WO2011133862A1 - Methods and compositions for promoting myelination - Google Patents

Abstract

Compositions and methods for increasing myelination and treating and/or inhibiting demyelinating diseases are disclosed.

Description

METHODS AND COMPOSITIONS FOR PROMOTING MYELINATION

By Larry S. Sherman

Marnie Preston

This application claims priority under 35 U.S.C. § 1 19(e) to U.S. Provisional Patent Application No. 61/327,344, filed on April 23, 2010. The foregoing application is incorporated by reference herein.

This invention was made with government support under Grant No. NS39550 awarded by the National Institutes of Health. The government has certain rights in the invention.

FIELD OF THE INVENTION

The present invention relates to the field of myelination. More specifically, the invention provides compositions and methods for the treatment of demyelinating disease.

BACKGROUND OF THE INVENTION

Several publications and patent documents are cited throughout the specification in order to describe the state of the art to which this invention pertains. Each of these citations is incorporated herein by reference as though set forth in full.

Demyelination is a significant cause of debilitation in patients with multiple sclerosis (MS). Demyelination can occur through multiple mechanisms that include the destruction of myelin and the death of oligodendrocytes (OLs) following inflammatory, ischemic, and traumatic insults in the brain and spinal cord (reviewed by Lassmann, H. (2001) Curr. Opin. Neurol., 14:253-8). The inhibition of OL progenitor cell (OPC) maturation in demyelinated lesions is an underlying cause of remyelination failure, leading to long-term neurological disability (Franklin et al. (2008) Nat. Rev. Neurosci., 9:839-55). Thus, defining strategies to promote OPC maturation is a significant aim in the development of therapies that prevent or reverse disability in patients with demyelinating conditions. SUMMARY OF THE INVENTION

In accordance with one aspect of the instant, methods for treating or inhibiting (reducing, suppressing, or slowing) a demyelinating disease or reducing the risk of developing a demyelinating disease (e.g., multiple sclerosis) in a patient in need thereof are provided. In a particular embodiment, the method comprises

administering at least one hyaluronidase inhibitor to the patient. In certain

embodiments, the method further comprises the administration of at least one other demyelinating disease therapeutic agent. The methods may also further comprise monitoring the patient to determine the state of demyelination/remyelination and, optionally, modifying treatment as necessary. In a particular embodiment, the hyaluronidase inhibitor inhibits neutral pH acting hylauronidases, such as PH20.

In accordance with another aspect of the instant invention, methods for increasing myelination in a patient in need thereof are provided. In a particular embodiment, the methods comprise administering at least one hyaluronidase inhibitor to the patient.

Compositions for increasing myelination and/or treating demyelinating diseases are also provided in the instant invention. In a particular embodiment, the composition comprises at least one hyaluronidase inhibitor and a pharmaceutically acceptable carrier. In certain embodiments, the compositions further comprise at least one other demyelinating disease therapeutic agent. In yet another embodiment, kits are provided which comprise a composition comprising at least one hyaluronidase inhibitor and a pharmaceutically acceptable carrier and, optionally, a second composition comprising at least one other demyelinating disease therapeutic agent and a pharmaceutically acceptable carrier.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1A provides images of OPCs grown with vehicle (left panels) or 100 U/ml bovine testicular hyaluronidase (right panels) for 72 hours. Hyaluronidase treated cultures had more platelet-derived growth factor receptor alpha positive (PDGF-R +) OPCs and few myelin basic protein positive (MBP+) OLs. Figure IB provides images of OPCs infected with control (GFP) or hyaluronidase (PH20 or HYAL5) expressing virus. OPC maturation was inhibited in hyaluronidase-infected cells. Nuclei were labeled with DAPI. Figure 1C provides a graph showing percent GFP+MBP+ cells ± SD for each treatment. Figure 2 provides images of OPCs that were grown for 72 hours in the presence of HMW HA that had been treated with 100 U/ml of either Streptomyces hyaluronidase (left) or bovine testicular hyaluronidase (BTH; right) and then immunolabeled with anti-MBP and anti-PDGF-Ra. Note that cells failed to mature into MBP+ OLs in the presence of BTH-treated HA, but not in the presence of Streptomyces hyaluronidase-treated HA.

Figure 3 A provides an image of a gel demonstrating RNA expression of various hyaluronidases. RNA was isolated from cultures of OPCs (left 5 lanes) or OLs (right 4 lanes) and analyzed for the expression of hyaluronidase transcripts as indicated. The far left lane is a DNA ladder. In OPCs, Hyall and Hyal2 were clearly amplified (major bands at 820 bp) and a weak PH20 band was also detected (100 bp). All of the transcripts were down-regulated in OLs. Figure 3B provides images of PH20 immunoreactivity in cultures of OPCs, thereby demonstrating strong PH20 expression in cell bodies and processes (PH20 and PDGF-Ra are shown). Figure 3C provides images of the lack of PH20 immunoreactivity in mature, MBP- immunolabeled OLs. Note the PH20 staining in neighboring OPCs.

Figures 4A-4D provide images showing PH20 is expressed by glial cells at the borders of chronic, cortical MS patient lesions. Ten micrometer sections through chronic, cortical MS patient lesions were immunolabeled with a polyclonal anti-PH20 antibody as described herein. PH20 immunoreactivity was enriched at the borders of lesions (arrows, Fig. 4A; see also higher power image in Fig. 4B). Numerous cells expressed PH20 at these borders (high magnification image, Fig. 4C), while small numbers of cells with the morphologies of reactive glia expressed PH20 in areas of complete demyelination (Fig. 4D).

Figure 5 provides a graph images demonstrating that N-(Pyridin-4yl)-[5- bromo-l-(4-fluorobenzyl)indole-3-yl]carboxamide inhibits hyaluronidase activity in OPC cultures. OPCs were grown for 72 hours in the presence and absence of 25 μΜ of the compound, and then assayed for MBP expression by immunocytochemistry.

Figure 6 shows that bovine testicular hyaluronidase inhibits OPC maturation, in vitro. OPCs were differentiated in T3/NAC alone (Fig. 6A) or in the presence of the HA synthase inhibitor 4-methylumbelliferone (4-MU; Fig. 6B) or BTH (Fig. 6C) for 72-96 hours, fixed and stained for the OL lineage markers: MBP, PDGFRa, and DAPI to visualize nuclei. The total percentage of PDGFRa+ and MBP+ cells are quantified in (Fig. 6D). Insets in (Figs. 6A-6C) show representative levels of HA (as assayed using HABP) for each treatment group. BTH treatment significantly inhibited OL maturation as assayed by decreased MBP expression compared to controls (<0.5% v. 54.1 % p = 0.00008) and increased PDGFRa expression compared to controls (97.8% v. 30.9%, p=0.00028). Inhibiting HA synthesis did not significantly alter OL maturation (Figs. 6B, 6D). Furthermore, mOPCs differentiated in the presence of chondroitinase ABC or the bacterial-derived Streptomyces hyaluronidase showed no significant changes in OL maturation as compared to controls (Fig. 6E).

Figure 7 shows that BTH breakdown products of HA block OL maturation and remyelination. Vehicle alone (Fig. 7A), HMW HA (100μg/mL)(Fig. 7B), BTH- degraded HA (100μg/mL) (Fig. 7C), or StrepH degraded HA (100μg/mL) (Fig. 7D) was injected into demyelinated corpus callsoum lesions 2 days following lysolecithin injections. Remyelination as assayed by recovery of MBP immunoreactivity (shown in white).

Figure 8 shows that OPCs express hyaluronidases and are capable of degrading HA as they mature. Total mRNA was isolated from mouse testes (as a positive control), OPCs and adult mouse corpus callosum, reverse transcribed and subjected to RT-PCR. OPCs express HYAL1 , HYAL2 and PH20 but not the testes- specific HYAL5 (Fig. 8A). The same pattern of HYAL expression was seen in the corpus callosum (Figs. 8D-8E). OPCs were plated onto coverslips coated with HA, allowed to mature for 24 (Figs. 8B-8C) or 72 (Figs. 8D-8E) hours and stained with 04, HABP and DAPI. Degradation of HA (Fig. 8B), as assayed by loss of HABP immunoreactivity, was seen around cell bodies and extending 04+ processes (Fig. 8C) at 24 hours. By 72 hours HA historeactivity was lost (Fig. 8D) in areas corresponding to the presence of 04+ membranes (Fig. 8E).

Figure 9 shows that inhibiting endogenous hyaluronidase activity promotes OL maturation in vitro and remyelination in vivo. OPCs were grown in OL differentiation medium alone (Fig. 9A) or with the pan hyaluronidase inhibitor VCPAL (6-O-Palmitoyl-L-ascorbic acid, 25μΜ; Fig. 9B) for 72-96 hours and stained with MBP, PDGFRa and DAPI. Total MBP+ and PDGFRa+ cells are quantified in Fig. 9C. Consistent with the hypothesis that hyaluronidases expressed by mOPCs generate HA fragments which inhibit OL maturation, blocking HYAL activity with VCPAL increased total percentage MBP+ cells compared to controls (69.31% v. 54.05% p= 0.00505) while decreasing total percentage PDGFRo+ cells (19.61% v. 30.98% in control, p=0.01719). Figures 9D and 9E show examples of lysolecithin lesions treated with HMW HA and vehicle or HMW HA with VCPAL (25 μΜ).

Figure 10 shows that elevated expression of PH20, the major hyaluronidase in BTH, blocks OL maturation in vitro. OPCs were infected with Antiviruses carrying GFP only (control; Fig. 1 OA), HYAL1.GFP, HYAL2.GFP, HYAL5.GFP or

PH20.GFP (Fig. 10B), allowed to differentiate for 72-96 hours and stained for GFP and MBP. Results are quantified in Fig. IOC. PH20 overexpression (Fig. 10B) robustly inhibited OL maturation compared to GFP only (Fig. 10A) as assayed by coexpression of MBP and GFP (35.91% of control, p=0.0008). Insets in Figs. 10A and 10B show complete degradation of HA in mOPCs overexpressing PH20.

HYAL2 overexpression partially inhibited OL maturation (78.48% of control, p=0.00148). HYAL5 overexpression also partially inhibited OL maturation (70.08% of control, p=0.0183) but is not expressed by OL lineage cells in vitro or in vivo and is thus unlikely to have any significant physiological relevance to remyelination. ·

Figure 1 1 shows that PH20 expression is seen on proliferating OPCs and mature OLs and in demyelinated lesions. PH20 expression was confirmed by immunocytochemisrty in OPCs (Figs. 1 1 A-l ID) and in maturing OLs (Figs. 1 1E- 1 1H). In OPCs, PH20 expression (Fig. 1 IB) was seen in cell bodies and processes of PDGFRoH- mOPCs (Fig. 1 1A). PH20 expression became confined to the cell body (Fig. 1 1 F) of MBP+ (Fig. 1 1 E) OLs. Inset in Fig. 1 1 D shows lack of staining in mOPCs incubated with preimmune serum. DAPI (Figs. 1 1C, 1 1G) was used to identify cell nuclei. Merged images are shown in Figs. 1 ID and 1 1H. Figs. 1 11- 1 1L show sections of lumbar spinal cord from mice with EAE, 21 days post-inoculation. PH20 immunoreactivity (Fig. 1 1 J) was elevated in areas where there was

demyelination, identified by loss of fluoromyelin staining (Fig. I ll) and increased DAPI labeling (Fig. 1 IK). Merged image is shown in Fig. 1 1L.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with the instant invention, methods and compositions for increasing myelination are provided. The methods and compositions comprise inhibitors of hyaluronidase. In a particular embodiment, the hyaluronidase inhibitor inhibits a neutral pH-acting hyaluronidase. In a specific embodiment, the neutral pH- acting hyaluronidase is PH-20 (see, e.g., GenBank Accession No. AAC60607.2; GenBank GenelD: 6677; Hajjaji et al. (2005) Arthritis Res. Ther., 7:R756-R768). In still another embodiment, the hyaluronidase inhibitor preferentially inhibits neutral pH-acting hyaluronidases to the general exclusion of acidic hyaluronidases (e.g., Hyall and Hyal2). The hyaluronidase inhibitor may inhibit neutral pH-acting hyaluronidases while having little to no inhibitory activity towards acidic

hyaluronidases. For example, the hyaluronidase inhibitor may inhibit neutral pH- acting hyaluronidases at least 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold or more than acidic hyaluronidases. In a certain embodiment, the hyaluronidase inhibitor may also inhibit hyaluronidases at neutral pH while having little to no inhibitory activity at acidic pH (e.g., a pH of 5 or lower, particularly about 3.5 or lower).

The hyaluronidase inhibitor of the instant invention may be any type of compound such as, without limitation, a nucleic acid (e.g., siRNA, shRNA, antisense, etc. directed to nucleic acids encoding the hyaluronidase), polypeptide/protein (e.g., antibodies (e.g., monoclonal antibodies; antibodies immunologically specific for PH- 20 (e.g., human PH-20)), ligands, antagonists), and chemical compound (e.g., small molecule (e.g., chemical compounds of less than lkDa, particularly less than 800 Da)). Examples of hyaluronidase inhibitors include, without limitation, those provided in Olgen et al. (Chem. Biol. Drug Des. (2007) 70:547-51), Kaessler et al. (J. Enzyme Inhib. Med. Chem. (2008) 23:719-27), and Olgen et al. (Z. Naturforsch.

(2010) 65c:445-450). Additionally, Hunnicutt et al. (Biol. Reprod. (1996) 55:80-86) describe a monoclonal antibody (mAb PH-21) which inhibits the hyaluronidase activity of PH-20. Kaessler et al. (Eur. J. Pharm. Sci. (201 1) 42: 138-47) provides an example of a method for screening for PH-20 inhibitors. In a particular embodiment, the hyaluronidase inhibitor is 6-O-palmitoyl-L-ascorbic acid (1-ascorbic acid 6- hexadecanoate; VCPAL). In a particular embodiment, the hyaluronidase inhibitor is an antibody or antibody fragment immunologically specific for PH-20 (e.g., human PH-20) that inhibits the hyaluronidase activity of PH-20 (e.g., binds the hyaluronidase domain of PH-20, particularly at or near the active site). In a particular embodiment, the hyaluronidase inhibitor is selected from the group consisting of N-(Pyridin-4yl)- [5-bromo-l -(4-fluorobenzyl)indole-3-yl]carboxamide, apigenin (Hunnicutt et al., Biol. Reprod. (1996) 55:80-86), and those provided in Olgen et al. (Chem. Biol. Drug Des. (2007) 70:547-51), Kaessler et al. (J. Enzyme Inhib. Med. Chem. (2008) 23:719- 27), and Olgen et al. (Z. Naturforsch. (2010) 65c:445-450). In a particular embodiment, the hyaluronidase inhibitor is an indole carboxamide. In a particular embodiment, the hyaluronidase inhibitor is N-(Pyridin-4yl)-[5-bromo-l -(4- fluorobenzyl)indole-3-yl]carboxamide.

According to one aspect of the instant invention, methods of treating a demyehnating disease in a patient are provided, wherein said method comprises the administration of at least one hyaluronidase inhibitor. The hyaluronidase inhibitor may be contained within a composition further comprising at least one

pharmaceutically acceptable carrier. The methods of the instant invention may also further comprise the administration of at least one other therapy for treating the demyehnating disease (e.g., the administration of at least one demyehnating disease therapeutic agent). Demyehnating disease therapeutic agents include, without limitation, beta interferons (e.g., interferon beta- 1 a (A vonex®, Rebif®), interferon beta- lb (Betaseron®, Betaferon®)), glatiramer acetate (Copaxone®), mitoxantrone (Novantrone®), natalizumab (Tysabri®; humanized monoclonal antibody against the cellular adhesion molecule a4-integrin), azathioprine (AZA), cyclosporine, methotrexate, cyclophosphamide, intravenous immunoglobulin, prednisone, methylprednisone, prednisolone, methylprednisolone, dexamethasone, adreno- corticotrophic hormone (ACTH), corticotropin, 2-chlorodexyadenosine (2-CDA, cladribine), inosine, interleukin-2 antibody (Zenapax®, daclizunab), leucovorin, teriflunomide, estroprogestins, desogestrel, pirfenidone, etinilestradiol, BHT-3009, ABT-874, Bacille Calmette-Guerin (BCG) Vaccine, T cell vaccination, CNTO 1275, Rituximab, N-acetylcysteine, minocycline, RO0506997, gelsolin, and statins (e.g., atorvastatin, lovastatin, pravastatin, fluvastatin, and simvastatin).

Compositions of the instant invention comprise at least one hyaluronidase inhibitor and at least one pharmaceutically acceptable carrier. The compositions may also comprise at least one other demyehnating disease therapeutic agent.

Alternatively, at least one other demyehnating disease therapeutic agent may be contained in a separate composition. The composition(s) may be provided in a kit, e.g., at least one composition comprising at least one hyaluronidase inhibitor and at least one composition comprising at least one demyehnating disease therapeutic agent.

I. Definitions The term "demyelinating disease" as used herein refers to any disease, disorder, or condition wherein the myelin sheath of neurons are damaged.

Demyelinating diseases may be caused by any factor including, without limitation, genetics, infectious agents, hypoxia, age, inflammation, ischemia, autoimmune reactions, chemical agents such as organophosphates, neuroleptics or antipsychotic drugs, traumatic injury, or unknown factors. Demyelination (as well as

remyelination) of nerve fibers can be detected by a variety of clinical methods well known in the art (e.g., evoked potentials (EP), computer-assisted tomography (CT), magnetic resonance imaging (MRI), and cerebrospinal fluid (e.g., via lumbar puncture or spinal tap procedures) may be examined for increased levels of gamma globulin and oligoclonal banding). Demyelinating diseases include, without limitation, demyelinating diseases of the central nervous system and demyelinating diseases of the peripheral nervous system. Examples of demyelinating disease include, without limitation, multiple sclerosis (MS), transverse myelitis, Devic's disease (also known as Devic's syndrome or neuromyelitis optica (NMO)), Alzheimer's disease, progressive multifocal leukoencephalopathy (PML), disseminated necrotizing leukoencephalopathy (DNL), optic neuritis, leukodystrophies, acute inflammatory demyelinating polyneuropathy (AIDP), acute disseminated encephalomyelitis, Guillain-Barre syndrome, chronic inflammatory demyelinating polyneuropathy, anti- myelin-associated glycoprotein (anti-MAG) peripheral neuropathy, Charcot-Marie- Tooth Disease (also known as Hereditary Motor and Sensory Neuropathy (HMSN), Hereditary Sensorimotor Neuropathy (HSMN), or Peroneal Muscular Atrophy), Schilder disease, central pontine myelinolysis (CPM), radiation necrosis, Binswanger disease (SAE), leukodystrophy, acute transverse myelitis, acute viral encephalitis, adrenoleukodystrophy (ALD), adrenomyeloneuropathy, AIDS-vacuolar myelopathy, experimental autoimmune encephalomyelitis (EAE), experimental autoimmune neuritis (EAN), HTLV-associated myelopathy, Leber's hereditary optic atrophy, subacute sclerosing panencephalitis, and tropical spastic paraparesis. Demyelinating diseases also include other conditions where demyelination occurs including, without limitation, stroke, ischemic conditions, inflammatory conditions, hypoxia conditions, age-related myelin damage, and traumatic insult (e.g., traumatic nervous system injury). In a particular embodiment, the demyelinating disease is MS.

The term "treat" as used herein refers to any type of treatment that imparts a benefit to a patient afflicted with a disease, including improvement in the condition of the patient (e.g., in one or more symptoms), delay in the progression of the condition, etc. In a particular embodiment, the treatment of a demyelinating disease results in at least an inhibition/reduction in the loss of myelination and, more preferably, in an increase in myelination (e.g., remyelination).

The phrase "effective amount" refers to that amount of therapeutic agent that results in an improvement in the patient's condition.

"Pharmaceutically acceptable" indicates approval by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.

A "carrier" refers to, for example, a diluent, adjuvant, preservative (e.g., Thimersol, benzyl alcohol), anti-oxidant (e.g., ascorbic acid, sodium metabisulfite), solubilizer (e.g., Tween 80, Polysorbate 80), emulsifier, buffer (e.g., Tris HCl, acetate, phosphate), water, aqueous solutions, oils, bulking substance (e.g., lactose, mannitol), excipient, auxilliary agent or vehicle with which an active agent or pharmaceutically acceptable salt thereof of the present invention is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water or aqueous saline solutions and aqueous dextrose and glycerol solutions may be employed as carriers. Suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E.W. Martin (Mack

Publishing Co., Easton, PA); Gennaro, A. R., Remington: The Science and Practice of Pharmacy, 20th Edition, (Lippincott, Williams and Wilkins), 2000; Liberman, et al., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, N.Y., 1980; and Kibbe, et al., Eds., Handbook of Pharmaceutical Excipients (3rd Ed.), American Pharmaceutical Association, Washington, 1999.

"Nucleic acid" or a "nucleic acid molecule" as used herein refers to any DNA or RNA molecule, either single or double stranded and, if single stranded, the molecule of its complementary sequence in either linear or circular form. In discussing nucleic acid molecules, a sequence or structure of a particular nucleic acid molecule may be described herein according to the normal convention of providing the sequence in the 5' to 3' direction. With reference to nucleic acids of the invention, the term "isolated nucleic acid" is sometimes used. This term, when applied to DNA, refers to a DNA molecule that is separated from sequences with which it is immediately contiguous in the naturally occurring genome of the organism in which it originated. For example, an "isolated nucleic acid" may comprise a DNA molecule inserted into a vector, such as a plasmid or virus vector, or integrated into the genomic DNA of a prokaryotic or eukaryotic cell or host organism.

When applied to RNA, the term "isolated nucleic acid" may refer to an RNA molecule encoded by an isolated DNA molecule as defined above. Alternatively, the term may refer to an RNA molecule that has been sufficiently separated from other nucleic acids with which it would be associated in its natural state (i.e., in cells or tissues). An isolated nucleic acid (either DNA or RNA) may further represent a molecule produced directly by biological or synthetic means and separated from other components present during its production.

With respect to single stranded nucleic acids, particularly oligonucleotides, the term "specifically hybridizing" refers to the association between two single-stranded nucleotide molecules of sufficiently complementary sequence to permit such hybridization under pre-determined conditions generally used in the art (sometimes termed "substantially complementary"). In particular, the term refers to hybridization of an oligonucleotide with a substantially complementary sequence contained within a single-stranded DNA molecule of the invention, to the substantial exclusion of hybridization of the oligonucleotide with single-stranded nucleic acids of non- complementary sequence. Appropriate conditions enabling specific hybridization of single stranded nucleic acid molecules of varying complementarity are well known in the art.

For instance, one common formula for calculating the stringency conditions required to achieve hybridization between nucleic acid molecules of a specified sequence homology is set forth below (Sambrook et al, 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press):

Tm = 81.5°C + 16.6Log [Na+] + 0.41(% G+C) - 0.63 (% formamide) - 600/#bp in duplex

As an illustration of the above formula, using [Na+] = [0.368] and 50% formamide, with GC content of 42% and an average probe size of 200 bases, the Tm is 57°C. The Tm of a DNA duplex decreases by 1 -1.5°C with every 1% decrease in homology. Thus, targets with greater than about 75% sequence identity would be observed using a hybridization temperature of 42°C.

The stringency of the hybridization and wash depend primarily on the salt concentration and temperature of the solutions. In general, to maximize the rate of annealing of the oligonucleotide with its target, the hybridization is usually carried out at salt and temperature conditions that are 20-25°C below the calculated Tm of the hybrid. Wash conditions should be as stringent as possible for the degree of identity of the probe for the target. In general, wash conditions are selected to be

approximately 12-20°C below the Tm of the hybrid. In regards to the nucleic acids of the current invention, a moderate stringency hybridization is defined as hybridization in 6X SSC, 5X Denhardt's solution, 0.5% SDS and 100 μg/ml denatured salmon sperm DNA at 42°C, and washed in 2X SSC and 0.5% SDS at 55°C for 15 minutes. A high stringency hybridization is defined as hybridization in 6X SSC, 5X Denhardt's solution, 0.5% SDS and 100 μg/ml denatured salmon sperm DNA at 42°C, and washed in IX SSC and 0.5% SDS at 65°C for 15 minutes. A very high stringency hybridization is defined as hybridization in 6X SSC, 5X Denhardt's solution, 0.5% SDS and 100 μg/ml denatured salmon sperm DNA at 42°C, and washed in 0.1 X SSC and 0.5% SDS at 65°C for 15 minutes.

The term "oligonucleotide," as used herein, refers to nucleic acid sequences, primers, and probes of the present invention, and is defined as a nucleic acid molecule comprised of two or more ribo or deoxyribonucleotides, preferably more than three. The exact size of the oligonucleotide will depend on various factors and on the particular application and use of the oligonucleotide.

The phrase "small, interfering RNA (siRNA)" refers to a short (typically less than 30 nucleotides long, more typically between about 21 to about 25 nucleotides in length) double stranded RNA molecule. Typically, the siRNA modulates the expression of a gene to which the siRNA is targeted. The term "short hairpin RNA" or "shRNA" refers to an siRNA precursor that is a single RNA molecule folded into a hairpin structure comprising an siRNA and a single stranded loop portion of at least one, typically 1 -10, nucleotide.

The term "RNA interference" or "RNAi" refers generally to a sequence- specific or selective process by which a target molecule (e.g., a target gene, protein or RNA) is downregulated via a double-stranded RNA. The double-stranded RNA structures that typically drive RNAi activity are siRNAs, shRNAs, microRNAs, and other double-stranded structures that can be processed to yield a small RNA species that inhibits expression of a target transcript by RNA interference.

The term "antisense" refers to an oligonucleotide having a sequence that hybridizes to a target sequence in an RNA by Watson-Crick base pairing, to form an RNA:oligonucleotide heteroduplex with the target sequence, typically with an mRNA. The antisense oligonucleotide may have exact sequence complementarity to the target sequence or near complementarity. These antisense oligonucleotides may block or inhibit translation of the mRNA, and/or modify the processing of an mRNA to produce a splice variant of the mRNA. Antisense molecules may be as long as the target sequence (e.g., mRNA). Antisense oligonucleotides are typically between about 5 to about 100 nucleotides in length, more typically, between about 7 and about 50 nucleotides in length, and even more typically between about 10 nucleotides and about 30 nucleotides in length.

An "antibody" or "antibody molecule" is any immunoglobulin, including antibodies and fragments thereof, that binds to a specific antigen. The term includes polyclonal, monoclonal, chimeric, single domain (Dab) and bispecific antibodies. As used herein, antibody or antibody molecule contemplates recombinantly generated intact immunoglobulin molecules and immunologically active portions of an immunoglobulin molecule such as, without limitation: Fab, Fab', F(ab')2, F(v), scFv, scFv2, scFv-Fc, and the like.

With respect to antibodies, the term "immunologically specific" refers to antibodies that bind to one or more epitopes of a protein or compound of interest, but which do not substantially recognize and bind other molecules in a sample containing a mixed population of antigenic biological molecules. In a particular embodiment, the antibody may be immunologically specific for PH-20 and fail to substantially recognize or bind other hyaluronidases, particularly acidic hyaluronidases.

II. Administration

In accordance with the instant invention, at least one hyaluronidase inhibitor is administered to a patient to increase myelination and/or treat a demyelinating disease and/or inhibit the onset or progression of a demyelinating disease. Hyaluronidase inhibitors will generally be administered to a patient (i.e., human or animal subject) in a composition with a pharmaceutically acceptable carrier. For example, the hyaluronidase inhibitor(s) may be formulated with an acceptable medium such as water, buffered saline, ethanol, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol and the like), dimethyl sulfoxide (DMSO), oils, detergents, suspending agents or suitable mixtures thereof. The concentration of the

hyaluronidase inhibitor in the chosen medium may be varied and the medium may be chosen based on the desired route of administration of the pharmaceutical preparation. Except insofar as any conventional media or agent is incompatible with the hyaluronidase inhibitor, its use in the pharmaceutical preparation is contemplated.^■ In yet another embodiment, the pharmaceutical compositions of the present invention can be delivered in a controlled release system, such as using an intravenous infusion, an implantable osmotic pump (e.g., a subcutaneous pump), a transdermal patch, liposomes, or other modes of administration. In a particular embodiment, a pump may be used (see Langer (Science (1990) 249: 1527-1533); Sefton, CRC Crit. Ref. Biomed. Eng. (1987) 14:201 ; Buchwald et al., Surgery (1980) 88:507; Saudek et al., N. Engl. J. Med. (1989) 321 :574). In another embodiment, polymeric materials may be employed (see Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Press: Boca Raton, Florida (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley: New York (1984); Ranger and Peppas, J. Macromol. Sci. Rev. Macromol. Chem. (1983) 23:61 ; see also Levy et al., Science (1985) 228: 190; During et al., Ann. Neurol. (1989) 25:351 ; Howard et al., J. Neurosurg. (1989) 71 : 105). In yet another embodiment, a controlled release system can be placed in proximity of the target tissues of the animal, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, in Medical Applications of Controlled Release, supra, (1984) vol. 2, pp. 1 15-138). In particular, a controlled release device can be introduced into an animal in proximity to the desired site. Other controlled release systems are discussed in the review by Langer (Science (1990) 249: 1527-1533).

The dose and dosage regimen of the hyaluronidase inhibitor(s) that are suitable for administration to a particular patient may be determined by a physician considering the patient's age, sex, weight, general medical condition, and the specific condition for which the hyaluronidase inhibitor is being administered and the severity thereof. The physician may also take into account the route of administration, the pharmaceutical carrier, and the biological activity of the administered hyaluronidase inhibitor. Selection of a suitable pharmaceutical preparation will also depend upon the mode of administration chosen. For example, hyaluronidase inhibitor may be administered by direct injection into an area of demyelination (e.g., demyelinated lesion). In this instance, a pharmaceutical preparation comprises the hyaluronidase inhibitor dispersed in a medium that is compatible with the site of injection. The hyaluronidase inhibitor may be administered by any method such as intravenous injection into the blood stream, oral administration, or by subcutaneous, intramuscular or intraperitoneal injection. Pharmaceutical preparations for injection are known in the art. If injection is selected as a method for administering the hyaluronidase inhibitor, steps should be taken to ensure that sufficient amounts of the molecules reach their target cells to exert a biological effect.

As stated hereinabove, inhibitors of hyaluronidases, particular inhibitors of neutral pH hyaluronidases such as PH-20, are administered to a subject. As explained herein, hyaluronidase inhibitors having significant inhibitory activity against acidic hyaluronidases such as HYAL1 and HYAL2 may have adverse side effects, particularly upon systemic administration. To lessen or avoid these potential adverse effects, hyaluronidase inhibitors which have significant inhibitory activity against acidic hyaluronidases may be administered locally (e.g., by direct injection to an area of demyelination or demyelinated lesion).

Pharmaceutical compositions containing a hyaluronidase inhibitor as the active ingredient in intimate admixture with a pharmaceutically acceptable carrier can be prepared according to conventional pharmaceutical compounding techniques. The carrier may take a wide variety of forms depending on the form of preparation desired for administration, e.g., intravenous, oral, direct injection, intracranial, and

intravitreal.

A pharmaceutical preparation of the invention may be formulated in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form, as used herein, refers to a physically discrete unit of the pharmaceutical preparation appropriate for the patient undergoing treatment. Each dosage should contain a quantity of active ingredient calculated to produce the desired effect in association with the selected pharmaceutical carrier. Procedures for determining the appropriate dosage unit are well known to those skilled in the art.

Dosage units may be proportionately increased or decreased based on the weight of the patient. Appropriate concentrations for alleviation of a particular pathological condition may be determined by dosage concentration curve calculations, as known in the art.

In accordance with the present invention, the appropriate dosage unit for the administration of the hyaluronidase inhibitor may be determined by evaluating the toxicity of the molecules in animal models. Various concentrations of the

hyaluronidase inhibitor in pharmaceutical preparations may be administered to mice (e.g., an EAE mouse) or other animals, and the minimal and maximal dosages may be determined based on the beneficial results and side effects observed as a result of the treatment. Appropriate dosage unit may also be determined by assessing the efficacy of the hyaluronidase inhibitor treatment, optionally, in combination with other standard drugs. The dosage units of the hyaluronidase inhibitor may be determined individually or in combination with each treatment according to the effect detected.

The pharmaceutical preparation comprising the hyaluronidase inhibitor may be administered at appropriate intervals, for example, at least twice a day or more until the pathological symptoms are reduced or alleviated, after which the dosage may be reduced to a maintenance level. The appropriate interval in a particular case would normally depend on the condition of the patient.

Pharmaceutical compositions comprising at least one hyaluronidase inhibitor may be administered in coordination with at least one other agent used to increase myelination and/or treat a demyelinating disease, as described hereinabove. The hyaluronidase inhibitor and the other demyelinating disease therapeutic agent may be administered together in a single composition or may be administered in separate compositions. Additionally, the hyaluronidase inhibitor and the other demyelinating disease therapeutic agent may be administered at the same time or on different schedules.

The following examples are provided to illustrate certain embodiments of the invention. They are not intended to limit the invention in any way.

EXAMPLE I

Hyaluronan (HA) is a non-sulfated, protein-free glycosaminoglycan found in nearly all extracellular matrices. In vertebrates, HA molecules are synthesized by at least three independent hyaluronan synthases (Has 1-3) that extrude HA into the extracellular matrix where they have distinct activities depending on their size (Spicer et al. (1999) Biochem. Soc. Trans., 27: 109-15). HA molecules are comprised of unbranched, repeating disaccharide units of glucuronic acid and N-acetyl-D- glucosamine that range in molecular mass from ~2 xlO⁵ to ~10xl0⁶ Da. HA turnover is controlled by the actions of hyaluronidases. There are six hyaluronidase-like (HYAL) genes in mammals. In humans, one of these genes, PHYAL1, is a pseudogene. Some hyaluronidases, such as PH20 (also called Sperm adhesion molecule- 1) are only expressed by a limited number of cell types, while others are more widely expressed. It has been proposed that two of these widely expressed enzymes, both of which optimally function at acidic pH, are involved in HA degradation (Stern, R. (2003) Glycobiology 13: 105R-1 15R). In this model, high molecular weight (HMW; e.g. >10⁶ Da) HA molecules bind to a transmembrane HA receptor called CD44 and to the GPI-anchored hyaluronidase Hyal2 in lipid raft structures (caveolae). HA molecules are reduced to cleavage products of about 50 disaccharide units by Hyal2 that are internalized and transported to lysosomes where they are further degraded by another hyaluronidase, Hyall , and by β-Ν- acetylglucosaminidase and β-glucuronidase. How HA degradation is regulated in the CNS is unclear.

It was previously discovered that HA accumulates in demyelinating lesions from patients with MS, in mice with experimental autoimmune encephalomyelitis (EAE; a demyelinating disease that mimics multiple sclerosis), and in rats with traumatic spinal cord injuries (Back et al. (2005) Nat. Med., 1 1 :966-72; Struve et al. (2005) Glia 52: 16-24). Aberrant HA accumulation is linked to elevated expression CD44 whose overexpression by OPCs leads to HA accumulation and dysmyelination (Tuohy et al. (2004) Glia 47:335-45; Back et al. (2005) Nat. Med., 1 1 :966-72). This HA appears to be synthesized predominantly by reactive astrocytes as a HMW molecule. Adding this form of HA to lysolecithin-demyelinated corpus callosum lesions inhibited remyelination and resulted in the accumulation of OPCs that failed to become myelin basic protein + (MBP+) OLs (Back et al. (2005) Nat. Med., 1 1 :966- 72). Furthermore, adding this form of HA to OPC cultures reversibly inhibited their maturation (Back et al. (2005) Nat. Med., 11 :966-72).

These findings supported the hypothesis that HMW HA itself prevented remyelination by inhibiting OPC maturation. This hypothesis was tested by determining if OPC maturation in HA-rich environments could be induced following chronic treatment of the cells with hyaluronidases. Both Streptomyces hyaluronidase, which, like mammalian Hyal l and Hyal2, is active at acidic pH, and bovine testicular hyaluronidase, which is comprised mostly of PH20 and Hyal5 that can function at neutral pH, were tested. Surprisingly, although Streptomyces hyaluronidase did not significantly influence OPC maturation, bovine testicular hyaluronidase inhibited maturation (Figure 1A). Overexpression of PH20 or Hyal5 via lentiviral infection similarly inhibited OPC maturation (Figures IB and 1 C; note lack of MBP in PH20+ cells). This effect was not observed following overexpression of Hyall or Hyal2.

These findings suggested that HA degradation products, and not high molecular weight HA, inhibited OPC maturation. This hypothesis was tested by treating HA with an average molecular weight of 10⁶ Da with 100 U/ml of either Streptomyces hyaluronidase or bovine testicular hyaluronidase for 2 hours, then heat inactivating the enzymes and treating OPCs with the resulting products in vitro. It was determined that cultures treated with Streptomyces hyaluronidase-degraded HA still matured into myelin basic protein (MBP)-immunoreactive OLs, while cells treated with bovine testicular hyaluronidase-treated HA failed to mature (Figure 2). Treatment with heat-inactivated enzymes alone had no effect on OPC maturation. Furthermore, bovine testicular hyaluronidase-degraded HA but not Streptomyces hyaluronidase-degraded HA inhibited remyelination in mice with lysolecithin-induced demyelinated lesions. Together, these studies indicate that HA degradation products of neutral but not acidic hyaluronidases can potently block OPC maturation and inhibit remyelination.

Given that both high molecular weight HA and HA degradation products can inhibit OPC maturation and remyelination, it was hypothesized that OPCs that are recruited to demyelinated lesions may express neutral hyaluronidases that degrade the high molecular weight HA found in demyelinated lesions. Enriched cultures of mouse OPCs express Hyall , Hyal2, Hya and PH20 but not Hyal5, while mature OLs express only low levels of Hyal2 as assessed by RT-PCR (Figure 3 A). As discussed above, Hyall and Hyal2, both acidic hyaluronidases, are believed to be mostly involved in HA degradation with Hyall predominantly localized to lysosomes. HyaB has no apparent hyaluronidase activity of its own but may modulate Hyall (Atmuri et al. (2008) Matrix Biol., 27:653-60; Hemming et al. (2008) Glycobiology 18:280-9). Thus, the only neutral pH-acting hyaluronidase that degrades extracellular HA which is expressed by OPCs is PH20. It was confirmed that OPCs but not OLs express high levels of PH20 by immunocytochemistry with two different PH20 antibodies (Figures 3B and 3C). Furthermore, when OPCs were plated on HA-coated coverslips, HA was digested as indicated by labeling with a biotinylated HA-binding protein. These data indicate that OPCs express active hyaluronidases, including a neutral pH-acting hyaluronidase whose expression is downregulated as OPCs mature into OLs.

OPCs accumulate at chronic demyelinated lesions in MS patients and in mice with experimentally induced demyelination, but fail to mature into OLs even when spared demyelinated axons are present (Scolding et al. (1998) Brain 121 :2221-8; Wolswijk, G. (1998) J. Neurosci., 18:601-9; Chang et al. (2000) J. Neurosci.,

20:6404-12; Wolswijk, G. (2002) Brain 125:338-49; Chang et al. (2002) N. Engl. J. Med., 346: 165-73). Therefore, if PH20 activity blocks OPC maturation in MS lesions, then one would expect to see elevated PH20 expression by OPCs or other cells in areas of chronic demyelination. PH20 immunoreactivity was examined in cortical lesions from 3 MS patients and elevated PH20 expression was observed in cells with the morphology of reactive glia at lesion borders (Figure 4).

Hyaluronidase expression was also examined in lumbar spinal cord lesions of mice with EAE at different times post-inoculation with a myelin-oligodendrocyte glycoprotein peptide. PH20 was upregulated in these lesions during the period of active demyelination. Collectively, these data indicate that PH20 expression is increased in demyelinated and demyelinating lesions.

Given that PH20 is chronically elevated in demyelinated lesions and is expressed by OPCs, and that PH20 degradation products inhibit OPC maturation and remyelination, it was predicted that inhibiting hyaluronidase activity would promote OPC maturation. This hypothesis was tested by treating OPCs with a previously characterized hyaluronidase inhibitor, Vcpal (L-ascorbic acid 6-hexadecanoate; Botzki et al. (2004) J. Biol. Chem., 279:45990-7) then assaying the numbers of cells expressing MBP (to label OLs) versus PDFGF-Ra (to label OPCs) as described for Figure 1. Vcpal blocks the activities of both Streptomyces hyaluronidase and bovine testicular hyaluronidase (Botzki et al. (2004) J. Biol. Chem., 279:45990-7). It was found that >80% of OPCs grown in the presence of HA and treated with Vcpal became MBP+ OLs while <12% of cells treated with vehicle demonstrated MBP immuno-reactivity. These data indicate that pharmacological inhibitors of hyaluronidases promote OPC maturation in HA-rich microenvironments such as those observed in chronic demyelinated lesions.

A possible concern about utilizing hyaluronidase inhibitors with broad specificity for any systemic therapy is that they could have significant side effects. This is especially a concern for Hyall and Hyal2 given that Hyall- and Hyal2-null mice have numerous aberrant phenotypes related to HA accumulation including skeletal, joint and hematological abnormalities (Martin et al. (2008) Hum. Mol.

Genet, 17: 1904-15; Jadin et al. (2008) FASEB J., 22:4316-26). A series of N- substituted indole-2- and 3-carboxamide derivatives have been synthesized and characterized that function as potent hyaluronidase inhibitors (Olgen et al. (2007) Chem. Biol. Drug Des., 70:547-51) especially at neutral pH (Olgen et al. (2007) Chem. Biol. Drug Des., 70:547-51 ; Kaessler et al. (2008) J. Enzyme Inhib. Med. Chem., 23:719-27). The most active compound at pH 7.0 identified so far is N- (Pyridin-4yl)-[5-bromo-l-(4-fluorobenzyl)indole-3-yl]carboxamide with an IC50 value of 46 μΜ (Kaessler et al. (2008) J. Enzyme Inhib. Med. Chem., 23:719-27). This compound was tested using OPCs grown in the presence of HA as described above. It was determined that, like Vcpal, it inhibited HA degradation by OPCs (Figure 5) and promoted >80% of treated cells to mature into MBP+ OLs at 25 μΜ. These findings indicate that particular indole carboxamide derivatives are effective inhibitors of hyaluronidases that can function at neutral pH, like PH20.

N-(Pyridin-4yl)-[5-bromo-l-(4-fluorobenzyl)indole-3-yl]carboxamide

All together, these data indicate that HMW HA accumulates in chronically demyelinated lesions, that OPCs and other cells within these lesions express elevated levels of the hyaluronidase PH20, that OPCs and other cells can degrade HMW HA into products that act to inhibit OPC maturation and remyelination, and that indole carboxamide derivatives and that agents that inhibit hyaluronidases effectively promote OPC maturation and remyelination. Accordingly, inhibitors of neutral pH hyaluronidases such as PH20 can be used for novel therapeutic strategies to promote remyelination in a wide range of diseases and conditions characterized by

demyelination.

EXAMPLE 2

The destruction of myelin sheaths that surround axons in the central nervous system (CNS) causes conduction deficits in affected neurons that can lead to motor, sensory and cognitive deficits. Demyelination occurs following numerous insults to the CNS and is the hallmark of multiple sclerosis (MS). Pre-myelinating

oligodendrocytes (OLs) and OL progenitors (OPCs) are recruited to demyelinated lesions of MS patients and of mice with experimental autoimmune encephalomyelitis (EAE), a demyelinating disease that mimics features of MS pathology (Nait- Oumesmar et al. (2008) J. Neurol. Sci., 265:26-31). OPCs can mature into OLs that remyelinate demyelinated axons. However, OPCs often accumulate at chronic demyelinated lesions and fail to give rise to myelinating OLs (Wolswijk, G. (2002) Brain 125:338-49; Chang et al. (2000) J. NeuroscL, 20:6404-12; Chang et al. (2002) N. Engl. J. Med., 346: 165-73; Wolswijk, G. (1998) J. Neurosci., 18:601 -9; Scolding et al. (1998) Brain 121 :2221-8; Maeda et al. (2001) Ann. Neurol., 49:776-85).

Strategies that promote OPC maturation within demyelinated lesions therefore have the potential to promote remyelination and functional recovery in affected individuals.

High molecular weight (HMW) forms of the glycosaminoglycan hyaluronan (HA) accumulate in demyelinating lesions and are linked to remyelination failure (Back et al. (2005) Nat. Med., 1 1 :966-72; Sloane et al. (2010) Proc. Natl. Acad. Sci., 107:1 1555-60). HA is a glycosaminoglycan synthesized by transmembrane synthases and composed of multiple disaccharide units of glucuronic acid and N- acetylglucosamine. HA molecules range in size from <2.5xl0⁵ Da to >4xl0⁶ Da. Different molecular weight forms of HA have distinct functions in the nervous system including inducing cell motility, regulating cell growth, and regulating cell differentiation (Sherman et al. (2008) Trends Neurosci., 31 :44-52). During inflammatory responses in the periphery, activated fibroblasts or other cells secrete hyaluronidases that degrade HA, generating HA oligosaccharides that act as immune regulators (Jiang et al. (201 1) Physiol. Rev., 91 :221-64). Reactive oxygen species at sites of inflammation further promote this degradation (Soltes et al. (2006) Biomacromolecules, 7:659-68). Although hyaluronidases are expressed in the CNS (Sloane et al. (2010) Proc. Natl. Acad. Sci., 107: 1 1555-60; Al'Qteishat et al. (2006) Brain 129:2158-76), it is unclear if HA is similarly degraded during CNS

inflammation where astrocytes are the principle source of HA (Marret et al. (1994) J. Neurochem., 62: 1285-95).

HMW HA blocks OPC maturation and prevents remyelination following lysolecithin-induced demyelination (Back et al. (2005) Nat. Med., 1 1 :966-72;

Sloane et al. (2010) Proc. Natl. Acad. Sci., 107: 1 1555-60). Degradation of HA in astrocyte-OPC co-cultures with Streptomyces hyaluronidase results in increased OL maturation and HMW HA pre-treated with Streptomyces hyaluronidase does not prevent remyelination suggesting that HMW HA itself signals the inhibition of OPC maturation (Back et al. (2005) Nat. Med., 1 1 :966-72). In contrast, treatment of OPCs with HA degraded by a mammalian hyaluronidase followed by β-glucuronidase blocked OPC maturation in vitro (see, e.g., Sloane et al. (2010) Proc. Natl. Acad. Sci., 107: 1 1555-60). It is possible, therefore, that specific HA degradation products in demyelinated lesions rather than HMW HA cause remyelination failure. Here, it is shown that a specific hyaluronidase, called PH20, is expressed by OPCs and astrocytes in demyelinated lesions and blocks OPC maturation. Degradation products of this hyaluronidase but not other hyaluronidases prevent remyelination, while inhibition of hyaluronidase activity promotes OPC maturation and remyelination. All together these data indicate that agents that block PH20 activity or expression could promote remyelination in demyelinated lesions with spared axons.

METHODS

Reagents

HMW HA (100 μg; 1.59 x 10⁶ Da, Seigaku, Japan) was dissolved in sterile PBS and HA fragments were generated with addition of BTH (Sigma, St. Louis, MO, 100 U/ml), StrepH (Sigma, 1-10 U/ml) or PBS vehicle for 1 hour at 37°C then incubated at 95-100°C for 30 minutes to heat inactivate enzymes. HA fragments were analyzed by electrophoresis on a 0.5% agarose gel, followed by detection of HA using the cationic dye Stains-All (Sigma) as previously described (Lee et al. (1994) Anal. Biochem., 219:278-87). 4-MU (Sigma) was dissolved in PBS at 37°C and added to cultures at a final concentration of 1 mM. VCPAL (Sigma) was dissolved in DMSO at a concentration of 100 mM and further diluted to a working concentration of 25μΜ for cell culture experiments and for co-injection into lysolecithin lesions. Turbidity assays for VCPAL activity and IC50 calculations were performed as previously described (Botzki et al. (2004) J. Biol. Chem., 279:45990-7). Lentiviral Construction and Infections

The open reading frame of PH20, HYAL1, HYAL2 and HYAL5 were cloned in front of the CMV promoter of a vector plasmid and packaged into a third generation lentiviral vector. Cells were plated at 4-5 x 10⁴ cells per coverslip and infected overnight using a MOI of 1 :50- 1 : 100.

Cell Culture

Neural stem cells were isolated from the medial and lateral ganglionic eminences of embryonic day 13.5 mouse (C57BL/6) embryos and expanded in epidermal growth factor and fibroblast growth factor-2 (10 ng/ml) as neurospheres for one week as previously described (Zhang et al. (1998) J. Neurocytol., 27:475-89). Neurospheres were dissociated into single cells in trypsin (0.05%, Invitrogen), washed in DMEM plus 10% fetal bovine serum and plated at 5 x 10⁶ cells/ml on uncoated polystyrene plates in DMEM/F12 media containing 0.1 % BSA, PDGF-AA and FGF2 (20 ng/ml each), B27 supplement minus vitamin A (GIBCO®), Nl supplement (Sigma) and D-Biotin (10 nM, Sigma) for conversion into OPCs. Small floating spheres of cells formed and were passaged once a week in following dissociation with accutase (Invitrogen, Carlsbad, CA). After 2-3 weeks mouse Oligospheres' were transferred to poly-L-ornithine-coated 100 mm dishes. After 1-2 passages, highly enriched populations (>95%) of PDGFRa+01ig2+04- OPCs (as assayed by immunocytochemistry) were obtained and further propagated for in vitro experiments. For maturation experiments, OPCs were plated at 4-5 lO⁴ cells per coverslip and differentiated in DMEM/F12, 0.1% BSA, plus triiodothyronine (30 nM, Sigma) and N-acetyl-L-cysteine (NAC, Sigma) as previously described (Zhang et al. (1998) J. Neurocytol., 27:475-89).

Lysolecithin Lesions

All animal experiments were approved by the Institutional Animal Care and Use Committee at the Oregon Health & Science University. Demyelination was induced in the rostral corpus callosum of 3-4 month old C57BL/6J mice by injection of lysolecithin (4% in PBS; Sigma) as previously described8 mixed with either vehicle (PBS), HMW HA, degraded HA, or VCPAL. Four days post-injection, the same volumes of PBS, HA, and VCPAL were re-injected. Brains were harvested, fixed and processed for immunohistochemistry as previously described (Back et al. (2005) Nat. Med., 1 1 :966-72).

Induction of EAE

EAE was induced in female C57BL/6J mice as previously described (Back et al. (2005) Nat. Med., 1 1 :966-72). Animals were anesthetized with isofluorane and perfussed transcardially with 100 U/ml sodium heparin ( Sigma- Aldrich) containing saline followed by 4% paraformaldehyde in PBS. Spinal cords were dissected and processed for immunohistochemistry as described below.

Immunohistochemistry

Cells were fixed for 30 minutes at room temperature in 4% paraformaldehyde and washed 3 times in PBS. Lumbar spinal cord tissue from mice with EAE was post-fixed for 12-16 hrs in 4% paraformaldehyde at 4°C, rinsed three times in PBS at room temperature, then cyroprotected in 30% sucrose overnight at 4°C. Tissues were embedded in Optimal Cutting Temperature (OCT) medium, rapidly frozen on dry ice and cryosectioned at a thickness of 10-12 μηα. Cells and tissues were pre-blocked in 10% heat-inactivated fetal bovine serum for 45 minutes. Primary antibodies were diluted in blocking buffer and cells or EAE tissue were incubated overnight at 4°C, rinsed in blocking buffer three times, then incubated with the appropriate species- specific fluoro-conjugated secondary antibodies (Alexa546 or Alexa488, Molecular Probes Inc., Carlsbad, CA) for 45 minutes. Antibodies used were: rat anti-PDGFR-a (1 :250, BD Pharmingen, San Diego, CA) mouse anti-04 (1 :500, Millipore, Billerica, MA) mouse anti MBP (1 : 1000, Sternburger Monoclonal, Baltimore, MD) rabbit anti GalC (1 : 100, Millipore) rabbit anti GFAP (1 : 1000, DAKO) rabbit anti MAP2 (1 : 1000, Millipore) rabbit anti OLIG2 (1 :500, Millipore). Polyclonal rabbit antibodies to PH20 were generously provided by James Overstreet (PH20, 1 : 1000) and Patricia DeLeon (msSPAM, 1 :400). HA was visualized by probing cells or tissues with biotinylated HA Binding Protein (HABP, 1 :200, Seigaku) followed by avidin- conjugated Cy3 (1 : 1000, Molecular Probes Inc.). Myelin was visualized with 30 minute incubation of EAE sections with Fluoromyelin (1 :400 in PBS, Invitrogen) following primary and secondary antibody application. Cell nuclei were visualized by DAPI staining (Hoeschst 33342, 1 : 15,000; Molecular Probes). Sections from mice with lysolecithin lesions were analyzed for MBP reactivity as previously described.

The use of tissues from individuals with MS was approved by the Human Subjects Committee at the University of Washington. For the analysis of MS patient lesions, paraffin sections from 3 MS patients were deparaffinized in xylene and then rehydrated in graded alcohols. Endogenous peroxidase activity was blocked with 0.3% (v/v) hydrogen peroxide in methanol before washing the slides in water. Slides were heated in citrate buffer (10 mmol/L, pH 6.0) for 5 minutes in a microwave for antigen retrieval. Sections were then pre-blocked in 5% normal goat serum. Primary antibody incubation was the same as indicated above, with the exception that rabbit anti PH20 was used at a 1 :500 dilution. Following biotinylated goat anti-rabbit secondary antibody binding, the avidin-biotin-peroxidase complex technique (Vectastain® ABC kit and NovaRED™, Vector Laboratories Inc.) were used for visualization. The substrate reaction was stopped by washing the slides in running water. Finally, the slides were dehydrated, and mounted with a permanent mounting medium. In separate experiments, sections were also double-labeled with anti-PH20 and either anti-MBP, anti-04, anti-GFAP, or anti-Ibal followed by fluorescence- conjugated secondary antibodies as above.

Image processing and cell counts were done in Photoshop® 3.0 and ImageJ, respectively. For cell counts, 10 fields were randomly selected and at least 500 cells counted per coverslip (3 coverslips per group). Mean cell numbers and standard deviations were calculated for each group. Student's t-test was used to determine statistical significance at <0.05% confidence.

RT-PCR and qRT-PCR

Total RNA was isolated from cells or tissues using Trizol (Invitrogen) following the manufacturer's instructions. mRNA was reverse transcribed into cDNA using random hex or oligo d(t) primers and a reverse transcriptase kit (Promega). mRNA sequences for each hyaluronidase were downloaded from the NCBI website (www.ncbi.nlm.nih.gov) and primers for RT-PCR were designed manually (supplied by Intergrated DNA Technologies). See Table 1 for primers used. RT-PCR was performed using GoTaq® or Superscript™ DNA transcriptase (Promega or

Invitrogen) in a Mastercycler® Gradient following the manufacturer's protocols. RT- PCR reaction products were analyzed by electrophoresis on a 1% agarose gel and amplicons visualized following staining with ethidimum bromide. Quantitative Real Time PCR (qRT-PCR) was preformed using predesigned primer and probe sets (Taqman® Assays, Applied Biosciences; HYAL1 : Mm00476206; HYAL2:

Mm0477731 ; and PH20 (SPAM1 : Mm00486329)), with an AB 7900HT fast PCR system using SDS 2.4 software. Data analysis was preformed using Microsoft Excel.

Table 1: PCR Primers and Products. SEQ ID NOs are provided in parentheses. Generation of embryonic neural stem cell-derived mouse OPC primary cultures Highly purified embryonic neural stem cell-derived mouse OPCs were generated to study the effects of HA on OPC differentiation and maturation. The earliest populations of OPCs in the mouse brain arise from the medial and lateral ganglionic emeninences (MGE and LGE); therefore, to enrich for OL progenitors, NSCs were isolated and expanded from embryonic day 14.5 ganglionic eminences to generate free floating 'neurospheres'. After 2 weeks in culture neurospheres where dissociated into single cells and transferred into media containing PDGF-AA and FGF2 to promote the generation of so-called 'oligospheres'. After approximately 2-4 weeks in OPC media, free-floating spheres became adherent and small migrating bipolar and tripolar cells co-expressing PDGFRa and OLIG2 began appearing.

Following several (4-6) passages, highly purified (>95%) stable populations of mouse oligodendrocyte progenitor cells, henceforth referred to as mOPCs, were obtained and further propagated for in vitro studies.

For OL differentiation and maturation, mOPCs were dissociated into single cells, plated on glass coverslips in Thyroid Hormone (T3) and N-acetylcystine (NAC) for 72-96 hours. Total percentage of bipolar and tripolar PDGFRalpha+ progenitors declined from greater than 95% to approximately 36% total cells over time coincident with the appearance of highly branched 04+ cells beginning 24 hours after exposure to T3/NAC indicating the differentiation of OPCs into immature pre-OLs. By 72-96 hours post T3/ AC exposure, numerous highly branched, membrane-rich 04+GalC+ and GalC+MBP+ cells were observed (>50%) consistent with previous reports describing the maturation of OL lineage cells. Contamination of mOPCs or maturing OLs by astrocytes (as assayed by GFAP+ immunoreactivity) or neurons (as assayed by DCX immunoreactivity) was less than 5%, indicating that a highly purified population of mouse OPCs capable of maturing into myelin producing

oligodendrocytes had been generated.

Mouse OPCs make HA as they differentiate and mature into myelianating OLs

In contrast to primary rat OPC cultures, which synthesize very low levels of HA, it was observed that embryonic NSC-derived primary mOPC cultures contained large quantities of HA under both proliferative and differentiating conditions as assayed by HA binding protein (HABP) cytoreactivity. As cells are continuously propogated and differentiated in the absence of serum, a source of exogenous HA, it was asked if mOPCs synthesize HA in vitro. mOPCs were plated in serum free media, fixed after 1 , 3, or 12 hours and stained for HABP and PDGFRa or OLIG2. HABP immunoreactivity was not observed during the first hour of plating yet HA clearly accumulated on the cell surface of and around PDGFRa+ and OLIG2+ progenitors by 3 hours, more particularly 12 hours, post plating. To confirm HA synthesis by mOPCs, total mRNA was isolated from mOPCs, reverse transcribed and RT-PCR performed. It was found that mOPCs contain transcripts for the all three HA synthases (HAS 1-3), the enzymes responsible for synthesizing HA. Hence, mouse neural stem cell derived OPCs are capable of making HA in vitro.

To examine the distribution of HA in differentiating OPC cultures, cells were fixed and stained for HABP and OL lineage markers at 24, 48, 72, and 96 hours following removal of PDGF-AA and FGF2 and addition of T3/NAC. Consistent with the hypothesis HMW HA blocks maturation of OPCs into mature myelin producing OLs, high concentrations of HA were seen surrounding immature platelet-derived growth factor receptor alpha positive (PDGFRa+) OPCs and 04+ pre-OLs, while HABP immunoreactivity is largely absent from the soma and processes of mature, membrane rich MBP+ OLs. HA accumulation around PDGFRa+ OPCs began 3 hours post plating. Less HA is associated with cells expressing 04 or MBP and having the morphology of maturing OLs.

Viral constructs

The Transgene expression vector (LV-intron -GFP) used for cloning is described (Dissen et al. (2009) Methods 49:70-77). PH20 and HYAL5 cDNA were from Stephan Reitinger (extracellular Matrix Research Group, Institute for

Biomedical Aging Research, Austrian Academy of Sciences, Innsbruck, Austria) Hyal 1 cDNA was as described (Atmuri et al. (2008) Matrix Biol., 27:653-660). Hyal 2 was obtained by RT-PCR using the forward primer: 5'-

GAGTTCCTGAGCTGCTACCA-3 ' (SEQ ID NO: 13) and the reverse primer: 5'- AGGGGGAGAGATCCCTCATA-3 ' (SEQ ID NO: 14).

RESULTS

Degradation of endogenous HA by a mammalian hyaluronidase blocks OPC maturation in vitro

To test how inhibition of HA synthesis or degradation of HA influence OPC maturation, a culture system was generated in which mouse neural stem cells are differentiated in vitro into mouse OPCs that can subsequently be differentiated into pre-OLs and mature OLs. Under media conditions that promote OPC maturation, less than 60% of cells in these cultures express myelin basic protein (MBP), a marker of mature OLs, after 72 hours. Given that HA can block OPC maturation, it was determined whether these OPCs synthesized HA. By 12 hours post-plating, elevated HA synthesis was observed in OPC cultures that increased over time. It was therefore tested if the prevention of HA synthesis or the removal of HA from these

differentiating OPC cultures would promote OL maturation. To block HA synthesis, OPCs were switched to media containing the HA synthase inhibitor 4- methylumbelliferone (4-MU, ImM). Unexpectedly, it was found that inhibiting HA synthesis with 4-MU had no significant effect on OL maturation compared to controls despite the nearly complete absence of HA in these cultures (Figure 6A, 6B, 6D).

Surprisingly, degradation of HA using bovine testicular hyaluronidase (BTH) potently inhibited OPC maturation (Fig. 6A, 6C, 6D). This inhibitory activity was reversed by heat inactivation of the enzyme. To test if the inhibitory effects of BTH on OPC maturation were the specific result of HA degradation as opposed to a more general breakdown of

glycosaminoglycans in the cultures, OPC maturation in cultures treated with BTH (as above) was compared with chondroitinase ABC (CS'ase; which degrades chondroitin sulfate into unsaturated disaccharides) or with Streptomyces hyaluronidase (StrepH), each at concentrations that were optimal for their substrates. OPC maturation was only inhibited in the BTH-treated cultures (Fig. 6E). It was found that StrepH generated different sizes (larger) of HA oligosaccharides compared to BTH. Taken together, these data indicate that specific HA degradation products generated by BTH inhibit OPC maturation.

HA degradation products are sufficient to inhibit remyelination in vivo

It was then determined whether the HA breakdown products generated by BTH activity are capable of blocking remyelination. Focal demyelination was induced in the corpus callosum of mice using lysolecithin as previously described (Back et al. (2005) Nat. Med., 1 1 :966-72). After 4 days, a second injection of either vehicle, HMW HA that had been incubated with vehicle, BTH-degraded HMW HA, or StrepH-degraded HMW HA, was delivered into the original lesion site and mice were allowed to recover for 6 days (n > 6 per group). OPC maturation and remyelination were assessed by analysis of MBP immunoreactivity in sections through lesions as previously described (Back et al. (2005) Nat. Med., 1 1 :966-72). Compared to vehicle controls (Fig. 7 A), lesions injected with HMW HA (Fig. 7B shows typical example) or BTH-degraded HA (Fig. 7C) failed to remyelinate as shown by the lack of MBP immunoreactivity at the injection site, while animals treated with StrepH-degraded HA remyelinated to the same degree as vehicle controls (Fig 7D). Based on these results, it is clear that the HA-degradation products produced by BTH are capable of blocking OL maturation in demyelinated lesions where HA accumulates. OPCs degrade HA and express hyaluronidases

The majority of the hyaluronidase activity in BTH is attributed to PH20 (also called sperm adhesion molecule- 1 or SPAM1) and HYAL5 with only very low activity from other hyaluronidases including HYAL1 and HYAL2 (Meyer et al. (1997) FEBS Lett., 413:385-8). Using only immunohisotchemistry with single antibodies showed that OPCs express multiple hyaluronidases, including PH20 (see, e.g., Sloane et al. (2010) Proc. Natl. Acad. Sci., 107: 1 1555-60). It was therefore hypothesized that OPCs that are recruited to demyelinating lesions expressing hyaluronidases that then degrade the HMW HA synthesized by reactive astrocytes in the lesion microenvironment. Total RNA was isolated from mouse testes (as a positive control for testicular hyaluronidases), OPCs grown in vitro, and from adult mouse corpus callosum, then performed RT-PCR using primers specific for the hyaluronidases with known hyaluronidase activity in BTH (HYAL1 , HYAL2, HYAL5 and PH20). It was found that HYAL1, HYAL2 and PH20 but not the testes- specific HYAL5 are expressed by OPCs and in white matter (Fig. 8A). HYAL1, HYAL2 and PH20 transcripts were also amplified from RNA isolated from whole brain, cortex and spinal cord.

It was then determined if OPCs are capable of degrading HA. OPCs were plated onto coverslips uniformly coated with HMW HA (approximately 1.59 MDa) and allowed to differentiate for 24 or 72 hours, then fixed and labeled with an anti-04 antibody and a biotinylated HA-binding protein (HABP). It was found that OL lineage cells are capable of degrading HA, as seen by loss of HABP reactivity around the soma and processes of 04+ cells at 24 hours (Fig. 8B, 8C) and as large holes in the HA coated surface after 72 hours (Fig. 8D) corresponding to the presence of 04+ membranes (Fig. 8E). Cells with the morphologies of mature OLs also demonstrated an absence of HA staining around their somas and processes, although this degradation of HA may have occurred as the cells matured into pre-myelinating OLs (Fig. 8E). Collectively, these data indicate that OPCs are capable of using hyaluronidases to degrade the HMW HA found within chronic demyelinated lesions.

Pharmacological inhibition of hyaluronidase activity promotes OL maturation and remyelination

Given that OPCs express multiple hyaluronidases and that degradation products of BTH inhibit OPC maturation, it was reasoned that blocking the activity of hyaluronidases, and therefore the generation of inhibitory HA breakdown products, would promote OL maturation. Hyaluronidase activity in OPC cultures was inhibited with the hyaluronidase inhibitor 6-O-Palmitoyl-L-ascorbic acid (VCPAL) for 72-96 hours. Consistent with previous studies, it was found that VCPAL inhibited BTH activity with an IC50 of 25-35 μΜ. Treated and control (vehicle) cultures were examined for changes in the expression of the OL lineage markers PDGFRa to label OPCs and MBP to label mature OLs. VCPAL treatment prevented HA degradation and significantly increased the proportion of cells that became mature OLs, assayed as the total percentage cells expressing MBP as compared to cultures treated with vehicle alone (Fig. 9A-9C).

To assess whether inhibition of hyaluronidase activity is sufficient to promote remyelination, VCPAL was co-injected with HMW HA into lysolecithin-induced corpus callosum lesions. In animals with lysolecithin-induced lesions, VCPAL resulted in elevated MBP immunoreactivity, overcoming the effects of HMW HA, while there was reduced MBP immunoreactivity in animals treated with HMW HA and vehicle (Figure 9D-9E). These findings indicate that the hyaluronidase activity of OPCs or other cells in demyelinating lesions contributes to the impairment of OL maturation leading to the remyelination failure seen in chronically demyelinated MS plaques, and indicate that hyaluronidase inhibition is an efficacious strategy to promote remyelination.

PH20 is the hyaluronidase expressed by OPCs that blocks OPC maturation

A gain-of-function strategy was used to assess which of the hyaluronidases found in BTH inhibit OPC maturation. The cDNAs from HYAL1 , HYAL2, HYAL5 and PH20 were cloned into bicistronic lentiviral expression vectors carrying the cDNA for enhanced green fluorescence protein (EGFP). OPCs were then infected with lentiviruses carrying these vectors and analyzed for changes in OPC maturation as described above. Cells infected with the PH20-carrying viruses demonstrated a dramatic and significant (p<0.0009) inhibition of OPC maturation, as assessed by quantification of MBP+ cells, compared to cultures infected with a virus carrying only the EGFP cDNA (Fig. 1 OA- IOC). Infection with the HYAL1 -bearing virus had no significant effect on OL maturation while cells transduced with HYAL2 and HYAL5 were able to weakly inhibit OL maturation (p<0.002 and p<0.02, respectively; Fig. IOC). Cells infected with hyaluronidase vectors demonstrated reduced levels of pericellular HA (e.g. inset, Fig. 10B) which was not observed in cells infected with EGFP alone (e.g. inset, Fig. 10A). Taken together these results indicate that overexpression of PH20, a major component of BTH, generates HA breakdown products capable of blocking OL maturation. PH20 expression is elevated in demyelinating lesions

Given the data demonstrating that PH20 transcripts are expressed by OPCs and in the corpus callosum, and that elevated PH20 expression is sufficient to inhibit OPC maturation, PH20 expression was further characterized in OPCs and mature OLs, and assess whether PH20 expression is altered in demyelinating lesions. To verify that the RT-PCR amplified transcripts were indeed mouse PH20, three distinct sets of primers were used to amplify separate regions of PH20 mRNA isolated from adult mouse brain and were sequenced, confirming that OPCs were expressing bona fide PH20 RNA.

PH20 protein expression was analyzed in proliferating OPCs and maturing

OLs by immunocytochemistry using two separate PH20 antibodies in combination with the OL lineage specific markers PDGFRa, 04 or MBP. Consistent with the notion that OL lineage cells are capable of degrading HA as they mature, high PH20 immunostaining was seen in the soma and processes of immature PDGFRoH- OPCs as well as 04+ pre-OLs (Fig. 1 1 A-l ID) but was less intense and restricted to the soma of mature MBP+ OLs (Fig. 1 lE-1 1H). All together, these data indicate that both the levels and localization of PH20 change as OPCs mature into OLs.

Given that HMW HA accumulates in chronic demyelinated lesions, that PH20 can degrade HMW HA, that PH20 breakdown products can inhibit OPC maturation and remyelination, and that PH20 is only found at low levels in the mature brain, it was hypothesized that PH20 expression may be upregulated in demyelinated lesions. In mice with EAE, PH20 was elevated in demyelinated spinal cord lesions (Fig. 1 11- 1 1L) where it was expressed by both reactive astrocytes and occasionally by OPCs. Furthermore in chronically demyelinated plaques of MS patients, PH20

immunoreactivity was enriched at the borders of lesions (Figure 4A, arrows).

Numerous cells expressed PH20 in the lesion borders (Figures 4B-4C), while small numbers of cells with the morphologies of reactive glia expressed PH20 in areas of complete demyelination (Figure 4D). Double labeling immunohistochemistry demonstrated that the majority of the PH20 staining in MS patients originated from reactive astrocytes and from 04+ OPCs. These data indicate that PH20 expression is elevated in demyelinating lesions.

It has been demonstrated for the first time that the PH20 hyaluronidase is elevated in astrocytes and OPCs in demyelinating lesions from MS patients and rodents with EAE; that elevated expression of PH20 by OPCs leads to the generation of HA breakdown products that inhibit OPC maturation; and that inhibiting hyaluronidase activity leads to enhanced OPC maturation and remyelination in vivo. These data indicate a model for remyelination failure in which HMW HA synthesized by reactive astrocytes and other cells within demyelinated lesions is degraded into specific classes of HA oligosaccharides by PH20 expressed by glial cells in the lesion microenvironment. These oligosaccharides in turn inhibit OPC maturation. Thus, inhibiting hyaluronidase activity or blocking signaling by hyaluronidase-generated HA oligosaccharides are potentially efficacious strategies for promoting

remyelination.

HA oligosaccharides may influence OPCs through a number of mechanisms. A study in Xenopus tadpoles demonstrated that glycogen synthase kinase-3

(GSK3 ), a serine/threonine protein kinase that is part of the Wnt signaling cascade, is activated by HA signaling (Contreras et al. (2009) Development 136:2987-96). The Wnt signaling cascade and GSK3P in particular has been implicated in OPC maturation (Fancy et al. (2009) Genes Dev., 23: 1571 -85; Feigenson et al. (2009) Mol. Cell Neurosci., 42:255-65; Azim et al. (201 1) Glia 59:540-53; Tawk et al. (201 1) J. Neurosci., 31 :3729-3742). Inhibition of GSK3P stimulates remyelination in adult mice (Azim et al. (201 1) Glia 59:540-53). It is possible therefore that HA

oligosaccharides inhibit OPC maturation at least in part through the activation of GSK3p. HA oligosaccharides may also signal through toll-like receptor-2 or -4 (Termeer et al. (2002) J. Exp. Med., 195:99-1 1 1 ; Taylor et al. (2004) J. Biol. Chem., 279: 17079-84; Jiang et al. (2005) Nat. Med., 1 1 : 1 173-9; Scheibner et al. (2006) J. Immunol., 177: 1272-81 ; Shimada et al. (2008) Development 135:2001-1 1), both of which can also influence GSK3P activation (Kim et al. (2010) FEBS J., 277:2830-7; Zhang et al. (2009) Mol. Immunol., 46:677-87). Toll-like receptors are expressed by OPCs and one study has suggested that HA-mediated inhibition of OPC maturation is dependent on toll-like receptor 2 (Sloane et al. (2010) Proc. Natl. Acad. Sci.,

107:1 1555-60). The contributions of other HA receptors, including CD44 and the receptor for hyaluronan-mediated motility in regulating OPC maturation can be determined.

HYAL1 and HYAL2, for example, are widely distributed in many different tissues (Stern et al. (2006) Chem. Rev., 106:818-39). Hyall -null mice develop osteoarthritis (Martin et al. (2008) Hum. Mol. Genet., 17: 1904-15) while humans with Hyal l mutations develop a lysosomal storage disorder, mucopolysaccharidosis (MPS) IX (Triggs-Raine et al. (1999) Proc. Natl. Acad. Sci., 96:6296-300). Hyal2-null mice develop skeletal and hematological abnormalities (Jadin et al. (2008) FASEB J., 22:4316-26). In contrast, although PH20 mRNA has been detected at high levels in testis and at low abundance in a limited number of other tissues, PH20-null mice do not display any significant pathological phenotypes (Baba et al. (2002) J. Biol. Chem., 277:30310-4). The finding that PH20 is specifically upregulated in demyelinating lesions and is sufficient to block OPC maturation suggest that agents that target PH20 and not other hyalurondiases will be both safe and efficacious as long-term therapies for the promotion of remyelination.

While certain of the preferred embodiments of the present invention have been described and specifically exemplified above, it is not intended that the invention be limited to such embodiments. Various modifications may be made thereto without departing from the scope and spirit of the present invention, as set forth in the following claims.

Claims

What is claimed is

1. A method for inhibiting a demyelinating disease or reducing the risk of developing a demyelinating disease in a patient in need thereof comprising

administering to said patient a composition comprising at least one hyaluronidase inhibitor and a pharmaceutically acceptable carrier.

2. The method of claim 1 , wherein said method further comprises the administration of at least one other demyelinating disease therapeutic agent.

3. The method of claim 2, wherein said demyelinating disease therapeutic agent is selected from the group consisting of interferon beta- l a, interferon beta- lb, glatiramer acetate, mitoxantrone, and natalizumab.

4. The method of claim 1 , further comprising monitoring said patient for the presence of demyelination.

5. The method of claim 1 , wherein said demyelinating disease is multiple sclerosis.

6. The method of claim 1 , wherein said demyelinating disease is a state of demyelination caused by ischemic conditions, conditions linked to hypoxia, inflammatory conditions, traumatic injury, and age-related myelin damage.

7. The method of claim 1 , wherein said hyaluronidase inhibitor inhibits neutral pH acting hylauronidases.

8. The method of claim 7, wherein said neutral pH acting hyaluronidase is

PH20.

9. The method of claim 1 , wherein said hyaluronidase inhibitor is N-(Pyridin- 4yl)-[5-bromo-l -(4-fluorobenzyl)indole-3-yl]carboxamide.

10. The method of claim 1 , wherein said hyaluronidase inhibitor is vcpal (6-0- palmitoyl-L-ascorbic acid).

1 1. The method of claim 1 , wherein said hyaluronidase inhibitor is an antibody or antibody fragment immunologically specific for PH-20.

12. The method of claim 1, wherein said hyaluronidase inhibitor is

administered directly to a demyelinated lesion by direct injection.

13. A method for increasing myelination in a patient in need thereof comprising administering to said patient a composition comprising at least one hyaluronidase inhibitor and a pharmaceutically acceptable carrier.

14. A composition comprising at least one hyaluronidase inhibitor, at least one other demyelinating disease therapeutic agent, and a pharmaceutically acceptable carrier.

15. The composition of claim 14, wherein said demyelinating disease therapeutic agent is selected from the group consisting of interferon beta- la, interferon beta- lb, glatiramer acetate, mitoxantrone, and natalizumab.

16. A kit comprising a first composition comprising at least one hyaluronidase inhibitor and a pharmaceutically acceptable carrier and a second composition comprising at least one other demyelinating disease therapeutic agent and a pharmaceutically acceptable carrier.