U.S. patent application number 14/191984 was filed with the patent office on 2014-12-04 for compositions and methods for treating alzheimer's disease.
This patent application is currently assigned to EIP Pharma, LLC. The applicant listed for this patent is EIP Pharma, LLC. Invention is credited to John Jahangir Alam.
Application Number | 20140357638 14/191984 |
Document ID | / |
Family ID | 47139618 |
Filed Date | 2014-12-04 |
United States Patent
Application |
20140357638 |
Kind Code |
A1 |
Alam; John Jahangir |
December 4, 2014 |
COMPOSITIONS AND METHODS FOR TREATING ALZHEIMER'S DISEASE
Abstract
The present invention provides compositions for reducing amyloid
plaque burden associated with Alzheimer's disease and methods of
using the same.
Inventors: |
Alam; John Jahangir;
(Cambridge, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
EIP Pharma, LLC |
Cambridge |
MA |
US |
|
|
Assignee: |
EIP Pharma, LLC
Cambridge
MA
|
Family ID: |
47139618 |
Appl. No.: |
14/191984 |
Filed: |
February 27, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13467519 |
May 9, 2012 |
8697627 |
|
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14191984 |
|
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61483919 |
May 9, 2011 |
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Current U.S.
Class: |
514/248 |
Current CPC
Class: |
C07D 213/82 20130101;
C07D 413/04 20130101; C07D 231/40 20130101; C07D 513/04 20130101;
C07D 211/16 20130101; C07D 401/04 20130101; C07D 403/04 20130101;
A61K 31/519 20130101; C07D 487/04 20130101; A61K 31/5025 20130101;
C07D 401/12 20130101; C07D 401/14 20130101; C07D 417/04 20130101;
A61P 25/28 20180101; C07D 403/12 20130101; C07D 471/04 20130101;
A61P 43/00 20180101; C07D 405/14 20130101 |
Class at
Publication: |
514/248 |
International
Class: |
A61K 31/5025 20060101
A61K031/5025 |
Claims
1-4. (canceled)
5. A method of reducing amyloid plaque burden, said method
comprising: (i) imaging the brain of a subject; (ii) determining
the number and/or area of the amyloid plaques; and (iii)
administering a therapeutically effective amount of a p38
mitogen-activated protein kinase (MAPK) inhibitor if the number
and/or area of the amyloid plaques exceeds a predetermined
threshold, wherein the p38 MAPK inhibitor is VX-745.
6. The method according to claim 5, wherein the subject is a
patient at risk of developing Alzheimer's disease.
7. The method according to claim 5, wherein the subject is a
patient suffering from Alzheimer's disease.
8. The method according to claim 5, wherein steps (i), (ii) and
(iii) are repeated at one or more predetermined intervals.
9. The method according to claim 8, wherein the predetermined
interval is about six (6) months.
10. The method according to claim 8, wherein the predetermined
interval is about one (1) year.
11. The method according to claim 5, wherein the brain of the
subject is imaged using a neuroimaging technique selected from the
group consisting of computerized axial tomography (CAT or CT),
single photon emission computed tomography (SPECT), positron
emission tomography (PET), magnetic resonance imaging (MRI) or
functional magnetic resonance imaging (fMRI).
12. The method according to claim 11, wherein the neuroimaging
technique is positron emission tomography (PET).
13. The method according to claim 12, further comprising an imaging
agent selected from amyvid or Pittsburgh compound B.
14. The method according to claim 5, wherein the p38 inhibitor is
administered for a period of less than six (6) months.
15. The method according to claim 14, wherein the p38 inhibitor is
administered for a period of less than four (4) months.
16. The method according to claim 14, wherein the p38 inhibitor is
administered for a period of less than two (2) months.
17. The method according to claim 14, wherein the p38 inhibitor is
administered for a period of less than one (1) month.
18. The method according to claim 14, wherein the p38 inhibitor is
administered for a period of about two (2) weeks.
19-21. (canceled)
22. A method of reducing the number and/or volume of amyloid
plaques in a patient suffering from Alzheimer's disease comprising
administering to the patient a therapeutically effective dose of a
p38 inhibitor, wherein the therapeutically effective dose is
between about 1 mg to about 100 mg.
23. The method according to claim 22, wherein the therapeutically
effective dose is between about 1 mg to about 20 mg.
24. The method according to claim 22, wherein the therapeutically
effective dose is between about 1 mg to about 10 mg.
25. The method according to claim 22, wherein the therapeutically
effective dose is between about 1 mg to about 5 mg.
26-29. (canceled)
30. The method according to claim 5, wherein the number and/or area
of the amyloid plaques is determined by comparing a neuroimage of
the subject to a reference image.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] The present application claims priority to U.S. Provisional
Application No. 61/483,919, filed May 9, 2011, which is hereby
incorporated by reference in its entirety.
BACKGROUND OF THE INVENTION
[0002] Alzheimer's disease is the most common cause of dementia,
and is characterized by the loss of intellectual and social
abilities severe enough to interfere with daily functioning. In
Alzheimer's disease, healthy brain tissue degenerates, causing a
steady decline in memory and mental abilities. Alzheimer's disease
is not a part of normal aging, but the risk of the disorder
increases with age. About 5 percent of people between the ages of
65 and 74 have Alzheimer's disease, while nearly half the people
over the age of 85 have Alzheimer's.
[0003] Two types of neuron pathology, plaques and tangles, are
common in patients with Alzheimer's disease. Extracellular plaques
are clumps of a normally harmless protein called beta-amyloid
(A.beta.) which may interfere with communication between brain
cells. Tangles are the internal support structure for brain cells
depends on the normal functioning of a protein called tau. In
people affected with Alzheimer's disease, threads of tau protein
undergo alterations that cause them to become twisted. Many
researchers believe this may seriously damage neurons, causing them
to die and leading to memory deficit.
BRIEF DESCRIPTION OF THE DRAWINGS
[0004] FIGS. 1A and 1B summarize the effects of VX-745 on the area
percentage of amyloid plaques in the cortex (FIG. 1A) and
hippocampus (FIG. 1B) following two-week administration of VX-745
(3 mg/kg BID).
[0005] FIG. 2 summarizes the effects of VX-745 on IL-1.beta. as
compared to wild-type and vehicle controls.
DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS OF THE INVENTION
Definitions
[0006] Carrier: The term "carrier" refers to any chemical entity
that can be incorporated into a composition containing an active
agent (e.g., a p38 inhibitor) without significantly interfering
with the stability and/or activity of the agent (e.g., with a
biological activity of the agent). In certain embodiments, the term
"carrier" refers to a pharmaceutically acceptable carrier. An
exemplary carrier herein is water.
[0007] Combination. As used herein, the term "combination,"
"combined," and related terms refers to a subject's simultaneous
exposure to two or more therapeutic agents in accordance with this
invention. For example, an agent of the present invention (e.g., a
p38 inhibitor) may be administered with another therapeutic agent
simultaneously or sequentially in separate unit dosage forms or
together in a single unit dosage form. Accordingly, the present
invention provides, among other things, dosing regimens that
involve administering at least an agent of the present invention
(e.g., a p38 inhibitor), an additional therapeutic agent, and a
pharmaceutically acceptable carrier, adjuvant, or vehicle (the
pharmaceutically acceptable carrier, adjuvant, or vehicle typically
being in association with one or both of the p38 inhibitor and the
additional therapeutic agent).
[0008] Formulation. The term "formulation" refers to a composition
that includes at least one active agent (e.g., a p38 inhibitor)
together with one or more carriers, excipients or other
pharmaceutical additives for administration to a patient. In
general, particular carriers, excipients and/or other
pharmaceutical additives are selected in accordance with knowledge
in the art to achieve a desired stability, release, distribution
and/or activity of active agent(s) and which are appropriate for
the particular route of administration.
[0009] Low dose. The term "low dose" as used herein refers to a
dose that is below the therapeutically effective amount of the
reference p38 inhibitor when administered to treat a disease other
than Alzheimer's disease. In some embodiments, the term "low dose"
refers to a dose that is one or more orders of magnitude lower than
the therapeutically effective amount of the reference p38 inhibitor
when administered to treat a disease other than Alzheimer's
disease. In some embodiments, the term "low dose" refers to a dose
that is one-half, one-third, one-fourth, one-fifth, one-sixth,
one-seventh, one-eighth or less than the therapeutically effective
amount of the reference p38 inhibitor when administered to treat a
disease other than Alzheimer's disease. For example, a
therapeutically effective unit dose of VX-745 for the treatment of
rheumatoid arthritis in humans is 250 mg. In some embodiments, a
"low dose" of VX-745 is within the range of about 1 mg to about 100
mg. In some embodiments, a "low dose" of VX-745 is within the range
of about 1 mg to about 50 mg. In some embodiments, a "low dose" of
VX-745 is within the range of about 1 mg to about 30 mg. In some
embodiments, a "low dose" of VX-745 is within the range of about 1
mg to about 10 mg. In some embodiments, a "low dose" of VX-745 is
within the range of about 1 mg to about 5 mg. In some embodiments,
a "low dose" of VX-745 is about 3 mg. In some embodiments, a "low
dose" of VX-745 is within the range of 5-10 mg. In some
embodiments, a "low dose" of VX-745 is within the range of 10-20
mg. In some embodiments, a "low dose" of VX-745 is within the range
of 20-30 mg.
[0010] Neuroimaging. As used herein, the term "neuroimaging" refers
to a technique which directly or indirectly images the structure or
function of the brain. In some embodiments, the term "neuroimaging"
refers to a technique selected from computerized axial tomography
(CAT or CT), single photon emission computed tomography (SPECT),
positron emission tomography (PET), magnetic resonance imaging
(MRI) or functional magnetic resonance imaging (fMRI). In some
embodiments, a neuroimaging technique employs one or more imaging
agents such as radioactive, fluorescent or other detectable
ligands. In some embodiments, a fluorescent ligand is Pittsburg
compound B
([N-Methyl-.sup.11C].sub.2-(4'-methylaminophenyl)-6-hydroxybenzothiazole)-
, a fluorescent analog of thioflavin T. In some embodiments, a
radioactive ligand is Amyvid.RTM. (florbetapir F18) or
18F-flutemetamol. In some embodiments, the neuroimaging technique
is PET scan using Pittsburgh compound B as an imaging agent. In
some embodiments, the neuroimaging technique is PET scan using
Amyvid.RTM. as an imaging agent. In some embodiments, the
neuroimaging technique is PET scan using 18F-flutemetamol as an
imaging agent.
[0011] Neuroimage. As used herein, the term "neuroimage" refers to
an image or picture generated by a neuroimaging technique. In some
embodiments, a "neuroimage" refers to one or more of CAT (or CT),
SPECT, PET, MRI or fMRI scans.
[0012] Parenteral. The term "parenteral" as used herein includes
subcutaneous, intravenous, intramuscular, intra-articular,
intra-synovial, intrasternal, intrathecal, intrahepatic,
intralesional and intracranial injection or infusion techniques.
Preferably, the compositions are administered orally,
intraperitoneally or intravenously. Sterile injectable forms of the
compositions of this invention may be aqueous or oleaginous
suspension. These suspensions may be formulated according to
techniques known in the art using suitable dispersing or wetting
agents and suspending agents. The sterile injectable preparation
may also be a sterile injectable solution or suspension in a
non-toxic parenterally acceptable diluent or solvent, for example
as a solution in 1,3-butanediol. Among the acceptable vehicles and
solvents that may be employed are water, Ringer's solution and
isotonic sodium chloride solution. In addition, sterile, fixed oils
are conventionally employed as a solvent or suspending medium.
[0013] Patient. The term "patient", as used herein, means a mammal
to which a formulation or composition comprising a formulation is
administered, and in some embodiments includes humans.
[0014] Pharmaceutically acceptable carrier, adjuvant, or vehicle.
The term "pharmaceutically acceptable carrier, adjuvant, or
vehicle" refers to a non-toxic carrier, adjuvant, or vehicle that
does not destroy the pharmacological activity of the compound with
which it is formulated. Pharmaceutically acceptable carriers,
adjuvants or vehicles that may be used in the compositions of this
invention include, but are not limited to, ion exchangers, alumina,
aluminum stearate, lecithin, serum proteins, such as human serum
albumin, buffer substances such as phosphates, glycine, sorbic
acid, potassium sorbate, partial glyceride mixtures of saturated
vegetable fatty acids, water, salts or electrolytes, such as
protamine sulfate, disodium hydrogen phosphate, potassium hydrogen
phosphate, sodium chloride, zinc salts, colloidal silica, magnesium
trisilicate, polyvinyl pyrrolidone, cellulose-based substances,
polyethylene glycol, sodium carboxymethylcellulose, polyacrylates,
waxes, polyethylene-polyoxypropylene-block polymers, polyethylene
glycol and wool fat.
[0015] Selective p38 Inhibitor. As used herein, the phrase
"selective p38 inhibitor" refers to an agent which elicits a
biological effect (i.e., an inhibitory or antagonistic effect) on
p38 mitogen-activated protein kinase (also referred to as p38 MAPK)
that is at least one order of magnitude greater than another
kinase. For example, in some embodiments, a selective p38 inhibitor
is an inhibitor which is selective for p38 MAPK over other protein
kinases or tyrosine kinases. In some embodiments, a selective p38
inhibitor is selective for one p38 MAPK isoform over another. For
example, in some embodiments, a selective p38 inhibitor refers to
an inhibitor that has greater antagonistic effect against one of
alpha (.alpha.), beta (.beta.), gamma (.gamma.) or delta (.delta.)
p38 MAPK isoforms over another isoform. In some embodiments, a
selective p38 inhibitor refers to an inhibitor that has greater
antagonistic effect against the p38a isoform of MPAK as compared to
the p38.beta., p38.gamma. and/or p38.delta. isoforms.
Representative selective p38 inhibitors include, but are not
limited to, RWJ 67657, SCIO 469, EO 1428, Org 48762-0, SD 169, SB
203580, SB 202190, SB 239063, SB 220025, VX 745, SB 242235, VX 702,
SD-282, PH-797804 and others.
[0016] Therapeutic agent. As used herein, the phrase "therapeutic
agent" refers to any agent that elicits a desired biological or
pharmacological effect when administered to an organism.
[0017] Therapeutically effective amount and effective amount. As
used herein, and unless otherwise specified, the terms
"therapeutically effective amount" and "effective amount" of an
agent refer to an amount sufficient to provide a therapeutic
benefit in the treatment, prevention and/or management of a
disease, disorder, or condition, e.g., to delay onset of or
minimize (e.g., reduce the incidence and/or magnitude of) one or
more symptoms associated with the disease, disorder or condition to
be treated. In some embodiments, a composition may be said to
contain a "therapeutically effective amount" of an agent if it
contains an amount that is effective when administered as a single
dose within the context of a therapeutic regimen. In some
embodiments, a therapeutically effective amount is an amount that,
when administered as part of a dosing regimen, is statistically
likely to delay onset of or minimize (reduce the incidence and/or
magnitude of) one or more symptoms or side effects of a disease,
disorder or condition. In some embodiments, a "therapeutically
effective amount" is an amount that enhances therapeutic efficacy
of another agent with which the composition is administered in
combination. In some embodiments, a therapeutically effective
amount for administration to a human corresponds to a reference
amount (e.g., a therapeutically effective amount in an animal model
such as a mouse model) adjusted for body surface area of a human as
compared with body surface area of the animal model, as is known in
the art (see, for example Reagan-Shaw et al., "Dose translation
from animal to human studies revisited," The FASEB Journal 22:
659-661 (2007), the entirety of which is herein incorporated by
reference). In some embodiments, the reference therapeutically
effective amount is an amount that is therapeutically effective in
a mouse model, for example, as described herein. In some
embodiments, the reference therapeutically effective amount is
within the range of about 0.0001 mg/kg to about 500 mg/kg. In some
embodiments, the reference therapeutically effective amount is
within the range of about 0.0001 mg/kg to about 0.001 mg/kg. In
some embodiments, the reference therapeutically effective amount is
within the range of about 0.001 mg/kg to about 0.01 mg/kg. In some
embodiments, the reference therapeutically effective amount is
within the range of about 0.01 mg/kg to about 0.1 mg/kg. In some
embodiments, the reference therapeutically effective amount is
within the range of about 0.1 mg/kg to about 0.5 mg/kg. In some
embodiments, the reference therapeutically effective amount is
within the range of about 0.5 mg/kg to about 1 mg/kg. In some
embodiments, the reference therapeutically effective amount is
within the range of about 1 mg/kg to about 2.5 mg/kg. In some
embodiments, the reference therapeutically effective amount is
within the range of about 2.5 mg/kg to about 10 mg/kg. In some
embodiments, the reference therapeutically effective amount is
within the range of about 10 mg/kg to about 50 mg/kg. In some
embodiments, the reference therapeutically effective amount is
within the range of about 50 mg/kg to about 100 mg/kg. In some
embodiments, the reference therapeutically effective amount is
within the range of about 100 mg/kg to about 250 mg/kg. In some
embodiments, the reference therapeutically effective amount is
within the range of about 250 mg/kg to about 500 mg/kg.
[0018] Treat or Treating. The terms "treat" or "treating," as used
herein, refer to partially or completely alleviating, inhibiting,
delaying onset of, reducing the incidence of, yielding prophylaxis
of, ameliorating and/or relieving a disorder, disease, or
condition, or one or more symptoms or manifestations of the
disorder, disease or condition.
[0019] Unit Dose. The expression "unit dose" as used herein refers
to a physically discrete unit of a formulation appropriate for a
subject to be treated (e.g., for a single dose); each unit
containing a predetermined quantity of an active agent selected to
produce a desired therapeutic effect when administered according to
a therapeutic regimen (it being understood that multiple doses may
be required to achieve a desired or optimum effect), optionally
together with a pharmaceutically acceptable carrier, which may be
provided in a predetermined amount. The unit dose may be, for
example, a volume of liquid (e.g., an acceptable carrier)
containing a predetermined quantity of one or more therapeutic
agents, a predetermined amount of one or more therapeutic agents in
solid form, a sustained release formulation or drug delivery device
containing a predetermined amount of one or more therapeutic
agents, etc. It will be appreciated that a unit dose may contain a
variety of components in addition to the therapeutic agent(s). For
example, acceptable carriers (e.g., pharmaceutically acceptable
carriers), diluents, stabilizers, buffers, preservatives, etc., may
be included as described infra. It will be understood, however,
that the total daily usage of a formulation of the present
invention will be decided by the attending physician within the
scope of sound medical judgment. The specific effective dose level
for any particular subject or organism may depend upon a variety of
factors including the disorder being treated and the severity of
the disorder; activity of specific active compound employed;
specific composition employed; age, body weight, general health,
sex and diet of the subject; time of administration, and rate of
excretion of the specific active compound employed; duration of the
treatment; drugs and/or additional therapies used in combination or
coincidental with specific compound(s) employed, and like factors
well known in the medical arts. In some embodiments, a unit dose of
a p38 inhibitor is about 1 mg, 3 mg, 5 mg, 10 mg, 15 mg, 20 mg, 25
mg, 30 mg, 35 mg, 40 mg, 45 mg or 50 mg.
[0020] Pathology of Alzheimer's Disease
[0021] Alzheimer's disease pathology is characterized by the
deposition of extracellular amyloid plaques in the brain parenchyma
and neurofibrillary tangles within neurons.
[0022] The primary component of extracellular amyloid plaques found
in the brains of Alzheimer's disease patients is abnormally folded
beta-amyloid protein (A.beta.), a 36- to 43-amino acid peptide
produced by proteolysis of amyloid precursor protein (APP) by
enzymes known as secretases. APP is an integral membrane protein
expressed in many tissues and concentrated in the synapses of
neurons. Its primary function is not known, though it has been
implicated as a regulator of synapse formation, neural plasticity
and iron export. The most common isoforms of A.beta. are
A.beta..sub.40 and A.beta..sub.42; the shorter form is typically
produced by cleavage that occurs in the endoplasmic reticulum,
while the longer form is produced by cleavage in the trans-Golgi
network. The A.beta..sub.40 form is the more common of the two, but
A.beta..sub.42 is the more fibrillogenic and is thus associated
with disease states. Mutations in APP associated with early-onset
Alzheimer's have been noted to increase the relative production of
A.beta..sub.42, and thus one suggested avenue of Alzheimer's
therapy involves modulating the activity of .beta.- and
.gamma.-secretases to produce mainly A.beta..sub.40.
[0023] In contrast, neurofibrillary tangles are intracellular
aggregates of microtubule-associated protein tau (MAPT). Tau
proteins, abundant in neurons in the central nervous system but
less common elsewhere, stabilize microtubules. Hyperphosphorylated
tau (hTau) associates with other threads of tau, eventually forming
neurofibrillary tangles inside nerve cell bodies. When this occurs,
the microtubules disintegrate, collapsing the neuron's transport
system, cite_note-pmid15615638-46 resulting in malfunctions in
biochemical communication between neurons and, eventually, cell
death.
[0024] Recent evidence suggests that neuroinflammatory processes
also contribute to the pathophysiology of Alzheimer's disease. See,
e.g., Hull et al., "Pathways of Inflammatory Activation in
Alzheimer's Disease: Potential Targets for Disease Modifying
Drugs," Curr. Med. Chem. 2002, 9, 83-88, the entirety of which is
incorporated herein by reference. Microglia, the resident
inflammatory cells of the brain, are found in a highly activated
state in the Alzheimer's disease brain, including morphological
alterations, proliferation, increased expression of cell surface
receptors, and secretion of inflammatory cytokines and chemokines
Microglia fulfill numerous different tasks within the central
nervous system (CNS) related to both immune response and the
maintenance of homeostasis. The main role of microglia is
phagocytosis, or the engulfing of various materials. Engulfed
materials include damaged neurons, plaques, cellular debris and
infectious agents such as viruses and bacteria. Microglia
accumulate at the site of newly formed A.beta. deposits in the
Alzheimer's disease brain and may help restrict plaque growth by
degrading A.beta..
[0025] Recent studies have also shown that overexpression of
IL-1.beta., an inflammatory cytokine, leads to reduced A.beta.
pathology in mouse models of Alzheimer's disease. However,
chronically activated microglia are also associated with
inflammatory cytokines including TNF.alpha. that can substantially
block the ability of the microglia to remove or degrade A.beta..
Thus, the role of microglia in the pathophysiology of Alzheimer's
disease is complex, with microglial activation exerting either a
beneficial or detrimental effect depending on local conditions.
[0026] One signaling pathway through which neurons and microglia
communicate is fractalkine (CX3CL1) and its cognate receptor
(CX3CR1), a unique, one-to-one ligand-receptor chemokine pair.
CX3CL1-CX3CR1 signaling has been demonstrated to play an important
role in neuroinflammation and neuroprotection. Notably, CX3CL1 is
highly expressed in neurons while CX3CR1 is exclusively expressed
in microglia. One recent study demonstrated that the inhibition or
deletion of the microglial receptor CX3CR1 leads to an amelioration
of the amyloid pathology in both rapid onset and gradual onset
transgenic mouse models of Alzheimer's disease. See Lee et al.,
"CX3CR1 Deficiency Alters Microglial Activation and Reduces
Beat-Amyloid Deposition in Two Alzheimer's Disease Mouse Models,"
The American Journal of Pathology, 177(5): 2549-2562 (2010), the
entirety of which is incorporated herein by reference. In fact,
CX3CR1-deficient mice exhibited a dose-dependent reduction in
A.beta. deposition in the APPPS1 mouse model of Alzheimer's
disease, suggesting that CX3CR1 deficiency harnesses the beneficial
effects of microglial activation in response to A.beta.. Moreover,
the number of plaque-associated microglia were decreased in the
knockout mice as compared to control. However, despite the
reduction in the number of microglia around the A.beta. deposits in
the CX3CR1-deficient animals, there was observed a significant
reduction in A.beta. deposition, consistent with an enhanced
capacity of microglia to remove A.beta.. Thus, CX3CR1 signaling
appears to inhibit microglial phagocytosis and prevent effective
A.beta. clearance. These results suggest that alterations in
CX3CL1-CX3CR1 signaling can lead to altered phagocytic capabilities
of microglia.
[0027] Microglial neuroinflammation also promotes MAPT
phosphorylation and aggregation through the overexpression of IL-1.
Recently, CX3CR1 deficiency has been shown to result in both
enhanced microglial activation and MAPT phosphorylation/aggregation
in humanized tau mice. Researchers observed that transgenic
humanized tau mice first develop hyperphosphorylated MAPT at 3
months of age, MAPT aggregates at 9 months of age, and neuronal
loss by 15 months of age. Significantly, by 12 months of age,
humanized tau mice exhibited microglia in the hippocampus with
shorter processes and rounder cell bodies consistent with
microglial activation.
[0028] Without wishing to be bound by any particular theory, it is
believed that the activation of microglia in response to such
inflammatory signals, particularly neuroinflammatory processes,
delays the accumulation of beta amyloid plaques associates with
Alzheimer's disease. Thus, inhibition or suppression of
inflammatory cascades is likely to prevent microglial activation,
leading to an increase in accumulation of beta amyloid plaques and
the progression of Alzheimer's disease.
[0029] p38 MAPK Inhibitors
[0030] Many extracellular stimuli, including pro-inflammatory
cytokines and other inflammatory mediators, elicit specific
cellular responses through the activation of mitogen-activated
protein kinase (MAPK) signaling pathways. MAPKs are
proline-targeted serine-threonine kinases that transduce
environmental stimuli to the nucleus. Once activated, MAPKs
activate other kinases or nuclear proteins through phosphorylation,
including potential transcription factors and substrates. The novel
mammalian reactivating protein kinase (p38/RK) MAPKs are
stress-activated protein kinases that mediate responses to cellular
stresses and inflammatory signals.
[0031] p38 MAPK activation occurs in the very early stages of
Alzheimer's disease and is an important contributor to the
inflammation of the brain. See, e.g., Bhasker et al., "Regulation
of Tau Pathology by the Microglial Fractalkine Receptor," Neuron
68:19-31 (2010), the entirety of which is incorporated herein by
reference. In fact, beta-amyloid fibrils in microglia stimulate
rapid, transient activation of p38 MAPK resulting in inflammatory
gene expression and upregulation of proinflammatory cytokines.
Thus, activation of the p38 MAPK pathway attenuates plaque
accumulation and stimulates microglial plaque degradation.
[0032] Moreover, researchers confirmed that enhancement of MAPT
phosphorylation could be blocked by preincubating neurons in vitro
with a specific MAPK inhibitor, SB203580, indicating that the
enhancement of MAPT phosphorylation occurred via a p38
MAPK-dependent pathway. Thus, research demonstrates that the role
of p38 MAPK in Alzheimer's disease is complex, as it both
stimulates microglial degradation of A.beta. plaques while
simultaneously promoting MAPT phosphorylation, a process which can
lead to neurofibrillary tangles and loss of neuronal function. See,
e.g., Munoz, et al., "Targeting p38 MAPK pathway for the treatment
of Alzheimer's disease," Neuropharmacology, 58(3):561-568 (2010),
incorporated herein by reference in its entirety.
[0033] The role of p38 MAPK in the various stages of inflammation
has prompted the discovery of several compounds capable of
inhibiting p38 (SB203580, RWJ 67657, L-167307, VX-745, RPR200765A
and others). See, e.g., Kumar et al., "p38 MAP Kinases: Key
Signaling Molecules as Therapeutic Targets for Inflammatory
Diseases," Nature Reviews, 2:717-726 (2003); Brown et al., "p38 MAP
kinase inhibitors as potential therapeutics for the treatment of
joint degeneration and pain associated with osteoarthritis," J.
Inflammation 5:22 (2008), the entirety of each of which is
incorporated herein by reference. These pharmacological inhibitors
are cytokine-suppressive anti-inflammatory drugs responsible for in
vitro and in vivo inhibition of lipopolysaccharide-induced tumor
necrosis factor-.alpha. (TNF-.alpha.) expression. Although p38 MAPK
inhibitors have long peaked the interest of Alzheimer's disease
researchers, the complexity of the disease has limited the use of
such agents. More particularly, while p38 inhibitors block tau
phosphorylation, the resulting decrease in the inflammatory
cascades are expected to increase A.beta. plaque accumulation due
to the lack of microglial activation.
[0034] It has now been surprisingly found that p38 MAPK inhibitors
reduce amyloid plaque burden within the central nervous system
(CNS). Accordingly, the present invention encompasses the
recognition that p38 MAPK inhibitors are effective for reducing
amyloid plaque burden associated with Alzheimer's disease. In some
embodiments, the present invention provides a method of reducing
amyloid plaque burden within the central nervous system (CNS). In
some embodiments, the present invention provides a method of
reducing amyloid plaque burden associated with Alzheimer's disease
comprising administering to a patient in need thereof a p38 MAPK
inhibitor.
[0035] Exemplary p38 MAPK Inhibitors
[0036] As generally described above, there has been extensive
research directed towards the discovery of p38 MAPK inhibitors for
the treatment of the various stages of inflammation.
[0037] Exemplary p38 MAPK inhibitors can be found, for example, in
Mayer et al., "p38 MAP kinase inhibitors: A future therapy for
inflammatory diseases," Drug Discovery Today: Therapeutic
Strategies 3(1): 49-54 (2006); and Regan et al., "Pyrazole
Urea-Based Inhibitors of p38 MAP Kinase: from Lead Compound to
Clinical Candidate," J. Med. Chem. 2002, 45, 2994-3008, the
entirety of each of which are incorporated herein by reference.
Table 1 lists representative p38 MAPK inhibitors.
TABLE-US-00001 TABLE 1 ##STR00001## ##STR00002## ##STR00003##
##STR00004## ##STR00005## ##STR00006## ##STR00007## ##STR00008##
##STR00009## ##STR00010## ##STR00011## ##STR00012## ##STR00013##
##STR00014## ##STR00015## ##STR00016## ##STR00017## ##STR00018##
##STR00019## ##STR00020## ##STR00021## ##STR00022## ##STR00023##
##STR00024## ##STR00025## ##STR00026## ##STR00027## ##STR00028##
##STR00029## ##STR00030## ##STR00031## ##STR00032## ##STR00033##
##STR00034## ##STR00035## ##STR00036## ##STR00037## ##STR00038##
##STR00039##
[0038] VX-745 is a selective small-molecule inhibitor of p38 MAPK
developed by Vertex Pharmaceuticals for the treatment of rheumatoid
arthritis (RA). The inhibition of MAPK by VX-745 blocks the
downstream synthesis of inflammatory cytokines TNF-.alpha.,
IL-1.beta. and IL-6. Because VX-745 exhibited significant
anti-inflammatory activity in rodent arthritis models, Vertex
initiated a clinical trial in human rheumatoid arthritis (RA).
However, patients treated with 250 mg VX-745 b.i.d. experienced
adverse events, including gastrointestinal effects such as diarrhea
and abdominal pain, and elevations in liver transaminases.
Moreover, VX-745 is known to penetrate the blood brain barrier
(BBB) in animals. In fact, animals subjected to high doses of
VX-745 experienced adverse neurological effects, although these
adverse events were not observed in humans. Despite validating the
proof-of-concept for the inhibition of p38 MAPK as a treatment for
RA, VX-745 was discontinued due to the potential for serious
adverse events.
[0039] Another study utilizing VX-745 as a reference compound in an
arthritis model demonstrated that a 10 mg/kg dose of VX-745 was not
as effective at inhibiting paw swelling as other compounds assayed.
See Chopra et al., "Pharmacological profile of AW-814141, a novel,
potent, selective and orally active inhibitor of p38 MAP kinase,"
International Immunopharmacology, 10: 467-473 (2010), the entirety
of which is incorporated herein by reference.
[0040] In an osteoarthritis model, VX-745 showed statistically
significant inhibition of knee degeneration compared to control
animals when administered to rats at 50 mg/kg. VX-745 was also
assayed in a hyperalgesia model and showed significant inhibition
of hyperalgesic response when administered to rats at doses of 30
mg/kg, 10 mg/kg and 3 mg/kg. The researchers discovered that the
mice exhibited hyperalgesia at the 3 mg/kg, 10 mg/kg and 30 mg/kg
doses. However, the researchers observed minimal effect at the 3
mg/kg dose. See Brown et al., "p38 MAP kinase inhibitors as
potential therapeutics for the treatment of joint degeneration and
pain associated with osteoarthritis," J. Inflamm., 5:22 (2008), the
entirety of which is incorporated herein by reference. Without
wishing to be bound by theory, it is believed that the clinical
failures of p38 inhibitors to treat chronic conditions such as
rheumatoid arthritis are due to redundancy of the inflammatory
pathway. Such redundancy results in the upregulation of feedback
loops when p38 is chronically inhibited, leading to an overall lack
of efficacy.
[0041] Methods of the Invention
[0042] As described above, in some embodiments, the present
invention provides a method of reducing amyloid plaque burden
within the CNS. In some embodiments, a method of reducing amyloid
plaque burden comprises administering to a patient in need thereof
a p38 MAPK inhibitor.
[0043] In some embodiments, a method of reducing amyloid plaque
burden comprises administering to a patient in need thereof a
selective p38 MAPK inhibitor.
[0044] In certain embodiments, the present invention provides a
method of reducing amyloid plaque burden by administering to a
patient in need thereof a low dose of a p38 MAPK inhibitor. In some
embodiments, a method of reducing amyloid plaque burden comprises
administering to a patient in need thereof VX-745. In some such
embodiments, a method of reducing amyloid plaque burden comprises
administering to a patient in need thereof a low dose of
VX-745.
[0045] An agent's therapeutic efficacy is affected by the degree to
which it binds blood plasma proteins. Only the fraction of unbound
agent exhibits any pharmacological effect because protein-bound
agents cannot traverse cell membranes or diffuse throughout the
body. Thus, the more highly bound a therapeutic agent is, the lower
the concentration of the agent available to elicit the desired
pharmacological response. However, because there is less protein in
the brain, a therapeutic agent which is capable of crossing the
blood-brain barrier will have a higher concentration of free agent
available to elicit the desired pharmacological response. Indeed,
although it is known that VX-745 is a highly protein-bound agent,
its brain levels in dogs is twice that of systemic levels.
[0046] In some embodiments, the present invention provides a method
of reducing amyloid plaque burden associated with Alzheimer's
disease comprising administering to a patient in need thereof a low
dose of a p38 MAPK inhibitor. In some embodiments, the present
invention provides a method of reducing amyloid plaque burden
associated with Alzheimer's disease comprising administering to a
patient in need thereof VX-745. In some embodiments, the present
invention provides a method of reducing amyloid plaque burden
associated with Alzheimer's disease comprising administering to a
patient in need thereof a low dose of VX-745.
[0047] In some embodiments, the present invention provides a method
of (i) reducing plaque burden and (ii) inhibiting MAPT
phosphorylation. In some embodiments, a method of (i) reducing
plaque burden and (ii) inhibiting MAPT phosphorylation comprises
administering to a patient in need thereof a p38 MAPK inhibitor. In
certain embodiments, a method of (i) reducing plaque burden and
(ii) inhibiting MAPT phosphorylation comprises administering to a
patient in need thereof a low dose of a p38 MAPK inhibitor. In some
embodiments, a method of (i) reducing plaque burden and (ii)
inhibiting MAPT phosphorylation comprises administering to a
patient in need thereof VX-745. In certain embodiments, a method of
(i) reducing plaque burden and (ii) inhibiting MAPT phosphorylation
comprises administering to a patient in need thereof a low dose of
VX-745.
[0048] As discussed above, while IL-1.beta. overexpression enhances
A.beta. clearance, chronic activation of microglia reduces the
ability of microglia to degrade A.beta.. Thus, in some embodiments,
the present invention provides a method of reducing plaque burden
without inducing neuroinflammation. In some embodiments, a method
of reducing plaque burden without inducing neuroinflammation
comprises administering to a patient in need thereof a p38 MAPK
inhibitor. In some embodiments, a method of reducing plaque burden
without inducing neuroinflammation comprises administering to a
patient in need thereof a low dose of a p38 MAPK inhibitor. In some
embodiments, a method of reducing plaque burden without inducing
neuroinflammation comprises administering to a patient in need
thereof VX-745. In some embodiments, a method of reducing plaque
burden without inducing neuroinflammation comprises administering
to a patient in need thereof a low dose of VX-745.
[0049] In some embodiments, the present invention provides a method
of reducing plaque burden without increasing expression and/or
levels of inflammatory cytokines. In some embodiments, a method of
reducing plaque burden without increasing expression and/or levels
of inflammatory cytokines comprises administering to a patient in
need thereof a p38 MAPK inhibitor. In some embodiments, a method of
reducing plaque burden without increasing expression and/or levels
of inflammatory cytokines comprises administering to a patient in
need thereof a low dose of a p38 MAPK inhibitor. In some
embodiments, a method of reducing plaque burden without increasing
expression and/or levels of inflammatory cytokines comprises
administering to a patient in need thereof VX-745. In some
embodiments, a method of reducing plaque burden without increasing
expression and/or levels of inflammatory cytokines comprises
administering to a patient in need thereof a low dose of
VX-745.
[0050] In some embodiments, the present invention provides a method
of reducing plaque burden without increasing IL-1.beta. expression
and/or levels. In some embodiments, a method of reducing plaque
burden without increasing IL-1.beta. expression and/or levels
comprises administering to a patient in need thereof a p38 MAPK
inhibitor. In certain embodiments, the present invention provides a
method of reducing plaque burden without increasing IL-1.beta.
expression and/or levels by administering to a patient in need
thereof a low dose of a p38 MAPK inhibitor. In some embodiments, a
method of reducing plaque burden without increasing IL-1.beta.
expression and/or levels comprises administering to a patient in
need thereof VX-745. In certain embodiments, the present invention
provides a method of reducing plaque burden without increasing
IL-1.beta. expression and/or levels by administering to a patient
in need thereof a low dose of VX-745.
[0051] In some embodiments, the present invention provides for the
administration of a p38 inhibitor, for example VX-745, once a day,
twice a day, once a week, twice a week or once a month. In some
embodiments, the present invention provides for the administration
of a p38 inhibitor at more frequent intervals, such as one, two,
three or four times per day, for up to one, two or three or more
weeks, followed by a tapered dosing schedule to maintain the
desired level of the p38 inhibitor. In some embodiments, the
present invention provides for the administration of a p38
inhibitor at intervals of one, two, three or four times per day,
for up to one, two or three or more months, followed by a tapered
dosing schedule to maintain the desired level of the p38 inhibitor.
More particularly, in some embodiments, the present invention
provides a dosing schedule for the administration of a p38
inhibitor at intervals sufficient to achieve therapeutic levels in
the brain, followed by a tapering of the dosage.
[0052] In some embodiments, the present invention provides a method
of reducing the number and/or volume of amyloid plaques in a
patient suffering from Alzheimer's disease comprising administering
to the patient a p38 inhibitor. In some embodiments, the present
invention provides a method of reducing the number and/or volume of
amyloid plaques in a patient suffering from Alzheimer's disease
comprising administering to the patient a low dose of a p38
inhibitor. In some embodiments, the present invention provides a
method of reducing the number and/or volume of amyloid plaques in a
patient suffering from Alzheimer's disease comprising administering
to the patient a therapeutically effective dose of VX-745. In some
embodiments, the present invention provides a method of reducing
the number and/or volume of amyloid plaques in a patient suffering
from Alzheimer's disease comprising administering to the patient a
low dose of VX-745.
[0053] In some embodiments, the present invention provides a method
of reducing the number and/or volume of amyloid plaque
A.beta..sub.42 in a patient suffering from Alzheimer's disease
comprising administering to the patient a p38 inhibitor. In some
embodiments, the present invention provides a method of reducing
the number and/or volume of amyloid plaque A.beta..sub.42 in a
patient suffering from Alzheimer's disease comprising administering
to the patient a low dose of a p38 inhibitor. In some embodiments,
the present invention provides a method of reducing the number
and/or volume of amyloid plaque A.beta..sub.42 in a patient
suffering from Alzheimer's disease comprising administering to the
patient VX-745. In some embodiments, the present invention provides
a method of reducing the number and/or volume of amyloid plaque
A.beta..sub.42 in a patient suffering from Alzheimer's disease
comprising administering to the patient a low dose of VX-745.
[0054] In some embodiments, the present invention provides a method
of reducing the number and/or volume of a beta amyloid plaque in a
patient suffering from Alzheimer's disease comprising administering
to the patient a therapeutically effective dose of a p38 inhibitor.
In some embodiments, the present invention provides a method of
reducing the number and/or volume of a beta amyloid plaque in a
patient suffering from Alzheimer's disease comprising administering
to the patient a therapeutically effective dose of VX-745. In some
embodiments, the present invention provides a method of reducing
the number and/or volume of amyloid plaque A.beta..sub.42 in a
patient suffering from Alzheimer's disease comprising administering
to the patient a low dose of a p38 inhibitor. In some embodiments,
the present invention provides a method of reducing the number
and/or volume of amyloid plaque A.beta..sub.42 in a patient
suffering from Alzheimer's disease comprising administering to the
patient a low dose of VX-745.
[0055] In some embodiments, the present invention provides a method
for preventing the accumulation of amyloid plaques comprising the
administration to a patient in need thereof a therapeutically
effective amount of a p38 inhibitor. In some embodiments, the
present invention provides a method for preventing the accumulation
of amyloid plaques comprising the administration to a patient in
need thereof a therapeutically effective amount of a low dose of a
p38 inhibitor. In some embodiments, the present invention provides
a method for preventing the accumulation of amyloid plaques
comprising the administration to a patient in need thereof a
therapeutically effective amount of VX-745. In some embodiments,
the present invention provides a method for preventing the
accumulation of amyloid plaques comprising the administration to a
patient in need thereof a therapeutically effective amount of a low
dose of VX-745.
[0056] In some embodiments, the present invention provides a method
of reducing the number and/or volume of amyloid plaques in a
patient suffering from Alzheimer's disease comprising administering
to the patient a therapeutically effective dose of a p38 inhibitor,
wherein the therapeutically effective dose is between about 1 mg to
about 50 mg. In some embodiments, the present invention provides a
method of reducing the number and/or volume of amyloid plaques in a
patient suffering from Alzheimer's disease comprising administering
to the patient a therapeutically effective dose of a p38 inhibitor,
wherein the therapeutically effective dose is between about 1 mg to
about 20 mg. In some embodiments, the present invention provides a
method of reducing the number and/or volume of amyloid plaques in a
patient suffering from Alzheimer's disease comprising administering
to the patient a therapeutically effective dose of a p38 inhibitor,
wherein the therapeutically effective dose is between about 1 mg to
about 10 mg. In some embodiments, the present invention provides a
method of reducing the number and/or volume of amyloid plaques in a
patient suffering from Alzheimer's disease comprising administering
to the patient a therapeutically effective dose of a p38 inhibitor,
wherein the therapeutically effective dose is between about 1 mg to
about 5 mg. In some embodiments, the present invention provides a
method of reducing the number and/or volume of amyloid plaques in a
patient suffering from Alzheimer's disease comprising administering
to the patient a therapeutically effective dose of a p38 inhibitor,
wherein the therapeutically effective dose is between about 5 mg to
about 10 mg. In some embodiments, the present invention provides a
method of reducing the number and/or volume of amyloid plaques in a
patient suffering from Alzheimer's disease comprising administering
to the patient a therapeutically effective dose of a p38 inhibitor,
wherein the therapeutically effective dose is between about 10 mg
to about 20 mg. In some embodiments, the present invention provides
a method of reducing the number and/or volume of amyloid plaques in
a patient suffering from Alzheimer's disease comprising
administering to the patient a therapeutically effective dose of a
p38 inhibitor, wherein the therapeutically effective dose is
between about 20 mg to about 30 mg. In some embodiments, the
present invention provides a method of reducing the number and/or
volume of amyloid plaques in a patient suffering from Alzheimer's
disease comprising administering to the patient a therapeutically
effective dose of a p38 inhibitor, wherein the therapeutically
effective dose is between about 30 mg to about 40 mg. In some
embodiments, the present invention provides a method of reducing
the number and/or volume of amyloid plaques in a patient suffering
from Alzheimer's disease comprising administering to the patient a
therapeutically effective dose of a p38 inhibitor, wherein the
therapeutically effective dose is between about 40 mg to about 50
mg.
[0057] In some embodiments, the present invention provides an
amyloid plaque clearance mechanism comprising administering to a
subject in need thereof a p38 MAPK inhibitor.
[0058] In some embodiments, the present invention provides a method
of reducing plaque burden in a patient in need thereof, said method
comprising administering to said patient a p38 MAPK inhibitor for a
period of less than about 6 months. In some embodiments, the
present invention provides a method of reducing plaque burden in a
patient in need thereof, said method comprising administering to
said patient a p38 MAPK inhibitor for a period of less than about 4
months. In some embodiments, the present invention provides a
method of reducing plaque burden in a patient in need thereof, said
method comprising administering to said patient a p38 MAPK
inhibitor for a period of less than about 2 months. In some
embodiments, the present invention provides a method of reducing
plaque burden in a patient in need thereof, said method comprising
administering to said patient a p38 MAPK inhibitor for a period of
less than about 1 month. In some embodiments, the present invention
provides a method of reducing plaque burden in a patient in need
thereof, said method comprising administering to said patient a p38
MAPK inhibitor for a period of less than about 2 weeks.
[0059] In some embodiments, the present invention provides a method
of reducing amyloid plaque burden, said method comprising: [0060]
(i) imaging the brain of a subject to produce a neuroimage; [0061]
(ii) comparing the neuroimage to a reference image to determine the
number and/or area of the amyloid plaques; and [0062] (iii)
administering a therapeutically effective amount of a p38 inhibitor
if the subject is determined to have an increased amount of amyloid
plaques when compared to the reference image.
[0063] In some embodiments, the present invention provides a method
of reducing amyloid plaque burden, said method comprising: [0064]
(i) imaging the brain of a subject; [0065] (ii) determining the
number and/or area of the amyloid plaques; and [0066] (iii)
administering a therapeutically effective amount of a p38 inhibitor
if the number and/or area of the amyloid plaques exceeds a
predetermined threshold.
[0067] In some embodiments, the reference image is an image of a
control subject. In some embodiments, the reference image is an
image of a subject having normal cognitive function. In some
embodiments, the reference image is a baseline image of the
subject's brain. In some such embodiments, the reference image is a
prior scan of the subject's brain. In some embodiments, the subject
is at risk for developing Alzheimer's disease.
[0068] In some embodiments, steps of (i) imaging, (ii) comparing
amyloid plaques to a reference image and/or determining the number
and/or area of amyloid plaques and (iii) administering a p38
inhibitor are repeated at one or more predetermined intervals. In
some such embodiments, a predetermined interval is one month, two
months, three months, four months, five months, six months, seven
months, eight months, nine months, ten months, eleven months,
twelve months. In some embodiments, a predetermined interval is one
year, two years, three years, four years or five years. In some
embodiments, the predetermined interval is six (6) months.
[0069] In some embodiments, the subject is a patient at risk of
developing or suffering from Alzheimer's disease.
[0070] In some embodiments, the brain of a subject is imaged using
one or more neuroimaging techniques. In some embodiments, the
neuroimaging technique is selected from the group consisting of
computerized axial tomography (CAT or CT), single photon emission
computed tomography (SPECT), positron emission tomography (PET),
magnetic resonance imaging (MRI) or functional magnetic resonance
imaging (fMRI). In some embodiments, the neuroimaging technique is
computerized axial tomography (CAT or CT). In some embodiments, the
neuroimaging technique is positron emission tomography (PET). In
some such embodiments, the imaging agent used in the PET scan is
selected from amyvid or Pittsburgh compound B. In some embodiments,
the neuroimaging technique is magnetic resonance imaging (MRI). In
some embodiments, the neuroimaging technique is functional magnetic
resonance imaging (fMRI).
[0071] A person of ordinary skill understands how to determine or
measure the number and/or area of the amyloid plaques in a
neuroimage. For example, see Zeman et al., "Diagnosis of Dementia
Using Nuclear Medicine Imaging Modalities," Chapter 8, 12 Chapters
on Nuclear Medicine, Gholamrezanezhad, Ed., 199-229 (Dec. 22, 2011)
and Hsiao et al., "Correlation of early-phase .sup.18F-florbetapir
(AV-45/Amyvid) PET images to FDG images: preliminary studies,"
European Journal of Nuclear Medicine and Molecular Imaging, 39(4),
613-620 (2012), the entirely of each of which is hereby
incorporated by reference in its entirely.
[0072] In some embodiments, a p38 inhibitor is administered for a
period of less than six (6) months. In some embodiments, a p38
inhibitor is administered for a period of less than four (4)
months. In some embodiments, a p38 inhibitor is administered for a
period of less than two (2) months. In some embodiments, a p38
inhibitor is administered for a period of less than one (1) month.
In some embodiments, a p38 inhibitor is administered for a period
of less than three (3) weeks. In some embodiments, a p38 inhibitor
is administered for a period of less than two (2) weeks. In some
embodiments, a p38 inhibitor is administered for a period of less
than one (1) week.
[0073] In some embodiments, a predetermined threshold is a baseline
for a particular subject. For example, in some embodiments, the
brain of a subject at risk for developing Alzheimer's disease is
imaged and the number and/or area of amyloid plaques is determined
and/or measured. The number and/or area of the plaques is that
subject's baseline or predetermined threshold against which all
later brain images are compared.
[0074] In some embodiments, a predetermined threshold is based on
the number and/or area of amyloid plaques typically found in an
Alzheimer's diseased brain. In some such embodiments, a
predetermined threshold is an average of the number and/or area of
amyloid plaques typically found in an Alzheimer's diseased
brain.
[0075] In some embodiments, the present invention provides a method
of reducing amyloid plaque burden in a subject suffering from or at
risk for developing Alzheimer's disease, said method comprising:
[0076] (iv) imaging the brain of a subject; [0077] (v) determining
the number and/or area of the amyloid plaques; and [0078] (vi)
administering a therapeutically effective amount of a p38 inhibitor
if the number and/or area of the amyloid plaques exceeds a
predetermined threshold.
[0079] In some embodiments, the present invention provides a method
of treating a subject suffering from amyloid plaques, wherein the
number and/or area of the amyloid plaques exceeds a predetermined
threshold, said method comprising administering to the subject a
therapeutically effective amount of a p38 inhibitor.
[0080] In some embodiments, the present invention provides a method
of treating a subject suffering from amyloid plaques, wherein the
number and/or area of the amyloid plaques exceeds a predetermined
threshold, said method comprising: [0081] (i) administering a
therapeutically effective amount of a p38 inhibitor for a period of
less than six (6) months; [0082] (ii) imaging the brain of the
subject at regular intervals; and [0083] (iii) administering a
therapeutically effective amount of a p38 inhibitor if the number
and/or area of the amyloid plaques exceeds the previously measured
amyloid plaque level.
[0084] In some such embodiments, the subject is administered a p38
inhibitor for a period of less than four (4) months, less than two
(2) months, less than one (1) month, or less than two (2)
weeks.
[0085] In some embodiments, the present invention provides a method
of treating a subject suffering from amyloid plaques, wherein the
number and/or area of the amyloid plaques exceeds a predetermined
threshold as measured by one or more neuroimaging techniques, said
method comprising administering a therapeutically effective amount
of a p38 inhibitor for a period of less than six (6) months. In
some such embodiments, the neuroimaging technique is a PET scan. In
some embodiments, the method further comprises (i) imaging the
brain of the subject at regular intervals; and (ii) administering a
therapeutically effective amount of a p38 inhibitor if the number
and/or area of the amyloid plaques exceeds the previously measured
amyloid plaque level.
[0086] Combination Therapies
[0087] In certain embodiments, the present invention provides a
method of treating Alzheimer's disease comprising administering to
a subject a therapeutically effective amount of a p38 inhibitor
together with one or more additional therapeutic agents. In some
embodiments, the present invention provides a method of treating
Alzheimer's disease comprising administering to a subject a
therapeutically effective amount of a p38 inhibitor together with
one or more additional therapeutic agents selected from
cholinesterase inhibitors, N-methyl-D-aspartate antagonists,
vitamin E, antidepressants, anxiolytics, antipsychotics, mood
stabilizers and sleep aids.
[0088] Representative cholinesterase inhibitors include, without
limitation, donepezil (Aricept.RTM.), rivastigmine (Exelon.RTM.),
galantamine (Razadyne.RTM.) and tacrine (Cognex.RTM.).
[0089] Representative antidepressants include, without limitation,
bupropion (Wellbutrin.RTM.), citalopram (Celexa.RTM.), fluoxetine
(Prozac.RTM.), mirtazapine (Remeron.RTM.), paroxetine (Paxil.RTM.),
sertraline (Zoloft.RTM.), trazodone (Desyrel.RTM.), venlafaxine
(Effexor.RTM.), nortriptyline (Pamelor.RTM.) and desipramine
(Norpramine.RTM.).
[0090] Representative anxiolytics include, without limitation,
lorazepam (Ativan.RTM.) and oxazepam (Serax.RTM.).
[0091] Representative antipsychotics include, without limitation,
aripiprazole (Abilify.RTM.), clozapine (Clozaril.RTM.), haloperidol
(Haldol.RTM.), olanzapine (Zyprexa.RTM.), quetiapine
(Seroquel.RTM.), risperidone (Risperdal.RTM.) and ziprasidone
(Geodon.RTM.).
[0092] Representative mood stabilizers include, without limitation,
carbamazepine (Tegretol.RTM.) and divalproex (Depakota.RTM.).
[0093] Representative sleep aids include, without limitation,
zolpidem, zaleplon and chloral hydrate.
[0094] Representative N-methyl-D-aspartate antagonists include,
without limitation, memantine (Namenda.RTM.).
[0095] In some embodiments, the present invention provides a method
of treating Alzheimer's disease comprising administering to a
subject a therapeutically effective amount of a p38 inhibitor
together with one or more additional therapeutic agents selected
from the group consisting of exenatide (Byetta.RTM.), varenicline,
PF-04360365, rivastigmine, LY450139, ST101, bryostatin, EVP-6124,
atomoxetine, HF0220, resveratrol, galantamine, PF-01913539,
semagacestat, 3APS, immunoglobulin, dimebon, alpha-tocopherol,
BAY85-8101, estrogen, progesterone, ACC-001, ginko biloba,
nicergoline, piracetam, NIC5-15, xaliproden (SR57746A),
indomethacin, DMXB-A, LY2062430, 11-C PIB, bapineuzumab,
etanercept, ramipril, interferon beta-1a, simvastatin, lipoic acid,
fish oil, curcumin, PF-04447943, folate, vitamin B6, vitamin B12,
leuprolide, INM-176, AH110690, tryptophan, SK-PC-B70M, BMS-708163,
escitalopram, TRx0014, BAY94-9172, cerebrolysin,
epigallocatechin-galate, SB-742457, lithium, rosiglitazone,
divalproex, SAR110894D, PRX-03140, CX516 (Ampalex), nicotinamide,
rasagiline, AC-1202 (Ketasyn.RTM.), enduramide, neramexane,
razadyne, NS 2330 (Tesofensine.RTM.), tamibarotene, acitretin,
methylphenidate, mifepristone, ZT-1, AFFITOPE AD01, AFFITOPE AD02,
GSK239512, GSK933776, SR57667B, PPI-1019, MPC-7869, AZD3480,
PAZ-417, solanezumab, masitinib (AB1010), BAY1006578,
docosahexaenoic acid, QS-21, MNI-558, reminyl retard, flutemetamol,
estradiol, medroxyprogesterone, valproate, T-817MA, AZD1446,
AAB-003 (PF-05236812), modafinil, raloxifene, atorvastatin,
doxycycline, trazadone, sodium oxybate, huperzine A, lutein,
zeaxanthin, AC-3933, dextroamphetamine, EPAX 1050TG, SRA-333,
MNI-168, CAD106, SGS742, NP031112, SSR180711C, GSI-953, prazosin,
MEM 1003, AndroGel, AVE1625, cyclophosphamate, TC-5619-238, MK0249,
lecozotan, circadin, MEM 3454, PPI-1019, UB 311, PF-04494700,
ABT-089, LY451395, E2020, Rofecoxib, PF-03654746, EHT 0202
etazolate, DCB-AD1, ONO-2506PO, EGb761.RTM., gantenerumab,
florbetapir, ELND005, prednisone, novasoy, ginseng, pioglitazone,
caprylidene, ABT-288, ABT-384, nefiracetam, AQW051, Pitavastatin,
naproxen sodium (Aleve.RTM.), lornoxicam, AN-1792, SR57667B,
melatonin, SAM-531, MK0952, MK0677, IFN-alpha2A, BAY 94-9172,
PYM50028, lecozotan SR, thalidomide, tramiprosate, FK962, IVIG,
RO5313534, bifeprunox, LNK-754, ELND005, NSA-789, ramelteon,
Florbetaben, SRA-444, VP4896, celecoxib, hydrocodone, GSI-136,
Zolpidem, MK3328, metformin, CTS21166, elontril, ibuprofen,
posiphen tartrate, JNJ-39393406, testosterone, BRL-049653,
BMS-708163, SAM-315, ketoconazole, fluconazole, warfarin, E2609,
AZD0328, LY2886721, CHF 5074, E2212, acetaminophen, LY2811376,
ABT-126, melatonin, GSK1034702, armodafinil, depakote, gemfibrozil,
AL-108, levetiracetam, and quinacrine.
[0096] Pharmaceutical Compositions
[0097] In some embodiments, the present invention provides a
pharmaceutical composition comprising a p38 MAPK inhibitor together
with one or more therapeutic agents and a pharmaceutically
acceptable carrier, adjuvant, or vehicle. In some embodiments, the
present invention provides a pharmaceutical composition comprising
a low dose of a p38 MAPK inhibitor together with one or more
therapeutic agents and a pharmaceutically acceptable carrier,
adjuvant, or vehicle. In some embodiments, the present invention
provides a pharmaceutical composition for treating Alzheimer's
disease comprising a p38 inhibitor and one or more pharmaceutically
acceptable excipients. In some embodiments, the present invention
provides a pharmaceutical composition for treating Alzheimer's
disease comprising a p38 inhibitor selected from VX-702, VX-745,
BIRB 796, TAK-715, SCIO 469, RWJ 67657, SB 681323, SB 242235, SB
203580, L-167307, RPR-203494, RPR-200765A, PD 169316, SB 200025, JX
401, CMPD1, SKF 86002, SX 011, SD 282, EO 1428, SD 169, SB 220025,
SB 202190, SB 239063, Org 48762-0, LY2228820, vinorelbine,
PH-797804 and asiatic acid, and one or more pharmaceutically
acceptable excipients. In some such embodiments, a pharmaceutical
composition for treating Alzheimer's disease comprises a low dose
p38 inhibitor. In some embodiments, the present invention provides
a pharmaceutical composition for treating Alzheimer's disease
comprising VX-745. In some such embodiments, a pharmaceutical
composition comprises a low dose of VX-745.
[0098] In some embodiments, the present invention provides a
pharmaceutical composition comprising VX-745 together with one or
more therapeutic agents and a pharmaceutically acceptable carrier,
adjuvant, or vehicle. In some embodiments, the present invention
provides a pharmaceutical composition comprising a low dose of
VX-745, one or more therapeutic agents and a pharmaceutically
acceptable carrier, adjuvant, or vehicle. In some embodiments, the
present invention provides a pharmaceutical composition comprising
a low dose of VX-745, one or more therapeutic agents selected from
donepezil (Aricept.RTM.), rivastigmine (Exelon.RTM.), galantamine
(Razadyne.RTM.), tacrine (Cognex.RTM.), bupropion
(Wellbutrin.RTM.), citalopram (Celexa.RTM.), fluoxetine
(Prozac.RTM.), mirtazapine (Remeron.RTM.), paroxetine (Paxil.RTM.),
sertraline (Zoloft.RTM.), trazodone (Desyrel.RTM.), venlafaxine
(Effexor.RTM.), nortriptyline (Pamelor.RTM.), desipramine
(Norpramine.RTM.), lorazepam (Ativan.RTM.), oxazepam (Serax.RTM.),
aripiprazole (Abilify.RTM.), clozapine (Clozaril.RTM.), haloperidol
(Haldol.RTM.), olanzapine (Zyprexa.RTM.), quetiapine
(Seroquel.RTM.), risperidone (Risperdal.RTM.), ziprasidone
(Geodon.RTM.), carbamazepine (Tegretol.RTM.), divalproex
(Depakota.RTM.), zolpidem, zaleplon, chloral hydrate, memantine
(Namenda.RTM.), exenatide (Byetta.RTM.), varenicline, PF-04360365,
rivastigmine, LY450139, ST101, bryostatin, EVP-6124, atomoxetine,
HF0220, resveratrol, galantamine, PF-01913539, semagacestat, 3APS,
immunoglobulin, dimebon, alpha-tocopherol, BAY85-8101, estrogen,
progesterone, ACC-001, ginko biloba, nicergoline, piracetam,
NIC5-15, xaliproden (SR57746A), indomethacin, DMXB-A, LY2062430,
11-C PIB, bapineuzumab, etanercept, ramipril, interferon beta-1a,
simvastatin, lipoic acid, fish oil, curcumin, PF-04447943, folate,
vitamin B6, vitamin B12, leuprolide, INM-176, AH110690, tryptophan,
SK-PC-B70M, BMS-708163, escitalopram, TRx0014, BAY94-9172,
cerebrolysin, epigallocatechin-galate, SB-742457, lithium,
rosiglitazone, divalproex, SAR110894D, PRX-03140, CX516 (Ampalex),
nicotinamide, rasagiline, AC-1202 (Ketasyn.RTM.), enduramide,
neramexane, razadyne, NS 2330 (Tesofensine.RTM.), tamibarotene,
acitretin, methylphenidate, mifepristone, ZT-1, AFFITOPE AD01,
AFFITOPE AD02, GSK239512, GSK933776, SR57667B, PPI-1019, MPC-7869,
AZD3480, PAZ-417, solanezumab, masitinib (AB1010), BAY1006578,
docosahexaenoic acid, QS-21, MNI-558, reminyl retard, flutemetamol,
estradiol, medroxyprogesterone, valproate, T-817MA, AZD1446,
AAB-003 (PF-05236812), modafinil, raloxifene, atorvastatin,
doxycycline, trazadone, sodium oxybate, huperzine A, lutein,
zeaxanthin, AC-3933, dextroamphetamine, EPAX 1050TG, SRA-333,
MNI-168, CAD106, SGS742, NP031112, SSR180711C, GSI-953, prazosin,
MEM 1003, AndroGel, AVE1625, cyclophosphamate, TC-5619-238, MK0249,
lecozotan, circadin, MEM 3454, PPI-1019, UB 311, PF-04494700,
ABT-089, LY451395, E2020, Rofecoxib, PF-03654746, EHT 0202
etazolate, DCB-AD1, ONO-2506P0, EGb761.RTM., gantenerumab,
florbetapir, ELND005, prednisone, novasoy, ginseng, pioglitazone,
caprylidene, ABT-288, ABT-384, nefiracetam, AQW051, Pitavastatin,
naproxen sodium (Aleve.RTM.), lornoxicam, AN-1792, SR57667B,
melatonin, SAM-531, MK0952, MK0677, IFN-alpha2A, BAY 94-9172,
PYM50028, lecozotan SR, thalidomide, tramiprosate, FK962, IVIG,
RO5313534, bifeprunox, LNK-754, ELND005, NSA-789, ramelteon,
Florbetaben, SRA-444, VP4896, celecoxib, hydrocodone, GSI-136,
Zolpidem, MK3328, metformin, CTS21166, elontril, ibuprofen,
posiphen tartrate, JNJ-39393406, testosterone, BRL-049653,
BMS-708163, SAM-315, ketoconazole, fluconazole, warfarin, E2609,
AZD0328, LY2886721, CHF 5074, E2212, acetaminophen, LY2811376,
ABT-126, melatonin, GSK1034702, armodafinil, depakote, gemfibrozil,
AL-108, levetiracetam, and quinacrine, and a pharmaceutically
acceptable carrier, adjuvant, or vehicle.
[0099] In certain embodiments, pharmaceutically acceptable
compositions of this invention are formulated for oral
administration. Pharmaceutically acceptable compositions of this
invention may be orally administered in any orally acceptable
dosage form including, but not limited to, capsules, caplets,
tablets, aqueous suspensions or solutions. In the case of tablets
for oral use, carriers commonly used include lactose and corn
starch. Lubricating agents, such as magnesium stearate, are also
typically added. For oral administration in a capsule form, useful
diluents include lactose and dried cornstarch. When aqueous
suspensions are required for oral use, the active ingredient is
combined with emulsifying and suspending agents. If desired,
certain sweetening, flavoring or coloring agents may also be
added.
[0100] The quantities of the compounds of the present invention
that are combined with the carrier materials to produce a
composition in a single dosage form will vary depending upon the
patient and the particular mode of administration. Preferably,
provided compositions should be formulated so that a dosage of
between 1-50 mg/day of the p38 inhibitor (ie, VX-745 or other p38
inhibitor) can be administered to a patient receiving these
compositions. Examples of compositions include compositions
formulated to administer dosages of between 1-10 mg, 10-25 mg or
25-50 mg per day of the p38 inhibitor to the patient receiving
these compositions. In other embodiments of the invention,
compositions include compositions formulated to administer dosages
of between 3-5 mg, 5-10 mg, 10-20 mg, 20-30 mg, 30-40 mg or 40-50
mg, per day of the inhibitor to the patient receiving these
compositions. In some embodiments, the composition is formulated
into doses containing 1 mg, 3 mg, 5 mg, 10 mg, 20 mg, 25 mg, 30 mg
or 50 mg of the active composition. Dosing regimens for these
formulations may include but are not limited to single
administration dosing, once, twice, or three times daily dosing,
weekly dosing, and monthly dosing.
[0101] In some treatment regimens, patients will be initially
treated with larger doses of the compounds of the present invention
("loading dose") for a certain period of time ("loading period") in
order to achieve a high tissue concentration of the drug, before
being treated with lower doses of active composition ("maintenance
dose") for a longer period of time ("maintenance period") in order
to maintain the serum or tissue concentration of the active
composition.
[0102] In some treatment regimens, administration of the inhibitor
to a patient is temporarily halted (a "drug holiday"). In some
examples, a patient may have cycles of daily doses of inhibitor for
a month followed by a one month holiday. In another example, a
patient might have daily dosing of an inhibitor for six months,
followed by a one month holiday. In another example, a patient
might have daily doses of an inhibitor for three weeks followed by
a one week holiday. In yet another example, a patient might have
daily doses of a drug for one week, followed by a three week
holiday. In another example, a patient might have cycles of weekly
doses of a drug for 6 weeks, followed by a three week holiday.
[0103] It should also be understood that a specific dosage and
treatment regimen for any particular patient will depend upon a
variety of factors, including the activity of the specific compound
employed, the age, body weight, general health, sex, diet, time of
administration, rate of excretion, drug combination, and the
judgment of the treating physician and the severity of the
particular disease being treated. The amount of a compound of the
present invention in the composition will also depend upon the
particular compound in the composition.
EXEMPLIFICATION
Example 1
[0104] The purpose of the study was to evaluate the effect of 2
week twice a day oral VX-745 treatment on beta amyloid (A.beta.)
accumulation and plaque load and inflammation in Alzheimer's
disease (AD) transgenic Tg2576 mouse model.
[0105] Animals.
[0106] Transgenic mice were treated either with vehicle or VX-745
for 2 weeks starting at 26 months of age. After 2 weeks of
treatment the animals were terminated, and the brains were used for
biochemical and immunohistological analyses for insoluble amyloid
beta levels and plaque load by A.beta.1-42 ELISA and A.beta.
immunohistochemistry. Inflammation was analyzed by ventral cortex
IL-1.beta. and TNF-.alpha. ELISA and microgliosis by CD11b
immunohistochemistry. Plasma was collected at end-point and sent to
client for PK analysis.
[0107] All animal experiments were carried out according to the
National Institute of Health (NIH) guidelines for the care and use
of laboratory animals, and approved by the State Provincial Office
of Southern Finland. Female transgenic Tg2576 mice (n=12) and
wild-type mice (n=5), purchased from Taconic, were used for the
experiment. Animals were housed at a standard temperature
(22.+-.1.degree. C.) and in a light-controlled environment (lights
on from 7 am to 8 pm) with ad libitum access to food and water. The
Tg2576 transgenic line was developed through insertion of the
hAPP695 construct with the `Swedish` double mutation and hamster
prion protein cosmid vector into a C57B6/J.times.SJL host; the
prion promoter limits overexpression of mutant APP to neurons in
the brain. Consequently, the Tg2576 mouse develops elevated brain
levels of soluble A.beta.1-40 and A.beta.1-42 by 6-8 months of age
and A.beta.-containing neuritic plaques in the neocortex and
hippocampus by 10-16 months. The mice were divided into treatment
groups as follows: [0108] 5 wild-type control mice treated with
Vehicle [0109] 6 Tg2576 mice treated with Vehicle [0110] 6 Tg2576
mice treated with VX-745 (3 mg/kg)
[0111] Compound Storage and Instructions for Formulation.
[0112] VX-745 was delivered to Cerebricon as dry compound by the
sponsor. The vehicle to be utilized was 1% Pluronic F108. The
storage and dissolving instructions were provided by the sponsor.
Material safety data sheet or similar document of the compound was
provided by the sponsor. The solutions were stored according to
instructions provided by the sponsor (storage conditions and
expiration day of solution). Vehicle was provided by the
sponsor/Cerebricon.
[0113] Drug Delivery.
[0114] The oral administration of VX-745 or vehicle by oral gavage
(10 ml/kg) was done BID starting at the age of 26 months and
continuing for 14 days. On the day of termination, treatment was
given 2 hours prior to termination.
[0115] General Health Status and Humane End-Points.
[0116] Animals were monitored twice-a-day by laboratory personnel
(8 am and 4 pm). In cases where general health status of an animal
significantly worsened, the mouse was terminated by an overdose of
CO.sub.2, decapitated and brains processed as detailed below.
Definitions of acceptable endpoints included: no spontaneous
movements and inability to drink or eat in 24-h observation period,
massive bleeding, spontaneous inflammation, missing anatomy,
swelling or tumors.
[0117] Collection of Plasma and Brain Samples.
[0118] Two hours after the last dosing the mice were deeply
anesthetized with sodium pentobarbital (60 mg/kg Mebunat, Orion
Pharma, Finland). The mice were subjected to cardiac puncture and
blood samples were collected into pre-cooled (ice bath) EDTA tubes.
The tubes were kept on ice and plasma was separated by
centrifugation at 2000 g (+4.degree. C.) as soon as possible.
150-200 .mu.A of plasma from each mouse was transferred into
pre-cooled polypropylene tubes and kept frozen at -80.degree. C.
until sent to the sponsor for PK analysis.
[0119] The brains were perfused with non-heparinized saline. Right
hemisphere was post-fixed by immersion in 4% PFA in 0.1 M PB. After
a brief wash with phosphate buffer, it was cryoprotected in 30%
sucrose in PB for 2-3 days, after which it was frozen on liquid
nitrogen and stored at -80.degree. C. for further analysis
(immunohistochemistry). Left hemisphere (dissected on ice to
hippocampus, ventral and dorsal cortex and the rest fractions) was
fresh-frozen on dry ice and stored at -80.degree. C. for
biochemical analysis (ELISA). Cerebellum was fresh-frozen and
stored at -80.degree. C. for optional future PK/other analysis.
[0120] Immunohistochemistry.
[0121] Twenty-.mu.m-thick coronal sections were prepared with a
cryostat and mounted on SuperFrost Plus glass slides from the
fixed, cryoprotected and frozen hemispheres. Selected sections were
used for immunohistochemical analyses. Plaque load and the degree
of amyloid aggregates in cortical and hippocampal structures were
analyzed with amyloid beta immunohistochemical staining
[0122] From the adjacent sections, degree of microgliosis was
analyzed with CD11b immunohistochemistry.
[0123] A.beta. and CD11b Immunohistochemistry:
[0124] Briefly, tissue sections used in immunohistochemistry were
thawed and air dried. After blocking the internal peroxidase
activity and unspecific binding, and washes, sections were reacted
overnight at RT with: [0125] anti-A.beta. (mouse
anti-A.beta.[4-10], the Genetics Company AB02, 1:20,000, clone
W0-2) [0126] anti-CD11b (rat anti-CD11b, AbD Serotec Inc. MCA711,
1:500)
[0127] Thereafter the sections were incubated with proper
biotinylated secondary antibody and avidin-biotin complex
(Vectastain Elite kit, Vector Laboratories, Burlingame, Calif.) for
2 h each. The peroxidase containing avidin-biotin complex was
visualized using nickel-enhanced DAB as a substrate. Finally, the
sections were rinsed, dehydrated, coverslipped and examined with a
Leica 3000RB microscope.
[0128] Image Analysis.
[0129] Equally spaced coronal tissue sections along the
antero-posterior axis of the hippocampus (3-4 tissue sections from
each animal) were analyzed for immunostaining intensity by ImagePro
Plus software. Images of immunoreactive staining were captured at
defined light and filter settings in a brightfield microscope
equipped with a color CCD-camera. The captured images of
A.beta.-immunoreactive plaque deposits and intraneuronal A.beta.
aggregates as well as CD11b immunoreactive images were converted to
grayscale images, processed with a delineation function to sharpen
edges to allow an accurate segmentation. The images were segmented
with an auto-threshold command (ImageProPlus, MediaCybernetics).
The results were expressed as area fraction (stained
area.sub.tot/measured area.sub.tot, expressed in %) and presented
as mean.+-.SEM among the tissue sections analyzed from each
individual transgenic mouse. Ventral cortex and dorsal hippocampus
were analyzed from the coronal sections (at the AP level of dorsal
hippocampus).
[0130] Insoluble and Soluble Amyloid Beta 1-42 ELISA.
[0131] Amyloid beta 1-42 ELISA analyses were applied to detect
insoluble and soluble form of A.beta..sub.1-42 in ventral
cortex.
[0132] The ventral cortex tissue sample was homogenized and samples
prepared according to the manufacturers detailed instructions (the
Genetics Company, Switzerland, hAmyloid B42 Brain ELISA). Briefly
the tissues were homogenized with a Dounce homogenizer (2.times.10
strokes on ice) in lysis buffer at a ratio of 1:10 (tissue
weight:lysis buffer). Lysis buffer was Tris-buffered saline (TBS;
20 mM Tris-base and 137 mM NaCl, pH7.4) with protease inhibitors.
The homogenate was centrifuged for 10 min at +4.degree. C. with
13,000 rpm and the supernatant was divided in aliqouts and stored
frozen at -20.degree. C. prior to analyses (=Soluble A.beta.).
[0133] The pellet was re-homogenized in cold 70% formic acid in
distilled water, sonicated for 10 min, neutralized with 15.times.
volume 1M Tris pH 7.4, and centrifuged for 10 min at +4.degree. C.
with 13,000 rpm. The supernatant was stored frozen at -20.degree.
C. (=Insoluble A.beta.).
[0134] A.beta..sub.1-42 levels in insoluble and soluble fractions
of brain tissue samples were analyzed with ELISA using Amyloid Beta
1-42 ELISA kits (hAmyloid B42 Brain ELISAs, The Genetics Company,
Switzerland) according to instructions of the manufacturer.
Standard curve range was from 25 to 500 pg/ml. FIGS. 1A and 1B
depict the area percentage of A.beta..sub.1-42 amyloid plaques of
transgenic mice following a two-week administration of VX-745 3
mg/kg BID. Of particular note, the present study was conducted on
older mice (26 months of age). Other studies attempt to prevent
amyloid plaque accumulation and are thus conducted on mice of about
4 and/or 8 months of age (see, for example, Zhu et al., J.
Neuroscience, 31(4): 1355-136 (2011), incorporated herein by
reference in its entirety). Tg2576 mice aged 26 months have
elevated brain levels of soluble amyloid plaque by 6-8 months of
age. The present experiments were designed to evaluate the amyloid
plaque clearing ability of a p38 inhibitor (i.e., VX-745).
Significantly, VX-745 showed a 32.5% decrease of amyloid plaque
area in the cortex as compared with vehicle (mean 27.7% amyloid
plaque area in control vs. mean 18.7% amyloid plaque area in
VX-745-treated animals). VX-745 showed a 61.8% decrease of amyloid
plaque area in the hippocampus as compared with vehicle (mean 13.6%
amyloid plaque area in control vs. mean 5.2% amyloid plaque area in
VX-745-treated animals).
[0135] IL-1.beta. and TNF-.alpha. ELISA.
[0136] IL-1.beta. and TNF-.alpha. levels were analyzed from the
soluble dorsal cortex brain tissue fraction with mouse IL-1.beta.
and TNF-.alpha. ELISA Kits (Quantikine M Cytokine mouse IL-1.beta.
and TNF-.alpha. ELISA kits, RND-Systems, MLB00 and MTA00, R&D
Systems, MN, USA) according to instructions of the manufacturer.
FIG. 2 depicts the IL-1.beta. levels in transgenic mice following a
two-week administration of VX-745 3 mg/kg BID. Increases in
inflammation, particularly neuroinflammation, are known to trigger
MAPT phosphorylation and aggregation through overexpression of
IL-1. FIG. 2 depicts the IL-1.beta. levels in treated mice vs.
control and wild type mice. Notably, the VX-745-treated mice showed
no increases in IL-1.beta. levels when compared to the wild-type or
control animals.
[0137] Statistical Analysis.
[0138] All data were presented as mean.+-.standard deviation (SD)
or standard error of mean (SEM), and differences were considered to
be statistically significant at the P<0.05 level. Statistical
analysis was performed using StatsDirect statistical software.
Differences between group means were analyzed by using un-paired
t-test.
* * * * *