U.S. patent application number 15/063256 was filed with the patent office on 2016-06-30 for methods of preventing the development of mucositis and related disorders.
This patent application is currently assigned to The United States of America, as represented by the Secretary, Department of Health and Human Serv. The applicant listed for this patent is The United States of America, as represented by the Secretary, Department of Health and Human Serv, The United States of America, as represented by the Secretary, Department of Health and Human Serv. Invention is credited to Ana P. Cotrim, J. Silvio Gutkind, Ramiro Iglesias-Bartolome, James B. Mitchell, Alfredo Molinolo, Vyomesh Patel.
Application Number | 20160184280 15/063256 |
Document ID | / |
Family ID | 50188371 |
Filed Date | 2016-06-30 |
United States Patent
Application |
20160184280 |
Kind Code |
A1 |
Gutkind; J. Silvio ; et
al. |
June 30, 2016 |
METHODS OF PREVENTING THE DEVELOPMENT OF MUCOSITIS AND RELATED
DISORDERS
Abstract
Disclosed is a method of preventing the development of mucositis
in a subject undergoing radiation therapy or chemotherapy for a
disease in need thereof comprising administering an effective
amount of a mammalian target of rapamycin (mTOR) inhibitor, such as
rapamycin, to the subject. Further disclosed is a method of
increasing the lifespan of a normal oral keratinocyte and/or
reducing oxidative stress in a normal epithelial cell, wherein the
method comprises administering an effective amount of an mTOR
inhibitor to a subject undergoing radiation therapy or chemotherapy
for a disease in need thereof.
Inventors: |
Gutkind; J. Silvio; (Del
Mar, CA) ; Iglesias-Bartolome; Ramiro; (Bethesda,
MD) ; Patel; Vyomesh; (Washington, DC) ;
Cotrim; Ana P.; (Bethesda, MD) ; Molinolo;
Alfredo; (Rockville, MD) ; Mitchell; James B.;
(Damascus, MD) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The United States of America, as represented by the Secretary,
Department of Health and Human Serv |
Bethesda |
MD |
US |
|
|
Assignee: |
The United States of America, as
represented by the Secretary, Department of Health and Human
Serv
Bethesda
MD
|
Family ID: |
50188371 |
Appl. No.: |
15/063256 |
Filed: |
March 7, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13798863 |
Mar 13, 2013 |
9278090 |
|
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15063256 |
|
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61696861 |
Sep 5, 2012 |
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Current U.S.
Class: |
514/291 |
Current CPC
Class: |
A61K 31/436
20130101 |
International
Class: |
A61K 31/436 20060101
A61K031/436 |
Claims
1. A method of preventing the development of mucositis in a subject
undergoing radiation therapy or chemotherapy for a disease in need
thereof comprising administering an effective amount of a mammalian
target of rapamycin (mTOR) inhibitor to the subject.
2. A method of preventing hair loss in a subject undergoing
radiation therapy or chemotherapy for a disease in need thereof
comprising administering an effective amount of a mammalian target
of rapamycin (mTOR) inhibitor to the subject.
3. A method of increasing the lifespan of a normal oral
keratinocyte in a subject undergoing radiation therapy or
chemotherapy for a disease in need thereof comprising administering
an effective amount of a mammalian target of rapamycin (mTOR)
inhibitor to the subject.
4. The method of claim 1, wherein epithelial progenitor cells are
preserved after radiation therapy relative to their condition prior
to radiation therapy or chemotherapy.
5. The method of claim 3, wherein the disappearance of the
epithelial progenitor marker p63 is decreased.
6. The method of claim 3, wherein the level of mitochondrial
superoxide dismutase (MnSOD) is increased in the normal oral
keratinocyte upon administration of mTOR inhibitor compared to the
level of MnSOD in the absence of mTOR inhibitor.
7. A method of reducing oxidative stress in a normal epithelial
cell compared to a cancerous epithelial cell in a subject
undergoing radiation therapy or chemotherapy for a disease in need
thereof comprising administering an effective amount of a mammalian
target of rapamycin (mTOR) inhibitor to the subject.
8. The method of claim 7, wherein the normal and cancerous
epithelial cells are from an oral mucosa.
9. The method of claim 7, wherein the amount of reactive oxygen
species (ROS) is reduced in a normal epithelial cell compared to a
cancerous epithelial cell.
10. The method of claim 1, wherein the disease is cancer.
11. The method of claim 10, wherein the cancer is head and neck
squamous cell carcinoma (HNSCC) or cancer of the tongue.
12. The method of claim 10, wherein the cancer is an oral
cancer.
13. (canceled)
14. The method of claim 1, wherein the mTOR inhibitor is selected
from the group consisting of rapamycin, prodrugs of rapamycin,
40-O-(2-hydroxyethyl)-rapamycin, 32-deoxorapamycin,
16-pent-2-ynyloxy-32-deoxorapamycin, 16-pent-2-ynyloxy-32 (S or
R)-dihydro-rapamycin, 16-pent-2-ynyloxy-32 (S or
R)-dihydro-40-O-(2-hydroxyethyl)-rapamycin,
40-[3-hydroxy-2-(hydroxy-methyl)-2-methylpropanoate]-rapamycin
(CCI779), 40-epi-(tetrazolyl)-rapamycin (ABT578),
42-O-(2-hydroxy)ethyl rapamycin (RAD001), AP23573, TAFA-93, and
biolimus.
15. The method of claim 14, wherein the mTOR inhibitor is selected
from rapamycin, AP23573, RAD001, CCI779, and TAFA-93.
16. The method of claim 15, wherein the mTOR inhibitor is
rapamycin.
17. The method of claim 1, wherein the mTOR inhibitor is orally
administered.
18. The method of claim 2, wherein the mTOR inhibitor is selected
from the group consisting of rapamycin, prodrugs of rapamycin,
40-O-(2-hydroxyethyl)-rapamycin, 32-deoxorapamycin,
16-pent-2-ynyloxy-32-deoxorapamycin, 16-pent-2-ynyloxy-32 (S or
R)-dihydro-rapamycin, 16-pent-2-ynyloxy-32 (S or
R)-dihydro-40-O-(2-hydroxyethyl)-rapamycin,
40-[3-hydroxy-2-(hydroxy-methyl)-2-methylpropanoatel-rapamycin
(CCI779), 40-epi-(tetrazolyl)-rapamycin (ABT578),
42-O-(2-hydroxy)ethyl rapamycin (RAD001), AP23573, TAFA-93, and
biolimus.
19. The method of claim 3, wherein the mTOR inhibitor is selected
from the group consisting of rapamycin, prodrugs of rapamycin,
40-O-(2-hydroxyethyl)-rapamycin, 32-deoxorapamycin,
16-pent-2-ynyloxy-32-deoxorapamycin, 16-pent-2-ynyloxy-32 (S or
R)-dihydro-rapamycin, 16-pent-2-ynyloxy-32 (S or
R)-dihydro-40-O-(2-hydroxyethyl)-rapamycin,
40-[3-hydroxy-2-(hydroxy-methyl)-2-methylpropanoatel-rapamycin
(CCI779), 40-epi-(tetrazolyl)-rapamycin (ABT578),
42-O-(2-hydroxy)ethyl rapamycin (RAD001), AP23573, TAFA-93, and
biolimus.
20. The method of claim 7, wherein the mTOR inhibitor is selected
from the group consisting of rapamycin, prodrugs of rapamycin,
40-O-(2-hydroxyethyl)-rapamycin, 32-deoxorapamycin,
16-pent-2-ynyloxy-32-deoxorapamycin, 16-pent-2-ynyloxy-32 (S or
R)-dihydro-rapamycin, 16-pent-2-ynyloxy-32 (S or
R)-dihydro-40-O-(2-hydroxyethyl)-rapamycin,
40-[3-hydroxy-2-(hydroxy-methyl)-2-methylpropanoatel-rapamycin
(CCI779), 40-epi-(tetrazolyl)-rapamycin (ABT578),
42-O-(2-hydroxy)ethyl rapamycin (RAD001), AP23573, TAFA-93, and
biolimus.
21. The method of claim 2, wherein the disease is cancer.
Description
BACKGROUND OF THE INVENTION
[0001] Both epidermis and mucosal epithelia are highly dependent on
resident self-renewing stem cells, making them particularly
vulnerable to physical and chemical insults compromising the
repopulating capacity of the epithelial stem cell compartment. This
is often the case in cancer patients receiving radiation or
chemotherapy, many of whom develop mucositis, a debilitating
condition involving painful and deep mucosal ulcerations as a
result of damage to the normal tissue (Sonis, Oral Oncol., 45,
1015-1020 (2009); Sonis, Oral Diseases, 16, 597-600 (2010)).
Mucositis causes distress to the patients and results also in
substantial increase in per-patient care cost (Nonzee et al.,
Cancer, 113, 1446-1452 (2008)). Radiotherapy is one of the most
widely used cancer treatments (Begg et al., Nat. Rev. Cancer, 11,
239-253 (2011)), and although technical advances now allow a more
targeted delivery of radiation to the cancer cells, indirect damage
to the normal surrounding tissues still remains a common and
frequently weakening side effect (Citrin et al., The Oncologist,
15, 360-371 (2010)). Hence, therapeutic interventions that can
reduce the deleterious effects of chemotherapy or radiation on
normal epithelial stem cells are needed that will improve on the
quality of life of cancer patients and treatment outcome.
BRIEF SUMMARY OF THE INVENTION
[0002] The present invention provides a method of preventing the
development of mucositis in a subject undergoing radiation therapy
or chemotherapy for a disease in need thereof comprising,
consisting essentially of, or consisting of administering an
effective amount of a mammalian target of rapamycin (mTOR)
inhibitor to the subject.
[0003] It has been surprisingly found that mTOR inhibition protects
normal epithelial progenitor cells (e.g., hair follicles and oral
epithelial cells) from ionizing radiation-induced epithelial stem
cell depletion. In addition, it has been found that inhibition of
mTOR increases the clonogenic capacity of primary human oral
keratinocytes and their resident self-renewing cells by preventing
stem cell senescence. Accordingly, the present invention provides a
method of increasing the lifespan of a normal oral keratinocyte in
a subject undergoing radiation therapy or chemotherapy for a
disease in need thereof comprising, consisting essentially of, or
consisting of administering an effective amount of an mTOR
inhibitor to the subject. Also provided is a method of preventing
(including minimizing or reducing) hair loss in a subject
undergoing radiation therapy or chemotherapy for a disease in need
thereof comprising, consisting essentially of, or consisting of
administering an effective amount of an mTOR inhibitor to the
subject.
[0004] The invention further provides a method of reducing
oxidative stress in a normal epithelial cell compared to a
cancerous epithelial cell (e.g., from an oral mucosa) in a subject
undergoing radiation therapy or chemotherapy for a disease in need
thereof comprising, consisting essentially of, or consisting of
administering an effective amount of an mTOR inhibitor to the
subject.
BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)
[0005] FIGS. 1A, 1B, and 1C show the surviving fractions and
individual colony areas from clonogenic assays of the HNSCC cell
lines HN12 (FIG. 1A) and Cal 27 (FIG. 1B) and human primary normal
oral keratinocytes (NOK) (FIG. 1C) (C=control, R=rapamycin). FIG.
1D shows the colony forming efficiency from clonogenic assays of
human primary NOK.
[0006] FIG. 2A shows .gamma.H2AX levels in irradiated control or
rapamycin treated human primary NOK at 30 min and 24 h after the
indicated dose of radiation in grays (Gy). FIG. 2B shows p16 levels
in irradiated control or rapamycin treated human primary NOK after
the indicated dose of radiation in grays (Gy). FIG. 2C shows the
senescence-associated .beta.-galactosidase (SA-.beta.gal) in
irradiated control (Con) or rapamycin treated human primary NOK
after the indicated dose of radiation in grays (Gy) 3 days after
rapamycin treatment.
[0007] FIG. 3A shows the cumulative population doubling from a
representative culture of human primary NOK in control conditions
or continuous presence of rapamycin. Each dot represents a passage
and dotted line indicates that cells could not be further expanded.
FIGS. 3B and 3C show the average number of days before replicative
senescence (FIG. 3B) and the maximum cumulative population
doublings (FIG. 3C) from three different cultures of NOK cultured
in control conditions (Con) or continuous presence of rapamycin
(Rap).
[0008] FIGS. 4A-4D show the analysis of reactive oxygen species
(ROS) levels by dihydroethidium hydroethidine (DHE) staining 24
hours after radiation in NOK and HN12 cells pretreated or not
(control) with rapamycin (FIGS. 4A and 4B) and fluorescent
activated cell sorting (FACS) analysis of ROS levels by DHE
staining in NOK after 10 or 40 days of continuous rapamycin
treatment (FIGS. 4C and 4D). FIG. 4E shows the quantification of
MnSOD protein expression levels by western blot in NOK and HN12
cells after 72 h of control conditions or rapamycin treatment. FIG.
4F shows p16 levels in control or rapamycin treated human primary
NOK transfected with control siRNA (siCon) or siRNA targeted
against MnSOD (siMnSOD). FIG. 4G shows colony forming efficiency
from clonogenic assays of human primary NOK transfected with
control siRNA (siCon) or siRNA targeted against MnSOD (siMnSOD).
Cells were pretreated or not (control) with rapamycin for 72 h
after siRNA transfection, irradiated with 0 or 3 Gy and 24 h later
subjected to clonogenic assays.
[0009] FIG. 5A shows a fractionated radiation and drug treatment
scheme for mice. FIG. 5B shows quantification of ulceration in
tongues stained with toluidine blue from irradiated animals on day
5 following the final radiation dose, receiving vehicle (Radiation)
or rapamycin (Radiation+Rapamycin). Lack of protective epithelial
barrier and therefore ulcer formation was indicated by deep, royal
blue staining (not shown) in epithelium defects. Quantification
represents at least 6 animals per group in 3 independent
experiments.
[0010] FIGS. 6A and 6B show the percentage of basal p63+ cells
(FIG. 6A) and the Ki-67 index (percentage of Ki67+ cells/total
epithelial cells) (FIG. 6B) in non-irradiated (No IRR) or
irradiated (IRR) mice at day 5 and day 10 under either control
conditions or rapamycin treatment. FIG. 6C shows the expression
levels of the stem cell marker p63 in tongues from non-irradiated
animals treated or not with rapamycin at the end of the treatment
period (day 5) or 5 days after the last dose of rapamycin (day 10).
FIG. 6C shows the average fluorescence value per nucleus in the
basal layer, expressed in arbitrary units.
DETAILED DESCRIPTION OF THE INVENTION
[0011] The present invention provides a method of preventing the
development of mucositis in a subject undergoing radiation therapy
or chemotherapy for a disease in need thereof comprising,
consisting essentially of, or consisting of administering an
effective amount of an mTOR inhibitor to the subject. In accordance
with an embodiment, rapamycin protects epithelial progenitor cells
from replicative senescence, extending their life span in vitro.
This protective effect of mTOR inhibition is believed to be
mediated by the increased expression of mitochondrial superoxide
dismutase (MnSOD), and the consequent suppression of oxidative
stress caused by the accumulation of reactive oxygen species (ROS)
in normal cells but not in cancer cells. In accordance with an
embodiment, the present method includes inhibition of mTOR to
prevent the loss of proliferative epithelial progenitor stem cells
upon radiation or chemotherapy. The proposed method enhances the
stem cells' tissue repopulating capacity, thereby preserving the
integrity of the oral mucosa and protecting from radiation-induced
mucositis in a subject. Moreover, the method enables prevention of
hair loss, including minimizing or reducing hair loss, in a subject
undergoing radiation therapy or chemotherapy for a disease in need
thereof.
[0012] The mTOR is a serine/threonine protein kinase that regulates
cell growth, cell proliferation, cell motility, cell survival,
protein synthesis, and transcription. mTOR integrates the input
from multiple upstream pathways, including insulin, growth factors
(such as IGF-1 and IGF-2), and mitogens. mTOR also functions as a
sensor of cellular nutrient and energy levels and redox status.
Dysregulation of mTOR pathways is a contributing factor to various
human disease processes, especially various types of cancer.
[0013] Additionally, in accordance with the present invention, it
has been found that inhibition of mTOR increases the repopulating
capacity of human epithelial stem cells, thereby preventing
epithelial stem cell senescence. Thus, the present invention
provides a method of increasing the lifespan of a normal oral
keratinocyte in a subject undergoing radiation therapy or
chemotherapy for a disease in need thereof comprising, consisting
essentially of, or consisting of administering an effective amount
of an mTOR inhibitor to the subject. As a result of mTOR
inhibition, e.g., epithelial progenitor cells are preserved after
radiation therapy relative to their condition prior to radiation
therapy. Moreover, the level of MnSOD is increased in the normal
oral keratinocyte upon administration of mTOR inhibitor compared to
the level of MnSOD in the absence of mTOR inhibitor. The
preservation of the normal oral keratinocyte stem cells can be
measured by, for example, a decrease in the disappearance of the
epithelial progenitor marker p63.
[0014] In an embodiment of the methods described herein, the mTOR
inhibitor decreases the release of cytokines that comprise the
"aging secretome," which are inflammatory mediators.
[0015] The invention further provides a method of reducing
oxidative stress in a normal epithelial cell compared to a
cancerous epithelial cell (e.g., from an oral mucosa) in a subject
undergoing radiation therapy or chemotherapy for cancer comprising,
consisting essentially of, or consisting of administering an
effective amount of an mTOR inhibitor to the subject. It has been
found that mTOR inhibition protects from oxidative stress by
increasing MnSOD expression. As a result of the mTOR inhibition,
the amount of ROS is reduced in a normal epithelial cell compared
to a cancerous epithelial cell.
[0016] In embodiments of the methods described herein, the subject
undergoing radiation therapy or chemotherapy for a disease in need
thereof has cancer. The type of cancer is not particularly limited,
as long as chemotherapy or radiation therapy is a prescribed
treatment therefor. Examples of cancer include cancers of the head
and neck, eye, skin, mouth, throat, esophagus, chest, bone, lung,
colon, sigmoid, rectum, stomach, prostate, breast, ovaries, kidney,
liver, pancreas, brain, intestine, heart, or adrenals. More
particularly, cancers include solid tumor, sarcoma, carcinomas,
fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic
sarcoma, chordoma, angiosarcoma, endotheliosarcoma,
lymphangiosarcoma, lymphangioendothelio sarcoma, synovioma,
mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma,
colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer,
prostate cancer, squamous cell carcinoma, basal cell carcinoma,
adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma,
papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma,
medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma,
hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal
carcinoma, Wilms' tumor, cervical cancer, testicular tumor, lung
carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial
carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma,
ependymoma, Kaposi's sarcoma, pinealoma, hemangioblastoma, acoustic
neuroma, oligodendroglioma, menangioma, melanoma, neuroblastoma,
retinoblastoma, a blood-borne tumor, acute lymphoblastic leukemia,
acute lymphoblastic B-cell leukemia, acute lymphoblastic T-cell
leukemia, acute myeloblastic leukemia, acute promyelocytic
leukemia, acute monoblastic leukemia, acute erythroleukemic
leukemia, acute megakaryoblastic leukemia, acute myelomonocytic
leukemia, acutenonlymphocyctic leukemia, acute undifferentiated
leukemia, chronic myelocytic leukemia, chronic lymphocytic
leukemia, hairy cell leukemia, or multiple myeloma. See, e.g.,
Harrison's Principles of Internal Medicine, Eugene Braunwald et
al., eds., pp. 491 762 (15th ed. 2001). In accordance with an
embodiment, the cancer is an oral cancer, such as cancer of the
head and/or neck (e.g., tissues of the mouth, including tongue,
gums, cheek linings, and palate, salivary glands, tonsils, or other
lymph nodes) and head and neck squamous cell carcinoma (HNSCC). In
another embodiment, the cancer is a cancer of the tongue.
[0017] In certain embodiments, the cancer is HNSCC, which includes
the squamous cell carcinomas of the oral cavity, pharynx and
larynx. The HNSCC can be, for example, a cancer of the tongue.
HNSCC progression involves the sequential acquisition of genetic
and epigenetic alterations in genes encoding tumor suppressors and
oncogenes, together with the aberrant activity of signaling
networks controlling cell proliferation, differentiation,
migration, survival, and death. The most frequent genetic
alterations in HNSCC include loss of heterozygosity and promoter
silencing of the p16 tumor suppressor gene, and inactivating
mutations in the p53 tumor suppressor gene. HNSCCs often
overexpress the epidermal growth factor receptor (EGFR) and some of
its active variants such as the truncated mutant form EGFR variant
III (EGFRvIII), which causes its constitutive activation.
[0018] Both hereditary and environmental factors are implicated in
head and neck carcinogenesis and their roles are difficult to
separate. Several cancer prone syndromes are associated with an
increased risk of head and neck cancer, including Lynch-II, Bloom
syndrome, Fanconi anemia, ataxia telangiectasia and Li-Fraumeni
syndrome. But genetic susceptibility to HNSCC is more likely to be
due to various degrees of DNA maintenance after exposure to tobacco
carcinogens. Mutagen sensitivity tests, polymorphism in DNA repair
enzymes or in carcinogens metabolizing enzymes supports the role of
heredity in HNSCC. GSTM1 and GSTT1 null phenotypes are associated
with an increased risk of HNSCC. Concerning XRCC1, the Arg allele
(Arg194Trp) and the Gln allele (Arg399Gln) are also linked to an
increased risk of oral and pharyngeal cancers.
[0019] In accordance with an embodiment, a method of preventing the
development of mucositis in a subject undergoing chemotherapy or
radiation therapy comprises reducing inflammation of the oral
mucosa and/or halting, ameliorating, or slowing the formation of
oral ulcers, for example, delaying the onset or severity of,
reducing the number, surface area, or size of ulcers in oral mucosa
(e.g., tissues of the soft palate, tongue, and/or cheeks). Further,
in accordance with an embodiment of the invention, the preventing
also includes preserving (i.e., not damaging) epithelial progenitor
cells (including hair follicles and oral epithelial cells) after
the subject's exposure to radiation therapy or chemotherapy
relative to the cells' condition prior to the therapy. When the
integrity of the epithelial progenitor cell is preserved, the
formation of ulcerous lesions is prevented and/or hair loss is
reduced or minimized.
[0020] The term "preventing" is meant any degree (10, 20, 30, 40,
50, 60, 70, 80, 90% or more) in inhibition of the onset of
radiation-or chemotherapy-induced oral ulcers/lesions and/or
mucositis, including complete inhibition of the development of
radiation-or chemotherapy-induced oral ulcers/lesions and/or
mucositis. Alternatively, or in addition, the onset of the
development of radiation-or chemotherapy-induced oral
ulcers/lesions and/or mucositis can be delayed with the methods
described herein by at least one week (e.g., at least 2 weeks, at
least 3 weeks, at least 1 month, at least 2 months, at least 3
months, at least 4 months).
[0021] In accordance with the invention, any suitable mTOR
inhibitor can be administered, for example, the mTOR inhibitor is
selected from rapamycin, prodrugs of rapamycin,
40-O-(2-hydroxyethyl)-rapamycin, 32-deoxorapamycin,
16-pent-2-ynyloxy-32-deoxorapamycin, 16-pent-2-ynyloxy-32 (S or
R)-dihydro-rapamycin, 16-pent-2-ynyloxy-32 (S or
R)-dihydro-40-O-(2-hydroxyethyl)- rapamycin,
40-[3-hydroxy-2-(hydroxy-methyl)-2-methylpropanoatel-rapamycin
(CCI779), 40-epi-(tetrazolyl)-rapamycin (ABT578),
42-O-(2-hydroxy)ethyl rapamycin (RAD001), AP23573, TAFA-93, and
biolimus, particularly, selected from rapamycin, AP23573, CCI779,
RAD001, and TAFA-93. Preferably, the mTOR inhibitor is
rapamycin.
[0022] Rapamycin, also known as sirolimus, is a relatively new
immunosuppressant drug used to prevent rejection in organ
transplantation, and is especially useful in kidney transplants.
Sirolimus is a macrolide first discovered as a product of the
bacterium Streptomyces hygroscopicus in a soil sample from an
island called Rapa Nui, better known as Easter Island. Rapamycin
inhibits mTOR through association with its intracellular receptor
FKBP12. The FKBP12-rapamycin complex binds directly to the
FKBP12-Rapamycin Binding (FRB) domain of mTOR.
[0023] The term "prodrug" denotes a derivative of a compound, which
derivative, when administered to warm-blooded animals, e.g. humans,
is converted into the compound (drug). The enzymatic and/or
chemical hydrolytic cleavage of the compounds of the present
invention occurs in such a manner that the proven drug form (parent
carboxylic acid drug) is released, and the moiety or moieties split
off remain nontoxic or are metabolized so that nontoxic metabolic
products are produced. Non-limiting examples include esters,
amides, ethers, and carbonates. Examples of prodrugs of rapamycin
include such as those disclosed in United States Patent Application
Publication No. 2008/0171763 A1, for example, paragraphs [0029] to
[0035], [0037], and [0038], which are incorporated by
reference.
[0024] In accordance with any of the embodiments, one or more than
one, e.g., two, three, or more mTOR inhibitors can be
administered.
[0025] In accordance with an embodiment, the methods described
herein comprise administering an mTOR inhibitor in the form of a
pharmaceutical composition. In particular, a pharmaceutical
composition will comprise, consist essentially of, or consist of at
least one mTOR inhibitor and a pharmaceutically acceptable carrier.
When the pharmaceutical composition consists essentially of at
least one mTOR inhibitor and a pharmaceutically acceptable carrier,
compounds that exert a biological effect (e.g., an
immunosuppressant, a matrix metalloprotease (MMP) inhibitor, an
anticancer drug, a non-steroidal anti-inflammatory drug (NSAID), a
.beta.-adrenergic receptor antagonist) in a subject are excluded
from the pharmaceutical composition. When the pharmaceutical
composition consists of at least one mTOR inhibitor and a
pharmaceutically acceptable carrier, the composition excludes any
other compounds. In an embodiment, the pharmaceutical composition
is free of a compound that exerts a biological effect selected from
an immunosuppressant, an MMP inhibitor, an anticancer drug, an
NSAID, and a .beta.-adrenergic receptor antagonist.
[0026] The pharmaceutically acceptable excipients described herein,
for example, vehicles, adjuvants, carriers or diluents, are
well-known to those who are skilled in the art and are readily
available to the public. It is preferred that the pharmaceutically
acceptable carrier be one that is chemically inert to the active
compounds and one that has no detrimental side effects or toxicity
under the conditions of use.
[0027] The pharmaceutical compositions can be administered orally
or parenterally. Thus the compositions can be administered as oral,
sublingual, transdermal, subcutaneous, topical, absorption through
epithelial or mucocutaneous linings, intravenous, intranasal,
intraarterial, intramuscular, intratumoral, peritumoral,
interperitoneal, intrathecal, rectal, vaginal, or aerosol
formulations.
[0028] In accordance with any of the embodiments, the mTOR
inhibitor can be administered orally to a subject in need thereof.
Formulations suitable for oral administration can consist of (a)
liquid solutions, such as an effective amount of the compound
dissolved in diluents, such as water, saline, or orange juice; (b)
capsules, sachets, tablets, lozenges, and troches, each containing
a predetermined amount of the active ingredient, as solids or
granules; (c) powders; (d) suspensions in an appropriate liquid;
and (e) suitable emulsions and gels. Liquid formulations may
include diluents, such as water and alcohols, for example, ethanol,
benzyl alcohol, and the polyethylene alcohols, either with or
without the addition of a pharmaceutically acceptable surfactant,
suspending agent, or emulsifying agent. Capsule forms can be of the
ordinary hard- or soft-shelled gelatin type containing, for
example, surfactants, lubricants, and inert fillers, such as
lactose, sucrose, calcium phosphate, and cornstarch. Tablet forms
can include one or more of lactose, sucrose, mannitol, corn starch,
potato starch, alginic acid, microcrystalline cellulose, acacia,
gelatin, guar gum, colloidal silicon dioxide, croscarmellose
sodium, talc, magnesium stearate, calcium stearate, zinc stearate,
stearic acid, and other excipients, colorants, diluents, buffering
agents, disintegrating agents, moistening agents, preservatives,
flavoring agents, and pharmacologically compatible carriers.
Lozenge forms can comprise the active ingredient in a flavor,
usually sucrose and acacia or tragacanth, as well as pastilles
comprising the active ingredient in an inert base, such as gelatin
and glycerin, or sucrose and acacia, emulsions, gels, and the like
containing, in addition to the active ingredient, such carriers as
are known in the art.
[0029] Formulations suitable for parenteral administration include
aqueous and non-aqueous, isotonic sterile injection solutions,
which can contain anti-oxidants, buffers, bacteriostats, and
solutes that render the formulation isotonic with the blood of the
intended recipient, and aqueous and non-aqueous sterile suspensions
that can include suspending agents, solubilizers, thickening
agents, stabilizers, and preservatives. The compound, or a prodrug,
salt, or solvate thereof, can be administered in a physiologically
acceptable diluent in a pharmaceutical carrier, such as a sterile
liquid or mixture of liquids, including water, saline, aqueous
dextrose and related sugar solutions, an alcohol, such as ethanol,
isopropanol, or hexadecyl alcohol, glycols, such as propylene
glycol or polyethylene glycol, glycerol ketals, such as
2,2-dimethyl-1,3-dioxolane-4-methanol, ethers, such as
poly(ethyleneglycol) 400, an oil, a fatty acid, a fatty acid ester
or glyceride, or an acetylated fatty acid glyceride with or without
the addition of a pharmaceutically acceptable surfactant, such as a
soap or a detergent, suspending agent, such as pectin, carbomers,
methylcellulose, hydroxypropylmethylcellulose, or
carboxymethylcellulose, or emulsifying agents and other
pharmaceutical adjuvants.
[0030] Oils, which can be used in parenteral formulations include
petroleum, animal, vegetable, or synthetic oils. Specific examples
of oils include peanut, soybean, sesame, cottonseed, corn, olive,
petrolatum, and mineral. Suitable fatty acids for use in parenteral
formulations include oleic acid, stearic acid, and isostearic acid.
Ethyl oleate and isopropyl myristate are examples of suitable fatty
acid esters. Suitable soaps for use in parenteral formulations
include fatty alkali metal, ammonium, and triethanolamine salts,
and suitable detergents include (a) cationic detergents such as,
for example, dimethyl dialkyl ammonium halides, and alkyl
pyridinium halides, (b) anionic detergents such as, for example,
alkyl, aryl, and olefin sulfonates, alkyl, olefin, ether, and
monoglyceride sulfates, and sulfosuccinates, (c) nonionic
detergents such as, for example, fatty amine oxides, fatty acid
alkanolamides, and polyoxyethylene-polypropylene copolymers, (d)
amphoteric detergents such as, for example,
alkyl-beta-aminopropionates, and 2-alkyl-imidazoline quaternary
ammonium salts, and (3) mixtures thereof.
[0031] The parenteral formulations will typically contain from
about 0.5 to about 25% by weight of the inhibitors in solution.
Suitable preservatives and buffers can be used in such
formulations. In order to minimize or eliminate irritation at the
site of injection, such compositions may contain one or more
nonionic surfactants having a hydrophile-lipophile balance (HLB) of
from about 12 to about 17. The quantity of surfactant in such
formulations ranges from about 5 to about 15% by weight. Suitable
surfactants include polyethylene sorbitan fatty acid esters, such
as sorbitan monooleate and the high molecular weight adducts of
ethylene oxide with a hydrophobic base, formed by the condensation
of propylene oxide with propylene glycol. The parenteral
formulations can be presented in unit-dose or multi-dose sealed
containers, such as ampoules and vials, and can be stored in a
freeze-dried (lyophilized) condition requiring only the addition of
the sterile liquid carrier, for example, water, for injections,
immediately prior to use. Extemporaneous injection solutions and
suspensions can be prepared from sterile powders, granules, and
tablets of the kind previously described.
[0032] The inhibitors may be made into injectable formulations. The
requirements for effective pharmaceutical carriers for injectable
compositions are well known to those of ordinary skill in the art.
See Pharmaceutics and Pharmacy Practice, J. B. Lippincott Co.,
Philadelphia, Pa., Banker and Chalmers, eds., pages 238-250 (1982),
and ASHP Handbook on Injectable Drugs, Toissel, 4th ed., pages
622-630 (1986).
[0033] Topically applied compositions are generally in the form of
liquids (e.g., mouthwash), creams, pastes, lotions and gels.
Topical administration includes application to the oral mucosa,
which includes the oral cavity, oral epithelium, palate, gingival,
and the nasal mucosa. In some embodiments, the composition contains
at least one active component and a suitable vehicle or carrier. It
may also contain other components, such as an anti-irritant. The
carrier can be a liquid, solid or semi-solid. In embodiments, the
composition is an aqueous solution, such as a mouthwash.
Alternatively, the composition can be a dispersion, emulsion, gel,
lotion or cream vehicle for the various components. In one
embodiment, the primary vehicle is water or a biocompatible solvent
that is substantially neutral or that has been rendered
substantially neutral. The liquid vehicle can include other
materials, such as buffers, alcohols, glycerin, and mineral oils
with various emulsifiers or dispersing agents as known in the art
to obtain the desired pH, consistency and viscosity. It is possible
that the compositions can be produced as solids, such as powders or
granules. The solids can be applied directly or dissolved in water
or a biocompatible solvent prior to use to form a solution that is
substantially neutral or that has been rendered substantially
neutral and that can then be applied to the target site. In
embodiments of the invention, the vehicle for topical application
to the skin can include water, buffered solutions, various
alcohols, glycols such as glycerin, lipid materials such as fatty
acids, mineral oils, phosphoglycerides, collagen, gelatin and
silicone based materials.
[0034] The inhibitors, alone or in combination with other suitable
components, can be made into aerosol formulations to be
administered via inhalation. These aerosol formulations can be
placed into pressurized acceptable propellants, such as
dichlorodifluoromethane, propane, nitrogen, and the like. They also
may be formulated as pharmaceuticals for non-pressured
preparations, such as in a nebulizer or an atomizer.
[0035] The dose administered to the mammal, particularly human and
other mammals, in accordance with the present invention should be
sufficient to affect the desired response. One skilled in the art
will recognize that dosage will depend upon a variety of factors,
including the age, condition or disease state, predisposition to
disease, genetic defect or defects, and body weight of the mammal.
The size of the dose will also be determined by the route, timing
and frequency of administration as well as the existence, nature,
and extent of any adverse side-effects that might accompany the
administration of a particular inhibitor and the desired effect. It
will be appreciated by one of skill in the art that various
conditions or disease states may require prolonged treatment
involving multiple administrations.
[0036] Suitable doses and dosage regimens can be determined by
conventional range-finding techniques known to those of ordinary
skill in the art. Generally, the preventive treatment is initiated
with smaller dosages that are less than the optimum dose of the
compound. Thereafter, the dosage is increased by small increments
until the optimum effect under the circumstances is reached. The
inventive method typically will involve the administration of about
0.01 mg to about 10 mg of one or more of the inhibitors described
above per kg body weight of the individual. For example, in
embodiments, the inhibitors can be administered in an amount from
about 0.05 mg/kg to about 5 mg/kg, from about 0.1 mg/kg to about 3
mg/kg, from about 0.5 mg/kg to about 2.5 mg/kg, from about 1 mg/kg
to about 2 mg/kg, from about 1.5 mg/kg to about 1.8 mg/kg, of
subject body weight per day, one or more times a day, to obtain the
desired therapeutic effect.
[0037] The subject to be treated is any patient with a disease that
requires chemotherapy and/or radiation therapy and typically is a
mammal. The term "subject" includes humans, sheep, horses, cattle,
pigs, dogs, cats, rats, and mice. In embodiments of the invention,
the subject is a human.
[0038] The following examples further illustrate the invention but,
of course, should not be construed as in any way limiting its
scope.
EXAMPLES
[0039] Cell Culture and siRNA Transfections
[0040] NOK isolation and culture was performed as described
(Leelahavanichkul and Gutkind, 2012. "Oral and Pharyngeal
Epithelial Keratinocyte Culture." In Method in Molecular Biology:
Epithelial Cell Culture Protocols (Humana Press)). Briefly, small
(0.1-0.3 cm) gingival biopsies obtained from healthy volunteers
under an NIH-approved clinical protocol (NIH-NIDCR, protocol
06D0144) were rinsed with phosphate buffer saline (PBS) and
incubated with trypsin (0.25%) solution overnight at 4.degree. C.
Next day the epithelium was peeled and scraped with forceps, finely
minced with scalpels and passed through a cell strainer (100
.mu.m). Cells were pelleted at 125 g and plated in 60-mm dish
coated with 0.3 mg/ml of collagen I in 1% acetic acid (BD
Biosciences, Franklin Lakes, N.J.). NOK were maintained in defined
keratinocyte serum free media (KSFM) (Life Technologies
Corporation, Carlsbad, Calif.) supplemented with antibiotics at
37.degree. C. in the presence of 5% CO.sub.2 and passed every 3-4
days. NOK between passages 2 to 4 were used for the experiments.
HN12 and Cal27 cells were maintained in Dulbecco's modified eagle
medium (DMEM) (Life Technologies, Carlsbad, Calif.) supplemented
with 10% fetal bovine serum (FBS) and antibiotics, at 37.degree. C.
in the presence of 5% CO.sub.2. siRNAs were purchased from
Thermo-Dharmacon (Lafayette, Colo.) (siGENOME SMARTpool siRNA for
MnSOD, catalog M-009784-02-0005; p16, catalog M-011007-03-0005; and
non-targeting control siRNA, catalog D-001206-13) and transfected
at a final concentration of 80 nM using LIPOFECTAMINE.TM. RNAiMAX
(Life Technologies Corporation, Carlsbad, Calif.), according to the
manufacturer's instructions.
Irradiation and Clonogenic Assays
[0041] Cells were plated in 3.5 cm dishes and treated with vehicle
or 20 nM rapamycin (LC Laboratories, Woburn, Mass.) for 3 days, and
then .gamma.-irradiated with 0 (control), 3 or 8 grays (Gy), and
kept with vehicle or rapamycin for 24 h. Afterwards, cells were
trypsinized, counted with an automated cell counter (Scepter
Scientific Inc., Livermore, Calif.; EMD Millipore Corporation,
Billerica, Mass.) and replated in duplicate in 6 well plates at
400, 800 or 1200 cells per well. Cells were grown for further 7 to
10 days and the resulting colonies were fixed in 3.2%
paraformaldehyde and stained with 0.5% of crystal violet in PBS for
30 min at room temperature. Plates were washed with running water
to remove excess of labeling and scanned. Colonies were counted and
measured with calibrated images in ImageJ with the analyze
particles function. Surviving fraction was calculated as previously
described (Franken et al., Nat. Protoc., 1, 2315-2319 (2006))
relative to the respective non-irradiated cells. Colony forming
efficiency was determined using the same protocol but expressed as
the proportion of plated cells that formed colonies relative to the
number of colonies formed by non-irradiated control cells. For
siRNA experiments, cells were transfected with the corresponding
siRNA 24 h before rapamycin treatment as described above.
Immunoblot Analysis
[0042] Cells were treated and irradiated as described above and
harvested 30 min or 24 h after radiation. Cells were lysed at
4.degree. C. in lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 1 mM
EDTA, 1% Nonidet P-40) supplemented with protease (protease
inhibitor cocktail, Sigma-Aldrich, St. Louis, Mo.) and phosphatase
inhibitors (1 mM Na.sub.3VO.sub.4 and 1 mM NaF). Equal amounts of
total cell lysate proteins were subjected to SDS-polyacrylamide gel
electrophoresis and transferred to nitrocellulose membranes. Bands
were detected by using near-infrared fluorescence (ODYSSEY.TM.
LI-COR Biosciences, Lincoln, Nebr.) with goat anti-mouse coupled to
IRDYE700CW.TM. (LI-COR Biosciences, Lincoln, Nebr.) or goat
anti-rabbit coupled to ALEXA FLUOR.TM. 680 (Life Technologies
Corporation, Carlsbad, Calif). The relative contribution of
individual bands was calculated using the ODYSSEY.TM. Application
Software v3 (LI-COR Biosciences, Lincoln, Nebr.). Primary
antibodies used were: phospho Akt 473 and 308 (1:500, Cell
Signaling Technology, Danvers, Mass.), Akt (1:500, Cell Signaling
Technology, Danvers, Mass.), S6 (1:500, Cell Signaling Technology,
Danvers, Mass.), phospho S6 (1:500, Cell Signaling Technology,
Danvers, Mass.), 4EBP (1:500,Cell Signaling Technology, Danvers,
Mass.), GAPDH (1:1000, Cell Signaling Technology, Danvers, Mass.),
.gamma.H2AX (1:500, Millipore Corporation, Billerica, Mass.), p16
(1:50, Santa Cruz Biotechnology, Santa Cruz, Calif.), p63 (1:500,
Santa Cruz Biotechnology, Santa Cruz, Calif.), .alpha.-tubulin
(1:2000, Sigma-Aldrich Co., St. Louis, Mo.), involucrin (1:2000,
Sigma-Aldrich Co., St. Louis, Mo.), human p53 (1:2000, Dako,
Carpinteria, Calif.), MnSOD (1:150, Santa Cruz Biotechnology, Santa
Cruz, Calif.), Cu-ZnSOD (1:150, Santa Cruz Biotechnology, Santa
Cruz, Calif.), Catalase (1:150, Santa Cruz Biotechnology, Santa
Cruz, Calif.).
Cytokine Measurements
[0043] Cells were plated in 3.5 cm dishes and treated with vehicle
or 20 nM rapamycin for 3 days in complete KSFM. Twenty-four hours
prior to cytokine measurement, media was changed to KSFM without
supplements. At the end of the 24 h period media and cells were
harvested. Media was filtered through 0.45 .mu.m PVDF low protein
binding filter, and cytokines were analyzed by the Cytokine Core
Laboratory, University of Maryland, using the LUMINEX.TM.
Multianalyte System. The cell lysates were used to quantify
cellular proteins. Values of cytokines were corrected to the
protein and expressed as percentage of the control. Media that was
not conditioned was completely absent of cytokines. Each value
corresponds to duplicate measurements of 3 independent samples of
NOK. Concentration of cytokines for cells under control conditions
were (expressed as pg. of cytokine/ml/10 .mu.g protein.+-.SE):
CXCL1 (GRO.alpha.), 67.+-.3; IL1.beta., 1.0.+-.0.2; IL6, 41.+-.7;
IL8, 11.8.+-.0.6; VEGFA, 12.+-.2; TNF-.alpha., 0.54.+-.0.04.
Example 1
[0044] This example illustrated that in vitro mTOR blockade by
rapamycin only slightly increased radiation-induced cancer cell
death in representative HNSCC cells lines.
[0045] Cell proliferation was evaluated using incorporation of
5-ethynyl-2'-deoxyuridine (EdU) with the CLICK-IT.TM. EdU Cell
Proliferation Assay Kit (Life Technologies, Carlsbad, Calif.).
Briefly, cells were treated with rapamycin 20 nM or vehicle for 3
days and then incubated with 10 .mu.M for 6 h before fixation,
permeabilization, and EdU staining, which were carried out
according to the kit's protocol. Cell nuclei were stained with
Hoechst 33342 (Life Technologies Corporation, Carlsbad, Calif.).
The proportion of cells incorporating EdU was determined by
fluorescence microscopy and quantified using ImageJ "nucleus
counter" plugin as described below. Eight different fields
containing approximately 60 cells each were quantified per
condition.
[0046] Using HN12 and Cal27 cells as typical examples of HNSCC
cells that are highly sensitive to the antitumoral activity of
rapamycin (Amomphimoltham et al., Cancer Research, 65, 9953-9961
(2005)), however, a limited synergistic effect on radiation-induced
cell death in vitro was detected, as judged by the surviving
fraction of cells exposed to clinically-relevant, sub-lethal doses
of radiation in the presence of rapamycin (FIGS. 1A and 1B). The
size of the colonies growing after radiation was also not
significantly affected by rapamycin, which completely inhibited
mTOR activity as assessed by the phosphorylation of S6 (FIGS. 1A
and 1B). Similar results were observed in a large collection of
HNSCC cells (not shown). Hence, these in vitro studies suggest that
inhibition of mTOR in HNSCC cells may only sensitize slightly to
radiation-induced cancer cell death.
Example 2
[0047] This example demonstrates that mTOR inhibition prevents
senescence in normal epithelial cells.
[0048] NOK from gingival biopsies from healthy volunteers were
isolated, and it was confirmed that these cultures lacked
fibroblast contamination and include epithelial stem cells
exhibiting tissue regenerative capacity, as judged by organotypic
co-cultures and by their ability to regenerate stratified
epithelium when grafted into nude mice. NOK were plated in collagen
coated LabTek chamber slides and treated with vehicle or 20 nM
rapamycin for 3 days. Cells were then y-irradiated or not, and
further treated with vehicle or rapamycin for 24 h. For TUNEL assay
cells were processed for staining immediately, for SA-.alpha.-gal
cells were passed and assessed 4 days after plating. Apoptosis was
detected using the CLICK-IT.TM. TUNEL ALEXA FLUOR.TM. 594 Imaging
Assay (Life Technologies, Carlsbad, Calif.) and SA-.beta.-gal
activity was measured using the SA-.beta.-gal kit (Cell Signaling
Technology, Danvers, Mass.) according to the manufacturer's
instructions. The proportion of positive cells was determined by
fluorescence or transmission microscopy and quantified using
ImageJ. Eight different fields containing approximately 60 cells
each were quantified per condition. For the calculation of
population doublings cells were cultured as described and treated
with vehicle or 20 nM rapamycin. Population doublings were
calculated every passage using the formula
x=[log10(NH/N1)/]/log10(2)] (Cristofalo et al., PNAS, 95,
10614-10619 (1998)), where N1 is the inoculum cell number and NH
the cell harvest number. To yield the cumulated doubling doublings,
the population doublings for each passage was calculated and then
added to the population doubling levels of the previous
passages.
[0049] A decrease in the surviving fraction of NOK undergoing
rapamycin treatment was not observed (FIG. 1C). On the contrary,
rapamycin enhanced the colony size of NOK compared with vehicle
treated cells, and this increase was maintained after radiation
(FIG. 1C). Along with the increase in colony size, rapamycin also
increased the colony forming efficiency of control and irradiated
human NOK (FIG. 1D). Since each large colony is expected to grow
from a single surviving self-renewing epithelial stem cell (Jensen
et al., Nat. Protoc., 5, 898-911 (2010)), these findings indicate
that rapamycin increases the survival and repopulating capacity of
epithelial progenitors, hence protecting from radiation-induced
loss of this tissue regenerative cell population.
[0050] Rapamycin-treated NOK exposed to radiation show reduced
levels of the DNA damage response marker .gamma.H2AX at early and
late time points (FIG. 2A). The activation of the DNA damage
response is one of the main mediators of cell senescence, which
results in irreversible growth arrest (Kuilman et al., Genes &
Development, 24, 2463-2479 (2010); Rodier et al., J. Cell. Biol.,
192, 547-556 (2011)). Accordingly, reduced levels of the senescence
marker p16 in rapamycin-treated NOK after radiation were observed
(FIG. 2B).
[0051] The percentage of cells positive for senescence-associated
beta-galactosidase (SA-.beta.gal) was measured, a known marker of
senescent cells (Debacq-Chainiaux et al., Nat. Protoc., 4,
1798-1806 (2009)). Compared with control cells, rapamycin treatment
reduced the accumulation of senescent cells in both control and
radiated cells (FIG. 2C). The activation of the RB tumor
suppressive pathway by the accumulation of p16 is often considered
a key mechanism triggering cell senescence in several systems,
including skin keratinocytes (Kuilman et al., Genes &
Development, 24, 2463-2479 (2010); Luis et al., Cell Stem Cell, 9,
233-246 (2011)). Knockdown of p16 in NOK resulted in an increase in
the colony forming efficiency of NOK under control conditions as
well as after ionizing radiation. Together, these findings suggest
that mTOR inhibition may increase the clonal proliferative capacity
of epithelial progenitor stem cells by preventing the activation of
specific cell senescence mechanisms.
[0052] Based on these results, it was then tested if mTOR
inhibition would also protect NOK from replicative senescence, a
process that involves the progressive depletion of progenitor cells
due to senescence and differentiation, and often serves as an in
vitro surrogate for aging (Zeng, Stem Cell Reviews and Reports, 3,
270-279 (2007)). Surprisingly, continuous treatment of human
primary NOK with rapamycin dramatically extended the lifespan of
the cells in culture (FIG. 3A). Specifically, rapamycin treated
cells almost tripled their lifespan and total cumulative population
doublings when compared to control cells (FIGS. 3B and 3C). It is
estimated that while in the absence of rapamycin a single cell will
give rise approximately to 10.sup.6 cells before undergoing
senescence, in the presence of rapamycin a single cell will give
rise to around 10.sup.17 cells. Similar results were observed in
multiple independent NOK isolates.
[0053] Western blot analysis revealed that cells continued to
respond to rapamycin along the entire treatment, as assessed by the
inhibition of phosphorylation of ribosomal protein S6 by rapamycin.
While significant changes in the phosphorylation status of Akt were
not observed, there was a clear reduction in the levels of p16 in
rapamycin-treated NOK when compared to the increasing p16
expression levels with time in control cells, concomitant with a
massive increase in SA-.beta.gal in control cells but not in
rapamycin treated cells.
[0054] As replicative senescence involves the progressive depletion
of progenitor cells in primary cultures, the effects of rapamycin
treatment on the levels of the epithelial stem cell marker p63 were
analyzed (Blanpain et al., Nat. Rev. Mol. Cell Biol., 10, 207-217
(2009)). As determined by western blot analysis, rapamycin
prevented the disappearance of the epithelial progenitor marker
p63, indicating that mTOR inhibition reduces the depletion of stem
cells from the culture. These results highlight the possibility
that mTOR inhibition may increase the proliferative capacity of
human primary NOK by preventing the senescence of their
repopulating stem cells.
Example 3
[0055] This example demonstrates that mTOR inhibition protects
epithelial cells from oxidative stress through increased MnSOD
expression in an embodiment of the invention.
[0056] ROS measurement was performed by incubating cells with
dihydroethidium hydroethidine (DHE) (5 mM stock solution stabilized
in DMSO, MOLECULAR PROBES.TM., Life Technologies, Carlsbad, Calif.)
at a final concentration of 1.5 .mu.M in culture media without
supplements or FBS for 10 min at 37.degree. C. Afterwards cells
were washed with PBS, detached and analyzed by fluorescent
activated cell sorting (FACS). FACS analysis was performed in a
FACSCALIBUR.TM. flow cytometer (BD Biosciences, Franklin Lakes,
N.J.). GSH and GSSG were measured in NOK and HN12 cells treated for
3 days with vehicle or 20 nM of rapamycin with the GSH/GSSG-GLO.TM.
Assay kit (Promega Corporation, Madison, Wisc.) according to the
manufacturer's instructions.
[0057] Total RNA was isolated from cultures and processed as
described (Martin et al., Oncogene, 27, 1844-1852 (2007)). One
.mu.g of cDNA was used as template for quantitative PCR using
IQ.TM. SYBR.TM. Green Supermix (Bio-Rad Laboratories, Hercules,
Calif.). Samples were analyzed using a Bio-Rad ICYCLER.TM. IQ.TM.
multicolor real-time PCR detection system. Oligonucleotides used
for amplification were from Quiagen (Valencia, Calif.) for human
MnSOD (catalog 330001 PPH01716B) and CuZnSOD (catalog 330001
PPH00234B). For immunopurification, proteins were extracted in
lysis buffer (50 mM Tris-Cl pH 7.5, 150 mM NaCl, 10% glycerol, 2 mM
MgCl.sub.2, and 1% NP40 ) supplemented with a complete protease
inhibitor cocktail (Sigma-Aldrich Co., St. Louis, Mo.). Protein
extracts were subjected to centrifugation at 14,000 rpm for 10 min
and then immunoprecipitated with specified antibodies overnight.
Immunoprecipitates were extensively washed with lysis buffer and
eluted with loading buffer. Antibodies used were: Acetyl-lysine
antibody Cell Signaling Technology, Danvers, Mass., and MnSOD
(Santa Cruz Biotechnology, Santa Cruz, Calif.).
[0058] By analyzing the levels of ROS by DHE staining, it was found
that rapamycin treatment prevented the induction of ROS after
radiation in primary NOK, while it failed to protect from ROS
accumulation in HNSCC cells (FIGS. 4A and 4B). Furthermore,
rapamycin prevented the remarkable increase in ROS preceding NOK
cell growth arrest (FIG. 4C, 10 days) as well as in cells
undergoing replicative senescence (FIG. 4D, 40 days). These
observations suggested that mTOR inhibition might limit the
accumulation of ROS in NOK by decreasing ROS formation or by
accelerating their inactivation. Regarding the latter, it was
hypothesized that if the protective effects of rapamycin is
mediated by an increase in the cellular ROS scavenging capacity,
mTOR inhibition should also protect from hydrogen peroxide
(H.sub.2O.sub.2) induced senescence. Indeed, pretreatment of
primary NOK with rapamycin prevented the appearance of the DNA
damage marker .gamma.H2AX and the expression of the senescence
marker p16 following H.sub.2O.sub.2 treatment, while it did not
prevent the increase in DNA damage in HNSCC cells. These results
further supported the possibility that rapamycin may prevent normal
cells from entering senescence and increase their replicative
capacity by suppressing ROS accumulation, and hence oxidative
stress.
[0059] The levels of different enzymes involved in the
detoxification of ROS were measured. Interestingly, among these,
rapamycin treatment did not affect the expression of catalase or
cytosolic superoxide dismutase (Cu-ZnSOD), but specifically
increased the protein levels of MnSOD, the mitochondrial-localized
superoxide dismutase, in NOK but not in HN12 cells (FIG. 4E). MnSOD
knockdown in NOK increased the basal levels of p16 expression and
DNA damage as measured by .gamma.H2AX, and this effect could only
be partially rescued by rapamycin (FIG. 4F). Furthermore, decreased
expression of MnSOD abrogated the increased clonogenic capacity of
rapamycin-treated NOK under normal conditions and after radiation
(FIG. 4G).
Example 4
[0060] This example demonstrates that mTOR inhibition prevents the
development of mucositis in vivo.
[0061] Female C3H mice, which were bred in the National Cancer
Institute Animal Production Area (Frederick, MD) and free of
genetic mutations, were used for this study. The mice were 7-9
weeks of age at the time of experimentation and weighed between 20
and 30 grams. All experiments were carried out under the aegis of a
protocol approved by the National Cancer Institute Animal Care and
Use Committee and were in compliance with the Guide for the Care
and Use of Laboratory Animal (1996), National Research Council. The
study was conducted in a blinded fashion. The head and neck area
was irradiated by placing each animal in a specially built
LUCITE.TM. jig in such a way that the animal could be immobilized
without the use of anesthetics (Vitolo et al., Clin. Cancer Res.,
10, 1807-1812 (2004)). Additionally, the jig was fitted with a
LUCITE.TM. cone that surrounded the head and prevented head
movement during radiation exposure. For single-dose radiation, mice
were once injected intraperitoneally with rapamycin and irradiated
at 15 Gy. For fractionated radiation, mice were injected
intraperitoneally every day for 5 days with rapamycin (5 mg/kg) and
irradiated at 6 Gy/day (see treatment scheme in FIG. 5A). Ionizing
radiation was delivered with a THERAPAX.TM. DXT300 X-ray irradiator
(Precision X-ray, North Branford, Conn.) by using 2.0 mm Al
filtration (300 kVp) at a dose rate of 1.9 Gy/minute. After
radiation, animals were removed from the jig, housed (4 or 5
animals per cage) in a climate- and light-controlled environment,
and allowed free access to food and water. Mice were euthanized at
day 5, one hour after the last dose of radiation, and at day 10, 5
days after the last radiation dose.
[0062] Some tongues were stained in a solution of 1% toluidine blue
in 10% acetic acid and analyzed macroscopically. Repeated wiping
with gauze soaked in acetic acid was continued until there was no
further recovery of dye. A negative result is indicated by no dye
uptake or light, diffusely stippled uptake of dye. A positive
result, identified as lack of epithelium and therefore an ulcer, is
indicated by deep, royal blue staining in epithelium defects. Image
quantifications were performed with NIH ImageJ software; tongues
were selected, an automatic threshold was applied and blue staining
was quantified as the average staining intensity value per tongue
(expressed in arbitrary units). Result corresponds to 6 animals and
is representative of 3 independent experiments. Finally, tongues
were processed and embedded in paraffin, 3-.mu.m sections were
stained with H&E and microscopic analysis was conducted.
Stained slides were scanned at 40.times. using an Aperio
SCANSCOPE.TM. CS (Aperio, Vista, Calif.). For immunofluorescence
staining, tongues were embedded in optimal cutting temperature
(OCT) compound and kept at -80.degree. C. for cryosection.
[0063] NOK were seeded on the coverslips coated with collagen,
treated and irradiated as described above. One hour after radiation
cells were washed with ice-cold PBS and fixed with 3.2%
paraformaldehyde in PBS. After washing three times with PBS, cells
were permeabilized with TritonX100 0.1% in glycine 200 mM in PBS
and nonspecific binding was blocked with 3 % of bovine serum
albumin (BSA) in PBS for 1 h. Fixed cells were incubated with the
primary antibody (anti .gamma.H2AX; 1: 800) overnight at 4.degree.
C., followed by 1.5 h incubation with the secondary antibody (goat
anti-mouse ALEXA FLUOR.TM. 488, Life Technologies, Carlsbad,
Calif.). Then nuclei were stained with Hoechst 33342 (1:2000 Life
Technologies, Carlsbad, Calif.) and actin was stained with ALEXA
FLUOR.TM. 546-phalloidin (Life Technologies, Carlsbad, Calif.)
according to the manufacturers' instructions. For tissue
immunostaining, cryosections were mounted in silanated slides and
fixed with 3.2% paraformaldehyde in PBS. After washing three times
with PBS, cells were permeabilized with TRITON.TM. X-100 0.5% in
glycine 200 mM in PBS and non-specific binding was blocked with 3%
of BSA or 10% FBS in PBS for 1 h. Slides were then incubated with
the primary antibody overnight at 4.degree. C., followed by a 1.5 h
incubation with the secondary antibody (goat anti-mouse and goat
anti-rabbit ALEXA FLUOR.TM. 488 or 546, 1:800; Life Technologies,
Carlsbad, Calif.). Then nuclei were stained with Hoechst 33342 and
actin was stained with ALEXA FLUOR.TM. 546 or 488-phalloidin (Life
Technologies, Carlsbad, Calif.) according to the manufacturers'
instructions. Primary antibodies used were .gamma.H2AX (1:800,
Millipore Corporation, Billerica, Mass.), .gamma.H2AX-FITC (1:400,
Millipore Corporation, Billerica, Mass.), pS6 (1:500, Cell
Signaling), mouse p53 (1:100, Dako, Carpinteria, Calif.), mouse
Ki-67 (1:100, Dako, Carpinteria, Calif.), MnSOD (1:100, Santa Cruz
Biotechnology, Inc., Santa Cruz, Calif.). Images were taken using
Zeiss Axio Imager Z1 microscope equipped with an Apotome device
(Carl Zeiss, Maple Grove, Minn.) and a motorized stage. Tissue
images were stitched from 3 to 5 different images taken with a
Zeiss Plan-Apochromat 20.times./0.8 na objective using AxioVision
4.8 software with MosaiX (Carl Zeiss, Maple Grove, Minn.). For p63,
Z scan was performed and final images are maximum intensity
projections (MIP) from 4 to 5 focal planes. Final images were
stitched and bright contrast adjusted with AxioVision 4.8 (Carl
Zeiss, Maple Grove, Minn.).
[0064] Five days of radiation (day 5) did not result in an increase
in mTOR activity in vehicle-treated animals as judged by
phosphorylation of S6. However, aberrant activation of mTOR
throughout the basal layer of the tongue epithelium 5 days after
the last dose of radiation (day 10) was observed, which was
prevented by rapamycin treatment during the radiation period.
Surprisingly, mTOR inactivation by rapamycin concomitant with
radiation almost completely prevented the appearance of
mucositis/ulcers in irradiated mice (FIG. 5B). Histological
analysis of the tongues revealed the preservation of the epithelial
layer in irradiated rapamycin-treated mice, although
radiation-associated tissue changes were observed. No differences
were observed in response to rapamycin in non-irradiated mice. In
contrast, extensive ulceration, up to 50% of the surface of the
tongue, was evident in the animals treated with vehicle, which was
absent in all rapamycin treated mice.
[0065] Aligned with the in vitro results, substantial differences
between control and rapamycin-treated mice in the visible
accumulation of p53 in basal epithelial cells after radiation were
not observed. However, a reduction in the levels of .gamma.H2AX and
increased levels of MnSOD were observed, indicating that rapamycin
may protect normal epithelial cells from DNA damage and oxidative
stress in vivo.
[0066] As shown in FIGS. 6A and 6B, it was found that rapamycin
prevented the radiation-induced disappearance of the epithelial
progenitor marker p63 and the basal cell marker cytokeratin 5 (FIG.
6A). Furthermore, by using the proliferation marker Ki-67, it was
found that rapamycin prevented also the radiation-induced block in
proliferation of the basal progenitor layer of the oral epithelia
at the end of the radiation regime, and preserved the proliferative
and tissue repopulating function of the basal epithelial cells 5
days after the last radiation dose (FIG. 6B). Rapamycin treatment
in non-irradiated mice did not affect the number and distribution
pattern of p65 positive cells (not shown); only a slight and yet
significant increase in the levels of p65 expression in basal
progenitor cells upon rapamycin treatment was observed (FIG. 6C).
Taken together, these observations indicate that rapamycin
treatment prevents the loss of proliferative capacity of the
epithelial progenitor and stem cell compartment after radiation,
which in turn, protects from epithelial stem cell depletion and the
consequent appearance of widespread ulcers and mucositis.
[0067] All references, including publications, patent applications,
and patents, cited herein are hereby incorporated by reference to
the same extent as if each reference were individually and
specifically indicated to be incorporated by reference and were set
forth in its entirety herein.
[0068] The use of the terms "a" and "an" and "the" and "at least
one" and similar referents in the context of describing the
invention (especially in the context of the following claims) are
to be construed to cover both the singular and the plural, unless
otherwise indicated herein or clearly contradicted by context. The
use of the term "at least one" followed by a list of one or more
items (for example, "at least one of A and B") is to be construed
to mean one item selected from the listed items (A or B) or any
combination of two or more of the listed items (A and B), unless
otherwise indicated herein or clearly contradicted by context. The
terms "comprising," "having," "including," and "containing" are to
be construed as open-ended terms (i.e., meaning "including, but not
limited to,") unless otherwise noted. Recitation of ranges of
values herein are merely intended to serve as a shorthand method of
referring individually to each separate value falling within the
range, unless otherwise indicated herein, and each separate value
is incorporated into the specification as if it were individually
recited herein. All methods described herein can be performed in
any suitable order unless otherwise indicated herein or otherwise
clearly contradicted by context. The use of any and all examples,
or exemplary language (e.g., "such as") provided herein, is
intended merely to better illuminate the invention and does not
pose a limitation on the scope of the invention unless otherwise
claimed. No language in the specification should be construed as
indicating any non-claimed element as essential to the practice of
the invention.
[0069] Preferred embodiments of this invention are described
herein, including the best mode known to the inventors for carrying
out the invention. Variations of those preferred embodiments may
become apparent to those of ordinary skill in the art upon reading
the foregoing description. The inventors expect skilled artisans to
employ such variations as appropriate, and the inventors intend for
the invention to be practiced otherwise than as specifically
described herein. Accordingly, this invention includes all
modifications and equivalents of the subject matter recited in the
claims appended hereto as permitted by applicable law. Moreover,
any combination of the above-described elements in all possible
variations thereof is encompassed by the invention unless otherwise
indicated herein or otherwise clearly contradicted by context.
* * * * *