Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/475—Growth factors; Growth regulators
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
  • A61P25/02—Drugs for disorders of the nervous system for peripheral neuropathies
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
  • A61P25/04—Centrally acting analgesics, e.g. opioids
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
  • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
  • A61P25/16—Anti-Parkinson drugs
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
  • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P27/00—Drugs for disorders of the senses
  • A61P27/02—Ophthalmic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
  • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides

Definitions

• the present invention relates to neurotrophic and analgesic peptides More particularly, the invention relates to cyclic peptides derived from the active region of saposin C which have neurotrophic and analgesic effects
• Neurotrophic factors are proteins or peptides capable of affecting the survival, target innervation and/or function of neuronal cell populations (Barde, Neuron 2 1525- 1534, 1989)
• nerve growth factor acts as a trophic factor for forebrain cholinergic, peripheral and sensory neurons (Hefti et al , Neurobiol Aging 10 515-533, 1989) and can reverse naturally-occurring as well as physical traumatic injuries to peripheral nerves (Rich et al , J Neurocytol 16 261-268, 1987)
• Brain- de ⁇ ved neurotrophic factor (BDNF) is a trophic factor for pe ⁇ pheral sensory neurons, dopaminergic neurons of the substantia nigra, central cholinergic neurons and retinal ganglia (Henderson et al , Restor Neurol Neurosci 5 15-28, 1993
• Prosaposin is the precursor of a group of four small heat-stable glycoproteins which are required for hydrolysis of glycosphingolipids by lysosomal hydrolases (Kishimoto et al , J Lipid Res 33 1255-1267, 1992) Prosaposin is proteolytically processed in lysosomes, generating saposins A, B, C and D (O'Brien et al , FASEB J 5 301-308, 1991) O'Brien et al (Proc Natl Acad Sa USA 91 9593-9596, 1994),
• U S Patent No 5,571,787 and published PCT Application No WO95/03821 disclose that prosaposin, saposin C stimulate neurite outgrowth and promote increased myelination
• U S Patent Nos 5,571,787, 5,696,080, 5,714,459 and published PCT application No WO95/03821 disclose that a 22-mer peptide (CEFLVKEVTKLLDNNK
• the active neu ⁇ togenic fragment was localized to a linear 12-mer sequence located within the amino terminus of saposin C (LIDNNKTEKEIL, SEQ LD NO: 3) (O'Brien et al, FASEB J. 9:681-685, 1994).
• the present invention provides cyclic prosaposin peptidomimetics which have good resistance to proteolytic degradation and are capable of crossing the blood brain barrier.
• One embodiment of the present invention is a cyclic neurotrophic and analgesic peptide having between about 1 1 and 25 amino acids, and including the sequence
• Xi is a hydrophobic amino acid (alanine, leucine, isoleucine, valine, tyrosine, tryptophan, phenylalanine or methionine);
• X 2 is a hydrophobic amino acid,
• X 3 is aspartic acid, glutamic acid, lysine or arginine;
• N is asparagine,
• X 4 is any amino acid;
• T is threonine;
• X 5 is glutamic acid or aspartic acid;
• X ⁇ is any amino acid, X is a hydrophobic amino acid; and
• X 8 is a hydrophobic amino acid.
• the peptide has the amino acid sequence shown in SEQ LD NO: 5 or 6.
• the present invention also provides a composition
• a composition comprising the peptide described above in a septum sealed vial, formulated with a controlled release material, in lyophilized form, in liposomal form, in a form suitable for topical administration or in unit dosage form.
• Another embodiment of the present invention is a method for inducing myelination or inhibiting demyelination in a mammal, comprising administering to a mammal afflicted with demyelination a pharmaceutically effective demyelination inhibiting amount of a cyclic peptide having between about 11 and 25 amino acids, and including the sequence X]X 2 X 3 NNX4TX X6X7X , wherein Xi is a hydrophobic amino acid (leucine, isoleucine, valine, tyrosine, tryptophan, phenylalanine or methionine); X 2 is a hydrophobic amino acid, X 3 is aspartic acid, glutamic acid, lysine or arginine; N is asparagine, is any amino acid; T is threonine; X 5 is glutamic acid or aspartic acid; X 6 is any amino acid, X 7 is a hydrophobic amino acid;
• the peptide is administered in a pharmaceutically acceptable carrier.
• the peptide is enclosed in a lamellar structure.
• the peptide has the sequence shown in SEQ LD NOS: 5 or 6.
• the mammal is a human.
• the present invention also provides a method for inhibiting neural degeneration or promoting neurite outgrowth in neural tissue, comprising: contacting neural tissue susceptible to such degeneration with an effective neural degeneration-inhibiting amount of a cyclic peptide having between about 11 and 25 amino acids, and including the sequence X]X 2 X 3 NNX TX 5 X6X 7 X 8 , wherein Xi is a hydrophobic amino acid (leucine, isoleucine, valine, tyrosine, tryptophan, phenylalanine or methionine); X 2 is a hydrophobic amino acid, X 3 is aspartic acid, glutamic acid, lysine or arginine; N is asparagine, X is any amino acid; T is threonine; X 5 is glutamic acid or aspartic acid; X ⁇ is any amino acid, X 7 is a hydrophobic amino acid; and X 8 is a hydrophobic amino acid.
• Xi
• the administration is intravenous, intramuscular, intradermal. subcutaneous, intracranial, intracerebrospinal or topical.
• the peptide is administered in a pharmaceutically acceptable carrier.
• the peptide is enclosed in a lamellar structure.
• the mammal is a human.
• Still another embodiment of the invention is a method for treating neuropathic pain in a mammal in need thereof, comprising the step of administering to a mammal suffering from neuropathic pain an amount of a cyclic peptide effective to inhibit neuronal degeneration, wherein said peptide peptide has between about 11 and 25 amino acids, and including the sequence X ⁇ X 2 X 3 NNX4TX 5 X6X 7 X , wherein Xi is a hydrophobic amino acid (leucine, isoleucine, valine, tyrosine, tryptophan, phenylalanine or methionine); X is a hydrophobic amino acid, X 3 is aspartic acid, glutamic acid, lysine or arginine; N is asparagine, X is any amino acid; T is threonine; X 5 is glutamic acid or aspartic acid; X 6 is any amino acid, X is a hydrophobic amino acid; and
• the peptide has the amino acid sequence shown in SEQ LD NOS: 5 or 6.
• the peptide is administered in a pharmaceutically acceptable carrier.
• the peptide is enclosed in a lamellar structure.
• the mammal is a human.
• the present invention also provides a cyclic peptide having between about 1 1 and 25 amino acids, and including the sequence X ⁇ X 2 X 3 NNX 4 TX 5 X 6 X X 8 , wherein Xi is a hydrophobic amino acid (leucine, alanine isoleucine, valine, tyrosine, tryptophan, phenylalanine or methionine); X 2 is a hydrophobic amino acid, X 3 is aspartic acid, glutamic acid, lysine or arginine; N is asparagine, X is any amino acid; T is threonine; Xs is glutamic acid or aspartic acid; X 6 is any amino acid, X 7 is a hydrophobic amino acid; and X 8 is a hydrophobic amino acid, for use in inducing myelination or inhibiting demyelination in a mammal.
• the peptide has the sequence shown in SEQ LD
• Another embodiment of the invention is a cyclic peptide having between about 1 1 and 25 amino acids, and including the sequence X ⁇ X X 3 NNX 4 TX 5 X6X 7 X 8 , wherein Xi is a hydrophobic amino acid (leucine, alanine isoleucine, valine, tyrosine, tryptophan, phenylalanine or methionine); X 2 is a hydrophobic amino acid, X 3 is aspartic acid, glutamic acid, lysine or arginine; N is asparagine, X is any amino acid; T is threonine; X5 is glutamic acid or aspartic acid; X ⁇ is any amino acid, X 7 is a hydrophobic amino acid; and X 8 is a hydrophobic amino acid for use in inhibiting neural degeneration or promoting neurite outgrowth.
• the peptide has the sequence shown in SEQ ID NO: a hydrophobic amino acid
• the present invention also provides a cyclic peptide having between about 11 and 25 amino acids, and including the sequence X ⁇ X X NNX TX 5 X6X 7 X 8 , wherein Xi is a hydrophobic amino acid (leucine, alanine isoleucine, valine, tyrosine, tryptophan, phenylalanine or methionine); X 2 is a hydrophobic amino acid, X 3 is aspartic acid, glutamic acid, lysine or arginine; N is asparagine, X 4 is any amino acid; T is threonine; X5 is glutamic acid or aspartic acid; X ⁇ is any amino acid, X 7 is a hydrophobic amino acid; and X 8 is a hydrophobic amino acid for use in treatment of neuropathic pain.
• Xi is a hydrophobic amino acid (leucine, alanine isoleucine, valine, tyrosine
• the peptide has the sequence shown in SEQ LD NOS: 5 or 6 Detailed Description of the Preferred Embodiment
• the present invention provides cyclic saposin C-derived peptides having between about 1 1 and 25 amino acids, and including the consensus sequence (SEQ LD NO: 4), wherein X, is a hydrophobic amino acid (alanine, leucine, isoleucine, valine, tyrosine, tryptophan, phenylalanine or methionine); X 2 is a hydrophobic amino acid, X 3 is aspartic acid, glutamic acid, lysine or arginine; N is asparagine, X4 is any amino acid; T is threonine; X5 is glutamic acid or aspartic acid; X ⁇ is any amino acid, X 7 is a hydrophobic amino acid; and X 8 is a hydrophobic amino acid.
• X is a hydrophobic amino acid (alanine, leucine
• the peptide has the sequence: cyclo-[LLDNNKTEKLY] (SEQ LD NO: 5) or cyclo-[LLDNNATEELL] (SEQ ID NO: 6). Due to their constrained structures, these cyclic prosaposin peptidomimetics are significantly more resistant to enzymatic degradation and are capable of crossing the blood brain barrier to a greater extent than corresponding linear peptides.
• the cyclic peptides of the invention are equally as effective or more effective in stimulating neurite outgrowth than a known highly-active linear prosaposin peptidomimetic ("prosaptide") having the sequence TXLIDNNATEEILY, wherein X is D-alanine (SEQ ID NO: 7) (Example 1).
• the cyclic peptides of the invention are more effective than prosaptide in preventing neural cell death in vitro (Example 2). These cyclic peptides lack free amino- and carboxy-terminal ends. Thus, they are more resistant to degradation in vivo by aminopeptidases and carboxypeptidases which degrade peptides from the amino-and carboxy-termini. Cyclic saposin C-derived peptides comprising the active 11-mer region (SEQ ID NO:
• neurotrophic analogs thereof have utility in promoting functional recovery after toxic, traumatic, ischemic, degenerative and inherited lesions to the peripheral and central nervous system.
• these peptides stimulate myelination and counteract the effects of demyelinating diseases.
• These peptides stimulate the outgrowth of neurons, promote myelination, promote neuroprotection and prevent programmed cell death in neuronal tissues in mammals, preferably humans.
• 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.
• a neuropathy is a functional disturbance or pathological change in the peripheral nervous system and is characterized clinically by sensory or motor neuron abnormalities.
• the peptides are also useful as analgesics, particularly for the treatment of neuropathic pain in a mammal, preferably a human, which can develop days or months after a traumatic injury and is often long-lasting or chronic.
• the second asparagine residue within the native prosaposin sequence (corresponding to second "N" in SEQ ID NO: 4 ) is known to be glycosylated with N-acetylglucosamine which may provide some resistance to proteolytic degradation.
• 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 cyclic 1 1-mer region shown in SEQ ID NO:4.
• Saposin C-derived cyclic peptide analogs of the invention can differ from saposin C sequences or SEQ ID NOS: 4-6, for example, by replacement of one or more lysine and/or arginine residues; replacement of one or more tyrosine and/or phenylalanine residues, deletion of one or more phenylalanine residues and/or conservative replacement of one or more amino acids within the peptide.
• the replacement or deletion of lysine/arginine and tyrosine/phenylalanine residues will reduce the susceptibility of peptide degradation by trypsin and chymotrypsin, respectively.
• the cyclic neurotrophic and myelinotrophic 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 11 and 25 amino acids and include therein the sequence shown in SEQ ID NO: 4.
• peptide sequences contemplated for use in the present invention include minor insertions, deletions and substitutions.
• 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, histidine); the acidic charged amino acids (aspartic acid, glutamic acid); the non-polar amino acids (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan); the uncharged polar amino acids (glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine); and the aromatic amino acids (phenylalanine, tryptophan and tyrosine).
• the basic charged amino acids lysine, arginine, histidine
• the acidic charged amino acids aspartic acid, glutamic acid
• non-polar amino acids alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan
• the uncharged polar amino acids glycine, asparagine, glutamine,
• 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, or a similar conservative replacement of an amino acid with a structurally related amino acid will not significantly affect the properties of the peptide.
• the cyclic saposin C-derived sequences including SEQ LD NO: 4 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, hydrophilicity and the like. Sequences outside the active neurotrophic region are not typically required for activity. Thus, in most instances, the subject peptide will be active regardless of the identity of these sequences. Again, any such peptide can be screened for such activity using the protocols described herein.
• any such cyclic peptide to stimulate neurite outgrowth, prevent neural cell death, promote myelination, inhibit demyelination, alleviate neuropathic pain and treat sensory neuropathy can easily be dete ⁇ nined by one of ordinary skill in the art using the procedures described in Examples 1-9. Methods for assaying the abilities of these peptides to promote myelination and to inhibit demyelination are set forth 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 cyclic synthetic peptides of the invention for in vitro use is contemplated.
• concentrations in the range of 0.1 ⁇ g/ml to about 10 ⁇ g/ml 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 vivo by directly administering the peptides of the invention to the cells. This can be done, for example, by culturing the cells in growth medium suitable for the particular cell type, followed by addition of the peptide to the medium.
• the composition can be administered by one of several techniques. Most preferably, the composition is injected directly into the blood or tissue in sufficient quantity to give the desired local concentration of peptide.
• the peptides of the invention may also be esterified with fatty acids to form peptide fatty acid esters using conventional acid-catalyzed esterification.
• 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 lauric, 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. Such modifications will facilitate the ability of the peptide to cross the blood brain barrier due to increased hydrophobicity.
• direct intracranial injection or injection into the cerebrospinal fluid may also be used in sufficient quantities to give the desired local concentration of neurotrophin.
• a pharmaceutically acceptable injectable carrier is used.
• Such carriers include, for example, phosphate buffered saline and Ringer's solution.
• 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, intramuscular, intradermal, subcutaneous, intracranial, intranasal, epidural, topical and oral.
• administration by direct intramuscular or intravenous injection is prefe ⁇ ed.
• the peptide compositions of the invention can be packaged and administered in unit dosage form, such as an injectable 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, demyelination or as an analgesic in general or for treatment of neuropathic pain are in the range of from about 0.01 to about 10,000 ⁇ g/kg. More preferably, daily systemic dosages are between about 0.1 and 1,000 ⁇ g/kg.
• daily systemic dosages are between about 10 and 100 ⁇ g/kg.
• dosages can be between about 0.7 and 700,000 ⁇ g daily; more preferably between about 7 and 70,000 ⁇ g daily; and most preferably between about 700 and 7,000 ⁇ g/kg.
• Daily dosages of locally administered material will be about an order of magnitude less. Oral administration is also contemplated.
• the neurotrophic peptides are administered locally to neural cells in vivo by implantation thereof.
• 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 be 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 (Radin et al., Meth. Enzymol, 98:613-618, 1983).
• 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 18-mer fragment derived from saposin C crosses the blood brain barrier. Peptides of the present invention having up to about 22 amino acids will also cross this barrier and can thus be administered intravenously, with greater transport occurring for shorter peptides.
• 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 demyelination in the manner described above in vivo, 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. Reversal of demyelination diseases or other neural damage is also contemplated.
• 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 are synthesized on a solid support using established methods and Fmoc chemistry using an automated solid-phase protocol well known in the art
• Example 1 is illustrative and are not intended to limit the scope of the present invention.
• NS20Y neuroblastoma cells were grown in DMEM 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 ml DMEM containing 0.5% FCS plus various concentrations of the peptide A, peptide B or TX14(A). Cells were cultured for an additional 24 hours, washed with PBS and fixed with Bouin's solution (saturated aqueous picric acid/formalin/acetic 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.
• FCS fetal calf serum
• T [ 2 (ED 50 ) which is the concentration of peptide resulting in 50% increased neurite outgrowth, was 1.0 ng/ml for TX14(A) and for peptide B.
• the T 1 2 for peptide A was 0.6 ng/ml.
• peptide B was as effective as TX14(A), while peptide A was more effective than TX14(A). This indicates that the cyclized peptides of the present invention have excellent activity in comparison to an established standard "prosaptide".
• Example 2 Prevention of cell death in vitro NS20Y cells were plated as described in Example 1 and grown on glass coverslips in 0.5%) fetal bovine serum for 2 days in the presence or absence of TX14(A), peptide A or peptide B Media was removed and 0.2% 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 5%. TX14(A), peptide A and peptide B reduced the number of trypan blue-positive (dead) cells. The Tin (ED 50 ) for prevention of neural cell death for TX 14(A) was 1.0 ng/ml.
• Peptides A and B were more potent, with T m values of 0.6 ng/ml and 0.8 ng/ml, respectively. This indicates that the cyclic peptides have exceptional activity in rescuing neural cells from programmed cell death in comparison to a standard linear "prosaptide".
• Example 3 Ex vivo myelination assay Newborn mouse cerebellar explants are prepared according to Satomi (Zool. Set, 9:127-137, 1992). Neurite outgrowth and myelination are observed over 22 days in culture, during the period when the newborn mouse cerebellum normally undergoes neuronal differentiation and myelination begins. A cyclic saposin C-derived peptide having between 11 and 25 amino acids and containing the sequence shown in SEQ ED NO: 4 (10 ⁇ g/ml) is added on the second day after preparation of the explants (three control and three treated explants), and outgrowth of neurites and myelination is assessed under a bright field microscope with a video camera.
• 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.
• the peptides of the invention induce increased myelination in differentiating cerebellum ex vivo.
• the prevention of Schwann cell death is correlated with prevention of demyelination.
• Schwann cells contain an extensive myelin sheath.
• the addition of. a cyclic saposin C-derived peptide having between 11 and 25 amino acids and containing the sequence shown in SEQ LD NO: 4 to Schwann cells in culture reduces Schwann cell death in a dose-dependent manner and stimulates the incorporation of sulfatides, myelin-specific lipids, into Schwann cells. This indicates that the cyclic peptides of the invention can prevent demyelination due to Schwann cell death.
• peptides in treating traumatic ischemic CNS lesions Humans with traumatic lesions to the spinal cord receive intracerebrospinal or direct injection of about 100 ⁇ g/ml of. a cyclic saposin C-derived peptide having between 11 and 25 amino acids and containing the sequence shown in SEQ LD NO: 4 in a sterile saline solution or in depot form to enable slow, continuous release of the peptide at the lesion site.
• Improvement is assessed by gain of motor nerve function (i.e. increased limb movement). Treatments continue until no further improvement occurs.
• Example 6 Use of peptides in treating demyelination disorders Patients diagnosed with early stage MS are given a cyclic saposin C-derived peptide having between 11 and 25 amino acids, and containing the sequence shown in SEQ LD NO: 4, by direct intravenous injection into the cerebrospinal fluid using the same dose range as in Example 3. Dosages are repeated daily every 2-5 days, or weekly and improvement in muscle strength, musculoskeletal coordination and myelination (as dete ⁇ nined by MRI) is observed.
• Example 7 Alleviation of neuropathic pain in Chung model rats This example describes the effects of bolus intrathecal injection of a cyclic saposin C- derived peptide having between 11 and 25 amino acids and containing the sequence shown in SEQ LD NO: 4 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 male 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 ⁇ g/rat.
• 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 neuropathy Mice are administered taxol in order to induce sensory neuropathy.
• Taxol-treated mice are administered 50 ⁇ g kg, 100 ⁇ g/kg or 250 ⁇ g/kg of a cyclic saposin C-derived peptide having between 11 and 25 amino acids and containing the sequence shown in SEQ D NO: 4.
• 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 preventing or retarding loss of thermal sensation in taxol-treated mice.
• the synthetic saposin C-derived peptides of the invention effectively inhibit sensory neuropathy.
• Rats are made diabetic by a single intraperitoneal injection of streptozotocin (50 mg/kg body weight, freshly dissolved in 0.9% sterile saline) to ablate pancreatic ⁇ cells and induce insulin deficiency as described by Calcutt et al. (Pain, 68:293-299, 1996). Two days later, diabetes is confirmed in streptozotocin-injected rats by measuring blood glucose levels. Streptozotocin-injected animals with a blood glucose concentration below 15 mmol/1 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 ⁇ l of 0.5%) 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 hyperalgesia in diabetic rats during phases Q and 2 (Calcutt et al., Ewr. J Pharmacol, 285:189-197, 1995). Animals are transferred to an observation chamber constructed to allow continuous visualization of the paws.
• 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, 1993). Comparisons of activity during each phase are made by surnming the flinches at measurement points within the phase, diabetic rats five an abnormal flinch response.
• Diabetic rats are divided in two groups of four animals each which are administered saline or a cyclic saposin C-derived peptide having between 11 and 25 amino acids and containing the sequence shown in S ⁇ Q LD NO: 4, respectively.
• Two hours before treatment with 0.5% formalin the diabetic rats are treated with saline or 200 ⁇ g/kg 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%.
• parenteral administration of peptide alleviates the pain from formalin injection and improves motor neuron function in a rat model of painful diabetic neuropathy.

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