U.S. patent application number 14/215982 was filed with the patent office on 2014-09-25 for use of phosphodiesterase inhibitors for treating multidrug resistance.
This patent application is currently assigned to St. John's University. The applicant listed for this patent is St. John's University. Invention is credited to Charles R. ASHBY, Zhe-Sheng CHEN, Zhi SHI.
Application Number | 20140288078 14/215982 |
Document ID | / |
Family ID | 46928026 |
Filed Date | 2014-09-25 |
United States Patent
Application |
20140288078 |
Kind Code |
A1 |
CHEN; Zhe-Sheng ; et
al. |
September 25, 2014 |
USE OF PHOSPHODIESTERASE INHIBITORS FOR TREATING MULTIDRUG
RESISTANCE
Abstract
The present invention relates to methods of treating multidrug
resistance in cancerous cells with phosphodiesterase (PDE)
inhibitors, e.g., PDE5 inhibitors. More specifically, the invention
relates to methods of treating multidrug resistance that arises,
e.g., during administration of chemotherapeutic/antineoplastic
(anticancer) agents for treatment of cancer, with a PDE5 inhibitor
(e.g., sildenafil, vardenafil, and tadalafil). The invention also
relates to methods of treating cancer, e.g., multidrug resistant
cancer, using a PDE5 inhibitor in combination with an
antineoplastic therapeutic agent. Further, the invention relates to
pharmaceutical compositions for treating multidrug resistant
cancers comprising a PDE5 inhibitor, or a combination of a PDE5
inhibitor and an antineoplastic agent.
Inventors: |
CHEN; Zhe-Sheng; (Belle
Mead, NJ) ; SHI; Zhi; (Guangzhou, CN) ; ASHBY;
Charles R.; (Sound Beach, NY) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
St. John's University |
Jamaica |
NY |
US |
|
|
Assignee: |
St. John's University
Jamaica
NY
|
Family ID: |
46928026 |
Appl. No.: |
14/215982 |
Filed: |
March 17, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13432315 |
Mar 28, 2012 |
8673914 |
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14215982 |
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61501984 |
Jun 28, 2011 |
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61465948 |
Mar 28, 2011 |
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Current U.S.
Class: |
514/243 ;
514/250; 514/252.16; 514/654 |
Current CPC
Class: |
A61P 35/00 20180101;
A61K 31/277 20130101; A61K 31/519 20130101; A61K 2300/00 20130101;
A61K 31/53 20130101; A61K 31/519 20130101; A61K 31/4985 20130101;
A61K 45/06 20130101 |
Class at
Publication: |
514/243 ;
514/250; 514/654; 514/252.16 |
International
Class: |
A61K 31/53 20060101
A61K031/53; A61K 31/277 20060101 A61K031/277; A61K 31/519 20060101
A61K031/519; A61K 31/4985 20060101 A61K031/4985 |
Goverment Interests
STATEMENT AS TO FEDERALLY SPONSORED RESEARCH
[0002] This invention was made partially with Government support
under NIH grant number 1R15CA143701. The Government has certain
rights in the invention.
Claims
1. A method of treating multidrug resistance in a cancer cell in a
subject in need thereof by inhibiting ABCB1 transporter activity,
comprising administering to the subject a therapeutically effective
amount of a phosphodiesterase 5 (PDE5) inhibitor.
2-21. (canceled)
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 13/432,315, filed Mar. 28, 2012, now U.S. Pat. No. 8,673,914,
which claims the benefit of U.S. Provisional Patent Application No.
61/465,948, filed Mar. 28, 2011, and U.S. Provisional Patent
Application No. 61/501,984, filed Jun. 28, 2011. The entire
contents of all the above-mentioned applications are herein
incorporated by reference.
BACKGROUND OF THE INVENTION
[0003] 1. Field of the Invention
[0004] The present invention relates to a method of treating
multidrug resistance with phosphodiesterase (PDE) inhibitors, e.g.,
PDE5 inhibitors. More specifically, the present invention relates
to a method of treating multidrug resistance, e.g., multidrug
resistance that arises, e.g., during administration of
chemotherapeutic or antineoplastic agents for treatment of cancer,
with a PDE5 inhibitor (e.g., vardenafil, sildenafil, and
tadalafil). In addition, the invention relates to a method of
treating cancer, e.g., multidrug resistant cancer, using a PDE5
inhibitor in combination with an anticancer agent, i.e., a
chemotherapeutic/antineoplastic agent. Further, the invention
relates to a pharmaceutical composition for treating a multidrug
resistant cancer comprising a PDE5 inhibitor, or a combination of a
PDE5 inhibitor and an antineoplastic agent.
[0005] 2. Description of Related Art
[0006] Multidrug resistance (MDR) is a phenomenon in which
disease-causing organisms or cells are able to evade treatment with
drugs designed to target them. Multidrug resistance to cancer
chemotherapy, a major hurdle to successfully treating cancer,
commonly arises due to the ability of one or more cancer cells to
resist treatment with a variety of agents, e.g., drugs that may be
distinct in one or both of the following: structure and mechanism
of action. Therefore, whereas a portion of cancer cells, i.e.,
drug-sensitive cancer cells, are killed with a chemotherapeutic
agent, drug-resistant cancer cells survive, such that the resultant
or remaining cancers consist primarily of drug-resistant (or
medicine-tolerant) cells.
[0007] One known cause of multidrug resistance to cancer
chemotherapy is the overexpression of ATP-binding cassette (ABC)
transporters in the membranes of cancers cells (Szakacs et al.
(2006) Nat. Rev. Drug Discov. 5:219-34). These pumps efflux agents,
including structurally and functionally unrelated chemotherapeutic
agents, out of cancer cells, significantly decreasing the chance of
successful treatment. In mammals, the ABC transporters have been
divided into seven subfamilies (i.e., subfamilies A-G), based on
genome sequence similarities (Gottesman et al. (2002) Nat. Rev.
Cancer 2:48-58). Studies to date have consistently shown that the
three major ABC transporters involved in multidrug resistance in
most cancer cells are P-glycoprotein (Pgp/ABCB1/MDR1), breast
cancer resistant protein (ABCG2/BCRP/ABCP), and members of the
multidrug resistance protein family, e.g., multidrug resistant
protein 1 (ABCC1/MRP1).
[0008] Overexpression of ABCB1 transporter occurs in 40-50% of all
cancers; therefore, there is a major ongoing effort to design a
strategy for inhibiting this transporter. Three generations of
compounds that inhibit ABCB1 have been studied. The first
generation of ABCB1 inhibitors included such drugs as verapamil,
quinine, and cyclosporine A; however, these first generation agents
produced undesirable adverse effects at concentrations necessary to
inhibit ABCB1 (Krishna et al. (2000) Eur. J. Pharm. Sci.
11:265-83). The second generation of ABCB1 inhibitors, e.g.,
valspodar and biricodar, produced unpredictable interactions with
other transport proteins and inhibited cytochrome P450 3A4
(CYP3A4), an enzyme responsible for metabolizing many xenobiotics,
including chemotherapeutic drugs. Therefore, the use of the second
generation ABCB1 inhibitors resulted in decreased clearance and
increased toxicity of many chemotherapeutic agents (Gottesman et
al., supra). The third generation of inhibitors, e.g., LY335979
(zosuquidar), GF120918 (elacridar), and MS-209 (dofequidar), were
derived from second generation agents, and had nanomolar affinity
for the ABCB1 transporter. However, to date third generation
inhibitors have not been approved for use in patients due to
several factors, including adverse side effects, unfavorable
pharmacokinetic profiles, and/or lack of significant efficacy in
late-phase clinical trials (Modok et al. (2006) Curr. Opin.
Pharmacol. 6:350-54; Wu et al. (2009) Curr. Mol. Pharmacol.
1:93-105). Thus, it would be beneficial to determine whether other
drugs, including drugs already used in the clinic, are capable of
inhibiting ABC transporters and, therefore, can be used for the
treatment of multidrug resistance.
[0009] Multidrug resistant protein 7 (MRP7; ABCC10), a member of
MRP subfamily, is similar in topology to other MRPs (including
MRP1) with two nucleotide-binding domains and three transmembrane
domains (Kruh et al. (2007) Pflugers Arch. 453:675-84; Chen et al.
(2003) Mol. Pharmacol. 63:351-58). ABCC10 is able to confer
resistance to several natural product chemotherapeutic drugs,
including taxanes and vinca alkaloids, which are also substrates of
Pgp (Hopper-Borge et al. (2004) Cancer Res. 64:4927-30). ABCC10 has
been reported to confer resistance to vinorelbine and paclitaxel in
non-small cell lung cancer cells (Bessho et al. (2009) Oncol. Rep.
21:263-68; Oguri et al. (2008) Mol. Cancer Ther. 7:1150-55) and to
vincristine in human salivary gland adenocarcinoma cells (Naramoto
et al. (2007) Int. J. Oncol. 30:393-401). The in vivo functions of
ABCC10 have recently been confirmed using an Mrp7 knockout mouse
model (Hopper-Borge et al. (2011) Cancer Res. 71(10):3649-57).
[0010] Phosphodiesterase type 5 (PDE5) inhibitors are widely used
in the treatment of male erectile dysfunction and in improving
breathing efficiency in pulmonary hypertension. As agents already
used in the clinic for other purposes, these drugs were
investigated for their effects on MDR and ABC transporters.
SUMMARY OF THE INVENTION
[0011] The present invention relates to methods of treating
multidrug resistance in a cancer cell in a subject in need thereof
by inhibiting ABCB1 transporter activity, comprising administering
to the subject a therapeutically effective amount of a
phosphodiesterase 5 (PDE5) inhibitor. In one embodiment, the PDE5
inhibitor is administered in combination with an antineoplastic
agent.
[0012] The present invention further relates to methods of treating
a cancer in a subject by inhibiting ABCB1 transporter activity,
comprising administering to the subject a therapeutically effective
amount of an antineoplastic agent and a therapeutically effective
amount of a PDE5 inhibitor. In one embodiment, the cancer is a
multidrug resistant cancer.
[0013] In some embodiments of the above methods of treating, the
subject is a mammal, e.g., a human.
[0014] In addition, the present invention relates to (1) methods of
increasing sensitivity of a multidrug resistant cancer cell to an
antineoplastic agent by inhibiting ABCB1 transporter activity,
comprising contacting the cancer cell with a PDE5 inhibitor; (2)
methods of inhibiting growth of a multidrug resistant cancer cell
by inhibiting ABCB1 transporter activity, comprising contacting the
cell with a combination of an antineoplastic agent and a PDE5
inhibitor; (3) methods of increasing accumulation of an
antineoplastic agent in a cancer cell by inhibiting ABCB1
transporter activity, comprising contacting the cell with a PDE5
inhibitor; and (4) methods of decreasing efflux of an
antineoplastic agent from a cancer cell by inhibiting ABCB1
transporter activity, comprising contacting the cell with a PDE5
inhibitor. In some embodiments, these methods are performed in
vivo. For example, the methods are performed in a mammalian
subject, e.g., a human subject. In further embodiments of the above
methods, both the antineoplastic agent and the PDE5 inhibitor are
administered in therapeutically effective amounts.
[0015] In some embodiments of the above methods related to ABCB1
transporter activity, the PDE5 inhibitor is selected from the group
consisting of sildenafil, vardenafil, tadalafil, lodenafil,
udenafil, benzamidenafil, mirodenafil, avanafil, zaprinast,
SLX-2101, UK-371,800, UK-122764, icariin, DA-8159,
3-[4-(2-hydroxyethyl)piperazin-1-yl]-7-(6-methoxypyridin-3-yl)-1-(2-propo-
xyethyl)pyrido[3,4-b]-pyrazin-2(1H)-one, and their analogs. In some
embodiments of the above methods, the antineoplastic agent is
selected from the group consisting of antineoplastic agents listed
in Table 1; as a nonlimiting example, the antineoplastic agent can
be selected from the group consisting of paclitaxel, vinblastine,
and vincristine.
[0016] The present invention also relates to methods of treating
multidrug resistance in a cancer cell in a subject in need thereof
by inhibiting ABCG2 transporter activity, comprising administering
to the subject a therapeutically effective amount of a PDE5
inhibitor. In one embodiment, the PDE5 inhibitor is administered in
combination with an antineoplastic agent.
[0017] The present invention further relates to methods of treating
a cancer in a subject by inhibiting ABCG2 transporter activity,
comprising administering to the subject a therapeutically effective
amount of an antineoplastic agent and a therapeutically effective
amount of a PDE5 inhibitor. In one embodiment, the cancer is a
multidrug resistant cancer.
[0018] In some embodiments of the above methods of treating, the
subject is a mammal, e.g., a human.
[0019] Further, the present invention relates to (1) methods of
increasing sensitivity of a multidrug resistant cancer cell to an
antineoplastic agent by inhibiting ABCG2 transporter activity,
comprising contacting the cancer cell with a PDE5 inhibitor; (2)
methods of inhibiting growth of a multidrug resistant cancer cell
by inhibiting ABCG2 transporter activity, comprising contacting the
cell with a combination of an antineoplastic agent and a PDE5
inhibitor; (3) methods of increasing accumulation of an
antineoplastic agent in a cancer cell by inhibiting ABCG2
transporter activity, comprising contacting the cell with a PDE5
inhibitor; and (4) methods of decreasing efflux of an
antineoplastic agent from a cancer cell by inhibiting ABCG2
transporter activity, comprising contacting the cell with a PDE5
inhibitor. In some embodiments, these methods are performed in
vivo. For example, the methods are performed in a mammalian
subject, e.g., a human subject. In further embodiments of the above
methods, both the antineoplastic agent and the PDE5 inhibitor are
administered in therapeutically effective amounts.
[0020] In some embodiments of the above methods related to ABCG2
transporter activity, the PDE5 inhibitor is selected from the group
consisting of sildenafil, vardenafil, tadalafil, lodenafil,
udenafil, benzamidenafil, mirodenafil, avanafil, zaprinast,
SLX-2101, UK-371,800, UK-122764, icariin, DA-8159,
3-[4-(2-hydroxyethyl)piperazin-1-yl]-7-(6-methoxypyridin-3-yl)-1-(2-propo-
xyethyl)pyrido[3,4-b]-pyrazin-2(1H)-one, and their analogs. In some
embodiments of the above methods, the antineoplastic agent is
selected from the group of antineoplastic agents listed in Table 2;
as a nonlimiting example, the antineoplastic agent can be selected
from the group consisting of SN-38, flavopiridol, mitoxantrone, and
methotrexate.
[0021] The present invention also relates to methods of treating
multidrug resistance in a cancer cell in a subject in need thereof
by inhibiting ABCC10 transporter activity, comprising administering
to the subject a therapeutically effective amount of a PDE5
inhibitor. In one embodiment, the PDE5 inhibitor is administered in
combination with an antineoplastic agent.
[0022] The present invention further relates to methods of treating
a cancer in a subject by inhibiting ABCC10 transporter activity,
comprising administering to the subject a therapeutically effective
amount of an antineoplastic agent and a therapeutically effective
amount of a PDE5 inhibitor. In one embodiment, the cancer is a
multidrug resistant cancer.
[0023] In some embodiments of the above methods of treating, the
subject is a mammal, e.g., a human.
[0024] Further, the present invention relates to (1) methods of
increasing sensitivity of a multidrug resistant cancer cell to an
antineoplastic agent by inhibiting ABCC10 transporter activity,
comprising contacting the cancer cell with a PDE5 inhibitor; (2)
methods of inhibiting growth of a multidrug resistant cancer cell
by inhibiting ABCC10 transporter activity, comprising contacting
the cell with a combination of an antineoplastic agent and a PDE5
inhibitor; (3) methods of increasing accumulation of an
antineoplastic agent in a cancer cell by inhibiting ABCC10
transporter activity, comprising contacting the cell with a PDE5
inhibitor; and (4) methods of decreasing efflux of an
antineoplastic agent from a cancer cell by inhibiting ABCC10
transporter activity, comprising contacting the cell with a PDE5
inhibitor. In some embodiments, these methods are performed in
vivo. For example, the methods are performed in a mammalian
subject, e.g., a human subject. In further embodiments of the above
methods, both the antineoplastic agent and the PDE5 inhibitor are
administered in therapeutically effective amounts.
[0025] In some embodiments of the above methods related to ABCC10
transporter activity, the PDE5 inhibitor is selected from the group
consisting of sildenafil, vardenafil, tadalafil, lodenafil,
udenafil, benzamidenafil, mirodenafil, avanafil, zaprinast,
SLX-2101, UK-371,800, UK-122764, icariin, DA-8159,
3-[4-(2-hydroxyethyl)piperazin-1-yl]-7-(6-methoxypyridin-3-yl)-1-(2-propo-
xyethyl)pyrido[3,4-b]-pyrazin-2(1H)-one, and their analogs. In some
embodiments of the above methods, the antineoplastic agent is
selected from the group of antineoplastic agents listed in Table 3;
as a nonlimiting example, the antineoplastic agent can be selected
from the group consisting of paclitaxel, docetaxel, vinblastine,
and vincristine.
[0026] The invention also provides methods of stimulating the
ATPase activity of an ABCB1 transporter in a cell comprising
contacting the cell with a PDE5 inhibitor. In one embodiment, the
PDE5 inhibitor is selected from the group consisting of sildenafil,
vardenafil, tadalafil, lodenafil, udenafil, benzamidenafil,
mirodenafil, avanafil, zaprinast, SLX-2101, UK-371,800, UK-122764,
icariin, DA-8159,
3-[4-(2-hydroxyethyl)piperazin-1-yl]-7-(6-methoxypyridin-3-yl)-1-(2-propo-
xyethyl) pyrido[3,4-b]-pyrazin-2(1H)-one, and their analogs.
[0027] The invention also provides methods of stimulating the
ATPase activity of an ABCG2 transporter in a cell comprising
contacting the cell with a PDE5 inhibitor. In one embodiment, the
PDE5 inhibitor is selected from the group consisting of sildenafil,
vardenafil, tadalafil, lodenafil, udenafil, benzamidenafil,
mirodenafil, avanafil, zaprinast, SLX-2101, UK-371,800, UK-122764,
icariin, DA-8159,
3-[4-(2-hydroxyethyl)piperazin-1-yl]-7-(6-methoxypyridin-3-yl)-1-(2-propo-
xyethyl) pyrido[3,4-b]-pyrazin-2(1H)-one, and their analogs.
[0028] The invention also contemplates methods of stimulating the
ATPase activity of an ABCC10 transporter in a cell comprising
contacting the cell with a PDE5 inhibitor. In one embodiment, the
PDE5 inhibitor is selected from the group consisting of sildenafil,
vardenafil, tadalafil, lodenafil, udenafil, benzamidenafil,
mirodenafil, avanafil, zaprinast, SLX-2101, UK-371,800, UK-122764,
icariin, DA-8159,
3-[4-(2-hydroxyethyl)piperazin-1-yl]-7-(6-methoxypyridin-3-yl)-1-(2-propo-
xyethyl) pyrido[3,4-b]-pyrazin-2(1H)-one, and their analogs.
[0029] Further, the present invention provides pharmaceutical
compositions for treating a multidrug resistant cancer by
inhibiting ABCB1 transporter activity in a subject comprising a
PDE5 inhibitor and a pharmaceutical excipient. In one embodiment,
the PDE5 inhibitor is selected from the group consisting of
sildenafil, vardenafil, tadalafil, lodenafil, udenafil,
benzamidenafil, mirodenafil, avanafil, zaprinast, SLX-2101,
UK-371,800, UK-122764, icariin, DA-8159,
3-[4-(2-hydroxyethyl)piperazin-1-yl]-7-(6-methoxypyridin-3-yl)-1-(2-propo-
xyethyl)pyrido[3,4-b]-pyrazin-2(1H)-one, and their analogs. In some
embodiments, the pharmaceutical composition of the invention
further comprises an antineoplastic agent. In some embodiments, the
antineoplastic agent is selected from the group consisting of
antineoplastic agents listed in Table 1.
[0030] Yet further, the present invention provides pharmaceutical
compositions for treating a multidrug resistant cancer by
inhibiting ABCG2 transporter activity in a subject comprising a
PDE5 inhibitor and a pharmaceutical excipient. In one embodiment,
the PDE5 inhibitor is selected from the group consisting of
sildenafil, vardenafil, tadalafil, lodenafil, udenafil,
benzamidenafil, mirodenafil, avanafil, zaprinast, SLX-2101,
UK-371,800, UK-122764, icariin, DA-8159,
3-[4-(2-hydroxyethyl)piperazin-1-yl]-7-(6-methoxypyridin-3-yl)-1-(2-propo-
xyethyl) pyrido[3,4-b]-pyrazin-2(1H)-one, and their analogs. In
some embodiments, the pharmaceutical composition further comprises
an antineoplastic agent. In some embodiments, the antineoplastic
agent is selected from the group consisting of antineoplastic
agents listed in Table 2.
[0031] Further, the present invention provides pharmaceutical
compositions for treating a multidrug resistant cancer by
inhibiting ABCC10 transporter activity in a subject comprising a
PDE5 inhibitor and a pharmaceutical excipient. In one embodiment,
the PDE5 inhibitor is selected from the group consisting of
sildenafil, vardenafil, tadalafil, lodenafil, udenafil,
benzamidenafil, mirodenafil, avanafil, zaprinast, SLX-2101,
UK-371,800, UK-122764, icariin, DA-8159,
3-[4-(2-hydroxyethyl)piperazin-1-yl]-7-(6-methoxypyridin-3-yl)-1-(2-propo-
xyethyl)pyrido[3,4-b]-pyrazin-2(1H)-one, and their analogs. In some
embodiments, the pharmaceutical composition of the invention
further comprises an antineoplastic agent. In some embodiments, the
antineoplastic agent is selected from the group consisting of
antineoplastic agents listed in Table 3. In some embodiments of the
pharmaceutical composition of the present invention, the subject is
a mammal, e.g., a human subject.
BRIEF DESCRIPTION OF THE DRAWINGS
[0032] FIG. 1 depicts the effects of vardenafil and tadalafil on
the accumulation (FIG. 1A) and efflux (FIG. 1B) of
[.sup.3H]-paclitaxel, as well as ABCB1 expression (FIG. 1C) in
ABCB1-overexpressing cells. FIG. 1A: The accumulation of
[.sup.3H]-paclitaxel was measured after cells were preincubated
with or without vardenafil, tadalafil, or verapamil for 1 h at
37.degree. C., and then incubated with 0.1 .mu.M
[.sup.3H]-paclitaxel for another 2 h at 37.degree. C. * and **
represent p<0.05 and p<0.01, respectively, for values for
[.sup.3H]-paclitaxel accumulation in samples treated with
vardenafil, tadalafil, and verapamil versus those in the control
group. FIG. 1B The percentage of paclitaxel released was plotted as
a function of time. KB-3-1 or KB-C2 cells received either no
reversal agent treatment or were treated with vardenafil and
verapamil throughout the experiment. Cells were pretreated for 1 h,
coincubated with [.sup.3H]-paclitaxel, washed, and reincubated in
paclitaxel-free medium. Cells were collected and levels of
[.sup.3H]-paclitaxel determined either at 0, 60, or 120 min. Data
points represent the means.+-.standard deviations (SD) of
triplicate determinations. FIG. 1C: Effect of vardenafil (left
panel) or tadalafil (right panel) on the expression of ABCB1 for 36
and 72 h, respectively. Independent experiments were performed at
least three times, and a representative experiment is shown.
[0033] FIG. 2 depicts the effects of vardenafil and tadalafil on
the Vi-sensitive ATPase activity of ABCB1, represented as a
function of ATP hydrolysis (FIG. 2A), and the photoaffinity
labeling of ABCB1 with [.sup.125I]-IAAP (iodoarylazidoprazosin),
represented as IAAP incorporation (FIG. 2B). Mean values are given,
and the error bars represent standard error from at least three
independent experiments.
[0034] FIG. 3A is ribbon diagram of open-to-the-cytoplasm
3D-structural conformation of a homology model of human ABCB1 based
on the crystal structure coordinates of mouse ABCB1. The docked
poses of tadalafil (shown on bottom), and vardenafil and IAAP
(shown on top; overlapping) as ball and stick models are shown
within the large hydrophobic cavity of ABCB1 at different inhibitor
binding sites (see also Ding et al. (2011) PLoS ONE 6(4):e19329).
FIG. 3B is the XP-Glide-predicted binding model of vardenafil (left
panel) and tadalafil (right panel). Important amino acids are
depicted as stick models, whereas the two inhibitors are shown as
ball and stick models. The short dotted lines indicate hydrogen
bonding interactions, whereas the long dotted lines indicate
interacting distances.
[0035] FIG. 4 demonstrates that sildenafil increased the
accumulation of [.sup.3H]-paclitaxel or [.sup.3H]-mitoxantrone in
ABCB1- or ABCG2-overexpressing cells. The accumulation of
[.sup.3H]-paclitaxel (FIG. 4A) or [.sup.3H]-mitoxantrone (FIGS. 4B,
4C, and 4D) was measured after cells were preincubated with or
without sildenafil, verapamil, or FTC for 1 h at 37.degree. C. and
then incubated with 0.1 .mu.M [.sup.3H]-paclitaxel or 0.02 .mu.M
[.sup.3H]-mitoxantrone for another 2 h at 37.degree. C. Data points
represent the means.+-.SD of triplicate determinations. * and **
represent p<0.05 and p<0.01, respectively, for values versus
those in the control group. Independent experiments were carried
out at least three times, and a representative experiment is
shown.
[0036] FIG. 5 demonstrates that sildenafil inhibited the efflux of
BODIPY-prazosin (FIGS. 5A-5D) and transport of
[.sup.3H]-E.sub.217.beta.G as well as [.sup.3H]-methotrexate (FIGS.
5E and 5F) by ABCG2. In FIGS. 5A-5D, flow cytometric data (measured
as cell numbers) from HEK293/pcDNA3.1, ABCG2-482-G2, ABCG2-482-R5,
and ABCG2-482-T7 cells are shown after incubation with 250 nM
BODIPY-prazosin alone (solid line) or with 50 .mu.M sildenafil
(dashed line), and 2.5 .mu.M FTC (shaded histogram). In FIGS. 5E
and 5F, membrane vesicles were prepared from HEK293/pcDNA3.1 and
ABCG2-482-R5 cells, and the rates of the uptake of
[.sup.3H]-E.sub.217.beta.G and [.sup.3H]-methotrexate into membrane
vesicles (10 .mu.g protein/reaction) were measured. Data points
represent the means.+-.SD of triplicate determinations. * and **
represent p<0.05 and p<0.01, respectively, for values versus
those in the control group. At least three independent experiments
were carried out, and a representative experiment is shown.
[0037] FIG. 6 depicts the effect of sildenafil on the ATPase
activity and [.sup.125I]-IAAP photoaffinity labeling of ABCB1 and
ABCG2. The Vi-sensitive ATPase activities (measured as a function
of ATP hydrolysis) of ABCB1 (FIG. 6A) and ABCG2 (FIG. 6B) in
membrane vesicles were determined with different concentrations of
sildenafil. Mean values are given, and the error bars represent
standard error from at least three independent experiments. The
photoaffinity labeling (measured as a function of IAAP
incorporation) of ABCB1 (FIG. 6C) and ABCG2 (FIG. 6D) with
[.sup.125I]-IAAP was conducted in the presence of different
concentrations of sildenafil. The autoradiograms and quantification
of incorporation of IAAP into ABCB1 and ABCG2 bands were obtained
from at least two independent experiments. Cyclosporine A (CSA) and
verapamil (Vera) were used as positive controls for inhibition of
[.sup.125I]-IAAP photolabeling of ABCB1, and FTC was used as a
positive control for ABCG2. * and ** represent p<0.05 and
p<0.01, respectively, for values versus those in the control
group.
[0038] FIG. 7 is a model for binding of sildenafil to ABCB1. FIG.
7A is a ribbon diagram of open-to-the-cytoplasm 3D-structural
conformation of a homology model of human ABCB1 based on the
crystal structure coordinates of mouse Mdr3. Sildenafil is shown as
a ball and stick model within the large hydrophobic cavity of ABCB1
characterized by the QZ59-RRR inhibitor binding site (see also Shi
et al. (2011) Cancer Research 71(8):1-13). FIG. 7B is an
XP-Glide-predicted binding model of sildenafil within the QZ59-RRR
binding site. Important amino acids are depicted as stick models,
whereas the inhibitor is shown as a ball and stick model. The short
dotted line indicates a hydrogen bonding interaction, whereas the
long dotted line indicates an interacting distance.
[0039] FIG. 8 represents survival curves at different
concentrations of colchicine (FIG. 8A), vinblastine (FIG. 8B),
paclitaxel (FIG. 8C), and cisplatin (FIG. 8D) for either KB-3-1
cells with (open squares) or without (filled squares) 10 .mu.M
sildenafil; KB-C2 cells with (open diamonds) or without (filled
diamonds) 10 .mu.M sildenafil; or KB-V1 cells with (open triangles)
or without (filled triangles) 10 .mu.M sildenafil. Cell survival
was determined by MTT assay; data points are the means.+-.SD of
triplicate determinations, with a representative experiment
shown.
[0040] FIG. 9 represents survival curves at different
concentrations of flavopiridol (FIG. 9A), mitoxantrone (FIG. 9B),
SN-38 (FIG. 9C), and cisplatin (FIG. 9D) for either S1 cells with
(open squares) or without (filled squares) 50 .mu.M sildenafil;
S1/FLV5000 cells with (open diamonds) or without (filled diamonds)
50 .mu.M sildenafil; or S1-M1-80 cells with (open triangles) or
without (filled triangles) 50 .mu.M sildenafil. Cell survival was
determined by MTT assay; data points are the means.+-.SD of
triplicate determinations, with a representative experiment
shown.
[0041] FIG. 10 represents survival curves at different
concentrations of flavopiridol (FIG. 10A), mitoxantrone (FIG. 10B),
SN-38 (FIG. 10C), and cisplatin (FIG. 10D) for either MCF-7 cells
with (open squares) or without (filled squares) 50 .mu.M
sildenafil; MCF-7/FLV10000 cells with (open diamonds) or without
(filled diamonds) 50 .mu.M sildenafil; or MCF-7/ADVP3000 cells with
(open triangles) or without (filled triangles) 50 .mu.M sildenafil.
Cell survival was determined by MTT assay; data points are the
means.+-.SD of triplicate determinations, with a representative
experiment shown.
[0042] FIG. 11 represents survival curves at different
concentrations of flavopiridol (FIG. 11A), mitoxantrone (FIG. 11B),
SN-38 (FIG. 11C), and cisplatin (FIG. 11D) for either HEK293/pcDNA3
cells with (open squares) or without (filled squares) 50 .mu.M
sildenafil; HEK293/ABCG2-G2 cells with (open diamonds) or without
(filled diamonds) 50 .mu.M sildenafil; HEK293/ABCG2-R5 cells with
(open triangles) or without (filled triangles) 50 .mu.M sildenafil,
or HEK293/ABCG2-T7 cells with (open circles) or without (filled
circles) 50 .mu.M sildenafil. Cell survival was determined by MTT
assay; data points are the means.+-.SD of triplicate
determinations, with a representative experiment shown.
[0043] FIG. 12 represents the effects of sildenafil treatment on
the growth of tumor xenografts. Mice were treated with either
vehicle, sildenafil alone, paclitaxel alone, or a combination of
sildenafil and paclitaxel. FIG. 12A demonstrates the sizes of the
excised tumors, and FIG. 12B demonstrates the tumor weight of the
excised tumors. * represents p<0.05 for values versus those
indicated.
[0044] FIG. 13 demonstrates the effects of sildenafil treatment on
tumor volume in mouse xenografts, measured on days 0, 1, 3, 6, 9,
12, 15, and 18 after transplantation. Mice were treated with either
vehicle, sildenafil alone, paclitaxel alone, or a combination of
sildenafil and paclitaxel. * represents p<0.05 for values versus
those in the vehicle group; *, # represents p<0.05 for values
versus those in the paclitaxel alone group.
[0045] FIG. 14 demonstrates the effects of PDE5 inhibitors on the
reversal of ABCC10-mediated drug resistance in HEK/MRP7 cells,
showing the survival curves of HEK/MRP7 cells in the presence or
absence of sildenafil, vardenafil, or tadalafil at 5 .mu.M and the
parental HEK293-pcDNA3.1 cells at different concentrations of
paclitaxel (FIG. 14A), docetaxel (FIG. 14B), vinblastine (FIG.
14C), and cisplatin (FIG. 14D). Cell survival was determined by MTT
assay as described herein. Data points represent the means.+-.SD of
triplicate determinations. Experiments were performed at least
three independent times.
[0046] FIG. 15 shows the effects of sildenafil, vardenafil, and
tadalafil on the accumulation of [.sup.3H]-paclitaxel in
HEK293-pcDNA3.1 and HEK/MRP7 cells. The intracellular accumulation
of [.sup.3H]-paclitaxel was measured by scintillation counting
after cells were preincubated with or without sildenafil,
vardenafil, tadalafil, or cepharanthine for 2 h at 37.degree. C.
and then incubated with 0.1 .mu.M [.sup.3H]-paclitaxel for another
2 h at 37.degree. C. Data points represent the means.+-.SD of
triplicate determinations. Experiments were performed at least
three independent times. ** represents p<0.01 for values versus
those in the control group.
[0047] FIG. 16 demonstrates the effects of sildenafil (FIG. 16A),
vardenafil (FIG. 16B), and tadalafil (FIG. 16C) on the efflux of
[.sup.3H]-paclitaxel in HEK293-pcDNA3.1 and HEK/MRP7 cells. Cells
were preincubated with or without sildenafil, vardenafil, or
tadalafil for 2 h at 37.degree. C., and further incubated with 0.1
.mu.M [.sup.3H]-paclitaxel for another 2 h at 37.degree. C. Cells
were then incubated in fresh medium with or without PDE5 inhibitors
for different time periods at 37.degree. C. Cells were then
collected, and the intracellular levels of [.sup.3H]-paclitaxel
were measured by scintillation counting. A time course versus
percentage of intracellular [.sup.3H]-paclitaxel was plotted (0,
30, 60, and 120 min). Data points represent the means.+-.SD of
triplicate determinations. Experiments were performed at least
three independent times.
[0048] FIG. 17 shows immunoblot detection (FIG. 17A) and
immunofluorescence detection (FIG. 17B) of MRP7 in HEK/MRP7 cells
following incubation with PDE5 inhibitors. Cell lysates were
prepared from HEK/MRP7 cells incubated with 5 .mu.M sildenafil,
vardenafil, and tadalafil for different time periods (0, 24, 48,
and 72 h). Immunoblot detection of MRP7 (FIG. 17A) was performed
using polyclonal anti-MRP7 antibody; GAPDH was used as an internal
control for equal loading. Equal amounts (40 .mu.g of protein) of
total cell lysates were used for each sample. The localization of
MRP7 by immunofluorescence (FIG. 17B) was performed on
paraformaldehyde-fixed cells using polyclonal antibody D19 against
MRP7 (1:200) and Alexa Flour.RTM. 488 donkey anti-goat IgG
(1:2000). Propidium iodide was used for nuclear counterstaining
Results from a representative experiment are shown; similar results
were obtained in two other trials.
DETAILED DESCRIPTION OF THE INVENTION
[0049] The present invention is based on the finding that
phosphodiesterase 5 (PDE5) inhibitors can be useful for treating
multidrug resistance in cells, e.g., cancer cells.
Methods of Treatment
[0050] The present invention provides a method of treating
multidrug resistance in cancer cells in a subject in need thereof,
comprising administering to the subject a therapeutically effective
amount of a PDE5 inhibitor.
[0051] As used herein, "multidrug resistance" (commonly abbreviated
as "MDR") refers to a phenomenon wherein disease-causing organisms
or cells are able to evade treatment with agents designed to target
them, often agents that are structurally or mechanistically
distinct. As such, multidrug resistance can be viewed as
simultaneous resistance to different drugs that have either the
same or different targets, and either similar or distinct chemical
structures. Organisms or cells may acquire a multidrug resistant
phenotype upon exposure to a single drug, e.g., a
chemotherapeutic/antineoplastic drug (also referred to herein as an
"anticancer drug"). Multidrug resistance in cancer, or multidrug
resistant cancer, refers to the situation wherein at least one
cancer cell becomes resistant, or nonresponsive, to treatment with
drugs, e.g., chemotherapeutic/antineoplastic drugs (agents,
compounds, etc.), designed to target it, e.g.,
chemotherapeutic/antineoplastic drugs that may be structurally
and/or mechanistically distinct. Therefore, cancer cells sensitive
to the treatment with chemotherapeutic or antineoplastic drugs are
killed, and the resistant cancer cell divides and propagates into a
drug-resistant cancer growth.
[0052] The multidrug resistant phenotype in cancer cells is usually
a result of either intrinsic and/or acquired overexpression of the
ATP-binding cassette transporters (ABC transporters). ABC
transporters are conserved proteins that translocate substrates
across cellular membranes. There are nearly fifty known ABC
transporters in the human genome, divided into seven families
(i.e., A-G), with a variety of transport functions, including
transport of biological compounds, toxins, xenobiotics and
drugs.
[0053] As used herein, "MDR transporters" refers to ABC
transporters involved in multidrug resistance in cancer cells. The
three MDR transporters with previously known predominant function
in multidrug resistance to cancer chemotherapeutics are ABCB1
(P-glycoprotein or Pgp), ABCC1 (multidrug resistance protein 1 or
MRP1), and ABCG2 (breast cancer resistant protein or BCRP), with
ABCB1 transporter overexpression occurring in nearly 50% of all
cancers. In addition, as disclosed herein, ABCC10 (MRP7) has now
been shown to function in multidrug resistance to cancer
chemotherapeutics.
[0054] The types of cancers or metastases that may be affected by
multidrug resistance, and thus may be treated using the methods of
the present invention, include but are not limited to the
following: cancers of the central nervous system, skin, respiratory
tract, oral cavity, eyes, bone, skin, connective tissue,
gastrointestinal tract, cardiovascular system (heart, vasculature,
etc.), ear, sinuses, salivary glands, urethra, lips, bile duct,
gall bladder, etc.; hematological-associated cancers (e.g.,
lymphomas, leukemias, myeloproliferative cancers, etc.); breast,
ovarian, cervical, vaginal, uterine, testicular, renal, penile,
bladder, prostate, nasopharyngeal, endocrine (thyroid, parathyroid,
pituitary, adrenal, pancreatic, pineal), splenic, etc. cancers; as
well as viral-associated cancers such as Kaposi's sarcoma, etc. In
one embodiment of the invention, the cancer type that is affected
by multidrug resistance and is thus to be treated with the methods
of the present invention is lung cancer.
[0055] A subject in need thereof, as used herein, refers to any
subject that is determined to be in need of therapy, e.g., therapy
for the treatment of multidrug resistance with PDE5 inhibitors, by
a treating physician (e.g., an oncologist). For example, a subject
in need thereof may be a subject for whom a treatment with one or
more chemotherapeutic agents was determined to be unsuccessful. The
success of a treatment with any particular therapy is evaluated
based on the ability of that therapy to eliminate or reduce tumor
burden, i.e., to eradicate or diminish the number of cancer cells.
The most suitable way to measure the elimination or reduction in
tumor burden may vary by the type of cancer involved. A treating
physician will know the most suitable way to measure elimination or
reduction in tumor burden, e.g., through obtaining and analyzing a
biopsy sample. A subject in need thereof may be a subject suffering
from cancer, e.g., multidrug resistant cancer. In one embodiment,
the subject in need thereof may be suffering from lung cancer.
[0056] A subject in need thereof may be a human or nonhuman animal.
The term "nonhuman animal" includes all vertebrates, such as
nonhuman primates, sheep, dogs, cows, chickens, amphibians,
reptiles, etc. A subject may be a mammalian subject.
[0057] As described herein, the present invention relates to the
treatment of multidrug resistance by administering a
phosphodiesterase inhibitor, e.g., a PDE5 inhibitor.
Phosphodiesterases are protein enzymes that function to break down
a phosphodiester bond. Cyclic nucleotide phosphodiesterases, a
subclass of phosphodiesterases, function to break down the
phosphodiester bond in cyclic nucleotides, e.g., converting cyclic
AMP (3'5'-cAMP; also commonly abbreviated as cAMP) and cyclic GMP
(3'5'-cGMP; also commonly abbreviated as cGMP) to 5'-AMP and
5'-GMP, respectively; thereby inactivating these important second
messengers and halting signal transduction cascades. Increased
levels of cyclic GMP are associated with smooth muscle
vasodilation, and thus increased blood flow. PDE5 is a
phosphodiesterase responsible for converting second messenger
cyclic GMP into 5'-GMP, thereby terminating its biological actions
and potentially leading to vasoconstriction and reduced blood flow.
Inhibitors of PDE5 increase cyclic GMP levels, and have been used
in the clinic to treat erectile dysfunction and pulmonary arterial
hypertension.
[0058] PDE5 inhibitors include, but are not limited to, vardenafil
(LEVITRA.RTM.); sildenafil (VIAGRA.RTM.); tadalafil (CIALIS.RTM.);
lodenafil (HELLEVA.RTM.); udenafil (ZYDENA.RTM.); benzamidenafil
(Zou et al. (2008) J. Pharm. Biomed. Anal. 47:255-59); SLX-2101;
mirodenafil; avanafil; UK-371,800 (Bunnage et al. (2008) Bioorg.
Med. Chem. Lett. 18:6033-36); UK-122,764; zaprinast;
3-[4-(2-hydroxyethyl)piperazin-1-yl]-7-(6-methoxypyridin-3-yl)-1-(2-propo-
xyethyl)pyrido[3,4-b]-pyrazin-2(1H)-one (Hughes et al. (2010) J.
Med. Chem. 53:2656-60); icariin and its derivatives (e.g.,
3,7-bis(2-hydroxyethyl)icaritin) (Dell'Agli et al. (2008) J. Nat.
Prod. 71:1513-17); DA-8159 (Oh et al. (2000) Arch. Pharm. Res. (NY)
23:471-76); and their analogs. These and other known PDE5
inhibitors are further described in Hughes et al. (2009) Biorg.
Med. Chem. Lett. 19:5209-13; Rotella et al. (2000) J. Med. Chem.
43:1257-63; Duan et al. (2009) Bioorg. Med. Chem. Lett. 19:2777-79;
Giovannoni et al. (2006) J. Med. Chem. 49:5363-71; Yoo et al.
(2007) Bioorg. Med. Chem. Lett. 17:4271-74; Daugan et al. (2003) J.
Med. Chem. 46:4525-32; Arnold et al. (2007) Bioorg. Med. Chem.
Lett. 17:2376-79; and Eros et al. (2008) Curr. Med. Chem.
15:1570-85, all incorporated by reference herein in their
entireties.
[0059] As taught herein, PDE5 inhibitors, e.g., inhibitors such as
sildenafil, tadalafil, and vardenafil, are useful in blocking
multidrug resistance in cancer, and thereby increasing the efficacy
of antineoplastic agents. Thus, because the present invention shows
that PDE5 inhibitors are capable of reversing multidrug resistance,
for the purposes of this invention, PDE5 inhibitors, as well as
other agents known to reverse multidrug resistance, are sometimes
referred to as "reversal agents." PDE5 inhibitors can increase the
efficacy of antineoplastic drugs in several ways. PDE5 inhibitors
can block the efflux of antineoplastic agents through MDR
transporters. For example, it is known that the ATPase activity of
ABC transporters, e.g., MDR transporters, is stimulated in the
presence of their transport substrates, e.g., either their natural
transport substrates or antineoplastic drugs. As shown herein, the
ATPase activity of the MDR transporters is stimulated in the
presence of PDE5 inhibitors, e.g., sildenafil, tadalafil, and
vardenafil. Therefore, in one embodiment, PDE5 inhibitors may act
as transport substrates of MDR transporters (e.g., competitive
inhibitors of MDR transporters), thereby reducing the ability of
MDR transporters to efflux antineoplastic agents. By increasing the
efficacy of antineoplastic agents, PDE5 inhibitors can decrease
cancer cell survival, thus having a positive effect on the outcome
of cancer treatment.
[0060] PDE5 inhibitors can also positively affect cancer treatment
because PDE5 inhibitors block cGMP hydrolysis, and thus increase
cGMP levels, which may lead to activation of protein kinase G
(PKG). Activation of PKG in turn leads to growth suppression or
apoptosis.
[0061] The methods and compositions of the present invention
encompass the use of specific or selective PDE5 inhibitors.
However, one skilled in the art will understand that a nonspecific
PDE5 inhibitor (e.g., a PDE inhibitor that blocks the function of
other cGMP phosphodiesterases, in addition to its ability to block
PDE5) may be effective in blocking MDR transporters, such as ABCB1,
ABCC10, and ABCG2. In addition, one skilled in the art will
understand that a PDE inhibitor that is able to block the function
of other cyclic nucleotide phosphodiesterases (e.g., cyclic GMP
phosphodiesterases), exclusive of an ability to block PDE5, may be
effective in blocking MDR transporters such as ABCB1, ABCC10, and
ABCG2. Examples of phosphodiesterases that are able to catalyze
cyclic GMP to 5'-GMP conversion include, but are not limited to,
PDE1, PDE1B, PDE1C, PDE2A, PDE6A, PDE6B, PDE6C, PDE9A, PDE10A, and
PDE11A.
[0062] MDR transporters are expressed in the membranes of various
cell types (both normal and cancer cells), including cell types
present at sites of drug absorption, e.g., intestinal epithelial
cells; therefore, in one embodiment of the invention, a PDE5
inhibitor may block multidrug resistance, at least in part, by
blocking the ability of MDR transporters expressed on epithelial
cells to efflux antineoplastic drugs, thereby aiding drug
absorption. In another embodiment of the invention, a PDE5
inhibitor blocks multidrug resistance by blocking the ability of
MDR transporters expressed on cancer cells to efflux antineoplastic
drugs.
[0063] Depending on the type of MDR transporter involved, it may be
preferable to use a certain PDE5 inhibitor to block antineoplastic
drug efflux. For example, when ABCG2 transporter-mediated efflux is
involved, it may be preferable to use, e.g., sildenafil, because
the ABCG2 transporter is relatively insensitive to inhibition by
other PDE5 inhibitors, e.g., vardenafil and tadalafil.
[0064] Numerous chemotherapeutic agents (antineoplastic agents)
have been developed for treating cancer. In the clinic, depending
on cancer diagnosis, different antineoplastic agents are preferred.
Antineoplastic agents used for treating cancers, or under
investigation for use in treating cancers, may include, but are not
limited to: (1) nitrogen mustards, such as mechlorethamine,
cyclophosphamide, ifosfamide, melphalan (L-sarcolysin, L-PAM),
uramustine, and chlorambucil; (2) ethylenimines and methylmelamines
(aziridines), such as thioTEPA, hexamethylmelamine (HMM,
altretamine), and triethylenemelamine (TEM); (3) nitrosoureas, such
as carmustine (BCNU), lomustine (CCNU), semustine (Methyl-CCNU),
fotemustine, and streptozotocin; (4) alkyl sulfonates, such as
busulfan; (5) azines and hydrazines (triazenes), such as
dacarbazine (DTIC) and procarbazine (MATULANE.RTM.); (6) platins,
such as cisplatin, carboplatin and oxaliplatin; (7) pteridines,
such as methotrexate; (8) pyrimidine analogs, such as
5-fluorouracil, fluorodeoxyuridine (floxuridine, FUDR.RTM.),
cytarabine (AraC), and gemcitabine (GEMZAR.RTM.); (9) purine
analogs and related inhibitors, such as 6-mercaptopurine (6-MP),
thioguanine (6-TG), and pentostatin; (10) antimitotic drugs, for
example, (a) vinca alkaloids, such as vinblastine, vincristine,
vindesine, and vinorelbine; and (b) taxanes, such as paclitaxel and
docetaxel; (11) camptothecins, such as topotecan, irinotecan,
diflomotecan, and 9-aminocamptothecin; (12) epipodophyllotoxins,
such as etoposide and teniposide; (13) antibiotics, for example,
(a) actinomycins, such as actinomycin D (DACTINOMYCIN.RTM.),
mitomycin, and plicamycin; (b) anthracyclines, such as daunorubicin
(daunomycin), doxorubicin, idarubicin, epirubicin, valrubicin,
bisantrene, and mitoxantrone; and (c) other antibiotics, e.g.,
azacitidine (5-azacytidine) and pentostatin (deoxycoformycin); (14)
hormones and antagonists, for example, (a) adrenocorticosteroids,
such as prednisone, dexamethasone, methylprednisolone, and mitotane
(o,p'-DDD); (b) progestins, such as hydroxyprogesterone
(17-hydroxyprogesterone), medroxyprogesterone, medroxyprogesterone
acetate, megestrol, and megestrol acetate; (c) estrogens, such as
DES and ethinyl estradiol; (d) antiestrogens, such as tamoxifen;
(e) antiandrogens, such as flutamide; (f) androgens, such as
testosterone and fluoxymesterone; (g) gonadotropin-releasing
hormone analogs, such as leuprorelin (leuprolide acetate); and (h)
aromatases, such as aminoglutethimide, anastrozole, letrozole, and
exemestane; (15) heavy metal compounds, such as arsenic trioxide;
(16) substituted ureas, such as hydroxyurea; (17) retinoids, such
as tretinoin (retinoic acid), alitretinoin, and isotretinoin; (18)
tyrosine kinase inhibitors, such as imatinib, gefitinib, lapatinib,
erlotinib, sunitinib, nilotinib, vandetanib, dasatinib, afatinib,
neratinib, axitinib, pazopanib, sorafenib, toceranib, lestaurtinib,
cediranib, regorafenib, semaxanib, bosutinib, tandutinib, aparinib,
AG1478, and crizotinib; (19) angiogenesis inhibitors, such as
marimastat, angiostatin, endostatin, and thalidomide; (20) enzymes,
such as L-asparaginase; (21) CDK inhibitors, such as flavopiridol,
and (22) biologic response modifiers, such as natural and synthetic
interferons. Exemplary antineoplastic agents of choice for various
cancer diagnoses are summarized in, e.g., Basic and Clinical
Pharmacology, pp. 945-46 (Bertram Katzung ed., Lange Medical
Books/McGraw-Hill 8.sup.th ed. 2001); and Goodman and Gilman's The
Pharmacological Basis of Therapeutics, pp. 1383-85 (Joel G.
Hardman, Lee E. Limbird, and Alfred Goodman Gilman eds.,
McGraw-Hill Medical Publishing Division 10.sup.th Ed. 2001). Some
sources use different groupings/classes to categorize the various
antineoplastic or chemotherapeutic agents (drugs, compounds, etc.),
but all appropriate antineoplastic agents are contemplated in the
present invention.
[0065] Different agents, e.g., antineoplastic agents, are known to
be substrates for different MDR transporters, e.g., ABCB1, ABCC1,
ABCC10, and ABCG2 transporters. A skilled artisan will know which
antineoplastic agents are substrates for a particular MDR
transporter. Exemplary antineoplastic agents that are substrates
for ABCB1 are presented in Table 1; exemplary antineoplastic agents
that are substrates for ABCG2 are presented in Table 2; and
exemplary antineoplastic agents that are substrates for ABCC10 are
presented in Table 3. For example, a skilled artisan will know that
paclitaxel, vinblastine, and vincristine are substrates for the
ABCB1 transporter, whereas cisplatin is not a substrate for the
ABCB1 transporter. Similarly, a skilled artisan will know that
flavopiridol, mitoxantrone, SN-38, and methotrexate are substrates
for the ABCG2 transporter, whereas cisplatin is not a substrate for
the ABCG2 transporter.
TABLE-US-00001 TABLE 1 Representative Antineoplastic Agents That
Are Substrates for the ABCB1 Transporter Classical Antineoplastic
Agents Tyrosine Kinase Inhibitors Vinblastine Tandutinib
Vincristine Erlotinib Vinorelbine Dasatinib Vindesine Imatinib
Paclitaxel Nilotinib Docetaxel Lapatinib Doxorubicin Sunitinib
Daunorubicin Apatinib Idarubicin AG1478 Bisantrene Mitoxantrone
Etoposide Teniposide Dactinomycin Mitomycin Plicamycin Methotrexate
Topotecan Irinotecan SN-38
TABLE-US-00002 TABLE 2 Representative Antineoplastic Agents That
Are Substrates for the ABCG2 Transporter Classical Antineoplastic
Agents Tyrosine Kinase Inhibitors Aza-anthrapyrazole Tandutinib
(BBR 3390) 9-Aminocamptothecin Erlotinib Bisantrene Dasatinib
Diflomotecan Gefitinib Doxorubicin Imatinib Daunorubicin Lapatinib
Epirubicin Sorafenib Etoposide Flavopiridol GV-196771 Irinotecan
(CPT-11) J-107088 Methotrexate and its polyglutamates Mitoxantrone
NB-506 Quinazoline SN-38 Teniposide Topotecan
TABLE-US-00003 TABLE 3 Representative Antineoplastic Agents That
Are Substrates for the ABCC10 Transporter Classical Antineoplastic
Agents Tyrosine Kinase Inhibitors Vinblastine Nilotinib Vincristine
Erlotinib Vinorelbine Lapatinib Paclitaxel Imatinib Docetaxel
Tandutinib Etoposide Masitinib Teniposide Dactinomycin SN-38
Gemcitabine Cytarabine Epothilone-B
[0066] In one embodiment, the present invention encompasses a
method of treating multidrug resistance in cancer cells in a
subject in need thereof by inhibiting ABCB1 transporter activity,
comprising administering to the subject a therapeutically
effective, nontoxic amount of a PDE5 inhibitor. The PDE5 inhibitor
may be selected from a variety of known PDE5 inhibitors. In one
embodiment, the PDE5 inhibitor is sildenafil, tadalafil, or
vardenafil. In another embodiment, the PDE5 inhibitor is
administered in combination with a chemotherapeutic agent(s). In a
further embodiment, the chemotherapeutic (antineoplastic) agent(s)
is selected from the group consisting of paclitaxel, vincristine,
and vinblastine.
[0067] In a different embodiment, the present invention encompasses
a method of treating multidrug resistance in cancer cells in a
subject in need thereof by inhibiting ABCG2 transporter activity,
comprising administering to the subject a therapeutically
effective, nontoxic amount of a PDE5 inhibitor. In one embodiment,
the PDE5 inhibitor is sildenafil. In another embodiment, the PDE5
inhibitor is administered in combination with a chemotherapeutic
agent(s). In a further embodiment, the chemotherapeutic
(antineoplastic) agent(s) is selected from the group consisting of
SN-38, flavopiridol, mitoxantrone, and/or methotrexate.
[0068] In a different embodiment, the present invention encompasses
a method of treating multidrug resistance in cancer cells in a
subject in need thereof by inhibiting ABCC10 transporter activity,
comprising administering to the subject a therapeutically
effective, nontoxic amount of a PDE5 inhibitor. In one embodiment,
the PDE5 inhibitor is sildenafil or vardenafil. In another
embodiment, the PDE5 inhibitor is administered in combination with
a chemotherapeutic agent(s). In a further embodiment, the
chemotherapeutic (antineoplastic) agent(s) is selected from the
group consisting of paclitaxel, docetaxel, vinblastine, and
vincristine.
[0069] An additional embodiment of the invention comprises a method
of treating cancer, e.g., multidrug resistant cancer, in a subject
by inhibiting MDR transporter activity, e.g., ABCB1, ABCC10, or
ABCG2 transporter activity, comprising administering to the subject
a therapeutically effective amount of a chemotherapeutic agent and
a PDE5 inhibitor. Exemplary embodiments of chemotherapeutic agents
include vincristine, vinblastine, paclitaxel, docetaxel, SN-38,
flavopiridol, mitoxantrone, and methotrexate. Exemplary embodiments
of PDE5 inhibitors include vardenafil, tadalafil, and sildenafil.
In one embodiment, the method comprises treating multidrug
resistant lung cancer using a combination of a PDE5 inhibitor and
paclitaxel. A "therapeutically effective amount" means an amount of
a compound, alone or in a combination, required to treat,
ameliorate, reduce or prevent multidrug resistance in cancer cells
of a subject. The therapeutically effective amount of an active
compound(s), e.g., a PDE5 inhibitor(s), varies depending upon the
route of administration, age, body weight, and general health of
the subject. Ultimately, the attending physician or veterinarian
will decide the appropriate amount and dosage regimen.
[0070] In addition to methods of treating cancer, the inventors
contemplate that PDE5 inhibitors may be effective in treating other
disorders with ABC transporter involvement (may be referred to as
ABC-associated disorders), e.g., infectious disorders. It is known
in the art that ABC transporters are localized in the membranes of
pathogenic organisms, and therefore, are able to efflux drugs
designed to target them. Therefore, one embodiment of the present
invention encompasses a method of treating a multidrug-resistant
infectious disease comprising administering to the subject a
therapeutically effective amount of a PDE5 inhibitor. Similarly,
the inventors contemplate that PDE5 inhibitors may be effective in
treating inflammatory conditions, e.g., gout.
Pharmaceutical Compositions and Methods of Administration
[0071] Certain embodiments of the invention include compositions
comprising PDE5 inhibitors. The compositions are suitable for
pharmaceutical use and administration to patients. The compositions
can comprise PDE5 inhibitors and pharmaceutical excipients. The
compositions can also comprise PDE5 inhibitors and chemotherapeutic
agents, i.e., antineoplastic agents, as well as pharmaceutical
excipients. As used herein, "pharmaceutical excipients" include
solvents, dispersion media, coatings, antibacterial and antifungal
agents, isotonic and absorption delaying agents, etc., that are
compatible with pharmaceutical administration. Use of these agents
for pharmaceutically active substances is well known in the art.
The compositions may also contain other active compounds providing
supplemental, additional, or enhanced therapeutic functions. The
pharmaceutical compositions may also be included in a container,
pack, or dispenser, together with instructions for
administration.
[0072] A pharmaceutical composition of the invention is formulated
to be compatible with its intended route of administration. Methods
to accomplish such administration are known to those of ordinary
skill in the art. Pharmaceutical compositions may be topically or
orally administered, or capable of transmission across mucous
membranes. Examples of administration of a pharmaceutical
composition include oral ingestion or inhalation. Administration
may also be intravenous, intraperitoneal, intramuscular,
intracavity, subcutaneous, cutaneous, or transdermal.
[0073] Solutions or suspensions used for intradermal or
subcutaneous application typically include at least one of the
following components: a sterile diluent, such as water, saline
solution, fixed oils, polyethylene glycol, glycerine, propylene
glycol, or other synthetic solvent; antibacterial agents, such as
benzyl alcohol or methyl parabens; antioxidants, such as ascorbic
acid or sodium bisulfite; chelating agents, such as
ethylenediaminetetraacetic acid (EDTA); buffers, such as acetate,
citrate, or phosphate; and tonicity agents, such as sodium chloride
or dextrose. The pH can be adjusted with acids or bases by methods
known in the art. Such preparations may be enclosed in, e.g.,
ampoules, disposable syringes, or multiple dose vials.
[0074] Solutions or suspensions used for intravenous administration
include a carrier such as physiological saline, bacteriostatic
water, CREMOPHOR EL.RTM. (BASF Corp., Ludwigshafen, Germany),
ethanol, or polyol. In all cases, the composition must be sterile
and fluid for easy syringability. Proper fluidity can often be
obtained using lecithin or surfactants. The composition must also
be stable under the conditions of manufacture and storage.
Prevention from introduction or growth of microorganisms can be
achieved with antibacterial and antifungal agents, e.g., parabens,
chlorobutanol, phenol, ascorbic acid, thimerosal, etc. In many
cases, isotonic agents (sugar), polyalcohols (e.g., mannitol and
sorbitol), or sodium chloride may be included in the composition.
Prolonged absorption of the composition can be accomplished by
adding an agent that delays absorption, e.g., aluminum monostearate
or gelatin.
[0075] Oral compositions include an inert diluent or edible
carrier. For the purpose of oral administration, PDE5 inhibitors,
or combinations of PDE5 inhibitors and antineoplastic agents, can
be incorporated with excipients and placed, e.g., in tablets,
troches, capsules, or gelatin. Pharmaceutically compatible binding
agents or adjuvant materials can be included in the composition.
The compositions may contain, for example, (1) a binder, such as
microcrystalline cellulose, gum tragacanth or gelatin; (2) an
excipient, such as starch or lactose; (3) a disintegrating agent,
such as alginic acid, PRIMOJEL.RTM., or corn starch; (4) a
lubricant, such as magnesium stearate; (5) a glidant, such as
colloidal silicon dioxide; and/or (6) a sweetening or flavoring
agent.
[0076] The composition may also be administered by a transmucosal
or transdermal route. Transmucosal administration can be
accomplished by lozenges, nasal sprays, inhalers, or suppositories.
Transdermal administration can be accomplished with
composition-containing ointments, salves, gels, or creams known in
the art. For transmucosal or transdermal administration, penetrants
appropriate to the barrier to be permeated are used. For
administration by inhalation, a PDE5 inhibitor, either alone or in
combination with a chemotherapeutic agent, may be delivered in an
aerosol spray from a pressured container or dispenser, which
contains a propellant (e.g., liquid or gas), or a nebulizer.
[0077] In certain embodiments, a PDE5 inhibitor, either alone or in
combination with a chemotherapeutic agent, is prepared with
carriers to protect the PDE5 inhibitor and/or the chemotherapeutic
agent against rapid elimination from the body. Biodegradable
polymers (e.g., ethylene vinyl acetate, polyanhydrides,
polyglycolic acid, collagen, polyorthoesters, and polylactic acid)
are often used. Methods for the preparation of such formulations
are known by those skilled in the art. Liposomal suspensions can
also be used as pharmaceutically acceptable carriers. The liposomes
can be prepared according to established methods known in the
art.
[0078] In some embodiments of the invention, PDE5 inhibitors and
antineoplastic agents are administered in a formulation and via a
route already utilized in the clinic for those particular agents.
For example, sildenafil, vardenafil, and tadalafil are administered
orally. In another example, sildenafil, vardenafil, and tadalafil
are administered intravenously.
[0079] The compositions of the invention are administered in
therapeutically effective amounts. Appropriate dosages can be
determined by a physician based upon clinical indications. For
example, the compositions may be given as a bolus dose or a
continuous infusion.
[0080] Examples of dosage ranges for PDE5 inhibitors that can be
administered to a subject include: 1 .mu.g/kg to 20 mg/kg, 1
.mu.g/kg to 10 mg/kg, 1 .mu.g/kg to 1 mg/kg, 10 .mu.g/kg to 1
mg/kg, 10 .mu.g/kg to 100 .mu.g/kg, 100 .mu.g/kg to 1 mg/kg, 250
.mu.g/kg to 2 mg/kg, 250 .mu.g/kg to 1 mg/kg, 500 .mu.g/kg to 2
mg/kg, 500 .mu.g/kg to 1 mg/kg, 1 mg/kg to 2 mg/kg, 1 mg/kg to 5
mg/kg, 5 mg/kg to 10 mg/kg, 10 mg/kg to 20 mg/kg, 15 mg/kg to 20
mg/kg, 10 mg/kg to 25 mg/kg, 15 mg/kg to 25 mg/kg, 20 mg/kg to 25
mg/kg, and 20 mg/kg to 30 mg/kg (or higher). In one embodiment, the
dosage range of PDE5 is 10 .mu.g/kg to 3 mg/kg. These dosages may
be administered daily, weekly, biweekly, monthly, or less
frequently, for example, biannually, depending on dosage, method of
administration, cancer(s) to be treated, and individual subject
characteristics.
[0081] In certain circumstances, it may be advantageous to
formulate compositions in dosage unit form for ease of
administration and uniformity of dosage. Dosage unit form as used
herein refers to physically discrete units suited for the patient.
Each dosage unit contains a predetermined quantity of PDE5
inhibitor, either alone or in combination with a chemotherapeutic
agent, calculated to produce a therapeutic effect in association
with the carrier. The dosage unit depends on the characteristics of
the PDE5 inhibitor and/or the chemotherapeutic agent, and the
particular therapeutic effect to be achieved.
Additional Applications of the Invention
[0082] One embodiment of the present invention comprises a method
of increasing sensitivity of a multidrug resistant cancer cell to a
chemotherapeutic agent by inhibiting MDR transporter activity,
e.g., ABCB1, ABCC10, or ABCG2 transporter activity, comprising
contacting the cancer cell with a PDE5 inhibitor. The method of
increasing sensitivity of a multidrug resistant cancer cell may be
performed in vivo, e.g., in a human or an animal cancer patient, or
in an animal cancer model (e.g., a cancer xenograft in a mouse).
The method of increasing sensitivity of a multidrug resistant
cancer cell may also be performed in vitro, e.g., in a cultured
cancer cell, e.g., a primary or immortalized cancer cell. A
cultured cancer cell may be a cell that overexpresses a particular
MDR transporter, e.g., ABCB1, ABCC10, or ABCG2 transporter.
Exemplary embodiments of PDE5 inhibitors include but are not
limited to sildenafil, vardenafil, and tadalafil.
[0083] Another embodiment of the present invention comprises a
method of inhibiting growth (also referred to as proliferation) of
a multidrug resistant cancer cell by inhibiting MDR transporter
activity, e.g., ABCB1, ABCC10, or ABCG2 transporter activity,
comprising contacting the cell with a combination of a
chemotherapeutic agent and a PDE5 inhibitor. Commonly, inhibition
of cell growth or proliferation is presented as the concentration
of the drug, e.g., an antineoplastic drug, required to inhibit the
growth of cells, e.g., drug resistant cancer cells, by 50%,
otherwise known as the IC.sub.50 of the drug. Assays for measuring
inhibition of cell growth, e.g., inhibition due to treatment with
an antineoplastic agent or a combination of an antineoplastic agent
and a PDE5 inhibitor, are known in the art. In one example, a
skilled artisan can use an MTT cytotoxicity assay described in the
Examples. In another example, a skilled artisan can use a colony
formation assay.
[0084] In yet another embodiment, the invention comprises a method
of increasing accumulation of a chemotherapeutic agent in a cancer
cell by inhibiting MDR transporter activity, e.g., ABCB1, ABCC10,
or ABCG2 transporter activity, comprising contacting the cell with
a PDE5 inhibitor.
[0085] In another embodiment, the invention comprises a method of
decreasing efflux of a chemotherapeutic agent from a cancer cell by
inhibiting MDR transporter activity, e.g., ABCB1, ABCC10, or ABCG2
transporter activity, comprising contacting the cell with a PDE5
inhibitor.
[0086] In another embodiment, the invention comprises a method of
stimulating ATPase activity of an MDR transporter, e.g., ABCB1,
ABCC10, or ABCG2 transporter, in a cell comprising contacting the
cell with a PDE5 inhibitor. The present invention teaches that PDE5
inhibitors can stimulate the ATPase activity of MDR transporters.
As described herein, the ability of PDE5 inhibitors to stimulate
ATPase activity of MDR transporters demonstrates that PDE5
inhibitors serve as substrates for the MDR transporters, thereby
inhibiting the efflux of other agents, e.g., chemotherapeutic
agents. One skilled in the art will know how to measure the ATPase
activity of a transporter, e.g., stimulation of the ATPase activity
of a transporter, based on, for example, Ambudkar et al. (1998)
Methods Enzymol. 292:504-14.
[0087] The invention also comprises a method of identifying a PDE5
inhibitor capable of blocking multidrug resistance in cancer cells
comprising administering a PDE5 inhibitor to a cancer cell, wherein
the PDE5 inhibitor is administered in a combination with a
chemotherapeutic agent, and determining at least one of the
following: (1) increased inhibition of growth of the cancer cell in
the presence of the PDE5 inhibitor, in comparison with the same
determination in the absence of the PDE5 inhibitor; (2) increased
accumulation of the chemotherapeutic agent in the cancer cell in
the presence of the PDE5 inhibitor, in comparison with the same
determination in the absence of the PDE5 inhibitor; and (3)
decreased efflux of the chemotherapeutic agent from the cancer cell
in the presence of the PDE5 inhibitor, in comparison with the same
determination in the absence of the PDE5 inhibitor. Specifically,
in one embodiment, the method of identifying a PDE5 inhibitor
capable of blocking multidrug resistance in a cancer cell
comprises: (a) a step of administering to the cancer cell a
chemotherapeutic agent known to induce multidrug resistance in that
cancer cell; (b) a step of measuring either (1) growth of the
cancer cell, (2) accumulation of the chemotherapeutic agent in the
cancer cell, or (3) efflux of the chemotherapeutic agent from the
cancer cell; (c) a step of administering a PDE5 inhibitor to the
cancer cell; (d) a step of measuring either (1) growth of the
cancer cell, (2) accumulation of the chemotherapeutic agent in the
cancer cell, or (3) efflux of the chemotherapeutic agent from the
cancer cell in the presence of the PDE5 inhibitor; and (e) a step
of comparing the measurements in steps (b) and (d), wherein a
determination of (1) decreased growth of the cancer call, (2)
increased accumulation of the chemotherapeutic agent in the cancer
cell, or (3) decreased efflux of the chemotherapeutic agent from
the cancer cell in the presence of the PDE5 inhibitor indicates
that the PDE5 inhibitor is capable of blocking multidrug
resistance. In addition to measuring (1) growth of the cancer cell,
(2) accumulation of the chemotherapeutic agent in the cancer cell,
or (3) efflux of the chemotherapeutic agent from the cancer cell, a
skilled artisan will know of other assays that may be used to
determine whether a PDE5 inhibitor has a beneficial effect on
multidrug resistance.
[0088] As used herein, the step of contacting the cell, e.g., the
cancer cell, is carried out in vivo, in vitro, or ex vivo. In one
embodiment, the step of contacting a cell is carried out in a human
or nonhuman vertebrate subject, e.g., a mammalian subject, e.g., a
human subject.
[0089] The entire contents of all references, patent applications,
and patents cited throughout this application are hereby
incorporated by reference herein.
EXAMPLES
[0090] The invention will be further illustrated in the following
nonlimiting examples. These Examples are set forth to aid in the
understanding of the invention but are not intended to, and should
not be construed to, limit its scope in any way. The Examples do
not include detailed descriptions of conventional methods that are
well known to those of ordinary skill in the art. Additionally, the
present invention is further illustrated in Ding et al. (2011) PLoS
ONE 6(4):e19329, and Shi et al. (2011) Cancer Research 71(8):1-13,
incorporated herein by reference in their entireties.
Example 1
Effects of Vardenafil and Tadalafil on
ABCB1/P-Glycoprotein-Mediated Multidrug Resistance
Example 1.1
Materials and Methods
Example 1.1.a
Reagents
[0091] Vardenafil and tadalafil were purchased from Toronto
Research Chemicals Inc. (Ontario, Canada). [.sup.3H]-paclitaxel
(37.9 Ci/mmol) was purchased from Moravek Biochemicals Inc (Brea,
Calif.). [.sup.125I]-Iodoarylazidoprazosin (IAAP) (2,200 Ci/mmol)
was obtained from Perkin Elmer Life Sciences (Boston, Mass.).
Monoclonal antibody C-219 (against ABCB1) was acquired from Signet
Laboratories Inc. (Dedham, Mass.). Anti-glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) monoclonal antibody (14C10) was obtained from
Cell Signaling Technology, Inc. (Danvers, Mass.). Fumitremorgin C
(FTC) was synthesized by Thomas McCloud Developmental Therapeutics
Program, Natural Products Extraction Laboratory, NCI, NIH
(Bethesda, Md.). ONO1078 was a gift from Dr. Akiyama (Kagoshima
University, Japan). Paclitaxel, vincristine (VCR), colchicine,
7-ethyl-10-hydroxy-20 (S)-camptothecin (SN-38), verapamil, dimethyl
sulfoxide (DMSO), 1-(4,5-dimethylthiazol-2-yl)-3,5-diphenylformazan
(MTT) and all other chemicals were purchased from Sigma Chemical
Company.
Example 1.1.b
Cell Lines and Cell Culture
[0092] The ABCB1/Pgp-overexpressing drug-resistant cell line KB-C2
was established in a cell culture medium by a step-wise selection
of the parental human epidermoid carcinoma cell line KB-3-1 using
colchicine at concentrations up to 2 .mu.g/ml. The KB-C2 and KB-3-1
were kindly provided by Dr. Akiyama (Kagoshima University, Japan).
HEK293-pcDNA3.1 and wild-type HEK/ABCG2 transfected cells were
established by selection with G418 after transfecting HEK293 with
either (1) empty pcDNA3.1 vector or (2) pcDNA3.1 vector containing
full length of ABCG2 coding arginine (R) at amino acid position
482, and were then cultured in a medium with 2 mg/ml of G418
(ABCG2-482-R2 cells). Similarly, the HEK/MRP1 (ABCC1) and HEK/ABCB1
cells were generated by transfecting the HEK293 cells with either
the MRP1 expression vector or the ABCB1 expression vector. All of
the cell lines were grown in Dulbecco's modified Eagle's medium
(DMEM) supplemented with 10% bovine serum, 100 units/ml penicillin,
and 100 mg/ml streptomycin in a humidified incubator containing 5%
CO.sub.2 at 37.degree. C.
Example 1.1.c
Cell Cytotoxicity by MTT Assay
[0093] The MTT assay was used to assess cytotoxicity. The cultured
cells were harvested with trypsin and resuspended in a final
concentration of 4.times.10.sup.3 cells/well for KB-3-1;
7.5.times.10.sup.3 cells/well for KB-C2; and 8.times.10.sup.3 for
all of the other cell lines used in this study. Cells were seeded
evenly in 96-well multiplates. In the reversal experiments,
different concentrations of chemotherapeutic drugs (20 .mu.l/well)
were added into designated wells after 1 h with or without exposure
to potential reversal compounds: vardenafil, tadalafil, verapamil,
ONO-1078, or FTC (20 .mu.l/well). After 68 h of incubation, 20
.mu.l of the MTT solution (4 mg/ml) was added to each well, and the
plate was further incubated for 4 h at 37.degree. C., allowing
viable cells to convert the yellow-colored MTT into dark-blue
formazan crystals. Subsequently, the medium was discarded, and 100
.mu.l of DMSO was added into each well to dissolve the formazan
crystals, generating purple color. The absorbance was determined at
570 nm by an OPSYS Microplate Reader from DYNEX Technologies, Inc.
(Chantilly, Va.). The degree of resistance was calculated by
dividing the IC.sub.50 (concentrations required to inhibit growth,
i.e., proliferation, by 50%) of a chemotherapeutic agent in the
resistant cells by the IC.sub.50 of the chemotherapeutic agent in
the parental sensitive cells. The degree of the reversal of
multidrug resistance (MDR) was calculated by dividing the IC.sub.50
of an anticancer drug, e.g., an antineoplastic or chemotherapeutic
drug (agent, compound), in a cell type in the absence of a reversal
agent by the IC.sub.50 of the anticancer drug in the same cell type
in the presence of the reversal agent. The IC.sub.50 values were
calculated from survival curves using the Bliss method.
Example 1.1.d
[.sup.3H]-Paclitaxel Accumulation and Efflux
[0094] The intracellular accumulation of [.sup.3H]-paclitaxel was
measured as follows. Confluent cells in 24-well plates were
preincubated with or without the reversal agents, e.g., vardenafil,
tadalafil, or verapamil, for 1 h at 37.degree. C. Intracellular
paclitaxel accumulation was measured by incubating cells with 0.1
.mu.M [.sup.3H]-paclitaxel for 2 h in the presence or absence of
the reversal agents at 37.degree. C. The cells were washed three
times with ice-cold PBS, then suspended in fresh medium with or
without reversal agents at 37.degree. C. Aliquots of the
extracellular medium (40 .mu.l) were collected at various time
points (0, 60, 120 min), and finally the cells were collected and
lysed in 10 mM lysis buffer (pH 7.4, containing 1% Triton X-100 and
0.2% SDS). Each sample was placed in scintillation fluid and the
radioactivity was measured in a Packard TRI-CARB1 1900CA liquid
scintillation analyzer from Packard Instrument Company, Inc.
(Downers Grove, Ill.).
Example 1.1.e
Western Blot and Immunofluorescence Analysis
[0095] To determine the effect of vardenafil or tadalafil on the
expression of ABCB1, KB-C2 cells were incubated with 10 .mu.M
vardenafil or tadalafil for 0, 36 and 72 h. Following incubation,
the cells were harvested and rinsed twice with ice-cold PBS, and
total cell lysates were collected with cell lysis buffer
(1.times.PBS, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS,
100 .mu.g/ml phenylmethylsulfonyl fluoride, 10 .mu.g/ml aprotinin,
10 .mu.g/ml leupeptin) for 30 min with gentle rocking, and
clarified by centrifugation at 12,000 rpm for 10 min at 4.degree.
C. Equal amounts (100 .mu.g of protein) of cell lysates were
resolved by sodium dodecyl sulfate polyacrylamide gel
electrophoresis (SDS-PAGE) and electrophoretically transferred onto
polyvinylidene fluoride (PVDF) membranes. After incubation in a
blocking solution containing 5% nonfat milk in TBST buffer (10 mM
Tris-HCL (pH 8.0), 150 mM NaCl, and 0.1% Tween 20) for 1 h at room
temperature, membranes were immunoblotted overnight with primary
monoclonal antibodies C219 (1:200) against ABCB1, or 14C10 (1:200)
against GAPDH, at 4.degree. C. Subsequently, the membranes were
washed three times for 15 min with TBST buffer and incubated at
room temperature for 2 h with HRP-conjugated secondary antibody at
1:1000 dilutions. The membranes underwent three additional washes
for 15 min with TBST buffer and the protein-antibody complexes were
visualized by the enhanced PHOTOTOPE.RTM. HRP Detection Kit (Cell
Signaling Technology, Inc.) and exposed to Kodak medical X-ray
processor (Eastman Kodak Co., Rochester, N.Y.). For the
immunofluorescence analysis, cells (2.times.10.sup.3) were seeded
in 24-well plates and, after 12 h of incubation at 37.degree. C. in
a humidified atmosphere of 5% CO.sub.2, vardenafil or tadalafil at
10 mM were added into the wells. After incubation for 72 h with
vardenafil or tadalafil, cells were washed with PBS and fixed with
4% paraformaldehyde for 15 min at room temperature and then rinsed
with PBS three times. The monoclonal antibody C219 against ABCB1
(1:500) (Signet Laboratories Inc., Dedham, Mass.) was added for
overnight incubation, followed by incubation for 1 h in Alexa Fluor
488 goat antimouse IgG (1:1000; Molecular Probe, Carlsbad, Calif.).
Propidium iodide was used for nuclear staining.
Example 1.1.f
ATPase Assay of ABCB1
[0096] The Vi-sensitive ATPase activity of ABCB1 in membrane
vesicles of High Five.TM. insect cells was measured as follows. The
membrane vesicles (10 .mu.g of protein) were incubated in ATPase
assay buffer (50 mM MES [pH 6.8], 50 mM KCl, 5 mM sodium azide, 2
mM EGTA, 2 mM dithiothreitol, 1 mM ouabain, and 10 mM MgCl.sub.2)
with or without 0.3 mM orthovanadate (freshly prepared) at
37.degree. C. for 5 min, then incubated with different
concentrations of drug at 37.degree. C. for 3 min. The ATPase
reaction was started by the addition of 5 mM ATP, and the total
volume was 0.1 ml. After incubation at 37.degree. C. for 20 min,
the reactions were stopped by the addition of 0.1 ml of 5% SDS
solution and vortexed and kept at room temperature. The liberated
Pi was measured as described in Ambudkar et al., supra.
Example 1.1.g
Photoaffinity Labeling of ABCB1 with [.sup.125I]-IAAP
[0097] The photoaffinity labeling of ABCB1 with [.sup.125I]-IAAP
was performed as described in Sauna et al. (2000) Proc. Natl. Acad.
Sci. USA 97:2515-20. The membrane vesicles from High Five.TM.
insect cells expressing ABCB1 (50 .mu.g of protein) were incubated
at room temperature with different concentrations of drugs in the
ATPase assay buffer with [.sup.125I]-IAAP (7 nM) for 5 min under
subdued light. The samples were photo cross-linked by using a 365
nm UV light source for 10 min at room temperature. Subsequently,
the samples were run in a 7% Tris-acetate NuPAGE gel on SDS-PAGE;
and the gels were dried and exposed to Bio-Max MR film (Eastman
Kodak Co., Rochester, N.Y.) at -70.degree. C. for 8-12 h. The
radioactivity incorporated into the ABCB1 band was quantified using
the STORM 860 PhosphorImager system and ImageQuaNT (Molecular
Dynamics, CA).
Example 1.1.h
Ligand-ABCB1 Structure Preparation
[0098] Vardenafil, tadalafil (modeled as R,R isomer), and IAAP were
constructed using the fragment dictionary of Maestro 9.0 and the
energy minimized by Macromodel program v9.7 (Schrodinger, Inc., New
York, N.Y.) using the OPLSAA force field (as described Jorgensen et
al. (1996) J. Am. Chem. Soc. 118:11225-36), with the steepest
descent followed by a truncated Newton conjugate gradient protocol.
Partial atomic charges were computed using the OPLS-AA force field.
The low-energy 3D structures of vardenafil, tadalafil, and IAAP
were generated with the following parameters present in LigPrep
v2.3: different protonation states at physiological pH, all
possible tautomers and ring conformations.
Example 1.1.i
Protein Structure Preparation
[0099] The X-ray crystal structure of mouse ABCB1 in the apoprotein
state (PDB ID: 3G5U) and in complex with inhibitors QZ59-RRR (PDB
ID: 3G6O) and QZ59-SSS (PDB ID: 3G61) was obtained from the RCSB
Protein Data Bank; these were used to build the homology model of
human ABCB1. The homology modeling was conducted using the default
parameters of Prime v2.1 as implemented in Maestro 9.0. The input
file for the amino acid sequence of human ABCB1 in the Prime
structure prediction application was obtained as a FASTA file
(UniProt Accession Number P08183.3) extracted from uniprot.org. The
cocrystal structures of ABCB1 from the mouse model in complex with
QZ59-RRR and QZ59-SSS inhibitors were used as templates for
modeling site-1 and site-2, respectively; while apoprotein-ABCB1
was used as a template for modeling site-3 and site-4. The
resultant alignment of human ABCB1 and mouse ABCB1 sequences
produced 87% sequence identity and 93% similarity. Based on the
resultant alignment that was constructed using default parameters,
the side chains were optimized and residues were minimized. The
initial structure thus obtained was refined by means of default
parameters mentioned in a protein preparation facility implemented
in Maestro v9.0 and Impact program v5.5 (Schrodinger, Inc., New
York, N.Y.), in which the protonation states of residues were
adjusted to the dominant ionic forms at pH 7.4. The refined human
ABCB1 homology model was further used to generate four different
receptor grids by selecting QZ59-RRR (site-1) and QZ59-SSS (site-2)
bound ligands, all amino acid residues known to contribute to
verapamil binding (site-3), and two residues known to be common to
the three previous sites (site-4), as shown in Table 7.
Example 1.1.j
Docking Protocol
[0100] The docking calculations were performed using the "Extra
Precision" (XP) mode of Glide program v5.5 (Schrodinger, Inc.) and
the default parameters. The top scoring pose-ABCB1 complex was then
subjected to energy minimization using Macromodel program v9.7
using the OPLS-AA force field and used for graphical analysis. All
computations were carried out on a Dell Precision 470n dual
processor with the Linux OS (Red Hat Enterprise WS 4.0).
Example 1.1.k
Statistical Analysis
[0101] All experiments were repeated at least three times and the
differences were determined by using the Student's t-test. The
statistical significance was determined at p<0.05.
Example 1.2
Vardenafil Enhances Drug Sensitivity of ABCB1- but Not ABCC1- or
ABCG2-Overexpressing Cancer Cells
[0102] Cytotoxic effects of vardenafil and tadalafil on different
cell lines using the MTT assay were tested, and neither vardenafil
nor tadalafil inhibited the growth of any cell lines tested at
concentrations of up to 20 .mu.M.
[0103] Subsequently, the effect of vardenafil or tadalafil on the
sensitivity of anticancer drugs in ABCB1-, ABCC1-, and
ABCG2-overexpressing MDR cells was tested. At 5 and 10 .mu.M,
vardenafil produced a concentration-dependent increase in
cytotoxicity of colchicine and paclitaxel in ABCB1-overexpressing
drug-selected cell line KB-C2, but not parental sensitive KB-C2
cells (Table 4). In order to eliminate the possibility of multiple
factors playing a role in drug-selected cell lines, the effect of
vincristine and paclitaxel cytotoxicity on ABCB1-transfected
HEK293/ABCB1 cells was also measured. Vardenafil increased the
sensitivity of ABCB1-transfected HEK/ABCB1 cells to vincristine and
paclitaxel (Table 5), but not the control cell line
HEK293/pcDNA3.1. Therefore, the effect of vardenafil was specific
to ABCB1-overexpressing cells, but had no significant toxic effects
on parental cells when combined with ABCB1 transporter substrate
anticancer drugs. Vardenafil did not reverse MDR induced by cells
expressing ABCC1 and ABCG2 (Table 6), or significantly alter the
IC.sub.50 values of cisplatin, which is not a substrate of ABCB1
(Tables 4 and 5).
TABLE-US-00004 TABLE 4 The Effects of Vardenafil and Tadalafil on
the Reversal of ABCB1-Mediated Resistance to Colchicine,
Paclitaxel, and Cisplatin in Drug-Selected Cell Line IC.sub.50 .+-.
SD (.mu.M) (fold reversal) Compounds KB-3-1 KB-C2 (ABCB1)
Colchicine 0.0063 .+-. 0.0013 (1.00) 2.9017 .+-. 0.6127 (1.00) +
Vardenafil 0.0070 .+-. 0.0014 (0.90) 0.1053 .+-. 0.0215** (27.6) 5
.mu.M + Vardenafil 0.0068 .+-. 0.0012 (0.93) 0.0157 .+-. 0.0063**
(184.8) 10 .mu.M + Tadalafil 0.0073 .+-. 0.0009 (0.86) 0.6923 .+-.
0.1518** (4.19) 5 .mu.M + Tadalafil 0.0071 .+-. 0.0015 (0.89)
0.4547 .+-. 0.1033** (6.38) 10 .mu.M + Verapamil 0.0064 .+-. 0.0011
(0.98) 0.0347 .+-. 0.0071** (83.6) 10 .mu.M Paclitaxel 0.0066 .+-.
0.0016 (1.00) 0.7354 .+-. 0.0141 (1.00) + Vardenafil 0.0074 .+-.
0.0015 (0.89) 0.0281 .+-. 0.0067** (26.2) 5 .mu.M + Vardenafil
0.0072 .+-. 0.0017 (0.92) 0.0136 .+-. 0.0025** (54.1) 10 .mu.M +
Tadalafil 0.0071 .+-. 0.0020 (0.93) 0.2911 .+-. 0.0669** (2.53) 5
.mu.M + Tadalafil 0.0065 .+-. 0.0028 (1.02) 0.1641 .+-. 0.0311**
(4.48) 10 .mu.M + Verapamil 0.0060 .+-. 0.0009 (1.10) 0.0132 .+-.
0.0035** (55.7) 10 .mu.M Cisplatin 1.9032 .+-. 0.0709 (1.00) 1.7877
.+-. 0.2171 (1.00) + Vardenafil 2.1316 .+-. 0.3653 (0.89) 2.0555
.+-. 0.7811 (0.87) 5 .mu.M + Vardenafil 2.0364 .+-. 0.6313 (0.93)
1.8651 .+-. 0.5409 (0.96) 10 .mu.M + Tadalafil 1.9899 .+-. 0.4975
(0.96) 1.7700 .+-. 0.7257 (1.01) 5 .mu.M + Tadalafil 1.7890 .+-.
0.6083 (1.06) 1.9221 .+-. 0.9034 (0.93) 10 .mu.M + Verapamil 1.8703
.+-. 0.2330 (1.02) 1.7890 .+-. 0.6472 (1.00) 10 .mu.M Cell survival
was determined by MTT assay as described in Example 1.1.c. Data are
expressed as the mean .+-. SD of at least three independent
experiments performed in triplicate. The fold-reversal values of
MDR (values given in parentheses) were calculated by dividing the
IC.sub.50 values of anticancer drugs in cells in the absence of an
inhibitor by those obtained in the same cell type in the presence
of the inhibitor. **represents p < 0.01, for values versus those
obtained in the absence of inhibitor.
TABLE-US-00005 TABLE 5 The Effects of Vardenafil and Tadalafil on
the Reversal of ABCB1-Mediated Resistance to Vincristine,
Paclitaxel, and Cisplatin in ABCBl-Transfected Cell Line IC.sub.50
.+-. SD (.mu.M) (fold reversal) Compounds HEK293/pcDNA3.1 HEK/ABCB1
(ABCB1) Vincristine 11.55 .+-. 1.59 (1.00) 169.84 .+-. 12.93 (1.00)
+ Vardenafil 5 .mu.M 13.13 .+-. 0.93 (0.88) 41.41 .+-. 6.12**
(4.10) + Vardenafil 10 .mu.M 9.72 .+-. 2.13 (1.19) 14.64 .+-.
2.36** (11.60) + Tadalafil 5 .mu.M 10.02 .+-. 1.24 (1.15) 112.94
.+-. 5.04 (1.50) + Tadalafil 10 .mu.M 8.57 .+-. 0.62 (1.35) 69.27
.+-. 3.48* (2.45) + Verapamil 10 .mu.M 8.23 .+-. 1.11 (1.40) 18.21
.+-. 4.82** (9.33) Paclitaxel 23.73 .+-. 5.21 (1.00) 219.14 .+-.
13.16 (1.00) + Vardenafil 5 .mu.M 21.85 .+-. 2.04 (1.09) 56.83 .+-.
7.74** (3.86) + Vardenafil 10 .mu.M 20.09 .+-. 3.06 (1.18) 25.23
.+-. 4.17** (8.69) + Tadalafil 5 .mu.M 24.04 .+-. 2.95 (0.99)
159.78 .+-. 11.52 (1.37) + Tadalafil 10 .mu.M 19.64 .+-. 3.17
(1.21) 96.92 .+-. 4.83* (2.26) + Verapamil 10 .mu.M 20.17 .+-. 2.52
(1.18) 31.03 .+-. 2.19** (7.06) Cisplatin 890.32 .+-. 33.92 (1.00)
850.84 .+-. 82.53 (1.00) + Vardenafil 5 .mu.M 839.03 .+-. 48.37
(1.06) 893.19 .+-. 32.60 (0.95) + Vardenafil 10 .mu.M 892.44 .+-.
19.26 (1.00) 782.82 .+-. 59.31 (1.09) + Tadalafil 5 .mu.M 901.29
.+-. 67.23 (0.99) 909.06 .+-. 98.44 (0.94) + Tadalafil 10 .mu.M
792.85 .+-. 92.60 (1.12) 783.60 .+-. 84.20 (1.09) + Verapamil 10
.mu.M 853.62 .+-. 61.04 (1.04) 725.71 .+-. 48.56 (1.17) Cell
survival was determined by MTT assay as described in Example 1.1.c.
Data are expressed as the mean .+-. SD of at least three
independent experiments performed in triplicate. The fold-reversal
values of MDR (values given in parentheses) were calculated by
dividing the IC.sub.50 values of anticancer drugs in cells in the
absence of an inhibitor by those obtained in the same cell type in
the presence of the inhibitor. **represents p < 0.01,
*represents p < 0.05, for values versus those obtained in the
absence of inhibitor.
TABLE-US-00006 TABLE 6 The Effect of Vardenafil and Tadalafil on
the Reversal of ABCG2-Mediated Resistance to SN-38 and
ABCC1-Mediated Resistance to Vincristine Compounds IC.sub.50 .+-.
SD (.mu.M) (fold reversal) HEK293/pcDNA3.1 .sup. ABCG2-482-R2
(ABCG2) SN-38 0.1157 .+-. 0.0151 (1.00) 3.6471 .+-. 0.5472 (1.00) +
Vardenafil 0.1049 .+-. 0.0186 (1.10) 3.7652 .+-. 0.6264 (0.97) 10
.mu.M + Tadalafil 0.1202 .+-. 0.0250 (0.96) 3.5173 .+-. 0.8198
(1.04) 10 .mu.M + FTC 0.1021 .+-. 0.0238 (1.13) 0.9484 .+-.
0.1634** (3.84) 10 .mu.M HEK293/pcDNA3.1 .sup. HEK/MRP1 (ABCC1)
Vincristine 0.0012 .+-. 0.0002 (1.00) 0.0106 .+-. 0.0022 (1.00) +
Vardenafil 0.0013 .+-. 0.0001 (0.92) 0.0099 .+-. 0.0016 (1.07) 10
.mu.M + Tadalafil 0.0011 .+-. 0.0002 (1.09) 0.0103 .+-. 0.0021
(1.03) 10 .mu.M + ONO-1078 0.0009 .+-. 0.0001 (1.33) 0.0018 .+-.
0.0003** (5.9) 10 .mu.M Cell survival was determined by MTT assay
as described in Example 1.1.c. Data are expressed as the mean .+-.
SD of at least three independent experiments performed in
triplicate. The fold-reversal values of MDR (values given in
parentheses) were calculated by dividing the IC.sub.50 values of
anticancer drugs in cells in the absence of an inhibitor by those
obtained in the same cell type in the presence of the inhibitor.
**represents p < 0.01, for values versus those obtained in the
absence of inhibitor.
Example 1.3
Vardenafil Increases Accumulation of Intracellular Paclitaxel in
ABCB1-Overexpressing Cells by Inhibiting Drug Efflux
[0104] To determine the reversal mechanism of vardenafil and
tadalafil for ABCB1 transporter, the accumulation of
[.sup.3H]-paclitaxel was measured after cells were preincubated
with or without vardenafil, tadalafil, or verapamil (control) for 1
h at 37.degree. C. and then incubated with [.sup.3H]-paclitaxel for
another 2 h at 37.degree. C. (FIG. 1A). The intracellular
concentration of paclitaxel in KB-C2 cells was approximately 55% of
that in the parental KB-3-1 cells. However, 10 .mu.M of vardenafil
significantly increased the intracellular accumulation of
paclitaxel in KB-C2 by 1.6-fold without altering the levels
accumulated in KB-3-1 cells. Although tadalafil at 10 .mu.M had
significant effects on paclitaxel accumulation, this accumulation
was reduced in comparison with vardenafil and verapamil
(control).
[0105] The effect of vardenafil on paclitaxel efflux was also
determined (FIG. 1B). The intracellular levels of paclitaxel were
measured over 2 h. A significantly higher concentration of
paclitaxel was effluxed from the KB-C2 cells compared to KB-3-1
cells, and the amount of effluxed paclitaxel increased with time;
at 1 h time point, 70% of accumulated paclitaxel was effluxed from
KB-C2 cells in the absence of vardenafil, and 10 .mu.M of
vardenafil significantly blocked ABCB1 efflux function, with 75% of
accumulated paclitaxel being retained inside KB-C2 cells. No
significant change in concentration of effluxed paclitaxel in
parental KB-3-1 cells in the absence or presence of vardenafil was
noted.
Example 1.4
Vardenafil does not Alter Membrane Expression of ABCB1
[0106] To determine whether reversal of ABCB1-mediated MDR occurred
by either decreasing ABCB1 expression or inhibiting ABCB1 activity,
the ABCB1-overexpressing KB-C2 cells were incubated with 10 .mu.M
vardenafil or tadalafil for 36 and 72 h and then assayed to
determine the difference in protein levels via Western blot.
According to FIG. 1C, the protein level of ABCB1 in KB-C2 cells was
not significantly altered after the cells were incubated with
vardenafil or tadalafil. It was possible that the ABCB1 transporter
on the membrane had translocated inside the cell as a result of
treatment, and the Western blot of whole lysates could not
distinguish this possibility. Therefore, an immunofluorescence
assay was performed to detect the location of the ABCB1
transporter. The results suggest that neither vardenafil nor
tadalafil altered the expression of ABCB1 in the membrane of the
KB-C2 cells after 72 h of incubation (not shown). Thus, vardenafil
and tadalafil altered neither the expression nor the localization
of ABCB1 transporter. These data are in agreement with the
conclusion that PDE5 inhibitors inhibit ABCB1 function rather than
its expression.
Example 1.5
The Effect of Vardenafil and Tadalafil on the ATPase Activity of
ABCB1
[0107] The drug-efflux function of ABCB1 is coupled to ATP
hydrolysis by the ATPase enzyme that is usually stimulated in the
presence of ABCB1 substrates. Therefore, to assess the effect of
vardenafil and tadalafil on the ATPase activity of ABCB1, the rate
of ABCB1-mediated ATP hydrolysis was measured in isolated membrane
vesicles in the presence of various concentrations of vardenafil
and tadalafil under conditions that suppressed activity of other
major ATPases. Vardenafil produced a concentration-dependent
increase in the ATPase activity of ABCB1 over a range of
concentrations (FIG. 2A), with the concentration of vardenafil
required for a 50% stimulation of ATPase activity being
2.69.+-.0.72 .mu.M. In contrast, tadalafil mildly stimulated ATPase
activity, such that at the highest concentration tested (25 .mu.M),
the concentration required for 50% stimulation was not reached.
Example 1.6
Effect of Vardenafil and Tadalafil on the Photoaffinity Labeling of
ABCB1 with [.sup.125I]-IAAP
[0108] In order to determine whether vardenafil and tadalafil
interacted with the substrate binding site of ABCB1, the effects of
vardenafil and tadalafil on photoaffinity labeling of ABCB1
transporter with [.sup.125I]-IAAP using membrane vesicles was
measured. Vardenafil inhibited the photoaffinity labeling of ABCB1
with [.sup.125I]-IAAP in a concentration-dependent manner (FIG.
2B). At concentrations of 0.69 .mu.M and 10 .mu.M, vardenafil
inhibited the [.sup.125I]-IAAP photolabeling of ABCB1 by 50% and
90%, respectively, whereas concentrations of up to 50 .mu.M
tadalafil did not produce a 50% inhibition of the [.sup.125I]-IAAP
photolabeling of ABCB1 transporter. These data demonstrate that the
tested PDE5 inhibitors interacted with the drug binding site of
ABCB1 and, as these agents stimulated the ATPase activity of ABCB1
(Example 1.5), they may be transport substrates for ABCB1.
Example 1.7
Model for Binding of Vardenafil and Tadalafil to ABCB1
[0109] In the Examples above, the PDE5 inhibitors vardenafil and
tadalafil are described for the first time as ABCB1 inhibitors.
Their predicted binding conformation within the large cavity of
ABCB1 required determination. Because the crystal structure of the
human ABCB1 remains to be elucidated and the binding conformation
of vardenafil and tadalafil within the large cavity of ABCB1
transporter is unknown, a homology model of human ABCB1, based on
the mouse ABCB1-QZ59-RRR cocrystal structure as a template (FIG.
3A), was utilized for the Glide docking study of vardenafil and
tadalafil. Four binding sites were reported in the crystal
structure of mouse ABCB1 as represented by the following sites:
ABCB1-QZ59-RRR (site-1), ABCB1-QZ59-SSS (site-2), ABCB1-verapamil
(site-3), and the site common to the above three sites (site-4).
Aller et al. (2009) Science 323:1718-22. As the photoaffinity
labeling data suggested that vardenafil displaces IAAP in a
concentration-dependent manner, IAAP was also docked to these sites
for comparison. These data also indicated that vardenafil and IAAP
share the same binding site, i.e., site-1; however, the tadalafil
binding site is somewhat different from the vardenafil docking
site--the Glide docking score predicts that it is site-4 (Table 7).
A comparison of the binding energy data for the docked poses of
vardenafil, tadalafil, and IAAP at each of the binding sites (Table
7) suggested that the more potent of the two ABCB1 inhibitors,
vardenafil, exhibited the most favorable binding energy within the
QZ59-RRR binding site of ABCB1, whereas tadalafil interacted most
favorably with site-4. Thus, the following section addresses the
bound conformation of vardenafil and tadalafil in site-1 and in
site-4, respectively.
TABLE-US-00007 TABLE 7 Binding Energies of Vardenafil, Tadalafil,
and IAAP within Each of the Predicted Binding Sites of ABCB1. Glide
score kcal/mol Ligands Site-1.sup.a Site-2.sup.b Site-3.sup.c
Site-4.sup.d ##STR00001## -8.56 -6.26 -5.13 -4.87 ##STR00002##
-6.26 -5.05 -7.35 -7.85 ##STR00003## -8.89 -5.79 -4.54 -5.16
.sup.a-Site represented by bound QZ59-RRR. .sup.b-Site represented
by bound ligand QZ59-SSS. .sup.c-Verapamil binding site.
.sup.d-Site grid generated using residues Phe728 and Val982, which
are known to be common to above three sites.
[0110] The XP-Glide-predicted binding mode of vardenafil indicates
the importance of hydrophobic and electrostatic interactions within
the large drug binding cavity of ABCB1 (FIG. 3B, left panel).
Whereas the N-ethylpiperazine (D-ring) of vardenafil forms
hydrophobic contacts with Met69, Phe336, Leu339, and Ile340, the
C-ring along with its ethoxy substituent enters into favorable
hydrophobic interactions with Phe72, Leu975, and Phe978. The
A-ring, along with its methyl and propyl substituents and the
B-ring, are engaged in hydrophobic interactions with the side
chains of Phe728, Ala729, Phe732, and Val982. In addition,
vardenafil also appears to form favorable electrostatic
interactions with residues Tyr953 and Tyr307. For example, the
sulfonyl oxygen atom forms a hydrogen bond with the hydroxyl group
of Tyr953 (--SO.sub.2--HO-Tyr953), whereas the carbonyl function of
the B-ring is located at a distance of 4.0 .ANG. from the side
chain hydroxyl group of Tyr307.
[0111] The XP-Glide-predicted binding mode of tadalafil in site-4
of the large drug binding cavity of ABCB1 is shown in FIG. 3B
(right panel). The A- and B-rings of the indole moiety bind to the
hydrophobic pocket formed by the side chains of Phe303, Leu304,
Tyr307, and Phe343. Moreover, Phe343 also has hydrophobic contacts
with the B-, C-, and E-rings. Both E- and F-rings of the
benzodioxole moiety are surrounded by the side chains of Phe336,
Leu339, Phe978, and Val982. The carbonyl oxygen atom (close to
E-ring) of the D-ring is stabilized by a hydrogen bonding
interaction with the side chain amide group of Gln725
(--CO--H.sub.2NOC-Gln725). The lower efficacy of tadalafil compared
to vardenafil may be due to the orientation of its hydrophobic
N-methyl substituent of the D-ring towards the unfavorable polar
backbone of Met986 and Gly989 and the polar side chain amide group
of Gln990.
Example 2
Effect of Sildenafil on ABCB1- and ABCG2-Mediated Multidrug
Resistance
Example 2.1
Materials and Methods
Example 2.1.a
Materials
[0112] [.sup.3H]-paclitaxel (37.9 Ci/mmol), [.sup.3H]-mitoxantrone
(4 Ci/mmol), and [.sup.3H]-methotrexate (23 Ci/mmol) were purchased
from Moravek Biochemicals, Inc. [.sup.3H]-E.sub.217.beta.G (40.5
Ci/mmol) and [.sup.125I]-IAAP (2,200 Ci/mmol) were obtained from
PerkinElmer Life Sciences. The fluorescent compound BODIPY-prazosin
was purchased from Invitrogen. Monoclonal antibodies C-219 (against
ABCB1) and BXP-21 (against ABCG2) were acquired from Signet
Laboratories, Inc. Sildenafil was purified from 100 mg VIAGRA.RTM.
tablets as described by Francis et al. (2003) J. Impot. Res.
15:369-72. Fumitremorgin C (FTC) was synthesized by Thomas McCloud
Developmental Therapeutics Program, Natural Products Extraction
Laboratory, National Cancer Institute (NCI), NIH. Other chemicals
were purchased from Sigma Chemical Co.
Example 2.1.b
Cell Lines and Cell Culture
[0113] The ABCB1-overexpressing drug-resistant cell line KB-C2 was
established by stepwise selection of the parental human epidermoid
carcinoma cell line KB-3-1 in increasing concentrations of
colchicine and was cultured in medium containing 2 .mu.g/mL of
colchicine. The ABCB1-overexpressing drug resistant cell line KB-V1
(generously provided by Dr. Gottesman, NCI, NIH) was established by
a stepwise concentration increase of vinblastine in the culture
medium of KB-3-1 cells and subsequently culturing these cells in
medium with 1 .mu.g/mL of vinblastine. An ABCC1-overexpressing MDR
cell line, KB-CV60, was also derived from KB-3-1 cells and was
maintained in medium with 1 .mu.g/mL of cepharanthine and 60 ng/mL
of vincristine. Both KB-C2 and KB-CV60 cell lines were kindly
provided by Dr. Akiyama (Kagoshima University, Japan).
HEK293/pcDNA3.1, ABCG2-482-R5, ABCG2-482-G2, and ABCG2-482-T7 cells
were established by selection with 2 mg/mL G418 after transfecting
HEK293 with either (1) empty pcDNA3.1 vector or (2) pcDNA3.1 vector
containing full length ABCG2 coding either (2a) wild-type arginine
(R) or mutant (2b) glycine (G) or (2c) threonine (T) at amino acid
position 482, respectively. The wild-type ABCG2-overexpressing
drug-resistant cell line MCF-7/Flv1000 was cultured in the medium
with 1 .mu.M of flavopiridol. The mutated ABCG2-overexpressing drug
resistant cell line MCF-7/ADVP3000 was maintained in the medium
with 5 .mu.g/mL of verapamil and 3 .mu.g/mL of doxorubicin. Another
G482 mutant ABCG2-overexpressing drug resistant cell line,
S1-M1-80, was maintained in the medium with 80 .mu.M of
mitoxantrone. The drug-resistant cell line S1/Flv5000, which does
not express ABCG2, was also generated from S1 by increasing the
amount of flavopiridol and was maintained in the medium with 5
.mu.M of flavopiridol. All the cell lines were grown as adherent
monolayers in flasks with DMEM (Dulbecco's modified Eagle's medium)
culture medium (Hyclone Co.) containing 10% bovine serum at
37.degree. C. in a humidified atmosphere of 5% CO.sub.2.
Example 2.1.c
MTT Cytotoxicity Data
[0114] Cells in 96-well plates were preincubated with or without
the reversal agents for 1 h and then different concentrations of
chemotherapeutic drugs were added into designated wells. After 68 h
of incubation, MTT solution (4 mg/mL) was added to each well, and
the plate was further incubated for 4 h, allowing viable cells to
change the yellow-colored MTT into dark-blue formazan crystals.
Subsequently the medium was discarded, and 100 mL of DMSO was added
into each well to dissolve the formazan crystals. The absorbance
was determined at 570 nm by an OPSYS MR Microplate Reader from
DYNEX Technologies, Inc.
Example 2.1.d
Paclitaxel and Mitoxantrone Accumulation
[0115] Cells in 24-well plates were preincubated with or without
the reversal agents for 1 h at 37.degree. C., then incubated with
0.1 .mu.M [.sup.3H]-paclitaxel or 0.2 .mu.M [.sup.3H]-mitoxantrone
for 2 h in the presence or absence of the reversal agents at
37.degree. C. After washing 3 times with ice-cold PBS, the cells
were trypsinized and lysed in 10 mM lysis buffer (pH 7.4,
containing 1% Triton X-100 and 0.2% SDS). Each sample was placed in
scintillation fluid and radioactivity was measured in a Packard
TRI-CARB 1900CA liquid scintillation analyzer from Packard
Instrument Company, Inc.
Example 2.1.e
Flow Cytometric Assays
[0116] Flow cytometric assays were carried out as follows. The
cells were trypsinized and then resuspended in complete media
(phenol red-free Iscove's modified Eagle's medium, IMEM, with 10%
fetal calf serum) containing 250 nM BODIPY-prazosin alone or with
various concentrations of the inhibitors for 30 min at 37.degree.
C. Cells were then washed once in cold complete medium and then
incubated for another 1 h at 37.degree. C. in substrate-free media
with or without the same described concentrations of the inhibitors
to generate the efflux histograms. Subsequently, cells were washed
twice with cold DPBS (Dulbecco's PBS) and placed on ice in a dark
environment until ready for analysis. Cells were analyzed on a
FACSort flow cytometer equipped with a 488-nm argon laser. For all
samples, at least 10,000 events were collected. Cell debris was
eliminated by gating on forward versus side scatter, and dead cells
were excluded on the basis of propidium iodide staining.
Example 2.1.f
In Vitro Transport Assays
[0117] Transport assays were carried out using the rapid filtration
method as described in Chen et al. (2003) Cancer Res. 63:4048-54.
Membrane vesicles were incubated with various concentrations of
inhibitors for 1 h on ice, and then transport reactions were
carried out at 37.degree. C. for 10 min in a total volume of 50
.mu.L of medium (membrane vesicles 10 .mu.g, 0.25 M sucrose, 10 mM
Tris-HCl, pH 7.4, 10 mM MgCl.sub.2, 4 mM ATP or AMP, 10 mM
phosphocreatine, 100 .mu.g/mL creatine phosphokinase, and 0.25
.mu.M [.sup.3H]-E.sub.217.beta.G, or 0.5 .mu.M
[.sup.3H]-methotrexate). Reactions were stopped by the addition of
3 mL of ice-cold stop solution (0.25 M sucrose, 100 mM NaCl, and 10
mM Tris-HCl, pH 7.4). During the rapid filtration step, samples
were passed through 0.22 .mu.m GVWP filters (Millipore) presoaked
in the stop solution. The filters were washed three times with 3 mL
of ice-cold stop solution. Radioactivity was measured by liquid
scintillation counting.
Example 2.1.g
ATPase Assay of ABCB1 and ABCG2
[0118] The vanadate (Vi)-sensitive ATPase activity of ABCB1 or
ABCG2 in the membrane vesicles of HIGH FIVE.TM. insect cells was
measured as described in Ambudkar et al., supra. The membrane
vesicles (100 .mu.g of protein/mL) were incubated in ATPase assay
buffer (50 mM MES, pH 6.8, 50 mM KCl, 5 mM sodium azide, 2 mM EGTA,
2 mM dithiothreitol, 1 mM ouabain, and 10 mM MgCl.sub.2) with or
without 0.3 mM vanadate at 37.degree. C. for 5 min, and then
incubated with different concentrations of drugs at 37.degree. C.
for 3 min. The ATPase reaction was incubated by the addition of 5
mM Mg-ATP. After incubating at 37.degree. C. for 20 min, the
reactions were stopped by adding 0.1 mL of 5% SDS solution. The
liberated inorganic phosphate was measured as described in Shukla
et al. (2006) Biochemistry 45:8940-51. In the inhibition assays,
the decrease in maximum Vi-sensitive ABCB1 or ABCG2 activity by
sildenafil was measured in the presence of verapamil at 50 .mu.M or
FTC at 10 .mu.M, respectively.
Example 2.1.h
Photoaffinity Labeling of ABCB1 and ABCG2 with [.sup.125I]-IAAP
[0119] The photoaffinity labeling of ABCB1 and ABCG2 with
[.sup.125I]-IAAP was conducted as described in Sauna et al., supra.
The crude membranes from HIGH FIVE.TM. insect cells expressing
ABCB1 and MCF7/FLV1000 cells expressing R482 ABCG2 (50 .mu.g of
protein) were incubated at room temperature with different
concentrations of drugs in 50 mM Tris-HCl (pH 7.5) with
[.sup.125I]-IAAP (5-7 nM) for 5 min under subdued light. The
samples were photo-cross-linked by exposure to a 365 nm UV light
for 10 min at room temperature. ABCB1 and ABCG2 were
immunoprecipitated using BXP-21 antibodies as described in Shukla
et al., supra. Both ABCB1 and ABCG2 samples were subjected to
SDS-PAGE in a 7% Trisacetate NuPAGE gel, the gel was dried, and
exposed to Bio-Max MR film (Eastman Kodak Co.) at -70.degree. C.
for 8 to 12 h. The radioactivity incorporated into the ABCB1 or
ABCG2 band was quantified using the STORM 860 PhosphorImager System
and ImageQuaNT (Molecular Dynamics).
Example 2.1.i
Ligand Structure Preparation
[0120] Sildenafil and IAAP structures were built using the fragment
dictionary of Maestro 9.0 and energy minimized by Macromodel
program v9.7 (2009; Schrodinger, Inc.) using the OPLS-AA force
field (Jorgensen et al., supra) with the steepest descent followed
by a truncated Newton conjugate gradient protocol. Partial atomic
charges were computed using the OPLS-AA force field. The low-energy
3D structures of sildenafil and IAAP were generated with the
following parameters present in LigPrep v2.3: different protonation
states at physiologic pH, all possible tautomers and ring
conformations.
Example 2.1.j
Protein Structure Preparation
[0121] The X-ray crystal structure of ABCB1 in apoprotein state
(PDB ID: 3G5U) and in complex with inhibitors QZ59-RRR (PDB ID:
3G6O) and QZ59-SSS (PDB ID: 3G61) obtained from the RCSB Protein
Data Bank were used to build the homology model of human ABCB1.
Aller et al., supra. Homology modeling was carried out using the
default parameters of Prime v2.1 as implemented in Maestro 9.0. The
input file for the amino acid sequence of human ABCB1 in the Prime
structure prediction application was obtained as a FASTA file
(UniProt Accession number P08183.3) extracted from www.uniprot.org.
The cocrystal structures of ABCB1 from the mouse model in complex
with QZ59-RRR and QZ59-SSS inhibitors were used as templates for
modeling site-1 and site-2, respectively; whereas apoprotein ABCB1
was used as a template for modeling site-3 and site-4. The
resultant alignment of human ABCB1 and mouse ABCB1 sequences
produced 87% sequence identity and 93% similarity. On the resultant
alignment built using default parameters, side chains were
optimized and residues were minimized. The initial structure thus
obtained was refined by means of default parameters mentioned in
protein preparation facility implemented in Maestro v9.0 and Impact
program v5.5 (2009; Schrodinger, Inc.), in which the protonation
states of residues were adjusted to the dominant ionic forms at pH
7.4. The refined human ABCB1 homology model was used further to
generate four different receptor grids by selecting QZ59-RRR
(site-1) and QZ59-SSS (site-2) bound ligands, all amino acid
residues known to contribute to verapamil binding (site-3), and two
residues known to be common to three previous sites (site-4), as
shown in Table 11.
Example 2.1.k
Docking Protocol
[0122] All docking calculations were done using the "Extra
Precision" (XP) mode of Glide program v5.5 (2009; Schrodinger,
Inc.) and the default parameters. The top-scoring pose-ABCB1
complex was then subjected to energy minimization using Macromodel
program v9.7 utilizing the OPLS-AA force field (Jorgensen et al.,
supra), and used for graphical analysis. All computations were
carried out on a Dell Precision 470n dual processor with the Linux
OS (Red Hat Enterprise WS 4.0).
Example 2.1.l
Statistical Analysis
[0123] All experiments were repeated at least three times and the
differences were determined by the Student's t test. The
statistical significance was determined at p<0.05.
Example 2.2
Sildenafil Sensitizes ABCB1- and ABCG2-Overexpressing Cells to
Chemotherapeutic Drugs
[0124] First, the sensitivity of ABCB1-, ABCG2-, and
ABCC1-overexpressing cells to sildenafil was assessed. The results
of the MTT assay showed that sildenafil did not inhibit the growth
of any of the cell lines used at concentrations of up to 50 .mu.M
(not shown). However, sildenafil increased the sensitivity of
ABCB1-overexpressing drug-resistant cells to substrate drugs. The
IC.sub.50 values of ABCB1 substrates colchicine, vinblastine, and
paclitaxel were much higher in ABCB1-overexpressing cells KB-C2 and
KB-V1 than parent KB-3-1 cells, but sildenafil decreased the
IC.sub.50 values of these drugs in KB-C2 and KB-V1 cells (Table 8).
At 2.5 .mu.M, sildenafil moderately increased the sensitivity (in
KB-C2 and KB-V1 cells) to all three drugs, and at 10 .mu.M,
sildenafil increased the sensitivity with efficacy comparable to
that of the equivalent concentration of verapamil. Neither
sildenafil nor verapamil altered the cytotoxicity of the three
drugs in parental KB-3-1 cells. The IC.sub.50 values of cisplatin,
which is not a substrate for ABCB1 and exhibited equal sensitivity
in KB-3-1, KB-C2, and KB-V1 cells, were not affected by either
sildenafil or verapamil in these three cell lines.
[0125] The effects of sildenafil on ABCC1- and ABCG2-mediated drug
resistance were determined. In the ABCC1-overexpressing KB-CV60
cells, sildenafil at 10 .mu.M showed no significant reduction in
the IC.sub.50 value of vincristine, a known ABCC1 substrate (not
shown).
[0126] It has been reported that mutations at amino acid 482 in
ABCG2 alter the substrate and antagonist specificity of ABCG2
(Robey et al. (2003) Br. J. Cancer 89:1971-78; Honjo et al. (2001)
Cancer Res. 61:6635-39); therefore, the reversing effects of
sildenafil on both wild type (R482) and mutant (R482G and R482T)
ABCG2-overexpressing cells were determined. Compared with parental
MCF-7 cells, MCF-7/Flv1000 and MCF-7/AdVp3000 cells exhibited high
levels of resistance to ABCG2 substrates flavopiridol,
mitoxantrone, and SN-38, but not to non-ABCG2 substrate cisplatin
(Table 9). Similarly, the IC.sub.50 values for flavopiridol,
mitoxantrone, and SN-38 in S1-M1-80 were significantly higher than
in parental S1 cells. At 50 .mu.M, sildenafil decreased the
IC.sub.50 values for flavopiridol, mitoxantrone, and SN-38 in the
ABCG2-expressing cell lines down to levels observed when
cytotoxicity assays were carried out in the presence of the known
specific ABCG2 inhibitor FTC at 2.5 .mu.M. However, the IC.sub.50
values of these ABCG2 substrate drugs were not significantly
different in the parental cells or the S1/FLV5000 cells (which do
not express ABCG2, but also are resistant to flavopiridol in the
presence or absence of sildenafil). Furthermore, when the non-ABCG2
substrate cisplatin was used, its IC.sub.50 values were not
affected by either sildenafil or FTC in any of the cell lines. A
similar phenomenon was observed in both wild-type and mutant
ABCG2-transfected HEK293 cells (Table 10). Representative cell
survival curves (FIGS. 8-11) show that the survival curves in the
presence of sildenafil were shifted remarkably to the left in the
ABCB1- or ABCG2-overexpressing cells. Based on the above results,
it appears that sildenafil significantly inhibits ABCB1-mediated
drug efflux and partially reverses ABCG2-mediated efflux.
TABLE-US-00008 TABLE 8 The Reversal Effect of Sildenafil and
Verapamil on ABCB1-Mediated Resistance.sup.a IC.sub.50 .+-.
SD.sup.b (.mu.M) Compounds KB-3-1 KB-C2 KB-V1 Colchicine 0.0057
.+-. 0.0015 (1.0).sup.c 4.292 .+-. 1.493 (747.5) 0.240 .+-. 0.035
(41.8) + Sildenafil (2.5 .mu.M) 0.0062 .+-. 0.0013 (1.1) 0.721 .+-.
0.236 (125.5) 0.059 .+-. 0.035 (10.3) + Sildenafil (5 .mu.M) 0.0058
.+-. 0.0021 (1.0) 0.163 .+-. 0.031 (28.4) 0.052 .+-. 0.022 (9.1) +
Sildenafil (10 .mu.M) 0.0051 .+-. 0.00148 (0.9) 0.060 .+-. 0.004
(10.5) 0.044 .+-. 0.024 (7.6) + Verapamil (2.5 .mu.M) 0.0054 .+-.
0.0016 (0.9) 0.264 .+-. 0.132 (46.0) 0.168 .+-. 0.036 (29.2) +
Verapamil (5 .mu.M) 0.0047 .+-. 0.0021 (0.8) 0.101 .+-. 0.026
(17.6) 0.081 .+-. 0.037 (14.1) + Verapamil (10 .mu.M) 0.0032 .+-.
0.0001 (0.6) 0.055 .+-. 0.009 (9.6) 0.055 .+-. 0.038 (9.6)
Vinblastine 0.0452 .+-. 0.0010 (1.0).sup.c 0.464 .+-. 0.234 (10.3)
5.427 .+-. 3.362 (120.1) + Sildenafil (2.5 .mu.M) 0.0445 .+-.
0.0114 (1.0) 0.139 .+-. 0.086 (3.1) 0.295 .+-. 0.211 (6.5) +
Sildenafil (5 .mu.M) 0.0443 .+-. 0.0076 (1.0) 0.092 .+-. 0.085
(2.0) 0.131 .+-. 0.091 (2.9) + Sildenafil (10 .mu.M) 0.0437 .+-.
0.0173 (1.0) 0.036 .+-. 0.016 (0.8) 0.084 .+-. 0.049 (1.9) +
Verapamil (2.5 .mu.M) 0.0400 .+-. 0.0148 (0.9) 0.123 .+-. 0.109
(2.7) 0.238 .+-. 0.085 (5.3) + Verapamil (5 .mu.M) 0.0250 .+-.
0.0038 (0.6) 0.056 .+-. 0.035 (1.2) 0.107 .+-. 0.015 (2.4) +
Verapamil (10 .mu.M) 0.0222 .+-. 0.0002 (0.5) 0.038 .+-. 0.021
(0.8) 0.057 .+-. 0.013 (1.3) Paclitaxel 0.0056 .+-. 0.0006
(1.0).sup.c 4.380 .+-. 0.802 (788.5) 5.997 .+-. 1.952 (1079.6) +
Sildenafil (2.5 .mu.M) 0.0059 .+-. 0.0014 (1.1) 0.056 .+-. 0.027
(10.0) 0.293 .+-. 0.337 (52.7) + Sildenafil (5 .mu.M) 0.0057 .+-.
0.0010 (1.0) 0.024 .+-. 0.016 (4.4) 0.144 .+-. 0.171 (25.8) +
Sildenafil (10 .mu.M) 0.0056 .+-. 0.0021 (1.0) 0.013 .+-. 0.007
(2.4) 0.071 .+-. 0.040 (12.8) + Verapamil (2.5 .mu.M) 0.0048 .+-.
0.0021 (0.9) 0.049 .+-. 0.039 (8.9) 0.237 .+-. 0.308 (42.7) +
Verapamil (5 .mu.M) 0.0042 .+-. 0.0011 (0.7) 0.012 .+-. 0.003 (2.1)
0.055 .+-. 0.044 (9.9) + Verapamil (10 .mu.M) 0.0034 .+-. 0.0007
(0.6) 0.009 .+-. 0.004 (1.7) 0.042 .+-. 0.017 (7.5) Cisplatin .sup.
1.745 .+-. 0.161 (1.0).sup.c 1.726 .+-. 0.083 (1.0) 1.756 .+-.
0.178 (1.0) + Sildenafil (2.5 .mu.M) 1.637 .+-. 0.088 (0.9) 1.884
.+-. 0.320 (1.1) 1.835 .+-. 0.071 (1.1) + Sildenafil (5 .mu.M)
1.601 .+-. 0.030 (0.9) 1.838 .+-. 0.206 (1.1) 1.865 .+-. 0.006
(1.1) + Sildenafil (10 .mu.M) 1.628 .+-. 0.157 (0.9) 1.651 .+-.
0.104 (0.9) 1.811 .+-. 0.081 (1.0) + Verapamil (2.5 .mu.M) 1.729
.+-. 0.231 (1.0) 1.790 .+-. 0.250 (1.0) 1.784 .+-. 0.165 (1.0) +
Verapamil (5 .mu.M) 1.623 .+-. 0.001 (0.9) 1.762 .+-. 0.142 (1.0)
1.747 .+-. 0.056 (1.0) + Verapamil (10 .mu.M) 1.457 .+-. 0.007
(0.8) 1.738 .+-. 0.042 (1.0) 1.797 .+-. 0.106 (1.0) .sup.aCell
survival was determined by MTT assay as described in the Example
2.1.c. .sup.bData are expressed as means .+-. SD of at least three
independent experiments carried out in triplicate.
.sup.cFold-resistance values (values in parentheses) were
calculated by dividing all IC.sub.50 values obtained for
colchicine, vinblastine, paclitaxel, and cisplatin by the
respective IC.sub.50 value for colchicine, vinblastine, paclitaxel,
and cisplatin in the KB-3-1 cells in the absence of a reversal
agent.
TABLE-US-00009 TABLE 9 The Reversal Effect of Sildenafil and FTC on
ABCG2-Mediated Resistance in Drug-Selected Cell Lines.sup.a
IC.sub.50 .+-. SD.sup.b (.mu.M) S1/ MCF-7/ MCF7/ Compounds S1
FLV5000 S1-M1-80 MCF-7 Flv1000 ADVP3000 Flavopiridol 0.0961 .+-.
0.0096 6.2046 .+-. 0.4631 0.6992 .+-. 0.0470 0.3443 .+-. 0.0021
6.8556 .+-. 0.4794 3.9005 .+-. 0.4139 (1.0).sup.c (64.9) (7.3)
(1.0).sup.c (19.9) (11.3) +Sildenafil 0.0854 .+-. 0.0291 5.4169
.+-. 0.1866 0.1310 .+-. 0.0192 0.3445 .+-. 0.0342 3.3609 .+-.
0.2038 2.0516 .+-. 0.2546 (10 .mu.M) (0.9) (56.4) (1.4) (1.0) (9.8)
(6.0) +Sildenafil 0.0767 .+-. 0.0292 5.1281 .+-. 0.3575 0.0951 .+-.
0.0351 0.3605 .+-. 0.0445 0.4531 .+-. 0.0151 0.5682 .+-. 0.0321 (50
.mu.M) (0.8) (53.4) (1.0) (1.0) (1.3) (1.7) +FTC 0.1239 .+-. 0.0388
5.6540 .+-. 0.1843 0.0948 .+-. 0.0148 0.3441 .+-. 0.0148 0.2777
.+-. 0.0197 0.4166 .+-. 0.0147 (2.5 .mu.M) (1.3) (58.8) (1.0) (1.0)
(0.8) (1.2) Mitoxantrone 0.3796 .+-. 0.0416 1.1218 .+-. 0.1437
223.60 .+-. 8.60 7.6649 .+-. 0.1934 511.35 .+-. 16.71 279.25 .+-.
6.03 (1.0).sup.c (3.0) (559.3) (1.0).sup.c (66.7) (36.4)
+Sildenafil 0.1917 .+-. 0.0006 0.4212 .+-. 0.1297 113.65 .+-. 12.80
2.6792 .+-. 0.0243 127.21 .+-. 2.62 96.77 .+-. 3.01 (10 .mu.M)
(0.5) (1.1) (284.3) (0.3) (16.6) (12.6) +Sildenafil 0.1085 .+-.
0.0344 0.3469 .+-. 0.0330 32.95 .+-. 2.33 1.3434 .+-. 0.0843 36.93
.+-. 2.12 24.39 .+-. 3.66 (50 .mu.M) (0.3) (0.9) (82.4) (0.2) (4.8)
(3.2) +FTC 0.3119 .+-. 0.0310 0.9986 .+-. 0.0218 3.55 .+-. 1.91
1.4795 .+-. 0.2029 18.45 .+-. 1.63 13.82 .+-. 0.67 (2.5 .mu.M)
(0.8) (2.6) (8.9) (0.2) (2.4) (1.8) SN-38 0.2449 .+-. 0.0170 0.3442
.+-. 0.0874 3.5533 .+-. 1.3962 1.2218 .+-. 0.2626 22.827 .+-. 2.001
14.301 .+-. 1.617 (1.0).sup.c (1.4) (14.4) (1.0).sup.c (18.7)
(11.3) +Sildenafil 0.2488 .+-. 0.0598 0.1647 .+-. 0.0063 1.4886
.+-. 0.5598 0.6009 .+-. 0.0071 10.639 .+-. 1.537 3.861 .+-. 0.173
(10 .mu.M) (1.0) (0.7) (6.0) (0.5) (8.7) (3.2) +Sildenafil 0.2070
.+-. 0.0244 0.1584 .+-. 0.0151 0.4924 .+-. 0.0281 0.3137 .+-.
0.0346 4.214 .+-. 0.277 1.784 .+-. 0.086 (50 .mu.M) (0.8) (0.6)
(2.0) (0.3) (3.4) (1.5) +FTC 0.2808 .+-. 0.0280 0.2933 .+-. 0.0265
0.1014 .+-. 0.0310 0.2993 .+-. 0.0272 2.580 .+-. 0.132 0.198 .+-.
0.049 (2.5 .mu.M) (1.1) (1.2) (0.4) (0.2) (2.1) (0.2) Cisplatin
14.70 .+-. 2.23 53.45 .+-. 5.80 18.00 .+-. 2.59 34.41 .+-. 1.76
35.12 .+-. 1.86 31.12 .+-. 3.20 (1.0).sup.c (3.6) (1.2) (1.0).sup.c
(1.0) (0.9) +Sildenafil 15.39 .+-. 2.94 56.27 .+-. 1.93 20.08 .+-.
1.43 34.76 .+-. 0.80 34.43 .+-. 1.32 30.72 .+-. 3.79 (10 .mu.M)
(1.0) (3.8) (1.4) (1.0) (1.0) (0.9) +Sildenafil 15.90 .+-. 3.35
56.88 .+-. 4.19 20.64 .+-. 2.24 32.27 .+-. 1.70 34.47 .+-. 5.58
28.01 .+-. 4.65 (50 .mu.M) (1.1) (3.9) (1.4) (1.0) (1.0) (0.8) +FTC
13.27 .+-. 0.52 47.35 .+-. 4.48 15.30 .+-. 1.38 33.24 .+-. 1.41
35.02 .+-. 1.04 31.13 .+-. 0.33 (2.5 .mu.M) (0.9) (3.2) (1.0) (1.0)
(1.0) (0.9) .sup.aCell survival was determined by MTT assay as
described in the Example 2.1.c. .sup.bData are expressed as means
.+-. SD of at least three independent experiments performed in
triplicate. .sup.cFold-resistance values (values in parentheses)
were calculated by dividing all IC.sub.50 values obtained for
flavopiridol, mitoxantrone, SN-38, and cisplatin by the respective
IC.sub.50 value for flavopiridol, mitoxantrone, SN-38, and
cisplatin in the S1 cells in the absence of a reversal agent.
TABLE-US-00010 TABLE 10 The Reversal Effect of Sildenafil and FTC
on ABCG2-Mediated Resistance in ABCG2-Transfected Cell Lines.sup.a
IC.sub.50 .+-. SD.sup.b (.mu.M) Compounds HEK293/pcDNA3
HEK/ABCG2-G2 HEK/ABCG2-R5 HEK/ABCG2-T7 Flavopiridol 0.1640 .+-.
0.0171 (1.0).sup.c 0.3976 .+-. 0.0910 (2.4) 0.2821 .+-. 0.0148
(1.7) 0.7578 .+-. 0.0423 (4.6) + Sildenafil (10 .mu.M) 0.1493 .+-.
0.0059 (0.9) 0.1216 .+-. 0.0112 (0.7) 0.1670 .+-. 0.0107 (1.0)
0.2256 .+-. 0.0108 (1.4) + Sildenafil (50 .mu.M) 0.1599 .+-. 0.0180
(1.0) 0.1122 .+-. 0.0117 (0.7) 0.1466 .+-. 0.0030 (0.9) 0.1916 .+-.
0.0316 (1.2) + FTC (2.5 .mu.M) 0.1276 .+-. 0.0033 (0.8) 0.0988 .+-.
0.0386 (0.6) 0.1643 .+-. 0.0236 (1.0) 0.1643 .+-. 0.0296 (1.0)
Mitoxantrone 0.0356 .+-. 0.0030 (1.0).sup.c 1.1042 .+-. 0.6023
(31.0) 0.4574 .+-. 0.3924 (12.8) 0.8424 .+-. 0.1858 (23.7) +
Sildenafil (10 .mu.M) 0.0115 .+-. 0.0007 (0.3) 0.0875 .+-. 0.0045
(2.5) 0.1142 .+-. 0.0004 (3.2) 0.1168 .+-. 0.0118 (3.3) +
Sildeuafil (50 .mu.M) 0.0057 .+-. 0.0006 (0.2) 0.0429 .+-. 0.0061
(1.2) 0.0784 .+-. 0.0207 (2.2) 0.0418 .+-. 0.0020 (1.2) + FTC (2.5
.mu.M) 0.0315 .+-. 0.0092 (0.9) 0.0696 .+-. 0.0054 (2.0) 0.0420
.+-. 0.0156 (1.2) 0.0919 .+-. 0.0208 (2.6) SN-38 0.0058 .+-. 0.0002
(1.0).sup.c 0.2042 .+-. 0.1362 (2.3) 0.1282 .+-. 0.0882 (22.1)
0.1171 .+-. 0.0704 (20.2) + Sildenafil (10 .mu.M) 0.0034 .+-.
0.0010 (0.6) 0.0101 .+-. 0.0021 (1.7) 0.0175 .+-. 0.0018 (3.0)
0.0488 .+-. 0.0186 (8.4) + Sildenafil (50 .mu.M) 0.0019 .+-. 0.0003
(0.3) 0.0044 .+-. 0.0100 (0.8) 0.0063 .+-. 0.0029 (1.1) 0.0139 .+-.
0.0025 (2.4) + FTC (2.5 .mu.M) 0.0036 .+-. 0.0092 (0.8) 0.0050 .+-.
0.0009 (1.9) 0.0081 .+-. 0.0051 (1.4) 0.0082 .+-. 0.0023 (1.4)
Cisplatin 1.6300 .+-. 0.1697 (1.0).sup.c 1.2305 .+-. 0.0841 (0.8)
1.3265 .+-. 0.2001 (0.8) 1.8435 .+-. 0.0233 (1.1) + Sildenafil (10
.mu.M) 1.5640 .+-. 0.0156 (1.0) 1.1371 .+-. 0.1577 (0.7) 1.3855
.+-. 0.0955 (0.9) 1.8115 .+-. 0.0403 (1.1) + Sildenafil (50 .mu.M)
2.0180 .+-. 0.4285 (1.2) 1.1993 .+-. 0.1429 (0.7) 1.9820 .+-.
0.0438 (1.2) 1.9680 .+-. 0.0665 (1.2) + FTC (2.5 .mu.M) 1.1535 .+-.
0.0488 (0.7) 1.0466 .+-. 0.028 (0.6) 1.3495 .+-. 0.2072 (0.8)
1.9530 .+-. 0.0834 (1.2) .sup.aCell survival was determined by MTT
assay as described in the Example 2.1.c. .sup.bData are expressed
as means .+-. SD of at least three independent experiments carried
out in triplicate. .sup.cFold-resistance values (values in
parentheses) were calculated by dividing all IC.sub.50 values
obtained for flavopiridol, mitoxantrone, SN-38, and cisplatin by
the respective IC.sub.50 value for flavopiridol, mitoxantrone,
SN-38, and cisplatin in the HEK293/pcDNA3 cells in the absence of a
reversal agent.
Example 2.3
Sildenafil Increases Accumulation of [.sup.3H]-Paclitaxel in
ABCB1-Overexpressing Cells, and [.sup.3H]-Mitoxantrone and
BODIPY-Prazosin in ABCG2-Overexpressing Cells
[0127] To investigate the potential mechanism by which sildenafil
sensitizes ABCB1- and ABCG2-overexpressing cells to
chemotherapeutic drugs, the effect of sildenafil on the
accumulation of chemotherapeutic drugs in ABCB1- or
ABCG2-overexpressing cells was examined. Intracellular
[.sup.3H]-paclitaxel was measured in ABCB1-overexpressing cells in
the presence or absence of sildenafil, and the results are shown in
FIG. 4A. After 2 h of incubation, the intracellular levels of
[.sup.3H]-paclitaxel in ABCB1-overexpressing KB-C2 and KB-V1 cells
were significantly lower than that of the parental KB-3-1 cells.
Sildenafil at 10 .mu.M significantly increased the intracellular
level of [.sup.3H]-paclitaxel, close to the effect of verapamil at
10 .mu.M in KB-C2 and KB-V1 cells. Neither sildenafil nor verapamil
altered the intracellular levels of [.sup.3H]-paclitaxel in
parental KB-3-1 cells. Similarly, the intracellular levels of
[.sup.3H]-mitoxantrone were measured in the ABCG2-overexpressing
cells in the presence or absence of sildenafil (FIG. 4B-D). The
intracellular levels of [.sup.3H]-mitoxantrone in cells expressing
either wild-type or mutant ABCG2 were significantly less than that
in parental cells. In the presence of sildenafil, either at 10 or
50 .mu.M, all ABCG2-overexpressing cell lines displayed elevated
intracellular [.sup.3H]-mitoxantrone levels, and intracellular
levels of [.sup.3H]-mitoxantrone increased with increasing
concentrations of sildenafil. However, the effects of sildenafil at
50 .mu.M were less than those observed for FTC at 2.5 .mu.M.
Neither sildenafil nor FTC affected the intracellular levels of
[.sup.3H]-mitoxantrone in parental cells.
[0128] In addition, the effect of sildenafil on the accumulation of
a known fluorescent substrate of ABCG2 in the ABCG2-overexpressing
cells was evaluated. Sildenafil at 50 .mu.M enhanced the
accumulation of BODIPY-prazosin in cells expressing either
wild-type or mutant ABCG2, but demonstrated weaker enhancement than
that of FTC at 2.5 .mu.M. Representative histograms for
BODIPY-prazosin are shown in FIGS. 5A-D. Taken together, these data
are in agreement with the cytotoxicity results and suggest that
sildenafil is able to inhibit the efflux function of ABCB1 and
ABCG2, leading to the significant increase of intracellular
accumulation of [.sup.3H]-paclitaxel in ABCB1-overexpressing cells
and [.sup.3H]-mitoxantrone as well as BODIPY-prazosin in
ABCG2-overexpressing cells.
Example 2.4
Sildenafil Inhibits Transport of E.sub.217.beta.G and Methotrexate
by ABCG2
[0129] To assess the potency of sildenafil as an in vitro inhibitor
of ABCG2, the ability of sildenafil to inhibit the transport
activity of ABCG2 was analyzed using the chemotherapeutic drug
substrate [.sup.3H]-methotrexate and the physiologic substrate
[.sup.3H]-E.sub.217.beta.G. In Shi et al. ((2007) Cancer Res.
97:11012-20), it was shown that only wild-type ABCG2 was able to
transport methotrexate and E.sub.217.beta.G by the in vitro
transport system. Thus, membrane vesicles prepared from
HEK293/pcDNA3.1 and ABCG2-482-R5 cells were used (FIGS. 5E-F).
Sildenafil significantly inhibited the rates of both methotrexate
and E.sub.217.beta.G uptake in the membrane vesicles of wild-type
ABCG2 in a concentration-dependent manner, but its inhibitory
effect was weaker than that of FTC at the same concentration. FTC
also moderately decreased the uptake rates of both methotrexate and
E.sub.217.beta.G in the membrane vesicles of HEK293/pcDNA3.1, but
sildenafil did not. These in vitro transport results suggest that
sildenafil is able to directly inhibit the transport function of
E.sub.217.beta.G and methotrexate in wild-type ABCG2-expressing
cells.
Example 2.5
Sildenafil Stimulates ATPase Activity and Affects the Photoaffinity
Labeling of ABCB1 and ABCG2 with [.sup.125I]-IAAP
[0130] Generally, the substrates of ABC transporters stimulate
their ATPase activity, and among the reversal agents, some (e.g.,
verapamil) stimulate the activity, whereas others (e.g.,
cyclosporine A) inhibit ATP hydrolysis (Ambudkar et al. (1999)
Annu. Rev. Pharmacol Toxicol. 39:361-98). To assess the effect of
sildenafil on the ATPase activity of ABCB1 and ABCG2, the membrane
vesicles of HIGH FIVE.TM. insect cells overexpressing ABCB1 or
ABCG2 were used in the presence of various concentrations of
sildenafil under conditions that suppressed the activity of other
major membrane ATPases. As shown in FIGS. 6A and 6B, sildenafil at
the indicated concentrations potently stimulated the ATPase
activity of ABCB1, but mildly stimulated the ATPase activity of
ABCG2. The concentrations of sildenafil required for 50%
stimulation of ATPase activity of ABCB1 and ABCG2 were 5-10 and
0.25-0.5 .mu.M, respectively. These results indicate that
sildenafil may be a substrate for ABCB1 and ABCG2.
[0131] The photoaffinity analogue of prazosin, [.sup.125I]-IAAP,
which is transported by both ABCB1 and ABCG2, has been widely used
to determine the binding regions of ABCB1 and ABCG2 that interact
with substrates and inhibitors. To test whether sildenafil
interacts at the prazosin binding site of ABCB1 or ABCG2, the
ability of sildenafil to prevent photolabeling of ABCB1 and ABCG2
with [.sup.125I]-IAAP was examined by using the membrane vesicles
from HIGH FIVE.TM. insect cells transfected with ABCB1 or ABCG2. As
shown in FIGS. 6C and 6D, sildenafil dose dependently inhibited the
photoaffinity labeling of ABCB1 or ABCG2 with [.sup.125I]-IAAP. The
50% inhibition of the photoaffinity labeling of ABCB1 and ABCG2
with [.sup.125I]-IAAP by sildenafil was observed at sildenafil
concentrations of 10 and 100 .mu.M, respectively. The ABCB1
inhibitors cyclosporine A (at 10 .mu.M) and verapamil (at 20 .mu.M)
inhibited the [.sup.125I]-IAAP photolabeling of ABCB1 up to about
95% and 65%, respectively, and the ABCG2 inhibitor FTC (at 10
.mu.M) inhibited the [.sup.125I]-IAAP photolabeling of ABCG2 to
about 40%, compared with membranes incubated with no inhibitor.
Thus, these data suggest that sildenafil competes with IAAP to bind
both ABCB1 and ABCG2, and that sildenafil interacts with the
transmembrane regions of both transporters.
Example 2.6
Model for Sildenafil Binding to ABCB1
[0132] PDE5A homology model of human ABCB1 based on the mouse
(Mdr3) ABCB1-QZ59-RRR cocrystal structure was used to predict
sildenafil-ABCB1 binding conformation. A human ABCB1 homology model
(FIG. 7A) was then used to dock sildenafil using Glide docking
software to investigate its potential binding mode. Three binding
sites were reported in the crystal structure of mouse Mdr3 as
represented by ABCB1-QZ59-RRR (site-1), ABCB1-QZ59-SSS (site-2),
and ABCB1-verapamil (site-3). Aller et al., supra. To identify a
favorable binding site on ABCB1 for sildenafil, docking experiments
using these sites were carried out. Because the photoaffinity
labeling data suggested that sildenafil displaces IAAP in a
dose-dependent manner, IAAP was also docked to these sites for
comparison. These data also indicated that sildenafil and IAAP
share the same binding site on ABCB1 (i.e., site-1). Binding energy
data for the docked poses of sildenafil and IAAP were compared at
each of the binding sites (Table 11). On the basis of binding
energy data analysis, both ligands (sildenafil and IAAP) were found
to bind most favorably within the QZ59-RRR binding site of ABCB1.
Bound conformation of sildenafil in context of site-1 is addressed
below.
TABLE-US-00011 TABLE 11 Binding Energies of Sildenafil and IAAP in
Their Ionized States within Each of the Predicted Binding Sites of
ABCB1 Glide score kcal/mol Ligands Site-1.sup.a Site-2.sup.b
Site-3.sup.c Site-4.sup.d ##STR00004## -8.48 -5.72 -5.20 -3.21
##STR00005## -8.89 -5.79 -4.54 -5.16 .sup.a-Site represented by
bound QZ59-RRR. .sup.b-Site represented by bound ligand QZ59-SSS.
.sup.c-Verapamil binding site. .sup.d-Site grid generated using
residues Phe728 and Val982, which are known to be common to the
above three sites.
[0133] The XP-Glide-predicted binding mode of sildenafil shows the
importance of hydrophobic interactions within the large drug
binding cavity of ABCB1 (FIG. 7B). The N-methylpiperazine (D-ring)
of sildenafil was found to be stabilized by hydrophobic contacts
with the side chains of Met69 of transmembrane (TM) 1 and Phe336,
Leu339, and Ile340 of TM6. The C-ring along with its ethoxy
substituent enters into favorable hydrophobic interactions with
residues Phe72 of TM1, Leu975 and Phe978 of TM12. The A-ring (along
with its methyl and propyl substituents) and the B-ring forms
hydrophobic interactions with the side chains of Phe728, Ala729,
and Phe732 of TM7 and Val982 of TM12. In addition to the
hydrophobic contacts, sildenafil also seems to be stabilized by
electrostatic interactions with key residues Tyr953 of TM11 and
Tyr307 of TM5. For example, the sulfonyl oxygen atom enters into a
hydrogen bonding interaction with the hydroxyl group of Tyr953
(--SO.sub.2--HO-Tyr953), whereas the carbonyl oxygen atom present
in the B-ring is located at a distance of 3.9 .ANG. from the side
chain hydroxyl group of Tyr307. Transmembrane domain numbering is
as reported previously (Aller et al., supra).
Example 3
Effect of Sildenafil on Antineoplastic Drug Treatment of Tumors in
a Mouse Model
Example 3.1
Materials and Methods
Example 3.1.a
Materials
[0134] Paclitaxel (as antineoplastic drug) and saline (as vehicle)
were purchased from Sigma Chemical Company. Sildenafil was purified
from 100 mg VIAGRA.RTM. tablets as described in Example 2.1.a.
Example 3.1.b
Cell Lines and Experimental Animals
[0135] KB-3-1 cells and the ABCB1/Pgp-overexpressing drug-resistant
cell line KB-C2 cells were obtained as described in Example
2.1.b.
[0136] Six-to-eight-week old male athymic NCR nude mice from
Taconic (Hudson, N.Y.) were used for all xenografts. The mice were
provided with sterilized food and water. The animal protocol was
reviewed and approved by the Institutional Animal Care and Use
Committee at St. John's University.
Example 3.1.c
Mouse Tumor Xenografts
[0137] KB-3-1 or KB-C2 cells were grown in flasks, harvested and
implanted (at 2.5.times.10.sup.6 cells in 0.2 mL for KB-3-1 cells
and 4.0.times.10.sup.6 cells in 0.2 mL for KB-C2 cells)
subcutaneously under the shoulder in the nude mice. When the tumors
reached 0.5 cm, the mice were randomized into four groups, six mice
per group: (1) vehicle, (2) sildenafil (given at 75 mg/kg p.o.),
(3) paclitaxel (given at 18 mg/kg i.p.), and (4) paclitaxel and
sildenafil, all treated every three days for a total of six
administrations. The mice were observed for 2-3 weeks, and the
tumor volumes estimated every three days from the perpendicular
diameters (A and B) according to the following formula:
V=.pi./6.times.((A+B)/2).sup.3 (see, e.g., Dai et al. (2008)
68:7509-14).
[0138] Mice were euthanized when the tumor weight exceeded 1 gram
in the control group, and the tumors were excised and weighed.
Example 3.2
Coadministration of Sildenafil and Paclitaxel Leads to Significant
Decrease in Tumor Weight and Volume in KB-C2 Mouse Xenografts
[0139] The efficacy of sildenafil in blocking MDR in vivo was
evaluated using mouse tumor xenografts. As demonstrated in FIGS. 12
and 13, there was a significant reduction in tumor weight and
volume in mice treated with both sildenafil and paclitaxel in
comparison with mice treated with paclitaxel alone.
[0140] Similar results were obtained from experiments on mouse
xenografts of human lung cancer cell line H460/MX20 (which is an
ABCG2-overexpressing cell line derived from parental H460
cells).
Example 4
Effects of Sildenafil, Vardenafil, and Tadalafil on
ABCC10/MRP7-Mediated Multidrug Resistance
Example 4.1
Materials and Methods
Example 4.1.a
Reagents
[0141] Sildenafil, vardenafil, and tadalafil were purchased from
Toronto Research Chemicals Inc. (Ontario, Canada). Cepharanthine
was provided by Daiichi Sankyo Pharmaceutical Co. Ltd (Tokyo,
Japan). Dulbecco's modified Eagle's medium (DMEM), fetal bovine
serum (FBS), penicillin/streptomycin and trypsin 0.25% were
products of Hyclone (Logan, Utah). Monoclonal antibody 14C10
(against GAPDH) was acquired from Cell Signaling Technology, Inc.
(Danvers, Mass.). Polyclonal antibody D-19 (against MRP7) was
obtained from Santa Cruz Biotechnology Inc (Santa Cruz, Calif.).
Alexa Flour 488 donkey anti-goat secondary antibody for
immunocytochemistry was purchased from Molecular Probes (Eugene,
Oreg.). [.sup.3H]-paclitaxel (46.5 Ci/mmol) was purchased from
Moravek Biochemicals Inc (Brea, Calif.). Paclitaxel, docetaxel,
vinblastine, DMSO, MTT, and other chemicals were purchased from
Sigma Chemical (St. Louis, Mo.).
Example 4.1.b
Cell Lines and Cell Culture
[0142] The MRP7 cDNA was generously provided by Dr. Gary Kruh
(University of Illinois, Chicago, Ill.) and inserted into the
pcDNA3.1 expression vector. The MRP7 expression vector and parental
plasmid were introduced into HEK293 cells by electroporation as
previously described (Chen et al. (2003) Mol. Pharmacol.
63:351-58). Individual colonies were selected in medium containing
G418 (1 mg/ml) and cultured for further analysis. All the cell
lines were grown as adherent monolayers in flasks with Dulbecco's
modified Eagle's medium (DMEM) supplemented with 10% bovine serum,
100 units/ml penicillin and 100 mg/ml streptomycin in a humidified
incubator containing 5% CO.sub.2 at 37.degree. C.
Example 4.1.c
Cytotoxicity Assay
[0143] MTT colorimetric assay was performed to analyze the drug
sensitivity as previously described (Shi et al. (2007) Cancer Res.
67:11012-20). HEK293-pcDNA3.1 and HEK/MRP7 cells were seeded into
96-well plates in triplicate at 6000 cells/well. After incubation
in DMEM supplemented with 10% bovine serum at 37.degree. C. for 24
h, three different nontoxic concentrations of sildenafil,
vardenafil, and tadalafil (1.25, 2.5, and 5 .mu.M) were added into
plates 1 h prior to the addition of the substrates of MRP7
(paclitaxel, docetaxel, and vinblastine).
[0144] After drug incubation of 68 h, 20 .mu.l MTT solution (4
mg/ml) was added into each well. The plates were further incubated
for 4 h, then the medium was discarded, and 100 .mu.l DMSO was
added into each well to dissolve the formazan crystals. The
absorbance was determined at 570 nm by an OPSYS Microplate Reader
from DYNEX Technologies, Inc. (Chantilly, Va.). The degree of
resistance was calculated by dividing the IC.sub.50 values
(concentrations required to inhibit growth by 50%) for the HEK/MRP7
cells by those of the parental HEK293-pcDNA3.1 cells. The Bliss
method was used to calculate the IC.sub.50 values according to
survival curves.
Example 4.1.d
[.sup.3H]-Paclitaxel Accumulation and Efflux
[0145] The effect of PDE5 inhibitors on the intracellular
accumulation of paclitaxel in HEK293-pcDNA3.1 and HEK/MRP7 cells
was measured using [.sup.3H]-paclitaxel. HEK293-pcDNA3.1 and
HEK/MRP7 cells were trypsinized and four aliquots from each cell
line were suspended in the medium, aliquots were preincubated with
medium-only (control), sildenafil, vardenafil, or tadalafil (5
.mu.M each) at 37.degree. C. for 2 h, then incubated with 0.1 .mu.M
[.sup.3H]-paclitaxel for another 2 h. For efflux studies, the cells
were treated the same as in the drug accumulation study, and then
washed three times with ice-cold PBS, and suspended in fresh medium
with or without PDE5 inhibitors. Aliquots were evenly collected at
various time points (0, 30, 60, and 120 min). Samples from both
accumulation and efflux experiments were washed with ice-cold PBS
three times and placed in scintillation fluid, and the
radioactivity was measured in a Packard TRI-CARB.RTM. 1900CA liquid
scintillation analyzer from Packard Instrument Company, Inc.
(Downers Grove, Ill.).
Example 4.1.e
Preparation of Total Cell Lysates and Immunoblotting Analysis
[0146] To determine the effect of PDE5 inhibitors on the expression
of MRP7, HEK/MRP7 cells were incubated with 5 .mu.M sildenafil,
vardenafil, or tadalafil for different time periods (0, 24, 48 and
72 h), then harvested and rinsed twice with cold PBS. The total
cell lysates were collected with radioimmunoprecipitation assay
(RIPA) buffer (1.times.PBS, 1% Nonidet P-40, 0.5% sodium
deoxycholate, 0.1% SDS, 100 .mu.g/ml phenylmethylsulfonyl fluoride,
10 .mu.g/ml aprotinin, 10 .mu.g/ml leupeptin) for 30 min with
occasional rocking followed by centrifugation at 12,000 rpm at
4.degree. C. for 15 min.
[0147] The protein concentration was determined by a bicinchoninic
acid (BCATM)-based protein assay (Thermo Scientific, Rockford,
Ill.). Equal amounts of total cell lysates (40 .mu.g of protein)
were resolved by 4-12% sodium dodecyl sulfate polyacrylamide gel
electrophoresis (SDS-PAGE) and electrophoretically transferred onto
PVDF membranes. After being incubated in blocking solution
containing 5% skim milk in TBST buffer (10 mM Tris-HCL, pH 8.0, 150
mM NaCl and 0.1% Tween 20) at room temperature for 1 h, the
membranes were immunoblotted overnight with primary antibodies to
anti-MRP7 (1:200 dilution) and anti-GAPDH (1:1000 dilution) at
4.degree. C.
[0148] Subsequently, the membranes were washed three times for 15
min with TBST buffer and incubated at room temperature for 2 h with
horseradish peroxide (HRP)-conjugated secondary antibody (1:2000
dilution). The protein-antibody complex was detected by the
enhanced Phototope TM-HRP Detection Kit (Cell Signaling Technology,
Inc.) and exposed to Kodak medical X-ray processor (Eastman Kodak
Co.). The protein expression was quantified by Scion Image software
(Scion Co., Frederick, Md.).
Example 4.1.f
Immunofluorescence Analysis
[0149] HEK/MRP7 cells (1.times.10.sup.4) were seeded in 24 well
plates and cultured overnight. Sildenafil, vardenafil, or tadalafil
at 5 .mu.M were added into the wells and then cultured at
37.degree. C. for 72 h in a humidified incubator containing 5%
CO.sub.2. Cells were washed with PBS and fixed with 4%
paraformaldehyde for 15 min at room temperature and then rinsed
with PBS three times. Nonspecific reaction was blocked with 1% BSA
for 1 h at room temperature. A polyclonal antibody D19 against MRP7
(1:200) was added and incubated overnight. Cells were then
incubated with Alexa Flour.RTM. 488 donkey anti-goat IgG (1:2000)
for 1 h at room temperature. DAPI was used for nuclear staining.
Immunofluorescent images were taken with an inverted microscope
(model IX70; Olympus, Center Valley, Pa.) with IX-FLA fluorescence
and CCD camera.
Example 4.1.g
Statistical Analysis
[0150] All experiments were repeated at least three times, and the
differences were determined by using the Student's t-test. The
statistical significance was determined at p<0.05.
Example 4.2
Effects of PDE5 Inhibitors on the Sensitivity of Antineoplastic
Drugs in HEK293-pcDNA3.1 and HEK/MRP7 Cells
[0151] In order to analyze the reversal efficacy of PDE5
inhibitors, the cytotoxicities of antineoplastic drugs (paclitaxel,
docetaxel, or vinblastine) alone and in combination with a PDE5
inhibitor (sildenafil, vardenafil, or tadalafil) were investigated
at nontoxic concentrations (1.25, 2.5, and 5 .mu.M) in
HEK293-pcDNA3.1 and HEK/MRP7 cells. As shown in Table 12 and FIG.
14, HEK/MRP7 cells compared to parental HEK293-pcDNA3.1 cells
exhibited a significant resistance to various MRP7 substrates such
as paclitaxel, docetaxel, and vinblastine, which is consistent with
previous reports (e.g., Shi et al. (2011) Cancer Res. 71:3029-41).
Sildenafil, vardenafil, and tadalafil dose-dependently decreased
the IC.sub.50 values of the above-mentioned MRP7 substrates for
HEK/MRP7 cells.
[0152] Tadalafil showed the least reversal effect of the three PDE5
inhibitors. Cepharanthine, a known MRP7 inhibitor used as a
positive control at 2.5 .mu.M, completely reversed the resistance
of HEK/MRP7 cells to paclitaxel, docetaxel, and vinblastine. In
contrast, sildenafil, vardenafil, or tadalafil did not
significantly reverse the resistance of HEK/MRP7 cells to
cisplatin, a nonsubstrate of MRP7 (Table 12, FIG. 14). In the
parental HEK293-pcDNA3.1, the IC.sub.50 values of paclitaxel,
docetaxel, and vinblastine in the presence or absence of
sildenafil, vardenafil, or tadalafil showed no significant
difference (Table 12, FIG. 14).
TABLE-US-00012 TABLE 12 The effects of PDE5 inhibitors on the
sensitivity of HEK293-pcDNA3.1 and HEK/MRP7 cells to paclitaxel,
docetaxel, vinblastine and cisplatin. IC.sub.50 .+-. SD.sup.a (nM)
Compounds HEK 293-pcDNA-3.1 HEK/MRP7 Paclitaxel 11.64 .+-. 1.33
(1.0)b 107.18 .+-. 11.25 (9.21) + Sildenafil 1.25 .mu.M 10.69 .+-.
1.04 (0.92) 45.47 .+-. 3.78** (3.91) + Sildenafil 2.5 .mu.M 9.96
.+-. 0.92 (0.86) 28.35 .+-. 2.41** (2.44) + Sildenafil 5 .mu.M 9.38
.+-. 0.86 (0.81) 13.37 .+-. 2.07** (1.15) + Vardenafil 1.25 .mu.M
10.85 .+-. 1.27 (0.93) 34.85 .+-. 3.36** (2.99) + Vardenafil 2.5
.mu.M 9.63 .+-. 0.89 (0.83) 19.86 .+-. 2.61** (1.71) + Vardenafil 5
.mu.M 9.14 .+-. 1.01 (0.79) 12.39 .+-. 1.54** (1.06) + Tadalafil
1.25 .mu.M 12.43 .+-. 0.95 (1.07) 93.78 .+-. 6.23 (8.06) +
Tadalafil 2.5 .mu.M 11.41 .+-. 1.22 (0.98) 81.25 .+-. 7.16* (6.98)
+ Tadalafil 5 .mu.M 10.84 .+-. 0.77 (0.93) 68.36 .+-. 5.83** (5.87)
+ Cepharanthine 2.5 .mu.M 8.97 .+-. 1.18 (0.77) 11.81 .+-. 0.82**
(1.01) Docetaxel 5.73 .+-. 0.65 (1.0) 64.81 .+-. 5.19 (11.31) +
Sildenafil 1.25 .mu.M 5.35 .+-. 0.47 (0.93) 38.29 .+-. 3.75**
(6.68) + Sildenafil 2.5 .mu.M 5.28 .+-. 0.41 (0.92) 22.37 .+-.
2.97** (3.90) + Sildenafil 5 .mu.M 4.74 .+-. 0.56 (0.83) 7.36 .+-.
0.83** (1.28) + Vardenafil 1.25 .mu.M 5.72 .+-. 0.38 (1.0) 31.85
.+-. 3.62** (5.56) + Vardenafil 2.5 .mu.M 4.88 .+-. 0.55 (0.85)
17.95 .+-. 2.57** (3.13) + Vardenafil 5 .mu.M 4.59 .+-. 0.39 (0.80)
6.84 .+-. 0.79** (1.19) + Tadalafil 1.25 .mu.M 6.26 .+-. 0.43
(1.09) 62.48 .+-. 5.03 (10.91) + Tadalafil 2.5 .mu.M 6.35 .+-. 0.59
(1.11) 55.21 .+-. 5.87 (9.64) + Tadalafil 5 .mu.M 5.86 .+-. 0.54
(1.02) 47.85 .+-. 3.98* (8.35) + Cepharanthine 2.5 .mu.M 4.07 .+-.
0.52* (0.71) 5.88 .+-. 0.41** (1.03) Vinblastine 11.19 .+-. 1.23
(1.0) 56.31 .+-. 4.61 (5.03) + Sildenafil 1.25 .mu.M 10.96 .+-.
0.95 (0.98) 36.19 .+-. 2.48** (3.23) + Sildenafil 2.5 .mu.M 10.27
.+-. 0.82 (0.92) 25.92 .+-. 3.04** (2.32) + Sildenafil 5 .mu.M 9.69
.+-. 0.97 (0.87) 14.37 .+-. 1.19** (1.28) + Vardenafil 1.25 .mu.M
10.73 .+-. 1.11 (0.96) 33.72 .+-. 2.89** (3.01) + Vardenafil 2.5
.mu.M 10.32 .+-. 1.03 (0.92) 21.45 .+-. 1.94** (1.92) + Vardenafil
5 .mu.M 9.48 .+-. 0.98 (0.85) 12.39 .+-. 1.05** (1.11) + Tadalafil
1.25 .mu.M 11.84 .+-. 0.88 (1.06) 51.28 .+-. 4.85 (4.58) +
Tadalafil 2.5 .mu.M 11.51 .+-. 1.19 (1.03) 47.95 .+-. 4.11 (4.29) +
Tadalafil 5 .mu.M 10.66 .+-. 1.08 (0.95) 40.54 .+-. 3.92* (3.62) +
Cepharanthine 2.5 .mu.M 8.92 .+-. 1.07 (0.80) 11.35 .+-. 0.93**
(1.01) Cisplatin 1574.26 .+-. 84.95 (1.0) 1552.22 .+-. 74.39 (0.99)
+ Sildenafil 5 .mu.M 1730.35 .+-. 63.89 (1.10) 1629.48 .+-. 81.76
(1.04) + Vardenafil 5 .mu.M 1681.45 .+-. 102.34 (1.07) 1714.60 .+-.
71.43 (1.09) + Tadalafil 5 .mu.M 1746.87 .+-. 75.02 (1.11) 1757.12
.+-. 109.15 (1.12) + Cepharanthine 2.5 .mu.M 1677.38 .+-. 59.32
(1.07) 1635.30 .+-. 62.78 (1.04) .sup.aIC.sub.50: concentration
that inhibited cell survival by 50%. Data are means .+-. SD of at
least three independent experiments performed in triplicate.
bFold-resistance was determined by dividing the IC.sub.50 values of
HEK/MRP7 cells by the IC.sub.50 values of HEK293-pcDNA3.1 cells in
the absence or presence of sildenafil, vardenafil, tadalafil or
verapamil. *represents p < 0.05; **represents p < 0.01
Example 4.3
PDE5 Inhibitors Increase the Intracellular Accumulation of
[.sup.3H]-Paclitaxel in HEK/MRP7 Cells
[0153] As further confirmation of the effects of PDE5 inhibitors on
the drug efflux function of MRP7, intracellular accumulation of
[.sup.3H]-paclitaxel studies were performed. The intracellular
concentration of [.sup.3H]-paclitaxel in HEK/MRP7 cells was
significantly lower (28.5%) than that in parental HEK293-pcDNA3.1
cells (100%, FIG. 15). After the cells were incubated with either
sildenafil, vardenafil, or tadalafil at 5 .mu.M for 2 h, the
intracellular accumulation of [.sup.3H]-paclitaxel in HEK/MRP7
cells was significantly increased by 3.3-, 3.7- and 2.1-fold,
respectively, as compared to 2.5 .mu.M of cepharanthine as a
positive control (3.8-fold; FIG. 15). Neither PDE5 inhibitors nor
cepharanthine significantly affected the intracellular levels of
[.sup.3H]-paclitaxel in HEK293-pcDNA3.1 cells (FIG. 15).
Example 4.4
PDE5 Inhibitors Inhibit the Efflux of [.sup.3H]-Paclitaxel Mediated
by MRP7 in HEK/MRP7 Cells
[0154] To ascertain whether the increase in the intracellular
[.sup.3H]-paclitaxel accumulation in the presence of sildenafil,
vardenafil, or tadalafil was due to the inhibition of
[.sup.3H]-paclitaxel efflux by MRP7 (ABCC10), a time course study
was designed to measure intracellular [.sup.3H]-paclitaxel levels
in the presence of sildenafil, vardenafil, or tadalafil. As shown
in FIG. 16, HEK/MRP7 cells significantly extruded a higher
percentage of intracellular [.sup.3H]-paclitaxel than that in
HEK293-pcDNA3.1 cells. However, in the presence of sildenafil,
vardenafil, or tadalafil at 5 .mu.M, there were significant
decreases in the efflux of intracellular [.sup.3H]-paclitaxel at
different time periods (0, 30, 60, and 120 min) from HEK/MRP7
cells, but not from the parental HEK293-pcDNA3.1 cells. The
intracellular accumulation of [.sup.3H]-paclitaxel at 0 min was set
as 100%, and at 30, 60 and 120 min, the percentages were 62.43%,
39.47%, and 23.44%, respectively, of the accumulated
[.sup.3H]-paclitaxel that remained in HEK/MRP7 cells in the absence
of PDE5 inhibitors. When HEK/MRP7 cells were incubated with
sildenafil, the percentage of the intracellular
[.sup.3H]-paclitaxel at 30, 60 and 120 min increased significantly
to 90.62%, 82.14%, and 62.35%, respectively (FIG. 16A). Vardenafil
significantly increased the percentages of the intracellular
[.sup.3H]-paclitaxel at 30, 60 and 120 min to 95.34%, 93.58%, and
63.92%, respectively (FIG. 16B). Tadalafil at 30, 60 and 120 min
increased significantly the percentage of [.sup.3H]-paclitaxel
accumulation to 76.44%, 63.41%, and 46.48%, respectively (FIG.
16C). Sildenafil and vardenafil were more potent than tadalafil,
which is consistent with the results in colorimetric growth assay
and [.sup.3H]-paclitaxel accumulation experiments.
Example 4.5
PDE5 Inhibitors do not Alter the Expression of MRP7
[0155] Reversal of MRP7-mediated MDR can be achieved by either
altering MRP7 expression or inhibiting MRP7 function. To evaluate
the effects of sildenafil, vardenafil, and tadalafil on MRP7
expression, HEK/MRP7 cells were treated with sildenafil,
vardenafil, or tadalafil at 5 .mu.M for 0, 24, 48, and 72 h and the
MRP7 expression levels were examined by Western blot analysis. The
results shown in FIG. 17A indicated that PDE5 inhibitors do not
significantly alter the protein expression levels of MRP7 in
HEK/MRP7 cells.
Example 4.6
PDE5 Inhibitors do not Alter the Localization of MRP7
[0156] Presumably, the ABC transporters could be downregulated if
they were translocated or dislodged from the plasma membrane to a
cytosolic region. To rule out this possibility, an
immunofluorescence assay was performed to examine whether the
location of MRP7 was altered after treatment with a PDE5 inhibitor.
As shown in FIG. 17, there was no alteration of MRP7 protein
localization in plasma membranes after the treatment with
sildenafil, vardenafil, or tadalafil at 5 .mu.M for 72 h. The
Western blotting (FIG. 17A) and immunocytochemical (FIG. 17B)
experiments suggested that all three PDE5 inhibitors do not alter
the expression and/or localization of the MRP7 transporter in
HEK/MRP7 cells.
EQUIVALENTS
[0157] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents of the specific embodiments of the invention described
herein. Such equivalents also are intended to be encompassed by the
following claims.
* * * * *
References