U.S. patent application number 13/022227 was filed with the patent office on 2011-12-15 for prosaposin as a neurotrophic factor.
This patent application is currently assigned to MYELOS CORPORATION. Invention is credited to Yasuo KISHIMOTO, John S. O'BRIEN.
Application Number | 20110306558 13/022227 |
Document ID | / |
Family ID | 22278796 |
Filed Date | 2011-12-15 |
United States Patent
Application |
20110306558 |
Kind Code |
A1 |
O'BRIEN; John S. ; et
al. |
December 15, 2011 |
PROSAPOSIN AS A NEUROTROPHIC FACTOR
Abstract
Prosaposin, saposin C and various peptide fragments of saposin C
stimulate neurite outgrowth in vitro. In addition, prosaposin and
saposin C promote increased myelination ex vivo. Prosaposin is
present in large neurons of the brain, including both upper and
lower motor neurons.
Inventors: |
O'BRIEN; John S.; (La Jolla,
CA) ; KISHIMOTO; Yasuo; (San Diego, CA) |
Assignee: |
MYELOS CORPORATION
East Brunswick
NJ
|
Family ID: |
22278796 |
Appl. No.: |
13/022227 |
Filed: |
February 7, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11787565 |
Apr 16, 2007 |
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13022227 |
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10464347 |
Jun 18, 2003 |
7524818 |
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11787565 |
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08756031 |
Nov 26, 1996 |
6590074 |
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10464347 |
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08483146 |
Jun 7, 1995 |
5696080 |
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08756031 |
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08100247 |
Jul 30, 1993 |
5571787 |
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08483146 |
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Current U.S.
Class: |
514/17.8 ;
435/375; 514/17.7; 514/17.9; 530/395 |
Current CPC
Class: |
E03B 7/074 20130101;
A61P 25/00 20180101; C07K 14/47 20130101; C07K 14/475 20130101;
C07K 14/71 20130101; C07K 14/715 20130101; Y02A 50/30 20180101;
C07K 7/06 20130101; C07K 14/52 20130101; A61K 9/0048 20130101; A61P
25/28 20180101; A61K 38/00 20130101; Y02A 50/465 20180101; A61K
9/0085 20130101; C07K 14/705 20130101; A61K 9/127 20130101; A61K
9/0051 20130101; A61K 9/19 20130101; C07K 7/08 20130101; A61K
9/0019 20130101; A61P 25/16 20180101 |
Class at
Publication: |
514/17.8 ;
435/375; 514/17.7; 514/17.9; 530/395 |
International
Class: |
A61K 38/17 20060101
A61K038/17; C07K 14/435 20060101 C07K014/435; A61P 25/16 20060101
A61P025/16; A61P 25/28 20060101 A61P025/28; C12N 5/0784 20100101
C12N005/0784; A61P 25/00 20060101 A61P025/00 |
Claims
1. A method for stimulating neural cell outgrowth or increased
myelination, comprising: contacting neuronal cells with a
composition comprising prosaposin or a fragment thereof having the
ability to promote increased neural outgrowth or increased
myelination activity.
2. The method of claim 1 wherein said prosaposin is native.
3. The method of claim 1 wherein said prosaposin is recombinantly
produced.
4. The method of claim 1 wherein said fragment is saposin C.
5. The method of claim 1 wherein said fragment is a peptide
comprising amino acids 8-29 of saposin C.
6. The method of claim 5 wherein said fragment consists essentially
of the active neurotrophic fragment located within amino acids 8-29
of SEQ ID NO: 1.
7. The method of claim 1 wherein said neuronal cells are
neuroblastoma cells.
8. The method of claim 7 wherein said neuroblastoma cells are
selected from the group consisting of: NS20Y, Neuro 2A and N1E 115
cells.
9. The method of claim 1 wherein said neuronal cells are contacted
in vitro.
10. The method of claim 1 wherein said neuronal cells are contacted
in vivo.
11. The method of claim 1 wherein said cells are from mouse
cerebellar explants.
12. A method for treatment of demyelination disorders in a mammal
comprising: identifying a mammal afflicted with said disorder; and
administering to said mammal a pharmaceutically effective
demyelination inhibiting amount of prosaposin or a neurotrophic
fragment thereof.
13. The method of claim 12 wherein said fragment comprises saposin
C.
14. The method of claim 12 wherein said demyelination disorder is
selected from the group consisting of: multiple sclerosis, acute
disseminated leukoencephalitis, progressive multifocal
leukoencephalitis and adrenal leukodystrophy.
15. The method of claim 12 wherein said administration is selected
from the group consisting of: intravenous, intramuscular,
intradermal, subcutaneous, intracranial, intracerebrospinal and
topical.
16. The method of claim 12 wherein said prosaposin or fragment
thereof is administered in a biologically compatible carrier.
17. The method of claim 12 wherein said prosaposin or fragment
thereof is enclosed in a lamellar structure.
18. A method for halting or slowing the progress of neural or
myelin degeneration in neural tissue, comprising: contacting
neuronal tissue susceptible to such degradation with prosaposin or
an active degradation-inhibiting fragment thereof.
19. The method of claim 18 wherein said fragment is saposin C.
20. The method of claim 18 wherein said tissue is in vitro.
21. The method of claim 18 wherein said tissue is in vivo.
22. A method for the treatment of neuronal degenerative diseases of
the central or peripheral nervous system, comprising administering
to a mammal suffering from said disease an amount of a prosaposin
fragment effective to retard or halt neuronal degeneration, wherein
said fragment includes the neurotrophic activity of the peptide of
SEQ ID NO:1.
23. The method of claim 22 wherein said administration is selected
from the group consisting of: intravenous, intramuscular,
intradermal, subcutaneous, intracranial, intracerebrospinal,
topical and oral.
24. The method of claim 22 wherein said disease is a disease of the
central nervous system and said fragment is selected to cross the
blood brain barrier.
25. The method of claim 24 wherein said disease is selected from
the group consisting of: Alzheimer's disease, Parkinson's disease,
stroke, post-polio syndrome and amyotrophic lateral sclerosis.
26. A method for retarding the progress of retinal neuropathy in a
patient by administering to the patient an effective amount of
prosaposin or a neurotrophic fragment thereof.
27. The method of claim 26 wherein said retinal neuropathy is
macular degeneration and said patient is a human over the age of
65.
28. The method of claim 26 wherein said administration is selected
from the group consisting of: topical, intravenous, intraocular and
oral.
29. A pharmaceutical composition comprising prosaposin or a
neurotrophic fragment thereof in unit dosage form.
30. A pharmaceutical composition comprising prosaposin or a
neurotrophic fragment thereof formulated with a controlled release
material.
31. A neural prosaposin receptor protein in isolated or purified
form.
32. The receptor protein of claim 31 wherein said receptor is
isolated from a P100 plasma membrane fraction by affinity
purification using a neurite growth-inducing peptide contained
within the saposin C sequence linked to a solid support.
33. The receptor protein of claim 31 wherein said receptor has a
molecular weight of approximately 20 kDa.
Description
FIELD OF THE INVENTION
[0001] This invention discloses the utilization of prosaposin, or
fragments thereof, as a cytokine, growth factor or neurotrophin.
More specifically, prosaposin may be a neurotrophic factor for
populations of neurons for which neurotrophins have yet to be
identified.
BACKGROUND OF THE INVENTION
[0002] Prosaposin, a 70 kilodalton glycoprotein, 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., (1992) J. Lipid Res., 33: 1255-1267)
Prosaposin is proteolytically processed in lysosomes to generate
saposins A, B, C, and D which exist as four adjacent tandem domains
in prosaposin (O'Brien and Kishimoto, (1991) FASEB J., 5: 301-308)
All four saposins are structurally similar to each other, including
the placement of six cysteines, a glycosylation site and conserved
proline residues.
[0003] Unprocessed prosaposin also exists as an integral membrane
protein and a secreted protein which is present in human milk,
cerebrospinal fluid and seminal plasma. The presence of high
concentrations of unprocessed prosaposin in the central nervous
system indicates that it may play a significant role in addition to
activation of lysosomal hydrolases.
[0004] Prosaposin binds membrane lipids called glycosphingolipids
which are sphingolipids consisting of a carbohydrate head group and
two hydrocarbon chains; a fatty acid and a sphingosine derivative.
Glycosphingolipids are important components of the myelin sheath, a
structure which protects and insulates nerve fibers. Demyelination
is a defect common to a number of central nervous system disorders,
the most common being multiple sclerosis (MS). MS, a chronic
disorder which may lead to total disability, is characterized by
damage to the myelin sheath leaving the axons mostly intact. It is
currently believed that autoimmune mechanisms, perhaps
virally-induced, may play a role in development of the disease.
There is currently no effective treatment for MS. Other central
nervous system disorders involving demyelination include acute
disseminated encephalomyelitis, amyotrophic lateral sclerosis,
acute necrotizing hemorrhagic leukodystrophy, progressive
multifocal leukoencephalitis, metachromatic leukodystrophy and
adrenal leukodystrophy. An example of a demyelinating disease of
the peripheral nervous system is Guillain-Barre syndrome
(Pathologic Basis of Disease, Robbins, S. L. and Cotran, R. S.,
eds, W. B. Saunders, Philadelphia, (1979), pp. 1578-1582).
[0005] Post-polio syndrome is characterized by muscle fatigue and
decreased endurance with accompanying muscle weakness and atrophy.
The disease is believed to be caused in part by the same type of
spinal cord motor neurons that are damaged in amyotrophic lateral
sclerosis.
[0006] Peripheral nerve injuries and peripheral neuropathies, such
as those resulting from diabetes or chemotherapy, comprise the most
prevalent peripheral nervous system disorders (see Table 1) Current
treatments for peripheral nerve disorders only treat the symptoms,
not the cause of the disease.
TABLE-US-00001 TABLE 1 Disease No. of U.S. patients Amyotrophic
Lateral 30,000 Sclerosis Spinal Muscular Atrophy 50,000 Post-Polio
Syndrome 250,000 Guillain-Barre Syndrome 20,000 Muscular
Dystrophies 175,000 Peripheral Neuropathies 1,000,000 Peripheral
Nerve Injuries 500,000 Total 2,150,000
[0007] Prosaposin binds glycosphingolipids such as gangliosides,
cerebrosides and sulfatides with high affinity and facilitates
their transfer from micelles to membranes (Sueda, et al. (1993) J.
Biol. Chem. in press; Hiraiwa et al., (1992) Proc. Natl. Acad. Sci.
USA., 89: 11254-11258). Gangliosides contain one or more sialic
acid residues and are most abundant in the plasma membrane of
neurons where they constitute approximately 6% of the total lipid
mass. Although the function of gangliosides is largely unknown,
they have been implicated in the stimulation of neuronal
differentiation, neuritogenesis and nervous system repair.
[0008] Neurotrophins may be defined as those proteins capable of
affecting the survival, target innervation and/or function of
neuronal cell populations (Barde, (1989) Neuron, 2: 1525-1534). The
efficacy of neurotrophins both in vitro and in vivo has been
well-documented. The most well-characterized of such proteins is
nerve growth factor (NGF) which is synthesized by target cells of
sympathetic and sensory neurons and acts as a trophic factor for
forebrain cholinergic, peripheral and sensory neurons (Hefti et
al., (1989) Neurobiol. Aging, 10: 515-533). In vivo experiments
indicate that NGF can reverse naturally-occurring as well as
physical traumatic injuries to peripheral nerves. For example,
local application of NGF has been shown to prevent the atrophy of
sensory ganglia resulting from transection of the sciatic nerve in
adult rats (Rich et al., (1987) J. Neurocytol., 16: 261-268). In
addition, NGF plays a role in the neural regenerative process since
it enhances neurite extension of developing sympathetic and sensory
neurons (Purves et al., (1988) Nature, 336: 123-128). Moreover,
since NGF supports the function of forebrain cholinergic neurons
which are lost in Alzheimer's patients, this indicates that NGF may
have a clinical use in treatment of this disease (Hefti et al.,
(1989) Neurobiol. Aging, 10: 515-533).
[0009] Brain-Derived Neurotrophic Factor (BDNF) is synthesized in
the central nervous system and is a trophic factor for peripheral
sensory neurons, dopaminergic neurons of the substantia nigra,
central cholinergic neurons and retinal ganglia (Henderson et al.,
(1993) Restor. Neurol. Neurosci., 5: 15-28). BDNF has also been
shown to prevent normally-occurring cell death both in vitro and in
vivo (Hofer and Barde, (1988) Nature, 331: 261-262).
[0010] Since NGF and BDNF share large regions of homology
(approximately 50%), degenerate oligonucleotide primers
corresponding to four of these regions were used in PCR reactions
to amplify novel related sequences. A related neurotrophic factor
called neurotrophin 3 (NT-3) was cloned (Maisonpierre et al.,
(1990) Science, 247: 1446-1451). NT-3 is found both centrally and
peripherally and is capable of promoting survival of sensory and
sympathetic neurons, including dorsal root ganglia (DRG)
explants.
[0011] The three neurotrophins described above have different
neuronal specificities. All three neurotrophins induced neurite
outgrowth from DRG explants. NGF induces neurite outgrowth from
sympathetic ganglia (SG) but not nodose ganglion (NG), whereas BDNF
induces neurite outgrowth from NG but not SG. NT-3 promotes neurite
outgrowth from NG and to a lesser extent from SG, suggesting a
broader specificity than either NGF or BDNF (Lindsay et al., (1991)
Restor. Neural. Neurosci., 2: 211-220).
[0012] Ciliary Neurotrophic Factor (CNTF) promotes survival of
chicken embryo ciliary ganglia in vitro and was also found to
support survival of cultured sympathetic, sensory and spinal motor
neurons (Ip et al., (1991) J. Physiol., Paris, 85: 123-130). Local
administration of this protein to the lesion site of newborn rats
has been shown to prevent the degeneration of the corresponding
motor neurons. CNTF also rescued motor neurons from developmental
cell death (Henderson et al., (1993) Restor. Neurol. Neurosci., 5:
15-28).
[0013] Fibroblast Growth Factor (FGF) can also promote in vitro
survival of embryonic neurons. Effects have also been observed on
neuronal survival after lesion in vivo. FGF apparently acts on a
wide variety of neurons (Hefti et al., (1989) Neurobiol. Aging, 10:
515-533).
[0014] The identification of prosaposin itself as a neurotrophic
factor which is present in the cell bodies of large populations of
neurons including upper and lower motor neurons, and its ability to
induce myelination in mouse cerebellar explants, represent
significant new functions for this protein. Additionally, the fact
that fragments of prosaposin retain neurotogenic activity has no
precedent in the literature. No reports have appeared on the use of
small active fragments of the aforementioned neurotrophic factors
to promote neuronal survival and differentiation. Prosaposin and
its, derivatives are therefore believed to have important
therapeutic potential in the treatment of neurodegenerative and
demyelination disorders.
BRIEF DESCRIPTION OF THE FIGURES
[0015] FIG. 1a is a graph illustrating the neurite outgrowth
response of NS20Y neuroblastoma cells treated with prosaposin,
saposin C, active 22-mer peptide (CEFLVKEVTKLIDNNKTEKEIL; SEQ ID
NO: 1) and iodine labeled 18 mer over the 0.01-0.5 .mu.g/ml range.
The concentration of effector protein, in .mu.g/ml, is shown on the
x-axis and the percentage of cells with neurites is shown on the
y-axis.
[0016] FIG. 1b is a bar graph showing the effect of 5 .mu.g/ml NGF
on neurite outgrowth in prosaposin and saposin C treated NS20Y
cells. The y-axis indicates the percentage of cells with
neurites.
[0017] FIG. 2a shows a hydropathy plot of the human saposin c
sequence. The amino acid residue position is shown on the x-axis
and the hydropathic index is shown on the y-axis.
[0018] FIG. 2b provides a sequence alignment of the active 22-mer
human saposin C sequence with the same sequence from four other
species. The consensus (completely conserved) residues are
indicated below the sequence alignment. The sequence of human
saposin A (which is inactive) in the same region is provided to
illustrate the divergence between the sequence of three of the
first four residues in the same hydrophilic region (18-29) in
saposin A but conservation of the remaining residues.
SUMMARY OF THE INVENTION
[0019] One embodiment of the present invention is a method for
stimulating neural cell outgrowth or increased myelination by
contacting neuronal cells with a composition including prosaposin
or a fragment thereof having the ability to promote increased
neural outgrowth or increased myelination activity. Preferably the
prosaposin is native. Most preferably the prosaposin is
recombinantly produced. The neurotrophic fragment may
advantageously be saposin C, a peptide comprising amino acids 8-29
of saposin C, or another peptide containing the same active region.
Most preferably, this fragment consists essentially of the active
neurotrophic fragment located within amino acids 8-29 of SEQ ID NO
1. Preferably the neuronal cells are either NS20Y, Neuro 2A or
N1E115 neuroblastoma cells. These neuronal cells are preferably
contacted in vitro and most preferably contacted in vivo. Also
within the scope of the invention are cells from mouse cerebellar
explants.
[0020] Another aspect of the present invention relates to a method
for treatment of demyelination disorders in a mammal by identifying
a mammal afflicted with the disorder, and administering to the
mammal a pharmaceutically effective demyelination inhibiting amount
of prosaposin or a neurotrophic fragment thereof. Preferably this
fragment is saposin C and the demyelination disorder is either
multiple sclerosis, acute disseminated leukoencephalitis,
progressive multifocal leukoencephalitis or adrenal leukodystrophy.
Preferably, the method of administration is either intramuscular,
intradermal, subcutaneous, intracranial, intracerebrospinal or
topical in a biologically compatible carrier. Most preferably, the
administration is intravenous. Moreover, the prosaposin or fragment
thereof may be advantageously enclosed in a lamellar structure.
[0021] The invention further comprises a method for halting or
slowing the progress of neural or myelin degeneration in neural
tissue, by contacting neuronal tissue susceptible to such
degradation with prosaposin or an active degradation-inhibiting
fragment thereof. Preferably, the fragment is saposin C and the
tissue is in vitro. Most preferably the fragment is the
neurotrophic peptide and the tissue is in vivo.
[0022] Another aspect of the present invention is a method for the
treatment of neuronal degenerative diseases of the central or
peripheral nervous system, by administering to a mammal suffering
from such a disease an amount of a prosaposin fragment effective to
retard or halt neuronal degeneration. Most preferably, this
fragment includes the neurotrophic activity of the peptide of SEQ
ID NO 1 and is able to cross the blood brain barrier. In addition,
the administration is intravenous or intradermal. It is envisioned
that the disease is a central nervous system disorder including
Alzheimer's disease, Parkinson's disease, stroke, post-polio
syndrome and amyotrophic lateral sclerosis
[0023] Further, the invention includes a method for retarding the
progress of retinal neuropathy (senile macular degeneration) in a
patient by administering to the patient an effective amount of
prosaposin or a neurotrophic fragment thereof. Preferably this
retinal neuropathy is senile macular degeneration, the patient is a
human over the age of 65, and the administration is either topical,
intravenous or intraocular.
[0024] Another aspect of the present invention is a pharmaceutical
composition comprising prosaposin or a neurotrophic fragment
thereof in unit dosage form.
[0025] Still another aspect of the present invention is a
pharmaceutical composition comprising prosaposin or a neurotrophic
fragment thereof formulated with a controlled release material.
[0026] Finally, the invention includes a neural prosaposin receptor
protein in isolated or purified form. Preferably, this receptor
protein is the same protein that can be isolated from a P100 plasma
membrane fraction by affinity purification using a neurite
growth-inducing peptide contained within the saposin C sequence
linked to a solid support, and has a molecular weight of
approximately 20 kDa.
DETAILED DESCRIPTION OF THE INVENTION
[0027] This invention discloses our discovery that prosaposin,
saposin C or a peptide comprising amino acids 8-29 of saposin C can
be used to stimulate neurite outgrowth and to promote increased
myelination.
[0028] Prosaposin or its derivatives possess significant
therapeutic applications in promoting functional recovery after
toxic, traumatic, ischemic, degenerative and inherited lesions to
the peripheral and central nervous system. In addition, prosaposin
or its derivatives may be used to counteract the effects of
demyelinating diseases.
[0029] Prosaposin and its derivatives are known to be present in
many types of neurons, are water soluble (in contrast to
glycosphingolipids) and are less immunogenic than ganglioside
micelles since for therapy in humans the human sequence will be
used which will not elicit an immune response.
[0030] Human prosaposin has the amino acid sequence set forth in
SEQ ID NO:2. Saposin C has the amino acid sequence set forth in SEQ
ID NO:3. The human cDNA sequence for prosaposin is set forth in SEQ
ID NO:4. An active 18-mer fragment derived from the active 22-mer
fragment is set forth as SEQ ID NO: 5.
[0031] As will be discussed in more specific detail in the
Examples, prosaposin, saposin C and amino acids of saposin C that
include at least amino acids 8-29 are active as neurotrophic
factors. In addition, a peptide including at least amino acids
12-29 (with a tyrosine substituted for valine at position 12) is
also an active neurotrophic factor. Similar active peptides, also
within the scope of this invention, can be prepared and screened as
described herein (See Example 2). These proteins and peptides
stimulate the outgrowth of neurites, promote myelination and
prevent programmed cell death in neuronal tissues.
[0032] One aspect of the present invention is a method for
facilitating outgrowth of neurites in differentiated or
undifferentiated neural cells. This method requires administration
of an effective, neurite-outgrowth facilitating amount of
prosaposin, saposin C, or the 18 or 22 amino acid fragment thereof
to the cells in question. A typical minimum amount of prosaposin
for the neurotrophic factor activity in cell growth medium is
usually at least about 1.4.times.10.sup.-11 M, or about 10 ng/ml.
This amount or more of saposin C or its active 18 or 22 amino acid
fragments may also be used. Usually concentrations in the range of
0.1 .mu.g/ml to about 10 .mu.g/ml of any of these materials will be
used. Effective amounts for any particular tissue can be determined
in accordance with Example 1.
[0033] The neural cells can be treated in vitro or ex vivo by
directly administering the neurotrophic factor of the present
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 neurotrophic factor to the medium.
[0034] When the cells to be treated are in vivo, typically in a
vertebrate, preferably a mammal or a bird, the composition can be
administered to the cells to be treated by one of several
techniques. Most preferably, the composition can be injected
directly into the blood in sufficient quantity to give the desired
concentration of neurotrophic factor since an iodinated 18-mer
peptide consisting of amino acids 12-29 of the 22-mer with a
substitution of tyrosine for valine at amino acid 12 (M.W=2000)
crosses the blood brain barrier and enters the central nervous
system as described in Example 7 (see Banks et al., (1992)
Peptides, 13: 1289-1294). The uptake by the brain was approximately
0.03% which is in the midrange of values for peptides of that
approximate size which will cross the blood brain barrier. This is
the only neurotrophic factor so far described which will cross the
blood brain barrier when administered intravenously.
[0035] 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 of well
known type can be used. Such carriers include, for example,
phosphate buffered saline (PBS). 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,
intramuscular, intradermal, subcutaneous, intracranial, epidural,
topical and oral administration.
[0036] The composition 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.
[0037] Since the molecular weight of the active 22-mer is
approximately 2600, and an iodinated 18-mer contained within this
sequence will cross the blood, brain barrier, then the 22-mer will
also most likely cross and enter the central nervous system (Banks
et al., (1992) Peptides, 13: 1289-1294). Appropriate daily systemic
dosages based on the body weight of the vertebrate are in the range
of from about 10 to about 100 .mu.g/kg, although dosages from about
0.1 to about 1000 .mu.g/kg are also contemplated. Daily dosages of
locally administered material will be about an order of magnitude
less. Oral administration may be possible if the peptide is stable
to gastrointestinal degradation and readily absorbed.
[0038] In one preferred embodiment of the invention, the
neurotrophic factor is administered locally to the neural cells in
vivo by implantation of the material. 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 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
transdermal delivery devices are also contemplated.
[0039] The neurotrophic factors of the present invention may also
advantageously 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 (i.e., Radin and Metz, (1983) Methods Enzymol., 98:
613-618).
[0040] There are currently no available pharmaceuticals able to
promote full functional regeneration and restoration of structural
integrity of neural systems. This is particularly true of the
central nervous system. Regeneration of peripheral nerves through
use of neurotrophic factors is a more immediately demonstrable
goal. Such treatment 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, prosaposin
and its active fragments may be therapeutically useful in the
treatment of Parkinson's disease.
[0041] 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. However, as previously mentioned, the active
iodinated 18-mer will cross and the active 22-mer will most likely
cross this barrier and would thus be administered intravenously.
Other neuronal populations, such as motor neurons, would also be
treated by intravenous injection, although direct injection into
the cerebrospinal fluid is also envisioned as an alternate
route.
[0042] Cells may be treated to facilitate myelin formation or to
prevent demyelination in the manner described above, both in vitro,
ex vivo and in vivo. There are several diseases that result in
demyelination of nerve fibers including multiple sclerosis, acute
disseminated leukoencephalitis, progressive multifocal
leukoencephalitis, metachromatic leukodystrophy and adrenal
leukodystrophy. These diseases can be treated, and the progression
of the demyelination can be slowed or halted, by administration of
the neurotrophic factors of the present invention to the cells
affected by the disease.
[0043] 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 in order to better enable growth of
neural cells in vitro.
[0044] The prosaposin used in the present invention may be obtained
from various sources, and may be, for example, naturally occurring
protein isolated from human milk or seminal plasma or recombinant
human prosaposin purified from spent media of Spodoptera frugiperda
(Sf9) cells infected with a baculovirus expression vector
containing full-length cDNA for human prosaposin as described
(Dewji et al., (1987) Proc. Natl. Acad. Sci. USA, 84: 8652-8656).
O'Brien et al., (1988) Science, 241: 1098-1101); Hiraiwa et al.,
(1993) Arch. Biochem. Biophys., 304, 110-116). Saposin C is
isolated in pure form from spleens of patients with Gaucher
disease, a lysosomal storage disorder (Morimoto et al., (1990)
Proc. Natl. Acad. Sci. USA, 87: 3493-3497). Saposin C (80 amino
acids) can also be chemically synthesized and refolded (Weiler et
al., (1993) J. Mol. Neurosci., in press).
[0045] The peptides corresponding to sequences within saposin C may
be synthesized using an automated solid-phase protocol on an
Applied Biosystems Model 430 peptide synthesizer. After synthesis,
peptides 1-40, 41-82, 1-27, 13-34 and 21-48 are desalted on a
Sephadex G-75 column prior to use.
Example 1
Effect of Prosaposin, Saposins and NGF on NS20Y Neurite
Outgrowth
[0046] NS20Y neuroblastoma cells were grown in Dulbecco's Modified
Eagle Medium (DMEM) containing 10% fetal calf serum (FCS) and 1 mM
sodium pyruvate. Cells were removed with trypsin and plated in 30
mm petri dishes onto glass coverslips. After 20-24 hours the medium
was replaced with DMEM containing 0.5% fetal calf serum plus
effector proteins. Cells were cultured for another 24 hours, washed
with phosphate buffered saline (PBS) and fixed with Bouin's
solution (saturated aqueous picric acid/formalin/acetic acid
15:5:1) for 30 minutes. Fixative was removed with PBS and neurite
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.
[0047] A dose-response curve (FIG. 1a) demonstrated that prosaposin
promoted reversible neurite outgrowth in NS20Y neuroblastoma cells.
The lowest concentration for activity was 1.4.times.10.sup.-11 M
(10 ng/ml) which is in the effective concentration range of other
neurotrophins. When prosaposin was removed, retraction of neurite
outgrowth was complete at hours, demonstrating that its continual
presence is necessary in order to maintain neurite outgrowth. In
addition, saposin C was the sole fragment of prosaposin found to
possess neurotogenic activity, as did the 22-mer and iodinated
18-mer peptides derived from the saposin C sequence.
[0048] Since nerve growth factor (NGF) acts on a variety of cell
types, we wanted to determine whether it was involved in
prosaposin-mediated outgrowth in neuroblastoma cells. NGF by itself
had no effect on neurite outgrowth in NS20Y cells and did not
augment the prosaposin response (FIG. 1b). When 5'-methyladenosine
(MeSAdo), which specifically inhibits NGF-induced neuritogenesis in
PC12M pheochromocytoma cells was added, MeSAdo did not inhibit
prosaposin-induced NS20Y neurite outgrowth. Additionally,
prosaposin failed to stimulate neurite outgrowth from
NGF-responsive PC12M cells at high concentrations (2 mg/ml). Since
NS20Y cells are not NGF responsive, this indicates that the NGF
response and the prosaposin response are different.
[0049] A set of synthetic peptides from different regions of
saposin C was utilized to further define the active sequence. An
amino terminal peptide (1-40) was active and a carboxy terminal
peptide (41-82) was inactive. Testing of four more peptides (Table
2) further narrowed the active sequence to a region between
residues 8-29, the most hydrophilic region in the saposin C domain
(FIG. 2a) which also contains the single glycosylation site (Asn
22). Higher concentrations of the active 22-mer (residues 8-29)
were required for activity but the extent of neurite outgrowth was
greater than with prosaposin or saposin C (FIG. 1a). The sequence
between residues 18 and 29 is highly conserved (FIG. 2b).
Interestingly, human saposin A is nearly identical to saposin C in
this region except for the first four residues, indicating that the
active sequence requires the presence of leucine 18 and asparagines
at residues 21 and 22 or both.
TABLE-US-00002 TABLE 2 Neurite outgrowth response of NS20Y cells
treated with human saposin C, saposin A and synthetic peptides from
the human saposin C domain at 5 .mu.g/ml. The dose response curve
for peptide 8-29 (active 22 mer) is given in FIG. 1a. Peptide Added
(5 .mu.g/ml) % Neurites after 24 hours Saposin C 40% 1-40 42% 41-82
17% 1-27 46% 13-34 50% 21-48 18% 8-29 56% Saposin A 20% None
18%
[0050] To test whether gangliosides were involved in the response,
a prosaposin-ganglioside GM1 complex (4:1) was generated by a
method well known in the art. When tested in the neurite outgrowth
assay, the complex had negligible activity. The same result was
obtained with a ganglioside GM3-saposin C complex. This indicated
that the neurotogenic effect was not the result of ganglioside
transport, but was instead due to the prosaposin and saposin C,
respectively.
[0051] In order to determine whether prosaposin or its fragments
would have an effect on neurite outgrowth in nontransformed cells,
newborn mouse cerebellar explants were used as described in the
following example:
Example 2
Effect of Prosaposin and its Active Fragments on Neurite Outgrowth
in Mouse Cerebellar Explants
[0052] Newborn mouse cerebellar explants were prepared according to
Satomi (Zool. Sci. 9, 127-137 (1992)). Neurite outgrowth and
myelination were observed over 22 days in culture, during the
period when the newborn mouse cerebellum normally undergoes
neuronal differentiation and myelination begins. Prosaposin (5
.mu.g/ml) and saposins A, B and C (10 .mu.g/ml) were added on the
second day after preparation of the explants (three control and
three treated explants) and outgrowth of neurites and myelination
were assessed under a bright field microscope with a video camera.
On the eighth day cultures containing prosaposin and saposin C
became thinner and more spread out than control cultures. On day
15, the prosaposin and saposin C treated cultures contained many
cells with long projections at the periphery of the explant which
were less prominent in controls or those treated with saposins A or
B. Saposin C treated cultures contained twice as many myelinated
axons in the subcortical white matter at 22 days as controls or
those treated with saposins A or B. Both the number of myelinated
fibers observed visually per optical field and the activity of the
myelin marker enzyme CNP were twice the control value. These
results demonstrate that the neurotrophic effect of prosaposin and
saposin C also occurs in differentiating cerebellum ex vivo. These
results further demonstrate the ability of prosaposin and saposin C
to induce increased myelination in differentiating cerebellum ex
vivo.
[0053] Since prosaposin appears to be active at the plasma membrane
it should be present in the plasma membranes of responsive cells as
shown in the following example:
Example 3
Western Blots of Prosaposin and Saposin C from NS20Y Cells
[0054] NS20Y cells were grown to confluence in 75 cm flasks in the
presence of growth medium. Cells were harvested by scraping and
surface membranes were isolated by the zinc ion method of Warren
and Glick (1969) using discontinuous gradients of 50, 48, 45, 43,
40 and 35% sucrose; surface membranes localize in the 40 and 43%
sucrose fraction. These fractions, as well as the infranatant and
supernatant fractions bounding them, were electrophoresed on 10%
SDS polyacrylamide gels along with the whole cell extracts,
transferred to nitrocellulose filters, and probed with a monoclonal
antibody to saposin C by methods well known in the art.
[0055] Examination of Western blots revealed that prosaposin,
migrating as a 68 kDa band on SOS polyacrylamide gels, was
localized to surface membrane fractions from both NS20Y and Neuro
2A cells. Mature saposin C and intermediate molecular weight
saposin derivatives were minor components of the membrane fractions
but were abundant in the whole cell extract. This demonstrates that
prosaposin is located in the plasma membrane of responsive
cells.
[0056] In order to localize prosaposin histochemically,
neuroblastoma cell lines were immunostained with a
prosaposin-specific antibody (JP-1) as illustrated in the following
example:
Example 4
Immunohistochemical Localization of Prosaposin
[0057] Cells were grown on glass cover slips, washed three times
with PBS and fixed with Bouin's solution for one hour at room
temperature. Bouin's solution was then rinsed out with 5 washes of
PBS and slips were incubated in 30% goat serum, 0.5% Tween 20 in
PBS to block nonspecific binding and, after rinsing, were incubated
in a 1:100 dilution of IgG purified rabbit JP-1 at 4.degree. C.
overnight. After rinsing with PBS containing 0.1% Triton X-100, the
preparations were incubated with either peroxidase conjugated goat
anti-rabbit IgG (Bio-Rad, 1:2000) or FITC-conjugated goat
anti-rabbit IgG (Cappel, 1:2000). After rinsing, peroxidase
immunostaining was detected using the
imidazole-diaminobenzidine-H.sub.2O.sub.2 reaction. Fluorescence
immunostaining was detected under a fluorescence microscope using
Nofade as a quenching deterrent. Preimmune rabbit IgG (1:100) was
used as a control for nonspecific binding. Immunostaining of
extended neurites, plasma membranes and growth cones were
observed.
[0058] A similar methodology was used to immunostain postmortem
human brain sections to detect reactive cell types. In frontal
cortex, intense staining of the perikarya of large and medium sized
Golgi type 1 neurons was observed. The surface of neuronal
perikarya and the proximal segment of axons at the hillock region
were also strongly stained as Were some extended axons. In the
cerebellum strong staining of Purkinje and stellate cells was
observed, as well as large neurons in the cerebellar nuclei
(dentate, emboliform and globose nuclei). Cerebellar granular cells
were moderately stained. In the mesencephalon, moderate staining
was observed in dopaminergic neurons of the substantia nigra. Large
neurons in the red nucleus, neurons in the oculomotor nucleus, the
amygdaloid nucleus and ependymal cells lining the lateral ventricle
were also moderately stained. In the hippocampus, pyramidal cells
and granule cells of the dentate gyrus were strongly stained. In
the spinal cord alpha motor neurons were intensely stained. This
survey indicated that prosaposin was localized to populations of
large neurons including upper and lower motor neurons.
[0059] Since all neurotrophins identified thus far exert their
effects by binding to a cell surface receptor and initiating a
kinase cascade, phosphorylation assays were performed in NS20Y
cells treated with prosaposin or its fragments as described in the
following example:
Example 5
Incorporation of 32P into NS20Y Proteins after Treatment with
Prosaposin or its Active Fragments
[0060] NS20Y cells were incubated in phosphate-free Hanks' balanced
salt solution containing 2.5 .mu.g/ml actinomycin D and 80-100
.mu.Ci/ml carrier-free [.sup.32P]-orthophosphate (New England
Nuclear) and effector proteins (0.5-1.0 .mu.g/ml) and incubated for
10-15 minutes at room temperature. Cells were solubilized in
SDS-PAGE sample buffer, analyzed by SDS-PAGE and
autoradiographed.
[0061] Prosaposin, saposin C and SEQ ID NO: 1 were found to
stimulate, phosphorylation of proteins of 148, 100, 80, 68, 50, 38
and 34 kDa to a greater extent than controls or cells treated with
similar concentrations of saposins A, B or D. This 148 kDa protein
may be phospholipase C-.gamma., a protein known to be involved in
phospholipid metabolism and which is phosphorylated on tyrosine
residues in response to a number of growth factors. Densitometric
analysis indicated a 3-5 fold stimulation of phosphorylation after
10 minutes. Treatment of gels with alkali revealed that the
prominent phosphorylated proteins were alkali-resistant, indicating
that they contain phosphotyrosine and/or phosphothreonine (located
next to proline) residues. These results indicate that prosaposin
and its active fragments bind to a cell surface receptor and
activate a kinase cascade, similar to other neurotrophins and
growth factors.
[0062] Since prosaposin-ganglioside GM1 or saposin C-ganglioside
GM3 complexes inhibit neuritogenesis, while prosaposin or saposin C
alone promote this process, this indicates that gangliosides may
abolish neurotogenic activity by masking a receptor binding site on
the neurotrophin. In addition, since prosaposin and its active
fragments induce tyrosine phosphorylation of cytoplasmic proteins
in responsive cells, most likely by activation of a tyrosine
kinase(s) similar to cytokines and growth factors, this provides
further evidence that a cell surface receptor is involved.
[0063] A 20 kDa protein has been identified as the putative
receptor for prosaposin as described in the following example:
Example 6
Isolation of the Prosaposin Receptor
[0064] The putative prosaposin receptor protein was isolated from
whole rat brain, rat cerebellum and mouse neuroblastoma cells using
the plasma membrane P-100 fraction. Briefly, cells or tissues were
solubilized and centrifuged at 14,000 rpm to remove debris. The
supernatant was centrifuged at 40,000 rpm for 1 hour at 4.degree.
C. The pellet, enriched in plasma membrane, was solubilized in RIPA
buffer (10 mM MOPS, pH 7.5, 0.3 M sucrose, 5 mM EDTA, 1% Trasylol,
10 .mu.M leupeptin and 10 .mu.M antipain). This P-100 fraction was
applied to an affinity column containing the bound, active 22-mer
fragment of saposin C. The column was washed with 0.25 M NaCl to
elute loosely-bound proteins followed by 1.0 M NaCl which eluted
the putative 20 kDa prosaposin receptor. In addition, it was
determined that the 20 kDa protein could be eluted using a 100 fold
excess of unbound peptide thus demonstrating specific elution. The
20 kDa protein was approximately 90% pure as judged by SDS-PAGE.
The protein was purified to homogeneity using HPLC and eluted at
50% acetonitrile in an acetonitrile/water gradient on a Vydac C4
column.
Example 7
In Vivo Peptide Uptake by the Central Nervous System
[0065] An 18-mer peptide consisting of amino acids 12-29 of saposin
C with a tyrosine substituted for valine at position 12 was
chemically synthesized on an Applied Biosystems Model 430 peptide
synthesizer. The peptide was then radioiodinated by the
lactoperoxidase method and 20.times.10.sup.6 cpm were injected into
the auricles of rats. The animals were sacrificed after one hour
and 24 hours and the hearts were perfused with isotonic saline in
order to remove the blood from the brain. The brain was then
counted in a gamma counter in order to determine the percentage of
peptide uptake. In addition, in the 24 hour experiment the brain
was homogenized and separated into a capillary rich fraction
(pellet) and a parenchymal brain fraction (supernatant) after
dextran centrifugation (Triguero et al., (1990) J. Neurochem., 54:
1882-1888). This method allows for the discrimination between
radiolabelled peptide within blood vessels and that within the
brain. In the 24 hour experiment, 0.017% of the injected peptide
was detected in whole brain; 75% of the label was in the
parenchymal fraction and 25% was in the capillary fraction. At 1
hour 0.03% of the injected dose was present in whole brain.
Example 8
Use of Prosaposin and its Active Fragments in Treating Traumatic
Ischemic Lesions to the CNS In Vivo
[0066] Rats with traumatic lesions to the spinal cord receive
direct or intravenous administration of prosaposin or its active
fragments in the 10 ng-10 mg/ml range in a sterile saline solution
or in a depot form to enable slow release. The same number of
animals receive only saline. After surgical partial transection of
the spinal cord or a crush injury, prosaposin or a neurotrophic
fragment thereof is directly injected into the lesion site using
the same dose range (control animals receive saline injections) and
improvement is assessed by gain of motor neuron function (i.e.,
increased limb movement). The treatments continue until no further
improvement occurs. Since prosaposin and its active fragments are
very water-soluble, no special delivery system for the preparation
is required. Injection of the 18 or 22 amino acid fragments is
preferred since there is less chance of degradation and diffusion
will be greater. Additionally, this fragment can be chemically
synthesized in large quantities.
Example 9
Use of Prosaposin and its Active Fragments in Treating
Demyelination Disorders
[0067] Patients diagnosed with early stage MS (or other
demyelination disorder) are given the active 18 or 22-mer fragment
(in saline) by direct intravenous injection or injection into the
cerebrospinal fluid using the same dose range as in Example 7.
Control patients receive only saline. The treatment is administered
weekly or monthly and any improvement is assessed by increased
muscle strength, musculoskeletal coordination, and assessing
myelination by magnetic resonance imaging.
Example 10
Use of Prosaposin or its Active Fragments in Treating Retinal
Neuropathy
[0068] Retinal neuropathy, an ocular neurodegenerative disorder
leading to loss of vision in the elderly, is believed to be a
disorder treatable by prosaposin or its active fragments.
Prosaposin or its active neurotrophic fragments are administered
either topically, systemically or intraocularly in an amount
sufficient to produce a local concentration of neurotrophin of
about 10 ng/ml to about 10 mg/ml. The administration is continued
weekly until visual loss is slowed or no further increase in vision
is noticed.
Sequence CWU 1
1
* * * * *