Skip to main content

Full text of "USPTO Patents Application 09804625"

See other formats


WORLD INTELLECTUAL PROPERTY ORGANIZATION 
International Bureau 




PCT 

INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) 



(51) International Patent Classification 6 : 
C12N 5/08, A61K 38/18 



Al 



(11) International Publication Number: WO 98/31788 

(43) International Publication Date: 23 July 1998 (23.07.98) 



(21) International Application Number: PCT/US98/0H43 

(22) International Filing Date: 21 January 1998 (21.01.98) 



(30) Priority Data: 

60/035,865 
09/009,085 



21 January 1997 (21.01.97) US 
20 January 1998 (20.01.98) US 



(71) Applicant: GENETICS INSTITUTE, INC. [US/US]; 87 Cam- 

bridgePark Drive, Cambridge, MA 02140 (US). 

(72) Inventors: VALENTIN-OPRAN, Alexandre; 72 Rutledge 

Road, Belmont, MA 02178 (US). TRIPPEL, Stephen, B.; 
59 Woodland Road, Newton, MA 02166 (US). WOZNEY, 
John, M.; 59 Old Bolton Road, Hudson, MA 01749 (US). 
SEEHERMAN, Howard; 307 Cabot Street, Newtonville, 
MA 02160 (US). TUREK, Thomas; 65 Bearse Avenue, 
Boston, MA 02124 (US). 

(74) Agent: LAZAR, Steven, R.; Genetics Institute, Inc., 87 
CambridgePark Drive, Cambridge, MA 02140 (US). 



(81) Designated States: AL, AM, AT, AU, AZ, BA, BB, BG, BR, 
BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, Fl, GB, GE, 
GH, GM, GW, HU, ID, IL, IS, JP, KE, KG, KP, KR, KZ, 
LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, 
MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, 
TM, TR, IT, UA, UG, UZ, VN, YU, ZW, ARIPO patent 
(GH, GM, KE, LS, MW, SD, SZ, UG, ZW), Eurasian patent 
(AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), European patent 
(AT, BE, CH, DE, DK, ES, Fl, FR, GB, GR, IE, IT, LU, 
MC, NL, PT, SE), OAPI patent (BF, BJ, CF, CG, CI, CM, 
GA, GN, ML, MR, NE, SN, TD, TG). 



Published 

With international search report. 

Before the expiration of the time limit for amending the 
claims and to be republished in the event of the receipt of 
amendments. 



(54) Title: INJECTABLE FORMULATIONS FOR TREATMENT OF OSTEOPOROTIC BONE 



(57) Abstract 



Methods and compositions are provided for the treatment of defects and disease involving osteoporosis, or osteopenic conditions. 
The methods comprise applying to the site of osteoporotic or osteopenic conditions a composition comprising an active agent, preferably 
from the TGF-/? superfamily of proteins. The advantages of the invention include a reduction or avoidance of the severity and/or incidence 
of bone fractures. Also, the methods of the present invention are advantageous in that administration is local, rather than systemic. 



FOR THE PURPOSES OF INFORMATION ONLY 



Codes used to identify States party to the PCT on the front pages of pamphlets publishing international applications under the PCT. 



AL 


Albania 


ES 


Spain 


LS 


Lesotho 


SI 


Slovenia 


AM 


Armenia 


FI 


Finland 


LT 


Lithuania 


SK 


Slovakia 


AT 


Austria 


FR 


France 


LU 


Luxembourg 


SN 


Senegal 


AU 


Australia 


GA 


Gabon 


LV 


Latvia 


sz 


Swaziland 


AZ 


Azerbaijan 


GB 


United Kingdom 


MC 


Monaco 


TD 


Chad 


BA 


Bosnia and Herzegovina 


GE 


Georgia 


MD 


Republic of Moldova 


TG 


Togo 


BB 


Barbados 


GH 


Ghana 


MG 


Madagascar 


TJ 


Tajikistan 


BE 


Belgium 


GN 


Guinea 


MK 


The former Yugoslav 


TM 


Turkmenistan 


BF 


Burkina Faso 


GR 


Greece 




Republic of Macedonia 


TR 


Turkey 


BG 


Bulgaria 


HU 


Hungary 


ML 


Mali 


TT 


Trinidad and Tobago 


BJ 


Benin 


IE 


Ireland 


MN 


Mongolia 


UA 


Ukraine 


BR 


Brazil 


IL 


Israel 


MR 


Mauritania 


UG 


Uganda 


BY 


Belarus 


IS 


Iceland 


MW 


Malawi 


US 


United States of America 


CA 


Canada 


IT 


Italy 


MX 


Mexico 


UZ 


Uzbekistan 


CF 


Central African Republic 


JP 


Japan 


NE 


Niger 


VN 


Viet Nam 


CG 


Congo 


KE 


Kenya 


NL 


Netherlands 


YU 


Yugoslavia 


CH 


Switzerland 


KG 


Kyrgyzstan 


NO 


Norway 


ZW 


Zimbabwe 


CI 


Cdie d'lvoire 


KP 


Democratic People's 


NZ 


New Zealand 






CM 


Cameroon 




Republic of Korea 


PL 


Poland 






CN 


China 


KR 


Republic of Korea 


PT 


Portugal 






CU 


Cuba 


KZ 


Kazaksian 


RO 


Romania 






CZ 


Czech Republic 


LC 


Saint Lucia 


RU 


Russian Federation 






DE 


Germany 


U 


Liechtenstein 


SD 


Sudan 






DK 


Denmark 


LK 


Sri Lanka 


SE 


Sweden 






EE 


Estonia 


LR 


Liberia 


SG 


Singapore 







WO 98/31788 



PCT/US98/01143 



INJECTABLE FORMULATIONS FOR TREATMENT OF 
OSTEOPOROTIC BONE 

FIELD OF THE INVENTION 

5 The present invention relates to the field of tissue repair, specifically, to the 

treatment of osteoporotic bone and/or prevention of osteoporosis. Osteoporotic or 
osteopenic bone is often characterized by suboptimal bone density. The osteoporotic 
condition may be related to diet, trauma or stress, or to degenerative or congenital 
disease. Thus, the present invention may be useful in the treatment and/or the 
10 prevention of osteoporosis, or the treatment of osteoporotic or osteopenic bone. 

BACKGROUND OF THE INVENTION 
Background of the incidence and etiology of need: Idiopathic osteoporosis is a 
disease of unknown etiology characterized by progressive loss of bone mass and 
increased fragility. It is a medical problem because it is associated with a marked 
15 increase in susceptibility to fractures. It is a public health problem for several reasons. 
First, it is among the most prevalent of all musculoskeletal disorders. Fifty-six percent 
of women over 45 years of age are afflicted. Praemer et ai, Musculoskeletal 
Conditions in the United States, American Academy of Orthopaedic Surgeons, Park 
Ridge, IL (1992). Second, its incidence increases with age. Because the percentage 
20 of elderly in the population is increasing, osteoporosis will become more common 
with time. Third, osteoporosis presently has no known cure, and is difficult to treat 
locally. Fourth, it is a major economic burden to individuals and to society. Fifth, and 
most significantly, osteoporosis is associated with a substantial morbidity and 
mortality. The most serious fracture resulting from osteoporosis is that the of the 
25 proximal femur in the region of the hip joint. With an annual incidence of 250,000, 
hip fractures are currently the most common fracture in the elderly. Praemer et aL 
Ibid. Estimates based on census projections indicate that this figure will increase to 
approximately 340,000 hip fractures per year by the year 2000. One out of every six 
Caucasian women will have a hip fracture during her lifetime (Cummings et al, Arch 
30 Intern Med 749:2455-2458 (1989), and for those who attain the age of 90, this figure 
becomes one in three. 



1 



WO 98/31788 



PCT/US98/0U43 



5 Of the patients who are independent and living at home at the time of hip 

fracture, approximately 20 percent remain in a long term care institution for at least 
one year following the fracture. During the first year following injury, the mortality 
rate is approximately 15% higher than for age and gender matched controls. Praemer 
et ai, Ibid. The financial toll of these fractures is also high. In 1988, hip fractures 

10 cost the United States 8.7 billion dollars. Praemer et al t Ibid, The increased 
incidence of proximal femur fracture observed in elderly patients is mainly related to 
two factors: (1) a decreased bone density of their proximal femora; (2) an increased 
propensity to fall. There is an inverse relationship between the age-related bone loss 
in the proximal femur and the risk of hip fracture. Each decrease of one standard 

15 deviation (SD) in femoral neck bone density increases the age-adjusted risk of hip 
fracture 2.6 times (95% CI 1.9 - 3.6); and (3) women with bone density in the lowest 
quartile have an 8.5-fold greater risk of hip fracture than those in the highest quartile. 
Cummings et ai, The Lancet 341:12-15 (1993). This relation between hip bone mass 
and hip fracture risk allows the screening and identification of patients at risk for 

20 fracture. Patients who are two standard deviations below peak hip bone mass have 
passed beneath the "fracture threshold." 

Bioactive agents that reduce the incidence of hip fractures or augment the 
healing of such fractures when they occur would substantially improve the health of 
the elderly. The availability of an injectable agent would permit its use in fracture 

25 prevention, such as hip fracture, without the costs and morbidity of surgical 
intervention, such as for femoral neck fractures. Such an agent would have 
application in fracture treatment, without increasing the extent or complexity of the 
surgical management. The inventors have provided a novel method using bioactive 
agents to decrease the occurrence and/or severity of fractures to osteoporotic bone, 

30 such as an osteoporotic hip. 

While several therapies for osteoporosis have been approved, there is none that 
addresses the localized treatment of proximal femur osteoporosis. Current therapies 
for osteoporosis are systemic. These include fluoride, bisphosphonates, calcitonin, 
estrogens and progestins, testosterone, vitamin D metabolites, and/or calcium. In the 

35 United States, only estrogens and alendronate, a bisphosphonate, are indicated for the 



2 



WO 98/31788 



PCT/US98/01143 



5 prevention of hip fractures in postmenopausal osteoporotic women. Each of these 
agents requires continuous administration over a time period of years. Estrogen has 
undesirable side effects (WHO Study Group on Assessment of Fracture Risk and its 
Application to Screening for Postmenopausal Osteoporosis. WHO Technical Report 
Series 843, Assessment of fracture risk and its application to screening for 

10 postmenopausal osteoporosis. Geneva, World Health Organization (1992)), 
compliance is poor, and it is not indicated in males. Alendronate requires carefully 
separated administration of the drug and sources of calcium and is not recommended 
in patients with reduced renal function. A recent study found a reduction in hip 
fractures in elderly women with involutional osteoporosis after eighteen months of 

15 vitamin D and calcium (Chapuy et ai New Engl J. Med. 527:1637-1642 (1992)), but 
this treatment has not been conformed by independent sources, and lacks United 
States regulatory approval. While several treatments have effectively arrested or 
decreased bone loss, only the use of fluoride has been associated with rapid increases 
in bone density. However, fluoride treatment increased bone density less in the 

20 femoral region as compared to the spine and was associated with poor bone quality. 
Hedlund et aL J. Bone Min. Res. 4:223-225 (1989). Fluoride treatment again has not 
received regulatory approval. Thus, no safe, effective, generally applicable local 
treatment is available which protects against the occurrence of hip fractures in 
osteoporosis by providing a reliable and durable increase in hip bone mass. 

25 Accordingly, despite substantial endeavors in this field, there remains a need for an 
effective method of repair and/or treatment of osteoporotic and osteopenic bone, and 
for treatment and/or prevention of osteoporosis. 

SUMMARY OF THE INVENTION 

30 Accordingly, the present invention provides methods and compositions for 

increasing bone mass and quality, and for minimizing or reducing the incidence or 
severity of osteoporosis-related fractures. Accordingly, the present invention provides 
methods and compositions useful for decreasing the incidence of fractures of 
osteoporotic or osteopenic bone. In particular, the present invention comprises 

35 methods of treating patients with osteoporosis, or with other evidence of osteoporosis 



3 



WO 98/31788 



PCT/US98/01143 



5 or osteopenic condition. Preferred embodiments where the present invention may 
prove particularly useful include treatment of metaphyseal bone, including proximal 
femur (hip), proximal humerus (upper arm), distal radius (wrist), and vertebral bodies 
(spine), particularly the vertebral body. 

- The method comprises administering to a site of osteopenic or osteoporotic 
10 bone, or a site of low bone mass or density, an effective amount of a composition 
comprising at least one active agent which is capable of inducing growth of bone or 
increasing the formation of bone tissue or reducing bone loss at the site. Bone mass 
is commonly designated "bone mineral content" or BMC and is measured in grams. 
Bone density is commonly designated "bone mineral density" or BMD and is 
15 expressed as grams per unit area or grams per unit volume. In a preferred 
embodiment, the mode of administration is by intraosseous injection. In preferred 
embodiments, the active agent is one or more proteins selected from the group of 
proteins known as the Transforming Growth Factors-Beta (TGF-p) superfamily of 
proteins, preferably selected from the Bone Morphogenetic Proteins (BMPs), the 
20 Growth and Differentiation Factors (GDFs), as well as other proteins, as described 
more fully herein. The methods and compositions of the present invention are 
advantageous in that they provide a localized treatment for osteoporosis or osteopenic 
bone, rather than systemic treatment. The present invention is further advantageous 
in that it utilizes as active agents osteogenic proteins, which may be produced via 
25 recombinant DNA technology, and therefore are of potentially unlimited supply. The 
methods and compositions of the present invention are further advantageous in that 
regeneration of the bone tissue increases the bone mass/density, increase the bone 
strength, and thereby reduce the severity of osteoporosis or incidence of osteoporotic 
lesions, ultimately lessening the incidence of bone fractures. 
30 In a preferred embodiment of the present invention, the active agents are 

administered locally through injection using a suitable buffer and/or carrier. One 
suitable buffer comprises glycine, sucrose, and glutamic acid hydrochloride, at a pH 
of less than 6.0. In a preferred embodiment of the invention, this formulation 
comprises about 2.5% glycine (g/ 100 ml (w/v)), about 0.5% sucrose (w/v), about 5 
35 mM glutamic acid hydrochloride (about 0.1% w/v), and about 0.01% (w/v) 



4 



WO 98/31788 



PCT/US98/01143 



5 polysorbate 80, at a pH of about 4.5. This buffer is referred to later in this application 
as "MFR 842." Further buffers suitable for use in the present invention are described 
in United State Patent 5,385,887, the disclosure of which is hereby incorporated by 
reference. Suitable carriers include collagen gels, hyaluronate, alginates and 
hyaluronic acids, injectable calcium phosphates, polyols, demineralized bone matrix 

10 and combinations of the above. Other carriers which may be useful for the present 
invention include blood as well as clotting proteins, such as fibrin or thrombin, and 
oils. 

In other preferred embodiments, the active agent further comprises, in addition 
to one or more proteins selected from the TGF-P superfamily of proteins, one or more 
15 auxiliary proteins, such as Hedgehog, Noggin, Chordin, Frazzled, Cerberus and 
Follistatin, soluble BMP receptors, or other protein or agent, as described further 
herein. 

In addition to healing of osteoporotic bone, compositions of the present 
invention may be useful for injectable formulations of BMPs for uses such as injection 
20 into joints for treatment and repair of osseous defects, cartilage defects, inhibition of 
cartilage degradation and to promote cartilage repair. The formulations may also be 
injected into tendons, ligaments and/or their attachment sites to bone. Injectable 
formulations of BMPs may also find application to other bone sites such as bone cysts, 
implants into bones, closed fractures and distraction osteogenesis. 

25 

DETAILED DESCRIPTION OF THE INVENTION 

According to the present invention, methods and compositions are provided 
for treatment of patients who exhibit signs of osteoporosis, or osteopenic conditions, 
including osteoporotic bone lesions. The identification of such patients may be 

30 accomplished by procedures which are well known in the art. These procedures 
include measurement of bone mass/density using dual-energy X-ray absorptiometry 
(DEXA), Kilgus et aL, J. Bone & Joint Surgery . 75-B:279-287 (1992); Markel et al., 
Acta Orthop Scand , 61:487-498 (1990); and quantitative computed tomography 
(QCT), Laval-Jeantet et al., J Comput Assist Tomogr , 17:915-921 (1993); Markel, 

35 Calcif Tissue Int , 49:427-432 (1991); single-photon absorptiometry, Markel et al. 



5 



WO 98/31788 



PCT/US98/01143 



5 Calcif Tissue Int . 48:392-399 ( 1991 ); ultrasound transmission velocity (UTV); Heaney 
et al., JAMA , 261 :2986-2990 (1989); Langton et al. f Clin Phvs Phvsiol Meas , 1 1 :243- 
249 (1990); and radiographic assessment, Gluer et al., J Bone & Mineral Res , 9:67 1 - 
677 (1994). Other methods of identification of patients at risk of bone fracture include 
assessment of age-related factors, such as cognisance, as well as prior occurrence of 

10 osteoporosis-related fractures. Porter et al., BMJ . 301 : 638-641 (1990); Hui et al., J 
Clin Invest , 81:1804-1809 (1988). The above publications are hereby incorporated 
by reference herein. 

The methods comprise applying to the osteoporotic or osteopenic site an 
amount of a composition comprising one or more purified osteogenic proteins which 

15 is effective to induce the formation and/or maintenance of bone. 

ACTIVE AGENT 

The active agent is preferably selected from the family of proteins known as 
the transforming growth factors-beta (TGF-P) superfamily of proteins, which includes 

20 the activins, inhibins and bone morphogenetic proteins (BMPs). Most preferably, the 
active agent includes at least one protein selected from the subclass of proteins known 
generally as BMPs, which have been disclosed to have osteogenic activity, and other 
growth and differentiation type activities. These BMPs include BMP proteins BMP-2, 
BMP-3, BMP-4, BMP-5, BMP-6 and BMP-7, disclosed for instance in United States 

25 Patents 5,108,922; 5,013,649; 5,1 16,738; 5,106,748; 5,187,076; and 5,141,905; BMP- 
8, disclosed in PCT publication WO91/18098; and BMP-9, disclosed in PCT 
publication WO93/00432, BMP- 10, disclosed in PCT application W094/26893; 
BMP-11, disclosed in PCT application' W094/26892, or BMP- 12 or BMP- 13, 
disclosed in PCT application WO 95/16035; BMP-15,BMP-15, disclosed in United 

30 States Patent 5,635,372; or BMP- 1 6, disclosed in co-pending patent application, serial 
no. 08/715,202, filed on September 18, 1996. Other TGF-P proteins which may be 
useful as the active agent in the present invention include Vgr-2, Jones et al., Mol. 
Endocrinol. , 6:1961-1968 (1992), and any of the growth and differentiation factors 
[GDFs], including those described in PCT applications W094/15965; W094/15949; 

35 WO95/01 801 ; WO95/01 802; W094/2 168 1 ; W094/1 5966; WO95/10539; 



6 



WO 98/31788 



PCT/US98/01143 



5 WO96/01 845; WO96/02559 and others. Also useful in the present invention may be 
BIP, disclosed in WO94/01557; HP00269, disclosed in IP Publication number: 
7-250688; and MP52, disclosed in PCT application WO93/16099. The disclosures 
of all of the above applications are hereby incorporated by reference. A subset of 
BMPs which are presently preferred for use in the present invention include BMP-2, 

10 BMP-4, BMP-5, BMP-6, BMP-7, BMP-10, BMP-12 and BMP-13. The active agent 
is most preferably BMP-2, the sequence of which is disclosed in United States Patent 
5,013,649, the disclosure of which is hereby incorporated by reference. Other BMPs 
and TGF-P proteins known in the art can also be used. 

The active agent may be recombinantly produced, or purified from a protein 

15 composition. The active agent, if a TGF-P such as a BMP, or other dimeric protein, 
may be homodimeric, or may be heterodimeric with other BMPs (e.g., a heterodimer 
composed of one monomer each of BMP-2 and BMP-6) or with other members of the 
TGF-P superfamily, such as activins, inhibins and TGF-pl (e.g., a heterodimer 
composed of one monomer each of a BMP and a related member of the TGF-P 

20 superfamily). Examples of such heterodimeric proteins are described for example in 
Published PCT Patent Application WO 93/09229, the specification of which is hereby 
incorporated herein by reference. 

The active agent may further comprise additional agents such as the Hedgehog, 
Frazzled, Chordin, Noggin, Cerberus and Follistatin proteins. These families of 

25 proteins are generally described in Sasai et al., CeH, 79:779-790 (1994) (Chordin)\ 
PCT Patent Publication WO94/05800 (Noggin); and Fukui et al., Developmental 
Biology , 159:131-139 (1993) {Follistatin). Hedgehog proteins are described in 
W096/16668; W096/17924; and W095/18856. The Frazzled family of proteins is 
a recently discovered family of proteins with high homology to the extracellular 

30 binding domain of the receptor protein family known as Frizzled. The Frizzled family 
of genes and proteins is described in Wang et al., J. Biol. Chem. , 271:4468-4476 
(1996). The active agent may also include other -soluble receptors, such as the 
truncated soluble receptors disclosed in PCT patent publication WO95/07982. From 
the teaching of WO95/07982, one skilled in the art will recognize that truncated 

35 soluble receptors can be prepared for numerous other receptor proteins. Such would 



7 



WO 98/31788 



PCI7US98/01143 



5 also be encompassed within the present invention. The above publications are hereby 

incorporated by reference herein. 

The amount of active agent useful herein is that amount effective to stimulate 

increased osteogenic activity of present or infiltrating progenitor or other cells, and 

will depend upon the size and nature of the defect being treated, as well as the carrier 
10 being employed. Generally, the amount of protein to be delivered is in a range of from 

about 0.1 to about 100 mg; preferably about 1 to about 100 mg; most preferably about 

10 to about 80 mg. 

CARRIER 

Materials which may be useful as the carrier in practicing the present invention 

1 5 include pharmaceutical^ acceptable materials having viscosity and polarity such that, 
when added to the bone morphogenetic protein, form a composition that possesses 
appropriate handling characteristics for injectable application to the site of 
osteoporotic or osteopenic bone. Adding the carrier to the bone morphogenetic 
protein allows the protein to remain in the diseased or lesioned site for a time 

20 sufficient to allow the protein to increase the otherwise natural rate of regenerative 
osteogenic activity of the infiltrating mammalian progenitor or other cells, and to form 
a space in which new tissue can grow and allow for ingrowth of cells. The carrier may 
also allow the bone morphogenetic protein to be released from the disease or lesion 
site over a time interval appropriate for optimally increasing the rate of regenerative 

25 osteogenic activity of the progenitor cells. The carrier may also supply a framework 
on which to induce new formation in severely osteoporotic bone. 

The most preferred family of carriers comprises collagenous materials. These 
are preferably in a form suitable for injection, such as a gel. Such gels may be cross- 
linked or non-cross-linked. Other forms of collagen, such as dispersions or fibrillar 

30 collagen, may also be useful in the methods of the present invention. Another 
preferred family of carriers is cellulosic materials such as alkylcellulose, including 
hydroxyalkylcellulose, methylcellulose, ethylcellulose, hydroxyethylcellulose, 
hydroxypropylcellulose, hydroxypropylmethylcellulose, and carboxymethylcellulose, 
the most preferred being the cationic salts of carboxymethylcellulose (CMC). 



8 



WO 98/31788 



PCT/US98/01143 



5 In the case of cellulosic carriers and collagen gels, it is preferred that the 

carrier be in the form of a hydrated cellulosic viscous gel. Viscosity may be increased 
through mechanical means, such as high agitation for a suitable period of time, 
followed by autoclaving, or chemically. The active agent and cellulosic carrier is 
preferably in a solution of suitable buffer. One preferred buffer solution is a 

10 composition comprising, in addition to the active agent, about 1.0 to about 10.0% 
(w/v) glycine, about 0.1 to about 5.0% (w/v) of a sugar, preferably sucrose, about 1 
to about 20 mM glutamic acid hydrochloride, and optionally about 0.01 to about 0.1% 
of a non-ionic surfactant, such as polysorbate 80. Preferred solutions are from about 
1% to about 20% w/v cellulosic carrier/buffer. If desired, a salt may be added. A 

15 preferred viscous gel carrier is described in Example 2 below. The amount of 
osteogenic protein useful with viscous gel carrier is generally in a range of from about 
0.1 to about 100 mg, preferably about 1 to about 100 mg; most preferably about 10 
to about 80 mg per cubic centimeter of implant material required. 

Another class of materials of particular interest for injectable carriers are 

20 resorbable hydroxyapatites as well as minerals, ceramics and phosphates. Resorbable 
hydroxyapatites, for example, can be formulated at various porosities with varying 
resorption rates; their handling characteristics vary from hard implantable types, to 
gel-like consistency, to those that are injectable but harden at body temperature. 
Suitable hydroxyapatite and ceramic carriers are described, for example in 

25 W096/36562; and United States Patents 5,543,019; 5,306,305; 5,258,044; 5,496,399; 
5,455,231; 5,336,264; 5,178,845; 5,053,212; 5,047,031; 5,129,905; 5,034,059; 
4,880,610; 5,290,763; and 5,563,124; the disclosures of which are incorporated herein 
by reference. 

Another preferred family of carriers for administration of the active agent of 
30 the present invention are injectable polymers, which may be viscous and which may 
optionally include a sequestering agent as well. Suitable polymers and sequestering 
agents include those described in United States Patent 5,171,579, the entire disclosure 
of which is incorporated herein by reference. Other polymers include the pluronics, 
such as Poloxamer 407 gel. Pluronics are a class of water soluble ABA type block 
35 surfactant copolymers which exhibit the unique property of reverse thermal gelation. 



9 



WO 98/31788 



PCT/US98/01143 



5 They are liquid (and hence syringeable) at 4°C and gel at body temperature. 
Poloxamer 407, MW 12,500, is excreted unchanged in the urine after systemic 
absorption and has supposedly been shown to be non-toxic in animals. Polylactides 
and/or polyethylene glycols, including poly(lactide)/poly(ethylene glycol) gels. 
Polylactides may be dissolved in polyethylene glycols, such as low molecular weight 

10 (2000) PLA dissolved in PEG to produce a syringeable solution that precipitates PLA 
upon injection into an aqueous environment, resulting in a relatively firm gel. In 
addition, the literature cites conjugates, such as Poly(lactic acid)-poly(ethylene glycol) 
conjugates, as appropriate carriers for BMPs (Miyamoto et al t Clin. Orthop. Rel. Res. 
294:333 (1993)). Also useful as the carriers are fibrin-based polymers, in liquid or gel 

15 form. Among the materials useful as sequestering agents are hyaluronic acid, sodium 
alginate, poly(ethylene glycol), polyoxyethylene oxide, carboxyvinyl polymer and 
polyvinyl alcohol), and cellulosic materials, such as hydroxycelluloses. One such 
preferred agent is carboxymethylcellulose. 

-The above materials disclosed to be useful as sequestering agents may 

20 themselves be useftil as carriers for injection. In addition, combinations of the above 
described materials may be used. 

It is also possible to use as injectable carriers solid materials which are nanrow 
enough to be administered through injection. In such cases, the solid material may be 
combined with a viscous liquid carrier for ease and uniformity of administration. 

25 In cases where the carrier may be of higher viscosity than optimal, the carrier 

may optionally be combined with a diluent, such as aqueous glycerol, preferably the 
carrier diluent would be present in concentrations of about 10 to about 80% (v/v). 
Also, the above materials may be combined in particular embodiments of the present 
invention. For example, polymers, such as porous particulate polymers, may be 

30 dissolved or suspended in cellulosic or gel carriers to increase viscosity. 

In a preferred embodiment of the present invention, the active agents are 
administered locally through injection using only a suitable buffer as carrier. One 
suitable buffer comprises glycine, sucrose, and glutamic acid hydrochloride, at a pH 
of less than 6.0. Preferred compositions of buffer solutions comprise about 1.0 to 

35 about 10.0% (w/v) glycine, about 0.1 to about 5.0% (w/v) of a sugar, preferably 



10 



WO 98/31788 



PCT/US98/01143 



5 sucrose, about 1 to about 20 mM glutamine, glutamic acid, or glutamic acid 
hydrochloride, and optionally about 0.01 to about 0.1 % of a non-ionic surfactant, such 
as polysorbate 80. In a preferred embodiment of the invention, this formulation 
comprises about 2.5% glycine (g/100 ml (w/v)), about 0.5% sucrose (w/v), about 5 
mM glutamic acid hydrochloride (about 0.1% w/v), and about 0.01% (w/v) 

10 polysorbate 80, at a pH of about 4.5. This buffer has been described as MFR 842. 
Further buffers suitable for use in the present invention are described in United State 
Patent 5,385,887, the disclosure of which is hereby incorporated by reference. 
Preferred solutions may also include combinations of buffer and other carrier, such as 
a combination of buffer and cellulosic carrier. Preferred ranges for this combination 

15 are from about 1% to about 20% w/v cellulosic carrier/buffer. If desired, a salt may 
be added. 

MODE OF ADMINISTRATION 

The injectable compositions of the present invention may be administered in 
any clinically acceptable manner of injection. A number of commercially available 

20 syringes may be suitable for use in the present invention, and for administration of the 
compositions of the present invention. For example, suitable syringes are available 
the Calasept R syringe (JS Dental Manufacturing, Ridgefield CT] comprises sterile 
calcium hydroxide paste in isotonic saline solution, in a non-aspirating or modified 
aspirating cartridge syringe; Henke-Ject Aspirating Syringe and Hypo Brand Dental 

25 Syringes/Needles [Smith & Nephew MPL, Franklin Park, IL]; intraosseous needles 
from MPL, Inc., Chicago DL; and Luer-Lok R Syringes {Becton Dickinson, Franklin 
Lakes, NJ], may all be appropriate syringes for use in the present invention. 

In another embodiment of the present invention, bone morphogenetic proteins 
are used as an osteoinductive agent in the process known as distraction osteogensis. 

30 This process is an alternative to segmental bone regeneration in response to implanted 
osteoinductive agents. In traditional segmental bone repair, the osteoinductive agent 
and carrier are placed in the defect created between the parent bone ends. For bone 
formation to occur, the osteoinductive agent has to have sufficient residence time in 
the defect to stimulate differentiation of sufficient numbers of bone forming cells to 

35 support new bone formation. The carrier also has to have sufficient residence time to 



11 



AVO 98/31788 



PCT/US98/01143 



5 serve as a scaffold for bone forming cells to attach (osteoconduction). In addition, the 
carrier also has to have sufficient porosity to allow cells and blood vessels to penetrate 
into the defect for bone formation to occur. In contrast, the process of distraction 
osteogenesis creates a regenerate construct between the distracted parent bone ends 
that is highly vascular and contains a large population of mesenchymal stem cells 

10 destined to become bone forming cells. As a result, the regenerate construct 
represents a much more ideal environment for cell differentiation growth factors such 
rhBMP-2 to stimulate rapid bone induction relative to induction of bone within a 
segmental defect. 

The process of distraction osteogenesis begins with an initial latency period 

15 allowing a Fibrous connection to form between the bone ends to be distracted. 
Following this latency period, the bone ends are slowly distracted at a controlled rate 
of up to 1 mm per day in human clinical cases. Once the regenerate forms and the 
bone ends are distracted to the appropriate length, a prolonged consolidation period 
is required to allow the regenerate to form bone. This prolonged consolidation period 

20 which can be on the order 4 to 6 months is associated with considerable morbidity. 
A frequent complication is the occurrence of pin track infections resulting from the 
extended length of time the external Fixator used to generate the distraction must 
remain in place. In addition, there are considerable psychological effects and life style 
alterations associated with wearing the external Fixator for prolonged periods of time. 

25 In addition to complications associated with the external Fixator, there are a number 
of patients where the regenerate does not form properly and a delayed union or 
non-union occurs. Since the regenerate contains a responsive cell population and is 
already highly vascularized following the initial distraction phase, the use of bone 
morphogenetic proteins may rapidly accelerate the rate of bone formation during the 

30 normally prolonged consolidation phase of distraction osteogenesis. Acceleration of 
the distraction phase is limited by stretching of the soft tissues associated with bone. 
The cells created using distraction osteogenesis may also be harvested in order to 
provide a source of cells which are primed for osteogenesis. These cells can be 
cultured to prepare immortalized cell lines. If desired, these cells can also be 

35 immunotolerized using agents such as CTLA4 receptors [U.S. Patent 5,434,131] or 



12 



WO 98/31788 



PCT/US98/01143 



5 CTLA4 ligands or B7 monoclonal antibodies [WO 96/40915]. Methods and materials 
for such immunotolerization are disclosed in the above references, and include co- 
transfection or treatment with these factors. The disclosure of these references is 
hereby incorporated herein by reference. 

The following examples further describe the practice of embodiments of the 

1 0 invention with rhBMP-2 in a buffer carrier. The examples are not limiting, and as will 
be appreciated by those skilled in the art, can be varied in accordance with the above 
specification. 

EXAMPLES 
15 Example 1: Oophorectomized Rat Model: 

The oophorectomized rat is a well-established model of osteoporosis and its use is 
recommended by the FDA as one of the animal models to be employed in 
demonstrating efficacy of potential therapeutic agents for osteoporosis. Preclinical 
studies testing the efficacy of rhBMP-2 in the oophorectomized rat model of 

20 osteoporosis demonstrated that 100 wg rhBMP-2 in MFR 842 buffer, injected into the 
intramedullary space of the tibial shaft, was associated with extensive bone formation. 
Further evaluation with this model is conducted at lower doses. 

In a similar study, five orariectomized rats were injected with 40 ug of rhBMP- 
2 in MFR 842 buffer solution into one side of the femoral neck, and MFR 842 buffer 

25 alone on the other side. After six weeks, the femora are harvested, analyzed by DEXA 
for bone density and by histology. 

In a larger study, six weeks after ovariectomy, rat tibiae are injected with 0, 10, 
or 50 «g of rhBMP-2 in 20 uL MFR 842 buffer. Controls include sham 
ovariectomized animals [surgery performed, but ovaries are not removed], buffer 

30 injections and sham surgeries. After six weeks, the femora are harvested and groups 
analyzed by DEXA, biomechanics, and histomorphometry. 

Example 2. Sheep Core Decompression Model 

Preclinical studies in support of the application of rhBMP-2 to the treatment 
35 of osteonecrosis of the hip at the time of core decompression demonstrated that 



13 



WO 98/31788 



PCT/US98/01143 



5 rhBMP-2 stimulates bone formation in the core decompression track within the 
femoral neck of normal (non-osteoporotic) sheep. In this study, defects in both the 
femoral head/neck and distal femur were created, the latter to facilitate DEXA 
imaging. rhBMP-2 in various formulations, including buffer, blood, blood clot and 
collagen dispersions was placed at the site of defect. 

10 Example 3. Human Core Decompression Study. 

Patients with osteonecrosis of the femoral head were treated by core 
decompression with or without implantation of blood clot containing rhBMP-2 into 
the core track. Patients who received rhBMP-2 experienced a reduction in the volume 
of necrotic bone compared to patients treated by core decompression alone. The 

15 treatment was well tolerated with complications limited to some bone formation 
adjacent to the cortical entry site. 
Example 4. Biodistribution of rhBMP-2. 

Four aged ewes underwent surgical placement of a cannulated needle into the 
femoral neck using uniplanar fluoroscopic guidance. A bolus injection of 

20 radiolabeled rhBMP-2 in buffer solution was delivered through the needle into the 
proximal femur. Local (bone) distribution of rhBMP-2 was monitored by gamma 
camera. Systemic distribution was assessed by periodic serum sampling. In vivo 
gamma camera images of the femur indicate that the injected rhBMP-2 enters the 
femoral head and neck and that part of the injected material is retained at these sites 

25 for at least 72 hours. Serum analysis of radiolabeled rhBMP-2 indicates that the 
majority (-85%) of the rhBMP-2 enters the systemic circulation within five minutes 
of administration. 

Example 5. Intra-Femoral Injections. 

Patients with osteoporosis who have sustained a fracture of the proximal femur 
30 are identified. Intraosseous administration of injectable rhBMP-2 is accomplished in 
the non-fractured (contralateral) femur using an operating room, biplanar fluoroscopy, 
and patient positioning on a fracture table. Each patient undergoes the necessary 
surgical treatment of the fractured femur. At the completion of surgical procedure, but 
before termination of the anesthesia, the lower extremities are repositioned on the 
35 fracture table to gain access to the uninjured contralateral hip. Under sterile 



14 



WO 98/31788 



PCT/US98/01143 



5 conditions and biplanar image intensifier visualization, the designated dose of rhBMP- 
2 is delivered by percutaneous, intraosseous route into the proximal femur. Follow-up 
evaluations include radiographic assessment, clinical evaluation of infection and 
fracture, and DEXA evidence of bone formation. 
Example 6: Rabbit Ulnar Osteotomy and Fracture Models. 

10 In the osteotomy model, the ulna is simply exposed, the periosteum is stripped, 

and a 2 to 5 mm osteotomy created using an oscillating saw. The incision is then 
closed, and the appendage bandaged to provide additional stability. Bilateral surgeries 
may be used in order to compare active agent against untreated or control substances. 
The rabbit ulnar fracture model is similar to the rat femoral fracture model. 

15 The rabbit ulnar fracture model relies on a weight-driven blunt instrument to create 
the fracture essentially using incident three-point bending. The fracture is created by 
fully extending and clamping the forelimb to an aluminum rod containing two vertical 
bars. In a modification from the rat apparatus, a 23G needle is placed in between the 
radius and ulna to serve as a guide pin. The blunt guillotine is then positioned, and 

20 a 850 gm weight raised to 45 cm and allowed to fall to create the fracture. 

Assessment of efficacy in both the rat femoral and rabbit ulnar fracture models 
can be accomplished primarily using biomechanics. Qualitative measures of fracture 
repair, such as radiology, can be obtained, but it is difficult to see subtle differences 
over normal fracture repair, and will not give a convincing demonstration that the 

25 injection is translating to accelerated fracture repair. 
Example 7: Rat Ectopic Implant Models 

In one experiment, several non-collagenous materials were tested in the rat ectopic 
implant model. The materials included in this study were: 
1. MFR 842 buffer; 
30 2. Poloxamer 407 gel. 

3. PoIy(lactide)/poly(ethylene glycol) gels. Low molecular weight (2000) 
PLA dissolved in PEG produces a syringeable solution that precipitates PLA upon 
injection into an aqueous environment, resulting in a relatively firm gel Two different 
percentages were tested. 
35 4. Poly(lactic acid)-poly(ethylene glycol) conjugates. 



15 



WO 98/31788 



PCT/US98/01143 



5 Each material was implanted with 0, 10, and 80 |jg rhBMP-2. Lyophilized 

rhBMP-2 was mixed with the gels using a mortar and pestle. The buffer, Pluronic 
gels, and PEG/PLA gels were first injected into blood clots. The PLA-PEG 
conjugates were implanted directly into the subcutaneous space. Six implants (three 
animals) were used per group. In addition, the intramuscular injection technique was 

10 investigated using the PLA/PEG gel and one Pluronic gel (both at the high dose only). 

The above were scored for bone formation. Bone score is a semi-quantitative 
measurement of the area of bone present in representative histological sections; the 
score ranges from 0 for no bone present, to 5 when the entire section consists of bone. 
Coupled with the wet weights and qualitative histological assessment of the sections, 

15 the following conclusions can be made. The Pluronics produced relatively little bone 
at the lower dose of rhBMP-2, and induced none when placed intramuscularly. 
Presumably the material does not have a long enough residence time, or does not 
sequester rhBMP-2 sufficiently. In addition, a significant inflammatory response was 
noted. The PLA/PEG gels produced reasonable quantities of bone, with some residual 

20 matrix observed (presumably the precipitated PLA) along with a foreign body giant 
cell response. The PLA-PEG conjugates produced well-defined ossicles of bone with 
bone marrow. Of the gels tested, this material performed best. Based on the samples 
tested, the intramuscular implantation appeared to be a more rigorous test of materials. 
In this experiment, rhBMP-2 in buffer simply injected into blood clot performed the 

25 best overall; presumably the rhBMP-2 is able to rapidly distribute and bind to the 
blood clot. 

In another rat ectopic study, several cross-linked and non-cross-linked collagen 
materials were tested. In addition, porosity was introduced into the PLA/PEG gels by 
the addition of MFR 842 buffer as an excipient, and a lower percentage of PLA was 
30 tested. Some of the collagen materials were tested in an aqueous (MFR 842) or non- 
aqueous (PLA/PEG gel) environment. The collagen materials tested included: 

1. Homogenized Helistat® bovine type I collagen cross-linked hemostatic 
sponge (currently in use with rhBMP-2 clinically) made into a suspension; 

2. Homogenized Helitene™ (fibrillar form). Only tested with PLA/PEG in this 
35 experiment; 



16 



WO 98/31788 



PCT/US98/01143 



5 3. Collagen dispersion (Integra LifeSciences, Plainsboro, N J). The non-cross- 

linked parent material of Helistat®. At the concentration tested <0.78%, as provided), 
it is not viscous at all. 

4. Zimmer collagen gel. The collagen component of the Collagraft® kit. 
All materials were tested with 10 \xg rhBMP-2 using the blood clot injection 

1 0 method. The samples were scored for histologic bone formation. The materials which 
performed best in this study were the Helistat® dispersion and the collagen gel, 
yielding bone scores of 3 to 4, and no adverse tissue reaction. All PLA/PEG 
containing materials showed much smaller amounts of bone, and significant 
inflammatory response (non-specific foreign body type). Addition of PLA/PEG to the 

15 collagen material descreased the bone formation observed. In this experiment, the 
rhBMP-2 in buffer placed in blood clot perform poorly; this may be because a larger 
gauge needle was used to place the material, and it flowed back along the needle track 
before it could distribute in the blood clot. Likewise, the DLS dispersion performed 
poorly, perhaps due to low viscosity. These results indicate relatively little promise 

20 for the PLA/PEG gels, unless use of very low molecular weight PLA accelerates its 
resorption time. The collagen materials stand out above the other materials tested to 
date using this rat ectopic assay system. 

Various collagen materials tested include 2% ILS dispersion, 2% Helitene™ 
dispersion, 2% Helistat® dispersion, the Zimmer collagen gel, and another collagen 

25 gel (6.5%) from Matrix Pharmaceuticals. All have been placed at 0, 20, and 80 pg 
rhBMP-2 and analyzed histologically. 

Example 8: Distraction Osteogenesis 

BMP-2 may accelerate the consolidation (bone formation or mineralization) 

30 phase in a rabbit model of limb lengthening. Osteotomies are created in rabbit tibia. 
The tibia are then be distracted 2 cm over a period of approximately ten days. 
Following the distraction phase, rhBMP-2 is administered. Two modes of application 
are tested. One set of animals has rhBMP-2/ACS (1.5 mg/mL; 0.075 mg/tibia) 
surgically placed within the regenerate. The second group of animals has 

35 rhBMP-2/MFR842 buffer (0.75 mg/mL; 0.075 mg/tibia) percutaneously injected into 



17 



WO 98/31788 



PCT/US98/01143 



5 three sites within the regenerate. A third group of control animals has surgical 
intervention similar to the rhBMP-2/ACS-treated animals, with placement of 
ACS/buffer within the regenerate. Additional sets of animals have distraction 
performed, with no surgical intervention. Groups of animals are sacrificed at 5, 14 
and 24 days. Radiographs are taken following the distraction phase and at sacrifice 

10 and are used to evaluate new bone formation in response to treatment and control. 
Bone density within the defect is determined with Dual Energy X-Ray Absorptiometry 
and Peripheral Quantitative Computed Tomography (pQCT). The three dimensional 
distribution of bone within the defect is evaluated with pQCT. Histological evaluation 
of non-decalcified specimens is used to quantify histomorphometric parameters. 

1 5 Results to date indicate a visually detectable acceleration of bone induction in 

rhBMP-2/ACS treated animals compared to untreated surgical controls at 5 days and 
14 days based on radiographic evaluation. The DXA and pQCT images confirm these 
findings. In addition the pQCT images indicate greater three-dimensional distribution 
of bone within the regenerate and evidence of early stages of corticalization in the 

20 rhBMP-2/ACS treated animals at 14 days. Further results may support the 
rhBMP-2/ACS treatment effect of significant acceleration of the consolidation phase 
of distraction osteogenesis. Formation of an intact cortex is the primary indicator for 
removal of the external fixator and associated return to unprotected full weight 
bearing. The rhBMP-2/MFR842 injection buffer treatment is the preferred route of 

25 administration having more wide spread application in primary distraction 
osteogenesis. 



18 



WO 98/31788 



PCT/US98/01143 



5 CLAIMS 

We claim: 

1 . A method for reducing the severity and/or incidence of fractures comprising 
administering to a site of osteoporotic or osteopenic bone tissue an injectable 
composition comprising an effective amount of an active agent in a suitable carrier. 
10 2. The method of claim 1, wherein the active agent is a member of the TGF-P 

superfamily of proteins. 

3. The method of claim 1, wherein the active agent is a member of the bone 
morphogenetic protein family of proteins. 

4. The method of claim 1 , wherein the active agent is selected from the group 
15 consisting of BMP-2, BMP-4, BMP-5, BMP-6, BMP-7, BMP-10, BMP-12 and BMP- 

13. 

5. The method of claim 1, wherein the active agent is BMP-2. 

6. The method of claim 1, wherein the carrier is selected from the group 
consisting of a suitable buffer, blood, collagen gel and injectable calcium phosphates. 

20 7. A method for healing bone fractures comprising implanting BMP into the 

region between ends of the bone fracture and allowing such rhBMP-2 to stimulate 
bone induction. 

8. A cell produced by the method of claim 7 and harvested from the region 
between bone ends. 



19 



INTERNATIONAL SEARCH REPORT 



Intern... .onal Application No 

PCT/US 98/01143 



A. CLASSIFICATION OF SUBJECT MATTER 

IPC 6 C12N5/08 A61K38/18 



According to International Patent Classification {IPC) or to both national classification and IPC 



B. FIELDS SEARCHED 



Minimum documentation searched (classification system followed by classification symbols) 

IPC 6 A61K 



Documentation searched other than minimum documentation to the extent that such documents are included in the fields searched 



Electronic data base consulted during the international search (name of data base and. where practical, search terms used) 



C. DOCUMENTS CONSIDERED TO BE RELEVANT 



Category a Citation of document with indication, where appropriate, of the relevant passages 



Relevant to claim No. 



X 
X 



US 5 597 897 A (RON EYAL ET AL) 28 
January 1997 

* see in particular examples 4,5 and col. 

5, 1. 58- col. 6, 1.7 * 

& WO 93 00050 A (GENETICS INST.) 7 January 

1993 

US 5 453 419 A (MURAKAMI KAZUO ET AL) 26 
September 1995 

*see in particular col. 7, 1.63 - col. 8, 
1. 12, and claims 1-2* 

US 5 166 058 A (WANG ELIZABETH A ET AL) 
24 November 1992 

*see in particular col. 5 1. 50 - col. 7, 
1.43 * 



■I- 



1-7 

1-7 
1-7 

1-7 



LD 



Further documents are listed in the continuation of box C. 



Patent family members are listed in annex. 



* Special categories of cited documents : 

"A* document defining the general state of the art which is not 

considered to be of particular relevance 
"E" earlier document but published on or after the international 

tiling date 

"L" document which may throw doubtB on priority claim(s) or 
whtch ta cited to establish the publication date of another 
citation or other special reason (as specified) 

'O* document referring to an oral disclosure, use, exhibition or 
other means 

"P" document published prior to the international filing date but 
later than the priority date olatmed 



"T" later document published after the international filing date 
or priority date and not in conflict with the application but 
cited to understand the principle or theory underlying the 
invention 

*X a document of particular relevance; the claimed invention 
cannot be considered novel or cannot be considered to 
involve an inventive step when the document is taken alone 

*Y° document of particular relevance; the claimed invention 

cannot be considered to involve en inventive step when the 
document is combined with one or more other such docu- 
ments, such combination being obvious to a person skilled 
in the art. 

*&" document member of the same patent family 



Date of the actual completion of the international search 



20 May 1998 



Date of mailing of the international search report 



06. 07.98 



Name and mailing address of the ISA 

European Patent Office. P. B. 5818 Patentlaan 2 
NL-2280 HV Rijswijk 
Tel. (+31*70) 340-2040, Tx. 31 651 epo nl, 
Fax: (+31-70) 340-3016 



Authorized officer 



Is€rt, B 



Farm PCT7ISA/210 (second steet) {July 1992) 



page 1 of 2 



INTERNATIONAL SEARCH REPORT 



Inteniutional Application No 

PCT/US 98/01143 



C.(Conlinuation) DOCUMENTS CONSIDERED TO BE RELEVANT 



Category * CSatbn of document, with indioation,where appropriate, ot the relevant passages 



Relevant to claim No. 



WANG: "Bone morphogenetic proteins 

(BHPS): Therapeutic potential in healing 

bony defects" 

TRENDS IN BIOTECHNOLOGY, 

vol. 11, no. 9, 1993, 

pages 379-383, XP0O2O65499 

* see in particular p. 381, 2nd paragraph 

-382 * 



REDDI ET AL. : 
p. 281-287)" 
1996 , ACADEMIC 
XP002065501 
*see in particular p 



"Osteoporosis (chapter 9, 
, SAN DIEGO, CALIF. 
285 * 



TAGAKI ET AL.: "The role of bone marrow 
in bone tnorphogenetic protein-induced 
repair of femoral massive diaphyseal 
defects" 

CLINICAL ORTHOPAEDICS AND RELATED 

RESEARCH, 

vol. 171, 1982, 

pages 224-231, XP002065500 

* see in particular p. 230 (summary) and 

Figures 1-5 n* 



1-7 



1-7 



1-3 



Fcrm PCT/ISA/2! 0 (continuation zl second « nee!. . July I9?2» 



page 2 of 2 



INTERNATIONAL SEARCH REPORT 



International application No. 

PCT/US 98/01143 



Box I Observations where certain claims were found unsearchable (Continuation of item 1 of first sheet) 

This International Search Report has not been established in respect of certain claims under Article I7(2)(a) for the following reasons: 
1. | | Claims Nos.: 

because they relate to subject matter not required to be searched by this Authority, namely: 



2. X Claims Nos.: 8 

because they relate to parts of the International Application that do not comply with the prescribed requirements to such 
an extent that no meaningful International Search can be carried out, specifically: 

Claim 8 relates to a cell "produced by the method of claim 7". However, 
claim 7 does not describe such a method but relates to a method of 
treatment. 

3. | | Claims Nos.: 

because they are dependent claims and are not drafted in accordance with the second and third sentences of Rule 6.4(a). 

Box II Observations where unity of invention is lacking (Continuation of item 2 of first sheet) 

This International Searching Authority found multiple inventions in this international application, as follows: 



1 . I I As all required additional search fees were timely paid by the applicant, this Internationa) Search Report covers all 
I — I searchable claims, 



2. | j As alt searchable claims could be searched without effort justifying an additional fee, this Authority did not invite payment 
of any additional fee. 



3. I I As only some of the required additional search fees were timely paid by the applicant, this International Search Report 
' ' covers only those claims for which fees were paid, specifically claims Nos.: 



4. | | No required additional search fees were timely paid by the applicant. Consequently, this International Search Report is 
restricted to the invention first mentioned in the claims; it is covered by claims Nos.: 



Remark on Protest [ | The additional search fees were accompanied by the applicant's protest. 

| | No protest accompanied the payment of additional search fees. 



Form PCT/ISA/21 0 (continuation of first sheet (1 )) (July 1 992) 



INTERNATIONAL SEARCH REPORT 

Information on patent family members 



Intentional Application No 

PCT/US 98/01143 



Patent document 
cited in search report 



Publication 
date 



Patent family 
member(s) 



Publication 
date 



US 5597897 



28-01-1997 



AU 
DE 
DE 
EP 
FI 
JP 
NO 
AT 
AU 
CA 
ES 
MX 
WO 



663328 B 
69213739 D 
69213739 
G591392 

935732 
6508777 

934573 

142460 
2254292 
2111199 
2094359 
9203083 
9300050 



US 5453419 A 



26-09-1995 



US 
AT 
CA 
DE 
DE 
EP 
JP 



5670338 A 
140965 T 

2024629 A 
69027961 D 
69027961 T 

0416578 A 

4154799 A 



US 5166058 



24-11-1992 



US 
US 
AT 
AU 
AU 
DE 
DE 
OK 
DK 
EP 
EP 
IE 
IL 
JP 
JP 
JP 
JP 
JP 



A 
A 
T 



5013649 
5631142 
141928 
613314 B 
7783587 A 
3751887 
3751887 
53497 
106288 
0313578 
0688869 
75881 B 
83003 A 
2729222 B 
6298800 A 
10070989 A 
2500241 T 



D 
T 
A 
A 
A 
A 



05- 10 
17-10 
20-02 
13-04 
20-12 

06- 10 
13-12 

15- 09 
25-01 

07- 01 

16- 01 
31-08 
07-01 



•1995 
■1996 
•1997 
•1994 
•1993 
■1994 
■1993 
■1996 
■1993 
■1993 
■1997 
•1994 
•1993 



23-09-1997 
15-08-1996 
07-03-1991 
05-09-1996 
09-01-1997 
13-03-1991 
27-05-1992 



2713715 B 



07-05- 
20-05- 

15- 09- 

01- 08- 
29-01- 

02- 10- 
06-03- 
09-05- 
28-04- 

03- 05- 
27-12- 

24- 09- 
31-07- 
18-03- 

25- 10- 
17-03- 
01-02- 

16- 02- 



1991 
1997 
1996 
1991 
1988 
1996 
1997 
1997 
1988 
1989 
1995 
1997 
1995 
1998 
1994 
1998 
1990 
1998 



Farm PCT.'ISA^tO {patent family anntu) (July 



page 1 of 2 



INTERNATIONAL SEARCH REPORT 

Information on patent family members 



International Application No 

PCT/US 98/01143 



Patent document 
cited in search report 

US 5166058 



Publication 
date 



Patent family 
member(s) 



Publication 
date 



l/D 


7/ UD 00 J 


D 
D 


18-04-1997 


MY 




D 
D 


??-flQ-1993 


DT 
r 1 


00 CCD 


D 
D 




WU 




A 
M 


-1Q88 


IK 




A 
M 




IK 




A 
M 




IK 




A 
n 




us 


5187076 


A 


16-02-1993 


us 


5618924 


A 


08-04-1997 


us 


5116738 


A 


26-05-1992 


us 


5366875 


A 


22-11-1994 


NO 


963788 


A 


17-02-1988 


NO 


963789 


A 


17-02-1988 


us 


5106748 


A 


21-04-1992 


us 


5141905 


A 


25-08-1992 


us 


5108922 


A 


28-04-1992 



Form PCT/lSA/210 (patent tomiiy anne*> iJuly 1992) 



page 2 of 2 



This Page is Inserted by IFW Indexing and Scanning 
Operations and is not part of the Official Record 

BEST AVAILABLE IMAGES 

Defective images within this document are accurate representations of the original 
documents submitted by the applicant. 

Defects in the images include but are not limited to the items checked: 

□ BLACK BORDERS 

□ IMAGE CUT OFF AT TOP, BOTTOM OR SIDES 
□f FADED TEXT OR DRAWING 
(^BLURRED OR ILLEGIBLE TEXT OR DRAWING 

□ SKEWED/SLANTED IMAGES 

□ COLOR OR BLACK AND WHITE PHOTOGRAPHS 

□ GRAY SCALE DOCUMENTS 

□ LINES OR MARKS ON ORIGINAL DOCUMENT 

□ REFERENCE(S) OR EXHIBIT(S) SUBMITTED ARE POOR QUALITY 

□ OTHER: 

IMAGES ARE BEST AVAILABLE COPY. 
As rescanning these documents will not correct the image 
problems checked, please do not report these problems to 
the IFW Image Problem Mailbox.