U.S. patent application number 11/794855 was filed with the patent office on 2009-03-19 for pde5 inhibitor compositions and methods for immunotherapy.
This patent application is currently assigned to The John Hopkins University. Invention is credited to Ivan M. Borrello, Vincenzo Bronte, Kimberly A. Noonan, Paolo Serafini.
Application Number | 20090074796 11/794855 |
Document ID | / |
Family ID | 36648248 |
Filed Date | 2009-03-19 |
United States Patent
Application |
20090074796 |
Kind Code |
A1 |
Borrello; Ivan M. ; et
al. |
March 19, 2009 |
Pde5 inhibitor compositions and methods for immunotherapy
Abstract
The invention features methods and compositions featuring a PDE5
inhibitor for treating or preventing immunological-mediated disease
in a subject.
Inventors: |
Borrello; Ivan M.;
(Baltimore, MD) ; Serafini; Paolo; (Baltimore,
MD) ; Noonan; Kimberly A.; (Owings Mills, MA)
; Bronte; Vincenzo; (Padova, IT) |
Correspondence
Address: |
EDWARDS ANGELL PALMER & DODGE LLP
P.O. BOX 55874
BOSTON
MA
02205
US
|
Assignee: |
The John Hopkins University
Baltimore
MD
|
Family ID: |
36648248 |
Appl. No.: |
11/794855 |
Filed: |
January 9, 2006 |
PCT Filed: |
January 9, 2006 |
PCT NO: |
PCT/US06/00699 |
371 Date: |
October 8, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60642029 |
Jan 7, 2005 |
|
|
|
Current U.S.
Class: |
424/184.1 ;
514/250; 514/262.1 |
Current CPC
Class: |
A61P 31/00 20180101;
A61K 31/53 20130101; A61P 35/00 20180101; A61K 31/519 20130101;
A61P 29/00 20180101; A61K 31/4985 20130101; G01N 2800/52 20130101;
G01N 33/5091 20130101; A61K 45/06 20130101; A61K 31/4985 20130101;
A61K 2300/00 20130101; A61K 31/519 20130101; A61K 2300/00 20130101;
A61K 31/53 20130101; A61K 2300/00 20130101 |
Class at
Publication: |
424/184.1 ;
514/262.1; 514/250 |
International
Class: |
A61K 39/00 20060101
A61K039/00; A61K 31/519 20060101 A61K031/519; A61K 31/4985 20060101
A61K031/4985 |
Claims
1. A method of treating or preventing cancer in a subject
comprising administration to the subject of a PDE-5 inhibitor
compound.
2. The method of claim 1, further comprising administration to the
subject of an additional anticancer agent.
3. The method of claim 2, wherein the additional anticancer agent
is an immunotherapeutic agent.
4. The method of claim 3, wherein the additional anticancer agent
is a vaccine.
5. The method of claim 1, wherein the subject is identified as in
need of treatment of cancer with a PDE-5 inhibitor compound.
6. The method of claim 1, wherein the PDE-5 inhibitor compound is
selected from sildenafil, vardenafil or tadalafil.
7. A method of modulating myeloid suppressor cells (MSCs) immune
suppression in a subject comprising administration to the subject
of a PDE-5 inhibitor compound.
8. The method of claim 7, wherein the modulating is
down-modulation.
9. A method of modulating arginase-1 activity in a subject
identified as in need of such treatment comprising administration
to the subject of a PDE-5 inhibitor compound.
10. The method of claim 9, wherein the modulating is
down-regulation.
11. A method of modulating nitric oxide synthase 2 (NOS2) activity
in a subject identified as in need of such treatment comprising
administration to the subject of a PDE-5 inhibitor compound.
12. The method of claim 11, wherein the modulating is
down-regulation.
13. A method of modulating interleukin-4R.alpha. (IL-4R.alpha.)
activity in a subject identified as in need of such treatment
comprising administration to the subject of a PDE-5 inhibitor
compound.
14. A method of modulating CD8+ T cells in a subject identified as
in need of such treatment comprising administration to the subject
of a PDE-5 inhibitor compound.
15. A method of improving the efficacy of immune-based treatment
protocols of malignancies in a subject comprising the step of
further administration to the subject of a PDE-5 inhibitor compound
in addition to the immune-based treatment steps.
16. The method of claim 15, wherein the immune-based treatment
protocol is administration of a vaccine.
17. A method of treating or preventing disease in a subject
comprising administration to the subject of a PDE-5 inhibitor
compound.
18. The method of claim 17, wherein the disease is a disease
mediated by myeloid suppressor cells (MSCs).
19. The method of claim 18, wherein the disease is cancer, chronic
infection, chronic inflammation, hematopoietic reconstitution
following chemotherapy.
20. The method of claim 1, further comprising the step of assessing
MSC levels in the subject.
21-32. (canceled)
Description
REFERENCE TO RELATED APPLICATIONS
[0001] This application claims benefit of U.S. Provisional
Application Ser. No. 60/642,029, filed on Jan. 7, 2005, the
contents of which are incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0002] Host immunity to cancers has been extensively documented
both in animal models and humans (1). In fact, there is strong
evidence that the immune surveillance plays a critical role in
limiting tumor outgrowth in the early stages of tumorigenesis (2,
3). However, the ability to prime tumor-specific T-cells and
sustain an immune response that imparts a measurable clinical
benefit, is limited in the setting of an established tumor burden
(4, 5). Taken together, these findings suggest numerous
requirements for effective immunotherapy. Tumor-specific T cells
must not only possess a sizeable precursor frequency and reach
sufficient numbers following activation, but they must also be able
to traffic to the tumor site and effectively kill their targets in
situ.
[0003] Growing tumors are able to modify their microenvironment and
render it more immunosuppressive. Such intratumoral changes include
altering the cytokine milieu, changing the extracellular matrix,
and recruiting immune cells with a suppressive function. In mice,
the CD11b.sup.+/Gr1.sup.+ MSCs represent one population of cells
within the tumor microenvironment responsible for the
immunosuppression accompanying tumor growth (6, 7). Their
elimination in tumor-bearing hosts restores CD8.sup.+ T cell
responsiveness (8, 9). This observation points to a reversible
process and supports the hypothesis that strategies aimed at the
pharmacologic inhibition of these pathways can be effective in
restoring immune responsiveness. L-Arginine metabolism is a key
pathway used by MSCs to blunt the anti-tumor response both in mice
and humans ((10, 11) and Serafini, Noonan unpublished data). Arg1
and NOS2, the main enzymes that catabolize L-arginine, can, in
fact, work either alone or synergistically in restrain T-cells
response (12). Through an understanding of these critical
suppressive pathways, it is possible to determine whether selective
immunopharmacologic targeting can augment anti-tumor immunity.
Nitroaspirin derivatives were recently shown to down-regulate NOS2
expression in tumor associated MSCs and to abrogate MSC-mediated
immune-suppression in vivo (13) but the mechanisms of these effects
were not defined. While the transcriptional and posttranscriptional
mechanisms regulating NOS2 expression have been extensively
studied, little is known about the pathways regulating Arginase
expression.
[0004] Agents increasing intracellular cGMP levels can induce
either positive or negative effects on NOS2 in a cell dependent
manner (14). In macrophages, for example, cGMP analogues inhibit
NOS2 expression (15). Phosphodiesterase-5 (PDE5) inhibitors such as
(sildenafil (Viagra.RTM.), vardenafil (Levitra.RTM.), tadalafil
(Cialis.RTM.)) increase intracellular concentrations of cGMP with
therapeutic implications that include the treatment of erectile
dysfunction, (16) pulmonary hypertension (17) and cardiac
hypertrophy (18). The results delineated herein relate to new
mechanisms and functions involving PDE inhibitors, thus providing
new therapeutic compositions and methods for treating or preventing
disease and disease symptoms.
SUMMARY OF THE INVENTION
[0005] The invention features methods and compositions for the
treatment and prevention of disease or disease symptoms. This
invention is based on the discovery that PDE5 plays an important
role in immune regulation.
[0006] In one aspect, the invention generally features a method of
treating or preventing disease, disease symptoms, or disease
progression in a subject (e.g., a human patient). The method
comprises administering to the subject an effective amount of a
PDE5 inhibitor.
[0007] In another aspect, the invention provides a method of
treating, preventing, reducing, or reversing cancer in a subject
(e.g., a human patient), the method comprising administering to the
subject an effective amount of a PDE5 inhibitor, where the
administration of the inhibitor treats, prevents, reduces or
reverses cancer.
[0008] In another aspect, the invention provides a method for
treating, preventing, reducing, or reversing disease in a subject
having or having a propensity to develop the disease, the method
comprising administering to the subject an effective amount of a
PDE5 inhibitor, where the inhibitor treats, prevents, reduces or
reverses the disease.
[0009] In another aspect, the invention provides a composition for
the treatment of a condition selected from the group consisting of
cancer (e.g., multiple myeloma, melanoma, breast, stomach, head and
neck, ovarian, colon, prostate, cervical cancer), chronic
infection, or hematopoietic reconstitution following chemotherapy,
the composition comprising a PDE5 inhibitor in a pharmaceutically
acceptable excipient, where administration of the composition to a
subject results in treatment of the cancer, chronic infection, or
hematopoietic reconstitution following chemotherapy.
[0010] In another aspect, the invention provides a composition for
the treatment of disease (e.g., any disease delineated herein), the
composition comprising at least 0.1-200 mg of a PDE5 inhibitor in a
pharmaceutically acceptable excipient.
[0011] In various embodiments of the above aspects, the composition
comprises at least 10, 20, 100, or 150 mg of a PDE5 inhibitor. In
yet other embodiments of the above aspects, the composition
provides for the sustained release of the PDE5 inhibitor In still
other embodiments, the composition provides for release of the PDE5
inhibitor over at least 4-8, 8-12, or 12-24 hours. In yet other
embodiments of the above aspects, the composition consists
essentially of a PDE5 inhibitor.
[0012] In another aspect, the invention provides pharmaceutical
pack comprising a composition comprising at least 5 mg of a PDE5
inhibitor in a pharmaceutically acceptable excipient, where the
pharmaceutical pack is labeled for use in the treatment or
prevention of disease (e.g., any disease delineated herein).
[0013] In a related aspect, the invention provides pharmaceutical
pack comprising a composition comprising at least 5 mg of a PDE5
inhibitor in a pharmaceutically acceptable excipient, where the
pharmaceutical pack is labeled for use in the treatment or
prevention of disease (e.g., any disease delineated herein).
[0014] In various embodiments of the previous aspects, the pack
comprises at least 10 mg, 20 mg, or 100 mg of a PDE5 inhibitor. In
other embodiments, the PDE5 inhibitor is provided in a sustained
release formulation. In other embodiments, the composition consists
essentially of a PDE5 inhibitor. In other embodiments, further
comprising written instructions for administering the composition
to a subject for the treatment or prevention of disease (e.g., any
disease delineated herein).
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] FIG. 1: In vivo PDE5 inhibition downregulates NOS2 in
tumor-associated MSCs. A cohort of mice were challenged with
0.5.times.10.sup.6 C26GM cells and treated (black bars) with
sildenafil (20 mg/kg/day) or left untreated (gray bars). After nine
days the mice were sacrificed, single cell suspensions were
obtained from the tumors through collagenase treatment, and the
tumor-associated CD11b.sup.+ cells were magnetically purified as
described in the Material and Methods. A) Intracellular
concentration of cGMP was measured on the lysate of 10.sup.6
CD11b.sup.+ cells through a competitive Enzyme ImmunoAssay (EIA)
kit. B) Western blot analysis was performed to detect NOS2, Arg-1
and .beta.-actin expression on 0.5.times.10.sup.6 magnetically
purified tumor-associated CD11b.sup.+ cells. C) NO production was
evaluated as the concentration of NO.sub.3--NO.sub.2 in the
supernatant of 10.sup.6 purified CD11b.sup.+ cells cultured for 24
h in DMEM. Arginase activity was determined on cell lysates and
normalized for the number of cells. D) Purified tumor-derived CD11b
cells were labeled with anti CD11b-APC and anti-IL4R.alpha.-PE. The
histogram is gated on CD11b.sup.+ live cells purified from either
the spleen of tumor free (no tumor) mice, untreated C26GM
tumor-bearing mice (no treatment) or sildenafil-treated
tumor-bearing mice (Sildenafil). Data are the average +/-SD of
IL4R.alpha..sup.+ cells from two independent experiments.
[0016] FIG. 2: PDE5 inhibition reverts MSC suppressive pathways.
Splenic CD11b.sup.+ cells were magnetically purified from BALB/c
mice challenged 9 days before with C26GM, and added to CFSE labeled
splenocytes containing either naive HA-specific CD8.sup.+ CL4 cells
(A) or naive HA specific CD4.sup.+ 6.5 cells (B). The cultures were
stimulated for 4 days with the relevant peptide in the presence or
in the absence of sildenafil (50 .mu.g/ml). The proliferation was
evaluated as CFSE dilution by cytoflorimetric analysis. C) Splenic
CD11b.sup.+ cells were magnetically purified from C57Bl/6
NOS.sup.+/+ or from C57Bl/6 NOS.sup.-/- challenged with the
melanoma B16GM 15 days before and added to CFSE labeled splenocytes
containing naive OVA specific CD4.sup.+ T cells. The cultures were
stimulated for 4 days with the relevant peptide in the presence or
in the absence of sildenafil (50 .mu.g/ml). The proliferation was
evaluated as CFSE dilution by flow cytometry. Data derived from one
of two independent experiments with similar results.
[0017] FIG. 3: PDE5 inhibition alone imparts a measurable
immune-system mediated antitumor effect. Balb/c (A) or BALB/c
Rag.sup.-/- mice (B) were challenged s.c. with 0.5.times.10.sup.6
cells of the indicated tumor. Sildenafil (20 mg/kg/day) was added
to the drinking water or given i.p. daily where indicated.
Tadalafil (2 mg/kg/day) was given ip. Tumor size is indicated as
the product of the two main perpendicular diameters measured with a
caliper. (C) GR-1.sup.+ cells were depleted where indicated by i.p.
injection of 200 .mu.g of anti-GR-1 depleting antibody (clone
RB6-8C5.3) on day 0, 3, 6 after tumor challenge. Best fit of the
data was obtained by four parameter sigmoid curves. Paired T test P
value (P.sup.T) or one way Anova pValue (P.sup.A) are reported.
[0018] FIG. 4: Adoptive cell transfer (ACT) efficacy is improved by
sildenafil treatment. BALB/c mice were challenged on day 0 with
0.5.times.10.sup.6 C26GM cells s.c. and were either given
sildenafil (20 mg/kg/day) in their drinking water or left
untreated. Where indicated, the mice received 20.times.10.sup.6
splenocytes from mice vaccinated 7 days before with 10.sup.6
.gamma.-irradiated C26GM. Tumor size is indicated as the product of
the two main perpendicular diameters measured with a caliper. One
way Anova P value (P.sup.A) is reported, The paired T-test (P
value=P.sup.T) was used to compare sildenafil vs sildenafil+ACT
groups.
[0019] FIG. 5: PDE5 inhibition improves the infiltration and
activation of tumor specific CD8.sup.+ T cells A) BALB/c mice were
challenged s.c. with 0.5.times.10.sup.6 CT26WT cells and given
sildenafil (20 mg/kg/day) in the drinking water or not. The mice
were sacrificed 15 days later. The tumors were surgically removed,
fixed with 10% neutral buffered formalin, and stained with
hematoxylin-eosin. B) BALB/c mice were challenged with
0.5.times.10.sup.6 C26GM cells s.c. Half of these mice were treated
with sildenafil starting on day 0. Where indicated, adoptive
transfer was performed utilizing 20.times.10.sup.6 splenocytes from
H2.sup.d pIL-2/GFP mice vaccinated s.c. one week earlier with
.gamma.-irradiated C26GM. After 9 days the tumors were surgically
removed, treated with collagenase, labeled with anti-CD4 and
anti-CD8 antibodies and analyzed by flow cytometry. The percentage
of CD8.sup.+ T cells was plotted against the tumor size at the time
of tumor harvest. Sigma plot was used to fit a 3 parameters
exponential decay curve (y=36.13+92xe.sup.-5.93x). Pearson
bivariate correlation: P=0.0000002. Data derived from 3 independent
experiments. Tumor single cells suspension were also labeled with
anti-CD25 or anti-CD69 antibodies (C). Data are expressed as
percentage of positive cells gated on the CD8.sup.+ population. D)
Since the splenocytes used for the ACT were derived from pIL-2/GFP
transgenic mice, IL-2 production in the C26GM-vaccinated T cells is
reported as percent of GFP positive, CD8.sup.+ T cells. Data are
derived from two independent experiments. Paired T test P value is
reported. E) BALB/c mice were challenged with 0.5.times.10.sup.6
C26GM cells on day 0. Where indicated the mice were treated with
either sildenafil, the anti-CD8.sup.+ depleting antibody (clone
2.43) on day 0, . . . 2, . . . 4, . . . and 6, or both treatments.
One way Anova P value is reported. Data is reported from one of two
similar experiments.
[0020] FIG. 6: The impaired lymphocyte proliferation in cancer
patients is restored by PDE5 inhibition. Ficoll-purified PBLs from
healthy donors, head and neck cancer patients or multiple myeloma
patients were stimulated with anti-CD3 and anti-CD28
antibody-coated beads at a 3:1 bead to T cell ratio in the presence
or absence of sildenafil (50 .mu.g/ml). CD4.sup.+ and CD8.sup.+ T
cell expansion was measured by flow cytometry 5 days later. Data
are reported as fold change calculated as: (number of positive
cells in the stimulated culture)/(number of positive cells in the
un-stimulated control).
[0021] FIG. 7: Sildenafil down-regulates IL4R.alpha. expression on
purified MSCs. CD11b+ cells were magnetically purified and cultured
in media with or without Sildenafil (50 .mu.g/ml). Where indicated,
INF-.gamma. (25 ng/ml) was added on day 2. The cells were harvested
at the indicated time, labeled with anti-CD11b and anti IL4R.alpha.
antibodies. The percentage of IL-4R.alpha..sup.+ cells was
determined by flow cytometry collecting 10000 CD11b.sup.+ cells.
7AAD and annexin V were used to exclude dead cells. Results are
expressed as the average of triplicate wells +/-the standard
deviation.
[0022] FIG. 8: PDE-5 inhibitors reduce tumor growth. Balb/c mice
were challenged s.c. with 0.5.times.10.sup.6 C26GM cells.
Sildenafil (20 mg/kg/day), tadalafil (2 mg/kg/day) or PBS were
given i.p daily. The tumors were surgically removed and
photographed on day 9.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
[0023] By "PDE5 inhibitor" is meant a compound that inhibits cGMP
hydrolysis by phosphodiesterase-5. PDE5 inhibitors preferably
reduce PDE5 enzymatic activity by at least 5% (e.g., 10%, 15%, 20%,
30%, 50%, 60%, 75%, 85%, 90% or 95%). Methods for assaying the
activity of a PDE5 inhibitor are known in the art and are described
herein.
[0024] By "treat" is meant decrease, suppress, attenuate, diminish,
arrest, or stabilize the development or progression of a
disease.
[0025] By "disease" is meant any condition or disorder that damages
or interferes with the normal function of a cell, tissue, or
organ.
[0026] By "modulation" or "modulating" is meant any alteration
(e.g., increase or decrease) in a biological function or
activity.
[0027] By "reduce" or "increase" is meant alter negatively or
positively, respectively, by at least 5%. An alteration may be by
5%, 10%, 25%, 30%, 50%, 75%, or even by 100%.
[0028] By "subject" is meant a mammal, such as a human patient or
an animal (e.g., a rodent, bovine, equine, porcine, ovine, canine,
feline, or other domestic mammal).
[0029] An "effective amount" is an amount sufficient to effect a
beneficial or desired clinical result.
[0030] The term "hydrate" means a compound of the present invention
or a salt thereof, which further includes a stoichiometric or
non-stoichiometric amount of water bound by non-covalent
intermolecular forces.
[0031] A "Marker" is any compound (e.g., molecule, protein, nucleic
acid) or portion thereof (e.g., atom, functional group) or physical
characteristic (e.g., radioactivity, binding, energy emission) that
is measurable and whose presence, absence, or quantification is
useful to provide an indication or correlation with an effect or
activity. The Marker can be any characteristic or identifier,
including for example, a chemical, a fluid, a protein, gene,
promoter, enzyme, protein, labeled molecule, tagged molecule,
antibody, and the like.
[0032] In this disclosure, "comprises," "comprising," "containing"
and "having" and the like can have the meaning ascribed to them in
U.S. patent law and can mean "includes," "including," and the like;
"consisting essentially of" or "consists essentially" likewise has
the meaning ascribed in U.S. patent law and the term is open-ended,
allowing for the presence of more than that which is recited so
long as basic or novel characteristics of that which is recited is
not changed by the presence of more than that which is recited, but
excludes prior art embodiments.
[0033] The compounds (e.g., PDE inhibitors, additional therapeutic
agents) described herein can also be any of salts, prodrugs and
prodrug salts of said compound, or any solvates, hydrates and
polymorphs of any of the foregoing. The compounds of this invention
may contain one or more asymmetric centers and thus occur as
racemates and racemic mixtures, single enantiomers, individual
diastereomers and diastereomeric mixtures. All such isomeric forms
of these compounds are expressly included in the present invention.
The compounds of this invention may also be represented in multiple
tautomeric forms, in such instances, the invention expressly
includes all tautomeric forms of the compounds described herein.
All crystal forms of the compounds described herein are expressly
included in the present invention.
[0034] As used herein, the term "salt" or "pharmaceutically
acceptable salt," is a salt formed from, for example, an acid and a
basic group of a compound of any one of the formulae disclosed
herein. Illustrative salts include, but are not limited, to
sulfate, citrate, acetate, chloride, bromide, iodide, nitrate,
bisulfate, phosphate, acid phosphate, lactate, salicylate, acid
citrate, tartrate, oleate, tannate, pantothenate, bitartrate,
ascorbate, succinate, maleate, besylate, fumarate, gluconate,
glucaronate, saccharate, formate, benzoate, glutamate,
methanesulfonate, ethanesulfonate, benzenesulfonate, and
p-toluenesulfonate salts. The term "pharmaceutically acceptable
salt" also refers to a salt prepared from a compound of any one of
the formulae disclosed herein having an acidic functional group,
such as a carboxylic acid functional group, and a pharmaceutically
acceptable inorganic or organic base. Suitable bases include, but
are not limited to, hydroxides of alkali metals such as sodium,
potassium, and lithium; hydroxides of alkaline earth metal such as
calcium and magnesium; hydroxides of other metals, such as aluminum
and zinc; ammonia, and organic amines, such as unsubstituted or
hydroxy-substituted mono-, di-, or trialkylamines;
dicyclohexylamine; tributyl amine; pyridine; N-methyl,
N-ethylamine; diethylamine; triethylamine; mono-, bis-, or
tris-(2-hydroxy-lower alkyl amines), such as mono-, bis-, or
tris-(2-hydroxyethyl)amine, 2-hydroxy-tert-butylamine, or
tris-(hydroxymethyl)methylamine, N,N,-di-lower alkyl-N-(hydroxy
lower alkyl)-amines, such as N,N-dimethyl-N-(2-hydroxyethyl)amine,
or tri-(2-hydroxyethyl)amine; N-methyl-D-glucamine; and amino acids
such as arginine, lysine, and the like. The term "pharmaceutically
acceptable salt" also refers to a salt prepared from a compound of
any one of the formulae disclosed herein having a basic functional
group, such as an amino functional group, and a pharmaceutically
acceptable inorganic or organic acid. Suitable acids include
hydrogen sulfate, citric acid, acetic acid, hydrochloric acid
(HCl), hydrogen bromide (HBr), hydrogen iodide (HI), nitric acid,
phosphoric acid, lactic acid, salicylic acid, tartaric acid,
ascorbic acid, succinic acid, maleic acid, besylic acid, fumaric
acid, gluconic acid, glucaronic acid, formic acid, benzoic acid,
glutamic acid, methanesulfonic acid, ethanesulfonic acid,
benzenesulfonic acid, and p-toluenesulfonic acid.
Methods of the Invention
[0035] The invention generally provides compositions comprising
PDE5 inhibitors that are useful for the prevention or treatment of
disease or disease symptoms (e.g., any disease delineated herein).
Compositions and methods of the invention are particularly useful
for the treatment or prevention of proliferative diseases, cancer,
or tumors. This invention is based, in part, on the discoveries
that PDE5 is useful for treating conditions delineated herein.
[0036] The methods herein include administering to the subject
(including a subject identified as in need of such treatment) an
effective amount of a compound described herein, or a composition
described herein to produce a beneficial effect to the subject.
Identifying a subject in need of such treatment can be in the
judgment of a subject or a health care professional and can be
subjective (e.g. opinion) or objective (e.g. measurable by a test
or diagnostic method).
[0037] As used herein, the terms "treat," treating," "treatment,"
and the like refer to reducing or ameliorating a disorder and/or
symptoms associated therewith. It will be appreciated that,
although not precluded, treating a disorder or condition does not
require that the disorder, condition or symptoms associated
therewith be completely eliminated.
[0038] As used herein, the terms "prevent," "preventing,"
"prevention," "prophylactic treatment" and the like refer to
reducing the probability of developing a disorder or condition in a
subject, who does not have, but is at risk of or susceptible to
developing a disorder or condition.
[0039] The therapeutic methods of the invention (which include
prophylactic treatment) in general comprise administration of a
therapeutically effective amount of a compound described herein,
such as a PDE5 inhibitor (e.g., vardenafil, tadalafil, or
sildenafil) to a subject (e.g., animal, human) in need thereof,
including a mammal, particularly a human. Such treatment will be
suitably administered to subjects, particularly humans, suffering
from, having, susceptible to, or at risk for a disease, disorder,
or symptom thereof. Determination of those subjects "at risk" can
be made by any objective or subjective determination by a
diagnostic test or opinion of a subject or health care provider
(e.g., genetic test, enzyme or protein marker, Marker (as defined
herein), family history, and the like). The compounds herein may be
also used in the treatment of any other disorders in which are
mediated by an immune response (e.g., anti-tumor immune
response).
[0040] In aspects of the methods herein, assays are used to monitor
the condition of a subject prior to, during, or following treatment
with a PDE5A inhibitor. Treatments can be used in conjunction with
one or more relevant diagnostic test(s) for determining the
efficacy, the progression, or the appropriate dosage in the methods
of the invention.
[0041] Any number of standard methods are available for assaying
certain markers. Methods for assaying include any one or more of
the following: tumor size, measurement, x-ray, CAT scan, magnetic
resonance imaging, protein expression, nucleic acid expression,
isotopologue, radiolabel, fluorescent probe, and the like.
Prophylactic and Therapeutic Applications
[0042] One aspect is a method of modulating myeloid suppressor
cells (MSCs) immune suppression in a subject comprising
administration to the subject of a PDE-5 inhibitor compound.
Another aspect is a method of modulating arginase-1 (Arg-1)
activity in a subject identified as in need of such treatment
comprising administration to the subject of a PDE-5 inhibitor
compound. Another aspect is a method of modulating nitric oxide
synthase 2 (NOS2) activity in a subject identified as in need of
such treatment comprising administration to the subject of a PDE-5
inhibitor compound. Another aspect is a method of modulating (e.g.,
down-regulating) interleukin-4R.alpha. (IL-4R.alpha.) activity in a
subject identified as in need of such treatment comprising
administration to the subject of a PDE-5 inhibitor compound. The
methods can be wherein the modulating is down-regulation.
[0043] Another aspect is a method of modulating (e.g., activating)
CD8+ T cells in a subject identified as in need of such treatment
comprising administration to the subject of a PDE-5 inhibitor
compound. Another aspect is a method of improving the efficacy of
immune-based treatment protocols of malignancies in a subject
comprising the step of further administration to the subject of a
PDE-5 inhibitor compound in addition to the immune-based treatment
steps.
[0044] Another aspect is a method of reducing tumor size in a
subject comprising administration to the subject an effective
amount of a PDE-5 inhibitor, wherein the subject is identified as
in need of anti-tumor treatment with a PDE-5 inhibitor
compound.
[0045] Another aspect is a method of modulating the suppressive
function of Arg-1 or NOS2 in a subject comprising administration to
the subject an effective amount of a PDE-5 inhibitor, wherein the
subject is identified as in need of such treatment with a PDE-5
inhibitor compound.
[0046] Another aspect is a method of reducing NO production in a
subject comprising administration to the subject an effective
amount of a PDE-5 inhibitor, wherein the subject is identified as
in need of such treatment with a PDE-5 inhibitor compound.
[0047] Another aspect is a method of modulating T-cell
proliferation in a subject comprising administration to the subject
an effective amount of a PDE-5 inhibitor, wherein the subject is
identified as in need of such treatment with a PDE-5 inhibitor
compound.
[0048] Another aspect is a method of modulating the efficacy of
adoptive T-cell immunotherapy in a subject comprising
administration to the T-cells an effective amount of a PDE-5
inhibitor, wherein the administration results in expansion of T
cells in vitro in the presence of PDE-5 inhibitors. The PDE-5
inhibitor is thus useful as an adjuvant in adoptive cell transfer
(ACT) protocols. Such pre-treatment of T-cells with a PDE-5
inhibitor can enhance the effectiveness of a therapeutic (e.g.,
vaccine) by modulating immunesuppression when administered to a
subject, thus allowing for greater efficacy of the vaccine
itself.
[0049] Another aspect is a method of modulating peripheral blood
lymphocyte (PBL) proliferation in a subject comprising
administration to the subject an effective amount of a PDE-5
inhibitor, wherein the subject is identified as in need of such
treatment with a PDE-5 inhibitor compound.
[0050] Another aspect is a method of modulating CD4.sup.+ or
CD8.sup.+ proliferation in a subject comprising administration to
the subject an effective amount of a PDE-5 inhibitor, wherein the
subject is identified as in need of such treatment with a PDE-5
inhibitor compound.
[0051] In other aspects, the compositions and methods for treating,
preventing or modulating disease herein are those wherein the
disease is a disease mediated by myeloid suppressor cells (MSCs).
The compositions and methods for treating, preventing or modulating
disease include diseases such as cancer (e.g., multiple myeloma,
lymphomas, melanoma, breast, stomach, head and neck, ovarian,
colon, prostate, lung, high grade gliomas, or cervical cancer),
chronic infection, chronic inflammation, and hematopoietic
reconstitution following chemotherapy. The methods can further
comprise the step of assessing MSC levels in the subject; or can
further comprise the steps of assessing MSC levels in the subject
before and after administration. The assessment can be made by
surface marker expression, by MSC number, or by measure of
immunosuppression function.
[0052] In each of the embodiments herein, an additional therapeutic
agent may be administered together with a PDE inhibitor compound of
this invention as part of a single dosage form or as separate
dosage forms. Alternatively, the additional agent may be
administered prior to, consecutively with, or following the
administration of a compound of this invention. In such combination
therapy treatment, both the compounds of this invention and the
second therapeutic agent(s) are administered by conventional
methods. The administering of the second therapeutic agent may
occur before, concurrently with, and/or after the administering of
the compound of this invention. When administration of the second
therapeutic agent occurs concurrently with a compound of this
invention, the two (or more) agents may be administered in a single
dosage form (such as a composition of this invention comprising a
compound of the invention and an second therapeutic agent as
described above), or in separate dosage forms. The administration
of a composition of this invention comprising both a compound of
the invention and a second therapeutic agent to a subject does not
preclude the separate administration of said second therapeutic
agent, any other therapeutic agent or any compound of this
invention to said subject at another time during a course of
treatment.
[0053] Examples of additional anticancer therapeutic agents
include, for example, an antiangiogenesis agent, selective
estrogen-receptor modulator (SERM), breast cancer therapeutic
agent, aromatase inhibitor, biologic response modifiers, hormonal
therapies agent, anthracycline, taxane, alkylating agent, taxol,
cis-platin, arabinofuranosyl cytosine (ara-C), 5-fluorouracil
(5-FU), altretamine, busulfan, chlorambucil, cyclophosphamide,
ifosfamide, mechlorethamine, melphalan, thiotepa, cladribine,
fluorouracil, floxuridine, gemcitabine, thioguanine, pentostatin,
methotrexate, 6-mercaptopurine, cytarabine, carmustine, lomustine,
streptozotocin, carboplatin, oxaliplatin, iproplatin, tetraplatin,
lobaplatin, JM216, JM335, fludarabine, aminoglutethimide,
flutamide, goserelin, leuprolide, megestrol acetate, cyproterone
acetate, tamoxifen, anastrozole, bicalutamide, dexamethasone,
diethylstilbestrol, prednisone, bleomycin, dactinomycin,
daunorubicin, doxirubicin, idarubicin, mitoxantrone, losoxantrone,
mitomycin-c, plicamycin, paclitaxel, docetaxel, CPI-11,
epothilones, topotecan, irinotecan, 9-amino camptothecan, 9-nitro
camptothecan, GS-211, etoposide, teniposide, vinblastine,
vincristine, vinorelbine, procarbazine, asparaginase, pegaspargase,
methoxtrexate, octreotide, estramustine, hydroxyurea, tamoxifen,
raloxifene, toremifene, exemestane, letrozole, anastrozole,
megestrol, trastuzumab, goserelin acetate, fulvestrant,
doxorubicin, epirubicin, or cyclophosphonamide and the like.
Immunotherapeutic agents are also useful in the embodiments
delineated herein. Examples of immune-based strategies include
cancer-specific vaccines such as DNA-based, protein-based, whole
cell tumor vaccines, dendritic cell based vaccines; adoptive T cell
therapy; strategies aimed at augmenting T cell function through
blockade or elimination of inhibitory or suppressor mechanisms such
as CTLA-4 blockade, elimination of regulatory T cells (Tregs) or
abrogation of myeloid suppressor mechanisms such as in this patent
or through activation of T cells such as anti-CD3/CD28 stimulation,
growth in IL-2.
PDE5 Inhibitors
[0054] PDE5 inhibitors are known in the art, and include, but are
not limited to, sildenafil (Compound 1), vardenafil (Compound 2),
tadalafil (Compound 3), EMD 360527, DA 8159, or analogs thereof, or
any other compound that inhibits cGMP hydrolysis by
phosphodiesterase-5 (PDE5).
##STR00001##
[0055] Certain compounds useful in the present invention can be
represented by the structure (Formula I):
##STR00002##
[0056] in which R.sup.1 is H, C.sub.1-C.sub.3 alkyl,
C.sub.3-C.sub.5 cycloalkyl or C.sub.1-C.sub.3 perfluoroalkyl;
R.sup.2 is H, C.sub.1-C.sub.6 alkyl optionally substituted by OH,
C.sub.1-C.sub.3 alkoxy or C.sub.3-C.sub.6 cycloalkyl, or
C.sub.1-C.sub.3 perfluoroalkyl; R.sup.3 is C.sub.1-C.sub.6 alkyl,
C.sub.3-C.sub.6 alkenyl, C.sub.3-C.sub.6 alkynyl, C.sub.3-C.sub.67
cycloalkyl, C.sub.1-C.sub.6 perfluoroalkyl or (C.sub.3-C.sub.6
cycloalkyl) C.sub.1-C.sub.6 alkyl; R.sup.4 taken together with the
nitrogen atom to which it is attached completes a
4-N--(R.sup.6)-piperazinyl group; R.sup.5 is H, C.sub.1-C.sub.4
alkyl, C.sub.1-C.sub.3 alkoxy, NR.sup.7R.sup.8, or
CONR.sup.7R.sup.8; R.sup.6 is H, C.sub.1-C.sub.6 alkyl,
(C.sub.1-C.sub.3 alkoxy) C.sub.2-C.sub.6 alkyl hydroxy
C.sub.2-C.sub.6 alkyl, (R.sup.7R.sup.8N)C.sub.2-C.sub.6 alkyl,
(R.sup.7R.sup.8NCO)C.sub.1-C.sub.6 alkyl, CONR.sup.7R.sup.8,
CSNR.sup.7R.sup.8 or C(NH)N R.sup.7R.sup.8; R.sup.7 and R.sup.8 are
each independently H, C.sub.1-C.sub.4 alkyl, (C.sub.1-C.sub.3
alkoxy) C.sub.2-C.sub.4 alkyl or hydroxy C.sub.2-C.sub.4 alkyl; and
pharmaceutically acceptable salts thereof.
[0057] Other preferred compounds for use in the present invention
are disclosed in U.S. Pat. No. 6,362,178 and can be represented by
the structure (Formula II):
##STR00003##
[0058] in which
[0059] R.sup.1 represents hydrogen or straight-chain or branched
alkyl having up to 4 carbon atoms,
[0060] R.sup.2 represents straight-chain alkyl having up to 4
carbon atoms,
[0061] R.sup.3 and R.sup.4 are identical or different and each
represents hydrogen or represents straight-chain or branched
alkenyl or alkoxy having in each case up to 8 carbon atoms, or
represents a straight-chain or branched alkyl chain having up to 10
carbon atoms which is optionally interrupted by an oxygen atom and
which is optionally mono- or polysubstituted by identical or
different substituents selected from the group consisting of
trifluoromethyl, trifluoromethoxy, hydroxyl, halogen, carboxyl,
benzyloxycarbonyl, straight-chain or branched alkoxycarbonyl having
up to 6 carbon atoms and/or by radicals of the formulae
--SO.sub.3H, -(A).sub.a-NR.sup.7R.sup.8,
--O--CO--NR.sup.7'R.sup.8', --S(O).sub.b--R.sup.9,
--P(O)(OR.sup.10)(OR.sup.11),
##STR00004##
in which
[0062] a and b are identical or different and each represents a
number 0 or 1,
[0063] A represents a radical CO or SO.sub.2,
[0064] R.sup.7, R.sup.7', R.sup.8 and R.sup.8' are identical or
different and each represents hydrogen, or represents cycloalkyl
having 3 to 8 carbon atoms, aryl having 6 to 10 carbon atoms, a 5-
to 6-membered unsaturated, partially unsaturated or saturated,
optionally benzo-fused heterocycle having up to 3 heteroatoms from
the group consisting of S, N and O, where the abovementioned ring
systems are optionally mono- or polysubstituted by identical or
different substituents selected from the group consisting of
hydroxyl, nitro, trifluoromethyl, trifluoromethoxy, carboxyl,
halogen, straight-chain or branched alkoxy or alkoxycarbonyl having
in each case up to 6 carbon atoms or by a group of the formula
--(SO.sub.2).sub.c--NR.sup.12R.sup.13, in which
[0065] c represents a number 0 or 1,
[0066] R.sup.12 and R.sup.13 are identical or different and each
represents hydrogen or straight-chain or branched alkyl having up
to 5 carbon atoms, or
[0067] R.sup.7, R.sup.7', R.sup.8 and R.sup.8' each represent
straight-chain or branched alkoxy having up to 6 carbon atoms, or
represents straight-chain or branched alkyl having up to 8 carbon
atoms which is optionally mono- or polysubstituted by identical or
different substituents selected from the group consisting of
hydroxyl, halogen, aryl having 6 to 10 carbon atoms, straight-chain
or branched alkoxy or alkoxycarbonyl having in each case up to 6
carbon atoms or by a group of the formula
--(CO).sub.d--NR.sup.14R.sup.15, in which
[0068] R.sup.14 and R.sup.15 are identical or different and each
represents hydrogen or straight-chain or branched alkyl having up
to 4 carbon atoms, and
[0069] d represents a number 0 or 1, or
[0070] R.sup.7 and R.sup.8 and/or R.sup.7' and R.sup.8' together
with the nitrogen atom form a 5- to 7-membered saturated
heterocycle which may optionally contain a further heteroatom from
the group consisting of S and O or a radical of the formula
--NR.sup.16, in which
[0071] R.sup.16 represents hydrogen, aryl having 6 to 10 carbon
atoms, benzyl, a 5- to 7-membered aromatic or saturated heterocycle
having up to 3 heteroatoms from the group consisting of S, N and O
which is optionally substituted by methyl, or represents
straight-chain or branched alkyl having up to 6 carbon atoms which
is optionally substituted by hydroxyl,
[0072] R.sup.9 represents aryl having 6 to 10 carbon atoms, or
represents straight-chain or branched alkyl having up to 4 carbon
atoms,
[0073] R.sup.10 and R.sup.11 are identical or different and each
represents hydrogen or straight-chain or branched alkyl having up
to 4 carbon atoms, and/or the alkyl chain listed above under
R.sup.3/R.sup.4 is optionally substituted by cycloalkyl having 3 to
8 carbon atoms, aryl having 6 to 10 carbon atoms or by a 5- to
7-membered partially unsaturated, saturated or unsaturated,
optionally benzo-fused heterocycle which may contain up to 4
heteroatoms from the group consisting of S, N and O or a radical of
the formula --NR.sup.17, in which
[0074] R.sup.17 represents hydrogen, hydroxyl, formyl,
trifluoromethyl, straight-chain or branched acyl or alkoxy having
in each case up to 4 carbon atoms, or represents straight-chain or
branched alkyl having up to 6 carbon atoms which is optionally
mono- or polysubstituted by identical or different substituents
selected from the group consisting of hydroxyl and straight-chain
or branched alkoxy having up to 6 carbon atoms, and where aryl and
the heterocycle are optionally mono- or polysubstituted by
identical or different substituents selected from the group
consisting of nitro, halogen, --SO.sub.3H, straight-chain or
branched alkyl or alkoxy having in each case up to 6 carbon atoms,
hydroxyl, trifluoromethyl, trifluoromethoxy and/or by a radical of
the formula --SO.sub.2--NR.sup.18R.sup.19, in which
[0075] R.sup.18 and R.sup.19 are identical or different and each
represents hydrogen or straight-chain or branched alkyl having up
to 6 carbon atoms, and/or
[0076] R.sup.3 or R.sup.4 represents a group of the formula
--NR.sup.20R.sup.21, in which R.sup.20 and R.sup.21 have the
meanings of R.sup.18 and R.sup.19 given above and are identical to
or different from them, and/or
[0077] R.sup.3 or R.sup.4 represents adamantyl, or represents
radicals of the formulae
##STR00005##
or represents cycloalkyl having 3 to 8 carbon atoms, aryl having 6
to 10 carbon atoms or represents a 5- to 7-membered partially
unsaturated, saturated or unsaturated, optionally benzo-fused
heterocycle which may contain up to 4 heteroatoms from the group
consisting of S, N and O, or a radical of the formula --NR.sup.22,
in which
[0078] R.sup.22 has the meaning of R.sup.16 given above and is
identical to or different from it, or represents carboxyl, formyl
or straight-chain or branched acyl having up to 5 carbon atoms, and
where cycloalkyl, aryl and/or the heterocycle are optionally mono-
or polysubstituted by identical or different substituents selected
from the group consisting of halogen, triazolyl, trifluoromethyl,
trifluoromethoxy, carboxyl, straight-chain or branched acyl or
alkoxycarbonyl having in each case up to 6 carbon atoms, nitro
and/or by groups of the formulae --SO.sub.3, --OR.sup.23,
(SO.sub.2).sub.eNR.sup.24R.sup.25, --P(O)(OR.sup.26)(OR.sup.27), in
which
[0079] e represents a number 0 or 1,
[0080] R.sup.23 represents a radical of the formula
##STR00006##
represents cycloalkyl having 3 to 7 carbon atoms, or represents
hydrogen or straight-chain or branched alkyl having up to 4 carbon
atoms which is optionally substituted by cycloalkyl having 3 to 7
carbon atoms, benzyloxy, tetrahydropyranyl, tetrahydrofuranyl,
straight-chain or branched alkoxy or alkoxycarbonyl having in each
case up to 6 carbon atoms, carboxyl, benzyloxycarbonyl or phenyl
which for its part may be mono- or polysubstituted by identical or
different substituents selected from the group consisting of
straight-chain or branched alkoxy having up to 4 carbon atoms,
hydroxyl and halogen, and/or alkyl which is optionally substituted
by radicals of the formulae --CO--NR.sup.28R.sup.29 or
--CO--R.sup.30, in which
[0081] R.sup.28 and R.sup.29 are identical or different and each
represents hydrogen or straight-chain or branched alkyl having up
to 8 carbon atoms, or
[0082] R.sup.28 and R.sup.29 together with the nitrogen atom form a
5- to 7-membered saturated heterocycle which may optionally contain
a further heteroatom from the group consisting of S and O, and
[0083] R.sup.30 represents phenyl or adamantyl, [0084] R.sup.24 and
R.sup.25 have the meanings of R.sup.18 and R.sup.19 given above and
are identical to or different from them,
[0085] R.sup.26 and R.sup.27 have the meanings of R.sup.10 and
R.sup.11 given above and are identical to or different from them
and/or cycloalkyl, aryl and/or the heterocycle are optionally
substituted by straight-chain or branched alkyl having up to 6
carbon atoms which is optionally substituted by hydroxyl, carboxyl,
by a 5- to 7-membered heterocycle having up to 3 heteroatoms from
the group consisting of S, N and O, or by groups of the formula
--SO.sub.2--R.sup.31, P(O)(OR.sup.32)(OR.sup.33) or
--NR.sup.34R.sup.35, in which
[0086] R.sup.31 represents hydrogen or has the meaning of R.sup.9
given above and is identical to or different from it,
[0087] R.sup.32 and R.sup.33 have the meanings of R.sup.10 and
R.sup.11 given above and are identical to or different from
them,
[0088] R.sup.34 and R.sup.35 are identical or different and each
represents hydrogen or straight-chain or branched alkyl having up
to 6 carbon atoms which is optionally substituted by hydroxyl or by
straight-chain or branched alkoxy having up to 4 carbon atoms,
or
[0089] R.sup.34 and R.sup.35 together with the nitrogen atom form a
5- to 6-membered saturated heterocycle which may contain a further
heteroatom from the group consisting of S and O, or a radical of
the formula --NR.sup.36, in which
[0090] R.sup.36 represents hydrogen, hydroxyl, straight-chain or
branched alkoxycarbonyl having up to 7 carbon atoms or
straight-chain or branched alkyl having up to 5 carbon atoms which
is optionally substituted by hydroxyl, or
[0091] R.sup.3 and R.sup.4 together with the nitrogen atom form a
5- to 7-membered unsaturated or saturated or partially unsaturated,
optionally benzo-fused heterocycle which may optionally contain up
to 3 heteroatoms from the group consisting of S, N and O, or a
radical of the formula --NR.sup.37, in which
[0092] R.sup.37 represents hydrogen, hydroxyl, formyl,
trifluoromethyl, straight-chain or branched acyl, alkoxy or
alkoxycarbonyl having in each case up to 4 carbon atoms, or
represents straight-chain or branched alkyl having up to 6 carbon
atoms which is optionally mono- or polysubstituted by identical or
different substituents selected from the group consisting of
hydroxyl, trifluoromethyl, carboxyl, straight-chain or branched
alkoxy or alkoxycarbonyl having in each case up to 6 carbon atoms,
or by groups of the formula -(D).sub.f-NR.sup.38R.sup.39,
--CO--(CH.sub.2).sub.g--O--CO--R.sup.40,
--CO--(CH.sub.2).sub.h--OR.sup.41 or --P(O)(OR.sup.42)(OR.sup.43),
in which
[0093] g and h are identical or different and each represents a
number 1, 2, 3 or 4, and
[0094] f represents a number 0 or 1,
[0095] D represents a group of the formula --CO or --SO.sub.2,
[0096] R.sup.38 and R.sup.39 are identical or different and each
has the meaning of R.sup.7 and R.sup.8 given above,
[0097] R.sup.40 represents straight-chain or branched alkyl having
up to 6 carbon atoms,
[0098] R.sup.41 represents straight-chain or branched alkyl having
up to 6 carbon atoms,
[0099] R.sup.42 and R.sup.43 are identical or different and each
represents hydrogen or straight-chain or branched alkyl having up
to 4 carbon atoms, or
[0100] R.sup.37 represents a radical of the formula --(CO).sub.i-E,
in which i represents a number 0 or 1,
[0101] E represents cycloalkyl having 3 to 7 carbon atoms or
benzyl, represents aryl having 6 to 10 carbon atoms or a 5- to
6-membered aromatic heterocycle having up to 4 heteroatoms from the
group consisting of S, N and O, where the abovementioned ring
systems are optionally mono- or polysubstituted by identical or
different constituents selected from the group consisting of nitro,
halogen, --SO.sub.3H, straight-chain or branched alkoxy having up
to 6 carbon atoms, hydroxyl, trifluoromethyl, trifluoromethoxy, or
by a radical of the formula --SO.sub.2--NR.sup.44R.sup.45, in
which
[0102] R.sup.44 and R.sup.45 have the meaning of R.sup.18 and
R.sup.19 given above and are identical to or different from them,
or
[0103] E represents radicals of the formulae
##STR00007##
and the heterocycle listed under R.sup.3 and R.sup.4, which is
formed together with the nitrogen atom, is optionally mono- or
polysubstituted, if appropriate also geminally, by identical or
different substituents selected from the group consisting of
hydroxyl, formyl, carboxyl, straight-chain or branched acyl or
alkoxycarbonyl having in each case up to 6 carbon atoms, nitro and
groups of the formulae --P(O)(OR.sup.46)(OR.sup.47),
##STR00008##
.dbd.NR.sup.48, or --C(O).sub.jNR.sup.49R.sup.50,
[0104] in which
[0105] R.sup.46 and R.sup.47 have the meanings of R.sup.10 and
R.sup.11 given above and are identical to or different from
them,
[0106] R.sup.48 represents hydroxyl or straight-chain or branched
alkoxy having up to 4 carbon atoms, j represents a number 0 or 1,
and
[0107] R.sup.49 and R.sup.50 are identical or different and have
the meanings of R.sup.14 and R.sup.15 given above, and/or the
heterocycle listed under R.sup.3 and R.sup.4, which is formed
together with the nitrogen atom, is optionally substituted by
straight-chain or branched alkyl having up to 6 carbon atoms which
is optionally mono- or polysubstituted by identical or different
substituents selected from the group consisting of hydroxyl,
halogen, carboxyl, cycloalkyl or cycloalkyloxy having in each case
3 to 8 carbon atoms, straight-chain or branched alkoxy or
alkoxycarbonyl having in each case up to 6 carbon atoms, or by a
radical of the formula --SO.sub.3H, --NR.sup.51R.sup.52 or
P(O)OR.sup.53OR.sup.54, in which
[0108] R.sup.51 and R.sup.52 are identical or different and each
represents hydrogen, phenyl, carboxyl, benzyl or straight-chain or
branched alkyl or alkoxy having in each case up to 6 carbon
atoms,
[0109] R.sup.53 and R.sup.54 are identical or different and have
the meanings of R.sup.10 and R.sup.11 given above, and/or the alkyl
is optionally substituted by aryl having 6 to 10 carbon atoms which
for its part may be mono- or polysubstituted by identical or
different substituents selected from the group consisting of
halogen, hydroxyl, straight-chain or branched alkoxy having up to 6
carbon atoms, or by a group of the formula --NR.sup.51'R.sup.52',
in which
[0110] R.sup.51' and R.sup.52' have the meanings of R.sup.51 and
R.sup.52 given above and are identical to or different from them,
and/or the heterocycle listed under R.sup.3 and R.sup.4, which is
formed together with the nitrogen atom, is optionally substituted
by aryl having 6 to 10 carbon atoms or by a 5- to 7-membered
saturated, partially unsaturated or unsaturated heterocycle having
up to 3 heteroatoms from the group consisting of S, N and O,
optionally also attached via a nitrogen function, where the ring
systems for their part may be substituted by hydroxyl or by
straight-chain or branched alkyl or alkoxy having in each case up
to 6 carbon atoms, or
[0111] R.sup.3 and R.sup.4 together with the nitrogen atom form
radicals of the formulae
##STR00009##
R.sup.5 and R.sup.6 are identical or different and each represents
hydrogen, straight-chain or branched alkyl having up to 6 carbon
atoms, hydroxyl or represents straight-chain or branched alkoxy
having up to 6 carbon atoms, and their salts, hydrates, N-oxides
and structural isomers.
[0112] Other suitable compounds include those of the following
Formula III:
##STR00010##
[0113] wherein in Formula III, R.sup.0 represents hydrogen,
halogen, or C.sub.1-6 alkyl;
[0114] R.sup.1 represents hydrogen, C.sub.1-6 alkyl, C.sub.2-6
alkenyl, C.sub.2-6 alkynyl, haloC.sub.1-6 alkyl, C.sub.3-8
cycloalkyl, C.sub.3-8 cycloalkylC.sub.1-3 alkyl, arylC.sub.1-3
alkyl, or heteroarylC.sub.1-3 alkyl;
[0115] R.sup.2 represents an optionally substituted monocyclic
aromatic ring selected from benzene, thiophene, furan, and
pyridine, or an optionally substituted bicyclic ring;
##STR00011##
attached to the rest of the molecule via one of the benzene ring
carbon atoms and wherein the fused ring A is a 5- or 6-membered
ring which may be saturated or partially or fully unsaturated and
comprises carbon atoms and optionally one or two heteroatoms
selected from oxygen, sulphur, and nitrogen; and
[0116] R.sup.3 represents hydrogen of C.sub.1-3 alkyl, or R.sup.1
and R.sup.3 together represent a 3- or 4-membered alkyl or alkenyl
chain; and pharmaceutically and salts and solvates (e.g., hydrates)
thereof.
[0117] Certain preferred compounds also include those of the
following Formula IV:
##STR00012##
[0118] wherein in Formula IV, R.sup.0 represents hydrogen, halogen,
or C.sub.1-6 alkyl;
R.sup.1 represents hydrogen, C.sub.1-6 alkyl, haloC.sub.1-6 alkyl,
C.sub.3-8 cycloalkylC.sub.1-3 alkyl, arylC.sub.1-3 alkyl, or
heteroarylC.sub.1-3 alkyl; and R.sup.2 represents an optionally
substituted monocyclic aromatic ring selected from benzene,
thiophene, furan, and pyridine, or an optionally substituted
bicyclic ring
##STR00013##
attached to the rest of the molecule via one of the benzene ring
carbon atoms, and wherein the fused ring A is a 5- or 6-membered
ring which can be saturated or partially or fully unsaturated and
comprises carbon atoms and optionally one or two heteroatoms
selected from oxygen, sulphur, and nitrogen; and pharmaceutically
acceptable salts and solvates (e.g., hydrates) thereof.
[0119] A further group of compounds preferred for use in the
invention are compounds of the following Formula V:
##STR00014##
[0120] wherein in Formula V:
R.sup.0 represents hydrogen, halogen, or C.sub.1-6 alkyl; R.sup.1
represents hydrogen or C.sub.1-6 alkyl; R.sup.2 represents the
bicyclic ring
##STR00015##
which can be optionally substituted by one or more groups selected
from halogen and C.sub.1-3 alkyl; and
[0121] R.sup.3 represents hydrogen or C.sub.1-3 alkyl; and
pharmaceutically acceptable salts and solvates (e.g., hydrates)
thereof.
[0122] In Formula IV above, with respect to R.sup.1, the term
"aryl" as part of an arylC.sub.1-3 alkyl group means phenyl or
phenyl substituted by one or more (e.g., 1, 2, or 3) substituents
selected from halogen, C.sub.1-6 alkyl, C.sub.1-6 alkoxy, and
methylenedioxy. The term "heteroaryl" as part of a
heteroarylC.sub.1-3 alkyl group means thienyl, furyl, or pyridyl,
each optionally substituted by one or more (e.g., 1, 2, or 3)
substituents selected from halogen, C.sub.1-6 alkyl, and C.sub.1-6
alkoxy. The term "C.sub.3-8 cycloalkyl" as a group or part of a
C.sub.3-8 cycloalkylC.sub.1-3 alkyl group means a monocyclic ring
comprising three to eight carbon atoms. Examples of suitable
cycloalkyl rings include the C.sub.3-6 cycloalkyl rings
cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.
[0123] In formula IV above, with respect to R.sup.2, optional
benzene ring substituents are selected from one or more (e.g., 1,
2, or 3) atoms or groups comprising halogen, hydroxy, C.sub.1-6
alkyl, C.sub.1-6 alkoxy, CO.sub.2 R.sup.b, haloC.sub.1-6 alkyl,
haloC.sub.1-6 alkoxy, cyano, nitro, and NR.sup.aR.sup.b, where
R.sup.a and R.sup.b are each hydrogen or C.sub.1-6 alkyl, or
R.sup.a also can represent C.sub.2-7 alkanoyl or C.sub.1-6
alkylsulphonyl. Optional substituents for the remaining ring
systems are selected from one or more (e.g., 1, 2, or 3 atoms or
groups comprising halogen, C.sub.1-6 alkyl, C.sub.1-6 alkoxy, and
arylC.sub.1-3 alkyl as defined above. The bicyclic ring
##STR00016##
can, for example, represent naphthalene, a heterocycle such as
benzoxazole, benzothiazole, benzisoxazole, benzimidazole,
quinoline, indole, benzothiophene, benzofuran, or
##STR00017##
wherein n is an integer 1 or 2 and X and Y each can represent
CH.sub.2, O, S, or NH.
[0124] See also U.S. Pat. Nos. 6,916,927, 6,911,542, 6,903,099,
6,878,711, 6,872,721, 6,858,620, 6,825,197, 6,774,128, 6,723,719,
6,699,870, 6,670,366, 5,859,006 and 5,250,534. Other PDE5
inhibitors useful in the methods of the invention are described in
WO 03/063875; WO 03/1012761 WO 2004/037183, and WO 98/38168. All of
these patents and patent applications are incorporated herein by
reference in their entirety.
[0125] Sildenafil is commercially available in three dosages of 25,
50, or 100 mg and has an IC.sub.50 of approximately 10 nM.
Effective plasma concentrations are between 1 nM and 250 nM, where
the bottom of the range is any integer between 1 and 249; and the
top of the range is any integer between 2 nM and 250 nM.
Preferably, an effective plasma concentration is between 5 nM and
100 nM, more preferably it is between 10 nM and 50 nM (e.g., 15 nM,
20 nM, 25 nM, 30 nM, 40 nM, or 45 nM).
[0126] Tadalafl is commercially available in three dosages of 5,
10, or 20 mg and has an IC.sub.50 of approximately 1 nM. Following
oral administration of a 20 mg dose of tadalafil to healthy
subjects, tadalafil is rapidly absorbed with the peak plasma
concentration of 378 ng/ml occurring two hours post-dose.
Preferably an effective plasma concentration is between 5 nM and
100 nM, more preferably it is between 10 nM and 50 nM (e.g., 15 nM,
20 nM, 25 nM, 30 nM, 40 nM, or 45 nM). Tadalafil has a relative
large apparent volume of distribution (Vd/F) of 62.6 L, and a low
apparent oral clearance (CL/F) of 2.48 L/h. As a result, the mean
elimination half-life of tadalafil is about 17.5 h, which is
substantially longer than that of sildenafil or vardenafil.
[0127] Vardenafil is commercially available in three dosages of 5
mg, 10 mg, and 20 mg and has an IC.sub.50 of 0.7 mM. Effective
plasma concentrations of vardenafil are between 0.1 and 5.0 nM.
[0128] The skilled artisan appreciates that any compound that
reduces the activity of PDE5 is useful in the methods of the
invention. Other exemplary compounds useful in the methods of the
invention include UK-343,664 (Walker et al., Xenobiotica, 31:
651-664), UK-427,387, UK-357903
[1-ethyl-4-{3-[3-ethyl-6,7-dihydro-7-oxo-2-(2-pyridylmethyl)-2H-
-pyrazolo[4,3-d]pyrimidin-5-yl]-2-(2-methoxyethoxy)-5-pyridylsulphonyl}pip-
erazine] (Gardiner et al. J Pharmacol Exp Ther. 2005; 312:
265-271), UK-371800 (Pfizer), UK-313794 (Pfizer) and UK-343664
(Abel et al., Xenobiotica. 2001 31:665-76); TA-1790 from Tanabe
Seiyaku; CP-248, CP-461 and exisulind (Deguchi et al., Molecular
Cancer Therapeutics 803-809, 2002), which are available from Osi
Pharmaceuticals; pyrazolinone; EMD82639
(4-(4-[2-ethyl-phenylamino)-methylene]-3-methyl-5-oxo-4,5-di-hyd-
ro-pyrazol-1-yl)-benzoic acid (Senzaki et al., FASEB Journal. 2001;
15:1718-1726);
[7-(3-Chloro-4-methoxy-benzylamino)-1-methyl-3-propyl-1H-pyrazolo[4,3-d]p-
yrimidin-5-ylmethoxy]-acetic acid (EMD360527),
4-[4-(3-Chloro-4-methoxy-benzylamino)-benzo[4,5]thieno[2,3-d]-pyrimidin-2-
-yl}-cyclohexanecarboxylic acid, ethanolamin salt (EMD221829) and
5-[4-(3-Chloro-4-methoxy-benzylamino)-5,6,7,8-tetrahydro-benzo[4,5]thieno-
[2,3-d]pyrimidin-2-yl]-pentanoic acid (EMD171827), which are
commercially available from Merck KgaA (Darmstadt, Del.) and are
described, for example, in Scutt et al. (BMC Pharmacol. 2004; 4:
10);
3-(1-Methyl-7-oxo-3-propyl-6,7-dihydro-1H-pyrazolo-[4,3-d]pyrimidin-5-yl)-
-N-[2-(1-methylpyrrolidin-2-yl)ethyl]-4-propoxybenzenesulfonamide
(DA-8259); E-4021 (Dukarm et al., Am. J. Respir. Crit. Care Med.,
1999, 160:858-865); pentoxifylline and FR22934 (Fujisawa).
[0129] The synthesized compounds can be separated from a reaction
mixture and further purified by a method such as column
chromatography, high pressure liquid chromatography, or
recrystallization. As can be appreciated by the skilled artisan,
methods of synthesizing the compounds of the formulae herein will
be evident to those of ordinary skill in the art. Additionally, the
various synthetic steps may be performed in an alternate sequence
or order to give the desired compounds. Synthetic chemistry
transformations and protecting group methodologies (protection and
deprotection) useful in synthesizing the compounds described herein
are known in the art and include, for example, those such as
described in R. Larock, Comprehensive Organic Transformations, 2nd.
Ed., Wiley-VCH Publishers (1999); T. W. Greene and P. G. M. Wuts,
Protective Groups in Organic Synthesis, 3rd. Ed., John Wiley and
Sons (1999); L. Fieser and M. Fieser, Fieser and Fieser's Reagents
for Organic Synthesis, John Wiley and Sons (1999); and L. Paquette,
ed., Encyclopedia of Reagents for Organic Synthesis, John Wiley and
Sons (1995), and subsequent editions thereof.
Pharmaceutical Compositions
[0130] The present invention features pharmaceutical preparations
comprising a PDE5 inhibitor (e.g., sildenafil, vardenafil,
tadalafil, or analogs thereof) together with pharmaceutically
acceptable carriers, where the compounds provide for the treatment
of disease or disease symptoms (e.g., cancer, any disease
delineated herein). Pharmaceutical preparations of the invention
have both therapeutic and prophylactic applications. In one
embodiment, a pharmaceutical composition includes an effective
amount of a PDE5 inhibitor. The compositions should be sterile and
contain a therapeutically effective amount of a PDE5 inhibitor in a
unit of weight or volume suitable for administration to a subject
(e.g., a human patient). The compositions and combinations of the
invention can be part of a pharmaceutical pack, where the PDE5
inhibitor is present in individual dosage amounts.
[0131] Pharmaceutical compositions of the invention to be used for
prophylactic or therapeutic administration should be sterile.
Sterility is readily accomplished by filtration through sterile
filtration membranes (e.g., 0.2 .mu.m membranes), by gamma
irradiation, or any other suitable means known to those skilled in
the art. Therapeutic compositions generally are placed into a
container having a sterile access port, for example, an intravenous
solution bag or vial having a stopper pierceable by a hypodermic
injection needle. These compositions ordinarily will be stored in
unit or multi-dose containers, for example, sealed ampoules or
vials, as an aqueous solution or as a lyophilized formulation for
reconstitution.
[0132] A PDE5 inhibitor may be combined, optionally, with a
pharmaceutically acceptable excipient. The term
"pharmaceutically-acceptable excipient" as used herein means one or
more compatible solid or liquid filler, diluents or encapsulating
substances that are suitable for administration into a human. The
term "carrier" denotes an organic or inorganic ingredient, natural
or synthetic, with which the active ingredient is combined to
facilitate administration. The components of the pharmaceutical
compositions also are capable of being co-mingled with a PDE5
inhibitor of the present invention, and with each other, in a
manner such that there is no interaction that would substantially
impair the desired pharmaceutical efficacy.
[0133] Compounds of the present invention can be contained in a
pharmaceutically acceptable excipient. The excipient preferably
contains minor amounts of additives such as substances that enhance
isotonicity and chemical stability. Such materials are non-toxic to
recipients at the dosages and concentrations employed, and include
buffers such as phosphate, citrate, succinate, acetate, lactate,
tartrate, and other organic acids or their salts;
tris-hydroxymethylaminomethane (TRIS), bicarbonate, carbonate, and
other organic bases and their salts; antioxidants, such as ascorbic
acid; low molecular weight (for example, less than about ten
residues) polypeptides, e.g., polyarginine, polylysine,
polyglutamate and polyaspartate; proteins, such as serum albumin,
gelatin, or immunoglobulins; hydrophilic polymers, such as
polyvinylpyrrolidone (PVP), polypropylene glycols (PPGs), and
polyethylene glycols (PEGs); amino acids, such as glycine, glutamic
acid, aspartic acid, histidine, lysine, or arginine;
monosaccharides, disaccharides, and other carbohydrates including
cellulose or its derivatives, glucose, mannose, sucrose, dextrins
or sulfated carbohydrate derivatives, such as heparin, chondroitin
sulfate or dextran sulfate; polyvalent metal ions, such as divalent
metal ions including calcium ions, magnesium ions and manganese
ions; chelating agents, such as ethylenediamine tetraacetic acid
(EDTA); sugar alcohols, such as mannitol or sorbitol; counterions,
such as sodium or ammonium; and/or nonionic surfactants, such as
polysorbates or poloxamers. Other additives may be included, such
as stabilizers, anti-microbials, inert gases, fluid and nutrient
replenishers (i.e., Ringer's dextrose), electrolyte replenishers,
and the like, which can be present in conventional amounts.
[0134] The compositions, as described above, can be administered in
effective amounts. The effective amount will depend upon the mode
of administration, the particular condition being treated and the
desired outcome. It may also depend upon the stage of the
condition, the age and physical condition of the subject, the
nature of concurrent therapy, if any, and like factors well known
to the medical practitioner. For therapeutic applications, it is
that amount sufficient to achieve a medically desirable result.
[0135] With respect to a subject having a disease or disorder
delineated herein, an effective amount is an amount sufficient to
stabilize, slow, or reduce a symptom associated with the condition.
Generally, doses of the compounds of the present invention would be
from about 0.01 mg/kg per day to about 1000 mg/kg per day. In one
embodiment, 25, 50, 75, 100, 125, 150 or 200 mg of a PDE5
inhibitor, such as sildenafil, is administered to a subject.
Preferably, 100 mg of a PDE5 inhibitor is administered. Effective
doses range from 0.1 nM to 200 nM, where the bottom of the range is
any integer between 1 and 199, and the top of the range is any
integer between 2 and 200. It is expected that doses ranging from
about 5 to about 2000 mg/kg will be suitable--depending on the
specific PDE5 inhibitor used. Lower doses will result from certain
forms of administration, such as intravenous administration and
pharmaceutical. In the event that a response in a subject is
insufficient at the initial doses applied, higher doses (or
effectively higher doses by a different, more localized delivery
route) may be employed to the extent that patient tolerance
permits. Multiple doses per day are contemplated to achieve
appropriate systemic levels of a composition of the present
invention.
[0136] A variety of administration routes are available. The
methods of the invention, generally speaking, may be practiced
using any mode of administration that is medically acceptable,
meaning any mode that produces effective levels of the active
compounds without causing clinically unacceptable adverse effects.
In one preferred embodiment, a composition of the invention is
administered orally. Other modes of administration include rectal,
topical, intraocular, buccal, intravaginal, intracisternal,
intracerebroventricular, intratracheal, nasal, transdermal,
within/on implants, or parenteral routes or possibly
intratumorally. The term "parenteral" includes subcutaneous,
intrathecal, intravenous, intramuscular, intraperitoneal, or
infusion. Intravenous or intramuscular routes are not particularly
suitable for long-term therapy and prophylaxis. They could,
however, be preferred in emergency situations. Compositions
comprising a composition of the invention can be added to a
physiological fluid, such as blood. Oral administration can be
preferred for prophylactic treatment because of the convenience to
the patient as well as the dosing schedule.
[0137] Pharmaceutical compositions of the invention can comprise
one or more pH buffering compounds to maintain the pH of the
formulation at a predetermined level that reflects physiological
pH, such as in the range of about 5.0 to about 8.0. The pH
buffering compound used in the aqueous liquid formulation can be an
amino acid or mixture of amino acids, such as histidine or a
mixture of amino acids such as histidine and glycine.
Alternatively, the pH buffering compound is preferably an agent
which maintains the pH of the formulation at a predetermined level,
such as in the range of about 5.0 to about 8.0, and which does not
chelate calcium ions. Illustrative examples of such pH buffering
compounds include, but are not limited to, imidazole and acetate
ions. The pH buffering compound may be present in any amount
suitable to maintain the pH of the formulation at a predetermined
level.
[0138] Pharmaceutical compositions of the invention can also
contain one or more osmotic modulating agents, i.e., a compound
that modulates the osmotic properties (e.g, tonicity, osmolality
and/or osmotic pressure) of the formulation to a level that is
acceptable to the blood stream and blood cells of recipient
individuals. The osmotic modulating agent can be an agent that does
not chelate calcium ions. The osmotic modulating agent can be any
compound known or available to those skilled in the art that
modulates the osmotic properties of the formulation. One skilled in
the art may empirically determine the suitability of a given
osmotic modulating agent for use in the inventive formulation.
Illustrative examples of suitable types of osmotic modulating
agents include, but are not limited to: salts, such as sodium
chloride and sodium acetate; sugars, such as sucrose, dextrose, and
mannitol; amino acids, such as glycine; and mixtures of one or more
of these agents and/or types of agents. The osmotic modulating
agent(s) may be present in any concentration sufficient to modulate
the osmotic properties of the formulation.
[0139] Pharmaceutical compositions of the invention can also be a
non-aqueous liquid formulation. Any suitable non-aqueous liquid may
be employed, provided that it provides stability to the active
agents (s) contained therein. Preferably, the non-aqueous liquid is
a hydrophilic liquid. Illustrative examples of suitable non-aqueous
liquids include: glycerol; dimethyl sulfoxide (DMSO);
polydimethylsiloxane (PMS); ethylene glycols, such as ethylene
glycol, diethylene glycol, triethylene glycol, polyethylene glycol
("PEG") 200, PEG 300, and PEG 400; and propylene glycols, such as
dipropylene glycol, tripropylene glycol, polypropylene glycol
("PPG") 425, PPG 725, PPG 1000, PPG 2000, PPG 3000 and PPG
4000.
[0140] Pharmaceutical compositions of the invention can also be a
mixed aqueous/non-aqueous liquid formulation. Any suitable
non-aqueous liquid formulation, such as those described above, can
be employed along with any aqueous liquid formulation, such as
those described above, provided that the mixed aqueous/non-aqueous
liquid formulation provides stability to the compound contained
therein. Preferably, the non-aqueous liquid in such a formulation
is a hydrophilic liquid. Illustrative examples of suitable
non-aqueous liquids include: glycerol; DMSO; PMS; ethylene glycols,
such as PEG 200, PEG 300, and PEG 400; and propylene glycols, such
as PPG 425, PPG 725, PPG 1000, PPG 2000, PPG 3000 and PPG 4000.
[0141] Suitable stable formulations can permit storage of the
active agents in a frozen or an unfrozen liquid state. Stable
liquid formulations can be stored at a temperature of at least
-70.degree. C., but can also be stored at higher temperatures of at
least 0.degree. C., or between about 0.1.degree. C. and about
42.degree. C., depending on the properties of the composition.
[0142] Other delivery systems can include time-release, delayed
release or sustained release delivery systems. Such systems can
avoid repeated administrations of compositions of the invention,
increasing convenience to the subject and the physician. Many types
of release delivery systems are available and known to those of
ordinary skill in the art. They include polymer base systems such
as polylactides (U.S. Pat. No. 3,773,919; European Patent No.
58,481), poly(lactide-glycolide), copolyoxalates,
polycaprolactones, polyesteramides, polyorthoesters,
polyhydroxybutyric acids, such as poly-D-(-)-3-hydroxybutyric acid
(European Patent No. 133,988), copolymers of L-glutamic acid and
gamma-ethyl-L-glutamate (Sidman, K. R. et al., Biopolymers 22:
547-556), poly (2-hydroxyethyl methacrylate) or ethylene vinyl
acetate (Langer, R. et al., J. Biomed. Mater. Res. 15:267-277;
Langer, R. Chem. Tech. 12:98-105), and polyanhydrides.
[0143] Other examples of sustained-release compositions include
semi-permeable polymer matrices in the form of shaped articles,
e.g., films, or microcapsules. Delivery systems also include
non-polymer systems that are: lipids including sterols such as
cholesterol, cholesterol esters and fatty acids or neutral fats
such as mono- di- and tri-glycerides; hydrogel release systems such
as biologically-derived bioresorbable hydrogel (i.e., chitin
hydrogels or chitosan hydrogels); sylastic systems; peptide based
systems; wax coatings; compressed tablets using conventional
binders and excipients; partially fused implants; and the like.
Specific examples include, but are not limited to: (a) erosional
systems in which the agent is contained in a form within a matrix
such as those described in U.S. Pat. Nos. 4,452,775, 4,667,014,
4,748,034 and 5,239,660 and (b) diffusional systems in which an
active component permeates at a controlled rate from a polymer such
as described in U.S. Pat. Nos. 3,832,253, and 3,854,480.
[0144] Another type of delivery system that can be used with the
methods and compositions of the invention is a colloidal dispersion
system. Colloidal dispersion systems include lipid-based systems
including oil-in-water emulsions, micelles, mixed micelles, and
liposomes. Liposomes are artificial membrane vessels, which are
useful as a delivery vector in vivo or iii vivo.
[0145] Liposomes can be targeted to a particular tissue by coupling
the liposome to a specific ligand such as a monoclonal antibody,
sugar, glycolipid, or protein. Liposomes are commercially available
from Gibco BRL, for example, as LIPOFECTIN.TM. and LIPOFECTACE.TM.,
which are formed of cationic lipids such as N-[1-(2,3
dioleyloxy)-propyl]-N,N,N-trimethylammonium chloride (DOTMA) and
dimethyl dioctadecylammonium bromide (DDAB). Methods for making
liposomes are well known in the art and have been described in many
publications, for example, in DE 3,218,121; Epstein et al., Proc.
Natl. Acad. Sci. (USA) 82:3688-3692 (1985); Hwang et al., Proc.
Natl. Acad. Sci. (USA) 77:4030-4034 (1980); EP 52,322; EP 36,676;
EP 88,046; EP 143,949; EP 142,641; Japanese Pat. Appl. 83-118008;
U.S. Pat. Nos. 4,485,045 and 4,544,545; and EP 102,324. Liposomes
also have been reviewed by Gregoriadis, G., Trends Biotechnol., 3:
235-241).
[0146] Another type of vehicle is a biocompatible microparticle or
implant that is suitable for implantation into a mammalian
recipient. Exemplary bioerodible implants that are useful in
accordance with this method are described in PCT International
application no. PCT/US/03307 (Publication No. WO 95/24929, entitled
"Polymeric Gene Delivery System"). PCT/US/0307 describes
biocompatible, preferably biodegradable polymeric matrices for
containing an exogenous gene under the control of an appropriate
promoter. The polymeric matrices can be used to achieve sustained
release of the exogenous gene or gene product in the subject.
[0147] The polymeric matrix preferably is in the form of a
microparticle such as a microsphere (wherein an agent is dispersed
throughout a solid polymeric matrix) or a microcapsule (wherein an
agent is stored in the core of a polymeric shell). Microcapsules of
the foregoing polymers containing drugs are described in, for
example, U.S. Pat. No. 5,075,109. Other forms of the polymeric
matrix for containing an agent include films, coatings, gels,
implants, and stents. The size and composition of the polymeric
matrix device is selected to result in favorable release kinetics
in the tissue into which the matrix is introduced. The size of the
polymeric matrix further is selected according to the method of
delivery that is to be used. Preferably, when an aerosol route is
used the polymeric matrix and composition are encompassed in a
surfactant vehicle. The polymeric matrix composition can be
selected to have both favorable degradation rates and also to be
formed of a material, which is a bioadhesive, to further increase
the effectiveness of transfer. The matrix composition also can be
selected not to degrade, but rather to release by diffusion over an
extended period of time. The delivery system can also be a
biocompatible microsphere that is suitable for local, site-specific
delivery. Such microspheres are disclosed in Chickering, D. E., et
al., Biotechnol. Bioeng., 52: 96-101; Mathiowitz, E., et al.,
Nature 386: 410-414.
[0148] Both non-biodegradable and biodegradable polymeric matrices
can be used to deliver the compositions of the invention to the
subject. Such polymers may be natural or synthetic polymers. The
polymer is selected based on the period of time over which release
is desired, generally in the order of a few hours to a year or
longer. Typically, release over a period ranging from between a few
hours and three to twelve months is most desirable. The polymer
optionally is in the form of a hydrogel that can absorb up to about
90% of its weight in water and further, optionally is cross-linked
with multivalent ions or other polymers.
[0149] Exemplary synthetic polymers which can be used to form the
biodegradable delivery system include: polyamides, polycarbonates,
polyalkylenes, polyalkylene glycols, polyalkylene oxides,
polyalkylene terepthalates, polyvinyl alcohols, polyvinyl ethers,
polyvinyl esters, poly-vinyl halides, polyvinylpyrrolidone,
polyglycolides, polysiloxanes, polyurethanes and co-polymers
thereof, alkyl cellulose, hydroxyalkyl celluloses, cellulose
ethers, cellulose esters, nitro celluloses, polymers of acrylic and
methacrylic esters, methyl cellulose, ethyl cellulose,
hydroxypropyl cellulose, hydroxy-propyl methyl cellulose,
hydroxybutyl methyl cellulose, cellulose acetate, cellulose
propionate, cellulose acetate butyrate, cellulose acetate
phthalate, carboxylethyl cellulose, cellulose triacetate, cellulose
sulphate sodium salt, poly(methyl methacrylate), poly(ethyl
methacrylate), poly(butylmethacrylate), poly(isobutyl
methacrylate), poly(hexylmethacrylate), poly(isodecyl
methacrylate), poly(lauryl methacrylate), poly(phenyl
methacrylate), poly(methyl acrylate), poly(isopropyl acrylate),
poly(isobutyl acrylate), poly(octadecyl acrylate), polyethylene,
polypropylene, poly(ethylene glycol), poly(ethylene oxide),
poly(ethylene terephthalate), poly(vinyl alcohols), polyvinyl
acetate, poly vinyl chloride, polystyrene, polyvinylpyrrolidone,
and polymers of lactic acid and glycolic acid, polyanhydrides,
poly(ortho)esters, poly(butic acid), poly(valeric acid), and
poly(lactide-cocaprolactone), and natural polymers such as alginate
and other polysaccharides including dextran and cellulose,
collagen, chemical derivatives thereof (substitutions, additions of
chemical groups, for example, alkyl, alkylene, hydroxylations,
oxidations, and other modifications routinely made by those skilled
in the art), albumin and other hydrophilic proteins, zein and other
prolamines and hydrophobic proteins, copolymers and mixtures
thereof. In general, these materials degrade either by enzymatic
hydrolysis or exposure to water in vivo, by surface or bulk
erosion.
Methods of Treatment
[0150] In one embodiment, the present invention provides a method
of inhibiting an anti-tumor immune response mediated pathway target
in a subject comprising the step of administering to the subject an
effective amount of a PDE5 inhibitor, preferably as part of a
composition additionally comprising a pharmaceutically acceptable
carrier. Preferably this method is employed to treat a subject
suffering from or susceptible to a condition selected from a
disease or disease symptom (e.g., cancer, tumor, any disease or
disorder delineated herein). Other embodiments include any of the
methods herein wherein the subject is identified as in need of the
indicated treatment.
[0151] In another aspect, the method further includes
administration of an additional therapeutic agent. The additional
therapeutic agent can be an anticancer agent, an anti-infective, or
a PDE inhibitor.
[0152] Another aspect of the invention is a compound herein (e.g.,
PDE inhibitor, compound of any of the formulae herein) for use in
the treatment or prevention in a subject of a disease, disorder or
symptom thereof delineated herein. Another aspect of the invention
is the use of a PDE5 inhibitor in the manufacture of a medicament
for a disease or disease symptom (e.g., cancer, tumor, any disease
or disorder delineated herein) in a subject. Preferably, the
medicament is used for treatment or prevention in a subject of a
disease, disorder or symptom set forth above.
Kits
[0153] The invention provides kits for the treatment or prevention
of a condition associated with a disease or disease symptom (e.g.,
cancer, tumor, anti-tumor immune response, any disease or disorder
delineated herein). In one embodiment, the kit includes a
pharmaceutical pack comprising an effective amount of a PDE5
inhibitor (e.g., a PDE5a inhibitor, such as sildenafil).
Preferably, the compositions are present in unit dosage form. In
some embodiments, the kit comprises a sterile container which
contains a therapeutic or prophylactic composition; such containers
can be boxes, ampules, bottles, vials, tubes, bags, pouches,
blister-packs, or other suitable container forms known in the art.
Such containers can be made of plastic, glass, laminated paper,
metal foil, or other materials suitable for holding
medicaments.
[0154] If desired compositions of the invention or combinations
thereof are provided together with instructions for administering
them to a subject having or at risk of developing a disease or
disease symptom (e.g., cancer, tumor, anti-tumor immune response,
any disease or disorder delineated herein). The instructions will
generally include information about the use of the compounds for
the treatment or prevention of a disease or disease symptom (e.g.,
cancer, tumor, anti-tumor immune response, any disease or disorder
delineated herein). In other embodiments, the instructions include
at least one of the following: description of the compound or
combination of compounds; dosage schedule and administration for
treatment of a disease or disease symptom (e.g., cancer, tumor,
anti-tumor immune response, any disease or disorder delineated
herein); precautions; warnings; indications; counter-indications;
overdosage information; adverse reactions; animal pharmacology;
clinical studies; and/or references. The instructions may be
printed directly on the container (when present), or as a label
applied to the container, or as a separate sheet, pamphlet, card,
or folder supplied in or with the container.
[0155] Here, it is demonstrated that PDE5 blockade represents a
pharmacologic target capable of down-regulating both Arg1 and NOS2
in tumor infiltrating MSCs. This down-regulation abrogates tumor
induced MSCs mediated immunosuppression and enhances tumor-specific
immunity that results in measurable anti-tumor activity.
[0156] This is the first demonstration that blocking MSC-mediated
immunosuppression via PDE5 inhibition imparts a measurable
anti-tumor immune effect. It is shown that PDE5-blockade increases
intracellular cGMP resulting in degradation of NOS2 and suppression
of NO production. Furthermore it also results in down-regulation of
the IL4R.alpha.-ARG-1 pathway. Thus it targets both pathways
critical to MSC function. These findings establish a new role for
PDE5 inhibition as a viable and effective immunological adjunct in
the treatment of various malignancies adding to its therapeutic
applications that already include the treatment of erectile
dysfunction, pulmonary hypertension (35, 36) and cardiac
hypertrophy (18).
[0157] Although NO production from tumor-associated macrophages has
both tumor-promoting and tumoricidal properties, the ultimate
effect of these free radicals is complex and likely dependent upon
their local concentration within the microenvironment. In fact, NO
exerts its tumoricidal action through modulation of p53 expression
(37). Interestingly, in a model in which human cancer cells were
modified to express high levels of NO, cells containing wild type
p53 demonstrated reduced tumor growth whereas cells with mutant p53
showed increased production of vascular endothelial growth factor
(VEGF), neovascularization and increased tumor growth (38). These
studies demonstrate the duality of NO-mediated effects and its
regulation by p53 within a tumor setting. Unfortunately, most human
tumors possess p53 mutations (39) making them resistant to
NO-mediated apoptosis. Furthermore, prolonged exposure to NO leads
to the selection of a more aggressive p53 mutant clone better able
to escape the tumoricidal action (40). The clinical importance of
NO-mediated antitumor efficacy is further limited by the fact that
many human tumors such as melanoma, breast, stomach, ovarian and
cervical cancers actually express NOS2. In fact, NO can promote
cancer growth not only in the early stages of tumor progression by
facilitating DNA mutations (40) but also in the later stages by
increasing tumor angiogenesis (41) and immunosuppression (42). With
regards to the immune escape mechanisms, it has been shown that
MSCs and/or tumor associated macrophages induce apoptosis or anergy
in CD8.sup.+ and CD4.sup.+ T cells through a NOS2-dependent
mechanisms (7, 20, 43). In fact, through inhibition of IL-2
signaling (20), NO production anergizes Th1 T-cells. Alternatively,
in a mixed Th1-Th2 environment where arginase-induced pathways also
mediate immunosuppression, MSCs produce NO and super-oxide radicals
to generate peroxynitrites that induce apoptosis of activated
CD8.sup.+ T cells (12). With the growing understanding of the role
of MSCs in tumor-induced immune dysfunction (6, 44, 45), targeted
pharmacologic interventions have significant appeal in overcoming
the suppressive mechanisms in immune-based therapeutic settings. It
is recently shown that nitroaspirin could abrogate the inhibitory
activity of NO. It restored immune responsiveness in tumor-bearing
hosts and enhanced the preventive and therapeutic efficacy of
antitumor vaccines (13). However, despite its use as a vaccine
adjuvant, nitroaspirin demonstrated no anti-tumor efficacy when
used alone.
[0158] PDE5 inhibition represents a novel immunopharmacologic
target that down-modulates the expression of both Arg1 and NOS2 in
MSCs. Interestingly, this approach more effectively reverses
MSC-induced immune suppression than does nitroaspirin by exerting a
significant in vivo anti-tumor effect. The augmented anti-tumor
effect can be attributed to the ability of PDE5 inhibition to
target the various suppressive pathways by which MSCs inhibit
T-cell function. To our knowledge, this is the first demonstration
that abrogation of MSC suppressive mechanisms alone is sufficient
to generate an antitumor immune response. The measurable anti-tumor
efficacy seen with PDE5 inhibitors but not with NO inhibitors is
likely due to the multi-target inhibition exerted by these agents.
Although results herein show that PDE5 inhibitors affect both Arg-1
and NOS2 pathways in MSCs, it is possible that additional as yet
undefined pathways capable of further abrogating MSC-mediated
immunosuppression may also be involved. For example, cGMP is also
capable of reducing VEGF production within the hypoxic
intra-tumoral environment (46).
[0159] One likely mechanism for the effect of PDE5-inhibitors on
reducing NO production involves the impact of these inhibitors on
mRNA stability. cGMP destabilizes NOS2 mRNA by reducing the
ubiquitous mRNA binding protein, human-antigen-R (HuR) (47). HuR
binds to AU-rich elements in the 3'-untranslated region (UTR)
thereby increasing the mRNA half-life (48). As such,
destabilization of NOS2 mRNA via PDE5 inhibition would abrogate
NO-mediated immunosuppression more effectively than would
competitive inhibition of NO itself.
[0160] Since Arg1 mRNA does not possess AU rich elements nor has it
been described to be stabilized by HUR, other mechanism(s) are
likely involved in PDE5-mediated down-regulation of Arg1. One
possible explanation is that high levels of cGMP induced by PDE5
blockade reduce the cytosolic Ca2+ concentration (49) leading to a
reduction of the calcium-dependent protein kinase C (PKC) activity
(50) that in turn prevents up-regulation of IL4R.alpha. (51). The
link between IL4R.alpha. and Arg-1 in MSCs is supported by recent
data demonstrating a direct correlation between ARG1 expression and
IL4R.alpha. expression. LysM.sup.CreIL4R.alpha..sup.-/flox mice in
which IL4R.alpha. expression is knocked-out in neutrophils and
macrophages subsequently challenged with C26GM completely rejected
the tumor when adoptively transferred with tumor-primed CD8+ T
cells (Gallina et al. manuscript submitted). These data support our
findings by demonstrating that PDE5 blockade down-regulates
IL4R.alpha. expression on tumor-infiltrating MSCs (FIG. 1) and
synergizes with the adoptive transfer of tumor-primed CD8+ T cells
(FIG. 4). This effect appears to specifically target MSCs since
IL4R.alpha. expression on isolated CD11b.sup.+ cells from
tumor-bearing mice is significantly reduced when co-cultured in the
presence of sildenafil. Furthermore, the addition of IFN-.gamma.,
which in vivo up-regulates IL4R.alpha. expression through both
autocrine and paracrine (presumably through activated T cells)
mechanisms, is significantly reduced in the presence of sildenafil
(FIG. 7). Taken together these data underscore the critical role of
the IL4R.alpha.-ARG1 pathway in MSCs as well as the use of PDE5
inhibitors as therapeutically effective drugs to overcome
tumor-induced immunosuppression.
[0161] Effective adoptive cellular therapy requires T cells with
the predetermined antigen specificity to be present in sufficient
numbers, traffic to the tumor site, and kill their target. Most
solid tumors are characterized by a lymphocytic infiltration that
is frequently unable to kill autologous tumor cells, indicating T
cell anergy (52, 53), the presence of regulatory T cells (Tregs)
(54) or the existence of a non-T cell immunosuppressive population.
It was recently shown that human prostate cancer anergic TILs can
be reactivated in vitro through the inhibition of NOS2 and Arg1.
These findings underscore importance of MSC-mediated
immunosuppression and identify putative targets of
immunosuppressive pathways used by MSCs to improve immune-based
therapeutic outcomes (11). These results are indicated by our in
vivo studies whereby the sildenafil treatment led to an increase in
intratumoral CD8.sup.+ T cell infiltration that inversely
correlated with tumor size (FIG. 5b), increased the percentage of
activated T cells (FIG. 5c, and d) and was the only condition in
which adoptive cell transfer resulted in a measurable anti-tumor
effect (FIG. 4).
[0162] The phenotype of human MSCs is still not well defined.
However, there is evidence to suggest that a non-lymphoid
CD34.sup.+ population plays a role in the hypo-responsiveness of
PBLs from head and neck cancer patients (55). A similar
unresponsiveness is seen in PBLs from multiple myeloma patients
(56). While the low proliferative capacity may be due to intrinsic
T cell defects, a likely explanation for T cell unresponsiveness is
the presence of a non-lymphoid suppressor accessory population
since PDE5-inhibition augments the proliferative index of
lymphocytes from unfractionated peripheral mononuclear cell
population but not of purified CD3.sup.+ cells (data not shown).
Moreover, results from our experiments suggest a prominent role of
both Arginase and NOS2 in MM PBLs unresponsiveness. The ability of
sildenafil to restore CD3/CD28-stimulated proliferation of PBLs
from both head and neck and myeloma patients suggests that the
mechanisms found in mice are also present in humans.
[0163] Although different drugs such L-NMMA, Nor-NOHA, NO-aspirin,
or Vitamin D3 (57) have been used in vitro and in mouse models to
alter the MSCs suppressive mechanisms, they have either not been
extensively tested in humans or found to be extremely toxic, as in
the case with L-NMMA (58). Moreover the cytokines present in tumor
microenvironment can be very different among patient and tumor
stage (59) and thus can promote different suppressive pathways on
MSCs. The use of safe and extensively tested PDE5 inhibitors such
as sildenafil, tadalafil, or vardenafil to overcome the different
MSCs immune suppressive pathways is demonstrated by results
described herein.
[0164] The following examples are provided to illustrate the
invention, not to limit it. Those skilled in the art will
understand that the specific constructions provided below may be
changed in numerous ways, consistent with the above described
invention while retaining the critical properties of the compounds
or combinations thereof.
EXAMPLES
PDE5 Inhibition Down-Regulates NOS2 Expression in Tumor-Associated
MSCs
[0165] The primary property of MSCs is their ability to suppress an
immune response. While this phenotype is an essential defining
feature, emerging data reveals varying degrees of immunosuppression
of MSCs isolated from different organs. Tumor-associated MSCs
express greater levels of NOS2 and Arg-1 than do splenic MSCs and,
thus, result in greater immune suppression (data not shown). Since
cGMP analogues can reduce NO generation in monocytes (15), this
investigation sought to determine whether NO production in
tumor-associated MSCs could be reduced with the in vivo treatment
of the PDE5 inhibitor, sildenafil (20 mg/kgday). BALB/c mice were
challenged subcutaneously with the colon carcinoma, CT26WT. Half
the mice were then treated with sildenafil. The mice were
sacrificed 15 days later and intratumoral MSCs were purified from
the single cell suspensions. MSCs derived from the sildenafil group
showed higher intracellular cGMP levels than the control group as
assessed by a competitive enzyme immune assay (FIG. 1a). No
significant differences were seen in the non-MSC population (data
not shown). Interestingly, higher cGMP concentrations correlated
with down-regulation of NOS2 (FIG. 1b) and lower NO production
(FIG. 1c) by the intratumoral MSCs. Surprisingly, sildenafil
treatment also down-regulated Arg-1 (FIG. 1b,c) the other gene
involved in MSC mediated immunosuppression. Arg-1 expression is
mainly regulated by the STAT6-IL4R.alpha. pathway (19) and data by
Gallina et al. recently correlated IL4R.alpha. expression on
CD11b/GR1 with the immunosuppressive phenotype (manuscript
submitted). IL4R.alpha. expression via flow cytometry on purified
tumor infiltrating MSCs from untreated or sildenafil treated mice
was analyzed. IL4R.alpha. is up-regulated in tumor infiltrating
MSCs as compared to splenic MSCs from tumor free mice. In contrast,
sildenafil treatment significantly reduced IL4R.alpha. expression
on the intratumoral MSCs and this correlated directly with the
decrease in Arg-1 expression and activity. In addition to
confirming the role of IL4R.alpha. in MSC mediated
immunosuppression, its expression in splenic MSCs cultured alone or
in presence of sildenafil (FIG. 7) was analyzed. Sildenafil not
only down-regulates IL4R.alpha. in cultured MSCs, but also prevents
IFN-.gamma. mediated IL4R.alpha. up-regulation--a necessary process
for MSC-mediated immune suppression (Gallina et al. submitted).
These findings reveal a novel mechanism by which MSCs suppressive
pathways can be pharmacologically regulated both in vivo and in
vitro: PDE5 inhibition up-regulates intracellular cGMP and
decreases NOS2 and Arg1 protein levels--the mediators of MSC
suppression.
In Vitro PDE5 Inhibition Abrogates MSC Immunosuppression.
[0166] Freshly isolated MSCs from tumor-bearing mice suppress the
in vitro proliferation of activated lymphocytes. The exact
mechanisms of suppression appear to be strain specific: in the Th-1
prone strain, C57Bl/6, it is mediated by NOS2 through NO production
(via the NOS2 catalytic domain) (20). Whereas, in the mixed
Th-1/Th2 Balb/c strain, suppression is mediated either by
peroxynitrite formation (via Arg1 and NOS2 co-expression) (10) or
by L-arginine depletion secondary to Arg1 over-expression (21). By
reducing both Arg1 and NOS2 expression, PDE5 inhibition affects all
these suppressive pathways resulting in reduced MSC-mediated
immunosuppression and enhances antigen-specific T cell
responsiveness. Tumor-derived CD11b.sup.+ MSCs were isolated from
BALB/c mice bearing the colon carcinoma, C26-GM. We utilized the
irradiated, C26 cell line retrovirally transduced to produce GM-CSF
since this cytokine has been shown to recruit MSCs more rapidly
than the unmodified CT26 cell line (10, 22, 23). We tested MSC
suppressive activity by admixing MSCs with CFSE-labeled
hemagluttinin (HA)-specific CD8.sup.+ (clone 4) or CD4.sup.+ (6.5)
T cells pulsed with their relevant peptide in the presence or
absence of sildenafil (FIGS. 2a and b). Whereas the addition of
tumor-derived MSCs significantly impaired antigen specific T cell
proliferation as demonstrated by the low percentage of CFSE.sup.lo
clonotypic T cells, sildenafil almost completely restored both
CD4.sup.+ and CD8.sup.+ responsiveness of antigen-specific T cells.
The absence of a sildenafil-mediated enhancement in T cell function
in the groups lacking CD11b cells underscores the targeted role of
sildenafil on the MSC population. In an effort to understand the in
vivo mechanisms mediating this effect, we utilized C57Bl/6 mice for
several reasons: 1) in this strain, inhibition of NOS2 is
sufficient to revert MSCs mediated immunosuppression (7); and 2)
NOS2.sup.-/- mice are available thus enabling us to examine the
effect of PDE5 blockade in the NO-mediated pathway of
immunosuppression. CD11b.sup.+ MSCs were isolated from either B16GM
melanoma-bearing C57BL/6-NOS2.sup.+/+ or B16GM melanoma-bearing
C57BL/6-NOS2.sup.-/- mice. A suppression assay was performed by
stimulating OVA-specific CD4.sup.+ T-cells with the relevant
peptide in the presence or absence of MSCs derived from either
NOS2.sup.+/+ or NOS2.sup.-/- mice (FIG. 2c). While the addition of
C57Bl/6-NOS2.sup.+/+-derived MSCs induced considerable T cell
suppression, no suppression was observed with MSCs from
NOS2.sup.-/- mice. Furthermore, while PDE5 inhibition reversed MSC
suppression in NOS2.sup.+/+ mice, the addition of sildenafil to the
NOS2.sup.-/--derived MSC suppression assay did not augment T cell
responsiveness. Taken together, these results confirm the role of
NOS2 in MSC-mediated T cell suppression (FIG. 2c) and demonstrate
that PDE5 inhibition can revert two different suppressive pathways
(Arg1 and NOS2) by which MSCs impair immune responsiveness.
In Vivo PDE5 Inhibition Delays Tumor Outgrowth by an Immune
Mediated Mechanism.
[0167] Having recently shown that the in vivo inhibition of the MSC
suppressive pathways by nitro-aspirin was ineffective as a single
agent but augmented the anti-tumor efficacy of vaccines on
established tumors (13), it was sought to determine whether PDE5
inhibition alone, by affecting both Arg-1 and NOS2 suppressive
activity, could impart a measurable anti-tumor effect. Mice were
challenged either with CT26-WT or with the more aggressive tumor,
C26GM and then treated with PDE5 inhibitors. As shown in FIG. 3a
and FIG. 8, sildenafil or tadalafil treatment alone significantly
delayed tumor outgrowth through an immune mediated mechanism as
evidenced by the lack of anti-tumor efficacy in the immune
deficient Rag.sup.-/- mice (FIG. 3b). However, the combination of
PDE inhibition with the MSC-depleting anti-GR-1.sup.+ antibody
conferred no synergistic effect (FIG. 3c). Taken together, these
data confirm that MSC-mediated immunosuppressive pathways function
via NOS2-Arg1 enzymatic activity produced by GR-1.sup.+ cells and
demonstrate the ability of PDE5 inhibition to abrogate their
activity in vivo. To the best of our knowledge, this is the first
demonstration that a direct anti-tumor effect can be obtained
through the pharmacologic inhibition of tumor-induced
immunosuppressive pathways.
PDE5 Inhibition Enhances Anti-Tumor Efficacy of Adoptive
Immunotherapy.
[0168] Adoptive immunotherapy of tumor-specific T cells offers much
promise as a therapeutic modality. Considerable progress has been
made in developing strategies to isolate, expand and activate tumor
specific cells in vitro. In the appropriate environment, these
lymphocytes can mediate significant tumor destruction. However, the
mere presence of tumor reactive T cells in the peripheral
circulation is not sufficient to induce tumor rejection (24). T
cells must also traffic to the tumor site and overcome the
intrinsic immunosuppressive barriers to effectively kill in situ.
To determine whether sildenafil inhibition of the MSC suppressive
pathways could improve the efficacy of adoptive immunotherapy,
20.times.10.sup.6 C26GM-primed splenocytes were transferred into
C26GM bearing animals. Following adoptive transfer, the mice were
either treated with sildenafil or left untreated. As shown in FIG.
4, adoptive transfer alone demonstrated no anti-tumor efficacy
whereas PDE5 inhibition showed a statistically significant
reduction in tumor outgrowth. However, coupling adoptive
immunotherapy with PDE5 inhibition yielded the greatest anti-tumor
efficacy. These data suggest that disruption of the MSC-mediated,
immunosuppressive microenvironment is critical to augment the
therapeutic efficacy of adoptive immunotherapy in cancer-bearing
hosts.
PDE5 Inhibition Increases the Number of Tumor Infiltrating
CD8.sup.+ Cells.
[0169] Tumor specific T-cells must be present in sufficient numbers
and capable of trafficking to their targets to exert a measurable
antitumor effect. In fact, a direct correlation exists between the
number of infiltrating lymphocytes and a favorable clinical outcome
in patients with metastatic ovarian cancer (25). Moreover, the
presence and the functionality of tumor infiltrating lymphocytes
(TILs) correlates with a favorable prognosis in various human
malignancies (25-29). Since PDE5 inhibition augments anti-tumor
immunity, whether sildenafil treatment altered the number and/or
the activation state of TILs was considered. Hematoxylin-eosin
staining revealed a greater intratumoral cellular infiltration in
the sildenafil-treated mice compared to the untreated controls
(FIG. 5a). To better evaluate these differences, tumor-bearing mice
either received tumor-primed T cells or no T cells followed by
sildenafil treatment or no additional therapy. The tumor was then
excised and single cell suspensions were obtained. The T cell
infiltration was analyzed by flow cytometry for CD4.sup.+ and
CD8.sup.+ T cells. This approach enabled us to accurately examine
the entire tumor mass and reliably quantify the infiltrating
lymphocytic population. Sildenafil treatment resulted in a greater
CD8.sup.+ tumor infiltration. Interestingly, no increase in
CD4.sup.+ T cells was observed with PDE5 blockade (FIG. 5b insert).
Moreover, sildenafil significantly activated the tumor-infiltrating
CD8.sup.+ T cells as revealed by up-regulation of both CD69 and
CD25 activation markers (FIG. 5c). There were no differences in
activation markers between the sildenafil-treated group and
sildenafil plus adoptive cell therapy (ACT). These data indicate
that the anti-tumor efficacy in the sildenafil+ACT group (FIG. 4
and FIG. 5b) is primarily attributable to sildenafil's ability to
abrogate the immunosuppressive mechanisms within the tumor
microenvironment.
[0170] Interestingly, in the advanced tumor setting, the percentage
of tumor infiltrating CD8.sup.+ T cells negatively correlated
(Spearman bivariate correlation P<0.001) with tumor size which
supports the concept of sildenafil's ability to create a more
permissive immune environment (FIG. 5b). The maximal therapeutic
effect was seen in the ACT group where a larger CD8.sup.+ T cell
infiltrate was present in the tumor. Moreover, tetramer staining
suggested that the tumor-infiltrating CD8.sup.+ more effectively
recognized the tumor associated antigen in the sildenafil-treated
group. In fact, 9.08%.+-.0.905 of tumor-infiltrating CD8.sup.+
cells were specific for AH1, one of the major C26GM-associated
antigens, whereas only 1.19.+-.1.180 were tetramer positive in the
untreated group (n=3).
[0171] IL-2 is required for the activation of naive T cells and
generates a lymphocyte population with heightened recall responses.
Furthermore, IL-2 production is associated with the persistence of
tumor specific CD8.sup.+ lymphocytes within the tumor
microenvironment and systemic administration of low doses of IL-2
improves the persistence and antitumor efficacy of transferred T
cells (30). It was previously shown that NO can alter IL-2
production in activated lymphocytes. To examine whether the
immunomodulatory effect of PDE5 inhibition affected T cell
activation within the tumor microenvironment, IL-2 production by
TIL was examined. To accomplish this, a transgenic mouse model of
green fluorescent protein (GFP) under an IL-2 promoter
(BALB/c-IL-2p/GFP) (31) was utilized. In this model, T cell
stimulation results in activation of the IL-2 and expression of the
reporter transgene GFP, easily detectable by flow cytometry.
C26GM-primed BALB/c-IL-2p/GFP splenocytes were adoptively
transferred to tumor-bearing recipients that were either left
untreated or treated with sildenafil for 9 days. Single cell
suspensions of the tumor-infiltrating CD8.sup.+ were analyzed by
FACS for GFP expression. Adoptively transferred, vaccine-primed T
cells were activated in the tumor microenvironment only with PDE5
inhibition whereas, in its absence, they were unable to release
IL-2, and hence were bona fide anergic T cells (FIG. 5d).
To further prove that these effects were dependent on CD8.sup.+ T
cells, mice were challenged with C26GM and either: 1) left
untreated; 2) given sildenafil; 3) an anti-CD8 depleting antibody;
or 4) both. Sildenafil treatment again demonstrated a statistically
significant reduction in tumor outgrowth, an effect completely
abolished by CD8.sup.+ depletion (FIG. 5e). These experiments
indicate that the in vivo MSC suppressive pathways limit T cell
infiltration, activation and anti-tumor efficacy. Abrogating these
suppressive mechanisms via PDE5 inhibition enhances the tumor
specific T cell response and generates a measurable anti-tumor
response.
T Cell Proliferation is Restored by PDE5 Inhibition in Multiple
Myeloma and Head and Neck Cancer Patients.
[0172] Head and neck cancers express high levels of GM-CSF
responsible for the intratumoral infiltration by CD34.sup.+ MSC
suspected of playing a major role in the immune suppression
observed in these patients (32). In fact, it has been shown, that
peripheral blood lymphocytes (PBLs) from these patients are
functionally impaired in that they fail to be activated and
proliferate poorly upon stimulation (33). This anergic state is in
large part due to the Arg1 and/or NOS-dependent suppressive
activity of MSCs (21, 34). Similar results were also seen in
prostate cancer (11) and in multiple myeloma patients (Noonan,
unpublished data). It was sought to determine whether one could
restore T cell proliferation of PBLs isolated from head and neck
and multiple myeloma patients and stimulated with anti-CD3 and
anti-CD28 antibody-coated beads, in the presence or absence of
sildenafil. While the addition of sildenafil to the culture had no
effect on PBLs from healthy donors, PDE5 inhibition significantly
restored CD4.sup.+ and CD8.sup.+ proliferation in all the examined
patients (FIG. 6). Interestingly, the addition of sildenafil to
isolated CD3.sup.+ T cells was unable to increase T cell
proliferation (data not shown). Taken together, these human data
confirm that the PDE5 inhibition augments immune responsiveness
through its effect on an accessory, non-T cell population.
Moreover, these data suggest that the same immunosuppressive
mechanisms found in mice are conserved in human malignancies and
that PDE5 can be a useful therapeutic target to improve anti-tumor
immunotherapy.
Cell Lines: CT26 and C26-GM are BALB/c colon carcinoma cells lines
previously described (10). B16-GM is a C57Bl/6 melanoma cell line
previously described (60). Cells were grown in DMEM (Invitrogen
Carlsbad, Calif.) or in RPMI medium 1640 (Invitrogen) supplemented
with 2 mM L-glutamine/10 mM Hepes/20 .mu.M 2-mercaptoethanol/150
units/ml streptomycin/200 units/ml penicillin/10% heat-inactivated
FBS (Harlan, Indianapolis Ind.). Drugs and cytokines: Sildenafil
(Pfizer, New York N.Y.) was dissolved in the drinking water (20
mg/kg/24 h), given intraperitoneally (ip) daily where indicated (20
mg/kg/24 h) or added to the cell cultures at a final concentration
of 50 mg/ml. Tadalafil (Lilly ICOS. Bothel Wash.) was given ip at a
concentration of 2 mg/kg/24 h. IFN-.gamma. (25 ng/ml)(Peprotech,
Rocky Hill, N.J.) was added where indicated. Mice and in vivo
experiments: 4-6 weeks old Balb/c mice were purchased from Harlan
C57Bl/6-NOS2.sup.-/- mice (strain B6; 129P2-Nos2.sup.tmILau) or the
control mice (strain B6129PF2/J 100903) were purchased by Jackson
Laboratories (Bar Harbor, Me.). Rag-/- were bred in the Johns
Hopkins animal facility. Balb/c-pIL2-GFP mice were a kind gift of
CT. Weaver (University of Alabama)(31). pCL4-TCR mice are
transgenic for an influenza virus HA512-520 peptide-specific, H-2
Kd-restricted TCR composed of V10 and V.beta.8.2 chains were
described before (23). All experiments involving the use of mice
were in accordance with protocols approved by the Animal Care and
Use Committee of the Johns Hopkins University School of Medicine.
Tumor measurements were performed in a blind fashion with a caliper
by measuring the two main diameters and tumor size is expressed as
their product. Mice were euthanized for ethical reasons when tumor
size was greater than 150 mm.sup.2. GR1 depletion was performed by
ip injection of 100 .mu.g of anti-GR-1 depleting antibody (clone
RB6.8C5-18). This clone was obtained by sub-cloning the originally
described RB6.8C5 to maximize the antibody production. CD8
depletion was performed by ip injection of 200 .mu.g of anti-CD8
depleting antibody (clone 2.43) on days 0, 2, 4, 6. The antibodies
were produced in vitro in protein-free medium (Invitrogen),
purified by G-protein affinity chromatography, and quantified
utilizing a Rat IgG2b ELISA (Bethyl Inc Montgomery, Tex.). Adoptive
cell transfer (ACT): Donor mice were tumor primed by subcutaneous
(sc) injection of 10.sup.6 .gamma.-irradiated C26GM into four limbs
one week prior to adoptive T cell transfer. For the adoptive
transfer experiments, lymph nodes and spleens were harvested and
mechanically disrupted to obtain single cell suspensions. The cells
then underwent RBC lysis with ACK Lysing buffer (Biosource,
Camarillo Calif.) and 20.times.10.sup.6 cells were injected i.v.
into each recipient. Collagenase treatment of tumors: Single cell
suspensions were obtained from the tumors by collagenase treatment.
Briefly, tumors were surgically removed, and incubated 30 min @ 37
C..degree. with a solution of collagenase (10 mg/ml Collagenase,
0.1 mM MgCl2 0.1 mM CaCl.sub.2) coupled with mechanical disruption.
The reaction was stopped with 10 vol. of medium containing 10%
fetal calf serum. The cells were washed, red blood cells lysed, and
the cell suspensions were passed through a cell strainer. For
hematoxylin-eosin staining, whole tumors were washed twice with PBS
and then incubated for 5 days with 10 volumes of 10% neutral
buffered formalin. Flow cytometry: Single cells suspensions from
spleens or tumors were stained with Phycoerythrin (PE)-conjugated
anti-mouse CD8 (CD8-PE) (BD-Pharmingen San Jose, Calif.),
Allophycocyanin (APC)-conjugated anti-mouse CD4 (CD4-APC)
(BD-Pharmingen) or with APC conjugated anti-mouse CD11b (BD) and
PE-conjugated anti-mouse Gr-1 (CD8-PE). IL4R.alpha. expression was
evaluated on purified CD11b.sup.+ cells by the use of an anti-mouse
CD124 PE-conjugated (BD-Pharmingen). Isotyped matched antibodies
were used as controls and living cells were gate based on 7AAD,
annexinV staining. A total of 100,000 events were collected for
each sample on a FACScalibur (BD) flow cytometer, and the data were
analyzed using FCS express v2.0 (De-novo software). Cell
purification: CD11b.sup.+ purification was performed with the Mouse
CD11b MicroBeads (Miltenyi Biotec, Bergish-Gladbach, Germany),
positive and negative fractions were sorted with the LS columns
following the manufacturer's instructions. Suppressive assay:
Purified splenic CD11b.sup.+ cells (2.times.10.sup.5) were added to
CFSE-labeled splenocytes (10.sup.6) derived from Cl4 transgenic
mice stimulated for 3 days with Class I HA.sub.512-520 peptide
(IYSTVASSL) in 96 flat bottomed plates. Sildenafil was added where
indicated. Proliferation assay: PBLs were obtained from head and
neck or multiple myeloma cancer patients having obtained informed
consent using an Institutional Review Board-approved protocol.
T-cell stimulation was performed by adding anti-CD3/CD28 antibody
coated Dynal beads to ficolled PBLs suspended in serum free media
at a 3:1 bead to T-cell ratio. The cells were cultured for 5 days
in a 96 round-bottom well plate at 37 C 5% CO2. The cells were then
labeled with CD4 and CD8 antibodies and analyzed by flow cytometry.
Sildenafil was added where indicated. Results are reported as fold
change (number of activated cells/number of unactivated cells).
cGMP was measured on purified CD11b.sup.+ cells using the "Cyclic
GMP EIA" Kit (Cayman Chemical Ann Arbor, Mich.). Data analysis was
performed with the workbooks available at
http://www.caymanchem.com/neptune/servlet/neptune/template/analysis%2CEIA-
.vm/a/z. Data are expressed as mean+/-SE of quadruplicate wells.
Western Blot Cells (10.sup.6) were purified and washed twice with
PBS, and the pellet was resuspended in PBS with 0.2% Triton X-100
and 2 mM EDTA, and incubated for 10 min at room temperature. Cell
lysates were centrifuged at 14,000 rpm for 1 min, and 1 vol of
Laemmli's sample buffer (4% SDS, 20% glycerol, 10% 2-ME, 4 mg/100
ml bromophenol blue, and 125 mM Tris-HCl; pH 6.8) was added to the
supernatant. After incubation at 95.degree. C. for 10 min, lysates
were subjected to SDS-PAGE, and proteins were transferred overnight
to PVDF membranes. The membranes were saturated at RT in PBS/0.05%
Tween 20 containing 2% nonfat milk (Sigma-Aldrich) for 1 hr. The
membranes were then incubated overnight at room temperature with
rabbit polyclonal anti-NOS2 (Santa Cruz Biotechnology Santa Cruz,
Calif.), mouse anti-Arg1 (a kind gift from Augusto C. Ochoa,
Lousiana State University, New Orleans, La.) or polyclonal rabbit
anti-actin antibody (Sigma-Aldrich), washed twice with PBS/0.05%
Tween 20, and incubated with the either mouse anti-mouse IGG or
donkey anti rabbit IGG HRP-linked secondary antibody (Amersham
Biosciences, Little Chalfont, U.K.) for 1 h at room temperature.
Proteins were detected using the Supersignal West
picochemiluminescent substrate kit (Pierce, Rockford, Ill.)
according to the manufacturer's instructions. NO measurement: NO
was measured using a nitrate/nitrite assay kit (Cayman) according
to the manufacturer's instructions. Results were normalized to
10.sup.6 cells. Data are from triplicate wells. Arginase assay:
CD11b.sup.+ cells were magnetically purified from the surgically
removed tumor, washed twice in PBS-BSA 1%, counted and lysed with
Triton X100, 0.1%. The arginase assay was performed as previously
described (10). Statistical analysis: Bivariate Pearson and ANOVA
analysis were performed using SPSS v7.0 All experiments were
repeated at least twice and all the P values were two-sided (T
test) or one-sided (Anova).
[0173] All references cited herein, whether in print, electronic,
computer readable storage media or other form, are expressly
incorporated by reference in their entirety, including but not
limited to, abstracts, articles, journals, publications, texts,
treatises, technical data sheets, internet web sites, databases,
patents, patent applications, and patent publications.
[0174] The recitation of a listing of chemical groups in any
definition of a variable herein includes definitions of that
variable as any single group or combination of listed groups. The
recitation of an embodiment for a variable herein includes that
embodiment as any single embodiment or in combination with any
other embodiments or portions thereof.
[0175] Other embodiments of the invention will be apparent to those
skilled in the art from consideration of the specification and
practice of the invention disclosed herein. It is intended that the
specification and examples be considered as exemplary only, with a
true scope and spirit of the invention being indicated by the
following claims.
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