JP2011144184A - Retro-inverso peptide derived from leukemia inhibitory factor - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a peptide having the same activity as native LIF (leukemia inhibitory factor), and also having neurotrophic activity. <P>SOLUTION: This invention relates to retro-inverso peptides derived from LIF having between 18 and about 40 amino acids, wherein the retro-inverso peptides include a specific amino acid sequence, and are less susceptible to proteolytic degradation in vivo because of their D-amino acid linkage, and are thus stable. This invention also relates to a composition including the retro-inverso peptides and a pharmaceutically acceptable carrier. This invention further relates to a method for promoting neurite outgrowth or myelination, including the step of administering the composition to a mammal. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to retro-inverso peptides derived from leukemia inhibitory factor (LIF). The peptides have activity similar to the natural parent protein and also neurotrophic activity.

  Cytokines are proteins that are produced during the innate and specific immune effector phases and serve to mediate and control immune and inflammatory responses. Cytokines, like other polypeptide hormones, initiate their action by binding to specific receptors on the surface of target cells. One of the best known cytokine families is interleukins that mediate innate immunity. For details on the structure and function of interleukins, see Abbas et al. Cellular and Molecular Immunology, W.B. Saunders Company, Philadelphia, pp. 225-243, 1991.

  Leukemia inhibitory factor (LIF), named after its activity to inhibit the proliferation of myeloid leukemia cell lines by inducing differentiation, is IL-6, Oncostatin M, Ciliary neurotrophic factor (CNTF) and Cardio It is a member of the ligand family that includes trophin-1 (Gearing, Adv. Immunol. 53: 31-58, 1993; Pennica et al., J. Biol. Chem. 270: 10915-10922, 1995; Patterson, Proc. Natl. Acad. Sci. USA 91: 7833-7835, 1994). These cytokines have very limited sequence homology, but exert very similar effects on various tissues. For example, several of the above proteins, including LIF, induce the same set of acute phase response proteins in the liver, support autonomous regeneration of cultured embryonic stem cells, inhibit lipid biosynthesis, and in cultured motor neurons Survival can be promoted. LIF is produced by various cell groups such as macrophages, synoviocytes and chondrocytes. When applied to peripheral nerves in vivo, LIF is transported backwards and helps damaged sensory neurons (Hendry et al., J. Neurosci. 12: 3427-3434, 1992; Cheema et al., J. Neurosci. Res. 37: 213-218, 1994). LIF also regulates the proliferation and differentiation of osteoblasts and endothelial cells. Increased vesicular fluid LIF levels around the ovulation phase indicate that LIF may play a role in ovulation, early embryogenesis and implantation (Senturk et al., Am. J. Reprod. Immunol). 39: 144-151, 1998; Stewart, Annals NY Acad. Sci. 157-165).

  Neurotrophins and neurotrophic factors are proteins or peptides that can affect the survival of neuronal populations, neural connectivity to targets and / or function (Barde, Neuron, 2: 1525-1534, 1989). The effects of neurotrophins both in vivo and in vitro are well documented. For example, ciliary neurotrophic factor (CNTF) promotes the survival of chick embryo ciliary ganglia in vitro and supports the survival of cultured sympathetic, sensory and spinal motor neurons ( Ip et al., J. Physiol. Paris, 85: 123-130, 1991).

  A major obstacle to the in vitro therapeutic use of peptides is that they are susceptible to proteolysis. A retro-inverso peptide is an isomer of a linear peptide whose sequence direction is opposite (retro) and whose chirality, D or L is reversed (inverso). There are also partially modified linear peptide isomers in which only some peptide bonds are in the opposite direction and the chirality of the amino acid residues in the opposite direction portion is reversed. The main advantage of such peptides is that their in vivo activity is enhanced by increased resistance to proteolysis (for review, Chorev et al., Trends Biotech., 13: 438-445, 1995). . Such retro-inverso analogs show increased metabolic stability, but their biological activity is often greatly reduced (Guichard et al., Proc. Natl. Acad, Sci. USA, 91: 9765-9769, 1994). ).

For example, Richman et al. (J. Peptide Protein Res., 25: 648-662) found that linear and cyclic leu-enkephalin analogs with modified amide bonds in Gly ³ -Phe ⁴ are 6-14 of natural leu-enkephalin. % Activity was determined. Chorev et al. (Supra) described one peptide that is 50,000 times less active than the parent isomer and 4000 than the parent cyclic peptide by retro-inversification of the peptide that inhibits the binding of vitronectin to its receptor. It was shown that another peptide with twice the activity was obtained. Guichard et al. (TIBTECH 14, 1996) state that retro-inverso (all D-retro) antigenic mimicry can only occur in peptides with random coils, loops or cyclic structures. In the case of a “spiral” peptide, an appropriate functional mimetic would be expected only if the helix is virtually lost under the solvent conditions used to evaluate the antigen mimetic.

  There is a need for a peptide derived from LIF and having neurotrophic activity that has increased metabolic stability but retains biological activity. The present invention solves this need.

One embodiment of the present invention is an isolated retro-inverso peptide having from 18 to about 40 amino acids and comprising the sequence shown in SEQ ID NO: 1. In one preferred embodiment, at least one basic charged amino acid in the sequence is replaced with a different basic charged amino acid. In another preferred embodiment, at least one acidic charged amino acid in the sequence is replaced with a different acidic charged amino acid. Advantageously, at least one nonpolar amino acid in said sequence is replaced by a different nonpolar amino acid. Preferably, at least one uncharged amino acid in the sequence is replaced with a different uncharged amino acid. In another preferred embodiment, at least one aromatic amino acid in the sequence is replaced with a different aromatic amino acid. Advantageously, the peptide is at its amino terminus, carboxy terminus, or both terminus, CH ₃ CO, CH ₃ (CH ₂ ) _n CO, C ₆ H ₅ CH ₂ CO and H ₂ N (CH ₂ ). _n CO, modified with a moiety independently selected from the group consisting of n = 1-10. Preferably, the peptide is glycosylated. In another preferred embodiment, one or more amide bonds of the peptide are reduced. Preferably, one or more nitrogens of the peptide are methylated. In yet another preferred embodiment, one or more carboxylic acid groups of the peptide are esterified. Preferably, the peptide has the amino acid sequence of SEQ ID NO: 1.

Another aspect of the invention is a composition comprising a retro-inverso peptide having from 18 to about 40 amino acids and comprising the sequence of SEQ ID NO: 1 and a pharmaceutically acceptable carrier. .
The invention also provides a method for promoting neurite outgrowth or myelination in a mammal in need thereof. The method comprises an effective amount of a composition comprising a peptide having 18 to about 40 amino acids and comprising the sequence of SEQ ID NO: 1, in an amount that promotes neurite growth or myelination. , Comprising the step of administering to the mammal. Preferably, the peptide has the amino acid sequence of SEQ ID NO: 1. Advantageously, the mammal is a human. In one preferred embodiment, the course of administration is direct local injection, systemic, intracranial, intracerebral spinal, topical or oral administration.

Another aspect of the invention has from 18 to about 40 amino acids and comprises the sequence of SEQ ID NO: 1 for use in promoting neurite outgrowth or myelination in a mammal in need thereof. It is a retro-inverso peptide. Preferably, the peptide has the amino acid sequence of SEQ ID NO: 1. Advantageously, the mammal is a human.
The present invention also includes 18 to about 40 amino acids and a sequence of SEQ ID NO: 1 for preparing a medicament for promoting neurite growth or myelination in a mammal in need thereof The use of retro-inverso peptides is provided. The peptide of claim 21 having the amino acid sequence of SEQ ID NO: 1 is useful. Preferably, the mammal is a human.

  The present invention is a retro-inverso (RI) peptide derived from leukemia inhibitory factor (LIF) that mediates effects similar to natural LIF, such as control of osteoblast and endothelial cell proliferation and differentiation The peptide is provided. The term “derived from” means that the peptide contains the active region of interleukin 6 or an analog thereof as defined below. These RI LIF-derived peptides induce the production of acute phase response proteins by the liver, support the self-renewal of cultured embryonic stem cells, inhibit lipid biosynthesis, promote the survival of cultured motor neurons, It controls the proliferation and differentiation of osteoblasts and endothelial cells. The ability of a particular LIF-derived retro-inverso peptide to mediate effects similar to its parent peptide can be determined by those skilled in the art by standard LIF tests, such as those described in Example 10.

  These RI LIF-derived peptides are also therapeutically applied in promoting functional recovery after injury, injury, ischemia, degenerative injury, and hereditary injury due to toxins to the peripheral and central nervous system Is done. These peptides are also useful to promote increased myelin formation and to counteract the effects of demyelinating diseases. The ability of any such peptide to stimulate neurite growth and myelination and to inhibit neuronal cell death can be readily determined by those skilled in the art by the methods described in Examples 1-9. it can. The use of these peptides will facilitate the treatment of various diseases because they are more stable and easier to synthesize than either natural or recombinant cytokines.

  Table 1 shows certain RI LIF-derived peptides of the present invention and the parent proteins from which they are derived. Its corresponding natural (non-retro-inverso) peptide is disclosed in US Pat. No. 5,700,909.

  As mentioned above, these RI LIF-derived peptides have the same activity as the corresponding full-length LIF and also have neurotrophic and myelinotropic activity. One aspect of the invention is a method of promoting neurite growth of differentiated or undifferentiated neurons, the method having 12 to about 40 amino acids and shown in SEQ ID NO: 1. By administering to the cell an RI peptide comprising an RI LIF-derived peptide, or an analog thereof having similar activity, in an effective amount, ie, an amount that promotes neurite growth. The analog includes, for example, one or more lysine and / or arginine residues substituted with alanine or another amino acid in the peptide; one or more lysine and / or arginine residues deleted Those in which one or more tyrosine and / or phenylalanine residues are substituted, those in which one or more phenylalanine residues are deleted, and those in which one or more amino acids are conservatively substituted. Substitution or deletion of lysine / arginine and tyrosine / phenylalanine residues will reduce the sensitivity of peptide degradation by trypsin and chymotrypsin, respectively.

  Further variations of these peptide sequences that can be used in the present invention also contemplate minor insertions and deletions. Conservative amino acid substitutions are possible. Such a substitution is, for example, a substitution performed in a group of amino acids related in the side chain chemistry. Amino acid groups include basic charged amino acids (lysine, arginine, histidine); acidic charged amino acids (aspartic acid, glutamic acid); nonpolar amino acids (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan); uncharged Polar amino acids (glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine); and aromatic amino acids (phenylalanine, tryptophan, tyrosine) are included. In particular, conservative amino acid substitutions consisting of a single substitution of leucine with isoleucine or valine, aspartic acid with glutamic acid or threonine with serine, or similar conservative substitutions of certain amino acids with structurally related amino acids It is generally accepted that it will not significantly affect the properties of Promotes neurite growth, myelination, suppresses and restores demyelination in the sequence of SEQ ID NO: 1, or any RI peptide comprising that sequence having an insertion, deletion or substitution, and neuronal cell death The ability to suppress this can be determined by the inspection shown in the following examples.

Various standard chemical modifications will improve the stability, biological activity, and ability to cross the brain blood barrier of the peptide. One such modification is an aliphatic amino terminal modification with a derivative of an aliphatic or aromatic acid to create an amide bond. Examples of the derivatives include CH ₃ CO, CH ₃ (CH ₂ ) _n CO (n = 1 to 10), C ₆ H ₅ CH ₂ CO, H ₂ N— (CH ₂ ) _n CO (n = 1 to 1). 10). Another modification is a modification of the carboxy terminus with an aliphatic or aromatic amine / alcohol derivative linked to the peptide via an amide / ester bond. Such derivatives include those listed above. The peptide may also have both amino-terminal and carboxy-terminal modifications, in which case the derivative is independently selected from those listed above. The peptide may be glycosylated, where either the alpha amino group or D-Asn, or both are modified with glucose or galactose. Another possible modification reduces the amide bond of the selected backbone (—NH—CH ₂ ). Other modifications include N-methylation of selected nitrogens within the amide bond and esterification by modifying at least one of the acid groups within the peptide as an aromatic or aliphatic ester. Any combination of the modifications is also contemplated.

  The typical minimum amount of a RI peptide of the invention required for LIF activity or neurotrophic activity in cell growth media is usually at least about 5 ng / ml. This amount or more of the RI peptide of the present invention is contemplated for in vitro use. Typically, the peptide will be used at a concentration of 0.1 g / ml to about 10 g / ml. An effective amount in a particular tissue can be determined according to Example 1.

  Cells can be treated in vitro or ex vivo by administering the RI peptides of the present invention directly to the cells. This can be done, for example, by culturing the cells in a growth medium suitable for that particular cell type and then adding the peptide to the medium. Where the cells to be treated are in vivo, typically in a vertebrate, preferably a mammal, the composition can be administered by several methods. Most preferably, the composition is injected directly into the blood in an amount sufficient to obtain the desired local peptide concentration. These RI peptides exist longer in vivo due to their D peptide bonds. Peptides lacking lysine and arginine residues have reduced proteolysis. Smaller peptides (ie 50 amino acids or less) will best cross the blood brain barrier and enter the central nervous system for the treatment of central nervous system diseases (Banks et al., Peptides, 13: 1289-1294, 1992).

  For the treatment of neurological disorders, direct injection into the skull or into the cerebrospinal fluid may be performed in an amount sufficient to obtain the desired local concentration of neurotrophin. In both cases, a pharmaceutically acceptable injectable carrier is used. Such carriers include, for example, phosphate buffered saline and Ringer's solution. Alternatively, the composition can be administered to peripheral nerve tissue by direct local injection or by systemic administration. Various traditional administration methods are contemplated, such as intravenous, intrapulmonary, intramuscular, intradermal, subcutaneous, intracranial, epidural, intradural, topical, and oral administration.

  A composition in which the peptide composition of the present invention is formulated and released as a unit dose such as an injectable composition or a topical formulation, at a dose equivalent to the dose administered to a patient per day, or in a controlled manner Can be administered as A sterile sealed vial containing a daily dose of the active ingredient in either a PBS solution or a lyophilized product is one example of a unit dose. In a preferred embodiment, the daily systemic dosage of the RI peptide of the present invention based on the vertebrate body weight required for the promotion of LIF-induced stroke, treatment of neurodegenerative disease or demyelinating disease is: It is in the range of about 0.01 to about 10,000 g / kg. More preferably, the daily systemic dosage is about 0.1 to 1000 g / kg. Most preferably, the daily systemic dosage is about 10-100 g / kg. The daily dose of topical agent will be almost an order of magnitude less. Oral administration is particularly preferred because of the resistance of the peptide to proteolysis in the gastrointestinal system.

  In one preferred embodiment of the present invention, the peptide is locally administered to nerve cells in vivo by transplantation. For example, polylactic acid, polygalactic acid, regenerated collagen, multilamellar liposomes and many other conventional retentive formulations can be formulated with biologically active neurotrophic peptide compositions. It comprises a bioerodible or biodegradable material that can. When implanted, these substances are gradually degraded and the active substance is released into the surrounding tissue. The present invention clearly contemplates bioerodible, biodegradable and other retention formulations. Combinations with infusion pumps, matrix capture systems, and transdermal delivery devices are also contemplated. The peptide can also be encapsulated in a polyethylene glycol structural coating prior to implantation as described in US Pat. No. 5,529,914.

  The RI LIF-derived peptide of the present invention can also be sealed in micelles or liposomes. Liposome encapsulation technology is well known. Liposomes can be targeted to a particular tissue, eg, to neural tissue, through the use of receptors, ligands or antibodies that can bind to the target tissue. The preparation of these compositions is well known in the art (Radin et al., Meth. Enzymol., 98: 613-618, 1983).

  Currently, there are no available drugs that can promote full functional regeneration and structural recovery of the nervous system. This is especially true in the central nervous system. The regeneration of peripheral nerves through the use of the neurotrophic factor is within the scope of the present invention. Furthermore, neurotrophic factors can be used therapeutically in the treatment of neurodegenerative diseases related to degeneration of nerve groups or specific regions in the brain. The main cause of Parkinson's disease is degeneration of nigral dopaminergic neurons. Since the anti-prosaposin antibody can immunohistochemically stain nigral dopaminergic neurons in human brain sections, the RI peptide of the present invention is therapeutically useful in the treatment of Parkinson's disease. I will. Retinal neuropathy, an ocular neurodegenerative disease that causes vision loss in the elderly, can also be treated with the RI peptides of the present invention.

  For a long time, it was believed that neurotrophic factors do not cross the brain blood barrier, so that they should be administered into the cerebrum to reach the nerve cells in the brain. US Pat. No. 5,571,787 discloses that an iodinated product of a neurotrophic 18-mer fragment derived from saposin C crosses the brain blood barrier. Thus, RI peptides with about 22 amino acids or less will also cross this barrier. It can therefore be administered intravenously. Other neuronal groups, such as motor neurons, can also be treated by intravenous injection. However, an alternative route may be direct injection into the cerebrospinal fluid.

  Cells can be treated in the methods described above in vivo, ex vivo or in vitro to promote myelin formation or to inhibit demyelination. Diseases leading to demyelination of nerve fibers such as MS, acute epidemic leukoencephalitis, trauma to the brain and / or spinal cord, progressive multifocal leukoencephalitis, metachromatic leukotrophy, adrenal leukotrophy, and White matter developmental disorder (periventricular leukomalacia) in premature infants can be delayed or stopped by administering the neurotrophic peptide of the present invention to cells affected by the disease.

The composition of the RI LIF-derived peptide of the present invention also supports the self-renewal of cultured embryonic stem cells, promotes the survival of cultured motor neurons, and neurotrophic factor and myelin-promoting substance It can also be used to determine the effect of. More practically, however, they are readily useful as research reagents and components of cell growth media to promote proliferation in vitro and to maintain neurons and stem cells.
The peptides of the present invention are synthesized by automated solid phase methods well known in the art. All peptides are purified by high performance liquid chromatography (HPLC) to a degree greater than about 95% before use.

  The following examples are given for illustration only and not for limitation.

Example 1: Stimulation of neurite growth NS20Y neuroblasts are cultured in DMEM containing 10% fetal calf serum (FCS). Cells are removed with trypsin and plated on coverslips in 30 mm Petri dishes. After 20-24 hours, the medium has 0.5% FCS and 0, 0.5, 1, 2, 4 or 8 ng / ml of 18 to about 40 amino acids and SEQ ID NO: 1. Replace with 2 ml of DMEM containing RI peptide containing sequence. Cells were further cultured for 24 hours, washed with PBS, and fixed with Bouin's solution (saturated aqueous picric acid / formalin / acetic acid 15: 5: 1) for 30 minutes. Fixative was removed with PBS and neurite growth was assessed under a phase contrast microscope. Cells with one or more clearly identified neurites that were equal to or longer than the cell diameter were rated positive. To determine the percentage of cells with neurites, at least 200 cells were evaluated in different parts of each dish. The inspection was performed twice.

Example 2: Inhibition of cell death NS20Y cells are seeded as described in Example 1 and are 8 ng / ml of RI containing 18 to about 40 amino acids and comprising the sequence of SEQ ID NO: 1. Incubate on coverslips for 2 days in 0.5% fetal calf serum in the presence or absence of peptide. Remove the media and add 0.2% trypan blue / PBS to each well. On an inverted microscope, dead cells that are stained blue are evaluated as a percentage of the total number by counting 400 cells in 4 regions on each well. The average error of 2 measurements was 5%.

Example 3: Promotion of neurite outgrowth ex vivo Dorsal root ganglia were removed from adult rats and sensory neurons were prepared as described in Kuffler et al. J. Neurobiol. 25: 1267-1282, 1994. . Nerve cells are treated with a RI peptide having 0.5 ng / ml of 18 to about 40 amino acids and comprising the sequence of SEQ ID NO: 1. Three days after treatment, the length of the longest neurite is measured on a micrometer grid. The longest neurites in neurons treated with RI peptides are about 3 times longer than cells treated with control (non-RI) peptides or untreated control cells. All cells respond similarly to nerve growth factor (NGF) in that multiple branches are observed after 48 hours of treatment. These results mean that the LIF-derived peptide promotes differentiation of sensory neurons.

Example 5: Recovery of demyelination in a rat model Experimental allergic encephalomyelitis (EAE) is a rat model of human multiple sclerosis (MS). In rats, EAE is induced by injection of a foreign protein (guinea pig spinal cord), resulting in white matter inflammation and demyelination after 11 days. This demyelination is similar to that seen in human MS injury with active demyelination (Liu et al., Multiple Sclerosis 1: 2-9, 1995).
In Lewis rats, EAE is induced by injection of guinea pig spinal cord and complete Freund's adjuvant (CFA) emulsion. On day 14, after weakening became apparent, treatment with a RI peptide having 18 to about 40 amino acids and containing the sequence of SEQ ID NO: 1 was initiated (200 g / kg intramuscularly) And continue this treatment daily for 8 days. Six rats are injected with carrier only. As a measure of muscle weakness, stride length is evaluated on days 14 and 22. In addition, on day 22, the number and size of demyelinating lesions (plaques) per mm ² in the spinal cord are evaluated. Finally, on day 22, the amount of cholesterol ester in the brain is evaluated as a marker for myelin degradation.

  On day 14, the stride length of both groups decreases, but after 8 days of treatment, animals treated with the LIF-derived peptide recover normally, whereas animals treated with the carrier do not. There is a significant decrease in cholesterol ester content in the brain of the peptide treated group. Furthermore, after 10 days of treatment with LIF-derived peptides, the number of spinal cord injuries is significantly reduced. Finally, the average size of the lesion is significantly reduced. There is no difference in weight loss between control and experimental animals. These results indicate a clinical, biochemical, and morphologically significant recovery of EAE after systemic treatment with LIF-derived peptides. This effect is different from the anti-inflammatory effects of current MS drugs that do not act directly on myelin repair.

Example 6: Examination of ex vivo myelin formation Explants of cerebellum of newborn mice are prepared according to Satomi, Zool. Sci. 9: 127-137, 1992. Observe neurite growth and myelination for 22 days in the culture. In the meantime, the cerebellum of the newborn mouse normally initiates neuronal differentiation and myelination. A RI peptide having 18 to about 40 amino acids and containing the sequence of SEQ ID NO: 1 is added on day 2 after preparing the explant (three control and three treated explants). ), And under a bright field microscope with a video camera, assess neurite growth and myelination. Saposin C at a concentration of about 1-10 g / ml is used as a positive control. LIF-derived peptides stimulate myelin formation to the same extent as saposin C.
Alternatively, myelin formation may be examined by incorporation of ³⁵ S into sulfolipids localized to myelin as described below.

Example 7: Incorporation of ³⁵ S into sulfolipids Primary cells of Schwann cells that form myelin were incubated in low sulfate medium (DMEM) containing 0.5% fetal bovine serum (FBS), then ³⁵ S methionine and an RI peptide having 18 to about 40 amino acids and containing the sequence of SEQ ID NO: 1 is added for 48 hours. Saposin C is used as a positive control. Cells are rinsed with PBS, harvested and sonicated in 100 l of distilled water. For protein analysis, an aliquot of cell lysate is removed and the remainder is extracted with 5 ml of chloroform / methanol (2: 1, v / v). Lipid extracts are chromatographed and immunostained with anti-sulfatide monoclonal antibodies as described in Hiraiwa et al., Proc. Natl. Acad. Sci. USA 94: 4778-4781. Similar amounts of sulfatide are observed after peptide and saposin C treatment.

Example 8: Use of RI peptides in the treatment of traumatic ischemic CNS injury For humans with traumatic injury in the brain or spinal cord, having about 100 g / kg, 18 to about 40 amino acids, and The RI peptide comprising the sequence of SEQ ID NO: 1 is given by systemic injection in the form of a sterile saline solution or in a retained form. The degree of improvement is assessed by an increase in sensory or motor function (ie, increased limb motility). Continue treatment until no further improvement is observed.

Example 9: Use of RI peptides in the treatment of demyelinating disease Patients diagnosed with early stage MS have 18 to about 40 amino acids and contain the sequence of SEQ ID NO: 1 The peptide is given by systemic injection using the same dosage as described in Example 8. Dosing is repeated daily or weekly and observed for increased muscle strength, musculoskeletal coordination and myelination (determined by MRI). Patients with chronic relapsed MS are treated in a similar manner when the next relapse occurs.

Example 10: Cell proliferation test by LIF 18 Incubation with cultured TF-1 cells (ATCC) of LIF activity in a RI peptide having about 40 to about 40 amino acids and containing the sequence of SEQ ID NO: 1 And by measuring the proliferation of TF-1 cells. TF-1 cells are human T cells whose proliferation in the presence of colony stimulating factor (CSF) is inhibited in response to LIF. TF-1 cells are washed 2-3 days after feeding and resuspended to a final concentration of 1 × 10 ⁶ cells / ml in RPMI 1640 containing 5% FCS. Reference titration with LIF is performed in triplicate in a 100 l volume. The baseline titration starts at 1 ng / ml and is serially diluted 2-fold to 0.1 pg / ml. Appropriate dilutions of RI LIF-derived peptides are performed in triplicate in 100 l volumes. Medium alone is used as a negative control.

Cell suspension (100 l) is added to each well and the plate is incubated for about 44 hours at 37 ° C. in a humidified CO ₂ incubator. Tritiated thymidine (0.5 Ci) is added to each well and the plate is incubated for about 4 hours. The contents of each well are collected on a filter mat and the amount of radiolabel is determined by liquid scintillation counting. Plot a standard curve of absorbance versus reference concentration. The LIF activity of the RI LIF-derived peptide is estimated by comparison with the standard curve.

  The present invention is not limited to the embodiments described in the detailed description. Any aspect that retains the intent of the invention is within the scope of the invention. However, the invention is limited only by the claims.

Claims (23)

  An isolated retro-inverso peptide having 18 to about 40 amino acids, said peptide comprising the sequence shown in SEQ ID NO: 1.   The peptide of claim 1, wherein at least one basic charged amino acid in the sequence is replaced by a different basic charged amino acid.   The peptide of claim 1 or 2, wherein at least one acidic charged amino acid in the sequence is replaced by a different acidic charged amino acid.   4. The peptide of any of claims 1-3, wherein at least one nonpolar amino acid in the sequence is substituted with a different nonpolar amino acid.   5. The peptide of any of claims 1-4, wherein at least one uncharged amino acid in the sequence is substituted with a different uncharged amino acid.   6. The peptide of any of claims 1-5, wherein at least one aromatic amino acid in the sequence is substituted with a different aromatic amino acid. The peptide may be CH ₃ CO, CH ₃ (CH ₂ ) _n CO, C ₆ H ₅ CH ₂ CO and H ₂ N (CH ₂ ) _n CO, provided that n = 1 to 10, it has been modified by selected portion independently from the group consisting of any of the peptides of claims 1-6.   8. A peptide according to any of claims 1 to 7, wherein the peptide is glycosylated.   The peptide according to any one of claims 1 to 8, wherein one or more amide bonds in the peptide are reduced.   The peptide according to any one of claims 1 to 9, wherein one or more nitrogens in the peptide are methylated.   The peptide according to any one of claims 1 to 10, wherein one or more carboxylic acid groups in the peptide are esterified.   The peptide according to any one of claims 1 to 11, wherein the peptide has the amino acid sequence shown in SEQ ID NO: 1.   A composition comprising a retro-inverso peptide having from 18 to about 40 amino acids and comprising the sequence shown in SEQ ID NO: 1 and a pharmaceutically acceptable carrier.   A method for promoting neurite outgrowth or myelination in a mammal in need thereof, having an effective amount of 18 to about 40 amino acids to promote neurite outgrowth or myelination. And administering to the mammal a composition comprising a retro-inverso peptide comprising the sequence set forth in SEQ ID NO: 1.   15. The method of claim 14, wherein the peptide has the amino acid sequence shown in SEQ ID NO: 1.   16. The method of claim 14 or 15, wherein the mammal is a human.   17. A method according to any of claims 14 to 16, wherein the course of administration is selected from the group consisting of direct local injection, systemic, intracranial, intracerebral spinal, topical and oral administration.   A retro-inverso peptide having from 18 to about 40 amino acids and comprising the sequence shown in SEQ ID NO: 1 for use in promoting neurite outgrowth or myelination in a mammal in need thereof .   19. The peptide of claim 18, wherein the peptide has the amino acid sequence shown in SEQ ID NO: 1.   20. A peptide according to claim 18 or 19, wherein the mammal is a human.   In preparing a drug for promoting neurite outgrowth or myelination in a mammal in need thereof, a retro- having 18 to about 40 amino acids and comprising the sequence shown in SEQ ID NO: 1 Use of inverso peptides.   The peptide of claim 21, wherein the peptide has the amino acid sequence set forth in SEQ ID NO: 1.   The peptide of claim 21 or 22, wherein the mammal is a human.