CA2284105A1 - Synthetic saposin c-derived neurotrophic peptides - Google Patents

Abstract

Non-naturally occurring synthetic peptide analogs derived from the active neurotrophic region of saposing C. The saposin C-derived peptides induce neurite outgrowth in vitro, promote myelination, promote neuroprotection and prevent programmed cell death and have an analgesic effect. They are useful in the treatment of central and peripheral nervous system disorders and pain management.

Description

SYNTHETIC SAPOSIN C-DERIVED NEUROTROPHIC PEPTIDES
Field of the Invention The present invention relates to neurotrophic peptides and their methods of use. More specifically, the invention relates to synthetic peptides related to the active neurotrophic fragment located within saposin C.
~ 5 Background of the Invention Demyelination is a defect common to a number of central nervous system (CNS) disorders, the most prevalent being multiple sclerosis (MS). MS, a chronic disorder which may lead to total disability. is characterized by damage to the myelin sheath, while leaving the axons mostly intact. There is currently no effective treatment for MS. Other central nervous system disorders involving demyelination include acute disseminated encephalomyelitis, amyotrophic lateral sclerosis, acute hemorrhagic leukodystrophy, progressive muhifocal leukoencephalitis, metachromatic leukodystrophy and adrenal leukodystrophy. The peripheral nervous system (PN51 can also be afflicted with demye6nation, such as that occurring in Guiliain-Barn syndrome (Pathologic Basis of Disease, Robbins et al.
eds., W.B. Sounders, Philadelphia, 1979, pp. i 578-1582).
Peripheral nerve injuries and peripheral neuropathies, such as those resulting from diabetes or chemotherapy, comprise the most prevalent peripheral nervous system disorders. Current treatments for peripheral nervous system disorders only treat the symptoms, not the cause of the disease.
Heurotrophins are proteins or peptides capable of affecting the survival, target innervation andlor function of neuronal cell populations (Bards, Neuron, 2:1525-1534. 1989). The efficacy of neurotrophins both in vivo and in vitro has been well documented. For example, nerve growth factor (NGF) acts as a trophic factor for forebrain cholinergic, peripheral and sensory neurons (Hefti et al., Neurobio. Aging, 10:515-533, 19891. In v'rvo experiments indicate that NGF can reverse naturally-occurring as well as physical traumatic injuries to peripheral nerves (Rich et al., J. Neurncytol., 16:261-268, 1987). Brain-derived neurotrophic factor (BDNF) is a trophic factor for peripheral sensory neurons, dopaminergic neurons of the substantia nigra, central choiinergic neurons and retinal ganglia (Henderson et al., Rector. Neurol. Neurosci., 5:15-28, 1993). BDNF has been shown to prevent normally-occurring cell death both in vitro and u~ viva fHofer et al., Nature, 331:262-262, 1988). Ciliary neurotrophic factor (CNTF) promotes survival of chicken embryo ciliary ganglia in vitro and supports survival of cuhured sympathetic, sensory and spinal motor neurons (Ip et al.. J. Physiol. Paris, 85:123-130, 1991 ).
Prosaposin is the precursor of a group of four small heat-stable glycoproteins which are required for hydrolysis of glycosphingolipids by lysosomal hydroiases (Kishimoto et al., J.
Lipid Res., 33:1255-1267, 1992).
- 30 Prosaposin is proteoiyticaby processed in lysosomes, generating saposins A, B, C and D (0'Brien et al., fASEB J., 5:301-308, 1991). 0'Brien et aL (Proc. Natl. Aced Sci. U.S.A., 91:9593-9596, 1994), U.S. Patent Nos. 5,571,787, 5,698,080, 5,714,459 and published PCT Application No. W095103821, disclose that prosaposin and saposin C
stimulate neurite outgrowth and promote increased myelination. In addition, these references disclose that a ZZ-mer peptide ICEFLVKEUTKLIONNKTEKEIL; SEO 10 N0: 1) consisting of amino acids 8-29 of human saposin C stimulated neurite outgrowth in both neuroblastoma cells and mouse cerebellar explants.
These references also disclose that an iodinated 1B-mer peptide (YKEVTKLIONNKTEKEIL; SEO 10 N0: 2) contained within the active 22-mer of saposin C iwith 11 replaced by Y1 also promoted neurite outgrowth and was able to cross the blood-brain barrier. 0'Brien et al. (fASEB J., 9:681-685, 19951 showed that the 22-mer stimulated choline acetyitransferase activity and prevented cell death in neuroblastoma cells in vitro. The active neuritogenic fragment was localized to a linear 12-mer located in the amino-terminal sequence of saposin C (LIDNNKTEKEIL; SEO ID
N0: 31.
There is a significant need for neurotrophic peptides having modified structural stability and~or activities.
The present invention addresses this need.
Summary of the Invention the present invention provides modified peptides based on the naturally.occurring saposin C sequence, and particularly based on neuritogenic fragments of saposin C. The modifications to these peptides can address issues of activity, stability and persistence.
One embodiment of the present invention is a neurotrophic, myelinotrophic or neuroprotective non-native peptide preferably having up to about 50 amino acids and including the sequence shown in SEO ID N0: 8, with the proviso that the peptide does not have the sequence shown in SEO ID N0: 4.
More preferably, the peptide has up to about 30 amino acids. More preferably, the peptide has between about 12 and 25 amino acids. Preferably, the amino acid at position 1 of SEO ID N0: 8 is isoleucine. Advantageously, the amino acid at position 3 of SEO ID
N0: 8 is not aspartic acid. In another aspect of this preferred embodiment, the amino acid at position 8 of SED ID
N0: 8 is not glutamic acid. Preferably, the amino acid at position 10 of SEO
ID N0: 8 is not glutamic acid.
Advantageously, the amino acid at position 11 of SEO 10 N0: 8 is not leucine.
preferably, the amino acid at position 12 of SEO ID N0: 8 is not leucine. Further, the peptides described above may be acetylated or esterified with a 2D fatty acid.
Another embodiment of the invention is a method of stimulating neural cell outgrowth, promoting neuroprotection or promoting increased myelination comprising the step of contacting neuronal cells with a composition comprising an effective neurotrophic and myelinotrophic concentration of a non-native peptide having up to about 50 amino acids and including the sequence shown in SEO ID N0: 8, with the proviso that the peptide does not have the sequence shown in SEO ID N0: 4. Preferably, the neuronal cells are neuroblastoma cells.
Advantageously, the neuroblastoma cells are NS20Y cells. According to one aspect of this preferred embodiment, the contacting step occurs in vitro. Alternatively, the contacting step occurs in viva.
The present invention also provides a method of treating neuropathic pain in a mammal in need thereof, comprising the step of administering an effective pain-treating amount of a non-native neurotrophic peptide fragment of saposin C, the peptide having up to about 50 amino acids and including the sequence shown in SEO ID N0: 8, with the proviso that the peptide does not have the sequence shown in SEO ID
N0: 4. Preferably, the administering step is intravenous, intramuscular, intradermal, subcutaneous, intracranial, epidural, topical, oral, transdermal, transmucosal or intranasal.
The present invention also provides a method of treating sensory or motor neuropathy in a mammal in need thereof, comprising administering an effective sensory or motor neuropathy-treating amount of a non-native neurotrophic peptide fragment of saposin C, the peptide having up to about 50 amino acids and including the I
r i WO 9$/42746 PCT/US98/05503 sequence shown in SEO 10 N0: 8, with the proviso that the peptide does not have the sequence shown in SEQ ID
N0: 4. Preferably, the administering step is intravenous, intramuscular, intradermal, subcutaneous, intracranial, epidural, topical, oral, transdermal, transmucosal or intranasal. Further, the peptide may be acetylated or esterified with a fatty acid.
Still another embodiment of the invention is a pharmaceutical composition comprising a non-native neurotrophic peptide fragment of saposin C having up to about 50 amino acids and including the sequence shown in SEO ID N0: 8, with the proviso that the peptide does not have the sequence shown in SEO ID N0: 4, in a pharmaceutically acceptable carrier. Preferably, the composition is a controlled release formulation. The composition may be in liposomal, lyophilized or unit dosage form.
Brief Descriotian of the Drawinos Figure 1 illustrates a NS20Y neuroblastoma neurite outgrowth assay using peptides TX14(A) (TXLIDNNATEEILY; X-D-alanine; SEO 10 N0: 4) and a rat 14-mer derived from the saposin C active sequence (SELIINNATEELLY; SEO ID N0: 5).
Figure 2 illustrates a cell death assay using NS20Y neuroblastoma cells. NS20Y
cells were grown for 48 hours in tow serum in the presence or absence of TX14(A) and dead cells were identified by Trypan blue staining.
Detailed Oescriotion of the Preferred Embodiments The present invention includes the discovery that non-naturally occurring variants encompassing the active neurotrophic region of saposin C stimulate neurite outgrowth, prevent neural cell death, promote myelination, inhibit demyeiination, promote neuroprotectian and can be used to treat various neuropathies. As used herein, a neuropathy is a functional disturbance or pathological change in the peripheral nervous system and is characterized clinically by sensory or motor neuron abnormalities.
A native 15-mer (TKLIDNNKTEKEILD; SEO ID N0: 6) contained within human saposin C and encompassing the active neurite-promoting region shown in SEO ID N0: 3 was modified as follows to decreases its susceptibility to proteolysis in rivo: Lys 2 was replaced with D-ale to increase resistance to exopeptidases; iys 8 was replaced with ale to increase resistance to trypsin digestion; and lys 11 was deleted to increased resistance to trypsin digestion. In addition, asp 15 was replaced with tyr to provide an iodination site. Thus, the resulting peptide, TX14(A), contained no cleavage sites for trypsin or chymotrypsin. Peptide TX141A) exhibited neuritogenic activ'tty in vitro neurite outgrowth assays. TX14(A) also prevented cell death in neuroblastoma cells in culture.
It is also contemplated that the leucine at position three andlor 14 can be changed to an isoleucine with retention of activity. This will prevent degradation by enkephalinase which cleaves at hydrophobic residues.
Replacement of leucine residues also helps minimize degradation by angiotensin converting enzyme (ACE) which also cleaves at hydrophobic residues. In another preferred embodiment, lys 8 is an amino acid other than aianine, lysine or arginine to prevent degradation by dipeptidyl pept~ase (alanine) or trypsin (lysine, arginine).
Saposin C-derived peptides comprising the active 12-mer region of the sequence shown in SEO iD N0: 3 (LIDNNKTEKEIL; SED ID N0: 7), and neurotrophic analogs thereof, possess significant therapeutic applications in promoting functional recovery after toxic, traumatic, ischemic, degenerative and inherited lesions to the peripheral and central nervous system. In addition, these peptides stimulate myelination and counteract the effects of demyefinating diseases. These peptides stimulate the outgrowth of neurons, promote myelination, promote neuroprotection and prevent programmed cell death in neuronal tissues. The peptides of the invention can also be used to treat various neuropathies including, but not limited to, motor, sensory, peripheral, taxol-induced and diabetic neuropathies. The peptides are also useful as analgesics, particularly for the treatment of neuropathic pain which can develop days or months after a traumatic injury and is often long-lasting or chronic.
SEO ID N0: 7 may be modified as follows and still retain neurotrophic activity: Leu 1 may be leu or ile;
Ile 2 is essential; asp 3 is any amino acid; asn 4 and asn 5 are essential;
lys 6 is any amino acid, preferably not lysine or arginine: thr 7 is essential, glu 8 is a charged amino acid; lys 9 is absent or a charged amino acid, 1 D preferably not lysine or arginine; glu 10 is any charged amino acid; ile 11 is any amino acid; leu 12 is any amino acid. These guidelines produce the following consensus sequence:
X,IX2NNX3TX4X5XsX~XB (SEO ID N0: 8) in which I is isoleucine; X, is leucine or isoleucine; XZ is any amino acid; N
is asparagine; X3 is any amino acid; X, is lysine, arginine, histidine, aspartic acid or glutamic acid; X5 is absent, lysine, arginine, histidine, aspartic acid, glutamic acid or glycine; X6 is lysine, arginine, histidine, aspartic acid or glutamic acid; X, is any amino acid; and X8 is any amino acid.
The second asparagine residue within the native prosaposin sequence (corresponding to second "N" in SEQ
ID N0: 81 is known to be glycosylated with N-acetylglucosamine which may provide some resistance to protealytic degradation. The synthetic modification of this asparagine residue within the instant non-native saposin C-derived peptides by standard methods (i.e. Merrifieid synthesis) with various carbohydrates, preferably glucose, is also within the scope of the present invention.
One embodiment of the present invention is a method of facilitating neurite outgrowth or increased myelination in differentiated or undifferentiated neural cells by administering to the cells an effective, neurite outgrowth or myelin-facilitating amount of a saposin C-derived peptide encompassing the active 12-mer region shown in SEQ ID N0: 7 )amino acids 18-29 of saposin C) or, more preferably, non-natural analogs thereof including the sequence shown in SEQ ID N0: 8.
Non-natural saposin C-derived peptide analogs of the invention further include, for example, replacement of one or more lysine and(or arginine residues; replacement of one or more tyrosine andlor phenylalanine residues, deletion of one or more phenylalanine residues and conservative replacement of one or more amino acids within the peptide. The replacement or deletion of lysinelarginine and tyrosinelphenylalanine residues will reduce the susceptibility of peptide degradation by trypsin and chymatrypsin, respectively. The non-native neurotrophic and myeiinotrophic peptide sequences of the invention preferably have up to about 50 amino acids; more preferably, up to about 30 amino acids; and most preferably, between about 12 and 25 amino acids and include therein the sequence shown in SEfl ID N0: 8.
In one preferred embodiment, the peptide does not contain the sequence shown in SEO ID N0: 4. In another preferred embodiment, the amino acid at position 6 of SEO ID N0: 8 is not alanine. In still another preferred embodiment, the amino acid at position 1 of SEQ ID N0: 8 is isofeucine. In yet another preferred embodiment, the amino acid at position 3 of SEO ID NO: 8 is not aspartic acid. In yet another preferred embodiment, the amino acid at position 8 of SEO ID ND: 8 is not giutamic acid. In additional preferred embodiments, the amino acids at positions 10, 11 and 12 of SEO ID N0: 8 are not giutamic acid, isoleucine and leucine, respectively.
Additional variations of these peptide sequences contemplated for use in the present invention include minor insertions, deletions and substitutions. For example, conservative amino acid replacements are contemplated. Such replacements are, for example, those that take place within a family of amino acids that are related in the chemical nature of their side chains. The families of amino acids include the basic charged amino acids (lysine, arginine, histidineh the acidic charged amino acids (aspartic acid, glutamic acid), the non-polar amino acids (alanine, valine, leucine, isoleucine, praline, phenylalanine, methionine, tryptophan); the uncharged polar amino acids (glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine); and the aromatic amino acids (phenyialanine, tryptophan and tyrosine). In particular, it is generally accepted that conservative amino acid replacements consisting of an isolated replacement of a leucine with an isoleucine or valine, or an aspartic acid with a glutamic acid, or a threonine with a serine, ar a similar conservative replacement of an amino acid with a structurally related amino acid will not significantly affect the properties of the peptide. The non-native saposin C
sequences containing SEO ID N0: 8 therein can be modified to attain various objectives such as increased activity and stability. Other amino acids can be present outside this consensus sequence including native saposin C
sequence, conservative substitutions of these native sequences, or unrelated peptide sequences to achieve objectives such as increased binding, hydrophobicity, hydrophiliciiy and the like. Sequences outside the active neurotrophic region are not typically required for activ'tty.
Thus, in most instances, the subject peptide will be active regardless of these sequences. Again, any such peptide can be screened for such activity using the protocols described herein.
The ability of any such peptide to stimulate neurite outgrowth, prevent neural cell death, promote myelination and inhibit demyelination can easily be determined by one of ordinary skill in the art using the procedures described in Examples 1-4. Methods for assaying the abilities of these non-naturally occurring peptides to promote myelination and to inhibit demyelination are set forth in in Examples 3 and 4 hereinbelow.
A typical minimum amount of the peptides of the invention for the neurotrophic activity in cell growth medium is usually at least about 5 ng/ml. This amount or more of the non-naturally occurring synthetic peptides of the invention for in vitro use is contemplated. Typically, concentrations in the range of 0.1 iuglml to about 10 pglml of these peptides will be used. Effective amounts for any particular tissue can be determined in accordance with Example 1.
The neural cells can be treated in vitro or ex v'rvo by directly administering the peptides of the invention to the cells. This can be done, for example, by cuhuring the cells in growth medium suitable for the particular cell type, followed by addition of the peptide to the medium. When the cells to be treated are in vivo, typically in a vertebrate, preferably a mammal, the composition can be administered by one of severe! techniques. Most preferably, the composition is injected directly into the blood or tissue in sufficient quantity to give the desired local concentration of peptide. In the peptides lacking lysine and arginine residues, proteolytic degradation is reduced.

The smaller peptides (i.e., 20-mer or less) will most likely cross the blood brain barrier and enter the central nervous system for treatment of CNS disorders (see Banks et al., Peptides, 13:1289-1294, 1992?.
The peptides of the invention may also be esterified with fatty acids to form peptide fatty acid esters using conventional acid-catalyzed esterification. Alternatively, the last amino acid added in the synthetic procedure is itself a commercially available esterified amino acid which obviates the need for the esterification reaction. Fatty acids contemplated for use in formation of peptide esters include fauric, myristic, palmitic, stearic, oleic and linoleic.
The subject peptides may also be acetylated by inclusion of commercially available acetylated lysine, arginine or asparagine residues during the synthetic procedure. These modified peptides retain the activity of the parent compound.
These modifications will facilitate the ability of the peptide to cross the blood brain barrier due to increased hydrophobicity.
For treatment of neural disorders, direct intracranial injection or injection into the cerebrospinal fluid may also be used in sufficient Quantities to give the desired local concentration of neurotrophin. In both cases, a pharmaceutically acceptable injectable carrier is used. Such carriers include, for example, phosphate buffered saline and Ringer's solution. Alternatively, the composition can be administered to peripheral neural tissue by direct local injection or by systemic administration. Various conventional modes of administration are contemplated, including intravenous, intracerebrospinal, intramuscufar, intradermal, subcutaneous, intracraniaf, intranasal, epidural, topical and oral. For use as an analgesic, administration by direct intramuscular or intravenous injection is preferred.
The peptide compositions of the invention can be packaged and administered in unit dosage farm, such as an injectabie composition or local preparation in a dosage amount equivalent to the daily dosage administered to a patient or as a controlled release composition. A septum sealed vial containing a daily dose of the active ingredient in either PBS or in lyophilized form is an example of a unit dosage.
Appropriate daily systemic dosages of the peptides of the invention based on the body weight of the vertebrate for treatment of neural diseases or as an analgesic are in the range of from about 10 to about 100 Ng/kg, although dosages from about 0.1 to about 1,000 Nglkg are also contemplated. Thus, for the typical 70 kg human, dosages can be between 7 and 70,000 dug daily, preferably between 700 and 7,000 Ng daily. Daily dosages of locally administered material will be about an order of magnitude less. Oral administration is also contemplated.
In one preferred embodiment of the invention, the neurotrophic peptides are administered locally to neural cells in vivo by implantation thereof. For example, polylactic acid, polygalactic acid, regenerated collagen, multilamellar liposomes and many other conventional depot formulations is expressly contemplated in the present invention. infusion pumps, matrix entrapment systems and combination with transdermal delivery devices are also contemplated. The peptides may also be encapsulated within a polyethylene glycol conformal coating as described in U.S. Patent No. 5,529,914 prior to implantation.
The neurotrophic peptides of the invention may also 6e enclosed in micelles or liposomes. Liposome encapsulation technology is well known. Liposomes may be targeted to specific tissue, such as neural tissue. through ,.

the use of receptors, ligands or antibodies capable of binding the targeted tissue. The preparation of these formulations is well known in the art (Radio et al., Meth. Enzymol., 98:613618, 19831.
There are currently no available pharmaceuticals capable of promoting full functional regeneration and restoration of the structural integrity of neural systems. This is particularly true of the CNS. Regeneration of peripheral nerves through the use of neurotrophic factors is within the scope of the invention. Moreover, neurotrophic factors can be therapeutically useful in the treatment of neurodegenerative diseases associated with the degeneration of neural populations or specific areas of the brain. The principal cause of Parkinson's disease is the degeneration of dopaminergic neurons of the substantia nigra. Since antibodies against prosaposin immunohistochemically stain the dopaminergic neurons of the substantia nigra in human brain sections, the neurotrophic peptides of the invention may be therapeutically useful in the treatment of Parkinson's disease. Retinal neuropathy, an ocular neurodegenerative disorder leading to loss of vision in the elderly, is also treatable using the peptides of the invention.
It has long been believed that in order to reach neuronal populations in the brain, neurotrophic factors would have to be administered intracerebrally since these proteins do not cross the blood brain barrier. U.S. Patent No. 5,571,787 discloses that an iodinated neurotrophic l8~mer fragment derived from saposin C crosses the blood brain barrier. Thus, the peptides having up to about 22 amino acids will also cross this barrier and can thus be administered intravenously. Other neuronal populations, such as motor neurons, can also be treated by intravenous injection, although direct injection into the cerebrospinal fluid is also envisioned as an alternate route.
Cells may be treated to facilitate myelin formation or to prevent demyeiination in the manner described above in v'rvo, ex vivo or in vitro. Diseases resulting in demyelination of nerve fibers including MS, acute disseminated leukoencephalitis, progressive multifocal leukoencephalitis, metachromatic leukodystrophy and adrenal leukodystrophy can be slowed or halted by administration of the neurotrophic peptides of the invention to the cells affected by the disease.
The compositions of the present invention can be used in vitro as research tools for studying the effects of neurotrophic factors and myelin facilitating materials. However, more practically, they have an immediate use as laboratory reagents and components of cell growth media for facilitating growth and maintaining neural cells in vitro.
The peptides of the invention can be synthesized using an automated sotid~phase protocol well known in the art on an Applied Biosystems Model 430 peptide synthesizer. All peptides were purified by high performance liquid chromatography (HPLC) on a llydac C4 column to an extent greater than 95% prior to use.
The following examples are illustrative and are not intended to limit the scope of the present invention.
Example 1 Stimulation of neurite outgrowth in vitro NS20Y neuroblastoma cells were grown in OMEM containing 10% fetal calf serum (FCS). Cells were removed with trypsin and plated in 30 mm petri dishes onto glass coverslips.
After 20-24 hours, the medium was replaced with 2 mI DMEM containing 0.5% FCS plus 0, 0.5, 1, 2, 4 or 8 nglml TX14(AI. Cells were cultured for an additional 24 hours, washed with PSS and fixed with Bouin's solution (saturated aqueous picric acidlformalinlacetic acid 15:5:1) for 30 minutes. Fixative was removed with PBS and neurite outgrowth was scored under a phase contrast microscope. Cells exhibiting one or more clearly defined neurites equal to or longer than one cell diameter were scored as positive. At least 200 cells were scored in different portions of each dish to determine the percentage of neurite bearing cells and assays were performed in duplicate.
As shown in Figure 1. TX14(A) and the rat 14-mer both induced neurite outgrowth in NS20Y ceps.
Increased neurite outgrowth was evident using as little as 0.5 nglml peptide resulting in a 7% increase for TX14(A) and rat 14-mer. At 1 mglml, TX14(A) and rat 14-mer resulted in a 109'o and 12%
increase, respectively, in neurite outgrowth. Both peptides stimulated neurite outgrowth to similar extents at 8 nglmi. This indicates that the peptides are biologically active.
Example 2 Prevention of cell death in vitro NS20Y cells were plated as described in Example 1 and grown nn glass coverslips in 0.5% fetal bovine serum for 2 days in the presence or absence of 8 nglml TX14(A). Media was removed and 0.2°Yo trypan blue in PBS
was added to each well. Blue-staining dead cells were scored as a percentage of the total on an inverted microscope, counting 400 cells in four areas of each well. The average error of duplicates was t5%. As shown in Figure 2, TX14(A) reduced the number of trypan blue-positive (dead) cells by about 7%. This indicates that the peptide can rescue neural cells from programmed cell death.
Example 3 fx vivo myelination assay Newborn mouse cerebellar explants are prepared according to Satomi (tool.
Sci., 9:127-137, 19821. Neurite outgrowth and myefination are observed over 22 days in culture, during the period when the newborn mouse cerebellum normally undergoes neuronal differentiation and myelination begins.
A 30~mer nonnative saposin C peptide containing the sequence shown in SEO ID N0: B (10 ~rglmll is added on the second day after preparation of the explants (three control and three treated expiants), and outgrowth of neurites and myelination is assessed under a bright field microscope with a video camera. On the eighth day, cultures containing the peptides are thinner and more spread out than control cultures. On day 15, peptide-treated cultures contain many cells with long projections at the periphery of the explant which are less prominent in untreated control cultures. Peptide-treated cultures contain significantly more myelinated axons in the subcortical white matter at 22 days compared to control explants.
Thus, the peptides of the invention induce increased myelination in differentiating cerebellum ex vivo.
Example 4 Prevention of demyeiination The prevention of Schwann cell death is correlated with prevention of demyelination. Schwann cells contain an extensive myelin sheath. The addition of a non-native 20 mer peptide containing the sequence shown in SEn ID
N0: 8 to Schwann cells in culture reduces Schwann cell death in a dose-dependent manner and stimulates the incorporation of sulfatide, myelin-specific lipids, into Schwann cells.

..... r i Example 5 Use of oeotides in treating traumatic ischemic CNS lesions Humans with traumatic lesions to the spinal cord receive intracerebrospinal or direct injection of about 100 Nglml TX14(A) or other peptide encompassed by SEO ID N0: 8 in a sterile saline solution or in depot form to enable stow, continuous release of the peptide at the lesion site. Improvement is assessed by gain of motor nerve function (i.e. increased limb movementl. Treatments continue until no further improvement occurs.
Example 6 Use of oeutides in treating demvelination disorders Patients diagnosed with early stage MS are given peptide TX14(A) or other peptide encompassed by SED
ID N0: 8 by direct intravenous injection into the cerebrospinal fluid using the same dose range as in Example 3.
Dosages are repeated daily or weekly and improvement in muscle strength, muscuioskeletal coordination and myelination (as determined by MRI) is gbserved.
Example 7 Alleviation of neurooathic yain in Chung model rats This example describes the effects of bolus intrathecal injection of TX 14(A) and other peptides encompassed by SEO iD N0: 8 in the Chung experimental model of peripheral neuropathic pain. Each peptide is chemically synthesized, purified, dissolved in sterile PBS and buffered to neutral pH.
The surgical procedure previously described by Kim et al. (Pain, 50:355, 1992) is performed on mate rats to induce an allodynic state. A spinal catheter is introduced two weeks after surgery, Five days later, the peptides are administered at 0.007, 0.07 and 0.7 pglrat.
Pressure thresholds are then determined using calibrated von Frey hairs. The longer the time taken for an animal to withdraw the paw in response to applied pressure, the less severe the neuropathic pain. The peptides significantly increase the threshold pressure, indicating a significant alleviation of neuropathic pain.
Example 8 Treatment of sensory neurooathv Mice are administered taxol in order to induce sensory neuropathy. Taxoi-treated mice are administered 50 ~uglkg, 100 auglkg or 250 Nglkg of TX14(A) or other peptide encompassed by SED ID N0: 8. The loss of thermal sensation is measured using a Hargreaves sensory testing apparatus as an indicator or sensory neuropathy. Each of the three doses of peptide is effective in inhib'tting loss of thermal sensation in taxohtreated mice. Thus, the synthetic saposin C-derived peptides of the invention effectively inhibit sensory neuropathy.
Example 9 Alleviation of neurooathic pain in diabetic rats This example describes the effects of intraperitoneal administration of TX141A) or other peptide encompassed by SEO ID N0: 8 in a rat model of diabetic neuropathy. .
Rats are made diabetic by a single intraperitoneal injection of streptozotgcin (50 mgJkg body weight, freshly dissolved in 0.9% sterile saline) to ablate pancreatic ,Q cells and induce insulin deficiency as described by Calcutt et ai. (Pain, 68:293-299, 19961. Two days later, diabetes is confirmed in streptozotocin-injected rats by measuring WO 98/42746 PCTlUS98/05503 blood glucose levels. Streptozotocin-injected animals with a blood glucose concentration below 15 mmoill were excluded from subsequent studies, according to the commonly accepted definition of non-fasting hyperglycemia in studies of diabetes in rats.
Both diabetic and control rats are studied at 8 weeks by analyzing the behavioral response to the noxious chemical formalin as an indicator of allodynia (Calcutt et al., supra, 1996).
Briefly, rats receive a subcutaneous injection of freshly-prepared formalin (50 NI of 0.59'a solution in sterile saline) into the dorsal surface of the right hind paw. This concentration of formalin induces sub-maximal behavioral responses in control rats and allows detection of hyperaigesia in diabetic rats during phases 0 and 2 (Calcutt et al., Eur. J. Pharmacvl., 285:189-197, 1995). Animals are transferred to an observation chamber constructed to allow continuous visualization of the paws.
The number of flinches during one minute periods is counted at 5 minute intervals for the next 60 minutes by an observer who is unaware of the treatment group of each animal. Phase 1 is defined as the initial measurement of flinching (1-2 and 5-6 minutes post injection); the Q (quiescent) phase as the measurements made at 10-11, 15-16 and 20-21 minutes; and Phase 2 as all subsequent measurements post-injection, as previously defined for studies of diabetic rats (see, for example, Malmberg et al., Neurosci. Lett., 161:45-48, 19931. Comparisons of activity during each phase are made by summing the flinches at measurement points within the phase. diabetic rats five an abnormal flinch response.
Diabetic rats are divided in two groups of four ananals each which are administered saline, TX141A) or another peptide encompassed by SEfl ID N0: 8, respectively. Two hours before treatment with 0.5% formalin, the diabetic rats are treated with saline or Z00 ~glkg peptide using intraperitoneal administration. Administration of peptide completely prevents the abnormal flinch response in Phase 1 and ameliorates the response in Phase 2 by 70%. Thus, parenteral administration of peptide alleviates the pain from formalin injection and improves motor neuron function in a rat model of painful diabetic neuropathy.
It should be noted that the present invention is not limited to only those embodiments described in the Detailed Description. Any embodiment which retains the spirit of the present invention should be considered to be within its scope. However, the invention is only limited by the scope of the following claims.
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SEQUENCE LISTING
(1) GENERAL INFORMATION
(i) APPLICANT: MYELOS NEUROSCIENCES CORP.
(ii) TITLE OF THE INVENTION: SYNTHETIC SAPOSIN C-DERIDED
NEUROTROPHIC PEPTIDES
fiii) NUMBER OF SEQUENCES: B
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Knobbe, Martens, Olson & Bear (B) STREET: 620 Newport Center Drive, 16th Floor (C) CITY: Newport Beach (D) STATE: CA
(EI COUNTRY: U.S.A.
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(A) MEDIUM TYPE: Diskette (B) COMPUTER: IBM Compatible (C) OPERATING SYSTEM: DOS
(D) SOFTWARE: FastSEO for Windows Version 2.0 (vi) CURRENT APPLICATION DATA:
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(B) FILING DATE:
fC) CLASSIFICATION:
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: 081823,425 (B) FILING DATE: 24-MAR-1997 (viii? ATTORNEYIAGENT INFORMATION:
(A) NAME: Bartfeld, Neil S
(B) REGISTRATION NUMBER: 39,901 ICI REFERENCEIDOCKET NUMBER: MYELOS.005VPC
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: 619-235-8550 (B) TELEFAX: 619-235-0176 (C) TELEX:
(2) INFORMATION FOR SEQ ID N0:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID N0:1:
Cys Glu Phe Leu Ual Lys Glu llal Thr Lys Leu Ile Asp Asn Asn Lys Thr Glu Lys Glu Ile Leu (2) INFORMATION FOR SEO ID N0:2:
(i) SEQUENCE CHARACTERISTICS:
(AI LENGTH: 18 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear Iii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEO ID N0:2:
Tyr Lys Giu Vai Thr Lys Leu Ife Asp Asn Asn Lys Thr Glu Lys Glu Ile Leu (2) INFORMATION FOR SEO ID N0:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID N0:3:
Leu Ile Asp Asn Asn Lys Thr Glu Lys Glu Ile Leu 1 5 t0 (2) INFORMATION FOR SEO ID N0:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (ix) FEATURE:
(A) NAMEIKEY: Modified amino acid (B) LOCATION: 2...2 (0) OTHER INFORMATION: D-alanine (xi) SEQUENCE DESCRIPTION: SEQ 10 N0:4:
Thr Xaa Leu Ile Asp Asn Asn Ala Thr Glu Glu Ile Leu Tyr (2) INFORMATION FOR SEQ ID N0:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID N0:5:
Ser Glu Leu Ile Ile Asn Asn Ala Thr Glu Glu Leu Leu Tyr (2) INFORMATION FOR SEQ lD N0:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids (B) TYPE: amino acid (C) STRANOEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID N0:6:
Thr Lys Leu Ile Asp Asn Asn Lys Thr Glu Lys Glu Ile Leu Asp 1 5 t0 15 (2) INFORMATION FOR SEQ ID N0:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 amino acids (B) TYPE: amino acid (C) STRANOEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRtPTtON: SEQ ID N0:7:

Leu Ile Asp Asn Asn Lys Thr Glu Lys Glu lle leu (2) INFORMATION FOR SEQ 10 NO:B:
(i) SEQUENCE CHARACTERISTICS:
(Al LENGTH: 12 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single fD) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (ix) FEATURE:
(A) NAMEIKEY: Modified amino acid (B) LOCATION: 1...1 (D) OTHER INFORMATION: leu, ile (AI NAMEIKEY: Modified amino acid (B) LOCATION: 3...3 (D) OTHER INFORMAT10N: any amino acid (A) NAMEIKEY: Modified amino acid (B) LOCATION: 6...6 (D) OTHER INFORMATION: any amino acid (A) NAMEIKEY: Modified amino acid (B) LOCATION: 8...8 (DI OTHER INFORMATION: fys, arg, his, asp, glu (A) NAME1KEY: Modified amino acid (B) LOCATION: 9...9 (D) OTHER INFORMATION: absent, lys, arg, his, asp, glu, gly (A) NAMEIKEY: Modified amino acid (B) LOCATION: 10...10 (D) OTHER INFORMATION: lys, arg, his, asp, glu (A) NAMEIKEY: Modified amino acid (B) LOCATION: 11...11 (D) OTHER INFORMATION: any amino acid (A) NAMEIKEY: Modified amino acid (B) LOCATION: 12...12 (D) OTHER INFORMATIDN: any amino acid (xi) SEQUENCE DESCRIPTION: SEQ ID N0:8:

Xaa Ile Xaa Asn Asn Xaa Thr Xaa Xaa Xaa Xaa Xaa

Claims (27)

1. An isolate neurotrophic, myelinotrophie or neuroprotective peptide having up to 50 amino acids and including the sequence shown in SEQ ID NO: 8, wherein said peptide is not present within the native sequence of prosaposin, saposin C or any native prosaposin-derived peptide, with the proviso that said peptide does not have the sequence shown in SEQ ID NO: 4.

2. The peptide of claim 1, wherein said peptide has up to 30 amino acids.

3. The peptide of claim 2, wherein said peptide has between 12 and 25 amino acids.

4. The peptide of claim 1, wherein the amino acid at position 1 of SEQ ID NO:
8 is Isoleucine.

5. The peptide of claim 1, wherein the amino acid of position 3 of SEQ ID NO:
8 is not aspartic acid.

6. The peptide of claim 1, wherein the amino acid at position 8 of SEQ ID NO:
8 is not glutamic acid.

7. The peptide of claim 1, wherein the amino acid at position 10 of SEQ ID NO:
8 is not glutamic acid.

8. The peptide of claim 1, wherein the amino acid at position 11 of SEQ ID NO:
8 is not isoleucine.

9. The peptide of claim 1, wherein the amino acid at position 12 of SEQ ID NO:
8 is not leucine.

10. The peptide of claim 1, wherein said peptide is acetylated or esterified with a fatty acid.

11. A method for stimulating neural cell outgrowth, promoting neuroprotection or promoting increased myslination, comprising the step of contacting neuronal cells with a composition comprising an effective neuritogenic, neuroprotective or myelinotrophic concentration of a peptide having up to 50 amino acids and including the sequence shown in SEQ ID NO: 8, wherein said peptide is not present within the native sequence of prosaposin, saposin C or any native prosaposin-derived peptide, with the proviso that said peptide does not have the sequence shown in SEQ ID NO: 4.

12. The method of claim 11, wherein said neuronal cells are neuroblastoma cells.

13. The method of claim 12, wherein said neuroblastoma cells are NS2OY cells.

14. The method of claim 11, wherein said contacting step occurs in vitro.c

15. The method of claim 11, wherein said contacting step occurs in vivo.

16. The method of claim 11, wherein said peptide is acetylated or esterified with a fatty acid.

17. A method for treating neuropathic pain in a mammal in need thereof, comprising the step of administering an effective pain-treating amount of an effective neuritogenic, neuroprotective or myelinotrophic concentration of a peptide having up to 50 amino acids and including the sequence shown is SEQ ID NO:8, wherein said peptide is not present within the native sequence of prosaposin, saposin C or any native prosaposin-derived peptide, with the proviso that said peptide does not have the sequence shown in SEQ ID NO:4.

18. The method of claim 17, wherein said administering step is selected from the group consisting of intravenous, intramuscular, introdermal, sub~utaneous, intracranial, epidural, topical, oral, transdermal, transmucosal end intranasal.

19. A peptide having up to 50 amino acids and including the sequence shown in SEQ ID
NO: 8, wherein said peptide is not present within the native sequence of prosaposin, saposin C or any native prosaposin-derived peptide, with the proviso that said peptide does not have the sequence shown in SEQ ID NO: 4, for treatment of neuropathic pain in a mammal.

20. A method for treating sensory or motor neuropathy in a mammal in need thereof, comprising the step of administering to said mammal an effective sensory or meter neuropathy-treating amount of a peptide having up to 50 amino acids and including the sequence shown in SEA ID NO:8, wherein said peptide is not present within the native sequence of prosaposin, saposin C or any native prosaposin-derived peptide, with the proviso that said peptide does not have the sequence shown in SEQ
ID NO: 4.

21. The method of claim 20, wherein said administering step is selected from the group consisting of intravenous, intramuscular, intradermal, subcutaneous, intracranial, epidural, topical, oral, transdermal, transmucosal and intranasal.

22. The method of claim 20, wherein said peptide is acetylated or esterified with a fatty acid.

23. A peptide having up to 50 amino acids and including the sequence shown in SEQ ID
NO: 8, wherein said peptide is not present within the native sequence of prosaposin, saposin C or any native prosaposin-derived peptide, with the proviso that said peptide does not have the sequence shown in SEQ ID NO: 4, for treatment of sensory or motor neuropathy in a mammal.

24. A pharmaceutical composition comprising the peptide of claim 1 in a pharmaceutically acceptable carrier.

25. The composition of claim 24 in liposomal form.

26. The composition of claim 24 in lyophilized form.

27. The composition of claim 24 in unit dosage form.