JPH07508979A - Targeted transport of growth factors for bone regeneration - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Targeted transport of growth factors for bone regeneration The present invention provides a specific target transport system, namely a chelate composition system for bone regeneration. Concerning the transport of growth-promoting factors to bone. Compositions containing these systems, and Also part of this invention are processing methods for making them.

Historically, intentional or accidental traumatic injury, osteoporosis, and periodontal disease There are many physical conditions and diseases that cause bone loss in mammals, including Exists. Therefore, stimulating and stimulating bone regeneration in mammals, including human patients. It is desirable in the fields of medicine and dentistry to provide compositions that will enhance It is often done.

One class of proteins that may be useful in bone regeneration is polypeptide growth factors. (GF), which is also described as a tissue growth promoting factor. Growth factors are A polypeptide that stimulates a specific population of target cells. This as a multifunctional molecule They can stimulate or inhibit cell proliferation and affect cell function as well. Yes, but this depends on the target cell type and the presence of other signal peptides . Examples of growth factors are platelet-derived growth factor (PDGF), insulin-like growth factor (IGF), beta type (TGF-β) and alpha type (TGF-β) Transforming growth factors (TGF) such as GF-α), epidermal growth factor (EGF), acidic fibroblast growth factor (aFGF) and basic fibroblast growth Fibroblast growth factor (FGF) including factor (bFGF), nerve growth factor ( NGF), and bone morphogenetic proteins including osteogenic and osteoinductive factors (BMP). A combination of tissue growth factors may be helpful in promoting bone regeneration. It may be beneficial. For example, PDGF and IGF-1, or PDGF and IC Combinations with F-11 promote bone regeneration and wound healing [e.g. ch et al, Pr.

c, Nat' 1. Acad, Sci, (USA) 84.7696-770 0 (1987), Lynch et al., J., Cl1n.

rnvest, 84, 640-646 (1989), Lynchet al,, J, Cl1n, Periodontol, 15.545 588 (1989), Lynch et al., J., Periodontol , 62.458-467 (1991), and U.S. Patent No. 4.861. No. 757 and No. 5.019,559].

PDGF is a polypeptide of approximately 28-35 kilodaltons (kD).

They are found within various types of cells in the body. human platelets PDGF derived from PDG contains two polypeptide sequences, namely PDG The polypeptides F-A and PDGF-B [H,N, Antonia des and M, Hunkapiller.

5science 220.963-965 (1983)].

PDGF-A is encoded by a gene located within chromosome 7 [C.

Betholtz et al, Nature 320.695-699 (1986) and PDGF-B is present within chromosome 22 [R, Dal la-Favera S Science 218, 686-688 (1982) ] s　is　s oncogene [R, Doo l’i t t Ie　et al　 , 5science λdan 1.275-277 (1983) ing.

The two polypeptide chains of P D G F are two different polypeptide chains that reside in separate chromosomes. Since human PDGF is encoded by a gene that contains disulfides, Heteronimer of bound PDGF-A and PDGF-B, or two Different homodimers (PDGF-A homodimer and PDGF-B homodimer) ), three forms occur. The role of PDGF in bone formation is unclear . Several studies have shown that PDGF enhances bone resorption [Ta 5hjian etal, Endocrinology Yoyo 1.118-1 24 (1982), Canaljs et al, J. Ce1l Phy. siol 1st A4, 530-537 (1989). Other studies have shown that PDG F stimulates osteoblast proliferation in vitro and in neonatal rats. New bone shapes in vivo when repeatedly administered via subperiosteal injections [Piche and Graves, Bone 10.131-138 (1989), C11nical and Exp J in erimental Δocroac391-416 (1991) oyce et al.

IGF, or somatomedin, has high homology to human proinsulin. It is a polypeptide of approximately 7.5 kD with [Humbel, + (ormonal Proteins and PeptideS 12.57-79 (198 4) Ko. IGF-1 and IGF-IJ have 62% sequence homology. These works The function is carried out through two different receptors. IGF-Type Rusebtar- Lucebutor (IGF-IR) and IC; II receptor (rGF-11R).

IGF-1 alone has been extensively studied for its effects on bone growth. ing. In vivo, a titanium chamber implanted into the tibia of an adult rabbit. Continuous and local application of IGF-I within the body does not significantly alter bone formation. There was no [Aspenberg etal,, Acta 0rthop, 5cand, 5dan, 6o7-10 (1989) 3.77 Tomezin-C (IG F-1) f) Repeated IUtJ systemic administration also results from femoral osteotomy in rats did not enhance bone wound repair [Kirkeby and Ekeland, Acta 0rthop, 5cand S 6-≦t 335-38 (19 90) Ko. Preliminary studies in a small number of animals have shown that older animals Within the arterial supply system (arterial 5uppI) of one hind leg in the Continuous injection of GF-1 for 14 days resulted in cortical bone formation in the hind foot. , but no such effect was seen in young rats.

This effect is likely the result of an increase in the number of osteoblasts and a decrease in the number of osteoclasts. [5pencer et al, Bone↓7.21-26 (199 1)]. The growth plate of a young hypophysectomized rat. ) Local adaptation of 1CF-1 to -characteristically small in longitudinal bone growth However, a significant effect was brought about [A λ1. Isgaard et al , J. Physiol λ] Dan, E367-372 (1986)]. ICF -1 and PDGF have also been isolated from bone matrix [Hausc hka et al SJ, Biol, Chem, 261.665-7 4 (1986), and Canal is et al., Cal.

In vitro, the effects of IGF-1 on bone cells are apparently contradictory. data exists. Pfeilshifter etal, [Endoc Rinology 1λU, 69-75 (1990) Culture of rat fetuses Report only modest effects of IGF-I alone on bone matrix adhesion on the skull It's just that. Significant effects on bone matrix formation showed that IGF-1, PDG In combination with F-BB, TGF-β, or both PDGF-BB and TGF-β It was observed when On the contrary, McCarthy et al. ocrinology, λA, 301-7 (1989). and IGF-11 significantly stimulated NA and collagen synthesis in bone cultures. It is reported that it gets worse. Hock et al, [Endocrinolo gy ↓λ2.254-60 (1988)] shows that IGF-1 is in vitro. first stimulate pre-osteoblast regeneration, and then collagen and bone matrix synthesis They found that it was stimulated independently of replication. Canalis et at, ( J, Ce1l Physiol Vol. 40.530-537 (1989)] ,] PDGF-BBI; interferes with the stimulatory effect of IGF-1 in the t:+Lagen combination However, IGF-1 interferes with the effect of PDGF on collagen degradation, and PD GFB and IGF-1 have additive effects on supracranial DNA synthesis 8 (1989)] Again, in the intro, ICF-1 is does not stimulate significant 3H-thymidine incorporation into bone-derived cells (and Furthermore, it has been reported that in this relationship, it does not enhance PDGF activity. There is. As a result of combining IGF-I with PDGF, EGF, and TGF-B, This uptake by bone cells approximates that achieved with 10% fetal bovine serum. became equal. Receptors for IGF-1 and II are found in fetal rat bone. [Centrella et al. al,, J, Ce1l Biol, (abstract) Yodandan, 62a (1 988)] o The role of IGF-1 in bone metabolism was discussed by Canalis et al. ,, [JEndocrinol, Invest, 12.577-84 (1 989)] has recently compiled a review.

Another class of growth factors whose effects on bone growth have been studied are FGFs. stomach In vivo, both aFGF and bFGF, as well as their respective mRNAs, have been detected at fracture sites [Joyce et al., (1991) Description. Both aFGF and bFGF have been isolated from bone tissue [Hausch Ka et al., (1986) supra]. bFGF and IGF-1 are effective in skin wounds. Used in combination to enhance wound healing [Lynch et a 1, J., Cl1n, Invest, (1989) supra].

In vitro, bFGF has been shown to induce 3H-thymidine uptake in bone fractures. It was not changed intentionally [Joyce et al., (1991) supra]. b FGF has been reported to enhance mitogenesis in fetal cranial tibia cultures. However, it did not directly stimulate the differentiated functions of osteoblasts [Can alis et al, JClin, Invest, 81.1572 (19 88 rivers. aFGF has been reported to have the same biological effects on bone as bFGF. [Canalis SJ, Cl 1n, Invest, 79-152-58 (1987). aFGF and bFGF both reduce substrate synthesis in a fetal rat supracranial model [Canalis et al., (1989) supra]. Bovine culture Osteocytes synthesize both bFGF and aFGF and store them in their own extracellular matrix. ]・Stored in the litox [C; 1 obus et al., Endocrin ology 124.1539 (1989). bFGF can be used in vitro. [N off et al,, F, E, B, S, Letters 250.619-2 1 (1989) Ko. Both aFGF and bFGF were detected in cultured cells from parietal bones. bFGF increases alpha 1 type 1 protein synthesis while increasing DNA synthesis in was a more potent stimulator of collagen mRNA [McCarthy e. t al, Endocrinology 25.2118-26 (198 9)]. These are mitogenic and chemotactic cells derived from the periodontal ligament. act on both, and these temporarily bond to pre-treated dentin [Terr anova et al, J, Periodontol, 293-3 01 (1989), Terranova et al., J., Perio dontol, 58.247-257 (1987), The Biology cal ~ iechanisms of Tooth Extraction and Root Re5orptionSDavidovitch Z, e d.

: Terranova in pp. 23-34 (1989)].

TGF-β-family proteins also have potential as regulators of bone growth. Seem. In vitro, TGF-β is produced by osteoblasts and stimulate proliferation and collagen synthesis by these cells [Robey et al. al., J. Ce1l Biol, Oct. 457-463 (1987), R. Osen et al., Exp385 (abstract) (1988)]. stomach In vivo, injection of TGF-β stimulates cartilage and bone formation [ Joyce et al, J, Ce1l Biol, 11th, 2195-2 207 (1990), Noda et al., Endocrinology. 4, 2991-2996 (1989) 3゜bone inductive protein (bone mo rhogenetic pr.

tein), osteogenin, and osteoinducible protein 1 (osteoinduc) Osteoinductive factors such as tive proteins can also stimulate bone formation. I can do it. These are often structurally similar to TGF-β. , and ectopic cartilage and cartilage when implanted subcutaneously or intramuscularly in mammals. Characterized by their ability to induce bone formation (U.S. Pat. No. 4,877) ．． No. 864, No. 4.619°989, No. 4.455.256, No. 4.596 ．． No. 574, and No. 4.563.489, ~Vozney et al. 5science λ''l, 1528-1534 (1988)].

target transport While growth factors appear to have the ability to regulate bone growth, therapeutic agents and The efficacy of these drugs is limited by the ability to transport them to the site of the bone defect. Mau. In diseases such as osteoporosis, these defects occur throughout the skeletal system at various times. Occurs at any time. Therefore, the skeletal tissue can be selectively applied to the site of the bone defect. It would be ideal to target the transport of growth factors to. polymer Current methods for protein delivery such as bone grafts, bone grafts, and liposomes are insufficient. The reason for this is that they require local transport without targeting. Otherwise, only systemic dissemination is possible.

Various metals coordinated to aminophosphonic acid or aminocarboxylic acid ligands It is known that aminophosphonic acid and aminocarboxylic acid complexes transport reagents to bone. [U.S. Pat. No. 4,508.704; ．． No. 560,548, No. 4.606,907, No. 4.897.254, No. 4 ．． No. 898.724, No. 5,059.412, No. 5.064.633, and and No. 5.066.478].

selectively transports the desired growth factor(s) to the bone site and It is clear that it would be desirable to retain their activity for the intended use. It is white. Selectively create the desired bone structure in areas of bone that have been wounded or removed. transports long factor(s) and still maintains its intended activity It would be more preferable to have the ability to maintain Wounded or removed selectively transports desired growth factors (one or more) to the affected bone site and “as needed” determined by the body’s natural bone homeostasis mechanisms. It has the ability to activate bone growth factors (one or more types) in a deaf manner. It would be most preferable to have one.

present invention The present invention is modified by linking with a polyamino methylene phosphonic acid ligand. bone growth-promoting factors, allowing them to be preferentially localized to skeletal tissue. a substance that stimulates and enhances bone growth by administering in a manner that induces Provide a compound. The compound for this transport system has the formula % formula % (1) [where GF is a growth-promoting factor or a combination thereof, and CL is GF an acid-cleavable linker covalently attached to Z, where Z is 0, 1, or 2, q is from 1 to the total number of amino groups present on natural GF, and L is the bonding moiety. is, and AP is a polyamino methylene phosphonic acid ligand].

Formulations for administration of compounds of formula ■ to mammals and targeted delivery to bone Methods for the use of compounds of formula I for and for preparing compounds of formula ■ Also contemplated by the present invention are methods of treating.

When using compositions or preparations containing compounds of formula The area of bone that was removed is treated, and the bone is regenerated.

Natural and recombinant tissue growth factors are available from R&D Systems (Minnea polisSMN), Col11aborative Re5earch11nc Company (BedfordSMA), Genzyme.

Inc. (Cambr. ID, MA), ICN Biomedicals , Inc. (C1eveland, OH), Peprotech, Inc. (Rocky Hi 11 NJ), and UBI (Lake Placi d, NY) as a commercial product.

Osteoinductive proteins can be purified from bone [Celeste et al. Proc, NAt1. Acad, Sc i, (USA) 87.9843- 9847 (1990), Wang et al, Proc, Nat 1. Acad, Set, (USA) 85.9484-9488 (1988 ), U.S. Pat. No. 4,455.256 and U.S. Pat. No. 4.619.989.

In compounds of formula I, any of the growth-promoting factors (GF) described above may be used. GF, PDGF, IGF, FGF.

It is strongly preferred to select from TGF or cartilage/bone inducing factor (BMP).

Preferably, PDGF is combined with IGF-1), but this factor is A shearing force X (L) applied alone or directly to the affected bone has been strongly shown to increase new bone formation when No. 61.757, and No. 5.019.559, and H, Antoniad. Filed September 13, 1990 by Es and S. Lynch, co-pending. No. 582,332).

PDGF contains 30 free amino groups per molecule, and these can be modified to improve bone function. Increasing the affinity of PDGF for PDGF (possibly force 1) F is obtained from R&D Systems and Genzyme Inc. be able to. PDGF is disclosed in U.S. Patent Nos. 4,861.757 and 5,019. , No. 559.

IGF-1 can be applied directly to diseased bones, alone or preferably in PD. It has been shown to increase new bone formation when applied in combination with GF. Ru. IGF-1 contains four free amino groups per molecule, and these can be modified to It may be possible to increase the affinity of PDGF for bone. I.G. F-1 was purchased from R&D Systems and Genzyme Inc. Hand can.

IGF-1 is R, E, Humbel, Eur, J, Biochem 190.4 45-462 (1990).

IGF-11 stimulates new bone formation when applied directly to diseased bone. has been shown to increase IGF-II has two free atoms per molecule. Contains min groups, which can be modified to increase PDGF's affinity for bone. It may be possible to do so. [GF-II is manufactured by R&D Systems and Genzyme Inc. IGF-II is R , E. Humbel.

According to Eur, J., Biochem ↓90.445-462 (1990) is described.

bFGF stimulates new bone formation when applied directly to diseased bone. has been shown to increase bFGF has 15 iHJ amino acids per molecule. groups, which can be modified to increase PDGF's affinity for bone. may be possible. bFGF was manufactured by R&D Systems and G. It can be obtained from enzyme Inc. bFGF is Gospod arowicz et al 5Endocrino1. Rev, 3.95-1 14 (1987).

aFGF stimulates new bone formation when the disease is applied directly to the underlying bone. It is a low molecular weight, homodimeric polypeptide that has been shown to increase aF GF contains 14 free amino groups per molecule, and these can be modified to improve bone function. It may be possible to increase the affinity of PDGF for. aFGF are obtained from R&D Systems and Genzyme Inc. be able to. a FGF is Gospodarowicz et al., En docrinol.

Rev. 3.95-114 (1987).

TGF-β1 stimulates new bone formation when applied directly to diseased bone. A low molecular weight (approximately 25 kDa) that has been shown to increase

It is a homodimeric polypeptide of amino acids). TGF-β1 is 1 per molecule Contains 8 free amine groups, which can be modified to improve PDGF's affinity for bone. It may be possible to increase the TGF-β1 is R&D Sys tems Inc. and Genzyme Inc.

It can be obtained from the company. TGF-β1 was reported by 5porn et al.

, J. Ce1l Biol, 105-11039-1045 (1987) It is described in.

BMP has been shown to increase new bone formation when applied within mesenchymal tissue. There is. BMP is Celeste et al, [ProcNatl, Acac L, Sci, (USA) 87.9843-9847 (1990)] Theories have been summarized and found to be as follows. .

BMP The polyamino methylene phosphonic acid ligand (AP of formula ■) is covalently bonded to GF of formula ■. (2=0) or a cleavable linker (L) is present (z=1 ). AP ligand linear or branched moiety, cyclic moiety, port Rimmer (Dense Star Polymers) La's denodori? (dendrimers) and puntron (dendron) s)) or aryl moieties; It has at least two, preferably three or more nitrogen atoms. child The glue gant is preferably the formula [In the formula, Each R1 is independently hydrogen, C, -C, alkyl, phenyl, hydroxy01-04 Alkyl, -CH3COOH, CH2P　O3H2, or L part, However, only one R1 can be an L part, and there must be one L part. and at least half of the total R1 must be -CH2P　Oa　H 2, and each R2 and R3 independently represents hydrogen, C, -C4 alkyl kill, or L moiety, provided that only one L moiety is present in Formula II The condition is that n is independently 2.3 or 4; no is independently 2.3 or 4; and m is from O to 10. [I engineering 1) [wherein R1, R2, R3, n, and m are defined as above] or a polyamino methylene phosphor in which R1 is defined as above; It is a phonic acid ligand.

The bonding moiety (the symbol in formula I) is represented by the formula (V) [Wherein, G is hydrogen, NH2, or R4 is an electronic group that can be linked to proteins, and R5 and R6 are hydrogen or -COOH, but if G is hydrogen, R5 is also Or one of R6 is C0OH, R7 is hydrogen, hydroxy, or CI C4 alkoxy, and y is 0.1.2.3 or 4, but if y is 1.2.3 or 4 If , only one of R1 or R6 can be C0OH is the condition] Represented by

In the present invention, the following terms are defined as shown below. The term “straight chain” "branched chain moieties" include ethyl, propyl, n-butyl, n-dodecane, etc. or isopropyl, tert-butylene, etc. le, and 2.5. 7-1-limethyldodecyl, etc. having from 1 to about 100 carbon atoms, which can be any branched moiety means an alkyl group. Both straight chain and branched chain parts have two nitrogen elementary atoms, preferably from 3 to 50 nitrogen atoms, and more preferably from 3 to 2 Must contain up to 5 nitrogen atoms. Some examples of these parts include [wherein, PDTMP=(N-propylcarboxyl)ethylenediamine-N.

N', N'-trimethylenephosphonic acid, APEDTMP=[N-(4-amino phenyl)ethyl]ethylenediamine-N,N',N'-trimethylenephos Fonic acid, CEDTMP'=1-(carboxyl)ethylenediamine-N, N , N’.

No -tetramethylenephosphonic acid, ABEDTMP=[1-(4-aminobenzyl)]]ethylenediamine-N N, N", No-tetramethylenephosphonic acid, AP I FTMP=N-(4 -aminophenyl)-N,N-bis-[propyl(iminodimethylenephosphonic acid) )], ΔPDTMP=N-[(4-aminophenyl)ethyl]-N,N-bis- [ethyl(iminodimethylenephosphonic acid)], and ABDTMP=N-[1- (4-aminobenzyl)-N,N’-ethylenediamine-N’,N”-ethyl Diamine-N, N, N’, N”-pentamethylenephosphonic acid] ΔBEDTMP and ΔBDTMP are preferred. These compounds are carried out Shown in the example.

The term "cyclic moiety" refers to at least two, more preferably from three to ten, Preferably, it has 3 to 8 nitrogen atoms, and about twice as many carbon atoms as nitrogen atoms. means an aliphatic saturated ring system present. Examples of these parts include 1. 4. 7. 10-tetraaza/lyrhododecane, 1. 5. 8゜12-tetraazancro Tetradecane, 2-4(4-aminobenzyl)-1,4,7,10-tetraaza /ryrhododecane]-1,4,7,10-tetramethylenephosphonic acid, 1-[(α -carboxyl)-4-amino-2-methoxybenzyl]-1,4,7,10- tetraazacyclododecane-4,7,10-trimethylenephosphonic acid, and 1 -[(α-phosphonyl)(4-aminophenyl)ethyl]-1,4,7,10- There is tetraazacyclododecane-4,7,10-1-dimethylenephosphonic acid.

The term "aryl moiety" includes one or more additional rings or aromatic rings, or an aromatic ring system that can have substituents with straight chain or branched moieties. means. The total number of atoms in the aryl ring nucleus is from 3 to 30, preferably 6. to 16, and more preferably from 8 to 16. The aryl part is small. at least 2 nitrogen atoms, preferably from 3 to 10 and more preferably 3 Contains up to 8 nitrogen atoms. Some examples of these moieties include pyrazolyl, 3 -Methylpyrazolyl, 5-methylpyrazolyl, imidazolyl, 4-methylimida Zolyl, 5-methylimidazolyl, 1,4-dimethylimidazolyl, 1,5-di Methylimidazolyl, pyridazinyl, pyriminyl, 2. 4. 6-1-Rime Tylpyrimidinyl, pyrazinyl, purinyl, buterdinyl, 3,6゜9.15- Tetraazabinclo[9,3,1]pentadeca-1 (15).

11.131-Tzen-3,6,9-trimethylenephosphonic acid (PCTMP), 6-(α-carboxy-4-aminobenzyl)-3,6,9゜15-tetra Azabincro[9,3,1]pentadeca-1 (15), 11.13-1 Lien- 3,9-dimethylenephosphonic acid (PCAPCDMP), 13-(4-aminoben nor)-3,6,9,15-tetraazapinochlor[9,3,1]pentadeca-1 (15), 11.13-triene-3. 6. 9-trimethylenephosphonic acid ( PCABTMP), and 6-[(α-phosphonyl-4-aminophenyl)ethyl Chill] -3,6,9゜15-tetraazabicyclo[9,3,1]pentadeca- 1 (15),.

11.13-triene-3,9-dimethylenephosboxylic acid (PCAPCTMP) , PCTMPSPCAPCDMP, and PCAPCTMP, and PCTMP and PCAPcTMP was submitted on December 10, 1991. Co-pending U.S. Patent Application No. 805.551 of Kiefer et al. (Assigned by unrecorded deed to Dow Chemical Company) ), the disclosure of which is incorporated herein by reference. Incorporated into the detailed document. Other heteroatoms such as oxygen can be present.

The term "polymer", including dense star polymer, was introduced on June 15, 1988. As described in published European Patent Application No. 0 271 180 , the disclosure of which is incorporated herein by reference. this Their "dense star polymer" is 5TARBUR5T (trademark) poly? -(Mi Chigan Molecular Institute, Midland, ML (trademark), or cited as 5TARBUR3T (trademark) denodrimer. and are described in the same European Patent Publication. Like The new 5TARBUR5T (trademark) denodrimer has aminomethylene phosphorus on the surface. It has a polyaminoimine group with an amino group converted to a ponate group, and on its surface (both with one 4-aminophenyl moiety. These 5TARBUR3 T (trademark) denodrimer is a European patent application published on June 15, 1988. manufactured as described in Publication No. 0271180 and published in this publication. The disclosures herein are incorporated by reference. "Polymer" also includes arborols (G, R, Newkome, J, Org, Chem, 50゜2004-2006 (1985)), and amines. (V, P, To rchilin et al, Byull, Eksp, Biol, Med, Yotan I, 63-65 (1986)).

In the definition of the term AP in formula ■, H, N-(CH3), -C(PO3H2)OH [wherein, n is 1-3] Also included. Such compounds are described in U.S. Pat. No. 5,039,819, No. 5.0. No. 19,651, No. 4.922,007, No. 4.621.077, No. 4,1 No. 34.969, No. 4,117.086, No. 4.108.962, and No. No. 3.962.432.

The term "cleavable linker" (CL in formula I) refers to polyamino methylene phosphonic acid (AP of formula ■) [bifunctional chelating reagent (BFCA) on the binding moiety (L of formula l) ] and the binding between GF and protein is reversible under certain physical conditions. cleavable or cleavable. Such a linker as taught in the art and includes thioureas, thioethers, peptides, etc. There are bonds containing , ester, and disulfide groups [C, F, Meares et al, , 1nt'1. J, CancerSSupp, 2.99-1 02 (1988) and references contained therein, U.S. Pat. 045. No. 312]. Bonds between molecules and proteins, including amidine bonds, are also applicable to this technology. taught in the technical field. These bonds connect imidoester-containing molecules to proteins. [0. R37-202, A, C, 01son and C.C.Cooney, eds, Plenum, New Y ork (1974). Similarly, amides, diesters, thioethers, hydrocarbons and disulfide bonds are taught in the art [C,H,Pa 1k et al, J, Nucl, Med, 30.1963-1701 ( 1989), and M., Haseman, Eur., J., Nucl, Med. .

Yol, 455-460 (1986)]. Cleavable diphosphonates and amino acids Donophosphonate linkers are taught in U.S. Patent No. 5.094.848. It will be done. These linkers are phosphogesterase, 5°-nucleotidase , and cleaved in situ by enzymes such as acid phosphaase. It will be done. Cleavable linker in effect on alkylidene hydrazide bond is also US patented No. 5.094.849. These bonds are carbonyl-containing It is formed by reacting the molecule with a molecule containing a hydrazide moiety. additionally Acetal glyconodes have been proposed as selectively cleavable bonds [L, F .

It is. The cleavage rate was lower at pH5 when compared to the rate at pH7. Acid-cleavable linker at least 10 times faster (both rates determined at 37°C) Particularly preferred are the following. Some examples of acid-cleavable linkers are described in U.S. Pat. No. 4,542. ．． No. 225 and No. 4.618,492 and described therein. citations, as well as our co-pending documents filed March 4, 1993. Disclosed in U.S. Patent Application No. 026,800, which is incorporated by reference. incorporated herein by. In this latter pending application, A preferred acid-cleavable linker is 4-isothiocyanate phthalic anhydride.

The term "group present to enable coupling to a protein," or "Electronic group (one or more) that can be linked to protein (one or more)" ``species or higher)'' refers to electrons that can be bonded to amino acids in proteins, such as GF. (R4 in the terminology of formula I).

Some examples known to those skilled in the art as suitable groups include amino, maleimide, silicon. American isothiocyanate, vinyl pyride, bromoacetamide, carboxyl, and N-hydroxysuccinimide active ester, but are not limited to these. Not possible. When these electronic groups are present, the ligand (formula 1 before coupling) L-AP) is BFCA.

The “polyaminomethylenephosphonic acid moiety” (AP of formula I) can be a wide variety of possible groups. are represented by cyclic moieties, straight or branched moieties as specified above, Polymers including aryl moieties, dense star polymers, and these two methylene (CH2), a group [where n is 2.3 or 4] between the nitrogen atoms of ] (polyaminomethylene group). one More than one such polyaminomethylene group can be present in this moiety.

This moiety is covalently linked to the polyamino methylene group via nitrogen It also contains at least two methylenephosphonic acid groups (CHzPOsHJ). The riaminomethylenephosphonic acid moiety is represented by formula II, III, or Although it is preferable to include, but is not limited to, polyamino methylene phosphone The acid moiety allows for coupling to proteins, as described herein. or electronic groups (a type of group) that can be linked to proteins. or two or more types). Polyamino methylene phosphonic acid moiety (represented by L-AP in formula functional linker (represented by CL in formula I), preferably an acid-cleavable linker. The protein "R" (GF in formula I) can be bound to the protein "R" (GF in formula I).

The terms "growth factor" and "tissue growth-promoting factor" refer to Any molecule that stimulates chemotaxis or chemotaxis. These factors are natural can be obtained from natural sources, or by recombinant DNA technology or chemical synthesis. can be created. It is preferable that these factors be purified.

The term "purified" as used herein refers to mixed with other growth factors. Previously, we have defined factors in which 90% or more of a particular protein is present by weight. taste (i.e., in its natural state it is in an associated state) substantially free of protein, fat, and carbohydrates). Purified protein preparation The samples are generally analyzed on polyacrylamide gels for each subunit. A single major band will result. Purification used in compositions of the invention Factors are best purified as determined by amino-terminal amino acid sequence analysis. preferable.

The ligand of the invention (AP of formula I) is in the form of a pharmaceutically acceptable salt. Can be done. The term "ligand" as used herein refers to these salts. be understood as including. The term "pharmaceutically acceptable salts" refers to means a cation that is allowed in These are used to achieve the desired effect. A cation that is not substantially toxic at the doses administered. explain in detail and these salts contain alkali metals such as sodium and potassium; Alkaline earth metals such as sium and magnesium, ammonium, aluminum Group IIIA light metals including Ni and triethylamine trialkylamine, dibenzylamine, N,N'-dibenzi diethylenediamine, dihydroabiethylamine, N-(C+-C4)al Organic primary, secondary, such as chilpiperidine, and any other suitable amine There are salts of secondary and tertiary amines. Sodium and potassium salts are preferred . The term "pharmaceutically acceptable" refers to warm-blooded humans, such as mammals, specifically humans. means suitable for administration to animals and suitable for pharmaceutical use; This includes being non-toxic, such as not being toxic to warm-blooded animals. Book Pharmaceutically acceptable salts of the compounds of the invention can be prepared by conventional ion exchange treatment methods. or prepared by treating a ligand or compound of formula I with a suitable base. Ru.

Method for producing polyamino methylene phosphonic acid Polyamino methylene phosphonic acid of the present invention Various methods are known for manufacturing parts. Two of these methods. The third one is described below.

A Production method from amines Polyamino methylene phosphonic acid derived from amines, especially straight or branched chain moieties There are many publications describing manufacturing methods. For example, U.S. Patent No. 2.599.80 No. 7 (the disclosure of which is incorporated herein by reference) at pH>10. An aqueous solution of the amine is diluted with chloromethane in the presence of a base such as sodium carbonate. Preparation of polyamino methylene phosphonic acid by heating with tyrene phosphonic acid He teaches the method of making. Other examples are given by the quotations given below, but these are shown in the table below (using various phosphonomethylation reagents, indicated as Ethylenediamine tetramethylene phosphone from ethylenediamine (EDA) A method for producing acid (EDTMP) is described.

Addition with process for producing cyclic polyamino methylene phosphonic acids from the corresponding amines A specific example is U.S. Pat. No. 937.333, andori, W, 5w1nke l set al,, R ecl, Trav, Chim, Pays-BASllo, 124-128 (1 991).

Intermediate aminomethylene phosphonate ester or mixed aminomethylene Preparation of polyaminomethylenephosphonic acid from amines via phosphonic acid ester The manufacturing method is described in International Publication No. 91107811 published on June 13, 1991. , the disclosure of which is incorporated herein by reference. Ru. This method uses phosphonomethylating reagents (e.g., formaldehyde, and dialkyl phosphite), but this reagent ralky+) phosphonate ester is formed. Then these esters , hydrolyzed to aminomethylenephosphonic acid. The cited references are based on strong bases (e.g. n -butyllithium) and alkyl halides or is formed on the carbon between nitrogen and phosphorus by alkylation with an aryl halide. Methods of forming kill or aryl substitutions are also described.

B. Production method of polyaminomethylenephosphonic acid from carboxylic acids Polyaminomethylene lenecarboxylic acid to the corresponding polyamino methylene phosphoric acid by various known methods. can be converted to phosphoric acid. Common reactions that convert the corresponding carboxylic acids The reaction requires a reagent capable of donating a phosphonic acid group. The example is ED Reaction of EDTA with PCl3 in nitrobenzene to yield TMP (for example, U.S. Pat. No. 3,832,392, the disclosure of which is incorporated by reference) (incorporated herein).

C Difunctionality with an amino group that can be converted to polyamino methylene phosphonic acids Production method of chelating reagent (BFCA) Simon et al. (Dow Ch. (transferred by unrecorded deed of transfer to the chemical company) 19 Co-pending U.S. Patent Application No. 565,379, filed August 9, 1990, A variety of linear or branched linear polyamides that can be linked to proteins This disclosure is incorporated herein by reference. incorporated into the statement. International Publication No. 8410 published on September 27, 1984 No. 3698 discloses an open chain point that can be phosphonomethylated to yield BFCA. Bifunctional chelating reagent intermediates based on lyamines are disclosed. linear pattern The synthesis of the branched polyalkylene polyphosphonate BFCΔ is also a US patent. No. 4.808.541, this disclosure is incorporated by reference. Incorporated herein.

U.S. Patent Nos. 3.994.966 and 4.622.420; Various references cited in et al. include A variety of products based on ethylenediamine and diethylenetriamine with various groups It describes how to make FCA, all of which are made from amines. Converted to polyaminomethylenephosphonic acid BFCΔ and linked to amine groups of proteins can do.

Ring-opened polyaminomethylenephosphonic acid BFC containing linkage through aminocarboxylic acid A is European patent application publication number 0 279 30 published on August 24, 1988. The corresponding polyamine intermediate BFCA described in No. 7 can be prepared from , the disclosure of which is incorporated herein by reference. U.S. Patent No. 4. No. 994,560, No. 5. No. 006゜643, and No. 5.　064,956 The number is also phosphonomethylated and the corresponding polyamino methylene phosphonic acid B Methods for making a variety of open-chain and cyclic amine-containing BFCAs that can yield FCA is listed.

Cyclic polyamine BFCA is a European patent application published on February 7, 1990. No. 0,353,450, and a number of various cyclic amine-containing B FCA is produced and then carboxymethylated to produce aminomethylene carboxylic acid. A method for producing kinrates is described. The amine content of these BFCAs Alternatively, the intermediate can be phosphonomethylated to form the corresponding polyamino methylene phosphatide. BFCA sulfonate can be produced. Similarly, U.S. Patent No. 4.8 No. 85,363 is based on various 1.4.7.10-tetraazacyclododecane ( describes amine-containing intermediates, which can be synthesized by phosphonomethylation into the corresponding polyamides. It can be converted to aminomethylenephosphonic acid BFCA. 1゜4, 7 ．． Based on 10-tetraazane clododecyltetramethylenebosphonic acid (cyclic B Open-chain BF based on FCA and diethylenetriaminopentamethylene phosponic acid CA is a preferred group of polyamino methyleneposboxylic acid BFCA ligands.

A linking group of polyamino methylene postponate BFCA to a protein is attached to a cyclic polyester. Can be substituted with aminomethylenephosphonic acid itself, and this is J, Ch em, Soc, Chem, Commun.

797 (1989) by J. P. L. Cox, Soshite J.

Amer, Chem, Soc, Kamijodan, 6266-6267 (1988) It is described in detail by M., Moi et al. similar The compound is disclosed in International Publication No. 89/11475, published on November 30, 1989. It is shown in

1.4.8.11-Tetraazacyclotetradecane-based cyclic amine compounds are , Inorg, Chem, λ6, 3458-3463 (1987) M., by Moi et al.

These are converted into equivalent compounds of tetraamino methylene phosphonic acid by the method described above. can be converted into Similar compounds that can be used in the present invention are described in US Pat. No. 4,678,667. cyclic polyamine Additional examples of the class are given in BioconjugateChem, 3(2), 108- T, J. 117 (1992).

As described by McMurry et al. The bifunctionality of these intermediates Polyamino methylene phosphonic acid obtained by phosphonomethylating cyclic amines BFCA (L-AP moiety of Formula I) can be obtained.

Hinoclopolyazamacronocrocarboxylic acid BFCΔ intermediate was released in December 1991. Klefer eL al, (Dow Chemical C Concurrently pending U.S. (transferred by unrecorded deed to It is disclosed in Japanese Patent Application No. 805.270, and this disclosure is incorporated by reference. As incorporated herein, by phosphonomethylating this, poly Aminomethylenephosphonic acid BFCA can be obtained.

Vinclopolyazamacrocyclophosphonic acid BFCA was released on December 10, 1991. Submitted to Kiefer et al, (Dow Chemical Com Concurrently pending U.S. patent application (assigned by unrecorded deed to No. 805.551, the disclosure of which is incorporated by reference. Incorporated herein is a polyamino methylene phosphonic acid BFCA. teaches methods for producing various bicyclopolyazamacrocyclophosphonic acids BFCA are doing.

D. Production of bifunctional chelating reagent (BFCA) in which the nitro group is reduced to an amine group manufacturing method Reduction of a nitro group, especially a nitrophenyl group, to the corresponding amino group is particularly useful in the previous section. If desired for the compound in C, this reduction is -417 (1970 ), pub, John Wiley & 5onS, and the references contained therein. These are the methods listed in the literature.

E. Production of bifunctional chelating reagent (BFCA) in which the amino group is converted to an electronic group law BFCA, the ligand of the present invention (for example, L in formula Linking to a group or protein present to enable pulling It has an electronic group that is capable of It is possible to link amino groups to proteins. Methods for converting certain electronic groups are well known in the art. . Some references that provide suitable methods include C. F. Meares et al. l,, Acc, Chem, Res ↓ Yu, 202-209 (1984) and references cited therein, D. Parker, Chem, Soc, R. ev.

19.271-291 (1990) and references cited therein, C. F. Meares et al., J. of ProteinChem, 2. 215-228 (1984) and references cited therein, C, F, M ares + Protein Tailoring Food Med, U ses”, Amer, Chem, Soc, Symp, 339-352 (1985), ed. R,E.

Feeney, and J. R. Whitaker, pub. Dekke r, N. Y, NY. An example of BFCA was given earlier. Additionally, in aqueous solution Many reagents are available for forming amide bonds in More amine-containing molecules are obtained together with carboxylate-containing molecules. For example, commercially available Commercially available water-soluble carbodiimides have been developed for this purpose. [J, V, 5 taros, Ana 1. Biochem, Yodan 6,2 20-222 (1986)].

Other methods for linking two nuclear reagents to each other include acryloyl chloride or By reaction using its chemical equivalent, one can produce a Michael acceptor, such as an amine. It is to transform it into a condition. This converts the amine group to an acrylamide group. , which can then be reacted with different nuclear amines (Ch em, Abst, 83/80295b).

Other methods for manufacturing drug-carrier conjugates are available from Pharmac.

M, J, P in 1, Rev. 36.278-336 (1984).

znansky and R,L, Jul 1ano, and International Publication No. 90/1 No. 4844.

F. Manufacturing method of BFCA by other methods Molecules for linking two nuclear moieties together are called bifunctional cross-linking reagents.

If the two reactive ends of the molecule are identical, they are "homonifunctional" It is called a crosslinking agent. If the two reactive ends of this molecule are different, they are These are referred to as "heteronifunctional" crosslinkers.

Chem, Pharm, Bul 1. λdan, 1130-1135 (1981) T. Kitagawa et al. , discloses a method for producing heterobifunctional crosslinkers for protein modification. like this The reagents include maleimide groups (which partially react with thiols) and N-hydroxy Succinimidyl esters (reacts with amine groups such as succinimidyl esters) It carries two selectively reactive groups such as One molecule contains an amine group; If the other molecule contains one or two thiol groups, these The reagent allows the two molecules to be combined with each other. maleimide group Similar compounds including 5ynthesis 819-821 (1991) :t=; was reported by O.Nelsen.

For linking BFCA or other electronic molecules to proteins or other nuclear materials. Other difunctional crosslinkers have been developed (U.S. Pat. No. 4,680,338). and references cited therein), dialdehyde crosslinkers [S, Avameas et al 5 Scand, J.

Immunol, 7-23 (1987)] and commercially available. Pierce 1989 Handbook and Gen eral　CatalogSl)I), 283-311 (Pierce, Ro ckford S IL).

Proteins and proteins that contain electronic groups such as amines (but also other similar electronic groups) and small molecules using Traut’s reagent, which is commercially available. s reagent) [1, PowerSNuc, Ac1d Res dan, 42 85-4291 (1981)] to modify the amino group. into a sulfhydryl group, which is then selectively converted into a maleimide group. can be reacted with.

G Cleavable linker The linker between polyamino methylene phosphonic acid BFCA and CF has a predetermined physiological Advantageously reversible or cleavable under certain conditions sometimes occurs. Such combinations are taught in the art and are An example is shown below. Particularly advantageous are acid-cleavable linkers. Ru. Some examples of acid-cleavable linkers are described in U.S. Patent No. 4.542.225 and and No. 4.618.492, as well as the references cited therein. It is. These cleavable linkers release a cyclic anhydride that reacts with the amine group. At one end of the linker, a maleimide group that reacts with the sulfhydryl group is added to the linker. It is held at the other end. The bond between the acid anhydride and the amino group (thereby forming a It cleaves easily at acidic pH (forming an amide with bonic acid). US Patent No. 4 ．． No. 764.368 also includes an acid-cleavable amide functional group between a small molecule and a large molecule. fil of cyclohexane-1,2-carboxylic anhydride as a method for introducing The purpose is described. WO 90/14844 discloses that under weakly acidic conditions describes the use of m derivatives as cleavable linkers.

Presence of H1 metal ion Surprisingly, the addition of metal ions can be applied to polyamino methylene on calcareous surfaces. Does not significantly inhibit phosphonic acid affinity. For example, calcium or The addition of samarium ions is related to the calcareous surface of hydroxyapatite. ．． 7. 10-tetraazacyclododecanetetramethylenephosphonic acid (DOT MP) affinity.

For example, DOTMP, in U.S. Pat. No. 14,976.950, , when complexed with holmium, gadolinium, or yttrium ions. In some cases, it has been shown to progress to calcific sites in vivo. Cal When complexed with ium or other metal ions, ethylenediaminetetra Methylene phosphonic acid (EDTMP) also progresses to calcific sites in vivo. (European patent publication published on December 27, 1991) Application Publication No. 0462787).

■ Cluster of aminophosphonic acid for linking to proteins In European Patent Application No. 0272180 published on June 15, 1988 Polyamino methylene polymers, including dense star polymers such as those described in Polymers with sulfonic acid can react with formaldehyde and amines in aqueous solution. It can be produced by reacting with di-alkyl phosphorous acid, thereby A veralkylposponate ester is formed. Then this ester is placed in a dense star. - Hydrolyzed on the polymer to aminomethylenephosphonic acid. specifically This dense star polymer has PAMAM morphology with amine groups on its surface. It has become. These amine groups are then reacted with a Bosbo methylation reagent to release the amine groups. formation of riaminomethylene phosphonic acid, which is then conjugated to GF. let The advantage of dense star as a bone exploration moiety is that it can be water soluble , the size can be adjusted, and there are groups with specificity, and GF has This means that the number of groups that can be used to form covalent bonds is the same.

Polymers of L-AP of formula I allow for linkage to proteins such as GF. arborols (G, R, Newkome, J, Org, Chem, 50, 2004-2006 (1985)). This Al Borol maintains a three-dimensional microenvironment with an outer surface covered with polar functional groups. Monocascade sphere (mon.

cascade 5phere). These arborols can be converted into esters. and known in the art for converting alcohols to amines. can be converted into polyamines (for example, 5u rvey of Organic 5 synthesis 1.411-417 (1970), pub, John Wiley and references cited therein) . This arborol polymer was converted into a polyamino methylene postbond by the method described above. can be converted to phosphoric acid. a suitable group such as p-nitrobenyl bromide. arborol by initiating the arborol cascade polymer using can contain a group capable of linking to a protein. Therefore, multiple polyamino methylene phosphonic acid groups, as well as stable covalent bonds or cleavable one group that can be linked to a protein by any of the following functional bonds: It is possible to produce arborool containing.

Contains multiple amines and one group capable of linking to proteins, and is a linear Other polymers that are either branched or branched have also been described. [V, P, Torchilin et al.

Byull, Eksp, Biol, Med, Yo02, 63-65 (198 6)]. Such a polyamine-containing polymer is prepared by the method described above. It can be converted to a polymer containing riaminomethylene phosphonic acid groups.

Protein conversion to reactive group-containing clusters of polyamino methylene phosphonic acid By adjusting the stoichiometry, the type of product can be varied. Also , by adjusting the stoichiometry during the step of introducing bonds containing reactive groups. Raster compounds can be proteins of similar or different molecules to each other Polyamide for non-critical amine groups on CF to be attached to two protein molecules Selective ligation of nomethylenephosponate BFCA first transfers GF to receptors from cells. Achieved by mixing with a soluble or solid support-immobilized form of the protein. GF-receptor aggregates will be formed.

This aggregate is then converted into an excess of the reactive form of polyaminomethylenephosboxylic acid BFCA. It can be reacted with the state. Since GF is bound to a receptor, GF-receptor Only amine groups not involved in receptor interaction will be modified. the CF modified in this manner then interacts with its receptor in vivo. It is possible to produce biological activity accordingly. After that, the modified CF and receiver This selectively modified CF aggregate between the receptor and the reusable receptor can be decomposed into , so that after this separation it remains fully active and will produce a modified GF that recognizes the receptor.

Compounds of formula I can be prepared in several different ways.

For example, an AP moiety may be reacted with L to form an L-AP moiety, and then this L-A The P moiety is linked to CL and then CL-L-AP is linked to GF. in another way reacts GF with CL to form a CF-CL moiety, and then this GF-CL is reacted with L-AP to form a compound of formula I. The degree of modification of CF is determined by the formula ■It is described by the term q of CF, CL, and L-AP molecules. carried out by adjusting the concentration, time, temperature, pH and stoichiometry of the reaction between achieved experimentally.

! agent The formulations of the invention are in solid or liquid form. These preparations are available for direct use. Either a single substance or the two components should be mixed at the appropriate time before use. provided as two or more substances (e.g. kit effects) can be provided. Whether pre-mixed or in a kit , this formulation may require a pharmaceutically acceptable carrier or adjuvant. .

The compounds of the invention may also be administered parenterally, i.e. subcutaneously, intravenously, intramuscularly, if or intraperitoneally with water, saline, aqueous dextrose, and related sugar solutions; Alcohols such as ethanol, isopropanol, or hexadenor alcohol Glycols such as coals, propylene glycol, or polyethylene glycol Recall neck, 2,2-dimethyl-1,3-dioxolane-4-methanol-like Ethers such as glycerol ketals, polyethylene glycol) 400 oils, fats, fatty acids, fatty acid esters, glycerides, or acetylation It should be a sterile liquid or mixture of liquids, such as diluted fatty acid glycerides. containing a certain pharmaceutical carrier and a pharmaceutically acceptable surfactant such as a soap or detergent. Surfactants, pectin, carbomers, methylcellulose , hydroxypropyl methylcellulose, or carboxymethylcellulose Add suspending or emulsifying reagents such as of the compound in a physiologically acceptable diluent with or without the addition of It can also be administered as an injectable dose.

Examples of fats and oils that can be used in parenteral formulations of the present invention include peanut oil , soybean oil, sesame oil, cotton oil, corn oil, olive oil, petroleum, and inorganic It may be of petroleum, animal, vegetable, or synthetic origin, such as oil. appropriate Common fatty acids include oleic acid, stearic acid, and isostearic acid. suitable Examples of suitable fatty acid esters include ethyl oleate and isopropyl myristate. be. Suitable soaps include aliphatic alkali metals, ammonium, and triethanol. and suitable detergents include halogenated dimethyldialkyl amine salts. Examples include ammonium, alkylpyrinnium halides, and alkylamine acetates. cationic detergents, alkyl sulfonates, aryl sulfonates, and sulfonates. Olefins phonates, alkyl sulfates, olefins sulfates, ether sulfates, and sulfuric acid Monoglycerides, as well as anionic detergents such as sulfoscutunatate, fats Amine oxides, fatty acid alkanolamides, and polyoxyethylene-polyamides Non-ionic detergents, such as propylene copolymers, and alkyl-baked aminopropionate, and 2-alkyl-imidacillin tetraammonium salt. Examples are amphoteric detergents, as well as mixtures thereof. The parenteral composition of the present invention is , typically from about 0.001% to about 10% by weight of the compound of formula I in solution. will include. Preservatives and buffers can also be used with advantage.

In order to minimize irritation at the injection site, such compositions may be Contains nonionic surfactants with a hydrophilic-lipophilic balance (HLB) of up to about 17. be able to. This surfactant may be a single component with a previous HBL. two or more components with the desired HBL. It can be a mixture of minutes. Surfactants used in parenteral formulations Examples include polyethylene sorbitan fatty acid esters such as sorbitan monooleate. It is in the tell category.

Other possible formulations for compounds of formula ■ include known oral pharmaceutical formulations (such as (some of which may contain excessive amounts of magnesium hydroxide) For oral use [P, J, Neuvonen etal, Eur, J, Cl 1n, Pharmacol, 35.495-501 (1988)], B, K ari, Diabetes, 217 (1986) 1. B.R.Mayer et a+, Cl1n, Pharmacol, Ther, 4th grade, 6 07 (1988) for transdermal delivery using known processing methods, activated carbon using the absorption of a compound onto a subatomic particle and subsequently implanting or injecting it. [W, A, Lee, BioPharm, 22-25 (Nov/Dec , 1990) or Wall 5treet J, Bl (August 16.1989)], and R. Langer, Sci.

Others by "λ" Dan, 1527-1533 (Sept, 28.1990) There is. Apply these preparations to the damaged or removed bone area. It can also be adapted locally by direct topical administration to the site of. The method described below requires surgery to expose the area of bone that has been damaged or removed Sometimes.

Bone regeneration using the compounds of the invention can be achieved without treatment (i.e., with exogenous agents). (in the absence of drug application) or unmodified growth promoters at similar levels. Treatment using children, ie formula! compared to that achieved by GF alone. Effective. GF production is relatively expensive and high doses are transported systemically Because CF has the ability to produce adverse toxic effects when to ensure delivery to the desired site and to reduce the total GF dose and mammalian It is also desirable to use ligands to reduce toxic effects.

In accordance with the present invention, in a method of bone regeneration in a mammal, particularly a human patient, the mammal for direct application to areas of damaged or removed bone. or the systemic circulatory system (parenterally, intramuscularly, or subcutaneously, as shown below). Compounds of formula administering an effective amount of the composition.

The amount of (CL), -L-AP of formula ■ required for the purpose of bone transport of GF Yes It can be an effective amount of either. In any case where such a transport system is desired The amount of compound of formula ■ administered for the purpose of treating a disease will depend on the specific dose utilized. unit, treatment period, age and gender of the patient being treated, nature and nature of the disease being treated. degree of infection, and other factors well known to those of ordinary skill in the medical arts. It can be varied widely according to the child. Moreover, the compound of formula ■ can be used to treat bone diseases. used in combination with other reagents known to be useful in the treatment of be able to.

The effective amount of a compound of formula (1) to be administered in accordance with the present invention will generally be about 0.005-50 mg/kg (patient's weight) and this - once per day or It can be administered multiple times. A compound of formula I, as previously described. It can be administered as a pharmaceutically acceptable formulation. Compounds of formula I can also be used at the same time. one or more different active compounds administered either sequentially or sequentially. and such compounds can be combined with other known active agents for bone regeneration. Can be administered with drugs.

The invention will become clearer by considering the examples given below. , and the examples are not intended to be merely a detailed description of the invention. be.

definition Terms used in this example and previously specified herein The missing terms are shown below.

BLIP - Bone-shaped tube forming protein USA=Bovine serum albumino Fe2-Uno fetal serum HP L C-High Performance Liquid Chromatography +13 A = Human Serum Albumin PBS = phosphate buffered saline RP-HPLC=Reverse phase HPLC 8CN-BDTMP=4-isothiosoanate benzyldiethylenetriaminobe tamethylene phosphonic acid All solvents and reagents were obtained from commercial sources and are specified below. The following were used without further purification.

All water is Barnstead NANOpure™ ion exchange/carbon bed It was passed through a water purification system and had a resistivity of about 18.5 megohms.

Acetic acid is greater than 9999%, from Aldrich Chemical Co. of glacial acetic acid.

1.0 in PBS (20mM phosphoric acid, pH 7, 4, 0, 15M NaCI) 1% If SA in mL aliquots, In5 titute.

Frozen from Mo1ecular Biology, Inc. (1M8 company) I received it in the same condition.

1251-PDGF (frozen in 135 μL aliquots, 100 μg Contains 265 μCi of IIJ-PDGF without radioactive ICF and carrier ), and purified recombinant PDGF-BB and IGF-1 (all O, LM in acetic acid) was obtained from 1M8.

Centricon™ for 10.000 and 3.000 molecular weight cutoffs ) Membrane filter (Amicon Division of ~V, R, Grace and Co., Ltd.) was used for washing and concentrating the aqueous protein solution.

General method Purified recombinant human PDGF-BB and recombinant human IGF-1 and each The radioiodinated form of Molecular Bi Supplied by Logic, Inc. Identical of PDGF-β and IGF-1 The identity was confirmed by the amino acid constituents and the amine terminal amino acid sequence. amino acid The constituent components are gas-phase hydrolysis of the protein and subsequent mainly Eveleigh and and Winter [J.

W, Eve Ieigh and G, D, Winter” Am1no A9 1-95 (1970)]: determined from 1. Ta.

The amine terminal amino acid sequences are mainly Hunkap i l l e r and Ho .

d [M, W, Hunkap i l l e r, E, j- food, Methods in Enzymology 91.486- 489 (1983 Edman degradation and subsequent formulation processing, according to the method of and PTH amino acid analysis using HPLC. Purity is Cooma Scanning denometre of 5DS-PAGE gel with blue and silver staining Determined using HPLC and RP-HPLC.

starting material Example A Method for producing PDGF-BB and IGF-1 Both PDGF-BB and IGF-1 are produced by standard recombinant DNA techniques and subjected to conventional chromatography. It was purified by The image processing method is also well known to those skilled in the art (PDGF, US Patent No. 4. No. 061.757 and No. 5.045.633, IGF-[:Y , 5ato et al., J.Biochem, 1011247-1252 ( 1987) and Wong et al., Gene 68.193-203. (1988)).

1 to 2o amines of PDGF-BB (supplied by IMB) Analysis of the terminal amino acid residues yields a single intact amine terminus, and this were identical to the residues predicted by the cDNA sequence.

Amine powders from 1 to 53 of human IGF-1 (produced by IMB) Sequence analysis of terminal residues reveals a single complete sequence homologous to residues predicted by the cDNA sequence. Complete amine termination was obtained. This amino acid component is incorporated into human IGF. It was found to be consistent with what would be expected by the -1 gene. both growth factors Both had a purity of over 95%.

Example B Biological activity of PDGF-BB and ICF-1 Efficacy or biological activity of each drug is the cell that specifies the effective dose at half maximal stimulation (ED, .) as a unit. Measured by culture mitogen assay. Both biological assays BALB/c 3 Based on measurements of [3H]-thymine incorporation into the DNA of T3 mouse fibroblasts. are doing. 3T3 cells were grown per well in DMEM supplemented with 10% FC3. 2500 cells were plated into a 96-well assay plate. A, say play The plates are incubated (37°C) for 7 days before use. This allows serum composition components become depleted and can induce a dormant state within the cell. . In the PDGF-BB bioassay, standard and test samples were in the afternoon for the triplicate assay and incubate it for 18 hours-overnight. let Cells were then exposed to 1.0 uCi of 31-1-demidine for 6 hours. , this thymidine is incorporated according to the biological activity of PDGF.

In biological assays for IGF-1, add test samples and standards. Cells are pre-incubated with PDGF-BB before treatment. This pre-line In the presence of epidermal growth factor (EGF), IGF Upon addition of ICF-1, cells become capable of responding to ICF-1. - Evening I After the incubation period, the cells were treated with 1.0 μC of 311-thymidine. According to the biological activity of IGF-1, this thymidine It is captured.

For both PDGF-BB and IGF-1 assays, cells were washed after extensive washing. and quantitate the sample in a liquid scintillation counter. . Plot the results to create a dose-response curve and calculate the unit activity from this curve. can be determined.

One unit produces 50% of the peak cell response (ED5o value) in the appropriate assay. is defined as the concentration of GF that induces PDGF and IGF-1 assembly The highest value of the reaction in B Defined as a value. Specific activity is unit activity based on mite/enoanosei is calculated by dividing by the mass determined by amino acid analysis. protein repair The efficacy of both PDGF-B10 and IGF-1 before one treatment was 11-3n/ mL (see Figure 3 for PDGF-B and lCF-1). (See Figure 4).

Typically the specific activity is in the range of 3-10 x 10 units per mg of protein. Ta.

Example C Radiolabeled “5l-PDGF” The “cold” PDGF described in Example A was -[+2J]-Portone-Hunter primarily labels the primary amine of the terminal residue Labeling was performed using the Bolton Hunter method. +2 51 PDGF is also available as a commercial product from NEN. NEN company is Usually in sodium citrate buffered saline solution containing 1% BS8 as carrier protein. 1251-PDGF. Before use, obtain this BSA as a commercially available product. INI-PDGF can be removed from INI-PDGF to efficiently modify PDFG. It is necessary to ``Antibody affinity clones for the purpose of producing the carrier free form of J-PDGF.'' chromatography was used. Immobilized anti-PDGF antibody (by IMB) supplied) has specific binding to isoforms of r'DGF that are AB and BB. and no binding to the AA homodimer.

42.6 μCi of 1251-PDGF in neutral pH buffer at 2°Oμ It was applied to a L anti-PDGF column. Add BSA to the column using PBS The solution was drained completely and the +2J pDGF was eluted in O,LM acetic acid.

Based on a specific activity of 35 μCi/μg, this particular type of +251-PDGF is Those with one [+25■] that connects 18 PDGF two-member dichromats on average It is rated as.

Examples Radiolabeled “5l-ICF” The previously described “cold” IGF was converted to iodotyro by iodizing the thyronone residue. [ +251]. +2'1-IGF-1 is a commercial product from NEN. It can be obtained from ``5l-ICF-1 is carrier-free and p i (lyophilized from 100 μL of 100 mM sodium citrate of 4,5 Supplied in condition. Based on the specific activity of 208μC4/μg, this lot's "5l" -IGF-1 is linked on average to every 1.4 IGF-1 proteins [+2 5　■Rated as having 3.

Example E Preparation method for two PDGF solutions A vial containing radioactive PDGF was adjusted for +251 into the wells of the teC dose calibrator. Vialbi containing 50gg of PDGF rinse with 200 μL of O, 1M acetic acid and replace this with 135 μL of O, 1M vinegar. Vials containing 100 μg of cold IGF and “’I-PDGF” in acid was added to the tube. Add a cold vial of PDGF to 200 μL of O,IM acetic acid. After 7 additional rinses, the rinses were combined to contain radioactive and non-radioactive P. One sample containing DGF as well as non-8i1-f active IGF was obtained. combined Mix 1 iffl of the sample Pyarhin, and add 7 ml of this solution. Two 50 μL aliquots were taken out. Label each aliquot as 1 and 2. placed on separate membranes of 10 centricons attached. . Select appropriate volume and gamma measurements before and after splitting the sample. , thereby confirming the capacity and radioactivity. Centrifuge two aliquot samples It was centrifuged in the machine, tared and placed in a new filtrate cup. .

Each membrane filter (1 and 2) was then washed with 0.1 mL of 3.0 M sodium bicarbonate. Treated with buffer and re-centrifuged. Take out these two cups and Tare the weight and place it in a new filtrate cup. holding the sample Both membranes showed values of 1o, 5 μCi.

The two membrane filters were then washed with an additional 1.0 mL of 3.0 M sodium bicarbonate. buffer and recentrifuged. Remove both filtrate cups and Then, the tare weight was measured and placed in a new filtrate cup. of two samples Each exhibited a value of 105 μCi on their respective membranes. Cleaning treatment as described above A procedure was performed to remove carrier and non-radioactive IGF that would have passed through the membrane.

The material remaining on the membrane was then immediately used for covalent bond formation.

Example F 1-(α-carboxylate), a preparative free base of H2N I3 D O3T M Silu-2-methokino-5-nitrohenol)-1,4,7,10-tetraazacyl Clododecane, 513 g (13 mmol), 776 mg (4.7 mmol) of triethyl phosphite and 141 mg (4.7 mmol) of paraformaldehyde. The mixture was added to the stirred slurry. The resulting slurry was heated to 95°C for 2 hours with stirring. Heat, cool to room temperature, and concentrate under reduced pressure to obtain a sticky oil. did. This oil was eluted using a basic alumina column and chloroform. After chromatography and evaporation of the solvent, a thin layer of the ester is 1-(α-carboxy-2-methquin-5-nitro) as a yellow sticky oil. benol)-1,4,7,10-tetraazacyclododecane-4,7,10-1 -rimethylenephosphonic acid hexaethyl ester was obtained (55%).

250 mg (0.3 mmol) of this ester was added using 3 mL of 1-thick HCI. , hydrolyzed at 100°C for 18 hours with stirring. Freeze this aqueous solution 1-(α-carboxy-2-methoxy/- 5-nitrohennol)-1,4,7,10-tetraazacyclododecane-4,7 , 10-trimethylenephosphonic acid (OzN-BDO3TMP) was obtained (80 %).

100 mg of 02N-BDO3TMP was dissolved in 20 mL of water.

After purging the system with nitrogen, 120 mg of 10% Pd/C was added. Then, the suspension was placed under a hydrogen atmosphere while constantly shaking vigorously. 3 hours The catalyst was later removed by filtration and the filtrate was lyophilized to yield 89.1 mg 11□N-[3DO3TMT' was obtained as a chocolate colored solid. 4. 2 (dissolve 89.1 mg of H2N-B　D　O3'r　MP in 3 mL of water) A 23.4 mM solution of this ligand was prepared by

Example G Preparation method of 5CN-[3DThlP (1,) In a 100 mL three-necked flask, 2. 0g (5, furomillimole) of 1-(4-nitrohennor)ene ethylenetriamine trihydrochloride [1r+org, %I, W, 1 in Cbem, 25.2772-2781 (1986) 3 r c c h +) i e l e t a l [prepared by removing the material] was added. 10.93g (0,108 mol) aIj Prepare a liquid solution of Cl and 6.91 g (0,086 mol) of phosphorous acid, and Added to previous reaction flask. Add a thermometer, reflux condenser, and stirrer to this flask. I put the child in. The reaction solution was refluxed with constant stirring. reaction fla 12.Additional funnel attached to Sco. 37% by weight of 0g (0°144 mol) of formaldehyde solution and dropwise into the heated mixture at a rate of approximately 1 mL/min. I let it go down. The reaction was left at reflux for an additional 16 hours and then removed under reduced pressure. The volume was reduced to obtain an amber semi-solid. Add this solid to about 2 mL of water. Dissolve and add dropwise to approximately 800 mL of methanol with vigorous shaking. It was added by The resulting white precipitate was removed by filtration and incubated at 45°C. and dried to yield 2.32 g (57%) of 1-(4- nitrobenzyl) diethylenetriaminepentamethylenephosphonic acid (02N-B DTMP) was obtained.

(2,) 500 mg of crude 1-(4-nitrobenzyl)diethylenetria Minpentamethylenephosphonic acid was added to 1.5 cm of Q-Sepharose (Sepha rose) (Pharmacia) anion exchange column. It was purified by elution using an ammonium acid concentration gradient solution. The desired product, 1- (4-nitrobenzyl)diethylenetriaminepentamethylenephosphonic acid is UV This is the peak at 280 nm with the longest retention time. eluted after l& When the peak was collected, approximately 43 mL of a solution containing the purified product was obtained. This capacity The volume was reduced to approximately 26 mL and treated with 200 mg of 10% Pd/C.

The suspension was placed under a hydrogen gas balloon with vigorous shaking for approximately 25 hours.

HPLC (anion exchange, 0-IN1 ammonium acetate over 30 min at 2 mL/min) monium concentration gradient solution), the reduction product has a retention time of It was shown that there was a slight shift towards shorter lengths. Then this suspension Filter and lyophilize the filtrate to obtain 259 mg of white glassy solid. did. HPLC showed approximately 92% purity. This product is Further purification was performed by ion exchange chromatography as described in 144 mg of 1-(4-aminobenzyl)diethylenetriamine pen as body When we obtained the ammonium salt of tamethylene phosphonic acid, anion exchange HP Purity by LC was >95%. For this product, as shown below: Detailed characterization was performed.

Decoupled P-31 NMR shows the expected ratio of 2.2.1. Three singlets were shown.

□HNMR (D, 01 δ2 S9J, 67 (m, 17+), 4. +3 ib, 2H], 6 ．． 91 (d, 2Hi, 7.21 (6,2 Niri (NMR (0,01 δ52.0.52.6.53.3.53.11.54. I, 549. SS , B, 57. I, 57. I, 58.3.58.3D65.2. + 20.5, Dl, l, +33.5.146.8 (3a) 1-(4-amino) in water ammonium salt of diethylenetriaminepentamethylenephosphonic acid A 0.09M solution of 146 mg of phosphonic acid in 1.193 mL of water It was prepared by decomposition. 328 μL of this solution (approximately 25 mg of phosphonic acid) acid) in a 10 mL vial containing 1 mL of water and 1 mL of chloroform. I put it in the bottle. Add 100 μL of thiophosgene to the formed emulsion for about 1 hour. The mixture was added with constant stirring. Remove the chloroform layer and add the aqueous portion to 1 Extracted with water using 4 portions of mL of chloroform. Product cleaned The aqueous solution was frozen in a dry ice/acetone bath and freeze-dried. 6.5 mg of fluffy white powder was obtained. 200μL of 0.5mg of water. 0. Dilute 6 μL of this solution with 3.8 mL of water and The UV spectrum obtained showed maximum absorption at 270 and 283 μm, This results in the product 1-(4-isothiocyanatebenzyl)diethyl Triamine pentamethylene phosphonic acid (SCN-BDTMP) was confirmed.

(3b,) 43mg (,51μM) of 1-(4-aminobenzyl)diethyle Dissolve triamine pentamethylene phosphonic acid in 2 mL of water and add 1 m It was mixed with L of chloroform. Add 150 μL of thiophosge to this stirred solution. added all at once. After stirring vigorously for 25 hours, remove the chloroform layer. and dilute the aqueous portion with water using three 3 mL portions of chloroform. Extracted. Freeze the washed aqueous solution of the product in a dry ice/acetone bath; and freeze-drying to obtain 45.4 mg of fluffy white powder. This 2 ．． A 4 mg portion was dissolved in 960 μL of water. Add 6 μL of this solution to 3.8 mL. diluted with water, and the resulting UV spectra showed polarities of 272 and 282 μm. A large absorption was shown, which led to the product 1-(4-isothionoanate base) diethylenetriaminepentamethylenephosphonic acid (SCN-BDTMP) ) was confirmed.

(3c,) Preparation method of 3.47 mM and 34.7 mM solutions of SCN-BDTMP 100 mg (1,389xlO-'mol) of SCN-BDTMP was added to 400 Dissolved in TL of 0.3M sodium bicarbonate buffer. The resulting solution has a pH 7,5, which was purified by addition of 5 μL of 50% (wt) sodium hydroxide. Adjusted to H9,48. The final solution was 34.7mM SCN-BDTMP. .

Add 45 μL of 5 μL of the 34.7 mM SCNBDTMP solution prepared as above. of O, 1M sodium bicarbonate and mixed thoroughly. Final melt The solution was 3.47mM SCN-BDTMP.

Example I ( How to prepare 4 bottles of IGF solution Freeze-dried radioactively labeled 1251-IGF-1 (from NEN) or A vial containing 100 μl of lyophilized recombinant ICF-1 (from IMB) Each sample was reconstituted with 100 μL of water and 0.25% USA was added as carrier protein. containing 0.5M sodium chloride in 20mM Tris-HCl with added A solution of 1 mg/mL [GF was obtained. Contents of a vial of recombinant IGF and 535 mg of 10 mM acetic acid. It was transferred into a glass serum vial containing certain iodinated IGF. tracer A solution of combined IGF containing 1251-IGF-1, which is Dialyzed against 3 portions of acetic acid (Spectrum Medical Indu 5pectra/Pro7™ membrane from Sjrics). For dialysis The contents of the Centric were then distributed into 4 equal volumes and 4 separate Centric on (enclosed) placed on the membrane of a membrane filter for a micro concentrator (3000 molecules m are off). The Centricon (trademark) is then centrifuged to concentrate the solution. The contents of the membrane were diluted with 3 ml of 0.3M sodium bicarbonate at pH=9.48. Washed a1 with thorium slowing solution-batch. This method contains IGF in the amount shown below. Four Centricon™ membranes were obtained.

ICF Approximate figures Example I Preparation method for two bottles of IGF solution Containing 300 μg of ICF-1 and 23.6 μCi of carrier in 0.1 M acetic acid. Frozen radiolabel as 150 μL aliquots containing no +251-IGF. The Bell-conjugated +251-IGF was thawed and then evaporated using 0°1M acetic acid to approx. Diluted to a final volume of 1.5 mL. Transfer 700 μL of this solution to two Cen plates. Placed on top of each of the tricon™ microconcentrator membrane filters (300 cutoff at 0 molecular weight). Then centrifuge this Centricon (trademark) The solution was concentrated and the contents of the membrane were dissolved in 1 mL of 0.3M at pH=9.48. Two consecutive washes were performed with sodium carbonate buffer. ICF (tracer) in the membrane number 1 Calculation I of 130 μg (1,7xlO- @mole) and in membrane number 2 was 127 μg.

Example J 1.3-Propanamine-N-(carboquine-propyl')-N,N'.

Method for producing N-trimethylene-phosphonic acid (PDTMP) Structure of compound PDTMP is expressed by the formula shown below.

Commercial grade 3-aminopropyl-2-pyrrolidinone (5.0 g) was dissolved in 6M hydrochloric acid ( was added slowly to a solution of phosphorous acid (9.7 g) in 50 mL). this The yellow solution was heated to reflux and formalin (9,2 The solution was treated by dropping 3 mL of 37% eL). an additional 18 hours of reflux Afterwards, the solution was cooled and the solvent was removed to obtain a sticky oil. this oil The mixture was dropped into 200 mL of methanol, and after filtration and drying, 5.15 PDTMP of g was obtained. This compound is a decoupled 31P-NM In the R spectrum, 8.2 ppm and 7.5 ppm (ratio to H3PO4 The two non-crust nodes preset in the comparison) are shown at a ratio of 2.1.

This compound can be defined as It can be used as a hard AP.

Example Ethylene/amine-N=(4-amino-phenethyl)-N, N', N' -Production method of trimethylene phosponic acid (8PEDTMP) Compound Δr’EDTMP The structure of is shown in the formula shown below.

120 mL of ethylene diamine in three 250 mL Erlenmeyer flasks. (EDA) and fitted with a stir bar and placed on top of the stirrer. Bromide Three 10 g samples of p-nitrophenethyl were weighed and incubated for 30 minutes. was added slowly over a period of time to the stirred flask containing EDA. p-nitro bromide After addition of phenethyl, the flask was allowed to stir overnight (16 hours) at room temperature. . Transfer the contents of the flask to a 500 mL round bottom flask and use a simple distillation apparatus. I connected it to

Excess EDA is distilled off from the desired product under reduced pressure at a temperature of 28-32°C. After removing most of the starting EDA, the sticky oil is dissolved in water and Extraction was performed three times with 75 mL of methylene chloride (CH2Cl2). CH2C1 The two layers were combined and concentrated in a rotary evaporator to yield 2934 grams of adhesive. A dark colored liquid was obtained.

Transfer all samples to a 500 mL round bottom flask along with 100 mL of 1.5 M MCI. Added to Sco. The aqueous layer was treated with activated carbon heated to 90°C, then filter paper was removed. filtered through. The aqueous layer was removed under reduced pressure and dried overnight in a vacuum dencator. Dry.

The resulting dry solid was washed with cold methanol and weighed 15°60g with i*a+. N-(p-nitro-phenethyl)ethylenediamine was obtained as the hydrochloride.

An additional 677 g of product was obtained by distilling the filtrate and washing with methanol. The combined yield was 72%.

20.0 g (0,063 mol) of N-(p -nitro-phenethyl) ethylenenoamine trihydrochloride.

This flask contains 90 g of deionized water in solution and 22.79 g (0.0 g) of deionized water in solution. , 219 mol) of monohydrochloric acid and 17.16 g (0,209 mol) of phosphorous acid. Added.

The flask was connected to a reflux condenser, fitted with a stirrer, and placed on a stirrer. Ta. 17.74 g (0,219 moles) of 37% formal in a 10 mL syringe Fill the dehyde solution and connect it to a syringe pump that has been calibrated in advance. Transport was carried out at a flow rate of 0.1 mL/min. Bring this reaction solution to reflux temperature. The formaldehyde was then added over a period of 3 hours with constant stirring. and slowly added to the flask.

The reaction was refluxed after addition of the formaldehyde solution and an additional 3 hours Stir for a while. The reaction was allowed to cool and the water was removed under reduced pressure. A sticky dark solid was obtained. Add this slowly to methanol A light brown solid was obtained. This precipitate was filtered and dried to give 20 g ( yield 65%) of N-(p-nitrophenethyl)-ethylenediamine-N,N' , N'-trimethylenephosphonic acid was obtained. 10g of this triphosphonic acid Dissolve the sample in 20 mL of water and use 2 mL of concentrated ammonium hydroxide. and neutralized it.

This crude ammonium salt was then purified with water using tlPLC for reverse phase purification. It was eluted and purified. N-(p-nitrophenethyl) purified in this manner Ethylene/amine = N, N', N' trimethylenephosphonic acid (710 mg , J,, 21 g mol) was dissolved in 50 mL of water and 235 mL of It was placed in a hydrogenation bottle with storage capacity. Loosely place the nitrogen plunket into the bottle. I let it blow in and replace it with air. Approximately 50 mg of 10% Pd on carbon was used as catalyst. and any catalyst on the side of the container was rinsed with water to submerge it. all After submerging the catalyst in water and filling the bottle with nitrogen, the bottle was placed in Parr's hydrogenation equipment. It was installed on the equipment.

After removing nitrogen through the suction line, hydrogen at normal pressure was introduced. This flushing was repeated to ensure all oxygen was removed. Pressure is increased by the hydrogen finally added. The power became 35 ps i. Hydrogenation is initiated by farming the bottle, The process was continued until hydrogen uptake stopped (2.5 hours). This pressure drop and the known hydrogen volume can be used to calculate the number of moles of hydrogen used. , in this case this value was 992% of the theoretical value. Then crow this reaction mixture Carefully filter the mixture through a vacuum filter and remove it from the hydrogenation equipment after removing the vacuum. ) and the catalyst thus separated was washed with four 25 ml portions of water. Ta.

The combined aqueous filtrates were then frozen and lyophilized overnight to give 580 mg (yield 85%) of N-(4-aminophenyl)-ethylenediamine-N, N', N '-trimethylenephosphonic acid with ammonium salt (APEDTMP) and obtained it.

The decoupled 32P-NMR spectrum of this compound shows the expected 8, One noncrunod at 0ppm (compared to H3PO4) and at 16.9ppm A single noncrusty throat was shown at a ratio of 12.

This compound is a ligator in formula I [wherein L is connected through the NlI2 group]. It can be used as a host AP.

Example 1-(Carboquino)ethylenediaminetetramethylenephosphonic acid (CEDTMP) ) manufacturing method The structure of compound CEDTMP is shown below.

To a solution of phosphorous acid (12.9 g) in 6M hydrochloric acid (50 mL) was added 5.0 g of 2.3 -aminobrobionic acid dihydrochloride compound (Aldrich Chemical C Company O, Miwaukee, Wl) was added.

The solution was heated to reflux and formalin (3.5% in water) was heated to reflux and over a period of 45 minutes. 7% formaldehyde (12.3 mL) was added dropwise. additional 18 hours After refluxing, the solvent was removed and the oil was dissolved in methanol (20 mL). I set it. This solution was then added to absolute ethanol (200 mL) with stirring. A fine precipitate formed. The solid was then filtered and dried to yield 737 g of C. Obtained EDTMP. The partial pressure of this solution was diluted 10 times, and the high performance liquid chromatography Analyzed by chromatography (HPLC). HPLC is AS-7 anion Dionex™ 2010 using a column exchange column (8mm x 25cm) i JonSupression Chromatography System m (Sunnyva Ie, CA) and at a flow rate of 0.6 mL/min. eluted with 0.03M nitric acid and monitored at 330 nm. It was performed using outside line detection. The retention time of this compound was 10°11 minutes.

This compound can be combined with the ligator of formula It can be used as a host AP.

Example M 1-(4-aminobenzyl)ethylene-diaminetetramethylenephosphonic acid (Δ BEDTMP) manufacturing method The structure of the compound ABEDTMP is of the formula It is shown in

In a 50 mL round bottom flask, add 0.6 g (0,2256 mol) of 1-(4-nito). lobennor) ethylenediamine, essential HCI (1.24g, 0゜0118mol ), followed by the addition of phosphorous acid (0,0925 g, 0.0113 mol) . This solution was brought to reflux and then over a period of 90 minutes 0.956 g ( 0,0118 mol) of 30% formalde, cooled to room temperature, and and then added dropwise to 100 mL of chilled methanol. Precipitate obtained was filtered and dried to give 1-(4-nitrobenyl)-ethylenediamine. Obtain tetramethylene phosphonic acid and add 60 mL of it in a hydrogenation flask. Dissolved in water. Add a 5Qmg sample of 5% Pd on carbon and add this Connect the lasco to the Parr hydrogenation system and wait until hydrogen uptake has stopped. Agricultural production was carried out in The solution is then filtered to remove the catalyst and the resulting clear filter The solution was frozen and lyophilized to yield 1.28 grams (86%) of 1-(4-alpha). (minobenzyl)-ethylenediaminetetramethylenephosphonic acid was obtained. this The compound showed the expected decoupled "P-NMR spectrum.

This compound has the formula 1 [wherein L l! N) Connected through 12 units It can be used as a liquid ΔP (can be used heavily).

Example N Process for producing N''-(p-aminophenyl)-norsperminone [His(Noanoech) p-aminophenylamine is described in U.S. Patent No. 2.809.985. Manufactured as described in . 9.0 g (42 mmol) of bisni-1-lyl ) sample in 90 mL of ethanol and ], 50% N a of Q m I- Dissolved in OH and treated with 6g Raney nickel (grade Xv-2) and The plant was placed under a hydrogen atmosphere at normal pressure while being produced. Until hydrogen uptake stops Farming was allowed to continue and the catalyst was removed by filtration when hydrogen uptake was stopped. child The volume of the filtrate was reduced under reduced pressure until it became 1,11 and 11 filtrate. Dissolve it in 40 mL of 50% \aOH and add 3 x 75 mL portions of chloroform. It was extracted separately. The combined chloroform layers were dried over sodium sulfate and dehydrated. 7°2 g ( The title compound was obtained with a yield of 77%. This product was characterized by proton NMR. I made a decision.

This compound is a ligator in the formula l [wherein L is connected through the NH2 group]. It can be used as a host AP.

Example O N'' (4-aminophenyl)-dipropylenetriamine-N', N'.

No”, No-tetramethylenephosphonic acid (API PMP) manufacturing method compound The structure of PIFMP is the formula It is shown by.

N'-(p-aminophenyl)norspermine (3 , 65 g, 16.4 tt mol) were added to 25 mL of water and 13 mL (1 63 mmol) in concentrated HCl. 2 g (75.6 mmol) of phosphorous acid treated with a solution of The resulting dark blue solution was heated to 100°C and 5. 9 g (73 mmol) of 37 weight percent formaldehyde at 2.75 hours The treatment was carried out dropwise over a period of time. Add this solution for an additional 15 hours. Leave to reflux, then cool to room temperature, and add 1:1 dropwise in ethyl alcohol. I let it go down. The resulting brown precipitate was vacuum filtered and dried under vacuum.3. Obtained 84 g (40% yield) of the title compound (API PMP) as a brown solid. did. The product thus obtained can be obtained by anion exchange H 5.50 minutes (small peak) and 1050 minutes (major peak) when analyzed on a PLC system. The retention time of

This compound is a ligand of the formula ■ [wherein L is NH, connected through a group] It can be used as a hard AP.

Example P N"-(4-aminophenethyl)-diethylenetriamine-N", No.

No”, No”-tetramethylenephosphonic acid tetrasodium salt (APDTMP) manufacturing method The structure of the compound △PDTMP is the formula It is shown by.

7 Ethylenetriamine (20 g, 0.045 mol) was dissolved in 200 mL of toluene. p-Nitrophen bromide in 150 mL of toluene for 5 minutes. Treated with a solution of netyl (10 g, 089 mol).

After 3 hours of stirring, the supernatant was decanted from the gummy solid and The mixture was extracted three times with 100 mL of water. Reduce the volume of the combined aqueous layer to a low volume under reduced pressure. volume and back-extracted with 100 mL of chloroform.

The chloroform was then evaporated under reduced pressure to yield 9.32 g (83%) of N'' -(p-nitrophenethyl)-diethylenetriamine was obtained.

A 5 g sample of this amine was then dissolved in 3N HCI to reduce the p tl occurred. The resulting solution was poured into excess methanol. The resulting sediment The precipitate was filtered and dried under reduced pressure to obtain 3 g of the corresponding hydrochloride salt. Ta. Dissolve 1.3 g (0,004 mol) of this salt in 20 mL of water, and Phosphorous acid (1.5 g, 0.018 mol) and concentrated hydrochloric acid (2.0 g, 0.019 mol) Processed with The solution was refluxed and 37% formaldehyde (1,5 g, 0.019 mol) was added in a dropwise manner. After an additional 2 hours of reflux , the solution was cooled to room temperature and evaporated under reduced pressure to form a sticky oil. occurred. Add this oil dropwise to 150 mL of methanol with vigorous stirring. It was added by The resulting white precipitate was filtered and dried under reduced pressure. 1.65 g (yield 66%) of N”-(4-nitrophenethyl)diethylenetri Obtained amine-N’, N’, N’”, No-tetramethylenephosphonic acid did. In the HPLC method described in the example, this product was showed a single peak with a retention time of .

A sample of 700 mg (1.1 mol) of this compound was added to 44 mL (4°4 mm). mol) of O, IN was dissolved in NaOH. Suspend this solution in 56 mL of water. Add 100 mg of 10% Pd on carbon and evaporate the entire reaction mixture. Place it under hydrogen while producing agricultural products properly. After hydrogen uptake has stopped, the catalyst is Filter, strain and evaporate the filtrate under reduced pressure to give 0.80 g (100% yield) N”-(4-aminophenethyl)diethylenetriamine-N’, N’, N’”, N’”-tetramethylenephosphonic acid tetrasodium salt (APDTM P) was obtained as a hygroscopic yellow solid. This title compound was prepared in the following example. The described HPLC system (showing a single peak with a retention time of 595 minutes) Shitko.

This compound has the formula I [where L is connected through N H2] (where L is connected through H2). It can be used as a node AP.

Example Q 4-isothionoanatophthalic anhydride (ACL-3, acid cleavable 1 noncar) manufacturing method 4-aminophthalic acid (2 7183 g, 15.00 mmol) and anhydrous potassium carbonate (8°75 g, Thiophosgene (2.30 mL, 30.18 mmol) was added to a slurry of thiophosgene (2.30 mL, 30.18 mmol). mol) was added. The reaction mixture was stirred at room temperature for 10 minutes, and First, heat for 1 hour and bring to reflux. After cooling [1] to room temperature (this reaction The product solution was filtered through Nurite and then under a vapor of dry nitrogen and Under effective toxic gas ventilation to avoid exposure to thiophosgenes. I was so sad that I had to eat 1 dried food. In this way, the crude 4-isothiocia Nephthalic acid was recovered.

The 4-isothionanatophthalic acid thus obtained was immediately heated to in a mixture of phthalic anhydride and methylene chloride for 2 hours under a nitrogen atmosphere. Refluxed. After cooling to room temperature, the reaction mixture was concentrated under reduced pressure. profit When the solid was recrystallized from 30 mL of carbon tetrachloride, 2.3778 g 4- as brownish-purple crystals in a yield of (77% of theory). Isocyanate phthalic anhydride was obtained. This title compound is 106-108° It melted at C.

NMR (3nOMHz, acetate:,"d61d8.1Sfd, 1=8.ll-1 z, It-11,13,06 (d, I=1.7Hx, It4P.7.97 fd d, J = 8.1. I, 782, + Ml: I 3 (NMR (75 MH (acet/・d61 d +63.3.162.6.D9.8.1348 ．． 13a, 6. P30.6.128.2.123.5゜ 111.6:IR[(H(1112010(b+l,+840.1740cm- 1; MSm/e205.161,133 (iQTll, 74 final generation Example 1 and Comparative Example A Conjugate binding of PDGF using SCN-BDTMP 34.7mM for 400μL SCN-BDTMP solution (1,389 x 1o-5 mol) (prepared in Example G) ) was added to aliquot 1 from Example E. At this stoichiometry, protein 794:1 molar ratio of isothionane per amino group on sample 1 cause

400 μL of 0.3 M sodium bicarbonate buffer from Example E as a control. (Comparison Δ).

Both samples 1 and 2 were mixed well. These samples are still on the membrane. The pull was left at room temperature for 1454 hours. Then run these samples at maximum speed for 1 hour. Samples were concentrated by centrifugation. After centrifugation, each sample was placed on the membrane at 10.0 The value of μCi is shown. 1 for 6 membranes. OmL of 0.1M sodium phosphate buffer Added. After swirling the container to mix, test these samples again at maximum speed. The mixture was centrifuged for 40 minutes at 30°C.

Remove the filtrate cup and add a new filtrate cup with a tare weight of ff111. Replaced it with a cup. Each sample showed a value of 99 μC1 on the membrane.

Add 740 μL of l-ISA solution to each sample to calculate the volume contained in the membrane. Adjusted to 800ttL. Mix each solution and tare weight. New +! ! Transferred to liquid cup. Wash each membrane unit with 500 μL of 1% H3A. Purified. Then close the cap of the Centricon(TM) unit and turn it upside down. and centrifuge at maximum speed for approximately 10 minutes to remove the membrane retentate. did. The washing solution was injected into the corresponding filtrate cup using a viper. . Centricon™ cups were also washed with 200 μm 7 min H3Δ solution; After that, this was transferred to the #formation cup. The final solution for each of these is Approximately 185 μg (1) or and 19011 g (comparison Δ).

Example 2 One S N C-B l) T M P conjugate bond 1011L per IGF (3,/17X]-0-'mol) of 3.47mM SCN-BDTMP solution ( prepared in Example G) and Centric of Example H.

No. 1 (contains 41.2 μg of IGF). additional 390 μL of pH=9.35 O, 1M sodium bicarbonate buffer was added.

The solution remaining on the Centricon™ membrane is then mixed well and It was left at room temperature for 14 hours. The unit is then turned upside down and centrifuged to remove the membrane contents. I took it out. 158μL aliquot of 0. 1MI [final addition of sodium carbonate] The volume was approximately 600 μL.

Example 3 20 μL of conjugated binding of two SCN-BDTMPs per IGF (6,94×lO -”mol) of 3.47 mM SCN-BDTMP solution (prepared in Example G). ) of Example H.

n(TM) number 2 (containing approximately 41.2 μg of IGF). additional 39 0 μL of 0.1 M sodium bicarbonate buffer pH=935 was added. Cen The solution remaining on the tr1con™ membrane is then mixed well and placed in a chamber. It was left warm for 14 hours. The unit is then turned upside down and centrifuged to remove membrane contents. I took it out. Add 86 μL aliquot of O, 1M sodium bicarbonate to final volume. was set to about 600μ■.

Example 4 400 uL of conjugated binding of 4 SCN-BDTMPs per IGF (1,39X1 0-5 mol) of 34.7 mM 5CII-BD TMP solution (Example G) Centricon™ No. 3 of Example H (approximately 457 μg containing IGF). Solution remaining on Centricon™ membrane was then mixed well and left at room temperature for 14 hours. After that, this unit, The membrane-containing material was taken out by turning it in the opposite direction and pulling it out 7 times. 208μL aliquot of O, 1M weight Sodium carbonate was added to bring the final volume to approximately 600 μL.

Comparative example B Control for Examples 2.3 and 4 Perform 400 μL of 0.1 M sodium bicarbonate solution at pH = 9.35. Example I] Centricon™ No. 4 (containing approximately 47.7 μg of IGF) ) was added. Additional 390 μL of ptl=9.35 0. IN1 sodium bicarbonate Thorium buffer was added. Remove the solution remaining on the Centricon™ membrane. Then mix well and leave at room temperature for 14 days.

Then, turn the unit upside down and centrifuge to remove the membrane contents. 23/ 41 - Add aliquots of 0.1 h sodium bicarbonate to bring the final volume to 600'J G Conjugate bond of 4 SCN-BDTMPs per F 40011[-(1,39 3/1.7mM 5CN-I3D TNI l) solution ( prepared in Example G) and Centricon™ No. 1 of Example I (approximately 130f1go':) Contains IGF) Test the solution remaining on the membrane (mix and leave at room temperature for 13 hours) . Thereafter, the protein 11 in the solution was concentrated by diluting the solution. So of i (Q, 5 mL of Q, 1 MIJ of sodium chloride buffer overnight (p H=7. /I) was washed with L1 by adding this buffer for 7 nights, and then and then centrifuged for 1 hour. Then apply Cen t r 1con (trademark) membrane” second To the residue, add 572 uL of 1/Mercent HS A solution and rotate the container. Then, I washed it with 1 bottle of bipenotein. this Wash the membrane with additional Q, 5 mL of 1-cent HS A to completely remove membrane contents. I took it out. The total volume of this solution was approximately 1.5 mL, which was collected together. This contains an estimated 9.5 μg/100 μL of modified IGF in approximately 1% H3A. It contained.

Comparative example C Control for Example 5 Example I of Centricon™ No. 2 (containing approximately 127 μg of IGF). Ta. The solution remaining on the Centricon™ membrane is then mixed well and The mixture was left at room temperature for 13 hours. The unit is then centrifuged to remove the protein from the solution. was concentrated. The residue on the membrane was then removed with 0.5 mL of 0.1 M sodium phosphate buffer. (pH = 7.4) and washed by adding this buffer, and It was then centrifuged for 1 hour.

The residue on the (&Centr icon (trademark) membrane) was Add 1% II S A solution, shake the container and pipette. Washed by washing. Wash the membrane with an additional 0.5 mL of 1% I-ISAS. The membrane contents were completely removed by cleaning. The final volume of the combined solution was approximately 1. 4 mL, which contains an estimated 95 μg/100 μL in approximately 1% HS A. there was.

Example 6 ACL-3 of IGF175IIL modified with BDT\IP using ACL-3 (12.1 mg ACL-3 in 200 μL trifluoroethanol, Example prepared in Q) in approximately 500 μL of 0.3 M bicarbonate buffer (pH = 9.5). Centricon™ containing approximately 100 μg of IGF. obtained The pH was 885. Then, after leaving it at room temperature for 15 minutes, Centricon (quotient 5) unit until the volume decreases to approximately 243 μL (weight of 243 mg) Centrifugation was performed, which required approximately 2 hours. Add 300 μL to this concentrated solution. ABDTMP solution (7.1 mg, p) of 300 μL of bicarbonate at I=9.5 11 (105 μmol) in heavy liquid was added. The p■1 obtained was 927.

This solution was left at room temperature for 46 hours. The pH was found to be 978; The volume was approximately 0485μ.

This was placed in a centrifuge (ClAy-Ad ams) for 6 hours, and the volume was reduced to 1 Reduced to 17 μL. Add 2 mL of 0.3 M sodium bicarbonate buffer and The solution was then centrifuged again. After 10 hours of centrifugation, the volume was approximately 49μ. . Add an additional 500 μL of 0.3 M sodium bicarbonate and icon™ units were centrifuged for 4 hours to a final volume of 52 μL. 54 Add 7 μL of O,LM sodium phosphate solution (pH = 7.02). , and mixed well. Total weight in Centricon(TM) unit is 5 It was 99 mg, or about 600 μm in volume. Estimated concentration in this sample The concentration is 100μg of ICF per 600μL of solution (16.7μg/100μ L).

comparative example Pair 1 IC for Example 6 Add 175 μl of trifluoroethanol to approximately 500 μl of 0.3 Mfi carbonate. Centricon containing about 100 μg of IGF in carbonated liquid 11 liquid = 9.5) (trademark). The resulting pH was 950. Then leave it at room temperature for 15 minutes After that, add a Centricon (trademark) unit to a volume of approximately 230 μL (23 Centrifugation was performed until the weight decreased to 0 mg), but this took approximately 2 hours. . 300 μL of 03-1 bicarbonate buffer was added to this acondensation solution. Obtained pH was 946. This solution was left at room temperature for 46 hours. pH is 9.79 It was found that the solution had a thickness of about 397μ. Put this in a centrifuge (C1a y-Adams) for 6 hours, and the volume was reduced to 27 μL. 2mL portion of 0.3M sodium bicarbonate buffer was added and the solution was centrifuged again.

After 10 hours of centrifugation, the volume was approximately 21 microns. Additional 500 μL of 0.3 Add M sodium bicarbonate and Centric.

n™ units were centrifuged for 4 hours to a final volume of 27 μL. 573 It Add I-min of 0.1M sodium phosphate buffer (pH=7.02) and Mix well. Total weight in Centricon(TM) unit is 603m The weight was about 600 μg, or about 600 μm in volume. The estimated a degree in this sample is 100 μg of IGF per 600 μL of solution (167 μg/100 μL) Ru.

Example 7 210 μL aliquot of IGFIGF solution modified with BDTMP using ACL-3. (100 μg of IGF containing trace amounts of +15I-labeled IGF in buffer solution) ) was placed on the membrane of a Centricon™ membrane filtration device. 0.3M sodium bicarbonate Add a 500 μL aliquot of thorium to both Centricon™ units. and then add it to the membrane so that less than 50 mg remains on the membrane. centrifuge until The content on the membrane (washed IGF) was removed in an amount greater than 500 μL. ゜Dissolved in 3M sodium bicarbonate and 300 μL of warmed trifluoroethylene. It was treated with 19.4 mg of ACL-3 dissolved in ethanol.

The sample was left for 15 minutes and centrifuged for 2 hours. This period has ended After that, a volume of 300 μL of ABHDP (357 μmol) was added to the modified IGF. and then add this solution to a Centricon (commercial t!J) unit containing The solution was allowed to stand for approximately 16 hours. The pH remained around 10 during this period. That(&C The entr icon™ was centrifuged for approximately 4 hours to reduce the volume. the The substance i (modified IGF) retained in the posterior membrane was dissolved in 500 μL of sodium bicarbonate. and reconcentrated by centrifugation. Amount of residue retained in the membrane was 221 mg. A volume of 1.279 mL of 0.1 M sodium phosphate (p H=7.02) was added to bring the final volume up to a total of 15. Then Centr The material remaining on the membrane was removed by centrifugation with the icon (trademark) in the opposite direction. child The material was used in the rat biodiffusion study shown in Example V.

sacrificial dan Control for Example 7 ACL-3 binding compounds are included upon addition of warmed trifluoroethanol. The same treatment method as in Example 7 was carried out, except that

Example 8 , of PDGFl)DGF solution modified with 13DTMP using \CL-3. A 100 tt L aliquot was placed on the membrane of a Centricon™ membrane filtration device. I put it on. This PDGF solution was prepared using 200 μL of 01N1 phosphorus at pH = 4.6. sodium citrate, 001M sodium citrate, 05N1 NaCl, and 1 10/lCi of 1251 labeled PDGF per % Uno blood/FI albumin (22μCi/μg).

Q. A 500 μL aliquot of 3h+ sodium bicarbonate was added to this Centri con(TM) units, and then this was added to the membrane with 50 mg remaining on the membrane. The mixture was centrifuged until it became clear. The content on the membrane (cleaned PDGF) is 50% Dissolved in >0 μL of 0.3M sodium bicarbonate. After that, it remains in the membrane. 36.7 by dissolving the substance in 300 μL of warmed trifluoroethanol. mg of ACL-3, left for 15 minutes, and then centrifuged for 2 hours. this At the end of the period, a volume of 300 μL of AIIHDP (357 μmol) was added to the Centricon™ unit containing a certain PDGF and this The solution was allowed to stand for about 6 hours. The pH remained around 10 during this period. Then C The entricon™ was centrifuged for approximately 4 hours to reduce the volume. Then on the membrane The remaining substance (modified PDGF) was dissolved in 500 μL of sodium bicarbonate. Diluted with buffer and reconcentrated by centrifugation. Amount of residue retained in the membrane was 329 mg. 1.171 mL volume of 0.1 M sodium phosphate (p t+=7.02) to bring the final volume up to a total of 1.5 mL. Then C Centrifuge the entricon (trademark) in the opposite direction to remove any substances remaining on the membrane. did. This material was used in the rat biodiffusion study presented in Example V.

Comparative example F Control for Example 8 ACL-3 binding compounds are included upon addition of warmed trifluoroethanol. The same treatment method as in Example 8 was carried out, except that

Biological Examples: Biodiffusion Example I +251-PDGF-(SCN-BDTMP) within the weight range of 175-230g Spraque-Dawley rats being weighed were allowed to acclimate for 5 days before injection. from Example 1 to the rat via the tail vein. 5 of both samples (Sample 1 and Comparative Example Δ) (approximately 200.000 cpm) 0 μL was injected. 2. After 6 and 18 hours, rats were sacrificed by cervical dislocation. , tissue samples were taken, body weight was measured, and the radioactivity in each tissue was measured using multiple channels. Non-chirenoyon measuring device equipped with a Na + ore detector connected to a metal analyzer It was determined by measuring within 5 minutes. Percentage of dose injected into each tissue For the purpose of determining the compared. Subtract the measured value at the tail from the standard value to find the value found at the tail. A percentage injection solution was obtained that was adjusted to the amount required. Background measurement value was obtained and subtracted from the histological measurements. percente in bone The dose is estimated by multiplying the percentage dose in the thigh by 25. did.

The values for muscle and blood are that muscle accounts for 43% of the rat's body weight; The sample was obtained assuming that the blood content was 65%. The amount of money I spent making this kind of preparation was Percentage doses for different body parts are acceptable for the rat model. It is a value. [W, F, Goeckeleret am, J, of Nucl M ed, 28(4), 495-504 (1987)]. These results are shown in Table I. However, in this table, the values for each data are based on five rats unless otherwise noted. Represents the average value.

Table■ Adjust the dose found in the caudal region of 4 rats for injection. Average value ・Comparison A PI) conjugated bonding of 5CN-[3DTMP with GF to It is easy to understand that a cherry-like setting of PDC;F is obtained. 5CN-B D PDGF, which is conjugated to TMP, is a natural, unmodified B Compared to DGF, it was 8 times more effective in reaching the bone. the Residence time in the humerus increased approximately 30 times.

Example II +251-IGF-(SCN-BDTMP) weighed within the weight range of 175-230g Spraque-Dawley rats to be weighed The animals were allowed to acclimatize for 5 days before injection. Example 2.3.4 was also administered to rats via the tail vein. Alternatively, 50 μL of one of the four samples from Comparison B was injected. 30 minutes later Next, rats were sacrificed by cervical dislocation, tissue samples were collected, body weights were determined, and Radioactivity in each tissue is connected to a multi-channel analyzer to detect Na+ ore. Determined by measuring for 5 minutes in a nonchirenoyon 81 instrument equipped with a . For the purpose of determining the percentage of dose in each tissue, Measurements were compared to measurements in a 50 μL standard. Standard measurements at the tail Percentage injections subtracted from values and adjusted to the members found in the tail. Got the dose. Obtain a bank ground measurement and subtract this from the tissue measurement. I pulled it. Percentage dose in bone is for percentage in thigh Estimated by multiplying the amount by 25. Values for muscle and blood are for rats. For the body weight, assuming that muscle is 43% and blood is 6.5% I got 11 points. These results are shown in Table II. In this table, the numerical values of each data are represents the average value of 5 rats.

Table■ Through % Injectable Type I AP which is adjusted to the dosage found in the caudal region. The degree of modification of IGF with respect to the number of bone-seeking groups added is based on the stoichiometry of the reactants. It is. These results are summarized in Table 111. In this table, each data Values in ta represent average values of 5 rats.

“Comparison B These results show that the target transport of IGF-1 to bone is It is clearly shown that this is achieved by conjugate binding of BDTMP. Table III odor As shown, the degree of targeting is directly related to the degree of modification. It's obvious.

Example II! 1251-ICF-(SCN-BDTMP) weighs within the weight range of 175-230g Spraque-Dawley rats to be weighed The animals were allowed to acclimatize for 5 days before injection. Example 5 or C was administered to the rat via the tail vein. 50 μL of the sample was injected. 2. After 6 and 18 hours, the rats were The layer was killed by dislocation, the tissue was collected, the body weight was measured, and the radioactivity in each tissue was determined. A non-condensing device equipped with a Nal concretion detector coupled to a multi-channel analyzer. It was determined by measuring for 5 minutes in a Chile Noyon meter.

Measurements within each tissue for the purpose of determining the percentage of dose injected into each tissue. was compared with the measured value in 50 μL of Sakura standard. Measured value at the tail from the base value Subtract the percentage injection dose adjusted to the amount found in the tail. Obtained. Obtain a background measurement and subtract this from the tissue measurement. I pulled it. Percentage dose in bone is percentage dose in thigh Estimated by multiplying by 25. Values for muscle and blood are for rats. For body weight, assume that muscle is 43% and blood is 6.5%. I got it. These results are shown in Table IV, where the numerical value of each data is 4. Represents the average value of 1 rat.

Table■ % injection dose adjusted to the dose found in the tail” Comparison C Example IV Biological activity of IGF-1-(SCN-BDTMP) Samples were measured in an IGF-1 mitotzene activity assay to determine their effectiveness. Determined sex. This first step of the experiment is based on the reaction capacity-continuation model; Assay cell lines are exposed to varying levels of I in a dose-dependent manner. React with CF-1. Cells from a mouse fibroblast cell line were analyzed in a 96-well assay. plate and grow until fusion and inducing quiescence. sump Pre-incubate the cells with PDGF-BB before adding cells and standards. This allows the cells to remain intact when added in the presence of EGF. cells become responsive to IGF-I. - After an overnight incubation period, The cells are exposed to 10 μC of 3H-thymidine, which is Uptake at varying levels depending on the amount of IGF-I added to Saywell . After extensive washing, lyse the cells and transfer the sample to a multichannel analyzer. Quantified in a scintillation meter equipped with a coupled Nal ore detector. do. Plot the results to create a dose-response curve and calculate the unit activity from this curve. gender can be determined.

One unit corresponds to 50% of the maximum cellular reactivity (EDa value) in the assay. It is identified as one degree of inducing IGF-1. Maximum response is based on 5% FCSI It is identified as a cellular response to The maximal response due to exogenous IGF-1 is It is usually 3-5 times the reference line.

Before making dilutions for the assay, the concentration of IGF-I is stored prior to performing the procedure. I-IGF-1) is added to the sample beforehand and determined via a racer. Ru. Measurement was performed by dispensing 25 μL of the sample for storage and dispensing 25 μL of the two samples. I went there. IGF-1 ratio per minute (CPM)/μg in storage samples was used to determine the concentration of IGF-1 in two samples for biological assays. Protection The average value for the remaining sample aliquots was 5.135 CPM/μg. Each sump Determine the average value for CPM per μL and save the value. sample aliquot of 5.135 CPM/μg. These measurements and The values shown below were obtained by counting and counting values, but the sample of Example 5 was 107.2μ. g/mL, and that of Example C was 110.8 μg/mL. this The results are shown in Table ■.

Table ■ 1 Comparison B 1' Comparison C Example■ 1251-IGF-ACL-3-BDTMP Material from Example 7 and Comparative Example E (150 μL) was injected into a 500 μL injection using a 28-gauge 7 needle. and injected into the tail vein of five rats. 1 barley for 6 hours The rats were sacrificed and dissected to examine the tissue diffusion of radiolabeled substances. Import Samples were counted for 10 minutes due to the low amount of radioactivity contained. Example 7 The final results found in the thighs of rats given injections with modified IGF substances. The number of measurements (below bank ground) is as shown in Table Vl shown below. sea urchins were found in the thighs of rats injected with the lamp substance of Comparative Example E. This was 3.8 times the number of measurements (below background).

Table ■ 12J-PDGF-ACL-3-BDTMP A dose (100 μL) of this substance was Insert a 28 gauge needle into the attached 500 μL syringe, and Cen t r i conl (for experiment) and Cen t r i con 2 (control) was injected into the tail vein of 5 rats each. 6 hours Rats were later sacrificed and dissected to examine tissue diffusion of radiolabeled substances.

Due to the low amount of incorporated radioactivity, samples were measured for 10 minutes. Ce nLr1c.

The best results found in the thighs of rats injected with nl (experimental) The final count (below background) is shown in Table V11 below. The rats injected with the substance Cen t r i con 2 (control) were 38 of the measurements (below background) found in the thighs of cats. It was double that.

Table ■ This data therefore demonstrates that GF using the techniques described in this invention The modification may result in targeted transport of biologically active CF to bone. shown.

The present invention is also described with respect to its preferred deaf configuration, and upon reading this disclosure, those skilled in the art will and the invention as previously described or hereafter claimed when understood. You may make changes and modifications without departing from the scope and spirit of be able to understand what is possible.

Continuation of front page (81) Designated countries EP (AT, BE, CH, DE.

DK, ES, FR, GB, GR, IE, IT, LU, MC, NL, PT, S E), AU, CA, FI, HU, JP, KR, No, NZ, US (72) Inventor: Lynch, Samuel E. Massachusetts, USA 0 1915 Beverly East Street 26 (72) Inventor Pribisch, James R. Michigan, USA 4 8640 Midland Linden Drive 4010

Claims (33)

[Claims] 1. formula GF-[(CL)z-L-AP]q(I) [wherein, GF is a tissue growth promoting factor or combination thereof, and CL is covalently linked to GF. z is 0, 1, or 2; q is from 1 to the total number of amino groups present in natural GF, and L is the linking moiety. Yes, and AP is a polyamino methylene phosphonic acid ligand. 2. GF is a platelet-derived growth factor, insulin-like growth factor, and fibroblast growth factor. epidermal growth factor, transforming growth factor, nerve growth factor, or soft The compound of claim 1 which is a bone/osteoinductive factor or a combination thereof. thing. 3. GF is a platelet-derived growth factor, insulin-like growth factor, and fibroblast growth factor. children, transforming growth factors, or cartilage/osteogenic factors, or The compound of claim 2 which is a combination thereof. 4. GF may be platelet-derived growth factor and insulin-like growth factor-I, or blood is a combination of platelet-derived growth factor and insulin-like growth factor-II, A compound according to claim 2. 5. AP is a linear or branched moiety, a cyclic moiety, a polymer, or an aryl moiety, and this ligand has at least two nitrogen atoms. The compound of claim 1 comprising. 6. 6. The compound of claim 5, wherein the polymer is a dense star polymer. 7. Claims where the dense star polymer is a dendrimer or dendron 6 compounds. 8. The embodiment of claim 5, wherein the ligand has three or more nitrogen atoms. Compound. 9. AP is the formula ▲There are mathematical formulas, chemical formulas, tables, etc.▼(II) [In the formula, Each R1 is independently hydrogen, C1-C4 alkyl, phenyl, hydroxyC1-C4 Alkyl, -CH2COOH, -CH2PO3H2, or L moiety, However, it can be only one part of R1, and there must be only one part of R1. and at least half of all R1 are -C The condition is that H2PO3H2, Each R2 and R3 is independently hydrogen, or C1-C4 alkyl, or the L portion Although it is a minute, However, the condition is that only one moiety exists in formula II, and n is 2, 3 , or 4, n' is 2, 3, or 4, and m is from 0 to 10] The compound of claim 5 which is. 10. AP is the formula ▲There are mathematical formulas, chemical formulas, tables, etc.▼(III) [In the formula, R1, R2, R3, n, and m are defined as in claim 9] The compound of claim 5 which is. 11. 6. The compound of claim 5, wherein AP is a linear or branched moiety. 12. The straight chain or branched chain part is (N-propylcarboxyl)ethylenediamine-N,N',N'-trimethylene phosphonic acid, [N-(4-aminophenyl)ethyl]ethylenediamine-N,N',N'-t rimethylenephosphonic acid, 1-(Carboxyl)ethylenediamine-N,N,N',N'-tetramethylene phosphonic acid, [1-(4-aminobenzyl)]ethylenediamine-N,N,N',N'-tet Ramethylene phosphonic acid, N-(4-aminophenyl)-N,N,-bis-[propyl(iminodimethylene) phosphonic acid)], N-[(4-aminophenyl)ethyl]-N,N-bis-[ethyl(iminodimethyl) tyrenephosphonic acid)], or N-[1-(4-aminobenzyl)-N,N' -Ethylenediamine-N',N''-Ethylenediamine-N,N,N',N' ′-Pentamethylenephosphonic acid The compound according to claim 11. 13. 6. The compound of claim 5, wherein AP is a cyclic moiety. 14. The cyclic part is 1,4,7,10-tetraazacyclododecane, 1,5,8,12-tetraaza Cyclotetradecane, 2-[(4-aminobenzyl)-1,4,7,10-tet raazacyclododecane]-1,4,7,10-tetramethylenephosphonic acid, 1- [(α-carboxyl)-4-amino-2-methoxybenzyl]-1,4,7, 10-tetraazacyclododecane-4,7,10-trimethylenephosphonic acid, Ruiha 1-[(α-phosphonyl)(4-aminophenyl)ethyl]-1,4,7,10 -tetraazacyclododecane-4,7,10-trimethylenephosphonic acid The compound according to claim 13. 15. 6. The compound of claim 5, wherein AP is an aryl moiety. 16. Atoms in the core of an aryl ring in which the aryl moiety is from 3 to 30 16. The compound of claim 15, which is an aromatic ring system having a total number of . 17. An aromatic ring system is one in which one or more additional rings or aromatic rings are present. or substituted with a linear or branched moiety. compound. 18. The aromatic ring system is 3,6,9,15-tetraazabicyclo[9,3,1]pentadeca-1 (15) , 11,13-triene-3,6,9-trimethylenephosphonic acid, 6-(α-carboxyl-4-aminobenzyl)-3,6,9,15-tetraa Zabicyclo[9,3,1]pentadeca-1(15),11,13-triene-3 , 9-dimethylenephosphonic acid, 13-(4-aminobenzyl)-3,6,9,1 5-tetraazabicyclo[9,3,1]pentadeca-1(15),11,13- Triene-3,6,9-trimethylenephosphonic acid, or 6-[(α-phosphonic acid) Nyl-4-aminophenyl)ethyl]-3,6,9,15-tetraazabinclo [9,3,1]pentadeca-1(15),11,13-triene-3,9-dime 17. The compound of claim 16, which is tyrenephosphonic acid. 19. L is the formula ▲There are mathematical formulas, chemical formulas, tables, etc.▼(V) [In the formula, G is hydrogen, NH2, or ▲Contains mathematical formulas, chemical formulas, tables, etc.▼ and R4 is an electrophilic group that can be linked to proteins, and R5 and R6 are independently , hydrogen or -COOH, provided that when G is hydrogen, R5 or One of R6 is COOH, and R7 is hydrogen, hydroxy, or C1- C4 alkoxy and y is 0, 1, 2, 3, or 4, but only If y is 1, 2, 3, or 4, one of R5 or R6 the scope of the claim, with the proviso that 1 compound. 20. 5. The compound of claim 1, wherein the L moiety is covalently attached to GF. 21. z is 1 or 2, and CL is thiourea, thioether, peptide de, ester, disulfide, amide, diester, thioether, hydrocarbon, Acetal glycoside or 4-isothiocyanate phthalic anhydride , the compound of claim 1. 22. L-AP moiety is 1-(α-carboxyl-2-methoxy-5-aminoben zyl)-1,4,7,10-tetraazacyclododecane-4,7,10-trimethane 2. The compound of claim 1 which is tyrene phosphonic acid. 23. L-AP moiety is 1-(4-isothiocyanatebenzyl)diethylene 2. The compound of claim 1 which is lyamine pentamethylene phosphonic acid. 24. GF is a platelet-derived growth factor, insulin-like growth factor, and fibroblast growth factor, transforming growth factor, or cartilage/osteogenic factor, or 24. The compound of claim 23 which is a combination thereof. 25. GF is platelet-derived growth factor, insulin-like growth factor, or 24. The compound of claim 23, wherein is a combination thereof. 26. formula GF-[(CL)z-L-AP]q(I) [wherein, GF, CL, L, AP, z, and q are as defined in claim 1 ] the compound in solid or liquid form, and a pharmaceutically acceptable carrier or is a pharmaceutical preparation, including adjuvants. 27. Supplied as two or more substances in kit form. 27. The formulation of claim 26. 28. 27. The formulation of claim 26, supplied as a single substance. 29. Injured or removed mammals requiring bone regeneration treatment. by direct application or by indirect application to the affected bone area. ,formula GF-[(CL)z-L-AP]q(I) [wherein, GF, CL, L, AP, z, and q are as defined in claim 1 ] in mammals by administering an effective amount of a composition comprising as an active ingredient a compound of How to regenerate bones. 30. The claimed effective amount is about 0.005-50 mg/kg (mammal body weight). Range 29 method. 31. 30. The method of claim 29, wherein more than one compound of Formula I is administered. 32. 30. The method of claim 29, wherein other active compounds are also administered. 33. The method of claim 29 for preventing or reducing further bone loss. .