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WO1999042123A1 - Procede pour favoriser la proliferation et la differentiation des neurones - Google Patents

Procede pour favoriser la proliferation et la differentiation des neurones Download PDF

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Publication number
WO1999042123A1
WO1999042123A1 PCT/US1999/003772 US9903772W WO9942123A1 WO 1999042123 A1 WO1999042123 A1 WO 1999042123A1 US 9903772 W US9903772 W US 9903772W WO 9942123 A1 WO9942123 A1 WO 9942123A1
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Prior art keywords
seq
group
tyr
ala
active agent
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PCT/US1999/003772
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English (en)
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Kathleen E. Rodgers
Gere Dizerega
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University Of Southern California
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Priority to EP99908331A priority Critical patent/EP1054684A1/fr
Priority to CA002321164A priority patent/CA2321164A1/fr
Priority to JP2000532137A priority patent/JP2002503702A/ja
Priority to AU27791/99A priority patent/AU750029B2/en
Publication of WO1999042123A1 publication Critical patent/WO1999042123A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/08—Peptides having 5 to 11 amino acids
    • A61K38/085—Angiotensins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/14—Angiotensins: Related peptides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0618—Cells of the nervous system
    • C12N5/0623—Stem cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/30—Hormones
    • C12N2501/32—Angiotensins [AT], angiotensinogen

Definitions

  • This present invention relates to methods and kits for accelerating the proliferation and/or differentiation of neuronal cells.
  • CNS central nervous system
  • CNS progenitor cells have a more restricted potential than a stem cell
  • CNS precursor cells comprise any non- ully differentiated CNS cell type (McKay, 1997).
  • Mammalian fetal precursor cells that give rise to neurons and glia have been isolated
  • the adult CNS also contains multipotential precursor cells for neurons, astrocytes and oligodendrocytes (McKay, 1997). Cultured cells from both the fetal and adult CNS that have proliferated in vitro can differentiate to show morphological and electrophysiological features characteristic of neurons (Gritti et al., J. Neurosci. 16:1091 (1996); Nicario-Abejon et al., Neuron 15:105 (1995)). These data show the multipotential nature of cells derived from the C ⁇ S.
  • Multipotential cells from the fetal brain have been demonstrated to be both homogenous and stable. (Johe et al, Genes Dev. 10:3129 (1996)). In vitro, these cells divide daily and efficiently generate neurons and glia for at least the first month of culture. These cells can be considered to be stem cells because they fulfill the criteria of multipotency and self-renewal.
  • EGF epidermal growth factor
  • bFGF basic fibroblast growth factor
  • Methods that increase the in vitro and ex vivo proliferation and differentiation of neuronal stem and progenitor cells will greatly increase the utility of neuron replacement therapy in various neurodegenerative conditions such as Parkinson's and Alzheimer's Diseases, and amyotrophic lateral sclerosis.
  • methods that increase in vivo proliferation and differentiation of neuronal stem and progenitor cells will enhance the utility of neuron replacement therapy by rapidly increasing local concentrations of neuronal stem and progenitor cell at the site of therapy.
  • the present invention provides methods that increase the proliferation or differentiation of neuronal stem and progenitor cells that are useful in rapidly providing a large population of such cells for use in neuron replacement therapy and for making a large population of transfected cells for use in neuron replacement therapy.
  • the present invention provides methods that promote neuronal cell proliferation or differentiation by contacting the cells with angiotensinogen, angiotensin I
  • Al Al analogues, M fragments and analogues thereof, angiotensin II (MI), All analogues,
  • an improved cell culture medium for the proliferation or differentiation of neuronal cells, wherein the improvement comprises addition to the cell culture medium of an effective amount of angiotensinogen, Al,
  • kits for the propagation or differentiation of neuronal cells comprising an effective amount of angiotensinogen, Al, Al analogues, and/or Al fragments and analogues thereof, All, All analogues, All fragments or analogues thereof, and/or All AT 2 type 2 receptor agonists, and
  • kits further comprise cell culture growth medium, a sterile container, and an antibiotic supplement.
  • neuronal cells include either primary cells or established cell lines with the potential to differentiate into neurons, astrocytes, and oligodendrocytes and to self renew, and also to differentiated cells derived therefrom, including fully differentiated CNS and peripheral nervous system (“PNS”) cell types.
  • PNS peripheral nervous system
  • Examples of neuronal stem and progenitor cells include, but are not limited to, those described in Gritti et al., J. of Neuroscience 16:1091-1100 (1996); Frederiksen et al., (1988); Reynolds and Weiss, (1992); Davis and Temple, (1994); McKay, (1997);.
  • active agents refers to the group of compounds comprising angiotensinogen, angiotensin I (M), M analogues, M fragments and analogues thereof, angiotensin II (All), All analogues, All fragments or analogues thereof and All AT 2 type 2 receptor agonists.
  • M angiotensinogen
  • M angiotensin I
  • M analogues
  • M fragments and analogues thereof angiotensin II
  • AT 2 type 2 receptor agonists AT 2 type 2 receptor agonists.
  • U.S. Patent No. 5,015,629 to DiZerega describes a method for increasing the rate of healing of wound tissue, comprising the application to such tissue of angiotensin II (All) in an amount which is sufficient for said increase.
  • the application of All to wound tissue significantly increases the rate of wound healing, leading to a more rapid re-epithelialization and tissue repair.
  • the term MI refers to an octapeptide present in humans and other species having the sequence Asp- Arg-Val-Tyr-Ile-His-Pro-Phe [SEQ ID NO:l].
  • the biological formation of angiotensin is initiated by the action of renin on the plasma substrate angiotensinogen.
  • angiotensin I M
  • angiotensinase which removes the C-terminal His-Leu residues from Al (Asp-Arg- Val-Tyr-He-His-Pro-Phe-His-Leu [SEQ ID NO:37]).
  • All is a known pressor agent and is commercially available.
  • the use of All analogues and fragments, AT2 agonists, as well as AIII and AIII .analogues and fragments in wound healing has also been described. (U.S. Patent No. 5,629,292; U.S. Patent No. 5,716,935; WO 96/39164; all references herein incorporated by reference in their entirety.)
  • AII(l-7) elicits some, but not the full range of effects elicited by AI Pfeilschifter, et al., Eur. J. Pharmacol. 225:57-62 (1992); Jaiswal, et al., Hypertension 19(Supp. II):II-49-II-55 (1992); Edwards and Stack, J. Pharmacol. Exper. Ther. 266:506-510 (1993); Jaiswal, et al., J. Pharmacol. Exper. Ther. 265:664-673 (1991); Jaiswal, et al., Hypertension 17:1115-1120 (1991); Portsi, et a., Br. J. Pharmacol. 111:652-654 (1994).
  • a peptide agonist selective for the AT2 receptor (All has 100 times higher affinity for AT2 than ATI) is p-aminophenylalanine6-AII ["(p- ⁇ H 2 -Phe)6-AII)"], Asp-Arg-Val-Tyr-Ile- Xaa-Pro-Phe [SEQ ID NO.36] wherein Xaa is p-NH 2 -Phe (Speth and Kim, BBRC 169:997- 1006 (1990).
  • This peptide gave binding characteristics comparable to AT2 antagonists in the experimental models tested (Catalioto, et al., Eur. J. Pharmacol. 256:93-97 (1994); Bryson, et al., Ewr.
  • R A is suitably selected from Asp, Glu, Asn, Acpc ( 1 -aminocyclopentane carboxylic acid), Ala, Me 2 Gly, Pro, Bet, Glu(NH 2 ), Gly, Asp(NH 2 ) and Sue,
  • R B is suitably selected from Arg, Lys, Ala, Orn, Ser(Ac), Sar, D-Arg and D- Lys;
  • R 3 is selected from the group consisting of Val, Ala, Leu, norLeu, He, Gly, Pro, Aib, Acpc, Lys, and Tyr;
  • R 4 is selected from the group consisting of Tyr, Tyr(PO ) 2 , Thr, Ser, homoSer, Ala, and azaTyr;
  • R 5 is selected from the group consisting of He, Ala, Leu, norLeu, Val and Gly; R° is His, Arg or 6-NH 2 -Phe; R 7 is Pro or Ala; and
  • R 8 is selected from the group consisting of Phe, Phe(Br), He and Tyr, excluding sequences including R 4 as a terminal Tyr group.
  • R A and R B are Asp-Arg, Asp-Lys, Glu-Arg and Glu-Lys.
  • Particularly preferred embodiments of this class include the following: AH,
  • AIH or AII(2-8) Arg-Val-Tyr-Ile-His-Pro-Phe [SEQ ID NO:2]; AII(3-8), also known as desl-
  • Asp-Arg-Val-Tyr [SEQ ID NOJ0]; and AII(l-3), Asp-Arg-Val [SEQ ID NOJ 1].
  • Other preferred embodiments include: Arg-norLeu-Tyr-Ile-His-Pro-Phe [SEQ ID NO: 12] and Arg-
  • Val-Tyr-norLeu-His-Pro-Phe [SEQ ID NO: 13]. Still another preferred embodiment encompassed within the scope of the invention is a peptide having the sequence Asp-Arg-Pro- Tyr-He-His-Pro-Phe [SEQ ID NO:31]. AII(6-8), His-Pro-Phe [SEQ ID NO: 14] and AH(4-8),
  • the present invention provides a method for promoting neuronal cell proliferation or differentiation comprising contacting neuronal cells with an amount effective to promote proliferation or differentiation of at least one active agent comprising a sequence consisting of the general formula: RI -ARG-VAL-TYR-R2-HIS-PRO-R3 wherein RI is selected from the group consisting of H or Asp; R2 is selected from the group consisting of He, Val, Leu, norLeu and Ala; R3 is either Phe or H.
  • the active agent is selected from the group consisting of SEQ ID NOJ, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NOJ3, SEQ ID NO:18, SEQ ID NOJ9, SEQ ID NO:26, and SEQ ID NO:34.
  • R 2 is selected from the group consisting of H, Arg, Lys, Ala, Orn, Ser(Ac), Sar, D-.Arg and D-Lys;
  • R 3 is selected from the group consisting of Val, Ala, Leu, norLeu, He, Gly, Pro, Aib, Acpc and Tyr;
  • R 4 is selected from the group consisting of Tyr, Tyr(PO 3 ) 2 , Thr, Ser, homoSer and azaTyr;
  • R 5 is selected from the group consisting of He, Ala, Leu, norLeu, Val and Gly;
  • R° is His, Arg or 6-NH 2 -Phe;
  • R 7 is Pro or Ala;
  • R 8 is selected from the group consisting of Phe, Phe(Br), He and Tyr.
  • a particularly preferred subclass of the compounds of general formula II has the formula
  • R 2 , R 3 and R 5 are as previously defined. Particularly preferred is angiotensin
  • Bet l-carboxy-N,N,N-trimethylmethanaminium hydroxide inner salt betaine
  • AH and its analogues adopt either a gamma or a beta turn (Regoli, et al., Pharmacological Reviews 26:69 (1974).
  • neutral side chains in position R 3 , R 5 and R 7 may be involved in maintaining the appropriate distance between active groups in positions R 4 , R° and R 8 primarily responsible for binding to receptors and/or intrinsic activity.
  • Hydrophobic side chains in positions R 3 , R 5 and R 8 may also play an important role in the whole conformation of the peptide and/or contribute to the formation of a hypothetical hydrophobic pocket.
  • Appropriate side chains on the amino acid in position R .2 may contribute to affinity of the compounds for target receptors and/or play an important role in the conformation of the peptide. For this reason, Arg and Lys are particularly preferred as R 2 .
  • R 3 may be involved in the formation of linear or nonlinear hydrogen bonds with R 5 (in the gamma turn model) or R 6 (in the beta turn model). R 3 would also participate in the first turn in a beta antiparallel structure (which has also been proposed as a possible structure). In contrast to other positions in general formula I, it appears that beta and gamma branching are equally effective in this position. Moreover, a single hydrogen bond may be sufficient to maintain a relatively stable conformation. Accordingly, R 3 may suitably be selected from Val, Ala, Leu, norLeu, He, Gly, Pro, Aib, Acpc and Tyr. In another preferred embodiment, R 3 is Lys.
  • R 4 is preferably selected from Tyr, Thr, Tyr (PO ) 2 , homoSer, Ser and azaTyr.
  • Tyr is particularly preferred as it may form a hydrogen bond with the receptor site capable of accepting a hydrogen from the phenolic hydroxyl (Regoli, et al. (1974), supra).
  • R 4 is Ala.
  • an amino acid with a ⁇ aliphatic or alicyclic chain is particularly desirable. Therefore, while Gly is suitable in position R 5 , it is preferred that the amino acid in this position be selected from He, Ala, Leu, norLeu, Gly and Val.
  • R° is His, Arg or 6-NH 2 -Phe.
  • R 13 aromatic character are believed to contribute to its particular utility as R ⁇ .
  • conformational models suggest that His may participate in hydrogen bond formation (in the beta model) or in the second turn of the antiparallel structure by influencing the orientation of R 7 .
  • R 7 should be Pro in order to provide the most desirable orientation of R 8 .
  • both a hydrophobic ring and an anionic carboxyl terminal appear to be particularly useful in binding of the analogues of interest to receptors; therefore, Tyr and especially Phe are preferred for purposes of the present invention.
  • Analogues of particular interest include the following: TABLE 2 Angiotensin II Analogues
  • polypeptides of the instant invention may be synthesized by methods such as those set forth in J. M. Stewart and J. D. Young, Solid Phase Peptide Synthesis, 2nd ed., Pierce Chemical Co., Rockford, 111. (1984) and J. Meienhofer, Hormonal Proteins and
  • these methods involve the sequential addition of protected amino acids to a growing peptide chain (U.S. Patent No. 5,693,616, herein incorporated by reference in its entirety). Normally, either the amino or carboxyl group of the first amino acid and any reactive side chain group are protected. This protected amino acid is then either attached to an inert solid support, or utilized in solution, and the next amino acid in the sequence, also suitably protected, is added under conditions amenable to formation of the amide linkage. After all the desired amino acids have been linked in the proper sequence, protecting groups and any solid support are removed to afford the crude polypeptide. The polypeptide is desalted and purified, preferably ohromatographically, to yield the final product.
  • a method of increasing in vivo, in vitro and ex vivo neuronal stem and progenitor cell proliferation by exposure to angiotensinogen, Al, Al analogues, Al fragments and analogues thereof, AH analogues, AH fragments or analogues thereof or MI AT 2 type 2 receptor agonists ("active agents") is disclosed.
  • Proliferation can be quantitated using any one of a variety of techniques well known in the art, including, but not limited to, bromodeoxyuridine incorporation (Vicario-Abejon et al., 1995), 3 H-thymidine incorporation (Fredericksen et al., 1988), or antibody labeling of a protein present in higher concentration in proliferating cells than in non-proliferating cells.
  • bromodeoxyuridine incorporation Vicario-Abejon et al., 1995
  • 3 H-thymidine incorporation Feredericksen et al., 1988
  • antibody labeling of a protein present in higher concentration in proliferating cells than in non-proliferating cells include, but not limited to, bromodeoxyuridine incorporation (Vicario-Abejon et al., 1995), 3 H-thymidine incorporation (Fredericksen et al., 1988), or antibody labeling of a protein present in higher concentration in proliferating cells than in
  • proliferation of neuronal stem and progenitor cells is assessed by reactivity to an antibody directed against a protein known to be present in higher concentrations in proliferating cells than in non-proliferating cells, including but not limited to proliferating cell nuclear antigen (PCNA, or cyclin; Zymed Laboratories, South San Francisco, California).
  • PCNA proliferating cell nuclear antigen
  • neuronal cells are isolated from primary cell masses according to standard methods (Jirikowski et al., 1984; Reynolds and Weiss, 1992; Johe et al., 1996; Gritti et al., 1996; Vicario-Abejon et al., 1995; Kordower et al., 1995; Nauert and Freeman, Cell Transplant 3:147-151, 1994; Freeman and Kordower, In: Lindvall et al., eds. Intracerebral Transplantation in Movement Disorders.
  • neuronal stem and progenitor cells are isolated from primary cells which are isolated from the adult rat mammalian forebrain or rat embryonic hippocampus (Johe et al., 1996). The cell mass is dissociated by either mechanical trituration or by incubating minced tissue in Hank's Buffered Saline Solution (HBSS).
  • HBSS Hank's Buffered Saline Solution
  • the cells are collected by centrifugation and resuspended in a serum-free medium containing Dulbecco's modified Eagle medium (DMEMVF12, glucose, glutamine, sodium bicarbonate, 25 ⁇ g/ml of insulin, 100 ⁇ g/ml of human apotransferrin, 20 nm progesterone, 100 ⁇ m putrescene, 30 nm sodium selenite (pH 7.2), plus 10 ng/ml of recombinant basic fibroblast growth factor (bFGF; R&D, Inc.) (Johe et al., 1996).
  • DMEMVF12 Dulbecco's modified Eagle medium
  • bFGF basic fibroblast growth factor
  • neuronal stem and progenitor cells are isolated from the dissociated cell mass by antibody-mediated cell capture ("panning"; Barres et al., Cell 70:31-46, 1992).
  • Antibodies that can be used to isolate neuronal stem and precursor cells include, but are not limited to, nestin antibody (Vicario-Abejon et al., 1995).
  • the cells are then treated as above.
  • the neuronal stem and progenitor cells exposed to the active agents as described above can be used for neuron replacement therapy, to treat disorders including, but not limited to, Parkinson's disease, Alzheimer's disease, and amyotrophic lateral sclerosis.
  • the cells are cultured in vitro or ex vivo as described above.
  • the cells are rinsed to remove all traces of culture fluid, resuspended in an appropriate medium and then pelleted and rinsed several times. After the final rinse, the cells are resuspended at between 0.7 x 10 6 and 50 x 10 6 cells per ml in an appropriate medium and used for transplantation according to previously described methods.
  • the neuronal stem and progenitor cells used for transplantation are transfected with an expression vector so as to express a therapeutic protein, including but not limited to glial-cell-line-derived neurotrophic factor (GD ⁇ F; Beck et al.
  • GD ⁇ F glial-cell-line-derived neurotrophic factor
  • the effect of the active agents on neuronal stem and progenitor cell differentiation is assessed by examination of changes in gene expression, phenotype, morphology, or any other method that distinguishes stem and/or progenitor cells from fully differentiated cells.
  • Examples of such differentiation markers against which antibodies are available include, but are not limited to, neuron-specific microtubule-associated protein 2 (MAP2; Vicario-Abejon et al., 1995; antibody available from Boehringer Manheim, Germany), astroglial-specific glial fibrillary acidic protein (GFAP; Vicario-Abejon et al., 1995; antibody available from Incstar,); neuron-specific tau protein (Johe et al., 1996; antibody available from Sigma, St. Louis, MO); neurofilaments L and M (Johe et al., 1996; antibody available from Boehringer Manheim); and oligodendrocyte-specific 04 and galactocerebroside (GalC; Johe et al., 1996).
  • MAP2 neuron-specific microtubule-associated protein 2
  • GFAP astroglial-specific glial fibrillary acidic protein
  • GFAP astroglial-specific glial fibrillary acidic protein
  • neuronal stem and progenitor cells are isolated and cultured as described above. Differentiation is initiated by contacting the cells with the active agents as described above, in serum-free medium in the absence of bFGF. Differentiation is assessed at various times by immunodetection of differentiation-specific markers, using the antibodies described above (Johe et al., 1996). Alternatively, differentiation is assessed morphologically by measurement of neurite outgrowth.
  • the active agents are used to increase in vivo neuronal stem and progenitor cell proliferation.
  • the active agents may be administered by any suitable route, including orally, parentally, by inhalation spray, rectally, or topically in dosage unit
  • parenteral as used herein includes, subcutaneous, intravenous, intra- arterial, intra-ventricular, intramuscular, intrasternal, intratendinous, intraspinal, intracranial, intrathoracic, infusion techniques or intraperitoneally.
  • the active agents may be made up in a solid form (including granules, powders or suppositories) or in a liquid form (e.g., solutions, suspensions, or emulsions) and may be subjected to conventional pharmaceutical operations such as sterilization and/or may contain conventional adjuvants, such as preservatives, stabilizers, wetting agents, emulsifiers, buffers etc.
  • active agents can be administered as the sole active agent, they can also be used in combination with one or more other compounds.
  • the active agents and other compounds can be formulated as separate compositions that are given at the same time or different times, or the active agents and other compounds can be given as a single composition.
  • active agents are ordinarily combined with one or more adjuvants appropriate for the indicated route of administration.
  • the compounds may be admixed with lactose, sucrose, starch powder, cellulose esters of alkanoic acids, stearic acid, talc, magnesium stearate, magnesium oxide, sodium and calcium salts of phosphoric and sulphuric acids, acacia, gelatin, sodium alginate, polyvinylpyrrolidine, and/or polyvinyl alcohol, and tableted or encapsulated for conventional administration.
  • the compounds of this invention may be dissolved in saline, water, polyethylene glycol, propylene glycol, carboxymethyl cellulose colloidal solutions, ethanol, corn oil, peanut oil, cottonseed oil, sesame oil, tragacanth gum, and/or various buffers.
  • Other adjuvants and modes of administration are well known in the pharmaceutical art.
  • the carrier or diluent may include
  • time delay material such as glyceryi monostearate or glyceryl distearate alone or with a wax, or other materials well known in the art.
  • Formulations suitable for topical administration include liquid or semi-liquid preparations suitable for penetration through the skin (e.g., liniments, lotions, ointments, creams, or pastes) and drops suitable for administration to the eye, ear, or nose.
  • liquid or semi-liquid preparations suitable for penetration through the skin e.g., liniments, lotions, ointments, creams, or pastes
  • drops suitable for administration to the eye, ear, or nose e.g., liniments, lotions, ointments, creams, or pastes
  • the dosage regimen for increasing in vivo proliferation or differentiation of neuronal stem and progenitor cell with the active agents of the invention is based on a variety of factors, including the type of injury or deficiency, the age, weight, sex, medical condition of the individual, the severity of the condition, the route of administration, and the particular compound employed. Thus, the dosage regimen may vary widely, but can be determined routinely by a physician using standard methods.
  • the active agents are administered by unilateral infusion directly into the mammalian brain lateral ventricle using an osmotic pump (Alza Palo Alto, CA) attached to a 30 gauge cannulae implanted at the injection coordinate, as described in Craig et al., J. of Neuroscience 16:2649-2658 (1996).
  • a suitable injected dose of active ingredient of the active agents is preferably between about 0J ng/kg and about 10 mg/kg administered twice daily.
  • the active ingredient may comprise from 0.001% to 10%) w/w, e.g., from 1% to 2% by weight of the formulation, although it may comprise as much as 10% w/w, but preferably not more than 5% w/w, and more preferably from 0.1%) to 1%. of the formulation.
  • an improved cell culture medium for the proliferation and differentiation of neuronal cells, wherein the improvement comprises addition to the cell culture medium of an effective amount of the active agents, as described above.
  • Any cell culture media that can support the growth of neuronal stem and progenitor cells can be used with the present invention.
  • Such cell culture media include, but are not limited to Basal Media Eagle, Dulbecco's Modified Eagle Medium, Iscove's Modified
  • the improved cell culture medium can be supplied in either a concentrated (ie: 10X) or non-concentrated form, and may be supplied as either a liquid, a powder, or a lyophilizate.
  • the cell culture may be either chemically defined, or may contain a serum supplement.
  • kits for the propagation of neuronal stem and progenitor cells wherein the kits comprise an effective amount of the active agents, as described above.
  • the kit further comprises cell culture growth medium.
  • Any cell culture media that can support the growth of neuronal stem and progenitor cells can be used with the present invention. Examples of such cell culture media are described above.
  • the improved cell culture medium can be supplied in either a concentrated (ie: 10X) or non-concentrated form, and may be supplied as either a liquid, a powder, or a lyophilizate.
  • the cell culture may be either chemically defined, or may contain a serum supplement.
  • the kit further comprises a sterile container.
  • the sterile container can comprise either a sealed container, such as a cell culture flask, a roller bottle, or a centrifuge tube, or a non-sealed container, such as a cell culture plate or microtiter plate (Nunc; Naperville, IL).
  • the kit further comprises an antibiotic supplement for inclusion in the reconstituted cell growth medium. Examples of appropriate antibiotic supplements include, but are not limited to actimonycin D, Fungizone®, kanamycin, neomycin, nystatin, penicillin, streptomycin, or combinations thereof (GEBCO).
  • NPMM Neural Progenitor Cell Maintenance Medium
  • the cells were seeded upon wells coated with 0.05% polyethyleneimine (PEI) substrate in borate buffer solution.
  • PEI polyethyleneimine
  • the wells of a 96 well plate were coated with 0.05 ml of this solution overnight at room temperature. After the incubation, the substrate was removed by aspiration, rinsed with sterile water and allowed to dry before seeding of the cells.
  • the effect of MI on the differentiation of neuronal progenitor cells was assessed. After adherence to PEI substrate, the cells cease proliferation and undergo differentiation and neurite outgrowth. One thousand cells were placed in each well in the presence and absence of various concentrations of AIL Four and seven days after initiation of culture, the size of the neurites (by measurement with an ocular micrometer) on the cells undergoing differentiation and the number of cells undergoing differentiation was assessed.
  • NPMM Neuronal Progenitor Cell Maintenance Medium
  • Basal Medium containing human recombinant fibroblast growth factor beta, human recombinant epidermal growth factor, neural survival factors, gentamycin, and amphotericin B).
  • the cells were thawed, diluted into NPMM and cultured for 24 hours in a 75 cm 2 flask. Until studies to assess differentiation, the cells were cultured in dedifferentiated spheroids.
  • the present invention by providing a method for enhanced proliferation of neuronal cells, will greatly increase the clinical benefits of neuronal stem and progenitor transplantation. This is true both for increased “self-renewal" of neuronal stem cells, which will provide a larger supply of stem cells capable of differentiation into various neuronal cell types, and for proliferation with differentiation, which will provide a larger supply of neuronal progenitor and differentiated cells at the appropriate site. Similarly, methods that increase in
  • the method of the present invention also increases the potential utility of neuronal stem and progenitor cells as vehicles for gene therapy in central and peripheral nervous system disorders by more efficiently providing a large number of such cells for transfection, and also by providing a more efficient means to rapidly expand transfected neuronal stem and progenitor cells.

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  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Peptides Or Proteins (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

La présente invention concerne des procédés, un bouillon de culture amélioré et des kits destinés à favoriser la prolifération et/ou la différentiation des neurones par le biais de la croissance en présence de l'angiotensinogène, d'AI, d'analogues d'AI, de fragments d'AI et de leurs analogues, d'AII, d'analogues d'AII, de fragments d'AII et de leurs analogues et/ou d'agonistes des récepteurs 2 d'AT2 d'AI, pris séparément ou en combinaison avec d'autres facteurs de croissance et cytokines.
PCT/US1999/003772 1998-02-19 1999-02-19 Procede pour favoriser la proliferation et la differentiation des neurones WO1999042123A1 (fr)

Priority Applications (4)

Application Number Priority Date Filing Date Title
EP99908331A EP1054684A1 (fr) 1998-02-19 1999-02-19 Procede pour favoriser la proliferation et la differentiation des neurones
CA002321164A CA2321164A1 (fr) 1998-02-19 1999-02-19 Procede pour favoriser la proliferation et la differentiation des neurones
JP2000532137A JP2002503702A (ja) 1998-02-19 1999-02-19 神経細胞の細胞増殖および分化を促進する方法
AU27791/99A AU750029B2 (en) 1998-02-19 1999-02-19 Method of promoting neuronal cell proliferation and diffrentiation

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US7523298P 1998-02-19 1998-02-19
US60/075,232 1998-02-19

Publications (1)

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WO1999042123A1 true WO1999042123A1 (fr) 1999-08-26

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PCT/US1999/003772 WO1999042123A1 (fr) 1998-02-19 1999-02-19 Procede pour favoriser la proliferation et la differentiation des neurones

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EP (1) EP1054684A1 (fr)
JP (1) JP2002503702A (fr)
AU (1) AU750029B2 (fr)
CA (1) CA2321164A1 (fr)
WO (1) WO1999042123A1 (fr)

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002006308A3 (fr) * 2000-07-13 2003-02-27 Univ Southern California Procedes favorisant la proliferation ou la differenciation des cellules dendritiques
US6730775B1 (en) 1999-03-23 2004-05-04 University Of Southern California Methods for limiting scar and adhesion formation
US6747008B1 (en) 2000-06-19 2004-06-08 University Of Southern California Methods for treating and preventing alopecia
WO2013091883A3 (fr) * 2011-12-23 2013-10-24 Medical Research Council Ligands de gpcr sélectifs
WO2014021942A1 (fr) * 2012-08-01 2014-02-06 University Of Southern California Procédés pour limiter le développement d'une neurodégénérescence
WO2014151338A1 (fr) * 2013-03-15 2014-09-25 University Of Southern California Méthodes de traitement de la sclérose en plaques
US9732074B2 (en) 2013-03-15 2017-08-15 University Of Southern California Methods, compounds, and compositions for the treatment of angiotensin-related diseases

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US5015629A (en) * 1989-06-26 1991-05-14 University Of Southern California Tissue repair
WO1998032457A2 (fr) * 1997-01-28 1998-07-30 University Of Southern California Procede stimulant la proliferation et la differentiation de cellules hematopoietiques et mesenchymateuses

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US5015629A (en) * 1989-06-26 1991-05-14 University Of Southern California Tissue repair
WO1998032457A2 (fr) * 1997-01-28 1998-07-30 University Of Southern California Procede stimulant la proliferation et la differentiation de cellules hematopoietiques et mesenchymateuses

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
L. LAFLAMME ET AL.: "Angiotensin II induction of neurite outgrowth by AT2 receptors in NG108-15 cells", JOURNAL OF BIOLOGICAL CHEMISTRY. (MICROFILMS), vol. 271, no. 37, September 1996 (1996-09-01), MD US, pages 22729 - 22735, XP002106039 *

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6730775B1 (en) 1999-03-23 2004-05-04 University Of Southern California Methods for limiting scar and adhesion formation
US6747008B1 (en) 2000-06-19 2004-06-08 University Of Southern California Methods for treating and preventing alopecia
WO2002006308A3 (fr) * 2000-07-13 2003-02-27 Univ Southern California Procedes favorisant la proliferation ou la differenciation des cellules dendritiques
WO2013091883A3 (fr) * 2011-12-23 2013-10-24 Medical Research Council Ligands de gpcr sélectifs
CN104040345A (zh) * 2011-12-23 2014-09-10 医疗研究局 血管紧张素ii受体的选择性配体
WO2014021942A1 (fr) * 2012-08-01 2014-02-06 University Of Southern California Procédés pour limiter le développement d'une neurodégénérescence
WO2014151338A1 (fr) * 2013-03-15 2014-09-25 University Of Southern California Méthodes de traitement de la sclérose en plaques
US9623084B2 (en) 2013-03-15 2017-04-18 University Of Southern California Methods for treating multiple sclerosis
US9732074B2 (en) 2013-03-15 2017-08-15 University Of Southern California Methods, compounds, and compositions for the treatment of angiotensin-related diseases
US10301298B2 (en) 2013-03-15 2019-05-28 University Of Southern California Methods, compounds, and compositions for the treatment of angiotensin-related diseases

Also Published As

Publication number Publication date
CA2321164A1 (fr) 1999-08-26
EP1054684A1 (fr) 2000-11-29
AU750029B2 (en) 2002-07-11
JP2002503702A (ja) 2002-02-05
AU2779199A (en) 1999-09-06

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