U.S. patent application number 15/920885 was filed with the patent office on 2018-09-27 for treatment of lymphangioleiomyomatosis.
This patent application is currently assigned to THE BRIGHAM AND WOMEN'S HOSPITAL, INC.. The applicant listed for this patent is THE BRIGHAM AND WOMEN'S HOSPITAL, INC.. Invention is credited to Chenggang LI, Jane YU.
Application Number | 20180271886 15/920885 |
Document ID | / |
Family ID | 51491836 |
Filed Date | 2018-09-27 |
United States Patent
Application |
20180271886 |
Kind Code |
A1 |
YU; Jane ; et al. |
September 27, 2018 |
TREATMENT OF LYMPHANGIOLEIOMYOMATOSIS
Abstract
Embodiments disclosed herein provide compositions and methods
for treating lymphangioleiomyomatosis (LAM) comprising inhibiting
COX overexpression and prostaglandin over production by
administering at least one COX inhibitor and/or prostaglandin
biosynthetic pathway inhibitors.
Inventors: |
YU; Jane; (Cincinnati,
OH) ; LI; Chenggang; (Maineville, OH) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
THE BRIGHAM AND WOMEN'S HOSPITAL, INC. |
Boston |
MA |
US |
|
|
Assignee: |
THE BRIGHAM AND WOMEN'S HOSPITAL,
INC.
Boston
MA
|
Family ID: |
51491836 |
Appl. No.: |
15/920885 |
Filed: |
March 14, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14771817 |
Sep 1, 2015 |
9925202 |
|
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PCT/US2014/020125 |
Mar 4, 2014 |
|
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15920885 |
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61851249 |
Mar 4, 2013 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 31/18 20130101;
A61K 31/405 20130101; A61K 31/192 20130101; A61K 31/616 20130101;
A61K 31/436 20130101; A61K 31/196 20130101; A61K 31/415 20130101;
A61K 45/06 20130101; A61K 31/42 20130101; A61K 31/12 20130101; A61P
11/00 20180101; A61K 31/365 20130101; A61K 31/12 20130101; A61K
2300/00 20130101; A61K 31/18 20130101; A61K 2300/00 20130101; A61K
31/192 20130101; A61K 2300/00 20130101; A61K 31/196 20130101; A61K
2300/00 20130101; A61K 31/365 20130101; A61K 2300/00 20130101; A61K
31/405 20130101; A61K 2300/00 20130101; A61K 31/415 20130101; A61K
2300/00 20130101; A61K 31/42 20130101; A61K 2300/00 20130101; A61K
31/616 20130101; A61K 2300/00 20130101; A61K 31/436 20130101; A61K
2300/00 20130101 |
International
Class: |
A61K 31/616 20060101
A61K031/616; A61K 31/436 20060101 A61K031/436; A61K 45/06 20060101
A61K045/06; A61K 31/42 20060101 A61K031/42; A61K 31/415 20060101
A61K031/415; A61K 31/12 20060101 A61K031/12; A61K 31/18 20060101
A61K031/18; A61K 31/192 20060101 A61K031/192; A61K 31/196 20060101
A61K031/196; A61K 31/365 20060101 A61K031/365; A61K 31/405 20060101
A61K031/405 |
Goverment Interests
GOVERNMENT SUPPORT
[0002] This invention was made with Government support under Grant
No.: RO1 HL098216-3 awarded by the National Institutes of Health,
and Grant No.: W81XBH-12-1 awarded by the Department of Defense.
The Government has certain rights in the invention.
Claims
1. A composition comprising at least one a cyclooxygenase (COX)
inhibitor and/or an inhibitor of the prostaglandin biosynthetic
pathway for the treatment of lymphangioleiomyomatosis (LAM).
2. The composition of claim 1, further comprising rapamycin.
3. (canceled)
4. The composition of claim 1, further comprising at least one
compound selected from the group consisting of SCH-202676
hydrobromide, danusertib (PHA-739358), AZ-960, nicardipine,
SB-590885, Thimerosal, ionomycin, U-73343, PAF C16, BX912, and
Chlorambucil.
5. (canceled)
6. A composition comprising at least one a cyclooxygenase (COX)
inhibitor and/or an inhibitor of the prostaglandin biosynthetic
pathway and at least one compounds selected from the group
consisting of nateglinide, Z-L-Phe chloromethyl ketone, clemastine
fumarate, supercinnamaldehyde, practolol, fluvastatin Na, sulindac,
BIO, amorolfine, spectinomucin, sibutramine HCl, nelfinavir
mesylate, moroxydine HCl, nicotine ditartrate, trequinsin,
megluime, tizanidine HCl, CGP-74514A hydrochloride, tioconazole,
afatinib, kasugamycin, flupentixol, fluphenazine, mephenytoin,
aminoglutethimide, betaxolol hydrochloride, salmeterol,
chelerythrine chloride, paroxetine, trifluoperazine, fluoxetine,
methiothepin, nortriptyline, A-77636, rapamycin, SCH-202676
hydrobromide, danusertib (PHA-739358), AZ-960, nicardipine,
SB-590885, Thimerosal, ionomycin, U-73343, PAF C16, BX912, and
Chlorambucil for the treatment of lymphangioleiomyomatosis
(LAM).
7. A composition comprising at least one a cyclooxygenase (COX)
inhibitor and/or an inhibitor of the prostaglandin biosynthetic
pathway and at least one compounds selected from the group
consisting of nateglinide, Z-L-Phe chloromethyl ketone, clemastine
fumarate, supercinnamaldehyde, practolol, fluvastatin Na, sulindac,
BIO, amorolfine, spectinomycin, sibutramine HCl, nelfinavir
mesylate, moroxydine HCl, nicotine ditartrate, trequinsin,
meglumine, tizanidine HCl, CGP-74514A hydrochloride, tioconazole,
afatinib, kasugamycin, flupentixol, fluphenazine, mephenytoin,
aminoglutethimide, betaxolol hydrochloride, salmeterol,
chelerythrine chloride, paroxetine, trifluoperazine, fluoxetine,
methiothepin, nortriptyline, A-77636, rapamycin, SCH-202676
hydrobromide, danusertib (PHA-739358), AZ-960, nicardipine,
SB-590885, Thimerosal, ionomycin, U-73343, PAF C16, BX912, and
Chlorambucil.
8. The composition of claim 1, further comprising at least one
pharmaceutically acceptable carrier.
9.-59. (canceled)
60. The composition of claim 1, further comprising rapamycin, and
at least one compound selected from the group consisting of
SCH-202676 hydrobromide, danusertib (PHA-739358), AZ-960,
nicardipine, SB-590885, Thimerosal, ionomycin, U-73343, PAF C16,
BX912, and Chlorambucil.
61. The composition of claim 6, further comprising at least one
pharmaceutically acceptable carrier.
62. The composition of claim 7, further comprising at least one
pharmaceutically acceptable carrier.
Description
CROSS REFERENCE TO RELATED APPLICATION
[0001] This application is a divisional application of U.S. patent
application Ser. No. 14/771,817 filed on Sep. 1, 2015, issued as
U.S. Pat. No. 9,925,202 on Mar. 27, 2018 and is a 35 U.S.C. .sctn.
371 National Phase Entry Application of International Application
No.: PCT/US2014/020125 filed Mar. 4, 2014, which designates the
U.S., and which claims benefit under 35 U.S.C. .sctn. 119(e) of the
U.S. provisional application No. 61/851,249 filed Mar. 4, 2013, the
contents of each of which are incorporated herein by reference in
their entirety.
TECHNICAL FIELD
[0003] The disclosure herein relates to compositions and methods
for the treatment of lymphangioleiomyomatosis (LAM) and
lymphangioleiomyomatosis in tuberous sclerosis complex
(TSC/LAM).
BACKGROUND
[0004] Lymphangioleiomyomatosis (LAM) is a rare lung disease. Some
LAM occurrences are associated with mutations in the tuberous
sclerosis complex (TSC) locus. LAM occurs almost exclusively in
women, usually of childbearing age. There are two types of LAM,
sporadic LAM and LAM/TSC which is LAM that frequently occurs in
patients who have TSC.
[0005] LAM is characterized by the proliferation of abnormal smooth
muscle-like cells throughout the lungs, in the bronchioles,
alveolar septa, perivascular spaces, and lymphatics, resulting in
the obstruction of small airways that leads to pulmonary cyst
formation and pneumothorax, and lymphatics that leads to chylous
pleural effusion.
[0006] TSC is a rare multi-system genetic disease that results in
the growth of non-malignant tumors in the brain and on other vital
organs such as the kidneys, heart, eyes, lungs, brain, and skin. A
combination of symptoms may include seizures, developmental delay,
behavioral problems, skin abnormalities, and lung and kidney
disease. TSC is caused by a mutation of either of two genes, TSC1
and TSC2, with the TSC2 mutation being the more typical TSC
mutation. The mutations in the TSC locus tend to be negative
mutations that lead to no or reduced amount of functional TSC 1 or
2 gene product. This in turn leads to a deregulation of the mTOR
signaling pathway.
[0007] The mammalian target of rapamycin (mTOR) signaling pathway
is a major player controlling cell growth and cell division. The
kinase, mTOR, is a master regulator of protein synthesis that
couples nutrient sensing to cell growth. Defects in the mTOR
signaling pathway can result in loss of control in cell growth and
cell division. For example, two proteins, hamartin and tuberin, are
known to be involved in the control of cell growth and cell
division via their effects on the mTOR signaling pathway. Hamartin
and tuberin function as a complex to interact with Rheb GTPase,
thereby sequestering it from activating mTOR signaling. Mutations
at the TSC1 and TSC2 loci which codes for hamartin and tuberin
respectively result in the deregulation of the mTOR signaling
pathway resulting in increased mTOR signaling. This in turn leads
to a loss of control of cell growth and cell division, and
subsequently a predisposition to forming tumors.
[0008] High percentages (60-80%) of TSC patients have benign tumors
in the kidneys called angiomyolipomas (AML) which frequently
causing hematuria. These tumors are composed of vascular tissue
(angio-), smooth muscle (-myo-), and fat (-lipoma). Although
benign, AML may grow such that kidney function is impaired or the
blood vessels may dilate and burst leading to catastrophic
hemorrhage either spontaneously or with minimal trauma. Large AML
can be treated with embolization.
[0009] In addition, TSC patients who have AML are predisposed to
develop LAM in the lungs. The proliferating smooth muscle that
occurs in the type of LAM seen in these patients (TSC-LAM) has been
shown to represent clones of the smooth muscle in those patients'
renal AML. It is believed to represent metastases of this "benign"
tumor.
[0010] Leading causes of death in TSC patients include renal
disease, brain tumor, LAM of the lung, and status epilepticus or
bronchopneumonia in those with severe mental handicap. There is no
current effective treatment for TSC or the consequential AML or
LAM; treatment is mainly symptomatic management, e.g., everolimus
(derivative of rapamycin) for the treatment of subependymal giant
cell astrocytoma (brain tumor), vigabatrin for infantile spasm,
ACTH for epilepsy and rapamycin for shrinking the tumors.
[0011] The clinical course of patients with LAM shows considerable
variation. The disease can progress slowly, but ultimately leads to
respiratory failure and death. The 10-year survival rate from the
start of symptoms has been reported to range from 47-79% depending
on the various studies. Current treatments include administration
of rapamycin (also known as sirolimus, an mTOR inhibitor) for
shrinking tumors, and therapies targeting the reproductive cycle of
the women, e.g., progesterone, oophorectomy, tamoxifen,
gonadotropin-releasing hormone (GnRH) agonists or analogues and
androgen therapy. Although the mTORC1 inhibitor rapamycin has been
shown to stabilize lung function and improved symptoms in these
patients, the lung function tend to declined when rapamycin was
discontinued. Improvement to the current repertoire of therapies
for LAM and LAM/TSC are needed.
SUMMARY
[0012] The methods and compositions provided herein relate, in
part, to the discovery of cancer cells and tumors of
lymphangioleiomyomatosis (LAM) are responsive to aspirin; they
decreased in size and reduced proliferation when treated with
aspirin. The inventors observed that the expression of
cyclooxygenase-2 (COX-2) and prostaglandin production are elevated
in a rapamycin-insensitive and Torin1-sensitive manner in
TSC2-deficient cells, but not in TSC2-reexpressing cells.
[0013] Accordingly, inhibitors of a COX or the prostaglandin
biosynthetic pathway are useful for inhibiting the cell
proliferation of cancer cells and tumors of LAM and therefore for
treating LAM.
[0014] Accordingly, it is the objective of this disclosure to
provide additional therapeutics to the existing repertoire of
therapies currently available for LAM. Compositions and methods are
provided for treating lymphangioleiomyomatosis (LAM) comprising
inhibiting COX overexpression and prostaglandin over production by
administering at least one COX inhibitor and/or prostaglandin
biosynthetic pathway inhibitors.
[0015] Accordingly, in one embodiment, provided herein is a method
for treating LAM in a subject in need comprising administering to a
subject therapeutically effective amount of a cyclooxygenase (COX)
inhibitor or an inhibitor of the prostaglandin biosynthetic
pathway.
[0016] In one embodiment, provided herein is a method for treating
LAM in a subject in need comprising selecting a subject having LAM
and administering to the subject therapeutically effective amount
of a COX inhibitor or an inhibitor of the prostaglandin
biosynthetic pathway.
[0017] In one embodiment, provided herein is a method for treating
LAM in a subject in need comprising selecting a subject having LAM
and has a mutation in the TSC locus, and administering to the
subject therapeutically effective amount of a COX inhibitor or an
inhibitor of the prostaglandin biosynthetic pathway.
[0018] In one embodiment, provided herein is a method for treating
LAM in a subject in need comprising selecting a subject having
cancer cells that are TSC-1 or TSC-2 deficient or both TSC1/2
deficient and administering to the subject therapeutically
effective amount of a COX inhibitor or an inhibitor of the
prostaglandin biosynthetic pathway.
[0019] In one embodiment, provided herein is a method for treating
LAM in a subject in need comprising selecting a subject having COX
overexpression and administering to the subject therapeutically
effective amount of a COX inhibitor or an inhibitor of the
prostaglandin biosynthetic pathway.
[0020] In one embodiment, provided herein is a method for treating
LAM in a subject in need comprising selecting a subject having
increased prostaglandin production and administering to the subject
therapeutically effective amount of a COX inhibitor or an inhibitor
of the prostaglandin biosynthetic pathway.
[0021] In one embodiment, provided herein is a method for treating
LAM in a subject in need comprising selecting a subject having a
mutation in the TSC locus and having a COX overexpression and/or
increased prostaglandin production, and administering to the
subject therapeutically effective amount of a COX inhibitor or an
inhibitor of the prostaglandin biosynthetic pathway.
[0022] In one embodiment, provided herein is a method for treating
LAM in a subject in need comprising selecting a subject having LAM
and having a COX overexpression and/or increased prostaglandin
production, and administering to the subject therapeutically
effective amount of a COX inhibitor or an inhibitor of the
prostaglandin biosynthetic pathway.
[0023] In one embodiment, provided herein is a method for treating
LAM in a subject in need comprising selecting a subject having a
mutation in the TSC locus and having increased prostaglandin
production, and administering to the subject therapeutically
effective amount of a COX inhibitor or an inhibitor of the
prostaglandin biosynthetic pathway.
[0024] In one embodiment, provided herein is a method for treating
LAM in a subject in need comprising selecting a subject having LAM
and having increased prostaglandin production and administering to
the subject therapeutically effective amount of a COX inhibitor or
an inhibitor of the prostaglandin biosynthetic pathway.
[0025] In one embodiment, provided herein is a method for treating
LAM in a subject in need comprising and having selecting a subject
having a mutation in the TSC locus and having a COX overexpression,
and administering to the subject therapeutically effective amount
of a COXinhibitor or an inhibitor of the prostaglandin biosynthetic
pathway.
[0026] In one embodiment, provided herein is a method for treating
LAM in a subject in need comprising and having selecting a subject
having having LAM and having a COX overexpression, and
administering to the subject therapeutically effective amount of a
COXinhibitor or an inhibitor of the prostaglandin biosynthetic
pathway.
[0027] In one embodiment, provided herein is a method for treating
LAM in a subject in need comprising first determining whether the
subject has one or more of the following: (a) a COX overexpression;
(b) a mutation in the TSC locus; (c) increased prostaglandin
production; (d) mTOR deregulation or hyperactivity ie., normal mTOR
regulation or activity; and (e) at least one cancer cell that is
insensitive to rapamycin; and if any is affirmative or positive,
administering to the subject therapeutically effective amount of a
COXinhibitor or an inhibitor of the prostaglandin biosynthetic
pathway.
[0028] In one embodiment of any method described, the method
further comprises determining whether the subject has a negative
mutation in the TSC gene 1 or 2.
[0029] In one embodiment of any method described, the method
further comprises selecting the subject having a mutation in the
TSC locus.
[0030] In one embodiment of any method described, the method
further comprises determining whether the cancer cells of the
subject are TSC-1 or TSC-2 or both TSC-1 and TSC-2 deficient.
[0031] In one embodiment of any method described, the method
further comprises selecting the subject having cancer cells that
are TSC-1 or TSC-2 or both TSC-1 and TSC-2 deficient.
[0032] In one embodiment of any method described, the method
further comprises selecting the subject having a COX
overexpression.
[0033] In one embodiment of any method described, the method
further comprises selecting the subject having increased
prostaglandin production.
[0034] In one embodiment of any method described, the subject has a
mutation in the TSC locus, for example, in the TSC1 or TSC2 or both
TSCs.
[0035] In one embodiment of any method described, the mutation is a
negative mutation that produces loss-of-function of the affected
gene. This in essence leads to cells that are deficient in TSC-1 or
TSC-2 or both proteins.
[0036] In one embodiment of any method described, the cancer cells
of the subject are TSC-1 or TSC-2 deficient.
[0037] In one embodiment of any method described, the subject has a
COX overexpression. In one embodiment of any method described, the
COX overexpression is COX-1 or COX-2 or both.
[0038] In one embodiment of any method described, the subject has
increased prostaglandin production.
[0039] In one embodiment of any method described, at least one
cancer cell of the subject is insensitive to rapamycin.
[0040] In one embodiment of any method described, at least one
cancer cell of the subject does not involve mTOR deregulation or
hyperactivity. This means that there is normal mTOR regulation or
activity in the cancer cell comparable to normal control cells.
[0041] In one embodiment of any method described, the subject is
further treated with an effective amount of one or more compounds
selected from the group consisting of nateglinide, Z-L-Phe
chloromethyl ketone, clemastine fumarate, supercinnamaldehyde,
practolol, fluvastatin Na, sulindac, 6-bromoindirubin-3'-oxime
(BIO), amorolfine, spectinomycin, sibutramine HCl, nelfinavir
mesylate, moroxydine HCl, nicotine ditartrate, trequinsin,
meglumine, tizanidine HCl, CGP-74514A hydrochloride, tioconazole,
afatinib, kasugamycin, flupentixol, fluphenazine, mephenytoin,
aminoglutethimide, betaxolol hydrochloride, salmeterol,
chelerythrine chloride, paroxetine, trifluoperazine, fluoxetine,
methiothepin, nortriptyline, and A-77636.
[0042] In one embodiment of any method described, the subject is
further treated with a therapeutically effective amount of
rapamycin and at least one compound selected from the group
consisting of SCH-202676 hydrobromide, danusertib (PHA-739358),
AZ-960, nicardipine, SB-590885, Thimerosal, ionomycin, U-73343, PAF
C16, BX912, and Chlorambucil.
[0043] In one embodiment of any method described, the subject is
further treated with at least one additional therapy.
[0044] In one embodiment of any method described, the at least one
additional therapy is a cancer therapy.
[0045] In one embodiment of any method described, the at least one
additional cancer therapy is selected from the group consisting of
radiation therapy, chemotherapy, immunotherapy and gene
therapy.
[0046] In one embodiment of any method described, the additional
cancer therapeutics are known drugs that are not currently being
use for the treatment of cancer, LAM, or TSC.
[0047] In one embodiment of any method described, subject is
further treated with hormone therapy such as progesterone,
oophorectomy, tamoxifen, gonadotropin-releasing hormone (GnRH)
agonists or analogues and androgen therapy.
[0048] In one embodiment of any method described, the subject is
human.
[0049] In one embodiment of any method described, the
therapeutically effective amount of the inhibitor is administered
by a route selected from the group consisting of: aerosol, direct
injection, local, systemic, intradermal, direct inhalation,
intravitreal, intramuscular, intraperitoneal, intravenous,
intrathecal, intrapleural, intrauterine, subcutaneous, epidural,
topical, oral, transmucosal, buccal, rectal, vaginal, transdermal,
intranasal, intrasynovial, intraocular/periocular, intraorgan,
intratumor, and parenteral administration.
[0050] In one embodiment of any method described, the COX inhibitor
is a COX-1 or COX-2 inhibitor.
[0051] In one embodiment of any method described, the COX inhibitor
is a selective COX-1 or a selective COX-2 inhibitor.
[0052] In one embodiment of any method described, the COX inhibitor
is selected from the group of rofecoxib, celecoxib, valdecoxib,
nimesulide, ibuprofen, diclofenac, nabumetone, naprosen, aspirin
and analogs thereof.
[0053] In one embodiment of any method described, the inhibitor of
the prostaglandin biosynthetic pathway is indomethacin and
flufenamic acid.
[0054] In one embodiment of any method described herein, a tumor in
the subject being treated is reduced in size by at least 5%.
[0055] In one embodiment of any method described herein, the
subject being treated has a reduction in prostaglandin production
by at least 5%.
[0056] In one embodiment of any method described herein, the
subject being treated has a reduction in COX overexpression by at
least 5%.
[0057] In one embodiment of any method described herein, the
subject being treated has an improvement of lung function by at
least 5%.
[0058] In one embodiment, provided herein is a composition
comprising at least one a cyclooxygenase (COX) inhibitor and/or an
inhibitor of the prostaglandin biosynthetic pathway for the
treatment of lymphangioleiomyomatosis (LAM).
[0059] In one embodiment, provided herein is a composition
comprising at least one a cyclooxygenase (COX) inhibitor and/or an
inhibitor of the prostaglandin biosynthetic pathway and rapamycin
for the treatment of lymphangioleiomyomatosis (LAM).
[0060] In one embodiment, provided herein is a composition
comprising at least one a COX inhibitor and/or an inhibitor of the
prostaglandin biosynthetic pathway, rapamycin and at least one
compound selected from the group consisting of SCH-202676
hydrobromide, danusertib (PHA-739358), AZ-960, nicardipine,
SB-590885, Thimerosal, ionomycin, U-73343, PAF C16, BX912, and
Chlorambucil for the treatment of LAM.
[0061] In one embodiment, provided herein is a composition
comprising at least one a COX inhibitor and/or an inhibitor of the
prostaglandin biosynthetic pathway and at least one compounds
selected from the group consisting of nateglinide, Z-L-Phe
chloromethyl ketone, clemastine fumarate, supercinnamaldehyde,
practolol, fluvastatin Na, sulindac, 6-bromoindirubin-3'-oxime
(BIO), amorolfine, spectinomycin, sibutramine HCl, nelfinavir
mesylate, moroxydine HCl, nicotine ditartrate, trequinsin,
meglumine, tizanidine HCl, CGP-74514A hydrochloride, tioconazole,
afatinib, kasugamycin, flupentixol, fluphenazine, mephenytoin,
aminoglutethimide, betaxolol hydrochloride, salmeterol,
chelerythrine chloride, paroxetine, trifluoperazine, fluoxetine,
methiothepin, nortriptyline, A-77636, rapamycin, SCH-202676
hydrobromide, danusertib (PHA-739358), AZ-960, nicardipine,
SB-590885, Thimerosal, ionomycin, U-73343, PAF C16, BX912, and
Chlorambucil for the treatment of LAM.
[0062] In one embodiment of any composition described, the
composition further comprises at least one pharmaceutically
acceptable carrier.
[0063] In one embodiment, provided herein is a use of at least one
a COX inhibitor and/or an inhibitor of the prostaglandin
biosynthetic pathway for the treatment of LAM.
[0064] In one embodiment, provided herein is a use of at least one
a COX inhibitor and/or an inhibitor of the prostaglandin
biosynthetic pathway for the manufacture of medicament for
treatment of LAM.
[0065] In one embodiment, provided herein is a combinatorial use of
at least one a COX inhibitor and/or an inhibitor of the
prostaglandin biosynthetic pathway and rapamycin for the treatment
of LAM.
[0066] In one embodiment, provided herein is a combinatorial use of
at least one a COX inhibitor and/or an inhibitor of the
prostaglandin biosynthetic pathway and rapamycin for the
manufacture of medicament for treatment of LAM.
[0067] In one embodiment, provided herein is a combinatorial use of
at least one a COX inhibitor and/or an inhibitor of the
prostaglandin biosynthetic pathway, rapamycin and at least one
compound selected from the group consisting of SCH-202676
hydrobromide, danusertib (PHA-739358), AZ-960, nicardipine,
SB-590885, Thimerosal, ionomycin, U-73343, PAF C16, BX912, and
Chlorambucil for the treatment of LAM.
[0068] In one embodiment, provided herein is a combinatorial use of
at least one a COX inhibitor and/or an inhibitor of the
prostaglandin biosynthetic pathway, rapamycin and at least one
compound selected from the group consisting of SCH-202676
hydrobromide, danusertib (PHA-739358), AZ-960, nicardipine,
SB-590885, Thimerosal, ionomycin, U-73343, PAF C16, BX912, and
Chlorambucil for the manufacture of medicament for the treatment of
LAM.
[0069] In one embodiment, provided herein is a combinatorial use of
at least one a COX inhibitor and/or an inhibitor of the
prostaglandin biosynthetic pathway and at least one compounds
selected from the group consisting of nateglinide, Z-L-Phe
chloromethyl ketone, clemastine fumarate, supercinnamaldehyde,
practolol, fluvastatin Na, sulindac, 6-bromoindirubin-3'-oxime
(BIO), amorolfine, spectinomycin, sibutramine HCl, nelfinavir
mesylate, moroxydine HCl, nicotine ditartrate, trequinsin,
meglumine, tizanidine HCl, CGP-74514A hydrochloride, tioconazole,
afatinib, kasugamycin, flupentixol, fluphenazine, mephenytoin,
aminoglutethimide, betaxolol hydrochloride, salmeterol,
chelerythrine chloride, paroxetine, trifluoperazine, fluoxetine,
methiothepin, nortriptyline, A-77636, rapamycin, SCH-202676
hydrobromide, danusertib (PHA-739358), AZ-960, nicardipine,
SB-590885, Thimerosal, ionomycin, U-73343, PAF C16, BX912, and
Chlorambucil for the treatment of LAM.
[0070] In one embodiment, provided herein is a combinatorial use of
at least one a COX inhibitor and/or an inhibitor of the
prostaglandin biosynthetic pathway and at least one compounds
selected from the group consisting of nateglinide, Z-L-Phe
chloromethyl ketone, clemastine fumarate, supercinnamaldehyde,
practolol, fluvastatin Na, sulindac, 6-bromoindirubin-3'-oxime
(BIO), amorolfine, spectinomycin, sibutramine HCl, nelfinavir
mesylate, moroxydine HCl, nicotine ditartrate, trequinsin,
meglumine, tizanidine HCl, CGP-74514A hydrochloride, tioconazole,
afatinib, kasugamycin, flupentixol, fluphenazine, mephenytoin,
aminoglutethimide, betaxolol hydrochloride, salmeterol,
chelerythrine chloride, paroxetine, trifluoperazine, fluoxetine,
methiothepin, nortriptyline, A-77636, rapamycin, SCH-202676
hydrobromide, danusertib (PHA-739358), AZ-960, nicardipine,
SB-590885, Thimerosal, ionomycin, U-73343, PAF C16, BX912, and
Chlorambucil for the manufacture of medicament for the treatment of
LAM.
[0071] In one embodiment, provided herein is a method for
inhibiting cell growth, the method comprising contacting a cell
with an effective amount of a COX inhibitor or an inhibitor of the
prostaglandin biosynthetic pathway. In some embodiments, the
inhibition of cell growth is measured in terms of apoptosis or cell
proliferation.
[0072] In one embodiment of any method described herein, each
inhibitor or additional compound is administered singly, i.e., each
each inhibitor or additional compound is administered independently
of the others. In another embodiment of any method described, the
each inhibitor or additional compounds are administered singly and
simultaneously. In another embodiment, the each inhibitor or
compounds are administered together, e.g., in a cocktail or
admixture.
[0073] In one embodiment of any method or composition described,
the inhibitor is singly administered by a route selected from the
group consisting of: aerosol, direct injection, local, systemic,
intradermal, direct inhalation, intravitreal, intramuscular,
intraperitoneal, intravenous, intrathecal, intrapleural,
intrauterine, subcutaneous, epidural, topical, oral, transmucosal,
buccal, rectal, vaginal, transdermal, intranasal, intrasynovial,
intraocular/periocular, intraorgan, intratumor, and parenteral
routes of administration. In one embodiment, where more than one
inhibitor or compound is used for treatment, the inhibitor and/or
compounds are administered simultaneously. In another embodiment,
where more than one inhibitor or compound is used for treatment,
the inhibitor and/or compounds are administered sequentially. The
inhibitor and/or compounds can be admixed prior to administration
and administered together, for example, in a single pharmaceutical
composition.
[0074] In some embodiments, the one or more inhibitor and/or
additional compound used for treatment is administering by nasal
inhalation such as via a nebulizer. For example, the inhibitor
and/or additional compound is formulated as a powder for delivery
via a nebulizer.
[0075] In one embodiment of any composition described, the
composition comprising at least one inhibitor and/or compound is
administered singly or in combination by a route selected from the
group consisting of: aerosol, direct injection, local, systemic,
intradermal, direct inhalation, intravitreal, intramuscular,
intraperitoneal, intravenous, intrathecal, intrapleural,
intrauterine, subcutaneous, epidural, topical, oral, transmucosal,
buccal, rectal, vaginal, transdermal, intranasal, intrasynovial,
intraocular/periocular, intraorgan, intratumor, and and parenteral
administration.
[0076] In one embodiment of any composition described, the
composition is formulated for administration by a route selected
from the group consisting of: aerosol, direct injection, local,
systemic, intradermal, direct inhalation, intravitreal,
intramuscular, intraperitoneal, intravenous, intrathecal,
intrapleural, intrauterine, subcutaneous, epidural, topical, oral,
transmucosal, buccal, rectal, vaginal, transdermal, intranasal,
intrasynovial, intraocular/periocular, intraorgan, intratumor, and,
and parenteral administration.
[0077] In one embodiment of any of the methods or compositions
described herein, derivatives or analogues of the known
drugs/compounds are included.
[0078] In one embodiment, provided herein is a method for treating
LAM in a subject in need comprising (a) determining whether the
subject has a mutation in the tuberous sclerosis TSC locus and (b)
administering to the subject therapeutically effective amount of
any composition described herein when the subject is determined to
have a TSC mutation.
[0079] In one embodiment, provided herein is a method for treating
LAM in a subject in need comprising (a) selecting a subject having
COX-1 or COX-2 overexpression; and (b) administering to the subject
therapeutically effective amount of any composition described
herein.
[0080] In one embodiment, provided herein is a method for treating
LAM in a subject in need comprising (a) selecting a subject having
increased prostaglandin production; and (b) administering to a
subject therapeutically effective amount of any composition
described herein.
[0081] In one embodiment, provided herein is a method for treating
LAM in a subject in need comprising (a) selecting the subject
having a negative mutation in the TSC locus and (b) administering
to a subject therapeutically effective amount of any composition
described herein.
[0082] In one embodiment, provided herein is a method for treating
LAM in a subject in need comprising (a) selecting a subject who has
at least one cancer cell that is insensitive to rapamycin; and (b)
administering to a subject therapeutically effective amount of any
composition described herein.
[0083] In one embodiment, provided herein is a method for treating
LAM in a subject in need comprising (a) selecting a subject who has
at least one cancer cell that does not have mTOR deregulation or
hyperactivity; and (b) administering to a subject therapeutically
effective amount of any composition described herein.
[0084] In one embodiment, provided herein is a method for treating
LAM in a subject in need comprising (a) selecting a subject having
LAM and (b) administering to the subject therapeutically effective
amount of any composition described herein.
[0085] In one embodiment, provided herein is a method for treating
LAM in a subject in need comprising selecting a subject having LAM
and has a mutation in the TSC locus, and administering to the
subject therapeutically effective amount of any composition
described herein.
[0086] In one embodiment, provided herein is a method for treating
LAM in a subject in need comprising selecting a subject having a
mutation in the TSC locus and having a COX overexpression and/or
increased prostaglandin production, and administering to the
subject therapeutically effective amount of any composition
described herein.
[0087] In one embodiment, provided herein is a method for treating
LAM in a subject in need comprising selecting a subject having LAM
and having a COX overexpression and/or increased prostaglandin
production, and administering to the subject therapeutically
effective amount of any composition described herein.
[0088] In one embodiment, provided herein is a method for treating
LAM in a subject in need comprising selecting a subject having a
mutation in the TSC locus and having increased prostaglandin
production, and administering to the subject therapeutically
effective amount of any composition described herein.
[0089] In one embodiment, provided herein is a method for treating
LAM in a subject in need comprising selecting a subject having LAM
and having increased prostaglandin production and administering to
the subject therapeutically effective amount of any composition
described herein.
[0090] In one embodiment, provided herein is a method for treating
LAM in a subject in need comprising and having selecting a subject
having a mutation in the TSC locus and having a COX overexpression,
and administering to the subject therapeutically effective amount
of any composition described herein.
[0091] In one embodiment, provided herein is a method for treating
LAM in a subject in need comprising and having selecting a subject
having having LAM and having a COX overexpression, and
administering to the subject therapeutically effective amount of
any composition described herein.
[0092] In one embodiment, provided herein is a method for treating
LAM in a subject in need comprising first determining whether the
subject has one or more of the following: (a) a COX overexpression;
(b) a mutation in the TSC locus; (c) increased prostaglandin
production; (d) mTOR deregulation or hyperactivity ie., normal mTOR
regulation or activity; and (e) at least one cancer cell that is
insensitive to rapamycin; and if any is affirmative or positive,
administering to the subject therapeutically effective amount of
any composition described herein. In some embodiments, the cancer
cells also have lower levels of phospho-Akt S473, higher levels of
phospho-MARK or phospho-S6 S235.
[0093] In one embodiment, provided herein is a method for treating
LAM in a subject in need comprising first determining whether the
cancer cells of the subject has one or more of the following
features: (a) a mutation in the TSC locus; (b) are TSC-1 or TSC-2
deficient; (c) have overexpression of COX-1 or COX-2; (d) have
lower levels of phosphos-Akt S473; (d) have higher levels of
phosphos-MAPK; (e) have higher levels of phospho-S6 S235; (f) have
increased production of prostaglandin; (g) insensitive to
rapamycin; and (h) do do not have mTOR deregulation or
hyperactivity; and if any is affirmative or positive, administering
to the subject therapeutically effective amount of any composition
or at least one COX inhibitor and/or an inhibitor of the
prostaglandin biosynthesis pathway or described herein.
[0094] In one embodiment, provided herein is a method for treating
LAM in a subject in need comprising selecting a subject who has the
cancer cells that have one or more of the following features: (a) a
mutation in the TSC locus; (b) are TSC-1 or TSC-2 deficient; (c)
have overexpression of COX-1 or COX-2; (d) have lower levels of
phosphos-Akt S473; (d) have higher levels of phosphos-MAPK; (e)
have higher levels of phospho-S6 S235; (f) have increased
production of prostaglandin; (g) insensitive to rapamycin; and (h)
do do not have mTOR deregulation or hyperactivity; and if any is
affirmative or positive, administering to the subject
therapeutically effective amount of any composition or at least one
COX inhibitor and/or an inhibitor of the prostaglandin biosynthesis
pathway or described herein.
BRIEF DESCRIPTION OF THE DRAWINGS
[0095] FIGS. 1a-1i show the identification of an estrogen-induced
prostaglandin biosynthesis signature in TSC2-deficient ELT3 cells
and xenograft tumors.
[0096] FIG. 1a shows the cellular metabolites profile of ELT3
(Tsc2-deficient rat uterus-derived) cells treated with 10 nM
estrogen for 24 hr. The cellular metabolites were profiled by mass
spectrometry (n=5).
[0097] FIG. 1b shows the Box-plots of PGE2 and PGD2 based on the
mass spectrometry profiles of ELT3 cells treated with estrogen.
[0098] FIG. 1c shows the immunoblot analysis of ELT3 cells treated
with estrogen for 2, 4, or 24 hr. Beta-actin was used as a loading
control.
[0099] FIG. 1d shows the secreted PGE2 levels measured in
conditioned media collected from ELT3 cells treated with estrogen
or control at the indicated times (n=3).
[0100] FIG. 1e shows the immunoblot analysis of 621-101 (LAM
patient-derived) cells treated with estrogen for 0.5, 4, or 24 hr.
Beta-actin was used as a loading control.
[0101] FIG. 1f shows the secreted PGE2 levels measured in
conditioned media collected from 621-101 cells treated with
estrogen or control at the indicated times (n=3).
[0102] FIG. 1g shows the cellular metabolite profile of xenograft
tumors from estrogen or placebo-treated mice. Cellular metabolites
extracted from xenograft tumors from estrogen or placebo-treated
mice and profiled by mass spectrometry (n=8).
[0103] FIG. 1h shows the Box-plots of PGE2 and PGD2 obtained in the
cellular metabolite profile of xenograft tumors from estrogen or
placebo-treated mice.
[0104] FIG. 1i shows the urinary PGE2 and creatinine from placebo
or estrogen-implanted ovariectomized female mice bearing xenograft
tumors was measured five-week post cell inoculation. PGE2 levels
were normalized to creatinine. * p<0.05, ** p<0.01.
[0105] FIGS. 2a-2l show that TSC2 negatively regulates COX-2
expression and prostaglandin production in a rapamycin-insensitive
manner in vitro and in vivo.
[0106] FIG. 2a shows the analysis of public expression data sets of
TSC2-deficient LAM patient-derived and TSC2-reexpressing cells
treated with 20 nM rapamycin or vehicle for 24 hours.
[0107] FIG. 2b shows the immunoblot analysis of tuberin, phospho-S6
(S235/236) and COX-2 protein in 621-101 cells.
[0108] FIG. 2c shows the secreted prostaglandin levels quantified
in conditioned media collected from 621-101 cells treated with 20
nM rapamycin for 24 hr or control (n=3).
[0109] FIG. 2d shows the immunoblot analysis of tuberin, phospho-S6
(S235/236) and COX-2 protein in ELT3 cells.
[0110] FIG. 2e shows the secreted prostaglandin levels quantified
in conditioned media collected from ELT3 cells treated with 20 nM
rapamycin for 24 hr or control (n=3).
[0111] FIG. 2f shows the cell variability of cells were treated
with 500 nM PGE2 or vehicle for four days. Cell variability was
measured using MTT assay.
[0112] FIG. 2g shows cell growth of HUVECs that were co-cultured
with TSC2- or TC2+ LAM patient-derived cells using 0.3 .mu.M
transwell inserts for 72 hr. Cell number was assayed using a
fluorescent dye.
[0113] FIG. 2h shows the immunoblot analysis of tuberin, phospho-S6
(S235/236) and COX-2 in xenograft tumors of ELT3 cells. Female
CB17-scid mice were inoculated with ELT3-V3 cells (TSC2-, vector
addback) or TSC2 addback (TSC2+) ELT3-T3 cells subcutaneously.
[0114] FIG. 2i shows the urinary levels of PGE2 and
6-keto-PGF1.alpha. normalized to creatinine levels in mice bearing
xenograft tumors (n=9).
[0115] FIG. 2j shows the immunoblot analysis of phospho-S6
(S235/236) and COX-2 in xenograft tumors.
[0116] FIG. 2k shows the immunoblot analysis of phospho-S6
(S235/236) and COX-2 in TSC2+/- renal cystadenoma (Cyst). N denotes
normal tissue.
[0117] FIG. 2l shows the immunohistochemical staining of COX-2 in
renal cystadenoma of Tsc2+/- mice treated with either vehicle or
rapamycin. Scale bar, 100 .mu.M. * p<0.05, ** p<0.01, ***
p<0.001.
[0118] FIGS. 3a-3g shows that aspirin treatment inhibits the growth
of TSC2-deficient cells in vitro and in vivo, and reduces urinary
levels of prostaglandins in vivo.
[0119] FIG. 3a shows the immunoblot of levels of COX-1, COX-2,
phospho-p44/42 MAPK and phospho-S6 in 621-101 cells treated with 5
.mu.M Sulindac, 50 .mu.M NS398, or 450 .mu.M aspirin for 24 hr.
Levels of COX-1, COX-2, phospho-p44/42 MAPK and phospho-S6 were
assessed by immunoblot.
[0120] FIG. 3b shows the PGE2 levels from conditioned media
measured using ELISA.
[0121] FIG. 3c shows the location of TSC2-deficient LAM
patient-derived cells that were intratracheally instilled into NCr
nu/nu mice. SPECT/CT imaging shows tumor development seven months
post-cell inoculation in mice treated with vehicle or aspirin for
30 days. Arrows indicate individual tumors.
[0122] FIG. 3d shows the tumor growth that was quantified using the
radiotracer 99mTcO4-uptake (n=3). Bioluminescent imaging was
performed weekly. Tumor area was normalized to the baseline before
drug administration.
[0123] FIG. 3e shows the table summarizing the recorded tumor
growth.
[0124] FIGS. 3f and 3g show the urinary PGE2 that was quantified
from mice treated with aspirin or vehicle for 7 and 30 days. *
p<0.05.
[0125] FIGS. 4a-4f shows the COX-2 expression and prostaglandin
production in LAM nodules and LAM patients.
[0126] FIG. 4a shows the transcript levels of PTGS2 (COX-2) that
were measured using real-time RT-PCR on RNA prepared from LAM lung
and clinically normal lung samples (n=3 each).
[0127] FIG. 4b shows the levels of COX-1, COX-2, or PLA2 proteins
from LAM or normal lungs (n=6) were determined by immunoblotting.
Beta-actin was included as a loading control.
[0128] FIG. 4c shows LAM lung tissues stained with smooth muscle
actin (SMA), phospho-S6 (P-S6) (S235/236), or COX-2 antibodies.
Scale bar, 250 .mu.M.
[0129] FIG. 4d shows serum levels of PGE2 in LAM (n=14) and healthy
women (N) (n=13). *p<0.05.
[0130] FIG. 4e shows serum levels of 6-keto PGF1.alpha. in LAM
(n=14) and healthy women [0131] (N) (n=13). *p<0.05.
[0132] FIG. 4f shows a schematic illustration of
rapamycin-insensitive COX-2 activation and prostaglandin production
in cells with mTORC1 activation. 1) TSC2 negatively regulates COX-2
expression and prostaglandin production; 2) Torin 1 suppresses
COX-2 activation and prostaglandin production specifically to
TSC2-deficient cells; 3) estrogen further increases COX-2
activation and prostaglandin production in TSC2-deficient cells; 4)
Aspirin treatment suppresses tumor growth and prostaglandin
production in a metastatic model of LAM; 5) LAM lesions express
COX-2 and LAM patients have higher serum prostaglandins relative to
healthy women, suggesting a potential biomarker for LAM.
[0133] FIGS. 5a-5c show that estrogen increases COX-2 expression in
a xenograft tumor model. TSC2-deficient ELT3 cells were
subcutaneously injected into female ovariectomized mice implanted
with estrogen or placebo pellets.
[0134] FIG. 5a shows the immunoblot analysis of phospho-MAPK
(T202/Y204) and phospho-S6 (S235/236) in xenograft tumors (n=2-3)
in host mice treated with estrogen and control vehicle.
[0135] FIG. 5b shows the transcript levels of Pgr (progesterone
receptor), PTGS1 (COX-1) and PTGS2 (COX-2), were measured using
real-time RT-PCR in primary tumors from placebo (PL) or
estrogen-treated (E2) mice (n=3). *p<0.05.
[0136] FIG. 5c shows the immunoblot analysis of COX-2 levels in
estrogen-treated xenograft tumor (n=4-5).
[0137] FIG. 6 shows a table of the analysis of published expression
array shows upregulation of genes involved in prostaglandin
biosynthesis.
[0138] FIG. 7 shows the gene products that regulate prostaglandin
biosynthesis in TSC2-deficient LAM patient-derived cells.
[0139] FIGS. 8a-8b show that rapamycin-insensitive expression of
COX-2 and prostaglandin production in cells with mTORC1
activation.
[0140] FIG. 8a shows the immunoblot analysis of phospho-S6
(S235/236) and COX-2 protein in 293, HeLa, MCF-5 OVCAR-5 and U2OS
cells treated with 20 nM rapamycin for 24 hr or control (n=3).
[0141] FIG. 8b shows the secreted prostaglandin levels quantified
in conditioned media collected from 293, HeLa, MCF-5 OVCAR-5 and
U2OS cells treated with 20 nM rapamycin for 24 hr or control
(n=3).
[0142] FIG. 9 shows the urinary levels of prostaglandins are not
elevated in LAM patients. Urinary levels of 6-keto PGF1.alpha. and
(b) PGE2 in LAM (n=4) and healthy women (N) (n=4) recruited at BWH
were compared. (c) Urinary levels of PGE2 in LAM (n=15) and healthy
women (N) (n=12) recruited at PUMC-China were compared.
DETAILED DESCRIPTION
Definitions
[0143] As used herein, the term "apoptosis" refers to a natural
process of self-destruction in certain cells that is initiated
and/or determined by activation of certain genes. Apoptosis can be
initiated by an external stimulus e.g., administration of an
inducer of apoptosis. Several biochemical events lead to
characteristic cell changes (morphology) and death. These changes
include, but are not limited to, cell blebbing, cell shrinkage,
nuclear fragmentation, chromatin condensation, and chromosomal DNA
fragmentation. Analysis of apoptosis can be performed by any method
known in the art; non-limiting examples include cell free apoptotic
assay, DNA fragmentation assay, DNA laddering assay, terminal
transferase dUTP nick end labeling (TUNEL) assay and Annexin A5 (or
annexin V) detection. The DNA can be labeled with propidium iodide
or 7-AAD and analyzed by flow cytometry.
[0144] A "cancer" in a subject refers to the presence of cells
possessing characteristics typical of cancer-causing cells, such as
uncontrolled proliferation, immortality, metastatic potential,
rapid growth and proliferation rate, loss of contact inhibition and
certain characteristic morphological features. Often, cancer cells
will be in the form of a tumor, but such cells may exist alone
within a subject, or may be a non-tumorigenic cancer cell.
[0145] As used herein, the term "tumor" means a mass of transformed
cells that are characterized by neoplastic uncontrolled cell
multiplication and at least in part, by containing angiogenic
vasculature. The abnormal neoplastic cell growth is rapid and
continues even after the stimuli that initiated the new growth has
ceased. The term "tumor" is used broadly to include the tumor
parenchymal cells as well as the supporting stroma, including the
angiogenic blood vessels that infiltrate the tumor parenchymal cell
mass. Although a tumor generally is a malignant tumor, i.e., a
cancer having the ability to metastasize (i.e., a metastatic
tumor), a tumor also can be nonmalignant (i.e., non-metastatic
tumor). Tumors are hallmarks of cancer, a neoplastic disease the
natural course of which is fatal. Cancer cells exhibit the
properties of invasion and metastasis and are highly
anaplastic.
[0146] As used herein, the term "cancer therapy" refers to a
therapy useful in treating cancer. In some embodiments, the cancer
therapy involves the use of anti-cancer therapeutic agents and
medical procedures. Non-limiting examples of cancer therapy and
anti-cancer therapeutic agents include, but are not limited to,
e.g., surgery, chemotherapeutic agents, immunotherapy, growth
inhibitory agents, cytotoxic agents, agents used in radiation
therapy, anti-angiogenesis agents, apoptotic agents, anti-tubulin
agents, and other agents to treat cancer, such as anti-HER-2
antibodies (e.g., HERCEPTIN), anti-CD20 antibodies, an epidermal
growth factor receptor (EGFR) antagonist (e.g., a tyrosine kinase
inhibitor), HER1/EGFR inhibitor (e.g., erlotinib (TARCEVA)),
platelet derived growth factor inhibitors (e.g., GLEEVEC' (Imatinib
Mesylate)), interferons, cytokines, antagonists (e.g., neutralizing
antibodies) that bind to one or more of the following targets
ErbB2, ErbB3, ErbB4, PDGFR-beta, BlyS, APRIL, BCMA or VEGF
receptor(s), TRAIL/Apo2, and other bioactive and organic chemical
agents, etc. Combinations thereof are also contemplated for use
with the methods described herein.
[0147] In one embodiment, "administration," "treating," and
"treatment," as it applies to a subject, refers to the contact of
an exogenous pharmaceutical, a drug, a compound, a therapeutic, or
a composition to the subject. In another embodiment,
"administration," "treating," and "treatment," as it applies to a
subject, refers to the contact of any one of the described
compounds or compositions to the subject.
[0148] Alternatively, the term "administering," refers to the
placement of an inhibitor, a combination of compound, or a
composition described herein for intended purposes such as treating
LAM, inhibiting cell growth, killing cells or inducing apoptosis,
into a subject by a method or route which results in at least
partial localization of the inhibitors, the combination of
compound, or the composition respectively at a desired site, i.e.,
cancer cells, tumor cells, tumor cells with TSC mutation(s) and/or
overexpression of COX and/or prostaglandin production in the
subject. The inhibitors, the combination of compounds, or the
composition(s) described herein can be administered by any
appropriate route which results in effective treatment of the
subject, i.e. administration results in delivery to a desired
location (e.g., directly to a tumor or near a tumor) in the subject
where at least a portion of the inhibitors delivered. The period of
time the inhibitors, the combination of compounds, or the
composition(s) is/are active depends on the half-life in vivo after
administration to a subject, and can be as short as a few hours,
e.g., twenty-four hours, to a few days, to as long as several
years. Modes of administration include injection, infusion,
instillation, suppository (e.g., for vaginal, cervical. rectal or
urethral insertion), percutaneous implantation or ingestion.
"Injection" includes, without limitation, intravenous,
intramuscular, intraarterial, intraventricular, intradermal,
intraperitoneal, subcutaneous, subcuticular injection and
infusion.
[0149] In one embodiment, as used herein, the term "treat" or
"treatment" refers to reducing or alleviating at least one adverse
clinical symptom associated with cancer, e.g., pain, swelling,
tumor size, tumor growth rate, low blood count etc. In another
embodiment, the term "treat" or "treatment" refers to slowing or
reversing the progression of neoplastic uncontrolled cell
multiplication, i.e., shrinking existing tumors and/or halting
tumor growth. In another embodiment, the term "treat" or
"treatment" refers to inducing apoptosis in cancer or tumor cells
in the subject.
[0150] As used herein, the term "a therapeutically effective
amount" or "an effective amount" refers to an amount sufficient to
achieve the intended purposes such as treating cancer, inhibiting
cell growth, killing cells or inducing apoptosis. In one
embodiment, a therapeutically effective amount of a compound, a
combination of compounds, a pharmaceutical formulation, or a
composition described herein for a method of treating cancer or TSC
is an amount of sufficient to induce apoptosis of cancer cells of
the subject as compared to the level of apoptosis/cell death in the
absence of the compound, the combination of compounds, the
pharmaceutical composition/formulation or the composition. In other
embodiments, the amount of the composition administered is
preferably safe and sufficient to treat, delay the development of a
tumor, and/or delay further growth of the tumor. In some
embodiments, the amount can thus cure or result in amelioration of
the symptoms of cancer and tumor growth, slow the course of cancer
progression, slow or inhibit a symptom of cancer, slow or inhibit
the establishment of secondary symptoms of cancer or inhibit the
development of a secondary symptom of the cancer. For example, an
effective amount of a compound, a combination of compounds, or a
composition described herein inhibits further tumor growth (e.g.,
LAM or AML), cause a reduction in size or even completely halt
tumor growth, shrink the size of a tumor(s), even initiate complete
regression of tumor, and reduce clinical symptoms associated with a
tumor. In one embodiment, an effective amount for treating cancer
or TSC is an amount of a compound, a combination of compounds, or a
composition described herein sufficient to result in a reduction or
complete removal of the symptoms of the disorder, disease, or
medical condition. In another embodiment, an effective amount for
treating or ameliorating a disorder, disease, or medical condition
is an amount sufficient to result in a reduction or complete
removal of the symptoms of the disorder, disease, or medical
condition. The effective amount of a given therapeutic agent will
vary with factors such as the nature of the agent, the route of
administration, the size and species of the animal to receive the
therapeutic agent, and the purpose of the administration. Thus, it
is not possible or prudent to specify an exact "therapeutically
effective amount." However, for any given case, an appropriate
"effective amount" can be determined by a skilled artisan according
to established methods in the art using only routine
experimentation.
[0151] Derivatives, as used herein, include a chemically modified
compound wherein the modification is considered routine by the
ordinary skilled chemist, such as additional chemical moieties
(e.g., an ester or an amide of an acid, protecting groups, such as
a benzyl group for an alcohol or thiol, and tert-butoxycarbonyl
group for an amine). Derivatives also include radioactively labeled
derivatives of the compounds described herein (e.g., biotin or
avidin, with enzymes such as horseradish peroxidase and the like,
with bioluminescent agents, chemoluminescent agents or fluorescent
agents). Additionally, moieties can be added to the compounds
described herein or a portion thereof to increase half-life in
vivo. Derivatives, as used herein, also encompasses analogs, such
as a compound that comprises a chemically modified form of a
specific compound or class thereof, and that maintains the
pharmaceutical and/or pharmacological activities characteristic of
the compound or class, are also contemplated herein. In one
embodiment, the term "derivatives", as used herein, also
encompasses prodrugs of the compounds described herein, which are
known to enhance numerous desirable qualities of pharmaceuticals
(e.g., solubility, bioavailability, manufacturing, etc.).
[0152] The term "analogue" or "analog", as used herein, refers to a
chemical compound having a structure similar to that of another but
differing from it in respect to a certain component, e.g., it can
have a similar action metabolically. In one embodiment, an analog
is a drug that is similar to the drug from which it is derived.
[0153] As used herein, the terms "drug" and "compound" are used
interchangeably and they refer to a known drug described
herein.
[0154] As used herein, the term "mTOR deregulation" with respect to
cancer cells or cells with neoplasia refers to increased or
decreased signaling of the mTOR pathway compared to normal cells or
cells without neoplasia. Increased or decreased signaling can be
analyzed by any method known in the art, e.g., by monitoring the
corresponding increase or decrease phosphorylation of the mTOR
downstream effectors molecules S6K1 and 4E-BP1. See L. Yan, 2006 J.
Biol. Chem., 281: 19793-19797.
[0155] As used herein, the term "mTOR hyperactivation" with respect
to cancer cells or cells with neoplasia refers to increased
signaling of the mTOR pathway compared to normal cells or cells
without neoplasia. Increased mTOR signaling can be analyzed by any
method known in the art, e.g., by monitoring the increase
phosphorylation of the mTOR downstream effectors molecules S6K1 and
4E-BP1. See L. Yan, 2006 J. Biol. Chem., 281: 19793-19797.
[0156] As used herein, the term "neoplasia" refers to the abnormal
proliferation of benign or malignant cells.
[0157] The terms "decrease", "reduced", "reduction", or "inhibit"
are all used herein to mean a decrease by a statistically
significant amount. In some embodiments, "reduce," "reduction" or
"decrease" or "inhibit" typically means a decrease by at least 10%
as compared to a reference level (e.g. the absence of a given
treatment) and can include, for example, a decrease by at least
about 10%, at least about 20%, at least about 25%, at least about
30%, at least about 35%, at least about 40%, at least about 45%, at
least about 50%, at least about 55%, at least about 60%, at least
about 65%, at least about 70%, at least about 75%, at least about
80%, at least about 85%, at least about 90%, at least about 95%, at
least about 98%, at least about 99%, or more. As used herein,
"reduction" or "inhibition" does not encompass a complete
inhibition or reduction as compared to a reference level. "Complete
inhibition" is a 100% inhibition as compared to a reference level.
A decrease can be preferably down to a level accepted as within the
range of normal for an individual without a given disorder.
[0158] As used herein the term "cell proliferation" or "cell
growth" refers to reproduction and increase in cell number, i.e.,
cell division.
[0159] As used herein in the context of a level of mTOR
deregulation or hyperactivity, a "detectable level" refers to a
level of deregulation and/or hyperactivity in a sample that allows
the regulation and/or activity of mTOR to be distinguished from a
reference level, e.g. the regulation and/or activity of mTOR in a
reference level (e.g., mTOR activity in a cancer free sample), by
at least one of the methods and/or assays for mTOR regulation
and/or activity described elsewhere herein. In some embodiments, a
detectable level of mTOR hyperactivity can be a level of mTOR
activity at least 10% greater than a reference level, e.g. 10%
greater, 20% greater, 50% greater, 100% greater, 200% greater, or
300% or greater.
[0160] As used herein the term "comprising" or "comprises" is used
in reference to compositions, methods, and respective component(s)
thereof, that are essential to the disclosure, yet open to the
inclusion of unspecified elements, whether essential or not.
[0161] As used herein the term "consisting essentially of" refers
to those elements required for a given embodiment. The term permits
the presence of additional elements that do not materially affect
the basic and novel or functional characteristic(s) of that
embodiment of this disclosure.
[0162] The term "consisting of" refers to compositions, methods,
and respective components thereof as described herein, which are
exclusive of any element not recited in that description of the
embodiment.
[0163] Unless otherwise explained, all technical and scientific
terms used herein have the same meaning as commonly understood by
one of ordinary skill in the art to which this disclosure belongs.
Definitions of common terms in molecular cell biology may be found
in Harvey Lodish et al., Molecular Cell Biology, 6.sup.th edition,
published by W. H. Freeman and Company, 2007 (ISBN 0716776014);
Kendrew et al. (eds.), The Encyclopedia of Molecular Biology,
published by Blackwell Science Ltd., 1994 (ISBN 0716776014); and
Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a
Comprehensive Desk Reference, published by VCH Publishers, Inc.,
1995 (ISBN 1-56081-569-8). Further, unless otherwise required by
context, singular terms shall include pluralities and plural terms
shall include the singular.
[0164] Unless otherwise stated, the technology and embodiments
thereof presented herein can be performed using standard procedures
known to one skilled in the art, for example, in Maniatis et al.,
Molecular Cloning: A Laboratory Manual, Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y., USA (1982); Sambrook et
al., Molecular Cloning: A Laboratory Manual (2 ed.), Cold Spring
Harbor Laboratory Press, Cold Spring Harbor, N.Y., USA (1989);
Davis et al., Basic Methods in Molecular Biology, Elsevier Science
Publishing, Inc., New York, USA (1986); Current Protocols in
Immunology (CPI) (John E. Coligan, et. al., ed. John Wiley and
Sons, Inc.), Current Protocols in Cell Biology (CPCB) (Juan S.
Bonifacino et. al. ed., John Wiley and Sons, Inc.), Culture of
Animal Cells: A Manual of Basic Technique by R. Ian Freshney,
Publisher: Wiley-Liss; 5th edition (2005), Animal Cell Culture
Methods (Methods in Cell Biology, Vol. 57, Jennie P. Mather and
David Barnes editors, Academic Press, 1st edition, 1998), and
Methods in Molecular biology, Vol. 180, Transgenesis Techniques by
Alan R. Clark editor, second edition, 2002, Humana Press, which are
all herein incorporated by reference in their entireties.
[0165] It should be understood that this technology is not limited
to the particular methodology, protocols, and reagents, etc.,
described herein and as such may vary. The terminology used herein
is for the purpose of describing particular embodiments only, and
is not intended to limit the scope of the present technology, which
is defined solely by the claims.
[0166] Other than in the operating examples, or where otherwise
indicated, all numbers expressing quantities of ingredients or
reaction conditions used herein should be understood as modified in
all instances by the term "about." The term "about" when used in
connection with percentages will mean.+-.1%.
[0167] All patents and publications identified are expressly
incorporated herein by reference for the purpose of describing and
disclosing, for example, the methodologies described in such
publications that might be used in connection with the present
technology. These publications are provided solely for their
disclosure prior to the filing date of the present application.
Nothing in this regard should be construed as an admission that the
inventors are not entitled to antedate such disclosure by virtue of
prior invention or for any other reason. All statements as to the
date or representation as to the contents of these documents is
based on the information available to the applicants and does not
constitute any admission as to the correctness of the dates or
contents of these documents.
[0168] The methods and compositions provided herein relate, in
part, to the discovery of cancer cells and tumors of LAM are
responsive to aspirin; they decreased in size and reduced
proliferation when treated with aspirin. The inventors observed
that the expression of cyclooxygenase-2 (COX-2) and prostaglandin
production are elevated in a rapamycin-insensitive and
Torin1-sensitive manner in TSC2-deficient cells, but not in
TSC2-reexpressing cells. Cells having a mutation in the TSC2 locus
are deficient in TSC2 or tuberin.
[0169] The inventors have discovered previously that estrogen
increases levels of circulating tumor cells and pulmonary
metastases of tuberin-deficient cells in a xenograft model of LAM.
Studies have demonstrated that estrogen induces COX-2-mediated
prostaglandin synthesis. COX-2 is a rate-limiting enzyme catalyzing
the conversion of arachidonate to prostaglandins. COX-2
overexpression has been documented in human tumors, and
prostaglandins may contribute to cancer development. Here, the
inventors discovered that estrogen enhances prostaglandin
production in TSC2-deficient cells. Surprisingly, loss of TSC2
increases COX-2 and prostaglandin biosynthesis in a
rapamycin-insensitive manner, indicatting an mTORC1-independent
pathway in LAM in addition to the mTORC1 pathway. Aspirin
suppresses tumor progression in a xenograft tumor model. In a
metastatic tumor model, Aspirin treatment decreased the growth of
tumors of LAM derived cells, correlated with reduction of urinary
prostaglandins. Quantitative measurement revealed that LAM patients
have significantly higher serum levels prostaglandins compared with
healthy women. Aspirin treatment improved lung function and reduced
prostaglandin levels in exhaled breath condensate in LAM patients.
This study indicates that targeting COX-2 with aspirin or related
drugs and/or targeting the prostaglandin biosynthetic pathway would
have therapeutic benefit in LAM and TSC-related diseases.
Treatment Methods of Lymphangioleiomyomatosis (LAM).
[0170] Accordingly, provided herein is a method for treating LAM in
a subject in need thereof comprising administering to a subject
therapeutically effective amount of a cyclooxygenase (COX)
inhibitor and/or an inhibitor of the prostaglandin biosynthetic
pathway.
[0171] In one embodiment, provided herein is a method for treating
LAM in a subject in need comprising selecting a subject having LAM
and administering to the subject therapeutically effective amount
of a COX inhibitor or an inhibitor of the prostaglandin
biosynthetic pathway.
[0172] In one embodiment, provided herein is a method for treating
LAM in a subject in need comprising selecting a subject having LAM
and has a mutation in the TSC locus, and administering to the
subject therapeutically effective amount of a COX inhibitor or an
inhibitor of the prostaglandin biosynthetic pathway.
[0173] In one embodiment, provided herein is a method for treating
LAM in a subject in need comprising selecting a subject having
cancer cells that are TSC-1 or TSC-2 deficient or both TSC1/2
deficient and administering to the subject therapeutically
effective amount of a COX inhibitor or an inhibitor of the
prostaglandin biosynthetic pathway.
[0174] In one embodiment, provided herein is a method for treating
LAM in a subject in need comprising selecting a subject having COX
overexpression and administering to the subject therapeutically
effective amount of a COX inhibitor or an inhibitor of the
prostaglandin biosynthetic pathway.
[0175] In one embodiment, provided herein is a method for treating
LAM in a subject in need comprising selecting a subject having
increased prostaglandin production and administering to the subject
therapeutically effective amount of a COX inhibitor or an inhibitor
of the prostaglandin biosynthetic pathway.
[0176] In one embodiment, provided herein is a method for treating
LAM in a subject in need comprising selecting a subject having a
mutation in the TSC locus and having a COX overexpression and/or
increased prostaglandin production, and administering to the
subject therapeutically effective amount of a COX inhibitor or an
inhibitor of the prostaglandin biosynthetic pathway.
[0177] In one embodiment, provided herein is a method for treating
LAM in a subject in need comprising selecting a subject having LAM
and having a COX overexpression and/or increased prostaglandin
production, and administering to the subject therapeutically
effective amount of a COX inhibitor or an inhibitor of the
prostaglandin biosynthetic pathway.
[0178] In one embodiment, provided herein is a method for treating
LAM in a subject in need comprising selecting a subject having a
mutation in the TSC locus and having increased prostaglandin
production, and administering to the subject therapeutically
effective amount of a COX inhibitor or an inhibitor of the
prostaglandin biosynthetic pathway.
[0179] In one embodiment, provided herein is a method for treating
LAM in a subject in need comprising selecting a subject having LAM
and having increased prostaglandin production and administering to
the subject therapeutically effective amount of a COX inhibitor or
an inhibitor of the prostaglandin biosynthetic pathway.
[0180] In one embodiment, provided herein is a method for treating
LAM in a subject in need comprising and having selecting a subject
having a mutation in the TSC locus and having a COX overexpression,
and administering to the subject therapeutically effective amount
of a COXinhibitor or an inhibitor of the prostaglandin biosynthetic
pathway.
[0181] In one embodiment, provided herein is a method for treating
LAM in a subject in need comprising and having selecting a subject
having having LAM and having a COX overexpression, and
administering to the subject therapeutically effective amount of a
COXinhibitor or an inhibitor of the prostaglandin biosynthetic
pathway.
[0182] In one embodiment, provided herein is a method for treating
LAM in a subject in need comprising first determining whether the
subject has one or more of the following: (a) a COX overexpression;
(b) a mutation in the TSC locus; (c) increased prostaglandin
production; (d) mTOR deregulation or hyperactivity ie., normal mTOR
regulation or activity; and (e) at least one cancer cell that is
insensitive to rapamycin; and if any is affirmative or positive,
administering to the subject therapeutically effective amount of a
COX inhibitor or an inhibitor of the prostaglandin biosynthetic
pathway.
[0183] In one embodiment, provided herein is a method for treating
LAM in a subject in need comprising first determining whether the
cancer cells of the subject has one or more of the following
features: (a) a mutation in the TSC locus; (b) are TSC-1 or TSC-2
deficient; (c) have overexpression of COX-1 or COX-2; (d) have
lower levels of phosphos-Akt 5473; (d) have higher levels of
phosphos-MAPK; (e) have higher levels of phospho-S6 S235; (f) have
increased production of prostaglandin; (g) insensitive to
rapamycin; and (h) do do not have mTOR deregulation or
hyperactivity; and if any is affirmative or positive, administering
to the subject therapeutically effective amount of any composition
or at least one COX inhibitor and/or an inhibitor of the
prostaglandin biosynthesis pathway or described herein.
[0184] In one embodiment, provided herein is a method for treating
LAM in a subject in need comprising selecting a subject who has the
cancer cells that have one or more of the following features: (a) a
mutation in the TSC locus; (b) are TSC-1 or TSC-2 deficient; (c)
have overexpression of COX-1 or COX-2; (d) have lower levels of
phosphos-Akt 5473; (d) have higher levels of phosphos-MAPK; (e)
have higher levels of phospho-S6 S235; (f) have increased
production of prostaglandin; (g) insensitive to rapamycin; and (h)
do do not have mTOR deregulation or hyperactivity; and if any is
affirmative or positive, administering to the subject
therapeutically effective amount of any composition or at least one
COX inhibitor and/or an inhibitor of the prostaglandin biosynthesis
pathway or described herein.
[0185] In one embodiment of any method described herein, the method
further comprises selecting a subject who has TSC or has been
diagnosed with a mutation at the TSC loci. The subject can be
genetically screened for TSC. A skilled physician will be able to
differentially diagnosis TSC using medical diagnostic methods known
within the art.
[0186] In one embodiment of any method described herein, the
subject is a mammal. In another embodiment, the subject is a
primate mammal. In one embodiment of any method described, the
subject is human.
[0187] In one embodiment of any method described, the method
further comprises determining whether the subject has a negative
mutation in the TSC locus. In one embodiment, the TSC1 or TSC2 or
both TSC1 and TSC2 have at least one negative mutation.
[0188] In one embodiment of any method described, the method
further comprises selecting the subject a negative mutation in the
TSC locus.
[0189] In one embodiment of any method described, the method
further comprising determining whether the cancer cells of the
subject are TSC-1 or TSC-2 deficient.
[0190] In one embodiment of any method described, the method
further comprises selecting the subject having cancer cells that
are TSC-1 or TSC-2 deficient.
[0191] In one embodiment of any method described, the method
further comprises selecting the subject having COX-1 or COX-2
overexpression.
[0192] In one embodiment of the method described, the method
further comprises selecting the subject having increased
prostaglandin production.
[0193] In one embodiment of any method described herein, a tumor in
the subject being treated is reduced in size by at least 5%.
[0194] In one embodiment of any method described herein, the
subject being treated has a reduction in prostaglandin production
by at least 5%.
[0195] In one embodiment of any method described herein, the
subject being treated has a reduction in COX overexpression by at
least 5%.
[0196] In one embodiment of any method described herein, the
subject being treated has an improvement of lung function by at
least 5%.
[0197] In one embodiment of any method described, the subject has a
mutation in the TSC locus. In one embodiment, the mutation is at
TSC 1 or TSC2 or at both TSC1 and TSC2. In one embodiment, the
mutation is a negative mutation that results in reduced or decrease
functional protein expression, for example, prematurely translation
that give rise to a truncated protein.
[0198] In one embodiment of any method described, the subject has a
mutation in at least one of the TSC loci. In one embodiment of any
method described, the cancer cells or tumor obtained from the
subject to be treated involves mutations in at least one of the TSC
loci. In one embodiment, the mutation is at the TSC1 locus. In
another embodiment, the mutation is at the TSC2 locus. In another
embodiment, the mutation is at both the TSC1 and TSC2 loci.
[0199] In one embodiment of any method described, the cancer cells
of the subject are TSC-1 or TSC-2 deficient. In other words, the
subject is hamartin-deficient or tuberin-deficient. Negative
mutations in the TSC1 and TSC2 that result in hamartin-deficiency
or tuberin-deficiency are known in the art, eg. small deletions or
insertions of DNA in the TSC gene that create a premature stop
signal in the transcribed mRNA. These mutations can be determined
by any method known in the art, eg. DNA sequencing.
Hamartin-deficient or tuberin-deficient status of cells can be
determined by any method known in the art, e.g., immunoassays.
[0200] In one embodiment of any method described, the subject has a
COX overexpression. In one embodiment, the COX overexpression is a
COX-1 or COX-2 overexpression. Cyclooxygenase (COX), also known as
prostaglandin-endoperoxide synthase (PTGS), is an enzyme (EC
1.14.99.1) that is responsible for formation of important
biological mediators called prostanoids, including prostaglandins,
prostacyclin and thromboxane. Overexpression of COX-1 or COX-2 can
be determined by any method known in the art, e.g., by quantitative
RT-PCR or Western blot analysis or immunoassays as described in the
Example.
[0201] In one embodiment of any method described, the cancer cells
of the subject are TSC-1 or TSC-2 deficient, and have
overexpression of COX-1 or COX-2.
[0202] In one embodiment of any method described, the cancer cells
of the subject have lower levels of phosphos-Akt 5473 by at least
5% compared to control cells from a healthy subject or control
reference for phosphos-Akt S473 in health cells. In some other
embodiments, the cancer cells of the subject have lower levels of
phosphos-Akt S473 by at least 10%, at least about 20%, at least
about 25%, at least about 30%, at least about 35%, at least about
40%, at least about 45%, at least about 50%, at least about 55%, at
least about 60%, at least about 65%, at least about 70%, at least
about 75%, at least about 80%, at least about 85%, at least about
90%, at least about 95%, at least about 98%, at least about 99%, or
more than a control healthy subject or a control reference for
phosphos-Akt S473 in health cells.
[0203] In one embodiment of any method described, the cancer cells
of the subject have higher levels of phosphos-MAPK or phospho-S6 by
at least 5% compared to control cells from a healthy subject or
control reference for phosphos-MAPK or phospho-S6 S235 respectively
in health cells. In some other embodiments, the cancer cells of the
subject have lower levels phosphos-MAPK or phospho-S6 S235 by at
least 10%, at least about 20%, at least about 25%, at least about
30%, at least about 35%, at least about 40%, at least about 45%, at
least about 50%, at least about 55%, at least about 60%, at least
about 65%, at least about 70%, at least about 75%, at least about
80%, at least about 85%, at least about 90%, at least about 95%, at
least about 98%, at least about 99%, or more than a control healthy
subject or a control reference for phosphos-MAPK or phospho-S6 S235
respectively in health cells.
[0204] In one embodiment of any method described, the cancer cells
of the subject are TSC-1 or TSC-2 deficient, and have lower levels
of phosphos-Akt S473 by at least 5% compared to control cells from
a healthy subject or control reference for phosphos-Akt S473 in
health cells.
[0205] In one embodiment of any method described, the cancer cells
of the subject are TSC-1 or TSC-2 deficient, and have higher levels
of phosphos-MAPK or phospho-S6 S235 by at least 5% compared to
control cells from a healthy subject or control reference for
phosphos-MAPK or phospho-S6 S235 respectively in health cells.
[0206] In one embodiment of any method described, the cancer cells
of the subject are TSC-1 or TSC-2 deficient, have lower levels of
phosphos-Akt 5473 and have higher levels of phosphos-MAPK or
phospho-S6 S235.
[0207] In one embodiment of any method described, the cancer cells
of the subject are TSC-1 or TSC-2 deficient, have lower levels of
phosphos-Akt S473 and have higher levels of phosphos-MAPK or
phospho-S6 S235.
[0208] In one embodiment of any method described, the cancer cells
of the subject are TSC-1 or TSC-2 deficient, have overexpression of
COX-1 or COX-2, have lower levels of phosphos-Akt S473 and have
higher levels of phosphos-MAPK or phospho-S6 S235.
[0209] In some embodiments, the overexpression of COX-1 or COX-2 is
increased by at least 5%, at least 10%, at least about 20%, at
least about 25%, at least about 30%, at least about 35%, at least
about 40%, at least about 45%, at least about 50%, at least about
55%, at least about 60%, at least about 65%, at least about 70%, at
least about 75%, at least about 80%, at least about 85%, at least
about 90%, at least about 95%, at least about 98%, at least about
99%, or more than a control subject or a control reference for
COX-1 or COX-2 respectively. In one embodiment, the control subject
is one who does not have LAM and/or is not TSC1- or TSC2-deficient.
In one embodiment, the control reference for COX-1 or COX-2
expression level is the level obtained for health subjects. For
example, the average amount of COX-1 or COX-2 found in a population
of health subjects not having been diagnosed with LAM and/or TSC 1
or 2 deficient or cells obtain therefrom.
[0210] In one embodiment of any method described, the subject has
increased prostaglandin production. Prostaglandins are products of
cyclooxygenases (COX-1/COX-2). The prostaglandins are a group of
lipid compounds that are derived enzymatically from fatty acids and
have important functions in the animal body. Every prostaglandin
contains 20 carbon atoms, including a 5-carbon ring. Prostaglandins
can be determined by any method known in the art, e.g., by using
enzyme immunoassay kits as described in the Example.
[0211] In one embodiment of any method described, the
prostaglandins include but are not limited to prostaglandin E1
(PGE1 or PGE1), prostaglandin 12 (PGI2 or PGI2) and prostaglandin
E2 (PGE2 or PGE.sub.2).
[0212] In one embodiment of any method described, the prostaglandin
level is determined from a biological sample obtained from the
subject, e.g., a body fluid of the subject. Examples of bodily
fluids that can be obtained for the measuring the prostaglandin
level include but are not limited to a blood sample, a peritoneal
sample, a urine sample, a bladder sample and a sweat sample. In
another embodiment, the biological sample is a blood sample. In one
embodiment, the blood sample is a plasma sample or a serum
sample.
[0213] In some embodiments, the increased in prostaglandin
production is at least 5%, at least 10%, at least about 20%, at
least about 25%, at least about 30%, at least about 35%, at least
about 40%, at least about 45%, at least about 50%, at least about
55%, at least about 60%, at least about 65%, at least about 70%, at
least about 75%, at least about 80%, at least about 85%, at least
about 90%, at least about 95%, at least about 98%, at least about
99%, or more than a control subject or a control reference for the
respective prostaglandins analysed. In one embodiment, the control
subject is one who does not have LAM and/or TSC 1 or 2 deficient.
In one embodiment, the control reference for prostaglandin is the
level obtained for health subjects. For example, the average amount
of the respective prostaglandin found in a population of health
subjects not having been diagnosed with LAM and/or TSC 1 or 2
deficient.
[0214] In one embodiment of any method described, the subject is
tested to determine whether the subject has at least one cancer
cell that is insensitive to rapamycin.
[0215] In one embodiment of any method described, at least one
cancer cell of the subject is insensitive to rapamycin. For
example, a sample of cancer cells or tumor is excised from the
subject in a biopsy. The sample can then be contacted with
rapamycin ex vivo or in vitro and monitored for the effect of
rapamycin on apoptosis and cell proliferation of the cancer cells
or tumor cells. Alternatively, the sample can be explanted into a
host subject, e.g., a mouse or rat, the host subject is treated
with rapamycin and monitored for the effect of rapamycin on changes
in sample size, growth etc. of the explanted sample.
[0216] In some embodiment, "insensitive to rapamycin" means that
treatment of the cancer cell with rapamycin does improve correct
the overexpression of COX-1 or COX-2, increased production of
prostaglandin, lower levels of phosphos-Akt 5473 and higher levels
of phosphos-MAPK or phospho-S6 S235.
[0217] In one embodiment, if there is no or neglible observable
decrease in sample size or growth or weight of the explanted sample
or tumor, the subject has at least one cancer cell that is
insensitive to rapamycin. In one embodiment, if there is no or
neglible observable increase in apoptosis and/or decrease in cell
proliferation of cancer or tumor cells contacted in ex vivo or in
vitro, the subject has at least one cancer cell that is insensitive
to rapamycin.
[0218] In another embodiment, if there is observable increase in
sample size or growth or weight of the explanted sample or tumor,
the subject has at least one cancer cell that is insensitive to
rapamycin. In one embodiment, if there is no or neglible observable
increase in cell proliferation of cancer or tumor cells contacted
in ex vivo or in vitro, the subject has at least one cancer cell
that is insensitive to rapamycin.
[0219] In some embodiment, no or neglible observable decrease in
sample size or growth, or decrease in cell proliferation of cancer
or tumor cells means a decrease less than 5.0%, less than 4.5%,
less than 4.0%, less than 3.5%, less than 3.0%, less than 2.5%,
less than 2.0%, less than 1.5%, less than 1.0%, less than 0.5%,
less than 0.1% or 0% compared to control cancer cells not contacted
with rapamycin or control host subject not treated with
rapamycin.
[0220] In one embodiment, no or neglible observable decrease in
sample size or growth means no detectable significant differences
in the changes in the size or weight of the explanted samples in
the rapamycin-treated host subject compared to non-rapamycin
treated control host subject.
[0221] In some embodiment, no or neglible observable increase in
apoptosis means an increase less than 5.0%, less than 4.5%, less
than 4.0%, less than 3.5%, less than 3.0%, less than 2.5%, less
than 2.0%, less than 1.5%, less than 1.0%, less than 0.5%, less
than 0.1% or 0% compared to control cancer cells not contacted with
rapamycin.
[0222] In one embodiment, an observable increase in increase in
sample size or growth or weight of the explanted sample or tumor,
or cell proliferation of cancer cells means at least 10%, at least
about 20%, at least about 25%, at least about 30%, at least about
35%, at least about 40%, at least about 45%, at least about 50%, at
least about 55%, at least about 60%, at least about 65%, at least
about 70%, at least about 75%, at least about 80%, at least about
85%, at least about 90%, at least about 95%, at least about 98%, at
least about 99%, or more than a control cancer cells or control
host subject not treated with rapamycin.
[0223] In one embodiment of any method described, at least one
cancer cell of the subject does not involve mTOR deregulation or
hyperactivity.
[0224] The mTOR signaling pathway is a major player in controlling
cell growth and cell division. Cancers associated with genetic
defects often have aberrant mTOR signaling. Some LAM cells have TSC
mutations. A subset of LAM occurs in conjunction with mutations in
TSC2, which encodes the protein tuberin (TSC2). The TSC1/TSC2
heterodimer, through inhibition of the Ras homolog enriched in the
brain protein (Rheb), negatively regulates the mammalian target of
rapamycin (mTOR) complex 1 (TORC1). Therefore, LAM patient lesions
have hyperactivation of TORC1. Rapamycin is a naturally occurring
macrolide that inhibits TORC1 actively and is effective in
shrinking kidney angio myolipomas (AML).
[0225] In one embodiment, the mTOR deregulation results in mTOR
hyperactivity.
[0226] In one embodiment of any method, the mTOR hyperactivity is
at least 10% higher compared to a control mTOR activity level. In
other embodiments, the mTOR hyperactivity is at least 15%, at least
20%, at least 25%, at least 30%, at least 35%, at least 40%, at
least 45%, at least 50%, at least 55%, at least 60%, at least 65%,
at least 70%, at least 75%, at least 80%, at least 85%, at least
90%, at least 95%, at least 99%, at least 100% over the mTOR
control. In other embodiment, the mTOR activity level is at least
2-fold, at least 5-fold, at least 10-fold, at least 100-fold, at
least 1000-fold or higher as compared to the control mTOR activity
level.
[0227] In one embodiment of any method described herein, the mTOR
control is an mTOR activity level in a population of normal
non-cancerous cells from the subject being treated. In another
embodiment, the mTOR control is an average mTOR activity level in a
population of healthy subjects. For example, normal non-cancer
cells can be taken from the subject being treated and analyzed for
cellular mTOR activity level. The normal non-cancer cells can be
taken from the same organ diagnosed with cancer or tumors, or the
normal non-cancer cells can be taken from other healthy organs that
are free from cancer or tumors in the subject to be treated.
[0228] Alternatively, healthy cells can be collected from a
population of healthy subjects, e.g., human subjects, the mTOR
activity for the cells of each subject is analyzed and the average
mTOR activity is calculated. The healthy cells collected from the
healthy subjects can be from the same organ where cancer or tumors
are diagnosed in the subject being treated. Alternatively, the
healthy cells can come from a variety of tissue types in a
subject.
[0229] In one embodiment of any method described, the cancer cells
or tumors to be treated in the subject do not involve mTOR
deregulation or hyperactivity.
[0230] In one embodiment of any method described, the cancer cells
or tumors in the subject comprise a mixture of cells, some cells
that involves mTOR deregulation or hyperactivity, and other cells
that do do not involve mTOR deregulation or hyperactivity.
[0231] For analyzing TSC mutation and/or mTOR activity, a tissue
sample is collected from the subject to be treated or healthy
volunteer subjects. Cancer cells can be obtained from a subject
diagnosed with or suspected of having cancer and/or tumors. For
example, cancer cells can be obtained from a tissue biopsy or an
excised tumor during a routine surgery to remove cancerous tumors.
During the biopsy, healthy, normal non-cancer cells can be taken
for analyzing the control cellular mTOR level. A skilled physician
or surgeon will be able to obtain a tissue biopsy or excise a tumor
from a subject. Alternatively, for TSC gene analysis, a sample of
blood from the subject can be used.
[0232] In one embodiment, the tissue sample is a tumor sample. In
another embodiment, the tissue sample contains cancerous cells.
[0233] As used herein, a "tissue sample" refers to a portion,
piece, part, segment, or fraction of a tissue which is obtained or
removed from an intact tissue of a subject, preferably a human
subject. In one embodiment, the tissue sample is a blood sample. In
another embodiment, the tissue sample is a bone marrow sample. In
one embodiment, the tissue sample is a cerebrospinal fluid
sample.
[0234] As used herein, a "tumor sample" refers to a portion, piece,
part, segment, or fraction of a tumor, for example, a tumor which
is obtained or removed from a subject (e.g., removed or extracted
from a tissue of a subject), preferably a human subject.
[0235] In one embodiment, the tissue sample is obtained from a
biopsy procedure in the subject. In another embodiment, the tissue
sample is obtained from a surgical procedure to remove a tumor mass
from the subject.
[0236] The cellular mTOR activity level of cancer cell and normal
non-cancer cells can be analyzed by any method known in the art,
for example, as described by Ikenoue T. et a., Methods Enzymol.
2009; 452:165-80; and by Jinhee Kim, et al., Methods in Molecular
Biology; 2012; 821:215-225. These references are incorporated
herein by reference in their entirety. Alternatively, the cellular
mTOR activity level can be determined by using any one of the
commercially available kits following the manufacturer's protocol,
for example, the K-LISA.TM. mTOR Activity Kit by Merck Millipore
Catalogs# CBA055 and CBA104).
[0237] For TSC loci gene analysis, the mutations in the TSC loci
can be analyzed by any known genomic method in the art. For
example, by single-strand conformation polymorphism analysis (SSCP)
coupled with DNA sequencing as described by Galina D. et al., Am.
J. Respir. Crit. Care Med.; 2001; 163:253-258; Hornigold N, et al.,
Oncogene; 1999; 18:2657-2661. Briefly, the coding exons of TSC1 or
2 are amplified by polymerase chain reaction (PCR) and the
amplified PCR products are then analyzed for variation on DNA gels
without glycerol and with 5% glycerol. As a good number of TSC loci
mutations result in chain-terminating, quantitative real-time
(RT-PCR) assays can be used to analyze the amount of TSC1/2 mRNA as
described in Kwiatkowska J. et al., Ann Hum Genet. 1998; 62:277-85.
Alternatively, commercial kits are available, e.g., RT.sup.2 qPCR
Primer Assay for Human TSC1 and TSC2 respectively from
SABIOSCIENCES.TM. catalog# PPH00244B-200 and PPH00245F. The PCR
primers for the human TSC1 and TSC2 can be purchased from BIORAD.
Alternatively, one skilled in the art can design PCR primers for
the human TSC1 and TSC2 with the following information regarding
the human TSC1 and TSC2 genes:
[0238] The gene symbol, TSC1 stands for the gene name tuberous
sclerosis 1. Aliases for TSC1 include; KIAA0243, LAM, MGC86987, and
TSC. The RefSeqs of TSC1 are NC 000009.11; NG_012386.1;
NT_035014.4. Ensembl: ENSG00000165699; Entrez: 7248; UniGene:
Hs.370854.
[0239] The gene symbol, TSC2, stands for the gene name tuberous
sclerosis 2. Aliases for TSC1 include FLJ43106, LAM, and TSC4. The
RefSeqs of TSC2 are: NC 000016.9; NG_005895.1; NG_008412.1;
NG_008617.1; and NT_010393.16. Ensembl: ENSG00000103197; Entrez:
7249; UniGene: Hs.90303.
[0240] In one embodiment of any method described, the contacted
cell or the cancer to be treated involving mTOR deregulation or
hyperactivity is LAM. In another embodiment, the cancer is LAM,
e.g., the cancer cells tested positive for mTOR hyperactivity or
increased mTOR pathway signaling.
[0241] In one embodiment of any method described, the method
further comprises determining whether the cancer cells of the
subject has one or more of the following features prior to
administering the treatment described herein when the selected
feature is present, the treatment being administering at least one
COX inhibitor and/or an inhibitor of the prostaglandin biosynthesis
pathway or any compositions described herein. The features being
determined in the cancer cells of the subject are: (a) a mutation
in the TSC locus; (b) being TSC-1 or TSC-2 deficient; (c) have
overexpression of COX-1 or COX-2; (d) have lower levels of
phosphos-Akt 5473; (d) have higher levels of phosphos-MAPK; (e)
have higher levels of phospho-S6 S235; (f) have increased
production of prostaglandin; (g) insensitive to rapamycin; and (h)
do do not have mTOR deregulation or hyperactivity.
[0242] In one embodiment of any method described, the method
further comprises selecting a subject who has cancer cells that
have one or more of the following features: (a) a mutation in the
TSC locus; (b) being TSC-1 or TSC-2 deficient; (c) have
overexpression of COX-1 or COX-2; (d) have lower levels of
phosphos-Akt S473; (d) have higher levels of phosphos-MAPK; (e)
have higher levels of phospho-S6 S235; (f) have increased
production of prostaglandin; (g) insensitive to rapamycin; and (h)
do do not have mTOR deregulation or hyperactivity, and
administering the treatment described herein when the selected
feature is present, the treatment being administering at least one
COX inhibitor and/or an inhibitor of the prostaglandin biosynthesis
pathway or any compositions described herein.
[0243] In one embodiment of any method described, the subject is
further treated with an effective amount of one or more compounds
selected from the group consisting of nateglinide, Z-L-Phe
chloromethyl ketone, clemastine fumarate, supercinnamaldehyde,
practolol, fluvastatin Na, sulindac, 6-bromoindirubin-3'-oxime
(BIO), amorolfine, spectinomycin, sibutramine HCl, nelfinavir
mesylate, moroxydine HCl, nicotine ditartrate, trequinsin,
meglumine, tizanidine HCl, CGP-74514A hydrochloride, tioconazole,
afatinib, kasugamycin, flupentixol, fluphenazine, mephenytoin,
aminoglutethimide, betaxolol hydrochloride, salmeterol,
chelerythrine chloride, paroxetine, trifluoperazine, fluoxetine,
methiothepin, nortriptyline, and A-77636.
[0244] In one embodiment of any method described, the subject is
further treated with a therapeutically effective amount of
rapamycin and at least one compound selected from the group
consisting of SCH-202676 hydrobromide, danusertib (PHA-739358),
AZ-960, nicardipine, SB-590885, Thimerosal, ionomycin, U-73343, PAF
C16, BX912, and Chlorambucil.
[0245] In one embodiment of any method described, the subject is
further treated with at least one additional therapy.
[0246] In one embodiment of any method described, the at least one
additional therapy is a cancer therapy.
[0247] In one embodiment of any method described, the at least one
additional cancer therapy is selected from the group consisting of
radiation therapy, chemotherapy, immunotherapy and gene
therapy.
[0248] In one embodiment of any method described, the subject is
further treated with hormone therapy. For example, progesterone,
oophorectomy, tamoxifen, gonadotropin-releasing hormone (GnRH)
agonists or analogues and androgen therapy.
[0249] In one embodiment of any method described, the subject is
further treated with an inhibitor of the mTOR pathway, for example,
rapamycin, temsirolimus, everolimus, ridaforolimus,
epigallocatechin gallate (EGCG), caffeine, curcumin, resveratrol
etc.
[0250] In one embodiment of any method described, the subject is
human.
[0251] In one embodiment of any method described, the
therapeutically effective amount of the COX inhibitor, the
prostaglandin pathway inhibitor and/or the compound is administered
by a route selected from the group consisting of: aerosol, direct
injection, local, systemic, intradermal, direct inhalation,
intravitreal, intramuscular, intraperitoneal, intravenous,
intrathecal, intrapleural, intrauterine, subcutaneous, epidural,
topical, oral, transmucosal, buccal, rectal, vaginal, transdermal,
intranasal, intrasynovial, intraocular/periocular, intraorgan,
intratumor, and parenteral administration.
[0252] In one embodiment of any method described, the one or more
inhibitor and/or additional compound used for treatment is
administering by nasal inhalation such as via a nebulizer. For
example, the inhibitor and/or additional compound is formulated as
a powder for delivery via a nebulizer.
[0253] In one embodiment of any method described, the COX inhibitor
is a COX-1 or COX-2 inhibitor. Exemplary of some COX inhibitors are
the non-steroidal anti-inflammatory drugs, such as aspirin and
ibuprofen.
[0254] In one embodiment of any method described, the COX inhibitor
is a selective COX-1 inhibitor.
[0255] In one embodiment of any method described, the COX inhibitor
is a selective COX-2 inhibitor.
[0256] In one embodiment of any method described, the COX inhibitor
is selected from the group of rofecoxib, celecoxib, valdecoxib,
nimesulide, ibuprofen, diclofenac, nabumetone, naprosen, aspirin
and analogs thereof.
[0257] In one embodiment of any method described, the inhibitor of
the prostaglandin biosynthetic pathway is indomethacin and
flufenamic acid.
[0258] In one embodiment of any method described herein, a tumor in
the subject being administered with the respective inhibitor or
additional drug compound combinations is reduced in size by at
least 10% compared to the tumor size prior to treatment with the
respective drugs or drug combinations. In other embodiments, the
tumor is reduced in size by at least 15%, at least 20%, at least
25%, at least 30%, at least 35%, at least 40%, at least 45%, at
least 50%, at least 55%, at least 60%, at least 65%, at least 70%,
at least 75%, at least 80%, at least 85%, at least 90%, at least
95%, at least 99%, at even 100% (i.e., below detectable limits)
compared to the tumor size prior to treatment with the respective
inhibitor or additional drug combinations.
[0259] In one embodiment of any method described herein, the
subject being treated with the respective inhibitor or additional
drug compound combinations has a reduction in prostaglandin
production by at least 10%. In other embodiments, the reduction in
prostaglandin production is reduced by at least 15%, at least 20%,
at least 25%, at least 30%, at least 35%, at least 40%, at least
45%, at least 50%, at least 55%, at least 60%, at least 65%, at
least 70%, at least 75%, at least 80%, at least 85%, at least 90%,
at least 95%, at least 99%, at even 100% (i.e., below detectable
limits) compared to the prostaglandin production prior to treatment
with the respective inhibitor or additional drug combinations.
[0260] In one embodiment of any method described herein, the
subject being treated with the respective inhibitor or additional
drug compound combinations has a reduction in COX overexpression by
at least 10%. In other embodiments, the reduction in COX
overexpression is reduced by at least 15%, at least 20%, at least
25%, at least 30%, at least 35%, at least 40%, at least 45%, at
least 50%, at least 55%, at least 60%, at least 65%, at least 70%,
at least 75%, at least 80%, at least 85%, at least 90%, at least
95%, at least 99%, at even 100% (i.e., below detectable limits)
compared to the COX overexpression prior to treatment with the
respective inhibitor or additional drug combinations.
[0261] In one embodiment of any method described herein, the
subject being treated with the respective inhibitor or additional
drug compound combinations has an improvement of lung function by
at least 10%. In other embodiments, the lung function is improved
by at least 15%, at least 20%, at least 25%, at least 30%, at least
35%, at least 40%, at least 45%, at least 50%, at least 55%, at
least 60%, at least 65%, at least 70%, at least 75%, at least 80%,
at least 85%, at least 90%, at least 95%, at least 99%, at even
100% (i.e., below detectable limits) compared to the lung function
prior to treatment with the respective inhibitor or additional drug
combinations.
Contacting a Cell with a Composition as Described Herein
[0262] In one embodiment, provided herein is a method for
inhibiting cell growth, the method comprising contacting a cell
with an effective amount of a cyclooxygenase (COX) inhibitor or an
inhibitor of the prostaglandin biosynthetic pathway. In some
embodiments, the inhibition of cell growth is measured in terms of
apoptosis or cell proliferation. Apoptosis or cell proliferation
can be determined by any method known in the art, for example, by
TUNEL DNA fragmentation assay, or by cell counting or as described
in the Example.
[0263] In one embodiment of the method described, the cell has a
mutation in the TSC locus. In one embodiment, the mutation is in
TSC1 or TSC2. In one embodiment, the mutation is a negative
mutation that results in a loss-of-function of the affected
gene.
[0264] In one embodiment of the method described, the cell is TSC-1
or TSC-2 deficient.
[0265] In one embodiment of the method described, the cell has a
COX overexpression. In one embodiment, the COX overexpression is a
COX-1 or COX-2 overexpression.
[0266] In one embodiment of the method described, the cell has
increased prostaglandin production.
[0267] In one embodiment of the method described, the cell is
insensitive to rapamycin.
[0268] In one embodiment of the method described, the cell does not
have mTOR deregulation or hyperactivity.
[0269] In one embodiment of the method described, the contacting
period is at least one hour. In one embodiment, the contact period
is at least one hour to 24 hours. In other embodiments, the contact
period is at least two, at least three, at least four, at least
five, at least six, at least seven, at least eight, at least nine,
at least 10, at least 11, at least 12, at least 13, at least 14, at
least 15, at least 16, at least 17, at least 18, at least 19, at
least 20, at least 21, at least 22, at least 23, or at least 24
hours. In one embodiment, the contact period is between one hour
and 24 hours. In other embodiments, the contact period is two,
three, four, five, six, seven, eight, nine, 10, 11, 12, 13, 14, 15,
16, 17, 18, 19, 20, 21, 22, 23, or 24 hours, including all the time
periods between one to 24 hours to the minute. In other
embodiments, the contacting period is between 24-72 hrs, including
all the time periods between 24-72 hours to the half hour.
[0270] In one embodiment of the method described, the cell is
further contacted with an effective amount of one or more compounds
selected from the group consisting of nateglinide, Z-L-Phe
chloromethyl ketone, clemastine fumarate, supercinnamaldehyde,
practolol, fluvastatin Na, sulindac, 6-bromoindirubin-3'-oxime
(BIO), amorolfine, spectinomycin, sibutramine HCl, nelfinavir
mesylate, moroxydine HCl, nicotine ditartrate, trequinsin,
meglumine, tizanidine HCl, CGP-74514A hydrochloride, tioconazole,
afatinib, kasugamycin, flupentixol, fluphenazine, mephenytoin,
aminoglutethimide, betaxolol hydrochloride, salmeterol,
chelerythrine chloride, paroxetine, trifluoperazine, fluoxetine,
methiothepin, nortriptyline, and A-77636.
[0271] In one embodiment of the method described, the cell is
further contacted with an effective amount of of rapamycin and at
least one compound selected from the group consisting of SCH-202676
hydrobromide, danusertib (PHA-739358), AZ-960, nicardipine,
SB-590885, Thimerosal, ionomycin, U-73343, PAF C16, BX912, and
Chlorambucil.
Compositions for Treating LAM
[0272] Compositions and combinatorial compositions for the
treatment of LAM are provided.
[0273] In one embodiment, provided herein is a composition
comprising at least one a cyclooxygenase (COX) inhibitor or an
inhibitor of the prostaglandin biosynthetic pathway for the
treatment of lymphangioleiomyomatosis (LAM).
[0274] In one embodiment, provided herein is a composition
comprising at least one a COX inhibitor or an inhibitor of the
prostaglandin biosynthetic pathway and rapamycin for the treatment
of LAM.
[0275] In one embodiment, provided herein is a composition
comprising at least one a COX inhibitor or an inhibitor of the
prostaglandin biosynthetic pathway, rapamycin and at least one
compound selected from the group consisting of SCH-202676
hydrobromide, danusertib (PHA-739358), AZ-960, nicardipine,
SB-590885, Thimerosal, ionomycin, U-73343, PAF C16, BX912, and
Chlorambucil for the treatment of LAM.
[0276] In one embodiment, provided herein is a composition
comprising at least one a COX inhibitor or an inhibitor of the
prostaglandin biosynthetic pathway and at least one compounds
selected from the group consisting of nateglinide, Z-L-Phe
chloromethyl ketone, clemastine fumarate, supercinnamaldehyde,
practolol, fluvastatin Na, sulindac, 6-bromoindirubin-3'-oxime
(BIO), amorolfine, spectinomycin, sibutramine HCl, nelfinavir
mesylate, moroxydine HCl, nicotine ditartrate, trequinsin,
meglumine, tizanidine HCl, CGP-74514A hydrochloride, tioconazole,
afatinib, kasugamycin, flupentixol, fluphenazine, mephenytoin,
aminoglutethimide, betaxolol hydrochloride, salmeterol,
chelerythrine chloride, paroxetine, trifluoperazine, fluoxetine,
methiothepin, nortriptyline, A-77636, rapamycin, SCH-202676
hydrobromide, danusertib (PHA-739358), AZ-960, nicardipine,
SB-590885, Thimerosal, ionomycin, U-73343, PAF C16, BX912, and
Chlorambucil for the treatment of LAM.
[0277] In one embodiment, provided herein is a composition
comprising at least one a COX inhibitor or an inhibitor of the
prostaglandin biosynthetic pathway and more than one compound
(e.g., 2, 3, 4, 5, 6, 7, or more) selected from the group
consisting of nateglinide, Z-L-Phe chloroethyl ketone, clemastine
fumarate, supercinnamaldehyde, practolol, fluvastatin Na, sulindac,
6-bromoindirubin-3'-oxime (BIO), amorolfine, spectinomycin,
sibutramine HCl, nelfinavir mesylate, moroxydine HCl, nicotine
ditartrate, trequinsin, meglumine, tizanidine HCl, CGP-7451A
hydrochloride, tioconazole, TOVOK.TM. (afatinib), kasugamycin,
flupentixol, fluphenazine, mephenytoin, aminoglutethimide,
betaxolol hydrochloride, salmeterol, chelerythrine chloride,
paroxetine, trifluoperazine, fluoxetine, methiothepin,
nortriptyline, A-77636, rapamycin, SCH-202676 hydrobromide,
danusertib (PHA-739358), AZ-960, nicardipine, SB-590885,
Thimerosal, ionomycin, U-73343, PAF C16, BX912, and Chlorambucil
for use treatment of LAM.
[0278] Alternatively, the compositions described herein are used
for the prevention of tumor formation, reducing the frequency of
tumor development, inducing apoptosis in a cell, killing a cell and
inhibiting cell growth.
[0279] In some embodiments, the compositions described herein are
for use in any of the methods described herein, e.g., treatment of
LAM, prevention of tumor formation, reducing the frequency of tumor
development, inducing apoptosis in a cell, killing a cell and
inhibiting cell growth.
[0280] In another embodiment of any composition described herein,
the composition is administered in conjunction with at least one
additional therapy to achieve a combination therapy.
[0281] In one embodiment of any composition described herein, the
composition or the combination of compounds are further
administered with a pharmaceutically acceptable carrier.
[0282] In one embodiment of any composition described, the
composition further comprising at least one pharmaceutically
acceptable carrier.
[0283] In one embodiment of any composition described, the
composition is formulated for nasal delivery such as nasal
inhalation.
[0284] In one embodiment, provided herein is a use of at least one
a COX inhibitor or an inhibitor of the prostaglandin biosynthetic
pathway for the treatment of LAM.
[0285] In one embodiment, provided herein is a use of at least one
a COX inhibitor or an inhibitor of the prostaglandin biosynthetic
pathway for the manufacture of medicament for treatment of LAM.
[0286] In one embodiment, provided herein is a combinatorial use of
at least one a COX inhibitor or an inhibitor of the prostaglandin
biosynthetic pathway and rapamycin for the treatment of LAM.
[0287] In one embodiment, provided herein is a combinatorial use of
at least one a COX inhibitor or an inhibitor of the prostaglandin
biosynthetic pathway and rapamycin for the manufacture of
medicament for treatment of LAM.
[0288] In one embodiment, provided herein is a combinatorial use of
at least one a COXinhibitor or an inhibitor of the prostaglandin
biosynthetic pathway, rapamycin and at least one compound selected
from the group consisting of SCH-202676 hydrobromide, danusertib
(PHA-739358), AZ-960, nicardipine, SB-590885, Thimerosal,
ionomycin, U-73343, PAF C16, BX912, and Chlorambucil for the
treatment of LAM.
[0289] In one embodiment, provided herein is a combinatorial use of
at least one a COX inhibitor or an inhibitor of the prostaglandin
biosynthetic pathway, rapamycin and at least one compound selected
from the group consisting of SCH-202676 hydrobromide, danusertib
(PHA-739358), AZ-960, nicardipine, SB-590885, Thimerosal,
ionomycin, U-73343, PAF C16, BX912, and Chlorambucil for the
manufacture of medicament for the treatment of LAM.
[0290] In one embodiment, provided herein is a combinatorial use of
at least one a cyclooxygenase (COX) inhibitor or an inhibitor of
the prostaglandin biosynthetic pathway and at least one compounds
selected from the group consisting of nateglinide, Z-L-Phe
chloromethyl ketone, clemastine fumarate, supercinnamaldehyde,
practolol, fluvastatin Na, sulindac, 6-bromoindirubin-3'-oxime
(BIO), amorolfine, spectinomycin, sibutramine HCl, nelfinavir
mesylate, moroxydine HCl, nicotine ditartrate, trequinsin,
meglumine, tizanidine HCl, CGP-74514A hydrochloride, tioconazole,
afatinib, kasugamycin, flupentixol, fluphenazine, mephenytoin,
aminoglutethimide, betaxolol hydrochloride, salmeterol,
chelerythrine chloride, paroxetine, trifluoperazine, fluoxetine,
methiothepin, nortriptyline, A-77636, rapamycin, SCH-202676
hydrobromide, danusertib (PHA-739358), AZ-960, nicardipine,
SB-590885, Thimerosal, ionomycin, U-73343, PAF C16, BX912, and
Chlorambucil for the treatment of LAM.
[0291] In one embodiment, provided herein is a combinatorial use of
at least one a COX inhibitor or an inhibitor of the prostaglandin
biosynthetic pathway and at least one compounds selected from the
group consisting of nateglinide, Z-L-Phe chloromethyl ketone,
clemastine fumarate, supercinnamaldehyde, practolol, fluvastatin
Na, sulindac, 6-bromoindirubin-3'-oxime (BIO), amorolfine,
spectinomycin, sibutramine HCl, nelfinavir mesylate, moroxydine
HCl, nicotine ditartrate, trequinsin, meglumine, tizanidine HCl,
CGP-74514A hydrochloride, tioconazole, afatinib, kasugamycin,
flupentixol, fluphenazine, mephenytoin, aminoglutethimide,
betaxolol hydrochloride, salmeterol, chelerythrine chloride,
paroxetine, trifluoperazine, fluoxetine, methiothepin,
nortriptyline, A-77636, rapamycin, SCH-202676 hydrobromide,
danusertib (PHA-739358), AZ-960, nicardipine, SB-590885,
Thimerosal, ionomycin, U-73343, PAF C16, BX912, and Chlorambucil
for the manufacture of medicament for the treatment of LAM.
COX Inhibitors
[0292] The COX-2 inhibitors can include, but are in no way limited
to, NS-398 (available from Cayman Chemical; Ann Arbor, Mich.),
celecoxib (available from Pfizer under the trade name "CELEBREX";
New York, N.Y.), rofecoxib (available from Merck under the trade
name "VIOXX"; Whitehouse Station, N.J.), valdecoxib (available from
Pfizer under the trade name "BEXTRA"), meloxicam (available from
Boebringer Ingelheim under the tradename "MOBICOX"; Burlington,
Ontario) and pharmaceutical equivalents, derivatives and salts, as
well as other functionally related compounds as will be readily
appreciated by those of skill in the art. Guidance as to additional
COX-2 inhibitors is provided in the literature and generally
available to practitioners in the art. See, e.g., U.S. Pat. No.
6,649,645 (describing the use of radiation with COX-2 inhibitors
for the treatment of cancer), which is incorporated by reference
herein in its entirety.
[0293] COX-2 selective inhibitors are well understood from the
patent literature. For example, compounds useful herein and methods
of synthesis are disclosed U.S. Pat. No. 5,861, U.S. Pat. No.
5,859,257 and PCT WO 97/38986. These publications are incorporated
by reference in their entirety. The compounds rofecoxib, MK-663,
celecoxib, valdecoxib and parecoxib have known structures, as well
as known dosages and dosage ranges.
[0294] Alternatively, a nonselective COX inhibitor may be used in
various embodiments described herein in the form of a nonsteriodal
anti-inflammatory drug (NSAID). A NSAID may include, but is in no
way limited to, Retodolac, aspirin, diclofenac, diflunisal,
etodolac, fenoprofen, floctafenine, flurbiprofen, ibuprofen,
indomethacin, ketoprofen, meclofenamate, mefenamic acid, meloxicam,
nabumetone, naproxen, oxaprozin, piroxicam, sunlindac, tenoxicam,
tiaprofenic acid, tolmetin, and pharmaceutical equivalents,
derivatives and salts, as well as other functionally related
compounds, although numerous other NSAIDs may be used, as will be
readily appreciated by those of skill in the art. For example,
guidance as to particular NSAIDS is provided in the literature and
generally available to practitioners in the art. See, e.g., U.S.
Pat. No. 6,761,913 and U.S. Pat. No. 6,759,056, both of which are
incorporated by reference in their entirety. In various embodiments
of the present invention, the COX-2 inhibitor can be formulated in
a pharmaceutical composition.
Prostaglandin Pathway Inhibitors
[0295] In the biosynthesis of prostaglandins (PG), it is known that
arachidonic acid is converted firstly into PGG.sub.2 by the action
of cyclooxygenase, and the PGG.sub.2 in turn is converted into
PGH.sub.2 and further to PGD.sub.2, PGE2, PHI.sub.2, etc.
Therefore, if the first step of this cascade is inhibited, the
biosynthesis of prostaglandins would be entirely inhibited.
Accordingly, the term "prostaglandins" to be referred to in the
phrase as "prostaglandin biosynthesis inhibitors(s)" used herein is
intended to include all prostaglandins which may be biologically
synthesized.
[0296] The term "prostaglandin biosynthesis inhibitors" or
"prostaglandin biosynthetic pathway inhibitors" are used
interchangeably and herein can include, but are in no way limited
to compounds which inhibit the formation of arachidonic acid from
arachidonic acid glyceride (phospholipase inhibitor) and those
which inhibit the formation of prostaglandin from arachidonic acid,
such as via the enzyme prostaglandin endoperoxide synthase (PGHS,
aka COX-2; EC 1.14.99.1). The former includes lipomodulin (also
known as macrocortin, or lipocortin), a kind of protein, and the
latter includes non-steroidal anti-inflammatory substances
(especially acidic ones). Specific examples of the latter are
salicylic acid derivatives such as acetylsalicylic acid, salicyl
salicylic acid, DL-lysine monoacetyl salicylate, etc., pyrazolone
derivatives such as Phenopyrazone, Nifenazone, Phenylbutazone,
Oxyphenbutazone, Ketophenylbutazone, Clofezone, Difenamizole, etc.,
anthranylic acid derivatives such as Mefenamic acid, Fulfenamic
acid, Niflumic acid, etc., phenyl acetic acid derivatives such as
Diclofenac, Ibufenac, Ibuprofen, Alclofenac, Kotoprofen, Fenbufen,
Flurbiprofen, etc., indol or indazole derivatives such as
Indomethacin, Sulindac, Benzydamine, etc., and further Naproxen,
Tiaramide, Bucolome, Metopyrimazole, Azapropazone, etc. and their
salts.
[0297] The prostaglandin biosynthetic pathway inhibitors may
include, but are in no way limited to inhibitors described in U.S.
Pat. Nos. 4,880,742; 5,593,994; 5,932,586; 5,973,191; 6,284,918;
8,337,914; 8,263,139; and 8,486,457, and International PCT Patent
Publication Nos: WO1999/010332; WO2000/024719; and WO2006042625;
these references are are incorporated by reference in their
entirety.
Additional Compounds
[0298] Additional compounds that used in the methods and
compositions described herein include nateglinide, Z-L-Phe
chloromethyl ketone, clemastine fumarate, supercinnamaldehyde,
practolol, fluvastatin Na, sulindac, 6-bromoindirubin-3'-oxime
(BIO), amorolfine, spectinomycin, sibutramine HCl, nelfinavir
mesylate, moroxydine HCl, nicotine ditartrate, trequinsin,
meglumine, tizanidine HCl, CGP-74514A hydrochloride, tioconazole,
afatinib, kasugamycin, flupentixol, fluphenazine, mephenytoin,
aminoglutethimide, betaxolol hydrochloride, salmeterol,
chelerythrine chloride, paroxetine, trifluoperazine, fluoxetine,
methiothepin, nortriptyline, A-77636, Kanamycin, SCH-202676
hydrobromide, danusertib (PHA-739358), AZ-960, nicardipine,
SB-590885, Thimerosal, ionomycin, U-73343, PAF C16, BX912, and
Chlorambucil.
[0299] It is contemplated that one or more of the disclosed
compounds may be used for the treatment of LAM in combination with
at least one COX inhibitor and/or with at least one inhibitor of
the prostaglandin biosynthetic pathway.
[0300] Z-L-PHE chloromethyl ketone (ZPCK) is a ketone containing
compound that has bioactivity in the NFAT and STAT signaling
pathways. Z-L-PHE chloromethyl ketone is also known in the art as
L-Carbobenzyloxyphenylalanyl chloromethyl ketone,
N-Carbobenzoxy-L-phenylalanyl-chloromethyl ketone, or
N-((Benzyloxy)carbonyl)-L-phenylalanine chloromethyl ketone. In one
embodiment, Z-L-PHE chloromethyl ketone has the structure of
Formula I.
##STR00001##
[0301] Clemastine fumarate is an antihistamine and anticholinergic
drug used for the treatment of allergy symptoms, for example,
sneezing, watery eyes, itching, wheezing etc. Clemastine fumarate
is also known in the art as meclastin, AGASTEN.TM., TAVEGIL.TM.,
and TAVEGYL.TM.. Clemastine fumarate can be obtained commercially
from e.g., NOVARTIS.TM. and TOCRIS BIOSCIENCE.TM.. In one
embodiment, clemastine fumarate has the structure of Formula
II.
##STR00002##
[0302] Supercinnamaldehyde is an organic compound derived from
cinnamon and that gives cinnamon its flavor and color. It is used
as a flavoring, a fungicide, an insecticide, an antimicrobial and
an anti-cancer agent. Supercinnamaldehyde is also known in the art
as 1,3-Dihydro-1-methyl-3-(2-oxopropylidene)-2H-Indol-2-one,
cinnamaldehyde, trans-Cinnamaldehyde, Cinnamic aldehyde, Cinnamal,
(E)-Cinnamaldehyde, 3-Phenylacrylaldehyde, Cinnamylaldehyde,
Phenylacrolein, Zimtaldehyde, and Cassia aldehyde, among others. In
one embodiment, supercinnamaldehyde has the structure of Formula
III.
##STR00003##
[0303] Practolol is a selective beta blocker used in the emergency
treatment of cardiac arrhythmias. Practolol is also known in the
art as ERALDIN.TM., DALZIC.TM., PRAKTOL.TM., CARDIOL.TM.,
PRALON.TM., CORDIALINA.TM., ERALDINA.TM., and TERANO.TM.. In one
embodiment, practolol has the formula of Formula IV.
##STR00004##
[0304] Fluvastatin (fluvastatin Na or fluvastatin sodium) is a
member of the statin family of compounds used for the treatment of
hypercholesterolemia and the prevention of heart disease.
Fluvastatin is also known in the art as LESCOL.TM., CANEF.TM., and
VASTIN.TM.. In one embodiment, fluvasatin has the formula of
Formula V.
##STR00005##
[0305] Sulindac is a Non-Steroidal Anti-Inflammatory Drug (NSAID)
used for the treatment of pain and inflammatory conditions, such as
rheumatoid arthritis, osteoarthritis, gout, dysmenorrhea,
metastatic bone pain, fever, headache, muscle stiffness and
migraine, among others. Sulindac can be obtained from MERCK.TM.
under the name CLINORIL.TM.. In one embodiment, sulindac has the
formula of Formula VI.
##STR00006##
[0306] Amorolfine is a morpholine antifungal drug that is used
primarily to treat fungal infections in toenails and fingernails.
Amorolfine is also known as CURANAIL.TM., LOCERYL.TM. LOCETAR.TM.,
and ODENIL.TM.. In one embodiment, amorolfine has the formula of
Formula VII.
##STR00007##
[0307] Spectinomycin is an antibiotic closely related to the
aminoglycoside antibiotics. Spectinomycin has been used as an
injected antibiotic for the treatment of gonorrhea. Spectinomycin
is also known in the art as TROBICIN.TM.. In one embodiment,
spectinomycin has the formula of Formula VIII.
##STR00008##
[0308] Sibutramine HCL or sibutramine is an oral anorexient used
for the treatment of obesity in conjunction with diet and exercise.
Sibutramine is also known in the art as REDUCTIL.TM., MERIDIA.TM.
and SIBUTREX.TM.. In one embodiment, sibutramine has the formula of
Formula IX.
##STR00009##
[0309] Nelfinavir mesylate is a protease inhibitor and
antiretroviral drug useful for treating HIV infection. Nelfinavir
is also known in the art as VIRACEPT.TM.. In one embodiment,
nelfinavir has the formula of Formula X.
##STR00010##
[0310] Moroxydine HCL or moroxydine is a heterocyclic biguanidine
antiviral drug useful for the treatment of influenza. Moroxydine is
also known by the following chemical synonyms: ABOB, VIRONIL.TM.,
moroxydine; moroxydinum, TIMTEC-BB, SBB003847,
1-(1-morpholinoformimidoyl)guanidine,
n-(aminoiminomethyl)-4-morpholinecarboximidamide;
4-morpholinecarboximidamide, and N-(aminoiminomethyl)-;
N4amino(imino)methyl]morpholine-4-carboximidamide hydrochloride. In
one embodiment, moroxydine has the formula of Formula XI.
##STR00011##
[0311] Nicotine ditartrate is a nicotine derivative that exhibits
vasoconstrictive, hypertensive and prothrombotic activity. Nicotine
ditartrate is also known by the following chemical synonyms:
nicotine tartrate, tartratedenicotine, nicotine bitartrate,
nicotine acid tartrate, nicotine,tartrate(1:2), (-)-Nicotine
dirartate, and nicotinehydrogentartrate. In one embodiment,
nicotine ditartrate has the formula of Formula XII.
##STR00012##
[0312] Trequinsin (also known as
9,10-Dimethoxy-2-mesitylimino-3-methyl-2,3,6,7-tetrahydro-4H-pyrimido-[6,-
1a]-isoquinolin-4-one) is a cGMP-inhibited phosphodiesterase III
inhibitor useful in the treatment of short term cardiac failure and
intermittent claudication. In one embodiment, trequinsin has the
formula of Formula XIII
##STR00013##
[0313] Meglumine is a sugar derived from sorbitol and is typically
used as an excipient in pharmaceutical compositions or with
iodinated organic compounds as a contrast medium. Meglumine is also
known in the art as: N-Methyl-D-glucamine, Meglumin,
N-Methylglucamine, 6284-40-8, 1-Deoxy-1-methylaminosorbitol,
Megluminum, Methylglucamin, Meglumina, and
1-Deoxy-1-(methylamino)-D-glucitol. In one embodiment, meglumine
has the formula of Formula XIV.
##STR00014##
[0314] Tizanidine HCl or tizanidine is used as a muscle relaxant in
the treatment of a variety of conditions including, but not limited
to, spasms, cramping, and tightness of muscles caused by medical
problems such as multiple sclerosis, spastic diplegia, back pain,
or injuries to the spine or central nervous system. Tizanidine is
also known in the art as ZANAFLEX.TM., and SIRDALUD.TM. In one
embodiment, tizanidine has the formula of Formula XV.
##STR00015##
[0315] CGP-74514A hydrochloride is also known in the art by the
following chemical synonyms: compound 13 hydrochloride, and
N2-(CIS-2-AMINOCYCLOHEXYL)-N6-(3-CHLOROPHENYL)-9-ETHYL-9H-PURINE-2,6-DIAM-
INE HYDROCHLORIDE. In one embodiment, CGP-74514A hydrochloride has
the formula of Formula XVI.
##STR00016##
[0316] Tioconazole is an antifungal compound useful for treating
vaginal yeast infections, ringworm, jock itch, athlete's foot, and
tinea versicolor. Tioconazole is also known in the art as
TROSYD.TM., and GYNO-TROSYD.TM., both of which can be obtained
commercially from PFIZER.TM.. In one embodiment, tioconazole has
the formula of Formula XVII.
##STR00017##
[0317] TOVOK.TM. (afatinib) is a next generation tyrosine kinase
inhibitor and an anti-cancer compound that may be useful in the
treatment of non-small cell lung carcinoma, breast cancer, prostate
cancer, glioma, and head and neck cancer. TOVOK.TM. is also known
in the art as TOMTOVOK.TM. and can be obtained commercially from
BOEHRINGER INGELHEIM.TM.. In one embodiment, TOVOK.TM. has the
formula of Formula XVIII.
##STR00018##
[0318] Kasugamycin is an aminoglycoside antibiotic also known as
kasumin,
3-042-Amino-4-[(carboxyiminomethyl)amino]-2,3,4,6-tetradeoxy-D-arabino-he-
xopyranosyl]-D-chiro-inositol, and 2-amino-2-[(2R,3
S,5S,6R)-5-amino-2-methyl-6-[(2R,3 S, 5
S,6S)-2,3,4,5,6-pentahydroxycyclohexyl]oxyoxan-3-yl]iminoacetic
acid. In one embodiment, kasugamycin has the formula of Formula
XIX.
##STR00019##
[0319] Nateglinide is a meglitinide compound used for reducing
blood glucose levels in the treatment of Type II diabetes.
Nateglinide is also known in the art as STARLIX.TM. and can be
obtained commercially from NOVARTIS.TM.. In one embodiment,
nateglinide has the structure of Formula XX.
##STR00020##
[0320] Methods for synthesizing the foregoing compounds are known
in the art. Moreover, the foregoing compounds are commercially
available, e.g. clemastine fumarate is available from NOVARTIS.TM.
as MECLASTIN.TM..
[0321] It is also contemplated that the methods described herein
can be used as prophylaxis. Since subjects with TSC are prone to
developing tumors in various organs, administration of the
inhibitors or compositions can help prevent tumor formation and
thereby reduce the frequency of these tumors in such
individuals.
[0322] A skilled physician will be able to diagnose LAM and/or
LAM/TSC, for example, based on known clinical symptoms, lung tissue
biopsy and genetic analysis of the TSC loci in the afflicted
subject.
[0323] In one embodiment, provided herein is a composition
comprising at least one COX inhibitor and/or a prostaglandin
biosynthesis pathway inhibitor and at least one compound (e.g., 2,
3, 4, 5, 6, 7, or more) selected from the group consisting of
nateglinide, Z-L-Phe chloroethyl ketone, clemastine fumarate,
supercinnamaldehyde, practolol, fluvastatin Na, sulindac,
6-bromoindirubin-3'-oxime (BIO), amorolfine, spectinomycin,
sibutramine HCl, nelfinavir mesylate, moroxydine HCl, nicotine
ditartrate, trequinsin, meglumine, tizanidine HCl, CGP-7451A
hydrochloride, tioconazole, TOVOK.TM. (afatinib), kasugamycin,
flupentixol, fluphenazine, mephenytoin, aminoglutethimide,
betaxolol hydrochloride, salmeterol, chelerythrine chloride,
paroxetine, trifluoperazine, fluoxetine, methiothepin,
nortriptyline, A-77636, Kanamycin, SCH-202676 hydrobromide,
danusertib (PHA-739358), AZ-960, nicardipine, SB-590885,
Thimerosal, ionomycin, U-73343, PAF C16, BX912, and Chlorambucil
for use in any of the methods described herein, e.g., treatment of
LAM and/or LAM/TSC, prevention of tumor formation, reducing the
frequency of tumor development, inducing apoptosis in a cell,
killing a cell and inhibiting cell growth.
[0324] In one embodiment, the method for treating LAM in a subject
comprises administering to a subject in need thereof a
therapeutically effective amount of any one of the composition
described herein. For example, a composition comprising at least
one COX inhibitor and/or a prostaglandin biosynthesis pathway
inhibitor and at least compound selected from the group consisting
of nateglinide, Z-L-Phe chloroethyl ketone, clemastine fumarate,
supercinnamaldehyde, practolol, fluvastatin Na, sulindac,
6-bromoindirubin-3'-oxime (BIO), amorolfine, spectinomycin,
sibutramine HCl, nelfinavir mesylate, moroxydine HCl, nicotine
ditartrate, trequinsin, meglumine, tizanidine HCl, CGP-7451A
hydrochloride, tioconazole, TOVOK.TM. (afatinib), and kasugamycin,
flupentixol, fluphenazine, mephenytoin, aminoglutethimide,
betaxolol hydrochloride, salmeterol, chelerythrine chloride,
paroxetine, trifluoperazine, fluoxetine, methiothepin,
nortriptyline, A-77636, Kanamycin, SCH-202676 hydrobromide,
danusertib (PHA-739358), AZ-960, nicardipine, SB-590885,
Thimerosal, ionomycin, U-73343, PAF C16, BX912, and
Chlorambucil.
[0325] In one embodiment, provided herein is a method for treating
LAM in a subject, the method comprising determining whether the
cancer cells of the subject or the subject comprises one or more of
the following: (a) a COX overexpression; (b) a mutation in the TSC
locus; (c) increased prostaglandin production; (d) absence of mTOR
deregulation or hyperactivity ie., normal mTOR regulation or
activity; and (e) at least one cancer cell that is insensitive to
rapamycin; and if any is affirmative or positive, administering to
the subject a therapeutically effective amount of a composition
comprising at least one of the composition described herein. For
example, a composition comprising at least one COX inhibitor and/or
a prostaglandin biosynthesis pathway inhibitor and at least
compound selected from the group consisting of nateglinide, Z-L-Phe
chloroethyl ketone, clemastine fumarate, supercinnamaldehyde,
practolol, fluvastatin Na, sulindac, 6-bromoindirubin-3'-oxime
(BIO), amorolfine, spectinomycin, sibutramine HCl, nelfinavir
mesylate, moroxydine HCl, nicotine ditartrate, trequinsin,
meglumine, tizanidine HCl, CGP-7451A hydrochloride, tioconazole,
TOVOK.TM. (afatinib), kasugamycin, flupentixol, fluphenazine,
mephenytoin, aminoglutethimide, betaxolol hydrochloride,
salmeterol, chelerythrine chloride, paroxetine, trifluoperazine,
fluoxetine, methiothepin, nortriptyline, A-77636, Kanamycin,
SCH-202676 hydrobromide, danusertib (PHA-739358), AZ-960,
nicardipine, SB-590885, Thimerosal, ionomycin, U-73343, PAF C16,
BX912, and Chlorambucil. For example, a composition comprising at
least one COX inhibitor and/or a prostaglandin biosynthesis pathway
inhibitor and rapamycing and at least and at least one compound
selected from the group consisting of SCH-202676 hydrobromide,
danusertib (PHA-739358), AZ-960, nicardipine, SB-590885,
Thimerosal, ionomycin, U-73343, PAF C16, BX912, and
Chlorambucil.
[0326] In some embodiments, a combination of more than one
compositions described herein can be administered for the treatment
of LAM. In one embodiment relating to combination therapy with more
than one compositions, the compositions are administered together,
e.g., in a cocktail, admixture or as a single pharmaceutical
composition.
[0327] In one embodiment of any method or composition described
herein, the COX inhibitiors, the prostaglandin biosynthesis pathway
inhibitors and compound(s) are administered by a route selected
from the group consisting of aerosol, direct injection, local,
systemic, intradermal, direct inhalation, intravitreal,
intramuscular, intraperitoneal, intravenous, intrathecal,
intrapleural, intrauterine, subcutaneous, epidural, topical, oral,
transmucosal, buccal, rectal, vaginal, transdermal, intranasal,
intrasynovial, intraocular/periocular, intraorgan, intratumor and
parenteral route.
[0328] In one embodiment of any method or composition described,
only one compound selected from the group consisting of
nateglinide, Z-L-Phe chloroethyl ketone, clemastine fumarate,
supercinnamaldehyde, practolol, fluvastatin Na, sulindac,
6-bromoindirubin-3'-oxime (BIO), amorolfine, spectinomycin,
sibutramine HCl, nelfinavir mesylate, moroxydine HCl, nicotine
ditartrate, trequinsin, meglumine, tizanidine HCl, CGP-7451A
hydrochloride, tioconazole, TOVOK.TM. (afatinib), kasugamycin,
flupentixol, fluphenazine, mephenytoin, aminoglutethimide,
betaxolol hydrochloride, salmeterol, chelerythrine chloride,
paroxetine, trifluoperazine, fluoxetine, methiothepin,
nortriptyline, A-77636, Kanamycin, SCH-202676 hydrobromide,
danusertib (PHA-739358), AZ-960, nicardipine, SB-590885,
Thimerosal, ionomycin, U-73343, PAF C16, BX912, and Chlorambucil
are administered. In other embodiments, at least two compounds, at
least three compounds, or more compounds selected from the group
consisting of nateglinide, Z-L-Phe chloroethyl ketone, clemastine
fumarate, supercinnamaldehyde, practolol, fluvastatin Na, sulindac,
6-bromoindirubin-3'-oxime (BIO), amorolfine, spectinomycin,
sibutramine HCl, nelfinavir mesylate, moroxydine HCl, nicotine
ditartrate, trequinsin, meglumine, tizanidine HCl, CGP-7451A
hydrochloride, tioconazole, TOVOK.TM. (afatinib), kasugamycin,
flupentixol, fluphenazine, mephenytoin, aminoglutethimide,
betaxolol hydrochloride, salmeterol, chelerythrine chloride,
paroxetine, trifluoperazine, fluoxetine, methiothepin,
nortriptyline, A-77636, Kanamycin, SCH-202676 hydrobromide,
danusertib (PHA-739358), AZ-960, nicardipine, SB-590885,
Thimerosal, ionomycin, U-73343, PAF C16, BX912, and Chlorambucilare
administered. For example, only practolol and fluvastatin Na are
administered. All possible two compound combinations derived from
the group consisting of nateglinide, Z-L-Phe chloroethyl ketone,
clemastine fumarate, supercinnamaldehyde, practolol, fluvastatin
Na, sulindac, 6-bromoindirubin-3'-oxime (BIO), amorolfine,
spectinomycin, sibutramine HCl, nelfinavir mesylate, moroxydine
HCl, nicotine ditartrate, trequinsin, meglumine, tizanidine HCl,
CGP-7451A hydrochloride, tioconazole, TOVOK.TM. (afatinib),
kasugamycin, flupentixol, fluphenazine, mephenytoin,
aminoglutethimide, betaxolol hydrochloride, salmeterol,
chelerythrine chloride, paroxetine, trifluoperazine, fluoxetine,
methiothepin, nortriptyline, A-77636, Kanamycin, SCH-202676
hydrobromide, danusertib (PHA-739358), AZ-960, nicardipine,
SB-590885, Thimerosal, ionomycin, U-73343, PAF C16, BX912, and
Chlorambucil are contemplated herein for use as a combination
therapy. All possible three compound combinations derived from the
group consisting of nateglinide, Z-L-Phe chloroethyl ketone,
clemastine fumarate, supercinnamaldehyde, practolol, fluvastatin
Na, sulindac, 6-bromoindirubin-3'-oxime (BIO), amorolfine,
spectinomycin, sibutramine HCl, nelfinavir mesylate, moroxydine
HCl, nicotine ditartrate, trequinsin, meglumine, tizanidine HCl,
CGP-7451A hydrochloride, tioconazole, TOVOK.TM. (afatinib),
kasugamycin, flupentixol, fluphenazine, mephenytoin,
aminoglutethimide, betaxolol hydrochloride, salmeterol,
chelerythrine chloride, paroxetine, trifluoperazine, fluoxetine,
methiothepin, nortriptyline, A-77636, Kanamycin, SCH-202676
hydrobromide, danusertib (PHA-739358), AZ-960, nicardipine,
SB-590885, Thimerosal, ionomycin, U-73343, PAF C16, BX912, and
chlorambucil are contemplated herein for use as a combination
therapy.
[0329] In one embodiment of any method or composition described
herein, the COX inhibitor and/or a prostaglandin biosynthesis
pathway inhibitor or composition is administered in conjunction
with at least one additional therapy to achieve a combination
therapy.
[0330] In one embodiment of any method or composition described
herein, the COX inhibitor and/or a prostaglandin biosynthesis
pathway inhibitor or composition is further administered with a
pharmaceutically acceptable carrier.
[0331] In one embodiment of any method or composition described,
the at least one additional therapy is a therapy that help the
subject cope with cancer treatment side effects. For example,
aromatherapy, exercise, hypnosis, massage, meditation, tai chi,
yoga, acupuncture, music therapy and relaxation techniques.
[0332] In one embodiment of any method or composition described
herein, the at least one additional cancer therapy is selected from
therapies such as one or more anti-cancer therapeutic agents
selected from the group consisting of growth inhibitory agents,
cytotoxic agents, anti-angiogenesis agents, apoptotic agents,
anti-tubulin agents, anti-HER-2 antibodies, anti-CD20 antibodies,
an epidermal growth factor receptor (EGFR) antagonist, a HER1/EGFR
inhibitor, a platelet derived growth factor inhibitor, a COX-2
inhibitor, an interferon, and a cytokine (e.g., G-CSF,
granulocyte-colony stimulating factor).
[0333] In one embodiment of any method or composition described,
the at least one additional therapy is a cancer therapy.
Non-limiting examples of anti-cancer therapeutic agents are
13-cis-retinoic acid, 2-CdA, 2-Chlorodeoxyadenosine, 5-Azacitidine,
azacytidine, 5-Fluorouracil, 5-FU, 6-Mercaptopurine, 6-MP, 6-TG,
6-Thioguanine, abiraterone acetate, Abraxane, Accutane
Actinomycin-D, Adriamycin Adrucil Afinitor Agrylin Ala-Cort
Aldesleukin, Alemtuzumab, ALIMTA, Alitretinoin, Alkaban-AQ Alkeran
All-transretinoic Acid, Alpha Interferon, Altretamine,
Amethopterin, Amifostine, Aminoglutethimide, Anagrelide, Anandron
Anastrozole, Arabinosylcytosine, Ara-C, Aranesp Aredia Arimidex
Aromasin Arranon.RTM., Arsenic Trioxide, Arzerra.TM., Asparaginase,
ATRA, Avastin Axitinib, Azacitidine, BCG, BCNU, Bendamustine,
Bevacizumab, Bexarotene, BEXXAR Bicalutamide, BiCNU, Blenoxane
Bleomycin, Bortezomib, Busulfan, Busulfex.RTM., C225, Cabazitaxel,
Calcium Leucovorin, Campath.RTM. Camptosar.RTM. Camptothecin-11,
Capecitabine, Caprelsa.RTM. Carac.TM. Carboplatin, Carmustine,
Carmustine Wafer, Casodex CC-5013, CCI-779, CCNU, CDDP, CeeNU,
Cerubidine Cetuximab, Chlorambucil, Cisplatin, Citrovorum Factor,
Cladribine, Cortisone, Cosmegen CPT-11, Crizotinib,
Cyclophosphamide, Cytadren Cytarabine, Cytarabine Liposomal,
Cytosar-U Cytoxan Dacarbazine, Dacogen, Dactinomycin, Darbepoetin
Alfa, Dasatinib, Daunomycin, Daunorubicin, Daunorubicin
Hydrochloride, Daunorubicin Liposomal, DaunoXome Decadron,
Decitabine, Delta-Cortef Deltasone.RTM., Denileukin Diftitox,
Denosumab, DepoCyt.TM., Dexamethasone, Dexamethasone Acetate,
Dexamethasone Sodium Phosphate, Dexasone, Dexrazoxane, DHAD, DIC,
Diodex, Docetaxel, Doxil Doxorubicin, Doxorubicin Liposomal,
Droxia.TM., DTIC, DTIC-Dome Duralone Eculizumab, Efudex
Eligard.TM., Ellence.TM., Eloxatin.TM., Elspar Emcyt Epirubicin,
Epoetin Alpha, Erbitux, Eribulin, Erlotinib, Erwinia
L-asparaginase, Estramustine, Ethyol, Etopophos Etoposide,
Etoposide Phosphate, Eulexin Everolimus, Evista Exemestane,
Fareston Faslodex Femara.RTM., Filgrastim, Floxuridine,
Fludara.RTM., Fludarabine, Fluoroplex Fluorouracil, Fluorouracil
(cream), Fluoxymesterone, Flutamide, Folinic Acid, FUDR
Fulvestrant, Gefitinib, Gemcitabine, Gemtuzumab ozogamicin, Gemzar,
Gleevec.TM., Gliadel.RTM. Wafer, Goserelin, Granulocyte-Colony
Stimulating Factor (G-CSF), Granulocyte Macrophage Colony
Stimulating Factor (GM-CSF), Halaven Halotestin Herceptin Hexadrol,
Hexalen Hexamethylmelamine, HMM, Hycamtin Hydrea Hydrocort
Acetate.RTM., Hydrocortisone, Hydrocortisone Sodium Phosphate,
Hydrocortisone Sodium Succinate, Hydrocortone Phosphate,
Hydroxyurea, Ibritumomab, Ibritumomab Tiuxetan, Idamycin.RTM.,
Idarubicin, Ifex.RTM., IFN-alpha, Ifosfamide, IL-11, IL-2, Imatinib
mesylate, Imidazole Carboxamide, Inlyta.RTM., Interferon alpha,
Interferon Alpha-2b (PEG Conjugate), Interleukin-2, Interleukin-11,
Intron A.RTM. (interferon alpha-2b), Ipilimumab, Iressa.RTM.,
Irinotecan, Isotretinoin, Ixabepilone, Ixempra.TM., Jevtana.RTM.,
Kidrolase (t), Lanacort.RTM., Lapatinib, L-asparaginase, LCR,
Lenalidomide, Letrozole, Leucovorin, Leukeran, Leukine.TM.,
Leuprolide, Leurocristine, Leustatin.TM., Liposomal Ara-C, Liquid
Pred.RTM., Lomustine, L-PAM, L-Sarcolysin, Lupron.RTM., Lupron
Depot.RTM., Matulane.RTM., Maxidex, Mechlorethamine,
Mechlorethamine Hydrochloride, Medralone.RTM., Medrol.RTM.,
Megace.RTM., Megestrol, Megestrol Acetate, Melphalan,
Mercaptopurine, Mesna, Mesnex.TM., Methotrexate, Methotrexate
Sodium, Methylprednisolone, Meticorten.RTM., Mitomycin,
Mitomycin-C, Mitoxantrone, M-Prednisol.RTM., MTC, MTX,
Mustargen.RTM., Mustine, Mutamycin.RTM., Myleran.RTM., Mylocel.TM.,
Mylotarg.RTM., Navelbine.RTM., Nelarabine, Neosar.RTM.,
Neulasta.TM., Neumega.RTM., Neupogen.RTM., Nexavar.RTM.,
Nilandron.RTM., Nilotinib, Nilutamide, Nipent.RTM., Nitrogen
Mustard, Novaldex.RTM., Novantrone.RTM., Nplate, Octreotide,
Octreotide acetate, Ofatumumab, Oncospar.RTM., Oncovin.RTM.,
Ontak.RTM., Onxal.TM., Oprelvekin, Orapred.RTM., Orasone.RTM.,
Oxaliplatin, Paclitaxel, Paclitaxel Protein-bound, Pamidronate,
Panitumumab, Panretin.RTM., Paraplatin.RTM., Pazopanib,
Pediapred.RTM., PEG Interferon, Pegaspargase, Pegfilgrastim,
PEG-INTRON.TM., PEG-L-asparaginase, PEMETREXED, Pentostatin,
Phenylalanine Mustard, Platinol.RTM., Platinol-AQ.RTM.,
Prednisolone, Prednisone, Prelone.RTM., Procarbazine, PROCRIT.RTM.,
Proleukin.RTM., Prolia.RTM., Prolifeprospan 20 with Carmustine
Implant, Provenge.RTM., Purinethol.RTM., Raloxifene, Revlimid.RTM.,
Rheumatrex.RTM., Rituxan.RTM., Rituximab, Roferon-A.RTM.
(Interferon Alfa-2a), Romiplostim, Rubex.RTM., Rubidomycin
hydrochloride, Sandostatin.RTM., Sandostatin LAR.RTM.,
Sargramostim, Sipuleucel-T, Soliris.RTM., Solu-Cortef.RTM.,
Solu-Medrol.RTM., Sorafenib, SPRYCEL.TM., STI-571, Streptozocin,
SU11248, Sunitinib, Sutent.RTM., Tamoxifen, Tarceva.RTM.,
Targretin.RTM., Tasigna.RTM., Taxol.RTM., Taxotere.RTM.,
Temodar.RTM., Temozolomide, Temsirolimus, Teniposide, TESPA,
Thalidomide, Thalomid TheraCys Thioguanine, Thioguanine
Tabloid.RTM., Thiophosphoamide, Thioplex Thiotepa, TICE.RTM.,
Toposar Topotecan, Toremifene, Torisel Tositumomab, Trastuzumab,
Treanda Tretinoin, Trexall.TM., Trisenox TSPA, TYKERB Valrubicin,
Valstar, vandetanib, VCR, Vectibix.TM., Velban Velcade Vemurafenib,
VePesid Vesanoid Viadur.TM., Vidaza Vinblastine, Vinblastine
Sulfate, Vincasar Pfs Vincristine, Vinorelbine, Vinorelbine
tartrate, VLB, VM-26, Vorinostat, Votrient, VP-16, Vumon Xalkori
capsules, Xeloda Xgeva.RTM., Yervoy.RTM., Zanosar Zelboraf,
Zevalin.TM., Zinecard Zoladex Zoledronic acid, Zolinza,
Zometa.RTM., and Zytiga.RTM..
[0334] In one embodiment of any method or composition described
herein, the at least one additional cancer therapy is selected from
the group consisting of radiation therapy, chemotherapy,
immunotherapy and gene therapy.
[0335] In one embodiment of any method described herein, the method
further comprises administering a drug that treats at least one
symptom of cancer or cancer therapy. For example, for low blood
count or anemia resulting from the chemo- or radiation therapy,
erythropoietin can be administered to promote de novo the
production of blood cells.
[0336] In one embodiment of any method described, each compound is
administered singly, i.e. each compound is administered
independently of the others. In another embodiment of any method
described, the compounds are administered singly and
simultaneously. In another embodiment, the compounds are
administered together, e.g., in a cocktail or a pharmaceutical
composition.
[0337] In one embodiment of any composition described, the
composition is formulated for administration by a route selected
from the group consisting of: aerosol, direct injection, local,
systemic, intradermal, direct inhalation, intranasal, intravenous,
intravitreal, intramuscular, subcutaneous, intradermal,
transdermal, topical, oral, intraperitoneal, intrathecal,
intrapleural, intrauterine, transmucosal, buccal, rectal, epidural,
vaginal, intrasynovial, intraocular/periocular, intraorgan,
intratumor, and parenteral administration.
[0338] In one embodiment of any method described herein, the method
further comprises selecting a subject who has LAM or has been
diagnosed with LAM. The subject can be further diagnosed with a
mutation in the TSC locus. The subject can be screened for LAM with
a combination with diagnostics such as, for example, additional
biomarkers, mammography, manual examination, MM, or tissue biopsy
and histopathological examination. A skilled oncologist or
physician will be able to differentially diagnose cancer using
medical diagnostic methods known within the art.
Formulation and Application
[0339] In one embodiment, the compounds or combination of compounds
are delivered with a pharmaceutically acceptable carrier.
[0340] In one embodiment, the term "pharmaceutically acceptable"
means approved by a regulatory agency of the Federal or a state
government or listed in the U.S. Pharmacopeia or other generally
recognized pharmacopeia for use in animals, and more particularly
in humans. Specifically, it refers to those compounds, materials,
compositions, and/or dosage forms which are, within the scope of
sound medical judgment, suitable for use in contact with the
tissues of human beings and animals without excessive toxicity,
irritation, allergic response, or other problem or complication,
commensurate with a reasonable benefit/risk ratio.
[0341] The term "carrier" refers to a diluent, adjuvant, excipient,
or vehicle with which the therapeutic is administered. Such
pharmaceutical carriers can be sterile liquids, such as water and
oils, including those of petroleum, animal, vegetable or synthetic
origin, such as peanut oil, soybean oil, mineral oil, sesame oil
and the like. Water is a preferred carrier when the pharmaceutical
composition is administered intravenously. Saline solutions and
aqueous dextrose and glycerol solutions can also be employed as
liquid carriers, particularly for injectable solutions. Suitable
pharmaceutical excipients include starch, glucose, lactose,
sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium
stearate, glycerol monostearate, talc, sodium chloride, dried skim
milk, glycerol, propylene, glycol, water, ethanol and the like. The
composition, if desired, can also contain minor amounts of wetting
or emulsifying agents, or pH buffering agents. These compositions
can take the form of solutions, suspensions, emulsion, tablets,
pills, capsules, powders, sustained-release formulations, and the
like. The composition can be formulated as a suppository, with
traditional binders and carriers such as triglycerides. Oral
formulation can include standard carriers such as pharmaceutical
grades of mannitol, lactose, starch, magnesium stearate, sodium
saccharine, cellulose, magnesium carbonate, etc. Examples of
suitable pharmaceutical carriers are described in Remington's
Pharmaceutical Sciences, 18th Ed., Gennaro, ed. (Mack Publishing
Co., 1990). The formulation should suit the mode of administration.
Additional carrier agents, such as liposomes, can be added to the
pharmaceutically acceptable carrier.
[0342] Therapeutic compositions contain a physiologically tolerable
carrier together with at least a compound or combination of
compounds as described herein, dissolved or dispersed therein as an
active ingredient. In one embodiment, the therapeutic composition
is not immunogenic when administered to a mammal or human patient
for therapeutic purposes. As used herein, the terms
"pharmaceutically acceptable", "physiologically tolerable" and
grammatical variations thereof, as they refer to compositions,
carriers, diluents and reagents, are used interchangeably and
represent that the materials are capable of administration to or
upon a mammal without the production of undesirable physiological
effects such as nausea, dizziness, gastric upset and the like. A
pharmaceutically acceptable carrier will not promote the raising of
an immune response to an agent with which it is admixed, unless so
desired. The preparation of a pharmacological composition that
contains active ingredients dissolved or dispersed therein is well
understood in the art and need not be limited based on formulation.
Compositions can be prepared as injectable either as liquid
solutions or suspensions, however, solid forms suitable for
solution, or suspensions; in liquid prior to use can also be
prepared. The preparation can also be emulsified or presented as a
liposome composition. The compounds or combination of compounds can
also be conjugated with lipids, e.g., amphipathic lipids, for
stability and delivery purposes. The conjugation bonds are
reversible and are broken or dissolved when the compounds or
combination of compounds are delivered to target destination.
Alternatively, the compounds or combination of compounds described
herein can be prepared as a solid or semi-solid or emulsion in
suppository, e.g., as microspheres. The microspheres can be
inserted as a solid into or targeted to a solid tumor. The
compounds or combination of compounds described herein can be mixed
with excipients which are pharmaceutically acceptable and
compatible with the active ingredient and in amounts suitable for
use in the therapeutic methods described herein. Specifically
contemplated pharmaceutical compositions are compounds or
combination of compounds in a preparation for delivery as described
herein above, or in references cited and incorporated herein in
that section. Suitable excipients include, for example, water,
saline, dextrose, glycerol, ethanol or the like and combinations
thereof. In addition, if desired, the composition can contain minor
amounts of auxiliary substances such as wetting or emulsifying
agents, pH buffering agents and the like which enhance the
effectiveness of the active ingredient. The therapeutic composition
comprising the compounds or combination of compounds described
herein can include pharmaceutically acceptable salts of the
components therein. Pharmaceutically acceptable salts include the
acid addition salts (formed with the free amino groups of the
polypeptide) that are formed with inorganic acids such as, for
example, hydrochloric or phosphoric acids, or such organic acids as
acetic, tartaric, mandelic and the like. Salts formed with the free
carboxyl groups can also be derived from inorganic bases such as,
for example, sodium, potassium, ammonium, calcium or ferric
hydroxides, and such organic bases as isopropylamine,
trimethylamine, 2-ethylamino ethanol, histidine, procaine and the
like. Physiologically tolerable carriers are well known in the art.
Exemplary liquid carriers are sterile aqueous solutions that
contain no materials in addition to the active ingredients and
water, or contain a buffer such as sodium phosphate at
physiological pH value, physiological saline or both, such as
phosphate-buffered saline. Still further, aqueous carriers can
contain more than one buffer salt, as well as salts such as sodium
and potassium chlorides, dextrose, polyethylene glycol and other
solutes. Liquid compositions can also contain liquid phases in
addition to and to the exclusion of water. Exemplary of such
additional liquid phases are glycerin, vegetable oils such as
cottonseed oil, and water-oil emulsions. The amount of compounds or
combination of compounds or composition used in the methods
described herein that will be effective in the treatment of a
particular disorder or condition will depend on the nature of the
disorder or condition, and can be determined by standard clinical
techniques.
[0343] The therapeutic compositions or pharmaceutical compositions
described herein can be formulated for passage through the
blood-brain barrier or direct contact with the endothelium. In some
embodiments, the compositions can be formulated for systemic
delivery. In some embodiments, the compositions can be formulated
for delivery to specific organs, for example but not limited to the
liver, spleen, the bone marrow, and the skin. The compositions or
pharmaceutical compositions can also be formulated for aerosol
application by inhalation into the lung. Alternatively, the
therapeutic compositions or pharmaceutical compositions can also be
formulated for a transdermal delivery, e. g. via a skin patch.
Therapeutic compositions or pharmaceutical compositions can be
enteric coated and formulated for oral delivery. Alternatively, the
compositions or pharmaceutical compositions can be encapsulated in
liposomes or nanoparticles and formulated for slow sustained
delivery in vivo. Alternatively, the therapeutic compositions or
pharmaceutical compositions is be formulated for targeted delivery,
eg., encapsulated in liposomes or nanoparticles that are designed
and feature targeting moiety to on the liposomes or
nanoparticles.
[0344] The therapeutic compositions or pharmaceutical compositions
described herein can be administered by any known route. By way of
example, the therapeutic compositions or pharmaceutical
compositions described herein can be administered by a mucosal,
pulmonary, topical, or other localized or systemic route (e.g.,
enteral and parenteral). The compositions may be administered by
any convenient route, for example by infusion or bolus injection,
by absorption through epithelial or mucocutaneous linings (e.g.,
oral mucosa, rectal and intestinal mucosa, etc.) and may be
administered together with other active agents.
[0345] Routes of administration include, but are not limited to
aerosol, direct injection, intradermal, transdermal (e.g., in slow
release polymers), intravitreal, intramuscular, intraperitoneal,
intravenous, subcutaneous, epidural, topical, oral, transmucosal,
buccal, rectal, vaginal, transdermal, intranasal and parenteral
routes.
[0346] "Parenteral" refers to a route of administration that is
generally associated with injection, including but not limited to
intraorbital/periocular, infusion, intraarterial, intracapsular,
intraocular, intracardiac, intraorgan, intradermal, intrahepatic,
intrarogan, intramuscular, intraperitoneal, intrapulmonary,
intratumor, intraspinal, intrasternal, intrathecal, intrauterine,
intravenous, subarachnoid, subcapsular, intrasynovial,
subcutaneous, transmucosal, or transtracheal. Any other
therapeutically efficacious route of administration can be used,
for example, infusion or bolus injection, absorption through
epithelial or mucocutaneous linings. In various embodiments,
administration can be inhaled in to the lung via aerosol
administration, e.g. with nebulization. Administration also can be
systemic or local, for example, intratumoral delivery is also
included.
[0347] In some embodiments, the one or more compounds used for
treatment or the therapeutic compositions or pharmaceutical
compositions described herein are administering via a nebulizer.
For example, the agent can be formulated as a powder for delivery
via a nebulizer.
[0348] For example, the one or more compounds used for treatment or
the therapeutic compositions or pharmaceutical compositions
described herein are formulated for delivery by nebulizer. Such
formulations are known in the art. For examples, nebulizer
formulations are described in O'Riordan TG. et al., 2002, Respir
Care. 47:1305-12; U.S. Patent publication US20070207091; and U.S.
Pat. No. 7,405,207; the contents of which are incorporated by
reference in their entirety. Other dary inhalation formulation are
known in the art, for example, in U.S. Pat. Nos. 5,983,956,
6,027,714, and 8,071,127, and U.S. Patent publication
US2003/0148925 and US2011/0236492, the contents of which are
incorporated by reference in their entirety.
[0349] The precise dose and formulation to be employed depends upon
the potency of the compounds or combination of compounds described
herein, and depends on the amounts large enough to produce the
desired effect, e.g., a reduction in size and/or growth of the
tumors in the subject. The dosage should not be so large as to
cause unacceptable adverse side effects. Generally, the dosage will
vary with the compounds or combination of compounds, and with the
age, condition, and size of the tumors in the subject are also
considered. Dosage and formulation of the compounds or combination
of compounds will also depend on the route of administration, and
the mass and number of tumors in the subject, and should be decided
according to the judgment of the practitioner and each subject's
circumstances. Effective doses can be extrapolated from
dose-response curves derived from in vitro or animal model test
systems.
[0350] The dosage can be determined by one of skill in the art and
can also be adjusted by the individual physician in the event of
any complication. Typically, the dosage ranges from 0.001 mg/kg
body weight to 5 g/kg body weight. In some embodiments, the dosage
range is from 0.001 mg/kg body weight to 1 g/kg body weight, from
0.001 mg/kg body weight to 0.5 g/kg body weight, from 0.001 mg/kg
body weight to 0.1 g/kg body weight, from 0.001 mg/kg body weight
to 50 mg/kg body weight, from 0.001 mg/kg body weight to 25 mg/kg
body weight, from 0.001 mg/kg body weight to 10 mg/kg body weight,
from 0.001 mg/kg body weight to 5 mg/kg body weight, from 0.001
mg/kg body weight to 1 mg/kg body weight, from 0.001 mg/kg body
weight to 0.1 mg/kg body weight, from 0.001 mg/kg body weight to
0.005 mg/kg body weight. Alternatively, in some embodiments the
dosage range is from 0.1 g/kg body weight to 5 g/kg body weight,
from 0.5 g/kg body weight to 5 g/kg body weight, from 1 g/kg body
weight to 5 g/kg body weight, from 1.5 g/kg body weight to 5 g/kg
body weight, from 2 g/kg body weight to 5 g/kg body weight, from
2.5 g/kg body weight to 5 g/kg body weight, from 3 g/kg body weight
to 5 g/kg body weight, from 3.5 g/kg body weight to 5 g/kg body
weight, from 4 g/kg body weight to 5 g/kg body weight, from 4.5
g/kg body weight to 5 g/kg body weight, from 4.8 g/kg body weight
to 5 g/kg body weight. In one embodiment, the dose range is from 5
g/kg body weight to 30 g/kg body weight. Alternatively, the dose
range will be titrated to maintain serum levels between 5 g/mL and
30 g/mL.
[0351] Administration of the doses recited above can be repeated
for a limited period of time. In some embodiments, the doses are
given once a day, or multiple times a day, for example but not
limited to three times a day. In one embodiment, the doses recited
above are administered daily for several weeks or months. The
duration of treatment depends upon the subject's clinical progress
and responsiveness to therapy, e.g., shrinkage of tumor sizes.
Continuous, relatively low maintenance doses are contemplated after
an initial higher therapeutic dose. As but one example, the
compounds or combination of compounds and a pharmaceutically
acceptable carrier can be formulated for direct application by
injection into the tumor in the subject.
[0352] Efficacy testing can be performed during the course of
treatment using the methods described herein, e.g., ultrasound, MRI
and CT to monitor the shrinkage in size of the tumors in the
treated subject. A decrease in size of the tumors during and after
treatment indicates that the treatment is effective in reducing
tumor size. Measurements of the degree of severity of a number of
symptoms associated with cancerous tumors are also noted prior to
the start of a treatment and then at later specific time period(s)
after the start of the treatment. A skilled physician will be able
to ascertain the tumor sizes and related symptoms by known methods
in the art and those described herein.
[0353] Those skilled in the art will recognize, or be able to
ascertain using not more than routine experimentation, many
equivalents to the specific embodiments of the disclosure described
herein. Such equivalents are intended to be encompassed by the
following claims.
[0354] The references cited herein and throughout the specification
are incorporated herein by reference.
[0355] Some embodiments of the technology described herein can be
defined according to any of the following numbered paragraphs:
[0356] [1] A composition comprising at least one a cyclooxygenase
(COX) inhibitor and/or an inhibitor of the prostaglandin
biosynthetic pathway for the treatment of lymphangioleiomyomatosis
(LAM). [0357] [2] A composition comprising at least one a
cyclooxygenase (COX) inhibitor and/or an inhibitor of the
prostaglandin biosynthetic pathway and rapamycin. [0358] [3] A
composition comprising at least one a cyclooxygenase (COX)
inhibitor and/or an inhibitor of the prostaglandin biosynthetic
pathway and rapamycin for the treatment of lymphangioleiomyomatosis
(LAM). [0359] [4] A composition comprising at least one a
cyclooxygenase (COX) inhibitor and/or an inhibitor of the
prostaglandin biosynthetic pathway, rapamycin and at least one
compound selected from the group consisting of SCH-202676
hydrobromide, danusertib (PHA-739358), AZ-960, nicardipine,
SB-590885, Thimerosal, ionomycin, U-73343, PAF C16, BX912, and
Chlorambucil. [0360] [5] A composition comprising at least one a
cyclooxygenase (COX) inhibitor and/or an inhibitor of the
prostaglandin biosynthetic pathway, rapamycin and at least one
compound selected from the group consisting of SCH-202676
hydrobromide, danusertib (PHA-739358), AZ-960, nicardipine,
SB-590885, Thimerosal, ionomycin, U-73343, PAF C16, BX912, and
Chlorambucil for the treatment of lymphangioleiomyomatosis (LAM).
[0361] [6] A composition comprising at least one a cyclooxygenase
(COX) inhibitor and/or an inhibitor of the prostaglandin
biosynthetic pathway and at least one compounds selected from the
group consisting of nateglinide, Z-L-Phe chloromethyl ketone,
clemastine fumarate, supercinnamaldehyde, practolol, fluvastatin
Na, sulindac, 6-bromoindirubin-3.sup.1-oxime (BIO), amorolfine,
spectinomycin, sibutramine HCl, nelfinavir mesylate, moroxydine
HCl, nicotine ditartrate, trequinsin, meglumine, tizanidine HCl,
CGP-74514A hydrochloride, tioconazole, afatinib, kasugamycin,
flupentixol, fluphenazine, mephenytoin, aminoglutethimide,
betaxolol hydrochloride, salmeterol, chelerythrine chloride,
paroxetine, trifluoperazine, fluoxetine, methiothepin,
nortriptyline, A-77636, rapamycin, SCH-202676 hydrobromide,
danusertib (PHA-739358), AZ-960, nicardipine, SB-590885,
Thimerosal, ionomycin, U-73343, PAF C16, BX912, and Chlorambucil
for the treatment of lymphangioleiomyomatosis (LAM). [0362] [7] A
composition comprising at least one a cyclooxygenase (COX)
inhibitor and/or an inhibitor of the prostaglandin biosynthetic
pathway and at least one compounds selected from the group
consisting of nateglinide, Z-L-Phe chloromethyl ketone, clemastine
fumarate, supercinnamaldehyde, practolol, fluvastatin Na, sulindac,
6-bromoindirubin-3.sup.1-oxime (BIO), amorolfine, spectinomycin,
sibutramine HCl, nelfinavir mesylate, moroxydine HCl, nicotine
ditartrate, trequinsin, meglumine, tizanidine HCl, CGP-74514A
hydrochloride, tioconazole, afatinib, kasugamycin, flupentixol,
fluphenazine, mephenytoin, aminoglutethimide, betaxolol
hydrochloride, salmeterol, chelerythrine chloride, paroxetine,
trifluoperazine, fluoxetine, methiothepin, nortriptyline, A-77636,
rapamycin, SCH-202676 hydrobromide, danusertib (PHA-739358),
AZ-960, nicardipine, SB-590885, Thimerosal, ionomycin, U-73343, PAF
C16, BX912, and Chlorambucil. [0363] [8] The composition of any one
of claims 1-7, further comprising at least one pharmaceutically
acceptable carrier. [0364] [9] Use of at least one a cyclooxygenase
(COX) inhibitor and/or at least one inhibitor of the prostaglandin
biosynthetic pathway for the treatment of lymphangioleiomyomatosis
(LAM). [0365] [10] Use of at least one a cyclooxygenase (COX)
inhibitor and/or at least one inhibitor of the prostaglandin
biosynthetic pathway for the manufacture of medicament for
treatment of lymphangioleiomyomatosis (LAM). [0366] [11] A
combinatorial use of at least one a cyclooxygenase (COX) inhibitor
and/or at least one inhibitor of the prostaglandin biosynthetic
pathway and rapamycin for the treatment of lymphangioleiomyomatosis
(LAM). [0367] [12] A combinatorial use of at least one a
cyclooxygenase (COX) inhibitor and/or at least one inhibitor of the
prostaglandin biosynthetic pathway and rapamycin for the
manufacture of medicament for treatment of lymphangioleiomyomatosis
(LAM). [0368] [13] A combinatorial use of at least one a
cyclooxygenase (COX) inhibitor and/or an inhibitor of the
prostaglandin biosynthetic pathway, rapamycin and at least one
compound selected from the group consisting of SCH-202676
hydrobromide, danusertib (PHA-739358), AZ-960, nicardipine,
SB-590885, Thimerosal, ionomycin, U-73343, PAF C16, BX912, and
Chlorambucil for the treatment of lymphangioleiomyomatosis (LAM).
[0369] [14] A combinatorial use of at least one a cyclooxygenase
(COX) inhibitor and/or an inhibitor of the prostaglandin
biosynthetic pathway, rapamycin and at least one compound selected
from the group consisting of SCH-202676 hydrobromide, danusertib
(PHA-739358), AZ-960, nicardipine, SB-590885, Thimerosal,
ionomycin, U-73343, PAF C16, BX912, and Chlorambucil for the
manufacture of medicament for the treatment of
lymphangioleiomyomatosis (LAM). [0370] [15] A combinatorial use of
at least one a cyclooxygenase (COX) inhibitor and/or an inhibitor
of the prostaglandin biosynthetic pathway and at least one
compounds selected from the group consisting of nateglinide,
Z-L-Phe chloromethyl ketone, clemastine fumarate,
supercinnamaldehyde, practolol, fluvastatin Na, sulindac,
6-bromoindirubin-3.sup.1-oxime (BIO), amorolfine, spectinomycin,
sibutramine HCl, nelfinavir mesylate, moroxydine HCl, nicotine
ditartrate, trequinsin, meglumine, tizanidine HCl, CGP-74514A
hydrochloride, tioconazole, afatinib, kasugamycin, flupentixol,
fluphenazine, mephenytoin, aminoglutethimide, betaxolol
hydrochloride, salmeterol, chelerythrine chloride, paroxetine,
trifluoperazine, fluoxetine, methiothepin, nortriptyline, A-77636,
rapamycin, SCH-202676 hydrobromide, danusertib (PHA-739358),
AZ-960, nicardipine, SB-590885, Thimerosal, ionomycin, U-73343, PAF
C16, BX912, and Chlorambucil for the treatment of
lymphangioleiomyomatosis (LAM). [0371] [16] A combinatorial use of
at least one a cyclooxygenase (COX) inhibitor and/or an inhibitor
of the prostaglandin biosynthetic pathway and at least one
compounds selected from the group consisting of nateglinide,
Z-L-Phe chloromethyl ketone, clemastine fumarate,
supercinnamaldehyde, practolol, fluvastatin Na, sulindac,
6-bromoindirubin-3.sup.1-oxime (BIO), amorolfine, spectinomycin,
sibutramine HCl, nelfinavir mesylate, moroxydine HCl, nicotine
ditartrate, trequinsin, meglumine, tizanidine HCl, CGP-74514A
hydrochloride, tioconazole, afatinib, kasugamycin, flupentixol,
fluphenazine, mephenytoin, aminoglutethimide, betaxolol
hydrochloride, salmeterol, chelerythrine chloride, paroxetine,
trifluoperazine, fluoxetine, methiothepin, nortriptyline, A-77636,
rapamycin, SCH-202676 hydrobromide, danusertib (PHA-739358),
AZ-960, nicardipine, SB-590885, Thimerosal, ionomycin, U-73343, PAF
C16, BX912, and Chlorambucil for the manufacture of medicament for
the treatment of lymphangioleiomyomatosis (LAM). [0372] [17] A
method for treating lymphangioleiomyomatosis (LAM) in a subject in
need comprising administering to a subject therapeutically
effective amount of a composition in any one of paragraphs 1-7.
[0373] [18] A method for treating lymphangioleiomyomatosis (LAM) in
a subject in need comprising administering to a subject
therapeutically effective amount of a cyclooxygenase (COX)
inhibitor or an inhibitor of the prostaglandin biosynthetic
pathway. [0374] [19] The method of claim 1, wherein the subject has
a negative mutation in the tuberous sclerosis complex (TSC) gene 1
or 2. [0375] [20] The method of paragraph 17, 18 or 19, wherein the
cancer cells of the subject have a mutation in the TSC locus.
[0376] [21] The method of paragraph 20, wherein the cancer cells of
the subject are TSC-1 or TSC-2 deficient. [0377] [22] The method of
any one of paragraphs 17-21, wherein the subject has COX-1 or COX-2
overexpression. [0378] [23] The method of any one of paragraphs
17-22, wherein the subject has an increased prostaglandin
production. [0379] [24] The method of any one of paragraphs 17-23,
wherein at least one cancer cell of the subject is insensitive to
rapamycin. [0380] [25] The method of any one of paragraphs 17-24,
wherein at least one cancer cell of the subject does not involve
mTOR deregulation or hyperactivity. [0381] [26] The method of any
one of paragraphs 17-25, further comprising determining whether the
subject has a negative mutation in the tuberous sclerosis complex
(TSC) gene 1 or 2. [0382] [27] The method of any one of paragraphs
17-26, further comprising selecting the subject having a negative
mutation in the tuberous sclerosis complex (TSC) gene 1 or 2.
[0383] [28] The method of any one of paragraphs 17-9, further
comprising determining whether the cancer cells of the subject are
TSC-1 or TSC-2 deficient. [0384] [29] The method of any one of
paragraphs 17-28, further comprising selecting the subject having
cancer cells that are TSC-1 or TSC-2 deficient. [0385] [30] The
method of any one of paragraphs 17-29, further comprising selecting
the subject having COX-1 or COX-2 overexpression. [0386] [31] The
method of any one of paragraphs 17-30, further comprising selecting
the subject having increased prostaglandin production. [0387] [32]
The method of any one of paragraphs 17-31, wherein the subject is
further treated with an effective amount of one or more compounds
selected from the group consisting of nateglinide, Z-L-Phe
chloromethyl ketone, clemastine fumarate, supercinnamaldehyde,
practolol, fluvastatin Na, sulindac, 6-bromoindirubin-3'-oxime
(BIO), amorolfine, spectinomycin, sibutramine HCl, nelfinavir
mesylate, moroxydine HCl, nicotine ditartrate, trequinsin,
meglumine, tizanidine HCl, CGP-74514A hydrochloride, tioconazole,
afatinib, kasugamycin, flupentixol, fluphenazine, mephenytoin,
aminoglutethimide, betaxolol hydrochloride, salmeterol,
chelerythrine chloride, paroxetine, trifluoperazine, fluoxetine,
methiothepin, nortriptyline, and A-77636. [0388] [33] The method of
any one of paragraphs 17-32, wherein the subject is further treated
with a therapeutically effective amount of rapamycin and at least
one compound selected from the group consisting of SCH-202676
hydrobromide, danusertib (PHA-739358), AZ-960, nicardipine,
SB-590885, Thimerosal, ionomycin, U-73343, PAF C16, BX912, and
Chlorambucil. [0389] [34] The method of any one of paragraphs
17-33, wherein the subject is further treated with at least one
additional therapy. [0390] [35] The method of any one of paragraphs
17-34, wherein the at least one additional therapy is a cancer
therapy. [0391] [36] The method of paragraph 35, wherein the at
least one additional cancer therapy is selected from the group
consisting of radiation therapy, chemotherapy, immunotherapy and
gene therapy. [0392] [37] The method of any one of paragraphs
17-36, wherein the subject is further treated with hormone therapy.
[0393] [38] The method of any one of paragraphs 17-37, wherein the
subject is human. [0394] [39] The method of any one of paragraphs
17-38, wherein the therapeutically effective amount of the
inhibitor and/or compound is administered by a route selected from
the group consisting of: aerosol, direct injection, local,
systemic, intradermal, direct inhalation, intravitreal,
intramuscular, intraperitoneal, intravenous, intrathecal,
intrapleural, intrauterine, subcutaneous, epidural, topical, oral,
transmucosal, buccal, rectal, vaginal, transdermal, intranasal,
intrasynovial, intraocular/periocular, intraorgan, intratumor, and
parenteral administration. [0395] [40] The method of any one of
paragraphs 17-39, wherein the COX inhibitor is a COX-1 or COX-2
inhibitor. [0396] [41] The method of paragraph 40, wherein the COX
inhibitor is selected from the group of rofecoxib, celecoxib,
valdecoxib, nimesulide, ibuprofen, diclofenac, nabumetone,
naprosen, aspirin and analogs thereof. [0397] [42] The method of
any one of paragraphs 17-41, wherein the inhibitor of the
prostaglandin biosynthetic pathway is indomethacin or flufenamic
acid. [0398] [43] A method for treating lymphangioleiomyomatosis
(LAM) in a subject in need comprising (a) determining whether the
subject has a mutation in the tuberous sclerosis complex (TSC)
locus and (b) administering to the subject therapeutically
effective amount of a composition in any one of paragraphs 1-7 when
the subject is determined to have a TSC mutation. [0399] [44] A
method for treating lymphangioleiomyomatosis (LAM) in a subject in
need comprising (a) selecting a subject having COX-1 or COX-2
overexpression; and (b) administering to the subject
therapeutically effective amount of a composition in any one of
paragraphs 1-7. [0400] [45] A method for treating
lymphangioleiomyomatosis (LAM) in a subject in need comprising (a)
selecting a subject having increased prostaglandin production; and
(b) administering to a subject therapeutically effective amount of
a composition in any one of paragraphs 1-7. [0401] [46] A method
for treating lymphangioleiomyomatosis (LAM) in a subject in need
comprising (a) selecting the subject having a negative mutation in
the tuberous sclerosis complex (TSC) locus and (b) administering to
a subject therapeutically effective amount of a composition in any
one of paragraphs 1-7. [0402] [47] A method for treating
lymphangioleiomyomatosis (LAM) in a subject in need comprising (a)
selecting a subject who has at least one cancer cell that is
insensitive to rapamycin; and (b) administering to a subject
therapeutically effective amount of a composition in any one of
paragraphs 1-7. [0403] [48] A method for treating
lymphangioleiomyomatosis (LAM) in a subject in need comprising (a)
selecting a subject who has at least one cancer cell that does not
have mTOR deregulation or hyperactivity; and (b) administering to a
subject therapeutically effective amount of a composition in any
one of paragraphs 1-7.
[0404] This disclosure is further illustrated by the following
example which should not be construed as limiting. The contents of
all references cited throughout this application, as well as the
figures and table are incorporated herein by reference.
Example
[0405] Lymphangioleiomyomatosis (LAM) is a female predominant
interstitial lung disease due to proliferating smooth muscle-like
(LAM) cells that typically have TSC2 mutation, leading to
mTORC1-activation (1-5). mTORC1 regulates cell growth, protein
translation and metabolism (6). In a randomized clinical trial the
mTORC1 inhibitor rapamycin stabilized lung function and improved
symptoms in LAM patients. However, lung function declined when
rapamycin was discontinued (7). The female predominance of LAM,
coupled with the genetic evidence indicating that estrogen promotes
the metastasis of cells with mTORC1 activation (1,4). It was
previously have discovered that estrogen increases levels of
circulating tumor cells and pulmonary metastases of
tuberin-deficient cells in a xenograft model of LAM (8). Studies
have demonstrated that estrogen induces COX-2-mediated
prostaglandin synthesis (9). COX-2 is a rate-limiting enzyme
catalyzing the conversion of arachidonate to prostaglandins. COX-2
overexpression has been documented in human tumors, and
prostaglandins may contribute to cancer development (10-13). In
this study, it was discovered that estrogen enhances prostaglandin
production in TSC2-deficient cells. Surprisingly, loss of TSC2
increases COX-2 and prostaglandin biosynthesis in a
rapamycin-insensitive manner, indicating an mTORC1-independent
pathway. Aspirin suppresses tumor progression in a xenograft tumor
model. This study indicates that targeting COX-2 with aspirin or
related drugs may have therapeutic benefit in LAM and TSC-related
diseases.
Methods
[0406] Cell culture and reagents. ELT-3 cells (Eker rat uterine
leiomyoma-derived) (22,23) and LAM patient-derived 621-101 cells
were cultured in IIA complete medium. HeLa, U2OS and OVARC5 cells
were cultured in DMEM supplemented with 10% FBS. 17-beta-estradiol
(10 nM), rapamycin (20 nM, Biomols), NS398 (50 .mu.M), aspirin (450
.mu.M Sigma), and 15-epi-LXA4 (100 nM) were used as indicated.
[0407] Animal studies. All animal work was performed in accordance
with protocols approved by the IACUC-BWH. Female intact CB17-SCID
mice were used as described previously (8, 24, 25). Aspirin (100
mg/kg/day, in drinking water) treatment was initiated five weeks
post-cell inoculation. Urine specimens were collected.
[0408] Quantitative RT-PCR. RNA from cultured cells and xenograft
tumors was isolated using RNeasy Mini Kit (QIAGEN). Gene expression
was quantified using One-Step qRT-PCR Kits (INVITROGEN) in the
Applied Biosystems Real-Time PCR System and normalized to
beta-actin.
[0409] Immunoblotting and antibodies. Cells were lysed in m-PER
buffer (PIERCE). Antibodies were used: COX-1, COX-2, phospho-MAPK
(T202/Y204), phospho-S6 (S235/236), Cleaved caspase 3, cleaved PARP
(CELL SIGNALING), tuberin and c-Myc (SANTA CRUZ), smooth muscle
actin (BIOGENEX), and beta-actin (SIGMA).
[0410] Immunohistochemistry. Sections were deparaffinized,
incubated with primary antibodies and biotinylated secondary
antibodies and counterstained with Gill's Hematoxylin.
[0411] Quantification of prostaglandin levels. PGE2,
6-keto-PGF1.alpha. and creatinine were measured using enzyme
immunoassay kits (Cayman Chemical). Levels of secreted
prostaglandins were normalized to protein concentrations and
expressed as pg/mg protein. Urinary levels of prostaglandins were
normalized to creatinine levels and expressed as ng/mL.
[0412] Statistical analyses. Statistical analyses were performed
using Student's t-test when comparing two groups for in vitro and
in vivo studies. Two-Way ANOVO test was performed in xenograft
tumor-aspirin studies. Mann Whitney tests were used for
prostaglandin quantification in clinical data.
[0413] Metabolomic profiling. 100 .mu.g of frozen biopsy tissue was
submitted to Metabolon, Inc. (Durham, N.C.) for sample extraction
and analysis. In brief, Metabolon performed cold methanol
extraction of mechanically disaggregated tissue samples and these
extracts were split into three aliquots. The reproducibility of the
extraction protocol was assessed by the recovery of xenobiotic
compounds spiked into every tissue sample prior to extraction.
These aliquots were processed and characterized by one of the three
analytical methods previously described: UHPLC-ESI-MS/MS in the
positive ion mode, UHPLC-ESI-MS/MS in the negative ion mode and
sialylation followed by GC-EI-MS. Chromatographic timelines were
standardized using a series of xenobiotics that elute at specified
intervals throughout each chromatographic run. The technical
variability of each analytical platform was assessed by repeated
characterization of a pooled standard that contained an aliquot of
each sample within the study.
Results
Identification of an Estrogen-Induced Prostaglandin Biosynthesis
Signature in TSC2-Deficient Cells and Xenograft Tumors
[0414] To examine the possible effects of estrogen on metabolic
pathways in Tsc2-deficient rat-uterus-derived ELT3 cells14, a
metabolomic screen was performed. A significant increase in
prostaglandins including PGE2, PGD2, and 6-keto-PGF1.alpha., was
seen in estrogen-treated cells (FIGS. 1a and 1b). Furthermore,
estrogen increased MAPK phosphorylation, COX-2 expression, and PGE2
levels in TSC2-deficient cells at 2 and 24 hr (FIGS. 1c and 1d).
Analysis of LAM patient-derived TSC2-deficient 621-101 cells15
showed that estrogen also increased COX-2 expression (FIG. 1e) and
PGE2 levels at 24 hr (FIG. 1f), confirming the results in the
Tsc2-deficient ELT3 cells.
[0415] To determine the effect of estrogen on cellular metabolomics
in vivo, xenograft tumors of Tsc2-deficient ELT3 cells (8) from
placebo or estrogen-implanted ovariectomized female mice were used.
MAPK phosphorylation was evident in xenograft tumors from
estrogen-treated mice (Data not shown). The metabolomic screen
showed that PGE2 and PGD2 levels were significantly increased in
xenograft tumors from mice treated with estrogen (FIGS. 1g and 1i).
Estrogen-treated mice bearing ELT3 xenograft tumors also exhibited
higher levels of urinary PGE2 and PGD2 relative to placebo controls
(FIG. 1j). These data demonstrate that estrogen stimulates
prostaglandin biosynthesis by TSC2-deficient cells in vitro and in
vivo.
TSC2 Negatively Regulates COX-2 Expression and Prostaglandin
Production in Rapamycin-Insensitive Manner In Vitro and In
Vivo.
[0416] To define the molecular mechanisms responsible for
estrogen-enhanced COX-2 expression and prostaglandin production, an
expression array of TSC2-deficient LAM patient-derived cells (16)
was analyzed (FIG. 2a) and it was found that prostacyclin synthase
(PTGIS) was elevated by 40-fold and COX-2 (PTGS2) by 2-fold.
Interestingly, these changes were rapamycin insensitive (FIG. 6).
COX-2 protein levels were more abundant in TSC2-deficient cells
compared to TSC2 reexpressing 621-101 cells (FIG. 2b). Rapamycin
treatment suppressed phosphorylation of the ribosomal protein p70S6
(S6); however, did not affect COX-2 expression (data not shown),
indicating that TSC2 regulates COX-2 expression in a
rapamycin-insensitive manner. Levels of PGE2, 6-keto-PGF1.alpha.,
PGF2.alpha. and thromboxane B2 were all significantly higher in
TSC2-deficient cells relative to TSC2-reexpressing cells, and were
also insensitive to rapamycin treatment (FIG. 2c). Similar results
on COX-2 expression were also seen in Tsc2-deficient ELT3 cells
(FIG. 2d). PGE2 levels were elevated by 2.5-fold in ELT3 cells in
comparison to TSC2 reexpressing cells (FIG. 2e), and this again was
not affected by rapamycin treatment (FIG. 2e). To determine whether
this phenomenon of rapamycin-insensitive prostaglandin production
was seen in other cells with intact TSC2 levels but mTORC1
activation, HeLa, U2OS and OVCAR5 cells were also examined.
Although PGE2 production was variable among these cell lines,
rapamycin did not affect COX-2 expression or PGE2 production though
reducing phospho-S6 (FIG. 8). Together, these data indicate that
upregulation of COX-2 and prostaglandin production is
rapamycin-insensitive in cells with mTORC1 activation.
[0417] To determine whether TSC2 regulates COX-2 and prostaglandin
production in vivo, xenograft tumors from mice inoculated with
TSC2-deficient ELT3-V3 (vector-control) cells and TSC2-addback
ELT3-T3 cells were studied. COX-2 levels were significantly higher
in the ELT3-V3 xenograft tumors with elevated phospho-S6 relative
to TSC2-addback ELT3-T3 tumors (FIG. 2h). In addition, urinary PGE2
and 6-keto-PGF1.alpha. levels were significantly higher in mice
with the ELT3-V3 tumors in comparison to mice with the TSC2-addback
ELT3-T3 tumors (FIG. 2i).
[0418] To further assess the effect of TSC2-loss on COX-2 levels in
vivo, the inventors examined a spontaneously-arising renal
cystadenoma from TSC2+/- mice (17), and found that COX-2 and
phospho-S6 was more abundant in the TSC2-deficient tumor compared
to normal kidney (FIG. 2k). Rapamycin treatment did not affect
COX-2 expression in these tumors (FIG. 2l). These data indicate
that COX-2 expression and prostaglandin production are enhanced in
cells and tumors lacking TSC2 in a rapamycin-insensitive
manner.
Aspirin Treatment Inhibits TSC2-Deficient Cell and Xenograft Tumor
Growth and Reduces Urinary Levels of Prostaglandins
[0419] To determine whether inhibition of COX-1 and/or COX-2
impacts the growth of LAM patient-derived cells, 621-101 cells were
treated with Sulindac (a COX-1 inhibitor), NS398 (a COX-2
inhibitor), or aspirin (an irreversible COX-1 and COX-2 inhibitor)
for 24 hr. NS398 and aspirin reduced COX-2 and COX-1 levels without
affecting phosphorylation of MAPK or S6 (FIG. 3a). Aspirin
significantly decreased PGE2 levels (FIG. 3b), and reduced
proliferation of 621-101 cells (FIG. 3c). The inventors next
assessed the possible benefit of aspirin in a xenograft tumor model
of TSC2-deficient ELT3-luciferase-expressing cells. Aspirin
treatment for two-three weeks decreased the intensity of
bioluminescence (FIG. 3c), reduced the growth of xenograft tumors
by 35% (FIG. 3d), and decreased the tumor size (FIG. 3e). Tumors
also had reduced expression of COX-2 and c-Myc, and increased
levels of cleaved-caspase-3 and cleaved-PARP (Data not shown).
Furthermore, aspirin-treated mice bearing ELT3 xenograft tumors had
markedly reduced urinary levels of PGE2 and 6-Keto-PGF1.alpha.
(Data not shown). These data suggest that aspirin has efficacy in
suppressing TSC2-deficient tumor progression.
Activation of COX-2 in LAM Nodules and Elevated Prostaglandin
Production in LAM Patient Serum.
[0420] To confirm that these observations were relevant to patients
with LAM, the inventors showed that LAM lungs expressed higher
levels of COX-2 (PTGS2) in comparison to control lungs (FIGS. 4a
and 4b). Further, COX-2 immunohistochemistry showed increased COX-2
expression in pulmonary LAM lesions (arrows), which were also
positive for smooth muscle actin (SMA) and phospho-S6 (FIG. 4c). To
examine the functional effect of COX-2 in LAM lungs, 15-epi-lipoxin
A4 (LXA4), a product of aspirin-acetylated COX-2, was measured in
exhaled breath condensate (EBC) from three LAM subjects (data not
shown). 15-epi-LXA4 was detected in EBC and the levels were
increased with aspirin (data not shown). Of interest, 15-epi-LXA4
decreases proliferation of A549 cells (Human lung adenocarcinoma)
(18) and also suppressed the proliferation of LAM patient-derived
621-101 cells (data not shown).
[0421] Urinary PGE2 levels were measured in 29 LAM patients and in
18 healthy women; however, these levels were not significantly
different between two groups (FIG. 9). Because renal prostaglandin
production can significantly influence urinary prostaglandin
levels, the inventors next measured PGE2 and 6-keto-PGF1.alpha.
levels in sera from 14 LAM patients and 13 healthy women. Of note,
the mean serum PGE2 levels of LAM patients (27.8.+-.1.8 pg/mL) were
higher than those of healthy women (19.6.+-.1.4 pg/mL, p=0.0021)
(FIG. 4d). In addition, the mean serum 6-keto-PGF1.alpha. levels of
LAM patients (192.2.+-.64.9 pg/mL) were also higher compared with
levels in healthy women (82.6.+-.6.3 pg/mL, p=0.0006) (FIG.
4e).
[0422] The inventors have found that estrogen enhances the
expression of COX-2 and induces production of PGE2 and
6-keto-PGF1.alpha. in TSC2-deficient cells in vitro and in vivo.
See FIG. 5. Furthermore, they identified a novel function of TSC2
as a negative regulator of COX-2 expression and prostaglandin
biosynthesis. Interestingly, this regulation appears to be
rapamycin-insensitive, indicating that it may be another
mTORC1-independent function of TSC2. The inventors also
demonstrated that COX-2 is abundant in LAM lesions, and that serum
levels of PGE2 and 6-keto-PGF1.alpha. are elevated in LAM patients.
Collectively, the data indicate that COX-2 plays an important role
in LAM pathogenesis.
[0423] Aspirin, the prototypical non-steroidal anti-inflammatory
drug, covalently modifies both COX-1 and COX-2 by acetylation. The
inventors demonstrated that aspirin treatment significantly reduced
the growth of xenograft tumors of TSC2-deficient ELT3 cells, and
led to reduced urinary levels of PGE2 and 6-keto-PGF1.alpha.,
consistent with loss of COX-1/COX-2 function. Furthermore,
xenograft tumors from aspirin-treated mice exhibited higher levels
of apoptosis compared to vehicle-treatment. While
aspirin-acetylated COX-2 inhibits prostaglandin formation, the
enzyme is not completely inactivated. Rather, aspirin-acetylated
COX-2 catalyzes the conversion of arachidonate to 15-epi-LXA418. In
this manner, aspirin both inhibits prostaglandin production and
triggers the formation of 15-epi-LXA4, a potent inhibitor of
malignant cell proliferation18. 15-epi-LXA4 was present in EBC from
LAM patients, and was increased with oral aspirin ingestion.
15-epi-LXA4 also decreased LAM-patient-derived cell proliferation.
Together, these findings indicate that aspirin have
rapamycin-insensitive protective actions in LAM.
[0424] LAM is often a progressive disease which leads to
respiratory failure and death in the absence of lung
transplantation. The recent demonstration that rapamycin has
clinical benefit in LAM is a major success. However, not all
patients respond to rapamycin, and upon rapamycin withdrawal, lung
function decline resumes (7). Hence lifelong treatment of LAM
patients with rapamycin may be required to maintain benefit, with
unknown long-term toxicities. The present findings suggest that
aspirin and/or other COX-1/COX-2 inhibitors may have significant
benefit in slowing LAM progression and possibly other neoplastic
conditions associated with mTORC1 hyperactivation. Since aspirin
has a well-known side-effect profile, it is of particular clinical
interest. Further clinical investigation is warranted to explore
these possibilities.
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