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WORLD INTELLECTUAL PROPERTY ORGANIZATION 
International Bureau 




per 

INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) 



(51) International Patent Gassiflcation ^ : 
A61L 27/00, A61K 37/02 



Al 



(11) Internationa] PubllcaUon Number: WO 93/06872 

(43) International Publication Date: 15 April 1993 (15.04.93) 



(21) Inteniational Application Number: PCT/US92/08628 

(22) International Filing Date: 9 October 1992 (09.10.92) 



(30) Priority data: 
776,514 



11 October 1991 (H.10.91) US 



(60) Parent Application or Grant 
(63) Related by Continuation 

US 776,514 (CIP) 

Filed on 1 1 October 1991 (1 1.10.91) 



(71) Applicant (for all designated States except US): GENETICS 
INSTITUTE, INC. [US/US]; 87 CambridgePark Drive, 
Cambridge, MA 02140 (US). 



(72) Inventors; and 

(75) Inventors/Applicants (for US only) : RON, Eyal (US/US]; 
71 Grant Street, Lexington, MA 02173 (US). SCHAUB, 
Robert, George [US/US]; 118 Jeremy Hill Road, Pel- 
ham. NH 03076 (US). TUREK, Thomas, Joseph [US/ 
US]; 65 Bearse Avenue, Boston, MA 02124 (US). 

(74) Agent: McDANIELS, Patricia, A.; Genetics Institute, 
Inc., 87 CambridgePark Drive, Cambridge, MA 02140 
(US). 



(81) Designated States: AU, BR, CA, n, JP, KR, NO, RU, US, 
European patent (AT, BE, CH, DE, DK, ES, FR, GB, 
GR, IE, IT, LU, MC, NL, SE). 



Published 

With international search report. 



(54) Title: FORMULATIONS OF BLOOD CLOT-POLYMER MATRIX FOR DELIVERY OF OSTOOGENIC PROTEINS 
(57) Abstract 



A composition comprising a phannaceutically acceptable admixture of an osteogenic protein ; a porous particulate polym- 
er matrix; and an osteogenic protein-sequestering amount of blood clot 



FOR THE PURPOSES OF INFORMATION ONLY 

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



AT 


Austria 


FR 


AU 


Australia 


GA 


BB 


BarbaUos 


GB 


BE 


Belgium 


GN 


BF 


Burlctna Faao 


GR 


BC 


Bulgaria 


HU 


aj 


Benin 


IE 


BR 


Brazil 


IT 


CA 


Canada 


JP 


CF 


Central Arrican Republic 


KP 


CC 


Congo 


KR 


CH 


Swtizcrland 


CI 


Coic d'l voire 


LI 


CM 


Cameroon 


LK 


CS 


Otechcsstovakia 


LU 


CZ 


Ceech Republic 


MC 


0£ 


Germany 


MG 


DK 


DenmarL 


ML 


ES 


Spain 


MN 


Fl 


Finland 





France 
Gabon 

United Kingdom 

Guinea 

Greece 

Hungary 

Ireland 

luily 

Japan 

Democratic People's Republic 
of Korea 

Republic of Korea 

IJechtenstcin 

Sri Lanka 

loiitembourg 

Monaco 

Madagascar 

Mali 

Mongolia 



MR 


Mauritania 


MW 


Malawi 


NL 


Netherlands 


NO 


Norway 


NZ 


New Zealand 


PL 


Poland 


PT 


Portugal 


RO 


Romania 


RU 


Russian Federation 


SD 


Sudan 


SE 


Sweden 


5K 


Slovak Republic 


SN 


Senegal 


SU 


Soviet Union 


TD 


Chad 


TG 


Togo 


UA 


Ukraine 


US 


Uiiiied Slates of America 


VN 


Viet Nam 



wo 93/06872 



PCT/US92/08628 



TITLE OF THE INVENTION 

5 FORMULATIONS OF BLOOD CLOT-POLYMER MATRIX 

FOR DELIVERY OF OSTEOGENIC PROTEINS 

BACKGROUND OF THE INVENTION 
The subject invention relates to the field of osteogenic 
10 proteins and pharmaceutical formulations thereof. More 
particularly, the subject invention involves pharmaceutical 
formulations designed to sequester osteogenic protein in-situ for 
a time sufficient to allow the protein to induce cartilage and/or 
bone formation • 

15 

Osteogenic proteins are those proteins capable of inducing, 
or assisting in the induction of, cartilage and/or bone 
formation. Many such osteogenic proteins have in recent years 
been isolated and characterized, and some have been produced by 

20 recombinant methods. For example, so-called bone morphogenic 
proteins (BMP) have been isolated from demineralized bone tissue 
(see e.g. Urist US 4,455,256) ; a nvimber of such BMP proteins have 
been produced by recombinant techniques (see e.g. Wang et al. US 
4,877,864 and Wang et al. US 5,013,549); a family of transforming 

25 growth factors (TGF-a and TGF-/8) has been identified as 
potentially useful in the treatment of bone disease (see e.g. 
Derynck et al., EP 154,434); a protein designated Vgr-1 has been 
found to be expressed at high levels in osteogenic cells (see 
Lyons et al. (1989) Proc. Nat'l. Acad. Sci. USA 86/ 4554-4558); 

30 and proteins designated OP-1, COP-5 and COP-7 have purportedly 
shown bone inductive activity (see Oppermann, et al. U.S. 
5,001,691). 

Various attempts have been made at developing formulations 
designed to deliver osteogenic proteins to a site where induction 
35 of bone formation is desired. For example, certain polymeric 
matrices such as acrylic ester polymer (Urist, US 4,526,909) and 
lactic acid polymer (Urist, US 4,563,489) have been utilized, but 
these formulations do not sequester the osteogenic protein for 
a time sufficient to optimally induce bone formation, and further 
40 have been found to erode too slowly for optimal bone formation. 

1 



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10 



A biodegradeable matrix of porous particles for delivery of 
an osteogenic protein designated as OP is disclosed in 
Kuberasampath, U.S. 5,108,753. While US 5,108,753 discloses that 
a successful carrier for OP must bind the protein, act as a slow 
release delivery system, accommodate each step of the cellular 
response during bone development, and protect the protein from 
nonspecific proteolysis, no formulations are suggested which 
contain components that specifically sequester the OP at the site 
where bone formation is desired. 

Okada et al. , US 4,652,441, US 4,711,782, US 4,917,893 and 
US 5,061,492 and Yamamoto et al., US 4,954,298 disclose a 
prolonged-release microcapsule comprising a polypeptide drug and 
a drug-retaining substance encapsulated in an inner aqueous layer 
surrounded by a polymer wall substance in an outer oil layer. 
15 Although bone morphpgenic protein is listed as a polypeptide 
capable of such a formation, microencapsulation of osteogenic 
proteins prevents controlled release of such protein sufficient 
for optimal bone formation. 

Collagen matrices have also been used as delivery vehicles 
20 for osteogenic proteins (see e.g. Jeffries, U.S. 4,394,370), but 
collagen frequently causes undesirable antigenic reactions in 
patients. Therefore, there remains a need for a pharmaceutical 
formulation capable of sequestering osteogenic proteins at a site 
where induction of bone formation is desired for a time 
25 sufficient to allow safe, effective induction of such bone 
formation. 

RtlMMARY QT THE INV ENTION 

Applicants have surprisingly discovered that osteogenic 
proteins can be sequestered at a site where bone inducing 

30 activity is desired using blood clot in the absence of an 
antifibrinolytic agent, provided that a porous particulate 
polymer matrix is incorporated into the formulation. Therefore, 
more particularly, the subject invention provides a composition 
comprising a pharmaceutically acceptable admixture of an 

35 osteogenic protein; a porous particulate polymer matrix and an 
osteogenic protein-sequestering amount of blood clot. 



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DETAILED DESCRIPTION OF THE INVENTION 
The osteogenic proteins useful in the practice of the 
subject invention are well known to those skilled in the art and 
include those discussed above • The preferred osteogenic proteins 
5 for use herein are those of the BMP class identified as BMP-1 
through BMP-8 in US 4,877,864; US 5,013,649; - WO 90/11366 
published October 4, 1990 and WO 91/18098 published November 28, 
1991. The most preferred is BMP-2, the full length cDNA sequence 
and the ultimate mature protein sequence described in detail in 

10 the '649 patent. Of course, combinations of two or more of such 
osteogenic proteins may be used, as may fragments of such 
proteins that also exhibit osteogenic activity. Such osteogenic 
proteins are known to be homodimeric species, but also exhibit 
activity as mixed heter odimer s . Heterodimeric forms of 

15 osteogenic proteins may also be used in the practice of the 
subject invention. Recombinant proteins are preferred over 
naturally occurring isolated proteins. The amount of osteogenic 
protein useful herein is that amount effective to stimulate 
increased osteogenic activity of infiltrating progenitor cells, 

20 and will depend upon the size and nature of defect being treated 
as discussed in more detail below, such amounts being orders of 
magnitude less than the amount of polymer matrix employed, 
generally in the range of 1-50 /xg of protein for each 10 mg of 
polymer matrix employed and more preferably in the range of 0.5- 

25 10 /xg protein for each milligram of polymer matrix employed 
(assuming 0.2 g/cc density). 

The osteogenic proteins can be utilized in the form of a 
pharmaceutically acceptable solution (including reconstitution 
from a lyophilized form) . It is optimal to solubilize the 

30 osteogenic protein at concentrations of at least about 1 mg/ml, 
preferably about 2 mg/ml, so that a pharmaceutically effective 
amount of protein can be delivered without undue volumes of 
carrier being necessary. Amino acids having a net positive 
charge (e.g. net 1+ species such as arginine, histidine, lysine 
35 and the ethyl esters of glycine and beta-alanine) , preferably a 
net 2+ charge (e.g. the ethyl ester of histidine, the methyl 
esters of lysine and arginine, and agmatine) , are useful in this 



3 



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WO 93/06872 

regard. Amino acids having a net zero charge are useful in this 
regard provided that the positive charge of the compound is 
sufficiently distant (at least 2-3 CH, units away) from the 
neutralizing negative charge (e.g. net neutral species such as 
5 gamma-amino butyric acid, beta-amino propionic acid, and glycine- 
glycine dipeptide). Other solubilizing agents useful herein 
include poly(sorbate) , dextran sulfate, guanidine, heparin and 
sodium chloride. For use in solubilizing BMP-2, the preferred 
solubilizing agents are arginine and histidine (including esters 

10 thereof) . Arginine is used in concentrations of 50-600 mM, 
preferably 300-500mM. Histidine may be added to arginine to 
solubilize BMP-2, in concentrations of about 1-100 mM, preferably 
10-50 mM. When histidine is used alone as a solubilizing agent, 
it is used in concentrations of about 50-600 mM, preferably 300- 

15 500 mM. various well known methods may be used to compound the 
osteogenic protein and solubilizing agents for use herein, 
including but not limited to ultrafiltration, dialysis, gel 
filtration, and hydrophobic interaction chromatography. 

The polymer matrix component useful in the practice of the 

20 subject invention is a polymeric material that can be formed into 
porous particles as described below thereby providing in-situ 
scaffolding for the osteogenic protein, while having 
biodegradable properties allowing for replacement by new bone 
growth. Examples are polymers of amino acids, orthoesters, 

25 anhydrides, propylene-co-f umarates , or a polymer of one or more 
a-hydroxy carboxylic acid monomers, (e.g. a-hydroxy acetic acid 
(glycolic acid) and/or a-hydroxy propionic acid (lactic acid)). 
The latter can be employed in its d- or 1- form, or as a racemic 
mixture, the racemic mixture being preferred. When a copolymer 

30 of lactic acid and glycolic acid is employed (PLGA) , the molar 
ratio of monomers can range from 1:99 to 99:1 depending upon the 
desired bioerosion lifetime which in turn depends upon the 
clinical indication being addressed, as more than 50% of either 
monomer gives longer bioerosion lifetime (slower biodegradation) . 

35 The molecular weight of the polymer can range from about 1,000 
to 100,000 (relative to polystyrene in CHCl,) with 30,000-50,000 
being preferred when a 50:50 copolymer is employed. The higher 



4 



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the molecular weight the slower the biodegradation. 

The polymeric matrix component of the subject invention is 
used in the form of highly porous to hollow (with surface 
porosity) particles, hereinafter collectively referred to as 
5 "porous particles," These porous particles are generally 
spherical having diameters of 150 to 850 microns, preferably 150- 
500 microns, most preferably 150-300 microns. This particle size 
creates sufficient spacing between particles to allow mammalian 
osteoprogenitor cells to infiltrate and be positively influenced 

10 by (evidenced by an increase in osteogenic activity/bone growth 
rate) the osteogenic protein. 

While it has generally been postulated that particles 
suitable as matrices for delivery of osteogenic proteins should 
be porous, the extent of porosity necessary to optimally induce 

15 bone formation has not previously been studied. The present 
inventors have discovered that the average surface area per 
porous particle is critical to optimize bone formation. 
Specifically, porous particles useful in bone formation according 
to the present invention should have an average surface area of 

20 from about 0.02 to 4 m^/g. The present inventors have further 
discovered that it is possible to produce porous particles having 
the desired surface area by introducing a "porosigen" 
(composition capable of imparting porosity by increasing particle 
surface area) into the solution used to produce the porous 

25 particles. It is also possible to control the bioerosion rate 
by subjecting the porous particles to sterilizing doses of 7 
radiation. The higher the 7 radiation dose^ the faster the 
bioerosion. 

Particles useful herewith have a porosity such that the 
30 surface area of the particles is increased about 2-250 fold over 
the surface area of non-porous particles of comparable size. 

A preferred method of production of the porous particles of 
the invention is, generally speaking, a solvent evaporation 
process comprising dissolving the polymer (in e.g. CH2CI2) , and 
35 adding a porosigen such as NaCl, mannitol or sucrose in solid 
and/or liquid form. When porosigen is added in solid form, the 
matrix-porosigen solution takes the form of a suspension. 



5 



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Another preferred method of production of the porous particles 
of the invention is a solvent extraction method, wherein the 
porosigen is added in liquid form with concomitant 
homogenization. When porosigen is added in liquid form with 
5 homogenization, the matrix-porosigen solution takes the form of 
an emulsion. With either method, the matrix-porosigen emulsion 
is added to an excess aqueous solution containing surfactant such 
as poly(vinyl alcohol) with controlled stirring and temperature. 
The resultant porous particles are hardened by extracting or 

10 evaporating residual solvent, and dried. PLGA particles useful 
in the subject invention made utilizing 50% NaCl as a porosigen 
have a surface area of between about 0.2 and 0.6 m^/g; and 
particles made using sucrose as a porosigen have a surface area 
of between about 0.04 and 0.09 mVg- P^GA particles of the 

15 present invention made using liquid porosigen with homogenization 
have a surface area of between about 0.02 and 4 mVg. 

The porous nature of the particles of the present invention 
creates sufficient surface area for protein adsorption and 
increases biodegradation, the desirable extent of both being 

20 dependent upon the clinical indication being addressed. Surface 
area can be measured by any conventional technique. For example, 
BET surface area analysis can be employed using a Micromeritics 
ASAP 2000 system, which measures surface area based upon 
adsorption and desorption of Krypton gas at the surface and 

25 within the pores of the solid sample. The unit calculates and 
prints out the 
surface area: 

1 = Crl (P/Po) + ^ 

VA[(Po/P)-l] V„C V„C 



30 



V = volume absorb at pressure P Po = saturation pressure 

P/Po = relative pressure P = pressure 

C = constant A = gas cross sectional area 
Vm = Monolayer Capacity 

By plotting i vs P/Po, the slope being 

VA((Po/P)-l 



6 



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PCT/US92/08628 



c-1 and the intercept being l . the surface area 

Si - VpNA where N = Avogadro's number and V = molar volume. 
5 V 

The amount of porous particles used to treat a particular defect 
will, of course, depend upon the size of the defect being 
treated, and on the effective amount required to adsorb the 

10 osteogenic protein. 

The protein-sequestering material useful in the practice of 
the subject invention is pharmaceutical ly acceptable human blood, 
preferably autologous blood. When added to an osteogenic 
protein/porous particle mixture, the blood clots to form a 

15 malleable composite wherein the adsorbed protein is sequestered 
within the matrix for a time sufficient to allow the protein to 
increase the otherwise natural rate of osteogenic activity of the 
infiltrating mammalian progenitor cells. In the absence of such 
blood clot, osteogenic protein desorbs from the PLGA particles 

20 in - situ at a rate such that the osteoinducing effect of the 
protein is not clinically significant. The ratio of blood to 
porous particles useful herein is 1:0.5 to 1:10 (v:v) , preferably 
1:5 (v:v) , and more preferably 1:2 (v:v), which ratio represents 
the amount necessary to prevent desorption from the polymer 

25 matrix, yet not so much that the progenitor cells are prevented 
from infiltrating the matrix, thereby providing the protein the 
opportunity to assist the osteogenic activity of the progenitor 
cells. For each 1 ml defect, the amount of blood required will 
therefore generally be about 0.5-1.0 ml. In cases where large 

30 doses of osteogenic protein are employed, clot facilitating 
agents such as thrombin may be employed to offset the dilution 
effect of the osteogenic protein. It is preferable to mix the 
blood component with the solution of osteogenic protein prior to 
addition of the porous particles. 

35 Additional optional components useful in the practice of the 

subject application include, e.g. cryogenic protectors such as 
mannitol, sucrose, lactose, glucose, or glycine (to protect from 
degradation during lyophilization) , antimicrobial preservatives 
such as methyl and propyl parabens and benzyl alcohol. 



PCr/US92/08628 

WO 93/06872 

antioxidants such as EDTA, citrate, and BHT (butylated 
hydroxytoluene) , and siarfactants such as poly (sorbates) and 
poly(oxyethylenes) , etc. Of course, the traditional preparation 
of formulations in phannaceutically acceptable form (i.e. pyrogen 
5 free, appropriate pH and isotonicity, sterility, etc.) is well 
within the skill in the art and is applicable to the formulations 
of the subject invention. The osteogenic protein and porous 
particles of the formulations may be provided to the clinic as 
a single vial formulation, either as a solution or in lyophilized 

10 form, or the formulation may be provided as a multicomponent kit 
wherein, e.g. the osteogenic protein is provided in one vial and 
the porous particles are provided in a separate vial. The blood 
to be used in the formulation is admixed at a time prior to use 
sufficient to allow clotting, generally 3d to 180 minutes prior 

15 to use, taking into account the well-known patient-to-patient 
variability in clotting time. 

The formulations of the subject invention provide malleable 
implants that allow therapeutically effective amounts of 
osteoinductive protein to be delivered to an injury site where 

20 cartilage and/or bone formation is desired. Such an implant may 
be used as a stibstitute for autologous bone graft in fresh and 
non-union fractures, spinal fusions, and bone defect repair in 
the orthopaedic field; in cranio/maxillofacial reconstructions; 
for prosthesis integration, especially as a surface coating to 

25 improve fixation of prosthetic implants such as hydroxylapatite 
coated prostheses; in osteomyelitis for bone regeneration; and 
in the dental field for erosion of the alveolar ridge and 
periodontal disease. When used to treat osteomyelitis, the 
osteogenic protein may be used in combination with porous 

30 particles and antibiotics, with the addition of autologous blood 
as a sequestering agent. The lower viscosity formulations may 
also be used as a percutaneous injection to accelerate healing 
of closed fractures. In certain of these uses, the compositions 
of the subject invention may be used in combination with various 

35 bone cements, including erodible bone cements such as 
poly(propylene-co-fumarate) . Also, certain of these uses will 
utilize bioerodible hardware such as erodible plates, screws. 



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etc. As alluded to above, the dosage regimen will be determined 
by the clinical indication being addressed, as well as by various 
patient variables (e.g. weight, age, sex) and clinical 
presentation (e.g. extent of injury, site of injury, etc.). 



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EXAMPLE I 
PREPARATION OF IMPLANT 
A 50:50 random copolymer of lactic acid and glycolic acid 
(PLGA) having an average molecular weight of 30-40 kD, a number 
5 average molecular weight of about 20 kD (by gel permeation 
chromatography relative to polystyrene standards) and an inherent 
viscosity of 0.35-0.45 dL/g was dissolved in CH^Clj (15% w/v) , and 
10 g porosigen (7.5% w/v) was suspended in this solution. The 
resulting solution was added to an excess poly (vinyl alcohol) 

10 aqueous solution (0.1% w/v) . After a few hours of stirring under 
partial vacuum (24 inches Hg) , the particles were hardened in 
excess cold ethanol (95%) . The resulting particles were washed 
with water for injection and vacuum dried to give a free-flowing 
product. BET surface area analysis was performed using a 

15 Micrometrics ASAP 2000 system as described above, and the 
particles had a surface area of between about 0.2 and 1.0 m^g: 
particles made using sucrose as a porosigen had a surface area 
of between about 0.04 and 0.09 m'/g- The porous particles are 
sterilized by 7 irradiation prior to use. For each implant 

20 intended for human use, 10 ml of porous particles are placed in 
a sterile cup. 

Lyophilized recombinant human BMP-2 (rhBMP-2) (2 mg) is 
reconstituted with 1 ml sterile water for injection (WFI) . 0.5 
ml of the rhBMP-2 solution (1 mg) is drawn into a 3 ml syringe, 
25 the needle of which is then replaced with a double female Luer 
Lok connector. 

5.5 ml of the patient's venous blood is drawn into a 10 
ml syringe (not through a heparinized line) , the needle of which 
is removed and immediately attached to the other side of the Luer 

30 Lok connector on the 3 ml syringe containing the rhBMP-2 
solution. The rhBMP-2 solution is gently transferred into the 
syringe containing the blood, and the resulting mixture is gently 
transferred between the syringes four times to assure uniform 
mixing, ending with the mixture in the 10 ml syringe. The empty 

35 3 ml syringe and Luer Lok connector is removed, and the 
blood/rhBMP-2 mixture is transferred into the cup containing the 
porous particles. If necessary, the entire mixture is gently 



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stirred with a stainless steel spatula to achieve uniform 
consistency. The cup is covered and allowed to sit at room 
temperature for about one hour. The implant mixture should be 
used within the following two hours. 
5 When the desired consistency of the malleable implant is 

obtained, the bony defect is exposed by surgical incision at the 
site of injury. The implant is applied by the surgeon by shaping 
the malleable composite of porous particles /rhBMP-2 /blood clot 
to span the bony defect which is desired to be treated. The 
10 incision is then closed using conventional methods. Healing is 
monitored by X-ray analysis of the defect site. 

EXAMPLE II 

CANINE CALVARIAL DEFECT IMPLANT ANALYSIS 
15 Two male and two female dogs were placed under general 

anaesthesia and four trephine holes of approximately 12 mm each 
were made in each skull using an Acra-cut DG II cranial drill, 
as shown below. 



20 




25 All holes were filled with an implant consisting of rhM!P-2, PLGA 
porous particles, and autologous blood which had been allowed to 
clot to form a moldable implant. The dose of rhBMP-2 was 
approximately 200 /xg/ml in each implant. One male and one female 
animal was sacrificed at 4 weeks and the remaining animals were 

30 sacrificed at 8 weeks after surgery. 

After sacrifice, the left rostral and right caudal calvarial 
sites of each animal were used for biomechanical testing, and the 
right rostral and left caudal calvarial sites were used for 
histopathology. Biomechanical testing revealed a similar stress 

35 resistance in the animals sacrificed at 4 and 8 weeks, which was 
not statistically significantly different from the untreated 
control dogs. At both 4 and 8 weeks following surgery. 



11 



15 



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histological examination revealed that bony ingrowth occurred 
into the implant sites which probably bridged the calvarial 
defects. Remodeling seemed to be occurring between 4 and 8 weeks 
with the trabeculae becoming thicker. The porous particulate 
5 matrix material was disappearing over the course of the study 
with little left after 8 weeks. 

EXAMPLE III 
RAT FEMORAL DEFECT IMPLANT ANALYSIS 
3^0 A critical-size defect (5 mm) was surgically created in the 

mid-diaphysis of the left femur of each of 56 male Long Evans 
retired breeder rats (450-550 grams) , by affixing a pre-drilled 
polyethylene plate to the anterior portion of the femur and 
excising a segment of bone with a carbide dental drill. A 
bioerodible implant was prepared by mixing rhBMP-2 (in varying 
amounts), PLGA porous paticles, and venous rat blood and allowing 
the blood to clot to form a moldable implant. Eight groups of 
seven animals each were implanted as follows: 0 fig rhBMP-2; 0.93 
ng rhBMP-2; 3.1 rhBMP-2; 9.3 /zg rhBMP-2; 0 ng rhBMP-2 + 2M c- 
amino caproic acid; 0.93 ng rhBMP-2 + 2M e-amino caproic acid; 
3.1 ng rhBMP-2 + 2M e-amino caproic acid; and 9.3 rhBMP-2 + 
2M c-amino caproic acid. (e-amino caproic acid is a hemostatic 
agent). The PLGA porous particles used in this study had a 
surface area of 0.217 mVg- 

Animals were radiographed at Day 0 and Weeks 3, 6, and 9. 
The animals were sacrificed at week 9, the tissues surrounding 
the polyethylene plates were removed for histological 
examination, and the implanted and contralateral non-implanted 
femurs were harvested. Two femurs in each group were 
histologically examined, and the remaining femurs were used for 
biomechanical testing. 

Animals receiving 0 ng rhBMP-2 were non-unions at Week 9, 
and no new bone formation was evident in those groups. Fifty 
(animals receiving e-amino caproic acid) to eighty (animals not 
35 receiving e-amino caproic acid) percent of the 0.93 ng dose 
groups achieved union at Week 9. All animals in the 3.1 fig dose 
groups achieved union at Week 9. Twelve of thirteen 9.3 ^g dose 



20 



25 



30 



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group animals which remained at Week 9 achieved union (one 9.3 
/xg animal had a plate failure at Week 6). 

c-amino caproic acid did not appear to modify the healing 
response. No bone formation was found in any of the soft tissue 
5 surrounding the implants. Some cartilage formation was seen in 
all implant groups (including the 0 rhBMP-2 groups) in the 
fibrous tissue surrounding the polyethylene plate. 

Femurs had a dose-dependent healing response at Week 9. 
Implants with no rhBMP-2 developed fibrous tissue in the defect 

10 area. The 0.93 iig rhBMP-2 dose groups had some fibrous tissue 
in the defect in addition to areas of poor alignment of the 
defect site with proximal and distal bone. The 3.1 and 9.3 /xg 
rhBMP-2 implants had little to no fibrous tissue, good alignment 
of proximal and distal segments and some callus formation. 

15 Callus formation was greater in the 9.3 jug groups. Bone marrow 
appeared normal in all rhBMP-2 dose groups. 

No PLGA porous particles were found in any of the rhBMP-2 
implant groups. Doses of rhBMP-2 at 3.1 and 9.3 /xg/i^plant gave 
the most acceptable bone growth. 



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What is claimed is: 

1. A composition comprising a pharmaceutically acceptable 

admixture of 

(i) an osteogenic protein; 
(ii) a porous particulate polymer matrix; and 
(iii) an osteogenic protein-sequestering amount of 
autologous blood clot. 

2 The composition of claim 1 wherein the osteogenic protein is 
selected from the group consisting of the members of the BMP- 
family- 

3. The composition of claim 2 wherein the osteogenic protein is 
BMP-2. 

4. The composition of claim 1, 2, or 3 wherein the admixture is 
free from antif ibrinolytic agents. 

5. The composition of claim 1, 2, or 3 wherein the polymer 
matrix component is selected from the group consisting of 
polydactic acid), poly (glycolic acid), and copolymers of lactxc 
acid and glycolic acid. 

6. The composition of claim 1, 2, 3, 4, or 5 wherein the polymer 
matrix component is PLGA. 

7 The composition of claim 1, 2, 3 or 4 wherein the polymer 
matrix component is selected from poly (orthoester) , polyanhydride 
and poly(propylene-co-fumarate) polymers. 

8. A composition comprising a pharmaceutically acceptable 

admixture of 

(i) BMP-2; 
(ii) a polymeric matrix component comprising polymeric 



14 



wo 93/06872 



PCr/US92/08628 



particles having a diameter of between about 150 
and 850 microns and a porosity such that the 
surface area of the particles is between about 
0*01 and 4.0 m^/g, wherein the polymer is selected 
from the group consisting of poly (lactic acid) , 
poly (glycolic acid), and copolymers of lactic 
acid and glycolic acid; and 
(iii) a protein sequestering amount of autologous blood 

clot. 

9. A kit for the repair of cartilage and/or bone injuries which 
comprises: 

(i) an osteogenic protein; and 

(ii) a porous particulate polymer matrix. 



15 



INTERNATIONAL SEARCH REPORT 

iDternationaJ AppUcatl»»- .Vo PCT/US 92/08628 



1. CLASSinCATlON OF SUBJECT MATTER (if several dassification symbols apply, indicate a»)^ 



According to International Patent aassification (IPQ or to both National Clas$mcation Md IPC 

Int. CI. 5 A 61 L 27/00 A 61 K 37/02 



n. FIELDS SEARCHED 



Minimum DocumentatioD Searched' 



aassification System 



Classtfication Symbols 



Int. CI. 5 



A 61 L 



A 61 K 



DooimentatioD Searched other than Minimum Docuroentailon 
to the Extent that such Documents are Included in the Fields Searched 



in. DOCUMENTS CONSIDERED TO BE RELEVANT' 



Category** 



Otation of Document," with indication, where appropriate, of the relevant passages » 



R^evant to Claim No.^ 



GB,A, 2215209 (OSMED INC.) 20 

September 1989, see page 24, lines 25-31; page 

25, lines 1-2; claims 

WO, A, 8504185 (A.I. CAPLAN et al.) 26 
September 1985, see claims 

US,A,4563489 (M.R. URIST) 7 January 
1986, see abstract; example (cited in the 
application) 

EP.A,0341007 (PROJECT HEAR) 8 
November 1989, see examples 1-5^ 



1-9 
1 

1-9 
1-9 



Special categories of cited documents : 

'A' document defioing the general state of the an whidi Is not 
considered to be of particular tdevaooe 
earlier document hot published on or after the intematioaal 
filing date 

«L* docomat which may throw doubts on priority daim(s) or 
which is cited to esubllsb the pubUcatioo date of another 
dtattoo or other spedal reason (as spedTied) 

'^O' docomeot referring to as oral disdosnre, use, cxhibHIoa or 
other means 

document published prior to the Intervationai filing date but 
later than the priority date dalmed 



'nr later document published after the International filing date 
or priority date and not in conflict with the apnUcatlon tot 
dted to tudetstand the prindple or theory antferiyiog the 
iovcntion 

V document of particular rdevanoe; the dalnjedtovtttiOT 
cannot be considered Dovd or cannot be considered to 
involve 80 inventive step 

'y document of particular rdewce; the claimed Invwjilon 
cannot be considered to involve an inventive step when tbe 
document is combined with one or more docj- 
ments, such combination bdng obvious to a person sUlled 
m tbeart. 

'A' document member of the same patent family 



IV, CERHnCATION 



Date of tbe Aoual Completion of tbe International Search 

14-12-1992 



Date of MatUoeof this loteraatioBal Search Report 



rfJMaiUneofthtel 

25.1)193 



International Searching Authority 

EUROPEAN PATENT OFFICE 



Signature of Authorized Officer 

Mme Oagmar FRANK 



Fon PCT/lSA/210 4KOWd ikeell Urn 



rl98S) 



Page 2 

iDwraational Atfllai^ No PCT/US 92/08628 



• ,n nnn..MENTSC0 NSIDO EDT0BEKEIXVANT (CONTLVUU) tKU.W .HE SECO^u .nEET) 

nt irtrn rf P "'"""fa"- «^ 



WO.A,8600526 (A.I. CAPLAN et a1.) 30 
January 1986, see claims 

US.A,4743259 (M.E. BOLANDER et al.) 
10 May 1988, see the whole document 

WO A 9015586 (MASSACHUSETTS 

INSTITUTE OF TECHNOLOGY) 27 December 1990, see 

claims 

W0,A,8603122 (CURATECH, INC.) 5 June 
1986 



Relevant to aaim No. 

1-9 
1-9 
1-9 

1-9 



ANNEX TO THE INTERNATIONAL SEARCH REPORT 

ON INTERNATIONAL PATENT APPLICATION NO. US 9208628 

SA 65710 

niis annex Ifets the patent family roembere relating to the patent documents cited in Ae above-mentioned international search report. 
The membeis are as contained in the European Patent Office EDP fUe on 05/01/93 , . * ^ 

The European Patent Office is in no way liable for these particulars which are merely given for the purpose of mfonnabon. 



Patent document 
cited in search report 



Pubfication 
date 



Patent family 
member(6) 



PiAtication 
date 



GB-A- 2215209 



20-09-89 


JP-A- 


1232967 


18-09-89 




US-A- 


5133755 


28-07-92 


26-09-85 


US-A- 


4609551 


02-09-86 




AU-A- 


4153985 


11-10-85 




EP-A- 


0175762 


02-04-86 



WO-A- 8504185 



US-A- 


4563489 


07-01-86 


None 






EP-A- 


0341007 


08-11-89 


JP-A- 


2071747 


12-03-90 


WO-A- 


8600526 


30-01-86 


US-A- 


4620327 


04-11-86 




AU-A- 


4601485 


10-02-86 








EP-A- 


0188552 


30-07-86 


US-A- 


4743259 


10-05-88 


US-A- 


4902296 


20-02-90 


WO-A- 


9015586 


27-12-90 


CA-A- 


2056384 


06-12-90 




EP-A- 


0476045 


25-03-92 


WO-A- 


8603122 


05-06-86 


AU-B- 


596954 


24-05-90 




AU-A- 


5094985 


18-06-86 








CA-A- 


1261259 


26-09-89 








CH-A- 


673774 


12-04-90 








DE-A- 


3586355 


20-08-92 








DE-T- 


3590594 


29-01-87 








EP-A,B 


0202298 


26-11-86 








EP-A- 


0383363 


22-08-90 








GB-A- 


2248777 


22-04-92 








JP-T- 


62501628 


02-07-87 








NL-T- 


8520384 


29-ll-«4 








SE-A- 


8603228 


25-07-86 








US-A- 


4957742 


18-09-90 



For more details about this annex : sec Official Journal off the European Patent Office, No. 12/82 



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