Title: Immunological compositions and methods of use to
transiently alter mammalian central nervous system myelin to promote
United States Patent: 6,969,516
Issued: November 29, 2005
Inventors: Steeves; John D. (North Vancouver, CA); Dyer;
Jason K. (North Vancouver, CA); Keirstead; Hans S. (Irvine, CA)
Assignee: MBM & Co. (Ottawa, CA)
Appl. No.: 530234
Filed: October 28, 1998
PCT Filed: October 28, 1998
PCT NO: PCT/CA98/00997
371 Date: July 6, 2000
102(e) Date: July 6, 2000
PCT PUB.NO.: WO99/21581
PCT PUB. Date: May 6, 1999
A method is described comprising the combined administration of a novel
composition comprising serum complement proteins with complement-fixing
antibodies to induce transient disruption of myelin and/or demyelination.
The antibodies specifically bind to one or more epitopes of myelin, and
complement proteins. A method is also described for promoting regrowth,
repair, and regeneration of neurons in a mammalian subject. The methods can
be used following immediate or chronic injury.
SUMMARY OF THE INVENTION
The purpose of the present invention is to provide a means of promoting
regrowth, repair, and regeneration of neurons in the mammalian CNS.
Accordingly, the invention provides compositions and methods of use for
promoting regrowth, repair, and/or regeneration of neurons in the CNS of a
mammalian subject, such as a human, in both chronic and acute disorders.
One embodiment of the present invention provides a composition comprising
therapeutically effective amounts of the following:
|(a) one or more complement-fixing antibodies or fragments thereof,
which specifically bind to an epitope of myelin; and |
|(b) one or more complement proteins or fragments thereof, |
wherein the binding of said antibodies to myelin causes transient
disruption and/or transient demyelination of myelin. The antibodies may be
monoclonal and/or polyclonal. The complement proteins or fragments thereof
may be derived from a species different from that species to which it is
administered. In a preferred embodiment, the complement proteins or
fragments thereof are human. The complement component may be a physically
distinct component from the antibody component, or it may be covalently or
noncovalently attached directly to the antibody component, such that
binding of the antibody to the surface of the myelin triggers the
endogenous immune system attack. One or more growth factors may be added
(in an appropriate sequence) to facilitate regrowth and regeneration.
In a specific embodiment, the epitope of myelin is a myelin sheath epitope,
such as galactocerebroside (GalC), 04, Myelin Oligodendrocyte Glycoprotein (MOG),
or Myelin Associated Glycoprotein (MAG), NOGO, NI22, NI-35/250, or arretin,
or fragments thereof. In a preferred embodiment, the epitope of myelin is
GalC. Another preferred embodiment is MOG.
In a preferred embodiment, the complement proteins or fragments thereof
include the C3 component or a fragment, variant, analog, or chemical
derivative thereof. In a preferred embodiment, the component C3b is used.
In another embodiment of the present invention, the composition further
comprises neurotrophins and growth factors, such as NT-3, CNTF, FGF-1, BDNF,
PDGF, GDNF, CT-1, or BNP.
The present invention also relates to the use of these compositions to
promote regrowth, repair, and/or regeneration of neurons in a subject by the
transient disruption and/or transient demyelination of myelin.
In one embodiment of the present invention, the compositions are used in
subjects requiring neuron repair and/or regeneration due to neuron
dysfunction. This neuron dysfunction may be a result of acute or chronic
injury to the CNS. It may also be a result of degenerative disease, such as
Alzheimer's or Parkinson's disease.
In another embodiment of the present invention, the compositions are used in
subjects to generate an environment within the CNS that is relatively
permissive to growth of transplanted cells The present invention also
relates to a method of promoting regrowth, repair, and regeneration of
neurons in mammalian CNS, wherein the damage resulted from either a chronic
or acute disorder. The method entails delivery of one or more
complement-fixing antibodies or fragments thereof, which specifically bind
to an epitope of myelin and delivery of one or more complement proteins or
fragments thereof, delivered either together or separately to effect
transient disruption and/or transient demyelination of myelin in the
neuronal zone requiring regeneration.
DETAILED DESCRIPTION OF THE INVENTION
The present invention resides in the unexpected discovery that a combination
of both antibody, which binds an epitope on a myelin-producing glial cell,
and complement can be used for disruption and demyelination of the myelin
sheath, such that repair and regeneration of mammalian neurological tissue
is enhanced. The composition of this invention is valuable as a therapeutic
agent in cases in which there is injury or disease of the mammalian nervous
system such that there is a need to facilitate neuronal plasticity and the
regrowth of neural connections. The neurological tissue is exposed to the
myelin disrupting composition, according to the invention, as soon as
possible following the injury, trauma, or disease. The nature of the
protocol to effect transient demyelination can be determined from Kierstead
and Blakemore, 1997, J. Neuropath. Expt. Neurol. 56:1191-1201; Kierstead et
al., 1998, Glia, 22:161-170.
The present invention provides compositions and methods of their use for
promoting regeneration of neurological tissue in a mammalian subject, such
as a human, with a nervous system dysfunction by contacting the neurological
tissue with a therapeutically effective amount of a composition comprising a
complement fixing antibody, which binds to myelin, and complement. Uses of
the composition in the field of veterinary medicine are also an embodiment
of the present invention.
The compositions of the present invention are comprised of one or more
antibodies or fragments thereof, which bind myelin, and one or more serum
complement proteins or fragments thereof.
The antibodies used in this invention can be any antibodies or fragments
there of that specifically bind to myelin, wherein said antibodies activate
the complement system. The preferred antibodies of the present invention
specifically bind a myelin sheath epitope such as galactocerebroside (GalC),
O4, Myelin Oligodendrocyte Glycoprotein (MOG), or Myelin Associated
Glycoprotein (MAG). Other preferred epitopes are NOGO (formerly NI 35/250)
and NI220 and arretin.
Generation of Antibodies
The antibodies of the present invention, or fragments thereof, can be:
|a) naturally occurring; |
|b) antibodies obtained from disease states such as B-cells from
multiple-sclerosis patients; |
|b) produced by recombinant DNA technology; |
|c) produced by biochemical or enzymatic fragmentation of larger
|d) produced by methods resulting from a combination of a) to c); or
|e) produced by any other means for producing antibodies. |
Human antibodies can be generated by a number of techniques known to those
skilled in the art, including the use of insect cells and transgenic plants
such as tobacco or corn seed (Cramer, C. L., Crop Tech Development Corp;
Reno, J., NeoRx—IVC's IV Annual Conference: Sep. 9-12, S. F., U.S.A.)
The antibodies of the present invention can also be made by traditional
technqiues such as monoclonal or polyclonal, although monoclonal antibodies
are preferred. In general, antibodies may be obtained by injecting the
desired immunogen into a wide variety of vertebrates or invertebrates in
accordance with conventional techniques. While rodents, particularly mice,
are preferred, other species may be employed, such as members of the bovine,
ovine, equine, porcine, or avian families. Immunization of these animals can
be readily performed and their lymphocytes, particularly splenocytes, may be
obtained for fusions.
Immunization protocols are well known and can vary considerably yet remain
effective (Goding, Monoclonal Antibodies: Principles and Practice
(2nd ed.) (Academic Press, 1986). Isolated proteins, synthetic peptides, and
bacterial fusion proteins which contain antigenic fragments of the myelin
molecule may be used as immunogens. Preferably the immunogen of peptides or
recombinant proteins will be enriched for proteins or fragments thereof
containing the epitopes to which antibody-producing B cells or splenocytes
Once the proteins or peptides thereof have been purified to the extent
desired, they may be suspended or diluted in an appropriate physiological
carrier for immunization, or may be coupled to an adjuvant. Immunogenic
amounts of antigenic preparations enriched in myelin, or antigenic portions
thereof, are injected, generally at concentrations in the range of 1 ug to
100 mg/kg of host. Administration may be by injection, such as
intramuscularly, peritoneally, subcutaneously, or intravenously.
Administration may be one or a plurality of times, usually atone to four
Immunized animals are monitored for production of antibody to the desired
antigens, then the spleens are removed and splenic B-lymphocytes isolated
and fused with a myeloma cell line or transformed. The B-lympocytes can also
be isolated from the blood. The transformation or fusion can be carried out
in conventional ways, the fusion technique being described in an extensive
number of patents, such as U.S. Pat. Nos. 4,172,124, 4,350,683; 4,363,799;
4,381,292, and 4,423,147. The manner of immortalization is not critical, but
the most common method is fusion with a myeloma fusion partner. Other
techniques of immortalization include EBV transformation, transformation
with bare DNA, such as oncogenes or retroviruses, or any other method that
provides for stable maintenance of the cell line and production of
monoclonal antibodies. The general process for obtaining monoclonal
antibodies has been described (Kohler and Milstein (1975) Nature
256:495-497). Human monoclonal antibodies may be obtained by fusion of the
spleen cells with an appropriate human fusion partner, such as WI-L2,
described in European Application No. 82.301103.6. A detailed technique for
producing mouse X-mouse monoclonal antibodies has been taught (Oi and
Herzenberg (1980) in Mishell and Shiigi (eds.) Selected Methods in
Cellular Immunology 351-372). The resulting hybridomas are screened to
isolate individual clones, each of which secretes a single antibody species
to the antigen.
The immortalized cell lines may be cloned and screened in accordance with
conventional techniques, and antibodies in the cell supernatants detected
that are capable of binding to myelin. The appropriate immortalized cell
lines may then be grown in vitro or injected into the peritoneal cavity of
an appropriate host for production of ascites fluid. Immortalized hybridoma
cell lines can be readily produced from a variety of sources. Alternatively,
these cell lines may be fused with other neoplastic B-cells, where such
other B-cells may serve as recipients for genomic DNA coding for the
The monoclonal antibody secreted by the transformed or hybrid cell lines may
be of any of the classes or subclasses of immunoglobulins, such as IgM, IgD,
IgA, IgG1-4, or IgE. As IgG is the most common isotype utilized
in diagnostic assays, it is often preferred.
To circumvent the possible antigenicity in a human host of a monoclonal
antibody derived from an animal other than human, chimeric antibodies may be
constructed. For example, the antigen binding fragment of an immunoglobulin
molecule (variable region) may be connected by peptide linkage to at least
part of another protein not recognized as foreign by humans, such as the
constant portion of a human immunoglobulin molecule. This can be
accomplished by fusing the animal variable region exons with human kappa or
gamma constant region exons. Various techniques are known to the skilled
artisan, such as those described in PCT 86/01533, EP 171496, and EP 173494.
As an alternative method of producing antibodies, U.S. Pat. No. 5,627,052
describes methods of producing proteins that replicate the binding
characteristics and desired function of particular antibodies. An example of
application of this method includes the isolation and characterization of a
human B-lymphocyte cell, producing a specific anti-myelin antibody, for
example from the blook of a patient with Multiple Sclerosis.
The antibodies may be used intact, or as fragments, such as Fv, Fab, and
F(ab′)2 as long as there is an Fc region present to bind
complement. Such antibody fragments provide better diffusion characteristics
in vivo than the whole antibody, due to their smaller size. The means for
engineering antibodies by recombinant DNA and chemical modification methods
are considered well-known in the art.
The antibodies may be fragmented to obtain highly immunoreactive F(ab′)2,
F(ab′), and Fab fragments using the enzyme pepsin by methods well known in
the art (see Colcher et al., (1983) Cancer Res. 43:736-742).
Due to the development of molecular cloning technqiues, it is now possible
to produce human monoclonal antibody fragments quickly by paning phage
display libraries against predefined antigenic specificities. For exemplary
techniques see: Pistillo et al., Human Immunology, 57(1): 19-26, 1997 Sep.
Antibodies or fragments thereof are also made into recombinant forms by
techniques of molecular biology well known in the art (see Rice et al.,
(1982) Proc. Natl. Acad. Sci. USA 79:7862-7865; Kurokawa et al.,
(1983) Nucleic Acids Res. 11:3077-3085, Oi et al., (1983) Proc.
Natl. Acad. Sci. USA 80:825-829, Boss et al., (1984) Nucleic Acids
Res. 12:3791-3806; Boulianne et al., (1984) Nature (London)
312:643-646; Cabily et al., (1984) Proc. Natl. Acad. Sci. USA
81:3273-3277; Kenten et al., (1984) Proc. Natl. Acad. Sci. USA
81.2955-2959; Liu et al., (1984) Proc. Natl. Acad. Sci. USA
81:5369-5373; Morrison et al., (1984) Proc. Natl. Acad. Sci. USA
81:6851-6855; Neuberger et al., (1984) Nature (London) 312 604-608; Potter
et al., (1984) Proc. Natl. Acad. Sci. USA 81:7161-7165; Neuberger et
al., (1985) Nature (London) 314:268-270, Jones et al., (1986)
Nature (London) 321:522-525; Oi et al., (1986) BioTechniques
44:214-221; Sahagan et al., (1986) J. Immunol. 137:1066-1074; Sun et
al., (1986) Hybridoma 5 (Supp. 1): S17-S20; and Sun et al., (1987)
Proc. Natl. Acad. Sci. USA 84:214-218).
More specifically, the antibodies and fragments thereof may be altered to a
chimeric form by substituting antibody fragments of another species, e.g.,
human constant regions (Fc domains) for mouse constant regions by
recombinant DNA techniques known in the art as described in the above cited
references. These Fc domains can be of various human isotypes, i.e., IgG,
IgG2, IgG3, IgG4, or IgM.
In addition, the antibodies and fragments thereof may be altered to an
affinity modified form, avidity modified form, or both, by altering binding
sites or altering the hinge region using recombinant DNA techniques well
known in the art as described in the above cited references.
The recombinant antibody forms may also be fragmented to produce
immunoreactive fragments F(ab′)2, F(ab′), and Fab in the same
manner as described.
Antibody fragments may also include Fv fragments, the smallest functional
modules of antibodies required to maintain the binding and specificity of
the whole antibody. Fv fragments are heterodimers composed of a variable
heavy chain and a variable light chain domain. Proteolytic digestion of
antibodies can yield isolated Fv fragments, but the preferred method of
obtaining Fvs is by recombinant technology (See Skerra and Pluckthun (1988)
Fvs can be noncovalently-associated VH and VL domains,
although these tend to dissociate from one another. Stable Fvs can be
produced by making recombinant molecules in which the VH and VL
domains are connected by a peptide linker so that the
antigen-combining site is regenerated in a single protein. These recombinant
molecules are termed single chain Fvs (scFvs). The means for preparing scFvs
are known in the art (See: Raag and Whitlow (1995) FASEB 9:73, Bird
et al., (1988) Science 242:423-426; Huston et al., (1988) Proc.
Natl. Acad. Sci. USA 85:5879-5883). Alternatively, the two variable
domains may be joined and stabilized by an engineered disulphide bond; these
are termed disulfide Fvs (dsFvs) (Reiter and Pastan (1996) Clin. Cancer
The Fc domain of an antibody is required for the activation of complement.
Fv fragments, which lack the Fc domain, cannot activate complement. In order
for Fv fragments to be useful in the present invention, they would have to
be designed with a novel activator of the complement cascade. As an example,
the Fv fragment could be designed to include the CH2 domain of an
IgG antibody. As an alternative example, a wholly synthetic molecule may be
linked to the Fv fragment to activate complement, or an activator of
complement familiar to those in the field may be linked to the Fv fragment.
The antibody may also be modified by the addition of such molecules as
polyethylene glycol (as described in U.S. Pat. No. 5,766,897) as to prolong
its biological half-life, potency, or the diffusion of the molecule in situ
(U.S. Pat. No. 5,747,446, Chinol et al., 98 Brit. J. Cancer, 78:189-197;
Francis et al., 98, Intl J. Hematol. 68:1-18).
Labeling of Antibodies or Fragments:
The antibodies of this invention, or fragments thereof, may be used without
modification or may be modified in a variety of ways, for example, by
labeling. Labeling is intended to mean joining, either covalently or
non-covalently, a label which directly or indirectly provides for a means of
detection of the antibody to enable monitoring of the progress of
therapeutic treatment using the composition.
A label can comprise any material possessing a detectable chemical or
physical property. A wide variety of labels is known, including
radionuclides, enzymes, enzyme substrates, enzyme cofactors, enzyme
inhibitors, ligands (particularly haptens), fluorescers, chromophores,
luminescers, and magnetic particles. These labels are detectable on the
basis of either their own physical properties (eg., fluorescers,
chromophores and radioisotopes), or their reactive or binding properties (eg.,
enzymes, substrates, cofactors and inhibitors). These materials are well
known to one skilled in the art. U.S. Pat. No. 4,671,958 teaches methods
that can be used for labelling antibodies or attaching complement to
The complement portion of the composition may be comprised of one or more
complement proteins, fragments, variants, analogs, and/or chemical
A fragment of a complement protein refers to any subset of the C molecule.
For example, fragments of C3 include C3b, iC3b, C3a, C3c, C3dg, and C3d.
A "variant" of a complement protein or fragments thereof refers to a
molecule substantially similar to either the entire protein or a fragment
thereof, which possesses biological activity that is substantially similar
to a biological activity of the complement protein or fragments thereof. A
molecule is said to be "substantially similar" to another molecule if both
molecules have substantially similar structures or if both molecules possess
a similar biological activity.
Variants of C3b, for example, include C3b dimers, and higher oligomers. When
C activation occurs at the cell-surface, multiple cycles of enzyme reactions
result in the deposition on the surface of C3b in multimeric form. C3b
dimers or higher oligomers indeed have higher affinity for the cell than do
Variants of complement protein or fragments thereof are produced by chemical
or recombinant means well-known in the art. Such variants include, for
example, deletions from, or insertions or substitutions of, amino acid
residues within the amino acid sequence. For example, at least one amino
acid residue may be removed and a different residue inserted in its place.
Substantial changes in functional or immunological properties are made by
selecting substitutions that are less conservative, ie. that differ more
significantly in their effect on maintaining (a) the structure of the
peptide backbone in the area of the substitution, for example, as a sheet or
helical conformation, (b) the charge or hydrophobicity of the molecule at
the target site, or (c) the bulk of the side chain. The substitutions that
in general are expected to induce greater changes are those in which (a)
glycine and/or proline is substituted by another amino acid or is deleted or
inserted, (b) a hydrophilic residue, e.g., seryl or threonyl, is substituted
for (or by) a hydrophobic residue, e.g., leucyl, isoleucyl, phenylalanyl,
valyl, or alanyl; (c) a cysteine residue is substituted for (or by) any
other residue; (d) a residue having an electropositive side chain, e.g.,
lysyl, arginyl, or histidyl, is substituted for (or by) a residue having an
electronegative charge, e.g., glutamyl or aspartyl; or (e) a residue having
a bulky side chain, e.g., phenylalanine, is substituted for (or by) one not
having such a side chain, e.g., glycine.
Most deletions, insertions, and substitutions are not expected to produce
radical changes in the characteristics of the protein molecule; however,
when it is difficult to predict the exact effect of the substitution,
deletion, or insertion in advance of doing so, one skilled in the art will
appreciate that the effect will be evaluated by routine screening assays.
For example, a change in the immunological character of the protein
molecule, such as binding to a given antibody, is measured by an immunoassay
such as a competitive type immunoassay.
An "analog" of a complement protein or fragment thereof refers to a
non-natural molecule substantially similar to either the entire protein or a
A "chemical derivative" of a complement protein or fragment thereof contains
additional chemical moieties that are not normally part of the protein or
fragment. Covalent modifications of the peptides are included within the
scope of this invention. Such modifications may be introduced into the
molecule by reacting targeted amino acid residues of the peptide with
organic derivatizing agents that are capable of reacting with selected side
chains or terminal residues, as is well-known in the art (T. E. Creighton
Proteins: Structure and Molecule Properties (San Francisco: W. H.
Freeman, 1983) at 70-86).
The complement portion of the composition may be a physically distinct
component from the antibody component. Alternatively, the complement
proteins or fragments thereof, may be covalently or noncovalently attached
directly to the antibody component, such that binding of the antibody to the
surface of the myelin triggers the endogenous immune system attack.
The complement components may be fractions that have been purified as well
as those that have been enriched in the proteins which comprise the
complement system. Such preparations should take into account the relative
lability of complement and provide a sufficient combination of factors to
allow complete activation of the complement cascade to allow transient
demyelination to occur.
The complement portion of the composition may be comprised of one or more
complement proteins, fragments, variants, analogs, and/or chemical
derivatives. It should be noted, however, that the C3 component of
complement plays a fundamental role either in opsonization or in the
propagation of the cascade to the lytic MAC. In a preferred embodiment, the
C3 component or a fragment, variant, analog, or chemical derivative thereof
should be included in the complement portion of the composition. In
situations targeted for demyelination, the C3 component should certaintly be
present for optimal results. In situations targeted for regeneration it is
less certainty required.
The complement portion of the composition may be derived from a subject's
own serum, from the serum of a donor, or from the pooled sera of a number of
donors, such as those available commercially, which are produced to
consistent, approved standards.
The complement components may be derived from species different from that
species to which it is administered due to the fact that the compositions
are introduced directly to the neural tissue (e.g., intrathecally).
The composition may optionally include other chemicals or drugs such as
growth factors and neurotrophins. It is known that the beneficial effects of
blocking CNS myelin-associated inhibitors on axonal regeneration can be
augmented by the concomitant application of neurotrophins, such as NT-3 (Bregman
et al., (1995) Nature 378:498-501, Schnell et al., (1994) Nature
367:170-173). FGF-1 can also be used (Chang et al., 1996, supra).
In a preferred embodiment, the composition is comprised of a GalC-specific
monoclonal antibody and human serum complement.
In another preferred embodiment, the composition is comprised of a MOG-specific
monoclonal antibody and human serum complement.
The compositions of the present invention can be used to promote regrowth,
repair, and/or regeneration of neurons in the CNS of a subject by
stimulating transient immunological disruption of myelin or transient
demyelination of axons. Preferably, the transient demyelination process of
the present invention occurs in the CNS, most preferably in the spinal cord.
The subject may be any mammal. In a preferred embodiment, the subject is
The compositions of the present invention can be used to promote regrowth,
repair, and/or regeneration of dysfunctional neurons in the CNS that have
been damaged as a result of injury, such as a spinal cord injury. The method
can be used following immediate or chronic injury.
The compositions of the present invention can also be used to promote
regrowth, repair, and/or regeneration of dysfunctional neurons in the CNS
that have been damaged as a result of disease, such as degenerative diseases
including Alzheimer's and Parkinson's disease.
The compositions of the present invention can also be used to generate an
environment within the mammalian CNS that is relatively permissive to growth
of transplanted cells. For example, if PNS cells are transplanted into a
site in the CNS that has been damaged, axons will be able to grow into the
transplanted tissue but will be unable to grow out of this tissue back into
the CNS due to the inhibitory effects of myelin. The compositions of the
present invention can be used to disrupt the myelin in the CNS to allow the
axons to extend into this area.
Preparations and Administration
Methods of using the compositions of the present invention comprise
administering a therapeutically effective amount of such a composition to
the subject. As used herein, the term "therapeutically effective amount"
refers to an amount of composition sufficient to effectively and transiently
disrupt and/or demyelinate the CNS so that repair and regeneration of
neurological tissue and neuronal connections is enhanced. Generally, the
therapeutic composition is administered at a range from about 0.03 mg
antibody to about 0.6 mg antibody in a 20% to 30% complement solution per kg
body weight. Preferably, the range is from 0.05 mg antibody to 0.4 mg
antibody in a 20% to 30% complement solution per kg body weight. Most
preferably, the range is from 0.1 mg antibody to 0.3 mg antibody in a 20% to
30% complement solution per kg body weight. The exact ratio of antibody to
complement will vary depending on the circumstances; however, since the
amount of complement activated is directly proportional to the number of
bound antibody molecules, it is possible to administer relatively high
concentrations of complement in excess of the relative concentration of
antibody. In addition, the particular concentration of antibody administered
will vary with the particular dysfunction and its severity, as well as with
such factors as the age, sex, and medical history of the patient. Those of
skill in the clinical arts will know of such factors and how to compensate
the dosage ranges of the composition accordingly.
The majority of spinal cord injuries result from damage to the vertebral
column surrounding the spinal cord. This damage includes fractures,
dislocations, or both. Much of the damage to the spinal cord is due to
secondary phenomena that occur within hours following the injury. At this
point, the resultant damage may be reversible; consequently, a critical
factor for recoverable CNS function is the amount of time that evolves
between injury and the institution of therapy. Most preferably, when the
nervous system dysfunction is a result of injury, administration of the
composition to the subject will be as close in time to the time of the
injury as possible.
A composition according to the method of the invention can be administered
to a subject parenterally by injection or by gradual infusion over time. For
example, the composition can be administered intrathecally or injected
directly into the spinal cord.
Preparations for parenteral administration are contained in a
pharmaceutically acceptable carrier that is compatible with both the
components of the composition and the patient. Such carriers include sterile
aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of
non-aqueous solvents include propylene glycol, polyethylene glycol,
metabolizable oils such as olive oil or squalane, and injectable organic
esters such as ethyl oleate. Aqueous carriers include water, alcoholic/acqueous
solutions, and emulsions or suspensions, including saline and buffered
media. Parenteral vehicles include sodium chloride solution, Ringer's
dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils.
Preservatives and other additives may also be present such as, for example,
antimicrobials, anti-oxidants, chelating agents, and inert gases and the
like. A preferred carrier is artificial cerebrospiral fluid.
The materials for use in the method of the invention are ideally suited for
the preparation of a kit. Such a kit may comprise a carrier means
compartmentalized to receive in close confinement one or more container
means, such as vials, tubes, and the like, each of the container means
comprising one of the separate elements to be used in the method. For
example, one of the container means may comprise a GalC-specific antibody.
Alternatively, the antibody and complement may be present in the same
container. The constituents may be present in liquid or lyophilized form, as
desired. Needles and/or other equipment that facilitates delivery of the
complement and antibody to the site of damage may include:
|a) silastic, Polyethylene, Tygon (Norton Performance Plastics) tubing,
|b) subcutaneous pumps, (such as the Medtronic pump system known for
the administration of baclofen intrathecally), |
|c) spinal needle for direct intraspinal administration, or for
short-term intrathecal administration. |
One example of a method using such a kit can be described as, a 14-gauge
Tuohy needle is inserted into the lumbar subarachnoid space. A 5-F catheter
is coaxially placed with the tip at L10 and tunneled to the flank (or
appropriate location). This type of instruction would be understood by one
familiar with the technique. Tubing is placed intrathecally, and connected
to the pump. The pump, containing a finite folume of the reagents is placed
under the skin this can be refilled in the Doctor's office, via a needle
inserted into a septa in the pump. Or the Infusaid pump may be used in the
Advantages Over Current Methods
The compositions and their uses of the present invention have a number of
advantages over methods currently available for the regeneration of neuronal
growth in the CNS.
Interventional therapies, including opiate antagonists, thyrotropin-releasing
hormone, local cord cooling, dextran infusion, adrenergic blockade,
corticosteroids, and hyperbaric oxygen, are targeted at reducing secondary
inflammatory damage after atraumatic injury to the spinal cord in order to
prevent the spread of damage to uninjured neurons. Unlike the present
invention, however, they do not promote regeneration of the damaged neurons.
Peripheral nerve transplants and the grafting of donor cells into the CNS
are useful in that axons can grow into them, however, the axons cannot grow
out of them into the surrounding CNS due to the inhibitory myelin present.
In contrast, the present invention disrupts the inhibitory myelin to allow
regrowth of neurons in the CNS.
Claim 1 of 5 Claims
1. A method for promoting neuron repair or regeneration in a human subject
suffering from spinal cord disruption by intrathecal administration of a
therapeutically effective amount of a composition comprising:
(a) one or more complement-fixing antibodies or complement-fixing
fragments thereof, which specifically bind to galactocerebroside (GalC);
(b) one or more complement proteins or fragments thereof, wherein at least
one of the complement proteins is a C3 protein;
wherein the combination of said antibodies and complement proteins or
fragments thereof causes activation of the complement system resulting in
disruption of myelin or demyelination, thereby promoting neuron repair or
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