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Title:  Immunological composition and its method of use to transiently disrupt mammalian central nervous system myelin to promote neuronal regeneration

United States Patent:  6,548,061

Issued:  April 15, 2003

Inventors:  Steeves; John D. (N. Vancouver, CA); Dyer; Jason K. (N. Van, CA); Keirstead; Hans S. (Vancouver, CA)

Assignee:  University of British Columbia (CA)

Appl. No.:  181719

Filed:  October 28, 1998

Abstract

Novel compositions are described comprising the combined administration of serum complement proteins with complement-fixing antibodies. The antibodies specifically bind to one or more epitopes of myelin, and complement proteins. These compositions are useful for promoting regrowth, repair, and regeneration of neurons in the CNS of a mammalian subject. The compositions and method can be used following immediate or chronic injury.

DETAILED DESCRIPTION OF THE INVENTION

The following terms and abbreviations are used throughout the specification and in the claims:

The term "antibodies or fragments thereof" includes recombinant, chimeric, and affinity modified forms made by techniques of molecular biology well known in the art;

"CNS" refers to the central nervous system;

The term "complement protein or fragment thereof" (C) refers to any of 13 whole serum proteins or any of more than 20 intermediates and complexes of the complement system, the primary humoral mediator of antigen-antibody reactions, and includes variants, analogs, and chemical derivatives thereof;

The term "composition" is used to indicate more than one component. The elements of the composition can be mixed together, however, it is not necessary that they be combined in the same solution. In an alternative embodiment, they do not need to be packaged, stored or even mixed together. The elements (antibody-type and complement-type) can be delivered to the site of nerve damage sequentially, or at the same time. The need for a therapeutically effective temporal sequence is understood by one skilled in the art. The concept of at least one complement fixing antibody or fragment thereof, plus at least complement protein or active fragment thereof equates with the concept of the composition. These elements are delivered to the site of damage to form a complex with an appropriate epitope present in myelin to be transiently demyelinated. Thus, the first two types of elements (of which there can be more than one member of each type of element, for example, two or more antibody or component proteins or fragments) are delivered to the site targeted for transient demyelination to form a complex in situ, in vivo with the epitope(s) on myelin.

The term "demyelination" refers to the removal or breakdown of myelin in neurological tissue. Demyelination consists of the removal of the myelin sheath, such as that surrounding neurons or neuronal projections (e.g., the axons). This process may be chemical or immunological in both the experimental and pathological states. This invention effects transient demyelination in order to promote repair and regrowth.

The term "disruption" refers to delamination or disruption of the three-dimensional conformation of myelin;

The term "dysfunction" when used to describe the therapeutic use of the invention encompasses any type of trauma to the nervous system and resulting loss of function. Such trauma can arise from either physical injury or disease;

The term "Fab" means an antibody fragment that is obtained by cleaving an antibody in the hinge region yielding two Fab fragments, each having the heavy and light chain domains of the antibody, along with an Fc region;

The term "Fc" means the constant region of the antibody, which may activate complement;

The term "Fv fragment" means a heterodimer of the heavy and light chain variable domain of an antibody. These variable domains may be joined by a peptide linker or by an engineered disulphide bond;

Growth factors are extracellular polypeptide signaling molecules that stimulate a cell to grow or proliferate. Examples are epidermal growth factor (EGF) and platelet-derived growth factor (PDGF). Most growth factors have other actions besides the induction of cell growth or proliferation. Growth factors can be divided into broad- and narrow-specificy classes. The broad-specificity factors, like PDGF and EGF affect any classes of cells. At the opposite extreme lie narrow-specificity factors. In intact animals proliferation of mot cell types depends on a specific combination of growth factors rather than a single growth factor. Thus a fairly smal number of growth factor families may serve, in different combinations, to regulate selectively the proliferation of each of the many types of cells in a higher animal.

Fibroblast Growth Factor (FGF) is any one of a group of proteins, usually intracellular, that have important angiogenic function and enhance would healing and tissue repair. Over-activity of these factors has been associate with neoplasia.

Neurotrophic factors are a family of substances that promote growth and regeneration of neurons. While growth factors elsewhere in the body promote and support cell division, neurons cannot divide; but they can regenerate after injury and neurotrophic factors promote this regeneration. They also promote the growth of axons and dendrites, the neuron branches that form connections with other neurons.

"GalC" refers to galactocerebroside;

"MAG" refers to myelin-associated glycoprotein;

"MBP" refers to myelin basic protein;

"MOG" refers to myelin oligodendrocyte glycoprotein;

The term "neurological tissue" refers to neurons and other cells typically situated in the region of the nervous system, such as the spinal cord of the CNS;

"PNS" refers to the peripheral nervous system;

The term "recombinant antibodies or fragments thereof" collectively includes chimeric or recombinant forms of the antibodies or fragments thereof wherein the Fc domain is substituted for an Fc domain of another species or isotype, affinity modified forms of the antibodies or fragments thereof wherein the binding sites are altered, avidity modified forms of the antibodies or fragments thereof wherein the hinge regions are altered, immunoreactive fragments thereof, and combinations thereof; and

The term "regeneration of neurological tissue" includes the regrowth of neurons that results in the reformation of neuronal connections, both anatomically and/or functionally.

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.

Antibodies

The antibodies used in this invention can be any antibodies or fragments thereof 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 molecules;

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., CropTech Development Corp; Reno, J., NeoRx-IVC's IV Annual Conference: Sept 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 are desired.

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 at one to four week intervals.

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 antibody.

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, EP171496, and EP173494.

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.

Antibody Engineering

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, Sep. 15, 1997).

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 4:214-221; 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., IgG1, 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) Science 240:1038-1041).

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 Res. 2:245-252).

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 CH 2 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 antibodies.

Complement

The complement portion of the composition may be comprised of one or more complement proteins, fragments, variants, analogs, and/or chemical derivatives.

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 C3b monomers.

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 fragment thereof.

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 certaintly 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).

Other Factors

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.

Uses

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 human.

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 demyclinate 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.

Kit

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 alternative.

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 a traumatic 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 17 Claims

We claim:

1. A kit for parenteral administration comprising one or more containers comprising

(a) one or more complement-fixing human, recombinant human or humanized antibodies or fragments thereof in a sterile pharmaceutical carrier, wherein said antibodies or fragments comprise a complement-binding Fc region of a complement-fixing antibody which specifically binds to an epitope of a mammalian myelin selected from the group consisting of galactocerebroside (GalC), O4, Myelin Associated Glycoprotein (MAG), NOGO, NI220, NI-35/250, myelin oligodendrocyte glycoprotein (MOG) and arretin; and

(b) complement protein in a sterile pharmaceutical carrier;

wherein when (a) and (b) are administered together are effective to cause in vivo transient disruption and/or transient demyelination of mammalian neurons.
 


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