U.S. patent application number 09/366021 was filed with the patent office on 2002-08-08 for method for the treatment of chemonucleolysis.
Invention is credited to AN, HOWARD, MASUDA, KOICHI, SAMPATH, KUBER T., THONAR, EUGENE J-M.A..
Application Number | 20020106362 09/366021 |
Document ID | / |
Family ID | 22293705 |
Filed Date | 2002-08-08 |
United States Patent
Application |
20020106362 |
Kind Code |
A1 |
MASUDA, KOICHI ; et
al. |
August 8, 2002 |
METHOD FOR THE TREATMENT OF CHEMONUCLEOLYSIS
Abstract
The invention relates to a method of treatment for a mammal in
need of chemonucleolysis. The method comprising the administration
of an effective proteoglycan cleaving amount of a
proteoglycan-degrading enzyme and an effective amount of a growth
factor effective in promoting the synthesis of a matrix component.
The proteoglycan-degrading enzyme is preferably chondroitinase. The
growth factor is preferably osteogenic protein. The
proteoglycan-degrading enzyme and the growth factor are preferably
administered simultaneously.
Inventors: |
MASUDA, KOICHI; (WILMETTE,
IL) ; THONAR, EUGENE J-M.A.; (LOCKPORT, IL) ;
AN, HOWARD; (RIVERWOOD, IL) ; SAMPATH, KUBER T.;
(HOLLISTON, MA) |
Correspondence
Address: |
FOLEY & LARDNER
150 EAST GILMAN STREET
P.O. BOX 1497
MADISON
WI
53701-1497
US
|
Family ID: |
22293705 |
Appl. No.: |
09/366021 |
Filed: |
August 2, 1999 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
60103161 |
Oct 6, 1998 |
|
|
|
Current U.S.
Class: |
424/94.6 ;
424/94.1 |
Current CPC
Class: |
A61K 38/1875 20130101;
A61L 27/227 20130101; A61K 38/51 20130101; A61K 2300/00 20130101;
A61K 2300/00 20130101; A61P 19/04 20180101; A61K 2300/00 20130101;
A61K 38/1841 20130101; A61K 38/51 20130101; A61L 2430/06 20130101;
A61K 38/1875 20130101; A61P 19/00 20180101; A61K 38/1841 20130101;
A61P 19/02 20180101 |
Class at
Publication: |
424/94.6 ;
424/94.1 |
International
Class: |
A61K 038/46 |
Goverment Interests
[0002] This invention was made with U.S. governmental support under
NIH grants 2-P50-AR39239 and AG04736. The U.S. government has
certain rights in the invention.
Claims
What is claimed is:
1. A method of treatment comprising administering to a mammal in
need of chemonucleolysis: (a) an effective proteoglycan cleaving
amount of a proteoglycan-degrading enzyme; and (b) an amount of a
growth factor effective for stimulating the formation of a matrix
component.
2. The method of claim 1 wherein said proteoglycan-degrading enzyme
is chondroitinase.
3. The method of claim 2 wherein said proteoglycan-degrading enzyme
is chondroitinase ABC.
4. The method of claim 2 wherein said proteoglycan-degrading enzyme
is chondroitinase AC.
5. The method of claim 2 wherein said proteoglycan-degrading enzyme
is chondroitinase B.
6. The method of claim 2 wherein said proteoglycan-degrading enzyme
is chondroitinase C.
7. The method of claim 1 wherein said growth factor is osteogenic
protein.
8. The method of claim 7 wherein said osteogenic protein is
OP-1.
9. The method of claim 1 wherein said growth factor is transforming
growth factor .beta..
10. The method of claim 1 wherein said proteoglycan-degrading
enzyme and said growth factor are administered simultaneously.
11. The method of claim 1 wherein said matrix component is
proteoglycan.
12. The method of claim 1 wherein said matrix component is
collagen.
13. A method of repairing the matrices of the nucleus pulposus
and/or annulus fibrosus in an intervertebral disk following
chemonucleolysis by a proteoglycan-degrading enzyme comprising
administering to a mammal an effective proteoglycan synthesizing
amount of a growth factor.
14. The method of claim 13 wherein said growth factor is osteogenic
protein.
15. The method of claim 14 wherein said osteogenic protein is
OP-1.
16. The method of claim 13 wherein said growth factor is
transforming growth factor .beta..
17. A method of replenishing proteoglycan in the nucleus pulposus
and/or annulus fibrosus in an intervertebral disk following
chemonucleolysis by a proteoglycan-degrading enzyme comprising
administering to a mammal an effective proteoglycan synthesizing
amount of a growth factor.
18. The method of claim 17 wherein said growth factor is osteogenic
protein.
19. The method of claim 18 wherein said osteogenic protein is
OP-1.
20. The method of claim 17 wherein said growth factor is
transforming growth factor .beta..
21. A composition of matter comprising a proteoglycan-degrading
enzyme and a growth factor.
22. The composition of matter of claim 21 wherein said
proteoglycan-degrading enzyme is chondroitinase.
23. The composition of matter of claim 22 wherein said
proteoglycan-degrading enzyme is chondroitinase ABC.
24. The composition of matter of claim 22 wherein said
proteoglycan-degrading enzyme is chondroitinase AC.
25. The composition of matter of claim 22 wherein said
proteoglycan-degrading enzyme is chondroitinase B.
26. The composition of matter of claim 22 wherein said
proteoglycan-degrading enzyme is chondroitinase C.
27. The composition of matter of claim 21 wherein said growth
factor is osteogenic protein.
28. The composition of matter of claim 27 wherein said osteogenic
protein is OP-1.
29. The composition of matter of claim 21 wherein said growth
factor is transforming growth factor .beta..
30. A kit comprising a proteoglycan-degrading enzyme and a growth
factor.
31. The kit of claim 30 wherein said proteoglycan-degrading enzyme
is chondroitinase.
32. The kit of claim 31 wherein said proteoglycan-degrading enzyme
is chondroitinase ABC.
33. The kit of claim 31 wherein said proteoglycan-degrading enzyme
is chondroitinase AC.
34. The kit of claim 31 wherein said proteoglycan-degrading enzyme
is chondroitinase B.
35. The kit of claim 31 wherein said proteoglycan-degrading enzyme
is chondroitinase C.
36. The kit of claim 30 wherein said growth factor is osteogenic
protein.
37. The kit of claim 36 wherein said osteogenic protein is
OP-1.
38. The kit of claim 30 wherein said growth factor is transforming
growth factor .beta..
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Ser. No.
60/103,161, filed Oct. 6, 1998, which is incorporated herein in its
entirety by reference.
FIELD OF THE INVENTION
[0003] The invention relates to a method of treatment for a mammal
in need of chemonucleolysis. More specifically, the invention
relates to a method of treatment comprising the administration of
an effective proteoglycan cleaving amount of a
proteoglycan-degrading enzyme and an effective proteoglycan
synthesizing amount of a growth factor.
BACKGROUND OF THE INVENTION
[0004] Low back pain constitutes a devastating economic burden for
individuals and society. In industry, it is the most frequent cause
of disability: the number of working days lost per year in the
United States is in excess of 100 million. Kramer, J.,
Intervertebral Disk Disease. Stuttgart, George Thieme Verlag
(1981). Over 500,000 workers are affected in the U.S. each year,
costing over 20 billion dollars. Kranzler, L. I., L. et al.,
Neurologic Clinics 3: 2 (1985). Low back pain is almost always
associated with pathological changes in one or more intervertebral
disks of the lumbar spine.
[0005] Intervertebral disks are a specialized fibrocartilaginous
connective tissue whose function is to resist the compressive,
rotational and tensile stresses applied to the vertebral column.
Humzah, M. D. et al., Anat. Rec. 220(4): 337-56 (1988). These disks
act as a hydrostatic shock absorber that cushions the forces
generated between two vertebrae and help these vertebrae articulate
smoothly with one another. The disk is a complex structure
consisting of two interdependent, but morphologically distinct
regions: the nucleus pulposus (NP), an inner gelatinous cushion
rich in proteoglycans (PGs), and an outer annulus fibrosus (AF)
made up of concentric lamellae rich in collagen fibers.
[0006] The metabolism of the cells that produce and maintain the
extracellular matrix of both the NP and AF is poorly understood.
The matrix in the NP is very similar to that found in articular
cartilage. Jahnke, M. R. et al., Biochemical Journal 251(2): 347-56
(1988). It is synthesized and maintained throughout adult life by
relatively few cells. More than 75% of NP cells are
chondrocyte-like, but a significant number of large notochordal
cells are present, especially prior to adult life. Maldonado, B. A.
et al., Journal of Orthopaedic Research 10(5): 677-90 (1992). It is
not clear if both NP cell types synthesize the
large-molecular-weight hydrophilic PG, termed aggrecan, that
constitutes the most abundant molecule in the tissue. As in
articular cartilage, these aggrecan molecules interact
extracellularly with long linear stands of hyaluronan (HA), forming
aggregates that become entangled in a fibrillar network made up
principally of type II collagen. Thonar, E. J. et al., Rheum. Dis.
Clin. North Am. 19(3): 635-57 (1993). The swelling, fluid- and
ion-transport properties, and the intrinsic mechanical properties
of the collagen-aggrecan solid matrix govern the deformational
behavior of the NP. The collagen network gives the tissue tensile
strength and hinders expansion of the viscoelastic, under-hydrated,
aggrecan molecules that provide compressive stiffness and enable
the tissue to undergo reversible deformation.
[0007] The AF contains a relatively homogeneous population of
chondrocyte-like cells (Maldonado, B. A. et al., Journal of
Orthopaedic Research 10(5): 677-90 (1992)) that synthesize a matrix
richer in collagen and poorer in PGs than cells from the NP.
Importantly, some of the cells synthesize PG and collagen molecules
not normally found in significant amounts in cartilage. Wu, J. J.
et al., Biochemical Journal 248(2): 373-81 (1987); Mayne, R. and
Brewton, R. G., "Extracellular matrix of cartilage collagen," in
Joint Cartilage Degradation. Basic and Clinical Aspects (Woessner,
J. R. and Howell, D. S., eds.), Marcel Dekker, Inc., New York, pp.
81-108 (1993); Thonar, E. J.-M. A. et al., "Body fluid markers of
cartilage changes in osteoarthritis," in Rheumatic Disease Clinics
of North America: Osteoarthritis (Moskowitz, R., ed.), W. B.
Saunders Co., Philadelphia, pp. 634-658 (1993). The AF is thus
usually classified as a fibrocartilage: it is built for strength
rather than to provide reversible deformation.
[0008] The metabolism of intervertebral disk cells is much less
well known than that of chondrocytes from articular cartilage.
Progress in this area has been limited by the costly nature of in
vivo experimental approaches and the restrictions imposed in the
past by the lack of an appropriate culture system to study the
metabolism of the cells. Chiba et al. (Chiba, K. et al., "Nucleus
pulposus and annulus fibrosus cells cultured in alginate:
characterization of matrix metabolism in different compartments,"
Transactions of 2.sup.nd Combined Meeting of the Orthopaedic
Research Societies of U.S.A., Japan, Canada and Europe, p. 32
(1995)) have developed a cell culture system that takes advantage
of an alginate bead culture system that was developed and refined
to study the metabolism of phenotypically-stable chondrocytes and
the turnover of the matrix they form de novo. Hauselmann, H. J., et
al., Matrix 12(2): 116-29 (1992); Huselmann, H. J. et aL, J Cell
Sci. 107: 17-27 (1994); Mok, S. S. et al., J. Biol. Chem. 269(52):
33021-7(1994); Petit, B., et al., Experimental Cell Research 225:
151-161 (1996). As in articular chondrocytes, intervertebral disk
cells entrapped in these alginate beads also reform an
extracellular matrix. Chiba, K., et al. Spine 22(24): 2885-93
(1997). This cell culture system can be used to study the effect of
compounds on the metabolism of both AF and NP cells. This cell
culture system can distinguish between changes occurring in the
metabolically active (cell-associated) and-inactive (further
removed) compartments of the matrix. Chiba et al. have also shown
that it is feasible to entrap a whole rabbit intervertebral disk in
alginate gel. Chiba, K., G. B. J. Andersson, et al., Ortho. Res.
Soc. Trans. 21:190 (1996). This approach, which more closely mimics
the in vivo situation, leads to improved retention of the disk
structure and promotes high metabolic activities.
[0009] Lumbar intervertebral disk herniation is one of the most
common causes of lower back pain. Disk herniation is initially
conservatively treated with physical therapy, but other more
invasive treatment modalities are sometimes required. For example,
chemonucleolysis, the dissolution of intervertebral disk tissue
using a locally injected enzyme, has been used for over thirty
years. Olmarker, et al. Clin. Orthopaedics and Related Res. 257:274
(1990). As PGs contribute most of the swelling pressure in the NP,
intradiscal injections of enzymes that degrade PGs (Bradford, D.
S., et al., Journal of Bone and Joint Surgery--American Volume
65(9): 1220-31 (1983); Hill, G. M. et al., Clinical Orthopaedics
and Related Researches 225: 229-233 (1987)) or collagens cause a
decompression of the nerve root entrapped by the herniated mass,
and thus help relieve pain. There is preliminary histological
evidence that the cells in disks treated with these enzymes can
replenish the NP with PGs, (Bradford, D. S., et al., Journal of
Bone and Joint Surgery--American Volume 65(9): 1220-31 (1983))
helping to reestablish the shock-absorbing properties of the
intervertebral cushion and, most importantly, to normalize forces
and stresses placed upon adjacent disks.
[0010] Complications can arise from chemonucleolysis when it is
performed with two commonly used enzymes, namely chymopapain and
collagenase. Kitchel and Brown report that chymopapain treatment
can lead to subarachnoid hemorrhage, paraplegia, anaphylaxis, and
even death. Clin. Orthopaedics and Related Res. 284:63 (1992).
Olmarker et al. note that the injected chymopapain is both
neurotoxic and allergenic, and that treatment with collagenase may
lead to neurologic deficits. Clin. Orthopaedics and Related Res.
257:274 (1990).
[0011] Because the painful sciatica associated with disk herniation
is often not relieved by conservative treatments, including
physical therapy and chemonucleolysis, many patients undergo disk
surgery. Hill, G. M. et al., Clinical Orthopaedics and Related
Researches 225: 229-233 (1987). However, surgical results are not
always satisfying and importantly, this approach is far from
optimal as removal of a lumbar intervertebral disk causes
significant destabilization of the lower spine and predisposes
adjacent intervertebral disks to degeneration in later years. Hill,
G. M. et al., Clinical Orthopaedics and Related Researches 225:
229-233 (1987).
[0012] Recently, newer treatment modalities for chemonucleolysis
have been developed that avoid some of the problems inherent in the
use of proteases such as chymopapain and collagenases, and may
avoid the need for surgical intervention. One such chemonucleolytic
enzyme is chondroitinase ABC, a product of Proteus vulgaris.
Chondroitinase ABC is an endo-N-acetyl-D-hexosaminidase that
degrades mucopolysaccharides such as chondroitin sulfate, dermatan
sulfate, chondroitin and hyaluronic acid. Takahashi et al., Spine
21:2405 (1996). Olmarker et al. note that chondroitinase ABC is
much less injurious to spinal tissue than chymopapain. Spine
21:1952 (1996).
[0013] Osteogenic protein-1 (OP-1), also known as Bone
Morphogenetic Protein-7 (BMP-7), is a member of the TGF-.beta.
superfamily that exerts potent effects on osteocyte and chondrocyte
differentiation and metabolism. Asahina, I., et al., J. Cell Biol.
123(4): 921-33 (1993). The bone morphogenetic proteins were shown
to induce new bone formation when injected subcutaneously in the
rat. Cook, S. D. et al., Clin. Orthop. 324: 29-38 (1996).
Recombinant human OP-1 (rhOP-1) has been shown to promote growth
and differentiation of osteoblasts in vitro. Sampath, T. K., et
al., J. Biol. Chem. 267: 20352-20360 (1992). It also causes
differentiation of mesenchymal stem cells along chondrogenic and
osteogenic pathways. Asahina, I., J. Cell. Biol. 123(4): 921-33
(1993). Recombinant human OP-1 also exerts specific effects on
chondrocytes. Chen, et al. demonstrated that OP-1 promotes growth
of chick sternal chondrocytes. Chen, P. et al., J. Cell. Sci.
108(Pt 1): 105-14 (1995). In this system, induction of synthesis of
type X collagen was noted, suggesting that OP-1 exerted an effect
on chondrocyte maturation as well. Bovine chondrocytes, in
contrast, did not express type X collagen in response to OP-1 but
did exhibit increased synthesis of PGs and type II collagen. Chen,
P. et al., Biochem. Biophys. Res. Commun. 197(3): 1253-9 (1993).
Growth factors are not used in existing chemonucleolysis treatment
utilizing protease enzymes such as chymopapain and collagenase
because these enzymes cleave or degrade the growth factors.
[0014] There is thus a need for a chemonucleolysis treatment that
not only provides relief from the symptoms of intervertebral disk
herniation, but also provides enhanced stability and repair of the
AF and NP. Similarly, there is a need for a chemonucleolysis
treatment that provides sufficient mechanical support to the spinal
column, and that obviates the need for invasive surgical
intervention. There is further a need to have a method of
chemonucleolytic treatment that requires only one treatment
modality for both dissolution of the intervertebral disk tissue
while providing both stability and repair of that tissue. The
treatment method of the present invention provides such a
method.
BRIEF SUMMARY OF THE INVENTION
[0015] A method of this invention involves a treatment comprising
administering to a mammal in need of chemonucleolysis an effective
proteoglycan cleaving amount of a proteoglycan-degrading enzyme;
and an amount of a growth factor effective for stimulating the
formation of a matrix component. In a preferred embodiment, the
matrix component is proteoglycan. In another preferred embodiment,
the matrix component is collagen.
[0016] In another aspect, the invention contemplates a method of
repairing the matrices of nucleus pulposus and/or annulus fibrosus
cells after chemonucleolysis by a proteoglycan-degrading enzyme
comprising administering to a mammal in need of such treatment an
effective proteoglycan synthesizing amount of a growth factor.
[0017] In yet another aspect, the invention contemplates a method
of replenishment of nucleus pulposus and/or annulus fibrosus cells
after chemonucleolysis by a proteoglycan-degrading enzyme
comprising administering to a mammal in need of such treatment an
effective proteoglycan synthesizing amount of a growth factor.
[0018] A preferred proteoglycan-degrading enzyme is chondroitinase.
Preferred chondroitinases include chondroitinase AC, chondroitinase
B, and chondroitinase C. A particularly preferred chondroitinase is
chondroitinase ABC.
[0019] A preferred growth factor is osteogenic protein. A
particularly preferred osteogenic protein is OP-1. Another
preferred growth factor is transforming growth factor .beta..
[0020] In a preferred embodiment, the proteoglycan-degrading enzyme
and the growth factor are administered simultaneously.
[0021] In another aspect, the present invention contemplates a
composition of matter comprising a proteoglycan-degrading enzyme
and a growth factor. Preferably, the proteoglycan-degrading enzyme
and the growth factor are formulated as a pharmaceutical
composition containing a pharmaceutically acceptable carrier and/or
vehicle.
[0022] In a still further aspect, the present invention
contemplates a kit comprising a proteoglycan-degrading enzyme and a
growth factor. In a preferred embodiment, the kit comprises a
vessel containing a proteoglycan-degrading enzyme and a vessel
containing a growth factor. In another preferred embodiment, the
kit comprises a vessel containing a proteoglycan-degrading enzyme
and a growth factor. Any of these kits optionally include
instructions for using the proteoglycan-degrading enzyme and the
growth factor in chemonucleolysis.
[0023] The present invention offers general benefits and
advantages. One advantage is that the deleterious effects of
chemonucleolysis on the structure of intervertebral disk matrices
can be counteracted by including as part of the treatment a growth
factor effective in promoting repair of the disk structures. A
benefit of the present invention is that the administration of a
growth factor is not impaired by the administration of proteolytic
chemonucleolysis enzymes. Another advantage is that the
administration of a chemonucleolytic enzyme can occur concurrently
with the administration of a growth factor protein since the enzyme
has no proteolytic activity and thus does not inactivate the growth
factor. A further benefit is that this method of treatment provides
fewer complications than known chemonucleolysis treatments.
[0024] Still further benefits and advantages of the present
invention will be apparent to a person of ordinary skill from the
description that follows.
BRIEF DESCRIPTION OF THE DRAWINGS
[0025] In the drawings forming a portion of this disclosure,
[0026] FIG. 1 illustrates the effects of OP-1 on the DNA content,
in micrograms per 9 alginate beads, of the annulus fibrosus ("AF")
treated with chondroitinase ABC ("C-ABC"). The open bars represent
data for cells not treated with C-ABC or OP-1 ("C-ABC(-),
OP-1(-)"). The hatched bars represent data for cells treated with
C-ABC only ("C-ABC(+), OP-1(-)"). The filled bars represent data
for cells treated with both C-ABC and OP-1 ("C-ABC(+), OP-1(+)").
The data are all presented with standard error bars. "N.S."
indicates that the difference in DNA content in the AF before and
after C-ABC treatment at time zero is not significant.
[0027] FIG. 2 illustrates the effects of OP-1 on the DNA content,
in micrograms per 9 alginate beads, of the nucleus pulposus ("NP")
treated with chondroitinase ABC ("C-ABC"). The open bars represent
data for cells not treated with C-ABC or OP-1 ("C-ABC(-),
OP-1(-)"). The hatched bars represent data for cells treated with
C-ABC only ("C-ABC(+), OP-1(-)"). The filled bars represent data
for cells treated with both C-ABC and OP-1 ("C-ABC(+), OP-1(+)").
The data are all presented with standard error bars. "N.S."
indicates that the difference in DNA content in the NP before and
after C-ABC treatment at time zero is not significant.
[0028] FIG. 3 illustrates the effects of OP-1 on total proteoglycan
("PG") accumulation, expressed as micrograms per 9 alginate beads,
of the annulus fibrosus ("AF") treated with chondroitinase ABC
("C-ABC"). The open bars represent data for cells not treated with
C-ABC or OP-1 ("C-ABC(-), OP1(-)"). The hatched bars represent data
for cells treated with C-ABC only ("C-ABC(+), OP-1(-)"). The filled
bars represent data for cells treated with both C-ABC and OP1
("C-ABC(+), OP-1(+)"). The data are all presented with standard
error bars. At time zero, the difference in total PG before and
after C-ABC treatment is statistically significant at p<0.05.
The * represents statistically significant differences at p<0.05
when C-ABC(+), OP-1(+) is compared to C-ABC(+), OP-1(-) at 7, 10,
14, 17 and 21 days after C-ABC treatment.
[0029] FIG. 4 illustrates the effects of OP-1 on total proteoglycan
("PG") accumulation, expressed as micrograms per 9 alginate beads,
of the nucleus pulposus ("NP") treated with chondroitinase ABC
("C-ABC"). The open bars represent data for cells not treated with
C-ABC or OP-1 ("C-ABC(-), OP-1(-)"). The hatched bars represent
data for cells treated with C-ABC only ("C-ABC(+), OP-1(-)"). The
filled bars represent data for cells treated with both C-ABC and
OP-1 ("C-ABC(+), OP-1(+)"). The data are all presented with
standard error bars. At time zero, the difference in total PG
before and after C-ABC treatment is statistically significant at
p<0.05. The * represents statistically significant differences
at p<0.05 when C-ABC(+), OP-1(+) is compared to C-ABC(+),
OP-1(-) at 10, 14, 17 and 21 days after C-ABC treatment.
[0030] FIG. 5 illustrates the effects of OP-1 on total proteoglycan
("PG") accumulation, expressed as micrograms PGs per microgram DNA,
of the annulus fibrosus ("AF") treated with chondroitinase ABC
("C-ABC"). The open bars represent data for cells not treated with
C-ABC or OP-1 ("C-ABC(-), OP-1(-)"). The hatched bars represent
data for cells treated with C-ABC only ("C-ABC(+), OP-1(-)"). The
filled bars represent data for cells treated with both C-ABC and
OP-1 ("C-ABC(+), OP-1(+)"). The data are all presented with
standard error bars. At time zero, the difference in total PG per
DNA content before and after C-ABC treatment is statistically
significant at p<0.05. The * represents statistically
significant differences at p<0.05 when C-ABC(+), OP-1(+) is
compared to C-ABC(+), OP-1(-) at 7, 10, 14, 17 and 21 days after
C-ABC treatment.
[0031] FIG. 6 illustrates the effects of OP-1 on total proteoglycan
("PG") accumulation, expressed as micrograms PGs per microgram DNA,
of the nucleus pulposus ("NP") treated with chondroitinase ABC
("C-ABC"). The open bars represent data for cells not treated with
C-ABC or OP-1 ("C-ABC(-), OP-1(-)"). The hatched bars represent
data for cells treated with C-ABC only ("C-ABC(+), OP-1(-)"). The
filled bars represent data for cells treated with both C-ABC and
OP-1 ("C-ABC(+), OP-1(+)"). The data are all presented with
standard error bars. At time zero, the difference in total PG
accumulation per DNA content before and after C-ABC treatment is
statistically significant at p<0.05. The * represents
statistically significant differences at p<0.05 when C-ABC(+),
OP-1(+) is compared to C-ABC(+), OP-1(-) at 7, 10, 14, 17 and 21
days after C-ABC treatment.
[0032] FIG. 7 illustrates the effects of OP-1 on proteoglycan
("PG") synthesis, expressed as counts per minute ("CPM") per 9
alginate beads, of the annulus fibrosus ("AF") treated with
chondroitinase ABC ("C-ABC"). The open bars represent data for
cells not treated with C-ABC or OP-1 ("C-ABC(-), OP-1(-)"). The
hatched bars represent data for cells treated with C-ABC only
("C-ABC(+), OP-1(-)"). The filled bars represent data for cells
treated with both C-ABC and OP-1 ("C-ABC(+), OP-1(+)"). The data
are all presented with standard error bars. The * represents
statistically significant differences at p<0.05 when C-ABC(+),
OP-1(+) is compared to C-ABC(+), OP-1(-) at 7, 14 and 21 days after
C-ABC treatment.
[0033] FIG. 8 illustrates the effects of OP-1 on proteoglycan
("PG") synthesis, expressed as counts per minute ("CPM") per 9
alginate beads, of the nucleus pulposus ("NP") treated with
chondroitinase ABC ("C-ABC"). The open bars represent data for
cells not treated with C-ABC or OP-1 ("C-ABC(-), OP-1(-)"). The
hatched bars represent data for cells treated with C-ABC only
("C-ABC(+), OP-1 (-)"). The filled bars represent data for cells
treated with both C-ABC and OP-1 ("C-ABC(+), OP-1(+)"). The data
are all presented with standard error bars. The * represents
statistically significant differences at p<0.05 when C-ABC(+),
OP-1(+) is compared to C-ABC(+), OP-1(-) at 7, 14 and 21 days after
C-ABC treatment.
[0034] FIG. 9 illustrates the effects of OP-1 on proteoglycan
("PG") synthesis, expressed as counts per minute ("CPM") per
microgram of DNA, of the annulus fibrosus ("AF") treated with
chondroitinase ABC ("C-ABC"). The open bars represent data for
cells not treated with C-ABC or OP-1 ("C-ABC(-), OP-1(-)"). The
hatched bars represent data for cells treated with C-ABC only
("C-ABC(+), OP-1(-)"). The filled bars represent data for cells
treated with both C-ABC and OP-1 ("C-ABC(+), OP-1(+)"). The data
are all presented with standard error bars. The * represents
statistically significant differences at p<0.05 when C-ABC(+),
OP-1(+) is compared to C-ABC(+), OP-1(-) at 7, 14 and 21 days after
C-ABC treatment.
[0035] FIG. 10 illustrates the effects of OP-1 on proteoglycan
("PG") synthesis, expressed as counts per minute ("CPM") per
microgram of DNA, of the nucleus pulposus ("NP") treated with
chondroitinase ABC ("C-ABC"). The open bars represent data for
cells not treated with C-ABC or OP-1 ("C-ABC(-), OP-1(-)"). The
hatched bars represent data for cells treated with C-ABC only
("C-ABC(+), OP-1(-)"). The filled bars represent data for cells
treated with both C-ABC and OP-1 ("C-ABC(+), OP-1(+)"). The data
are all presented with standard error bars. The * represents
statistically significant differences at p<0.05 when C-ABC(+),
OP-1(+) is compared to C-ABC(+), OP-1(-) at 7 and 14 days after
C-ABC treatment.
[0036] FIG. 11 illustrates the effects of recombinant human OP-1
("rhOP-1") on proteoglycan ("PGs") and collagen synthesis in the
annulus fibrosus ("AF"; open bars) and the nucleus pulposus ("NP",
filled bars). In the control ("Cont"), no rhOP-1 was added. PG
synthesis is expressed as the percentage of .sup.35S-PGs
synthesized when 100 ng/ml rhOP-1 or 200 ng/ml rhOP-1 was added,
compared to the control. Collagen synthesis is expressed as the
percentage of .sup.3H-hydroxyproline synthesized when 100 ng/ml
rhOP-1 or 200 ng/ml rhOP-1 was added, compared to the control. The
data are all presented with standard error bars.
[0037] FIG. 12 represents replicate studies illustrating the
effects of OP-1 on accumulation of proteoglycans in the annulus
fibrosus ("AF", left panel) and nucleus pulposus ("NP", right
panel), expressed as micrograms per 9 alginate beads, following
chondroitinase ABC ("C-ABC") treatment. The open bars represent
data for cells not treated with C-ABC or OP-1 ("W/O C-ABC W/O
OP-1"). The hatched bars represent data for cells treated with
C-ABC only ("with C-ABC W/O OP-1"). The filled bars represent data
for cells treated with both C-ABC and OP-1 ("with C-ABC, with
OP-1"). The data are all presented with standard error bars.
DETAILED DESCRIPTION OF THE INVENTION
[0038] A method of the invention contemplates treatment of a mammal
in need of chemonucleolysis. A mammal in need of such treatment is
identified by, for example, differential diagnosis following
presentation with lower back pain. Intervertebral disk herniation
can be diagnosed by, for example, complaints of a history of lower
back pain, followed by physical examination of the lower back.
Diagnostic radiographic imaging, such as MRI, can be performed to
confirm the diagnosis. Chemonucleolysis is usually indicated in
mammals that are refractory to physical therapy where there is
bulging of the disk herniation.
[0039] Chemonucleolysis is typically performed by direct injection
into the intervertebral space of an enzyme that causes the
dissolution of intervertebral disk tissue by protein hydrolysis.
When non-proteolytic proteoglycan-degrading enzymes are used to
disrupt the intervertebral disk tissue, the glycosaminoglycan
chains of proteoglycans present in the AF and NP are enzymatically
cleaved while the core protein of the proteoglycan remains intact.
In other words, such non-proteolytic proteoglycanases depolymerize
the glycosaminoglycan chains of the proteoglycan component of the
AF and NP.
[0040] A number of proteoglycan-degrading enzymes can be used in
the treatment of the present invention, including chondroitinase
(chondroitin lyase), which depolymerizes the glycosaminoglycans of
chondroitin sulfate (particularly chondroitin-4-sulfate and
chondroitin-6-sulfate) by the elimination of 1,4-hexosaminidic
bonds, hyaluronidase, hyaluronoglucosaminidase,
hyaluronoglucuronidase, N-acetylglucosaminidase- , hyaluronate
lyase, chondroitinsulphatase, chondro-4-sulphatase, and
chondro-6-sulphatase. A particularly preferred chondroitinase is
chondroitinase ABC (chondroitin ABC lyase; EC 4.2.2.4), which is an
endo-.beta.-N-acetyl-D-hexosaminidase. Other preferred
chondroitinases include chondroitinase AC (chondroitin AC lyase),
chondroitinase B (chondroitin B lyase), and chondroitinase C
(chondroitin C lyase). As can be seen, a proteoglycan-degrading
enzyme includes any glycosidase that can degrade chondroitin
sulfate or hyaluronic acid.
[0041] The amount of proteoglycan-degrading enzyme used in a method
of the present invention is an effective proteoglycan cleaving
amount. An effective proteoglycan cleaving amount of a particular
proteoglycan-degrading enzyme can be determined by measuring the
formation of reaction endproducts over time when the
proteoglycan-degrading enzyme is incubated in the presence of its
substrate. Thus, for example, one unit (U) of chondroitinase ABC is
defined as the amount of enzyme that catalyzes the formation of 1
mmole of unsaturated disaccharide from chondroitin-6-sulfate per
minute at 37.degree. C. at pH 8.0. An effective proteoglycan
cleaving amount of a proteoglycan-degrading enzyme is in the range
of about 0.01 U/disk to about 10 U/disk, preferably from about 0.05
U/disk to about 5 U/disk, and more preferably from about 0.1 U/disk
to about 10 U/disk. The amount of proteoglycan-degrading enzyme
administered to a host mammal can also be expressed as an amount
per unit volume. Thus, an effective proteoglycan cleaving amount of
a proteoglycan-degrading enzyme is in the range of about 0.0001
U/mL to about 100 U/mL, preferably from about 0.05 U/mL to about 50
U/mL, and more preferably from about 0.1 U/mL to about 20 U/mL.
[0042] A method of the present invention also involves
administering to a mammal in need of chemonucleolysis an amount of
a growth factor effective for stimulating the formation of a matrix
component. An effective proteoglycan synthesizing amount of a
growth factor can be administered, or alternatively an effective
collagen synthesizing amount of a growth factor can be
administered.
[0043] A wide variety of growth factors can be used in the present
invention. Growth factors are proteins that can activate cellular
proliferation and/or differentiation. Many growth factors are
pleuripotent and can lead to cell growth and/or differentiation in
a variety of cell types. Growth factors useful in the present
invention include those growth factors that stimulate matrix
synthesis by stimulating the production of proteoglycan and
collagen. A suitable growth factor for use in a method of the
invention includes any growth factor that has the potential to
stimulate matrix synthesis, such as for example, the synthesis of
proteoglycan and collagen. Such a growth factor repairs the
matrices of the nucleus pulposus and/or annulus fibrosus following
chemonucleolysis treatment. Such a growth factor also replenishes
the proteoglycan and/or collagen components of NP and/or AF after
chemonucleolysis with a proteoglycan-degrading enzyme. Thus,
administration of a growth factor restores mechanical strength to
the intervertebral disk following chemonucleolysis. In this way,
fusion of the intervertebral disk can be delayed or even
avoided.
[0044] Preferred growth factors include members of the transforming
growth factor .beta. family, which family has proliferative effects
on many mesenchymal and epithelial cell types. Members of the
transforming growth factor .beta. family that are preferred include
bone morphogenetic protein 2 (BMP-2); bone morphogenetic protein 4
(BMP-4); and transforming growth factors .beta.-1, .beta.-2, and
.beta.-3 (potent keratinocyte growth factors). Other useful members
of the transforming growth factor .beta. family include BMP-3,
BMP-5, BMP-6, BMP-9, DPP, Vg1, Vgr, 60A protein, GDF-1, GDF-3,
GDF-5, GDF-6, GDF-7, CDMP-1, CDMP-2, CDMP-3, BMP-10, BMP-11,
BMP-13, BMP-15, Univin, Nodal, Screw, ADMP, Neural, and amino acid
sequence variants thereof. Other preferred growth factors include
epidermal growth factor (EGF), which induces proliferation of both
mesodermal and ectodermal cells, particularly keratinocytes and
fibroblasts; platelet-derived growth factor (PDGF), which exerts
proliferative effects on mesenchymal cells; fibroblast growth
factor (FGF), both acidic and basic; and insulin-like growth factor
1 (IGF-1) and 2 (IGF-2), which mediate the response to growth
hormone, particularly in bone growth. Further preferred growth
factors include osteogenic proteins. A particularly preferred
osteogenic protein is OP-1, also known as bone morphogenetic
protein 7 (BMP-7). OP-1 is a member of the transforming growth
factor .beta. gene superfamily. It is a 139 amino acid residue long
homodimer of MW 36,000. OP-1 induces new bone formation in vivo and
promotes the repair of diaphyseal segmental bone defects.
[0045] As disclosed in co-pending U.S. application Ser. No.
60/103,161, filed Oct. 6, 1998, and whose disclosure is
incorporated herein in its entirety by reference, useful osteogenic
proteins include those having an amino acid sequence sharing at
least 70% sequence homology or "similarity", and preferably 80%
homology, with a reference morphogenic protein selected from the
group of naturally-occurring proteins. A candidate amino acid
sequence thought to be functionally equivalent to a reference amino
acid sequence can be aligned therewith using the method of
Needleman et al., J. Mol. Biol. 48:443-453 (1970), implemented
conveniently by computer programs such as the Align program
(DNAstar, Inc.). Internal gaps and amino acid insertions in the
candidate sequence are ignored for purposes of calculating the
defined relationship, conventionally expressed as a level of amino
acid sequence homology or identity, between the candidate and
reference sequences.
[0046] "Amino acid sequence homology" is understood herein to
include both amino acid sequence identity and similarity.
Homologous sequences share identical and/or similar amino acid
residues, where similar residues are conservative substitutions
for, or "allowed point mutations" of, corresponding amino acid
residues in an aligned reference sequence. Thus, a candidate
polypeptide sequence that shares 70% amino acid homology with a
reference sequence is one in which any 70% of the aligned residues
are either identical to, or are conservative substitutions of, the
corresponding residues in a reference sequence.
[0047] As used herein, "conservative substitutions" are residues
that are physically or functionally similar to the corresponding
reference residues, e.g., that have similar size, shape, electric
charge, chemical properties including the ability to form covalent
or hydrogen bonds, or the like. Particularly preferred conservative
substitutions are those fulfilling the criteria defined for an
accepted point mutation in Dayhoff et al. (1978), 5 Atlas of
Protein Sequence and Structure, Suppl. 3, ch. 22 (pp. 354-352),
Natl. Biomed. Res. Found., Washington, D.C. 20007. Examples of
conservative substitutions include the substitution of one amino
acid for another with similar characteristics, e.g., substitutions
within the following groups are well-known: (a) valine, glycine;
(b) glycine, alanine; (c) valine, isoleucine, leucine; (d) aspartic
acid, glutamic acid; (e) asparagine, glutamine; (f) serine,
threonine; (g) lysine, arginine, methionine; and (h) phenylalanine,
tyrosine. The term "conservative variant" or "conservative
variation" also includes the use of a substituted amino acid in
place of an unsubstituted parent amino acid in a given polypeptide
chain, provided that antibodies having binding specificity for the
resulting substituted polypeptide chain also have binding
specificity (i.e., "crossreact" or "immunoreact" with) the
unsubstituted or parent polypeptide chain.
[0048] Useful osteogenically active proteins can also have
polypeptide chains with amino acid sequences comprising a sequence
encoded by a nucleic acid that hybridizes, under low, medium or
high stringency hybridization conditions, to DNA or RNA encoding
reference osteogenic sequences, e.g., C-terminal sequences defining
the conserved seven cysteine domains of OP-1, OP-2, BMP-2, BMP-4,
BMP-5, BMP-6, 60A, GDF-3, GDF-5, GDF-6, GDF-7, CDMP-1, CDMP-2,
CDMP-3 and the like. As used herein, high stringent hybridization
conditions are defined as hybridization according to known
techniques in 40% formamide, 5.times.SSPE, 5.times.Denhardt's
Solution, and 0.1% SDS at 37.degree. C. overnight, and washing in
0.1.times.SSPE, 0.1% SDS at 50.degree. C. Standard stringency
conditions are well characterized in commercially available,
standard molecular cloning texts. See, for example, Molecular
Cloning A Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch and
Maniatis (Cold Spring Harbor Laboratory Press: 1989); DNA Cloning,
Volumes I and II (D. N. Glover ed., 1985); Oligonucleotide
Synthesis (M. J. Gait ed., 1984): Nucleic Acid Hybridization (B. D.
Hames & S. J. Higgins eds. 1984); and B. Perbal, A Practical
Guide To Molecular Cloning (1984).
[0049] An effective proteoglycan synthesizing amount of a growth
factor can be determined by, for example, assaying for the amount
of sulfated proteoglycans in a growth factor treated sample using
the DMMB dye assay. Hauselmann, et al., J. Cell. Sci. 107:17-27
(1994). Alternatively, proteoglycan synthesis can be measured by
monitoring the uptake of sulfur-35 (.sup.35S) by cells in a growth
factor treated sample. The proteoglycan synthesizing growth factor
can be administered to a host mammal in single or divided daily
doses of about 100 .mu.g/disk to about 10 mg/disk daily, preferably
about 200 .mu.g/disk to about 1 mg/disk daily and more preferably
about 200 .mu.g/disk to about 500 .mu.g/disk. The amount of
proteoglycan synthesizing growth factor administered to a host
mammal in a single or divided daily dose can also be expressed as
an amount per unit volume. Thus, the proteoglycan stimulating
growth factor can be administered to a host mammal in single or
divided daily doses of about 50 ng/mL to about 5 mg/mL daily,
preferably about 200 ng/mL to about 500 .mu.g/mL, and more
preferably about 200 ng/mL to about 500 ng/mL. Dosage unit
compositions can contain such amounts or submultiples thereof to
make up the daily dose. A suitable dose can be administered in
multiple sub-doses per day. Multiple doses per day can also
increase the total daily dose, should such dosing be desired by the
person prescribing the drug.
[0050] The dosage regimen for treating a disease condition with a
compound and/or composition useful in this invention is selected in
accordance with a variety of factors, including the type, age,
weight, sex, diet and medical condition of the patient, the
severity of the disease, the route of administration,
pharmacological considerations such as the activity, efficacy,
pharmacokinetic and toxicology profiles of the particular compound
employed, whether a drug delivery system is utilized and whether
the compound is administered as part of a drug combination. Thus,
the dosage regimen actually employed can vary widely and therefore
can deviate from the preferred dosage regimen set forth above.
[0051] It is contemplated that the growth factors can be
administered either singly or in a "cocktail" comprising more than
one growth factor. For example, a mixture of OP-1 and BMP-4 can be
administered simultaneously to provide an effective proteoglycan
synthesizing amount of growth factor. To facilitate the
simultaneous administration of a cocktail of growth factors, the
growth factor cocktail can be formulated as a pharmaceutical
composition containing conventional pharmaceutically acceptable
carriers and vehicles, as described elsewhere herein.
[0052] It is also contemplated that the effective proteoglycan
cleaving amount of a proteoglycan-degrading enzyme and the
effective proteoglycan synthesizing amount of a growth factor can
be administered simultaneously to a mammal in need of
chemonucleolysis. The effect of a proteoglycan-degrading enzyme
such as chondroitinase ABC is maximal within 24 hours after
injection, although approximately 2% of the administered amount can
still be found in the NP 10 days post-injection. Takahashi et al.,
Spine 21:2405-2411 (1996). Growth factors typically require at
least 24 hours to induce an effect through the growth factor
cascade. OP-1, for example, exhibits its maximal effect on the
metabolism of chondrocytes approximately 72 hours after
administration. To facilitate the simultaneous administration of a
proteoglycan-degrading enzyme and a growth factor, the combination
of these two compounds can be prepared as a composition of matter
comprising a proteoglycan-degrading enzyme and a growth factor.
This combination can be formulated as a pharmaceutical composition
containing conventional pharmaceutically acceptable carriers and
vehicles, as described elsewhere herein.
[0053] The effective proteoglycan cleaving amount of a
proteoglycan-degrading enzyme and the effective proteoglycan
synthesizing amount of a growth factor can also be administered
sequentially to a mammal in need of chemonucleolysis. The
proteoglycan-degrading enzyme administration can be followed with
growth factor administration at any time point after the
administration of the proteoglycan-degrading enzyme, preferably
within about 24 hours after completion of the administration of the
proteoglycan-degrading enzyme. However, it is to be understood that
growth factor administration can be repeated on multiple occasions
after the administration of the proteoglycan-degrading enzyme.
[0054] A mammal in need of chemonucleolysis can receive one or more
administrations of proteoglycan-degrading enzyme until the desired
clinical outcome is achieved. Thus, for example, multiple doses of
proteoglycan-degrading enzyme can be administered until there is a
reduction in reported pain, or until diagnostic analyses, such as
by MRI, demonstrate that the degree of herniation of the
intervertebral disk has lessened. Growth factor administration can
then proceed thereafter, and can also utilize multiple
administrations until the desired clinical outcome is achieved. For
example, repair of the AF and NP following administration of a
growth factor can be confirmed by diagnostic analyses, such as by
MRI. The skilled practitioner will appreciate that the
administration of growth factor following administration of
proteoglycan-degrading enzyme is controlled so as to prevent
recurrence of symptomology or to prevent recurrence of the
herniation.
[0055] The administration of a proteoglycan-degrading enzyme is
preferably performed by direct injection into the intervertebral
disk space. Similarly, the administration of a growth factor is
preferably done by direct injection into the intervertebral disk
space. However, it is also contemplated that the growth factor can
be administered by continuous infusion into the intervertebral
space using, for example, an implantable or external continuous
infusion pump fitted with an appropriate intervertebral
catheter.
[0056] The treatment method of the invention is used for treating a
host mammal such as a mouse, rat, rabbit, dog, horse, primate such
as a monkey, chimpanzee or human that has a condition requiring the
treatment method.
[0057] A compound useful in the present invention can be formulated
as a pharmaceutical composition. Such a composition can then be
administered parenterally in dosage unit formulations containing
conventional nontoxic pharmaceutically acceptable carriers and
vehicles as desired. The term parenteral as used herein includes
intervertebral injections, or local infusion techniques.
Formulation of drugs is discussed in, for example, Hoover, John E.
(ed.), Remington's Pharmaceutical Sciences (18.sup.th Edition),
Mack Publishing Co., Easton, Pa., 1990 and Liberman, H. A. and
Lachman, L., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New
York, N.Y., 1980. As discussed elsewhere herein, the administration
of a proteoglycan-degrading enzyme or growth factor of the
invention is typically by direct local injection or local infusion
into the intervertebral disk space.
[0058] Injectable preparations, for example, sterile injectable
aqueous or oleaginous suspensions can be formulated according to
the known art using suitable dispersing or wetting agents and
suspending agents. The sterile injectable preparation can also be a
sterile injectable solution or suspension in a nontoxic
parenterally acceptable diluent or solvent, for example, as a
solution in 1,3-butanediol. Among the acceptable vehicles and
solvents that can be employed are water, Ringer's solution, and
isotonic sodium chloride solution. In addition, sterile, fixed oils
are conventionally employed as a solvent or suspending medium. For
this purpose any bland fixed oil can be employed including
synthetic mono- or diglycerides. In addition, fatty acids such as
oleic acid find use in the preparation of injectables. Dimethyl
acetamide, surfactants including ionic and non-ionic detergents,
polyethylene glycols can be used. Mixtures of solvents and wetting
agents such as those discussed above are also useful.
[0059] For therapeutic purposes, formulations for parenteral
administration can be in the form of aqueous or non-aqueous
isotonic sterile injection solutions or suspensions. These
solutions and suspensions can be prepared from sterile powders or
granules having one or more of the carriers or diluents used in
formulations for oral administration, as is well known in the art.
The compounds can be dissolved in water, polyethylene glycol,
propylene glycol, ethanol, corn oil, cottonseed oil, peanut oil,
sesame oil, benzyl alcohol, sodium chloride, and/or various
buffers. Other adjuvants and modes of administration are well and
widely known in the pharmaceutical art.
[0060] A compound useful in the present invention can also be
formulated into liposomes, as discussed in Hoover, John E. (ed.),
Remington's Pharmaceutical Sciences (18.sup.th Edition), Mack
Publishing Co., Easton, Pa., 1990, p. 1691. Liposomes are formed by
dispersing phospholipids in an aqueous medium. Water- or
lipid-soluble substances can be entrapped in the aqueous space
within a liposome, or within the lipid bilayers of the liposome,
respectively. Thus, a growth factor can be formulated into
liposomes for use in a method of the invention using techniques
that are well known in the art.
[0061] As noted above, the amount of active ingredient that can be
combined with the carrier materials to produce a single dosage form
varies depending upon the mammalian host treated and the particular
mode of administration.
[0062] In another embodiment, the present invention contemplates a
kit comprising a proteoglycan-degrading enzyme and a growth factor.
Such a kit contains a proteoglycan-degrading enzyme of the
invention packaged together with a growth factor of the invention.
The kit is packaged in a conventional manner, as is well known in
the art.
[0063] A kit of the invention preferably comprises a vessel
containing a proteoglycan-degrading enzyme and a vessel containing
a growth factor. Such a vessel can be a glass vial or container, a
plastic vial or container, or other suitable container, as is well
known in the art. In another preferred embodiment, the kit
comprises a vessel containing a proteoglycan-degrading enzyme and a
growth factor. Such a kit is especially suited for simultaneous
administration of a proteoglycan-degrading enzyme and a growth
factor according to a method of the invention.
[0064] Any of the above-described kits optionally include
instructions for using the proteoglycan-degrading enzyme and the
growth factor in chemonucleolysis. For example, the instructions
provide details on using the components of the kit in a method of
the present invention.
[0065] The following examples are offered to further illustrate,
but not limit the present invention.
EXAMPLE 1
[0066] Upregulation of Extracellular Matrix Metabolism by Rabbit
Annulus Fibrosus and Nucleus Pulposus Cells Using Recombinant
Osteogenic Protein-1
MATERIALS AND METHODS
[0067] Cell Culture: Lumbar intervertebral disks (IVDs) were
aseptically dissected from the spine of New Zealand white rabbits
after euthanasia. The NP and AF were separated by blunt dissection
and separately pooled. Cells were released from each tissue by
sequential enzyme digestion (Pronase 1 hour, and Collagenase P and
DNAase II for 16 hours) and the isolated cells encapsulated in 1.2%
low-viscosity alginate at 2 million/mL as previously described.
Chiba, K. et al, Spine 22:2885 (1997). The cultures were maintained
in DMEM/F12 plus 10% FBS with a daily change of media.
[0068] Measurement of the Effect Of rhOP-1 on Cell Proliferation
and PG and Collagen Synthesis: On day 7 of culture, the cells in
alginate were incubated in the presence of various concentrations
of the growth factor recombinant human OP-1 (rhOP-1) for 72 hours.
The culture was also treated as follows:
[0069] (1) To measure cell proliferation, MTT (5%) was added to the
medium of these cultures during the last 60 minutes. After
dissolving the beads with calcium chelating agents, the cells were
lysed, centrifuged and the absorbance of the supernatant at 550 nm
was measured. Mosmann, T. J., Immunol. Methods 65:55 (1984).
[0070] (2) To measure PG synthesis, .sup.35S-sulfate (20 .mu.Ci/mL)
was added to the medium during the last 4 hours of the 72 hour
period of incubation in the presence of rhOP-1. After removing the
medium, the beads were dissolved and the two compartments
[cell-associated matrix (CM) and further removed matrix (FRM)]
separated by mild centrifugation. Mok, S. S. et al., J. Biol. Chem.
269:33021 (1994). Each fraction was solubilized with papain and
incorporation of .sup.35S-sulfate determined using a rapid
filtration assay to recover .sup.35S-PGs precipitated by alcian
blue. Masuda, K. et al., Anal. Biochem. 217:167 (1994).
[0071] (3) To measure collagen synthesis, the cultures were labeled
with .sup.3H-proline in DMEM+2% FBS during the last 16 hours of
treatment with rhOP-1. After removing the medium, collagen in the
beads was extracted. Each extract was dialyzed against water
followed by pepsinization and dialysis. .sup.3H-hydroxyproline (as
a measure of .sup.3H-collagen) was quantified after acid hydrolysis
followed by separation of the radiolabeled imino acids by HPLC on a
cation exchange column. Hauselmann, H. J. et al., J.Cell Sci.
107:17 (1994). PG and Collagen Accumulation in Alginate Beads: The
cells were cultured with DMEM/F12+10% FBS in the continuous absence
or presence of rhOP-1 (100 ng/mL). At various time points, the
beads were treated with papain at 60.degree. C. and the papain
digests were analyzed for contents of PG (by the DMMB procedure),
hydroxyproline (using reverse-phase HPLC after acid hydrolysis and
PITC labeling) and DNA (by a fluorometric method using Hoechst
33258 dye). Chiba, K. et al., Spine 22:2885 (1997).
[0072] Statistical Analyses: Statistical analyses were performed by
one way ANOVA with Fisher's PLSD test as a post hoc test.
RESULTS
[0073] Cell Proliferation and DNA Content: The MTT analyses
revealed that rhOP-1 had a significant mitogenic effect at high
concentrations only (AF: 100 and 200 ng/mL, P<0.01; NP: 100
ng/mL, P<0.01). Treatment with rhOP-1 (100 ng/mL) was associated
at all time points studied with an increase in the total DNA
content of AF cultures (% of control: day 3=135%; day 6=134%; Day
11 =126%; Day 14=126%) but not of NF cultures (p>0.05).
[0074] PG Synthesis: rhOP-1-treatment resulted in a significant
dose-dependent increase in PG synthesis by both the AF and NP
cultures maintained in the presence of 10% FBS (see FIG. 1,
p<0.001). The rate of PG synthesis remained significantly
elevated in both AF and NP cultures when expressed per .mu.g DNA:
NP cells were more responsive than AF cells to rhOP-1 (rate of
synthesis as % of control: AF=230%; NP=460%). It should be noted
that in the presence or absence of rhOP-1, AF cells produced more
.sup.35S-PGs than NP cells.
[0075] Collagen Synthesis: rhOP-1 stimulated collagen synthesis by
both NP cells and AF cells in a dose-dependent manner (see FIG. 11,
P<0.0 1), but the magnitude of the response was less marked than
in the case of PG synthesis (see FIG. 11, P<0.01). Although
rhOP-1 had a significant mitogenic effect at high doses, the rate
of collagen synthesis remained significantly elevated when
expressed per .mu.g DNA. As was observed in the case of PG
synthesis, AF cells produced under all conditions more
.sup.3H-collagen than NP cells.
[0076] Matrix Accumulation: The addition of rhOP1 at 100 ng/ml to
the medium resulted in a marked increase in the total content of PG
and collagen in both the AF and NP beads. In both the AF and NP,
this increase was only minor during the first week, but dramatic
during the second week of culture with rhOP-1 (data not shown).
This delay was especially evident in the case of collagen
accumulation.
DISCUSSION
[0077] These data show the ability of growth factor rhOP-1 to
stimulate the metabolism of both AF and NP cells. Although this
morphogenetic protein only had a moderate mitogenic effect upon the
cells, it stimulated them to synthesize PGs and collagen at faster
rates than in the presence of 10% FBS alone. The stimulation of PG
synthesis was especially great in the case of the NP cells that are
normally much less effective than articular chondrocytes or AF
cells to reform a cell-associated matrix in vitro. Interestingly,
the stimulatory effects of rhOP-1 on PG synthesis were more
apparent than those on collagen metabolism.
[0078] In human disks, the PG content of the NP decreases with age
and drops rapidly in degenerative disk disease (Gower, W. E. et
al., J. Bone Joint Surg. 51-A:1154 (1969), when longitudinal and
circular tears, that reflect the disruption of the collagen network
structure, are often observed by MRI. The data demonstrate that
growth factor rhOP-1 is useful as a therapeutic agent in promoting
synthesis and repair of the matrix of both the AF and NP elements
of degenerating human IVDs by increasing the synthesis of
proteoglycan and collagen.
EXAMPLE 2
[0079] Osteogenic Protein-1 Stimulation of Proteoglycan Synthesis
by Nucleus Pulposus and Annulus Fibrosus Following Chondroitinase
ABC-Induced Chemonucleolysis Treatment
MATERIALS AND METHODS
[0080] Lumbar spines were removed en bloc, under aseptic
conditions, from adolescent New Zealand white rabbits weighing
3-4.0 Kg (IACUC approval # 94-053). The lumbar disks were dissected
and the NP separated in each case from the AF. Cells were
separately isolated from the two tissues by sequential enzyme
digestion and resuspended in 1.2% low viscosity sterile alginate at
2 million/mL. Chiba, K., et al., Spine 22:2885-2893 (1997). Beads
were formed by expressing this solution dropwise, through a 22
gauge needle, into a 102 mM CaCl.sub.2 solution. The NP and AF
cells in the beads were maintained in batch culture in DMEM/F-12
medium containing 10% FBS, 25 .mu.g/mL ascorbate and 50 .mu.g/mL
gentamicin. This complete medium was changed daily throughout the
study.
[0081] On day 14 of culture, beads were divided into three groups.
The first comprised beads cultured for an additional 12 days in
complete medium (Group 1--control cultures). The beads in the other
two groups were first cultured for 2 hours in the presence of the
proteoglycan-degrading enzyme chondroitinase ABC (C-ABC) (0.1
U/mL), to deplete PGs from the matrix, followed by 3.times.30
minute washes before culturing the beads for the next 12 days in
complete medium either in the presence (Group 2) or absence (Group
3) of growth factor OP-1 at 200 ng/mL.
[0082] At various times, the matrix in the beads was solubilized by
digestion with papain at 60.degree. C. The digests were analyzed
for content of DNA, using the Hoechst 33258 dye and fluorometry,
and sulfated PGs, using the DMMB dye assay. Hauselmann, H. J. et
al., J. Cell Sci. 107:17-27 (1994). For each time point, all
analyses of NP and AF beads were performed on 3 sets of 9 beads
each. Statistical analyses were performed by one way ANOVA with
Fisher's PLSD test as a post hoc test.
RESULTS
[0083] The DNA content did not decrease during the treatment of NP
or AF cells with C-ABC. DNA content rose moderately in all groups
of NP and AF cells during the next nine days before reaching a
plateau. Although the increase was not as pronounced in NP and AF
beads cultured with OP-1, this difference was not statistically
significant. FIGS. 1 and 2.
[0084] Similar observations and conclusions were drawn when PG
contents were expressed per bead or per .mu.g DNA. FIGS. 3-10, 12.
Consequently, all values for PG contents are reported here only as
.mu.g PG per 9 beads. As shown in FIG. 12, the sulfated PG contents
of the NP and AF beads not exposed to C-ABC increased progressively
over the time of culture. Treatment with C-ABC caused the PG
content to drop by more than 85% in both NP and AF beads. Although
matrix recovery was clearly evident in both NP and AF beads
returned to complete medium without OP-1, the matrix thus formed
never reached the PG content seen in beads not treated with
C-ABC.
[0085] OP-1 at 200 ng/mL appeared to have no significant effect
upon matrix accumulation during the first 3 days after treatment
with C-ABC. In marked contrast, between days 3 and 6, both NP and
AF cells exposed to OP-1 reestablished a matrix as rich in sulfated
PGs as that of control cells not exposed to C-ABC. At later times,
OP-1 continued to exert its stimulatory effect upon the
C-ABC-treated cells: the PG contents reached values that were
significantly higher than those not only of the C-ABC-treated cells
not exposed to OP-1 but also of the control cells (P<0.01). FIG.
12.
DISCUSSION
[0086] Theses data demonstrate the effectiveness of growth factor
OP-1 in stimulating matrix repair by NP and AF cells after their
matrix had been nearly totally depleted of sulfated
glycosaminoglycans by a proteoglycan-degrading enzyme. The results
are unexpected for several reasons. First, the data show that NP
and AF cells are as responsive as articular chondrocytes to
OP-1-induced stimulation of PG synthesis. While not wishing to be
bound by theory, because OP-1 is much more effective in
upregulating the synthesis of aggrecan than of small nonaggregating
PGs in articular cartilage (Huch, K., et al., Arthritis Rheum.
40:2157-2161 (1997)), it is most likely that the enrichment in PGs
reported here reflects principally the incorporation into the
repairing matrix of newly-synthesized aggrecan molecules.
[0087] Second, the results indicate that OP-1 or related growth
factors with similar modes of action are useful to stimulate matrix
repair in vivo, not only after clinically-induced chemonucleolysis
but also in the therapeutic treatment of disk degeneration.
[0088] Third, the data show that the combination of a
proteoglycan-degrading enzyme and a growth factor provides a method
of chemonucleolysis that is superior to the use of a
proteoglycan-degrading enzyme such as chondroitinase ABC alone.
EXAMPLE 3
[0089] Osteogenic Protein-1 Stimulation of Cartilage Matrix Repair
by Nucleus Pulposus and Annulus Fibrosus
[0090] This Example demonstrates the efficacy of osteogenic protein
in stimulating cartilage matrix repair by cells, specifically
nucleus pulposus ("NP") and annulus fibrosus ("AF") cells, isolated
from intervertebral discs ("IVDs").
[0091] In this Example, lumbar discs were isolated from New Zealand
white rabbit and NP tissue was separated from AF tissue by
dissection. NP and AF cells were separately isolated from the two
tissues by sequential enzyme digestion and resuspended in 1.2% low
viscosity sterile alginate, which was then formed into beads. The
cells were separately cultured in DMEM/F-12 medium containing 10%
FBS, with the medium being changed daily. After 7 days, each
culture was subdivided into three groups. The first group was a
control group that was not treated with OP-1. The second and third
groups were grown in the presence of OP-1 for 72 hours, the second
group being treated with 100 ng/ml of OP-1, and the third group
being treated with 200 ng/ml of OP-1. Radiolabelled .sup.3H-proline
was added to the cultures for the last 4 hours of incubation with
OP-1. After the incubation, collagen was extracted from the
cultures, and the rate of collagen production was determined by
measuring the radiolabel's incorporation into the extracts.
Collagen production is associated with growth and repair of
cartilage matrix. To determine the rate of cell proliferation, the
content of each group's DNA was measured using Hoechst 33258
dye.
[0092] Osteogenic protein increased collagen production in both NP
and AF cell cultures in a concentration-dependent manner. The third
group incorporated more radiolabel than the second group, which in
turn incorporated more radiolabel than the first control group.
Osteogenic protein had a significant mitogenic effect at high
concentrations, which accounts for some of the elevation in
collagen production. Nonetheless, the rate of collagen synthesis
was significantly increased even when increased cell proliferation
is accounted for. These results suggest that osteogenic protein
stimulates the growth and repair of extracellular matrix.
EXAMPLE 4
[0093] Development of Animal Models for Intervertebral Disc
degeneration. Assessment of Morphologic, Biomechanical and
Biochemical Changes in the Disc Due to Degeneration.
[0094] From among numerous animal models for disc degeneration, two
rabbit models are selected, the stab-wounding of the annulus
fibrosus model (Lipson, S. J. and Muir H, Spine 6:194-210 (1981) as
well as the C-ABC intradiscal injection model (Kato, F. et al.,
Clin. Orthop. 253:301-308 (1990). These models are reproducible and
biochemical changes have been described at different stages of
degeneration.
[0095] White New Zealand rabbits, each weighing about 2.5 kg are
used. Anesthesia is done with intramuscular ketamine 45 mg/kg and
maintained with IV 5-10 mg/kg as needed and by oxygen/nitrous oxide
by mask. Following prepping and draping, the posterolateral
retroperitoneal approach is used to expose the anterior and lateral
aspects of the discs. For the stab-wounding group, a transverse
incision is made into the ventral annulus fibrosus using a number
11 blade. For the chemonucleolysis group, 0.25 cc of C-ABC (187
U/ml) is injected into the disc with a 28 gauge needle attached to
a microsyringe. Each animal has two discs treated by stab-wound or
chemonucleolysis and two control discs. The levels are randomly
chosen. The wound is closed in layers and normal recovery is
monitored 1 day after surgery. Three animals are sacrificed from
both the stab-wound and chemonucleolysis groups at 2, 4, B, 12 and
24 weeks (a total of 30 rabbits).
[0096] Morphologic Assessment
[0097] MRI is obtained one day prior to sacrifice to determine the
grade of disc degeneration. Additionally, to assess histological
changes in the AF and NP, routine histology is performed on one
treated and one control disc from each time period. For histology,
the motion segments are fixed in phosphate buffered formalin,
decalcified, embedded in parafin, sectioned, and stained with H
& E. The MRI and histology results are graded by Thompson's
criteria.
[0098] For MRI, the motion segments prior to loading are imaged at
room temperature in a 1.5 Tesla cryomagnet (Signa scanner, General
Electric Medical Systems, Milwaukee, Wis.) with a 4 or 5 inch
diameter solenoid coils (Medical Advances. Inc., Milwaukee).
Conventional spin echo (SE) sequences are used. T2-weighted images
are obtained of each motion segments in the sagittal plane with
conventional spin echo sequence. T2-weighted axial and parasagittal
images are obtained with TE 33 and 80 msec., TR 2000 msec., 2 NEX,
1.0 mm slice thickness, 8 sq.cm. display field of view and
512.times.256 matrix.
[0099] The MRI images are examined for signal changes of the NP and
inner AF (normal, dark, intermediate) and the presence of annular
tears. Additionally, any structural changes such as disc height,
involution of the outer annulus, the presence of myxoid
degeneration, separation of the NP from the end-plate, vacuum
phenomenon, Schmorl's nodes, and herniation are noted. Degenerative
changes are graded from I to V based on Vemon-Roberts (Table 1).
With the radial tear in mind, the discs are classified as I:
normal, II: transverse tears or circumferential tears, III: radial
tears, IV: advanced degeneration with loss of disc height. The
sagittal images of the histologic sections are analyzed and graded
from Thompson I to V (Table 2). The MRI and histology sections are
correlated.
1TABLE 1 MRI Grading (Vemon-Roberts) Disc Nucleus Annulus Vertebral
Grade Pulposus Fibrosus End-Plate Body I Homogeneous Homogenous
Single Margins bright, dark gray dark line rounded distinct margin
II Horizontal dark Areas of Increase in Tapering of bands toward
brighter spots central margins the annulus concavity III Decreased
Indistinguishable Line less Small dark signal from nucleus distinct
projections intensity pulposus from margins Gray tone with
stippling IV Proportions of Bright and Focal Osteophytes gray
signal reduced dark defects <2 mm Bright and dark signals
regions larger contiguous with nucleus pulposus V Gross loss of
Signals Defects Osteophytes disc Height contiguous and areas >2
mm Dark signals with nucleus of dominant pulposus thickening
[0100]
2TABLE 2 Description of the Morphologic Grades by Thompson Disc
Nucleus Annulus Vertebral Grade Pulposus Fibrosus End-Plate Body I
Bulging gel Discrete Hyaline Margins lamella fibrous and rounded
uniform thickness II White fibrous Mucinous Thickness Margins
tissue material irregular pointed peripherally between lamellas III
Consolidated Extensive Focal Early fibrous tissue mucinous defects
osteophytes infiltration at margins Indistinct margins IV
Horizontal Focal Focal Osteophytes clefts disruptions sclerosis
<2 mm and fibro- cartilage to bone V Clefts extend through
nucleus Diffuse Osteophytes pulposus, and the annulus fibrosus
sclerosis >2 mm
[0101] Biomechanical Tests
[0102] The goal is to determine changes in the dynamic axial and
torsional stiffness of the disc and in the hysteresis response due
to the various stages of disc degeneration when compared to normal
disc. Biomechanical tests are performed within 12 hours after
euthanasia of all animals, to preserve the biochemical composition
as much as possible.
[0103] Protocol: Immediately after euthanizing animals, the lumbar
motion segments are obtained, and extraneous soft tissues and
posterior elements are removed leaving the vertebral
body-disc-vertebral body units intact. The superior and inferior
part of the each unit is cut by a slow speed diamond bone saw such
that the resulting superior and posterior surfaces are parallel to
the mid-transverse plane of the disc. The prepared specimen is
subjected to a constant axial compressive load of 50 N for 30 min.
Axial displacement resulting from the constant load is measured to
investigate if there is any creep behavior. Then, the cyclic loads
(50+20 N) is applied at frequencies of 0.5 and 5 Hz. The maximum
load of 20 N is selected not to cause a permanent deformation of
the disc tissue, failure of the end-plate or subchondral bone, or
delamination or tears in the annulus. Load-unload cycles are
repeated 20 times to ensure saturation for successive cycles, and a
time interval of 5 minutes is allowed between each loading
condition to ensure intradiscal pressure recovery. Following the
compression tests, the specimen is kept in load-free condition for
30 minutes and is subjected to torsional moments applied in a
similar manner. The mean torsional moment and the amplitude is 1.5
Nm and 0.5 Nm, respectively, and the loading frequencies are 0.1
and 1.0 Hz. While loading, signals from the load cell, LVDT (RVDT
in the case of torsional tests), and the pressure sensor are
sampled at 10, 50, 100, or 500, Hz. Vertebral body-disc-vertebral
body units obtained from the experimental animals undergo cyclic
compression and torsion tests once. However, the biomechanical
tests are repeated twice on the vertebral body-disc-vertebral body
units obtained first with intact discs and the second with stab
wound discs. Biomechanical tests of the vertebral body-disc-body
units with stab wounds are performed to investigate the
biomechanical responses immediately after injury.
[0104] Load-displacement curves are obtained and analyzed to
determine the dynamic stiffness and hysteresis. The dynamic
stiffness is determined as the ratio of the input peak-to-peak load
to the peak-to-peak displacement at the 20th cycle whereas the
hysteresis is defined by the ratio of the area of the envelope of
the loading and unloading paths to the area under the loading path.
Load-intradiscal pressure curves are also obtained and analyzed to
investigate the relationship between the applied load and
intradiscal pressure responses. The intradiscal pressure slope
dp/dl (KPa/N) is determined by dividing the peak-to-peak pressure
by the peak-to-peak load, and the phase shifts (time lag between
controlled load and intradiscal pressure) are represented as the
area defined by the envelope of the loading and unloading paths.
Creep behavior is described by the amount of axial and torsional
displacement measured from the displacement-time curve obtained
under constant static loading conditions, and the intradiscal
pressure changes during the same time period is also determined
from the corresponding intradiscal pressure-time curve.
[0105] Biochemical Studies
[0106] The purpose of these studies is to identify over time,
changes in biochemical composition resulting from the stab-wounding
or the C-ABC intradiscal injection rabbit disc degeneration models
and to correlate the biomechanical characteristics with the
biochemical composition of the disc. Following the biomechanical
tests, from each vertebral body-disc-vertebral body unit, the disc
is excised and biochemical tests that are described above are also
performed. The entire lumber spinal segment is removed. Half of the
specimens are analyzed for biochemical tests. The biochemical
composition of the discs is quantified in terms of the
proteoglycan, collagen (hydroxyproline) and collagen stable
crosslink contents. The other half is fixed in 4% paraformaldehyde
in PBS, decalcified. embedded in paraffin, sectioned and assessed
by histology and immunostaining. Sagittal sections (5-8 .mu.m) of
each disc are stained with hematoxylin and eosin, as well as
Safranin-O. Serial sections from each animal are tested for type II
collagen and type I collagen immunoreactivity. Mouse monoclonal
antibodies against human type II collagen and type I collagen are
used as the primary antibodies. Sections are counterstained with
Meier's hematoxylin prior to mounting.
EXAMPLE 5
[0107] Determination of OP-1 Mediated In Vivo Repair of
Intervertebral Discs in Animal Models for Intervertebral Disc
Degeneration.
[0108] This Example tests the potential usefulness of OP-1 injected
intradiscally at the same time as the C-ABC solution or at the time
of AF puncture, in promoting repair of the NP and AF.
[0109] Protocol: Twelve New Zealand rabbits weighing 3 kilograms
are used for stab-wounding and stab-wounding+OP-1. Each rabbit has
one disc that receives stab-wounding alone and another disc that
receives stab-wounding+OP-1. An additional twenty-four rabbits are
divided into 4 groups (control-saline injection, OP-1 alone, C-ABC
alone, C-ABC+OP-1). Each rabbit has only two discs injected. After
intravenous administration of sodium pentobarbital (25 mg/Kg),
baseline radiographs are taken. Following inhalation of isoflurane,
each rabbit is placed in a lateral prone position and the anterior
surface of the lumbar disc is exposed through a posterolateral
retroperitoneal approach. Chondroitinase-ABC (187 U/ml) alone, OP-1
(2 .mu.g) alone or C-ABC premixed with OP-1 (2 .mu.g) is injected
into the two lower lumbar intervertebral discs (30 .mu.l of
solution per disc) with a 28 gauge needle attached to a
microsyringe. For the control group, an equal volume of saline is
injected. The wound is washed several times with sterile saline
containing antibiotics and then closed with layered sutures. Three
rabbits in each group are euthanized with an excess dose of
pentobarbital at 2, 4, 8 and 16 weeks after injection. After
euthanasia, radiographs are taken to evaluate disc space narrowing.
The entire lumbar spinal segment is removed, fixed in 4%
paraformaldehyde in PBS, decalcified, and embedded in paraffin.
Sagittal sections (5-8 .mu.m) of each disc are stained with
hematoxylin and eosin, as well as Safranin-O. Serial sections from
each animal are tested for type II collagen and, type I collagen
immunoreactivity. Mouse monoclonal antibodies against human type II
collagen and type I collagen are used as the primary antibodies,
Sections are counterstained with Meier's hematoxylin prior to
mounting.
[0110] From the foregoing, it will be observed that numerous
modifications and variations can be effected without departing from
the true spirit and scope of the present invention. It is to be
understood that no limitation with respect to the specific examples
presented is intended or should be inferred. The disclosure is
intended to cover by the appended claims modifications as fall
within the scope of the claims.
* * * * *