Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
~
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CYCLIC PROSAPOSIN-DERIVED PEPTIDES AND USES THEREOF
Field of the Invention
The present invention relates to neurotrophic and analgesic peptides. More
particularly, the invention relates to cyclic peptides derived from the active
region of
saposin C which have neurotrophic and analgesic effects.
Background of the Invention
Neurotrophic factors are proteins or peptides capable of affecting the
survival,
target innervation and/or function of neuronal cell populations (Barde, Neuron
2:1525
1534, 1989). The efficacy of neurotrophic factors both in vitro and in vivo
have been
well-documented. For example, nerve growth factor (NGF) acts as a trophic
factor for
forebrain cholinergic, peripheral and sensory neurons (Hefti et al.,
Neurobiol. Aging
10:515-533, 1989) and can reverse naturally-occurring as well as physical
traumatic
injuries to peripheral nerves (Rich et al., J. Neurocytol. 16:261-268, 1987).
Brain-
I S derived neurotrophic factor (BDNF) is a trophic factor for peripheral
sensory neurons,
dopaminergic neurons of the substantia nigra, central cholinergic neurons and
retinal
ganglia (Henderson et al., Restor. Neurol. Neurosci. 5:1 S-28, 1993). BDNF has
been
shown to prevent naturally-occurring cell death both in vitro and in vivo
(Hofer et al.,
Nature 331:262-262, 1988). Ciliary neurotrophic factor (CNTF) promotes
survival of
chicken embryo ciliary ganglia in vitro and supports survival of cultured
sympathetic
sensory and spinal motor neurons (Ip et al., .l. Physiol. Paris 85:122-130,
1991).
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 multifocal
leukoencephalitis,
metachromatic leukodystrophy and adrenal leukodystrophy. The peripheral
nervous system
(PNS) can also be afflicted with demyelination, such as that occurring in
Guillain-Barre
syndrome (Pathologic Basis of Disease, Robbins et al. eds., W.B. Saunders,
Philadelphia,
1979, pp. 1578-1582).
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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.
Neuropathic pain results from nerve injury, such as a nerve compression or
crush
and traumatic injury to the spinal cord, and is often long-lasting or chronic.
Most
traumatic nerve injuries also cause the formation of neuromas, in which pain
occurs as a
result of aberrant nerve regeneration. In addition, cancer-related neuropathic
pain results
when tumor growth compresses adjacent nerves, brain or spinal cord.
Neuropathic pain
is also associated with diseases including diabetes and alcoholism. In most
cases,
neuropathic pain is resistant to current drugs. These drugs also have serious
side-effects.
U.S. Patent Application Serial No. 08/611,307 provides a method for
alleviating or
preventing neuropathic pain by administering to an individual an effective
amount of an
active fragment of prosaposin.
Prosaposin is the precursor of a group of four small heat-stable glycoproteins
which are required for hydrolysis of glycosphingolipids by lysosomal
hydrolases
(Kishimoto et al., J. Lipid Res. 33:1255-1267, 1992). Prosaposin is
proteolytically
processed in lysosomes, generating saposins A, B, C and D (O'Brien et al.,
FASEB J.
5:301-308, 199I). O'Brien et al. (Proc. Natl. Acad. Sci. U.S.A. 91:9593-9596,
1994),
U.S. Patent No. 5,571,787 and published PCT Application No. W095/03821
disclose
that prosaposin, saposin C stimulate neurite outgrowth and promote increased
myelination. In addition, U.S. Patent Nos. 5,571,787, 5,696,080, 5,714,459 and
published PCT application No. W095/03821 disclose that a 22-mer peptide
(CEFLVKEVTKLIDNNKTEKEIL; SEQ ID NO: 1 ) consisting of amino acids 8-29 of
human saposin C stimulates neurite outgrowth. These references also disclose
that an
18-mer peptide (YKEVTKLIDNNKTEKEIL; SEQ ID NO: 2) contained within the
active 22-mer of saposin C (with V replaced by Y) also promotes neurite
outgrowth and
is capable of crossing the blood brain barrier. O'Brien et al. (FASEB J. 9:681-
685,
1995) show that the 22-mer stimulates choline acetyltransferase activity and
prevented
neuronal cell death. The active neuritogenic fragment was localized to a
linear 12-mer
sequence located within the amino terminus of saposin C (LIDNNKTEKEIL; SEQ m
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NO: 3) (O'Brien et al., FASEB J. 9:681-685, 1994). Liepinsh et al. (Nature
Struct. Biol.
4:793-795) indicated that the loop conformation of a neurotrophically active
peptide
segment of NK-lysin (residues 17-30) could be mimicked by a circular peptide.
A major obstacle to the in vivo therapeutic use of peptides is their
susceptibility
to proteolytic degradation. The present invention provides cyclic prosaposin
peptidomimetics which have good resistance to proteolytic degradation and are
capable
of crossing the blood brain barrier.
Summary of the Invention
One embodiment of the present invention is a cyclic neurotrophic and analgesic
peptide having between about 11 and 25 amino acids, and including the sequence
X,XzX3NNX4TX5X6X~Xg, wherein X, is a hydrophobic amino acid (alanine, leucine,
isoleucine, valine, tyrosine, tryptophan, phenylalanine or methionine); Xz is
a
hydrophobic amino acid, X3 is aspartic acid, glutamic acid, lysine or
arginine; N is
asparagine, X4 is any amino acid; T is threonine; XS is glutamic acid or
aspartic acid; Xb
is any amino acid, X, is a hydrophobic amino acid; and X8 is a hydrophobic
amino acid.
Preferably, the peptide has the amino acid sequence shown in SEQ ID NO: 5 or
6.
The present invention also provides a composition comprising the peptide
described above in a septum sealed vial, formulated with a controlled release
material, in
lyophilized form, in liposomal form, in a form suitable for topical
administration or in
unit dosage form.
Another embodiment of the present invention is a method for inducing
myelination or inhibiting demyelination in a mammal, comprising administering
to a
mammal afflicted with demyelination a pharmaceutically effective demyelination
inhibiting amount of a cyclic peptide having between about 11 and 25 amino
acids, and
including the sequence X,XZX3NNX.4TXSX6X~Xg, wherein X~ is a hydrophobic amino
acid (leucine, isoleucine, valine, tyrosine, tryptophan, phenylalanine or
methionine); Xz is
a hydrophobic amino acid, X~ is aspartic acid, glutamic acid, lysine or
arginine; N is
asparagine, X4 is any amino acid; T is threonine; XS is glutamic acid or
aspartic acid; X6
is any amino acid, X~ is a hydrophobic anuno acid; and Xx is a hydrophobic
amino acid.
In one aspect of this preferred embodiment, the demyelination is due to
multiple
sclerosis, ischemic injury or traumatic injury. Preferably, the administration
is
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intravenous, intramuscular, intradermal. subcutaneous, intracranial,
intracerebrospinal or
topical. Advantageously, the peptide is administered in a pharmaceutically
acceptable
carrier. In another aspect of this preferred embodiment, the peptide is
enclosed in a
lamellar structure. Preferably, the peptide has the sequence shown in SEQ m
NOS: 5 or
6. In another aspect of this preferred embodiment, the mammal is a human.
The present invention also provides a method for inhibiting neural
degeneration
or promoting neurite outgrowth in neural tissue, comprising: contacting neural
tissue
susceptible to such degeneration with an effective neural degeneration-
inhibiting amount
of a cyclic peptide having between about 11 and 25 amino acids, and including
the
sequence X,X2X3NNX4TXSX6X~X~, wherein X, is a hydrophobic amino acid (leucine,
isoleucine, valine, tyrosine, tryptophan, phenylalanine or methionine); XZ is
a
hydrophobic amino acid, X3 is aspartic acid, glutamic acid, lysine or
arginine; N is
asparagine, X4 is any amino acid; T is threonine; XS is glutamic acid or
aspartic acid; X6
is any amino acid, X~ is a hydrophobic amino acid; and X8 is a hydrophobic
amino acid.
1 S Preferably, the peptide has the amino acid sequence shown in SEQ )D NOS: 5
or 6. .
Preferably, the administration is intravenous, intramuscular, intradermal.
subcutaneous,
intracranial, intracerebrospinal or topical. Advantageously, the peptide is
administered in
a pharmaceutically acceptable carrier. In another aspect of this preferred
embodiment,
the peptide is enclosed in a lamellar structure. Preferably, the mammal is a
human.
Still another embodiment of the invention is a method for treating neuropathic
pain in a mammal in need thereof, comprising the step of administering to a
mammal
suffering from neuropathic pain an amount of a cyclic peptide effective to
inhibit
neuronal degeneration, wherein said peptide peptide has between about 11 and
25 amino
acids, and including the sequence X,X2X3NNX.~TX5X6X~Xg, wherein X, is a
hydrophobic amino acid (leucine, isoleucine, valine, tyrosine, tryptophan,
phenylalanine
or methionine); XZ is a hydrophobic amino acid, X3 is aspartic acid, glutamic
acid, lysine
or arginine; N is asparagine, X4 is any amino acid; T is threonine; XS is
glutamic acid or
aspartic acid; X6 is any amino acid, X~ is a hydrophobic amino acid; and Xg is
a
hydrophobic amino acid. Preferably, the administering step is intravenous,
intramuscular, intradermal, subcutaneous, intracranial, intracerebrospinal,
topical or oral.
Advantageously, the peptide has the amino acid sequence shown in SEQ B7 NOS: 5
or
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6. Advantageously, the peptide is administered in a pharmaceutically
acceptable carrier.
In another aspect of this preferred embodiment, the peptide is enclosed in a
lamellar
structure. Preferably, the mammal is a human.
The present invention also provides a cyclic peptide having between about 11
and
25 amino acids, and including the sequence X,XZX3NNX4TXSX6X7X~, wherein X, is
a
hydrophobic amino acid (leucine, alanine isoleucine, valine, tyrosine,
tryptophan,
phenylalanine or methionine); XZ is a hydrophobic amino acid, X~ is aspartic
acid,
glutamic acid, lysine or arginine; N is asparagine, X4 is any amino acid; T is
threonine; XS
is glutamic acid or aspartic acid; X6 is any amino acid, X, is a hydrophobic
anuno acid;
and XR is a hydrophobic amino acid, for use in inducing myelination or
inhibiting
demyelination in a mammal. Preferably, the peptide has the sequence shown in
SEQ m
NOS: 5 or 6.
Another embodiment of the invention is a cyclic peptide having between about
11
and 25 amino acids, and including the sequence X,XZX3NNX4TXSX6X7X&, wherein X,
is
1 S a hydrophobic amino acid (leucine, alanine isoleucine, valine, tyrosine,
tryptophan,
phenylalanine or methionine); XZ is a hydrophobic amino acid, X3 is aspartic
acid,
glutamic acid, lysine or arginine; N is asparagine, XQ is any amino acid; T is
threonine; Xs
is glutamic acid or aspartic acid; X6 is any amino acid, X~ is a hydrophobic
amino acid;
and XR is a hydrophobic amino acid for use in inhibiting neural degeneration
or
promoting neurite outgrowth. Preferably, the peptide has the sequence shown in
SEQ
ID NOS: 5 or 6.
The present invention also provides a cyclic peptide having between about 11
and
amino acids, and including the sequence X,XZX3NNX4TXSX6X~Xg, wherein X, is a
hydrophobic axnino acid (leucine, alanine isoleucine, valine, tyrosine,
tryptophan,
25 phenylalanine or methionine); XZ is a hydrophobic amino acid, X3 is
aspartic acid,
glutamic acid, lysine or arginine; N is asparagine, X4 is any amino acid; T is
threonine; XS
is glutamic acid or aspartic acid; X6 is any amino acid, X~ is a hydrophobic
amino acid;
and XR is a hydrophobic amino acid for use in treatment of neuropathic pain.
Preferably,
the peptide has the sequence shown in SEQ ID NOS: 5 or 6
Detailed Description of the Preferred Embodiment
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The present invention provides cyclic saposin C-derived peptides having
between
about 11 and 25 amino acids, and including the consensus sequence
X,XZX3NNX4TXSX6X,Xg (SEQ ID NO: 4), wherein X, is a hydrophobic amino acid
(alanine, leucine, isoleucine, valine, tyrosine, tryptophan, phenylalanine or
methionine);
XZ is a hydrophobic amino acid, X3 is aspartic acid, glutamic acid, lysine or
arginine; N is
asparagine, X4 is any amino acid; T is threonine; XS is glutamic acid or
aspartic acid; X6
is any amino acid, X~ is a hydrophobic amino acid; and XR is a hydrophobic
amino acid.
In a preferred embodiment, the peptide has the sequence: cyclo-[LLDNNKTEKLYJ
(SEQ ID NO: S) or cyclo-[LIDNNATEEILJ (SEQ ID NO: 6). Due to their
constrained structures, these cyclic prosaposin peptidomimetics are
significantly more
resistant to enzymatic degradation and are capable of crossing the blood brain
barrier to
a greater extent than corresponding linear peptides. The cyclic peptides of
the invention
are equally as effective or more effective in stimulating neurite outgrowth
than a known
highly-active linear prosaposin peptidomimetic ("prosaptide") having the
sequence
TXLIDNNATEEILY, wherein X is D-alanine (SEQ ID NO: 7) (Example 1 ). Moreover,
the
cyclic peptides of the invention are more effective than prosaptide in
preventing neural cell
death in vitro (Example 2). These cyclic peptides lack free amino- and carboxy-
terminal
ends. Thus, they are more resistant to degradation in vivo by aminopeptidases
and
carboxypeptidases which degrade peptides from the amino-and carboxy-termini.
Cyclic saposin C-derived peptides comprising the active 11-mer region (SEQ 11?
NO:
4), and neurotrophic analogs thereof, have utility in promoting functional
recovery after toxic,
traumatic, ischemic, degenerative and inherited lesions to the peripheral and
central nervous
system. In addition, these peptides stimulate myelination and counteract the
effects of
demyelinating diseases. These peptides stimulate the outgrowth of neurons,
promote
myelination, promote neuroprotection and prevent programmed cell death in
neuronal tissues
in mammals, preferably humans. 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. As used herein, a neuropathy is a functional
disturbance or
pathological change in the peripheral nervous system and is characterized
clinically by sensory
3 0 or motor neuron abnormalities. The peptides are also useful as analgesics,
particularly for the
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treatment of neuropathic pain in a mammal, preferably a human, which can
develop days or
months after a traumatic injury and is often long-lasting or chronic.
The second asparagine residue within the native prosaposin sequence
(corresponding
to second "N" in SEQ >D NO: 4 ) is known to be glycosylated with N-
acetylglucosamine
which may provide some resistance to proteolytic degradation. The synthetic
modification of
this asparagine residue within the instant non-native saposin C-derived
peptides by standard
methods (i.e. Merrifield 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
neurai cells by
administering to the cells an effective, neurite outgrowth or myelin-
facilitating amount of a
saposin C-derived peptide encompassing the active cyclic 11-mer region shown
in SEQ )D
N0:4.
Saposin C-derived cyclic peptide analogs of the invention can differ from
saposin C
sequences or SEQ >D NOS: 4-6, for example, by replacement of one or more
lysine and/or
arginine residues; replacement of one or more tyrosine and/or phenylalanine
residues, deletion
of one or more phenylalanine residues and/or conservative replacement of one
or more amino
acids within the peptide. The replacement or deletion of lysine/arginine and
tyrosine/phenylalanine residues will reduce the susceptibility of peptide
degradation by trypsin
and chymotrypsin, respectively. The cyclic neurotrophic and myelinotrophic
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 11 and 25 amino acids
and include
therein the sequence shown in SEQ >D NO: 4.
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,
histidine); the acidic charged amino acids (aspartic acid, glutamic acid); the
non-polar amino
acids (alanine, vaiine, leucine, isoleucine, proline, phenylalanine,
methionine, tryptophan); the
uncharged polar amino acids (glycine, asparagine, glutamine, cysteine, serine,
threonine,
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tyrosine); and the aromatic amino acids (phenylalanine, 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, or a similar conservative
replacement of an amino
acid with a structurally related amino acid will not significantly affect the
properties of the
peptide. The cyclic saposin C-derived sequences including SEQ )D NO: 4 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, hydrophilicity and the like.
Sequences outside the
active neurotrophic region are not typically required for activity. Thus, in
most instances, the
subject peptide will be active regardless of the identity of these sequences.
Again, any such
peptide can be screened for such activity using the protocols described
herein.
The ability of any such cyclic peptide to stimulate neurite outgrowth, prevent
neural
1 S cell death, promote myelination, inhibit demyelination, alleviate
neuropathic pain and treat
sensory neuropathy can easily be determined by one of ordinary skill in the
art using the
procedures described in Examples 1-9. Methods for assaying the abilities of
these peptides to
promote myelination and to inhibit demyelination are set forth 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 S ng/ml. This amount
or more of the
cyclic synthetic peptides of the invention for in vitro use is contemplated.
Typically,
concentrations in the range of 0.1 p,g/ml to about 10 pg/ml 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 vivo by directly administering
the
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 vivo, 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 or tissue in sufficient
quantity to give the
desired local concentration of peptide. In the peptides lacking lysine and
arginine residues,
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proteolytic degradation is reduced. 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
fonmation of peptide esters include lauric, myristic, palinitic, 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. Such
modifications will
facilitate the ability of the peptide to cross the blood brain burner due to
increased
hydrophobicity.
For treatment of neural disorders, direct intracranial injection or injection
into the
1 S cerebrospinal fluid may also be used in su~cient 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, intracerebrospina.l, intramuscular,
intradermal,
subcutaneous, intracranial, intranasal, epidural, topical and oral. For use as
an anals~esic.
administration by direct intramuscular or intravenous injection is preferred.
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. Appropriate daily
systemic
dosages of the peptides of the invention based on the body weight of the
vertebrate for
treatment of neural diseases, demyelination or as an analgesic in general or
for treatment of
neuropathic pain are in the range of from about 0.01 to about 10,000 p.g/kg.
More
preferably, daily systemic dosages are between about 0.1 and 1,000 p.g/kg.
Most preferably,
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daily systemic dosages are between about 10 and 100 ~g/lcg. Thus, for the
typical 70 kg
human, dosages can be between about 0.7 and 700,000 pg daily; more preferably
between
about 7 and 70,000 pg daily; and most preferably between about 700 and 7,000
pg/kg. 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 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., 98:613-6I8, 1983).
There are currently no commercially available pharmaceuticals capable of
promoting
firll firnctional regeneration and restoration of the structural integnty 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
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cross 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.
Peptides of the present invention having up to about 22 amino acids will also
cross this barrier
and can thus be administered intravenously, with greater transport occurring
for shorter
peptides. 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, 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. Reversal of demyelination diseases or other neural damage is
also
contemplated.
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 are synthesized on a solid support using
established
methods and Fmoc chemistry using an automated solid-phase protocol well known
in the art
(Traeciak et al., Tetrahedron Letf. 33:4557-4561, 1992) on a Protein
Technologies
Symphony peptide synthesizer. The cyclized peptides were cleaved from the
sofid support
using TFA/water/triisopropylsilane (95:2.5:2.5). The peptides were purified
using reverse
phase HPLC on a C-18 column eluting with 0.3% triffuoroacetic acid (TFA) in
acetonitrile.
Mass spectral analysis of the peptides shown in SEQ 1D NOS: 5 (peptide A) and
6 (peptide
B) confirmed that cyclic peptides had been synthesized [SEQ )D NO: 5: Mfi'1333
(expected), 1333 (observed); SEQ m NO: 6 MH'1127 (expected), 1127 (observed)].
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
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NS20Y neuroblastoma cells were grown in DMEM containing i 0% 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 ml DMEM
containing 0.5%
FCS plus various concentrations of the peptide A, peptide B or TX14(A). Cells
were cultured
for an additional 24 hours, washed with PBS and fixed with Bouin's solution
{saturated
aqueous picric acid/formalin/acetic acid I 5: S : I ) 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 di$'erent portions of each dish to
determine the
I 0 percentage of neurite bearing cells and assays were performed in
duplicate.
All three peptides induced neurite outgrowth in NS20Y cells. The Tt,~ (EDso),
which
is the concentration of peptide resulting in 50% increased neurite outgrowth,
was 1.0 nglml
for TX14(A) and for peptide B. The Tin for peptide A was 0.6 ng/ml. Thus,
peptide B was
as effective as TX14{A), while peptide A was more effective than TX14(A). This
indicates
that the cyclized peptides of the present invention have excellent activity in
comparison to an
established standard "prosaptide".
Example 2
Prevention of cell death in vitro
NS20Y cells were 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 TX14(A),
peptide A or
peptide B Media was removed and 0.2% 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 S%.
TX14(A), peptide A and peptide B reduced the number of trypan blue-positive
(dead) cells.
The TI,~ (EDso) for prevention of neural cell death for TX14(A) was 1.0 ng/ml.
Peptides A
and B were more potent, with Tln values of 0.6 nglml and 0.8 ng/ml,
respectively. This
indicates that the cyclic peptides have exceptional activity in rescuing
neural cells from
programmed cell death in comparison to a standard linear "prosaptide".
Example 3
F.x vivo myelination assay
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Newborn mouse cerebellar explants are prepared according to Satomi (Zool.
Sci.,
9:127-137, 1992). Neurite outgrowth and myelination are observed over 22 days
in culture,
during the period when the newborn mouse cerebellum normally undergoes
neuronal
differentiation and myelination begins. A cyclic saposin C-derived peptide
having between 11
and 25 amino acids and containing the sequence shown in SEQ D7 NO: 4 ( 10
p,g/mI) is added
on the second day after preparation of the explants (three control and three
treated explants),
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
1 S Prevention of dem~elination
The prevention of Schwann cell death is correlated with prevention of
demyelination.
Schwann cells contain an extensive myelin sheath. The addition of. a cyclic
saposin C-derived
peptide having between 11 and 25 amino acids and containing the sequence shown
in SEQ
ID NO: 4 to Schwann cells in culture reduces Schwann cell death in a dose-
dependent
manner and stimulates the incorporation of sulfatides, myelin-specific lipids,
into Schwann
cells. This indicates that the cyclic peptides of the invention can prevent
demyelination due to
Schwann cell death.
Example 5
Use of nentides in treatine traumatic ischemic CNS lesions
Humans with traumatic lesions to the spinal cord receive intracerebrospinal or
direct
injection of about 100 pg/ml of. a cyclic saposin C-derived peptide having
between 11 and 25
amino acids and containing the sequence shown in SEQ )D NO: 4 in a sterile
saline solution
or in depot form to enable slow, continuous release of the peptide at the
lesion site.
Improvement is assessed by gain of motor nerve function (i.e. increased limb
movement).
Treatments continue until no fizrther improvement occurs.
Example 6
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Use of nentides in treating dem e~lination disorders
Patients diagnosed with early stage MS are given a cyclic saposin C-derived
peptide
having between 11 and 25 amino acids, and containing the sequence shown in SEQ
B7 NO:
4, by direct intravenous injection into the cerebrospinal fluid using the same
dose range as in
Example 3. Dosages are repeated daily every 2-5 days, or weekly and
improvement in
muscle strength, musculoskeletal coordination and myelination (as determined
by MRI) is
observed.
Example 7
Alleviation of neuropathic yain in Chun~ model rats
This example describes the effects of bolus intrathecal injection of a cyclic
saposin C-
derived peptide having between 11 and 25 amino acids and containing the
sequence shown in
SEQ 1'D NO: 4 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 male 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
pg/rat. 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 neuropathy
Mice are administered taxol in order to induce sensory neuropathy. Taxol-
treated
mice are administered 50 p,g/kg, 100 ~g/kg or 250 p.g/kg of a cyclic saposin C-
derived
peptide having between 11 and 25 amino acids and containing the sequence shown
in SEQ
m NO: 4. 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 preventing or retarding loss of thermal sensation in taxol-
treated mice. Thus, the
synthetic saposin C-derived peptides of the invention effectively inhibit
sensory neuropathy.
Example 9
Alleviation of neuropathic pain in diabetic rats
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This example describes the effects of kg of a cyclic saposin C-derived peptide
having
between 1 l and 25 amino acids and containing the sequence shown in SEQ D7 NO:
4 by
intraperitoneal administration in a rat model of diabetic neuropathy.
Rats are made diabetic by a single intraperitoneal injection of streptozotocin
(50
mg/kg body weight, freshly dissolved in 0.9% sterile saline) to ablate
pancreatic j3 cells and
induce insulin deficiency as described by Calcutt et al. (Pain, 68:293-299,
1996). Two days
later, diabetes is confirmed in streptozotocin-injected rats by measuring
blood glucose levels.
Streptozotocin-injected animals with a blood glucose concentration below 15
mmol/1 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 allodvnia
(Calcutt et al., supra,
1996). Briefly, rats receive a subcutaneous injection of freshly-prepared
formatin (50 p,l of
0.5% solution in sterile saline) into the dorsal surface of the right hind
paw. This
concentration of forznalin induces sub-maximal behavioral responses in control
rats and
allows detection of hyperalgesia in diabetic rats during phases Q and 2
(Calcutt et al., Eur. J.
Pharmacol., 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-? and 5-6 minutes post injection); the Q (quiescent) phase as
the
measurements made at 10-i l, 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, Malinberg et al., Neurosci. Lett., 161:45-48, 1993). 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 animals each which are
administered
saline or a cyclic saposin C-derived peptide having between 11 and 25 amino
acids and
containing the sequence shown in SEQ m NO: 4, respectively. Two hours before
treatment
with 0.5% formalin, the diabetic rats are treated with saline or 200 ~g/kg
peptide using
intraperitoneal administration. Administration of peptide completely prevents
the abnormal
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WO 00/12553 PCT/US99/19378
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.
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SEQUENCE LISTING
( 1 ) GENERAL INFORMATION:
(i) APPLICANT: MYELOS CORPORATION
{ii) TITLE OF INVENTION: CYCLIC ANALGESIC AND NEUROTROPHIC
PEPTIDES
(iii) NUMBER OF SEQUENCES: 6
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Knobbe, Martens, Olson & Bear
(B) STREET: 620 Newport Center Drive, 16th Floor
(C) CTTY: Newport Beach
(D) STATE: CA
(E) COUNTRY: U.S.A.
(F) ZIP: 92660
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Diskette
(B) COMPUTER: IBM Compatible
(C) OPERATING SYSTEM: Windows
(D) SOFTWARE: FastSEQ for Windows Version 2.Ob
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(C) CLASSIFICATION:
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION I~1LJMBER: 60/098,359
(B) FILING DATE: 28-AUG-1998
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Bartfeld, Neil S
(B) REGISTRATION NUMBER: 39,901
{C) REFERENCE/DOCKET NUMBER: MYELOS.014PR
(ix) TELECOWJNICATION INFORMATION:
(A) TELEPHONE: 619-235-8550
(B) TELEFAX: 619-235-0176
(C) TELEX:
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CA 02341810 2001-02-28
WO 00/12553 PCT/US99/19378
(2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ 1D NO:1:
Cys Glu Phe Leu Val Lys Glu Val Thr Lys Leu Ile Asp Asn Asn Lys
1 5 10 15
Thr Glu Lys Glu Lys
(2) INFORMATION FOR SEQ 1D N0:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ Ifl N0:2:
Tyr Lys Glu Val Thr Lys Leu Ile Asp Asn Asn Lys Thr Glu Lys Glu
1 S 10 15
Ile Leu
(2) INFORMATION FOR SEQ B7 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:
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WO 00/12553 PCT/US99/19378
Leu lle Asp Asn Asn Lys Thr Glu Lys Glu lle Leu
1 5 10
(2) INFORMATION FOR SEQ ID N0:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 11 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
{ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/ICEY: Other
(B) LOCATION: 1...2
(D) OTHER INFORMATION: A,L,I,V,Y,W,F OR M
(A) NAME/KEY: Other
(B) LOCATION: 3...3
(D) OTHER INFORMATION: D,E,K OR R
(A) NAMEJKEY: Other
(B) LOCATION: 6...6
(D) OTHER INFORMATION: ANY AMINO ACID
(A) NAMEJKEY: Other
(B) LOCATION: 8...8
(D) OTHER INFORMATION: D OR E
(A) NAME/KEY: Other
{B) LOCATION: 9...9
(D) OTHER INFORMATION: ANY AMINO ACID
(A) NAME/KEY: Other
{B) LOCATION: 10...11
(D) OTHER INFORMATION: A,L,I,V,Y,W,F OR M
(xi) SEQUENCE DESCRIPTION: SEQ ll~ N0:4:
Xaa Xaa Xaa Asn Asn Xaa Thr Xaa Xaa Xaa Xaa
1 5 10
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(2) INFORMATION FOR SEQ ID NO:S:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 11 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: cyclic
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:S:
Leu Leu Asp Asn Asn Lys Thr Glu Lys Leu Tyr
1 5 10
(2) INFORMATION FOR SEQ ID N0:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 11 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: cyclic
{ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:6:
Leu Ile Asp Asn Asn Ala Thr Glu Glu Ile Leu
1 S 10
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