Note: Descriptions are shown in the official language in which they were submitted.
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RETRO-INVERSO PEPTIDES DERIVED FROM LEUKEMIA INHIBITORY FACTOR
Field of the Invention
The present invention relates to retro-inverso peptides derived from leukemia
inhibitory factor (LIF). These
peptides have activities similar to that of the native parent protein, and
also have neurotrophic activity.
Back4round of the Invention
Cytokines are proteins which are produced during the effector phases of
natural and specific immunity and serve
to mediate and regulate 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 most well known families of cytokines are
the interleukins which mediate natural immunity. For a detailed description of
the structure and function of the
interleukins, see Abbas et al. Cellular and Mo%cular lmmuno%gy , W. B.
Sounders Company, Philadelphia, pp. 225-243,
1991.
Leukemia inhibitory factor (LIF), named for its ability to inhibit
proliferation of a myeloid leukemic cell line by
inducing differentiation, is a member of a family of ligands that includes IL-
6, oncostatin M, ciliary neurotrophic factor
(CNTF) and cardiotrophin-1 (Gearing, Adv. Immunol. 53:31-58, 1993; Pennica et
al., J. Biol. Chem. 270:10915-10922,
1995; Patterson, Proc. Nat/. Acad Sci. U.S.A. 91:7833-7835, 1994). Although
these cytokines share only very limited
sequence homology, they exert very similar effects on a variety of tissues.
For example, several of these proteins, including
LIF, can induce the same set of acute-phase response proteins in liver,
support the self renewal of cultured embryonic stem
cells, inhibit lipogenesis and enhance the survival of cultured motor neurons.
LIF is produced by diverse cell populations,
including macrophages, synoviocytes and chondrocytes. When applied to
peripheral nerves in vivo, LIF is retrogradely
transported and rescues 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 growth and
differentiation of osteoblasts and endothelial cells.
The rising follicular fluid LIF level around the time of ovulation indicates
that LIF may play a role in ovulatory events, early
embryonic development and implantation (Senturk et al., Am. J. Reprod lmmunol.
39:144-151, 1998; Stewart, Annals
N. Y. Acad Sci. 157-1651.
Neurotrophins and neurotrophic factors are proteins or peptides capable of
affecting the survival, target
innervation andlor function of neuronal cell populations (Barde, Neuron,
2:1525-1534, 19891. The efficacy of
neurotrophins both in vivo and in vitro has been well documented. For example,
ciliary neurotrophic factor (CNTF) promotes
survival of chicken embryo ciliary ganglia in vitro and supports survival of
cultured sympathetic, sensory and spinal motor
neurons (1p et al., J. Physiol. Paris, 85:123-130, 19911.
A major obstacle to the in vivo therapeutic use of peptides is their
susceptibility to proteolytic degradation.
Retro-inverso peptides are isomers of linear peptides in which the direction
of the sequence is reversed (retro) and the
chirality, D or L, of each amino acid is inverted (inverso). There are also
partially modified retro-inverso isomers of linear
peptides in which only some of the peptide bonds are reversed and the
chirality of the amino acid residues in the reversed
portion is inverted. The major advantage of such peptides is their enhanced
activity in vivo due to improved resistance to
proteolytic degradation (For review, see Chorev et al., Trends Biotech.,
13:438-445, 1995). Although such retro-inverso
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analogs exhibit increased metabolic stability, their biological activity is
often greatly compromised (Guichard et al., Pioc.
Nat/. Acad Sci. U.S.A., 91:9765-9769, 1994). Far example, Richman et al. (J.
Peptide Protein Res., 25:648-662)
determined that analogs of linear and cyclic leu-enkephalin modified at the
GIy3-Phe4 amide bond had activities ranging from
6%-14 % of native leu-enkephalin. 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. (TlBTECH 14, 19961,
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 helicity was, in
fact, absent under the solvent conditions used for assessing antigenic
mimicry.
There is a need for LIF-derived and neurotrophic peptides exhibiting increased
metabolic stability while retaining
biological activity. The present invention addresses this need.
Summary of the Invention
One embodiment of the present invention is an isolated retro-inverso peptide
having between 18 and about 40
amino acids, wherein the peptide includes the sequence shown in SEO ID N0: 1.
In one aspect of this preferred
embodiment, at least one basic charged amino acid of the sequence is replaced
with a different basic charged amino acid.
In another aspect of this preferred embodiment, at least one acidic charged
amino acid of said sequence is replaced with a
different acidic charged amino acid. Advantageously, at least one non-polar
amino acid of said sequence is replaced with a
different non-polar amino acid. Preferably, at least one uncharged amino acid
of the sequence is replaced with a different
uncharged amino acid. In another aspect of this preferred embodiment, at least
one aromatic amino acid of said sequence
is replaced with a different aromatic amino acid. Advantageously, the peptide
is modified at the amino terminus, carboxy
terminus, or both amino and carboxy terminus with a moiety independently
selected from the group consisting of CH3C0,
CH3ICHz)~CO, C6H5CHZC0 and HzNICHz)~CO, wherein n=1-10. Preferably, the
peptide is glycosylated. In another aspect of
this preferred embodiment, one or more amide bonds of the peptide is reduced.
Preferably, one or more nitrogens in said
peptide is methylated. In still another aspect of this preferred embodiment,
one or more carboxylic acid groups in the
peptide is esterified. Preferably, the peptide has the amino acid sequence
shown in SEO ID N0: 1.
Another embodiment of the invention is a composition comprising a retro-
inverso peptide having between 18 and
about 40 amino acids, wherein the peptide includes the sequence shown in SEO
ID N0: 1, and a pharmaceutically
acceptable carrier.
The present invention also provides a method for promoting neurite outgrowth
or myelination in a mammal in
need thereof, comprising the step of administering to the mammal an effective,
neurite outgrowth or myelination-
facilitating amount of a composition comprising a retro-inverso peptide having
between 18 and about 40 amino acids,
wherein the peptide includes the sequence shown in SEO ID N0: 1. Preferably,
the peptide has the amino acid sequence
shown in SEO ID N0: 1. Advantageously, the mammal is a human. In one aspect of
this preferred embodiment, the
administering step is direct local injection, systemic, intracranial,
intracerebrospinal, topical or oral.
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Another embodiment of the invention is a retro-inverso peptide having between
18 and about 40 amino acids,
wherein the peptide includes the sequence shown in SEO ID N0: 1 for use in
promoting neurite outgrowth or myelination in
a mammal in need thereof.
Preferably, the peptide has the amino acid sequence shown in SEO ID N0: 1.
Advantageously, the mammal is a human.
The present invention also provides the use of a retro-inverso peptide having
between 18 and about 40 amino
acids, wherein the peptide includes the sequence shown in SED ID N0: 1 in the
preparation of a medicament for promoting
neurite outgrowth or myelination in a mammal in need thereof. Advantageously,
the peptide of Claim 21, wherein said
peptide has the amino acid sequence shown in SEO ID N0: 1. Preferably, the
mammal is a human.
Detailed Description of the Preferred Embodiments
The present invention provides retro-inverso (Rp peptides derived from
leukemia inhibitory factor (LIF) which
mediate similar effects to native LIF, including regulation of growth and
differentiation of osteoblasts and endothelial cells.
The term "derived from" indicates that the peptides include the active region
of interleukin-6, or analogs thereof as defined
below. These RI LIF-derived peptides induce production by the liver of acute
phase response proteins, support self renewal
of cultured embryonic stem cells, inhibit lipogenesis, enhance survival of
cultured motor neurons, and regulate growth and
differentiation of osteoblasts and endothelial cells. The ability of a
particular LIF-derived retro-inverso peptide to mediate
an effect similar to the parent peptide can be determined by a person of
ordinary skill in the art using standard LIF assays
such as the one described in Example 10 below.
These RI LIF-derived 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 in promoting increased myelination and for counteracting the
effects of demyelinating diseases. The ability of
any such peptide to stimulate neurite outgrowth and myelination, and to
prevent neural cell death, can easily be determined
by one of ordinary skill in the art using the procedures described in Examples
1-9 hereinbelow. 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
recombinant cytokines.
A particular RI LIF-derived 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.
Table 1
Protein Name seguence SEO ID N0:
human LIF YTAOGEPFPNNVEKLCAP 1
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As discussed above, these RI LIF-derived peptides have the same activities as
the corresponding full-length LIF,
and also possess neurotrophic and myelinotrophic activity. One embodiment of
the present invention is a method of
facilitating neurite outgrowth in differentiated or undifferentiated neural
cells by administering to the cells an effective,
neurite outgrowth-facilitating amount of a RI peptide having between 12 and
about 40 amino acids, and encompassing the
RI LIF-derived peptide shown in SEO ID N0: 1, or analogs thereof which have
similar activity. Such analogs include, for
example, replacement of one or more lysine andlor arginine residues with
alanine or another amino acid; deletion of one or
more lysine andlor arginine residues; replacement of one or more tyrosine
andlor phenylalanine residues, deletion of one or
more phenylalanine residues and conservative replacements of one or more amino
acids within the peptide. The
replacement or deletion of lysinelarginine and tyrosinelphenylalanine residues
will reduce the susceptibility of peptide
degradation by trypsin and chymotrypsin, respectively.
Additional variations of these peptide sequences contemplated for use in the
present invention include minor
insertions and deletions. Conservative amino acid replacements are
contemplated. Such replacements are, for example,
those that take place within a family of amino acids that are related in the
chemical nature of their side chains. The
families of amino acids include the basic charged amino acids (lysine,
arginine, histidinel; the acidic charged amino acids
(aspartic acid, glutamic acid); the non-polar amino acids (alanine, valine,
leucine, isoleucine, proline, phenylalanine,
methionine, tryptophan); the uncharged polar amino acids (glycine, asparagine,
glutamine, cysteine, serine, threonine,
tyrosinel; and the aromatic amino acids (phenylalanine, tryptophan and
tyrosinel. In particular, it is generally accepted that
conservative amino acid replacements consisting of an isolated replacement of
a leucine with an isoleucine or valine, or an
aspartic acid with a glutamic acid, or a threonine with a serine, or a similar
conservative replacement of an amino acid with
a structurally related amino acid will not have a major effect on the
properties of the peptide. The ability of any RI peptide
comprising the sequence shown in SEO ID N0: 1, or insertions, deletions or
substitutions thereof, to promote neurite
outgrowth, myelination, reverse demyelination and prevent neural cell death
can be determined using the assays provided in
the examples presented below.
Various standard chemical modifications will improve the stability,
bioactivity and ability of the peptide to cross
the blood brain barrier. One such modification is aliphatic amino terminal
modification with a derivative of an aliphatic or
aromatic acid, forming an amide bond. Such derivatives include, for example,
CH3C0, CH3(CH21~C0 (n=1-101, C6H5CH2C0,
HZN-ICHz)~CO (n=1-10). Another modification is carboxy terminal modification
with a derivative of an aliphatic or aromatic
aminelalcohol coupled to the peptide via an amidelester bond. Such derivatives
include those listed above. The peptides
may also have both amino and carboxy terminal modifications, wherein the
derivatives are independently selected from
those listed above. The peptides may also be glycosylated, wherein either the
alpha amino group or a D-Asn, or both, are
modified with glucose or galactose. In another contemplated modification,
selected backbone amide bonds are reduced (-
NH-CHZ). 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.
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A typical minimum amount of the RI peptides of the invention for the LIF or
neurotrophic activity in cell growth
medium is usually at least about 5 nglml. This amount or more of the RI
peptides of the invention for in vitro use is
contemplated. Typically, concentrations in the range of 0.1 glml to about 10
glml of these peptides will be used.
Effective amounts for any particular tissue can be determined in accordance
with Example 1.
Cells can be treated in vitro or ex vivo by directly administering the RI
peptides of the invention to the cells. This
can be done, for example, by culturing the cells in growth medium suitable for
the particular cell type followed by addition
of the peptide to the medium. When the cells to be treated are in viva,
typically in a vertebrate, preferably a mammal, 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 RI peptides persist longer in vivo
due to the D peptide bonds. In the peptides lacking lysine and arginine
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).
For treatment of neural disorders, direct intracranial injection or injection
into the cerebrospinal fluid may also be
used in sufficient quantities to give 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 neural tissue
by direct local injection or by systemic
administration. Various conventional modes of administration are contemplated
including intravenous, pulmonary,
intramuscular, intradermal, subcutaneous, intracranial, epidural, intrathecal,
topical and oral.
The peptide compositions of the invention can be packaged and administered in
unit dosage form such as an
injectable 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. In a preferred embodiment,
daily systemic dosages of the RI peptides of
the invention based on the body weight of the vertebrate for promotion of LIF-
induced processes, treatment of
neurodegenerative diseases or demyelination diseases are in the range of from
about 0.01 to about 10,000 glkg. More
preferably, daily systemic dosages are between about 0.1 and 1,000 glkg. Most
preferably, daily systemic dosages are
between about 10 and 100 glkg. 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.
In one preferred embodiment of the invention, the peptides are administered
locally to neural cells in vivo by
implantation thereof. For example, 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. The use of bioerodible,
biodegradable and other depot formulations is
expressly contemplated in the present invention. Infusion pumps, matrix
entrapment systems and combination with
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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 RI LIF-derived 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).
There are currently no available pharmaceuticals able to promote full
functional regeneration and restoration of
the structural integrity of neural systems. This is particularly true of the
CNS. Regeneration of peripheral nerves through
the use of neurotrophic factors is within the scope of this invention.
Moreover, 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. The principal cause of Parkinson's disease is the
degeneration of dopaminergic neurons of the
substantia nigra. Since antibodies against prosaposin immunohistochemically
stain the dopaminergic neurons of the
substantia nigra in human brain sections, the RI 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 RI 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 crass the blood
brain barrier. U.S. Patent No. 5,571,787
discloses that an iodinated neurotrophic 18-mer fragment derived from saposin
C crosses the blood brain barrier. Thus, the
RI peptides having up to about 22 amino 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 cerebrospinal fluid is also envisioned as an alternate route.
Cells may be treated to facilitate myelin formation or to prevent
demyelination in the manner described above in
vivo, ex vivo or in vitro. Diseases resulting in demyelination of nerve fibers
including MS, acute disseminated
leukoencephalitis, trauma to brain andlor spinal cord, progressive multifocal
leukoencephalitis, metachromatic
leukodystrophy, adrenal leukodystrophy and maldevelopment of the white matter
in premature infants (periventricular
leucomalacia) can be slowed or halted by administration of the neurotrophic
peptides of the invention to the cells affected
by the disease.
The RI LIF-derived peptide compositions of the present invention can also be
used for supporting the self renewal
of cultured embryonic stem cells, to enhance the survival of cultured motor
neurons and for determining the effects of
neurotrophic factors and myelin 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 neural cells and stem
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) to an
extent greater than about 95% prior to use.
The following examples are merely illustrative and are not intended to limit
the scope of the present invention.
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Example 1
Stimulation of neurite outgrowth
NS20Y neuroblastoma cells are grown in DMEM containing 10% fetal calf serum
(FCSI. Cells are removed with
trypsin and plated in 30 mm petri dishes onto glass coverslips. 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 nglml of an RI peptide having
between 18 and about 40 amino acids, and
including the sequence shown in SEO ID N0: 1. Cells were cultured for an
additional 24 hours, washed with PBS and fixed
with Bouin's solution (saturated aqueous picric acidlformalinlacetic acid
15:5:1 ) for 30 minutes. Fixative was removed
with PBS and neurite outgrowth was scored under a phase contrast microscope.
Cells exhibiting one or more clearly
defined neurites equal to or longer than one cell diameter were scored as
positive. At least 200 cells were scored in
different portions of each dish to determine the percentage of neurite bearing
cells and assays were performed in duplicate.
Example 2
Prevention of cell death
NS20Y cells are plated as described in Example 1 and grown on glass coverslips
in 0.5% fetal bovine serum for 2
days in the presence or absence of 8 nglml of an RI peptide having between 18
and about 40 amino acids, and including the
sequence shown in SE0 ID N0: 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%.
Example 3
Promotion of neurite outnrowth ex vivo
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 nglml of an
RI peptide having between 18 and about
40 amino acids, and including the sequence shown in SEO ID N0: 1. After three
days of treatment, the length of the
longest neuritic projections are measured on a micrometer grid. The longest
neurites in neurons treated with RI peptide are
approximately three times longer than those treated with a control (non-RI)
peptide or in untreated controls. After a 48
hour treatment, all cells respond similarly to nerve growth factor (NGF) in
that extensive branching is observed. These
results indicate that the LIF-derived peptides promote the differentiation of
sensory neurons.
Example 5
Reversal 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 injecting foreign protein (guinea pig spinal cord) which results in
inflammation and demyelination in white matter
11 days later. This demyelination resembles that seen in actively
demyelinating human MS lesions (Liu et al., Multiple
Sclerosis 1:2-9, 19951.
EAE is induced in Lewis rats by injection of an emulsion of guinea pig spinal
cord and complete Freund's adjuvant
(CFAI. At day 14, when weakness is evident, treatment with RI peptides having
between 18 and about 40 amino acids,
and including the sequence shown in SE0 ID N0: 1, is begun (200 glkg
intramuscularly) and continued for 8 days every
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day. Six rats are injected with vehicle only. Stride length, a measure of
muscle weakness, is scored on days 14 and 22. In
addition, the number and size of demyelinating lesions (plaques) in the spinal
cord at day 22 per mm2 is scored. Lastly, the
amount of cholesterol ester in brain, a marker of myelin breakdown, is scored
at day 22.
The stride length of both groups is decreased at day 14, whereas after
treatment for 8 days, the LIF-derived
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. Moreover, the number
of spinal cord lesions is significantly reduced
after 10 days of treatment with LIF-derived peptide. Lastly, the average
lesion size is significantly reduced. There is no
difference in weight loss between the control and experimental animals. These
results indicate a significant clinical,
biochemical and morphological reversal of EAE after systemic treatment with
LIF-derived peptides. This action differs
from the anti-inflammatory effect of current MS drugs which do not act
directly upon myelin repair.
Example 6
Ex vivo myelination assay
Newborn mouse cerebellar explants are prepared according to Satomi (Zoo/. Sci.
9:127-137, 1992). Neurite
outgrowth and myelination are observed for 22 days in culture, during the
period when the newborn mouse cerebellum
normally undergoes neuronal differentiation and myelination begins. An RI
peptide having between 18 and about 40 amino
acids, and including the sequence shown in SEO ID N0: 1, is added on the
second day after preparation of the explants
(three control and three treated explants) and outgrowth of neurites and
myelination 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. Myelination is stimulated by the LIF-derived peptides to a similar
extent as with saposin C.
Alternatively, myelination may be assayed by incorporation of 35S into
sulfolipids which are exclusive to myelin as
described below.
Example 7
Incorporation of 35S into sulfolipids
Primary myelin~containing Schwann cells are incubated in low sulfate media
(DMEM) containing 0.5% fetal
bovine serum (FBSI, followed by addition of 35s_methionine and an RI peptide
having between 18 and about 40 amino acids,
and including the sequence shown in SEQ ID N0: 1, for 48 hours. Saposin 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 chloroformlmethanol 12:1, vlu). Lipid
extracts are chromatographed and
immunostained with anti-sulfatide monoclonal antibody as described (Hiraiwa et
al., Proc. Nat/. Acad Sci. U.S.A. 94:4778
4781 ). Similar amounts of sulfatide are observed after peptide and saposin C
treatment.
Example 8
Use of RI peptides in treating traumatic ischemic CNS lesions
Humans with traumatic lesions to the brain or spinal cord receive systemic
injections of about 100 glkg of an RI
peptide having between 18 and about 40 amino acids, and including the sequence
shown in SEQ ID N0: 1, in a sterile saline
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solution or in depot form. Improvement is assessed by gain of sensory or motor
nerve function /i.e. increased limb
movementl. Treatments continue until no further improvement occurs.
Example 9
Use of RI peptides in treatinn demyelination disorders
Patients diagnosed with early stage MS are given an RI peptide having between
18 and about 40 amino acids,
and including the sequence shown in SEO ID N0: 1 by systemic injection using
the same dose range as in Example 8.
Dosages are repeated daily or weekly and improvement in muscle strength,
musculoskeletal coordination and myelination
(as determined by MRI) is observed. Patients with chronic relapsing MS are
treated in the same manner when subsequent
relapses occur.
Example 10
LIF cell proliferation assay
An RI peptide having between 18 and about 40 amino acids, and including the
sequence shown in SEQ ID N0: 1,
is assayed for LIF activity by incubation with TF-1 cells IATCC) in culture
and measurement of TF-1 cell proliferation. TF1
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 to 3 days after feeding and resuspended to a final
concentration of 1 x 106 cellslml in RPMI 1640
containing 5% FCS. Titrations of an LIF standard are distributed in 100 I
volumes in triplicate. The titration of the
standard is started at 1 nglml and serial two-fold dilutions are made down to
0.1 pglml. Appropriate dilutions of the RI LIF
peptides are made in 100 I volumes in triplicate. Medium alone is included as
a negative control. Cell suspension (100 I) is
added to each well and plates are incubated for about 44 hours at 37 C in a
humidified COz incubator. Tritiated thymidine
(0.5 Ci) is added to each well and plates are incubated for about 4 hours. The
contents of each well are harvested onto
filter mats and the amount of radiolabel is determined by liquid scintillation
counting. A standard curve of absorbance
versus concentration of standard is plotted. LIF activity of the RI LIF-
derived peptides is estimated by comparison with the
standard curve.
It should be noted that the present invention is not limited to only those
embodiments described in the Detailed
Description. Any embodiment which retains the spirit of the present invention
should be considered to be within its scope.
However, the invention is only limited by the scope of the following claims.
.g.
