Title: Methods for making and
delivering Rho-antagonist tissue adhesive formulations to the injured
mammalian central and peripheral nervous systems and uses thereof
United States Patent: 7,491,692
Issued: February 17, 2009
McKerracher; Lisa (Ile des Soeurs, Quebec, CA)
Filed: November 24, 2003
Pharm Bus Intell
& Healthcare Studies
The present invention provides methods
for making, delivering and using formulations that combine a
therapeutically active agent(s) (such as for example a Rho antagonist(s))
and a flowable carrier component capable of forming a therapeutically
acceptable matrix in vivo (such as for example tissue adhesives), to
injured nerves to promote repair and regeneration and regrowth of injured
(mammalian) neuronal cells, e.g. for facilitating axon growth at a desired
lesion site. Preferred active agents are known Rho antagonists such as for
example C3, chimeric C3 proteins, etc. or substances selected from among
known trans-4-amino(alkyl)-1-pyridylcarbamoylcyclohexane compounds or Rho
kinase inhibitors. The system for example may deliver an antagonist(s) in
a tissue adhesive such as, for example, a fibrin glue or a collagen gel to
create a delivery matrix in situ. A kit and methods of stimulating
neuronal regeneration are also included.
Description of the
SUMMARY OF THE INVENTION
As discussed herein in accordance with the present invention a
therapeutically active agent for facilitating axon growth may be delivered
(in a flowable matrix forming substance) to a (nerve) lesion site, for
example, by injection using known syringe type glue or sealant devices
modified as necessary or desired (e.g. by addition of a further substance
container); examples of known delivery devices, systems, mechanisms, matrix
forming compositions, and the like are shown for example in U.S. Pat. No.
5,989,215, U.S. Pat. No. 4,978,336, U.S. Pat. No. 4,631,055, U.S. Pat. No.
4,359,049, U.S. Pat. No. 4,974,368, U.S. Pat. No. 6,121,422, U.S. Pat. No.
6,047,861, U.S. Pat. No. 6,036,955, U.S. Pat. No. 5,945,115, U.S. Pat. No.
5,900,408 , U.S. Pat. No. 6,124,273, U.S. Pat. No. 5,922,356, and in
particular U.S. Pat. No. 6,117,425; the entire contents of each of these
patents is incorporated herein by reference.
A sufficient amount of a therapeutically active agent for facilitating axon
growth may be dispersed in a stable flowable (known) type of (proteinaceous)
matrix forming material. Once delivered to the desired lesion site the
resulting in situ or in vivo matrix (e.g. gel or crosslinked substances)
inhibits the migration or diffusion of the agent from the site of injection,
so as to maintain the primary effect of the agent in the region of
injection, i.e. in the area of the lesion. In any event the active agent is
to be present in an amount effective to facilitate axon growth.
A substantially uniform dispersion of the active agent may be initially be
formed so as to provide a concentrated amount of active agent in a
physiologically acceptable matrix forming material. The matrix forming
material may be comprised of any (known) individual or combination of
peptides, proteins etc. which provides for stable placement, or combinations
thereof. Of particular interest is a collagen material, a fibrinogen
material, or derivatives thereof; other high molecular weight
physiologically acceptable biodegradable protein matrix forming materials
may if desired be used. The active agent may, for example, be incorporated
in a sufficient concentration so as to provide the desired or effect the
desired sustained release.
Typically when estimating doses in different animal species, the same weight
ratio is used. It is for example possible to apply 40 ug protein per 20 gm
mouse. Therefore, we anticipate that the ideal dose should be approximately
3 gm per 60 kg person. We expect that the dose necessary will depend on the
size of the lesion and the time of application (acute or chronic) spinal
cord injury. In cases of chronic injury, there is often a necrotic center in
the spinal cord, and higher doses may be required.
The matrix forming material may be a one-component adhesive or sealant type
material (e.g. collagen material); alternatively it may be a multi-component
adhesive or sealant (e.g. a fibrinogen based material). The matrix may be a
human protein matrix or if necessary or desired a non-human protein matrix;
preferably a human protein matrix.
The (proteinaceous) matrix forming material is flowable for injection, but
once in vivo it provides for stable placement, of the active agent in the
lesion area; i.e. after injection, the active agent is released into the
immediate environment the matrix providing a medium for prolonged contact
between a lesion site and the active agent (i.e. axon growth facilitator or
The matrix forming material(s) is (are) of course to be chosen on the basis
that the materials and resultant formed matrix will be capable on the one
hand of holding the active agent for release in situ and on the other
without preventing the therapeutic effect thereof, i.e. the matrix is to be
therapeutically acceptable. The choice of active agent may be determined
empirically through appropriate or suitable assays keeping in mind that the
matrix etc. are to be therapeutically acceptable.
The present invention in an aspect relates to a biocompatible, (supplemented
tissue sealant or adhesive) composition comprising: (i) at least one
supplement selected from the group consisting of therapeutically active
agents for facilitating axon growth; and (ii) a flowable carrier component
capable of forming a pharmaceutically or therapeutically acceptable matrix
(in vivo)--i.e. a nerve lesion site; wherein said supplement is releasable
from said matrix into the adjacent external environment (e.g. for a
sustained period of time).
The present invention in another aspect relates a method for the preparation
of a flowable biocompatible composition comprising admixing (i) at least one
supplement selected from the group consisting of therapeutically active
agents for facilitating axon growth and (ii) a flowable carrier component
capable of forming a therapeutically acceptable matrix in vivo at a nerve
lesion site; wherein said supplement is releasable from said matrix into the
adjacent external environment.
By way of example only in accordance with the present invention a method of
applying an supplemented solution of polymerizable fibrin to a desired
lesion site, may comprise a) affixing a cartridge containing immobilized
thrombin to a syringe containing a solution of fibrinogen, b) contacting the
solution of fibrinogen with immobilized thrombin under conditions resulting
in an activated solution of polymerizable fibrin by passing the solution of
fibrinogen through the cartridge containing immobilized thrombin, c) adding
to the fibrinogen solution or to the activated solution a supplement (i) at
least one supplement selected from the group consisting of therapeutically
active agents for facilitating axon growth; and c) delivering the
supplemented activated solution of polymerizable fibrin to the desired
lesion site (e.g. a central nervous system (CNS) lesion site or a peripheral
nervous system (PNS) lesion site) under conditions which result in
polymerized fibrin at the lesion site having dispersed therein the
supplement wherein said supplement is released from said fibrin matrix into
the adjacent external environment.
In accordance with another aspect the present invention relates to a kit
comprising, in suitable container means (e.g. separate means): (a) a first
pharmaceutical composition or substance comprising a biological agent
capable of facilitating axon growth; and (b) a second pharmaceutically or
therapeutically acceptable component comprising a single flowable carrier
component or two or more separate components capable once intermingled of
forming a flowable carrier component, said flowable carrier components each
being capable of forming a pharmaceutically or therapeutically acceptable
matrix (e.g. proteinaceous matrix, i.e. a proteinaceous glue, proteinaceous
sealant, proteinaceous gel, etc.; e.g. a human derived proteinaceous matrix)
in vivo at a (nerve) lesion site.
In particular the present invention provides a (axon growth stimulation) kit
comprising a) a first container means (e.g. one or more separate containers)
for containing a flowable carrier component(s) or two or more separate
components capable once intermingled of forming a flowable carrier
component, said flowable carrier components each being capable of forming a
pharmaceutically or therapeutically acceptable matrix (e.g. proteinaceous
matrix, i.e. a proteinaceous glue, proteinaceous sealant, proteinaceous gel,
etc.; i.e. a human derived proteinaceous matrix) in vivo at a (nerve) lesion
site (e.g. a central nervous system (CNS) lesion site or a peripheral
nervous system (PNS) lesion site) and b) a second container means for
containing a therapeutically active agent for facilitating axon growth at
the lesion site wherein said therapeutically active agent supplement is
releasable from said in vivo matrix into the adjacent external environment
(e.g. for a sustained period of time). Alternatively, if desired or as
necessary, the first and second container means may be the same, (e.g. a
container may hold collagen and C3). The kit may if desired or necessary
additionally comprise means for dispersing (i.e. co-mingle, blend, etc.) the
therapeutically active agent in said flowable carrier component so as to
form a flowable axon growth stimulation composition as well as means for
delivering the flowable axon growth stimulation composition to the lesion
site (e.g. syringe needle). The pharmaceutically acceptable matrix may as
discussed herein be a collagen matrix or a fibrin matrix.
In accordance with the present invention the therapeutically active agent
for facilitating axon growth may for example be a Rho antagonist which may
be identified by an assay method comprising the following steps: a)
culturing neurons on inhibitory substrate or a substrate that incorporates a
growth-inhibitory protein. b) Exposing the cultured neuron of step a) to a
candidate Rho antagonist in an amount and for a period sufficient to permit
growth of neurites, and determining if the candidate has elicited neurite
growth from the cultured neurons of step a), the appearance of neurites
being suggestive or indicative of a Rho antagonist.
A compound can be confirmed as a Rho antagonist in one of the following
ways: a) Cells are cultured on a growth inhibitory substrate as above, and
exposed to the candidate Rho antagonist; b) Cells of step a) are homogenized
and a pull-down assay is performed. This assay is based on the capability of
GST-Rhotektin to bind to GTP-bound Rho. Recombinant GST-Rhotektin or GST
rhotektin binding domain (GST-RBD) is added to the cell homogenate made from
cells cultured as in a). It has been found that inhibitory substrates
activate Rho, and that this activated Rho is pulled down by(GST-RBD). Rho
antagonists will block activation of Rho, and therefore, an effective Rho
antagonist will block the detection of Rho when cell are cultured as
described by a) above; c) An alternate method for this pull-down assay would
be to use the GTPase activating protein, Rho-GAP as bait in the assay to
pull down activated Rho, as described (Diekmann and Hall, 1995. In Methods
in Enzymology Vol. 256 part B 207-215).
Another method to confirm that a compound is a Rho antagonist is as follows:
When added to living cells antagonists that inactivate Rho by ADP-ribosylation
of the effector domain can be identified by detecting a molecular weight
shift in Rho (Lehmann et al, 1999 IBID). The molecular weight shift can be
detected after treatment of cells with Rho antagonist by homogenizing the
cells, separating the proteins in the cellular homogenate by SDS
polyacrylamide gel electrophoresis. The proteins are transferred to
nitrocellulose paper, then Rho is detected with Rho-specific antibodies by a
Western blotting technique.
Another method to confirm that compound is a Rho-kinase antagonist is as
follows: a) Recombinant Rho kinase tagged with myc epitope tag, or a GST tag
is expressed in HeLa cells or another suitable cell type by transfection. b)
The kinase is purified from cell homogenates by immunoprecipation using
antibodies directed against the myc tag or the GST tag. c) The recovered
immunoprecipitates from b) are incubated with [32P] ATP and histone type 2
as a substrate in the presence or absence of the Rho kinase. In the absence
of Rho kinase activity the Rho kinase antigens is able to block the
phosphorylation activity of Rho kinase (i.e. phosphorylation of hislore),
and as such identified the compound as a Rho kinase antagonist.
The present invention is in particular,concerned with a delivery system and
kit to apply for example, known C3, chimeric C3, or Y-27632 type compounds
(e.g. Y-27632, Y-30141 and the like) or a Rho kinase inhibitor to injured
regions of the CNS that include injured spinal cord or brain, and regions of
the CNS injured by stroke. The nature of C3 is discussed herein; Y-27632 is
for example mentioned above.
In the context of the present invention, the ability of C3 to stimulate
(axon) regeneration in vivo was examined. Thus adult rat optic nerves were
crushed an C3 applied at the same time, directly at the lesion site (Lehmann,
et al. (1999) J. Neurosci. 19:7537-7547). It was found that large numbers of
axons traversed the lesion to grow in the distal optic nerve. In particular
there was for example examined the delivery of C3 to optic nerve through the
use of gelfoam an Elvax, a slow release matrix (Lehmann, et al. (1999) J.
It has also been found that the combination of collagen gels and C3 was able
to allow axons to into the site of the glial scar. Based on experiments with
fibrin glue (see below), it is believed that delivery of C3 in collagen may
be improved by the addition of protease inhibitors to prevent lysis of the
gel and C3.
However, the present invention as mentioned above is directed to the
delivery system of a therapeutically active agent (such as for example a Rho
antagonist--C3, Y-27632, etc.) in a protein matrix that holds the active
agent (e.g. Rho antagonist) at the site of application. This delivery system
retains the active agent (e.g. Rho antagonist) at the site of CNS injury,
allows large doses to be given at the site of injury, and prevents large
amounts of the active agent (e.g. Rho antagonist) from leaking into the
systemic circulation. The protein matrix can either be based on the fibrin,
a protein of the coagulation pathway, or it can be based on collagen, a
protein of the extracellular matrix. Both proteins when applied under
specific conditions form protein networks when polymerized. These proteins
can be applied in soluble form with the additional components necessary for
polymerization, together with the Rho antagonist. When the components are
mixed immediately before use, polymerization occurs after application to the
body site, in our case after application to the CNS.
The present invention as mentioned above in particular relates to a kit
suitable for use in the above-described method of delivering fibrin sealant
components to a wound site. The kit comprises individually packaged
component solutions provided in separate bottles to prevent mixing before
use, and an applicator designed so as to permit mixing of the
fibrinogen/Factor XIII and thrombin with C3 at the body site. The kit
provides pre-measured amounts of the fibrinogen and factor XIII in one
bottle, the thrombin in another bottle, a C3 solution in another bottle. The
contents of the bottles would be mixed in a prescribed order, as detailed in
the example below. The kit can also include one or more other storage
containers which are any necessary reagents including solvents, buffers,
calcium chloride, protease inhibitors etc. The kit could be sold as
lyophilized or frozen components to preserve the activity of C3 or other Rho
antagonist added to the kit.
Rho antagonist delivery system may be used in conjunction cell
transplantation. Many different cell transplants have been extensively
tested for their potential to promote regeneration and repair. These
include, but are not restricted to, Schwann cells (Xu, et al. (1996) Exp.
Neurol. 134:261-272, Guest (1997) Exp. Neurol. 148:502-522, Tuszynski, et
al. (1998) Cell Transplant. 7:187-96), fibroblasts modified to express
trophic factors (Liu, et al. (1999) J Neurosci. 19:4370-87, Blesch, et al.
(1999) J Neurosci. 19:3556-66, Tuszynski, et al. (1994) Exp Neurol.
126:1-14, Nakahara, et al. (1996) Cell Transplant. 5:191-204), fetal spinal
cord transplants (Diener and Bregman (1998) J. Neurosci. 18:779-793, Bregman
(1993) Exp. Neurol. 123:2-16), macrophages (Lazarov-Spiegler, et al. (1996)
FASEB. J. 110:1296-1302), embryonic stem cells (McDonald, et al. (1999) Nat
Med. 5:1410-2), and olfactory ensheathing glia (Li, et al. (1997) Science.
277:2000-2002, Ramon-Cueto, et al. (1998) J Neurosci. 18:3803-15, Ramon-Cueto,
et al. (2000) Neuron. 25:425-35).
Rho GTPases include members of the Rho, Rac and Cdc42 family of proteins.
Our invention concerns Rho family members of the Rho class. Rho proteins
consist of different variants encoded by different genes. For example, PC12
cells express RhoA, RhoB and RhoC (Lehmann et al 1999 IBID). To inactivate
Rho proteins inside cells, Rho antagonists of the C3 family type are
effective because they inactivate all forms of Rho (e.g. RhoA, Rho B etc.).
In contrast, gene therapy techniques, such as introduction of a dominant
negative RhoA family member into a diseased cell, will only inactivate that
specific RhoA family member.
Compounds of the C3 family from Clostridium botulinum inactivate Rho by ADP-ribosylation.
Recombinant C3 proteins, or C3 proteins that retain the ribosylation
activity are also effective in our delivery system and are covered by this
invention. In addition, Rho kinase is a well-known target for active Rho,
and inactivating Rho kinase has the same effect as inactivating Rho, at
least in terms of neurite or axon growth (Kimura and Schubert (1992) Journal
of Cell Biology. 116:777-783, Keino-Masu, et al. (1996) Cell. 87:175-185,
Matsui, et al. (1996) EMBO J. 15:2208-2216, Matsui, et al. (1998) J. Cell
Biol. 140:647-657, Ishizaki (1997) FEBS Lett. 404:118-124), the biological
activity that concerns this invention. Therefore, chemical compounds such as
Y-27632, any other compound are covered by this invention as a preferred
delivery in a tissue adhesive system. Numerous references describing C3 type
compounds can be found in Methods in Enzymology, Vol. 256, Part B, Eds.: W.
E. Balch, C. H. Der, and A. Hall; Academic Press, 1995, for e.g. Pgs.
196-206, 207 et seq, 184-189, and 174 et seq. In any event C3 may for
example be selected from the group consisting of ADP-ribosyl transferase
derived from Clostridium botulinum and a recombinant ADP-ribosyl transferase.
On the other hand any compound or molecule that does not have a direct
action on Rho itself but works to decrease the function of Rho such as
anti-sense oligos to Rho, anti-Rho kinase antibodies, and the like. Such Rho
antagonists that can be delivered in a tissue adhesive system are also
covered by our invention. The C3 polypeptides of the present invention
include biologically active fragments and analogs of C3; fragments encompass
amino acid sequences having truncations of one or more amino acids, wherein
the truncation may originate from the amino terminus, carboxy terminus, or
from the interior of the protein. Analogs of the invention involve an
insertion or a substitution of one or more amino acids. Fragments and
analogs will have the biological property of C3 that is capable of
inactivation Rho GTPases. Also encompassed by the invention are chimeric
polypeptides comprising C3 amino acid sequences fused to heterologous amino
acid sequences. Said heterologous sequences encompass those which, when
formed into a chimera with C3 retain one or more biological or immunological
properties of C3. A host cell transformed or transfected with nucleic acids
encoding C3 protein or c3 chimeric protein are also encompassed by the
invention. Any host cell which produces a polypeptide having at least one of
the biological properties of a C3 may be used. Specific examples include
bacterial, yeast, plant, insect or mammalian cells. In addition, C3 protein
may be produced in transgenic animals. Transformed or transfected host cells
and transgenic animals are obtained using materials and methods that are
routinely available to one skilled in the art. Host cells may contain
nucleic acid sequences having the full-length gene for C3 protein including
a leader sequence and a C-terminal membrane anchor sequence (see below) or,
alternatively, may contain nucleic acid sequences lacking one or both of the
leader sequence and the C-terminal membrane anchor sequence. In addition,
nucleic acid fragments, variants and analogs which encode a polypeptide
capable of retaining the biological activity of C3 may also be resident in
host expression systems.
The Rho antagonist that is a recombinant proteins can be made according to
methods present in the art. The proteins of the present invention may be
prepared from bacterial cell extracts, or through the use of recombinant
techniques. In general, C3 proteins according to the invention can be
produced by transformation (transfection, transduction, or infection) of a
host cell with all or part of a C3-encoding DNA fragment in a suitable
expression vehicle. Suitable expression vehicles include: plasmids, viral
particles, and phage. For insect cells, baculovirus expression vectors are
suitable. The entire expression vehicle, or a part thereof, can be
integrated into the host cell genome. In some circumstances, it is desirable
to employ an inducible expression vector.
Those skilled in the field of molecular biology will understand that any of
a wide variety of expression systems can be used to provide the recombinant
protein. The precise host cell used is not critical to the invention. The C3
protein can be produced in a prokaryotic host (e.g., E. coli or B. subtilis)
or in a eukaryotic host (e.g., Saccharomyces or Pichia; mammalian cells,
e.g., COS, NIH 3T3, CHO, BHK, 293, or HeLa cells; or insect cells).
Proteins and polypeptides can also be produced by plant cells. For plant
cells viral expression vectors (e.g., cauliflower mosaic virus and tobacco
mosaic virus) and plasmid expression vectors (e.g., Ti plasmid) are
suitable. Such cells are available from a wide range of sources (e.g., the
American Type Culture Collection, Rockland, Md.). The methods of
transformation or transfection and the choice of expression vehicle will
depend on the host system selected.
The host cells harbouring the expression vehicle can be cultured in
conventional nutrient media adapted as need for activation of a chosen gene,
repression of a chosen gene, selection of transformants, or amplification of
a chosen gene. One expression system is the mouse 3T3 fibroblast host cell
transfected with a pMAMneo expression vector (Clontech, Palo Alto, Calif).
pMAMneo provides an RSV-LTR enhancer linked to a dexamethasone-inducible
MMTV-LTR promoter, an SV40 origin of replication which allows replication in
mammalian systems, a selectable neomycin gene, and SV40 splicing and
polyadenylation sites. DNA encoding a C3 protein would be inserted into the
pMAMneo vector in an orientation designed to allow expression. The
recombinant C3 protein would be isolated as described below. Other
preferable host cells that can be used in conjunction with the pMAMneo
expression vehicle include COS cells and CHO cells (ATCC Accession Nos. CRL
1650 and CCL 61, respectively).
C3 polypeptides can be produced as fusion proteins. For example, expression
vectors can be used to create lacZ fusion proteins. The pGEX vectors can be
used to express foreign polypeptides as fusion proteins with glutathione S-transferase
(GST). In general, such fusion proteins are soluble and can be easily
purified from lysed cells by adsorption to glutathione-agarose beads
followed by elution in the presence of free glutathione. The pGEX vectors
are designed to include thrombin or factor Xa protease cleavage sites so
that the cloned target gene product can be released from the GST moiety.
Another strategy to make fusion proteins is to use the His tag system.
In an insect cell expression system, Autographa californica nuclear
polyhedrosis virus AcNPV), which grows in Spodoptera frugiperda cells, is
used as a vector to express foreign genes. A C3 coding sequence can be
cloned individually into non-essential regions (for example the polyhedrin
gene) of the virus and placed under control of an AcNPV promoter, e.g., the
polyhedrin promoter. Successful insertion of a gene encoding a C3
polypeptide or protein will result in inactivation of the polyhedrin gene
and production of non-occluded recombinant virus (i.e., virus lacking the
proteinaceous coat encoded by the polyhedrin gene). These recombinant
viruses are then used to infect spodoptera frugiperda cells in which the
inserted gene is expressed (see, Lehmann et al for an example of making
recombinant MAG protein).
In mammalian host cells, a number of viral-based expression systems can be
utilised. In cases where an adenovirus is used as an expression vector, the
C3 nucleic acid sequence can be ligated to an adenovirus
transcription/translation control complex, e.g., the late promoter and
tripartite leader sequence. This chimeric gene can then be inserted into the
adenovirus genome by in vitro or in vivo recombination. Insertion into a
non-essential region of the viral genome (e.g., region E1 or E3) will result
in a recombinant virus that is viable and capable of expressing a C3 gene
product in infected hosts.
Specific initiation signals may also be required for efficient translation
of inserted nucleic acid sequences. These signals include the ATG initiation
codon and adjacent sequences. In cases where an entire native C3 gene or
cDNA, including its own initiation codon and adjacent sequences, is inserted
into the appropriate expression vector, no additional translational control
signals may be needed. In other cases, exogenous translational control
signals, including, perhaps, the ATG initiation codon, must be provided.
Furthermore, the initiation codon must be in phase with the reading frame of
the desired coding sequence to ensure translation of the entire insert.
These exogenous translational control signals and initiation codons can be
of a variety of origins, both natural and synthetic. The efficiency of
expression may be enhanced by the inclusion of appropriate transcription
enhancer elements, transcription terminators.
In addition, a host cell may be chosen which modulates the expression of the
inserted sequences, or modifies and processes the gene product in a
specific, desired fashion. Such modifications (e.g., glycosylation) and
processing (e.g., cleavage) of protein products may be important for the
function of the protein. Different host cells have characteristic and
specific mechanisms for the post-translational processing and modification
of proteins and gene products. Appropriate cell lines or host systems can be
chosen to ensure the correct modification and processing of the foreign
protein expressed. To this end, eukaryotic host cells that possess the
cellular machinery for proper processing of the primary transcript,
glycosylation, and phosphorylation of the gene product can be used. Such
mammalian host cells include, but are not limited to, CHO, VERO, BHK, HeLa,
COS, MDCK, 293, 3T3, W138, and in particular, choroid plexus cell lines.
Alternatively, a C3 protein can be produced by a stably-transfected
mammalian cell line. A number of vectors suitable for stable transfection of
mammalian cells are available to the public; methods for constructing such
cell lines are also publicly available. In one example, cDNA encoding the C3
protein can be cloned into an expression vector that includes the
dihydrofolate reductase (DHFR) gene. Integration of the plasmid and,
therefore, the C3 protein-encoding gene into the host cell chromosome is
selected for by including 0.01-300 .mu.M methotrexate in the cell culture
medium (as described in Ausubel et al., supra). This dominant selection can
be accomplished in most cell types.
Recombinant protein expression can be increased by DHFR-mediated
amplification of the transfected gene. Methods for selecting cell lines
bearing gene amplifications are known in the art; such methods generally
involve extended culture in medium containing gradually increasing levels of
methotrexate. DHFR-containing expression vectors commonly used for this
purpose include pCVSEII-DHFR and pAdD26SV(A). Any of the host cells
described above or, preferably, a DHFR-deficient CHO cell line (e.g., CHO
DHFR cells, ATCC Accession No. CRL 9096) are among the host cells preferred
for DHFR selection of a stably-transfected cell line or DHFR-mediated gene
A number of other selection systems can be used, including but not limited
to the herpes simplex virus thymidine kinase, hypoxanthine-guanine
phosphoribosyltransferase, and adenine phosphoribosyltransferase genes can
be employed in tk, hgprt, or aprt cells, respectively. In addition, gpt,
which confers resistance to mycophenolic acid; neo, which confers resistance
to the aminoglycoside G-418; and hygro, which confers resistance to
hygromycin can be used.
Alternatively, any fusion protein can be readily purified by utilising an
antibody specific for the fusion protein being expressed. For example, a
system described in Janknecht et al. (1981) Proc. Natl. Acad. Sci. USA 88,
8972, allows for the ready purification of non-denatured fusion proteins
expressed in human cell lines. In this system, the gene of interest is
subcloned into a vaccinia recombination plasmid such that the gene's open
reading frame is translationally fused to an amino-terminal tag consisting
of six histidine residues. Extracts from cells infected with recombinant
vaccinia virus are loaded onto Ni2+ nitriloacetic acid-agarose columns, and
histidine-tagged proteins are selectively eluted with imidazole-containing
Alternatively, C3 or a portion thereof, can be fused to an immunoglobulin Fc
domain. Such a fusion protein can be readily purified using a protein A
It is envisioned that small molecule mimetics of the above described
antagonists are also encompassed by the invention.
In the following a method to identify active Rho antagonists will be
To test Rho antagonists for activity, a tissue culture bioassay system was
used. This bioassay is used to define activity of Rho antagonists that will
be effective in promoting axon regeneration in spinal cord injury, stroke or
Neurons do not grow neurites on inhibitory myelin substrates. When neurons
are placed on inhibitory substrates in tissue culture, they remain rounded.
When an effective Rho antagonist is added, the neurons are able to grow
neurites on myelin substrates. The time that it takes for neurons to growth
neurites upon the addition of a Rho antagonist is the same as if neurons had
been plated on growth permissive substrate such as laminin or polylysine,
typically 1 to 2 days in cell culture. The results can be scored visually.
If needed, a quantitative assessment of neurite growth can be performed.
This involved measuring the neurite length in a) control cultures where
neurons are plated on myelin substrates and left untreated b) in positive
control cultures, such as neurons plated on polylysine c) or treating
cultures with different concentrations of the test antagonist.
To test C3 in tissue culture, it has been found that the best concentration
is 25-50 ug/ml. Thus, high concentrations of this Rho antagonist are needed
as compared to the growth factors used to stimulate neurite outgrowth.
Growth factors, such as nerve growth factor (NGF) are used at concentrations
of 1-100 ng/ml in tissue culture. However, growth factors are not able to
overcome growth inhibition by myelin. Our tissue culture experiments are all
performed in the presence of the growth factor BDNF for retinal ganglion
cells, or NGF for PC12 cells. When growth factors have been tested in vivo,
typically the highest concentrations possible are used, in the ug/ml range.
Also they are often added to the CNS with the use of pumps for prolonged
delivery (e.g. Ramer et al, IBID). For in vivo experiments the highest
concentrations possible was used when working with C3 stored as a frozen 1
mg/ml solution. The concentration that was chosen does not prevent the
fibrin matrix from polymerizing.
For test purposes it was decided to dilute a 1 mg/ml solution of C3 with 1/3
volume thrombin and 1/3 volume fibrinogen solutions (contain calcium and
aprotinin). In order to increase the concentration of C3, it would be
possible to lyophylize C3 and then resuspend it in the fibrinogen solution.
Lyophilized C3 has been tested and found to be active. The Rho antagonist C3
is stable at 37 C for at least 24 hours. The stability of C3 was tested in
tissue culture with the following experiment. The C3 was diluted in tissue
culture medium, left in the incubator at 37 C for 24 hours, then added to
the bioassay system described above, using retinal ganglion cells as the
test cell type. These cells were able to extend neurites on inhibitory
substrates when treated with C3 stored for 24 hours at 37 C. Therefore, the
minimum stability is 24 hours. This is in keeping with the stability
projections based on amino acid composition (see sequence data, below).
In the following various tissue Adhesives and Formulations used to make them
will be discussed.
Different types of tissue adhesive can be made. Examples include collagen
gels, fibrin tissue adhesives. Other examples are matrigel, laminin
networks, and adhesives based on a composition of basement membrane proteins
that contain collagen.
Fibrin sealant has three basic components: fibrinogen concentrate, calcium
chloride and thrombin. Other components can be added to affect the time of
clot formation, and the size of the protein network that is formed.
Generally when the components mix, a fibrin coagulum is formed in that the
fibrinogen molecule is cleaved through the action of thrombin to form fibrin
monomers which spontaneously will polymerize to form a three-dimensional
network of fibrin, largely kept together by hydrogen bonding. This
corresponds to the last phase of the natural blood clotting cascade, the
coagulation rate being dependent on the concentration of thrombin used. In
order to improve the tensile strength, covalent crosslinking between the
fibrin chains is provided for by including Factor II in the sealant
composition. In the presence of calcium ions, thrombin activates factor XIII
to factor XIIIa. Activated factor XIIIa together with thrombin catalyzes the
cross-linkage of fibrin and increases the strength of the clot. The strength
of the fibrin clot is further improved by the addition of fibronectin to the
composition, the fibronectin being crosslinked and bound to the fibrin
network formed. During wound healing the clot material undergoes gradual
lysis and is completely absorbed. To prevent a too early degradation of the
fibrin clot by fibrinolys, the fibrin sealant composition may comprise a
plasminogen activator inhibitor or a plasmin inhibitor, such as aprotinin.
Such an inhibitor will also reduce the fibrinolytic activity resulting from
any residual plasminogen in the fibrinogen composition. Similarly,
compositions may include hyaluronic acid (or other polysaccharides), and
these may also comprise a hyaluronidase inhibitor such as one or more
flavonoids (or corresponding inhibitors for other polysaccharides) in order
to prevent degradation (i.e. to prolong the duration) of the hyaluronic acid
component by hyaluronidase which is always present in the surrounding
tissues. The hyaluronic acid may, as mentioned above, be crosslinked, a
commercially available example being Hylan..RTM..TM.(trademark, available
from Biomatrix, Ritchfield, N.Y., USA). The hyaluronic acid compositions may
e.g. have the form of gels, solutions, etc.
Fibrin clots in any one of the above described embodiments, may be used for
the application of a pharmaceutically active substance. By incorporating a
drug, such as an antibiotic, a growth factor, etc. into the tissue adhesive
it will be enclosed in the fibrin network formed upon application of the
tissue adhesive. It will thereby be ensured that the drug is kept at the
site of application while being controllably released from the composition.
Fibrin sealant products prepared from human plasma fibrinogen/Factor XIII
are available commercially. One product is a tissue glue called Tisseel
Fibrin Sealant (Baxter Hyland Immuno Corporation). (Tissucol/Tisseel, Immuno
AG, Vienna) and another Beriplast P, Hoechst, West Germany. A frozen
formulation of a fibrin glue delivered with a 2 syringe system is Hemaseel
made by Hemacure Inc. (Kirkland, Quebec).
In the following methods for making Tissue Adhesive Delivery kits will be
In a preferred embodiment, the kit includes the solutions provided in
separate bottles to prevent mixing before use, and an applicator designed so
as to permit mixing of the fibrinogen/Factor XIII and thrombin with C3 at
the body site. The kit would provide pre-measured amounts of the fibrinogen
and factor XIII in one bottle, the thrombin in another bottle, a calcium
chloride solution in third bottle, and a C3 solution in a fourth bottle. The
contents of the bottles would be mixed in a prescribed order, as detailed in
the example below. The kit can also include one or more other storage
containers which are any necessary reagents including solvents, buffers,
etc. The kit could be sold as lyophilized or frozen components to preserve
the activity of C3 or other Rho antagonist added to the kit.
The applicator can, for example, take the form of a glass or plastic syringe
with disposable needles. With a single syringe system, the components of the
kit would be mixed immediately before application to the injury site.
A more elaborate system would allow two syringes to be attached, so that the
mixing could take place in the syringe or a mixing compartment of the
syringe, before injection. One example of a two syringe system is a Luer
lock syringe, such as used for mixing adjuvants. For this a 3-way stopcocks,
such as commercially available (Bio-Rad cat #7328103) is attached to the
syringe so that the solution can be passed back and forth before attaching
the injection needle to the third port of the 3-way stopcock. These are
plastic, sterile, and disposable.
Another method of application could be through the use of a clip to hold two
syringes, and the clip would have a common plunger to ensure that equal
volumes of the thrombin and fibrinogen components are mixed in a chamber
with the calcium chloride and C3, before being ejected trough the needle.
Other Ingredients for the Tissue Adhesive Rho Antagonist Delivery System are
Other components can be added to the tissue adhesive to improve efficacy of
the treatments. Such additions include growth factors, protease inhibitors,
cytokines, anti-inflammatory compounds, cell transplant systems. Agents that
prevent cell death, such as agents that affect the apoptosis pathway could
be added components to the delivery system.
Methods of Packaging Delivery System are discussed hereinafter.
In the preferred formulation, Rho antagonist, fibrinogen and thrombin are
mixed together just before application, so that polymerization of the gel
occurs in the injured CNS. Therefore, it is important that the fibrinogen
and thrombin are package separately. However, the C3 can be packaged
separately, or added to either the thrombin or fibrinogen bottles. In
another formulation, the fibrinogen, thrombin and C3 are packaged together,
but help at low pH, which prevents polymerization of the gel. Polymerization
would be induced by mixing this formulation with a basic component that
would neutralize the pH to induce coagulation of the adhesive. In another
formulation, the Rho antagonist could be added separately to the
fibrinogen/thrombin mix in the form of liposomes or other similar delivery
system. Living cells could that secrete C3 could be added as Rho antagonist.
A Method of Applying Rho Antagonist in Vivo is discussed hereinafter.
Tissue adhesive formulations are typically applied to wound sites with a
syringe and needle. The shape of the need determine the type of surface that
is formed when the adhesive polymerizes. In some cases, adhesives can be
sprayed onto the wound surface, or into the desired region. This invention
covers all types of syringes and needles used to apply fibrin plus Rho
antagonists to injured regions of the CNS. In addition, it covers the
addition of previously polymerized tissue adhesives with C3 to the wound.
For example, fibrin can be polymerized in a teat tube, and forceps used to
remove the gel and place it in the body cavity. Similarly, collagen can be
applied by pre-polymerization and application by using forceps to place the
gel in the injured spinal cord. One example of this is more fully explained
in the example section of this application.
Tests were done with Gelfoam.TM., a surgical collagen-based sponge, and
Elvax, a slow release plastic (Lehmann et al 1999, IBID) for the ability to
deliver biologically effective concentrations of C3. Neither of these two
delivery systems was effective. Therefore, only tissue adhesive formulations
(i.e. the matrix forming formulations discussed herein) have efficacy in the
delivery of C3 to the injured CNS in vivo.
Therapeutic Applications/Medical Uses will be discussed below.
The tissue adhesive system for the delivery of Rho antagonists may be useful
in many other conditions that affect the central and peripheral nervous
system. Treatments that are effective in eliciting sprouting from injured
axons are equally effective in treating some types of stroke (Boston Life
Sciences, Sep. 6, 2000 Press Release). Since it has been determined that it
is possible to elicit sprouting (using a kit of the present invention), it
is obvious that the treatments can be extended to stroke. Similarly,
although the subject of this invention is related to delivery of Rho
antagonists to the traumatically damaged nervous system, this invention also
pertains to damage from neurodegeneration, such as during Parkinson's
disease, Alzheimer's disease, prion diseases or other diseases of the CNS
were axons are damaged in the CNS environment. In such cases, small volumes
of the tissue adhesive with C3 could be injected into the affected region
with the use of a syringe. The treatment will cause local sprouting to
restore function of neurons whose axon processes had retracted in the course
of the neurodegeneration.
Claim 1 of 12 Claims
1. An axon sprouting stimulation kit
comprising a first container comprising a flowable collagen matrix, a
second container comprising a matrix-releasable therapeutically active
agent, a mixing means for intermingling the flowable collagen matrix and
the matrix-releasable therapeutically active agent into a therapeutically
acceptable matrix, and; a delivery means, wherein the matrix-releasable
therapeutically active agent is selected from the group consisting of C3
and Y-27632 for facilitating axon sprouting at a nerve lesion site.
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