U.S. patent application number 12/701309 was filed with the patent office on 2010-08-12 for agents, compositions and methods for enhancing neurological function.
This patent application is currently assigned to University of Sourthern California. Invention is credited to Roberta Diaz Brinton, Jun Ming Wang.
Application Number | 20100204192 12/701309 |
Document ID | / |
Family ID | 42540931 |
Filed Date | 2010-08-12 |
United States Patent
Application |
20100204192 |
Kind Code |
A1 |
Brinton; Roberta Diaz ; et
al. |
August 12, 2010 |
AGENTS, COMPOSITIONS AND METHODS FOR ENHANCING NEUROLOGICAL
FUNCTION
Abstract
Neuro-enhancing agents, compositions and methods are disclosed
herein. Preferred neuro-enhancing agents of the present invention
include progesterone and metabolites of progesterone, such as
3.alpha.-hydroxy-5.alpha.-pregnan-20-one (THP). These agents yield
neuro-enhancing effects on neural cells that include neural
progenitor and/or stem cells, whereby the agents stimulate mitosis
of neural progenitor cells, stimulate neurite growth and
organization, protect against neural loss, or one or more of these
neural processes. Thus, the neuro-enhancing agents, compositions
and methods disclosed herein are useful to reverse or prevent
neurological disease or defects associated with neural loss or
degeneration, such as Alzheimer's disease, neurological injuries,
including injuries resulting from radiation therapy, and
age-related neurological decline, including impairments in memory
and learning.
Inventors: |
Brinton; Roberta Diaz;
(Rancho Palos Verdes, CA) ; Wang; Jun Ming;
(Pearl, MS) |
Correspondence
Address: |
Pabst Patent Group LLP
1545 PEACHTREE STREET NE, SUITE 320
ATLANTA
GA
30309
US
|
Assignee: |
University of Sourthern
California
|
Family ID: |
42540931 |
Appl. No.: |
12/701309 |
Filed: |
February 5, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12526604 |
Aug 10, 2009 |
|
|
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PCT/US08/66558 |
Jun 11, 2008 |
|
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12701309 |
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61150159 |
Feb 5, 2009 |
|
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60943187 |
Jun 11, 2007 |
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Current U.S.
Class: |
514/182 |
Current CPC
Class: |
A61K 9/06 20130101; A61P
25/28 20180101; A61K 31/57 20130101; A61P 25/00 20180101; A61K
9/0043 20130101; A61P 25/16 20180101; A61K 47/32 20130101 |
Class at
Publication: |
514/182 |
International
Class: |
A61K 31/57 20060101
A61K031/57; A61P 25/28 20060101 A61P025/28; A61P 25/00 20060101
A61P025/00; A61P 25/16 20060101 A61P025/16 |
Claims
1. A pharmaceutical composition for transdermal administration
comprising a compound selected from the group consisting of
3.alpha.-hydroxy-5.alpha.-pregnan-20-one, a derivative or analog
thereof, or a pharmaceutically acceptable salt thereof, and a
carrier for transdermal administration.
2. The composition of claim 1, wherein the compound is
3.alpha.-hydroxy-5.alpha.-pregnan-20-one.
3. The composition of claim 2, wherein the composition is in the
form of a gel.
4. The composition of claim 3, wherein the gel comprises a
thickening agent.
5. The composition of claim 4, wherein the thickening agent is a
cross linked acrylic acid polymer.
6. The composition of claim 5, wherein the crosslinked acrylic acid
polymer is carbomer 940.
7. The composition of claim 3, wherein the gel further comprises a
solvent.
8. The composition of claim 7, wherein the solvent is selected from
the group consisting of diglycol monoethyl ether; ethylene glycol;
propylene glycol; dimethyl isosorbide; isopropyl alcohol; and
ethanol.
9. The composition of claim 8, wherein the solvent is ethanol.
10. The composition of claim 3, wherein the gel further comprises
one or more penetration enhancers.
11. The composition of claim 1, wherein the compound is present in
an amount effective to reverse the learning and/or memory deficits
in an individual suffering from a neurodegenerative disease,
defect, or injury.
12. The composition of claim 1, wherein the compound is present in
an amount effective to reduce .beta.-amyloid expression.
13. The composition of claim 11 or 12, wherein the amount of
neuro-enhancing agent present in the composition is from about 0.1
to about 1000 mg, preferably from about 0.1 to about 500 mg, more
preferably from about 0.1 to about 100 mg.
14. The composition of claim 11 or 12, wherein the concentration of
the agent is 10 mg/kg.
15. A method for reversing the learning and/or memory deficits in
an individual suffering from a neurodegenerative disease, defect,
or injury the method comprising administering an effective amount
of the composition of claim 1.
16. The method of claim 15, wherein the composition is administered
for a period of at least one month.
17. The method of claim 15, wherein the composition is administered
for a period of at least three months.
18. The method of claim 15, wherein the composition is administered
for a period of at least six months.
19. The method of claim 18, wherein the composition is administered
once a week for a period of six months.
20. The method of claim 15, wherein the amount of
3.alpha.-hydroxy-5.alpha.-pregnan-20-one or a derivative or analog
thereof in the composition is from about 0.1 to about 1000 mg,
preferably from about 0.1 to about 500 mg, more preferably from
about 0.1 to about 100 mg.
21. The method of claim 15 or 19, wherein the amount of
3.alpha.-hydroxy-5.alpha.-pregnan-20-one or a derivative or analog
thereof is 10 mg/kg.
22. The method of claim 15, wherein the neurodegenerative disease
or disorder is Alzheimer's disease.
23. The method of claim 15, wherein the neurodegenerative disease
or disorder is Parkinson's disease.
24. The method of claim 15, wherein the neurodegenerative disease
or disorder is traumatic brain injury.
25. A pharmaceutical composition for intranasal administration
comprising a compound selected from the group consisting of
3.alpha.-hydroxy-5.alpha.-pregnan-20-one, a derivative or analog
thereof, or a pharmaceutically acceptable salt thereof, and a
carrier for intranasal administration.
26. The composition of claim 25, wherein the compound is present in
an amount effective to reverse the learning and/or memory deficits
in an individual suffering from a neurodegenerative disease,
defect, or injury.
27. The composition of claim 25, wherein the compound is present in
an amount effective to reduce .beta.-amyloid expression.
28. A method for reversing the learning and/or memory deficits in
an individual suffering from a neurodegenerative disease, defect,
or injury the method comprising administering an effective amount
of the composition of claim 25.
29. The method of claim 28, wherein the composition is administered
for a period of at least one month.
30. The method of claim 28, wherein the composition is administered
for a period of at least three months.
31. The method of claim 28, wherein the composition is administered
for a period of at least six months.
32. The method of claim 28, wherein the composition is administered
once a week for a period of six months.
33. The method of claim 28, wherein the neurodegenerative disease
or disorder is Alzheimer's disease.
34. The method of claim 28, wherein the neurodegenerative disease
or disorder is Parkinson's disease.
35. The method of claim 28, wherein the neurodegenerative disease
or disorder is traumatic brain injury.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation-in-part of U.S. Ser. No.
12/526,604 entitled "Allopregnanolone in a Method for Enhancing
Neurological Functions", filed on Aug. 10, 2009, which is a 371 of
International Application No. PCT/US2008/066558, filed on Jun. 11,
2008, which claims priority to U.S. Ser. No. 60/943,187, filed on
Jun. 11, 2007. This application also claims priority to U.S. Ser.
No. 61/150,159 entitled "Agents, Compositions and Methods for
Enhancing Neurological Function" by Roberta Diaz Brinton and Jun
Ming Wang, filed on Feb. 5, 2009.
FIELD OF THE INVENTION
[0002] This invention is in the field of pharmaceutical
compositions for enhancing neurological function and methods of use
thereof, particularly compositions containing allopregnanolone or a
derivative or analogue thereof.
BACKGROUND OF THE INVENTION
[0003] The mammalian nervous system includes a peripheral nervous
system (PNS) and a central nervous system (CNS), including the
brain and spinal cord, and is composed of two principal classes of
cells, namely neurons and glial cells. The glial cells fill the
spaces between neurons, nourishing them and modulating their
function. During development, differentiating neurons from the
central and peripheral nervous systems send out axons that grow and
make contact with specific target cells. In some cases, axons must
cover enormous distances with some growing into the periphery,
whereas others are confined within the central nervous system. In
mammals, this stage of neurogenesis is thought to be complete
during the embryonic phase of life. Further, neuronal cells are
generally thought not to multiply once they have fully
differentiated.
[0004] A host of neuropathies, including neurodegenerative
diseases, have been identified that affect the nervous system of
mammals. These neuropathies, which may affect neurons themselves or
associated glial cells, may result from cellular metabolic
dysfunction, infection, injury, exposure to toxic agents,
autoimmunity, malnutrition, and/or ischemia or may be due to
age-related neurological changes. In some cases, the neuropathy is
thought to induce cell death directly. In other cases, the
neuropathy may induce sufficient tissue necrosis to stimulate the
body's immune/inflammatory system and the immune response to the
initial injury then destroys neural pathways. Also, neuronal tissue
may be lost as a result of physical insult or trauma.
[0005] Loss of neurons, either directly or indirectly, was thought
to be irreversible in the adult human brain, as it was long held
that the generation of new neurons did not occur in the mature
brain. In most brain regions, the generation of neurons is
generally confined to a discrete developmental period. However,
notable exceptions are found in the dentate gyrus and the
subventricular zone of several species, where it has been shown
that new neurons are generated well into the postnatal and adult
period. Granule neurons are generated throughout life from a
population of continuously dividing neural progenitor cells
residing in the subgranular zone of the dentate gyrus in the rodent
brain.
[0006] "Newborn" neurons generated from these neural progenitor
cells migrate into the granule cell layer, differentiate, extend
axons and express neuronal marker proteins. The mechanisms and
appropriate stimuli that promote the generation of new neurons,
however, are largely unknown.
[0007] Attempts to counteract the effects of acute or
neurodegenerative lesions of the brain and/or spinal cord have
primarily involved implantation of embryonic neurons in an effort
to compensate for lost or deficient neural or neurological
function. However, human fetal cell transplantation research is
severely restricted. Administration of neurotrophic factors, such
as nerve growth factor and insulin-like growth factor, also has
been suggested to stimulate neuronal growth within the CNS.
[0008] To date, however, no satisfactory agents or treatment
methods exists to repair, or counteract, the neuronal damage
associated with neuropathies, such as Parkinson's disease and
Alzheimer's disease, neurological injury or neurological
age-related decline or impairment. Accordingly, there is a need for
new treatment modalities directed to improving the adverse
neurological conditions associated with neuropathies, neurological
injuries and age-related neurological decline or impairment.
[0009] Therefore, it is an object of the invention to provide
compositions for the treatment or prevention of neuronal damage
associated with neuropathies, such as Parkinson's disease and
Alzheimer's disease, neurological injury or neurological
age-related decline or impairment, and methods of making and using
thereof.
SUMMARY OF THE INVENTION
[0010] Compositions for the treatment or prevention of neuronal
damage associated with neuropathies, such as Parkinson's disease
and Alzheimer's disease, neurological injury or neurological
age-related decline or impairment, and methods of making and using
thereof are described herein. In one embodiment, the composition
contains .alpha.-hydroxy-5.alpha.-pregnan-20-one (also referred to
as allopregnanolone, THP, or AP.alpha.), a derivative, analogue or
prodrug thereof, a pharmaceutically acceptable salt thereof, or
combinations thereof. Suitable analogues or derivatives of THP
include, but are not limited to, 3-beta-phenylethynyl derivatives
of 3.alpha.-hydroxy-5.alpha.-pregnan-20-one; analogues or
derivatives of 3.alpha.-hydroxy-5.alpha.-pregnan-20-one that
exhibit substantially equivalent neuro-enhancing activity as
3.alpha.-hydroxy-5.alpha.-pregnan-20-one; progesterone; and
progesterone-like molecules, which are either natural metabolites
of progesterone or synthetic variants of progesterone, and exhibit
substantially equivalent neuro-enhancing activity as
3.alpha.-hydroxy-5.alpha.-pregnan-20-one.
[0011] Effective therapeutic amounts of the neuro-enhancing agents
will depend on the neurological disease or defect being targeted,
but generally range from about 0.1 mg to 1000 mg, preferably from
about 0.1 to 500 mg, more preferably from about 0.1 to about 100
mg. In one embodiment, the compositions contain at least about 10
mg or greater of the pharmaceutically active form of
3.alpha.-hydroxy-5.alpha.-pregnan-20-one or an analogue,
derivative, or prodrug thereof.
[0012] The compositions can be administered in a single dose or
multiple doses. The compositions can be administered on a daily
basis or less frequently, for example, every other day, once a
week, once a month, etc. The effective administration periods
depend on the particular neurological disease or defect being
targeted. Generally effective administration periods are about one
month or longer, but can be about six months to about one year or
longer. In a preferred embodiment, 10 mg/kg of allopregnanolone is
administered once per week. This dosing regimen maximizes
neurogenesis and minimizes pathology burden. FIG. 1 shows the
optimal allopregnanolone therapeutic regimen. The compositions are
typically administered for an extended period of time, for example,
at least about 10 weeks, preferably at least about 30 weeks, more
preferably at least about 60 weeks, even more preferably at least
about 72 weeks, and most preferably as long as the patient is
receiving noticeable benefit from the treatment method. In one
embodiment, the composition is administered once a week for at
least 6 months.
[0013] The compositions can be formulated for oral, enteral,
topical, or transdermal administration. The compositions can
further contain one or more pharmaceutically acceptable excipients,
carriers, and/or additives. In one embodiment, the compositions are
formulated for oral administration. Suitable oral dosage forms
include, but are not limited to, tablets, soft or hard, gelatin, or
non-gelatin capsules, caplets, solutions, syrups, and suspensions.
In a preferred embodiment, allopregnanolone is administered in a
transdermal gel containing CARBOMER.RTM. 940, and ethanol. In a
particularly preferred embodiment, the gel contains 10 mg/kg
allopregnanolone and is administered once a week for a period of at
least 3 months, preferably at least 6 months. Studies showed that
the transdermal gel was as effective as subcutaneous administration
(0.1% ethanol/phosphate buffered saline). In another embodiment,
allopregnanolone is administered intranasally.
[0014] In one embodiment, the compositions are administered to
enhance neurological function in an individual with a neurological
disease, neurological injury or age-related neuronal decline or
impairment. The compositions are administered over a period of time
effective to stimulate neural mitosis, to prevent neuronal loss, or
combination thereof. Target neurological dysfunctions and disease
states include Alzheimer's disease and Parkinson's disease;
neurological injuries, such as those following radiation therapy
for brain-related cancers or traumatic brain injuries; and
age-related memory decline and age-related learning impairments. In
one embodiment, the compositions are administered to reduce
.beta.-amyloid accumulation in the brain, which is associated with
Alzheimer's disease. The methods for enhancing neurological
function in an individual can be practiced in-vivo and/or
ex-vivo.
[0015] The compositions can also be administered to improve or
restore neurological function by inducing or stimulating the
generation of new neurons, protecting against neuronal loss,
stimulating or inducing neurite outgrowth and organization or
protecting against loss of neurites and neural networks, or
combination thereof.
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] FIG. 1 is a graphical representation of the optimal
allopregnanolone therapeutic regimen.
[0017] FIG. 2 is a bar graph showing the percent of the total
number of hippocampal neural cells exhibiting a mitotic appearance
(i.e. a doublet form cell body indicative of mitosis) in control
(clear rectangle), or following administration of 10 (nanomolar,
nM) or 100 (nanomolar, nM) 3.alpha.-hydroxy-5.alpha.-pregnan-20-one
(THP) (hatched rectangles).
[0018] FIG. 3 is a bar graph showing the percent of the hippocampal
neurons relative to the number of control neurons with a mitotic
appearance (i.e. a doublet form cell body indicative of mitosis) in
control (clear rectangle), or following administration of 10
(nanomolar, nM), 100 (nanomolar, nM), 250 (nanomolar, nM), or 500
(nanomolar, nM) 3.alpha.-hydroxy-5.alpha.-pregnan-20-one (THP)
(hatched rectangles).
[0019] FIG. 4 is a bar graph showing the ratio of relative
intensity of the cell division control protein cdc2 and actin in
control hippocampal neurons (clear rectangles) and hippocampal
neurons treated with 250 (nanomolar, nM), or 500 (nanomolar, nM)
3.alpha.-hydroxy-5.alpha.-pregnan-20-one (THP). Neurons were
collected following 24 hrs of THP exposure.
[0020] FIG. 5 is a bar graph showing the effect of
3.alpha.-hydroxy-5.alpha.-pregnan-20-one (THP, nM) on the total
number of hippocampal neurons.
[0021] FIG. 6 is a graph showing the effect of
3.alpha.-hydroxy-5.alpha.-pregnan-20-one (THP) on neuron number as
assessed in MuLV-GFP infected mouse neurons; the effect of THP on
HT-22 cells proliferation was detected on MuLV infected cells; left
panel shows the FACS profile of vehicle; right panel shows the FACS
profile of THP treated MuLV-GFP infected cells; the accompanying
table (Table 1) summarizes the FACS results. V=vehicle; THP (250
nM).
[0022] FIG. 7 is a line graph showing 3H-thymidine incorporation (%
of Basal) in hippocampal neural cells as a function of
3.alpha.-hydroxy-5.alpha.-pregnan-20-one (THP, nM) dosage.
[0023] FIG. 8 is a bar graph showing 3H-thymidine incorporation (%
of Basal) in hippocampal neural cells treated with
3.alpha.-hydroxy-5.alpha.-pregnan-20-one (THP), or other
structurally or chemically similar steroids.
[0024] FIG. 9 is a line graph showing depicts the time course of
3.alpha.-hydroxy-5.alpha.-pregnan-20-one (THP)-induced 3H-thymidine
incorporation (% of basal) in hippocampal neural cells as function
3.alpha.-hydroxy-5.alpha.-pregnan-20-one (THP) dosage (nM) at 1
hour (clear diamond), 8 hour (clear square) and 24 hour (clear
triangle) time intervals.
[0025] FIG. 10 shows the number of rat neural stem/progenitor cells
(cell counts (10.sup.5)) from neural spheres (generated from the
periventricular area and hippocampus of embryonic day 18 rat
embryos) treated with control, 250 nM
3.alpha.-hydroxy-5.alpha.-pregnan-20-one (THP), 500 nM
3.alpha.-hydroxy-5.alpha.-pregnan-20-one (THP), or .beta.(THP) with
(rectangles hatched from bottom left to top right), or without
EGF/FGF (rectangles hatched from top left to bottom right).
[0026] FIG. 11 is a graph showing percent of vehicle control BrdU
incorporation in human neural stem cells treated with various
concentrations of 3.alpha.-hydroxy-5.alpha.-pregnan-20-one
(AP.alpha.) (1, 10, 100, 250, 200, 1000 nM) or with bFGF (20
ng/ml)+heparin (5 llg/ml).
[0027] FIGS. 12A-D show the effect of
3.alpha.-hydroxy-5.alpha.-pregnan-20-one (AP.alpha.) on cell
proliferation in mouse hippocampus. FIG. 12A is a bar graph showing
the stereological analyses of mouse dentate gyrus subgranular zone
(SGZ) 24 hours following one subcutaneous dose of
3.alpha.-hydroxy-5.alpha.-pregnan-20-one (AP.alpha.) (10 mg/kg) or
vehicle control. Data is presented as total BrdU positive cells as
a function of time (months) in nonTg and 3.times.Tg mice treated
with vehicle or AP.alpha. (THP). Moving left to right across the
x-axis, Non-Tg/vehicle, Non-Tg/AP.alpha., 3.times.Tg/vehicle, and
3.times.Tg/AP.alpha. are clustered for 3, 6, 9, and 12 month time
points. FIG. 12B is a bar graph showing optical density reading
ratio of PCNA to .beta.-actin immunoblots in 3 month old nonTg
treated with vehicle or AP.alpha. (THP), and 3.times.Tg mice
treated with vehicle or AP.alpha. (THP). Immunoblots are shown
below. FIG. 12C is a bar graph showing optical density reading
ratio of PCNA to .beta.-actin immunoblots in 6 month old nonTg
treated with vehicle or AP.alpha. (THP), and 3.times.Tg mice
treated with vehicle or AP.alpha. (THP). Immunoblots are shown
below. FIG. 12D is a bar graph showing optical density reading
ratio of PCNA to .beta.-actin immuno blots in 3 month nonTg mice,
and 3, 6, and 9 month Tg mice treated with vehicle. The data
reveals that basal level (vehicle) of PCNA expression decreases
with age and pathology in 3.times.TgAD mice hippocampus compared to
3 month background strain non-Tg mice.
[0028] FIG. 13A is a schematic showing the experimental design of
the learning and memory experiments. FIG. 13B is a graph showing
the results of 5 days training. FIG. 13C is a bar graph showing the
results of the memory tests 9 days following the learning trial in
Non-Tg and 3.times.TgAD mice with or without
3.alpha.-hydroxy-5.alpha.-pregnan-20-one (AP.alpha.) treatment.
Data is represented as % of conditioned response in Non-Tg/vehicle
treated, Non-Tg/AP.alpha. treated, 3.times.TgAD/vehicle treated,
3.times.TgAD/AP.alpha. treated (from left to right across the
x-axis) mice.
[0029] FIG. 14A is a bar graph showing trace eye-blinking
conditioning (% conditioned response) as a function of training
days for 6 month old non-Tg mice treated with vehicle (solid line)
or 10 mg/kg 3.alpha.-hydroxy-5.alpha.-pregnan-20-one (AP.alpha.)
(dashed line). FIG. 14B is a bar graph showing trace eye-blinking
conditioning (% conditioned response) as a function of training
days for 6 month old 3.times.TgAD mice treated with vehicle (bottom
line) or 10 mg/kg 3.alpha.-hydroxy-5.alpha.-pregnan-20-one
(AP.alpha.) (top line). FIG. 14C is a bar graph showing trace
eye-blinking conditioning (% conditioned response) as a function of
training days for 9 month old non-Tg mice treated with vehicle
(solid line) or 10 mg/kg 3.alpha.-hydroxy-5.alpha.-pregnan-20-one
(AP.alpha.) (dashed line). FIG. 14D is a bar graph showing trace
eye-blinking conditioning (% conditioned response) as a function of
training days for 9 month old 3.times.TgAD mice treated with
vehicle (bottom line) or 10 mg/kg
3.alpha.-hydroxy-5.alpha.-pregnan-20-one (AP.alpha.) (top line).
FIG. 14E is a bar graph showing trace eye-blinking conditioning (%
conditioned response) as a function of training days for 12 month
old non-Tg mice treated with vehicle (solid line) or 10 mg/kg
3.alpha.-hydroxy-5.alpha.-pregnan-20-one (AP.alpha.) (dashed line).
FIG. 14F is a bar graph showing trace eye-blinking conditioning (%
conditioned response) as a function of training days for 12 month
old 3.times.TgAD mice treated with vehicle or 10 mg/kg
3.alpha.-hydroxy-5.alpha.-pregnan-20-one (AP.alpha.). FIG. 14G is a
bar graph showing trace eye-blinking conditioning (% conditioned
response) as a function of training days for 15 month old non-Tg
mice treated with vehicle (solid line) or 10 mg/kg
3.alpha.-hydroxy-5.alpha.-pregnan-20-one (AP.alpha.) (dashed line).
FIG. 14H is a bar graph showing trace eye-blinking conditioning (%
conditioned response) as a function of training days for 15 month
old 3.times.TgAD mice treated with vehicle or 10 mg/kg
3.alpha.-hydroxy-5.alpha.-pregnan-20-one (AP.alpha.).
[0030] FIG. 15 is a series of graphs showing that the
administration of THP increases neural progenitor cell
proliferation in hippocampus of 3.times.TgAD mice in an age
dependent manner. The effect is expressed as the total BrdU
cell/hippocampus in vehicle (left bar) and
3.alpha.-hydroxy-5.alpha.-pregnan-20-one (AP.alpha.) treatment
(right bar) for 3, 6, 9, and 12 month aged mice.
[0031] FIG. 16 is a bar graph showing the concentration of
A.beta..sub.1-42 (pg/mg) as a function of age of male mice (6, 9,
12, 15 months) transgenic for Alzheimer's disease
(3.times.Tg-AD).
[0032] FIG. 17 is a graph showing the effect of 10 mg/kg
3.alpha.-hydroxy-5.alpha.-pregnan-20-one (AP.alpha., THP)
administered once a week for 6 months, on the level of Abeta*56
(average intensity) in the cerebral cortex of 9- and 12-month male
mice transgenic for Alzheimer's disease. Data is presented as
average intensity for 9 month old 3.times.Tg/vehicle treated, 9
month old 3.times.Tg/AP.alpha. treated, 12 month old
3.times.Tg/vehicle treated, 12 month old 3.times.Tg/AP.alpha.
treated (from left to right across the x-axis) mice.
[0033] FIG. 18 is a bar graph showing that
3.alpha.-hydroxy-5.alpha.-pregnan-20-one (AP.alpha., THP) reduces
beta-amyloid 56 KD expression and overall amyloid burden in 9- and
12-month old male 3.times.TgAD mice following once per week
exposure for 6 months. Data is presented as % of control intensity
from an immunoblot of the 56 kD band detected with .beta.-amyloid
antibody 6E10 (which recognizes the abnormally processed isoforms
as well as precursor forms of beta-amyloid protein), in 9 month
vehicle treated (control) and AP.alpha. treated, and 12 month
vehicle treated (control) and AP.alpha. treated (from left to right
across the x-axis) mice. Immunoblot is found below.
[0034] FIG. 19A is a bar graph showing the immunocytochemical
detection of beta amyloid (A.beta.) (% of IR intensity vs. control)
in mice treated with vehicle (control) or
3.alpha.-hydroxy-5.alpha.-pregnan-20-one (AP.alpha., THP). FIG. 19B
is a bar graph showing the immunocytochemical detection of
phosphorylated tau (ptau) (% of IR intensity vs. control) in mice
treated with vehicle (control) or
3.alpha.-hydroxy-5.alpha.-pregnan-20-one (AP.alpha., THP).
[0035] FIG. 20 is a schematic of the treatment protocol for
evaluating the effects of long term
3.alpha.-hydroxy-5.alpha.-pregnan-20-one (AP.alpha., THP)
administration on neural progenitor cell proliferation in
hippocampus of 3.times.TgAD mice. FIGS. 20B and 20C are graphs
showing the amount of positive cells per 10,000 cells as a function
of the BrdU, IdU, and CIdU for 3 month old mice treated for 6
months (FIG. 20B) and 6 month old mice treated 6 months (FIG.
20C).
[0036] FIGS. 21A-D show the effect of
.alpha.-hydroxy-5.alpha.-pregnan-20-one (AP.alpha., THP) dose and
method of administration on the promotion of neural progenitor cell
proliferation in three month old 3.times.TgAD mouse hippocampus.
FIG. 21A is a bar graph showing the effect of
.alpha.-hydroxy-5.alpha.-pregnan-20-one (alloprenanoione,
AP.alpha., or THP) administered subcutaneously (0.1%
ethanol/phosphate buffered saline) on the promotion of neural
progenitor cell proliferation. Data is presented as BrdU
cells/hippocampus as a function of AP.alpha. concentration (0
mg/kg, or 10 mg/kg). FIG. 21B is a bar graph showing the effect of
10 mg/kg .alpha.-hydroxy-5.alpha.-pregnan-20-one (AP.alpha., or
THP) administered in a transdermal gel containing ethanol and
Carbomer 940 on the promotion of neural progenitor cell
proliferation. Data is presented as BrdU cells/hippocampus as a
function of AP.alpha. concentration (0 mg/kg, 5 mg/kg, 10 mg/kg or
50 mg/kg). FIG. 21C is a bar graph showing relative protein
intensity (% vs. vehicle from western blot analysis) of PCNA from
three month old 3.times.TgAD mice treated with 0 mg/kg, or 10 mg/kg
.alpha.-hydroxy-5.alpha.-pregnan-20-one (alloprenanoione,
AP.alpha., or THP) administered subcutaneously (0.1%
ethanol/phosphate buffered saline), or 0 mg/kg, 5 mg/kg, 10 mg/kg
or 50 mg/kg .alpha.-hydroxy-5.alpha.-pregnan-20-one
(alloprenanoione, AP.alpha., or THP) administered transdermally.
FIG. 21D is a bar graph showing relative protein intensity (% vs.
vehicle from western blot analysis) of total cdc2 from three month
old 3.times.TgAD mice treated with 0 mg/kg, or 10 mg/kg
.alpha.-hydroxy-5.alpha.-pregnan-20-one (alloprenanoione,
AP.alpha., or THP) administered subcutaneously (0.1%
ethanol/phosphate buffered saline), or 0 mg/kg, 5 mg/kg, 10 mg/kg
or 50 mg/kg .alpha.-hydroxy-5.alpha.-pregnan-20-one
(alloprenanoione, AP.alpha., or THP) administered
transdermally.
[0037] FIG. 22 is a graph showing quantification of 28 kDa
.beta.-amyloid oligomer band detected with .beta.-amyloid antibody
6E10 (percent of vehicle intensity) in 3 month old 3.times.Tg-AD
mice following vehicle, or 10 mg/kg
.alpha.-hydroxy-5.alpha.-pregnan-20-one (alloprenanoione,
AP.alpha., or THP) administered transdermally, THP (10 mg/kg) once
a week for 6 months. Non-treatment is also shown.
DETAILED DESCRIPTION OF THE INVENTION
I. Definitions
[0038] The term "analogue", as used herein, refers to a chemical
compound with a structure similar to that of another (reference
compound) but differing from it in respect to a particular
component, functional group, atom, etc.
[0039] The term "derivative", as used herein, refers to compounds
which are formed from a parent compound by one or more chemical
reaction(s).
[0040] The term "prodrug", as used herein, refers to an active drug
chemically transformed into a per se inactive derivative which, by
virtue of chemical or enzymatic attack, is converted to the parent
drug within the body before or after reaching the site of action.
Prodrugs are frequently (though not necessarily) pharmacologically
inactive until converted to the parent drug. Methods for converting
to drugs to prodrugs are known in the art. Suitable examples of
prodrugs include, but are not limited to, ester and amide prodrugs;
polyethylene glycol prodrugs with or without a linker; carbonate
prodrugs; and dihydroxypropyl prodrugs.
[0041] "Pharmaceutically acceptable salt", as used herein, refers
to the modification of the parent compound by making the acid or
base salts thereof. Example of pharmaceutically acceptable salts
include, but are not limited to, mineral or organic acid salts of
basic residues such as amines and alkali or organic salts of acidic
residues such as carboxylic acids. The pharmaceutically acceptable
salts include the conventional non-toxic salts or the quaternary
ammonium salts of the parent compound formed, for example, from
non-toxic inorganic or organic acids. Such conventional non-toxic
salts include those derived from inorganic acids such as
hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, and
nitric acids; and the salts prepared from organic acids such as
acetic, propionic, succinic, glycolic, stearic, lactic, malic,
tartaric, citric, ascorbic, pamoic, maleic, hydroxymaleic,
phenylacetic, glutamic, benzoic, salicylic, sulfanilic,
2-acetoxybenzoic, fumaric, toluenesulfonic, naphthalenesulfonic,
methanesulfonic, ethane disulfonic, oxalic, and isethionic
salts.
[0042] The pharmaceutically acceptable salts of the compounds can
be synthesized from the parent compound, which contains a basic or
acidic moiety, by conventional chemical methods. Generally, such
salts can be prepared by reacting the free acid or base forms of
these compounds with a stoichiometric amount of the appropriate
base or acid in water or in an organic solvent, or in a mixture of
the two; generally, non-aqueous media like ether, ethyl acetate,
ethanol, isopropanol, or acetonitrile are preferred. Lists of
suitable salts are found in Remington's Pharmaceutical Sciences,
20th ed., Lippincott Williams & Wilkins, Baltimore, Md., 2000,
p. 704; and "Handbook of Pharmaceutical Salts: Properties,
Selection, and Use," P. Heinrich Stahl and Camille G. Wermuth,
Eds., Wiley-VCR, Weinheim, 2002.
[0043] As generally used herein "pharmaceutically acceptable"
refers to those compounds, materials, compositions, and/or dosage
forms which are, within the scope of sound medical judgment,
suitable for use in contact with the tissues of human beings and
animals without excessive toxicity, irritation, allergic response,
or other problems or complications commensurate with a reasonable
benefit/risk ratio.
[0044] Modified release dosage form: A modified release dosage form
is one for which the drug release characteristics of time, course
and/or location are chosen to accomplish therapeutic or convenience
objectives not offered by conventional dosage forms such as
solutions, ointments, or promptly dissolving dosage forms. Delayed
release, extended release, and pulsatile release dosage forms and
their combinations are types of modified release dosage forms.
[0045] Delayed release dosage form: A delayed release dosage form
is one that releases a drug (or drugs) at a time other than
promptly after administration.
[0046] Extended release dosage form: An extended release dosage
form is one that allows at least a twofold reduction in dosing
frequency as compared to the drug presented as a conventional
dosage form (e.g. as a solution or prompt drug-releasing,
conventional solid dosage form).
[0047] Pulsatile release dosage form: A pulsatile release dosage
form is one that mimics a multiple dosing profile without repeated
dosing and allows at least a twofold reduction in dosing frequency
as compared to the drug presented as a conventional dosage form
(e.g. as a solution or prompt drug-releasing, conventional solid
dosage form). A pulsatile release profile is characterized by a
time period of no release (lag time) or reduced release followed by
rapid drug release.
II. Compositions
[0048] A. Neuro-Enhancing Agents
[0049] The compositions described herein contain one or more
neuro-enhancing agents. In one embodiment, the one or more
neuro-enhancing agents are selected from progesterone or an
analogue or derivative thereof, such as precursors of progesterone,
progesterone metabolites and progesterone derivatives in its
metabolic pathway, as well as the salts or hydrates of these
analogues and derivatives. In a preferred embodiment, the
compositions contains a naturally occurring metabolite of
progesterone, 3.alpha.-hydroxy-5.alpha.-pregnan-20-one (AP.alpha.),
also known as tetrahydroprogesterone (THP), as well as the
pharmaceutically acceptable salts and hydrates thereof.
3.alpha.-hydroxy-5.alpha.-pregnan-20-one (THP) is generally
classified as a neurosteroid as it is produced in the central
nervous system and previously has been found to be an allosteric
modulator of GABA receptors.
[0050] Other suitable analogs and derivatives include variant
molecules of 3.alpha.-hydroxy-5.alpha.-pregnan-20-one or
substituted derivatives of
3.alpha.-hydroxy-5.alpha.-pregnan-20-one, such as 3.alpha.-oxy
derivatives, 3.alpha.-alkyl derivatives, 3.alpha.-alkenyl
derivatives, 3.alpha.-ester derivatives, 3.alpha.-ether
derivatives; 3ss-phenylethynyl derivatives of
3.alpha.-hydroxy-5.alpha.-pregnan-20-one, and 3p-phenylethynyl
derivatives of 3.alpha.-hydroxy-5.alpha.-pregnan-20-one, as
described in Hawkinson, et al. J. Pharmacology & Experimental
Therapeutics 287: 198-207 (1998); as well as steroids derivatives
of the 5.alpha. pregnan-20-one series such as those described in
U.S. Pat. Nos. 5,925,630; 6,143,736; and 6,277,838.
[0051] Analogs or derivatives of
3.alpha.-hydroxy-5.alpha.-pregnan-20-one include progesterone-like
molecules that are either natural precursors or metabolites of
progesterone or synthetic variants of progesterone that exhibit
substantially equivalent neurogenic activity as
3.alpha.-hydroxy-5.alpha.-pregnan-20-one. Substantially equivalent
neuro-enhancing activity is defined as approximately 30% to
approximately 300% of the neuro-enhancing activity of
3.alpha.-hydroxy-5.alpha.-pregnan-20-one.
[0052] The neuro-enhancing agents are administered at dosages and
for periods of time effective to stimulate or induce neural
proliferation and/or to protect against neural loss in an
individual. Dosage regimes may be adjusted for purposes of
improving the therapeutic response to the particular composition
administered. For example, several divided doses may be
administered daily or the dose may be proportionally reduced as
indicated by the exigencies of the therapeutic situation. The
dosages of the one or more neuro-enhancing agents is in the range
of about 0.1 mg to about 1000 mg, more preferably in the range of
about 1 mg to about 500 mg, most preferably in the range from about
10 mg to about 100 mg. However, the particular dose depends on the
particular neurological disease or defect being targeted and can be
readily determined by the treating physician.
[0053] The compounds described herein may have one or more chiral
centers and thus exist as one or more stereoisomers. Such
stereoisomers can exist as a single enantiomer, a mixture of
diastereomers or a racemic mixture. As used herein, the term
"stereoisomers" refers to compounds made up of the same atoms
having the same bond order but having different three-dimensional
arrangements of atoms which are not interchangeable. The
three-dimensional structures are called configurations. As used
herein, the term "enantiomers" refers to two stereoisomers which
are non-superimposable mirror images of one another. As used
herein, the term "optical isomer" is equivalent to the term
"enantiomer". As used herein the term "diastereomer" refers to two
stereoisomers which are not mirror images but also not
superimposable. The terms "racemate", "racemic mixture" or "racemic
modification" refer to a mixture of equal parts of enantiomers. The
term "chiral center" refers to a carbon atom to which four
different groups are attached. Choice of the appropriate chiral
column, eluent, and conditions necessary to effect separation of
the pair of enantiomers is well known to one of ordinary skill in
the art using standard techniques (see e.g. Jacques, J. et al.,
"Enantiomers, Racemates, and Resolutions", John Wiley and Sons,
Inc. 1981).
[0054] B. Additional Active Agents
[0055] The compositions can further contain one or more additional
active agents. In one embodiment, the additional active agent is a
steroid. Suitable steroids include biologically active forms of
vitamin D3 and D2, such as those described in U.S. Pat. Nos.
4,897,388 and 5,939,407. The steroids may be co-administered to
further aid in neurogenic stimulation or induction and/or
prevention of neural loss, particularly for treatments of
Alzheimer's disease. Estrogen and estrogen related molecules also
may be co-administered with the neuro-enhancing agents to enhance
neuroprotection as described in Brinton (2001) Learning and Memory
8 (3): 121-133.
[0056] Other neuroactive steroids, such as various forms of
dehydroepiandrosterone (DHEA) as described in U.S. Pat. No.
6,552,010, can also be co-administered to further aid in neurogenic
stimulation or induction and/or prevention of neural loss. Other
agents that cause neural growth and outgrowth of neural networks,
such as Nerve Growth Factor (NGF) and Brain-derived Neurotrophic
Factor (BDNF), can be administered either simultaneously with or
before or after the administration of THP. Additionally, inhibitors
of neural apoptosis, such as inhibitors of calpains and capases and
other cell death mechanisms, such as necrosis, can be
co-administered with the neuro-enhancing agents o further prevent
neural loss associated with certain neurological diseases and
neurological defects.
[0057] C. Formulations
[0058] Depending upon the manner of introduction, the
neuro-enhancing agents described herein may be formulated in a
variety of ways. Formulations containing THP or other substantially
equivalent variant molecules can be prepared in various
pharmaceutical forms, such as granules, tablets, capsules,
suppositories, powders, controlled release formulations,
suspensions, emulsions, creams, gels, ointments, salves, lotions,
or aerosols and the like.
[0059] In one embodiment, the neuro-enhancing agent are formulated
as solid dosage forms suitable for simple, and preferably oral,
administration of precise dosages. Solid dosage forms for oral
administration include, but are not limited to, tablets, soft or
hard gelatin or non-gelatin capsules, and caplets. However, liquid
dosage forms, such as solutions, syrups, suspension, shakes, etc.
can also be utilized.
[0060] In another embodiment, the formulation is administered
topically or transdermally. Suitable topical and transdermal
formulations include, but are not limited to, lotions, ointments,
creams, and gels. In a preferred embodiment, the transdermal
formulation is a gel. "Topical", as used herein, generally refers
to formulations for local delivery of an active agent, for example,
via a gel, lotion, cream, ointment, or patch. "Transdermal", as
used herein, generally refers to systemic delivery of a drug
through the unbroken skin, for example, via a gel, lotion, cream,
ointment, or patch.
[0061] In another embodiment, the formulation is administered
intranasally. Examples of intranasal formulations include aqueous
preparations, preparations containing one or more inhalants, and
dry powder formulations. The nasal mucosa is highly vascularized;
the delivery of a thin layer of medication across a broad surface
area can result in rapid transmucosal absorption of the medication
into the blood stream and cerebral spinal fluid. This can result in
more rapid achievement of therapeutic drug levels compared to oral
or parenteral formulations.
[0062] Formulations containing one or more of the compounds
described herein may be prepared using a pharmaceutically
acceptable carrier composed of materials that are considered safe
and effective and may be administered to an individual without
causing undesirable biological side effects or unwanted
interactions. The carrier is all components present in the
pharmaceutical formulation other than the active ingredient or
ingredients. As generally used herein "carrier" includes, but is
not limited to, diluents, binders, lubricants, disintegrators,
fillers, pH modifying agents, preservatives, antioxidants,
solubility enhancers, and coating compositions.
[0063] Carrier also includes all components of coating compositions
which may include plasticizers, pigments, colorants, stabilizing
agents, and glidants. Delayed release, extended release, and/or
pulsatile release dosage formulations may be prepared as described
in standard references known in the art. These references provide
information on carriers, materials, equipment and process for
preparing tablets and capsules and delayed release dosage forms of
tablets, capsules, and granules.
[0064] Examples of suitable coating materials include, but are not
limited to, cellulose polymers such as cellulose acetate phthalate,
hydroxypropyl cellulose, hydroxypropyl methylcellulose,
hydroxypropyl methylcellulose phthalate and hydroxypropyl
methylcellulose acetate succinate; polyvinyl acetate phthalate,
acrylic acid polymers and copolymers, and methacrylic resins that
are commercially available under the trade name EUDRAGIT.RTM. (Roth
Pharma, Westerstadt, Germany), zein, shellac, and
polysaccharides.
[0065] Additionally, the coating material may contain conventional
carriers such as plasticizers, pigments, colorants, glidants,
stabilization agents, pore formers and surfactants.
[0066] Optional pharmaceutically acceptable excipients present in
the drug-containing tablets, beads, granules or particles include,
but are not limited to, diluents, binders, lubricants,
disintegrants, colorants, stabilizers, and surfactants.
[0067] Diluents, also referred to as "fillers," are typically
necessary to increase the bulk of a solid dosage form so that a
practical size is provided for compression of tablets or formation
of beads and granules. Suitable diluents include, but are not
limited to, dicalcium phosphate dihydrate, calcium sulfate,
lactose, sucrose, mannitol, sorbitol, cellulose, microcrystalline
cellulose, kaolin, sodium chloride, dry starch, hydrolyzed
starches, pregelatinized starch, silicone dioxide, titanium oxide,
magnesium aluminum silicate and powdered sugar.
[0068] Binders are used to impart cohesive qualities to a solid
dosage formulation, and thus ensure that a tablet or bead or
granule remains intact after the formation of the dosage forms.
Suitable binder materials include, but are not limited to, starch,
pregelatinized starch, gelatin, sugars (including sucrose, glucose,
dextrose, lactose and sorbitol), polyethylene glycol, waxes,
natural and synthetic gums such as acacia, tragacanth, sodium
alginate, cellulose, including hydroxypropylmethylcellulose,
hydroxypropylcellulose, ethylcellulose, and veegum, and synthetic
polymers such as acrylic acid and methacrylic acid copolymers,
methacrylic acid copolymers, methyl methacrylate copolymers,
aminoalkyl methacrylate copolymers, polyacrylic
acid/polymethacrylic acid and polyvinylpyrrolidone.
[0069] Lubricants are used to facilitate tablet manufacture.
Examples of suitable lubricants include, but are not limited to,
magnesium stearate, calcium stearate, stearic acid, glycerol
behenate, polyethylene glycol, talc, and mineral oil.
[0070] Disintegrants are used to facilitate dosage form
disintegration or "breakup" after administration, and generally
include, but are not limited to, starch, sodium starch glycolate,
sodium carboxymethyl starch, sodium carboxymethylcellulose,
hydroxypropyl cellulose, pregelatinized starch, clays, cellulose,
alginine, gums or cross linked polymers, such as cross-linked PVP
(Polyplasdone XL from GAF Chemical Corp).
[0071] Stabilizers are used to inhibit or retard drug decomposition
reactions which include, by way of example, oxidative
reactions.
[0072] Surfactants may be anionic, cationic, amphoteric or nonionic
surface active agents. Suitable anionic surfactants include, but
are not limited to, those containing carboxylate, sulfonate and
sulfate ions. Examples of anionic surfactants include sodium,
potassium, ammonium of long chain alkyl sulfonates and alkyl aryl
sulfonates such as sodium dodecylbenzene sulfonate; dialkyl sodium
sulfosuccinates, such as sodium dodecylbenzene sulfonate; dialkyl
sodium sulfosuccinates, such as sodium
bis-(2-ethylthioxyl)-sulfosuccinate; and alkyl sulfates such as
sodium lauryl sulfate. Cationic surfactants include, but are not
limited to, quaternary ammonium compounds such as benzalkonium
chloride, benzethonium chloride, cetrimonium bromide, stearyl
dimethylbenzyl ammonium chloride, polyoxyethylene and coconut
amine. Examples of nonionic surfactants include ethylene glycol
monostearate, propylene glycol myristate, glyceryl monostearate,
glyceryl stearate, polyglyceryl-4-oleate, sorbitan acylate, sucrose
acylate, PEG-150 laurate, PEG-400 monolaurate, polyoxyethylene
monolaurate, polysorbates, polyoxyethylene octylphenylether,
PEG-1000 cetyl ether, polyoxyethylene tridecyl ether, polypropylene
glycol butyl ether, Poloxamer.RTM. 401, stearoyl
monoisopropanolamide, and polyoxyethylene hydrogenated tallow
amide. Examples of amphoteric surfactants include sodium
N-dodecyl-.beta.-alanine, sodium N-lauryl-.beta.-iminodipropionate,
myristoamphoacetate, lauryl betaine and lauryl sulfobetaine.
[0073] If desired, the tablets, beads, granules, or particles may
also contain minor amount of nontoxic auxiliary substances such as
wetting or emulsifying agents, dyes, pH buffering agents, or
preservatives.
[0074] The proportion of pharmaceutically active neuro-enhancing
agent to carrier and/or other substances may vary from about 0.5 to
about 100 wt % (weight percent). For oral use, the pharmaceutical
formulation will generally contain from about 5 to about 100% by
weight of the active material. For other uses, the pharmaceutical
formulation will generally have from about 0.5 to about 50 wt. % of
the active material.
[0075] 2. Transdermal Formulations
[0076] Suitable transdermal formulations include lotions,
ointments, creams, gels, and patches. A "lotion" is a low- to
medium-viscosity liquid formulation. A lotion can contain finely
powdered substances that are in soluble in the dispersion medium
through the use of suspending agents and dispersing agents.
Alternatively, lotions can have as the dispersed phase liquid
substances that are immiscible with the vehicle and are usually
dispersed by means of emulsifying agents or other suitable
stabilizers. In one embodiment, the lotion is in the form of an
emulsion having a viscosity of between 100 and 1000 centistokes.
The fluidity of lotions permits rapid and uniform application over
a wide surface area. Lotions are typically intended to dry on the
skin leaving a thin coat of their medicinal components on the
skin's surface.
[0077] A "cream" is a viscous liquid or semi-solid emulsion of
either the "oil-in-water" or "water-in-oil type". Creams may
contain emulsifying agents and/or other stabilizing agents. In one
embodiment, the formulation is in the form of a cream having a
viscosity of greater than 1000 centistokes, typically in the range
of 20,000-50,000 centistokes. Creams are often time preferred over
ointments as they are generally easier to spread and easier to
remove.
[0078] An emulsion is a preparation of one liquid distributed in
small globules throughout the body of a second liquid. The
dispersed liquid is the discontinuous phase, and the dispersion
medium is the continuous phase. When oil is the dispersed liquid
and an aqueous solution is the continuous phase, it is known as an
oil-in-water emulsion, whereas when water or aqueous solution is
the dispersed phase and oil or oleaginous substance is the
continuous phase, it is known as a water-in-oil emulsion. The oil
phase may consist at least in part of a propellant, such as an HFA
propellant. Either or both of the oil phase and the aqueous phase
may contain one or more surfactants, emulsifiers, emulsion
stabilizers, buffers, and other excipients. Preferred excipients
include surfactants, especially non-ionic surfactants; emulsifying
agents, especially emulsifying waxes; and liquid non-volatile
non-aqueous materials, particularly glycols such as propylene
glycol. The oil phase may contain other oily pharmaceutically
approved excipients. For example, materials such as hydroxylated
castor oil or sesame oil may be used in the oil phase as
surfactants or emulsifiers. A sub-set of emulsions are the
self-emulsifying systems. These drug delivery systems are typically
capsules (hard shell or soft shell) comprised of the drug dispersed
or dissolved in a mixture of surfactant(s) and lipophilic liquids
such as oils or other water immiscible liquids. When the capsule is
exposed to an aqueous environment and the outer gelatin shell
dissolves, contact between the aqueous medium and the capsule
contents instantly generates very small emulsion droplets. These
typically are in the size range of micelles or nanoparticles. No
mixing force is required to generate the emulsion as is typically
the case in emulsion formulation processes. Self generating
emulsions are known to enhance the absorption of drugs as shown in
the following table.
[0079] In one embodiment, the formulation is a transdermal gel. A
"gel" is a semisolid system containing a dispersion of the active
agent, i.e., allopregnanolone, in a liquid vehicle that is rendered
semisolid by the action of a thickening agent or polymeric material
dissolved or suspended in the liquid vehicle. The liquid may
include a lipophilic component, an aqueous component or both. Some
emulsions may be gels or otherwise include a gel component. Some
gels, however, are not emulsions because they do not contain a
homogenized blend of in components. "Lipophilic" refers to
compounds having an affinity for lipids.
[0080] The gelling agent can be natural, semi-synthetic, or
synthetic. Suitable thickening or gelling agents include, but are
not limited to, acacia, acrylates/steareth-20 methacrylate
copolymer, agar, algin, alginic acid, ammonium acrylate copolymers,
ammonium alginate, ammonium chloride, ammonium sulfate,
amylopectin, attapulgite, bentonite, C.sub.9-C.sub.15 alcohols,
calcium acetate, calcium alginate, calcium carrageenan, calcium
chloride, caprylic alcohol, vinyl polymers such as cross linked
acrylic acid polymers with the name carbomer, such as but not
limited to carbomer 910, carbomer 934, carbomer 934P, carbomer 940,
carbomer 941, carboxymethyl hydroxyethylcellulose, carboxymethyl
hydroxypropyl guar, carrageenan, cellulose, cellulose gum, cetearyl
alcohol, cetyl alcohol, corn starch, damar, dextrin, dibenzylidine
sorbitol, ethylene dihydrogenated tallowamide, ethylene dioleamide,
ethylene distearamide, gelatin, guar gum, hydroxypropyltrimonium
chloride, hectorite, hyaluronic acid, hydrated silica, hydroxybutyl
methylcellulose, hydroxyethylcellulose, hydroxyethyl
ethylcellulose, hydroxyethyl stearamide-MIPA,
hydroxypropylcellulose, hydroxypropyl guar, hydroxypropyl
methylcellulose, isocetyl alcohol, isostearyl alcohol, karaya gum,
kelp, lauryl alcohol, locust bean gum, magnesium aluminum silicate,
magnesium silicate, magnesium trisilicate, methoxy PEG-22/dodecyl
glycol copolymer, methylcellulose, microcrystalline cellulose,
montmorillonite, myristyl alcohol, oat flour, oleyl alcohol, palm
kernel alcohol, pectin, PEG-2M is also known as Polyox WSR.RTM.
N-IO, which is available from Union Carbide and as PEG-2,000;
PEG-5M is also known as Polyox WSR.RTM. N-35 and Polyox WSR.RTM.
N-80, both available from Union Carbide and as PEG-5,000 and
Polyethylene Glycol 300,000; PEG-7M is also known as Polyox
WSR.RTM. N-750 available from Union Carbide; PEG 9-M is also known
as Polyox WSR.RTM. N-3333 available from Union Carbide; PEG-14M is
also known as Polyox WSR.RTM. N-3000 available from Union Carbide.,
polyacrylic acid, polyvinyl alcohol, potassium alginate, potassium
aluminum polyacrylate, potassium carrageenan, potassium chloride,
potassium sulfate, potato starch, propylene glycol alginate, sodium
acrylate/vinyl alcohol copolymer, sodium carboxymethyl dextran,
sodium carrageenan, sodium cellulose sulfate, sodium chloride,
sodium polymethacrylate, sodium silicoaluminate, sodium sulfate,
stearalkonium bentonite, stearalkonium hectorite, stearyl alcohol,
tallow alcohol, TEA-hydrochloride, tragacanth gum, tridecyl
alcohol, tromethamine magnesium aluminum silicate, wheat flour,
wheat starch, xanthan gum, and mixtures thereof.
[0081] The concentration of gelling agent can be adjusted to change
the viscosity of the gel. For example, in some embodiments the
formulation includes 10%, 20%, 30%, 40%, 50%, 60%, or 70% w/v of a
gelling agent. Alternatively, the gelling agent can be in a range
of 1-80% w/v.
[0082] Suitable solvents in the liquid vehicle include, but are not
limited to, diglycol monoethyl ether; alklene glycols, such as
propylene glycol; dimethyl isosorbide; alcohols, such as isopropyl
alcohol and ethanol. The solvents are typically selected for their
ability to dissolve the drug.
[0083] The concentration of the solvent can also be adjusted. For
example, in some embodiments the formulation includes 10%, 20%,
30%, 40%, or 50% v/v of solvent. Alternatively, the solvent can be
in a range of 1-50% v/v.
[0084] The gel may contain one or more penetration enhancers, for
example to cross the barrier of the stratum corneum. Suitable
enhancer include, but are not limited to, urea, (carbonyldiamide),
imidurea, N,N-diethylformamide, N-methyl-2-pyrrolidine,
1-dodecal-azacyclopheptane-2-one, calcium thioglycate,
2-pyyrolidine, N,N-diethyl-m-toluamide, oleic acid and its ester
derivatives, such as methyl, ethyl, propyl, isopropyl, butyl, vinyl
and glycerylmonooleate, sorbitan esters, such as sorbitan
monolaurate and sorbitan monooleate, other fatty acid esters such
as isopropyl laurate, isopropyl myristate, isopropyl palmitate,
diisopropyl adipate, propylene glycol monolaurate, propylene glycol
monooleatea and non-ionic detergents such as BRIJ.RTM. 76 (stearyl
poly(10 oxyethylene ether), BRIJ.RTM. 78 (stearyl
poly(20)oxyethylene ether), BRIJ.RTM. 96 (oleyl poly(10)oxyethylene
ether), and BRIJ.RTM. 721 (stearyl poly (21) oxyethylene ether)
(ICI Americas Inc. Corp.).
[0085] Other additives, which improve the skin feel and/or
emolliency of the formulation, may also be incorporated. Examples
of such additives include, but are not limited, isopropyl
myristate, ethyl acetate, C12-C15 alkyl benzoates, mineral oil,
squalane, cyclomethicone, capric/caprylic triglycerides, and
combinations thereof.
[0086] The gel may also contain a preservative. Preservatives can
be used to prevent the growth of fungi and microorganisms. Suitable
antifungal and antimicrobial agents include, but are not limited
to, benzoic acid, butylparaben, ethyl paraben, methyl paraben,
propylparaben, sodium benzoate, sodium propionate, benzalkonium
chloride, benzethonium chloride, benzyl alcohol, cetylpyridinium
chloride, chlorobutanol, phenol, phenylethyl alcohol, and
thimerosal.
[0087] Transdermal formulations can be prepared to provide
sustained or extended release of the neuro-enhancing agents.
[0088] In a preferred embodiment, the gel contains ethanol as a
solvent and carbomer 940 as the gelling agent.
[0089] 3. Intranasal Formulations
[0090] In one embodiment, the compounds described herein are
formulated for intranasal administration for delivery of the
compounds to the brain. The olfactory mucosa are in direct contact
with the brain and CSF. Therefore, medications absorbed across the
olfactory mucosa directly enter the CSF. This provides a rapid,
direct route for drug delivery to the brain. Bioavailability for
drugs can be much higher when administered intranasally versus
other routes of administration. Further, intranasal administration
avoids the gut thereby bypassing first pass metabolism by the
liver.
[0091] The compounds can be formulated as solutions or suspensions
in an aqueous or organic solvent or as a dry powder. For
suspensions and dry powder formulations, particles sizes of 10-50
microns adhere best to the nasal mucosa, as smaller particles may
pass on to the lungs and larger particles can form droplets and run
out of the nose. Atomized drugs are typically more effective than
liquids since they provide larger surface area coverage and the
smaller particle size provides a thin layer to cover the
mucosa.
[0092] Compounds can be administered intranasally in the form of
drops which are administered using a syringe or dropper, sprays or
atomized formulations which provide a unit dose, such as a via
syringe or a unit dose pump, or nebulized formulations. Devices for
administering drugs intranasally are well known in the art.
[0093] Intranasal formulations may contain one or more excipients,
such as penetration enhancers, surfactants, preservatives, etc.
[0094] 4. Enteral Formulations
[0095] Pharmaceutical compositions for oral administration can be
liquid or solid. Liquid dosage forms suitable for oral
administration include, but are not limited to, pharmaceutically
acceptable emulsions, microemulsions, solutions, suspensions,
syrups and elixirs. In addition to an encapsulated or
unencapsulated HDAC inhibitor, the liquid dosage forms may contain
inert diluents commonly used in the art such as, for example, water
or other solvents, solubilizing agents and emulsifiers such as
ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate,
benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene
glycol, dimethylformamide, oils (in particular, cottonseed,
groundnut, corn, germ, olive, castor and sesame oils), glycerol,
tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters
of sorbitan and mixtures thereof. Besides inert diluents, the oral
compositions can also include adjuvants, wetting agents,
emulsifying and suspending agents, sweetening, flavoring and
perfuming agents.
[0096] Solid dosage forms for oral administration include, but are
not limited to, capsules, tablets, caplets, dragees, powders and
granules. In such solid dosage forms, the encapsulated or
unencapsulated compound is typically mixed with at least one inert,
pharmaceutically acceptable excipient or carrier such as sodium
citrate or dicalcium phosphate and/or (a) fillers or extenders such
as starches, lactose, sucrose, glucose, mannitol and silicic acid,
(b) binders such as, for example, carboxymethylcellulose,
alginates, gelatin, polyvinylpyrrolidinone, sucrose and acacia, (c)
humectants such as glycerol, (d) disintegrating agents such as
agar-agar, calcium carbonate, potato or tapioca starch, alginic
acid, certain silicates and sodium carbonate, (e) solution
retarding agents such as paraffin, (f) absorption accelerators such
as quaternary ammonium compounds, (g) wetting agents such as, for
example, cetyl alcohol and glycerol monostearate, (h) absorbents
such as kaolin and bentonite clay and (i) lubricants such as talc,
calcium stearate, magnesium stearate, solid polyethylene glycols,
sodium lauryl sulfate and mixtures thereof. In the case of
capsules, tablets and pills, the dosage form may also contain
buffering agents.
[0097] Solid compositions of a similar type may also be employed as
fill materials in soft and hard-filled gelatin capsules using such
excipients as lactose or milk sugar as well as high molecular
weight polyethylene glycols and the like. The solid dosage forms of
tablets, dragees, capsules, pills and granules can be prepared with
coatings and shells such as enteric coatings and other coatings
well known in the pharmaceutical formulating art.
[0098] 5. Modified Release Formulations
[0099] The compositions described herein can be formulation for
modified or controlled release. Examples of controlled release
dosage forms include extended release dosage forms, delayed release
dosage forms, pulsatile release dosage forms, and combinations
thereof.
[0100] Extended Release Dosage Forms
[0101] The extended release formulations are generally prepared as
diffusion or osmotic systems, for example, as described in
"Remington-The science and practice of pharmacy" (20th ed.,
Lippincott Williams & Wilkins, Baltimore, Md., 2000). A
diffusion system typically consists of two types of devices, a
reservoir and a matrix, and is well known and described in the art.
The matrix devices are generally prepared by compressing the drug
with a slowly dissolving polymer carrier into a tablet form. The
three major types of materials used in the preparation of matrix
devices are insoluble plastics, hydrophilic polymers, and fatty
compounds. Plastic matrices include, but are not limited to, methyl
acrylate-methyl methacrylate, polyvinyl chloride, and polyethylene.
Hydrophilic polymers include, but are not limited to, cellulosic
polymers such as methyl and ethyl cellulose, hydroxyalkylcelluloses
such as hydroxypropyl-cellulose, hydroxypropylmethylcellulose,
sodium carboxymethylcellulose, and Carbopol.RTM. 934, polyethylene
oxides and mixtures thereof. Fatty compounds include, but are not
limited to, various waxes such as carnauba wax and glyceryl
tristearate and wax-type substances including hydrogenated castor
oil or hydrogenated vegetable oil, or mixtures thereof.
[0102] In certain preferred embodiments, the plastic material is a
pharmaceutically acceptable acrylic polymer, including but not
limited to, acrylic acid and methacrylic acid copolymers, methyl
methacrylate, methyl methacrylate copolymers, ethoxyethyl
methacrylates, cyanoethyl methacrylate, aminoalkyl methacrylate
copolymer, poly(acrylic acid), poly(methacrylic acid), methacrylic
acid alkylamine copolymer poly(methyl methacrylate),
poly(methacrylic acid) (anhydride), polymethacrylate,
polyacrylamide, poly(methacrylic acid anhydride), and glycidyl
methacrylate copolymers.
[0103] In certain preferred embodiments, the acrylic polymer is
comprised of one or more ammonio methacrylate copolymers. Ammonio
methacrylate copolymers are well known in the art, and are
described in NF XVII as fully polymerized copolymers of acrylic and
methacrylic acid esters with a low content of quaternary ammonium
groups.
[0104] In one preferred embodiment, the acrylic polymer is an
acrylic resin lacquer such as that which is commercially available
from Rohm Pharma under the tradename Eudragit.RTM.. In further
preferred embodiments, the acrylic polymer comprises a mixture of
two acrylic resin lacquers commercially available from Rohm Pharma
under the tradenames Eudragit.RTM. RL30D and Eudragit.RTM. RS30D,
respectively. Eudragit.RTM. RL30D and Eudragit.RTM. RS30D are
copolymers of acrylic and methacrylic esters with a low content of
quaternary ammonium groups, the molar ratio of ammonium groups to
the remaining neutral (meth)acrylic esters being 1:20 in
Eudragit.RTM. RL30D and 1:40 in Eudragit.RTM. RS30D. The mean
molecular weight is about 150,000. Edragit.RTM. S-100 and
Eudragit.RTM. L-100 are also preferred. The code designations RL
(high permeability) and RS (low permeability) refer to the
permeability properties of these agents. Eudragit.RTM. RL/RS
mixtures are insoluble in water and in digestive fluids. However,
multiparticulate systems formed to include the same are swellable
and permeable in aqueous solutions and digestive fluids.
[0105] The polymers described above such as Eudragit.RTM. RL/RS may
be mixed together in any desired ratio in order to ultimately
obtain a sustained-release formulation having a desirable
dissolution profile. Desirable sustained-release multiparticulate
systems may be obtained, for instance, from 100% Eudragit.RTM. RL,
50% Eudragit.RTM. RL and 50% Eudragit.RTM. RS, and 10%
Eudragit.RTM. RL and 90% Eudragit.RTM. RS. One skilled in the art
will recognize that other acrylic polymers may also be used, such
as, for example, Eudragit.RTM. L.
[0106] Alternatively, extended release formulations can be prepared
using osmotic systems or by applying a semi-permeable coating to
the dosage form. In the latter case, the desired drug release
profile can be achieved by combining low permeable and high
permeable coating materials in suitable proportion.
[0107] The devices with different drug release mechanisms described
above can be combined in a final dosage form comprising single or
multiple units. Examples of multiple units include, but are not
limited to, multilayer tablets and capsules containing tablets,
beads, or granules.
[0108] An immediate release portion can be added to the extended
release system by means of either applying an immediate release
layer on top of the extended release core using a coating or
compression process or in a multiple unit system such as a capsule
containing extended and immediate release beads.
[0109] Extended release tablets containing hydrophilic polymers are
prepared by techniques commonly known in the art such as direct
compression, wet granulation, or dry granulation. Their
formulations usually incorporate polymers, diluents, binders, and
lubricants as well as the active pharmaceutical ingredient. The
usual diluents include inert powdered substances such as starches,
powdered cellulose, especially crystalline and microcrystalline
cellulose, sugars such as fructose, mannitol and sucrose, grain
flours and similar edible powders. Typical diluents include, for
example, various types of starch, lactose, mannitol, kaolin,
calcium phosphate or sulfate, inorganic salts such as sodium
chloride and powdered sugar. Powdered cellulose derivatives are
also useful. Typical tablet binders include substances such as
starch, gelatin and sugars such as lactose, fructose, and glucose.
Natural and synthetic gums, including acacia, alginates,
methylcellulose, and polyvinylpyrrolidone can also be used.
Polyethylene glycol, hydrophilic polymers, ethylcellulose and waxes
can also serve as binders. A lubricant is necessary in a tablet
formulation to prevent the tablet and punches from sticking in the
die. The lubricant is chosen from such slippery solids as talc,
magnesium and calcium stearate, stearic acid and hydrogenated
vegetable oils.
[0110] Extended release tablets containing wax materials are
generally prepared using methods known in the art such as a direct
blend method, a congealing method, and an aqueous dispersion
method. In the congealing method, the drug is mixed with a wax
material and either spray-congealed or congealed and screened and
processed.
[0111] Delayed Release Dosage Forms
[0112] Delayed release formulations are created by coating a solid
dosage form with a polymer film, which is insoluble in the acidic
environment of the stomach, and soluble in the neutral environment
of the small intestine.
[0113] The delayed release dosage units can be prepared, for
example, by coating a drug or a drug-containing composition with a
selected coating material. The drug-containing composition may be,
e.g., a tablet for incorporation into a capsule, a tablet for use
as an inner core in a "coated core" dosage form, or a plurality of
drug-containing beads, particles or granules, for incorporation
into either a tablet or capsule. Preferred coating materials
include bioerodible, gradually hydrolyzable, gradually
water-soluble, and/or enzymatically degradable polymers, and may be
conventional "enteric" polymers. Enteric polymers, as will be
appreciated by those skilled in the art, become soluble in the
higher pH environment of the lower gastrointestinal tract or slowly
erode as the dosage form passes through the gastrointestinal tract,
while enzymatically degradable polymers are degraded by bacterial
enzymes present in the lower gastrointestinal tract, particularly
in the colon. Suitable coating materials for effecting delayed
release include, but are not limited to, cellulosic polymers such
as hydroxypropyl cellulose, hydroxyethyl cellulose, hydroxymethyl
cellulose, hydroxypropyl methyl cellulose, hydroxypropyl methyl
cellulose acetate succinate, hydroxypropylmethyl cellulose
phthalate, methylcellulose, ethyl cellulose, cellulose acetate,
cellulose acetate phthalate, cellulose acetate trimellitate and
carboxymethylcellulose sodium; acrylic acid polymers and
copolymers, preferably formed from acrylic acid, methacrylic acid,
methyl acrylate, ethyl acrylate, methyl methacrylate and/or ethyl
methacrylate, and other methacrylic resins that are commercially
available under the tradename Eudragit.RTM. (Rohm Pharma;
Westerstadt, Germany), including Eudragit.RTM. L30D-55 and L100-55
(soluble at pH 5.5 and above), Eudragit.RTM. L-100 (soluble at pH
6.0 and above), Eudragit.RTM. S (soluble at pH 7.0 and above, as a
result of a higher degree of esterification), and Eudragits.RTM.
NE, RL and RS (water-insoluble polymers having different degrees of
permeability and expandability); vinyl polymers and copolymers such
as polyvinyl pyrrolidone, vinyl acetate, vinylacetate phthalate,
vinylacetate crotonic acid copolymer, and ethylene-vinyl acetate
copolymer; enzymatically degradable polymers such as azo polymers,
pectin, chitosan, amylose and guar gum; zein and shellac.
Combinations of different coating materials may also be used.
Multi-layer coatings using different polymers may also be
applied.
[0114] The preferred coating weights for particular coating
materials may be readily determined by those skilled in the art by
evaluating individual release profiles for tablets, beads and
granules prepared with different quantities of various coating
materials. It is the combination of materials, method and form of
application that produce the desired release characteristics, which
one can determine only from the clinical studies.
[0115] The coating composition may include conventional additives,
such as plasticizers, pigments, colorants, stabilizing agents,
glidants, etc. A plasticizer is normally present to reduce the
fragility of the coating, and will generally represent about 10 wt.
% to 50 wt. % relative to the dry weight of the polymer. Examples
of typical plasticizers include polyethylene glycol, propylene
glycol, triacetin, dimethyl phthalate, diethyl phthalate, dibutyl
phthalate, dibutyl sebacate, triethyl citrate, tributyl citrate,
triethyl acetyl citrate, castor oil and acetylated monoglycerides.
A stabilizing agent is preferably used to stabilize particles in
the dispersion. Typical stabilizing agents are nonionic emulsifiers
such as sorbitan esters, polysorbates and polyvinylpyrrolidone.
Glidants are recommended to reduce sticking effects during film
formation and drying, and will generally represent approximately 25
wt. % to 100 wt. % of the polymer weight in the coating solution.
One effective glidant is talc. Other glidants such as magnesium
stearate and glycerol monostearates may also be used. Pigments such
as titanium dioxide may also be used. Small quantities of an
anti-foaming agent, such as a silicone (e.g., simethicone), may
also be added to the coating composition.
[0116] Pulsatile Release
[0117] The formulation can provide pulsatile delivery of the one or
more neuro-protective agents. By "pulsatile" is meant that a
plurality of drug doses are released at spaced apart intervals of
time. Generally, upon ingestion of the dosage form, release of the
initial dose is substantially immediate, i.e., the first drug
release "pulse" occurs within about one hour of ingestion. This
initial pulse is followed by a first time interval (lag time)
during which very little or no drug is released from the dosage
form, after which a second dose is then released. Similarly, a
second nearly drug release-free interval between the second and
third drug release pulses may be designed. The duration of the
nearly drug release-free time interval will vary depending upon the
dosage form design e.g., a twice daily dosing profile, a three
times daily dosing profile, etc. For dosage forms providing a twice
daily dosage profile, the nearly drug release-free interval has a
duration of approximately 3 hours to 14 hours between the first and
second dose. For dosage forms providing a three times daily
profile, the nearly drug release-free interval has a duration of
approximately 2 hours to 8 hours between each of the three
doses.
[0118] In one embodiment, the pulsatile release profile is achieved
with dosage forms that are closed and preferably sealed capsules
housing at least two drug-containing "dosage units" wherein each
dosage unit within the capsule provides a different drug release
profile. Control of the delayed release dosage unit(s) is
accomplished by a controlled release polymer coating on the dosage
unit, or by incorporation of the active agent in a controlled
release polymer matrix. Each dosage unit may comprise a compressed
or molded tablet, wherein each tablet within the capsule provides a
different drug release profile. For dosage forms mimicking a twice
a day dosing profile, a first tablet releases drug substantially
immediately following ingestion of the dosage form, while a second
tablet releases drug approximately 3 hours to less than 14 hours
following ingestion of the dosage form. For dosage forms mimicking
a three times daily dosing profile, a first tablet releases drug
substantially immediately following ingestion of the dosage form, a
second tablet releases drug approximately 3 hours to less than 10
hours following ingestion of the dosage form, and the third tablet
releases drug at least 5 hours to approximately 18 hours following
ingestion of the dosage form. It is possible that the dosage form
includes more than three tablets. While the dosage form will not
generally include more than a third tablet, dosage forms housing
more than three tablets can be utilized.
[0119] Alternatively, each dosage unit in the capsule may comprise
a plurality of drug-containing beads, granules or particles. As is
known in the art, drug-containing "beads" refer to beads made with
drug and one or more excipients or polymers. Drug-containing beads
can be produced by applying drug to an inert support, e.g., inert
sugar beads coated with drug or by creating a "core" comprising
both drug and one or more excipients. As is also known,
drug-containing "granules" and "particles" comprise drug particles
that may or may not include one or more additional excipients or
polymers. In contrast to drug-containing beads, granules and
particles do not contain an inert support. Granules generally
comprise drug particles and require further processing. Generally,
particles are smaller than granules, and are not further processed.
Although beads, granules and particles may be formulated to provide
immediate release, beads and granules are generally employed to
provide delayed release.
III. Methods of Use
[0120] The compositions described herein provide an effective
amount of one or more neuro-enhancing agents upon administration to
an individual. As used in this context, an "effective amount" of
one or more neuro-enhancing agents is an amount that is effective
to improve or ameliorate one or more symptoms associated with a
particular neurological disease, neurological defect or age-related
neurological decline or impairment. Such a therapeutic effect is
generally observed within about 4 to about 6 weeks of initiating
administration of a composition containing an effective amount of
one or more neuro-enhancing agents, although the therapeutic effect
may be observed in less than 4 weeks or greater than 6 weeks.
[0121] The individual is preferably a mammal, and more preferably
the mammal is a human who has lost some amount of neurological
function as a result of neurological disease, neurological injury
or age-related neurological decline or impairment. Generally neural
loss implies any neural loss at the cellular level, including loss
in neurites, neural organization or neural networks. Examples of
other subjects who can be treated include humans, dogs, cats, rats,
and mice. Lower mammal models using, for example, rats or mice can
be used to predict modes of general brain aging and associated
neuronal loss in higher mammals, such as humans.
[0122] The compositions can be administered daily, weekly, or less
frequently in an amount to provide a therapeutically effective
increase in the blood level of the one or more neuro-enhancing
agents described herein. For example, the total daily dosage will
be at least about 10 mg and more preferably at least about 50 mg to
about 500 mg or 1000 mg, when administered orally. Capsules or
tablets for oral delivery can contain up to a full daily oral dose,
e.g., 100 mg or more.
[0123] Where the administration is by other than an oral route, the
neuro-enhancing agents or compositions may be delivered over an
extended period, e.g., 3-10 days, in an amount effective to produce
at least an average daily dose of, e.g., 50 mg. Alternatively, the
compositions can be formulated for controlled release, wherein the
composition is administered once a day, once a week, or once a
month.
[0124] In a preferred embodiment, the dosage of allopregnanolone is
10 mg/kg administered once a week. Therefore, a balance between
optimal neurogenesis and optimal anti-amyloidogenic effects is
predicted to be achievable with a once per week dosing schedule.
The compositions are typically administered for an extended period
of time, for example, at least about 10 about, preferably at least
about 30 weeks, more preferably at least about 60 weeks, even more
preferably at least about 72 weeks, and most preferably as long as
the patient is receiving noticeable benefit from the treatment
method.
[0125] In a preferred embodiment, the composition containing one or
more neuro-enhancing agents is administered to an individual at a
dose and for a period effective to produce an improvement in at
least one criterion set forth as indicative of an improvement in
the neurological disease, neurological defect or neurological
age-related decline or impairment, such as an improvement in
cognitive abilities, memory, motor skills, learning or the like,
preferably an improvement is observed in at least two such
criteria.
[0126] Criteria for assessing improvement in a particular
neurological disease, neurological injury or age-related
neurological change include methods of evaluating cognitive skills,
motor skills, memory capacity or the like, as well as methods for
assessing physical changes in selected areas of the central nervous
system, such as magnetic resonance imaging (MRI) and computed
tomography scans (CT) or other imaging methods. Such methods of
evaluation are well known in the fields of medicine, neurology,
psychology and the like, and can be appropriately selected to
diagnosis the status of a particular neurological impairment. To
assess a change in a neurological disease, neurological injury or
age-related neurological change, the selected assessment or
evaluation test, or tests, are given prior to the start of
administration of the neuro-enhancing agents or compositions of the
present invention. Following this initial assessment, treatment
methods for the administration of the neuro-enhancing agents of the
present invention are initiated and continued for various time
intervals. At a selected time interval subsequent to the initial
assessment of the neurological defect impairment, the same
assessment or evaluation test (s) is again used to reassess changes
or improvements in selected neurological criteria.
[0127] The compositions described herein can be administered in a
variety of ways, such as orally, parenterally (e.g., subcutaneous,
intravenous, intramuscular, intraarterial, intraperitoneal,
intrathecal, intracardiac, or intrasternal), transcutaneously,
transmucosally, subcutaneously, by inhalation, infusion,
particularly via intracerebroventricular infusion, although oral
administration is generally preferred. Depending on the route of
administration, the compositions may be coated with or in a
material to protect it from the natural conditions which may
detrimentally affect its ability to perform its intended function.
A particularly convenient method of administering compositions of
the present invention is via oral administration.
[0128] A. Diseases and Disorders to be Treated
[0129] Neuro-enhancement resulting from the administration of the
compositions described herein includes the stimulation or induction
of neural mitosis leading to the generation of new neurons, i.e.,
exhibiting a neurogenic effect, prevention or retardation of neural
loss, including a decrease in the rate of neural loss, i.e.,
exhibiting a neuroprotective effect, or one or more of these modes
of action. The term "neuroprotective effect" is intended to include
prevention, retardation, and/or termination of deterioration,
impairment, or death of an individual's neurons, neurites and
neural networks. Administration of the compositions described
herein leads to an improvement, or enhancement, of neurological
function in an individual with a neurological disease, neurological
injury, or age-related neuronal decline or impairment.
[0130] Neural deterioration can be the result of any condition
which compromises neural function which is likely to lead to neural
loss, Neural function can be compromised by, for example, altered
biochemistry, physiology, or anatomy of a neuron, including its
neurite. Deterioration of a neuron may include membrane, dendritic,
or synaptic changes which are detrimental to normal neuronal
functioning. The cause of the neuron deterioration, impairment,
and/or death may be unknown. Alternatively, it may be the result of
age-, injury- and/or disease-related neurological changes which
occur in the nervous system of an individual.
[0131] When neural loss is described herein as "age-related", it is
intended to include neural loss resulting from known and unknown
bodily changes of an individual that are associated with aging.
When neural loss is described herein as "disease-related", it is
intended to include neural loss resulting from known and unknown
bodily changes of an individual which are associated with disease.
When neural loss is described herein as "injury-related", it is
intended to include neural loss resulting from known and unknown
bodily changes of an individual which are associated with injury or
trauma. Examples of trauma include brain injuries due to
explosions, for example from explosive devices, or other traumas,
such as gun shots and/or stabbings. It should be understood,
however, that these terms are not mutually exclusive and that, in
fact, many conditions that result in the loss of neural cells
and/or neural connections can be related to age, disease and/or
injury.
[0132] Some of the more common age-related neuropathies associated
with neural loss and changes in neural morphology include, for
example, Alzheimer's disease, Pick's disease, Parkinson's disease,
vascular disease, Huntington's disease, and Age-Associated Memory
Impairment. In Alzheimer's patients, neural loss is most notable in
the hippocampus, frontal, parietal, and anterior temporal cortices,
amygdala, and the olfactory system. The most prominently affected
zones of the hippocampus include the CA1 region, the subiculum, and
the entorhinal cortex. Memory loss is considered the earliest and
most representative cognitive change because the hippocampus is
well known to play a crucial role in memory.
[0133] Pick's disease is characterized by severe neural
degeneration in the neocortex of the frontal and anterior temporal
lobes which is sometimes accompanied by death of neurons in the
striatum. Parkinson's disease can be identified by the loss of
neural cells in the substantia nigra and the locusceruleus.
Huntington's disease is characterized by degeneration of the
intrastriatal and corticalcholmergic neural cells and GABA-ergLc
neural cells. Rarkmson's and Huntington's diseases are usually
associated with movement disorders, but often show cognitive
impairment (memory loss) as well.
[0134] Age-Associated Memory Impairment (AAMI) is another
age-associated disorder that is characterized by memory loss in
healthy, elderly individuals in the later decades of life.
Presently, the neural basis for AAMI has not been precisely
defined. However, neural death with aging has been reported to
occur in many species in brain regions implicated in memory,
including cortex, hippocampus, amygdala, basal ganglia, cholinergic
basal forebrain, locus ceruleus, raphe nuclei, and cerebellum.
[0135] Animal Models for Evaluating Neurogenesis and/or Reducing
Expression of Beta-Amyloid
[0136] Aging rodent brains do not develop senile plaques and
neurofibrillary tangles. Most recent studies suggest, however, that
loss or shrinkage of neurons, dendrites, and/or synapses is more
closely correlated with either dementia or aging than are plaques
and tangles. Aging rats exhibit neural cell loss in the pyramidal
cells of the hippocampus, especially in field CA1, as well as cell
loss or dendritic/synaptic changes in some other brain regions.
Moreover, aging rodents show extensive hippocampalastrocyte
hypertrophy just as do aging humans. In addition, loss of neural
cells in field CA1 of the hippocampus is a consistent correlate of
aging across species, and is also prominent in human
neurodegenerative diseases, such as Alzheimer's disease. For these
reasons, the study of neural loss in aging rats, for example, is
predictive of general mechanisms of brain aging and associated
neural loss in humans due to diseases such as Alzheimer's
diseases.
[0137] Animal models, such as the models described in U.S. Pat. No.
5,939,407 and Haughey et al., J. Neurochem. 83: 1509-1524 (2002),
represent improvement in models for age-associated disease and
decline because they relate to an intact animal, which is generally
preferred over tissue culture models. Further, the animal model
described in U.S. Pat. No. 5,939,407 employs a strain of rat that
was developed by the National Institute on Aging as a premier model
of mammalian aging. The particular rat strain (Brown Norway/Fischer
344 F1 cross rats) was selected due to its normal pattern of aging,
with few indications of abnormal pathology. This strain also loses
neural cells in field CA1 of the hippocampus with aging and
exhibits memory loss. This system represents one of the most
natural animal models of neural degeneration and/or deterioration
because it reflects a gradual loss of neural cells. Furthermore,
the neural loss is not provoked by experimental intervention or
abnormal pathology. Its brain aging pattern is also highly
analogous to human and other mammalian species brain aging
patterns.
[0138] In one embodiment, an animal model can be used to evaluate
the effect of a neuro-enhancing agent in beta-amyloid expression
and/or neurogenesis in an animal transgenic for Alzheimer's
disease. Suitable models include the animal model described by
Borchelt et al. (1996) Neuron 17: 1005-1013 and Haughey et al.
(2002) J. Neurochemistry 83: 1509-1524. Male mice (12-14 months
old) overexpressing a mutant form of amyloid precursor protein
(APP) are maintained on a 12 hour light/12 hour dark cycle with
free access to food and water. This line of mice exhibits increased
levels of soluble amyloid beta protein and develops amyloid
deposits in an age-dependent manner with diffuse deposits first
appearing at about 12 months of age and plaque-like deposits
developing later, typically by 18-22 months of age.
[0139] Neural loss through disease, age-related decline or physical
insult leads to neurological disease and impairment. The
compositions described herein can counteract the deleterious
effects of neural loss by promoting development of new neurons, new
neurites and/or neural connections, resulting in the
neuroprotection of existing neural cells, neurites and/or neural
connections, or one or more these processes. Thus, the
neuro-enhancing properties of the compositions described herein
provide an effective strategy to generally reverse the neural loss
associated with degenerative diseases, aging and physical injury or
trauma.
[0140] The administration of
3.alpha.-hydroxy-5.alpha.-pregnan-20-one, or a substantially
equivalent variant molecule, to an individual who is undergoing or
has undergone neural loss, as a result of a disease, defect,
injury, or age-related decline, can generally provide an effective
therapeutic strategy for the treatment of neurological conditions
caused by neural loss. The defects and diseases that can benefit
from administering the agents, compositions and methods of the
present invention include, but are not limited to, spinal cord
injury, stroke, head injury, epilepsy, Parkinson's disease and
Alzheimer's disease. Moreover, given that
3.alpha.-hydroxy-5.alpha.-pregnan-20-one, and substantially
equivalent variant molecules, possess neuro-enhancing activities,
these agents and compositions may also be administered to improve
age-related memory and learning impairments.
[0141] The examples demonstrate that administration of
.alpha.-hydroxy-5.alpha.-pregnan-20-one or tetrahydroprogesterone
(THP or AP.alpha.) reverses the learning deficits of mice
transgenic for Alzheimer's disease (3.times.TgAD mice). The data
indicate that at 3 months, 3.times.TgAD mice exhibit a learning
deficit relative to the performance of normal non-Tg mice. In the
normal high functioning non-Tg mice, with a concomitant high level
of neurogenesis, THP did not augment the learning performance. In
contrast, THP significantly increased the learning performance of
3.times.TgAD mice to a level comparable to non-Tg mice such that
the performance of THP treated 3.times.TgAD mice was not
statistically different from the normal non-Tg mouse. One week
following the learning trial, mice were tested for memory of the
learned association. Non-Tg mice exhibited slightly less than 50%
of the conditioned response compared to a 28% response rate of
3.times.TgAD mice. THP did not significantly augment the memory
performance of non-Tg mice. However, THP treated 3.times.TgAD mice
exhibited a significant increase in memory to a level comparable to
the normal non-Tg mice.
[0142] As also shown in the examples,
3.alpha.-hydroxy-5.alpha.-pregnan-20-one or tetrahydroprogesterone
(THP), a naturally occurring metabolite of progesterone, was found
to induce or stimulate the formation of new hippocampal neurons.
Results of these analyses demonstrate that the number of mitotic
neural cells was approximately doubled in the presence of
tetrahydroprogesterone.
[0143] The examples also demonstrate that the administration of
3.alpha.-hydroxy-5.alpha.-pregnan-20-one (THP) reduces age-related
.beta.-amyloid expression. The development of intraneuronal beta
amyloid is typically seen in 6 and 9 month old animals and the
development of plaques in 12 month old animals. Plaques are rarely
seen in 9 month old animals. THP (10 mg/kg/week, administered once
a week for 6 months) was administered to 9- and 12-month male mice
transgenic for Alzheimer's disease (3.times.Tg-AD). The 9 month old
animals were started on THP at 3 months of age prior to the
development of beta amyloid accumulation in the whereas the 12
month old animals were started on THP at 6 months of age when beta
amyloid had already begun to accumulate within neurons. The results
indicate that administration of THP significantly decreased the
amount of beta amyloid in the cerebral cortex of male mice
transgenic for Alzheimer's disease.
[0144] Western Blot analysis showed a form of beta amyloid termed
Abeta*56, which is the oligomer (multiple amyloid beta peptides
joined together) that, in animal studies, leads to memory loss in
both transgenic Alzheimer mouse models and in rats injected with
Abeta*56. In the 12 month old animals, the level of Abeta*56 was
much lower, likely due to the development of beta amyloid plaques
in these animals, which reduces the amount of Abeta*56.
Immunocytochemical detection of beta amyloid showed that
administration of THP substantially decreases Abeta*56 in
hippocampal neurons. THP also decreases the immunoreactivity of
phosphorylated tau, which is the basis for neurofibrillary
tangles.
[0145] Several treatment regimens of THP at 10 mg/kg were evaluated
including 24 hours following a single dose. Further,
allopregnanolone in three long-term treatment paradigms was also
tested: 2) single dose followed by cognitive testing over three
week duration and biochemical analyses at endpoint; 3) once per
week for six months; and 4) every other day dosing for three
months. An optimal treatment regimen was found to be THP 10 mg/kg
once per week beginning at middle age (6 month old mice) to
significantly decrease amyloid deposits.
[0146] Allopregnanolone has been shown to: (1) be a proliferative
factor for human, rat, and mouse neural progenitor cells; (2)
promote neurogenesis in both proliferative zones of the brain; and
(3) promote neurogenesis in a mouse model of Alzheimer's disease
which restored learning and memory function to normal.
Allopregnanolone violates none of the Lipinski rules for
drugability, referred to as the Rule of 5 (RO5). The RO5 states
that the MW.ltoreq.500, ClogP.ltoreq.5, the number of H-bond
donors.ltoreq.5, and the number of H-bond acceptors (sum of N and O
atoms) .ltoreq.10. Additional criteria include that the polar
surface area.ltoreq.140 A.sup.2 or the sum of H-bond donors and
acceptors.ltoreq.12 and the number of rotatable
bonds.ltoreq.10.
[0147] The compositions described herein may also be effective for
the treatment of neural damage caused by therapies aimed at
combating certain cancers that affect the brain. For instance,
cranial radiation therapy is crucial to the successful treatment of
many primary brain tumors, cancers metastatic to the brain, CNS
involvement of leukemia/lymphoma, and head and neck cancers. Such
irradiation that involves the cerebrum causes a debilitating
cognitive decline in both children and adults. Experiments have
shown that hippocampus-dependent learning and memory are strongly
influenced by the--activity of neural progenitor- and/or stem cells
and their--proliferative progeny. Since the hippocampal granule
cell layer undergoes continuous renewal and structuring by the
addition of new neurons, radiation at much lower does than that
needed to injure the more resistant post-mitotic neurons and glia
of the brain, has been found to affect these highly proliferative
progenitors and/or stem cells severely. The progenitor and/or stem
cell, therefore, is considered to be so sensitive to radiation that
a single low dose to the cranium of a mature rat is sufficient to
ablate hippocampal neurogenesis. Recent experiments have further
found that progressive learning and memory deficits following
irradiation may be caused by the accumulating hippocampal
dysfunction that results from a long-term absence of normal
progenitor and/or stem cell activity. Thus, given the neurogenic
effect compositions described herein on hippocampal cell cultures,
therapeutic methods utilizing these compositions may benefit
individuals who are undergoing or have undergone radiation therapy
for brain-related cancers.
IV. Kits
[0148] The compositions described herein can be packaged in kit.
The kit can include a single dose or a plurality of doses of a
composition containing one or more neuro-enhancing agents, and
instructions for administering the compositions. Specifically, the
instructions direct that an effective amount of the composition be
administered to an individual with a particular neurological
disease, defect or impairment as indicated. The composition can be
formulated as described above with reference to a particular
treatment method and can be packaged in any convenient manner.
[0149] The instructions can be affixed to the packaging material or
can be included as a package insert. While the instructions
typically comprise written or printed materials they are not
limited to such. Any medium capable of storing such instructions
and communicating them to an end user is contemplated by this
invention. Such media include, but are not limited to, electronic
storage media (e.g., magnetic discs, tapes, cartridges, chips),
optical media (e.g., CD ROM), and the like. As used herein, the
term "instructions" can include the address of an internet site
that provides the instructions. Embodiments of the present
invention also include the use of the above-described
pharmaceutical products for the treatment of a human patient with a
neurological disease, neurological defect or age-related
neurological decline or impairment.
[0150] The following examples are put forth so as to provide those
of ordinary skill in the art with a complete disclosure of how to
make, to use and to evaluate the therapeutic agents, compositions
and methods of the present invention, and are not intended to limit
the scope of what is regarded as the invention. Efforts have been
made to ensure accuracy with respect to numbers presented (e, g,
amounts, concentrations, etc.), but some experimental errors and
deviations should be allowed for.
EXAMPLES
Materials and Methods
[0151] The animals studies described below for evaluating
neurogenesis and/or reduction of beta-amyloid expression were done
using a triple transgenic Alzheimer's disease (AD) mouse model
(3.times.TgAD). The 3.times.TgAD mouse carries mutations (APPSwe,
PS1M146V and tauP301L) of three human familial AD genes and
manifests age-dependent neuropathology of both .beta.-amyloid
plaque formation and neurofibrillary tangles. In addition to
expressing neuropathological markers of AD, the 3.times.TgAD mouse
exhibits learning and memory deficits as early as 4 months, but not
2 months.
[0152] Breeding pairs of the triple transgenic Alzheimer's disease
mouse (3.times.TgAD, homozygous mutant of human APPswe, tauP301L,
and PS1M146V) and its background (129/Sv.times.C57BL/6) were
obtained from Dr. Frank Laferla (University of California at
Irvine) and the colonies were established at the University of
Southern California. The characterization of amyloid and tau
pathologies and synaptic dysfunction in this line of mice has been
described previously and confirmed in our laboratory. Mice were
genotyped regularly to confirm the purity of the colony.
[0153] Using this AD model, the following were assessed: 1) THP
concentration, neurogenic and cognitive status at 3 months of age;
2) impact of THP on both neurogenic and cognitive status using
unbiased stereology, phenotype immunocytochemistry, real-time
RT-PCR, Western blot, and eyeblink trace conditioning training and
memory.
[0154] Experiments were performed using 6, 9, 12, and 15-month-old
male 3.times.TgAD and non-Tg mice. The number of mice per condition
is indicated within the results section. Mice were maintained under
a 12 hr light/dark cycle with continuous access to food and
water.
[0155] THP stock solution was prepared in pure ethanol and diluted
in PBS before injection (with a final ethanol concentration of
0.002% of the body weight). THP transdermal gels were prepared
using techniques known in the art.
[0156] Mice of each genotype and age received a subcutaneous (s.c.)
injection of THP at a concentration of 10 mg/kg body weight (BW),
an optimal dose of THP-based on our previous studies. One hour
after THP injection, mice were intraperitoneally (i.p.) injected
with 100 mg/kg BW bromodeoxyuridine (BrdU). Two experimental
paradigms were used: 1) THP treatment for behavior and cell
survival assessment, in which animals received a single shot of
THP/vehicle, followed by a 7-day neurogenesis and migration phase,
followed by a 5-day trace-conditioning test followed 9 days later
by a 1-day memory test, and scarified 21 days after a single THP
injection at which time FACS analysis of BrdU positive cells was
performed to assess cell survival; 2).
[0157] Acute THP treatment for cell proliferation assessment, in
which animals received a single injection of THP/vehicle and were
sacrificed 24 hr later followed by quantitative unbiased stereology
of BrdU positive cells. Timing of THP and BrdU injections, training
diagrams, and perfusion were based on previous studies showing that
learning enhances the survival of newly born cells generated 1 week
before training and from our previous analysis indicating that
THP-induced neurogenesis significantly increased learning of trace
eyeblink conditioning in 3 month old 3.times.TgAD male mice
following an injection of THP one week prior to behavioral
testing.
[0158] All experiments strictly conformed to the Animal Welfare
Act, Guide to Use and Care of Laboratory Animals, and the U.S.
Government Principles of the Utilization and Care of Vertebrate
Animals Used in Testing, Research, and Training guidelines on the
ethical use of animals. In addition, the minimal number of required
animals was used for these experiments and pain was minimized.
[0159] Trace Eyeblink Conditioning
[0160] Under deep anesthesia by intraperitoneal (i.p.) injection
with ketamine (100 mg/kg i.p.) and xylazine (25 mg/kg i.p.), a 4
pin head stage (DIGI-KEY) was cemented to the skull of 3 month old
male mouse with dental acrylic. The connector has four
Teflon-coated stainless steel wires and one bare stainless steel
wire (0.003'' bare and 0.0055'' coated, A-M Systems, Inc.). The
bare wire was attached via a gold pin (Time Electronics) to the
head stage. Coated wires were implanted s.c. in the orbicularis
oculi dorsal to the left upper eyelid to record the EMG and s.c.
periorbitally to deliver the shock US (3). All animals were then
placed on a warm isothermal pad after surgery to recovery for 30
min. After surgery, mice were individually housed, provided with ad
libitum access to food and water, and maintained on a 12 hr
light/dark cycle.
[0161] After one week of acclimation to the colony room, mice with
no obvious adverse responses to surgery were randomly assigned to
an experimental condition. Mice were injected subcutaneously (s.c.)
with 10 mg/kg THP or vehicle followed one hour later with an IP
injection of BrdU (100 mg/kg). Following injection of test
compound, mice were returned to their home cage for 7 days prior to
onset of behavioral testing.
[0162] During the first day of training, mice were placed within
Plexiglas cylinders in a sound-attenuated chamber and were
habituated to the test environment for one session consisting of 30
stimulus-free trials at 30-60 sec inter-trial intervals while
spontaneous eye-blink activity was recorded using electromyographic
(EMG) activity recorded from the obicularis oculi dorsal to the
orbit during each trial. EMG activity was rectified, and integrated
using custom designed computational Labview routines.
[0163] Following habituation to the test environment mice underwent
a learning phase and were trained for five days. Mice were trained
by pairing delivery of a tone (CS, 250 msec, 2 kHz, 85 dB) as the
conditioned stimulus followed by a 250 msec period of no stimuli,
followed by the periobital shock as the unconditioned stimulus (US,
100 msec). Mice received two blocks of 30 trials per day (30-60-sec
inter-trial intervals, 3-4-h inter-block intervals). This trace
eye-blink conditioning paradigm is subthreshold for inducing
neurogenesis. The unpaired group received random tone at the same
magnitude as the paired conditioning and shock with 15-30 sec as
inter-trial intervals and a total of 60 trials for one session per
day. Shock intensity was adjusted daily for each mouse to elicit a
head-movement response. Following the learning phase, mice were
returned to their home cage for eight days followed on the ninth
day by a single session to assess memory of the conditioned
response. The percentage of CR was computed as the ratio of the
number of CRs to the total number of valid trials. Animals were
perfused at the end of the memory trial day.
[0164] Animal Dissection and Tissue Collection
[0165] Mice were sacrificed 24 h after THP administration for cell
proliferation assessment or at the end of memory test for cell
survival assessment. On the day of sacrifice mice were deeply
anesthetized with a combination of ketamine (100 mg/kg) and
xylazine (10 mg/kg), and perfused with PBS. Brains were dissected
into two hemispheres, and one hemisphere was fixed immediately in
cold 4% paraformaldehyde and was used for either stereological
analysis (cell proliferation) or FACS analysis (cell survival).
[0166] Nuclei Extraction and Flaw Cytometry Counting
[0167] Hippocampus was dissected from the fixed hemispheres from
cell survival assessment experiment using consistent anatomical
landmarks as criteria for dissection as described in the
literature. The rostral 1/3 of the hippocampus lobe was removed to
avoid the subventricular zone and rostral migratory stream
proliferative pools. The extracted hippocampi were homogenized
using Next advance 24 sample homogenizer (Next Advance Inc., NY)
for 3 minutes on speed 7. This procedure lyses the plasma lemma
while preserves the nuclear envelope intact. The nuclei sample was
collected into a regular 1.5 mL microcentrifuge tube by washing the
beads and tube 4 times using 200 .mu.L of PBS, and then centrifuged
for 10 minutes at 10,000 rpm. Once all of the nuclei were collected
in a pellet, the supernatant was discarded. The pellet was then
re-suspended in 600 .mu.L of PBS plus 0.5% Triton x-100. The number
of nuclear density was estimated by counting the propidium iodide
(PI), a fluorescent molecule stoichiometrically binds to DNA by
intercalating between the bases with no sequence preference,
positive particles. Aliquots of 25 .mu.l were re-suspended in 200
.mu.l of a 0.2 M solution of boric acid, pH 9.0, and heated for 1 h
at 75.degree. C. for epitope retrieval. After washed in PBS, the
nuclei were incubated for 24 hours at 4.degree. C. with primary
mouse monoclonal anti-BrdU antibody (1:100, Abeam, Ab12219) and
subsequently with FITC-conjugated goat anti-mouse IgG secondary
antibody (1:100 in PBS; Vector Labs, FI-2001). The remainder of
cell suspension is diluted to 500 .mu.L and sent for flow cytometry
assay using Beckman FC 500 System with CXP Software. Propidium
iodide (PI) cells were first gated on a histogram; the expressing
cells were visualized on a forward/side scatter plot. PI cells were
`back-gated` on the forward/side scatter plot to eliminate debris
prior to analysis; this also eliminated auto fluorescence of the
sample. Gates were always set using dissociates with cell aliquots
which lack of the first antibody, but which were incubated with
second antibody and processed alongside the experimental procedure.
PI-labeled cells in a fixed volume were gated, and the number of
cells showing BrdU signal was analyzed. Data were expressed as
total positive cells per hippocampus.
[0168] Western Blot
[0169] Protein was extracted from mouse hippocampus and separated
by SDS gel as described in the literature. After transfer, PVDF
membrane was plotted with monoclonal antibody for proliferating
cell nuclear antigen (PCNA, 1:500, Zymed Laboratories Inc, San
Francisco, Calif.) and then incubated with a horseradish peroxidase
conjugated secondary antibody which is complementary to the primary
antibody. Results were visualized by the ECL Plus Western Blotting
Detection System (GE Healthcare, Amersham, Buckinghamshire, UK)
followed by TMB development. Optical density was and analyzed by
BioRad Quantity One software. The percent protein expression vs.
control was normalized by loading control .beta.-actin.
[0170] Unbiased Stereology
[0171] The fixed hemispheres from cell proliferation assessment
experiment were sectioned into a series of 40 .mu.m coronal
sections and every sixth section in the series was processed for
BrdU histolabeling by NeuroScience Associates (Knoxville, Tenn.).
Prior to labeling, all slides were coded and the codes were not
broken until analyses were completed. The number of BrdU labeled
cells was determined by unbiased stereology (optical dissector).
Systematic samplings of unbiased counting frames of 50 .mu.m on a
side with a 200 .mu.m matrix spacing were produced using a
semiautomatic stereology system (Zeiss Axiovert 200M fluorescent
microscope as part of the 3iMarianas digital microscopy and a
60.times. SPlan apochromat oil objective (1.4 numerical aperture).
Positive cells that intersected the uppermost focal (exclusion)
plane and those that intersected the exclusion boundaries of the
unbiased sampling frame were excluded from analysis. Cells that met
analysis criteria through a 20 .mu.m axial distance were counted
according to the optical dissector principle. The granule cell
layer reference volume was determined by summing the traced SGZ,
granule cell areas for each section multiplied by the distance
between sections sampled. The mean granule cell number per
dissector volume was multiplied by the reference volume to estimate
the total granule cell number. The stereologically determined
number of BrdU-positive cells was related to the granule cell layer
sectional volume and multiplied by the reference volume to estimate
the total number of BrdU-positive cells.
[0172] Statistical Analysis
[0173] Data were analyzed using a one-way ANONA followed by
Neuman-Keuls post hoc analysis. Data displayed in graphs were
reported as mean.+-.SEM or fold change.+-.SEM. P-values of <0.05
were considered to be significant regardless of the statistical
test used.
Example 1
The Effect of 3.alpha.-hydroxy-5.alpha.-pregnan-20-one (THP) on
Hippocampal Neural Cells
[0174] Hippocampal neural cells were obtained from an embryonic day
18 rat hippocampus. The samples contained approximately 12,000
neurons/sample. The sample was 95% neuronal. No selection for
neuronal subtypes was conducted. Hippocampal neurons were treated
with 3a-hydroxy-5a-pregnan-20-one (THP).
3.alpha.-hydroxy-5.alpha.-pregnan-20-one (THP) was added to two
samples containing hippocampal neural cells at a concentration of
either 10 nanomolar (nM) and 100 nanomolar (nM). The cells were
incubated for 24 hrs at 37.degree. C. Neurons were grown in a
defined medium, Neurobasal+B27 supplement in the absence (control)
or presence of THP or other test molecule (experimental). The
samples with added THP were compared with a control sample
containing only hippocampal neural cells. Changes in the mitotic
appearance of the neural cells were observed. A mitotic appearance
of a particular neural cell is defined as a doublet form in the
cell body of the neural cell. The doublet form is indicative of a
neural cell undergoing mitosis. A graphic comparison among the
three samples studied is shown in FIG. 2. These data reveal that
there is an approximate 2 fold increase in the mitotic phenotype of
the neural cells studied at either 10 nm THP or 100 nm THP, as
compared with the control sample. Data are expressed as percent of
the total number of neurons exhibiting mitotic phenotype, mean+SEM,
**p<0.01, ***p<0.001.
[0175] The experiment described above was repeated only THP was
added to three samples containing hippocampal neural cells at a
concentration of 100 nanomolar (nM), 250 nM and 500 nM. The samples
with added THP were compared with a control sample containing only
hippocampal neural cells. A graphic comparison among the four
samples studied is shown in FIG. 3. These data reveal that there is
an approximate 2-3 fold increase in the mitotic phenotype of the
neural cells studied, as compared with the control sample. The
greatest effect in induction of the mitotic phenotype was observed
at 500 nm THP. Data are expressed as percent of mean.+-.SEM,
**p<0.01, ***p<0.001.
Example 2
Effect of 3.alpha.-hydroxy-5.alpha.-pregnan-20-one (THP) on the
Expression of Cell Proliferating Markers
[0176] THP also was shown to increase the expression of cell
proliferating markers. Expression of cell cycle proteins have been
successfully used to evaluate cellular proliferation. One such
protein is the nuclear proliferation protein, Ki-67, which is
expressed during the G1, S, G2, and M phases of the cell cycle, but
is not expressed during the Go (resting) phase. Because Ki-67
antigen has a short half-life, it can be used as a marker of
actively proliferating cells. Another cell cycle protein is cell
division control protein 2 (cdc2) which is a cyclin dependent
kinase (also called CDK1) which plays a crucial role in the G1/S
and G2/M phase. If THP induces neuronal proliferation, cell
proliferation markers should be elevated.
[0177] Hippocampal neurons were treated with THP at a concentration
of 250 nM for 72 hours and immunostained with antibodies for the
nuclear proliferation marker, ki-67 antigen, which appears yellow.
The results indicate that THP induces the expression of the nuclear
proliferation marker Ki-67. This is supported by the fact that the
cytoplasm of the donor and the daughter cells did not completely
separate. The cell cycle protein cdc2 is also observed in a dose
dependent fashion (see FIG. 4). As shown in the figure, THP
increases expression of cell division control protein 2 (cdc2) in
hippocampal neurons.
[0178] For this experiment, neurons were collected following 24 hrs
of THP exposure. Forty .mu.g protein of the total cell lysate was
loaded and separated by 12% SDS-gel using antibody (Ahcom)
specifically against cdc2- and analyzed using Un-Scan-It image
software (Ilk Scientific Corp.). This figure shows a representative
Western blot from one of three different experiments which have the
similar results.
Example 3
Effect of 3.alpha.-hydroxy-5.alpha.-pregnan-20-one (THP) on the
Production of Neurons
[0179] Having determined that THP increases the expression of the
cell proliferating markers, a determination was sought as to
whether the increase in cell proliferation markers translated into
an increase in neuronal number. It was found that THP induced
neuronal proliferation by increasing the total cell number and the
dividing speed. As shown in FIG. 5, THP increased the neuron number
by approximately 30%. These results are highly consistent across
different experiments and are also comparable to the results
obtained using the mouse hippocampal neuron cell line (HT-22) (FIG.
6 and Table 1 (Example 12)). As shown in FIG. 6 and Table 1, THP
increases neuron number as assessed in MuLV-GFP infected mouse
neurons. The effect of THP on HT-22 cells proliferation was
detected on MuLV infected cells. The left panel shows the FACS
profile of the vehicle. The right panel shows the FACS profile of
THP treated MuLV-GFP infected cells.
[0180] The table summarizes the FACS results. V=vehicle; THP (250
nM). THP treatment increased the dividing cell number 22% as
determined by fluorescent associated cell sorting (FACS).
Therefore, the data demonstrate that THP can increase the
proliferation of neuronal cells either in primary cultured cells or
continuous cell lines, from rat and mouse.
Example 4
Effect of 3.alpha.-hydroxy-5.alpha.-pregnan-20-one (THP) on
3H-Thymidine Uptake
[0181] Biochemical analyses of 3H-thymidine uptake, as a measure of
DNA synthesis, were used as the experimental vehicle to confirm the
morphological observations described in examples 1 and 2. As shown
in FIG. 7, THP induced a 80% increase in 3H-thymidine uptake
relative to control (F=12.31, df3, 19, p<0.0001) from about 0.1
inn THP to about 250 nm THP. Thus, the range for the neurogenic
effect of THP on neural cells is quite is sensitive and quite
broad. Furthermore, DNA synthesis is specifically induced in the
presence of THP (F=9.15, df6, 27, p<0.0001), as compared with
other structurally and chemically similar steroids, as shown in
FIG. 8.
[0182] For these experiments, cultured hippocampal neural cells,
derived from embryonic day 18 rat fetuses, were allowed to adhere
to polylysine coated plastic cover-slips for 40 min in serum
containing medium. Following adhesion, neurons were exposed to 1
Ci/ml 3H-thymidine in the presence or absence of 100-500 nM THP and
allowed to incubate at 37.degree. C. for 24 hours in the absence or
presence of the indicated steroids. Data are expressed as
mean.+-.SEM, *p<0.05, **p<0.01, ***p<0.001. The results
demonstrated that THP induction of 3H-thymidine incorporation is
highly specific. Progesterone induced a modest increase in
3H-thymidine incorporation; however, the stereoisomers of THP,
i.e., 5 cc, 3ss-THP and 5ss, 3ss-THP, as well as 5.alpha.,
3P-pregnen-3-one, showed no effect.
[0183] Additionally, 5.alpha.,3.alpha.-pregnan-diol;
5.alpha.,3.alpha.-pregnan-triol and pregnenolone sulfate (PS),
which are known to increase morphological differentiation, induced
a significant decrease in 3H-thymidine incorporation which is
consistent with their differentiation effect. The steroid
specificity analysis provides evidence for the specificity of
THP-induced mitogenesis. Moreover, consistent with this evidence is
the observation that differentiation factors have an effect
opposite to that of THP in that these agents cause a decrease in
3H-thymidine incorporation.
[0184] The time course of THP-induced 3H-thymidine incorporation in
hippocampal neuronal cells is shown in FIG. 9 where cultured
hippocampal nerve cells, derived from embryonic day 18 rat fetuses,
were allowed to adhere to polylysine coated plastic cover-slips for
40 min in serum containing medium. Following adhesion, serum
containing medium in the presence or absence of 10-250 nM THP plus
1 pCi/ml 3H-thymidine and allowed to incubate at 37 for 1, 8 or 24
hours. Data are expressed as mean.+-.SEM, *p<0.05, **p<0.01,
***p<0.001
Example 5
Effect of 3.alpha.-hydroxy-5.alpha.-pregnan-20-one (THP) on the
Production of Neural Stem Cells
[0185] Experiments to determine whether THP promotes neural stein
cells growth were also performed. In FIG. 10, neural spheres were
generated from the periventricular are and hippocampus of embryonic
day 18 rat embryos. 5 rat embryos were treated with THP alone or
with EGF and FGF-2 as mitogens. The approximately third passage of
neural spheres were collected and randomly disturbed evenly to each
dish. Dishes were treated with reagents as labeled in the absence
of progesterone for 36 hours. Cells were then collected and
trypsinized in to single cells. The cell numbers were counted blind
using a hemacytometer and plotted in Excel.
[0186] Another experiment was performed to assess the neurogenic
effects of THP administration on human neural stem cells. The
results are shown in FIG. 11. In this experiment, neural stem cells
derived from human fetal cortex were treated with varying
concentrations of THP [1-1000 nM] or with bFGF [20 ng/ml]+heparin
[5 llg/ml] as a THP positive control. The proliferation marker,
BrdU [10 uM] was added simultaneously with test molecules and the
cells were incubated at 37.degree. C. for 24 hours. Quantitative
Elisa chemiluminescence of BrdU signal was conducted at 24 hrs
following addition of substrate and chemiluminescence determined by
LMax microplate luminometer (Molecular Devices) (Roche Diagnostics
Corp., Cell Proliferation ELISA, BrdU (chemiluminescence). THP at
250 and 500 nM, significantly increased BrDU chemiluminescence
relative to vehicle control condition and was consistently greater
than the positive control bFGF+heparin. Data are presented as
mean.+-.SEM and are derived from three separate experiments.
Example 6
Neural Progenitor Proliferation in Dentate Granular Zone (DGZ) is
Deficient in 3 Month Old Male 3.times.TgAD Mice Prior to Onset of
Visible AD Pathology
[0187] BrdU immunohistochemistry (IHC) was preformed on adjacent
sections of IHC-labeled for A.beta.. Sections were immunostained
with BrdU antibody (Novus Biologicals) and imaged using 3I Marianas
Imaging System with Zeiss Axiovert 200M interfaced with a Sony
ICX-285 CCD CoolSnap HQ camera and Xenon 2-Gal Fast Excitation
Source equipped with SlideBook unbiased quantitative stereology
software. Results of un-biased quantitative stereological analyses
indicate that at 3 months prior to the appearance of markers of
Alzheimer's (AD) pathology, the BrdU-positive cell number was
significantly lower in the 3.times.TgAD mouse dentate relative to
non-Tg mouse dentate. This finding indicates basal neurogenesis in
3.times.TgAD mice DGZ is reduced prior to development of overt AD
pathology. These results also suggest that the early neurogenic
deficits, which were evident prior to visible A.beta. and ptau, may
contribute to etiology of AD.
Example 7
3.alpha.-hydroxy-5.alpha.-pregnan-20-one (THP) Reverses the
Neurogenic Deficits in 3.times.TgAD Mice Hippocampal Dentate
Gyrus
[0188] THP increased BrdU incorporation in both non-Tg and
3.times.TgAD mice. A more pronounced and significant increase was
observed in 3.times.TgAD mice with the greatest increase, 55+18%
greater vs vehicle control group, which occurred at 10 mg/kg BW.
Analysis of total number of cells generated indicated that THP
restored proliferation to that of normal nontransgenic mice thereby
reversing the neurogenic deficit.
[0189] Cortices from brain hemisection of mice treated with THP or
vehicle were collected at time of sacrifice for measurement of THP
by GC/MS. Plasma was also collected at time of sacrifice.
Three-month-old male non-Tg and 3.times.TgAD mice were
subcutaneously injected with THP (1, 10, and 20 mg/kg BW) or
vehicle (0.1% ethanol in PBS, n=4 in each group). The mice were
sacrificed 24 hours later. THP concentration in plasma and brain
were measured by GC/MS. THP was detectable in plasma and cortex in
a linear dose dependent manner. An interesting finding is that
3.times.TgAD mice exhibited a consistently lower level of THP in
both plasma and cortex relative to nontransgenic THP treated mice.
In the 3.times.TgAD mouse cortex, a 10 mg/kg dose of THP results in
a cortical level of 20 ng/g protein 24 hrs post injection.
Example 8
3.alpha.-hydroxy-5.alpha.-pregnan-20-one (THP) Increased Expression
of Proliferating Cell Nuclear Antigen (PCNA) and Cyclin Dependent
Kinase 1 (CDK1/cdc2) in the Hippocampus of 3.times.TgAD and Non-Tg
Mice
[0190] We sought to identify a medium throughput marker of
proliferation that would allow us to detect proliferative efficacy
in hippocampus with greater speed relative to unbiased
stereological analyses. Thus, we conducted biochemical analyses in
parallel to the stereological analyses to determine whether two
well defined cell cycle related proteins, PCNA and CDK1/cdc2, would
serve as biochemical indicators of proliferation in the
hippocampus.
[0191] Brain samples derived from the same brains that underwent
unbiased stereological analysis and GC/MC for THP detection were
also analyzed by real-time RT-PCR and Western blot for expression
of PCNA and CDK1 mRNA and protein. Results of these analyses
indicate that THP induced a dose-dependent increase in PCNA mRNA in
a pattern consistent with the stereological results in the
3.times.TgAD mice. Results of Western blot analyses indicate that
10 mg/kg THP induced greater PCNA and CDK1 protein expression in
the hippocampi of 3.times.TgAD relative to the non-Tg mice which is
consistent with the stereological data. Importantly, these data
indicate that either mRNA or protein expression of PCNA can serve
as an indicator of proliferation within the hippocampus to permit a
first pass medium throughput analysis of the proliferative efficacy
of THP across multiple doses and topical or transdermal
formulations.
[0192] FIG. 12A shows the stereological analyses of mouse dentate
gyrus subgranular zone (SGZ) performed 24 hours following one
subcutaneous dose of THP (10 mg/kg) or vehicle control. Total BrdU
labeled cells were quantified for 3, 6, 9, and 12 months non-Tg and
3.times.TgAD male mice. The serial brain sections were collected to
analyze the effects of THP on BrdU incorporation by unbiased
stereological analysis. Data are represented as mean.+-.SEM
(n.gtoreq.4 in each group). Data demonstrated that THP-induced
increase in proliferation reversed the neurogenic deficit of the
3.times.TgAD mouse SGZ and restored normal levels of neurogenesis.
PCNA, an S-phase cell cycle marker, was increased in THP compared
to vehicle control in 3.times.TgAD mice in three month (FIG. 12B)
and six month (FIG. 12C) mice as shown by immunoblot. FIG. 12D is a
graph of the PCNA immunoblot data which shows that the basal level
(vehicle) of PCNA expression decreases with age and pathology in
3.times.TgAD mice hippocampus compared to 3 month background strain
non-Tg (positive control).
[0193] Aging Differently Affects Neurogenic Activities in
3.times.TgAD and Control Mice in Hippocampal Subgranular Zone
(SGZ)
[0194] Results showed that 21 days after THP administration an
increase in BrdU+ cell number was observed at all ages, indicating
an increase in neurogenesis after THP treatment. Neurogenesis
includes cell proliferation and cell survival; it is thus important
to determine whether increased neurogenesis after THP treatment is
due to increased cell proliferation or increased cell survival. To
decipher the THP effect, it is critical to first determine the
basal level of cell proliferation in those animals. Hence, a
comparative analysis of BrdU incorporation was conducted in both
3.times.TgAD and non-Tg mice at 6, 9, 12-months of age to determine
basal level of cell proliferation in the SGZ. The distribution of
the BrdU-positive (BrdU+) cells within the 3.times.TgAD and non-Tg
mice was consistent with that observed in both rat and mouse
dentate gyrus. Interestingly, two distinct populations of BrdU+
cells were observed in both 3.times.TgAD and non-Tg mice across all
three ages: normal BrdU+ cells with regular cell shape and even
BrdU staining representing normal proliferating cells, and
irregular BrdU+ cells with either condensed or fragmented nuclei.
The later were consistent with the observations of pyknotic cells
reported by other groups, and are referred to as pyknotic BrdU+
cells.
[0195] Results of unbiased quantitative stereological analyses
indicated that unlike 3-months-old mice, there was no significant
difference in proliferating cell number between non-Tg and
3.times.TgAD mice at 6, 9, and 12-months of age (p=0.27, p=0.39 and
p=0.22, respectively; N=6-10). Basal level proliferations in non-Tg
mice at 6, 9, 12-months were 792.0.+-.284.41, 456.0.+-.226.42,
456.0.+-.153.68, respectively while the basal level proliferations
in 3.times.TgAD mice at 6, 9, 12-months were 600.0.+-.117.23,
537.6.+-.168.84, 326.4.+-.81.21, respectively. Consistent with
findings reported in the literature, both mouse genotypes showed a
trend of age-dependent decline in normal BrdU+ cell number; while
the only statistical significant was observed between 6- and
12-month-old 3.times.Tg-AD mice (p<0.05, N=6-10), consistent
with previous western blot result on PCNA expression level.
Notably, significant reduction in basal level proliferation was
found between 3- and 6-months old animals in both mouse genotypes,
with 3-months-old non-Tg mice showing a basal level of 4560.+-.1089
BrdU+ cells and 3.times.TgAD mice showing 2625.+-.426 BrdU+ cells
(p<0.05, N=6-10).
[0196] In contrast, analysis from the pyknotic cell population
indicated that 3.times.TgAD mice exhibited a significant higher
number of pyknotic cells at 6-months of age, almost 9-folds higher
than that of age-matched non-Tg mice. Pyknotic BrdU+ cell counts in
non-Tg mice at 6, 9, 12-months of age were 48.0.+-.48.00,
192.0.+-.81.13, 48.0.+-.31.42, respectively. On the other hand,
pyknotic BrdU+ cell counts in 3.times.TgAD mice at 6, 9, 12-months
of age were 456.0.+-.125.37, 172.8.+-.66.82, 96.0.+-.51.60,
respectively. The pyknotic cell number in 3.times.TgAD mice dropped
to a level comparable to non-Tg animals at older ages (9&12
months) and no significant difference were found between the two
mouse genotypes. The findings of abnormally high pyknotic cell
population indicated that in addition to reduced basal cell
proliferation, 3.times.TgAD mice might also exhibit varied forms of
neurogenic deficiencies at different ages including cell survival
and cell death.
[0197] THP Specifically Reverses the Neurogenic Deficit in
3.times.TgAD Mice and Had No Effect on Non-Tg Mice
[0198] To decipher the THP effect in neurogenesis, cell
proliferation assessment experiments were conducted. Following
animal sacrifice and brain section staining, unbiased stereology
was conducted in both non-Tg and 3.times.TgAD mice at three
different ages. Consistent with previous results that no
significant differences were found in basal level cell
proliferation between non-Tg and 3.times.TgAD at all ages, THP
showed no significant effect on cell proliferation in both mouse
genotypes. Specifically, THP had no effect on cell proliferation in
non-Tg mice at all ages (p=0.40, p=0.27, and p=0.35, respectively).
In 3.times.TgAD mice, THP showed no significant effect in cell
proliferation although a trend was observed that THP tended to
bring the cell proliferation level more comparable to that of
non-Tg mice (p=0.07, p=0.39 and p=0.32 respectively).
[0199] In contrast, THP had a significant effect on the pyknotic
cell population in both mouse genotypes at 6-months of age, when a
dramatic difference was observed in the basal level cell pyknosis.
While 3.times.TgAD mice exhibited a 9-fold higher basal level
pyknosis, treatment with AP.alpha. significantly reduced it to the
level of non-Tg mice, the pyknotic BrdU+ cell count before and
after THP treatment were 456.0.+-.125.36 and 48.0.+-.48.00,
respectively. Surprisingly, THP treatment in non-Tg mice
significantly increased the pyknotic BrdU+ cell count. The numbers
before and after THP treatment were 48.0.+-.48.00 and
576.0.+-.185.49, respectively. The increase in pyknotic cell
population was unexpected; however it could be explained by a
"proliferative sensor" mechanism which tightly controls cell
proliferation in normal animals. At 9- and 12-months of age, no
difference was observed between vehicle- and THP-treated groups in
both non-Tg and 3.times.TgAD mice.
[0200] To confirm the stereological analyses results, Western blot
analyses of cell proliferation marker PCNA were carried out using
samples derived from the same brains which underwent unbiased
stereological analysis. PCNA, or Proliferating Cell Nuclear
Antigen, is a protein that acts as a processivity factor for DNA
polymerase delta subunit in eukaryotic cells and was induced in
vitro by THP in cultured rat hippocampal progenitor cells.
Consistent with stereological analyses, results of western blot
indicated that no significant differences in basal PCNA expression
level exist between non-Tg and 3.times.TgAD mice at 6- and 9-months
of age (p=0.40 and p=0.32, respectively; N=5). Treatment with THP
did not significantly affect PCNA expression in either mouse
genotype, although a trend was observed that AP.alpha. tended to
bring the PCNA expression level more comparable to that of non-Tg
mice.
[0201] Together with our earlier findings, these results indicated
that THP could specifically modulate the neurogenic activities
(mainly cell proliferation) in 3.times.TgAD mice, and this effect
will only be shown when there is an intrinsic
deficiency/abnormality in these mice. A.alpha.has limited effect on
the neurogenic activities in non-Tg mice, indicating that the
effect of THP is specifically related to the Alzheimer's disease
transgenes. Further, regardless of the age of the mice, THP tends
to bring the neurogenic activity level in 3.times.TgAD mice more
comparable to that of non-Tg mice.
Example 9
Phenotype of Newly Formed Cells in
3.alpha.-hydroxy-5.alpha.-pregnan-20-one (THP) Treated 3.times.TgAD
Mouse Dentate Gyrus are Neuronal and Astrocytic
[0202] To verify the phenotype of the BrdU-positive cells in vivo,
double or triple immunolabeling of BrdU-positive cells with
neuronal markers Tuj1, MAP2, NeuN and astrocyte marker, GFAP, were
performed in the 3.times.TgAD mouse hippocampi, which were treated
with 10 mg/kg THP at 3 months and survived for 3-12 weeks. Under
lower magnification, the majority of the BrdU-positive cells are
observed in the SGZ or Hilus. The distribution of the newly formed
cells is consistent with that observed by previous studies. Imaging
showed co-localization of BrdU in NeuN positive cells, indicating
that newly generated cells exhibit an early neuronal phenotype.
Imaging also showed a newly formed granular cell layer integrated
neuron with nuclear co-localization of BrdU and NeuN and a glial
cell with BrdU positive nuclear and GFAP positive cytosol.
[0203] Neurons and glia are generated throughout adulthood from
proliferating cells in two regions of the rat brain, the
subventricular zone (SVZ) and the hippocampal SGZ. We
stereologically analyzed the SVZ from 3.times.TgAD mice in the
control and 10 mg/kg THP groups. Results of these analyses
indicated that THP induced a 58% increase in BrdU-positive cells
relative to the 3.times.TgAD untreated group. These data indicate
THP increase BrdU incorporation in both SVZ and SGZ, but was more
pronounced in SVZ.
Example 10
3.alpha.-hydroxy-5.alpha.-pregnan-20-one (THP) Reversed the
Learning Deficits of 3.times.TgAD Mice
[0204] To determine if there was a functional consequence of
THP-induced neurogenesis, the impact of THP on both the learning
and memory of a behavioral task shown to be dependent upon the
generation of new neurons in the dentate gyrus, delayed trace
conditioning was assessed. 3.times.TgAD and non-Tg background mice
were prepared for behavioral testing and received a single s.c.
injection of THP (10 mg/kg once) or vehicle 7 days prior to start
of the learning trial. After injection, mice were housed for 7 days
before the training process started. The rationale for the 7 day
interim between exposure to THP and the start of the behavioral
experiment was to allow time for the proliferation, migration and
integration of newly generated neurons into the dentate gyrus.
[0205] Following the 7 day interim period, the behavioral testing
commenced with a 5 day (2.times.30 trials/day) training learning
phase to assess rate and magnitude learning performance. After the
learning trial, mice were returned to their home cage for another 9
day period and subsequently tested for memory of the learned
association. Paired trace eyeblink paradigm: In each trial, mice
were first exposed to a conditioned stimulation of a 85 dB tone for
250 ms; followed by a 250 ms delay (no stimuli), followed by the
unconditioned 60 Hz shock for 100 ms. Introduction of the 250 ms
delay between the conditioned and unconditioned stimuli requires
the hippocampus to acquire the learned association between tone and
shock. Unpaired paradigm: The unpaired diagram (no conditioned) was
applied to serve as control for conditioned trace eye blink
experiment. The experimental diagram is shown in FIG. 13A. The
sequences of conditioned stimuli and the unconditioned shock were
applied randomly. The results are shown in FIGS. 13B and 13C.
[0206] The results of the 5 days training are shown in FIG. 13Ba.
The data indicate that at 3 months, 3.times.TgAD mice exhibit a
learning deficit relative to the performance of normal non-Tg mice
(p=0.038, FIG. 13Bb). In the normal high functioning non-Tg mice,
with a concomitant high level of neurogenesis, THP did not augment
the learning performance (p=0.97). In contrast, THP significantly
increased the learning performance of 3.times.TgAD mice (p=0.04) to
a level comparable to non-Tg mice such that the performance of THP
treated 3.times.TgAD mice was not statistically different from the
normal non-Tg mouse (p=0.87, FIG. 13Bd).
[0207] THP Reversed the Memory Deficits of 3.times.TgAD Mice
[0208] The results of the memory test are shown in FIG. 13C. Nine
days following the learning trial, mice were tested for memory of
the learned association. Non-Tg mice exhibited slightly less than
50% of the conditioned response compared to a 28% response rate of
3.times.TgAD mice. THP did not significantly augment the memory
performance of non-Tg mice. However, THP treated 3.times.TgAD mice
exhibited a significant increase in memory to a level comparable to
the normal non-Tg mice. Multivariant ANOVA analysis indicated
significant differences for learning and memory in genotype
(p=0.004) and days of training (0.04). No interaction occurred
between days of training and genotype (p=0.997). Results of the
behavioral analyses indicate that AP.alpha. enhanced the rate of
learning in 3.times.TgAD mice, increased magnitude of the learning
performance and reversed the memory deficit of 3.times.TgAD
mice.
[0209] THP Reverses Learning and Memory Deficits in Aged
3.times.TgAD Mice
[0210] 3, 6, 9, and 12 month aged mice were tested by trace
eye-blinking conditioning following a single dose of THP (10
mg/kg). The results are shown in FIG. 14. Day 5 was a measure of
learning (black bars) and day 14 was a measure of memory (white
bars). A significant effect of THP was observed in 3 month aged
mice (p<0.05).
[0211] FIGS. 14A and 14B show significant effects of THP in 6
month-age mice (P<0.01). The non-Tg group showed a modest level
of learning (highest level: 31.3.+-.6.1%) and THP did not augment
the learning. The 3.times.TgAD group showed a reduced basal level
of learning when compared to the non-Tg group (but not
significant). THP treatment significantly increased both the rate
of learning and the highest CR % compared to vehicle-treated
3.times.TgAD mice, and the final level was comparable to the non-Tg
group. THP significantly enhanced the memory retention in 6-month
old 3.times.TgAD mice and showed no effect on the age-matched
non-Tg mice. THP-treated non-Tg mice achieved a maximal conditioned
response rate of 30.4.+-.7.7% which was not statistically different
than vehicle-treated (F(1, 20)=0.010; p>0.05). There was no
significant difference observed during the course of training
within the vehicle-treated and THP-treated groups (F(1, 108)=0.292;
p>0.05). Learning performance was mirrored in their memory
performance 9 days post learning phase which was not statistically
different than vehicle-treated non-Tg mice (F(1, 16)=0.038;
p>0.05).
[0212] In contrast, THP significantly increased learning
performance of 6-months-old 3.times.TgAD mice from a basal learning
level of 17.4.+-.3.3% to 39.2.+-.9.2% (F(1, 12)=4.943; p<0.05)
at the end of training. During the entire course of training,
AP.beta.-treated 3.times.TgAD mice performed significantly better
than vehicle-treated mice (F(1, 68)=31.072; p<0.000001)
THP-treated 3.times.TgAD mice performed comparable to the
vehicle-treated non-Tg mice at the end of the training (F(1,
15)=0.538; p>0.05). THP also significantly increased the
retention of the conditioned response rate in the memory test
conducted 9 days after the acquisition phase in 3.times.TgAD mice
(F(1, 10)=5.560; p<0.05).
[0213] FIGS. 14 C and D show significant effects in 9 month-age
mice (P<0.001). The non-Tg group showed a modest level of
learning (highest level: 31.3.+-.6.1%) and THP did not augment the
learning. The 3.times.TgAD group showed a reduced basal level of
learning when compared to non-Tg group. THP treatment significantly
increased both the rate of learning and the highest CR % compared
to vehicle-treated 3.times.TgAD mice, and the final level was
comparable to the non-Tg group. THP significantly enhanced the
memory retention in 9-months-old 3.times.TgAD mice with no effect
on the age-matched non-Tg mice.
[0214] FIGS. 14 E and F show the effects in 12 month-age mice.
12-month-old non-Tg mice showed low learning rate and maximum level
(highest level: 23.8.+-.5.0%) during the training. Treatment with
THP did not significantly alter the learning of non-Tg mice
although a positive trend was observed in the initial phase of
training. 12-month-old 3.times.TgAD mice showed almost no learning
with the highest levels of learning remaining around 15% at the end
of the acquisition phase. THP affected neither the learning rate
nor the final levels of learning after 5 days of training.
12-months-old 3.times.TgAD mice exhibited significant lower memory
retention than age-matched non-Tg mice (p<0.05). THP did not
affect the retention of the learned responses in both 3.times.TgAD
and non-Tg mice.
[0215] Data was also obtained from 15-month age mice. The date is
shown in FIGS. 14 G and H. 15-months-old non-Tg mice showed low
learning rate and maximum level (highest level: 20.3.+-.3.4%)
during the training. Treatment with THP did not significantly alter
the learning of non-Tg mice although a trend towards an increase at
all time points was observed throughout the training phase with a
maximum level of 29.6.+-.5.5%. 15-month-old 3.times.TgAD mice
showed almost no learning with the highest levels of learning
around 15% at the beginning of the acquisition phase. THP did not
affect the learning rate nor the final levels of learning after 5
days of training.
[0216] To determine whether cognitive performance was correlated
with survival of neuroprogenitor cells, hippocampi from behavioral
test animals were analyzed for total number of BrdU positive cells
using fluorescence activated cell sorting (FACS). As BrdU was
administered one hour post THP injection at the start of the
behavioral experiment, FACS analysis detected the total number of
surviving BrdU+ cells and thus can be used as a marker of neural
progenitor cells that survived and integrated into the hippocampal
neuronal network. In 6-months-old vehicle treated non-Tg mice,
BrdU+ cell survival was 2003.6.+-.229.5. In THP-treated non-Tg
mice, 2772.2.+-.534.9 BrdU+ cells survived which was not
significantly different from vehicle-treated (F(1, 13)=1.142;
p>0.05). 6-months-old 3.times.TgAD mice exhibited a
significantly lower number of surviving BrdU+(574.1.+-.136.9)
compared to non-Tg mice (F(1, 15)=27.937; p<0.001). THP
significantly increased BrdU+ in cell survival (1372.5.+-.326.2)
(F(1, 19)=4.069; p=0.05) to promote cell survival by greater than 2
fold in 3.times.TgAD mice.
[0217] At 9-months of age, greater intraneuronal A.beta.
accumulation is apparent in the hippocampus and in very rare
instances A.beta. plaques have developed. Similar to the effects in
6-months-old animals, THP reversed the learning and neurogenic
deficits of 3.times.TgAD mice with no effect in non-Tg mice.
9-months-old non-Tg mice were impaired in their learning in the
initial phase (first 2 days) but improved to 27.8.+-.6.1% with
continuous practice over 5-days of training. Treatment with THP had
no significant effect on learning (F(1, 17)=0.915; p>0.05)
(final level of learning was 20.6.+-.4.1%) although an enhancing
trend was observed in the initial phase. Similarly, no significant
effect in the performance was observed in the memory test between
vehicle- and THP-treated 9-months-old non-Tg mice (F(1, 16)=0.149;
P>0.05). Vehicle-treated 3.times.TgAD mice showed a deficit in
the final level of learning as compared to the vehicle-treated
non-Tg mice. Vehicle-treated 9-months-old 3.times.TgAD mice
exhibited almost no learning with a response rate of 12.5.+-.2.4%.
Compared to the vehicle-treated group the final learning of
THP-treated group was significantly increased (F(1, 25)=4.820;
p<0.05) to a level of 23.6.+-.4.0%. Similar to effects observed
in 6-months-old 3.times.TgAD mice, THP significantly increased the
learning of 9-months-old 3.times.TgAD mice during the entire course
of training (F(1, 129)=24.837; p<0.00001). The enhancement in
learning by THP was not a transient effect as it persisted during
the whole acquisition course, and was confirmed by the
significantly better memory performance after 9 days relative to
vehicle-treated 3.times.TgAD mice F(1, 24)=5.141; p<0.05).
Vehicle-treated 3.times.TgAD mice showed a significant deficit in
the memory test as compared to the vehicle-treated non-Tg mice
(F(1, 20)=15.237; p=0.001), which was reversed by treatment with
THP. THP-treated 3.times.TgAD mice performed comparable to
vehicle-treated non-Tg mice (F(1, 21)=0.282; p>0.05).
[0218] THP induced a significant increase in BrdU+ cell survival in
9-months-old 3.times.TgAD mice similar to what occurred in the
6-months-old 3.times.tgAD mice. Vehicle-treated 9-months-old non-Tg
mice exhibited survival of BrdU+ cells (2288.2.+-.557.9) which was
not significantly different from that of 6-months-old non-Tg mice
(F(1, 13)=0.214; p>0.05. In the 9-months-old non-Tg mice, THP
had no effect on BrdU+ cell survival (2255.3.+-.662.4) as compared
to the vehicle-treated group (F(1, 14)=0.001; p>0.05). In
9-months-old vehicle-treated 3.times.TgAD mice, BrdU+ cell survival
(243.7.+-.30.3) was significantly reduced as compared to
vehicle-treated non-Tg mice (F(1, 15)=17.294; p=0.001). THP
significantly increased the number of surviving BrdU+ cells to
899.3.+-.313.1 (F(1, 19)=7.234; p<0.05) as compared to the
vehicle-treated group. However the treatment with THP in
3.times.TgAD mice was not able to reverse the levels of BrdU+ cells
back to that of vehicle-treated non-Tg mice (F(1, 17)=7.288;
p<0.05).
[0219] At 12-months of age, maximal A.beta. intraneuronal
accumulation is apparent in the hippocampus and A.beta. plaques are
widespread. In contrast to the cognitive and neurogenic efficacy of
THP in the 6 and 9 months-old 3.times.TgAD mice, THP treatment in
12-months-old 3.times.TgAD mice had little effect. In parallel to
the loss of efficacy in 3.times.TgAD mice, emergence of trends
towards an THP effect in 12 months-old non-Tg mice was observed. In
12-months-old non-Tg mice a profound decline in associative
learning ability was apparent during the initial phase (first 2
days) which subsequently improved to a level of 23.8.+-.5% with 5
days of training. The final level of learning in THP-treated non-Tg
mice was 25.4.+-.6%, which was not significantly different from the
vehicle-treated group. THP-treated non-Tg mice did not show a
significant increase in their learning during the course of
training as compared to the vehicle-treated group (F(1, 83)=2.449;
p>0.05). THP-treated non-Tg mice showed a positive trend towards
increased learning on day 2 as compared to the vehicle-treated
group but it was not significant (F(1, 15)=3.750; p>0.05).
THP-treated mice showed a modest improved memory performance which
was not significantly better than vehicle-treated group (F(1,
15)=1.115; p>0.05). Vehicle-treated 3.times.TgAD mice showed a
deficit in the final level of learning as compared to the
vehicle-treated non-Tg mice. Vehicle-treated 12-months-old
3.times.TgAD mice exhibited almost no learning with a final
response rate of 17.5.+-.2.6% (FIG. 14D). Compared to the
vehicle-treated group the final learning of THP-treated group was
not significantly increased and the final level was 14.9.+-.3.6%.
The 12-months-old 3.times.TgAD mice exhibited no improvement in
learning over the 5 days of training (F(1, 113)=0.632; p>0.05)
which was also evident in the memory performance (F(1, 21)=0.019;
p>0.05). Vehicle-treated 3.times.TgAD mice showed a significant
deficit in the memory test as compared to the vehicle-treated
non-Tg mice (F(1, 17)=4.963; p<0.05), which was not reversed by
treatment with THP. THP-treated 3.times.TgAD mice showed an equally
significant deficit to vehicle-treated non-Tg mice (F(1, 19)=4.287;
p=0.05).
[0220] As in the learning and memory analyses, THP induced a modest
but not statistically significant trend towards an increase in
BrdU+ cell survival. Basal level of BrdU+ cell survival was
significantly decreased in 12-months-old 3.times.TgAD mice
(201.1.+-.51.5) as compared to age-matched non-Tg mice (F(1,
16)=135.58; p<0.00000001) (1460.8.+-.99.3). In cell survival
experiment, basal level of BrdU+ cells in 12-months old non-Tg mice
was not significantly different from that of 6-months old (F(1,
14)=4.209; p>0.05) and 9-months old (F(1, 14)=2.438; p>0.05)
non-Tg mice. THP exerted no significant effect on BrdU+ cell
survival in 12-months-old non-tg mice (F(1, 15)=0.329; p>0.05)
nor 12-months-old 3.times.TgAD mice (F(1, 19)=2.977;
p>0.05).
[0221] Western blot analysis was performed to determine
proliferating cell nuclear antigen (PCNA) expression levels in the
hippocampus from the same animals that underwent behavior
experiments. Results indicated that significant difference was only
found between the vehicle-treated 6- and 12-month-old animals in
the basal PCNA expression level (p<0.01, N=6). Twenty-one (21)
day after THP injection, no significant difference was found
between vehicle- and THP-treated animals at either 6, 9 or
12-month-old of age (p=0.34, p=0.10, and p=0.69, respectively;
N=6), indicating AP.alpha. had no sustained effect on cell
proliferation in 3.times.TgAD mice at all ages tested. Further,
these data provided evidence that aging male 3.times.TgAD mice
retained the capacity to undergo 5-phase of the cell cycle, and an
age-related decline in such capacity is obvious at 12-month-old of
age.
[0222] THP Increases Neural Progenitor Cell Proliferation in
Hippocampus of 3.times.TgAD Mice in an Age Dependent Manner
[0223] Total cell proliferation in hippocampus following one dose
of THP (10 mg/kg) and 23 days of learning and memory (trace
eye-blink conditioning) in 3, 6, 9, and 12 month aged mice was
measured. FACS analysis detected immunopositive BrdU labeled cells
by Beckman Coulter Cytomics FC 500 fluorescent flow cytometry.
Significant THP-induced proliferation occurred in 3 and 6 month
aged 3.times.TgAD mice and declined with age thereafter. The data
for BrdU positive cells are summarized in FIG. 15. Non-Tg mice, in
contrast, did not show statistical significance between treatment
and vehicle groups. The data indicates an age dependent decrease of
neural progenitor cell proliferation in mice hippocampus from 3 to
12 month old mice and a genotype dependent decrease of neural
progenitor cell proliferation in 3.times.TgAD mice at any age
tested, particularly at 3 months old, indicating an early
neurogenic deficit in 3.times.TgAD mice.
Example 11
3.alpha.-hydroxy-5.alpha.-pregnan-20-one (THP) Significantly
Increased Survival of BrdU Labeled Cells
[0224] We sought to determine whether cell survival was directly
attributable to THP exposure or whether cell survival was dependent
on the training experience. Thus, the relationship between survival
of BrdU positive cells and cognitive performance on the memory
phase of the behavioral testing was analyzed. It was first
determined whether the conditioning paradigm contributed to an
increase in BrdU positive cells as this has been observed in
behavioral paradigms in which the learning trials were 6-7 times
greater than our 35/trials/day; eg >200+ trials/day. To
determine the effect of training/learning, 3.times.TgAD mice were
trained for 35 trials/day for 5 days and subsequently sacrificed at
the end of the learning phase and brains processed for BrdU
analysis by unbiased stereological analysis. The data demonstrate
that the training/learning paradigm used in our behavioral analyses
did not induce an increase in BrdU positive cells. In contrast, THP
treated 3.times.TgAD mice exhibited a near doubling in the number
of surviving cells generated 20 days prior to sacrifice. These data
indicate that the mechanism of THP action is independent of
training condition and is specific to THP.
[0225] THP Enhancement of Memory Function is Highly Correlated to
the Number of Newly Formed BrdU Positive Cell Numbers
[0226] We sought to determine the relationship between survival of
BrdU positive cells and cognitive performance on the memory phase
of the behavioral testing. Correlational analysis indicated a
highly significant correlation between the number of surviving BrdU
positive cells and memory performance for both the vehicle treated
3.times.TgAD mice and the THP treated 3.times.TgAD mice (Table
1).
TABLE-US-00001 TABLE 1 Correlation of survived BrdU cells with
conditioned response (CR) BrdU cells % CR CR/BrdU R value Vehicle
(n = 10) 652.8 28.33 0.0434 0.58 AP.alpha. (n = 12) 1088 53.96
0.0496 0.68
Example 12
3.alpha.-hydroxy-5.alpha.-pregnan-20-one (THP) Reduces
Immunocytochemically Detectable .beta.-Amyloid and Ptau Expression
in the 6 Month Old 3.times.TgAD Male Mouse CA1 Region of the
Hippocampus
[0227] Triple transgenic mice were sacrificed at different ages as
indicated, brain sections immunostained with anti-Amyloid .beta.42
antibody and observed with peroxidase-DAB. Results of our
assessment of pathology development replicate those of the LaFerla
group and indicate that results from our laboratory are consistent
with previously published characterization. At 3 months, cellular
A.beta. immunoreactivity (IR) was barely visible. At 6, 9 and 12
months, intracellular A.beta. IR was apparent and intensity
increased with age. Extraneuronal A.beta. IR was rarely observed in
9-month-old 3.times.TgAD hippocampi but was consistently present in
the hippocampus of 12-month-old 3.times.TgAD mice. Preliminary
results indicate an age-dependent increase of A.beta. levels in the
cortex which is also in agreement with published reports.
[0228] In a pilot project to determine the impact of longterm
exposure to THP, the impact of THP, 10 mg/kg s.c. thrice weekly for
3 months, on the progression of Alzheimer's disease (AD) pathology
in 3-6 month old 3.times.TgAD mice was investigated. Mouse brain
hemisphere sections were immunostained with specific antibody
(6E10) against A.beta. or HT7, which recognizes tau and PHF-tau
between residue 159 and 163 and visualized with FITC conjugated
second antibody. Observation of immunoreactivity indicated that
both A.beta. and ptau IR were primarily localized within neuronal
cell bodies. Quantitative analysis using SlideBook supported color
mask and automatic color-cell counting system (3i Intelligent
Imaging System) indicated that THP reduced the level of AD
pathology markers in 3.times.TgAD mouse hippocampal CA1. Results of
this pilot project indicate that THP reduced AD pathology burden in
the subicular region of the hippocampus.
[0229] In another experiment, THP (10 mg/kg/week, administered once
a week for 6 months) was administered to 9- and 12-month male mice
transgenic for Alzheimer's disease (3.times.Tg-AD). The 9 month old
animals were started on THP at 3 months of age prior to the
development of beta amyloid, whereas the 12 month old animals were
begun at 6 months of age when beta amyloid had already begun to
accumulate within neurons. Typically, the development of
intraneuronal beta amyloid is seen in 6 and 9 month old animals and
the development of plaques in 12 month old animals. Plaques are
rarely seen in 9 month old animals. This is shown in FIG. 16.
[0230] The results of THP administration are shown in FIG. 17. The
graph shows that THP significantly decreased the amount of beta
amyloid in the cerebral cortex of male mice transgenic for
Alzheimer's disease. Western Blot analysis showed a form of beta
amyloid termed Abeta*56, which is the oligomer (multiple amyloid
beta peptides joined together) that, in animal studies, leads to
memory loss in both transgenic Alzheimer mouse models and in rats
injected with Abeta*56 (Lesne et al., Nature, 440, 352-357 (Mar.
16, 2006)). Thus, reducing Abeta*56 has the potential for
preventing or reversing memory loss. In the 12 month old animals,
the level of Abeta*56 was much lower, likely due to the development
of beta amyloid plaques in these animals, which reduces the amount
of Abeta*56. Western Blot analysis was also conducted for
.beta.-amyloid antibody 6E10 which recognizes the abnormally
processed isoforms, as well as precursor forms of beta-amyloid
protein. .beta.-Amyloid expression from treated mice brain was
shown as the labeled of 56 KD. This band at 56 KD corresponding to
amyloid 12-mer oligomer. The results are shown in FIG. 18A.
Treatment with THP partially reduced A.beta. in both ages. THP
decreased A.beta. level by 25.+-.4%, p=0.004 versus vehicle
treatment in 9-month old 3.times.Tg-AD mice. In 12-month old
3.times.Tg-AD mice, AP.alpha. induced a reduction 15.+-.4%, p=0.05.
Allopregnanolone was shown to attenuate A.beta. accumulation. Brain
sections were stained with beta amyloid antibody 6E10. Imaging
showed 6E10 immunoreactivity was lower in THP-treated brain region
CA1, Cortex and Amygdala than vehicle treated-brain, but not in the
subiculum.
[0231] Immunocytochemical detection of beta amyloid showed that
administration of THP substantially decreases Abeta*56 in
hippocampal neurons. THP also decreases the immunoreactivity of
phosphorylated tau, which is the basis for neurofibrillary tangles.
Results of a study of 3 and 6 month old mice administered 10 mg/kg
THP once a week for six months are shown in FIG. 19. The
quantitation of the immunofluorescent signals for Abeta*56 and
phosphorylated tau are shown in FIGS. 19A and 19B,
respectively.
[0232] In yet another study, THP (10 mg/kg once a week for 6
months) was administered to mice beginning at 3 months or 6 months
of age. The cell proliferation marker, 5-bromodeoxyuridine (BrdU)
was injected (100 mg/kg) one hour after initial THP treatment and
once per day for the next four days to detect cell proliferation
and cell survival over the six month treatment period. After 3
months treatment, the mice were injected 5 times with another
nucleotide analog iododeoxyuridine (one per day). After 6 months
treatment, the mice were injected with chlorodeoxyuridine (CldU) 5
times (once per day). The treatment protocol is shown in FIG. 20A.
Using specific antibodies, BrdU, IdU, and CldU incorporated cells
will identify the 6 month survival, 3 month survival, and newly
formed cells respectively. The mice were then sampled for
biochemical, IHC, and flow cytometry assay. The results from mice
with THP treatment initiated at 3 months old are shown in FIG. 20B
and initiated at 6 months old are shown in FIG. 20C. The aim was to
determine whether or not desensitization to THP was occurring. The
results in FIGS. 20B and 20C suggest that the brain is still
responsive to THP at 3 and 6 months.
Example 13
Dose-Response and Time Course of
3.alpha.-hydroxy-5.alpha.-pregnan-20-one (THP)-Induced
Proliferation in Human Neural Progenitor Cells (hNPC)
[0233] To determine the impact of THP on hNPC cell proliferation, a
dose response (pM-nM) experiment was performed. Results of these
analyses indicate:
[0234] a. THP, at doses within an achievable therapeutic range,
increased human neural progenitor cell proliferation by 50%.
Results of dose response analyses indicate that THP promoted human
neural stem cell proliferation in a biphasic dose dependent
fashion.
[0235] b. THP-induced human neural stem cell proliferation was
first evident at 1 nM and maximal at 100 nM. Maximal proliferative
efficacy was asymptotic at 100 nM, sustained at 250 and 500 nM and
diminished at 1000 nM.
[0236] c. THP-induced hNPC proliferation was linear and evident at
3 hours and reached maximum at 6 hours.
[0237] d. Efficacy of THP as a neurogenic factor exceeded that of
bFGF+heparin in promoting human neural progenitor cell
proliferation.
[0238] e. Findings derived from hNPCs replicate results of
THP-induced proliferation of rodent hippocampal NPCs.
Example 14
3.alpha.-hydroxy-5.alpha.-pregnan-20-one (THP) Increases hNPC
Proliferation while not Changing Neuronal Phenotype
[0239] To determine the impact of THP on the stability of hNPC
phenotype, hNPCs were double labeled with BrdU, Tuj1 and MAP2, or
GFAP. Quantitative analysis of phenotype (DAPI-positive blue nuclei
as marker of total cell number) indicated that THP significantly
increased the number of BrdU positive hNPCs while not changing the
proportion of Tuj1, MAP2 or GFAP cells vs. vehicle treated
hNPCs.
[0240] AP.alpha.-induced hNPC Cell Proliferation is Blocked by
GABAAR Antagonist
[0241] Previously, we demonstrated that THP-induced rat NPC
proliferation is mediated by GABAAR, as the GABAAR antagonist
bicuculline abolished the THP-induced intracellular calcium
concentration increase, required for THP-induced rNPC cell
proliferation. To determine whether the same mechanisms of
THP-induced proliferation in rodent derived NPCs could be
generalized to humans, we determined the requirement of the GABAAR
by antagonizing the GABAAR with bicucculline followed by assessment
of THP-induced hNPC cell proliferation. Results of those analyses
indicate that 250 nM THP was as efficacious a proliferative factor
as the positive control, bFGF. Both vehicles, alcohol and DMSO, had
no significant effect on basal hNPC proliferation. Bicuculline
completely antagonized, THP-induced hNPC proliferation. These
results indicate that as in rNPCs, THP-induced proliferation
requires the GABAAR.
[0242] Human Neural Progenitor Cells (hNPC) Express a Specific
Combination of GABAAR (GBRC) Subunits.
[0243] Activation of GABAAR in mature neurons leads to
hyperpolarization via an influx of chloride. In contrast, in
immature neurons and neural progenitors activation of the GABAAR
leads to a depolarization through an efflux of chloride. It is
hypothesized that hNPCs responsive to THP will exhibit a GABAAR
phenotype that is comparable to extrasynaptic GABAAR. The tonic
conductances of the extrasynaptic GABAARs may be more conducive to
depolarization required for opening voltage dependent L-type
calcium channels and downstream signaling cascades required for
cell cycle activation.
[0244] To determine GABAAR receptor subunit expression in hNPC
cells, reverse transcriptase PCR (RT-PCR) using total RNA extracted
from cultured hNPC and human fetal brain was performed, the latter
used as a control to verify all the primers used are functional in
RT-PCR. cDNA from total human fetal brain total RNA showed positive
amplification of GBRC subunits. In contrast, .alpha.2 and .alpha.5,
but not .alpha.1 and .alpha.4 were expressed in hNPC. In addition,
a much higher expression of .delta. subunit in hNPC was also
observed. These results are consistent with recent data in the
literature showing that the THP binding pocket required for direct
activation of the GBRC requires a pocket formed by the interface
between .alpha. and .beta. subunits in which THP spans the
interface between the 2 subunits.
Example 15
Transdermal THP Formulation Promotes Neural Progenitor Cell
Proliferation in Three Month Old Male 3.times.TgAD Mouse
Hippocampus
[0245] Flow cytometry assay detected total BrdU labeled cells 24
hours following THP administration via (A) subcutaneous and (B)
transdermal routes. The optimal dose to significantly increase BrdU
cell numbers was found to be 10 mg/kg for both routes and confirmed
previous results obtained by unbiased stereology. BrdU (100 mg/kg)
was administered by intraperitoneal injection one hour after THP
administration. Transdermal gel THP dose-response for 5, 10, and 50
mg/kg (FIG. 21B) was compared to 0 mg/kg control vehicle (negative
control) and subcutaneous injection of 10 mg/kg (positive control)
(FIG. 21A). THP significantly increases BrdU cell numbers in 10
mg/kg dose. Immunoblots of proliferating cell nuclear antigen
(PCNA), an S-phase marker and cyclin dependent kinase 1
(CDK1/cdc2), an M-phase marker, are shown in FIGS. 21C and 21D,
respectively. Hippocampus protein expression of PCNA and CDK1/cdc2
was normalized to .beta.-tubulin loading control and compared
relative to the vehicle control by immunoblot. Both bands were
incorporated in the analysis for CDK1/cdc2; the upper band
represents the inactive form, while the lowe band represents the
active form. The data were analyzed by one way ANOVA followed by at
test (two samples assuming equal variance) vs. vehicle. Data are
presented as the mean.+-.SEM, n=5, *=P<0.05, and
**=P<0.01.
Example 16
Effect of Transdermal THP on .beta.-Amyloid Expression in 9-Month
Male 3.times.Tg-AD Mice
[0246] THP (10 mg/kg) or vehicle was transdermal treated once a
week to 3-month-old 3.times.Tg-AD mice for 6 months. Western blot
analysis was conducted using brain protein from hippocampus with
beta amyloid antibody 6E10, which recognizes the abnormally
processed isoforms, as well as precursor forms of beta amyloid
protein. Three major immunoreactive bands were detected in
nitrocellulose membrane from 9-month 3.times.Tg-AD mice brain
(hippocampus), but the band in age-matched nonTg mice sample was
not detectable. Two full-length APP bands around 100 kDa and
oligomer band around .about.28 kDa were detected with 6E10
antibody. Quantification of 28 kDa .beta.-amyloid oligomer band
intensity level (relative to .beta.-actin level) shows that
treatment with THP partially reduced .beta.-amyloid expression. THP
decreased intensity level by 39.+-.13% versus vehicle treatment in
9-month old 3.times.Tg-AD mice (n=10, p<0.05, FIG. 22).
Example 17
Chronic Administration of THP Decreases Alzheimer Disease Pathology
in Triple Transgenic Mice
[0247] THP (10 mg/kg) or vehicle was subcutaneously administered
once a month, once per week for 6 months, or once every other day
for 3 months. Brain sections of treated mice were stained with beta
amyloid antibody 6E10 which recognizes the abnormally processed
isoforms, as well as precursor forms of beta amyloid protein. The
representative image shows that different treatment paradigm
reveals different effects. THP significantly decreases 6E10
immunoreactivity when treatment every other day for 3 months or
once a week for 6 months, but no effect when treated once a
month.
[0248] Further studies were conducted using a dosing regime of once
a week for six months administered subcutaneously. Three and six
months old 3.times.Tg-AD and age-matched non-Tg mice were chosen
for the experiments, since AD pathology (A.beta.) starts
development at 3-month and abundant accumulation at 6-months.
Allopregnanolone was subcutaneously (s.c.) administrated with 10
mg/kg once per week for 6 months. For neurogenesis and
neurosurvival study, BrdU, IdU and CIdU were intraperitoneal (i.p.)
injected at the first 5 days, mid 5 days and 5 days before
sacrifice during 6-month treatment. After 6 months of
allopregnanolone treatment, mice, at 9- and 12-month old, were
sacrificed.
[0249] THP Reduces .beta.-Amyloid 27 and 56 kDa Expression in 9-
and 12-Month Male 3.times.Tg-AD Mice
[0250] Western blot analysis was conducted using brain protein from
frontal-parietal-temporal cortex with beta amyloid antibody 6E10,
which recognizes the abnormally processed isoforms, as well as
precursor forms of beta amyloid protein. The expression of
different forms of beta amyloid in 3.times.Tg-AD mice was
characterized. Three major immunoreactive bands were detected in
the samples from 9- and 12-month 3.times.Tg-AD mice brain
(frontal-parietal cortex), but the bands in age-matched non-Tg mice
were very faint even though the intensity of the bands increased
with aging.
[0251] The bands around .about.27 kDa and 56 kDa oligomers were
detected with 6E10 antibody as hexamers (6-mer) and dodecamers
(12-mer). One 27-month 3.times.Tg brain sample was included as a
positive control. The oligomer bands intensity was much stronger
for older mice. Quantification of these two oligomer bands
intensity level (relative to b-actin level) shows that treatment
with AP.alpha. partially reduced A.beta.*56 in both ages. THP
decreased intensity level by 25.+-.4% versus vehicle treatment in
9-month old 3.times.Tg-AD mice (n=5, p<0.01). In 12-month old
3.times.Tg-AD mice, THP induced a reduction of 15.+-.4% (n=3-4,
p=0.05. A.beta. 6-mer at 27 kDa band intensity was also reduced by
THP-treatment, 35.+-.10%, p<0.05, but no difference in 12-month
old 3.times.Tg mice. Immunohistochemistry analyses showed a
widespread reduction of A.beta. staining in THP-treated brain
sections. These results show that intraneuronal A.beta.
immunoreactivity is lower in THP-treated 9-month old 3.times.Tg-AD
mice hippocampus, cortex, and amygdala than in vehicle-treated
brain, but not in subiculum.
[0252] Such inhibition could be highly significant for AD
treatment, because oligomeric A.beta. species in the brain
currently is considered as a major risk factor for the onset and
progression of cognitive decline in AD. Although A.beta. plaques
are the most visible and well characterized amyloid pathology in
the AD brain, recent studies indicate that A.beta. oligomers,
especially A.beta.*56, is mainly responsible for AD dementia and
memory deficits. Thus, allopregnanolone-induced oligomer
attenuation may improve cognitive function.
[0253] THP Reduces ABAD Expression in 3.times.Tg-AD Mice
[0254] Beta amyloid binding alcohol dehydrogenase (ABAD) was probed
with antibody ERAB (1:500, Abeam). THP-treatment decreased ABAD
level by 30.+-.4% (p<0.05) and 20.+-.7% (p=0.07) respectively,
in 9-month and 12-month 3.times.Tg mice relative to the
vehicle-treated control. Immunofluorescent staining using goat
polyclonal anti-ERAB (Santa Cruz) confirmed the results obtained by
Western Blot analysis. The concentration of ABAD immunoactive cells
is less in THP-treated brains than in the brains treated with the
vehicle.
[0255] The interaction of A.beta. and ABAD disrupts mitochondrial
function. It has been reported that the increase in mitochondrial
A.beta. correlated with the increase in ABAD level in the
3.times.Tg-AD mouse brain which resulted in neurotoxicity due to
the formation of mitochondrial deposits of A.beta.. THP
significantly decreased ABAD expression in 3.times.Tg-AD mice which
should reduce the interaction of and ABAD. Minimizing the
interaction of A.beta. and ABAD should be beneficial for improving
mitochondria function in AD patients.
[0256] THP Modulates Phospho-Tau Expression in 3.times.Tg-AD
Mice
[0257] Western blot analysis was conducted with monoclonal
phospho-tau antibody (AT8, PIERCE) which recognizes phosphorylated
tau serine 202. Immunoblotting data did not show a significantly
reduction of AT8 immunoreactivity in mouse brains treated with THP.
However, immunostaining with AT8 showed lower levels of
immunoreactivity in THP-treated hippocampal CA1, cortex and
amygdala.
[0258] A direct relationship between A.beta. and tau pathologies in
3.times.Tg-AD mice has be reported. Specifically, it has been
reported that A.beta. causes tau accumulation and subsequent
phosphorylation. Removal of intraneuronal A.beta. via immunotherapy
leads to tau clearance from neurons following the A.beta. itself
clearance. Therefore, the reduction of phosphor-tau level with AP
appears to closely related to the reduction of intracellular
A.beta. levels.
[0259] THP Treatment Inhibited Microglial Reaction
[0260] Western blot analysis was conducted with rabbit polyclonal
anti-CD11b/c (OX42) which recognizes reactive microglial cells. The
band intensities trended towards a decrease in THP treated mouse
brains, but there are no significant differences in 9-month
3.times.Tg p-0.07 and 12-month p=0.10. The immunofluorescent
staining with rabbit polyclonal anti-Ibal (1:1000, Woka) showed
lower levels of immunoactivity in THP-treated hippocampal CA1.
[0261] Microglial cells become activated in AD brains. Activated
microglia cells generate the wide panel neurotoxic agents, inducing
neurodegeneration and neuronal loss. Therefore, suppression of
microglial activation should be beneficial for treatment of this
disease. Our results show that THP treatment inhibited microglia
activation, suggesting that THP could be one a potential candidate
drugs for AD prevention and therapy.
[0262] THP Increased Myelination in Mouse Brain
[0263] Western blot analysis using anti-CNPase antibody, an
oligodendrocyte marker which generates myelin, shows THP increased
CNPase expression in 9-month nonTg mice (p<0.01) and
3.times.TgAD mice (p<0.05). In 12-month non-Tg mice, there were
no significant difference, but CNPase expression trended towards an
increase (p=0.045) in 3.times.TgAD mice. Immunofluorescent staining
shown the immunoreactivity increased in CA1, entorhinal cortex, and
primary somatosensory cortex.
[0264] Overall, the results discussed above indicate that THP
treatment reduces A.alpha. oligomer accumulation, paralleled with a
reduction in ABAD expression and microglia activation.
Collectively, these findings indicate that in a mouse model of AD,
THP induces a profile consistent with reduction and or delay in
progression of Alzheimer's pathology. These findings have important
implications for THP as a therapeutic candidate for the treatment
of Alzheimer's disease pathology.
[0265] All patent and non-patent references cited in this
specification are herein incorporated by reference as if each
individual patent or non-patent reference were specifically and
individually indicated to be incorporated by reference.
[0266] Although the foregoing embodiments of the invention has been
described in some detail by way of illustration and example for
purposes of clarity and understanding, it will be readily apparent
to those of ordinary skill in the art in light of the teachings of
this invention that certain changes and modifications may be made
thereto without departing from the scope of the appended
claims.
[0267] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
herein. Such equivalents are intended to be encompassed by the
following claims.
* * * * *