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EuropSisches Patentamt 
European Patent Office 
Office europden des brevets 

^ Publication number: 0 512 844 A1 


@ Application number : 92304142.0 

@\ni CI ^A61K 47/48 

g) Date of filing : 08.05.92 

(So) Priority : 10.05.91 US 698467 

@ Date of publication of application : 
11.11.92 Bulletin 92/46 

@ Designated Contracting States : 



3055 Patrick Henry Drive 

Santa Clara, CA 95052-8203 (US) 

@ Inventor : Bentz, Hanne 
36125 Toulouse Street 
Newaric California 9450 (US) 
Inventor : Rosen, David 
3141 Valmaine Court 
San Jose, California 95135 (US) 

@ Representative : Gotdin, Douglas Michael et al 
J.A. KEMP & CO. 14, South Square Gray's Inn 
London WC1R 5LX (GB) 

@ Targeted delivery of bone growth factors. 

(57) Methods and compositions for using conju- 
gates of bone growth factors and targeting 
molecules in bone repair and augmentation are 






Jouve, 18, rue Saint-Denis, 75001 PAR^S 


EP 0 512 844 A1 


Technical Field 

This Invention relates to the fields of protein en- 
gineering and medical treatment. More particularly, 
this invention relates to conjugates of bone growth 
factors and targeting molecules and their uses in bone 
repair and augmentation and the like. 

Background of the Invention 

Transfomiing growth factor-p" (TGF-p) repre- 
sents a family of proteins which are evolution aril y 
highly conserved, and which affect a broad spectrum 
of cell types. Two fomis, TGF-p1 and TGF-p2. have 
been identified in platelet releasates and bone. Both 
proteins exist as 26 kD disulfide-linked homodimers. 
Even though there are 14 amino acid differences in 
the first 36 amino acids residues of the two forms, their 
biological activities are similar (Cherfetz et al.. Cell 
(1987) 48:409-415; S. Seyedin et al.. J. Biol. Chem. 
(1987) 262:1946-1949). Although TGF-pi and TGF- 
P2 apparently bind to common cell surface receptors, 
the amino acid sequence homology is only about 
70%. TGF-P3 exhibits about 76% homology with 
TGF-pi, and about 79% homology with TGF-P2. 

Additional TGF-ps have also been described. 
U.S. Patent No. 4,886.747 describes the identification 
of TGF-ps and its nucleotide sequence, and de- 
scribes a method for recovery of TGF-pS from recom- 
binant cell cultures. S.B. Jakowlew et al.. Molec. En- 
docrinol. (1988) 2:1186-1195 describes TGF-p4 and 
its nucleotide sequence, identified by cDNA charac- 
terization. A.B. Roberts et al.. Growth Factors , Vol. 2 
(1990), pp. 135-147. describes the purification of 
TGF-p5 from Xenopus-conditioned medium. 

TGF-p was first identified as a factor which per- 
mitted anchorage-independent grov^h of primary cell 
cultures. In vivo, TGF-p promotes the deposition of 
connective tissue, and induces cell growth for cells of 
mesenchymal origin. TGF-pi and TGF-p2 affect the 
proliferation and differentiation of cells of the immune 
system including macrophages (Wall et al.. Proc. 
Natl. Acad. Sci. U.S.A. (1987) 84:5788). pre-B cells 
(Lee et al.. J. Exp. Med. (1987) 166:1290). hemato- 
poietic stem cells (Ohta et al., Nature (1987) 329:539; 
Ishibashi et al.. Blood (1 987) 69:1737; Keller et al., J. 
Exp. Med. (1988) in press; Sing et al.. Blood (1988) 
in press), and NK cells (Rook et al.. J. Immunol. 
(1986) 136:3916). Bentz et al.. U.S. Patent No. 
4.806,523. disclosed that TGF-p may also be admin- 
istered to suppress Inflammatory responses, includ- 
ing immune suppressk)n. McPherson et al.. U.S. Pa- 
tent No. 4,816.442 disclosed that TGF-p may also be 
administered to suppress hyperproliferation, for ex- 
ample, cancer and leukemia. 

Activins are dimeric proteins structurally similar to 
inhibin. TGF-pi, TGF-p2. and other proteins that 
makeup a family of proteins structurally related to 

TGF-pi. These proteins exhibit the chromatographic 
properties of TGF-ps, In addition to having homology 
with respect to the amino acid sequences, activins ex- 
hibit conservation of cysteine positions characteristic 

5 of the TGF-ps. Activins exhibit a molecular weight of 
25 kD under nonreducing conditions by SDS-PAGE 
(and a molecular weight of 14 kD under reducing con- 
ditions). There are two known forms of the activin sub- 
units, which have been termed pA or pB. Homodime- 

10 ric forms PAA and pBB and a heterodimeric fonm pAB 
have been described in the literature. Activin subunits 
have about a 30% homology to TGF-pi and TGF-P2 
chains in temns of their amino acid sequences. Inhib- 
ins are polypeptides which are also structurally relat- 

15 ed to activins. Inhibins are heterodimers of the activin 
PA or pB subunit and a separate a subuniL Inhibins 
exhibit activity essentially opposite to activin. 

The activin pA homodimer and pAB heterodimer 
have been purified from porcine follicular fluid, and 

20 have been shown to stimulate the release of follicle 
stimulating hormone (FSH) from rat pituitary celts in 
vitro (W. Vale et al.. Nature (1 986) 321 :776-79). Other 
reported activities include stimulation of oxytocin re- 
lease from neurosecretory neurons (P.E. Sawchem- 

25 ko, et al.. Nature (1988) 334:615-17; W. Vale et al., 
"Recent Progress in Hormone Research" (1988) 44:1- 
34); stimulation of insulin secretion from pancreatic is- 
lets (Y. Totsuka et al., Biochem. & Biophys. Res. 
Comm. (1988) 156:335-39); and stimulation of ery- 

30 throid and multipotential progenitor cell colony forma- 
tion in bone marrow culture (J. Yu et al., Nature (1 987) 
330:765-67; H.E. Broxmeyer et al., Proc. Natl. Acad. 
Sci. U.S.A. (1988) 85:9052-56). Activin pA is appa- 
rently identical to erythroid d ifferentiation factor (EDF) 

35 (M. Murata et a!., Proc. Natl.. Acad. Sci. U.S.A. (1988) 

Despite the fact that activin is similar in amino 
acid sequence to TGF-p, activin does not compete 
with TGF-p for binding to TGF-p receptors types I, II. 

40 or IN present on fibroblasts and epithelial cells. How- 
ever, activin has been reported to compete against 
binding of TGF-pi to rat pituitary tumor cells (S. Chei- 
fetz et al.. J. Biol. Chem. (1 988) 263:17225-28). TGF- 
P1 and TGF-P2 have been reported to induce forma- 

45 tion of endochondral bone in vivo (M.E. Joyce et al., 
J. Cell Biol. (1990) 110:2195-2207, H. Bentz. et al. 
(1989) J. Biol. Chem. . 264:20805-10). 

A "bone morphogenetic protein" (BMP) was ex- 
tracted from demineralized bone using urea or guani- 

50 dine hydrochloride and reprecipitated according to 
the disclosures in U.S. Patent Nos. 4,294,753 and 
4,455,256 to Urist. Urist subsequently reported (Urist, 
M. R., Clin. Orthop. Rel. Res. (1 982) 162:21 9) that ion 
exchange purification of this crude protein mixture 

55 yielded an activity which was unadsorbed to carbox- 
ymethyl cellulose resin (CMC) at pH 4.8. Urist's re- 
ports in Science (1983) 220:680-685 and Proc. Natl. 
Acad. Sci. U.S.A. (1984) 81:371-375 describe BMPs 



£P 0 512 844 Ai 


having molecular weights of 17,500 and 18,500 dat- 
tons. Urist's patent publication, EPA Publication No. 
0212474, describes BMP fragments of 4,000 to 7,000 
daltons obtained by limited proteolysis of BMP. 

U.S. Patent No. 4,608.199 describes a bone- 
derived protein of 30,000-32,000 daltons. The protein 
is described as being water soluble and having no af- 
finity for concanavalin A. 

WO 88/00205 reports four proteins, designated 
BMP-1. BMP-2 Class I rBMP-2"), BMP-3. and BMP- 
2 Class II ("BMP-4"). that are alleged to have osteo- 
genic activity. It is not known whether these BMPs 
have osteogenic activity by themselves or require 
combination with other factors. 

J.M. Wozney. in Growth Factor Research. Vol. 1 
(1989), pp: 267-280, describes three additional BMP 
proteins closely related to BMP-2, and which have 
been designated BMP-5. BMP-6 and BMP-7. 

WO 89/09787 and 89/09788 describe a protein 
called "OP-r, now known to be BMP-7. The cloning 
of BMP-7 is described in E. Ozkaynak et al.. EMBO 
Journal (1990) 9:2085-2093, and the purification of 
BMP-7 Is described in T.K. Sampath et al., J. Biol. 
Chem. (1990) 265:13198-13205. 

U.S. Patent No. 4,434,094 to Seyedin and Tho- 
mas reported the partial purification of a bone gener- 
ation-stimulating, bone-derived protein by extraction 
with chaotropic agents, fractionation on anion and ca- 
tion exchange columns, and recovery of the activity 
from a fraction adsorbed to CMC at pH 4.8. This new 
protein fraction was termed "osteogenic factor" (OF) 
and was characterized as having a molecular weight 
below about 30,000 daltons. 

There are several references in the art to proteins 
modified by covalent conjugation to polymers, to alter 
the solubility, antigenicity and biological clearance of 
the protein. For example. U.S. Patent No. 4,261,973 
disclosed the conjugation of several allergens to PEG 
(polyethylene glycol) or PPG (polypropylene glycol) to 
reduce the proteins' immunogenicity. U.S. Patent No. 
4,301,144 disclosed the conjugation of hemoglobin 
with PEG and other polymers to increase the protein's 
oxygen carrying capability. EPO 98,110 disclosed 
coupling an enzyme or interferon to a polyoxy ethy- 
lene- polyoxypropylene (POE-POP) block polymer in- 
creases the protein's halflife in serum. U.S. Patent No. 
4,1 79,337 disclosed conjugating hydrophilic enzymes 
and insulin to PEG or PPG to reduce immunogenicity. 
Davis et al.. Lance t (1 981 ) 2:281-83 disclosed the en- 
zyme uricase nrK}difted by conjugation with PEG to 
provide uric acid metabolism in serum having a long 
halflife and low immunogenicity. Nishida et al., 
Pharm. Phannacol. (1 984) 36:354-55 disclosed PEG- 
uricase conjugates administered orally to chickens, 
demonstrating decreased serum levels of uric acid. 
Inada etal.. Biochem. & Biophys. Res. Comm. (1984) 
122:845-50 disclosed lipoprotein lipase conjugation 
with PEG to render it soluble in organic solvents. Ta- 

kahashi et al., Biochem. & Biophys. Res. Comm. 
(1984) 121:261-65 disclosed HRP conjugated with 
PEG to render the enzyme soluble in benzene. Abu- 
chowski et al., Cancer Biochem. Biophys . (1984) 

5 7:175-86, disclosed that enzymes such as asparagi- 
nase, catalase, uricase. arginase, trypsin, superoxide 
dismutase, adenosine deaminase, phenylalanine am- 
monia-lyase. and the like, conjugated with PEG ex- 
hibit longer half-lives in serum and decreased immu- 

10 nogenicity. U.S. Patent No. 4,830,847. and European 
Patent Application Ser. No. 207,557 disclose diphos- 
phonate-derivatized macromolecules which may be 
labeled with techniunrv99m. 

Both antibodies and liposomes have been used 

IS to direct drugs to target sites (see. for example. Tyle 
and Ram, eds.. Targeted Therapeutic Systems , Mar- 
cel Dekker, New York. 1 990.) 

Targeted delivery of bone growth factors may re- 
duce hamiful or undesirable effects of those mole- 

20 cules. allow the use of tower doses because relatively 
higher doses can be delivered to the site of interest, 
and may prolong the effect at the site of interest. In ad- 
dition, the use of a targeting molecule that influences 
bone metabolism, including bone resorption and for- 

25 matioh, may result in an additive or synergistic effect. 

Summary of the Invention 

It is an object of the invention to provide a conrv 
30 position comprising a bone growth factor and a target- 
ing molecule having affinity for a tissue of interest, 
where the bone growth factor and targeting molecule 
are chemically conjugated to a crosslinker. The cross- 
linker is preferably a synthetic hydrophilic polymer. 
35 The bone growth factor is preferably TGF-p, activin, 
or bone morphogenic protein (BMP). Targeting mole- 
cules preferably have an affinity for bone, such as tet- 
racycline, calcein, bisphosphonate. polyaspartic acid, 
polyglutamic acid, aminophosphosugars. or estro- 
40 gen. 

It is another object of the invention to provide a 
method for augmenting bone formation in a subject. 

It is another object of the invention to provide a 
method for treating bone loss in a subject. 


Modes of Carrying Out The Invention 
A. Definitions 

so The tenm "bone growth factor" refers to the famil- 
ies of proteins which affect bone formation. Examples 
of bone growth factors include transforming growth 
factor-beta (TGF-P), activin. and bone nrarphogenic 
factor (BMP). 

55 The term "TGF-p" refers to beta-type transfonrv 

ing growth factors, including TGF-pl. TGF-P2. TGF- 
p3, TGF-p4, TGF-P5. heterodimers of the TGF-p poly- 
peptide chains (e.g. TGF-pi .2), and fragments there- 



EP 0 512 844 A1 


of, synthetic peptides, and homologous proteins hav- 
ing substantially equivalent biological activity in any of 
the nunnerous known TGF-p assays, such as the one 
described in Methods for Preparation of Media, Sup- 
plenr^ents, and Substrate for Serum-free Animal Cell 
Culture (1984) pp. 181-194. Alan R. Liss, Inc. This 
particular assay determines ability to induce anchor- 
age-dependent growth in no n- neoplastic nomrial rat 
kidney (NRK) fibroblasts by measuring the formation 
of cell colonies in soft agar. Other known TGF-f ac- 
tivity assays include but are not limited to stimulation 
of osteoblast proliferation, inhibition of keratinocyte 
proliferation, stimulation of collagen synthesis in a va- 
riety of cells, inhibition of mitogen-stimulated T-cell 
proliferation, and stimulation of chemotactic activity. 
Preparation of TGF-pi and TGF-p2 is described in 
U.S. Patent No. 4,774,322, incorporated herein by ref- 
erence. Additional TGF-ps have also been described. 
U.S. Patent No. 4,886,747 describes the identification 
of TGF-P3 and its nucleotide sequence, and de- 
scribes a method for recovery of TGF-p from recom- 
binant cell cultures. S. B. Jakowlew et al., Molec. En- 
docrinol. (1988) 2:1186-1195, describes TGF-P4 and 
its nucleotide sequence, identified by cDNA charac- 
terization. A.B. Roberts et al., Growth Factors , Vol. 2 
(1990) pp. 135-147, describes the purification of TGF- 
P5 from Xe no pus- conditioned medium. 

The term "TGF-p" as used herein is also intended 
to include the heterodimer TGF-p2.3, disclosed in co- 
pending patent application U.S. Ser. No. 614,306, 
filed 1 1/1 6/90. TGF-p2.3 may be prepared from an e^t- 
tract of bone by pooling side fractions from peaks of 
column chromatography, subjecting those fractions to 
reverse phase HPLC and recovering those fraction 
which migrate more slowly than TGF-p2 by SDS-PA- 
GE, subjecting those slower migrating fractions to 
FPLC and recovering those that migrate during a pH 
4.6 to 6.7 gradient, subjecting the pH 4.6 to 6.7 eluant 
to reverse phase HPLC or gel electrophoresis, and re- 
covering substantially pure TGF-p2.3. 

The tenm TGF-p" as used herein is also intended 
to include any other synthetic molecule whose mech- 
anism of action is mediated through the TGF-p recep- 
tor or second messenger pathway. 

Because these proteins are non-species-specific 
in their activity they maybe used to treat subjects in 
general, including sport, pet, and farm animals, and 

As used herein, "bone morphogenetic protein" 
(BMP) refers to a class of bone-derived proteins ca- 
pable of inducing bone fonmation, as measured by ac- 
tivity in an in vivo rat bone fonmation assay. BMPs in- 
clude BMP-1, BMP-2, BMP-3, BMP-4. BMP-5. BMP- 
6 and BMP-7, and fragments, deletions, additions, 
substitutions, mutations and modifications thereof 
which retain the biological characteristics of the natu- 
ral BMP. The BMP may be of human, bovine, or other 
species origin. 

The tenm "activin" as used herein refers to activin 
PAA, activin pAB, activin pBB, and fragments thereof, 
synthetic peptides, and proteins having similar activ- 
ity in a standard cell culture assay where the protein 

5 stimulates the release of follicular stimulating hor- 
mone (FSH) from rat pituitary cells (Vale, et al., Nature 
(1986) 321:776). Briefly, in this assay, anterior pitui- 
taries from adult male Sprague-Dawley rats (200-220 
g) are dissociated by collagenase and plated at a con- 

10 centration of 0.33 X 1 0» cells per well in 24 well tissue 
culture dishes. The cells are allowed to recover for 72 
hr in medium containing 2% fetal bovine serum (FBS). 
Following the recovery period, the cells are washed 
twice in fresh medium containing 2% FBS. All treat- 

15 ments are added at this time and the cells are incu- 
bated for 72 hr. The media is then collected and the 
FSH levels are detenmined using a radioimmunoas- 
say kit provided by the National Honmone and Pitui- 
tary program of NIADDK. 

20 As used herein, the term "targeting molecule" re- 
fers to a molecule that binds to the tissue of interest 
or that influences metabolism of that tissue of interest. 
So, for example, bone-targeting molecules may in- 
clude bone-seeking molecules such as tetracycline, 

25 calcein. bisphosphonates. chelators, phosphates, 
polyaspartic acid, polyglutamic acid, aminophospho- 
sugars, peptides known to be associated with the min- 
eral phase of bone such as osteonectin, bone sialo- 
protein and osteopontin, proteins with bone mineral 

30 binding domains, and the like. Bone-targeting mole- 
cules may also include molecules which affect bone 
resorptk)n and bone formation rates, such as bisphos- 
phonates, and estrogens and other steroids. These 
bone-targeting molecules may bring the bone growth 

35 factors to the bone and/or result in a synergistic or ad- 
ditive effect on bone resorption or fonmation. 

The tenm "hydrophilic polymer" as used herein re- 
fers to a synthetic or natural polymer having an aver- 
age molecular weight and composition which renders 

40 the polymer essentially water-soluble. Most hydro- 
philic polymers achieve this property by incorporating 
a sufficient number of oxygen (or less frequently nitro- 
gen) atoms available for fonming hydrogen bonds in 
aqueous solution. Hydrophilic polymers used herein 

45 will generally be propylene glycol, polyoxyethylene, 
polyethylene glycol, polytrimethylene glycols, poly- 
lactic acid, or derivatives thereof. Other suitable poly- 
mers include polyoxyethylene-polyoxypropylene 
block polymers and copolymers. Naturally occurring 

50 polymers such as starch, heparin and the like are also 
included within the scope of this invention. All suitable 
polymers will be non-toxic and non-inflammatory 
when administered subcutaneously. and will prefer- 
ably be essentially non-degradable in vivo over a per- 

55 iod of several months. Presently prefenred hydrophilic 
polymers are synthetic, preferably polyethylene gly- 
cols {PEG) having an average molecular weight be- 
tween about 200 and about 10,000. more preferably 



EP 0 512 844 A1 


between about 600 and about 8,000, and nnost prefer- 
ably about 3400. 

The PEG is preferably difunctional. I.e., a cross- 
linker between the bone growth factor and the target- 
ing molecule, consisting of homobifunctionat groups 5 
such as in PEG 600 diglycidylether, or of heterofunc- 
tional groups. Branched polyfuncttonal PEG cross- 
linkers may also be used. The nature of the functional 
groups on the crosslinker depends on the reactivities 
of the molecules to be conjugated. io 

The term "chemically conjugated" as used herein 
means attached through a covalent chemical bond. In 
the practice of the invention, a hydrophilic polymer 
and a bone growth factor may be chemically conjugat- 
ed by using a linking radical, so that the polymer and is 
the bone growth factor are each bound to the radical, 
but not directly to each other. 

Those of ordinary skill in the art will appreciate 
that polymers such as polyethylene glycol cannot 
practically be prepared having exact molecular 20 
weights, and that the tenm "molecular weight" as used 
herein refers to the average molecular weight of a 
number of molecules in any given sample, as com- 
monly used in the art Thus, a sample of PEG 2000 
might contain polymer molecules ranging in weight 25 
from, for example, 1,200 to 2,500 daltons. Specifica- 
tion of a molecular weight range indicates that the 
average molecular weight may be any value between 
the limits specified, and may include molecules out- 
side those limits. Thus, a molecular weight range of 30 
about 800 to about 20,000 indicates an average mo- 
lecular weight of at least about 800, ranging to about 
20 kD. 

The term "available lysine residue" as used here- 
in refers to lysine side chains exposed on the outer 35 
surface of the bone growth factor or targeting mole- 
cule, which are positioned in a manner allowing reac- 
tion with activated PEG. In general, 10-100%, and 
more preferably, 10-50% of available lysine residues 
may be used. The number of available lysine residues 40 
may be determined by reaction with sodium 2,4,6-tri- 
nitrobenzenesulfonate (TNBS). Bone growth factor 
genetic variants can be constructed so as to substi- 
tute lysine residues at specific sites in the molecule, 
thus improving the efficiency of the crosslinking. In 45 
practice, such substitution will be conservative so as 
not to interfere with the receptor binding of growth fac- 
tor and will be within exterior or more hydrophilic re- 
gions of the growth factor. 

The term "treat" or "treatment" as used herein re- so 
fer to repair, prevention, or alleviation of bone defects, 
especially defects due to loss of bone. Treatment of 
bone defects includes augmentation or restoration of 
lost bone as well as the prevention of further bone loss 
due to metabolic disease, chronic inflammatory proc- ss 
esses (e.g., osteoporosis, osteoarthritis, Paget's dis- 
ease, osteomalacia, osteohalisteresis, multiple mye- 
loma and other fonms of cancer, and age-related loss 

of bone mass). Compositions of the invention may in- 
clude additional biologically active factors to aid in 
healing or regrowth of normal tissue. 

The temn "tissue of interest" as used herein refers 
to a desired target in the body for treatment or for 
placement of the bone growth factor. Tissues of inter- 
est may include bone, cartilage, or other tissues or 
cell types to which bone growth factors may be target- 

B. General Methods 

The compositions of the invention comprise a 
bone growth factor chemically conjugated to a target- 
ing molecule. Examples of chemistries for conjugat- 
ing or coupling proteins have been summarized in the 
art (see, for example, P.Tyle and B. Ram, eds., Tar- 
geted Therapeutic Systems , Marcel Dekker, New 
York, 1990; and Means and Feeney, "Chemical Mod- 
ifications of Proteins, History and Applications," Bio- 
conjugate Chem. 1:2-12 (1990), hereby incorporated 
by reference in their entirety). 

One such method includes chemical conjugation 
of the bone growth factor to a selected synthetic hy- 
drophilic polymer or polymers, which in turn is linked 
to a targeting molecule. Thus, for example, a reactive 
group on the bone growth factor may be conjugated 
through a crosslinker to a polymer, and the targeting 
molecule may be conjugated to the bone growth fac- 
tor-polymer through a second crosslinker, where the 
second crosslinker may be of the same or different 
species as the first, depending on the reactive groups 
involved in the targeting molecule. Examples of reac- 
tive groups on proteins include but are not limited to 
the carboxyl groups of the C-temiinus or of aspartic 
and glutamic acid; amino groups of N-tenminal and ly- 
sine residues; imidazole of histidine and phenolic 
functions of tyrosine; sulfhydryl groups of cysteine; 
and guantdine groups of arginine. 

For example, TGF-p contains a number of avail- 
able amino, carboxyl, and hydroxy groups which may 
be used to bind the synthetic hydrophilic polymer. The 
polymer may be connected using a "linking group", as 
the native hydroxy or amino groups in TGF-p and in 
the polymer frequently require activation before they 
can be linked. One may thus employ compounds such 
as dicarboxylic anhydrides (e.g., glutaric or succinic 
anhydride) to fonm a polymer derivative (e.g., succin- 
ate), which may then be activated by esterification 
with a convenient leaving group, for example, N-hy- 
droxysuccininriide, N,N'-disuccinimidyl oxalate, N,N'- 
disuccinimidyl carbonate, and the like. Presently pre- 
fered dicarboxylic anhydrides that are used to form 
polymer-glutarate compositions include glutaric an- 
hydride, adipic anhydride, 1,8-naphthalene dicarbox- 
ylic anhydride, and 1,4,5,&-naphthalenetetracarbox- 
ytic dian hydride. The polymer thus activated is then 
allowed to react with the TGF-p, fomning a TGF-p- 



EP 0 512 844 A1 


polymer conjugate. 

TGF-p is difficult to dissolve In solutions of appro- 
priate pH for coupling to hydrophilic polymers. In a 
presently preferred method, the TGF-p Is lyophillzed 
in the absence of a carrier protein, and dissolved in a 
mild acid, preferably about 10 mM HCI. The solution 
is neutralized by adding a strong base, preferably 
NaOH (1 N solution) in buffered saline, additionally in- 
cluding a solubillzing amount of DMSO. The final sol- 
ution preferably contains about 50% DMSO or CH3CN 
to solubilize the TGF-p and to prevent aggregation. 

in one embodiment, monomethylpolyethyiene 
glycol (mPEG) (mw 5.000) Is reacted with glutaric an- 
hydride to fonm mPEG glutarate. The glutarate deriv- 
ative is then reacted with N-hydroxysuccinlmide to 
form a succlnimidyl monomethylpolyethyiene glycol 
glutarate. The succlnimidyl ester is then capable of re- 
acting with free amino groups present on a bone 
growth factor (lysine residues) to form a bone growth 
factorPEG conjugate, which may then be further con- 
jugated to a targeting molecule. Other polymers may 
be substituted for the mPEG, as described above. 
Similarly, the coupling reaction may be canrled out us- 
ing any known method for derivatizing proteins and 
synthetic polymers. 

The activated mPEG may be replaced, In whole 
or In part, by bifunctional activated PEG (I.e., non-me- 
thylated PEG which Is then activated at each end), 
thus providing a crosslinked or partially crossllnked 
bone growth factor composition. The character of the 
composition may be adjusted as desired, by varying 
the amount of brfunctbnal PEG included during the 

The resulting bone growth factor-targeting mole- 
cule conjugates may be purified by standard techni- 
ques, preferably by reverse-phase HPLC (RP-HPLC). 
size-exclusion HPLC (SEC-HPLC; e.g., tetrahydro- 
gel-HPLC) or ion-exchange chromatography. RP- 
HPLC is preferably perfonmed using a C1 8 column us- 
ing 90% acetonitrlle or 90% isopropanot with 0.1% tri- 
fluoroacetic acid (TFA) as the B solvent. For SEC- 
HPLC, a preferred running buffer can be 5 mM sodium 
acetate at pH 5.5. 

Whether a homobifu notional group or a heterobi- 
functlonat group is used depends on the nature of the 
reactive groups on the bone growth factor and the tar- 
geting molecule. For example, homoblfunctlonal re- 
agents may have the general formula R-PEG-R. 
where R is glycldylether (e.g.. to form diglycidyl-PEG 
600). succlnimidyl succinate, p-nitrophenytcarbonate 
(e.g., to fomn bis(p-nltrophenylcarbonate) PEG3400, 
imidazoylcarbonyl. and the like. These and similar 
crossi Inkers are available from Signna. Union Carbide, 
and Polyscience. Shorter crosslinkers such as carbo- 
dilmide. not containing the PEG bridging part, have 
well described chemistries In the art and are available 
commercially (e.g.. Pierce). 

The bone growth factor-targeting compositions of 

the invention are preferably administered by parent- 
eral routes, intravenous injection, intranasal or bron- 
chial aerosol, and the like. Sustained release devices 
may be surgically implanted under the skin or within 

5 the peritoneal cavity. 

Phamnaceutical formulations of the Invention 
which include a bone growth factor and a targeting 
molecule for administration will generally include an 
osteogenlcally effective amount of the bone growth 

10 factor to promote bone growth, in addition to a phar- 
maceutical ly acceptable exclplent. Suitable exci- 
pients Include most caniers approved for parenteral 
administration. Including water, saline. Ringer's solu- 
tion, Hank's solution, and solutions of glucose, lac- 

15 tose. dextrose, ethanol, glycerol, albumin, and the 
tike. These compositions may optionally Include sta- 
bilizers, antioxidants, antimicrobials, preservatives, 
buffering agents, surfactants, and other accessory 
additives. The bone growtii factor- targeting composi- 

20 tlon may also be del ivered In a slow release form from 
a suitable carrier. 

A presently preferred vehicle comprises about 1 
mg/ml serum albumin (species-specific) in phos- 
phate-buffered saline (PBS) or Isotonic citrate buffer. 

25 A thorough discussion of suitable vehicles for parent- 
eral administration may be found in E.W. Martin, "Re- 
mington's Phamnaceutical Sciences" (Mack Pub. Co., 
current edition sections relating to the exclplent vehi- 
cles and formulating being incorporated herein by ref- 

30 erence to disclose such). Such fonmulations are gen- 
erally known to those skilled In the art and are admin- 
istered systemlcally to provide systemic treatment. 

The "effective amount" of bone growth factor tar- 
geting composition necessary to effect treatment will 

35 depend upon the growth factor, the nature and sever- 
ity of the condition to be treated, the age and general 
health of the subject, the specific activity of the conrv 
position prepared, and other factors which may be de- 
termined by the practitioner of ordinary skill in the art. 

40 As a general guide, however, an effective amount of 
bone growth factor-targeting molecule conjugate for 
treating a bone loss or defect will range from about 0.1 
^g/kg to about 25 ^g/kg. 

The bone growth factor - targeting compositions 

45 of the invention may be formulated as solutions or 
suspensions, or may be lyophillzed for later reconsth 

C. Examples 


The examples presented below are provided as 
a further guide to the practitioner of ordinary skill in the 
art, and are not to be construed as limiting the inven- 
tion in any way. 



EP 0 512 844 A1 


Example 1 

(TGF-p-PEG-Tetracydine Conjugate Preparation) 

Tetracycline (2 ^mol; Aldrich Chem. Co.) Is dis- 
solved in 1 lAmol bIs-epoxy-PEG (Polyscience) and 
reacted at 90^0 under nitrogen for 24 hr with gentle 
stinrlng. The resulting tetracycllne-PEG conjugate Is 
then reacted with TGF-p2 by adding 100 ^ig TGF-P2 
dissolved in 0.02 M sodium borate, 0.02% SDS. 50% 
acetonitrlle, pH 9.0 to 52 ^l of the tetracycline-PEG 
conjugate. The mixture Is Incubated at C for 48 hr. 
The resulting TGF-p-PEG-tetracydlne conjugates 
are then purified by molecular sieve chromatography 

Example II 

(Conjugation of TGF-p2 to 3-amino-1- 
hydroxypropytidene-1,1-bisphosphonates (ADP) via 
bis-epoxy PEG 600). 

Lyophilized TGF-p Is dissolved In 50% wa- 
ter/acetonitrile (final-concentration 200-500 jig/ml). 
The pH is adjusted to 7-8 with phosphate or borate 
buffer. A 1-20 molar excess of bis-epoxy-PEG 600 or 
tetra-epoxy PEG 1 700 (Polyscience) Is added and the 
mixture is reacted for 15-20 hours at pH 7.0-8.0 at 
40''C. Amicon ultrafiltration may be used to remove 
unattached PEG. The pH Is raised to 9.5 with 1% de- 
oxycholate, pH 9.5, 0.01 N NaOH, orO.1 M borate buf- 
fer. The 3-amlno 1-hydroxypropyIldene 1,1-bisphos- 
phonate (APD. CIBA-GEIGY) is dissolved in 1% de- 
oxycholate, pH 9.5. at 2 mg/ml. The bisphosphonate 
is then added in a 5-200 molar excess and stirred 
overnight at 40*='C. The reaction mixture is acidified 
and filtered through a 0.45 ^m low protein binding syr- 
inge filter prior to chromatography. Purification is per- 
formed by gel filtration. Ion exchange (Mono-S) and 
reverse- phase HPLC. 

Example III 

(Conjugation of TGF-p to 3-amino-1- 
hydroxypropy!idene-1,1-bisphosphonate (APD) 

Lyophilized TGF-p (200 ^g) is dissolved in 0.1 M 
MES (2-(N-Morpholino) ethanesulfonic acid) buffer. 
50% acetonitrlle. at pH 4.7. Sulfo-NHS (N-hydroxysul- 
fosuccininiide; Pierce) is dissolved in aqueous 50% 
acetonitrile and 2-10 ^moles are added to the TGF-p 
solution. The water soluble 1-ethyl-3-(3-dimethylami- 
nopropyl)carbodlimide HCL (EDC. Pierce) Is dis- 
solved in 0.1 MES buffer. pH 4.7. and added in 2-50 
molar amounts. Finally, the bisphosphonate (ADP, 
CIBA-GEIGY) dissolved in 0.1 N HCI (5 mg/100 ^1) is 
added in 2-50 ^molar amounts. The pH Is adjusted to 

4.7-5.5 with 0.01 N NaOH. The mixture is reacted 
overnight at room temperature. The TGF-p/APD con- 
jugate is purified by ion-exchange (Mono-S) chroma- 
tography and reverse-phase HPLC. 


Example IV 

(Conjugation of TGF-p to Tetracycline (Einhorn 


Tetracycline dissolved in methanol is reacted with 
38% fonmaldehyde. TGF-p dissolved in a 1:5 solution 
of water:methanol is added dropwise. The reaction 
mixture is stirred gently for 2 hr at room temperature. 
15 The resulting TGF-p- N-lysinemethyltetracycline can 
be separated from other reaction products by size- 
exclusion and RP-HPLC. (Bernardelli, et al. Liebigs 
Ann. Chem. 706:243-249 (1967)). 

20 Example V 

(Conjugation of TGF-p to Tetracycline) 

Alkoxyalkyltetracydines can be obtained by con- 

25 densing a tetracydine with an aldehyde in alcohol. For 
example, tetracydine, mono-hydroxy-methoxy PEG- 
2000 (MeO-PEG 2000-OH). and fomialin are added 
in equlmolar amounts to a round bottom flask fitted 
with a reflux condenser. The mixture is dissolved in 

30 tert butyl alcohol by heating to approximately SO^'C (oil 
bath). The temperature is increased to 115°C and 
held for 50 hours. This reaction is conducted under ni- 
trogen. The system is then attached to a water aispir- 
ator to remove water from the reaction mixture, after 

35 which the mixture is allowed to cool to room temper- 
ature. Ethanol is added to dissolve the tert butyl alco- 
hol and the mixture is filtered. The remaining wine-red 
precipitate is further purified by acetone extractbn. 
The so-obtained intermediate can be further purified 

40 by ion exchange and size exclusion chromatography. 
After activation, the tetracydine-PEG conjugate can 
then be coupled to the TGF-p, preferably in an organic 
solvent such as methylene chloride, dimethylforma- 
mide. DMSO, and the like. 



1. A composition comprising a bone growth factor 
50 and a targeting molecule having affinity for a tis- 
sue of interest, wherein said bone growth factor 
and targeting molecule are chemically conjugat- 
ed to a crosslinker. 

55 2. A composition according to daim 1 , wherein the 
bone growth factor is TGF-p. 

3. A composition according to daim 1 , wherein the 



EP 0 512 844 A1 


bone growth factor is an activin. 

4. A composition according to claim 1, wherein the 
bone growth factor is a bone nfwrphogenic protein 

5. A composition according to any one of claims 1 to 

4, wherein the crosslinker is polyfunctional. 

6. A composition according to any one of claims 1 to 

5, wherein the crosslinker is a synthetic hydrophil- 
ic polymer, 

7. A composition according to claim 6, wherein said 
polymer is polyethylene glycol. 

8. A composition according to claim 7, wherein said 
polyethylene glycol has a molecular weight of 
about 200 to about 10,000. 

9. A composition according to claim 6, wherein said 
synthetic hydrophilic polymer is bound to an avail- 
able lysine residue on said bone growth factor. 

10. A composition according to claim 6, wherein said 
growth factor has a plurality of available lysine re- 
sidues and molecules of said synthetic hydrophil- 
ic polymer are bound to 10-100% of said available 
lysine residues. 

11. A composition according to claim 6, wherein said 
growth factor has a plurality of available lysine re- 
sidues and molecules of said synthetic hydrophil- 
ic polymer are bound to 10-50% of said available 
lysine residues. 

12. A composition according to any one of the pre- 
ceding claims, wherein said targeting molecule 
has an afTinity for bone. 

13. A composition according to any one of claims 1 to 
1 1 , wherein said targeting molecule is a protein. 

18. A composition according to any one of claims 1 to 
11, wherein said targeting molecule is polyglu- 
tamic acid. 

5 19, A composition according to any one of clainns 1 to 
11, wherein said targeting molecule is an amino- 

20. A composition according to any one of clainns 1 to 
10 11, wherein said targeting molecule is a steroid. 

21. A composition according to any one of claims 1 to 
11, wherein said targeting molecule is an estro- 


22. A composition according to any one of the pre- 
ceding claims which contains a sustained-re- 
lease vehicle. 

20 23. A composition according to any one of the pre- 
ceding claims comprising a pharmaceutically ac- 
ceptable canrier. 

24. A composition according to any one of the pre- 
25 ceding claims for use in a method of treating the 

human or animal body for augmenting the forma- 
tion of mature bone. 

25. A composition according to claim 23 for use in a 
30 method, wherein the bone loss is osteoporosis- 
related or osteomalacia-related or osteohaltster- 
esis-related or age-related or is related to steroid 
therapy or is osteoarthritis- related or is related to 
Pagef s disease or is related to cancer, e.g. mul- 

35 tiple myeloma or wherein the treatment is prophy- 



14. A composition according to any one of claims 1 to 
1 1 , wherein said targeting molecule is tetracy- 45 

1 5. A composition according to any one of claims 1 to 
11, wherein said targeting molecule is calcein. 

1 6. A composition according to any one of claims 1 to 
1 1 , wherein said targeting molecule is a bisphos- 

17. A composition according to any one of claims 1 to 55 
1 1 , wherein said targeting molecule is polyaspart- 
ic acid. 


EP 0 512 844 A1 

European Patent 


Application Nomba 

EP 92 30 4142 



CiUtioD of doauDcnt with indicatioat where appropriate, 
♦f fdcvant passagos 

to daim 



WO-A-9 005 755 (COLLAGEN CORP,) 

* Page 6, line 21 - page 7, line 21; 
page 8, line 15 - page 9, line 25; page 
11, lines 7-34; page 14, line 33 - page 
15, line 29; claim 42 * 

AN=90272939; C.C. JOHNSTON. Jr, : 
"Treatment of osteoporotic patients", & 
REF: 17 

* Whole abstract * 


* Page 5, column 2; page 7, column 3; 
claims 1,25 * 

AN=92163668; C.H. TURNER: "Toward a 
cure for osteoporosis: reversal of 
excessive bone fragility", & OSTEOPOROS 
INT., (1991 OCTOBER), 2(1). 12-9, REF: 

* Whole abstract * 


A 61 K 47/48 



SEARCHED (Int. Cl5) 

A 61 K 

Tbe preseat search report has been drawn up for all claims 


Me of coHpledu of Ikr lemb 




X : particularly rdevam K taken alone 

Y : parttcalarty relevatit If combined with another 

document of the same catcgoiy 
A : technok>glcal background 
O : aon-writlen itisclosDre 
P : intennediatc tfocumeot 

T : theory or principle underlying tbe invention 
E : earlier patent document, hut published on. or 

after tbe filing date 
D : document dted In tbe application 
L : docnment dted for other reasons 

& : menber of tbe same patent family, CDrrespondiog 


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