U.S. patent application number 16/636299 was filed with the patent office on 2020-06-25 for treatment of lung diseases using pharmaceutical agents that eliminate senescent cells.
The applicant listed for this patent is Unity Biotechnology, Inc. Buck Institute for Research on Aging. Invention is credited to Scott Armstrong, Anne-Marie Beausoleil, Jamie Dananberg, Nathaniel David, Ryan Hudson, Remi-Martin Laberge, Nick Vlahakis.
Application Number | 20200199103 16/636299 |
Document ID | / |
Family ID | 68841874 |
Filed Date | 2020-06-25 |
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United States Patent
Application |
20200199103 |
Kind Code |
A1 |
Vlahakis; Nick ; et
al. |
June 25, 2020 |
Treatment of Lung Diseases Using Pharmaceutical Agents that
Eliminate Senescent Cells
Abstract
This invention is based on the discovery that many lung diseases
associated with aging are mediated at least in part by cells
bearing a senescent phenotype. Senescent cells accumulate with age,
and express factors that contribute to the pathophysiology of age
related conditions. The severity of age-related conditions
typically correlates with the abundance of senescent cells: thus,
clearing senescent cells can help abrogate the condition: providing
symptomatic relief, and potentially inhibiting disease progression.
In accordance with this invention, a family of Bcl protein
inhibitors has been developed for the treatment of lung diseases.
These senolytic agents have an appropriate dose and specificity
profile to be effective in the clinical management of previously
intractable pulmonary diseases.
Inventors: |
Vlahakis; Nick; (Brisbane,
CA) ; Armstrong; Scott; (Brisbane, CA) ;
Dananberg; Jamie; (Brisbane, CA) ; Hudson; Ryan;
(Brisbane, CA) ; Beausoleil; Anne-Marie;
(Brisbane, CA) ; David; Nathaniel; (Brisbane,
CA) ; Laberge; Remi-Martin; (Brisbane, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Unity Biotechnology, Inc.
Buck Institute for Research on Aging |
Brisbane
Novato |
CA
CA |
US
US |
|
|
Family ID: |
68841874 |
Appl. No.: |
16/636299 |
Filed: |
August 13, 2018 |
PCT Filed: |
August 13, 2018 |
PCT NO: |
PCT/US2018/046567 |
371 Date: |
February 3, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
62684681 |
Jun 13, 2018 |
|
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07D 207/34 20130101;
C07F 9/65583 20130101; A61P 9/10 20180101; C07D 401/12 20130101;
A61P 43/00 20180101; A61P 19/02 20180101; A61P 27/00 20180101; A61P
11/00 20180101 |
International
Class: |
C07D 401/12 20060101
C07D401/12; A61P 11/00 20060101 A61P011/00 |
Claims
1. A compound according to Formula (II): ##STR00097## wherein:
X.sup.1 is --Cl; X.sup.2 is --COOH or --SO.sub.2CH.sub.3; X.sup.3
is --SO.sub.2CF.sub.3, --SO.sub.2CH.sub.3, or --NO.sub.2; X.sup.5
is --F or --H; R.sup.1 is --CH(CH.sub.3).sub.2; R.sup.2 is
--CH.sub.3; R.sup.3 and R.sup.4 are both --H; n is 2; R.sup.6 is
selected from --OH, --OR.sup.7, ##STR00098## and R.sup.7 is
--PO(OH).sub.2, or a salt or a stereoisomer thereof.
2. The compound of claim 1, wherein X.sup.2 is --COOH.
3. The compound of claim 1, wherein X.sup.2 is
--SO.sub.2CH.sub.3.
4. The compound of claim 1, wherein X.sup.3 is
--SO.sub.2CF.sub.3.
5. The compound of claim 1, wherein X.sup.3 is
--SO.sub.2CH.sub.3.
6. The compound of claim 1, wherein X.sup.3 is --NO.sub.2.
7. The compound of any of claims 1 to 6, wherein X.sup.5 is
--F.
8. The compound of any of claims 1 to 6, wherein X.sup.5 is
--H.
9. The compound of any of claims 1 to 8, wherein X.sup.6 is
--OH.
10. The compound of any of claims 1 to 8, wherein X.sup.6 is
--OR.sup.7.
11. The compound of any of claims 1 to 8, wherein X.sup.6 is
##STR00099##
12. The compound of any of claims 1 to 8, wherein X.sup.6 is
##STR00100##
13. The compound of any of claims 1 to 8, wherein X.sup.6 is
##STR00101##
14. The compound of any of claims 1 to 13, wherein the carboxyl
group in X.sup.2 is phosphorylated.
15. The compound of claim 1, wherein the compound is selected from
the group consisting of: ##STR00102##
16. The compound of any preceding claim, which has pro-apoptotic
activity.
17. The compound of any preceding claim, which specifically kills
senescent cells compared with non-senescent cells, said senescent
cells being defined as non-cancerous cells that express p16.
18. The compound of any preceding claim, which specifically kills
cancer cells compared with non-cancer cells of the same tissue
type.
19. The compound of any preceding claim, which has an IC.sub.50 for
Bcl-xL of 1 nM or less.
20. The compound of any preceding claim, which has an IC.sub.50 for
Bcl-2 of 10 nM or less.
21. The compound of any preceding claim, which has an IC.sub.50 for
Bcl-xL of 1 nM or less and an IC.sub.50 for Bcl-2 of 10 nM or
less.
22. A pharmaceutical composition comprising a compound according to
any preceding claim in a pharmaceutically compatible excipient.
23. A method of selectively removing senescent cells and/or cancer
cells from a mixed cell population or tissue, comprising contacting
a cell, a cell population or a tissue with a compound according to
any of claims 1 to 21 or a pharmaceutical composition according to
claim 22.
24. A method of treating a senescence related condition in a tissue
in a subject, wherein the senescence related condition is
characterized as being caused or mediated at least in part by
senescent cells, or is characterized as having an overabundance of
senescent cells in or around the tissue, in comparison with
unaffected tissue, the method comprising: administering to a tissue
of a subject in need thereof, an amount of a compound according to
any of claims 1 to 21 or a pharmaceutical composition according to
claim 22 that is effective to selectively remove senescent cells
from the tissue, thereby relieving or ameliorating one or more
signs or symptoms of a senescence related condition in the
subject.
25. A unit dose of a pharmaceutical composition comprising: an
amount of a compound that inhibits Bcl function configured for use
in the treatment of a senescence associated condition that is
caused or mediated at least in part by senescent cells, wherein the
compound is a compound according to any of claims 1 to 21, wherein
the pharmaceutical composition contains a formulation of the
compound configured for administration to a target tissue in a
subject that manifests the senescence associated condition, and
wherein the formulation and the amount of the compound in the unit
dose configure the unit dose to be effective in selectively
removing senescent cells in or around the tissue in the subject,
thereby decreasing the severity of one or more signs or symptoms of
the condition without causing adverse effects in the subject when
administered to the tissue as a single dose.
26. The unit dose of claim 25, packaged with an informational
insert describing the use and attendant benefits of the drugs in
treating the senescent cell associated condition.
27. A compound according to any of claims 1 to 21 or a
pharmaceutical composition according to claim 24 for use in
selectively eliminating senescent cells from a tissue or mixed cell
population or for use in treating a senescence-related
condition.
28. Use of a compound according to any of claims 1 to 21 in the
manufacture of a medicament for treating a senescence-related
condition.
29. The method, unit dose, or use of any of claims 24 to 28,
wherein the condition is osteoarthritis.
30. The method, unit dose, or use of any of claims 24 to 28,
wherein the condition is an ophthalmic condition.
31. The method, unit dose, or use of any of claims 24 to 28,
wherein the condition is a pulmonary disease.
32. A method of treating cancer, comprising administering to a
tissue of a subject in need thereof an amount of a compound
according to any of claims 1 to 21 or a pharmaceutical composition
according to claim 22 effective to selectively remove cancer cells
from the tissue.
33. A compound according to any of claims 1 to 21 or a
pharmaceutical composition according to claim 22 for use in
selectively eliminating cancer cells from a tissue or mixed cell
population or for use in treating cancer.
34. A method of treating a pulmonary disease in a subject,
comprising administering to the subject in need thereof a
therapeutically effective amount of a pharmaceutical composition
comprising: a compound of Formula (I): ##STR00103## wherein:
X.sup.1 is --Cl; X.sup.2 is --COOH or --SO.sub.2CH.sub.3; X.sup.3
is --SO.sub.2CF.sub.3; --SO.sub.2CH.sub.3; or --NO.sub.2; X.sup.5
is --F or --H; R.sup.1 is --CH(CH.sub.3).sub.2; R.sup.2 is
--CH.sub.3; R.sup.3 and R.sup.4 are both --H; n is 2; and R.sup.6
is selected from --OR.sup.7, ##STR00104## and R.sup.7 is --H or
--PO(OH).sub.2, or a salt or a stereoisomer thereof; and a
pharmaceutically compatible excipient.
35. The method of claim 34, wherein X.sup.2 is --COOH.
36. The method of claim 34, wherein X.sup.2 is
--SO.sub.2CH.sub.3.
37. The method of claim 34, wherein X.sup.3 is
--SO.sub.2CF.sub.3.
38. The method of claim 34, wherein X.sup.3 is
--SO.sub.2CH.sub.3.
39. The method of claim 34, wherein X.sup.3 is --NO.sub.2.
40. The method of any of claims 34 to 39, wherein X.sup.5 is
--F.
41. The method of any of claims 34 to 39, wherein X.sup.5 is
--H.
42. The method of any of claims 34 to 39, wherein R.sup.6 is
--OR.sup.7.
43. The method of any of claims 34 to 39, wherein R.sup.6 is
##STR00105##
44. The method of any of claims 34 to 39, wherein R.sup.6 is
##STR00106##
45. The method of any of claims 34 to 44, wherein R.sup.7 is
--H.
46. The method of any of claims 34 to 44, wherein R.sup.7 is
--PO(OH).sub.2.
47. The method of any of claims 34 to 46, wherein the carboxyl
group in X.sup.2 is phosphorylated.
48. The method of claim 34, wherein the compound is selected from
the group consisting of: ##STR00107## ##STR00108##
49. The method of claim 34, wherein the pulmonary disease is
idiopathic pulmonary fibrosis (IPF).
50. The method of claim 34, wherein the pulmonary disease is
chronic obstructive pulmonary disease (COPD).
51. The method of claim 48, wherein the pulmonary disease is
idiopathic pulmonary fibrosis (IPF).
52. The method of claim 48, wherein the pulmonary disease is
chronic obstructive pulmonary disease (COPD).
53. The method of claim 34, wherein the administration of the
pharmaceutical composition is by inhalation as an aerosol.
54. The method of claim 48, wherein the administration of the
pharmaceutical composition is by inhalation as an aerosol.
Description
RELATED APPLICATIONS
[0001] This application claims priority to U.S. patent application
Ser. No. 15/675,171, filed Aug. 11, 2017, which is a
continuation-in-part of U.S. patent application Ser. No.
15/611,589, filed Jun. 1, 2017, which is a continuation of
International patent application no. PCT/US16/16894, filed Feb. 5,
2016, which claims priority to U.S. provisional patent application
No. 62/113,227, filed Feb. 6, 2015. This application also claims
priority to U.S. provisional patent application No. 62/684,681,
filed Jun. 13, 2018. The disclosures of each of the
above-referenced applications are incorporated herein by reference
in their entireties.
FIELD OF THE INVENTION
[0002] The technology disclosed and claimed below relates generally
to the field of lung disease leading to impaired respiration
capacity. This disclosure provides a family of compounds and
techniques that can be used for treating pulmonary disease by
eliminating senescent cells implicated in the underlying
pathophysiology and symptomatology.
BACKGROUND
[0003] Recent WHO data in 2015 show that respiratory diseases make
up three of the top five causes of death worldwide
(http://www.who.int/mediacentre/factsheets/fs310/en/). Chronic
obstructive pulmonary disease (COPD) and lower respiratory
infections are third and fourth on this list and both are diseases
most prevalent in the elderly. Furthermore, the NHLBI published a
white paper in 2017 highlighting the association of age with lung
disease, including inflammatory pulmonary fibrosis (IPF) and COPD,
and underscoring the potential for understanding and developing
therapeutics related to ageing biology. Historically, therapies for
these diseases have been non-specific in their mode of action,
either anti-inflammatory (e.g. corticosteroids) or
immunosuppressive (e.g., cyclophosphamide) or purely supportive in
nature e.g., supplemental oxygen, anti-tussives and diuretics.
[0004] There is growing evidence of senescent cells playing a role
in such non-oncologic pulmonary diseases, including data disclosed
herein. Thus, the goal of the compounds of the invention is to not
just interrupt specific pathogenic pathways but specifically target
senescent cells and in turn inhibit multiple pathogenic
pathways.
[0005] The invention provided herein provides novel compounds for
the treatment of respiratory disease (primary or secondary
etiology), and extra-pulmonary effects arising from or associated
with such lung diseases, through the elimination of senescent cells
implicated in the pathophysiology of diseases of the pulmonary
system. The disclosure that follows outlines its implementation and
use and describes many of the ensuing benefits.
SUMMARY
[0006] This invention is based in part on the discovery that many
pulmonary diseases and conditions associated with aging are
mediated at least in part by cells bearing a senescent phenotype.
Senescent cells accumulate with age, which is why conditions
mediated by senescent cells occur more frequently in older adults.
Senescent cells express factors that contribute to the
pathophysiology of the age related and senescence-associated
conditions. Different types of stress on pulmonary tissues may
promote the emergence of senescent cells and the phenotype they
express. Cell stressors include oxidative stress, metabolic stress,
DNA damage (for example, because of environmental ultraviolet light
exposure or genetic cause), oncogene activation, and telomere
shortening (resulting, for example, from hyperproliferation).
[0007] This invention is also based in part on new acyl
sulfonamides that are Bcl inhibitors. Some of the Bcl inhibitors in
this family are particularly effective senolytic agents for lung
diseases. Contacting senescent cells in vitro or in vivo with the
compounds and compositions of the invention selectively modulates
or eliminates such cells. These inhibitors can be used for
administration to a diseased lung tissue in a subject having an
age-related lung disease, thereby selectively eliminating senescent
cells in or around the diseased lung tissue and relieving one or
more symptoms or signs of the disease. Selected compounds from the
family can be formulated and marketed as chemotherapeutic
agents.
BRIEF DESCRIPTION OF THE DRAWINGS
[0008] FIG. 1 shows a general synthetic scheme for chemically
synthesizing exemplary compounds according to this invention.
[0009] FIGS. 2A and 2B show immunohistochemical staining for p16 in
human IPF lung tissue to demonstrate the presence of senescent
cells. FIG. 2A shows a macro-view of the IPF lung tissue having p16
positive staining FIG. 2B is an enlarged view of an area of the IPF
lung tissue of FIG. 2A. The senescent cells were predominately
epithelial in origin and located in areas of fibrosis and at the
leading edge of the disease. See Example 1.
[0010] FIG. 3 shows the quantification of p16 positive cells in all
normal human lung tissues sampled as compared to all human IPF lung
tissues sampled. Increased presence of p16 positive cells in human
lung tissue with significant fibrotic area was indicative of a
significant role in disease progression (****p<0.0001 for group
difference among means by one-way ANOVA). See Example 1.
[0011] FIGS. 4A, 4B and 4C show immunohistochemical staining for
p16 in human scleroderma lung tissue to demonstrate the presence of
senescent cells. FIG. 4A shows a macro-view of the human
scleroderma lung tissue having p16 positive staining. FIG. 4B is an
enlarged view of an area of the scleroderma lung tissue of FIG. 4A.
FIG. 4C is a further enlarged view of an area of the scleroderma
lung tissue of FIG. 4B. The p16 positive senescent cells were
fibrotic in origin and located in honeycomb areas of the lung. See
Example 1.
[0012] FIG. 5 shows the quantification of p16 positive cells in
normal human lung tissue as compared to human scleroderma lung
tissue. Increased presence of p16 positive cells in human lung
tissue with significant fibrotic area was indicative of a
significant role in disease progression (****p<0.0001 for group
difference among means by one-way ANOVA). See Example 1.
[0013] FIG. 6 shows a concentration-response curve demonstrating
the selectivity of Compound 1 for senescent lung epithelial cells
(SnC--solid lines) in contrast to non-senescent lung epithelial
cells (NsC--dashed lines). See Example 2.
[0014] FIG. 7 shows a concentration-response curve, using one-way
ANOVA, of relative p16 gene expression changes of 8%, 14% and 27%
upon treatment with 0.1 mg/ml, 0.3 mg/ml and 1.0 mg/ml of Compound
1, respectively, in mice challenged with bleomycin (+) to induce
senescence in the lung. See Example 4.
[0015] FIG. 8 shows the ability of Compound 1 to eliminate p16
positive lung epithelial cells in mice challenged with bleomycin
(+) to induce senescence in the lung. See Example 4.
DETAILED DESCRIPTION
Definitions
[0016] The terms "enantiomerically enriched" and
"stereoisomerically enriched" denote that the compound of the
invention comprises 75%, 80%, 85%, 90%, 95%, 98%, or 99% or more by
weight of the enantiomer or the stereoisomer.
[0017] The term "ex vivo" refers to experimentation or manipulation
done in or on living tissue in an artificial environment outside
the organism.
[0018] The term "pharmaceutically acceptable carrier" or
"pharmaceutically acceptable excipient" includes any and all
solvents, dispersion media, coatings, antibacterial and antifungal
agents, isotonic and absorption delaying agents and the like. The
use of such media and agents for pharmaceutically active substances
is well known in the art. Except insofar as any conventional media
or agent is incompatible with the active ingredient, its use in the
therapeutic compositions is contemplated. Supplementary active
ingredients can also be incorporated into the compositions.
[0019] The term "pharmaceutically acceptable salt" refers to salts
that retain the biological effectiveness and properties of the
compounds of this invention and, which are not biologically or
otherwise undesirable. In many cases, the compounds of this
invention are capable of forming acid and/or base salts by virtue
of the presence of amino, phosphate, and/or carboxyl groups or
groups similar thereto. Pharmaceutically acceptable acid addition
salts can be formed with inorganic acids and organic acids.
Inorganic acids from which salts can be derived include, for
example, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric
acid, phosphoric acid, and the like. Organic acids from which salts
can be derived include, for example, acetic acid, propionic acid,
naphtoic acid, oleic acid, palmitic acid, pamoic (emboic) acid,
stearic acid, glycolic acid, pyruvic acid, oxalic acid, maleic
acid, malonic acid, succinic acid, fumaric acid, tartaric acid,
citric acid, ascorbic acid, glucoheptonic acid, glucuronic acid,
lactic acid, lactobioic acid, tartaric acid, benzoic acid, cinnamic
acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid,
p-toluenesulfonic acid, salicylic acid, and the like.
Pharmaceutically acceptable base addition salts can be formed with
inorganic and organic bases. Inorganic bases from which salts can
be derived include, for example, sodium, potassium, lithium,
ammonium, calcium, magnesium, iron, zinc, copper, manganese,
aluminum, and the like; particularly preferred are the ammonium,
potassium, sodium, calcium and magnesium salts. Organic bases from
which salts can be derived include, for example, primary,
secondary, and tertiary amines, substituted amines including
naturally occurring substituted amines, cyclic amines, basic ion
exchange resins, and the like, specifically such as isopropylamine,
trimethylamine, diethylamine, triethylamine, tripropylamine,
histidine, arginine, lysine, benethamine, N-methyl-glucamine, and
ethanolamine Other acids include dodecylsufuric acid,
naphthalene-1,5-disulfonic acid, naphthalene-2-sulfonic acid, and
saccharin.
[0020] A "phosphorylated" form of a compound is a compound in which
one or more --OH or --COOH groups have been substituted with a
phosphate group which is either --OPO(OH).sub.2 or
-alkyl-OPO(OH).sub.2 (where alkyl is C.sub.1-6 alkyl), such that
the phosphate group may be removed in vivo (for example, by
enzymolysis). A non-phosphorylated or dephosphorylated form has no
such phosphate group.
[0021] "Prodrug" refers to a derivative of an active agent that
requires a transformation within the body to release the active
agent. The transformation can be an enzymatic transformation.
Prodrugs are frequently, although not necessarily,
pharmacologically inactive until converted to the active agent.
[0022] A "senescent cell" is generally thought to be derived from a
cell type that typically replicates, but as a result of aging or
other event that causes a change in cell state, can no longer
replicate. It remains metabolically active and commonly adopts a
senescence associated secretory phenotype (SASP) that includes
chemokines, cytokines and extracellular matrix and fibrosis
modifying proteins and enzymes. The nucleus of senescent cells is
often characterized by senescence-associated heterochromatin foci
and DNA segments with chromatin alterations reinforcing senescence.
Without implying any limitation on the practice of what is claimed
in this disclosure that is not explicitly stated or required, the
invention is premised on the hypothesis that senescent cells cause
or mediate certain conditions associated with tissue damage or
aging. For the purpose of practicing aspects of this invention,
senescent cells can be identified as expressing at least one marker
selected from p16, senescence-associated .beta.-galactosidase, and
lipofuscin; sometimes two or more of these markers, and other
markers of SASP such as but not limited to, interleukin 6 (IL-6),
and inflammatory, angiogenic and extracellular matrix modifying
proteins.
[0023] A "senescence associated", "senescence related" or "age
related" disease, disorder, or condition is a physiological
condition that presents with one or more symptoms or signs, wherein
a subject having the condition needs or would benefit from a
lessening of such symptoms or signs. The condition is senescence
associated if it is caused or mediated in part by senescent cells,
which may be induced by multiple etiologic factors including age,
DNA damage, oxidative stress, genetic defects, etc. Lists of
senescence associated disorders that can potentially be treated or
managed using the methods and products taught in this disclosure
include those discussed in this disclosure and the previous
disclosures to which this application claims priority.
[0024] A compound of the invention is typically referred to as
"senolytic" if it eliminates senescent cells, compared with
replicative cells of the same tissue type, or quiescent cells
lacking SASP markers. Alternatively, or in addition, compounds of
the invention may effectively be used according to this invention
if it decreases the release of pathological soluble factors or
mediators as part of the senescence associated secretory phenotype
that play a role in the initial presentation or ongoing pathology
of a condition or inhibit its resolution. In this respect, the term
"senolytic" is exemplary, with the understanding that compounds
that work primarily by inhibiting rather than eliminating senescent
cells (senescent cell inhibitors) can be used in a similar fashion
with ensuing benefits.
[0025] "Small molecule" Bcl inhibitors according to this invention
have molecular weights less than 20,000 daltons, and are often less
than 10,000, 5,000, or 2,000 daltons. Small molecule inhibitors are
not antibody molecules or oligonucleotides, and typically have no
more than five hydrogen bond donors (the total number of
nitrogen-hydrogen and oxygen-hydrogen bonds), and no more than 10
hydrogen bonds.
[0026] Successful "treatment" of a lung disease according to this
invention may have any effect that is beneficial to the subject
being treated. This includes decreasing severity, duration, or
progression of a condition, or of any adverse signs or symptoms
resulting therefrom. In some circumstances, senolytic agents can
also be used to prevent or inhibit presentation of a condition for
which a subject is susceptible, for example, because of an
inherited susceptibility of because of medical history.
[0027] A "therapeutically effective amount" is an amount of a
compound of the present disclosure that (i) treats the particular
disease, condition, or disorder, (ii) attenuates, ameliorates, or
eliminates one or more symptoms of the particular disease,
condition, or disorder, (iii) prevents or delays the onset of one
or more symptoms of the particular disease, condition, or disorder
described herein, (iv) prevents or delays progression of the
particular disease, condition or disorder, or (v) at least
partially reverses damage caused by the condition prior to
treatment.
[0028] Unless otherwise stated or required, all the compound
structures referred to in the invention include conjugate acids and
bases having the same structure, crystalline and amorphous forms of
those compounds, pharmaceutically acceptable salts, and dissolved
and solid forms thereof, including, for example, polymorphs,
solvates, hydrates, unsolvated polymorphs (including anhydrates),
conformational polymorphs, and amorphous forms of the compounds, as
well as mixtures thereof. Except where otherwise stated or
required, other terms used in the specification have their ordinary
meaning.
[0029] The half maximal inhibitory concentration (IC.sub.50) is a
measure of the potency of a compound in inhibiting a specific
biological or biochemical function. Specifically, for compounds of
the invention, IC.sub.50 is the measure of the amount of a compound
required to achieve 50% inhibition of the activity of the target
Bcl. For example, compounds of the invention have a demonstrated
IC.sub.50 for Bcl-xL of less than 10 nM, less than 5 nM, or less
than 1 nM. Compounds of the invention have a demonstrated IC.sub.50
for Bcl-xL of between 1 nM to 10 nM, between 1 nM and 5 nM, between
5 nM to 10 nM, or between 0.1 nM to 1 nM. Compounds of the
invention have also demonstrated an IC.sub.50 for Bcl-2 of less
than 15 nM, less than 10 nM, less than 5 nM, or less than 1 nM.
Compounds of the invention have also demonstrated an IC.sub.50 for
Bcl-2 of between 1 nM to 10 nM, between 1 nM and 5 nM, between 5 nM
to 10 nM, or between 0.1 nM to 1 nM.
[0030] "Alkyl" refers to monovalent saturated aliphatic hydrocarbyl
groups having from 1 to 10 carbon atoms and preferably 1 to 6
carbon atoms. This term includes, by way of example, linear and
branched hydrocarbyl groups such as methyl (--CH.sub.3), ethyl
(--CH.sub.2CH.sub.3, n-propyl (--CH.sub.2CH.sub.2CH.sub.3),
isopropyl (--CH(CH.sub.3).sub.2), n-butyl
(--CH.sub.2CH.sub.2CH.sub.2CH.sub.3), isobutyl
(--CH.sub.2CH(CH.sub.3).sub.2), sec-butyl
(--CH(CH.sub.2CH.sub.3)(CH).sub.3), t-butyl (--C(CH.sub.3).sub.3),
n-pentyl (--CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.3), and
neopentyl (--CH.sub.2C(CH.sub.3).sub.3), etc. In certain
embodiments, an "alkyl" group can be substituted, where the term
"substituted," when used to modify a specified group or radical,
means that one or more hydrogen atoms of the specified group or
radical are each independently replaced with the same or different
substituent groups as defined below.
[0031] Substituent groups for substituting for one or more
hydrogens (any two hydrogens on a single carbon can be replaced
with .dbd.O, .dbd.NR.sup.50, .dbd.N--OR.sup.50, .dbd.N.sub.2 or
.dbd.S) on carbon atoms in the specified group or radical are,
unless otherwise specified, --R.sup.60, halo, .dbd.O, --OR.sup.50,
--SR.sup.50, --NR.sup.50R.sup.50, trihalomethyl, --CN, --OCN,
--SCN, --NO, --NO.sub.2, .dbd.N.sub.2, --N.sub.3,
--SO.sub.2R.sup.50, --SO.sub.2OR.sup.50, --OSO.sub.2R.sup.50,
--OSO.sub.2OR.sup.50, --P(O)(OH).sub.2, --P(O)(OR.sup.50)OH,
--P(O)(OR.sup.50).sub.2, --C(O)R.sup.50,
--C(S)R.sup.50)--C(NR.sup.50)R.sup.50, --C(O)OR.sup.50,
--C(S)OR.sup.50, --C(O)NR.sup.50R.sup.50,
--C(NR.sup.50)NR.sup.50R.sup.50, --OC(O)R.sup.50, --OC(S)R.sup.50,
--OC(O)OR.sup.50, --OC(S)OR.sup.50, --NR.sup.50C(O)R.sup.50,
--NR.sup.50 C(S)R.sup.50, --NR.sup.50CO.sub.2R.sup.50,
--NR.sup.50C(S)OR.sup.50, --N R.sup.50C(O)NR.sup.50R.sup.50,
--NR.sup.50C(NR.sup.50)R.sup.50 and
--NR.sup.50C(NR.sup.50)NR.sup.50R.sup.50, where R.sup.60 is
selected from optionally substituted alkyl, cycloalkyl,
heteroalkyl, heterocycloalkylalkyl, cycloalkylalkyl, aryl,
arylalkyl, heteroaryl and heteroarylalkyl, and each R.sup.50 is
independently hydrogen or R.sup.60.
[0032] "Heteroalkyl" refers to a saturated or unsaturated group
having a single ring or multiple condensed rings, including fused
bridged and spiro ring systems, and having from 3 to 20 ring atoms,
including 1 to 10 hetero atoms. These ring atoms are selected from
nitrogen, sulfur, or oxygen, where, in fused ring systems, one or
more of the rings can be cycloalkyl, aryl, or heteroaryl. In
certain embodiments, a "heteroalkyl" group can be substituted, as
defined above.
DESCRIPTION OF THE INVENTION
[0033] It is a premise of this disclosure that many or most lung
diseases that are age-related or are characterized by a pathogenic
senescent biology that may be present at any age, are caused or
mediated at least in part by senescent cells, which accumulate with
age or in a disease-specific manner, and with deleterious impact on
respiratory system pulmonary tissues. Senescent cells are typically
cells that no longer have replicative capacity, but remain in the
tissue of origin, eliciting a senescence-associated secretory
phenotype (SASP). Senescent cells are thought to derive from
proliferative cells of a variety of tissue types, including cells
that reside in and around the lung. SASP factors include molecules
that are angiogenic, inflammatory, proliferative, fibrotic, and
extracellular matrix modifying molecules (Acosta et al., 2013).
Some factors implicated in pulmonary pathologies are part of the
constellation of factors produced by senescent cells. For this
reason, elimination or control of senescent cells provides a means
by which to treat lung disease, not only through the elimination of
senescent cells but also through reduction of their associated SASP
factors and impact on surrounding cells.
[0034] Different lung diseases present in the clinic with different
signs and symptoms and have different types of pathophysiologic
mechanisms. The heterogeneity of lung disease is consistent with
the role of senescent cells in the disease pathology because
senescent cells may be from different cell lineages, induced by
different stressors, reside in different pulmonary tissues, and
interact with surrounding cells in a different fashion.
Nevertheless, senescent cells in the various tissues of the lung
have a related secretory phenotype that contributes to disorders
throughout the respiratory system.
[0035] The role of senescent cells in promoting or mediating the
spectrum of lung diseases provides an approach to treatment with a
number of advantages for the managing clinician. Since senescent
cells are non-proliferative, eliminating senescent cells has the
potential for a clinically beneficial effect that persists for an
extended time between episodes of treatment. Features of the
condition mediated by senescent cells can resolve at least until
senescent cells re-accumulate. Since senescent cells and the burden
of their pathogenic effect are likely to accumulate slowly, as the
nature of age related diseases is to evolve over a period of many
years, the effects of a single treatment or treatment cycle may
last for weeks, months, or years.
[0036] The specific clearance of senescent cells from tissue is
referred to in this disclosure as senolysis. Small molecule
compounds capable of senolysis are referred to as senolytic agents,
and clear senescent cells irrespective of mechanism of senescence
induction, SASP profile or cell lineage. To the extent that
senescent cells exacerbate the underlying extracellular pathogenic
mechanisms, the long-lasting effect of senolysis provides a window
in which such pathology is held at bay, potentially giving the
tissue a chance for repair. This means that senescent cell medicine
has the potential not just to halt progression of pulmonary
diseases and conditions but allow some degree of reversal of the
disease and its symptoms for the benefit of the patient.
[0037] Since senescent cells in different parts of the lung respond
to the same senolytic agents, several different lung diseases can
be treated in the same patient at the same time. For example, a
patient may present to the clinician with several concurrent active
disease processes already under way: such as fibrosis and chronic
obstructive lung disease. It may be possible to administer a single
senolytic agent in a treatment protocol that addresses the disease
and its symptoms of each of the multiple conditions. Beyond the
convenience of this approach, it has the added benefit of lowering
the risk of side effects that may result from multiple drugs being
given in combination to treat each of the conditions individually.
Furthermore, it is possible that factors elicited by cells in one
part of the lung may impact other parts of the lung such that
treating senescence in two locations in the lung may have a
beneficial effect on both lung diseases.
[0038] Senolytic medicines can be an important adjunct to other
types of therapies, such as for example, standard of care, to
relieve the symptoms that result from the condition(s). The two
modes of therapy can work synergistically to reduce the burden,
frequency and side effects of either mode administered
separately.
[0039] Bcl Inhibitors
[0040] The technology described and claimed below represents the
first description of a new class of Bcl inhibitors that can be used
to selectively eliminate senescent cells from a target tissue for
purposes of treatment of age-related conditions.
[0041] The Bcl protein family (TC#1.A.21) includes
evolutionarily-conserved proteins that share Bcl-2 homology (BH)
domains. Bcl proteins are most notable for their ability to up- or
down-regulate apoptosis, a form of programmed cell death, at the
mitochondrion. The following explanation is provided to assist the
user in understanding some of the scientific underpinnings of the
compounds of this invention. These concepts are not needed to
practice the invention, nor do they limit the use of the compounds
and methods described here in any manner beyond that which is
expressly stated or required. In the context of this invention, the
Bcl proteins of particular interest are those that downregulate
apoptosis. Anti-apoptotic Bcl proteins contain BH1 and BH2 domains,
some of them contain an additional N-terminal BH4 domain (Bcl-2,
Bcl-x(L) and Bcl-w (Bcl-2L2), Inhibiting these proteins increases
the rate or susceptibility of cells to apoptosis. Thus, an
inhibitor of such proteins can be used to help eliminate cells in
which the proteins are expressed.
[0042] In the mid-2000s, Abbott Laboratories developed a novel
inhibitor of Bcl-2, Bcl-xL and Bch w, known as ABT-737
(Navitoclax). This compound is part of a group of BH3 mimetic small
molecule inhibitors (SMI) that target these Bcl-2 family proteins,
but not Al or Mcl-1. ABT-737 is superior to previous BCL-2
inhibitors given its higher affinity for Bcl-2, Bcl-xL and Bcl-w.
In vitro studies showed that primary cells from patients with
B-cell malignancies are sensitive to ABT-737. In human patients,
ABT-737 is effective against some types of cancer cells but is
subject to dose-limiting thrombocytopenia.
[0043] U.S. Application Publication No. 2016/0339019 (Laberge et
al.) describes treatment of certain age-related conditions using
MDM2 inhibitors, Bcl inhibitors, and Akt inhibitors. U.S.
Application Publication No. 2017/0266211 (David et al.) describes
the use of particular Bcl inhibitors for treatment of age-related
conditions. U.S. Pat. Nos. 8,691,184, 9,096,625, and 9,403,856
(Wang et al.) describe Bcl inhibitors in a small-molecule library.
It has now been discovered that the compounds described here fit
into the active site of Bcl protein to provide strong Bcl
inhibition and/or promote apoptosis of target cells. These
compounds can be developed as highly potent and specific drugs to
target senescent cells and cancer cells, as described in the
sections that follow.
[0044] Generally, the compounds of the present disclosure have a
structure that falls within the scope of the structure according to
Formula (I) shown below.
##STR00001##
wherein: [0045] X.sup.1 is --Cl; [0046] X.sup.2 is --COOH or
--SO.sub.2CH.sub.3; [0047] X.sup.3 is --SO.sub.2CF.sub.3;
--SO.sub.2CH.sub.3; or --NO.sub.2; [0048] X.sup.5 is --F or --H;
[0049] R.sup.1 is .sup.--CH(CH.sub.3).sub.2; [0050] R.sup.2 is
--CH.sub.3; [0051] R.sup.3 and R.sup.4 are both --H; [0052] n is
2;
##STR00002##
[0052] and [0053] R.sup.6 is selected from --OR.sup.7, and [0054]
R.sup.7 is --H or --PO(OH).sub.2, [0055] or a salt or a
stereoisomer thereof; and [0056] a pharmaceutically compatible
excipient.
[0057] As described above, in compounds of Formula (I), X.sup.1 is
--Cl, R.sup.1 is --CH(CH.sub.3).sub.2, R.sup.2 is --CH.sub.3,
R.sup.3 is H, R.sup.4 is --H, and n is 2.
[0058] In certain embodiments,X.sup.2 is --COOH or
--SO.sub.2CH.sub.3. For example, X.sup.2 can be --COOH, or X.sup.2
can be --SO.sub.2CH.sub.3. As used herein, the dash symbol ("-")
indicates the point of attachment of the moiety of interest to the
remainder of the compound being described.
[0059] In certain embodiments, X.sup.3 is --SO.sub.2CF.sub.3,
--SO.sub.2CH.sub.3, or --NO.sub.2. For example, X.sup.3 can be
--SO.sub.2CF.sub.3, or X.sup.3 can be --SO.sub.2CH.sub.3, or
X.sup.3 can be --NO.sub.2.
[0060] In certain embodiments, X.sup.5 is --F or --H. For example,
X.sup.5 can be --F, or X.sup.5 can be --H.
[0061] In certain embodiments, R.sup.6 is selected from
--OR.sup.7,
##STR00003##
For example, R.sup.6 can be
##STR00004##
or R.sup.6 can be
##STR00005##
[0062] or R.sup.6 can be
[0063] As used herein, a wavy line ("") indicates the point of
attachment or the bond where the moiety of interest is attached to
the remainder of the compound being described.
[0064] In certain embodiments, R.sup.7 is --H or --PO(OH).sub.2.
For example, R.sup.7 can be --H, or R.sup.7 can be
--PO(OH).sub.2.
[0065] Any of the various combinations of the X.sup.2, X.sup.3,
X.sup.5, R.sup.6, and R.sup.7 substituents are possible for
compounds of Formula (I).
[0066] In combination with any of the aforelisted options, the
--COOH group of X.sup.2 may be phosphorylated as well as or instead
of the hydroxyl group at the R.sup.6 position, at the user's
option.
[0067] Examples of the compounds of Formula (I) are shown in TABLE
1.
TABLE-US-00001 TABLE 1 Com- pound No. Compound Structure and Name 1
##STR00006## 2 ##STR00007## 3 ##STR00008## 4 ##STR00009## 5
##STR00010## 6 ##STR00011## 7 ##STR00012## 8 ##STR00013## 9
##STR00014## 10 ##STR00015## 11 ##STR00016## 12 ##STR00017## 13
##STR00018## 14 ##STR00019## 15 ##STR00020## 16 ##STR00021## 17
##STR00022## 18 ##STR00023## 19 ##STR00024## 20 ##STR00025## 21
##STR00026## 22 ##STR00027## 23 ##STR00028## 24 ##STR00029## 25
##STR00030## 26 ##STR00031## 27 ##STR00032## 28 ##STR00033## 29
##STR00034## 30 ##STR00035## 31 ##STR00036## 32 ##STR00037## 33
##STR00038## 34 ##STR00039## 35 ##STR00040## 36 ##STR00041##
[0068] Compounds of Formula (I) find use in methods of treating a
pulmonary disease in a subject as described herein. For example,
the pulmonary disease can be idiopathic pulmonary fibrosis (IPF),
or the pulmonary disease can be chronic obstructive pulmonary
disease (COPD).
[0069] In certain embodiments, the compounds of the present
disclosure have a structure that falls within the scope of the
structure according to Formula (II) shown below.
##STR00042##
wherein: [0070] X.sup.1 is --Cl; [0071] X.sup.2 is --COOH or
--SO.sub.2CH.sub.3; [0072] X.sup.3 is --SO.sub.2CF.sub.3,
--SO.sub.2CH.sub.3, or --NO.sub.2; [0073] X.sup.5 is --F or --H;
[0074] R.sup.1 is --CH(CH.sub.3).sub.2; [0075] R.sup.2 is
--CH.sub.3; [0076] R.sup.3 and R.sup.4 are both --H; [0077] n is 2;
[0078] R.sup.6 is selected from --OH, --OR.sup.7,
##STR00043##
[0078] and [0079] R.sup.7 is --PO(OH).sub.2, [0080] or a salt or a
stereoisomer thereof.
[0081] As described above, in compounds of Formula (II), X.sup.1 is
--Cl, R.sup.1 is --CH(CH.sub.3).sub.2, R.sup.2 is --CH.sub.3,
R.sup.3 is H, R.sup.4 is --H, n is 2, and R.sup.7 is
--PO(OH).sub.2.
[0082] In certain embodiments, X.sup.2 is --COOH or
--SO.sub.2CH.sub.3. For example, X.sup.2 can be --COOH, or X.sup.2
can be --SO.sub.2CH.sub.3.
[0083] In certain embodiments, X.sup.3 is --SO.sub.2CF.sub.3,
--SO.sub.2CH.sub.3, or --NO.sub.2. For example, X.sup.3 can be
--SO.sub.2CF.sub.3, or X.sup.3 can be --SO.sub.2CH.sub.3, or
X.sup.3 can be --NO.sub.2.
[0084] In certain embodiments, X.sup.5 is --F or --H. For example,
X.sup.5 can be --F, or X.sup.5 can be --H.
[0085] In certain embodiments, R.sup.6 is selected from --OH,
--OR.sup.7,
##STR00044##
For example, R.sup.6 can be --OH, or R.sup.6 can be --, or R.sup.6
can be
##STR00045##
or R.sup.6 can be
##STR00046##
[0086] or R.sup.6 can be
##STR00047##
[0088] Any of the various combinations of the X.sup.2, X.sup.3,
X.sup.5, and R.sup.6 substituents are possible for compounds of
Formula (II).
[0089] In combination with any of the aforelisted options, the
--COOH group of X.sup.2 may be phosphorylated as well as or instead
of the hydroxyl group at the R.sup.6 position, at the user's
option.
[0090] Examples of the compounds of Formula (II) are shown in TABLE
2.
TABLE-US-00002 TABLE 2 Com- pound No. Compound Structure and Name 1
##STR00048## 2 ##STR00049## 3 ##STR00050## 4 ##STR00051## 5
##STR00052## 6 ##STR00053## 7 ##STR00054## 8 ##STR00055## 9
##STR00056## 10 ##STR00057## 11 ##STR00058## 12 ##STR00059## 13
##STR00060## 14 ##STR00061## 15 ##STR00062## 16 ##STR00063## 17
##STR00064## 18 ##STR00065## 19 ##STR00066## 20 ##STR00067## 21
##STR00068## 26 ##STR00069## 27 ##STR00070## 28 ##STR00071## 29
##STR00072## 30 ##STR00073## 31 ##STR00074## 32 ##STR00075## 33
##STR00076## 34 ##STR00077## 35 ##STR00078## 36 ##STR00079##
[0091] In certain embodiments, the compounds of the present
disclosure have a structure that falls within the scope of the
structure according to Formula (III) shown below.
##STR00080##
wherein: [0092] R.sup.1 and R.sup.2 are independently C.sub.1 to
C.sub.4 alkyl; [0093] R.sup.3, R.sup.4 and R.sup.5 are
independently --H or --CH.sub.3; [0094] R.sup.8 is --OH or
--N(R.sup.6)(R.sup.7), wherein R.sup.6 and R.sup.7 are
independently alkyl or heteroalkyl, and are optionally cyclized;
[0095] X.sup.1 is --F, --Cl, --Br, or --OCH.sub.3; [0096] X.sup.2
is --SO.sub.2R' or --CO.sub.2R', where R' is --H, --CH.sub.3, or
--CH.sub.2CH.sub.3; [0097] X.sup.3 is --SO.sub.2CF.sub.3;
--SO.sub.2CH.sub.3; or --NO.sub.2; and [0098] X.sup.5 is --F, --Br,
--Cl, --H, or --OCH.sub.3.
[0099] In certain embodiments, R.sup.1 and R.sup.2 are
independently C.sub.1 to C.sub.4 alkyl. For example, R.sup.1 can be
C.sub.1 to C.sub.4 alkyl, and R.sup.2 can be C.sub.1 to C.sub.4
alkyl.
[0100] In certain embodiments, R.sup.3, R.sup.4 and R.sup.5 are
independently --H or --CH.sub.3. For example, R.sup.3 can be --H or
--CH.sub.3. In some instances, R.sup.4 is --H or --CH.sub.3. In
some instances, R.sup.5 is --H or --CH.sub.3.
[0101] In certain embodiments, R.sup.8 is --OH or
--N(R.sup.6)(R.sup.7), where R.sup.6 and R.sup.7 are independently
alkyl or heteroalkyl, and are optionally cyclized. For example,
R.sup.8 can be --OH. In some instances, R.sup.8 is
--N(R.sup.6)(R.sup.7). In these instances, R.sup.6 can be alkyl or
heteroalkyl, and R.sup.7 can be alkyl or heteroalkyl. In some
instances, R.sup.6 and R.sup.7 together with the nitrogen to which
they are attached are cyclized. For example, in embodiments where
R.sup.6 and R.sup.7 together with the nitrogen to which they are
attached are cyclized, the resulting R.sup.8 group can be a
heterocyclyl. For instance, in embodiments where R.sup.6 and
R.sup.7 together with the nitrogen to which they are attached are
cyclized, the resulting R.sup.8 group can be
##STR00081##
where m is an integer selected from 1, 2, and 3, and X.sup.4 is
--OH, --COOH, or --CH.sub.2OH.
[0102] In some instances, the R.sup.8 heterocyclyl is selected from
a group such as, but not limited to, pyrrolidinyl, pyrazolidinyl,
imidazolidinyl, piperidinyl, piperazinyl, morpholinyl, and the
like. In some cases, where R.sup.6 and R.sup.7 together with the
nitrogen to which they are attached are cyclized, the resulting
R.sup.8 group is piperidinyl. In some cases, where R.sup.6 and
R.sup.7 together with the nitrogen to which they are attached are
cyclized, the resulting R.sup.8 group is morpholinyl. In some
cases, where R.sup.6 and R.sup.7 together with the nitrogen to
which they are attached are cyclized, the resulting R.sup.8 group
is optionally substituted with one or more substituent groups. For
example, the optional substituent on the R.sup.8 group can be --OH,
--COOH, or --CH.sub.2OH.
[0103] In certain embodiemnts, X.sup.1 is --F, --Cl, --Br, or
--OCH.sub.3. For example, X.sup.1 can be --F, or X.sup.1 can be
--Cl, or X.sup.1 can be --Br, or X.sup.1 can be --OCH.sub.3.
[0104] In certain embodiments, X.sup.2 is --SO.sub.2R' or
--CO.sub.2R', where R' is --H, --CH.sub.3, or --CH.sub.2CH.sub.3.
For example, X.sup.2 can be --SO.sub.2R', or X.sup.2 can be
--CO.sub.2R'. In some instances, R' is --H, --CH.sub.3, or
--CH.sub.2CH.sub.3. For example, R' can be --H, or R' can be
--CH.sub.3, or R' can be --CH.sub.2CH.sub.3.
[0105] In certain embodiments, X.sup.3 is --SO.sub.2CF.sub.3,
--SO.sub.2CH.sub.3, or --NO.sub.2. For example, X.sup.3 can be
--SO.sub.2CF.sub.3, or X.sup.3 can be --SO.sub.2CH.sub.3, or
X.sup.3 can be --NO.sub.2.
[0106] In certain embodiments, X.sup.5 is --F, --Br, --Cl, --H, or
--OCH.sub.3. For example, X.sup.5 can be --F, or X.sup.5 can be
--Br, or X.sup.5 can be --Cl, or X.sup.5 can be --H, or X.sup.5 can
be --OCH.sub.3.
[0107] In certain embodiments, the compound of Formula (III) is
phosphorylated. For example, compounds of Formula (III) can be
phosphorylated on the R.sup.8 group.
[0108] In certain embodiments, the compounds of Formula (III) also
include salts or stereoisomers thereof.
[0109] Compounds of Formula (III) find use in methods of treating a
pulmonary disease in a subject as described herein. For example,
the pulmonary disease can be idiopathic pulmonary fibrosis (IPF),
or the pulmonary disease can be chronic obstructive pulmonary
disease (COPD).
Evaluating Compounds for Senolytic Activity
[0110] These and other compounds can be evaluated on the molecular
level for their ability to perform as candidate senolytic agents
for use according to this invention. For example, where the therapy
includes triggering apoptosis of senescent cells by way of Bcl-2,
Bcl-xL, Bcl-w, or other Bcl family proteins, compounds of the
invention can be tested for their ability to inhibit binding
between one or more Bcl proteins and their respective cognate
ligand and thereby cause senolysis.
[0111] For example, a suitable assay can be a homogeneous assay (an
assay that does not require a separation step) for purposes of
determining binding to the Bcl isoforms, which is based on oxygen
channeling that is marketed by PerkinElmer Inc., Waltham,
Massachusetts: see Eglin et al., Current Chemical Genomics, 2008,
1, 2-10. The test compound is combined with the target Bcl protein
and a peptide representing the corresponding cognate ligand,
labeled with biotin. The mixture is then combined with streptavidin
bearing luminescent donor beads and luminescent acceptor beads,
which proportionally reduces luminescence if the compound has
inhibited the peptide from binding to the Bcl protein.
[0112] Alternatively, or in addition, compounds of the invention
can be evaluated for an ability to kill senescent cells
specifically, as described herein in Example 2. Compounds can be
screened for biological activity in an assay using senescent cells.
Cultured cells are contacted with the compound, and the degree of
cytotoxicity or inhibition of the cells is determined. The ability
of the compound to kill or inhibit senescent cells can be compared
with the effect of the compound on normal cells that are freely
dividing at low density, and normal cells that are in a quiescent
state at high density. By way of example, cultured cells, such as,
for example, human target tissue fibroblast IMR90 cell lines and
HUVEC cells, are contacted with the test compound, and the degree
of cytotoxicity or inhibition of the cells is determined using, for
example, a thermostable luciferase to enable reaction conditions
that generate a stable luminescent signal while simultaneously
inhibiting endogenous ATPase released during cell lysis.
Lung Diseases --Classification of Lung Disease According to
Underlying Pathophysiology
[0113] As a guide to treating pulmonary diseases in accordance with
this invention, the diseases can be classified according to the
primary underlying pathophysiology. Diseases that fall within the
same classification are amenable to applying senolytic medicine
with the same principles and with similar objectives.
[0114] Pulmonary diseases suitable for treatment are discussed in
more detail below, within the following classifications: [0115]
TYPE 1: Restrictive: result from diseases that cause a reduction in
lung compliance and in turn reduction in lung vital capacity and
total lung volume commonly a result of thickening of the lung
interstitium exemplified by fibrotic diseases. [0116] TYPE 2:
Obstructive: result from diseases that cause air trapping in the
lung and in turn a reduction in expiratory volume and increased
total lung volume commonly a result of airway obstruction or
destruction exemplified by diseases such as COPD and asthma. [0117]
TYPE 3: Vascular: result from a disruption of the normal
functioning of the blood vessels resulting from disorders affecting
the cellular components of the vessel including but not limited to
the endothelium. These disruptions may result in diseases
characterized by vascular inflammation and increased vessel tone
(e.g. ANCA-vasculitis and pulmonary hypertension) ultimately
leading to dysfunction of the physiologic function of the lung and
heart. [0118] TYPE 4: Genetic: result from genetic abnormalities
that result in lung disease that affects multiple anatomic
components of the lung and extra-pulmonary organs. These include
but are not limited to cystic fibrosis and alpha-1 antitrypsin.
[0119] TYPE 5: Infections: result from pathogenic microorganisms,
such as bacteria, viruses, parasites or fungi, may infect any
anatomical location including the airways, alveoli and pleura. They
may result in symptomatic (e.g. pneumonia) or asymptomatic latent
disease (e.g. tuberculosis).
[0120] This classification is provided to assist in understanding
and applying the invention to a particular patient and is not meant
to limit application of this technology. Certain conditions may
invoke several of these categories: for example, an inflammatory
process may contribute to pathological processes having other
underlying causes. Similarly, the SASP may trigger additional
pathologic processes regardless of the primary insult.
Pulmonary Diseases Suitable for Treatment with the Compounds of the
Invention:
[0121] Provided in the sections that follow is a discussion of
specific lung diseases arranged by broad etiologic category as
discussed above, that are candidates for treatment with a senolytic
agent in accordance with this invention. The degree to which a
particular pulmonary disease will be amenable to treatment with a
senolytic agent will depend on the degree and extent senescent
cells play a role in disease pathology or symptomatology. The
treatment protocol and patient management are within the judgment
of the managing clinician. The efficacy of the therapy can be
determined with clinical, physiological and radiological
evaluation.
[0122] In certain embodiments, compounds of Formula (I) as
described herein find use in methods of treating a pulmonary
disease in a subject as described herein. In certain embodiments,
compounds of Formula (II) as described herein find use in methods
of treating a pulmonary disease in a subject as described herein.
In certain embodiments, compounds of Formula (III) as described
herein find use in methods of treating a pulmonary disease in a
subject as described herein.
TYPE 1: Restrictive
[0123] Physiologic restriction of the lung result from diseases
that cause a reduction in lung compliance and in turn reduction in
lung vital capacity and total lung volume commonly a result of
thickening of the lung interstitium exemplified by fibrotic
diseases, such as, for example, idiopathic pulmonary fibrosis (IPF)
and connective tissue disease-associated lung fibrosis such as
systemic sclerosis (SSc).
[0124] IPF is a chronic and progressive fibrotic lung disease
characterized by stiffening and scarring of the lung, which can
lead to respiratory failure, pulmonary hypertension and increases
the risk for lung cancer, and heart failure. Fibrosis is associated
with dysfunctional repair of the lung interstitium 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.
[0125] The general approach and objectives of senolytic therapy for
restrictive conditions are based on elimination of senescent cells
from the area of the lung tissue that is central to the fibrogenic
process. For example, based on Unity data in human IPF lung tissue,
this includes the distal lung epithelium which has been
demonstrated to express the senescence marker p16.
[0126] Other subjects at risk of developing fibrotic diseases, for
example, include but are not limited to, those exposed to
environmental or occupational pollutants, such as asbestosis and
silicosis, those who have a connective tissue diseases such as RA,
SLE, scleroderma, sarcoidosis, those who take certain medications,
including, for example, amiodarone, bleomycin, busufan,
methotrexate, and nitrofurantoin; those subject to radiation
therapy to the chest; and those whose family member have pulmonary
fibrosis.
TYPE 2: Obstructive
[0127] Physiologic obstruction of the lung result from diseases
that cause air trapping in the lung and in turn a reduction in
expiratory volume and increased total lung volume commonly a result
of airway obstruction or destruction exemplified by diseases such
as, for example, chronic obstructive pulmonary diseases (COPD) and
asthma.
[0128] 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
can disintegrate the extracellular matrix of alveolar structures,
resulting in enlarged air spaces and loss of respiratory capacity.
COPD can be caused by, for example, tobacco smoke, cigarette smoke,
cigar smoke, secondhand smoke, pipe smoke, occupational exposure,
exposure to dust, smoke, fumes, and pollution, occurring over
decades thereby implicating aging as a risk factor for developing
COPD.
[0129] The processes that cause lung damage include, for example,
oxidative stress produced by the high concentrations of free
radicals in tobacco smoke, cytokine release due to the 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 can also
contribute to the disease. 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. Alpha-1-antitrypsin
is normally secreted into the bloodstream to help protect the
lungs.
[0130] Symptoms of COPD can include any one of shortness of breath,
wheezing, chest tightness, having to clear one's throat first thing
in the morning because of excess mucus in the lungs, a chronic
cough that produces sputum that can be clear, white, yellow or
greenish, cyanosis, frequent respiratory infections, lack of
energy, and unintended weight loss. Subjects with COPD can also
experience exacerbations, during which symptoms worsen and persist
for days or longer. Symptoms of pulmonary fibrosis include, for
example, shortness of breath, particularly during exercise; dry,
hacking cough; fast, shallow breathing; gradual, unintended weight
loss; fatigue; aching joints and muscles; and clubbing of the
fingers or toes.
TYPE 3: Vascular
[0131] These conditions are characterized by a disruption of the
normal functioning of the blood vessels resulting from disorders
affecting the cellular components of the vessel including, but not
limited to, the endothelium. These disruptions may result in
diseases characterized by such findings as vascular inflammation,
increased vessel tone (vasoconstriction) and restricted blood flow
ultimately leading to damage and physiologic dysfunction of the
lung and heart. It also includes local deficiencies that arise in a
given part of a body resulting from issues affecting blood flow but
not the vessel itself, such as vasoconstriction, thrombosis, or
embolism. Examples of vascular pulmonary diseases include, but are
not limited to, pulmonary hypertension and vasculitis such as
Wegener's granulomatosis.
[0132] The general approach and objectives of senolytic therapy for
ischemic or vascular conditions are based on elimination of
senescent cells from the vasculature and decrease the associated
SASP factor impact on surrounding cells or area of the lung tissue
that is central to the effects of vascular dysfunction. Thus, the
senolytic agent can be delivered either systemically or directly in
the vasculature.
[0133] Pulmonary hypertension (PH) is a pathophysiological disorder
that may involve multiple clinical conditions and can complicate
many pulmonary and cardiovascular diseases. It is defined
physiologically by the resulting hemodynamic change of a mean
pulmonary artery pressure (mPAP) at rest greater than 25 mmHg The
clinical pathogenic categories of PH are described based on broad
etiologies: pulmonary arteries (including pulmonary veno-occlusion
and pulmonary capillary dysfunction), left heart disease, lung
disease/hypoxia, artery obstruction and from unclear or
multi-factorial causes (2015 ESC/ERS Guidelines; Galie N. et al.,
Eur Respir J 2015; 46:903). The overall treatment goal in patients
with PH is to maintain good exercise capacity, good quality of
life, good Right Ventricular function and a low mortality risk.
Specifically, this means bringing and/or keeping the patient in
WHO-FC II whenever possible.
[0134] TYPE 4: Genetic
[0135] Genetic respiratory conditions are characterized as a
disease that is caused by a mutation, deletion, or insertion in an
individual's DNA sequence. Genetic disorders can be grouped into
three main categories: (1) Single gene disorders: disorders caused
by defects in one particular gene, often with simple and
predictable inheritance patterns such as dominant, recessive and
x-linked; (2) Chromosome disorders: disorders resulting from
changes in the number or structure of the chromosomes; and (3)
Multifactorial disorders (complex diseases): disorders caused by
changes in multiple genes, often in a complex interaction with
environmental and lifestyle factors such as diet or cigarette
smoke. Examples of genetic pulmonary diseases include cystic
fibrosis (CF) and alpha-1 antitrypsin deficiency (A1AT).
[0136] CF is a monogenic autosomal recessive disease that is caused
by mutations in CFTR, located on chromosome 7. The CFTR protein is
an ion channel that regulates transport of chloride ions (Cl--) in
epithelial cells in the airways, as well as in the pancreas, liver,
intestine and skin. The various CFTR mutations cause different CFTR
protein defects, which impair transport of chloride and sodium
across epithelial surfaces, leading to thick viscous secretions
(e.g. mucus or phlegm).
[0137] Consequently, dysfunction of the exocrine glands throughout
the body leads to elevated sweat chloride, pancreatic
insufficiency, recurrent pulmonary infection, hepatobiliary
disease, and infertility. The diagnosis of CF relies primarily on
clinical evidence and is confirmed by elevated sweat chloride or
CFTR mutations in two alleles. CFTR modulators and potentiators are
drugs that modify the function of CFTR to improve lung function and
reduce symptoms and pulmonary exacerbations.
[0138] The general approach and objectives of senolytic therapy for
genetic conditions are based on the following: Genetic disorders of
the pulmonary system can affect all anatomic layers and are
associated with cellular defects that may lead to an accelerated
aging phenotype, caused or mediated at least in part by senescent
cells. An inheritable susceptibility to certain lung diseases
suggests that the accumulation of disease-mediating senescent cells
may directly or indirectly be influenced by genetic components,
which again may lead to earlier presentation. Genetic disorders
demonstrate a multifactorial cascade with senescent cells and SASP
production contributing to ongoing cell dysfunction and
degeneration/death. These disorders can benefit from senolytic
therapy because senescent cells and their associated SASP factors
mediate associated contributions to ongoing cell dysfunction, cell
loss, and disease progression via blockage of the angiogenic,
inflammatory, fibrotic, and extracellular matrix-modifying proteins
present in the pathophysiology.
TYPE 5: Infectious Pulmonary Diseases
[0139] These are diseases caused by pathogenic microorganisms, such
as bacteria, viruses, parasites or fungi; the diseases can be
spread, directly or indirectly, from one person to another.
Infectious pulmonary diseases can be caused by numerous infectious
agents, including but not limited to Streptococcus, Myocobacteria,
Pneumocystis, Blastomyces, Paragonimus and human immunodeficiency
virus (HIV). These infections present with either or both acute and
chronic clinical features including cough, lung infiltrates,
hemoptysis and respiratory distress which may become life
threatening and in many cases require long term anti-infective
therapies with significant associated comorbidities.
[0140] The general approach and objectives of senolytic therapy for
infections conditions can be based upon the following: Infectious
disorders of the pulmonary system may impact all anatomic locations
of the lung and appear to occur more frequently in the elderly,
suggesting senescence. Infectious agents may contribute to the
induction of senescence and a multifactorial cascade with senescent
cells and SASP production contributing to ongoing cell dysfunction.
Once present, senescent cells may in turn impact the ability to
fight infection.
[0141] Senescent cells have an impaired ability to control viral
replication (Kim et al., Enhanced Viral Replication by Cellular
Replicative Senescence., Immune Network., 2016 October;
16(5):286-295), which is in line with the known increased
susceptibility to infection that occurs with age. Senescence and
the ability to respond to infectious agents are a category of lung
disease that can be significantly impacted by senolytic therapy.
Elimination of senescent cells and their associated SASP factors
can ameliorate damage to the cellular microenvironment.
Routes of Administration
[0142] In one embodiment, the pharmaceutical senolytic compositions
of the invention are formulated for administration by inhalation.
Suitable pharmaceutical compositions for administration by
inhalation will typically be in the form of an aerosol or a powder.
[0143] Aerosol Delivery:
[0144] Compounds of the invention may be directly administered as
an aerosol to a site of pulmonary pathology as described above. The
aerosol may also be delivered to the pulmonary compartment for
absorption into the pulmonary vasculature for therapy or
prophylaxis of extra-pulmonary pathologies such as fibrosis for
example, or pulmonary or intra-nasal delivery for extra-pulmonary
or extra-nasal cavity diseases.
[0145] When administered by inhalation using a pressurized
container, the pharmaceutical compositions of the invention will
typically comprise the active ingredient and a suitable propellant,
such as, for example, dichlorodifluoromethane,
trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide
or other suitable gas. Additionally, the pharmaceutical composition
may be in the form of a capsule or cartridge (made, for example,
from gelatin) comprising a compound of the invention and a powder
suitable for use in a powder inhaler. Suitable powder bases
include, by way of example, lactose or starch.
[0146] Compositions of the invention may be administered using
well-known delivery devices, such as a metered-dose inhaler, a dry
powder inhaler, a nebulizer, a vaporizer, or a similar delivery
device. [0147] A. Meter Dose Inhaler (MDI):
[0148] A propellant driven inhaler (pMDI) releases a metered dose
of medicine upon each actuation. The medicine is formulated as a
suspension or solution of a drug substance in a suitable propellant
such as a halogenated hydrocarbon. pMDIs are described in, for
example, Newman, S. P., Aerosols and the Lung, Clarke et al., eds.,
pp. 197-224 (Butterworths, London, England, 1984). [0149] B. Dry
Powder Inhaler (DPI):
[0150] There are two major designs of dry powder inhalers. One
design is the metering device in which a reservoir for the drug is
placed within the device and the patient adds a dose of the drug
into the inhalation chamber. The second is a factory-metered device
in which each individual dose has been manufactured in a separate
container. Both systems depend upon the formulation of drug into
small particles of mass median diameters from about 1 to about 5
micron, and usually involve co-formulation with larger excipient
particles (typically 100 micron diameter lactose particles). Drug
powder is placed into the inhalation chamber (either by device
metering or by breakage of a factory-metered dosage) and the
inspiratory flow of the patient accelerates the powder out of the
device and into the oral cavity. Non-laminar flow characteristics
of the powder path cause the excipient-drug aggregates to
decompose, and the mass of the large excipient particles causes
their impaction at the back of the throat, while the smaller drug
particles are deposited deep in the lungs. [0151] C.
Nebulizers:
[0152] Any known inhalation nebulizer suitable to provide delivery
of a medicament as described herein may be used in the various
embodiments and methods described herein. Such nebulizers include,
e.g., jet nebulizers, ultrasonic nebulizers, pulsating membrane
nebulizers, nebulizers with a vibrating mesh or plate with multiple
apertures, and nebulizers comprising a vibration generator and an
aqueous chamber (e.g., ParieFlow.RTM.). [0153] D. Vaporizers:
[0154] Any known vaporizer suitable to provide delivery of a
medicament as described herein may be used in the various
embodiments and methods described herein. For example, a vaporizer
may be used to vaporize a pharmaceutical composition as described
herein, such that the vaporized components of the pharmaceutical
composition can be inhaled by a user. In some instances, the
vaporizer applies sufficient heat to the pharmaceutical composition
to vaporize one or more components of the pharmaceutical
composition, such as the active agent or drug substance. The
pharmaceutical composition for use in a vaporizer may be provided
in solid form or liquid form prior to vaporization.
Formulation of Medicaments
[0155] A pulmonary preparation can be prepared by mixing a
senolytic agent with a pharmaceutically acceptable base or carrier
and as needed one or more pharmaceutically acceptable
excipients.
[0156] Ingredients acceptable in a pulmonary formulation are
excipients or carriers that cause little to no pulmonary
irritation, provide suitable preservation if needed, and deliver
one or more agents in a suitable volume. Examples of a base or
carrier include water; an aqueous solvent such as a polar solvent;
a polyalcohol; a vegetable oil; and an oily base. Examples of the
base or carrier for an intrapulmonary injection include water for
injection and physiological saline.
[0157] For pulmonary delivery, a senolytic agent may be combined
with acceptable excipients for use in and around the lung, such as
a surfactant, preservatives, co-solvents, a flavor or cooling
agent, an antiseptic, a bactericide or antibacterial agent, a pH
adjusting agent, a tonicity agent, a chelating agent, a buffering
agent, a stabilizer, an antioxidant, viscosity enhancers,
penetration enhancers, sodium chloride and a thickening agent. In
some cases, a composition for intrapulmonary injection may contain
one or more of a solubilizing agent, a suspending agent, a tonicity
agent, a buffering agent, a soothing agent, a stabilizer, and an
antiseptic. The pulmonary composition carrier and excipients can be
combined to form an aqueous, sterile pulmonary suspension,
solution, or viscous or semi-viscous gels or other types of solid
or semisolid composition such as an ointment.
[0158] Exemplary excipients and additives that can be used include
surfactants (for example, polyoxyethylene and block copolymers);
buffers and pH adjusting agents (for example, hydrochloric acid,
sodium hydroxide, phosphate, citrate, and sodium cyanide); tonicity
agents (for example, sodium bisulfite, sodium sulfite, glycerin,
and propylene glycol); chelating agents (for example, ascorbic
acid, sodium edetate, and citric acid); flavors; coloring agents;
antiseptics; bactericides; antibacterial agents; and the like.
[0159] Pulmonary solution formulations may be prepared by
dissolving the agent in a physiologically acceptable isotonic
aqueous buffer. Further, the pulmonary solution may include an
acceptable surfactant to assist in dissolving the agent. Viscosity
building compounds, such as hydroxymethyl cellulose, hydroxyethyl
cellulose, methylcellulose, polyvinylpyrrolidone may be added to
improve the retention of the compound.
[0160] Sterile pulmonary gel formulations may be prepared by
suspending the agent in a hydrophilic base prepared from the
combination of, for example, CARBOPOL.RTM.-940. VISCOAT.RTM. (Alcon
Laboratories, Inc., Fort Worth, Tex.) may be used for
intrapulmonary injection. Other compositions of the present
invention may contain penetration enhancing materials such as
CREMOPHOR.RTM. (Sigma Aldrich, St. Louis, Mo.) and TWEEN.RTM. 80
(polyoxyethylene sorbitan monolaureate, Sigma Aldrich), in the
event the agents of the present invention are less penetrating in
the lung.
[0161] This invention provides commercial products that are kits
that enclose one or more unit doses of one or more of the agents or
compositions described in this disclosure. Such kits typically
comprise a pharmaceutical preparation in one or more containers.
The preparatoins may be provided as one or more unit doses (either
combined or separate). The kit may contain a device such as a
syringe for administration of the agent or composition in or around
the lung of a subject in need thereof. The product may also contain
or be accompanied by an informational package insert describing the
use and attendant benefits of the drugs in treating the senescent
cell associated lung disease, and optionally an appliance or device
for delivery of the composition.
[0162] A unit dose refers to a physically discrete unit suitable as
a single dosage for a subject in need thereof, where each unit dose
contains a predetermined quantity of a compound of the invention in
an amount sufficient to produce the desired therapeutic effect. The
compound may be provided in the usit dose in association with a
pharmaceutically acceptable diluent, carrier and/or vehicle. The
amount of the compound in each unit dose may depend on the
particular compound employed and the effect to be achieved, and the
pharmacodynamics associated with the compound in the user.
Combination of Senolytic Agents with Approved Standard-of-Care
Therapies
[0163] Senolytic agents for treating pulmonary diseases and
conditions can be combined with other pharmaceutical agents that
are approved for clinical use. Since the removal of senescent cells
works by a different mechanism from current therapies, the two
agents can operate synergistically or additively to minimize the
administration schedule and improve outcomes. The senolytic agent
will remove senolytic cells in the lung that are promoting
persistence and progression of disease-related pathophysiology.
[0164] The methods of this invention for treating or reducing the
likelihood of a pulmonary disease or condition can also be used for
treating a subject who is aging and has loss of pulmonary function,
or degeneration of pulmonary tissue. The respiratory system can
undergo 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 can predispose older
adults to increased susceptibility to toxic environmental exposure
and accelerated lung function decline. Oxidative stress exacerbates
inflammation during aging. Alterations in redox balance and
increased oxidative stress during aging precipitate the expression
of cytokines, chemokines, and adhesion molecules, and enzymes.
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.
[0165] The effects of the treatment can be determined using
techniques that evaluate mechanical functioning of the lung, for
example, techniques that measure lung capacitance, elastance, and
airway hypersensitivity can be performed. To determine lung
function and to monitor lung function throughout treatment, any one
of numerous measurements can be obtained, for example, 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 (SpO2) can also be measured;
normal oxygen levels are typically between 95% and 100%. An SpO2
level below 90% suggests the subject has hypoxemia. Values below
80% are considered critical and require intervention to maintain
brain and cardiac function and avoid cardiac or respiratory
arrest.
Treatment Design
[0166] Senescent cells accumulate with age, which is why conditions
mediated by senescent cells occur more frequently in older adults.
In addition, different types of stress on pulmonary tissues may
promote the emergence of senescent cells and the phenotype they
express. Cell stressors include oxidative stress, metabolic stress,
DNA damage (for example, as a result of environmental ultraviolet
light exposure or genetic disorder), oncogene activation, and
telomere shortening (resulting, for example, from
hyperproliferation). Pulmonary tissues that are subject to such
stressors may have a higher prevalence of senescent cells, which in
turn may lead to presentation of certain lung diseases at an
earlier age, or in a more severe form. An inheritable
susceptibility to certain lung diseases suggests that the
accumulation of disease-mediating senescent cells may directly or
indirectly be influenced by genetic components, which can lead to
earlier presentation.
[0167] To treat a particular pulmonary disease with a senolytic
agent according to this invention, the therapeutic regimen will
depend on the location of the senescent cells, and the
pathophysiology of the disease.
[0168] In some embodiments, one or more doses of a compound or
pharmaceutical composition of the invention are administered to a
subject in need thereof. The frequency of administration of the
compound or pharmaceutical composition can vary depending on any of
a variety of factors, e.g., severity of the symptoms, condition of
the subject, etc. For example, in some embodiments, the compound or
pharmaceutical composition is administered once per month, twice
per month, three times per month, every other week (qow), once per
week (qw), twice per week (biw), three times per week (tiw), four
times per week, five times per week, six times per week, every
other day (qod), daily (qd), twice a day (qid), or three times a
day (tid), or the like.
[0169] Compounds that may be useful for clearing senescent cells in
or around the lung for purposes of treating pulmonary diseases
according to this invention include Bcl-2 inhibitors, Bcl-xL
inhibitors, MDM2 inhibitors, and Akt inhibitors. See U.S. Pat. Nos.
8,691,184, 9,096,625, and 9,403,856; published applications WO
2015/017159, WO 2015/116740, WO 2016/127135, WO 2017/008060, and
WO/2017/101851.
[0170] Candidate senolytic agents that act as Bcl 2, Bcl w, and Bcl
xL inhibitors can be characterized as a benzothiazole-hydrazone, an
amino pyridine, a benzimidazole, a tetrahydroquinolin, or a
phenoxyl compound. Examples of compounds that inhibit Bcl isoforms
include WEHI 539, A 1155463, ABT 737, and ABT 263 (Navitoclax).
INCORPORATION BY REFERENCE
[0171] For all purposes in the United States and in other
jurisdictions where effective, each and every publication and
patent document cited in this disclosure is hereby incorporated
herein by reference in its entirety for all purposes to the same
extent as if each such publication or document was specifically and
individually indicated to be incorporated herein by reference.
[0172] U.S. Application Publication No. 2016/0339019 (Laberge et
al.) and International application publication no. WO 2016127135
(David et al.) are hereby incorporated herein by reference in their
entireties for all purposes, including but not limited to the
identification, formulation, and use of compounds capable of
eliminating or reducing the activity of senescent cells and
treating pulmonary diseases and conditions. U.S. Pat. Nos.
8,691,184, 9,096,625, and 9,403,856 (Wang et al.) are hereby
incorporated herein by reference in their entireties for all
purposes, including the features of compounds in the Bcl library,
their preparation and use.
EMBODIMENTS OF THE INVENTION
[0173] Embodiments of the invention of the present disclosure can
be described by the following clauses.
[0174] Clause 1. A compound according to Formula (II):
##STR00082##
wherein: [0175] X.sup.1 is --Cl; [0176] X.sup.2 is --COOH or
--SO.sub.2CH.sub.3; [0177] X.sup.3 is --SO.sub.2CF.sub.3,
--SO.sub.2CH.sub.3, or --NO.sub.2; [0178] X.sup.5 is --F or --H;
[0179] R.sup.1 is --CH(CH.sub.3).sub.2; [0180] R.sup.2 is
--CH.sub.3; [0181] R.sup.3 and R.sup.4 are both --H; [0182] n is 2;
[0183] R.sup.6 is selected from --OH, --OR.sup.7,
##STR00083##
[0183] and [0184] R.sup.7 is --PO(OH).sub.2, [0185] or a salt or a
stereoisomer thereof.
[0186] Clause 2. The compound of Clause 1, wherein X.sup.2 is
--COOH.
[0187] Clause 3. The compound of Clause 1, wherein X.sup.2 is
--SO.sub.2CH.sub.3.
[0188] Clause 4. The compound of Clause 1, wherein X.sup.3 is
--SO.sub.2CF.sub.3.
[0189] Clause 5. The compound of Clause 1, wherein X.sup.3 is
--SO.sub.2CH.sub.3.
[0190] Clause 6. The compound of Clause 1, wherein X.sup.3 is
--NO.sub.2.
[0191] Clause 7. The compound of any of Clauses 1 to 6, wherein
X.sup.5 is --F.
[0192] Clause 8. The compound of any of Clauses 1 to 6, wherein
X.sup.5 is --H.
[0193] Clause 9. The compound of any of Clauses 1 to 8, wherein
X.sup.6 is --OH.
[0194] Clause 10. The compound of any of Clauses 1 to 8, wherein
X.sup.6 is --OR.sup.7.
[0195] Clause 11. The compound of any of Clauses 1 to 8, wherein
X.sup.6 is
##STR00084##
[0196] Clause 12. The compound of any of Clauses 1 to 8, wherein
X.sup.6 is
##STR00085##
[0197] Clause 13. The compound of any of Clauses 1 to 8, wherein
X.sup.6 is
##STR00086##
[0198] Clause 14. The compound of any of Clauses 1 to 13, wherein
the carboxyl group in X.sup.2 is phosphorylated.
[0199] Clause 15. The compound of Clause 1, wherein the compound is
selected from the group consisting of:
##STR00087##
[0200] Clause 16. The compound of any preceding Clause, which has
pro-apoptotic activity.
[0201] Clause 17. The compound of any preceding Clause, which
specifically kills senescent cells compared with non-senescent
cells, said senescent cells being defined as non-cancerous cells
that express p16.
[0202] Clause 18. The compound of any preceding Clause, which
specifically kills cancer cells compared with non-cancer cells of
the same tissue type.
[0203] Clause 19. The compound of any preceding Clause, which has
an IC.sub.50 for Bcl-xL of 1 nM or less.
[0204] Clause 20. The compound of any preceding Clause, which has
an IC.sub.50 for Bcl-2 of 10 nM or less.
[0205] Clause 21. The compound of any preceding Clause, which has
an IC.sub.50 for Bcl-xL of 1 nM or less and an IC.sub.50 for Bcl-2
of 10 nM or less.
[0206] Clause 22. A pharmaceutical composition comprising a
compound according to any preceding Clause in a pharmaceutically
compatible excipient.
[0207] Clause 23. A method of selectively removing senescent cells
and/or cancer cells from a mixed cell population or tissue,
comprising contacting a cell, a cell population or a tissue with a
compound according to any of Clauses 1 to 21 or a pharmaceutical
composition according to Clause 22.
[0208] Clause 24. A method of treating a senescence related
condition in a tissue in a subject, wherein the senescence related
condition is characterized as being caused or mediated at least in
part by senescent cells, or is characterized as having an
overabundance of senescent cells in or around the tissue, in
comparison with unaffected tissue, the method comprising: [0209]
administering to a tissue of a subject in need thereof, an amount
of a compound according to any of Clauses 1 to 21 or a
pharmaceutical composition according to Clause 22 that is effective
to selectively remove senescent cells from the tissue, thereby
relieving or ameliorating one or more signs or symptoms of a
senescence related condition in the subject.
[0210] Clause 25. A unit dose of a pharmaceutical composition
comprising: [0211] an amount of a compound that inhibits Bcl
function configured for use in the treatment of a senescence
associated condition that is caused or mediated at least in part by
senescent cells, [0212] wherein the compound is a compound
according to any of Clauses 1 to 21, [0213] wherein the
pharmaceutical composition contains a formulation of the compound
configured for administration to a target tissue in a subject that
manifests the senescence associated condition, and [0214] wherein
the formulation and the amount of the compound in the unit dose
configure the unit dose to be effective in selectively removing
senescent cells in or around the tissue in the subject, thereby
decreasing the severity of one or more signs or symptoms of the
condition without causing adverse effects in the subject when
administered to the tissue as a single dose.
[0215] Clause 26. The unit dose of Clause 25, packaged with an
informational insert describing the use and attendant benefits of
the drugs in treating the senescent cell associated condition.
[0216] Clause 27. A compound according to any of Clauses 1 to 21 or
a pharmaceutical composition according to claim 24 for use in
selectively eliminating senescent cells from a tissue or mixed cell
population or for use in treating a senescence-related
condition.
[0217] Clause 28. Use of a compound according to any of Clauses 1
to 21 in the manufacture of a medicament for treating a
senescence-related condition.
[0218] Clause 29. The method, unit dose, or use of any of Clauses
24 to 28, wherein the condition is osteoarthritis.
[0219] Clause 30. The method, unit dose, or use of any of Clauses
24 to 28, wherein the condition is an ophthalmic condition.
[0220] Clause 31. The method, unit dose, or use of any of Clauses
24 to 28, wherein the condition is a pulmonary disease.
[0221] Clause 32. A method of treating cancer, comprising
administering to a tissue of a subject in need thereof an amount of
a compound according to any of Clauses 1 to 21 or a pharmaceutical
composition according to Clause 22 effective to selectively remove
cancer cells from the tissue.
[0222] Clause 33. A compound according to any of Clauses 1 to 21 or
a pharmaceutical composition according to claim 22 for use in
selectively eliminating cancer cells from a tissue or mixed cell
population or for use in treating cancer.
[0223] Clause 34. A method of treating a pulmonary disease in a
subject, comprising administering to the subject in need thereof a
therapeutically effective amount of a pharmaceutical composition
comprising: [0224] a compound of Formula (I):
##STR00088##
[0224] wherein: [0225] X.sup.1 is --Cl; [0226] X.sup.2 is --COOH or
--SO.sub.2CH.sub.3; [0227] X.sup.3 is --SO.sub.2CF.sub.3;
--SO.sub.2CH.sub.3; or --NO.sub.2; [0228] X.sup.5 is --F or --H;
[0229] R.sup.1 is --CH(CH.sub.3).sub.2; [0230] R.sup.2 is
--CH.sub.3; [0231] R.sup.3 and R.sup.4 are both --H; [0232] n is 2;
and [0233] R.sup.6 is selected from --OR.sup.7,
##STR00089##
[0233] and [0234] R.sup.7 is --H or --PO(OH).sub.2, [0235] or a
salt or a stereoisomer thereof; and [0236] a pharmaceutically
compatible excipient.
[0237] Clause 35. The method of Clause 34, wherein X.sup.2 is
--COOH.
[0238] Clause 36. The method of Clause 34, wherein X.sup.2 is
--SO.sub.2CH.sub.3.
[0239] Clause 37. The method of Clause 34, wherein X.sup.3 is
--SO.sub.2CF.sub.3.
[0240] Clause 38. The method of Clause 34, wherein X.sup.3 is
--SO.sub.2CH.sub.3.
[0241] Clause 39. The method of Clause 34, wherein X.sup.3 is
--NO.sub.2.
[0242] Clause 40. The method of any of Clauses 34 to 39, wherein
X.sup.5 is --F.
[0243] Clause 41. The method of any of Clauses 34 to 39, wherein
X.sup.5 is --H.
[0244] Clause 42. The method of any of Clauses 34 to 39, wherein
R.sup.6 is --OR.sup.7.
[0245] Clause 43. The method of any of Clauses 34 to 39, wherein
R.sup.6 is
##STR00090##
[0246] Clause 44. The method of any of Clauses 34 to 39, wherein
R.sup.6 is
##STR00091##
[0247] Clause 45. The method of any of Clauses 34 to 44, wherein
R.sup.7 is --H.
[0248] Clause 46. The method of any of Clauses 34 to 44, wherein
R.sup.7 is --PO(OH).sub.2.
[0249] Clause 47. The method of any of Clauses 34 to 46, wherein
the carboxyl group in X.sup.2 is phosphorylated.
[0250] Clause 48. The method of Clause 34, wherein the compound is
selected from the group consisting of:
##STR00092## ##STR00093##
[0251] Clause 49. The method of Clause 34, wherein the pulmonary
disease is idiopathic pulmonary fibrosis (IPF).
[0252] Clause 50. The method of Clause 34, wherein the pulmonary
disease is chronic obstructive pulmonary disease (COPD).
[0253] Clause 51. The method of Clause 48, wherein the pulmonary
disease is idiopathic pulmonary fibrosis (IPF).
[0254] Clause 52. The method of Clause 48, wherein the pulmonary
disease is chronic obstructive pulmonary disease (COPD).
[0255] Clause 53. The method of Clause 34, wherein the
administration of the pharmaceutical composition is by inhalation
as an aerosol.
[0256] Clause 54. The method of Clause 48, wherein the
administration of the pharmaceutical composition is by inhalation
as an aerosol.
[0257] Clause 55. The method of Clause 34, wherein the pulmonary
disease is a restrictive pulmonary disease.
[0258] Clause 56. The method of Clause 55, wherein the restrictive
pulmonary disease is idiopathic pulmonary fibrosis (IPF) or
systemic sclerosis (SSc).
[0259] Clause 57. The method of Clause 34, wherein the pulmonary
disease is an obstructive pulmonary disease.
[0260] Clause 58. The method of Clause 57, wherein the obstructive
pulmonary disease is chronic obstructive pulmonary diseases (COPD)
or asthma.
[0261] Clause 59. The method of Clause 34, wherein the pulmonary
disease is a vascular pulmonary disease.
[0262] Clause 60. The method of Clause 59, wherein the vascular
pulmonary disease is pulmonary hypertension or vasculitis.
[0263] Clause 61. The method of Clause 34, wherein the pulmonary
disease is a genetic pulmonary disease.
[0264] Clause 62. The method of Clause 61, wherein the genetic
pulmonary disease is cystic fibrosis (CF) or alpha-1 antitrypsin
deficiency (A1AT).
[0265] Clause 63. The method of Clause 34, wherein the pulmonary
disease is an infectious pulmonary disease.
[0266] Clause 64. The method of Clause 63, wherein the infectious
pulmonary disease is pneumonia or tuberculosis.
[0267] Clause 65. A method of treating a pulmonary disease in a
subject, comprising administering to the subject in need thereof a
therapeutically effective amount of a compound of Formula (III) or
a phosphorylated form thereof:
##STR00094##
wherein: [0268] R.sup.1 and R.sup.2 are independently C.sub.1 to
C.sub.4 alkyl; [0269] R.sup.3, R.sup.4 and R.sup.5 are
independently --H or --CH.sub.3; [0270] R.sup.8 is --OH or
--N(R.sup.6)(R.sup.7), wherein R.sup.6 and R.sup.7 are
independently alkyl or heteroalkyl, and are optionally cyclized;
[0271] X.sup.1 is --F, --Cl, --Br, or --OCH.sub.3; [0272] X.sup.2
is --SO.sub.2R' or --CO.sub.2R', where R' is --H, --CH.sub.3, or
--CH.sub.2CH.sub.3; [0273] X.sup.3 is --SO.sub.2CF.sub.3;
--SO.sub.2CH.sub.3; or --NO.sub.2; and [0274] X.sup.5 is --F, --Br,
--Cl, --H, or --OCH.sub.3.
[0275] Clause 66. The method of Clause 65, wherein: [0276] R.sup.1
and R.sup.2 are independently C.sub.1 to C.sub.4 alkyl; [0277]
R.sup.3 and R.sup.4 are independently --H or --CH.sub.3; [0278]
R.sup.5 is --H; [0279] R.sup.8 is --OH or
[0279] ##STR00095## [0280] X.sup.1 is --F, --Cl, --Br, or
--OCH.sub.3; [0281] X.sup.2 is --SO.sub.2R' or --CO.sub.2R', where
R' is --H, --CH.sup.3, or --CH.sup.2CH.sup.3; [0282] X.sup.3 is
--SO.sub.2CF.sub.3, --SO.sub.2CH.sub.3, or --NO.sub.2; [0283]
X.sup.4 is --OH, --COOH or --CH.sub.2OH; [0284] X.sup.5 is --F,
--Cl, or --H; and [0285] m is 1, 2, or 3.
[0286] Clause 67. The method of Clause 65, wherein: [0287] X.sup.3
is --SO.sub.2CF.sub.3 or --NO.sub.2; and [0288] R.sup.8 is
--N(R.sup.6)(R.sup.7), wherein R.sup.6 and R.sup.7 are
independently alkyl or heteroalkyl, and are optionally
cyclized.
[0289] Clause 68. The method of Clause 66, wherein: [0290] X.sup.3
is --SO.sub.2CF.sub.3 or --NO.sub.2; and [0291] R.sup.8 is
##STR00096##
[0291] wherein X.sup.4 is --OH or --COOH.
EXAMPLES
Example 1
Cellular Senescence Burden in Diseased Human Lung Tissue
[0292] Senescent cells associated with areas of active disease in
lung tissue was tested in human idiopathic pulmonary fibrosis (IPF)
tissues and human scleroderma tissues taken from afflicted human
patients. Human IPF tissues were procured from the University of
Michigan, courtesy of Dr. Eric White. Human scleroderma tissues
were procured from the Medical University of South Carolina,
courtesy of Dr. Carol Browstick. The samples included both upper
and lower lung lobe sections from 9 normal, 12 human IPF patients
and 7 human scleroderma patients.
[0293] Immunohistochemistry staining for p16 was performed as
follows. All slides were processed using a standard p16 substrate
chromogen, 3,3'-diaminobenzidine tetrahydrochloride hydrate (DAB)
protocol and a Leica Bond Refine Detection kit which contains a
peroxide block, post primary, polymer reagent, DAB chromogen and
hematoxylin counterstain. Specifically, specimen slides were
incubated with hydrogen peroxide for 5 minutes to quench endogenous
peroxidase activity, and then washed. A p16 primary antibody
(ClNtec.RTM. p16, Roche Diagnostics) was applied for 15 minutes and
then washed. A Post Primary IgG linker reagent was applied for 8
minutes, and subsequently a polymer-HRP IgG reagent was applied,
before washing. The substrate chromogen, 3,3'-diaminobenzidine
tetrahydrochloride hydrate (DAB) was applied for 10 minutes to
visualize the complex via a brown precipitate before washing.
Hematoxylin (blue) counterstaining for 1 minute allowed the
visualization of cell nuclei.
[0294] Immunohistochemistry staining for p16 in human IPF lung
tissue demonstrated the presence of senescent cells, see FIGS. 2A
and 2B. These cells were predominantly epithelial in origin and
located in areas of fibrosis and at the leading edge of the
disease, which indicated accessibility by inhalation
therapeutics.
[0295] Next, quantitation of the extent of fibrosis in the lung in
human IPF was measured relative to normal lung tissue. Assignment
of percent of observed fibrosis was made as follows: <25%
fibrosis, 25-49% fibrosis, 50-74% fibrosis, and 75-100% fibrosis. A
p<0.0001 for group difference among means by one-way ANOVA was
performed. All human IPF samples tested positive for p16
expression, therefore confirming that an increase in p16 correlates
with the progression of IPF, see FIG. 3.
[0296] Immunohistochemistry staining for p16 in human scleroderma
lung tissue demonstrated the presence of senescent cells,
particularly in honeycomb areas, see FIGS. 4A, 4B and 4C, as
compared with normal lung, which had no p16 staining detected.
Furthermore, the extent of fibrosis in the lung in human
scleroderma was compared to normal lung tissue. All human
scleroderma samples tested positive for p16 expression, therefore
confirming that the presence of p16 correlates with scleroderma,
see FIG. 5.
Example 2
Effect of A Senolytic Agent on Senescent Airway Epithelial
Cells
[0297] The ability of candidate agents to eliminate senescent cells
or senescent-like lung epithelial cells was measured directly in
the following assay. Primary human small airway epithelial cells
(SAEC) and bronchial epithelial cells (BEC) were obtained from
Lonza.RTM., ATCC.RTM., and Promocell.RTM.. Cells were maintained
and propagated at <75% confluency in Airway Epithelial Cell
Growth Medium or Small Airway Epithelial Cell Growth Medium
(Promocell.RTM.; Heidelberg, Germany) at 20% O.sub.2, 5% CO.sub.2,
and .about.95% humidity. To make primary cells senescent, x-ray
irradiation was employed. On day 0, SAEC/BEC cells were covered
with TrypLE trypsin-containing reagent (Thermofisher Scientific,
Waltham, Mass.) and incubated for 8 min until the cells rounded up
and began to detach from the plate. Cells were dispersed, counted,
and prepared in medium at a concentration of 188,800, 94,400,
47,200, and 23,600 cells per mL. This cell suspension was plated in
384-well plates at a volume of 25 .mu.L per well (4720, 2360, 1180,
and 590 cells/well (c/w) respectively). Within 24-hours after cell
plating, the 384-well plates were irradiated at 12 Gy to generate
senescent cells (SnC). In addition, control 384-well plates were
processed in parallel that were not irradiated and served as a
control and represent normal, non-senescent cells (NsC). On day 3
the medium in each well was aspirated and replaced with 25 .mu.L
fresh medium. On day 7, an exemplary test compound of the
invention, Compound 1 was combined with the cells as follows. A
DMSO dilution series of the test compound was prepared at 1000
times the final desired concentration in a 384-well plate.
Immediately before use, the DMSO stocks were diluted 1:1000 into
prewarmed complete medium. Medium was aspirated from the cells in
each well, and 25 .mu.L/well of the compound containing medium was
added. Compound 1 was cultured with the cells for 3 days. The assay
system used the properties of a thermostable luciferase to enable
reaction conditions that generated a stable luminescent signal
while simultaneously inhibiting endogenous ATPase released during
cell lysis. On day 10, the end of the culture period, the plates
were removed from the incubator and allowed to equilibrate at room
temperature for 15 minutes, then 25 .mu.L of CellTiter-Glo.RTM.
reagent (Promega.RTM. Corp., Madison, Wis.) was added to each of
the wells. The cell plates were placed for 30 seconds on an orbital
shaker and then allowed to stand at room temperature for 30 minutes
before measuring luminescence. The luminescence readings were
normalized to determine % cell survival/growth and plotted against
test compound concentrations, and potencies (EC50 values) for
Compound 1 were determined by non-linear curve fitting in Graphpad
Prism.RTM..
[0298] FIG. 6 shows the results. The concentration-response curve
demonstrated sensitivity of senescent lung epithelial cell (SnC)
survival to incubation with different amounts of Compound 1,
whereas non-senescent cells (NsC) have very limited potency. These
data show that compounds of the invention are capable of
selectively eliminating senescent lung airway cells in culture.
Example 3
Measuring Senolysis in PCLS
[0299] Precision-cut lung slices (PCLS) are functional 3D organ
models that can be used to ex-vivo determine effective senolysis in
tissue slices obtained from normal and IPF human patients. Human
PCLS will be prepared as follows. Lungs from normal, healthy
individuals or IPF patients will be gently inflated with warm 1.5%
agarose-DMEM mix. Afterwards, lung explants will be macroscopically
assessed by an experienced pulmopathologist to identify regions of
interest and exclude previously unknown medical conditions (e.g.
neoplasias or infections). Next, sections (.0.4-8 mm) will be
sliced in cold EBSS using a Krumdieck Tissue Slicer (Alabama
Research and Development.sup.SM, Munford, Ala., USA) into approx.
250-300 .mu.m thin slices. PCLS will be washed thoroughly before
cultivation in DMEM (2 slice per 500 .mu.l) under normal immersion
culture conditions (37.degree. C., 5% CO2, and >95% air
humidity) for up to 15 days. PCLS will be treated for 1 h with 1%
Triton X-100 to serve as a dead, negative control reference.
[0300] As shown above in Example 1 and FIG. 2, senescence marker
p16 expression is upregulated in idiopathic pulmonary fibrosis
(IPF), therefore any significant reduction of p16 expression as
measured by qPCR or abundance of p16 as measured by IHC in IPF PCLS
following administration of compounds of the invention will
indicate effective senolysis. Determination of the beneficial
effects of compounds of the invention on reducing the senescence
burden in PCLS obtained from IPF patients will be performed as
follows. In each experiment three dose levels (10 .mu.M, 1 .mu.M
and 0.1 .mu.M) of a DMSO formulation of compounds of the invention
will be tested, in addition to a vehicle control sample. PCLSs will
be exposed to such compounds for three days, followed by a media
wash-out and a three-day recovery period. Upon harvest, PCLSs will
be collected for staining and analysis of p16/senescence and
fibrosis, or flash frozen for RNA-seq/qPCR detection of relevant
markers of senescence, fibrosis and epithelial regeneration.
Supernatants will be harvested for studying changes in SASP factors
through a Luminex.RTM. or MSD.RTM. analysis. Upon observing
senolysis through p16 reduction, markers of fibrosis will be
evaluated using established methodologies, including collagen level
determination (picrosirius red staining) and gene expression
changes of fibrosis markers, such as FN1, SERPINE1, COL1A1, CTGF,
MMPI, and ACTA2, via qPCR.
Example 4
Effect of senolytic Agents In an In Vivo Pharmacodynamic Model
[0301] The ability of candidate agents to eliminate senescent mouse
epithelial-enriched cells induced by locally administered bleomycin
was measured directly in the following assay. On day 0, male
C57BL/6 mice (The Jackson Laboratory) were administered 2.2 U/kg of
bleomycin or its vehicle (PBS) by oral aspiration (50 mL). On day
11, each mouse received either vehicle (2.5% glycerin in PBS) or
increasing concentrations of Compound 1 via oral aspiration (50
mL). On day 14, mice were euthanized, exsanguinated, and perfused
PBS, 1 mL of dispase, and 0.2 mL of a 1% low melt agarose.
Individual lobes of the lungs were collected for either single cell
isolation, cell enrichment, and qPCR or for fixing, staining, and
IHC analysis.
[0302] Left lung lobes were collected for epithelial cell
enrichment as follows. Lung lobes were placed in 2 mL of dispase on
a rocker for 45 minutes. To obtain isolated cells, a serological
pipet was then used to dissociate the lung tissue prior to adding
10 mL of sort buffer containing 50U/ml of DNase. Next, samples were
incubated on a rocker for 10 minutes at 37.degree. C. and then
passed through both 100 mm and 70 mm cell strainers. The collected
suspension was then spun at 550 g for 5 minutes at 4.degree. C. to
pellet the cells. The supernatant was then removed, and 1 mL cold
RBC lysis buffer was added at room temperature for 20 seconds.
Next, 5 mL of sort buffer was added, and the sample was spun at 550
g for 5 minutes at 4.degree. C. The supernatant was then removed
and 5 mL MACS buffer was added. The sample was next passed through
a 40 mm cell strainer. Cells were counted using a cell counter then
re-spun (550 g for 5 minutes) and suspended in MACS buffer
containing 10% CD45+ microbeads at a volume of 100 uL per
1.times.10.sup.7 cells. Cells were mixed and incubated for 15
minutes at 4.degree. C. Next, 2 mL of MACS buffer was added and
then the cells spun at 350 g for 5 minutes at 4.degree. C. The
supernatant was removed, cells were resuspended in 500 uL MACS
buffer and added to a MACS separator cell column. The column was
washed 4 times with 500 uL MACS buffer and the effluent (CD45-)
collected. Cells were then counted with a cell counter. The sample
was then spun at 350 g for 5 minutes at 4.degree. C. The
supernatant was removed, and the cells resuspended in MACS buffer
containing 10% CD326+microbeads at a volume of 100 uL per
1.times.10.sup.7 cells. Cells were mixed and incubated for 15
minutes at 4.degree. C. Next, 2 mL of MACS buffer was added and
then the cells spun at 350 g for 5 minutes at 4.degree. C. The
supernatant was removed, cells were resuspended in 500 uL MACS
buffer and added to a MACS separator cell column. The column was
washed 4 times with 500 uL MACS buffer and the effluent (EpCAM-)
collected. The column was then removed from the magnet and placed
on a new collection tube. Finally, 3 mL of MACS buffer was added to
the column through to collect remaining cells (CD45-, EpCAM+). The
cells were then spun down (550 g for 5 minutes) and resuspended in
500 uL TRIzol.RTM.(Thermo Fisher Scientific) for storage at
-80.degree. C.
[0303] Quantification of p16 mRNA by qPCR was performed as follows.
Desired frozen cells (CD45-, EpCAM+) were thawed, 20% chloroform
added, and sample vortexed until milky in appearance. Cells were
then spun at 15,000 g for 15 minutes. The supernatant was removed,
collected into a new tube, diluted with equal volume 100% ethanol,
and added to the Zymo column (Zymo Research Direct-zol RNA
MicroPrep kit; Cat #R2063). Column was spun at 15,000 g for 1
minute. Next, 400 uL wash buffer was added to each sample and
respun at 15,000 g for 1 minute. Next, 40 uL DNase reaction mix (5
uL DNase I+35uL DNA digestion buffer) was added to the column
matrix and incubated for 15 minutes at room temperature. Next, 400
uL pre-wash buffer was added to each sample and respun at 15,000 g
for 1 minute. This step was repeated and then the cell pellet was
resuspended in 700 uL wash buffer followed by another spin. The
flow-through was discarded and the column was respun at 15,000 g
for 2 minutes. Next, the column was transferred to a Rnase-free
tube and 20 uL of DNase/RNase free water was added to the column.
The column was then respun at 15,000g for 1 minute and samples were
analyzed for RNA quality on the Agilent TapeStation.RTM. 2200
(Agilent Technologies.RTM.).
[0304] The extracted RNA was then used to generate single-stranded
cDNA using the kit and protocol available from Applied
Biosystems.RTM. (High Capacity cDNA Reverse Transcription kit; Cat
#4368813). Finally, Real-time PCR was performed using the
QuantStudio.RTM. 7 PCR System with Taqman.RTM. gene-specific
primers for p16.sup.INK4a. All signals were normalized to
.beta.-actin. Relative gene expression was calculated by the
.DELTA..DELTA.Ct method where the ACt was calculated using the
.beta.-actin reference gene. AACt was calculated relative to the
PBS/Vehicle control group.
[0305] FIG. 7 shows the results. The concentration-response curve
for Compound 1, demonstrates sensitivity of mouse lung epithelial
cell p16 mRNA induced by bleomycin to a senolytic molecule
following local (OA) administration. These data show that senolytic
agents are capable of selectively eliminating senescent lung airway
epithelial cells in vivo.
[0306] Right lung lobes were collected for p16 immunohistochemistry
analysis was performed as follows. Lung lobes were fixed in 4%
paraformaldehyde overnight, dehydrated in increasing concentrations
of ethanol, and embedded in paraffin. Five micron sections were cut
from the paraffin block and collected on glass slides. Slides were
dewaxed using BOND Dewaxing Solution at 70.degree. C. for 30
seconds and washed with TBST 3 times. Antigen retrieval was done
using BOND ER2 solution (EDTA buffer pH 9.0) for 20 minutes at
95.degree. C. and then 3% H.sub.2O.sub.2 was added for 10 minutes
at room temperature followed by 3 successive washes. Slides were
then incubated with SEA BLOCK.RTM. blocking buffer (Thermo Fisher
Scientific Cat #37527) for 30 minutes at room temperature and
washed with TBST 3 times. Slides were then incubated in mouse IgG
block for 20 minutes and then incubated in primary p16 antibody (BD
Biosciences Cat #550834) at a 1:50 dilution in TBST for 1 hour.
Next, slides were washed with TBST 3 times and incubated with goat
anti-mouse poly-HRP secondary antibody (Thermo Fisher Scientific
Cat #B40961) for 30 minutes at room temperature. Next, slides were
washed with TBST 3 times and a DAB substrate solution was added and
incubated for 10 min at room temperature. Next, slides were washed
with dH.sub.2O 3 times and then placed in hematoxylin for 1 minute.
Slides were then washed thoroughly in dH.sub.2O for several
exchanges, placed in a bluing reagent for 30 seconds, and then
rinsed in tap water for 5 minutes. Finally, slides were mounted
using water-based mounting medium and left to dry overnight.
Quantification was performed with the number of p16+ foci
determined in reference to the total number of cells.
[0307] FIG. 8 demonstrates sensitivity of mouse lung epithelial
cell p16+ cells, induced to senesce by bleomycin, to Compound 1
following local (OA) administration. These data show that compounds
of the invention are capable of selectively eliminating senescent
lung airway epithelial cells in vivo.
Example 5
Synthesis
[0308] Compounds of this invention were prepared by using or
adapting the synthetic scheme shown in FIG. 1.
[0309] The several hypotheses presented in this disclosure provide
a premise by way of which the reader may understand the invention.
This premise is provided for the enrichment and appreciation of the
reader. Practice of the invention does not require detailed
understanding or application of the hypothesis. Except where stated
otherwise, features of the hypothesis presented in this disclosure
do not limit application or practice of the claimed invention. For
example, except where the elimination of senescent cells is
explicitly required, the compounds of this invention may be used
for treating the conditions described regardless of their effect on
senescent cells. Although many of the pulmonary diseases and
conditions referred to in this disclosure occur predominantly in
older patients, the invention may be practiced on patients of any
age having the condition indicated, unless otherwise explicitly
indicated or required.
[0310] While the invention has been described with reference to the
specific examples and illustrations, changes can be made and
equivalents can be substituted to adapt to a particular context or
intended use as a matter of routine development and optimization
and within the purview of one of ordinary skill in the art, thereby
achieving benefits of the invention without departing from the
scope of what is claimed.
* * * * *
References