CN1092076A - The target release of growth factors for bone regeneration - Google Patents

Abstract

The composition system that special target discharges, wherein somatomedin can adopt or not adopt sour cleavable connector to be connected with polyamino methylene phosphonic acid part, and is particularly suitable for discharging to the bone target.Said composition is activated in the somatomedin of bony site when having sour cleavable connector, and it in vivo circulation time be inactivation.The present invention has also instructed this compound of preparation and method for compositions.

Description

The target release of growth factors for bone regeneration

The present invention relates to specific target body release system; Promptly be, for osteanagenesis discharges positive growth factor by the chelant composite system to bone.The composition and their method of preparation that contain these systems also are parts of the present invention.

Be used to consider that the many physical appearance and diseases of mammalian bone loss of causing have, for example, that have a mind to and outer wound, osteoporosis and periodontal disease chance.Therefore, need provide the composition that stimulates and improve Mammals (for example patient) osteanagenesis usually in medical science and dental field.

Polypeptide growth factor (GF) is the protein that a class comes in handy for osteanagenesis, and it also is described as tissue growth and promotes the factor.Somatomedin is the polypeptide that stimulates the targeted cell population that limits.As polyfunctional molecule, they can stimulate or suppress cell proliferation and influence cell function, depend on the type of target cell and the existence of other single peptide.The example of somatomedin has platelet-derived somatomedin (PDGF), rhIGF-1 (IGF), transforming growth factor (TGF) is as (the TGF-β) of β and (the TGF-α) of α, Urogastron (EGF), fibroblast growth factor (FGF) comprises acid fibroblast growth factor (aFGF) and Prostatropin (bFGF), nerve growth factor (NGF), and bone morphogenetic protein (BMP), comprise skeletonization and bone-inducing factor.The combination of tissue growth factor can be of value to the promotion osteanagenesis.For example the combination of PDGF and IGF-I or PDGF and IGF-II promote osteanagenesis and wound healing [referring to, for example, Lynch et al., Proc.Nat ' l.Acad.Sci.(USA) 84,7696-7700(1987); Lynch et al., J.Clin.Invest.84,640-646(1989); Lynch et al., J.Clin.Periodontol.16,545-588(1989); Lynch et al., J.Periodontol.62,458-467(1991); With United States Patent (USP) 4,861,753 and 5,019,559].

PDGF is the polypeptide of about 28-35 kilodalton (KD).They are found in the many in vivo cell type.The PDGF that obtains from human blood platelets contains two peptide species sequences, PDGF-A and PDGF-B polypeptide [referring to H.N.Antoniaces and M.Hunkapiller, Science 220,963-965(1983)].PDGF-A is by genes encoding [the C.Betsholtz et al. that is positioned on the karyomit(e) 7, Nature 320,695-699(1986)], and PDGF-B by be positioned on the chromosome 22 [R.Dalla-Favera, Science 218,686-688(1982)] ill oncogene coding (R.Doolittle et al., Science 221,275-277(1983), and Waterfield et al., Nature 304,35-39(1983)].

Because the two peptide species chains of PDGF are by being positioned at different chromosomal two kinds of different genes codings, so people PDGF occurs with three kinds of forms, the heterodimer that the disulphide of PDGF-A and PDGF-B is connected, or two kinds of different homodimers (homodimer of PDGF-A and the homodimer of PDGF-B).The effect of PDGF in bone forming is unclear.Some studies show that its promote bone resorption [Tashjian et al., Endocrinology 111,118-124(1982); Canalis et al., J.Cell Physiol.140,530-537(1989)].Other studies show that PDGF is stimulating the scleroblast breeding of exsomatizing, when using for the duplicate injection of newborn rat subperiosteum, new bone forms [Piche and Graves in vivo, Bone 10,131-138(1989), Joyce et al., Clinical and Experimental Approaches to Dermal and Epidermal Repair:Normal and Chronic Wounds, pp.391-416(1991)].

IGF, or somatomedin are that having of about 7.5KD has 62% sequence homology with the polypeptide of the strong homology of proinsulin human [Humbel, Hormonal Proteins and Peptides 12,57-59(1984)] IGF-I and IGF-II.Their effect is regulated by two visibly different acceptors.IGF-I acceptor is called as I receptor (IGF-I R), and IGF-II acceptor is called as II receptor (IGF-II R).

At large studied the effect of IGF-I itself already to osteogenesis.In vivo, use by continuous and local in the rabbit shin bone of growing up and implant the formation [Aspenberg et al., Acta Orthop.Scand., 60,607-10(1989)] that the indoor IGF-I of peptide does not significantly change bone.Systematically using somatomedin C (IGF-I) continuously also fails to promote because recovery [Kirkeby and Ekeland, the Acta Orthop.Scand. of the bone fracture that the femur sacrotomy causes at the mouse body; 61; 335-38(1990)].The preliminary study of a small amount of animal shows, continues the IGF-I to be injected the artery of a rear foot continuously in 14 days, and the cortical bone that causes increasing at this pin of older mouse (but not young mouse) forms.This acts on the result [Spencer et al., Bone 12,21-26(1991)] that seemingly scleroblast quantity increases and amount of osteoclast reduces.The aufwuchsplate result that IGF-I part is imposed on HPX young mouse make one-sided produce on vertically little but significant osteogenesis effect (A.M.Isgaard et al., J.Physiol.250, E367-372(1986)].Isolate IGF-I and PDGF[Hanschka et al. from ground substance of bone, J.Biol.chem.261,665-74(1986); With Canalis et al., Cal.Tiss.Internatl.43,346-51(1988)].

Obviously inconsistent in external IGF-I to the data of osteocyte effect.Pfeilschifler etc. [Endocrinology 127,69-75(1990)] report, independent IGF have only slight effect during the ground substance of bone at the tire mouse braincap of cultivating is engaged.As IGF-I and PDGF-BB, TGF-β combination, or when combining, ground substance of bone is formed visible significantly effect with PDGF-BB and TGF-β.On the contrary, McCarthy etc. [Endocrinology 124,307-7(1989)] report IGF-I and IGF-II obvious stimulation DNA and collagen in the bone culture is synthetic.Hock etc. [Endocrinology 122,254-60(1988)] find that the IGF-I duplicates and stimulate with cellular replication irrelevant to collagen and ground substance of bone synthetic at external main stimulation preosteoblast.The anti-IGF-I of Canalis etc. [J.Cell.Physiol.140,530-537(1989)] report PDGF-BB is to collagen synthetic hormesis, and the IGF-I prevents that the effect of PDGF in the collagen degradation and PDGF-BB and IGF-I are to the synthetic other effect of tool of braincap DNA.Piche and Graves[Bone 10,131-8(1989)] also report, stimulate important in external IGF-I ³The H-thymidine mixes in the bone derived cell, does not also improve the activity of PDGF in this.IGF-I and PDGF, EGF and TGF-B combination causes by the picked-up of osteocyte almost equal in being obtained by 10% foetal calf serum.The acceptor of IGF-I and II is proved to be [Centrella et al., J.Cell Biol(summary) 107,62a(1988)] in the rich scleroblast culture of tire mouse bone.Recently Canalis etc. has been used as comment [J.Endocrinol.Invest.12,577-84(1989)] to the IGF-I in bone metabolism.

The somatomedin that another kind of osteogenesis to it has been used as research is FGF.In vivo, in fracture site aFGF and bFGF and their corresponding mRNA have been done detection [Joyce et al., (1991) are the same].AFGF all separates [Hauschka et al., (1986) are the same] from ground substance of bone with bFGF.Once bFGF and IGF were used in combination to promote the healing [Lynch et al., J.Clin.Invest.(1989) the same] of skin wound.

External, bFGF does not obviously change ³The H-thymidine mixes in the fracture callus [Joyce etc., (1991) are the same].Reported that bFGF improves mitogenesis in the tire cranium culture but the direct differentiation function of stimulating osteoblast [Canalis et al., J.Clin.Invest.81,1572(1988)] not.As bFGF, the aFGF biological action of pair bone that is in the news equally, but generally need higher concentration [Canalis, J.Clin.Invest.79,52-58(1987)].AFGF and bFGF all are tending towards reducing synthetic [the Canalis et al., (1989) are the same] of tire mouse braincap model mesostroma.The ox bone cell of cultivating can synthesize bFGF and aFGF, and storage [Globus et al., Endocrinology 124,1539(1989)] in its extracellular matrix.Reported that bFGF forms the ability [Noff et al., F.E.B.S.Letters 250,619-21(1989)] of bone sample joint at external raising medullary cell.The DNA that aFGF and bFGF all increase in the parietal bone cultured cells is synthetic, and bFGF is the stronger stimulant [McCarthy et al., Endocrinology 125,2118-26(1989)] of α 1-1 type tropocollagen mRNA simultaneously.They all are mitogenetic and chemotactic concerning the periodontal ligament derived cell and are attached on the pretreated dentine [Terranova et al., J.Periodontol.60,293-301(1989)]; Terranova et al., J.Periodontol.58,247-257(1987); Terranova, In the Biological Mechanisms ofTooth Extraction and Root Resorption, Davidovitch Z.ed.; Pp.23-34(1989)].

TGF-β family protein also shows the potentiality that have as the osteogenesis conditioning agent.External TGF-β produce by scleroblast and stimulate these cell proliferations and collagen synthetic [Robey et al., J.Cell Biol.105,457-463(1987); Rosen et al., Exper.Cell Res.165,127-138(1986); Hock et al., Cal.Tissue Int.32,385(summary) (1988)].Injection TFG-β stimulation cartilage formation and bone forming in the body [Joyce et al., J.Cell Biol.110,2195-2207(1990); Noda et al., Endocrinology 124,2991-2996(1989)].

Bone-inducing factor as bone morphogenetic protein osteogenin and osteoinductive protein matter 1, also can stimulate bone forming.Similar in appearance to TGF-β, its feature when subcutaneous or intramuscular are implanted into Mammals, has the dystopy of inducing cartilage and osteoplastic ability (United States Patent (USP) 4,877,864 on their common structures; 4,619,989; 4,455,256; 4,596,574; With 4,563,489; Wozney et al., Science 242,1528-1534(1988)].

Target release

Have the ability of regulating osteogenesis though somatomedin shows, its effectiveness as therapeutical agent is subjected to it is discharged into the restriction of the ability in the damaged site of bone.In disease such as osteoporosis, thisly damagedly appear at whole Skeletal system in the various times.Therefore, it is desirable to discharge somatomedin preferably for the damaged place of bone target to bone tissue.Discharge at present protein (as, polymer, bone graft and liposome) method target ground is local to be discharged or the whole body distribution owing to just have, and is not satisfied.

Known various aminophosphonic acid with a kind of metal that is coupled to aminophosphonic acid or aminocarboxylic acid ligand and aminocarboxylic acid mixture are to bone release medicine [United States Patent (USP) 4,508,704; 4,515,767; 4,560,548; 4,606,907; 4,897,254; 4,898,724; 5,059,412; 5,064,633; With 5,066; 478].

Very clear, people need preferentially be discharged into required somatomedin position of bone point and keep the purposes that its activity reaches expection.More preferably required somatomedin preferentially can be discharged into the position of bone point of damage or loss and still keep the activity of its expection.Most preferably required somatomedin preferentially can be discharged into the position of bone point of damage or loss and activate bone growth factor according to " required " that the Homeostatic mechanism by bone natural in the body determines.The present invention can satisfy above-mentioned purpose.

The invention provides by using the compound of bone growth promoting factor stimulation and enhance bone growth.Described bone growth promoting factor is to allow its mode that preferentially is positioned bone tissue link to each other with a kind of poly-aminomethy-lenephosphonic acids title complex and modified with a kind of.The compound that is used for this release system is expressed from the next:

GF-[（CL）z-L-AP]q（Ⅰ）

Wherein: GF is positive growth factor or its composition;

CL is that covalence key is incorporated into the sour cleavable linking group on the GF;

Z is 0,1 or 2;

Q is the sum of the amino that exists in 1 to natural GF;

L is the connection portion; With

AP is poly-aminomethy-lenephosphonic acids ligand.

The present invention has also considered formula I compound is imposed on mammiferous preparation and is released to the method for bone and the method for preparation with formula I compound target tropism.

When use contained formula I compound compositions or preparation, injured or loss bone place obtained medical treatment, and osteanagenesis.Tissue growth factor natural and reorganization can be buied everywhere by following: R ﹠amp; D Systems(Minneapolis, MN), Collaborative Research, Inc.(Bedford, MA), Genzyme, Inc(Cambridge, MA), ICN Biomedicals, INC, (Cleveland, OH), Peprotech, Inc.(Rocky Hill is NJ) with UBI(Lake Placid, NY).Osteoinductive protein matter can be from bone purifying [Celeste et al., Proc.Natl.Acad.Sci., (USA) 87,9843-9847(1990); Nang et al., Proc.Natl.Acad.Sci(USA) 85,9484-9488(1988); United States Patent (USP) 4,455,256 and 4,619,989].

Though above-mentioned arbitrary positive growth factor (GF) all can adopt in the formula I compound, GF is selected from PDGF preferably, IGF, FGF, TGF or cartilage/bone inducible factor (BMP).

PDGF preferably combines with the IGF-I, and showing when imposing on the disease bone separately or directly increases new bone forming (United States Patent (USP) 4,861,757 and 5,019,559 and unsettled U.S. Patent application 582, No. 332, on September 13 nineteen ninety, H.Antoniades and S.Lynch submit to).The PDGF per molecule contains 30 free amine groups, and thereby it may be modified the avidity of increase PDGF to bone.PDGF can be from R ﹠amp; D Systems and Genzyme, Inc. buys.PDGF is at United States Patent (USP) 4,861, is described in 757 and 5,019,559.

When independent or preferred and PDGF combination, when directly the IGF-I being applied to the disease bone, demonstrating increases new bone forming.Per molecule IGF-I contains 4 may be modified from increasing the free amine group of PDGF to the bone avidity.The IGF-I can be from R ﹠amp; D Systems and Genzyme, Inc. buys.The IGF-I, is described in 445-462(1990) at Eur.J.Biochem.190 by R.E.Humbel.

The IGF-II demonstrates when directly being applied to the disease bone increases new bone forming.Thereby containing 2, per molecule IGF-II may be modified the free amine group of increase PDGF to the bone avidity.The IGF-II can be from R ﹠amp; D Systems and Genzyme, Inc. buys.The IGF-II can be by R.E.Humbel at Eur.J.Biochem.190, is described in 445-462(1990).

BFGF demonstrates when directly being applied to the disease bone increases new bone forming.Thereby containing 15, per molecule bFGF may be modified the free amine group of increase PDGF to the bone avidity.BFGF can be by R ﹠amp; D Systems and Genzyme, Inc. buys.BFGF, is described in 95-114(1987) at Endocrinol.Rev.8 by Gospodarowicz etc.

AFGF is a kind of low-molecular-weight homodimer polypeptide, and when directly being applied to the disease bone, it demonstrates increases new bone forming.Thereby containing 14, the aFGF per molecule may be modified the free amine group of increase PDGF to the bone avidity.AFGF can be from R ﹠amp; D Systems and Genzyme, Inc. buys.AFGF, is described in 95-114(1987) at Endocrinol.Rev.8 by Gospodarowicz etc.

TGF-β ₁Be a kind of lower molecular weight (about 25KDa, amino acid) homodimer polypeptide, when directly being applied to the disease bone, it demonstrates increases new bone forming.Per molecule TGF-β ₁Thereby contain 18 and may be modified the free amine group of increase PDGF the bone avidity.TGF-β ₁Can be from R ﹠amp; D Systems and Genzyme, Inc. buys.TGF-β ₁, be described in 1039-1045(1987) at J.Cell Biol.105 by Sporn etc.

Demonstrating when using BMP in stroma increases new bone forming.BMP has done commentary by Celeste etc. in [Proc.Natl.Acad.Sci.(USA) 87,9843-9847(1990)], and the result is as follows:

BMP

BMP	The quantity of free amine group	KDa is about
			2	10	12.9
3	11	14.5
			4	8	13.1
5	11	15.6
			6	8	15.7
7	9	15.7

Poly-aminomethy-lenephosphonic acids title complex (AP of formula I) or be covalently bound on the GF of formula I (Z=0), or have and can cut linking group (L) and have (Z=1).This AP title complex can be the straight or branched part, circular part, polymkeric substance (comprises dense star polymer (dense star polymer), its tree (dendrimer) and dendron (dendron)), or aryl moiety, its title complex contains 2 at least, and preferred 3 or more, nitrogen-atoms.The poly-aminomethy-lenephosphonic acids title complex of one of preferred following formula of this title complex:

Wherein:

Each R ¹Be hydrogen independently of one another, C ₁-C ₄Alkyl, phenyl, hydroxyl C ₁-C ₄Alkyl ,-CH ₂COOH ,-CH ₂PO ₃H ₂Or L part;

Precondition is to have only a R ¹Can be L part and must have a L partly exist with precondition be half the R that has sum at least ¹For-CH ₂PO ₃H ₂;

Each R ²And R ³Be hydrogen independently of one another, C _1-4Alkyl or L part; Precondition is to have only a L partly to exist in the formula II;

N is 2,3 or 4 independently of one another;

N ' is 2,3 or 4 independently of one another; With

M is 0 to 10; Or

Wherein: R ¹, R ², R ³, n and m are as defined above; Or

Wherein: R ¹As defined above.

(L in the formula I) is expressed from the next in the connection portion:

Wherein: G is a hydrogen, NH ₂Or

R ⁴Be the electrophilic group that can be connected with protein;

R ⁵And R ⁶Be independently of one another hydrogen or-COOH;

Precondition is when G is hydrogen, then R ⁵Or R ⁶One of be COOH;

R ⁷Be hydrogen, hydroxyl or C ₁-C ₄Alkoxyl group; With

Y is 0,1,2,3 or 4;

Precondition be when y be 0,1,2,3 or 4 o'clock, R then ⁵Or R ⁶Have only one can be COOH.

Following term among the present invention is defined as follows.Term " straight or branched part " is meant to have 1 alkyl to about 100 carbon atoms, it can be linear fraction as, for example, ethyl, propyl group, normal-butyl, n-dodecane and other, or a chain portion as, for example, sec.-propyl, the tertiary butyl, 2,5,7-trimethyldodecane base or the like.The straight or branched part all must contain at least 2 nitrogen-atoms, preferred 3 to 50 nitrogen-atoms, and more preferably 3 to 25.Some examples of these parts comprise:

Wherein:

PDTMP=(N-propyl group carboxyl) ethylene diamine-N, N ', N '-trimethylene phosphonic;

The APEDTMP=[N-(4-aminophenyl) ethyl] ethylene diamine-N, N ', N '-trimethylene phosphonic;

The CEDTMP=1-(carboxyl) ethylene diamine-N, N, N ', N '-tetramethylene phosphonic acid;

ABEDTMP=[1-(4-amino-benzene methyl)] ethylene diamine-N, N, N ', N '-tetramethylene phosphonic acid;

The APIPTMP=N-(4-aminophenyl)-N, N-pair-[propyl group-(imido grpup dimethylene phosphonic acids)];

The APDTMP=N-[(4-aminophenyl) ethyl]-N, N-pair-[ethyl-(imido grpup dimethylene phosphonic acids]; With

The ABDTMP=N-[1-(4-aminobenzyl)-and N, N '-ethylene diamine-N ', N " ethyleneamines-N, N, N ', N '-pentamethylene phosphonic acids;

Preferred ABEDTMP and ABDTMP.These compounds are shown among the embodiment.

Term " loop section " is meant to have at least 2 nitrogen-atoms, preferred 3 to 10, and most preferably 3 to 8 nitrogen-atoms and carbon atom that exist doubles the aliphatic saturated ring system of nitrogen-atoms approximately.Some examples of this part comprise 1,4,7,10-tetraazacyclododecanand, 1; 5,8,12-tetraazacyclododecane tetradecane, 2-[(4-aminobenzyl)-1,4; 7, the 10-tetraazacyclododecanand]-1,4, the 7-10-tetramethylene phosphonic acid; 1-[(α-carboxylic acid)-and 4-amino-2-methoxybenzyl]-1,4,7,10-tetraazacyclododecanand-4; 7,10-trimethylene phosphonic and 1-[(α-phosphono) (4-aminophenyl) ethyl]-1,4; 7,10-tetraazacyclododecanand-4,7,10-trimethylene phosphonic.

Term " aryl moiety " is meant to have one or more additional rings or aromatic ring or by straight chain or by the aromatic ring of substitution in side chain be.Total atom number in this aromatic ring frame is 3 to 30, and is preferred 6 to 16, and most preferably 8 to 16.Aryl moiety contains 2 nitrogen-atoms at least, and preferred 3 to 10, and most preferably 3 to 8.Some examples of this part comprise pyrazolyl, 3-methylpyrazole base, 5-methylpyrazole base, imidazolyl, the 4-methylimidazole base, 5-methylimidazolyl, 1,4-methylimidazole base, 1,5-methylimidazole base, pyridazinyl, pyrimidyl, 2,4,6-trimethylammonium pyrimidyl, pyrazinyl, purine radicals, pteridine radicals, 3,6,9,15-4-azabicyclo [9.3.1] pentadecane-1(15), and 11,13-triolefin-3,6,9-trimethylene phosphonic (PCTMP), 6-(α-carboxyl-4-aminobenzyl)-3,6,9,15-4-azabicyclo [9.3.1] pentadecane-1(15), 11,13-triolefin-3,9-dimethylene phosphonic acids (PCAPCDMP), 13-(4-aminobenzyl-3,6,9,15-4-azabicyclo [9.3.1] pentadecane-1(15), 11,13-triolefin-3,6,9-trimethylene phosphonic (PCABTMP) and 6-[(α-phosphono-4-aminophenyl) ethyl]-3,6,9,15-4-azabicyclo [9.3.1] pentadecane-1(15), 11,13-triolefin-3,9-dimethylene phosphonic acids (PCAPCTMP).PCTMP, PCAPCDMP and PCAPCTMP, preferred PCTMP and PCAPCTMP, and unsettled U.S. Patent application 805 at Kiefer etc., No. 551, tell about in October 10 1991 applying date (entrust certificate by not record transfers The Dow Chemical Company), the description of this article is by reference and be incorporated into this paper.Also can there be other heteroatoms such as oxygen.

Term " polymer " comprises the compact star shaped polymer " as disclosed european patent application 0271180 on June 16th, 1988, described definition, the description of this article is incorporated into this paper by reference." compact star shaped polymer " also can be with reference to " STARBURST ^TM" polymkeric substance (Michigan Molecular Institute, Midland, the trade mark of MI) or STARBURST ^TMTree is also described in same European patent is open.Preferred STARBURST ^TMTree has poly-amino imino group, and wherein the surface has the amino that changes into the aminomethy-lenephosphonic acids group and has at least 1 4-aminophenyl part from the teeth outwards.This STARBURST ^TMTree is by the method preparation of describing in the disclosed european patent application 0271180 on June 15th, 1988, and is by reference, that this article is incorporated herein." polymkeric substance " also comprises Arborol(G.R.Newkome, J.Org.Chem.50,2004-2006(1985) and contain amine the straight or branched polymkeric substance (V.P.Torchilin et al., Byull.Eksp.Biol.Med.102,63-65(1986).

Following formula is included in the scope of AP term definition of formula I equally:

H ₂N-（CH ₂）n-C（PO ₃H） ₂OH

Wherein n is 1-3.

Such compound is described in United States Patent (USP) 5,039,819; 5,019,651; 4,922,007; 4,621,077; 4,134,969; 4,117,086; 4,108,962; With 3,962, in 432.

Term " can be cut connector ", and (CL of formula I) is meant that having being bonded under some physiological condition between the protein of bifunctional chelants (BFCA) and GF at poly-aminomethy-lenephosphonic acids (AP of formula I) [at connection portion (L of formula I)] is that reversible maybe can be cut.Instructed such bonding in the industry and comprise and contain thiocarbamide, thioether, peptide, the bonding of ester and disulfide group [C.F.Meares et al., Int ' l J.Cancer, Supp.2 is 99-102(1988) with the reference that comprises herein, United States Patent (USP) 5,045,312].Also instructed the linking group that contains the amidine bonding between molecule and protein in the industry.These linking groups can be by the molecule of imido-ester and the amino prepared in reaction [O.R.Zaborsky in the protein, Immobilized Enzymes in Food and Microbial Processes, P187-202, A.C.Olson and C.C.Cooney, eds., Plenum, New York(1974)].Same instruction has an acid amides in the industry, and diester, thioether, hydro carbons and disulfide linkage close [C.H.Paik et al., J.Nucl.Med.30,1693-1701(1989) and M.K.Haseman, Eur.J.Nucl.Med.12,455-460(1986)].At United States Patent (USP) 5,094, instructed the bisphosphate and the amidated bisphosphate linking group of cleavable in 848.These linking groups are at the scene by enzyme such as phosphodiesterase, 5 '-phosphonuclease and acid phosphatase excision.United States Patent (USP) 5,094 has also been instructed the cleavable linking group of alkylidene group hydrizide key form in 849.These linking groups form by molecule that contains carbonyl and the molecular reaction that contains the hydrazides part.In addition, the acetyl glucosides was once proposed as cleavable linking group optionally [L.F.Tietze, Nachr.Chem., Tech.Lab.36,728-737(1988)].The join domain of acid cleavable can particularly advantageous.The big acid 10 times or more of excision speed when particularly preferably being PH5 during at least than PH7 can be cut connector, and these two kinds of speed are all 37 ℃ of mensuration.United States Patent (USP) 4,542,225 and 4,618,492 and the reference mentioned herein and with reference to and be combined in and described the example that number acid can be cut connector in herein our No. 026,800, the unsettled U.S. Patent application of application in 4 days March in 1993.In last a patent application, preferred sour cleavable linking group is the different sulphur cyanato-of a 4-phthalate anhydride.

Term " existence can be coupled to the group on the protein " and " can be connected to the electrophilic group on the protein " are meant the electrophilic group (R among the L of formula I on the amino acid that can be bonded on the protein (for example GF) ⁴).Some examples of the known suitable group of industry those of skill in the art include, but not limited to amino, dimaleoyl imino, azido-, different sulphur cyanato, vinyl pyridine base, acetobrom amido, carboxyl and N-hydroxy-succinamide base active ester.When these electrophilic groups existed, title complex (L-AP of formula I, the preferential connection) was BF-CA.

Term " poly-aminomethy-lenephosphonic acids part " (AP of formula I) represented by various possible groups, as circular part, and the straight or branched part, aryl moiety comprises the polymkeric substance of dense star-shape polymer (as above definition).They have at least one such part, promptly contain a methylene radical (CH between 2 nitrogen-atoms ₂-) n(wherein n be 2,3,4) (poly-aminomethylene).In this part, can exist more than 1 so poly-aminomethylene.This part also contains at least 2 through the covalently bound methylene phosphonic acid base (CH to poly-aminomethylene of nitrogen-atoms ₂-PO ₃H ₂) poly-aminomethy-lenephosphonic acids part preferably by formula II, III or IV representative but be not limited thereto.Poly-aminomethy-lenephosphonic acids part preferably has one of existence as described herein and can be coupled to protein and maybe can be connected to group on the proteinic electrophilic group.Poly-aminomethy-lenephosphonic acids part (being represented by L-AP in the formula I) can be connected in protein (GF in the formula I) by cutting connector (being represented by CL in the formula I), and acid preferably as described herein can be cut connector.

Term " somatomedin " and " the tissue growth promotion factor " meaning are any stimulation mammalian cell breedings, differentiation, metabolism or mobile molecule.These factors can be obtained or be made by recombinant DNA technology or chemosynthesis by natural origin.These factors be purifying cross for well.

The term that this paper adopted " purifying " is meant, with before other somatomedin is mixed, this factor accounts for 90% or higher (that is, it is other protein of association natural with it not basically, lipid, and carbohydrate) of specified protein weight.The protein Preparation thing of purifying produces the single big bands of a spectrum of each subunit usually on polyacrylamide gel.The pure factor that the factor of used purifying is most preferably judged by the amino terminal amino acid sequential analysis in the present composition.

Title complex of the present invention (AP of formula I) can be the form of its medicinal salt.The term that is adopted " title complex " can be understood and comprises these salt herein.Term " medicinal salt " meaning is medicinal admissible positively charged ion.Be used to reach under the dosage of required effect, these positively charged ions are nontoxic on substantially.Illustrate, these salt comprise alkali-metal, as sodium and potassium; Alkaline-earth metal as calcium and magnesium; Ammonium; The light metal of III A family comprises aluminium; With organic the primary, secondary and tertiary amine, as trialkylamine, comprise triethylamine, PROCAINE HCL, PHARMA GRADE, dibenzyl amine, N, N-dibenzyl ethylene diamine, dihydro Ah must ethylamine (dihydroabieehylamide), N-(C ₁-C ₄) salt of Alkylpiperidine and any amine that other is fit to.Preferred sodium and sylvite.Term " medicinal " meaning is to warm-blooded animal for example to be suitable for, Mammals, and particularly the people uses, and comprises nontoxic, for example, suitable medicinal and nontoxic to warm-blooded animal.The pharmaceutical salts of The compounds of this invention prepares by the conventional ion exchanged form or with suitable alkaline purification title complex or formula I compound.

The preparation of poly-aminomethy-lenephosphonic acids

The known method that the poly-aminomethy-lenephosphonic acids part of the various the present invention of preparation is arranged.Below to wherein several do discussion.

A. prepare by amine

Many document descriptions have been arranged by amine preparation poly-aminomethy-lenephosphonic acids, particularly straight or branched part.For example, United States Patent (USP) 2,599,807, the description of this article is incorporated into this paper by reference, has instructed by the poly-aminomethy-lenephosphonic acids of heating amine aqueous solution and chloro methylene phosphonic acid preparation under the condition that PH in the presence of alkali such as the yellow soda ash＞10 are arranged.Below shown in other example of providing of reference described and adopt the various phosphonomethyl reagent shown in the following table from corresponding ethylene diamine (EDA) preparation ethylene diamine tetramethylene phosphonic acid (EDTMP).

The preparation of EDTMP

* EDTA=ethylene diamine tetramethylene carboxylic acid

Disclosing of *=this article is incorporated herein by reference

United States Patent (USP) 4,937,333, (this paper with reference to and combine the description of this article), with the Recl.Trav.Chim.Pays-BAS 110 of D.W.Swinkels etc., as seen prepare the other example of the poly-aminomethy-lenephosphonic acids of ring-type in 124-128(1991) by corresponding amine.

Be found in disclosed WO91/07911 on June 13rd, 1991 through intermediate aminomethy-lenephosphonic acids ester or blended aminomethy-lenephosphonic acids ester from the poly-aminomethy-lenephosphonic acids of amine preparation, this paper is with reference to the description that combines this article.This method adopts the aqueous solution of phosphonic acids methylating reagent (for example: formaldehyde and dialkyl phosphite) to form phosphonic acids all alkyl ester.Then this ester is hydrolyzed into aminomethy-lenephosphonic acids.The document has also been described and how have been used highly basic (for example, n-Butyl Lithium) and with haloalkyl or this ester of arylalkyl processing alkyl or aryl is substituted on the carbon between nitrogen and the phosphorus.

B. by the poly-aminomethy-lenephosphonic acids of carboxylic acid preparation

Can change into corresponding poly-aminomethy-lenephosphonic acids with gathering the aminomethylene carboxylic acid by various known method.The reaction of the general corresponding carboxylic acid of conversion relates to the reagent that can supply with phosphonyl group.For example, EDTA and PCl ₃Reaction in oil of mirbane for example provides EDTMP(, United States Patent (USP) 3,832,392) this paper with reference to and combine the description of this article.

C. the preparation that has the bifunctional chelants (BFCA) that can change into poly-aminomethy-lenephosphonic acids of amido

The unsettled U.S. Patent application 565 of (per power of attorney by not record transfers The Dow Chemical Company) application on August 9 nineteen ninety of people such as Simon, instructed for No. 379 preparation can be connected in the method for the poly-aminomethy-lenephosphonic acids of proteinic various straight or branched, this paper is with reference to the description that combines this article.On September 27th, 1984 disclosed WO 84/03698, having described can be by the bifunctional chelants intermediate of the open chain polyamine of phosphono methylene radical productization generation BFCA alkalization.Synthesizing in the U.S. 4,808,541 of straight or branched polyalkylene polyphosphonic acid BFCA also is described, and this paper is with reference to the description that combines this article.

United States Patent (USP) 3,994,966 and 4,622,420 and the various document descriptions that provide herein how to prepare the various BFCA that have the not isoplastic ethylene diamine that can be connected in the protein amido and diethylenetriamine alkalization.Thereby all above-mentioned BFCA can change into poly-aminomethy-lenephosphonic acids BFC by amine and can be connected on the proteinic amido.

Contain by the poly-aminomethy-lenephosphonic acids BFCA of the open chain of aminocarboxylic acid bonding passable, described on August 24th, 1988 disclosed european patent application 0279307, from corresponding polyamine intermediate preparation, this paper with reference to and combine the description of this article.United States Patent (USP) 4,994,560; 5,006,643 and 5,064,956 have also described open chain amine or the cyclammonium that how to prepare the various BFCA of containing.It can be produced corresponding poly-aminomethy-lenephosphonic acids BFCA by phosphonomethylization.

Instructed ring polyamine BFCA February 7 nineteen ninety in the disclosed european patent application 0353450, and described how to prepare many different cyclammonium that contain BFCA, can again its carboxymethylation be produced the aminomethylene carboxylate then.This amine that contains the BFCA intermediate in addition can be by the poly-accordingly aminomethy-lenephosphonic acids BFCA of phosphonomethyl generation.Equally, United States Patent (USP) 4,885,363 have described various 1,4,7 of the intermediates that contain, the amine of 10-tetraazacyclododecanand alkalization.This intermediate can turn into changing into poly-aminomethy-lenephosphonic acids BFCA by phosphonomethyl.With 1,4,7,10-tetraazacyclododecanand tetramethylene phosphonic acid is the ring BFCA of skeleton and is that the open chain BFCA of skeleton is the preferred group of poly-aminomethy-lenephosphonic acids BFCA title complex with diethylidene triamino pentamethylene phosphonic acids.

The proteinic group of connection on the poly-aminomethy-lenephosphonic acids BFCA can be replaced by the poly-aminomethy-lenephosphonic acids of ring-type itself, and by J.P.L.Cox at J.Chem.Soc., Chem.Commun.797(1989), with M.K.Moi etc. at J.Amer.Chem.Soc.110, exemplify out in 6266-6267(1988).It is open on November 30th, 89/11475,1989 that same compound is shown in WO.

With 1,4,8, the 11-tetraazacyclododecane tetradecane be the cyclic amine compound of skeleton by M.K.moi etc. at Inorg.Chem.26, be described in 3458-3463(1987).They can change into the mapping compound of tetramino methylene phosphonic acid from method above-mentioned.Can be used for similar compound of the present invention and be described in United States Patent (USP) 4,678, in 667.T.J.McMurry etc. are at Bioconjugate Chem.3(2), the example of other ring polyamine has been described in 108-117(1992).These intermediate double official can cyclammonium can phosphonomethylization and obtain the L-AP part of poly-aminomethy-lenephosphonic acids BFCA(formula I accordingly).

Many nitrogen heterocyclic rings of dicyclo carboxylic acid BFCA intermediate, be disclosed in the unsettled U.S. Patent application of submitting in (per power of attorney by not record transfers Tne Dow Chemical Company) on December 10th, 1991 of Kiefer etc. 805, in No. 270, this paper can be obtained poly-aminomethy-lenephosphonic acids BFCA by phosphonomethylization with reference to the description that combines this article.

Many nitrogen heterocyclic rings of dicyclo carboxylic acid BFCA, be disclosed in the unsettled U.S. Patent application of submitting in (per power of attorney by not record transfers Tne Dow Chemical Company) on December 10th, 1991 of Kiefer etc. 805, No. 551, this paper has instructed to prepare various methods with bicyclopolyazamacrocacidshosphonic acidshosphonic BFCA of poly-aminomethy-lenephosphonic acids BFCA with reference to the description that combines this article.

D. the preparation that has the bifunctional chelants (BFCA) of the nitro that is reduced into amido

When needs with nitro, particularly nitrophenyl is reduced into accordingly when amino, especially for the compound of describing in the C section in the above, this reduction is easy to be finished by the known method of the industry.For example, be listed in John Wiley ﹠amp; The Survey of Organic Synthesis 1 that Sons publishes, 411-417(1970) and the method in the document that comprises in this literary composition.

E. the preparation that has the bifunctional chelants (BFCA) of the amino that changes into electrophilic group

The BFCA of ligand of the present invention (for example L of formula I) has and can be coupled to proteinic group and maybe can be connected in proteinic electrophilic group.It is that the industry is known that this amino is changed into the method that can be connected in proteinic electrophilic group.Below some documents the method that is fit to is provided: C.F.Meares et al., Acc.Chem.Res.17,202-209(1984) and the reference that provides of this literary composition; D.Parker, Chem.Soc.Rev.19,271-291(1990) and the reference that provides of this literary composition; C.F.Meares et al., J.of Protein Chem.2,215-228(1984) and the reference that provides of this literary composition; C.F.Meares " Protein Tailoring Food Med Uses ", Amer.Chem.Soc.Symp., 339-352(1985), ed.R.E.Feeney and J.R.Whitaker, pub.Dekker, NY, NY.The front has provided the example of BFCA.In addition, many reagent can be used to be formed on the amido linkage that molecule that will contain amine in the aqueous solution and the molecule that contains carboxylate combine.For example, the water-soluble carbonyl diurethane imines that can buy on the market has been developed used as said purpose [J.V.Staros, Anal.Biochem.156,220-222(1986)].

The another kind of method that two nucleophilic groups are connected together is by reacting with aroyl chloride or its chemical equivalent, one of them being changed into the Micheal acceptor as amido.Like this amino being changed into can be again and the aromatic amide (Chem.Abst.83:802956) of different nucleophilic amine reactions.

M.J.Poznansky and R.L.Juliano be at Pharmacol.Rev.36,278-336(1984) and described other the method for preparing the pharmaceutical carrier binding substances among the WO 90/14844.

F. prepare BFCA by other method

The molecule that two nucleophilics are partly connected together is called the bifunctional cross-linker.If two reactions of molecule are terminal identical, they are called as " same difunctionality " linking agent.If two reactions of molecule are terminal different, they are called as " Heterobifunctional " linking agent.

T.Kitagawa etc. are at Chem.Pharm.Bull.29,1130-1135(1981) and in the reference that provides of this article disclose how to prepare the Heterobifunctional linking agent that is used for protein modification.Such reagent has two selective reaction groups such as dimaleoyl imino (it and thiol moiety reaction) and N-hydroxy-succinamide base ester (it reacts as Methionin with amino).Another molecule contains thiol group if molecule contains amido, and mentioned reagent can allow two molecules combine.The compound that similarly contains dimaleoyl imino by O.Nielsen at Synthesis 819-821(1991) in made report.

The bifunctional cross-linker who has developed other be used for BFCA or other close electric molecule be connected in protein or other nucleophilic substrate (United States Patent (USP) 4,680,338 and this article in the reference that provides); Dialdehyde crosslinking agent [S.Avamead et al., Scand.J.Immunol.8,7-23(1978)]; With the linking agent that can buy on the market referring to Pierce 1989 Handbook and General Cataloq.pp283-311(Pierce, Rockford, IL).

Contain the protein of electrophilic group such as amine (but also comprising other similar electrophilic group) and small molecules can by with market on the Traut reagent [I.Wower that can buy, Nuc.Acid Res.9,4285-4291(1981)] reaction with amino be converted into Cheng Kezai optionally with the Sulphydryl group of maleimide radical reaction.

G. can cut connector

At reversible poly-aminomethy-lenephosphonic acids BFCA that maybe can cut and the bonding between the GF under some physiological condition is favourable sometimes.The industry and above the example that provides instructed such bonding.Particularly advantageous is the connector that acid can be cut.At United States Patent (USP) 4,542,225 and 4,618,492 and the reference wherein mentioned in the example that acid can be cut connector has been described.These can be cut connector and have with the cyclic anhydride of amido reaction with at the dimaleoyl imino another end and the Sulphydryl reaction of connector at an end of connector.Strong being combined between acid anhydride and amino (forming acid amides at this and carboxylic acid) is easy under the acid PH cut.United States Patent (USP) 4,764,368 have also described application hexamethylene-1, the method that the 2-dicarboxylic anhydride can be cut the acid amides functional group as a kind of acid of introducing between small molecules and the macromole.WO 90/14844 has described and has used sugar derivatives as the connector cut under the mild acid conditions.

H. the existence of metal ion

Beat all is that the interpolation of metal ion does not significantly suppress the avidity of poly-aminomethy-lenephosphonic acids to the calcium surface.For example, the interpolation of calcium or samarium ion is disturbed one not, and 4,7,10-tetraazacyclododecanand tetramethylene phosphonic acid (DOTMP) is to the avidity on the calcification surface of hydroxylapatite.

For example, United States Patent (USP) 4,976, in 950 when and samarium, holmium, DOTMP demonstrates at live body and moves to the calcification site when gadolinium or ytterbium ion chelating.Ethylene diamine tetramethylene phosphonic acid (EDTMP) also demonstrates at live body and moves to calcification site (european patent application is open on December 27th, 0462787,1991) when with calcium or other metal ion-chelant equally.

I. connect proteinic aminophosphonic acid bunch

Polymkeric substance with poly-aminomethy-lenephosphonic acids, comprise as the dense star polymer described on June 15th, the 1988 disclosed european patent application 027180, can form phosphonic acids all alkyl ester by amine and formaldehyde and dialkyl phosphite reactant aqueous solution and prepare.This phosphonic acid ester of hydrolysis becomes the aminomethy-lenephosphonic acids of dense star polymer again.Particularly, this compact star superpolymer is for having the PAMAM form of amido on its surface.Above-mentioned amido forms poly-aminomethy-lenephosphonic acids with the phosphonomethyl reagent react again, is attached on the GF again.The advantage of dense star polymer of looking for (seeking) part as bone can be water-soluble for it, has controlled size and can be bonded to specific group on the GF and the amount of group.

The polymkeric substance of the L-AP of formula I also comprises the arborol(G.R.Newkome that can be connected on the protein (for example GF), J.Org.Chem.50,2004-2006(1985)).Arborol is the monocascade ball, has outside surface by the topped three-dimensional microenvironment of polar functional group.Can be (for example by the known method that ester and alcohol are transformed amine of the industry, the Survey of Organic Synthesis 1 that John Wiley publishes is 411-417(1970) with the reference that wherein provides) above-mentioned arborol is changed into polyamine.This arborol polyamine can change into poly-aminomethy-lenephosphonic acids by aforesaid method.Is raw material to have suitable group as the arborol cascade superpolymer to the nitrobenzyl bromide, and this arborol can contain can be connected in proteinic group.Thereby can prepare and contain poly-aminomethy-lenephosphonic acids group and maybe can cut the arborol that key is connected in proteinic group by stablizing covalent linkage.

Contain other polymkeric substance of amine and straight or branched that can be connected in proteinic group and also once did description [V.P.Torchilin et al., Byull.Eksp.Biol.Med.102,63-65(1986)].So polyamine-containing polymkeric substance can be changed into the polymkeric substance that contains poly-aminomethy-lenephosphonic acids group by above-described method.

By regulating the stoichiometry of protein, can change proteinic type to the reactive group that contains poly-aminomethy-lenephosphonic acids bunch.By regulate introducing the stoichiometry during the step that contains the reactive group key compound, cluster compound can be connected on two identical or different protein molecules.

J. alternative method

Be connected in unessential amido on the GF selectively in order to gather aminomethy-lenephosphonic acids BFCA, can finish by earlier GF being mixed formation GF-receptor clustering body with acceptor from the fixed form of the solubility of cell or solid support.This aggregate can be again and the poly-aminomethy-lenephosphonic acids BFCA reaction of excessive reaction formation then.Because GF is bonded on the acceptor, therefore have only the amido that does not relate to the GF acceptor interaction to be modified.The GF of Xiu Shiing produces biological activity at live body and its receptor acting by this way.At GF and the reusable acceptor that the GF that modifies and the aggregate between the acceptor then can split into new selective modification, its after separating can produce still can identification receptor full activity modifying GF.

Formula I compound can several different approaches preparations.For example, AP part and L reaction can be formed the L-AP part; L-AP partly links on the CL then; Again the CL-L-AP group is connected in GF.Under the another kind of situation, GF and CL reaction are formed the GF-CL part; This GF-CL reacts with L-AP and forms formula I compound.The degree of modification of GF is described by the q item of formula I, and it is by regulating GF, the concentration of reacting between CL and the L-AP molecule, time, temperature, PH and stoichiometry and tentatively obtain.

Formulation

Formulation of the present invention is solid or liquid form.The single material that these preparations can become direct use provides, or opportune moment before use two kinds of two or more materials (for example, kit form) of composition blended are provided.No matter be premix or medicine box, formulation all can need pharmaceutically acceptable carrier or auxiliary agent.

The all right parenterai administration of compound of the present invention, promptly subcutaneous, intravenously, intramuscular, or intraperitoneal become the injectable agent that can accept compound in the thinner on the physiology that has pharmaceutical carrier.Described pharmaceutical carrier can be following sterilised liq or mixtures of liquids, as water, and salt solution; D/W and relevant sugar aqueous solution, pure as ethanol, Virahol; or hexadecanol, glycerine such as propylene glycol or polyoxyethylene glycol, glycerol ketals is as 2; 2-dimethyl-1,3-diox-4-methyl alcohol, ether is as poly-(ethylene glycol) 400; oil; lipid acid, fatty acid ester or glyceryl ester, or acetylize glycerin fatty acid ester.Above-mentioned carrier adds or does not add pharmaceutically acceptable tensio-active agent such as soap or washing composition; Suspension agent such as pectin, carbon polymers (carbomer), methylcellulose gum, Vltra tears, or carboxymethyl cellulose; Or emulsifying agent and other medicines auxiliary agent.

The example that can be used on the oil of parenteral formulation of the present invention is an oil, animal, and vegetables oil, or synthetic oil, for example, peanut oil, soya-bean oil, sesame oil, Oleum Gossypii semen, Semen Maydis oil, sweet oil, Vaseline, and mineral oil.The lipid acid that is fit to comprises oleic acid, stearic acid, and Unimac 5680.The fatty acid ester that is fit to is, for example, and ethyl oleate and Isopropyl myristate.The soap that is fit to comprises fatty basic metal, and aluminium and triethanolamine salt and the washing composition that is fit to comprise cationic detergent, for example, and dimethyl dialkyl ammonium halogenide, alkyl pyridine halogenide and acetate alkylamine; Anionic detergent, for example, the alkyl of sulfonic acid, aryl and alkene ester, the alkyl of sulfuric acid and sulfo-succsinic acid, alkenyl, ether and direactive glyceride; Nonionic detergent, for example, fatty amine oxide, fatty acid alkyl amide and polyethylene oxide polypropylene copolymer, and ampholytic detergent, for example, alkyl-β-An Jibingsuan and 2-alkyl-tetrahydroglyoxaline quaternary amine, and composition thereof.Non-enteron aisle composition of the present invention generally contains about by weight 0.001% to about 10% the formula I compound that is dissolved in solution.It also is useful adopting sanitas and buffer reagent.For reducing or eliminate the excitement of injection site, said composition can contain the nonionogenic tenside that tool is about 12 to about 17 hydrophile-lipophile balance value (HLB).This tensio-active agent can be that the single composition with above-mentioned HLB maybe can be two or more mixture of ingredients with required HLB.The example that is used in the tensio-active agent in the parenteral formulation is a polyethylene fatty acid esters of sorbitan class, for example, and polyoxyethylene-sorbitan mono-oleate.

Other possible formula I compound formulation comprises: oral preparation, adopt known oral drug preparation, some of them can contain excessive magnesium hydroxide [P.J.Neuvonen et al., Eur.J.Clin.Pharmacol.35,495-501(1988)], adopt known B.Kari, Diabetes 35,217(1986), and B.R.Meyer et al., Clin.Pharmacol.Ther.44, the transdermal release of method 607(1988); Employing absorbs compound on the activated carbon granule, transplant again or the migration agent of injection [A.Hagiware, Gan to Kagaku Ryoho 15,1038-1042(1988) or Chem.Abst.109:156158n(1988)]; Discharge with intranasal, or make up separately or with permeation promoter [W.A.Lee.Bio Pharm.22-25(Nov/Dec, 1990) or Wall Street J.B1, (on August 16th, 1989)] and by R.Langer, Sci 249,1527-1583(1990 September 28) described.Preparation also can impose on injured or loss bone place by direct topical application method part.A kind of method in back needs the surgeon to expose position injured or the loss bone.

Adopt the osteanagenesis of The compounds of this invention not handle (that is, not using the external source medicament) or handle the more effective of gained with the unmodified positive growth factor (that is, being the GF of formula I) of same level than.Since GF product expense is high relatively and when discharges with the high dosage whole body GF produce the potential ability of unfavorable toxic effect, also need to use title complex with guarantee to be released into required site and minimizing GF total dose with its to mammiferous toxic action.

Method according to the regeneration bone of Mammals of the present invention (particularly patient), when giving the Mammals dispenser or by directly imposing on injured or loss bone district, or non-ly directly use, for example, comprise formula I compound compositions through systemic circulation (as following non-enteron aisle, intramuscular or subcutaneous injection) significant quantity.

Be used to help (CL) of the formula I of release in the GF bone ₂The requirement of-L-AP can be any significant quantity.The amount of the formula I compound of using for the disease for the treatment of this release system of any needs can have very big variation according to following factor: the concrete dose unit that is adopted, the course of treatment, by treatment patient's age and sex, the nature and extent of the illness of being treated and the factor that other medical professional knows.In addition, formula I compound can be used in combination with other known medicament that is used for the treatment of osteopathia.

The significant quantity of the formula I compound of using according to the present invention is generally about 0.005 to 50mg/Kg weight in patients, and the number of times of using can be once a day or repeatedly.Formula I compound can be subjected to the formulation administration as above-mentioned medical thing.Formula I compound can have one or more different activities compound, administration simultaneously or sequentially; And these compounds can be with other known regeneration bone active medicament administration.

The present invention will further be illustrated by the Consideration of the following example, and it is intended to explain purely the present invention.

Definition

The embodiment of the invention and not as follows at term defined above:

BMP=bone morphogenetic protein matter

The BSA=bovine serum albumin

The FCS=foetal calf serum

The HPLC=high pressure liquid chromatography

The HSA=human serum albumin

The PBS=phosphate buffered saline (PBS)

The RP-HPLC=reversed-phase HPLC

The different sulfo-cyanato-of SCN-BDTMP=4-benzyl diethylidene triamino pentamethylene phosphonic acids

Except that following explanation, all solvents and reagent obtain not repurity from commercial producer.

All water all passes through Barnstead NANOpure ^TMIon-exchange/carbon bed water purification system also has the resistivity of about 18.5 megaohms

Acetate for the purity that gets by Aldrich Chemical Co. greater than 99.99% glacial acetic acid

The 1.0ml equal portions of 1%HSA are at PBS(20mM phosphoric acid salt, and PH 7.4,0.15M NaCl) in sample be from Institute of Molecular Biology.Inc.(IMB) and freezing thing

¹²⁵I-PDGE is obtained by IMB that (freezing with the 135ml sample aliquot, it contains 100 μ g on-radiation IGF and 26.5 μ Ci carrier frees ¹²⁵I-PDGF), and the purification of Recombinant PDGF-BB and the IGF-I that do not demarcate, all be dissolved in the 0.1M acetate more than

Be used for washing or 10,000 and 3,000 weight shutoffs of the proteins concentrate aqueous solution be Centricon ^TMFilter membrane (Amicon Division of W.R.Grace and Co.)

Growth factor modified

General method

The recombinant human PDGF-BB of purifying and recombinant human IGF-I and divide other radioiodine form by Institute of Molecular Billogy, Inc. provides.By amino-acids composition and amine not terminal amino acid sequence determine the characteristic of PDGF-β and IGF-I.Basically according to method [the J.W.eveleigh and G.D.Winter of Eveleigh and Winter, " Amino Acid Composition Determination " Protein Sequence Determination, pp.91-95(1970)] then determine amino-acids composition by vapor phase hydrolysis protein with RPHPLC.Basically according to method [the M.W.Hunkapiller and L.E.Hood of Hunkapiller and Hood, Methodsin Enzymology 91,486-489, (1983)] determine the amine terminal amino acid sequence by HPLC preparation and analysis PTH amino acid then by the Edman degraded.The scan light densometer of employing Coomassie blue and the painted SDSPAGF gel of silver color and RP-HPLC determine purity.

Raw material

Embodiment A

The preparation of PDGF-BB and IGF-I

Produce also chromatography purification PDGF-BB and IGF-I routinely by the standard recombinant dna technology.Two kinds of methods all are that industry those of skill in the art know.(PDGF: United States Patent (USP) 4,061,757 and 5,045,633; IGF-I:Y.Sato et al., J.Biochem.101,1247-1252(1987) with Wong et al., Gene, 68,193-203(1988).

PDGF-BB(is provided by IMB) 1 to 20 amine terminal amino acid residue analysis obtain that single, complete amine is not held and consistent with those residues of estimating by the cDNA sequence.

People IGF-I(is provided by IMB) the sequential analysis of 1 to 53 the amine terminal residue consistent amine end of estimating with the cDNA sequence of those residues that produces single whole.Find this amino-acids composition with estimate by complete people IGF-I gene consistent.Two kinds of somatomedin purity are all greater than 95%.

Embodiment B

The biological activity of PDGF-BB and IGF-I

The effectiveness of every kind of factor or biologos are measured by cell cultures mitogenesis test (cell culture Mitogenic assay), wherein with half maximal stimulation effective dose [ED ₅₀] be defined as a unit.Two kinds of biometric establish a capital be according to measure [ ³H]-thymidine mixes the DNA of BALB/C3T3 l cell.3T3 cell among the DMEM of interpolation 10%FCS is gone into 96 hole assay plate with 2500 cell inoculations in every hole.Before using assay plate was cultivated (37 ℃) seven days.This makes the serum composition removal and causes in cell static.Added standard substance and specimen in the 7th day afternoon in the PDGF-BB biological assay, triplicate, and allow its overnight incubation continue 18 hours.Then with cellular exposure in 1.0 μ Ci's ³H-thymidine 6 hours, this thymidine are to mix according to the biological activity of FDGF.

In the biological assay of IGF-I, before adding specimen and standard substance, cell and PDGF-BB are cultivated in advance.Pre-cultivation can make cell when having Urogastron (EGF) to have adding IGF-I IGF-I be reacted.After a whole night incubation period, with cell and the 1.0 μ Ci that mix according to the biological activity of IGF-I ³The H-thymidine mixes.

In two kinds of mensuration of PDGF-BB and IGF-I, after the rough washing, cell carried out cytolysis and on liquid scintillation counter quantitatively.The data of gained plot dose response curve, can finish units activity thus and measure.

(the ED of 50% maximum cell reaction will be caused in suitable the mensuration ₅₀Value) GF concentration is defined as a unit.Maximum in PDGF and the IGF-I mensuration is reacted the cell response that is defined as 5% foetal calf serum (GCS) standard substance.The Mass Calculation of measuring divided by amino acid analysis according to mitogenetic units goes out special activity.PDGF-BB before the protein modification and the effectiveness of IGF-I are 1 to 3ng/mL(PDGF-BB referring to Fig. 3, and IGF-I is referring to Fig. 4).Typically, specific activity is 3 to 10 * 10 ⁵In the proteinic scope of unit/every milligram.

Embodiment C

Radiolabeled ¹²⁵I-PDGF

" cold " PDGF usefulness of describing in the embodiment A [ ¹²⁵I]-Bolton Hunter method mark, the primary amine of come-at-able Methionin of this method main mark and N-terminal residue.Also can buy in market and obtain from NEN ¹²⁵I-PDGF.NEN provides usually ¹²⁵I-PDGF is in containing the Trisodium Citrate buffered saline solution that 1%BSA makes carrier protein.Before the use, must buy from the market ¹²⁵IPDGF removes this BSA so that PDGF effectively modifies.DNAcarrier free for producing ¹²⁵I-PDGF adopts the antibody affinity chromatography.The anti-PDGF antibody of fixed (being provided by IMB) shows specific combination to AB and BB PDGF isomer, and is not joined on the AA homodimer.With 42.6 μ Ci ¹²⁵The neutral PH damping fluid of I-PDGF is added on the anti-PDGF post of 200 μ L.BSA cleaning down from the post is fallen and with 0.1M acetate wash-out with PBS ¹²⁵I-PDGF.According to the specific activity of 35 μ Ci/ μ g, estimate ¹²⁵Per 1.8 the PDGF dimers of the concrete probability average out to of I-PDGF connect one [ ¹²⁵I].

Embodiment D

Radiolabeled ¹²⁵I-IGF

Adopt lactoperoxidase or chloramine-T method (this method forms iodine tyrosyl product with the iodate of casein residue) to above-mentioned " cold " IGF carry out [ ¹²⁵I] mark. ¹²⁵I-IGF-I can buy from NEN.Provided ¹²⁵I-IGF-I is DNAcarrier free, and lyophilize from the 100mm Trisodium Citrate of 100 μ LPH45.According to the specific activity of 208 μ Ci/ μ g, right ¹²⁵The I-IGF-I probability averages appraisal, per 1.4 IGF-I protein chains be connected with one [ ¹²⁵I].

Embodiment E

The preparation of two kinds of PDGF solution

The bottle that will contain radioactivity PDGF is put into and has been carried out ¹²⁵In the hole of the Capintec dose calibrator that I regulates.Contain the bottle of 50 μ g PDGF with 200 μ L 0.1M acetate washings, and add to the cold IGF of 100 μ g that contains in 135 μ L 0.1M acetate and ¹²⁵In the bottle of I-PDGF.Use the bottle that contains cold IGF more than 7 parts 200 μ L0.1M acetate washing, merge washings, obtain containing the sample of radioactivity and active PDGF of non-radioactive and the active IGF of non-radioactive.The sample flasket that jolting merges shifts out two equal portions solution, every part 750 μ l to mixing.Duplicate samples such as every is placed on the separatory membrane of centricon10, put on 1 and 2.Take out suitable volume and γ reading, and before separating this sample and afterwards the volume and the radioactivity of taking-up sample are tested.With the centrifugal rotations of duplicate samples such as two, and put into the new filtering cup of demarcating weight.

Handle every part of membrane filtration thing (1 and 2) with 1.0mL 3.0M sodium bicarbonate buffer liquid then, and recentrifuge.Remove two filtering cups, replace with the new filtering cup of demarcating weight.The sample reading of two film reservations is 10.5 μ Ci.Handle two parts of membrane filtration things with other 1.0mL 3.0M sodium bicarbonate buffer liquid then, and recentrifuge.Removing two filtering cups also replaces with the new filtering cup of demarcating weight.The reading of two kinds of samples is 10.5 μ Ci on the film.Carry out above-mentioned washing step to remove the active IGF of membrane-permeable carrier non-radioactive.Remaining material is easy conjugated on the film.

Embodiment F

Preparation H ₂N-BDO3TMP

With 513g(1.3mmol) free alkali 1-(α-carboxyl-2-methoxyl group-5-nitrobenzyl)-1,4,7, the 10-tetraazacyclododecanand adds to the 776mg(4.7mmol of stirring) triethyl-phosphite and 141mg(4.7mmol) in the slurry of paraformaldehyde.With the gained slurry under agitation be heated to 95 ℃ 2 hours, be cooled to room temperature and vacuum concentration, obtain heavy-gravity oil.With the alkali alumina post this oil is carried out chromatogram, use the chloroform wash-out, evaporating solvent obtains faint yellow thickness buttery ester (55%) then, be 1-(α-carboxyl-2-methoxyl group-5-nitrobenzyl)-1,4,7,10-tetraazacyclododecanand-4,7,10-trimethylene phosphonic six ethyl esters.

With the 3mL concentrated hydrochloric acid at 100 ℃ with 250mg(0.3mmol) this ester stirs hydrolysis 18 hours.With this aqueous solution freeze-drying, look solid 1-(α-carboxyl-2-methoxyl group-5-nitrobenzyl obtains suckling)-1,4,7,10-tetraazacyclododecanand-4,7,10-trimethylene phosphonic (O ₂N-BDO 3TMP) (80%).

Section H with 100mg ₂N-BDO 3TMP is dissolved in the 20ml water.After the nitrogen purge reaction system, add 120mg 10%Pd/C, and under the vigorous stirring that continues, this suspension is placed under the nitrogen atmosphere.Freeze-drying filtrate obtained 89.1mg chocolate solid H after remove by filter catalyzer in 3 hours ₂N-BDO 3TMP.With 89.1mg H ₂N-BDO 3TMP is dissolved in the 4.26mL water, prepares the 23.4mM solution of this part.

Embodiment G

Preparation SCN-BDTMP

(1) the 1-(4-nitrobenzyl 2.0g(5.76mmol that in the 100mL three-necked flask, packs into))-Diethylenetriamine tri hydrochloride [adopt people such as M.W.Brechbiel, 2772-2781(1986) described in method preparation] at Inorg.Chem.25.Prepare 10.93g(0.108mol in addition) dense HCl and 6.91g(0.086mol) phosphorous acid solution and add in the reaction flask.Reaction flask is equipped with thermometer, reflux exchanger and splash bar.Continuing that reaction soln is refluxed.Abnormal pigmentary deposit on the skin is gone into 12.0g(0.144mol in addition funnel) 37%(weight) formaldehyde solution, funnel is connected on the reaction flask, drips formaldehyde solution with about 1mL/ minute speed in the mixture of heating.Reactant kept refluxing 16 hours again, reduced its volume then under vacuum, obtained amber semisolid.This solid is dissolved in about 2mL water, and under vigorous stirring, drops in about 800mL methyl alcohol.Remove by filter the gained white precipitate and, obtain 2.32g(57% 45 ℃ of dryings) the thick product 1-(4-of beige solid nitrobenzyl)-diethylenetriamine pentamethylenophosphonic acid(DTPP) (O ₂N-BDTMP).

(2) at 1.5cm Q-Sepharose(Pharmacia) the 1-(4-nitrobenzyl of purifying 500mg on the anion-exchange column) Diethylenetriamine pentamethylene phosphonic acids part crude product, with 0-1M ammonium acetate gradient elution.Required product 1-(4-nitrobenzyl) diethylenetriamine pentamethylenophosphonic acid(DTPP) has the maximum peak that keeps at UV280nm.Collect last elution peak, obtain the solution that about 43mL contains pure products.This volume is decreased to about 26mL and uses 200mg 10%Pd/C to handle.This suspension places under the nitrogen atmosphere and about 2.5 hours of vigorous stirring.HPLC(anionresin, through 30 minutes with 2mL/ minute speed with 0-1M ammonium acetate gradient elution) show that reduzate shifted to shorter retention time slightly.Then with suspension filtered, freeze in filtrate and obtain 259mg white glass shape solid.HPLC shows that its purity is 92%.Be further purified this product through above-mentioned preparation anion-exchange chromatography, obtain 144mg white solid 1-(4-aminobenzyl) ammonium salt of diethylenetriamine pentamethylenophosphonic acid(DTPP), anionresin HPLC shows its purity＞95%.The further feature of this product is as follows.

The P31NMR that uncouples has shown that 3 of 2: 2: 1 ratios of expection are unimodal.

¹H NMR（D ₂O）

δ 2.59-3.67（m，17H），4.13（b，2H），6.91（d，2H），7.21（d，2H）.

¹³CNMR（D ₂O）

δ 52.0，52.6，53.3，53.4，54.1，54.9，55.8，57.1，57.1，58.3，58.3，65.2，120.5，131.1，133.5，146.8.

(3a) with 146mg 1-(4-aminobenzyl) diethylenetriamine pentamethylenophosphonic acid(DTPP) is dissolved in the 1.913mL water ammonium salt solution that preparation 0.09M should acid.This solution of part (containing this phosphonic acids of about 25mg) of 328 μ L is placed the 10mL bottle that contains 1mL water and 1mL chloroform.In formed emulsion, add 100 μ L thiophosgenes, and about 1 hour of vigorous stirring.Remove chloroform layer, and partly use the chloroform extraction of 4 parts of 1mL with the aqueous solution of water extraction.Freezing and the freeze-drying in dry ice/acetone batch of the wash water solution of this product is obtained the loose white powder of 26.5mg.0.5mg wherein is dissolved in the 200 μ L water.Dilute 6 these solution of μ L with 3.8mL water, and the bright maximum absorption of gained UV stave has confirmed that 270 and the 283nm place this product is a 1-(4-isothiocyano benzyl) diethylenetriamine pentamethylenophosphonic acid(DTPP) (SCN-BDTMP).

(3b) with 43mg(51 μ M) part 1-(4-aminobenzyl) diethylenetriamine pentamethylenophosphonic acid(DTPP) is dissolved in the 2mL water, and mixes with the 1mL chloroform.In this solution that is stirring, once add 150 μ L thiophosgenes.Vigorous stirring was removed chloroform layer after 2.5 hours, and with the water of water extraction partly, with three parts of 3mL chloroform extractions.The product water solution of washing is freezing in dry ice/acetone batch, and lyophilize obtains the loose white powder of 45.4mg.Wherein 2.4mg is dissolved in the 960 μ L water.Dilute 6 these solution of μ L with 3.8mL water, and the bright peak value of gained UV stave has confirmed that 272 and the 282nm place this product is a 1-(4-isothiocyano benzyl) diethylenetriamine pentamethylenophosphonic acid(DTPP).

(3c) the SCN-BDTMP solution of preparation 3.47mM and 34.7mM

Part (1.389 * 10 with 100mg ^-5Mol) SCN-BDTMP is dissolved in the 400 μ L 0.3M sodium bicarbonate buffer liquid.The gained solution PH is 7.51, by adding 5 μ L 50%(weight) sodium hydroxide transfers to 9.48 with PH.Whole solution is 34.7mM SCN-BDTMP.

Add in the 45 μ L0.1M sodium bicarbonates 34.7mM SCNBDTMP solution of 5 μ L such as above-mentioned preparation and thoroughly mixing.Whole solution is 3.47mM SCN-BDTMP.

Embodiment H

Prepare four kinds of IGF solution

Again preparing every bottle 100 μ L with 100 μ L water respectively freezes in radio-labeled ¹²⁵I-IGF-I(derives from NEN) or freeze-drying reorganization IGF-I(derive from IMB), obtain 1mg/mL IGF solution, this solution contains 0.5M sodium-chlor in 20mM Tris-HCl, and contains 0.25%BSA and make carrier proteins.Content in the reorganization IGF bottle is dissolved in the 10mM acetate, and changes over to and contain in the glass serum bottle that is dissolved in the iodate IGF in the 535mg 10mM acetate.With the trace that contains that merges ¹²⁵The IGF solution of I-IGF-I is dialysed with the 10mM acetate of each 1L, and three times (Spectra/Por 7 ^TMFilm derives from Spectrum Medical Industries).Content in the dialysis tubing is divided into four parts of equal volumes, and is placed on 4 isolating Centricon ^TM(can see through 3000 molecular weight) in the microcon-centrator film filter and rotate Centricon then ^TMMake solution concentration, once with the content in the 1.6mL 0.3M sodium bicarbonate buffer liquid washing film of PH=9.48.This method is made 4 kinds of Centricon ^TMFilm unit, the amount of contained IGF is as follows:

IGF

Centricon TM	IGF μ g on the film *
		1	41.2
2	41.7
		3	45.7

* about

The embodiment I

Prepare two kinds of IGF solution

Freezing radiolabeled with every equal portions 150 μ L ¹²⁵I-IGF(wherein contains 300 μ g IGF-I and 23.6 μ Ci carrier frees in 0.1M acetate ¹²⁵I-IGF) thaw, be diluted to the about 1.5mL of final volume with 0.1M acetate then.This solution of every equal portions 700 μ L is placed two Centricon respectively ^TM(can see through 3000 molecular weight) on the microconcentrator film filter and rotate this Centricon then ^TMMake this solution concentration, the 0.3M sodium bicarbonate buffer liquid of every part of 1mL of the double PH=9.48 of using washs the content in the film.The calculated amount of IGF (comprises trace ¹²⁵I-IGF) be 130 μ g(1.7 * 10 on No. 1 film ^-8Mol), be 127 μ g on No. 2 films.

Embodiment J

Preparation 1,3-propylene diamine-N-(carboxyl propyl group)-N, N ', N '-trimethylene phosphonic (PDTMP)

The structure of Compound P DTMP is shown below:

3-aminopropyl-2-the tetramethyleneimine (5.0g) of commercial grade is slowly added in phosphorous acid (9.7g) solution in 6M hydrochloric acid (50mL).This yellow solution is heated to backflow, and dripped formalin (9.23ml 37% solution) with 2 hours and handle.With the solution cooling and except that desolvating, obtained heavy-gravity oil after refluxing again 18 hours.This oil droplet is gone in the 200mL methyl alcohol, filter also and obtain 5.15g PDTMP after the drying.This compound is being uncoupled ³¹The 8.2ppm of P-NMR spectrum and 7.5ppm(are with respect to H ₃PO ₄) ratio of locating to demonstrate expection is that 2: 1 two are unimodal.

This method can be as the part AP in the formula I, and wherein L is connected with the COOH group.

Embodiment K

Preparation quadrol-N-(4-amino-benzene ethyl)-and N, N ', N '-trimethylene phosphonic (APEDTMP)

The structure of compd A PEDTMP is shown below:

The 120mL quadrol (EDA) of packing in three 250mL Erlenmeyer flasks, flask is equipped with the rod of mixing that is contained in the stirring.Weigh up the p-nitrophenyl monobromoethane sample of three parts of 10g, and it is slowly added in the flask that is stirring that contains EDA with 30 minutes.After adding the p-nitrophenyl monobromoethane, with these flasks in stirred overnight at room temperature (16 hours).Change over to the content in the flask in the 500mL round-bottomed flask and connect the sample water distilling apparatus.Under 28-32 ℃ and vacuum from required product the excessive EDA of distillation.After most of initial EDA are removed, that heavy-gravity oil is soluble in water and with 75mL methylene dichloride (CH ₂Cl ₂) extract three times.Merge CH ₂Cl ₂Layer and rotary evaporation concentrate, and obtain 29.34 gram heavy-gravity dark oil liquid.

All these samples are added in the 500mL round-bottomed flask with 100mL 1.5M HCl.With gac, be heated to 90 ℃ and handle water layers, filter through paper filter then.The coupling vacuum stripping water layer and in vacuum drier dried overnight.

With cold methanol washing gained exsiccant solid and filtration, obtain 15.60g N-(p-nitrophenyl ethyl) ethylenediamine-hydrochloride.Evaporated filtrate also obtains other 6.77g product with methanol wash, and overall yield is 72%.

20.0g(0.063moles packs in a 250mL three-necked flask) N-(p-nitrophenyl ethyl) ethylenediamine-hydrochloride.In this flask, add the 9.0g deionized water and at 17.16g(0.209moles) 22.79g(0.219moles in the solution of phosphorous acid) concentrated hydrochloric acid.

This flask is connected with a reflux exchanger, loads onto a splash bar that is contained on the agitator.17.74g(0.219moles packs in a 10mL syringe) 37% formaldehyde solution and is connected with the syringe pump of demarcating in advance, make its flow velocity transmission with 0.1mL/ minute.Make reaction soln reach reflux temperature, then under continue stirring with in this flask, slowly adding formaldehyde solution in 3 hours.

After adding formaldehyde solution reaction is refluxed, and restir 3 hours.Reaction cooling and vacuum are removed anhydrated, obtain the heavy-gravity black solid.It is slowly added to obtain the light brown solid in the methyl alcohol.With this sedimentation and filtration and dry, obtain 20g(productive rate 65%) N-(p-nitrophenyl ethyl) quadrol-N, N ', N '-trimethylene phosphonic.This tri methylene phosphonic acid sample of 10g is dissolved in the 20mL water also with the dense ammonium hydroxide neutralization of 2mL.Then with anti-phase this crude product ammonium salt of preparation HPLC purifying, water wash-out.With the N-(p-nitrophenyl ethyl of purifying according to said method) quadrol-N, N ', N '-trimethylene phosphonic (710mg, 1.21 μ moles) sample is dissolved in the 50mL water and places the hydrogen cylinder that can hold the 235mL volume.In this bottle, be blown into nitrogen gradually to replace air.Add about 50mg10% palladium on carbon as catalyzer, water is washed the catalyzer of container side off.In case all catalyzer are positioned under water, the bottle that will be full of nitrogen places on the Parr hydrogenation oscillator arrangement.Vacuum is introduced hydrogen after removing nitrogen.Repeating this flushing is removed to guarantee all oxygen.Charging into hydrogen at last makes pressure reach 35psi.Begin hydrogenation and continue jolting by this bottle of jolting until stopping to absorb hydrogen (2.5 hours).Can use this pressure drop and known hydrogen volume to calculate the mole number of used hydrogen, be 99.2% of theoretical value in this example.Reaction mixture (shifting out the back from the decompression hydrogenation apparatus) through glass funnel vacuum filtration carefully, is used the isolating catalyzer of 25mL water washing institute four times at every turn.Freezing and freeze-drying is spent the night with the filter liquor that merges then, obtains 580mg(productive rate 86%) the N-(4-aminophenyl) ethylethylenediamine-N, N ', the ammonium salt of N '-trimethylene phosphonic (APEDTMP).

Uncoupling of this compound ³¹P-NMR spectrum demonstrate respectively expection 1: 2 ratio at 8.0ppm(with respect to H ₃PO ₄) locate unimodal and unimodal at the 16.9ppm place.

This compound can be as the part AP in the formula I, wherein L and NH ₂Group connects.

Embodiment L

Preparation 1-(carboxyl) ethylenediamine tetramethylene phosphonic acid (CEDTMP)

The structure of Compound C EDTMP is as follows:

In phosphorous acid (12.9g) solution in 6M hydrochloric acid (50mL), add 5.0g 2, and 3-diaminopropionic acid dihydrochloride (Aldrich Chemical Co., Milwaukee, WI).This solution is heated to backflow, and (37% formalin is handled 12.3mL) to add formalin with 45 minutes.After refluxing again 18 hours, remove and desolvate, oil is dissolved in the methyl alcohol (20mL).While stirring this solution is added in the dehydrated alcohol (200mL) then, carefully precipitated.Filter then and dry this solid, obtain 7.37g CEDTMP.This solution dilution of portion is also analyzed by high performance liquid chromatography (HPLC) for 10 times.HPLC is at Dionex ^TM2010; Ion Supression Chromatography System(Sunnyvale, CA) go up with the AS-7 anion-exchange column (8mm * 25cm) carry out, and with 0.6mL/ minute flow velocity with 0.03M nitric acid wash-out, carry out ultraviolet determination at the 330nm place and monitor.The hold-time of this compound is 10.11 minutes.

This compound can be as the part AP in the formula I, and wherein L is connected with the COOH group.

Embodiment M

Preparation 1-(4-aminophenyl) ethylenediamine tetramethylene phosphonic acid (ABEDTMP)

The structure of compd A BEDTMP is shown below:

In the 50mL round-bottomed flask, add 0.6g(0.2256moles) the 1-(4-nitrobenzyl) quadrol and dense HCl(1.24g, 0.0118mole), add then phosphorous acid (0.0925g, 0.0113mole).This solution being refluxed, then with 90 minutes dropping 0.956g(0.0118mole) 30% formaldehyde handles.Reaction keep to be refluxed spend the night, drop in the 100mL cold methanol after being cooled to room temperature.Filter and dry gained precipitation, obtain the 1-(4-nitrobenzyl) ethylenediamine tetramethylene phosphonic acid, it is dissolved in the 60mL water in the hydrogenation bottle.Add 50mg5% palladium on carbon sample, this bottle is connected also jolting until stopping to absorb hydrogen with the Parr hydrogenation apparatus.Filter this solution to remove catalyzer, freezing and freeze-drying obtains 1.28g(86% with the clarifying filtrate of gained) the 1-(4-aminobenzyl) ethylenediamine tetramethylene phosphonic acid.What this compound obtained expecting uncouples ³¹The P-NMR spectrum.

This compound can be as the part AP in the formula I, wherein L and NH ₂Group connects.

Embodiment N

Preparation N ⁴-(p-aminophenyl)-fall spermidine (norspermidine)

Preparation [two (cyano ethyl)] (p-aminophenyl) amine described in US2809985.With 9.0g(42mmol) this dintrile sample is dissolved among 90mL ethanol and the 10mL50%NaOH, and handle with 6g Raney nickel (w-2 level), place under the nitrogen atmosphere and jolting.Continue jolting until stopping to absorb hydrogen, at this moment filtration catalizer.The volume that vacuum reduces filtrate becomes heavy-gravity oil, and it is dissolved in 40mL 50%NaOH, and uses the 75mL chloroform extraction three times at every turn.With the chloroform layer that dried over sodium sulfate merges, decant and rotary evaporation in vacuo obtain 7.2g(productive rate 77%) title compound.Make product represent its feature by proton N MR.

This compound can be as the part AP in the formula I, wherein L and NH ₂Group connects.

Embodiment O

Preparation N " (4-aminophenyl)-dipropylenetriamine-N ', N ', N ' ", N ' " tetramethylene phosphonic acid (APIPMP)

The structure of compd A PIPMP is shown below:

With the N for preparing among the embodiment N ⁴-(p-aminophenyl) falls spermidine (3.65g, 16.4 μ moles) and adds in the 25mL water, uses 6.2g(75.6mmoles) phosphorous acid is at 13mL(163mmoles) solution among the dense HCl handles.The gained dark blue solution is heated to 100 ℃, and with 2.75 hours Dropwise 5 .9g(73mmoles) 37%(weight) formalin handles.Make solution keep again refluxing 15 hours, be cooled to room temperature then and splash in the ethanol.With the vacuum filtration of gained brown precipitate and splash in the ethanol.With vacuum filtration of gained brown precipitate and vacuum-drying, obtain 3.84g(productive rate 40%) brown solid title compound (APIPMP).When analyzing in anionresin HPLC system as described in embodiment 1, the retention time of products therefrom is 5.50 minutes (small peak) and 10.50 minutes (main peak).

This compound can be as the part AP in the formula I, wherein L and NH ₂Group connects.

Embodiment P

Preparation N " (4-amino-benzene ethyl) Diethylenetriamine-N ', N ', N ' ", N ' " tetramethylene phosphonic acid tetra-na salt (APDTMP)

The structure of compd A PDTMP is represented with following formula:

(20g 0.045mole) is dissolved in the 200mL toluene, and (10g, 0.89mole) solution-treated in 150mL toluene is 5 minutes with the p-nitrophenyl monobromoethane with Diethylenetriamine.Stir after 3 hours that decant goes out supernatant liquor from gummy solid, and with the water extraction of every part of 100mL three times.The water layer vacuum that merges is decreased to very little volume, and strips with the 100mL chloroform.The vacuum-evaporation chloroform obtains 9.32g(83% then) N " (p-nitrophenyl ethyl) Diethylenetriamine.

This amine sample of 5g is dissolved among the 3N HCl, makes PH less than 2.Gained solution is injected excessive methanol.Filter and vacuum-drying gained solution, obtain the corresponding hydrochloride of 3g.With 1.3g(0.004mole) this salt of part be dissolved in the 20mL water, and with phosphorous acid (1.5g, 0.018mole) and concentrated hydrochloric acid (2.0g 0.019moles) handles.This solution is refluxed and drip 37% formaldehyde (1.5g, 0.019moles).After refluxing again two hours this solution is cooled to room temperature and vacuum-evaporation, obtains heavy-gravity oil.Under vigorous stirring, this oil droplet is added in the 150mL methyl alcohol.Filter and vacuum-drying gained white precipitate, obtain 1.65g(productive rate 66%) N " (4-oil of mirbane ethyl) Diethylenetriamine-N ', N ', N ' ", N ' " tetramethylene phosphonic acid.In the described HPLC method of embodiment L, it is 16.9 minutes unimodal that this product demonstrates retention time.

With 700mg(1.1mmole) this compound sample is dissolved in 44mL(4.4mmole) among the 0.1N NaOH.In this solution, add 100mg 10% palladium on carbon that is suspended in the 56mL water, the entire reaction mixture is being placed under the nitrogen atmosphere under the vigorous stirring.After stopping to absorb hydrogen, filtration catalizer and vacuum-evaporation filtrate obtain 0.80g(productive rate 100%) the yellow solid N " (4-amino-benzene ethyl) diethylenetriamine-N ', N ', N ' " of moisture absorption, N ' " tetramethylene phosphonic acid tetra-na salt (APDTMP).To demonstrate retention time be 5.95 minutes unimodal to this title compound in the described HPLC of embodiment 1 system.

This compound can be as the part AP in the formula I, wherein L and NH ₂Group connects.

Embodiment Q

Preparation 4-isothiocyano Tetra hydro Phthalic anhydride (ACL-3, sour cleavable key)

To the 4-aminophthalic acid (2.7183g, 15.00mmol) and Anhydrous potassium carbonate (8.75g, 63.3mmol) add in the slurry in the 70mL tetrahydrofuran (THF) thiophosgene (2.30mL, 30.18mmol).Stirring at room 10 minutes, reflux was 1 hour then with reaction mixture.After being cooled to room temperature, with reaction product solution through diatomite filtration, then under the exsiccant nitrogen gas stream and in efficient fuming cupboard (avoiding exposing thiophosgene) be concentrated into dried.So obtain crude product 4-isothiocyano phthalic acid; ¹H NMR(300 MHz, the d 10.22(br s of acetone-d6), 2H), 7.81(br s, 1H), 7.61(s, 1H), 7.48(br s, 1H); 13c NMR(75 MHz, the d 167.8,167.7,138.3,135.8,134.4,131.7,131.7,128.6 of acetone-d6), 126.5.

With gained 4-isothiocyano phthalic acid immediately in the mixture of trifluoroacetic anhydride and methylene dichloride, under nitrogen atmosphere reflux 2 hours.Be cooled to concentrating under reduced pressure reaction mixture after the room temperature.Recrystallization gained solid from the 30mL tetracol phenixin obtains brownish purple crystal 4-isothiocyano Tetra hydro Phthalic anhydride, output be the 2.3778g(theoretical value 77%).The fusing point of this title compound is 106-108 ℃;

¹H NMR(300 MHz, the d 8.15(d of acetone-d6), J=8.1 Hz, 1H), and 8.06(d, J=1.7 Hz, 1H), and 7.97(dd, J=8.1,1.7 Hz, 1H); 13c NMR(75 MHz, the d 163.3,162.6 139.8 134.8,134.6,130.6,128.2,123.5,111.6 of acetone-d6); IR(CHCL ₃) 2010(br), 1840,1740 cm-1; MS m/e 205,161,133(alkali), 74.

End product

Embodiment 1 and Comparative examples A

The conjugation of PDGF and SCN-BDTMP

Part 34.7mM SCNBDTMP solution (1.389 * 10 with 400 μ L ^-5Moles) (prepared among the embodiment G) adds in 1 part of embodiment E product.This stoichiometry obtains the amino (sample 1) on isothiocyanate group/each protein of 794: 1 mol ratios.

The part 0.3M sodium bicarbonate buffer liquid of 400 μ L is added in the duplicate samples E products such as 2 (Comparative examples A) in contrast.

With sample 1 and 2 thorough mixing.Sample is stayed film last 14.5 hour in room temperature.Made sample concentration 1 hour with sample is at full speed centrifugal then.After centrifugal, the reading of every duplicate samples on film is 10.0 μ Ci.On every film, add 1.0mL 0.1M sodium phosphate buffer.After turn mixed, centrifugal this sample of full speed was 40 minutes once more.Remove this filtrate cup and with new, demarcate the filtering cup replacement of weight.The reading of each sample on film is 9.9 μ Ci.

Adding 740 μ L HSA solution in each sample makes the film upper volume reach 800 μ L.Each solution mixed and change over to new, demarcate in the filtering cup of weight.Wash each film unit with 500 μ L 1%HSA.With Centricon ^TMUnit adds a cover, turns around and centrifugal about 10 minutes at full speed, and the retention on the film is changed in the lid.Washings is sucked in the corresponding filtering cup.Use 200 μ L HSA solution washing Centricon again ^TMLid changes it in filtering cup over to then.Various final solutions contain the 18.5 μ g(1 that have an appointment respectively in 1.42mL HSA and 0.15M sodium-chlor) and 19.0 μ g(Comparative examples A).

Embodiment 2

The conjugation of each IGF and a SCN-BDTMP

With 10 μ L(3.47 * 10 ^-8Mole) 3.47mM SCN-BDTMP solution adds to (preparing among the embodiment G) Centricon of embodiment H ^TMNo.1(contains about 41.2 μ g IGF) in.The 390 μ L 0.1M sodium bicarbonate buffer liquid that add pH=9.35 in addition.Thorough mixing remains in Centricon then ^TMSolution on the film, and room temperature placement 14 hours.Upset rotation film unit is to shift out the composition on the film then.Make final volume reach about 600 μ L to wherein adding 158 μ L 0.1M sodium bicarbonates.

Embodiment 3

The conjugation of each IGF and two SCN-BDTMP

With 20 μ L(6.94 * 10 ^-8Mole) 3.47mM SCN-BDTMP solution adds to (preparing among the embodiment G) Centricon of embodiment H ^TMNo.2(contains about 41.7 μ g IGF) in.The 390 μ L 0.1M sodium bicarbonate buffer liquid that add pH=9.35 in addition.Thorough mixing remains in Centricon then ^TMSolution on the film, and room temperature placement 14 hours.Upset rotation film unit is to shift out the composition on the film then.Make final volume reach about 600 μ L to wherein adding 86 μ L 0.1M sodium bicarbonates.

Embodiment 4

The conjugation of each IGF and four SCN-BDTMP

With 400 μ L(1.39 * 10 ^-5Mole) 34.7mM SCN-BDTMP solution adds to (preparing among the embodiment G) Centricon of embodiment H ^TMNo.3(contains about 45.7 μ g IGF) in.Thorough mixing remains in Centricon then ^TMSolution on the film, and room temperature placement 14 hours.Upset rotation film unit is to remove the composition on the striping then.Make final volume reach about 600 μ L to wherein adding 208 μ L 0.1M sodium bicarbonates.

Comparative example B

Embodiment 2,3 and 4 contrast

The 400 μ L 0.1M sodium bicarbonate buffer liquid of pH=9.35 are added to the Centricon of embodiment H ^TMNo.4(contains about 47.7 μ g IGF) in.The 0.1M sodium bicarbonate buffer liquid that adds 390 μ L pH=9.35 in addition.Thorough mixing remains in Centricon then ^TMSolution on the film, and room temperature placement 14 hours.Upset rotation film unit is to shift out the composition on the striping then.Make final volume reach about 600 μ L to wherein adding 23 μ L 0.1M sodium bicarbonates.

Embodiment 5

The conjugation of each IGF and four SCN-BDTMP

With 400 μ L(1.39 * 10 ^-5Mole) 34.7mM SCN-BDTMP solution adds to (preparing among the embodiment G) Centricon of example I ^TMNo.1(contains about 130 μ g IGF) in.Thorough mixing remains in Centricon then ^TMSolution on the film, and room temperature placement 13 hours.Centrifugal this film unit concentrates protein soln.The residue of adding damping fluid on 0.5mL 0.1M sodium phosphate buffer (pH=7.4) the washing film, centrifugal then 1 hour.Add 572 μ L 1%HSA solution, turn and suction then and remove Centricon ^TMOn residue.Wash this film to remove the composition on the film fully with 0.5mL 1%HSA again.The merging solution final volume that is reclaimed is about 1.5mL, estimates to contain in about 1%HSA the IGF that 9.5 μ g/100 μ L modify.

Comparative example C

The contrast of embodiment 5

400 μ L 0.3M sodium bicarbonate buffer liquid (pH=9.48) are added to the Centricon of example I ^TMNo.2(contains about 127 μ g IGF) in.Thorough mixing remains in Centricon then ^TMSolution on the film, and room temperature placement 13 hours.Rotating this film unit concentrates protein soln.By adding damping fluid, on whizzer, rotated 1 hour then with the residue on 0.5mL 0.1M sodium phosphate buffer (pH=7.4) the washing film.By adding 855 μ L 1%HSA solution, reverberating and suction removes Centricon ^TMResistates on the film.Wash this film to remove the composition on the film fully with 0.5mL 1%HSA again.The merging solution final volume that is reclaimed is about 1.4mL, estimates to contain in about 1%HSA 9.5 μ g/100 μ L.

Embodiment 6

The IGF that adopts ACL-3 to modify with BDTMP

ACL-3 with 175 μ L ACL-3(12.1mg in 200 μ L trifluoroethanols prepares among the embodiment Q) add to the Centricon that in about 500 μ L 0.3M bicarbonate buffers (pH=9.5), contains about 100 μ g IGF ^TMOn.Gained pH is 8.85.In room temperature after 15 minutes, centrifugal Centricon ^TMUnit reduces to about 243 μ L(243mg until volume and weighs), this process need be carried out 2 hours approximately.In this spissated solution, add about 300 μ L ABDTMP solution (7.1mg, 10.5 μ moles in the 300 μ L bicarbonate buffers of pH=9.5).Gained pH is 9.27.This solution was placed 46 hours in room temperature.Recording pH is 9.78, and volume is about 0.485 μ L.Be placed in the whizzer (Clay-Adams) 6 hours, and made volume reduce to 117 μ L.Add 2mL 0.3M sodium bicarbonate buffer liquid, and this solution of recentrifuge.Volume is about 49 μ L after centrifugal 10 hours.Add 500 μ L 0.3M sodium bicarbonates and centrifugal Centricon again ^TMUnit 4 hours obtains 52 μ L final volume.Add 547 μ L 0.1M sodium phosphate buffer (pH=7.02) and thorough mixing.Centricon ^TMGross weight in the unit is 599mg, and perhaps its volume is about 600 μ L.Estimate that the concentration in this sample is per 600 μ L solution, 100 μ g IGF(16.7 μ g/100 μ L).

Comparative Example D

The contrast of embodiment 6

175 μ L trifluoroethanols are added to the Centricon that in about 500 μ L 0.3M bicarbonate buffers (pH=9.5), contains about 100 μ g IGF ^TMIn.Gained pH is 9.50.In room temperature after 15 minutes, centrifugal Centricon ^TMUnit reduces to about 230 μ L(230mg until volume and weighs), this process need be carried out about 2 hours approximately.In this spissated solution, add 300 μ L 0.3M sodium bicarbonate buffer liquid.Gained pH is 9.46.It was placed 46 hours in room temperature.Recording pH is 9.79, and volume is about 397 μ L.Be placed in the whizzer (Clay-Adams) 6 hours, and made volume reduce to 27 μ L.Add 2mL 0.3M sodium bicarbonate buffer liquid, this solution of recentrifuge.Volume is about 21 μ L after centrifugal 10 hours.Add 500 μ L 0.3M sodium bicarbonates and centrifugal Centricon in addition ^TMUnit 4 hours obtains 27 μ L final volume.Add 573 μ L 0.1M sodium phosphate buffer (pH=7.02) and thorough mixing.Centricon ^TMGross weight in the unit is 603mg, and perhaps its volume is about 600 μ L.The concentration of estimating IGF in this sample is per 600 μ L solution, 100 μ g IGF(16.7 μ g/100 μ L).

Embodiment 7

The IGF that adopts ACL-3 to modify with BDTMP

210 μ L equal portions IGF solution (are contained trace ¹²⁵The 100 μ g IGF in damping fluid of the IGF of I mark) place Centricon ^TMOn the film of membrane filter appts.500 μ L 0.3M sodium bicarbonates are added to Centricon ^TMIn the unit, centrifugal then making is left to be no more than 50mg on the film.Composition on the film (IGF of washing) is dissolved in the 0.3M sodium bicarbonate more than 500 μ L, and handles with the 19.4mg ACL-3 that is dissolved in the warm trifluoroethanol of 300 μ L.This sample was placed after 15 minutes centrifugal 2 hours.Centrifugal one finishes the ABHDP(357 μ moles with 300 μ L volumes) add to the Centricon of the IGF that contains modification ^TMIn the unit, just this solution was placed about 16 hours.PH is maintained at about 10 at this moment.Then with Centricon ^TMCentrifugal about 4 hours to reduce volume.With last material (IGF of modification) on the 500 μ L sodium bicarbonate buffer liquid dilution film, and by centrifugal concentrated once more.The volume of last resistates is 221mg on the film.To wherein adding 1.279mL 0.1M sodium phosphate (pH=7.02), make final volume amount to 1.5mL.Then with Centricon ^TMThe upset rotation is to remove remaining material on the film.This material is used for the mouse biodistribution research shown in the EXAMPLE V.

Comparative Example E

The contrast of embodiment 7

Adopt the method identical, just add warm trifluoroethanol and do not contain the compound that connects ACL-3 with embodiment 7.

Embodiment 8

The PDGF that adopts ACL-3 to modify with BDTMP

100 μ L PDGF solution are placed Centricon ^TMOn the film of membrane filter appts.This PDGF solution contains 10 μ Ci's in per 200 μ L 0.1M sodium phosphates, 0.01M Trisodium Citrate, 0.5M NaCl and 1% bovine serum albumin (pH=4.6) ¹²⁵The PDGF(22 μ Ci/ μ g of I mark).To Centricon ^TMAdd 500 μ L 0.3M sodium bicarbonates in the unit, centrifugal then to only surplus 50mg on film.Composition on the film (PDGF of washing) is dissolved in more than in the 500 μ L 0.3M sodium bicarbonates.The ACL-3 that is dissolved in the warm trifluoroethanol of 300 μ L with 36.7mg handles last material on the film then, places 15 minutes and centrifugal 2 hours.Centrifugal one finishes 300 μ L ABHDP(357 μ moles) add to and contain the Centricon that modifies PDGF ^TMIn the unit, and placed this solution about 16 hours.This moment, pH was maintained at about 10.Centrifugal then Centricon ^TMAbout 4 hours to reduce volume.With last material (PDGF of modification) on the 500 μ L sodium bicarbonate buffer liquid dilution film, and by centrifugal concentrated once more.The volume of last resistates is 329mg on the film.To Centricon ^TMAdd 1.171mL 0.1M potassiumphosphate (pH=7.02) on the film, make final volume amount to 1.5mL.Then with Centricon ^TMThe upset rotation is to remove last material on the film.This material is used for the mouse biodistribution research shown in the embodiment VI.

Comparative Example E

The contrast of embodiment 8

Adopt the method identical, just add warm trifluoroethanol and do not contain the compound that connects ACL-3 with embodiment 8.

Biology embodiment: bio distribution

The embodiment I

¹²⁵I-PDGF-（SCN-BDTMP）

Use the Spraque-Daawley mouse of heavy 175-230g, adapted to water and soil in preceding 5 days in injection.By the tail vein give two kinds of samples (sample 1 and Comparative examples A) that these mouse inject 50 μ L embodiment 1 (about 200,000cpm).After 2,6 and 18 hours, put to death mouse, take out and respectively organize, weigh, and measured radioactivity amount in each tissue in 5 minutes by numeration in the scintillometer that is equipped with the multichannel analyzer of Na I crystallization coupling by neck dislocation.This number in each tissue and the number in the 50 μ L standard substance are compared, to determine the percentage ratio of injected dose in each tissue.From standard substance, deduct the number in the tail, the adjusted percent dose of being injected of the amount of recording in the tail.Obtain the background number, and from organize number the subtracting background number.By the percent dose in the femur being multiply by the percent dose in the 25 estimation bones.Suppose muscle account for the mouse body weight 43% and blood accounts for 6.5% of mouse body weight, can obtain muscle and blood number.These percent dose that each body is partly adjusted are the acceptable values of mouse model.[people such as W.F.Goeckeler, J of Nucl.Med.28(4), 495-504(1987)].Provided this result in the table I, except as otherwise noted, wherein each data point is represented the mean value of five mouse.

The table I

Regulated the percentage injected dose that records dosage in the tail

^*The mean number of 3 mouse

^*The mean number of 4 mouse

* be comparison A

Obviously SCN-BDTMP and PDGF conjugation cause the PDGF target in bone.Compare with PDGF natural, unmodified, the effect that SCN-BDTMP conjugated PDGF enters bone exceeds 8 times.In addition, the residence time in bone has increased about 30 times.

The embodiment II

¹²⁵I-IGF-（SCN-BDTMP）

Use the Spraque-Daawley mouse of heavy 175-230g, adapted to water and soil in preceding 5 days in injection.Inject 50 μ L embodiment 2,3,4 or compare one of kind sample of B to these mouse by the tail vein.After 30 minutes, put to death mouse, take out and respectively organize, weigh, and measured radioactivity amount in each tissue in 5 minutes by numeration in the scintillometer that is equipped with the multichannel analyzer of Na I crystallization coupling by neck dislocation.This number in each tissue and the number in the 50 μ L standard substance are compared, to determine the percentage ratio of injected dose in each tissue.From standard substance, deduct the number in the tail, the adjusted percent dose of being injected of the amount of recording in the tail.Obtain the background number, and from organize number the subtracting background number.By the percent dose in the femur being multiply by the percent dose in the 25 estimation bones.Suppose muscle account for the mouse body weight 43% and blood accounts for 6.5% of mouse body weight, can obtain muscle and blood number.Provided this result in the table II, wherein each data point is represented the mean value of five mouse.

The table II

Regulated the percentage injected dose that records dosage in the tail

The AP of through type I adds that the degree of modification of IGF of the number of the bone that detects depends on the stoichiometry of reactant.The results are summarized in the table III, wherein each data point is represented the mean number of five mouse.

The table III

Obviously this result proves that IGF-I discharges and can realize by SCN-BDTMP and IGF conjugation to the bone target.As show as shown in the III, the degree of target is obviously directly related with degree of modification.

The embodiment III

¹²⁵I-IGF-（SCN-BDTMP）

The Spraque-Daawley mouse of heavy 175-230g adapted to water and soil in preceding 5 days in injection.Inject the sample of 50 μ L embodiment 5 or C for these mouse by the tail vein.After 2,6 and 18 hours, put to death mouse, take out and respectively organize, weigh, and measured radioactivity amount in each tissue in 5 minutes by numeration in the scintillometer that is equipped with the multichannel analyzer of Na I crystallization coupling by neck dislocation.This number in each tissue and the number in the 50 μ L standard substance are compared, to determine the percentage ratio of injected dose in each tissue.From standard substance, deduct the number in the tail, the adjusted percent dose of being injected of the amount of recording in the tail.By the percent dose in the femur being multiply by 25 times of percent dose in the estimation bone.Suppose muscle account for the mouse body weight 43% and blood accounts for 6.5% of mouse body weight, can obtain muscle and blood number.Provided this result in the table IV, wherein each data point is represented the mean value of four mouse.

The table IV

Regulated the percentage injected dose that records dosage in the tail

The embodiment IV

IGF-I-(SCN-BDTMP) biological activity

Each sample to the foregoing description carries out the analysis of IGF-I mitogenic activity to measure its usefulness.This scheme is based on the progressive model of ability, and analysis of cells system is replied the IGF-I product of various content in dosage dependence mode in this model.With the cell inoculation of mouse fibroblast cell system in 96 well analysis plates and grow to merge cause static.Before adding sample and standard substance, cultivate in advance with the PDGF-BB pair cell, it can be produced IGF-I when adding IFG-I in the presence of EGF reply.After the overnight incubation cell placed 1.0 μ Ci ³In the H-thymidine, measure according to the amount adding difference that adds to the IGF-I in the analysis hole ³The H-thymidine.Behind the thorough washing, make cytolysis, and in the scintillation counter of the multichannel analyzer that Na I crystallization coupling is housed, measure.With the drawing of gained data, obtain dose response curve, can carry out units activity by this curve and measure.

The IGF-I concentration (ED of a unit definition in above-mentioned analysis, causing 50% maximum cell to be replied ₅₀Value).Maximum is replied to be defined as the 5%FCS standard substance is produced cell response.The 3-5 that the maximum that is caused by exogenesis IGF-I is replied baseline normally doubly.

Before this analyzes dilution, by before operation, tracer having been inserted the deposit sample ¹²⁵The concentration of I-IGF-I tracer determination IGF-I is to these two kinds of sample numerations of 25 μ L deposit sample and each 25 μ L.The ratio of per minute numeration (CPM)/μ g of IGF-I is used for measuring the concentration of these two kinds of sample IGF-I to carry out bioanalysis in the deposit sample.The mean value of each equal portions deposit sample is 5.135CPM/ μ g.Measure the value of each sample and remove this number by CPM/ μ L with every part of value 5.135CPM/ μ g that lays in sample.These measure with calculating following value: embodiment 5 samples is 107.2 μ g/mL; Embodiment C is 110.8 μ g/mL.This results are shown in the table V.

The table V

* compare B

* is C relatively

The embodiment V

¹²⁵I-IGF-ACL-3-BDTMP）

The 500 μ L syringes that to be furnished with No. 28 entry needles of bore suck the material of potion (150 μ L) embodiment 7 and Comparative Example E, and 5 mouse are given the tail vein injection of every mouse.With mouse anesthesia and dissection, distribute after 6 hours with the organ that obtains the radio-labeled thing.Because included radioactivity amount is low, only to sample numeration 10 minutes.The final numeration (subtracting background) that records in the mouse femur of the modification IGF material of having injected embodiment 7 is to have injected 3.8 times of the numeration of surveying (subtracting background) in the mouse bone thigh of the contrast material of Comparative Example E, shown in following table VI.

The table VI

The embodiment VI

¹²⁵I-PDGF-ACL-3-BDTMP）

The 500 μ L syringes that to be furnished with No. 28 entry needles of bore suck this material of potion (100 μ L), and give injection Centicon 1(experiment in the tail vein of every mouse to 5 mouse) and Centicon 2(contrast).With mouse anesthesia and dissection, distribute after 6 hours with the organ that obtains the radio-labeled thing.Because included radioactivity amount is low, only to sample numeration 10 minutes.Injecting Centricon 1(experiment) the final numeration (subtracting background) that records in the mouse femur of material is to have injected Centricon 2(contrast) 3.8 times of the numeration of surveying (subtracting background) in the mouse bone thigh of material, shown in the following table VII.

The table VII

These data show to modify with technology GF of the present invention and cause biological activity GF bone target to discharge.

Although invention has been described for the preferred concrete scheme of reference, those of ordinary skill in the art obviously can not deviate from the change and the modification of the present invention's spirit mentioned above or hereinafter desired and scope to it after reading and having understood this specification sheets.

Claims (33)

1, following formula: compound

GF-[(CL)z-L-AP] _q(Ⅰ)

Wherein:

GF is that tissue growth promotes the factor or its composition;

CL is covalently bound to the sour cleavable linking group of GF;

Z is 0,1 or 2;

Q is the sum of the amino that exists in 1 to natural GF;

L is the connection portion;

AP is poly-aminomethy-lenephosphonic acids ligand.

2, the compound of claim 1, wherein GF is somatomedin, insulin-like growth factor, fibroblast growth factor, Urogastron, transforming growth factor, nerve growth factor or cartilage/bone incitant or its combination that thrombocyte produces.

3, the compound of claim 2, wherein GF is somatomedin, insulin-like growth factor, fibroblast growth factor, transforming growth factor or cartilage/bone incitant or its combination that thrombocyte produces.

4, the compound of claim 2, wherein GF is the somatomedin of thrombocyte generation and combining or the somatomedin of thrombocyte generation and combining of IGF-of IGF-.

5, the compound of claim 1, wherein AP is a part, and this part is straight or branched part, loop section, polymkeric substance or aryl moiety, and described part contains at least two nitrogen-atoms.

6, the compound of claim 5, wherein polymkeric substance is thick star-shape polymer.

7, the compound of claim 6, wherein thick celestial body polymkeric substance are branch polymers (dendrimer) or tree-shaped dendron.

8, the compound of claim 5, wherein part contains three or more nitrogen-atoms.

9, the compound of claim 5, wherein AP as shown in the formula:

Wherein:

Each R ¹Be hydrogen, C independently ₁-C ₄Alkyl, phenyl, hydroxyl C ₁-C ₄Alkyl ,-CH ₂COOH ,-CH ₂PO ₃H ₂Or L part;

Condition is to have only a R ¹Can be L part and essentially have a L part, and account for half R of sum at least ¹Be-CH ₂PO ₃H ₂;

Each R ²And R ³Be hydrogen or C independently ₁-C ₄Alkyl or L part;

Condition is only to have a L part in II;

N is 2,3 or 4;

N ' is 2,3 or 4; And

M is 0-10.

10, the compound of claim 5, wherein AP as shown in the formula:

R wherein ¹, R ², R ³, n and m in the claim definition.

11, the compound of claim 5, wherein AP is the straight or branched part.

12, the compound of claim 11, wherein straight or branched partly is

(N-propyl hydroxy) quadrol-N, N ', N '-trimethylene phosphonic;

[the N-(4-aminophenyl) ethyl] quadrol-N, N ', N '-trimethylene phosphonic;

The 1-(hydroxyl) quadrol-N, N, N ', M-tetramethylene phosphonic acid;

[the 1-(4-aminobenzyl)] quadrol-N, N, N ', N '-tetramethylene phosphonic acid;

The N-(4-aminophenyl)-and N, N-two [propyl group (imino--methylene phosphonic acid)];

The N-[(4-aminophenyl) ethyl]-N, N-two [ethyl (imino-diacetic methylene phosphonic acid)]; Or

The N-[1-(4-aminobenzyl)-and N, N-ethylenediamine-N ', N " quadrol-N, N, N ', N " pentamethylene phosphonic acids.

13, the compound of claim 5, wherein AP is a loop section.

14, the compound of claim 13, wherein loop section is: 1,4,7, the 10-tetraazacyclododecanand;

1,5,8, the 12-tetraazacyclododecane tetradecane;

The 2-[(4-aminobenzyl)-1,4,7, the 10-tetraazacyclododecanand]-1,4,7, the 10-tetramethylene phosphonic acid;

The 1-[(Alpha-hydroxy)-and 4-amino-2-methoxy-benzyl]-1,4,7,10-tetraazacyclododecanand-4,7,10-trimethylene phosphonic; Or

1-[(α-phosphono) (4-aminophenyl) ethyl]-1,4,7,10-tetraazacyclododecanand-4,7,10-trimethylene phosphonic.

15, the compound of claim 5, wherein AP is an aryl moiety.

16, the compound of claim 15, wherein aryl moiety is to have the aromatic ring system that adds up to 3-30 carbon atom on aromatic ring frame.

17, the compound of claim 16, wherein aromatic ring is fastened and is existed one or more additional rings or aromatic ring system partly to be replaced by straight or branched.

18, the compound of claim 16, wherein aromatic ring system is

3,6,9,15-four azabicyclos [9.3.1] 15 carbon-1(15), and 11,13-triolefin-3,6,9-trimethylene phosphonic;

6-(Alpha-hydroxy-4-aminobenzyl)-3,6,9,15-four azabicyclos [9.3.1] 15 carbon-1(15), and 11,13-triolefin-3,9-dimethylene phosphonic acids;

The 13-(4-aminobenzyl)-3,6,9,15-four azabicyclos [9.3.1] 15 carbon-1(15), and 11,13-triolefin-3,6,9-trimethylene phosphonic; Or

6-[(α-phosphono-4-aminophenyl) ethyl]-3,6,9,15-four azabicyclos [9.3.1] 15 carbon-1(15), and 11,13-triolefin-3,9-dimethylene phosphonic acids.

19, the compound of claim 1, wherein L as shown in the formula

Wherein G is hydrogen, NH ₂Or

R ⁴It is the electron-withdrawing group that can be connected with protein;

R ⁵And R ⁶Be independently hydrogen or-COOH; Condition is when G is hydrogen, R ⁵And R ⁶In one be-COOH;

R ⁷Be hydrogen, hydroxyl or C ₁-C ₄Alkoxyl group; And

Y is 0,1,2,3 or 4;

Condition is when y is 1,2,3 or 4, R ⁵Or R ⁶In one can be-COOH.

20, the compound of claim 1, wherein L part and GF covalent attachment.

21, the compound of claim 1, wherein z is 1 or 2, and CL is thiocarbamide, thioether, peptide, ester, disulphide, acid amides, diester, hydrocarbon, acetal glycosides or 4-isothiocyano phthalic anhydride.

22, the compound of claim 1, wherein L-AP partly is 1-(alpha-hydroxy-2-methoxyl group-5-aminobenzyl) 1,4,7,10-tetraazacyclododecanand-4,7,10-trimethylene phosphonic.

23, the compound of claim 1, wherein L-AP partly is a 1-(4-isothiocyano benzyl) Diethylenetriamine-pentamethylene phosphonic acids.

24, the compound of claim 23, wherein GF is somatomedin, insulin-like growth factor, fibroblast growth factor, transforming growth factor or cartilage/bone incitant or its combination that thrombocyte produces.

25, the compound of claim 23, wherein GF is somatomedin or insulin-like growth factor or its combination that thrombocyte produces.

26, pharmaceutical preparation, said preparation contains following formula: compound

GF-[（CL）z-L-AP]q （Ⅰ）

Wherein:

GF, CL, L, AP, z and q such as claim 1 definition; And contain pharmaceutically acceptable carrier and adjuvant; Said preparation is solid or liquid form.

27, the preparation of claim 26, said preparation is provided with two or more materials in medicine box.

28, the preparation of claim 26 provides with one matter.

29, mammalian bone regenerated method, what comprise administration to this treatment of needs (perhaps be directly used in damage or the bone zone that consumes or use indirectly) significant quantity contains following formula: compound as composition of active components

GF-[（CL）z-L-AP]q （Ⅰ）

Wherein GF, CL, L, AP, z and q are as defined in claim 1.

30, the method for claim 29, wherein significant quantity is about 0.005-50mg/Kg weight of mammal.

31, the method for claim 29 is wherein used more than a kind of formula I compound.

32, the method for claim 29 is wherein also used other active compounds.

33, the method for claim 29 wherein further prevents or reduces bone loss.