WO1999012559A1 - Inhibition de l'apoptose au moyen d'agonistes du recepteur de la prosaposine - Google Patents

Inhibition de l'apoptose au moyen d'agonistes du recepteur de la prosaposine Download PDF

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WO1999012559A1
WO1999012559A1 PCT/US1998/019216 US9819216W WO9912559A1 WO 1999012559 A1 WO1999012559 A1 WO 1999012559A1 US 9819216 W US9819216 W US 9819216W WO 9912559 A1 WO9912559 A1 WO 9912559A1
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seq
prosaposin
apoptosis
cells
cell
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PCT/US1998/019216
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John S. O'brien
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The Regents Of The University Of California
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Priority to EP98951917A priority Critical patent/EP1011709A4/fr
Priority to CA002304108A priority patent/CA2304108A1/fr
Priority to JP2000510456A priority patent/JP2001515866A/ja
Priority to AU97746/98A priority patent/AU9774698A/en
Publication of WO1999012559A1 publication Critical patent/WO1999012559A1/fr

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Definitions

  • This invention relates generally to apoptosis, and more specifically to the use of prosaposin receptor agonists to inhibit apoptosis upregulation of downstream cellular signaling molecules, such as Akt and Bcl-2 that act to inhibit caspase-mediated apoptosis.
  • Prosaposin is the precursor of a group of four heat-stable glycoproteins that are required for glycosphingolipid hydrolysis by lysosomal hydrolases.
  • Prosaposin a 70 kilodalton (kDa) glycoprotein, is proteolytically processed to generate saposins A, B, C, and D.
  • the saposins exist as 4 tandem domains in prosaposin before proteolysis. All 4 saposins are structurally similar to each other, having a similar placement of six cysteines, a glycosylation site and conserved proline residues.
  • Unprocessed prosaposin also exists as an integral membrane protein and as a secreted protein that is present in human milk, cerebrospinal fluid and seminal plasma.
  • Prosaposin, saposin C, and prosaposin-derived peptides have therapeutic applications in promoting functional recovery after toxic, traumatic, myocardial ischemic, degenerative and inherited lesions to the peripheral and central nervous system. See, U.S. Patent No. 5,571,787.
  • Prosaposin and prosaptides can also be used to counteract the effects of demyelinating diseases by inducing neurite outgrowth stimulating myelination. The neurotrophic and myelinotrophic activity of prosaposin has been localized to amino acids 18-29 of saposin C.
  • Tumor necrosis factor ⁇ is a proinflammatory cytokine.
  • TNF ⁇ induces a proinflammatory response in many disorders, including rheumatoid arthritis, Crohn's disease, irritable bowel syndrome, asthma, stroke cardiac infarction, and congestive heart failure.
  • TNF ⁇ therapy was identified as a potential therapeutic target for rheumatoid arthritis
  • antibodies to TNF ⁇ were shown to be effective in both animal models and human patients.
  • a similar approach was taken in animal models of inflammatory bowel disease.
  • injection of TNF ⁇ into the subperineural space in the sciatic nerve immediately proximal to the sciatic notch produces neuropathic pain in vivo.
  • TNF ⁇ -injected animals displayed a significant hyperalgesia compared to vehicle-injected animals, in which hyperalgesia lasted for 5 days.
  • TNF ⁇ also induces programmed cell death (apoptosis) in several neural cell types, including cortical neurons, oligodendrocytes, and oligodendrocyte precursor cells.
  • Apoptosis accounts for most of the programmed cell death in tissue remodeling and for the cell loss that accompanies atrophy of adult tissues following withdrawal of endocrine and other trophic factor stimuli.
  • abnormal apoptosis is responsible for many human diseases after injury, including traumatic, chemical, myocardial ischemic, and genetic causes.
  • the proinflammatory cytokine interferon ⁇ is a potent inducer of oligodendrocyte apoptosis. Oligodendrocyte apoptosis has been observed at the advancing margins of chronic active multiple sclerosis (MS) plaques. IFN ⁇ may therefore be a factor in the pathogenesis of multiple sclerosis by activating apoptosis in oligodendrocytes.
  • proinflammatory cytokines such as TNF ⁇ and IFN ⁇ are likely factors in the abnormal apoptosis underlying the pathogenesis in many demyelination disorders.
  • the present invention provides a method for using prosaposin receptor agonists to inhibit apoptosis.
  • inhibition of apoptosis associated with caspase activation is inhibition of apoptosis associated with caspase activation.
  • Caspase activation resulting in apoptosis may be induced, for example, by proinflammatory cytokines, as well as by Alzheimer's disease, stroke, myocardial ischemia, increased intracellular Ca ++ levels, and increased levels of the neurotransmitter glutamate.
  • the invention is thus useful for treating a proinflammatory cytokine-induced disease, such as multiple sclerosis, rheumatoid arthritis, irritable bowel syndrome, AIDS neuropathy and encephalitis, progressive multifocal leukoencephalitis, chronic myocardial atrophy, Alzheimers disease, and cell death of any type due to cytokine-induced apoptosis.
  • a proinflammatory cytokine-induced disease such as multiple sclerosis, rheumatoid arthritis, irritable bowel syndrome, AIDS neuropathy and encephalitis, progressive multifocal leukoencephalitis, chronic myocardial atrophy, Alzheimers disease, and cell death of any type due to cytokine-induced apoptosis.
  • a proinflammatory cytokine-induced disease such as multiple sclerosis, rheumatoid arthritis, irritable bowel syndrome, AIDS neuropathy and encephalitis, progressive multifocal leukoencephalitis, chronic myocardial
  • Akt dissociates complexes of Bcl-2 family members, such as BAD-Bcl-2, releasing Bcl-2 and its family members which inhibit caspases, thereby inhibiting apoptosis.
  • the activation (phosphorylation) of Akt by the action of prosaposin receptor agonists is a key event in the prevention of caspase-mediated apoptosis.
  • the inhibition of apoptosis by prosaposin receptor agonists is a unique method of inhibiting apoptosis, because many other inhibitors of apoptosis inhibit caspase- mediated apoptosis at stages of the caspase proteolytic cascade different from the stage influenced by prosaposin receptor agonists.
  • the use of prosaposin receptor agonists to inhibit caspase-mediated apoptosis represents a significant new function for these compositions.
  • prosaposin receptor agonists inhibit apoptosis is by blocking activation of JNK, a proapoptotic signaling component. Within several minutes after binding to the receptor, prosaposin receptor agonists block JNK activation induced by TNF ⁇ . The activation of JNK by TNF ⁇ is another well known mechanism for TNF ⁇ -induced, as well as other proinflammatory cytokine-induced, apoptosis.
  • the invention provides a method for inhibiting JNK-mediated and caspase- mediated apoptosis by contacting cells at risk of such apoptosis with an apoptosis- inhibiting amount of a prosaposin receptor agonist. The cells may be contacted in vivo or ex vivo.
  • the cells are oligodendrocytes, neurons, Schwann cells, or myocytes.
  • the prosaposin receptor agonist is a peptide selected from SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:l 1, and SEQ ID NO:12.
  • FIG. 1 is the polynucleotide sequence of human prosaposin cDNA.
  • FIG. 2 is the polypeptide sequences of prosaposin and saposin C.
  • FIG. 3 is the polypeptide sequence of several prosaposin-derived peptides.
  • FIG. 4 illustrates that the survival factor-promoted activation of Akt requires PI 3-kinase. Survival factor binding to the cognate receptor activates PI 3-kinase and other kinases. PI 3 -kinase activates the serine/threonine kinase Akt. Subsequently, Akt phosphorylates specific targets, including the Bcl-2 family member BAD. Phosphorylation inactivates BAD, causing other BCL-2 family members to inhibit cell death (apoptosis) and allow cell survival.
  • FIG. 5 illustrates that prosaposin receptor agonist TX14(A) binding to prosaposin receptor acts to inhibit caspace-mediated apoptosis.
  • Proinflammatory cytokine TNF ⁇ binds to TNF-Rto activate adaptor molecules, such as TRADD.
  • TRADD activates the caspase proteolytic cascade, causing apoptosis.
  • Prosaposin receptor agonist binding to prosaposin receptor activates PI 3-kinase and other kinases.
  • PI 3-kinase activates the serine/threonine kinase Akt. Subsequently, Akt phosphorylates specific targets, including the Bcl-2 family member BAD.
  • FIG. 6 shows that (A) prosaposin and (B) prosaposin receptor agonist TX 14(A) prevent TNF ⁇ -induced viability loss in NS20Y cells.
  • NS20Y cells were incubated for 48 hr in DMEM containing 0.5% fetal bovine serum (FBS) and 100 ng/ml TNF ⁇ ⁇ 2-fold dilutions of prosaposin (0 - 5 nM) or prosaptide (0 - 50 nM). Cell viability was assessed using MTT reduction. Results are mean ⁇ SEM. Asterisk (*) indicates that mean is significantly different to TNF ⁇ treated cells; p ⁇ 0.05.
  • FIG. 7 shows the time course of TNF ⁇ -induced viability loss and prevention by prosaposin receptor agonists.
  • NS20Y cells were incubated in DMEM containing 0.5% FBS without (solid bar) or with (hatched bar) 100 ng/ml TNF ⁇ and 5 nM prosaposin (grey bar) or 50 nM prosaptide (hollow bar) for 48-96 hours.
  • MTT was used to assess cell viability. Results are mean ⁇ SEM. Asterisk (*) indicates that mean is statistically different to TNF ⁇ treated cells; p ⁇ 0.05.
  • FIG. 8 shows that prosaposin receptor agonist TX14(A) prevents TNF ⁇ -induced death of NS20Y cells.
  • NS20Y cells were treated with TNF ⁇ in DMEM containing 0.5%) FBS with or without increasing doses of prosaptide.
  • At 48 hr cells were stained with trypan blue to assess cell death. Results are mean ⁇ SEM.
  • FIG. 9 shows that prosaposin receptor agonist TX 14(A) does not cause proliferation of NS20Y cells.
  • NS20Y cells were seeded at 10,000/well in 96-well plates and grown in DMEM containing 0.5% FBS and 2-fold dilutions of prosaptide (P; solid bar) or in DMEM containing 2-fold dilutions of FCS (S; hatched bar).
  • P prosaptide
  • S hatched bar
  • FIG. 10 shows inhibition of JNK2 phosphorylation in primary Schumann cells by a prosaposin derived peptide, TX14(A).
  • Schwann cells were stimulated for 5 minutes with TNF ⁇ +/- TX 14(A).
  • Equal amounts of proteins from cell lysates were analyzed by SDS-PAGE and inununoblotted using a polyclonal antibody that recognizes phosphorylated JNK2 (Promega, Madison, WI). Proteins were detected by ECL (Amersham, Arlington Heights). Autoradiographs were scanned using ImageQuantTM (Molecular Dynamics, Sunnyvale, CA). Data are shown as representative data of two independent experiments.
  • FIG. 10 shows inhibition of JNK2 phosphorylation in primary Schumann cells by a prosaposin derived peptide, TX14(A).
  • Schwann cells were stimulated for 5 minutes with TNF ⁇ +/- TX 14(A).
  • Equal amounts of proteins from cell lysates were analyzed by SDS-PAGE and inununo
  • FIG. 11 shows inhibition of pl 10 poly(ADP-ribose), PARP, cleavage by TX14(A).
  • Primary Schwann cells were placed in low serum media (0.25%) FBS) for 1 hour +/- TX14(A).
  • Equal amounts of proteins from cell lysates were analyzed by SDS- PAGE and immunoblotted using a polyclonal antibody rhar recognizes PARP (Upstate Biotechnology, Lake Placid, NY). Proteins were detected by ECL (Amersham, Arlington Heights). Autoradiographs were scanned using ImageQuantTM (Molecular Dynamics, Sunnyvale, CA). Data are expressed as a mean ratio of pl 10 to p85 PARP ⁇ SEM of two independent experiments.
  • FIG. 12 shows the effect of various doses of TX14(A) peptide (prosaptide; SEQ ID NO: 7) on TNF ⁇ -induced Schwann cell death.
  • the peptide concentration is shown on the x-axis and the percentage of trypan blue-stained cells is shown on the y- axis.
  • FIG. 13 shows the effect of prosaposin and TX14(A) (SEQ ID NO:7) on proinflammatory cytokine-induced cell death in undifferentiated CG4 oligodendrocytes.
  • FIG. 13A shows the effect of prosaposin and TX14(A) (SEQ ID NO:7) on TNF ⁇ - induced cell death in undifferentiated CG4 oligodendrocytes.
  • FIG. 13B shows the effect of prosaposin and TX14(A) on IFN ⁇ -induced cell death in undifferentiated CG4 oligodendrocytes.
  • FIG. 14 shows that prosaposin receptor agonist TX 14(A) inhibits proinflammatory cytokine TNF ⁇ -induced apoptosis in L6 myoblasts.
  • L6 myoblasts cells were incubated for 96 hours either in media (control); media with 10 ng/ml TNF ⁇ (TNF
  • FIG. 15 is a chart depicting the effect of prosaptide in vivo on thermal hyperalgesia following endoneurial injection of TNF ⁇ .
  • Prosaptide 200 ⁇ g/kg was injected subcutaneously 3 hr before injection of 10 ⁇ l TNF ⁇ (2.5 pg/ml).
  • DETAILED DESCRIPTION OF THE INVENTION 200 ⁇ g/kg was injected subcutaneously 3 hr before injection of 10 ⁇ l TNF ⁇ (2.5 pg/ml).
  • the present invention provides a method for inhibiting apoptosis.
  • the invention provides a method for inhibiting caspase-mediated apoptosis using a prosaptide, prosaposin, or saposin C.
  • Caspases can be activated by several factors, including cytokines, anticancer drugs, growth factor deprivation, myocardial ischemia, metabolic toxins, and Ca ++ toxicity.
  • the method of the invention involves administering an apoptosis-inhibiting amount of a prosaposin receptor agonist to cells.
  • prosaposin receptor agonist refers to a molecule that binds to any site on a cell to which prosaposin can bind, and to thereby alter the cell's function in the same manner to prosaposin.
  • Examples of prosaposin receptor agonists include prosaposin, prosaptides, and saposin C.
  • a receptor agonist is a substance that mimics the receptor ligand, is able to attach to that receptor, and thereby produces a same action that the ligand usually produces.
  • Drugs are often designed as receptor agonists to treat diseases and disorders caused when the ligand, such as a hormone, is missing or depleted in a subject.
  • Prosaposin is a 70 kDa glycoprotein that is the precursor of a group of 4 small heat-stable glycoproteins that are required for hydrolysis of glycosphingolipids by lysosomal hydrolases.
  • Prosaposin is a 517 amino acid protein, originally identified as the precursor of 4 sphingolipid activator proteins, as described in U.S. Patent No. 5,571 ,787.
  • Four adjacent tandem domains in prosaposin are proteolytically processed in lysosomes to generate saposins A, B, C, and D, that activate hydrolysis of glycosphingolipids by lysosomal hydrolases.
  • prosaposin The unprocessed form of prosaposin is found in high concentrations in human and rat brain, where it is localized within neuronal surface membranes. During embryonic development, prosaposin mRNA is abundant in brain and dorsal root ganglia. Furthermore, prosaposin binds with high affinity to gangliosides, to stimulate neurite outgrowth, and promote transfer of gangliosides from micelles to membranes.
  • Prosaposin receptor agonists can be identified both structurally and functionally.
  • a prosaposin receptor agonist has a structure that is similar to the region of prosaposin that, when bound to the prosaposin receptor, induces a prosaposin receptor activity.
  • the prosaposin receptor agonist can have a structure that is similar to the amino acid sequence LeuIleXaa ] AsnAsnXaa 1 ThrXaa 2 Xaa 3 Xaa 2 Xaa 1 , where Xaa, is any amino acid; Xaa- is a charged amino acid; and Xaa 3 is optionally present and, when present, is a charged amino acid.
  • a prosaposin receptor agonist induces a prosaposin receptor activity, for example, second messenger signaling, neurite outgrowth or myelination, decreased neuropathic pain, inhibition of proinflammatory cytokine-induced apoptosis, or inhibition of apoptosis caused by other agents.
  • the prosaposin agonist is prosaposin itself.
  • the prosaposin may be either prosaposin from native sources or prosaposin that is produced by recombinant methods, such as recombinant human prosaposin purified from spent media o ⁇ Spodopterafrugiperda (Sf9) cells infected with a baculovirus expression vector containing full-length cDNA for human prosaposin.
  • Human prosaposin has the amino acid sequence set forth in SEQ ID NO:2.
  • the human cDNA sequence for prosaposin is SEQ ID NO: 1. When the subject to be treated is human, human prosaposin and saposin sequence may more particularly be used.
  • the prosaposin agonist is saposin C.
  • saposin C refers to the proteolytic cleavage product from the third tandem domain of prosaposin. Saposin C can be isolated in pure form from spleens of patients with Gaucher disease, a lysosomal storage disorder, by the method of Morimoto et al. (Proc. Natl. Acad. Sci. USA, 87: 3493-3497, 1990). Human saposin C has the amino acid sequence set forth in SEQ ID NO:3.
  • the prosaposin agonist is a peptide including amino acids 18-28 of saposin C.
  • the term "prosaptide” includes a peptide comprising amino acids 18-28 of saposin C (SEQ ID NO:4), peptides that have the activity of prosaptide comprising amino acids 18-28 of saposin C, or conservative variations of these amino acid sequences that retain a bioactivity of amino acids 18-28 of saposin C.
  • a "conservative variation,” as used herein, denotes the replacement of an amino acid residue by another, biologically similar residue.
  • conservative variation also includes the use of a substituted amino acid in place of an unsubstituted parent amino acid provided that antibodies raised to the substituted polypeptide also immunoreact with the unsubstituted polypeptide. Generally, only conservative amino acid alterations are undertaken, using amino acids that have the same or similar properties.
  • Illustrative amino acid substitutions include the changes of: alanine to serine; arginine to lysine; asparagine to glutamine or histidine; aspartate to glutamate; cysteine to serine; glutamine to asparagine; glutamate to aspartate; glycine to proline; histidine to asparagine or glutamine; isoleucine to leucine or valine; leucine to valine or isoleucine; lysine to arginine, glutamine, or glutamate; methionine to leucine or isoleucine; phenylalanine to tyrosine, leucine or methionine; serine to threonine; threonine to serine; tryptophan to tyrosine; tyrosine to tryptophan or phenylalanine; valine to isoleucine or leucine.
  • deletion of one or more amino acids can also result in a modification of the structure of the resultant molecule without significantly altering its activity. This can lead to the development of a smaller active molecule.
  • An active octadecamer (18 amino acid) peptide fragment is set forth as SEQ ID NO:5.
  • An active docosanamer (22 amino acid) peptide fragment is set forth as SEQ ID NO: 6.
  • prosaptides of the invention have a length of at least about 11 amino acid residues, for example, at least about 14 amino acid residues.
  • Prosaptides of the invention comprise about 80 or fewer amino acid residues, for example, no more than about 40 amino acid residues or no more that about 22 amino acid residues.
  • the prosaposin receptor agonist is a prosaptide which has about 11 amino acids to about 80 amino acids (the full-length of saposin C) and the amino acid sequence LeuIleXaa 1 AsnAsnXaa 1 ThrXaa 2 Xaa 3 Xaa 2 Xaa 1 Xaa I , where Xaa, is any amino acid; Xaaj is a charged amino acid; and Xaa 3 is optionally present and, when present, is a charged amino acid.
  • the prosaposin receptor agonist may be a prosaposin-derived peptide.
  • the prosaposin receptor agonist may have the polypeptide sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7.
  • the polypeptide sequence LeuIleAspAsnAsnLysThrGluLysGluIleLeu corresponds to amino acids 18 to 29 of saposin C.
  • the polypeptide sequence ThrDAlaLeuIleAspAsnAsnAlaThrGluGluIleLeuTyr corresponds to amino acids 16 to 29 of saposin C modified by a D-alanine for lysine substitution at position 2; an alanine for lysine substitution at position 8; a deletion of lysine at position 11 and the addition of a C-terminal tyrosine residue. See, TABLE 1. Such modifications can be useful for increasing peptide stability or uptake across the blood-brain barrier as described in EXAMPLE 6.
  • D-alanine can be represented by D-Ala or X.
  • the prosaposin receptor agonist can also be an active fragment derived from another mammalian prosaposin.
  • active fragment of prosaposin is synonymous with "prosaptide.”
  • an active fragment of mouse prosaposin, rat prosaposin, guinea pig prosaposin or bovine prosaposin such as SEQ ID NOS: 8 through 11 is a prosaposin receptor agonist.
  • amino acid sequence of an active fragment of human prosaposin that corresponds to amino acids 8 to 29 of saposin C (docosanomer; SEQ ID NO:6), is well conserved among other species, as shown in TABLE 2.
  • adjacent asparagine (N) residues are conserved among human, mouse, rat, guinea pig and bovine prosaposins.
  • a leucine (L) residue is conserved 3 to 4 residues toward the N-terminus of the 2 asparagine residues and one or more charged residues (aspartic acid (D), lysine (K), glutamic acid (E) or arginine (R)) are conserved 2 to 8 residues toward the C-terminus of the 2 asparagine residues.
  • D partic acid
  • K lysine
  • E glutamic acid
  • R arginine
  • the prosaposin receptor agonist is selected from a population of peptides related in amino acid sequence to SEQ ID NO:6 by having the conserved asparagine residues, a leucine/isoleucine residue, and one or more charged residues at the positions corresponding to the positions in which these residues are found in SEQ ID NO:6, but also having one or more amino acids that differ from the amino acids of SEQ ID NO:6.
  • a prosaposin receptor agonist can be identified by screening a large collection, or library, of random peptides or peptides of interest using assays that detect prosaposin receptor agonist function, for example, one of a number of animal models of apoptosis or inflammation known to those of skill in the art.
  • a prosaposin receptor agonist can be isolated or synthesized using methods well known in the art. Such methods include recombinant DNA methods and chemical synthesis methods for production of a peptide. Recombinant methods of producing a peptide through expression of a nucleic acid sequence encoding the peptide in a suitable host cell are well known in the art and are described, for example, in Sambrook et al. (Molecular Cloning: A Laboratory Manual, 2nd Ed., Vols. 1 to 3, Cold Spring Harbor Laboratory Press, New York, 1989).
  • a prosaposin receptor agonist also can be produced by chemical synthesis, for example, by the solid phase peptide synthesis method of Merrifield et al. (J. Am. Chem. Soc. 55:2149, 1964).
  • Standard solution methods well known in the art also can be used to synthesize a peptide useful in the invention. See, for example, Bodanszky ⁇ Principles of Peptide Synthesis, Springer-Verlag, Berlin, 1984) and Bodanszky (Peptide Chemistry, Springer-Verlag, Berlin, 1993).
  • the chemically synthesized peptide may be prepared on an Applied Biosystems Model 430 peptide synthesizer using an automated solid-phase protocol provided by the manufacturer. Peptides may then be purified by high performance liquid chromatography (HPLC) on a Vydac C4 column to an extent greater than 95%.
  • HPLC high performance liquid chromatography
  • a newly synthesized peptide can be purified, for example, by high performance liquid chromatography (HPLC), and characterized using, for example, mass spectrometry or amino acid sequence analysis.
  • a particularly useful modification of a prosaposin receptor agonist is one that confers, for example, increased stability, by incorporation of one or more D-amino acids or substitution or deletion of lysine can increase the stability of a prosaposin receptor agonist by protecting against peptide degradation.
  • the prosaposin-derived tetradecamer SEQ ID NO: 7 has an amino acid sequence derived from amino acids 16 to 29 of saposin C, but which has been modified by substitution or deletion of each of the 3 naturally occurring lysines and the addition of a C-terminal tyrosine residue.
  • the prosaposin-derived tetradecamer SEQ ID NO:7 has a D-alanine for lysine substitution at position 2; an alanine for lysine substitution at position 8 and a deletion of lysine at position 11.
  • the D-alanine substitution at position 2 confers increased stability by protecting the peptide from endoprotease degradation, as is well known in the art. See, for example, Partridge (Peptide Drug Delivery to the Brain, Raven Press, New York, 1991 , page 247).
  • the substitution or deletion of a lysine residue confers increased resistance to trypsin-like proteases, as is well known in the art. See, Partridge, supra.
  • the prosaposin receptor agonist can also be made as a cyclic peptide for increased stability.
  • a useful modification to a prosaposin receptor agonist can also be one that promotes peptide passage across the blood-brain barrier, such as a modification that increases lipophilicity or decreases hydrogen bonding.
  • a tyrosine residue added to the C-terminus of the prosaposin-derived peptide (SEQ ID NO:7) increases hydrophobicity and permeability to the blood-brain barrier. See, for example, Banks et al. (Peptides 73.
  • prosaposin receptor refers to a site on a cell to which prosaposin or a prosaposin receptor agonist can bind, thereby acting to alter the cell's function.
  • the prosaposin receptor is a G-protein-coupled cell surface receptor of 54-60 kDa, isolated from baboon brains, pig brains, whole rat brain, and mouse neuroblastoma cells. This receptor protein can be isolated from a P100 plasma membrane fraction by affinity purification using a neurite growth-inducing peptide contained within the saposin C sequence linked to a solid support.
  • the 54-60 kDa protein crosslinks irreversibly to saposin C. The isolation of the putative prosaposin receptor is described in EXAMPLES 6 and 7.
  • Apoptosis refers to the cellular process of programmed cell death.
  • Apoptosis encompasses a group of characteristic structural and molecular events, in which a cell specifically and precisely controls its fate in a mixed cell population. Endogenous nucleases cleave chromatin between nucleosomes and reduce the content of intact DNA in cells undergoing apoptosis.
  • Apoptosis accounts for most of the programmed cell death in tissue remodeling and for the normal cell loss that accompanies atrophy of adult tissues following withdrawal of endocrine and other growth stimuli.
  • apoptosis is similar to proliferation in that both processes are tightly regulated and essential for the homeostasis of renewable tissues.
  • Apoptosis is also responsible, however, for the abnormal cell death that occurs in many diseases.
  • Apoptosis can be recognized by a characteristic pattern of morphological, biochemical and molecular changes in apoptotic cells. These changes can be broadly assigned to 3 stages: In the early stage, there is decreased cell size (cell dehydration), alterations in cell membranes, large (50 kilobase [kb]) DNA strand breaks, and an increase in cellular calcium levels. In the intermediate stage, DNA is cleaved into 180-200 bp fragments, giving the characteristic "laddering" on a DNA gel, further decrease in cell size, and a decreased cell pH. In the late stage, there is a loss of membrane function and the formation of apoptotic bodies.
  • Methods of detecting apoptosis can be based on the measurement of DNA content, altered membrane permeability, or the detection of endonucleolysis as characterized by DNA strand breaks. Such techniques are well known to those of skill in the art and can be readily performed without undue experimentation.
  • the apoptotic process can also be assayed by determining the activity of prosaposin receptor, Akt, Bcl-2 family members, associated PI 3 -kinase pathway components, and JNK.
  • the research tools that can be used are the well-known techniques involving antibodies and the various technologies (i.e., immunoprecipitation, immunoblotting, and immunoaffinity chromatography) that use these molecular probes. See, Kohler et al.
  • caspase refers to any of the aspartate-specific cysteine proteases, sharing a conserved active site that cleaves proteins at a highly specific site, to induce apoptosis. All caspases cleave their substrates after aspartate (Asp) residues. Caspases promote apoptosis through proteolytic degradation of cellular components, a process which is amplified by autocatalysis of the various caspases. Different members of the caspase superfamily (formerly known as the ICE family) have slightly different substrate specificities and may thus be involved in different aspects of the apoptotic pathway. Caspases generally function in the distal portions of the proteolytic cascades involved in apoptosis ⁇ see, FIG. 5).
  • Caspases are processed from a single-chain zymogen to a two-chain active enzyme by cleavage at internal Asp residues. Caspases with large prodomains are generally regulatory caspases, whereas those with small prodomains are generally effector caspases. Thus, active caspases can activate other caspases following an initial activating stimulus to form a proteolytic cascade, with the initial activation of a regulatory caspase serving to activate by proteolytic cleavage the downstream effector caspases.
  • caspases break down cellular proteins, causing severe morphological changes and cell shrinkage. Effector caspases, particularly caspase-3, cleave substrates such as poly(ADP-ribose) polymerase, actin, fodrin, and lamin.
  • the chromosomal DNA is cleaved by a DNase enzyme.
  • the enzyme caspase-3 activated DNase (CAD) cleaves chromosomal DNA. CAD does not control apoptosis itself.
  • the CAD inhibitor of caspase-3 activated DNase acts as a chaperone for CAD during CAD synthesis, remaining complexed with CAD to inhibit CAD DNase activity until the reactivity is triggered by appropriate apoptotic stimuli.
  • Caspase-3 when activated by apoptotic stimuli, cleaves ICAD to release the DNase activity, allowing CAD, which carries a nuclear-localization signal, to enter the nucleus, and degrade chromosomal DNA in nuclei, causing the characteristic DNA fragmentation. See, Sakahira et al. (Nature 391(6662): 96-99, 1998); and Enari et al. (Nature 391(6662): 43-50, 1998). Thus, activation of CAD downstream of the caspase cascade is responsible for characteristic DNA degradation during apoptosis.
  • the method of the invention involves administering an apoptosis-inhibiting amount of a prosaposin receptor to cells.
  • the invention provides a method for inhibiting caspace-mediated apoptosis due to a proinflammatory cytokine.
  • the proinflammatory cytokine that induces apoptosis may be TNF ⁇ , one of the most intensively studied caspase activators.
  • TNF ⁇ induces apoptosis in many cell types, including neurons, oligodendrocytes and oligodendrocyte precursor cells. While TNF ⁇ has been known to be important in proinflammatory responses for 20 years, the particular biochemical steps have been incompletely understood until recently.
  • the TNF ⁇ is now a paradigm that has been applied to the other activators of caspace- mediated apoptosis.
  • TNF ⁇ effects are mediated through binding of TNF ⁇ to two types of receptor, the 75 kDa TNF-R1 and a 55 kDa TNF-R2.
  • TNF ⁇ binding to a TNF-R initiates a variety of biological responses.
  • the cellular signaling in apoptosis begins at the TNF-R1 and moves downstream in a series of biochemical reactions.
  • TNF-R1 has 3 separate responses: (1) apoptosis; (2) the activation of NF- ⁇ B, a transcription factor that inhibits apoptosis; and (3) the activation of JNK, a protein kinase.
  • TNF ⁇ signal transduction pathway directly regulates caspase activation through recruitment of adaptor molecules and caspases to the cytoplasmic domain of TNF-R.
  • TNF-R contains a cytoplasmic death domain (DD) that activates the apoptotic process by interacting with the DD-containing adaptor proteins TNF-R-associated DD protein (TRADD) and Fas-associated DD protein (FADD/MORT1), leading to the activation of cysteine proteases of the caspase family.
  • TRADD TNF-R-associated DD protein
  • FADD/MORT1 Fas-associated DD protein
  • the TRADD protein has two distinct functional domains. The protein has a DD body and a tail. The tail of TRADD binds to TRAF2, eventually resulting in activation of NF- ⁇ B.
  • the body binds to FADD, another intracellular signaling protein, which then activates apoptosis.
  • Another DD- containing protein that binds to TNF-R is caspase-8. Binding of TNF ⁇ stimulates TNF-R, leading to the formation of a receptor-bound death-inducing signaling complex (DISC), consisting of FADD and two different forms of caspase- 8. As a result, activation of the caspase proteolytic cascade begins.
  • DISC receptor-bound death-inducing signaling complex
  • Fas Another intensively studied caspase activator is Fas.
  • Fas Autoimmune disorders are associated with defects in Fas pathway function. Inappropriate expression of the Fas ligand (FasL) can enable tumor cells to escape immune surveillance.
  • the Fas signal transduction pathway also directly regulates caspase activation through recruitment of adaptor molecules and caspases to the cytoplasmic domain of the receptor.
  • Fas (Apol; CD95) also contain a cytoplasmic death domain (DD) that activates the apoptotic process by interacting with TRADD and FADD, leading to caspase activation. Stimulation of Fas leads to the formation of a receptor-bound death-inducing signaling complex (DISC), consisting of FADD and two different forms of caspase-8.
  • DD cytoplasmic death domain
  • Fas-associated death domain protein (FADD) recruited caspase-8 to the Fas signaling complex by virtue of caspase-8 ability to bind the adapter molecule FADD established that this protease has a role in initiating the death pathway.
  • the method of the invention using a prosaposin receptor agonist is effective in inhibition of apoptosis in proinflammatory cytokine-susceptible cells that contain the prosaposin receptor, the downstream signaling elements of PI 3 -kinase, Akt, and Bcl-2, and the caspase-mediated cell death mechanism (See, FIG. 4 and 5. See also, Hemmings, Science 275: 628-630, 1997; Franke et al, Nature 390: 116-117, 1997; Datta et al, Cell i: 231-241, 1997).
  • Prosaposin receptor agonists stimulate several different signal transducers after binding to the prosaposin receptor. These signal transducers include mitogen- activated protein kinase (MAPK), PI 3 -kinase, and the non-receptor tyrosine kinase p60 Src . Signal transduction following prosaposin receptor agonist binding to prosaposin receptor has been shown in neuronal cells, Schwann cells, and myoblasts. Prosaposin receptor agonists utilize a pertussis toxin sensitive G-protein pathway to activate MAPK proteins. Furthermore, Akt is upregulated within minutes of cellular exposure to prosaposin receptor agonist. Akt activates an apoptosis-inhibiting Bcl-2 family member, which then inhibits the action of caspases. Thus, prosaposin receptor agonists prevent caspase-mediated apoptosis.
  • MAPK mitogen- activated protein kinase
  • PI 3 -kinase PI 3 -kin
  • Mitogen-activated protein kinase is a general name for a family of serine/threonine kinases that play an important role in cell signaling by a variety of ligands and receptors, including receptor tyrosine kinases and G-protein coupled receptors.
  • the extracellular signal-regulated protein kinases, ERKl and ERK2 are part of the MAPK family.
  • ERKl is the 44 kDa protein (p44 MAPK ).
  • ERK2 is the 42 kDa protein (p42 MAPK ).
  • Signaling proteins such as phosphatidylinositol-3 -kinase (PI 3 -kinase) and protein kinase C (PKC) phosphorylate ERK proteins, either independently or in association with the guanosine triphosphate (GTP)-binding protein Ras (p21 Ras ) pathway.
  • GTP guanosine triphosphate
  • Ras p21 Ras
  • Prosaposin receptor agonists bind to the prosaposin receptor with high affinity to activate ERKl and ERK2 phosphorylation in PC 12 cells, Schwann cells, and oligodendrocytes. Prosaposin receptor agonists also activate ERK activity by a pertussis toxin-sensitive mechanism involving the adapter protein She, p60 Src , and PI 3-kinase.
  • Akt has a critical role in the PI 3- kinase pathway.
  • the activation of the serine/threonine protein kinase Akt is a key event in apoptosis prevention. Modules made of protein kinases control cellular processes, including apoptosis. After growth factors bind to their cognate growth factor receptor tyrosine kinases, PI 3-kinases are recruited and activated. Inositol lipids are phosphorylated by PI 3-kinases to act as second messengers.
  • the serine/threonine protein kinase Akt protein kinase B; PKB
  • PKB protein kinase B
  • Akt dissociates a complex of Bcl-2 family members, activating an apoptosis- inhibiting Bcl-2 family member, which then inhibits caspases to prevent apoptosis.
  • the activation of Akt is a key event in cell death prevention by the PI 3 -kinase pathway.
  • Akt is a proto-oncogene with a pleckstrin homology domain.
  • the pleckstrin homology domains can bind lipids, providing a mechanism linking the activation of PI 3-kinase and Akt activity.
  • PI 3-kinase activity can be inhibited by wortmannin and by the inhibitor LY294002. Both of these inhibitors inhibit the rapid activation of Akt by growth factors, such as platelet-derived growth factor (PDGF), epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), insulin, and insulin-like growth factor- 1 (IGF-1).
  • PDGF platelet-derived growth factor
  • EGF epidermal growth factor
  • bFGF basic fibroblast growth factor
  • IGF-1 insulin-like growth factor- 1
  • Activation of Akt by protein phosphatase inhibitors is, however, relatively insensitive to wortmannin and LY294002.
  • the lipid kinase activity of PI 3-kinase
  • Th e PI 3-kinase-derived second messengers phosphatidylinositol-3 ,4-bisphosphate (PtdIns-3 ,4-P2) and phosphatidylinositol-3,4,5-triphosphate (PtdIns-3,4,5-PI 3), promote activation of Akt in 3 steps: (i) the translocation of the kinase to the membrane, (ii) the attachment to the membrane by means of pleckstrin homology domain binding to phospholipid, and (iii) phosphorylation.
  • Akt The high-affinity association of Akt with PtdIns-3,4-P2 and PtdIns-3,2, promotes a conformational change leading to an increase of kinase activity.
  • PtdIns-3,4-P2 and PtdIns-3,4-P3 which accumulate transiently upon cell stimulation by growth factors, also bind to the pleckstrin homology domain of Akt and promote the association of Akt with the membrane.
  • PI 3-kinase activity further leads to an increase in Akt kinase by promoting Akt phosphorylation of Akt at 2 sites (Thr 308 and Ser 473 ) by an upstream kinase, known as PKBK. Both phosphorylation events can be inhibited by wortmannin in vivo.
  • IP3 inositol trisphosphate
  • IGF- 1 protects cerebellar neurons from apoptosis by activating Akt.
  • Nerve growth factor (NGF) also promotes Akt activation in pheochromocytoma PC 12 cells, showing that kinase activation is also involved in the survival promoted by NGF.
  • the Akt signaling pathway can prevent apoptosis of neurons.
  • the Akt signaling pathway for suppressing apoptosis then proceeds to the phosphorylation of the Bcl-2 family member BAD, thereby inactivating BAD promotion of apoptosis and promoting cell survival. See, Datta et al. ⁇ Cell 91(2): 231-24, 1997).
  • Akt phosphorylates BAD in vitro and in vivo to inhibit BAD-induced apoptosis.
  • the inactivation of BAD allows other, apoptosis-inhibiting Bcl-2 family members to inhibit caspases.
  • the mammalian Bcl-2 family has members that are potent inhibitors of programmed cell death and inhibit activation of caspases in cells ⁇ e.g., Bcl-2, Bcl-x L , and Bag). Other members of the Bcl-2 family promote apoptosis ⁇ e.g. Bax, Bcl-x s , BAD, and Bak). However, Bcl-2 family members have several different mechanisms of function which need not be mutually exclusive. Bcl-2 family proteins either suppress or promote apoptosis by interacting with and functionally antagonizing each other. The regulation of apoptosis by Bcl-2 family members involves several regulatory processes including dimerization and phosphorylation.
  • Bcl-2 family members form homodimers and heterodimers to interact with one another, altering the balance between cell survival and apoptosis. Phosphorylation can also change the activity state of many Bcl-2 family members. For example, phosphorylation of BAD by Akt causes BAD to be sequestered and inactivated.
  • BH domains are (1) BH1 and BH2, which in apoptosis inhibitors allows heterodimerization with Bax to repress apoptosis; (2) BH3, which in the apoptosis promoters, Bax and Bak, allows heterodimerization with Bcl-x L and Bcl-2 to promote apoptosis; and (3) BH4 which conserved in apoptosis inhibitors, e.g. Bcl-x L , but absent in apoptosis agonists except Bcl-x s .
  • the BH4 domain allows interaction with apoptosis regulatory proteins such as Raf-1 and BAD.
  • All members of the Bcl-2 family (except BAD and Bid) contain a hydrophobic C-terminus (transmembrane, TM) domain which anchors the Bcl-2 protein to the cell membrane.
  • BAD lacks this sequence and is, therefore, located throughout the cytoplasm.
  • Bcl-2 and Bcl-x L localize predominantly to the outer mitochondrial membrane, but also to the nuclear and endoplasmic reticulum membranes.
  • the GTP -binding protein Raf-1 translocates Bcl-2 family members to the mitochondrial membrane.
  • Apoptosis-inhibiting members such as Bcl-2 and Bcl-x L form dimers with the apoptosis-inducing activity of Bax and BAD to block Bax and BAD activity.
  • the ratio of apoptosis-inhibiting Bcl-2 family members to apoptosis- inducing Bcl-2 family members is important in determining whether apoptosis will proceed. Excess apoptosis-inhibiting Bcl-2 family members promotes survival whereas excess apoptosis-inducing Bcl-2 family members promotes apoptosis.
  • Bcl-x L homodimers are required to actively suppress apoptosis or to actively promote survival. Therefore, Bcl-x L /Bax heterodimerization promotes apoptosis.
  • Bax homodimers are required to actively promote apoptosis or to actively inhibit survival.
  • Bcl-x L /Bax heterodimerization inhibits apoptosis. If Bcl-2 levels are higher that those of Bax, for example, then survival generally prevails, whereas the opposite circumstance is associated with cell death.
  • the Bcl-2 family member apoptosis inhibitors inhibit caspase activation. For example, there is a direct interaction between caspases and Bcl-x L .
  • the loop domain of Bcl-x L is cleaved by caspases in vitro and in cells induced to undergo apoptotic death. Interaction of Bcl-x L with caspases may be an important mechanism of inhibiting cell death.
  • the C-terminal fragment of Bcl-x potently induces apoptosis.
  • the recognition/cleavage site of Bcl-x L protects against apoptosis by acting at the level of caspase activation; cleavage of Bcl-x L during the execution phase of programmed cell death converts Bcl-x L from a protective to a lethal protein.
  • prosaposin receptor agonists By inhibiting caspase activity in cells, prosaposin receptor agonists inhibit the apoptotic pathway and allow cell survival. Prosaposin receptor agonists inhibit apoptosis in proinflammory cytokine-susceptible cells that contain the prosaposin receptor, the downstream signaling elements of PI 3 -kinase, Akt, and Bcl-2, and the caspase-mediated cell death mechanism. The inhibition of apoptosis by prosaposin receptor agonists occurs at the level of caspase activation, which is a unique method of inhibiting apoptosis. Known apoptosis inhibitors block caspace-mediated apoptosis at stages of the caspase proteolytic cascade different from the stage influenced by prosaposin agonists.
  • TNF ⁇ therapy was identified as a potential therapeutic target for rheumatoid arthritis, antibodies to TNF ⁇ were shown to have efficacy in both animal models and human patients. See, Eigler et al. ⁇ Immunol Today 18(10): 487-492, 1997).
  • Prosaposin receptor agonist treatment can be an effective alternative therapeutic agent for the treatment of Crohn's disease and rheumatoid arthritis because prosaposin receptor agonists are administered more easily than anti-T ⁇ F ⁇ antibodies, and animal studies demonstrate no antibodies against prosaptides after months of administration.
  • activation of the transcription factor ⁇ F- ⁇ B inhibits apoptotic signaling through the transcriptional activation of survival-promoting genes.
  • Prosaposin receptor agonists may also act through an alternative pathway to additionally promote survival.
  • caspase inhibitors In still other therapies, regulation of caspase-mediated apoptosis induction can be accomplished by expression of caspase inhibitors.
  • Peptides that inhibit caspases are commercially available.
  • the peptides Caspase-3 Inhibitor I (DEVD-CHO; a highly specific, potent, reversible, and cell-permeable inhibitor of caspase-3); Caspase-3 Inhibitor III (Ac-DEVD-CMK; a potent, cell-permeable and irreversible inhibitor of caspase-3); and Caspase-4 Inhibitor I (Ac-LEVD-CHO; a caspase-4 inhibitor) are available from Calbiochem (San Diego, CA).
  • Prosaposin receptor agonists do not compete with these peptides.
  • Prosaposin receptor agonists may also act to inhibit apoptosis through activation of a family of proteins known as Inhibitors of Apoptosis Proteins (IAPs).
  • IAPs were first identified on the basis of sequence similarity to the insect baculovirus which infects cells and inhibits apoptosis. These molecules contain four conserved regions which have death antagonizing properties: (1) three baculovirus inhibitory repeats (BIR); and (2) a ring zinc finger domain. Both regions are likely involved in mediating protein-protein interactions.
  • One IAP gene Neuronal Apoptosis Inhibitor Protein (NAIP), is selectively expressed in surviving neurons. NAIP was discovered to be the gene deleted in spinal muscular atrophy, a genetic disorder which causes spinal motor neuron degeneration and muscular atrophy leading to the death of newborn children. The IAPs act by preventing the activity or activation of caspases.
  • cells may be identified as susceptible to proinflammatory cytokine-induced apoptosis by one or a combination of the following analyses: identifying cells as undergoing apoptosis during events associated with contacting cells with proinflammatory cytokine; identifying proinflammatory cytokine during the events that cause cells to undergo apoptosis; preventing apoptosis by removing proinflammatory cytokine during the events that cause cells to undergo apoptosis; and inducing apoptosis by reintroducing proinflammatory cytokine or ending the removal of proinflammatory cytokine during the events that cause cells to undergo apoptosis. More particularly, cells may be identified as susceptible to proinflammatory cytokine-induced apoptosis by laboratory methods described by Vartanian et al. (Molecular Medicine 1(7): 732, 1995).
  • oligodendrocytes Expressly included as cells that are susceptible to proinflammatory cytokine- induced apoptosis are oligodendrocytes, neurons, Schwann cells, or myocytes. Since it is known that TNF ⁇ induces apoptosis in several neural cell types, including cortical neurons, oligodendrocytes and oligodendrocyte precursor cells, an identification of a cell as a neural cell is also an identification of that cell as susceptible to TNF ⁇ -mediated apoptosis. Since it is known that IFN ⁇ induces oligodendrocyte apoptosis, an identification of a cell as an oligodendrocyte is also an identification of that cell as susceptible to IFN ⁇ -mediated apoptosis.
  • Prosaposin receptor agonists are useful in treating diseases that involve cell death or that are mediated by proinflammatory cytokines. Prosaposin receptor agonists are therefore useful in treating degenerative diseases such as neurodegenerative diseases ⁇ e.g., Alzheimer's disease, post-polio syndrome, Parkinson's disease, amyotrophic lateral sclerosis, Huntington's disease), ischemic disease of the heart ⁇ e.g., myocardial infarction), traumatic brain and spinal cord injury, pain syndromes, alopecia, AIDS, and toxin mediated liver disease. See, Nicholson (Nature Biotechnology 14: 297, 1996).
  • neurodegenerative diseases ⁇ e.g., Alzheimer's disease, post-polio syndrome, Parkinson's disease, amyotrophic lateral sclerosis, Huntington's disease
  • ischemic disease of the heart e.g., myocardial infarction
  • traumatic brain and spinal cord injury traumatic brain and spinal cord injury
  • pain syndromes alopecia,
  • a disease may be identified as being a proinflammatory cytokine-induced disease by one or a combination of the following analyses: (1) identifying the disease as occurring during events associated with the proinflammatory cytokine; (2) identifying proinflammatory cytokine during the apoptotic disease events that cause inflammation; (3) preventing apoptosis by removing proinflammatory cytokine during the disease events; and (4) inducing apoptosis by reintroducing proinflammatory cytokine or ending the removal of proinflammatory cytokine during the disease events.
  • a subject to be treated according to the invention may be identified as being at risk for having a proinflammatory cytokine-induced disease at a future time.
  • the experimental subjects were identified as having a TNF ⁇ -mediated disease at the time of, or even before, the TNF ⁇ injection and therefore before the onset of hyperalgesia, a characteristic feature of experimental neuropathic pain states.
  • the treatment method of the invention therefore includes both treating existing proinflammatory cytokine-induced disease and prophylactically reducing the severity of future proinflammatory cytokine-induced disease.
  • Prosaposin receptor agonists are therefore useful in treating many disorders in which TNF ⁇ is known to be involved, including rheumatoid arthritis (Feldman et al. (Annals of the New York Academy of Sciences 766: 272-278, 1995); Feldman et al (Journal of Inflammation 47: 90-96, 1996)), Crohn's disease (Stokkers et al. (Journal of Inflammation 47: 97-103, 1996)), irritable bowel syndrome, asthma, stroke cardiac infarction, and congestive heart failure. See, Eigler et al ⁇ Immunol Today 18(10): 487-492, 1997); MacLellan et al. ⁇ Circ. Res.
  • TNF ⁇ induces apoptosis in several neuronal cell types, including cortical neurons, oligodendrocytes and oligodendrocyte precursor cells.
  • the use of prosaposin receptor agonist for the treatment of any of these disorders is within the scope of the present invention. These agonists can be administered either alone or as an adjunct to conventional anti-inflammatory therapies such as steroid administration.
  • the proinflammatory cytokine IFN ⁇ is also a potent inducer of oligodendrocyte apoptosis. Oligodendrocyte apoptosis has been observed at the advancing margins of chronic active multiple sclerosis (MS) plaques (Vartanian et al, Molecular Medicine 1(7): 732, 1995). IFN ⁇ may therefore be a factor in the pathogenesis of multiple sclerosis by activating apoptosis in oligodendrocytes.
  • the method of the invention may be used for halting or slowing the progress of the IFN ⁇ -mediated diseases associated with neural or myelin degeneration in neural tissue, by contacting neuronal tissue susceptible to such degradation with a prosaposin receptor agonist.
  • TNF ⁇ and IFN ⁇ may further be factors in the death of oligodendrocyte cells which underly the pathogenesis in many demyelination disorders.
  • a patient diagnosed as having a demyelination disease would also be expressly identified as having a proinflammatory cytokine-induced disease that may be treated by the method of the invention.
  • Prosaposin receptor agonists can also be used in the treatment of Alzheimer's disease. Caspase inhibition is relevent to neurodegenerative disease and inhibition of caspase activity may have an impact on the clinical course of neurodegenerative diseases, such as Alzheimer's disease. See, Holtzman et al. ⁇ Nature Medicine 3(9): 954-955, 1997). Prosaposin receptor agonists, especially prosaposin receptor agonists that cross the blood-brain barrier, treatment can be a therapeutically effective agent for treating neurodegenerative diseases of the central nervous system.
  • Apoptosis is a contributing cause of cardiac myocyte loss in ischemia reperfusion injury, myocardial infarction, and long-standing heart failure. See, MacLellan et al. ⁇ Circ. Res. 81(2): 137-144, 1997). Insights into the molecular circuitry controlling apoptosis suggest the potential to protect heart muscle from apoptosis through one or more of these pathways by pharmacological means.
  • Cytokines that are expressed within the myocardium in response to environmental injury are important for initiating and integrating homeostatic responses during cardiovascular disease. See, Mann ⁇ Cytokine Growth Factor Rev. 7(4): 341-354, 1996). For example, the failing human heart expresses TNF ⁇ . See, Kubota et al. ⁇ Circ. Res. 81(4): 627-635, 1997) in the development of congestive heart failure.
  • TNF ⁇ and its p55 TNF-R have been well documented in experimental animal models of arthritis and in transgenic mice expressing wild-type or mutant transmembrane human TNF ⁇ proteins in their joints. See, Alexopoulou et al. ⁇ Eur. J. Immunol. 27(10): 2588-2592, 1997).
  • Prosaposin receptor agonist administration can provide an effective therapy for treatment of arthritis, because prosaposin receptor agonists inhibit the effects of proinflammatory cytokines downstream of the interactions between TNF ⁇ and TNF-R.
  • administering prosaposin receptor agonists to inhibit caspase- mediated apoptosis includes the use of such agonists in the treatment of diseases such as rheumatoid arthritis, Crohn's disease, irritable bowel syndrome, asthma, cardiac infarction, congestive heart failure, multiple sclerosis, acute disseminated inflammatory (AIDS) leukoencephalitis, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, post-polio syndrome, Huntington's disease, ischemic heart disease, traumatic brain injury, traumatic spinal cord injury, alopecia, AIDS dementia, cerebral malaria, HTLV neuropathy, Guillain-Barre disease, AIDS neuropathy, inflammatory neurodegenerative diseases, and toxin-induced liver disease.
  • diseases such as rheumatoid arthritis, Crohn's disease, irritable bowel syndrome, asthma, cardiac infarction, congestive heart failure, multiple sclerosis, acute disseminated inflammatory (AIDS) leuk
  • apoptosis-inhibiting amount means the amount of prosaposin receptor agonist needed to inhibit apoptosis in a target cell.
  • the amount of prosaposin receptor agonist that inhibits apoptosis can easily be determined by one of skill in the art using standard methods for assaying apoptosis.
  • the activity of a prosaposin receptor agonist in inhibiting apoptosis can correlate with neurotrophic activity and activity in alleviating neuropathic pain or inducing neurotrophic activity.
  • the prosaposin-derived docosanomer (SEQ ID NO:6) and the prosaposin-derived tetradecamer (SEQ ID NO: 7) alleviate neuropathic pain and have neurotrophic activity.
  • the prosaposin-derived dodecamer peptide (SEQ ID NO:4), which has the conserved adjacent asparagines, leucine and charged residues described above, is active as a neurotrophic factor.
  • a typical minimum amount of prosaposin for the neurotrophic factor activity in cell growth medium is usually about 1.4 x 10 " " M, or about 10 ng/ml.
  • This amount or more of prosaposin receptor agonists may also be used to inhibit apoptosis or reduce inflammation. Typically concentrations in the range of 0.1 ⁇ g/ml to about 10 ⁇ g/ml of any of these materials will be used.
  • the contact between the prosaposin receptor agonist and the cells may be performed ex vivo or in vivo.
  • Cells can be treated ex vivo by directly administering prosaposin receptor agonists to the cells.
  • cells can be treated ex vivo by culturing the cells in growth medium suitable for the particular cell type followed by addition of the agonist to the medium.
  • Such ex vz ' v ⁇ -treated cells can then be administered to a patient.
  • Cells are treated in vivo by administering the agonist by any effective method that will result in contact between the prosaposin receptor agonist and the cell.
  • the method of administration of an apoptosis-inhibiting amount of prosaposin receptor agonist may be by conventional modes of administration, including intravenous, intramuscular, intradermal, pulmonary, nasal, mucosal, subcutaneous, epidural, intraocular, topical in a biologically compatible carrier and oral administration.
  • the composition may be injected directly into the blood in sufficient quantity to give the desired in vivo concentration.
  • Direct intracranial injection or injection into the cerebrospinal fluid also may be used provided sufficient quantities can be given such that the desired local concentration is achieved.
  • a pharmaceutically acceptable injectable carrier of well known type can be used. Such carriers include, for example, phosphate buffered saline (PBS) or lactated Ringer's solution.
  • PBS phosphate buffered saline
  • lactated Ringer's solution lactated Ringer's solution.
  • the composition can be administered to peripheral neural tissue by direct local injection or by systemic administration.
  • One skilled in the art can readily assay the ability of a prosaposin receptor agonist to cross the blood-brain barrier in vivo, for example, as disclosed in EXAMPLE 6.
  • an active fragment of prosaposin can be tested for its ability to cross the blood-brain barrier using an in vitro model of the blood-brain barrier based on a brain microvessel endothelial cell culture system, for example such as that described by Bowman et al. (Ann. Neurol. 74:396-402, 1983) or Takahura et al. (Adv. Pharmacol. 22:137-165, 1992).
  • An octadecamer (18 amino acid; SEQ ID NO:5) peptide consisting of amino acids 12-29 of the docosanamer (SEQ ID NO: 6) with a substitution of tyrosine for valine at amino acid 12 (with a molecular weight of 2 kDa) crosses the blood-brain barrier and enters the central nervous system. Conditions under which a peptide can cross the blood-brain barrier and enter the nervous system are described by Banks et al. (Peptides 13: 1289-1294, 1992).
  • neuronal populations such as motor neurons
  • Other neuronal populations can be treated by intravenous injection, although direct injection into the cerebrospinal fluid is also envisioned as an alternate route.
  • prosaposin receptor agonist is resistant to gastrointestinal degradation and readily absorbable.
  • substitution, for example, of one or more D-amino acids can confer increased stability to a prosaposin receptor agonist useful in the invention.
  • Retroinverso peptidomimetics that are stable and retain bioactivity can also be devised, as described by Brugidou et al. ⁇ Biochem. Biophys. Res. Comm. 214(2): 685-693, 1995) and Chorev et al. ⁇ Trends Biotechnol 13(10): 438-445, 1995).
  • the prosaposin receptor agonists 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 phosphate-buffered saline or in lyophilized form is an example of a unit dosage.
  • Appropriate daily systemic dosages of agonist based on the body weight for treatment of caspase-mediated apoptosis 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.
  • a systemic dosage can be between about 7 and about 70,000 ⁇ g daily and, alternatively, between about 700 and about 7,000 ⁇ g daily.
  • a daily dosage of locally administered material will be about an order of magnitude less than the systemic dosage.
  • a prosaposin receptor agonist also can be administered in an inhalant form.
  • Inhalant drug delivery has been successfully used for ⁇ -agonist and corticosteroid drugs of emphysema. See, Pingleton ⁇ JAMA, June 19, 1996).
  • Mask ventilation is now a first-line therapy for patients who have an exacerbation of chronic obstructive pulmonary disease.
  • Similar methods of inhalant drug delivery can be used to deliver prosaposin receptor agonists.
  • a prosaposin receptor agonist also can be administered in a sustained release form. The sustained release of a prosaposin receptor agonist has the advantage of inhibiting apoptosis over an extended period of time without the need for repeated administrations of the active fragment.
  • Sustained release can be achieved, for example, with a sustained release material such as a wafer, an immunobead, a micropump or other material that provides for controlled slow release of the prosaposin receptor agonist.
  • a sustained release material such as a wafer, an immunobead, a micropump or other material that provides for controlled slow release of the prosaposin receptor agonist.
  • Such controlled release materials are well known in the art and available from commercial sources (Alza Corp., Palo Alto CA; Depotech, La Jolla CA. See also, Pardoll (Ann. Rev. Immunol. 13: 399-415, 1995)).
  • a bioerodible or biodegradable material that can be formulated with a prosaposin receptor agonist, such as polylactic acid, polygalactic acid, regenerated collagen, lipo somes, or other conventional depot formulations, can be implanted to slowly release the active fragment of prosaposin.
  • the use of infusion pumps, matrix entrapment systems, and transdermal delivery devices also are contemplated in the present invention.
  • the invention also provides a method for inhibiting apoptosis or alleviating inflammation in a subject by transplanting into the subject a cell genetically modified to express and secrete a prosaposin receptor agonist. Transplantation can provide a continuous source of a prosaposin receptor agonist and, thus, sustained alleviation of neuropathic pain. For a subject suffering from prolonged apoptosis, such a method has the advantage of obviating or reducing the need for repeated administration of an active fragment of prosaposin.
  • a cell can be readily recombinanfiy modified, such as by transfection with an expression vector containing a nucleic acid encoding a prosaposin receptor agonist. See, Chang ⁇ Somatic Gene Therapy, CRC Press, Boca Raton, 1995). Following transplantation into the brain, for example, the transfected cell expresses and secretes a prosaposin receptor agonist and, thus, inhibits apoptosis. Such a method can be useful to alleviate neuropathic pain as described for the transplantation of cells that secrete substances with analgesic properties. See, for example, Czech and Sagen (Prog. Neurobiol. 46:501-529, 1995).
  • the transfected cell should be immunologically compatible with the subject. Consequently, autologous cells are particularly useful for recombinant modification. Non-autologous cells also can be useful if protected from immune rejection using, for example, microencapsulation or immunosuppression.
  • Useful microencapsulation membrane materials include alginate-poly-L-lysine alginate and agarose (See, for example, Goosen ⁇ Fundamentals of Animal Cell Encapsulation and Immobilization, CRC Press, Boca Raton, 1993); Tai and Sun ⁇ FASEBJ. 7:1061, 1993); Liu et al. (Hum. Gene Ther. 4:291, 1993); and Taniguchi et al. (Transplant. Proc. 24: 2977, 1992)).
  • the cell can be a human cell, although a non-human mammalian cell also can be useful.
  • a human fibroblast, muscle cell, glial cell, neuronal precursor cell or neuron can be transfected with an expression vector to express and secrete an active fragment of prosaposin such as SEQ ID NO:4.
  • a primary fibroblast can be obtained, for example, from a skin biopsy of the subject to be treated and maintained under standard tissue culture conditions.
  • a primary muscle cell also can be useful for transplantation. Considerations for neural transplantation are described, for example, in Chang, supra.
  • a cell derived from the central nervous system can be particularly useful for transplantation to the central nervous system, since the survival of such a cell is enhanced within its natural environment.
  • a neuronal precursor cell is particularly useful in the method of the invention since a neuronal precursor cell can be grown in culture, transfected with an expression vector and introduced into an individual, where it is integrated. The isolation of neuronal precursor cells that are capable of proliferating and differentiating into neurons and glial cells is described in Renfranz et al. (Cell 55:713-729, 1991).
  • a retroviral vector is preferred.
  • a replication-defective herpes simplex virus type 1 (HSV-1) vector is useful. See, During etal (Soc. Neurosci. Abstr. 77:140, 1991) and Sable et al. (Soc. Neurosci. Abstr. 77:570, 1991).
  • a nucleic acid encoding an active fragment of prosaposin can be expressed under the control of one of a variety of promoters well known in the art, including a constitutive promoter or inducible promoter. See, for example, Chang, supra.
  • a particularly useful constitutive promoter for high level expression is the Moloney murine leukemia virus long-terminal repeat (MLV-LTR), the cytomegalovirus immediate-early (CMV-IE) or the simian virus 40 early region (SV40).
  • the invention provides a method of alleviating neuropathic pain by administering a neuropathic pain-alleviating amount of a prosaposin receptor agonist to a subject who is suffering from neuropathic pain caused by proinflammatory cytokine.
  • the invention is therefore useful for treating neuropathic pain component of inflammatory disease, although the pain relief is not due to an anti-inflammatory effect.
  • the prosaposin receptor agonist activation of Akt described supra for inhibition of caspace-mediated apoptosis is relevant to the alleviation of neuropathic pain.
  • TNF ⁇ In an animal model, injection of TNF ⁇ into the subperineural space in the sciatic nerve immediately proximal to the sciatic notch produces neuropathic pain in vivo. See, Wagner et al. (NeuroReport 7: 2897-2901, 1996). Using behavioral testing of either mechanical or thermal hyperalgesia, TNF ⁇ -injected animals suffer significant hyperalgesia compared to vehicle-injected animals, whose algesia lasted for 5 days. The pain is due to nerve damage. Administration of prosaposin receptor agonist prevents the thermal hyperalgesia that occurs upon injection of TNF ⁇ into the sciatic nerve.
  • EXAMPLE 6 An example of the alleviation of neuropathic pain by prosaposin receptor agonist in the rat TNF ⁇ -injection model is provided in EXAMPLE 6.
  • a reduction in pain can be determined by behavioral measurements assessing the response to thermal or mechanical stimulii. When the subject is human, the subject can report a reduction in pain. Reduction in pain can also be detrmined in animal models.
  • animal models of neuropathic pain have been developed, including the Chung rat model, the streptozotocin-induced insulin-deficient diabetic rat model, the Seltzer rat model, the neuroma model, and several primate models. See, Myers (N7H Workshop on Low Back Pain (J. Weinstein, S.
  • an alleviation of neuropathic pain by aclministration of prosaposin receptor agonist that is successful in any one animal model of neuropathic pain may be extrapolated to all models and to all types of human neuropathic pain, as prosaposin receptor agonists operate at a fundamental convergent step in the pathogenesis of pain arising from nerve injury.
  • An effective concentration of prosaposin receptor agonist may also be determined by comparison with the concentrations of prosaposin receptor agonist recommended for other conditions.
  • Activation of immune cells by pathogens also induces the release of a proinflammatory cytokines. See, Watkins et al. ⁇ Brain Res. 692(1-2): 244-250, 1995).
  • the activated immune system communicates to the brain by release of proinflammatory cytokines. See, Watkins et al. ⁇ Pain 63(3): 289-302, 1995).
  • Proinflammatory cytokines mediate a variety of common neuropathic pain states. Illness responses in the brains of those suffering from neuropathic pain cause dramatic changes in neural functioning. For example, IL-l ⁇ can alter brain function, resulting in a variety of illness responses including increased sleep, decreased food intake, fever, etc. IL-l ⁇ also produces neuropathic pain. This IL-l ⁇ -induced neuropathic pain is mediated by activation of subdiaphragmatic vagal afferents in the brain.
  • the physiological basis of IL-l ⁇ -induced neuropathic pain is representative of a general physiological basis for proinflammatory cytokine-induced neuropathic pain.
  • TNF ⁇ produces dose-dependent neuropathic pain as measured by the tailflick test.
  • This TNF ⁇ -induced neuropathic pain is further mediated by the induced release of IL-l ⁇ .
  • this TNF ⁇ -induced hyperalgesia (as well as most illness responses) is also mediated by activation of subdiaphragmatic vagal afferents.
  • the effects of subdiaphragmatic vagotomy cannot be explained by a generalized depression of neural excitability. See, Watkins et al. ⁇ Brain Res. 692(1-2): 244-250, 1995).
  • Proinflammatory cytokines and the neural circuits that they activate are therefore involved in the neuropathic pain states produced by irritants, inflammatory agents, and nerve damage.
  • Prosaposin receptor agonists that cross the blood-brain barrier are especially useful for treatment of illness response and other proinflammatory cytokine-induced neuropathic pain components in the central nervous system.
  • This EXAMPLE was show that prosaposin and a peptide derived from prosaposin could prevent TNF ⁇ neurotoxicity.
  • TNF ⁇ treatment for 48 hr or more caused up to 50% loss of viability in a neuronal cell line, NS20Y, as demonstrated by MTT reduction.
  • Prosaposin and prosaptide TX 14(A) prevented the loss of viability dose dependently, with maximal protection seen at 5 nM and 50 nM, respectively.
  • Trypan blue exclusion and BrdU incorporation assays showed that prosaptide increased viability by preventing cell death and did not cause cell proliferation.
  • the prevention of TNF ⁇ -induced death by prosaposin receptor agonists was not inhibited by pertussis toxin.
  • Cell culture reagents were purchased from Gibco-BRL (Grand Island, NY).
  • the mouse neuroblastoma cell line, NS20Y was a gift from Drs. T. Taketomi and K. Uemura (Shinshu University, Matsumoto, Japan).
  • Cells were maintained in DMEM (high glucose) containing 10% fetal calf serum (FCS), 100 U/ml penicillin, 100 ⁇ g/ml streptomycin and 1.1 mg/ml sodium pyruvate, at 37°C under humidified 5% CO 2 .
  • FCS fetal calf serum
  • For cell viability assays cells were seeded at lxl0 4 /well in 96-well plates in complete media and allowed to grow overnight.
  • TNF ⁇ and prosaposin or prosaptide were applied to cells in DMEM containing penicillin, streptomycin, sodium pyruvate and 0.5% FCS. Cells were then incubated for 24-96 hr. Cell viability, as indicated by reduction of a tetrazolium salt (MTT) to a purple formazan product, was assessed using the CellTiter 96TM kit (Promega, Madison, WI) according to the manufacturer's instructions. Standard curves were constructed to ensure that optical density measurements were within a linear range and to allow optical density readings to be converted to cell number.
  • MTT tetrazolium salt
  • TNF ⁇ Treatment of NS20Y cells with TNF ⁇ resulted in a loss of viability as demonstrated by a decrease in MTT reduction.
  • the effect of TNF ⁇ was dose dependent with maximal diminuition at 100 ng/ml TNF ⁇ . No loss of viability was seen at 24 hr.
  • TNF ⁇ was administered at 100 ng/ml for 48 or more hours.
  • FIG. 6A shows that cultures which received TNF ⁇ for 48 hr demonstrated a 35%) reduction in the number of viable cells as compared to controls.
  • untreated cultures were 65% as viable as control cultures whereas cells treated with 5 nM prosaposin were 85%) as viable.
  • TNF ⁇ continued to result in decreased cell viability and at 96 hr viability was approximately 40%> as compared to controls whereas prosaposin treated cells were approximately 54%> as viable.
  • the protective effect of prosaptide was maintained at its maximum at all time points.
  • prosaptide-treated cells were as viable as controls.
  • FIG. 8 shows that 25% of cells treated for 48 hr with 100 ng/ml TNF ⁇ were trypan blue positive (dead) as compared to 7% positive in control cultures; the increase in cell death was completely prevented in a dose-dependent manner by prosaptide at a maximal concentration of InM.
  • FIG. 9 shows that at 48 hr prosaptide did not induce cell proliferation at any dose. The proliferative capacity of the cells was confirmed by demonstrating a dose-dependent stimulation of proliferation by serum. Additionally, prosaptide did not stimulate proliferation at 24, 72 or 96 hr.
  • This EXAMPLE demonstrates that prosaposin and a prosaposin-derived peptide of 14 amino acids, TX 14(A), prevented the TNF ⁇ -induced death of a neuronal cell line.
  • the neuroprotective action of prosaposin receptor agonists was dose-dependent. At the maximally effective doses the protection was almost complete.
  • Prosaposin was able to protect cells from TNF ⁇ -induced death at a 10-fold lower molar concentration than TX14(A). This is likely due to a difference in the binding affinity of the two ligands for the putative prosaposin receptor.
  • the Kd of prosaposin binding to PC12 cells was 2.5 nM while the Kd of prosaptide (TX14(A)) binding was 18.3nM.
  • Prosaposin receptor agonists can stimulate tyrosine phosphorylation in NS20Y cells, iSC cells and primary Schwann cells.
  • primary Schwann cells prosaptide-induced ERK phosphorylation is inhibited by pertussis toxin suggesting that the putative prosaposin receptor is linked to a heterotrimeric G-protein containing G oa or G ia subunits.
  • Hiraiwa et al. ⁇ Proc. Natl. Acad. Sci. USA 94: 4778-4781, 1997) have recently presented data to suggest that the association is with G 0 ⁇ .
  • Prosaposin receptor agonist-induced enhancement of sulfatide levels has been demonstrated in Schwann cells (See,Cam ⁇ ana et al, FASEB J. 12: 307-314, 1998) and neurite outgrowth in NS20Y cells (See, Misasi et ⁇ /., 1998).
  • PI3-kinase is known to play an integral role in the prevention of neuronal cell death by neurotrophic factors including BDNF, IGF, and NGF and the prevention of death of other cell types.
  • TNF ⁇ can be neurotoxic. This multifunctional cytokine can also be neuroprotective. Whether TNF ⁇ acts in a protective or toxic capacity may well be determined by which neuronal cell line is being studied or the regimen of TNF ⁇ treatment used. Under similar conditions NS20Y, PC 12 and SK-N-MC cells are susceptible to the cytotoxic effects of TNF ⁇ , whereas SH-SY5 Y and Neuro2A cultures do not lose viability when treated with 100 ng/ml TNF ⁇ for up to 96 hr. Furthermore, the susceptibility of SK-N-MC cells to TNF ⁇ changes with their differentiation state.
  • This EXAMPLE shows that prosaposin receptor agonist TX14(A) inhibits JNK2 phosphorylation in Schwann cells after a 5 minute treatment ⁇ see, FIG. 10). Additionally, prosaposin receptor agonist TX 14(A) enhances Schwann cell production of pl00 PARP after one hour in low serum media ⁇ see, FIG. 11).
  • This EXAMPLE shows that treatment of primary Schwann cells and an immortalized Schwann cell line, iSC, with a 14-mer prosaptide, TX14(A), (10 nM) enhanced phosphorylation of mitogen-activated kinases, ERKl (p44 MAPK ; extracellular signal -regulated kinase 1) and ERK2 (p42 MAPK ; extracellular signal-regulated kinase 2) within 5 minutes that was blocked by 4 hour pretreatment with pertussis toxin. Furthermore, incubation of Schwann cells with the non-hydrolyzable GDP analog, GDP- ⁇ S, inhibited TX14(A)-induced ERK phosphorylation.
  • TX14(A) 14-mer prosaptide
  • TX14(A) enhanced the sulfatide content of primary Schwann cells 2.5-fold which was inhibited by pretreatment with pertussis toxin or the synthetic MEK inhibitor, PD098059.
  • TX14(A) increased the tyrosine phosphorylation of all 3 isoforms of the adapter molecule, She, which coincided with the association of p60 Src and PI 3-kinase. Inhibition of PI 3-kinase by wortmannin blocked TX14(A)-induced ERK phosphorylation.
  • This EXAMPLE demonstrates that TX14(A) uses a pertussis toxin sensitive G-protein pathway to activate ERKs that is essential for enhanced sulfatide synthesis in Schwann cells.
  • TX14(A) (SEQ ID NO:7) was synthesized commercially to 98%> purity (AnaSpec, San Jose, CA).
  • Platelet Derived Growth Factor (PDGF) was purchased from Genzyme (Cambridge, MA ).
  • GDP- ⁇ S, PD098059, wortmannin (WT) and pertussis toxin (PT) were purchased from CalBiochem (San Diego, CA).
  • Anti-phosphotyrosine monoclonal Ab, anti-Src monoclonal Ab, anti-PI 3 -kinase polyclonal Ab and anti-She polyclonal Ab were purchased from Upstate Biotechnology Incorporated (Lake Placid, New York).
  • Schwann cell cultures Two Schwann cell cultures were used; (1) a spontaneously transformed cell line, iSC, from rat primary Schwann cells, as described by Bolin et al. (J Neurosci. Res. 33: 231-238, 1992), and primary Schwann cells that were prepared from neonatal rats as described by Assouline et al. (In A Dissection and Tissue Culture Manual of the Nervous System (Shahar et al, eds), Wiley-Liss, New York, 1989) pp. 247-250. At the first passage, Schwann cells were further selected from fibroblasts using an anti-fibronectin antibody and rabbit complement.
  • iSC spontaneously transformed cell line
  • iSC cells were maintained in DME/F 12 containing 10%) horse serum and P/S (100 U/mL penicillin and 100 g/mL streptomycin).
  • Primary Schwann cells were maintained in DMEM containing 10%) fetal bovine serum (FBS), P/S, 21 g/mL bovine pituitary extract and 4 mM forskolin. All cells were incubated at 37°C under humidified 1.5% CO 2 .
  • Schwann cells were prepared as described above, stimulated with effectors for 5 minutes and lysed in 20 mM Tris (pH 7.5), 150 mM NaCI, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, ImM ⁇ -glycerolphosphate, 1 mM sodium vanadate, I ⁇ g/mL leupeptin and 1 mM PMSF. Protein content of each sample was determined as above.
  • Immunoprecipitates were incubated at 30°C for 30 minutes in kinase buffer containing 1 ⁇ g ELK-1 fusion protein and 100 ⁇ M ATP. Reactions were terminated by the addition of 25 ⁇ l 3X SDS sample buffer. Samples were boiled for 5 minutes and proteins were resolved by SDS-PAGE). Proteins were electroblotted onto nitrocellulose membrane and ERK activity was identified by immunoblotting with a phospho-ELK-1 antibody followed by detection with ECL (Amersham, Arlington Heights, IL). iSC cells (approximately 2.0 x 10 7 ) were incubated in DMEM/F12 without serum 18 hours prior to stimulation with TX 14(A) for 5 minutes at 37°C.
  • TX 14(A) increased both ERKl and ERK2 phosphorylation in Schwann cells. There was a larger increase in the ratio of ERKl phosphorylation to total ERKl protein (18-fold that of controls) than that of ERK2 (3-fold greater than controls).
  • iSC cells were preincubated with pertussis toxin, which catalyzes the ADP- ribosylation of G,/G 0 ⁇ subunits of G-proteins, TX14(A)-induced ERK phosphorylation was inhibited. Similar results were also observed in primary Schwann cells.
  • PDGF which binds to a tyrosine kinase receptor and stimulates proliferation of Schwann cells, stimulated ERKl (4-6 fold) and ERK2 (2 fold) phosphorylation but was not inhibited by pertussis toxin pretreatment.
  • ERK phosphorylation by TX14(A) involved G-proteins the iSC cells were incubated with GDP- ⁇ S. This treatment also blocked TX14(A)-induced ERK phosphorylation.
  • ERK protein kinases are activated by phosphorylation of tyrosine and threonine residues and both are required for full protein kinase activity. Because the antibody used only recognized the phosphorylated tyrosine residue on ERKs, the TX14(A)-induced phosphorylation of ERK was correlated with ERK catalytic activity. Kinase activity was also increased in both primary Schwann cells and iSC cells after treatment with PDGF and TX 14(A). The activation of the adapter protein, She, was also examined in TX 14(A) signaling. iSC cells expressed all 3 isoforms: p46 Shc , p3__ c , and p66 Shc .
  • iSC cell lysates were immunoprecipitated with an antibody to p60 Src and Western blotted with an antibody to phosphotyrosine; this showed enhanced tyrosine phosphorylation of PI 3-kinase after treatment with TX14(A). Furthermore, preincubation of iSC cells with wortmannin completely blocked TX14(A)-induced ERK phosphorylation. In unstimulated cells, wortmannin treatment reduced ERK phosphorylation below control levels.
  • TX 14(A) stimulates synthesis of sulfatide in Schwann cells.
  • primary Schwann cells were preincubated with either pertussis toxin or the synthetic inhibitor of MEK, PD098059, before TX14(A) stimulation.
  • the anti-sulfatide monoclonal antibody identified only sulfatide that had the same mobility as purified sulfatide in all samples.
  • TX 14(A) treatment increased the sulfatide content 2.5-fold over controls.
  • Pretreatment with either pertussis toxin or PD098059 inhibited TX14(A)-induced sulfatide synthesis.
  • the viability of Schwann cells treated with either PD098059 or pertussis toxin after 48 hours did not differ from controls as determined by trypan blue exclusion.
  • TX14(A) increased the phosphorylation of ERKs, however, the magnitude of the increase was less than what was observed in iSC cells.
  • the same dose of PD098059 (50 ⁇ M) used in the sulfatide experiments blocked TX14(A)-induced phosphorylation of ERK in primary Schwann cells.
  • PD098059 decreased ERKl and ERK2 phosphorylation below control levels.
  • Timecourse experiments of TX14(A)-induced phosphorylation of ERKs in iSC cells demonstrated that TX14(A) rapidly activates ERKl and ERK2 within 5 minutes and returned to baseline levels by 30 minutes.
  • TX14(A) dose-dependently stimulates ERK phosphorylation in both iSC and primary Schwann cells. After quantification and expression of the data as a ratio of phosphorylated ERKs to total ERK proteins, TX 14(A) preferentially phosphorylated ERKl, although Schwann cells contained a greater amount of immunoreactive ERK2 protein. The same phenomenon has been observed in PC 12 cells and ERKl is preferentially activated in oligodendrocytes.
  • TX14(A)-stimulated ERK phosphorylation was blocked by pertussis toxin treatment which indicated that the primary mechanism of activation involved one or more pertussis toxin sensitive G proteins such as G ; or G 0 , both of which are abundantly expressed in Schwann cells.
  • ERK activation is associated with pertussis toxin-sensitive G-protein signaling in COS-7 cells, CHO cells) and Swiss 3T3 cells.
  • cell surface binding assays using radio-labeled TX 14(A) gave a single high affinity constant for binding to iSC cells with a Kd of 10 nM.
  • This EXAMPLE demonstrates that inhibition of MEK by PD098059 completely blocked TX14(A)-enhanced synthesis of sulfatide, an essential myelin lipid component of both central and peripheral nervous system myelin, in Schwann cells.
  • This concentration of PD098059 50 ⁇ M specifically inhibits MEK and not other kinases such as PKC, PI 3-kinase or p38 MAP kinase.
  • PD098059 did decrease ERK phosphorylation below controls suggesting that primary Schwann cells in culture contain autocrine regulated ERKs.
  • TX 14(A) signaling involved the adapter protein, She, and the non-receptor tyrosine kinase, p60 Src .
  • This EXAMPLE demonstrates that She associated with p ⁇ O ⁇ 0 following TX14(A) stimulation, which coincided with increased tyrosine phosphorylation of She.
  • the association of p60 Src and She has been observed previously in COS -7 cells after lysophosphatidate (LPA) stimulation and has been proposed to be involved in early activation of ERKs by pertussis toxin-sensitive G-protein coupled receptors.
  • PI 3-kinase has been shown previously to specifically inhibit PI 3 -kinase activity in Swiss 3T3 fibroblasts and L6 rat myoblasts.
  • PI 3-kinase has been shown to activate ERKs by a p21 Ras -independent mechanism and by linkage with G-protein coupled receptors showing that TX 14(A) signaling involves multiple and perhaps novel pathways leading to ERK activation.
  • Prosaposin is not only a neurotrophic factor, but an essential factor for events involved in myelination, including prevention of Schwann cell and oligodendrocyte death and synthesis of a myelin lipid, sulfatide.
  • prosaposin-deficient transgenic mice have severe hypomyelination in both the central and peripheral nervous system which was apparently due to failure of myelin synthesis, rather than demyelination. The deficiency of myelin in these animals and in prosaposin deficient humans is due to the lack of a myelinotropic effect of prosaposin during development.
  • TX 14(A) encompassing the neurotrophic region of prosaposin, appeared to exert its trophic effect by binding to a high affinity receptor which activated a pertussis toxin-sensitive G-protein and signaled through ERKs to up regulate the synthesis of sulfatide in Schwann cells. Inhibition of ERK activation blocked enhanced synthesis of sulfatide implicating ERKs as a key signaling component in myelin lipid synthesis.
  • EXAMPLE 4 EFFECT OF PROSAPOSIN AND TX14(A. ON PROINFLAMMATORY CYTOKINE-INDUCED OLIGODENDROCYTE CELL DEATH
  • This EXAMPLE demonstrates that prosaposin or the prosaposin-derived peptide TX 14(A) (SEQ ID NO: 7), can inhibit proinflammatory cytokine-induced apoptosis.
  • Undifferentiated CG4 oligodendrocytes were grown in DMEM containing 10% fetal calf serum. Cells were removed with trypsin and plated in 30 mm petri dishes onto glass coverslips in 0.5%> fetal bovine serum for 2 days in the presence of absence of the following effectors: 200 ng/ml TNF ⁇ alone or in the presence of 1 nM prosaposin, 5 nM prosaposin, 10 nM TX14(A) or 50 nM TX14(A).
  • the same experiment was also performed using 200 ng/ml IFN ⁇ alone or in the presence of 1 nM prosaposin, 5 nM prosaposin, 10 nM TX14(A) or 50 nM TX14(A).
  • the MTT cell death assay was then performed using a kit (Promega, Madison, WI). This assay measures the MTT dye reduced by mitochondria. In the presence of TNF ⁇ and IFN ⁇ , the MTT absorbance decreases due to increased cell death and greater reduction of the MTT dye by mitochondria that are released from lysed cells.
  • TNF ⁇ -induced apoptosis is completely reversed by prosaposin (1 nM and 5 nM) and TX14(A) (10 nM and 50 nM).
  • An inhibitory effect was also observed with IFN ⁇ , albeit not as strong as that obtained with TNF ⁇ . See, FIG. 13B. Therefore, prosaposin and TX14(A) inhibit TNF ⁇ and IFN ⁇ -induced apoptosis in oligodendrocytes.
  • This EXAMPLE demonstrates that TX14(A) inhibits proinflammatory cytokine TNF ⁇ -induced apoptosis in L6 myoblasts.
  • L6 myoblasts cells were incubated for 96 hours either in media (control); media with 10 ng/ml TNF ⁇ (TNF Category); or media with 10 ng/ml TNF ⁇ and 200 ng/ml TX14(A). See, FIG. 14.
  • Cell death was measured by trypan blue assay as described in EXAMPLE 3.
  • Cell death was inhibited in L6 myoblasts incubated with TNF ⁇ and TX 14(A), as compared to L6 myoblasts incubated with TNF ⁇ only (approximately 60% cell death). Therefore, prosaposin receptor agonist treatment inhibits TNF ⁇ -induced apoptosis in myoblasts.
  • This EXAMPLE demonstrates that a prosaposin-derived peptide, TX14(A) peptide (prosaptide; SEQ ID NO:7), was effective in treating TNF ⁇ -induced inflammation.
  • the inflammatory component of peripheral nerve injury may affect the development of local neuropathologic changes as well as the onset of hyperalgesia, the characteristic features of experimental neuropathic pain states. See, Wagner et al.
  • TNF ⁇ (2.5 pg/ml) was injected directly into the endoneurial space of normal rat nerves.
  • TX 14(A) (200 ⁇ g/kg) was injected subcutaneously prior to injection of TNF- ⁇ . The resulting effects on behavior were monitored for 1 week.
  • TX14(A) peptide dramatically reduced TNF ⁇ - induced thermal hyperalgesia in the rat model. Therefore, TX 14(A) peptide (prosaptide) inhibits TNF ⁇ -induced neuropathic pain in the endoneurial space of normal rat nerves.
  • This EXAMPLE demonstrates that prosaposin-derived peptides cross the blood-brain barrier.
  • An octadecamer (SEQ ID NO: 5) consisting of amino acids 12-29 of saposin C with a tyrosine substituted for valine at position 12 was chemically synthesized on an Applied Biosystems Model 430 peptide synthesizer. The peptide was then radioiodinated by the lactoperoxidase method; 20 x 10 6 cpm radiolabeled peptide were injected into the auricles of rats. The animals were sacrificed after 1 hr and 24 hr, and the hearts were perfused with isotonic saline in order to remove the blood from the brain.
  • the brain was then counted in a gamma counter.
  • the brain was homogenized and fractionated into a capillary rich fraction (pellet) and a parenchymal brain fraction (supernatant) after dextran centrifiigation. See, Triguero et al. (J. Neurochem., 54:1882-1888, 1990). This method allows for the discrimination between radiolabeled peptide within blood vessels and that within the brain.
  • 0.017% of the injected peptide SEQ ID NO:5 was detected in whole brain; 15% of the label was in the parenchymal fraction and 25% was in the capillary fraction.
  • the prosaposin-derived peptide SEQ ID NO:7 also was assayed for ability to cross the blood-brain barrier as follows.
  • a female Sprague-Dawley rat was anesthesized with methoxyflurane, and approximately 20 ⁇ g peptide SEQ ID NO:2 (3.2 x 10 8 cpm) was injected into the tail vein. After 40 minutes, the rat was sacrificed by ether anesthesia and perfused with about 250 ml PBS through the heart. The total amount of peptide in brain, liver and blood was calculated as a percentage of the injected material as shown in TABLE 3.
  • a 54 kDa protein has been identified as the receptor for prosaposin as described in this EXAMPLE:
  • a prosaposin receptor protein was isolated from whole rat brain, rat cerebellum and mouse neuroblastoma cells using the plasma membrane P-100 fraction. Briefly, cells or tissues were solubilized and centrifuged at 14,000 rpm to remove debris. The supernatant was centrifuged at 40,000 rpm for 1 hr at 4°C. The pellet, enriched in plasma membrane, was solubilized in RIPA buffer (10 mM MOPS, pH 7.5, 0.3 M sucrose, 5 mM EDTA, 1% Trasylol, 10 ⁇ M leupeptin and 10 ⁇ M pepstatin).
  • This P-100 fraction was applied to an affinity column containing the bound, active 14-mer fragment of saposin C, TX14(A).
  • the column was washed with 0.05 M NaCI to elute loosely-bound proteins followed by 0.25 M NaCI that eluted the putative 54 kDa prosaposin receptor.
  • it was determined that the 54-60 kDa protein could be eluted using a 100-fold excess of unbound peptide thus demonstrating specific elution.
  • the 54 kDa protein was approximately 90%> pure as judged by SDS-PAGE.
  • the protein was purified to homogeneity using HPLC and eluted at 50% acetonitrile in an acetonitrile/water gradient on a Vydac C4 column. After treatment with the cross-linking reagent disuccinimidyl suberate (DSS; Pierce, Rockford, IL), the 54 kDa protein bound irreversibly to 125 I labeled saposin C as evidenced by the 66 kDa molecular weight of the complex (54 kDa + 12 kDa).
  • DSS cross-linking reagent disuccinimidyl suberate
  • the prosaposin receptor was partially purified from baboon brain membranes by affinity chromatography using a saposin C-column.
  • the purified preparation gave a single major protein band with an apparent molecular weight of 54 kDa on SDS-PAGE.
  • Affinity cross-linking of 11 kDa I25 I-saposin C demonstrated the presence of a 66 kDa product, indicative of an apparent molecular weight of 55 kDa for the receptor.
  • a GTP ⁇ S-binding assay using cell membranes from SHSY5Y neural cells demonstrated agonist stimulated binding of [ 35 S]GTP ⁇ S upon treatment with prosaptide TX 14(A) a peptide from the neurotrophic region; maximal binding was obtained at 2 nM.
  • TX 14(A) stimulated binding was abolished by prior treatment of SHSY5Y cells with pertussis toxin and by a scrambled and an all D-amino acid- derivative of the 14-mer.
  • a 14-mer mutant prosaptide competed with TX 14(A) with a Ki of 0.7 nM.
  • TX14(A) also bound to PC12 cells and iSC Schwann cells with Kd values of 18.3 nM and 10 nM and increased phosphorylation of MAP kinase (15,16).
  • These findings suggested the presence of a specific receptor for prosaposin which triggers a MAP kinase cascade.
  • a prosaposin receptor is characterized from baboon brain membranes and SHSY5 Y cells as a G-protein associated receptor.
  • Affinity purification of the receptor All purification procedures were carried out at 4°C unless specified.
  • Baboon brains (1.6 kg) from Southwest Research Institute (San Antonio, TX) were washed in chilled 10 mM MOPS, pH 7.5, containing 0.3 M sucrose and a cocktail of protease inhibitors (5 mM EDTA, 1 mM PMSF, and 5 ⁇ g/ml of leupeptin, aprotinin, and pepstatin) (Buffer I).
  • the brains were homogenized in a teflon-glass homogenizer in 3 volumes of Buffer I and centrifuged at 1,500 X g for 20 min.
  • the supernatant was then centrifuged at 100,000 X g for 60 min and the pellet was washed with Buffer I. Then, the pellet was suspended in lysis buffer (10 mM Tris-HCI, pH 7.5, containing 1%> sodium deoxycholate, 1%> Triton X-100, and the same protease inhibitor cocktail as in Buffer I) and incubated for 60 min on ice with shaking. After removal of the insoluble materials by centrifugation, the supernatant was mixed with 20 ml of saposin C-beads and rotated for 12 hr.
  • lysis buffer 10 mM Tris-HCI, pH 7.5, containing 1%> sodium deoxycholate, 1%> Triton X-100, and the same protease inhibitor cocktail as in Buffer I
  • the beads were packed into a column and washed with 1 mM sodium phosphate buffer, pH 7.5, containing 0.1 %> Triton X-l 00 until protein was not detected in the eluate.
  • the proteins bound to the beads were eluted with 0.1 M glycine-HCl buffer, pH 3.0, at room temperature (affinity purified preparation).
  • the affinity purified preparation (4 ⁇ g of protein) was dialyzed against 4 changes of 1 liter of 1 mM sodium phosphate buffer, pH 7.5, containing 0.1 %> Triton X-l 00 and then concentrated to 1.4 ml by ultrafiltration using
  • the product cross-linked with saposin C was immunoprecipitated by an affinity purified anti saposin C antibody (2 ⁇ g), recovered by Protein A-insoluble (Sigma) then subjected to SDS-PAGE. After protein staining, the gel was dried and then exposed to a Kodak film, BioMax, at -80°C. GTP ⁇ S-binding.
  • the SHSY5Y assay was essentially carried out as described by Campana et al. ⁇ Biochem. Biophys. Res. Commun 229: 706-712, 1996), using SHSY5Y cell membrane preparations.
  • the reaction was performed by incubation of the membrane preparations (50-100 ⁇ g protein) with 125 ⁇ Ci of [ 35 S]-GTP ⁇ S (New England Nuclear Biolabs, 1250 Ci/mmol). In our experiments, a concentration of 3 ⁇ M GDP was added to amplify the difference between ligand stimulated and background binding. Unlabelled GTP ⁇ S (10 nM) was also added to define non-specific binding and this value was subtracted from specific binding. All assays were performed in duplicate.
  • a putative prosaposin receptor was partially purified from baboon brain membranes.
  • a solubilized membrane preparation was purified by affinity chromatography using a saposin C-column. From 1.6 kg of baboon brain, about 25 ⁇ g of the purified preparation was obtained. The purified preparation gave one major band with a molecular weight of 54 kDa on SDS-PAGE. Similar electrophoretic patterns were also observed in purified preparations from membrane fractions of human brain, pig brain and PC 12 cells. Cross-linking experiment using !25 I-saposin C and the purified preparation demonstrated the presence of a 66 kDa band.
  • MAP kinase phosphorylation induced by prosaposin receptor agonists in primary Schwann cells is blocked by treatment with pertussis toxin.
  • a GTP ⁇ S-binding assay was performed using TX14(A) and membrane preparations from SHSY5Y cells. As shown in FIG. 13 A, agonist-stimulated binding was increased by 50-60%) above control values in a dose-dependent manner at a maximal concentration of 2 nM. Activation peaked at 2 nm and was bimodal similar to MAP kinase activation in PC 12 cells.
  • saposin C 0.3 nM
  • SELIINNATEELLY SEQ ID NO: 12
  • Pretreatment of SHS Y5 Y cells for 4 hr with pertussis toxin ( 100 ng/ml) prior to membrane preparation abolished the agonist-stimulated binding of TX 14(A).
  • TX 14(A) An all D-amino acid-derivative of TX 14(A) and a scrambled peptide derivative of TX 14(A) were inactive. Furthermore, the purified receptor preparation was analyzed for G-proteins by western blotting using an antibody against G 0 ⁇ . The purified preparation contained cross-reacting material of 40 kDa indicating that G 0ct copurified with the receptor.
  • This EXAMPLE shows that the prosaposin receptor has a molecular weight of 55 kDa and is a G 0 protein-associated receptor.
  • Saposin C also interacts with a 56 kDa lysosomal protein, glucocerebrosidase, to stimulate the enzymic hydrolysis of glucocerebroside.
  • Western blot analysis using an antibody against purified human glucocerebrosidase gave no cross-reacting material in the purified receptor preparation.
  • Prosaposin receptor agonists induced MAP kinase phosphorylation in Schwann cells, and this phosphorylation was blocked both by the treatment with pertussis toxin and a non- hydrolyzable GDP analog, GDP ⁇ S.
  • GTP ⁇ S-binding to cell membranes has been utilized to characterize agonist-promoted activation of several G-protein associated receptors including opioid receptors and 5-hydroxy tryptophane receptors.
  • the assay relies upon agonist-promoted GDP/GTP exchange occurring at the G-protein level within the receptor/G-protein complex; [ 35 S]GTP ⁇ S binding is used to assess receptor activation since GTP ⁇ S is only slowly hydrolyzed by the intrinsic GTPase activity of the G-protein.
  • SHSY5Y membrane preparations agonist stimulated binding by ⁇ -opioid agonists was about twice the control level whereas we obtained about 50-70% augmentation using TX 14(A) and saposin C.
  • the alanine at position 2 is a D amino acid.

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Abstract

L'invention concerne un procédé permettant d'inhiber l'apoptose liée à la caspase, par l'administration d'agonistes du récepteur de la prosaposine. L'apoptose constitue un facteur causal majeur dans des maladies telles que la polyarthrite rhumatoïde, le syndrome du côlon irritable, l'insuffisance cardiaque globale, la sclérose en plaques, la maladie d'Alzheimer, la maladie de Parkinson, l'infarctus du myocarde et l'ischémie coronarienne.
PCT/US1998/019216 1997-09-09 1998-09-09 Inhibition de l'apoptose au moyen d'agonistes du recepteur de la prosaposine WO1999012559A1 (fr)

Priority Applications (4)

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EP98951917A EP1011709A4 (fr) 1997-09-09 1998-09-09 Inhibition de l'apoptose au moyen d'agonistes du recepteur de la prosaposine
CA002304108A CA2304108A1 (fr) 1997-09-09 1998-09-09 Inhibition de l'apoptose au moyen d'agonistes du recepteur de la prosaposine
JP2000510456A JP2001515866A (ja) 1997-09-09 1998-09-09 プロサポシン受容体アゴニストを利用したアポトーシスの阻害
AU97746/98A AU9774698A (en) 1997-09-09 1998-09-09 Inhibition of apoptotis using prosaposin receptor agonists

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US5835297P 1997-09-09 1997-09-09
US8812998P 1998-06-04 1998-06-04
US60/088,129 1998-06-04
US60/058,352 1998-06-04

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Cited By (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999064062A1 (fr) * 1998-06-08 1999-12-16 Pharmacia & Upjohn Ab Nouvelle utilisation therapeutique d'amplificateurs de l'activite de la proteine kinase b (picb)
WO2000014113A2 (fr) * 1998-09-09 2000-03-16 Myelos Corporation Procede pour stimuler l'activite du recepteur de la prosaposine
WO2000020025A2 (fr) * 1998-10-02 2000-04-13 St. Elizabeth's Medical Center, Inc. Compositions akt augmentant la survie de cellules
EP1072609A2 (fr) * 1999-06-30 2001-01-31 Masahiro Sakanaka Agents cytoprotectifs contenant analogues de peptides de prosaposin
EP1097165A1 (fr) * 1998-07-13 2001-05-09 Parkash S. Gill Nouveaux inhibiteurs de l'angiogenese et de la croissance tumorale
WO2001055198A2 (fr) * 2000-01-26 2001-08-02 Memorec Medical Molecular Research Cologne Stoffel Gmbh Activateur de sphingolipide specifique de la peau et de l'estomac
WO2002012338A2 (fr) * 2000-08-03 2002-02-14 Grünenthal GmbH Procede de criblage
WO2005037304A1 (fr) * 2003-10-17 2005-04-28 Crucell Holland B.V. Traitement et prevention d'escarres de decubitus
US7368420B1 (en) 1998-10-02 2008-05-06 Caritas St. Elizabeth's Medical Center Of Boston, Inc. Akt compositions for enhancing survival of cells
US7524818B2 (en) 1993-07-30 2009-04-28 Myelos Corporation Prosaposin as a neurotrophic factor
USRE43372E1 (en) 1999-03-05 2012-05-08 Duke University C16 unsaturated FP-selective prostaglandins analogs
US8906962B2 (en) 2000-03-31 2014-12-09 Duke University Compositions and methods for treating hair loss using non-naturally occurring prostaglandins
US9346837B2 (en) 2000-03-31 2016-05-24 Duke University Cosmetic and pharmaceutical compositions and methods using 2-decarboxy-2-phosphinico derivatives
EP3127549A1 (fr) * 2007-06-22 2017-02-08 Children's Medical Center Corporation Procédés et utilisations d'un fragment de saposine a
US10646541B2 (en) 2014-03-26 2020-05-12 Children's Medical Center Corporation Cyclic prosaposin peptides and uses thereof

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1677667A2 (fr) * 2003-10-24 2006-07-12 Medtronic, Inc. Techniques de traitement de troubles neurologiques par attenuation de la production de mediateurs pro-inflammatoires

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WO1995003821A1 (fr) * 1993-07-30 1995-02-09 The Regents Of The University Of California Prosaposine et peptides derives de la cytokine utilises comme agents therapeutiques
WO1997032895A1 (fr) * 1996-03-05 1997-09-12 Regents Of The University Of California Methodes de soulagement de la douleur nevropathique a l'aide de peptides derives de prosaposine

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US5571787A (en) * 1993-07-30 1996-11-05 Myelos Corporation Prosaposin as a neurotrophic factor

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WO1995003821A1 (fr) * 1993-07-30 1995-02-09 The Regents Of The University Of California Prosaposine et peptides derives de la cytokine utilises comme agents therapeutiques
WO1997032895A1 (fr) * 1996-03-05 1997-09-12 Regents Of The University Of California Methodes de soulagement de la douleur nevropathique a l'aide de peptides derives de prosaposine

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* Cited by examiner, † Cited by third party
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See also references of EP1011709A4 *

Cited By (28)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7524818B2 (en) 1993-07-30 2009-04-28 Myelos Corporation Prosaposin as a neurotrophic factor
WO1999064062A1 (fr) * 1998-06-08 1999-12-16 Pharmacia & Upjohn Ab Nouvelle utilisation therapeutique d'amplificateurs de l'activite de la proteine kinase b (picb)
EP1097165A1 (fr) * 1998-07-13 2001-05-09 Parkash S. Gill Nouveaux inhibiteurs de l'angiogenese et de la croissance tumorale
WO2000014113A2 (fr) * 1998-09-09 2000-03-16 Myelos Corporation Procede pour stimuler l'activite du recepteur de la prosaposine
WO2000014113A3 (fr) * 1998-09-09 2000-11-16 Myelos Corp Procede pour stimuler l'activite du recepteur de la prosaposine
US7368420B1 (en) 1998-10-02 2008-05-06 Caritas St. Elizabeth's Medical Center Of Boston, Inc. Akt compositions for enhancing survival of cells
WO2000020025A2 (fr) * 1998-10-02 2000-04-13 St. Elizabeth's Medical Center, Inc. Compositions akt augmentant la survie de cellules
WO2000020025A3 (fr) * 1998-10-02 2000-07-06 St Elizabeths Medical Ct Compositions akt augmentant la survie de cellules
USRE43372E1 (en) 1999-03-05 2012-05-08 Duke University C16 unsaturated FP-selective prostaglandins analogs
EP1072609A2 (fr) * 1999-06-30 2001-01-31 Masahiro Sakanaka Agents cytoprotectifs contenant analogues de peptides de prosaposin
EP1072609A3 (fr) * 1999-06-30 2002-04-03 Masahiro Sakanaka Agents cytoprotectifs contenant analogues de peptides de prosaposin
WO2001055198A2 (fr) * 2000-01-26 2001-08-02 Memorec Medical Molecular Research Cologne Stoffel Gmbh Activateur de sphingolipide specifique de la peau et de l'estomac
WO2001055198A3 (fr) * 2000-01-26 2002-03-14 Memorec Medical Molecular Res Activateur de sphingolipide specifique de la peau et de l'estomac
US8906962B2 (en) 2000-03-31 2014-12-09 Duke University Compositions and methods for treating hair loss using non-naturally occurring prostaglandins
US9675539B2 (en) 2000-03-31 2017-06-13 Duke University Cosmetic and pharmaceutical compositions and methods using 2-decarboxy-2-phosphinico derivatives
US9579270B2 (en) 2000-03-31 2017-02-28 Duke University Compositions and methods for treating hair loss using non-naturally occurring prostaglandins
US9346837B2 (en) 2000-03-31 2016-05-24 Duke University Cosmetic and pharmaceutical compositions and methods using 2-decarboxy-2-phosphinico derivatives
WO2002012338A2 (fr) * 2000-08-03 2002-02-14 Grünenthal GmbH Procede de criblage
EP1469316A1 (fr) * 2000-08-03 2004-10-20 Grünenthal GmbH Procédé de criblage
WO2002012338A3 (fr) * 2000-08-03 2002-12-19 Gruenenthal Gmbh Procede de criblage
WO2005037304A1 (fr) * 2003-10-17 2005-04-28 Crucell Holland B.V. Traitement et prevention d'escarres de decubitus
EP3127549A1 (fr) * 2007-06-22 2017-02-08 Children's Medical Center Corporation Procédés et utilisations d'un fragment de saposine a
US10267799B2 (en) 2007-06-22 2019-04-23 Children's Medical Center Corporation Saposin-A derived peptides and uses thereof
US10670600B2 (en) 2007-06-22 2020-06-02 Children's Medical Center Corporation Saposin-A derived peptides and uses thereof
EP3666284A1 (fr) * 2007-06-22 2020-06-17 Children's Medical Center, Corp. Procédés et utilisations d'un fragment de saposine a
US10646541B2 (en) 2014-03-26 2020-05-12 Children's Medical Center Corporation Cyclic prosaposin peptides and uses thereof
US11191805B2 (en) 2014-03-26 2021-12-07 Children's Medical Center Corporation Cyclic prosaposin peptides and uses thereof
US11654178B2 (en) 2014-03-26 2023-05-23 Children's Medical Center Corporation Cyclic prosaposin peptides and uses thereof

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AU9774698A (en) 1999-03-29
EP1011709A1 (fr) 2000-06-28

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