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J 



EuropSisches Patentamt 
® Oil) European Patent Office © Publication number: 0 128 041 

Office europten des brevets A2 



© EUROPEAN PATENT APPLICATION 

© Application number: 84303782.1 © Int. a 3 : C 07 G 7/00 

^ A 61 K 37/02, C 12 N 5/00 

© Date of filing: 05.06.84 ' 



® Priority: 06.06.83 US 501329 


©Applicant: Baylink, David Jeston 
1534 Fern Avenue West 


© Date of publication of application: 


Redlands California 92373{US) 


12.12.84 Bulletin 84/50 


© Inventor: Baylink, David Jeston 


© Designated Contracting States: 


1534 Fem Avenue West 


Redlands California 923731US) 

© Representative: Harrison, David Christopher et al, 
MEWBURN EUJS & CO 2/3 CursHor Street 
London EC4A1BQIGB) 


AT BE CH DE FR GB IT LI LU NL SE 



© Polypeptides exhibiting skeletal growth factor activity. 



© Polypeptide compositions are provided which when 
introduced into a host enhance bone growth, as well as 
alkaline and acid phosphatase levels in vivo. The polypeptides 
may also be used in diagnostics for prepa ration of reagents or 
antibodies. The polypeptides range in molecular weight from 
about 10,000 to about 200,000dal. 



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Croydon Priming Company Ltd. 



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0128041 



POLYPEPTIDES EXHIBITING SKELETAL GROWTH FACTOR ACTIVITY 

5 For several years, scientists have suspected 

that there was localized regulation of resorption of 
bone and growth of bone cells. See Farley et al. , 
Program and Abstracts of 61st Annual Meeting of the 
Endocrine Society (1979); Howard and Baylink, Clinical 

10 Research (1980) 28:50Ay and Howard et al . f Calcif . 

Tissue Int . (Suppl.) (1980) 31:53 as illustrative. The 
possibility of there being a localized factor which 
controls bone growth and resorption offers the 
opportunity to isolate a material which could be 

15 effective in the therapeutic control of bone growth in 
a wide variety of situations. Such a growth factor 
might be useful in the treatment of osteoporosis, in 
conjunction with bone implants, for the production of 
reagents and antibodies in diagnostic assays, and in 

20 the investigation of bone growth, bone replacement and 
bone deterioration. 

It is therefore of great importance to be 
able to provide compounds capable of being used for 
diagnosis and therapy of bone in the mammalian host. 

25 Description of the Prior Art 

Farley and Baylink, Biochemistry (1982) 
21:3502-3507; and Farley et al. , ibid . (1982) 
21:3508-3513 describe skeletal growth factor from human 
bone, its isolation and characterization. See also, 

30 Baylink and Farley, Abstract of Paper given at the XVII 
European Symposium on Calcified Tissues, April 11-14, 
1983 describing the isolation and partial purification 
of a putative coupling factor from human bone. Other 
abstracts which are cumulative of the above disclosures 

35 include Howard et al . , Calcif . Tissue Int . (Suppl.) 
(1982) 34:S30; Murphy et al . , ibid . (1982) 34:S26; 
Farley et al . , ibid . (1982) 34:S38; and Wergedal and 



0128041 

2 

Baylink, ibid , (1982) 34:516. Urist et al . , Clin. 
Orthopaedics and Rel . Res , (1982) 162 :219-231 and 
Sampath et al. : Role of extracellular matrix in local 
bone induction. In: Current Advances in 
5 Skeletogenesis. Eds. Silbermann and Slavkin, 

Amsterdam, Exerpta medica (1982) describe small factors 
which differ in properties or activities from the 
subject peptides. 

SUMMARY OF THE INVENTION 
10 Novel polypeptides ranging over a wide 

molecular weight range are provided having 
physiological activity in vitro and in vivo as skeletal 
growth factors. The polypeptides are found to enhance 
bone cell growth, while also enhancing levels of bone 
15 and serum alkaline and serum acid phosphatase. The 
compounds can be used to promote the proliferation of 
bone cells, in vitro and in vivo , potentially in the 
treatment of osteoporosis, in treatment to increase 
longitudinal bone growth in children with short 
20 statute, and for the preparation of reagents for use in 
diagnosis of the level of skeletal growth factors in 
serum or other tissue. 

DESCRIPTION OF SPECIFIC EMBODIMENTS 
Novel polypeptides of varying molecular 
25 weights are provided which when administered to bone 
cells induce a number of physiological effects. Bone 
cell proliferation occurs, which in vivo enhances bone 
formation and matrix apposition rate. Osteoclastic 
resorption is increased, as well as an increase in 
30 forming surface. Serum levels of alkaline phosphatase 
and tartrate insensitive acid phosphatase are enhanced, 
which increases are consistent with an increase in 
osteoblast number and bone formation. The polypeptides 
are active with vertebrates, such as mammals and birds, 
35 quadrapeds and bipeds, e.g. humans, rats, chickens, 
etc., as well as with fish. There appears, therefore, 



0128041 

3 

to be a similar mechanism in bone growth regulation 
over a wide spectrum of species. 

The polypeptides include naturally occurring 
polypeptides derived from native bone, analogs of such 
5 polypeptides demonstrating one or more of the 
physiological characteristics of the native 
polypeptides and fragments thereof. 

The subject polypeptides may be divided into 
three molecular weight categories: (1) about 
10 100-200kdal (kilodaltons) , usually 150-200kdal; 
(2) about 80-85kdal; and (3) about 10-20kdal. 

The "high" molecular weight (150-200kdal) or 
large skeletal growth factor ("SGF") may be obtained 
from bone, particularly portions of hip bone, such as 
15 femoral heads or other portions of the hip. The bone 
parts are cleaned, pulverized in liquid nitrogen, 
demineralized and extracted with a chelating agent, 
e.g. ethylenediaminetetraacetic acid (EDTA) , 
conveniently in combination with a biostatic or 
20 biocidal agent. Particularly, from about 5% to 25% 
EDTA with about 0.01 to 0.1% sodium azide may be 
employed, more particularly from about 10% to 20% EDTA 
and from about 0.03 to 0.05% sodium azide. 
Alternatively, the bone can be demineralized with HCl 
25 (0.1 to 1.0M) and extracted with aqueous solution. The 
extracts may then be concentrated and desalted, at 
least in part. Purification procedures then involve 
one or more of the following steps: Chromatography, 
e.g. on Sephadex or DEAE Sephacel; gel filtration, e.g. 
30 on Agarose, particularly Agarose 0.5M; and HPLC ion 
exchange chromatography. 

The large SGF may be characterized by 
polyacrylamide gel electrophoresis (PAGE) under 
non-denaturing conditions, where it migrates as a 
35 single major band with bovine serum albumin tetramer, 
while providing multiple bands in the 50 to 90kdal 
range on SDS PAGE. Also, multiple bands are observed 



0128041 

4 

on isoelectric focusing in the acidic range, pH 4 to 6. 
At about physiologic pH (pH 7.2) the high molecular 
weight SGF binds to hydroxy apatite, QAE cellulose, and 
DE-52, but not CM-cellulose . The large SGF is stable 
to denaturing agents, such as dithiothreitol, urea and 
guanidine-HCl, while losing its activity upon 
hydrolysis with trypsin. 

The "middle" molecular weight or medium SGF 
may be prepared in part as described above, except 
differing in that after treatment with EDTA, the 
dispersion is heated for a short period at an elevated 
temperature, generally in the range of about 75 to 85 °C 
for about 15 to 30min, cooled, and insoluble protein 
separated and discarded. The solution is then 
subjected to acid precipitation (pH -3) , and the acid 
soluble protein neutralized to substantially neutral 
pH, concentrated and chromatographed (gel filtered) on ■ 
Sephadex, e.g. Sephadex G-200. 

The procedure may be found in greater detail 
in Farley and Baylink (1982) , supra . 

The middle molecular weight SGF gives a 
single band that migrates just behind bovine serum 
albumin monomer on PAGE under non-denaturing 
conditions, multiple bands on SDS PAGE in the 50-70kdal 
range and an isoelectric point of 6.5. The middle 
molecular weight SGF is stable to collagenase, 
6-mercaptoethanol and butanol, but loses it activity 
upon treatment with trypsin or urea. 

The "low" molecular weight or small SGF is 
obtained in substantially the same manner as the large 
SGF. The amount of the small SGF may be enhanced by 
preincubation prior to HPLC gel filtration and is found 
to be unbound or weakly bound to DEAE Sephacel and 
binds to hydroxyapatite. This small SGF fraction is 
not observed in the preparation of human SGF, but is 
found in the preparation of bovine and chicken SGF. 
The compositions obtained from bovine and chicken are 



0128041 

5 

believed to contain a protease which results in the 
fragmentation of the large SGF to provide for the small 
SGF fractions. Treatment of human SGF with the 
protease found in other species or modifying the 
5 isolation procedure may provide for the small SGF 
observed with the other species. 

The subject compositions can be used in a 
wide variety of ways f both for diagnosis and therapy; 
For diagnosis r the compounds may be used as antigens 

10 for the production of antibodies specific for the 

particular types of growth factor. The antibodies may 
then find application both in diagnosis and therapy. 
Alternatively, the polypeptides may be used as reagents 
in diagnostic assays, where the polypeptides may be 

15 labeled or unlabeled. Similarly, for use in diagnosis, 
the antibodies may be labeled or unlabeled. A wide 
variety of assays can be employed using one or both, 
the antigen or antibody, either labeled or unlabeled. 
Various labels include fluorescers, enzymes, 

20 chemiluminescers, radionuclides, substrates, cof actors, 
or the like. Assays in which the reagents may be 
employed include hemagglutination, radioimmunoassay, 
enzyme immunoassay, both homogeneous and heterogeneous, 
fluorescence immunoassays, substrate-linked 

25 immunofluorescence assays, or the like. These assays 
may be found in numerous U.S. patents, such as U.S. 
Patent Nos. 3,817,837; 4,133,639; 4,134,792; 4,160,016; 
and 4,172,117. The relevant disclosures of these 
patents are incorporated herein by reference as to the 

30 preparation and use of the various reagents. 

The subject compounds may also be used for 
the study of bone-related cells in the determination of 
available receptors, responsive cells to the presence 
of the SGF, or the like. Toward this end, the subject 

35 compounds can be used in vitro for enhancing the 
proliferation of bone cells responsive to the SGF. 



0128041 

6 

In situations where there is a deficiency of 
SGF in the serum or the patient is suffering from 
osteoporosis or other bone disease or when implanting 
bone # where it is desirable to enhance the bone 
5 proliferation in a particular area, the subject 

compounds can be locally administered or administered 
intraperitoneally, intravenously or subcutaneously. 
Administration of the SGF or antibodies tc SGF may be 
in any convenient formulation in accordance with the 
10 purpose of such administration and the manner of 

administration. Where the protein administered will be 
generally distributed in the blood system, the dosage 
will generally be in the range of about 0.005 to about 
0.5mg/g of host r depending upon the efficiency of the 
15 SGF, duration in the host and the potential for 

localization of the SGF in the host. The SGF may be 
administered neat or as a solution of from about 0.01 
to 10 weight percent in a physiologically acceptable 
carrier, e.g. water, PBS-saline, or the like. 
20 The subject compounds can also be used for 

determining receptor populations of bone cells and to 
modulate the growth rate of bone cells in vitro . 

The following examples are offered by way of 
illustration and not by way of limitation. 
25 EXPERIMENTAL 

The preparation of human skeletal growth 
factor was performed as follows. Human femoral heads, 
(obtained at hip replacement surgery) were frozen in 
liquid nitrogen, crushed with a Wiley mill and 
30 repeatedly rinsed with 25mM phosphate buffer, pH 7.2, 
to remove all contaminating serum and marrow. The 
washed bone fragments were then suspended in a small 
volume of 10% EDTA, 0.04% sodium azide, at pH 7.2 
(about lOmL/lOOg original weight of bone) and dialysed 
35 against the same solution at 4°C. In order to obtain 
the high molecular weight hSGF, the retentate is 
desalted with Sephadex G-25, chromatographed on DE-52 



7 



01 28041 



(DEAE Sephacel) and then subjected to Biogel A (0.5M) 
gel filtration after which time the material is treated 
to HPLC ion exchange chromatography followed by HPLC 
TSK 3000 gel filtration. 
5 The resulting product which is found to have 

a molecular yeight in a range from about 150,000 to 
200,000dal migrates as a single major band on PAGE 
under non-denaturing conditions, migrating with bovine 
. serum albumin tetramer, while multiple bands in the 

10 SOkdal to 90kdal range are observed on SDS PAGE and on 
isoelectric focusing in the acidic range (pH 4 to 6). 
At pH 7.2, the high molecular weight hSGP binds to 
hydroxyapatite, QAE cellulose and DEAE Sephacel, but 
not carboxymethyl cellulose. The large hSGF is stable 

15 to denaturing agents such as dithiothreitol, urea and 
guanidine HC1, while activity is lost upon treatment 
with trypsin. 

The 83kdal hSGF is obtained by somewhat 
different conditions as described in Farley and Baylink 

20 (1982) , supra . 

Bovine skeletal growth factor (bSGF) can be 
prepared from the hip bones from freshly slaughtered 
cows. The bones are mechanically scraped clean of soft 
tissue and cut into 2cm 3 sections. The bone sections 

25 are frozen in liquid nitrogen and ground in a Wiley 
mill to yield a bone powder that is washed with warm 
water to remove fat and serum protein, demineralized 
and extracted using 20% EDTA, 0.04% sodium azide as 
described above. The EDTA extracts are concentrated 

30 and partially desalted by Amiccn ultrafiltration. The 
remaining EDTA is removed by desalting with Sephadex 
G-25. Three alternative methods can be used for 
further purification of the desalted crude extracts. 
(1) Gel filtration on Agarose yields an active bSGF 

35 fraction of 150 to 200kdal. (2) Direct purification of 
the crude extract on DEAE Sephacel yields a large bSGF 
fraction that is modestly bound. In addition, active 



0128041 

8 

bSGF fractions that are unbound or weakly bound to DEAE 
Sephacel are obtained in the range of 10 to 20kdal. 
(3) Purification of desalted crude extract on 
hydroxyapatite yields a non-active fraction that elutes 
5 with 0.15M phosphate in an active fraction that is 

recovered by dissolving the hydroxyapatite support with 
EDTA. The tightly bound fraction yields approximately 
egual amounts of large bSGF and small bSGF. 

The large partially purified bSGF tends to 

10 become small on further manipulation. Evidence 

suggests that an endogenous bone protease is present in 
the bone extracts and is activated during the 
purification process. This protease is evident in the 
bovine bone extracts and as will be indicated 

15 subsequently in chicken extracts, but is not observed 
in the purification of the human extracts. 

Chicken skeletal growth factor (cSGF) is 
prepared from the tibia and femur of adult chickens. 
The bones are mechanically scraped clean of soft 

20 tissue , the cartilage ends are cut off , the bones are 
frozen in dry ice and smashed into 3mm 3 pieces. Serum 
proteins and fat are removed by extensive washing of 
the bone pieces in 0.03M tris (acetate) , 0.15M NaCl, pH 
7.4, with vigorous agitation. The bone is then 

25 subjected to demineralization and extraction with 10% 
EDTA, 0.04% sodium azide. The EDTA extracts are 
concentrated and partially desalted by Amicon 
ultrafiltration and the remaining EDTA removed by 
Sephadex G-25 chromatography. Two approaches are used 

30 to further purify the cSGF. (1) Gel filtration of the 
desalted crude extract on Agarose 0.05M yields 
predominately a large form of cSGF (100 to 200kdal) , as 
well as small quantities of the small ~cSGF in the 10 to 
20kdal range. (2) DE-52 chromatography of the desalted 

35 crude bone extract yields a cSGF fraction that is 

weakly bound to the matrix. The weakly bound fraction 
behaves like large cSGF when chroma tographed on a TSK 



0128041 

9 

3000 HPLC gel filtration column shortly after 
isolation. Preincubation of the weakly bound fraction 
before HPLC gel filtration results in a shift of large 
cSGF to the smaller cSGF. This change in apparent size 
is believed to be a result of the presence of a 
protease which is detected in the fraction weakly bound 
to DE-52. cSGF activity in the desalted crude extracts 
binds to hydroxy apatite, QAE cellulose and DE-52, but 
does not bind to collagen linked to Agarose or to 
carboxymethyl Sepharose. 

Pig, salmon, and rat bones can also be 
processed as described above for the chicken bones. 
The desalted crude extracts from these three species 
causes a substantial increase in cellular proliferation 
when tested on bone cells from chick calvaria. 

The extracts were then employed in a number 
of tests to demonstrate their activity as compared to 
other known growth factors as well as to determine 
their intrinsic activity. 

The first study was a comparison of the SGF 
growth factors with a number of other growth factors. 
(See Table 1.) 



0128041 

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0128041 



References for Table 1 
l a . Mohan et al,, Unpublished observations. 

2 b . Farley, J.R. and Baylink, D.J.: Purification of a skeletal 
growth factor from human bone. Biochemistry (1982) 21 ; 3502-3507. 

3. Cohen, S. and Taylor, J.M.: Epidermal Growth Factor: 
Chemical and biological characterization. Recent Prog . Horm . Res . 
(1974) 30:533-550. 

4. Gospodarowiscz: Purification of a Fibroblast Growth Factor 
from Bovine Pituitary. J. Biol . Chem . (1975) 250(7) :2515-2520. 



5. Lemmon et al ., Bovine Fibroblast Growth Factor: Comparison 
of brain and pituitary preparations, jj. Cell Biol . (1982) 
95:162-169. 

6. Hall, K. and Van Wyk, J.J., Somatomedin In: Current topics 
in experimental endocrinology. James, V.H. and Martin, L. Eds., 
Vol. 2, New York, Academic Press (1974). 

7. Daughaday et al., Studies on rat somatomedin In: 
Somatomedins and growth. Giordano, G., Van Wyk, J.J. and Minuto, 
F. Eds., New York, Academic Press (1979). 

8. Antoniades et al . , Purification of human platelet-derived 
growth factor. Proc . Natl . Acad . Sci. (1979) 76(4) : 1809-1813. 

9. Maciag et al ., High and low molecular weight forms of 
endothelial cell growth factor. J_. Biol . Chem . (1982) 
257(10): 5333-5336. 

10. Burgess et al., Purification and properties of colony 
stimulating factor from mouse lung-conditioned medium. J_. Biol . 
Chem . (1977) 252:1998-2003. 

11. Stanley et al., Factors regulating macrophage production and 
growth. Identity of colony-stimulating factor and macrophage 
growth factor. J. Exp . Med . (1976) 143:631-647. 

12. Koroly, M.J. and Young, M., Nerve growth factor In: Tissue 
Growth Factor. Baserga, R. Ed., Handbook of Experimental 
Pharmacology, Vol. 57, New York, Springer Verlag (1981). 

13. Todaro et al., Sarcoma growth factor and other transforming 
peptides produced by human cells: Interactions with membrane 
receptors. Fed. Proc . (1982) 41 (13): 2996-3003. 

14. Ozanne et al., Cells transformed by KNA and DNA tumor viruses 
produce transforming factors. Fed. Proc . (1982) 41 (13) :3004-3007. 



0128041 

13 

The above Table 1 compares a number of 
physical properties of the various growth factors 
showing similarities and differences between the 
various growth factors. 

In the next study , the SGF was tested in 
combination with a number of other growth factors or 
mitogens on 3T3 cells using 3 H-thymidine incorporation 
as a measure of cell proliferation. The following 
Table 2 indicates the results. 



Table 2: Effects of Combination of hSGF with Other 
Polypeptide Growth Factors 



% of hSGF Activity 
Factor -hSGF +hSGF Significance 



Control 


9.4 


+ 


2. 


4 


100.0 


+ 


19 


p<0.001 


EGF (lOng/ml) 


13.7 


+ 


3. 


0 


92.0 


+ 


27 


p<0.001 


FGF (lOng/ml) 


70.9 


+ 


13. 


2 


136.6 


+ 


46.6 


p<0.01 


IGF (25ng/ml) 


30.2 


+ 


4. 


6 


84.3 


+ 


13.7 


p<0.001 


PDGF(20ng/ml) 


34.8 




2. 


3 


75.3 


+ 


9.9 


p<0.001 



3T3 cells were plated at 70,000 cells/well in DMEM in 24 
well culture dishes. 24 hours after plating, the factors 
were added and incubated for an additional 24 hours. The 
cells were then pulse labeled for 2 hours with 2pCi of 
^-TdK and TCA precipitable radioactivity was quant it ated. 
Incorporation is expressed as % of maximum incorporation 
stimulated by hSGF alone and is the average of 6 repli- 
cates. 



Comparisons are made between the maximum 
stimulation of the same cells with hSGF alone to the 
maximum stimulation by the four mitogens in the 
presence and absence of hSGF. hSGF alone stimulates 
cell proliferation seven-fold greater than EGF, 



01 28041 



14 

three-fold greater than IGF-I and PDGF, and 40% greater 
than FGF. In addition, the maximum stimulation of the 
four mitogens was significantly elevated by the 
addition of hSGF. These results indicate that hSGF is 
5 non-competitive and different from the four mitogens 
tested. 

In a similar experiment, stimulation of chick 
embryonic calvarial cell proliferation by fetal calf 
serum at different serum concentrations (0.05%, 0.1%, 

10 0.5%, 1.0%, and 2.0%) was significantly elevated by the 
addition of hSGF. The maximum stimulatory effect of 
serum (1050% ± 133% of control with 1.0% added serum) 
increased by 27% (p<0.001) in the presence of hSGF. 

The above results suggest that hSGF is 

15 distinct from the other known growth factors that were 
tested and from the growth promoting agents in the 
serum (polypeptide growth factors as well as 
f ibronectin and transferrin) . 

Experiments were carried out in vivo with 

20 rats. Hats were given intraperitoneal ly daily 

injections of bovine bone extract containing 2.5mg of 
protein for a total of ten days. Biochemical and 
quantitative histological comparisons were made between 
the control rats and rats treated with bovine bone 

25 extract. ~ The results of this experiment are summarized 
in Table 3. 



15 



0128041 



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0128041 

17 

This experiment was performed with five rats 
in the control group and three rats in the treated 
group. Serum calcium and final body weight were not 
altered by the treatment. The femur alkaline 
5 phosphatase level, a probable index of bone formation, 
and the tartrate insensitive acid phosphatase, a 
possible index of osteoclastic cell number, were both 
increased in the treated rats. Quantitative 
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10 results of the enzymatic analysis. Tibia periosteal 
bone formation as weli as the matrix apposition rate 
were clearly increased in the treated rats. Tibia 
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was also increased in the treated rats. In addition, 

15 histological analysis of the vertebral sections gave a 
highly significant increase in forming surface of the 
treated rats. 

The next study was a more detailed study on 
the effects of bovine bone extract in vivo , in this 

20 study, ten rats were used in both the control and 
treated groups. (See Table 4.) 



0128041 



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0128041 

20 

Serum calcium and body weight did not change 
with treatment. However, both serum alkaline 
phosphatase (p<0.001) and serum phosphate (p<0.05) were 
significantly increased in the treated rats. The 
levels of alkaline phosphatase in the femur, skull and 
sternum of treated rats were elevated (p<0.001). 
Tartrate insensitive acid phosphatase was also 
increased in the femur, and skull (p<0.001), but not the 
sternum. The results of the alkaline phosphatase 
analysis in the femur, skull and sternum are consistent 
with an increase in osteoblast number and bone 
formation. The alkaline phosphatase data agrees very 
well with the quantitative histology of the tibiae of 
treated rats, where a 60% increase (p<0.001) in the 
periosteal bone formation (mm 3 ) and a 40% increase in 
the matrix apposition rate (pM/day) were observed. 



Table 5: Experiment 2 - Quantitative Histological 

Comparisons of Tibiae Sections from Control Rats 
and Rats Treated vith Bovine Bone Extract 



a I 

Parameter Control Treated p 



Tibiae Periosteal Bone 0.«(±0.02) 0.72(±0.04) <0.001 
Formation (mm 2 ) 

Tibiae Periosteal Bone 8.58(+0.33) 12.07(10.51) <0.001 
Apposition Rate (uM/day) 



a All data reported as mean t S.E.M. and based on 
observations from at least 10 rats in each group. 

b Comparisons of treated versus control were by the 
students T test. 



The increase in tartrate sensitive acid 
phosphatase in the femur and skull is consistent with 
an increase in bone resorption. 



-21- 



0128041 



A low molecular weight SGF (approximately 11 
kdal) was isolated and purified from both human and 
bovine bones , as follows. Human femoral heads obtained 
during hip replacement surgery and bovine bone (pelvis 
5 and femur) obtained from a local abattoir were cleaned 
to remove the adhering soft tissue, cut into small 
pieces with a band saw, washed with water to remove 
marrow and fat, ground in a Wiley mill, washed with 
deionized water containing protease inhibitors to 

10 remove blood, and then treated with 4M guanidine 
hydrochloride (pH 7.4) with protease inhibitors to 
extract the soluble cellular constituents, serum 
proteins and soluble collagen. The resulting human 
bone residues were then demineralized, and the 

15 noncollagenous proteins extracted 4-5 times with 10% 
EDTA containing 4M guanidine hydrochloride and protease 
inhibitors (EDTA-guanidine) . 

Purification of the 11 kdal SGF from the 
human bone EDTA guanidine extracts was as follows. The 

20 extracts were subjected to hydroxylapatite 

chromatography which, in the presence of 4M guanidine, 
will pass the SGF while binding other noncollagenous 
proteins. The unbound fraction from the 
hydroxylapatite chromatography was then subjected to 

25 high pressure liquid chromatography (HPLC) on a 

preparative gel filtration column. Using 4M guanidine 
hydrochloride in 30mM Tris acetate (pH 7.4) as an 
eluent, it was found that the 11 kdal SGF eluted after 
myoglobin (17.5 kdal) and before aproteinin (6.2 kdal). 

30 The HPLC fraction containing the 11 kdal SGF was then 
desalted and purified by HPLC reverse phase chroma- 
tography using a silica-based C^-bonded phase column 
which separates proteins based on their hydrophobicity. 
Proteins bound to the silica were eluted by increasing 

35 the concentration of acetonitrile in the eluent 



0128041 

-22- 

(water:trifluoroacetic acidracetonitrile) . The above 
extraction ana purification procedures are specifically 
for the human skeletal growth factor. Purification of 
the bovine skeletal growth factor involved only minor 
5 modifications of the procedure used for the human 
factor. 

The purity of the 11 kdal human SGF vas 
measured at each stage of purification, and the results 
are set forth in Table 6 below. 



10 



Table 6: Human Skeletal Growth Factor Purification Table 



Step 



Protein Specific Activity 

(Mg) Activity Recovered 

(Units/pg Protein) (Z) 



Guanidine EDTA 322 

Hydroxylapatite 168 
(Fast Flow) 

15HPLC Gel 43.5 
Filtration 

HPLC Reverse 1.86 
Phase 1 

HPLC Reverse 0.21 
Phase II 



14 

25.5 
75.0 
945 
4000 



100 
95 

72 

39 

18.6 



360 g of human femoral head bones vere used as starting 
material. Activity of SGF vas assayed by its effect on bone cell 
20 Proliferation. SGF stimulates the incorporation of H-Thymidine 
(Tdr) into TCA precipitate material in embryonic chick calvaria 
cells. 100Z increase in "TI-Tdr incorporation over unstimulated 
control was defined as one activity unit. 

The homogeneity of both the human and bovine 
25 11 kdal SGF fractions was demonstrated by the following 
observations. The SGF was shown to elute as a single 
symmetric peak upon HPLC reverse phase separation. The 
SGF was shown to migrate as a single band upon 
polyacrylamide-sodium deodecyl sulfate (SDS)-urea gel 



0128041 

-23- 

electrophoresis which effects separation based on 
molecular weight. The SGF was also shown to migrate as 
a single band upon electrophoresis using a 5-15% 
polyacrylamide gradient gel where separation is 
5 effected based on differences in molecular size. 
Finally, the SGF was shown to migrate as a single band 
upon isoelectric focusing on a 7.5% polyacrylamide gel 
where separation depends on differences in the isoelec- 
tric points. Thus, the material in the 11 kdal SGF 
10 fraction was shown to have uniform molecular weight, 
molecular- size, and isoelectric point. 

The molecular weight of the purified SGF 
species was determined to be 11±1 kdal by polyacryl- 
amide-SDS-ufea gel electrophoresis and HPLC gel fil- 
15 tration using 4M guanidine hydrochloride (pH 7.4) as an 
eluent. The large molecular weight (83 kdal) species 
of SGF can be dissociated into the 11 kdal species and 
a carrier protein in the presence of 4M guanidine 
hydrochloride. The isoelectric point of the 11 kdal 
20 sgf was determined to be 6.810.2, and was shown to be 
sensitive to reducing agents such as dithiothreitol. 
The 11 kdal SGF was shown to be sensitive to trypsin, 
but resistant to treatment with collagenase and with 
acid (0.1% trifluoroacetic acid, pH 2.5), 6M urea, and 
25 4M guanidine hydrochloride. 

The 11 kdal SGF was shown to be a very potent 
mitogen for bone cells with a half-maximum activity 

reached at 2 to 3 ng/rol. The mitogenic activity was 

3 

assayed by observing the incorporation of H-Tdr into 
30TCA precipitable material (DNA) in embryonic chick 
calvarial (bone) cells. hSFG at 0.1 ng/ml, the 
activity (expressed as a percentage incorporation of 
H-Tdr to that of untreated control) was approximately 
150%. The activity reached a maximum of approximately 
35 400% at between 5 and 10 ng/ml. The maximum activity 
did not further increase as the concentration was 
increased to 100 ng/ml. bSFG showed similar 



0128041 

-24- 

activities. Preliminary studies of the amino acid 
composition of bovine SGF indicate that the 11 kdal SGF 
is rich in aspartic acid/ glutamic acid, leucine, and 
proline. The amino acid analysis is set forth in Table 
5 7 below. 



Table 7: Amino Acid Analysis of Bovine SGF (Residues/Molecule) 





Lysine 


6 




Hlstidine 


3 




Arginine 


3 


10 


Aspartic Acid 


9 




Threonine 


3 




Serine 


5 




Glutamic Acid 


9 




Proline 


6 


15 


Glycine 


5 




Alanine 


4 




Valine 


5 




Isoleucine 


4 




Leucine 


7 


20 


Tyrosine 


2 




Phenylalanine 


3 


Total No. 


of Amino Acid Residues 


75 



Comparisons of the molecular weight, chemical 
characteristics, and amino acid composition of the 11 kdal 

25 SGF with other known polypeptide growth factors indicate 
that the 11 kdal SGF is a unique mitogen different from 
other known growth factors* 

The 11 kdal SGF has also been demonstrated to 
enhance bone formation in vivo in each of four separate 

30 experiments with ten rats per group in each experiment. 
Eleven kdal SGF which had been purified by EDTA extraction 
and hydroxylapatite chromatography from a bovine bone 
extract was administered to groups of rats by daily intra- 
peritoneal injection for a period of 10 days. Control rats 

35 received BSA injections, and (in order to assess the rate of 
bone formation) all animals were injected with tetracycline 
on the first and tenth days. Tetracycline is a highly 
fluorescent substance that tightly binds to bone as it 



0128041 

-25- 

mineralizes. Thus, only skeletal surfaces that are 
undergoing bone formation become fluorescent. Animals were 
sacrificed after the tenth day, and one tibia from each rat 
was histologically evaluated for bone formation, including 
5 measurements of surface fluorescence. A portion of the 

other tibia was extracted with a butanol solution to release 
alkaline phosphatase from the plasma membranes of the 
osteoblasts. Alkaline phosphatase is regarded as a specific 
marker for the osteoblasts which are bone forming cells. 

10 The amount of tetracycline labelled bone surface 

was increased in the tibiae of rats that received daily 
injections of the 11 kdal SGF compared to the BSA controls. 
The percentage of increase varied from a low of approxi- 
mately 140% of control for one experiment to a high of 

15 approximately 240% of control for another experiment. The 
rate of bone formation on the external (periosteal) surface 
of the tibia was shown to be greater in rats injected with 
the 11 kdal SGF than in controls. The skeletal alkaline 
phosphatase activity in the bone samples taken from rats 

20 treated with 11 kdal SGF were also shown to be increased 
from a low approximately 140% in one experiment to a high of 
approximately 210% in another experiment. Taken together, 
these results clearly demonstrate that the 11 kdal SGF is a 
potent mitogen capable of systemically enhancing bone 

25 formation in mammals. 



0128041 



-26- 

It is evident from the above results that the 
novel skeletal growth factors provided by the subject 
invention can be used for a multitude of purposes in 
enhancing the proliferation of bone cells, both in vivo 
and in vitro , diagnosing for the presence of these 
skeletal growth factors in serum or other sites, and in 
the treatment of various bone diseases, where there is 
a need for stimulation of bone growth. 

Although the foregoing invention has been 
described in some detail by way of illustration and 
example for purposes of clarity of understanding, it 
will be obvious that certain changes and modifications 
may be practiced. 



0128041 

-27- 



CLAIMS: 



1. A vertebrate skeletal growth factor of 
from about 100,000 to 200,000dal or fragment thereof, 
capable of stimulating a proliferation of bone cells. 

2. A skeletal growth factor according to 
Claim 1, wherein said vertebrate is mammal, and said 
growth factor is of from about 150,000 to 200,000dal. 

3. A skeletal growth factor according to 
Claim 1, wherein said mammal is human and said skeletal 
growth factor is of from about 150,000 to 200,000dal. 

4. A skeletal growth factor according to 
Claim 1, wherein said skeletal growth factor is a 
fragment of from about 10,000 to 20,000dal. 

5. A skeletal growth factor according to 
Claim 1, wherein said vertebrate is human. 

6. A skeletal growth factor according to 
Claim 1, wherein said vertebrate is bovine. 

7. A skeletal growth factor according to 
Claim 1, wherein said vertebrate is chicken. 

8. A method for enhancing the proliferation 
of bone, said method comprising: 

contacting bone cells with an effective 
amount of a skeletal growth factor according to Claim 
1. 

9. A method according to Claim 8, wherein 
said vertebrate is human. 



10. Antibodies capable of binding to a 
vertebrate skeletal growth factor according to Claim 1. 



0128041 

-28- 

11. Antibodies according to Claim 10, 
conjugated with a label capable of providing a 
detectable signal. 

12. A method for determining the presence of 
a vertebrate skeletal growth factor according to Claim 
1, which comprises: 

combining a sample suspected of containing 
said skeletal growth factor with an antibody according 
to Claim 10, and determining the occurrence of binding 
of said antibody to said skeletal growth factor. 



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