CN1317045A - Pharmaceutical uses of NAB1 and NAB2 - Google Patents
Pharmaceutical uses of NAB1 and NAB2 Download PDFInfo
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- CN1317045A CN1317045A CN99810616A CN99810616A CN1317045A CN 1317045 A CN1317045 A CN 1317045A CN 99810616 A CN99810616 A CN 99810616A CN 99810616 A CN99810616 A CN 99810616A CN 1317045 A CN1317045 A CN 1317045A
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- nab2
- nab1
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
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- A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
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Abstract
The invention relates to the use, particularly in gene therapy, of an NAB1 or NAB2 polypeptide or a biologically active fragment thereof, and to nucleic acid molecules encoding such polypeptides, in the manufacture of a medicament for the treatment of cell proliferation disorders associated with wound healing in a mammal, including human.
Description
The present invention relates to the application of gene therapy technology in wound healing.More particularly, it relates to the new purposes of the polynucleotide of coding NAB1 or NAB2 transcription repressor, this new purposes is used for regulating cell proliferation disorders downwards, and the skin healing that especially slows down forms, alleviates psoriatic, suppresses the propagation through restenosis, the calcification of regulating vessel wall and the inhibition cancer cell of skin behind the tube chamber coronary angioplasty to prevent hypertrophic scar and keloid scars.
Skin healing relates to various cells, molecule, physiology and biological process.In the skin healing process, cell migration is to wound location, in wound location propagation and synthesize the extracellular matrix composition to rebuild and not damage the closely similar tissue of fundamental weave.Agglutination as described in the medium of the peripheral emiocytosis of damage is regulated as Thr6 PDGF BB (PDGF), Urogastron (EGF), transforming growth factor-beta (TGF β) and other cytokine.Beneficial effect (Moulin summary, the Eur.J.Cell Biol.68 of these factor pair cells have all been confirmed in vitro and in vivo; 1-7,1995), be included in the beneficial effect (J.Surg.Res.56 such as Brown that gives PDGF in the rat diabetes model; 562-570,1994).
Verified many somatomedins are in external acceleration cell proliferation and promote wound healing in animal model in the past 5 years.TGF β is the most noticeable aspect injury recovery, because it promotes cell proliferation, differentiation and matrix to produce.In animal model, part or system give TGF β all can quicken the speed that skin injury recovers.(Nature Medicine such as Ashcroft, 3; 1209-1215,1997; Sporn and Roberts J.Cell Biol.119; 1017-1021,1997; J.Clin.Invest.92 such as Beck; 2841-2849,1993).Reported that equally, PDGF promotes epithelium in ischemic tissue and the diabetic animal to form with blood vessel to form (Langenbecks Archiv fur Chirurgie-Supplement-Kongressband 114 such as Uhl again again; 705-708,1997 and Dirks and Bloemers summary be stated from Mol.Biol.Reports 22; 1-24,1996).
Transcription factor Egr-1 (early growth response gene) is effective conditioning agent of the gene more than 30, and plays a role in cell proliferation, growth and differentiation that (Liu etc. summarize Crit.Rev.Oncogenesis 7; 101-125,1996; Khachigian and Collins Circ.Res.81; 457-461,1997).Induce Egr-1 (Science such as Khachigian for example during blood vessel endothelium injury; 271,1427-1431,1996) and the transcription activating target be numerous gene, comprise that EGF, blood are little, plate derivative growth factor-A (PDGF-A), Prostatropin (bFGF), and induce PDGF A, Thr6 PDGF BB-B (PDGF B), TGF β, bFGF, u-PA (u-PA), tissue factor and rhIGF-1-2 (IGF-2).Blocking-up Egr-1 induction type TGF β can be used for preventing cicatrization.Can alleviate cicatrization (the J.Cell Science such as Shah of rodent otch damage at the antibody of TGF β; 107; 1137-1157; Lancet such as Shah; 339,213-214,1992).
The transcription complex of mediation induction of vascular endothelial growth factor (VEGF) directly depends on AP2 rather than Egr-1 (EMBO J16 such as Gille; 750-759,1997).Yet PDGF B directly raises vegf expression (Finkenzeller Oncogene 15; 669-676,1997).Many factors comprise that PDGF B, bFGF, keratinocyte growth factor (KGF), EGF, tumour necrosis factor (TNF) α and TGF β 1 strengthen VEGF mRNA and transcribe.The passivation that has confirmed the metal stent that VEGF drives in animal model can suppress new tunica intima and form, quickens the formation again of endothelium and strengthen vasomotor activity (Circulation such as Asahara; 94,3291-3302).
Reported that VEGF expresses in healing wound and psoriatic skin, TGF α and its ligands, EGF r are to adjusted in both cases.The induced expression Egr-1 of EGF (Am.J.Physiol.270 such as Iwami; H2100-2107,1996; Calcified Tissue International 57 such as Fang; 450-455,1995; J.Neuroscience Res.36; 58-65,1993).Existing interesting evidence (anecdotal eviderve) is, Egr-1 can activate intercellular adhesion molecule-1 (ICAM-1) and express (Mol.Cell.Biol such as Maltzman in the bone-marrow-derived lymphocyte that fluorine ripple ester stimulates; 16; 2283-2294,1996) and can activate TNF alpha expression (Biochim.Biophys.Acta 1219 such as Kramer by means of the Egr-1 binding site that in TNF α promotor, exists; 413-421,1994).At last, (Lee etc. 273 for Egr-1 inactivation mouse apogeny and shortage lutropin (LH); 1219-1221,1996), this means that the LH promotor also may be an Egr-1 activation target.Angiosteosis is to be similar to osteoplastic active adjustment process, relates to the known cell important in regulating bone metabolism and the factor (summary such as Dermer Trends Cardiovasc.Med.4; 45-49,1994).The degree that the conditioning agent of scleroblast generation (osteoblastogenesis) and/or osteoclast generation (osteoclastogenesis) can be regulated angiolithic degeneration.NAB albumen NAB1 and NAB2 (NGFI-A in conjunction with auxilliary repressor) interact with the conservative R1 territory of Egr-1 and Egr-2 trans-activator (EMBO J. such as Svaren, 17; 6010-6019), 1998).Confirmed in the past that NAB2 can prevent differentiation (Qu, the J.Cell Biol.142 such as Z of NGF inductive PC12 cell; 1075-1082,1998) and the basic FGF of Egr-1 mediation activate (EMBO J. such as Svaren, 17; 6010-6019,1998).
A problem that runs in wound healing is to form hypertrophic scar and keloid scars.This is extremely undesirable, especially after cosmetic surgery.Tissue fibrosis (for example fibrosis of kidney, liver or skin) shows as gathering of extracellular matrix.Many somatomedins to small part is regulated the dysregulation generation of the extracellular matrix on the basis that constitutes the tissue fibrosis development, and described somatomedin mainly is but is not limited to TGF β (Muir Eur.J.Plast.Surg.21; 1-7,1998), PDGF isotype (J.Pathol.186 such as Katou; 201-208,1998, Heldin etc., the molecule of wound repair and cytobiology, Clak edits; 249-264,1996 Plenum Press) and VEGF (Frontiers in Bioscience 4 such as Jones; D303-309,1999).Reduce but whether eliminate somatomedin and the healing that scar reduces tissue is had material impact, and can improve quality of life in the methods of treatment of the expression of wound location.
International patent application no PCT.GB99.01722 describes and uses Egr-1 transcription factor promotion wound healing.The inventor finds now, give encoding transcription repressor NAB1 or NAB2 polynucleotide can: (a) at the growth factor activation of vitro inhibition Egr-1 mediation; (b) in the basal level of the expression of vitro inhibition growth factor gene; (c) it can reduce TGF-β 1 in the expression of rodent wound site subsequently and expresses, rather than reduces TGF-β and express, and therefore can be used for alleviating cicatrization in the healing (J.Cell Science 107 such as Shah for example; 1137-1157,1994).
So, regulate Egr-1 downwards and can slow down agglutination and reduce the generation hypertrophic scar and formation of keloid scars and psoriatic incidence.Regulate Egr-1 downwards and can also be used for the treatment of other wound healing diseases associated as breeding through skin restenosis, adjusting angiolithic degeneration and inhibition cancer cell behind the tube chamber coronary angioplasty.
Therefore, according to one aspect of the present invention, the nucleic acid molecule of the sequence that comprises coding NAB1 or NAB2 polypeptide or its bioactive fragment is provided, and its purposes is to produce the medicine that is used for the treatment of the cell proliferation disorders that comprises that human Mammals wound healing is relevant.
According to one side more of the present invention, the method that provides treatment to comprise the cell proliferation disorders that human Mammals wound healing is relevant, this method comprise that the nucleic acid molecule that will comprise the sequence of coding NAB1 or NAB2 polypeptide or its bioactive fragment gives described Mammals.
The invention provides the nucleic acid molecule of the sequence that comprises coding NAB1 or NAB2 polypeptide or its bioactive fragment more on the one hand according to of the present invention, its purposes is the relevant cell proliferation disorders of treatment wound healing.
Again on the one hand, the further combined utilization of the present invention comprises the nucleic acid molecule and the another kind of nucleic acid molecule that comprises the sequence of coding NAB2 polypeptide of the sequence of coding NAB1 polypeptide.
A preferred aspect of the present invention, the relevant cell proliferation disorders of wound healing are selected from hypertrophic scar and the formation of keloid scars, psoriatic, breed through skin restenosis, angiolithic degeneration and cancer cell behind the tube chamber coronary angioplasty.The particularly preferred aspect of the present invention, described cell proliferation disorders are that hypertrophic scar forms and the keloid scars forms.
The therepic use of polynucleotide in wound healing process of NAB1 or NAB2 so the present invention relates to encode.The invention still further relates to this therepic use in wound healing process of NAB1 or NAB2, as more detailed description hereinafter.
The present invention relates to use NAB1 or NAB2 polypeptide and any source of coding or the NAB1 of species or the nucleotide sequence of NAB2 polypeptide.Described human DNA sequence lists in Genbank with registration number U47007,486 amino acid whose nucleoprotein of this sequence encoding.Rat NAB1 is 570 amino acid whose nucleoprotein and is described in PNAS USA92,1995, the 6873-6877 pages or leaves.Described rat dna sequence dna is listed in Genbank with registration number U17253.
Mouse and people NAB2 protein sequence are described in Molecular and Cellular Biology, 199616,3545-3553.Described human DNA sequence lists in Genbank with registration number U48361.
NAB1 that hereinafter touches upon or NAB polypeptide and polynucleotide generally are applicable to the especially above-mentioned human sequence of sequence in any source.As mentioned below, term NAB1 or NAB2 also comprise NAB1 or NAB2 varient, fragment and analogue.
Provide following illustrative to explain to help to understand some term used herein.Provide and explain it is for the ease of understanding the present invention rather than restriction the present invention.
NAB1 as referred to herein or NAB2 bioactive fragment are to have the active fragment of Egr-1 transcription repression.
" gene " generally is meant the polynucleotide in the district that comprises coded polypeptide, or refer to regulate and duplicate, transcribe or translate or other expresses the polynucleotide district of important process for described polypeptide in host cell, or the district that comprises the peptide species of encoding simultaneously is connected, regulates the polynucleotide in the district of expressing with one with its operability.Gene can be included in the carrier that duplicates as additional factor (promptly being totally independent of the molecule of host cell gene group physically).They can be included in the plasmid.Gene also can be included in the host cell gene group; Be not its native state, but with comprising such as separate, the clone and introduce the operation of host cell after the purify DNA form or be present in form in the carrier.
" host cell " is to have transformed or transfection or can be transformed or cells transfected by exogenous polynucleotide sequence.
" identity " according to the understanding of this area, is meant two or more peptide sequences by the sequence comparative measurement, the relation between two or more polynucleotide sequences.In the art, identity also refers to the sequence degree of correlation between polypeptide or the polynucleotide sequence as the case may be, and described sequence degree of correlation is determined according to the coupling between the string of such sequence.Identity calculates easily that (A.M. edits, OxfordUniversity Press, New York, 1988 for Computational Molecular Biology, Lesk; Biocomputing:Informatics and GenomeProjects, Smith, D.W. edits, Academic Press, New York, 1993; ComputerAnalysis of Sequence Data, Part I, Griffin, A.M. and Griffin, H.G. edits, Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; With Sequence Analysis Primer, Gribskov, M. and Devereux, J. edits, M Stockton Press, New York, 1991).Though there is the method for the identity between two kinds of polynucleotide of many detections or the two peptide species sequences, but this term is those of skill in the art to be known (Sequence Analysis in MolecularBiology, von Heinje, G., Academic Press, 1987; Sequence Analysis Primer, Gribskov, M. and Devereux, J. edits, M Stockton Press, New York, 1991; And Carillo, H. and Lipman, D., SIAM J.Applied Math., 48:1073 (1988).The method that generally is used to measure the identity between the sequence includes but not limited to Carillo, H. and Lipman, D., SIAM J.Applied Math., 48:1073 (1988) disclosed method.Design is measured the preferred method of identity to obtain the maximum match between institute's cycle tests.Measure the method for identity with computer program code.The preferred computer program technic of measuring the identity between two kinds of sequences includes but not limited to GCG routine package (Devereux, 387 (1984)), BLASTP, BLASTN and FASTA (Atschul J. wait Nucleic AcidsResearch12 (1):, S.F. etc., J.Molec.Biol.215:403 (1990)).
" isolating " is meant that " artificially " change its native state, if promptly natural existence, by its original environment change or taking-up, perhaps two kinds of situations all exist.For example natural polynucleotide or the polypeptide that exists with its native state naturally in the organism that lives is not " isolating ", but with identical polynucleotide or polypeptide that the coexisting substances of its native state separates is " isolating ", and term used herein comes to this.As isolating integral part or after separation, described polynucleotide can be connected with other polynucleotide such as DNA forming fusion rotein, and purpose is to carry out mutagenesis and for example in host's breeding or express.Isolating polynucleotide can be separately or are combined with other polynucleotide sequence in the host cell that (for example carrier format) import the host cell cultivated or complete organism.Behind host cell that importing is cultivated or the host cell of complete organism, this DNA still should be isolating, as used herein this term, because they are not its natural forms or are not to be in its natural composition environment, wherein still are isolating polynucleotide or the polypeptide in term used herein " isolating " implication.
" polynucleotide " generally are meant any polyribonucleotide or polydeoxyribonucleotide, and they can be the RNA or the DNA of unmodified, or the RNA or the DNA that modify, or cDNA.Therefore, polynucleotide used herein are for example comprising the DNA of the mixture of single stranded DNA and double-stranded DNA, strand and double stranded region or strand, two strands and three sequences, the RNA of the mixture of single stranded RNA and double-stranded RNA and strand and double stranded region comprises and can be strand or be more typically two strands or the DNA of the mixture of three chains or strand and double-stranded section and the hybrid molecule of RNA.In addition, polynucleotide used herein be meant the RNA that comprises three sequences or DNA or RNA and DNA the two.Chain in the described section can be from same molecular or differing molecular.Described section can comprise the whole of one or more molecules, but a more common section that includes only some molecule.It usually is oligonucleotide that a triple helices is distinguished son.Term polynucleotide used herein comprise above-mentioned DNA or the RNA that contains one or more modified bases.Therefore, DNA or the RNA that has for stability or the main chain modified of other purpose is this paper term indication " polynucleotide ".And, comprise rare base such as inosine or modified base such as tritylation base-these two examples that just exemplify-DNA or RNA be term polynucleotide used herein.Should be appreciated that as is known to persons skilled in the art, DNA and the RNA that meets many useful purposes carried out a large amount of modifications.Term polynucleotide used herein comprise that described chemistry, zymetology or metabolism modify the DNA and the RNA of the chemical species of the polynucleotide that form and virus and cell (particularly including unicellular or many cells) feature.Polynucleotide comprise the short polynucleotide that are commonly referred to oligonucleotide.
" polypeptide " used herein comprises all polypeptide of the following stated.The basic structure of polypeptide is well-known, and has been described in the countless textbooks in this area and other publication.In this respect, term used herein is meant and comprises two or more amino acid whose any peptide or albumen that connect with straight chain each other by peptide bond.Term used herein had both comprised also long-chain of short chain, and described short chain this area is also referred to as for example peptide, oligopeptides and oligomer, and the general this area of described long-chain is called protein, and had many types.Should be realized that polypeptide contains the amino acid of 20 seed amino acids that are not to be commonly referred to 20 kinds of natural amino acids usually, and in particular peptide, comprise end amino acid many amino acid can by natural process as processing and other posttranslational modification, but also can modify by the known chemical modification technology in this area.Even how the natural common modification that is present in polypeptide also must be difficult to enumerate in this total number, but they have sufficient description in basic books and more detailed monograph and many research documents, and they are that those skilled in the art are known.
Can be present in the known modification that is used for polypeptide of the present invention and comprise-exemplify some illustrative example-acetylizes; acidylate; the ADP-ribosylation; amidation; the covalent attachment of flavine; the covalent attachment of heme moiety; the covalent attachment of Nucleotide or nucleotide derivative; the covalent attachment of lipid or lipid derivate; the covalent attachment of phosphatidylinositols; crosslinked; cyclisation; disulfide linkage forms; demethylation; form covalent cross-linking; form Gelucystine; form Pyrrolidonecarboxylic acid; formylation; γ-carboxylated; glycosylation; the GPI anchor formation; hydroxylation; iodate; methylate; the Semen Myristicae acidylate; oxidation; proteolysis processing; phosphorylation; isoprenylation; racemization; selenizing; sulphating; amino acid being added in the protein of transfer-RNA mediation as arginineization and ubiquitination.Such modification is that those skilled in the art are known and very detailed description arranged in scientific literature.The γ of several common especially for example glycosylations of modification, lipid combination, sulphating, glutaminic acid residue-carboxylated, hydroxylation and ADP-ribosylation have description in most basic textbook, PROTEINS-STRUCTURE AND MOLECULARPROPERTIES for example, second edition, T.E.Creighton, W.H.Freeman and Company, NewYork (1993).Can utilize many more detailed summaries about this theme, Wold for example, F., Posttranslational Protein Modifications:Perspectives and Prospects, be stated from the 1-12 page or leaf of POSTTRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B.C.Johnson edits, Academic Press, New York (1983); Seifter etc., Meth.Enzymol.182:626-646 (1990) and Rattan etc., Protein Synthesis:Posttranslational Modifications and Aging, Ann.N.Y.Acad.Sci.663:48-62 (1992).As everyone knows and as mentioned above, should be realized that polypeptide is always linear fully.For example polypeptide is because the effect of translation back generally can be non-linear, and described translation back effect comprises the natural process process and is not the process of the manual operation of natural generation.The synthetic method of annular, side chain and ramose annular polypeptide can be the untranslated natural process, also can be synthetic method completely.Any position of polypeptide all can be modified, and comprises peptide main chain, amino acid side chain and N-terminal or C-terminal.In fact, the amino or the carboxyl of covalent modification sealing polypeptide, perhaps two kinds of all sealings all are common at natural polypeptides and synthetic polypeptide, also can there be this modification in polypeptide of the present invention.For example the n terminal residue with the polypeptide of intestinal bacteria or other cell preparation was always the N-methylmethionine before proteolysis.In the posttranslational modification of peptide, can lack the methionine residue of amino-end.Therefore, the present invention designs to use and contains the aminoterminal protein variants of the present invention of methionine(Met) and do not have the aminoterminal varient of methionine(Met).The modification that polypeptide takes place has its effect how to prepare of reflection.For example for the polypeptide by the preparation of cloning by expression gene in the host, the nature and extent of modification depends on host cell posttranslational modification ability to a great extent and is present in the modification signal of polypeptid acid sequence.For example well-known glycosylation can not betide host bacterium such as intestinal bacteria usually.Therefore, when the needs glycosylation, polypeptide should be expressed in the glycosylation host, is generally eukaryotic cell.Insect cell carries out the post-translational glycosylation identical with mammalian cell usually, for this reason, has developed the insect cell expression system that effective expression has the mammalian proteins of natural type glycosylation itself.Similarly consider to be used for other modification.Will be appreciated that same type modifies several sites that can identical or different degree be present in specific polypeptide.In addition, specific polypeptide can contain the modification of many types.In general, term polypeptide used herein comprises the modification that all are such, thereby especially is present in by the modification in the polypeptide that is re-combined at the host cell expression polynucleotide.
" varient " of term polynucleotide used herein or polypeptide is for being different from the polynucleotide or the polypeptide of reference polynucleotide or polypeptide respectively.Hereinafter locate to describe the varient of this meaning in more detail disclosed by the invention with it.(1) is different from the polynucleotide of another reference polynucleotide nucleotide sequence.In general, difference is limited, thereby makes that the nucleotide sequence of reference sequence and varient is closely similar generally, and many districts are identical.As described below, it can be reticent that the varient nucleotide sequence changes.Be the amino acid that they can not change described polynucleotide encoding.When change only limits to such reticent variation, varient will encode and the polypeptide of reference sequence same acid sequence.As described below, the varient nucleotide sequence changes the amino acid sequence of polypeptide that also can change described reference polynucleotide encoding.Such Nucleotide changes aminoacid replacement, interpolation, disappearance, fusion and the brachymemma that can cause described reference sequence encoded polypeptides, as discussed below.(2) be different from the polypeptide of another reference polypeptid acid sequence.In general, difference is limited, thereby makes reference sequence and varient sequence closely similar generally, and many districts are identical.The different of varient and reference amino acid sequence of polypeptide can be one or more replacements, interpolation, disappearance, fusion and brachymemma, and these differences can exist in any combination.
The present invention relates to contain the therepic use of nucleic acid molecule of the sequence of coding NAB1 or NAB2 polypeptide or its composition.The invention still further relates to the therepic use of the varient of the fragment of described polynucleotide sequence of bioactive fragment of coding NAB1 or NAB2 polypeptide or described polynucleotide sequence, described polynucleotide sequence variations body is bioactive fragment by means of the function that has of genetic code degeneracy coding NAB1 or NAB2, and the invention still further relates to the allelic variation body of functional equivalent and the therepic use that has the correlated series of NAB2 or the active polypeptide of NAB2 by the coding that single or multiple bases replace, add and/or disappearance is modified.
Can obtain these sequences with standard cloning process well known by persons skilled in the art.
The polynucleotide of coding NAB1 or NAB2 transcription repressor can be DNA, cDNA or rna form, for example by the mRNA of clone's acquisition or the mRNA that produces by chemical synthesising technology.Described DNA can be short chain or double-stranded DNA.Single stranded DNA can be coding strand or sense strand, and perhaps it can be noncoding strand or antisense strand.For therepic use, described polynucleotide have the NAB1 of function or the form of NAB2 transcription repressor to exist expressing in the patient's of needs treatments wound site.Described polynucleotide also can be used for produced in vitro NAB1 or NAB2 polypeptide to carry out administration aspect the treatment more of the present invention as detailed below.
The polynucleotide of the present invention of coding NAB1 or NAB2 polypeptide include but not limited to the encoding sequence of NAB1 or NAB2 polypeptide or its bioactive fragment.Therefore, the encoding sequence of the interpolation of the amino acid (providing functional amino acid of interpolation as those) that described polynucleotide can add with the non-coding sequence and the coding of interpolation provides, the non-coding sequence of described interpolation comprises, such as but not limited to non-coding 5 ' and 3 ' sequence, rrna binding member, mRNA stable element, described non-coding 5 ' and 3 ' sequence as transcribing of in transcribing, working but the sequence of not translating (comprise, for example, termination signal).Polynucleotide of the present invention also include but not limited to comprise NAB1 or NAB2 structure gene and its natural relevant genic polynucleotide.
According to the above, term used herein " polynucleotide of coded polypeptide " comprises the polynucleotide of the sequence that contains coding NAB1 or NAB2 polypeptide.Described term comprises the single continuum of containing coding said polypeptide or locus of discontinuity (for example be integrated plasmid or insertion sequence or editor is interrupted) and the polynucleotide in the district that adds, and coding and/or non-coding sequence can also be contained in described interpolation district.
The invention still further relates to the varient of the polynucleotide mentioned above of coding said polypeptide fragment, analogue and derivative.The varient of described polynucleotide can be naturally occurring varient, as naturally occurring allelic variation body, perhaps can be unknown naturally occurring varient.This non-natural of described polynucleotide exists varient to prepare by induced-mutation technique, comprises the induced-mutation technique that is applicable to polynucleotide, cell or organism.
The varient that is different from above-mentioned polynucleotide because Nucleotide replaces, lacks or adds belongs to the varient of this respect.Described replacement, disappearance or interpolation can relate to one or more Nucleotide.Varient can be in the coding region or non-coding region changes or all changes in the two.The change of coding region may produce conservative or non-conserved amino acid and replace, lacks or add.
A preferred embodiment more of the present invention in its whole length with coding have the identical polynucleotide of the polynucleotide at least 70% of polypeptide of above-mentioned aminoacid sequence part and with this polynucleotide complementary polynucleotide.On the other hand, most preferred polynucleotide are the polynucleotide that comprise such district: described district has at least 80% identical at its whole length and the polynucleotide of code book invention polypeptide.In this respect, the identical polynucleotide of the polynucleotide at least 90% of preferred especially its whole length and code book invention polypeptide.The polynucleotide that in these particularly preferred polynucleotide, especially preferably have 95% identity at least.And at the polynucleotide of preferred at least 97% identity of the sequence camber of at least 95% identity, and in the sequence of at least 98% identity especially highly preferably at least 99%, at least 99% more preferably.
In addition, preferred embodiment in this respect is that coding keeps and the ripe NAB1 of above-mentioned dna sequence dna (Genbank registration number U47007 and U48361) coding or the polynucleotide of essentially identical biological function of NAB2 polypeptide or active polypeptide.
The invention still further relates to polynucleotide with the above-mentioned sequence hybridization of this paper.About this, The present invention be more particularly directed under stringent condition polynucleotide with the above-mentioned multi-nucleotide hybrid of this paper.Term used herein " stringent condition " is meant if the condition that exists at least 95% identity, preferred at least 97% identity just to hybridize between described sequence.Preferably encode with the sequence of sequence hybridization of the present invention by this way and have NAB1 or the bioactive polypeptide of NAB2.
Described polynucleotide may be encoded as described maturation protein and add extra N-terminal or the amino acid whose polypeptide of C-terminal.Such additional sequences can have effect, for example comprises prolonging or to shorten the operation that the protein transformation period maybe can help analysis of protein or production.The same with situation in the general body, remove described additional amino acid by cellular enzymes from maturation protein processing.
The polynucleotide that are used for gene therapy of the present invention aspect can provide separately, or provide as the integral part of carrier such as virus vector, and the example is that this area is known.
NAB1 or NAB2 coded polynucleotide can therapeutic be used for the inventive method by means of gene therapy, and wherein said polynucleotide are administered to it in wound site or other tissue that needs healing in the segmental mode that can instruct original position to produce NAB1 or NAB2 or its biologically active.
Preferably make polynucleotide for example with the form of recombinant DNA molecules such as the expression vector patient's expression in vivo in the needs treatment in the method that gives of polynucleotide described in the gene therapy, described recombinant DNA molecules contains operability and is connected to the coding NAB1 of the nucleotide sequence that control expresses or the polynucleotide of NAB2.Therefore such carrier will contain the suitable control signal of transcribing, and comprise the promoter region that can express described encoding sequence, and described promotor can work in patient's body of needs treatment.So for the human gene therapy, the condition of described promotor-promotor is not only to contain the guide RNA polysaccharase to be bonded to the necessary sequence of transcription initiation site, and suitable words contain other operation or control sequence (comprising enhanser)-preferably promotor from the Human genome or the gene of expressing the mankind usually, for example promotor of people CMV.The known promoter in eukaryote of Shi Heing has CMV immediate early promoter, HSV thymidine kinase promotor, early stage and late period SV40 promotor, the promotor of retrovirus LTR such as the promotor of Rous sarcoma virus (" RSV ") in this respect, and metallothionein promoter such as mouse metallothionein(MT)-1 promotor.
The polynucleotide sequence and the transcriptional control sequence that are cloned into replicating plasmid vector can be provided, and described replicating plasmid vector perhaps can be by the conventional known open method of using by the available plasmid construction based on plasmid that is commercially available such as pBR322.
Described carrier can also contain the control signal of transcribing that is positioned at NAB1 or NAB2 encoding sequence 3 ', but also can contain the discernible polyadenylation signal of patient for the treatment of at needs, for example derive from the corresponding sequence of virus (when for example carrying out the human treatment, using SV40 virus).Can use the known transcriptional control sequence in other this area.
Described expression vector can also comprise for example antibiotics resistance of selective marker, and it makes described carrier to breed.
Can be directly with physical method can the synthetic NAB1 of original position or the expression vector of NAB2 import damage location.Such example comprises local " naked " nucleic acid carrier that uses in suitable solvent, for example pharmaceutically acceptable excipient solution of described suitable solvent such as phosphate buffered saline(PBS) (PBS), or with physical method such as particle bombardment (being also referred to as ' particle gun ' technology), give described carrier according to methods known in the art, described currently known methods is the method described of U.S. Patent number 5371015 for example, and wherein the speed that sees through damage location surface (for example skin cells) from launching device with the inert particle (as gold bead) that is enough to use described carrier bag quilt by means of high pressure is quickened inert particle as described in the emission.
That other physical method that gives the recipient with described dna direct comprises is ultrasonic, electricity irritation and electroporation.
The NAB1 or the NAB2 nucleic acid sequence encoding that are used for therapy of the present invention also can give by transmitting carrier.These carriers comprise the viral carrier, and adenovirus as known in the art or retrovirus transmit carrier.
Other non-viral carrier comprises that lipid known in the art transmits carrier, comprises that liposome transmits solvent.
NAB1 or NAB2 nucleic acid sequence encoding also can give damage location by transformed host cells.Such cell comprises the cell that described patient collects, and wherein said nucleotide sequence imports in this cell by gene transfer method known in the art, cultivates described transformant then and be transplanted to described patient in culture.
Can several different methods be used for therapy of the present invention such as above-mentioned expression constructs.Therefore, they can directly be administered to patient's damage location, or they can be used to preparation reorganization NAB1 or NAB2 polypeptide itself, then described polypeptide are administered to damage location, and more detailed discussion sees below.The invention still further relates to host cell, this host cell is used and is comprised genetically engineered the making of genic construction of NAB1 or NAB2 polynucleotide or polynucleotide of the present invention or above-mentioned definition, and the present invention relates to these carriers and the application of cell in methods of treatment of the present invention.These constructions itself can be used for methods of treatment of the present invention, and perhaps they can be used to prepare NAB1 or the NAB2 polypeptide that uses in the methods of treatment hereinafter of the present invention in greater detail.
Whether directly be administered to damage location (being synthetic NAB1 of original position or NAB2) or be not used for synthetic reorganization NAB1 or NAB2 according to described carrier, described carrier can be, for example, plasmid vector, strand or double stranded phage carrier, strand or double-stranded RNA or dna viral vector.Initial plasmid disclosed herein or be commercially available or the obtainable plasmid of the public, or the available conventional known open method of using is by obtainable plasmid construction.Can many used according to the present invention plasmids be known, and those skilled in the art obtain easily with other clone and expression vector.
Generally speaking, the carrier that is used for NAB1 of the present invention or NAB2 polypeptide in order to expression comprise operability be connected to polynucleotide that needs express, in the cis acting control region of host's effective expression.Described host, complementary carrier or in importing described host the time described carrier the suitable cis acting factor is provided itself.
In some embodiment aspect this, described carrier provides specific expressed.For the generation of reorganization NAB1 or NAB2, so specific expressed can be inducible expression only in some cell type, express or be inducible expression be again cell specific expression.Particularly preferred carrier is can be by the carrier of environmental factors abduction delivering in the induction type carrier, wherein said environmental factors easy handling, for example temperature and nutritional additive.Comprise that the composing type that is used for prokaryotic organism and eukaryote host and the various carriers that are applicable to this aspect of the present invention of inducible expression vector are known, and conventional these carriers that use of those skilled in the art.
Can use the expression vector of numerous kinds to express NAB1 of the present invention or NAB2.Such carrier is particularly including the carrier of karyomit(e), episome and viral source, as producing carrier: bacterial plasmid, phage, transposon, yeast episome, insertion element, yeast chromosomal element, virus (as baculovirus, papovavirus as (SV40), vaccinia virus, adenovirus, fowlpox virus, pseudorabies virus and retrovirus) from following structure, and the carrier of the carrier of their combination results such as plasmid and the generation of phage gene, as clay and phagemid, all these carriers can be used for meeting the expression of this aspect of the present invention.In general, be fit to keep, breed or express polynucleotide and can be used for expression aspect this with any carrier at the host expresses polypeptide.
Any technology in available numerous known routine techniques such as Sambrook etc. be at MOLECULAR CLONING, A LABORATORY MANUAL, second edition; ColdSpring Harbor Laboratory Press, Cold Spring Harbor, the method described in the New York (1989) is inserted described carrier with suitable dna sequence dna.
Nucleotide sequence operability in the expression vector is connected to suitable expression control sequenc, comprises the promotor that for example instructs mRNA to transcribe.Typical this promotor includes but not limited to the SV40 that is used for recombinant expressed lambda particles phage PL promotor, intestinal bacteria lac, trp and tac promotor and is used for expressed in situ early stage and late promoter and retrovirus LTR promotor.
In general, expression constructs contains transcription initiation and ends the site, and contains the translation ribosome bind site at transcriptional domain.The encoding part of the mature transcript of being expressed by construction contains translation initiation AUG at initial position, contains terminator codon at the correct position of the polypeptide end of needs translation.
In addition, construction can contain the control region of regulating and causing expression.Generally speaking, according to many conventional methods of using, such district will be by comprising that (for example transcription factor, repressor combining site) transcribed in control and termination is worked.
The carrier that is used to breed and expresses generally comprises selective marker and amplification region, Sambrook etc. for example, MOLECULAR CLONING, ALABORATORY MANUAL, second edition; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, selective marker and the amplification region described in the NewYork (1989).
The representative instance that is used for the suitable host of recombinant expressed NAB1 or NAB2 comprises bacterial cell such as suis, staphylococcus, intestinal bacteria, streptomycete and bacillus subtilis cell; Fungal cell such as yeast cell and Aspergillus cell; Insect cell such as fruit bat S2 and fall army worm Sf9 cell; Zooblast such as CHO, COS, HeLa, C127,3T3, BHK, 293 and the Bowes melanoma cells; And vegetable cell.
Exemplify the following carrier that is commercially available.The carrier that wherein is preferred for bacterium has: pQE70, pQE60 and pQE-9 that Qiagen is on sale; PBS carrier, Phagescript carrier, Bluescript vector, pNH8A, pNH16a, pNH18A, pNH46A that Stratagene is on sale; Ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 that Pharmacia is on sale, and pBR322 (ATCC 37017).Preferred eukaryote carrier has pWLNEO, pSV2CAT, pOG44, pXT1 and the pSG that can derive from Stratagene; Can derive from pSVK3, pBPV, pMSG and the pSVL of Pharmacia.These not only can be used for carrier recombinant expressed but also that can be used for expressed in situ only enumerates by exemplifying many carriers of knowing that are commercially available, and they are carriers of knowing that those skilled in the art can be used for meeting this aspect of the present invention.Should be understood that any other plasmid that is suitable for for example importing, keep, breed or express polynucleotide of the present invention or polypeptide in the host or carrier all can be used for the present invention aspect this.
The carrier example that is used for this aspect of the present invention comprises expression vector, and wherein NAB1 or NAB2 cDNA sequence are inserted in the plasmid, so the early stage immediately cytomegalovirus enhanser of people-promoters driven genetic expression (Foecking and Hofstetter, Cell, 45,101-105,1986).Such expression plasmid can contain SV40 RNA processing signal, as polyadenylation signal and termination signal.The expression constructs that adopts the CMV promotor and be commercially available has pCDM8, pcDNA1 and derivative, pcDNA3 and derivative (Invitrogen).Spendable other obtainable expression vector is SV40 promotor and mRNA splice site and polyadenylation signal (pSVK3) and the SV40 VP1 processing signal (pSVL that contains from SV40; The Pharmacia carrier) pSVK3 and pSVL.
Can select promoter region according to any goal gene, wherein use and contain the report transcription unit carrier that lacks promoter region, E.C. 2.3.1.28 (" CAT ") transcription unit for example, it is positioned at the restriction site downstream that is used to import candidate's promoter fragment (fragment that promptly contains promotor).As everyone knows, the fragment that will contain promotor at the restriction site of cat upstream region of gene imports carrier and causes producing the CAT activity, the active available standards CAT of CAT analyzing and testing.The carrier that is applicable to this purpose is well-known and is easy to obtain, as pKK232-8 and pCM7.The promotor that is used to express polynucleotide of the present invention not only comprises promotor well-known but also that be easy to obtain, and comprises and be easy to the promotor that adopts reporter gene to obtain by above-mentioned technology; For expressed in situ, it is desirable to such promotor and in treatment patient body, be identified.
The known promoter in prokaryote that is fit to express according to the present invention polynucleotide and polypeptide has intestinal bacteria lacI and lacZ promotor, T3 and T7 promotor, gpt promotor, λ PR, PL promotor and trp promotor.
Recombinant expression vector comprise replication orgin for example, preferably derive from cance high-expression gene instruct promotor that the downstream configurations sequence transcribes and the selective marker that after being exposed to described carrier, allows to separate carrier-containing cell.
General employing standard technique is inserted the polynucleotide of the present invention of the allos structure sequence of code book invention polypeptide in the described carrier, and its operability is connected to express with described promotor.The position of described polynucleotide makes described transcription initiation site suitably be positioned at ribosome bind site 5 ' end.Described ribosome bind site is positioned at the 5 ' end of the AUG of the polypeptide that opens the beginning accurate translation.
In general, do not originate in initiator codon-be generally AUG-and other open reading-frame (ORF) between ribosome bind site and initiator codon.In addition, in general, there is translation stop codon, and in construction, has the polyadenylation signal that is used for the eukaryote host at described polypeptide end.The transcription termination signal that suitably is arranged in 3 ' end of described transcriptional domain also can be included in described polynucleotide construction.
The protein excretion of translating in order to make is gone into endoplasmic, periplasmic space or born of the same parents' external environment, suitable secretion signal can be added in the polypeptide expressed when being re-combined into.These signals can be that the endogenous signal of described polypeptide or they are heterology signals.
Described polypeptide can be with modification type such as expressing fusion protein, and not only can comprise secretion signal and comprise other allos functional zone.Therefore, for example can add other amino acid at the N-terminal or the C-terminal of described polypeptide, the district of charge residue especially, thus improve its stability and persistence in described cell in purifying or processing subsequently and storage.In addition, can in described polypeptide, add the section that helps purifying.Such section was removed before finally making described polypeptide.In polypeptide, add peptide moiety so that its secretion or drainage, raising stability or promotion purifying are routine techniquess well known in the art.Preferred fusion protein comprises and is used for the immunoglobulin (Ig) allos district of solubilising or purified polypeptide.Then usually by centrifugal collecting cell,, keep the crude extract that obtains and be further purified being used to physics or chemical process smudge cells.
The broken available any ordinary method that is used for the microorganism cells of expressing protein comprises freeze-thaw cycle, supersound process, Mechanical Crushing or uses the lysis agent that these methods are well known to those skilled in the art.
Mammalian expression vector can comprise replication orgin, suitable promotor and enhanser, and the non-transcribed sequence that also comprises 5 ' essential flank of any essential ribosome bind site, polyadenylation district, donor splicing site and acceptor splicing site, transcription termination sequence and expression.
To be used for NAB1 of the present invention or NAB2 polypeptide in order preparing, can to use the host cell of genetic engineering.The method that polynucleotide import described host cell has transfection, transposition, microinjection, the transfection of cation lipid mediation, electroporation, transduction, the scraping of calcium sulfate transfection, the mediation of DEAE-dextran to load (scrape loading), attack importing (ballistic introduction), infect or other method.These class methods have description in many standard laboratory handbooks, Davis etc. for example, BASIC METHODS IN MOLECULAR BIOLOGY, (1986) and Sambrook etc., MOLECULAR CLONING:ALABORATORY MANUAL, second edition, ColdSpring Haror Laboratory Press, Cold Spring Harbor, N.Y. (1989).
Maturation protein can be expressed in host cell under suitable promotor control, and described host cell comprises mammalian cell, for example Chinese hamster ovary celI, yeast cell, bacterial cell or other cell.Also can use cell free translation system, the RNA that utilizes DNA construction of the present invention to produce produces this proteinoid.Sambrook etc. are at MOLECULAR CLONING:ALABORATORY MANUAL, second edition, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor has described suitable clone and the expression vector that is used for prokaryotic organism and eukaryote host among the N.Y. (1989).
Can reclaim and the described polypeptide of purifying from the reconstitution cell culture by known method, described method comprises ammonium sulfate precipitation or ethanol sedimentation, acid extraction, negatively charged ion or cation-exchange chromatography, phosphorylated cotton chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyapatite chromatography and lectin chromatography.Most preferably use high performance liquid chromatography to carry out purifying.When separating and/or purifying during during described polypeptide sex change, use known refolding albumen technology regeneration activity conformation.
Use technology known in the art, for example by means of Sambrook etc. at MOLECULARCLONING:A LABORATORY MANUAL, second edition, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. column chromatography of describing in (1989), but the NAB1 or the NAB2 coded polynucleotide of for example recombinant vectors form that purifying is used for the treatment of.
As mentioned above, can NAB1 or NAB2 polypeptide be administered to damage location with NAB1 or NAB2 coding nucleic acid with the form of gene therapy, this transcribes described coding nucleic acid and translation becomes NAB1 or NAB2 in wound site, perhaps can directly give described transcription repressor itself.
So, use the medicine of NAB1 or NAB2 polypeptide or its bioactive fragment production for treating Mammals (comprising the mankind) cell proliferation disorders according to one side more of the present invention.
Provide the method that comprises human mammalian cell proliferation disease for the treatment of again on the one hand according to of the present invention, it comprises NAB1 or NAB2 polypeptide or its bioactive fragment that gives described Mammals treatment significant quantity.
Therefore in view of one side more of the present invention, the invention provides the purposes that NAB1 or NAB2 polypeptide or its bioactive fragment are used for the treatment of damage and wound healing.
Term used herein " NAB1 or NAB2 polypeptide " comprises NAB1 or NAB2 natural and that reorganization produces, its natural, synthetic and biologically active polypeptides analogue or varient or derivative or its bioactive fragment, and described segmental varient, derivative and analogue.
Can produce and/or separate the NAB1 or the NAB2 protein product of the bioactive fragment that comprises NAB1 or NAB2 with ordinary skill known in the art.
Can extract NAB1 or NAB2 and above-mentioned fragment and the derivative that is used for the present invention's treatment from natural resource with methods known in the art.These class methods comprise the oligonucleotide in conjunction with DNA of using identification NAB1 or NAB2, utilize Briggs etc. at Science 234,47-52, and the method for describing in 1986 is carried out purifying by the sequence specific DNA affinity chromatography.Use above-mentioned recombinant DNA technology method known in the art (being that above-mentioned construction is expressed) and also can prepare described polypeptide in host cell.Perhaps with the synthetic production polypeptide of the present invention of conventional peptide synthesizer.
The invention still further relates to the purposes of NAB1 or NAB2 fragment, analogue and derivative.Term " fragment ", " derivative " are meant biological function or the active polypeptide that basic reservation is identical with described peptide with " analogue ".Therefore analogue comprises preceding albumen, can produce active mature polypeptide by the described protein part activation of cutting.
The fragment of described polypeptide, derivative or analogue can be (ⅰ) the described polypeptide fragment, derivative or the analogue that are replaced by conservative or non-conservative amino acid residues (preferred conservative amino acid residues) of wherein one or more amino-acid residues, and the amino-acid residue of described replacement can be or can not be described genetic code amino acids coding residue; Or (ⅱ) wherein one or more amino-acid residues contain substituent described polypeptide fragment, derivative or analogue; Or (ⅲ) wherein said mature polypeptide and another kind of compound for example strengthen described polypeptide fragment, derivative or the analogue that the compound (for example polyoxyethylene glycol) of described polypeptide transformation period merges; Or (ⅳ) wherein said other amino acid merges described polypeptide fragment, derivative or analogue to described mature polypeptide, and described other amino acid is for example for leading or secretion sequence or be used for the sequence of described mature polypeptide of purifying or preceding protein sequence.According to this paper content, think that this class fragment, derivative and analogue are in those skilled in the art's technical scope.
Wherein preferred varient has the varient that is different from natural NAB1 or NAB2 because conserved amino acid replaces.This class is substituted by another replacement with aminoacid replacement polypeptide specific amino acids of similar characteristics.The common conservative mutual replacement that is substituted with aliphatic amino acid Ala, Val, Leu and Ile; The mutual replacement of hydroxyl residue Ser and Thr, the exchange of acidic residues Asp and Glu, the replacement between amide residues Asn and the Gln, the exchange of alkaline residue Lys and Arg, and the replacement between aromatic moieties Phe, the Try.
This aspect further preferred especially wherein several to several 5-10,1-5,1-3,2,1 or do not have varient, analogue, derivative and the fragment of the polypeptid acid sequence that amino-acid residue replaces, lacks, adds with any combination, and described segmental varient, analogue and derivative.Wherein particularly preferably be and can not change polypeptide character of the present invention and active reticent replacement, add and lack.In also preferred especially conservative replacement the aspect this.
Particularly preferred fragment is a bioactive fragment, promptly possesses the fragment of the wound healing characteristic of parental generation polypeptide.
Preferably provide polypeptide of the present invention and polynucleotide, and preferably be purified to homogeneity with divergence type.
Being used for NAB1 of the present invention or NAB2 polypeptide comprises NAB1 or NAB2 polypeptide and has at least 70% identity, preferably at least 80% identity, the more preferably polypeptide of at least 90% even more preferably at least 95% similarity (even more preferably at least 95% identity) with this paper aforementioned polypeptides sequence, but also comprise the part of this class polypeptide, and this part of described polypeptide generally contains at least 30 amino acid, more preferably at least 50 amino acid.
Can use the fragment or the part of polypeptide of the present invention and synthesize the corresponding full-length polypeptide of generation by peptide; So described fragment can be as the intermediate that produces full-length polypeptide.The fragment of polynucleotide of the present invention or part can be used for synthesizing total length polynucleotide of the present invention.
The invention still further relates to NAB1 or the fragment of NAB2 polypeptide and the fragment of varient and derivative thereof of using above-mentioned definition.
Aspect this, fragment is part rather than whole identical polypeptide of aminoacid sequence of its aminoacid sequence and NAB1 or NAB2 polypeptide and varient or derivative.
Such fragment can be " free ", promptly be not other amino acid or polypeptide part or with its fusion, perhaps they can be included in their and form one partly or among the bigger polypeptide in a district.In the time of in being contained in bigger polypeptide, the present fragment of discussing most preferably constitutes single continuous section.But several fragments can be included in the single bigger polypeptide.For example, some embodiment preferred relates to the fragment that is contained in the polypeptide of the present invention in the polypeptide precursor, described polypeptide precursor design is expressed in host cell, and the district that adds has been merged at described segmental C-terminal in polypeptide and propolypeptide district before described segmental N-terminal has merged allos.So this paper specifies the fragment of an aspect of implication to be meant the fusion polypeptide of polypeptide generation of the present invention or a part or a plurality of part of fusion rotein.
In the present invention is aspect this, preferably have the structural performance of polypeptide of the present invention or the fragment of functional performance in addition.In this regard, the preferred embodiment of the invention comprises and contains fragment and these the segmental combination of polypeptide of the present invention with inferior segment: α spiral and α spiralization district, βZhe Die and βZhe Die form that district, corner and corner form district, spiral and spiralization district, hydrophilic area, hydrophobic region, α amphiphilic district, β amphiphilic district, flex region, the surface forms district, substrate land and high antigenic index region.
Preferred district is the district of mediation polypeptide active of the present invention.Wherein most preferred fragment is to have chemiluminescent polypeptide of the present invention, biology or other active fragment, comprises active similar or active raising or the unwanted active fragment that reduces.Further preferred polypeptide fragment is the fragment that comprises or contain especially human antigen of animal or immunogen decision family.
Should be realized that, the invention still further relates to and comprise the above-mentioned segmental polynucleotide of coding, with the polynucleotide of polynucleotide, especially hybridize under stringent condition of the described segmental multi-nucleotide hybrid of coding, and the polynucleotide (as the PCR primer) that are used for the described segmental polynucleotide of amplification coding.Wherein preferred polynucleotide are with respect to preferred segmental polynucleotide discussed above.
In the further embodiment of the present invention aspect this, comprise varient, analogue or derivatives thereof or its fragment (fragment that comprises described varient, analogue and derivative) useful on biology, prevention, the clinical or therapeutics and the composition that contains them.Biological activity varient, analogue and fragment comprise within the scope of the present invention.
The invention still further relates to the composition that contains polynucleotide discussed above or polypeptide.So, polynucleotide of the present invention or polypeptide can with pharmaceutically acceptable one or more carrier combined utilization.
Such carrier includes but not limited to salt solution, buffer salt solution, glucose, water, glycerine, ethanol and their combination.
Being used for polypeptide of the present invention and polynucleotide can use separately, or uses as the treatment compound in conjunction with other compound.
The administering mode of described medicinal compositions can be the efficient targeting wound location any effectively and make things convenient for mode, for example comprise part, intravenously, intramuscular, nose is interior or intradermal routes gives.
In treatment or prevention, described active medicine can be with for example aseptic aqueous dispersion of injectable composition, be preferably the isotonicity aqueous dispersion gives individuality.
Perhaps described composition can be prepared and be used for topical application, for example following form: ointment, creme, lotion, eye ointment, eye drop, [, mouth wash shua, dipping dressing and suture and aerosol, and described composition can contain suitable conventional additives, comprises for example sanitas, the solvent of ancillary drug infiltration and the softener in ointment and the creme.Such topical application preparation can also contain the conventional carrier of coupling, for example ethanol or the oleyl alcohol used of creme or ointment base and lotion.This class carrier can account for about 98% (weight) of about 1%-of described preparation; The more common height that they account for described preparation is to about 80% (weight).
Especially human in order to give Mammals, the dosage level of wishing described active medicine is 0.01mg/kg-10mg/kg, about usually 1mg/kg.Under any circumstance the attending doctor will determine the most suitable individuality and the different actual dose with age, body weight and the reaction of particular individual.Above-mentioned dosage is represented general case.Certainly may have the individual instances that dosage range should be higher or lower, they also belong to category of the present invention.In a word, the dosage selected of technician has the effect that reduces cell proliferation and can not hinder wound healing.
Again on the one hand, provide and contain NAB1 or NAB2 polypeptide or contain the nucleic acid molecule of sequence of coding NAB1 or NAB2 polypeptide and the medicinal compositions of its one or more pharmaceutically acceptable carrier.
The treatment advantage that transcription repressor is used for wound healing is to make a plurality of target gene inactivations that impel healing to accelerate.Natural activation NAB1 or NAB2 in injury response, strengthening this natural response also is a kind of advantage.Described treatment is based on DNA, and it provides reliably and repeatably transfer system.
When NAB1 or NAB2 polynucleotide were used for methods of treatment of the present invention, described polynucleotide were as the integral part of expression constructs (for example expression vector form).In such method, described construction imports damage location, produces NAB1 or NAB2 in this original position.Used construction can be standard vector and/or gene delivery system, for example liposome, receptor-mediated transfer system and virus vector.
The present invention is applicable to the wound healing (comprising cicatrization after diabetic limbs ulcer and peripheral occlusive arterial disease, the operation, burn) of all aspects, psoriatic, inhibition is regulated the angiolithic degeneration effect and is suppressed cancer cell propagation through the restenosis of skin behind the tube chamber coronary angioplasty.
As mentioned above, use any ordinary method and for example use topical administration, can be with NAB1 of the present invention or NAB2 polypeptide or nucleic acid topical administration to the tissue injury position.The preferred method that transmits nucleic acid product is to use gene gun technology, and wherein for example the Egr-1 isolated nucleic acid molecule of cDNA form or expression vector is fixed on the gold grain, and directly shoots damage location.Therefore, a preferred aspect of the present invention provides the nucleic acid molecule that will comprise the sequence of coding NAB1 or NAB2 polypeptide with particle gun and is used for the treatment of the cell proliferation disorders of following wound healing.In addition, provide the composition that is applicable to the particle gun treatment that comprises the NAB1 that is fixed on the gold grain or NAB2 encoding sequence.
Exemplify following non-limiting example referring now to accompanying drawing and describe the present invention, wherein:
Fig. 1 a)-1d) shows the activation of the PDGF-AB, TGF β, HGF and the VEGF that prevent the Egr-1 mediation respectively;
Fig. 2 describes the HGF generation that NAB2 trans-repression Egr-1 mediates in HVSMC;
Fig. 3 describes the influence of NAB2 to the vasculogenesis of Egr-1 driving;
Fig. 4 a is described in the influence that damage NAB2 transfection in back 7 days is shunk linear cuts;
Fig. 4 b describes the growth factor levels in the epidermis is hindered in the NAB2 transfection to 7 days rat otch influence;
Fig. 4 c describes the growth factor levels in the granulation tissue is hindered in the NAB2 transfection to 7 days rat otch influence; With
Fig. 4 d describes the influence of NAB2 transfection to the vasculogenesis of 7 days rat otch wounds.
Embodiment
Suggestion cultivator vascular smooth muscle (HVSMC according to the manufacturer; Clonetics).Culturing cell is to carry out transfection in 6 hole microtiter plates (Costar).To contain human cytomegalic inclusion disease virus promotor (hCMV with FUGENE (BoehringerMannheim) with the diagram ratio; Mol.Cell.Biol. such as Svaren, 16; 3545-3553,1996) expression plasmid of the NAB2 cDNA that drives and the expression plasmid that contains Egr-1 cDNA (Arterioscler.Thromb.Vasc.Biol. such as Houston, 19; 281-289,1999) be transfected among the HVSMC together.3: 1 ratio (v/w) FUGENE:DNA be used for all tests, and the beta-galactosidase enzymes expression plasmid normalization method transfection that drives with CMV.Adopt suitable contrast, use ELISA (R﹠amp; D Systems) the secretor type somatomedin in the detection tissue culture medium (TCM).1.2 result
Fig. 1 a)-1d) shows the activation of the PDGF-AB, TGF β, HGF and the VEGF that prevent the Egr-1 mediation respectively.The independent transfection of 6 μ g Egr-1 expression plasmids (Fig. 1 a, Fig. 1 b and Fig. 1 d) or with 500,100 or 25ng NAB2 cotransfection (Fig. 1 a and Fig. 1 d).When the independent transfection of Egr-1, during detection by quantitative secretor type somatomedin, PDGF-AB, TGF β and VEGF have only a little increase.During with the NAB2 cotransfection, eliminated fully Egr-1 activated PDGF-AB is replied and reduces basal expression, (Fig. 1 a) to make the ultimate production of PDGF-AB reduce by 5 times.The NAB2 transfection has been eliminated fully Egr-1 activated TGF β has been replied, and makes TGF β ultimate production reduce by 30% (Fig. 1 b).With the DNA concentration shown in the figure, the Egr-1 transfection makes HGF output increase by 50%, and the output that the cotransfection of low dosage NAB2 is partly blocked HGF increases, and the output that the cotransfection of high dosage NAB2 is blocked HGF fully increases (Fig. 1 c).Observed the maximum activation effect on the 1st day after the transfection, but the retarding effect of NAB2 at the 2nd and 3 day obviously.The same with TGF β with PDGF-AB, the activation VEGF of Egr-1 mediation has been blocked in the NAB2 transfection, and makes the VEGF ultimate production reduce by 40% (Fig. 1 d).1.3 conclusion
These data show that the somatomedin of the repressor NAB2 of Egr-1 Egr-1 mediation capable of blocking activates, and are the same with situation about seeing at the acute injury position.
Cell cultures, transfection and somatomedin detect described with above 1.1 parts.NAB2 expression vector transfection final concentration is 0,250,500,1000 or 3000ng.2.2 result
Shown in Fig. 2 a, NAB2 is transfected into and causes PDGF-AB to produce sharply minimizing among the HVSMC.The NAB2 of maximum dose level makes PDGF-AB reduce by 10 times.2.3 conclusion
These data show that the Egr-1 repressor can reduce the basal level of the somatomedin that is produced by two kinds of PDGF-AB promotors.
Embodiment 3 uses 3.1 methods of inducing of NAB vitro inhibition vasculogenesis
(Svaren etc., EMBO are J.17 in the description of existing NAB2 expression constructs (wild-type and dominant) about the hCMV promoters driven; 6010-6019,1998).We verified when by the hCMV promoter expression transfection of Egr-1 transcription factor promote vasculogenesis (patent application PG3412).In these trials, DNA is transfected in the vasculogenesis test kit, according to manufacturer's suggestion supply and maintenance (TCS Biologicals).Vegf protein (2ng/ml) is as positive control, and Suramine (20 μ M) is as angiogenesis inhibitor.Triplicate on 24 hole microtiter plates with Mirus Transit reagent (Cambridge Biosciences) with 2: 1 (v/w) DNA, by 0.5 μ g/ hole transfection CMV Egr-1 DNA.In order to analyze the potentiality that NAB2 prevents the vasculogenesis of Egr-1 mediation, with 0.5mg Egr-1 DNA and every hole 10,25 or 100ngNAB2 expression plasmid in shortage or exist under the 100ng NAB2 dominant expression plasmid and carry out cotransfection.After cultivating altogether in 11 days, the endothelial cell marker PECAM-1 by staining cell also manifests with the BCIP/NBT substrate, determines vasculogenesis.
Catch and handle typical case's image of the tubular formation of the Egr-1 expression plasmid that adopts whole 4 dosage and VEGF (positive control) and Suramine (negative control) with Quantimet 600 Quantimets and related software.3.2 result
International patent application no PCT.GB99.01722 shows that Egr-1 DNA promotes vasculogenesis (pro-angiogemc) (seeing the figure post 4 of Fig. 3 a).Shown in Fig. 3 a,, partly eliminate this dose-dependently with the dominant negative cotransfection of NAB2 and reduce with the dose-dependently reduction that the cotransfection of NAB2 produces Egr-1 induction of vascular generative capacity.3.3 conclusion
NAB2 can be used to block the vasculogenesis under the acute injury situation, and the example is the growth factor-induced of Egr-1.
Embodiment 4 uses the cicatrization 4.1 method 4.1.1 influence of the NAB2 transfection of particle gun to the growth factor levels of rat otch wound that NAB2 reduces rodent otch wound model
NAB2 is transfected into the direct repression that rodent otch wound expresses by its cicatrization somatomedin to key (being TGF β 1) and has the potentiality of anti-cicatrization.In this test, with the Biorad particle gun NAB2 cDNA is transfected into during the rat otch hinders, and with conventional organization and the immunocytochemistry evaluation effect to wound healing and growth factor levels.4.1.2 the transgenosis of particle mediation
8 male Sprague Dawley rats, the about 250g of body weight anaesthetizes under isoflorane with 2: 1 oxygen/nitrous oxide mixtures.At first wipe out fur, use the razor scraping then, prepare 2 transfection positions (from basis cranii 5cm, the 1.5cm of any side of median line) and 2 contrast positions (from basis cranii 8cm, the 1.5cm of any side of median line) at rat back.Carry out a transfection at each transfection position, the plasmid/Au composite that quickens NAB2 or Egr-1 (somatomedin activated positive control) with 350psi enters skin.0.5-1.5 μ g DNA is transmitted in each transfection usually.At a contrast position, quicken goldc grains (not having DNA) with 350psi and enter skin; Keep somewhere another contrast position inoperation.At each other animal clockwise rotation transfection position and contrast position, with the difference before and after the wound healing of contrast rodent.4.1.3 otch recovery matched moulds type
24 hours anesthetized animals after the transfection use the scalper edge of a knife at the linear omnidistance skin otch of the parallel preparation with backbone in the accurate position of transfection 1cm.Make animal normal, keep somewhere wound and under situation about not sewing up, heal by anesthesia recovery.Dissection was also collected damaged tissue and is carried out conventional organization and immunohistochemistry detection with all 8 sacrifice of animal in the 7th day after damage.4.1.4 histologic analysis
After the dissection with the horizontal five equilibrium of each damaged tissue of each time point.Half placed 4% paraformaldehyde 24 hours, and processing is used for wax biopsy tissues.Cut into slices from each damaged tissue section 5mm with slicing machine, and will cut into slices with van Geison dyeing.With the described section of optics microscopic and estimate the effect of NAB2 to healing reaction.4.1.5 immunohistochemistry
In OCT when freezing, second half of each damaged tissue cut into slices with 7mm with cryostat.Two sections of each damaged tissue are fixing in ice-cold acetone, carry out fluorescence immunization coloration by following scheme with the first antibody of anti-Egr-1, PDGF, TGF β 1, TGF β 3 and vWF.
1.PBS washed
2. applied the 30ml first antibody 1 hour
3. with PBS washing 3 * 5 minutes
4. apply 30ml second antibody (FITC directly connects) 45 minutes
5. with PBS washing 3 * 5 minutes
6. with the immobilized slide of sealing fluid-tight
Behind immunostaining, immediately each slide glass is placed under the fluorescent microscope, amplify 100 times and catch damaged area.Integrate image and setting threshold and make the background minimum.Measure painted area and intensity with image analysis and mapping.4.2 as a result 4.2.1 NAB2 to the rat otch recover from injury close influence 4.2.1.1 damage 7 days contraction of wound and histologic analysis
According to Fig. 4 a, the contraction of wound of handling with NAB2 is to the width similar with control wounds to the wound of Egr-1 transfection, and histology shows similar granulation tissue ripening degree.Conclusion
NAB2 transmits the healing speed that can not influence rodent otch recovery matched moulds type.4.2.1.2 somatomedin profile
Freezing microtome section at the damage location skin histology carries out immunostaining to Egr-1, TGF β 1, TGF β 3 and PDGF-AB, detects the staining power of each somatomedin with image analysis.Carry out independently immunohistochemistry mensuration twice at damage location.Detect in the section near the growth factor expression in epidermis on the damage location and the damage location (granulation tissue).A) the just dyeing of Urogastron
Shown in Fig. 4 b, compare with independent goldc grains contrast with Egr-1, significantly reduce the expression of TGF β 1 in epidermis in NAB2 transfection in the 7th day after the damage.Compare with the contrast of not operation, the NAB2 transfection reduces the expression of TGF β 1 in epidermis, and Egr-1 and goldc grains transmission all activate the generation of TGF β 1.Compare with the contrast of not operation with independent goldc grains, the NAB2 transfection increases the level of TGF β 3 in epidermis.NAB2 can obviously not change epidermis and express Egr-1 and PDGF.B) the just dyeing of somatomedin in the granulation tissue
Shown in Fig. 4 c, the NAB2 transfection is to damaging Egr-1, PDGF-AB and the not influence of TGF β 1 level in the 7th day the granulation tissue in back.But compare with the contrast of not operation with independent goldc grains, NAB2 all increases TGF β 3 levels in the granulation tissue.Conclusion
After damage the 7th day, the NAB2 transfection that otch is hindered reduced epidermis TGFb1 level, and increased TGF β 3 levels in epidermis and the granulation tissue.TGF β 1 is known cicatrization agent, and TGF β 3 has anti-cicatrization character (J.Cell Science 107 such as Shah; 1137-1157,1994).So transmit the effect that NAB2 can have anti-cicatrization.4.2.2 NAB2 is to the effect of vasculogenesis
The 7th day the time, the dyeing skin biopsy is also estimated vWF with immunohistochemistry and image analysis and is expressed after damage.Use is measured orthochromatic area in the damage location to the von Willebrand factor immunostaining and the image analysis of wound freezing microtome section, thus quantitative vasculogenesis.Shown in Fig. 4 d, compare with contrast (goldc grains handle wound tissue), after damage the 7th day, the neovascularity of the wound of NAB2 transfection was less.Egr-1 promotes vasculogenesis in the body, and this result supports the external discovery of embodiment 3.Conclusion
NAB2 stops the activation of vasculogenesis in the body that Egr-1 stimulates, and this result supports the effect of the repressor of the growth factor activation that NAB2 causes as acute irritation, and the example is that Egr-1 activates and produces somatomedin.
Claims (22)
1. comprise the purposes of nucleic acid molecule in the preparation medicine of the sequence of coding NAB1 or NAB2 polypeptide or its bioactive fragment, described medicine is used for the treatment of and comprises the human mammiferous cell proliferation disorders relevant with wound healing.
2. the claimed purposes of claim 1, NAB1 shown in it or NAB2 polypeptide are people NAB1 or NAB2 polypeptide.
3. each claimed purposes among the claim 1-2, the wherein said cell proliferation disorders relevant with wound healing are that hypertrophic scar forms and the formation of keloid scars.
4. each claimed purposes among the claim 1-3, wherein said nucleic acid molecule is connected with the nucleotide sequence operability that control is expressed.
5. each claimed purposes among the claim 1-4, wherein said nucleic acid molecule are identical with NAB1 or NAB2 polynucleotide sequence at least 70% or 80% or 90% or 95% on total length.
6. according to each purposes among the claim 1-5, this purposes comprises the combination of the nucleic acid molecule of the sequence that comprises not only encode NAB1 polypeptide but also encode NAB2 polypeptide or its bioactive fragment.
7. each claimed purposes among the claim 1-5, wherein said nucleic acid molecule comprise the sequence of coding NAB2 polypeptide or its bioactive fragment.
8. each claimed purposes among the claim 1-7 is wherein handled described nucleic acid molecule to give described Mammals by physical method.
9. the claimed purposes of claim 8 is wherein handled described nucleic acid molecule to give described Mammals by particle bombardment.
10. the claimed purposes of claim 9, wherein said nucleic acid molecule is fixed on the goldc grains.
11. the purposes that claim 8 is claimed is wherein handled described nucleic acid molecule to give by trace plantation (microseeding).
12. each claimed purposes among the claim 1-7, wherein said nucleic acid molecule is in carrier.
13. each claimed purposes among the claim 1-7, wherein said nucleic acid molecule is in cell.
14. be used for the nucleic acid molecule of the sequence that comprises coding NAB1 or NAB2 polypeptide or its bioactive fragment of gene therapy.
15. a medicinal compositions, it comprises nucleic acid molecule and its one or more pharmaceutically acceptable carrier of the sequence that contains coding NAB1 or NAB2 polypeptide or its bioactive fragment.
16. treatment comprises the method for the cell proliferation disorders that human Mammals is relevant with wound healing, this method comprises and gives the nucleic acid molecule that described Mammals contains the sequence of encode NAB1 or NAB2 polypeptide or its bioactive fragment.
17.NAB1 or NAB2 polypeptide or its bioactive fragment purposes in producing medicine, described medicine is used for the treatment of and comprises the human Mammals cell proliferation disorders relevant with wound healing.
18. the purposes that claim 17 is claimed, wherein said NAB1 or NAB2 polypeptide or its bioactive fragment are natural, synthetic or reorganization produces.
19. claim 17 or 18 claimed purposes, wherein said NAB1 or NAB2 polypeptide are people NAB1 or NAB2 polypeptide.
20. each claimed purposes among the claim 17-19, wherein said polypeptide are identical with NAB1 or NAB2 polynucleotide sequence at least 70% or 80% or 90% or 95% on total length.
21. treatment comprise human Mammals relevant with wound healing the method for cell proliferation disorders, this method comprises and gives NAB1 or NAB2 polypeptide or its bioactive fragment that described Mammals is treated significant quantity.
22. a medicinal compositions, it comprises NAB1 and/or NAB2 polypeptide or its bioactive fragment and its one or more pharmaceutically acceptable carrier.
Applications Claiming Priority (4)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| GBGB9814989.1A GB9814989D0 (en) | 1998-07-11 | 1998-07-11 | Gene therapy method |
| GB9814989.1 | 1998-07-11 | ||
| GBGB9819826.0A GB9819826D0 (en) | 1998-09-12 | 1998-09-12 | Gene therapy method |
| GB9819826.0 | 1998-09-12 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN1317045A true CN1317045A (en) | 2001-10-10 |
Family
ID=26314007
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN99810616A Pending CN1317045A (en) | 1998-07-11 | 1999-07-09 | Pharmaceutical uses of NAB1 and NAB2 |
Country Status (22)
| Country | Link |
|---|---|
| EP (1) | EP1097200A1 (en) |
| JP (1) | JP2002520020A (en) |
| KR (1) | KR20010071832A (en) |
| CN (1) | CN1317045A (en) |
| AP (1) | AP2001002038A0 (en) |
| AU (1) | AU763713B2 (en) |
| BR (1) | BR9912018A (en) |
| CA (1) | CA2336805A1 (en) |
| EA (1) | EA200100028A1 (en) |
| EE (1) | EE200100020A (en) |
| HR (1) | HRP20010025A2 (en) |
| HU (1) | HUP0102835A3 (en) |
| ID (1) | ID27742A (en) |
| IL (1) | IL140533A0 (en) |
| IS (1) | IS5788A (en) |
| NO (1) | NO20010166L (en) |
| NZ (1) | NZ509174A (en) |
| PL (1) | PL345507A1 (en) |
| SK (1) | SK382001A3 (en) |
| TR (1) | TR200100622T2 (en) |
| WO (1) | WO2000003014A1 (en) |
| YU (1) | YU1901A (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB9928430D0 (en) * | 1999-12-01 | 2000-01-26 | Glaxo Group Ltd | Screening |
| AU2001291019A1 (en) | 2000-09-15 | 2002-03-26 | Genvec, Inc. | Method of modulating neovascularization |
| WO2004083435A1 (en) * | 2003-03-17 | 2004-09-30 | Julius-Maximilians-Uni Versität Würzburg | Nab proteins and their use in diagnostic and therapeutic applications in heart disease |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5036006A (en) * | 1984-11-13 | 1991-07-30 | Cornell Research Foundation, Inc. | Method for transporting substances into living cells and tissues and apparatus therefor |
| US5697901A (en) * | 1989-12-14 | 1997-12-16 | Elof Eriksson | Gene delivery by microneedle injection |
-
1999
- 1999-07-09 EA EA200100028A patent/EA200100028A1/en unknown
- 1999-07-09 CA CA002336805A patent/CA2336805A1/en not_active Abandoned
- 1999-07-09 CN CN99810616A patent/CN1317045A/en active Pending
- 1999-07-09 ID IDW20010345A patent/ID27742A/en unknown
- 1999-07-09 BR BR9912018-6A patent/BR9912018A/en not_active IP Right Cessation
- 1999-07-09 EE EEP200100020A patent/EE200100020A/en unknown
- 1999-07-09 YU YU1901A patent/YU1901A/en unknown
- 1999-07-09 EP EP99931378A patent/EP1097200A1/en not_active Withdrawn
- 1999-07-09 KR KR1020017000413A patent/KR20010071832A/en not_active Application Discontinuation
- 1999-07-09 WO PCT/GB1999/002199 patent/WO2000003014A1/en not_active Application Discontinuation
- 1999-07-09 JP JP2000559235A patent/JP2002520020A/en active Pending
- 1999-07-09 NZ NZ509174A patent/NZ509174A/en unknown
- 1999-07-09 AP APAP/P/2001/002038A patent/AP2001002038A0/en unknown
- 1999-07-09 TR TR2001/00622T patent/TR200100622T2/en unknown
- 1999-07-09 PL PL99345507A patent/PL345507A1/en not_active Application Discontinuation
- 1999-07-09 HU HU0102835A patent/HUP0102835A3/en unknown
- 1999-07-09 AU AU47914/99A patent/AU763713B2/en not_active Ceased
- 1999-07-09 SK SK38-2001A patent/SK382001A3/en unknown
- 1999-07-09 IL IL14053399A patent/IL140533A0/en unknown
-
2000
- 2000-12-22 IS IS5788A patent/IS5788A/en unknown
-
2001
- 2001-01-10 NO NO20010166A patent/NO20010166L/en not_active Application Discontinuation
- 2001-01-11 HR HR20010025A patent/HRP20010025A2/en not_active Application Discontinuation
Also Published As
| Publication number | Publication date |
|---|---|
| NO20010166D0 (en) | 2001-01-10 |
| TR200100622T2 (en) | 2001-10-22 |
| AU763713B2 (en) | 2003-07-31 |
| ID27742A (en) | 2001-04-26 |
| EP1097200A1 (en) | 2001-05-09 |
| AU4791499A (en) | 2000-02-01 |
| NZ509174A (en) | 2003-08-29 |
| EE200100020A (en) | 2002-06-17 |
| YU1901A (en) | 2005-06-10 |
| BR9912018A (en) | 2006-01-31 |
| KR20010071832A (en) | 2001-07-31 |
| EA200100028A1 (en) | 2001-08-27 |
| SK382001A3 (en) | 2001-09-11 |
| HUP0102835A3 (en) | 2003-09-29 |
| CA2336805A1 (en) | 2000-01-20 |
| NO20010166L (en) | 2001-03-06 |
| JP2002520020A (en) | 2002-07-09 |
| HRP20010025A2 (en) | 2001-12-31 |
| HUP0102835A2 (en) | 2001-11-28 |
| IS5788A (en) | 2000-12-22 |
| AP2001002038A0 (en) | 2001-03-31 |
| PL345507A1 (en) | 2001-12-17 |
| WO2000003014A1 (en) | 2000-01-20 |
| IL140533A0 (en) | 2002-02-10 |
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| C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
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