CN101232898A - Use of VEGF for wound healing - Google Patents

Abstract

Methods for accelerating and/or improving wound healing in a subject by administering vascular endothelial growth factor (VEGF) are provided.

Description

VEGF is used for the purposes of wound healing

Related application

The application requires the priority and the rights and interests of U.S. Provisional Application serial number of submitting on June 17th, 2,005 60/691,909 and the U.S. Provisional Application serial number of submitting on April 21st, 2,006 60/794,008, in this their description of complete income.

Invention field

The present invention relates to by using the method that VEGF (VEGF) quickened or improved wound healing.

Background of invention

Wound healing is the process of a complexity, comprise inflammation phase, granulation tissue form phase and tissue remodeling phase (Singer and Clark, Cutaneous Wound Healing, N.Engl.J.Med.341:738-46 (1999)).These incidents are that the cytokine and the somatomedin that are discharged by damage location trigger.Many factors can cicatrize a wound and complicate or disturb normal, suitable wound healing.For example, this type of factor comprises age, infection, malnutrition, immunosuppressant, drug treating, radiation, diabetes, peripheral vascular disease, systemic disease, smoking, pressure etc.

For diabetics (diabetes are a kind of chronic, diseases of making people's weakness, in 2005 about 2,000 ten thousand people of u.s. influence), forming diabetic foot ulcers (being called wound again) is a kind of common complication.Chronic ulcer is defined as and is not that the wound of generation anatomy and functional completeness (is consulted for example Lazarus et al. via orderly and in good time repair process development Definitions and guidelines For assessment of wounds and evaluation of healing, Arch.Dermatol.130:489-93 (1994)).With regard to its essence with regard to, the diabetic foot ulcers be a kind of chronic wounds (AmericanDiabetes Association (american diabetes association), Consensus development conference on Diabetic foot wound care (about the common research and development symposium of diabetic foot wound care), Diabetes Care 22 (8): 1354-60 (1999)).Because skin is serving as the effect of the primary barrier of environment to external world, so wound open, refractory may be catastrophic; Can cause great deformity (comprise and lose extremity) and even dead.Foot ulcers is that the omen of about 85% lower extremity amputation among the diabetes patient (is consulted for example Apelqvist et al. What is the most effective way to reduce incidence of Amputation in the diabetic foot?Diabetes Metab Res.Rev.16 (1 Suppl.): S75-S83 (2000)).

According to reports, there are every year the skin wounds that need to intervene above 35,000,000 examples (to consult for example Tonnesen et al. in the U.S. Angiogenesis in Wound Healing, JID Symposium Proceedings5 (1): 40-46 (2000)).Present wound care therapy also is not very successful, because their effect and expense are disappointing.Therefore, need improve and optimize the wound healing therapy for the experimenter.The present invention is devoted to solve these demands and other demand, as after reading following summary of the invention with conspicuous.

Summary of the invention

The invention provides the method that is used for accelerating wound healing, for example acute (acute) of described wound (for example burn (burn), surgical wound (surgical wound) etc.), chronic (chronic) (for example diabetic ulcer (diabetic ulcer), pressure ulcer (pressure ulcer), decubital ulcer (decubitus ulcer), varicose ulcer (venous ulcer) etc.) or normal (normal) wound.Also provide by using VEGF (VEGF) and improved the method that wound healing reduces ulcer recurrence quantity simultaneously.Described method comprises the method for for example quickening the wound healing among the experimenter, wherein a kind of method comprises the VEGF that wound is used effective dose, the VEGF's of effective dose uses to cicatrize a wound and quickens compared with the control greater than 50% under this situation, perhaps be equal to or greater than 60%, be equal to or greater than 70%, be equal to or greater than 74%, be equal to or greater than 75%,, be equal to or greater than 80%, be equal to or greater than 85%, be equal to or greater than 90%, be equal to or greater than 95%, be equal to or greater than 100%, be equal to or greater than 110% or more.Contrast is not including, but not limited to for example implementing the experimenter who handles, perhaps use the experimenter of the VEGF that is lower than the treatment consumption, perhaps implement the experimenter of another kind of treatment of wounds, perhaps use the experimenter of placebo, or possesses or do not possess good wound care (Good Wound Care, GWC), perhaps only implement the experimenter of GWC.GWC can be including, but not limited to for example debridement (debridement), cleaning (cleaning)/dressing (dressings), ease off the pressure (pressure relief), control infection (infection control) and/or its combination.In one embodiment, the method of quickening the wound healing among the experimenter comprises the VEGF that wound is used effective dose, the using to cicatrize a wound and quicken greater than 60% compared with the control of the VEGF of effective dose wherein, and wherein said wound had existed about 4 weeks or longer time on the experimenter before using the VEGF of effective dose.In one embodiment, the method for the wound healing among the acceleration human experimenter comprises the rhVEGF that the diabetic wound is used effective dose ₁₆₅, the rhVEGF of effective dose under this situation ₁₆₅Use to cicatrize a wound and quicken greater than 60% compared with the control.In certain embodiments, the VEGFR agonist can replace or unite the VEGF use in described method.

The assessment of wound healing can be determined by % or complete wound closure that for example wound area dwindles.Wound area can be measured by quantitative analysis, for example planimetry trace of the area measurement of wound, wound (planimetric tracings) etc.Fully wound closure can be determined by for example not having drain and cover the wound closure of wrapping up in needs.The photo of wound, the physical examination of wound etc. also can be used for the assess wound healing.The acceleration of wound healing can be stated % as and quicken, and the risk of perhaps stating the healing required time as is than (Hazardratio) (for example ratio of VEGF and reference examples such as placebo) etc.In certain embodiments of the invention, risk than (HR) more than or equal to 1.75, perhaps more than or equal to 1.8, perhaps more than or equal to 1.85, perhaps more than or equal to 1.87, perhaps more than or equal to 1.9, perhaps more than or equal to 1.95, perhaps more than or equal to 1.98, perhaps more than or equal to 2.0, perhaps more than or equal to 2.1 or bigger.

In one embodiment, wound further comprises infection.In another embodiment, wound is the ischemic wound.In one embodiment, the wound area before the treatment is about 0.4cm ²Or bigger, perhaps about 1.0cm ²Or bigger, perhaps at about 1.0cm ²With about 10.0cm ²Between, perhaps at about 1.0cm ²With about 6.5cm ²Between, perhaps at about 1.0cm ²With about 5.0cm ²Between.In another embodiment, wound area is mensuration before handling with VEGF but after the debridement.In one embodiment, wound had existed about 4 weeks or longer time, perhaps 6 weeks or longer time on the experimenter before using VEGF.Aspect some, the experimenter accepts or had accepted processing of the present invention, and wherein said processing has delayed wound healing or invalid to wound healing.In another embodiment, the experimenter suffers from second kind of illness (secondary condition), and second kind of illness wherein delayed wound healing or invalid to wound healing.In another embodiment, described second kind of illness is diabetes.

In one embodiment, the VEGF that uses is VEGF ₁₆₅(for example Chong Zu people VEGF, for example people VEGF ₁₆₅).In one embodiment, VEGF is that use on surface (topically).In certain embodiments, other factor (for example angiogenesis factor or medicament, wound healing medicament or rules, somatomedin etc.) of VEGF and accelerating wound healing is co-administered.VEGF can be formulated in for example agent for slow releasing type, gel dosage form, binder or the dressing etc.In certain embodiments, the experimenter is the people.In one embodiment, the effective dose of the VEGF that is used is about 20 μ g/cm ²To about 250 μ g/cm ²In certain embodiments, the effective dose of being used is about 24 μ g/cm ², perhaps 24 μ g/cm ²In certain embodiments, the effective dose of being used is about 72 μ g/cm ², perhaps 72 μ g/cm ²In certain embodiments, the effective dose of being used is about 216 μ g/cm ², perhaps 216 μ g/cm ²In one embodiment, the effective dose of the VEGF that is used is 20 μ g/cm ²To 250 μ g/cm ²In certain embodiments, the effective dose of being used is about 24 μ g/cm ²To about 216 μ g/cm ², perhaps 24 μ g/cm ²To 216 μ g/cm ²In certain embodiments, the effective dose of being used is about 24 μ g/cm ²To about 72g/cm ², perhaps 24 μ g/cm ²To 72 μ g/cm ²In certain embodiments, the effective dose of being used is about 72 μ g/cm ²To about 216 μ g/cm ², perhaps 72 μ g/cm ²To 216 μ g/cm ²In certain embodiments, the effective dose of being used is about 216 μ g/cm ²To about 250 μ g/cm ², perhaps 216 μ g/cm ²To 250 μ g/cm ²

The VEGF of effective dose use can be once a day or an optional week for several times, for example at least biweekly, perhaps at least one Wednesday time, perhaps at least one Thursday time, perhaps at least one Friday time, perhaps at least one Saturday time.In one embodiment, VEGF used at least 6 weeks, perhaps was longer than for 6 weeks, and perhaps about at least 12 weeks are perhaps until wound fully closed (for example this can determine according to the skin closure that does not have drain or dressing needs).In one embodiment, VEGF uses a course of treatment that was less than for 20 weeks.

Method of the present invention also comprises the method for improving the wound healing in the population of subjects.For example, a kind of method comprises the VEGF that the experimenter's of described colony wound is used effective dose, and the wound healing that causes in this colony of using of the VEGF of effective dose is compared with control population and improved greater than 10% under this situation, perhaps greater than 12%, or 14%, or 15%, or 17%, or 20%, or 25%, or 30%, or 33%, or 35%, or 40%, or 45%, or 50% or bigger.For example, control population is not including, but not limited to for example implementing the experimenter who handles, perhaps use the experimenter of the VEGF that is lower than the treatment consumption, perhaps implement the experimenter of another kind of treatment of wounds, perhaps use the experimenter of placebo, or possess or do not possess good wound care (GWC), the experimenter who perhaps only implements GWC.In one embodiment, the wound healing of improvement is assessed by complete wound healing.In certain embodiments of the invention, described colony comprises having the impaired experimenter of wound healing.In one embodiment, described colony is the diabetics with chronic injury, and about 4 weeks or longer time are for example arranged before treatment.

The present invention also provides the method that reduces ulcer recurrence.For example, a kind of method comprises the VEGF that ulcer is used effective dose, and the incidence rate of ulcer decreases compared with the control by using VEGF in this case.For example, contrast is not including, but not limited to for example implementing the experimenter who handles, perhaps use the experimenter of the VEGF that is lower than the treatment consumption, perhaps implement the experimenter of another kind of treatment of wounds method, perhaps use the experimenter of placebo, or possess or do not possess good wound care (GWC), the experimenter who perhaps only implements GWC.

The accompanying drawing summary

Fig. 1 has described experimental design, and for example VGF 2763g supplies to use rhVEGF with treatment diabetic wound.

Fig. 2 has described to add in the 14th day dosage-response curve of rhVEGF in the ischemic ear wound model of rabbit.

Fig. 3 has described to add in the 8th day dosage-response curve of rhVEGF in the diabetic mice model.

Detailed Description Of The Invention

Definition

Before describing the present invention in detail, should understand and the invention is not restricted to specific composition or biology system, they can change certainly to some extent. Should also be clear that term used herein only for the purpose of describing specific embodiments, is not that intention limits it. When being used for this specification and claims, singulative " ", " a kind of ", " being somebody's turn to do " and " described " comprise the object of plural number, unless expressly stated otherwise. Therefore, for example, mention " one/kind molecule " optional combination that comprises two/kind or more/kind of this quasi-molecule, like that.

Term " VEGF " (being also referred to as VEGF-A) refers to vascular endothelial growth factor albumen when being used for this paper. Term " people VEGF " (being also referred to as people VEGF-A) refers to 165 amino acid whose human vascular endothelial growth factors when being used for this paper, and relevant 121,145,183,189 and the vascular endothelial growth factor of 206 amino acid (and other isoform (isoform)), as described in Leung et al. Science 246:1306 (1989) and Houck et al.Mol.Endocrin.5:1806 (1991), and natural allelic form and the form processing of existing of those growth factors.

" native sequences " polypeptide comprises and the polypeptide that has same acid sequence derived from natural polypeptide. So, the native sequences polypeptide can have from any mammiferous natural amino acid sequence that has polypeptide. This type of native sequences polypeptide can separate from nature, perhaps can generate by restructuring or synthesizing mean. Term " native sequences " polypeptide is clearly contained naturally occurring brachymemma or secreted form (for example ectodomain sequence), naturally occurring variant form (for example alternative splicing form) and the naturally occurring allelic variant of this polypeptide.

Polypeptide " variant " means to have biologically active polypeptides at least about 80% amino acid sequence identity with corresponding native sequences polypeptide or its fragment. This type of variant comprises for example at the N-of polypeptide end and/or the terminal polypeptide that adds or delete one or more amino acid residues of C-. Usually, variant and native sequences polypeptide or its fragment will have the amino acid sequence identity at least about 80%, perhaps at least about 90% amino acid sequence identity, perhaps at least about 95% or more amino acid sequence identity. Analog or variant are defined as the molecule that amino acid sequence, glycosylation or the further feature of natural VE GF have covalently or non-covalently been modified.

Term " VEGF variant " refers to variant mentioned above and/or comprises the VEGF of a place or many places amino acid mutation in natural VE GF sequence when being used for this paper. Optional is that a described place or many places amino acid mutation comprise amino acid replacement. Being used for VEGF of the present invention and variant thereof can prepare by several different methods well-known in the art. The amino acid sequence variant of VEGF can prepare by the sudden change among the VEGF DNA. This type of variant comprises that for example the VEGF amino acid sequence is (for example by US 5,332,671; 5,194,596; Or 5,240, the human amino acid sequence of nucleic acid coding shown in 848) interior residue deletion, insertion or alternative. Any combination that can delete, insert and substitute is to obtain to have the final construction of expectation active (for example VEGF is active, for example accelerating wound healing). The sudden change that to carry out in the DNA of coding variant must not place sequence outside the reading frame, and preferably can not produce the complementation district that can produce secondary mRNA structure. EP 75,444A. Can be active to VEGF variant assessment VEGF, for example by the cell proliferating determining method. For example, the cell proliferating determining method is included in external or improves in vivo cell with respect to untreated cell or alleviate growth and/or the breeding degree of processing cell (reduced treated cell). The rising of cell proliferation can be by detecting cell count before being exposed to molecules of interest and afterwards during cell was cultivated. The propagation degree can be converged degree by microscopic examination and come quantitatively. Cell proliferation also can be mixed determination method with thymidine and be come quantitatively.

The preparation of VEGF variant is optional by nucleotide site directed mutagenesis or display technique of bacteriophage among the DNA of coding natural VE GF, produces thus the DNA of coding variant, then expresses this DNA in the recombinant cell culture thing.

Be predetermined although be used to introduce the site of variant amino acid sequence, yet sudden change itself needn't be predetermined.For example,, can carry out random mutagenesis at target codon or zone in order to optimize the consequence of specifying the site sudden change, and to the expressed active best of breed of VEGF variant screening expectation.It is well-known being used for substituting the technology of suddenling change in the site that the DNA with known array is predetermined, such as for example site-specific mutagenesis.The preparation of VEGF variant described herein can realize by display technique of bacteriophage, such as what put down in writing among those PCT publications WO 00/63380.

After selecting a such clone, can take out the protein district after the sudden change and insert the carrier that is applicable to protein production, generally be the expression vector that can be used for transforming suitable host's the sort of type.

The scope of aminoacid sequence deletion generally is about 1-30 residue, optional 1-10 residue, and it is individual or still less to choose 1-5 wantonly, and normally successive.

Aminoacid sequence inserts the fusion that comprises amino and/or carboxyl terminal, the unrestricted in essence polypeptide from a residue to length, and insert in the sequence of single or multiple amino acid residues.The scope of inserting (being the insertion in the natural VE GF sequence) in the sequence generally can be about 1-10 residue, optional 1-5, or optional 1-3.The terminal example that inserts is included in the terminal signal sequence (no matter be allogenic or homologous to host cell) that merges of N-so that secreted by recombinant host.

Other VEGF variant have those wherein at least one amino acid residue of natural VE GF eliminated and inserted different residues in its position.This type of substitutes can be according to carrying out shown in the table 1.The VEGF variant also can comprise alpha-non-natural amino acid as herein described.

Aminoacid can be according to the following grouping of the similarity of its side chain characteristic (A.L.Lehninger, Biochemistry, the 2nd edition, pp.73-75, Worth Publishers, New York, (1975)):

(1) nonpolar: Ala (A), Val (V), Leu (L), Ile (I), Pro (P), Phe (F), Trp (W), Met (M)

(2) uncharged, polar: Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gln (Q)

(3) tart: Asp (D), Glu (E)

(4) alkalescence: Lys (K), Arg (R), His (H)

Perhaps, the natural residue that exists can be based on the following grouping of common side chain characteristic:

(1) hydrophobic: nor-leucine, Met, Ala, Val, Leu, Ile;

(2) neutral, hydrophilic: Cys, Ser, Thr, Asn, Gln;

(3) tart: Asp, Glu;

(4) alkalescence: His, Lys, Arg;

(5) influence the residue of chain orientation: Gly, Pro;

(6) aromatic: Trp, Tyr, Phe.

Table 1

Original residue	Illustration substitutes	Preferred substituting
			Ala(A)	Val；Leu；Ile	Val
Arg(R)	Lys；Gln；Asn	Lys
			Asn(N)	Gln；His；Asp；Lys；Arg	Gln
Asp(D)	Glu；Asn	Glu
			Cys(C)	Ser；Ala	Ser
Gln(Q)	Asn；Glu	Asn
			Glu(E)	Asp；Gln	Asp
Gly(G)	Ala	Ala

His(H)	Asn；Gln；Lys；Arg	Arg
			Ile(I)	Leu; Val; Met; Ala; Phe; Nor-leucine	Leu
Leu(L)	Nor-leucine; Ile; Val; Met; Ala; Phe	Ile
			Lys(K)	Arg；Gln；Asn	Arg
Met(M)	Leu；Phe；Ile	Leu
			Phe(F)	Trp；Leu；Val；Ile；Ala；Tyr	Tyr
Pro(P)	Ala	Ala
			Ser(S)	Thr	Thr
Thr(T)	Val；Ser	Ser
			Trp(W)	Tyr；Phe	Tyr
Tyr(Y)	Trp；Phe；Thr；Ser	Phe
			Val(V)	Ile; Leu; Met; Phe; Ala; Nor-leucine	Leu

" naturally occurring amino acid residue " (promptly by genetic code amino acids coding residue) can be selected from: alanine (Ala); Arginine (Arg); Agedoite (Asn); Aspartic acid (Asp); Cysteine (Cys); Glutamine (Gln); Glutamic acid (Glu); Glycine (Gly); Histidine (His); Isoleucine (Ile); Leucine (Leu); Lysine (Lys); Methionine (Met); Phenylalanine (Phe); Proline (Pro); Serine (Ser); Threonine (Thr); Tryptophan (Trp); Tyrosine (Tyr); And valine (Val)." non-natural exist amino acid residue " refers to above listed natural the existence beyond the amino acid residue, can be in polypeptide chain and the covalently bound residue of contiguous amino acid residue.Non-natural exists the example of amino acid residue to comprise for example nor-leucine, ornithine, norvaline, homoserine and other amino acid residue analog, such as what put down in writing in those Ellman et al.Meth.Enzym.202:301-336 (1991) and U.S. Patent application publication 20030108885 and 20030082575.In brief, these rules involve with non-natural and exist amino acid residue activation to prevent type tRNA, follow external or body in transcribe and translate RNA.Referring to for example U.S. Patent application publication 20030108885 and 20030082575; Noren et al.Science 244:182 (1989); And Ellman et al.supra.

" percentage ratio (%) amino acid sequence identity " is defined as the contrast sequence in this article and introduces breach where necessary with after obtaining largest percentage sequence homogeneity, and not with any conservative substituting when being considered as sequence homogeneity a part of, the percentage rate of the amino acid residue identical in the candidate sequence with amino acid residue in the selected sequence.For the contrast of the measuring percentage ratio amino acid sequence identity purpose multiple mode in can the art technology scope is carried out, for example use the available computer software of the public, such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software.Those skilled in the art can determine to be used to measure correlated suitable parameter, comprise institute's comparative sequences total length is obtained the required any algorithm of maximum contrast.Yet, being the object of the invention, % amino acid sequence identity value is relatively computer program ALIGN-2 acquisition of use sequence as described below.ALIGN-2 sequence comparison computer program is write by Genentech company, submit to (the US CopyrightOffice of U.S. Copyright Bureau together with customer documentation, Washington D.C.20559), and register with U.S. copyright registration TXU510087, the public can pass through Genentech company, and (South San Francisco California) obtains.The ALIGN2 program should be compiled in UNIX operating system, uses on the preferred digital UNIX V4.0D.The all sequences comparative parameter is by ALIGN-2 program setting and constant.

Be the object of the invention, given aminoacid sequence A with respect to (to), with (with) or at the % amino acid sequence identity of (against) given aminoacid sequence B (perhaps can be expressed as have or comprise with respect to, with or at the given aminoacid sequence A of a certain % amino acid sequence identity of given aminoacid sequence B) following calculating:

Mark X/Y takes advantage of 100

Wherein X is to be the total number of atnino acid of identical match by sequence contrast program ALIGN-2 scoring in the A of this program and B contrast, and wherein Y is an amino acid residue sum among the B.Can understand, if the length of the length of aminoacid sequence A and aminoacid sequence B is unequal, then A will be not equal to the % amino acid sequence identity of B with respect to A with respect to the % amino acid sequence identity of B.

Term " vegf receptor " or " VEGFR " refer to the cell receptor of VEGF when being used for this paper, the cell surface receptor that normally finds on vascular endothelial cell, and the variant of reservation and VEGF binding ability.

The molecule that term " VEGFR agonist " refers to activate vegf receptor or improves its expression.The VEGFR agonist includes but not limited to for example ligand agonist, VEGF variant, antibody and the active fragment of VEGFR.VEGF is the VEGFR agonist, but in this article it is separately enumerated and censures.Term " anti-VEGFR antibody " refers to enough affinitys and specificity and the bonded antibody of VEGFR.In one embodiment, anti-VEGFR agonistic antibody of the present invention can be used as the therapeutic agent in the treatment wound.In another embodiment, the VEGF variant can be used as the therapeutic agent in the treatment wound.

Term " antibody " uses with broad sense, comprise monoclonal antibody (comprising monoclonal antibody total length or complete), polyclonal antibody, multivalent antibody, multi-specificity antibody (for example bi-specific antibody), reach antibody fragment, as long as they show desired biological activity.

Except as otherwise noted, statement " multivalent antibody " is used in reference to the antibody that comprises three or more antigen binding sites.Multivalent antibody is transformed into usually has three or more antigen binding sites, and generally is not native sequences IgM or IgA antibody.

" antibody fragment " only comprises the part of complete antibody, generally comprises the antigen binding site of complete antibody, therefore kept and the bonded ability of antigen.The example of the antibody fragment that this definition is contained comprises: (i) Fab fragment, and it has VL, CL, VH and CH1 domain; (ii) Fab ' fragment, it is the Fab fragment that has one or more cysteine residues at the C-of CH1 domain end; (iii) Fd fragment, it has VH and CH1 domain; (iv) Fd ' fragment, it has one or more cysteine residues of VH and CH1 domain and CH1 domain C-end; (v) Fv fragment, it has the VL and the VH domain of antibody single armed; (vi) dAb fragment (Ward et al.Nature 341:544-546 (1989)), it is made up of the VH domain; (vii) isolating CDR district; (viii) F (ab ') ₂Fragment comprises by the continuous segmental bivalence fragment of two Fab ' of the disulfide bond of hinge region; (ix) single-chain antibody molecule (strand Fv for example; ScFv) (Birdet al.Science 242:423-426 (1988) and Huston et al.PNAS (USA) 85:5879-5883 (1988)); (x) " double antibody ", it has two antigen binding sites, is included in heavy chain variable domain (VH) continuous in same the polypeptide chain and light chain variable territory (VL) (referring to for example EP 404,097; WO93/11161; With Hollinger et al.Proc.Natl.Acad.Sci.USA 90:6444-6448 (1993)); (xi) " linear antibody ", it comprises the Fd section (VH-CH1-VH-CH1) of pair of series, form a pair of antigen binding domain (Zapata et al.Protein Eng.8 (10): 1057-1062 (1995) and United States Patent (USP) 5,641,870) with complementary light chain polypeptide.

Term " monoclonal antibody " refers to that when being used for this paper each antibody that promptly constitutes colony is identical from a group antibody that obtains of the antibody of homogeneity basically, except may be with indivisible possible natural the existence the sudden change that exists.Monoclonal antibody is a high degree of specificity, at single antigen.In addition, with the polyclonal antibody prepared product difference that typically comprises at the different antibodies of different determinants (epi-position), every kind of monoclonal antibody is at the single determinant on the antigen.Modifier " monoclonal " should not be construed as requirement and generates antibody by any ad hoc approach.For example, to can prepare by the hybridoma method of putting down in writing by Kohler et al.Nature 256:495 (1975) at first according to the monoclonal antibody that the present invention uses, perhaps can prepare (referring to for example U.S. Patent No. 4,816,567) by recombinant DNA method." monoclonal antibody " also can use the technology of record among Clackson et al.Nature 352:624-628 (1991) for example or the Marks et al.J.Mol.Biol.222:581-597 (1991) to separate from phage antibody library.

Monoclonal antibody clearly comprises " chimeric " antibody in this article, wherein the part of heavy chain and/or light chain with derived from specific species or belong to the identical or homology of corresponding sequence in the antibody of specific antibodies classification or subclass, and the remainder of chain with derived from another species or belong to the identical or homology of corresponding sequence in the antibody of another antibody classification or subclass, and the fragment of this antibody-like, as long as they show desired biological activity (U.S. Patent No. 4,816,567 and Morrison et al.Proc.Natl.Acad Sci.USA 81:6851-6855 (1984)).

" humanization " form of inhuman (for example Mus) antibody refers to that bottom line comprises the chimeric antibody derived from the sequence of non-human immunoglobulin.Largely, humanized antibody refers to the immunoglobulin that the hypervariable region residue among human normal immunoglobulin's (receptor antibody) is replaced with the hypervariable region residue of inhuman species (donor antibody) such as mice, rat, rabbit or non-human primate with expectation specificity, affinity and ability.In some situation, human normal immunoglobulin's framework region (FR) residue is replaced with corresponding inhuman residue.In addition, humanized antibody can be included in the receptor antibody or the residue that does not find in donor antibody.Carrying out these modifications is in order further to improve the performance of antibody.Generally speaking, humanized antibody will comprise at least one, common two whole basically following variable domains, wherein all or basically all hypermutation rings corresponding to the hypermutation ring of non-human immunoglobulin, and all or basically all FR are FR of human normal immunoglobulin's sequence.Optional partial immunity immunoglobulin constant district (Fc), normally human normal immunoglobulin's the constant region at least of also will comprising of humanized antibody.More details are referring to Jones et al.Nature 321:522-525 (1986); Riechmann et al.Nature 332:323-329 (1988); And Presta, Curr.Op.Struct.Biol.2:593-596 (1992).

" people's antibody " refers to have and the aminoacid sequence corresponding amino acid sequence of the antibody that is generated by the people and/or the antibody that uses any technology that is used to generate people's antibody disclosed herein to generate.This definition clear-cut of people's antibody is got rid of and is comprised the humanized antibody of non-human antigen in conjunction with residue.People's antibody can use multiple technologies known in the art to generate.In one embodiment, people's antibody is selected from phage library, this phage library expressing human antibody (Vaughan et al.Nature Biotechnology 14:309-314 (1996); Sheets et al.PNAS (USA) 95:6157-6162 (1998); Hoogenboom andWinter, J.Mol.Biol.227:381 (1991); Marks et al.J.Mol.Biol.222:581 (1991)).People's antibody also can by human immunoglobulin gene's seat is imported endogenous immunoglobulin gene partially or completely the transgenic animal of deactivation for example mice generate.When under attack, observe people's antibody and generate, it in all respects in human body, see extremely similar, comprise gene rearrangement, assembling and antibody complete or collected works.This method is recorded in for example U.S. Patent No. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016, and following scientific publication thing: Marks et al.Bio/Technology 10:779-783 (1992); Lonberg et al.Nature 368:856-859 (1994); Morrison, Nature 368:812-13 (1994); Fishwild et al.Nature Biotechnology14:845-51 (1996); Neuberger, Nature Biotechnology 14:826 (1996); Lonberg andHuszar, Intern.Rev.Immunol.13:65-93 (1995).Perhaps, people's antibody can prepare (this type of bone-marrow-derived lymphocyte can reclaim from individuality, perhaps can in external immunity) by generating immortalization at the human B lymphocyte of the antibody of target antigen.Referring to for example Cole et al.Monoclonal Antibodies andCancer Therapy, Alan R.Liss, p.77 (1985); Boerner et al.J.Immunol.147 (1): 86-95 (1991); U.S. Patent No. 5,750,373.

Diabetes are the chronic diseases that influence carbohydrate, fat and protein metabolism in animal.Diabetes are the main causes that cause blind, renal failure and lower extremity amputation in the adult, and are the principal risk factors of cardiovascular disease and apoplexy.

Type i diabetes (or insulin dependent diabetes mellitus (IDDM) (" IDDM ") or young hair style diabetes) accounts for about 10% of all diabetes cases.This disease is characterised in that the gradual forfeiture insulin secretion function of pancreatic beta cell.This feature is also shared by the Fei Tefa that is derived from pancreatic diseases or " Secondary cases " diabetes.Type i diabetes is relevant with following clinical sign or symptom, for example continues the plasma glucose concentration or the hyperglycemia that raise; Polyuria; Polydipsia and/or hyperphagia; Chronic microvascular complication is such as retinopathy, nephropathy and neuropathy; And the trunk complication, such as hyperlipemia and hypertension, can cause losing one's sight, latter stage nephropathy, amputation and myocardial infarction.

Type ii diabetes (non-insulin-dependent diabetes mellitus or NIDDM) is to involve glucose metabolism imbalance and the impaired metabolic disorder of insulin sensitivity.Type ii diabetes forms in adult age usually, and can not utilize with health or synthetic enough insulin relevant.Except observed insulin resistant in target tissue, the patient who suffers from type ii diabetes has relative insulin deficit--that is to say that patient's insulin level is compared the low of given plasma glucose concentration prediction.Type ii diabetes is characterised in that following clinical sign or symptom, for example continues the plasma glucose concentration or the hyperglycemia that raise; Polyuria; Polydipsia and/or hyperphagia; Chronic microvascular complication is such as retinopathy, nephropathy and neuropathy; And the trunk complication, such as hyperlipemia and hypertension, can cause losing one's sight, latter stage nephropathy, amputation and myocardial infarction.

Be the object of the invention, " experimenter " refers to any animal.Animal refers generally to mammal.Be the object of the invention, " mammal " aim is gone into mammiferous any animal, comprises the people, and domestic animal and domestic animal, and zoo, motion or pet animals are such as dog, horse, cat, cattle, sheep, pig etc.Mammal is often referred to the people.

Term " accelerating wound healing " or " acceleration of wound healing " refer to the to heal rising of speed, for example the closed fully time that needs of wound shortens or wound area % dwindles the time that needs and shortens.

Term " effective dose " or " treatment effective dose " refer to the amount that medicine effectively quickens or improves the wound healing among the experimenter or prevent ulcer recurrence among the experimenter.Therapeutic dose refers to the experimenter is represented the dosage of therapeutic effect, and inferior therapeutic dose refers to the processing experimenter is not represented the dosage of therapeutic effect.

Use with one or more other therapeutic agents " associating " and to comprise that simultaneously (jointly) uses and/or the sequential of any order used.

" processing " and " treatment " (treatment) refer to that therapeutic is disposed and preventative or precaution measure the two.Need the experimenter of treatment comprise those suffer from for a long time disease and those to prevent disease.

" wound healing " refers to benefit from the situation of the treatment of using molecule of the present invention.

" chronic wounds " (chronic wound) refers to the wound that can not heal.Referring to for example Lazarus et al. Definitions and guidelines for assessment of wounds and evaluation of healing, Arch.Dermatol.130:489-93 (1994).Chronic wounds includes but not limited to for example ulcer of artery (arterial ulcer), diabetic ulcer, pressure ulcer, varicose ulcer etc.Acute wounds (acute wound) can develop into chronic wounds.Acute wounds includes but not limited to the wound that caused by for example thermal burn, wound, operation, large area skin carcinectomy, deep fungal and bacterial infection, vasculitis, scleroderma, pemphigus toxic epidermal necrolysis etc.Referring to for example Buford, Wound Healing and Pressure Sores, Healing Well.com is published in October 24 calendar year 2001." normal wound " (normalwound) refers to take place the wound that normal wound healing is repaired.

" good wound care " (Good Wound Care GWC) refers to the step of nuring wound.For example, the practice of good wound care includes but not limited to the next item down or multinomial: debridement (for example operation/rapid (sharp), machinery, self-dissolving or chemistry/enzymatic), clean (for example conventional wound cleans, and uses for example saline), dressing, ease off the pressure (pressure relief) (for example unloading the pressure (off-loading pressure to the foot) to foot), keep moist wound environment and/or control infection (for example antibiotic ointment or pill).Other step is optional to be comprised and makes that the experimenter feels comfortably cool, the footwear of cushioning, nutritional support, keeps blood-glucose control, manages other risks and assumptions (for example body weight, smoking) etc.GWC can comprise one or more practices.

Statement " influence the wound of blood vessel endothelium " refer to animal or human, preferred mammal, optimum choose suffered to blood vessel or heart, comprise the wound (trauma) of the blood vessel network of each organ, such as damaging (injury).The example of this type of wound comprises tear (laceration) of wound (wound), otch (incision) and ulcer or blood vessel or heart.Wound comprise the situation that causes by internal event and apply by external factor, can be by the situation that promotes that vascular endothelial cell growth improves.

" angiogenesis factor " or " blood vessel propellant " refers to stimulate vascular development, for example promotes blood vessel that the somatomedin of (angiogenesis), endothelial cell growth, blood vessel stability and/or angiogenesis (vasculogenesis) etc. takes place.For example, angiogenesis factor includes but not limited to that for example member, PlGF, PDGF family, fibroblast growth family (FGF), TIE part (angiogenin), the liver of VEGF and VEGF family are joined albumen, ANGPTL3, ANGPTL4 etc.It also comprises such as growth hormone, insulin like growth factor-1 (IGF-I), VIGF, epidermal growth factor (EGF), CTGF and family member thereof, and the factor of TGF-α and TGF-β.Referring to for example Klagsbrun and D ' Amore, Annu.Rev.Physiol.53:217-39 (1991); Streit and Detmar, Oncogene 22:3172-3179 (2003); Ferrara﹠amp; Alitalo, Nature Medicine 5 (12): 1359-1364 (1999); Tonini et al.Oncogene 22:6549-6556 (2003) (for example enumerating the table 1 of angiogenesis factor); And Sato, Int.J.Clin.Oncol.8:200-206 (2003).

Wound healing

Wound healing is the process of a complexity, relates to three Main Stage: inflammation, granulation formative tissue and tissue remodeling.Have many activities carrying out in wound location, for example migration/contraction, matrix metalloproteinase (MMP) generation, propagation and blood vessel take place.Exist and shrink (wound dwindles), epithelium formation (new epithelial cell produces) and connective tissue deposition to cicatrize a wound.Consult Singer﹠amp; Clark, Cutaneous Wound Healing, N.Engl.J.Med 341:738-46 (1999).A target of wound therapy is to promote granulation to form substrate, needs competent blood supply under this situation.Yet, risks and assumptions, usually relevant with morbid state, (for example including, but not limited to age, infection, malnutrition, immunosuppressant, drug treating, radiation, diabetes, peripheral vascular disease, systemic disease, smoking, pressure etc.) have caused the challenge to wound healing.

The example of some risks and assumptions of diabetic foot ulcers comprises peripheral neuropathy, and its influences the motion of foot and feels two kinds of functions, and the restriction joint mobilization makes foot deformity, and foot pressure distributes unusual, little wound repeatedly, and weakening visual sensitivity.Consult for example Boyko et al. A prospective study of Risk factors for diabetic foot ulcer, The Seattle Diabetic Foot Study(prospective study of the risks and assumptions of relevant diabetic foot ulcers, the research of Seattle diabetic foot), Diabetes Care22:1036-42 (1999) and Apelqvist et al. International consensus and practical Guidelines on the management and the prevention of the diabetic foot, International Working Group on the Diabetic Foot(about the international common of the processing of diabetic foot and prevention with use policy, international diabetic foot seminar), Diabetes Metab Res.Rev.16 (1Suppl): S84-S92 (2000).Esthesioneurosis is a principal element on every side.About 45%-60% of all diabetic ulcers is neuropathic, and has nerve and ischemic composition concurrently up to 45%.Because foot is sensation not, the patient the walking of life every day and can not perception between active stage because footgear discomfort repeated injury that foot is caused for example.Neuropathy combines with altered walking biomechanics, causes repeated blunt wound and abnormal pressure burden that foot is subject to divide in the traumatic part are distributed, and causes callus to form and skin erosion.In case formation ulcer, indolence usually can continue to enlarge, and offers an opportunity for part or system infect, and need carry out comprehensively medical treatment and operation nursing promotes to heal.

Also exist and be used for the challenge of accelerating wound healing, for example quicken the chronic wounds healing, for example the diabetic foot ulcers setting up protein protective.Wound area is a kind of hostile environment (proteolytic enzyme, (superimposed infection) infected in the natural generation inhibitor of protein active and stack), wherein usually under morbid state (for example in diabetes), host factor be altered (for example, in diabetes, have that vegf expression is suppressed, VEGF replys that weakening, cell metabolism change, immunity/inflammatory reaction is suppressed etc. to histanoxia).For example, based in vitro study, keratinocyte and fibroblast from diabetes (db/db) mice show the selectivity weakening that normal structure is repaired necessary cellular activity, and the db/db fibroblast demonstrates significantly reduced cell migration and somatomedin changes.Consult for example Frank et al. Regulation of vascular endothelial growth factor Expression in cultured keratinocytes, Implications for normal and impaired Wound healing(regulation and control of the keratinocyte medium vessels endothelial growth factor expression of artificial culture are with the relation of normal and impaired wound healing), J.Biol.Chem.270:12607-13 (1995) and Lerman et al. Cellular dysfunction in the diabetic fibroblast:impairment in Migration, vascular endothelial growth factor production, and response to Hypoxia(the fibroblastic cell dysfunction of diabetic: migration, VEGF generation and the weakening that anoxia is replied), Am.J.Pathol.162:303-12 (2003).

This paper provides the method for quickening and/or improve wound healing by VEGF that uses effective dose or VEGF agonist.For example, a kind of method comprises the VEGF that experimenter's wound is used effective dose, and using of the VEGF of effective dose quickened wound healing under this situation.Described method also comprises the method for improving the wound healing in the population of subjects.For example, a kind of method comprises the VEGF that the experimenter's of colony wound is used effective dose, wherein the wound healing that causes in this colony of using of the VEGF of effective dose improves compared with the control greater than 10%, or greater than 12% or 14% or 15% or 17% or 20% or 25% or 30% or 33% or 35% or 40% or 45% or 50% or more.The method that is used to reduce ulcer recurrence also is provided.For example, a kind of method comprises the VEGF that ulcer is used effective dose, and wherein VEGF uses the incidence rate of ulcer recurrence has been descended compared with the control.

Described method also is applicable to the experimenter who is accepting to handle or accepted processing, and described processing has delayed wound healing or invalid to wound healing under this situation.Processing can be including, but not limited to drug treating, radiate, cause the downtrod processing of immune system etc.Optional is, experimenter of the present invention suffers from second kind of illness, and second kind of illness wherein delayed wound healing or invalid to wound healing.Second kind of illness including, but not limited to for example diabetes, peripheral vascular disease, infection, autoimmune or collagen vascular disorder, cause the downtrod morbid state of immune system etc.

The acceleration of wound healing can be described as wound healing and quickens percentage ratio (%) and/or risk ratio.In certain embodiments, the using to cicatrize a wound of the VEGF of effective dose quickened compared with the control greater than 50%, or is equal to or greater than 60%, is equal to or greater than 70%, be equal to or greater than 74%, be equal to or greater than 75%, be equal to or greater than 80%, be equal to or greater than 85%, be equal to or greater than 90%, be equal to or greater than 95%, be equal to or greater than 100%, be equal to or greater than 110% or more.In certain embodiments, the VEGF's of effective dose uses to cicatrize a wound and quickens compared with the control greater than between 60% and 110%.In certain embodiments, the acceleration of wound healing is described as the risk ratio, it is equal to or greater than 1.75, or be equal to or greater than 1.80, or be equal to or greater than 1.85, or be equal to or greater than 1.95, or be equal to or greater than 2.0, or be equal to or greater than 2.1, or be equal to or greater than 2.2, or be equal to or greater than 2.3 or bigger.In certain embodiments, the acceleration of wound healing is described as the risk ratio, and it is between 1.75 and 2.3.

Experimenter of the present invention has at least one place wound.Described wound can be chronic, acute or normal wound.In one embodiment, the wound of being treated is a 1A phase wound.Consult the wound stage of table 2.Wound of the present invention can be chosen wantonly and comprise infection or ischemia, perhaps comprises and infecting and ischemia.In one embodiment, wound is the diabetic foot ulcers.In one embodiment, wound had existed about 4 weeks or longer time on the experimenter before using VEGF, perhaps about 6 weeks or longer time.

Table 2

	Wound rank/degree of depth
					The stage/with sick	0	?1	?2	?3
A	Before or after the ulcer damage that epithelium is completed into	The skin ulcer that does not relate to tendon, capsule or bone	Go deep into the ulcer of tendon or capsule	Go deep into the ulcer in bone or joint
					B	With infection	With infection	With infection	With infection
C	With ischemia	With ischemia	With ischemia	With ischemia
					D	With infecting and ischemia	With infecting and ischemia	With infecting and ischemia	With infecting and ischemia

Can come assess wound healing with quantitative analysis, for example measure wound area and dwindle percentage ratio (%), perhaps wound closure (for example by do not have drain or cover wrap up in the skin closure that needs and measure) fully.By method known to those skilled in the art before handling, during and assess wound area afterwards.For example, can be by for example quantitatively planimetry (consulting for example Robson et al.Arch.Surg.135:773-77 (2000)), photograph, physical examination wait and assess.Can be before handling, during and measure wound area afterwards.In one embodiment, wound area can be by measuring the length L of wound, and promptly the longest edge is to edge length, with for example centimetre (cm) expression, and width W, with L the longest vertical edge to border width, with centimetre (cm) expression, the multiplication LxW surface area (cm that obtains estimating then ²).The size of pending wound can change.In one embodiment of the invention, handling preceding wound area is about 0.4cm ²Or bigger, or about 1.0cm ²Or bigger, or about 0.4cm ²With about 10cm ²Between, or about 1cm ²With about 10cm ²Between, or about 1cm ²With about 6.5cm ²Between, or about 1cm ²With about 5cm ²Between, or greater than 4.0cm ²Area can be measured before or after debridement.

VEGF

The VEGF that uses effective dose in method provided herein promotes wound healing that quicken or improvement.The VEGF gene family is one of them member for its VEGF, is one of crucial modulator of vascular system growth.The VEGF gene family comprises VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E and placental growth factor (PlGF).Consult for example Ferrara, Role of Vascular endothelial Growth factor in physiologic and pathologic angiogenesis:therapeutic Implications(effect of VEGF in physiological and pathologic vessels generation: the treatment enlightenment), Sem Oncol.29 (suppl 16): 10-14 (2002) and Veikkola and Alitalo, VEGFs Receptors and angiogenesis(vegf receptor and blood vessel take place), Semin Cancer Biol.9:211-220 (1999).VEGF-A is called VEGF again, is the main modulator that normal blood vessels takes place, and comprises that normal wound healing and knitting and unusual blood vessel take place, such as the blood vessel hyperplasia (for example degeneration that the age is relevant, diabetic retinopathy) in tumor and the eye disease.Consult for example Ferrara, Vascular Endothelial Growth Factor:Basic Science and Clinical Progress(VEGF: basic science and clinical progress), Endocrine Reviews 25 (4): 581-611 (2004); Ferrara and Henzel, Pituitary follicular cells secrete a novel Heparin-binding growth factor specific for vascular endothelial cells(the follicular cell secretion is a kind of to the specific heparin binding growth factor of vascular endothelial cell), Biochem.BiophysRes Comm 161:851-58 (1989); With Leung et al. Vascular endothelial growth Factor is a secreted angiogenic mitogen(VEGF is a kind of secreting type blood vessel generation mitogen), Science 246:1306-9 (1989).Substitute or associating VEGF uses the agonist with the active VEGF variant of VEGF and vegf receptor for example VEGFR1 and/or VEGFR2 agonist be within the scope of the invention.

There is at least 6 kinds of isoforms (isoform) (VEGF in people's VEGF ₁₂₁, VEGF ₁₄₅, VEGF ₁₆₅, VEGF ₁₈₃, VEGF ₁₈₉And VEGF ₂₀₆), they by the mRNA alternative splicing that is positioned at the term single gene that is organized into 8 exons on the chromosome 6 produce (consult for example Ferrara N, Davis Smyth T, Endocr Rev18:1-22 (1997) and Henry and Abraham, Review of Preclinical and Clinical Results With Vascular Endothelial Growth Factors for Therapeutic Angiogenesis(about being used for the treatment of property of VEGF blood vessel take place clinical before and the review of clinical effectiveness ),CurrentInterventional Cardiology Reports 2:228-241 (2000)).Also can consult U.S. Patent No. 5,332,671 and 6,899,882.In one embodiment, use VEGF in the method for the invention ₁₆₅Common end user VEGF ₁₆₅(Chong Zu people VEGF for example ₁₆₅).VEGF ₁₆₅, the abundantest isoform, be a kind of alkalescence, with the covalency glycoprotein of heparin-bounding, dimerization, molecular weight is about 45,000 dalton (Id).VEGF ₁₆₅Homodimer is made up of two 165 amino acid whose chains.This protein has two particular structure territories: receptors bind domain (1-110 position residue) and heparin binding structural domain (110-165 position residue).Described domain is stable by 7 intramolecular disulfide bonds, and monomer is connected to form natural homodimer by two intermolecular disulfide bonds.VEGF ₁₂₁Lack heparin binding structural domain (consulting for example U.S. Patent No. 5,194,596), and VEGF ₁₈₉(consult for example U.S. Patent No. 5,008,196; 5,036,003; With 5,240,848) and VEGF ₂₀₆Detain in extracellular matrix.Consult for example Ferrara, VEGF and the quest for tumor angiogenesis factors(VEGF and to the searching of tumor angiogenic factor), Nature Rev.Cancer 2:795-803 (2002).

The biological action of VEGF mediates by the high-affinity tyrosine kinase receptor.The agonist of vegf receptor also can be used for method of the present invention.Two kinds of vegf receptor tyrosine kinases have been identified, VEGFR1 and VEGFR2 (Shibuya et al.Oncogene 5:519-24 (1990); Matthews et al.Proc Natl Acad Sci USA 88:9026-30 (1991); Terman et al.Oncogene 6:1677-83 (1991); Terman et al.Biochem Biophys Res Commun 187:1579-86 (1992); DeVries et al.Science 255:989-91 (1992); Millauer et al.Cell 72:835-46 (1993); With Quinn et al.Proc Natl Acad Sci USA 90:7533-7 (1993)).VEGFR1 has the highest affinity to VEGF, Kd is about 10-20pM (de Vries et al.Science 255:989-91 (1992)), and VEGFR2 has low slightly affinity to VEGF, and Kd is about 75-125pM (Termanet al.Oncogene 6:1677-83 (1991); Millauer et al.Cell 72:835-46 (1993); And Quinn et al.Proc Natl Acad Sci USA 90:7533-7 (1993)).Identified the third tyrosine kinase receptor, VEGFR3, it relates to the regulation and control that lymphatic vessel takes place.VEGFR3 is the receptor of VEGF-C and VEGF-D (they also can combine with VEGFR2).These receptors are made up of with born of the same parents' intracellular domain (comprising the element relevant with tyrosine kinase pathway) ectodomain (comprise 7 immunoglobulin-like districts, 1 stride the film district).VEGF-B and PlGF be not in conjunction with VEGFR1 but in conjunction with VEGFR2.VEGF ₁₆₅Also in conjunction with neuropil albumen-1, the receptor of a kind of adjusting neurocyte guiding (guidance).With the VEGFR2 coexpression time, neuropil albumen-1 strengthens VEGF ₁₆₅With combining and the chemotaxis of VEGF mediation of VEGFR2.Other research has been grown neuropil albumen 2 (NP2) and has been connected with lymphatic vessel.Consult for example Ferrara, Vascular Endothelial Growth Factor:Basic Science and Clinical Progress(VEGF: basic science and clinical progress), Endocrine Reviews254 (4): 581-611.With after VEGF-A combines, the tyrosine autophosphorylation takes place in VEGFR2, causes subsequently blood vessel generation, vascular permeability rising, mitosis and chemotaxis.

VEGF has several biological functions, comprise regulation and control VEGF gene expression (Ferrara N under the hypoxia condition, Davis Smyth T, Endocr Rev 18:1-22 (1997)), to mitogenic activity (the Ferrara N of blood capillary and trunk endotheliocyte, Henzel WJ, Biochem Biophys ResCommun 161:851-8 (1989); Leung et al.Science 246:1306-9 (1989); Connolly etal.J Clin Invest 84:1470-8 (1989a); Keck et al.Science 246:1309-12 (1989); Plouet et al.EMBO J 8:3801-6 (1989); Conn et al.Proc Natl Acad Sci USA87:2628-32 (1990); And Pepper et al.Exp Cell Res 210:298-305 (1994)) and induce the expression (Pepper et al.Biochem Biophys Res Commun181:902-6 (1991)) of plasminogen activator and collagenase.During hypoxia, hypoxia inducible factor-1 (HIP-1) raises, and (consults for example Wang et al. in conjunction with the promoter region and the activated transcription of VEGF gene Hypoxia-inducible factor 1 Is a basic-helix-loop-helix-PAS heterodimer regulated by cellular oxygen tension(but hypoxia inducible factor 1 is by the pressure controlled basic helix-loop-helix of cell oxygen-PAS heterodimer), PNAS USA 92:5510 (1995)).Other agonist that can raise VEGF comprises cytokine (IL-6) and other somatomedin (comprising EGF, PDGF, bFGF).

Except polytype solid tumor, VEGF is produced by the extremely multiple normal cell type that spreads all over whole body (for example, keratinocyte, platelet, macrophage, fibroblast, retina cell, gonad cell).The important function (Ferrara et al.Endocr Rev18:4-25 (1997)) of VEGF (VEGF) in regulating normal and abnormal vascular generation determined in the work of finishing in the past few years.VEGF for normal embryonic blood vessel generate, myocardial cell grows and (to consult for example Ferrara et al. Heterozygous embryonic lethality induced by targeted inactivation Of the VEGF gene(the heterozygosis embryonic death that the deactivation of VEGF gene target is brought out), Nature380:439-42 (1996)), normal endochondral bone formation (is consulted for example Gerber et al. VEGF Couples hypertrophic cartilage remodeling, ossification, and angiogenesis during Enchondral bone formation(VEGF coupling proliferative cartilage is reinvented, is ossify and the blood vessel generation during the endochondral bone formation), Nat Med 5:623-28 (1999)), tissue repair is a kind of somatomedin of necessity, and in the physiology of female reproductive tract-endocrine function of folliculus growth and corpus luteum depends on new propagation capillaceous and (consult for example Phillips et al. Vascular endothelial growth factor is Expressed in rat corpus luteum(VEGF is expressed in the rat corpus luteum), Endocrinology, 127:965-67 (1990)).In addition, to have demonstrated be the crucial medium (Ferrara et al.) of the neovascularization relevant with the ophthalmic disease with tumor to VEGF.Great majority are crossed VEGF expression mRNA (Berkman et al.J Clin Invest 91:153-159 (1993) through people's tumor of checking; Brown etal.Human Pathol 26:86-91 (1995); Brown et al.Cancer Res 53:4727-4735 (1993); Mattern et al.Brit J Cancer 73:931-934 (1996); And Dvorak et al.Am JPathol 146:1029-1039 (1995)).

VEGF is called vascular permeability factor again, based on the ability of its induction of vascular seepage in animal model.Consult for example Senger et al. Tumor cells secrete a vascular permeability factor that Promotes accumulation of ascites fluid(tumor cell secretion promotes the vascular permeability factor that ascites is gathered) Science 219:983-89 (1983).It is committed step during blood vessel takes place to the rising of proteinic permeability that Senger and colleague thereof propose blood capillary.For example, the formation of the seepage of inductive plasma proteins and born of the same parents' outer fiber protein-colloid can become sufficient substrate for endothelial cell growth; The effect of mitogenesis somatomedin can be to advance this process.Leukocyte, somatomedin and oxygen can move during also needing blood vessel to make the granulation tissue of wound healing form.

In wound healing, VEGF during the skin wound healing blood vessel take place induce in bring into play pivotal role.It is a kind of strong mitogen for dermal microvascular endothelial cell, and (consults for example Nissen et al. by the keratinocyte expression of the wound in the healing Vascular endothelial growth Factor mediates angiogenic activity during the proliferative phase of wound Healing(VEGF takes place active at the proliferative phase intermediary emissary vein of wound healing) AmJ Pathol 152:1445-52 (1998); Corral et al. Vascular endothelial growth factor is More important than basic fibroblast growth factor during ischemic wound Healing(VEGF is more important than basic fibroblast growth factor in the ischemic wound healing process) Arch Surg 134:200-5 (1999); Frank et al. Regulation of vascular Endothelial growth factor expression in cultured keratinocytes, Implications for Normal and impaired wound healing(regulation and control of the keratinocyte medium vessels endothelial growth factor expression of artificial culture are to the enlightenment of normal and impaired wound healing) J Biol Chem270:12607-13 (1995); Ballaun et al. Human keratinocytes express the three Major splice forms of vascular endothelial growth factor(people's keratinocyte is expressed three kinds of main splicing forms of VEGF) J Invest Dermatol 104:7-10 (1995).It also acts on the epidermis blood capillary in the mode of paracrine, causes the vascularity of skin to increase and the formation of granulation substrate.Consult for example Corral et al.supra and Romano de Peppe et al. Adenovirus-mediated VEGF165 gene transfer enhances wound healing by Promoting angiogenesis in CD1 diabetic mice(adenovirus mediated VEGF165 gene transfer has strengthened wound healing by the promotion blood vessel in the CD1 diabetic mice) Gen Ther9:1271-7 (2002).When changing, VEGF level and other angiogenesis factor see for example defective of wound healing etc. of tissue repair.Consult for example Howdieshell et al. Antibody neutralization of Vascular endothelial growth factor inhibits wound granulation tissue formation(the antibody neutralization of VEGF suppresses the wound granulation tissue and forms) J Surg Res96:173-82 (2001); Street et al. Vascular endothelial growth factor stimulates Bone repair by promoting angiogenesis and bone turnover(VEGF takes place and bone turnover stimulation bone reparation by promoting blood vessel) PNAS USA 99:9656-61 (2002); Tsouet al. Retroviral delivery of dominant-negative vascular endothelial growth factor Receptor type 3 to murine wounds inhibits wound angiogenesis(retrovirus is delivered dominant negative 3 type vascular endothelial growth factor receptors to Mus wound and suppressed the generation of wound blood vessel) Wound Repair Regen 10:222-9 (2002); Frank et al. Regulation of vascular Endothelial growth factor expression in cultured keratinocytes, Implications for Normal and impaired wound healing(regulation and control of the keratinocyte medium vessels endothelial growth factor expression of artificial culture are to the enlightenment of normal and impaired wound healing) J Biol Chem270:12607-13 (1995); And Lerman et al. Cellular dysfunction in the diabetic Fibroblast:impairment in migration, vascular endothelial growth factor Production, and response to hypoxia(cell dysfunction in the diabetic fibroblast: migration, VEGF generation and the reduction that hypoxia is replied) Am J Pathol 162:303-12 (2003).In some animal model, external source VEGF promotes wound healing.Consult for example Galliano et al. Topical Vascular Endothelial Growth Factor Accelerates Diabetic Wound Healing Through increased angiogenesis and by Mobilizing and recruiting bone Marrow-derived cells(the vascular surface endothelial cell growth factor (ECGF) takes place to reach by mobilization via the blood vessel that raises and raises bone marrow derived cell and quickened the diabetic wound healing) American Journal ofPathology 164 (6): 1935-1947 (2004); Romano de Peppe S et al. Adenovirus-mediated VEGF165 gene transfer enhances wound healing by Promoting angiogenesis in CD1 diabetic mice(adenovirus mediated VEGF165 gene transfer has strengthened wound healing by the promotion blood vessel in the CD1 diabetic mice) Gen Ther9:1271-7 (2002); And U.S. Patent application US20030180259.

As mentioned above, the challenge that protein protective comes accelerating wound healing is set up in existence.We have described the clinical trial with VEGF recombinant protein therapy processes ulcer in this article, cause the acceleration of wound healing.Referring to embodiment 1 herein.

Other medicament

Quicken and/or improve in the wound healing VEGF therapy and following one or more therapies are joined together to strengthen VEGF activity also within the scope of the invention, for example, (for example other member of VEGF family, somatomedin such as cited herein, nerve growth factor (NGF), the positive vessels generation factor or medicament or activator, assimilation steroid, bioengineered tissue substitute (for example Apligraph , Dermagraft for good wound care therapy (for example GWC), other new or conventional therapy ^TMDeng), hyperbaric oxygen, vacuum therapy).Consult for example Meier and Nanney, Emerging New Drugs for Wound Repair(the emerging novel drugs that is used for wound repair) Expert Opin Emerging Drugs11 (1): 23-37 (2006).

Six main growth factor families (EGF, FGF, IGF, PDGF, TGF and VEGF) are relevant with wound healing.Consult for example Nagai and Embil, Becaplermin:recombinant platelet Derived growth factor, a new treatment for healing diabetic foot ulcers(becaplermin: the recombinant platelet derivative growth factor is used to the new treatment that the diabetic foot ulcers is healed) Exprt Opin Biol Ther 2:211-18 (2002).The example of this type of somatomedin comprises platelet derived growth factor (PDGF-A, PDGF-B, PDGF-C and PDGF-D), insulin like growth factor (IGF-I and IGF-II), acidity and basic fibroblast growth factor (aFGF and bFGF), α and β transforming growth factor (TGF-α and TGF-β (for example TGF-β 1, TGF β 2, TGF β 3)), epidermal growth factor (EGF) and other.Consult above.The mitosis of one or more related cells and can uniting in these factors stimulated growth wound healings with VEGF.

Can with other positive vessels propellant of VEGF associating including, but not limited to for example (Folkman et al.J Biol Chem267:10931-10934 (1992) such as HGF, TNF-α, angiogenin (angiogenin), IL-8; Klagsbrun et al, Annu Rev Physiol 53:217-239 (1991)), blood vessel generation activator, angiogenesis factor described herein and the medicament etc. in the table 3.

The example of table 3 blood vessel generation activator

Blood vessel takes place
	Angiogenin (Angiopoietin) 1 and 2
Tie-2
	α-5 integrin
Matrix metalloproteinase
	Nitric oxide (NO)
COX-2
	TGF β and receptor
VEGF and receptor

In addition, following medicament also can be handled associating with the VEGF wound healing, and for example (for example becaplermin (Becaplermin) is (rhPDGF-BB) such as Regranex , Johnson﹠amp for platelet derived growth factor (PDGF); Johnson; Consult for example U.S. Patent No. 5,457,093; 5,705,485 and 5,427,778; Perry, BH et al. A meta-analytic approach to an integrated summary of efficacy: A case study of becamplemin gelThe colloidal case study of becaplermin) Cont Clin Trials23:389-408 (2002)), adenosine-A2A receptor stimulating agent (MRE0094 for example (to the element method of the comprehensive summary of effect:, King Pharmaceuticals), keratinocyte growth factor (KGF-2, repifermin, Human Genome Sciences), lactoferrin (LF) (Agennix, Inc.), extrasin beta-4 (T β 4, ReGeneRx Biopharmaceuticals), thrombin derivative activation receptor peptide (TP508; Chrysalin , Chrysalis Biotechnology, Inc.), the coding platelet derived growth factor adenovirus vector (PDGF-B) (GAM501) (Selective Genetics), autologous bone marrow stem cell (BMSC) (are consulted for example Badiavas﹠amp; Falanga, Treatment of chronic wounds with bone Marrow-derived cellsAnd the living tissue graft of through engineering approaches (for example Apligraf etc.) (handling chronic wounds) Arch Dermatol139:510-16 (2003)), with bone marrow derived cell.Antibiotic and antiseptical ulcer agent also can be used associating with VEGF.VEGF uses and can also handle (for example corticosteroid, radiotherapy, chemotherapy) together with immunosuppressant or cancer treatment method is used.

Like this common healing potion or rules must not be included in the compositions of the present invention in essence, although this is can be easily under the proteinaceous situation at described medicament.Such mixture is with mode identical with independent use VEGF and identical suitable the using of purpose.Effective molar ratio of VEGF and such second treatment factor normally 1: 0.1-10 uses about equimolar amount in one embodiment of the invention.

Dosage and using

The dosage of Pharmaceutical composition of the present invention and expectation drug level can change according to the concrete purposes of being envisioned.Determining fully in gengral practitioner's technical scope of suitable dose of using or approach.Zoopery can be the effective dose that is identified for human body therapy reliable guidance is provided.Convergent-divergent can be followed the principle that document drafts and carries out between the species of effective dose: Mordenti, J.and Chappell, W. The use of Interspecies scaling in toxicokinetics(purposes of convergent-divergent in toxicokinetics between species) in Toxicokinetics and New Drug Development, people such as Yacobi compile, Pergamon Press, New York 1989, pp.42-96.VEGF uses visible Fig. 2 of example of the dosage-response curve of animal wound model, and it is the dosage-response curve that is applied to the courageous and upright ear of cleft lip wound about VEGF.Fig. 3 is the dosage-response curve that is applied to the diabetic mice wound about VEGF.For prevention or treatment disease or wound type, type (as hereinbefore defined), severity of disease and the course of disease, the medicament that the suitable dose of VEGF will depend on disease to be treated for prevent still be use for therapeutic purposes, previous therapy, patient's clinical history and to the replying of medicament, and attending doctor's judgement.VEGF prepares and takes in the mode that meets good medical practice, need consider delivery position, mode of administration, and the other factors known of practitioner of situation, the VEGF of concrete disease to be treated, individual patients.

The dosage that adopts is depended on factor as herein described.In certain embodiments of the invention, the type and the seriousness that depend on experimenter's illness, the VEGF of about 1 μ g/kg to 50mg/kg (for example 0.1-20mg/kg) and/or other medicament are the candidate's dosage that is applied to the patient, no matter be for example to pass through the one or many separate administration, or pass through continuous application.Document is consulted in guidance about concrete dosage and delivering method.In one embodiment, the effective dose of the VEGF that uses is about 20 μ g/cm ²To about 250 μ g/cm ²In certain embodiments, the effective dose of being used is about 24 μ g/cm ², or 24 μ g/cm ²In certain embodiments, the effective dose of being used is about 72 μ g/cm ², or 72 μ g/cm ²In certain embodiments, the effective dose of being used is about 216 μ g/cm ², or 216 μ g/cm ²In one embodiment, the effective dose of the VEGF that uses is 20 μ g/cm ²To 250 μ g/cm ²In certain embodiments, the effective dose of being used is about 24 μ g/cm ²To about 216 μ g/cm ², or 24 μ g/cm ²To 216 μ g/cm ²In certain embodiments, the effective dose of being used is about 24 μ g/cm ²To about 72 μ g/cm ², or 24 μ g/cm ²To 72 μ g/cm ²In certain embodiments, the effective dose of being used is about 72 μ g/cm ²To about 216 μ g/cm ², or 72 μ g/cm ²To 216 μ g/cm ²In certain embodiments, the effective dose of being used is about 216 μ g/cm ²To about 250 μ g/cm ², or 216 μ g/cm ²To 250 μ g/cm ²Described medicament is by a series of processing or the disposable suitable experimenter that is applied to.

Repetitive administration for continuing a couple of days or longer time depends on illness, keeps treatment and is suppressed for example complete closure of wound or reducing of wound area until the disease symptoms that expectation occurs.Yet other dosage may be effective.Usually, the clinician will use molecule of the present invention until reaching the dosage that can obtain to expect biology effect.Can be once a day or use the VEGF of effective dose an optional week for several times, for example at least biweekly, or at least one Wednesday time, or at least one Thursday time, or at least one Friday time, or at least one Saturday time.In one embodiment, VEGF used at least 6 weeks, or about at least 12 weeks or until wound fully closed (for example this can wrap up in the skin closure that needs and determine by not having drain or cover).Aspect some, VEGF uses and was less than for 20 weeks of the present invention.The progress of therapy of the present invention can be easy to monitoring by routine techniques and algoscopy.

The common surface applied of therapeutic composition of the present invention is in the experimenter.In one embodiment of the invention, VEGF is in surperficial colloid dosage form, for example in pre-syringe or container of filling.In certain embodiments, also other therapeutic agent of surface applied.Other of also optional use VEGF and/or other therapeutic agent used the path, for example use by any suitable means, in parenteral, subcutaneous, intraperitoneal, lung, in the marrowbrain, in subcutaneous, the intraarticular, synovial membrane, in the film/sheath in, oral and intranasal administration.The parenteral infusion comprises intramuscular, intravenous, intra-arterial, intraperitoneal or subcutaneous administration.

As described herein, VEGF can unite with one or more other therapeutic agents or rules.Use co-administered comprising altogether, uses preparaton or single medicinal proportional preparation separately, and the sequential of arbitrary order used.Use various medicaments to be also included among the present invention.For example, VEGF can be before using other therapeutic agent, use afterwards or alternately, perhaps can give simultaneously with it.In one embodiment, two kinds of (or all) activating agents are brought into play its biologic activity simultaneously for some time.In the conjoint therapy scheme, use compositions of the present invention with treatment effective dose or the collaborative amount of treatment.When being used for this paper, the treatment effective dose refers to use jointly VEGF and one or more other therapeutic agents, perhaps implements a kind of rules, the amount that causes target disease or illness to alleviate or suppress.The collaborative amount of treatment refers to work in coordination with or significantly quicken and/or improve the amount of the necessary VEGF of wound healing and one or more other therapeutic agents (for example as herein described).

Pharmaceutical composition

Molecule of the present invention according to the present invention's use, for example VEGF or with the treatment of other therapeutic agent of VEGF associating with preparaton by having the molecule of expectation purity, for example polypeptide and optional pharmaceutics acceptable carrier, excipient or stabilizing agent (Remington ' s Pharmaceutical Sciences, the 16th edition, Osol, A. compile (1980)) mix, with the form preparation of freeze-dried formulation or aqueous solution for storing.Acceptable carrier, excipient or stabilizing agent are nontoxic at dosage that is adopted and concentration to the receiver, comprise buffer agent, such as phosphate, citrate and other organic acid; Antioxidant comprises ascorbic acid and methionine; Antiseptic is (such as octadecyl dimethyl benzyl ammonium chloride; Chlorination hexane diamine; Benzalkonium chloride, benzethonium chloride; Phenol, butanols or benzyl alcohol; Alkyl paraben is such as methyl parahydroxybenzoate or propyl ester; Catechol; Resorcinol; Hexalin; The 3-amylalcohol; And metacresol); Low-molecular-weight (being less than about 10 residues) polypeptide; Protein is such as serum albumin, gelatin or immunoglobulin; Hydrophilic polymer is such as polyvinylpyrrolidone; Aminoacid is such as glycine, glutamine, agedoite, histidine, arginine or lysine; Monosaccharide, disaccharide and other carbohydrate comprise glucose, mannose or dextrin; Chelating agen is such as EDTA; Saccharide is such as sucrose, mannitol, trehalose or sorbitol; The salify gegenion is such as sodium; Metal composite (for example Zn-protein complex); And/or non-ionic surface active agent, such as TWEEN ^TM, PLURONICS ^TMOr Polyethylene Glycol (PEG).

In certain embodiments, the preparaton that will be used for using in the body is aseptic.This can be easy to realize by aseptic membrane filtration.VEGF can be with lyophilized form or aqueous solution or colloidal form storage.The pH of VEGF preparation can be about 5 to 9, although higher or lower pH value may also suit in some cases.The use that should understand some excipient, carrier or stabilizing agent can cause the formation of the salt of VEGF.

Active component also can wrap and be stated from for example by (for example being respectively hydroxy methocel or gelatin microcapsule and poly-(methyl methacrylate) microcapsule) in condensation technique or the microcapsule by the interfacial polymerization preparation, in gluey drug delivery system (for example liposome, albumin microsphere, microemulsion, nano-particle and Nano capsule), or in macro emulsion.This type of technology is disclosed in Remington ' s PharmaceuticalSciences, and the 16th edition, Osol, A. compiles (1980).Also can be referring to Johnson et al.Nat.Med.2:795-799 (1996); Yasuda, Biomed.Ther.27:1221-1223 (1993); Hora et al.Bio/Technology 8:755-758 (1990); Cleland, Design and Production of Single Immunization Vaccines Using Polylactide Polyglycolide Microsphere Systems, in Vaccine Design:The Subunit and Adjuvant Approach, Powell and Newman compiles, Plenum Press:New York, 1995, pp.439-462; WO 97/03692, and WO 96/40072, WO96/07399; And U.S. Patent No. 5,654,010.

Usually for wound healing, the VEGF preparation is used for locus specificity delivers.When surface applications, suitable unites VEGF with other composition such as carrier and/or adjuvant.For the character of described other composition without limits, just they must be that pharmaceutics is acceptable and to use for their expection be effectively, and can not reduce the activity of active component in the compositions.The example of suitable media comprises ointment, cream, gel, spray or suspensoid, contains or do not contain the collagen of purification.Compositions also can immerse aseptic dressing, transdermal subsides, plaster and binder, and is optional with liquid or semi-liquid form.Also can use oxidation, regenerated cellulose/collagen stroma, for example Promogran ^TMMatrix WoundDressing or Promogran Prisma Matrix ^TM

Can prepare extended release preparation.The suitable example of extended release preparation comprises the solid hydrophobic polymer semi permeability substrate that contains polypeptide of the present invention, and this substrate is the form of approved product, for example thin film or microcapsule.The example that continues release matrix comprises polyester, hydrogel (for example poly-(2-ethoxy-methacrylate) or poly-(vinyl alcohol)), polyactide (U.S. Patent No. 3,773,919), the copolymer of L-glutamic acid and L-glutamic acid, gamma-ethyl ester, nondegradable ethene-vinyl acetate copolymer, degradable lactic acid-ethanol copolymer are such as LUPRON DEPOT ^TM(the Injectable microspheres body that constitutes by lactic acid-ethanol copolymer and leuprorelin acetate), polylactic acid-altogether-glycolic (PLGA) polymer and poly--D-(-)-3-hydroxybutyric acid.Reach more than 100 days though can discharge molecule such as polymer such as ethane-acetic acid ethyenyl and lactic acid-ethanols, the time of some hydrogel release protein is shorter.When encapsulated antibody was kept in vivo for a long time, their may degeneration or gathering owing to be exposed to 37 ℃ wet environment, the loss and immunogenic may the variation that cause biologic activity.Can come the rational strategy of design stabilityization according to related mechanism.For example, if finding aggregation of multiple is to form via the intermolecular S-S key that mercaptan-disulphide exchanges, so can be by modifying sulfhydryl residue, realizing stablizing by acid solution lyophilizing, controlled humidity, the suitable additive of employing and exploitation particular polymers base composition.

In order to obtain the gelinite preparaton, the VEGF for preparing in fluid composition can be mixed to form the gelinite that viscosity is suitable for surface applications with the water soluble polysaccharide or synthetic polymer such as the Polyethylene Glycol of effective dose.Spendable polysaccharide comprises for example cellulose derivative, such as the etherified cellulose derivant, comprise alkylcellulose, hydroxy alkyl cellulose and alkane hydroxy alkyl cellulose, for example methylcellulose, hydroxyethyl-cellulose, carboxymethyl cellulose, hydroxypropyl emthylcellulose and hydroxypropyl cellulose; Starch and classification starch; Agar; Alginic acid and alginate; Arabic gum; Pullullan; Agarose; Carrageenin; Dextran; Dextrin; Levan; Inulin; Mannan; Xylan; Arabinan; Chitosan; Glycogen; Glucosan; With synthetic biopolymer; And colloid, such as xanthan gum; Guar gum; Tracasol; Radix Acaciae senegalis; Tragacanth; And karaya; And derivant and mixture.In one embodiment of the invention, gellant herein is to have for example gel of following characteristic, for example to biological systems be inert, nontoxic, preparation is simple and/or not too runny or not too glue, and the stability of the VEGF that can not destroy wherein to be contained.

In certain embodiments of the invention, described polysaccharide is the cellulose derivative of etherificate, it is clear that determine, purification and be listed among the USP in another embodiment, for example methylcellulose and hydroxyalkyl cellulose derivative are such as hydroxypropyl cellulose, hydroxyethyl-cellulose and hydroxypropyl emthylcellulose.In one embodiment, methylcellulose is exactly said polysaccharide.

In order to obtain suitable viscosity, can be used for the normally mixture of low-molecular-weight and high molecular weight polyethylene glycol of agglomerative Polyethylene Glycol.For example, the Polyethylene Glycol of the Polyethylene Glycol of molecular weight 400-600 and molecular weight 1500 will be effective with the mixture that correct proportions mixes when obtaining paste to this purpose.

Term " water solublity " is intended to comprise colloidal solution and dispersion when being applied to polysaccharide and Polyethylene Glycol.Generally speaking, the dissolubility of cellulose derivative is the level determinations that replaces by ether, and can be used for stabilisation derivant of the present invention and should have this type of ether of sufficient amount so that described derivant is water miscible in each the glucoside unit in cellulose chain.It generally is enough that the ether of each at least 0.35 ether in glucoside unit replaces degree.In addition, described cellulose derivative can be the form of alkali metal salt, for example Li, Na, K or Cs salt.

If adopt methylcellulose in gelinite, then for example it accounts for about 2-5% or about 3% or about 4% or about 5% of gelinite, and VEGF exists with the amount of the about 100-2000 μ of every ml gelinite g.

VEGF and/or other medicament also can be applied to wound by gene therapy.Gene therapy refers to the therapy by carrying out for experimenter's administration of nucleic acid.In the application of gene therapy,, for example, gene is introduced cell in order to replace dcc gene for synthetic in the body of realizing treating the efficient gene product." gene therapy " comprises by single treatment and realizes traditional gene therapy of lasting effect and relate to once or gene therapeutic agents that effective DNA or mRNA are gone up in the repetitive administration treatment is used the two.Can modified oligonucleotide to strengthen their picked-up, for example by with uncharged their electronegative phosphodiester group of group replacement.Comprehensive review about the method for gene therapy is consulted for example Goldspiel et al.ClinicalPharmacy 12:488-505 (1993); Wu and Wu, Biotherapy 3:87-95 (1991); Tolstoshev, Ann.Rev.Pharmacol.Toxicol.32:573-596 (1993); Mulligan, Science260:926-932 (1993); Morgan and Anderson, Ann.Rev.Biochem.62:191-217 (1993); And May, TIBTECH 11:155-215 (1993).Method that the recombinant DNA technology field is generally known, operable is consulted people's volume (1993) Current Protocols in MolecularBiology such as Ausubel, John Wiley﹠amp; Sons, NY and Kriegler (1990) Gene Transfer andExpression, A Laboratory Manual, Stockton Press, NY.

There are multiple technologies to can be used for nucleic acid is introduced living cells.Described technology can be to change at the external cell that is transferred to cultured cells or is transferred to the expection host in vivo according to nucleic acid.Be suitable for comprising liposome, electroporation, microinjection, cell fusion, DEAE-dextran, the calcium phosphate precipitation method etc. used in the external technology that nucleic acid is transferred to mammalian cell.Current preferred vivo gene transfer technology comprises transfection and the virus capsid protein-liposome-mediated transfection of carrying out with virus (being typically retrovirus) carrier (Dzau et al.Trends in Biotechnology 11:205-210 (1993)).For example, the nucleic acid in vivo transfer techniques comprises with viral vector (such as adenovirus, I herpes simplex virus type, slow virus, retrovirus or adeno associated virus) and the transfection carried out based on the system of lipid (lipid that can be used for the gene transfer of lipid mediation has for example DOTMA, DOPE and DC-Chol).In gene therapy, use the example of viral vector can consult Clowes et al.J.Clin.Invest.93:644-651 (1994); Kiemet al.Blood 83:1467-1473 (1994); Salmons and Gunzberg, Human GeneTherapy 4:129-141 (1993); Grossman and Wilson, Curr.Opin.in Genetics andDevel.3:110-114 (1993); Bout et al.Human Gene Therapy 5:3-10 (1994); Rosenfeld et al.Science 252:431-434 (1991); Rosenfeld et al.Cell 68:143-155 (1992); Mastrangeli et al.J.Clin.Invest.91:225-234 (1993); And Walsh et al.Proc.Soc.Exp.Biol.Med.204:289-300 (1993).

Wishing in some situation provides the nucleic acid source with the medicament of targeting wound cell, such as pair cell surface membrane protein or the specific antibody of target cell, at the part of receptor on the target cell etc.If adopt liposome, then can use the protein bound protein targeting of the cell surface membrane relevant and/or promote picked-up with endocytosis, for example to the capsid protein of particular cell types aeoplotropism or its fragment, at location in the proteinic antibody of experience internalization in circulation, the targeting born of the same parents and prolong the protein of half-life in the born of the same parents.The technology of receptor-mediated endocytosis is consulted for example Wu et al.J.Biol.Chem.262:4429-4432 (1987) and Wagner et al.Proc.Natl.Acad Sci.USA 87:3410-3414 (1990).Consult Anderson et al.Science256:808-813 (1992) about the summary of genetic marker and gene therapy scheme.

Covalent modification to polypeptide of the present invention

Polypeptide of the present invention, for example VEGF or comprise within the scope of the invention with the covalent modification of other other therapeutical peptide medicament of VEGF associating.They can carry out by chemosynthesis or by enzymatic or the described polypeptide of chemical cleavage if suitably.Other type covalent modification of polypeptide is introduced molecule, its by with the target amino acid residue of polypeptide with can with organic derivatization reagent reaction of selected side chain or N-or the reaction of C-terminal residue, perhaps mix the polypeptide chain that is increasing, for example Ellman et al.Meth.Enzym.202:301-336 (1991) by the aminoacid after will modifying or non-natural aminoacid; Noren etal.Science 244:182 (1989); And U.S. Patent application 20030108885 and 20030082575.

Cysteinyl residue is modal to be and alpha-halogen acetic acid (salt/ester) (with corresponding amine) reaction, such as monoxone or chloroacetamide, to obtain carboxymethyl or carboxamide methyl (carboxyamidomethyl) derivant.Cysteinyl residue can also by with bromo trifluoroacetone, alpha-brominated-β-(5-imidozoyl) propanoic acid, chloracetyl phosphoric acid (salt/ester), N-alkyl maleimide, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyl disulfide, parachloromercuribenzoic acid (salt/ester), 2-chloromercuri-4-nitrophenols or (4-) chloro-7-nitro benzo-2-oxygen-1, the reaction of 3-diazole and derivatization.

The histidyl-residue is by reacting and derivatization at pH 5.5-7.0 with diethyl-Jiao-carbonic acid (salt/ester), because this reagent is specific to the histidyl-side chain relatively.The PBPB thing also is useful; This reaction is carried out in the 0.1M of pH 6.0 sodium dimethylarsonate usually.

Lysyl and n terminal residue and succinic anhydrides or the reaction of other carboxylic acid anhydrides.Has the effect that reverses the lysyl-residue electric charge with the derivatization of these reagent.Be suitable for other reagent that derivatization contains alpha-amino residue and comprise imino esters, such as picoline imidic acid methyl ester (methyl picolinimidate), pyridoxal 5-phosphate ester, 2-methyl-3-hydroxy-4-formyl-5-hydroxymethylpyridine., chlorine boron hydride, trinitro-benzene-sulfonic acid, adjacent methyl-isourea, 2, the 4-pentanedione and with transaminase's catalytic reaction of glyoxalic acid (glyoxylate).

The arginyl residue wherein has phenylglyoxal (phenylglyoxal), 2,3-diacetyl, 1,2-cyclohexanedione and 1,2,3-indantrione monohydrate by modifying with a kind of or several conventional reagent reactings.The derivatization of arginine residues is because guanidine functional group's high pK _aSo need under alkali condition, react.In addition, these reagent can react with lysine and arginine epsilon-amino.

The tyrosyl residue can carry out specificity to be modified, interested in especially introducing spectroscopic tags in the tyrosyl residue, and it is by reacting with aromatic diazo compound or tetranitromethane.Modal is to use N-acetyl imidazole and tetranitromethane to form adjacent acetyl group tyrosyl kind and 3-nitro-derivative respectively.With ¹²⁵I or ¹³¹I iodate tyrosyl residue uses for radioimmunoassay with the protein of preparation tape label.

Carboxylic side-chain group (aspartoyl or glutamy) by with carbodiimide (R-N=C=N-R ') reaction carrying out selective modification; R in the carbodiimide is different alkyl with R '; such as 1-cyclohexyl-3-(2-morpholinyl-4-ethyl) carbodiimide or 1-ethyl-3-(4-azonia-4,4-dimethyl amyl group) carbodiimide.In addition, by aspartoyl and glutamy residue being transformed into asparaginyl-and glutamine acyl residue with the ammonium ion reaction.

Glutamine acyl and asparaginyl-residue be deacylated tRNA amine usually, becomes corresponding glutamy and aspartoyl residue respectively.These residues are deacylated tRNA amine under neutrality or alkali condition.The deacylated tRNA amine form of these residues falls into scope of the present invention.

Other modification comprises the hydroxylating of proline and lysine, the phosphorylation of seryl or threonyl residue hydroxyl, alpha-amino (the T.E.Creighton that methylates of lysine, arginine and histidine side chain, Proteins:Structure and Molecular Properties, W.H.Freeman﹠amp; Co.San Francisco, pp.79-86 (1983)), the acetylation of N-terminal amine, and the amidatioon of any C-terminal carboxyl.

The covalent modification of another type comprises to chemiluminescent polypeptide of the present invention or enzymatic coupling glucosides.The advantage of these rules is that they need not connect in the host cell of glycosylated glycosylation ability and produce polypeptide having N-or O-.Depend on employed coupling pattern, sugar can be attached to (a) arginine and histidine, (b) free carboxy, (c) sulfydryl of free sulfhydryl groups such as cysteine, (d) hydroxyl of free hydroxyl group such as serine, threonine or hydroxyproline, (e) aromatic residue such as phenylalanine, tyrosine or tryptophan, or (f) amide groups of glutamine.These methods can be consulted the WO87/05330 and the Aplin and Wriston of JIUYUE in 1987 announcement on the 11st, CRC Crit.Rev.Biochem.pp.259-306 (1981).

The elimination of any carbohydrate module that exists on the polypeptide of the present invention can realize by chemistry or enzymatic method.The de-glycosylation effect of chemistry need make polypeptide be exposed to chemical compound trifluoromethanesulfonic acid or equivalent chemical compound.This processing causes the most of or whole sugar except that connecting sugar (linking sugar) (N-acetyl-glucosamine or N-acetylgalactosamine) to obtain excision, and polypeptide is kept perfectly.Hakimuddin et al.Arch.Biochem.Biophys.259:52 (1987) and Edge et al.Anal.Biochem.118:13 (1981) are consulted in the de-glycosylation effect of chemistry.For example on the antibody, the cutting of the enzymatic of carbohydrate module can realize by using multiple inscribe and exoglycosidase, consults Thotakura et al.Meth.Enzymol.138:350 (1987).

The covalent modification of another type of polypeptide of the present invention comprises polypeptide chain is connected to one of multiple nonprotein character polymer, and for example Polyethylene Glycol, polypropylene glycol or polyoxyalkylene are with U.S. Patent No. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337 listed modes.

Carrier, host cell and recombination method

Polypeptide of the present invention can use the technology and the material that can obtain easily, recombinant production.

For recombinant production polypeptide of the present invention, for example VEGF or with other treatment polypeptide medicament of VEGF associating, separate its nucleic acid of coding and also insert replicable vector, be used for further clone (DNA cloning) or expression.Can utilize many carriers.Support element generally comprise but be not limited to following one or more: control sequence, signal sequence, origin of replication, one or more marker gene, enhancer element, promoter and transcription terminator.

Statement " control sequence " refers to express the necessary DNA sequence of coded sequence that can be operatively connected in the specific host organism.For example, be suitable for procaryotic control sequence and comprise promoter, optional operator sequence and ribosome binding site.The known genuine nucleus utilizes promoter, polyadenylation signal and enhancer.

If one section nucleic acid and another section nucleotide sequence are in the functional mutual relation, then it is " can be operatively connected ".For example, if presequence (presequence) or the DNA that secretes leading (secretory leader) be expressed as participate in polypeptide excretory before protein (preprotein), then the DNA of it and this polypeptide can be operatively connected; If promoter or enhancer influence transcribing of coded sequence, then it and this sequence can be operatively connected; Perhaps, if the position of ribosome binding site promotes translation, then it and coded sequence can be operatively connected.Generally speaking, " can be operatively connected " means that continuous DNA sequence is adjacent, and means adjacent in the leading situation of secretion and be in readable state.Yet enhancer needn't be adjacent.Connection can be by realizing in the coupled reaction at restriction site place easily.If there is not this type of site, then use synthetic oligonucleotide adapter or joint according to conventional practice.

The DNA of code book invention polypeptide can use conventional rules to be easy to separate and/or order-checking.For example, the DNA of coding VEGF can separate with the bonded oligonucleotide probe of gene specific of coding VEGF and check order by for example using." isolating " nucleic acid molecules refer to identify and with the natural origin of polypeptide-nucleic acid in the nucleic acid molecules that separates of related with it usually at least a contaminative nucleic acid molecules.Isolated nucleic acid molecule is different from form or the background when occurring in nature finds it.Therefore isolated nucleic acid molecule has any different with the nucleic acid molecules that is present in the n cell.Yet isolated nucleic acid molecule comprises the nucleic acid molecules that is comprised in the cell of common this polypeptide of expression, for example when the chromosome mapping of described nucleic acid molecules in described cell is different from its chromosome mapping in n cell.

The signal sequence member

Polypeptide of the present invention is direct recombinant production not only, and can be used as the fused polypeptide with heterologous polypeptide, and wherein said heterologous polypeptide normally has signal sequence or other polypeptide of specificity cleavage site in the N-end of mature protein or polypeptide.Selected allos signal sequence normally is subjected to host cell identification and processing (promptly being subjected to the signal peptidase cutting).Prokaryotic host cell for nonrecognition and processing natural polypeptides signal sequence replaces described signal sequence with the prokaryotic signal sequence that for example is selected from alkali phosphatase, penicillinase, lpp or thermally-stabilised enterotoxin 1 I targeting sequencing.For yeast secretary, can replace the natural signals sequence with for example signal described in yeast invertase targeting sequencing, alpha factor targeting sequencing (comprising saccharomyces and Crewe Vickers Saccharomyces alpha factor targeting sequencing) or acid phosphatase targeting sequencing, white candida mycoderma glucoamylase targeting sequencing or the WO 90/13646.In mammalian cell expression, can utilize mammalian signal sequence and viral secretory targeting sequencing, for example herpes simplex gD signal.

The DNA of this type of prosoma is connected in the reading frame of DNA of code book invention polypeptide.

The origin of replication member

Expression and cloning vehicle all comprise the nucleotide sequence that carrier is duplicated in one or more selected host cells.Generally speaking, in cloning vehicle, this sequence does not rely on the sequence that host chromosome DNA duplicates for making carrier, comprises origin of replication or autonomous replication sequence.This type of sequence of well-known various bacteria, yeast and virus.Origin of replication from plasmid pBR322 is suitable for most of gram negative bacteria, and 2 μ plasmid starting points are suitable for yeast, and various viral starting point (SV40, polyoma virus, adenovirus, VSV or BPV) can be used for the cloning vehicle in the mammalian cell.Generally speaking, mammalian expression vector does not need origin of replication member (the SV40 starting point may only just be used because of comprising early promoter usually).

Select gene component

Expression and cloning vehicle can comprise the selection gene, are also referred to as selection marker.The typical following protein of gene code of selecting: (a) give antibiotic or other toxin resistance, for example ampicillin, neomycin, methotrexate or tetracycline; (b) supply auxotrophy; Or (c) the supply crucial nutrient that can not obtain by complex medium, for example be the gene of bacillus cereus encoding D-alanine racemase.

An example of selection scheme utilizes medicine to block the growth of host cell.Give the protein of drug resistance with those cells generations that heterologous gene successfully transforms, thereby survive selection scheme.The example that this type of dominance is selected uses medicine neomycin, mycophenolic acid and hygromycin.

Another example that is suitable for the selection marker of mammalian cell is the selection marker that can identify the cell of the picked-up nucleic acid of having the ability, such as DHFR, thymidine kinase, metallothionein-I and-II (normally primates metallothionein gene), ADA Adenosine deaminase, ODC Ornithine decarboxylase etc.

For example, at first, all transformants identify the cell of selecting gene transformation through DHFR in the culture medium that contains methotrexate (Mtx) (a kind of competitive antagonist of DHFR) by being cultivated.When adopting wild type DHFR, suitable host cells is Chinese hamster ovary (CHO) cell line of the active defective of DHFR.

Perhaps, can by contain at selective agent such as the aminoglycoside antibiotics of selection marker for example in the culture medium of kanamycin, neomycin or G418 cultured cell select encoded polypeptide of the present invention, wild type dhfr protein matter and another kind of selection marker such as aminoglycoside 3 '-DNA sequence of phosphotransferase (APH) transforms or the host cell (the wild type host who particularly comprises endogenous DHFR) of cotransformation.Consult U.S. Patent No. 4,965,199.

Be applicable to that zymic selection gene is the trp1 gene (Stinchcomb et al.Nature 282:39 (1979)) that is present among the yeast plasmid Yrp7.The trp1 gene is for lacking the yeast mutant of energy for growth in tryptophan, and for example ATCC No.44076 or PEP4-1 provide selection marker (Jones, Genetics 85:12 (1977)).Exist trp1 infringement to provide in the yeast host cell genome to be used for by not existing growth under the tryptophan to detect the effective environment of conversion thereupon.Similarly, supply Leu2 defective yeast bacterial strain (ATCC 20,622 or 38,626) with the known plasmid that carries the Leu2 gene.

In addition, the carrier derived from 1.6 μ m cyclic plasmid pKD1 can be used for transforming Crewe Vickers Saccharomyces yeast.Perhaps, reported the expression system (Van den Berg, Bio/Technology 8:135 (1990)) that is used at lactic acid Crewe Vickers yeast large-scale production reorganization calf chymosin.Also disclosed and be applicable to by the industrial strain of Crewe Vickers Saccharomyces and secrete the albuminised stable multicopy expression vector of ripe recombinant human serum (Fleer et al.Bio/Technology 9:968-975 (1991)).

The promoter member

Express and cloning vehicle comprises usually and is subjected to the promoter that host organisms is discerned, and the nucleic acid of it and code book invention polypeptide can be operatively connected.The promoter that is applicable to prokaryotic hosts comprises that PhoA promoter, beta-lactamase and lactose promoter systems, alkali phosphatase, tryptophan (trp) promoter systems and hybrid promoter are such as the tac promoter.Yet other known antibacterial promoter also is suitable.The promoter that is used for bacterial system also will comprise Shine-Dalgamo (S.D.) sequence that the DNA with code book invention polypeptide can be operatively connected.

Known eukaryotic promoter sequence.In fact, all eukaryotic genes all have the AT of being rich in district, are positioned at about 25 to 30 base places, initial upstream, site of transcribing.The another kind of sequence that finds at 70 to 80 base places, many gene transcription starting points upstream is the CNCAAT district, and wherein N can be any nucleotide.At 3 of most of eukaryotic genes ' end is the AATAAA sequence, and it may be the signal that adds poly A tail to 3 of coded sequence ' end.In the suitable insertion carrier for expression of eukaryon of all these sequences.

The example that is applicable to the promoter sequence of yeast host comprises the promoter of glycerol 3-phosphate acid kinase or other glycolytic ferment, such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvic carboxylase, phosphofructokinase, G-6-P isomerase, 3-phoshoglyceric acid mutase, pyruvate kinase, phosphotriose isomerase, glucose phosphate isomerase and glucokinase.

As other Yeast promoter of the inducible promoter with the additional advantage of being transcribed by growth conditions control is alcoholdehydrogenase 2, different cell pigment C, acid phosphatase, the digestive enzyme relevant with nitrogen metabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase, and the promoter region of the enzyme of responsible maltose and galactose utilization.The carrier and the promoter that are applicable to yeast expression further are recorded in EP 73,657.The yeast enhancer also can be favourable use with Yeast promoter.

In mammalian host cell, for example be subjected to by virus such as polyoma virus, fowlpox virus, adenovirus (such as adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, retrovirus, hepatitis B virus and common simian virus 40 (SV40) genome, by for example actin promoter or immunoglobulin promoter, and the control of the promoter that obtains by the heat shock promoter of allos mammalian promoter, if the compatible words of this type of promoter and host cell systems by carrier transcript invention polypeptide.

Obtain the early stage and late promoter of SV40 virus easily with the form of SV40 restriction fragment, this fragment also comprises SV40 virus replication starting point.Obtain the immediate early promoter of human cytomegalic inclusion disease virus easily with the form of HindIII E restriction fragment.U.S. Patent No. 4,419 has disclosed the system of bovine papilloma virus as carrier expressible dna in mammalian hosts that use in 446.U.S. Patent No. 4,601 has been put down in writing a kind of improvement of this system in 978.About in mouse cell under from the control of the thymidine kinase promoter of herpes simplex virus expressing human beta-interferon cDNA also can consult Reyes et al.Nature 297:598-601 (1982).Perhaps, can use the rous sarcoma virus long terminal repeat as promoter.

The enhancer element member

Usually improve higher eucaryotic cells transcribing to the DNA of code book invention polypeptide by in carrier, inserting enhancer sequence.Known many enhancer sequence from mammalian genes (globulin, elastoser, albumin, α-fetoprotein and insulin).Usually use enhancer from eukaryotic cell virus.Example comprises enhancer (bp 100-270), the sub-enhancer of cytomegalovirus early promoter of SV40 origin of replication side in late period one, the enhancer and the adenovirus enhancer of polyoma virus origin of replication side in late period one.Also can consult Yaniv, Nature 297:17-18 (1982) about the enhancing element that activates eukaryotic promoter.Enhancer can montage in carrier, be positioned at 5 of polypeptid coding sequence ' or 3 ' position, but be usually located at 5 ' site of promoter.

The tanscription termination member

The expression vector that is used for eukaryotic host cell (yeast, fungus, insecticide, plant, animal, people or from the nucleated cell of other multicellular organisms) also will comprise and stop transcribing and the necessary sequence of stable mRNA.This type of sequence can be obtained by 5 of eucaryon or viral DNA or cDNA ' end and 3 ' end untranslated region once in a while usually.These zones are included in the untranslated part of mRNA of code book invention polypeptide and are transcribed into the segmental nucleotide section of polyadenylation.A kind of useful tanscription termination member is bovine growth hormone polyadenylation district.The expression vector of consulting WO 94/11026 and wherein disclosing.

The selection of host cell and conversion

The host cell that is suitable for cloning or express the DNA of the code book invention polypeptide in this paper carrier is above-described prokaryote, yeast or higher eucaryotic cells.The prokaryote that is suitable for this purpose comprises eubacteria, such as Gram-negative or gram-positive organism, enterobacteriaceae for example, such as Escherichia (Escherichia) colon bacillus (E.coli) for example, Enterobacter (Enterobacter), Erwinia (Erwinia), Klebsiella (Klebsiella), proteus (Proteus), Salmonella (Salmonella) is Salmonella typhimurium (Salmonella typhimurium) for example, Serratia (Serratia) is serratia marcescens (Serratia marcescans) for example, and Shigella (Shigella), and bacillus (Bacilli) such as bacillus subtilis (B.subtilis) and Bacillus licheniformis (B.licheniformis) (for example Bacillus licheniformis 41P that discloses among the DD 266,710 that announced on April 12nd, 1989), Rhodopseudomonas (Pseudomonas) is such as Pseudomonas aeruginosa (P.aeruginosa), and streptomyces (Streptomyces).Usually, the escherichia coli cloning host is escherichia coli 294 (ATCC 31,446), although other bacterial strain such as escherichia coli B, escherichia coli X1776 (ATCC 31,537) and escherichia coli W3110 (ATCC 27,325) also are suitable.These examples are illustration and unrestricted.

Except prokaryote, eukaryotic microorganisms also is the suitable clone or the expressive host of the carrier of code book invention polypeptide such as filamentous fungi or yeast.Wine brewing sugar yeast (Saccharomyces cerevisiae) or bakery yeast commonly used are the most frequently used eucaryon host microorganisms such as low.Yet, can obtain many other genus, kind and bacterial strain usually and can be used for the present invention, such as foxtail millet wine fragmentation sugar yeast (Schizosaccharomyces pombe); Kluyveromyces (Kluyveromyces) host such as for example Kluyveromyces lactis (K.lactis), (ATCC 12 for Kluyveromyces fragilis (K.fragilis), 424), (ATCC 16 for Bulgarian kluyveromyces (K.bulgaricus), 045), (ATCC 24 for Brunswick kluyveromyces (K.wickeramii), 178), (ATCC 56 for K.waltii, 500), fruit bat kluyveromyces (K.drosophilarum) (ATCC 36,906), heat-resisting kluyveromyces (K.thermotolerans) and marxianus yeast (K.marxianus); Inferior sieve Saccharomyces (Yarrowia) (EP 402,226); Pichia pastoris phaff (Pichia pastoris) (EP 183,070); Mycocandida (Candida); Rui Shi Trichoderma spp. (Trichoderma reesia) (EP 244,234); Neuraspora crassa (Neurospora crassa); Permitted all so prosperous yeast of prosperous Saccharomyces (Schwanniomyces) (Schwanniomyces occidentalis); With filamentous fungi such as for example Neurospora (Neurospora), Penicillium (Penicillium), the curved mould genus of neck (Tolypocladium) and aspergillus (Aspergillus) host such as aspergillus nidulans (A.nidulans) and aspergillus niger (A.niger).

Be applicable to that the host cell of expressing glycosylated polypeptide of the present invention is derived from multicellular organisms.The example of invertebral zooblast comprises plant and insect cell.Identified many baculovirus strains and variant and the corresponding insect host cell that allows, they are from such as hosts such as fall army worm Spodoptera frugiperda (caterpillar), Aedes aegypti Aedes aegypti (mosquito), Aedes albopictus Aedes albopictus (mosquito), Drosophila melanogaster Drosophila melanogaster (fruit bat) and silkworm Bombyx mori.The public can obtain multiple Strain and be used for transfection, the for example Bm-5 strain of the L-1 variant of autographa california Autographa californica NPV and silkworm NPV, and this viroid can be according to the virus of the present invention as this paper, especially for transfection fall army worm cell.Also can utilize the plant cell cultures of cotton, corn, Rhizoma Solani tuber osi, Semen sojae atricolor, petunia, Fructus Lycopersici esculenti and Nicotiana tabacum L. as the host.

Yet most interested is vertebrate cells, and the breeding of vertebrate cells has become conventional rules in the cultivation (tissue culture).The example of useful mammalian host cell line is the monkey kidney CV1 system (COS-7, ATCC CRL 1651) that transforms with SV40; Human embryo kidney (HEK) system (293 or for growth in suspension culture 293 cells of sub-clone, Graham et al.J.Gen Virol.36:59 (1977)); Baby hamster kidney cell (BHK, ATCC CCL 10); Chinese hamster ovary cell/-DHFR (CHO, Urlaub et al.Proc.Natl.Acad.Sci.USA 77:4216 (1980)); Mice Sai Tuoli (sertoli) cell (TM4, Mather, Biol.Reprod 23:243-251 (1980)); Monkey-kidney cells (CV1, ATCCCCL 70); African green monkey kidney cell (VERO-76, ATCC CRL-1587); Human cervical carcinoma cell (HELA, ATCC CCL2); Madin-Darby canine kidney(cell line) (MDCK, ATCC CCL 34); Cattle Mus (buffalorat) hepatocyte (BRL 3A, ATCC CRL 1442); Human pneumonocyte (W138, ATCC CCL 75); Human liver cell (Hep G2, HB 8065); MMT (MMT 060562, ATCC CCL 51); TRI cell (Mather et al.Annals N.Y.Acad.Sci.383:44-68 (1982)); The MRC5 cell; The FS4 cell; With people's hepatoma system (Hep G2).

With production polypeptide expression of the present invention or the cloning vehicle transformed host cell of being used for mentioned above, and in the conventional Nutrient medium of the gene of expecting sequence for evoked promoter, selection transformant or amplification coding and suitably change, cultivate.

Cultivate host cell

Can in multiple culture medium, cultivate the host cell that is used for production polypeptide of the present invention.Commercially available culture medium is such as HamShi F10 (Sigma), minimum essential medium (MEM, Sigma), the EagleShi culture medium of RPMI-1640 (Sigma), DulbeccoShi improvement (DMEM, Sigma), be used for hepatocellular normal growth medium (Cambrex), be used for before the growth medium (Cambrex) etc. of adipose cell be suitable for cultivating host cell.In addition, can use any culture medium of putting down in writing in the following document culture medium: Ham et al.Meth.Enz.58:44 (1979) as host cell; Barnes et al.Anal.Biochem.102:255 (1980); U.S. Patent No. 4,767,704; 4,657,866; 4,927,762; 4,560,655; Or 5,122,469; WO 90/03430; WO 87/00195; Or United States Patent (USP) reexamination 30,985.Any of these culture medium as required supplementing hormone and/or other somatomedin (such as insulin, transferrin or epidermal growth factor), salt (such as sodium chloride, calcium, magnesium and phosphate), buffer agent (such as HEPES), nucleotide (such as adenosine and thymidine), antibiotic (such as GENTAMYCIN ^TMMedicine), trace element (being defined as the common inorganic compound that exists with the final concentration of micro-molar range) and the glucose or the equivalent energy.Can also with debita spissitudo comprise those skilled in the art will know that any other must fill-in.Condition of culture such as temperature, pH etc. before selected to be used for host cell for expression, and this is obvious for those of ordinary skill.

Peptide purification

When using recombinant technique, can in cell, in periplasmic space, generate polypeptide of the present invention, VEGF or use the polypeptide medicament for example with other treatment that VEGF unites, perhaps direct secretion is in culture medium.Can reclaim and/or separate polypeptide of the present invention from the culture fluid of culture or from the molten cytosol of host cell." isolating " polypeptide refer to identify and with/the polypeptide that separates and/or reclaim by a kind of composition of its natural surroundings.The contaminative composition of the natural surroundings of polypeptide refers to disturb the material of its diagnosis or therapeutic use, can comprise the solute of enzyme, hormone and other protein properties or nonprotein character.In certain embodiments, with peptide purification to (1) mensuration according to the Lowry method, polypeptide weight surpass 95% or weight surpass 99%, (2) be enough to by using the rotary-cup type sequenator to obtain the N-end of at least 15 residues or the degree of internal amino acid sequence, or (3) reach homogeneity according to the SDS-PAGE that uses under Coomassie blue or silver-colored painted reproducibility or the irreducibility condition.Since at least a composition of the natural surroundings of polypeptide can not exist, so isolating polypeptide comprises the original position polypeptide in the reconstitution cell.Yet isolating polypeptide prepares by at least one purification step usually.

Can adopt the whole bag of tricks of protein purification, these methods are known in the art and are recorded in for example Deutscher, Methods in Enzymology, 182 (1990); Scopes, Protein Purification:Principles and Practice, Springer-Verlag, New York (1982).The selection of purification step will be depended on for example character and the concrete polypeptide of the present invention that is produced of employed production method.If membrane-bound, polypeptide so of the present invention can discharge from film with suitable detergent solution (for example Triton-X 100) or by enzymatic lysis.The cell that is adopted in the polypeptide expression of the present invention can be by various physics or chemical means fragmentation, such as freeze-thaw cycle, supersound process, Mechanical Crushing or lysis agent.Following rules are examples of suitable purification rules: by the classification on the ion exchange column; Ethanol precipitation; Reversed-phase HPLC; Chromatography on the tripoli, heparin SEPHAROSE ^TMOn chromatography, the chromatography on anion or the cation exchange resin (such as poly-aspartate post, DEAE etc.); Chromatofocusing; SDS-PAGE; Ammonium sulfate precipitation; Utilize for example gel filtration of Sephadex G-75; Impurity with protein A Sepharose post removal such as IgG; Reach with of the present invention polypeptide of metal chelating column in conjunction with the epi-position mark pattern.

For example, can use for example heparin chromatography, gel electrophoresis and dialysis to carry out purification from the VEGF compositions of cell preparation.Also can utilize other purified technology of protein.

Goods

In another embodiment of the invention, the goods that comprise the material that can be used for method mentioned above and treatment of wounds are provided.Described goods comprise container, label and package insert.Suitable containers comprises for example medicine bottle, pencil, syringe, dressing, binder etc.Described container can be made with various materials, such as glass, plastics, nylon, Cotton Gossypii, polyester etc.Described container is equipped with the compositions of the described illness of effective treatment and can has aseptic access port, perhaps can be the pipe that a plurality of dosage are housed, and perhaps can be the syringe that has indicated the activating agent of having measured.At least a activating agent in the described compositions is contained in the described container.Be attached on the container or the label relevant with it indicates described compositions and be used for quickening or improving wound healing.Described goods also can comprise second container, and the acceptable buffer of pharmaceutics wherein is housed, such as normal saline, phosphate buffered saline (PBS), woods Ge Shi (Ringer) solution and dextrose solution or gel solution.It also can comprise other required material on commercial and the user's position, comprises other buffer agent, diluent, filter, dressing, binder, applicator, gauze, barrier (barrier), semipermeable barrier, glossocatochus, syringe needle and syringe.Optional is, comprises the one group instruction relevant with dosage with the usage that VEGF is applied to wound described herein, generally is written instructions.The instruction that test kit comprised generally comprises dosage, the dosage regimen about treatment for diseases and uses the information in path.The container of VEGF can be unit dose, in bulk/a large amount of packing (for example multiple-unit container) or subunit dosage.

Think that this description is enough to make those skilled in the art can implement the present invention.In fact, those contents of and record shown except this paper, various modifications of the present invention are described according to preamble to those skilled in the art and will will be conspicuous and fall into the scope of claims.

Embodiment

Should understand embodiment described herein and embodiment and only be used for the illustration purpose, and those skilled in the art will remember various modifications or variation according to it that they are included in the application's spirit and the boundary and in the scope of claims.

Embodiment 1: the surperficial VEGF in the wound healing VGF2763g clinical trial

Carry out double blinding (for example the pharmacists knows the inside story, and the doctor is ignorant and the patient is ignorant) clinical trial to determine whether surface applications VEGF can improve the wound healing among the human experimenter who suffers from diabetic ulcer.Table 4 is the charts about patient's the baseline state of an illness (baseline disease characteristics) in the research of using rhVEGF (being called " Telbermin " at this) treatment diabetic wound.Research design as shown in Figure 1.

The table 4 baseline state of an illness

	Placebo (N=26)	Telbermin(N＝29)
			Mean age, year (scope)	59.3(38-81)	59.5(42-74)
Average glucose, mg/dL (scope) *	225.8(77-465)	179.1(29-593)
			Average HbA1C, % (scope) 	8.4(5.5-13.6)	8.3(5.6-13.6)
The ulcer of debridement during examination, whether N (%)	21(80.8) 5(19.2)	27(93.1) 2(6.9)
			Average ulcer area, cm 2The 1st day area measurement of area measurement  during length x width examination during (scope) examination	1.85(1.08-2.90) 1.14(0.50-2.24) 1.05(0.62-2.34)	1.92(0.96-4.08) 1.35(0.59-3.51) 1.15(0.44-2.97)

^*The experimenter of a placebo treatment is because of lacking the eliminating from gather of glucose numerical value.

The experimenter that telbermin of  handles is because of lacking the eliminating from gather of HbA1C numerical value.

The experimenter of two placebo treatment of  does not have the assessment of baseline planimetry.

The processing stage that 24 patients with placebo treatment having finished, finished the observation stage for 22.The processing stage that 27 patients that handle with telbermin (surface reorganization VEGF) having finished, finished the observation stage for 22.Handle in 6 weeks (totally 18 doses) with VEGF or inferior on every Wendesdays the continuing of placebo at the 1st day time 〉=0.4cm ²And≤4.0cm ²Debridement after the ulcer area be the I type of superficial cut (for example be in UT stage 1a (non-bone, muscle, tendon), see Table 2) or type ii diabetes patient (controlled, glycosylated hemoglobin A lc (HbA1c)≤12%).Processing be per 48 hours (+/-24 hours) once but be no more than 3 doses weekly.The amount of each VEGF that handles is 72 μ g/cm ²VEGF is on-the-spot the preparation.From medicine bottle, take out 0.22ml VEGF (5mg/ml) or placebo (buffering media) and add to about 5% methylcellulose (for example 4.7%) (Methocel A4M high-quality methylcellulose (The Dow Chemical Company for example of preparation in 5mM succinate pH of buffer 5.0; Midland, MI)) preparaton.VEGF or placebo were mixed with gel 2 hours, and this has improved viscosity and has reduced the loss of dosed administration material when using.Last 0.06%VEGF gel (final gel 3% methylcellulose) in 5mM succinate pH of buffer 5.0 (for example at VEGF 1.8mg/ml time contain 0.0036% polysorbate20 and 100mM dehydration trehalose) is final dosed administration material.Final dosed administration material is used with the 1.0ml tuberculin syringe, and the final dosed administration material of 0.6mL for example is housed.

Usually, ulcer is chronic ulcer.The persistent period of ulcer thoughtfully is less than 6 months more than or equal to 4 before handling.Do not have active infection and experimenter to have drug of topical application limb at the foot of being studied: ankle-arm index (ankle-brachial index, ABI) 〉=0.6 and≤1.2.During handling, two groups of VEGF or placebo have been carried out good wound care and assessment weekly, physical examination, planimetry trace and/or 35mm photo (Food and Drug Administration (FDA) Guidance for Industry 2000 (Food and Drug Administration (FDA) 2000 industry guide) for example for example, Chronic Cutaneous Ulcer and BurnWounds-Developing Products for Treatment (chronic skin ulcer that is used for the treatment of and burn wound research and development product), in June, 2000).

The terminal point that solves is the closed fully incidence rate of wound, it comprises does not have drain or covers the skin closure (for example assessing in back three months in closure) of wrapping up in needs and the wound healing that quickens, wherein speed has reflected that for example reaching the needed time of complete closure has clinically shortening significantly, and the time (for example reaching the time of complete closure) that reaches event analysis.Main effect terminal point is the percentage rate that total ulcer surface area dwindles when baseline the 43rd day (can late to the 49th day), and this quantitative analysis that is the planimetry trace by ulcer carries out is measured.Accessory effect terminal point comprises: compared the percentage rate that total ulcer surface area dwindles with baseline (for example the 1st day numerical value) during with the 84th day on the 29th day, the 29th, the incidence rate that ulcer heals fully in the time of 43 and 84 days, reach the time (for example natural law) that ulcer heals fully, the time (for example natural law) of experimenter's ulcer recurrence that ulcer healed fully before processing finished, compare the incidence rate that total ulcer surface area increases (＞15%) with baseline, advance the ulcer phase (advancing ulcerstage) incidence rate of (for example＞UT 1a), and the 1st, 8, the microcirculatory perfusion (microcirculatory perfusion) of ulcer bed surface in the time of 22 and 43 days (ulcer bed).

The safety issue of being monitored comprise clinically significant hypotension (before for example being defined as when handling after (the 1st, 3 and 5 day) uses every dose of research medicine between the period 1 60 minutes with respect to medication systolic pressure descend 〉=35mmHg), significant ulcer infects the generation of (ejection that for example is defined as ulcer increases and exudate frowziness, heating (temperature 〉=38.6 ℃) and＞10,000/μ of leukocyte (WBC) counting L), VEGF antibody etc. clinically.Measuring blood pressure between the period 1 before each medication and after each medication in 60 minutes.

Each gel cumulative volume of handling application is 0.12-0.48mL (72 μ g-288 μ g VEGF).The application quantity of gel is measured (LxW) based on wound, wherein L be the longest edge represented with cm to edge length, and W be represent with cm with L the longest vertical edge to the border width (surface area (cm that LxW=estimates ²)).For example, use gel by using aseptic spatula, wherein the gel total amount thickness in whole ulcer surface applications is 1/16 ".Wound covers with aseptic semipermeable barrier (for example film dressing of Shi Heing) and wraps up in gauze (for example Kerlix) wraparound.During the infra single treatment, remove dressing and wash ulcer with aseptic normal saline gentleness.Check the ulcer surface once more, use the gel of suitable dosage, and redress ulcer.

The result: surperficial VEGF looks like safe and well-tolerated.The incidence rate of adverse events is suitable between processed group (telbermin group and placebo group).None is drug induced by research for viewed adverse events or serious adverse events.Two patients are because serious adverse events has stopped the research (1 people → infected skin ulcer in the telbermin group; 1 people in the placebo group → localized infection).Have 1 patient to handle death in back 4 days the last time in the telbermin group, but death is not because the research medicine.In arbitrary processed group, all do not observe significant clinically hypotension case.

Data declaration the evidence of biologic activity.Do not observe in test the safety signal of aphtha with UT phase 1a size.Intermediate value % about wound area dwindles, fully the experimenter of healing % and reach healing time the results are summarized in table 5.Handling on every Wendesdays time in the diabetic subjects in 6 weeks with VEGF, population of subjects demonstrate compare with placebo with VEGF handle 6 week the back fully wound healing increased 14-25%.Test shows that VEGF is to the fast approximately 75-100% of the speed-up ratio placebo of healing.Referring to table 6, it has described the time that ulcer first heals fully that reaches in the patient with Telbermin (rhVEGF) or placebo treatment.Reaching the time that ulcer heals has fully quickened in the patient who handles with VEGF, for example reaches fully the time of the time (25 percentage points) of healing and be 32.5 days to 43.0 days.

Table 5

The intermediate value of wound area (total ulcer surface)

The experimenter that % heals fully

Reach the time (VEGF is to placebo) 4 of healing *

		% dwindles (VEGF is to placebo) 2*	(VEGF is to placebo) 3*
					43 days (the 6th week) the 84th day (the 12nd week) of the mat woven of fine bamboo strips	Safety can be assessed effect 1Safety can be assessed and effect can be assessed 1Can assess	95% pair 85% (p=0.67) 100% pair 88% (p=0.17) 100% pair 92% (p=0.49) 100% pair 93% (p=0.05)	41% pair 27% (p=0.39) 52% pair 27% (p=0.13) 52% pair 35% (p=0.28) 71% pair 38% (p=0.06)	HR?1.75(p＝0.18)HR?1.98(p＝0.12)HR?1.87(p＝0.13)HR?2.10(p＝0.08)

¹Effect can be assessed experimenter's (specifying) before knowing the inside story:

Remove the major programme violator

Lack three successive experimenters during available the last time administration and do not have LOCF (missing data is not put into) through the dosed administration checked

²P value: Wilcoxon grade-summation inspection

³P value: FisherShi Precision Test

⁴P value: Log-rank test

^*Appraisal procedure-quantitatively planimetry analysis

Table 6

Reach the time of healing fully *	Placebo (N=26)	Telbermin(N＝29)
			25 percentage points, day	43.0	?32.5
50 percentage points, day	ND	?58.0

ND=detect less than

^*Estimate with Kaplan Meier method.

For the experimenter who has realized that ulcer heals fully, finish or the recurrence of assessment ulcer between time of stopping in time that ulcer first heals fully and research.Realized that ulcer heals fully, can assess among the experimenter of safety, ulcer recurrence (log-grade p value=0.57) has taken place in experimenter's (15 philtrums have 4 people) that 26.7% usefulness telbermin handles and experimenter's (9 philtrums have 3 people) of 33.3% usefulness placebo treatment.Relatively the experimenter who handles with telbermin is 0.63 (95%CI:0.13,3.15) with the risk ratio that ulcer with the experimenter of placebo treatment recurs.

Embodiment 2: the surperficial VEGF in the wound healing

Estimation ulcer area (estimated ulcer area) after rapid debridement of when beginning treatment (sharp debridement) for example 〉=1.0cm ²And≤6.5cm ²The experimenter, the patient who for example suffers from I type or type ii diabetes, handle totally 12 weeks (can reach 84 doses altogether) by surface reorganization VEGF (example gel dosage form) every day or until wound closed fully (for example not having drain or cover to wrap up in the skin closure that needs), be as the criterion with person formerly.Can observe 12 weeks of experimenter or longer time at the treatment after date.The experimenter is each 24 μ g/cm that accept in treatment once a day ², 72 μ g/cm ²Or 216 μ g/cm ²VEGF.Estimate the area (cm on ulcer surface ²), for example length (L (cm)) is got the longest edge of ulcer to edge metering, and width (W (cm)) is taken from the longest edge with the vertical axle of length to edge metering.Estimate that then surface area is LxW.Can wait the evaluation process effect by analyze trace, photograph, physical examination via the planimetry at the measurement of the ulcer surface girth of trace, ulcer edge.The VEGF that uses should be 1.8,0.6 and 0.2mg/mlVEGF, 3% methylcellulose (Methocel A4M high-quality methylcellulose (The DowChemical Company for example; Midland, MI)), at 5mM, preparation in the succinate buffer of pH5.0 (for example when VEGF 1.8mg/ml, containing 0.0036% polysorbate20 and 100mM dehydration trehalose).

Think that this description is enough to make those skilled in the art can implement the present invention.Be to be understood that embodiment described herein and embodiment are only for the illustration purpose.In fact, except those this paper shown and describe, it is conspicuous and within the scope of the appended claims that various modifications of the present invention are described according to preamble to those skilled in the art.

Claims (25)

1. quicken the method for the wound healing among the experimenter, this method comprises: wound is used the VEGF of effective dose, and wherein the VEGF's of effective dose uses to cicatrize a wound and quickens greater than 60% compared with the control.

2. the process of claim 1 wherein that wound healing acceleration compared with the control is equal to or greater than 74%.

3. the process of claim 1 wherein that the acceleration of wound healing is to assess by the % that wound area dwindles.

4. the method for claim 3, wound area wherein is about 0.4cm before processing ²Or it is bigger.

5. the method for claim 3, wound area wherein is about 1.0cm before processing ²Or it is bigger.

6. the process of claim 1 wherein that the acceleration of wound healing is to assess by the speed that wound heals fully.

7. the wound that the process of claim 1 wherein is the diabetic foot ulcers.

8. the process of claim 1 wherein and use three times at least one week of VEGF of effective dose.

9. the process of claim 1 wherein that the VEGF of effective dose used for six weeks at least.

10. the process of claim 1 wherein that the VEGF that uses effective dose is closed fully until wound occurring.

11. the VEGF that the process of claim 1 wherein is VEGF ₁₆₅

12. the method for claim 1 or 11, VEGF wherein is the people VEGF of reorganization.

13. using of the process of claim 1 wherein is surperficial.

14. the VEGF that the process of claim 1 wherein is in supplying the preparaton of surface applied.

15. the wound that the process of claim 1 wherein is a chronic wounds.

16. the wound that the process of claim 1 wherein is pressure ulcer, decubital ulcer, varicose ulcer, burn, surgical wound or normal wound.

17. the experimenter who the process of claim 1 wherein is accepting to handle or accepted processing, processing has wherein delayed wound healing or invalid to wound healing.

18. the experimenter who the process of claim 1 wherein suffers from second kind of illness, second kind of illness wherein delayed wound healing or invalid to wound healing.

19. the method for claim 18, second kind of illness wherein is diabetes.

20. the process of claim 1 wherein that the effective dose of VEGF is about 20 μ g/cm ²To about 250 μ g/cm ²

21. the method for claim 20, wherein the effective dose of VEGF is about 24 μ g/cm ²

22. the method for claim 20, wherein the effective dose of VEGF is about 72 μ g/cm ²

23. the method for claim 20, wherein the effective dose of VEGF is about 216 μ g/cm ²

24. the experimenter who the process of claim 1 wherein is the people.

25. quicken the method for the wound healing among the human experimenter, this method comprises the VEGF that wound is used effective dose, the using to cicatrize a wound and quicken greater than 60% compared with the control of the VEGF of effective dose wherein, and wherein said wound had existed about 4 weeks or longer time on the experimenter before using the VEGF of effective dose.

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