Biological materials and their use
SUBSTANCE: peptide of DGSVVVNKVSELPAGHGLNVNTLSYGDLAAD structure is used for suppression of allergic inflammation of respiratory passages, for prophylaxis and treatment of arthritis, as well as for pain relief. A peptide is effective as an adjuvant and for stimulation of IL-12 products in a cell.
EFFECT: peptide allows increasing IL-12 products by 10 times relative to normal levels of IL-12 cellular production.
19 cl, 25 dwg, 10 ex
The invention relates to polypeptides derived from the chaperonin 60.1, and to their use for the treatment of diseases and as a means to mitigate pain.
The heat shock polypeptides are the family of molecules that are common in all organisms, the function of which is to support biological processing and stability of biological molecules (Zugel & Kauffman (1999) Role of heat shock polypeptides in protection from and pathogenesis of infection diseases. Clin. Environ. Rev. (12)1: 19-39; Ranford et al. (2000) Chaperonins are cell signalling polypeptides: the unfolding biology of molecular chaperones. Exp. Rev. Mol. Med., 15 September, www.ermn.cbcu.cam.ac.uk/00002015h.htm).
The heat shock polypeptides are located in each cellular compartment and have the ability to interact with a wide range of biological molecules. In particular, heat shock polypeptides contribute to the folding of the polypeptide and the translocation of the polypeptide, and affect them, at any time after Assembly by disassembly of the polypeptide and any of its complexes. Help nature polypeptides heat shock led to the fact that they are also known as molecular chaperones (Laskey et al. (1978) Nucleosomas are assembled by an acidic polypeptide, which binds histones and transfers them to DNA. Nature (275): 416-420).
The heat shock polypeptides are synthesized by cells in response to the external environment, which include, but are not limited to, temperature changes (both increases and is the lower) and pathophysiological signals, such as cytokines. In response to the external environment polypeptides heat shock use their ability to carry out the processing of other polypeptides to protect such polypeptides from any denaturation, which can occur due to the impact. This mechanism serves to protect cells that contain this protein.
The polypeptides of chaperonins are a subset of heat shock polypeptides, whose role in the folding of polypeptides are well known. There are two families of polypeptides of chaperonins, family chaperonin 60 (approximately 60 kDa) and chaperonin 10 (priblisitelno 10 kDa) (Ranford, 2000). The most characterized chaperonine are chaperonine originating from E. coli, which was established characteristic structure of chaperonin 60 and chaperonin 10. Complexes of chaperonins most other organisms also essentially correspond to this characteristic structure.
The characteristic structure of chaperonin is a complex formed from two heptameric rings (consisting of seven monomers chaperonin 60), which are facing each other and covered heptamer ring consisting of monomers chaperonin 10.
Usually chaperonin contribute to the folding of the polypeptide when the polypeptide-target penetrates into the core ring heptameron, and at last the subsequent release of energy from ATP polypeptide-target is released from the core by conformational changes in the structure of chaperonin (Ranson et al. (1998) Review Article: Chaperones. Biochem. J (333): 233-242).
Mycobacterium tuberculosis (M. tuberculosis) produce chaperonin 60.1 (cpn 60.1), polypeptide, which is so named based on the identity of its amino acid sequence with other known chaperonine. Other polypeptides of chaperonins M. tuberculosis are chaperonin 10 (cpn 10) and chaperonin 60.2 (cpn 60.2). Chaperonin 60.2 shows to 59.6% identity of amino acid sequence and 65.6% identity nucleic acid sequence with cpn 60.1.
The present invention relates to the use of fragments of chaperonin 60.1 or functionally equivalent molecules from Mycobacterium tuberculosis or similar prokaryotes for the prevention and/or treatment of malignant and non-malignant conditions. Examples of non-malignant conditions include autoimmune disorders, osteoporosis, allergic disorders, or the state of immunoactivity, in particular asthma and/or condition, a typical representative of which is the immune response by type of T-helper lymphocyte 2 (Th2), and/or status, associated with eosinophilia and ways of stimulating the production of mediators of the immune response, such as cytokines, in vitro or in vivo.
Autoimmunity reflects the loss of tolerance to "own"that leads to inadequate destruction of normal cells or tissue. Many States are detected autoantibodies, however they can from the mirror effect, and not the cause of the disease. However, in some diseases, autoantibodies are the first, the only amenable to the detection of a violation. One of the classes involved in this respect molecules are chaperonin, which are highly immunogenic. Chaperonin belong to the group of proteins called molecular chaperones that bind negativnye proteins and help them, ATP-dependent catalytic process, to fold into the correct three-dimensional shape required for a functional protein.
I believe that chaperonine stimulate the immune system at many levels simultaneously, including monocytes, macrophages, fibroblast-like cells, and possibly other types of cells, and T cells. Immune protection in mammals can be divided into "innate" and "adaptive" protection. The one that is already in scope, for example, phagocytes, natural killer cells and complement, consider congenital. When stimulus, adaptive immunity is activated in the form of B - and T-lymphocytes. It is known that chaperonine act directly on the mechanisms of innate protection, in particular, on the phagocytes. They also stimulate potent adaptive immune response, namely the production of antibodies and stimulation of T-lymphocytes, which in some cases can be protective. It is noteworthy that they induce secrete the cytokines, which is considered important for the protection of the owner. However, in some instances, it is believed that the presence of chaperonins can be damaging to the host.
The role of chaperonins in autoimmune disease is controversial. Although infection/immunity in the case of chaperonin containing organisms are ubiquitous, and healthy people have T-cell responses to native chaperonin, including products chaperonin-specific antibodies, classic autoimmune disease is extremely rare. Thus, the presence of immune reactions on chaperonin can be random and insignificant.
However, theory of molecular mimicry suggests the involvement of chaperonins in autoimmune disease and is based on the high levels of conservatism amino acid sequence in chaperoning of microorganisms and mammals. theory assumes that in the process of infection by a wide range of microorganisms, the epitopes of chaperonins that are shared between microorganisms and mammals, stimulate T-lymphocytes. According to this theory, a high level representation of chaperonine epitopes of chaperonins violates the tolerance to a self-chaperoning, and developing an autoimmune disease.
It was found that chaperonine derived from tumors, leading to necrotic EF is the projects on these tumors. It is assumed that this can happen by strengthening the immune recognition of tumor antigens, although the mechanisms for this are unknown. Thus, apparently, chaperonin induce protective adaptive immunity against bacterial infections and malignant tumors.
Allergic reactions such as asthma, refer respectively to inadequate or improperly directed immune responses. The prevalence of, for example, asthma, increases and effective treatment of all cases has not yet been found. In the modern treatment of commonly used immunosuppressive corticosteroids, beta-agonists, cromoglicate, leukotriene modifiers, etc. that have many side effects.
When such allergic reactions are revealed high levels of IgE and immune responses of T-helper lymphocytes-2 (Th2) prevail over Th1 responses, which leads to an inflammatory response. I believe that Th1 responses, mainly, are protective against microbial infection and are run by cytokines, particularly interleukin-12 (IL-12), IL-2 and interferon-γ. In contrast, Th2 responses in the appropriate genetic background associated with harmful allergic tissue damage.
However, it has been suggested that other conditions, such as autoimmune disorders, for example, adjuvant arthritis, variative Th1 responses are the cause of the violation. Thus, the conversion of Th1 responses in Th2 or Th2 to Th1 may be suitable for treatment of the above disorders.
Although it is known that bacteria such as L. monocytogenes, M. bovis and M. tuberculosis, can convert the responses of Th2 to Th1, the molecules that are responsible for this transformation have been identified.
However, existing in this area assumptions apply heat shock protein, hsp65 from M. leprae, which is able to induce Th1 responses (Lowrie et al., 1999, Nature, 400, p269-271; Bonato et al., 1998, Infect. Immun., 66, p169-175). Homolog hsp65 from M. tuberculosis has the ability to stimulate monocytes synthesis of proinflammatory cytokines and activate monocytes and endothelial cells of vessels (Friedland et al., 1993, Clin. Exp. Immunol., 91, p5862; Peetermans et al., 1995, Infect. Immun., 63, p3454-3458; Verdegaal, et al., 1996, J. Immunol., 157, p369-376).
Also the present invention relates to the use of fragments of chaperonin 60.1 or functionally equivalent molecules from Mycobacterium tuberculosis or related prokaryotes to mitigate pain.
Easing the pain usually perform oral or parenteral treatment. Effective mitigation of pain can be achieved in most cases by using a widely known drug to alleviate pain, such as paracetamol, aspirin and other nonsteroidal anti-inflammatory drug (NSAID)such as ibuprofen, and selek is active inhibitors of cyclooxygenase-2 (CSI). Narcotic analgesics act on certain receptors in the Central nervous system (CNS). Codeine and Dihydrocodeine are narcotic analgesics moderate steps and have a low potential for addiction. Other stronger narcotic analgesics, such as morphine and methadone, can be used to combat severe pain.
There are many problems with the currently known means to mitigate pain. These drugs have a relatively short-term effect, and the pain lasts for only a few hours. Usually for dealing with pain requires multiple doses of the drug. Another common problem is suboptimal pain mitigation, leading to higher patient doses or change medications. In the case of NSAID common unpleasant gastrointestinal side effects, such as dyspepsia and ulcers, and approximately two-thirds of consumers change their NSAID at least one cut due to adverse side effects and low efficacy (Steinfeld S. and Bjorke P.A. Results from a patient survey to assess gastrointestinal burden of non-steroidal anti-inflammatory agent therapy contrasted with a review of data from EVA to determine satisfaction with rofecoxib. Rheumatology (Oxford) 2002, 41 (S1), 23-27.). In addition, NSAID and CSI can lead to cardiovascular complications (Hillis W. S, (2000)Areas of emerging interest in analgesia: cardiovascular complications. Am. J. Then 9 (3) 259-69). Aspirin can cause Reye's syndrome a small proportion of children, and thus, aspirin is unsuitable for use in children. Paracetamol should be used with caution, since overdose is hepatotoxic (Cranswick, N., D. Coghlan Paracetamol efficacy and safety in children: the first 40 years (2000) Am. J. Ther. 7(2) 135-41). Narcotic analgesics have many side effects, including drowsiness, constipation, nausea, headache and dizziness. Repeated introduction of potent narcotic analgesics such as morphine, can cause dependency.
The advantage of chaperonins as a means to mitigate pain over modern medicines to alleviate pain is that they may have less side effects. An estimated two billion people are carriers of M. tuberculosis without the development of Tuberculosis. Carriage of M. tuberculosis is not associated with side effects that are observed in the case of the widely known drug therapy, such as gastrointestinal side effects, cardiovascular complications, hepatotoxicity, Reye's syndrome or dependency.
Another advantage over the previously known means to mitigate the pain is the fact that the analgesic effect of chaperonins can last longer.
Against this background, the authors of this izopet is of unexpectedly identified peptides and polypeptide fragments of the protein chaperonin 60.1 (also called Cpn60.1) Mycobacterium tuberculosis, you can treat both malignant and non-malignant conditions, and to ensure mitigation of pain in vivo and in vitro.
Thus, in the first aspect of the invention relates to the selection or recombinant peptide molecule containing a polypeptide sequence or consisting of a polypeptide sequence selected from the group:
(xi) a polypeptide sequence that has more than 66%, or 70%, or 75%, or 80%, or 85%, or 90%, or 95% identity with the polypeptide sequence defined in any of (i)to(x) and has the function equivalent to the polypeptide sequence defined in any of (i)to(x); and
(xii) the fragment of the polypeptide sequence defined in any of (i)to(xi), which has the function equivalent to the polypeptide sequence defined in any of (i)to(xi).
In "polypeptide" also includes peptides, proteins, and peptidomimetics compounds. The term "peptidomimetic" refers to a compound that mimics the conformation and the desired attributes of a specific peptide as medicines, but in which no undesirable features.
"The function equivalent to the polypeptide sequence defined in any of (i)-(x)," includes any peptide, polypeptide and/or fragment, which have unctionally activity identical or substantially similar to any of the functions shown a particular polypeptide sequence or peculiar to it. For example, the polypeptide sequence defined in (i)-(x), can exhibit anti-inflammatory properties (as illustrated in the accompanying examples), allowing their use for the prevention and/or treatment of a non-cancerous condition or to alleviate pain.
Functional equivalence can be measured using, for example, the methods described in the accompanying examples (for example, by measuring the latent period for pads on the heated plate; or measuring the release of inflammatory cytokines in vivo or in vitro).
The terms "polypeptide" or "peptide" or "amino acid sequence" refer to an Oligopeptide, peptide, polypeptide, or protein sequence, or their fragments and to naturally occurring or synthetic molecules. A polypeptide "fragment", "portion" or "segment" is a stretch of amino acid residues of at least about 5 amino acids, preferably at least about 7 amino acids, more preferably at least about 9 amino acids, and most preferably at least about 17 or more amino acids. The peptide preferably is has no more than about 200 amino acids, more preferably less than 150 amino acids and most preferably less than 100 amino acids. Preferably the peptide has from about 5 to about 200 amino acids. He was active, any polypeptide must have sufficient length for the manifestation of biological and/or immunological activity.
The term "naturally occurring polypeptide" refers to polypeptides produced by cells that are created by means of genetic engineering, and specifically covers a variety of polypeptides that appear after post-translational modifications of the polypeptide including, but not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation and acylation.
The term "derivative" refers to polypeptides chemically modified by such methods as ubiquitination, tagging (e.g., with radionuclides or various enzymes), covalent joining of polymers, such as pegylation (conversion to derived from polyethylene glycol) and the incorporation of amino acids or substitution of amino acids such as ornithine, which do not occur in normal proteins in person, by chemical synthesis.
The term "variant" or "analog"refers to any polypeptide differing from naturally occurring polypeptides UGT what ukami, deletions and substitutions of amino acids made using, for example, methods of recombinant DNA. Those amino acid residues that can be replaced, added or deleted without eliminating interest activity can be determined by comparing the sequence of the particular polypeptide with sequences of homologous peptides and minimize changes in the amino acid sequence introduced into a region of high homology (conservative regions) or by replacing amino acid consensus sequence.
Alternative it is possible to synthesize or to select recombinant variants encoding these or similar polypeptides, using the "redundancy" in the genetic code. To optimize cloning into a plasmid or viral vector or expression in a particular prokaryotic or eukaryotic system, you can make a variety of replacement codons, such as the silent changes which form the different parts of the restriction. Mutations in the polynucleotide sequence can affect the polypeptide or domains of other peptides added to the polypeptide to modify the properties of any part of the polypeptide in order to change characteristics such as binding affinity of ligand miapocca affinity or rate of degradation/update.<> Preferably the amino acid "substitutions" are the result of replacing one amino acid with another amino acid having similar structural and/or chemical properties, i.e. conservative amino acid substitutions. "Conservative" amino acid substitutions can be performed on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or amphipatic nature of the residues involved. For example, nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, Proline, phenylalanine, tryptophan, and methionine; polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine; positively charged (basic) amino acids include arginine, lysine and histidine; and negatively charged (acidic) amino acids include aspartic acid and glutamic acid. The size of "insertions" or "deletions" is preferably in the range from approximately 1 to 20 amino acids, more preferably from 1 to 10 amino acids. Valid change can be experimentally determined systematically making insertions, deletions or substitutions of amino acids in the polypeptide molecule using recombinant DNA and analysis of the activity of resulting recombinant variants.
Alternatively, when it is desired to change options and, to obtain the modified polypeptides can be made by methods engineering insertion, deletion or non-conservative substitutions. Such changes may, for example, alter one or more biological functions or biochemical characteristics of the polypeptides according to the invention. For example, such changes may change the characteristics of the polypeptide, such as the binding affinity of the ligand, miapocca affinity or rate of degradation/update. In addition, such alterations can be selected so as to obtain polypeptides that are better suited for expression, scale, etc. in the cells of the host selected for expression. For example, cysteine residues can be deleted or replaced by another amino acid residue to eliminate disulfide bonds.
The protein fragments of the present invention, which is able to show biological activity, are also covered by the present invention. Fragments of the protein may have a linear form or they may be closed using known methods, for example, as described in H. U. Saragovi, et al., Bio/Technology 10, 773-778 (1992) and in R. S. McDowell, et al., J. Amer. Chem. Soc. 114, 9245-9253 (1992), both of which are incorporated herein as references. Such fragments can be subjected to fusion with the molecules of media, such as immunoglobulins, for many purposes, including increasing the shaft is nutnosti binding sites of the protein.
In addition, the invention relates to the selection or the recombinant nucleic acid molecule containing a polynucleotide sequence or consisting of a polynucleotide sequence selected from the group:
(a) a polynucleotide sequence that encodes a polypeptide sequence selected from the group consisting of:
(b) a polynucleotide sequence that has more than 66%, or 70%, or 75%, or 80%, or 85%, or 90%, or 95% identity with a polynucleotide sequence specified in (a); or a polynucleotide sequence that hybridizes with a polynucleotide sequence specified in (a) in 2xSSC, 65°C, which encodes the polypeptide sequence having the function equivalent to the polypeptide sequence defined in any of (i)to(x); and
(c) fragment of a polynucleotide sequence specified in (a) or (b)that encodes a polypeptide sequence having the function equivalent to the polypeptide sequence defined in any of (i)to(x).
The terms "nucleotide sequence" or "nucleic acid" or "polynucleotide" or "oligonucleotide" are used interchangeably, and they refer to heteropolymer nucleotides or PEFC is the sequences of these nucleotides. These phrases also refer to DNA or RNA of genomic or synthetic origin which may be single-stranded or double-stranded and may represent a sense or antisense chain peptide nucleic acid (PNA), or to any DNA-like or RNA-like material. In the sequence A represents adenine, C represents cytosine, T is thymine, G represents guanine, and N represents A, C, G or T (U). It is envisaged that when polynucleotide represents RNA, T (thymine) in the sequence presented in this document, replaced by U (uracil). Typically, segments of nucleic acids according to the invention can be assembled from fragments of the genome and short oligonucleotide linkers, or from a series of oligonucleotides, or from individual nucleotides, to obtain a synthetic nucleic acid, which can be expressed in a recombinant transcriptional element containing regulatory elements derived from a microbial or viral operon, or a eukaryotic gene.
Polynucleotide according to the invention include naturally occurring or wholly or partially synthetic DNA, for example, cDNA, genomic DNA, and RNA, for example mRNA. Polynucleotide may include the entire coding region cDNA or movetreestate a portion of the coding region cDNA.
The present invention also relates to the genes corresponding to the cDNA sequences described herein. These genes can be distinguished by known methods using the information in the sequence described herein. Such methods include receiving probes or primers based on the information described sequence for identification and/or amplification of genes in appropriate genomic libraries and other sources of genomic material. In addition, the 5'- and 3'-sequence can be obtained using methods known in this field. For example, a full-sized cDNA or genomic DNA that corresponds to any of polynucleotides according to the invention can be obtained by screening appropriate cDNA libraries or genomic DNA in a suitable hybridization conditions using any of polynucleotides according to the invention or parts thereof as a probe. Alternative polynucleotide according to the invention can be used as the basis for suitable primer(s), which allows the identification and/or amplification of genes in appropriate genomic DNA libraries or cDNA.
The nucleic acid sequences according to the invention can be assembled from EST sequences (including sequences of cDNA and genomic posledovatel the property), obtained from one or more public databases, such as dbEST, gbpri and UniGene. The EST sequence can provide identification information sequence, of information representative of a fragment or segment, or information of a new segment for full-length gene.
To polynucleotides according to the invention also include polynucleotide, including nucleotide sequences that are essentially equivalent to polynucleotides above. Polynucleotide according to the invention can have, for example, at least about 65%, at least about 70%, at least about 75%, at least about 80%, 81%, 82%, 83%, 84%, more specifically at least about 85%, 86%, 87%, 88%, 89%, more specifically at least about 90%, 91%, 92%, 93%, 94%, and even more specifically at least about 95%, 96%, 97%, 98%, 99% the sequence identity with the above polynucleotides.
The sequences of the nucleic acids according to the invention included fragments of the sequences of nucleic acids that hybridize under strict conditions with any of the nucleotide sequences according to the invention, or the complement of sequences, where the fragment is greater than about 5 nucleotides, preferably 7 nucleotides, more is predpochtitelno 9 nucleotides and most preferably 17 nucleotides. Provides fragments, such as 15, 17 or 20 nucleotides or more, which are selective with respect to any of polynucleotides according to the invention (i.e. specific hybridize with them). Probes capable of specific to gibridizatsiya with polynucleotides can distinguish the polynucleotide sequence according to the invention from other polynucleotide sequences in the same family of genes, or they can distinguish human genes genes from other species, and preferably they are based on the unique nucleotide sequences.
Sequences that are in the scope of the present invention is not limited to these specific sequences, but they also have their allelic and species variants. Allelic and species variations can be defined in a common manner by comparing the sequences according to the invention, a representative fragment or a nucleotide sequence at least 90% identical, preferably 95% identical with a sequence from another isolate of the same species. Moreover, to account for variability codons invention includes nucleic acid molecule encoding the same amino acid sequence as specific ORF described herein. In other words, in the coding region of ORF explicitly provides for the replacement of the and one other codon by codon, which encodes the same amino acid.
The term "simple" is used to denote conditions that are generally understood in the field as strict. Stringent conditions can include highly stringent conditions (i.e., the hybridization with the associated filter DNA in 0.5 M NaHPO4, 7% sodium dodecyl sulfate (SDS), 1 mm EDTA at 65°C, and washing in 0.1x SSC/0,1% SDS at 68°C) and moderately stringent conditions (i.e. washing in 0.2X SSC/0,1% SDS at 42°C). Other illustrative of the hybridization conditions described in this document in the section "Examples".
In cases of hybridization deoxyoligonucleotide, additional illustrative stringent hybridization conditions include washing in 6x SSC/0,05% sodium pyrophosphate at 37°C (for oligonucleotides 14 bases), 48°C (for oligonucleotides of 17 bases), 55°C (for oligonucleotide of 20 bases) and 60°C (for oligonucleotides of 23 bases).
SSC is defined as 0.15 M NaCl, 0.015 g M sodium citrate, pH 7,2.
Under "identity" means the quantity or percentage (depending on the presentation of the results) amino acid residues or residues of the nucleic acid sequence of the candidate that are identical with amino acid residues or residues of the nucleic acid of interest sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the Maxi is the real percentage of sequence identity, and not considering any conservative substitutions as part of the identity of the sequence.
The percentage sequence identity between the two polynucleotide or polypeptides can be determined using suitable computer programs, for example, the GAP program of the University of Wisconsin Genetic Computing Group, and it is clear that the percentage of identity is calculated in respect of the polypeptide, the sequence of which is aligned optimally. Alternative alignment can be performed using the program Clustal W (Thompson et al., (1994) Nucleic Acids Res. 22, 4673-80). Used the following options are available: settings for fast pairwise alignment: the size of the K-tuple (word); 1, the window size; 5, the penalty for deletion; 3, number of top diagonals; 5. method of evaluation: x percent; multiple alignment parameters: the penalty for making deletions; 10, the penalty for continued deletions; 0,05, evaluation matrix: BLOSUM.
As used herein, "essentially equivalent" or "essentially similar" may refer to both nucleotide and amino acid sequences, for example, a mutant sequence, which differs from the reference sequence by one or more substitutions, deletions or insertions, the net effect of which does not result in an adverse functional dissimilarity between etal is authorized and considered sequences. Typically, this is essentially equivalent to the sequence differs from the sequences provided herein, not more than about 35% (i.e., the number of substitutions, insertions and/or deletions of individual residues in a substantially equivalent sequence, as compared with the corresponding reference sequence, divided by the total number of residues in a substantially equivalent sequence is about 0.35 or less). Indicate that this sequence has 65% sequence identity with the sequence. In one embodiment, essentially equivalent, for example, a mutant sequence according to the invention differs from the listed sequence by no more than 30% (70% sequence identity); in the version of this case for not more than 25% (75% sequence identity); and in the next version of this case for not more than 20% (80% sequence identity), and in the next version of this case for not more than 10% (90% sequence identity), and in the next version of this case for not more than 5% (95% sequence identity). Essentially equivalent, for example, mutant amino acid follower of the spine according to the invention preferably have at least 80% sequence identity with the amino acid sequence, more preferably at least 85% sequence identity, more preferably at least 90% sequence identity, more preferably at least 95% sequence identity, more preferably at least 98% sequence identity, and most preferably at least 99% sequence identity. Essentially equivalent to the nucleotide sequence according to the invention can have a lower percent identity of the sequences, taking into account, for example, redundancy or degeneracy of the genetic code. Preferably the nucleotide sequence has at least about 65% identity, more preferably at least about 75% identity, more preferably at least about 80% sequence identity, more preferably at least 85% sequence identity, more preferably at least 90% sequence identity, more preferably at least about 95% sequence identity, more preferably at least 98% sequence identity, and most preferably at least 99% sequence identity. For the purposes of the present invention, the sequence is lnasty, having essentially equivalent biological activity and essentially equivalent expression characteristics, considered essentially equivalent. For the determination of equivalence, the shortening of the Mature sequence (e.g., through mutation, which generates a random stop-codon) should be neglected. The sequence identity can be determined, for example, using the method of Jotun Hein (Hein, J. (1990) Methods Enzymol. 183: 626-645). The identity between sequences can also be defined in other ways known in this field, for example, by varying hybridization conditions.
In addition, the invention relates to a peptide molecule according to the invention for use in medicine and/or the nucleic acid molecule according to the invention for use in medicine.
Preferably the invention relates to a peptide molecule according to the invention and/or the nucleic acid molecule according to the invention for use in preventing and/or treating a non-cancerous condition or malignant condition.
In addition, the invention relates to the use of peptide molecules or nucleic acid molecules according to the invention for the manufacture of a medicine for the prevention and/or treatment of a non-cancerous condition or malignant condition.
The following aspect of izaberete the s relates to pharmaceutical compositions, containing the peptide molecule according to the invention and a pharmaceutically acceptable excipient, or consisting of them.
The following aspect of the invention relates to pharmaceutical compositions containing the nucleic acid molecule according to the invention and a pharmaceutically acceptable excipient, or consisting of them.
The molecules, medicaments and pharmaceutical compositions of the present invention can be delivered using an injectable systems for the delivery of delayed release. They are designed specifically to reduce the frequency of injections. An example of such system is Nutropin Depot, which encapsulates recombinant human growth hormone (rhGH) in biodegradable microspheres that after the injection of slow-release rhGH for a long period. Preferably the delivery spend intramuscularly (i.m.) and/or subcutaneously (s.c.) and/or intravenous (i.v.).
The molecules, medicaments and pharmaceutical compositions of the present invention can be entered using a surgically implanted device that releases the drug directly to the desired area. For example, Vitrasert releases ganciclovir straight in the eye for the treatment of CMV-retinitis. Direct application of this toxic means in the affected area provides effective therapy without substantially the x systemic side effects of the drug.
Also for the introduction of substances, medicaments and pharmaceutical compositions according to the invention can be used system for therapy by electroporation (EPT). A device that delivers pulsed electric field to the cells, increases the permeability of cell membranes for drugs, which leads to a significant increase of intracellular delivery of the drug.
The molecules, medicaments and pharmaceutical compositions according to the invention can also be delivered by electroencephalogra (EI). EI occurs when small particles up to 30 micrometers in diameter on the surface of the skin affected by electrical impulses that are identical or similar to the pulses used for electroporation. When EI these particles are directed through the outer layer and deeper layers of the skin. The particles can be loaded or coated with drugs or genes, or may simply act as "shells", which form pores in the skin through which can pass the drug.
An alternative method of delivery of the molecules, medicaments and pharmaceutical compositions according to the invention is an injectable system ReGel, which is heat-sensitive. At temperatures below body temperature ReGel is an injectable LM the bone, and at body temperature, it immediately forms a gel-like capacity, which slowly disintegrates and dissolves to a known, safe biodegradable polymers. The active substance is delivered over time as the dissolution of biopolymers.
The molecules, medicaments and pharmaceutical compositions according to the invention can also be delivered orally. The method uses the natural process of oral absorption of vitamin B12and/or vitamin D in the body for the joint delivery of proteins and peptides. By moving through the system the absorption of vitamin B12and/or vitamin D, nucleic acids, molecules and pharmaceutical formulations according to the invention can move through the intestinal wall. Synthesize complexes analogues of vitamin B12and/or analogs of vitamin D and medicines that save as pronounced affinity to the internal factor (IF) in the part of vitamin B12/part of the vitamin D complex, and expressed biological activity of the active substance complex.
The molecules, medicaments and pharmaceutical compositions according to the invention can be introduced in the cell by "Trojan peptides". They represent a class of polypeptides called penetratingly that have the properties of translocation and able to endure hydrophilic soy is inane across plasma membrane. This system allows direct targeting of oligopeptides in the cytoplasm and the nucleus, and may be non-specific to the type of cells and highly efficient. Cm. Derossi et al. (1998), Trends Cell Biol 8, 84-87.
Preferably, the drug and/or pharmaceutical composition of the present invention is a unit dosage containing a daily dose or unit, daily curioso or its relevant part, of the active ingredient.
The molecules, medicaments and pharmaceutical compositions according to the invention typically can be administered orally or by any parenteral way, in the form of pharmaceutical compositions containing the active ingredient, optionally in the form of non-toxic organic or inorganic acid or base or addition salt, in a pharmaceutically acceptable dosage form. Depending on the violation and the patient being treated, and the method of administration, the compositions can be administered in a number of doses.
In human therapy the molecules, medicaments and pharmaceutical compositions according to the invention can be entered separately, however, as a rule, you can enter them in a mixture with suitable pharmaceutical excipient, diluent or carrier selected with regard to the intended route of administration and standard pharmaceutical practice.
For example, they say cooly, medicaments and pharmaceutical compositions according to the invention can be administered orally, buccal or sublingual tablets, capsules, suppositories, elixirs, solutions or suspensions, which may contain flavouring or colouring agents, for immediate -, delayed-or controlled-release. The molecules, medicaments and pharmaceutical compositions according to the invention can also be administered by vnutrikavernozno injection.
Such tablets may contain excipients such as microcrystalline cellulose, lactose, sodium citrate, calcium carbonate, dibasic calcium phosphate and glycine, dezintegriruetsja tools such as starch (preferably corn, potato or manioc starch), sodium starch glycolate, croscarmellose sodium and certain complex silicates, and granulated binders, such as polyvinylpyrrolidone, hypromellose (HPMC), hydroxypropylcellulose (HPC), sucrose, gelatin and gum Arabic. Additionally, there may be included a lubricating agents such as magnesium stearate, stearic acid, glycerinated and talc.
Also as fillers in gelatin capsules it is possible to use solid compositions of a similar type. Preferred excipients in this regard include lacto is zu, starch, cellulose, milk sugar or high molecular weight polyethylene glycols. For aqueous suspensions and/or elixirs means according to the invention can be combined with various sweetening or flavouring additives, coloring substances or pigments, with emulsifying and/or suspendresume agents and with diluents such as water, ethanol, propylene glycol and glycerin, and combinations thereof.
The molecules, medicaments and pharmaceutical compositions according to the invention can also be entered parenterally, for example intravenously, intraarterially, intraperitoneally, intrathecal, intraventricular, nutrigrain, intracranially, intramuscularly or subcutaneously, or they can be administered by methods of infusion. The best way you can use them in the form of a sterile aqueous solution which may contain other substances, for example enough salts or glucose to make the solution isotonic with blood. Aqueous solutions must be suitable manner buffered (preferably to pH 3 to 9), if necessary. Suitable parenteral formulations can be easily obtained in sterile conditions standard methods of pharmacy well known to specialists in this field.
Medicines and pharmaceutical compositions suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions, which may contain antioxidants, buffers, bacteriostatic and dissolved substances, which make the solution isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions that can include suspendresume substances and thickeners. Medicines and compositions can be presented in containers for single or multiple doses, for example, sealed ampoules and vials, and can be stored in a freeze dried (lyophilized) condition requiring only the addition of sterile liquid carrier, for example water for injections, immediately prior to use. From sterile powders, granules and tablets of the type indicated above can be obtained injection solutions and suspensions for immediate use.
For oral and parenteral administration to patients-the people, the daily dosage level of the molecules, medicaments and pharmaceutical compositions according to the invention will usually be from 0.1 to 100 mg per adult human per day, which is administered in single or divided doses.
Thus, for example, tablets or capsules molecules according to the invention may contain from 0.1 mg to 100 mg of active means for the introduction of one or two or more tablets and capsules at one time, depending on the situation. The doctor in each case may determine and the true dosage, which is the most suitable for any particular patient, and it can vary depending on age, weight and response of the individual patient. The above dosages are illustrative for the average case. Of course, there may be isolated cases are appropriate higher or lower ranges of dosages, and are within the scope of this invention.
The molecules, medicaments and pharmaceutical compositions according to the invention can also be entered intranasally or by inhalation, and conveniently delivered in the form of dry inhaler dry powder or by aerosol spray presentation from a pressurized container, pump, spray or device for spraying with the use of a suitable propellant, e.g. DICHLORODIFLUOROMETHANE, trichloromethane, dichlorotetrafluoroethane, hydrofluroalkane, such as 1,1,1,2-Tetrafluoroethane (HFA 134A or 1,1,1,2,3,3,3-Heptafluoropropane (HFA 227EA), carbon dioxide or other suitable gas. In the case of an aerosol under pressure dosage unit can be determined by providing a valve to deliver a measured quantity. The pressurized container, pump, spray or spraying unit may contain a solution or suspension of the active means, for example, when used as will dissolve the La mixture of ethanol and propellant, which, in addition, may contain a lubricating substance, such as sorbitan trioleate. Capsules and cartridges (made, for example, from gelatin) for use in an inhaler or insufflator can be produced so as to contain a powder mix means according to the invention and a suitable powder base, such as lactose or starch.
The compositions of aerosols or dry powders are preferably such that each metered dose or "release" contained at least 0.1 mg of a molecule according to the invention for delivery to the patient. It is clear that the total daily dose in the case of an aerosol can vary from patient to patient, and can be entered in a single dose or, more often divided doses during the day.
Alternative molecules, medicaments and pharmaceutical compositions according to the invention can be introduced in the form of a suppository or pessary, or they can be applied topically in the form of a lotion, solution, cream, gel, ointment or spray powder. The molecules, medicaments and pharmaceutical compositions according to the invention can also be entered transdermal, for example, using a skin patch. They can also enter the eye by, inter alia, for the treatment of eye diseases.
For ophthalmic applications molecule pharmaceuticals to the notizie according to the invention can be produced as micronized suspensions in isotonic sterile saline solution with adjusted pH, or, preferably, as solutions in isotonic sterile saline solution with adjusted pH, optionally in combination with a preservative such as chloride benzylamine. Alternative can be made in the form of ointment, such as vaseline ointment.
For local application on the skin of the molecules, medicaments and pharmaceutical compositions according to the invention can be manufactured as a suitable ointment containing the active compound suspended or dissolved in, for example, in a mixture with one or more of the following components: mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene-polyoxypropylene agent, emulsifying wax and water. Alternative they can produce as a suitable lotion or cream, suspended or dissolved in, for example, mixtures of one or more of the following components: mineral oil, sorbitan the monostearate, a polyethylene glycol, liquid paraffin, Polysorbate 60, wax-based atilovykh esters, Cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water.
Formulations suitable for local injection in the mouth include lozenges, containing the active ingredient in a flavor, usually sucrose and gum Arabic or tragacanth gum; mints containing active the ingredient in an inert basis, such as gelatin and glycerin, or sucrose and gum Arabic; and means for rinsing the mouth, containing the active ingredient in a suitable liquid carrier.
Typically, a person's oral or parenteral administration of the molecules, medicaments and pharmaceutical compositions according to the invention is the preferred method, which is the most convenient.
For veterinary use molecules, medicaments and pharmaceutical compositions according to the invention is administered as a suitable acceptable composition in accordance with normal veterinary practice and the veterinary surgeon will determine the dosing and route of administration which will be most suitable for a particular animal.
Convenient to have the composition represented a pharmaceutical composition. Mainly, the composition is a veterinary composition.
Preferably the pharmaceutical composition is intended for use in the prevention or treatment of a non-cancerous condition.
The following aspect of the invention relates to a method of prevention and/or treatment of non-cancerous or cancerous condition in a patient, comprising the stage of introduction to a patient in need, an effective amount of the peptide molecules of the invention or the nucleic acid molecule according to the invention,or pharmaceutical compositions according to the invention.
By "therapeutically effective amount" or "effective amount" refers to the number of molecules of the peptide according to the invention or nucleic acid molecules according to the invention that, when administered to a subject, either alone or in combination with other means, alleviate the symptoms of diseases, disorders or conditions.
"Autoimmune disease" includes the cases when it can be shown that the autoimmune process leading to disease pathogenesis. Such diseases usually associated with immune response by type of T-helper lymphocytes 1 (Th-1).
"Allergic conditions include a condition associated with immune response by type of T-helper lymphocytes-2 (Th-2). When allergic reactions have high levels of IgE and immune responses Th-2 is dominated by the response of Th-1, which leads to an inflammatory response. Examples of allergic conditions include the following conditions: asthma, rhinitis/hay fever, eczema and anaphylaxis.
By "adjuvant" means a substance that, when combined or introduction simultaneously with the antigen, enhances the immune response.
Typically, non-cancerous condition is selected from the group consisting of, or consisting of the following: autoimmune disorders, such as hemolytic anemia, thrombocytopenia, thyroiditis, pernicious anemia, a disease Addie is she, autoimmune diabetes, myasthenia gravis, rheumatoid arthritis, systemic lupus erythematosus, atherosclerosis, and autoimmune encephalitis; allergic conditions such as eczema, dermatitis, allergic rhinitis, allergic conjunctivitis; allergic diseases of the respiratory tract; symptoms of hypereosinophilia; contact dermatitis, food Allergy; and respiratory disease characterized by eosinophilic airway inflammation and increased airway reactivity, such as allergic asthma, hereditary asthma, allergic bronchopulmonary aspergillosis, eosinophilic pneumonia, allergic bronchitis bronchiectasis, occupational asthma, reactive respiratory syndrome, interstitial lung disease, hypereosinophilic syndrome, parasitic lung disease.
The following examples of autoimmune conditions are, for example, connective tissue disease, multiple sclerosis, systemic lupus erythematosus, rheumatoid arthritis, autoimmune inflammation of the lungs, Guillain-Barre syndrome, autoimmune thyroiditis, insulin dependent diabetes mellitus, myasthenia gravis, graft-versus host disease and autoimmune inflammatory eye disease. Such a protein (or its antagonists, including antibodies) of the present invention can also be used to treat allergic the fir reactions and conditions (for example, anaphylaxis, serum sickness reactions to medications, food allergies, allergies to insect venom, mastocytosis, allergic rhinitis associated with hypersensitivity pneumonitis, urticaria, angioedema, eczema, atopic dermatitis, allergic contact dermatitis, poly-form of erythema, Stevens-Johnson, allergic conjunctivitis, atopic keratoconjunctivitis, venereal keratoconjunctivitis, giant papillary conjunctivitis and contact allergies, such as asthma (particularly allergic asthma) or other respiratory problems. Also using a protein (or antagonists) according to the present invention it is possible to treat other conditions, which is desirable immune suppression (including, for example, organ transplantation). therapeutic effects of the polypeptides or antagonists for allergic reactions can be assessed in animal models in vivo, such as test cumulative contact enhancement (Lastbom et al., Toxicology 125: 59-66, 1998), injectable skin test (Hoffmann et at., Allergy 54: 446-54, 1999), test for skin sensitization Guinea pig (Vohr et al., Arch. Toxocol. 73: 501-9) and local analysis of lymph nodes of a mouse (Kimber et al., J. Toxicol. Environ. Health 53: 563-79).
Suitable assays cytotoxicity of thymocytes and splenocytes include, but are not limited to the mi, the analyses described in: Current Protocols in Immunology, Ed by J. E. Coligan et al., Strober, Pub. Greene Publishing Associates and Wiiey-Interscience (Chapter 3, In vitro assays for Mouse Lymphocyte Function 3.1-3.19; Chapter 7, Immunologic studies in Humans); Herrmann et al., Proc. Natl. Acad. Sci. USA 78: 2488-2492, 1981; Herrmann et al., J. Immunol. 128: 1968-1974, 1982; Handa et al., J. Immunol. 135: 1564-1572, 1985; Takai et al., J. Immunol. 137: 3494-3500, 1986; Takai at al., J. Immunol. 140: 508-512, 1988; Bowman et al., J. Virology 61: 1992-1998; Bertagnolli et al., Cellular Immunology 133: 327-341, 1991; Brown et al., J. Immunol. 153: 3079-3092, 1994.
Analyses of the dependent T-cell responses of immunoglobulins and switch isotypes (which will identify, among others, proteins that modulate dependent T-cell or antibody-based test answers that affect the profiles of Th1/Th2) include, but are not limited to, the assays described in: Maliszewski, J. Immunol. 144: 3028-3033, 1990; and analyses of the function of B-cells: In vitro antibody production, Mond, J.J. and Brunswick, M., Current Protocols in Immunology. J. E. Coligan et al., eds. Vol 1 pp. 3.8.1-3.8.16, John Wiley and Sons, Toronto. 1994.
Analysis of the mixed reactions of lymphocytes (MLR) (which identifies, among others, proteins that carry out mainly Th1 and CTL responses) include, but are not limited to, the assays described in: Current Protocols in Immunology, Ed by J. E. Coligan et al., Pub. Greene Publishing Associates and Wiley-lnterscience (Chapter 3, In vitro assays for Mouse Lymphocyte Function 3.1-3.19; Chapter 7, Immunologic studies in Humans); Takai et al., J. Immunol. 137: 3494-3500, 1986; Takai et al., J. Immunol. 140: 508-512, 1988; Bertagnolli et al., J. Immunol. 149: 3778-3783, 1992.
Dependent dendritic cell assays (which will identify, among others, proteins that Express the dummy dendritic cells, which can activate naive T-cells) include, but are not limited to, the assays described in: Guery et al., J. Immunol. 134: 536-544, 1995; Inaba et al., J. Experimental Medicine 173: 549-559, 1991; Macatonia et al., J. Immunol. 154: 5071-5079, 1995; Porgador et al., J. Experimental Medicine 182: 255-260, 1995; Hair et al., J. Virology 67: 4062-4069, 1993; Huang et al., Science 264: 961-965, 1994; Macatonia et al., J. Experimental Medicine 169: 1255-1264, 1989; Bhardwaj et al., J. Clinical Investigation 94: 797-807, 1994; and Inaba et al., J. Experimental Medicine 172: 631-640, 1990.
Analyses of survival of lymphocytes/apoptosis (which will identify, among others, proteins that prevent apoptosis after induction by superantigens, and proteins that regulate the homeostasis of lymphocytes include, but are not limited to, the assays described in: Darzynkiewicz et al., Cytometry 13: 795-808, 1992; Gorczyca et al., Leukemia 7: 659-670, 1993; Gorczyca et al., Cancer Research 53: 1945-1951, 1993; Itoh et al., Cell 66: 233-243, 1991; Zacharchuk, J. Immunol. 145: 4037-4045, 1990; Zamai et al., Cytometry 14: 891-897, 1993; Gorczyca et al., Int. J. Oncol. 1: 639-648, 1992.
Analyses of proteins that affect early stages of commitirovannah and development of T-cells include, but are not limited to, the assays described in: Antica et al., Blood 84: 111-117, 1994; Fine et al., Cellular Immunology 155: 111-122, 1994; Galy et al., Blood 85: 2770-2778, 1995; Toki et al., Proc. Natl. Acad Sci. USA 88: 7548-7551, 1991.
The compositions of the present invention may also have anti-inflammatory activity. Anti-inflammatory activity can be achieved by providing a stimulus to the cells involved in the inflammatory response by inhibiting or run the intercellular interactions (for example, such as adhesion of cells)by inhibiting or start chemotaxis of cells involved in the inflammatory process, inhibiting or start extravasation of cells, or by stimulating or suppressing the production of other factors that are more directly inhibit or stimulate an inflammatory response. Compositions with such activities can be used to treat inflammatory conditions including chronic or acute conditions), including, but not limited to, signs associated with infection (such as septic shock, sepsis or systemic inflammatory response syndrome (SIRS)), damage during ischemia-reperfusion, lethality from endotoxin, arthritis, mediated by complement Verhoture rejection, nephritis, cytokine or chemokine-induced lung injury, inflammatory bowel disease, Crohn's disease, or resulting from the production of cytokines, such as TNF or IL-1. The composition of the invention can also be suitable for the treatment of anaphylaxis and hypersensitivity to an antigenic substance or material. The compositions of this invention can be used to prevent or treat conditions such as, but not limited to, sepsis, acute pancreatitis, endotoxin shock induced by cytokines shock, rheumatoid arthritis, chronic inflammatory and the trits, damage to the cells of the pancreas in diabetes mellitus type 1, graft versus host, inflammatory bowel disease, inflammation associated with pulmonary disease, other autoimmune disease or inflammatory disease, an antiproliferative agent, such as a remedy for acute or chronic myelogenous leukemia, or for the prevention of preterm birth, secondary to intrauterine infections.
In a preferred embodiment, the non-cancerous condition is asthma; alternative non-cancerous condition is arthritis.
Immunosuppressive effects of the compositions according to the invention against rheumatoid arthritis is determined in an experimental model system in animals. Experimental model system is an adjuvant-induced arthritis in rats, the Protocol for which is described in J. Holoshitz, et al., 1983, Science, 219: 56, or B. Waksman et al., 1963, Int. Arch. Allergy Appl. Immunol., 23: 129. Induction of the disease may be caused by a single injection, mainly, intradermal, suspension of killed Mycobacterium tuberculosis in complete Freund's adjuvant (CFA). The method of injection may vary, however, rats can be injected into the base of the tail with adjuvant mixture. The polypeptide is administered in phosphate-buffered saline (PBS) at a dose of approx is Ino 1-5 mg/kg The control consists of an introduction only PBS.
The way to test the effects of test compounds may consist of an intradermal injection of killed Mycobacterium tuberculosis in CFA followed by immediate introduction of the test compounds and the subsequent introduction of a time in two days to 24 days. 14, 15, 18, 20, 22 and 24 days after the injection of CFA Mycobacterium you can get the overall score for the evaluation of arthritis, as described in J. Holoskitz above. Data analysis may reveal that the test compound may have a strong effect on the swelling of the joints when determining the reduction rate for arthritis.
Treatment of malignant tumors provides regression of tumors by inhibiting the proliferation of tumor cells, inhibition of angiogenesis (growth of new blood vessels, which is necessary for the maintenance of tumor growth and/or prevention of metastasis by reducing the motility of tumor cells or invasiveness. Therapeutic compositions according to the invention may be effective in cancer in adults and children, including solid tumors/malignancies, locally developed tumors, soft tissue sarcoma in humans, metastatic malignancy, including lymphatic metastases, malignant tumors of blood cells, including multiple myeloma, acute and chronic leukemias, and lymphomas, head and neck cancer including oral cancer, larynx cancer and thyroid cancer, lung cancer, including small cell carcinoma and non-small cell lung cancer, breast cancer, including small cell carcinoma and carcinoma of the ducts, gastrointestinal cancer, including esophageal cancer, stomach cancer, colon cancer, cancer of the colon, rectum and polyps associated with colorectal neoplasia, pancreatic cancer, liver cancer, urological cancer, including bladder cancer and prostate cancer, malignant tumors of the female genital tract, including cancer of the ovary, cancer of the uterus (including endometrial cancer), and solid tumor of the ovarian follicles, kidney cancer, including renal cell carcinoma, malignant brain tumors, including congenital brain tumors, neuroblastoma, astrocytic brain tumors, gliomas, metastatic invasion of tumor cells into the Central nervous system, bone cancer, including osteomas, skin cancers, including malignant melanoma, tumor progression of keratinocytes of human skin, squamous cell carcinoma, basal cell carcinoma, hemangiopericytoma and Kaposi's sarcoma.
Polypeptides, polynucleotide or modulators of the polypeptides of the invention (including inhibitors and stimulators of the biological activity of the polypeptide according to the invention) can be entered for the treatment of malignant whom the tumor. Therapeutic compositions can be entered in therapeutically effective dosages alone or in combination with adjuvant therapy of malignant tumors, such as surgery, chemotherapy, radiotherapy, thermotherapy and laser therapy, and they can provide a beneficial effect, for example, reduction in tumor size, a reduction in the rate of tumor growth, inhibition of metastasis, or other improvement in overall clinical condition, not necessarily eliminating malignant tumor.
The composition can also be entered in therapeutically effective amounts as part of a "cocktail" against malignant tumors. "Cocktail" against malignant tumors is a mixture of the polypeptide or modulator according to the invention with one or more drugs against malignant tumors in addition to the pharmaceutically acceptable carrier for delivery. The use of "cocktails" against malignant tumors as a tool for the treatment of malignant tumors is generally accepted. Drugs against malignant tumors, which are well known in the field and which can be used for treatment in combination with the polypeptide or modulator of the invention include: actinomycin D, aminoglutetimid, asparaginase, bleomycin, osullivan, carboplatin, carmustine, chlorambucil, cisplatin (cis-DDP), cyclophosphamide, cytarabine HCl (citizenoriented), dacarbazine, dactinomycin, daunorubicin HCl, doxorubicin HCl, estramustine sodium phosphate, etoposide (V16-213), floxuridine, 5-fluorouracil (5-Fu), flutamide, hydroxyurea (hydroxycarbamide), ifosfamide, interferon Alfa-2a, interferon Alfa-2b, leuprolide acetate (similar to releasing factor LHRH), lomustin, mechlorethamine HCl (nitrogen mustard), melphalan, mercaptopurine, mesna, methotrexate (MTX), mitomycin, mitoxantrone HCl, octreotide, plicamycin, procarbazine HCl, streptozocin, tamoxifen citrate, tioguanin, thiotepa, vinblastine sulfate, vincristine sulfate, amsacrine, azacytidine, hexamethylmelamine, interleukin-2, mitoguazone, pentostatin, semustine, teniposide and vindesine sulfate.
In addition, therapeutic compositions according to the invention can be used for prophylactic treatment of malignant tumors. In this area known genetic conditions and/or situations in the environment (for example, exposure to carcinogens)that predispose individuals to the development of malignant tumors. In these circumstances, it may be useful to treatment these individuals therapeutically effective dose of the polypeptide according to the invention to reduce the risk of development of malignant tumors.
The in vitro model, you can use the La to determine the effective dose of the polypeptide according to the invention as a potential treatment for malignant tumors. These models in vitro include analyses of proliferation of cultured tumor cells, growth of cultured tumor cells in soft agar (see Freshney, (1987) Culture of Animal Cells: A Manual of Basic Technique, Wily-Uss, New York, NY Ch 18 and Ch 21), tumor systems in nude mice, as described in Giovanella et al., J. Natl. Can. Inst, 52: 921-30 (1974), motility and invasive potential of tumor cells in the analysis Boyden Chamber as described in Pilkington et al., Anticancer Res., 17: 4107-9 (1997), and analyses of angiogenesis, such as induction of vascularization chorioallantoic membrane chicken or induction of migration of vascular endothelial cells, as described in Ribatta et al., Intl. J. Dev. Biol., 40: 1189-97 (1999) and Li et al., Clin. Exp. Metastasis, 17: 423-9 (1999), respectively. Suitable tumor cell lines are available, for example, from the catalogues of the American Type Tissue Culture Collection.
Leukemia and related disorders can be treated or prevented by the introduction of a drug that stimulates or inhibits the function of polynucleotides and/or polypeptides according to the invention. Such leukemias and related disorders include, but are not limited to, acute leukemia, acute lymphocytic leukemia, acute military leukemia, myeloblastic, promyelocytic, myelomonocytic, monocytic leukemia, erythroleucus, chronic leukemia, chronic miliitary (granulocytic) leukemia and chronic lymphocytic leukemia (for an overview of such n the violations, see Fishman et al., 1985, Medicine, 2d Ed., J. B. Lippincott Co., Philadelphia).
The following aspect of the invention relates to a peptide molecule according to the invention or the nucleic acid molecule according to the invention for use to alleviate pain.
The following aspect of the invention relates to a pharmaceutical composition which is intended for use to alleviate pain. Preferred pharmaceutical compositions according to the invention described above and in the accompanying examples.
The following aspect of the invention relates to the use of the peptide molecule according to the invention or nucleic acid molecules according to the invention for the manufacture of drugs to alleviate pain.
"Application for easing pain" includes any introduction, which affects the pain which is felt by the individual, and this effect involves the slow emergence, reducing the severity, reducing the duration and/or eliminate the sensation of pain (and/or analgesia and/or hyperalgesia) the patient-the person or patient is an animal. "Pain" also includes analgesia and/or hyperalgesia - "hyperalgesia" includes an earlier appearance, increase the weight, increase the duration and/or increased sensitivity to pain.
In a preferred embodiment, the drug further includes at least one additive to the method is twolane or enhance the effects of the molecules of the peptide or nucleic acid molecule. Typically, the additive is chosen from at least one of the of paracetamol, aspirin, ibuprofen, and other nonsteroidal anti-inflammatory drugs (NSAID), selective inhibitors of cyclooxygenase-2 (CSI), opiates.
"Additive" includes ingredient, which provide in addition to the main medicinal product and which is pharmacologically active, either independently or in conjunction with the main drug, where its presence in the medicine helps or enhances the action of the main drugs.
Preferably, the drug provides a long-term or slow easing of pain.
Mostly, when applying the invention, the daily level of dosage may range from 0.0001 to 100,000 mg, administered once or divided doses; preferably daily level of dosing from 0.0001 to 1000 mg
Preferred pharmaceutical compositions include compositions in which the active ingredient is present in amounts of at least 1% (e.g. at least 10%, preferably at least 30% and most preferably at least 50%) by weight. Thus, the ratio of the active ingredient and other components (i.e. adding adjuvant, diluent and carrier) in pharmaceutical the second composition is at least 1:99 (for example at least 10:90, preferably at least 30:70 and most preferably at least 50:50) by weight.
Typically, the time between the introduction of the dose to the patient is between six and twelve hours; in a preferred embodiment, the time between dose to the patient is between nine and twelve hours after the previous dose; more preferably the time between the dose to the patient is between twelve hours and twelve days; even more preferably the time between the dose to the patient is between twelve days and six months.
In a preferred embodiment, the invention relates to the application, where the medicinal product according to the invention are used to alleviate pain in the patient-the person or patient is an animal.
Preferably the pharmaceutical composition or the medicinal agent according to the invention is made so as to ensure the introduction of at least one method selected from the group consisting of, or consisting of the following: intranasal; oral; parenteral; local; eye way; by using a suppository, pessary, or way of inhalation. Formulations suitable for such techniques, well known to experts in the field of pharmaceuticals and medicine, and illustrative of the compositions described above and in prilagaemyi.
Preferably pain selected from the group consisting of, or consisting of the following: back pain; headache; toothache; ear pain; arthritis; gout; soft tissue injury; traumatic injury of the ligaments and/or tendons; bone fractures; malignant tumor; postoperative pain; menstrual pain; the pain of childbirth, the pain of the urinary tract, pain in the internal organs; burns; abscesses and other infections.
The following aspect of the invention relates to a method of alleviating pain in a patient, comprising the stage of introduction to a patient in need, an effective amount of a molecule of peptide according to the invention and/or nucleic acid molecules according to the invention and/or pharmaceutical compositions according to the invention.
The following aspect of the invention relates to the use of peptide molecules according to the invention and/or nucleic acid molecules according to the invention as an adjuvant.
The following aspect of the invention relates to adjuvant system containing (i) a molecule of peptide according to the invention and/or the nucleic acid molecule according to the invention and (ii) the antigen.
Preferably the antigen is selected from the group consisting of, or consisting of the following: antigen anthrax; antigen cholera; diphtheria antigen; antigen Haemophilus influenza b (Hib); hepatitis be antigen A, antigen of hepatitis B; influenza antigen; na is the Egan Japanese encephalitis; antigen measles, mumps and rubella (MMR); meningococcal antigen; pertussis antigen; pneumococcal antigen; antigen poliomyelitis; rabies antigen; antigen rubella; smallpox antigen and/or ospowiki; antigen tetanus; antigen tick-borne encephalitis; antigen tuberculosis; typhoid antigen; antigen varicella/herpes simplex virus; yellow fever antigen and antigen vaccines.
The following aspect of the invention relates to a method of stimulating the production of cytokines in the cell, including the stage of introduction of the molecules of the peptide according to the invention or nucleic acid molecules according to the invention.
Preferably cytokine production increases at least 10-fold relative to normal levels. Normal levels for a particular cell type can be easily determined using a control sample which was not subjected to the polypeptide or polynucleotide according to the invention in any of the analyses discussed below, or as described in the examples.
The polypeptide of the present invention may be activity associated with cytokine, cell proliferation (either induction or inhibiting) or cell differentiation (either induction or inhibition), or may induce production of other cytokines in certain populations of cells. Polynucleotide according to the invention may code the encoded polypeptide, with such properties. Many protein factors that are open to the present time, including all known cytokines, have shown activity in one or more factor-dependent analyses of cell proliferation, and thus, these analyses serve as a convenient confirmation of the activity of cytokines. The activity of therapeutic compositions of the present invention determines one of the many conventional analyses of the factor-dependent cell proliferation for cell lines including, but not limited to, 32D, DA2, DA1G, T10, B9, B9/11, BaF3, MC9/G, M+(preB M+), 2E8, RB5, DA1, 123, T1165, HT2, CTLL2, TF-1, Mo7e, CMK, HUVEC and Caco.
Analyses of proliferation of T-cells or thymocytes include, but are not limited to, the assays described in: Current Protocols in Immunology, Ed by J. E. Coligan et al., Pub. Greene Publishing Associates and Wiley-lnterscience (Chapter 3, In vitro assays for Mouse Lymphocyte Function 3.1-3.19; Chapter 7, Immunologic studies in Humans); Takai et al., J. Immunol. 137: 3494-3500, 1986; Bertagnolli et al., J. Immunol. 145: 1706-1712, 1990; Bertagnolli et al., Cellular Immunology 133: 327-341, 1991; Bertagnolli et al., J. Immunol. 149: 3778-3783, 1992; Bowman et al., J. Immunol. 152: 1756-1761, 1994.
Analyses of cytokine production and/or proliferation of spleen cells, cells of the lymph nodes or thymocytes include, but are not limited to, the assays described in: Polyclonal T cell stimulation, Kruisbeek, A. M. and Shevach, E. M., Current Protocols in Immunology. J. E. Coligan eds. Vol 1 pp. 3.12.1-3.12.14, John Wiley and Sons, Toronto. 1994; and Measurement of mouse and human interleukin-γ, Schreiber, R. D. Current Protocols in Immunolog. J. E. Coligan eds. Vo1 1 pp. 6.8.1-6.8.8, John Wiley and Sons, Toronto, 1994.
Analysis of proliferation and differentiation of hematopoietic and lymphopoietic cells include, but are not limited to, the assays described in: Measurement of Human and Murine Interleukin 2 and Interleukin 4, Bottomly, K., Davis, L. S. and Lipsky, P. E. Current Protocols in Immunology. J. E. Coligan eds. Vol. 1 pp. 6.3.1-6.3.12, John Wiley and Sons, Toronto. 1991; deVries et al., J. Exp. Med. 173: 1205-1211, 1991; Moreau et al., Nature 336: 690-692, 1988; Greenberger et al., Proc. Natl. Acad. Sci. USA 80: 2931-2938, 1983; Measurement of mouse and human Interleukin 6-Nordan, R. Current Protocols in Immunology. J. E. Coligan eds. Vol. 1 pp. 6.6.1-6.6.5, John Wiley and Sons, Toronto. 1991; Smith et al., Proc. Natl. Acad. Sci. U.S.A. 83: 1857-1861, 1986; Measurement of human Interleukin 11-Bennett, F., Giannotti, J., Clark, S. C. and Turner, K. J. Current Protocols in Immunology. J. E. Coligan eds. Vol. 1 pp. 6.15.1 John Wiley and Sons, Toronto. 1991; Measurement of mouse and human Interleukin 9-Ciarletta, A., Giannotti, J., Clark, S. C. and Turner, K. J. Current Protocols in Immunology. J. E. Coligan eds. Vo1 1 pp. 6.13.1, John Wiley and Sons, Toronto. 1991.
Analyses of T-cell responses clones to antigens (which will identify, among others, proteins that affect APC interaction of T-cells, as well as direct effects of T-cells by measuring proliferation and cytokine production) include, but are not limited to, the assays described in: Current Protocols in Immunology, Ed by J. E. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach, W. Strober, Pub. Greene Publishing Associates and Wiley-Interscience (Chapter 3, In vitro assays for Mouse Lymphocyte Function; Chapter 6, Cytokines and their cellular receptors; Chapter 7, Immunologic studies in Humans); Weinberger et al., Proc. Natl. Acad. Sci. USA 77: 6091-6095, 1980; Weinberger et al., Eur. J. Immun. 11: 405-411, 1981; Takai et al., J. Immunol. 137: 3494-3500, 1986; Takai et al., J.Immunol. 140: 508-512, 1988.
In a preferred embodiment, the cytokine is selected from the group consisting of, or consisting of: IL-1β; IL-2; IL-6; IL-8; IL-10; IL-12; TNF-α; interferon-γ; GM-CSF.
In addition, the invention relates to a method for assessing the presence and/or quantity of the sample molecule peptide according to the invention or nucleic acid molecules according to the invention, which includes stages or consisting of them:
i) providing a test sample;
ii) contacting the test sample with the cage;
iii) measurement and/or detection of the level of production of one or more cytokines cage;
iv) comparing the level of production of one or more cytokines stage (iii) with the level of production of one or more cytokines in the control sample, where a higher level of production of one or more cytokines induced in the test sample compared to a control sample indicates the presence and/or amount in the test sample molecules peptide according to the invention or nucleic acid molecules according to the invention.
The terms "MT60.1", "Mtcpn60.1", "cpn60.1", "60.1" and "chaperonin 60.1" used in the description interchangeably to refer to the amino acid sequence represented in figure 25.
The listing or discussion it is clear previously published document in this description is not necessarily perceived as PR is knowing, the document is in part a claims-this field or is common General knowledge.
Preferred non-limiting examples, which are the implementation of some aspects of the invention, further described using the following figures:
Figure 1. The involvement of eosinophils in the Airways after 24 h after repeated load inhibited ovalbumin in mice which were injected Cpn60.1, Cpn10, but not Cpn60.2. Vertical lines represent standard error of the mean (SEM) for 4-12 (Cpn60.1), 3-5 (Cpn60.2), 4-10 (Cpn10) animals per group. *Significant reduction in the percentage of eosinophils compared with ovalbumin separately.
Figure 2. Increased bronchial atachement on metafolin 24 h after repeated load ovalbumin was reduced in mice that were injected Cpn60.1, Cpn10, but not Cpn60.2 (n=16-17). Vertical bars represent SEM.
Figure 3. The levels of cytokines in BAL fluid 24 h after repeated load inhibited ovalbumin in mice which were injected Cpn60.1. Vertical bars represent SEM. *Significant decrease in levels of cytokines compared with ovalbumin alone (n=8-10).
Figure 4. The migration of dendritic cells, pre-treated Cpn10 (10 μg/ml) in vitro, OVA sensitized recipient mice significantly inhibited the migration of eosinophils into the lung. VERTIC is further bars represent SEM. *A significant decrease in the number of eosinophils compared to sensitized mice, which were instillation raw DC (n=9-15).
Figure 5. Dendritic cells (DC)treated Cpn60.1 within 48 hours, IL12 produced dose-dependent manner. Pre-processing dendritic cells Cpn10 (10 µg/ml) induced low levels of IL12. Cells stimulated by chaperonine within 48 h and tested in quadruplicate, and each column presents the average of two experiments. Vertical bars represent standard deviation (SD).
Figure 6. Pre-incubation of dendritic cells with Cpn60.1 within 24 hours inhibits IL12 production induced by LPS. Cells stimulated by chaperonine for 248 h and LPS over the next 24 hours at All concentrations tested in quadruplicate, and each column presents the average of two experiments. Vertical bars represent SD.
Figure 7. Pre-processing dendritic cells Cpn60.1 or Cpn10 (10 µg/ml) reduced the levels of IL4 in coculture dendritic cells and T-cells RD11.10. IL5 and IL10 was not detected in this culture. Dendritic cells stimulated by chaperonine within 24 h before cocultivation their cells DO.11. Coculture maintained for 6 days and cells stimulated with antibody against CD3 and the antibody is against CD28 in the next 24 hours. Supernatant collected and tested in four copies. Vertical bars represent SD.
Figure 8. Pretreatment of spleen cells of mice C57B1/6 different concentrations of Cpn60.1 and its peptides induces the release of IL-12 in vitro. Medium containing 5 μg/ml polymyxin b b Cells stimulated by chaperonine within 24 h and supernatant tested in four copies. Each line corresponds to the average value for the two experiments.
Figure 9. The spleen cells collected from mice and TLR4 KO showed a reduced response to chaperonin 60.1 and its peptides compared to similar mice wild-type C57b1/6. Mouse TLR2 KO showed inhibition of the response to chaperonin. All concentrations were tested in four copies. Vertical bars represent SD.
Figure 10. The involvement of the total cells in the respiratory tract after 24 h after repeated load inhibited ovalbumin in mice which were injected Cpn60 and Cpn60.1-peptide 4. Vertical bars represent SEM for 5-9 animals per group. *A significant decrease in the number of total cells compared to simulating group. **A significant decrease in the number of total cells compared with ovalbumin separately.
Figure 11. The involvement of eosinophils in the Airways after 24 h after repeated load inhibited ovalbumin in mice, which enter and Cpn60 and Cpn60.1-peptide 4. Vertical bars represent SEM for 5-9 animals per group. *A significant decrease in the number of total cells compared to simulating group. **A significant decrease in the number of total cells compared with ovalbumin separately.
Figure 12. The levels of IL-5 in BAL fluid 24 h after repeated load ovalbumin not inhibited at significant levels in mice which were injected Cpn60.1 or Cpn60.1-peptide 4. Vertical bars represent SEM.
Figure 13. The levels of total IgE circulating in the serum 24 h after repeated load ovalbumin, not inhibited at significant levels in mice which were injected Cpn60.1-peptide 4 (0,005 µg/mouse). Vertical bars represent SEM. **Significantly increased levels of total IgE compared with ovalbumin separately.
Figure 14. The effect of Cpn60.1 on mechanical hyperalgesia in rats, which were injected with CFA (n=6).
Figure 15. The effect of Cpn60.1 on thermal hyperalgesia in rats, which were injected with CFA (n=6).
Figure 16. The effect of Cpn60.1 on mechanical hyperalgesia in rats that were treated with UV-radiation (n=6).
Figure 17. The effect of Cpn60.1 on thermal hyperalgesia in rats that were treated with UV-radiation (n=6).
Figure 18. Pre-treatment of monocytes in human peripheral blood Cpn60.1 suppresses LPS induced secretion of TNF-alpha. The data demonstrate the weakening of the secretion of TNF-α in old age the years of concentration Cpn60.1. Maximum inhibition at a concentration of 1 ng/ml was not significant. Data represent mean ± SEM (n=5).
Figure 19. Pre-treatment of monocytes in human peripheral blood Cpn60.1 suppresses LPS induced secretion of TNF-alpha in the presence of polymyxin b-B. the Data show a weakening of the secretion of TNF-α with increasing concentrations of Cpn60.1. Maximum inhibition was concentration of 1 ng/ml Data represent mean ± SEM (n=5).
Figure 20. Pre-treatment of monocytes in human peripheral blood Cpn10 not suppress LPS induced secretion of TNF-alpha. Cpn10 had no effect on the inhibition of the secretion of TNF-alpha at any concentration compared to LPS. Data represent mean ± SEM (n=5).
Figure 21. Pre-treatment of monocytes in human peripheral blood Cpn60.1 or peptides Cpn60.1 inhibits the release of TNF-alpha induced by LPS. Data are provided as mean values of two separate experiments in triplicate, expressed as percentage of control values.
Figure 22. The total number of neutrophils isolated from lavage of mice loaded with increasing concentrations of LPS (1 µg/ml) relative to the saline solution, after 24 hours (n=4).
Figure 23. The total number of neutrophils isolated from love the MS mice, which were administered LPS (1 μg/ml), and mice, which were injected Cpn60.1, after 24 hours (n=4).
Figure 24. The total number of neutrophils isolated from lavage of mice loaded with increasing concentrations of LPS (1 µg/ml) relative to the saline solution, after 24 hours (n=4). In animals that were injected Cpn60.1, was reduced LPS induced neutrophilia. The inhibition was significant compared with the group that was administered LPS (*p<0,05).
Figure 25. Peptides spanning the Equatorial domain of Mycobacterium tuberculosis Cpn60.1. The Equatorial domain is marked by a solid black lines and the arrows above the sequence.
Peptides 1, 2, 5, 7, 8, 9, 10 are highly exposed on the surface and/or represent a high dissimilarity between Cpn60.1 and Cpn60.2, and thus, they should be the most interesting peptides. Peptides 3 and 9 are exposed on the surface, but in the direction of the inner surface of the alleged oligomeric structure, and are highly interesting for research. Peptides 4 and 6 are partially submerged in the structure and may represent a relatively lower potential interest.
The objective of the experiments of the authors of the present invention was to study the anti-inflammatory properties of chaperonins in the model allerg the ical inflammation. The results extend previous data of the authors of the present invention, showing that Cpn60.1 and Cpn10 inhibit allergic inflammatory responses in vivo, and at the cellular level can inhibit the function of dendritic cells, which may explain its anti-inflammatory properties of these molecules in vivo.
Structural determinants of this anti-inflammatory properties Cpn60.1 is not yet established. Thus, different peptide sequence Cpn60.1 investigated in relation to any potential anti-inflammatory properties in vitro and in vivo. It turned out that a number of these peptides has immunomodulatory activity in vitro and anti-inflammatory properties in vivo, indicating that the structural determinants of activity in chaperoning, answer this question from the point of view of the discovery of new anti-inflammatory drugs.
Example 1 - Effect of Cpn60.1 and 10 in a model of allergic inflammation in mice:
Allergic diseases, including asthma, consider associated with excessive activity of Th2-lymphocytes, and, thus, the strategy designed for the induction of immune deviation, can be used to suppress allogeneic inflammation in asthma. It has been shown that M. tuberculosis and vaccine M. bovis BCG, which antigen is highly similar to Mycobacterium tuberculosis (Harboe M et al. Scand J. Immunol. 197; 9: 115-24), can suppress allergic inflammation of the lungs. Data for Helperby Therapeutics confirmed that M. tuberculosis has three GroEL, namely 60.1, 60.2 and 10. Moreover, the authors of the present invention previously demonstrated that Cpn10 and 60.1, but not 60.2, inhibit inflammation of the lung in a model of asthma in mice (Riffo-Vasquez, Y. et al., Clin. Exp. Allergy, 2004, 34: 712-19).
Allergic inflammation of the lungs induce 2 intraperitoneal injections of 10 μg of ovalbumin (OVA) in 1 mg of alum with an interval between them, amounting to 7 days. Control animals injected only alum. On the 14th day of all animals exposed to OVA aerosol for 25 minutes, once a day for 3 consecutive days. All the measurements were carried out 24 h after the last antigen load.
The authors of the present invention showed that intratracheal injection of 10 µg Cpn60.1 or Cpn10, for 24 h before the second injection of OVA and one hour before each load antigen, can significantly inhibit the inflammation of the Airways (figure 1), the excess atachement (figure 2) and the release of cytokines in the lung (figure 3). However, the mechanism by which these chaperonin have this effect in vivo is not yet installed.
Example 2 - the Effect of Cpn10 and Cpn60.1 on the function of dendritic cells mouse
It has been shown that heat shock proteins (hsp) mycobacteria are able to enhance antigen processing and presentation by dendritic cells (DC) exogenous proteins to T cells without the need for complex formation between hsp and protein (Chen K., et al., J. Leuk. Biol., 2004, 75: 1-7).
To clarify the mechanism by which Cpn10 and 60.1 regulate allergic inflammation of the lung in vivo, the authors of the present invention investigated the effect of these chaperonins on the function of dendritic cells from bone marrow in vitro.
The bone marrow cells of Balb/c mice were collected and cultured for 6 days in complete modified method of Dulbecco environment Needle containing 8 ng/ml GM-CSF mouse. On the 6th day of cultivation, the cells were collected, re-sown for 1 hour to remove attached cells were washed and were sown in the number 2x106cells/well in 6-well plates to analyze the expression of surface markers and experiments on transfer in the amount of 105cells/well in 96-well plates for determination of cytokines. In other experiments cells were incubated in 106cells/well for 24 h with chaperonine, and then another 24 h with LPS (10 ng/ml).
DC pre-treated in vitro for 24 h by Cpn10 (10 μg/ml), and then locally injected into the lungs OVA sensitized recipient mice. This treatment significantly inhibited the migration of eosinophils into the lung (figure 4), reproducing the effect observed in vivo with chaperoning.
Pre-treatment of DC in vitro through the Cpn60.1 (0.1 to 30 μg/ml) induced the release of IL-12 dose-dependent manner. However, pre-treatment of DC through Cpn10 (10 µg/ml) induced lower levels of release of IL-12 compared with LPS and Cpn60.1 (figure 5).
Pre-incubation of DC with Cpn60.1, but not with Cpn10, within 24 hours inhibited the production of IL12 induced by LPS (figure 6).
DC pre-treated by Cpn60.1 (10 μg/ml) or Cpn10 (10 μg/ml) for 24 h inhibited the release of IL4 T-cells RD11.10 in coculture DC/T cells. After culturing for 6 days, the cells were stimulated with antibody against CD3 and antibody against CD28 in the next 24 hours and supernatant collected. IL5 and IL10 was not detected in the culture (figure 7).
Example 3 - Effect of peptides chaperonin 60.1 in vitro
To identify the structural determinants chaperonin 60.1 responsible for studying biological effects, the authors of the present invention examined the effects of 5 different peptide sequences, designated as peptide 1, 2, 3, 4, and 6 in various biological assays in vitro in this study.
Party Cpn60.1 used in these experiments: Cpn60.1 1_04-03/1.
The authors of the present invention investigated the effect of these peptides in the culture of spleen cells. The spleen cells were obtained from mice C57B1/6 or from mice, TLR4 and TLR2 KO. The bodies were soaked in sterile conditions, and the cell suspension skipping the if through a sieve with a mesh size of 70 μm. Erythrocytes were removed by osmotic shock using sterile water. The remaining cells were washed and were sown in quantity of 106/hole for measurement of IL-12. Cells were incubated with various concentrations of peptides or chaperonin 60.1 (0.001 to 30 mg/ml). Supernatant collected after 24 h and stored at -20°C. IL-12 was measured by conventional ELISA.
Experimental observations of the authors of the present invention showed that Cpn60.1 and all peptides induce the release of IL-12 in the culture of splenocytes. Peptide 4 is more effective than Cpn60.1, at lower concentrations (0.001 to 0.1 ág/ml). However, at higher concentrations (0.1 to 30 μg/ml), this peptide showed a reduced effect compared to the Cpn60.1. Peptide 6 has a similar effect at higher concentrations compared with Cpn60, however, has a greater effect at lower concentrations (figure 8). All culturing was performed in the presence of polymyxin b B to exclude any effect of LPS contamination on the functional responses that were defined.
In addition, the authors present invention additionally investigated the role of toll-receptor (TLR) response of spleen cells to these peptide sequences. The spleen cells obtained from mice and TLR4 KO, were largely refractory in terms of their ability to produce IL-12 in response to shapero is in 60.1 and its peptides compared to similar mice wild-type C57b1/6. Spleen cells taken from mice TLR2 KO were also refractory on their ability to produce IL-12 in response to chaperonin and its peptides.
This in vitro study demonstrates that the biological activity of these peptide fragments is mediated by the activation of TLR2 and TLR4 (figure 9).
Example 4 - Effect of peptides of chaperonins in vivo
Next, the authors present invention investigated the effect of low concentrations of Cpn60.1 and Cpn60.1-peptide 4 on allergic airway inflammation in light of the results obtained from in vitro studies, as described earlier.
For these experiments, the authors present invention slightly modified their Protocol of immunization. Allergic inflammation in the lung induce 2 by intranasal instillation of 10 μg of ovalbumin (OVA) in 100 μg of alum with an interval of 7 days between them. Control animals were injected only alum. At 14 days, all animals were subjected to OVA aerosol for 25 minutes, once a day for 3 consecutive days. All the measurements were carried out 24 h after the last antigen load.
The authors of the present invention showed that intranasal introduction Cpn60.1 (1 µg) significantly inhibited the total number of cells involved in lungs after acute loading with antigen (figure 10). The effect of Cpn60.1 and peptide 4 on the involvement of eosinophils in the airway b is l more pronounced (figure 11). As Cpn60.1 low dose (0.1 and 1 µg)and peptide 4 (0,005 µg) caused a significant reduction in the attraction of eosinophils, which was not caused by an obstruction in the production of IL-5 from inflammatory cells (figure 12). Interestingly, low doses of peptide 4 was influenced on the production of total IgE in this model, allergies (figure 13).
Example 5 - Cpn60.1 in models of pain
The effect of Cpn60.1 investigated in 2 models of pain: induced complete adjuvant's adjuvant (CFA) and induced hyperalgesia burn ultraviolet radiation hyperalgesia. Mechanical hyperalgesia was measured using yarns Von Frey applied with increasing force to the rear paw. Thermal hyperalgesia was measured using the time that took OTDELENIE hind legs from the heat source. Cpn60.1 (50 and 500 mg/kg) long was administered to male Wistar rats (200 g) subcutaneously for 7 days. Times in two days was recorded behavioral responses to mechanical and thermal allodynia. After registration of the original levels, a rear leg were injected with CFA for the induction of hyperalgesia and conducted behavioral tests. No dose Cpn60.1 is not reduced CFA induced hyperalgesia compared with control, ibuprofen (100 mg/kg) (figures 14 and 15). Tested another model of hyperalgesia, this time using UV-burn for the induction of allodynia. Again, Cpn60.1 had a small e the effect of inhibition of hyperalgesia compared with group, which was introduced ibuprofen (figures 16 and 17).
Example 6 - in vitro Studies on inflammatory cells
The authors of the present invention have developed the analysis of human monocytes to compare the effectiveness of Cpn60.1 and various peptide sequences of this protein. The monocytes were obtained dependent on density centrifugation from peripheral venous blood from healthy volunteers. Then monocytes were isolated and purified using cells, brought to a density of 2×106cells/ml, which were incubated for 1 hour 30 minutes. Then the tablets were washed twice with Hanks solution. Then to each well was added 0.9 ml of RPMI medium, and then polymyxin-B (5 µg/ml). After 30 minutes, the wells were added 0.1 ml of Cpn in the amount of 0.0001-1000 ng/ml or peptides. The cells are then incubated at 37°C, 5% CO2within 1 hour. The medium was removed and cells were washed. To each well was added to 0.9 ml of medium, and then 0.1 ml of LPS (1 ng/ml) and left to incubated for 24 hours. Supernatant was collected from each well and frozen for enzyme-linked immunosorbent assay (ELISA).
The authors of the present invention showed that Cpn60.1 separately can stimulate human monocytes to induce secretion of TNF-alpha, but adding to the already applied inflammatory stimulus (i.e. in the presence of LPS), Cpn60.1 inhibited the secretion of TNF-al is in low doses (1 ng/ml) (figure 18). On the contrary, Cpn10 not inhibited the secretion of TNF-alpha from human monocytes (figure 20). However, squirrels, apparently, had some degree of contamination with LPS, in the presence of polymyxin b B this contamination has decreased (figure 19). This potential Association with LPS contamination was avoided by using peptide sequences Cpn60.1. It was found that the peptides also inhibit the secretion of TNF-alpha at low concentrations (figure 21).
Example 7 - Cpn60.1 models non-allergic inflammation
Due to the inhibitory effect on inflammatory cells, the authors of the present invention investigated whether Cpn60.1 to inhibit the attraction of neutrophils in vivo. To further confirm this, Riffo-Vasquez et al. have recently shown that chaperones M. tuberculosis can inhibit the attraction of eosinophils and excess atachement bronchi in a model of allergic inflammation in mice (Riffo-Vasquez y, Spina D., Page, C. et al. Effect of Mycobacterium tuberculosis chaperonins on bronchial eosinophilia and hyper-responsiveness in a murine model of allergic inflammation. Clin. Exp Allergy, 2004; 34(5); 712-719).
Female Balb/c mice previously administered intranasally Cpn60.1 (1 μg/ml) for 3 days in the absence of pre-treatment with polymyxin B or after pre-treatment with polymyxin B (5 µg/ml) to exclude any effect of LPS contamination. 30 minutes after the last dose Cpn60.1 mice enter the whether LPS (1 μg/ml). The influx of neutrophils and the release of TNF-alpha in the lungs were determined using bronchoalveolar lavage 24 hours. LPS induced a dose-dependent increase of neutrophilia compared with control (figure 22). Interestingly, the group, which had previously been introduced Cpn60.1, showed suppression of neutrophilia, however, the secretion of TNF-alpha was not significantly weakened (figure 24). Administration to mice of polymyxin b B did not change the ability of Cpn60.1 to suppress the attraction of neutrophils, which indicates that this effect cannot be explained by LPS contamination. In mice which were injected Cpn60.1 separately, were not observed attract neutrophils, which then allows us to assume that no LPS contamination does not cause neutrophilia (figure 23).
Example 8 is an Illustrative pharmaceutical compositions
Although you can type molecule according to the invention separately, it is preferable to present it as a pharmaceutical composition together with one or more acceptable carriers. The carrier(s) must be "acceptable" from the point of view that it should be compatible with the tool according to the invention and is not harmful to the recipient. Typically, the media are water or saline solution that is sterile and free of pyrogens.
The examples below illustrate medicines, pharmaceutical and the definition of the composition according to the invention, in which the active ingredient is a molecule according to the invention.
Preferably the molecule according to the invention is provided in an amount of from 10 μg to 500 mg Clear that it is possible to obtain the following illustrative medicines and pharmaceutical compositions containing the number of molecules according to the invention, comprising from 10 μg to 500 mg, for Example, a molecule according to the invention can be present in 10's or 100's or 200's or 500's shares from the number,presented in the following illustrative drug and pharmaceutical compositions and amounts of the other ingredients change accordingly.
Example a: the Pill
|The active ingredient||1 mg|
|Magnesium stearate||4 mg|
Tablets produced from the above ingredients by wet granulation followed by compression.
Example B: Ophthalmic solution
|The active ingredient||1 mg|
|Sodium chloride, chemically pure||0.9 g|
|Purified water to||100 ml|
|the pH value is brought to||7,5|
Example C: Formulations tablets
The following compositions A and B get wet granulation of the ingredients with a solution of povidone, followed by addition of magnesium stearate and pressed.
|mg tablet||mg tablet|
|(a) Active ingredient||1||1|
|(b) Lactose B.P.||210||26|
|(c) Povidone B.P.||15||9|
|(d) Sodium starch glycolate||20||12|
|(e) Article is Arat magnesium||5||3|
|mg tablet||mg tablet|
|(a) Active ingredient||1||1|
|(C) Avicel PH 101 ®||60||26|
|(d) Povidone B.P.||15||9|
|(e) Sodium starch glycolate||20||12|
|(f) magnesium Stearate||5||3|
The composition of
|The active ingredient||1|
The following formulations D and E, obtained by direct pressing of the mixed ingredients. Lactose, used as part of E represents the lactose for direct compression.
|The active ingredient||1|
|Pregelatinization starch NF15||150|
|The active ingredient||1|
The composition F (controlled release)
This part get wet granulation of the ingredients (below) with a solution of povidone followed by the addition of magnesium stearate and pressed.
|(a) Active ingredient||1|
|(b) Hypromellose (Methocel K4M Premium)®||112|
|(C) Lactose B.P.||53|
|(d) Povidone VRS||28|
|(e) magnesium Stearate||7|
The release of lcars the governmental funds occurs within a period of approximately 6-8 hours, and it ends in 12 hours.
Example D: Formulations capsules
The capsule is produced by mixing the ingredients of the composition D according to example C, above, and filling their gelatin hard capsule of two parts. Part B (below) are obtained in a similar manner.
|(a) Active ingredient||1|
|(b) Lactose B.P.||143|
|(C) Sodium starch glycolate||25|
|(d) magnesium Stearate||2|
|(a) Active ingredient||1|
|(b) Macrogol 4000 BP||350|
Capsules produced by melting Macrogol 4000 BP, dispersing the active ingredient in the melt and filling the melt hard gelatin capsules of two parts.
|The active ingredient||1|
Capsules produced by dispersing the active ingredient in the lecithin and peanut oil and filling the variance of the soft elastic gelatin capsules.
Composition E (Capsule with controlled-release)
The following composition capsules controlled release is obtained by extrusion of the ingredients a, b and c, using an extruder, followed by giving the extrudate spherical shape and drying. Then the dried granules cover control release membrane (d) and fill them hard gelatin capsule of two parts.
|(a) Active ingredient||1|
|(b) Microcrystalline cellulose||125|
|(C) Lactose B.P.||125|
Example E: Injectable composition
|The active ingredient||1 mg|
|Sterile, does not contain pyrogens|
phosphate buffer (pH 7.0)
|to 10 ml|
The active ingredient is dissolved in most parts of phosphate buffer (35-40°C), then brought to volume and filtered through a sterile filter with micropores in a sterile vial of yellow glass with a volume of 10 ml (type 1), and cover with a sterile tube and additional sealing means.
Example F: Intramuscular injection
|The active ingredient||1 mg|
|Benzyl alcohol :||0.10 g|
|Glucofurol 75®||1.45 g|
|Water for injections to||3,00 ml|
The active ingredient is dissolved in glucotrole. Then add benzyl alcohol and dissolve, and add water to 3 ml of the mixture is Then filtered through a sterile filter with micropores and closed in a sterile glass vials with a volume of 3 ml (type 1).
Example G: Suspension in the form of a syrup
|The active ingredient||1 mg|
|The solution of sorbitol||1,5000 g|
|Dispersible cellulose||0,0750 g|
|Sodium benzoate||0,0050 g|
|Flavoring, peach 17.42.3169||of 0.0125 ml|
|Purified water to||5,0000 ml|
Sodium benzoate is dissolved in parts of purified water and add a solution of sorbitol. Add the active ingredient and is dispersed. In the glycerol of disperser the Ute thickener (dispersible cellulose). The two dispersions are mixed and brought to the desired volume of purified water. If necessary, further thickening reach by an additional shift efforts on suspension.
Example H: Suppository
|Active ingredient (63 μm)*||1|
|Solid lubricant, BP (Witepsol H15-Dynamit Nobel)||1700|
|*The active ingredient is used as a powder, where at least 90% of the particles have a diameter of 63 μm or less.|
One fiftieth part of the Witepsol H15 is melted in a vessel with a steam jacket and a maximum at 45°C. the Active ingredient is sifted through a 200 μm sieve and added to the molten base with mixing, using a silverson, equipped with a cutting head, to achieve a uniform dispersion. Maintaining the mixture at 45°C, in the slurry, add the remaining Witepsol H15 and stirred to ensure a homogeneous mixture. The entire suspension is passed through a 250 μm stainless steel sieve, and, when the permanent paramasivan and, allow it to cool to 40°C. At a temperature of from 38°C to 40°C 2,02 g of the mixture is filled into suitable plastic moulds. Suppositories allow to cool to room temperature.
Example I: Pessaries
|The active ingredient||1|
The above ingredients right mix and get pessaries by direct compression of the mixture.
Example 9 Treatment of non-malignant disorders with the use of a molecule according to the invention
The patient with arthritis injected 1 mg means according to the invention per day intramuscularly or by using the product of prolonged action, delivering the dose according to the methods of the invention.
Example 10 - ways of mitigating the pain
Molecules according to the invention can effectively reduce pain in the following cases the Oli: back pain, headache, toothache, ear pain, arthritis, gout, soft tissue injury, traumatic injury of the ligaments/tendons, broken bones, cancer, postoperative pain, menstrual pain, pain during labor, pain in urinary tract, pain in the internal organs, burns, abscesses and other infections.
The intended route of administration and the regime for the treatment of any of these conditions are the introduction from 0.1 mg to 1 gram once per 12 hours by inhalation, delivered through an inhaler. However, a qualified expert may know that the most suitable mode of treatment may depend on the individual and severity of perceived pain.
1. Dedicated or recombinant molecule of the peptide having the activity of inhibiting allergic airway inflammation or arthritis, comprising the amino acid sequence:
2. Molecule peptide according to claim 1 for use in medicine.
3. Molecule peptide according to claim 1, where the arthritis selected from rheumatoid arthritis or atherosclerosis, and allergic airway inflammation is selected from allergic rhinitis, allergic airway disease; syndrome hypereosinophilia; and respiratory disease characterized by eosinophilic airway inflammation and increased reactivity of dehat the selected paths, such as allergic asthma, hereditary asthma, allergic bronchopulmonary aspergillosis, eosinophilic pneumonia, allergic bronchitis bronchiectasis, occupational asthma, reactive respiratory syndrome, interstitial lung disease, hypereosinophilic syndrome, parasitic lung disease.
4. Molecule peptide according to claim 3, where the allergic inflammation is an asthma.
5. The use of molecules of the peptide according to claim 1 for the manufacture of a medicine for the prevention or treatment of allergic airway inflammation or arthritis.
6. The use according to claim 5, where the arthritis selected from rheumatoid arthritis or atherosclerosis, and allergic airway inflammation is selected from allergic rhinitis, allergic airway disease; syndrome hypereosinophilia; and respiratory disease characterized by eosinophilic airway inflammation and increased airway reactivity, such as allergic asthma, hereditary asthma, allergic bronchopulmonary aspergillosis, eosinophilic pneumonia, allergic bronchitis bronchiectasis, occupational asthma, reactive respiratory syndrome, interstitial lung disease, hypereosinophilic syndrome, parasitic lung disease.
7. The use according to claim 5, dealerlink inflammation of the respiratory tract represents the asthma.
8. The use of molecules of a peptide having the sequence (A) DGSVVVNKVSELPAGHGLNVNTLSYGDLAAD, or nucleic acid encoding the above peptide, and optionally at least one additive for the manufacture of a medicinal product for the relief of pain.
9. The use of claim 8, where the medicinal product further comprises at least one additive to facilitate or enhance the effects of the peptide molecules.
10. The use according to claim 9, where the additive is selected from at least one of the of paracetamol, aspirin, ibuprofen, and other nonsteroidal anti-inflammatory drugs (NSAID), selective inhibitors of cyclooxygenase-2 (CSI), opiates.
11. The use according to any one of p-10, where the pain is selected from the group consisting of, or consisting of the following: back pain, headache, toothache, ear pain, arthritis, gout, soft tissue injury, traumatic injury of the ligaments/tendons, broken bones, cancer, postoperative pain, menstrual pain, pain during labor, pain in urinary tract, pain in the internal organs, burns, abscesses and other infections.
12. The use of molecules of the peptide molecule is a peptide having the sequence (A) DGSVVVNKVSELPAGHGLNVNTLSYGDLAAD as adjuvant.
13. Pharmaceutical composition for use in prevention or treatment of allergic is asplenia respiratory tract, containing a therapeutic amount of molecules of a peptide having the sequence (A) DGSVVVNKVSELPAGHGLNVNTLSYGDLAAD, or nucleic acid coding thereof, and pharmaceutically acceptable excipient.
14. Pharmaceutical composition for use in prevention or treatment of arthritis, containing a therapeutic amount of molecules of a peptide having the sequence (A) DGSVVVNKVSELPAGHGLNVNTLSYGDLAAD, or nucleic acid coding thereof, and pharmaceutically acceptable excipient.
15. Pharmaceutical composition for use for relief of pain containing a therapeutic amount of molecules of a peptide having the sequence (A) DGSVVVNKVSELPAGHGLNVNTLSYGDLAAD, or nucleic acid coding thereof, and pharmaceutically acceptable excipient.
16. The pharmaceutical composition according to item 15, where the pain is selected from the group consisting of, or consisting of the following: back pain, headache, toothache, ear pain, arthritis, gout, soft tissue injury, traumatic injury of the ligaments/tendons, broken bones, cancer, postoperative pain, menstrual pain, pain during labor, pain in urinary tract, pain in the internal organs, burns, abscesses and other infections.
17. Adjuvant system containing (i) a molecule peptide having the sequence (A) DGSVVVNKVSELPAGHGLNVNTLSYGDLAAD, or nucleic acid, coderush the th, and (ii) the antigen.
18. Method of stimulating the production of IL-12 in the cell, including the use of molecules of a peptide having the sequence (A) DGSVVVNKVSELPAGHGLNVNTLSYGDLAAD, or nucleic acid that encodes it.
19. The method according to p, where the production of IL-12 increases at least 10-fold relative to normal levels.
SUBSTANCE: invention relates to microbiology and molecular genetics and pertains to a recombinant polypeptide A2, DNA coding said polypeptide, a strain which produces polypeptide A2 and methods of using such a recombinant polypeptide. The disclosed recombinant polypeptide A2 is characterised by an amino acid sequence of 346 amino acids in which the first 13 amino acids are coded by the plasmid sequence pQE 32 and are covalently bonded with the next 333 amino acids which are coded by a sequence of the HSA-binding fragment of chromosomal DNA of the strain DG 13 of streptococci of the group G-CFG.
EFFECT: group of inventions can be used in diagnosis, eg, when making a test system for determining microalbuminuria based on a laboratory criterion of the preclinical phase of diabetic nephropathy, as well as a reagent for separating human serum albumin by affinity chromatography and for freeing serum from HAS, which enables to determine other proteins present in the serum in lower concentrations.
9 cl, 11 dwg, 1 tbl, 9 ex
SUBSTANCE: inventions refer to polynucleotide sequence coding the structured pertactin protein (Prn); a vector including such a sequence and compositions containing protein or vector. The characterised polynucleotide sequence codes 300 first amino acids that are the closest to N-end of this type of natural mature Prn (PrnX300), and amino-acid sequence including 620 last amino acids that are the closest to C-end of this type of natural mature Prn (PrnY620) so that structured pertactin PrnX300-PrnY620 of Bordetella class is obtained. Structured molecules Prn include polymorphisms of different B. Pertussis strains and cause immune responses with increased protective ability and opsonophagocytic activity, which exceed the corresponding properties of the preceding vaccines.
EFFECT: protein obtained as per the presented invention can be used in medicine and veterinary as a component of antibacterial vaccines against Bordetella pertusis.
12 cl, 3 dwg, 2 tbl, 4 ex
SUBSTANCE: present inventions relate to protein engineering, plant molecular biology and pest control, as well as a hybrid insecticide protein and use thereof. Described is a hybrid insecticide protein which includes from the N-end to the C-end an N-end portion of Cry3A protein which is fused with the C-end portion of Cry1Ab protein, wherein the position of the crossover of the Cry3A protein and the Cry1Ab protein is located in a conservative block 2, in a conservative block 3 or in a conservative block 4 and has anti-western corn rootworm activity. Also disclosed are nucleic acid molecules which code the novel proteins, methods of producing proteins, methods for use thereof, as well as transgenic plants and seeds thereof which contain such proteins.
EFFECT: inventions enable to obtain cheap means of controlling Diabrotica worms.
39 cl, 8 dwg, 9 tbl, 46 ex
FIELD: medicine, pharmaceutics.
SUBSTANCE: given invention refers to medicine, biopharmaceutics, and may be used to prepare vaccines. For this purpose, the immunogenic composition contains: 1) an antigen Neisseria autotransporter protein representing NadA or Hsf; 2) an antigen Neisseria protein involved in iron absorption and representing Lipo28 or low-molecular or high-molecular TbpA; and 3) a vesicle preparation of an outer membrane containing immunotype L3 Neisseria lipopolysaccharide (LPS); and wherein the above antigens if present in the outer membrane vesicle are positively regulated in accordance with recombinant technology in the above outer membrane vesicle.
EFFECT: using the given vaccine that is a combination of the various class Neisseria antigens generates the immune response which occurs to be stronger if expressed in bactericidal units.
17 cl, 11 dwg, 21 ex
SUBSTANCE: what is presented is a nucleic acid construct for treating tumours comprising at least two open reading frames that include sequences encoding a cytotoxic or cytostatic gene product, in particular diphtheria toxin functionally related to various tumour-specific promoters: H19-specific promoter, IGF-II P3 or P4 promoter. What is described is a eukaryotic expression vector comprising the above nucleic acid construct, and methods of treating and inhibiting the tumour development in a human subject by administering the nucleic acid construct under the invention.
EFFECT: invention can find further application in therapy of cancer.
33 cl, 12 ex, 40 dwg
FIELD: medicine, pharmaceutics.
SUBSTANCE: invention refers to biotechnology, namely a method for differentiation of tularemia microbial subspecies. The invention may be used in laboratory diagnosis of tularemia. The method involves PCR amplification with the use of gene-specific iglC and chromosome regions containing E. coli-like Chi-sequences of the primers FiglC-AAGGATAAGACCTGTCTG, RiglC-TTGAAACCATACCGGGTA and Chi If CTAGG-GCTGGTGG-G. It is followed by electrophoresis of the amplification products to be differentiated by comparing electrophoretic mobility of the produced amplicons and mobility of DNA marker fragments. If observing a specific light-producing strip at the level of 986 base pairs, the strain data are recorded as Francisella gen. The strip distribution pattern within the range of ~190 to ~830 base pairs enables differentiating the analysed samples at the level of tularemia microbial subspecies: ~190 base pairs for subsp. novicida, ~500-570 base pairs for subsp. mediasiatica, ~570 base pairs for subsp. holarctica, whereas subsp. tularensis is characterized by the fragment of ~500 base pairs.
EFFECT: presented invention enables accurate, instant and high-specific method for differentiation of F tularensis subspecies.
SUBSTANCE: invention relates to recombinant plasmid pVA891-2[mutR], a method of producing mutR gene-mutant strains of Streptococcus pyogenes and recombinant strains of Streptococcus pyogenes. The plasmid pVA891-2[mutR] is constructed based on the vector pVA891-2 which is incapable of replicating in cells of gram-positive microorganisms and contains an erythromycin resistance marker gene, by introducing a Pstl-Sacl fragment of the mutR gene of the Streptococcus pyogenes SF370 strain with size of 520 bps, shown on fig.1. To obtain mutant avirulent strains of Streptococcus pyogenes (SF3 70[mutR], No.97[mutR], No.152[mutR), the obtained plasmid is introduced into corresponding strains of S. pyogenes, which results in violation of the structural region of the mutR gene in the genome of such strains. Selection of clones with an inactivated mutR gene enabled to obtain strains in which exhibition of virulent properties is absent or considerably reduced.
EFFECT: group of inventions can be used in preventive medicine when producing vaccines.
5 cl, 8 dwg
SUBSTANCE: there are presented versions of recombinant virulent polypeptides and nucleic molecules coding them with the sequences disclosed in the description, used to prepare a vaccine composition for A/E-pathogen. What is described is a vaccine composition for inducing a response on A/E-pathogen in animals containing said nucleic acid molecule; a cell culture supernatant containing said polypeptide; and/or a bacterium or its preparation having a mutation in its genome in said nucleic acid molecule inducing virulence attenuation, or having nleA gene deletion in a combination with a physiologically acceptable carrier. What is presented is a method for virulence attenuation of A/E-pathogen, involving nleA gene deletion in A/E-pathogen or deletion of one or more said nucleic acids in the A/E-pathogen genome.
EFFECT: invention provides producing the effective vaccine.
24 cl, 2 tbl, 27 dwg, 9 ex
SUBSTANCE: invention discloses RumC1, RumC2 and RumC3 peptides with antimicrobial activity, particularly on Clostridium perfringens, having molecular weight ranging from 4000 to 4600 Da and isolated from a mutant strain of Ruminococcus gnavus, deposited under number CNCM I-3705. The invention describes polynucleotides which code said peptides, an expression cassette which includes a promoter, said polynucleotide and a terminator sequence, as well as a polynucleotide-based cloning and expression vector. Described is a host organism which expresses said peptides, a composition having antimicrobial activity on Clostridium perfringens and containing a peptide, a host organism or a CNCM I-3705 strain, animal feed which is a food base and a composition.
EFFECT: use of the invention enables to produce a medicinal agent for preventing and treating diseases, particularly necrotic enteritis in pigs or poultry, and human gastrointestinal diseases.
16 cl, 9 dwg, 7 tbl
SUBSTANCE: recombinant plasmid DNA pTB323 under the invention coding the hybrid polypeptide glutathione-8-transferase (GST) and a shorter version of the protein MPT64 (rΔMPT64), has an average molecular weight 3.6 MDa, size 5574 base pairs, consists of: a) EcoRI-BamHI-fragment of the vector plasmid pGEX-2T of size 4938 base pairs containing the β-lactamase gene inducing tac-promotor, the internal gene Iaclg coding the lactose operone repressor protein, a glutathione-5-transferase gene fragment from S. japonicum with a multiple sites of gene cloning (MSC) in 3'-terminal part of this gene and a nucleotide sequence coding a thrombine proteolysis site and found in front of the MSC; b) EcoRI-BamHI-fragment of 636 base pairs containing a truncated gene MPT64 flanked by EcoRI and BamHI restriction endonuclease sites and prepared by amplification of the gene-related fragment with genome DNA M. tuberculosis; c) a genetic marker - β-lactamase gene determining resistance of pTB323 plasmid transformed cells E. coli to the antibiotic ampicillin; d) unique restriction sites: BamHI - 930/934, EcoRI ~ 1566/1570. The recombinant bacterial strain Escherichia coli BL21/pTB323 - producer of hybrid polypeptide GST-ΔMPT64 with the properties of the mycobacterial antigen ΔMPT64 is deposited in the Collection of Microorganisms of Federal State Research Institution State Science Centre Vector, No. B-1028. The recombinant polypeptide GST-ΔMPT64 produced by the recombinant strain under the invention contains as a carrier protein the N-terminal polypeptide fragment glutathione-S-transferase S.j. (226 amino acid residues, 26.31 kDa) and has a complete amino acid sequence (431 amino acid residues, 48.76 kDa) presented in the description.
EFFECT: using the invention enables developing the high-purity polypeptide in the preparation amounts with the preserved immunogenic properties and provided separation of the target protein from the amino acid sequence of the carrier protein for studying of the immunogenic properties of the target protein.
3 cl, 4 dwg, 4 tbl, 6 ex
SUBSTANCE: microorganism-producent is cultivated in a suitable nutrient medium with subsequent extraction and treatment of target protein. At the same time the producent is yeast strain Saccharomyces cerevisiae, transformed with the expression vector, which contains an area of initiation of replication of endogenic 2-mcm plasmid of yeast Saccharomyces cerevisiae, and also a yeast promotor GAL1, which controls gene expression, including a DNA sequence SEQ ID NO: 1, coding a fused protein, the composite parts of which are aminoacid sequences of the protein E7-HSP70 and the protein of ubiquitin of yeast Saccharomyces cerevisiae, occupying within the fused protein an N-end position ending with a processing site, which separates it from the sequence of the protein E7-HSP70 and recognised natural ubiquitin-specific yeast proteinases. The yeast strain Saccharomyces cerevisiae VKPM Y-3853 - producent of the protein E7-HSP70 - is produced by transformation with the expression vector pPDX3U-E7-HSP70 of the yeast strain Saccharomyces cerevisiae D702.
EFFECT: group of inventions provides for high level of biosynthesis and yield of treated protein E7-HSP70, production of a water-soluble correctly processed protein E7-HSP70, principal absence of toxic and pyrogenic bacterial LPS in preparations of the protein E7-HSP70.
2 cl, 4 dwg, 12 ex
SUBSTANCE: invention describes mutations of the amino acid sequence of the protein of atpE mycobacteria (M. tuberculosis and M. smegmatis), which determine their resistance to DARQ J. Obtained are coding mutant forms of atpE nucleic acids, vectors containing said forms and host cells expressing mutant proteins.
EFFECT: invention discloses use of said host cells in a method of identifying compounds which can be used as antimicrobial agents for mycobacteria strains resistant to existing drugs.
5 cl, 4 dwg, 7 tbl
SUBSTANCE: Amycolatopsis fructiferi subsp. ristomycini 8765 strain comes from mutant transformation of Proactinomyces fructiferi var. ristomycini 5339 strain properties as a result of consequent three-step actions on the stock strain by methylnitrosoguanidine, ultraviolet and gamma-radiation of 60Co with following selection of mutated strain possessing increased antibiotic activity. Obtained strain differs from already known one with growth form on diagnostic agar media, carbon sources assimilation rate (saccharose, starch, rhamnose, and mannose) chromogenic ability and antibiotic activity which is 5 times higher of Proactinomyces fructiferi var. ristomycini 5339 strain activity and is equal to 2000 mcg/ml. Strain is deposited to All Russian Collection of Industrial Microorganisms, collection # S1085.
EFFECT: invention can be used in medical industry for antibacterial antibiotic ristomycin manufacture; ristomycin is effective in treatment of infections induced by gram-positive microorganisms.
FIELD: chemistry; pharmacology.
SUBSTANCE: present invention pertains to biotechnology. Description is given of urease deficient cell Mycobacterium, a fused polypeptide producer, containing a recombinant nucleic acid molecule, which codes the fused polypeptide, containing a minimum of one polypeptide domain, capable of triggering an immune response in mammals, and an output domain from phagolysosomes. A pharmaceutical composition is invented, which is a live vaccine, containing the above described cell as the active agent, providing for protective immunity agains tuberculosis. A live vaccine method is proposed. A method of preparing the Mycobacterium cell described above is invented as well.
EFFECT: obtaining a safer vaccine for tuberculosis.
35 cl, 3 ex, 4 dwg
FIELD: biotechnology and gene engineering.
SUBSTANCE: invention relates to recombinant plasmide encoding hybrid GST-ESAT-6 polypeptide having activities of species-specific mycobacterial ESAT-6 antigen. Plasmide has molecular mass of 3.45 MD and size of 5315 n.p. Recombinant E.coli polypeptide containing such plasmide and recombinant GST-ESAT-6 polypeptide also are disclosed. Recombinant protein has activities of species-specific antigen protein Mycobacterium tuberculosis ESAT-6. Method of present invention makes in possible to simplify purification process of recombinant polypeptide and to produce protein of high purity hawing activities of mycobacterial ESAT-6 antigen without degradation thereof.
EFFECT: earlier species-specific diagnosis of tuberculosis infection.
3 cl, 3 dwg, 4 tbl, 5 ex
SUBSTANCE: present invention relates to organic chemistry and specifically to 5-phenyl-1H-pyrazin-2-one derivatives of general formula II or pharmaceutically acceptable salts thereof, where R denotes -R1 or - R1-R2-R3; R1 denotes aryl or heteroaryl, and is optionally substituted with one or two R1'; where each R1' independently denotes C1-6alkyl, halogen or C1-6halogenalkyl; R2 denotes -C(=O), -CH2-; R3 denotes R4; where R4 denotes an amino group or heterocycloalkyl, and is optionally substituted with one or two substitutes selected from C1-6alkyl, hydroxy group, oxo group, C1-6hydroxyalkyl, C1-6alkoxy group; Q denotes CH2; Y1 denotes C1-6alkyl; Y2 denotes Y2b; where Y2b denotes C1-6alkyl, optionally substituted with one Y2b'; where Y2b' denotes a hydroxy group, n and m are equal to 0; Y4 denotes Y4c or Y4d; where Y4c denotes lower cycloalkyl, optionally substituted with halogen; and Y4d denotes an amino group, optionally substituted with one or more C1-6alkyl; where "aryl" denotes phenyl or naphthyl, "heteroaryl" denotes a monocyclic or bicyclic radical containing 5 to 9 atoms in the ring, which contains at least one aromatic ring containing 5 to 6 atoms in the ring, with one or two N or O heteroatoms, wherein the remaining atoms in the ring are carbon atoms, under the condition that the binding point of the heteroaryl radical is in the aromatic ring, "heterocycloalkyl" denotes a monovalent saturated cyclic radical consisting of one ring containing 5 to 6 atoms in the ring, with one or two ring heteroatoms selected from N, O or SO2. The invention also relates to use of the compound of formula II or a pharmaceutical composition based on the compound of formula II.
EFFECT: obtaining novel compounds that are useful for modulating Btk activity and treating diseases associated with excessive activity of Btk.
7 cl, 2 tbl, 53 ex
SUBSTANCE: present invention relates to novel compounds of formula I: or salts thereof, where: A1 denotes hydrogen, CN, CI, F, Br, OMe, (1-4C alkyl) or cyclopropyl; A2 denotes hydrogen, Cl, Br, F, (1-4C alkyl) or cyclopropyl; W denotes -C(=O)NR1- or -NR2C(=O)-; each of R1 and R2 denotes hydrogen or methyl; L denotes a chemical bond, -(CR3R4)n-(CRaRb)m-(CR5R6)-*, (2-4C)alkenylene, -O(1-4C alkyl)-*, -(1-4C alkyl)-O-*, -(1-4C alkyl)-S-*, (3-6C)cycloalkylene or hetCyc1, where the symbol "*" indicates the binding position of G, under the condition that if W denotes -C(=O)NR2-, then L is not -(CH=CH)-; m equals 0, 1 or 2; n equals 0 or 1; Ra and Rb are independently selected from hydrogen and (1-4C alkyl); R3 denotes hydrogen, (1-4C alkyl) or CH2OH; R4 denotes hydrogen or methyl; R5 denotes hydrogen, (1-4C alkyl), OH, -O(1-4C alkyl) or F; R6 denotes hydrogen, F or methyl; or R5 and R6 together with the carbon atom with which they are bonded form a cyclopropyl ring, hetCyc1 denotes a group of formula where t equals 1 or 2 and p equals 0 or 1, and the symbol "*" indicates the position of binding with G; G denotes Ar1, Ar2, naphthyl, benzo-condensed (5-6C)cycloalkyl ring, optionally substituted with one or more substitutes independently selected from Cl and OMe, benzo-condensed 5-6-member heterocyclic ring with 1-2 heteroatoms independently selected from O and N, (3-6C)cycloalkyl ring, optionally substituted with one or more substitutes independently selected from (1-4C)alkyl, oxaspirononanyl ring or t-butyl; Ar1 denotes phenyl, optionally substituted with one or more substitutes independently selected from F, Cl, Br, CF3, (1-4C)alkyl, OH, -O(1-4C alkyl), -S(1-3C alkyl), -SCF3, cyclopropyl, -CH2N(1-3C alkyl)2, -O-(2-3C)fluoroalkyl, -O-(1-3C)difluoroalkyl-O-(1-3C)trifluoroalkyl, -OCH2(cyclopropyl) and (3-4C)alkynyl; Ar2 denotes phenyl, substituted with Ar3, -O-Ar4, hetAr1 or -O-hetAr2, where Ar2 is further optionally substituted with one or more substitutes independently selected from F, O or CF3; Ar3 denotes phenyl, optionally substituted with one or more substitutes independently selected from F, CI, Br and (1-4C alkyl); Ar4 denotes phenyl, optionally substituted with one or more substitutes independently selected from F, CI, Br and (1-4C alkyl); hetAr1 denotes a 6-member heteroaryl with 1-2 nitrogen atoms, optionally substituted with one or more substitutes independently selected from (1-4C alkyl); hetAr2 denotes a 6-member heteroaryl with 1-2 nitrogen atoms, optionally substituted with one or more substitutes independently selected from (1-4C alkyl) and CF3; R7a, R7 and R8 each independently denotes hydrogen or methyl; R9 denotes hydrogen, methyl, fluorine or NO2; and R10 denotes hydrogen, methyl or fluorine; where A1, A2, W, L, G, R7a, R7b, R8, R9 and R10 assume values given in the description, which are DP2 receptor modulators which are effective in treating immunological diseases.
EFFECT: inventions relate to a method of producing compounds of formula 1, a pharmaceutical composition based on said compounds and a method of treatment.
30 cl, 1 tbl, 239 ex
SUBSTANCE: group of inventions relates to biotechnology. A method for preparing the vaccine provides crossing of two different species of the four: T.spiralis, T.nativa, T.pseudospiralis and T.britovi, and selecting the line having the lower pathogenic and higher immunogenic properties by infecting with murine Trichinella larvae and controlling the embryogenesis; 1.5-2 months later, infected mouse's muscular tissue is separated, and the invasion larvae are recovered to be used for re-infection in a dose of 200-300 larvae; a degree of infection is determined, while the vaccine is prepared of the Trichinella line that leads to a mildest disease, while the re-infection causes no disease. Milk of the animals immunised with the vaccine prepared as described above is used for the purpose of prevention and treatment of the immune disorders. The vaccine described above is also used to prepare the serum. The vaccine is orally administered in a dose of 5000 to 20000 living larvae starting from the 10-14th day from the administration of the preparation; a usual therapeutic correction is required as that for trichinellosis, while the blood is sampled 30 days after the administration of the preparation. The prepared serum is administered into an individual according to the following schedule: either orally starting from 0.05 ml per one intake 3 times a day and increased to 1.2 ml by adding 0.05 ml to each intake, and then decreased in reverse order, or in the form of injections parenterally in a dose of 1-2 ml every 2 days, and in a dose of 10-15 ml into an animal three times to prevent and treat the immune disorders.
EFFECT: using the inventions described above enables more effective prevention and treatment of the immune disorders.
4 cl, 9 ex
SUBSTANCE: present invention generally refers to immunology, particularly to bolstering the immune system in the elderly age and represents a composition. The composition for treating and preventing the diseases related to the altered immune system specified in a group consisting of infections, particularly bacterial, viral, mycotic and/or parasitic infections; the autoimmune diseases; the inborn immunity diseases, e.g. NK cell deficiency, phagocyte deficiency, such as leukocyte adhesion deficiency, cyclic neutropenia; and combinations thereof containing a protein fraction comprising acetylneuraminic acid related to a threonine-rich peptide/protein frame.
EFFECT: invention provides bolstering the immune system more effectively.
13 cl, 2 dwg