WO2006019365A1 - Nouveaux polypeptides et polynucléotides analogues de l’humanine, et méthodes d’utilisation de ces composés - Google Patents

Nouveaux polypeptides et polynucléotides analogues de l’humanine, et méthodes d’utilisation de ces composés Download PDF

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WO2006019365A1
WO2006019365A1 PCT/US2004/007733 US2004007733W WO2006019365A1 WO 2006019365 A1 WO2006019365 A1 WO 2006019365A1 US 2004007733 W US2004007733 W US 2004007733W WO 2006019365 A1 WO2006019365 A1 WO 2006019365A1
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polypeptide
humanin
cells
disease
amino acid
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PCT/US2004/007733
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Kekuda Ramesh
Qian Xiaozhong
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Curagen Corporation
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4711—Alzheimer's disease; Amyloid plaque core protein

Definitions

  • the instant invention relates to novel humanin-like polypeptides and polynucleotides encoding the same, and anti-humanin-like antibodies and their variants.
  • the invention also describes the therapeutic activity of humanin in the treatment of neurodegenerative diseases.
  • AD Alzheimer's disease
  • Symptoms usually start with forgetfulness and progress to difficulties in speaking and understanding.
  • AD is a slow disease, starting with mild memory problems and ending with severe brain damage. The course the disease takes and how fast changes occur vary from person to person.
  • AD patients live from 8 to 10 years after they are diagnosed, though the disease can last for as long as 20 years.
  • Alzheimer's patients Abnormal clumps (amyloid plaques) and tangled bundles of fibers (neurofibrillary tangles) are seen in the brains of Alzheimer's patients, along with reduced levels of neurotransmitters in cortical cells. Despite current therapeutics, all Alzheimer's patients will eventually require total care. If the individual has no other serious illness, the loss of brain function itself will cause death.
  • Tacrine Cognex
  • donepezil Aricept
  • rivastigmine Exelon
  • galantamine Reminyl
  • NSAIDs, vitamin E, and estrogen may also be of limited use to slow disease progress, but recent studies have not proved encouraging.
  • Parkinson's disease is a neurodegenerative disease that is clinically characterized by decrease in spontaneous movements, gait difficulty, postural instability, rigidity and tremor. Parkinson's disease affects one of every 100 persons over the age of 60. Parkinson's disease occurs when dopaminergic neurons of the substantial nigra begin to malfunction and eventually die. Dopamine is a neurotransmitter that is involved in control of movement initiation and coordination. The degeneration of dopaminergic neurons creates a shortage of the neurotransmitter dopamine, causing the movement impairments that characterize the disease. While several treatment options are currently available, none prevent the progressive changes of the brain typical of Parkinson's disease. Hashimoto ⁇ t al. (Proc. Nat. Acad. Sci.
  • AD Alzheimer's disease
  • HN humanin
  • APP amyloid precursor protein
  • PS1 presenilin-1
  • PS2 presenilin-2
  • Cerebral amyloid beta-protein angiopathy is a pathological feature of AD and related disorders.
  • Jung and Van Nostrand J Neurochem. 2003; 84(2):266-72 used human cerebrovascular smooth muscle cells as a model of CAA. They demonstrated that humanin could protect human cerebrovascular smooth muscle cells from A-beta-induced toxicity.
  • the novel humanin-like polypeptides (CuraGen accno: CG202524) of the present invention differ from the humanin polypeptides of the prior art (GenBank Ace. No. AY029066; PCT Publication WO200121787; European Publication EP01221480 A1) as follows.
  • CG202524-02 has a Leucine to Serine amino acid substitution at position 12 (L12S).
  • CG202524-03 has 2 amino acid substitutions: L12S, and an Alanine to Threonine substitution at position 24 (A24T).
  • CG202524-04 has 3 amino acid substitutions: L12S, an Arginine to Leucine amino acid substitution at position 23 (R23L), and an Alanine to Leucine substitution at position 24 (A24L). Furthermore, CG202524-04 has 4 additional amino acids (SSVF) at positions 25 to 28, as compared to humanin (AAK50430). Thus, 3 novel CG202524 polypeptides have a Serine at position 12 (S12).
  • S12 Serine at position 12
  • a systematic site-directed mutagenesis analysis of Humanin has been disclosed in EP01221480, showing that replacement of Leucine at position 12 (L12) with Ala abolished the protective function of Humanin. The inventor concluded that the L12 residue is essential for protection against cell death.
  • CG202524-02, -04 and -03 each having S12 protected neurons from cell death under various conditions.
  • another humanin-like polypeptide (CG202524-08) has 5 amino acid substitutions: S12L and T24A, S14T, E15A and I16T as compared to the known humanin (GenBank AY029066).
  • the instant application further describes the use of the novel humanin-like compositions (polypeptide, polynucleotide and antibodies) in neurodegenerative diseases including, but not limited to, Parkinson's disease and Alzheimer's disease.
  • the present invention discloses a novel protein and nucleic acids, herein generally referred to by the identifier CG202524, bearing sequence similarity to Humanin, fragments thereof, and antibodies that bind immunospecifically to a protein of the invention.
  • One of the aspects of the present invention is to describe the therapeutic use of the polypeptide in preventing or inhibiting death of neuronal or cerebrovascular smooth muscle cells. More specifically, the novel compositions detailed in the present invention are useful in the prevention of cell death associated with neurodegenerative diseases.
  • Another aspect of the present invention is to describe a method of treating or preventing patients suffering from, or believed to be at risk of, a neurodegenerative disease or a condition of the central nervous system associated with neuronal death.
  • the disease or disorder may include but is not limited to Alzheimer's disease, Parkinson's disease, Huntington's disease, multiple sclerosis, spinocerebellar ataxia, amyotrophic lateral sclerosis, muscular dystrophy, peripheral neuropathy, traumatic head or spinal cord injury, and stroke.
  • Another aspect of the invention is to provide a method of preventing, reducing, or slowing the progression of, symptoms of neurodegenerative disease, or symptoms of a condition of the central nervous system associated with neuronal death.
  • symptoms may include but are not limited to cognitive impairment, learning deficit, memory deficit, memory loss, motor function impairment and mood disorder.
  • Figure 1 depicts a time course of cell death in rat PC2 cell cultures in response to serum withdrawal.
  • Cell death was assessed by a fluorescent assay of lactate dehydrogenase concentration in the culture supernatants. The results show that serum starvation causes a time dependent increase in PC-12 cell death.
  • Figure 2a depicts the effect of different concentrations of HN-01 (CG202524-01 ) on cell death in rat PC2 cell cultures in response to serum withdrawal.
  • HN-01 CG202524-01 rescues PC12 cells from serum withdrawal-induced cell death in a dose-dependent manner.
  • Figure 2b depicts the effect of different concentrations of HN-06 (CG202524-02) on cell death in rat PC2 cell cultures in response to serum withdrawal.
  • HN-06 CG202524-02 rescues PC12 cells from serum withdrawal-induced cell death in a dose-dependent manner.
  • Figure 2c depicts the effect of different concentrations of HN-Q3 (CG202524-03) on cell death in rat PC2 cell cultures in response to serum withdrawal.
  • HN-03 CG202524-03 rescues PC12 cells from serum withdrawal-induced cell death in a dose-dependent manner.
  • Figure 2d depicts the effect of different concentrations of HN-07 (CG202524-04) on cell death in rat PC2 cell cultures in response to serum withdrawal.
  • HN-07 (CG202524-04) rescues PC12 cells from serum withdrawal-induced cell death in a dose-dependent manner.
  • Figure 3 depicts the effects of novel humanin-like polypeptides on PC12 survival after serum withdrawal. Relative fluorescence units were normalized to the maximal fluorescence units observed under no HN treatment. All humanin-like polypeptides tested showed similar efficiency in protecting PC12 cells from serum withdrawal-induced cell death.
  • Figure 4 depicts the effects of novel humanin-like polypeptides on Dopamine-induced cell death.
  • Relative fluorescence units indicate LDH level in supernatant released from dead cells, and were normalized to the maximal fluorescence units observed under no HN treatment. All humanin-like polypeptides tested showed similar efficiency in protecting PC12 cells from dopamine-induced toxicity.
  • Figure 5 depicts the effects of novel humanin-like polypeptides on CREB-3-mediated cell death. Relative fluorescence units indicate LDH level in supernatant released from dead cells. All humanin-like polypeptides tested showed similar efficiency in protecting PC12 cells from CREB-3-mediated cell death.
  • the present invention is based upon the discovery of novel humanin-like polypeptides and polynucleotides encoding them.
  • the identifier CG202524 herein generally refers to the novel humanin-like polypeptides and polynucleotides encoding them.
  • Table 1 provides a summary of the CG202524 nucleic acids and their encoded polypeptides.
  • Sequence homology search strategies were used to identify several additional chromosomal gene loci coding for humanin-like polypeptides. Five additional loci encoding humanin-like polypeptides were found in human genomic sequences from chromosomes 3, 6, 11 , 17 and 5. The sequences of Ace. No. CG202524-02, CG202524-03 and CG202524-04, CG202524-06, CG202524-07 and CG202524-08 were derived by in silico prediction of the sequence based on sequences available in the public human sequence databases.
  • novel humanin-like polypeptides of the present invention differ from the humanin polypeptides of the prior art (see GenBank Acc.No. AY029066; WO200121787; European Publication EP01221480 A1).
  • the novel humanin-like gene loci on chromosome 3 and 11 each encode a polypeptide (CG202524-02), which has a Leucine to Serine amino acid substitution at position 12 (L12S).
  • a locus on chromosome 17 encodes another humanin related polypeptide (CG202524-03), which has 2 amino acid substitutions: L12S, and an Alanine to Threonine substitution at position 24 (A24T).
  • a locus on chromosome 5 encodes yet another humanin like polypeptide (CG202524-04) with 3 amino acid substitutions: L12S, an Arginine to Leucine amino acid substitution at position 23 (R23L), and an Alanine to Leucine substitution at position 24 (A24L). Furthermore, CG202524-04 has 4 additional amino acids (SSVF) at positions 25 to 28, as compared to humanin (AAK50430). Thus, each of the three 3 novel polypeptides of the invention have a Serine at position 12 (S12).
  • a systematic site-directed mutagenesis analysis of Humanin has been disclosed in EP01221480, showing that replacing Leucine at position 12 (L12) with Alanine abolished the protective function of Humanin.
  • the current invention is unique in that CG202524-02, -04 and - 03 (having a Serine residue at position 12) has shown protection of neurons from cell death under various conditions. This is supported by the data presented in the instant application.
  • CG202524-08 An additional locus was found in human genomic sequence from chromosome 6, which encodes a humanin- like polypeptide (CG202524-08) having 5 amino acid substitutions, S12L and T24A, S14T, E15A and I16T, as compared to the known humanin GenBank AY029066. CG202524-08 retains the Ser in the 12 th position as compared to Leucine in the known humanin.
  • Table 2A provides the specific DNA and amino acid sequences for the nucleic acids and polypeptides of Table 1.
  • CG202524-02 and -06 are novel humanin-like polypeptides and polynucleotides encoding them. CG202524-02 and -06 have the same protein sequences.
  • CG202524-03 is a novel humanin-like polypeptide and the polynucleotide encoding it.
  • CG202524-04 and -07 are novel humanin-like polypeptides and polynucleotides encoding them.
  • CG202524-04 and -07 have the same protein sequences.
  • CG202524-08 is a novel humanin-like polypeptide and the polynucleotide encoding it.
  • CG202524-01 and -05 are the known humanin (GenBank: AY029066).
  • the CG202524-07 protein according to the present invention was found to have homology to the proteins shown in the BLASTP data in Table 2D.
  • nucleic acid or amino acid sequence homologous to the CG202524 sequences disclosed herein could be used in the method of the invention.
  • "Homologous” refers to sequences characterized by a homology at the nucleotide level or amino acid level and include sequences coding for isoforms such as those expressed in different tissues of the same origin encoded for example by alternative splicing of RNA. Alternatively, isoforms can be encoded by different genes.
  • homologous sequences include CG202524 polypeptides and the nucleotide sequences encoding them, of species other than humans, including, but not limited to vertebrates, and thus can include, e.g., frog, mouse, rat, rabbit, dog, cat, cow, horse, and other organisms. Homologous sequences also include naturally occurring allelic variations and mutations of the sequences set forth herein. Homologous sequences include those sequences having conservative amino acid substitutions and the nucleotides encoding them.
  • a polypeptide having a biologically active portion of a CG202524 polypeptide has an activity of CG202524 as measured in a particular biological assay (such as those described herein).
  • biologically active polypeptides can be used in the method of the invention.
  • a nucleic acid fragment encoding a biologically-active polypeptide can be prepared by isolating a portion of the nucleotide that encodes a polypeptide having a CG202524 biological activity, expressing the encoded portion of CG202524 (e.g., by recombinant expression in vitro) and assessing the activity of the encoded portion of CG202524.
  • nucleotide sequences that may lead to changes in the amino acid sequences of the encoded CG202524 protein, without altering the functional ability of that protein.
  • Such proteins also have utility in the method of the invention.
  • nucleotide substitutions leading to amino acid substitutions at "non-essential" amino acid residues can be made in the sequence.
  • a "non-essential" amino acid residue is a residue that can be altered from the wild-type sequence of the protein without altering its biological activity, whereas an "essential" amino acid residue is required for such biological activity.
  • Amino acids for which conservative substitutions can be made are well-known within the art.
  • Polypeptides that contain changes in amino acid residues of CG202524 polypeptides that are not essential for activity may also be used in the method of the invention.
  • Chimeric or fusion proteins including CG202524 polypeptides may be used in the method of the invention.
  • a "chimeric protein” or “fusion protein” comprises a CG202524 polypeptide operatively-linked to a non-CG202524 polypeptide.
  • the CG202524 polypeptide can correspond to all or a portion of a CG202524 protein.
  • a CG202524 fusion protein comprises at least one biologically-active portion of a CG202524 protein.
  • the CG202524 polypeptide and the non-CG202524 polypeptide are "operatively-linked", that is they are fused in-frame with one another.
  • the non-CG202524 polypeptide can be fused to the N-terminus or C-terminus of the CG202524 polypeptide.
  • the fusion protein may be a CG202524 protein containing a heterologous signal sequence at its N-terminus. In certain host cells (e.g., mammalian host cells), expression and/or secretion of CG202524 can be increased through use of a heterologous signal sequence.
  • the fusion protein is a CG202524-immunoglobulin fusion protein in which the CG202524 sequences are fused to sequences derived from a member of the immunoglobulin protein family.
  • the CG202524-immunoglobulin fusion proteins can be incorporated into pharmaceutical compositions and administered to a subject to inhibit an immunological response according to the present invention.
  • a CG202524 chimeric or fusion protein for use in the method of the invention may be chemically modified for the purpose of improving bioavailability, and increasing efficacy, solubility and stability.
  • the protein may be covalently or non-covalently linked to polyethylene glycol (PEG), or by other well-known methods in the art.
  • PEG polyethylene glycol
  • a CG202524 chimeric or fusion protein for use in the method of the invention can be produced by standard recombinant DNA techniques.
  • DNA fragments coding for the different polypeptide sequences may be ligated together in-frame in accordance with conventional techniques, e.g., by employing blunt-ended or stagger-ended termini for ligation, restriction enzyme digestion to provide for appropriate termini, filling-in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and enzymatic ligation.
  • the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers.
  • PCR amplification of gene fragments can be carried out using anchor primers that give rise to complementary overhangs between two consecutive gene fragments that can subsequently be annealed and reamplified to generate a chimeric gene sequence [see, e.g., Ausubel, etal. (eds.) CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, (1992)].
  • anchor primers that give rise to complementary overhangs between two consecutive gene fragments that can subsequently be annealed and reamplified to generate a chimeric gene sequence
  • the chimeric or fusion protein may be produced synthetically.
  • the fusion protein can be a CG202524 protein fused to a His tag or epitope tag (e.g. V5) to aid in the purification and detection of the recombinant CG202524 protein.
  • the present invention provides CG202524 proteins, analogs and homologs that can be incorporated into pharmaceutical compositions suitable for administration for patient use.
  • Such compositions comprise the CG202524 protein, either alone or together with one or more conventional pharmaceutically acceptable carriers, such as solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration.
  • conventional pharmaceutically acceptable carriers such as solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like.
  • the use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound which is the CG202524 protein as described herein, use thereof in the compositions is contemplated. Supplementary active compounds may also be incorporated into the compositions.
  • the method uses a pharmaceutical composition formulated to be compatible with its intended route of administration.
  • routes of administration include but not limited to parenteral i.e intravenous, intravenous, intracranial and intracerebral, transmucosal or transdermal administration.
  • Solutions or suspensions used for parenteral, intradermal, or subcutaneous application may include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates or phosphates, and agents for the adjustment of tonicity such as sodium chloride or dextrose.
  • the pH may be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.
  • the parenteral preparation may be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
  • intranasal administration it is meant that the compounds are delivered in the form of an aerosol spray from pressured container or dispenser, which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.
  • Systemic administration may also be by transmucosal or transdermal means.
  • penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives.
  • Transmucosal administration may be accomplished through the use of nasal sprays or suppositories.
  • the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.
  • composition that is utilized for the administration into a mammal, or more specifically human.
  • Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
  • suitable carriers include physiological saline, bacteriostatic water, or phosphate buffered saline (PBS).
  • PBS phosphate buffered saline
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof.
  • the proper fluidity may be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • Prevention of the action of microorganisms may be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars, polyalcohols such as manitol, sorbitol, and sodium chloride in the composition
  • composition is prepared with carriers that protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems.
  • a controlled release formulation including implants and microencapsulated delivery systems.
  • Biodegradable, biocompatible polymers may be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art.
  • Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) may also be used as pharmaceutically acceptable carriers. These may be prepared according to methods well-known to those skilled in the art.
  • the present invention provides a novel Humanin-like polypeptides and polynucleotides encoding them that prevent cell death associated with neurodegenerative diseases, and use of the same. Furthermore, the present invention describes compositions comprising a therapeutic polypeptide, CG202524, useful in the prevention of cell death. More specifically, the novel compositions detailed in the present invention are useful in the prevention of cell death associated with neurodegenerative diseases.
  • the present invention provides a method of treating subjects suffering from, or believed to be at risk of, a neurodegenerative disease or a condition of the central nervous system associated with neuronal death.
  • the disease or condition may include but is not limited to Alzheimer's disease, Parkinson's disease, Huntington's disease, multiple sclerosis, spinocerebellar ataxia, amyotrophic lateral sclerosis, muscular dystrophy, peripheral neuropathy, traumatic head or spinal cord injury, and stroke.
  • Another aspect of the invention is a method of reducing, or slowing the progression of, symptoms of neurodegenerative disease. These symptoms may include but are not limited to cognitive impairment, learning deficit, memory deficit, memory loss, motor function impairment, tremor and mood disorder.
  • a method of administering a pharmaceutical composition comprising humanin polypeptides to reduce neuronal death resulting from ischemic insult, stroke, or prion-induced disease and to treat or prevent cerebral amyloid beta-protein ((Abeta) angiopathy in Alzheimer's disease (AD) and related disorders are also described herein.
  • PC 12 cell is a rat pheochromocytoma cell line that has been widely used as a cell system for neuronal signaling and differentiation due to its ability to alter its phenotype to a sympathetic neuron-like cell in response to nerve growth factor or fibroblast growth factor. Therefore, determining whether the CG202524 variants rescued PC12 from cell death induced by serum withdrawal provided insight on the role of these proteins in neuronal protection.
  • LDH assays A rapid, fluorescence-based LDH assays was used to measure the release of lactate dehydrogenase from cells with a damaged membrane. Briefly, PC12 cells were plated in 96 wells plate in complete serum. After 24 hours, cells were washed with serum-free media twice and were cultured in serum-free media together with a CG202524 variants at various concentrations. Cell supernatants were collected and subjected to LDH assays (CytoTox- ONETM, Promega, Wl) at 24 or 48 hrs after treatment. The reactions were terminated by adding a stop buffer and the signal measured using a fluorescence reader. Higher read of LDH signal indicated more cell death occurring during the incubation time.
  • Figure 1 shows the cell death response pattern of PC-12 upon serum starvation.
  • the data presented indicates that serum starvation results in a time dependent increase in the PC-12 cell death.
  • Humanin HN-01 CG202524-01 , known form
  • Humanin HN-01 CG202524-01 , known form
  • Figure 2a Similar results were obtained with the novel humanin-like polypeptides HN-06 (CG202524-02 also known as -06), HN-03 (CG202524-03), HN-07 (CG202524-04 also known as -07) as seen in Figures 2b, 2c and 2d respectively.
  • the effect of survival of PC-12 was comparable in the presence of all the novel variants identified ( Figure 3).
  • Dopamine induces cell toxicity in primary neurons and several neuronal cell lines, such as PC12 cells. It has been implicated that Dopamine toxicity is partially responsible for neuronal cell death in Alzheimer's, Parkinson's disease and several other neurodegenerative diseases. Therefore, determining whether a Humanin-like variants described herein, rescues PC12 cell death induced by Dopamine will provide important information about the role of the proteins in neuron protection.
  • a rapid, fluorescence-based LDH assay was used to measure the release of lactate dehydrognease from PC-12 cells with a damaged membrane as described in Example 1.
  • the goal of this experiment was to assess the survival of PC-12 cells in the presence of Humanin-like polypeptides (CG202524-01 , CG202524-02, CG202524-04).
  • Cells were transiently cotransfected with a dominant interfering CREB-3 plasmid and plasmids encoding different isoforms of HNs.
  • LDH level in supernatant released from dead cells was determined by LDH assay as described in Examples 1 and 2.
  • the control without humanin polypeptide showed elevated levels of cell death.
  • cell death was decreased suggesting the protective role of the humanin-like polypeptides of the present invention in CREB-3 mediated cytotoxicity (Figure 5).
  • compositions comprising CG202524 polypeptides are administered to rodents intravenously (IV), intracranial ⁇ or intracerebral ⁇ (IC), or intranasal ⁇ (IN).
  • IV intravenously
  • IC intracranial ⁇ or intracerebral ⁇
  • IN intranasal ⁇
  • Humanin concentration is measured in the cerebrospinal fluid (CSF) at various time intervals following administration.
  • CSF cerebrospinal fluid
  • BBB blood-brain barrier
  • Humanin concentration is measured by immunological or radiological methods well known in the art.
  • IC administration may be achieved by one of several methods known in the art, including, i.e. by injection of humanin polypeptide in a suitable pharmacological carrier, by sustained release of humanin polypeptide from a polymer implant, or by use of a mini-osmotic pump (see Br J Cancer 2000, 82(1):74-80). Long-term IC administration may also be achieved by transplanting recombinant humanin-secreting fibroblasts into the CNS (see Clin Neurosci.1995-96, 3(5):268-74).
  • Intranasal administration allows therapeutic polypeptides to cross the blood-brain barrier, (see Nat Neurosci 2002, 5(6):514-6; and Eur J Pharm Sci 2000, 11(1):1-18).
  • Intranasal administration may be achieved by delivering the therapeutic polypeptides in the form of an aerosol, in a pharmaceutically suitable carrier.
  • the aerosol may be produced by a variety of devices known to those skilled in the art, including but not limited to inhaler devices, nebulizers and vaporizers.
  • CG202524 is detected in CSF by one of several methods including specific anti- humanin antibodies, epitope tagging, biotinylation, and radiolabelling, all of which are techniques readily recognized by one skilled in the art. Stability of humanin in blood may also be determined by adding humanin polypeptide to blood serum in vitro, and measurement of Humanin concentration at various time intervals.
  • humanin-like polypeptides of the invention are administered to appropriate in vivo disease models.
  • Animal models of neurodegenerative diseases are well known to those skilled in the art and exemplified by Examples 5A and 5B below.
  • the polypeptide is administered to a rodent model of the disease.
  • PD-like symptoms are induced by unilateral stereotaxic injection of 6-hydroxy-dopamine into specific regions of the brain, such as the substantia nigra or the striatum (see Brain Res. 1992, 595(2):316-26).
  • Humanin polypeptide is administered at a dose of 0.1 ⁇ M, 1 ⁇ M, 10 mM or 10OmM via one or more of the routes detailed in Example 4.
  • Therapeutic outcome is measured by immunohistochemical measurement of tyrosine hydroxylase.
  • Therapeutic outcome is further assessed by measurements of animal behavior, such as T-maze spontaneous alternation.
  • CG202524 polypeptides are administered to a rodent model of the disease.
  • doubly transgenic mice overexpressing mutant APP and PS1 transgenes develop AD-like phenotypes including amyloid deposits and behavioral impairment as early as 10 weeks of age (see Neurobiol Dis. 1999, 6(4):231-44).
  • CG202524 polypeptide is administered at a dose of 0.1 ⁇ M, 1 ⁇ M, 10 mM or 10OmM via one or more of the routes detailed in Example 4.
  • Therapeutic outcome is assessed by measurements of amyloid plaques, and behavioral studies such as the Y - maze.
  • markers of oxidative stress are measured by methods well known to one skilled in the art.
  • this invention is capable of variation and modification, and we therefore do not wish to be limited to the precis terms set forth, but desire to avail our of such changes and alterations which may.be made for adapting the invention t various usages and conditions.
  • Such alterations and changes may include, for different compositions for the administration of the polypeptides according to the present invention to a mammal; different amounts of the polypeptide; different times and means of administration; different materials contained in the administration dose including, for example combinations of different peptides, or combinations of peptides with different biologically active compounds.
  • Such changes and alterations also are intended to include modifications in the amino acid sequence of the specific polypeptides described herein in which such changes alter the sequence in a manner as not to change the functionality of the polypeptide, but as to change solubility of the peptide in the composition to be administered to the mammal, absorption of the peptide by the body, protection of the polypeptide for either shelf life or within the body until such time as the biological action of the peptide is able to bring about the desired effect, and such similar modifications. Accordingly, such changes and alterations are properly intended to be within the full range of equivalents, and therefore within the purview of the following claims.

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  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

La présente invention a pour objet de nouveaux composés de structure semblable à celle de l’humanine : il s’agit de polynucléotides, de polypeptides et d’anticorps. Cette invention présente également les variantes de l’humanine qui sont associées à ces composés. L’utilisation de ces nouveaux composés (polynucléotides, polypeptides et anticorps) est décrite, en même temps que les diverses méthodes dérivées entrant dans la prévention et le traitement des troubles qui entraînent la mort des cellules neuronales.
PCT/US2004/007733 2003-05-09 2004-03-12 Nouveaux polypeptides et polynucléotides analogues de l’humanine, et méthodes d’utilisation de ces composés WO2006019365A1 (fr)

Applications Claiming Priority (4)

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US47054403P 2003-05-09 2003-05-09
US60/470,544 2003-05-09
US51796103P 2003-11-06 2003-11-06
US60/517,961 2003-11-06

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WO2006019365A1 true WO2006019365A1 (fr) 2006-02-23

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009040017A2 (fr) * 2007-09-11 2009-04-02 Mondobiotech Laboratories Ag Utilisation d'un peptide comme agent thérapeutique
US8309525B2 (en) 2007-05-30 2012-11-13 Albert Einstein College Of Medicine Of Yeshiva University Treatment of type 2 diabetes, metabolic syndrome, myocardial injury and neurodegeneration with humanin and analogs thereof

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
HASHIMOTO, Y.: "A Rescue Factor Abolishing Neuronal Cell Death By A Wide Spectrum Of Familial Alzheimer's Disease Genes And A-Beta", PNAS, vol. 98, 22 May 2001 (2001-05-22), pages 6336 - 6341, XP002190962 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8309525B2 (en) 2007-05-30 2012-11-13 Albert Einstein College Of Medicine Of Yeshiva University Treatment of type 2 diabetes, metabolic syndrome, myocardial injury and neurodegeneration with humanin and analogs thereof
US9034825B2 (en) 2007-05-30 2015-05-19 Albert Einstein College Of Medicine Of Yeshiva University Treatment of myocardial injury with humanin analogs
WO2009040017A2 (fr) * 2007-09-11 2009-04-02 Mondobiotech Laboratories Ag Utilisation d'un peptide comme agent thérapeutique
WO2009040017A3 (fr) * 2007-09-11 2009-09-03 Mondobiotech Laboratories Ag Utilisation d'un peptide comme agent thérapeutique

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