U.S. patent application number 12/419131 was filed with the patent office on 2009-12-03 for spinal nucleus pulposus implant.
This patent application is currently assigned to SCIL TECHNOLOGY GMBH. Invention is credited to CAROLA DONY, KLAUS HELLERBRAND, ELISABETH HUSTERT, SUSANNE PIPPIG, RAINER SIGL.
Application Number | 20090297580 12/419131 |
Document ID | / |
Family ID | 38910893 |
Filed Date | 2009-12-03 |
United States Patent
Application |
20090297580 |
Kind Code |
A1 |
DONY; CAROLA ; et
al. |
December 3, 2009 |
SPINAL NUCLEUS PULPOSUS IMPLANT
Abstract
The present invention relates to a spinal nucleus pulposus
implant for use in the treatment of the intervertebral disc and in
particular, to the use of a CD-RAP protein therefore.
Inventors: |
DONY; CAROLA; (MUNCHEN,
DE) ; HELLERBRAND; KLAUS; (MOORENWEIS (EISMERSZELL),
DE) ; HUSTERT; ELISABETH; (GERMERING, DE) ;
PIPPIG; SUSANNE; (MUNCHEN, DE) ; SIGL; RAINER;
(PUCHHEIM, DE) |
Correspondence
Address: |
ARENT FOX LLP
1050 CONNECTICUT AVENUE, N.W., SUITE 400
WASHINGTON
DC
20036
US
|
Assignee: |
SCIL TECHNOLOGY GMBH
MARTINSRIED
DE
|
Family ID: |
38910893 |
Appl. No.: |
12/419131 |
Filed: |
April 6, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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PCT/EP2007/008660 |
Oct 5, 2007 |
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12419131 |
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Current U.S.
Class: |
424/423 ;
514/1.1 |
Current CPC
Class: |
A61K 9/0019 20130101;
A61P 25/00 20180101; A61K 38/1875 20130101; A61P 19/00 20180101;
A61K 9/19 20130101; A61P 19/08 20180101; A61P 25/28 20180101; A61P
19/02 20180101; A61K 38/18 20130101; A61P 25/02 20180101; A61K
9/127 20130101; A61K 9/1272 20130101; A61P 29/00 20180101; A61K
9/0085 20130101; A61P 37/00 20180101; A61K 35/35 20130101; A61P
37/02 20180101 |
Class at
Publication: |
424/423 ; 514/2;
514/12 |
International
Class: |
A61K 9/00 20060101
A61K009/00; A61K 38/00 20060101 A61K038/00; A61K 38/18 20060101
A61K038/18; A61P 25/00 20060101 A61P025/00 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 6, 2006 |
EP |
06021093.7 |
Claims
1. A spinal nucleus pulposus implant or formulation comprising a
cartilage differentiation and maintenance factor which is a
non-antibody or non-receptor molecule.
2. The spinal nucleus pulposus implant or formulation of claim 1,
wherein the implant is injectable or implantable transdiscally.
3. The spinal nucleus pulposus implant or formulation of claim 1,
wherein the cartilage differentiation and maintenance factor is a
chondrogenic morphogen.
4. The spinal nucleus pulposus implant or formulation of claim 1,
wherein the cartilage differentiation and maintenance factor has
anabolic activity for cartilage regeneration.
5. The spinal nucleus pulposus implant or formulation of claim 1,
wherein the cartilage differentiation and maintenance factor is a
member of the MIA (melanoma inhibitory activity) family.
6. The spinal nucleus purposes implant or formulation of claim 1,
wherein the cartilage differentiation and maintenance factor is
selected from the group of a) chondrocyte proteins comprising the
mature sequence of CD-RAP according to Seq ID No. 1, or a
contiguous sequence thereof having at least 20 amino acids, b)
proteins having at least 70% amino acid sequence homology with the
C-terminal four cysteine skeleton of CD-RAP, amino acids 12 to 107
of Seq. ID No. 1, or c) proteins having any of the generic
sequences 1 to 3 according to Seq ID Nos 2, 3 and 4.
7. The spinal nucleus pulposus implant or formulation of claim 1,
wherein the implant or formulation comprises a carrier.
8. The spinal nucleus pulposus implant or formulation of claim 1,
wherein the implant or formulation comprises a sustained release
device.
9. The spinal nucleus pulposus implant or formulation of claim 1,
wherein the cartilage differentiation and maintenance factor is
encapsulated in liposomes.
10. The spinal nucleus pulposus implant or formulation of claim 1,
wherein the spinal nucleus pulposus implant comprises nucleus
pulposus tissue or cells.
11. Use of a cartilage differentiation and maintenance factor which
is a non-antibody or non-receptor molecule for the manufacture of a
pharmaceutical composition for the treatment of a spinal
disorder.
12. Use of claim 11, wherein the cartilage differentiation and
maintenance factor is a chondrogenic morphogen.
13. Use of claim 11, wherein the spinal disorder is idiopathic low
back pain, disc herniation, internal disc disruption or fissured
discs, radiculopathy, spinal stenosis, herniated nucleus
pulposus-induced sciatica, sciatica, idiopathic scoliosis or
myelopathy.
14. Use of claim 11, wherein the pharmaceutical composition
comprises a carrier.
15. A method for the treatment of a spinal disorder in a mammal
comprising administering an effective dose of a cartilage
differentiation and maintenance factor which is a non-antibody or a
non-receptor molecule to a mammal in need of such treatment.
16. Use of claim 11, wherein the cartilage differentiation and
maintenance factor has anabolic activity for cartilage
regeneration.
17. Use of claim 11, wherein the cartilage differentiation and
maintenance factor is a member of the MIA (melanoma inhibitory
activity) family.
18. Use of claim 11, wherein the cartilage differentiation and
maintenance factor is selected from the group of a) chondrocyte
proteins comprising the mature sequence of CD-RAP according to Seq
ID No. 1, or a contiguous sequence thereof having at least 20 amino
acids, b) proteins having at least 70% amino acid sequence homology
with the C-terminal four cysteine skeleton of CD-RAP, amino acids
12 to 107 of Seq. ID No. 1, or c) proteins having any of the
generic sequences 1 to 3 according to Seq ID Nos 2, 3 and 4.
19. Use of claim 11, wherein the pharmaceutical composition
comprises a sustained release device.
20. Use of claim 11, wherein the cartilage differentiation and
maintenance factor is encapsulated in liposomes.
21. Use of claim 11, wherein the pharmaceutical composition
comprises nucleus pulposus tissue or cells.
Description
[0001] The present invention relates to a spinal nucleus pulposus
implant for the treatment of the intervertebral disc and, in
particular, to the use of a CD-RAP protein therefore.
[0002] Degeneration of the intervertebral disc (IVD) is a
multifactorial process involving mechanical, genetic and biological
factors. Although the pathophysiological mechanism remains unclear,
resultant changes in structure and function of the disc have been
well described. Unlike articular cartilage, the IVD is composed of
different tissues. The healthy IVD is a well-encapsulated,
avascular organ which contains a jelly-like nucleus pulposus (NP)
surrounded by a fibrous annulus fibrosus (AF), which provides
mobility and a cushion between the vertebrae. The nucleus pulposus
is located at the center of each disc and is composed of
chondrocytes which produce an extracellular matrix containing a
high percentage of proteoglycans (PG) and type II collagen in the
adult. The nucleus pulposus is surrounded by the annulus fibrosus
which consists of highly organized, directionally oriented collagen
fibers oriented in concentric lamellae, and extracellular matrix.
The inner annulus fibrosus is thicker than the outer and has a
fibrocartilaginous matrix that lacks the lamellar structure. A thin
distinct region, the transition zone (TZ), divides the inner
annulus from the nucleus pulposus.
[0003] During the aging process, the reduction in proteoglycan
content of the nucleus leads to decreased hydration and evidence of
degeneration, including reduction in disc height and increased load
on the surrounding structures of the spine. At the biological level
it reflects an imbalance between the normal anabolic and catabolic
function of the nucleus pulposus cells. In some cases of
degenerative disc disease (DDD), gradual degeneration of IVD is
caused by mechanical instabilities. Increased load and pressure on
the nucleus pulposus cause the cells or invading macrophages to
produce larger amounts of cytokines or toxic amounts of
metalloproteinases (MMPs). As DDD progresses, toxic levels of
cytokines and MMPs degrade the extracellular matrix and lead to a
destruction of the proteoglycans, thereby reducing the
water-retaining capabilities with resulting dehydration of the
nucleus pulposus. In the following the flexibility of the nucleus
pulposus is reduced and delamination of the annulus fibrosus might
be the consequence, eventually developing internal fissures
spreading out towards the periphery. These alterations cause even
more mechanical instability and induction of cytokine production,
which progress the DDD and the disc begins to bulge (herniated disc
disease) and ultimately ruptures, with nerve irritation and low
back pain.
[0004] Unfortunately, most current therapies for disc-related low
back pain are targeted to obtain symptomatic relief rather than
repairing the underlying degenerative process. Conservative
treatment consists of physical measures, the use of analgetics,
muscle relaxants, non-steroidal anti-inflammatory drugs, systemic
corticosteroids, epidural injections and injections of cytokine
antagonists. In later stages of therapy the treatment of a
degenerated disc is either removal of the degenerated disc and
fusion of adjacent vertebrae on either side of the disc or a
replacement of the disc by a synthetic disc material.
[0005] Disc replacement or fusion do not restore normal disc
height, physiology or mechanical properties and might lead to
further symptoms at either the site of surgery or adjacent discs.
Therefore, future treatment methods are needed which inhibit or
reverse the cellular disturbances underlying the degeneration and
restore the biological function of the vertebral disc. Many
researchers worldwide are seeking biological ways to repair
degenerated intervertebral discs.
[0006] Chemonucleolytic agents, such as chymopapain, have been used
in the past as a method of treating herniated IVDs. Pain relief was
linked to the ability to degrade PGs, thereby decreasing
intradiscal pressure and relieving compression of the affected
nerve roots. However, associated complications such as anaphylaxis,
neurological injury and infection diminished the use of
chymopapain. More recently, chondroitinase ABC (C-ABC) has been
suggested as an alternative for chemonucleolysis because it lacks
protease activity and possesses a narrower substrate spectrum.
Although the regeneration of the cartilaginous matrix occurs
earlier after treatment with C-ABC than with chymopapain, disc
height and PG content do not recover sufficiently and the IVD is
left with altered suboptimal biochemical properties thus
accelerating the degenerative cascade of events (Takegami et al.,
2005; Masuda et al., 2004; Masuda and An, 2004).
[0007] WO2005/000283 and references therein describe methods
treating degenerative disc disease including injecting an
antagonist such as high affinity antagonists of MMP, highly
specific cytokine antagonists, a highly specific p38 kinase
inhibitor, anti-inflammatory drugs, a cycline compound,
anti-proliferative or anti-apoptotic agents into a diseased
intervertebral disc. These compounds are believed to inhibit
catabolic processes within the DDD by inhibition of
pro-inflammatory cytokines, MMPs, prostaglandin regulation or
reduction in pro-inflammatory effects or inhibit chondrocyte
proliferation or apoptosis, but no anabolic activity has been
described.
[0008] WO2006/086105 describes methods for treating and/or
reversing disorders of the intervertebral disc using transcription
factor inhibitors which target transcription factors such as
NF-.sub.kB, E2F, GATA-3 and STATs.
[0009] WO2006/031376 describes a method of treating degenerative
disc disease comprising administration of an antioxidant into the
intervertebral disc either alone or in combination with an
additional therapeutic agent such as fibrin, hyaluronic acid, stem
cells, bone marrow, or a growth factor.
[0010] In other studies exogenous growth factors like transforming
growth factor beta-1 (TGF-.beta.1), insulin like growth factor-1
(IGF-1), bone morphogenetic protein-2 (BMP-2), growth and
differentiation factor-5 (GDF-5) and osteogenic protein (OP-1) have
been administered to intradiscal cells or injected intradiscally to
stimulate synthesis of proteoglycans and collagen to slow down or
reverse the degeneration of the IVD (Levicoff et al., 2005;
Sobajima et al., 2004; Takegami et al., 2005; Kawakami et al.,
2005). However, these growth factors are rather unspecific having
the risk to induce osteogenic genes and therefore may induce
undesired bone formation. Another limitation of the application of
this growth factor technology is the relatively short biological
half-life of the exogenous growth factor that would enable only
transient biological effects after their delivery.
[0011] Therefore, the object of the present invention is to provide
a spinal nucleus pulposus implant which improves or restores the
biomechanical properties of the vertebral disc and/or inhibits
further progression of diseases affecting the vertebral disc.
[0012] Another object of the present invention is to provide a
nucleus pulposus implant comprising a cartilage differentiation and
maintenance factor able to reverse disease processes affecting the
vertebral disc, to restore the properties of the vertebral disc or
to inhibit further progression of the disease.
[0013] Another object of the present invention is to modify
cartilage homeostasis by stimulating anabolic processes on the
expense of catabolic processes within the IVD for providing a new
approach for treating chronic conditions such as DDD.
[0014] Another object of the present invention is to provide a
nucleus pulposus implant comprising a cartilage differentiation and
maintenance factor for treatment of localized degeneration of the
vertebral disc, in which the cartilage differentiation and
maintenance factor provides the patient with a better opportunity
to heal, slow disease progression, and/or otherwise improve
patient's health.
[0015] Another object is to provide a spinal nucleus pulposus
implant to increase the disc height and proteoglycan content in the
IVD tissue.
[0016] Another object of the present invention is to provide a
nucleus pulposus implant comprising a cartilage differentiation and
maintenance factor for providing suppression and inhibition of the
action of specific cytokines in humans to treat chronic conditions
such as DDD in addition to cartilage maintenance.
[0017] Another object of the present invention is to provide a
spinal nucleus pulposus implant which may be implanted or injected
by minimal invasive procedures or endoscopically.
[0018] Another object of the present invention is to provide a
nucleus pulposus implant delivery system capable of providing more
prolonged levels or sustained controlled release of the therapeutic
agent ensuring that the agent is available at the site of
degenerated IVD for a longer time frame.
[0019] Another object of the present invention is to provide a
spinal nucleus pulposus implant which is a delivery system capable
of providing more prolonged levels of a therapeutic agent.
[0020] To achieve the objects of the invention, a spinal nucleus
pulposus implant or formulation is provided which comprises a
cartilage differentiation and maintenance factor which is a
non-antibody or non-receptor molecule. The implant, in particular,
is injectable or implantable transdiscally. For clarity,
non-antibody means that the cartilage differentiation and
maintenance factor is not an antibody such as for example a
monoclonal, polyclonal or chimeric antibody against TNF alpha.
Receptor molecule means a receptor molecule with a high specificity
against pro-inflammatory cytokines such as TNF.alpha., truncated
forms thereof or functional equivalents thereof.
[0021] The present inventors have developed a number of procedures
for treating pathological spinal disorders such as degenerative
disc disease by application of an effective amount of a functional
biomolecule into a pathological spinal disorder (e.g. DDD). In
accordance with the present invention, one embodiment encompasses a
method of treating an intervertebral disc in which a cartilage
differentiation and maintenance factor is administered
transdiscally preferably by one or repeated injections in a
therapeutically effective amount. In one embodiment the cartilage
differentiation and maintenance factor is selected from the group
of highly cartilage specific proteins such as CD-RAP or active
fragments thereof able to stimulate the differentiation and
maintenance of IVD cells while inhibiting undesired bone
formation.
[0022] Such cartilage differentiation and maintenance factors like
CD-RAP inhibit the activity of cytokines and MMPs and/or reduces
the expression of such cytokines and MMPs while stimulating
anabolic processes resulting in partial or complete restoration of
the degenerated tissue e.g. fibrocartilage and are therefore able
to reverse or stop the disease process. Thereby an injection of the
cartilage differentiation and maintenance factor assists in
arresting the aging process of the degenerating disc. Accordingly,
the present invention enables to treat a degenerative disc at an
earlier stage and thereby prevents degradation of the extracellular
matrix.
[0023] In addition, the cartilage differentiation and maintenance
factor alleviates or prevents cartilage degradation and preserves
or improves the structure and function of the IVD preferably
without undesired bone formation.
[0024] Another embodiment of the present invention relates to the
use of a cartilage determination and maintenance factor for
manufacturing of a pharmaceutical composition for treating a spinal
disorder in a mammal in need of such treatment.
[0025] In a preferred embodiment the spinal disorder is idiopathic
low back pain, disc herniation, internal disc disruption or
fissured discs, radiculopathy, spinal stenosis, herniated nucleus
pulposus-induced sciatica, sciatica, idiopathic scoliosis or
myelopathy.
[0026] Yet another aspect of the present invention comprises the
use of the spinal nucleus implant or formulation which comprises a
cartilage differentiation and maintenance factor which is a
non-antibody or non-receptor molecule, preferably wherein the
implant or formulation is injectable or implantable transdiscally,
for manufacturing of a pharmaceutical composition for treating a
spinal disorder in a mammal in need of such treatment.
[0027] For the purpose of the present invention, the term "spinal
nucleus pulposus implant" means a device or a preparation which is
to be administered in the intervertebral disc, in particular, into
the nucleus pulposus (NP) of the intervertebral disc. Preferably,
the implant can be administered directly into the intervertebral
disc through the AF or deposited directly into the NP of the disc.
It might be injected into the disc through a needle or other means
of minimal invasive application. It is also meant that the spinal
nucleus pulposus implant is a pharmaceutical preparation which can
be injected into the nucleus pulposus, intradiscal space or
intervertebral space. The implant can be a solid, e.g. a sponge or
a solid carrier, which comprises a cartilage differentiation and
maintenance factor. Preferably, the implant is liquid which allows
for easy application thereof. The implant, for example, can be a
liquid comprising a cartilage differentiation and maintenance
factor, optionally together with other drugs, formulation aids or
carriers. In instances of applying a larger size of a carrier
material partial or total removement of the disc might be
preferred.
[0028] For the purpose of the present invention, the term
"transdiscal administration" includes but is not limited to
injection of the spinal nucleus pulposus implant into an
intervertebral disc, in particular, into the NP of an
intervertebral disc which includes an intact disc, a degenerated
disc of different stages, a herniated disc, a ruptured disc, a
delaminated disc or a fissured disc. If the volume to be injected
might cause pressure of the NP, at least part of the NP can be
removed prior to injection or application of the implant for the
spinal column. In some cases the volume of the removed material is
about the amount of volume.+-.20% to be applied. The term
transdiscal administration also includes an injection of the spinal
nucleus pulposus implant into the AF of a degenerating or intact
disc as described above for the NP. In instances of applying a
larger size of a carrier material partial or total removement of
the disc might be necessary before application of the spinal
nucleus pulposus implant. It further includes providing the implant
into a location outside but closely adjacent to the AF wall or
endplate of an adjacent vertebral body, this might avoid the
puncture of the AF and therefore potential burden on the disc.
[0029] The term "degenerative disc disease (DDD)" is a chronic
process characterized in part by progressive loss of proteoglycan
and water content in the nucleus pulposus that can become manifest
in multiple disorders such as idiopathic low back pain, disc
herniation, internal disc disruption or fissured discs,
radiculopathy, spinal stenosis, herniated nucleus pulposus-induced
sciatica, sciatica, idiopathic scoliosis and/or myelopathy. The
disc degeneration grade can be ranked by analysis of preoperative
MRI.
[0030] The term "cartilage differentiation and maintenance factor"
means one or more cartilage differentiation factors that might have
mitogenic capability but are characterized by their ability to
increase and/or maintain the chondrocyte-specific phenotype of the
cell (e.g. anabolic activity) without undesired bone formation.
Chondrocyte-specific characteristics are for example the production
of aggrecan, type II collagen, SOX-9 and proteoglycans.
Chondrogenic morphogens may reverse the dedifferentiated or
fibrotic phenotype of disc cells to a fibrochondrocytic phenotype
comparable to disc nucleus cells of younger and normal adult discs.
It may also have an anabolic effect on annulus cells and/or
endplate cells of the disc. The cartilage differentiation and
maintenance factor is preferably a secreted molecule and hence can
potentially act in autocrine, paracrine or endocrine fashion.
[0031] "Mesenchymal stem cells (MSCs)" according to the present
invention are primitive or resting cell populations that reside in
many mature skeletal tissues as uncommitted mesenchymal progenitor
cells. MSCs are flexible and have the ability to differentiate
towards several mature tissue types, including cartilage, bone, fat
and other tissue, depending on the environment and biological
signals provided to these resting cells. MSCs are available from
many autologous sources, including bone marrow, blood, muscle
tissue and fat that can be harvested to isolate these cells without
significant donor site morbidity or immunogenic potential.
[0032] MSCs can be precursor cells of NP cells or AF cells,
chondrocytes, or other living cells that could function like AF or
NP cells or could differentiate into cells or build a functional NP
and/or AF.
[0033] "Treating or treatment" as used herein, means an alleviation
of symptoms associated with a disorder or disease, halt of further
progression or worsening of the symptoms, prevention or prophylaxis
of the disease or disorder.
[0034] The invention is based on the finding that a cartilage
differentiation and maintenance factor such as a member of the MIA
(melanoma inhibitory activity factor) family like CD-RAP can affect
or modify a spinal disorder by anabolic effects (e.g. regeneration
or restoration of fibrocartilage within the vertebral column) while
arresting or inhibiting catabolic degenerative processes (e.g.
degeneration of the extracellular matrix) within the spinal
disc.
[0035] Such an effect of a cartilage differentiation and
maintenance factor on spinal disorders is surprising, since the
cartilage of the intervertebral disc (IVD) is quite different from
other common cartilage.
[0036] In contrast to articular cartilage which is referred to
hyaline cartilage the cartilage of IVDs consists of fibrocartilage
which is a special type of cartilage. Particularly the AF of the
IVD is considered fibrocartilaginous and consists primarily of
lamellae composed of highly oriented collagen fibers. The NP
contains a higher content of type II collagen that is randomly
orientated, with a much higher concentration of proteoglycans.
Cells of the AF have shown to orientate along the predominant
collagen fiber direction of the lamella. Cells of the innermost AF
and NP region are more rounded and accumulate more type II and VI
collagens and proteoglycans.
[0037] There are significant physical and chemical differences
between fibrocartilage and other types of cartilage such as
articular/hyaline cartilage (WO2005/091960 and references therein).
For example fibrocartilage differs in having much type I collagen
in its matrix mainly in the annulus. Type II collagen from
fibrocartilage such as IVD cartilage, has a substantially higher
level of hydroxylation and glycosylation than type II collagen from
articular cartilage and aggrecan is more highly substituted. These
posttranslational modifications affect the structure and physical
function of the fibrocartilage collagen and IVD. Antenatal
differentiation of the fibrocartilaginous IVD also differs from
that of the articular cartilage in a synovial joint. The IVD has a
complex developmental history and contains notocord derived cells
which have no equivalence in articular cartilage.
[0038] Until the present invention, cartilage differentiation and
maintenance factors of the present invention such as CD-RAP had not
been shown to have biological effects to prevent fibrocartilage or
fibrochondrocytes degeneration in vitro or in vivo. The cartilage
differentiation and maintenance factors of the present invention
induce and maintain cartilage anabolism in the IVD (e.g. synthesis
of proteoglycan and aggrecan) while the pathogenesis of
degeneration and cartilage catabolism including breakdown of
matrix, synthesis of abnormal proteoglycans and collagens is
partially or fully arrested, inhibited or even reversed.
[0039] Abnormal disc degeneration, increased apoptosis and
diminished matrix molecule synthesis are at least partially
mediated by cytokines such as IL-1 which is shown to switch
chondrocytes from anabolism to catabolism, inducing cartilage
breakdown at molecular and morphological level. Inhibition of the
activity of cytokines and MMPs, known to be involved in disc
degeneration and/or reduction of the expression of such cytokines
and MMPs mediated by the present invention aids in re-synthesis of
normal disc matrix and influences discal cell function. Thereby an
injection of the cartilage differentiation and maintenance factors
assists in arresting or reversing the aging or degenerative process
of the degenerating disc. Accordingly, the present invention
enables to treat a degenerative disc at an earlier stage and
thereby prevents degradation of the extracellular matrix and
preserves the structure and function of the IVD.
[0040] Preferably, the cartilage differentiation and maintenance
factor of the present invention is a non-antibody and non-receptor
molecule. This means that the factor used according to the
invention is not an antibody and not a receptor molecule either.
The cartilage differentiation and maintenance factor is preferably
a non-transcription factor (e.g. is not SOX-9) or preferably is an
extracellular protein. More preferably, the cartilage
differentiation and maintenance factor is not selected from the
group of transforming growth factor .beta.(TGF-.beta.e.g.
TGF-.beta.1)-, bone morphogenetic (BMP)- and insulin like growth
factor (IGF)-family proteins (e.g. IGF-1). BMP proteins are
described by Wozney et al. (Wozney and Rosen, 1998) and include for
example BMP-2, BMP-7 and growth and differentiation factors such as
GDF-5, GDF-6 and GDF-7.
[0041] Preferably, the cartilage differentiation and maintenance
factor is a chondrogenic morphogen, more preferably a chondrogenic
morphogen which is a protein, preferably a cartilage specific
protein, with anabolic activity for cartilage regeneration
(anabolic factor) and maintenance. The anabolic factor in contrast
to catabolic factors such as metalloproteinases, apoptotic factors,
interleukins, prostaglandins, proteolytic and degradative enzymes,
oxygen free radicals, nitric oxide and fibronectin fragments which
induce degradation of the nucleus pulposus region, increase the
chondrocyte-specific phenotype of cells within the vertebral disc.
Preferably, the chondrogenic morphogen is a cartilage determination
factor preferably specific for cartilage tissue controlling
cartilage formation and maintenance, while avoiding or inhibiting
formation of bone.
[0042] In one embodiment, the cartilage differentiation factor has
a molecular weight of less than 80 kDa, preferably of .ltoreq.30
kDa, more preferably of .ltoreq.15 kDa, most preferably between 10
and 15 kDa.
[0043] The cartilage differentiation and maintenance factor used
according to the invention, in particular, is a factor which
induces the synthesis of extracellular matrix proteins such as
proteoglycan, aggrecan and/or collagen. Further, preferably, the
factor results in a reduction of the amount of cytokines and
MMPs.
[0044] In one embodiment, the cartilage differentiation and
maintenance factor is selected from the group of proteins with an
SH3-domain or with a domain which adopts an SH3-like domain fold
such as CD-RAP. SH3-domain or SH3-like domain are described for
example in Stoll et al. (Stoll et al., 2001 b) and can be
determined by the prediction of an SH3-fold by an 3D-PSSM Web
server published in Kelley et al. (Kelley et al., 2000).
SH3-domains also, called Src homology domains, are protein
molecules that are found in many intracellular proteins. So far, no
SH3-domain proteins were described to be useful in treatment of
spinal disorders.
[0045] In another embodiment the cartilage differentiation and
maintenance factor is a protein which specifically can bind to
fibronectin, fibronectin fragments and/or proline rich sequences as
for example described in the literature (Stoll et al., 2001a;
Homandberg and Hui, 1996; Homandberg et al., 1997).
[0046] In one embodiment, the cartilage differentiation and
maintenance factor comprises a fibronectin or integrin binding
domain. Binding of the cartilage differentiation and maintenance
factor to extracellular proteins such as fibronectin or fibronectin
fragments as well as integrins can be determined for example by
ELISA. Fibronectin, fragments or integrins thereof can be coated on
plastic surfaces and are exposed to the cartilage differentiation
and maintenance factor. The amount of binding can be determined by
a peroxidase-linked monoclonal antibody against the cartilage
differentiation and maintenance factor. Integrin binding can also
be determined as described by Bauer et al. herewith incorporated by
reference.
[0047] Preferably, the cartilage differentiation and maintenance
factor is selected from the group of a) chondrocyte proteins
comprising or having the mature sequence of CD-RAP (Seq ID No. 1)
and functional fragments or variants thereof, b) proteins having at
least 63%, preferably 80%, more preferably 90% amino acid sequence
homology with the C-terminal four cysteine skeleton of CD-RAP,
amino acids 12 to 107 of Seq ID No 1, or c) proteins having any of
the generic sequences 1 to 3 defined herein (Seq ID No. 2, 3 and
4).
[0048] Functional fragments having the same biological function as
CD-RAP preferably have a length of at least 20, in particular, at
least 40 and more preferably at least 50, most preferably 80
contiguous amino acids of the sequence shown in Seq ID No 1.
Preferably, the functional fragments comprise the amino acids from
position 1 to 50, 1 to 70, 1 to 80, 20 to 80, 20 to 107 of Seq ID
No. 1.
TABLE-US-00001 Mature CD-RAP sequence
GPMPKLADRKLCADQECSHPISMAVALQDYMAPDCRFLTIHRGQVVYVFS (SeqID No. 1)
KLKGRGRLFWGGSVQGDYYGDLAARLGYFPSSIVREDQTLKPGKVDVKTD KWDFYCQ Generic
sequence 1 C X.sub.4 C X.sub.17 C X.sub.12 V X.sub.11-13 W
X.sub.7-18 F X.sub.4 V X.sub.21 C X (SeqID No. 02) Generic sequence
2 K X C X D X E C X.sub.11 D X.sub.3 P D C X.sub.12 V X.sub.2 K L
X.sub.7-9 W X G S X.sub.5-13 G Y F P X.sub.3 V (SeqID No. 03)
X.sub.18 D F X C X Generic sequence 3 K X C X D X.sub.2 C X.sub.8 A
X.sub.2 D X.sub.3 P D C R F X.sub.5 G X V X.sub.5 K L X.sub.7 W X G
S V X.sub.12 G (SeqID No. 04) Y F P X.sub.22 D F X C Q
wherein "X" at each occurrence independently represents any amino
acid and the number in lowercase the number of any amino acid.
Preferably, "X" independently represents a naturally occurring
amino acid and, in particular, A, R, N, D, B, C, Q, E, Z, G, H, I,
L, K, M, F, P, S, T, W, Y or V.
[0049] Particularly preferably, the cartilage differentiation and
maintenance factor is CD-RAP (Cartilage derived retinoic acid
sensitive protein), also named MIA (melanoma inhibitory activity),
OTOR (fibrocyte derived protein, FDP, MIA-like, MIAL) and TANGO 130
which belongs to a class of secreted proteins (Bosserhoff et al.,
2004; Bosserhoff and Buettner, 2003; Bosserhoff et al., 1997;
WO00/12762). CD-RAP or MIA is a 130 amino acid protein (EP 0710248,
EP 1146897, fully incorporated herein by reference) that is a
highly specific marker for chondroid differentiation. Gene
expression is activated at the beginning of chondrogenesis
throughout cartilage development (Dietz and Sandell, 1996; Sakano
et al., 1999). In case of cartilage damage due to osteoarthritis,
CD-RAP is expressed with increasing levels at the onset of disease
at which time a strong anabolic effect is observed and will decline
once the disease worsens (Saito et al., 2002).
[0050] The protein contemplated herein can be expressed from intact
or truncated genomic DNA or cDNA or from synthetic DNAs, in
prokaryotic or eukaryotic host cells. Proteins can be isolated from
the culture media or inclusion bodies and/or refold to form
biological active compositions. See e.g. EP 0710248 and Lougheed et
al. (Lougheed et al., 2001) for exemplary protocols for recombinant
protein purification of CD-RAP. Detailed description of how to test
the activity (e.g. chondrogenesis) of such isolated proteins is
described in Tscheudschilsuren et al. and Stoll et al.
(Tscheudschilsuren et al., 2005; Stoll et al., 2003), the
disclosures of which is incorporated by reference herein. A
bioassay for cartilage induction is described in example 2 to 5 in
EP 1146897, incorporated by reference herein. Example 5 describes a
mouse ectopic implant assay for cartilage induction. Alternatively
cartilage induction and maintenance can be determined in a partial
or full thickness articular cartilage repair model.
[0051] In one embodiment of the present invention, the spinal
nucleus pulposus implant further comprises one or more additional
active agents, preferably anticatabolics (e.g. TIMP-1 and TIMP-2),
mitogens (e.g. IGF-1, PDGF, EGF, FGF), bone morphogenetic proteins
such as GDF-5, BMP antagonists such as noggin or chordin and/or
intracellular regulators (e.g. LMP-1, SOX-9) or combinations
thereof. The addition of anticatabolics further increases matrix
synthesis mediated by the cartilage differentiation and maintenance
factor or chondrogenic morphogen for example by inhibition of
degradative enzymes. Mitogenic molecules are growth factors which
increase the rate of mitosis of cells and might also increase PG
synthesis to various degrees depending on the region of the disc
where the cells are derived from and therefore further support the
effect of the cartilage differentiation and maintenance factor or
chondrogenic morphogen. By combining a cartilage differentiation
and maintenance factor with an intracellular regulator
up-regulation of the cartilage differentiation and maintenance
factor and/or PG synthesis can be achieved in in vitro
experiments.
[0052] In another embodiment, the spinal nucleus pulposus implant
further comprises one or more anti-metalloproteinases, cycline
compounds, cytokine antagonists, TNF inhibitors, IL-inhibitors,
anti-angiogenic substances, inhibitors of proteolytic enzymes,
anti-inflammatory drugs including infliximab, etanercerpt,
adalimulab, nerelimonmab, lenercerpt and the like, or combinations
thereof.
[0053] In another embodiment, the spinal nucleus pulposus implant
is co-administered or administered after injection or application
of chemonucleolytic agents such as C-ABC or those described in US
2005/0031666 in order to prevent a long-term structural damage of
the disc.
[0054] Preferably, the spinal nucleus implant is injectable or
implantable transdiscally. Preferably, the injection is local or
non-systemic injection. An advantage for injection or implantation
transdiscally is that higher concentrations of the cartilage
differentiation and maintenance factor can be used while causing
minimal systemic toxicity.
[0055] The cartilage differentiation and maintenance factor or IVD
disc repairing agent can directly be implanted or injected in an
acceptable solvent or vehicle, for example, but not limited to
physiological saline solution, sterilized water, Ringer's solution.
Preferably, it is administered into the intradiscal or NP space.
Administration can be achieved with a single or repetitive
injection(s), preferably with a percutaneous injection or
percutaneously via a catheter. The intradiscal injection of the
chondrogenic protein preferably CD-RAP will increase intervertebral
disc height by stimulating intervertebral disc cells to upregulate
proteogycan, aggrecan and/or collagen synthesis. Clinical
application can thus be accomplished by minimal invasive
techniques, significantly saving costs and the likelihood of
complications relative to other procedures such as partial
disectomy or vertebral fusion.
[0056] While it is possible to add the spinal nucleus pulposus
implant of the invention to the intervertebral disc, it is also
possible to remove part of the intervertebral disc and replace it
by the implant of the invention.
[0057] The present invention, thus, also provides for replacing an
amount of the NP removed, for example, in a nucleotomy or partial
nucleotomy procedure, or for supplementing a NP that has become
degenerated by reason of age, injury or the like with a nucleus
pulposus implant of the present invention. The degenerating or
degenerated disc or a part thereof may be removed with standard
techniques, with a laser, shaver, or other surgical instrument.
[0058] The degenerating disc can be an intact disc or a ruptured
disc. The degenerating disc can be delaminated, can have fissures
or can be herniated.
[0059] The spinal nucleus pulposus implant may be combined with a
minimal invasive stabilizing procedure. In severe cases of advanced
disc degeneration where continuous stimulation results in
production of undesired factors such as catabolic factors a minimal
invasive stabilizing procedure can further support regeneration or
inhibit progression of the degenerated disc.
[0060] In one embodiment of the invention, the implant or
formulation further comprises a carrier or a drug delivery device.
The carrier or drug delivery device used in the invention is
biocompatible in that it is not toxic and does not elicit
inflammatory reactions in the body. The carrier can include a
matrix or scaffold structure. The carrier may be solid, a liquid, a
gel, a paste or other injectable form. Preferably, the carrier
comprises a hydrogel as for example described in WO2005/113032, in
particular injectable hydrogels, sulphated hyaluronic acid, a
highly substituted carboxymethylcellulose and salts thereof,
alginate, hydroxypropylalginate, chitosan, hydroxethylstarch,
collagen, gelatin, reverse thermal gels (e.g. Pluronic F128), a
chitosan based thermosensitive copolymer (e.g.
chitosan-Pluronic.RTM. hydrogel), a porous silk scaffold, a
plurality of microspheres, a lipososmal formulation and a
hydroxyapatite fiber mesh. The carrier is an appropriate substrate
for cells suited for ingrowth, proliferation and residence of IVD
cells.
[0061] The carrier can comprise a polymer such as Pluronics e.g.
pluronic 168, a block copolymer of ethylene oxide and propylene
oxide such as those described in WO2005/034800.
[0062] Preferably, the carrier comprises chondroitin sulfate,
gelatin, hyaluronan and/or sodium hyaluronate or a mixture thereof
including tri-copolymers such as
gelatin/chondroitin-6-sulfate/hyaluoran tri-copolymer. Preparations
of tri-copolymers are described in Yang et al. (Yang et al., 2005)
which is herewith fully incorporated as reference.
[0063] The carrier may comprise a fibrin gel composed of
platelet-rich plasma, platelet enriched plasma with biodegradable
gelatin hydrogel, fibrin/hyaluronic acid composites, factor
encapsulated gelatine hydrogel microspheres, injectable
biodegradable hydrogel composites comprising for example polymers
such as oligo(poly(ethyleneglycol) fumarate, polylactide
(PLA)/polyglycolicacid (PGA) and poly epsilon capronolactone.
[0064] Preferably, the carrier comprises a chitosan-glycerol
phosphate or an in situ blood clot or blood clot stabilized with
chitosan-glycerol phosphate solution.
[0065] In one embodiment of the invention, the spinal nucleus
pulposus implant further comprises a sustained release device e.g.
a sustained release device comprising a hydrogel, polyanionic
hydrogel, a plurality of microspheres, a liposomal formulation and
a hydroxyapatite fiber mesh. The sustained release device, in
particular, enables controlled release.
[0066] In one embodiment, the sustained release device provides
continuous release, in another embodiment, the sustained release
device provides intermittent release.
[0067] In one embodiment, the cartilage differentiation and
maintenance factor is encapsulated in liposomes. The liposomes have
the advantage over a crystalline solution in that a mechanical
irritation in the intervertebral disc can be avoided and hence a
therapy induced inflammation can be avoided in addition to a longer
duration of the therapeutic agent and slower clearance at the site
of application.
[0068] One approach for improving efficacy of delivery of
therapeutic compounds and other agents has been the encapsulation
in a lipid structure such as liposomes. Liposomes generally
comprise an enclosed lipid droplet having a core typically
containing a compound in an aqueous medium. In certain embodiments,
the compound is chemically bound to a lipid component or simply
contained within the aqueous inside compartment of the
liposome.
[0069] A pharmaceutical composition or spinal nucleus pulposus
implant provided according to the present invention comprising the
cartilage differentiation and maintenance factor is preferably
provided as dried liposomal compositions that can be reconstituted
to produce liposomes encapsulating the cartilage differentiation
and maintenance factor. Preferably, the liposomal preparation are
dried reconstituted vesicles (DRVs) which upon reconstitution in an
aqueous solution form cartilage differentiation and maintenance
factor encapsulated liposomes. The liposomal composition used
herein is for example a dry granular product which upon addition of
water disperses to form multi-lamellar liposomal formulations
comprising the biological active component. Advantageously,
stability problems such as aggregation or oxidation of the active
agent and/or liposomes are avoided by using dried liposomes.
[0070] Suitable lipids for use in the formulations which are
present individually or in mixtures include neutral or positively
charged lipids such as cholesterol, phosphatidylcholine,
hydrogenated phosphatidylcholine, distearoylphosphatidylcholine,
sphingomyelin, dioleyl phosphatidylcholine,
dioleylphosphatidylglycerol, phosphatidylglycerol,
dimyristoylphosphatidylcholine, dipamlitoylcholine, gangliosides,
ceramides, phosphatidylinositol, phosphatic acids,
dicetylphosphate, dimyrylstoyl phosphatidylcholine, stearylamine,
dipalmitoyl phosphatidylglycerol and other similar lipids.
Preferably the lipid mix is charged. The liposomal formulation is
typically a mixture of at least two lipids such as cholesterol and
phosphatidylcholine and more usually a mixture of three or more
lipids.
[0071] In another embodiment, the cartilage differentiation and
maintenance factor of the present invention is pegylated. This
modified cartilage differentiation and maintenance factor has a
biological half-life time greater than the unmodified agent and
therefore can improve the efficacy of the agent for medical
treatment of spinal disorders. Pegylation can increase the size of
the protein, improve stability, increase solubility of the protein,
reduce proteolysis and decrease dosing frequency. In addition,
tendency towards aggregation of the protein can be reduced.
[0072] Pegylation can be achieved via stable covalent bonds between
an amino or sulfhydryl group on the protein and a chemically
reactive group (carbonate, ester, aldehyde, or tresylate) on the
polyethylenglycol (PEG). The resulting structure may be linear or
branched. PEG reagents are for example described in Roberts et al.
(Roberts et al., 2002). Other pegylation agents are but are not
limited to methoxypoly(ethylene glycol) (mPEG), methyl PEO.sub.12
maleimide PEG, amine-reactive, methyl-capped polyethylene oxide
(PEO)-containing modification agents (Methyl PEO.sub.n-NHS Esters,
n=4, 8, 12).
[0073] In another embodiment, the spinal nucleus implant of the
present invention comprises nucleus pulposus tissue or cells,
preferably cells derived from mesenchymal stem cells (MSCs).
[0074] MSCs or autologous stem cells isolated from the donor tissue
(e.g. bone marrow stroma) can be cultured in or on a
three-dimensional biodegradable scaffold material such as
hyaluronic acid, silk, collagen, collagen/hyaluronan scaffolds,
hydrogels, chitosan, chitosan gel, injectable cross-linkable
polymeric preparations, degradable polymer gels or scaffolds,
polylactide, gelatin/chondroitin-6-sulfate/hyaluronan scaffold,
ester or derivatives of hyaluronic acid such as Hyaff 11, a
hydroxyapatite fiber mesh and fibrin glue in the presence of a
cartilage differentiation and maintenance factor either alone or in
combination with other morphogens or growth factors like IGF-1
members or BMPs, which support the differentiation of those cells
into nucleus pulposus-like cells and stimulate PG synthesis
preferably under oxygen tension. Methods for cultivation are for
example described in Honda et al., 2000, which are herewith
incorporated by reference (Honda et al., 2004). The thus cultured
cells or neotissue, preferably, along with the scaffold material,
will then be transplanted or injected into the affected disc to
achieve regeneration of the NP.
[0075] It is also possible to isolate and expand MSCs in monolayers
culture for example under hypoxic conditions in vitro, as occurs in
the NP region of the intervertebral disc. MSCs progressing to
transplantation can be transfected with one or more cartilage
differentiation and maintenance factor required to stimulate IVD
healing or can be stimulated with a chondrogenic induction medium
containing such cartilage differentiation and maintenance factor
(e.g. CD-RAP or active fragments thereof). Preferably, a
transfection can be performed by an expression vector encoding such
cartilage differentiation and maintenance factor like the CD-RAP
protein or active fragments thereof. Transfected and/or stimulated
MSCs or cultured MSCs preferably embedded in a biomaterial such as
collagen, atelocollagen gel, gelatine, alginate,
hydroxypropylalginate, carboxymethylcellulose or hydroyethylstarch
can be transplanted into the degenerative disc through injection
such as an insulin microinjector (Sakai et al., 2005) or another
application device. Cell density can be for example between
1.times.10.sup.4 cells/ml to 1.times.10.sup.7 cells/ml, preferably
between 1.times.10.sup.5 cells/ml to 1.times.10.sup.6 cells/ml.
[0076] MSCs also can be cultured in alginate, pellet, micromass or
aggregate cell cultures in the presence of the cartilage
differentiation and maintenance factor either alone or in
combination with other morphogens or growth factors like TGF-.beta.
members and/or BMPs (e.g. BMP-2), which support the differentiation
of those cells into NP-like cells.
[0077] In one embodiment, the delivery device comprises MSCs or AF
cells in combination with a 3-D porous silk scaffold. The porous
silk scaffold may be derived from silk fibroin extracted from
Bombyx mori. The cells can be expanded undifferentiated and will be
induced into chondrocytes by culturing with the cartilage
differentiation and maintenance factor. Silk scaffolds can either
directly or loaded with the cartilage differentiation and
maintenance factor be seeded with MSCs or AF cells and can be
cultured in medium supplemented with or without the cartilage
differentiation and maintenance factor alone or in combination with
other factors described above. Silk fibroin scaffolds from Bombyx
mori silkworm cocoons can be extracted as for example described in
Sofia et al. and Karageorgiou et al. (Sofia et al., 2001;
Karageorgiou and Kaplan, 2005).
[0078] In one embodiment, AF, NP or MSC cells can be transfected ex
vivo with at least one gene for a cartilage differentiation and
maintenance factor to provide both the cells and the protein or
proteins required to stimulate IVD healing and are reimplanted with
or without culturing in for example monolayer cultures, alginate
beads or a three-dimensional biodegradable scaffold material into
the targeted host tissue. As an example, isolated NP cells are
seeded as monolayer, followed by transfection with the cartilage
differentiation and maintenance factor comprising for example an
expression vector or viral gene therapy vector such as the
adeno-virus or adeno-associated virus (AAV) using a transfection
agent such as the FuGene6 reagent. After several days of culture
(e.g. 7 days), cells are passaged and encapsulated in alginate e.g.
preferably between about 0.5 to 2% alginate. Expression of the gene
can be analyzed by standard methods such as RT-PCR or ELISA. These
transfected nucleus pulposus cells in alginate beads can be used
for tissue engineering of the IVD and treatment for the
degenerative disc disease. Further gene therapy methods are
described for example in Wells, 2004; Paul et al., 2003 and
Sobajima et al., 2004.
[0079] In one embodiment cells such as NF cells, MSCs or autologous
chondrocytes preferably of human origin are temporarily
immortalized using for example a recombinant Simian Virus 40
adenovirus vector or baculovirus vector encoding for the cartilage
differentiation and maintenance factor required to stimulate IVD
healing.
[0080] In another embodiment degenerate human IVD cells (e.g. NP
cells) can be transfected with an adenovirus vector carrying the
exogenous factor such as the cartilage differentiation and
maintenance factor. A subsequent step would then be to inject these
modified cells back into the diseased IVD.
[0081] The present invention is also directed to the use of a
cartilage differentiation and maintenance factor of the present
invention for culturing mesenchymal stem cells for manufacturing of
a pharmaceutical composition for treating a spinal disorder in a
mammal, in particular, in a human in need of such treatment.
[0082] The invention is further illustrated by the Figures and
Examples.
[0083] FIG. 1 illustrates the stability of the liposomal
formulation comprising CD-RAP over several days (triangles).
Stability of the liposomal formulation was determined according to
example 3. The upper curve (squares) shows the stability of CD-RAP
in buffer at 37.degree. C. to determine stability of the protein
under these conditions.
[0084] FIG. 2 shows the immobilization of 50 .mu.g CD-RAP in the
fibrin clot system after 24 h at 37.degree. C. [0085] A:
Tachotop.RTM., 14 mm diameter, 4 mm height from Nycomed; B: porcine
type I collagen sponge, BiomUp; C: rehydrated Hyalofill-F.
[0086] FIG. 3 illustrates the percent (%) proteoglycan (GAG)
induction of cultured rabbit IVD cells upon stimulation with CD-RAP
(100 ng/ml) related to the negative control without addition of
CD-RAP and BMP-2 (n=3).
EXAMPLES
Example 1
Annulus Fibrosus Puncture Model
[0087] In this example, an injection of CD-RAP is effective in
partially restoring the disc height in a rabbit annular puncture
model.
[0088] Disc degeneration can be induced in adolescent New Zealand
White Rabbits by puncture of the annulus fibrosus of the disc using
defined needle gauges (Singh et al., 2005). After provision of a
local anaesthetic by injection of lidocain to the dorsal region of
the disc lateral plain radiographs are obtained to determine
preinjection baseline values for IVD heights. Subsequently the
rabbits are placed into a lateral prone position and a
posterolateral retroperitoneal approach is used to expose the
lumbar IVDs. In each rabbit the AF will be punctured with an 18 G
needle. After four, eight and 10 weeks the animal receive an
injection of buffered saline (arginine phosphate or PBS) or vehicle
liposomes as a control or protein solution of 2.5 mg/ml to 10 mg/ml
CD-RAP (in PBS) or liposomal encapsulated CD/RAP (2.5 mg/ml) into
the nucleus pulposus. The animals are followed for 8 or 12 weeks
(induction and treatment period). Preclinical outcome is analyzed
by magnetic resonance imaging (MRI) scans of the lumbar spine, IVD
height is monitored by radiological observation measured with a
custom program using Imaging software and the % DHI (postoperative
DHI/preoperative DHI.times.100) is calculated. For histological
analysis of the IVDs, sections are stained with Hematoxylin Eosin
and Safranin O. Differences among groups are assessed for
statistical significance by using a one-way analysis of variance
(ANOVA).
[0089] An alternative slow progressive and reproducible animal
model of disc degeneration therapies is a classical "stab model" of
Lipson and Muir as described in Sobajima et al. (Sobajima et al.,
2005). For the stab method, an incision will be made in an AF of
New Zealand White rabbits. Each rabbit will have one disc treated
with CD-RAP, the other with saline solution or vehicle.
[0090] The annulus needle puncture model results in disc narrowing
with the 12 week observation period. Saline treated discs exhibit
extensive degeneration of the disc. However, the % DHI reveals a
tendency of a preserved disc height in the CD-RAP group compared to
the saline or vehicle injected discs. Histological analysis shows
an increase of proteoglycan synthesis and protection against
degenerative changes compared to the control group.
Example 2
Preparation of CD/RAP Containing Liposomes
[0091] For preparation of a sustained liposomal preparation 750 mg
phosphatidylcholine and 250 mg cholesterol were solved in 20 ml
ethanol in a round bottom flask. The solvent was removed in a
rotary evaporator quantitatively. The generated thin lipid film was
rehydrated in 10 ml water to get liposomes (10% (w/v) lipid) by
gentle stirring at room temperature. Unilamellar vesicles (SUV)
were prepared with a diameter of approximately 100 nm by subsequent
sonification. 300 .mu.l of SUV were mixed with 250 .mu.l CD-RAP
solution (3 mg/ml in 420 mM/I Arg/PO.sub.4 pH 7.5) and were
subsequently lyophilized. Multilamellar liposomes (MLVs)
encapsulating the protein were generated by reconstitution of the
lyo cake with destined water shortly before application of the
dried reconstituted vesicles. This rehydration led to an entrapment
efficacy of about 50% or more into MLVs with an average diameter of
about 1.5 .mu.m or more without chemical alteration of the
entrapped drug.
Example 3
Stability of Liposomal Formulation Comprising CD-RAP
[0092] This example illustrates the stability of the liposomal
formulation comprising CD-RAP over several days. Stability of the
liposomal formulation was determined as follows: 120 .mu.g of
CD-RAP were encapsulated in 300 .mu.l liposomal suspension as
described above. An aliquot of 100 .mu.l was diluted with 300 .mu.l
bidestilled water and separated by centrifugation for 15 min at
16000 rcf. To determine the encapsulation efficacy, the
non-encapsulated CD-RAP was quantified by RP-HPLC using a standard
curve. A six-fold repetition of a resuspension and centrifugation
step did not show increase of the free CD-RAP.
[0093] The release was described by measuring the free
concentration of CD-RAP in the supernatant within a time period of
seven days, separated by centrifugation (15 min at 16000 rcf). At
each time point the amount of free CD-RAP was measured by RP-HPLC
using a standard curve.
[0094] The results shown in FIG. 1 indicate a high encapsulation
efficacy of more than 50% (time 0 [d]). During the following
observation time period of up to 7 days at 37.degree. C. a plateau
was achieved with maintenance of an encapsulation of about 45%,
therefore indicating stability of the reconstituted CD-RAP
comprising liposomes.
Example 4
CD-RAP Immobilized Implants
[0095] This example illustrates CD-RAP immobilized implants in
collagen or hyaluronic acid based scaffolds.
[0096] 50 .mu.g of CD-RAP were formulated in 125 .mu.l 20 mM
KH.sub.2PO4, 150 mM KCl, KOH, pH 7.5, 0.01% Tween 80. The solution
was soaked into a collagen sponge (A: Tachotop.RTM. (A), 14 mm
diameter, 4 mm height from Nycomed; (B) porcine type I collagen
sponge, BiomUp; or mixed with 500 .mu.l Hyaff gel (rehydrated
Hyalofill-F (C), 30 mg in 0.5 ml bidest water, 3 h, 5.degree. C.)
to adsorb CD-RAP. Subsequently the water was removed by freeze
drying.
[0097] To determine the immobilization kinetic of CD-RAP adsorbed
onto collagen or Hyaff, the CD-RAP impregnated specimen was fixed
within 500 .mu.l bovine fibrin clot (4.9 mg fibrinogen, 0.3 U
thrombin in 50 mM sodium citrate, 150 mM sodium chloride, 10 mM
calcium chloride, pH 6.4) as described by Meyenburg et al.
(Meyenburg et al., 2000). The clot was then covered completely by 2
ml acceptor medium (phosphate buffered saline, 0.02% Tween 80). The
amount of free CD-RAP was quantified after 24 h by RP-HPLC using a
standard curve.
[0098] The results shown in FIG. 2 indicate differences in
immobilization efficacy of various biomaterials with material A and
C showing strong binding properties for the recombinant CD-RAP
protein. These binding properties can be used for local targeting
and retention of the cartilage determination and maintenance factor
within the site of defect e.g. the degenerated disc avoiding a high
initial burst of CD-RAP and providing long-term maintenance at the
site of regeneration.
Example 5
Isolation of Human and Animal Intervertebral Disc Cells
[0099] This example illustrates methods to prepare IVD cells useful
for analyzing the effect of CD-RAP on production of extracellular
matrix components specific for cartilage cell stimulation and
anabolic activity of CD-RAP.
[0100] Human disc cells can be isolated from human disc tissue
recovered by disectomy performed in the treatment of patients with
degenerative disc disease. The specimen (nucleus pulposus or
annulus fibrosus) are rinsed with PBS to remove residual blood or
extracellular matrix. After mincing of the tissue cells are
released from the extracellular matrix with collagenase solution
(0.5 mg/ml in PBS) at 37.degree. C. for 45 min and cells can be
isolated by centrifugation (see Klagsburn, "Methods in Enzymology",
Vol VII). After removal of the supernatant harvested cells are
grown on six well plates in Eagle minimal essential medium with or
without fetal calf serum.
[0101] Bovine IVD cells from animal tissue are isolated by
sequential enzymatic digestion. Cells are cultivated with daily
medium changes of DMEM/F12 medium supplemented with 10% fetal
bovine serum, 25 .mu.g/ml ascorbate, 360 .mu.g/ml L-glutamine and
50 .mu.g/ml gentamicin in a humidified atmosphere at 37.degree. C.
with 5% CO.sub.2 until they have reached about 80% confluence.
[0102] Rabbit IVD cells from animal tissue were isolated by
sequential enzymatic digestion. Cells were cultivated in DMEM
medium supplemented with 10% FCS, 1% Penicillin/Streptavidin and 50
ng/ml ascorbic acid in a humidified atmosphere at 37.degree. C.
with 5% CO.sub.2 until they reached about 90% confluence.
Thereafter they were passaged and cultivated for additional 7
days.
Example 6
Culturing of Intervertebral Disc Cells in Alginate Beads
[0103] Alginate beads are formed by expressing 60 .mu.l 1.2%
alginate in 0.15 NaCl with IVD cells of example 5 into a 102 mmol/L
calcium chloride solution, forming a semisolid bead. The beads are
washed twice and placed in 12 well plates with 1 ml medium (Aota et
al., 2005). The resulting alginate beads can be used for analyzing
the influence of fibronectin fragments (120 kDa fragment (Chemicon,
Cat. No. F1904), 70 kDa fibronectin proteolytic fragment from human
plasma (Sigma, Cat. No. F0287)) with or without addition of CD-RAP
for proteoglycan synthesis and aggrecan expression.
Example 7
CD-RAP Mediated Induction of Aggrecan and Proteoglycan Synthesis in
IVD Cells
[0104] This example illustrates the use of the spinal implant to
demonstrate that CD-RAP when added to IVD cells significantly
increases proteoglycan synthesis and aggrecan expression of IVD
cell during culturing.
[0105] On day 7 IVD cells of example 5 are placed in 24 well plates
to study proteoglycan synthesis and expression of aggrecan. Cells
are cultured for 7 more days in serum free medium with 0.5 .mu.M
and 0.1 .mu.M of a 70 kDa fragment of fibronectin (FO297 Sigma).
For analyzing the influence of CD-RAP on proteoglycan synthesis and
expression of aggrecan different concentrations of CD-RAP (1, 5,
10, 20 ug/ml) are added two days after culturing with fibronectin.
After 7 days aggrecan expression is analyzed by Lightcycler
analysis (SybrGreen).
[0106] To release the cells from the beads, the bead are
solubilized in a buffer containing 55 mmol/L sodium citrate, 30
mmol/L Na.sub.2EDTA, 0.15M sodium chloride pH 6.8. The cell pellets
are washed and resuspended in lysis buffer for RNA extraction
(Qiagen). RNA isolation is performed using the RNeasy Mini Kit
(Qiagen). CDNA synthesis is performed according to the instruction
of the Superscript II RT kit of Invitrogen. .beta.-actin is used as
a control. Primers used for amplification are the following: a)
bovine .beta.-actin primers 5' GGA AAT CGT CCG TGA CAT CAA 3'; 5'
AAG GAA GGC TGG AAG AGA GC 3'; Aggrecan primers were: 5' AAG AGA
GCC AAA CAG CCG A 3'; 5' CTG GTA GTC CTG GGC ATT GT 3'.
[0107] Proteoglycan synthesis is measured using the
dimethylmethylene blue (DMMB) colorimetric assay according to the
method as described in Farndale et al. (Farndale et al., 1982). The
culture medium can be concentrated with a centricon filter for 10
to 20 minutes at 5000 rev/min. The GAG content can be determined by
mixing 20 .mu.l of concentrated or diluted culture medium with 200
.mu.l of DMMB solution and measuring the optical density at 525-530
nm. For standardization chondroitin sulfate (chondroitinsulfat A
94%, bovine trachea, ICN) can be used. The mean of all measures are
calculated per microgram of DNA or cell number.
Example 8
Proteoglycan Induction in Rabbit Intervertebral Disc Cells
[0108] When rabbit IVD cells isolated according to example 5
reached 90% confluence cells were placed in 6 well plates to study
proteoglycan synthesis. Cells were cultured for 5 more days. For
analyzing the influence of CD-RAP on proteoglycan synthesis the
medium was changed to 1% FCS and BMP-2 (100 ng/ml) alone or in
combination with CD-RAP (10 ng/ml) was added to the culture. After
5 days proteoglycan synthesis was measured in the cell culture
supernatant using the dimethylmethylene blue (DMMB) calorimetric
assay as described in example 7. The results of GAG stimulation of
three independent experiments are summarized in FIG. 3. These data
indicate that the addition of CD-RAP to BMP-2 stimulated IVD cells
led to an increase of GAG in cultured rabbit IVD cells.
[0109] All references disclosed herein are specifically
incorporated by reference thereto in their entireties.
[0110] While preferred embodiments have been illustrated and
described, it should be understood that modifications can be made
in accordance with ordinary skill in the art without departing from
the invention in its broader aspect as defined by the claims.
REFERENCE LIST
[0111] Aota, Y., An, H. S., Homandberg, G., Thonar, E. J.,
Andersson, G. B., Pichika, R., and Masuda, K. (2005). Differential
effects of fibronectin fragment on proteoglycan metabolism by
intervertebral disc cells: a comparison with articular
chondrocytes. Spine 30, 722-728. [0112] Bauer, R., Humphries, M.,
Fassier, R., Winklmeieri, A., Craig, S. E., and Bosserhoff, A. K.
(2006). Regulation of integrin activity by MIA. J Biol Chem 281,
11669-11677. [0113] Bosserhoff, A. K. and Buettner, R. (2003).
Establishing the protein MIA (melanoma inhibitory activity) as a
marker for chondrocyte differentiation. Biomaterials 24, 3229-3234.
[0114] Bosserhoff, A. K., Kondo, S., Moser, M., Dietz, U. H.,
Copeland, N. G., Gilbert, D. J., Jenkins, N. A., Buettner, R., and
Sandell, L. J. (1997). Mouse CD-RAP/MIA gene: structure,
chromosomal localization, and expression in cartilage and
chondrosarcoma. Dev. Dyn. 208, 516-525. [0115] Bosserhoff, A. K.,
Moser, M., and Buettner, R. (2004). Characterization and expression
pattern of the novel MIA homolog TANGO. Gene Expr. Patterns. 4,
473-479. [0116] Dietz, U. H. and Sandell, L. J. (1996). Cloning of
a retinoic acid-sensitive mRNA expressed in cartilage and during
chondrogenesis. J. Biol. Chem. 271, 3311-3316. [0117] Farndale, R.
W., Sayers, C. A., and Barrett, A. J. (1982). A direct
spectrophotometric microassay for sulfated glycosaminoglycans in
cartilage cultures. Connect. Tissue Res. 9, 247-248. [0118]
Homandberg, G. A. and Hui, F. (1996). Association of proteoglycan
degradation with catabolic cytokine and stromelysin release from
cartilage cultured with fibronectin fragments. Arch. Biochem.
Biophys. 334, 325-331. [0119] Homandberg, G. A., Hui, F., Wen, C.,
Purple, C., Bewsey, K., Koepp, H., Huch, K., and Harris, A. (1997).
Fibronectin-fragment-induced cartilage chondrolysis is associated
with release of catabolic cytokines. Biochem. J 321 (Pt 3),
751-757. [0120] Honda, M. J., Yada, T., Ueda, M., and Kimata, K.
(2004). Cartilage formation by serial passaged cultured
chondrocytes in a new scaffold: hybrid 75:25
poly(L-lactide-epsilon-caprolactone) sponge. J Oral Maxillofac Surg
62, 1510-1516. [0121] Karageorgiou, V. and Kaplan, D. (2005).
Porosity of 3D biomaterial scaffolds and osteogenesis. Biomaterials
26, 5474-5491. [0122] Kawakami, M., Matsumoto, T., Hashizume, H.,
Kuribayashi, K., Chubinskaya, S., and Yoshida, M. (2005).
Osteogenic protein-1 (osteogenic protein-1/bone morphogenetic
protein-7) inhibits degeneration and pain-related behavior induced
by chronically compressed nucleus pulposus in the rat. Spine 30,
1933-1939. [0123] Kelley, L. A., MacCallum, R. M., and Sternberg,
M. J. (2000). Enhanced genome annotation using structural profiles
in the program 3D-PSSM. J Mol. Biol 299, 499-520. [0124] Levicoff,
E. A., Gilbertson, L. G., and Kang, J. D. (2005). Gene therapy for
disc repair. Spine J 5, 287S-296S. [0125] Lougheed, J. C., Holton,
J. M., Alber, T., Bazan, J. F., and Handel, T. M. (2001). Structure
of melanoma inhibitory activity protein, a member of a recently
identified family of secreted proteins. Proc. Natl. Acad. Sci.
U.S.A 98, 5515-5520. [0126] Masuda, K. and An, H. S. (2004). Growth
factors and the intervertebral disc. Spine J 4, 330S-340S. [0127]
Masuda, K., Oegema, T. R., Jr., and An, H. S. (2004). Growth
factors and treatment of intervertebral disc degeneration. Spine
29, 2757-2769. [0128] Meyenburg, S., Lilie, H., Panzner, S., and
Rudolph, R. (2000). Fibrin encapsulated liposomes as protein
delivery system. Studies on the in vitro release behavior. J
Control Release 69, 159-168. [0129] Paul, R., Haydon, R. C., Cheng,
H., Ishikawa, A., Nenadovich, N., Jiang, W., Zhou, L., Breyer, B.,
Feng, T., Gupta, P., He, T. C., and Phillips, F. M. (2003).
Potential use of Sox9 gene therapy for intervertebral degenerative
disc disease. Spine 28, 755-763. [0130] Roberts, M. J., Bentley, M.
D., and Harris, J. M. (2002). Chemistry for peptide and protein
PEGylation. Adv. Drug Deliv. Rev 54, 459-476. [0131] Saito, S.,
Kondo, S., Mishima, S., Ishiguro, N., Hasegawa, Y., Sandell, L. J.,
and Iwata, H. (2002). Analysis of cartilage-derived
retinoic-acid-sensitive protein (CD-RAP) in synovial fluid from
patients with osteoarthritis and rheumatoid arthritis. J Bone Joint
Surg Br 84, 1066-1069. [0132] Sakai, D., Mochida, J., Iwashina, T.,
Watanabe, T., Nakai, T., Ando, K., and Hofta, T. (2005).
Differentiation of mesenchymal stem cells transplanted to a rabbit
degenerative disc model: potential and limitations for stem cell
therapy in disc regeneration. Spine 30, 2379-2387. [0133] Sakano,
S., Zhu, Y., and Sandell, L. J. (1999). Cartilage-derived retinoic
acid-sensitive protein and type II collagen expression during
fracture healing are potential targets for Sox9 regulation. J. Bone
Miner. Res. 14, 1891-1901. [0134] Singh, K., Masuda, K., and An, H.
S. (2005). Animal models for human disc degeneration. Spine J 5,
267S-279S. [0135] Sobajima, S., Kim, J. S., Gilbertson, L. G., and
Kang, J. D. (2004). Gene therapy for degenerative disc disease.
Gene Ther. 11, 390-401. [0136] Sobajima, S., Kompel, J. F., Kim, J.
S., Wallach, C. J., Robertson, D. D., Vogt, M. T., Kang, J. D., and
Gilbertson, L. G. (2005). A slowly progressive and reproducible
animal model of intervertebral disc degeneration characterized by
MRI, X-ray, and histology. Spine 30, 15-24. [0137] Sofia, S.,
McCarthy, M. B., Gronowicz, G., and Kaplan, D. L. (2001).
Functionalized silk-based biomaterials for bone formation. J Biomed
Mater Res 54, 139-148. [0138] Stoll, R., Renner, C., Buettner, R.,
Voelter, W., Bosserhoff, A. K., and Holak, T. A. (2003). Backbone
dynamics of the human MIA protein studied by (15) N NMR relaxation:
implications for extended interactions of SH3 domains. Protein Sci.
12, 510-519. [0139] Stoll, R., Renner, C., Zweckstetter, M.,
Bruggert, M., Ambrosius, D., Palme, S., Engh, R. A., Golob, M.,
Breibach, I., Buettner, R., Voelter, W., Holak, T. A., and
Bosserhoff, A. K. (2001a). The extracellular human melanoma
inhibitory activity (MIA) protein adopts an SH3 domain-like fold.
EMBO J. 20, 340-349. [0140] Stoll, R., Renner, C., Zweckstetter,
M., Bruggert, M., Ambrosius, D., Palme, S., Engh, R. A., Golob, M.,
Breibach, I., Buettner, R., Voelter, W., Holak, T. A., and
Bosserhoff, A. K. (2001 b). The extracellular human melanoma
inhibitory activity (MIA) protein adopts an SH3 domain-like fold.
Embo J 20, 340-349. [0141] Takegami, K., An, H. S., Kumano, F.,
Chiba, K., Thonar, E. J., Singh, K., and Masuda, K. (2005).
Osteogenic protein-1 is most effective in stimulating nucleus
pulposus and annulus fibrosus cells to repair their matrix after
chondroitinase ABC-induced in vitro chemonucteolysis. Spine J 5,
231-238. [0142] Tscheudschilsuren, G., Bosserhoff, A. K., Schlegel,
J., Vollmer, D., Anton, A., Schnettler, R., Brandt, J., and
Proetzel, G. (2005). Regulation of mesenchymal stem cell and
chondrocyte differentiation by MIA. Experimental Cell Research
1-10. [0143] Wells, D. J. (2004). Gene therapy progress and
prospects: electroporation and other physical methods. Gene Ther.
11, 1363-1369. [0144] Wozney, J. M. and Rosen, V. (1998). Bone
morphogenetic protein and bone morphogenetic protein gene family in
bone formation and repair. Clin Orthop 346, 26-37. [0145] Yang, S.
H., Chen, P. Q., Chen, Y. F., and Lin, F. H. (2005). An in-vitro
study on regeneration of human nucleus pulposus by using
gelatin/chondroitin-6-sulfate/hyaluronan tri-copolymer scaffold.
Artif. Organs 29, 806-814.
Sequence CWU 1
1
81107PRTUnknownDescription of Unknown Unknown mature CD-RAP
polypeptide 1Gly Pro Met Pro Lys Leu Ala Asp Arg Lys Leu Cys Ala
Asp Gln Glu1 5 10 15Cys Ser His Pro Ile Ser Met Ala Val Ala Leu Gln
Asp Tyr Met Ala 20 25 30Pro Asp Cys Arg Phe Leu Thr Ile His Arg Gly
Gln Val Val Tyr Val 35 40 45Phe Ser Lys Leu Lys Gly Arg Gly Arg Leu
Phe Trp Gly Gly Ser Val 50 55 60Gln Gly Asp Tyr Tyr Gly Asp Leu Ala
Ala Arg Leu Gly Tyr Phe Pro65 70 75 80Ser Ser Ile Val Arg Glu Asp
Gln Thr Leu Lys Pro Gly Lys Val Asp 85 90 95Val Lys Thr Asp Lys Trp
Asp Phe Tyr Cys Gln 100 105298PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 2Cys Xaa Xaa Xaa Xaa Cys
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa1 5 10 15Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25 30Xaa Xaa Xaa Xaa
Val Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 35 40 45Xaa Xaa Trp
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 50 55 60Xaa Xaa
Xaa Xaa Xaa Phe Xaa Xaa Xaa Xaa Val Xaa Xaa Xaa Xaa Xaa65 70 75
80Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
85 90 95Cys Xaa3100PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 3Lys Xaa Cys Xaa Asp Xaa Glu Cys Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa1 5 10 15Xaa Xaa Xaa Asp Xaa Xaa Xaa Pro
Asp Cys Xaa Xaa Xaa Xaa Xaa Xaa 20 25 30Xaa Xaa Xaa Xaa Xaa Xaa Val
Xaa Xaa Lys Leu Xaa Xaa Xaa Xaa Xaa 35 40 45Xaa Xaa Xaa Xaa Trp Xaa
Gly Ser Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 50 55 60Xaa Xaa Xaa Xaa Xaa
Gly Tyr Phe Pro Xaa Xaa Xaa Val Xaa Xaa Xaa65 70 75 80Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Asp 85 90 95Phe Xaa
Cys Xaa 100498PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 4Lys Xaa Cys Xaa Asp Xaa Xaa Cys Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa1 5 10 15Ala Xaa Xaa Asp Xaa Xaa Xaa Pro
Asp Cys Arg Phe Xaa Xaa Xaa Xaa 20 25 30Xaa Gly Xaa Val Xaa Xaa Xaa
Xaa Xaa Lys Leu Xaa Xaa Xaa Xaa Xaa 35 40 45Xaa Xaa Trp Xaa Gly Ser
Val Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 50 55 60Xaa Xaa Xaa Gly Tyr
Phe Pro Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa65 70 75 80Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Asp Phe Xaa 85 90 95Cys
Gln521DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 5ggaaatcgtc cgtgacatca a 21620DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
6aaggaaggct ggaagagagc 20719DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 7aagagagcca aacagccga
19820DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 8ctggtagtcc tgggcattgt 20
* * * * *