U.S. patent application number 15/616563 was filed with the patent office on 2017-09-21 for system and method for multiphasic release of growth factors.
This patent application is currently assigned to Induce Biologics Inc. The applicant listed for this patent is Induce Biologics Inc. Invention is credited to Cameron M. L. CLOKIE, Sean A.F. PEEL.
Application Number | 20170266259 15/616563 |
Document ID | / |
Family ID | 47008753 |
Filed Date | 2017-09-21 |
United States Patent
Application |
20170266259 |
Kind Code |
A1 |
CLOKIE; Cameron M. L. ; et
al. |
September 21, 2017 |
System and Method for Multiphasic Release of Growth Factors
Abstract
A system for multiphasic delivery of at least one growth factor
at a treatment site comprises a delivery vehicle for releasing at
least one growth factor in an initial release profile and a carrier
for releasing at least one growth factor in a sustained release
profile. The initial release profile releases at least one growth
factor over a period of hours to days, wherein the growth factor is
released in a large amount initially, with the remainder being
released in progressively lower amounts. The sustained release
profile releases at least one growth factor over a period of days
to weeks, wherein the growth factor is released at a generally
constant amount over such period. The system of the invention is
particularly suited for applications on bioimplants. The invention
also comprises methods and kits for multiphasic delivery of at
least one growth factor.
Inventors: |
CLOKIE; Cameron M. L.;
(Toronto, CA) ; PEEL; Sean A.F.; (Oakville,
CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Induce Biologics Inc |
Toronto |
|
CA |
|
|
Assignee: |
Induce Biologics Inc
Toronto
CA
|
Family ID: |
47008753 |
Appl. No.: |
15/616563 |
Filed: |
June 7, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14111518 |
Dec 17, 2013 |
9675670 |
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PCT/CA2012/050234 |
Apr 11, 2012 |
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15616563 |
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61474049 |
Apr 11, 2011 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 9/1611 20130101;
A61K 9/1682 20130101; A61L 27/46 20130101; A61K 38/1866 20130101;
A61L 27/46 20130101; A61K 9/1641 20130101; A61P 43/00 20180101;
A61K 47/6923 20170801; A61K 47/34 20130101; A61K 9/0024 20130101;
A61K 38/18 20130101; A61K 38/1858 20130101; A61K 38/1825 20130101;
A61K 47/02 20130101; A61P 19/08 20180101; A61K 38/1875 20130101;
A61K 9/19 20130101; A61L 27/46 20130101; A61K 38/1841 20130101;
A61K 47/10 20130101; A61P 17/02 20180101; A61L 27/54 20130101; A61K
9/5084 20130101; C08L 71/02 20130101; C08L 67/04 20130101 |
International
Class: |
A61K 38/18 20060101
A61K038/18; A61K 47/02 20060101 A61K047/02; A61K 47/10 20170101
A61K047/10; A61K 47/34 20170101 A61K047/34; A61K 9/00 20060101
A61K009/00; A61K 9/16 20060101 A61K009/16; A61K 9/19 20060101
A61K009/19; A61K 9/50 20060101 A61K009/50; A61L 27/46 20060101
A61L027/46; A61L 27/54 20060101 A61L027/54 |
Claims
1. A composition for multiphasic release of growth factors at a
treatment site, the composition comprising: a delivery vehicle
comprising at least one first growth factor; and a carrier
comprising at least one second growth factor; wherein: the delivery
vehicle is a polymer liquid or gel that is in a flowable form to be
applied on the carrier and adapted to release about 80% of the at
least one first growth factor within a first time period of 72
hours; and, the carrier consists of a plurality of particles that
are adapted to release the at least one second growth factor in a
sustained release profile over a second time period.
2. The composition according to claim 1, wherein the polymer gel is
formed from poloxamer 407.
3. The composition according to claim 1, wherein the second time
period comprises several days or weeks.
4. The composition according to claim 1, wherein the at least one
first growth factor and the at least one second growth factor are
the same or different and at least one of the first and second
growth factors is chosen from: growth factors of the transforming
growth factor beta superfamily; insulin-like growth factors (IGFs);
fibroblast growth factors (FGFs); platelet derived growth factors
(PDGFs); and vascular endothelial growth factors (VEGFs).
5. The composition according to claim 1, wherein the at least one
first growth factor and the at least one second growth factor are,
independently, BMP-2 or BMP-7.
6. The composition according to claim 1, wherein the delivery
vehicle delivers at least 10% of the total amount of the at least
one first growth factor and the carrier delivers at least 50% of
the total amount of the at least one second growth factor.
7. The composition according to claim 1, wherein the plurality of
particles is dispersed within a polymeric matrix.
8. The composition according to claim 7, wherein the polymeric
matrix comprises polylactic acid (PLA) or polylactic-co-glycolic
acid (PLGA).
9. The composition according to claim 1, wherein the at least one
second growth factor of the carrier is provided on the surfaces of
the particles or is mixed within the particles.
10. The composition according to claim 1, wherein the at least one
first growth factor comprises at least 10% of the total amount of
growth factors provided in the composition and the at least one
second growth factor comprises at least 50% of the total amount of
growth factors provided in the composition.
11. The composition according to claim 1, wherein the delivery
vehicle comprises a reverse phase polymer.
12. The composition according to claim 1, wherein the composition
comprising the delivery vehicle and the carrier is in the form of a
gel or putty when the carrier is mixed within the delivery
vehicle.
13. The composition according to claim 1, wherein the ratio of the
carrier to the delivery vehicle is from 1:1 to 2:1 (v/v).
14. A kit for multiphasic delivery of growth factors, the kit
comprising: a delivery vehicle component; at least one first growth
factor associated with the delivery vehicle and adapted to be
delivered by the delivery vehicle; a carrier component; and at
least one second growth factor associated with the carrier and
adapted to be delivered by the carrier; wherein: the delivery
vehicle is a polymer liquid or gel that is in a flowable form to be
applied on the carrier and adapted to release about 80% of the at
least one first growth factor within a first time period of 72
hours; and the carrier consists of a plurality of particles that
are adapted to release the at least one second growth factor in a
sustained release profile over a second time period.
15. The kit according to claim 14, wherein: the at least one first
growth factor and the at least one second growth factor are the
same or different and at least one of the first and second growth
factors is chosen from: growth factors of the transforming growth
factor beta superfamily; insulin-like growth factors (IGFs);
fibroblast growth factors (FGFs); platelet derived growth factors
(PDGFs); and vascular endothelial growth factors (VEGFs).
16. The kit according to claim 14, wherein the at least one first
growth factor and the at least one second growth factor are,
independently, BMP-2 or BMP-7.
17. The kit according to claim 14, wherein the plurality of
particles is dispersed within a polymeric matrix.
18. The kit according to claim 14, wherein the at least one second
growth factor of the carrier is provided on the surfaces of the
particles or is mixed within the particles.
19. The kit according to claim 14, wherein the at least one first
growth factor comprises at least 10% of the total amount of growth
factors provided in the kit and the at least one second growth
factor comprises at least 50% of the total amount of growth factors
provided in the kit.
20. The kit according to claim 14, wherein the delivery vehicle
comprises a reverse phase polymer.
21. The kit according to claim 14, wherein the delivery vehicle and
the carrier are adapted to form a gel or putty when the carrier is
mixed with the delivery vehicle.
22. The kit according to claim 14, wherein the ratio of the carrier
to the delivery vehicle is from 1:1 to 2:1 (v/v).
23. A method of multiphasic release of growth factors, the method
comprising: delivering at least one first growth factor in an
initial release profile over a first time period with a delivery
vehicle, wherein the delivery vehicle is adapted to release about
80% of the at least one first growth factor within a first time
period of 72 hours; delivering at least one second growth factor in
a sustained release profile over a second time period with a
carrier, wherein the carrier consists of a plurality of particles
that are adapted to release the at least one second growth factor
in a sustained release profile over a second time period.
24. The method according to claim 23, wherein the second time
period comprises several days or weeks.
25. The method according to claim 23, wherein the at least one
first growth factor and the at least one second growth factor are
the same or different and at least one of the first and second
growth factors is chosen from: growth factors of the transforming
growth factor beta superfamily; insulin-like growth factors (IGFs);
fibroblast growth factors (FGFs); platelet derived growth factors
(PDGFs); and vascular endothelial growth factors (VEGFs).
26. The method according to claim 23, wherein the at least one
first growth factor and the at least one second growth factor are,
independently, BMP-2 or BMP-7.
27. The method according to claim 23, wherein the delivery vehicle
delivers at least 10% of the total amount of the growth factors and
the carrier delivers at least 50% of the total amount of the growth
factors.
28. The method according to claim 23, wherein the carrier comprises
a plurality of calcium phosphate particles.
29. The method according to claim 23, wherein the carrier comprises
a plurality of calcium phosphate particles dispersed within a
polymeric matrix.
Description
CROSS REFERENCE TO PRIOR APPLICATIONS
[0001] This application is a Continuation of U.S. application Ser.
No. 14/111,518, filed on Apr. 11, 2012, which is a national stage
application of PCT International Patent Application Number
PCT/CA2012/050234, filed Apr. 11, 2012, which claims priority under
the Paris Convention from U.S. Application No. 61/474,049, filed on
Apr. 11, 2011. The entire contents of the aforementioned
applications are incorporated herein by reference.
FIELD OF THE INVENTION
[0002] This invention relates to systems and methods for releasing
biological substances. In particular, the invention relates to the
release of growth factors associated with bioimplants. More
particularly, the invention provides a system and method for
producing a multiphasic release profile of at least one growth
factor to improve the performance of the bioimplant.
BACKGROUND OF THE INVENTION
[0003] Growth factors (GFs) are peptides and proteins that
stimulate the growth and/or differentiation of cells via the
interaction of the GFs with specific cell surface receptors. Growth
factors play an integral role in the repair and regeneration of
tissues and exogenous application of GFs can be used to stimulate
the repair of various tissues and organs including bone, cartilage,
skin and mucosa and to enhance repair of tissues through the
stimulation of angiogenesis at the repair site.
[0004] The transforming growth factor beta (TGF.beta.) superfamily
of secreted growth and differentiation factors in mammals has over
30 members. These dimeric proteins are characterized by a conserved
seven cystine knot-based structure. They regulate the
proliferation, differentiation and migration of many cell types,
and have important roles in morphogenesis, organogenesis, tissue
maintenance and wound healing. The TGF.beta. superfamily of growth
factors can be subdivided into several subfamilies including the
transforming growth factor beta subfamily, the bone morphogenetic
protein (BMP) and growth and differentiation factor (GDF) family
(also called the BMP subfamily), and the inhibin and activin
subfamily.
[0005] The BMP subfamily of the TGF.beta. superfamily comprises at
least twenty proteins, including BMP-2, BMP-3 (also known as
osteogenin), BMP-3b (also known as growth and differentiation
factor 10, GDF-10), BMP-4, BMP-5, BMP-6, BMP-7 (also known as
osteogenic protein-1, OP-1), BMP-8 (also known as osteogenic
protein-2, OP-2), BMP-9, BMP-10, BMP-11 (also known as growth and
differentiation factor 8, GDF-8, or myostatin), BMP-12 (also known
as growth and differentiation factor 7, GDF-7), BMP-13 (also known
as growth and differentiation factor 6, GDF-6), BMP-14 (also known
as growth and differentiation factor 5, GDF-5), and BMP-15 (for a
review, see e.g., Azari et al. Expert Opin Invest Drugs
2001;10:1677-1686).
[0006] BMPs have been shown to stimulate matrix synthesis in
chondroblasts; stimulate alkaline phosphatase activity and collagen
synthesis in osteoblasts, induce the differentiation of early
mesenchymal progenitors into osteogenic cells (osteoinduction),
regulate chemotaxis of monocytes and mesenchymal cells, and
regulate the differentiation of neural cells (for a review, see
e.g., Azari et al. Expert Opin Invest Drugs 2001;10:1677-1686 and
Hoffman et al. Appl. Microbiol. Biotech 2001;57:294-308).
[0007] One of the many functions of BMP proteins is to induce
cartilage, bone, and connective tissue formation in vertebrates.
The most osteoinductive members of the BMP subfamily are BMP-2,
BMP-4, BMP-6, BMP-7, BMP-8 and BMP-9 (see, e.g., Hoffman et al.,
Appl. Microbiol Biotech 2001, 57-294-308; Yeh et al., J Cellular
Biochem., 2005; 95-173-188; and Boden, Orthopaedic Nursing
2005,24:49-52). This osteoinductive capacity of BMPs has long been
considered very promising for a variety of therapeutic and clinical
applications, including fracture repair; spine fusion; treatment of
skeletal diseases, regeneration of skull, mandibular, and bone
defects; and in oral and dental applications such as dentogenesis
and cementogenesis during regeneration of periodontal wounds,
extraction socket grafting, alveolar ridge augmentation, and sinus
augmentation. Currently, recombinant human BMP-2 sold as
INFUSE.RTM. by Medtronic FDA approved for use in spinal fusion
surgery, for repair of fracture non-unions and for use in oral
surgery, while and recombinant human BMP-7 sold as OP-1.RTM. by
Stryker is approved as an alternative to autograft in recalcitrant
long bone nonunion and for revision posterolateral
(intertransverse) lumbar spine fusions, where autograft and bone
marrow harvest are not feasible or are not expected to promote
fusion
[0008] Other recombinant growth factors that have been used
exogenously to enhance bone repair include various TGF.beta.s (see
Clokie & Bell, J. Craniofacial Surg. 2003, 14:268-77), members
of the fibroblast growth factor superfamily (FGFs) (see Kawaguchi
et al., (2007) J. Orthopaedic Res. 25(4): 480-487), members of the
platelet derived growth factor superfamily (PDGFs) (see Hollinger
et al., 2008 JBJS 90(s1):48-54), and vascular endothelial growth
factor (VEGF) (Street et al., 2002 PNAS 99:9656-61)
[0009] For these growth factors to be effective they must be active
and available at a sufficient concentration at the time when
critical densities of the appropriate responsive cells are present
in the repair site. The short half-life, thermal instability,
sensitivity to proteases and/or solubility of the GFs requires
their administration in combination with a carrier to achieve this
requirement.
[0010] A number of carriers have been evaluated for the delivery of
GFs. These include fibrous collagen sponges, gelatin hydrogels,
fibrin gels, heparin, reverse phase polymers such as the
poloxamers, scaffolds composed of poly-lactic acid (PLA),
poly-glycolic acid (PGA) or their co-polymers (PLGA),
heparin-conjugated PLGA scaffolds, and inorganic materials such as
calcium phosphates. For example the bioimplant (GEM-21S.RTM.) which
is used for periodontal regeneration uses beta tricalcium phosphate
(.beta.-TCP) as the carrier for rhPDGF-BB.
[0011] However, these carriers are of limited effectiveness, due to
loss of growth factor activity when associated with the carrier,
inefficient release of the GF at the implantation site, and/or poor
protection from proteolysis and degradation. For example the
bioimplant Infuse.RTM. uses a type I collagen sponge as the carrier
for rhBMP-2. The rhBMP-2 is released in a burst from the carrier
and the half life of the BMP within the wound site is 1-3 days
(Winn et al., 1998, Adv. Drug Del. Rev. 31:303; Friess et. al.,
1999, Intl. J. Pharm.,187:91). By the time the mesenchymal stem
cells which regenerate bone have migrated into the wound site only
fractions of a percent of the original amount of BMP loaded is
present to stimulate these cells to make bone. The current solution
to ensure an effective level of BMP remaining at these later times
is to significantly increase the amount of BMP that is initially
loaded. These increased doses increase the risk of complications
including bone formation beyond the implant site, autoimmune
responses and potentially cancer. Further this dramatically
increases the cost of the implant.
[0012] Therefore, a need exists in the art for materials and
methods which release growth factors with a profile which minimizes
the amount of growth factor that needs to be loaded to achieve the
required therapeutic effect.
[0013] One strategy is to encapsulate the GF in a biodegradable
polymeric matrix that releases the GF with a sustained release
profile over many days. For example BMPs have been combined with
poly-lactic acid (PLA) or poly-lactic co-glycolic acid (PLGA) to
produce sustained release profiles. However the incorporation of
the BMP in the PLA or PLGA can denature the BMP reducing its
activity and it is difficult to manipulate the release profile to
optimize the effectiveness of the bioimplant. Further the
degradation rate of these carriers is typically such that large
amounts of GF remain locked away long after healing is
complete.
[0014] Another strategy is to chemically immobilize the GF directly
onto the carrier retain it at the implant site. However this may
also result in partial or complete loss of activity of the GF, and
restricts the GF activity such that only those cells directly in
contact with the carrier are able to interact with the GF and
respond (see Steinmuller-Nethl, D. et al., Biomaterials, 2006, 27:
4547-56) which is not desirable as the effect is limited to the
immediate interface with the carrier and not throughout the wound
site.
[0015] In nature during wound healing multiple GF are present
within the wound site and surrounding tissue at varying
concentrations at different times. For example, immediately
following bone fracture, platelets at the injury site will
initially release large amounts of PDGF, with a sharp decline in
protein levels within the fracture site over the following days
(see Tyndall et al., Clinical Orthopaedics and Related Research,
2003, 408: 319-330). Conversely BMP-2 is expressed at all stages of
the fracture healing process (see Rasubala et al. British Journal
of Oral and Maxillofacial Surgery, 2003, 41: 173-178). The
concentration of these growth factors is estimated to be orders of
magnitude lower than those used during therapeutic application of
exogenous GF due to matching of the concentration to the cellular
requirements and synergistic effects of the multiple growth
factors. Producing a system that allows the delivery of growth
factors with multiphasic release profiles and the release of
multiple growth factors with different release profiles would
permit the use of bioimplants with GF release profiles that more
closely mimic GF release during the natural healing process than
current bioimplants that release a single growth factor in a burst
or with sustained release.
[0016] This background information is provided for the purpose of
making known information believed by the applicant to be of
possible relevance to the present invention. No admission is
necessarily intended, nor should be construed, that any of the
preceding information constitutes prior art against the present
invention.
SUMMARY OF THE INVENTION
[0017] The present invention provides, in one aspect, a system,
method and kit for the multiphasic release of at least one growth
factor at, for example a treatment site. For this purpose, the
system of the invention may be provided as a bioimplant or the
like. In one aspect, the method of the invention delivers at least
one growth factor in an initial release followed by the delivery of
at least one growth factor in a "sustained release profile". The
invention utilizes a delivery system for the initial release and a
carrier for the sustained release.
[0018] In one aspect, the same growth factor is released in the
initial and sustained release profiles. In another aspect,
different growth factors are released, with a first growth factor
released in an initial profile and a second growth factor released
in a sustained release profile. As will be known to persons skilled
in the art, the release of two different growth factors in such
differing manners is believed to more closely mimic the natural
growth factor release system at a treatment site.
[0019] In accordance with one aspect of the invention, there is
provided a carrier that provides a sustained release of at least
one growth factor, combined with a delivery vehicle that provides
an initial release of at least one growth factor. The combination
of the carrier and the delivery vehicle results in a multiphasic
release profile of the growth factor(s).
[0020] In preferred embodiments the growth factor ("GF") is a
member of the transforming growth factor beta (TGF.beta.)
superfamily. In particularly preferred embodiments the growth
factor is a bone morphogenetic protein (BMP).
[0021] In one aspect of the present invention, the carrier ("CAR")
is formed of calcium phosphate particles dispersed within a polymer
scaffold or matrix. In one aspect, the scaffold or matrix is
further coated with a hydroxyapatite layer.
[0022] In one embodiment the at least one growth factor is/are
applied as a liquid to the calcium particles and are then
lyophilized onto the particles before combining with the polymer
matrix.
[0023] In another aspect of the present invention the carrier is
formed by mixing one or more calcium phosphate powders with a
liquid solution containing at least one growth factor to produce a
calcium phosphate cement. In one aspect, the cement is then ground
into particles.
[0024] In preferred embodiments the delivery vehicle is a reverse
phase polymer. In particularly preferred embodiments the reverse
phase polymer is a poloxamer, more particularly poloxamer 407 (also
called Pluronic.TM. F127).
[0025] As indicated above, in one aspect, the carrier and the
delivery vehicle are adapted to release the same growth factor
while in another aspect, the carrier and delivery vehicle are
adapted to release different growth factors. In yet another aspect
of the invention, the carrier and delivery vehicle are each adapted
to release combinations of two or more growth factors, with the
combination released by each being the same or different.
[0026] Thus, in one aspect, the invention provides a system for
multiphasic release of growth factors at a treatment site, the
system comprising: [0027] a delivery vehicle comprising at least
one first growth factor; and [0028] a carrier comprising at least
one second growth factor;
[0029] wherein: [0030] the delivery vehicle is adapted to release
the at least one first growth factor in an initial release profile
over a first time period; [0031] the carrier is adapted to release
the at least one second growth factor in a sustained release
profile over a second time period.
[0032] In another aspect, the invention provides a method of
multiphasic release of growth factors, the method comprising:
[0033] delivering at least one first growth factor with an initial
release profile; [0034] delivering at least one second growth
factor in a sustained release profile.
[0035] In a further aspect, the invention provides a kit for
multiphasic delivery of growth factors, the kit comprising: [0036]
a delivery vehicle component; [0037] at least one first growth
factor associated with the delivery vehicle; [0038] a carrier
component; and [0039] at least one second growth factor associated
with the carrier.
BRIEF DESCRIPTION OF THE DRAWINGS
[0040] The invention will now be described with reference to the
appended figures, which are briefly described below.
[0041] FIG. 1 illustrates a sustained release profile exhibited by
the carrier of the invention.
[0042] FIG. 2 illustrates the initial release profile exhibited by
the delivery vehicle of the invention.
[0043] FIG. 3 illustrates differing release profiles based on an
amount of growth factor in the delivery vehicle and carrier. A
multiphasic release profile is observed when growth factors are
incorporated into both the delivery vehicle and carrier
(50-50).
[0044] FIG. 4 illustrates the in vivo activity of the bioimplants
where a growth factor is released as shown in FIG. 3 according to
the method of the invention.
[0045] FIG. 5 illustrates the formation of new bone (Bone) onto
calcium phosphate particles (CaP) when a bioimplant produced
according to the method of the invention was implanted into a
mouse.
[0046] FIG. 6 illustrates the histological appearance of the new
bone (Bone) formed on a carrier (Carrier) when bioimplant produced
according to the method of the invention was implanted into a
mouse
[0047] FIG. 7 illustrates a short sustained growth factor release
profile produced by a carrier produced according to the method of
the invention
[0048] FIG. 8 illustrates how a sustained release profile can be
altered by changing the properties of the carrier produced
according to the method of the invention
DETAILED DESCRIPTION OF THE INVENTION
[0049] Growth factors (GF) play an integral role in the repair and
regeneration of tissues and exogenous GFs can be used to stimulate
the repair of various tissues and organs. For exogenous growth
factors to be effective in stimulating repair they must be retained
at the site requiring repair, and be protected from inactivation,
sequestration or degradation. To achieve this carriers are used.
However the release of growth factors from these carriers is not
ideal and cannot be easily modified. The current invention is based
on the discovery that the multiphasic release of growth factors
from a bioimplant increases the efficacy of the implant.
[0050] The present inventors have developed methods and materials
for enhancing the efficacy of, for example, bioimplants by
improving the release kinetics or release profile of growth factors
at sites of implantation, while maintaining the activity of the
growth factors. In one aspect, the present invention provides a
growth factor delivery system and method comprising a carrier
containing at least one growth factor, combined with a delivery
vehicle also containing at least one growth factor. The at least
one growth factor released by the carrier and delivery vehicle may
be the same or different.
[0051] The system and method of the invention can be used for a
variety of therapeutic and clinical applications, including:
fracture repair; bone grafts; spine fusion; and regeneration of
skull, mandibular, and bone defects. For such applications, the
system of the invention is preferably provided on, or in the form
of a bioimplant.
[0052] Definitions
[0053] Unless defined otherwise below, all technical and scientific
terms used herein have the same meaning as commonly understood by
one of ordinary skill in the art to which this invention
belongs.
[0054] As used herein the term "bioimplant" refers to a material
which is suitable for implantation and contains an exogenous growth
or biologically active factor. As discussed further herein, the
system of the present invention is preferably used by applying same
to a bioimplant. The bioimplant is then provided within a body of a
subject wherein the system releases at least one growth factor in a
multiphasic release profile.
[0055] As used herein the term "growth factor" refers to peptides
and proteins that stimulate the growth and/or differentiation of
cells via the interaction of the GFs with specific cell surface
receptors. Examples of growth factors include the bone
morphogenetic proteins (BMPs), transforming growth factor beta
(TGF.beta.), the insulin-like growth factors (IGF), the fibroblast
growth factors (FGFs), platelet derived growth factor (PDGF) and
vascular endothelial growth factor. In preferred embodiments the
growth factors are BMPs. p By "recombinant" is meant a protein
produced by a transiently transfected, stably transfected, or
transgenic host cell or animal as directed by an expression
construct containing the cDNA for that protein. The term
"recombinant" also encompasses pharmaceutically acceptable salts of
such a polypeptide
[0056] As used herein, the term "polypeptide" or "protein" refers
to a polymer of amino acid monomers that are alpha amino acids
joined together through amide bonds. Polypeptides are therefore at
least two amino acid residues in length, and are usually longer.
Generally, the term "peptide" refers to a polypeptide that is only
a few amino acid residues in length. A polypeptide, in contrast
with a peptide, may comprise any number of amino acid residues.
Hence, the term polypeptide included peptides as well as longer
sequences of amino acids.
[0057] As used herein, the terms "bone morphogenetic protein" or
"bone morphogenic protein" or "BMP" are used interchangeably and
refer to any member of the bone morphogenetic protein (BMP)
subfamily of the transforming growth factor beta (TGF.beta.)
superfamily of growth and differentiation factors, including BMP-2,
BMP-3 (also known as osteogenin), BMP-3b (also known as growth and
differentiation factor 10, GDF-10), BMP-4, BMP-5, BMP-6, BMP-7
(also known as osteogenic protein-1, OP-1), BMP-8 (also known as
osteogenic protein-2, OP-2), BMP-9, BMP-10, BMP-11 (also known as
growth and differentiation factor 8, GDF-8, or myostatin), BMP-12
(also known as growth and differentiation factor 7, GDF-7), BMP-13
(also known as growth and differentiation factor 6, GDF-6), BMP-14
(also known as growth and differentiation factor 5, GDF-5), and
BMP-15.
[0058] The terms "bone morphogenetic protein" and "BMP" also
encompass allelic variants of BMPs, function conservative variants
of BMPs, and mutant BMPs that retain BMP activity. The BMP activity
of such variants and mutants may be confirmed by any of the methods
well known in the art (see the section Assays to measure BMP
activity, below) or as described in Example 1
[0059] In preferred embodiments, the BMP is BMP-2, BMP-4, BMP-5,
BMP-6, BMP-7, BMP-8 or BMP-9. In particularly preferred embodiments
the BMP is BMP-2, BMP-4 or BMP-7.
[0060] In preferred embodiments the BMP is a mammalian BMP (e.g.,
mammalian BMP-2 or mammalian BMP-7). In particularly preferred
embodiments, the BMP is a human BMP (hBMP) (e.g. hBMP-2 or
hBMP-7).
[0061] As used herein the term "scaffold" refers to a material
whose purpose is to provide a structure which supports cell
adhesion, migration and ingrowth into a tissue repair site.
[0062] As used herein the term "carrier" refers to a material
comprising single or multiple components and is adapted to release
at least one growth factor at a treatment site in a "sustained
release" profile over a period of time. In one aspect, the period
of time taken by the carrier to release the at least one growth
factor is between several days and several weeks. Preferably, the
carrier is adapted to release the at least one growth factor over a
period of weeks.
[0063] In preferred embodiments the carrier also acts as a scaffold
or matrix. As discussed above, in one aspect of the invention, the
carrier is formed of calcium phosphate particles dispersed within a
macroporous polymer scaffold or matrix. In one aspect, the scaffold
or matrix is further coated with a hydroxyapatite layer. In one
embodiment the at least one growth factor is applied as a liquid to
the calcium particles and are then lyophilized onto the particles
before combining with the polymer matrix.
[0064] As used herein the term "delivery vehicle" refers to a
material which comprises or contains at least one growth factor and
is adapted to release the at least one growth factor at a treatment
site in an initial release profile over a time period. In one
aspect, the material forming the delivery vehicle is in the form of
a gel. In one aspect, the period of time taken by the delivery
vehicle to release the at least one growth factor is between
several hours and several days. In a preferred embodiment of the
invention, the delivery vehicle releases the majority of the at
least one growth factor in an "initial release" or "initial release
profile" that lasts a period of hours. Preferably, the delivery
vehicle is adapted to release at least 80% of the growth factor(s)
contained therein (or associated therewith) within a period of 72
hours.
[0065] In preferred embodiments the delivery vehicle is a reverse
phase polymer. As used herein the term "reverse phase" refers to
the property whereby the polymer undergoes a reversible temperature
dependent transition from a liquid to a gel. In one aspect the
transition temperature is between 15.degree. C. and 37.degree. C.
Preferably the transition temperature is between 15.degree. C. and
25.degree. C. As would be known to persons skilled in the art,
"normal phase" materials increase their viscosity with a decline in
temperature. In contrast, reverse phase materials show a decline in
viscosity as the temperature drops below their transition
temperature.
[0066] In particularly preferred embodiments the reverse phase
polymer is a poloxamer, more particularly Pluronic.TM. F127 (also
known as poloxamer 407).
[0067] As used herein the term "sustained release" or "sustained
release profile" refers to the release of at least one growth
factor, by the carrier, over a period of several days or weeks with
the amount released over an initial period being similar to or less
than the amount released over the same period after several days or
weeks of implantation. Preferably, a sustained release profile
lasts at least one week. As will be understood by persons skilled
in the art, typically, the amount of growth factor released in a
sustained release profile over the first three days will be less
than the amount released over the following seven days.
[0068] As used herein the term "initial release" or "initial
release profile" refers to the initial release, by the delivery
vehicle, of a large amount of at least one growth factor followed
by progressively smaller amounts released over a period of hours or
days. In one aspect, an initial release profile results in the
delivery of at least 80% of the loaded growth factor(s) within a
period of roughly 72 hours. An initial release profile is
illustrated in FIG. 2.
[0069] As used herein the term "multiphasic release" refers to an
initial release of the at least one growth factor over an initial
period of time, followed by "sustained" release of the at least one
growth factor over a second period of time. Preferably, the initial
period of time is roughly several hours and the second period of
time is roughly several days to weeks. Such a release profile may
also be referred to as "biphasic release" since it occurs in two
stages. In preferred embodiments, the initial release is provided
by the delivery system of the invention and the "sustained" release
is provided by he carrier of the invention.
[0070] In one aspect of the invention, the delivery vehicle
component comprises at least 10% and not more than 50% of the total
amount of growth factor(s) delivered by the system of the invention
and the carrier component comprises at least 50% of the total
amount of growth factor(s) delivered by the system.
[0071] Assays to Measure BMP Activity
[0072] Assays to characterize in vitro and in vivo function of
recombinant BMPs are well known in the art, (see, e.g., U.S. Pat.
No. 4,761,471; U.S. Pat. No. 4,789,732; U.S. Pat. No. 4,795,804;
U.S. Pat. No. 4,877,864; U.S. Pat. No. 5,013,649; U.S. Pat. No.
5,166,058; U.S. Pat. No. 5,618,924; U.S. Pat. No. 5,631,142; U.S.
Pat. No 6,150,328; U.S. Pat. No. 6,593,109; Clokie and Urist,
Plast. Reconstr. Surg. 2000; 105:628-637; Kirsch et al., EMBO J
2000; 19:3314-3324; Vallejo et al., J. Biotech. 2002; 94:185-194;
Peel et al., J. Craniofacial. Surg. 2003; 14:284-291; and Hu et
al., Growth Factors, 2004; 22:29-33).
[0073] Such assays include: in vivo assays to quantify
osteoinductive activity of a BMP following implantation (e.g., into
hindquarter muscle or thoracic area) into a rodent (e.g. a rat or a
mouse) (see, for example, U.S. Pat. No. 4,761,471; U.S. Pat. No.
4,789,732; U.S. Pat. No. 4,795,804; U.S. Pat. No. 4,877,864; U.S.
Pat. No. 5,013,649; U.S. Pat. No. 5,166,058; U.S. Pat. No.
5,618,924; U.S. Pat. No. 5,631,142; U.S. Pat. No 6,150,328; U.S.
Pat. No. 6,503,109; Kawai and Urist., Clin. Orthop. Relat. Res.,
1988; 222:262-267; Clokie and Urist, Plast. Reconstr. Surg.,
2000;105:628-637; and Hu et al., Growth Factors, 2004;22:29-33); in
vivo assays to quantify activity of a BMP to regenerate skull
trephine defects in mammals (e.g., rats, dogs, or monkeys) (see,
for example, U.S. Pat. No. 4,761,471 and U.S. Pat. No. 4,789,732);
in vitro assays to quantify activity of a BMP to induce
proliferation of in vitro cultured cartilage cells (see, for
example, U.S. Pat. No. 4,795,804); in vitro assays to quantify
activity of a BMP to induce alkaline phosphatase activity in in
vitro cultured muscle cells (e.g., C2C12 cells, ATCC Number
CRL-1772) or bone marrow stromal cells (e.g., murine W-20 cells,
ATCC Number CRL-2623) (see, for example, U.S. Pat. No. 6,593,109;
Ruppert et al., Eur J Biochem 1996;237:295-302; Kirsch et al., EMBO
J, 2000;19:3314-3324; Vallejo et al., J Biotech, 2002;94:185-194;
Peel et al., J Craniofacial Surg., 2003;14:284-291; and Hu et al.,
Growth Factors, 2004;22:29-33); in vitro assays to quantify
activity of a BMP to induce FGF-receptor 2 (FGFR3) expression in
cultured mesenchymal progenitor cell lines (e.g., murine C3H10T1-2
cells) (see, for example, Vallejo et al. J Biotech
2002;94:185-194); in vitro assays to quantify activity of a BMP to
induce proteoglycan synthesis in chicken limb bud cells (see, for
example, Ruppert et al., Eur J Biochem 1996;237:295-302); and in
vitro assays to quantify activity of a BMP to induce osteocalcin
treatment in bone marrow stromal cells (e.g., murine W-20 cells;
ATCC Number CRL-2623) (see, for example, U.S. Pat. No.
6,593,109).
[0074] Assays to Measure BMP Binding and Release
[0075] Various assays can be used to measure binding and release of
recombinant BMP from a carrier. For example, the amount of
recombinant BMP protein can be quantified by any of the techniques
well known in the art, including dot blots, immunoassay (e.g.,
enzyme linked immunosorbent assays, ELISA), measurement of the
increase in radioactivity present in the release buffer when the
bioimplant incorporates radiolabeled BMP and chromatography (e.g.,
high pressure liquid chromatography, HPLC and ion-exchange
chromatography).
[0076] Such methods are well known in the art (See for example,
Harlow and Lane, Using Antibodies: A Laboratory Manual, Cold Spring
Harbor Laboratory Press. 1999; Gosling, ed., Immunoassays: A
Practical Approach, Oxford University Press. 2000; Oliver, ed.,
HPLC of Macromolecules: A Practical Approach., Oxford University
Press, 1998; Millner, ed., High Resolution Chromatography: A
Practical Approach. Oxford University Press, 1999; Hockfield et
al., Selected Methods for Antibody and Nucleic Acid Probes. Cold
Spring Harbor Laboratory Press. 1993; Gore, ed., Spectrophotometry
and Spectrofluorimetry: A Practical Approach. Oxford University
Press, 2000).
[0077] For example, protocols for radioimmunoassay analysis of BMP
proteins have been described (see, for example, U.S. Pat. No.
4,857,456). For example, protocols for immunoblot analysis of BMP
proteins have been described (see, for example, Wang et al. Proc
Natl Acad Sci USA 1990; 87:2220-2224). For example, ELISA kits for
the quantitation of protein levels of human, rat, or mouse BMP-2
are commercially available, for example, from R&D Systems
(catalog #DBP200, PDBP200, or SBP200). For example, ELISA kits for
the quantitation of protein levels of human BMP-7 are commercially
available, for example, from R&D Systems (catalog #DY354 or
DY354E).
[0078] Kits
[0079] In one aspect, the invention provides a kit for containing
the system described herein. In one embodiment, the kit comprises
the necessary components for making the delivery vehicle and the
carrier as well as the needed growth factors. That is, the kit of
the invention would comprise the necessary components for making
the delivery vehicle and the carrier as well as least one growth
factor associated with the delivery vehicle and at least one growth
factor associated with the carrier.
[0080] Preferably, the delivery vehicle and the associated growth
factor(s) are maintained in separate containers, to be combined at
the time of use. This would be particularly preferable in cases
where the delivery vehicle may comprise a liquid or a gel. In such
case, the associated growth factor(s) may be kept in a separate
container as a lyophilized powder. At the time of use, the growth
factor(s), in powder form, may be combined with the liquid or gel
delivery vehicle.
[0081] The kit preferably comprises a further container comprising
the carrier onto which may be loaded or coated the associated
growth factor(s). In one embodiment, the carrier material may also
be maintained separate from the associated growth factor(s).
[0082] In a preferred embodiment, the kit of the invention would
comprise at least three containers for each of the following: 1)
the delivery vehicle component; 2) the at least one first growth
factor (i.e. the growth factor(s) associated with the delivery
vehicle); and, 3) the carrier and the least one second growth
factor (i.e. the growth factor(s) associated with the carrier). In
use, the at least one second growth factor, in powder form, is
combined with the liquid or gel form delivery vehicle and the
mixture is then applied to the carrier onto which the at least one
second growth factor was pre-loaded.
[0083] In one aspect, the kit of the invention may comprise any
necessary reagents and/or instructions and/or vessels as may be
needed.
EXAMPLES
[0084] The present invention will now be described by means of the
following examples. These examples illustrate the novel findings by
the inventors that a multiphasic release profile of a growth
factor, such as rhBMP-2 produced by loading part of the BMP within
a carrier that releases BMP with a sustained release and part of
the BMP within a delivery vehicle that releases BMP with an initial
release is more effective than carriers that only produce a burst
release or a sustained release.
[0085] As will be obvious to one skilled in the art it is possible
to place one growth factor within the carrier and a different
growth factor within the delivery vehicle, resulting in different
release profiles of each growth factor.
[0086] It will be understood that the examples provided herein are
intended solely to illustrate the present invention and not to
limit the scope of the invention in any way. Likewise, the
invention is not limited to any particular preferred embodiments
described herein. Indeed, many modifications and variations of the
invention may be apparent to those skilled in the art upon reading
the present specification. The invention is therefore to be limited
only by the terms of the appended claims, along with the full scope
of equivalents to which the claims are entitled.
Example 1
Manufacture of a Sustained Release Composite Carrier Containing Bmp
by Encapsulation in PLGA.
[0087] This example demonstrates how to form a carrier containing
rhBMP-2 and which releases the growth factor in a sustained release
profile.
[0088] Materials and Methods
[0089] PLGA 75/25 with inherent viscosity of 1.33 dL/g
(MW=205,000-210,000) was purchased from Birmingham Polymers Inc.
(Birmingham, Ala.). Tetracalcium phosphate (TTCP) was obtained from
Taihei Chemical Industrial Co. (Osaka, Japan) and dicalcium
phosphate anhydrous (DCPA) and dimethyl sulfoxide (DMSO) were
obtained from Sigma Chemical Co. (MO, USA). Sugar particles were
purchased from Tate & Lyle North America Inc. (Toronto,
Canada).
[0090] Resorbable calcium phosphate particles were prepared by
mixing equimolar TTCP and DCPA with deionized distilled water
(ddH2O) at 100% relative humidity for 24 h. The reactant was ground
and sieved through 45 .mu.m sieve.
[0091] Recombinant human BMP-2 (rhBMP-2, Induce Biologics Inc) in
was prepared in formulation buffer (1.5 mg/ml, pH 4.5; 5 mm
glutamic acid, 2.5% glycine, 0.5% sucrose and 0.01% Tween.TM. 80
with ddH.sub.2O). The protein solution was added to vials
containing CaP powder and agitated for at least 15 minutes. The
powder was then frozen and lyophilized.
[0092] Particles with (CaP-BMP) or without (CaP) BMP were then used
to make CaP particulate-PLGA scaffold blocks by
phase-inversion/particle leaching as follows: PLGA was dissolved in
DMSO at a concentration of 11.5% (w/v). To this solution, the CaP
and CaP-BMP particles were thoroughly mixed according to a CaP/PLGA
ratio of 2:1 (w/w). Sugar crystals with size ranges of 0.85-1.18 mm
were dispersed in the CaP/PLGA and the mixture was solidified at
-18.degree. C. in a mold. The PLGA was precipitated and the sugar
crystals leached out by soaking in three changes of ddH.sub.2O.
[0093] A layer of hydroxyapatite was deposited onto and throughout
the macroporous composite scaffolds as follows: dry PLGA/CaP
cylinders, measuring 2 mm in diameter and 2 mm in length, were
pre-wetted in 70% ethanol and immersed in 60 ml of 3.times.SBF for
a period of 9 days at 37.degree. C. SBF was prepared as follows: to
1.8L of ddH2O under vigorous stirring the following salts were
added sequentially 29.711 g NaCl, 2.206 g CaCl.sub.2-2H.sub.2O, 10
ml 1M HCl, 0.852 Na.sub.2HPO.sub.4. The final volume was brought to
2L. The SBF solution was changed daily. Following coating, the 3PCC
samples were rinsed in ddH.sub.2O and air dried.
[0094] This resulted in the formation of a macroporous composite
carrier (3PS) that is able to release rhBMP-2 with a sustained
release profile over at least seven days. These results are
illustrated in FIG. 1.
Example 2
Manufacture of a Sustained Release Carrier Containing BMP by
Encapsulation in a Calcium Phosphate Cement
[0095] The present example demonstrates how to form a calcium
phosphate cement (CPC) carrier containing rhBMP-2 that has a
sustained release profile.
[0096] Materials and Methods
[0097] Tetracalcium phosphate (TTCP) was obtained from Taihei
Chemical Industrial Co. (Osaka, Japan) and dicalcium phosphate
anhydrous (DCPA) was obtained from Sigma Chemical Co. Macroporous
biphasic calcium phosphate granules (Eclipse) were purchased from
Citagenix (Laval Qc, Canada). Recombinant human BMP-2 (rhBMP-2,
Induce Biologics Inc) was prepared in formulation buffer (1.5
mg/ml, pH 4.5; 5 mm glutamic acid, 2.5% glycine, 0.5% sucrose and
0.01% Tween.TM. 80 with ddH2O).
[0098] Resorbable calcium phosphate cement particles were prepared
by mixing equimolar TTCP and DCPA with rhBMP-2 solution. The
reactant was ground and sieved through a 300 and 100 .mu.m sieve
and particles between 100 and 300 .mu.m, retained.
[0099] This resulted in the formation of calcium phosphate cement
carrier particles into which the rhBMP-2 was incorporated. Upon
implantation into an animal BMP is released in a sustained manner
over a period of at least several weeks.
[0100] To produce a CPC based sustained release carrier that also
acted as a macroporous scaffold CPC particles (0.1 to 0.3 mm) were
mixed macroporous calcium phosphate granules (1-2 mm) in a 1:1
ratio (w/w).
Example 3
Manufacture of a Sustained Release Carrier Containing BMP by use of
a Coating that binds BMP.
[0101] The present example demonstrates how to form a carrier that
has a sustained release profile by applying a BMP binding coating.
One such method is to coat a scaffold with an antibody or BMP
binding protein as described in our co-pending application number
U.S. application Ser. No. 13/002,444 (the entire content of which
is incorporated herein by reference).
[0102] Materials and Methods
[0103] Purified polyclonal rabbit anti-human BMP-2 antibodies were
purchased from Cell Sciences, (Canton Mass., Cat #PA0025).
Macroporous biphasic calcium phosphate (BCP) granules (Eclipse)
were purchased from Citagenix (Laval, Qc, Canada.)
[0104] Sterile BCP granules were weighed out in a biosafety cabinet
and placed in sterile TPP tubes (Mandel Scientific, Guelph ON,
Canada). The antibody solution was diluted in phosphate buffered
saline to final concentration of 150, 300 and 600 ng of antibody in
1 ml PBS, filter sterilized and applied to the scaffold at a 1:1
v/v ratio. The samples were agitated for at least 15 minutes at
room temperature, before being frozen and lyophilized. BMP solution
was then applied to the granules, allowed to soak for 15 minutes at
room temperature and then frozen and re-lyophilized.
[0105] This resulted in the formation of a BCP granules coated with
antibody that bound and slowly released the rhBMP-2 in a sustained
fashion.
[0106] The amount of rhBMP-2 that can be bound can be increased by
increasing the amount of antibody used. The rate of release can be
increased by using antibodies with lower affinity or avidity.
Example 4
Production of a BMP Containing Delivery Vehicle using F127
[0107] The present example demonstrates how to prepare a delivery
vehicle containing rhBMP-2 using F127.
[0108] Materials and Methods
[0109] Poloxamer was prepared as follows: 100 ml of distilled water
was chilled to 4.degree. C. and various amounts of poloxamer 407
were added slowly with stirring over a period of several hours,
until all the solid prill was dissolved making a final solution
ranging between 12 and 33%. The poloxamer solution was then
sterilized in an autoclave (121.degree. C., 20 minutes, 30 psi).
Following sterilization, the poloxamer solution was kept at
4.degree. C. until use.
[0110] Lyophilized recombinant human BMP-2 powder (rhBMP-2, Induce
Biologics Inc) was added to the poloxamer solution and was slowly
mixed.
[0111] Alternatively rhBMP-2 was added from solution (1 mg/ml, pH
4.5; 5 mm glutamic acid, 2.5% glycine, 0.5% sucrose and 0.01% Tween
80) at a 1/10 or 1/20 ratio (v/v).
[0112] This resulted in the formation of a delivery vehicle that
released more than 80% of the rhBMP-2 over the first two days (as
illustrated in FIG. 2).
Example 5
Production of a Bioimplant with a Multiphasic Release Profile
[0113] The present example demonstrates how to form a 3PS-F127
bioimplant containing rhBMP-2 that releases the rhBMP-2 with a
multiphasic release profile.
[0114] Materials and Methods
[0115] The 3PS carrier (as described in Example 1) containing 0,
4.55 or 9.1 .mu.g of rhBMP-2 per 5 mg of carrier was prepared and
stored in Eppendorf tubes. A delivery vehicle containing 0, 4.55 or
9.1 .mu.g of rhBMP-2 in 45.5 .mu.l F127 (prepared as described in
Example 4) was stored in Eppendorf tubes at 4.degree. C.
Immediately prior to use, the F127 was pipetted onto the 3PS
carrier and the carrier was mixed into the delivery vehicle.
[0116] This 3PS-F127 bioimplant was then used to measure BMP
release in vitro and bone formation activity in vivo as described
below.
[0117] The ratios of carrier to delivery vehicle can be varied to
produce gel (1:1 ratio v:v) or putties (2:1 ratio v:v). Further the
ratio of BMP to carrier or the particle size of the carrier can be
varied to alter the sustained release profile. Finally the amount
of rhBMP-2 in the carrier and the delivery vehicle can be varied to
alter the amount of rhBMP-2 released initially over the first few
hours compared to amount released over the following weeks.
Example 6
An In Vitro Assay for Release of BMPs from Bioimplants.
[0118] The present example describes how to measure the release of
rhBMP-2 from the various bioimplants described in Examples 1 to
5.
[0119] Materials & Methods
[0120] Bioimplants containing known amounts of rhBMP-2 prepared as
in Examples 1 to 5 were transferred to Eppendorf tubes. The total
amount of rhBMP-2 used was 9.1 .mu.g of rhBMP-2 per 5 mg of carrier
and 45.5 .mu.l of F127, or 20 .mu.g of rhBMP-2 to 10 mg of carrier
to 100 .mu.l of F127.
[0121] Samples were then incubated under agitation with a 1 ml
solution of release buffer comprising phosphate buffered saline
(PBS)+1% BSA at 37.degree. C. The buffer was removed and replaced
with fresh release buffer after various times (e.g. 1, 2, 5, 7 and
10 days) and the collected solutions were stored with 1.5 ml vials
at -20.degree. C. for further analysis.
[0122] The amount of BMP-2 released into the buffer was measured
using a commercial ELISA (Quantikine.TM. hBMP-2 ELISA, RnD
Systems). The ELISA was carried out according to the manufacturer's
instructions.
[0123] Results
[0124] No BMP was detectable in release buffer collected from any
of the bioimplants which had not been loaded with BMP. The carrier
samples which had been loaded with rhBMP-2 demonstrated a sustained
release of rhBMP-2 over the period of the study, while samples in
the delivery vehicle alone were released in an "initial release
profile".
[0125] When the carrier and delivery vehicle were combined, various
release profiles were obtained depending on which component the BMP
was loaded into. When 100% of the rhBMP-2 (9.1 .mu.g) was loaded
within the 3PS (5mg) carrier which was then mixed with 33% F127
(45.5 .mu.l), the BMP release profile matched the sustained
pattern, where the amount of BMP released over the first 2 days was
5 ng, between days 3 and 5 it was 8 ng and between days 5 and 7 it
was 10 ng (FIG. 3, 100-0).
[0126] When 100% of the rhBMP-2 (9.1 .mu.g) was loaded within 33%
F127 (45.5 .mu.l ) and then was then mixed with the 3PS carrier (5
mg) which had no BMP within it, the BMP was released where the
amount of BMP released over the first 1 day was 2363 ng, over the
second day was 381 ng and then 12 ng on the third day (FIG. 3;
0-100).
[0127] When the BMP was distributed between the carrier and the
delivery vehicle the bioimplant demonstrated a biphasic release
profile, with an intermediate initial release followed by sustained
release of BMP (FIG. 3; 50-50).
Example 7
An In Vitro Assay to test the Activity of Released BMPs.
[0128] The present example describes how to determine whether the
rhBMP-2 released from the bioimplants retains its activity. To
demonstrate that the released rhBMP is biologically active,
responsive cells can be cultured in with the releasate and their
response to the growth factor measured. Such assays are known in
the art (see Peel et al., J. Craniofac. Surg. 2003,
14:284-291).
[0129] Materials & Methods
[0130] Materials with or without rhBMP-2 as described in Examples 1
to 5 were prepared. Releasates were prepared as described in
Example 3 except the buffer was alpha minimal essential medium with
15% fetal bovine serum and antibiotics (aMEM+15%FBS+AB)
[0131] C2C12 cells were seeded into 24 well tissue culture plates
at 0.5.times.10.sup.5 cells/ml, 1 ml alpha MEM+15%FBS per well.
After various periods between 24 and 72 hours the media was removed
and the various releasates were applied. Negative controls included
C2C12 cells cultured with fresh aMEM+15%FBS+AB. Positive controls
included C2C12 cells incubated with aMEM+15%FBS+AB containing 25,
50 and 100 ng/ml rhBMP-2. After 48 hours the cells were lysed in 1
ml cell lysis buffer (Cellytic Sigma Aldrich) and the alkaline
phosphatase (ALP) activity of the cell lysates measured using the
para-nitrophenol phosphate assay (Sigma Aldrich). The cell protein
content of the lysates was measured using Coomassie Plus Reagent
(Fisher) and was used to normalize ALP activity to the number of
cells in each well.
[0132] Generally, to determine whether there has been any loss in
activity of the BMP when associated with the carrier or delivery
vehicle, a standard activity curve of ALP/PTN results for rhBMP-2
standards which have not been associated with the carrier or
delivery vehicle is determined. The concentration of active rhBMP-2
in the releasates can be determined from this standard curve and
this is expressed as a percentage of the total the amount of
rhBMP-2 present in the releasates as determined by ELISA.
Example 8
Evaluation of Osteoinductive Activity of Multiphasic BMP
Implants
[0133] The present example describes how to determine the
osteoinductive activity of BMP containing bioimplants in vivo. To
evaluate the ability of bioimplants to induce bone formation the
mouse muscle pouch assay was used. In this model the bioimplant is
placed in a muscle pouch made in the hind limbs of the mouse and
the size of the induced bone formed is proportional to the amount
of BMP tested. Such assays are known in the art (see for example
Barr et al., Oral Surg. Oral Med. Oral Pathol. Oral Radiol. Endod.,
2010; 109:531-40.)
[0134] Materials and Methods
[0135] Bioimplants were prepared as described in Examples 1 and 5.
Under anesthesia bilateral pouches were made in the thigh muscles
of the hind limbs of male CD-1 mice aged 37-42 days, by blunt
dissection. The bioimplants were then placed into sterile gelatin
capsules which had been placed into the muscle pouch. The muscle
was pulled together and the skin closed with Mitchel clips.
[0136] The animals were euthanized on post-op day 28. The hind
limbs were harvested and fixed with 10% buffered formalin.
Following fixation, the specimens were imaged using a microCT
scanner (General Electric Healthcare eXplore.TM. Locus SP). Samples
were scanned and reconstructed using the manufactures software at a
resolution of 59 .mu.m. Following image reconstruction, a region of
interest (ROI) was determined. This area encompassed all areas
containing the bioimplant induced bone. These can be easily
distinguished from the skeletal bones based on location and
density.
[0137] In order to analyze the quantity and quality of bone within
the ROI, the voxels of the mCT images were segmented into bone and
non-bone phases. Segmentation was achieved by determining a
threshold value for the voxel grayscale at which the voxel was
counted as bone. The total volume (TV), bone volume (BV), mineral
density of the total volume (TV-MD), mineral density of the bone
volume (BV-MD), mineral content of the total volume (TV-MC),
mineral content of the bone volume (BV-MC) and bone volume fraction
(BVF) of the ROI were determined for each sample. Values were
adjusted for the presence of calcium due to the carrier by using an
upper threshold value that selected only carrier and subtracting it
from the values obtained using a lower threshold which included
carrier plus new bone (see Humber et al., Oral Surgery, Oral
Medicine, Oral Pathology, Oral Radiology, and Endodontology. 2010.
109:372-384).
[0138] Following completion of the microCT analysis, the specimens
were either embedded in spurs resin or decalcified in formic acid
and embedded in wax. Resin embedded samples were evaluated by
backscatter SEM while wax embedded samples were cut and stained
with hematoxylin and eosin (H&E) and examined by light
microscopy to evaluate the tissue types present at the implantation
site.
[0139] Results
[0140] A carrier and a delivery vehicle were combined as described
in Example 5.
[0141] MicroCT analysis showed that bioimplants with all of the BMP
within the 3PS carrier, which had a sustained BMP release profile,
produced the smallest ossicles of bone (FIG. 4; 100-0), bioimplants
with all of the BMP within the F127 delivery vehicle, which had a
burst BMP release profile produced intermediate sized ossicles
(FIG. 4; 0-100), while bioimplants with 50% of the BMP loaded into
the carrier and 50% loaded into the delivery vehicle, which had a
multiphasic BMP release profile, produced the largest ossicles of
bone (FIG. 4; 50-50).
[0142] Backscatter SEM showed that by 28 days bone formed
throughout the bioimplant and onto the calcium phosphate
particulate that had been incorporated into the PLGA (FIG. 5).
Histology confirmed the tissue formed was bone (FIG. 6).
[0143] Example 9
An In Vivo Assay for Release of BMPs from Bioimplants
[0144] The present example describes how to measure the release of
rhBMP-2 from the various bioimplants described in Examples 1, 2, 3,
4 or 5 following implantation into an animal. Methods to do this
are well known in the art. For example see Uludag et al. J Biomed
Mater Res, 46, 193-202, 1999.
[0145] Materials & Methods
[0146] Recombinant hBMP-2 is radiolabeled with Iodine125 (I-125) by
Perkin Elmer. The radiolabelled rhBMP-2 (hot) is mixed with
unlabeled rhBMP-2 (cold) to produce a hot cold mixture of
1:100.
[0147] Bioimplants containing known amounts of rhBMP-2 are prepared
as in Examples 1 to 5. These bioimplants are then implanted into
animals as described in Example 8. At various times the animals are
sacrificed and the implant site is dissected out. The dissected
tissue is then weighed, and the amount of radioactivity determined
using a gamma counter.
[0148] To determine whether the counts are associated with protein,
the tissue is homogenized in 0.5 m1 PBS+0.5% BSA. Two mis of ice
cold 10% trichloroacetic acid are added to the homogenate and is
then held for at least 1 hour at 4.degree. C. The homogenate is
then centrifuged and the supernatant removed. The radioactivity of
the precipitate is then measured using a gamma counter.
[0149] The radioactivity associated with implants is corrected for
the decay and the total amount of BMP remaining in the implant is
estimated.
Example 10
Production of a Carrier with a Short Sustained Release Profile
[0150] The present example describes means of producing a carrier
that releases a growth factor with a short sustained release
profile.
[0151] Materials & Methods
[0152] Macroporous biphasic calcium phosphate (BCP) granules
(Eclipse) were purchased from Citagenix (Laval, Qc, Canada.)
Recombinant human BMP-2 (rhBMP-2, Induce Biologics Inc.) was
prepared in formulation buffer (1.5 mg/ml, pH 4.5; 5 mm glutamic
acid, 2.5% glycine, 0.5% sucrose and 0.01% Tween.TM. 80 with
ddH2O).
[0153] Sterile rhBMP-2 solution was incubated with sterile BCP
granules at a ratio of 9.1 .mu.g per 5 mg or 4.55 .mu.g per 5 mg
(BMP per BCP) for 15 minutes under shaking. The samples were then
frozen and lyophilized aseptically.
[0154] Following lyophilization the carriers were weighed into 5 mg
aliquots and placed in sterile epindorf tubes. Some tubes had 33%
F127 (45.5 .mu.l added). The BMP release profile was then
determined as described in Example 6.
[0155] Results
[0156] Carriers that were not coated with F127 (BCP) showed a burst
release profile with the largest amount of BMP released over the
first day and then decreasing amounts of BMP released at each
subsequent time point. Mixing the BCP within the F127 (BCP-Pol)
resulted in a short sustained release profile where similar amount
of BMP were collected each day over the first 4 days (FIG. 7).
Example 11
Altering the Sustained Release Profile of the Carrier
[0157] The present example describes a means of altering the
release profile from a carrier.
[0158] Materials & Methods
[0159] PLGA with differing inherent viscosities and molecular
weights were purchased from Birmingham Polymers Inc. (Birmingham,
Ala.). Carriers were then made using these PLGAs as described in
Example 1. The BMP release profile from these carriers was
determine according to the method of Example 6.
[0160] Results
[0161] All carriers produced sustained release profiles. However
the amount of BMP released differed depending on the
viscosity/molecular weight of the PLGA used. The carriers made with
low viscosity PLGA (Pol-1) released more rhBMP-2 than those using
the high viscosity (Pol-2) PLGA over the 12 week duration of the
study (FIG. 8).
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