U.S. patent application number 10/040776 was filed with the patent office on 2003-01-02 for method for identifying compounds for inhibition of neoplastic lesions.
Invention is credited to Pamukcu, Rifat, Piazza, Gary A., Thompson, W. Joseph.
Application Number | 20030004093 10/040776 |
Document ID | / |
Family ID | 26724247 |
Filed Date | 2003-01-02 |
United States Patent
Application |
20030004093 |
Kind Code |
A1 |
Piazza, Gary A. ; et
al. |
January 2, 2003 |
Method for identifying compounds for inhibition of neoplastic
lesions
Abstract
This invention provides a method to identify compounds
potentially useful for the treatment of neoplasia in mammals. The
phosphodiesterase inhibitory activity of a compound is determined
along with COX inhibitory activity. Growth inhibitory and apoptosis
inducing effects on cultured tumor cells are also determined.
Compounds that exhibit phosphodiesterase inhibiton, growth
inhibition and apoptosis induction, but not substantial
prostaglandin inhibitory activity, are desirable for the treatment
of neoplasia.
Inventors: |
Piazza, Gary A.;
(Doylestown, PA) ; Pamukcu, Rifat; (Spring House,
PA) ; Thompson, W. Joseph; (Mobile, AL) |
Correspondence
Address: |
Cell Pathways, Inc.
702 Electronic Drive
Horsham
PA
19044
US
|
Family ID: |
26724247 |
Appl. No.: |
10/040776 |
Filed: |
January 7, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10040776 |
Jan 7, 2002 |
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09664035 |
Sep 18, 2000 |
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09664035 |
Sep 18, 2000 |
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09046739 |
Mar 24, 1998 |
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09046739 |
Mar 24, 1998 |
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08866027 |
May 30, 1997 |
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5858694 |
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Current U.S.
Class: |
514/1 ; 435/4;
435/7.23 |
Current CPC
Class: |
G01N 33/5011 20130101;
C12Q 1/26 20130101; C12Q 1/44 20130101; A61P 35/00 20180101; C12Q
1/533 20130101 |
Class at
Publication: |
514/1 ; 435/4;
435/7.23 |
International
Class: |
A61K 031/00; C12Q
001/00; G01N 033/574 |
Claims
We claim:
1. A method for identifying a compound with potential for treating
neoplasia, comprising: treating neoplastic cells with the compound
to be evaluated at a concentration between about 200 .mu.M to 200
pM; determining the intracellular amount of cGMP in the treated
cells; determining the intracellular amount of cAMP in the treated
cells; wherein about a three-fold or greater increase in the ratio
of cGMP/cAMP in the treated cells compared to the ratio of
cGMP/cAMP in untreated neoplastic cells is indicative that the
compound has potential for treating neoplasia.
2. The method of claim 1 wherein an intracellular amount of cGmp in
the treated cells greater than 500 fmol/mg protein is indicative
that the compound has potential for treating neoplasia.
3. The method of claim 1 wherein an intracellular amount of cAMP in
the treated cells less than 4000 fmol/mg protein is indicative that
the compound has potential for treating neoplasia.
4. The method of claim 3 wherein an intracellular amount of cAMP in
the treated cells less than 4000 fmol/mg protein is indicative that
the compound has potential for treating neoplasia.
Description
BACKGROUND OF THE INVENTION
[0001] This invention provides a method for identifying compounds
potentially useful for the treatment and prevention of
pre-cancerous and cancerous lesions in mammals. This application is
a continuation-in-part of U.S. patent application Ser. No.
08/866,027 to Piazza et al. Filed May 30, 1997.
[0002] Familial adenomatous polyposis ("FAP") is an inherited
disease where the victim's colon contains many polyps or adenomas
virtually uncountable in most instances. Because such patients
develop so many polyps or adenomas each of which has a significant
risk of developing into a cancer--the typical treatment is surgical
removal of the colon. In about 1983, Waddell discovered that the
nonsteroidal anti-inflammatory drug ("NSAID") sulindac would cause
colonic polyps (a type of pre-cancerous lesion) to regress and
prevent their recurrence when that drug was administered to
patients with FAP. Waddell's experience with sulindac in FAP
patients was confirmed in several subsequent studies.
Unfortunately, since sulindac and other NSAIDS aggravate the
digestive tract (not to mention side effects involving kidney and
interference with normal blood clotting) of patients to whom it has
been chronically administered, it is not a practical treatment for
FAP or any other cancer or precancerous indication (i.e.,
neoplasia) requiring long-term administration.
[0003] Waddell originally hypothesized that the mechanism of action
of sulindac on colonic polyps involved the inhibition of the
synthesis of prostaglandin (PG). (Waddell, W. R. et al., "Sulindac
for Polyposis of the Colon," Journal of Surgical Oncology,
24:83-87, 1983). Prostaglandin ("PG") synthesis inhibition results
from the inhibition of cyclooxygenase (COX) caused by NSAIDs. A
common benefit of NSAIDs is the reduction of inflammation, which is
known to be caused by the reduction of PG levels. Since NSAIDs are
known to inhibit COX, which inhibits PG synthesis, it is widely
believed that the regression of colonic polyps is attributed to
this property. In fact, notwithstanding recent discoveries to the
contrary, it has become conventional wisdom that administration of
an inhibitor of PG synthesis (e.g., an NSAID) to a patient with FAP
or other precancerous or cancerous lesion will result in the
regression of the lesion due to a reduction of PG levels.
[0004] Recent discoveries, however, are leading scientists in a
completely different direction--that it is not necessary to inhibit
COX to treat neoplasia patients successfully. Pamukcu et al., in
U.S. Pat. No. 5,401,774, disclosed that sulfonyl compounds, that
were previously reported to be inactive as PG synthesis inhibitors
(and therefore not an NSAID or an anti-inflammatory compound)
unexpectedly inhibited the growth of a variety of neoplastic cells,
including colon polyp cells. These sulfonyl derivatives have proven
effective in rat models of colon carcinogenesis, and one variant
(now referred to as exisulind) has proven effective in preliminary
human clinical trials with FAP patients.
[0005] The importance of this discovery--and the de-linking of
anti-neoplasitic activity and COX inhibition--cannot be overstated.
If those two phenomena were related, there would be little hope for
a safe NSAID therapy for FAP patients because the side effects of
NSAIDs, such as gastric irritation, are also caused by COX
inhibition. Prostaglandins play a protective function in the lining
of the stomach. When NSAIDs are administered, COX is inhibited and
PG levels are reduced: gastric irritation is a common result. Those
side effects may not manifest themselves in short-term (acute)
NSAID therapy. However, during long-term (chronic) NSAID therapy,
gastric irritation, bleeding and ulceration are very common. In
significant numbers of cases, NSAID therapy must be stopped due to
the severity of those side effects and other potentially lethal
side effects. Furthermore, the severity of such side effects
increases with age, probably because natural PG levels in gastric
mucosa falls with age. Thus, useful compounds for treating
neoplastic lesions should desirably inhibit neoplastic cell growth,
but should not inhibit COX.
[0006] Conventional methods for screening compounds may be used to
find improved compounds that inhibit neoplastic cell growth. Under
this scenario, drugs may be screened using in vitro models. But
conventional in vitro screening methods could pass many compounds
that later are shown to be ineffective in animal models because of
a number of unanticipated problems, one of which may be that the in
vitro screen is not predictive of efficacy. Animal model studies
are time consuming and expensive. Therefore, a more precise in
vitro screening method that provides predictive information for
treating neoplasia is needed to screen compounds prior to human
testing. Knowledge of a specific target for inhibiting human cancer
would allow for greater precision and efficiency whereby highly
effective and safe compounds can be identified prior to animal
testing.
[0007] Presently, rational drug discovery methods are being applied
in the pharmaceutical industry to improve methods for identifying
clinically useful compounds. Typically, rational drug discovery
methods relate to a "lock and key" concept whereby structural
relationships between a therapeutic target molecule (lock) and
pharmaceutical compounds (key) are defined. Such methods are
greatly enhanced by specialty computer software that accesses
databases of compounds to identify likely geometric fits with the
target molecule. Unfortunately, to use these systems, one has to
have insight to the target molecule (lock). The target may be an
enzyme, a protein, a membrane or nuclear receptor, or a nucleic
acid sequence, for example.
[0008] In complex diseases, such as neoplasia, scientists have
identified a number of potential targets. However, many of the
drugs available for the treatment of neoplasia are non-specific and
toxic to normal tissues, and are not indicated for precancer and
used only when neoplastic cells progress to cancer. Greater
understanding of the mechanisms involved in cancer may lead
scientists on the path towards designing more specific
antineoplastic drugs--drugs that can safely be administered earlier
in the disease process.
SUMMARY OF THE INVENTION
[0009] This invention relates to a novel in vitro method for
screening test compounds for their ability to treat and prevent
neoplasia, especially pre-cancerous lesions, safely. In particular,
the present invention provides a method for identifying test
compounds that can be used to treat and prevent neoplasia,
including precancerous lesions, with minimal side effects
associated with COX inhibition and other non-specific
interactions.
[0010] In one embodiment of this invention, therefore, the
screening method involves determining the COX inhibition activity
of a test compound. Because the inventors have discovered a
relationship between inhibition of cancer and inhibition of
phosphodiesterase Type-5 isoenzyme ("PDE5"), this invention
includes determining the PDE5 inhibition activity of the compound.
Preferably, the screening method of this invention farther includes
determining whether the compounds inhibit the growth of tumor cells
in a cell culture.
[0011] In an alternate embodiment, the screening method of this
invention involves determining the COX inhibition activity of the
compound, determining the PDE5 inhibition activity of the compound
and determining whether the compound induces apoptosis in tumor
cells.
[0012] By screening compounds in this fashion, potentially
beneficial and improved compounds can be identified more rapidly
and with greater precision than possible in the past. Further
benefits will be apparent from the detailed description that
follows.
BRIEF DESCRIPTION OF THE DRAWINGS
[0013] FIG. 1 illustrates the effect of the sulfide derivative of
sulindac and the sulfone derivative of sulindac (a.k.a. exisulind)
on purified cyclooxygenase activity.
[0014] FIG. 2 illustrates the effects of test compounds B and E on
COX inhibition.
[0015] FIG. 3 illustrates the inhibitory effects of sulindac
sulfide and exisulind on PDE-4 and PDE5 purified from cultured
tumor cells.
[0016] FIG. 4 illustrates the effects of sulindac sulfide on cyclic
nucleotide levels in HT-29 cells.
[0017] FIG. 5 illustrates the phosphodiesterase inhibitory activity
of compound B.
[0018] FIG. 6 illustrates the phosphodiesterase inhibitory activity
of compound E.
[0019] FIG. 7 illustrates the effects of sulindac sulfide and
exisulind on apoptosis and necrosis of HT-29 cells.
[0020] FIG. 8 illustrates the effects of sulindac sulfide and
exisulind on HT-29 cell growth inhibition and apoptosis induction
as determined by DNA fragmentation.
[0021] FIG. 9 illustrates the apoptosis inducing properties of
compound E.
[0022] FIG. 10 illustrates the apoptosis inducing properties of
compound B.
[0023] FIG. 11 illustrates the effects of sulindac sulfide and
exisulind on tumor cell growth.
[0024] FIG. 12 illustrates the growth inhibitory and
apoptosis-inducing activity of sulindac sulfide and control
(DMSO).
[0025] FIG. 13 illustrates the growth inhibitory activity of
compound E.
[0026] FIG. 14 illustrates the inhibition of pre-malignant,
neoplastic lesions in mouse mammary gland organ culture by sulindac
metabolites.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0027] The method of this invention is useful to identify compounds
that can be used to treat or prevent neoplasms, and which are not
characterized by the serious side effects of conventional
NSAIDs.
[0028] Cancer and precancer may be thought of as diseases that
involve unregulated cell growth. Cell growth involves a number of
different factors. One factor is how rapidly cells proliferate, and
another involves how rapidly cells die. Cells can die either by
necrosis or apoptosis depending on the type of environmental
stimuli. Cell differentiation is yet another factor that influences
tumor growth kinetics. Resolving which of the many aspects of cell
growth is affected by a test compound is important to the discovery
of a relevant target for pharmaceutical therapy. Screening assays
based on this selectivity can be combined with tests to determine
which compounds having growth inhibiting activity.
[0029] This invention is the product of several important
discoveries. First, the present inventors discovered that desirable
inhibitors of tumor cell growth induce premature death of cancer
cells by apoptosis (see, Piazza, G. A., et al., Cancer Research,
55(14), 3110-16, 1995). Second, the present inventors unexpectedly
discovered that compounds that selectively induce apoptosis without
substantial COX inhibition also inhibit phosphodiesterase ("PDE").
In particular, and contrary to leading scientific studies,
desirable compounds for treating neoplastic lesions selectively
inhibit Type 5 isoenzyme form of phosphodiesterase ("PDE5") (EC
3.1.4.17). PDE5 is one of at least seven isoenzymes of
phosphodiesterase. PDE5 is unique in that it selectively degrades
cyclic GMP, while the other types of PDE are either non-selective
or degrade cyclic AMP. Preferably, desirable compounds do not
substantially inhibit other phosphodiesterase types.
[0030] A preferred embodiment of the present invention involves
determining the cyclooxygenase inhibition activity of a given
compound, and determining the PDE5 inhibition activity of the
compound. The test compounds are scored for their probable ability
to treat neoplastic lesions either directly by assessing their
activities against specific cutoff values or indirectly by
comparing their activities against known compounds useful for
treating neoplastic lesions. A standard compound that is known to
be effective for treating neoplastic lesions without causing
gastric iritation is 5-fluoro-2-methyl-1-(p-methylsulfonylbenzyli-
dene)-3-indenylacetic acid ("exisulind"). Other useful compounds
for comparative purposes include those that are known to inhibit
COX, such as indomethacin and the sulfide metabolite of sulindac:
5-fluoro-2-methyl-1-(p-methylsulfinylbenzylidene)-3-indenylacetic
acid ("sulindac sulfide"). Other useful compounds for comparative
purposes include those that are known to inhibit PDE5, such as
1-(3-chloroanilino)-4-phenyphthalazine ("MY5445").
[0031] A test compound is clearly determined to be a promising
candidate if it performs better than or comparable to exisulind and
does not inhibit COX. In general, desirable compounds are those
that inhibit PDE5 and inhibit cell growth and induce apoptosis, but
do not inhibit COX at pharmacologically accepted doses.
[0032] As used herein, the term "precancerous lesion" includes
syndromes represented by abnormal neoplastic, including dysplastic,
changes of tissue. Examples include dysplastic growths in colonic,
breast, prostate or lung tissues, or conditions such as dysplastic
nevus syndrome, a precursor to malignant melanoma of the skin.
Examples also include, in addition to dysplastic nevus syndromes,
polyposis syndromes, colonic polyps, precancerous lesions of the
cervix (i.e., cervical dysplasia), esophagus, lung, prostatic
dysplasia, prostatic intraneoplasia, breast and/or skin and related
conditions (e.g., actinic keraosis), whether the lesions are
clinically identifiable or not.
[0033] As used herein, the term "carcinoma" or "cancer" refers to
lesions which are cancerous. Examples include malignant melanomas,
breast cancer, prostate cancer and colon cancer. As used herein,
the terms "neoplasia" and "neoplasms" refer to both cancerous and
pre-cancerous lesions.
[0034] As used herein, the abbreviation PG represents
prostaglandin; PS represents prostaglandin synthetase; PGE.sub.2
represents prostaglandin E.sub.2; PDE represents phosphodiesterase;
COX represents cyclooxygenase; RIA
represents--radioimmunoassay.
[0035] As used herein, "PDE5" refers to that enzyme and any of its
isoforms that exhibit cGMP specific hydrolytic enzyme activities
and high affinity cGMP binding.
[0036] In another aspect of the invention, there is a method for
treating patients in need of treatment for neoplasia by identifying
compounds that exhibit substantial PDE5 inhibitory activity at
pharmacologically acceptable doses, and administering one or more
of those compounds to a patient in need thereof with neoplasia
sensitive to the compound.
SCREENING PROTOCOLS
[0037] The following screening protocols, and alternative
protocols, are provided to aid in the understanding of the
preferred methods used to screen test compounds to determine their
potential to treat or prevent neoplasia, especially pre-cancerous
lesions.
[0038] 1. Determining COX Inhibitory Activity
[0039] COX inhibition can be determined by either of two methods.
One method involves measuring PGE.sub.2 secretion by intact HL-60
cells following exposure to the compound being screened. The other
method involves measuring the activity of purified cyclooxygenases
(COXs) in the presence of the compound. Both methods involve
protocols previously described in the literature.
[0040] 1.A. PGE.sub.2 Secretion
[0041] Compounds of this can be evaluated to determine whether they
inhibited the production of prostaglandin E.sub.2 ("PGE.sub.2"),
according to procedures known in the art. For example, PGE.sub.2
secreted from a cell can be measured using an enzyme immunoassay
(EIA) kit for PGE.sub.2, such as commercially available from
Amersham, Arlington Heights, Ill. USA. Suitable cells include those
which make an abundance of PG, such as HL-60 cells. HL-60 cells are
human promyelocytes that are differentiated with DMSO in mature
granulocytes. (See, Collins, S. J., Ruscetti, F. W., Gallagher, R.
E. and Gallo, R. C., "Normal Functional Characteristics of Cultured
Human Promyelocytic Leukemia Cells (HL-60) After Induction of
Differentiation By Dimethylsulfoxide", J. Exp. Med., 149:969-974,
1979). These differentiated cells produce PGE.sub.2 after
stimulation with a calcium ionophore A23187 (see, Kargman, S.,
Prasit, P. and Evans, J. F., "Translocation of HL-60 Cell
5-Lipoxygenase", J. Biol. Chem., 266: 23745-23752, 1991). HL-60 are
available from the American Type Culture Collection (ATCC:CCL240).
They can be crown in a RPMI 1640 medium supplemented with 20%
heat-inactivated fetal bovine serum, 50 U/ml penicillin and 50
.mu.g/ml streptomycin in an atmosphere of 5% CO.sub.2 at 37.degree.
C. To induce myeloid differentiation, cells are exposed to 1.3%
DMSO for 9 days and then washed and resuspended in Dulbecco's
phosphate-buffered saline at 3.times.10.sup.6 cells/ml.
[0042] The differentiated HL-60 cells (3.times.10.sub.6 cells/ml)
can be incubated for 15 min at 37.degree. C. in the presence of the
compounds tested at the desired concentration. Cells are then
stimulated by A23187 (5.times.10.sup.-6M) for 15 min. PGE.sub.2
secreted into the external medium is measured as described
above.
[0043] 1.B Purified Cyclooxygenases
[0044] Two different forms of cyclooxygenase (COX-I and COX-2) have
been reported in the literature to regulate prostaglandin
synthesis. It is known that COX-2 represents the inducible form of
COX while COX-I represents a constitutive form. COX-I activity can
be measured using the method described by Mitchell et al.
("Selectivity of Nonsteroidal Anti-inflammatory Drugs as Inhibitors
of Constitutive and Inducible Cyclooxygenase," Proc. Natl. Acad.
Sci. USA., 90:11693-11697, 1993, which is incorporated herein by
reference) using COX-I purified from ram seminal vesicles as
described by Boopathy & Balasubramanian, "Purification And
Characterization Of Sheep Platelet Cyclooxygenase" (Biochem. J.,
239:371-377, 1988, which is incorporated herein by reference).
COX-2 activity can be measured using COX-2 purified from sheep
placenta as described by Mitchell et al., 1993, supra.
[0045] The cyclooxygenase inhibitory activity of a drug can be
determined by methods known in the art. For example, Boopathy &
Balasubramanian, 1988, supra, described a procedure in which
prostaglandin H synthase 1 (Cayman Chemical, Ann Arbor, Mich.) is
incubated at 37.degree. C. for 20 min with 100 .mu.M arachidonic
acid (Sigma Chemical Co.), cofactors (such as 1.0 mM glutathione,
1.0 mM hydroquinone, 0.625 .mu.M hemoglobin and 1.25 mM CaCl.sub.2
in 100 mM Tris-HCl, pH 7.4) and the drug to be tested. Following
incubation, the reaction can be terminated with trichloroacetic
acid. Enzymatic activity can then be measured
spectrophotometrically at 530 nm after stopping the reaction by
adding thiobarbituric acid and malonaldehyde.
[0046] Obviously, a compound that exhibits minimal COX-I or COX-2
inhibitory activity in relation to its greater PDE5 inhibitory
activity may not be entirely undesirable.
[0047] 1.C. Analyzing Results
[0048] The amount of inhibition is determined by comparing the
activity of the cyclooxygenase in the presence and absence of the
test compound. Residual or no COX inhibitory activity (i.e., less
than about 25%) at a concentration of about 100 .mu.M is indicative
that the compound should be evaluated further for usefulness for
treating neoplasia. Preferably, the IC.sub.50 concentration should
be greater than 1000 .mu.M for the compound to be further
considered potential use.
[0049] 2. Determining Phosphodiesterase (PDE5) Inhibition
Activity
[0050] Compounds can be screened for inhibitory effect on
phosphodiesterase activity using either the enzyme isolated from
any tumor cell line such as HT-29 or SW-480, or recombinant
HS-PDE5, for example, or measuring cyclic nucleotide levels in
whole cells.
[0051] 2.A. Enzyme Assay
[0052] Phosphodiesterase activity can be determined using methods
known in the art, such as a method using radioactive .sup.3H cyclic
GMP (cGMP)(cyclic 3',5'-guanosine monophosphate) as the substrate
for PDE5 enzyme. (Thompson, W. J., Teraski, W. L., Epstein, P. M.,
Strada, S. J., Advances in Cyclic Nucleotide Research, 10:69-92,
1979, which is incorporated herein by reference). In brief, a
solution of defined substrate .sup.3H-cGMP specific activity (0.2
.mu.M; 100,000 cpm; containing 40 mM Tris-HCl (pH 8.0), 5 mM
MgCl.sub.2 and 1 mg/ml BSA) is mixed with the drug to be tested in
a total volume of 400 .mu.l. The mixture is incubated at 30.degree.
C. for 10 minutes with partially purified PDE5 isolated from HT-29
cells. Reactions are terminated, for example, by boiling the
reaction mixture for 75 seconds. After cooling on ice, 100 .mu.l of
0.5 mg/ml snake venom (O. Hannah venom available from Sigma) is
added and incubated for 10 min at 30.degree. C. This reaction is
then terminated by the addition of an alcohol, e.g. 1 ml of 100%
methanol. Assay samples are applied to a anion chromatography
column (1 ml Dowex, from Aldrich) and washed with 1 ml of 100%
methanol. The amount of radioactivity in the breakthrough and the
wash from the columns in then measured with a scintillation
counter. The degree of PDE5 inhibition is determined by calculating
the amount of radioactivity in drug-treated reactions and comparing
against a control sample (a reaction mixture lacking the tested
compound).
[0053] 2.B. Cyclic Nucleotide Measurements
[0054] Alternatively, the ability for desirable compounds to
inhibit PDE5 is reflected by an increase in cGMP in neoplastic
cells exposed to a compound being screened. The amount of PDE5
activity can be determined by assaying for the amount of cyclic GMP
in the extract of treated cells using radioimmunoassay (RIA). In
this procedure, HT-29 or SW-480 cells are plated and grown to
confluency. The test compound is then incubated with the cell
culture at a concentration of compound between about 200 .mu.M to
about 200 pM. About 24 to 48 hours thereafter, the culture media is
removed from the cells, and the cells are solubilized. The reaction
is stopped by using 0.2N HCl/50%-MeOH. A sample is removed for
protein assay. Cyclic GMP is purified from the acid/alcohol
extracts of cells using anion-exchange chromatography, such as a
Dowex column. The cGMP is dried, acetylated according to published
procedures, such as using acetic anhydride in triethylamine,
(Steiner, A. L., Parker, C. W., Kipnis, D. M., J. Biol Chem.,
247(4): 1106-13, 1971, which is incorporated herein by reference).
The acetylated cGMP is quantitated using radioimmunoassay
procedures (Harper, J., Brooker, G., Advances in Nucleotide
Research, 10:1-33, 1979, which is incorporated herein by
reference). Iodinated ligands (tyrosine metheyl ester) of
derivatized cyclic GMP are incubated with standards or unknowns in
the presence of antisera and appropriate buffers. Antiserum may be
produced using cyclic nucleotide-haptene directed techniques. The
antiserum is from sheep injected with succinyl-cGMP-albumin
conjugates and diluted 1/20,000. Dose-interpolation and error
analysis from standard curves are applied as described previously
(Seibert, A. F., Thompson, W. J., Taylor, A., Wilbourn, W. H.,
Barnard, J. and Haynes, J., J. Applied Physio., 79:389-395, 1992,
which is incorporated hereinby reference).
[0055] In addition, the culture media may be acidified, frozen
(-70.degree. C.) and also analyzed for cGMP and cAMP.
[0056] In addition to observing increases in content of cGMP caused
by desirable test compounds, decreases in content of cAMP have been
observed. It has been observed that a particularly desirable
compound (i.e. one that selectively induces apoptosis in neoplastic
cells, but not substantially in normal cells) follows a time course
consistent with PDE5 inhibition as one initial action resulting in
an increased cGMP content within minutes. Secondarily, treatment of
neoplastic cells with a desirable anti-neoplastic compound leads to
decreased cAtIP content within 24 hours. The intracellular tarcets
of drug actions are being studied further, but current data
supports the concept that both the initial rise in cGMP content
followed by the subsequent fall in cAMP content precede apoptosis
in neoplastic cells exposed to desirable compounds.
[0057] The change in the ratio of the two cyclic nucleotides may be
a more accurate tool for evaluating desirable PDE5 inhibition
activity of test compounds, rather than measuring only the absolute
value of cGMP, only PDE5 inhibition, or only the absolute value of
cGMP. In neoplastic cells not treated with anti-neoplastic
compounds, the ratio of cGMP content/cAMNP content is in the
0.03-0.05 range (i.e., 300-500 fmol/mg protein cGMP content over
6000-8000 fmol/mg protein cAMP content). After exposure to
desirable anti-neoplastic compounds, that ratio increases several
fold (preferably at least about a three-fold increase) as the
result of an initial increase in cyclic GMP and the later decrease
in cyclic AMP.
[0058] Specifically, it has been observed that particularly
desirable compounds achieve an initial increase in cGMP content in
treated neoplastic cells to a level of cGMP greater than about 500
fmol/mg protein. In addition, particularly desirable compounds
cause the later decrease in cAMP content in treated neoplastic
cells to a level of cAMP less than about 4000 fmol/mg protein.
[0059] To determine the content of cyclic AMP, radioimmunoassay
techniques similar to those described above for cGMP are used.
Basically, cyclic nucleotides are purified from acid/alcohol
extracts of cells using anion-exchange chromatography, dried,
acetylated according to published procedures and quantitated using
radioimmunoassay procedures. Iodinated ligands of derivatized
cyclic AMP and cyclic GMP are incubated with standards or unknowns
in the presence of specific antisera and appropriate buffers.
[0060] Verification of the cyclic nucleotide content may be
obtained by determining the turnover or accumulation of cyclic
nucleotides in intact cells. To measure inteact cell cAMP,
.sup.3H-adenine prelabeling is used according to published
procedures (Whalin M. E., R. L. Garrett Jr., W. J. Thompson, and S.
J. Strada, "Correlation of cell-free brain cyclic nucleotide
phosphodiesterase activities to cyclic AMP decay in intact brain
slices", Sec. Mess. and Phos. Protein Research, 12:31 1-325, 1989,
which is incorporated herein by reference). The procedure measures
flux of labeled ATP to cyclic AMP and can be used to estimate
intact cell adenylate cyclase or cyclic nucleotide
phosphodiesterase activities depending upon the specific protocol.
Cyclic GMP accumulation was too low to be studied with intact cell
prelabeling according to published procedures (Reynolds, P. E., S.
J. Strada and W. J. Thompson, "Cyclic GMP accumulation in pulmonary
microvascular endothelial cells measured by intact cell
prelabeling," Life Sci., 60:909-918, 1997, which is incorporated
herein by reference).
[0061] 2.C. Tissue Sample Assay
[0062] The PDE5 inhibitory activity of a test compound can also be
determined from a tissue sample. Tissue samples, such as mammalian
(preferably rat) liver, are collected from subjects exposed to the
test compound. Briefly, a sample of tissue is homogenized in 500
.mu.l of 6% TCA. A known amount of the homogenate is removed for
protein analysis. The remaining homogenate is allowed to sit on ice
for 20 minutes to allow for the protein to precipitate. Next, the
homogenate is centrifuged for 30 minutes at 15,000 g at 4.degree.
C. The supernatant is recovered and the pellet recovered. The
supernatant is washed four times with five volumes of water
saturated diethyl ether. The upper ether layer is discarded between
each wash. The aqueous ether extract is dried in a speed vac. Once
dried, the sample can be frozen for future use, or used
immediately. The dried extract is dissolved in 500 .mu.l of assay
buffer. The amount of PDE5 inhibition is determined by assaying for
the amount of cyclic nucleotides using an enzyme immunoassay (EIA),
such as the Biotrak EIA system acetylation protocol (available from
Amersham, Arlington Heights, Ill., USA). Alternatively, RIA
procedures as detailed above may be used.
[0063] 2.D. Analyzing Results
[0064] The amount of inhibition is determined by comparing the
activity of PDE5 in the presence and absence of the test compound.
Inhibition of PDE5 activity is indicative that the compound is
useful for treating neoplasia. Significant inhibitory activity
greater than that of the benchmark, exisulind, preferably greater
than 50% at a concentration of 10 .mu.M or below, is indicative
that a compound should be further evaluated for antineoplastic
properties. Preferably, the IC.sub.50 value for PDE5 inhibition
should be less than 50 .mu.M for the compound to be further
considered for potential use.
[0065] 3. Determining Whether a Compound Reduces the Number of
Tumor Cells
[0066] In an alternate embodiment, the screening method of the
present invention involves further determining whether the compound
reduces the growth of tumor cells. Various cell lines can be used
in the sample depending on the tissue to be tested. For example,
these cell lines include: SW-480--colonic adenocarcinoma;
HT-29--colonic adenocarcinoma, A-427--lung adenocarcinoma
carcinoma; MCF-7--breast adenocarcinoma; and UACC-375--melanoma
line; and DU145--prostrate carcinoma. Cytotoxicity data obtained
using these cell lines are indicative of an inhibitory effect on
neoplastic lesions. These cell lines are well characterized. and
are used by the United States National Cancer Institute in their
screening program for new anti-cancer drugs.
[0067] 3A. Tumor Inhibition in HT-29 Cell Line
[0068] A compound's ability to inhibit tumor cell growth can be
measured using the HT-29 human colon carcinoma cell line obtained
from ATCC (Bethesda, Md.). HT-29 cells have previously been
characterized as relevant colon tumor cell culture model (Fogh, J.,
and Trempe, G. In: Human Tumor Cells in Vitro, J. Fogh (eds.),
Plenum Press, New York, pp. 115-159, 1975). HT-29 cells are
maintained in RPMI media supplemented with 5% fetal serum (Gemini
Bioproducts, Inc., Carlsbad, Calif.) and 2 mm glutamine, and 1%
antibiotic-antimycotic in a humidified atmosphere of 95% air and 5%
CO.sub.2 at 37.degree. C. Briefly, HT-29 cells are plated at a
density of 500 cells/well in 96 well microtiter plates and
incubated for 24 hours at 37.degree. C. prior to the addition of
compound. Each determination of cell number involved six
replicates. After six days in culture, the cells are fixed by the
addition of cold trichloroacetic acid to a final concentration of
10% and protein levels are measured using the sulforhodamine B
(SRB) colorimetric protein stain assay as previously described by
Skehan, P., Storeng, R., Scudiero, D., Monks, A., McMahon, J.,
Vistica, D., Warren, J. T., Bokesch, H., Kenney, S., and Boyd, M.
R., "New Colorimetric Assay For Anticancer-Drug Screening," J.
Natl. Cancer Inst. 82: 1107-1112, 1990, which is incorporated
herein by reference.
[0069] In addition to the SRB assay, a number of other methods are
available to measure growth inhibition and could be substituted for
the SRB assay. These methods include counting viable cells
following trypan blue staining, labeling cells capable of DNA
synthesis with BrdU or radiolabeled thymidine, neutral red staining
of viable cells, or MTT staining of viable cells.
[0070] 3.B. Analyzing Results
[0071] Significant tumor cell growth inhibition greater than about
50% at a dose of 100 .mu.M or below is further indicative that the
compound is useful for treating neoplastic lesions. Preferably, an
IC.sub.50 value is determined and used for comparative purposes.
This value is equivalent to the concentration of drug needed to
inhibit tumor cell growth by 50% relative to the control.
Preferably, the IC.sub.50 value should be less than 100 .mu.M for
the compound to be considered further for potential use for
treating neoplastic lesions.
[0072] 4. Determining Whether a Compound Induces Apoptosis
[0073] In a second alternate embodiment, the screening method of
the present invention further involves determining whether the
compound induces apoptosis in cultures of tumor cells.
[0074] Two distinct forms of cell death may be described by
morphological and biochemical criteria: necrosis and apoptosis.
Necrosis is accompanied by increased permeability of the plasma
membrane; the cells swell and the plasma membrane ruptures within
minutes. Apoptosis is characterized by membrane blebbing,
condensation of cytoplasm and the activation of endogenous
endonucleases.
[0075] Of the two, apoptosis is the most common form of eukaryotic
cell death. It occurs naturally during normal tissue turnover and
during embryonic development of organs and limbs. Apoptosis also is
induced by cytotoxic T-lymphocytes and natural killer cells, by
ionizing radiation and certain chemotherapeutic drugs.
Inappropriate regulation of apoptosis is thought to play an
important role in many pathological conditions including cancer,
AIDS, Alzheimer disease, etc. Compounds can be screened for
induction of apoptosis using cultures of tumor cells maintained
under conditions as described above. Treatment of cells with test
compounds involves either pre- or post-confluent cultures and
treatment for two to seven days at various concentrations.
Apoptotic cells are measured in both the attached and "floating"
compartments of the cultures. Both compartments are collected by
removing the supernatant, trypsinizing the attached cells, and
combining both preparations following a centrifugation wash step
(10 minutes, 2000 rpm). The protocol for treating tumor cell
cultures with sulindac and related compounds to obtain a
significant amount of apoptosis has been described in the
literature. (See, Piazza, G. A., et al., Cancer Research,
55:3110-16, 1995, which is incorporated herein by reference). The
novel features include collecting both floating and attached cells,
identification of the optimal treatment times and dose range for
observing apoptosis, and identification of optimal cell culture
conditions.
[0076] 4.A. Morphological Observation of Apoptosis
[0077] Following treatment with a test compound, cultures can be
assayed for apoptosis and necrosis by florescent microscopy
following labeling with acridine orange and ethidium bromide. The
method for measuring apoptotic cell number has previously been
described by Duke & Cohen, "Morphological And Biochemical
Assays Of Apoptosis," Current Protocols In Immunology, Coligan et
al., eds., 3.17.1-3.17.16 (1992, which is incorporated herein by
reference).
[0078] For example, floating and attached cells can be collected by
trypsinization and washed three times in PBS. Aliquots of cells can
be centrifuged. The pellet can then be resuspended in media and a
dye mixture containing acridine orange and ethidium bromide
prepared in PBS and mixed gently. The mixture can then be placed on
a microscope slide and examined.
[0079] 4.B. Analysis of Apoptosis by DNA Fragmentation
[0080] Apoptosis can also be quantified by measuring an increase in
DNA fragmentation in cells which have been treated with test
compounds. Commercial photometric EIA for the quantitative in vitro
determination of cytoplasmic histone-associated-DNA-fragments
(mono- and oligonucleosomes) are available (Cell Death Detection
ELISA.sup.okys, Cat. No. 1,774,425, Boehringer Mannheim). The
Boehringer Mannheim assay is based on a sandwich-enzyme-immunoassay
principle using mouse monoclonal antibodies directed against DNA
and histones, respectively. This allows the specific determination
of mono-and oligonucleosomes in the cytoplasmatic fraction of cell
lysates.
[0081] According to the vendor, apoptosis is measured in the
following fashion. The sample (cell-lysate) is placed into a
streptavidin-coated microtiter plate ("MTP"). Subsequently, a
mixture of anti-histone-biotin and anti-DNA peroxidase conjugate
are added and incubated for two hours. During the incubation
period, the anti-histone antibody binds to the histone-component of
the nucleosomes and simultaneously fixes the immunocomplex to the
streptavidin-coated MTP via its biotinylation. Additionally, the
anti-DNA peroxidase antibody reacts with the DNA component of the
nucleosomes. After removal of unbound antibodies by a washing step,
the amount of nucleosomes is quantified by the peroxidase retained
in the immunocomplex. Peroxidase is determined photometrically with
ABTS7 (2,2'-Azido-[3 -ethylbenzthiazolin-suffonate])* as
substrate.
[0082] For example, SW-480 colon adenocarcinoma cells are plated in
a 96-well MTP at a density of 10,000 cells per well. Cells are then
treated with test compound, and allowed to incubate for 48 hours at
37.degree. C. After the incubation, the MTP is centrifuged and the
supernatant is removed. The cell pellet in each well is then
resuspended in lysis buffer for 30 minutes. The lysates are then
centrifuged and aliquots of the supernatant (i.e. cytoplasmic
fraction) are transferred into streptavidin coated MTP. Care is
taken not to shake the lysed pellets (i.e. cell nucleii containing
high molecular weight, unfragmented DNA) in the MTP. Samples are
then analyzed.
[0083] Fold stimulation (FS=OD.sub.max/OD.sub.veh), an indicator of
apoptotic response, is determined for each compound tested at a
given concentration. EC.sub.50 values may also be determined by
evaluating a series of concentrations of the test compound.
[0084] 4.C. Analyzing Results
[0085] Statistically significant increases of apoptosis (i.e.,
greater than 2 fold stimulation at a concentration of 100 .mu.M)
are further indicative that the compound is useful for treating
neoplastic lesions. Preferably, the EC.sub.50 value for apoptotic
activity should be less than 100 .mu.M for the compound to be
further considered for potential use for treating neoplastic
lesions. EC.sub.50 is herein defined as the concentration that
causes 50% induction of apoptosis relative to vehicle
treatment.
[0086] 5. Validation--Mammary Gland Organ Culture Model Tests
[0087] Test compounds identified by the above methods can be tested
for antineoplastic activity by their ability to inhibit the
incidence of preneoplastic lesions in a mammary gland organ culture
system. This mouse mammary gland organ culture technique has been
successfully used by other investigators to study the effects of
known antineoplastic agents such as NSAIDs, retinoids, tamoxifen,
selenium, and certain natural products, and is useful for
validation of the screening method of the present invention.
[0088] For example, female BALB/c mice can be treated with a
combination of estradiol and progesterone daily, in order to prime
the glands to be responsive to hormones in vitro. The animals are
sacrificed and thoracic mammary glands are excised aseptically and
incubated for ten days in growth media supplemented with insulin,
prolactin, hydrocortisone, and aldosterone. DMBA
(7,12-dimethylbenz(a)anthracene) is administered to induce the
formation of premalignant lesions. Fully developed glands are then
deprived of prolactin, hydrocortisone, and aldosterone, resulting
in the regression of the glands but not the premalignant
lesions.
[0089] The test compound is dissolved in DMSO and added to the
culture media for the duration of the culture period. At the end of
the culture period, the glands were fixed in 10% formalin. stained
with alum carmine, and mounted on glass slides. The incidence of
forming mammary lesions is the ratio of the glands with mammary
lesions and glands without lesions. The incidence of mammary
lesions in test compound treated glands is compared with that of
the untreated glands.
[0090] The extent of the area occupied by the mammary lesions can
be quantitated by projecting an image of the gland onto a
digitation pad. The area covered by the gland is traced on the pad
and considered as 100% of the area. The space covered by each of
the unregressed structures is also outlined on the digitization pad
and quantitated by the computer.
EXPERIMENTAL SECTION
[0091] A number of test compounds were examined in the various
protocols and screened for potential use in treating neoplasia. The
results of these tests are reported below. The test compounds are
hereinafter designated by a letter code that corresponds to the
following:
[0092]
A--rac-threo-(E)-1-(N,N'-diethylaminoethanethio)-1-(butan-1',4'-oli-
do)-[3',4':1,2]-6-fluoro-2-methyl-3-(p-methylsulfonylbenzylidene)-indan;
[0093]
B--(Z)-5-Fluoro-2-methyl-1-(3,4,5-trimethoxybenzylidene)-3-acetic
acid;
[0094] C--(Z)-5-Fluoro-2-methyl-1-(p-chlorobenzylidene)-3-acetic
acid;
[0095] D--rac-(E)-1-(butan-1',4'-olido)-[3',
4':1,2]-6-fluoro-2-methyl-3-(-
p-methylsulfonylbenzylidene)-1S-indanyl-N-acetylcysteine;
[0096]
E--(Z)-5-Fluoro-2-methyl-1-(3,4,5-trimethoxybenzylidene)-3-indenyla-
cetamide, N-benzyl;
[0097]
F--(Z)-5-Fluoro-2-methyl-1-(p-methylsulfonylbenzylidene)-3-indenyla-
cetamide, N,N'-dicyclohexyl;
[0098]
G--ribo-(E)-1-Triazolo-[2',3':1",3"]-1-(butan-1',4'-olido)-[3',4':1-
,2]-6-methyl-3-(p-methylsulfonylbenzylidene)-indan; and
[0099]
H--rac-(E)-1-(butan-1',4-olido)-[3',4':1,2]-6-fluoro-2-methyl-3'-(p-
-methylsulfonylbenzylidene)-1S-indanyl-glutathione).
EXAMPLE 1
COX Inhibition Assay
[0100] Reference compounds and test compounds were analyzed for
their COX inhibitory activity in accordance with the protocol for
the COX assay of section 1.B. supra. FIG. 1 shows the effect of
various concentrations of either sulindac sulfide or exisulind on
purified cyclooxygenase (Type 1) activity. Cyclooxygenase activity
was determined using purified cyclooxygenase from ram seminal
vesicles as described previously (Mitchell et al, supra). The IC-50
value for sulindac sulfide was calculated to be approximately 1.76
.mu.M, while that for exisulind was greater than 10,000 .mu.M.
These data show that sulindac sulfide, but not exisulind, is a
COX-I inhibitor. Similar data was obtained for the COX-2 isoenzyme.
(Thompson, et al., Journal of the National Cancer Institute, 87:
1259-1260, 1995).
[0101] FIG. 2 shows the effect of test compounds B and E on COX
inhibition. COX activity was determined as for the compounds shown
in FIG. 1. The data show that both test compound B and E do not
significantly inhibit COX-I.
1TABLE 1 Cyclooxygenase inhibitory activity among a series of
compounds % Inhibition at 100 .mu.M Reference compounds
Indomethacin 95 MY5445 94 Sulindac sulfide 97 Exisulind <25 Test
compounds A <25 B <25 C 87 D <25 E <25
[0102] In accordance with the protocol of section 1.B., supra,
compounds A through E were evaluated for COX inhibitory activity as
reported in Table 1 above. Compound C was found to inhibit COX
greater than 25% at a 100 .mu.M dose, and therefore, would not be
selected for further screening.
EXAMPLE 2
PDE5 Inhibition Assay
[0103] Reference compounds and test compounds were analyzed for
their PDE5 inhibitory activity in accordance with the protocol for
the assay of section 2.A., supra. FIG. 3 shows the effect of
various concentrations of sulindac sulfide and exisulind on either
PDE-4 or PDE5 activity purified from human colon HT-29 cultured
tumor cells, as described previously (W. J. Thompson et al.,
supra). The IC.sub.50 value of sulindac sulfide for inhibition of
PDE4 was 41 .mu.M, and for inhibition of PDE5 was 17 .mu.M. The
IC.sub.50 value of exisulind for inhibition of PDE4 was 181 .mu.M,
and for inhibition of PDE5 was 56 .mu.M. These data show that both
sulindac sulfide and exisulind inhibit phospohodiesterase activity.
Both compounds show selectivity for the PDE5 isoenzyme form.
[0104] FIG. 4 shows the effects of sulindac sulfide on either cGMP
or cAMP production as determined on cultured HT-29 cells in
accordance with the assay of section 2.B., supra. HT-29 cells were
treated with sulindac sulfide for 30 minutes and cGMP or cAMP was
measured by conventional radioimmunoassay method. As indicated,
sulindac sulfide increased the levels of cGMP by greater than 50%
with an EC.sub.50 value of 7.3 .mu.M (top). Levels of cAMP were
unaffected by treatment, although a known PDE4 inhibitor, rolipram,
increased cAMP (bottom). The data demonstrate the pharmacological
significance of inhibiting PDE5, relative to PDE4.
[0105] FIG. 5 shows the effect of the indicated dose of test
compound B on either PDE5 or PDE4 isozymes of phosphodiesterase.
The calculated IC.sub.50 value for PDE5 was 18 .mu.M and 58 .mu.M
for PDE4.
[0106] FIG. 6 shows the effect of the indicated dose of test
compound E on either PDE4 or PDE5. The calculated IC.sub.50 value
was 0.08 .mu.M for PDE5 and greater than 25 .mu.M for PDE4.
2TABLE 2 PDE5 inhibitory activity among a series of compounds %
Inhibition at 10 .mu.M Reference compounds Indomethacin 34 MY5445
86 Sulindac sulfide 97 Exisulind 39 Test compounds A <25 B
<25 C <25 D 36 E 75
[0107] The above compounds in Table 2 were evaluated for PDE
inhibitory activity, as described in the protocol of section 2.A;
supra. Of the compounds that did not inhibit COX, only compound E
was found to cause greater than 50% inhibition at 10 .mu.M. As
noted in FIG. 11, compound B showed inhibition of greater than 50%
at a dose of 20 .mu.M. Therefore, depending on the dosage level
used in a single dose test, some compounds may be screened out that
otherwise may be active at slightly higher dosages. The dosage used
is subjective and may be lowered after active compounds are found
at certain levels to identify even more potent compounds.
EXAMPLE 3
Apoptosis Assay
[0108] Reference compounds and test compounds were analyzed for
their PDE5 inhibitory activity in accordance with the protocol for
the assay of section 4.A. and 4.B., supra. In accordance with the
assay of 4.A., FIG. 7 shows the effects of sulindac sulfide and
exisulind on apoptotic and necrotic cell death. HT-29 cells were
treated for six days with the indicated dose of either sulindac
sulfide or exisulind. Apoptotic and necrotic cell death was
determined previously (Duke and Cohen, In: Current Protocols in
Immunology, 3.17.1-3.17.16, New York, John Wiley and Sons, 1992).
The data shows that both sulindac sulfide and exisulind are capable
of causing apoptotic cell death without inducing necrosis. All data
were collected from the same experiment.
[0109] In accordance with the assay of 4.B., FIG. 8 shows the
effect of sulindac sulfide and sulfone on tumor growth inhibition
and apoptosis induction as determined by DNA fragmentation. Top
figure; growth inhibition (open symbols, right axis) and DNA
fragmentation (closed symbols, left axis) by exisulind. Bottom
figure; growth inhibition (open symbols) and DNA fragmentation
(closed symbols) by sulindac sulfide. Growth inhibition was
determined by the SRB assay after six days of treatment. DNA
fragmentation was determined after 48 hours of treatment. All data
was collected from the same experiment.
[0110] FIG. 9 shows the apoptosis inducing properties of compound
E. HT-29 colon adenocarcinoma cells were treated with the indicated
concentration of compound E for 48 hours and apoptosis was
determined by the DNA fragmentation assay. The calculated EC.sub.50
value was 0.05 .mu.M.
[0111] FIG. 10 shows the apoptosis inducing properties of compound
B. HT-29 colon adenocarcinoma cells were treated with the indicated
concentration of compound B for 48 hours and apoptosis was
determined by the DNA fragmentation assay. The calculated EC.sub.50
value was approximately 175 .mu.M.
3TABLE 3 Apoptosis inducing activity among a series of compounds
Fold induction at 100 .mu.M Reference compounds Indomethacin
<2.0 MY5445 4.7 Sulindac sulfide 7.9 Exisulind <2.0 Test
compounds A <2.0 B 3.4 C 5.6 D <2.0 E 4.6
[0112] In accordance with the protocol of section 4.B., supra, the
compounds A through E were tested for apoptosis inducing, activity,
as reported in Table 3 above. Compounds B, C and E showed
significant apoptotic inducing activity, graeter than 2.0 fold, at
a dosage of 100 .mu.M. Of these three compounds, at this dosage
only B and E did not inhibit COX and inhibited PDE5.
[0113] The apoptosis inducing activity for a series of
phosphodiesterase inhibitors was determined. The data are shown in
Table 4 below. HT-29 cell were treated for 6 days with various
inhibitors of phospohodiesterase. Apoptosis and necrosis were
determined morphologically after acridine orange and ethidium
bromide labelling in accordance with the assay of section 4.A.,
supra. The data show that PDE5 is useful for screening compounds
that induce apoptosis of HT-29 cells.
4TABLE 4 Apoptosis Inducing Data for PDE Inhibitors Inhibitor
Reported Selectivity % Apoptosis % Necrosis Vehicle 8 6
8-methoxy-IBMX PDE1 2 1 Milrinone PDE3 18 0 RO-20-1724 PDE4 11 2
MY5445 PDE5 80 5 IBMX Non-selective 4 13
EXAMPLE 4
Growth Inhibition Assay
[0114] Reference compounds and test compounds were analyzed for
their PDE5 inhibitory activity in accordance with the protocol for
the assay of section 3.A., supra. FIG. 11 shows the inhibitory
effect of various concentrations of sulindac sulfide and exisulind
on the growth of HT-29 cells. HT-29 cells were treated for six days
with various doses of exisulind (triangles) or sulfide (squares) as
indicated. Cell number was measured by a sulforhodamine assay as
previously described (Piazza et al., Cancer Research, 55:
3110-3116, 1995). The IC.sub.50 value for the sulfide was
approximately 45 .mu.M and 200 .mu.M for the sulfone. The data
shows that both sulindac sulfide and exisulind are capable of
inhibiting tumor cell growth.
[0115] FIG. 12 shows the growth inhibitory and apoptosis-inducing
activity of sulindac sulfide. A time course experiment is shown
involving HT-29 cells treated with either vehicle, 0.1% DMSO (open
symbols) or sulindac sulfide, 120 .mu.M (closed symbols). Growth
inhibition (top) was measured by counting viable cells after trypan
blue staining. Apoptosis (bottom) was measured by morphological
determination following staining with acridine orange and ethidium
bromide as described previously (Duke and Cohen, In: Current
Protocols in Immunology, 3.17.1-3.17.16, New York, John Wiley and
Sons. 1992). The data demonstrate that sulindac sulfide is capable
of inhibiting tumor cell growth and that the effect is accompanied
by an increase In apoptosis. All data were collected from the same
experiment.
[0116] FIG. 13 shows the growth inhibitory activity of test
compound E. HT-29 colon adenocarcinoma cells were treated with the
indicated concentration of compound E for six days and cell number
was determined by the SRB assay. The calculated IC.sub.50 value was
0.04 .mu.M.
5TABLE 5 Growth inhibitory activity among a series of compounds %
Inhibition at 100 .mu.M Reference compounds Indomethacin 75 MY5445
88 Sulindac sulfide 88 Exisulind <50 Test compounds A 68 B 77 C
80 D 78 E 62
[0117] In accordance with the screening protocol of section 3.A.,
supra, compounds A through B were tested for growth inhibitory
activity, as reported in Table 5 above. All the test compounds
showed activity exceeding the benchmark exisulind at a 100 .mu.M
single does rest.
[0118] The growth inhibitory activity for a series of
phosphodiesterase inhibitors was determined. The data are shown in
Table 6 below. HT-29 cell were treated for 6 days with various
inhibitors of phospohodiesterase. Cell growth was determined by the
SRB assay in accordance with section 3.A., supra. The data show
that inhibitors of PDE5 were effective for inhibiting tumor cell
growth.
6TABLE 6 Growth Inhibitory Data for PDE Inhibitors Growth
inhibition Inhibitor Reported Selectivity (IC.sub.50, .mu.M)
8-methoxy-IBMX PDE1 >200 .mu.M Milrinone PDE3 >200 .mu.M
RO-20-1724 PDE4 >200 .mu.M MY5445 PDE5 5 .mu.M IBMX
Non-selective >100 .mu.M
[0119] To show the effectiveness of this screening method on
various forms of neoplasia, compounds were tested on numerous cell
lines. The effects of sulindac sulfide and exisulind on various
cell lines was determined. The data is shown in table 7 below. The
IC.sub.50 values were determined by the SRB assay. The data shows
the broad effectiveness of these compounds on a broad range of
neoplasia, with effectiveness at comparable dose range. Therefore,
compounds identified by this invention should be useful for
treating multiple forms of neoplasia.
7TABLE 7 Growth Inhibitory Data of Various Cell Lines Cell Type/
IC.sub.50 (.mu.M) Tissue specificity Sulindac sulfide Exisulind
HT-29, Colon 60 120 HCT116, Colon 45 90 MCF7/S, Breast 30 90
UACC375, Melanoma 50 100 A-427, Lung 90 130 Bronchial Epithelial
Cells (normal) 30 90 NRK, Kidney (normal) 50 180 KNRK, Kidney
(transformed) 60 240 Human Prostate Carcinoma PC3 82
EXAMPLE 5
Activity in Mammary Gland Organ Culture Model
[0120] FIG. 14 shows the inhibition of premalignant lesions in
mammary gland organ culture by sulindac metabolites. Mammary gland
organ culture experiment were performed as previously described
(Mehta and Moon, Cancer Research, 46: 5832-5835, 1986). The results
demonstrate that sulindac and exisulind effectively inhibit the
formation of premalignant lesions, while sulindac sulfide was
inactive. The data support the hypothesis that cyclooxygenase
inhibition is not necessary for the anti-neoplastic properties of
desired compounds.
ANALYSIS
[0121] To identify compounds that have potential use for treating
neoplasia, this invention provides a rationale for comparing
experimental data of test compounds from several protocols. Within
the framework of this invention, test compounds can be ranked
according to their potential use for treating neoplasia in humans.
Those compounds having desirable effects may be selected for more
expensive and time consuming animal studies that are required to
get approval before initiating human clinical trials.
[0122] Qualitative data of various test compounds and the several
protocols are shown in Table 8 below. The data show that exisulind,
compound B and compound E exhibit the appropriate activity to pass
the screen of four assays: lack of COX inhibition, PDE inhibition,
growth inhibition and apoptosis induction. The activity of these
compounds in the mammary gland organ culture validates the
effectiveness of this invention. The qualitative valuations of the
screening protocols rank compound E best, then compound B and then
exisulind.
8TABLE 8 Activity Profile of Various Compounds Mammary COX PDE5
Growth Gland Organ Compound Inhibition Inhibition Inhibition
Apoptosis Culture Exisulind - ++ ++ ++ +++ Sulindac sulfide ++++
+++ +++ +++ - MY5445 ++++ +++ +++ +++ + A - - +++ ++ ++ B - +++ +++
+++ ++ D - - ++ - - E - ++++ ++++ ++++ ++++ F - - ++ + - G - - +++
++ +++ H - - ++ - - Table 8 Code: Activity of compounds based on
evaluating a series of experiments involving tests for maximal
activity and potency. -Not active +Slightly active ++Moderately
active +++Strongly active ++++Highest activity ever recorded
[0123] Obviously, numerous modifications and variations of the
present invention are possible in light of the above teachings. It
is therefore to be understood that within the scope of the appended
claims, the invention may be practiced otherwise than as
specifically described herein.
* * * * *