U.S. patent application number 12/323183 was filed with the patent office on 2009-03-19 for platelet-derived growth factor compositions and methods of use thereof.
Invention is credited to Samuel E. LYNCH.
Application Number | 20090074753 12/323183 |
Document ID | / |
Family ID | 36203442 |
Filed Date | 2009-03-19 |
United States Patent
Application |
20090074753 |
Kind Code |
A1 |
LYNCH; Samuel E. |
March 19, 2009 |
PLATELET-DERIVED GROWTH FACTOR COMPOSITIONS AND METHODS OF USE
THEREOF
Abstract
A method for promoting growth of bone, periodontium, ligament,
or cartilage in a mammal by applying to the bone, periodontium,
ligament, or cartilage a composition comprising platelet-derived
growth factor at a concentration in the range of about 0.1 mg/mL to
about 1.0 mg/mL in a pharmaceutically acceptable liquid carrier and
a pharmaceutically-acceptable solid carrier.
Inventors: |
LYNCH; Samuel E.; (Franklin,
TN) |
Correspondence
Address: |
MORRISON & FOERSTER LLP
755 PAGE MILL RD
PALO ALTO
CA
94304-1018
US
|
Family ID: |
36203442 |
Appl. No.: |
12/323183 |
Filed: |
November 25, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11159533 |
Jun 23, 2005 |
7473678 |
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12323183 |
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10965319 |
Oct 14, 2004 |
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11159533 |
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Current U.S.
Class: |
424/130.1 ;
424/184.1; 424/532; 514/1.1; 514/44R |
Current CPC
Class: |
A61L 2430/06 20130101;
A61P 19/08 20180101; A61F 2210/0004 20130101; A61K 38/1858
20130101; A61L 27/425 20130101; A61L 2430/10 20130101; A61L 2400/06
20130101; A61L 27/12 20130101; A61P 19/04 20180101; A61L 27/54
20130101; A61P 1/02 20180101; A61F 2/28 20130101; A61L 2430/12
20130101; A61P 19/00 20180101; A61F 2002/2835 20130101; A61L 27/56
20130101; A61L 2300/414 20130101; A61L 27/58 20130101; A61K 9/0063
20130101; A61L 27/40 20130101; A61L 27/227 20130101; A61L 27/24
20130101; A61L 27/025 20130101; A61L 2430/02 20130101 |
Class at
Publication: |
424/130.1 ;
514/12; 514/44; 514/2; 424/184.1; 424/532 |
International
Class: |
A61K 38/00 20060101
A61K038/00; A61P 19/00 20060101 A61P019/00; A61K 38/18 20060101
A61K038/18; A61K 39/00 20060101 A61K039/00; A61K 35/14 20060101
A61K035/14; A61K 39/395 20060101 A61K039/395; A61K 31/70 20060101
A61K031/70 |
Claims
1. A method for promoting growth of bone, periodontium, ligament,
or cartilage of a mammal comprising administering to said mammal an
implant material comprising platelet-derived growth factor (PDGF)
at a concentration in the range of about 0.1 mg/mL to about 1.0
mg/mL in a pharmaceutically acceptable liquid carrier and a
pharmaceutically acceptable solid carrier, wherein said implant
material promotes the growth of said bone, periodontium, ligament,
or cartilage.
2. The method of claim 1, wherein said PDGF has a concentration of
about 0.3 mg/mL.
3. The method of claim 2, wherein said PDGF has a concentration of
0.3 mg/mL.
4. The method of claim 1, wherein said pharmaceutically acceptable
solid carrier comprises one or more of the following: a
biocompatible binder, a bone substituting agent, or a gel.
5. The method of claim 4, wherein said biocompatible binder is a
natural or synthetic polymer.
6. The method of claim 5, wherein said natural or synthetic polymer
is selected from polysaccharides, nucleic acids, carbohydrates,
proteins, polypeptides, collagen, poly(.alpha.-hydroxy acids),
poly(lactones), poly(amino acids), poly(anhydrides),
poly(orthoesters), poly(anhydride-co-imides),
poly(orthocarbonates), poly(.alpha.-hydroxy alkanoates),
poly(dioxanones), poly(phosphoesters), polylactic acid,
poly(L-lactide) (PLLA), poly(D,L-lactide) (PDLLA), polyglycolic
acid, polyglycolide (PGA), poly(lactide-co-glycolide (PLGA),
poly(L-lactide-co-D, L-lactide), poly(D,L-lactide-co-trimethylene
carbonate), polyhydroxybutyrate (PHB),
poly(.epsilon.-caprolactone), poly(.delta.-valerolactone),
poly(.gamma.-butyrolactone), poly(caprolactone), polyacrylic acid,
polycarboxylic acid, poly(allylamine hydrochloride),
poly(diallyldimethylammonium chloride), poly(ethyleneimine),
polypropylene fumarate, polyvinyl alcohol, polyvinylpyrrolidone,
polyethylene, polymethylmethacrylate, carbon fibers, poly(ethylene
glycol), poly(ethylene oxide), poly(vinyl alcohol),
poly(vinylpyrrolidone), poly(ethyloxazoline), poly(ethylene
oxide)-co-poly(propylene oxide) block copolymers, poly(ethylene
terephthalate)polyamide, and copolymers and mixtures thereof.
7. The method of claim 5, wherein said natural or synthetic polymer
is selected from collagen, polyglycolic acid, polylactic acid, and
polymethylmethacrylate.
8. The method of claim 4, wherein said biocompatible binder is
selected from alginic acid, arabic gum, guar gum, xantham gum,
gelatin, chitin, chitosan, chitosan acetate, chitosan lactate,
chondroitin sulfate, N,O-carboxymethyl chitosan, a dextran, fibrin
glue, glycerol, hyaluronic acid, sodium hyaluronate, a cellulose, a
glucosamine, a proteoglycan, a starch, lactic acid, a pluronic,
sodium glycerophosphate, collagen, glycogen, a keratin, silk, and
derivatives and mixtures thereof.
9. The method of claim 4, wherein said biocompatible binder is
sodium hyaluronate or derivatives thereof.
10. The method of claim 9, wherein said biocompatible binder is
hyaluronic acid.
11. The method of claim 4, wherein said biocompatible binder is
selected from methylcellulose, carboxymethylcellulose,
hydroxypropyl methylcellulose, or hydroxyethyl cellulose.
12. The method of claim 10, wherein said biocompatible binder is
carboxymethylcellulose.
13. The method of claim 8, wherein said dextran is
.alpha.-cyclodextrin, .beta.-cyclodextrin, .gamma.-cyclodextrin, or
sodium dextran sulfate.
14. The method of claim 8, wherein said starch is hydroxyethyl
starch or starch soluble.
15. The method of claim 4, wherein said bone substituting agent is
selected from a calcium phosphate, calcium sulfate, or
demineralized bone.
16. The method of claim 15, wherein said calcium phosphate is
selected from tricalcium phosphate, hydroxyapatite, poorly
crystalline hydroxyapatite, amorphous calcium phosphate, calcium
metaphosphate, dicalcium phosphate dihydrate, heptacalcium
phosphate, calcium pyrophosphate dihydrate, calcium pyrophosphate,
and octacalcium phosphate.
17. The method of claim 15, wherein said calcium phosphate is
provided as a paste or putty that forms a hardened calcium
phosphate upon in vivo administration.
18. The method of claim 15, wherein said calcium phosphate is
provided as a hardened calcium phosphate.
19. The method of claim 15, wherein said calcium phosphate is
bioresorable.
20. The method of claim 16, wherein said tricalcium phosphate is
.beta.-tricalcium phosphate (.beta.-TCP).
21. The method of claim 20, wherein said .beta.-TCP comprises a
matrix of micron- or submicron-sized particles.
22. The method of claim 21, wherein said .beta.-TCP particles have
a size of less than about 5000 .mu.m.
23. The method of claim 21, wherein said .beta.-TCP particles have
a size in the range of about 100 to about 5000 .mu.m.
24. The method of claim 23, wherein said .beta.-TCP particles have
a size in the range of about 100 to about 3000 .mu.m.
25. The method of claim 24, wherein said .beta.-TCP particles have
a size in the range of about 250 to about 2000 .mu.m.
26. The method of claim 21, wherein said .beta.-TCP particles are
porous.
27. The method of claim 26, wherein said .beta.-TCP particles are
greater than 40% porous.
28. The method of claim 27, wherein said .beta.-TCP particles are
greater than 65% porous.
29. The method of claim 28, wherein said .beta.-TCP particles are
greater than 90% porous.
30. The method of claim 20, wherein said .beta.-TCP is provided in
a shape suitable for implantation.
31. The method of claim 30, wherein said shape is selected from a
sphere, a cylinder, and a block.
32. The method of claim 15, wherein said demineralized bone is
cortical or cancellous bone.
33. The method of claim 1, wherein said pharmaceutically acceptable
liquid carrier is selected from water, a physiologically acceptable
buffer, or a cell culture medium.
34. The method of claim 33, wherein said physiologically acceptable
buffer is sodium acetate buffer.
35. The method of claim 1, wherein said composition further
comprises a biologically active agent.
36. The method of claim 35, wherein said biologically active agent
is selected from an antibody, an antibiotic, a polynucleotide, a
polypeptide, a protein, an anti-cancer agent, a growth factor, an
anti-inflammatory agent, and a vaccine.
37. The method of claim 36, wherein said protein is an osteogenic
protein.
38. The method of claim 37, wherein said osteogenic protein is
selected from insulin-like growth factor I (IGF-I), insulin-like
growth factor II (IGF-II), transforming growth factor-.beta.1
(TGF-.beta.1), transforming growth factor-.beta.2 (TGF-.beta.2),
transforming growth factor-.alpha. (TGF-.alpha.), a bone
morphogenetic protein (BMP), or osteogenin.
39. The method of claim 1, wherein said implant material further
comprises autologous bone marrow or autologous platelet
extracts.
40. The method of claim 1, wherein said PDGF is partially or
substantially purified.
41. The method of claim 1, wherein said PDGF is obtained from a
natural source or a recombinant source.
42. The method of claim 41, wherein said natural source comprises
blood, platelets, serum, platelet concentrate, platelet-rich plasma
(PRP), or bone marrow.
43. The method of claim 41, wherein said natural source is
platelet-rich plasma (PRP).
44. The method of claim 1, wherein said implant material delivers
said PDGF to said bone, periodontium, ligament, or cartilage for at
least 1 day following administration.
45. The method of claim 1, wherein said implant material delivers
said PDGF to said bone, periodontium, ligament, or cartilage for
less than about 28 days following administration.
46. The method of claim 1, wherein said implant material delivers
said PDGF to said bone, periodontium, ligament, or cartilage for
less than about 21 days following administration.
47. The method of claim 1, wherein said implant material delivers
said PDGF to said bone, periodontium, ligament, or cartilage for
less than about 14 days following administration.
48. The method of claim 1, wherein said implant material delivers
said PDGF to said bone, periodontium, ligament, or cartilage from
about 1 day to about 14 days following administration.
49. The method of claim 1, wherein said bone, periodontium,
ligament, or cartilage is damaged.
50. The method of claim 1 further comprising the step of allowing
said bone, periodontium, ligament, or cartilage to grow.
51. The method of claim 50 further comprising the steps of exposing
said bone, periodontium, ligament, or cartilage by producing a
surgical flap of skin prior to administering said implant material,
and replacing said flap after administering said implant
material.
52. The method of claim 51 further comprising, following the step
of producing a surgical flap of skin to expose said bone,
periodontium, or ligament, but prior to step (a), the step of
planing said bone or periodontium to remove organic matter from
said bone or periodontium.
53. The method of claim 1, wherein said PDGF is released from the
implant material upon administration at an average rate of less
than or equal to 300 .mu.g/day.
54. The method of claim 1, wherein said PDGF is released from the
implant material upon administration at an average rate of less
than 100 .mu.g/day.
55. The method of claim 1, wherein said PDGF is released from the
implant material upon administration at an average rate of less
than 50 .mu.g/day.
56. The method of claim 1, wherein said PDGF is released from the
implant material upon administration at an average rate of less
than 10 .mu.g/day.
57. The method of claim 1, wherein said PDGF is released from the
implant material upon administration at an average rate of less
than 1 .mu.g/day.
58. The method of claim 1, wherein said pharmaceutically acceptable
liquid carrier is sterile.
59. The method of claim 1, wherein said PDGF is PDGF AA, PDGF BB,
PDGF CC, or PDGF DD, or combinations or derivatives thereof.
60. The method of claim 59, wherein said PDGF is PDGF-BB.
61. The method of claim 59, wherein said PDGF is PDGF-AB.
62. A method for promoting growth of bone, periodontium, ligament,
or cartilage of a mammal comprising (a) administering to said
mammal an implant material comprising platelet-derived growth
factor (PDGF) at a concentration in the range of less than or equal
to 0.3 mg/mL in a pharmaceutically acceptable liquid carrier and a
pharmaceutically acceptable solid carrier, wherein said implant
material promotes the growth of said bone, periodontium, ligament,
or cartilage.
63. A vial comprising platelet-derived growth factor (PDGF) at
a/concentration in the range of about 0.1 mg/mL to about 1.0 mg/mL
in a pharmaceutically acceptable liquid.
64. The vial of claim 63, wherein said liquid is sterile sodium
acetate buffer.
65. The vial of claim 63 comprising PDGF at a concentration of
about 0.3 mg/mL.
66. The vial of claim 63, wherein said PDGF is PDGF-BB.
67. The vial of claim 64, wherein said PDGF is stable in said
buffer for at least 36 months when stored at a temperature in the
range of 2.degree. C. to 80.degree. C.
68. The vial of claim 64, wherein said PDGF is stable for at least
24 months when stored at a temperature in the range of 2.degree. C.
to 80.degree. C.
69. The vial of claim 64, wherein said PDGF is stable for at least
18 months when stored at a temperature in the range of 2.degree. C.
to 80.degree. C.
70. The vial of claim 64, wherein said PDGF is stable for at least
12 months when stored at a temperature in the range of 2.degree. C.
to 80.degree. C.
71. An implant material comprising a porous calcium phosphate
having adsorbed therein a liquid comprising platelet-derived growth
factor (PDGF) at a concentration in the range of about 0.1 mg/mL to
about 1.0 mg/mL.
72. The implant material of claim 71, wherein the concentration of
PDGF is about 0.3 mg/mL.
73. The implant material of claim 71, wherein said calcium
phosphate is selected from tricalcium phosphate, hydroxyapatite,
poorly crystalline hydroxyapatite, amorphous calcium phosphate,
calcium metaphosphate, dicalcium phosphate dihydrate, heptacalcium
phosphate, calcium pyrophosphate dihydrate, calcium pyrophosphate,
and octacalcium phosphate.
74. The implant material of claim 71, wherein said PDGF is provided
in a sterile liquid.
75. The implant material of claim 74, wherein said liquid is sodium
acetate buffer.
76. A method of preparing an implant material comprising saturating
a calcium phosphate material in a sterile liquid comprising
platelet-derived growth factor (PDGF) at a concentration in the
range of about 0.1 mg/mL to about 1.0 mg/mL.
77. The method of claim 76, wherein the concentration of PDGF is
about 0.3 mg/mL.
78. The method of claim 76, wherein said calcium phosphate is
selected from tricalcium phosphate, hydroxyapatite, poorly
crystalline hydroxyapatite, amorphous calcium phosphate, calcium
metaphosphate, dicalcium phosphate dihydrate, heptacalcium
phosphate, calcium pyrophosphate dihydrate, calcium pyrophosphate,
and octacalcium phosphate.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation-in-part of, and claims
priority from, U.S. patent application Ser. No. 10/965,319, filed
Oct. 14, 2004, which is incorporated herein by reference in its
entirety.
FIELD OF THE INVENTION
[0002] This invention relates to the healing of bone and connective
tissues.
BACKGROUND OF THE INVENTION
[0003] Growth factors are proteins that bind to receptors on a cell
surface, with the primary result of activating cellular
proliferation and/or differentiation. Many growth factors are quite
versatile, stimulating cellular division in numerous different cell
types; while others are specific to a particular cell-type.
Examples of growth factors include platelet-derived growth factor
(PDGF), insulin-like growth factors IGF-I and II), transforming
growth factor beta (TGF-.beta.), epidermal growth factor (EGF), and
fibroblast growth factor (FGF). PDGF is a cationic, heat stable
protein found in a variety of cell types, including the granules of
circulating platelets, vascular smooth muscle cells, endothelial
cells, macrophage, and keratinocytes, and is known to stimulate in
vitro protein synthesis and collagen production by fibroblasts. It
is also known to act as an in vitro mitogen and chemotactic agent
for fibroblasts, smooth muscle cells, osteoblasts, and glial
cells.
[0004] Recombinant human PDGF-BB (rhPDGF-BB) has been shown to
stimulate wound healing and bone regeneration in both animals and
humans. It is approved in both the United States and Europe for
human use in topical applications to accelerate healing of chronic
diabetic foot sores. Recombinant hPDGF-BB has also been shown to be
effective either singly or in combination with other growth factors
for improving periodontal regeneration, i.e., regrowth of bone,
cementum, and ligament around teeth (see, e.g., U.S. Pat. No.
5,124,316, incorporated herein by reference).
SUMMARY OF THE INVENTION
[0005] We have now demonstrated that a low dose of rhPDGF
(.about.0.1 to 1.0 mg/mL) promotes repair of bone, periodontium,
ligament, and cartilage. A low amount of rhPDGF can be adsorbed to
.beta.-TCP, which can be implanted at the site of repair, such that
the rhPDGF is released in vivo. Addition of rhPDGF to .beta.-TCP
has been shown to enhance osteoblast cell attachment and
proliferation compared to untreated .beta.-TCP.
[0006] In a first aspect, the invention features a method for
promoting bone, periodontium, ligament, or cartilage growth in a
mammal, e.g., a human, by administering an implant material
containing platelet-derived growth factor (PDGF) at a concentration
of less than about 1.0 mg/ml, such that the implant material
promotes growth of the bone, periodontium, ligament, or cartilage.
In an embodiment, the PDGF is administered in an amount of less
than or equal to 0.3 mg/ml. In another embodiment, the PDGF is
administered in an amount in the range of about 0.1 to about 1.0
mg/ml. In several embodiments, the PDGF is administered in an
amount of between about 0.2 to about 0.75 mg/ml, about 0.25 to
about 0.6 mg/ml, and about 0.25 to about 0.5 mg/ml. In an
embodiment, the PDGF is administered in an amount of about 0.1
mg/ml, 0.3 mg/ml, or 1.0 mg/ml, preferably 0.3 mg/mL. In another
embodiment, the PDGF is either partially or substantially purified.
In yet a further embodiment, the PDGF is isolated or purified from
other contaminants. In a further embodiment, the PDGF is released
from the implant material upon administration at an average rate of
0.3 mg/day. In another embodiment, the PDGF is released from the
implant material upon administration at an average rate of 300
.mu.g/day. In still further embodiments, the PDGF is released from
the implant material at an average rate of less than 100 .mu.g/day,
less than 50 .mu.g/day, less than 10 .mu.g/day, or less than 1
.mu.g/day. Preferably, the PDGF is delivered over a few days, e.g.,
1, 2, 5, 10, 15, 20, or 25 days, or up to 28 days or more.
[0007] A second aspect of the invention features a method for
promoting bone, periodontium, ligament, or cartilage growth in a
mammal, e.g., a human, by administering an implant material
containing an amount of platelet-derived growth factor (PDGF) of
less than about 1.0 mg/ml and a pharmaceutically acceptable carrier
such that the implant material promotes the growth of the bone,
periodontium, ligament, or cartilage, and allowing the bone,
periodontium, ligament, or cartilage to grow. Preferably, the PDGF
is equal to or less than about 0.3 mg/ml. In an embodiment, the
PDGF is administered in a range of about 0.1 to 1.0 mg/ml. In other
embodiments, the amount of PDGF is about 0.1 mg/ml, 0.3 mg/ml, or
1.0 mg/ml, preferably 0.3 mg/mL. In another embodiment, the PDGF is
either partially or substantially purified. In yet a further
embodiment, the PDGF is isolated or purified from other
contaminants. Prior to administering the implant material to the
mammal, the method can additionally include the step of producing a
surgical flap of skin to expose the bone, periodontium, ligament,
or cartilage, and following the administration step, replacing the
flap. In yet another embodiment, after producing the surgical flap,
but prior to administering the implant material to the bone,
periodontium, ligament, or cartilage, the method can additionally
include the step of planing the bone or periodontium to remove
organic matter from the bone or periodontium. In yet another
embodiment, the method promotes the growth of damaged or diseased
bone, periodontium, ligament, or cartilage. In yet another
embodiment, the method promotes the growth of bone in locations
where new bone formation is required as a result of surgical
interventions, such as, e.g., tooth extraction, ridge augmentation,
esthetic grafting, and sinus lift.
[0008] A third aspect of the invention features an implant material
for promoting the growth of bone, periodontium, ligament, or
cartilage in a mammal, e.g., a human. The implant material includes
a pharmaceutically acceptable carrier (e.g., a biocompatible
binder, a bone substituting agent, a liquid, or a gel) and
platelet-derived growth factor (PDGF), which is present at a
concentration of less than about 1.0 mg/mL. Preferably, the PDGF is
present in the implant material at a concentration equal to or less
than about 0.3 mg/ml. In an embodiment, the PDGF is administered in
a range of about 0.1 to 1.0 mg/ml. In other embodiments, the amount
of PDGF is about 0.1 mg/ml, 0.3 mg/ml, or 1.0 mg/ml, preferably 0.3
mg/mL. In an embodiment, the pharmaceutically acceptable carrier of
the implant material includes a scaffold or matrix consisting of a
biocompatible binder (e.g., carboxymethylcellulose) or a bone
substituting agent (.beta.-TCP) that is capable of absorbing a
solution that includes PDGF (e.g., a solution containing PDGF at a
concentration in the range of about 0.1 mg/mL to about 1.0 mg/mL).
In another embodiment, the pharmaceutically acceptable carrier is
capable of absorbing an amount of the PDGF solution that is equal
to at least about 25% of its own weight. In other embodiments, the
pharmaceutically acceptable carrier is capable of absorbing an
amount of the PDGF solution that is equal to at least about 50%,
75%, 100%, 200%, 250%, or 300% or its own weight. In an embodiment,
the PDGF is absorbed by the pharmaceutically acceptable carrier of
the implant material by soaking the pharmaceutically acceptable
carrier in a solution containing PDGF. Preferably, the PDGF is
present in the solution at a concentration of less than about 1.0
mg/mL. In another embodiment, the PDGF is present in the solution
at a concentration equal to or less than about 0.3 mg/ml. In
another embodiment, the PDGF is present in the solution at a
concentration in the range of about 0.1 to 1.0 mg/ml. In yet other
embodiments, the PDGF is present in the solution in an amount of
about 0.1 mg/ml, 0.3 mg/ml, or 1.0 mg/ml, preferably 0.3 mg/mL. In
another embodiment, the PDGF is either partially or substantially
purified. In yet a further embodiment, the PDGF is isolated or
purified from other contaminants.
[0009] A fourth aspect of the invention features a method for
preparing an implant material for promoting growth of bone,
periodontium, ligament, or cartilage in a mammal, e.g., a human.
The method includes the step of combining partially purified or
purified platelet-derived growth factor (PDGF) in an amount of less
than about 1.0 mg/mL with a pharmaceutically acceptable carrier
substance. Preferably, the PDGF is combined with a pharmaceutically
acceptable carrier substance at a concentration equal to or less
than about 0.3 mg/ml. In an embodiment, the PDGF is combined with a
pharmaceutically acceptable carrier substance in an amount in the
range of about 0.1 to 1.0 mg/ml. In other embodiments, PDGF is
mixed in the amount of 0.1 mg/ml, 0.3 mg/ml, or 1.0 mg/ml. In
another embodiment, PDGF is mixed in the amount of 0.3 mg/ml. In
yet another embodiment, the PDGF is absorbed by the
pharmaceutically acceptable carrier to produce the implant
material.
[0010] A fifth aspect of the invention features a vial having
platelet-derived growth factor (PDGF) at a concentration in the
range of about 0.1 mg/mL to about 1.0 mg/mL in a pharmaceutically
acceptable liquid. In an embodiment of this aspect of the
invention, the liquid is sterile sodium acetate buffer. In another
embodiment, the vial contains PDGF at a concentration of about 0.3
mg/mL. In yet another preferred embodiment, the PDGF is PDGF-BB. In
yet other embodiments, the PDGF is stable in the sodium acetate
buffer for at least about 12 months, preferably at least about 18
months, more preferably at least about 24 months, and most
preferably at least about 36 months when stored at a temperature in
the range of about 2.degree. C. to 80.degree. C.
[0011] A sixth aspect of the invention features an implant material
that includes a porous calcium phosphate having adsorbed therein a
liquid containing platelet-derived growth factor (PDGF) at a
concentration in the range of about 0.1 mg/mL to about 1.0 mg/mL.
In several embodiments, the concentration of PDGF is about 0.3
mg/mL, the calcium phosphate is selected from tricalcium phosphate,
hydroxyapatite, poorly crystalline hydroxyapatite, amorphous
calcium phosphate, calcium metaphosphate, dicalcium phosphate
dihydrate, heptacalcium phosphate, calcium pyrophosphate dihydrate,
calcium pyrophosphate, and octacalcium phosphate, and the PDGF is
provided in a sterile liquid, for example, sodium acetate
buffer.
[0012] A seventh aspect of the invention features a method of
preparing an implant material by saturating a calcium phosphate
material in a sterile liquid that includes platelet-derived growth
factor (PDGF) at a concentration in the range of about 0.1 mg/mL to
about 1.0 mg/mL. In several embodiments, the concentration of PDGF
is about 0.3 mg/mL, and the calcium phosphate is selected from
tricalcium phosphate, hydroxyapatite, poorly crystalline
hydroxyapatite, amorphous calcium phosphate, calcium metaphosphate,
dicalcium phosphate dihydrate, heptacalcium phosphate, calcium
pyrophosphate dihydrate, calcium pyrophosphate, and octacalcium
phosphate.
[0013] In an embodiment of all aspects of the invention, PDGF
includes PDGF homo- and heterodimers, for example, PDGF-AA,
PDGF-BB, PDGF-AB, PDGF-CC, and PDGF-DD, and combinations and
derivatives thereof.
[0014] In an embodiment of all aspects of the invention, the
pharmaceutically acceptable carrier substance of the implant
material is or additionally includes one or more of the following:
a biocompatible binder (e.g., a natural or synthetic polymer), a
bone substituting agent, a liquid, and a gel. In another preferred
embodiment, the implant material includes PDGF present in a
pharmaceutically acceptable liquid carrier which is adsorbed by a
pharmaceutically acceptable solid carrier.
[0015] In another embodiment of all aspects of the invention, the
implant material is prepared by combining isolated, partially
purified, substantially purified, or purified PDGF in an amount in
the range of 0.1 to 1.0 mg/ml, more preferably 0.1 mg/ml, 0.3
mg/ml, or 1.0 mg/ml, most preferably 0.3 mg/ml, or even less than
0.1 mg/ml, with a pharmaceutically acceptable carrier substance,
e.g., a biocompatible binder, such as a natural or synthetic
polymer (e.g., collagen, polyglycolic acid, and polylactic acid), a
bone substituting agent (e.g., a calcium phosphate (e.g.,
tricalcium phosphate or hydroxyapatite), calcium sulfate, or
demineralized bone (e.g., demineralized freeze-dried cortical or
cancellous bone), or a commercially available gel or liquid (i.e.,
a viscous or inert gel or liquid).
[0016] In several embodiments, the carrier substance of the implant
material is, or additionally includes, one or more biocompatible
binders. A biocompatible binder is an agent that produces or
promotes cohesion between the combined substances. Non-limiting
examples of suitable biocompatible binders include polymers
selected from polysaccharides, nucleic acids, carbohydrates,
proteins, polypeptides, poly(.alpha.-hydroxy acids),
poly(lactones), poly(amino acids), poly(anhydrides),
poly(orthoesters), poly(anhydride-co-imides),
poly(orthocarbonates), poly(.alpha.-hydroxy alkanoates),
poly(dioxanones), poly(phosphoesters), polylactic acid,
poly(L-lactide) (PLLA), poly(D,L-lactide) (PDLLA), polyglycolide
(PGA), poly(lactide-co-glycolide (PLGA), poly(L-lactide-co-D,
L-lactide), poly(D,L-lactide-co-trimethylene carbonate),
polyglycolic acid, polyhydroxybutyrate (PHB),
poly(.epsilon.-caprolactone), poly(.delta.-valerolactone),
poly(.gamma.-butyrolactone), poly(caprolactone), polyacrylic acid,
polycarboxylic acid, poly(allylamine hydrochloride),
poly(diallyldimethylammonium chloride), poly(ethyleneimine),
polypropylene fumarate, polyvinyl alcohol, polyvinylpyrrolidone,
polyethylene, polymethylmethacrylate, carbon fibers, poly(ethylene
glycol), poly(ethylene oxide), poly(vinyl alcohol),
poly(vinylpyrrolidone), poly(ethyloxazoline), poly(ethylene
oxide)-co-poly(propylene oxide) block copolymers, poly(ethylene
terephthalate)polyamide, and copolymers and mixtures thereof.
Additional binders include alginic acid, arabic gum, guar gum,
xantham gum, gelatin, chitin, chitosan, chitosan acetate, chitosan
lactate, chondroitin sulfate, N,O-carboxymethyl chitosan, a dextran
(e.g., .alpha.-cyclodextrin, .beta.-cyclodextrin,
.gamma.-cyclodextrin, or sodium dextran sulfate), fibrin glue,
glycerol, hyaluronic acid, sodium hyaluronate, a cellulose (e.g.,
methylcellulose, carboxy methylcellulose, hydroxypropyl
methylcellulose, or hydroxyethyl cellulose), a glucosamine, a
proteoglycan, a starch (e.g., hydroxyethyl starch or starch
soluble), lactic acid, a pluronic, sodium glycerophosphate,
collagen, glycogen, a keratin, silk, and derivatives and mixtures
thereof. In some embodiments, the biocompatible binder is
water-soluble. A water-soluble binder dissolves from the implant
material shortly after its implantation in vivo, thereby
introducing macroporosity into the implant material. This
macroporosity increases the osteoconductivity of the implant
material by enhancing the access and, consequently, the remodeling
activity of the osteoclasts and osteoblasts at the implant
site.
[0017] The biocompatible binder may be added to the implant
material in varying amounts and at a variety of stages during the
preparation of the composition. Those of skill in the art will be
able to determine the amount of binder and the method of inclusion
required for a given application.
[0018] In an embodiment, the carrier substance is or includes a
liquid selected from water, a buffer, and a cell culture medium.
The liquid may be used in any pH range, but most often will be used
in the range of pH 5.0 to pH 8.0. In an embodiment, the pH will be
compatible with the prolonged stability and efficacy of the PDGF
present in the implant material, or with the prolonged stability
and efficacy of another desired biologically active agent. In most
embodiments, the pH of the liquid will be in the range of pH 5.5 to
pH 7.4. Suitable buffers include, but are not limited to,
carbonates, phosphates (e.g., phosphate buffered saline), and
organic buffers such as Tris, HEPES, and MOPS. Most often, the
buffer will be selected for its biocompatibility with the host
tissues and its compatibility with the biologically active agent.
For most applications in which nucleic acids, peptides, or
antibiotics are included in the implant material, a simple
phosphate buffered saline will suffice.
[0019] In another embodiment of all aspects of the invention, the
carrier substance of the implant material is, or additionally
includes, one or more bone substituting agents. A bone substituting
agent is one that can be used to permanently or temporarily replace
bone. Following implantation, the bone substituting agent can be
retained by the body or it can be resorbed by the body and replaced
with bone. Exemplary bone substituting agent include, e.g., a
calcium phosphate (e.g., tricalcium phosphate (e.g., .beta.-TCP),
hydroxyapatite, poorly crystalline hydroxyapatite, amorphous
calcium phosphate, calcium metaphosphate, dicalcium phosphate
dihydrate, heptacalcium phosphate, calcium pyrophosphate dihydrate,
calcium pyrophosphate, and octacalcium phosphate), calcium sulfate,
or demineralized bone (e.g., demineralized freeze-dried cortical or
cancellous bone)). In an embodiment, the carrier substance is
bioresorbable. In another embodiment, the bone substituting agent
is provided as a matrix of micron- or submicron-sized particles,
e.g., nano-sized particles. The particles can be in the range of
about 100 .mu.m to about 5000 .mu.m in size, more preferably in the
range of about 200 .mu.m to about 3000 .mu.m, and most preferably
in the range of about 250 .mu.m to about 2000 .mu.m, or the
particles can be in the range of about 1 nm to about 1000 nm,
preferably less than about 500 nm, and more preferably less than
about 250 nm. In another embodiment, the bone substituting agent
has a porous composition. Porosity of the composition is a
desirable characteristic as it facilitates cell migration and
infiltration into the composition so that the cells can secrete
extracellular bone matrix. It also provides access for
vascularization. Porosity also provides a high surface area for
enhanced resorption and release of active substances, as well as
increased cell-matrix interaction. Preferably, the composition has
a porosity of greater than 40%, more preferably greater than 65%,
and most preferably greater than 90%. The composition can be
provided in a shape suitable for implantation (e.g., a sphere, a
cylinder, or a block) or it can be sized and shaped prior to use.
In a preferred embodiment, the bone substituting agent is a calcium
phosphate (e.g., .beta.-TCP).
[0020] The bone substituting agent can also be provided as a
flowable, moldable paste or putty. Preferably, the bone
substituting agent is a calcium phosphate paste that self-hardens
to form a hardened calcium phosphate prior to or after implantation
in vivo. The calcium phosphate component of the invention may be
any biocompatible calcium phosphate material known in the art. The
calcium phosphate material may be produced by any one of a variety
of methods and using any suitable starting components. For example,
the calcium phosphate material may include amorphous, apatitic
calcium phosphate. Calcium phosphate material may be produced by
solid-state acid-base reaction of crystalline calcium phosphate
reactants to form crystalline hydroxyapatite solids. Other methods
of making calcium phosphate materials are known in the art, some of
which are described below.
[0021] The calcium phosphate material can be poorly crystalline
apatitic PCA) calcium phosphate or hydroxyapatite (HA). PCA
material is described in application U.S. Pat. Nos. 5,650,176;
5,783,217; 6,027,742; 6,214,368; 6,287,341; 6,331,312; and
6,541,037, all of which are incorporated herein by reference. HA is
described, for example, in U.S. Pat. Nos. Re. 33,221 and Re.
33,161. These patents teach preparation of calcium phosphate
remineralization compositions and of a finely crystalline,
non-ceramic, gradually resorbable hydroxyapatite carrier material
based on the same calcium phosphate composition. A similar calcium
phosphate system, which consists of tetracalcium phosphate (TTCP)
and monocalcium phosphate (MCP) or its monohydrate form (MCPM), is
described in U.S. Pat. Nos. 5,053,212 and 5,129,905. This calcium
phosphate material is produced by solid-state acid-base reaction of
crystalline calcium phosphate reactants to form crystalline
hydroxyapatite solids.
[0022] Crystalline HA materials (commonly referred to as dahllite)
may be prepared such that they are flowable, moldable, and capable
of hardening in situ (see U.S. Pat. No. 5,962,028). These HA
materials (commonly referred to as carbonated hydroxyapatite) can
be formed by combining the reactants with a non-aqueous liquid to
provide a substantially uniform mixture, shaping the mixture as
appropriate, and allowing the mixture to harden in the presence of
water (e.g., before or after implantation). During hardening, the
mixture crystallizes into a solid and essentially monolithic
apatitic structure.
[0023] The reactants will generally consist of a phosphate source,
e.g., phosphoric acid or phosphate salts, substantially free of
water, an alkali earth metal, particularly calcium, source,
optionally crystalline nuclei, particularly hydroxyapatite or
calcium phosphate crystals, calcium carbonate, and a
physiologically acceptable lubricant, such as any of the
non-aqueous liquids described herein. The dry ingredients may be
pre-prepared as a mixture and subsequently combined with the
non-aqueous liquid ingredients under conditions where substantially
uniform mixing occurs.
[0024] The calcium phosphate material is characterized by its
biological resorbability, biocompatibility, and its minimal
crystallinity. Its crystalline character is substantially the same
as natural bone. Preferably, the calcium phosphate material hardens
in less than five hours, and substantially hardens in about one to
five hours, under physiological conditions. Preferably, the
material is substantially hardened within about 10-30 minutes. The
hardening rate under physiological conditions, may be varied
according to the therapeutic need by modifying a few simple
parameters as described in U.S. Pat. No. 6,027,742, which is
incorporated herein by reference.
[0025] In an embodiment, the resulting bioresorbable calcium
phosphate material will be "calcium deficient," with a calcium to
phosphate molar ratio of less than about 1.6 as compared to the
ideal stoichiometric value of approximately 1.67 for
hydroxyapatite.
[0026] Desirable calcium phosphates are capable of hardening in a
moist environment, at or around body temperature in less than 5
hours and preferably within 10-30 minutes. Desirable materials are
those that, when implanted as a 1-5 g pellet, are at least 80%
resorbed within one year. Preferably, the material can be fully
resorbed.
[0027] In several embodiments of all aspects of the invention, the
implant material additionally may include one or more biologically
active agents. Biologically active agents that can be incorporated
into the implant materials of the invention include, without
limitation, organic molecules, inorganic materials, proteins,
peptides, nucleic acids (e.g., genes, gene fragments, gene
regulatory sequences, and antisense molecules), nucleoproteins,
polysaccharides, glycoproteins, and lipoproteins. Classes of
biologically active compounds that can be incorporated into the
implant materials of the invention include, without limitation,
anti-cancer agents, antibiotics, analgesics, anti-inflammatory
agents, immunosuppressants, enzyme inhibitors, antihistamines,
anti-convulsants, hormones, muscle relaxants, anti-spasmodics,
ophthalmic agents, prostaglandins, anti-depressants, anti-psychotic
substances, trophic factors, osteoinductive proteins, growth
factors, and vaccines.
[0028] Anti-cancer agents include alkylating agents, platinum
agents, antimetabolites, topoisomerase inhibitors, antitumor
antibiotics, antimitotic agents, aromatase inhibitors, thymidylate
synthase inhibitors, DNA antagonists, farnesyltransferase
inhibitors, pump inhibitors, histone acetyltransferase inhibitors,
metalloproteinase inhibitors, ribonucleoside reductase inhibitors,
TNF alpha agonists/antagonists, endothelin A receptor antagonists,
retinoic acid receptor agonists, immuno-modulators, hormonal and
antihormonal agents, photodynamic agents, and tyrosine kinase
inhibitors.
[0029] Any of the biologically active agents listed in Table 1 can
be used.
TABLE-US-00001 TABLE 1 Alkylating agents cyclophosphamide lomustine
busulfan procarbazine ifosfamide altretamine melphalan estramustine
phosphate hexamethylmelamine mechlorethamine thiotepa streptozocin
chlorambucil temozolomide dacarbazine semustine carmustine Platinum
agents cisplatin carboplatinum oxaliplatin ZD-0473 (AnorMED)
spiroplatinum, lobaplatin (Aeterna) carboxyphthalatoplatinum,
satraplatin (Johnson Matthey) tetraplatin BBR-3464 (Hoffmann-La
Roche) ormiplatin SM-11355 (Sumitomo) iproplatin AP-5280 (Access)
Antimetabolites azacytidine tomudex gemcitabine trimetrexate
capecitabine deoxycoformycin 5-fluorouracil fludarabine floxuridine
pentostatin 2-chlorodeoxyadenosine raltitrexed 6-mercaptopurine
hydroxyurea 6-thioguanine decitabine (SuperGen) cytarabin
clofarabine (Bioenvision) 2-fluorodeoxy cytidine irofulven (MGI
Pharma) methotrexate DMDC (Hoffmann-La Roche) idatrexate
ethynylcytidine (Taiho) Topoisomerase amsacrine rubitecan
(SuperGen) inhibitors epirubicin exatecan mesylate (Daiichi)
etoposide quinamed (ChemGenex) teniposide or mitoxantrone gimatecan
(Sigma-Tau) irinotecan (CPT-11) diflomotecan (Beaufour-Ipsen)
7-ethyl-10-hydroxy-camptothecin TAS-103 (Taiho) topotecan
elsamitrucin (Spectrum) dexrazoxanet (TopoTarget) J-107088 (Merck
& Co) pixantrone (Novuspharma) BNP-1350 (BioNumerik)
rebeccamycin analogue (Exelixis) CKD-602 (Chong Kun Dang) BBR-3576
(Novuspharma) KW-2170 (Kyowa Hakko) Antitumor dactinomycin
(actinomycin D) amonafide antibiotics doxorubicin (adriamycin)
azonafide deoxyrubicin anthrapyrazole valrubicin oxantrazole
daunorubicin (daunomycin) losoxantrone epirubicin bleomycin sulfate
(blenoxane) therarubicin bleomycinic acid idarubicin bleomycin A
rubidazone bleomycin B plicamycinp mitomycin C porfiromycin
MEN-10755 (Menarini) cyanomorpholinodoxorubicin GPX-100 (Gem
Pharmaceuticals) mitoxantrone (novantrone) Antimitotic paclitaxel
SB 408075 (GlaxoSmithKline) agents docetaxel E7010 (Abbott)
colchicine PG-TXL (Cell Therapeutics) vinblastine IDN 5109 (Bayer)
vincristine A 105972 (Abbott) vinorelbine A 204197 (Abbott)
vindesine LU 223651 (BASF) dolastatin 10 (NCI) D 24851 (ASTAMedica)
rhizoxin (Fujisawa) ER-86526 (Eisai) mivobulin (Warner-Lambert)
combretastatin A4 (BMS) cemadotin (BASF) isohomohalichondrin-B
(PharmaMar) RPR 109881A (Aventis) ZD 6126 (AstraZeneca) TXD 258
(Aventis) PEG-paclitaxel (Enzon) epothilone B (Novartis) AZ10992
(Asahi) T 900607 (Tularik) IDN-5109 (Indena) T 138067 (Tularik)
AVLB (Prescient NeuroPharma) cryptophycin 52 (Eli Lilly)
azaepothilone B (BMS) vinflunine (Fabre) BNP-7787 (BioNumerik)
auristatin PE (Teikoku Hormone) CA-4 prodrug (OXiGENE) BMS 247550
(BMS) dolastatin-10 (NIH) BMS 184476 (BMS) CA-4 (OXiGENE) BMS
188797 (BMS) taxoprexin (Protarga) Aromatase aminoglutethimide
exemestane inhibitors letrozole atamestane (BioMedicines)
anastrazole YM-511 (Yamanouchi) formestane Thymidylate pemetrexed
(Eli Lilly) nolatrexed (Eximias) synthase inhibitors ZD-9331 (BTG)
CoFactor .TM. (BioKeys) DNA antagonists trabectedin (PharmaMar)
mafosfamide (Baxter International) glufosfamide (Baxter
International) apaziquone (Spectrum Pharmaceuticals) albumin + 32P
(Isotope Solutions) O6 benzyl guanine (Paligent) thymectacin
(NewBiotics) edotreotide (Novartis) Farnesyltransferase arglabin
(NuOncology Labs) tipifarnib (Johnson & Johnson) inhibitors
lonafarnib (Schering-Plough) perillyl alcohol (DOR BioPharma)
BAY-43-9006 (Bayer) Pump inhibitors CBT-1 (CBA Pharma) zosuquidar
trihydrochloride (Eli Lilly) tariquidar (Xenova) biricodar
dicitrate (Vertex) MS-209 (Schering AG) Histone tacedinaline
(Pfizer) pivaloyloxymethyl butyrate (Titan) acetyltransferase SAHA
(Aton Pharma) depsipeptide (Fujisawa) inhibitors MS-275 (Schering
AG) Metalloproteinase Neovastat (Aeterna Laboratories) CMT-3
(CollaGenex) inhibitors marimastat (British Biotech) BMS-275291
(Celltech) Ribonucleoside gallium maltolate (Titan) tezacitabine
(Aventis) reductase inhibitors triapine (Vion) didox (Molecules for
Health) TNF alpha virulizin (Lorus Therapeutics) revimid (Celgene)
agonists/antagonists CDC-394 (Celgene) entanercept (Immunex Corp.)
infliximab (Centocor, Inc.) adalimumab (Abbott Laboratories)
Endothelin A atrasentan (Abbott) YM-598 (Yamanouchi) receptor
antagonist ZD-4054 (AstraZeneca) Retinoic acid fenretinide (Johnson
& Johnson) alitretinoin (Ligand) receptor agonists LGD-1550
(Ligand) Immuno- interferon dexosome therapy (Anosys) modulators
oncophage (Antigenics) pentrix (Australian Cancer Technology) GMK
(Progenics) ISF-154 (Tragen) adenocarcinoma vaccine (Biomira)
cancer vaccine (Intercell) CTP-37 (AVI BioPharma) norelin (Biostar)
IRX-2 (Immuno-Rx) BLP-25 (Biomira) PEP-005 (Peplin Biotech) MGV
(Progenics) synchrovax vaccines (CTL Immuno) .beta.-alethine
(Dovetail) melanoma vaccine (CTL Immuno) CLL therapy (Vasogen) p21
RAS vaccine (GemVax) Hormonal and estrogens prednisone antihormonal
conjugated estrogens methylprednisolone agents ethinyl estradiol
prednisolone chlortrianisen aminoglutethimide idenestrol leuprolide
hydroxyprogesterone caproate goserelin medroxyprogesterone
leuporelin testosterone bicalutamide testosterone propionate;
fluoxymesterone flutamide methyltestosterone octreotide
diethylstilbestrol nilutamide megestrol mitotane tamoxifen P-04
(Novogen) toremofine 2-methoxyestradiol (EntreMed) dexamethasone
arzoxifene (Eli Lilly) Photodynamic talaporfin (Light Sciences)
Pd-bacteriopheophorbide (Yeda) agents Theralux (Theratechnologies)
lutetium texaphyrin (Pharmacyclics) motexafin gadolinium
(Pharmacyclics) hypericin Tyrosine Kinase imatinib (Novartis)
kahalide F (PharmaMar) Inhibitors leflunomide (Sugen/Pharmacia)
CEP-701 (Cephalon) ZD1839 (AstraZeneca) CEP-751 (Cephalon)
erlotinib (Oncogene Science) MLN518 (Millenium) canertinib (Pfizer)
PKC412 (Novartis) squalamine (Genaera) phenoxodiol ( ) SU5416
(Pharmacia) trastuzumab (Genentech) SU6668 (Pharmacia) C225
(ImClone) ZD4190 (AstraZeneca) rhu-Mab (Genentech) ZD6474
(AstraZeneca) MDX-H210 (Medarex) vatalanib (Novartis) 2C4
(Genentech) PKI166 (Novartis) MDX-447 (Medarex) GW2016
(GlaxoSmithKline) ABX-EGF (Abgenix) EKB-509 (Wyeth) IMC-1C11
(ImClone) EKB-569 (Wyeth)
[0030] Antibiotics include aminoglycosides (e.g., gentamicin,
tobramycin, netilmicin, streptomycin, amikacin, neomycin),
bacitracin, corbapenems (e.g., imipenem/cislastatin),
cephalosporins, colistin, methenamine, monobactams (e.g.,
aztreonam), penicillins (e.g., penicillin G, penicillin V,
methicillin, natcillin, oxacillin, cloxacillin, dicloxacillin,
ampicillin, amoxicillin, carbenicillin, ticarcillin, piperacillin,
mezlocillin, azlocillin), polymyxin B, quinolones, and vancomycin;
and bacteriostatic agents such as chloramphenicol, clindanyan,
macrolides (e.g., erythromycin, azithromycin, clarithromycin),
lincomyan, nitrofurantoin, sulfonamides, tetracyclines (e.g.,
tetracycline, doxycycline, minocycline, demeclocyline), and
trimethoprim. Also included are metronidazole, fluoroquinolones,
and ritampin.
[0031] Enzyme inhibitors are substances which inhibit an enzymatic
reaction. Examples of enzyme inhibitors include edrophonium
chloride, N-methylphysostigmine, neostigmine bromide, physostigmine
sulfate, tacrine, tacrine, 1-hydroxy maleate, iodotubercidin,
p-bromotetramisole, 10-(alpha-diethylaminopropionyl)-phenothiazine
hydrochloride, calmidazolium chloride,
hemicholinium-3,3,5-dinitrocatechol, diacylglycerol kinase
inhibitor I, diacylglycerol kinase inhibitor II,
3-phenylpropargylamine, N.sup.6-monomethyl-L-arginine acetate,
carbidopa, 3-hydroxybenzylhydrazine, hydralazine, clorgyline,
deprenyl, hydroxylamine, iproniazid phosphate,
6-MeO-tetrahydro-9H-pyrido-indole, nialamide, pargyline,
quinacrine, semicarbazide, tranylcypromine,
N,N-diethylaminoethyl-2,2-diphenylvalerate hydrochloride,
3-isobutyl-1-methylxanthne, papaverine, indomethacind,
2-cyclooctyl-2-hydroxyethylamine hydrochloride,
2,3-dichloro-a-methylbenzylamine (DCMB),
8,9-dichloro-2,3,4,5-tetrahydro-1H-2-benzazepine hydrochloride,
p-aminoglutethimide, p-aminoglutethimide tartrate, 3-iodotyrosine,
alpha-methyltyrosine, acetazolamide, dichlorphenamide,
6-hydroxy-2-benzothiazolesulfonamide, and allopurinol.
[0032] Antihistamines include pyrilamine, chlorpheniramine, and
tetrahydrazoline, among others.
[0033] Anti-inflammatory agents include corticosteroids,
nonsteroidal anti-inflammatory drugs (e.g., aspirin,
phenylbutazone, indomethacin, sulindac, tolmetin, ibuprofen,
piroxicam, and fenamates), acetaminophen, phenacetin, gold salts,
chloroquine, D-Penicillamine, methotrexate colchicine, allopurinol,
probenecid, and sulfinpyrazone.
[0034] Muscle relaxants include mephenesin, methocarbomal,
cyclobenzaprine hydrochloride, trihexylphenidyl hydrochloride,
levodopa/carbidopa, and biperiden.
[0035] Anti-spasmodics include atropine, scopolamine, oxyphenonium,
and papaverine.
[0036] Analgesics include aspirin, phenybutazone, idomethacin,
sulindac, tolmetic, ibuprofen, piroxicam, fenamates, acetaminophen,
phenacetin, morphine sulfate, codeine sulfate, meperidine,
nalorphine, opioids (e.g., codeine sulfate, fentanyl citrate,
hydrocodone bitartrate, loperamide, morphine sulfate, noscapine,
norcodeine, normorphine, thebaine, nor-binaltorphimine,
buprenorphine, chlornaltrexamine, funaltrexamione, nalbuphine,
nalorphine, naloxone, naloxonazine, naltrexone, and naltrindole),
procaine, lidocain, tetracaine and dibucaine.
[0037] Ophthalmic agents include sodium fluorescein, rose bengal,
methacholine, adrenaline, cocaine, atropine, alpha-chymotrypsin,
hyaluronidase, betaxalol, pilocarpine, timolol, timolol salts, and
combinations thereof.
[0038] Prostaglandins are art recognized and are a class of
naturally occurring chemically related, long-chain hydroxy fatty
acids that have a variety of biological effects.
[0039] Anti-depressants are substances capable of preventing or
relieving depression. Examples of anti-depressants include
imipramine, amitriptyline, nortriptyline, protriptyline,
desipramine, amoxapine, doxepin, maprotiline, tranylcypromine,
phenelzine, and isocarboxazide.
[0040] Growth factors are factors whose continued presence improves
the viability or longevity of a cell. Trophic factors include,
without limitation, neutrophil-activating protein, monocyte
chemoattractant protein, macrophage-inflammatory protein, platelet
factor, platelet basic protein, and melanoma growth stimulating
activity; epidermal growth factor, transforming growth factor
(alpha), fibroblast growth factor, platelet-derived endothelial
cell growth factor, insulin-like growth factor (IGF, e.g., IGF-I or
IGF-II), glial derived growth neurotrophic factor, ciliary
neurotrophic factor, nerve growth factor, bone
growth/cartilage-inducing factor (alpha and beta), bone
morphogenetic proteins (BMPs), interleukins (e.g., interleukin
inhibitors or interleukin receptors, including interleukin 1
through interleukin 10), interferons (e.g., interferon alpha, beta
and gamma), hematopoietic factors, including erythropoietin,
granulocyte colony stimulating factor, macrophage colony
stimulating factor and granulocyte-macrophage colony stimulating
factor; tumor necrosis factors, transforming growth factors (beta),
including beta-1, beta-2, beta-3, transforming growth factors
(alpha), inhibin, and activin; and bone morphogenetic proteins such
as OP-1, BMP-2 and BMP-7.
[0041] Hormones include estrogens (e.g., estradiol, estrone,
estriol, diethylstibestrol, quinestrol, chlorotrianisene, ethinyl
estradiol, mestranol), anti-estrogens (e.g., clomiphene,
tamoxifen), progestins (e.g., medroxyprogesterone, norethindrone,
hydroxyprogesterone, norgestrel), antiprogestin (mifepristone),
androgens (e.g, testosterone cypionate, fluoxymesterone, danazol,
testolactone), anti-androgens (e.g., cyproterone acetate,
flutamide), thyroid hormones (e.g., triiodothyronne, thyroxine,
propylthiouracil, methimazole, and iodixode), and pituitary
hormones (e.g., corticotropin, sumutotropin, oxytocin, and
vasopressin). Hormones are commonly employed in hormone replacement
therapy and/or for purposes of birth control. Steroid hormones,
such as prednisone, are also used as immunosuppressants and
anti-inflammatories.
[0042] The biologically active agent is also desirably selected
from the family of proteins known as the transforming growth
factors-beta (TGF-.beta.) superfamily of proteins, which includes
the activins, inhibins, and bone morphogenetic proteins (BMPs). In
an embodiment, the active agent includes at least one protein
selected from the subclass of proteins known generally as BMPs,
which have been disclosed to have osteogenic activity, and other
growth and differentiation type activities. These BMPs include BMP
proteins BMP-2, BMP-3, BMP-4, BMP-5, BMP-6 and BMP-7, disclosed for
instance in U.S. Pat. Nos. 5,108,922; 5,013,649; 5,116,738;
5,106,748; 5,187,076; and 5,141,905; BMP-8, disclosed in PCT
publication WO91/18098; and BMP-9, disclosed in PCT publication
WO93/00432, BMP-10, disclosed in PCT application WO94/26893;
BMP-11, disclosed in PCT application WO94/26892, or BMP-12 or
BMP-13, disclosed in PCT application WO 95/16035; BMP-14; BMP-15,
disclosed in U.S. Pat. No. 5,635,372; or BMP-16, disclosed in U.S.
Pat. No. 5,965,403. Other TGF-.beta. proteins which may be useful
as the active agent in the calcium phosphate compositions of the
invention include Vgr-2, Jones et al., Mol. Endocrinol. 6:1961
(1992), and any of the growth and differentiation factors (GDFs),
including those described in PCT applications WO94/15965;
WO94/15949; WO95/01801; WO95/01802; WO94/21681; WO94/15966;
WO95/10539; WO96/01845; WO96/02559 and others. Also useful in the
invention may be BIP, disclosed in WO94/01557; HP00269, disclosed
in JP Publication number: 7-250688; and MP52, disclosed in PCT
application WO93/16099. The disclosures of all of the above
applications are incorporated herein by reference. A subset of BMPs
which can be used in the invention include BMP-2, BMP-4, BMP-5,
BMP-6, BMP-7, BMP-10, BMP-12, BMP-13, BMP-14, and MP52. The active
agent is most preferably BMP-2, the sequence of which is disclosed
in U.S. Pat. No. 5,013,649, the disclosure of which is incorporated
herein by reference. Other osteogenic agents known in the art can
also be used, such as teriparatide (Forteo.TM.), Chrysalin.RTM.,
prostaglandin E2, LIM protein, osteogenin, or demineralized bone
matrix (DBM), among others.
[0043] The biologically active agent may be synthesized chemically,
recombinantly produced, or purified from a source in which the
biologically active agent is naturally found. The active agent, if
a TGF-.beta., such as a BMP or other dimeric protein, may be
homodimeric, or may be heterodimeric with other BMPs (e.g., a
heterodimer composed of one monomer each of BMP-2 and BMP-6) or
with other members of the TGF-.beta. superfamily, such as activins,
inhibins and TGF-.beta.1 (e.g., a heterodimer composed of one
monomer each of a BMP and a related member of the TGF-.beta.
superfamily). Examples of such heterodimeric proteins are described
for example in Published PCT Patent Application WO 93/09229, the
specification of which is incorporated herein by reference.
[0044] Additional biologically active agents include the Hedgehog,
Frazzled, Chordin, Noggin, Cerberus, and Follistatin proteins.
These families of proteins are generally described in Sasai et al.,
Cell 79:779-790 (1994) (Chordin); PCT Patent Publication WO94/05800
(Noggin); and Fukui et al., Devel. Biol. 159:131 (1993)
(Follistatin). Hedgehog proteins are described in WO96/16668;
WO96/17924; and WO95/18856. The Frazzled family of proteins is a
recently discovered family of proteins with high homology to the
extracellular binding domain of the receptor protein family known
as Frizzled. The Frizzled family of genes and proteins is described
in Wang et al., J. Biol. Chem. 271:4468-4476 (1996). The active
agent may also include other soluble receptors, such as the
truncated soluble receptors disclosed in PCT patent publication
WO95/07982. From the teaching of WO95/07982, one skilled in the art
will recognize that truncated soluble receptors can be prepared for
numerous other receptor proteins. The above publications are
incorporated by reference herein.
[0045] The amount of the biologically active protein, e.g., an
osteogenic protein, that is effective to stimulate a desired
activity, e.g., increased osteogenic activity of present or
infiltrating progenitor or other cells, will depend upon the size
and nature of the defect being treated, as well as the carrier
being employed. Generally, the amount of protein to be delivered is
in a range of from about 0.1 to about 100 mg; preferably about 1 to
about 100 mg; most preferably about 10 to about 80 mg.
[0046] Standard protocols and regimens for delivery of the
above-listed agents are known in the art. Biologically active
agents are introduced into the implant material in amounts that
allow delivery of an appropriate dosage of the agent to the implant
site. In most cases, dosages are determined using guidelines known
to practitioners and applicable to the particular agent in
question. The exemplary amount of biologically active agent to be
included in the implant material of the invention is likely to
depend on such variables as the type and extent of the condition,
the overall health status of the particular patient, the
formulation of the active agent, and the bioresorbability of the
delivery vehicle used. Standard clinical trials may be used to
optimize the dose and dosing frequency for any particular
biologically active agent.
[0047] In an embodiment of all aspects of the invention, the
composition can additionally contain autologous bone marrow or
autologous platelet extracts.
[0048] In another embodiment of all of the above aspects, the PDGF
and/or other growth factors can be obtained from natural sources,
(e.g., platelets), or more preferably, produced by recombinant DNA
technology. When obtained from natural sources, the PDGF and/or
other growth factors can be obtained from a biological fluid. A
biological fluid includes any treated or untreated fluid (including
a suspension) associated with living organisms, particularly blood,
including whole blood, warn or cold blood, and stored or fresh
blood; treated blood, such as blood diluted with at least one
physiological solution, including but not limited to saline,
nutrient, and/or anticoagulant solutions; blood components, such as
platelet concentrate (PC), apheresed platelets, platelet-rich
plasma (PRP), platelet-poor plasma (PPP), platelet-free plasma,
plasma, serum, fresh frozen plasma (FFP), components obtained from
plasma, packed red cells (PRC), buffy coat (BC); blood products
derived from blood or a blood component or derived from bone
marrow; red cells separated from plasma and resuspended in
physiological fluid; and platelets separated from plasma and
resuspended in physiological fluid. The biological fluid may have
been treated to remove some of the leukocytes before being
processed according to the invention. As used herein, blood product
or biological fluid refers to the components described above, and
to similar blood products or biological fluids obtained by other
means and with similar properties. In an embodiment, the PDGF is
obtained from platelet-rich plasma (PRP). The preparation of PRP is
described in, e.g., U.S. Pat. Nos. 6,649,072, 6,641,552, 6,613,566,
6,592,507, 6,558,307, 6,398,972, and 5,599,558, which are
incorporated herein by reference.
[0049] In an embodiment of all aspects of the invention, the
implant material delivers PDGF at the implant site for a duration
of time greater than at least 1 day. In several embodiments, the
implant material delivers PDGF at the implant site for at least 7,
14, 21, or 28 days. Preferably, the implant material delivers PDGF
at the implant site for a time between about 1 day and 7, 14, 21,
or 28 days. In another embodiment, the implant material delivers
PDGF at the implant site for a time greater than about 1 day, but
less than about 14 days.
[0050] By "bioresorbable" is meant the ability of the implant
material to be resorbed or remodeled in vivo. The resorption
process involves degradation and elimination of the original
implant material through the action of body fluids, enzymes or
cells. The resorbed materials may be used by the host in the
formation of new tissue, or it may be otherwise re-utilized by the
host, or it may be excreted.
[0051] By "differentiation factor" is meant a polypeptide,
including a chain of at least 6 amino acids, which stimulates
differentiation of one or more target cells into cells with
cartilage or bone forming potential.
[0052] By "nanometer-sized particle" is meant a submicron-sized
particle, generally defined as a particle below 1000 nanometers. A
nanometer-sized particle is a solid particle material that is in an
intermediate state between molecular and macron substances. A
nanometer is defined as one billionth of a meter (1
nanometer=10.sup.9 m). Nanometer material is known as the powder,
fiber, film, or block having nanoscale size.
[0053] By "periodontium" is meant the tissues that surround and
support the teeth. The periodontium supports, protects, and
provides nourishment to the teeth. The periodontium consists of
bone, cementum, alveolar process of the maxillae and mandible,
periodontal ligament, and gingiva. Cementum is a thin, calcified
layer of tissue that completely covers the dentin of the tooth
root. Cementum is formed during the development of the root and
throughout the life of the tooth and functions as an area of
attachment for the periodontal ligament fibers. The alveolar
process is the bony portion of the maxilla and mandible where the
teeth are embedded and in which the tooth roots are supported. The
alveolar socket is the cavity within the alveolar process in which
the root of the tooth is held by the periodontal ligament. The bone
that divides one socket from another is called the interdental
septum. When multirooted teeth are present, the bone is called the
interradicular septum. The alveolar process includes the cortical
plate, alveolar crest, trabecular bone, and the alveolar bone
proper.
[0054] By "promoting growth" is meant the healing of bone,
periodontium, ligament, or cartilage, and regeneration of such
tissues and structures. Preferably, the bone, periodontium,
ligament, or cartilage is damaged or wounded and requires
regeneration or healing.
[0055] By "promoting periodontium growth" is meant regeneration or
healing of the supporting tissues of a tooth including alveolar
bone, cementum, and interposed periodontal ligament, which have
been damaged by disease or trauma.
[0056] By "purified" is meant a growth or differentiation factor,
e.g., PDGF, which, prior to mixing with a carrier substance, is 95%
or greater by weight, i.e., the factor is substantially free of
other proteins, lipids, and carbohydrates with which it is
naturally associated. The term "substantially purified" refers to a
lesser purity of factor, having, for example, only 5%-95% by weight
of the factor, preferably 65-95%. A purified protein preparation
will generally yield a single major band on a polyacrylamide gel.
Most preferably, the purified factor used in implant materials of
the invention is pure as judged by amino-terminal amino acid
sequence analysis. The term "partially purified" refers to PDGF
that is provided in the context of PRP, PPP, FFP, or any other
blood product that requires collection and separation, e.g., by
centrifugation, to produce.
[0057] By way of example, a solution having .about.1.0 mg/mL of
PDGF, when .about.50% pure, constitutes .about.2.0 mg/mL of total
protein.
[0058] The implant materials of this invention aid in regeneration
of periodontium, at least in part, by promoting the growth of
connective tissue, bone, and cementum. The implant materials can be
prepared so that they directly promote the growth and
differentiation of cells that produce connective tissue, bone, and
cementum. Alternatively, the implant materials can be prepared so
that they act indirectly by, e.g., attracting cells that are
necessary for promoting the growth of connective tissue, bone, and
cementum. Regeneration using a composition of this invention is a
more effective treatment of periodontal diseases or bone wounds
than that achieved using systemic antibiotics or surgical
debridement alone.
[0059] The PDGF, polypeptide growth factors, and differentiation
factors may be obtained from human tissues or cells, e.g.,
platelets, by solid phase peptide synthesis, or by recombinant DNA
technology. Thus, by the term "polypeptide growth factor" or
"differentiation factor," we mean tissue or cell-derived,
recombinant, or synthesized materials. If the factor is a dimer,
e.g., PDGF, the recombinant factor can be a recombinant
heterodimer, made by inserting into cultured prokaryotic or
eukaryotic cells DNA sequences encoding both subunits of the
factor, and then allowing the translated subunits to be processed
by the cells to form a heterodimer (e.g., PDGF-AB). Alternatively,
DNA encoding just one of the subunits (e.g., PDGF B-chain or
A-chain) can be inserted into cells, which then are cultured to
produce the homodimeric factor (e.g., PDGF-BB or PDGF-AA
homodimers). PDGF for use in the methods of the invention includes
PDGF homo- and heterodimers, for example, PDGF-AA, PDGF-BB,
PDGF-AB, PDGF-CC, and PDGF-DD, and combinations and derivatives
thereof.
[0060] The concentration of PDGF or other growth factors of the
invention can be determined by using, e.g., an enzyme-linked
immunoassay, as described in, e.g., U.S. Pat. Nos. 6,221,625,
5,747,273, and 5,290,708, incorporated herein by reference, or any
other assay known in the art for determining protein concentration.
When provided herein, the molar concentration of PDGF is determined
based on the molecular weight of PDGF dimer (e.g., PDGF-BB;
MW=approximately 25 kDa).
[0061] The methods and implant materials of the invention can be
used to heal bony wounds of mammals, e.g., fractures, implant
recipient sites, and sites of periodontal disease. The implant
materials promote connective tissue growth and repair and enhance
bone formation compared to natural healing (i.e., no exogenous
agents added) or healing supplemented by addition of systemic
antibiotics. Unlike natural healing, conventional surgical therapy,
or antibiotics, the implant materials of the invention prompt
increased bone, connective tissue (e.g., cartilage and ligament),
and cementum formation when applied to damaged or diseased tissues
or to periodontal disease affected sites. The restoration of these
tissues leads to an improved prognosis for the affected areas. The
ability of these factors to stimulate new bone formation also makes
it applicable for treating bony defects caused by other types of
infection or surgical or accidental trauma.
[0062] Other features and advantages of the invention will be
apparent from the following description of the embodiments thereof,
and from the claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0063] FIGS. 1A-1G are photomicrographs showing the effect on bone
formation 8 weeks following treatment. FIG. 1A is a photomicrograph
showing the effect of surgery alone on bone formation. FIG. 1B is a
photomicrograph showing the effect of .beta.-TCP alone on bone
formation. FIG. 1C is a photomicrograph showing the effect of
.beta.-TCP+0.3 mg/mL PDGF on bone formation. FIG. 1D is a
photomicrograph showing the effect of .beta.-TCP+1.0 mg/mL PDGF on
bone formation. FIG. 1E is a photomicrograph showing the effect of
demineralized freeze dried bone allograft (DFDBA) alone on bone
formation. FIG. 1F is a photomicograph showing the effect of
demineralized freeze dried bone allograft (DFDBA)+0.3 mg/mL PDGF on
bone formation. FIG. 1G is a photomicrograph showing the effect of
demineralized freeze dried bone allograft (DFDBA)+1.0 mg/mL on bone
formation.
[0064] FIGS. 2A-2C are photomicrographs showing the effect on bone
formation 16 weeks following treatment. FIG. 2A is a
photomicrograph showing the effect of .beta.-TCP alone on bone
formation. FIG. 2B is a photomicrograph showing the effect of
.beta.-TCP+0.3 mg/mL PDGF on bone formation. FIG. 2C is a
photomicrograph showing the effect of .beta.-TCP+1.0 mg/mL PDGF on
bone formation.
DETAILED DESCRIPTION
[0065] We now describe several embodiments of the invention. Two
examples demonstrating the use of PDGF as a bone and periodontum
healing agent are presented below.
EXAMPLES
Example I
Preparation of PDGF
[0066] Osseous wounds, e.g., following periodontal disease or
trauma, are treated and periodontium, including bone, cementum, and
connective tissue, are regenerated, according to the invention by
combining partially purified or purified PDGF with any of the
pharmaceutically acceptable carrier substances described above.
Purified PDGF can be obtained from a recombinant source or from
human platelets. Commercially available recombinant PDGF can be
obtained from R&D Systems Inc. (Minneapolis, Minn.), BD
Biosciences (San Jose, Calif.), and Chemicon, International
(Temecula, Calif.). Partially purified and purified PDGF can also
be prepared as follows:
[0067] Five hundred to 1000 units of washed human platelet pellets
are suspended in 1M NaCl (2 ml per platelet unit) and heated at
100.degree. C. for 15 minutes. The supernatant is then separated by
centrifugation and the precipitate extracted twice with the 1m
NaCl.
[0068] The extracts are combined and dialyzed against 0.08M
NaCl/0.01M sodium phosphate buffer (pH 7.4) and mixed overnight at
4.degree. C. with CM-Sephadex C-50 equilibrated with the buffer.
The mixture is then poured into a column (5.times.100 cm), washed
extensively with 0.08M NaCl/0.01M sodium phosphate buffer (pH 7.4),
and eluted with 1M NaCl while 10 ml fractions are collected.
[0069] Active fractions are pooled and dialyzed against 0.3M
NaCl/0.01M sodium phosphate buffer (pH 7.4), centrifuged, and
passed at 4.degree. C. through a 2.5.times.25 cm column of blue
sepharose (Pharmacia) equilibrated with 0.3M NaCl/0.01M sodium
phosphate buffer (pH 7.4). The column is then washed with the
buffer and partially purified PDGF eluted with a 1:1 solution of 1M
NaCl and ethylene glycol.
[0070] The partially purified PDGF fractions are diluted (1:1) with
1M NaCl, dialyzed against 1M acetic acid, and lyophilized. The
lyophilized samples are dissolved in 0.8M NaCl/0.01M sodium
phosphate buffer (pH 7.4) and passed through a 1.2.times.40 cm
column of CM-Sephadex C-50 equilibrated with the buffer. PDGF is
then eluted with a NaCl gradient (0.08 to 1M).
[0071] The active fractions are combined, dialyzed against 1M
acetic acid, lyophilized, and dissolved in a small volume of 1M
acetic acid. 0.5 ml portions are applied to a 1.2.times.100 cm
column of Biogel P-150 (100 to 200 mesh) equilibrated with 1M
acetic acid. The PDGF is then eluted with 1M acetic acid while 2 mL
fractions are collected.
[0072] Each active fraction containing 100 to 200 mg of protein is
lyophilized, dissolved in 100 mL of 0.4% trifluoroacetic acid, and
subjected to reverse phase high performance liquid chromatography
on a phenyl Bondapak column (Waters). Elution with a linear
acetonitrile gradient (0 to 60%) yields pure PDGF.
PDGF Made by Recombinant DNA Technology can be Prepared as
Follows:
[0073] Platelet-derived growth factor (PDGF) derived from human
platelets contains two polypeptide sequences (PDGF-B and PDGF-A
polypeptides; Antoniades, H. N. and Hunkapiller, M., Science
220:963-965, 1983). PDGF-B is encoded by a gene localized on
chromosome 7 (Betsholtz, C. et al., Nature 320:695-699), and PDGF-A
is encoded by the sis oncogene (Doolittle, R. et al., Science
221:275-277, 1983) localized on chromosome 22 (Dalla-Favera, R.,
Science 218:686-688, 1982). The sis gene encodes the transforming
protein of the Simian Sarcoma Virus (SSV) which is closely related
to PDGF-2 polypeptide. The human cellular c-sis also encodes the
PDGF-A chain (Rao, C. D. et al., Proc. Natl. Acad. Sci. USA
83:2392-2396, 1986). Because the two polypeptide chains of PDGF are
coded by two different genes localized in separate chromosomes, the
possibility exists that human PDGF consists of a disulfide-linked
heterodimer of PDGF-B and PDGF-A, or a mixture of the two
homodimers (PDGF-BB homodimer and PDGF-AA homodimer), or a mixture
of the heterodimer and the two homodimers.
[0074] Mammalian cells in culture infected with the Simian Sarcoma
Virus, which contains the gene encoding the PDGF-A chain, were
shown to synthesize the PDGF-A polypeptide and to process it into a
disulfide-linked homodimer (Robbins et al., Nature 305:605-608,
1983). In addition, the PDGF-A homodimer reacts with antisera
raised against human PDGF. Furthermore, the functional properties
of the secreted PDGF-A homodimer are similar to those of
platelet-derived PDGF in that it stimulates DNA synthesis in
cultured fibroblasts, it induces phosphorylation at the tyrosine
residue of a 185 kD cell membrane protein, and it is capable of
competing with human (.sup.125I)-PDGF for binding to specific cell
surface PDGF receptors (Owen, A. et al., Science 225:54-56, 1984).
Similar properties were shown for the sis/PDGF-A gene product
derived from cultured normal human cells (for example, human
arterial endothelial cells), or from human malignant cells
expressing the sis/PDGF-2 gene (Antoniades, H. et al., Cancer Cells
3:145-151, 1985).
[0075] The recombinant PDGF-B homodimer is obtained by the
introduction of cDNA clones of c-sis/PDGF-B gene into mouse cells
using an expression vector. The c-sis/PDGF-B clone used for the
expression was obtained from normal human cultured endothelial
cells (Collins, T., et al., Nature 216:748-750, 1985).
Use of PDGF
[0076] PDGF alone or in combination with other growth factors is
useful for promoting bone healing, bone growth and regeneration or
healing of the supporting structures of teeth injured by trauma or
disease. It is also useful for promoting healing of a site of
extraction of a tooth, for mandibular ridge augmentation, or at
tooth implant sites. Bone healing would also be enhanced at sites
of bone fracture or in infected areas, e.g., osteomyelitis, or at
tumor sites. PDGF is also useful for promoting growth and healing
of a ligament, e.g., the periodontal ligament, and of cementum.
[0077] In use, the PDGF or other growth or differentiation factor
is applied directly to the area needing healing or regeneration.
Generally, it is applied in a resorbable or non-resorbable carrier
as a liquid or solid, and the site then covered with a bandage or
nearby tissue. An amount sufficient to promote bone growth is
generally between 500 ng and 5 mg for a 1 cm.sup.2 area, but the
upper limit is really 1 mg for a 1 cm.sup.2 area, with a preferred
amount of PDGF applied being 0.3 mg/mL.
Example II
Periodontal Regeneration with rhPDGF-BB Treated Osteoconductive
Scaffolds
[0078] The effectiveness of PDGF in promoting periodontium and bone
growth is demonstrated by the following study.
In Vivo Dog Study
[0079] The beagle dog is the most widely used animal model for
testing putative periodontal regeneration materials and procedures
(Wikesjo et al., J. Clin. Periodontol. 15:73-78, 1988; Wikesjo et
al., J. Clin. Periodontol. 16:116-119, 1999; Cho et al., J.
Periodontol. 66:522-530, 1995; Giannobile et al., J. Periodontol.
69:129-137, 1998; and Clergeau et al., J. Periodontol. 67:140-149,
1996). Plaque and calculus accumulation can induce gingival
inflammation that may lead to marginal bone loss and the etiology
of periodontitis in dogs and humans can be compared. In naturally
occurring disease, however, there is a lack of uniformity between
defects. Additionally, as more attention has been given to oral
health in canine breeder colonies, it has become impractical to
obtain animals with natural periodontal disease. Therefore, the
surgically-induced horizontal Class III furcation model has become
one of the most commonly used models to investigate periodontal
healing and regeneration.
[0080] Beagle dogs with horizontal Class III furcation defects were
treated using PDGF compositions of the invention. Fifteen adult
beagle dogs contributed 60 treated defects. Forty-two defects were
biopsied two months after treatment and fifteen defects/were
biopsied four months after treatment
Defect Preparation
[0081] The "critical-size" periodontal defect model as described by
numerous investigators was utilized (see, e.g., Wikesjo, 1988 and
1999, supra; Giannobile, supra, Cho, supra, and Park et al., J.
Periodontol. 66:462-477, 1995). Both mandibular quadrants in 16
male beagle dogs (2-3 years old) without general and oral health
problems were used. One month prior to dosing, the animals were
sedated with a subcutaneous injection of atropine (0.02 mg/kg) and
acepromazine (0.2 mg/kg) approximately 30 minutes prior to being
anesthetized with an IV injection of pentobarbital sodium (25
mg/kg). Following local infiltration of the surgical area with
Lidocaine HCl plus epinephrine 1:100,000, full thickness
mucoperiosteal flaps were reflected and the first and third
premolars (P1 and P3) were extracted. Additionally, the mesial
portion of the crown of the 1st molar was resected.
[0082] Alveolar bone was then removed around the entire
circumference of P2 and P4, including the furcation areas using
chisels and water-cooled carbide and diamond burs. Horizontal bone
defects were created such that there was a distance of 5 mm from
the fornix of the furcation to the crest of the bone. The defects
were approximately 1 cm wide, depending on the width of the tooth.
The roots of all experimental teeth were planed with curettes and
ultrasonic instruments and instrumented with a tapered diamond bur
to remove cementum. After the standardized bone defects were
created the gingival flaps were sutured to achieve primary closure.
The animals were fed a soft diet and received daily chlorhexidine
rinses for the duration of the study.
[0083] Application of Graft Material
[0084] The periodontal defects of P2 and P4 in each mandibular
quadrant of the 15 animals were randomized prior to treatment using
sealed envelopes. About four weeks after defect preparation,
animals were re-anesthetized as described above and full thickness
flaps were reflected in both mandibular quadrants. A notch was
placed in the tooth root surfaces at the residual osseous crest
using a 1/2 round bur to serve as a future histologic reference
point. The sites were irrigated with sterile saline and the roots
were treated with citric acid as described previously for the
purpose of decontamination and removal of the smear layer (See,
e.g., Cho, supra, and Park, supra). During this period an amount of
.beta.-TCP or DFDBA sufficient to fill the periodontal defect was
saturated with a solution of rhPDGF-BB solution (0.3 or 1.0 mg/ml)
and the rhPDGF-BB/graft mixture was allowed to sit on the sterile
surgical stand for about ten minutes. The rhPDGF-BB saturated graft
was then packed into the defect with gentle pressure to the ideal
level of osseous regeneration.
[0085] After implantation of the graft material, the mucoperiosteal
flaps were sutured approximately level to the cementoenamel
junction (CEJ) using interproximal, interrupted 4.0 expanded
polytetrafluoroethylene (ePTFE) sutures. Following suturing of the
flaps chlorhexidine gluconate gel was gently placed around the
teeth and gingivae.
[0086] Treatment and Control Groups
[0087] Defects Received Either:
[0088] 1. .beta.-TCP
[0089] 2. .beta.-TCP plus rhPDGF-BB (0.3 mg/ml rhPDGF-BB)
[0090] 3. .beta.-TCP plus rhPDGF-BB (1.0 mg/ml rhPDGF-BB)
[0091] 4. Dog DFDBA
[0092] 5. Dog DFDBA plus rhPDGF-BB (0.3 mg/ml rhPDGF-BB)
[0093] 6. Dog DFDBA plus rhPDGF-BB (1.0 mg/ml rhPDGF-BB)
[0094] 7. Sham surgery (treated by open flap debridement only, no
graft)
[0095] Six defects per treatment group were biopsied at two months
(42 total sites). In addition, five defects in treatment groups 1,
2, and 3 were biopsied at four months (15 total sites).
TABLE-US-00002 TABLE 2 Experimental design NO. OF GROUP TEST NO.
SITES TREATMENT TIME POINTS 1 11 .beta.-TCP alone 8 & 16 weeks
n = 6 for 8 wk n = 5 for 16 wk 2 11 .beta.-TCP + 0.3 mg/ml 8 &
16 weeks rhPDGF-BB n = 6 for 8 wk n = 5 for 16 wk 3 11 .beta.-TCP +
1.0 mg/ml 8 & 16 weeks rhPDGF-BB n = 6 for 8 wk n = 5 for 16 wk
4 6 DFDBA alone 8 weeks 5 6 DFDBA + 0.3 mg/ml 8 weeks rhPDGF-BB 6 6
DFDBA + 1.0 mg/ml 8 weeks rhPDGF-BB 7 6 Surgery, no graft 8
weeks
[0096] Accordingly, at 8 weeks there are 7 groups divided among 42
sites in 11 dogs. At 16 weeks, there are 3 groups divided among 15
sites in 4 dogs (one dog received two treatment surgeries staggered
eight weeks apart and thus contributed two sites to each the 8 and
16 week time points).
Post-Surgical Treatment
[0097] The surgical sites were protected by feeding the dogs a soft
diet during the first 4 weeks post-operative. To insure optimal
healing, systemic antibiotic treatment with penicillin G benzathine
was provided for the first two weeks and plaque control was
maintained by daily irrigation with 2% chlorhexidine gluconate
throughout the experiment. Sutures were removed after 3 weeks.
[0098] Data Collection
[0099] Rationale for Data Collection Points
[0100] The eight week time point was chosen because this is the
most common time point reported for this model in the literature
and therefore there are substantial historical data. For example,
Wikesjo et al., supra, and Giannobile et al., supra, also chose 8
weeks to assess the regenerative effects of BMP-2 and OP-1,
respectively, in the same model. Additionally, Park et al., supra,
evaluated the effect or rhPDGF-BB applied directly to the
conditioned root surface with and without GTR membranes in the
beagle dog model at 8 weeks. These studies, strongly suggest that
the 8 week period should be optimal for illustrating potential
significant effects among the various treatment modalities.
[0101] The sixteen week time point was chosen to assess long-term
effects of growth factor treatment. Previous studies (Park et al.,
supra) suggest that by this time there is substantial spontaneous
healing of the osseous defects. Nevertheless, it is possible to
assess whether rhPDGF-BB treatment leads to any unusual or abnormal
tissue response, such as altered bone remodeling, tumorgenesis or
root resorption.
[0102] Biopsies and Treatment Assessments
[0103] At the time of biopsy, the animals were perfused with 4%
paraformaldehyde and sacrificed. The mandibles were then removed
and placed in fixative. Periapical radiographs were taken and the
treated sites were cut into individual blocks using a diamond saw.
The coded (blinded) blocks were wrapped in gauze, immersed in a
solution of 4% formaldehyde, processed, and analyzed.
[0104] During processing the biopsies were dehydrated in ethanol
and infiltrated and embedded in methylmethacrylate. Undecalcified
sections of approximately 300 .mu.m in thickness were obtained
using a low speed diamond saw with coolant. The sections were glued
onto opalescent acrylic glass, ground to a final thickness of
approximately 80 .mu.m, and stained with toludine blue and basic
fuchsin. Step serial sections were obtained in a mesiodistal
plane.
[0105] Histomorphometric analyses were performed on the masked
slides. The following parameters were assessed:
[0106] 1. Length of Complete New Attachment Apparatus (CNAA):
Periodontal regeneration measured as the distance between the
coronal level of the old bone and the coronal level of the new
bone, including only that new bone adjacent to new cementum with
functionally oriented periodontal ligament between the new bone and
new cementum.
[0107] 2. New Bone Fill (NB): Measured as the cross-sectional area
of new bone formed within the furcation.
[0108] 3. Connective Tissue fill (CT): Measured as the area within
the furcation occupied by gingival connective tissue.
[0109] 4. Void (VO): The area of recession where there is an
absence of tissue.
[0110] Results
[0111] A. Clinical Observations
[0112] Clinically, all sites healed well. There was an impression
that the sites treated with rhPDGF-BB healed more quickly, as
indicated by the presence of firm, pink gingivae within one week
post-operatively. There were no adverse events experienced in any
treatment group as assessed by visual inspection of the treated
sites. There appeared to be increased gingival recession in groups
that received .beta.-TCP or DFDBA alone.
[0113] B. Radiographic Observations
[0114] Radiographically, there was evidence of increased bone
formation at two months as judged by increased radiopacity in
Groups 2, 3 (.beta.-TCP+rhPDGF-BB 0.3 and 1.0 mg/ml, respectively)
and 6 (DFDBA+rhPDGF-BB 1.0 mg/ml) compared to the other groups
(FIGS. 1A-G). At four months, there was evidence of increased bone
formation in all groups compared to the two month time point. There
was no radiographic evidence of any abnormal bone remodeling, root
resorption, or ankylosis in any group.
TABLE-US-00003 TABLE 3 Radiographic results. Rank order.
QUALITATIVE ASSESSMENT OF BONE FILL AT 8 WKS* TREATMENT 6
.beta.-TCP alone 1 .beta.-TCP + 0.3 mg/ml rhPDGF 2 .beta.-TCP + 1.0
mg/ml rhPDGF 7 DFDBA alone 5 DFDBA + 0.3 mg/ml rhPDGF 3 DFDBA + 1.0
mg/ml rhPDGF 4 Surgery, no graft *1 = most fill; 7 = least fill
[0115] C. Histomorphometric Analyses:
[0116] Histomorphometric assessment of the length of new cementum,
new bone, and new periodontal ligament (CNAA) as well as new bone
fill, connective tissue fill, and void space were evaluated and are
expressed as percentages. In the case of CNAA, values for each test
group represent the CNAA measurements (length in mm)/total
available CNAA length (in mm).times.100%. Bone fill, connective
tissue fill and void space were evaluated and are expressed as
percentages of the total furcation defect area.
[0117] One-way analysis of variance (ANOVA) was used to test for
overall differences among treatment groups, and pairwise
comparisons were made using the student's t-test. Significant
differences between groups were found upon analyses of the coded
slides. Table 4 shows the results at two months.
TABLE-US-00004 TABLE 4 Two month histometric analyses % CNAA %
GROUP PERIODONTAL % CONNECTIVE % NO. TREATMENT REGENERATION BONE
FILL TISSUE FILL VOID 1 .beta.-TCP alone 37.0 .+-. 22.8** 28.0 .+-.
29.5 36.0 .+-. 21.5 12.0 .+-. 17.9 2 .beta.-TCP + 0.3 mg/ml 59.0
.+-. 19.1*, .dagger. 84.0 .+-. 35.8.dagger., .dagger-dbl. 0.0 .+-.
0.0 8.0 .+-. 17.9 rhPDGF 3 .beta.-TCP + 1.0 mg/ml 46.0 .+-. 12.3*
74.2 .+-. 31.7.dagger-dbl. 0.0 .+-. 0.0 0.0 .+-. 0.0 rhPDGF 4 DFDBA
alone 13.4 .+-. 12.0 6.0 .+-. 8.9 26.0 .+-. 19.5 30.0 .+-. 27.4 5
DFDBA + 0.3 mg/ml 21.5 .+-. 13.3 20.0 .+-. 18.7 36.0 .+-. 13.4 18.0
.+-. 21.7 rhPDGF 6 DFDBA + 1.0 mg/ml 29.9 .+-. 12.4 46.0 .+-.
23.0.noteq. 26.0 .+-. 5.48 8.0 .+-. 13.04 rhPDGF 7 Sham Surgery,
27.4 .+-. 15.0 34.0 .+-. 27.0 48.0 .+-. 35.64 10.0 .+-. 22.4 no
graft *Groups 2 and 3 significantly greater (p < 0.05) than
Groups 4 and 7. **Group 1 significantly greater (p < 0.05) than
Group 4. .dagger.Group 2 significantly greater (p < 0.05) than
Group 5. .dagger-dbl.Groups 2 and 3 significantly greater than
Groups 1, 4 and 7. .noteq.Group 6 significantly greater than Group
4.
[0118] The mean percent periodontal regeneration (CNAA) in the
surgery without grafts and surgery plus .beta.-TCP alone groups
were 27% and 37%, respectively. In contrast, .beta.-TCP groups
containing rhPDGF-BB exhibited significantly greater periodontal
regeneration (p<0.05) than surgery without grafts or DFDBA alone
(59% and 46% respectively for the 0.3 and 1.0 mg/ml concentrations
versus 27% for surgery alone and 13% for DFDBA alone). Finally, the
.beta.-TCP group containing 0.3 mg/ml rhPDGF-BB demonstrated
significantly greater periodontal regeneration (p<0.05) than the
same concentration of rhPDGF-BB combined with allograft (59% versus
21%).
[0119] Bone fill was significantly greater (p<0.05) in the
.beta.-TCP+0.3 mg/ml rhPDGF-BB (84.0%) and the .beta.-TCP+1.0 mg/ml
rhPDGF-BB (74.2%) groups than in the .beta.-TCP alone (28.0%),
surgery alone (34%) or DFDBA alone (6%) treatment groups. There was
also significantly greater bone fill (p<0.05) for the
.beta.-TCP+0.3 mg/ml rbPDGF-BB group compared to the DFDBA+0.3
mg/ml rbPDGF-BB group (84% and 20% respectively).
[0120] The group of analyses examining the 8-week data from the
DFDBA groups and the surgery alone group (Groups 4, 5, 6, and 7)
demonstrated no statistically significant differences between the
DFDBA groups and surgery alone for periodontal regeneration (CNAA).
There was a trend toward greater regeneration for those sites
treated with the 1.0 mg/ml rhPDGF-BB enhanced DFDBA versus DFDBA
alone. There was significantly greater bone fill (p<0.05) for
sites treated with DFDBA+1.0 mg/ml rhPDGF-BB than DFDBA alone (46
and 6% respectively). There was a trend toward greater bone fill
for sites treated with DFDBA containing 0.3 mg/ml rhPDGF-BB
compared to DFDBA alone or surgery alone. However, sites treated
with DFDBA alone demonstrated less bone fill into the defect than
surgery alone (6 and 34%, respectively), with most of the defect
being devoid of any fill or fill consisting of gingival (soft)
connective tissue.
[0121] At four months following treatment, there remained
significant differences in periodontal regeneration. .beta.-TCP
alone, as a result of extensive ankylosis, resulted in 36%
regeneration, while the sites treated with .beta.-TCP containing
rhPDGF-BB had a mean regeneration of 58% and 49% in the 0.3 and 1.0
mg/ml rhPDGF-BB concentrations. Substantial bone fill was present
in all three treatment groups. .beta.-TCP alone resulted in 70%
bone fill, .beta.-TCP plus 0.3 mg/ml rhPDGF yielded 100% fill while
the 1.0 mg/ml rhPDGF group had 75% fill.
[0122] D. Histologic Evaluation
[0123] Histologic evaluation was performed for all biopsies except
one, in which evaluation was not possible due to difficulties
encountered during processing.
[0124] Representative photomicrographs are shown in FIGS. 1A-G and
2A-C. FIG. 1A shows results from a site treated with surgery alone
(no grafts). This specimen demonstrates limited periodontal
regeneration (new bone (NB), new cementum (NC), and periodontal
ligament (PDL)) as evidenced in the area of the notches and
extending only a short distance coronally. The area of the
furcation is occupied primarily by dense soft connective tissue
(CT) with minimal new bone (NB) formation.
[0125] For sites treated with .beta.-TCP alone (FIG. 1B) there is
periodontal regeneration, similar to that observed for the surgery
alone specimen, that extends from the base of the notches for a
short distance coronally. As was seen in the surgery alone
specimens, there was very little new bone formation with the
greatest area of the furcation being occupied by soft connective
tissue.
[0126] In contrast, FIG. 1C illustrates results obtained for sites
treated with .beta.-TCP+0.3 mg/ml rhPDGF-BB. Significant
periodontal regeneration is shown with new bone, new cementum, and
periodontal ligament extending along the entire surface of the
furcation. Additionally, the area of the furcation is filled with
new bone that extends the entire height of the furcation to the
fornix.
[0127] Representative results for sites treated with .beta.-TCP+1.0
mg/ml rhPDGF-BB are shown in FIG. 1D. While there is significant
periodontal regeneration in the furcation, it does not extend along
the entire surface of the furcation. There is new bone formation
present along with soft connective tissue that is observed at the
coronal portion of the defect along with a small space which is
void of any tissue (VO) at the fornix of the furcation.
[0128] FIGS. 2A, 2B, and 2C illustrate results obtained for the
allograft treatment groups. Representative results for the DFDBA
alone group (FIG. 2A) shows very poor periodontal regeneration that
is limited to the area of the notches extending only slightly in a
coronal direction. New bone formation is limited and consists of
small amounts of bone formation along the surface of residual DFDBA
graft material (dark red staining along lighter pink islands).
Additionally, the new bone is surrounded by extensive soft
connective tissue that extends coronally to fill a significant area
within the furcation. Finally, a large void space extends from the
coronal extent of the soft connective tissue to the fornix of the
furcation.
[0129] Histologic results for the DFDBA+0.3 and 1.0 mg/ml rhPDGF-BB
are shown in FIGS. 2B and 2C, respectively. Both groups demonstrate
greater periodontal regeneration compared to DFDBA alone with a
complete new attachment apparatus (new bone, new cementum, and
periodontal ligament) extending from the base of the notches in the
roots for a short distance coronally (arrows). They also had
greater bone fill within the area of the furcation, although there
was significant fill of the furcation with soft connective
tissue.
[0130] Conclusions
[0131] Based on the results of the study, treatment of a
periodontal defect using rhPDGF-BB at either 0.3 mg/mL or 1.0 mg/mL
in combination with a suitable carrier material (e.g., .beta.-TCP)
results in greater periodontal regeneration than the current
products or procedures, such as grafts with .beta.-TCP or bone
allograft alone, or periodontal surgery without grafts.
[0132] Treatment with the 0.3 mg/mL and 1.0 mg/mL concentration of
rhPDGF resulted in periodontal regeneration. The 0.3 mg/ml
concentration of rhPDGF demonstrated greater periodontal
regeneration and percent bone fill as compared to the 1.0 mg/ml
concentration of rhPDGF when mixed with .beta.-TCP.
[0133] .beta.-TCP was more effective than allograft when mixed with
rhPDGF-BB at any concentration. The new bone matured (remodeled)
normally over time (0, 8, and 16 weeks) in all groups. There was no
increase in ankylosis or root resorption in the rhPDGF groups. In
fact, sites receiving rhPDGF-BB tended to have less ankylosis than
control sites. This finding may result from the fact that rhPDGF-BB
is mitogenic and chemotactic for periodontal ligament cells.
Materials and Methods
Materials Utilized: Test and Control Articles
[0134] The .beta.-TCP utilized had a particle-size (0.25 mm-1.0 mm)
that was optimized for periodontal use. Based on studies using a
canine model, administered .beta.-TCP is 80% resorbed within three
months and is replaced by autologous bone during the healing
process.
[0135] The DFDBA was supplied by Musculoskeletal Transplant
Foundation (MTF). The material was dog allograft, made by from the
bones of a dog that was killed following completion of another
study that tested a surgical procedure that was deemed to have no
effect on skeletal tissues.
[0136] Recombinant hPDGF-BB was supplied by BioMimetic
Pharmaceuticals and was manufactured by Chiron, Inc, the only
supplier of FDA-approved rhPDGF-BB for human use. This rhPDGF-BB
was approved by the FDA as a wound healing product under the trade
name of Regranex.RTM..
[0137] One ml syringes containing 0.5 ml of sterile rhPDGF-BB at
two separate concentrations prepared in conformance with FDA
standards for human materials and according to current applicable
Good Manufacturing Processes (cGMP). Concentrations tested included
0.3 mg/ml and 1.0 mg/ml.
[0138] .beta.-TCP was provided in vials containing 0.5 cc of
sterile particles.
[0139] DFDBA was provided in 2.0 ml syringes containing 1.0 cc of
sterile, demineralized freeze-dried dog bone allograft.
Material Preparation
[0140] At the time of the surgical procedure, the final implanted
grafts were prepared by mixing the rhPDGF-BB solution with the
matrix materials. Briefly, an amount of TCP or allograft sufficient
to completely fill the osseous defect was placed into a sterile
dish. The rhPDGF-BB solution sufficient to completely saturate the
matrix was then added, the materials were mixed and allowed to sit
on the surgical tray for about 10 minutes at room temperature prior
to being placed in the osseous defect.
[0141] A 10 minute incubation time with the .beta.-TCP material is
sufficient to obtain maximum adsorption of the growth factor (see
Appendix A). This is also an appropriate amount of time for
surgeons in a clinical setting to have prior to placement of the
product into the periodontal defect. Similarly, in a commercial
market, the rhPDGF-BB and the matrix material can be supplied in
separate containers in a kit and that the materials can be mixed
directly before placement. This kit concept would greatly simplify
product shelf life/stability considerations.
Example III
Use of PDGF for the Treatment of Periodontal Bone Defects in
Humans
[0142] Recombinant human PDGF-BB (rhPDGF-BB) was tested for its
effect on the regeneration of periodontal bone in human subjects.
Two test groups were administered rhPDGF-BB at either 0.3 mg/mL
(Group I) or 1.0 mg/mL (Group II). rhPDGF-BB was prepared in sodium
acetate buffer and administered in a vehicle of beta-tricalcium
phosphate (.beta.-TCP). The control group, Group III, was
administered .beta.-TCP in sodium acetate buffer only.
[0143] The objective of clinical study was to evaluate the safety
and effectiveness of graft material comprising .beta.-TCP and
rhPDGF-BB at either 0.3 mg/mL or 1.0 mg/mL in the management of one
(1) to three (3) wall intra-osseous periodontal defects and to
assess its regenerative capability in bone and soft tissue.
Study Design and Duration of Treatment
[0144] The study was a double-blind, controlled, prospective,
randomized, parallel designed, multi-center clinical trial in
subjects who required surgical intervention to treat a bone defect
adjacent to the natural dentition. The subjects were randomized in
equal proportions to result in three (3) treatment groups of
approximately 60 subjects each (180 total). The duration of the
study was six (6) months following implantation of the study
device. The study enrolled 180 subjects.
Diagnosis and Main Entry Criteria
[0145] Male and female subjects, 25-75 years of age, with advanced
periodontal disease in at least one site requiring surgical
treatment to correct a bone defect were admitted to the study.
Other inclusion criteria included: 1) a probing pocket depth
measuring 7 mm or greater at the baseline visit; 2) after surgical
debridement, 4 mm or greater vertical bone defect (BD) with at
least 1 bony wall; 3) sufficient keratinized tissue to allow
complete tissue coverage of the defect; and, 4) radiographic base
of defect at least 3 mm coronal to the apex of the tooth. Subjects
who smoked up to 1 pack a day and who had teeth with Class I &
II furcation involvement were specifically allowed.
Dose and Mode of Administration
[0146] All treatment kits contained 0.25 g of .beta.-TCP (an active
control) and either 0.5 mL sodium acetate buffer solution alone
(Group III), 0.3 mg/mL rhPDGF-BB (Group I), or 1.0 mg/mL rhPDGF-BB
(Group II).
[0147] Following thorough debridement and root planing, the test
solution was mixed with .beta.-TCP in a sterile container, such
that the .beta.-TCP was fully saturated. Root surfaces were
conditioned using either tetracycline, EDTA, or citric acid. The
hydrated graft was then packed into the osseous defect and the
tissue flaps were secured with interdental sutures to achieve
complete coverage of the surgical site.
[0148] Effectiveness Measurement
[0149] The primary effectiveness measurement included the change in
clinical attachment level (CAL) between baseline and six months
post-surgery (Group I vs. Group III). The secondary effectiveness
measurements consisted of the following outcomes: 1) linear bone
growth (LBG) and % bone fill (% BF) from baseline to six months
post-surgery based on the radiographic assessments (Group I and
Group II vs. Group III); 2) change in CAL between baseline and six
months post-surgery (Group II vs. Group III); 3) probing pocket
depth reduction (PDR) between baseline and six months post-surgery
(Group I and Group II vs. Group III); 4) gingival recession (GR)
between baseline and six months post-surgery (Group I and Group II
vs. Group III); 5) wound healing (WH) of the surgical site during
the first three weeks post-surgery (Group I and Group II vs. Group
III); 6) area under the curve for the change in CAL between
baseline and three (3) and six (6) months (Group I and Group II vs.
Group III); 7) the 95% lower confidence bound (LCB) for % BF at six
(6) months post-surgery (Groups I, II, and III vs. demineralized
freeze-dried bone allograft (DFDBA) as published in the literature;
Parashis et al., J. Periodontol. 69:751-758, 1998); 8) the 95% LCB
for linear bone growth at six (6) months post-surgery (Groups I,
II, and III vs. demineralized freeze-dried bone allograft (DFDBA)
as published in the literature; Persson et al., J. Clin.
Periodontol. 27:104-108, 2000); 9) the % LCB for the change in CAL
between baseline and six (6) months (Groups I, II, and II vs.
EMDOGAIN.RTM.-PMA P930021, 1996); and 10) the 95% LCB for the
change in CAL between baseline and six (6) months (Groups I, II and
III vs. PEPGEN P-15.TM.-PMA P990033, 1999).
[0150] Statistical Methods
[0151] Safety and effectiveness data were examined and summarized
by descriptive statistics. Categorical measurements were displayed
as counts and percents, and continuous variables were displayed as
means, medians, standard deviations and ranges. Statistical
comparisons between the test product treatment groups (Groups I and
II) and the control (Group III) were made using Chi-Square and
Fisher's Exact tests for categorical variables and t-tests or
Analysis of Variance Methods (ANOVA) for continuous variables.
Comparisons between treatment groups for ordinal variables were
made using Cochran-Mantel-Haenszel methods. A p<0.05 (one sided)
was considered to be statistically significant for CAL, LBG and %
BF.
[0152] Safety data were assessed by the frequency and severity of
adverse events as evaluated clinically and radiographically. There
were no significant differences between the three treatment groups
at baseline. There were also no statistically significant
differences observed in the incidence of adverse events (AEs; all
causes) among the three treatment groups. The safety analysis did
not identify any increased risk to the subject due to implantation
of the graft material.
Summary of Effectiveness Results
[0153] The results from the statistical analyses revealed both
clinically and statistically significant benefits for the two
treatment groups (Groups I and II), compared to the active control
of .beta.-TCP alone (Group III) and historical controls including
DFDBA, EMDOGAIN.RTM., and PEPGEN P-15.TM..
[0154] At three months post-surgery, a statistically significant
CAL gain from baseline was observed in favor of Group I versus
Group III (p=0.041), indicating that there are significant early
benefits of PDGF on the gain in CAL. At six months post-surgery,
this trend continued to favor Group I over Group III, although this
difference was not statistically significant (p=0.200). The area
under the curve analysis (AUC) which represents the cumulative
effect (i.e. speed) for CAL gain between baseline and six months
approached statistical significance favoring Group I in comparison
to Group III (p=0.054). Further, the 95% lower confidence bound
(LCB) analyses for all treatment groups substantiated the
effectiveness of Groups I and II compared to the CAL gains observed
at six (6) months for EMDOGAIN.RTM. and PEPGEN P-15.TM..
[0155] In addition to the observed clinical benefits of CAL,
radiographic analyses including Linear Bone Growth (LBG) and
Percent Bone Fill (% BF), revealed statistically significant
improvement in bone gain for Groups I and II vs. Group III. % BF
was defined as the percent of the original osseous defect filled
with new bone as measured radiographically. LBG showed significant
improvement in Group I (2.5 mm) when compared to Group III (0.9 mm,
p<0.001). LBG was also significant for Group II (1.5 mm) when
compared to Group III (p=0.021).
[0156] Percent Bone Fill (% BF) was significantly increased at six
months post-surgical in Group I (56%) and Group II (34%) when
compared to Group III (18%), for a p<0.001 and p=0.019,
respectively. The 95% lower bound of the confidence interval at six
months post-surgery, for both linear bone growth and % bone fill,
substantiated the effectiveness of Groups I and II compared to the
published radiographic results for DFDBA, the most widely used
material for periodontal grafting procedures.
[0157] At three months, there was significantly less Gingival
Recession (GR) (p=0.041) for Group I compared to Group III
consistent with the beneficial effect observed with CAL. No
statistically significant differences were observed in PDR and GR
at six months. Descriptive analysis of the number of sites
exhibiting complete wound healing (WH) at three weeks revealed
improvements in Group I (72%) vs. Group II (60%) and Group III
(55%), indicating a trend toward improved healing.
[0158] To assess the cumulative beneficial effect for clinical and
radiographic outcomes, a composite effectiveness analysis was
performed to determine the percent of patients with a successful
outcome as defined by CAL>2.7 mm and LBG>1.1 mm at six (6)
months. The CAL and LBG benchmarks of success were established by
the mean levels achieved for these parameters by the implanted
grafts, as identified in the "Effectiveness Measures" section
above. The results showed that 61.7% of Group I patients and 37.9%
of Group II patients met or exceeded the composite benchmark for
success compared to 30.4% of Group III patients, resulting in a
statistically significant benefit of Group I vs. Group III
(p<0.001). % BF revealed similar benefits for Group I (70.0%)
vs. Group III (44.6%) for p-value of 0.003.
[0159] In summary, Group I achieved statistically beneficial
results for CAL and GR at three (3) months as well as LBG and % BF
at six (6) months, compared to the .beta.-TCP alone active control
group (Group III). The clinical significance of these results is
further confirmed by comparison to historical controls. It is
concluded that PDGF-containing graft material was shown to achieve
clinical and radiographic effectiveness by six months for the
treatment of periodontal osseous defects.
TABLE-US-00005 TABLE 5 Summary of PDGF Graft Effectiveness ENDPOINT
GROUP I GROUP II GROUP III CAL Gain (mm): 3 months 3.8 3.4 3.3 (p =
0.04) (p = 0.40) CAL: AUC Analysis (mm .times. wk) 67.5 61.8 60.1
(p = 0.05) (p = 0.35) CAL (mm): 95% LCB 6 months 3.3 3.2 3.1 (vs
2.7 mm for EMDOGAIN & 1.1 mm for PEPGEN) GR (mm): 3 months 0.5
0.7 0.9 (p = 0.04) (p = 0.46) LBG (mm): 6 months 2.5 1.5 0.9 (p
< 0.001) (p = 0.02) % BF: 6 months 56.0 33.9 17.9 (p < 0.001)
(p = 0.02) Composite CAL-LBG 61.7% 37.9% 30.4% Analysis (p <
0.001) (p = 0.20) (% Success) CAL-% BF 70.0% 55.2% 44.6% (p =
0.003) (p = 0.13)
[0160] Graft material (i.e., .beta.-TCP) containing PDGF at 0.3
mg/mL and at 1.0 mg/mL was shown to be safe and effective in the
restoration of alveolar bone and clinical attachment around teeth
with moderate to advanced periodontitis in a large, randomized
clinical trial involving 180 subjects studied for up to 6 months.
These conclusions are based upon validated radiographic and
clinical measurements as summarized below.
[0161] Consistent with the biocompatibility data of the
PDGF-containing graft material, discussed above, and the historical
safe use of each individual component (i.e., .beta.-TCP alone or
PDGF alone), the study revealed no evidence of either local or
systemic adverse effects. There were no adverse outcomes
attributable to the graft material, which was found to be safe.
Conclusion
[0162] Implantation of .beta.-TCP containing PDGF at either 0.3
mg/mL or 1.0 mg/mL was found to be an effective treatment for the
restoration of soft tissue attachment level and bone as shown by
significantly improved CAL at 3 months compared to the active
control. Our findings are also consistent with the AUC analysis
that showed an improvement in CAL gain between baseline and six
months. Implantation of .beta.-TCP containing PDGF at either 0.3
mg/mL or 1.0 mg/mL was also found to be an effective treatment
based on significantly improved LBG and % BF compared to the active
control. Significantly improved clinical outcomes as shown by the
composite analysis of both soft and hard tissue measurements
compared to the .beta.-TCP alone active control also demonstrate
the effectiveness of the treatment protocol described above.
Finally, the results of administering .beta.-TCP containing PDGF at
either 0.3 mg/mL or 1.0 mg/mL were found to exceed established
benchmarks of effectiveness both clinically and
radiographically.
[0163] The results of this trial together with extensive and
confirmatory data from in vitro, animal and human studies
demonstrate that PDGF-containing graft material stimulates soft and
hard tissue regeneration in periodontal defects, although the
effects were more significant when PDGF in the range of 0.1 to 1.0
mg/mL (e.g., 0.1 mg/mL, 0.3 mg/mL, or 1.0 mg/mL) was administered
in the graft material. Moreover, PDGF administered in the graft
material in the amount of 0.3 mg/mL effectively regenerated soft
tissue and bone.
[0164] Other embodiments are within the following claims.
* * * * *