WO2000077033A1 - Peptides rétro-inverses dérivés de l'interleukine 6 - Google Patents

Peptides rétro-inverses dérivés de l'interleukine 6 Download PDF

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Publication number
WO2000077033A1
WO2000077033A1 PCT/US2000/040227 US0040227W WO0077033A1 WO 2000077033 A1 WO2000077033 A1 WO 2000077033A1 US 0040227 W US0040227 W US 0040227W WO 0077033 A1 WO0077033 A1 WO 0077033A1
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Prior art keywords
peptide
seq
sequence shown
ammo
mammal
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PCT/US2000/040227
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English (en)
Inventor
David E. Wright
D. Elliot Parks
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Myelos Corporation
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Publication date
Priority to IL14709400A priority Critical patent/IL147094A0/xx
Priority to EP00952761A priority patent/EP1183267A4/fr
Priority to CA002376479A priority patent/CA2376479A1/fr
Priority to JP2001503890A priority patent/JP4786843B2/ja
Priority to AU65405/00A priority patent/AU6540500A/en
Priority to US10/048,305 priority patent/US7135461B1/en
Application filed by Myelos Corporation filed Critical Myelos Corporation
Publication of WO2000077033A1 publication Critical patent/WO2000077033A1/fr
Priority to IL147094A priority patent/IL147094A/en
Priority to US11/544,507 priority patent/US7754688B2/en
Priority to IL190034A priority patent/IL190034A0/en
Priority to US12/751,192 priority patent/US8063016B2/en
Priority to US13/284,617 priority patent/US20120172305A1/en

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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]
    • C07K14/5412—IL-6
    • 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
    • A61P25/02—Drugs for disorders of the nervous system for peripheral neuropathies
    • 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
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A61P25/16—Anti-Parkinson drugs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/02—Linear peptides containing at least one abnormal peptide link
    • 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 inverso peptides derived from interleukin 6 (IL 6). These peptides have activities similar to that of the native parent protein, and also have neurotrophic activity.
  • IL 6 interleukin 6
  • IL 6 is a multifunctional cytokme having a molecular weight of 26 kDa which is produced by both lymphoid and non-lymphoid cells and regulates immune responses, acute-phase reactions and hematopoiesis.
  • 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 Diego, CA, 1998, and in Barton, Clm Immunol. Immunopathol. 85:16 20, 1997. Because many cells are capable of both producing and responding to IL 6, it is capable of being an autocnne regulator of growth and/or differentiation in many systems. Within the immune system, it has been shown to be an autocnne activator of peripheral
  • IL 6 is also involved in T cell activation, growth and differentiation.
  • IL-6 induces IL 3 receptor (Tac antigen) expression in one T cell line (Noma et al , 1987) and in thymocytes, and functions as a second signal for IL 2 production by T cells (Garman et al , 1987).
  • IL 6 promotes the growth of human T cells stimulated with PHA or mouse peripheral T cells.
  • IL 6 also inhibits several key inflammatory responses including the synthesis of LPS induced IL 1 and TNF in vitro and in vivo (Aderka et al., J. Immunol. 143:3517 3523, 1989, Ulich et al., J. Immunol. 146:2316-2323, 1991).
  • IL-6 has also been found to protect against lung damage in disease models of pulmonary inflammation (Chen et al., Infect. Immun.
  • Neurotrophms and neurotrophic factors are proteins or peptides capable of affecting the survival, target innervation and/or function of neuronal cell populations (Barde, Neuron, 2:1525 1534, 1989)
  • ciliary neurotrophic factor CNTF
  • CNTF ciliary neurotrophic factor
  • Retro inverso 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 (inverso).
  • retro inverso isomers of linear peptides in which only some of the peptide bonds are reversed and the chirality of the ammo acid residues in the reversed portion is inverted.
  • Chorev et al. [ibid.) showed that retro-inversion of a peptide which inhibits binding of vitronectin to its receptor resulted in one peptide which was less potent than the parent isomer by a factor of 50,000, and another peptide which was 4,000 fold more potent than the parent cyclic peptide.
  • Guichard et al. ( TIBTECH 14, 1996), teach that retro inverso (all D retro) antigenic mimicry may only occur with peptides in random coil, loop or cyclic conformations. In the case of "helical" peptide, adequate functional mimicry would be expected only if the he city was, in fact, absent under the solvent conditions used for assessing antigenic mimicry
  • One embodiment of the present invention is an isolated retro inverso peptide having between 17 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.
  • 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 carboxy c acid groups in the peptide is estenfied.
  • the peptide has the ammo acid sequence shown in SEQ ID NO: 1.
  • compositions 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, and a pharmaceutically acceptable carrier.
  • the present invention also provides a method for promoting neunte outgrowth or mye nation in a mammal in need thereof, comprising the step of administering to the mammal an effective, neunte outgrowth or myehnation facilitating amount of a composition comprising a retro inverso 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.
  • the mammal is a human
  • the administering step is direct local injection, systemic, intracranial, intracerebrospmal, topical or oral
  • the present invention also provides a method for promoting T cell activation, comprising contacting T cells with an effective, T cell activation promoting amount of a composition comprising a retro inverso peptide having between 17 and about 40 ammo acids, wherein the peptide includes the sequence shown in SEQ ID NO. 1.
  • a retro inverso peptide having between 17 and about 40 ammo acids, wherein the peptide includes the sequence shown in SEQ ID NO. 1 , for use in promoting neunte outgrowth or myehnation 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 inverso peptide having between 17 and about 40 ammo acids, wherein the peptide includes the sequence shown in SEQ ID NO 1 , for use in promoting T cell activation in a mammal in need thereof
  • the peptide has the sequence shown in SEQ ID NO. 1
  • the mammal is a human.
  • Still another embodiment of the present invention is the use of a retro inverso 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 promoting neunte outgrowth or myehnation in a mammal in need thereof.
  • the peptide has the ammo acid sequence shown in SEQ ID NO 1
  • the mammal is a human
  • the present invention also provides the use of a retro inverso peptide having between 17 and about 40 am o acids, wherein the peptide includes the sequence shown in SEQ ID NO: 1, in the preparation of a medicament for promoting T cell activation in a mammal in need thereof.
  • the peptide has the ammo acid sequence shown in SEQ ID N0'1.
  • the mammal is a human.
  • the present invention provides retro inverso (Rl) peptides derived from interleukin 6 (IL 6) which mediate similar effects to native IL 6, including regulation of immune responses, acute phase reactions and hematopoiesis.
  • IL 6 interleukin 6
  • the term "derived from” indicates that the peptides include the active region of interleukin 6, or analogs thereof as defined below.
  • Rl IL 6 derived peptides also activate peripheral T and NK cells, promote IgG secretion by activated B cells, induce the liver to produce acute phase proteins, promote growth of human T cells, inhibit inflammatory responses including synthesis of hpopolysaccharide (LPS) induced IL 1 and tumor necrosis factor (TNF)
  • LPS hpopolysaccharide
  • TNF tumor necrosis factor
  • IL 6 derived peptides are also useful in mediating similar effects to native IL 6
  • 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 6 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 6-der ⁇ ved peptides have the same activities as the corresponding full length IL-6 protein, and also possess neurotrophic and myelinotrophic activity.
  • One embodiment of the present invention is a method for promoting T cell activation by administering to T cells an effective, T cell activating amount of an Rl peptide having between 17 and about 40, ammo acids, and encompassing the Rl IL 6 derived 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 argmine residues with alanme or another a mo acid; deletion of one or more lysine and/or argmine residues; replacement of one or more tyrosme and/or phen ⁇ lalamne 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 lysme/argmine and t ⁇ rosine/phen ⁇ lalanme residues will reduce the susceptibility of peptide degradation by trypsin and chymotrypsm, respectively.
  • peptide sequences contemplated for use in the present invention include minor insertions and deletions Conservative am o 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.
  • ammo acids include the basic charged ammo acids (lysine, argmine, histidine); the acidic charged ammo acids (aspartic acid, glutamic acid); the non-polar ammo acids (alanme, valme, leucine, isoleucme, prohne, phenylalanme, methionme, tryptophan); the uncharged polar ammo acids (glyci ⁇ e, asparagme, glutamine, cysteme, senne, threonine, tyrosme); and the aromatic ammo acids (phenylalanme, tryptophan and tyrosme).
  • the basic charged ammo acids lysine, argmine, histidine
  • the acidic charged ammo acids aspartic acid, glutamic acid
  • non-polar ammo acids alanme, valme, leucine, isoleucme, prohne, phenylalanme, methionme, tryptophan
  • Various chemical modifications will improve the stability, bioactivity and ability of the peptide to cross the blood brain barrier.
  • One such modification is 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 amine/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 neunte outgrowth in differentiated or undifferentiated neural cells by administering to the cells an effective, neunte outgrowth facilitating amount of a Rl peptide having between 17 and about 40 ammo acids, and encompassing the Rl IL 6-der ⁇ ved peptide shown in SEQ ID NO: 1 , or analogs thereof which have similar activity as described above.
  • Rl IL 6-der ⁇ ved peptide to mediate the same activity of native IL 6 can be determined using standard assays for the parent peptide as discussed in Examples 10 12.
  • 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 T cells, B cells 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 cultunng 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 argmine 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).
  • direct intracramal injection or injection into the cerebrospmal fluid may also be used in sufficient quantities to give the desired local concentration of neurotrophm
  • 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, intracramal, epidural, mtrathecal, topical and oral.
  • administration by direct intravenous injection is preferred.
  • 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 lyophihzed 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 6 effects such as T cell activation, or for treatment of neurodegenerative diseases or demyehnation diseases are in the range of from about 0.01 to about 10,000 g/kg.
  • 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, pol ⁇ galactic acid, regenerated collagen, multilamellar hposomes and many other conventional depot formulations comprise bioerodible or biodegradable materials that can be formulated with biologically active neurotrophic peptide compositions.
  • peptides 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 combination with transdermal delivery devices are also contemplated
  • the peptides may also be encapsulated within a polyethylene glycol co ⁇ formal coating as described in U.S. Patent No. 5,529,914 prior to implantation.
  • 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., Nleth 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
  • 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 It has long been believed that in order to reach neuronal populations in the brain, neurotrophic factors would have to be administered intracerebrally since these proteins do not cross the blood brain barrier.
  • U.S. Patent No. 5,571,787 discloses that an lodmated neurotrophic 18-mer fragment derived from saposm C crosses the blood brain barrier.
  • the Rl peptides having up to about 22 ammo acids will also cross this barrier and can thus be administered intravenously
  • Other neuronal populations, such as motor neurons, can also be treated by intravenous injection, although direct injection into the cerebrospmal fluid is also envisioned as an alternate route
  • Cells may be treated to facilitate mye n formation or to prevent demye nation in the manner described above in vivo, ex vivo or in vitro Diseases resulting in demyehnation of nerve fibers including MS, acute disseminated leukoencephahtis, trauma to brain and/or spinal cord, progressive multifocal leukoencephalitis, metachromatic leukodystrophy, adrenal leukodystrophy and maldevelopment of the white matter in premature infants (penventncular 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 6 derived peptide compositions of the present invention can also be used to support T cell activation, to enhance the survival of cultured motor neurons and to determine the effects of neurotrophic factors and mye n facilitating materials. However, more practically, they have an immediate use as laboratory reagents and components of cell growth media in order to facilitate growth and maintain T cells and neural cells in vitro.
  • 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 neunte outgrowth NS20Y neuroblastoma cells are grown in DMEM containing 10% fetal calf serum (FCS) Cells are removed with trypsm and plated in 30 mm pet ⁇ 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 an Rl IL 6-der ⁇ ved peptide having between 17 and about 40 ammo acids, and including the sequence shown in SEQ ID NO: 1.
  • FCS fetal calf serum
  • Example 2 Prevention of cell death 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 ng/ml of an Rl IL 6 derived peptide having between 17 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%
  • Dorsal root ganglia are removed from adult rats and sensory neurons were prepared as described by Kuffler et al. (J. Neurobiol. 25:1267-1282, 1994) Neurons are treated with 0.5 ng/ml of an Rl IL 6 derived peptide having between 17 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 neu ⁇ tes in neurons treated with Rl peptide are approximately three times longer than those treated with a control (non Rl) 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).
  • CFA complete Freund's adjuvant
  • the amount of cholesterol ester in brain is scored at day 22.
  • the stride length of both groups is decreased at day 14, whereas after treatment for 8 days, the IL 6-der ⁇ ved peptide-treated animals return to normal, but the vehicle treated animals do not.
  • a significant reduction of cholesterol ester content is observed in the brains of the treated group.
  • the number of spinal cord lesions is significantly reduced after 10 days of treatment with IL-6-der ⁇ ved peptide.
  • the average lesion size is significantly reduced.
  • Example 5 Ex vivo myehnation assay Newborn mouse cerebellar explants are prepared according to Satomi [Zool. Sci 9.127-137, 1992). Neunte 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 6 derived peptide having between 17 and about 40 am o 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 neu ⁇ tes and myehnation are assessed under a bright field microscope with a video camera.
  • Saposin C is used as a positive control at a concentration of between about 1 and 10 g/ml Myehnation is stimulated by the IL 6 derived peptides to a similar extent as with saposin C
  • myehnation may be assayed by incorporation of 35 S into sulfohpids which are exclusive to myehn as described below.
  • Example 6 Incorporation of 35 S into sulfohpids
  • Primary myehn containing Schwann cells are incubated in low sulf ate media (DMEM) containing 0.5% fetal bovine serum (FBS), followed by addition of 35S -meth ⁇ on ⁇ ne and an Rl IL 6 derived peptide having between 17 and about 40 ammo acids, and including the sequence shown in SEQ ID NO 1 Saposin C is used as a positive control.
  • DMEM low sulf ate media
  • FBS fetal bovine serum
  • Example 7 Use of Rl peptides in treating traumatic ischemic CNS lesions
  • Humans with traumatic lesions to the brain or spinal cord receive systemic injections of about 100 g/kg of an Rl IL-6 derived peptide having between 17 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 8 Use of Rl peptides in treating demyehnation disorders Patients diagnosed with early stage MS are given an Rl IL 6 derived peptide having between 17 and about 40 ammo acids, and including the sequence shown in SEQ ID NO- 1, by systemic injection using the same dose range as in 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.
  • Example 9 Use of Rl peptides in treating demyehnation disorders Patients diagnosed with early stage MS are given an Rl IL 6 derived peptide having between 17 and about 40 ammo acids, and including the sequence shown in SEQ ID NO- 1, by systemic injection using the same dose range as in 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
  • the IL 6 dependent murine hybridoma cell line B9 provides a reliable and sensitive assay for measuring mammalian IL-6.
  • B9 cells are cultured in RPMI 1640 medium supplemented with 5% FCS and approximately 100 pg/ml (10 lU/ml) of IL-6 in 75 cm 2 flasks.
  • Cultures are split 1 :5 to 1 :10 every 2 to 3 days and refed with IL 6 when the cell density reaches approximately 5 x 10 5 cells/ml Cultures are maintained at 37 C in a humidified C0 2 incubator. B9 cells are washed 2 days after feeding and resuspended to a density of 5 x 10" cells/ml in RPMI 1640 medium supplemented with 5% FCS.
  • An IL 6 standard is distributed as a serial two fold dilution series in triplicate in 100 I volumes in 96-well icrotitration plates. The titration of standard is started at 100 pg/ml (10 lU/ml) and diluted down to 0.1 pg/ml (0.01 lU/ml).
  • Appropriate dilutions of an Rl IL-6 derived peptide having between 17 and about 40 ammo acids, and including the sequence shown in SEQ ID NO. 1, to be measured for IL-6 activity are made in triplicate in 100 I volumes.
  • As a negative control culture medium is included alone Cell suspension (100 I) is added to each well and the plates are incubated for about 72 hours at 37 C in a humidified C0 2 incubator. The tetrazo um salt MTT (10 I) is added to each well and the plates are incubated for an additional 4 5 hours.
  • Acid sodium dodecyl sulfate (SDS) 25 I
  • SDS Acid sodium dodecyl sulfate
  • 25 I Acid sodium dodecyl sulfate
  • the plates are incubated at 37 C in a humidified C0 2 incubator overnight and the absorbance is determined at 620 nm using a plate reader.
  • a standard curve of absorbance versus concentration of IL 6 is plotted.
  • test results are compared with the standard curve to determine whether the particular peptide has IL 6 activity.
  • Example 10 IL-6 IgG secretion assay IL 6 can be assayed by its ability to enhance differentiation and IgG secretion in EBV-transformed human lymphoblastoid cell lines such as CESS (ATCC TIB 190).
  • CESS cells are harvested from a vigorous log phase growth culture. The cells are subcultured for 24-48 hours beforehand. Cultures which contain many dead cells, or are growing slowly, will not perform well in this assay. Cells are washed once and resuspended to 10 6 cells/ml in culture medium. Cells (100 I) are added to give a final cell concentration in six replicate microtiter wells ranging from 10 3 to 10 s cells/ml.
  • IL-6 100 I
  • Rl IL 6 derived peptide having between 17 and about 40 ammo acids, and including the sequence shown in SEQ ID NO 1
  • medium is added to one set of six wells as a negative control.
  • Cells are incubated for 5 7 days at 37 C in an atmosphere of 5% C0 2 in air.
  • Cell supernatants are harvested and assayed for immunoglobuhn content.
  • Affinity purified goat anti-human IgG (75 I) is dispensed in bicarbonate coating buffer and incubated at room temperature overnight. Individual batches of antisera are pre titrated to determine the optimal signal to noise ratio with high, medium and low concentrations of IgG. Wells are washed three times with PBS/Tween-20. To block remaining protein binding sites, 100 I PBS/BSA/Tween 20 is added to each well and incubated at room temperature for 30-90 minutes. Normal goat serum (4%) diluted in PBS can also be used in this step. Wells are washed three times with PBS/Tween, and 75 I test supernatant and standards are added in duplicate.
  • An 1 1 point standard curve using doubling dilutions of either pooled normal human serum, or partially purified IgG at 1,000 ng/ml in PBS/BSA/Tween-20 is set up in duplicate on each plate along with a buffer only zero standard. Plates are incubated at room temperature for 1 -2 hours. Wells are washed three times with PBS/Tween-20. To each well is added 75 I of horseradish peroxidase (HPO) of alkaline phosphatase (AP)-conjugated affinity purified goat anti-human IgG diluted in PBS/BSA/Tween-20. Individual batches of antisera are titrated to determine the optimal dilution.
  • HPO horseradish peroxidase
  • AP alkaline phosphatase
  • Macrophages are activated by the addition of bacterial lipopolysaccharide (LPS), resulting in release of TNF into the culture medium which can be assayed using an enzyme linked immunosorbent assay (ELISA).
  • LPS bacterial lipopolysaccharide
  • ELISA enzyme linked immunosorbent assay
  • IL-6 is known to inhibit the LPS induced release of TNF (Aderka et al., J. Immunol. 143:3517-3523, 1989). In cultures treated with the IL-6-de ved peptide prior to LPS stimulation, the amount of TNF released is significantly reduced compared to cultures not given the peptide.

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  • Biomedical Technology (AREA)
  • Public Health (AREA)
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  • Immunology (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Gastroenterology & Hepatology (AREA)
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  • Peptides Or Proteins (AREA)

Abstract

Ces peptides rétro-inverses dérivés de l'interleukine 6 (IL-6) possèdent entre 17 et 40 acides aminés environ et renferment la séquence illustrée par SEQ ID N°: 1. Ces peptides, qui ont la même activité que l'IL-6 endogène, ont également une activité neurotrophique. La présence de la liaison génétique aminoacide de type D dans ces peptides les rend moins sujets à un dégradation protéolytique in vivo.
PCT/US2000/040227 1999-06-16 2000-06-16 Peptides rétro-inverses dérivés de l'interleukine 6 WO2000077033A1 (fr)

Priority Applications (11)

Application Number Priority Date Filing Date Title
EP00952761A EP1183267A4 (fr) 1999-06-16 2000-06-16 Peptides r tro-inverses d riv s de l'interleukine 6
CA002376479A CA2376479A1 (fr) 1999-06-16 2000-06-16 Peptides retro-inverses derives de l'interleukine 6
JP2001503890A JP4786843B2 (ja) 1999-06-16 2000-06-16 インターロイキン−６から誘導されるレトロ−インベルソペプチド
AU65405/00A AU6540500A (en) 1999-06-16 2000-06-16 Retro-inverso peptides derived from interleukin-6
US10/048,305 US7135461B1 (en) 2000-06-16 2000-06-16 Retro-inverso peptides derived from interleukin-6
IL14709400A IL147094A0 (en) 1999-06-16 2000-06-16 Retro-inverso peptides derived from interleukin-6
IL147094A IL147094A (en) 1999-06-16 2001-12-13 Retro-inverso peptides derived from interleukin-6
US11/544,507 US7754688B2 (en) 1999-06-16 2006-10-05 Methods of using retro-inverso peptides derived from interleukin-6
IL190034A IL190034A0 (en) 1999-06-16 2008-03-09 Use of retro-inverso peptides derived from interleukin-6
US12/751,192 US8063016B2 (en) 1999-06-16 2010-03-31 Retro-inverso peptides derived from interleukin-6
US13/284,617 US20120172305A1 (en) 1999-06-16 2011-10-28 Retro-inverso peptides derived from interleukin-6

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US13968799P 1999-06-16 1999-06-16
US60/139,687 1999-06-16

Related Child Applications (2)

Application Number Title Priority Date Filing Date
US10048305 A-371-Of-International 2000-06-16
US11/544,507 Continuation US7754688B2 (en) 1999-06-16 2006-10-05 Methods of using retro-inverso peptides derived from interleukin-6

Publications (1)

Publication Number Publication Date
WO2000077033A1 true WO2000077033A1 (fr) 2000-12-21

Family

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Application Number Title Priority Date Filing Date
PCT/US2000/040227 WO2000077033A1 (fr) 1999-06-16 2000-06-16 Peptides rétro-inverses dérivés de l'interleukine 6

Country Status (6)

Country Link
EP (1) EP1183267A4 (fr)
JP (1) JP4786843B2 (fr)
AU (1) AU6540500A (fr)
CA (1) CA2376479A1 (fr)
IL (3) IL147094A0 (fr)
WO (1) WO2000077033A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7524818B2 (en) 1993-07-30 2009-04-28 Myelos Corporation Prosaposin as a neurotrophic factor

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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
WO1998039357A1 (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

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
ARCOLEO ET AL.: "Effect of partially modified retro-inverso analogues derived from C-reactive protein on the induction of nitric oxide synthesis in peritoneal macrophages", BRITISH JOURNAL OF PHARMACOLOGY, vol. 120, 1997, pages 1383 - 1389, XP002933304 *
OSTANKOVITCH ET AL.: "A partially modified retro-inverso pseudopeptide modulates the cytokine profile of CTL specific for an influenza virus epitope", THE JOURNAL OF IMMUNOLOGY, vol. 161, 1998, pages 200 - 208, XP002933305 *
See also references of EP1183267A4 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7524818B2 (en) 1993-07-30 2009-04-28 Myelos Corporation Prosaposin as a neurotrophic factor

Also Published As

Publication number Publication date
CA2376479A1 (fr) 2000-12-21
EP1183267A1 (fr) 2002-03-06
AU6540500A (en) 2001-01-02
JP2003502343A (ja) 2003-01-21
EP1183267A4 (fr) 2002-09-18
IL147094A0 (en) 2002-08-14
JP4786843B2 (ja) 2011-10-05
IL190034A0 (en) 2011-08-01
IL147094A (en) 2011-04-28

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