Note: Descriptions are shown in the official language in which they were submitted.
CA 02284105 1999-09-13
WO 98/42746 PCT/US98/05503
SYNTHETIC SAPOSIN C-DERIVED NEUROTROPHIC PEPTIDES
Field of the Invention
The present invention relates to neurotrophic peptides and their methods of
use. More specifically, the
invention relates to synthetic peptides related to the active neurotrophic
fragment located within saposin C.
~ 5 Background of the Invention
Demyelination is a defect common to a number of central nervous system (CNS)
disorders, the most
prevalent being multiple sclerosis (MS). MS, a chronic disorder which may lead
to total disability. is characterized
by damage to the myelin sheath, while leaving the axons mostly intact. There
is currently no effective treatment
for MS. Other central nervous system disorders involving demyelination include
acute disseminated encephalomyelitis,
amyotrophic lateral sclerosis, acute hemorrhagic leukodystrophy, progressive
muhifocal leukoencephalitis,
metachromatic leukodystrophy and adrenal leukodystrophy. The peripheral
nervous system (PN51 can also be afflicted
with demye6nation, such as that occurring in Guiliain-Barn syndrome
(Pathologic Basis of Disease, Robbins et al.
eds., W.B. Sounders, Philadelphia, 1979, pp. i 578-1582).
Peripheral nerve injuries and peripheral neuropathies, such as those resulting
from diabetes or chemotherapy,
comprise the most prevalent peripheral nervous system disorders. Current
treatments for peripheral nervous system
disorders only treat the symptoms, not the cause of the disease.
Heurotrophins are proteins or peptides capable of affecting the survival,
target innervation andlor function
of neuronal cell populations (Bards, Neuron, 2:1525-1534. 1989). The efficacy
of neurotrophins both in vivo and
in vitro has been well documented. For example, nerve growth factor (NGF) acts
as a trophic factor for forebrain
cholinergic, peripheral and sensory neurons (Hefti et al., Neurobio. Aging,
10:515-533, 19891. In v'rvo experiments
indicate that NGF can reverse naturally-occurring as well as physical
traumatic injuries to peripheral nerves (Rich et
al., J. Neurncytol., 16:261-268, 1987). Brain-derived neurotrophic factor
(BDNF) is a trophic factor for peripheral
sensory neurons, dopaminergic neurons of the substantia nigra, central
choiinergic neurons and retinal ganglia
(Henderson et al., Rector. Neurol. Neurosci., 5:15-28, 1993). BDNF has been
shown to prevent normally-occurring
cell death both in vitro and u~ viva fHofer et al., Nature, 331:262-262,
1988). Ciliary neurotrophic factor (CNTF)
promotes survival of chicken embryo ciliary ganglia in vitro and supports
survival of cuhured sympathetic, sensory
and spinal motor neurons (Ip et al.. J. Physiol. Paris, 85:123-130, 1991 ).
Prosaposin is the precursor of a group of four small heat-stable glycoproteins
which are required for
hydrolysis of glycosphingolipids by lysosomal hydroiases (Kishimoto et al., J.
Lipid Res., 33:1255-1267, 1992).
- 30 Prosaposin is proteoiyticaby processed in lysosomes, generating saposins
A, B, C and D (0'Brien et al., fASEB J.,
5:301-308, 1991). 0'Brien et aL (Proc. Natl. Aced Sci. U.S.A., 91:9593-9596,
1994), U.S. Patent Nos. 5,571,787,
5,698,080, 5,714,459 and published PCT Application No. W095103821, disclose
that prosaposin and saposin C
stimulate neurite outgrowth and promote increased myelination. In addition,
these references disclose that a ZZ-mer
peptide ICEFLVKEUTKLIONNKTEKEIL; SEO 10 N0: 1) consisting of amino acids 8-29
of human saposin C stimulated
neurite outgrowth in both neuroblastoma cells and mouse cerebellar explants.
These references also disclose that
an iodinated 1B-mer peptide (YKEVTKLIONNKTEKEIL; SEO 10 N0: 2) contained
within the active 22-mer of saposin
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WO 98/42746 PCTIUS98/05503
C iwith 11 replaced by Y1 also promoted neurite outgrowth and was able to
cross the blood-brain barrier. 0'Brien
et al. (fASEB J., 9:681-685, 19951 showed that the 22-mer stimulated choline
acetyitransferase activity and
prevented cell death in neuroblastoma cells in vitro. The active neuritogenic
fragment was localized to a linear 12-
mer located in the amino-terminal sequence of saposin C (LIDNNKTEKEIL; SEO ID
N0: 31.
There is a significant need for neurotrophic peptides having modified
structural stability and~or activities.
The present invention addresses this need.
Summary of the Invention
the present invention provides modified peptides based on the
naturally.occurring saposin C sequence, and
particularly based on neuritogenic fragments of saposin C. The modifications
to these peptides can address issues
of activity, stability and persistence.
One embodiment of the present invention is a neurotrophic, myelinotrophic or
neuroprotective non-native
peptide preferably having up to about 50 amino acids and including the
sequence shown in SEO ID N0: 8, with the
proviso that the peptide does not have the sequence shown in SEO ID N0: 4.
More preferably, the peptide has up
to about 30 amino acids. More preferably, the peptide has between about 12 and
25 amino acids. Preferably, the
amino acid at position 1 of SEO ID N0: 8 is isoleucine. Advantageously, the
amino acid at position 3 of SEO ID
N0: 8 is not aspartic acid. In another aspect of this preferred embodiment,
the amino acid at position 8 of SED ID
N0: 8 is not glutamic acid. Preferably, the amino acid at position 10 of SEO
ID N0: 8 is not glutamic acid.
Advantageously, the amino acid at position 11 of SEO 10 N0: 8 is not leucine.
preferably, the amino acid at position
12 of SEO ID N0: 8 is not leucine. Further, the peptides described above may
be acetylated or esterified with a
2D fatty acid.
Another embodiment of the invention is a method of stimulating neural cell
outgrowth, promoting
neuroprotection or promoting increased myelination comprising the step of
contacting neuronal cells with a
composition comprising an effective neurotrophic and myelinotrophic
concentration of a non-native peptide having up
to about 50 amino acids and including the sequence shown in SEO ID N0: 8, with
the proviso that the peptide does
not have the sequence shown in SEO ID N0: 4. Preferably, the neuronal cells
are neuroblastoma cells.
Advantageously, the neuroblastoma cells are NS20Y cells. According to one
aspect of this preferred embodiment,
the contacting step occurs in vitro. Alternatively, the contacting step occurs
in viva.
The present invention also provides a method of treating neuropathic pain in a
mammal in need thereof,
comprising the step of administering an effective pain-treating amount of a
non-native neurotrophic peptide fragment
of saposin C, the peptide having up to about 50 amino acids and including the
sequence shown in SEO ID N0: 8,
with the proviso that the peptide does not have the sequence shown in SEO ID
N0: 4. Preferably, the administering
step is intravenous, intramuscular, intradermal, subcutaneous, intracranial,
epidural, topical, oral, transdermal,
transmucosal or intranasal.
The present invention also provides a method of treating sensory or motor
neuropathy in a mammal in need
thereof, comprising administering an effective sensory or motor neuropathy-
treating amount of a non-native
neurotrophic peptide fragment of saposin C, the peptide having up to about 50
amino acids and including the
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sequence shown in SEO 10 N0: 8, with the proviso that the peptide does not
have the sequence shown in SEQ ID
N0: 4. Preferably, the administering step is intravenous, intramuscular,
intradermal, subcutaneous, intracranial,
epidural, topical, oral, transdermal, transmucosal or intranasal. Further, the
peptide may be acetylated or esterified
with a fatty acid.
Still another embodiment of the invention is a pharmaceutical composition
comprising a non-native
neurotrophic peptide fragment of saposin C having up to about 50 amino acids
and including the sequence shown
in SEO ID N0: 8, with the proviso that the peptide does not have the sequence
shown in SEO ID N0: 4, in a
pharmaceutically acceptable carrier. Preferably, the composition is a
controlled release formulation. The composition
may be in liposomal, lyophilized or unit dosage form.
Brief Descriotian of the Drawinos
Figure 1 illustrates a NS20Y neuroblastoma neurite outgrowth assay using
peptides TX14(A)
(TXLIDNNATEEILY; X-D-alanine; SEO 10 N0: 4) and a rat 14-mer derived from the
saposin C active sequence
(SELIINNATEELLY; SEO ID N0: 5).
Figure 2 illustrates a cell death assay using NS20Y neuroblastoma cells. NS20Y
cells were grown for 48
hours in tow serum in the presence or absence of TX14(A) and dead cells were
identified by Trypan blue staining.
Detailed Oescriotion of the Preferred Embodiments
The present invention includes the discovery that non-naturally occurring
variants encompassing the active
neurotrophic region of saposin C stimulate neurite outgrowth, prevent neural
cell death, promote myelination, inhibit
demyeiination, promote neuroprotectian and can be used to treat various
neuropathies. As used herein, a neuropathy
is a functional disturbance or pathological change in the peripheral nervous
system and is characterized clinically by
sensory or motor neuron abnormalities.
A native 15-mer (TKLIDNNKTEKEILD; SEO ID N0: 6) contained within human saposin
C and encompassing
the active neurite-promoting region shown in SEO ID N0: 3 was modified as
follows to decreases its susceptibility
to proteolysis in rivo: Lys 2 was replaced with D-ale to increase resistance
to exopeptidases; iys 8 was replaced
with ale to increase resistance to trypsin digestion; and lys 11 was deleted
to increased resistance to trypsin
digestion. In addition, asp 15 was replaced with tyr to provide an iodination
site. Thus, the resulting peptide,
TX14(A), contained no cleavage sites for trypsin or chymotrypsin. Peptide
TX141A) exhibited neuritogenic activ'tty
in vitro neurite outgrowth assays. TX14(A) also prevented cell death in
neuroblastoma cells in culture.
It is also contemplated that the leucine at position three andlor 14 can be
changed to an isoleucine with
retention of activity. This will prevent degradation by enkephalinase which
cleaves at hydrophobic residues.
Replacement of leucine residues also helps minimize degradation by angiotensin
converting enzyme (ACE) which also
cleaves at hydrophobic residues. In another preferred embodiment, lys 8 is an
amino acid other than aianine, lysine
or arginine to prevent degradation by dipeptidyl pept~ase (alanine) or trypsin
(lysine, arginine).
Saposin C-derived peptides comprising the active 12-mer region of the sequence
shown in SEO iD N0: 3
(LIDNNKTEKEIL; SED ID N0: 7), and neurotrophic analogs thereof, possess
significant therapeutic applications in
promoting functional recovery after toxic, traumatic, ischemic, degenerative
and inherited lesions to the peripheral
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and central nervous system. In addition, these peptides stimulate myelination
and counteract the effects of
demyefinating diseases. These peptides stimulate the outgrowth of neurons,
promote myelination, promote
neuroprotection and prevent programmed cell death in neuronal tissues. The
peptides of the invention can also be
used to treat various neuropathies including, but not limited to, motor,
sensory, peripheral, taxol-induced and diabetic
neuropathies. The peptides are also useful as analgesics, particularly for the
treatment of neuropathic pain which
can develop days or months after a traumatic injury and is often long-lasting
or chronic.
SEO ID N0: 7 may be modified as follows and still retain neurotrophic
activity: Leu 1 may be leu or ile;
Ile 2 is essential; asp 3 is any amino acid; asn 4 and asn 5 are essential;
lys 6 is any amino acid, preferably not
lysine or arginine: thr 7 is essential, glu 8 is a charged amino acid; lys 9
is absent or a charged amino acid,
1 D preferably not lysine or arginine; glu 10 is any charged amino acid; ile
11 is any amino acid; leu 12 is any amino
acid. These guidelines produce the following consensus sequence:
X,IX2NNX3TX4X5XsX~XB (SEO ID N0: 8)
in which I is isoleucine; X, is leucine or isoleucine; XZ is any amino acid; N
is asparagine; X3 is any amino acid; X,
is lysine, arginine, histidine, aspartic acid or glutamic acid; X5 is absent,
lysine, arginine, histidine, aspartic acid,
glutamic acid or glycine; X6 is lysine, arginine, histidine, aspartic acid or
glutamic acid; X, is any amino acid; and
X8 is any amino acid.
The second asparagine residue within the native prosaposin sequence
(corresponding to second "N" in SEQ
ID N0: 81 is known to be glycosylated with N-acetylglucosamine which may
provide some resistance to protealytic
degradation. The synthetic modification of this asparagine residue within the
instant non-native saposin C-derived
peptides by standard methods (i.e. Merrifieid synthesis) with various
carbohydrates, preferably glucose, is also within
the scope of the present invention.
One embodiment of the present invention is a method of facilitating neurite
outgrowth or increased
myelination in differentiated or undifferentiated neural cells by
administering to the cells an effective, neurite
outgrowth or myelin-facilitating amount of a saposin C-derived peptide
encompassing the active 12-mer region shown
in SEQ ID N0: 7 )amino acids 18-29 of saposin C) or, more preferably, non-
natural analogs thereof including the
sequence shown in SEQ ID N0: 8.
Non-natural saposin C-derived peptide analogs of the invention further
include, for example, replacement of
one or more lysine and(or arginine residues; replacement of one or more
tyrosine andlor phenylalanine residues,
deletion of one or more phenylalanine residues and conservative replacement 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 chymatrypsin,
respectively. The non-native neurotrophic and
myeiinotrophic peptide sequences of the invention preferably have up to about
50 amino acids; more preferably, up
to about 30 amino acids; and most preferably, between about 12 and 25 amino
acids and include therein the
sequence shown in SEfl ID N0: 8.
In one preferred embodiment, the peptide does not contain the sequence shown
in SEO ID N0: 4. In
another preferred embodiment, the amino acid at position 6 of SEO ID N0: 8 is
not alanine. In still another preferred
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embodiment, the amino acid at position 1 of SEQ ID N0: 8 is isofeucine. In yet
another preferred embodiment, the
amino acid at position 3 of SEO ID NO: 8 is not aspartic acid. In yet another
preferred embodiment, the amino acid
at position 8 of SEO ID ND: 8 is not giutamic acid. In additional preferred
embodiments, the amino acids at positions
10, 11 and 12 of SEO ID N0: 8 are not giutamic acid, isoleucine and leucine,
respectively.
Additional variations of these peptide sequences contemplated for use in the
present invention include minor
insertions, deletions and substitutions. For example, 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,
histidineh the acidic charged amino acids (aspartic acid, glutamic acid), the
non-polar amino acids (alanine, valine,
leucine, isoleucine, praline, phenylalanine, methionine, tryptophan); the
uncharged polar amino acids (glycine,
asparagine, glutamine, cysteine, serine, threonine, tyrosine); and the
aromatic amino acids (phenyialanine, tryptophan
and tyrosine). 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, ar a similar conservative replacement of an amino acid with a
structurally related amino acid will not
significantly affect the properties of the peptide. The non-native saposin C
sequences containing SEO ID N0: 8
therein can be modified to attain various objectives such as increased
activity and stability. Other amino acids can
be present outside this consensus sequence including native saposin C
sequence, conservative substitutions of these
native sequences, or unrelated peptide sequences to achieve objectives such as
increased binding, hydrophobicity,
hydrophiliciiy and the like. Sequences outside the active neurotrophic region
are not typically required for activ'tty.
Thus, in most instances, the subject peptide will be active regardless of
these sequences. Again, any such peptide
can be screened for such activity using the protocols described herein.
The ability of any such peptide to stimulate neurite outgrowth, prevent neural
cell death, promote
myelination and inhibit demyelination can easily be determined by one of
ordinary skill in the art using the procedures
described in Examples 1-4. Methods for assaying the abilities of these non-
naturally occurring peptides to promote
myelination and to inhibit demyelination are set forth in in Examples 3 and 4
hereinbelow.
A typical minimum amount of the 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 non-
naturally occurring synthetic peptides of
the invention for in vitro use is contemplated. Typically, concentrations in
the range of 0.1 iuglml to about 10 pglml
of these peptides will be used. Effective amounts for any particular tissue
can be determined in accordance with
Example 1.
The neural cells can be treated in vitro or ex v'rvo by directly administering
the peptides of the invention
to the cells. This can be done, for example, by cuhuring 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 vivo, typically in a
vertebrate, preferably a mammal, the composition can be administered by one of
severe! techniques. Most preferably,
the composition is injected directly into the blood or tissue in sufficient
quantity to give the desired local
concentration of peptide. In the peptides lacking lysine and arginine
residues, proteolytic degradation is reduced.
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The smaller peptides (i.e., 20-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?.
The peptides of the invention may also be esterified with fatty acids to form
peptide fatty acid esters using
conventional acid-catalyzed esterification. Alternatively, the last amino acid
added in the synthetic procedure is itself
a commercially available esterified amino acid which obviates the need for the
esterification reaction. Fatty acids
contemplated for use in formation of peptide esters include fauric, myristic,
palmitic, stearic, oleic and linoleic.
The subject peptides may also be acetylated by inclusion of commercially
available acetylated lysine, arginine
or asparagine residues during the synthetic procedure. These modified peptides
retain the activity of the parent
compound.
These modifications will facilitate the ability of the peptide to cross the
blood brain barrier due to increased
hydrophobicity.
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, intracerebrospinal, intramuscufar, intradermal, subcutaneous,
intracraniaf, intranasal, epidural, topical and
oral. For use as an analgesic, administration by direct intramuscular or
intravenous injection is preferred.
The peptide compositions of the invention can be packaged and administered in
unit dosage farm, such as
an injectabie 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.
Appropriate daily systemic dosages of the
peptides of the invention based on the body weight of the vertebrate for
treatment of neural diseases or as an
analgesic are in the range of from about 10 to about 100 Ng/kg, although
dosages from about 0.1 to about 1,000
Nglkg are also contemplated. Thus, for the typical 70 kg human, dosages can be
between 7 and 70,000 dug daily,
preferably between 700 and 7,000 Ng daily. Daily dosages of locally
administered material will be about an order
of magnitude less. Oral administration is also contemplated.
In one preferred embodiment of the invention, the neurotrophic 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 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 conformal coating as described
in U.S. Patent No. 5,529,914 prior to implantation.
The neurotrophic peptides of the invention may also 6e enclosed in micelles or
liposomes. Liposome
encapsulation technology is well known. Liposomes may be targeted to specific
tissue, such as neural tissue. through
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the use of receptors, ligands or antibodies capable of binding the targeted
tissue. The preparation of these
formulations is well known in the art (Radio et al., Meth. Enzymol.,
98:613618, 19831.
There are currently no available pharmaceuticals capable of promoting 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 the 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
neurotrophic 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
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 iodinated neurotrophic l8~mer fragment derived
from saposin C crosses the blood
brain barrier. Thus, the 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
demyeiination in the manner described
above in v'rvo, ex vivo or in vitro. Diseases resulting in demyelination of
nerve fibers including MS, acute
disseminated leukoencephalitis, progressive multifocal leukoencephalitis,
metachromatic leukodystrophy and adrenal
leukodystrophy can be slowed or halted by administration of the neurotrophic
peptides of the invention to the cells
affected by the disease.
The compositions of the present invention can be used in vitro as research
tools for studying 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 for facilitating
growth and maintaining neural cells in vitro.
The peptides of the invention can be synthesized using an automated
sotid~phase protocol well known in
the art on an Applied Biosystems Model 430 peptide synthesizer. All peptides
were purified by high performance
liquid chromatography (HPLC) on a llydac C4 column to an extent greater than
95% prior to use.
The following examples are illustrative and are not intended to limit the
scope of the present invention.
Example 1
Stimulation of neurite outgrowth in vitro
NS20Y neuroblastoma cells were grown in OMEM containing 10% fetal calf serum
(FCS). Cells were
removed with trypsin and plated in 30 mm petri dishes onto glass coverslips.
After 20-24 hours, the medium was
replaced with 2 mI DMEM containing 0.5% FCS plus 0, 0.5, 1, 2, 4 or 8 nglml
TX14(AI. Cells were cultured for
an additional 24 hours, washed with PSS and fixed with Bouin's solution
(saturated aqueous picric
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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.
As shown in Figure 1. TX14(A) and the rat 14-mer both induced neurite
outgrowth in NS20Y ceps.
Increased neurite outgrowth was evident using as little as 0.5 nglml peptide
resulting in a 7% increase for TX14(A)
and rat 14-mer. At 1 mglml, TX14(A) and rat 14-mer resulted in a 109'o and 12%
increase, respectively, in neurite
outgrowth. Both peptides stimulated neurite outgrowth to similar extents at 8
nglmi. This indicates that the
peptides are biologically active.
Example 2
Prevention of cell death in vitro
NS20Y cells were plated as described in Example 1 and grown nn glass
coverslips in 0.5% fetal bovine
serum for 2 days in the presence or absence of 8 nglml TX14(A). Media was
removed and 0.2°Yo trypan blue in PBS
was added to each well. Blue-staining dead cells were 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 t5%. As shown
in Figure 2, TX14(A) reduced the number of trypan blue-positive (dead) cells
by about 7%. This indicates that the
peptide can rescue neural cells from programmed cell death.
Example 3
fx vivo myelination assay
Newborn mouse cerebellar explants are prepared according to Satomi (tool.
Sci., 9:127-137, 19821. Neurite
outgrowth and myefination are observed over 22 days in culture, during the
period when the newborn mouse
cerebellum normally undergoes neuronal differentiation and myelination begins.
A 30~mer nonnative saposin C peptide
containing the sequence shown in SEO ID N0: B (10 ~rglmll is added on the
second day after preparation of the
explants (three control and three treated expiants), and outgrowth of neurites
and myelination is assessed under a
bright field microscope with a video camera. On the eighth day, cultures
containing the peptides are thinner and
more spread out than control cultures. On day 15, peptide-treated cultures
contain many cells with long projections
at the periphery of the explant which are less prominent in untreated control
cultures. Peptide-treated cultures
contain significantly more myelinated axons in the subcortical white matter at
22 days compared to control explants.
Thus, the peptides of the invention induce increased myelination in
differentiating cerebellum ex vivo.
Example 4
Prevention of demyeiination
The prevention of Schwann cell death is correlated with prevention of
demyelination. Schwann cells contain
an extensive myelin sheath. The addition of a non-native 20 mer peptide
containing the sequence shown in SEn ID
N0: 8 to Schwann cells in culture reduces Schwann cell death in a dose-
dependent manner and stimulates the
incorporation of sulfatide, myelin-specific lipids, into Schwann cells.
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Example 5
Use of oeotides in treating traumatic ischemic CNS lesions
Humans with traumatic lesions to the spinal cord receive intracerebrospinal or
direct injection of about 100
Nglml TX14(A) or other peptide encompassed by SEO ID N0: 8 in a sterile saline
solution or in depot form to enable
stow, continuous release of the peptide at the lesion site. Improvement is
assessed by gain of motor nerve function
(i.e. increased limb movementl. Treatments continue until no further
improvement occurs.
Example 6
Use of oeutides in treating demvelination disorders
Patients diagnosed with early stage MS are given peptide TX14(A) or other
peptide encompassed by SED
ID N0: 8 by direct intravenous injection into the cerebrospinal fluid using
the same dose range as in Example 3.
Dosages are repeated daily or weekly and improvement in muscle strength,
muscuioskeletal coordination and
myelination (as determined by MRI) is gbserved.
Example 7
Alleviation of neurooathic yain in Chung model rats
This example describes the effects of bolus intrathecal injection of TX 14(A)
and other peptides encompassed
by SEO iD N0: 8 in the Chung experimental model of peripheral neuropathic
pain. Each peptide is chemically
synthesized, purified, dissolved in sterile PBS and buffered to neutral pH.
The surgical procedure previously described
by Kim et al. (Pain, 50:355, 1992) is performed on mate rats to induce an
allodynic state. A spinal catheter is
introduced two weeks after surgery, Five days later, the peptides are
administered at 0.007, 0.07 and 0.7 pglrat.
Pressure thresholds are then determined using calibrated von Frey hairs. The
longer the time taken for an animal
to withdraw the paw in response to applied pressure, the less severe the
neuropathic pain. The peptides significantly
increase the threshold pressure, indicating a significant alleviation of
neuropathic pain.
Example 8
Treatment of sensory neurooathv
Mice are administered taxol in order to induce sensory neuropathy. Taxoi-
treated mice are administered
50 ~uglkg, 100 auglkg or 250 Nglkg of TX14(A) or other peptide encompassed by
SED ID N0: 8. The loss of thermal
sensation is measured using a Hargreaves sensory testing apparatus as an
indicator or sensory neuropathy. Each
of the three doses of peptide is effective in inhib'tting loss of thermal
sensation in taxohtreated mice. Thus, the
synthetic saposin C-derived peptides of the invention effectively inhibit
sensory neuropathy.
Example 9
Alleviation of neurooathic pain in diabetic rats
This example describes the effects of intraperitoneal administration of
TX141A) or other peptide
encompassed by SEO ID N0: 8 in a rat model of diabetic neuropathy. .
Rats are made diabetic by a single intraperitoneal injection of streptozotgcin
(50 mgJkg body weight, freshly
dissolved in 0.9% sterile saline) to ablate pancreatic ,Q cells and induce
insulin deficiency as described by Calcutt
et ai. (Pain, 68:293-299, 19961. Two days later, diabetes is confirmed in
streptozotocin-injected rats by measuring
9
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WO 98/42746 PCTlUS98/05503
blood glucose levels. Streptozotocin-injected animals with a blood glucose
concentration below 15 mmoill were
excluded from subsequent studies, according to the commonly accepted
definition of non-fasting hyperglycemia in
studies of diabetes in rats.
Both diabetic and control rats are studied at 8 weeks by analyzing the
behavioral response to the noxious
chemical formalin as an indicator of allodynia (Calcutt et al., supra, 1996).
Briefly, rats receive a subcutaneous
injection of freshly-prepared formalin (50 NI of 0.59'a solution in sterile
saline) into the dorsal surface of the right
hind paw. This concentration of formalin induces sub-maximal behavioral
responses in control rats and allows
detection of hyperaigesia in diabetic rats during phases 0 and 2 (Calcutt et
al., Eur. J. Pharmacvl., 285:189-197,
1995). Animals are transferred to an observation chamber constructed to allow
continuous visualization of the paws.
The number of flinches during one minute periods is counted at 5 minute
intervals for the next 60 minutes by an
observer who is unaware of the treatment group of each animal. Phase 1 is
defined as the initial measurement of
flinching (1-2 and 5-6 minutes post injection); the Q (quiescent) phase as the
measurements made at 10-11, 15-16
and 20-21 minutes; and Phase 2 as all subsequent measurements post-injection,
as previously defined for studies
of diabetic rats (see, for example, Malmberg et al., Neurosci. Lett., 161:45-
48, 19931. Comparisons of activity
during each phase are made by summing the flinches at measurement points
within the phase. diabetic rats five
an abnormal flinch response.
Diabetic rats are divided in two groups of four ananals each which are
administered saline, TX141A) or
another peptide encompassed by SEfl ID N0: 8, respectively. Two hours before
treatment with 0.5% formalin, the
diabetic rats are treated with saline or Z00 ~glkg peptide using
intraperitoneal administration. Administration of
peptide completely prevents the abnormal flinch response in Phase 1 and
ameliorates the response in Phase 2 by
70%. Thus, parenteral administration of peptide alleviates the pain from
formalin injection and improves motor neuron
function in a rat model of painful diabetic neuropathy.
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.
,.
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WO 98/42746 PCT/US98105503
SEQUENCE LISTING
(1) GENERAL INFORMATION
(i) APPLICANT: MYELOS NEUROSCIENCES CORP.
(ii) TITLE OF THE INVENTION: SYNTHETIC SAPOSIN C-DERIDED
NEUROTROPHIC PEPTIDES
fiii) NUMBER OF SEQUENCES: B
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Knobbe, Martens, Olson & Bear
(B) STREET: 620 Newport Center Drive, 16th Floor
(C) CITY: Newport Beach
(D) STATE: CA
(EI COUNTRY: U.S.A.
(F) ZIP: 92660
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Diskette
(B) COMPUTER: IBM Compatible
(C) OPERATING SYSTEM: DOS
(D) SOFTWARE: FastSEO for Windows Version 2.0
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
fC) CLASSIFICATION:
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: 081823,425
(B) FILING DATE: 24-MAR-1997
(viii? ATTORNEYIAGENT INFORMATION:
(A) NAME: Bartfeld, Neil S
(B) REGISTRATION NUMBER: 39,901
ICI REFERENCEIDOCKET NUMBER: MYELOS.005VPC
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: 619-235-8550
(B) TELEFAX: 619-235-0176
(C) TELEX:
(2) INFORMATION FOR SEQ ID N0:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 amino acids
11
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WO 98/42746 PCT/US98/05503
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:1:
Cys Glu Phe Leu Ual Lys Glu llal Thr Lys Leu Ile Asp Asn Asn Lys
1 5 10 15
Thr Glu Lys Glu Ile Leu
(2) INFORMATION FOR SEO ID N0:2:
(i) SEQUENCE CHARACTERISTICS:
(AI LENGTH: 18 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
Iii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEO ID N0:2:
Tyr Lys Giu Vai Thr Lys Leu Ife Asp Asn Asn Lys Thr Glu Lys Glu
1 5 10 15
Ile Leu
(2) INFORMATION FOR SEO ID N0:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:3:
Leu Ile Asp Asn Asn Lys Thr Glu Lys Glu Ile Leu
1 5 t0
(2) INFORMATION FOR SEO ID N0:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
12
CA 02284105 1999-09-13
WO 98/4746 PCT/US98/05503
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAMEIKEY: Modified amino acid
(B) LOCATION: 2...2
(0) OTHER INFORMATION: D-alanine
(xi) SEQUENCE DESCRIPTION: SEQ 10 N0:4:
Thr Xaa Leu Ile Asp Asn Asn Ala Thr Glu Glu Ile Leu Tyr
1 5 10
(2) INFORMATION FOR SEQ ID N0:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:5:
Ser Glu Leu Ile Ile Asn Asn Ala Thr Glu Glu Leu Leu Tyr
1 5 10
(2) INFORMATION FOR SEQ lD N0:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(C) STRANOEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:6:
Thr Lys Leu Ile Asp Asn Asn Lys Thr Glu Lys Glu Ile Leu Asp
1 5 t0 15
(2) INFORMATION FOR SEQ ID N0:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 amino acids
(B) TYPE: amino acid
(C) STRANOEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRtPTtON: SEQ ID N0:7:
13
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WO 98/42746 PCT/US98/05503
Leu Ile Asp Asn Asn Lys Thr Glu Lys Glu lle leu
1 5 10
(2) INFORMATION FOR SEQ 10 NO:B:
(i) SEQUENCE CHARACTERISTICS:
(Al LENGTH: 12 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
fD) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAMEIKEY: Modified amino acid
(B) LOCATION: 1...1
(D) OTHER INFORMATION: leu, ile
(AI NAMEIKEY: Modified amino acid
(B) LOCATION: 3...3
(D) OTHER INFORMAT10N: any amino acid
(A) NAMEIKEY: Modified amino acid
(B) LOCATION: 6...6
(D) OTHER INFORMATION: any amino acid
(A) NAMEIKEY: Modified amino acid
(B) LOCATION: 8...8
(DI OTHER INFORMATION: fys, arg, his, asp, glu
(A) NAME1KEY: Modified amino acid
(B) LOCATION: 9...9
(D) OTHER INFORMATION: absent, lys, arg, his, asp, glu, gly
(A) NAMEIKEY: Modified amino acid
(B) LOCATION: 10...10
(D) OTHER INFORMATION: lys, arg, his, asp, glu
(A) NAMEIKEY: Modified amino acid
(B) LOCATION: 11...11
(D) OTHER INFORMATION: any amino acid
(A) NAMEIKEY: Modified amino acid
(B) LOCATION: 12...12
(D) OTHER INFORMATIDN: any amino acid
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:8:
14
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WO 98/42746 PCT/US98/05503
Xaa Ile Xaa Asn Asn Xaa Thr Xaa Xaa Xaa Xaa Xaa
10
