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WO1998042746A9 - Peptides neurotrophiques de synthese derives de la saposine c - Google Patents

Peptides neurotrophiques de synthese derives de la saposine c

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Publication number
WO1998042746A9
WO1998042746A9 PCT/US1998/005503 US9805503W WO9842746A9 WO 1998042746 A9 WO1998042746 A9 WO 1998042746A9 US 9805503 W US9805503 W US 9805503W WO 9842746 A9 WO9842746 A9 WO 9842746A9
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peptide
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native
amino acids
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Priority to EP98926027A priority patent/EP0971956A2/fr
Priority to AU77956/98A priority patent/AU7795698A/en
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Publication of WO1998042746A3 publication Critical patent/WO1998042746A3/fr
Publication of WO1998042746B1 publication Critical patent/WO1998042746B1/fr
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  • 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. Background of the Invention
  • Demyeiination is a defect common to a number of central nervous system (CNS) disorders, the most prevalent being multiple sclerosis (MS).
  • MS central nervous system
  • a chronic disorder which may lead to total disability is characterized by damage to the myelin sheath, while leaving the axons mostly intact.
  • CNS central nervous system
  • Other central nervous system disorders involving demyeiination include acute disseminated encephalomyelitis, amyotrophic lateral sclerosis, acute hemorrhagic leukodystrophy, progressive multifocal leukoencephalitis, metachromatic leukodystrophy and adrenal leukodystrophy.
  • the peripheral nervous system can also be afflicted with demyeiination, such as that occurring in Guillain-Barr ⁇ syndrome (Pathologic Basis of Disease, Robbi ⁇ s et al. eds., W.B. Saunders, Philadelphia, 1979, pp. 1578-1582).
  • Peripheral nerve injuries and peripheral neuropathies 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.
  • Neurotrophins are proteins or peptides capable of affecting the survival, target innervation and/or function of neuronal cell populations (Barde, Neuron, 2:1525-1534, 1989).
  • the efficacy of neurotrophins both in vivo and in vitro has been well documented.
  • nerve growth factor acts as a trophic factor for forebrain choiinergic, peripheral and sensory neurons (Hefti et al., Neurobio. Aging, 10:515-533, 1989).
  • NGF nerve growth factor
  • Brain-derived neurotrophic factor is a trophic factor for peripheral sensory neurons, dopa i ⁇ ergic neurons of the substantia nigra, central choiinergic neurons and retinal ganglia (Henderson et al., Restor. Neurol. Neurosci., 5:15-28, 1993). BDNF has been shown to prevent normally-occurring cell death both in vitro and in vivo (Hofer et al., Nature, 331:262-262, 1988). Ciliary neurotrophic factor (CNTF) promotes survival of chicken embryo ciliary ganglia in vitro and supports survival of cultured sympathetic, sensory and spinal motor neurons (Ip et al., J. Physio/. Paris, 85:123-130, 1991).
  • CNTF Ciliary neurotrophic factor
  • 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. Nat Acad. Sci. U.S.A., 91:9593-9596, 1994), U.S. Patent Nos.
  • the present invention addresses this need.
  • 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 SEQ ID NO: 8, with the proviso that the peptide does not have the sequence shown in SEQ ID NO: 4. More preferably, the peptide has up to about 30 amino acids. More preferably, the peptide has between about 12 and 25 amino acids.
  • the amino acid at position 1 of SEQ ID NO: 8 is isoleucine.
  • the amino acid at position 3 of SEQ ID NO: 8 is not aspartic acid. In another aspect of this preferred embodiment, the amino acid at position 8 of SEQ ID NO: 8 is not glutamic acid.
  • the amino acid at position 10 of SEQ ID NO: 8 is not glutamic acid.
  • the amino acid at position 11 of SEQ ID NO: 8 is not leucine.
  • the amino acid at position 12 of SEQ ID NO: 8 is not leucine.
  • the peptides described above may be acetylated or esterified with a 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 SEQ ID NO: 8, with the proviso that the peptide does not have the sequence shown in SEQ ID NO: 4.
  • the neuronal cells are neuroblastoma cells.
  • the neuroblastoma cells are NS20Y cells.
  • the contacting step occurs in vitro.
  • the contacting step occurs in vivo.
  • 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 SEQ ID NO: 8, with the proviso that the peptide does not have the sequence shown in SEQ ID NO: 4.
  • the administering step is intravenous, intramuscular, intradermai, 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 sequence shown in SEQ ID NO: 8, with the proviso that the peptide does not have the sequence shown in SEQ ID NO: 4.
  • the administering step is intravenous, intramuscular, intradermai, subcutaneous, intracranial, epidural, topical, oral, transdermal, transmucosal or intranasal.
  • the peptide may be acetylated or esterified with a fatty acid.
  • Still another embodiment of the invention is a pharmaceutical composition
  • 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 SEQ ID NO: 8, with the proviso that the peptide does not have the sequence shown in SEQ ID NO: 4, in a pharmaceutically acceptable carrier.
  • the composition is a controlled release formulation.
  • the composition may be in liposomal, lyophilized or unit dosage form.
  • Figure 1 illustrates a NS20Y neuroblastoma neurite outgrowth assay using peptides TX 14(A) (TXLIDNNATEEILY; X-D-alanine; SEQ ID NO: 4) and a rat 14-mer derived from the saposin C active sequence (SELIINNATEELLY; SEQ ID NO: 5).
  • Figure 2 illustrates a cell death assay using NS20Y neuroblastoma cells.
  • NS20Y cells were grown for 48 hours in low serum in the presence or absence of TX 14(A) and dead cells were identified by Trypan blue staining.
  • 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 neuroprotection and can be used to treat various 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.
  • a native 15-mer contained within human saposin C and encompassing the active neurite-promoting region shown in SEQ ID NO: 3 was modified as follows to decreases its susceptibility to proteolysis in vivo: Lys 2 was replaced with D ata to increase resistance to exopeptidases; lys 8 was replaced with ala 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 TX 14(A) exhibited neuritogenic activity in vitro neurite outgrowth assays. TX 14(A) also prevented cell death in neuroblastoma cells in culture.
  • the leucine at position three and/or 14 can be changed to an isoleucine with retention of activity. This will prevent degradation by enkephalinase which cleaves at hydrophobic residues.
  • lys 8 is an amino acid other than alanine, lysine or arginine to prevent degradation by dipeptidyl peptidase (alanine) or trypsin (lysine, argi ⁇ ine).
  • Saposin C-derived peptides comprising the active 12-mer region of the sequence shown in SEQ ID NO: 3 (LIDNNKTEKEIL; SEQ ID NO: 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.
  • 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.
  • 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.
  • SEQ ID NO: 7 may be modified as follows and still retain neurotrophic activity: Leu 1 may be leu or ile; lle 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 argini ⁇ e; thr 7 is essential, glu 8 is a charged amino acid; lys 9 is absent or a charged amino acid, preferably not lysine or arginine; glu 10 is any charged amino acid; ile 11 is any amino acid; leu 12 is any amino acid.
  • X 1 IX 2 NNX 3 TX 4 X 5 X 6 X 7 X 8 (SEQ ID NO: 8) in which I is isoieucine; X, is leucine or isoleucine; X 2 is any amino acid; N is asparagine; X 3 is any amino acid; X 4 is lysine, arginine, histidine, aspartic acid or glutamic acid; X 5 is absent, lysine, arginine, histidine, aspartic acid, glutamic acid or glycine; X 6 is lysine, arginine, histidine, aspartic acid or glutamic acid; X 7 is any amino acid; and X 8 is any amino acid.
  • the second asparagine residue within the native prosaposin sequence (corresponding to second "N" in SEQ ID NO: 8) is known to be glycosylated with N-acetylgiucosamine 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 myeii ⁇ -facilitating amount of a saposin C-derived peptide encompassing the active 12-mer region shown in SEQ ID NO: 7 (amino acids 18-29 of saposin C) or, more preferably, non-natural analogs thereof including the sequence shown in SEQ ID NO: 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 and/or 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 lysine/argi ⁇ ine and tyrosine/phe ⁇ ylalanine residues will reduce the susceptibility of peptide degradation by trypsin and ch ⁇ motrypsin, respectively.
  • the non-native 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 12 and 25 amino acids and include therein the sequence shown in SEQ ID NO: 8.
  • the peptide does not contain the sequence shown in SEQ ID NO: 4.
  • the amino acid at position 6 of SEQ ID NO: 8 is not alanine.
  • the amino acid at position 1 of SEQ ID NO: 8 is isoleucine.
  • the amino acid at position 3 of SEQ ID NO: 8 is not aspartic acid.
  • the amino acid at position 8 of SEQ ID NO: 8 is not glutamic acid.
  • the amino acids at positions 10, 11 and 12 of SEQ ID NO: 8 are not glutamic 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, 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 non-native saposin C sequences containing SEQ ID NO: 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, 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 these sequences. Again, any such peptide can be screened for such activity using the protocols described herein.
  • 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 ⁇ 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. 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.
  • 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, m ⁇ ristic, palmitic, stearic, oleic and li ⁇ oleic.
  • 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.
  • 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, intracerebrospinai, intramuscular, intradermai, subcutaneous, intracranial, intra ⁇ asai, epidural, topical and oral.
  • administration by direct intramuscular or intravenous injection is preferred.
  • 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 or as an analgesic are in the range of from about 10 to about 100 ⁇ g/kg, although dosages from about 0.1 to about 1,000 ⁇ g/kg are also contemplated. Thus, for the typical 70 kg human, dosages can be between 7 and 70,000 ⁇ g daily, preferably between 700 and 7,000 ⁇ g daily. 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 giycol 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 therapeuticall ⁇ 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.
  • Cells may be treated to facilitate myelin formation or to prevent demyeiination in the manner described above in vivo, ex vivo or in vitro.
  • Diseases resulting in demyeiination of nerve fibers including MS, acute disseminated leukoe ⁇ cephalitis, progressive multifocal leukoencephaiitis, 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.
  • 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 solid-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 Vydac C4 column to an extent greater than 95% prior to use.
  • HPLC high performance liquid chromatography
  • 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 coversiips. After 20-24 hours, the medium was replaced with 2 ml DMEM containing 0.5% FCS plus 0, 0.5, 1, 2, 4 or 8 ng/ml TX 14(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.
  • FCS fetal calf serum
  • TX 14(A) and the rat 14-mer both induced neurite outgrowth in NS20Y cells.
  • 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 8 ng/ml TX14(A). 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%. As shown in Figure 2, TX 14(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 Ex vivo myelination assay Newborn mouse cerebellar explants are prepared according to Satomi (Zool. Sci., 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 30-mer non-native saposin C peptide containing the sequence shown in SEQ ID NO: 8 (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.
  • Schwann cells contain an extensive myelin sheath.
  • the addition of a non-native 20 mer peptide containing the sequence shown in SEQ ID NO: 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.
  • 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 TX 14(A) or other peptide encompassed by SEQ ID NO: 8 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 demyeiination disorders Patients diagnosed with early stage MS are given peptide TX 14(A) or other peptide encompassed by SEQ ID NO: 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, musculoskeletal coordination and myelination (as determined by MRI) is observed.
  • Example 7 Alleviation of neuropathic pain in Chung model rats
  • This example describes the effects of bolus intrathecal injection of TX 14(A) and other peptides encompassed by SEQ ID NO: 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 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
  • 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/l 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., Eur. 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 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 animals each which are administered saline, TX 14(A) or another peptide encompassed by SEQ ID NO: 8, 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%. Thus, parenteral administration of peptide alleviates the pain from formalin injection and improves motor neuron function in a rat model of painful diabetic neuropathy.
  • MOLECULE TYPE peptide
  • SEQUENCE DESCRIPTION SEQ ID NO:1: Cys Glu Phe Leu Val Lys Glu Val Thr Lys Leu He Asp Asn Asn Lys 1 5 10 15
  • MOLECULE TYPE peptide
  • MOLECULE TYPE peptide
  • xi SEQUENCE DESCRIPTION: SEQ ID N0:3: Leu He Asp Asn Asn Lys Thr Glu Lys Glu He Leu 1 5 10
  • MOLECULE TYPE peptide
  • MOLECULE TYPE peptide
  • SEQUENCE DESCRIPTION SEQ ID N0:6: Thr Lys Leu He Asp Asn Asn Lys Thr Glu Lys Glu He Leu Asp 1 5 10 15

Abstract

L'invention concerne des analogues de peptides de synthèse non naturels dérivés de la région neurotrophique active de la saposine C. Les peptides dérivés de la saposine C induisent des excroissances des axones, favorisent la myélinisation et la neuroprotection, empêchent la mort cellulaire programmée et possèdent un pouvoir analgésique. Ils sont utiles dans le traitement des troubles du système nerveux périphérique et central et dans le traitement de la douleur.
PCT/US1998/005503 1997-03-24 1998-03-20 Peptides neurotrophiques de synthese derives de la saposine c Ceased WO1998042746A2 (fr)

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CA002284105A CA2284105A1 (fr) 1997-03-24 1998-03-20 Peptides neurotrophiques de synthese derives de la saposine c
EP98926027A EP0971956A2 (fr) 1997-03-24 1998-03-20 Peptides neurotrophiques de synthese derives de la saposine c
AU77956/98A AU7795698A (en) 1997-03-24 1998-03-20 Synthetic saposin c-derived neurotrophic peptides

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US08/823,425 1997-03-24

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WO1998042746B1 WO1998042746B1 (fr) 1999-02-11
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US5571787A (en) 1993-07-30 1996-11-05 Myelos Corporation Prosaposin as a neurotrophic factor
US6271196B1 (en) * 1996-03-05 2001-08-07 Regents Of The University Of Ca Methods of alleviating neuropathic pain using prosaposin-derived peptides
EP0979238A4 (fr) * 1997-03-05 2000-08-23 Univ California Procedes pour soulager les douleurs neuropathiques
US7834147B2 (en) 2003-04-28 2010-11-16 Childrens Hospital Medical Center Saposin C-DOPS: a novel anti-tumor agent
KR100973641B1 (ko) * 2008-05-09 2010-08-02 성균관대학교산학협력단 해마 신경 세포, 해마 조직 및 대뇌 피질 조직에서ｂｄｎｆ의 단백질 발현을 증가시키는 신규 펩타이드

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US5700909A (en) * 1993-07-30 1997-12-23 The Regents Of The University Of California Prosaposin and cytokine-derived peptides
US6271196B1 (en) * 1996-03-05 2001-08-07 Regents Of The University Of Ca Methods of alleviating neuropathic pain using prosaposin-derived peptides

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