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WORLD INTELLECTUAL PROPERTY ORGANIZATION 
International Bureau 




INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCI) 



(51) International Patent Classification 5 

A61F2/02, 2/28, 2/44 
A61K 9/14, 37/12, B32B 5/16 



Al 



(11) International Publication Number: WO 93/00050 

(43) Internationa) Publication Date: 7 January 1993 (07.01.93) 



(21) International Application Number: PCT/US92/05309 

(22) International Filing Date: 22 June 1992 (22.06.92) 



(30) Priority data: 
718,721 



21 June 1991 (21.06.91) 



US 



(71) Applicant (for all designated States except US): GENETICS 

INSTITUTE, INC. [US/US]; 87 Cambridge Park 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). TUREK, Tho- 
mas, J. [US/US]; 65 Bearse Avenue, Boston, MA 02124 
(US). ISAACS, Benjamin, S. [US/US]; 75 William G. 
Drive, Tewksbury, MA 01876 (US). PATEL, Himakshi 
[US/US]; 21 Clever Lane, Tewksbury, MA 01876 (US). 
KENLEY, Richard, A. [US/US]; 10 Westminster Road, 
Andover, MA 01810 (US). 



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



(81) Designated States: AU, BR, CA, FI, JP 5 KR, NO, RU, US, 
European patent (AT, BE, CH, DE, DK, ES, FR, GB, 
GR, IT, LU, MC, NL, SE). 



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: PHARMACEUTICAL FORMULATIONS OF OSTEOGENIC PROTEINS 



(57) Abstract 

A composition comprising a pharmaceutically acceptable admixture of an osteogenic protein; a polymer matrix compo- 
nent selected from the group consisting of polyflactic acid), poly(glycolic acid), and copolymers of lactic acid and glycolic acid; 
and an osteogenic protein-sequestering material. 



FOR THE PURPOSES OF INFORMATION ONLY 



Codes used 10 identify State, party lo the PCI* on the fiont pages of pamphlets publishing international 
applications under ;he PCI*. 



AT Austria 

AU Australia 

BC Barbados 

BE Belgium 

BF Burkina t-asu 

BC Bulgaria 

BJ Benin 

BR Brazil 

CA Canada 

CF Central African Republic 

CC Congo 

CH Swit/cilantl 

CI Cote d'lvuirc 

CM Cameroon 

G> ( >cchtx£ lovaL ia 

DE Cicrniany 

UK Denmark 

ES Spain 



Fl l*inlaiui 

FR Fiance 

CA Gabon 

CB United Kingdom 

CN Guinea 

CR Greece 

HU Hungary 

IE Ireland 

IT Italy 

JP Japan 

KP Democratic People** Republic 

of Korea 

KR Republic of Korea 

LI 1 lecliiemlein 

LK Sri Unla 

LU Ijixemhourg 

MC Monaco 

MC Madagascar 



Ml 


Mali 


MN 


Mongolia 


MR 


Mauritania 


MW 


Malawi 


NI- 


Netherlands 


NO 


Norway 


PL 


Poland 


RO 


Romania 


RU 


Russian Federation 


SD 


Sudan 


SE 


Sweden 


SN 


Senegal 


su 


Soviet Union 


TO 


Chad 


TC 


Togo 


US 


United States of America 



WO 93/00050 



PCI7US92/05309 



5 

TITLE OF THE INVENTION 
PHARMACEUTICAL FORMULATIONS OF OSTEOGENIC PROTEINS 

BACKGROUND OF THE INVENTION 
" 10 The subject invention relates to the field of osteogenic 

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 

15 bone formation. 

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 recombinant 

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

25 factors (TGF-a and TGF-0) 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); and proteins designated OP-1, 

30 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 
of bone formation is desired. For example, certain polymeric 

35 matrices such as acrylic ester polymer (Urist, U.S. 4,526,909) and 
lactic acid polymer (Urist, U.S. 4,563,489) have been utilized, but 
these formulations do not sequester the osteogenic protein for a 



1 



WO 93/00050 



PCT/US92/0S309 



time sufficient to optimally induce bone formation and further have 
been found to erode too slowly for optimal bone formation. 

A biodegradeable matrix of porous particles for delivery of an 
osteogenic protein designated as OP is disclosed in Kuberasampath, 
5 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 
10 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- 

15 retaining substance encapsulated in an inner aqueous layer 
surrounded by a polymer wall substance in an outer oil layer. 
Although bone morphogenic protein is listed as a polypeptide 
capable of such a formation, microencapsulation of osteogenic 
proteins prevents controlled release of such protein sufficient for 

20 optimal bone formation. 

Collagen matrices have also been used as delivery vehicles 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 

25 formulation capable of sequestering osteogenic proteins at a site 
where induction of bone formation is desired for a time sufficient 
to allow safe, effective induction of such bone formation. 

SUMMARY OV THE INVENTION 
30 In one embodiment, the subject invention provides a 

composition comprising a pharmaceutical^ acceptable admixture of 
an osteogenic protein; a polymer matrix component selected from the 
group consisting of poly(lactic acid), poly(glycolic acid), and 
copolymers of lactic acid and glycolic acid; and an osteogenic 
35 protein-sequestering alkylcellulose. 

2 



WO 93/00050 



PCT/US92/05309 



In another embodiment, the subject invention provides a 
composition comprising a pharmaceutical^ acceptable admixture of 
an osteogenic protein; a polymer matrix component selected from the 
group consisting of poly(lactic acid), poly (glycolic acid), and 
5 copolymers of lactic acid and glycolic acid; and an osteogenic 
protein-sequestering agent selected from the group consisting of 
hyaluronic acid, alginate, poly (ethylene glycol), polyoxyethylene 
oxide, carboxyvinyl polymer, and poly (vinyl alcohol) . 

In another embodiment, the subject invention provides a 
10 composition comprising polymeric particles having a spherical 
diameter of between about 150 and 850 microns and a porosity such 
that the surface area of the particles is between about 0.02 and 4 
m2 /9# wherein the polymer is selected from the group consisting of 
poly (lactic acid), poly (glycolic acid), and copolymers of lactic 
15 acid and glycolic acid; optionally in admixture with osteogenic 
protein. 

In yet another embodiment, the subject invention provides a 
composition comprising a pharmaceutical^ acceptable admixture of 
osteogenic protein and an effective solubilizing amount of a member 
20 selected from the group consisting of arginine, histidine, dextran 
sulfate, gamma-amino butyric acid, beta-amino propionic acid, 
glycine-glycine, glycine ethyl ester, histidine ethyl ester, lysine 
methyl ester, arginine methyl esteiv guanidine, sodium chloride, 
heparin, lysine, beta-alanine ethyl ester and agmatine. 

25 

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 
30 those discussed above. The preferred osteogenic proteins for use 
herein are those of the BMP class identified as BMP-1 through BMP-8 
in U.S. 4,877,864; U.S. 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 mature protein sequence beginning with the 



3 



WO 93/00050 



PCT/US92/05309 



amino acid Gin at nucleotide 1202 and ending with the amino acid 
Arg at nucleotide 1543, as described in detail in 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 
5 osteogenic activity and heterodimeric forms of such proteins. 
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, and will depend upon the size and 
10 nature of the defect being treated as discussed in more detail 
below, such amounts being orders of magnitude less than the amount 
of polymer matrix employed, preferably in the range of 1-50 ftg of 
protein for each 10 mg of polymer matrix employed and more 
preferably in the range of 0.5 - 5 ng protein for each mg of 
15 polymer matrix employed. 

The osteogenic proteins, can be utilized in the form of a 
pharmaceutically acceptable solution or in lyophilized form. In 
either case it is optimal to stabilize and solubilize the 
osteogenic protein, preferably at concentrations of at least 1 
20 mg/ml so that a pharmaceutically effective amount of protein can be 
delivered without undue volumes of carrier solution being 
necessary. However, a problem exists in that osteogenic proteins, 
particularly those of the BMP family, have proven to be difficult 
to solubilize. As detailed in the examples below, it has been 
25 discovered that amino acids having a net positive charge (e.g. net 
1+ species such as arginine, histidine, lysine 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 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 2 
units away) from the neutralizing negative charge (e.-g. net neutral 
species such as gamma-amino butyric acid, beta-amino propionic acid 
and glycine-glycine dipeptide) . Other solubilizing agents useful 



30 



4 



WO 93/00050 



PCT/US92/0S309 



herein include dextran sulfate, guanidine, heparin and sodium 
chloride. For use in solubilizing BMP-2, the preferred 
solubilizing agents are arginine and histidine (including esters 
thereof) . Arginine is used in concentrations of about 50-600 mM, 
5 preferably 300-500 mM. 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 300-500 
mM. Various well known methods may be used to compound the 

10 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 
subject invention is a polymeric material that can be formed into 

15 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, anhydrides, propylene-co- 
fumarates, or a polymer of one or more a-hydroxy carboxylic acid 

20 monomers, e.g. a-hydroxy acetic acid (glycolic acid) and/or o- 
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 61* lactic acid and glycolic acid 
is employed (PLGA) , the molar ratio of monomers can range from 1:99 

25 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) . The molecular weight of the polymer can range 
from about 1,000 to 100,000 with 30,000-50,000 being preferred when 

30 a 50:50 copolymer is employed. The higher 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 "porous 

35 particles." These porous particles are generally spherical having 



, AM , A PCT/US92/0S309 
WO 93/00050 



diameters of 150 to 850 microns. This particle size creates 
sufficient spacing between particles to allow mammalian 
osteoprogenitor cells to infiltrate and be positively influenced by 
the osteogenic protein (evidenced by an increase in osteogenic 
5 activity/bone growth rate) . 

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 bone formation 
has not previously been studied. The present inventors have 

10 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 from about 0.02 to 
4 m 2 /g. The present inventors have further discovered that it is 

15 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 particles. It is also possible to 
control the bioerosion rate by subjecting the porous particles to 

20 sterilizing doses of y radiation. The higher the y radiation dose, 
the faster the bioerosion. 

The method of producing porous particles in accordance with 
the subject- invention and discifssed hereinbelow results in 
particles having a porosity such that the surface area of the 

25 particles is increased about 2-250 fold over the surface area of 
non-porous particles of comparable size. More specifically, e.g., 
non-porous PLGA particles having an average size of 400/nn have a 
surface area of 0.018 m 2 /g. In contrast, PLGA particles useful in 
the subject invention made utilizing 50% NaCl as a porosigen have 

30 a surface area of between about 0.2 and 0.6 m 2 /g; and particles made 
using sucrose as a porosigen have a surface area of between about 
0.04 and 0.09 m 2 /g as described in Example 1. PLGA particles of the 
present invention made using liquid porosigen with homogenization 
as described in Example 2 have a surface area of between about 0.02 



6 



WO 93/00050 PCT/US92/05309 
and 4 m 2 /g, 

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. CH 2 C1 2 ) , and adding a 
5 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. 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 

10 with concomitant homogenization. When porosigen is added in liquid 
form with 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 

15 temperature. The resultant porous particles are hardened by 
extracting or evaporating residual solvent, and dried. 

The porous nature of the particles of the present invention 
creates sufficient surface area for protein adsorption and 
increases bi ©degradation, 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 Microroeritics 
ASAP 2000 system as is explained irt more detail in Examples 1 and 
2 below. The amount of porous particles used to treat a particular 

25 defect will, of course, depend upon the size of the defect being 
treated, and on the effective amount required to adsorb the 
osteogenic protein. 

The osteogenic protein-sequestering material useful in the 
practice of the subject invention is a pharmaceutical^ acceptable 

30 material having a viscosity and polarity such that, when added to 
an osteogenic protein/porous particle combination, a malleable 
(putty-like) composite is formed that handles appropriately for 
surgical implantation into an injury site. Adding the sequestering 
agent to the combination of bioerodible porous particles plus 



7 



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PCT/US92/05309 



osteogenic protein contains the adsorbed protein 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. The sequestering material further 
5 allows the osteogenic protein to diffuse from the malleable 
composite over a time interval appropriate for optimally increasing 
the rate of osteogenic activity of the progenitor cells. In the 
absence of such a sequestering material, osteogenic protein desorbs 
from the PLGA particles in-situ at a rate such that the 

10 osteoinducing effect of the protein is not clinically significant. 

A preferred family of sequestering agents is cellulosic 
materials such as alkylcellulose (including hydroxyalkylcellulose) , 
including methylcellulose, ethylcellulose, hydroxyethylcellulose, 
hydroxypropylcellulose, hydroxypropyl-methylcellulose, and 

15 carboxymethylcellulose, the most preferred being the cationic salts 
of carboxymethylcellulose (CMC). Other preferred sequestering 
agents include hyaluronic acid, sodium alginate, poly (ethylene 
glycol) , polyoxyethylene oxide, carboxyvinyl polymer and poly (vinyl 
alcohol) . The amount of sequestering agent useful herein is 0.5-20 

20 wt%, preferably 1-10 wt% based on total formulation weight, which 
represents the amount necessary to prevent desorbtion of the 
osteogenic protein from the polymer matrix and to provide 
appropriate handling of the composition, yet not so much that the 
progenitor cells are prevented from infiltrating the matrix, 

25 thereby providing the protein the opportunity to assist the 
osteogenic activity of the progenitor cells. 

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 the 

30 osteogenic protein from degradation during lyophilization) , 
antimicrobial preservatives such as methyl and propyl parabens and 
benzyl alcohol; antioxidants such as EDTA, citrate and BHT 
(butylated hydroxytoluene) ; and surfactants such as poly (sorbates) 
and poly (oxyethylenes) ; etc. 

35 According to the present invention, the osteogenic protein is 



8 



WO 93/00050 PCT/US92/0S309 

not included in the PLGA polymerization solution or encapsulated in 
PLGA microcapsules but is added to the already polymerized porous 
particles. It is preferable to add the porous particles to the 
solution of osteogenic protein prior to addition of sequestering 
5 agent in order to allow the protein to adsorb onto the particles. 
Of course, the traditional preparation of formulations in 
pharmaceutically acceptable form (i.e. pyrogen 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. 

10 The formulations may be provided to the clinic as a single vial 
formulation, either as a solution or in lyophilized 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 and sequestering agent are provided in a separate vial or 

15 vials. 

As seen in Examples 4 and 5 below, 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 cartilage and/or bone formation 

20 is desired. Such an implant may be used as a substitute 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; in osteomyelitis for bone 

25 regeneration, and in the dental field for erosion of the alveolar 
ridge and periodontal disease. In certain of these uses, the 
compositions of the subject invention may be used in combination 
with various bone cements, including erodible bone cements such as 
poly (propylene-co-fumarate) . The lower viscosity formulations may 

30 also be used as a percutaneous injection to accelerate healing of 
closed fractures. 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.). 

35 Currently, fresh autogeneic bone is widely used as a bone 

9 



WO 93/00050 



PCT/US92/0S309 



10 



30 



graft material. The limited supply of autogeneic bone, along with 
the necessity for an additional harvesting surgical procedure, 
constitute major disadvantages in use of autogeneic bone for bone 
grafting. In accordance with the present invention, the porous 
particles of the invention may be added to autogeneic bone to 
extend the amount of material available for bone grafting. The 
porous particles may also be used in combination with a 
sequestering agent as a substitute for bone wax at the site of a 
bony injury to act as a bioerodible hemostat. 



EXAMPLES 

All components utilized in these examples are pharmaceutical 
grade. The polymeric particle component was made from a 50:50 
(molar) random copolymer of lactic acid and glycolic acid (PLGA) 

15 having a weight average molecular weight of 30,000-50,000, a number 
average molecular weight of about 20,000 (by gel permeation 
chromatography relative to polystyrene standards) , and an inherent 
viscosity of 0.35-0.45 dL/g. The osteogenic protein utilized was 
rBMP-2. The production and characterization of rBMP-2 is described 

20 in detail in the above-referenced US 5,013,649. The sequestering 
agents utilized included carboxymethyl-cellulose, hydroxy- 
propylmethyl cellulose, sodium alginate, hyaluronic acid, and 
poly (ethylene glycol). The carboxy*»ethylcellulose (CMC) utilized 
contained 0.7 degree of substitution (carboxymethyl groups per 

25 hydroxy group on cellulose and had a viscosity of 2480 cps. 

Tj' VftMPT.T! 1 - PPTCP&PATION OF POROUS PARTICLES 
BV ROT .VENT* TCVAPORAT TOW TECHNIQUE 



PLGA was dissolved in CH 2 C1 2 (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) 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 
35 (95%). The resulting particles were washed with water for 



10 



WO 93/00050 



PCT/US92/0S309 



injection and vacuum dried to give a free-flowing product. BET 
surface area analysis was performed using a Micrometrics ASAP 2000 
system. The measurement of surface area is based upon adsorption 
and desorption of Krypton gas at the surface and within the pores 
of the solid sample. The unit calculates and prints out the 
surface area: 

1 = £zl (P/P 0 ) + 



VA[(P 0 /P)-1J V m C V„C 

10 V = volume absorb at pressure P P 0 = saturation pressure 

P/P 0 = relative pressure P = pressure 

C = constant A = gas cross sectional area 

Vm = Monolayer Capacity 

By plotting 1 vs P/P 0 , the slope being 

15 VA((P 0 /P)-1 

Ol and the intercept being l the surface area 
V C V C 

20 S t = V m NA where N = Avogadro's number and V = molar volume. 
V 



25 



Reactant details and results are depicted in Table 1 and Table 2, 
respectively. 



11 



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Table 1 





Batch 
No. 


PLGA 
(crams) 


CH2CL2 
fmLl 




PVA 


Impellers Stirring 
(toD/btm) frpm) 


5 


1 


10 


67 


NaCl/50 


1200 


(2rshtn/A-100) 


215 




2 


10 


67 


NaCl/80 


1200 


(2rshtn/A-100) 


215 


10 


3 


6.7 


67 


suc/50 


1200 


(2rshtn/A-100) 


215 




4 


6.7 


67 


NaCl/50 


1200 


(2rshtn/A-100) 


235 




5 


16 


106 


suc/50 


2000 


(A-310/A-310) 


140 


15 


6 


20 


133 


suc/50 


2000 


(A-310/A310) 


140 




7 


20 


133 


suc/50 


2000 


(A-310/A-310) 


140 


20 


8 


20 


133 


suc/50 


2000 


(8.5rsh/A-310) 


100 




9 


20 


133 


suc/50 


2000 


(8.5rsh/A-310) 


140 




10 


20 


133 


suc/50 


2000 


(2rshtn/A-100) 


140 



25 



12 



WO 93/00050 



PCT/US92/05309 



Table 2 



Batch Surface Area Yield % Density 



5 



* 10 



No. 


Mn 


Mw 




(m 2 /a) 


250-710ixm 


(ams/cc) 


1 


17500 


30800 


1.75 


0.54 


27.3 


0.41 




19400 


31700 


1.64 


0.037 


71. 6 


0.67 


3 


19700 


40900 


2.07 


0.089 


92.7 


0.70 


A 
H 




57700 


1.86 


0.28 


69. 5 


0 37 


C 






1 • 60 


0. 035 


N/A 


N/A 


6 


20200 


37700 


1.86 


0.079 


79.5 


0.64 


7 








0.060 


85 


0.76 


8 








0.038 


85 


0.86 


9 








0.057 


65 


0.71 


10 


20200 


37700 


1.86 


0.060 


64 


0.68 



25 



f 



WO 93/00050 



PCT/US92/05309 



TiYAMPT.E 2 - PREPAR&TTON OF P OROUS PARTICLES 
bv gnT.VENT EXTPRCTIOW T ECHNIQUE 

A 100-g sample of PLGA was dissolved in 670 mL of CH 2 C1 2 . 

5 The porosigen solution was prepared by dissolving 5g NaCl in 50 

mL of a 0.2% aqueous solution of polyvinyl alcohol). A 50-mL 

aliquot of the porosigen solution was added to a homogenizer and 

agitated at 3300 rpm. The PLGA solution was then added to the 

homogenizer (with agitation). A 77 mM NaCl solution in 10 liter 

10 of 0.2% aqueous poly (vinyl alcohol) was added to a 12-liter 

reactor and agitated at 175 rpm. The PLGA/ porosigen suspension 
was added to the reactor over 90 minutes. A 77 mM NaCl solution 
in 0.2% aqueous poly (vinyl alcohol) was then pumped into and out 
of the 12-liter reactor, thereby extracting methylene chloride 

15 from the mixture. After solvent extraction was complete, 
agitation was stopped, the porous particles are allowed to 
settle, the supernatant was decanted, and the particles were 
hardened with ethanol (95%) followed by washing with water or a 
solution of polysorbate 20 (0.05%) in water. The washed . 

20 particles were dried by vacuum or convection techniques. 

Particles prepared according to this procedure typically have 
0.09 g/cc bulk density, and total surface area = 4 m 2 /g- The 
dried particles can be sterilized by ethylene oxide exposure or 7 
irradiation. As noted above, the y radiation dose influences 

25 bioerosion rate. Table 3 gives examples of porous particles 
prepared by the above process. 



14 



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PCT/US92/0S309 



Table 3 



10 



15 



20 



25 



30 



35 



Batch 
No. 


Homogenizer 
(mm) 


Porosigen Soln, 


Density 
(a/cc) 


Yield 
150-500 


1 


10, 000 


50 


0.09 


80 


2 


3, 500 


50 


0.15 


80 


3 


6,000 


50 


0.09 


95 


4 


3,000 


50 


0.10 


95 


5 


1,900 


50 


0.29 


90 


6 


2,200 


50 


0.10 


95 


7 


2,000 


50 


0.09 


99 


8 


2,000 


25 


0.14 


90 


9 


2,000 


12.5 


0.24 


90 


10 


2, 000 


20 


0.28 


<75 


11 


2 , 000 


2U 


U • io 


^ / O 


12 


2,000 


17.5 


0.16 


95 


13 


2,000 


15.5 


0.21 


94 


14 


2,500 


15.5 


0.17 


90 


15 


2,200 


15.5 


0.19 


90 



15 



WO 93/00050 PCT/US92/0S309 



FY AMPLE * - SOI.UBT T.T7ATIOW OF PROTEIN 

Solubility of rBMP-2 in the excipients listed in Table 4 
below was determined by dialysis in accordance with the 
5 following. Concentrations were determined by absorbencies at 280 
nm using an extinction coefficient of 1.62. Protein solution (1 
ml) containing 2-3 mg/ml of protein, 0.5M arginine and lOmM 
phosphate (pH 6.5) was dialyzed against 1000 ml of buffer 
containing 0.5M excipient of choice (see Table 3) and 0.5M 

10 arginine, pH 6.5. The dialysis was carried out at room 

temperature. The excipients were allowed to equilibrate with the 
protein solution. The protein solution was then dialyzed twice 
against 1000 ml of an arginine-less buffer solution of otherwise 
identical composition. Solubility results are presented in Table 

15 4. Unless otherwise indicated, excipients were tested at 
standard concentrations of 500mM. 



16 



WO 93/00050 PCT/US92/05309 

yable 4 

NET CHARGE SOLUBILITY 

EXCIPIENT f«* neutral Pin fma7ml^ 

e-amino caproic acid 0 <0.4 

6- amino valeric acid o <0.4 

7- amino butyric acid 0 >1«7 
0-aroino propionic acid 0 >1.1 
Glycine-Glycine dipeptide 0 >1.8 
Glycine 0 <0 . 4 
Arginine 1+ >5 . 4 
Lysine 1+ >0.9 
Guanidine 1+ >1.8 
Glycine (ethyl ester) 1+ >2.2 
Histidine (ethyl ester) 2+ >2.2 
Lysine (methyl ester) 2+ >2.2 
Arginine (methyl ester) 2+ >2.2 
Histidine 1+ >2.2 
Dextran sulfate 

1.0M NaCl 0 >1.8 



17 



WO 93/00050 



PCI7US92/05309 



10 



■PYAMPLE 4 - TMPIAN T ANALYSIS 

rBMP-2 (22 /ig) , mannitol (8 mg) , and epsilon aminocaproic 
acid (2M, 20M1J were lyophilized onto the PLGA particles (10 mg, 
20% porosity, 325 mm). CMC (5.5 mg, -9%) was added and the solid 
powder was sterilized using ethylene oxide. Water for Injection 
(60 mD was added to form a malleable implant of the composite. 
As a control, the same formulation was made without the CMC, in 
which case a gelatin capsule was used to hold the formulation in 
place. Both formulations were implanted in a 5 mm rat femur 
defect. The rats were sacrificed after twelve weeks. Ex-ylyo 
analysis of the new bone was performed radiographically relative 
to the contralateral femur. Surprisingly, 83% (10 of 12) of the 
femur defects showed union utilizing the formulations of the 
subject invention, compared with only 50% (4 of 8) for the 
15 control. 

gy&MPT.T! 5 - T WPTAWT ANALYSIS 

A 300-/iL aliquot of 0.12 mg/mL rBMP2 solution (in 0.25 M 
arginine, 10 mM histidine, pH 6.5, 'plus 20 mM calcium chloride) 
was added to 9.6 mg of porous particles (0.16 g/cc density, 

20 surface area = approximately 0.8 m*/g, sterilized by 2.5 Mrad 7 
radiation) . To this mixture was added 15 mg sodium alginate. 
Gentle mixing provided a malleable composite. Similar 
formulations were made using 9 mg of hydroxypropylmethylcellulose 
or 9 mg carboxymethylcellulose except that the 0.12 mg/mL rBMP2 

25 was solubilized in 0.25 M arginine plus 10 mM histidine, pH 6.5 

(no added calcium chloride). As controls, malleable formulations 



18 



WO 93/00050 



PCIYUS92/05309 



were prepared with 0,25 M arginine plus lOmM histidine containing 
0 mg/mL rBMP2. The formulations were implanted into 8 mm 
diameter critical size circular defects in rat calvarium. After 
21 days the animals were sacrificed and bone regeneration 
5 assessed by radiomorphometry (X-OMATL high contrast X-ray film, 
using Cambridge 520 Image Analysis System) .* The control samples 
for alginate, CMC, and hydroxypropylmethyl-cellulose 
formulations, respectively, showed only 18%, 10% and 10%, 
radiopacity. By comparison the alginate, CMC and 
10 hydroxypropylmethylcellulose formulations (with added rBMP2) 
showed, respectively, 72%, 70% and 67% radiopacity indicating 
significant new bone growth. 



19 



WO 93/00050 



PCT/US92/05309 



What is claimed is: 

1. A composition comprising a pharmaceutical ly acceptable 
admixture of 

(i) an osteogenic protein; 

(ii) a polymer matrix component selected from the group 
consisting of poly (lactic acid) , -poly (glycolic acid), 
and copolymers of lactic acid and glycolic acid; and 

(iii) an osteogenic protein-sequestering alkylcellulose. 

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 2 wherein the cellulosic material 
is selected from hydroxypropylmethylcellulose and 
carboxymethylcellulose . 

5. The composition of claim 3 wherein the cellulosic material 
is selected from hydroxypropylmethylcellulose and 
carboxymethylcellulose. 

6. The composition of claim 5, wherein the polymer matrix 
component is a copolymer of lactic acid and glycolic acid. 

20 



WO 93/00050 



PCT/US92/05309 



7. The composition of claim 1 wherein the polymer matrix 
component is in the form of porous particles. 

8. The composition of claim 2 wherein the polymer matrix 
component is in the form of porous particles. 

9. The composition of claim 3 wherein the polymer matrix 
component is in the form of porous particles. 

10. The composition of claim 4 wherein the polymer matrix 
component is in the form of porous particles. 

11. The composition of claim 5 wherein the polymer matrix 
component is in the form of porous particles. 

12. The composition of claim 6 wherein the polymer matrix 
component is in the form of porous particles. 

13. A composition comprising a pharmaceutical ly acceptable 
admixture of 

(i) BMP-2; 

(ii) a polymeric matrix component comprising polymeric 
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 0.02 and 4 m 2 /g, 



WO 93/00050 



PCT/US92/0S309 



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 
carboxymethylcellulose . 

14. The composition of claim 1 wherein the osteogenic protein is 
TGF-/3. 

15. The composition of claim 1 wherein the osteogenic protein is 
Vgr-1. 

16. The composition of claim 1 wherein the osteogenic protein is 
OP-1. 

17. The composition of claim 1 wherein the osteogenic protein is 
selected from COP-5 and COP-7. 

18. A composition comprising polymeric particles having a 
spherical diameter of between about 150 and 850 microns and a 
porosity such that the surface area of the particles is between 
about 0.02 and 4 m 2 /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. 

19. A composition comprising polymeric particles as defined in 

22 



WO 93/00050 



PCT/US92/0S309 



claim 18 in admixture with osteogenic protein. 

20. A composition comprising a pharmaceutical^ acceptable 
admixture of osteogenic protein and an effective solubilizing 
amount of a member selected form the group consisting of 
arginine, histidine, dextran sulfate, gamma-amino butyric acid, 
beta-amino propionic acid, glycine-glycine, glycine ethyl ester, 
histidine ethyl easter, lysine methyl ester, arginine methyl 
ester, guanidine, sodium chloride, heparin, lysine, beta-alanine 
ethyl ester and agmatine. 

21. A composition comprising a pharmaceutical ly acceptable 
admixture of 

(i) an osteogenic protein; 
(ii) a polymer matrix component selected from the group 
consisting of poly (lactic acid), poly (glycolic acid), and 
copolymers of lactic acid and glycolic acid; and 

(iii) an osteogenic protein-sequestering agent selected 
from the group consisting of hyaluronic acid, sodium alginate, 
poly (ethylene glycol), polyoxyethylene oxide, carboxyvinyl 
polymer, and poly(vinyl alcohol). 



23 



INTERNATIONAL SEARCH REPORT 



International application No. 
PCT/US92/05309 



A. CLASSIFICATION OF SUBJECT MATTER 

IPC(5) :Pletae See Extra Sheet. 

US CL :Please See Extra Sheet. 
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) 
U.S. : 424/ 422, 423. 426, 484, 486, 489, 490, 497, 498; 514/2, 21, 773; 428/402.2, 402.21; 530/353, 840; 623/16, 17 



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 practicable, search terms used) 



DOCUMENTS CONSIDERED TO BE RELEVANT 



Category* 



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



Relevant to claim No. 



US, A, 4,917,893 (OKADA et al) 17 April 1990, col. 1-2; col. 2, line. 64-65; col. 5, lines 
5 and 22-24 

US, A, 4,637,931 (SCHMITZ) 20 January 1987, col. 3, lines 47-52 



1-20 



1-20 



Further documents are listed in the continuation of Box C. Q See patent family annex. 



* Special categories of cited document*: 

"A" document defeuni the general state of the art which is not considered 

to be part of particular relevance 

*E* earlier documeol publiihed on or after the international filing date 

"L* document which may throw doubt* oo priority claan(a) or which it 

cited to establish the publication dale of another citation or other 
special reason (as specified) 

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

"P° docunicm published prior to the tnternatiooal filing date but later than 
the priority date churned - 



later document published after the tnternatiooal filing date or priority 
date and not in conflict with the application but ched to understand the 
principle or theory underlying the invention 

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 

document of particular relevance; the claimed invention cannot be 
consklcred to involve an inventive step when the document is 
combined with one or more other such documents, 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 
06 August 1992 



Date of mailing of the international search report 



03 N 





Name and mailing address of the ISA/ 
Commissioner of Patent! and Trademark* 
Box PCT 

Washington, D.C. 20231 
Facsimile No. NOT APPLICABLE 



Authorized officer 

CARLOS. 
Telephone No. (703) 308-0237 



Form PCT/1SA/210 (second sheetXJuly 1992)* 



INTERNATIONAL SEARCH REPORT 



International application No. 
PCT/US92/Q5309 



A. CLASSIFICATION OF SUBJECT MATTER: 
IPC (5): 

A 61 F 2/02, 2/28, 2/44; A 61 K 9/14, 37/12; B 32 B 5/16 

A. CLASSIFICATION OF SUBJECT MATTER: 
USCL : 

424/ 422, 423, 426, 484, 486, 489, 490, 497, 498; 514/2, 21, 773; 428/402.2, 402.21; 530/353 , 840; 623/16, 



Form PCT/ISA/210 (extra shcct)(July 1992> 



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