MXPA01001966A - Cyclic prosaposin-derived peptides and uses thereof - Google Patents

Abstract

Cyclic neurotrophic and analgesic peptides derived from the active region of prosaposin having the consensus sequence X1X2X3NNX4TX5X6X7X8 (SEQ ID NO:4), wherein X1 is a hydrophobic amino acid (leucine, isoleucine, valine, tyrosine, tryptophan, phenylalanine or methionine);X2 is a hydrophobic amino acid, X3 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;X6 is any amino acid, X7 is a hydrophobic amino acid;and X8 is a hydrophobic amino acid. The peptides are useful in the promotion of neural outgrowth, prevention of cell death, promotion of myelination, treatment of neurodegenerative disorders and treatment and prevention of neuropathic pain.

Description

CYCLIC PEPTICIDES DERIVED FROM PROSAPOSINE AND YOUR EMPLOYMENT Field of the Invention 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 has neurotrophic and analgesic effects.

Background of the Invention Neurotrophic factors are proteins or peptides, capable of affecting survival, target innervation and / or function of neuronal cell populations (Barde, Neuron 2: 1525-1534, 1989). The efficacy of neurotrophic factors, both in vi tro and in vivo have been well documented. For example, nerve growth factor ("NGF") acts as a trophic factor for the anterior cholinergic brain, peripheral and sensory neurons (Hefti et al., Neurobiol, Agings 10: 515-533, 1989) and can reverse lesions. they occur naturally as well as physical traumatic ones in the peripheral nerves (Rich et al., J. Neurocytol., 16: 261-268, 1987). The neurotrophic factor derived from the brain ("BDNF") is a trophic factor for peripheral sensory neurons, dopaminergic neurons of the gray matter, central cholinergic neurons and retinal ganglia (Henderson et al., Restor, Neurol, Neurosci, 5: 15-28, 1993). BDNF has been shown to prevent cell death, which occurs naturally, both in vitro and in vivo (Hofer et al., Nature 331: 262-262, 1988). The ciliary neurotrophic factor ("CNTF") promotes the survival of ciliary ganglia of chicken embryos in vi tro and supports the survival of cultured sympathetic sensory neurons and the spinal motor (Ip et al., J. Physiol. 85: 122-130, 1991). Demyelination is a common defect in a number of disorders of the Central Nervous System ("CNS"), the most prevalent is multiple sclerosis ("MS").

MS, a chronic disorder that can lead to total disability, is characterized by damage to the myelin sheath, while leaving the axes mostly intact. There is currently no effective treatment for MS. Other disorders of the central nervous system that involve demyelination include acute disseminated encephalomyelitis, amyotrophic lateral sclerosis, acute hemorrhagic leukodystrophy, progressive multifocal leukoencephalitis, metachromatic leukodystrophy, and adrenal leukodystrophy. The Peripheral Nervous System ("PNS") can also be affected with demyelination, such as that which occurs in Guilllain syndrome.

Barré (Pathologic Basis of Disease, Robbins et al., Eds. B. Saunders, Philadelphia, 1979, pages 1578-1582). 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 disorders of the peripheral nervous system only treat the symptoms, not the cause of the disease. Neuropathic pain results from nerve injury, such as compression or crushing of the nerve, and traumatic injury to the spinal cord, and is often a long-term or chronic injury. Most traumatic nerve injuries also cause the formation of neuromas, in which pain occurs as a result of aberrant nerve regeneration. In addition, neuropathic pain related to cancer results when the ^ Tumor growth compresses the adjacent nerves, brain or spinal cord. Neuropathic pains are also associated with diseases, which include diabetes and alcoholism. In most cases, neuropathic pain is resistant to current drugs. These drugs also have serious side effects. The patent application of E.U.A :, Serial No. 08 / 611,307, provides a method for alleviating or preventing pains neuropathic drugs by administering to an individual an effective amount of an active prosaposin fragment. Prosaposin is the precursor of a group of four small glycoproteins, heat stable, which are required for the hydrolysis of glycosphingolipids by lysosomal hydrolases (Kishimoto et al., J. Lipid Res. 33: 1255-1267, 1991). Prosaposin is proteolytically processed in lysosomes, which generate saposins A, B, C and D (O'Brien et al., FASEB J. 5: 301-308, 1991). O'Brien et al (Proc. Nati, Acad. Sci U. S.A. 91: 9593-9596, 1994). U.S. Patent No. 5,571,787 and the PCT patent application, published, No. WO 95/03821, disclose that prosaposin, saposin C stimulates the growth of neurites and promotes increased myelination. Furthermore, U.S. Patent Nos. 5,571,787, 5,696,080, 5,714,459 and PCT patent application, published, No. WO 95/03821, disclose that the peptide 22 -mer (CEFLVKEVTKLIDNNKTEKEIL, SEQ ID NO: 1), which consists of of amino acids 8-29 of a human saposin C, stimulates the growth of neurites. These references also reveal that an 18-mer peptide (YKEVTKLIDNNKTEKEIL; SEQ ID NO: 2), contained within the active 22-mer of saposin C (with V replaced by Y), also promotes the growth of neurites and is able to cross the blood barrier of the brain. O'Brien et al (FASEB J. 9: 681-685, 1995) shows that 22-mer stimulates activity of acetyltransferase and prevents neuronal cell death. The active neuritogenic fragment was localized to a linear 12mer sequence located within the amino terminus of saposin C (LIDNNKTEKEIL; SEQ ID NO: 3) (O'Brien et al., FASEB J. 9: 681-685, 1994), Liepinsh et al (Nataure Struct. Biol., 4 793-795) indicates that the loop conformation of a neurotrophically active peptide segment of NK-lysine (residues 17-30) can be mimicked by a circular peptide. A major obstacle to the in vivo therapeutic use of peptides is their susceptibility to proteolytic degradation. The present invention provides the peptidomimetics of cyclic prosaposin which has good resistance to proteolytic degradation and is capable of crossing the blood barrier of the brain.

SUMMARY OF THE INVENTION One embodiment of the present invention relates to a cyclic, neurotrophic and analgesic peptide, having between about 11 and 25 amino acids, and includes the sequence of XXX2X-3NNX4R 5X6X7X8, wherein Xi is a hydrophobic amino acid (alanine) , leucine, isoleucine, valine, tyrosine, tryptophan, phenylalanine or methionine); X2 is a hydrophobic amino acid, X3 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; X6 is any amino acid, X7 is a hydrophobic amino acid; and X8 is a hydrophobic amino acid. Preferably, the peptide has the amino acid sequence shown in SEQ ID NO: 5 or 6. The present invention also provides a composition comprising the peptide described above in a bottle sealed with a septum (septum), formulated with a controlled release material. , in lyophilized form, in liposomal form, in a form suitable for topical administration or in a unit dosage form. Another embodiment of the present invention relates to a method for inducing myelination or inhibiting demyelination in a mammal, which comprises administering to the mammal afflicted with demyelination, a pharmaceutically effective amount, which inhibits the demyelination, of a cyclic peptide, which has between 11 and 25 amino acids, approximately, and including the sequence X, X2X3 NX4RX5X6X7X8, where Xi is a hydrophobic amino acid (leucine, isoleucine, valine, tyrosine, tryptophan, phenylalanine or methionine); X2 is a hydrophobic amino acid, X3 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; X6 is any amino acid, X7 is an amino acid hydrophobic; and X8 is a hydrophobic amino acid. In one aspect of this preferred embodiment, demyelination is due to multiple sclerosis, ischemic lesions or traumatic injuries. Preferably, the administration is intravenous, intramuscular, intradermal, subcutaneous, intracranial, intracerebro-spinal or topical. Advantageously, the peptide is administered in a pharmaceutically acceptable carrier. In another aspect of this preferred embodiment, the peptide is enclosed in a laminar structure. Preferably, the peptide has the sequence shown in SEQ ID NOS: 5 or 6. In another aspect of this preferred embodiment, the mammal is a human being. The present invention also provides a method for inhibiting neural degeneration or promoting the growth of neurites in neural tissue, comprising contacting the neural tissue, susceptible to such degeneration, with a neural, effective amount, which inhibits degeneration, of a cyclic peptide, which has between approximately 11 and 25 amino acids, and includes the sequence: X? X2X3NNX4RX5XsX7X8, where Xi is a hydrophobic amino acid (leucine, isoleucine, valine, tyrosine, tryptophan, phenylalanine or methionine); X2 is a hydrophobic amino acid, X3 is aspartic acid, glutamic acid, lysine or arginine; N is asparagine, X4 is any amino acid; T is threonine; X5 is glutamic acid or acid aspartic; X6 is any amino acid, X7 is a hydrophobic amino acid; and X8 is a hydrophobic amino acid. Preferably, the peptide has the amino acid sequence shown in SEQ ID NOS: 5 or 6. Preferably, administration is intravenous, intramuscular, intradermal, subcutaneous, intracranial, intracerebro-spinal or topical. Advantageously, the peptide is administered in a pharmaceutically acceptable carrier. In another aspect of this preferred embodiment, the peptide is enclosed in a laminar structure. Preferably, the mammal is a human being. Yet another embodiment of the invention relates to a method for treating neuropathic pain in a mammal in need, this method comprises the step of administering to the mammal suffering from neuropathic pain, an amount of a cyclic peptide, effective to inhibit neuronal degeneration , wherein said peptide has between about 11 and 25 amino acids, and includes the sequence of: X? X2X3NNX4RX5X6X7X8 where Xi is a hydrophobic amino acid (leucine, isoleucine, valine, tyrosine, tryptophan, phenylalanine or methionine); X2 is a hydrophobic amino acid, X3 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; X6 is any amino acid, X7 is an amino acid hydrophobic; and X8 is a hydrophobic amino acid. Preferably, the administration is intravenous, intramuscular, intradermal, subcutaneous, intracranial, intracerebro-spinal, topical or oral. Advantageously, the peptide has the sequence shown in SEQ ID NOS: 5 or 6. Advantageously, the peptide is administered in a pharmaceutically acceptable carrier. In another aspect of this preferred embodiment, the peptide is enclosed in a laminar structure. Preferably, the mammal is human. The present invention also provides a cyclic peptide, having between about 11 and 25 amino acids, and includes the sequence of: X? X2X3NNX4RX5X6X7X8, wherein Xx is a hydrophobic amino acid (leucine, isoleucine, valine, tyrosine, tryptophan, phenylalanine or methionine); X2 is a hydrophobic amino acid, X3 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; X6 is any amino acid, X7 is a hydrophobic amino acid; and X8 is a hydrophobic amino acid, for use in inducing myelination or inhibiting demyelination in a mammal. Preferably, the peptide has the sequence shown in SEQ ID NOS: 5 or 6. Another embodiment of the invention is a cyclic peptide having between about 11 and 25 amino acids, and includes the sequence of: X? X2X3NNX4RX5X6X7X8, where Xi is a hydrophobic amino acid (leucine, isoleucine, valine, tyrosine, tryptophan, phenylalanine or methionine); X2 is a hydrophobic amino acid, X3 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; X6 is any amino acid, X7 is a hydrophobic amino acid; and X8 is a hydrophobic amino acid, for use in inhibiting neural degeneration or promoting neurite growth. Preferably, the peptide has the sequence shown in SEQ ID NOS: 5 or 6. The present invention also provides a cyclic peptide, which has approximately between 11 and 25 amino acids, and includes the sequence of: X? X2X3NNX4RX5X6X7X8, where Xi is an amino acid hydrophobic (leucine, isoleucine, valine, tyrosine, tryptophan, phenylalanine or methionine); X2 is a hydrophobic amino acid, X3 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; X6 is any amino acid, X7 is a hydrophobic amino acid; and X8 is a hydrophobic amino acid, for use in the treatment of neuropathic pain. Preferably, the peptide has the sequence shown in SEQ ID NOS: 5 or 6.

Detailed Description of the Preferred Modality The present invention provides cyclic peptides derived from saposin C, which have approximately 11 to 25 amino acids, and include the consensual sequence of X1X2X3NNX4RX5X6X7X8, (SEQ ID NO: 4), where Xx is a hydrophobic amino acid (alanine) , leucine, isoleucine, valine, tyrosine, tryptophan, phenylalanine or methionine); X2 is a hydrophobic amino acid, X3 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; X6 is any amino acid, X7 is a hydrophobic amino acid; and X8 is a hydrophobic amino acid. In a preferred embodiment, the peptide has the sequence: cyclo- [LLDNNKTEKLY] (SEQ ID NO: 5) or cyclo- [LIDNNATEEIL] (SEQ ID NO: 6). Due to their limited structures, these peptidomimetics of the cyclic prosaposin are significantly more resistant to enzymatic degradation and are capable of crossing the blood brain barrier to a greater extent than the corresponding linear peptides. The cyclic peptides of the invention are equally as effective or more in stimulating the growth of neurites than a known peptidomimetic of highly active linear prosaposin ("prosaptide"), which has the sequence TXLIDNNATEEILY, where X is D-alanine (SEQ ID NO. : 7) (Example 1). Also, the cyclic peptides of the invention are more effective than the prosaptide in preventing the death of neural cells in vi tro (Example 2). These cyclic peptides lack terminal amino and carboxyl termini. Thus, they are more resistant to in vivo degradation by aminopeptidases and carboxypeptidases, which degrade the peptides of the amino and carboxy terminals. The cyclic peptides derived from saposin C, which comprise the active 11-mer region (SEQ ID NO: 4) and its neutrophic analogs, have utility in promoting functional recovery after toxic, traumatic, ischemic, degenerative and inherited lesions, at central and peripheral nervous system. In addition, these peptides stimulate myelination and counteract the effects of demyelinating diseases. These peptides stimulate the growth of neurons, promote myelination, promote neuroprotection and prevent programmed cell death in neuronal tissues in mammals, preferably humans. The peptides of the invention can be used to treat various neuropathies, including, but not limited to, motor, sensory, peripheral neuropathies, taxol-induced and diabetic neuropathies. As used herein, a neuropathy is a functional disturbance or pathological change in the peripheral nervous system and is clinically characterized by neuronal sensory or motor abnormalities. The peptides are also useful as analgesics, particularly for the treatment of neuropathic pain in mammals, preferably human, which may develop days or months after a traumatic injury and is often prolonged or chronic in nature. The second asparagine residue, within a native prosaposin sequence (corresponding to the second "N" in SEQ ID NO: 4), will be glycosylated with N-acetylglucosamine, which may provide some resistance to proteolytic degradation. Synthetic modification of this asparagine residue, within the saponin C derived peptides, present, non-native, by standard methods (ie, Merrifield synthesis) with several carbohydrates, preferably glucose, is also found within the scope of the present invention. One embodiment of the present invention is a method for facilitating neurite outgrowth or increased myelination in differentiated or undifferentiated neural cells by administering to the cells an amount effective for neurite growth or facilitating myelin, a peptide derived from the saposin C, which covers the active cyclic 11-mer region, shown in SEQ ID NO: 4. The cyclic peptide derived from the saposin C of the invention, may differ from the saposin C sequences or SEQ ID NOS: 4-6, for example, by the replacement of one or more lysine and / or arginine residues; the replacement of one or more residues of tyrosyria and / or phenylalanine, the deletion of one or more residues of the phenylalanine and / or the 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 trypsin chimer, respectively. The cyclic sequences of the neurotrophic and myelotrophic peptide of the invention preferably have up to about 50 amino acids; more preferably, up to about 30 amino acids, and, especially preferred, between about 11 to 25 amino acids, and includes the sequence shown in SEQ ID N0: 4. Additional variants of these peptide sequences, considered for use in the present invention, include minor insertions, deletions and substitutions. For example, conservative amino acid replacements are considered. These 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 amino acid families include the basic charged amino acids (lysine, arginine, histidine); the acid charged amino acids (aspartic acid, glutamic acid); the non-polar amino acids (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan); uncharged polar amino acids (glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine); and the aromatic amino acids (phenylalanine, 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, or a similar conservative replacement of a amino acid with a structurally related amino acid that does not significantly affect the properties of the peptide. The cyclic sequences, derived from saponin C, which include SEQ ID NO: 4, can be modified to obtain various objectives, such as increased activity and stability. Other amino acids may be present outside of this consensus sequence, which include the native sequence of saposin C, conservative substitutions of these native sequences, or unrelated peptide sequences, to achieve the targets, such as increased binding, hydrophobicity, hydrophilicity and similar. Sequences outside the active neurotrophic region are typically not required for activity. Thus, in most cases, the present peptide will be active, regardless of the identity of these sequences. From new, any peptide can be classified for such activity, using the protocols described herein. The ability of any such cyclic peptides to stimulate neurite growth, prevent neural cell death, promote myelination, inhibit demyelination, alleviate neuropathic pain and treat sensory neuropathy, can be determined by one of ordinary skill in the art, using the procedures described in Examples 1.9. Methods for testing the abilities of these peptides to promote myelination and to inhibit demyelination are set forth in Examples 3 and 4 below. A typical minimum amount of the peptides of the invention for neurotrophic activity in the cell growth medium is usually at least about 5 ng / ml. This amount or greater of the synthetic cyclic peptides of the invention, for in vi tro use is considered. Typically, concentrations in the ranges of 0.1 to 10 μg / ml, approximately, of these peptides will be used. Effective amounts for any particular tissue can be determined according to Example 1. The neural cells can be treated in vi tro or ex vivo by directly administering the peptides of the invention to the cells. This can be done, for example, by cultivating the cells in an appropriate growth medium for the particular cell type, followed by the 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 several techniques. More preferably, the composition is injected directly into the blood or tissue, in an amount sufficient to give the desired local concentration of the peptide. In peptides lacking lysine and arginine residues, proteolytic degradation is reduced. Smaller (ie 20-mer or less) peptides are more likely to cross the blood brain barrier and enter the central nervous system for the treatment of CNS disorders (see Banks et al., Peptides, 13: 1289-1294, 1992). The peptides of the invention can also be esterified with fatty acids, to form the fatty acid esters of peptides, using a conventional esterification catalyzed with acid. Alternatively, the last amino acids added in the synthetic process, is itself an esterified amino acid, commercially available, which obviates the need for the esterification reaction. The fatty acids considered for use in the formation of peptide esters include lauric, myristic, plastid, stearic, oleic and linoleic acids.

The present peptides can also be acetylated by the inclusion of acetylated residues of lysine, arginine or asparagine, commercially available, during the synthetic process. These modified peptides retain the activity of the main compound. Such modifications will facilitate the ability of the peptide to cross the brain blood barrier, due to the increased hydrophobicity. For the treatment of neural disorders, direct intracranial injection or injection into the cerebrospinal fluid can also be used, in amounts sufficient to give the desired local concentration of the neurotrophin. In both cases, an injectable, pharmaceutically acceptable carrier is used. Such carriers include, for example, a phosphate buffered saline solution and the Inger solution. Alternatively, the composition can be administered to peripheral neural tissue by direct local injection or by systemic administration. Several conventional modes of administration are considered, which include intravenous, intracerebroespinal, intramuscular, intradermal, subcutaneous, intracranial, intranasal, epidural, topical and oral administration. For use as an analgesic, administration by intramuscular or intravenous injection is preferred.

The peptide compositions of the invention can be packaged and administered in unit dosage forms, such as an injectable composition or the local preparation in a dose amount equivalent to the daily dose administered to a patient or as a controlled release composition. A bottle sealed with septum, containing a daily dose of the active ingredient in PBS or lyophilized form, is an example of a unit dose. Appropriate daily systemic doses of the peptides of the invention, based on the vertebrate body weight, for the treatment of neural diseases, demyelination or as an analgesic, in general, or for the treatment of neuropathic pains, are in the approximate range of 0.01 up to 10,000 μg / ml. More preferably, the daily systemic doses are between about 0.1 and 1,000 μg. Especially preferred, the systemic daily doses are between about 10 and 100 μg / ml. Thus, for a typical 70 kg human being, doses may be between about 0.7 and 700,000 μg daily, more preferably between about 7 and 70,000 μg daily; and especially preferred between about 700 and 7,000 μg / kg. The daily doses of the locally administered material will be approximately one order of magnitude less. Oral administration is also considered.

In a preferred embodiment of the invention, neurotrophic peptides are administered locally to neural cells in vivo by their implantation. For example, polylactic acid, polygalactic acid, regenerated collagen, mulilamellar liposomes and many other conventional depot formulations are expressly considered in the present invention. Infusion pumps, matrix entrapment systems and their combination with transdermal devices are also considered. The peptides may also be encapsulated within a coating conforming to the polyethylene glycol, as described in U.S. Patent No. 5,529,914, before implantation. The neurotrophic peptides of the invention can also be enclosed by micelles or liposomes. The encapsulation technology with liposomes is well known. Liposomes can be targeted to specific tissues, such as neural tissue, through the use of receptors, ligatures or antibodies, capable of binding to the target tissue. The preparation of these formulations is well known in the art (Radin et al., Meth. Enzymol., 98: 513-618, 1983). There is currently no product, commercially available, capable of promoting the functional regeneration and restoration of the structural integrity of the systems neuronal This is particularly true in the CNS. The regeneration of peripheral nerves through the use of neurotrophic factors is within the scope of the invention. Likewise, neurotrophic factors may be useful therapeutically in the treatment of neurodegenerative diseases associated with the degeneration of neuronal populations or specific areas of the brain. The main cause of Parkinson's disease is the degeneration of the dopaminergic neurons of the gray matter. Some antibodies against prosaposin immunohistochemically stain dopaminergic neurons of the subtantia nigra in sections of the human brain, the neurotrophic peptides of the invention may be therapeutically useful in the treatment of Parkinson's disease. Retinal neuropathy, an ocular neurodegenerative disorder, which leads to loss of vision in old age, is also treatable with the use of the peptides of the invention. It has long been believed that in order to reach neuronal populations in the brain, neurotrophic factors will 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 barrier of blood of the brain. The peptides of the present invention, which have up to about 22 amino acids, will also cross this barrier and can thus be administered intravenously, with increased transport occurring for the shorter peptides. Other neuronal populations, such as motor neurons, can also be treated by intravenous injection, although direct injection into the cerebrospinal fluid can be treated by intravenous injection, although direct injection into the cerebrospinal fluid is also considered as an alternative route. . The cells can be treated to facilitate the formation of myelin or to prevent demyelination, in the manner described above, in vivo, ex vivo or in vi tro. Diseases that result in the demyelination of nerve fibers, including MS, acute disseminated leukoencephalitis, progressive multifocal leukoencephalitis, metachromatic leukodystrophy and adrenal leukodystrophy, can be decreased or stopped by the administration of neurotrophic peptides of the invention to the affected cells. the illness. The reversal of demyelinating diseases or other neural damage is also considered. The compositions of the present invention can be used in vi tro as research tools to study the effects of neurotrophic factors and materials that facilitate myelin. However, more practically, they have an immediate use as laboratory reagents and components of the cell growth medium, to facilitate growth and maintain the neural cells in vivo. The peptides of the invention are synthesized on a solid support, using the established methods and chemistry of Fmoc, using an automatic solid phase protocol, well known in the art (Traeciak et al., Tetrahedron Lett., 33: 4557- 4561, 1992) on a Protein Technologies Symphony peptide synthesizer. The cyclized peptides are cleaved from the solid support, using TFA / water / triisopropylsilane (95: 2.5: 2.5). The peptides are purified using reverse phase HPLC chromatography on a C-18 column, eluting with 0.3% trifluoroacetic acid (TFA) in acetonitrile. The mass spectral analysis of the peptides, shown in SEQ ID NO: 5 (peptide A) and 6 (peptide B), confirmed that the cyclic peptides have been synthesized [SEQ ID NO: 5, MH + 1333 (expected), 1333 (observed; SEQ ID NO: 6 MH + 1127 (expected), 1127 (observed)). The following examples are illustrative and are not intended to limit the scope of the present invention.

EXAMPLE 1 Stimulus of Neurite Growth in vi tro NS20Y neuroblastoma cells grown in DMEM containing 10% fetal calf serum (FCS). The cells were removed with trypsin and plated in 30 mm Petri dishes on glass cover strips. After 20-24 hours, the medium was replaced with 2 ml of DMEM containing 0.5% FCS plus various concentrations of peptide A, peptide B or TX14 (A). The cells were cultured for a further 24 hours, washed with PBS and fixed with Bouin's solution (saturated aqueous picric acid / formalin / acetic acid, 15: 5: 1) for 30 minutes. The fixative was removed with PBS and the growth of neurites was classified under a phase contrast microscope. Cells exhibiting one or more neurites, clearly defined, equal to or greater than a cell diameter, were classified as positive. At least 2000 cells were sorted into different portions of each Petri dish to determine the percentage of neurites carrying cells and the assays were performed in duplicate. All three peptides induced neurite growth in NS20Y cells. The ED50 (T), which is the concentration of the peptide that results in 50% increased neurite growth, was 1.0 ng / ml for TX14 (A) and for peptide B. The T ^ for peptide A was 0.6 ng / ml. Thus, 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 as compared to an established standard "prosaptide".

Example 2 Prevention of cell death, in vi tro NS20Y cells were plated, as described in Example 1, and grown on glass cover strips 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. The dead cells stained blue were classified as a percentage of the total in an inverted microscope, which counted 400 cells in four areas of each cavity. The average error of duplicates was 5%. TX14 (A), peptide A and peptide B reduced the number of blue (dead) positive cells of trypan. The T (ED50) for the prevention of the neural cell death for R14 (A) was 1.0 ng / ml. Peptides A and B were more potent, with T values of 0.6 ng / ml and 0.8 ng / ml, respectively. This indicates that the cyclic peptides had exceptional activity in neural cells rescued from the programmed cell dead, compared to a standard linear "prosáptido".

EXAMPLE 3 Ex vivo Myelination Assay Brain explants from newborn mice were prepared according to Satorni (Zool, Sci., 9: 127-137, 1992). Neurite growth and myelination were observed in 22 days in culture, during the period when the cerebellum of the newborn mouse normally undergoes neuronal differentiation and begins myelination. A cyclic peptide, derived from saposin C, having between 11 and 25 amino acids and containing the sequence shown in SEQ ID NO: 4 (10 μg / ml) was added on the second day after the preparation of the explants (three explants control and three treated), and neurite growth and myelination were evaluated under a bright field microscope with a video camera. At day eight, the cultures containing the peptides were thinner and more disseminated than the control cultures. At day 15, cultures treated with peptides contain many cells with long projections on the periphery of the explant, which are less prominent in untreated control cultures. Cultures treated with peptides significantly contain more myelinated neuraxes in the subcortical white matter in 22 days, compared to control explants. Thus, the peptides of the invention induce increased myelination in the ex vivo differentiation cerebellum.

Example 4 Prevention of demyelination The prevention of Schwann cell death was correlated with the prevention of diesmelination. Schwann cells contain an extensive myelin sheath. The addition of a cyclic peptide derived from saposin C, which is between 11 and 25 amino acids and contains the sequence shown in SEQ ID NO: 4, to Schwann cells in the culture reduces the death of Schwann cells in a manner dependent on dose and stimulates the incorporation of sulfatides, myelin-specific lipids, in Scwann cells. This indicates that the cyclic peptides of the invention can prevent demyelination due to death of Schwann cells.

Example 5 Use of peptides in the treatment of ischemic, traumatic lesions of the Central Nervous System Human beings, with traumatic lesions in the spinal cord, received intracerebro-spinal or direct injections of approximately 100 μg / ml of a peptide cyclic derivative of saposin C, which has between 11 and 25 amino acids and contains the sequence shown in SEQ ID NO: 4, in sterile saline or in the form of a deposit, to enable the slow, continuous release of the peptide at the site of the injury. Improvement was assessed by the gain of motor nerve function (ie, increased limb movement). The treatments continued until a further improvement no longer occurs.

Example 6 Use of peptides in treating demyelination disorders Patients, diagnosed with early stage MS, were given a cyclic peptide derived from saposin C, which is between 11 and 25 amino acids, and which contains the sequence shown in SEQ ID NO: 4, by direct intravenous injection into the cerebrospinal fluid, using the same dose range as in Example 3. Doses were repeated daily every 2-5 days or weekly and improvement in muscle strength, musculoskeletal coordination and myelination (determined by MRI).

Example 7: Relief of neuropathic pain in Chung model rats This example describes the effects of intrathecal bolus injection of a cyclic peptide derived from the saposin C, which is between 11 and 25 amino acids and which contains the sequence shown in SEQ ID NO: 4, in the experimental Chung model of peripheral neuropathic pain. Each peptide was chemically synthesized, purified, dissolved in a sterile PBS solution and regulated to a neutral pH. The surgical procedure previously described by Kim et al (Pain, 50: 355, 1992) was performed on male rats to induce an allodynic state. A spinal catheter was inserted two weeks before surgery. Five days later, the peptides were administered at 0.07, 0.07 and 0.7 μg / rat. The pressure thresholds are then determined using calibrated von Frey hair. The longer the time taken by an animal to remove the paw in response to the applied pressure, the less severe the neuropathic pain is. Peptides significantly increase threshold pressures, indicating significant relief of neuropathic pain.

Example 8 Treatment of sensory neuropathy Taxol was administered to mice in order to induce sensory neuropathy. The mice treated with taxol were administered with 50, 100 or 250 μg / kg of the cyclic peptide derived from saposin C, which has between 11 and 25 amino acids and contains the sequence shown in SEQ ID NO: 4. The loss of thermal sensation was measured using a Hargreaves sensory test apparatus, as an indicator of sensory neuropathy. Each of the three doses of the peptide is effective in preventing or delaying the loss of thermal sensation in mice treated with taxol. Thus, the synthetic peptides derived from the saposin C of the invention effectively inhibit sensory neuropathy.

Example 9: Relief of neuropathic pain in diabetic rats. This example describes the effects of a cyclic peptide derived from saposin C, having between 11 and 25 amino acids and containing the sequence shown in SEQ ID NO: 4, by intraperitoneal administration in a rat model of diabetic neuropathy. The rats were made diabetic by the simple intraperitoneal injection of streptozotocin (50 mg / kg body weight, dissolved in 0.9% fresh sterile saline), to remove pancreatic β-cells and induce insulin deficiency, as described by Calcutt et al (Pain 68; 293-299, 1996). Two days later, diabetes was confirmed in the rats injected with streptozotocin, by measuring the levels of glucose in the blood. Animals injected with streptozotocin with a blood glucose concentration below 1.5 mmol / liter, 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 were studied in 8 weeks, analyzing the behavioral response to noxious chemical formalin, as an indicator of allodynia (Calcutt et al., Sup, 1996). In brief, the rats received a subcutaneous injection of freshly prepared formalin (50 μl of a 0.5% solution in sterile saline) on the dorsal surface of the right hind paw. This concentration of formalin induces sub-maximal behavioral responses in the control rats and allows the detection of hyperalgesia in diabetic rats during phases Q and 2 (Calcutt et al., Eur. J. Pharmacol. 285: 189- 197, 1995). The animals were transferred to an observation chamber constructed to allow continuous visualization of the legs. The number of reculsions during periods of one minute was counted in 5 minute intervals for the next 60 minutes by an observer, who does not know the treatment group of each animal. Phase 1 was defined as the initial measurement of retreats (1-2 and 5-6 minutes after the injection), Phase Q (quiet) is according to the measurements made at 10-11, 15-16 and 20- 21 minutes; and Phase 2 is compliant measurements after injection as previously defined for studies of diabetic rats (see, for example, Malmberg et al., Neuosci, Lett 161: 45-48, 1993), Comparisons of activity during each phase were made by adding the reclining at the measurement points within the phase, an abnormal retreat response is five in diabetic rats. The diabetic rats were divided into two groups of four animals each, which were administered a saline solution or the cyclic peptide derived from saposin C, which is between 11 and 25 amino acids and which contains the sequence shown in SEQ ID NO: 4, respectively. Two hours before treatment with 0.5% formalin, the diabetic rats were treated with saline or with 200 μg / kg of the peptide, using intraperitoneal administration. Administration of the peptide completely prevents the abnormal retreat response in Phase 1 and decreases the response in Phase 2 by 70%. Thus, parenteral administration of the peptide alleviates the pain of formalin injection and improves the function of motor neurons in a rat model of painful diabetic neuropathy.

LIST OF SEQUENCES (1) GENERAL INFORMATION (i) APPLICANT: MYELOS CORPORATION (ii) TITLE OF THE INVENTION: CYCLICAL, ANALGESIC AND NEUROTROPHIC PEPTIDES (iii) NUMBER OF SEQUENCES: 6 (iv) ADDRESS FOR CORRESPONDENCE: (A) RECIPIENT: Knobbe, Martens, Olson & Bear (B) STREET: 620 Newport Center Drive, 16th Floor (C) CITY: Newport Beach (D) STATE: CA (E) COUNTRY: E.U.A. (F) POSTAL ZONE: 92660. (v) HOW TO READ BY COMPUTER: (A) TYPE OF MEDIUM: Diskette (B) COMPUTER: IBM Compatible (C) OPERATING SYSTEM: Windows (D) SOFTWARE: FastSEQ for Windows, Version 2.0b (vi) DATA OF THE CURRENT APPLICATION (A) APPLICATION NUMBER: (B) DEPOSIT DATE: (C) CLASSIFICATION: (vii) DATA FROM THE PREVIOUS APPLICATION: (A) APPLICATION NUMBER: 60 / 098,359 (B) DEPOSIT DATE: August 28, 1998 (viii) INFORMATION OF THE EMPLOYEE / AGENT: (A) NAME: Bartfield, Neil S. (B) REGISTRATION NUMBER: 39,901 (C) REFERENCE NUMBER / FILE: MYELOS.014PR (ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: 619-235-8550 (B) TELEFAX: 619-235-0176 (C) TELEX (2) INFORMATION FOR SEQ ID NO. 1: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 21 amino acids (B) TYPE: amino acid (C) CHAIN STYLE: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO. 1: Cys Glu Phe Leu Val Lys Glu Val Thr Lys Leu lie Asp Asn Lys 1 5 10 15 Thr Glu Lys Glu Lys 20 (2) INFORMATION FOR SEQ ID NO. 2: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 18 amino acids (B) TYPE: amino acid (C) CHAIN STYLE: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO. 2 Tyr Lys Glu Val Thr Lys Leu lie Asp Asn Asn Lys Glu Lys Glu 1 5 10 15 lie Leu (2) INFORMATION FOR SEQ ID NO. 3: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 12 amino acids (B) TYPE: amino acid (C) CHAIN STYLE: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO. 3 Leu lie Asp Asn Asn Lys Thr Glu Lys Glu lie Leu 1 5 10 (2) INFORMATION FOR SEQ ID NO. 4: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 11 amino acids (B) TYPE: amino acid (C) CHAIN STYLE: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (ix) CHARACTERISTICS: (A) NAME / KEY: Other (B) LOCATION: 1 ... 2 (C) OTHER INFORMATION: A, L, I, V, Y, W, F OR M (A) NAME / KEY: Other (B) LOCATION: 3 ... 3 (C) OTHER INFORMATION: D, E, K, or R (A) NAME / KEY: Other (B) LOCATION: 6 ... 5 (C) OTHER INFORMATION: any amino acid (A) NAME / KEY: Other (B) LOCATION: 8 ... 8 (C) OTHER INFORMATION: D or E (A) NAME / KEY: Other (B) LOCATION: 9 ... 9 (C) OTHER INFORMATION: ANY AMINO ACID (A) NAME / KEY: Other (B) LOCATION: 10 ... 11 (C) OTHER INFORMATION: A, L, I, V, Y, W, F or M (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO Four.

Xaa Xaa Xaa Asn Xaa Rhr Xaa Xaa Xaa Xaa 1 5 10 (2) INFORMATION FOR SEQ ID NO. 5: (ii) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 11 amino acids (B) TYPE: amino acid (C) CHAIN STYLE: simple (D) TOPOLOGY: cyclic (ii) TYPE OF MOLECULE: peptide (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO. 5 Leu Leu Asp Asn Asn Lys Thr Glu Lys Leu Tyr 1 2 10 (2) INFORMATION FOR SEQ ID NO. 6: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 11 amino acids (B) TYPE: amino acid (C) CHAIN STYLE: simple (D) TOPOLOGY: cyclic (ii) TYPE OF MOLECULE: peptide (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO. 6 Leu lie Asp Asn Asn Ala Thr Glu Glu lie Leu 1 5 10

Claims (33)

1. CLAIMS 1. A cyclic peptide, having between about 11 and 25 amino acids and including the sequence X1X2X3NNX4RX5X6X7X8, wherein Xi is a hydrophobic amino acid (leucine, alanine, isoleucine, valine, tyrosine, tryptophan, phenylalanine or methionine); X2 is a hydrophobic amino acid, X3 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; X6 is any amino acid, X7 is a hydrophobic amino acid; and X8 is a hydrophobic amino acid.
2. 2. The peptide of claim 1, wherein said peptide has the amino acid sequence shown in SEQ ID NOS: 5 or 6.
3. 3. A composition comprising the peptide of claim 1, in a bottle sealed by a septum.
4. 4. A composition comprising the peptide of claim 1, formulated with a controlled release material.
5. 5. A composition comprising the peptide of claim 1, in lyophilized form.
6. 6. A composition comprising the peptide of claim 1, in liposomal form.
7. 7. A composition comprising the peptide of claim 1, in a form suitable for topical administration.
8. 8. A composition comprising the peptide of claim 1, in a unit dose form.
9. 9. A method for inducing myelination or inhibiting demyelination in a mammal, this method comprises: administering to a mammal, afflicted with demyelination, a pharmaceutically effective, inhibitory amount of demyelination of a cyclic peptide, having between about 11 and 25 amino acids and including the sequence Xa ^ XsNNX-tRXBXgXvXs, in which Xi is an amino acid hydrophobic (leucine, isoleucine, valine, tyrosine, tryptophan, phenylalanine or methionine); X2 is a hydrophobic amino acid, X3 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; X6 is any amino acid, X7 is a hydrophobic amino acid; and X8 is a hydrophobic amino acid.
10. 10. The method of claim 9, wherein said demyelination is due to multiple sclerosis, ischemic lesions or traumatic injuries.
11. 11. The method? Claim 9, wherein the administration is selected from the group consisting of intravenous, intramuscular, intradermal, subcutaneous, intracranial, intracerebroespinal and topical administration.
12. 12. The method of claim 9, wherein said peptide is administered in a pharmaceutically acceptable carrier.
13. 13. The method of claim 9, wherein the peptide is enclosed in a laminar structure.
14. 14. The method of claim 9, wherein the peptide has the sequence shown in SEQ ID NOS: 5 or 6.
15. 15. The method of claim 9, wherein the mammal is a human being.
16. 16. A method to inhibit neural degeneration or promote the growth of neurites in a neural tissue, this method comprises: contacting the neural tissue, susceptible to said degeneration, with an effective amount that inhibits the neural degeneration, of a cyclic peptide, which has between approximately 11 and 25 amino acids, and includes the sequence X1XX3NNXRX5X6X7X8, in which Xi is a hydrophobic amino acid ( leucine, isoleucine, valine, tyrosine, tryptophan, phenylalanine or methionine); X2 is a hydrophobic amino acid, X3 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; X6 is any amino acid, X7 is a hydrophobic amino acid; and X8 is a hydrophobic amino acid.
17. 17. The method of claim 16, wherein the administration is selected from the group consisting of intravenous, intramuscular, intradermal, subcutaneous, intracranial, intracerebro-spinal, and topical administration.
18. 18. The method of claim 16, wherein said peptide is administered in a pharmaceutically acceptable carrier.
19. 19. The method of claim 16, wherein said peptide is enclosed in a laminar structure.
20. 20. The method of claim 16, wherein said peptide has the amino acid sequence shown in SEQ ID NOS: 5 or 6.
21. 21. The method of claim 16, wherein the mammal is a human being.
22. 22. A method for treating neuropathic pain in a mammal in need, this method comprises the step of administering to a mammal suffering from a neurodegenerative disease, an amount of a cyclic peptide, effective for treating neuropathic pains, wherein said peptide contains between about 11 and 25 amino acids, and includes the sequence X? X2X3NNX4RX5X6X7X8, wherein Xi is a hydrophobic amino acid (leucine, isoleucine, valine, tyrosine, tryptophan, phenylalanine or methionine); X2 is a hydrophobic amino acid, X3 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; X6 is any amino acid, X7 is a hydrophobic amino acid; and X8 is a hydrophobic amino acid.
23. 23. The method of claim 22, wherein the administration is selected from the group consisting of intravenous, intramuscular, intradermal, subcutaneous, intracranial, intracerebro-spinal, topical and oral administration.
24. 24. The method of claim 22, wherein said peptide is administered in a pharmaceutically acceptable carrier.
25. 25. The method of claim 22, wherein said peptide is enclosed in a laminar structure.
26. 26. The method of claim 22, wherein said peptide has the amino acid sequence shown in SEQ ID NOS: 5 or 6.
27. 27. The method of claim 22, wherein the mammal is a human being.
28. 28. A cyclic peptide, having between about 11 and 25 amino acids and including the sequence X1X2X3NNX4RX5X6X7X8, wherein Xx is a hydrophobic amino acid (leucine, alanine, isoleucine, valine, tyrosine, tryptophan, phenylalanine or methionine); X2 is a hydrophobic amino acid, X3 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; X6 is any amino acid, X7 is a hydrophobic amino acid; and X8 is a hydrophobic amino acid, for use in inducing myelination or inhibiting demyelination in a mammal.
29. 29. The peptide of claim 28, wherein said peptide has the sequence shown in SEQ ID NOS: 5 or 6.
30. 30. A cyclic peptide, having between about 11 and 25 amino acids and including the sequence X? X2X3NNXRX5X6X7X8, in which Xi is a hydrophobic amino acid (leucine, alanine, isoleucine, valine, tyrosine, tryptophan, phenylalanine or methionine); X2 is a hydrophobic amino acid, X3 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; X6 is any amino acid, X7 is a hydrophobic amino acid; and X8 is a hydrophobic amino acid, for use in inhibiting neural degeneration or promoting neurite growth.
31. 31. The peptide of claim 30, wherein said peptide has the sequence shown in SEQ ID NOS: 5 or 6.
32. 32. A cyclic peptide, having between about 11 and 25 amino acids and including the sequence X? X2X3NNX4RX5X6X7X8, in which Xi is a hydrophobic amino acid (leucine, alanine, isoleucine, valine, tyrosine, tryptophan, phenylalanine or methionine); X2 is a hydrophobic amino acid, X3 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; X5 is any amino acid, X7 is a hydrophobic amino acid; and X8 is a hydrophobic amino acid, for use in the treatment of neuropathic pain.
33. 33. The peptide of claim 32, wherein said peptide has the sequence shown in SEQ ID NOS: 5 or 6.