indicates the location of the nuclei of cells.

FIG diagram showing the configuration of the various active sites of MMPs (SI-pocket).

FIG.9 is a diagram of the synthesis Imisdp.

Figure 10 shows the amino acid sequence of the antibodies of the present invention with selected areas CDR.

FIGA-D - pictures and models, showing that S binds only the active conformation MMP and MMP.

FIGA: Detection of active MMP, which is purified together with S from ascitic fluid of mice. Monoclonal antibody (10 μg), purified from the ascitic fluid of mice containing MMP, was subjected to the Western blotting (WB) using commercial antibodies to MMP. Unbound monoclonal antibody IgG, which was purified in the same way served as a neg is negative control monoclonal antibodies). Human Pro-MMP purified from transformed cells Hilla, served as a molecular weight marker for identification of the active species. Purification was performed by affinity chromatography using pellet protein G, which linked monoclonal antibody through his constant domain, leaving the binding site of the antigen free to interact with the antigen.

FIGV WITH: S monoclonal antibody, immobilizovannoi to pellet protein And analyzed for its ability to pull catalytic fragment Rhome, Rhome or MMP (in the absence of hemopexin and pradamano) from solution. MAbs S (10 µg), immobilizovannoi to the granules sepharose protein And incubated with the catalytic fragment MMP (1 µg) - FIGU, Rhome - PIGS top or Rhome (2 µg) PIGS down, for 2 hours at 20°C. the Bound granules complex monoclonal antibody was separated by centrifugation and washed three times, separated on the gel SDS/PAGE and visualized by staining Kumasi. Immunoprecipitate (S) and unbound fractions were separated on the gel SDS/PAGE and visualized by staining Kumasi. As a negative control for non-specific absorption of the enzyme incubated with beads of sepharose protein A.

.11D: three-Dimensional structure MMP without domain hemopexin with Pro-domain (bottom) and without predomina (top) shown in surface the om representation (PDB ID: 1CK7). Catalytic and fibronectin domains are shown by cyan, and propeptide shown in red. Ion catalytic zinc is shown as an orange sphere; it is associated with three conservative histidine shown as yellow bars. As shown, propeptide domain sterile blocks the active site.

FIGA-IN - graphics and data related to the mechanism of inhibition of MMP-9 using monoclonal antibodies S.

FIGA: recombinant catalytic fragment of MMP-9 (without hemopexin and predomina) were incubated with different amounts of monoclonal antibodies. Residual enzyme activity was measured after adding fluorogenic peptide substrate (10 Microm). Ki was estimated by fitting to the equation for competitive inhibition (vi/vo=Km+[S]/(Km(1+I/Ki)+[S]) Km=9,14±0,8) (Box). Active MMP-9 (at a fixed concentration of 2 nmol) pre-incubated for 60 minutes at 37°C in the absence (•) or presence of 0.7 (■) or 2 µm (A) monoclonal antibodies in 100 mmol NaCl, 10 mmol CaCl2, 100 mmol Tris pH 7.5. Then added fluorogenic peptide substrate (Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2) to obtain the final concentration (S) in the range of 0-30 µm, and the initial rate of hydrolysis of the substrate was determined by measuring the increased fluorescence. The values of apparent Km and Vmax were obtained by fitting the experimental data is x to the equation Michaelis-Menten. The obtained values were used for reconstructing the double reverse curve of Liveware-Berka, where the intersection point indicates competitive inhibition of MMP-9 using S.

FIGW: Different MSEs were pre-incubated with varying amounts of monoclonal antibodies. Residual enzyme activity was measured after adding fluorogenic peptide substrate (10 Microm). Ki was estimated by fitting to the equation for competitive inhibition (vi/vo=Km+[S]/(Km(1+I/Ki)+[S]) Km=2.46±0,34 for full length MMP, purified from cells Hila, Km=16±1 for the catalytic domain MT-MMP). Effective inhibition S also were detected using the full-length MMP-2 and MMP-9 (data not shown).

FIG - structural overlay of different MMP showing the total conservative topology of the active site changes, mainly in the peripheral loops. MMP (PDB 1GKC) - cyan, MN (PDB 1QIB) - bright red, MT-MMP (PDB 1BUV) - orange, MN (PDB 1MMQ) - red, TAS (PDB 2147) - yellow. Conservative histidine shown as rods, ion catalytic zinc - like the orange ball. It is noticeable that the General topography of the peripheral loops of MMP-2 and MMP-9 are similar. This may explain the selectivity S to MMP-2 and MMP-9 in the tested group of enzymes.

FIGA WITH a fluorescent micrograph illustrating that S inhibits the activity gelatina the s on the surface of cells. Representative fluorescent micrographs (generated direct demograficheskim analysis) cells NT, placed on a cover glass coated with DQ-gelatin, in the absence (FIGA) or presence (PIGV) 5 µmol monoclinal antibodies or 15 µmol SB-3CT-based mechanism nanomolar inhibitor gelatinas (FIGS). Activity gelatinase on the cell surface was analyzed as a measure of fluorescence emitted by decomposing gelatin. Untreated cells showed significant activity gelatinase on the surface (green), which significantly inhibited in the presence of monoclonal antibodies.

FIGA WITH graphs illustrating the impact S-processing on the various manifestations of acute colitis, induced by DSS in mice C57BL/6. The disease was induced by 2%DSS for 5 days. Treatment S in the amount of 1.5 mg/mouse was administered daily intraperitoneal injections, starting from day 0. FIGA: Clinical indicator was assessed by daily monitoring DAI (i.e., the combined weight, rectal bleeding and stool consistency, on a scale from 0 to 4). Data are expressed as the distribution of points the mean value for each animal on days 6-10. FIGW: Length of the colon. FIGS: Mortality. The data presented are the combined results from the two e is sperimental, with 15 mice in the group.*, a significant effect compared to mice with colitis who did not receive treatment (p<0,05).

FIG - graph of the results of x-ray absorption spectroscopy at the K-edge of the zinc active MMP (black) and inhibited complex MMP-S (red). The results are presented in the form of the radial distribution from the zinc ion. The regional location of the complex (red) catalytic domain of MMP-9 - monoclonal antibody is shifted towards higher energy with respect to active MMP-9 (insert), indicating binding to ion catalytic zinc. Structural data analysis x-ray spectroscopy shows that S directly binds a zinc ion and forms a five-axis complex of zinc-protein. It is noticeable that this mode of binding is similar to the binding of TIMP in the active site of MMP.

DESCRIPTION of the PREFERRED embodiments

The present invention relates to antibodies and their fragments, which can be used for inhibiting the activity of metalloproteins. Specifically, the antibodies of the present invention can be used for the treatment of diseases associated with an unbalanced activity matrix metalloprotease, such as multiple sclerosis, autoimmune diseases, and metastatic cancers.

The principles and effect of the present invention can is be better understood with reference to the drawings and the corresponding description.

Before to explain in detail at least one alternative implementation, it is necessary to say that the invention is not limited in application details set out in the following description or Examples. The invention can be implemented in other embodiments. You must also understand that applied here phraseology and terminology is intended for descriptive purposes and should not be construed as limiting.

Matrix metalloprotease involved in many biological processes, from proliferation and differentiation of cells and remodeling of the extracellular matrix (ACM) to vascularization and cell migration. These processes require a precise balance between the functions of the matrix metalloprotease (MMPs) and their natural tissue inhibitors (TIMP). Loss of balance is a symptom of numerous pathological conditions, including metastatic tumors, neurodegenerative disease and osteoarthritis.

There are many known inhibitors of MMP, including small peptide inhibitors, such as hydroxamate, nemikrobnoy tetracyclines and monoclonal antibodies. Although the first is limited by high production costs, high decay rate, low oral availability and lack of specificity, none of the latter has not demonstrated therapeutic efficacy in vivo./p>

The authors of the present invention previously found that antibodies that recognize electronic and structural determinants of the catalytic site of metallothionen, can be used as potent inhibitors. The use of haptens, imitating associated with metal catalytic site metallothionen, as immunogens helped to create a highly effective therapeutic antibodies that can be used for the treatment of clinical conditions characterized by increased activity of metalloproteins (see WO2004/087042 authors of the present invention).

When implementing the present invention into practice, the authors have developed a new heptenone connection, which accurately simulates the local structure and conformation of the reactive site zinc in MMP. This compound, [2-(2-aminoacylchromones)-ethoxymethyl]-Tris-[2-(N-(3-imidazol-1-yl-propyl))ethoxymethyl]methane, abbreviated Imisdp (see FIGURE 1), can simulate the 4-coordinate geometry and force field, similar to that induced by zinc ion coordinated trachyscelini matrix and water. Almost rectangular conformation formed by three imidazole bases and a water molecule as the fourth ligand. On FIGA shows the overlay constructed three-dimensional model compounds Imisdp on the catalytic site of MMP-9 (PDB 1GKC), to the which has been modified for to represent the rectangular geometry of the ligands of zinc. Modifications include replacement of the ligand present in the x-ray structure (hydroxamate inhibitor), a water molecule and the full optimization of the enzyme to a local minimum of the multi-layered approach QM/MM (see materials and methods). There is a high similarity between the computed location histidinol zinc in MMP-9 and Imisdp in terms of distances ε-nitrogen of histidine from zinc ions (2,04+0,06 and 2.02, respectively) and the relative orientation of the three histidines against the metal.

As illustrated below in the description and Examples, the authors present invention were immunized mice drug Imisdp and spent their screening for antibody to MMP, cross-reacting with MMP-2 and MMP-9. This antibody was named S (see FIGURE 10 and Examples 1-2). It was found that S binds MMP-2/9 and competitively inhibits the activity of MMP-9, MMP-2 (Ki-range 1 µm - 5 µm) and MT-MMP (Ki 15 µmol, see table 4 below). Binding and inhibition of MMP-9 and MMP-2 has been demonstrated in vitro and in situ using biochemical and biophysical tools (see Examples 4-7 and 9). Importantly, S binds only to the activated form of MMP-9 and MMP-2 (see Example 3 and Example 8). In this form of the enzyme lacking prodomain, which protects the catalytic zinc, in parts of the enzyme. The authors show the and, the antibodies generated in this way are able to connect in vivo MMP-9 (FIGA). In addition, the authors showed that the antibodies of the present invention have therapeutic potential for the treatment of inflammatory bowel disease (Example 10).

These data support the use of Imisdp as an important reagent (platform) for the production of inhibitors of metalloproteinase and S and derived peptides and peptidomimetics as a valuable therapeutic agent.

These results demonstrate the potential use of these antibodies as a platform for developing selective peptide inhibitors for individual MSEs by displaying bacteriophages and point mutations of monoclonal antibodies or their fragments.

Thus, according to one aspect of the present invention proposed a compound having General formula (I):

where:

m and n are independently integers from 1 to 6;

X1-X3 and Y1-Y3 are independently O or S;

R1-R3 independently are selected from the group consisting of hydrogen, alkyl and cycloalkyl; and R is-(CH2)x-C(=O)NR'-(CH2)y-NR'r R"

because:

x and y are independently integers from 1 to 6; and

R and R" are independently selected from the group consisting of hydrogen, alkyl and cycloalkyl.

According to a preferred variant osushestvlyaetsya aspect of the present invention, this compound is [2-(2-aminoacylchromones)-ethoxymethyl]-Tris-[2-(N-(3-imidazol-1-yl-propyl)-ethoxymethyl] methane, named, Imisdp, which has the General formula (II):

where R=-CH2-C(=O)NH-CH2-CH2-NH2

Synthesis Imisdp described in Example 7.

Because Imisdp simulates the local structure and transitional conformation of the reactive site zinc of MMP-9 and MMP-2, it can be used for the production of inhibitors of metalloproteinase.

Thus, according to one aspect of the present invention, a method for production of an inhibitor of metalloproteinase.

The method is carried out by creating antibodies or fragments of antibodies directed to the above-described compound (i.e., Imisdp). See Examples 1-2 and section "Materials and methods" section "Examples".

The term "metalloprotein" the present invention relates to metal-associated protein, in which the site of bonding with metal forms part of the catalytic domain of the enzyme, which electronically and structurally resembles that of the Imisdp.

Metalloprotein this aspect of the present invention preferably is metalloproteases - MMP (for example, gelatinases, such as MMP-2 and MMP-9).

It will be clear that all members of the MMP family are perceived as latent enzymes, which after activation is converted to active farms is, for example, in which the metal ion in the active site is available for communication with the substrate. For example, to explain the activation of MMP in vitro was previously proposed model of transition cysteine". Model transition cysteine suggests that after activation site education due to latent zinc is converted into the site of the formation of ties with the catalytic zinc by dissociation of the thiol propeptide s (Cys) from the zinc atom. Splitting this propeptide leads to the destruction produmannoy structure of the enzyme, and the loss of protect ion of the catalytic zinc. Therefore, the metal ion and the pocket of the active site is available for communication with the substrate and hydrolysis [van Warth and Birkedal-Hansen (1990) Proc. Natl. Acad. Sci. USA 87, 5578-5582].

Antibodies and antibody fragments created by the present invention, serve as potent inhibitors of MMP because of their ability to bind metal ion and the coordinating amino acids in the catalytic site of zinc, this specifically inhibiting the active conformation of these enzymes that are directly involved in the above-mentioned pathological processes.

Used herein, the term "antibody" refers to intact molecules antibodies, and the phrase "antibody fragment" refers to its functional fragment, such as Fab, F(ab')2 and Fv, which are capable to communicate with macrophages. These functional fragments of antibodies to them shall have the following definitions: (i) Fab, the fragment which contains a monovalent fragment molecules are antibodies that establishes a connection with the antigen can be obtained by digestion of whole antibody with the enzyme papain to obtain intact light chain and a portion of one heavy chain; (ii) Fab', the fragment of the antibody molecules, which can be obtained by treating whole antibody with pepsin, followed by reduction to obtain the intact light chain and part of the heavy chain; two Fab fragment' can be derived from antibody molecules; (iii) (Fab')2 fragment of an antibody, which can be obtained by treating whole antibody with the enzyme pepsin without subsequent recovery; F(ab')2 is a dimer of two Fab fragments', held together by two disulfide bonds; (iv) Fv, defined as the obtained genetic engineering fragment containing the variable region light chain and the variable region of the heavy chain expressed as two chains; and (v) single-stranded antibody ("SCA"), a molecule, obtained by genetic engineering, contains a variable region light chain and the variable region of the heavy chain associated with a suitable polypeptide linker as a genetically fused molecule with one chain; and (vi) a peptide coding for a single defining complementarity region (CDR).

Well-known ways of generating antibodies (i.e., monoclonal and polyclonal). EN is the body can be created by any known method, which can be applied induction for the production of antibody molecules in vivo, by screening immunoglobulin libraries or panels of highly specific binding reagents [Orlandi D.R. and others (1989) Proc. Natl. Acad. Sci. 86:3833-3837, winter, G. and others (1991) Nature 349:293-299] or create molecules of monoclonal antibodies by continuous cell lines in culture. They include, without limitation method hybridoma, how hybridoma human b-cells and method of hybridoma Epstein-Bar virus (EBV) [Kohler, and others (1975) Nature 256:495-497, Kosbor D., and others (1985) J. Immunol. Methods 81:31-42, Cote R.J. and others (1983) Proc. Natl. Acad. Sci. 80:2026-2030, S. p. Cole and others (1984) Mol. Cell. Biol. 62:109-120].

In those cases where the compounds of the present invention are too small to cause a strong immunogenic response, such antigens (haptens) can be connected with antigen-neutral media, such as media shell of hemocyanin (KLH) or serum albumin (e.g., bovine serum albumin (BSA)) (see U.S. patent 5,189,178 and 5,239,078 and Example 2). The connection with the carrier can be carried out using well known methods. For example, can be made a direct connection with the amine groups and then, if desired, can be accomplished restore formed aminokwasy. Alternatively, the carrier can be connected with the use of condensing agents, such is how developerirrational or other substances, dehydrating carbodiimide. For communication can also be used linker compounds; homobifunctional and generalfunctions.php linkers offers company Pierce Chemical Company, Rockford, Illinois. Received immunogenic complex can then be introduced suitable mammals, such as mice, rabbits, and other Suitable protocols include re-injection of the immunogen in the presence of adjuvants according to the schedule, which accelerates the production of antibodies in the serum. The titers of the immune serum can easily be measured by well-known procedures for immunological analysis.

The obtained antisera can be used directly, or can be obtained monoclonal antibodies, as described above.

Antibody fragments can be obtained by well known methods. (See, for example, Harlow and lane, "Antibodies: a Laboratory Handbook", Cold Spring Harbor Laboratory, new York, 1988, incorporated herein by reference). For example, fragments of antibodies according to the present invention can be prepared by proteolytic hydrolysis of the antibody or by expression in E. coli or mammalian cells (such as cell culture Chinese hamster ovary or other protein expression systems) of DNA encoding the fragment.

Alternatively, antibody fragments can be obtained PU is eating digestion of whole antibody with pepsin or papain by known methods. For example, antibody fragments can be produced by enzymatic cleavage of antibodies with pepsin to obtain 5S fragment denoted F(ab')2. This fragment can be further cleaved using a thiol reducing agent, and optionally a blocking group for sulphhydryl groups, obtained by cleavage of the disulfide bond, to receive a monovalent fragments 3.5S Fab'. Alternatively, an enzymatic cleavage using pepsin produces two monovalent fragment Fab' and fragment Fc. These methods are described, for example, Mikhail, in U.S. patents 4,036,945 and 4,331,647 and in the indicated reference materials, and these patents are incorporated herein in full by reference. See also porter P.P., Biochem. J., 73:119-126, 1959. Can also be used in other ways cleavage of antibodies, such as separation of heavy chains to the formation of monovalent fragments of light-heavy chain, further cleavage of fragments, or other enzymatic, chemical or genetic methods, in which the fragments form a connection with the antigen that is recognized by the intact antibody.

The Fv fragments contain communication circuits VHand VL. This relationship may be non-covalent, as described by Inbar and others, Proc. NAT'l Acad. Sci. USA 69:2659-25 62, 1972. Alternatively, the variable chains can be linked to imolecular by a disulfide bond or sewn chemicals such as glutaraldehyde. Preferably, the Fv fragments contain chain VHand VLUnited peptide linker. These proteins (sFv) with one chain that establishes a connection with the antigen is prepared by constructing a structural gene containing DNA sequences encoding domains of the VHand VLconnected by the oligonucleotide. This structural gene is introduced into an expression vector, which is then injected into the cell host, such as E. coli. Recombinant cell hosts synthesize single polypeptide chain with peptide-linker connecting the two domains V. Methods of production sFv described, for example, Whitlow and Filpula, Methods, 2:97-105, 1991; Baird and others, Science 242:423-426, 1988; Peck and others, Bio/Technology 11:1271-77, 1993; Ladnerom and others, U.S. patent 4,946,778.

The CDR peptides ("the smallest unit of recognition") can be obtained by constructing genes encoding the CDR of the corresponding antibodies. Such genes are prepared, for example, by the polymerase chain reaction to synthesize the variable region from RNA of cells that produce antibodies. See, for example, Larrick and fry, "Methods", 2:106-10, 1991.

It will be clear that for the treatment or diagnosis of people prefer to use humanized antibodies. Humanized forms of inhuman (e.g., murine) antibodies are chimeric molecules of immunoglobulins, immunoglobulin CE and or fragments (such as Fv, Fab, Fab', F(ab')2 or other sequences of antibodies that creates a connection with the antigens)that contain minimal sequence derived from a nonhuman immunoglobulin. Humanized antibodies include human immunoglobulins (antibody-recipient), in which residues, which form a complementary determining region (CDR) of the recipient are replaced by residues from a CDR of nonhuman species (donor antibody)such as mouse, rat or rabbit having the desired specificity, affinity and capacity. In some cases, the remains of a skeleton Fv of the human immunoglobulin are replaced by corresponding inhuman remnants. Humanized antibodies may also contain residues that are not contained either in the antibody-recipient, nor in the imported CDR sequences or frame. Humanitariannet antibody typically will contain essentially all of at least one, and typically two, variable domains, in which all or essentially all areas of the CDRs correspond to those of non-human immunoglobulin and all or essentially all areas of FR are those of the consensus sequence of human immunoglobulin. Optimally, humanitariannet antibody will also include at least a portion of the immunoglobulin constant region (Fc), typically that of a human body, control the immunoglobulin [Jones and others, Nature, 321:522-525 (1986); Richman and others, Nature, 332:323-329 (1988); Prest, Curr. Op.Struct. Biol., 2:593-596 (1992)].

Methods of humanizing the inhuman antibodies are well known. Humanitariannet antibody usually has one or more amino acid residues introduced into it from a source that is not a person. These inhuman amino acid residues are often referred to as import residues, which are usually taken from the import variable domain. Humanization can be performed according to the method of winter and his colleagues [Jonsi other, Nature, 321:522-525 (1986); Rehmann and other, Nature 332:323-327 (1988); Verhoeyen and others, Science, 239:1534-1536 (1988)], by substituting rodent CDR or CDR sequences instead of the corresponding sequences of a human antibody. Accordingly, such humanized antibodies are chimeric antibodies (U.S. patent 4,816,567), which is significantly less than an intact human variable domain is replaced by the corresponding sequence from nonhuman species. In practice humanitarianism antibodies are typically human antibodies in which some CDR residues and possibly some FR residues replaced by residues from analogous sites in rodent antibodies.

Human antibodies can also be produced by various known methods, including libraries of phages [Hoogenboom and winter, J. Mol. iol., 227:381 (1991); Marx and others, J. Mol. Biol., 222:581 (1991)]. How Cole and others, börner, etc. can also be used for the preparation of human monoclonal antibodies (Cole and others, "Monoclonal antibodies and cancer therapy, Alan R. Liss, p.77 (1985) and boerner and others, J. Immunol., 147(1):86-95 (1991)]. Similarly, human antibodies can be produced by introducing genes of human immunoglobulin in transgenic animals such as mice in which the endogenous immunoglobulin genes have been partially or completely deactivated. After replacing, you can observe the production of human antibodies, which is very similar in all respects to what happens in humans, including rearrangeable genes, Assembly, and range of antibodies. This approach is described, for example, in U.S. patents 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016 and in the following scientific publications: marks, etc., Bio/Technology 10, 779-783 (1992); lonberg and other, Nature 368 856-859 (1994); Morrison, Nature 368 812-13 (1994); Fishwild and other, Nature Biotechnology 14, 845-51 (1996); Neuberger, Nature Biotechnology 14, 826 (1996); lonberg and Huszar, Intern. Rev. Immunol. 13 65-93 (1995).

After receiving the antibodies they can be tested for the inhibitory metalloprotein activity. Relevant conditions described by knight and others, FEBS Letters 296(3):263-266(1992), Koston and other, Anal. Biochem, 99:340-345 (1979), Koston and other, Methods in Enzymology 80:771 et seq. (1981); Couston and others, Biochem. J., 195:159-165 (1981), Weingarten and others, Biochem. Biophys. Res. Comm., 139:184-1187 (1984) and in U.S. patents 4,743,587 and 5,240,958.

As mentioned, using the above methodology, the authors present invention was able to produce antibody-inhibitor matrix metalloprotease (MMP) MMP-2 and MMP-9, named S, the sequence of which is presented in SEQ ID NO:1. The CDR sequence presented in SEQ ID NO.7, 8, 9, 10, 11 and 12.

Thus, the present invention provides for any (poly)peptide sequence, which contains at least one of the above sequences of the CDR and its homologues and fragments while preserving its inhibitory metalloprotein activity (specific inhibition of catalytic activity of metalloproteins). An example of such a polypeptide is an antibody (see above).

Used herein, the term "polypeptide" includes natural peptides (decomposition products synthesized peptides or recombinant peptides) and peptidomimetics (usually synthesized peptides),as well as peptide and polypeptide, which are analogues of the peptides, which may be, for example, modification, making the peptides more stable in the body or more capable of penetrating into cells. Such modifications include, without limitation, modification of the N-end modification of the C-end, modified peptide bonds, including, without limitation, modification of the cores CH2-NH, CH2-S, CH2-S=O, O=C-NH, CH2-O, CH2-CH2, S=C-NH, CH=CH or CF=CH and mi is ificatio residues. Methods of preparation peptidomimetic compounds are well known and defined, for example, in the publication "Quantitative drug development", Kahraman DG., Chapter 17.2, F. Choplin Pergamon Press (1992), which is incorporated herein in full by reference. Further details in this regard are given below.

Peptide bonds (-CO-NH-) within the peptide may be substituted, for example, N-methylated bonds (-N(CH3)-CO-), ether linkages (-C(R)H-C-O-O-C(R)-N-), kilometrovymi bonds (-CO-CH2-), alkyla-nitrogen bonds (-NH-N(R)-CO-), where R is any alkyl, e.g., stands, carbon bonds (-CH2-NH-), hydroxyethylene bonds (-CH(OH)-CH2-), thioamide bonds (-CS-NH-), refinable double bonds (-CH=CH-), setrolename bonds (-NH-CO-), peptide derivatives (-N(R)-CH2-CO-), where R is the "normal" side chain, naturally presented on the carbon atom.

These modifications can occur on any of the links along the peptide chain, and even a few (2-3) at the same time.

Natural aromatic amino acids, Trp, Tyr and Phe, may be substituted for synthetic non-natural acid such as phenylglycine, teak, nafcillin (RAL), finalization, threonine, derivatives of Phe ring methylation, halogenated derivatives of Phe or o-methyl-Tyr.

In addition, the peptides of the present invention can also uklocation or more modified amino acids or one or more deamination monomers (for example, fatty acids, complex carbohydrates etc).

Used in this description and in the claims, the term "amino acid" or "amino acid" includes naturally occurring amino acids; amino acids often modified after translation in vivo, including, for example, hydroxyproline, phosphoserine and posttraining; and other unusual amino acids including, without limitation, 2-aminoadipic acid, hydroxylysine, isodesmosine, Norvaline, norleucine and ornithine. In addition, the term "amino acid" includes D - and L-amino acids.

In tables 1 and 2 below lists the naturally occurring amino acids (table 1) and non-conventional or modified amino acids (e.g., synthetic, table 2), which can be used with the present invention.

Table 1
Amino acidA three-letter abbreviationA one-letter symbol
AlanineAlaA
ArginineArgR
AsparagineAsnN
Aspartic acidAspD
CysteineCys
GlutamineGlnQ
Glutamic acidGluE
GlycineGlyG
HistidineHisH
IsoleucineIleI
LeucineLeuL
LysineLysTo
MethionineMetM
PhenylalaninePheF
ProlineProP
SerineSerS
ThreonineThr T
TryptophanTrpw
TyrosineTyrY
ValineValV
Any amino acid of the aboveXaaX

Table 2
Unconventional amino-CodeUnconventional amino-Code
α-aminobutyric acidAbuL-N-methylalanineNmala
α-amino-a-methylbutyrateVlgabuL-N-methylarginineNmarg
aminocyclopropane-CproL-N-metalsprayNmasn
carboxylateL-N-metilparabenaNfmasp
aminoadamantane acidAibL-N-methylcysteineNmcys
aminocarbonyl-NorbL-N-methylglucamineNmgin
carboxylateL-N-methylglucamine keyNmglu
cyclohexylaminChexaL-N-vtnbkubcnblbyNmhis
cyclopentylamineCpenL-N-methylisoleucineNmile
D-alanineDalL-N-metallationNmleu
D-argininDargL-N-methyllysineNmlys
D-aspartic acidDaspL-N-methylmethaneNmmet
D-cysteineDcysL-N-mernorialNmnle
D-glutamineDglnL-N-methylmorpholinNmnva
D-glutamic acidDgluL-N-meteoritenNmorn
D-histidineDhisL-N-methylphenylamineNmphe
D-isoleucineDileL-N-methylpropanNmpro
D-leucineDleuL-N-metalsinNmser
D-lysineDlysL-N-methylthionineNmthr
D-methionineDmetL-N-methyltryptophanNmtr
D-ornithineDornL-N-methyltyrosineNmtyr
D-phenylalanineDpheL-N-methylvalineNmval
D-Proline DproL-N-methylethylketonNmetg
D-serineDserL-N-methyl-t-butylglycolNmtbug
D-threonineDthrL-norleucineNle
D-tryptophanDtrpL-NorvalineNva
D-tyrosineDtyrα-methyl-aminoisobutyricMaib
D-valineTo dvalα-methyl-γ-aminobutyrateMgabu
D-α-methylalanineDmalaα-methylcyclohexylamineMchexa
D-α-methylarginineDmargα-methylcyclopentadienylMcpen
D-α-metalsprayDmasnα-methyl-α-nafcillinManap
D-α-methylaspartate Dmaspα-methylpenicillinMpen
D-α-methylcysteineDmcysN-(4-aminobutyl)glycineNglu
D-α-methylglucamineDmglnN-(2-amino-ethyl)glycineNaeg
D-α-methylhistidineDmhisN-(3-aminopropyl)glycineNorn
D-α-methylisoleucineDmileN-amino-α-methylbutyrateNmaabu
D-α-metallationDmleuα-nafcillinAnap
D-α-methyllysineDmlysN-benzylglycineNphe
D-α-methylmethaneDmmetN-(2-carbamylethyl)glycineNgln
D-α-meteoritenDmornN-(carbamoylmethyl)glycineNasn
D-α-methylphenylethylDmpheN-(2-carboxyethyl)glycineNglu

D-α-methylpropanDmproN-(carboxymethyl)glycineNasp
D-α-metalsinDmserN-cyclobutylmethylNcbut
D-α-methylthionineDmthrN-cyclohexylglycineNchep
D-α-methyltryptophanDmtrpN-cyclohexylglycineNchex
D-α-methyltyrosineDmtyM-cyclodecylNcdec
D-α-methylvalineDmvalN-cyclododecylNcdod
D-α-methylalanineDnmalaN-cyclooctylaminoNcoct
D-α-methylarginine N-cyclopropylmethylNcpro
D-α-metalsprayDnmasnN-cyclonicallyNcund
D-α-methylaspartateDnmaspN-(2,2-diphenylether)glycineNbhm
D-α-methylcysteineDnmcysN-(3,3-Nbhe
D-N-metallationDnmleuN-(3-indoleacetic) glycineNhtrp
D-N-methyllysineDnmlysN-methyl-γ-aminobutyrateNmgabu
N-methylcyclohexylamineNmchexaD-N-methylmethaneDnmmet
D-N-meteoritenDnmornN-methylcyclopentadienylNmcpen
N-methylglycineNalaD-N-methylphenylamineDnmphe
N-methylaminomethylNmaibD-N-methylpropanDnmpro
N-(1-methylpropyl " glycineNileD-N-metalsinDnmser
N-(2-methylpropyl " glycineNileD-N-metalsinDnmser
N-(2-methylpropyl " glycineNleuD-N-methylthionineDnmthr
D-N-methyltryptophanDnmtrpN-(1-methylethyl)glycineNva
D-N-methyltyrosineDnmtyrN-methyl-nafcillinNmanap
D-N-methylvalineDnmvalN-methylpenicillinNmpen
γ-aminobutyric acidGabuN-(p-gidroksifenil)glycineNhtyr
L-t-butylglycolTbugN-(thiomethyl)is Lizin Ncys
L-ethylglycineEtgpenicillaminePen
L-homophenylalanineHpheL-α-methylalanineMala
L-α-methylarginineMargL-α-metalsprayMasn
L-α-methylaspartateMasDL-α-methyl-t-bagillionMtbue
L-α-methylcysteineMcysL-methylethylketonMetg
L-α-methylglucamineMglnL-α-methylglutaricMglu
L-α-methylhistidineMhisL-α-methylhomopiperazineMhphe
L-α-methylisoleucineMileN-(2-methylthioethyl)glycineNmet
D-N-methylglucamineDnmgln N-(3-Narg
D-N-methylglucamineDnmgluN-(1-hydroxyethyl)glycineNfhr
D-N-methylhistidineDnmhisN-(hydroxyethyl)glycineNser
D-N-methylisoleucineDnmileN-(imidazolylalkyl)glycineNhis
D-N-metallationDnmleuN-(3-indoleacetic)glycineNhtro
D-N-methyllysineDnmlysN-methyl-γ-aminobutyrateNmgabu
N-methylcyclohexylamineNmchexaD-N-methylmethaneDNmmet
D-N-meteoritenDnmornN-methylcyclopentadienylNmcpen

N-methylglycineNalaD-N-methylphenylamineN-methylaminomethylNmaibD-N-methylpropanDNmpro
N-(1-methylpropyl " glycineNileD-N-metalsinDNmser
N-(2-methylpropyl " glycineNleuD-N-methylthionineDnmthr
D-N-methyltryptophanDnmtrpN-(1-methylethyl)glycineNval
D-N-methyltyrosineDnmtyrN-methyl-nafcillinNmanap
D-N-methylvalineDnmvalN-methylpenicillinNmpen
γ-aminobutyric acidGabuN-(p-hydroxyphenyl)glycineNhtyr
L-t-butylglycolTbugN-(thiomethyl)glycineNcys
L-ethylglycineEtg penicillaminePen
L-homophenylalanineHpheL-α-methylalanineMala
L-α-methylarginineMargL-α-metalsprayMasn
L-α-methylaspartateMaspL-a-methyl-t-butylglycolMtbug
L-α-methylcysteineMcysL-methylethylketonMetg
L-α-methylglucamineMglnL-α-methylglutaricMglu
L-α-methylhistidineMhisL-α-methylhomopiperazineMhphe
L-α-methylisoleucineMileN-(2-methylthioethyl)glycineNmet
L-α-metallationMleuL-α-methyllysineMlys
L-α-methylmethane MmetL-α-methylpiperazinMnle
L-α-metallosalenMnvaL-α-meteoritenMorn
L-α-methylphenylethylMpheL-α-methylpropanMpro
L-α-metalsinmserL-α-methylthionineMthr
L-α-methylvalineMtrpL-α-methyltyrosineMtyr
L-a-metallationMvalL-N-methylhomopiperazineNmhphe
N-(N-(2,2-diphenylether)N-(N-(3,3-diphenylpropyl)
carbonylmethyl-glycineNnbhmcarbonylmethyl(1)glycineNnbhe
1-carboxy-1-(2,2-diphenylethylamine)cyclopropeneNmbc

Peptides otluchennym affinity to the corresponding metalloprotease or enhanced biological activity can be created by well-known methods, including phage display and computational biology.

The peptides of the present invention can be synthesized by any means known to experts in the field of peptide synthesis. For solid-phase synthesis of peptides a summary of the many methods can be found in the publication: Stewart, J. M. and young, J. D. (1963), "solid-phase Synthesis of peptides", W.H.Freeman Co. (San Francisco); and Meienhofer, I. (1973). "Hormonal proteins and peptides", Vol. 2, page 46, Academic Press (new York). Review of classical synthesis in solution, refer to the publication: Schroeder, G. and lupke, K. (1965). "The Peptides", Vol. 1, Academic Press (New York).

Recombinant methods, refer to the reference materials below.

It also discusses the nucleic acid sequences that encode the above-described polypeptide sequence (see SEQ ID NO. 13, 14, 15,16,17 and 18).

As mentioned above, one specific use of the antibodies of the present invention is the prevention or treatment of disease associated with imbalanced or abnormal activity of metalloproteins, such as metalloprotease.

Examples of such diseases include, without limitation arthritis, such as osteoarthritis (OA), rheumatoid arthritis (RA), staticheskii arthritis, soft tissue rheumatism, polyhedric and tendonitis; metastatic tumors, diseases of the periodontium; ulceration of the cornea, caused for example, alkali or other burns, radiation, vitamin E or retinoid failure; glomerular disease, such as proteinuria, the bubble bullosa; diseases associated with bone resorption, such as osteoporosis, Paget's disease, hyperparathyroidism and cholesteatoma; contraceptive measures preventing ovulation or implantation; angiogenesis related to tumor growth or neovascularization associated with diabetic retinopathy and macular degeneration; coronary thrombosis associated with the rupture of atherosclerotic plaques; emphysema, the treatment of wounds and HIV infection.

As illustrated in Example 10, the authors of the present invention showed that the antibodies of the present invention can be used to treat irritation of the colon.

Inflammatory bowel disease (IBD) are serious disorders of the gastrointestinal tract characterized by inflammation of the intestines and remodeling of tissues, the frequency of which increases and can lead to disability of patients. The main forms of IBD, ulcerative colitis (UC) and Crohn's disease are chronic recurrent conditions that are clinically characterized by abdominal pain, diarrhoea, rectal bleeding and fever.

Thus, according to another aspect of the present invention pre is a false way of inhibiting the activity of matrix metalloprotease in need of this object.

Preferred individual objects according to the present invention are animals such as mammals (e.g. dogs, cats, sheep, pigs, horses, cows, primates), preferably, humans.

The method includes providing the object therapeutically effective amount of a MMP inhibitor of the present invention (i.e., antibodies or fragments of antibodies, described above).

As discussed in more detail below, the MMP inhibitor may be given by direct injection (e.g., orally or by injection) or can be expressed from a polynucleotide constructs that are delivered to the target cells of the individual.

The MMP inhibitors of the present invention can be provided to the individual as such or as part of a pharmaceutical composition where they are mixed with a pharmaceutically acceptable carrier.

Used herein, the term "pharmaceutical composition" refers to a preparation of one or more of the active ingredients described herein, with other chemical components such as physiologically suitable carriers and excipients. The purpose of the pharmaceutical composition is to facilitate the introduction of compounds into the body.

Used herein, the term "active ingredient" refers to the drug antibody, which is responsible for the biological effect.

Use the on going here, the phrases "physiologically acceptable carrier" and "pharmaceutically acceptable carrier", which can be used one instead of the other, belong to the carrier or diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the input connections. In the meaning of these phrases included adjuvant. One of the ingredients included in the pharmaceutically acceptable carrier may be, for example, polyethylene glycol (PEG), a biocompatible polymer with a wide range of solubility in organic and aqueous media (mutter and others (1979).

Used herein, the term "filler" refers to an inert substance added to a pharmaceutical composition to further facilitate the introduction of the active ingredient. Examples, without limitation, of excipients include calcium carbonate, calcium phosphate, various sugars and starches, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.

Methods of preparation and administration of drugs can be found in the publication "Pharmaceutical Sciences Remington", Mack Publishing Co., Easton, PA, latest edition, which is incorporated herein by reference.

Suitable routes of administration can be, for example, oral, rectal, cresselly, especially transnasal, intestinal or parenterally delivery, including intramuscular, subcutaneous and intramedullary injections as well as intrathecal,direct intraventricular, intravenous, intraperitoneally, intranasal, or intraocular injections.

Alternatively, you can enter the drug locally, not systemically, for example, by injection of the preparation directly into a specific area of a patient's body.

The pharmaceutical compositions of the present invention can be manufactured by well known methods, for example by conventional mixing, dissolving, granulating, production drops, grinding into powder, emulsifying, encapsulating, capture or freeze-drying.

Pharmaceutical compositions for use in accordance with the present invention can be prepared traditionally using one or more physiologically acceptable carriers containing fillers and auxiliaries, which facilitate processing of the active ingredients into preparations which can be used for pharmaceutical purposes. Proper preparation depends on the selected route of administration.

For injection, the active ingredients of the invention can be prepared in aqueous solutions, preferably in physiologically compatible buffer solution such as a solution of Hank, ringer's solution, or physiological saline. For crosslisted introduction when making use of wetting agents, podhodjashaja passing through the corresponding barrier. Such reagents are well known.

For oral administration the compounds can be easily prepared by combining the active compounds with pharmaceutically acceptable carriers well known in this area. Such carriers enable the production of compounds of the invention in the form of tablets, pills, coated tablets, capsules, liquids, gels, syrups, casic, suspensions, etc. for oral use by the patient. Pharmaceutical preparations for oral use can be made using a solid excipient, optionally grinding the resulting mixture and processing the mixture of granules, after adding suitable auxiliaries, if they desired, to obtain tablets or shell bean. Suitable fillers are, in particular, are sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations, for example, corn starch, wheat starch, rice starch, potato starch, gelatin, tragacanth gum, methylcellulose, hydroxy-propylmalonate, sodium carboxymethyl cellulose; and/or physiologically acceptable polymers such as polyvinylpyrrolidone (PVP). Optional, can be added depleting substances, such as cross-linked polyvinylpyrrolidone, agar or alginic acid or its salt, such as sodium alginate.

Shell beans sleep which reflect the appropriate coatings. For this purpose you can use concentrated sugar solutions which may optionally contain gum Arabic, talc, polyvinylpyrrolidone, gel of carbopol, polyethylene glycol, titanium dioxide, solutions varnishes and suitable organic solvents or solvent mixtures. Dyes or pigments can be added to the tablets or dragee coatings for identification or distinguishing of different combinations of doses of active compounds.

Pharmaceutical compositions that can be used orally include composite capsules made of gelatin, and also soft sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. Composite capsules can contain the active ingredients in a mixture with fillers, such as lactose, binders, such as starch, and lubricating agents such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active ingredients can be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, can be added stabilizers. All preparations for oral administration should take the dosage that is appropriate for the selected route of administration.

For transbukkalno the introduction of the composition can have the ü the form of tablets or lozenges, made in the traditional way.

For administration by nasal inhalation, the active ingredients according to the present invention is conveniently made in the form of an aerosol spray in a pressurized container or a nebulizer with the use of a suitable propellant, e.g. DICHLORODIFLUOROMETHANE, trichloromethane, dichlorotetrafluoroethane or carbon dioxide. In the case of an aerosol unit dosage can be determined by valve, measuring the required amount. Capsules and their packaging, for example, gelatin for use in machine-dispenser can be manufactured with a powder mix of the compound and a suitable base such as lactose or starch.

This preparations can be made for parenteral injection, for example, by introducing a ball or continuous infusion. Formulations for injection may be presented in the form of a unit dosage, e.g., in ampoules or containers containing multiple doses, optionally with the addition of preservative. Compositions can take the form of suspensions, solutions or emulsions in oily or aqueous media and may contain suspendida, stabilizing and/or dispersing agents.

Pharmaceutical compositions for parenteral injection include aqueous solutions of the active drug in a water soluble form. In addition, WM is ansii active ingredients can be manufactured in the form of an oil or aqueous suspensions for injection. Suitable lipophilic solvents or carriers include fatty oils such as sesame oil, or synthetic fatty acid esters, such as etiloleat, triglycerides, or liposomes.

Aqueous suspension for injection may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of active ingredients, allowing you to produce solutions of high concentration.

Alternatively, the active ingredient may be in powder form for connection before use with a suitable vehicle, e.g. sterile solution based pyrogen free and no water.

The drug of the present invention can also be manufactured in rectal compositions, such as candles or enemas, using, for example, the traditional foundations of candles, such as cocoa butter or other glycerides.

Pharmaceutical compositions suitable for use in the context of the present invention include compositions where the active ingredients are contained in amounts effective to achieve the intended purpose. More specifically, a therapeutically effective amount means an amount of active ingredients, effectively preventing, the mind is nausea or eliminates the symptoms or conducive to the recovery of the patient. Determination of therapeutically effective amounts well known to experts in this field.

For any preparation used in the methods of the invention, therapeutically effective amount or dose can initially be determined by in vitro tests. For example, the dose can be tested in animal models, and such information can be used for more accurate determination of the doses that are suitable for people.

Toxicity and therapeutic efficacy described here, the active ingredients can be determined by standard pharmaceutical procedures in vitro, in cell cultures or experimental animals. Data obtained from in vitro on cell cultures and animal studies can be used to develop a range of dosage for human use.

The dosage may vary depending on the form and route of administration. The exact composition, route of administration and dosage can be chosen by a physician considering the patient's condition. (See for example, Fingle and others, (1975) "Pharmacological basis of clinical medicine", p.1 D.1).

Depending on the complexity and response of the patient dose can be single or multiple, with course of treatment lasting from several days to several weeks to cure or minimize the painful conditions

A number of compositions for administration will, of course, depend on the patient, the severity of the disease, the route of administration, the views of the prescribing physician, etc.

Compositions comprising a preparation of the present invention, made with a compatible pharmaceutical carrier may also be manufactured, placed in an appropriate container and labeled for treatment of a particular condition.

Compositions of the present invention can, if desired, be contained in the packaging or distributing device, such as a set, approved by the U.S. food and drug administration, which may contain one or more doses of active ingredient. The package may, for example, contain a metal or plastic foil, i.e. take the form of blisters. Packaging or distributing device may be attached to the application instructions. Packaging or distributing device may be attached notice in the form prescribed by the state body that regulates the manufacture, use or sale of pharmaceuticals, which must be stated that the authority has approved this form of composition to assign people or animals. Such notification may be in the form approved by the U.S. food and drug administration for drugs, prescription, or in the approved form to be placed in the product.

As mentioned above, inhibitors, antibodies of the present invention can be isolated from the structure of nucleic acids.

It will be clear that polynucleotide encoding antibodies of the present invention preferably additionally encode a signal peptide that allows for the secretion or the passage of antibodies in interest subcellular or extracellular location.

For example, when the target metalloproteinase is MMP, the secretory signal peptide is preferably linked in frame with a segment of the antibody coding for polynucleotide.

In addition, it becomes clear that the recombinant single-stranded Fv fragments (ScFv) preferably may be selected because of their considerably less complicated structure compared to a complete antibody molecules. As mentioned above, ScFv proteins are composed of polypeptide chains VLand VHantibodies are synthesized as a single chain with carboxyl end of the VLrelated peptide is an offshoot of the N-end VH. Methods of recombinant production of these peptides is well known in this area (see Byrd and others, Science 242:423-426 (1988); Haston and others, Proc. NAT'l Acad. Sci. USA 85:5879-5883 (1988); and de-Kruif etc., J. Mol. Biol. 248:97-105 (1995)). According to variants of the implementation of this aspect of the present invention, after immunization compounds of the present invention, Selestat the th mRNA is collected from an immunized animal and used to produce cDNA library in the bacteriophage, which has the ScFv fragments. The phage particles can then review to identify those which specifically interact with, and preferably with an activated form of interest metalloprotein. Of these phage particles isolated segments of ScFv and clone into expression design (see U.S. patent 5,800,814).

Design nucleic acid of this aspect of the present invention may be introduced into the target cells of an individual object (i.e., gene therapy in vivo).

Alternatively, the design of nucleic acid is introduced into a suitable cell by a suitable method/means of gene delivery (transfection, transduction, homologene recombination, etc.) and expression system if necessary, and then the modified cells to expand in culture and returned to the patient (i.e., gene therapy ex vivo).

Cellular expression of antibodies or fragments of antibodies of the present invention, the design of the nucleic acids of the present invention also includes at least one current in the CIS-position of the regulatory element. Used here, the phrase "acting in CIS-position regulatory element" refers to a polynucleotide sequence, preferably a promoter which binds acting outside the regulator and regulates the transcription of the coding sequence, R is sporogenous after him.

In this methodology, you can use any available promoter. In a preferred embodiment of the present invention, the promoter used in the design nucleic acid of the present invention, active in transforming specific cell population. Examples of promoters that are specific to cell types and/or specific to the tissues, include such as albumin, which is specific to the liver [Pinkert and others, (1987) Genes Dev. 1:268-277], promoters specific to lymphoid [Kalam and others, (1988) Adv. Immunol. 43:235-275]; in particular promoters of receptors of T lymphocytes [Winoto and others, (1989) EMBO J. 8:729-733] and immunoglobulins; [Banuri and others (1983) Cell 33729-740], neuron-specific promoters such as the promoter of neurofilaments [Byrne and others (1989) Proc. Natl. Acad. Sci. USA 86:5473-5477], promoters, specific to the pancreas [Adlung and others (1985) Science 230:912-916] or promoters that are specific to the mammary gland, such as the promoter of whey (U.S. Patent 4,873,316 and published European application No. 264,166). The design of the nucleic acids of the present invention can also include gene-amplifier, which can be located near or far from the promoter sequence and can function to regulate its transcription.

The design of this methodology preferably also include a suitable choice is aemy marker and/or Replicator. Preferably, the design is a Shuttle vector that can replicate in E. coli (when the design contains suitable selectable marker and Replicator) and is compatible for propagation in cells, or integration into a gene and the selected fabric.

The design according to the present invention can be, for example, a plasmid, bacmids, fahmida, kosmidou, a phage, a virus or an artificial chromosome.

The currently favored methods of transfer of nucleic acids in vivo include transfection with viral or non-viral constructs, such as adenovirus, lentivirus, the virus I, herpes simplex virus, or adeno-associated virus (AAV) and systems based on lipids. Suitable lipids for lipid transport genes are, for example, DOTMA, DOPE and DC-Choi [Tonkinson, etc, Cancer Investigation, 14(1):54-65 (1996)]. The most preferred structures for use in gene therapy are viruses, most preferably adenoviruses, AAV, lentiviruses or retroviruses. Viral structure, such as a retroviral construct that includes at least one promoter/activator of transcription or element determining locus or other elements that regulate gene expression by other means such as alternate splicing, nuclear RNA export or post-translational, modify the tion messenger. Such vector constructs also include a packaging signal, long terminal repeats (LTR sequences or parts thereof, as well as the binding sites of primers positive and negative circuits corresponding to the virus, if it is not already represented in the viral construct. In addition, this design typically includes a signal sequence for secretion of the peptide or antibody from the host cell into which it is placed. Preferably, the signal sequence for this purpose is the signal sequence of the mammal. Additionally, the design may also include a signal that directs the polyadenylation, as well as one or more restriction sites and sequence of the end of the broadcast. For example, such constructs will typically include a 5' LTR, a tRNA binding site, a packaging signal, the beginning of the synthesis of the second DNA strand and the 3' LTR or a portion of it. Can be used and other vectors that are non-viral, such as cationic lipids, polylysine and dendrimers.

Preferred modes for implementation of protocols of gene therapy are presented in the publications of the IMO and Verma [(2000) Nature Reviews 1:91-99], Isner (2002) "Gene therapy myocardium. Nature 415:234-239; Hai (2001) "Gene therapy: Prospects in 2001. Haemophilia 7:23-27; and Hammond and MacKinnon (2001) "Angiogenic gene therapy is in heart disease: a review of animal studies and clinical trials". 49:561-567.

Because of the ability of the antibodies of the present invention differentially recognize the activated form of metalloprotein (see Example 3), they can be used as a powerful diagnostic and prognostic tool, for example by monitoring the activity of MMP in a biological sample [i.e., any sample from the body (serum or plasma), saliva, ascitic fluid, pleural effuse, urine, biopsy samples, isolated cells and/or preparation of cell membranes]. This is of particular importance when assessing the metastatic characteristics of cancer cells, when unbalanced activation of MMP facilitates penetration of the tumor. Similarly, antibodies of the present invention can be used to monitor therapeutic dosage of MMP inhibitors. For such applications, the antibodies of the present invention preferably mark each of any radioactive, fluorescent, biological or enzymatic tags, which are normally used in this field. The use of such labels is discussed in U.S. patents 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149 and 4,366,241.

It will be understood that such methods of detection can also be used for rapid screening of new MLM. Briefly, numerous biological sample can be introduced into contact with the antibodies of the present invention, where the activated M Is P can contact them. Efforts to use biological samples that include activated MMP, such as obtained from lines of tumor cells. Typically, the radioactive label is used to reduce the sample volume for analysis.

Alternatively, antibodies of the present invention can be used for purification of active metallothionen from biological samples.

In this area there are many methods to purify proteins. For example, antibodies or antibody fragments of the present invention can be used in affinity chromatography for isolation of metallothionen. You can prepare the column, where antibodies are associated with a solid substrate, e.g., particles, such as agarose, Sephadex, and the like, and a biological sample, such as cell lysate, can be omitted through the column, then the column is washed, and then increase the concentration of mild denaturing means is released whereby the purified metallothermic.

Antibodies or fragments thereof that are created according to the present invention may be included in a diagnostic or therapeutic kit. Antibodies or antibody fragments can be Packed in one or more containers with appropriate buffers and preservatives and used for diagnosis or to guide treatment.

Thus, antibodies Il is their fragments can be mixed in one container or placed in individual containers. Preferably, the containers have a label. Suitable containers include, for example, bottles, vials, syringes, and test tubes. Containers can be made from different materials, such as glass or plastic.

In addition, can also be added other additives such as stabilizers, buffer solutions, blockers, etc. Antibodies in these sets may also be attached to a solid support such as beads, the matrix substrate (e.g., chips), etc. and used for diagnostic purposes. The kit may also include instructions for determining, suffers if the test object from, or has the risk of some conditions, disorders or diseases associated with expression of the corresponding MSEs.

Other objectives, advantages and new features of the present invention will become clear to the average person skilled in the art after studying the following Examples, which should not be construed as limiting. In addition, each of the embodiments and aspects of the present invention that are described above and included in the claims, has experimental support in the following Examples.

EXAMPLES

Now will be considered examples, which together with the above description illustrate the invention a non-limiting manner.

In General, used here the nomenclature and laboratory procedures in the present invention include molecular, biochemical, microbiological and DNA recombinant methods. These methods are explained in detail in the literature. See, for example, "Molecular cloning: a laboratory Handbook" Sam brook and others, (1989); "Current protocols in molecular biology"Volumes I-III edited by Ausubel, R. M., (1994); Ausubel and others, "Current protocols in molecular biology", John Wiley & Sons, Baltimore, Maryland (1989); Perbal, "a Practical guide to molecular cloning", John Wiley & Sons, New York (1988); Watson and others, "Recombinant DNA, Scientific American Books, New York; Birren and other "genome Analysis: a Series of laboratory manuals", V.1-4, Cold Spring Harbor Laboratory Press, New York (1998); the methodology of U.S. patents 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057; "Cell biology: a laboratory Handbook", Vol I-III edited by Cellis, J. E., (1994); "Current protocols in immunology" So I-III edited Coligan j. E., (1994); State and others, "basic and clinical immunology" (8th edition), Appleton & Lange, Hopwalk, CT (1994); mishell and Siege, "Selected methods in cellular immunology", W. H. Freeman & Co., New York (1980); available immunoassays are widely described in the patent and scientific literature, see, for example, U.S. patents 3,791,932; 3,839,153; 3,850,752; 3.850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281,521; "the Synthesis of oligonucleotides" edited by Jordan, M. J., (1984);

"Hybridization of nucleic acids" Hams, D. and X is ggins C. J. (1985); "Transcription and translation" Hams, D. and Higgins S. J. (1984); Culture of animal cells", edited by Fresno, R.I., (1986); "Immobilized cells and enzymes", IRL Press, (1986); "a Practical guide to molecular clone", Perbal, B., (1984) and "Methods of fermentology", V.1-317, Academic Press; "PCR Protocols: a Guide to methods and applications", Academic Press, San Diego, CA (1990); Marshak and others, "Strategies purification and characterization of protein - Laboratory course", CSHL Press (1996); which are all incorporated by reference as if they were opened here in full. This document contains, and other General references. It is believed that the procedures are well known in this field and presents for the reader's convenience. All information contained therein are incorporated herein by reference.

Materials and methods

Recombinant enzymes - Catalytic domain of MMP-2 (amino acids 110-467 of the gene Bank with the access number NP032636.1) was expressed from the T7 promoter in the cells of BL-21. These cells were induced with 1 mm isopropyl-P-D-thiogalactopyranoside for 5 hours. The cell sediment was re-suspended in 50 mmol buffer solution Tris, pH 8.0, 0.5 mmol etc, 50 mmol NaCl, 5% glycerol and 1% Triton X-100 in the ratio of 1:25 with the buffer solution to the original volume of the culture. The suspension was centrifuged to use the e 10 min at 15,000 rpm and the precipitate was dissolved in 50 mmol Tris, pH 8.0, 0.5 mmol etc, 50 mm NaCI, 5% glycerol and 0.2% Sarkosyl then conducted incubation on ice for 30 minutes the Supernatant was loaded onto a column of 5 ml gelatin-separate (ready, the company Amersham Biosciences), pre-balanced and washed dialysis buffer solution (50 mmol Tris, pH 8.0, 50 mmol NaCl, 5 mmol CaCl2, 10 µmol ZnCl2, of 0.02% Brij). Protein was suirable using 50 mmol Tris, pH 8.0, 1 mol NaCl, 5 mm CaCl2, 10 µmol ZnCl2, of 0.02% Brij and 15% Me2SO [Rosen, O., "Inhibition of MMP monoclonal antibodies", 2001] and analyzed using SDS-PAGE, and its catalytic activity was measured by degradation plurigenera peptide [knight, K. J., F. Willenbrock and j. Murphy, "a New peptide, labeled with coumarin, for sensitive continuous assays of the matrix metalloproteinases". FEBS Lett, 1992. 296(3): R-6].

Pro-MMP-9 [with no hinge region and domain generacin, the precursor of matrix metalloproteinase-9 (MMP-9) Alal-Gly424 |P14780|9_HUMAN (EC 3.4.24.35)] was expressed in Escherichia coli ER2566 in the expression vector pTWIN and purified to homogeneity from inclusions, as stated above [Bjorklund, M., P. Heikkila and E. Koivunen, "Peptide inhibition of catalytic and non-catalytic activity of matrix metalloproteinase-9 blocks migration and invasion of tumor cells". J Biol Chem, 2004. 279(28): p.29589-97]. The precursor of the MP-9 activated 1 mmol of acetate p-aminophenylacetate (ARMA, the company ICN Biomedicals Inc., Ohio, USA) and was dissolved in 200 mmol Tris for 30 min at 37°C.

Recombinant human Pro-MMP-2 and Pro-MMP-9 expressed in cells HeLa S3 infected with the appropriate recombinant virus cowpox, and purified to homogeneity, as stated above [Olson, M.V., and free Wi-Fi, DK, Mobashery, S. and Friedman, R. (1997) J. Biol. Chem. 272, 29975-29983; Friedman, R., Fuerst, TR, bird, RA, Hoyhtya, M., Alcock, T.M., Kraus, S., period, D., Liotta, L.A., Berman, M., Stetler-Stevenson, U. j. (1992) J. Biol. Chem. 267, 15398-15405].

Tetracarboxylates Co(II)/Zn(II) (CoTCPP/ZnTCPP) - ZnTCPP synthesized by the reaction of ZnCl2and TSR in N,N-dimethylformamide (DMF) as described in the publication [Harada, A. and others, "Control of photoinduced electron transfer from the zinc porphyrin to methylviologen by supramolecular formation between a monoclonal antibody and zinc-porphyrin". Photochem Photobiol, 1999. 70(3): p.298-302]. Satsr synthesized by reaction With(SLA)2-4H2O and TSR in DMF as described in the publication [Harada, A. and others, "Control of photoinduced electron transfer from the zinc porphyrin to methylviologen by supramolecular formation between a monoclonal antibody and zinc-porphyrin"] and purified.

Synthesis Imisdp Is Described in Example 7, below.

The coupling of the hapten to protein - Haptens (4 mg) activated for coupling by adding ,1'-carbonyldiimidazole in DMF (molar ratio 1:1) and incubated for 1 h The activated hapten in the amount of 1-50 µm was added to 20 mg/ml BSA or shellfish to hemocyanine (KLH) in 0.1-molar carbonate buffer solution at pH 8. The solution was stirred at room temperature for 3 h and then extensively deliberately relatively phosphate-saline buffer solution (PBS).

Immunization and fusion - each of adjuvant (KLH), coupled with Satsr, ZnTCPP or Imisdp, used for immunization of BALB/c mice. Immunization and subsequent fusion with the cell line NSO myeloma was performed according to standard procedures [Harlow, E. and lane, D., Using antibodies: a Laboratory manual - Mobile Protocol I". 1998].

Screening of antibodies

ELISA Supernatant growing hybridomas were analyzed for antibodies reacting with ZnTCPP, CoTCPP or Imisdp using direct ELISA in which the corresponding hapten-BSA (3 mg/ml of phosphate-saline buffer solution) was applied to the plates Nunc Maxisorp. The base coat stood at 4°C overnight and incubated with antibodies at 20°C for 1 h Associated peroxidase from horseradish roots monoclonal antibody (mAb) for mice (Sigma) was used as secondary antibody, and 2,2'-Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid, ABTS, Sigma) was used as substrate. Phosphate-saline buffer solution (PBS)containing 0.005 volume. % Tween 20 (PBST), used in the quality of the ve wash reagent. Buffer for dissolving served PBS. And602registered tablet reader SPECTRAFluor Plus (Tecan, Austria). To control the supernatant incubated with tablets coated with BSA, using the same procedure. The values of absorption capacity above 0.5 ellipticheskoi density was considered to be positive.

Competitive ELISA Diluted to varying degrees supernatant liquid hybridoma incubated with tablets, coated with hapten-BSA, according to the above steps.

Was prepared by the titration curve and the dilution titers were determined by 50% of the binding.

Supernatant is diluted to a concentration of title, pre-incubated with soluble compounds ZnTCPP, CoTCPP or Imisdp for 30 min and then transferred hapten coated microtiter plates after the steps described above. Estimated dissociation constant was the concentration of the soluble hapten required to achieve 50% binding.

Production and purification - Selected hybridoma were subcloned twice limited dilutions, followed by large-scale production of ascites tumors, the seed of which served as pristine (2,6,10,14 tetramethylpentane), enter the BALB/c mice. Monoclonal antibodies were purified by affinity chromatography on protein G sepharose 4 (Amersham Biosciences). Ascites CE who was trippynova at 12000 g for 15 min to remove insoluble particles and lipid. Ascites in the amount of 1 ml was diluted 5 times by volume, using PBS, and then loaded into the column in a volume of 5 ml protein G sepharose. Peak elution were analyzed by SDS-PAGE.

Determination of the isotype - Supernatant culture fluids obtained from cloned hybridomas expressed in flasks with culture, was used as source of antibodies. The isotype of each antibody was determined using a kit for determining the isotype of the monoclonal antibody mouse (HyCult biotechnology B.V.., Netherlands).

The Western blot turns purified antibody Purified antibody was separarely in 8% SDS-polyacrylamide gel, transferred to NC membrane (Bio-Rad) and subsequently subjected to Western blot turns using antibodies against MMP-9 (Sigma). Goat IgG-intimissi, peroxidase conjugated horseradish root (Sigma)was used as secondary antibody. Signals were detected using ECL (Pierce).

The analysis of binding using purified proteins - antibodies (10 μg) were incubated with agarose pellets IgG-intimissi (Sigma) overnight at 4°C in PBS. After washing unbound antibody was added to the purified Pro-MMP-2, Pro-MMP-9, catalytic domain of MMP-2 catalytic domain MT or TACE (2 μg), and then incubated for 2 h at room temperature. Pellets were collected by centrifugation and washed 3 times with PBS. Proteins that remained associated with the pellet and, was suirable buffer solution of SDS was fractionally SDS-PAGE and detected by staining blue Kumasi.

Immunoprecipitation and Western blotting - Cells NT were sown in Petri dishes. After 80% confluence, the medium (DMEM, supplemented with 10% FCS, nonessential amino acids, penicillin, streptomycin, sodium pyruvate and L-glutamine) was replaced by containing no serum medium (without FCS). After another 24 h of incubation, the conditioned medium (COP) was collected from fused cells and concentrated using Millipore Cemricon-10 (Bedford, Massachusetts). Concentrated supernatant was used for immunoprecipitation. KS incubated with anti-1 (Atsr) monoclonal antibody (mAb) (15 μg/ml) overnight at 4°C. Protein And Sepharose (CL-4B, Amersham Biosciences) was added to the samples and was stirred for 2 h at room temperature. Pellets were washed 3 times with PBS, suspended in SDS buffer solution and heated to 95°C for 3 min Immunoprecipitate was collected by centrifugation and subjected to SDS/PAGE. After separation the proteins were transferred to nitrocellulose (NC) membrane and tested antibody anti-MMP-2.

To activate Pro-MMP-2 produced by the cells NT, 1 mmol 4-aminophenylacetate (ARMA) was added to concentrated COP, and incubated for 6 h at 37°C. After activation, the COP were dialyzed (X3) against PBS at 4°C to remove ARMA. Immunoprecipitation aktivirovannoi environment has done so, as said above.

Linking to active MMP-9 using direct ELISA Catalytic domain of MMP-9 (2 μg/ml) immobilizerpower in the wells on the microtiter plate. Monoclonal antibodies (1 mg/ml) was added to the wells after the procedure described for the screening ELISA, antibody against MMP-9 (Sigma) served as a positive control, and the affinity of IgG for an unrelated mouse, purified from ascites, served as a negative control.

Kinetic analysis of the Enzymatic activity of MMP was measured as previously described (Solomon, A. and others, "Pronounced difference in the electronic and chemical properties between the catalytic sites of the zinc enzyme that converts alpha-tumor necrosis factor, and matrix metalloproteinases despite their high structural similarity". J Biol Chem, 2004. 279(30): str-54). The activity of MMP-9, MMP-2 and MT-MMP was measured by monitoring the degradation fluorogenic peptide Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2 when γex=340 nm and γem=390 nm, as described by knight and others (FEBS Lett, 1992. 296(3): R-6), purchased the company Calbiochem-Novabiochem AG. The standard mixture for assays contained 50 mmol buffer solution Tris, pH 7.5, 200 mm NaCl, 5 mm CaCl2, 20 µmol ZnCl2and 0.05% Brij. The enzymatic activity of TACE was measured by monitoring the degradation fluorogenic peptide QF-45 (Mca-Ser-Pro-Leu-Ala-Gln-Ala-Val-Arg-Ser-Ser-Ser-Arg-Lys(dinitrophenyl)-NH2), PMC is built in the company Calbiochem-Novabiochem AG.

Zymography in situ For localization clean gelatinolytic activity of MMP-demografia in situ DQ-gelatin, marked fluorescein-isothiocyanato, which was intramolecular blocked (molecular probes)was used as a substrate for degradation gelatinase. Proteolysis gelatinase gives cleaved peptides gelatin - fluorescein of isothiocyanate, and localization of the fluorescence has sites clean gelatinolytic activity. Briefly, cells NT human fibrosarcoma (which produce MMP-2, MMP-9 and MT-MMP) were placed on glass cover 12 mmol. After incubation for 24 h the cells were treated with 1 µmol antibodies A for 30 min at 37°C. Untreated cells in this experiment was used as negative control. Cells were washed in PBS and then incubated with the buffer solution for demografie (0.05 mol Tris-HCl, 0.15 mol NaCl, 5 mm CaCl2and 0.2 mm NaN3, pH of 7.6, a high concentration of the azide phagocytal gelatin and allowed to get gelatinolytic activity on the surface of cells)containing 60 μg/ml DQ gelatin at 37°C over night. Buffer solution for demografie contained 1 mmol antibodies against Atsr for treated cells. At the end of the incubation period without commit or further leaching gelatinolytic the activity of MMP localized, fotografi is ovale using a fluorescent microscope, receiving image digital camera Spot.

EXAMPLE 1

Confirmations classes mimicry active site zinc small organometallics connections

Of the zinc ion in the active site of MMP evenly coordinated by three conservative histidine residues. During activation of zymogen and proteolysis of substrate coordination of zinc changing from 4-coordinate, rectangular geometry, non-catalytic stages on 5-axis, a triangular bipyramid [old, D.S., "the coordination of the zinc in the biochemical sites of zinc". Biometals, 2001. 14(3-4); R-313] on the catalytic stages. Conservative histidine so we can make different geometry with respect to the zinc ion. For a sample of these conformations selected two compounds as models for mimicry environment zinc - Imisdp and Co/ZnTCPP (FIGURE 1). Connection Imisdp (synthesis described in Example 7, below) can simulate the 4-coordinate geometry. In this case, almost rectangular conformation is formed by three imidazole bases and a water molecule as the fourth ligand.

On FIGA shows the overlay constructed 3D models of molecules Imisdp on the catalytic site of MMP-9 (PDB 1GKC) [Rosell, S. and others, "the crystal Structure of human MMP in combination with the reverse inhibitor of hydroxamate", J Mol Biol, 2002. 319(1): R-81], which was modified to represent even rejuvaline geometry of the ligands of zinc. Modifications included the replacement of the ligand present in the x-ray structure (inhibitor of hydroxamate), the water molecule and the optimization of the enzyme to a local minimum by a multilayered approach QM/UM (see materials and methods). There is a high similarity between the computed fragment zinc histidine in MMP-9 and Imisdp in terms of distances 6-nitrogen histidinol from zinc ions (2,04±0,06 and 2.02, respectively) and the relative orientation of the three histidines to metal. The second molecule, Zn/CoTCPP, has four imidazole Foundation in coordination with zinc or similar metal cobalt in co-planar conformation with respect to the metal ion [Steven, ED, "Electronic structure of metalloporphyrins. 1. The experimental electron density distribution (meso-tetraphenylporphine)cobalt (II)". J. Am. Chem. SOC, 1981. 103(17): p.5087-5095]. This configuration mimics the conformation of two of the three histidines in 5-axis, triangular bipyramidal geometry, where the metal is almost coplanar two histidines that form the base of the pyramid. On FIGU shows the crystal structure of MMP-9 (PDB 1GKC), where the zinc is coordinated 5 ligands (two additional ligand contributes inhibitor of hydroxamate), the orientation of the two histidines at the base of the pyramid and their distances from the ion zinc (2,2±0,02, 2,03±0.04 and 1,95 for MMP-9, ZnTCPP and Satsr, respectively) compare the molecules Co/ZnTCPP.

EXAMPLE 2

The creation and selection of monoclinal antibodies

Monoclonal antibodies against Satsr, ZnTCPP and Imisdp (FIGURE 1) were obtained by immunization of mice and selection of specific antibodies by screening ELISA with the corresponding connection as coated antigen. Three antibodies were selected for extensive research. These clones were chosen because they had the best affinity for the immunizing their hapten on the basis of a competitive ELISA screening. Of their binding constants in the range of 0.01-0.09 Microm, (table 3, below), characteristic of mAbs with high affinity. Monoclonal antibodies were propagated as ascites in mice and purified granules of protein G.

Table 3
Isotypes and the results of the competitive ELISA monoclonal antibodies against Satsr, ZnTCPP and Imisdp.
Immunizing haptenThe isotype antibodiesKd [µm] * Name
SatsrIgG2b0,09E
ZnTCPPIgG2a0,01E
ImisdpIgG2a0,09S
* - Affinity binding (Kd) of the antibody with the immunizing their hapten was determined by competitive ELISA (see details in "Materials and methods").

EXAMPLE 3

Monoclonal antibodies cross-react with MMP-2 and MMP-9

To determine whether antibodies against synthetic compounds that mimic the conformation of the histidine zinc in the catalytic site of MMP, cross-react with an open fragment of histidine zinc in the active site of MMP-2 and MMP-9 monoclonal antibody is first tested on the binding of MMP-9 using direct ELISA.

Three antibodies bound catalytic domain of MMP-9, directly adsorbed to the wells of the microtiter tablet (commercial antibody against MMP-9 was used as a positive control, and an unrelated IgG served as a negative control). Interestingly, antibodies that multiplied as ascites in mice and were purified with active MMP-9 was present in the ascitic fluid of mice. Western Western blot turns only purified antibodies with antibody against MMP-9 as the primary antibody showed pure range corresponding to the expected molecular weight of approximately 82 Amu for active MMP-9. Thus,antibodies formed a complex in vivo with native enzyme.

Monoclonal antibodies are further analyzed for binding of MMP-2 using immunoaffinity analysis. Antibodies were incubated with the catalytic domain of MMP-2 (MMP-2cat) in vitro, and then pulled using beads of agarose IgG anti-mouse. As shown in FIGA, all antibodies bound MMP-2cat.

To establish that the binding occurs through direct interaction with the active site, the antibodies were analyzed for their ability to bind Pro-MMP-2 and Pro-MMP-9. In the latent enzymes predomina structure protects the catalytic groove. Therefore, the blocking of the active site produmannoy structure should prevent binding of the antibody, provided that it recognizes the fragment zinc histidine in the active site. In the same conditions did not reveal the binding of proenzymes (PIGV). This mode of binding to the active MMP-2, but not with Pro-MMP-2 was further validated environment in vivo in native MMP-2 full length secreted by the cell cultures (NT) human fibrosarcoma. Immuno-precipitation of the environment, air-conditioned cells NT with antibody against Satsr, after which was performed Western Western blot turns, showed binding to the active MMP-2, but not with Pro-MMP-2 (FIGS). These results demonstrate that all three antibodies cross-react with MMP-2 and MMP-9. The opening of the deepening of the active site is larger the f value for binding of the antibody, confirming that the monoclonal antibodies directly interact with the active site of MMP-2 and 9.

EXAMPLE 4

Antibodies against Atsr and Imisdp inhibit MMP-2 and MMP-9 in vitro

Antibodies against Imisdp and Satsr inhibited the proteolytic activity of MMP-2 and MMP-9 in the micromolar range (FIGURE 5). Kinetic analysis of the inhibition of MMP antibodies was performed in a constant fluorometrically analysis hydrated fluorescent peptide substrate. Surprisingly, the antibody against ZnTCPP showed no inhibitory effect.

To determine the nature of inhibition by antibodies against Atsr conducted experiments using enzymes in the presence or absence of antibodies at various concentrations of the fluorescent peptide substrate. The data presented in the graph of Leinweber-Burke, shown in FIGA, characterize the profile of competitive inhibition with Ki values of 13 µm and 24 µm for MMP-9 and MMP-2, respectively. Profile competitive inhibition showed that the antibody is associated with the same site as the peptide substrate. This mode of inhibition is further confirmed direct interaction with the active site. It should be noted that the antibody against Imisdp showed depend on the concentration of the inhibiting effect of focused on MMP-2 and MMP-9, suggesting a competitive inhibition calculated the values of Ki was 5.8 µm and 3 µm for MMP-9 and MMP-2, respectively (FIGURE 5). Because antibodies recognize the binding site of MMP-2 and MMP-9, further optimization of the interface complementarities between antibodies and MSE, both structural and electrostatic obtainable by the methods of affinity maturation (Paul J. Cartier, Nature Reviews Immun. Vol.6 2006 343-357). The use of this approach can lead to highly specific inhibitors that will use the characteristics of specificity, inside or outside the active site.

EXAMPLE 3

Zymography in situ

To confirm the inhibitory activity of antibodies against Atsr at the cellular level, the effect of the antibodies was studied on gelatinolytic activity of cells NT human fibrosarcoma, which constitutively secrete MMP-2 and 9, the method of demografie in situ. For localization gelatinolytic activity of MMP method demografie in situ gelatin, labeled with fluorescein by isothiocyanates, which was repaid intramolecular (DQ-gelatin)was used as substrate. Proteolysis gelatinase gave derived peptides of fluorescein isothiocyanate - gelatin, and localization of the fluorescence indicates the sites clean gelatinolytic activity. Untreated cells NT human fibrosarcoma (FIGA) showed a significant gelatinolytic activity of the cell surface. In prisutstvie and 1 µmol antibodies (PIGV), gelatinase activity was reduced as compared to that observed in control cells. These results demonstrate that anti CoTCPP mAb inhibited MMP-2 nd MMP-9 at the cellular level.

EXAMPLE 6

The selectivity of monoclonal antibodies of the present invention

The selectivity of the antibodies was tested by examining the binding and inhibitory effect of monoclonal antibodies against CoTCPP and Imisdp against MMP-14 (MT-MMP) and TNF-α converting enzyme (TACE), is dependent on zinc metalloproteinases relating to kindred the family of ADAM (disintegrin and metalloproteinase) (ADAM-17). Inhibitory effect against MT-MMP and TACE was verified by in vitro analysis of the enzymatic activity of fluorescence with the relevant peptide substrates. Monoclonal antibody against CoTCPP showed no inhibitory effect against MT-MMP or TAS. To determine whether it binds TAS and MT-MMP without subsequent inhibition were performed immunoaffinity experiments with purified enzymes, but binding have been identified. In contrast, monoclonal antibody against CoTCPP, monoclonal antibody against Imisdp inhibited MT-MMP when the value of Ki 10 µmol, but showed no inhibitory effect against TAS. The results are presented in table 4, below.

Table 4
MLMS (IC50µmol)E (IC50µmol)E (IC50µmol)
MMP-23±0,224±1NO*
MMP-94,5±0,215±0,8NO*
MT-MMP14,4±0,7NO*NO*
TASNO*NO*NO*
*NO - Not inhibited at concentrations up to... (more illegible}

Among the members of the family of MMP and TACE there is a high structural similarity in the active site, specifically, the three-dimensional structural elements surrounding the binding site of zinc, almost identical, due to the need to accommodate the frame of peptide substrates and the presence of a conservative fragment EXXHXXGXXH bind zinc [Solomon, A. and others, "Expressed a variety of electronic and chemical properties of the sites of the catalytic zinc in the enzyme that converts alpha-tumor necrosis factor, and matrix the x metalloproteinases, despite their high structural similarity". J Biol Chem, 2004. 279(30): p.31646-54; Lukacova, W. and others, "comparison of the binding sites of matrix metalloproteinases and the enzyme that converts alpha-tumor necrosis factor: implications for selectivity". J Med Chem, 2005. 48(7): R-70]. Therefore, it is not expected that the selectivity of monoclonal antibodies to MMP based solely on the recognition of a conservative fragment zinc histidine. However, unlike synthetic inhibitors of low molecular weight antibody, which is a large protein molecule, should have limited access to the recess in the active site, which is hidden in the frame of the protein. In particular, since the monoclonal antibodies specifically interact with the ion catalytic zinc, the degree of openness of zinc ions to the solution should be critical for binding by the antibody. MT-MMP and more TAS differ deep SI-pocket correlated with relatively hidden ion catalytic zinc, which show the structure of their crystals. The difference in the depth of the active site can be the reason for the lack of inhibitory effect of antibodies against TAS. These results suggest that selectivity can be achieved on the basis of the degree of openness ion catalytic zinc. Another important factor, which must have ativates when comparing MMP and TACE, are differences in the pocket of the active site in the sense of chemistry, such as hydrophobicity and polarity (see FIG). The active site of TACE, for example, significantly more polar than the active sites of most MMP. Solomon and others have demonstrated that such a change in the polarity of the active site directly affects the orientation of the imidazole rings of histidine active site relative to the ion catalytic zinc [Solomon, A. and others, "Expressed a variety of electronic and chemical properties of the sites of the catalytic zinc in the enzyme that converts alpha-tumor necrosis factor, and matrix metalloproteinases, despite their high structural similarity". J Biol Chem, 2004. 279(30): R-54].

The selectivity of antibodies against Atsr and Imisdp was further tested for their cross-reactivity with unrelated enzymes depend on zinc - carbon anhydrase (KA) and alcohol dehydrogenase of brockii (TbADH). Like active MMP, KA contains a zinc ion, which is quadrangular coordinated to three histidine residues and a water molecule, TbADH contains ion catalytic zinc, which is quadrangular coordinated with four different amino acid residues, histidine, cysteine, aspartate and glutamate. Corresponding experiments of functional inhibition in vitro, and similar-monoamine experimentally performed to study the cross-reactivity of these enzymes, but neither binding nor inhibition was not observed. Monoclonal antibody against Satsr was also tested for its cross-reactivity with related physiological porphyrins such as Heme group in myoglobin and hemoglobin and vitamin. In competitive ELISA and immunoaffinity the analysis of cross-reactivity have been identified.

Carboxylic anhydrase and alcohol dehydrogenase have enough hidden active sites, like the porphyrin component in myoglobin and hemoglobin is not open.

Vitamin B12 contains the metal in the center of the glider imidazole structure, but the axial ligands can interfere with the binding of monoclonal antibodies. Together, these results demonstrate that monoclonal antibody against Atsr recognizes a relatively open configuration of the metal-imidazole without interfering residues that coordinate axial metal.

EXAMPLE 7

Synthesis of [2-(2-aminoacylchromones)-ethoxymethyl]-Tris-[2-(N-(3-imidazol-1-yl-propyl)-ethoxymethyl]memantine (II) (3), FIG.9

(i) Synthesis of Tetra (2-pentachloro-phenoxycarbonyl-ethoxymethyl)methane

Synthesis pentachlorophenol-substituted Tetra-active complex ester was carried out according to the procedure Khaimah Weizmann and others, JACS 1996, 118, 12368-12375.

(a) Preparation of monosubstituted three active ester: Tetra-active ester (1) (1 g, 0.69 mmol who) and BocNHCH 2CH2NH2(100 mg, of 0.62 mmol) was dissolved in 20 ml of dry dichloromethane. The solution was stirred over night, maintaining a pH ~8 with triethylamine. The solution was concentrated and purified flash chromatography with CHCl3and ethyl acetate (90:10) to obtain (152 mg, yield 15%).1H NMR 250 MHz (CDCl3) δ: 1,4 (s, S, Boc)and 2.4 (t, 2H, J=6 Hz, -CH2-CH2-CONH); 2,9 (t, 6H, 3=6 Hz, -CH2-CHrCOOPCP); 3,2 (q, 2H, J=6 Hz, -CONH-CH2-CH2-NHBoc); and 3.31 (t, 2H, J=6 Hz, -CONH-CH2-CH2-NHBOC); to 3.38 (s, 2H, -C-CH2-O-CH2-CH2-CONH-); of 3.42 (s, 6H, -C-CH2-O-CH2-CH2-COOPCP); 3,61 (t, 2H, J=6 Hz, -C-CH2-O-CH2-CH2-CONH-); of 3.78 (t, 6H, J=6 Hz, -C-CH2-O-CH2-CH2-CONH-); to 5.03 (t, 1H, NH); 6,7 (t, 1H, NH).

(b) Preparation of Tris(imidazole): monosubstituted triactive ester (150 mg, 0.11 mmol) and 1-(3-aminopropyl)-imidazole (33 μl, 0,39 mmol) was dissolved in dry THF (20 ml) and was stirred over night at room temperature. Solution white was concentrated and purified column chromatography using silica (0,063-0,200 mmol) with CHCl3:methanol (50-90%) to obtain (45 mg, yield 44%). *H NMR 250 MHz (CDCl3/MeOD) δ: 1,45 (s, 9H, Boc); 2,0 (m, 6H, J=6 Hz, -CONH-CH2-CH2-CH2them); 2,4 (t, 6H, J=6 Hz, -O-CHz-CHz-CONH-); 2,5 (t, 2H, J=6 Hz, -CH2-CH2-CONH-CH2-CH2-NHBoc); 3,0 (m, 8H, J=6 Hz, -CONH-CH2-CH2-CH2- ,- CH2-CH2-CONH-CH2-CH2-NHBoc); 3,1 (t, 2H, J=6 Hz, -CONH-CH2-CH2/sub> -NHBoc); 3,4 (b, 8H, -C-CH2-O-CH2-CH2-CONH - CH2-CH2-NHBoc, -C-CH2-O-CH2-CH2-CONH-); 3,6 (m, 8H, J=6 Hz, -C-CH2-O-CH2-CH2-CONH-, -C-CH2-O-CH2-CH2-CONH - CH2-CH2-NHBoc,); 4,0 (t, 6H, J=6 Hz, -CONH-CH2-CH2-CH2them), and 5.5 (t, 1H, NH); 6,98 (s, 3H, Them); 7,06 (s, 3H, Them); to 7.32 (t, 3H, NH); EUR 7.57 (s, 3H, Them). ESI-PC: 910.87[M+Na]+, 925.98 [M+K]+.

(c) Preparation of Tris(imidazole) with the free amine (2): Tris (imidazole) (40 mg, 0.045 mmol) was dissolved in 6 ml of a mixture of dichloromethane and triperoxonane acid (2:1) and stirred for one hour. The reaction mixture was concentrated and evaporated several times with carbon tetrachloride and dried under high vacuum to remove triperoxonane acid from the mixture to obtain (30 mg, yield 85%, b). 'H NMR 250 MHz (CDCl3/MeOD) δ: 1,9 (m, 6H, J=6 Hz, -CONH-CH2-CH2-CH2them); 2,3 (m, 8H, J=6 Hz, -O-CH2-CH2-CONH-, -CH2-CH2-CONH-CH2-CH2-NH2); to 2.9 (t, 2H, J=6 Hz, - CONH-CH2-CH2-CH2them); 3,0 (t, 2H, J=14 Hz, -CONH-CH2-CH2-NH2); and 3.31 (t, 2H, J-6 Hz, -CH2-CH2-CONH-CH2-CH2-NH2); 3,4 (b, 8H, -C-CH2-O-CH2-CH2-CONH-CH2-CH2-NH2, -C-CH2-O-CH2-CH2-CONH-); 3,6 (m, 8H, J=6 Hz, -C-CH2-O-CH2-CH2-CONH-, -C-CH2-O-CH2-CH2-CONH - CH2-CH2-NH2); 4,0 (t, 6H, J=6 Hz, -CONH-CH2-CH2-CH2them); 7,26 (s, 3H, Them); to 7.32 (s, 3H, Them); 8,82 (s, 3H, Them).

3. Preparation of the complex Tris(imidazole)-Zn(II) (3): Compound 2 (30 mg, of 0.038 mmol) was dissolved in 1 ml of methanol. Added 2-3 drops of 1 normal NaOH solution and ZnCl2(5 mg, 0.04 mmol) and was stirred for half an hour. The precipitated white oveta was filtered to obtain (12 mg, yield 37%). *H NMR 250 MHz (MeOD/D2O) δ: 1.8 m (m, 6H, J=6 Hz, -CONH-CH2-CH2-CH2them)and 2.4 (m, 8H, J=6 Hz, -O-CH2-CH2-CONH-, -CH2-CH2-CONH-CH2-CH2-NH2); 3,0 (t, 2H, J=6 Hz, -CONH-CH2-CH2-CH2them); 3,0 (t, 2H, J=6 Hz, -CONH-CH2-CH2-NH2); 3,31 (b, 2H, -CH2-CH2-CONH-CH2-CH2-NH2); 3,4 (b, 8H, -C-CH2-O-CH2-CH2-CONH-CH2-CH2-NH2, -C-CH2-O-CH2-CH2- CONH-); 3,6 (m, 8H, -C-CH2-O-CH2-CH2-CONH-, -S-CH2-O-CH2-CH2-CONH-CH2-CH2-NH2); 4,2 (b, 6H, -CONH-CH2-CH2-CH2them); 7,19 (s, 3H, Them); 7,28 (s, 3H, Them); 8,55(s, 3H, Them). ESI-PC:852.09[M+1]+.

R=O-CH2-CH2-CONH-CH2-CH2-NH2

[2-(2-aminoacylchromones)-ethoxymethyl]-Tris-[2-(N-(3-imidazol-1-yl-propyl)-ethoxymethyl]methane.

EXAMPLE 8

Cross-reaction S with catalytic sites gelatinas

It was found that a number S together with active MMP-9 was purified from ascitic fluid. Prisutstviezhdrugih quantities MMP in ascitic tumor, induced in mice for breeding monoclonal antibodies were identified Western immunoblotting and demografia gelatin (data not shown). Complex MMP-antibody was purified from ascitic fluid of mice using affinity chromatography, protein G (protein G binds to the constant domain of the antibody, giving a variable domain of freedom of interaction with the antigen). As shown in FIGA jointly purified MMP was detected by Western by Western blot turns purified complex S-MMP using commercially available antibodies against MMP. Was defined band with a molecular mass of ~82 Amu, corresponding active MMP without predomina. This band was not detected in the irrelevant control monoclonal mouse body, which was purified and analyzed in the same way. These results showed that S formed a specific complex in vivo with endogenous active MMP-9 mouse.

To further validate the binding of the active form of highly homologous enzyme MMP similar experiments were thus performed in vitro. 6C6 incubated with purified fragment MN in a molar ratio of 3:1. Analysis of SDS-PAGE of immunoprecipitates sepharose protein And revealed the formation of the specific complex S with an active catalytic fragment MMP (PIGV). Only protein granules And could not p is ivesti to thus MMP. Next, it was tested the binding of inactive simagename (latent) forms MMP and 9. Since all MSEs were produced as inactive Imogene, they have propeptide N-end approximately 80-90 amino acids that block the active sites [Bode, W. and K. Maskos, Biol Chem, 2003. 384(6): p.863-72] (.11D). The experiments thus Pro-MMP and 9 were performed in the same way. Importantly, the antibody did not correlate with the latent enzyme (FIGS). Antibody S been associated only with the active conformation of the enzyme, in which the complex of protein and zinc in the active site is open to the solution.

These results confirmed that the antibody IS obtained and analyzed relative to bioinorganic of hapten that mimic the active site, cross-reacted with the active sites of the protein MMP and 9. Obviously, the hapten triangle zinc able to simulate the three-dimensional structure of the corresponding epitope zinc-histidine in the native protein. It is worth noting that the recognition of this minimal structural epitope of metal-protein sufficient to identify the ability to cross-react with the native enzyme. Linking only with active enzymes, but not with their latent form in which prodomain blocked access to protein epitope of the catalytic zinc (.11D)showed direct interaction S site kata is imicheskogo zinc. Native MMP-9 associated in vivo with S demonstrated that this antibody can form a specific complex with this enzyme in complex protein environment.

The difference between the activated species of the enzymes from the latent form is unique and valuable functional property S. This activity is unique to S in contrast to other antibodies against MMP. This is because immunization proteins usually gives epitopes directed to surface loops, whereas the catalytic amino acids are mostly hidden inside crevices on the surface of the enzyme. This part of the molecule is considered to have low immunogenicity. Therefore, neutralizing monoclonal antibodies obtained by traditional methods (relative to the native proteins or fragments of proteins) usually interacts with regions adjacent to the active site, and not with the catalytic residues of the active site and inhibits the mechanism of steric hindrance. The quantum antibodies are usually associated with the inactive precursor, as well as with the active form. This immunization approach mimics the active site of hapten can be expected to generate antibodies that recognize the protein residues of the catalytic metal in the MMP, which cannot be achieved by traditional approaches to immunization proteins.

EXAMPLE 9

S with the collective inhibits gelatinase in vitro and in situ

To determine the ability IS to inhibit the enzymes in relation MMP and MMP were conducted analyses of inhibition using small fluorogenic peptide substrates (7 amino acids), which cover the recess gelatinase in the active site. Initial reaction rates were measured for several concentrations of monoclonal antibodies. S inhibited the catalytic activity of both enzymes (FIGA-IN). A competitive mechanism of inhibition was determined by analyzing the activity MMP in the presence of various concentrations of inhibitory antibodies as a function of the substrate concentration. The data shown in FIGA in the form of a double reverse graph of Liveware-Burke, demonstrate the profile of competitive inhibition. Was obtained fitting the data of inhibition to the equation for competitive inhibition systems, Ki 1±0.1 µm and 1.4±0.16 µmol for MMP and 2, respectively. Also has determined that S did not split MMP-9 after incubation overnight with MMP-9 in high concentrations (30 µm), demonstrating that the observed inhibition MMP antibody S was not due to cleavage of a competitive substrate. Kinetic analysis MMP was taken as representative mechanism of inhibition of antibody S, as it is, by design, must recognize the same epitope in different MSEs. The inhibitory effect was about inoculum for kinds of catalytic fragments MMP and 9, and forms of the enzymes, full length gelatinase. More specifically, the catalytic fragments of recombinant MMP and MMP containing the catalytic domain and the domain of fibronectin, but not the domain hemopexin, as well as recombinant minimal catalytic element MMP containing only the catalytic domain and do not contain domains of fibronectin and hemopexin were ingibirovany in a similar manner along the entire length (activated p-aminophenylacetate (ARMA)) Gelatines, purified from the media of S3 HeLa cells infected with recombinant vaccinia virus, cDNA encoding full length human Pro-MMP and 9, as stated above [Olson, MU etc., J Biol Chem, 2000. 275(4): p.2661-8]. These results confirmed that the inhibition is mediated by direct interaction with the catalytic domain and does not depend on interaction with domains hemopexin or fibronectin. Profile competitive inhibition provided further indication of direct interaction with the catalytic site of zinc. Irrelevant monoclonal antibody, prepared in a similar manner, did not prevent photolytic activity of the enzyme.

Thus, the observed inhibition was not due to the trace together cleaned of pollutants. Antibodies for these experiments were purified from tissue culture and did not contain the detected amounts of the active MMP in a fraction of the purified antibodies.

To study the selectivity S its reactivity was examined in relation to various subgroups of matrix metalloproteinases, including matrilysin (MMP), membrane-type MMP (MT-MMP) and related disintegrin (ADAM), the enzyme which converts α-tumor necrosis factor (TAS). Nuclear structure of these enzymes are highly similar, varying mainly in the peripheral loops. More specifically, the cell frame zinc-histidine good conservative, showing a typical helix followed by a loop which serves as a cell scaffold for three histidine residues that coordinate ion catalytic zinc (FIG).

Such inhibition assays were performed with suitable fluorogenic peptide substrates. Interestingly, neither MMP-7 or TAS were not ingibirovany in any measurable extent after incubation with S in concentrations up to 30 µm, indicating a significant level of selectivity in relation to gelatins. MT-MMP was ingibirovany S, to a lesser extent, with a Ki of 14.4±0.75 µm. Interestingly, the source of this selectivity cannot be explained on the basis of only the design of antibodies to recognize conservative cell frame zinc-histidine, because of the structure of nuclei, especially in the active site is highly similar. Changes in the sequences, mainly in the peri is erinya loops dictate differences in the degree of openness of the fragment zinc-histidine, in the form of the active site electrostatics its surface may be responsible for this selective inhibition model.

S also tested for cross-reactivity with different, depending on the zinc metalloprotease, carbon anhydrase and alcohol dehydrogenase. Similarly, MMP, carboxylic anhydrase (KA) is ion catalytic zinc, rectangular coordinated to three histidine ligands and a water molecule. Therefore, several potent inhibitors small molecule MMP (type from sulphonated of hydroxamate amino acids) act as effective inhibitors of the CA and Vice versa. Some JV-hydroxysulfonic, previously studied as inhibitors of the CA, also have inhibiting properties to MMP [Scozzafava, A. and K.T. Suburan, J Med Chem, 2000. 43(20): p.3677-87]. The active site of alcohol dehydrogenase from thermophilic bacteria (TbADH) includes another component zinc-protein, in which the zinc is associated with histidine, cysteine, aspartate and glutamate, located in the cleft. Corresponding experiments on the functional inhibition in the presence of antibodies in concentrations up to 30 µm showed no inhibitory effect against both enzymes. It should be noted that the active site of CA, located in the Central areas and 10-filament, twisted R-list, consists of crevices cone shape with a depth of 15 angstroms with a quadrangular ion Zn2+at the bottom of the cleft. Unlike inhibitors, small molecules, ion zinc should be too deeply hidden for interaction with the antibody. Importantly, these experiments further demonstrate selective inhibitory profile S.

In the cellular environment inhibitory effect S against gelatinase was tested demografia in situ with natural substrate gelatinase - gelatin.

The cells of the human fibrosarcoma, NT grown membrane expressing culture associated with MT-MMP and secreting MMP-2 and 9 [Jeanbernard, T.A. and others, Matrix Biol, 1998. 16(8): p.483-96] were placed on gelatin conjugated with fluorescein (DQ-gelatin). As shown in FIGA, untreated cells NT showed significant gelatinolytic activity on the cell surface. Treatment with monoclonal antibody in an amount of 5 mmol significantly reduced surface gelatinolytic activity, similar to the inhibition observed in inhibitor gelatinase-based mechanism, SB-ST. SB-3CT has a similar inhibitory profile, as it inhibits and gelatinase, and MT-MMP (the values of the Ki - 28, 400, and 110 nm for MMP, MMP and MT-MMP, respectively).

Briefly, IS inhibited and the cleft of the synthetic peptide is IDA in vitro, and natural macromolecular substrate in situ. 6C6 showed a competitive mode of inhibition against MMP, a similar mechanism of inhibition of TIMP. Competitive inhibitory profile is a further indication of direct interaction with a component of the catalytic zinc. It is important that 6C6 showed selective inhibitory profile against gelatinase. The source of this selectivity cannot be explained by the orientation of antibodies on a conservative fragment zinc-histidine. These results suggest that the antibody interacts with additional determinants on the surface of the enzyme that are responsible for the observed specificity.

EXAMPLE 10

The exposure processing by the monoclinal antibody 6C6 on DSS-induced colitis in mice

There is a growing amount of evidence that MMPs are involved in remodeling and tissue destruction associated with several inflammatory conditions, including inflammatory bowel disease (IBD) [BAFA, PPM and other, Gastroenterology, 1999. 117(4): p 814-22; Heiskell, RB and others, Gut, 2000. 47(1): R-62; Background LAMPE, B. and others, Gut, 2000. 47(1): R-73; Kirkegaard, T. and others, Gut, 2004. 53(5): R-9].

Therefore, the authors of the present invention investigated ingibiruyushee action 6C6 against gelatinase in vivo in mice with experimental model of inflammatory bowel disease.

For research ingibiruet is her activity 6C6 was studied the ability of processing a monoclonal antibody to improve the condition of DSS-induced acute colitis. Specifically, 2% DSS were introduced highly susceptible strain of C57BL/6 mice for five days. Treatment with 6C6 conducted daily by intra-peritoneal injection at a dose of 1.5 or 5 mg/mouse, starting from the day of induction. In mice subjected to induction of 2% DSS, developed symptoms of acute colitis with diarrhea, rectal bleeding and severe weight loss.

The effect of treatment with monoclonal antibody to the controlled daily the disease activity index (DAI) (joint assessment of body weight, bleeding and stool consistency) shown in FIGA. In mice treated with monoclonal antibody, the activity of the disease was less than in the control group (sncase with day 6). Additional macroscopic manifestation of DSS-induced colitis is a reduction in the length of the colon (PIGV). So, a 30% reduction in the length of the colon was found in untreated mice compared to animals that were not previously used in experiments 11 days after administration of DSS. In contrast, only an average reduction of 22% or 16% was obtained in mice treated S at doses of 1.5 and 5 mg/mouse, respectively. Protective effects S were also confirmed by the mortality rate from the disease. The mortality rate of 60% was detected in untreated mice 11 days after induction, whereas the mortality rate tol is to 33% was observed in mice receiving S (FIGS). Thus, treatment of C57BL/6 mice by antibody S has led to increased survival rate in addition to reduced manifestations of DSS-induced colitis.

In General, these results demonstrated therapeutic potential S as inhibitor gelatinase.

EXAMPLE 11

Characterization of complex MMP-S x-ray absorption spectroscopy

For further investigation of the differences between active MMP and inhibited complex MMP-S was performed x-ray absorption spectroscopy. On FIG shows data collected for fluorescence. These data are presented in the form of spectra Fourier transform to represent the radial distribution of the different atoms in the first and second coordination shells of the ion catalytic zinc in ME. The apparent change in the spectra of the radial distribution for the free and inhibited enzyme can be observed above the noise level. These spectral changes indicate that the local environment of the ion catalytic zinc undergoes structural changes upon binding S. The observed deviation in the spatial distribution and intensities of the peaks of the spectral characteristics of the Fourier transform between the active and inhibited the enzyme clearly indicates that the local structure of the ion is atleticheskogo zinc changes after the complex formation of antibodies.

It is clear that certain features of the invention, which, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which, for brevity, described in the context of one possible implementation can be provided separately or in any suitable combination.

Although the invention is described in connection with specific variants of implementation, it is clear that professionals in this field will be obvious, many alternatives, modifications, and changes. Accordingly, it is intended to embrace all such alternatives, modifications and changes which fall within the meaning and broad scope of the attached claims. All publications, patents and patent applications mentioned in this specification, is included in full by reference to the same extent as if each individual publication, patent or patent application was specifically and individually indicated as incorporated herein by reference. In addition, citation or identification of any reference in this application shall be construed as an admission that such reference is prior art against the present invention.

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10. Kleifeld, O., Kotra, L.P., Gervasi, D.C., Brown, S., Bernardo, M.M., Fridman, R., Mobashery, S., and Sagi, I. (2001). X-ray Absorption Studies of Human Matrix Metallo-proteinase-2 (MMP-2) Bound to a Highly Selective Mechanism-based Inhibitor. Comparison with the latent and active forms of the enzyme. J. Biol. Chem. 276, 17125-17131.

11. Korkhin, Y., Kalb (Gilboa), A.J., Peretz, M., Bogin, O., Burstein, Y., and Frolow, F. (1998). NADP-dependent Bacterial Alcohol Dehydrogenases: Crystal Structure, Co-factor-binding and Cofactor Specificity of the ADHs of Clostridium beijerinkii and Thermoanaerobacter brockii. J. Mol. Biol. 278, 967-981.

12. Morgunova, E., Tuuttila, A., Bergmann, U., Isupov, M., Lindqvist, Y., Schneider, G., and Tryggvason, K. (1999). Structure of Human Pro-Matrix Metalloproteinase-2: Activation Mechanism Revealed. Science 284, 1667-1670.

13. Bode, W., Femandez-Catalan, With, Tschesche, H., Grams, F., Nagase, H., and Maskos, K. (1999). Structural properties of matrix metalloproteinases. Cell. Mol. Life. Sci. 55, 639-652.

14. Netzel-Arnett, S., Mallya, S.K., Nagase, H., Birkedal-Hansen, H., and Van Wart, H.E. (1991). Continuously recording fluorescent assays optimized for five human matrix metalloproteinases. Anal. Biochem. 195, 86-92.

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16. Reponen, P., Sahlberg, C, Huhtala, P., Hurskainen, T., Theslef F, L, and Tryggvason, K. (1992). Molecular cloning of murine 72-kDa type IV collagenase and its expression during mouse development. J. Biol. Chem. 267, 7856-7862.

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18. Van Wart, H., and Birkedal-Hansen, H. (1990). The Cisteine Switch: A Principle of Regulation of Activity with Potential Applicability to the Entire Matrix Metalloproteinase Gene Family. Proc. Natl. Acad. Sci. USA 87, 5578-5582.

19. Will, H., Atkinson SJ, Butler, G.S., Smith, C., and Murphy, G. (1996). The soluble catalytic domain of membrane type 1 matrix metalloproteinase cleaves the pro-peptide of progelatinase A and initiates autoproteolytic activation. J. Biol. Chem. 271, 17119-17123.

20. Zabinsky, S.I., Rehr, J.J., Ankudinov, A., Albers, R.C., and Eller, M. J. (1995). Multiple-scattering calculations of x-ray-absorption spectra. Phys. Rev. 52, 2995-3009.

1. The compound having General formula (I):

where m and n are independently integers from 1 to 6;
each of X1-X3and Y1-Y3is About;
R1-R3each independently selected from the group consisting of hydrogen which any alkyl; and
R is O-(CH2)x-C(=O)NR'-(CH2y-other',
where x and y are each independently integers from 1 to 6; and
R' is chosen from the group consisting of hydrogen and alkyl.

2. The compound according to claim 1, having the formula (II):

where R=O-CH2-C(=O)NH-CH2-CH2-NH2.

3. Antibody containing region recognition of antigen, is able to specifically bind the compound according to claim 1 or 2, and obtained by using the compounds according to claim 2, and a compound capable to inhibit the activity of MMP-2 and MMP-9 inhibition constant Ki of less than 5 microns.

4. The antibody according to claim 3, which contains the amino acid sequence hypervariable regions indicated in SEQ ID NO:7, 8, 9, 10, 11 and 12.

5. The use of compounds according to claim 1 or 2 to obtain antibodies that detect MMP-2 and MMP-9.

6. A pharmaceutical composition comprising a therapeutic amount of the antibody according to claim 3 or 4, and pharmaceutically acceptable carrier for the treatment of diseases associated with unbalanced and abnormal activity of MMP-2 or MMP-9.

7. The use of antibodies according to claim 3 or 4 for the preparation of medicaments for the treatment of a disease associated with imbalanced or abnormal activity of metalloproteins.

8. The use according to claim 7, where the disease is inflammatory bowel disease.

9. How inhibi the Finance activity of MMP-2 or MMP-9 in the sample, moreover, the method includes the contact cells in vitro with any one of the antibodies according to claim 3 or 4, this inhibiting activity matrix activity of MMP-2 or MMP-9 in the sample.

10. Antibodies according to claim 3 or 4, attached to a solid substrate.

11. The composition for determination of MMP-2 or MMP-9, containing an effective amount of the antibody according to claim 3 and buffer solution.



 

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FIELD: medicine, pharmaceutics.

SUBSTANCE: present invention refers to biochemistry and immunology, and may be used in pharmacology for preparing medicines with anticancer activity. What is described is a peptide representing a fragment (351-359) of polypeptide 1 associated with the cell division cycle (CDCA1), and possessing ability to induce cytotoxic (killer) T cells, as well as analogues thereof, including a replacement of an amino acid the second next to an N-terminal by methionine and/or an C-terminal amino acid by valine or leucine and preserving its inducing action on killer T cells.

EFFECT: what is presented is using the peptides according the invention both directly, and as an ingredient of immunogenic compositions (vaccines), for preventing and/or treating cancer diseases associated with HLA-A2 and CDCA1 expression.

21 cl, 1 tbl, 16 dwg, 2 ex

FIELD: medicine.

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EFFECT: use of the invention can find further application in treating various S1P-related diseases.

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The invention relates to the field of analysis

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention describes methods of treating an angiogenesis regulated disorder, involving the administration of an effective amount of the human protein tyrosine phosphatase beta (HPTPβ) binding antibody to an individual. Binding the antibody to HPTPβ improves Tie-2 signal transmission and thereby regulates (increases or decrease) angiogenesis depending on the disease. The used antibody is produced by the hybridoma ATCC №PTA-7580. The antibody provides a basis to prepare a pharmaceutical composition for treating the angiogenesis regulated disease which contains both the antibody, and a pharmaceutically acceptable carrier.

EFFECT: methods according to the invention are used to treat the disorders accompanied by decreased or increased angiogenesis, particularly the diseases such as musculoskeletal or myocardial ischemia, stroke, coronary artery and peripheral vascular disease, sickle-cell anaemia, Paget disease, Lyme disease and others.

7 cl, 12 dwg, 1 tbl, 6 ex

FIELD: biotechnologies.

SUBSTANCE: medicinal agent for inhibition of MASP-2-dependent complement is an agent containing an antibody or its fragment, bound with a full-size polypeptide MASP-2, but not bound with a MASP-2 N-terminal fragment containing CUBI-EGF-CUBII domains and not bound with a MASP-2 C-terminal fragment, containing of CCPII-SP domains.

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9 cl, 39 dwg, 7 tbl, 31 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to medicine. What is presented is an antibody produced of hybridoma ATCC No. PTA-7580 specific to human protein tyrosine phosphatase beta (HPTPβ). What is described is a Fab version of said antibody, as well as versions of method of treating and pharmaceutical compositions based on the use of the antibodies. Binding the antibody and HPTPβ intensifies Tie-2 signal transmission, and thereby increases angiogenesis, whereas binding the Fab antigen-binding antibody fragment and HPTPβ inhibits Tie-2 signal transmission, and thereby reduces angiogenesis.

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21 cl, 12 dwg, 1 tbl, 5 ex

FIELD: medicine.

SUBSTANCE: invention relates to biotechnology and represents antibody, which is bound with human hepatocyte growth factor activator (HGFA). Also described is method of treating disease associated with impaired regulation of HGF/c-met-mediated signal that applies antibody.

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FIELD: biology, biotechnologies.

SUBSTANCE: invention can be used in the food-processing industry. For obtaining of 6-O-αD-(1,6-GPS) isomaltulose would submit to the reactionary solution containing enzyme, possessing ability of catalyzing transformation of isomaltulose in 1,6-GPS from which allocate a target product after an incubation at temperature of 20-40°C. Before or during incubation in a reactionary solution add regenerative equivalents. Allocate enzyme with means of anion exchange and two affine chromatography from a crude extract of a microorganism of sort Gluconobacter containing a gene of enzyme, possessing ability of catalyzing transformation of isomaltulose in 1,6-GPS. The nucleic acid coding enzyme, possessing ability to catalyze transformation of isomaltulose in 1,6-GPS is obtained. The vector containing given nucleic acid, is intended for provision of an expression of this enzyme in a host cell.

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26 cl, 2 dwg, 1 tbl, 4 ex

FIELD: biotechnological methods.

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14 cl, 3 tbl, 11 ex

FIELD: biotechnology, biochemistry, enzymes.

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11 cl, 3 tbl, 10 dwg

FIELD: biotechnology.

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27 cl, 8 ex, 3 tbl, 5 dwg

FIELD: genetic engineering, biotechnology, biochemistry, medicine.

SUBSTANCE: invention represents a polypeptide of new family of phosphodiesterases and a polynucleotide encoding thereof. Invention relates to the development of methods for detecting partners in specific binding indicated polypeptide and polynucleotide involving stages for their contacting with a compound, detection for binding and detection a compound as a partner for specific binding. Also, invention proposes the constructed expression construction that is used in the method for preparing polypeptide of new family of phosphodiesterases for preparing a cell-producer. Also, monoclonal and polyclonal antibodies raised to this polypeptide have been prepared. Invention describes anti-sense polynucleotide for regulation of expression of polypeptide of new family of phosphodiesterases. Using the invention provides additional pharmacological approaches in treatment of states associated with disturbance of metabolic ways of cyclic nucleotides.

EFFECT: valuable medicinal and biochemical properties of polypeptide.

27 cl, 3 tbl, 11 ex

FIELD: genetic engineering, in particular genes for cell cycle controlling point.

SUBSTANCE: polynucleotide encoding rad3 polypeptide ATR homologue is cloned into expression vector, having functionality in eucariotic cells. Polypeptide of rad3 polypeptide ATR homologue is obtained by cultivation of eucariotic cell culture, transformed by vector. Monoclonal antibody to rad3 polypeptide ATR homologue is obtained by hybridoma technologies. Polyclonal antibodies are obtained by inoculation of rad3 polypeptide ATR homologue in host animal. Polynucleotide presence in animal tissue sample is detected by contacting of this sample containing DNA or RNA with polynucleotide encoding rad3 polypeptide ATR homologue under hybridization conditions. Polypeptide in biological sample is detected by sample contact with monoclonal or polyclonal antibodies. Substances having anticancer activity are screened on the base of reduced activity of ATR polypeptide on substrate or reduced chelating of ATR homologue in presence of candidate substance. Present invention makes it possible to produce human or S.pombe rad3 polypeptide ATR homologue and is useful in investigation ATR role as gene for cell cycle controlling point in cell culture in vivo or in vitro.

EFFECT: new anticancer substances.

24 cl, 1 dwg

FIELD: chemistry.

SUBSTANCE: invention relates to molecular complexes of zinc and cadmium bis(1-phenyl-3-methyl-4-formyl-5-pyrazolonate) with amino-derivatives of nitric heterocycles of general formula (I) (NH2-Het)n, where NH2-Het is 1-aminoisoquinoline, 3-aminoquinoline, 6-aminoquinoline, 5-amino-4,6-dimethylquinoline, 2-aminopyridine, 2-amino-5-bromopyridine, 3-amino-5-methylisoxazole, 2-amino-1-ethylbenzimidazole, M is Zn, Cd, n=1, 2.

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14 ex

FIELD: chemistry.

SUBSTANCE: invention relates to electroluminescent substances and specifically to zinc (II) bis{3-methyl-1-phenyl-4-[(quinoline-3-imino)-methyl]1-H-pyrazol-5-onato} of general formula I . Also disclosed is an electroluminescent device which contains zinc (II) bis{3-methyl-1-phenyl-4-[(quinoline-3-imino)-methyl]1-H-pyrazol-5-onato} of general formula I.

EFFECT: compound exhibits electroluminscent properties in the yellow spectral region with high brightness.

2 cl, 4 ex

FIELD: chemistry.

SUBSTANCE: method involves reaction of a zinc halide with trialkylaluminium containing a hydride and trialkylaluminium with one or more polymerised alkyl groups. Content of hydride in trialkylaluminium ranges from 0.01 wt % to 0.10 wt %. After reaction, dialkylzinc containing not more than 10 ppmw aluminium is separated from the reaction products and dialkylaluminium monohalide containing not more than 10 ppmw zinc is then separated.

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7 cl, 2 dwg, 1 tbl, 5 ex

FIELD: chemistry.

SUBSTANCE: invention relates to novel substituted metal phthalocyanines, which can be used as direct and acid dyes for dyeing cotton and protein fibre. Disclosed are tetra-4-[(4'-carboxy)phenylamino]phthalocyanine metal complexes of formula, where M=Cu, Ni, Zn.

EFFECT: metal phthalocyanines expand the colour gamma of the current light-blue copper tetra-4-carboxyphthalocyanine, which is the closest on structure and application, to a blue-green and green colour.

6 dwg, 5 ex

FIELD: chemistry.

SUBSTANCE: invention relates to a method of producing zinc meso-tetra-aminotetrabenzoporphyrinate. The method involves reaction of a phthalic anhydride with aminoacetic acid to obtain N-carboxymethylphthalimide, from which while heating with zinc oxide, a zinc salt of N-carboxymethylphthalimide is obtained. The obtained salt reacts with phthalimide at high temperature from 240°C to 320°C for 2-2.5 hours, followed by treatment of the reaction mass with hydrazine hydrate in pyridine solution at boiling point thereof for 1.5-2 hours.

EFFECT: high safety of the process owing to prevention of formation of dangerous and toxic acetic and nitric acids, high cost-effectiveness of the process and obtaining an end product of high purity.

3 dwg

FIELD: chemistry.

SUBSTANCE: disclosed are novel phthalocyanines, which are quaternised derivates of zinc and aluminium tetra(3-thiophenyl)phthalocyanines of formula MPc(SPh)4Rn, where: MPc(SPh)4Rn is zinc or aluminium tetra(3-thiopheny)phthalocyanine, M=Zn, AlX; X=Cl, HO, , , n=4-9. The disclosed phthalocyanines are sensitising agents for formation of singlet oxygen under the effect of visible light. Also disclosed is a method of treating water using said phthalocyanines or mixture thereof with acridine, rhodamine or phenothiazine dyes and visible light in the presence of oxygen.

EFFECT: efficient treatment of water from bacterial contamination.

2 cl, 1 dwg, 3 tbl, 7 ex

FIELD: chemistry.

SUBSTANCE: disclosed is a method for synthesis of compounds of general formula R1-M1-AdzLix (I) by reacting compound R1 - A (III) with element M1, where M1 denotes Mn, Cu, Zn, Li, Bi, in the presence of lithium salts. Also disclosed is a method for synthesis of a compound of general formula R1m - M3 - TnzLix (II) by reacting compound R1 - A (III) with an M3-containing compound in the presence of lithium salts and in the presence of an elementary metal M2 - Li, Na, Mg. Metal M3 can be selected from Al, Mg, B.

EFFECT: improved method.

14 cl, 1 tbl

FIELD: chemistry.

SUBSTANCE: invention relates to novel derivatives of 1-allylimidazole with metal salts , where R denotes allyl, E denotes a metal, e.g. Zn (II) or Co (II), An denotes chlorine or acetate, n equals 2.

EFFECT: novel 1-allylimidazole derivatives having antihypoxic activity are obtained.

1 cl, 7 tbl, 2 ex

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