Peptides of directed action of vegfr-1/nrp-1
FIELD: medicine, pharmaceutics.
SUBSTANCE: invention refers to molecular medicine and a target-directed drug delivery. There are presented new peptide sequences sized 10 or less amino acids containing at least a continuous amino acid sequence D(LPR) which selectively influence on the VEGFR-1 and NRP-1 expressing cells.
EFFECT: target-directed molecules according to the inventions are applicable for treating and diagnosing the neovascular and angiogenic VEGF-associated disorders, such as cancer, obesity, diabetes, asthma, arthritis, cirrhosis and eye diseases.
16 cl, 4 dwg, 2 ex
The present invention was created with the financial support of the U.S. government grants CA103056 and CA100632 the National Institute of Health. Thus, the U.S. government has certain rights in the present invention.
This application claims the priority of provisional application U.S. No. 60/954750, registered on August 8, 2007, the description of which is incorporated herein by reference.
The SCOPE of the INVENTION
The present invention concerns the field of molecular medicine and target-oriented delivery of therapeutic agents. In particular, the present invention relates to the identification of new peptide sequences that selectively affect VEGFR-1 and NRP-1 as a therapeutic target for the treatment and detection of neovascular and angiogenic VEGF-associated disorders, including, among other things, cancer, obesity, diabetes, asthma, arthritis, liver and eye diseases.
Blood vessels are an integral part of the body, delivering oxygen and nutrients to almost all organs and tissues. Most vessels are formed during embryogenesis and in the adult organism, the formation of new blood vessels (a process called angiogenesis) limited and mainly occurs during wound healing and normalgenerator cycle in women. It helps in therapy, as some diseases progress only in the case of inducing them to form new blood vessels, cancer, obesity, diabetes, asthma, arthritis, liver and eye diseases are attributed to the majority of diseases, for which, apparently, you can slow down or stop using inhibitors of angiogenesis.
Vascular endothelial growth factor (VEGF) is a key molecule that controls angiogenesis, and three anti-VEGF drug were approved by the Department for supervision of food and drugs U.S. for the treatment of certain types of cancer with good, but not perfect, therapeutic effect (Kamba and McDonald, 2007). Thus, the development of new generation medications, selectively acting on VEGF-mediated signaling pathway, obviously, will make a significant contribution to the regulation of methods of treatment of various diseases. VEGF is a key regulator of angiogenesis and stimulates the division and migration of endothelial cells by binding with tyrosinekinase VEGF receptor (VEGFR-1 and -2) on the cell surface and neuropilins (NRP). Because VEGFR-2 is the major mediator of intracellular mitogenic activity of VEGF, most of the drugs in the clinic today aimed at directly is whether the indirect effect on this specific receptor. On the other hand, VEGFR-1 and NRP-1 was originally considered as a trap or acceptor VEGF (VEGFR-1) or a modulator of the activity of VEGFR-2 (NRP-1). However, studies over the last few years suggest otherwise. Both receptors play a significant role in angiogenesis (Carmeliet et al., 2001; Autiero et al., 2003; Luttun et al., 2004; Kaplan et al., 2005; Wu et al., 2006; Pan et al., 2007) and are important targets in the treatment of angiogenesis. For example, monoclonal antibodies against VEGFR-1 and NRP-1, demonstrate outstanding results in their use as anticancer agents, especially in combination with chemotherapy (Wu et al., 2006; Pan et al., 2007).
Many anti-VEGF drugs, such as bevacizumab (Avastin®) and ranibizumab (Lucentis®), are used in clinical practice and have a certain degree of effectiveness in the treatment and management for neovascular disorders, including various types of cancer and neovascular age-related disorders, age-related macular degeneration. Unfortunately, when conducting such non-specific anti-VEGF therapy were identified significant side effects, including, in particular, the toxic effect on the heart muscle (for example, chest pain, angina, mini, acute heart failure and myocardial infarction, bleeding, proteinuria, hypertension, congestive behold the Dechen failure, arterial embolism and perforation of the gastrointestinal tract). Studies have linked these side effects with the fact that drugs of this class, that is, drugs that directly affect vascular endothelial growth factor (VEGF-A; VEGF165in contrast to selective effects on receptors, can adversely affect the normal VEGF-mediated signaling pathways (Betsholtz et al., 2006). VEGF-A has a special position among the many molecules involved in the regulation of the formation of blood vessels. During embryogenesis, this factor controls many processes, ranging from the proliferation of undifferentiated precursor cells of blood vessels to control the proliferation and migration of endothelial cells, restructuring vessels, arteriovenous differentiation (Ferrara, 2004). Should the protein level of VEGF-A is absolutely critical for the development of blood vessels, because the reduction of its expression half or such increase in two times fatal to the mouse embryo.
Toxicity associated with VEGF-based therapy appears to be caused by the disappearance of the normal capillary bed and heart cells along with VEGF, which is required for normal cell function and vascularization. Interestingly, VEGF-dependent capillaries similar is the features that they exhibit high expression of VEGF receptors, such as receptor type 2 and 3 (VEGFR-2 and VEGFR-3, respectively), and have a small expression of the receptor VEGFR-1 or non-existent. However, VEGFR-1 receptors are expressed in tissues that are relevant targets in a particular disease, including the blood vessels of the retina and the blood vessels of the tumor. Thus, it would be desirable to develop medicinal anti-VEGF drugs, selectively acting on the receptors VEGFR-1, with reduced toxicity, but keeping the desired therapeutic activity. The present invention aims at the creation of peptide ligands selectively targeted to VEGFR-1.
The present invention overcomes the deficiencies of the preceding prior art by providing methods and compositions for selective effects on VEGFR-1 and NRP-1 (hereinafter referred to as VEGFR-1/NRP-1") by using motive LPR (Leu-Pro-Arg) and more preferredD(LPR). Selective directional effect on VEGFR-1/NRP-1 by use of the motif LPR suitable, for example, for treating cancer or other diseases associated with angiogenesis or the growth of blood vessels, such as obesity, diabetes, asthma, arthritis, liver and eye diseases.
In certain embodiments implementing the invention concerns a dedicated LPR peptides, and what about the peptides directed action, which include continuous LPR sequence, for example, located at the amino - or carboxy end of the peptide or inside it. Although the location of the LPR sequence at the end of the peptide is most preferred, it is assumed that the internal location of the LPR will provide, however, the possibility of directed action on VEGFR-1/NRP-1. For convenience in the preparation and use of such a variant embodiment of the invention aims to selected peptides of 10 or less amino acids, containing at least the contiguous amino acid sequence Leu Pro Arg. Because of this, even shorter peptides with a size 7 or 5 amino acids or less, and even by itself LPR Tripeptide most preferred. Thus, the peptides of the directional effects of the present invention can contain 3, 4, 5, 6, 7, 8, 9 or 10 amino acids, in which continuous LPR sequence or such in SEQ ID NO:1 is located inside.
In other embodiments of the invention, the authors propose a specific peptides containing LPR sequences that can be created in a circular form as, for example, peptides having cysteine residue (C) at both ends and turning, where necessary, in cyclic form through education dicysteine (i.e. cystine). An example of such a peptide is IDA is Cys Leu Pro Arg Cys (SEQ ID NO:1). Such cyclic peptides can be of great importance, since disulfide bonds in peptides gives them a strong resistance to chemical, thermal or enzymatic decomposition. Such cyclic peptides can be of particular importance in therapeutic and diagnostic applications, where reduced availability, susceptibility to proteolysis and a short time half-life in vivo.
In other embodiments, the invention describes the use of D-amino acids for the preparation of all or part of the above-mentioned peptides. Peptides containing D-amino acids, have certain advantages over those that contain L-amino acids, in that the use of D amino acids attached to the peptides directed action, in most cases, resistance to proteases and peptidases. In particular, from this point of view, the preferred peptides directed action, consisting entirely of D-amino acids, such asD(Leu Pro Arg) andD(Cys Leu Pro Arg Cys) (SEQ ID NO:1).
In certain embodiments of the invention the above-mentioned LPR molecules can be functionally associated with the second molecule or substance. In preferred embodiments of the invention the connection between the molecule is covalent, examples of which are the chemical conjugate (the way the van through a chemical linker) or fused constructs (formed by the merger of the coding region of the above-mentioned peptide with the coding region of the target protein or peptide, needed to interact with VEGFR-1/NRP-1, no shift of the reading frame). In the case of target-specific peptides or proteins, peptides directed action can be located near or directly at the amino - or carboxy-late (i.e. within the first or last 20 amino acids) of a protein or peptide, subject to interaction with the target.
In various embodiments of the invention the second molecule or substance is a diagnostic tool, a drug, a chemotherapeutic agent, a radioisotope, an anti-angiogenic agent, a Pro-apoptotic agent, a cytotoxic agent, a peptide, protein, hormone, growth factor, cytokine, an antibiotic, an antibody or its fragment or single-chain antibody, an imaging agent, the survival factor, an anti-apoptotic agent, a hormone antagonist or antigen. These molecules or substances represent almost any molecule that can have therapeutic or diagnostic effect in the treatment of cancer, can be attached to LPR molecule directional and/or assigned to the patient in the framework of the invention.
On this basis, if the molecules of the subject, interaction, are Pro-apoptotic substances, their typical representatives are this is posed, ceramide, sphingomyelin, Bcl-2, Bax, Bid, Bik, Bad, caspase-3, caspase-8, caspase-9, fas, fas ligand, fadd, fap-1, tradd, faf, rip, Reaper, apoptin, interleukin-2 converting enzyme, annexin V, (KLAKLAK)2(SEQ ID NO:2); (KLAKKLA)2(SEQ ID NO:3); (KAAKKAA)2(SEQ ID NO:4); or (KLGKKLG)3(SEQ ID NO:5). It should be noted that as with all peptides of the present invention, the above sequence can be either a D-or L-form. For example, proapoptotic peptides (e.g., SEQ ID NO:2-5) both D - and L-forms, apparently, have similar proapoptotic activity, while the D-form has a much longer half-life due to its relative proteinases sustainability. Although in practice, in some cases, preference is given to the L-form in connection with potentially reduced toxic side effects (due to its shorter half-life).
In addition, in embodiments of the invention, where the molecules due to the interaction, are anti-angiogenic substances, their typical representatives are thrombospondin, angiostatin as, for example, angiostatin 5, angiotensin, peptides of laminin, fibronectin peptides, inhibitors of plasminogen activator inhibitors of tissue metalloproteinases, interferons, cytokines such as interleukin 12, platelet factor 4, IP-10, Gro-β, 2-way shall estradiol, protein associated with proliferin, carboxamidates, CM101, marimastat, pentosanpolysulfate, angiopoietin 2 (Regeneron), herbimycin AND PNU145156E, a fragment of prolactin 16K, linomide, thalidomide, pentoxifylline, genistein, TNP-470, endostatin as, for example, endostatin XVII and XV, paclitaxel, docetaxel, polyamine, proteasome inhibitor, kinase inhibitor, signal peptide, accutan, cidofovir, vincristine, bleomycin, AGM-1470, minocycline, C-terminal hemopexin domain of matrix metalloproteinase-2, the Kringle domain 5 of human plasminogen, protein of endostatin and angiostatin, a fused protein of endostatin and the Kringle domain 5 of human plasminogen, minocin induced by interferon gamma (Mig), a protein of Mig and IP10, soluble FLT-1 (fins-like tyrosinekinase receptor 1), kinase insertion domain-containing receptor (KDR), factor pigment epithelium, interferon-alpha, an inhibitor of the signaling pathway (SU5416, SU6668, SUGEN, South San Francisco, California).
In other preferred embodiments of the invention, where the molecules due to the interaction, cytokines are typical representatives are interleukin 1 (IL-1), IL-2, IL-5, IL-10, IL-11, IL-12, IL-18, IL-24, interferon-γ (INF-γ), INF-α, INF-β, a tumor necrosis factor such as, for example, TNF-α or GM-CSF (granulocyte-macrophage colony-stimulating factor).
The above examples t is Auda only representative and do not imply the exclusion of other proapoptotic substances, anti-angiogenic substances or cytokines known in the art.
In other embodiments of the invention, the selected peptides can be attached to a macromolecular complex. In preferred variants of the invention, the macromolecular complex is a virus, a bacteriophage, a bacterium, a liposome, a microparticle, a nanoparticle (e.g., gold nanoparticles), magnetic bead, a yeast cell, a mammalian cell or a bacterial cell. In the case of viruses, particularly preferred bacteriophage, lentivirus, papovavirus, adenovirus, retrovirus, AAV, vaccinia virus, herpes virus. These examples are only representative and macromolecular complexes in the framework of the present invention can include virtually any complex, which can be attached to the peptide directional and assigned to the patient. In other preferred embodiments of the invention, the selected peptide can be attached to eukaryotic expression vectors, among which preferred is a vector for gene therapy.
In the following embodiment of the invention, the selected peptide can be attached to the solid media, among which more preferred magnetic beads, the beads sepharose, agarose beads, nitrocellulose the second membrane, nylon membrane, matrix column chromatography, matrix high performance liquid chromatography (HPLC), matrix high-speed liquid chromatography (FPLC), plate microtitration or microchip.
In the following embodiments of the invention are considered merged constructs proteins comprising any of the aforementioned LPR peptide directed action, coupled with the selected protein with the formation of the fused protein construct, in which the resulting merged the construct of the protein, with the additional inclusion of LPR molecules directed action, which is an artificial and not a natural protein. In General, in such preferred embodiments of the invention such fused constructs of proteins can be created using any of the above-mentioned class of molecules.
In the following embodiments, the invention describes the creation of a construct aimed at interaction with VEGFR-1/NRP-1, which includes obtained LPR peptide directed action, as described above, and the connecting peptide molecules, with the creation of the construct primarily by covalent crosslinking. As mentioned above, when the molecule is subject to interaction, is a protein or peptide, the preferred target-oriented constructs are those in which peptideprophet attached near or directly at the amino - or carboxy-end of such a molecule.
The present invention also aims at a method of the target-oriented delivery of a molecule or protein to cells expressing VEGFR-1 or NRP-1, where the method is to obtain LPR peptide directional or construct fused proteins, as described above, or a target-oriented construct, as described above, and the introduction of the peptide or the fused construct proteins in the cell population that contains cells expressing VEGFR-1 or NRP-1, order delivery, thereby, molecules or protein to specific cells. In General, if the conjugated or fused construct proteins will be used in diagnostic or therapeutic purposes in the subject, such as a person, conjugated or fused construct proteins create in the form of a pharmaceutically acceptable composition that is administered to the subject.
It is assumed that in the case of therapeutic treatment of patients with the disease or disorders, the subject will usually require the carrying out of anti-angiogenic therapy. Such diseases or disorders include hyperproliferative diseases, impaired weight gain, obesity, diabetes, asthma, arthritis, cirrhosis or eye diseases. Typical of hyperproliferative diseases considered for therapy with the use of therapeutic conjugates in accordance with the invention, are rawmat idny arthritis, inflammatory bowel disease, osteoarthritis, leiomyomata, adenoma, lipoma, hemangioma, fibroma, occlusion of vessels, restenosis, atherosclerosis, pre-neoplastic lesions (such as glandular hyperplasia, intraepithelial neoplasia of the prostate gland), carcinoma in situ, hairy leukoplakia of the mouth or psoriasis.
The invention also considers that the conjugates of the present invention will be suitable for the treatment of various cancers, particularly those species that are extremely angiogene. Typical types of cancer are the cancer of the gums, tongue, lung, skin, liver, kidneys, eyes, brain, leukemia, mesothelioma, neuroblastoma, cancer of the head, neck, breast, pancreatic, prostate, renal, bone, testicular, ovarian, cervix, esophagus, uterus, bladder, gastrointestinal, lymphoma, colon cancer, sarcoma, stomach cancer.
The following aspects of the invention considered that the subject to be treated, have eye diseases or disorders characterized intraocular cellular proliferation or neovascularization. Typical violations include age-related macular degeneration, proliferative diabetic retinopathy, retinopathy of prematurity, glaucoma, proliferative vitreoretinopathy, neovascularization due to ocular ischemic syndrome is a, neovascularization due to occlusion of the branches of the veins of the retina, neovascularization due to occlusion of the Central retinal vein or neovascularization due to retinopathy in sickle cell anemia.
In other aspects of the invention is seen that the conjugates of the present invention are suitable for treating disorders weight, such as obesity.
BRIEF DESCRIPTION of FIGURES
Figa, 1B.D(LPR) inhibits neovascularization in vivo. A representative illustration of the matrix of the Matrigel containing 500 μg/mlD(LPR) or control peptide after 7 days of implantation (a, lower panel). The matrix of the Matrigel was separated and angiogenesis was quantified by measuring the hemoglobin content in the matrix of the Matrigel. The histogram is constructed for representative animals from the same experiment (a, top panel). (b) the Number of vessels positive for von Willebrand factor human (P<0,01).
Figa, 2B, 2C. Inhibition of retinal angiogenesis induced by ischemia, under the actionD(LPR). (a) Neovascularization of the retina was induced in newborn mice C57B6 under exposure to 75% oxygen with subsequent therapyD(LPR) (daily injections at a dosage of 20 mg/kg). (b) Stained with hematoxylin and eosin sections of the retina (day P19) showed a significant reduction is obrazovaniya of new blood vessels on the inner surface of the retina (marked by arrows) compared with the control animals. (C) quantification of nuclei of endothelial cells on the inner surface P19 day.
Figa, 3V. Treatment of mice with a tumor, usingD(LPR) slows tumor growth. Mouse Balb/c mice with a tumor originating from EF43.fgf4 were grouped (N=7) and were treated daily with 50 mg/kgD(LPR) or its cyclic formD(CLPR), control peptide or carrier. (a) After five days of treatment, the animals treated withD(LPR) or its cyclic formD(CLPRC), showed reduced tumor volume compared to control animals. (b) the histogram shows the median and variance. The difference in tumor volume between animals treated withD(LPR) or its cyclic formD(CLPRC), was statistically significantly (P<0,02). Two independent experiment were performed with similar results.
Figa, 4B, 4C. Obesity treatment using VEGF-like connectionD(CLPRC). Obese mice (C57BL/6), fed on high-calorie and fat rich diet (weight from 40 to 50 grams)was used in this study. The animals were divided into four groups and conducted daily treatment with (a) VEGF-like peptideD(CLPRC), injectable administered intraperitoneally (50 mg/kg); (b) peptide fat burner (CKGGRAKDC-GG-D(KLAKLAK)2; Kolonin et al., 2004), injectable subcutaneously in to the iruke 1 mg/kg in combination with VEGF-like peptide D(CLPRC) at a dose of 50 mg/kg; or (C) of fat burner in the dosage of 3 mg/kg Animals in the control group were injected with only medium (phosphate-saline buffer solution, PBS).
DESCRIPTION of ILLUSTRATIVE embodiments
Peptides identified using combinatorial libraries, are leaders in the design and synthesis of drugs. They can be quickly synthesized and easily modified with a wide variety of functional groups, providing science and medicine effective tools for creating and directional effects of medicines. Due to their small molecular weight compared with macromolecules, such as antibodies, peptides have the advantage of tissue permeability, and biodistribution, which makes them exceptional leading substances in the field of discovery and development of medicines (review article Falciani et al., 2005). Indeed, the peptides identified by the method of phage display technique, have been used successfully in vivo in target-oriented therapy for delivery of chemotherapeutic drugs (Arap, 1998#10), proapoptotic peptides (Ellerby, 1999#69) or deliver viruses to visualize and gene therapy (Hajitou, 2006#6744). However, their use in drug development has been hampered due to the presence of problems.
Pepti the s by themselves are not suitable for use as medicines as they rapidly break down by proteases and removed from the plasma. The expression of proteases is often elevated in those biological processes that require cell proliferation, migration and tissue restructuring (common to most of the pathological processes such as angiogenesis), leading to increased local proteolytic activity and degradation of the peptide. From the point of view of creation of a medicinal product, peptides often exhibit significant conformational variability, determining the complexity and structural complexity research (Giordano et al., 2005). Thus, the creation of peptidomimetics based on the peptides identified by the method of phage display can be a complex task and is usually limited to pharmaceutical companies or laboratories with synthetic capabilities and access to a large chemical libraries.
Angiogenesis is the sprouting of new blood vessels from pre-existing, and it is an integral component of the growth and metastasis of the tumor (Folkman, 1971) as well as of a number of pathological disorders, such as diabetes, psoriasis, obesity and rheumatoid arthritis (Carmeliet, 2005). In the period of puberty angiogenesis occurs during wound healing, pregnancy and the menstrual cycle, and, thus, drugs aimed at the Academy of Sciences of johannie blood vessels, likely to have important clinical applications in many diseases (Carmeliet et al., 2005). Vascular endothelial growth factor (VEGF) and its receptors have been the focus in this branch of science because of their leading role in the formation of blood vessels. VEGF exerts its action by binding with tyrosinekinase receptors (VEGFR-1, VEGFR-2) and neuropilin-1 (NRP-1) (Olsson et al., 2006). The majority of intracellular signaling pathways and mitogenic effects of VEGF-mediated VEGFR-2, and several drugs targeting this metabolic pathway are currently under research in the clinic (Cardones & Banez, 2006; Schneider & Sledge, 2007). Although VEGFR-1 and NRP-1 play an important role in this process, at first they were accepted without enthusiasm as a possible therapeutic agents. Everything has changed, and studies conducted over the past few years suggest that both receptors play a significant role in angiogenesis (Luttun et al., 2004; Wu et al., 2006; Pan et al., 2007). Studies of gene deletions showed that VEGFR-1 and NRP-1 is required during the formation of blood vessels. Both molecules are receptors for VEGF and placental growth factor (PlGF), and the last together with VEGFR-1 is involved in pathological angiogenesis (Carmeliet et al., 2001), tumor growth (Luttun et al., 2002), increasing the signal in the cell by perekrestnolistaja VEGFR-1/VEGFR-2 (Autiero et al., 2003) and the recruitment of progenitor cells from the bone marrow during neovascularization (Jin et al, 2006; Li et al., 2006). Recent studies also suggested that recruiting VEGFR1+ hematopoietic precursor cells is important for the initiation of tumor metastasis (Kaplan et al., 2005). In addition, NRP-1 not only increases the binding of VEGF to VEGFR-2 (Soker et al., 2002), but also induces the contacts and the migration of endothelial cells independently of the activation of VEGFR-2 (Wang et al., 2003; Murga et al., 2005). Monoclonal antibodies directed against VEGF-binding domain of NRP-1 and against VEGFR-1, reduce both angiogenesis and tumor growth (Wu et al., 2006; Pan et al., 2007). This implies that drug aimed at a signaling pathway through VEGFR-1 and NRP-1, is likely to find important application in the clinic.
The present invention represents a unique angiogenesis inhibitors and substances directed action on VEGFR-1, LPR, andD(LPR), which demonstrated a significant reduction in angiogenesis in three different tests.D(LPR) also inhibit the neovascularization in two animal models after systemic application. Whereas resistanceD(LPR) to degradation under the action of a mixture of pancreatic enzymes, these data indicate that this compound and longer patterns of peptides containing this sequence, veroia is but will survive in the gastrointestinal tract and can be administered orally to patients. Thus, the present invention overcomes the deficiencies of the preceding prior art by identifying LPR motive for the preparation of compounds directed action on VEGFR-1/NRP-1, a therapeutic and/or diagnostic agents, e.g. for the treatment and/or detection of neovascular or angiogenic VEGF-associated disorders, including, without limitation, cancer, obesity, diabetes, asthma, arthritis, liver and eye diseases.
In certain embodiments of the invention are considered specific molecules directed actions that are subject to interaction with cells expressing VEGFR-1/NPR-1, including, more generally, peptides, polypeptides and proteins with modifications in the form of an insert LPR motive either inside or, more preferably, at the N - or C-end of the peptide or protein, or in the vicinity of these ends. It is noteworthy that although the D-form amino acids are more preferable due to its significant resistance to degradation by proteases, the present invention does not exclude the use of less preferred L-form or a mixture of D - and L-amino acids. Certain embodiments of the invention relate to molecules directed action on VEGFR-1/NPR-1, which is functionally linked to a therapeutic or diagnostic tool for what they means. In certain embodiments the invention, therapeutic agent is a virus that can be constructed for expression, or to insert or connect peptides directed action on VEGFR-1/NPR-1 viral envelope protein or fiber protein. Target-oriented viruses may then be used for gene therapy for the treatment of various diseases, including cancer. The ability to selectively act on VEGFR-1/NPR-1 in the vascular system around the tumor and/or the use of peptides, modified peptides, antibodies, viruses and/or other affinity reagents provides a significant advantage in the treatment of cancer, which can lead to increased efficiency and effectiveness.
Used in this description, the singular may mean one or more. Used in this description of the formulas(s) of the invention of the word in the singular in combination with the word "contains" can mean one or more than one. Used in this document the word "another" may mean at least a second or more.
The term "molecule directed action" covers various types of affinity reagents that can be used to improve localization or binding substance in a hearth in animals, the key bodies, tissue specific cell types, diseased tissue or tumor. Molecules directed action can be peptides, peptide mimetics, polypeptides, antibodies such as antibody molecules, nucleic acids, aptamers, and fragments of the above. Molecules directed action are also small molecules. In certain embodiments of the invention molecules directed action can improve the localization of substances in cells expressing VEGFR-1/NRP-1 extracellular, i.e. VEGFR-1/NRP-1 associated with the cell surface or associated with the surrounding extracellular matrix. Selective binding molecules directed action of the present invention, for example, peptide directed action, as well as variants and fragments of the above, is called the binding molecules directed action with molecule-target (e.g., VEGFR-1/NRP-1) in the absence of significant binding with other proteins. It is believed that the molecule directional selectively bind, even if it also binds to other proteins that are substantially non-homologous to the target, provided that such proteins have homology with a fragment or domain of the target peptide antibodies. In this case, it will be clear that the binding molecules directed action target is selection is m, despite some degree of cross-reactivity. As a rule, it is possible to assess the degree of cross-reactivity and to differentiate it from binding with the target.
"Peptide directed action" is a peptide consisting of a sequence of contiguous amino acids LPR and characterized by selective localization in an organ, tissue or cell type, which consists in a specific binding with extracellular protein or molecule that is specifically expressed or formed in a specific tissue or type(s) of cells. Selective localization can be determined, for example, the methods presented below, in which the purified peptide sequence directional built-in protein presented on the outer surface of the phage.
The term "subject" means, primarily, a mammal. In certain embodiments of the invention, the subject is a mouse, rabbit, pig, horse, cow, cat, dog, sheep, goat or monkey. In one aspect of the invention, the subject is the man.
3. Proteins and peptides
In certain embodiments implementing the present invention relates to new compositions comprising at least one protein or peptide. Used in this description of the invention the protein or peptide, mostly, are in the Isla other protein larger than 200 amino acids up to the full sequence, translated from a gene; a polypeptide of approximately more than 100 amino acids; and/or a peptide ranging in size from about 3 to about 100 amino acids. For convenience, the terms "protein", "polypeptide" and "peptide" are used herein interchangeably.
In certain embodiments of the invention the size of the at least one protein or peptide is, among other things, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, about 110, about 120, about 130, about 140, about 150, about 160, about 170, about 180, about 190, about 200, about 210, about 220, about 230, about 240, about 250, about 275, about 300, about 325, about 350, about 375, about 400, about 425, about 450, about 475, about 500, about 525, about 550, about 575, about 600, about 625, about 650, about 675, about 700, about 725, about 750, about 775, about 800, priblizitelen the 825, about 850, about 875, about 900, about 925, about 950, about 975, about 1000, about 1100, about 1200, about 1300, about 1400, about 1500, about 1750, about 2000, about 2250, about 2500 or greater amino acid residues, or any other number of amino acid residues derived from the above (for example, from about 200 to about 2500 amino acid residues).
Used in this description, the term "amino acid residue" refers to any naturally occurring amino acid, any derivative of an amino acid or any mimetic amino acids known in the art. In certain embodiments the invention, the amino acid residues of the protein or peptide serial without any interruption amino acid sequence is amino acids. In other embodiments, the implementation sequence may include one or more non-amino acid groups. In certain embodiments of the implementation of the sequence of amino acid residues of the protein or peptide can be interrupted one or more non-amino acid group.
Thus, the term "protein or peptide" means an amino acid sequence comprising, p is at least one of the 20 common amino acids found in nature, or at least one modified or not typical amino acid, including, without limitation, Aad, 2-aminoadipic acid; EtAsn, N-ethylasparagine; Baad, 3-aminoadipic acid; Hyl, hydroxylysine; Bala, β-alanine, β-aminopropionic acid; AHyl, ALLO-hydroxylysine; Abu, 2-aminobutyric acid; 3Hyp, 3-hydroxyproline; 4Abu, 4-aminobutyric acid, piperidine acid; 4Hyp, 4-hydroxyproline; Acp, 6-aminocapronic acid; Ide, isodesmosine; Ahe, 2-aminoheptanoic acid; AIle, ALLO-isoleucine; Aib, 2-aminoisobutyric acid; MeGly, N-methylglycine, sarcosine; Baib, 3-aminoisobutyric acid; MeIle, N-methylisoleucine; Apm, 2-aminopimelic acid; MeLys, 6-N-methyllysine; Dbu, 2,4-diaminobutyric acid; MeVal, N-methylvaline; Des, desmosine; Nva, Norvaline; Dpm, 2,2'-diaminopimelic acid; Nle, norleucine; Dpr, 2,3-diaminopropionic acid; Orn, ornithine; and EtGly, N-ethylglycine.
Proteins or peptides can be obtained using any technique known to the skilled in the art, including standard molecular biological techniques for the expression of proteins, polypeptides or peptides, separation of proteins or peptides from natural sources or by chemical synthesis of proteins or peptides. Nucleotide and protein, polypeptide and peptide sequences corresponding to different the m genes, were previously identified and can be found in computer databases, known to specialists in this field of technology. Some of these databases are Genbank and GenPept databases of the National center of Biotechnology (search the Internet ncbi.nlm.nih.gov). Coding region of known genes may be amplified and/or expressed using the techniques described herein or well-known experts in this field. Alternatively, various commercial preparations of proteins, polypeptides and peptides known to specialists in this field of technology.
4. Slit proteins
Other aspects of embodiments of the invention protein conjugates concern fused proteins. These molecules, in General, have all or a significant portion of the protein directional (for example, LPR peptide directional)connected to the N - or C-end with the whole polypeptide or protein or part thereof. For example, fused proteins can contain a leader sequence from other biotypes for the ability of recombinant protein expression in heterologous hosts. In another used fused protein is immunologically active domain, such as the epitope of the antibody, for example, to facilitate purification of fused protein. The accession of the cleavage site to the place of soedineniya or next to it will facilitate the removal of foreign polypeptide after purification. Part of other used fused proteins are found attached functional domains, such as active sites of enzymes, glycosylation domains, signals directed to a cell or transmembrane region. In preferred embodiments, the implementation of the fused protein of the present invention consist of LPR peptide coupled to a therapeutic protein or peptide. Examples of proteins or peptides that may be part of the fused protein, are cytotoxic proteins, cytotoxic proteins, proapoptotic substances, angiogenic agents, hormones, cytokines, growth factors, peptide drugs, antibodies, Fab fragments of antibodies, antigens, receptor proteins, enzymes, lectins, MHC proteins, cell adhesion proteins and protein binding. The list of these examples is not limited and it is believed that in the framework of the present invention virtually any protein or peptide could be incorporated into a protein, including protein directional. Methods of obtaining fused proteins is well known to specialists in this field of technology. Such proteins can be formed, for example, by chemical compounds using bifunctional cross-linking reagents, by synthesis de novo whole fused protein, or by joining a DNA sequence coding for the soup peptide directed action, and the DNA sequence that encodes a second peptide or protein, followed by expression of the intact fused protein.
5. Purification of proteins
In certain embodiments the invention, the protein or peptide can be selected or cleared. Methods of protein purification well known to specialists in this field of technology. These methods include, in the first phase, homogenization and coarse fractionation of cells, tissue or organ to the polypeptide and polypeptide not fractions. Interesting protein or polypeptide can be further purified chromatographic and electrophoretic way to achieve partial or complete purification (or homogeneous treatment). To analytical methods that are particularly suitable for the preparation of purified proteins include ion exchange chromatography, gel filtration chromatography, polyacrylamide gel electrophoresis, affinity chromatography, immunoaffinity chromatography and isoelectric focusing. An example of purification of the receptor protein affinity chromatography is discussed in U.S. patent No. 5206347, the content of which in full is included in this document by reference. The most effective method of purifying peptides is a high-speed liquid chromatography (FPLC) or even high-performance liquid chromatography (HPLC).
Purification of the protein and the peptide is aimed at the creation of the composition, purified from other components, in which the protein or peptide is purified to any degree relative to its condition encountered in nature. Selected or purified protein or peptide therefore also refers to proteins or peptide free from the environment in which it can exist in nature. In generally, "purified" will refer to the composition of the protein or peptide, which has been subjected to fractionation to remove various other components, and the composition substantially retains its expressed biological activity. Used the term "substantially purified" will refer to a composition in which the protein or peptide is a major component of the composition, such as constituting about 50%, about 60%, about 70%, about 80%, about 90%, about 95% or more of the proteins in the composition.
Various methods for quantifying the degree of purification of the protein or peptide known to experts in the art in light of the present invention. They include, for example, determining the specific activity of an active fraction, or assessing the number of polypeptides in the fraction by the method of SDS/PAGE analysis. The preferred method for assessing the purity of a fraction is to calculate the specific activity of this fraction, its comparison with ecifically activity of the original extract and thus, calculation of the degree of purity in it, estimated "the number of the multiplicity of purification". The actual units used to Express the amount of activity will, of course, depend on the method of analysis selected for cleaning, and whether or not the expressed protein or peptide to be determined activity.
Various techniques suitable for use in protein purification well known to specialists in this field of technology. They include, for example, precipitation with ammonium sulfate, PEG, antibodies and the like, or denaturation by heating, followed by: centrifugation; the stages of chromatography, such as ionoobmennaya chromatography, gel filtration, chromatography in reversed-phase chromatography hydroxylapatite and affinity chromatography; isoelectric focusing; gel electrophoresis; and combinations of such and other techniques. It is known in the art, it is believed that the order of conducting the various purification stages can be changed, or that certain steps may be omitted, and, nevertheless, the result is a suitable method for the preparation of substantially purified protein or peptide.
There is not a primary requirement that the protein or peptide is provided in its most purified form. Indeed, regulatives the use of less refined products in certain embodiments of the invention. Partial purification can be performed using a smaller combination of stages or by using different variants of the basic Protocol treatment. For example, take into account the fact that the cation-exchange column chromatography performed using HPLC equipment, will usually generate a great "multiplicity" clear than those methods using chromatography low pressure. Methods, showing a lower degree of relative purification may have advantages in the allocation of the total number of protein product, or in maintaining the activity of the expressed protein.
Affinity chromatography is a method of chromatography, based on the specific affinity between a substance to be selection, and the molecule with which it can specifically bind. It is a ligand-receptor type interactions. The column material is synthesized by covalent crosslinking of one of the participants of complexing with an insoluble matrix. The material of the column then can specifically adsorb a substance from a solution. Elution is achieved by changing conditions in comparison with those in which there is no binding (e.g., changing pH, ionic strength, temperature and others). The matrix must be prophetic is the your, which in itself does not adsorb molecules to any significant extent, and which has a wide range of chemical, physical and thermal stability. The ligand must be connected to the matrix so that it had no effect on binding activity. The ligand should also provide a relatively tight binding. And should be provided with the possibility of elution of a substance without destroying the sample or ligand.
6. Synthetic peptides
Because of the relatively small size of the peptides directed action of the present invention can be synthesized in solution or on a solid substrate in accordance with conventional methods. Various automatic synthesizers are commercially available and can be used in accordance with known protocols. See, for example, Stewart and Young, 1984; Tam et al., 1983; Merrifield, 1986; Barany and Merrifield, 1979, the contents of each are incorporated as references. Short peptide sequences, typically ranging in size from approximately 6 to approximately 35-50 amino acids, can be easily synthesized by such methods. Alternatively, the method can be applied recombinant DNA in which the nucleotide sequence encoding the peptide of the present invention, is inserted in the expression vector, transformed or transfairusa in approach is appropriate cell host and cultivated under conditions suitable for the expression.
7. Therapeutic or diagnostic conjugates
Molecules directional identified using these methods, can be connected or attached to various substances, including therapeutic or diagnostic tools for the selective delivery of the conjugate into the desired organ, tissue or cell type in the mouse model system. For example, target-oriented delivery of chemotherapeutic agents and proapoptotic peptides to receptors located in the tumor angiogenic vascular system, leads to a noticeable increase in the effectiveness of treatment and reducing systemic toxicity in containing the tumor mouse model (Arap et al., 1998; Ellerby et al., 1999).
Embodiments of the invention directed to the treatment of neovascularization associated with various diseases, such as vascular tumors. In addition to tumor growth, angiogenesis is an important component of other diseases. Uncontrolled angiogenesis contributes to the progression of rheumatoid arthritis, diabetic retinopathy, endometriosis, age-related macular degeneration and psoriasis. The growth of blood vessels leads to the formation of hemangiomas and arteriovenous malformations, causing various clinical manifestations, nachinaet cosmetic complications to life-threatening hemorrhages. Additional embodiments of the invention directed to the treatment of these common diseases, and other associated with neovascularization diseases.
Alternatively, increased expression of VEGFR-1/NRP-1 or stimulating angiogenesis compounds or substances can be used to stimulate angiogenesis. Increased expression of VEGFR-1/NRP-1 can be achieved by the delivery of transgenes VEGFR-1 (i.e., Flt-1 or NRP-1, which, in turn, can be delivered with different vectors for gene therapy, known to specialists in this field of technology.
A. cytokines and chemokines
In certain embodiments of the invention it would be desirable to combine specific bioactive substance with one or more molecules directional target-oriented delivery to the organ, tissue or cell type. Such substances include, without limitation, cytokines, chemokines, proapoptotic factors and angiogenic factors. The term "cytokine" is a generic term for proteins released by one cell population and acting on other cells as intercellular mediators.
Examples of such cytokines are lymphokines, Monokini, growth factors and classic polypeptide hormones. The cytokines are growth hormones such as human growth hormone, N-meteo the silt human growth hormone and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prolactin; glycoprotein hormones such as, for example, follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), and luteinizing hormone (LH); hepatic growth factor; prostaglandin; fibroblast growth factor; prolactin; placental lactogenic, S protein; tumor necrosis factor-α and-β; müller inhibiting substance; mouse associated with gonadotropin peptide; inhibin; activin; vascular endothelial growth factor; integrin; thrombopoietin (TPO); nerve growth factor such as NGF-β; platelet-derived growth factor; transforming growth factors (TGF) - type TGF-α and TGF-β; insulin-like growth factor-I and-II; erythropoietin (EPO); stimulates the growth of bone tissue factor; interferons such as interferon-α, -β, and-γ; colony stimulating factors (CSF) such as, for example, macrophage-CSF (M-CSF), granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF); interleukins (IL) as for example, IL-1, IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, LIF, G-CSF, GM-CSF, M-CSF, EPO, kit-ligand or FLT-3, angiostatin, thrombospondin, endostatin, tumor necrosis factor and LT. Used herein, the term cytokine includes proteins from natural sources or from recombinant cell line and biologically active equivalents of the native placentas is Teleostei cytokines.
Chemokines typically act as chemoattractants for the recruitment of effector cells to the site of expression of the chemokine. Preferred is the expression of certain chemokine in combination with, for example, the genome of the cytokine with the aim of increasing the recruitment of other immune system components to the site of treatment. Chemokines include, among other things, RANTES, MCAF, MIP1-alpha, MIP1-beta and IP-10. The person skilled in the art recognizes that certain cytokines also have chemoattractant properties and could be classified under the term chemokines.
B. Visualizing substances and radioisotopes
In certain embodiments of the implementation of the molecules directed action of the present invention can be attached to the used imaging agents for imaging and diagnosis of various diseased organs, tissues or cell types. Many appropriate imaging compounds are known in the art as methods for their accession to the proteins or peptides (see, for example, U.S. patent No. 5021236 and 4472509, the contents of each are included as references in the present document). Some methods for the formation of compounds consist in the use of the metal-chelate complex with the use of, for example, an organic chelating agent such as DTPA attached to the protein or is eptide (U.S. patent No. 4472509). Proteins or peptides can also react with the enzyme in the presence of a coupling agent such as glutaraldehyde or periodate. Conjugates with fluoresceine markers are prepared in the presence of these binding agents or during the reaction with isothiocyanates.
Unlimited examples of paramagnetic ions, used as imaging agents include chromium(III), manganese(II), iron(III), iron(II), cobalt(II), Nickel(II), copper(II), neodymium(III), samarium(III), ytterbium(III), gadolinium(III), vanadium(II), terbium(III), dysprosium(III), holmium(III) and erbium(III), with the most preferred use of gadolinium. Ions used in other methods, such as radiography, include, inter alia, lanthanum(III), gold(III), lead(II) and, especially, bismuth(III).
Radioisotopes of potential use as imaging or therapeutic substances include211astatine,14carbon,51chrome,36chlorine,57cobalt,58cobalt,67copper,152Eu67gallium,3hydrogen,123iodine,125iodine,131iodine,111indium,59iron,32phosphorus,186rhenium,188rhenium,75selenium,35sulfur,99mtechnetium and90yttrium.125I often preferred for use in certain embodiments of the invention, and99mtechnetium and111and the Dios also often prefer to use due to their low energy and possible applications for a long time.
Radioactively labeled proteins or peptides of the present invention can be obtained according to well known methods in the art. For example, they can be jodirovanic by interacting with sodium iodide or potassium iodide and a chemical oxidant such as sodium hypochlorite, or enzymatic oxidant, such as lactoperoxidase. Proteins or peptides in accordance with the present invention can be tagged with99mtechnetium during the reaction of the exchange of ligands, for example, by restoring pertechnitat solution containing bivalent tin, chelation restored technetium on a column with Sephadex and applying the peptide to this column or by direct labeling, for example, by incubation pertechnitat, reductant, such as SNCl2, buffer solution, such as phthalate, sodium-potassium, and peptide. Intermediate functional groups which are generally used to bind radioisotopes which exist in the form of metal ions, peptides, are diethylenetriaminepentaacetic acid (DTPA) and ethylenediaminetetraacetic acid (EDTA). Also discusses the use of fluorescent labels, including rhodamine, fluorescein isothiocyanate and renografin.
In certain embodiments of the invention the claimed proteins isopeptide can be connected with the second binding ligand or to an enzyme (an enzyme tag), to form a colored product upon contact with a chromogenic substrate. Examples of the used enzyme is urease, alkaline phosphatase, hydrogen peroxide (horseradish) and glucosidase. Preferred second binding ligands are compounds of Biotin and avidin or streptavidin. The use of such labels is well known to experts in the art and described, for example, in U.S. patents№№3817837; 3850752; 3939350; 3996345; 4277437; 4275149 and 4366241; the contents of each are incorporated herein by reference.
In other embodiments of the invention, the molecule directed action can be easily connected to the nanoparticle. The nanoparticles include, among other things, colloidal gold and silver nanoparticles. Metal nanoparticles exhibits a color in the visible part of the spectrum. Obviously, these colors are the result of resonant excitation of surface plasmons in metallic particles and are extremely sensitive to size, shape, and aggregation state of the particles; the dielectric properties of the environment; adsorption of ions on the surface of the particles (see, for example, an application for U.S. patent No. 20040023415, the contents of which are incorporated herein by reference).
Bifunctional cross-linking reagents are widely used for various whole is th, including preparation of affinity matrices, modification and stabilization of heterogeneous structures, identification of binding sites of the ligand and receptor, and structural studies. Homobifunctional reagents, carrying two identical functional groups proved to be highly effective in inducing cross-linkage between identical and different macromolecules or subunits of the macromolecule, and the connection of polypeptide ligands to their specific binding sites. Heterobifunctional reagents contain two different functional groups. By using different reactivity of the two different functional groups, cross-linking can be controlled selectively and sequentially. Bifunctional cross-linking reagents can be classified regarding the specificity of their functional groups such as amino, sulfhydryl, guanidino, indole, carboxyl-specific groups. Among them, reagents, aimed at free amino groups, have become especially popular because of their commercial availability, easy synthesis and mild reaction conditions in which they are applied. Most heterobifunctional cross-linking reagents contain primary amino-reactive group and thiol-reactive group.
Typical methods for using plumage is Resto-linking ligands to liposomes are described in U.S. patent No. 5603872 and 5401511, the contents of each are incorporated herein by reference. Various ligands can be covalently bound to the surface of liposomes via cross-linking of amine residues. Liposomes, in particular, multilamellar vesicles (MLV) or homogeneous lamellar vesicles, such as microemulsion liposomes (MEL) and large homogeneous lamellar liposomes (LUVET), each containing phosphatidylethanolamine (PE)were prepared with conventional methods. The inclusion of RE in the liposome provides the presence of active functional residue of the primary amine on the liposomal surface for cross-stitching. Ligands such as, for example, epidermal growth factor (EGF), have been successfully connected with PE liposomes. Ligands covalently bind to discrete sites on the liposomal surface. The number and density of these sites on the surface is determined by the liposomal composition and type of liposomes. Liposomal surface can also be sites for non-covalent bonds. For the formation of covalent conjugates of ligands and liposomes, cross-linking reagents have been studied for effectiveness and biocompatibility. To cross-linking ligands include glutaraldehyde (GAD), a bifunctional oxirane (OXR), air diglycidyl-ether (EGDE), and water-soluble carbodiimide, prefer the LNO 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC). Through a complex chemistry of cross-stitching have been contacted amine residues detectable substance and liposomes.
In another example described heterobifunctional cross-linking reagents and methods of using cross-linking reagents (U.S. Patent No. 5889155, the contents of which are incorporated herein by reference). Cross-linking reagents linked nucleophilic residue of the hydrazide with electrophilic residue of maleimide, providing the binding of aldehydes with free thiols in one of the examples. Cross-linking reagent can be modified to cross-stitching the various functional groups.
8. Nucleic acids
Nucleic acids according to the present invention can encode a peptide directional antibody directed action, fragment antibodies directed action, therapeutic polypeptide, protein or other protein or peptide. The nucleic acid may be derived from genomic DNA, complementary DNA (cDNA or synthetic DNA.
The term "nucleic acid"used in this document, see single-stranded and double-stranded molecules as well as DNA, RNA, chemically modified nucleic acids and nucleic acid analogues. It is assumed that the nucleic acid is in the framework of the present invention may be almost any size, determined partly by the length of the encoded protein or peptide.
It is assumed that the peptides directional and fused proteins can be encoded by any nucleic acid sequence that encodes the appropriate amino acid sequence. Design and synthesis of nucleic acids that encode the necessary amino acid sequence, are well known to experts in the art using standard table of codons. In preferred embodiments of the invention selected codons encoding each amino acid can be modified to optimize expression of the nucleic acid in the targeted cell host.
9. Target-oriented delivery of gene therapy vectors
There are several ways in which gene therapy vectors can be delivered into cells. In certain embodiments of the invention, the vector gene therapy consists of the virus. The ability of certain viruses to enter cells via receptor-mediated endocytosis, to integrate into the genome of the host cell or to remain episomal and Express viral genes stably and efficiently makes them appropriate candidates for transfer of foreign genes into mammalian cells (Ridgeway, 1988; Nicolas and Rubinstein, 1988; Baichwal and Sugden, 1986; Temin, 1986). Suppose the equipment vectors in gene therapy are mainly viral vector. For DNA viruses used as vectors in gene therapy include papovavirus (for example, a monkey virus 40, bovine papilloma virus and polyomavirus) (Ridgeway, 1988; Baichwal and Sugden, 1986) and adenoviruses (Ridgeway, 1988; Baichwal and Sugden, 1986).
One of the preferred methods of delivery in vivo includes the use of an adenovirus expression vector. Although it is known that adenovirus vectors of the low capability of integration into genomic DNA, this property is compensated by the high efficiency of gene transfer characteristic of these vectors. "Adenovirus expression vector" means a construct that contains, among other things, adenovirus sequences sufficient to (a) ensure the packaging of the construct and (b) the expression of antisense or sense polynucleotide, which was cloned in it.
Adenoviral vector was used for expression of eukaryotic genes (Levrero et al., 1991; Gomez-Foix et al., 1992) and the creation of vaccines (Grunhaus and Horwitz, 1992; Graham and Prevec, 1991). Research on the use of recombinant adenovirus to different tissues include trachea instillation (Rosenfeld at al., 1991; Rosenfeld at al., 1992), muscle injection (Ragot et al., 1993), peripheral intravenous injections (Herz and Gerard, 1993) and stereotactic inoculation into the brain (Le Gal La Salle et al., 1993).
In preferred embodiments of the invention it is possible to get some ol the property from the joining of therapeutic molecules or substances to LPR molecules directed action, aimed at vascular network of the affected tissues, such as tumors or neovascular bed. In particular, molecules aimed at a tumor vascular network were connected with cytoxicity drugs or proapoptotic peptides to produce compounds, more effective and less toxic than the parent compound in experimental mouse models bearing tumor xenotransplants (Arap et al., 1998; Ellerby et al., 1999). Insert RGD-4C peptide in the surface protein of adenovirus forms an adenoviral vector, which can be used for target-based gene therapy of tumors (Arap et al., 1998).
The term "fiber-protein" in accordance with the present invention means preferably an adenoviral fiber protein. Any of the serotypes of human or non-human adenovirus (e.g., chimeric fiber protein) can be used as a source of fiber protein or gene fiber protein. Optimal adenoviruses, however, is an Ad2 or Ad5 adenoviruses (see U.S. patent No. 6649407, the contents of which are incorporated herein by reference).
The fiber protein is a "chimeric" because it includes amino acid residues that are not normally found in protein in the allocation of wild-type adenovirus (i.e., having a composition of the active protein or protein wild type). The fiber protein, thus, includes "negativnuu amino acid sequence". "Anatevka amino acid sequence" means a sequence of any suitable length, preferably from about 3 to about 200 amino acids, optimally from about 3 to about 30 amino acids. It is desirable that anatevka amino acid sequence was inserted into the fiber protein at the level of gene expression (i.e., by introducing a "nucleic acid sequence that encodes negativnuu amino acid sequence"). This anatevka amino acid sequence or entered into an adenoviral sequence or in addition to the adenoviral sequences. Regardless of the nature of the introduction, its integration into an adenoviral fiber protein at the level of either DNA or protein leads to the formation of a peptide motif (i.e., peptide binding motif) in the resulting chimeric fiber-protein.
The peptide motif allows aiming to influence the target cell, for example, due to the presence in its composition of molecules directed action of the present invention and/or the ligand for the binding site on the cell surface. Optimally, the peptide motif may include other elements directed action on the cell (for example, tnotebook the offered sequence antibodies). The peptide motif binding can include an insert or, for example, from native and negativnyh sequences, or can be completely constructed from negativnyh sequences. The peptide motif created by inserting negativnoi amino acid sequence in the chimeric fiber protein, can be either high-affinity peptide (i.e., which links them recognizable binding site, for example, VEGFR-1/NRP-1, when used in relatively low concentrations) or low-affinity peptide (i.e., which links them recognizable binding site, for example, VEGFR-1/NRP-1, when used in relatively high concentrations). Preferably, however, when the resulting peptide motif is a high-affinity motif, in particular, which has high affinity to a distinctive binding site due to its conclusion in the adenovirus fiber protein.
Other vectors for gene transfer can be designed on the basis of retroviruses (Coffin, 1990). In order to construct a retroviral vector, a nucleic acid encoding a protein of interest, is inserted into the viral genome in the location of certain viral sequences to produce a virus with impaired replication. In order to create the virions, we construct a packaging cell line containing the genes gag, pol and env, but without TR and packaging components (Mann et al., 1983). When a recombinant plasmid containing a cDNA, together with the retroviral LTR and packaging sequences is introduced into this cell line (by calcium phosphate precipitation for example), the packaging sequence allows the RNA transcript of the recombinant plasmid to be packaged into viral particles, which are then secreted into the culture media (Nicolas and Rubenstein, 1988; Temin, 1986; Mann et al., 1983). The medium containing recombinant retroviruses, and then is collected, optionally concentrated and used for gene transfer. Retroviral vectors are capable of infecting a wide variety of cell types. However, integration and stable expression require separation of host cells (Paskind et al., 1975).
Other viral vectors can be used as vectors for the target-based gene therapy. Can be used with vectors derived from viruses, such as vaccinia virus (Ridgeway, 1988; Baichwal and Sugden, 1986), adeno-associated virus (AAV) (Ridgeway, 1988; Baichwal and Sugden, 1986; Hermonat and Muzycska, 1984) and herpes viruses.
In another embodiment of the invention the construct for gene therapy can be enclosed in the liposome. Liposome-mediated delivery of nucleic acids and expression of foreign DNA in vitro has been very successful. Wong et al. (1980) demonstrated the feasibility liposome-mediated to Tawke and expression of foreign DNA in cultured cells of the embryo chick, HeLa and hepatoma. Nicolau et al., (1987) have successfully completed liposome-mediated gene transfer in rats after intravenous injection.
Vectors for gene therapy of the present invention can contain various transgenes, which are usually encoded in DNA or RNA expression vector. Gene therapy can be used for expression of therapeutic gene, the expression of VEGFR-1/NRP-1 to increase neovascularization or for inhibiting the expression of VEGFR-1/NRP-1 for the treatment of diseases associated with neovascularization. DNA may be in the form of cDNA, synthesized in vitro DNA, plasmid DNA, parts of a plasmid DNA, the genetic material derived from a virus, linear DNA, vectors (P1, PAC, BAC, YAC, artificial chromosomes), expression cassettes, chimeric sequences, recombinant DNA, chromosomal DNA, oligonucleotide, antisense DNA, or derivatives of these groups. RNA may be in the form of oligonucleotide RNA, tRNA (transfer RNA), sn (small nuclear RNA), grnc (ribosomal RNA), mRNA (messenger RNA), synthesized in vitro RNA, recombinant RNA, chimeric sequences, antisense RNAS, siRNAs (small interfering RNA), ribozymes, or derivatives of these groups. "Antisense polynucleotide is polynucleotide, which inhibits the function of DNA and/or RNA. To antimicrob the m polynucleotides are among other things, morpholines, 2'-O-methylphenylamine, DNA, RNA, etc. SiRNAs consist of a double-stranded structure, usually containing 15-50 base pairs and preferably 21-25 base pairs and having a nucleotide sequence that is identical or almost identical to the expressed gene target or RNA inside the cell. Interference can lead to the suppression of expression. In addition, DNA and RNA can be single-stranded, double-stranded, triplex or chetyrekhstoechnymi.
10. The pharmaceutical composition
The pharmaceutical compositions of the present invention contain an effective amount of one or more compositions including a group of directed action, as described herein, dissolved or dispersed in a pharmaceutically acceptable carrier. The phrase "pharmaceutical or pharmacologically acceptable" refer to molecular compounds and compositions that do not lead to the opposite, allergic or other untoward reactions when assigning an animal, such as man. The preparation of a pharmaceutical composition that contains at least one composition according to the present invention (for example, LPR molecules directional) or additional active ingredient, known to experts in the art the light of the present description of the invention, an example of which is given in the "Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, the contents of which are incorporated herein by reference. Moreover, in the appointment of the animal (e.g. human) need to understand that the composition must meet the standards of sterility, progenote, General safety and purity as required by the FDA Department of biological standards.
Used herein "pharmaceutically acceptable carrier" includes any and all solvents, dispersants, coatings, surfactants, antioxidants, preservatives (antibacterial agents, antifungal agents), isotonic agents, preventing the absorption of nutrients, salts, drugs, stabilizers medications, gels, binders, excipients, disintegrators, lubricants, sweeteners, flavorings, colorants, such materials and combinations thereof, known to experts in the art (see, for example, Remington's Pharmaceutical Sciences, 18th Ed., 1990, the contents of which are incorporated herein by reference). Except when any conventional carrier is incompatible with the active ingredient, is considered its use in therapeutic or pharmaceutical compositions.
Therapeutic and diagnostic compositions of the present invention may contain the hypoxia media types depending on, will they be used in solid, liquid or aerosol form, and whether there is a need to be sterile for such applications. Provided that the composition of the present invention can be applied by any method known to experts in the art, such as orally, intravenously, intradermally, intraarterially, spinal, intraocular, subconjunctival, subretinal, intravitreal, in the anterior chamber of the eyeball, in nasceranno space eyes, topically administered intraperitoneally, into the affected tissue, intracranial, intraarticular, intrapleural intratrahealno, inside the tumor, intramuscularly, subcutaneously, intravesicular, by inhalation (e.g. aerosol inhalation), injection, infusion, continuous infusion, local perfusion for treatment of target cells directly, via a catheter, via a lavage, in lipid compositions (e.g., liposomes), or by another method, or a combination of the above known experts in the art (see, for example, Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, the contents are included as references in the present document).
The actual dosage amount of a composition of the present invention, assigned to the subject, there may be physical or physiological factors, and as the weight of the body, the severity of the condition, the type of disease being treated, previous or concomitant treatment activities, individual characteristics of the patient and route of administration of drugs. The practitioner responsible for the treatment, will, in any case, to determine the concentration of active ingredient(s) in the composition and a suitable dose(s) for the individual subject.
In certain embodiments of the invention the pharmaceutical compositions may contain, for example, at least about 0.1% of active substance. In other embodiments, the implementation, the active ingredient may be from about 2% to about 75% by weight of units or from about 25% to about 60%, for example, and any other received in this regard range. In other unlimited examples: approximately 1 mg protein/kg body weight, about 5 mg/kg body weight, about 10 mg/kg body weight, about 50 mg/kg body weight, about 100 mg/kg body weight, about 200 mg/kg of body weight or more per one intake and any other received in this regard range. Unlimited examples of the range obtained from the above figures, the preferred range is from about 1 mg/kg body weight to about 100 mg/kg of body weight, and especially preferred range of 20 to 50 mg/khepri repeated daily application (like ibuprofen, aspirin, etc. every 4-8 hours).
In any case, the composition may contain various antioxidants to retard oxidation of one or more components. In addition, the prevention of the action of microorganisms can be caused by preservatives such as, for example, antibacterial and antifungal agents, including, among other things, parabens (such as methylparaben, propylparaben), chlorobutanol, phenol, sorbic acid, thimerosal or combinations thereof.
In embodiments of the invention, where the composition is provided in liquid form, the carrier can be a solvent or dispersion medium consisting, inter alia, water, ethanol, polyol (e.g. glycerol, propylene glycol, liquid polyethylene glycol), lipids (e.g., triglycerides, vegetable oils, liposomes) and their combinations. In many cases, it is more preferable would be to include isotonic agents as, for example, sugars, sodium chloride or combinations thereof.
Sterile injectable solutions are prepared by dissolving LPR molecules directional or their conjugates in the required amount of the appropriate solvent with various, as required, other ingredients enumerated above, followed by sterilization by filtration. Typically, the dispersion is prepared by mixing various sterilized who's active ingredients into a sterile filler, contains the basic dispersion medium and/or other ingredients. In the case of sterile powders for the preparation of sterile injectable solutions, suspensions or emulsions, the preferred method of cooking is the method of vacuum drying or freeze-drying, which allows to obtain a powder of the active ingredient plus any additional desired ingredient from a previously filtered by the above liquid medium. Liquid medium, if necessary, must be properly buffered, and the liquid solvent to injections first converted into isotonic with the help of a sufficient amount of salt or glucose. The preparation of compositions for direct injection is also considered, where the use of DMSO as solvent leads to extremely rapid penetration, delivering high concentrations of active substances in small areas.
The composition should be stable under conditions of manufacture and storage, protected from contaminating action of microorganisms, such as bacteria and fungi. Whereas that endotoxin contamination should be kept to the minimum safe level, for example, less than 0.5 ng/mg protein.
Above the optimal composition can include one or more auxiliary therapeutic substances, healthy lifestyles is the R for the treatment or prevention of any of the aforementioned diseases.
11. Therapeutic substances
In certain embodiments the invention, therapeutic substance may be functionally related peptide directional or fused protein for selective delivery, for example, in the vascular network of tumors expressing VEGFR-1/NRP-1. Agents or factors suitable for use may include any chemical compound that induces apoptosis, cell death, the rest of the cells and/or anti-angiogenesis.
A. Regulators of programmed cell death
Apoptosis, or programmed cell death, is an essential process for normal embryogenesis, maintenance of homeostasis of adult tissues and suppression of carcinogenesis (Kerr et al., 1972). Proteins of Bcl-2 and ICE-like proteases, as shown, are important regulators and effectors of apoptosis in other systems. Bcl-2 protein, which opened in connection with follicular lymphoma, plays a significant role in the control of apoptosis and increased cell survival in response to various apoptotic stimuli (bakhshi for et al., 1985; Cleary and Sklar, 1985; Cleary et al., 1986; Tsujimoto et al., 1985; Tsujimoto and Croce, 1986). Admittedly, evolutionary conservative protein Bcl-2 is a member of a family of related proteins that can be classified as agonists death or antagonists of death.
After him from whom ryte been shown that Bcl-2 acts as a suppressor of cell death initiated by different stimuli. In addition, it is now clear that there exists a family of Bcl-2 proteins-regulators of cell death, which share homology in structure and sequence. These different members of the family, as it turns out, are similar to Bcl-2 functions (for example, BclXL, BclW, BclS, Mcl-1, A1, Bfl-1) or prevent the action of Bcl-2 and stimulate cell death (e.g., Bax, Bak, Bik, Bim, Bid, Bad, Harakiri).
B. Angiogenic inhibitors
In certain embodiments of the implementation of the present invention relates to a destination of molecules directional functionally related to anti-angiogenic substances, such as angiotensin, peptides of laminin, fibronectin peptides, inhibitors of plasminogen activator inhibitors of tissue metalloproteinases, interferons, interleukin 12, platelet factor 4, IP-10, Gro-β, thrombospondin, 2-methoxyestradiol, proliferin-associated protein, carboxamidates, SM, marimastat, pentosan polysulphate, angiopoietin 2 (Regeneron), interferon-alpha, herbimycin AND PNU145156E, a fragment of prolactin 16K, linomide, thalidomide, pentoxifylline, genistein, TNP-470, endostatin, paclitaxel, accutan, angiostatin, cidofovir, vincristine, bleomycin, AGM-1470, platelet factor 4 or minocycline.
Proliferat the I cell tumors depends heavily on the extensive vascularization of the tumor, which accompanies the development of cancer. Thus, inhibition of the formation of new blood vessels with anti-angiogenic substances and directed destruction of existing blood vessels were taken into consideration as an effective and relatively non-toxic approach to the treatment of tumors (Arap et al., 1998; Arap et al., 1998; Ellerby et al., 1999). Known for a variety of anti-angiogenic agents and/or inhibitors of blood vessels (e.g., Folkman, 1997; Eliceiri and Cheresh, 2001).
C. Cytotoxic substances
Chemotherapeutic (cytotoxic) drugs can be used to treat various diseases, including cancer. Chemotherapeutic (cytotoxic) substances of possible applications include, among others, 5-fluorouracil, bleomycin, busulfan, camptothecin, carboplatin, chlorambucil, cisplatin (CDDP), cyclophosphamide, dactinomycin, daunorubicin, doxorubicin, substances that bind the estrogen receptor, etoposide (VP16), inhibitors farnesyl-protein transferase, gemcitabine, ifosfamide, mechlorethamine, melphalan, mitomycin, navelbine, nitrosurea, plicamycin, procarbazine, raloxifene, tamoxifen, Taxol, temozolomide (aqueous solution DTIC), transplatinum, vinblastine and methotrexate, vincristine, or any analog or derivative the above. The majority of chemotherapeutic substances falls under the cat is the Gory alkylating substances, antimetabolites, antitumor antibiotics, corticosteroid hormones, mitotic inhibitors and nitrosoanatabine, hormonal substances, mixed substances and any analog or derivative of the above.
Chemotherapeutic agents and methods of use, dosages, etc. are well known to experts in the art (see as example the "Physicians Desk Reference", Goodman & Gilman''s "The Pharmacological Basis of Therapeutics" and "Remingthon''s Pharmaceutical Sciences" 15ththed., str-1038 and 1570-1580, the contents of which are included in the relevant parts of this document by reference) and can be correlated with the invention in light of the disclosure of the information presented here. Some adjustment of dosage is required depending on the condition to be treated of the subject. The person responsible for the appointment of the drug, will, in any case, to find the appropriate dose for the individual subject. Of course, all dosages and substances described in this document are illustrative rather than limiting, and other dosage or substances can be used by a qualified technician for a specific patient or application. Any dosage within the given interval or received here range also be used in the invention.
D. Alkylating substances is and
Alkylating agents are medicine that interacts directly with genomic DNA to prevent cell proliferation. This category of chemotherapeutic drug is a substance acting on all phases of the cell cycle, i.e. they are not phasespecific. Alkylating substance may include, among other things, nitrogen mustard, ethylenimine, methylmelamine, alkylsulfonate, nitrosoanatabine or triazine. They include, inter alia: busulfan, chlorambucil, cisplatin, cyclophosphamide (cytoxan), dacarbazine, ifosfamide, mechlorethamine (mustargen) and melphalan.
Antimetabolites interrupt the synthesis of DNA and RNA. Unlike alkylating substances, they specifically affect the cell cycle during the S-phase. Antimetabolites can be divided into different categories, such as analogs of folic acid, analogs of pyrimidine and purine analogues and related inhibitors. Antimetabolites include, among others, 5-fluorouracil (5-FU), cytarabine (Ara-C), fludarabine, gemcitabine, and methotrexate.
F. Natural products
Natural products usually refers to compounds originally isolated from a natural source and having pharmacological activity. Such compounds, analogs and derivatives thereof can be separated from the th source, chemically synthesized or recombinante created by any method known to the person skilled in the art. Natural products include categories such as mitotic inhibitors, antitumor antibiotics, enzymes and biological response modifiers.
Mitotic inhibitors include plant alkaloids and other natural substances that can inhibit either protein synthesis necessary for cell division, or mitosis. They operate during a certain phase during the cell cycle. Mitotic inhibitors include, for example, docetaxel, etoposide (VP16), teniposide, paclitaxel, Taxol, vinblastine, vincristine and vinorelbine.
Taxaide represent a class of related compounds isolated from the bark of the ash tree, Taxus brevifolia. Taxaide include, inter alia, compounds such as docetaxel and paclitaxel. Paclitaxel binds to tubulin (site differs from that used by the Vinca alkaloid) and starts the Assembly of microtubules.
Vinca alkaloids are a type of plant alkaloid, which revealed the pharmacological activity. They include compounds such as vinblastine (VLB) and vincristine.
Certain antibiotics have both antimicrobial and cytotoxic activity. These drugs also interfere cDNA, chemically inhibiting the enzymes and mitosis, or by changing the cell membrane. These substances are not phasespecific, because they work in all phases of the cell cycle. Examples of cytotoxic antibiotics include, among other things, bleomycin, dactinomycin, daunorubicin, doxorubicin (Adriamycin), plicamycin (mithramycin) and idarubitsin.
H. Mixed content
Mixed cytotoxic substances, which are not included in the previous category include, among other things, coordination complexes of platinum, anthracenedione, substituted urea derivatives methylhydrazine, amsacrine, L-asparaginase and tretinoin. Coordination complexes include platinum compounds such as carboplatin and cisplatin (cis-DDP). Typical anthracenediones is mitoxantrone. Typical substituted urea is hydroxyurea. Typical derivative methylhydrazine is procarbazine (N-methylhydrazine, MIH). These examples are not limiting, and it is envisaged that any known cytotoxic, cytostatic or destroying the cells of the substance can be combined with peptides directed action and put in the target organ, tissue or cell type within the present invention.
The following examples are included to demonstrate preferred variants of the invention. Specialists in this field is subramania admittedly, the methodology presented in the examples should be considered as methods developed by the authors to the best of their use in practice of the present invention, and thus can be considered as the preferred methods for their practical use. However, experts in the field of the invention should be recognized in the light of the present description of the invention that numerous changes may be made in certain embodiments of the invention presented here, and, nevertheless, will be received the same or similar results without deviating from the essence and scope of the invention.
Example 1: Anti-angiogenic compounds, targeted to VEGF signaling pathway
1. Materials and methods
Reagents and peptides. Peptides were synthesized and purified by HPLC according to our technical specifications with a purity of more than 95%: L-Arg-L-Pro-L-Leu (RPL), D-Leu-D-Pro-D-Arg [D(LPR)], D-Cys-D-Ala-D-Pro-D-Ala-D-Cys [D(CAPAC); SEQ ID NO:6] in the laboratory of the Polypeptides (Torrence, CA) and D-Ala-D-Pro-D-Ala [D(APA)] Genemed Synthesis Inc. (San Francisco, CA). Recombinant receptors (VEGFR-1 and NPR-1) and growth factors (human VEGF165) were obtained from R&D Systems (Minneapolis, MN). Heparin, reagent Drabkina, human hemoglobin, brij-35 were obtained from (Sigma-Aldrich, St. Louis, MO).
Animals. Experiments on mice were approved by the Committee on the protection and use of W the animal Cancer center, Anderson University of Texas. Mouse C57BL/6 and Balb/c mice were purchased commercially (Harlan, Indianapolis). This study adheres to the Declaration of the Association for research in vision and ophthalmology for the use of animals in research of vision and ophthalmology.
Phage analysis. Phages were prepared by infection K91kan E. coli cell culture during the log phase, the night growing in the medium Luria-Bertani (LB) supplemented with kanamycin (100 μg/ml) and tetracycline (20 μg/ml) at 37°C and 250 rpm Phages were precipitated from the supernatant medium by the method of PEG/NaCl, and phage titer was determined by serial dilution and counting colonies (Giordano, 2001). For the method for binding phage and competitive analysis of VEGFR-1, NRP-1 or BSA (10 μg/ml in PBS) were immobilized on microtiter plates overnight at 4°C. the Tablets were washed twice blocked with PBS 3% BSA for 2 h at room temperature and incubated in PBS 3% BSA in the presence of 109TU CPQPRPLC or phage (Fd-tet) with missing insert, which serves as a negative control. After 1 h at room temperature tablets were washed 10 times in PBS, and the phage bound to the immobilized receptor was extracted using a bacterial infection (Giordano, 2001).
Analysis of resistance to proteases.D(LPR) and RPL peptides were diluted to 500 μg/ml in PBS and incubated with increasing concentrations of Pancreatin (Sigma-Aldrich, St. Louis, MO) in those who tell 2 h at 37°C. Then the samples were analysed by mass spectroscopy (MALDI-TOF).
Analysis of angiogenesis. The authors used the method of analysis of angiogenesis in vivo in the Matrigel, in which the growth factor of the reduced matrix of Matrigel (BD Biosciences, Bedford, MA), impregnated with recombinant human VEGF165(1 μg/ml) and heparin (10 u/ml), whether or not containing peptidomimetics (500 µg), implanted in vivo subcutaneously (0.5 ml) in the dorsal part of mouse Balb-c.D(CAPAC) was used as control peptidomimetic. After 7 days mice were killed, dissected seals with Matrigel, photographed, homogenized in a solution of Brij 0,35% by using a Dounce homogenizer, centrifuged 5 min at 13,000 g. The supernatant was used twice: for the measurement of hemoglobin (Hb) reagent Drabkina and Hb concentration, calculated by simultaneously measuring the standard Hb.
Murine SCID model of angiogenesis of human. In mice implanted 106microengineering of human skin cells (HDMEC) in the matrix of the Matrigel per substrate 2 substrate on the mouse, one on each side of the mouse. On day 12 after implantation, the mice were injected dailyD(LPR) or control peptidomimeticD(APA) (100 μl intraperitoneal injection of a solution of 2 mg/ml). Drugs were prepared by dilution in DMSO to the main concentration of 20 mg/ml for the latter is affected dilutions, and by prepare fresh dilutions (1/10) in PBS before each injection. The substrates were taken out and fixed in 10% formalin/PBS. The tissue sections were stained with antibodies to von Willebrand factor (Neomarkers, Freemont, CA), and visualized using AEC (DABCO) and contractionary with hematoxylin. The calculations were performed in the light microscope, six fields of view on the wafer at 200x magnification (n=6 toD(LPR) and n=4 for control peptidomimetic). Statistical analysis was performed using Sigmastat.
Analysis neovascularization angiogenesis in the retina. For analysis neovascularization angiogenesis of the retina, the authors used mice C57BL/6 and their nursing mothers. Mouse (P7) were exposed to 75% oxygen for 5 days, then room air (20.8% of O2)(R) and they were administered daily injectionsD(LPR), the control peptidomimeticD(CAPAC; SEQ ID NO:6) (20 mg/kg in phosphate-buffered saline, used as a solvent or solvent within seven days (R-P18). For histological analysis mice were euthanized at the peak of angiogenesis (P19), eyes nuclearman, fixed, prepared serial sections and stained with hematoxylin and eosin (H&E). The nucleus of endothelial cells on the vitreal surface of the inner edge of the membrane were counted. A minimum of 10 H&E-stained sections of one chap who and were analyzed and counted the average number of nuclei, per 4-6 eyes for each experiment.
Tumor growth. Cells EF43.fgf4 cultured in medium Dulbecco Modified Eagle Medium with the addition of fetal bovine serum, glutamine and antibiotics. Cells were collected before reaching confluent layer and injected subcutaneously in infrapatellar fat body of the mammary gland of the mouse Balb/c mice. After 10 days the tumors reached ~50-80 mm3animals were divided into four groups of seven animals (N=7). Animals in each group were lechene or only the solvent (phosphate-saline buffer), or control peptideD(CAPAC) (SEQ ID NO:6), orD(LPR) (50 mg/kg), or cyclic formD(CLPRC)(SEQ ID NO:7) (25 mg/kg). Tumor volume was calculated by measuring the length of the long (L) and short (S) sides of each tumor (V=S2×L×0,04).
The statistical analysis. For in vivo experiments, the statistical significance of differences was calculated using test Kruskal-Wallis (nonparametric one-way ANOVA method), p<0.05 for each day of the treatment. Rank Wilcoxon test was used for further calculations of pairwise differences when comparing groups on the day of treatment, showing a statistical significance test of Kruskal-Wallis. Multiple comparisons were performed using the method of Benjamini &Hochberg. All statistical analysis was ispolnen using the R Project for statistical computing (version 2.4.1).
2. Experimental results
To assess whether peptidomimeticsD(LPR) in binding to VEGFR-1 and NRP-1, experiments were carried out on competitive binding. Linking parental phage CPQPRPLC (SEQ ID NO:8) c immobilized VEGF receptors, NRP-1 and VEGFR-1 was performed in the presence of increasing concentrations of peptide RPL or peptidomimeticD(LPR) of the present invention. As expected from previous works of the authors (Giordano et al., 2005), the peptide RPL completely prevented binding of phage CPQPRPLC (SEQ ID NO:8) with both receptors. Interestingly, peptidomimeticD(LPR) also inhibited the binding of phage at very similar concentrations when compared with RPL. Both peptide, RPL andD(LPR) inhibited the binding of phage dose-dependent way, whereas the control peptide used in the highest concentration (100 μm) had no effect on the binding of phage CPQPRPLC (SEQ ID NO:8). Since the concentration of peptide required to inhibit 50% of binding phage (IC50), is inversely proportional to the affinity of the peptide to the receptor, our data suggest that both RPL andD(LPR) have a higher affinity to VEGFR-1 and NRP-1 when compared with peptide CPQPRPLC (SEQ ID NO:8) (Giordano et al., 2001). IC50 D(LPR) for both receptors is much lower (1-10 PM) when compared with IC50CPQPRPLC (SEQ ID NO:8), as described earlier,for VEGFR-1 (~1 nm) or NRP-1 (~50-100 nm) (Giordano et al., 2001).
Then was tested resistanceD(LPR) to proteolytic degradation. As RPL, and soD(LPR) were incubated with increasing concentrations of Pancreatin (a mixture of various digestive enzymes from the pancreas), and the degradation products were analysed by mass spectroscopy. No cleavage products were observed at peptidomimeticD(LPR) at the highest investigated the ratio of concentrations of the enzyme and peptide (400 PG/nmol). However, the peptide RPL showed significant degradation in the presence of the products of cleavage of the dipeptide PL. In summary, these data show thatD(LPR) is similar to RPL that are less susceptible to proteolytic degradation, binds with high affinity to VEGFR-1 and NRP-1 and is the best candidate among the leading drugs for the study of the action of the motive RPL in angiogenesis.
After identification and characteristics of peptidomimeticD(LPR) was investigated actionD(LPR) on the formation of new blood vessels. For this purpose we have used two animal models of angiogenesis: in vivo with Matrigel and growth of human vascular endothelial cells in the mouse host (Nor et al., 2001). First conducted research using the model analysis with Matrigel in vivo, in which mice were subcutaneously injected with Matrigel, soderjashie VEGF165/sub> and peptidomimeticD(LPR). After 7 days of implantation, the matrix of the Matrigel impregnatedD(LPR), showed reduced vascularization compared with the matrix of the Matrigel containing only VEGF165and serving as a positive control; no effect on neovascularization were observed in matrices containing the control peptidomimetic (figa).
Then it was investigated the effectD(LPR) in the SCID mouse model of angiogenesis of human. In this model, endothelial cells were cultured in vivo in polymer implants and grew to form microvessels, which are then combined with mouse capillaries. The newly formed vessels were functional, stood in line with endothelial cells expressing markers of angiogenesis, and transported in the blood cells of the mouse. To assess actionsD(LPR) in this animal model of angiogenesis in mice implanted substrate containing endothelial cells and grew them for 12 days. During the 12-day period endothelial cells formed a non-tubular structures containing empty openings and which slowly matured to a fully operational vessels containing blood cells mouse (Nor et al., 2001). Ranging from 12 to 21 days, the mice were injectedD(LPR) or control peptidomimetic (25 m is/kg/day). Peptidomimetics were injected with daily administered intraperitoneally, and at the end of treatment, the substrates were removed and it was determined the number of endothelial cells that form functional blood vessels. On day 21, all animals were formed functional vessels with the expected density of cells; these vessels were functional and positive for markers of angiogenesis. The authors observed a reduction in the formation of vessels by 36.7% in the group of animals treated with peptideD(LPR), compared to animals treated with control peptidomimetics (32.1 vessels in sight of the enlarged image against 20,3 in the presence of aD(LPR). (pigv). Taken together, these data indicate that peptidomimeticD(LPR) inhibits the formation and maturation of blood vessels in vivo in two different animal models of angiogenesis.
Then conducted research to ascertain whether peptidomimeticsD(LPR) to be used as a drug for the treatment of pathophysiological angiogenesis. With this purpose were selected animal models of the two diseases in which angiogenesis is known to play an important role in the progression of the disease: retinopathy of prematurity (ROP) and cancer. To study ROP was used mouse model of oxygen-induced retinopathy (Smith et al., 1994; Lahdenranta et al. 2001). In this case, newborn mice (7 days old) were subjected to exposure to high oxygen levels (75%) for 5 days and returned to room conditions (20.8% of the oxygen in the air). Changes in the level of oxygen was caused by relative hypoxia in animals that endothelial cells respond by activating the oxygen-sensitive elements, such as hypoxia-inducible factor-1 (HIF-1) and VEGF (Smith et al., 1994; Lahdenranta et al., 2001). After return to room air (12 day) animals daily was administeredD(LPR) or control peptidomimetic within 7 days (20 mg/kg). At the end of treatment (day 19) eyes were nuclearman and investigated in order to determine the level of neovascularization by counting the number of blood vessels and endothelial cells, projecting from the retina (figa). A significant decrease in angiogenesis (68,5%) was observed in animals treatedD(LPR), compared to animals treated only with medium or control peptidomimetics (FIGU, 2C) (29,8±3.8 kernels/retin-a in animals treated only with the media; to 29.3±6.0 cores/retin-a in animals treated control peptidomimetics; 9,4±1,0 cores/retin-a in animals treatedD(LPR)).
Then conducted research to determine whether peptideD(LPR) to influence neovascularization, induserve the percent tumor. For these studies was selected highly angiogenic mouse model of breast cancer EF43.fgf4 (Deroanne et al., 1997; Hajitou et al., 1998). In this model, cancer cells synthesize and secrete fibroblast growth factor-4 (FGF-4), which induces autocrine production of VEGF, leading to the formation of highly vascularizing tumors. Animals were injected with the breast cancer cells subcutaneously in infrapatellar fatty tissue of the breast and gave the opportunity to grow tumors to small size (50-80 mm3). Animals with tumor, then daily was administered administered intraperitoneally injectionD(LPR) (50 mg/kg), control peptidomimetic or one medium. After 5 days of treatment was observed a clear reduction of tumor volume in animals treatedD(LPR) (figure 3). For these studies was used circular shape peptidomimeticD(LPR)D(CLPRC). Animals with tumor and treatedD(CLPRC) (25 mg/kg), also revealed a significant decrease in tumor volume (figure 3). No effect on tumor growth was not observed in animals treated control peptidomimetics.
Taken together, these data demonstrate that peptidomimeticD(LPR) and its cyclic form are a new class of inhibitors of angiogenesis and target-oriented funds, to the verge should find important applications in the clinic, and at a later stage, such as the formation and maturation of blood vessels (SCID murine model of angiogenesis).D(LPR) with the systemic administration in mice significantly reduced the formation of blood vessels during pathological angiogenesis (the mouse model of ROP), as well as angiogenesis induced by tumor.
Example 2: Targeted action on adipose tissue/Research obesity
Diet and lifestyle contribute to the high incidence of obesity in the developed world. In the United States, approximately 65% of the adult population are overweight with a body mass index greater than or equal to 25 kg/m2and over 30% are obese (body mass index greater than or equal to 30 kg/m2). Obesity is associated with an increased risk of developing diabetes, cancer and heart disease, and usually causes a reduction of the duration of human life. Advances in the treatment of obesity have so far been very limited use of certain medicines to control the abnormal accumulation of fat (Clapham et al., 2001). Most anti-obesity based on the change in energy balance and appetite by acting on receptors in the brain. Some medicinal substances of this class (such as fenfluramine) were removed from the sales due to unexpected toxicity. Modern attempts p is the development of compounds inhibiting the absorption of fat in the gastrointestinal tract (such as orlistat, marketed by Roche under the brand name Xenical®), can improve the treatment of obesity. Still, even the most effective drugs can reduce weight by just 5%, and further weight loss required strict diet (Clapham et al., 2001; Padwal & Majumdar, 2007).
It has been shown that is not related to neoplasia tissue growth (e.g., adipose tissue) also depends on the formation of new blood vessels (angiogenesis). For example, in mice of different models of obesity treated with anti-angiogenic substance, was detected in a dose-dependent treatment of reversible weight loss and loss of adipose tissue (Rupnick et al., 2002). These studies demonstrate that the mass of adipose tissue is sensitive to inhibitors of angiogenesis.
Thus, the authors conducted a study to assess the ability LPR peptides directed action on VEGFR-1 according to the present invention purposefully to influence adipose tissue, thereby reducing the weight of the suffering of dietary obese mice. To this end, suffering from alimentary obesity mice were divided into groups and were treated with peptide directed action on VEGFR-1/NRP-1. Mouse C57BL/6 J-60% DIO (at the age of 36 weeks) were purchased from Jackson Laboratory. These animals were fed on high-calorie diets is (J-60%) to obtain a phenotype alimentary obesity (DIO). Animals were divided into groups and every day received the following treatment: [Group 1]D(CLPRC) (SEQ ID NO:7) (N=5); [Group 2] CKGGRAKDC-GG-D(KLAKLAK)2(SEQ ID NO:9) (N=3); [Group 3]D(CLPRC) in combination with CKGGRAKDC-GG-D(KLAKLAK)2(SEQ ID NO:10) (N=4); [Group 4] only the media (N=4). All animals were gidratirovana intraperitoneally the introduction of 1 ml of saline solution (0.9% sodium chloride solution, quality USP) for 30 minutes before treatment. Mice were daily subjected to treatment by introducing aD(CLPRC), dissolved in phosphate-saline buffer media (PBS) (total volume injection 100 μl) at a dose of 50 mg/kg body weight (Group 1 and 3); CKGGRAKDC-GG-D(KLAKLAK)2(SEQ ID NO:9) in PBS was introduced subcutaneously (total injection 100 μl) at a dose of 3 mg/kg (Group 2) or 1 mg/kg in combination withD(CLPRC) (Group 3), 5 days per week (Monday to Friday). Mice were weighed once a week.
The results are shown at figa-4C. In the group of obese mice treatedD(CLPRC) by daily intraperitoneal injection (50 mg/kg), it can be seen that during the period of treatment was quite a significant weight loss of approximately 3 grams (approximately 6% of body mass), while the control mice was observed relative weight gain (figa). Then, to confirm the effect of weight loss under Asteem D(CLPRC), was a planned experiment in mixed therapy. Mice suffering from obesity, have introduced the peptide selectively activating the VEGFR-1/NRP-1 in conjunction with the remedy of obesity, which burns fat. In previous studies the authors showed that burning white fat can be achieved by selective purposeful influence on the vasculature of adipose tissue using a target peptide CKGGRAKDC (SEQ ID NO:11), conjugated with proapoptotic peptideD(KLAKLAK)2(SEQ ID NO:2) (Kolonin et al., 2004). This connection, called the "fat burner" (sequence CKGGRAKDC-GG-D(KLAKLAK)2) (SEQ ID NO:9), induces ablation of fat in therapeutic doses equal to or greater than 3 mg/kg/body weight. However, for mixed experiment peptide directed actionD(CLPRC) (SEQ ID NO:7) to VEGFR-1/NRP-1 was combined with subtherapeutic dose of fat burner (1 mg/kg), waiting for what the anti-angiogenic effectD(CLPRC) will be synergistic with the effect tissue ablation of fat burner.
Indeed, observed a significant weight loss of approximately 8 grams (up to 16% of body weight), when animals were treated with both drugs (pigv). Animals treated with the mixed therapy, showed weight loss, similar to that in the group of mice treated with only one fat burner in optimal therapeutic e is ze 3 mg/kg (figs), and component of approximately 10 grams (20% of body weight). Taken together, these data demonstrate that peptides directed action on VEGFR-1/NRP-1 induce weight loss in mice, obese, and that they are able to act synergistically with other medications for obesity. Peptides directed action on VEGFR-1/NRP-1 can find important applications in the treatment of obesity of a person.
All compositions and methods discussed and claimed herein can be made and executed with proper experimentation in light of the present invention. Although the compositions and methods of the present invention have been described in the framework of the preferred embodiments, specialists in the art it is obvious that changes may be made in the compositions and methods and in the steps or in the sequence of steps of the methods described herein, without deviating from the concept, nature and scope of the invention. In particular, it is obvious that certain substances, which are both chemically and physiologically related may be substituted for the substances covered in this document, when that will be received the same or similar results. All such substitutions and modifications are obvious to experts in the art, are treated in accordance with the nature, volume and koncepcia the present invention as defined in the attached claims.
LIST of references
The following list of literary sources, to the extent that it provides a standard procedure, or other details supplementary to the provisions set forth herein, in a special way are incorporated herein as references.
1. Peptide size 10 or less amino acids, containing at least the contiguous amino acid sequence D(Leu Pro Arg)used for the treatment of diseases or disorders, a part of which is angiogenesis, the individual.
2. The peptide according to claim 1, where the size of the peptide of 7 amino acids or less, or 5 amino acids or less.
3. The peptide according to claim 1, further characterized in that it contains D (Cys Leu Pro Arg Cys) (SEQ ID NO: 1).
4. The peptide according to claim 3, further characterized in that it is a cyclic peptide.
5. Conjugate or design containing the peptide according to any one of claims 1 to 4, for the treatment of diseases or disorders, a part of which is angiogenesis, the individual, where the specified peptide anywhereman or merged with
(a) protein and peptide anywhereman protein with the formation of the protein conjugate; or
(b) molecule, which is a drug, a chemotherapeutic agent, diag is eticheskim means, radioisotope, a Pro-apoptotic agent, an anti-angiogenic agent, a hormone, a cytokine, a growth factor, a cytotoxic agent, a peptide, a protein, an antibiotic; an antibody, fragment or single-chain antibody; visualizing matter, the survival factor, an anti-apoptotic agent, a hormone antagonist or antigen.
6. Conjugate or design according to claim 5, where this molecule is
(a) a Pro-apoptotic agent selected from the group consisting of gramicidin; magainin; melitina; defensin; cecropin; (KLAKLAK)2(SEQ ID NO: 2); (KLAKKLA)2(SEQ ID NO: 3); (KAAKKA)2(SEQ ID NO: 4); (KLGKKLG)3(SEQ ID NO: 5); Bcl-2; Bad; Bak; Bax and Bik; or
(b) an anti-angiogenic agent selected from the group consisting of thrombospondin, angiostatin, factor pigment epithelium, angiotensin, peptides of laminin, fibronectin peptides, inhibitors of plasminogen activators, inhibitors of tissue metalloproteinases, interferons, interleukin 12, platelet factor 4, IP-10, Gro-β, 2-methoxyestradiol, a protein associated with proliferin, carboxamidates, SM, marimastat, pentosanpolysulfate, angiopoietin 2, herbimycin A, PNU145156E, fragment of prolactin 16K, linomide, thalidomide, pentoxifylline, genistein, TNP-470, endostatin, paclitaxel, docetaxel, polyamines, proteasome inhibitor, an inhibitor of the kinase, signal peptide, accutane, cidofovir, vincristine, bleomycin, AGM-1470, minocycline, endostatin XVIII, endostatin XV, C-terminal hemopexin domain of matrix metalloproteinase-2, the Kringle domain 5 of human plasminogen, fused protein of endostatin and angiostatin, fused protein of endostatin and the Kringle domain 5 of human plasminogen, monokine induced by interferon gamma (Mig), fused protein of Mig and IP-10, soluble FLT-1 (fins-like tyrosinekinase receptor 1) and kinase insertion domain-containing receptor (KDR); or
(c) a cytokine selected from the group consisting of interleukin 1 (IL-1), IL-2, IL-5, IL-10, IL-11, IL-12, IL-18, interferon-γ (IF-γ), IF-α, IF-β, tumor necrosis factor and GM-CSF (granulocyte-macrophage colony-stimulating factor).
7. Conjugate or design according to claim 5, where the specified peptide attached to a macromolecular complex or solid media.
8. Conjugate or design according to claim 1, where the specified complex is a virus, a bacteriophage, a bacterium, a liposome, a microparticle, a magnetic busoi, a yeast cell or a mammalian cell.
9. Conjugate or construction of claim 8, where the virus is lentiviruses, papovavirus, adenovirus, retrovirus, AAV, vaccinia virus or herpes virus.
10. Fused protein structure containing the peptide according to any one of claims 1 to 4, which is first fused to the selected protein with the formation of the fused protein design,
where fused protein design is not a natural protein, and selected protein is a Pro-apoptotic agent, an anti-angiogenic agent, a hormone, a cytokine, a growth factor, a cytotoxic agent, a protein antibiotic, an antibody, or its fragment, or single chain anti-apoptotic agent, a hormone antagonist or antigens
for the treatment of diseases or disorders, a part of which is angiogenesis, the individual.
11. Fused protein structure of claim 10, where the specified protein is selected
(a) a Pro-apoptotic agent selected from the group consisting of gramicidin; magainin; melitina; defensin; cecropin; (KLAKLAK)2(SEQ ID NO: 2); (KLAKKLA)2(SEQ ID NO: 3); (KAAKKAA)2(SEQ ID NO: 4); (KLGKKLG)3(SEQ ID NO: 5); Bcl-2; Bad; Bak; Bax and Bik; or
(b) an anti-angiogenic agent selected from the group consisting of thrombospondin, angiostatin, factor pigment epithelium, angiotensin, peptides of laminin, fibronectin peptides, inhibitors of plasminogen activators, inhibitors of tissue metalloproteinases, interferons, interleukin 12, platelet factor 4, IP-10, 2-methoxyestradiol, a protein associated with proliferin, angiopoietin 2, a fragment of prolactin 16K, endostatin XVIII, endostatin XV, C-terminal hemopexin domain of matrix metalloproteinase-2, the Kringle-the house is and 5 of human plasminogen, fused protein of endostatin and angiostatin, fused protein of endostatin and the Kringle domain 5 of human plasminogen, monokine induced by interferon gamma (Mig), fused protein of Mig and IP10, soluble FLT-1 (fins-like tyrosinekinase receptor 1), kinase insertion domain-containing receptor (KDR); or
(c) a cytokine selected from the group consisting of interleukin 1 (IL-1), IL-2, IL-5, IL-10, IL-11, IL-12, IL-18, interferon-γ (IF-γ), IF-α, IF-β, tumor necrosis factor and GM-CSF (granulocyte-macrophage colony-stimulating factor).
12. A method of obtaining a design aimed at VEGFR-1/NRP-1 construct that obtains a peptide according to any one of claims 1 to 4 and attaching the peptide to a molecule with obtaining the specified design.
13. Pharmaceutical composition for treating diseases or disorders, a part of which is angiogenesis, the individual containing an effective amount of a conjugate or design on any of pp.5-9, fused protein structure of claim 10 or 11, or targeted design obtained by the method according to item 12, and a pharmaceutically acceptable carrier.
14. The pharmaceutical composition according to item 13, where the disease or disorder is rheumatoid arthritis, inflammatory bowel disease, osteoarthritis, leiomyomas, adenoma, lipoma, hemangioma, fibroma, occlusion of vessels, restano the Ohm, atherosclerosis, pre-neoplastic lesions (such as glandular hyperplasia and intraepithelial neoplasia of the prostate, carcinoma in situ, hairy leukoplakia of the mouth, psoriasis, malignant neoplasm, impaired weight gain, obesity, diabetes, asthma, arthritis, cirrhosis, or eye disease.
15. The pharmaceutical composition according to 14, where a malignant tumor selected from the group consisting of cancer of the gums, tongue, lung, skin, liver, kidneys, eyes, brain, leukemia, mesothelioma, neuroblastoma, cancer of the head, neck, breast, pancreatic, prostate, renal, bone, testicular, ovarian, cervical, gastrointestinal, lymphoma, colon cancer, sarcoma and cancer of the urinary bladder.
16. The pharmaceutical composition according to 14, where the eye disease is selected from the group consisting of age-related macular degeneration, proliferative diabetic retinopathy, retinopathy of prematurity, glaucoma and proliferative vitreoretinopathy.
SUBSTANCE: disclosed are peptides which are isolated from the FOXM1 protein and are capable of activating cytotoxic (killer) human T cells by forming an antigen-presenting complex with a HLA-A2 molecule. Disclosed are compositions which contain the disclosed peptides, use of a peptide to produce an agent for inducing cancer immunity, treating and preventing cancer, and for producing antibodies which selectivity bind the disclosed peptides. The invention describes an exosome and an isolated antigen-presenting cell, which present a complex of the disclosed peptide with a HLA-A2 molecule, for inducing cytotoxic T cells, methods of inducing a antigen-presenting cell and a cytotoxic T cell, as well as a method of damaging FOXM1 and HLA-A2 expressing cells.
EFFECT: invention can further be used in treating tumours that are characterised by high FOXM1 expression.
14 cl, 2 ex, 1 dwg, 1 tbl
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
SUBSTANCE: there are presented versions of the peptide (A) or (B) with the amino acid sequence of SEQ ID NO: 1 or 2 respectively presented in this description. The peptide has activity to induce a cytotoxic T-cell when an antigen-presenting cell carrying HLA-A2 (A*0201) presents it. There are described the versions of the peptide antibodies prepared by immunisation by the proper peptide. There are presented: an agent, methods for inducing: a cytotoxic (killer) T-cell, an antigen-presenting cell, CDH3 expressing cancer immunity; as well as a method of treating CDH3 expressing cancer on the basis of the peptide. What is presented is an isolated cytotoxic T-cell induced by said method on the basis of the peptide. There are described: the antigen-presenting cell and exosome presenting the complex containing the peptide and HLA-A2 (A*0201).
EFFECT: higher effectiveness of the use of the invention in treating CDH3 expressing cancer.
15 cl, 5 dwg, 2 tbl, 5 ex
SUBSTANCE: peptide is presented, which is produced from protein WT1, which is able to bind with a molecule HLA-A*1101 and to induce CTL, having a sequence SEQ ID NO:6 or SEQ ID NO:7, presented in the description. Besides, a peptide dimer is described, which is used for the same purposes and comprises two peptide monomers selected from a group of peptides that includes SEQ ID NO:7, SEQ ID NO:3, SEQ ID NO:8 and SEQ ID NO:9, available in the description. A nucleic acid is presented, which codes the specified peptide and the expression vector, which contains the specified nucleic acid. A pharmaceutical composition is described for treatment or prevention of cancer of an individual, positive by HLA-A*1101, which contains the specified peptide, nucleic acid or vector. A WT1-specific CTL is described, which is induced by the specified peptide or dimer, and an antigen-presenting cell that presents the peptide. There is data on the method and set for induction of the WT1-specific CTL and for induction of the antigen-presenting cell. The method of in vitro diagnostics of cancer in an individual positive by HLA-A*1101 is presented, which includes incubation of the specified CTL or the antigen-presenting cell with a sample received from an individual positive by HLA-A*1101, and detection of the quantity of the specified CTL or antigen-presenting cell.
EFFECT: invention makes it possible to expand assortment of peptides that bind with HLA-A*1101 and are produced from the antigen WT1.
14 cl, 1 tbl, 14 dwg, 1 ex
SUBSTANCE: invention relates to a method of producing a peptide, which is characterised by that it involves conversion of the -SH group of the peptide which contains an amino acid residue having an -SH group to an -OH group, said method comprising the following steps from (a) to (c): (a) reaction of the -SH group in the peptide with a methylating agent to convert the -SH group to an -SMe group; (b) reaction of the -SMe group formed at step (a) with a cyanating agent to obtain an intermediate reaction product in form of an ester; (c) converting the intermediate reaction product obtained at step (b) to a peptide which contains an amino acid residue having an -OH group in more basic conditions than conditions at step (b).
EFFECT: improved method.
20 cl, 1 ex
FIELD: medicine, pharmaceutics.
SUBSTANCE: invention refers to biochemistry. What is presented is a peptide having an ability to induce cytotoxic T-cells wherein the peptide consists of amino acid sequences SEQ ID NO:3-5, 7-9, 12, 15-19, 22, 24, 27-30, 37, 67 or 74, what is also presented is a peptide having an ability to induce cytotoxic T-cells wherein the peptide consists of amino acid sequences SEQ ID NO:3-5, 7-9, 12, 17, 67 or 74 wherein 1 or 2 amino acids are substituted or added. What is presented is a peptide having an ability to induce cytotoxic T-cells wherein the peptide consists of amino acid sequences SEQ ID NO:15-19, 22, 24, 27-30 and 37 wherein 1 or 2 amino acids are substituted or added. What is presented is a pharmaceutical agent used for regulation of T-reg cells containing 1 or more types of said peptides. What is disclosed is a method for inducing antigen-presenting cells. What is presented is a recovered cytotoxic cell and an antigen-presenting cell. What is disclosed is a method for regulation of T-reg cells; what is also disclosed is the used of said peptides for producing a vaccine. There are presented polynucleotides coding said peptides.
EFFECT: invention enables producing the peptides used in the pharmaceutical agent for regulation of T-reg cells.
21 cl, 10 dwg, 4 tbl, 1 ex
SUBSTANCE: invention relates to biologically active peptide complexes with immunomodulating and antiviral activity. The disclosed peptide complexes have a three-dimensional structure wherein X1 is absent or contains at least 1 amino acid; R1 and R2 are peptide chains which contain amino acid residues, His or Cys, capable of reacting with transition metal ions, wherein R1 contains up to 5 amino acid residues or is absent; R2 contains up to 3 amino acid residues or is absent.
EFFECT: peptide complexes rich in histidine and, primarily alloferon peptides with Zn ions, enable to produce preparations with a directed mechanism of action and enable their design in accordance with the understanding of the structure of the medicinal target.
3 cl, 7 dwg, 2 tbl, 6 ex
SUBSTANCE: present invention relates to peptides having antibacterial and endotoxin-neutralising activity, having general formula (Xaa1)M-(Xaa2)o-Xaa3-(Xaa4)p-(Xaa5)Q-(Xaa6)M-(Xaa7)R-(Xaa8)S.
EFFECT: obtaining peptides with antibacterial and endotoxin-neutralising activity.
43 cl, 10 ex
SUBSTANCE: invention refers to peptides of general formula Rr-AA-R2 which can regulate neuron exocytosis, their mixtures and their cosmetically and pharmaceutically acceptable salts wherein AA represents a sequence of 6-40 neighbouring amino acids contained in the amino acid sequence of the protein SNAP-25 R1 is specified in a group consisting of H or acetyl, or saturated or unsaturated, linear, branched or cyclic C3-C24acyl, or a polyethylene glycol polymer; R2 is specified in a group consisting of amino, unsubstituted or substituted by saturated linear or branched, or cyclic C2-C24aliphatic groups provided when R1 represents H or acetyl, R2 is other tuhan unsubstituted amino. The invention also refers to cosmetic or pharmaceutical compositions containing such peptides, and use of such for treating the conditions which require regulation of neuron exocytosis, preferentially for skin treatment.
EFFECT: higher clinical effectiveness.
12 cl, 1 dwg, 8 ex
SUBSTANCE: invention relates to biotechnology and specifically to a peptide for inhibiting fractalkine-stimulated migration of monocytic cells. The peptide is selected from H-PKEQWVKD-NH2 or H-DAMQHLDRQAAA-NH2.
EFFECT: invention enables synthesis of a peptide without secondary reactions, which inhibits fractalkine-stimulated migration of monocytic cells.
1 dwg, 1 tbl, 3 ex
SUBSTANCE: invention refers to a compound having the structure of formula (I), or its pharmaceutically acceptable salt, wherein specified radicals are presented in the description, and also concerns a compound representing or its pharmaceutically acceptable salt. The present invention declares a pharmaceutical composition possessing inhibitory activity in the relation to 20S proteasome containing a pharmaceutically acceptable carrier or a diluent and a therapeutically effective amount of the compound, and also the invention refers to methods of treating the immune diseases, such as inflammatory intestinal disease, to treating cancer, to treating infection, to treating proliferative diseases, to treating neurodegenerative disease or asthma.
EFFECT: higher clinical effectiveness.
34 cl, 21 ex, 2 dwg
FIELD: medicine, pharmaceutics.
SUBSTANCE: in claim described are organic compounds of formula I where radicals are given in description, which are applicable for elimination, prevention or alleviation of one or more symptoms, associated with HCV disorders.
EFFECT: obtaining pharmaceutical composition which possesses inhibiting activity with respect to NS3-4 HCV serinprotease, including formula I compound and pharmaceutically acceptable carrier.
30 cl, 25 ex, 2 tbl
FIELD: medicine, pharmaceutics.
SUBSTANCE: invention refers to the application of a biologically active peptide which represents the amino acid sequence SEQ ID No.1.
EFFECT: preparation of a drug for modulation of at least one of the following conditions: fatigue, liver glycogen level and blood lactic acid level.
30 tbl, 14 ex
SUBSTANCE: invention relates to production of biologically active substances of peptide nature, having cartilage-derived morphogenetic protein CDMP-1 activity on chondrocyte proliferation. Through in silico construction, an oligopeptide of general formula I is obtained: X1-X2-X3-X4 (I), where X1 denotes M; X2 denotes A or G; X3 denotes W; X4 denotes W or is absent.
EFFECT: invention enables to obtain an oligopeptide having cartilage-derived morphogenetic protein CDMP-1 activity on chondrocyte proliferation, thereby widening the range of effective therapeutic agents which accelerate regeneration of cartilage tissue of joints.
5 dwg, 1 tbl, 4 ex
SUBSTANCE: invention relates to α',β'-epoxides of peptides of formulae (III) and (IV) which inhibit chymotrypsin-like activity of 20S proteasome.
EFFECT: increased effectiveness of the compounds.
19 cl, 29 ex
FIELD: chemistry; pharmacology.
SUBSTANCE: present invention refers to bioactive compounds of formula (Ic) , pharmaceutical compositions and application at cancer treatment, where R2-R7, X2, R, Q, G, J, L and M represent values estimated in invention formula and description.
EFFECT: production of compounds which can be used for anticancer medical product.
55 cl, 19 ex
FIELD: medicine; pharmacology.
SUBSTANCE: offered are compositions of formula 1 , where PG represents hydrogen or formyl group. R1 and R2 together with nitrogen atom to which they are attached, form heterocycle chosen from piperidine or morpholine and their pharmaceutically acceptable salts.
EFFECT: high haemostatic activity.
FIELD: organic chemistry, biochemistry.
SUBSTANCE: invention describes heterocyclic compounds represented by the general formula (I): and possessing elastase-inhibitory activity, and intermediate compounds for synthesis of such compounds. In the formula (I) R1 represents heterocyclic group represented by the formula (II): wherein A represents presence or absence of benzene ring; X represents oxygen atom, sulfur atom or -NH; Y represents nitrogen atom or -CH. Indicated heterocyclic group can be substituted with 1-3 substitutes that can be similar or different and they are chosen from group consisting of lower alkyl, lower alkoxy group and phenyl that can be optionally substituted with halogen-containing lower alkyl, lower alkoxy group or halogen atom; each among R2 and R3 represents hydrogen atom or hydroxyl, or R2 and R3 can be combined to form oxo group under condition that both are not hydrogen atoms.
EFFECT: valuable biochemical property of compounds.
8 cl, 7 tbl
FIELD: medicine, biochemistry.
SUBSTANCE: invention describes compounds that inhibit function of NS3-protease encoded by hepatitis C virus.
EFFECT: valuable medicinal properties of inhibitors.
6 cl, 2 tbl, 472 ex
FIELD: medicine, pharmaceutics.
SUBSTANCE: what is presented is a composition for nicotinic immunonanotherapy containing synthetic nanocarriers having a polymeric surface conjugated with a variety of nicotine residues with the variety of the nicotinic residues on the nanocarrier form an immunogenic surface providing a low affinity, a high-avidity binding of the nicotinic residues to the surfaces of an antigen presenting cell (APC) compared with an antibody binding, and a pharmaceutically acceptable excipient. The invention provides the nanocarriers capable to stimulate an immune response in T-cells and/or B cells and to produce the antinicotin antibodies with the humoral and cellular response to be achieved in the absence of an exogenous adjuvant.
EFFECT: invention provides the absence of the non-specific response on an inflammation caused by an adjuvant.
17 cl, 37 dwg