U.S. patent application number 15/113723 was filed with the patent office on 2017-08-03 for killing senescent cells and treating senescence-associated conditions using a src inhibitor and a flavonoid.
The applicant listed for this patent is Mayo Foundation for Medical Education and Research. Invention is credited to James L. Kirkland, Nathan K. LeBrasseur, Jordan D. Miller, Allyson K. Palmer, Tamar Tchkonia, Yi Zhu.
Application Number | 20170216286 15/113723 |
Document ID | / |
Family ID | 53757700 |
Filed Date | 2017-08-03 |
United States Patent
Application |
20170216286 |
Kind Code |
A1 |
Kirkland; James L. ; et
al. |
August 3, 2017 |
KILLING SENESCENT CELLS AND TREATING SENESCENCE-ASSOCIATED
CONDITIONS USING A SRC INHIBITOR AND A FLAVONOID
Abstract
Provided herein are methods and uses for treatment or
prophylaxis of a senescent cell associated disease or disorder by
administering a senolytic combination comprising dasatinib and
quercetin or an analog thereof to a subject in need thereof. In
certain embodiments, the senescent cell associated disease or
disorder is a cardiovascular disease or disorder, inflammatory
disease or disorder, a pulmonary disease or disorder, a
neurological disease or disorder, or a metabolic disease or
disorder.
Inventors: |
Kirkland; James L.;
(Rochester, MN) ; Tchkonia; Tamar; (Rochester,
MN) ; Zhu; Yi; (Rochester, MN) ; Palmer;
Allyson K.; (Rochester, MN) ; LeBrasseur; Nathan
K.; (Rochester, MN) ; Miller; Jordan D.;
(Rochester, MN) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Mayo Foundation for Medical Education and Research |
Rochester |
MN |
US |
|
|
Family ID: |
53757700 |
Appl. No.: |
15/113723 |
Filed: |
January 28, 2015 |
PCT Filed: |
January 28, 2015 |
PCT NO: |
PCT/US15/13376 |
371 Date: |
July 22, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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61932704 |
Jan 28, 2014 |
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61932711 |
Jan 28, 2014 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 31/506 20130101;
A61K 31/506 20130101; A61K 31/352 20130101; A61K 31/7048 20130101;
A61K 31/353 20130101; A61K 45/06 20130101; A61K 2300/00 20130101;
A61K 31/665 20130101; A61K 31/353 20130101; A61K 2300/00 20130101;
A61K 2300/00 20130101; A61K 2300/00 20130101; A61K 2300/00
20130101; A61K 31/7048 20130101; A61K 31/352 20130101; A61K 31/665
20130101 |
International
Class: |
A61K 31/506 20060101
A61K031/506; A61K 31/352 20060101 A61K031/352 |
Goverment Interests
STATEMENT OF GOVERNMENT INTEREST
[0001] This invention was made with government support under Grant
No. AG41122 and AG046061 awarded by the National Institutes of
Health. The government has certain rights in this invention.
Claims
1. A method for treating a senescence associated disease or
disorder in a subject comprising administering to the subject a
senolytic combination, wherein the senescence associated disease or
disorder is not cancer; and wherein the senolytic combination
includes a src inhibitor and a flavonoid.
2. The method of claim 1, wherein the senolytic combination is
administered once every 0.5-12 months; provided that if the
senescence associated disease or disorder is a senescence
associated metabolic disorder, the senolytic combination is
administered once every 4-12 months.
3. (canceled)
4. The method of claim 1, wherein the senescence associated disease
or disorder is atherosclerosis.
5. The method of claim 1, wherein the senescence associated disease
or disorder is osteoarthritis.
6. The method of claim 1, wherein the senescence associated disease
or disorder is idiopathic pulmonary fibrosis or chronic obstructive
pulmonary disease.
7. (canceled)
8. The method of claim 1, wherein the senescence associated disease
or disorder is selected from diabetes, metabolic syndrome, and
obesity.
9.-10. (canceled)
11. A method of killing a senescent cell, comprising contacting the
senescent cell with a src inhibitor and a flavonoid.
12. The method of claim 11, wherein the senescent cell is selected
from a senescent fibroblast, a senescent pre-adipocyte, a senescent
epithelial cell, a senescent chondrocyte, a senescent neuron, and a
senescent endothelial cell.
13. The method of claim 11, wherein the senescent cell is a
senescent pre-adipocyte.
14. The method of claim 11, wherein the src inhibitor is
dasatinib.
15. The method of claim 11, wherein the flavonoid is a compound
having a structure of the following formula (I): ##STR00014## or a
pharmaceutically acceptable salt thereof, wherein R.sub.1 is --OH
or --H; R.sub.2 is --OH or --H; R.sub.3 is --OH, --H, --R.sub.6, or
--OCH.sub.2PO(OH).sub.2; R.sub.4 is --OH, --OPO(OH).sub.2,
--OCH.sub.3, --OCH.sub.2PO(OH).sub.2, --R.sub.6, --H, or
--OSO.sub.3H; and R.sub.5 is --OH, --H, --R.sub.6, or --OCH.sub.3,
wherein R.sub.6 is ##STR00015##
16. (canceled)
17. The method of claim 15 wherein R.sub.3 is --OH.
18.-19. (canceled)
20. The method of claim 15 wherein R.sub.4 is --OH or
--OSO.sub.3H.
21. The method of claim 15 wherein R.sub.5 is --OH.
22. The method of claim 15 wherein the compound of Formula (I) is
selected from the following structures and pharmaceutically
acceptable salts thereof: ##STR00016## ##STR00017##
23. The method of claim 1, wherein the senolytic combination is
administered during a treatment course of 1-7 days once every
0.5-12 months.
24. The method of claim 1, wherein the senescence associated
disease or disorder is a senescence associated metabolic disorder,
and the senolytic combination is administered during a treatment
course of 1-7 days once every 4-12 months.
25. The method of claim 1, wherein the src inhibitor is
dasatinib.
26. The method of claim 1, wherein the flavonoid is a compound
having a structure of the following formula (I): ##STR00018## or a
pharmaceutically acceptable salt thereof, wherein R.sub.1 is --OH
or --H; R.sub.2 is --OH or --H; R.sub.3 is --OH, --H, --R.sub.6, or
--OCH.sub.2PO(OH).sub.2; R.sub.4 is --OH, --OPO(OH).sub.2,
--OCH.sub.3, --OCH.sub.2PO(OH).sub.2, --R.sub.6, --H, or
--OSO.sub.3H; and R.sub.5 is --OH, --H, --R.sub.6, or --OCH.sub.3;
wherein R.sub.6 is ##STR00019##
27. The method of claim 26, wherein the compound of Formula (I) is
selected from the following structures and pharmaceutically
acceptable salts thereof: ##STR00020## ##STR00021##
Description
BACKGROUND
[0002] Technical Field
[0003] The disclosure herein relates generally to methods for
treatment and prophylaxis of senescent cell-associated diseases and
disorders.
[0004] Description of the Related Art
[0005] Senescent cells accumulate in tissues and organs of
individuals as they age and are found at sites of age-related
pathologies. Senescent cells are believed important to inhibiting
proliferation of dysfunctional or damaged cells and particularly to
constraining development of malignancy (see, e.g., Campisi, Curr.
Opin. Genet. Dev. 21:107-12 (2011); Campisi, Trends Cell Biol.
11:S27-31 (2001); Prieur et al., Curr. Opin. Cell Biol. 20:150-55
(2008)); nevertheless, the presence of senescent cells in an
individual may contribute to aging and aging-related dysfunction
(see, e.g., Campisi, Cell 120:513-22 (2005)). Given that senescent
cells have been causally implicated in certain aspects of
age-related decline in health and may contribute to certain
diseases, and are also induced as a result of necessary
life-preserving chemotherapeutic and radiation treatments, the
presence of senescent cells may have deleterious effects to
millions of patients worldwide. However, identifying and developing
treatments of such diseases and conditions by selective elimination
of senescent cells has been an arduous undertaking. The present
disclosure addresses these needs and offers related advantages.
BRIEF SUMMARY
[0006] Provided herein are methods and agents for selective killing
of senescent cells that are associated with numerous pathologies
and diseases, including age-related pathologies and diseases.
Described herein are the following embodiments.
[0007] In one embodiment, a method is provided for treating a
senescence associated disease or disorder in a subject comprising
administering to the subject a senolytic combination, which
senolytic combination comprises (a) a first agent that alters
either one or both of a cell survival signaling pathway and an
inflammatory pathway; and (b) a second agent that alters either one
or both of a cell survival signaling pathway and an inflammatory
pathway; wherein the senescence associated disease or disorder is
not a cancer, wherein the first agent and second agent are
different, and wherein the senolytic combination is administered
during a treatment course of 1-7 days every 0.5-12 months; provided
that if the senescence associated disease or disorder is a
senescence associated metabolic disorder, the senolytic combination
is administered during a treatment course of 1-7 days every 4-12
months. In certain embodiments, the senolytic combination is
administered once every 0.5-12 months; provided that if the
senescence associated disease or disorder is a senescence
associated metabolic disorder, the senolytic combination is
administered once every 4-12 months. In other certain embodiments,
the senescent cell-associated disease or disorder is a
cardiovascular disease or disorder, inflammatory disease or
disorder, a pulmonary disease or disorder, a neurological disease
or disorder. In a specific embodiment, the cardiovascular disease
or disorder is atherosclerosis. In another specific embodiment, the
inflammatory disease or disorder is osteoarthritis. In another
specific embodiment, the pulmonary disease or disorder is
idiopathic pulmonary fibrosis or chronic obstructive pulmonary
disease. In another specific embodiment, the neurological disease
or disorder is selected from mild cognitive impairment; motor
neuron dysfunction; Alzheimer's disease; Parkinson's disease; and
macular degeneration. In another specific embodiment, the
senescence associated metabolic disease or disorder is selected
from diabetes, metabolic syndrome, and obesity. In another specific
embodiment, the senescence-associated disease or disorder is a
dermatological disease or disorder is selected from eczema,
psoriasis, hyperpigmentation, nevi, rashes, atopic dermatitis,
urticaria, diseases and disorders related to photosensitivity or
photoaging, rhytides; pruritis; dysesthesia; eczematous eruptions;
eosinophilic dermatosis; reactive neutrophilic dermatosis;
pemphigus; pemphigoid; immunobullous dermatosis; fibrohistocytic
proliferations of skin; cutaneous lymphomas; and cutaneous
lupus.
[0008] In one embodiment, a method is provided for treating a
senescence-associated metabolic disease or disorder in a subject
comprising administering to the subject a senolytic combination,
which senolytic combination comprises (a) a first agent that alters
either one or both of a cell survival signaling pathway and an
inflammatory pathway; and (b) a second agent that alters either one
or both of a cell survival signaling pathway and an inflammatory
pathway, wherein the first agent and second agent are different,
wherein the senolytic combination is administered during a
treatment course of 1-7 days every 4-12 months, and wherein the
metabolic disease or disorder is selected from diabetes, metabolic
syndrome, and obesity. In a specific embodiment, the senolytic
combination is administered once every 4-12 months. In another
specific embodiment, the senescent cell is selected from a
senescent fibroblast, a senescent pre-adipocyte, a senescent
epithelial cell, a senescent chondrocyte, a senescent neuron, and a
senescent endothelial cell. In another specific embodiment, the
senescent cell is a senescent pre-adipocyte.
[0009] In the above embodiments and the embodiments described
herein, the first agent is a src inhibitor and the second agent is
a flavonoid. In more specific embodiments, the first agent is
dasatinib and the second agent is a compound having a structure of
the following formula (I):
##STR00001##
[0010] or a pharmaceutically acceptable salt, stereoisomer,
tautomer, or prodrug thereof, wherein
[0011] R.sub.1 is --OH or H;
[0012] R.sub.2 is --OH or H;
[0013] R.sub.3 is --OH, H, R.sub.6, Or --OCH.sub.2PO(OH).sub.2;
[0014] R.sub.4 is --OH, --OPO(OH)(O.sup.-), --OCH.sub.3,
--OCH.sub.2PO(OH).sub.2, R.sub.6, H, or --OSO.sub.3.sup.-; and
[0015] R.sub.5 is --OH, H, R.sub.6, or --OCH.sub.3,
[0016] wherein R.sub.6 is
##STR00002##
[0017] In a specific embodiment, R.sub.3 is --OH, R.sub.6, or
--OCH.sub.2PO(OH).sub.2.
[0018] In a specific embodiment, R.sub.3 is --OH.
[0019] In a specific embodiment, R.sub.4 is --OH,
--OPO(OH)(O.sup.-), --OCH.sub.3, --OCH.sub.2PO(OH).sub.2, R.sub.6,
or --OSO.sub.3.sup.-.
[0020] In a specific embodiment, R.sub.4 is --OH,
--OPO(OH)(O.sup.-), --OCH.sub.2PO(OH).sub.2, R.sub.6, or
--OSO.sub.3.sup.-.
[0021] In a specific embodiment, R.sub.4 is --OH, R.sub.6, or
--OSO.sub.3.sup.-.
[0022] In a specific embodiment, R.sub.5 is --OH or R.sub.6.
[0023] In a specific embodiment, the compound of Formula (I) is
selected from:
##STR00003## ##STR00004##
[0024] In one embodiment, a method is provided for treating,
reducing the likelihood of occurrence of, or delaying onset of a
senescent cell-associated disease or disorder in a subject who has
a senescent cell-associated disease or disorder or who has at least
one predisposing factor for developing the senescent
cell-associated disease or disorder, comprising administering to
the subject a senolytic combination that comprises (a) a first
agent that alters either one or both of a cell survival signaling
pathway and an inflammatory pathway in the senescent cell, and (b)
a second agent that alters either one or both of a cell survival
signaling pathway and an inflammatory pathway in the senescent
cell, thereby promoting death of the senescent cell, wherein the
senescent cell-associated disease or disorder is a cardiovascular
disease or disorder, inflammatory disease or disorder, a pulmonary
disease or disorder, a neurological disease or disorder, with the
proviso that if the subject has a cancer, neither the first agent
nor the second agent is a primary therapy for treating the cancer,
and wherein the first agent is administered once every 0.5-12
months and the second agent is administered once every 0.5-12
months, and wherein the first and second agents are different.
[0025] In another embodiment, a method is provided for treating,
reducing the likelihood of occurrence of, or delaying onset of a
senescent cell-associated disease or disorder in a subject who has
a senescent cell-associated disease or disorder or who has at least
one predisposing factor for developing the senescent
cell-associated disease or disorder, comprising administering to
the subject a senolytic combination comprising (a) a first agent
that alters either one or both of a cell survival signaling pathway
and an inflammatory pathway in the senescent cell, and (b) a second
agent that alters either one or both of a cell survival signaling
pathway and an inflammatory pathway in the senescent cell, thereby
promoting death of the senescent cell, wherein the senescent
cell-associated disease or disorder is a metabolic disorder
selected from diabetes, metabolic syndrome, and obesity, and
wherein the senolytic combination is administered once every 4-12
months, and wherein the first and second agents are different.
[0026] In another embodiment, a method is provided for treating,
reducing the likelihood of occurrence of, or delaying onset of a
senescent cell-associated condition or disorder in a subject who
has a senescent cell-associated disease or disorder or who has at
least one predisposing factor for developing the senescent
cell-associated disease or disorder, comprising administering to
the subject a senolytic combination comprising (a) a first agent
that alters either one or both of a cell survival signaling pathway
and an inflammatory pathway in the senescent cell, and (b) a second
agent that alters either one or both of a cell survival signaling
pathway and an inflammatory pathway in the senescent cell, thereby
promoting death of the senescent cell, and wherein the senescent
cell-associated disease or disorder is selected from
atherosclerosis, osteoarthritis, idiopathic pulmonary fibrosis, and
chronic obstructive pulmonary disease, and wherein the senolytic
combination is administered once every 0.5-12 months, and wherein
the first and second agents are different.
[0027] In another embodiment, a method is provided for killing a
senescent cell comprising contacting the senescent cell and a
senolytic combination comprising (a) a first agent that alters
either one or both of a cell survival signaling pathway and an
inflammatory pathway in the senescent cell and (b) a second agent
that alters either one or both of a cell survival signaling pathway
and an inflammatory pathway in the senescent cell, thereby
promoting death of the senescent cell, wherein the senescence cell
is present in a subject who has a senescent cell-associated disease
or disorder or who has at least one predisposing factor for
developing the senescent cell-associated disease or disorder, with
the proviso that if the subject has a cancer, neither the first
agent nor the second agent of the combination is a primary therapy
for treating the cancer, wherein the senescent cell-associated
disease or disorder is a cardiovascular disease or disorder,
inflammatory disease or disorder, a pulmonary disease or disorder,
a neurological disease or disorder, wherein the combination is
administered once every 0.5-12 months, and wherein the first and
second agents are different.
[0028] In certain embodiments of the methods described above and
herein, the senescent cell-associated disease or disorder is
selected from atherosclerosis; osteoarthritis; idiopathic pulmonary
fibrosis; chronic obstructive pulmonary disease; mild cognitive
impairment; motor neuron dysfunction; Alzheimer's disease;
Parkinson's disease; and macular degeneration.
[0029] In another embodiment, a method is provided for killing a
senescent cell comprising contacting the senescent cell and a
senolytic combination comprising (a) a first agent that alters
either one or both of a cell survival signaling pathway and an
inflammatory pathway in the senescent cell and (b) a second agent
that alters either one or both of a cell survival signaling pathway
and an inflammatory pathway in the senescent cell, thereby
promoting death of the senescent cell, wherein the senescence cell
is present in a subject who has a senescent cell-associated disease
or disorder or who has at least one predisposing factor for
developing the senescent cell-associated disease or disorder,
wherein the senescent cell-associated disease or disorder is a
metabolic disorder selected from diabetes, metabolic syndrome, and
obesity, and wherein the combination is administered once every
4-12 months, and wherein the first and second agents are
different.
[0030] In another embodiment, a method is provided for treating or
reducing the likelihood of occurrence of atherosclerosis in a
subject who has atherosclerosis or who has at least one
predisposing factor for developing atherosclerosis, comprising
administering to the subject a senolytic combination comprising (a)
a first agent that alters either one or both of a cell survival
signaling pathway and an inflammatory pathway in the senescent
cell, and (b) a second agent that alters either one or both of a
cell survival signaling pathway and an inflammatory pathway in the
senescent cell, thereby promoting death of the senescent cell,
wherein the combination is administered once every 0.5-12 months,
and wherein the first and second agents are different.
[0031] In certain embodiments of the methods described above and
herein, the senescent cell is selected from a senescent fibroblast,
a senescent pre-adipocyte, a senescent epithelial cell, a senescent
chondrocyte, a senescent neuron, a senescent smooth muscle cell, a
senescent mesenchymal cell, a senescent macrophage, and a senescent
endothelial cell. In certain specific embodiments, the senescent
cell is a senescent pre-adipocyte.
[0032] In certain embodiments of the methods described above and
herein, at least one of the first agent and the second agent
inhibits Src kinase (e.g., dasatinib) and the second agent is a
flavonoid, (e.g., quercetin or an analog thereof).
[0033] In certain embodiments of the methods described above and
herein, the first agent of the combination is dasatinib and the
second agent is a compound having a structure of the following
formula (I) (i.e., quercetin or an analog thereof) as described
above and in greater detail herein.
[0034] In the following description, certain specific details are
set forth in order to provide a thorough understanding of various
embodiments. However, one skilled in the art will understand that
the invention may be practiced without these details. In other
instances, well-known structures have not been shown or described
in detail to avoid unnecessarily obscuring descriptions of the
embodiments. Unless the context requires otherwise, throughout the
specification and claims which follow, the word "comprise" and
variations thereof, such as, "comprises" and "comprising," are to
be construed in an open, inclusive sense, that is, as "including,
but not limited to." In addition, the term "comprising" (and
related terms such as "comprise" or "comprises" or "having" or
"including") is not intended to exclude that in other certain
embodiments, for example, an embodiment of any composition of
matter, composition, method, or process, or the like, described
herein, may "consist of" or "consist essentially of" the described
features. Headings provided herein are for convenience only and do
not interpret the scope or meaning of the claimed embodiments.
[0035] Reference throughout this specification to "one embodiment"
or "an embodiment" means that a particular feature, structure or
characteristic described in connection with the embodiment is
included in at least one embodiment. Thus, the appearances of the
phrases "in one embodiment" or "in an embodiment" in various places
throughout this specification are not necessarily all referring to
the same embodiment. Furthermore, the particular features,
structures, or characteristics may be combined in any suitable
manner in one or more embodiments.
[0036] Also, as used in this specification and the appended claims,
the singular forms "a," "an," and "the" include plural referents
unless the content clearly dictates otherwise. Thus, for example,
reference to "a non-human animal" may refer to one or more
non-human animals, or a plurality of such animals, and reference to
"a cell" or "the cell" includes reference to one or more cells and
equivalents thereof (e.g., plurality of cells) known to those
skilled in the art, and so forth. When steps of a method are
described or claimed, and the steps are described as occurring in a
particular order, the description of a first step occurring (or
being performed) "prior to" (i.e., before) a second step has the
same meaning if rewritten to state that the second step occurs (or
is performed) "subsequent" to the first step. The term "about" when
referring to a number or a numerical range means that the number or
numerical range referred to is an approximation within experimental
variability (or within statistical experimental error), and thus
the number or numerical range may vary between 1% and 15% of the
stated number or numerical range. For example, the use of "about X"
shall encompass +/-1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%,
12%, 13%, 14% and 15% of the value X. It should also be noted that
the term "or" is generally employed in its sense including "and/or"
unless the content clearly dictates otherwise. The term, "at least
one," for example, when referring to at least one compound or to at
least one composition, has the same meaning and understanding as
the term, "one or more."
BRIEF DESCRIPTION OF THE DRAWINGS
[0037] FIG. 1 shows an immunoblot showing the level of different
cellular proteins in senescent and non-senescent human abdominal
subcutaneous preadipocytes. Senescence was induced as described in
Example 1. Lysates were prepared at several time points after
induction of senescence, and the level of each protein in the
lysates detected at 24 hours and at days 3, 5, 8, 11, 15, 20, and
25 (D3, D5, D8, D11, D15, D20, and D25).
[0038] FIGS. 2A-2D illustrate pathways affected by the agents
described herein. FIG. 2A shows possible target proteins affected
indirectly by quercetin, which alters a cell signaling pathway.
FIG. 2B illustrates downstream components of the Src kinase
pathway. FIG. 2C shows the downstream components of the PI3K
pathway. FIG. 2D illustrates downstream components of the Akt
pathway. A solid arrow between two components indicates that the
component at the source of the solid line has a direct role in
upregulating the component to which the arrow is pointing.
[0039] FIG. 3 depicts the percent survival of irradiated IMR90
fibroblast cells after exposure to quercetin. Senescence of IMR90
cells was induced by exposure to radiation. Survival of senescent
IMR90 cells (SenIR IMR90, white bars) and non-senescent IMR90 cells
(NS IMR90, black bars) is shown after treatment with quercetin.
[0040] FIGS. 4A-4F illustrates the effect on senescent (SEN) and
proliferating endothelial cells (HUVEC) by exposure to quercetin,
dasatinib, and dasatinib+quercetin. The HUVEC cells were exposed to
radiation to induce senescence. The data are presented as mean
viability (ATP (.mu.M)).+-.SEM of 4. FIG. 4A presents the effect of
quercetin on senescent and proliferating, endothelial cells at the
concentrations shown. FIG. 4B illustrates the effect of dasatinib
alone on senescent and proliferating endothelial cells at the
concentrations shown. FIG. 4C illustrates the effect of dasatinib
in combination with quercetin on senescent and proliferating
endothelial cells at the concentrations shown. FIG. 4D presents the
effect of enzastaurin on senescent and proliferating, endothelial
cells at the concentrations shown; FIG. 4E illustrates the effect
of dasatinib in combination with enzastaurin on senescent and
proliferating endothelial cells at the concentrations shown. FIG.
4F illustrates the effect of enzastaurin in combination with
quercetin on senescent and proliferating endothelial cells at the
concentrations shown.
[0041] FIGS. 5A-5F illustrates the effect on senescent,
proliferating, or differentiated preadipocytes cells by exposure to
quercetin, dasatinib, dasatinib+quercetin, enzastaurin,
enzastaurin+dasatinib, and enzastaurin+quercetin. Human primary
abdominal subcutaneous preadipocytes were obtained with consent
from donors. The preadipocytes (sample S12-018) were exposed to
radiation to induce senescence. The data are presented as mean
viability (ATP (.mu.M)).+-.SEM of 4. FIG. 5A presents the effect of
quercetin on senescent, proliferating, and differentiated
preadipocytes at the concentrations shown. FIG. 5B illustrates the
effect of dasatinib alone on senescent, proliferating, and
differentiated preadipocytes at the concentrations shown. FIG. 5C
illustrates the effect of dasatinib in combination with quercetin
on senescent, proliferating, and differentiated preadipocytes at
the concentrations shown. FIG. 5D presents the effect of
enzastaurin on senescent and proliferating preadipocytes cells at
the concentrations shown; FIG. 5E illustrates the effect of
dasatinib in combination with enzastaurin on senescent and
proliferating preadipocytes cells at the concentrations shown. FIG.
5F illustrates the effect of enzastaurin in combination with
quercetin on senescent and proliferating preadipocytes cells at the
concentrations shown.
[0042] FIG. 6 presents the design of an animal study with
transgenic p16-3MR mice. Senescence was induced by administering
doxorubicin. Test drugs (dasatinib, quercetin, dasatinib+quercetin)
and ganciclovir (control) were administered as described in Example
5 ten days after administration of doxorubicin. Luminescence
imaging of the mice was performed on the day the test drugs were
administered (Imaging 1) and again at 5 days after administration
of the test drugs (Imaging 2).
[0043] FIGS. 7A-7B present the effects in animal when treated with
dasatinib or quercetin according to the design of the animal model
presented in FIG. 6. Groups of transgenic p16-3MR mice (9 animals
per group) received quercetin (Q), dasatinib (D), quercetin in
combination (D+Q) or ganciclovir (GCV) (control) or vehicle. FIG.
7A illustrates the percent luminescence (y-axis) for each animal
treatment group. FIG. 7B presents the luminescent images of
representative mice in each group.
[0044] FIG. 8 presents the acetylcholine (ACH) dose response as a
determination of endothelial function in the isolated aortas from
24 month old animals treated with quercetin (Drug Q); dasatinib
(Drug D); or dasatinib+quercetin (Drug D+Q); or vehicle (CTRL). The
concentration of ACH ranged from 10.sup.-9 M to 10.sup.-4 M
(depicted by -9 to -4 on the x-axis). BL=baseline
[0045] FIG. 9 presents the sodium nitroprusside (SNP) dose response
as a determination of smooth muscle function in the isolated aortas
from 24 month old animals treated with quercetin (Drug Q);
dasatinib (Drug D); or dasatinib+quercetin (Drug D+Q); or vehicle
(CTRL). The concentration of SNP ranged from 10.sup.-9 M to
10.sup.-4 M (depicted by -9 to -4 on the x-axis). BL=baseline
[0046] FIGS. 10A-G illustrate the effect on senescent or
non-senescent preadipocytes cells by exposure to dasatinib plus
quercetin or vehicle. Human primary abdominal subcutaneous
preadipocytes were obtained with consent from donors. The
preadipocytes from 6 subjects were pooled and exposed to radiation
to induce senescence. The data are presented as mean cell
survival.+-.SEM of 6. FIG. 10A presents the effect of
dasatinib+quercetin on senescent or non-senescent cells. FIG. 10B
shows DAPI stained non-senescent cells treated with
dasatinib+quercetin; FIG. 10D shows DAPI stained senescent cells
treated with vehicle; and FIG. 10G presents DAPI stained senescent
cells treated with dasatinib+quercetin, respectively. TUNEL stained
non-senescent cells treated with dasatinib+quercetin are shown in
FIG. 10C; senescent cells treated with vehicle in FIG. 10E, and
senescent cells treated with dasatinib+quercetin in FIG. 10G.
Treatment with dasatinib+quercetin induced apoptosis in senescent
(FIG. 10G) but not in non-senescent cells (FIG. 10C).
[0047] FIG. 11 depicts the level of senescent cells present in
inguinal and epididymal adipose tissue of old (26 month) male mice.
Animals were given a single dose of the senolytic combination
dasatinib and quercetin (solid bars) or vehicle (open bars) by oral
gavage 4 days before analysis (means.+-.SEM; N=5; ANOVA). Senescent
cells were detected by staining with SA-.beta.-gal.
[0048] FIG. 12 shows the level of mRNA of senescent cell markers
p16 and p21 in legs of mice exposed to 10 Gy collimated cesium
radiation. Mice were treated once with the combination, dasatinib
and quercetin, or vehicle. After 4 days, the levels of p21 and p16
mRNA were assayed by RT-PCR in muscle tissue from the radiated leg
(N=5; T tests).
[0049] FIG. 13 illustrates mRNA expression of senescence markers
(p16, p21, PAI-1) in inguinal fat of 24-month old male mice treated
with a single dose of vehicle, quercetin, dasatinib, or combination
of quercetin and dasatinib. The level of mRNA was assayed by RT-PCR
in inguinal adipose tissue obtained five days after treatment.
(p=0.049; Kruskal-Wallis Test, non-parametric ANOVA; n=8)
[0050] FIGS. 14A and 14B illustrate the percent positive
SA-.beta.-gal staining and p16 mRNA level in mice (n=14) treated
with a single dose of vehicle or combination of quercetin and
dasatinib. Five days after treatment, samples of inguinal fat were
obtained and stained with the SA-.beta.-gal (FIG. 14A) or analyzed
for p16 mRNA expression by RT-PCR (FIG. 14 B). The data were
analyzed by the Mann-Whitney test. SA-.beta.-gal staining:
p<0.0012; p16 mRNA expression: P<0.01
[0051] FIG. 15 illustrates the level of SA-.beta.-gal positive
cells in mice fed a high fat diet (HFD) vs. chow diet (Chow) for 4
month. Adipose tissue (inguinal (Ing) and epididymal (Epi) was
stained for SA-.beta.-gal.
[0052] FIG. 16 shows that groups of p16-3MR mice (n=6) fed a high
fat diet (high fat) for four months have increased numbers of
senescence cells compared with mice fed a regular chow diet (chow
fed) (n=6).
[0053] FIG. 17 illustrates decrease of senescent cells in adipose
tissue of p16-3MR mice fed a high fat diet for four months and then
treated with ganciclovir or vehicle. The presence of senescent
cells in perirenal, epididymal (Epi), or subcutaneous inguinal
(Ing) adipose tissue was detected by SA-.beta.-Gal staining.
[0054] FIG. 18A-C shows the effect of ganciclovir treatment on
glucose tolerance in p16-3MR mice fed a high fat diet. A bolus of
glucose was given at time zero, and blood glucose was monitored for
up to 2 hours to determine efficacy of glucose disposal (FIG. 18A).
This is quantified as area under the curve (AUC), with a higher AUC
indicating glucose intolerance. The glucose tolerance test (GTT)
AUC's of mice treated with ganciclovir is shown in FIG. 18B.
Hemoglobin A1c is shown in FIG. 18C. n=9; ANOVA.
[0055] FIGS. 19A-19B show insulin sensitivity (Insulin Tolerance
Testing (ITT)) of p16-3MR mice fed a high fat diet after
ganciclovir administration. Blood glucose levels were measured at
0, 14, 30, 60, and 120 minutes after the administration of glucose
bolus at time zero (see FIG. 19A). A change in insulin tolerance
testing when ganciclovir was administered to wild-type mice was not
observed (see FIG. 19B).
[0056] FIG. 20 illustrates results of glucose tolerance testing of
wild-type diet-induced obese (DIO) mice treated with the
combination of dasatinib and quercetin (DIO treat) or vehicle (DIO
vehicle). Control non-obese animals were also treated with vehicle
(Chow vehicle) or the combination (Chow treat).
[0057] FIG. 21 depicts glucose tolerance testing results in
diet-induced obese (DIO) mice. 3-month old INK-ATTAC;p16-3MR mice
fed a high fat diet for 4 months and then treated with ganciclovir
(HF Treated) or vehicle (HF Vehicle). DIO mice were treated with
ganciclovir on September 1. Weights, fasting glucose levels, and
areas under the curve in intraperitoneal glucose tolerance tests
were higher in DIO mice than chow-fed controls, indicating glucose
intolerance.
[0058] FIG. 22 illustrates glucose tolerance in animals. Groups of
mice (n=10) were treated with a high fat diet for 4 months.
Weights, fasting glucose levels, and areas under the curve in
intra-peritoneal glucose tolerance tests were significantly higher
in DIO than chow-fed controls. Animals received six doses of the
combination of dasatinib and quercetin (D+Q) (Senolytic) or vehicle
weekly. (P<0.01; ANOVA for repeated measures). Fasting glucose
levels were lower in DIO mice after 6 doses of D+Q, each dose given
weekly (P<0.05; N=10 mice/group; T test).
[0059] FIG. 23A-23B: FIG. 23A presents fat depot size in
diet-induced obese (DIO) animals and chow fed animals treated with
D+Q or vehicle. Mice were treated with dasatinib and quercetin
(D+Q) once per week (5 mg/kg D, 100 mg/kg Q) at 4 months of age.
Mice were sacrificed after 28 weeks of treatment. Fat depot weights
were measured at time of sacrifice and are expressed as percent of
whole body weight. Epi=epididymal fat; Mes=mesenteric fat;
Peri=perirenal fat. An increase in subscapular fat depot weight was
seen in diet-induced obese (DIO) mice treated with D+Q (n=6)
compared with vehicle-treated mice (n=10). No difference in
chow-fed mice fat depot weights were seen between treatment and
vehicle groups (n=9). The weights of other organs obtained from the
animals are shown FIG. 23B.
DETAILED DESCRIPTION
[0060] Aging is a risk factor for most chronic diseases,
disabilities, and declining health. Senescent cells, which are
cells in replicative arrest, accumulate as an individual ages and
may contribute partially or significantly to cell and tissue
deterioration that underlies aging and age related diseases. Cells
may also become senescent after exposure to an environmental,
chemical, or biological insult or as a result of a disease.
Provided herein are methods and agents for use in combination to
selectively kill senescent cells that are associated with numerous
pathologies and diseases, including age-related pathologies and
diseases. Senescent cell associated diseases and disorders (also
called herein senescence-associated diseases and disorders) may be
treated or prevented (i.e., the likelihood of occurrence is
reduced) by administering a senolytic agent (one that selectively
kills senescent cells alone) or a combination of agents that
together selective kill senescent cells. Examples of senolytic
combinations include dasatinib and quercetin or analogs thereof. In
certain embodiments as described herein, agents are administered in
combination to provide a senolytic effect, that is, selectively
killing senescent cells over non-senescent cells. The agents may be
compounds that alter either a cell survival signaling pathway or an
inflammatory pathway or may alter both the cell survival signaling
pathway and the inflammatory pathway in a senescent cell. Selective
killing of senescent cells may occur when at least two different
agents are used in combination (called senolytic combination). In
certain instances, agents of a senolytic combination may have
minimal, if any, observed selective killing of senescent cells when
used alone. In particular embodiments, a senolytic combination
provides a greater senolytic effect than when either agent is used
alone.
[0061] The senescent cell-associated disease or disorder treated or
prevented by the agents and methods described herein include a
cardiovascular disease or disorder, inflammatory disease or
disorder, a pulmonary disease or disorder, a neurological disease
or disorder, a chemotherapeutic side effect, a radiotherapy side
effect, or metastasis, or a metabolic disease, all of which are
described in greater detail herein. In some embodiments, a
senescent cell-associated disease or disorder does not include
cancer. In particular embodiments, each of the at least two agents
may be minimally or not senolytic when used alone. For convenience,
when two or more agents are described herein as being used in
combination, one agent will be called a first agent, and the other
agent will be called the second agent. The adjectives, first,
second, third, and such, in this context are used for convenience
only and are not to be construed as describing order or
administration, preference, or level of activity or other parameter
unless expressly described otherwise herein. A single dose of the
senolytic combination described herein is sufficient to kill
senescent cells.
Senolytic Combination
[0062] A senolytic combination "selectively" (preferentially or to
a greater degree) destroys or kills a senescent cell. In other
words, the senolytic combination destroys or kills a senescent cell
in a biologically, clinically, and/or statistically significant
manner compared with its capability to destroy or kill a
non-senescent cell. A senolytic combination is used in an amount
and for a time sufficient that selectively kills established
senescent cells but is insufficient to kill (destroy, cause the
death of) a non-senescent cell in a clinically significant or
biologically significant manner. In certain embodiments, the
senolytic combination described herein alters at least one
signaling pathway in a manner that induces (initiates, stimulates,
triggers, promotes) and results in (i.e., causes, leads to) death
of the senescent cell. The senolytic combination may alter, for
example, either or both of a cell survival signaling pathway (e.g.,
Akt pathway) or an inflammatory pathway, for example, by
antagonizing a protein within the cell survival and/or inflammatory
pathway.
[0063] Without wishing to be bound by a particular theory, the
mechanism by which the combination described herein selectively
kills senescent cells is by inducing (activating, stimulating,
removing inhibition of) an apoptotic pathway that leads to cell
death. Non-senescent cells may be proliferating cells or may be
quiescent cells. In certain instances, exposure of non-senescent
cells to the senolytic combination as used in the methods described
herein may temporarily reduce the capability of non-senescent cell
to proliferate; however, an apoptotic pathway is not induced and
the non-senescent cell is not destroyed.
[0064] The methods described herein are useful for treating a
senescence-associated disorder or disease that is not a cancer. The
method used for treating a senescence associated disease or
disorder with a senolytic combination described herein may comprise
one or more of a decreased daily dose, decreased cumulative dose
over a single treatment cycle, or decreased cumulative dose of the
agent from multiple treatment cycles than the dose of an agent
required for cancer therapy, which methods are described in greater
detail herein. The reduced doses of the agents may also result in
the likelihood of decreased adverse effects (i.e., side effects).
In other words, when treating a senescent cell associated disease
or disorder (that is not cancer) in a subject, including a subject
who has a cancer, by using the methods described herein, the
senolytic combination may be used in a manner inconsistent with a
primary therapy for treating the cancer. To further reduce
toxicity, a senolytic combination may be administered at a site
proximal to or in contact with senescent cells (not tumor cells).
Localized delivery of a senolytic combination is described in
greater detail herein. A "primary therapy for cancer" as used
herein means that when an agent, which may be used alone or
together with one or more agents, is intended to be or is known to
be an efficacious treatment for the cancer as determined by a
person skilled in the medical and oncology arts, administration
protocols for treatment of the cancer using the agent have been
designed to achieve the relevant cancer-related endpoints.
[0065] The senolytic combination described herein alter (i.e.,
interfere with, affect) one or more cellular pathways that are
activated during the senescence process of a cell. Senolytic
combinations may alter either a cell survival signaling pathway or
an inflammatory pathway or alter both a cell survival signaling
pathway and an inflammatory pathway. Activation of certain cellular
pathways during senescence decreases or inhibits the cell's
capability to induce, and ultimately undergo apoptosis. Without
wishing to be bound by theory, the mechanism by which a senolytic
combination selectively kills senescent cells is by inducing
(activating, stimulating, removing inhibition of) an apoptotic
pathway that leads to cell death. A senolytic combination may alter
one or more signaling pathways by interacting with one, two, or
more target proteins in the one or more pathways, which results in
removing or reducing suppression of a cell death pathway, such as
an apoptotic pathway. Contacting or exposing a senescent cell to a
senolytic combination to alter one, two, or more cellular pathways
in the senescent cell, restores the cell's mechanisms and pathways
for initiating apoptosis. In certain embodiments, a senolytic
combination alters a signaling pathway, which in turn inhibits
secretion and/or expression of one or more gene products important
for survival of a senescent cell. The senolytic combination may
inhibit a biological activity of the gene product(s) important for
survival of the senescent cell. Alternatively, the decrease or
reduction of the level of the gene product(s) in the senescent cell
may alter the biological activity of another cellular component,
which triggers, initiates, or stimulates an apoptotic pathway or
removes or reduces suppression of the apoptotic pathway. As
described herein, neither agent of the senolytic combination is
necessarily linked or conjugated to a cytotoxic moiety (e.g., a
toxin or cytotoxic peptide or cytotoxic nucleic acid). The
senolytic combination is also active in selectively killing
senescent cells in the absence of linkage or conjugation of either
one or both agents of the combination to a targeting moiety (e.g.,
an antibody or antigen-binding fragment thereof; cell binding
peptide) that selectively binds senescent cells.
[0066] Two alternative modes of cell death can be distinguished,
apoptosis and necrosis. The term apoptosis was initially used by
Kerr and colleagues (Br. J. Cancer 26:239-57 (1972)) to describe
the phenomenon as a mode of cell death morphologically distinct
from coagulative necrosis. Apoptosis is typically characterized by
the rounding of the cell, chromatin condensation (pyknosis),
nuclear fragmentation (karyorhexis), and engulfment by neighboring
cells (see, e.g., Kroemer et al., Cell Death Differ. 16:3-11
(2009)). Several molecular assays have been developed and are used
in the art; however, the morphological changes, which are detected
by light and electron microscopy, are viewed in the art as the
optimal techniques to differentiate the two distinct modes of cell
death (see, e.g., Kroemer et al., supra). Alternative cell death
modes, such as caspase-independent apoptosis-like programmed cell
death (PCD), autophagy, necrosis-like PCD, and mitotic catastrophe,
have also been characterized (see, e.g., Golstein, Biochem. Sci.
32:37-43 (2007); Leist et al., Nat. Rev. Mol. Cell Biol. 2:589-98
(2001)). See, e.g., Caruso et al., Rare Tumors 5(2): 68-71 (2013);
published online 2013 June 7. doi: 10.3081/rt.2013.e18. Techniques
and methods routinely practiced in the art and described herein
(e.g., TUNEL) may be used to show that apoptotic cell death results
from contact with the senolytic combination described herein.
[0067] In certain embodiments, a senolytic combination as used in
the methods described herein is comprises two small molecule
compounds. In certain embodiments, an agent of the combination is a
small molecule that may be activated or that is a pro-drug that is
converted to the active form by enzymes within the cell. In a more
specific embodiment, the enzymes that convert a pro-drug to an
active form are those expressed at a higher level in senescent
cells than in non-senescent cells.
[0068] Methods are provided herein for treating or preventing a
senescence-associated disease by administering to a subject in need
thereof a senolytic combination that comprises two small molecule
compounds, such as dasatinib and quercetin or an analog thereof.
When used alone, at least one or both of the small molecule
compounds in a senolytic combination has insufficient senolytic
activity to selectively kill senescent cells and provide a
therapeutic effect. Each compound may alter either a cell survival
signaling pathway or an inflammatory pathway or both the cell
survival signaling pathway and the inflammatory pathway in a
senescent cell. For convenience, when two or more compounds are
described herein as being used in combination, one compound may be
called a first agent or first compound, and another compound may be
called the second agent or second compound, etc. In other certain
embodiments, the methods described herein comprise administering at
least three compounds (a first agent, second agent, and third
agent). The adjectives, first, second, third, and such, in this
context are used for convenience only and are not to be construed
as describing order or administration, preference, or level of
activity or other parameter unless expressly described otherwise.
Use of the at least two compounds results in significantly
increased killing of senescent cells compared with use of each
compound alone.
[0069] As described in greater detail herein, a senolytic
combination may alter a signaling pathway such as a cell survival
signaling pathway. Each agent of the combination may alter one or
more cell survival signaling pathways, for example, a Src kinase
signaling pathway, a PI3K/Akt pathway, PI3K/Akt/mTor pathway,
p38/MAPK pathway, ERK/MAPK pathway, mTOR pathway, insulin/IGF-1
signaling pathway, or a TGF-.beta. signaling pathway. The agent may
instead alter or also alter an inflammatory pathway. Examples of
inflammatory pathways that may be altered by an agent of a
senolytic combination include one or more of a p38/MAPK signaling
pathway, ERK/MAPK pathway, a Src kinase signaling pathway, or an
NF-.kappa.B pathway. In certain embodiments, an agent of the
combination or the combination alters one or more of a p38/MAPK
signaling pathway, ERK/MAPK pathway, or a Src kinase signaling
pathway. Depending on the specific cellular polypeptide with which
an agent of the combination directly interacts in the p38/MAPK
signaling pathway, ERK/MAPK pathway, or Src kinase signaling
pathway, the pathway affected may be either one or both of a cell
survival pathway or an inflammatory pathway.
[0070] Senolytic combinations described herein that may alter at
least one signaling pathway may comprise an agent that inhibits an
activity of at least one of a src kinase, Akt kinase, ERK MAPK, p38
MAPK, histone deacetylase (HDAC), polar auxin transporter, monamine
oxidase (MAO), protein kinase C-beta, calcineurin, and calmodulin.
In certain embodiments, an agent of the combination can alter at
least one or at least two signaling pathways by inhibiting two or
more signaling pathway components. The agent or combination of
agents may inhibit two or more of a src kinase, Akt kinase, ERK
MAPK, p38 MAPK, histone deacetylase (HDAC), polar auxin
transporter, monoamine oxidase (MAO), calcineurin, and
calmodulin.
[0071] In certain embodiments, methods are provided wherein the
senolytic combination alters either a cell survival signaling
pathway or an inflammatory pathway or alters both the cell survival
signaling pathway and the inflammatory pathway in a senescent cell.
In other particular embodiments, methods comprise use of a
senolytic combination that comprises at least two agents wherein at
least one agent and a second agent are each different and
independently alter either one or both of a survival signaling
pathway and an inflammatory pathway.
[0072] Senolytic combinations that may be used in the methods for
treating or preventing a senescence cell associated disorder
described herein include, but are not limited to, small organic
molecules. A small molecule compound of interest may be
derivatized, either randomly or by SAR, to obtain analog compounds
with an improved bioavailability, pharmacokinetic characteristic,
or other characteristic (e.g., solubility, stability). Small
organic molecules typically have molecular weights less than
10.sup.5 daltons, less than 10.sup.4 daltons, or less than 10.sup.3
daltons. In certain embodiments, a small molecule compound does not
violate the following criteria more than once: (1) no more than 5
hydrogen bond donors (the total number of nitrogen-hydrogen and
oxygen-hydrogen bonds); (2) not more than 10 hydrogen bond
acceptors (all nitrogen or oxygen atoms); (3) a molecular mass less
than 500 daltons; (4) an octanol-water partition coefficient[5] log
P not greater than 5.
[0073] In yet another embodiment, the senolytic combination used in
the methods described herein for treating a senescence associated
disease or disorder comprises a flavonoid, such as quercetin or an
analog thereof and a src inhibitor (e.g., dasatinib). In a
particular embodiment, the small molecule compound has a structure
of formula (I) as described below, which includes quercetin and
analogs thereof.
##STR00005##
[0074] or a pharmaceutically acceptable salt, stereoisomer,
tautomer, or prodrug thereof, wherein
[0075] R.sub.1 is --OH or H;
[0076] R.sub.2 is --OH or H;
[0077] R.sub.3 is --OH, H, R.sub.6, or --OCH.sub.2PO(OH).sub.2;
[0078] R.sub.4 is --OH, --OPO(OH)(O.sup.-), --OCH.sub.3,
--OCH.sub.2PO(OH).sub.2, R.sub.6, H, or --OSO.sub.3.sup.-; and
[0079] R.sub.5 is --OH, H, R.sub.6, or --OCH.sub.3,
[0080] wherein R.sub.6 is
##STR00006##
[0081] In certain embodiments, R.sub.1 is --OH. In other certain
embodiments, R.sub.1 is H.
[0082] In certain embodiments, R.sub.2 is --OH. In other certain
embodiments, R.sub.2 is H.
[0083] In certain embodiments, R.sub.3 is --OH. In other certain
embodiments, R.sub.3 is H. In still other specific embodiments,
R.sub.3 is --OCH.sub.2PO(OH).sub.2.
[0084] In other specific embodiments, R.sub.4 is --OH,
--OPO(OH)(O.sup.-), --OCH.sub.3, --OCH.sub.2PO(OH).sub.2, R.sub.6,
or --OSO.sub.3.sup.-. In another particular embodiment, R.sub.4 is
--OH, --OPO(OH)(O.sup.-), --OCH.sub.2PO(OH).sub.2, R.sub.6, or
--OSO.sub.3.sup.-. In certain particular embodiments, R.sub.4 is
--OH, R.sub.6, or --OSO.sub.3.sup.-. In a more specific embodiment,
R.sub.4 is --OH. In other specific embodiments, R.sub.4 is
--OPO(OH)(O.sup.-). In yet other specific embodiments, R.sub.4 is
--OSO.sub.3.sup.-. In other specific embodiments, R.sub.4 is
--OCH.sub.2PO(OH).sub.2. In still a more specific embodiment,
R.sub.4 is R.sub.6. In still another embodiment, R.sub.4 is
--OCH.sub.3.
[0085] In other particular embodiments, R.sub.5 is --OH or
--OCH.sub.3. In certain particular embodiments, R.sub.5 is --OH. In
another embodiment, R.sub.5 is H. In other particular embodiments,
R.sub.5 is R.sub.6.
[0086] In one embodiment, the compound of formula (I) has the
structure of formula (Ia):
##STR00007##
[0087] In another embodiment, the compound of formula (I) has the
structure of formula (Ib):
##STR00008##
[0088] In another specific embodiment, the compound of formula (I)
has the structure of formula (Ic):
##STR00009##
[0089] In still another specific embodiment, the compound of
formula (I) has the structure of formula (Id):
##STR00010##
[0090] In a particular embodiment, the pharmaceutically acceptable
salt of the compound of structure (Id) is a sodium salt.
[0091] In another specific embodiment, the compound of formula (I)
has the structure of formula (Ie):
##STR00011##
[0092] In another specific embodiment, the compound of formula (I)
has the structure of formula (If):
##STR00012##
[0093] In a particular embodiment, the pharmaceutically acceptable
salt of the compound of structure (If) is a potassium salt.
[0094] In another particular embodiment, the compound of formula I
has the structure of formula (Ig):
##STR00013##
[0095] This compound may also be referred to herein as
quercetin-3'-glucuronide. In a certain particular embodiment, the
pharmaceutically acceptable salt of the compound of structure (Ig)
is a sodium salt.
[0096] Quercetin, fisetin, and luteolin (compounds of structure
(Ia), (Ib), and (Ic), respectively) are flavonoids found in plants
and have been studied for their potential therapeutic properties.
Quercetin is a flavonoid that is present in fruits and vegetables
including apples; black, green and buckwheat tea; onions; red
grapes; cherries; raspberries; and citrus fruits and in some plants
including ginkgo biloba and St. John's Wort. A naturally occurring
analog of quercetin that may be used in a senolytic combination in
the methods described herein is fisetin, which is found in a
variety of trees, shrubs, fruits, and vegetables, herbs, and teas.
Luteolin is a flavone and is also found in plants. Reports suggest
that luteolin has anti-inflammatory activity; however, its
biological activities have been less extensively studied than those
of quercetin or fisetin. With respect to the methods and uses for
treating senescence cell associated diseases and disorders as
described herein, the methods are not intended to include
administration of natural occurring compounds, e.g., quercetin or
fisetin, or luteolin, by administering plants, foods, or drinks
that naturally contain quercetin.
[0097] Quercetin has been described in the art as being capable of
inhibiting src kinase, Akt kinase, histone deacetylase (HDAC),
aldose reductase, and low-density lipoprotein oxidation. Quercetin
can act as a calmodulin antagonist and it also inhibits
cyclooxygenase and lipooxygenase and certain phospholipases.
Fisetin acts as a sirtuin-activating compound (STAC), and thus has
an effect on sirtuins, a group of enzymes that use NAD+ to remove
acetyl groups from proteins. Fisetin may act as a caloric
restriction mimetic. The compounds of formula (I), (i.e., quercetin
and analogs described herein) may alter any one or more of a Src
kinase signaling pathway, a PI3K/Akt pathway, a P38/MAPK pathway,
and an insulin/IGF-1 signaling pathway.
[0098] Isolated compounds of formula (I) (e.g., compounds of
formula (Ia), (Ib), (Ic), (Id), (Ie), (If), and (Ig)) may be
prepared by chemical synthetic methods according to any one or more
synthesis methods known and routinely practiced in the chemical
art. Alternatively, quercetin or other natural analogs, such as
fisetin and luteolin, may be isolated from a plant, fruit, or
vegetable to obtain highly purified quercetin (e.g., >95%, 96%,
97%, 98% or greater than 99% purity).
[0099] With regard to stereoisomers of quercetin and its analogs,
the compounds may have one or more chiral (or asymmetric) centers,
and may thus give rise to enantiomers, diastereomers, and other
stereoisomeric forms that may be defined, in terms of absolute
stereochemistry, as (R)- or (S)-. When the compounds described
herein contain olefinic double bonds or other centers of geometric
asymmetry, and unless specified otherwise, it is intended that the
compounds include both E and Z geometric isomers (e.g., cis or
trans). Likewise, unless otherwise specified, all possible isomers,
as well as their racemic and optically pure forms, and all
tautomeric forms are also intended to be included. It is therefore
contemplated that various stereoisomers and mixtures thereof and
includes "enantiomers," which refers to two stereoisomers whose
molecules are nonsuperimposeable mirror images of one another.
Thus, the compounds may occur in any isomeric form, including
racemates, racemic mixtures, and as individual enantiomers or
diastereomers. A "tautomer" refers to a proton shift from one atom
of a molecule to another atom of the same molecule. Some
embodiments of the compounds include tautomers of the compound.
[0100] Quercetin or an analog thereof may be used as a "prodrug,"
meant to indicate a compound that may be converted under
physiological conditions or by solvolysis to a biologically active
compound. Thus, the term "prodrug" refers to a metabolic precursor
of a compound that is pharmaceutically acceptable. A prodrug may be
inactive when administered to a subject in need thereof, but is
converted in vivo to an active compound as described herein.
Prodrugs are typically rapidly transformed in vivo to yield the
parent compound, for example, by hydrolysis in blood. The prodrug
compound often offers advantages of solubility, tissue
compatibility or delayed release in a mammalian organism (see,
e.g., Bundgard, H., Design of Prodrugs (1985), pp. 7-9, 21-24
(Elsevier, Amsterdam). A discussion of prodrugs is provided in
Higuchi, T., et al., "Pro-drugs as Novel Delivery Systems," A.C.S.
Symposium Series, Vol. 14, and in Bioreversible Carriers in Drug
Design, ed. Edward B. Roche, American Pharmaceutical Association
and Pergamon Press, 1987, both of which are incorporated in full by
reference herein.
[0101] The term "prodrug" is also meant to include any covalently
bonded carriers which release the active compound as described
herein in vivo when such prodrug is administered to a mammalian
subject. Prodrugs of a compound may be prepared by modifying
functional groups present in the compound in such a way that the
modifications are cleaved, either in routine manipulation or in
vivo, to the parent compound described herein. Prodrugs include
compounds wherein a hydroxy, amino or mercapto group is bonded to
any group that, when the prodrug of the compound is administered to
a mammalian subject, cleaves to form a free hydroxy, free amino or
free mercapto group, respectively. Examples of prodrugs include,
but are not limited to, ester and amide derivatives of hydroxy,
carboxy, mercapto or amino functional groups in the compounds and
the like.
[0102] To form a senolytic combination, quercetin or an analog
thereof may be combined with a small molecule compound that alters
a protein tyrosine kinase pathway. In certain embodiments, the
protein kinase tyrosine pathway is altered by a compound that
directly inhibits a protein tyrosine kinase (e.g., Src, Lck, Yes,
Fyn). In certain embodiments, the compound used in the senolytic
combination inhibits a Src protein kinase. An example of such a Src
kinase inhibitor is dasatinib
((N-(2-chloro-6-methylphenyl)-2-[[6-[4-(2-hydroxyethyl)-1-piperazinyl]-2--
methyl-4-pyrimidinyl]amino]-5-thiazole carboxamide monohydrate)
(SPRYCEL) (Bristol-Myers Squibb, New York, N.Y.). Dasatinib
inhibits the Bcr-abl and src family of kinases, which includes Src,
Lck, Yes, and Fyn. It is also described as affecting c-Kit (CD117),
Pha2, and inhibits PDGFR-beta. Dasatinib has been approved by
regulatory agencies for treating Philadelphia chromosome+chronic
myelogenous leukemia (CP-CML); and for treating chronic,
accelerated or myeloid or lymphoid blast phase Ph+ CML
resistant/intolerant to prior therapy that included imatinib; and
Ph+ ALL (acute lymphoblastic leukemia) with resistance to
intolerance prior to therapy. Dasatinib analogs include compounds
described in U.S. Pat. Nos. 6,596,746; 7,125,875; 7,153,856;
7,491,725, which patents are all herein incorporated by reference
in their entirety. Processes for making dasatinib and analogs
thereof may be performed by persons skilled in the art using
methods and techniques routinely practiced in the art and as
described in U.S. Pat. Nos. 6,596,746; 7,125,875; 7,153,856;
7,491,725. Dasatinib may affect any one or more of a Src kinase
signaling pathway, a PI3K/Akt pathway, a PI3K pathway, a P38/MAPK
pathway, and an ERK/MAPK pathway. Other compounds classified as
BCR-ABL inhibitors, such as imatinib and sorafenib, are not Src
inhibitors and are significantly less effective for use in the
methods described herein.
[0103] Another example of a small molecule agent that may be
included in a senolytic combination is enzastaurin, which is a
protein kinase C-beta (PKC.beta.) inhibitor. Enzastaurin
(3-(1-Methylindol-3-yl)-4-[1-[1-(pyridin-2-ylmethyl)piperidin-4-yl]indol--
3-yl]pyrrole-2,5-dione) (LY317615; Eli Lilly and Company,
Indianapolis, Ind.) (see U.S. Pat. No. 5,668,152) is a
serine/threonine kinase inhibitor that inhibits protein kinase C
beta (PKC-.beta.) and inhibits the AKT pathway, which induces
apoptosis of cancer cells. PKC-.beta. mediates VEGFR2 signaling
through MEK and MAP kinase activation. By activating Akt through
phosphorylation, PKC-.beta. interacts with the phosphatase and
tensin homolog (PTEN)/PI3K/Akt pathway. In a particular embodiment,
a PKC-.beta. inhibitor, such as enzastaurin, is combined with a
compound of formula I, such as quercetin or an analog thereof, for
use in the methods for treating or preventing a senescence cell
associated disease or disorder, which in certain embodiments is not
a cancer.
[0104] Also as described herein, in particular embodiments, when
two or more agents are used in combination for treatment or
prophylaxis of a senescence-associated disease or disorder, any one
of the compounds of formula (I) (e.g., a compound of formula (Ia),
(Ib), (Ic), (Id), (Ie), (If), and (Ig)) may be combined with a
second compound, such as by way of non-limiting example, dasatinib
or an analog thereof, or enzastaurin or an analog thereof. In other
specific embodiments, any one of the compounds of formula (I)
(e.g., a compounds of formula (Ia), (Ib), (Ic), (Id), (Ie), (If),
and (Ig)) may be combined with a second and a third compound, such
as by way of non-limiting example, dasatinib or an analog thereof,
and enzastaurin or an analog thereof.
[0105] Quercetin analogs that may be used in the methods for
treating a senescent cell associated disease or disorder described
herein include a compound having a structure of formula (Ib)
(fisetin), (Ic) (luteolin), (Id), (Ie), (If), or (Ig). In certain
embodiments, two or more compounds of formula (I) may be used in
these methods in combination with a third agent, such as dasatinib.
By way of example, the two or more compounds of formula (I) may
include a combination of quercetin (Ia) with any one of a compound
having the structure of formula (Ib), (Ic), (Id), (Ie), (If), or
(Ig) and a third agent that is not quercetin or an analog thereof.
In other embodiments, the methods described herein may comprise use
of any two compounds of formula (I), independently selected from
compounds of formula (Ia), (Ib), (Ic), (Id), (Ie), (If), and (Ig)
and a third agent that is not quercetin or an analog thereof. In
still other embodiments, any two or all three of quercetin,
fisetin, and luteolin may be used in these methods in combination
with a non-quercetin agent, such as dasatinib. In other
embodiments, a combination of quercetin and fisetin and a third
agent that is not quercetin or an analog thereof, such as dasatinib
is used in the methods described herein.
[0106] The compounds described herein may generally be used as the
free acid or free base. Alternatively, the compounds may be used in
the form of acid or base addition salts. Acid addition salts of the
free base amino compounds may be prepared according to methods well
known in the art, and may be formed from organic and inorganic
acids. Suitable organic acids include (but are not limited to)
maleic, fumaric, benzoic, ascorbic, succinic, methanesulfonic,
acetic, oxalic, propionic, tartaric, salicylic, citric, gluconic,
lactic, mandelic, cinnamic, aspartic, stearic, palmitic, glycolic,
glutamic, and benzenesulfonic acids. Suitable inorganic acids
include (but are not limited to) hydrochloric, hydrobromic,
sulfuric, phosphoric, and nitric acids. Base addition salts of the
free acid compounds of the compounds described herein may also be
prepared by methods well known in the art, and may be formed from
organic and inorganic bases. Suitable inorganic bases included (but
are not limited to) the hydroxide or other salt of sodium,
potassium, lithium, ammonium, calcium, magnesium, iron, zinc,
copper, manganese, aluminum, and the like, and organic bases such
as substituted ammonium salts. Thus, the term "pharmaceutically
acceptable salt" of compounds described herein is intended to
encompass any and all pharmaceutically suitable salt forms.
[0107] Compounds may sometimes be depicted as an anionic species.
One of ordinary skill in the art will recognize that the compounds
exist with an equimolar ratio of cation. For instance, the
compounds described herein can exist in the fully protonated form,
or in the form of a salt such as sodium, potassium, ammonium or in
combination with any inorganic base as described above. When more
than one anionic species is depicted, each anionic species may
independently exist as either the protonated species or as the salt
species. In some specific embodiments, the compounds described
herein exist as the sodium salt. In other specific embodiments, the
compounds described herein exist as the potassium salt.
[0108] Furthermore, some of the crystalline forms of any compound
described herein may exist as polymorphs, which are also included
and contemplated by the present disclosure. In addition, some of
the compounds may form solvates with water or other organic
solvents. Often crystallizations produce a solvate of the disclosed
compounds. As used herein, the term "solvate" refers to an
aggregate that comprises one or more molecules of any of the
disclosed compounds with one or more molecules of solvent. The
solvent may be water, in which case the solvate may be a hydrate.
Alternatively, the solvent may be an organic solvent. Thus, the
presently disclosed compounds may exist as a hydrate, including a
monohydrate, dihydrate, hemihydrate, sesquihydrate, trihydrate,
tetrahydrate and the like, as well as the corresponding solvated
forms. Certain embodiments of the compounds may be true solvates,
while in other cases, some embodiments of the compounds may merely
retain adventitious water or be a mixture of water plus some
adventitious solvent.
[0109] Specific and analogous reactants may also be identified
through the indices of known chemicals prepared by the Chemical
Abstract Service of the American Chemical Society, which are
available in most public and university libraries, as well as
through on-line databases (the American Chemical Society,
Washington, D.C., may be contacted for more details). Chemicals
that are known but not commercially available in catalogs may be
prepared by custom chemical synthesis houses, where many of the
standard chemical supply houses (e.g., those listed above) provide
custom synthesis services. A reference for the preparation and
selection of pharmaceutical salts of the present disclosure is P.
H. Stahl & C. G. Wermuth "Handbook of Pharmaceutical Salts,"
Verlag Helvetica Chimica Acta, Zurich, 2002. Methods known to one
of ordinary skill in the art may be identified through various
reference books and databases. Suitable reference books and
treatises detail the synthesis of reactants useful in the
preparation of compounds described herein, or provide references to
articles that describe the preparation.
[0110] In general, the compounds used in the methods described
herein may be made according to organic synthesis techniques known
to those skilled in this art, starting from commercially available
chemicals and/or from compounds described in the chemical
literature.
[0111] Assays and techniques for identifying senolytic combinations
are described in greater detail herein. In addition, identifying
and selecting small compounds for use in senolytic combinations, a
person skilled in the medicinal chemistry art may also consider
other properties of a small molecule, such as solubility,
bioavailability, pharmacokinetics, Lipinski Rule of 5, and the
like.
Senescent Cells
[0112] A senolytic agent and a senolytic combination selectively
kills or destroys senescent cells in a clinically significant or
biologically significant manner. A senolytic combination may
selectively kill one or more types of senescent cells (e.g.,
senescent preadipocytes, senescent endothelial cells, senescent
fibroblasts, senescent neurons, senescent epithelial cells,
senescent chondrocytes, senescent mesenchymal cells, senescent
macrophages, senescent smooth muscle cells). In certain
embodiments, a senolytic combination is capable of selectively
killing at least senescent preadipocytes. Senolytic combinations
that selective kill preadipocytes may be useful for treatment or
prophylaxis of diabetes (particularly type 2 diabetes), metabolic
syndrome, or obesity. In other embodiments, a senolytic combination
is capable of selectively killing at least senescent endothelial
cells. Such senolytic combinations may be useful for treatment or
prophylaxis or a cardiovascular disease (e.g., atherosclerosis). In
other particular embodiments, a senolytic combination is capable of
selectively killing at least senescent fibroblasts. In still
another embodiment, a senolytic combination may selectively kill at
least senescent neurons, including dopamine-producing neurons. In
still another embodiment, a senolytic combination may kill at least
senescent retinal pigmented epithelial cells or other senescent
epithelial cells (e.g., pulmonary senescent epithelial cells or
senescent kidney epithelial cells). Selective killing of pulmonary
epithelial cells may be useful for treating pulmonary diseases,
such as chronic obstructive pulmonary disease. In yet other
embodiments, a senolytic combination may selectively kill at least
senescent immune cells (e.g., senescent macrophages). In still
another embodiment, a senolytic combination may kill at least
chondrocytes, which may be useful for treatment or prophylaxis of
an inflammatory disorder, such as osteoarthritis.
[0113] A senescent cell may exhibit any one or more of the
following characteristics. (1) Senescence growth arrest is
essentially permanent and cannot be reversed by known physiological
stimuli. (2) Senescent cells increase in size, sometimes enlarging
more than twofold relative to the size of non-senescent
counterparts. (3) Senescent cells express a senescence-associated
.beta.-galactosidase (SA-.beta.-gal), which partly reflects the
increase in lysosomal mass. (4) Most senescent cells express
p16INK4a, which is not commonly expressed by quiescent or
terminally differentiated cells. (5) Cells that senesce with
persistent DDR signaling harbor persistent nuclear foci, termed DNA
segments with chromatin alterations reinforcing senescence
(DNA-SCARS). These foci contain activated DDR proteins and are
distinguishable from transient damage foci. DNA-SCARS include
dysfunctional telomeres or telomere dysfunction-induced foci (TIF).
(6) Senescent cells express and may secrete molecules associated
with senescence, which in certain instances may be observed in the
presence of persistent DDR signaling, which in certain instances
may be dependent on persistent DDR signaling for their expression.
(7) The nuclei of senescent cells lose structural proteins such as
Lamin B1 or chromatin-associated proteins such as histones and
HMGB1. See, e.g., Freund et al., Mol. Biol. Cell 23:2066-75 (2012);
Davalos et al., J. Cell Biol. 201:613-29 (2013); Ivanov et al., J.
Cell Biol. DOI: 10.1083/jcb.201212110, page 1-15; published online
Jul. 1, 2013; Funayama et al., J. Cell Biol. 175:869-80
(2006)).
[0114] Senescent cells and senescent cell associated molecules can
be detected by techniques and procedures described in the art. For
example, the presence of senescent cells in tissues can be analyzed
by histochemistry or immunohistochemistry techniques that detect
the senescence marker, SA-beta galactosidase (SA-.beta. gal) (see,
e.g., Dimri et al., Proc. Natl. Acad. Sci. USA 92: 9363-9367
(1995)). The presence of the senescent cell-associated polypeptide
p16 can be determined by any one of numerous immunochemistry
methods practiced in the art, such as immunoblotting analysis.
Expression of p16 mRNA in a cell can be measured by a variety of
techniques practiced in the art including quantitative PCR. The
presence and level of senescence cell associated polypeptides
(e.g., polypeptides of the SASP) can be determined by using
automated and high throughput assays, such as an automated Luminex
array assay described in the art (see, e.g., Coppe et al., PLoS
Biol 6: 2853-68 (2008)).
[0115] The presence of senescent cells can also be determined by
detection of senescent cell-associated molecules, which include
growth factors, proteases, cytokines (e.g., inflammatory
cytokines), chemokines, cell-related metabolites, reactive oxygen
species (e.g., H.sub.2O.sub.2), and other molecules that stimulate
inflammation and/or other biological effects or reactions that may
promote or exacerbate the underlying disease of the subject.
Senescent cell-associated molecules include those that are
described in the art as comprising the senescence-associated
secretory phenotype (SASP, i.e., which includes secreted factors
which may make up the pro-inflammatory phenotype of a senescent
cell), senescent-messaging secretome, and DNA damage secretory
program (DDSP). These groupings of senescent cell associated
molecules, as described in the art, contain molecules in common and
are not intended to describe three separate distinct groupings of
molecules. Senescent cell-associated molecules include certain
expressed and secreted growth factors, proteases, cytokines, and
other factors that may have potent autocrine and paracrine
activities (see, e.g., Coppe et al., supra; Coppe et al. J. Biol.
Chem. 281:29568-74 (2006); Coppe et al. PLoS One 5:39188 (2010);
Krtolica et al. Proc. Natl. Acad. Sci. U.S.A. 98:12072-77 (2001);
Parrinello et al., J. Cell Sci. 118:485-96 (2005). ECM associated
factors include inflammatory proteins and mediators of ECM
remodeling and which are strongly induced in senescent cells (see,
e.g., Kuilman et al., Nature Reviews 9:81-94 (2009)). Other
senescent cell-associated molecules include extracellular
polypeptides (proteins) described collectively as the DNA damage
secretory program (DDSP) (see, e.g., Sun et al., Nature Medicine
published online 5 Aug. 2012; doi:10.1038/nm.2890). Senescent
cell-associated proteins also include cell surface proteins (or
receptors) that are expressed on senescent cells, which include
proteins that are present at a detectably lower amount or are not
present on the cell surface of a non-senescent cell.
[0116] Senescence cell-associated molecules include secreted
factors which may make up the pro-inflammatory phenotype of a
senescent cell (e.g., SASP). These factors include, without
limitation, GM-CSF, GRO.alpha., GRO.alpha.,.beta.,.gamma., IGFBP-7,
IL-1.alpha., IL-6, IL-7, IL-8, MCP-1, MCP-2, MIP-1.alpha., MMP-1,
MMP-10, MMP-3, Amphiregulin, ENA-78, Eotaxin-3, GCP-2, GITR, HGF,
ICAM-1, IGFBP-2, IGFBP-4, IGFBP-5, IGFBP-6, IL-13, IL-10, MCP-4,
MIF, MIP-3a, MMP-12, MMP-13, MMP-14, NAP2, Oncostatin M,
osteoprotegerin, PIGF, RANTES, sgp130, TIMP-2, TRAIL-R3, Acrp30,
angiogenin, Axl, bFGF, BLC, BTC, CTACK, EGF-R, Fas, FGF-7, G-CSF,
GDNF, HCC-4, I-309, IFN-.gamma., IGFBP-1, IGFBP-3, IL-1 R1, IL-11,
IL-15, IL-2R-.alpha., IL-6 R, I-TAC, Leptin, LIF, MMP-2, MSP-a,
PAI-1, PAI-2, PDGF-BB, SCF, SDF-1, sTNF RI, sTNF RII,
Thrombopoietin, TIMP-1, tPA, uPA, uPAR, VEGF, MCP-3, IGF-1,
TGF-.beta.3, MIP-1-delta, IL-4, FGF-7, PDGF-BB, IL-16, BMP-4, MDC,
MCP-4, IL-10, TIMP-1, Fit-3 Ligand, ICAM-1, Axl, CNTF, INF-.gamma.,
EGF, BMP-6. Additional identified factors, which include those
sometimes referred to in the art as senescence messaging secretome
(SMS) factors, some of which are included in the listing of SASP
polypeptides, include without limitation, IGF1, IGF2, and IGF2R,
IGFBP3, IDFBP5, IGFBP7, PAl1, TGF-.beta., WNT2, IL-1.alpha., IL-6,
IL-8, and CXCR2-binding chemokines. Cell-associated molecules also
include without limitation the factors described in Sun et al.,
Nature Medicine, supra, and include, including, for example,
products of the genes, MMP1, WNT16B, SFRP2, MMP12, SPINK1, MMP10,
ENPP5, EREG, BMP6, ANGPTL4, CSGALNACT, CCL26, AREG, ANGPT1, CCK,
THBD, CXCL14, NOV, GAL, NPPC, FAM150B, CST1, GDNF, MUCL1, NPTX2,
TMEM155, EDN1, PSG9, ADAMTS3, CD24, PPBP, CXCL3, MMP3, CST2, PSG8,
PCOLCE2, PSG7, TNFSF15, C17orf67, CALCA, FGFJ8, IL8, BMP2, MATN3,
TFP1, SERPINI 1, TNFRSF25, and IL23A. Senescent cell-associated
proteins also include cell surface proteins (or receptors) that are
expressed on senescent cells, which include proteins that are
present at a detectably lower amount or are not present on the cell
surface of a non-senescent cell.
Methods for Characterizing and Identifying Senolytic
Combinations
[0117] Characterizing a senolytic combination can be determined
using one or more cell-based assays and one or more animal models
described herein or in the art and with which a person skilled in
the art will be familiar. A senolytic combination may selectively
kill one or more types of senescent cells (e.g., senescent
preadipocytes, senescent endothelial cells, senescent fibroblasts,
senescent neurons, senescent epithelial cells, senescent
mesenchymal cells, senescent smooth muscle cells, senescent
macrophages, or senescent chondrocytes).
[0118] A person skilled in the art will readily appreciate that
characterizing a combination of agents and determining the level of
killing by the combination can be accomplished by comparing the
activity of a test agent or combination with appropriate negative
controls (e.g., vehicle only and/or a composition or compound known
in the art not to kill senescent cells) and appropriate positive
controls. In vitro cell-based assays for characterizing senolytic
combinations also include controls for determining the effect of an
agent and combination comprising the agent on non-senescent cells
(e.g., quiescent cells or proliferating cells). A senolytic
combination reduces (i.e., decreases) percent survival of a
plurality of senescent cells (i.e., in some manner reduces the
quantity of viable senescent cells in the animal or in the
cell-based assay) compared with one or more negative controls.
Conditions for a particular in vitro assay include temperature,
buffers (including salts, cations, media), and other components,
which maintain the integrity of the test agent and reagents used in
the assay, and which are familiar to a person skilled in the art
and/or which can be readily determined.
[0119] The source of senescent cells for use in assays may be a
primary cell culture, or culture adapted cell line, including but
not limited to, genetically engineered cell lines that may contain
chromosomally integrated or episomal recombinant nucleic acid
sequences, immortalized or immortalizable cell lines, somatic cell
hybrid cell lines, differentiated or differentiatable cell lines,
transformed cell lines, and the like. In a particular embodiment,
the senescent cell is isolated from biological sample obtained from
a host or subject who has a senescent cell associated disease or
disorder. In other embodiments, non-senescent cells, which may be
obtained from a subject or may be a culture adapted line may be
used and senescence induced by methods described herein and in the
art, such as by exposure to irradiation or a chemotherapeutic agent
(e.g., doxorubicin). The biological sample may be a blood sample,
biopsy specimen, body fluids (e.g., lung lavage, ascites, mucosal
washings, synovial fluid), bone marrow, lymph nodes, tissue
explant, organ culture, or any other tissue or cell preparation
from a subject. The sample may be a tissue or cell preparation in
which the morphological integrity or physical state has been
disrupted, for example, by dissection, dissociation,
solubilization, fractionation, homogenization, biochemical or
chemical extraction, pulverization, lyophilization, sonication, or
any other means for processing a sample derived from a subject or
biological source. The subject may be a human or non-human
animal.
[0120] Transgenic animal models as described herein and in the art
may be used to determine killing or removal of senescent cells
(see, e.g., Baker et al., supra; Nature, 479:232-36 (2011); Int'l
Patent Application Publication No. WO/2012/177927; Int'l Patent
Application Publication No. WO 2013/090645). Exemplary transgenic
animal models contain a transgene that includes a nucleic acid that
allows for controlled clearance of senescence cells (e.g.,
p16.sup.ink4a positive senescent cells) as a positive control. The
presence and level of senescent cells in the transgenic animals can
be determined by measuring the level of a detectable label or
labels that are expressed in senescent cells of the animal. The
transgene nucleotide sequence includes a detectable label, for
example, one or more of a red fluorescent protein; a green
fluorescent protein; and one or more luciferases to detect
clearance of senescent cells.
[0121] Animal models that are described herein or in the art
includes art-accepted models for determining the effectiveness of a
senolytic combination to treat or prevent (i.e., reduce the
likelihood of occurrence of) a particular senescence associated
disease or disorder, such as atherosclerosis models, osteoarthritis
models, COPD models, and IPF models. As described herein, pulmonary
disease murine models, such as a bleomycin pulmonary fibrosis
model, and a chronic cigarette smoking model are applicable for
diseases such as COPD and may be routinely practiced by a person
skilled in the art. Animal models for determining the effectiveness
of a senolytic combination to treat and/or prevent (i.e., reduce
the likelihood of occurrence of) chemotherapy and radiotherapy side
effect models or to treat or prevent metastasis are described in
International Patent Application Publication Nos. WO 2013/090645
and WO 2014/205244, which are incorporated herein by reference in
their entirety. Animal models for determining the effectiveness of
combinations for treating eye diseases, particularly age-related
macular degeneration are also routinely used in the art (see, e.g.,
Pennesi et al., Mol. Aspects Med. 33:487-509 (2012); Zeiss et al.,
Vet. Pathol. 47:396-413 (2010); Chavala et al., J. Clin. Invest.
123:4170-81 (2013)).
[0122] By way of non-limiting example and as described herein,
osteoarthritis animal models have been developed. Osteoarthritis
may be induced in the animal, for example, by inducing damage to a
joint, for example, in the knee by surgical severing, incomplete or
total, of the anterior cruciate ligament. By way of another
non-limiting example and as described herein, atherosclerosis
animal models have been developed. Atherosclerosis may be induced
in the animal, for example, by feeding animals a high fat diet or
by using transgenic animals highly susceptible to developing
atherosclerosis. In still another example, and as described herein,
mouse models in which animals are treated with bleomycin has been
described (see, e.g., Peng et al., PLoS One 2013; 8(4):e59348. doi:
10.1371/joumal.pone.0059348. Epub 2013 Apr. 2; Mouratis et al.,
Curr. Opin. Pulm. Med. 17:355-61 (2011)) for determining the
effectiveness of an agent for treating IPF.
[0123] In pulmonary disease animals models (e.g., a bleomycin
animal model, smoke-exposure animal model, or the like),
respiratory measurements may be taken to evaluate the usefulness of
the senolytic combination. For all the disease models described
herein, immunohistology; assays for determining the level of
inflammatory molecules (e.g., IL-6) (e.g., immunochemistry,
molecular biology techniques); and assays (e.g., immunochemistry,
molecular biology techniques) for determining the level of
senescence markers as noted above may all be performed according to
methods described herein and that may be routinely practiced by the
skilled artisan.
[0124] Determining the effectiveness of a senolytic combination to
selectively kill senescent cells as described herein in an animal
model may be performed using one or more statistical analyses with
which a skilled person will be familiar. By way of example,
statistical analyses such as two-way analysis of variance (ANOVA)
may be used for determining the statistical significance of
differences between animal groups treated with an agent and those
that are not treated with the agent (i.e., negative control group,
which may include vehicle only). Statistical packages such as SPSS,
MINITAB, SAS, Statistika, Graphpad, GLIM, Genstat, and BMDP are
readily available and routinely used by a person skilled in the
animal model art.
[0125] A person skilled in the art will readily appreciate that
characterizing a senolytic combination and determining the level of
killing by the combination can be accomplished by comparing the
activity of a test agent or combination with appropriate negative
controls (e.g., vehicle only and/or a composition, agent, or
compound known in the art not to kill senescent cells) and
appropriate positive controls. In vitro cell-based assays for
characterizing the combination also include controls for
determining the effect of the combination on non-senescent cells
(e.g., quiescent cells or proliferating cells). A senolytic
combination that is useful reduces (i.e., decreases) percent
survival of senescent cells (i.e., in some manner reduces the
quantity of viable senescent cells in the animal or in the
cell-based assay) compared with one or more negative controls.
Accordingly, a senolytic combination selectively kills senescent
cells compared with killing of non-senescent cells (which may be
referred to herein as selectively killing senescent cells over
non-senescent cells). In certain embodiments (either in an in vitro
assay or in vivo (in a human or non-human animal)), the senolytic
combination kills at least 20% of the senescent cells and kills no
more than 5% of non-senescent cells. In other particular
embodiments (either in an in vitro assay or in vivo (in a human or
non-human animal)), the senolytic combination kills at least about
25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, or 65% of the senescent
cells and kills no more than about 5% or 10% of non-senescent
cells. In other particular embodiments (either in an in vitro assay
or in vivo (in a human or non-human animal)), the senolytic
combination kills at least about 30%, 35%, 40%, 45%, 50%, 55%, 60%,
or 65% of the senescent cells and kills no more than about 5%, 10%,
or 15% of non-senescent cells. In other particular embodiments
(either in an in vitro assay or in vivo (in a human or non-human
animal)), the senolytic combination kills at least about 40%, 45%,
50%, 55%, 60%, or 65% of the senescent cells and kills no more than
about 5%, 10%, 15%, 20%, or 25% of non-senescent cells. In other
particular embodiments (either in an in vitro assay or in vivo (in
a human or non-human animal)), the senolytic combination kills at
least about 50%, 55%, 60%, or 65% of the senescent cells and kills
no more than about 5%, 10%, 15%, 20%, 25%, or 30% of non-senescent
cells. Stated another way, a senolytic combination has at least
5-25, 10-50 or 10-100 times (5.times.-25.times.,
10.times.-50.times. or 10.times.-100.times.) greater selectively
for killing senescent cells than for non-senescent cells (e.g., at
least 5.times., 10.times., 20.times., 25.times., 30.times.,
40.times.. 50.times., 60.times., 75.times., 80.times., 90.times.,
or 100.times.). With respect to specific embodiments of the methods
described herein for treating a senescence-associated disease or
disorder, the percent senescent cells killed may refer to the
percent senescent cells killed in a tissue or organ that comprises
senescent cells that contribute to onset, progression, and/or
exacerbation of the disease or disorder. By way of non-limiting
example, tissues of the brain, tissues and parts of the eye,
pulmonary tissue, cardiac tissue, arteries, joints, skin, and
muscles may comprise senescent cells that may be reduced in percent
as described above by the senolytic combinations described herein
and thereby provide a therapeutic effect. Moreover, selectively
removing at least 20% or at least 25% of senescent cells from an
affected tissue or organ can have a clinically significant
therapeutic effect. In certain particular embodiments, in the
methods for treating the cardiovascular disease, such as
atherosclerosis, as described herein, the senolytic combination
kills at least 20% of the senescent cells and kills no more than 5%
of non-senescent cells in the artery. In other particular
embodiments, the senolytic combination selectively kills at least
25% of the senescent cells in the arteriosclerotic artery. In
another embodiment, with respect to the methods described herein
for treating osteoarthritis by administering a senolytic
combination, the percent senescent cells killed may refer to the
percent senescent cells killed in an osteoarthritic joint versus
non-senescent cells killed in the osteoarthritic joint. In certain
particular embodiments, in the methods for treating osteoarthritis
as described herein, the at least one senolytic combination kills
at least 20% of the senescent cells and kills no more than 5% of
non-senescent cells in the osteoarthritic joint. In other
particular embodiments, the senolytic combination selectively kills
at least 25% of the senescent cells in the osteoarthritic joint. In
still another embodiment, with respect to the methods described
herein for treating senescence associated pulmonary disease or
disorder (e.g., COPD, IPF) by administering a senolytic
combination, the percent senescent cells killed may refer to the
percent senescent cells killed in affected pulmonary tissue versus
non-senescent cells killed in the affected pulmonary tissue of the
lung. In certain particular embodiments, in the methods for
treating senescence associated pulmonary diseases and disorders as
described herein, a senolytic combination kills at least 20% of the
senescent cells and kills no more than 5% of non-senescent cells in
the affected pulmonary tissue. In other particular embodiments, the
senolytic combination selectively kills at least 25% of the
senescent cells in the affected pulmonary tissue.
[0126] In certain embodiments, methods are provided for identifying
(i.e., screening for) combinations that are useful for treating or
preventing (i.e., reducing the likelihood of occurrence of) a
senescence associated disease or disorder. In one embodiment, a
method for identifying a senolytic combination for treating such
diseases and disorders, comprises inducing cells to senesce to
provide established senescent cells. Methods for inducing cells to
senesce are described herein and in the art and include, for
example, exposure to radiation (e.g., 10 Gy is typically
sufficient) or a chemotherapeutic agent (e.g., doxorubicin or other
anthracycline). After exposure to the agent, the cells are cultured
for an appropriate time and under appropriate conditions (e.g.,
media, temperature, CO.sub.2/O.sub.2 level appropriate for a given
cell type or cell line) to allow senescence to be established. As
discussed herein, senescence of cells may be determined by
determining any number of characteristics, such as changes in
morphology (as viewed by microscopy, for example); production of,
for example, senescence-associated 3-galactosidase (SA-.beta.-gal),
p16INK4a, p21, or any one or more SASP factors (e.g., IL-6, MMP3).
A sample of the senescent cells is then contacted with a candidate
combination (i.e., mixed with, combined, or in some manner
permitting the cells and the agent to interact). Persons skilled in
the art will appreciate that the assay will include the appropriate
controls, negative and positive, either historical or performed
concurrently. For example, a sample of control non-senescent cells
that have been cultured similarly as the senescent cells but not
exposed to a senescence inducing combination are contacted with the
candidate agent. The level of survival of the senescent cells is
determined and compared with the level of survival of the
non-senescent cells. A senolytic combination is identified when the
level of survival of the senescent cells is less than the level of
survival of the non-senescent cells.
[0127] Determining the effectiveness of an agent to kill senescent
cells as described herein in an animal model may be performed using
one or more statistical analyses with which a skilled person will
be familiar. By way of example, statistical analyses such as
two-way analysis of variance (ANOVA) may be used for determining
the statistical significance of differences between animal groups
treated with an agent and those that are not treated with the agent
(i.e., negative control group). Statistical packages such as SPSS,
MINITAB, SAS, Statistika, Graphpad, GLIM, Genstat, and BMDP are
readily available and routinely used by a person skilled in the
animal art.
[0128] Animal models for these methods and purposes may include
non-human primate models, dog models, rodent models, or other
animal models appropriate for determining the safety and efficacy
of a senolytic agent.
Cell Survival Signaling Pathway and Inflammatory Pathway
[0129] The senolytic combinations described herein alter (i.e.,
interfere with, affect) one or more cellular pathways that are
activated during the senescence process of a cell. As described
herein, senolytic combinations alter either a cell survival
signaling pathway or an inflammatory pathway or alter both a cell
survival signaling pathway and an inflammatory pathway in a
senescent cell. Without wishing to be bound by theory, activation
of certain cellular pathways during senescence decreases or
inhibits the cell's capability to induce and ultimately undergo
apoptosis. A senolytic combination may alter one or more signaling
pathways (e.g., a cell survival pathway and an inflammatory
pathway) by interacting with one, two, or more target proteins in
the one or more pathways, which results in removing or reducing
suppression of a cell death pathway, such as an apoptotic pathway.
Contacting or exposing a senescent cell to a senolytic combination
to alter one, two, or more cellular pathways in the senescent cell,
restores the cell's mechanisms and pathways for initiating
apoptosis.
[0130] The senolytic combinations described herein alter either a
cell survival signaling pathway or an inflammatory pathway or alter
both the cell survival signaling pathway and the inflammatory
pathway that induces (i.e., initiates, triggers, stimulates or in
some manner removes or inhibits suppression of) a cell death
pathway, such as an apoptotic pathway, in the senescent cell. Cell
survival signaling pathways and inflammatory pathways that are
activated during senescence include PI3K/Akt, Src signaling
pathway, p38/MAPK, ERK/MAPK pathway, NF-.kappa.B signaling,
insulin/IGF-1 signaling pathway, TGF-.beta. signaling, mTOR
pathway, PI3K/Akt/mTor pathway, mTOR/protein translation pathways,
among others. A cell survival signaling pathway may include any one
or more of a Src kinase signaling pathway, a PI3K/Akt pathway,
PI3K/Akt/mTor pathway, p38/MAPK pathway, ERK/MAPK pathway, mTOR
pathway, insulin/IGF-1 signaling pathway, or a TGF-.beta. signaling
pathway, for example. Inflammatory pathways include, by way of
non-limiting example, a p38/MAPK signaling pathway, ERK/MAPK
pathway, a Src kinase signaling pathway, or an NF-.kappa.B
pathway.
[0131] A cell survival pathway includes the Src signaling pathway,
which is involved in regulation of cell proliferation,
differentiation, apoptosis, cell adhesion, and stress responses
(see, e.g., Wang, Oncogene 19:5643-50 (2000); Thomas et al., Annu.
Rev. Cell Dev. Biol. 13:513-609 (1997)). The Src pathway is also
involved in inflammatory responses, including macrophage mediated
immune responses (see, e.g., Byeon et al., Mediators of
Inflammation Vol. 2012, Article ID 512926, doi:10.1155/2012/512926
(2012)) and acute inflammatory responses (see, e.g., Okutani et
al., Am. J. Physiol. Lung Cell Mol. Physiol. 291:L129-L141 (2006)).
Accordingly, a senolytic combination that alters a cell survival
pathway that includes altering a Src signaling pathway may also
alter an inflammatory pathway.
[0132] Altering a cell signaling pathway or altering an
inflammatory pathway may alter or affect a function of one or more
downstream target proteins or may affect the interaction of the one
or more downstream target proteins with another component of the
respective cell signaling or inflammatory pathway. For example, a
senolytic combination that alters a Src kinase pathway or a
PI3K/Akt pathway may alter a function of one or more downstream
target proteins in the respective pathway or may affect the
interaction of the one or more downstream target proteins with
another component of the respective pathway (see, e.g., Example 1;
FIGS. 2B-2D). Exemplary target proteins that are upregulated in
senescent cells include P38/MAPK, ERK1/2, and PI3K (complex). In
certain embodiments, the PI3K/Akt pathway, which is a cell
signaling pathway, is activated during senescence and a senolytic
combination described herein inhibits the pathway, which may
enhance induction of apoptosis.
Senescence-Associated Disease or Disorder
[0133] Methods are provided herein for treating or preventing
(i.e., reducing the likelihood of occurrence) conditions, diseases,
or disorders related to, associated with, or caused by cellular
senescence, including age-related diseases and disorders in a
subject in need thereof. In certain embodiments of the methods
described herein, the senescent cell associated disease or disorder
is not cancer. Senescent cell associated diseases and disorders
include, for example, cardiovascular diseases and disorders,
inflammatory diseases and disorders, pulmonary diseases and
disorders, neurological diseases and disorders, chemotherapeutic
side effects, radiotherapy side effects, and metastasis. A
prominent feature of aging is a gradual loss of function, or
degeneration that occurs at the molecular, cellular, tissue, and
organismal levels. Age-related degeneration gives rise to
well-recognized pathologies, such as sarcopenia, atherosclerosis
and heart failure, osteoporosis, pulmonary insufficiency, renal
failure, neurodegeneration (including macular degeneration,
Alzheimer's disease, and Parkinson's disease), and many others.
Although different mammalian species vary in their susceptibilities
to specific age-related pathologies, collectively, age-related
pathologies generally rise with approximately exponential kinetics
beginning at about the mid-point of the species-specific life span
(e.g., 50-60 years of age for humans) (see, e.g., Campisi, Annu.
Rev. Physiol. 75:685-705 (2013); Naylor et al., Clin. Pharmacol.
Ther. 93:105-16 (2013)).
[0134] Exemplary conditions, disorders, or diseases that may be
treated or prevented by administering a senolytic combination
described herein include, without limitation, cognitive diseases
(e.g., mild cognitive impairment (MCI), Alzheimer's disease and
other dementias); cardiovascular disease (including
atherosclerosis); metabolic diseases and disorders (e.g., obesity,
diabetes, metabolic syndrome); motor function diseases and
disorders (e.g., Parkinson's disease, motor neuron dysfunction
(MND)); cerebrovascular disease; emphysema; osteoarthritis;
peripheral vascular disease; cardiac diastolic dysfunction; benign
prostatic hypertrophy; aortic aneurysm; idiopathic pulmonary
fibrosis; chronic obstructive pulmonary disease; osteoarthritis;
and macular degeneration. In certain embodiments, any one or more
of the diseases or disorders described above or herein may be
excluded.
[0135] Subjects (i.e., patients, individuals (human or non-human
animals)) who may benefit from use of the methods described herein
that comprise administering a senolytic combination include those
who may also have a cancer. The subject treated by these methods
may be considered to be in partial or complete remission (also
called cancer remission). As discussed in detail herein, the
senolytic combinations for use in methods for selective killing of
senescent cells are not intended to be used as a treatment for
cancer, that is, in a manner that kills or destroys the cancer
cells in a statistically significant manner. Therefore, the methods
disclosed herein do not encompass use of the senolytic agents in a
manner that would be considered a primary therapy for the treatment
of a cancer. Even though a senolytic agent, alone or with other
chemotherapeutic or radiotherapy agents, are not used in a manner
that is sufficient to be considered as a primary cancer therapy,
the methods and senolytic agents described herein may be used in a
manner (e.g., a short term course of therapy) that is useful for
inhibiting metastases. In other certain embodiments, the subject to
be treated with the senolytic agent does not have a cancer (i.e.,
the subject has not been diagnosed as having a cancer by a person
skilled in the medical art).
[0136] Cardiovascular Diseases and Disorders.
[0137] In another embodiment, the senescent cell-associated disease
or disorder treated or prevented by the methods described herein
comprising administering a senolytic combination is cardiovascular
disease. The cardiovascular disease may be any one or more of
angina, arrhythmia, atherosclerosis, cardiomyopathy, congestive
heart failure, coronary artery disease (CAD), carotid artery
disease, endocarditis, heart attack (coronary thrombosis,
myocardial infarction [MI]), high blood pressure/hypertension,
hypercholesterolemia/hyperlipidemia, mitral valve prolapse,
peripheral artery disease (PAD), aortic aneurysm, brain aneurysm,
cardiac fibrosis, cardiac diastolic dysfunction, cardiac stress
resistance, and stroke.
[0138] Atherosclerosis is characterized by patchy intimal plaques
(atheromas) that encroach on the lumen of medium-sized and large
arteries; the plaques contain lipids, inflammatory cells, smooth
muscle cells, and connective tissue. Atherosclerosis can affect
large and medium-sized arteries, including the coronary, carotid,
and cerebral arteries, the aorta and its branches, and major
arteries of the extremities. Atherosclerosis is characterized by
patchy intimal plaques (atheromas) that encroach on the lumen of
medium-sized and large arteries; the plaques contain lipids,
inflammatory cells, smooth muscle cells, and connective tissue.
[0139] Subjects suffering from cardiovascular disease can be
identified using standard diagnostic methods known in the art for
cardiovascular disease. Generally, diagnosis of atherosclerosis and
other cardiovascular disease is based on symptoms (e.g., chest pain
or pressure (angina), numbness or weakness in arms or legs,
difficulty speaking or slurred speech, drooping muscles in face,
leg pain, high blood pressure, kidney failure and/or erectile
dysfunction), medical history, and/or physical examination of a
patient. Subjects at risk of developing cardiovascular disease
include those having a family history of cardiovascular disease and
those having other risk factors such as high blood pressure, high
cholesterol, diabetes, obesity and/or smoking. In a certain
embodiment, the cardiovascular disease that is a senescence cell
associated disease/disorder is atherosclerosis. In a certain
specific embodiment, a cardiovascular disease or disorder treated
or prevented according to the methods and uses described herein
does not include stenosis and restenosis.
[0140] The effectiveness of a senolytic combination for treating or
preventing (i.e., reducing or decreasing the likelihood of
developing or occurrence of) a cardiovascular disease (e.g.,
atherosclerosis) can readily be determined by a person skilled in
the medical and clinical arts. One or any combination of diagnostic
methods, including physical examination, assessment and monitoring
of clinical symptoms, and performance of analytical tests and
methods described herein and practiced in the art (e.g.,
angiography, electrocardiography, stress test, non-stress test),
may be used for monitoring the health status of the subject. The
effects of the treatment of a senolytic combination can be analyzed
using techniques known in the art, such as comparing symptoms of
patients suffering from or at risk of cardiovascular disease that
have received the treatment with those of patients without such a
treatment or with placebo treatment.
[0141] Inflammatory Diseases and Disorders.
[0142] In certain embodiments, a senescent cell-associated disease
or disorder is an inflammatory disease or disorder, such as by way
of non-limiting example, osteoarthritis, that may be treated or
prevented (i.e., likelihood of occurrence is reduced) according to
the methods described herein that comprise administration of a
senolytic combination. Other inflammatory or autoimmune diseases or
disorders that may be treated by administering a senolytic
combination described herein include osteoporosis, psoriasis, oral
mucositis, rheumatoid arthritis, inflammatory bowel disease,
eczema, kyphosis, herniated intervertebral disc, and the pulmonary
diseases, COPD and idiopathic pulmonary fibrosis.
[0143] Osteoarthritis degenerative joint disease is characterized
by fibrillation of the cartilage at sites of high mechanical
stress, bone sclerosis, and thickening of the synovium and the
joint capsule. Fibrillation is a local surface disorganization
involving splitting of the superficial layers of the cartilage. The
early splitting is tangential with the cartilage surface, following
the axes of the predominant collagen bundles. Collagen within the
cartilage becomes disorganized, and proteoglycans are lost from the
cartilage surface. In the absence of protective and lubricating
effects of proteoglycans in a joint, collagen fibers become
susceptible to degradation, and mechanical destruction ensues.
Predisposing risk factors for developing osteoarthritis include
increasing age, obesity, previous joint injury, overuse of the
joint, weak thigh muscles, and genetics. It is a common cause of
chronic disability in the elderly. Symptoms of osteoarthritis
include sore or stiff joints, particularly the hips, knees, and
lower back, after inactivity or overuse; stiffness after resting
that goes away after movement; and pain that is worse after
activity or toward the end of the day. Osteoarthritis may also
affect the neck, small finger joints, the base of the thumb, ankle,
and big toe.
[0144] Chronic inflammation is thought to be the main age-related
factor that contributes to osteoarthritis. In combination with
aging, joint overuse and obesity appear to promote
osteoarthritis.
[0145] The effectiveness a senolytic combination described herein
for treatment or prophylaxis of osteoporosis and monitoring of a
subject who receives the senolytic combination can readily be
determined by a person skilled in the medical and clinical arts.
One or any combination of diagnostic methods, including physical
examination (such as determining tenderness, swelling or redness of
the affected joint), assessment and monitoring of clinical symptoms
(such as pain, stiffness, mobility), and performance of analytical
tests and methods described herein and practiced in the art (e.g.,
determining the level of inflammatory cytokines or chemokines;
X-ray images to determine loss of cartilage as shown by a narrowing
of space between the bones in a joint; magnetic resonance imaging
(MRI), providing detailed images of bone and soft tissues,
including cartilage), may be used for monitoring the health status
of the subject. The effects of the treatment of a senolytic
combination can be analyzed by comparing symptoms of patients
suffering from or at risk of an inflammatory disease or disorder,
such as osteoarthritis, who have received the treatment with those
of patients without such a treatment or with placebo treatment.
[0146] In certain embodiments, senolytic agents may be used for
treating and/or preventing (i.e., decreasing or reducing the
likelihood of occurrence) rheumatoid arthritis (RA). Dysregulation
of innate and adaptive immune responses characterize rheumatoid
arthritis (RA), which is an autoimmune disease the incidence of
which increases with age. Certain features of senescence of the
immune system and immune cells, such as the decrease in T-cell
generation and diversity, may contribute to the development of RA.
Older adults may therefore be more susceptible to RA. In young
adults, premature immunosenescence may contribute to development of
the disease. See, for example, Lindstrom et al., Journal of the
American Geriatrics Society, 19 Jul. 2010 DOI:
10.1111/j.1532-5415.2010.02965.x). In certain embodiments, RA is
excluded.
[0147] Chronic inflammation may also contribute to other
age-related or aging related diseases and disorders, such as
kyphosis and osteoporosis. Kyphosis is a severe curvature in the
spinal column, and it is frequently seen with normal and premature
aging (see, e.g., Katzman et al. (2010) J. Orthop. Sports Phys.
Ther. 40: 352-360). Age-related kyphosis often occurs after
osteoporosis weakens spinal bones to the point that they crack and
compress. A few types of kyphosis target infants or teens. Severe
kyphosis can affect lungs, nerves, and other tissues and organs,
causing pain and other problems. Kyphosis has been associated with
cellular senescence. Characterizing the capability of a senolytic
combination for treating kyphosis may be determined in pre-clinical
animal models used in the art. By way of example, TTD mice develop
kyphosis (see, e.g., de Boer et al. (2002) Science 296: 1276-1279);
other mice that may be used include BubR1.sup.H/H mice, which are
also known to develop kyphosis (see, e.g., Baker et al. (2011)
Nature 479: 232-36). Kyphosis formation is visually measured over
time. The level of senescent cells decreased by treatment with the
senolytic combination can be determined by detecting the presence
of one or more senescent cell associated markers such as by
SA-.beta.-Gal staining.
[0148] Osteoporosis is a progressive bone disease that is
characterized by a decrease in bone mass and density that may lead
to an increased risk of fracture. Bone mineral density (BMD) is
reduced, bone microarchitecture deteriorates, and the amount and
variety of proteins in bone are altered. Osteoporosis is typically
diagnosed and monitored by a bone mineral density test.
Post-menopausal women or women who have reduced estrogen are most
at risk. While both men and women over 75 are at risk, women are
twice as likely to develop osteoporosis than men. The level of
senescent cells decreased by treatment with the senolytic
combination can be determined by detecting the presence of one or
more senescent cell associated markers such as by SA-.beta.-Gal
staining.
[0149] In still other embodiments, an inflammatory/autoimmune
disorder that may be treated with the senolytic combinations
described herein includes irritable bowel syndrome (IBS) and
inflammatory bowel diseases, such as ulcerative colitis and Crohn's
disease. Inflammatory bowel disease (IBD) involves chronic
inflammation of all or part of the digestive tract. In addition to
life-threatening complications arising from IBD, the disease can be
painful and debilitating. Ulcerative colitis is an inflammatory
bowel disease that causes long-lasting inflammation in part of the
digestive tract. Symptoms usually develop over time, rather than
suddenly. Ulcerative colitis usually affects only the innermost
lining of the large intestine (colon) and rectum. Crohn's disease
is an inflammatory bowel disease that causes inflammation anywhere
along the lining of your digestive tract, and often extends deep
into affected tissues. This can lead to abdominal pain, severe
diarrhea, and malnutrition. The inflammation caused by Crohn's
disease can involve different areas of the digestive tract.
Diagnosis and monitoring of the diseases is performed according to
methods and diagnostic tests routinely practiced in the art,
including blood tests, colonoscopy, flexible sigmoidoscopy, barium
enema, CT scan, MRI, endoscopy, and small intestine imaging.
[0150] In other embodiments, the methods described herein may be
useful for treating a subject who has herniated intervertebral
discs. Subjects with these herniated discs exhibit elevated
presence of cell senescence in the blood and in vessel walls (see
e.g., Roberts et al. (2006) Eur. Spine J. 15 Suppl 3: S312-316).
Symptoms of a herniated intervertebral disc may include pain,
numbness or tingling, or weakness in an arm or leg. Increased
levels of proinflammatory molecules and matrix metalloproteases are
also found in aging and degenerating discs tissues, suggesting a
role for senescence cells (see e.g., Chang-Qing et al. (2007)
Ageing Res. Rev. 6: 247-61). Animal models may be used to
characterize the effectiveness of a senolytic combination in
treating herniated intervertebral discs; degeneration of the
intervertebral disc is induced in mice by compression and disc
strength evaluated (see e.g., Lotz et al. (1998) Spine
(Philadelphia Pa. 1976). 23:2493-506).
[0151] Other inflammatory or autoimmune diseases that may be
treated or prevented (i.e., likelihood of occurrence is reduced) by
using a senolytic combination include eczema, psoriasis,
osteoporosis, and pulmonary diseases (e.g., chronic obstructive
pulmonary disease (COPD), idiopathic pulmonary fibrosis (IPF),
asthma), inflammatory bowel disease, and mucositis (including oral
mucositis, which in some instances is induced by radiation).
Certain fibrosis or fibrotic conditions of organs such as renal
fibrosis, liver fibrosis, pancreatic fibrosis, cardiac fibrosis,
skin wound healing, and oral submucous fibrosis may be treated with
using the senolytic combination.
[0152] In certain embodiments, the senescent cell associated
disorder is an inflammatory disorder of the skin, such as by way of
a non-limiting examples, psoriasis and eczema that may be treated
or prevented according to the methods described herein that
comprise administration of a senolytic combination. Psoriasis is
characterized by abnormally excessive and rapid growth of the
epidermal layer of the skin. A diagnosis of psoriasis is usually
based on the appearance of the skin. Skin characteristics typical
for psoriasis are scaly red plaques, papules, or patches of skin
that may be painful and itch. In psoriasis, cutaneous and systemic
overexpression of various proinflammatory cytokines is observed
such as IL-6, a key component of the SASP. Eczema is an
inflammation of the skin that is characterized by redness, skin
swelling, itching and dryness, crusting, flaking, blistering,
cracking, oozing, or bleeding. The effectiveness of senolytic
combinations for treatment of psoriasis and eczema and monitoring
of a subject who receives such a senolytic combination can be
readily determined by a person skilled in the medical or clinical
arts. One or any combination of diagnostic methods, including
physical examination (such as skin appearance), assessment of
monitoring of clinical symptoms (such as itching, swelling, and
pain), and performance of analytical tests and methods described
herein and practiced in the art (i.e., determining the level of
pro-inflammatory cytokines).
[0153] Other immune disorders or conditions that may be treated
with a senolytic combination include conditions resulting from a
host immune response to an organ transplant (e.g., kidney, bone
marrow, liver, lung, or heart transplant), such as rejection of the
transplanted organ. The senolytic combination may be used for
treating or reducing the likelihood of occurrence of graft-vs-host
disease.
[0154] Pulmonary Diseases and Disorders.
[0155] In one embodiment, methods are provided for treating or
preventing (i.e., reducing the likelihood of occurrence) a
senescence-associated disease or disorder that is a pulmonary
disease or disorder by killing senescent cells (i.e., established
senescent cells) associated with the disease or disorder in a
subject who has the disease or disorder by administering a
senolytic combination. Senescence associated pulmonary diseases and
disorders include, for example, idiopathic pulmonary fibrosis
(IPF), chronic obstructive pulmonary disease (COPD), asthma, cystic
fibrosis, bronchiectasis, and emphysema.
[0156] COPD is a lung disease defined by persistently poor airflow
resulting from the breakdown of lung tissue (emphysema) and the
dysfunction of the small airways (obstructive bronchiolitis).
Primary symptoms of COPD include shortness of breath, wheezing,
chest tightness, chronic cough, and excess sputum production.
Elastase from cigarette smoke-activated neutrophils and macrophages
disintegrates the extracellular matrix of alveolar structures,
resulting in enlarged air spaces and loss of respiratory capacity
(see, e.g., Shapiro et al., Am. J. Respir. Cell Mol. Biol. 32,
367-372 (2005)). COPD is most commonly caused by tobacco smoke
(including cigarette smoke, cigar smoke, secondhand smoke, pipe
smoke), occupational exposure (e.g., exposure to dust, smoke or
fumes), and pollution, occurring over decades thereby implicating
aging as a risk factor for developing COPD.
[0157] The processes involved in causing lung damage include, for
example, oxidative stress produced by the high concentrations of
free radicals in tobacco smoke; cytokine release due to
inflammatory response to irritants in the airway; and impairment of
anti-protease enzymes by tobacco smoke and free radicals, allowing
proteases to damage the lungs. Genetic susceptibility likely
contributes to the disease because only about 20% of smokers
develop COPD. In about 1% percent of people with COPD, the disease
results from a genetic disorder that causes low level production of
alpha-1-antitrypsin in the liver. The enzyme is normally secreted
into the bloodstream to help protect the lungs.
[0158] Pulmonary fibrosis is a chronic and progressive lung disease
characterized by stiffening and scarring of the lung, which may
lead to respiratory failure, lung cancer, and heart failure.
Fibrosis is associated with repair of epithelium. Fibroblasts are
activated, production of extracellular matrix proteins is
increased, and transdifferentiation to contractile myofibroblasts
contribute to wound contraction. A provisional matrix plugs the
injured epithelium and provides a scaffold for epithelial cell
migration, involving an epithelial-mesenchymal transition (EMT).
Blood loss associated with epithelial injury induces platelet
activation, production of growth factors, and an acute inflammatory
response. Normally, the epithelial barrier heals and the
inflammatory response resolves. However, in fibrotic disease the
fibroblast response continues, resulting in unresolved wound
healing. Formation of fibroblastic foci is a feature of the
disease, reflecting locations of ongoing fibrogenesis. As the name
connotes, the etiology of IPF is unknown. The involvement of
cellular senescence in IPF is suggested by the observations that
the incidence of the disease increases with age and that lung
tissue in IPF patients is enriched for SA-.beta.-Gal-positive cells
and contains elevated levels of the senescence marker p21 (see,
e.g., Minagawa et al., Am. J. Physiol. Lung Cell. Mol. Physiol.
300:L391-L401 (2011); see also, e.g., Naylor et al., supra). Short
telomeres are a risk factor common to both IPF and cellular
senescence (see, e.g., Alder et al., Proc. Natl. Acad. Sci. USA
105:13051-56 (2008)). Without wishing to be bound by theory, the
contribution of cellular senescence to IPF is suggested by the
report that SASP components of senescent cells, such as IL-6, IL-8,
and IL-1.beta., promote fibroblast-to-myofibroblast differentiation
and epithelial-mesenchymal transition, resulting in extensive
remodeling of the extracellular matrix of the alveolar and
interstitial spaces (see, e.g., Minagawa et al., supra).
[0159] Subjects at risk of developing pulmonary fibrosis include
those exposed to environmental or occupational pollutants, such as
asbestosis and silicosis; who smoke cigarettes; having some typical
connective tissue diseases such as rheumatoid arthritis, SLE and
scleroderma; having other diseases that involve connective tissue,
such as sarcoidosis and Wegener's granulomatosis; having
infections; taking certain medications (e.g., amiodarone,
bleomycin, busufan, methotrexate, and nitrofurantoin); those
subject to radiation therapy to the chest; and those whose family
member has pulmonary fibrosis.
[0160] Symptoms of COPD may include any one of shortness of breath,
especially during physical activities; wheezing; chest tightness;
having to clear your throat first thing in the morning because of
excess mucus in the lungs; a chronic cough that produces sputum
that may be clear, white, yellow or greenish; blueness of the lips
or fingernail beds (cyanosis); frequent respiratory infections;
lack of energy; unintended weight loss (observed in later stages of
disease). Subjects with COPD may also experience exacerbations,
during which symptoms worsen and persist for days or longer.
Symptoms of pulmonary fibrosis are known in the art and include
shortness of breath, particularly during exercise; dry, hacking
cough; fast, shallow breathing; gradual unintended weight loss;
tiredness; aching joints and muscles; and clubbing (widening and
rounding of the tips of the fingers or toes).
[0161] Subjects suffering from COPD or pulmonary fibrosis can be
identified using standard diagnostic methods routinely practiced in
the art. Monitoring the effect of a senolytic combination
administered to a subject who has or who is at risk of developing a
pulmonary disease may be performed using the methods typically used
for diagnosis. Generally, one or more of the following exams or
tests may be performed: physical exam, patient's medical history,
patient's family's medical history, chest X-ray, lung function
tests (such as spirometry), blood test (e.g., arterial blood gas
analysis), bronchoalveolar lavage, lung biopsy, CT scan, and
exercise testing.
[0162] Other pulmonary diseases or disorders that may be treated by
using a senolytic combination include, for example, emphysema,
asthma, bronchiectasis, and cystic fibrosis (see, e.g., Fischer et
al., Am J Physiol Lung Cell Mol Physiol. 304(6):L394-400 (2013)).
These diseases may also be exacerbated by tobacco smoke (including
cigarette smoke, cigar smoke, secondhand smoke, pipe smoke),
occupational exposure (e.g., exposure to dust, smoke or fumes),
infection, and/or pollutants that induce cells into senescence and
thereby contribute to inflammation. Emphysema is sometimes
considered as a subgroup of COPD.
[0163] Bronchiectasis is results from damage to the airways that
causes them to widen and become flabby and scarred. Bronchiectasis
usually is caused by a medical condition that injures the airway
walls or inhibits the airways from clearing mucus. Examples of such
conditions include cystic fibrosis and primary ciliary dyskinesia
(PCD). When only one part of the lung is affected, the disorder may
be caused by a blockage rather than a medical condition.
[0164] The methods described herein for treating a senescence
associate pulmonary disease or disorder may also be used for
treating a subject who is aging and has loss (or degeneration) of
pulmonary function (i.e., declining or impaired pulmonary function
compared with a younger subject) and/or degeneration of pulmonary
tissue. The respiratory system undergoes various anatomical,
physiological and immunological changes with age. The structural
changes include chest wall and thoracic spine deformities that can
impair the total respiratory system compliance resulting in
increased effort to breathe. The respiratory system undergoes
structural, physiological, and immunological changes with age. An
increased proportion of neutrophils and lower percentage of
macrophages can be found in bronchoalveolar lavage (BAL) of older
adults compared with younger adults. Persistent low grade
inflammation in the lower respiratory tract can cause proteolytic
and oxidant-mediated injury to the lung matrix resulting in loss of
alveolar unit and impaired gas exchange across the alveolar
membrane seen with aging. Sustained inflammation of the lower
respiratory tract may predispose older adults to increased
susceptibility to toxic environmental exposure and accelerated lung
function decline. (See, for example, Sharma et al., Clinical
Interventions in Aging 1:253-60 (2006)). Oxidative stress
exacerbates inflammation during aging (see, e.g., Brod, Inflamm Res
2000; 49:561-570; Hendel et al., Cell Death and Differentiation
(2010) 17:596-606). Alterations in redox balance and increased
oxidative stress during aging precipitate the expression of
cytokines, chemokines, and adhesion molecules, and enzymes (see,
e.g., Chung et al., Ageing Res Rev 2009; 8:18-30). Constitutive
activation and recruitment of macrophages, T cells, and mast cells
foster release of proteases leading to extracellular matrix
degradation, cell death, remodeling, and other events that can
cause tissue and organ damage during chronic inflammation (see,
e.g., Demedts et al., Respir Res 2006; 7: 53-63). By administering
a senolytic combination to an aging subject (which includes a
middle-aged adult who is asymptomatic), the decline in pulmonary
function may be decelerated or inhibited by killing and removing
senescent cells from the respiratory tract.
[0165] The effectiveness of a senolytic combination can readily be
determined by a person skilled in the medical and clinical arts.
One or any combination of diagnostic methods, including physical
examination, assessment and monitoring of clinical symptoms, and
performance of analytical tests and methods described herein, may
be used for monitoring the health status of the subject. The
effects of the treatment of a therapeutic agent or pharmaceutical
composition can be analyzed using techniques known in the art, such
as comparing symptoms of patients suffering from or at risk of
pulmonary fibrosis that have received the treatment with those of
patients without such a treatment or with placebo treatment. In
addition, methods and techniques that evaluate mechanical
functioning of the lung, for example, techniques that measure lung
capacitance, elastance, and airway hypersensitivity may be
performed. To determine lung function and to monitor lung function
throughout treatment, any one of numerous measurements may be
obtained, expiratory reserve volume (ERV), forced vital capacity
(FVC), forced expiratory volume (FEV) (e.g., FEV in one second,
FEV1), FEV1/FEV ratio, forced expiratory flow 25% to 75%, and
maximum voluntary ventilation (MVV), peak expiratory flow (PEF),
slow vital capacity (SVC). Total lung volumes include total lung
capacity (TLC), vital capacity (VC), residual volume (RV), and
functional residual capacity (FRC). Gas exchange across alveolar
capillary membrane can be measured using diffusion capacity for
carbon monoxide (DLCO). Peripheral capillary oxygen saturation
(SpO.sub.2) can also be measured; normal oxygen levels are
typically between 95% and 100%. An SpO.sub.2 level below 90%
suggests the subject has hypoxemia. Values below 80% are considered
critical and requiring intervention to maintain brain and cardiac
function and avoid cardiac or respiratory arrest.
[0166] Neurological Diseases and Disorders.
[0167] Senescent cell-associated diseases or disorders treatable by
administering a senolytic combination described herein include
neurological diseases or disorders. Such senescent cell associated
diseases and disorders include Parkinson's disease, Alzheimer's
disease (and other dementias), motor neuron dysfunction (MND), mild
cognitive impairment (MCI), Huntington's disease, and diseases and
disorders of the eyes, such as the neurodegenerative
disease/disorder, macular degeneration. Other diseases of the eye
that are associated with increasing age are glaucoma, vision loss,
and cataracts.
[0168] Parkinson's disease is the second most common
neurodegenerative disease. It is a disabling condition of the brain
characterized by slowness of movement (bradykinesia), shaking,
stiffness, and in the later stages, loss of balance. Many of these
symptoms are due to the loss of certain nerves in the brain, which
results in the lack of dopamine. This disease is characterized by
neurodegeneration, such as the loss of about 50% to 70% of the
dopaminergic neurons in the substantia nigra pars compacta, a
profound loss of dopamine in the striatum, and/or the presence of
intracytoplasmic inclusions (Lewy bodies), which are composed
mainly of alpha-synuclein and ubiquitin. Parkinson's disease also
features locomotor deficits, such as tremor, rigidity,
bradykinesia, and/or postural instability. These motor
manifestations can also be accompanied by nonmotor symptoms such as
olfactory deficits, sleep impairment, and neuropsychiatric
disorders. Generally, diagnosis of Parkinson's disease is based on
symptoms, medical history, and neurological and/or physical
examination of a patient. Subjects at risk of developing
Parkinson's disease include those having a family history of
Parkinson's disease and those exposed to pesticides (e.g., rotenone
or paraquat), herbicides (e.g., agent orange), or heavy metals.
Senescence of dopamine-producing neurons is thought to contribute
to the observed cell death in PD through the production of reactive
oxygen species (see, e.g., Cohen et al., J. Neural Transm. Suppl.
19:89-103 (1983)); therefore, the methods and senolytic
combinations described herein are useful for treatment and
prophylaxis of Parkinson's disease.
[0169] Methods for detecting, monitoring or quantifying
neurodegenerative deficiencies and/or locomotor deficits associated
with Parkinson's diseases are known in the art, such as
histological studies, biochemical studies, and behavioral
assessment (see, e.g., U.S. Application Publication No.
2012/0005765). Symptoms of Parkinson's disease are known in the art
and include, but are not limited to, difficulty starting or
finishing voluntary movements, jerky, stiff movements, muscle
atrophy, shaking (tremors), and changes in heart rate, but normal
reflexes, bradykinesia, and postural instability. There is a
growing recognition that people diagnosed with Parkinson's disease
may have cognitive impairment, including mild cognitive impairment,
in addition to their physical symptoms.
[0170] Alzheimer's disease (AD) is a neurodegenerative disease that
shows a slowly progressive mental deterioration with failure of
memory, disorientation, and confusion, leading to profound
dementia. Age is the single greatest predisposing risk factor for
developing AD, which is the leading cause of dementia in the
elderly (see, e.g., Hebert, et al., Arch. Neurol. 60:1119-1122
(2003)). Early clinical symptoms show remarkable similarity to mild
cognitive impairment (see below), which is characterized by
difficulty in remembering recent life experiences or people's
names. As the disease progresses, impaired judgment, confusion,
behavioral changes, disorientation, and difficulty in walking and
swallowing occur.
[0171] Alzheimer's disease is characterized by the presence of
neurofibrillary tangles and amyloid (senile) plaques in
histological specimens. The disease predominantly involves the
limbic and cortical regions of the brain. The argyrophilic plaques
containing the amyloidogenic A.beta. fragment of amyloid precursor
protein (APP) are scattered throughout the cerebral cortex and
hippocampus. Neurofibrillary tangles are found in pyramidal neurons
predominantly located in the neocortex, hippocampus, and nucleus
basalis of Meynert. Other changes, such as granulovacuolar
degeneration in the pyramidal cells of the hippocampus, and neuron
loss and gliosis in the cortex and hippocampus, are observed. The
vast majority of Alzheimer's disease cases are sporadic; however, a
small percentage of patients have an autosomal dominant form of the
disease called early onset familial Alzheimer's disease that may be
a predisposing factor for developing the disease. Most autosomal
dominant familial AD can be attributed to mutations in APP,
presenilin 1, or presenilin 2. Subjects at risk of developing
Alzheimer's disease include those of advanced age, those with a
family history of Alzheimer's disease, those with genetic risk
genes (e.g., ApoE4) or deterministic gene mutations (e.g., APP,
PS1, or PS2), and those with history of head trauma or
heart/vascular conditions (e.g., high blood pressure, heart
disease, stroke, diabetes, high cholesterol).
[0172] A number of behavioral and histopathological assays are
known in the art for evaluating Alzheimer's disease phenotype, for
characterizing therapeutic agents, and assessing treatment.
Histological analyses are typically performed postmortem.
Histological analysis of A.beta. levels may be performed using
Thioflavin-S. Congo red, or anti-A.beta. staining (e.g., 4G8, 10D5,
or 6E10 antibodies) to visualize A.beta. deposition on sectioned
brain tissues (see, e.g., Holcomb et al., 1998, Nat. Med. 4:97-100;
Borchelt et al., 1997, Neuron 19:939-945; Dickson et al., 1988, Am.
J. Path. 132:86-101). In vivo methods of visualizing A.beta.
deposition in transgenic mice have been also described. BSB
((trans,
trans)-1-bromo-2,5-bis-(3-hydroxycarbonyl-4-hydroxy)styrylbenzene)
and PET tracer .sup.11C-labelled Pittsburgh Compound-B (PIB) bind
to A.beta. plaques (see, e.g., Skovronsky et al., 2000, Proc. Natl.
Acad. Sci. USA 97:7609-7614; Klunk et al., 2004, Ann. Neurol.
55:306-319). .sup.19F-containing amyloidophilic Congo red-type
compound FSB
((E,E)-1-fluoro-2,5-bis-(3-hydroxycarbonyl-4-hydroxy)styrylbenzene)
allows visualization of A.beta. plaques by MRI (see, e.g., Higuchi
et al., 2005, Nature Neurosci. 8:527-533). Radiolabeled,
putrescine-modified amyloid-beta peptide labels amyloid deposits in
vivo in a mouse model of Alzheimer's disease (see, e.g., Wengenack
et al., 2000, Nat. Biotechnol. 18:868-872).
[0173] Increased glial fibrillary acidic protein (GFAP) by
astrocytes is a marker for astroglial activation and gliosis during
neurodegeneration. A.beta. plaques are associated with
GFAP-positive activated astrocytes, and may be visualized via GFAP
staining (see, e.g., Nagele et al. 2004, Neurobiol. Aging
25:663-674; Mandybur et al., 1990, Neurology 40:635-639; Liang et
al., 2010, J. Biol. Chem. 285:27737-27744). Neurofibrillary tangles
may be identified by immunohistochemistry using thioflavin-S
fluorescent microscopy and Gallyas silver stains (see, e.g., Gotz
et al., 2001, J. Biol. Chem. 276:529-534; U.S. Pat. No. 6,664,443).
Axon staining with electron microscopy and axonal transport studies
may be used to neuronal degeneration (see, e.g., Ishihara et al.,
1999, Neuron 24:751-762).
[0174] Subjects suffering from Alzheimer's disease can be
identified using standard diagnostic methods known in the art for
Alzheimer's disease. Generally, diagnosis of Alzheimer's disease is
based on symptoms (e.g., progressive decline in memory function,
gradual retreat from and frustration with normal activities,
apathy, agitation or irritability, aggression, anxiety, sleep
disturbance, dysphoria, aberrant motor behavior, disinhibition,
social withdrawal, decreased appetite, hallucinations, dementia),
medical history, neuropsychological tests, neurological and/or
physical examination of a patient. Cerebrospinal fluid may also be
for tested for various proteins that have been associated with
Alzheimer pathology, including tau, amyloid beta peptide, and
AD7C-NTP. Genetic testing is also available for early-onset
familial Alzheimer disease (eFAD), an autosomal-dominant genetic
disease. Clinical genetic testing is available for individuals with
AD symptoms or at-risk family members of patients with early-onset
disease. In the U.S., mutations for PS2, and APP may be tested in a
clinical or federally approved laboratory under the Clinical
Laboratory Improvement Amendments. A commercial test for PS1
mutations is also available (Elan Pharmaceuticals).
[0175] The effectiveness of a senolytic combination described
herein and monitoring of a subject who receives the senolytic
combination can readily be determined by a person skilled in the
medical and clinical arts. One or any combination of diagnostic
methods, including physical examination, assessment and monitoring
of clinical symptoms, and performance of analytical tests and
methods described herein, may be used for monitoring the health
status of the subject. The effects of administering a senolytic
combination can be analyzed using techniques known in the art, such
as comparing symptoms of patients suffering from or at risk of
Alzheimer's disease that have received the treatment with those of
patients without such a treatment or with placebo treatment.
[0176] Mild Cognitive Impairment (MCI).
[0177] MCI is a brain-function syndrome involving the onset and
evolution of cognitive impairments beyond those expected based on
age and education of the individual, but which are not significant
enough to interfere with this individual's daily activities. MCI is
an aspect of cognitive aging that is considered to be a
transitional state between normal aging and the dementia into which
it may convert (see, Pepeu, Dialogues in Clinical Neuroscience
6:369-377, 2004). It is characterized by subtle memory impairment,
mild neuropathological changes (e.g., astogliosis, few deposit of
beta-amyloid, diffuse amyloid in the neocortex, neurofibrillary
tangles in the medial temporal lobe), and changes in the
cholinergic system (e.g., loss of cholinergic neurons, decrease in
acetyl choline release). MCI that primarily affects memory is known
as "amnestic MCI." A person with amnestic MCI may start to forget
important information that he or she would previously have recalled
easily, such as recent events. Amnestic MCI is frequently seen as
prodromal stage of Alzheimer's disease. MCI that affects thinking
skills other than memory is known as "nonamnestic MCI." This type
of MCI affect thinking skills such as the ability to make sound
decisions, judge the time or sequence of steps needed to complete a
complex task, or visual perception. Individuals with nonamnestic
MCI are believed to be more likely to convert to other types of
dementias (e.g., dementia with Lewy bodies).
[0178] Persons in the medical art have a growing recognition that
people diagnosed with Parkinson's disease may have MCI in addition
to their physical symptoms. Recent studies show 20-30% of people
with Parkinson's disease have MCI, and that their MCI tends to be
non-amnestic. Parkinson's disease patients with MCI sometimes go on
to develop full blown dementia (Parkinson's disease with
dementia).
[0179] Methods for detecting, monitoring, quantifying or assessing
neuropathological deficiencies associated with MCI are known in the
art, including astrocyte morphological analyses, release of
acetylcholine, silver staining for assessing neurodegeneration, and
PiB PET imaging to detect beta amyloid deposits (see, e.g., U.S.
Application Publication No. 2012/0071468; Pepeu, 2004, supra).
Methods for detecting, monitoring, quantifying or assessing
behavioral deficiencies associated with MCI are also known in the
art, including eight-arm radial maze paradigm,
non-matching-to-sample task, allocentric place determination task
in a water maze, Morris maze test, visuospatial tasks, and delayed
response spatial memory task, olfactory novelty test (see,
id.).
[0180] Motor Neuron Dysfunction (MND).
[0181] MND is a group of progressive neurological disorders that
destroy motor neurons, the cells that control essential voluntary
muscle activity such as speaking, walking, breathing and
swallowing. It is classified according to whether degeneration
affects upper motor neurons, lower motor neurons, or both. Examples
of MNDs include, but are not limited to Amyotrophic Lateral
Sclerosis (ALS), also known as Lou Gehrig's Disease, progressive
bulbar palsy, pseudobulbar palsy, primary lateral sclerosis,
progressive muscular atrophy, lower motor neuron disease, and
spinal muscular atrophy (SMA) (e.g., SMA1 also called
Werdnig-Hoffmann Disease, SMA2, SMA3 also called Kugelberg-Welander
Disease, and Kennedy's disease), post-polio syndrome, and
hereditary spastic paraplegia. In adults, the most common MND is
amyotrophic lateral sclerosis (ALS), which affects both upper and
lower motor neurons. It can affect the arms, legs, or facial
muscles. Primary lateral sclerosis is a disease of the upper motor
neurons, while progressive muscular atrophy affects only lower
motor neurons in the spinal cord. In progressive bulbar palsy, the
lowest motor neurons of the brain stem are most affected, causing
slurred speech and difficulty chewing and swallowing. There are
almost always mildly abnormal signs in the arms and legs. Patients
with MND exhibit a phenotype of Parkinson's disease (e.g., having
tremor, rigidity, bradykinesia, and/or postural instability).
Methods for detecting, monitoring or quantifying locomotor and/or
other deficits associated with Parkinson's diseases, such as MND,
are known in the art (see, e.g., U.S. Application Publication No.
20120005765).
[0182] Methods for detecting, monitoring, quantifying or assessing
motor deficits and histopathological deficiencies associated with
MND are known in the art, including histopathological, biochemical,
and electrophysiological studies and motor activity analysis (see,
e.g., Rich et al., J Neurophysiol 88:3293-3304, 2002; Appel et al.,
Proc. Natl. Acad. Sci. USA 88:647-51, 1991). Histopathologically,
MNDs are characterized by death of motor neurons, progressive
accumulation of detergent-resistant aggregates containing SOD1 and
ubiquitin and aberrant neurofilament accumulations in degenerating
motor neurons. In addition, reactive astroglia and microglia are
often detected in diseased tissue. Patients with an MND show one or
more motor deficits, including muscle weakness and wasting,
uncontrollable twitching, spasticity, slow and effortful movements,
and overactive tendon reflexes.
[0183] Macular degeneration is a neurodegenerative disease that
causes the loss of photoreceptor cells in the central part of
retina, called the macula. Macular degeneration generally is
classified into two types: dry type and wet type. The dry form is
more common than the wet, with about 90% of age-related macular
degeneration (ARMD) patients diagnosed with the dry form. The wet
form of the disease usually leads to more serious vision loss.
While the exact causes of age-related macular degeneration are
still unknown, the number of senescent retinal pigmented epithelial
(RPE) cells increases with age. Age and certain genetic factors and
environmental factors (see, e.g., Lyengar et al., Am. J. Hum.
Genet. 74:20-39 (2004) (Epub 2003 Dec. 19); Kenealy et al., Mol.
Vis. 10:57-61 (2004); Gorin et al., Mol. Vis. 5:29 (1999)) are risk
factors for developing ARMD. Environment predisposing factors
include omega-3 fatty acids intake (see, e.g., Christen et al.,
Arch Ophthalmol. 129:921-29 (2011)); estrogen exposure (see, e.g.,
Feshanich et al., Arch Ophthalmol 126(4):519-24) (2008)); and
increased serum levels of vitamin D (see, e.g., Millen, et al.,
Arch Ophthalmol. 129(4):481-89 (2011)). Genetic predisposing risk
factors include reduced levels Dicer1 (enzyme involved in
maturation of micro RNA) in eyes of patients with dry AMD, and
decreased micro RNAs contributes to a senescent cell profile; and
DICER1 ablation induces premature senescence (see, e.g., Mudhasani
J. Cell. Biol. (2008)).
[0184] Dry ARMD is associated with atrophy of RPE layer causes loss
of photoreceptor cells. The dry form of ARMD may result from the
aging and thinning of macular tissues, and deposition of pigment in
the macula. Senescence appears to inhibit both replication and
migration of RPE, resulting in permanent RPE depletion in the
macula of dry AMD patients (see, e.g., Iriyama et al., J. Biol.
Chem. 283:11947-953 (2008)). With wet ARMD, new blood vessels grow
beneath the retina and leak blood and fluid. This abnormal leaky
choroidal neovascularization causes the retinal cells to die,
creating blind spots in central vision. Different forms of macular
degeneration may also occur in younger patients. Non-age related
etiology may be linked to heredity, diabetes, nutritional deficits,
head injury, infection, or other factors.
[0185] Declining vision noticed by the patient or by an
ophthalmologist during a routine eye exam may be the first
indicator of macular degeneration. The formation of exudates, or
"drusen," underneath the Bruch's membrane of the macula is often
the first physical sign that macular degeneration may develop.
Symptoms include perceived distortion of straight lines and, in
some cases, the center of vision appears more distorted than the
rest of a scene; a dark, blurry area or "white-out" appears in the
center of vision; and/or color perception changes or diminishes.
Diagnosing and monitoring of a subject with macular degeneration
may be accomplished by a person skilled in the ophthalmic art
according to art-accepted periodic eye examination procedures and
report of symptoms by the subject.
[0186] Glaucoma.
[0187] In certain embodiments, the senescence associated disease or
disorder is glaucoma. Glaucoma is a broad term used to describe a
group of diseases that causes visual field loss, often without any
other prevailing symptoms. The lack of symptoms often leads to a
delayed diagnosis of glaucoma until the terminal stages of the
disease. Even if subjects afflicted with glaucoma do not become
blind, their vision is often severely impaired. Normally, clear
fluid flows into and out of the front part of the eye, known as the
anterior chamber. In individuals who have open/wide-angle glaucoma,
this fluid drains too slowly, leading to increased pressure within
the eye. If left untreated, this high pressure subsequently damages
the optic nerve and can lead to complete blindness. The loss of
peripheral vision is caused by the death of ganglion cells in the
retina. Ganglion cells are a specific type of projection neuron
that connects the eye to the brain. When the cellular network
required for the outflow of fluid was subjected to SA-.beta.-Gal
staining, a fourfold increase in senescence has been observed in
glaucoma patients (see, e.g., Liton et al., Exp. Gerontol.
40:745-748 (2005)).
[0188] For monitoring the effect of a therapy on inhibiting
progression of glaucoma, standard automated perimetry (visual field
test) is the most widely used technique. In addition, several
algorithms for progression detection have been developed (see,
e.g., Wesselink et al., Arch Ophthalmol. 127(3):270-274 (2009), and
references therein). Additional methods include gonioscopy
(examines the trabecular meshwork and the angle where fluid drains
out of the eye); imaging technology, for example scanning laser
tomography (e.g., HRT3), laser polarimetry (e.g., GDX), and ocular
coherence tomography); ophthalmoscopy; and pachymeter measurements
that determine central corneal thickness.
[0189] Cataracts.
[0190] Cataracts are a clouding of the lens of an eye, causing
blurred vision, and if left untreated can result in blindness.
Surgery is effective and routinely performed to remove cataracts.
Administration of a senolytic combination described herein may
result in decreasing the likelihood of occurrence of a cataract or
may slow or inhibit progression of a cataract. The presence and
severity of a cataract can be monitored by eye exams using methods
routinely performed by a person skilled in the ophthalmology
art.
[0191] Metabolic Disease or Disorder.
[0192] Senescent cell-associated diseases or disorders treatable by
administering a senolytic combination described herein include
metabolic diseases or disorders. Such senescent cell associated
diseases and disorders include diabetes, metabolic syndrome,
diabetic ulcers, and obesity.
[0193] Diabetes is characterized by high levels of blood glucose
caused by defects in insulin production, insulin action, or both.
The great majority (90 to 95%) of all diagnosed cases of diabetes
in adults are type 2 diabetes, characterized by the gradual loss of
insulin production by the pancreas. Diabetes is the leading cause
of kidney failure, nontraumatic lower-limb amputations, and new
cases of blindness among adults in the U.S. Diabetes is a major
cause of heart disease and stroke and is the seventh leading cause
of death in the U.S. (see, e.g., Centers for Disease Control and
Prevention, National diabetes fact sheet: national estimates and
general information on diabetes and prediabetes in the United
States, 2011 ("Diabetes fact sheet")). A senolytic combination
described herein may be used for treating and/or preventing type 2
diabetes, particularly age-, diet- and obesity-associated type 2
diabetes.
[0194] Involvement of senescent cells in metabolic disease, such as
obesity and type 2 diabetes, has been suggested as a response to
injury or metabolic dysfunction (see, e.g., Tchkonia et al., Aging
Cell 9:667-684 (2010)). Fat tissue from obese mice showed induction
of the senescence markers SA-.beta.-Gal, p53, and p21 (see, e.g.,
Tchkonia et al., supra; Minamino et al., Nat. Med. 15:1082-1087
(2009)). A concomitant upregulation of proinflammatory cytokines,
such as tumor necrosis factor-.alpha. and Ccl2/MCP1, was observed
in the same fat tissue (see, e.g., Minamino et al., supra).
Induction of senescent cells in obesity potentially has clinical
implications because proinflammatory SASP components are also
suggested to contribute to type 2 diabetes (see, e.g., Tchkonia et
al., supra). A similar pattern of upregulation of senescence
markers and SASP components are associated with diabetes, both in
mice and in humans (see, e.g., Minamino et al., supra).
Accordingly, the methods described herein that comprise
administering a senolytic combination may be useful for treatment
or prophylaxis of type 2 diabetes, as well as obesity and metabolic
syndrome. Without wishing to be bound by theory, contact of
senescent preadipocytes with a senolytic combination described
herein thereby killing the senescent preadipocytes may provide
clinical and health benefit to a person who has any one of
diabetes, obesity, or metabolic syndrome.
[0195] Subjects suffering from type 2 diabetes can be identified
using standard diagnostic methods known in the art for type 2
diabetes. Generally, diagnosis of type 2 diabetes is based on
symptoms (e.g., increased thirst and frequent urination, increased
hunger, weight loss, fatigue, blurred vision, slow-healing sores or
frequent infections, and/or areas of darkened skin), medical
history, and/or physical examination of a patient. Subjects at risk
of developing type 2 diabetes include those who have a family
history of type 2 diabetes and those who have other risk factors
such as excess weight, fat distribution, inactivity, race, age,
prediabetes, and/or gestational diabetes.
[0196] The effectiveness of a senolytic combination can readily be
determined by a person skilled in the medical and clinical arts.
One or any combination of diagnostic methods, including physical
examination, assessment and monitoring of clinical symptoms, and
performance of analytical tests and methods, such as those
described herein, may be used for monitoring the health status of
the subject. A subject who is receiving a senolytic combination
described herein for treatment or prophylaxis of diabetes can be
monitored, for example, by assaying glucose and insulin tolerance,
energy expenditure, body composition, fat tissue, skeletal muscle,
and liver inflammation, and/or lipotoxicity (muscle and liver lipid
by imaging in vivo and muscle, liver, bone marrow, and pancreatic
.beta.-cell lipid accumulation and inflammation by histology).
Other characteristic features or phenotypes of type 2 diabetes are
known and can be assayed as described herein and by using other
methods and techniques known and routinely practiced in the
art.
[0197] Subjects who have type 2 diabetes or who are at risk of
developing type 2 diabetes may have metabolic syndrome. Metabolic
syndrome in humans is typically associated with obesity and
characterized by one or more of cardiovascular disease, liver
steatosis, hyperlipidemia, diabetes, and insulin resistance. A
patient with metabolic syndrome may present with a cluster of
metabolic disorders or abnormalities which may include, for
example, one or more of hypertension, type-2 diabetes,
hyperlipidemia, dyslipidemia (e.g., hypertriglyceridemia,
hypercholesterolemia), insulin resistance, liver steatosis
(steatohepatitis), hypertension, atherosclerosis, and other
metabolic disorders.
[0198] Obesity and obesity-related are used to refer to conditions
of subjects who have a body mass that is measurably greater than
ideal for their height and frame. Body Mass Index (BMI) is a
measurement tool used to determine excess body weight, and is
calculated from the height and weight of a subject. A human is
considered overweight when the person has a BMI of 25-29; a person
is considered obese when the person has a BMI of 30-39, and a
person is considered severely obese when the person has a BMI of
.gtoreq.40. Accordingly, the terms obesity and obesity-related
refer to human subjects with body mass index values of greater than
30, greater than 35, or greater than 40. A category of obesity not
captured by BMI is called "abdominal obesity" in the art, which
relates to the extra fat found around a subject's middle, which is
an important factor in health, even independent of BMI. The
simplest and most often used measure of abdominal obesity is waist
size. Generally abdominal obesity in women is defined as a waist
size 35 inches or higher, and in men as a waist size of 40 inches
or higher. More complex methods for determining obesity require
specialized equipment, such as magnetic resonance imaging or dual
energy X-ray absorptiometry machines.
[0199] A condition or disorder associated with diabetes and
senescence is a diabetic ulcer (i.e., diabetic wound). An ulcer is
a breakdown in the skin, which may extend to involve the
subcutaneous tissue or even muscle or bone. These lesions occur,
particularly, on the lower extremities. Patients with diabetic
venous ulcer exhibit elevated presence of cellular senescence at
sites of chronic wounds (see, e.g., Stanley et al. (2001) J. Vas.
Surg. 33: 1206-1211). Chronic inflammation is also observed at
sites of chronic wounds, such as diabetic ulcers (see, e.g., Goren
et al. (2006) Am. J. Pathol. 7 168: 65-77; Seitz et al. (2010) Exp.
Diabetes Res. 2010: 476969), suggesting that the proinflammatory
cytokine phenotype of senescent cells has a role in the
pathology.
[0200] Renal Dysfunction:
[0201] Nephrological pathologies, such as glomerular disease, arise
in the elderly. Glomerulonephritis is characterized by inflammation
of the kidney and by the expression of two proteins, IL1.alpha. and
IL1.beta. (see, e.g., Niemir et al. (1997) Kidney Int. 52:393-403).
IL1.alpha. and IL1.beta. are considered master regulators of SASP
(see, e.g., Coppe et al. (2008) PLoS. Biol. 6: 2853-68). Glomerular
disease is associated with elevated presence of senescent cells,
especially in fibrotic kidneys (see, e.g., Sis et al. (2007) Kidney
Int. 71:218-226).
[0202] Dermatological Disease or Disorder.
[0203] Senescence-associated diseases or disorders treatable by
administering a senolytic combination described herein include
dermatological diseases or disorders. Such senescent cell
associated diseases and disorders include psoriasis and eczema,
which are also inflammatory diseases and are discussed in greater
detail above. Other dermatological diseases and disorders that are
associated with senescence include rhytides (wrinkles due to
aging); pruritis (linked to diabetes and aging); dysesthesia
(chemotherapy side effect that is linked to diabetes and multiple
sclerosis); psoriasis (as noted) and other papulosquamous
disorders, for example, erythroderma, lichen planus, and lichenoid
dermatosis; atopic dermatitis (a form of eczema and associated with
inflammation); eczematous eruptions (often observed in aging
patients and linked to side effects of certain drugs). Other
dermatological diseases and disorders associated with senescence
include eosinophilic dermatosis (linked to certain kinds of
hemotologic cancers); reactive neutrophilic dermatosis (associated
with underlying diseases such as inflammatory bowel syndrome);
pemphigus (an autoimmune disease in which autoantibodies form
against desmoglein); pemphigoid and other immunobullous dermatosis
(autoimmune blistering of skin); fibrohistocytic proliferations of
skin, which is linked to aging; and cutaneous lymphomas that are
more common in older populations. Another dermatological disease
that may be treatable according to the methods described herein
includes cutaneous lupus, which is a symptom of lupus
erythematosus. Late onset lupus may be linked to decreased (i.e.,
reduced) function of T-cell and B-cells and cytokines
(immunosenescence) associated with aging.
[0204] Metastasis.
[0205] In a particular embodiment, methods are provided for
treating or preventing (i.e., reducing the likelihood of occurrence
or development of) a senescence cell associated disease (or
disorder or condition), which is metastasis. The senolytic agents
described herein may also be used according to the methods
described herein for treating or preventing (i.e., reducing the
likelihood of occurrence of) metastasis (i.e., the spreading and
dissemination of cancer or tumor cells) from one organ or tissue to
another organ or tissue in the body. Even though an agent as used
in the combinations and methods described herein is not used in a
manner that is sufficient to be considered as a primary cancer
therapy, the methods and senolytic combinations described herein
may be used in a manner (e.g., a short term course of therapy) that
is useful for inhibiting metastases.
[0206] By way of explanation and example, one of the agents
described herein for use in a senolytic combination is dasatinib,
which is a compound used for treating chronic myelogenous leukemia
(chronic Philadelphia chromosome+CML (CP-CML)) and acute
lymphoblastic leukemia (ALL, Philadelphia chromosome positive). To
treat these leukemias, dasatinib is administered to the patient
daily at a dose between 100 and 140 mg/kg per day depending on the
leukemia to be treated (see, e.g., SPRYCEL product label). In
contrast, as an agent included in a senolytic combination, and as
described in greater detail herein, dasatinib is administered
significantly less often, such as for example, once every week or
any number of days between 1-7 days every week; or once every two
weeks or any number of days between 1-7 days every two weeks at
minimum (e.g., once per 0.5-12 months) together with a second
agent. In addition, dasatinib may be administered for treating the
senescence associated diseases and disorders described herein at a
lower dose per day, lower cumulative dose per treatment course,
lower cumulative dose per treatment cycle, and a lower cumulative
dose over 2 or more treatment cycles.
[0207] In one embodiment, methods are provided for preventing
(i.e., reducing the likelihood of occurrence of), inhibiting, or
retarding metastasis in a subject who has a cancer by administering
a senolytic combination as described herein. In a particular
embodiment, the senolytic combination is administered on one or
more days within a treatment window (i.e., treatment course) of no
longer than 1 day, 7 days, or 14 days. In other embodiments, the
treatment course is no longer than 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,
12, 13, 14, 15, 16, 17, 18, 19, 20, or no longer than 21 days. In
other embodiments, the treatment course is a single day. In certain
embodiments, the senolytic combination is administered on two or
more days within a treatment window of no longer than 7 or 14 days,
on 3 or more days within a treatment window of no longer than 7 or
14 days; on 4 or more days within a treatment window of no longer
than 7 or 14 days; on 5 or more days within a treatment window of
no longer than 7 or 14 days; on 6, 7, 8, 9, 10, 11, 12, 13, or 14
days within treatment window of no longer than 14 days. In certain
embodiments, when the treatment window is 3 days or more, the
treatment may be administered every 2.sup.nd day (i.e., every other
day). In other certain embodiments when the treatment window is 4
days or more, the treatment may be administered every 3.sup.rd day
(i.e., every other third day).
[0208] Because cells may be induced to senesce by cancer therapies,
such as radiation and certain chemotherapy drugs (e.g.,
doxorubicin; paclitaxel; gemcitabine; pomalidomide; lenalidomide),
a senolytic combination described herein may be administered after
the chemotherapy or radiotherapy to kill (or facilitate killing) of
these senescent cells. As discussed herein and understood in the
art, establishment of senescence, such as shown by the presence of
a senescence-associated secretory phenotype (SASP), occurs over
several days (see, e.g., Laberge et al., Aging Cell 11:569-78
(2012); Coppe et al., PLoS Biol 6: 2853-68 (2008); Coppe et al.
PLoS One 5:39188 (2010); Rodier et al., Nat. Cell Biol. 11:973-979;
Freund et al., EMBO J. 30:1536-1548 (2011)). Therefore,
administering a senolytic combination to kill senescent cells, and
thereby reduce the likelihood of occurrence or reduce the extent of
metastasis, is initiated when senescence has been established. As
discussed herein, the following treatment courses for
administration of the senolytic combination may be used in methods
described herein for treating or preventing (i.e., reducing the
likelihood of occurrence, or reducing the severity) a chemotherapy
or radiotherapy side effect.
[0209] In certain embodiments, when chemotherapy or radiotherapy is
administered in a treatment cycle of at least one day on-therapy
(i.e., chemotherapy or radiotherapy)) followed by at least 3, 4, 5,
6, 7, 8, 9, 10, 11, 12, 13, 14 (or about 2 weeks), 15, 16, 17, 18,
19, 20, 21 (or about 3 weeks) days, or about 4 weeks (about one
month) off-therapy (i.e., off chemo- or radio-therapy), the
senolytic combination is administered on one or more days during
the off-therapy time interval (time period) beginning on or after
the second day of the off-therapy time interval and ending on or
before the last day of the off-therapy time interval. By way of
illustrative example, if n is the number of days off-therapy, then
the senolytic combination is administered on at least one day and
no more than n-1 days of the off-therapy time interval. In a
certain particular embodiment when chemotherapy or radiotherapy is
administered in a treatment cycle of at least one day on-therapy
(i.e., chemotherapy or radiotherapy)) followed by at least one week
off-therapy, the senolytic combination is administered on one or
more days during the off-therapy time interval beginning on or
after the second day of the off-therapy time interval and ending on
or before the last day of the off-therapy time interval. In a more
specific embodiment, when chemotherapy or radiotherapy is
administered in a treatment cycle of at least one day on-therapy
(i.e., chemotherapy or radiotherapy)) followed by at least one week
off-therapy, the senolytic combination is administered on one day
that is the sixth day of the off-therapy time interval. In other
specific embodiments, when chemotherapy or radiotherapy is
administered in a treatment cycle of at least one day on-therapy
(i.e., chemotherapy or radiotherapy)) followed by at least two
weeks off-therapy, the senolytic combination is administered
beginning on the sixth day of the off-chemo- or radio-therapy time
interval and ending at least one day or at least two days prior to
the first day of a subsequent chemotherapy or radiation therapy
treatment course. By way of example, if the off-chemo- or
radio-therapy time interval is two weeks, a senolytic combination
may be administered on at least one and on no more than 7 days
(i.e., 1, 2, 3, 4, 5, 6, or 7 days) of the off-therapy time
interval beginning on the sixth day after the chemotherapy or
radiotherapy course ends (i.e., the sixth day of the off
chemo-radio-therapy interval). When the off-chemo- or radio-therapy
time interval is at least three weeks, a senolytic combination may
be administered on at least one day and on no more than 14 days
(i.e., 1-14 days: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14
days) of the off-therapy time interval beginning on the sixth day
after the chemotherapy or radiotherapy course ends. In other
embodiments, depending on the off-chemo-radio-therapy interval, the
senolytic combination treatment course is at least one day and no
longer than 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,
18, 19, 20, or no more than 21 days (i.e., 1-21 days), provided
that administration of the senolytic combination is not concurrent
with the chemotherapy or radiotherapy. In certain embodiments, the
senolytic combination treatment course is a single day. In certain
embodiments, the senolytic combination is administered on two or
more days within a treatment window of no longer than 14 days, on 3
or more days within a treatment window of no longer than 14 days;
on 4 or more days within a treatment window of no longer than 14
days; on 5 or more days within a treatment window of no longer than
14 days; on 6, 7, 8, 9, 10, 11, 12, 13, or 14 days within treatment
window of no longer than 14 days. In certain embodiments, when the
senolytic combination is administered to a subject during a
treatment course of 3 days or more, the combination may be
administered every 2.sup.nd day (i.e., every other day). In other
certain embodiments when the senolytic combination is administered
to a subject during a treatment course of 4 days or more, the
combination may be administered every 3.sup.rd day (i.e., every
other third day).
[0210] Many chemotherapy and radiotherapy treatment regimens
comprise a finite number of cycles of on-drug therapy followed by
off-drug therapy or comprise a finite timeframe in which the
chemotherapy or radiotherapy is administered. Such cancer treatment
regimens may also be called treatment protocols. The protocols are
determined by clinical trials, drug labels, and clinical staff in
conjunction with the subject to be treated. The number of cycles of
a chemotherapy or radiotherapy or the total length of time of a
chemotherapy or radiotherapy regimen can vary depending on the
patient's response to the cancer therapy. The timeframe for such
treatment regimens is readily determined by a person skilled in the
oncology art. In another embodiment for treating metastasis, a
senolytic combination may be administered after the treatment
regimen of chemotherapy or radiotherapy has been completed. In a
particular embodiment, the senolytic combination is administered
after the chemotherapy or radiotherapy has been completed on one or
more days within treatment window (i.e., senolytic combination
treatment course) of no longer than 14 days. In other embodiments,
the senolytic combination treatment course is no longer than 2, 3,
4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or no
more than 21 days. In other embodiments, the treatment course is a
single day. In certain embodiments, the senolytic combination is
administered on two or more days within a treatment window of no
longer than 14 days, on 3 or more days within a treatment window of
no longer than 14 days; on 4 or more days within a treatment window
of no longer than 14 days; on 5 or more days within a treatment
window of no longer than 14 days; on 6, 7, 8, 9, 10, 11, 12, 13, or
14 days within treatment window of no longer than 14 days. In
certain embodiments, when the senolytic combination is administered
to a subject after chemotherapy or radiotherapy for a treatment
window of 3 days or more, the combination may be administered every
2.sup.nd day (i.e., every other day). In other certain embodiments
when the senolytic combination is administered to a subject for a
treatment window of 4 days or more, the combination may be
administered every 3.sup.rd day (i.e., every other third day). In
one embodiment, the treatment with the senolytic combination may be
initiated at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14
days or later after the cancer treatment regimen has been
completed. In a more particular embodiment, the treatment with the
senolytic combination may be initiated at least 6, 7, 8, 9, 10, 11,
12, 13, or 14 days or later after the cancer treatment regimen has
been completed. Any of the additional treatment courses and
treatment cycles for administration of a senolytic combination
described herein may be followed for inhibiting metastasis in a
subject after a chemotherapy or radiotherapy protocol has been
completed.
[0211] A chemotherapy may be referred to as a chemotherapy,
chemotherapeutic, or chemotherapeutic drug. Many chemotherapeutics
are compounds referred to as small organic molecules. Chemotherapy
is a term that is also used to describe a combination
chemotherapeutic drugs that are administered to treat a particular
cancer. As understood by a person skilled in the art, a
chemotherapy may also refer to a combination of two or more
chemotherapeutic molecules that are administered coordinately and
which may be referred to as combination chemotherapy. Numerous
chemotherapeutic drugs are used in the oncology art and include,
without limitation, alkylating agents; antimetabolites;
anthracyclines, plant alkaloids; and topoisomerase inhibitors.
[0212] Metastasis of a cancer occurs when the cancer cells (i.e.,
tumor cells) spread beyond the anatomical site of origin and
initial colonization to other areas throughout the body of the
subject. A cancer that may metastasize may be a solid tumor or may
be a liquid tumor (e.g., a leukemia). Cancers that are liquid
tumors are classified in the art as those that occur in blood, bone
marrow, and lymph nodes and include generally, leukemias (myeloid
and lymphocytic), lymphomas (e.g., Hodgkin lymphoma), and melanoma
(including multiple myeloma). Leukemias include for example, acute
lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic
lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), and
hairy cell leukemia. Cancers that are solid tumors and occur in
greater frequency in humans include, for example, prostate cancer,
testicular cancer, breast cancer, brain cancer, pancreatic cancer,
colon cancer, thyroid cancer, stomach cancer, lung cancer, ovarian
cancer, Kaposi's sarcoma, skin cancer (including squamous cell skin
cancer), renal cancer, head and neck cancers, throat cancer,
squamous carcinomas that form on the moist mucosal linings of the
nose, mouth, throat, etc.), bladder cancer, osteosarcoma (bone
cancer), cervical cancer, endometrial cancer, esophageal cancer,
liver cancer, and kidney cancer. In certain specific embodiments,
the senescent cell-associated disease or disorder treated or
prevented (i.e., likelihood of occurrence or development is
reduced) by the methods described herein is metastasis of melanoma
cells, prostate cancer cells, testicular cancer cells, breast
cancer cells, brain cancer cells, pancreatic cancer cells, colon
cancer cells, thyroid cancer cells, stomach cancer cells, lung
cancer cells, ovarian cancer cells, Kaposi's sarcoma cells, skin
cancer cells, renal cancer cells, head or neck cancer cells, throat
cancer cells, squamous carcinoma cells, bladder cancer cells,
osteosarcoma cells, cervical cancer cells, endometrial cancer
cells, esophageal cancer cells, liver cancer cells, or kidney
cancer cells.
[0213] The methods described herein are also useful for inhibiting,
retarding or slowing progression of metastatic cancer of any one of
the types of tumors described in the medical art. Types of cancers
(tumors) include the following: adrenocortical carcinoma, childhood
adrenocortical carcinoma, aids-related cancers, anal cancer,
appendix cancer, basal cell carcinoma, childhood basal cell
carcinoma, bladder cancer, childhood bladder cancer, bone cancer,
brain tumor, childhood astrocytomas, childhood brain stem glioma,
childhood central nervous system atypical teratoid/rhabdoid tumor,
childhood central nervous system embryonal tumors, childhood
central nervous system germ cell tumors, childhood
craniopharyngioma brain tumor, childhood ependymoma brain tumor,
breast cancer, childhood bronchial tumors, carcinoid tumor,
childhood carcinoid tumor, gastrointestinal carcinoid tumor,
carcinoma of unknown primary, childhood carcinoma of unknown
primary, childhood cardiac (heart) tumors, cervical cancer,
childhood cervical cancer, childhood chordoma, chronic
myeloproliferative disorders, colon cancer, colorectal cancer,
childhood colorectal cancer, extrahepatic bile duct cancer, ductal
carcinoma in situ (dcis), endometrial cancer, esophageal cancer,
childhood esophageal cancer, childhood esthesioneuroblastoma, eye
cancer, malignant fibrous histiocytoma of bone, gallbladder cancer,
gastric (stomach) cancer, childhood gastric (stomach) cancer,
gastrointestinal stromal tumors (gist), childhood gastrointestinal
stromal tumors (gist), childhood extracranial germ cell tumor,
extragonadal germ cell tumor, gestational trophoblastic tumor,
glioma, head and neck cancer, childhood head and neck cancer,
hepatocellular (liver) cancer, hypopharyngeal cancer, kidney
cancer, renal cell kidney cancer, Wilms tumor, childhood kidney
tumors, Langerhans cell histiocytosis, laryngeal cancer, childhood
laryngeal cancer, leukemia, acute lymphoblastic leukemia (all),
acute myeloid leukemia (aml), chronic lymphocytic leukemia (cll),
chronic myelogenous leukemia (cml), hairy cell leukemia, lip
cancer, liver cancer (primary), childhood liver cancer (primary),
lobular carcinoma in situ (lcis), lung cancer, non-small cell lung
cancer, small cell lung cancer, lymphoma, aids-related lymphoma,
burkitt lymphoma, cutaneous t-cell lymphoma, Hodgkin lymphoma,
non-Hodgkin lymphoma, primary central nervous system lymphoma
(cns), melanoma, childhood melanoma, intraocular (eye) melanoma,
Merkel cell carcinoma, malignant mesothelioma, childhood malignant
mesothelioma, metastatic squamous neck cancer with occult primary,
midline tract carcinoma involving NUT gene, mouth cancer, childhood
multiple endocrine neoplasia syndromes, mycosis fungoides,
myelodysplastic syndromes, myelodysplastic neoplasms,
myeloproliferative neoplasms, multiple myeloma, nasal cavity
cancer, nasopharyngeal cancer, childhood nasopharyngeal cancer,
neuroblastoma, oral cancer, childhood oral cancer, oropharyngeal
cancer, ovarian cancer, childhood ovarian cancer, epithelial
ovarian cancer, low malignant potential tumor ovarian cancer,
pancreatic cancer, childhood pancreatic cancer, pancreatic
neuroendocrine tumors (islet cell tumors), childhood
papillomatosis, paraganglioma, paranasal sinus cancer, parathyroid
cancer, penile cancer, pharyngeal cancer, pheochromocytoma,
pituitary tumor, plasma cell neoplasm, childhood pleuropulmonary
blastoma, prostate cancer, rectal cancer, renal pelvis transitional
cell cancer, retinoblastoma, salivary gland cancer, childhood
salivary gland cancer, ewing sarcoma family of tumors, Kaposi
Sarcoma, osteosarcoma, rhabdomyosarcoma, childhood
rhabdomyosarcoma, soft tissue sarcoma, uterine sarcoma, sezary
syndrome, childhood skin cancer, nonmelanoma skin cancer, small
intestine cancer, squamous cell carcinoma, childhood squamous cell
carcinoma, testicular cancer, childhood testicular cancer, throat
cancer, thymoma and thymic carcinoma, childhood thymoma and thymic
carcinoma, thyroid cancer, childhood thyroid cancer, ureter
transitional cell cancer, urethral cancer, endometrial uterine
cancer, vaginal cancer, vulvar cancer, Waldenstrom
Macroglobulinemia.
[0214] Chemotherapeutic and Radiotherapy Side Effects.
[0215] In another embodiment, the senescence cell associated
disorder or condition is a chemotherapeutic side effect or a
radiotherapy side effect. A senolytic combination administered as
described herein may be used for treating and/or preventing (i.e.,
reducing the likelihood of occurrence of) a chemotherapeutic side
effect or a radiotherapy side effect. Removal or destruction of
senescent cells may ameliorate acute toxicity, including acute
toxicity comprising energy imbalance, of a chemotherapy or
radiotherapy. Acute toxic side effects include but are not limited
to gastrointestinal toxicity (e.g., nausea, vomiting, constipation,
anorexia, diarrhea), peripheral neuropathy, fatigue, malaise, low
physical activity, hematological toxicity (e.g., anemia),
hepatotoxicity, alopecia (hair loss), pain, infection, mucositis,
fluid retention, dermatological toxicity (e.g., rashes, dermatitis,
hyperpigmentation, urticaria, photosensitivity, nail changes),
mouth, gum or throat problems, or any toxic side effect caused by a
chemotherapy or radiotherapy. For example, toxic side effects
caused by radiotherapy or chemotherapy (see, e.g., National Cancer
Institute web site) may be ameliorated by the methods described
herein. Accordingly, in certain embodiments, methods are provided
herein for ameliorating (reducing, inhibiting, or preventing
occurrence (i.e., reducing the likelihood of occurrence)) acute
toxicity or reducing severity of a toxic side effect (i.e.,
deleterious side effect) of a chemotherapy or radiotherapy or both
in a subject who receives the therapy, wherein the method comprises
administering to the subject an agent that selectively kills,
removes, or destroys or facilitates selective destruction of
senescent cells. Administration of a senolytic combination for
treating or reducing the likelihood of occurrence, or reducing the
severity of a chemotherapy or radiotherapy side effect may be
accomplished by the same treatment courses described above for
treatment/prevention of metastasis. As described for treating or
preventing (i.e., reducing the likelihood of occurrence of)
metastasis, the senolytic combination is administered during the
off-chemotherapy or off-radiotherapy time interval or after the
chemotherapy or radiotherapy treatment regimen has been
completed.
[0216] In a more specific embodiment, the acute toxicity is an
acute toxicity comprising energy imbalance and may comprise one or
more of weight loss, endocrine change(s) (e.g., hormone imbalance,
change in hormone signaling), and change(s) in body composition. In
certain embodiments, an acute toxicity comprising energy imbalance
relates to decreased or reduced ability of the subject to be
physically active, as indicated by decreased or diminished
expenditure of energy than would be observed in a subject who did
not receive the medical therapy. By way of non-limiting example,
such an acute toxic effect that comprises energy imbalance includes
low physical activity. In other particular embodiments, energy
imbalance comprises fatigue or malaise.
[0217] In one embodiment, a chemotherapy side effect to be treated
or prevented (i.e., likelihood of occurrence is reduced) by a
senolytic combination described herein is cardiotoxicity. In one
embodiment, the cardiotoxicity results from administration of
doxorubicin. Doxorubicin is an anthracycline topoisomerase is
approved for treating patients who have ovarian cancer after
failure of a platinum based therapy; Kaposi's sarcoma after failure
of primary systemic chemotherapy or intolerance to the therapy; or
multiple myeloma in combination with bortezomib in patients who
have not previously received bortezomib or who have received at
least one prior therapy. Doxorubicin may cause myocardial damage
that could lead to congestive heart failure if the total lifetime
dose to a patient exceeds 550 mg/m.sup.2. Cardiotoxicity may occur
at even lower doses if the patient also receives mediastinal
irradiation or another cardiotoxic drug. See drug product inserts
(e.g., DOXIL, ADRIAMYCIN).
[0218] In other embodiments, a senolytic combination may be used in
the methods as provided herein for ameliorating chronic or long
term side effects. Chronic toxic side effects typically result from
multiple exposures to or administrations of a chemotherapy or
radiotherapy over a longer period of time. Certain toxic effects
appear long after treatment (also called late toxic effects) and
result from damage to an organ or system by the therapy. Organ
dysfunction (e.g., neurological, pulmonary, cardiovascular, and
endocrine dysfunction) has been observed in patients who were
treated for cancers during childhood (see, e.g., Hudson et al.,
JAMA 309:2371-81 (2013)). Without wishing to be bound by any
particular theory, by destroying senescent cells, particular normal
cells that have been induced to senescence by chemotherapy or
radiotherapy, the likelihood of occurrence of a chronic side effect
may be reduced, or the severity of a chronic side effect may be
reduced or diminished, or the time of onset of a chronic side
effect may be delayed. Chronic and/or late toxic side effects that
occur in subjects who received chemotherapy or radiation therapy
include by way of non-limiting example, cardiomyopathy, congestive
heart disease, inflammation, early menopause, osteoporosis,
infertility, impaired cognitive function, peripheral neuropathy,
secondary cancers, cataracts and other vision problems, hearing
loss, chronic fatigue, reduced lung capacity, and lung disease.
[0219] Administration of a senolytic combination for treating or
reducing the likelihood of occurrence, or reducing the severity of
a chemotherapy or radiotherapy side effect may be accomplished by
the same treatment courses described above for treatment/prevention
of metastasis. As with treating or preventing metastasis, the
senolytic combination is administered during the off-chemotherapy
or off-radiotherapy time interval or after the chemotherapy or
radiotherapy treatment regimen has been completed.
[0220] Age-Related Diseases and Disorders.
[0221] A senolytic combination may also be useful for treating or
preventing (i.e., reducing the likelihood of occurrence) of an
age-related disease or disorder that occurs as part of the natural
aging process or that occurs when the subject is exposed to a
senescence inducing agent or factor (e.g., irradiation,
chemotherapy, smoking tobacco, high-fat/high sugar diet, other
environmental factors). An age-related disorder or disease or an
age-sensitive trait may be associated with a senescence-inducing
stimulus. The efficacy of a method of treatment described herein
may be manifested by reducing the number of symptoms of an
age-related disorder or age-sensitive trait associated with a
senescence-inducing stimulus, decreasing the severity of one or
more symptoms, or delaying the progression of an age-related
disorder or age-sensitive trait associated with a
senescence-inducing stimulus. In other particular embodiments,
preventing an age-related disorder or age-sensitive trait
associated with a senescence-inducing stimulus refers to preventing
(i.e., reducing the likelihood of occurrence) or delaying onset of
an age-related disorder or age-sensitive trait associated with a
senescence-inducing stimulus, or reoccurrence of one or more
age-related disorder or age-sensitive trait associated with a
senescence-inducing stimulus. Age related diseases or conditions
include, for example, renal dysfunction, kyphosis, herniated
intervertebral disc, frailty, hair loss, hearing loss, vision loss
(blindness or impaired vision), muscle fatigue, skin conditions,
skin nevi, diabetes, metabolic syndrome, and sarcopenia. Vision
loss refers to the absence of vision when a subject previously had
vision. Various scales have been developed to describe the extent
of vision and vision loss based on visual acuity. Age-related
diseases and conditions also include dermatological conditions, for
example without limitation, treating one or more of the following
conditions: wrinkles, including superficial fine wrinkles;
hyperpigmentation; scars; keloid; dermatitis; psoriasis; eczema
(including seborrheic eczema); rosacea; vitiligo; ichthyosis
vulgaris; dermatomyositis; and actinic keratosis.
[0222] Frailty has been defined as a clinically recognizable state
of increased vulnerability resulting from aging-associated decline
in reserve and function across multiple physiologic systems that
compromise a subject's ability to cope with every day or acute
stressors. Frailty has been may be characterized by compromised
energetics characteristics such as low grip strength, low energy,
slowed waking speed, low physical activity, and/or unintentional
weight loss. Studies have suggested that a patient may be diagnosed
with frailty when three of five of the foregoing characteristics
are observed (see, e.g., Fried et al., J. Gerontol. A Biol. Sci.
Med, Sci. 2001; 56(3):M146-M156; Xue, Clin. Geriatr. Med. 2011;
27(1):1-15). In certain embodiments, aging and diseases and
disorders related to aging may be treated or prevented (i.e., the
likelihood of occurrence of is reduced) by administering a
senolytic combination. The senolytic combination may inhibit
senescence of adult stem cells or inhibit accumulation, kill, or
facilitate removal of adult stem cells that have become senescent.
See, e.g., Park et al., J. Clin. Invest. 113:175-79 (2004) and
Sousa-Victor, Nature 506:316-21 (2014) describing importance of
preventing senescence in stem cells to maintain regenerative
capacity of tissues.
[0223] The effectiveness of a senolytic combination with respect to
treating a senescence-associated disease or disorder described
herein can readily be determined by a person skilled in the medical
and clinical arts. One or any combination of diagnostic methods
appropriate for the particular disease or disorder, which methods
are well known to a person skilled in the art, including physical
examination, patient self-assessment, assessment and monitoring of
clinical symptoms, performance of analytical tests and methods,
including clinical laboratory tests, physical tests, and
exploratory surgery, for example, may be used for monitoring the
health status of the subject and the effectiveness of the senolytic
combination. The effects of the methods of treatment described
herein can be analyzed using techniques known in the art, such as
comparing symptoms of patients suffering from or at risk of a
particular disease or disorder that have received the
pharmaceutical composition comprising a senolytic combination with
those of patients who were not treated with the senolytic
combination or who received a placebo treatment.
Methods of Use
[0224] Provided herein are methods for selectively killing
senescent cells that result in treating a senescence-associated
disease or disorder and comprises use of a senolytic combination as
described herein. As described herein, the senolytic combination is
administered in a manner that would be considered ineffective for
treating a cancer. Because the method used for treating a
senescence associated disease with a senolytic combination
described herein comprises one or more of a decreased daily dose or
each compound of the combination, decreased cumulative dose over a
single therapeutic cycle, or decreased cumulative dose of the
senolytic combination over multiple therapeutic cycles compared
with the amount of the combination (or each compound alone)
required for cancer therapy, the likelihood is decreased that one
or more adverse effects (i.e., side effects) will occur, which
adverse effects are associated with treating a subject according to
a regimen optimized for treating a cancer.
[0225] The methods and senolytic combinations for selective killing
of senescent cells may be used for treating and/or preventing
(i.e., decreasing the likelihood of occurrence) of numerous
age-related pathologies and diseases as described herein. As
disclosed herein, senescent cell associated diseases and disorders
may be treated or prevented (i.e., the likelihood of occurrence of
is reduced) by administering a senolytic combination comprising at
least two agents, and which agents alter either a cell survival
signaling pathway or an inflammatory pathway or alters both the
cell survival signaling pathway and the inflammatory pathway. The
senolytic combinations described herein, and pharmaceutical
compositions comprising the combinations, are useful for treating,
reducing the likelihood of occurrence of, or delaying onset of a
senescent cell-associated disease or disorder in a subject who has
a senescent cell-associated disease or disorder or who has at least
one predisposing factor for developing the senescent
cell-associated disease or disorder. The combinations that comprise
at least two agents (e.g., dasatinib and quercetin or an analog
thereof), and at least one agent (for convenience, referred to
herein as a first agent) and a second agent are different and each
independently alters either one or both of a survival signaling
pathway and an inflammatory pathway.
[0226] In one embodiment, a method is provided for treating a
subject who has a senescent cell associated disease or disorder or
for prophylaxis (e.g., for reducing the likelihood of occurrence of
or delaying onset) of a senescent cell-associated disease or
disorder in a subject who has at least one predisposing factor for
developing the senescent cell-associated disease or disorder, which
method comprises administering to the subject a senolytic
combination that alters either one or both of a cell survival
signaling pathway and an inflammatory pathway in the senescent
cell. Also provided is a method for killing a senescent cell,
comprising contacting a senescent cell and the agents of the
combination, which agents alters either one or both of a cell
survival signaling pathway and an inflammatory pathway in the
senescent cell. In a particular embodiment, the senescent cell is
present in a subject who has a senescent cell associated disease or
disorder or who has at least one predisposing factor for developing
the senescent cell-associated disease or disorder. In certain
embodiments, the senescent cell-associated disease or disorder is a
cardiovascular disease or disorder (e.g., atherosclerosis),
inflammatory disease or disorder (e.g., osteoarthritis), a
pulmonary disease or disorder (e.g., idiopathic pulmonary fibrosis;
chronic obstructive pulmonary disease), a neurological disease or
disorder (e.g., Parkinson's disease, Alzheimer's disease, macular
degeneration, MCI, MND), a chemotherapeutic side effect (e.g., an
acute or chronic toxic effect; cardiotoxicity), a radiotherapy side
effect (e.g., an acute or chronic toxic effect), or metastasis of
any tumor type described herein. In certain other embodiments, the
senescent cell-associated disease or disorder is a metabolic
disease or disorder, including for example, diabetes, obesity, or
metabolic syndrome.
[0227] In certain embodiments, a senolytic agent or a senolytic
combination is administered within a treatment cycle, which
treatment cycle comprises a treatment course followed by a
non-treatment interval. A treatment course of administration refers
herein to a finite time frame over which one or more doses of the
senolytic combination on one or more days are administered. The
finite time frame may be also called herein a treatment window. In
one embodiment, the treatment course is between about 1-7 days
(i.e., 1, 2, 3, 4, 5, 6, or 7 days) every 0.5-12 months; provided
that if the senescence associated disease or disorder is a
senescence associated metabolic disorder, the senolytic combination
is administered during a treatment course of about 1-7 days (i.e.,
1, 2, 3, 4, 5, 6, or 7 days) every 4-12 months. In certain
embodiments, the treatment course is only one day. In other
specific embodiments, the senolytic combination may be administered
at time intervals as follows: once every 1, 2, 3, 4, 5, or 6 days
or every week, every 2 weeks (or once per 0.5 month), every 3
weeks, every 4 weeks (or once per month), every 2 months, every 3
months, every 4 months, every 5 months, every 6 months, every 7
months, every 8 months, every 9 months, every 10 months, every 11
months, or every 12 months (or once a year) or once at longer
intervals. The time interval when the senolytic combination is not
administered is also called herein a non-treatment interval or
off-treatment. In certain embodiments, the senolytic combination is
administered once every 0.5 month-12 months (i.e., once every 2
weeks (about 0.5 month), once every 2 months, once every 3 months,
once every 4 months, once every 5 months, once every 6 months,
every 7 months, once every 8 months, once every 9 months, once
every 10 months, once every 11 months, or once every 12 months (or
once a year)). In other particular embodiments, the senolytic
combination is administered once every 4-12 months (i.e., once
every 4 months, once every 5 months, once every 6 months, every 7
months, once every 8 months, once every 9 months, once every 10
months, once every 11 months, or once every 12 months). In other
specific embodiments, a senolytic combination may be administered
daily for one week, one month, 6 weeks, or 2 months; a next
treatment course of daily dosing for one week, one month, 6 weeks,
or 2 months may be initiated 2 days, 3 days, 4 days, 5 days, 6
days, one week, two weeks, three weeks, one month, 2 months, 3
months or longer after the treatment course is completed. In other
words, the non-treatment interval is at least about 2 days, 3 days,
4 days, 5 days, 6 days, one week, two weeks, three weeks, one
month, 2 months, 3 months or longer. In certain embodiments, the
non-treatment interval is 4 months, 5 months, 6 months, 7 months, 8
months, 9 months, 10 months, 11 months, or a year. In other
embodiments, a senolytic combination may be administered during a
treatment course of once or twice daily for 8, 9, 10, 11, 12, 13,
or 14 days followed by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,
or 14 days with no administration of the combination (i.e.,
non-treatment interval) followed by another treatment course of
once or twice daily administration of the combination for 8, 9, 10,
11, 12, 13, or 14 days. These cycles of administering a
combination, followed by a non-treatment interval may be repeated
as needed for treatment or prophylaxis of the particular subject's
disease or disorder. The amount of the dose administered according
to any of the aforementioned dosing regimens may administered as
single total dose on the day of administration, or the total dose
for the day's administration may be divided into multiple aliquots,
such as 2, 3, 4, or 5 separate doses on the day of administration.
The dosing regimens (e.g., the time interval between doses) can be
reviewed and modified or adjusted, continued or discontinued, as
determined by a person skilled in the art, depending on the
responsiveness of the subject to the therapy, the stage of the
disease, the general health of the subject, and other factors that
are described herein and in the art.
[0228] In a more particular embodiment, methods are provided that
comprise administering a senolytic agent or a senolytic combination
once every 0.5-12 months to a subject who has a senescent
cell-associated disease or disorder or who has at least one
predisposing risk factor for developing the disease or disorder,
which disease or disorder is any one of a cardiovascular disease or
disorder, inflammatory disease or disorder, a pulmonary disease or
disorder, a neurological disease or disorder, a chemotherapeutic
side effect, a radiotherapy side effect, or metastasis. In another
particular embodiment, a method is provided for killing a senescent
cell by contacting the senescent cell with a senolytic combination,
in a subject by administering the senolytic combination once every
0.5-12 months. In other particular embodiments, the combination is
administered on 1, 2, 3, 4, 5, 6, or 7 days every 0.5-12
months.
[0229] In still another embodiment, a method of treatment or
prophylaxis of a senescent cell associated disease or disorder is
provided wherein a senolytic agent or a senolytic combination is
administered once every 4-12 months to a subject who has a
senescent cell-associated disease or disorder or who has at least
one predisposing risk factor for developing the disease or
disorder, which may be any one of a cardiovascular disease or
disorder, inflammatory disease or disorder, a pulmonary disease or
disorder, a neurological disease or disorder, a metabolic disease
or disorder, a chemotherapeutic side effect, a radiotherapy side
effect, or metastasis. In an even more specific embodiment, the
senescent cell associated disease or disorder is a metabolic
disease such as obesity, metabolic syndrome, or diabetes. In other
particular embodiments, the treatment is administered on 1, 2, 3,
4, 5, 6, or 7 days every 4-12 months.
[0230] In yet other embodiments, a method is provided for killing a
senescent cell by contacting the senescent cell with a senolytic
agent or a senolytic combination, in a subject by administered once
every 4-12 months to a subject who has a senescent cell-associated
disease or disorder or who has at least one predisposing risk
factor for developing the disease or disorder, which may be any one
of a cardiovascular disease or disorder, inflammatory disease or
disorder, a pulmonary disease or disorder, a neurological disease
or disorder, a metabolic disease or disorder, a chemotherapeutic
side effect, a radiotherapy side effect, or metastasis. In an even
more specific embodiment, the senescent cell associated disease or
disorder is a metabolic disease such as obesity, metabolic
syndrome, or diabetes. In other particular embodiments, the
combination is administered on 1, 2, 3, 4, 5, 6, or 7 days every
4-12 months.
[0231] The agents of the combination may be administered together
at the administered on the same day (i.e., concurrently), at the
same time or at different times. For example, one agent of the
combination may be administered before meals and the second agent
of the combination administered after meals, or one agent may be
administered in the morning and the second agent in the evening.
The first or second agents, or both, administered according to any
of the aforementioned dosing regimens may administered as single
total dose on the day of administration, or the total dose for the
day's administration may be divided into multiple aliquots, such as
2, 3, 4, or 5 separate doses on the day of administration. Each
agent of the combination may be administered via the same
administrative route or by different administrative routes.
[0232] The treatment cycles of administering the senolytic
combination may be repeated as needed for treatment or prophylaxis
of the particular subject's disease or disorder. The treatment
course and non-treatment interval can be reviewed and modified or
adjusted, continued or discontinued, by a person skilled in the
art, depending on the responsiveness of the subject to the therapy,
the stage of the disease, the general health of the subject, and
other factors that are described herein and in the art.
Accordingly, in certain embodiments, one cycle of treatment is
followed by a subsequent cycle of treatment. Each treatment course
of a treatment cycle or each treatment course of two or more
treatment cycles are typically the same in duration and dosing of
the senolytic agent. In other embodiments, the duration and dosing
of the senolytic agent during each treatment course of a treatment
cycle may be adjusted as determined by a person skilled in the
medical art depending, for example, on the particular disease or
disorder being treated, the senolytic agent being administered, the
health status of the patient and other relevant factors, which are
discussed in greater detail herein. Accordingly, a treatment course
of a second or any subsequent treatment cycle may be shortened or
lengthened as deemed medically necessary or prudent. In other
words, as would be appreciated by a person skilled in the art, each
treatment course of two or more treatment cycles and each
non-treatment interval may be independent and the same or
different.
[0233] In other embodiments, when two agents are administered in
combination for treatment or prophylaxis of a senescent cell
associated disease or disorder or for killing a senescent cell, the
dosing regimen of the first agent may be different than the dosing
regimen of the second agent, such as sequential administration. For
example, sequential administration may include administering a
first agent and then 1 or 2 days later the second agent is
administered; in such embodiments, the time interval between dosing
of each respective agent would be the same so that the time
interval between administration of the first and second agents is
maintained. Administration of two agents of the combination must be
sufficiently close in time so that the two agents act together to
selectively kill senescent cells.
[0234] In embodiments when a third agent is administered, the third
agent may be administered according to the same dosing regimen as
the first agent or the second agent.
[0235] In particular embodiments, as described herein, such as when
the subject has a cancer, the senolytic combination is not intended
to be used as a primary therapy or as a component of a primary
therapy for the treatment of the cancer. By way of illustration,
dasatinib is administered daily for treatment of certain leukemias;
however, as described herein, dasatinib in combination with a
second agent is not be used in the same manner as when it is a
primary therapy for the treatment of the cancer. For example, a
subject who has a cancer and is being treated with an anthracycline
(such as doxorubicin, daunorubicin) may receive a senolytic
combination described herein that reduces, ameliorates, or
decreases the cardiotoxicity of the anthracycline. As is well
understood in the medical art, because of the cardiotoxicity
associated with anthracyclines, the maximum lifetime dose that a
subject can receive is limited even if the cancer is responsive to
the drug. Administration of a senolytic combination may reduce the
cardiotoxicity such that additional amounts of the anthracycline
can be administered to the subject, resulting in an improved
prognosis related to cancer disease.
[0236] Therapeutic and/or prophylactic benefit for subjects to whom
the senolytic combination is administered, includes, for example,
an improved clinical outcome, wherein the object is to prevent or
slow or retard (lessen) an undesired physiological change
associated with the disease, or to prevent or slow or retard
(lessen) the expansion or severity of such disease. As discussed
herein, effectiveness of a senolytic combination may include
beneficial or desired clinical results that comprise, but are not
limited to, abatement, lessening, or alleviation of symptoms that
result from or are associated with the disease to be treated;
decreased occurrence of symptoms; improved quality of life; longer
disease-free status (i.e., decreasing the likelihood or the
propensity that a subject will present symptoms on the basis of
which a diagnosis of a disease is made); diminishment of extent of
disease; stabilized (i.e., not worsening) state of disease; delay
or slowing of disease progression; amelioration or palliation of
the disease state; and remission (whether partial or total),
whether detectable or undetectable; and/or overall survival. The
effectiveness of the senolytic combinations described herein may
also mean prolonging survival when compared to expected survival if
a subject were not receiving the combination that selectively kills
senescent cells.
[0237] One or any combination of diagnostic methods appropriate for
the particular disease or disorder, which methods are well known to
a person skilled in the art and described herein, including
physical examination, patient self-assessment, assessment and
monitoring of clinical symptoms, performance of analytical tests
and methods, including clinical laboratory tests, physical tests,
and exploratory surgery, for example, may be used for monitoring
the health status of the subject and the effectiveness of the
treatment. The effects of the methods of treatment described herein
can be analyzed using techniques known in the art, such as
comparing symptoms of patients suffering from or at risk of a
particular disease or disorder that have received the
pharmaceutical composition comprising a senolytic combination, for
example, with those of patients who were not treated with the
senolytic combination or who received a placebo treatment.
[0238] As understood by a person skilled in the medical art, the
terms, "treat" and "treatment," refer to medical management of a
disease, disorder, or condition of a subject (i.e., patient) (see,
e.g., Stedman's Medical Dictionary). In general, an appropriate
dose and treatment regimen provide the therapeutic agent (e.g., a
senolytic combination) in an amount sufficient to provide
therapeutic and/or prophylactic benefit. Therapeutic benefit for
subjects to whom the treatments (e.g., senolytic combinations)
described herein are administered, includes, for example, an
improved clinical outcome, wherein the object is to prevent or slow
or retard (lessen) an undesired physiological change associated
with the disease, or to prevent or slow or retard (lessen) the
expansion or severity of such disease. As discussed herein,
effectiveness of the combinations and compositions described herein
may include beneficial or desired clinical results that comprise,
but are not limited to, abatement, lessening, or alleviation of
symptoms that result from or are associated with the disease to be
treated; decreased occurrence of symptoms; improved quality of
life; longer disease-free status (i.e., decreasing the likelihood
or the propensity that a subject will present symptoms on the basis
of which a diagnosis of a disease is made); diminishment of extent
of disease; stabilized (i.e., not worsening) state of disease;
delay or slowing of disease progression; amelioration or palliation
of the disease state; and remission (whether partial or total),
whether detectable or undetectable; and/or overall survival.
Effectiveness of the treatments described herein may also mean
prolonging survival when compared to expected survival if a subject
were not receiving the treatment that selectively kills senescent
cells.
[0239] Administration of a treatment (e.g., a senolytic
combination) described herein can prolong survival when compared to
expected survival if a subject were not receiving treatment.
Subjects in need of treatment include those who already have the
disease or disorder as well as subjects prone to have or at risk of
developing the disease or disorder, and those in which the disease,
condition, or disorder is to be treated prophylactically. A subject
may have a genetic predisposition for developing a disease or
disorder that would benefit from clearance of senescent cells or
may be of a certain age wherein receiving a senolytic combination
would provide clinical benefit to delay development or reduce
severity of a disease, including an age-related disease or
disorder.
[0240] A subject, patient, or individual in need of treatment may
be a human or may be a non-human primate or other non-human animal
(i.e., veterinary use) who has developed symptoms of a senescence
cell-associated disease or disorder or who is at risk for
developing a senescence cell-associated disease or disorder.
Examples of non-human primates and other animals include but are
not limited to farm animals, pets, and zoo animals (e.g., horses,
cows, buffalo, llamas, goats, rabbits, cats, dogs, chimpanzees,
orangutans, gorillas, monkeys, elephants, bears, large cats,
etc.).
Pharmaceutical Compositions
[0241] Also provided herein are pharmaceutical compositions
comprising the senolytic combinations described herein. A
pharmaceutical composition may be a sterile aqueous or non-aqueous
solution, suspension or emulsion, which additionally comprises a
physiologically acceptable excipient (pharmaceutically acceptable
or suitable excipient or carrier) (i.e., a non-toxic material that
does not interfere with the activity of the active ingredient). The
excipients described herein are merely exemplary and are in no way
limiting. An effective amount or therapeutically effective amount
refers to an amount of an agent or combination of agents or a
composition comprising the one or more agents administered to a
subject, either as a single dose or as part of a series of doses,
which is effective to produce a desired therapeutic effect.
[0242] When a senolytic combination is administered to a subject
for treatment of a disease or disorder described herein, each of
the agents of the combination may be formulated into separate
pharmaceutical compositions. A pharmaceutical preparation may be
prepared that comprises each of the separate pharmaceutical
compositions (which may be referred to for convenience, for
example, as a first pharmaceutical composition and a second
pharmaceutical composition comprising each of the first and second
agents, respectively). Each of the pharmaceutical compositions in
the preparation may be administered at the same time and via the
same route of administration or may be administered at different
times by the same or different administration routes.
Alternatively, two or more agents of the combination may be
formulated together in a single pharmaceutical composition.
[0243] Subjects may generally be monitored for therapeutic
effectiveness using assays and methods suitable for the condition
being treated or prevented, which assays will be familiar to those
having ordinary skill in the art and are described herein. The
level of an agent that is administered to a subject may be
monitored by determining the level of the agent in a biological
fluid, for example, in the blood, blood fraction (e.g., serum),
and/or in the urine, and/or other biological sample from the
subject. Any method practiced in the art to detect the agent may be
used to measure the level of agent during the course of a
therapeutic regimen.
[0244] The dose of each agent of a combination described herein for
treating a senescence cell associated disease or disorder may
depend upon the subject's condition, that is, stage of the disease,
severity of symptoms caused by the disease, general health status,
as well as age, gender, and weight, and other factors apparent to a
person skilled in the medical art. Pharmaceutical compositions may
be administered in a manner appropriate to the disease to be
treated as determined by persons skilled in the medical arts. A
suitable duration and frequency of administration may also be
determined by such factors as the condition of the patient, the
type and severity of the patient's disease, the particular form of
the active ingredient, and the method of administration. Optimal
doses of each agent of the combination may generally be determined
using experimental models and/or clinical trials. The optimal dose
may depend upon the body mass, weight, or blood volume of the
subject. The use of the minimum dose that is sufficient to provide
effective therapy is usually preferred. Design and execution of
pre-clinical and clinical studies for a combination (including when
administered for prophylactic benefit) described herein are well
within the skill of a person skilled in the relevant art. The
optimal dose of a senolytic combination may depend upon the body
mass, weight, or blood volume of the subject. Each agent of the
combination may be of an amount between 0.01 mg/kg and 1000 mg/kg
(e.g., about 0.1 to 1 mg/kg, about 1 to 10 mg/kg, about 10-50
mg/kg, about 50-100 mg/kg, about 100-500 mg/kg, or about 500-1000
mg/kg) body weight.
[0245] The pharmaceutical compositions may be administered to a
subject in need by any one of several routes that effectively
deliver an effective amount of the combination. Pharmaceutical
compositions comprising a senolytic combination can be formulated
in a manner appropriate for the delivery method by using techniques
routinely practiced in the art. The composition may be in the form
of a solid (e.g., tablet, capsule), semi-solid (e.g., gel), liquid,
or gas (aerosol). In other certain specific embodiments, the
senolytic combination (or pharmaceutical composition comprising
same) is administered as a bolus infusion. In certain embodiments
when the senolytic combination is delivered by infusion, the
senolytic combination is delivered to an organ or tissue comprising
senescent cells to be killed via a blood vessel in accordance with
techniques routinely performed by a person skilled in the medical
art.
[0246] Pharmaceutical acceptable excipients are well known in the
pharmaceutical art and described, for example, in Rowe et al.,
Handbook of Pharmaceutical Excipients: A Comprehensive Guide to
Uses, Properties, and Safety, 5.sup.th Ed., 2006, and in Remington:
The Science and Practice of Pharmacy (Gennaro, 21.sup.st Ed. Mack
Pub. Co., Easton, Pa. (2005)). Exemplary pharmaceutically
acceptable excipients include sterile saline and phosphate buffered
saline at physiological pH. Preservatives, stabilizers, dyes,
buffers, and the like may be provided in the pharmaceutical
composition. In addition, antioxidants and suspending agents may
also be used. In general, the type of excipient is selected based
on the mode of administration, as well as the chemical composition
of the active ingredient(s). Alternatively, compositions described
herein may be formulated as a lyophilizate, or the agent may be
encapsulated within liposomes using technology known in the art. A
composition described herein may be lyophilized or otherwise
formulated as a lyophilized product using one or more appropriate
excipient solutions for solubilizing and/or diluting the agent(s)
of the composition upon administration. In other embodiments, the
agent may be encapsulated within liposomes using technology known
and practiced in the art. Pharmaceutical compositions may be
formulated for any appropriate manner of administration described
herein and in the art.
[0247] A pharmaceutical composition may be delivered to a subject
in need thereof by any one of several routes known to a person
skilled in the art. By way of non-limiting example, the composition
may be delivered orally, intravenously, intraperitoneally, by
infusion (e.g., a bolus infusion), subcutaneously, enteral, rectal,
intranasal, by inhalation, buccal, sublingual, intramuscular,
transdermal, intradermal, topically, intraocular, vaginal, rectal,
or by intracranial injection, or any combination thereof. In
certain particular embodiments, administration of a dose as
described above is via intravenous, intraperitoneal, directly into
the target tissue or organ, or subcutaneous route. In certain
embodiments, a delivery method includes drug-coated or permeated
stents for which the drug is the senolytic agent. Formulations
suitable for such delivery methods are described in greater detail
herein.
[0248] A pharmaceutical composition (e.g., for oral administration
or delivery by injection) may be in the form of a liquid. A liquid
pharmaceutical composition may include, for example, one or more of
the following: a sterile diluent such as water for injection,
saline solution, preferably physiological saline, Ringer's
solution, isotonic sodium chloride, fixed oils that may serve as
the solvent or suspending medium, polyethylene glycols, glycerin,
propylene glycol or other solvents; antibacterial agents;
antioxidants; chelating agents; buffers and agents for the
adjustment of tonicity such as sodium chloride or dextrose. A
parenteral preparation can be enclosed in ampoules, disposable
syringes or multiple dose vials made of glass or plastic. The use
of physiological saline is preferred, and an injectable
pharmaceutical composition is preferably sterile.
[0249] In certain particular embodiments, a senolytic combination
(which may be combined with at least one pharmaceutically
acceptable excipient to form a pharmaceutical composition) is
administered directly to the target tissue or organ comprising
senescent cells that contribute to manifestation of the disease or
disorder. In specific embodiments when treating osteoarthritis, the
senolytic combination is administered directly to an osteoarthritic
joint (i.e., intra-articularly) of a subject in need thereof. In
other specific embodiments, a senolytic combination may be
administered to the joint via topical, transdermal, intradermal, or
subcutaneous route. In other certain embodiments, methods are
provided herein for treating a cardiovascular disease or disorder
associated with arteriosclerosis, such as atherosclerosis by
administering directly into an artery. In another particular
embodiment, a senolytic combination (which may be combined with at
least one pharmaceutically acceptable excipient to form a
pharmaceutical composition) for treating a senescent-associated
pulmonary disease or disorder may be administered by inhalation,
intranasally, by intubation, or intracheally, for example, to
provide the senolytic combination more directly to the affected
pulmonary tissue. By way of another non-limiting example, the
senolytic combination (or pharmaceutical composition comprising the
senolytic combination) may be delivered directly to the eye either
by injection (e.g., intraocular or intravitreal) or by conjunctival
application underneath an eyelid of a cream, ointment, gel, or eye
drops. In more particular embodiments, the senolytic combination or
pharmaceutical composition comprising the senolytic combination may
be formulated as a timed release (also called sustained release,
controlled release) composition or may be administered as a bolus
infusion.
[0250] A pharmaceutical composition (e.g., for oral administration
or for injection, infusion, subcutaneous delivery, intramuscular
delivery, intraperitoneal delivery or other method) may be in the
form of a liquid. A liquid pharmaceutical composition may include,
for example, one or more of the following: a sterile diluent such
as water, saline solution, preferably physiological saline,
Ringer's solution, isotonic sodium chloride, fixed oils that may
serve as the solvent or suspending medium, polyethylene glycols,
glycerin, propylene glycol or other solvents; antibacterial agents;
antioxidants; chelating agents; buffers and agents for the
adjustment of tonicity such as sodium chloride or dextrose. A
parenteral composition can be enclosed in ampoules, disposable
syringes or multiple dose vials made of glass or plastic. The use
of physiological saline is preferred, and an injectable
pharmaceutical composition is preferably sterile. In another
embodiment, for treatment of an ophthalmological condition or
disease, a liquid pharmaceutical composition may be applied to the
eye in the form of eye drops. A liquid pharmaceutical composition
may be delivered orally.
[0251] For oral formulations, the senolytic combinations described
herein can be used alone or in combination with appropriate
additives to make tablets, powders, granules or capsules, and if
desired, with diluents, buffering agents, moistening agents,
preservatives, coloring agents, and flavoring agents. The compounds
of the combination, each alone or together, may be formulated with
a buffering agent to provide for protection of the compound from
low pH of the gastric environment and/or an enteric coating. A
senolytic combination (or each agent of the combination) included
in a pharmaceutical composition may be formulated for oral delivery
with a flavoring agent, e.g., in a liquid, solid or semi-solid
formulation and/or with an enteric coating.
[0252] A pharmaceutical composition comprising the senolytic
combinations described herein may be formulated for sustained or
slow release (also called timed release or controlled release).
Such compositions may generally be prepared using well known
technology and administered by, for example, oral, rectal,
intradermal, or subcutaneous implantation, or by implantation at
the desired target site. Sustained-release formulations may contain
the compounds of the combination dispersed in a carrier matrix
and/or contained within a reservoir surrounded by a rate
controlling membrane. Excipients for use within such formulations
are biocompatible, and may also be biodegradable; preferably the
formulation provides a relatively constant level of active
component release. The amount of active agent contained within a
sustained release formulation depends upon the site of
implantation, the rate and expected duration of release, and the
nature of the condition, disease or disorder to be treated or
prevented.
[0253] In certain embodiments, the pharmaceutical compositions
comprising a senolytic combination are formulated for transdermal,
intradermal, or topical administration. The compositions can be
administered using a syringe, bandage, transdermal patch, insert,
or syringe-like applicator, as a powder/talc or other solid,
liquid, spray, aerosol, ointment, foam, cream, gel, paste. This
preferably is in the form of a controlled release formulation or
sustained release formulation administered topically or injected
directly into the skin adjacent to or within the area to be treated
(intradermally or subcutaneously). The active compositions can also
be delivered via iontophoresis. Preservatives can be used to
prevent the growth of fungi and other microorganisms. Suitable
preservatives include, but are not limited to, benzoic acid,
butylparaben, ethyl paraben, methyl paraben, propylparaben, sodium
benzoate, sodium propionate, benzalkonium chloride, benzethonium
chloride, benzyl alcohol, cetypyridinium chloride, chlorobutanol,
phenol, phenylethyl alcohol, thimerosal, and combinations
thereof.
[0254] Pharmaceutical compositions comprising a senolytic
combination can be formulated as emulsions for topical application.
An emulsion contains one liquid distributed the body of a second
liquid. The emulsion may be an oil-in-water emulsion or a
water-in-oil emulsion. Either or both of the oil phase and the
aqueous phase may contain one or more surfactants, emulsifiers,
emulsion stabilizers, buffers, and other excipients. The oil phase
may contain other oily pharmaceutically approved excipients.
Suitable surfactants include, but are not limited to, anionic
surfactants, non-ionic surfactants, cationic surfactants, and
amphoteric surfactants. Compositions for topical application may
also include at least one suitable suspending agent, antioxidant,
chelating agent, emollient, or humectant.
[0255] Ointments and creams may, for example, be formulated with an
aqueous or oily base with the addition of suitable thickening
and/or gelling agents. Lotions may be formulated with an aqueous or
oily base and will in general also contain one or more emulsifying
agents, stabilizing agents, dispersing agents, suspending agents,
thickening agents, or coloring agents. Liquid sprays may be
delivered from pressurized packs, for example, via a specially
shaped closure. Oil-in-water emulsions can also be used in the
compositions, patches, bandages and articles. These systems are
semisolid emulsions, micro-emulsions, or foam emulsion systems.
[0256] In some embodiments, the senolytic combination can be
formulated with oleaginous bases or ointments to form a semisolid
composition with a desired shape. In addition to the senolytic
combination, these semisolid compositions can contain dissolved
and/or suspended bactericidal agents, preservatives and/or a buffer
system. A petrolatum component that may be included may be any
paraffin ranging in viscosity from mineral oil that incorporates
isobutylene, colloidal silica, or stearate salts to paraffin waxes.
Absorption bases can be used with an oleaginous system. Additives
may include cholesterol, lanolin (lanolin derivatives, beeswax,
fatty alcohols, wool wax alcohols, low HLB (hydrophobellipophobe
balance) emulsifiers, and assorted ionic and nonionic surfactants,
singularly or in combination.
[0257] Controlled or sustained release transdermal or topical
formulations can be achieved by the addition of time-release
additives, such as polymeric structures, matrices, that are
available in the art. For example, the compositions may be
administered through use of hot-melt extrusion articles, such as
bioadhesive hot-melt extruded film. The formulation can comprise a
cross-linked polycarboxylic acid polymer formulation. A
cross-linking agent may be present in an amount that provides
adequate adhesion to allow the system to remain attached to target
epithelial or endothelial cell surfaces for a sufficient time to
allow the desired release of the compound.
[0258] An insert, transdermal patch, bandage or article can
comprise a mixture or coating of polymers that provide release of
the active agents at a constant rate over a prolonged period of
time. In some embodiments, the article, transdermal patch or insert
comprises water-soluble pore forming agents, such as polyethylene
glycol (PEG) that can be mixed with water insoluble polymers to
increase the durability of the insert and to prolong the release of
the active ingredients.
[0259] Transdermal devices (inserts, patches, bandages) may also
comprise a water insoluble polymer. Rate controlling polymers may
be useful for administration to sites where pH change can be used
to effect release. These rate controlling polymers can be applied
using a continuous coating film during the process of spraying and
drying with the active compound. In one embodiment, the coating
formulation is used to coat pellets comprising the active
ingredients that are compressed to form a solid, biodegradable
insert.
[0260] A polymer formulation can also be utilized to provide
controlled or sustained release. Bioadhesive polymers described in
the art may be used. By way of example, a sustained-release gel and
the combination (or each agent of the combination) may be
incorporated in a polymeric matrix, such as a hydrophobic polymer
matrix. Examples of a polymeric matrix include a microparticle. The
microparticles can be microspheres, and the core may be of a
different material than the polymeric shell. Alternatively, the
polymer may be cast as a thin slab or film, a powder produced by
grinding or other standard techniques, or a gel such as a hydrogel.
The polymer can also be in the form of a coating or part of a
bandage, stent, catheter, vascular graft, or other device to
facilitate delivery of the senolytic combination. The matrices can
be formed by solvent evaporation, spray drying, solvent extraction
and other methods known to those skilled in the art.
[0261] In certain embodiments of a method described herein for
treating a cardiovascular disease associated with or caused by
arteriosclerosis, a senolytic combination may be delivered directly
into a blood vessel (e.g., an artery) via a stent. In a particular
embodiment, a stent is used for delivering a senolytic combination
to an atherosclerotic blood vessel (an artery). A stent is
typically a tubular metallic device, which has thin-metal
screen-like scaffold, and which is inserted in a compressed form
and then expanded at the target site. Stents are intended to
provide long-term support for the expanded vessel. Several methods
are described in the art for preparing drug-coated and
drug-embedded stents. For example, a senolytic combination may be
incorporated into polymeric layers applied to a stent. A single
type of polymer may be used, and one or more layers of the
senolytic combination permeated polymer may be applied to a bare
metal stent to form the senolytic combination-coated stent. The
senolytic combination may also be incorporated into pores in the
metal stent itself, which may also be referred to herein as a
senolytic combination-permeated stent or senolytic
combination-embedded stent. In certain particular embodiments, a
senolytic combination may be formulated within liposomes and
applied to a stent. Placement of stents in an atherosclerotic
artery is performed by a person skilled in the medical art.
[0262] In one particular embodiment, the senolytic combination
administered to a subject who has an ophthalmic senescence
associated or disease or disorder may be delivered intraocularly or
intravitreally. In other specific embodiments, a senolytic
combination may be administered to the eye by a conjunctival route,
applying the senolytic combination to the mucous membrane and
tissues of the eye lid, either upper, lower, or both. Any of these
administrations may be bolus infusions. In other particular
embodiments, a pharmaceutical composition comprising a senolytic
combination described herein may be formulated for sustained or
slow release (which may also be called timed release or controlled
release), which formulations are described in greater detail
herein. In certain embodiments, methods are provided herein for
treating or preventing (i.e., reducing the likelihood of occurrence
of, delaying the onset or development of, or inhibiting, retarding,
slowing, or impeding progression or severity of) an ocular disease,
disorder, or condition (e.g., cataracts, macular degeneration); and
for selectively killing senescent cells in an eye of a subject in
need thereof by administering a senolytic combination (which may be
combined with at least one pharmaceutically acceptable excipient to
form a pharmaceutical composition) directly to an eye.
[0263] Kits with unit doses of one or more of the agents described
herein, usually in oral or injectable doses, are provided. Such
kits may include a container containing the unit dose, an
informational package insert describing the use and attendant
benefits of the drugs in treating the senescent cell associated
disease, and optionally an appliance or device for delivery of the
composition.
EXAMPLES
Example 1
Identification of Senescence Associated Pathways
[0264] Proteomic analyses by nano LC MS/MS were performed on
lysates on human abdominal subcutaneous preadipocytes that were
senescent or non-senescent. Preadipocytes, one of the most abundant
cell types in humans susceptible to senescence, were extracted from
fat tissues of nine different healthy kidney transplant donors by
collagenase digestion. Prior consent from the donors was obtained.
Senescence was induced by 10 Gy radiation or by serial
subculturing. Bioinformatics methods were used to identify pathways
that were susceptible to existing drugs and that could mediate cell
death.
[0265] Senescence-associated .beta.-galactosidase (SA-.beta. gal)
activity was used to assess the percentage of senescent cells
present in the irradiated cell cultures. To be considered a
senescent culture, 75% or more of the cells needed to demonstrate
SA-.beta. gal activity. Both whole cell lysates and cellular
supernatants were collected. Proteins were separated on 1D
SDS-PAGE. Sections of the gels were destained, reduced, alkylated,
and trypsin-digested. Extracted peptides were analyzed by
nano-LC-MS/MS on a THERMO SCIENTIFIC.TM. Q Exactive mass
spectrometer. LC Progenesis software (Nonlinear Dynamics, UK) was
used to identify and quantify proteins. The data were then
submitted to Ingenuity, Metacore, Cytoscape, and other software for
pathway and protein network analysis. Among the pathways identified
during senescence were those involved in cell survival signaling
and inflammatory pathways. These pathways include at least
PI3K/AKT, Src kinase signaling, insulin/IGF-1 signaling, p38/MAPK,
NF-.kappa.B signaling, TGF.beta. signaling, and mTOR/protein
translation (see FIGS. 2A-2D).
[0266] FIG. 1 shows a confirmatory Western immunoblot of proteins
involved in these and related pathways at various times (24 hr, 3,
6, 8, 11, 15, 20, and 25 days) after radiation. Phosphorylated
polypeptides in the senescent cell samples were detecting using
horse-radish peroxidase labeled antibodies (Cell Signaling
Technology, Danvers, Mass.) specific for the polypeptides indicated
in FIG. 1. Senescence is fully established at day 25 to 30 in these
cells.
[0267] Approximately 20 compounds were screened for killing
senescent human cultured preadipocytes and were detected by
phase-contrast microscopy. Dasatinib, enzastaurin, and quercetin
were included in the 20 compounds and were chosen for further
study. Dasatinib inhibits at least Src kinase activity; quercetin
is described in the art as inhibiting Src kinase, histone
deacetylase (HDAC), Akt kinase, p38 MAPK, and ROS (reactive oxygen
species); and enzastaurin inhibits at least protein kinase C-beta.
FIGS. 2A-2D illustrate pathways affected by the agents described
herein. FIG. 2A shows possible target proteins affected by
quercetin altering a cell survival pathway. Without wishing to be
bound by theory, the components shown are likely indirectly
affected (indicated by non-solid lines) when quercetin alters
either a cell survival signaling pathway or an inflammatory
pathway. Downstream components of the Src kinase pathway, the PI3K
pathway, and the Akt pathway are illustrated in FIGS. 2B-2D,
respectively. A solid arrow between two components indicates that
the component at the source of the solid line has a direct role in
upregulating the component to which the arrow is pointing. Included
in FIG. 2C is LY284992, which is a potent selective
phosphatidylinositol 3-kinase (PI3K) inhibitor.
Example 2
Selective Killing of Senescent Fibroblasts
[0268] Senescence of human primary lung fibroblasts (IMR90) (IMR-90
(ATCC.RTM. CCL-186.TM., Mannassas, Va.) was induced by irradiation.
IMR90 cells in culture were subjected to 10 Gy radiation (Day -7).
Four days after irradiation (Day -3), the culture media was
changed. Three days after the media change (Day 0), the cells were
exposed to media containing quercetin. Non-irradiated IMR90 cells
were included as controls. Non-irradiated and irradiated IMR90
cells were exposed to quercetin at concentrations of 5, 10, 15, and
45 .mu.M. Percent survival was determined four days after exposure
to the drugs (Day 4). The number of viable cells was determined by
using ATPLITE (Perkin-Elmer, Waltham, Mass.). The results are
presented in FIG. 3.
[0269] In a separate experiment, IMR90 cells were irradiated as
described above to induce senescence. Approximately 20 days after
irradiation, the senescent cells and proliferating IMR90 cells were
exposed to enzastaurin at concentrations of 0.25 .mu.M, 0.5 .mu.M,
1 .mu.M, and 2 .mu.M. Enzastaurin at these concentrations did not
selectively kill the senescent cells and did not kill the
proliferating cells. Enzastaurin may have a slight potentiating
effect of dasatinib (see FIG. 4E). Enzastaurin in combination with
quercetin killed senescent cells but also killed the non-senescent
proliferating cells (see FIG. 4F).
[0270] Imatinib and sorafenib were also tested for the capability
to selectively kill senescent cells. Some senolytic effect was
observed with imatinib at 50 .mu.M, which is not considered to be a
physiologically acceptable concentration of this drug. Microscopic
examination of senescent cells and non-senescent cells treated with
sorafenib suggested that both senescent cells and non-senescent
cells were killed.
Example 3
Selective Killing of Senescent Endothelial Cells
[0271] Human umbilical endothelial cells (HUVEC) (Lonza Group,
Basel, Switzerland) were induced to senescence by exposure to 10 Gy
radiation. Twenty days after irradiation, markers of cellular
senescence (SA-.beta. Gal) and growth arrest, determined by
incorporation of BRdU, were evident. Non-senescent HUVEC cells used
as control were plated at low density in culture media so that the
cells were proliferating when exposed to the test drug. Senescent
HUVEC cells and non-senescent, proliferating HUVEC cells were
treated for 48 hours with quercetin, dasatinib, and enzastaurin as
follows: (1) quercetin alone at 3.75, 7.5 and 15 .mu.M; (2)
dasatinib alone at 25, 50, 100, and 200 nM; (3) enzastaurin alone
at concentrations of 0.25 .mu.M, 0.5 .mu.M, 1 .mu.M, and 2 .mu.M;
(4) dasatinib at 100 nM plus quercetin at 7.5 .mu.M; (5) with
dasatinib at 50 nM plus quercetin at 7.5 .mu.M; (6) dasatinib at
100 nM plus enzastaurin at 1.0 .mu.M; (7) dasatinib at 100 nM plus
enzastaurin at 0.5 .mu.M; (8) dasatinib at 50 nM plus enzastaurin
at 1.0 .mu.M; (9) dasatinib at 50 nM plus enzastaurin at 0.5 .mu.M;
(10) quercetin at 15 .mu.M and enzastaurin at 1.0 .mu.M; (11)
quercetin at 15 .mu.M and enzastaurin at 1.0 .mu.M; and (12)
quercetin at 7.5 .mu.M and enzastaurin at 1.0 .mu.M. The number of
viable cells was determined by ATPLITE (Perkin-Elmer, Waltham,
Mass.). The data are presented in FIGS. 4A-4F as mean viability
(ATP (.mu.M)).+-.SEM of 4.
Example 4
Selective Killing of Senescent Preadipocyte Cells
[0272] Human primary abdominal subcutaneous preadipocytes were
obtained with consent from donors. The preadipocytes were exposed
to radiation and senescence determined as described in Example 3.
After 20 days in culture, senescent preadipocytes and control
proliferating preadipocytes and control differentiated
preadipocytes from the same donor were treated for 48 hours with
quercetin at 7.5, 15, 30, and 60 .mu.M; with dasatinib at 50, 100,
200 nM, and 400 nM; with enzastaurin at 0.25 .mu.M, 0.5 .mu.M, 1
.mu.M, and 2 .mu.M; with dasatinib at 100 nM plus quercetin at 15
and 30 .mu.M; with dasatinib at 200 nM plus quercetin at 15 and 30
.mu.M; with dasatinib at 200 nM plus enzastaurin at 1 .mu.M and 2
.mu.M; with dasatinib at 100 nM plus enzastaurin at 1 .mu.M and 2
.mu.M; with quercetin at 30 .mu.M plus enzastaurin at 1 .mu.M and 2
.mu.M; and with quercetin at 15 .mu.M plus enzastaurin at 1 .mu.M
and 2 .mu.M. The number of viable cells was determined by ATPLITE
(Perkin-Elmer, Waltham, Mass.). The data are presented in FIGS.
5A-5F as mean viability (ATP (.mu.M)).+-.SEM of 4. The combination
of dasatinib and quercetin exhibited selective killing of senescent
cells. An effective combination included dasatinib at 200 nM and
quercetin at 30 .mu.M (see FIG. 5C).
Example 5
Removal of Senescent Cells In Vivo by Dasatinib and Quercetin
[0273] The capability of dasatinib alone, quercetin alone, and the
combination of dasatinib and quercetin to remove senescent cells in
vivo was determined in transgenic p16-3MR mice (see International
Application Publication No. WO 2013/090645). The transgenic mouse
comprises a p16.sup.Ink4a promoter (see, e.g., operatively linked
to a trimodal fusion protein for detecting senescent cells and for
selective clearance of senescent cells in these transgenic mice.
The promoter, p16.sup.Ink4a which is transcriptionally active in
senescent cells but not in non-senescent cells (see, e.g., Wang et
al., J. Biol. Chem. 276:48655-61 (2001); Baker et al., Nature
479:232-36 (2011)), was engineered into a nucleic acid construct.
The p16.sup.Ink4a gene promoter (approximately 100 kilobase pairs)
was introduced upstream of a nucleotide sequence encoding a
trimodal reporter fusion protein. Alternatively, a truncated
p16.sup.Ink4a promoter may be used (see, e.g., Baker et al.,
Nature, supra; International Application Publication No.
WO/2012/177927; Wang et al., supra). The trimodal reporter protein
is termed 3MR and consists of renilla luciferase (rLUC), monomeric
red fluorescent protein (mRFP) and a truncated herpes simplex virus
thymidine kinase (tTK) (see, e.g., Ray et al., Cancer Res.
64:1323-30 (2004)). Thus, the expression of 3MR is driven by the
p16.sup.Ink4a promoter in senescent cells only. The detectable
markers, rLUC and mRFP permitted detection of senescent cells by
bioluminescence and fluorescence, respectively. The expression of
tTK permitted selective killing of senescent cells by exposure to
the pro-drug ganciclovir (GCV), which is converted to a cytotoxic
moiety by tTK. Transgenic founder animals, which have a C57B16
background, were established and bred using known procedures for
introducing transgenes into animals (see, e.g., Baker et al.,
Nature 479:232-36 (2011)).
[0274] Two-month old male and five-month old female p16-3MR mice
were randomized into groups of nine animals per group. Senescence
was induced by administering doxorubicin at 10 mg/kg to the mice
ten days prior to administration of the test drug(s) (D-10).
Quercetin (50 mg/kg), dasatinib (5 mg/kg), or combination of
quercetin and dasatinib (50 mg/kg and 5 mg/kg, respectively) were
administered once by oral gavage on Day 0. Ganciclovir (GCV) (25
mg/kg) was the positive control and was administered daily
beginning at Day 0 for five days by intraperitoneal injection.
Luminescence imaging was performed at Day 0 and at Day 5. A
schematic of this model is presented in FIG. 6.
[0275] Luminescence imaging of the mice was performed on the day
the test drugs were administered (Imaging 1) and again at 5 days
after administration of the test drugs (Imaging 2). Reduction of
luminescence (L) was calculated as: L=(Imaging 2)/(Imaging 1)%. If
L.gtoreq.100, no reduction in the number of senescent cells. If
L<100, reduction in in the number of senescent cells. Every
mouse was calculated independently, and background was subtracted
from each sample. The results are presented in FIGS. 7A and 7B.
Example 6
The Effect of Dasatinib and Quercetin on Vascular Function
[0276] Vasomotor function of aorta was evaluated ex vivo by
measurement of isometric tension (see, e.g., Roos et al., Am. J.
Physiol. Heart Cir. Physiol. 305:H1428-H1439 (2013)). Twenty-four
month old male mice (C57BL/6J) were used in the experiment. Groups
of mice (10 animals per group) were treated with a single dose of
quercetin, dasatinib, quercetin+dasatinib, or vehicle only
(control). After treatment, mice were sacrificed and the aorta was
excised. Connective and adipose tissue were removed from the aorta,
which was then placed in oxygenated Krebs buffer. The aorta samples
were suspended between two triangular hooks in an organ bath, and
isometric tension was measured. Responses to acetylcholine (Ach)
(endothelium dependent) and sodium nitroprusside (SNP) (endothelium
independent) were examined after preconstriction of the vessel to
.about.50-60% of its maximal force. The concentration of ACH and
SNP ranged from 10.sup.-9 M to 10.sup.-4 M (depicted by -9 to -4 on
the x-axis of FIG. 8 and FIG. 9, respectively). The acetylcholine
dose response data are shown in FIG. 8, and the nitroprusside dose
response is illustrated in FIG. 9.
Example 7
Dasatinib and Quercetin Cause Increased Apoptosis of Senescent than
Non-Senescent Primary Human Preadipocytes
[0277] Human primary abdominal subcutaneous preadipocytes were
obtained from six subjects with consent from donors and pooled. The
preadipocytes were exposed to radiation and senescence determined
as described in Example 3. After 20 days in culture, senescent
preadipocytes and control proliferating preadipocytes and control
differentiated preadipocytes from the same donor were treated for
24 hours with dasatinib alone (200 nM), quercetin alone (30 .mu.M),
or dasatinib (200 nM) plus quercetin (30 .mu.M). The number of
viable cells was determined by ATPLITE (Perkin-Elmer, Waltham,
Mass.). Experiments were performed in six replicates, and seven
separate experiments were performed, which each yielded similar
results for the dasatinib plus quercetin treated senescent cells. A
two-tailed t-test was used to evaluate the loss of treated
senescent cells relative to the loss of control cells. The
combination of dasatinib plus quercetin treatment resulted in
.about.50% fewer senescent cells after 3 days than were plated
(solid bar), while numbers of proliferating control cells increased
slightly (open bar) as shown in FIG. 10A.
[0278] In a second experiment, the human primary abdominal
subcutaneous preadipocytes were exposed to radiation and senescence
determined as described in Example 3. After 20 days in culture,
senescent preadipocytes and control non-senescent preadipocytes
from the same donor were treated for 24 hours with dasatinib (200
nM) plus quercetin (30 .mu.M). Senescent cells were exposed to
vehicle were a control. Preadipocytes were counterstained with DAPI
for nuclear visualization and analyzed by terminal deoxynucleotidyl
transferase dUTP nick end labeling (TUNEL) to visualize apoptotic
cells. Treatment with dasatinib plus quercetin induced apoptosis in
the senescent preadipocyte cells but not the non-senescent
preadipocyte cells. The data are presented in FIG. 10B-G.
Example 8
Dasatinib and Quercetin Treatment Decreases Presence of Senescent
Cells in Adipose Tissue
[0279] Old male mice (26 month) (n=5) were given a single dose of
the combination, dasatinib (5 mg/kg) and quercetin (50 mg/kg), or
vehicle by oral gavage. Four days after treatment, inguinal and
epididymal adipose tissue were obtained from the animals. Senescent
cells were detected by staining with SA-.beta.-gal. Data are
presented as means.+-.SEM; n=5; ANOVA. The data are shown in FIG.
11. The extent to which the combination caused removal of senescent
cells varied among adipose depots.
Example 9
Reduction of Senescence Markers in Animals by Dasatinib and
Quercetin Combination Treatment
[0280] One leg of mice that were exposed to 10 Gy collimated cesium
radiation two months. Two months after exposure, the animals
developed grey hair and senescent cell accumulation in the radiated
leg. Mice were treated once with the combination, dasatinib and
quercetin, or vehicle. After 4 days, the level of p21 and p16 mRNA
was assayed by RT-PCR in muscle tissue from the radiated leg (N=5;
T tests). The senolytic combination resulted in a decrease muscle
p16 and p21 in legs of radiation-exposed mice. p16 and p21 are
senescent cell markers. The results are presented in FIG. 12.
Example 10
Reduction of Senescence Markers in Animals by Dasatinib and
Quercetin Combination Treatment
[0281] The level of mRNA expression of senescence markers (p16,
p21, PAI-1) in inguinal fat of 24-month old male mice treated with
a single dose of vehicle, quercetin (50 mg/kg), dasatinib (5
mg/kg), or combination of quercetin (50 mg/kg) and dasatinib (5
mg/kg) was determined. mRNA was assayed by RT-PCR in inguinal
adipose tissue obtained five days after treatment. Kruskal-Wallis
Test, non-parametric ANOVA; n=8. As shown in FIG. 13, the
combination of quercetin and dasatinib reduced expression of p16
(p=0.049).
[0282] In a second experiment, groups of 24 month old mice (n=14)
were treated with a single dose of vehicle or combination of
quercetin and dasatinib. Five days after treatment, samples of
inguinal fat were obtained and stained with the SA-.beta.-gal or
analyzed for p16 mRNA expression by RT-PCR. The results are
presented in FIGS. 14A and B. The data were analyzed by the
Mann-Whitney test. SA-.beta.-gal staining: p<0.0012; p16 mRNA
expression: P<0.01
Example 11
Effect of Dasatinib and Quercetin Combination Treatment on Presence
of Senescent Cells in Fat Tissue of Progeroid Mice
[0283] BubR1.sup.H/H mice have accelerated development of
aging-like phenotypes. Four days after receiving 3 daily doses of
the combination of dasatinib and quercetin (D+Q), SA-.beta.-gal
activity was lower in paraovarian and inguinal fat of older (8
months old -10-12 month is maximum lifespan in these mice) female
BubR1.sup.H/H mice vs. vehicle-treated controls. In these mice,
inguinal fat on one side was biopsied 10 days before D+Q was given
and analyzed at autopsy 4 days after. Levels of mRNA were
determined by RT-PCR. p16 mRNA was decreased by D+Q (0.49.+-.0.17
fold vs. baseline [=1]; P=0.07), as were p21 (0.37.+-.0.15 fold;
P<0.03); PAI-1 (0.37.+-.0.01; P<0.00001), and IGFBP2
(0.30.+-.0.15; P<0.02). In vehicle-treated mice, p16, p21,
PAI-1, and IGFBP2 were not significantly lower vs. baseline
(1.24.+-.0.14, 1.25.+-.0.41, 1.16.+-.0.12, and 1.09.+-.0.13,
respectively; all p=NS).
Example 12
Cellular Senescence Increases in Diet-Induced Obese (DIO) Mice
[0284] An increased senescent cell burden was found in obese
compared to lean, age-matched controls. Mice were fed a high fat
vs. chow diet for 4 month. Adipose tissue (inguinal and epididymal)
was stained for SA-.beta.-gal. FIG. 15 shows that a higher burden
of senescent cells are present in high-fat fed, obese animals than
chow-fed controls.
Example 13
High Fat Feeding-Induced Senescence Reduced by Ganciclovir in
p16-3MR Mice
[0285] Groups of p16-3MR mice (n=6) were fed a high fat diet (60%
fat) for four months mice or a regular chow diet. The presence of
senescence cells was determined by measuring luminescence (i.e.,
p16 positive cells). As shown in FIG. 16, animals fed a high fat
diet have increased numbers of senescence cells compared with the
regular chow fed animals.
[0286] Animals were then treated with ganciclovir or vehicle to
determine if removal of senescent cells reduced the presence of
senescent cells in adipose tissue. Groups of animals were treated
with ganciclovir (GCV) or vehicle. Ganciclovir (25 mg/kg) was
administered daily for five consecutive days. The presence of
senescent cells in perirenal, epididymal, or subcutaneous inguinal
adipose tissue was detected by SA-.beta.-Gal staining. Data were
analyzed by ANOVA. The results are presented in FIG. 17. A
significant reduction in presence of senescent cells was observed
in epididymal fat. p=<0.004.
Example 14
Clearance of Senescent Cells Improves Glucose Tolerance and Insulin
Sensitivity
[0287] Groups of p16-3MR mice (n=9) were fed a high fat diet for
four months mice or a regular chow diet. Animals were then treated
with ganciclovir (3 rounds of 25 mg/kg ganciclovir administered
daily for five consecutive days) or vehicle. A glucose bolus was
given at time zero, and blood glucose was monitored at 20, 30, 60,
and 120 minutes after delivering glucose to determine glucose
disposal (see FIG. 18A). This was also quantified as "area under
the curve" (AUC) (see FIGS. 18B and 18C), with a higher AUC value
indicating glucose intolerance. AUCs of mice treated with
ganciclovir were significantly lower than their vehicle-treated
counterparts although not as low as chow-fed animals. Hemoglobin
A1c was lower in ganciclovir-treated mice (see FIG. 18C),
suggesting that the animals' longer-term glucose handling was also
improved.
[0288] Insulin sensitivity was also determined (Insulin Tolerance
Testing (ITT)). The results are presented in FIG. 19.
Ganciclovir-treated mice showed a greater decrease in blood glucose
at 0, 14, 30, 60, and 120 minutes after the administration of
glucose bolus at time zero (see FIG. 19A), suggesting that
senescent cell clearance improved insulin sensitivity. A change in
insulin tolerance testing when ganciclovir was administered to
wild-type mice was not observed (see FIG. 19B).
[0289] Changes in weight, body composition, and food intake were
also monitored. Treatment by ganciclovir did not alter body weight,
body composition monitored by measuring percent of fat, or food
intake (measured in grams per week).
Example 15
Effect of Treatment with Dasatinib and Quercetin on Glucose
Tolerance and Insulin Sensitivity
[0290] The effect of treating wild-type diet-induced obese (DIO)
mice with the combination of dasatinib and quercetin (D+Q) was
determined. Glucose tolerance testing was performed. The results
are presented in FIG. 20.
Example 16
Removal of Senescent Cells from DIO Mice Decreases Glucose
Intolerance
[0291] Three-month old INK-ATTAC;p16-3MR mice were fed a high fat
diet for 4 months. The animals were then treated with ganciclovir
or vehicle. DIO mice were treated with ganciclovir on September 1
(see FIG. 21). Glucose tolerance testing was performed, and the
results are presented in FIG. 21. Weights, fasting glucose levels,
and areas under the curve in intraperitoneal glucose tolerance
tests were determined and were significantly higher in DIO mice
than chow-fed controls, indicating glucose intolerance. Decreasing
senescent cells in DIO mice by treating with ganciclovir led to
sustained and improved glucose tolerance, as apparent from lower
areas under the curve in glucose tolerance tests, compared to
vehicle-treated DIO mice (lower vs. upper lines after 16-Sep,
respectively).
Example 17
Treatment of DIO Mice with Dasatinib and Quercetin
[0292] Diet-induced obese wild-type (DIO) mice were treated with
the combination of dasatinib (5 mg/kg) and quercetin (50 mg/kg)
(D+Q). Groups of mice (n=10) were treated with a high fat diet for
4 months. Weights, fasting glucose levels, and areas under the
curve in intra-peritoneal glucose tolerance tests were
significantly higher in DIO than chow-fed controls. Animals
received six doses of D+Q weekly. The results are presented in FIG.
22. Treating with the combination D+Q once weekly led to improved
glucose tolerance, as shown by lower areas under the curve in
glucose tolerance tests compared to vehicle-treated DIO mice (lower
vs. upper lines after 20-Oct, respectively; P<0.01; ANOVA for
repeated measures). Fasting glucose levels were significantly lower
in DIO mice after 6 doses of D+Q, each dose given weekly
(P<0.05; N=10 mice/group; T test).
[0293] In a second experiment, diet-induced obese (DIO) mice
treated with D+Q or vehicle. DIO mice were purchased from Jackson
Laboratory at 8 weeks of age and maintained on a 60% fat (% cal.)
diet throughout the study. Mice were treated with D+Q once per week
(5 mg/kg D, 100 mg/kg Q) at 4 months of age. Mice were sacrificed
after 28 weeks of treatment. Fat depot weights were measured at
time of sacrifice and were expressed as percent of whole body
weight. The results are presented in FIG. 23A. An increase in
subscapular fat depot weight was seen in diet-induced obese (DIO)
mice treated with D+Q (n=6) compared with vehicle-treated mice
(n=10). No difference in chow-fed mice fat depot weights were seen
between treatment and vehicle groups (n=9). Epididymal fat: p=0.34;
Brown fat p=0.23. The weights of other organs obtained from the
animals are shown FIG. 23B.
[0294] The various embodiments described above can be combined to
provide further embodiments. All U.S. patents, U.S. patent
application publications, U.S. patent applications, foreign
patents, foreign patent applications and non-patent publications
referred to in this specification and/or listed in the Application
Data Sheet, including U.S. Provisional Patent Application No.
61/932,711 filed Jan. 28, 2014 and U.S. Provisional Patent
Application No. 61/932,704 filed Jan. 28, 2014 are incorporated
herein by reference, in their entirety. Aspects of the embodiments
can be modified, if necessary to employ concepts of the various
patents, applications and publications to provide yet further
embodiments.
[0295] These and other changes can be made to the embodiments in
light of the above-detailed description. In general, in the
following claims, the terms used should not be construed to limit
the claims to the specific embodiments disclosed in the
specification and the claims, but should be construed to include
all possible embodiments along with the full scope of equivalents
to which such claims are entitled. Accordingly, the claims are not
limited by the disclosure.
* * * * *