Title: Method of treating brain
United States Patent: 7,427,597
Issued: September 23, 2008
Inventors: Li; Hung
(Taipei, TW), Shyu; Woei-Cherng (Taipei, TW), Lin; Shinn-Zong (Hualien, TW)
Assignee: Academia Sinica
Appl. No.: 11/284,796
Filed: November 21, 2005
Master of Science in Law
A method of treating brain tissue damage,
including administering to a subject in need thereof an effective amount
of secretoneurin. Disclosed are methods of promoting angiogenesis or
neurogenesis in the brain of subject. Also disclosed are a method of
homing of stem cells to the brain of a subject and a method of protecting
a neuronal cell from cell death.
Description of the
Brain tissue damage, resulting either from injuries or disorders (e.g.,
neurodegenerative and cerebrovascular diseases), is a leading cause of
long-term disability. Due to their pluripotency, embryonic stem cells (ES
cells) hold a great promise for treating brain tissue damage (Taguchi et
al., 2004, J. Clin. Invest.; 114(3):330-338). However, ethical and
logistical considerations have hampered their use (Barinaga, 2000,
Science, 287(5457):1421-1422). Use of non-ES pluripotent cells has also
been exploited. They include adult bone marrow mesenchymal stem cells or
stromal cells (Sanchez-Ramos et al., 2000, Exp. Neurol., 164(2):247-256
and Woodbury et al., 2000, J. Neurosci. Res., 61(4):364-370) and umbilical
cord blood cells (Galvin-Parton et al., 2003, Pediatr. Transplant. 2003;
7(2):83-85 and Ha et al., 2001 Neuroreport., 2(16):3523-3527).
Nonetheless, requirements for in vitro expansion and HLA-matching have
limited clinical applications of these cells. Thus, there is a need for an
alternative method of treating brain tissue damage.
This invention is based, at least in part, on the discovery that brain
tissue damage can be repaired by administration of secretoneurin.
Accordingly, one aspect of this invention features a method of treating
brain tissue damage. The method includes administering to a subject in
need thereof an effective amount of secretoneurin. The method can be used
to treat brain tissue damage caused by a cerebral ischemia, e.g., stroke,
or a neurodegenerative disease, e.g., Alzheimer's disease, epilepsy,
Huntington's disease, Parkinson's disease, or Spinocerebellar disease. The
method may further include administering to the subject an effective
amount of stem cells. Suitable stem cells include hematopoietic stem
cells, umbilical cord-derived mesenchymal stem cells, and stem cells that
are c-kit.sup.+ or CD34.sup.+. Preferably, the stem cells are autologous
to the subject. For example, the cells can be enriched from the blood or
bone marrow from the subject.
"Treating" refers to administration of a compound or composition to a
subject, who is suffering from or is at risk for developing brain tissue
damage or a disorder causing such damage, with the purpose to cure,
alleviate, relieve, remedy, prevent, or ameliorate the damage/disorder,
the symptom of the damage/disorder, the disease state secondary to the
damage/disorder, or the predisposition toward the damage/disorder. An
"effective amount" refers to an amount of the compound or composition that
is capable of producing a medically desirable result, e.g., as described
above, in a treated subject. The treatment method can be performed alone
or in conjunction with other drugs or therapies.
This invention also features a method of promoting cerebral angiogenesis
in a subject. The method includes administering intracerebrally to a
subject in need thereof an effective amount of secretoneurin. The method
further includes measuring a level of cerebral angiogenesis in the subject
before and after administering secretoneurin to confirm promotion of
cerebral angiogenesis. The method can also include administering to the
subject an effective amount of stem cells, such as those described above.
Also within the scope of this invention is a method of promoting
neurogenesis in the brain of a subject. The method includes administering
intracerebrally to a subject in need thereof an effective amount of
secretoneurin. The method further includes measuring a level of
neurogenesis in the brain of the subject before and after administering
promotion to confirm promotion of neurogenesis. For this purpose, one can
use standard neurological behavioral measurements including those
described in the examples below. The method can also include administering
to the subject an effective amount of stem cells, such as those mentioned
This invention further features a method of promoting homing of stem cells
to the brain of a subject by administering intracerebrally to a subject in
need thereof an effective amount of secretoneurin. Suitable stem cells
include those described above.
A method of preserving a neuronal cell is also contemplated in this
invention. The method of claim includes contacting the cell with
The details of one or more embodiments of the invention are set forth in
the description below. Other features, objects, and advantages of the
invention will be apparent from the description and from the claims.
The present invention relates to treating brain tissue damage using
secretoneurin). Secretoneurin, a 33-amino acid neuropeptide (TNEIVEEQYTPQSLATLESVFQELGKLTGPNNQ:
SEQ ID NO: 1), is derived from endoproteolytic processing of chromogranin/secretogranins
(Cg/Sg) family protein. It is a abundant protein within large dense core
vesicles in the endocrine tissue and nervous system (Kirchmair et al.
1993, Neuroscience, 53, 359-365; Saria et al., 1993, Neuroscience, 54,
1-4; Kahler et al., 1996, Eur. J. Pharmacol., 304, 135-139; Fischer-Colbrie
et al., 1995, Prog. Neurobiol. 46, 49-70; and Cozzi et al., 1989,
Neuroscience 28, 423-41.) As described in the examples below,
secretoneurin unexpectedly enhanced the targeting of stem cells to injured
brain, protected neurons from cell death, and promoted neurogenesis and
angiogenesis in injured brain. Not only did it protect existing neurons or
glial cells in the brain, it also facilitated neural regeneration to
replace damaged neurons glial cells.
While many secretoneurin preparations can be used to practice this
invention, highly purified secretoneurin is preferred. Examples include
mammalian secretoneurin (e.g., human, mouse, and rat secretoneurins) or
non-mammalian secretoneurin having substantially the same biological
activity as mammalian secretoneurin. Naturally occurring secretoneurin,
chemically synthesized secretoneurin, and genetically engineered
secretoneurin all can be used. Secretoneurin obtained by chemical
synthesis or genetic engineering may be that having the same amino acid
sequence as naturally occurring secretoneurin or an functionally
equivalent there of. A "functional equivalent" refers to a polypeptide
derivatives of a naturally occurring secretoneurin (SEQ ID NO: 1), e.g., a
protein having one or more point mutations, insertions, deletions,
truncations, a fusion protein, or a combination thereof. It possesses one
or more of the activities of secretoneurin, e.g., the ability to protect
cells from cell death, to promote angiogenesis or neurogenesis, and to
mobilize stem cells from bone marrow into the peripheral blood or into the
brain. The term "secretoneurin" also covers chemically modified
secretoneurin. Examples of chemically modified secretoneurin include
secretoneurin subjected to conformational change, addition or deletion of
a sugar chain. Once purified and tested by standard methods, secretoneurin
can be administered to a subject as described below.
To practice the treatment method of this invention, one can administer
secretoneurin via intracerebral injection. A sterile injectable
composition (e.g., aqueous or oleaginous suspension) can be formulated
according to techniques known in the art using suitable dispersing or
wetting agents (such as, for example, Tween 80) and suspending agents. The
sterile injectable preparation can also be a sterile injectable solution
or suspension in a non-toxic parenterally acceptable diluent or solvent,
for example, as a solution in 1,3-butanediol. Among the acceptable
vehicles and solvents that can be employed are mannitol, water, Ringer's
solution and isotonic sodium chloride solution. In addition, sterile,
fixed oils are conventionally employed as a solvent or suspending medium
(e.g., synthetic mono- or di-glycerides). Fatty acids, such as oleic acid
and its glyceride derivatives are useful in the preparation of injectables,
as are natural pharmaceutically-acceptable oils, such as olive oil or
castor oil, especially in their polyoxyethylated versions. These oil
solutions or suspensions can also contain a long-chain alcohol diluent or
dispersant, or carboxymethyl cellulose or similar dispersing agents.
A carrier in a pharmaceutical composition must be "acceptable" in the
sense of being compatible with the active ingredient of the formulation
(and preferably, capable of stabilizing it) and not deleterious to the
subject to be treated. For example, solubilizing agents, such as
cyclodextrins (which form specific, more soluble complexes with one or
more of active compounds of the extract), can be utilized as
pharmaceutical excipients for delivery of the active compounds. Examples
of other carriers include colloidal silicon dioxide, magnesium stearate,
cellulose, sodium lauryl sulfate, and D&C Yellow # 10.
Suitable in vitro assays can be used to preliminarily evaluate the
efficacy of a secretoneurin preparation. For example, one can measure the
neuroprotective effect of the preparation. More specifically, the
preparation can be added to a suitable cell culture (e.g., a primary
cortical cell culture of rat or mouse neuron-glial cells as described in
Example 1 below) and the level of protection is determined. One then
compares the level with a control level obtained in the absence of the
preparation. If the level of interest is higher than the control, the
preparation is identified as being active for treating brain tissue
damage. One can also evaluate the efficacy of a secretoneurin preparation
by examining the preparation's effects on cell death according to standard
methods. For example, one can measure the level of a protein involved in
cell-death (e.g., caspase). If the level is lower than that obtained in
the absence of the preparation, the preparation is determined to be
The preparation can further be examined for its efficacy in mobilizing
bone marrow-derived stem cells to the peripheral blood or the brain by an
in vivo assay. For example, the preparation can be evaluated in an animal
(e.g., a mouse or rat model). The level of mobilized stem cells in the
peripheral blood or the brain is determined by standard methods.
The preparation can also be administered to an animal model having brain
tissue damage or a disorder causing such damage. The therapeutic effects
of the preparation are then evaluated according to standard methods (e.g.,
those described in Examples 2-8 below). To confirm efficacy in promoting
cerebrovascular angiogenesis, one can examine the animal before and after
the treatment by standard brain imaging techniques, such as computed
tomography (CT), Doppler ultrasound imaging (DUI), magnetic resonance
imaging (MRI), and proton magnetic resonance spectroscopy (.sup.1H-MRS).
One can also measure the expression level of a neuronal marker, a vacular
marker, a glial marker, a trophic factor, or a cell death-related protein
in a sample (e.g., cerebrospinal fluid) obtained from the animal before or
after administration of secretoneurin to confirm efficacy. The expression
level can be determined at either the mRNA level or the protein level.
Methods of measuring mRNA levels in a tissue sample or a body fluid are
well known in the art. To measure mRNA levels, cells can be lysed and the
levels of mRNA in the lysates, whether purified or not, can be determined
by, e.g., hybridization assays (using detectably labeled gene-specific DNA
or RNA probes) and quantitative or semi-quantitative RT-PCR (using
appropriate gene-specific primers). Alternatively, quantitative or
semi-quantitative in situ hybridization assays can be carried out on
tissue sections or unlysed cell suspensions using detectably (e.g.,
fluorescent or enzyme) labeled DNA or RNA probes. Additional mRNA-quantifying
methods include the RNA protection assay (RPA) method and the serial
analysis of gene expression (SAGE) method, as well as array-based
Methods of measuring protein levels in a tissue sample or a body fluid are
also well known in the art. Some of them employ antibodies (e.g.,
monoclonal or polyclonal antibodies) that bind specifically to a target
protein. In such assays, the antibody itself or a secondary antibody that
binds to it can be detectably labeled. Alternatively, the antibody can be
conjugated with biotin. Its presence can be determined by detectably
labeled avidin (a polypeptide that binds to biotin). Combinations of these
approaches (including "multi-layer sandwich" assays) can be used to
enhance the sensitivity of the methodologies. Some protein-measuring
assays (e.g., ELISA or Western blot) can be applied to body fluids or to
lysates of cells, and others (e.g., immunohistological methods or
fluorescence flow cytometry) can be applied to histological sections or
unlysed cell suspensions. Appropriate labels include radionuclides (e.g.,
.sup.125I, .sup.131I, .sup.35S, .sup.3H, or .sup.32P) enzymes (e.g.,
alkaline phosphatase, horseradish peroxidase, luciferase, or .beta.-glactosidase),
fluorescent/luminescent agents (e.g., fluorescein, rhodamine,
phycoerythrin, GFP, BFP, and Qdot.TM. nanoparticles supplied by the
Quantum Dot Corporation, Palo Alto, Calif.). Other applicable methods
include quantitative immunoprecipitation or complement fixation assays.
Based on the results from the assays described above, an appropriate
dosage range and administration route can be determined. The dosage
required depends on the choice of the route of administration; the nature
of the formulation; the nature of the patient's illness; the subject's
size, weight, surface area, age, and sex; other drugs being administered;
and the judgment of the attending physician. Suitable dosages are in the
range of 0.001-100 mg/kg. Dosage variations are necessary in view of the
variety of compounds available and the different efficiencies of various
routes of administration. The variations can be adjusted using standard
empirical routines for optimization as is well understood in the art. In
general, secretoneurin can be administered at 10 to 500 .mu.g/day/kg body
weight for 2-10 days; preferably, 20 to 200 .mu.g/day/kg body weight for
3-8 day; and, more preferably, 50 to 100 .mu.g/day/kg body weight for 4 to
The treatment method of this invention optionally includes administering
to a subject an effective amount of stem cells. Both heterologous and
autologous stem cells can be used. In the former case, HLA-matching should
be conducted to avoid or minimize host reactions. In the latter case,
autologous cells are enriched and purified from a subject to be treated
before the cells are introduced back to the subject.
In both cases, secretoneurin can be used as the active ingredient to
mobilize hematopoietic stem cells (HSCs) out of bone marrow so as to
increase the number of stem cells in the peripheral blood, which home to
the brain (HSCs, once in the peripheral blood, are called peripheral blood
stem cells or PBSC).
Other factors, such as granulocyte-colony stimulating factor (G-CSF), can
also be used to mobilize HSCs. In one embodiment, PBSCs are obtained from
a subject as follows: A subject is first administered G-CSF to mobilize
HSCs from bone marrow into the peripheral blood. After this enriching
step, peripheral blood are collected and PBSCs purified. To prepare PBSCs,
G-CSF is administered at 10 to 200 .mu.g/day/kg body weight for 2-10 days.
The G-CSF can be administered to a subject via any suitable routes.
Examples include injection subcutaneously, intramuscularly, and
intraperitoneally. PBSCs are generally purified based on their physical
and biochemical properties. For example, peripheral blood cells may be
concentrated for hematopoietic stem cells by centrifugation,
counter-current elutriation, selection with cell surface markers (e.g.,
CD34.sup.+ or stem cell related antibodies), or removal of lineage
positive (committed) hematopoietic cells. Such methods are well known in
the art. See e.g., U.S. Pat. Nos. 5,061,620; 5,087,570; 5,061,620;
4,714,680; 4,965,204; and 5,035,994. Details of using G-CSF to obtain
greater than 90% purity of PBSCs can be found U.S. application Ser. No.
Stem cells other than PBSC can be used as long as they possess the
potential to differentiate into cells of vascular or neural-glial lineage.
Methods of preparing and testing such cells are well-known in the art.
Examples includes embryonic stem cells (Thomson et al., 1995, Proc. Natl.
Acad. Sci. U.S.A. 92:7844-7844; 1996, Biol. Reprod. 55:254-259; Shamblott
et al., 1998, Proc. Natl. Acad. Sci. U.S.A. 95:13726; and U.S. Pat. Nos.
6,921,632 and 6,875,607), bone marrow-derived mesenchymal stem cells (Tse,
et al., 2000, Journal of the American Society of Hematology, vol. 96, No.
11; Woodbury et al., 2002, J. Neuroscience Res. 96:908-917; Sanchez-Ramos
et al., 2000, Exp. Neurol., 164(2):247-256; Woodbury et al., 2000, J.
Neurosci. Res., 61(4):364-370); and U.S. Pat. Nos. 5,486,359, 6,355,239),
and umbilical cord-derived stem cells (Mitchell et al., 2003, Stem Cells.
2003;21(1):50-60; Galvin-Parton et al., 2003, Pediatr. Transplant.
2003;7(2):83-85; Ha et al., 2001 Neuroreport., 2(16):3523-3527; and U.S.
Provisional Application No. 60/706,377.
Purified stem cells are tested and stored by standard techniques. They can
be administered intracerebrally to a subject in need thereof. In general,
1.times.10.sup.4 and 1.times.10.sup.6 (e.g., 5.times.10.sup.4 to
8.times.10.sup.6 and more preferably 1.times.10.sup.5 to 6.times.10.sup.5)
cells are administered. Multiple sites can be used depending on the site
and nature of particular damage. Examples below describes approximate
coordinates for administering cells in a rat or a mouse. Coordinates for
other disorders in other species can be determined accordingly based on
comparative anatomy. Before or after the treatment, a subject can be
examined to confirm treatment efficacy. To this end, one can use suitable
standard tests or techniques described above and in the examples below (see Original Patent).
Claim 1 of 9 Claims
1. A method of treating brain tissue
damage, comprising administering to a subject in need thereof an effective
amount of secretoneurin, wherein the secretoneurin is administered
intracerebrally into an affected area and the brain tissue damage is
caused by cerebral ischemia or stroke.
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