EP1185552A1 - Peptides retro-inverso derives de l'interleukine-3 - Google Patents

Peptides retro-inverso derives de l'interleukine-3

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Publication number
EP1185552A1
EP1185552A1 EP00939960A EP00939960A EP1185552A1 EP 1185552 A1 EP1185552 A1 EP 1185552A1 EP 00939960 A EP00939960 A EP 00939960A EP 00939960 A EP00939960 A EP 00939960A EP 1185552 A1 EP1185552 A1 EP 1185552A1
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European Patent Office
Prior art keywords
peptide
seq
sequence shown
ammo
retro
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EP00939960A
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German (de)
English (en)
Inventor
David E. Wright
D. Elliot Parks
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Myelos Corp
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Myelos Corp
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Publication of EP1185552A1 publication Critical patent/EP1185552A1/fr
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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
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/5403—IL-3
    • 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
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to retro-mverso peptides derived from interleukin 3 (IL-3). These peptides have activities similar to that of the native parent protein, and also have neurotrophic activity.
  • IL-3 interleukin 3
  • Cytokmes are proteins which are produced during the effector phases of natural and specific immunity and serve to mediate and regulate immune and inflammatory responses. Cytokmes, like other polypeptide hormones, initiate their action by binding to specific receptors on the surface of target cells.
  • Cytokmes One of the most well known families of cytokmes are the mterleukins which mediate natural immunity. For a detailed description of the structure and function of the mterleukins, see Abbas et al. Cellular and Molecular Immunology, W. B. Saunders Company, Philadelphia, pp. 225-243,
  • IL 3 acts on numerous target cells within the hematopoietic system. A detailed review of the structure and function of this cytokme may be found in The Cytokme Handbook, Third Edition, Thomson, A. Ed., Academic Press, San
  • IL 3 is a glycoprotein having broad structural similarities with other mterleukins and hematopoietic growth factors.
  • Murme IL-3 contains 140 ammo acids, while human IL 3 contains 133 ammo acids.
  • the ammo acid sequences of mouse and human IL-3 exhibit only 30% identity, reflecting the lack of cross-species biologic activity (Yang et al, Cell 47:3-10, 1986).
  • IL-3 has the broadest target specificity of any of the hematopoietic growth factors.
  • the range of target cells includes progenitor cells of every lineage derived from the plu ⁇ potential hematopoietic stem cells.
  • IL 3 can stimulate the generation and differentiation of macrophages, neutrophils, eosinophils, basophiis, mast cells, megakaryocytes and erythroid cells.
  • hematopoietic stem and progenitor cells rapidly die if cultured in tissue culture medium alone.
  • IL-3 prevents death by apoptosis and promotes survival in vitro (Williams et al., Nature
  • IL-3 induced extramedullary hematopoiesis at sites of subcutaneous injection (Khan et al., Toxicol. Pathol. 24:391 -397, 1996). IL-3 may have particular utility in stimulating platelet production (Ganser et al., Blood 76:666-676, 1990). In addition, clinical trials suggest that sequential administration of IL 3 and G CSF or GM CSF may provide optimal stimulation of myelopoiesis
  • Neurotrophms and neurotrophic factors are proteins or peptides capable of affecting the survival, target mnervation and/or function of neuronal cell populations (Barde, Neuron, 2:1525 1534, 1989).
  • ciliary neurotrophic factor CNTF
  • CNTF ciliary neurotrophic factor
  • Retro mverso peptides are isomers of linear peptides in which the direction of the sequence is reversed (retro) and the chira ty, D or L, of each ammo acid is inverted (mverso)
  • Retro mverso isomers of linear peptides in which only some of the peptide bonds are reversed and the chirahty of the ammo acid residues in the reversed portion is inverted.
  • One embodiment of the present invention is an isolated retro mverso peptide having between 12 and about 40 ammo acids, wherein said peptide includes the sequence shown in SEQ ID NO. 1
  • at least one basic charged ammo acid of said sequence is replaced with a different basic charged ammo acid.
  • At least one acidic charged ammo acid of said sequence is replaced with a different acidic charged ammo acid
  • at least one non polar ammo acid of said sequence is replaced with a different non polar ammo acid
  • at least one uncharged ammo acid of said sequence is replaced with a different uncharged ammo acid
  • at least one aromatic ammo acid of said sequence is replaced with a different aromatic ammo acid.
  • the peptide is glycosylated.
  • one or more amide bonds of the peptide is reduced.
  • one or more nitrogens in said peptide is methylated
  • one or more carboxylic acid groups in the peptide is este ⁇ fied.
  • the peptide has the ammo acid sequence shown in SEQ ID NO: 1
  • the present invention also provides a method for promoting neu ⁇ te outgrowth or myelinatio ⁇ in a mammal in need thereof, comprising the step of administering to the mammal an effective, neu ⁇ te outgrowth or myeli ⁇ ation facilitating amount of a composition comprising a retro-mverso peptide having between 17 and about 40 ammo acids, wherein the peptide includes the sequence shown in SEQ ID NO: 1.
  • the peptide has the ammo acid sequence shown in SEQ ID NO. 1
  • the mammal is a human.
  • the administering step is direct local injection, systemic, mtracranial, intracerebrospinal, topical or oral.
  • the present invention also provides a method for stimulating hematopoiesis, comprising contacting plu ⁇ potential hematopoietic stem cells with an effective, hematopoiesis stimulating amount of a composition comprising a retro-mverso peptide having between 12 and about 40 ammo acids, wherein the peptide includes the sequence shown in SEQ ID NO: 1.
  • the method results in the generation and differentiation of macrophages, neutrophils, eosinophils, basophiis, mast cells, megakaryocytes or erythroid cells
  • a retro mverso peptide having between 17 and about 40 ammo acids wherein said peptide includes the sequence shown in SEQ ID NO: 1 , for use in promoting neu ⁇ te outgrowth or mye nation in a mammal
  • the peptide has the ammo acid sequence shown in SEQ ID NO: 1.
  • the mammal is a human.
  • the present invention also provides a retro mverso peptide having between 17 and about 40 ammo acids, wherein the peptide includes the sequence shown in SEQ ID NO 1, for use in stimulating hematopoiesis of plu ⁇ potential hematopoietic stem cells.
  • the peptide has the sequence shown in SEQ ID NO: 1.
  • Another embodiment of the present invention is the use of a retro-mverso peptide having between 17 and about 40 ammo acids, wherein yhr peptide includes the sequence shown in SEQ ID NO. 1, in the preparation of a medicament for promoting neu ⁇ te outgrowth or myehnation in a mammal in need thereof
  • the peptide has the sequence shown in SEQ ID NO. 1
  • the mammal is a human
  • the present invention also provides the use of a retro mverso peptide having between 17 and about 40 ammo acids, wherein the peptide includes the sequence shown in SEQ ID NO: 1 , in the preparation of a medicament for stimulating hematopoiesis of plu ⁇ potential hematopoietic stem cells in a mammal in need thereof.
  • the peptide has the sequence shown in SEQ ID NO: 1.
  • the mammal is a human.
  • the present invention provides retro mverso (Rl) peptides derived from mterleukin 3 (IL 3) which mediate similar effects to native IL 3, including stimulation of hematopoiesis and platelet production
  • Rl retro mverso
  • IL 3 mterleukin 3
  • the term "derived from” indicates that the peptides include an active region of mterleukin 3, or analogs thereof as defined below
  • These Rl IL 3-der ⁇ ved peptides stimulate the growth and differentiation of macrophages, neutrophils, eosinophils, basophiis, mast cells, megakaryocytes and erythroid cells.
  • peptides also have therapeutic applications in promoting functional recovery after toxic, traumatic, ischemic, degenerative and inherited lesions to the peripheral and central nervous system
  • These peptides are also useful for promoting increased myehnation and for counteracting the effects of demye nating diseases.
  • the ability of a particular retro-mverso peptide to mediate an effect similar to the parent peptide can be determined by a person of ordinary skill in the art using standard IL-3 assays as described in the examples below.
  • the use of these peptides will facilitate treatment of various disorders since they will be more stable and easier to synthesize than either the native or recombmant cytokmes.
  • Rl IL-3-der ⁇ ved peptide of the invention and the parent protein from which it is derived, is shown in Table 1.
  • the corresponding native (non-retro-inverted) peptides is disclosed in U.S. Patent No. 5,700,909, the entire contents of which are hereby incorporated by reference.
  • Rl IL-3-der ⁇ ved peptides have the same hematopoietic activities as the corresponding full length IL 3 protein, and also possess neurotrophic and myehnotrophic activity
  • One embodiment of the present invention is a method for stimulating the generation and differentiation of plu ⁇ potential hematopoietic stem cells into cells such as macrophages, neutrophils, eosinophils, basophiis, mast cells, megakaryocytes and erythroid cells by administering to the cells an effective, hematopoiesis-facihtating amount of a Rl peptide having between 12 and about 40 ammo acids, and encompassing the Rl IL 3-der ⁇ ved peptide shown in SEQ ID NO: 1, or analogs thereof which have similar activity.
  • Such analogs include, for example, replacement of one or more lysine and/or arginme residues with alanine or another ammo acid; deletion of one or more lysine and/or arginme residues; replacement of one or more tyrosme and/or phenylalanme residues, deletion of one or more phenylalanme residues and conservative replacements of one or more ammo acids within the peptide.
  • the replacement or deletion of lysine/arginme and tyrosine/phenylalanine residues will reduce the susceptibility of peptide degradation by trypsin and chymotrypsm, respectively Additional variations of these peptide sequences contemplated for use in the present invention include minor insertions and deletions.
  • Conservative ammo acid replacements are contemplated. Such replacements are, for example, those that take place within a family of ammo acids that are related in the chemical nature of their side chains.
  • the families of ammo acids include the basic charged ammo acids (lysine, arginme, histidme); the acidic charged ammo acids (aspartic acid, glutamic acid); the non-polar ammo acids (alanine, vahne, leucme, isoleucine, prolme, phenylalanme, methionme, tryptophan), the uncharged polar ammo acids (glycme, asparagine, glutamme, cysteme, se ⁇ ne, threomne, tyrosme); and the aromatic ammo acids (phenylalanme, tryptophan and tyrosme).
  • aliphatic ammo terminal modification with a derivative of an aliphatic or aromatic acid, forming an amide bond.
  • Another modification is carboxy terminal modification with a derivative of an aliphatic or aromatic amme/alcohol coupled to the peptide via an amide/ester bond.
  • Such derivatives include those listed above.
  • the peptides may also have both ammo and carboxy terminal modifications, wherein the derivatives are independently selected from those listed above
  • the peptides may also be glycosylated, wherein either the alpha ammo group or a D Asn, or both, are modified with glucose or galactose.
  • selected backbone amide bonds are reduced ( NH-CH 2 ).
  • Other modifications include N methylation of selected nitrogens in the amide bonds and esters in which at least one of the acid groups on the peptide are modified as aromatic or aliphatic esters Any combination of the above modifications is also contemplated.
  • Another embodiment of the present invention is a method of facilitating neurite outgrowth in differentiated or undifferentiated neural cells by contacting the cells with an effective, hematopoiesis-facilitatmg amount of a Rl peptide having between 12 and about 40 ammo acids, and encompassing the Rl IL 3 derived peptide shown in SEQ ID NO: 1, or analogs thereof which have similar activity as described above.
  • a typical minimum amount of the Rl peptides of the invention for the neurotrophic activity in cell growth medium is usually at least about 5 ng/ml. This amount or more of the Rl peptides of the invention for in vitro use is contemplated. Typically, concentrations in the range of 0.1 g/ml to about 10 g/ml of these peptides will be used. Effective amounts for any particular tissue can be determined in accordance with Example 1
  • the hematopoietic or neural cells can be treated in vitro or ex vivo by directly administering the Rl peptides of the invention to the cells This can be done, for example, by cultu ⁇ ng the cells in growth medium suitable for the particular cell type followed by addition of the peptide to the medium.
  • the composition can be administered by one of several techniques. Most preferably, the composition is injected directly into the blood in sufficient quantity to give the desired local concentration of peptide.
  • These Rl peptides persist longer in vivo due to the D peptide bonds. In the peptides lacking lysine and arginme residues, proteolytic degradation is reduced. The smaller peptides (i e. 50 mer or less) will most likely cross the blood brain barrier and enter the central nervous system for treatment of CNS disorders (see Banks et al., Peptides, 13-1289 1294, 1992).
  • a pharmaceutically acceptable mjectable carrier is used.
  • Such carriers include, for example, phosphate buffered saline and Ringer's solution.
  • the composition can be administered to peripheral neural tissue by direct local injection or by systemic administration.
  • Various conventional modes of administration are contemplated including intravenous, pulmonary, intramuscular, intradermal, subcutaneous, mtracranial, epidural, mtrathecal, topical and oral.
  • compositions of the invention can be packaged and administered in unit dosage form such as an mjectable composition or local preparation in a dosage amount equivalent to the daily dosage administered to a patient or as a controlled release composition.
  • a septum sealed vial containing a daily dose of the active ingredient in either PBS or in lyophilized form is an example of a unit dosage.
  • daily systemic dosages of the Rl peptides of the invention based on the body weight of the vertebrate for promoting IL 3 effects such as stimulation of hematopoiesis, and for treatment of neurodegenerative diseases or demyehnation diseases are in the range of from about 0.01 to about 10,000 g/kg More preferably, daily systemic dosages are between about 0 1 and 1 ,000 g/kg Most preferably, daily systemic dosages are between about 10 and 100 g/kg Daily dosages of locally administered material will be about an order of magnitude less. Oral administration is particularly preferred because of the resistance of the peptides to proteolytic degradation in the gastrointestinal system.
  • the peptides are administered locally to neural cells in vivo by implantation thereof.
  • polylactic acid, polygalactic acid, regenerated collagen, multilamellar liposomes and many other conventional depot formulations comprise bioerodible or biodegradable materials that can be formulated with biologically active neurotrophic peptide compositions. These materials, when implanted, gradually break down and release the active material to the surrounding tissue.
  • bioerodible, biodegradable and other depot formulations is expressly contemplated in the present invention Infusion pumps, matrix entrapment systems and combinations with transdermal delivery devices are also contemplated.
  • the peptides may also be encapsulated within a polyethylene glycol conformal coating as described in U.S. Patent No. 5,529,914 prior to implantation.
  • the peptides of the invention may also be enclosed in micelles or liposomes.
  • Liposome encapsulation technology is well known. Liposomes may be targeted to specific tissue, such as neural tissue, through the use of receptors, ligands or antibodies capable of binding the targeted tissue. The preparation of these formulations is well known in the art (Radin et al., Meth. Enzymol , 98.613 618, 1983)
  • neurotrophic factors can be therapeutically useful in the treatment of neurodegenerative diseases associated with the degeneration of neural populations or specific areas of the brain. Any degree of retardation or halting or reversing such degeneration is within the scope of the present invention.
  • the principal cause of Parkinson's disease is the degeneration of dopaminergic neurons of the substantia nigra
  • the Rl peptides of the invention may be therapeutically useful in the treatment of Parkinson's disease.
  • Retinal neuropathy an ocular neurodegenerative disorder leading to loss of vision in the elderly, is also treatable using the Rl peptides of the invention.
  • Cells may be treated to facilitate myehn formation or to prevent demyehnation in the manner described above in vivo, ex vivo or in vitro.
  • Diseases resulting in demyehnation of nerve fibers including MS, acute disseminated ieukoencephahtis, trauma to brain and/or spinal cord, progressive multifocal leukoencephalitis, metachromatic leukodystrophy, adrenal leukodystrophy and maldevelopment of the white matter in premature infants (perivent ⁇ cular leucomalacia) can be slowed or halted by administration of the neurotrophic peptides of the invention to the cells affected by the disease.
  • the Rl IL 3-der ⁇ ved peptide compositions of the present invention can also be used to support hematopoieses, to enhance the survival of cultured motor neurons and to determine the effects of neurotrophic factors and myelm facilitating materials. However, more practically, they have an immediate use as laboratory reagents and components of cell growth media in order to facilitate hematopoiesis and maintain neural cells in vitro.
  • the peptides of the invention are synthesized using an automated solid-phase protocol well known in the art. All peptides are purified by high performance liquid chromatography (HPLC) on a reverse-phase column to an extent greater than about 95% prior to use.
  • HPLC high performance liquid chromatography
  • Example 1 Stimulation of neurite outgrowth NS20Y neuroblastoma cells are grown in DMEM containing 10% fetal calf serum (FCS). Cells are removed with trypsin and plated in 30 mm petn dishes onto glass covershps. After 20-24 hours, the medium is replaced with 2 ml DMEM containing 0.5% FCS plus 0, 0.5, 1, 2, 4 or 8 ng/ml of a Rl IL-3-der ⁇ ved peptide having between 12 and about 40 ammo acids and including the sequence shown in SEQ ID NO: 1.
  • FCS fetal calf serum
  • NS20Y cells are plated as described in Example 1 and grown on glass covershps in 0.5% fetal bovine serum for 2 days in the presence or absence of 8 ⁇ g/ml of an Rl IL-3-der ⁇ ved peptide having between 12 and about 40 ammo acids and including the sequence shown in SEQ ID NO: 1. Media is removed and 0.2% trypan blue in PBS is added to each well.
  • Blue-staining dead cells are scored as a percentage of the total on an inverted microscope, counting 400 cells in four areas of each well. The average error of duplicates was 5%
  • Neurons are treated with 0.5 ng/ml of an Rl IL-3-der ⁇ ved peptide having between 12 and about 40 ammo acids and including the sequence shown in SEQ ID NO: 1.
  • Rl IL-3-der ⁇ ved peptide having between 12 and about 40 ammo acids and including the sequence shown in SEQ ID NO: 1.
  • the length of the longest neu ⁇ tic projections are measured on a micrometer grid.
  • the longest neuntes in neurons treated with Rl peptide are approximately three times longer than those treated with a control (non-RI) peptide or in untreated controls.
  • NGF nerve growth factor
  • EAE Experimental allergic encephalomyehtis
  • MS multiple sclerosis
  • EAE is induced in Lewis rats by injection of an emulsion of guinea pig spinal cord and complete Freund's adjuvant
  • CFA demyelinating lesions
  • Newborn mouse cerebellar explants are prepared according to Satomi ⁇ Zoo/. Sci 9:127-137, 1992) Neurite outgrowth and myehnation are observed for 22 days in culture, during the period when the newborn mouse cerebellum normally undergoes neuronal differentiation and myehnation begins.
  • An Rl IL-3-der ⁇ ved peptide having between 12 and about 40 ammo acids, and including the sequence shown in SEQ ID NO: 1, is added on the second day after preparation of the explants (three control and three treated explants) and outgrowth of neuntes and myehnation are assessed under a bright field microscope with a video camera.
  • Saposm C is used as a positive control at a concentration of between about 1 and 10 g/ml.
  • Myehnation is stimulated by the IL 3 derived peptides to a similar extent as with saposm C.
  • myehnation may be assayed by incorporation of 35 S into sulf o pids which are exclusive to myehn as described below
  • DMEM low sulfate media
  • FBS fetal bovine serum
  • 35S methionme 35S methionme
  • Rl IL 3 derived peptide having between 12 and about 40 ammo acids, and including the sequence shown in SEQ ID NO. 1, for 48 hours.
  • Saposm C is used as a positive control.
  • Cells are rinsed with PBS, harvested and sonicated in 100 I distilled water. An aliquot of cell lysate is removed for protein analysis and the remainder is extracted with 5 ml chloroform/methanol (2:1, v/v). Lipid extracts are chromatographed and immunostained with anti-sulfatide monoclonal antibody as described (Hiraiwa et al., Proc. Natl. Acad. Sci. U.S.A. 94:4778- 4781). Similar amounts of sulfatide are observed after peptide and sapos C treatment.
  • IL 3-der ⁇ ved peptide having between 12 and about 40 ammo acids, and including the sequence shown in SEQ ID NO: 1, in a sterile saline solution or in depot form Improvement is assessed by gam of sensory or motor nerve function (i.e. increased limb movement). Treatments continue until no further improvement occurs.
  • Example 7 Dosages are repeated daily or weekly and improvement in muscle strength, musculoskeletal coordination and myehnation (as determined by MRI) is observed. Patients with chronic relapsing MS are treated in the same manner when subsequent relapses occur.
  • IL 3 is assayed by its ability to stimulate the formation of colonies of differentiated cells from progenitor cells of the bone marrow in soft agar.
  • IL-3 can be assayed for its prohferative effect on cell lines derived from human leukemias such as TF-1 and M0-7e.
  • Detailed protocols for IL-3 assays may be found in Cytokmes, a Practical approach, Balkwill, F., ed., IRL Press, New York, second edition, 1995, pp. 247-268, 372-377, the entire contents of which are incorporated be reference.
  • IL-3 assay protocols are provided in the following example.
  • IL 3 stimulates the formation of mixed colonies of different cell types. Colonies of more than 50 cells are counted and analyzed after 7-14 days by eye using a binocular microscope. The number of colonies is usually related to the specific activity or concentration of IL-3 in the agar culture. Morphological analysis by staining of dried and fixed gels allows proper identification of the colony type (Metcalf, The Hemopoietic Colony Stimulating Factors, Elsevier Press, Amsterdam, 1984).
  • Cell suspensions are prepared from human bone marrow by collecting bone marrow aspirates in sterile tubes containing 5 10 ml of iscove's modified Dulbecco's medium (IMDM) plus 400 units of preservative-free hepa ⁇ n. Cells are centnfuged at 600 1,000 x g for 10 mm, the supernatant is discarded and the cells are resuspended in IMDM plus 2% fetal calf serum (FCS), then mixed gently by pipetting. Cells are counted in a hemacytometer and the concentration is adjusted as needed.
  • IMDM iscove's modified Dulbecco's medium
  • FCS fetal calf serum
  • the following protocol details the steps for the assay of Mix-CFC from human bone marrow.
  • This assay allows the growth of mixed colonies, which may contain several myeloid lineages (neutrophils, eosinophils, basophiis, erythroid cells, monocytes-macrophages and megakaryocytes) resulting from clonal proliferation of Mix CFC.
  • Three aliquots of 1 ml containing 5 x 10" human bone marrow cells each each are placed in 3 cm diameter Pet ⁇ dishes.
  • the plating mixture is made as follows, to a total value of 3.3 ml (to allow 0.3 ml for waste):
  • the agar (3.3%) is placed into a boiling water bath to melt. While the agar is melting, the plating mixture may be warmed to 37 C in a water bath to prevent the agar from setting too quickly when added.
  • Agar (0.30 ml) is added, mixed thoroughly but gently, and 1 ml is plated per dish Dishes are placed on a tray and allowed to set. The dishes can be placed in a refrigeration for about 2 mm to speed the process The plates are then placed into a fully humidified gas incubator at 37 C and incubated for 14 days. Colonies are scored under about 40X magnification using a microscope with a zoom lens, or an inverted microscope. Individual colonies are picked up for cytological examination using a Pasteur pipette and suspended in 0.1 ml of medium (plus a source of protein, either 1 % serum or 0.1 % BSA, for standard cytospin preparations.
  • IL-3 dependent cell lines can be employed to determine whether a peptide has IL 3 activity.
  • the cell lines FDCP 1, FDCP 2, 32DCL23, TF 1 (human erythroleukemia), AML 193 (human acute myeloid leukemia; ATCC CRL 9589); M0-7e (human megakaryoblastic leukemia) and DAM1 (human megakaryoblastic cells; Chen et al., Br. J. Hae atol. 88:481-490, 1994) are all IL-3-dependent.
  • a particular Rl IL-3-der ⁇ ved peptide having between 12 and about 40 ammo acids, and including the sequence shown in SEQ ID NO: 1, has IL-3 activity the peptide or analog thereof such as a peptide having one or more conservative am o acid replacements, is assayed for IL-3 activity by incubation with cells as follows
  • Cells are cultured in suspension until they reach an exponential growth rate, then harvested by spinning at 800 x g for 5 minutes on a bench centrifuge using a swing out rotor The cell pellet is suspended in the appropriate medium, centnfuged again, and resuspended a further two times Cells are resuspended at a concentration of 1 2 x 10 6 /ml in Fischer's medium (Gibco-plus glutamme) plus horse serum (10% v/v) and plated out in a total volume of 100 I at trial concentrations of 1-2 x 10 6 cells/ml with either dilutions of the Rl IL-3 derived peptide under test or dilutions of a standard preparation of IL-3.
  • Fischer's medium Gibco-plus glutamme
  • horse serum 10% v/v
  • the final concentration of horse serum (or, if the cells are normally cultured in it, fetal calf serum) is 10 20% (v/v). Cells are incubated in a C0 2 incubator at 37 C. After 24 or 48 hours, the effects of the growth factors on survival and proliferation are assessed using the
  • Trypan blue exclusion assay in which the cell suspension is mixed 1:1 with Try pan blue solution and the viable cells which exclude the dye are counted
  • Another method for assessment of cell proliferation involves the measurement of [ 3 H]thym ⁇ d ⁇ ne incorporation. After 24 or 48 hours, [ 3 H]thym ⁇ d ⁇ ne (37 kBq) is added to each well and the incubation continued for 4 hours. The cells are removed from the incubator and serially transferred to GF/C filters on a Milhpore cell harvester or similar apparatus. The cells are washed three times with t ⁇ chloroacetic acid (TCA) and the TCA precipitable material retained on the filter counted in a liquid scintillation counter. The relative growth promoting activities of the standard and the diluents of the Rl IL 3 derived peptides are compared to quantify the growth promoting activity of the Rl IL 3 derived peptides.
  • TCA t ⁇ chloroacetic acid

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Abstract

L'invention concerne des peptides rétro-inverso dérivés de l'interleukine-3 (IL-3), qui possèdent entre 12 et 40 acides aminés et comprennent la séquence montrée en SEQ ID NO: 1. Ces peptides possèdent la même activité que l'interleukine-3 (IL-3), d'origine et également l'activité neurotrophique. Grâce à la liaison D amino-acide présente dans ces peptides, ceux-ci sont moins susceptibles de subir une dégradation protéolytique in vivo.
EP00939960A 1999-06-16 2000-06-16 Peptides retro-inverso derives de l'interleukine-3 Withdrawn EP1185552A1 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US13968899P 1999-06-16 1999-06-16
US139688P 1999-06-16
PCT/US2000/016759 WO2000077028A1 (fr) 1999-06-16 2000-06-16 Peptides retro-inverso derives de l'interleukine-3

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US5571787A (en) 1993-07-30 1996-11-05 Myelos Corporation Prosaposin as a neurotrophic factor
CA2492542A1 (fr) * 2002-07-30 2004-02-05 Stem Cell Therapeutics Inc. Production d'oligodendrocyte a partir de cellules souches neurales multipotentes
WO2004035086A2 (fr) 2002-10-16 2004-04-29 Hunter Samuel F Technique de traitement par demyelinisation de maladie du systeme nerveux central
US7695723B2 (en) * 2002-12-31 2010-04-13 Sygnis Bioscience Gmbh & Co. Kg Methods of treating neurological conditions with hematopoietic growth factors

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US5700909A (en) * 1993-07-30 1997-12-23 The Regents Of The University Of California Prosaposin and cytokine-derived peptides
US6271196B1 (en) * 1996-03-05 2001-08-07 Regents Of The University Of Ca Methods of alleviating neuropathic pain using prosaposin-derived peptides
CA2282827A1 (fr) * 1997-03-05 1998-09-11 The Regents Of The University Of California Procedes pour soulager les douleurs neuropathiques
US6458357B1 (en) * 1997-09-09 2002-10-01 Myelos Corporation Retro-inverso neurotrophic and analgesic peptides

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CA2376394A1 (fr) 2000-12-21
JP2003502340A (ja) 2003-01-21
AU5496500A (en) 2001-01-02
IL147093A (en) 2011-04-28
JP2011137019A (ja) 2011-07-14
WO2000077028A1 (fr) 2000-12-21
IL147093A0 (en) 2002-08-14

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