U.S. patent application number 10/252983 was filed with the patent office on 2003-10-09 for methods for identifying compounds for inhibition of neoplastic lesions, and pharmacetical compositions containing such compounds.
Invention is credited to Pamukcu, Rifat, Piazza, Gary A..
Application Number | 20030190686 10/252983 |
Document ID | / |
Family ID | 26724247 |
Filed Date | 2003-10-09 |
United States Patent
Application |
20030190686 |
Kind Code |
A1 |
Pamukcu, Rifat ; et
al. |
October 9, 2003 |
Methods for identifying compounds for inhibition of neoplastic
lesions, and pharmacetical compositions containing such
compounds
Abstract
This invention provides pharmaceutical compositions containing
compounds 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 prefereably not substantial
prostaglandin inhibitory activity, are desirable for the treatment
of neoplasia.
Inventors: |
Pamukcu, Rifat;
(Springhouse, PA) ; Piazza, Gary A.; (Doylestown,
PA) |
Correspondence
Address: |
Cell Pathways, Inc.
702 Electronic Drive
Horsham
PA
19044
US
|
Family ID: |
26724247 |
Appl. No.: |
10/252983 |
Filed: |
September 24, 2002 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10252983 |
Sep 24, 2002 |
|
|
|
09414625 |
Oct 8, 1999 |
|
|
|
6500610 |
|
|
|
|
09414625 |
Oct 8, 1999 |
|
|
|
09046739 |
Mar 24, 1998 |
|
|
|
09046739 |
Mar 24, 1998 |
|
|
|
08866027 |
May 30, 1997 |
|
|
|
5858694 |
|
|
|
|
Current U.S.
Class: |
435/7.23 ;
514/1 |
Current CPC
Class: |
G01N 33/5011 20130101;
C12Q 1/44 20130101; A61P 35/00 20180101; C12Q 1/533 20130101; C12Q
1/26 20130101 |
Class at
Publication: |
435/7.23 ;
514/1 |
International
Class: |
A61K 031/00; G01N
033/574 |
Claims
We claim:
1 A method of treating neoplasia in a patient, comprising
administering to the patient a pharmacologically effective amount
of a compound selected by: determining COX inhibitory activity of
the compound, and determining PDE-5 inhibitory activity of said
compound, wherein the compound that is selected for administration
to the patient inhibits COX activity no more than 25% at a
concentration of 100 uM and inhibits PDE-5 activity to a greater
extent than exisulind, with the proviso that the compound is not
sulindac or sulindac sulfide; and providing written material to the
patient describing said compound as having PDE-5 inhibitory
activity.
2. A method for treating neoplasia in a patient, wherein said
neoplasia is selected from the group consisting of colo-rectal,
breast and prostate neoplasia, comprising administering to a
patient a pharmacologically effective amount of a compound selected
by: determining COX inhibitory activity of the compound, and
determining PDE-5 inhibitory activity of said compound, wherein the
compound that is selected for administration to the patient
inhibits COX activity no more than 25% at a concentration of 100 uM
and inhibits PDE-5 activity to a greater extent than exisulind,
with the proviso that the compound is not sulindac or sulindac
sulfide.; and providing written material to the patient describing
said compound as having PDE-5 inhibitory activity.
Description
BACKGROUND OF THE INVENTION
[0001] This application claims priority under 35 U.S.C. .sctn.120
to U.S. patent application Ser. Nos. 09/414,625, 08/866,027 and
09/046,739, filed Oct. 8, 1999, May 30, 1997 and Mar. 24, 1998,
respectively.
[0002] This invention relates to the use of one or more forms of
phosphodiesterase type 2 ("PDE2") and phosphodiesterase type 5
("PDE5") and/or protein kinase G to identify compounds useful for
the treatment and prevention of pre-cancerous and cancerous lesions
in mammals, and to pharmaceutical compositions containing such
compounds, as well as to therapeutic methods of treating neoplasia
with such compounds.
[0003] Currently, non-surgical cancer treatment involves
administering one or more highly toxic chemotherapeutics or
hormonal therapies to the patient after her cancer has progressed
to a point where the therapeutic benefits of chemotherapy/hormonal
outweigh its very serious side effects. Such side effects are well
known to any oncologist, and vary from drug to drug. However,
standard chemotherapeutics are typically used only for short
periods of time, often alternating chemotherapy with periods off
treatment, so as not to overwhelm the patient with drug side
effects. Thus, given the risk-benefit trade-off, side effects
typically preclude starting chemotherapy when patients exhibit
precancerous lesions, or continuing chemotherapy or hormonal
therapy on a chronic basis after frank cancer has been eliminated
in an attempt to prevent its re-occurrence.
[0004] Beginning a decade or so ago, a glimmer of hope began to
appear from an unexpected source: non-steroidal anti-inflammatory
drugs ("NSAIDs"). Cancer and precancer research is replete with
publications that describe various biochemical molecules that are
over-expressed in neoplastic tissue, leading one group after
another to research whether specific over-expressed molecules are
responsible for the disease, and whether, if such over-expression
were inhibited, neoplasia could be alleviated. For example, in
familial adenomatous polyposis ("FAP"), Waddell in 1983 (Waddell,
W. R. et al., "Sulindac for Polyposis of the Colon," Journal of
Surgical Oncology, 24:83-87, 1983) hypothesized that since
prostaglandins were over-expressed in such polyps, non-steroidal
anti-inflammatory drugs ("NSAIDs") should alleviate the condition
because NSAIDs inhibited prostaglandin synthetase (PGE.sub.2)
activity. Thus, he administered the nonsteroidal anti-inflammatory
drug ("NSAID") sulindac (an inhibitor of PGE.sub.2) to several FAP
patients. Waddell discovered that polyps regressed and did not
recur upon such therapy. PGE.sub.2 inhibition results from the
inhibition of cyclooxygenase (COX) by NSAIDs. The success by
Waddell with sulindac and the PGE.sub.2/COX relationship seemingly
confirmed the role of two other biochemical targets--PGE.sub.2 and
COX--in carcinogenesis, and the subsequent literature reinforced
these views.
[0005] The glimmer of hope for patients suffering from neoplasia
was that sulindac certainly exhibited far fewer side effects than
conventional chemotherapeutics or hormonals, and opened up the
possibility of treating cancer at earlier stages of the disease,
and for longer periods of time as compared with conventional
chemotherapeutics. However, such a hope had to be tempered with the
open question of whether a compound such as sulindac could be used
to treat frank cancer, given that Waddell had only administered
sulindac to patients with a pre-cancerous condition, FAP.
[0006] That hope was also tempered by NSAIDs own sets of side
effects. Sulindac and other NSAIDs when chronically administered,
aggravate the digestive tract where PGE.sub.2 plays a protective
role. In addition, when taken chronically, they exhibit side
effects involving the kidney and interference with normal blood
clotting. As Waddell unfortunately experienced, some of his
sulindac patients stopped taking drug because of side effects (see
Waddell, W. R. et al., "Sulindac for Polyposis of the Colon," The
American Journal of Surgery, 157: 175-79, 1989), most likely
returning to additional surgical interventions to control polyp
formation. Thus, for neoplasia patients, such drugs are not a
practical chronic treatment, e.g., for FAP, sporadic polyps or men
post-prostatectomy with rising PSAs (a rising PSA in such men
indicates the recurrence of disease, which may not yet present as a
frank, visible cancer). These side effects also limit NSAIDs' use
for any other neoplasia indication requiring long-term drug
administration. More recently, some have suggested that the COX-2
specific NSAIDs such as celecoxib be used. However, zthe renal and
other side effects of such compounds are believed to limit the
dosing and length of treatment with such compounds for long-term
anti-neoplastic indications. In addition, recently published data
indicate that very high doses are needed for drugs like celecoxib
to achieve a marginal effect on colon polyps in only pre-defined
regions of the colorectum. Perhaps more significant to colon cancer
treatment is that it has been reported that certain colonic
neoplasias (e.g., HCT-116) do not express COX-2, and that such
inhibitors are ineffective against such neoplasias (see, Sheng, et
al., "Inhibition of Human Colon Cancer Cell Growth By Selective
Inhibition of Cyclooxygenase-2," J. Clin. Invest., 99(9):2254-9,
1997).
[0007] Recent discoveries have lead scientists away from the
COX/PGE.sub.2 targets, since those targets may not be the primary
(or perhaps even secondary targets) to treat neoplasia patients
successfully on a chronic basis. Pamukcu et al., in U.S. Pat. No.
5,401,774, disclosed that sulfonyl compounds, that have been
reported to be practically devoid of PGE2 and COX inhibition (and
therefore not NSAIDs or anti-inflammatory compounds) 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 human clinical
trials with FAP patients, and even more remarkably has shown effect
in a frank cancer: prostate cancer itself, in a controlled clinical
study presented below. Furthermore, very recent research has
convincingly established that COX I and/or COX II are not expressed
substantially in all neoplasias, diminishing the hope that a COX I
or COX II specific inhibitor would be broadly therapeutically
useful in neoplasia treatment (see, Lim et al., "Sulindac
Derivatives Inhibit Growth and Induce Apoptosis in Human Prostate
Cancer Cell Lines," Biochem. Pharmacology, Vol. 58, pp. 1097-1107
(1999) in press).
[0008] Thus, like so many other proteins over-expressed in
neoplasias, PGE.sub.2/COX over-expression may not be a cause of
some neoplasias, rather a consequence of some of them. But the
combination of such discoveries, however, has raised the question
about how do compounds such as exisulind (that have a range of
activity against both COX and non-COX expressing neoplasias) act?
What do such compounds do to neoplastic cells?
[0009] Piazza, et al. (in U.S. patent application Ser. Nos.
08/866,027 and 09/046,739) discovered that compounds (such as
exisulind) inhibited cyclic-specific GMP phosphodiesterase (e.g.,
PDE5), and that other such compounds could be screened using that
enzyme, which could lead to the discovery of still other compounds
that could be developed and formulated into anti-neoplastic
pharmaceutical compositions. Such pharmaceutical compositions can
be highly anti-neoplastic, and can be practically devoid of side
effects associated with conventional chemotherapeutics, or even the
side effects of COX or PGE2 inhibition, if one wanted to avoid such
side effects. In addition, anti-neoplastic cGMP-specific
PDE-inhibiting compounds can induce apoptosis (a form of programmed
cell death or suicide) in neoplastic cells, but not in normal
cells. Thus, such new compounds have become referred to as a new
class of antineoplastics known as selective apoptotic
anti-neoplastic drugs ("SAANDs"). Accordingly, SAANDs have
challenged several matters of conventional wisdom: (1) that
anti-neoplastic compounds cannot be effective without also killing
normal cells; (2) that COX's are responsible for neoplasia; and (3)
that prevention of colonic neoplasia by NSAIDs is likely mediated
by the inhibition of one or both types of COX.
[0010] New research presented below has, however, shown that not
all compounds exhibiting classic PDE5 inhibition induce apoptosis
in neoplastic cells. For example, the well-known PDE5 inhibitors,
zaprinast and sildenafil, do not singly induce apoptosis, or even
inhibit neoplastic cell growth in our hands. However, because
pro-apoptotic PDE5 inhibitors induced apoptosis selectively (i.e.,
in neoplastic but not in normal cells), and could do so without
substantial COX inhibition, the usefulness of PDE5 as a screening
tool for desirable anti-neoplastic compounds is unquestioned.
[0011] However, an enhancement to the PDE5 screening method to find
anti-neoplastic, pro-apoptotic but safe compounds is desirable so
that new pharmaceutical compositions can be formulated for
therapeutic use in the treatment of neoplasia, including pre-cancer
and cancer.
SUMMARY OF THE INVENTION
[0012] In the course of researching why some PDE5 inhibitors singly
induced apoptosis while others did not, we uncovered a form of
cyclic GMP-specific phosphodiesterase activity, not previously
described. This new phosphodiesterase activity was previously
uncharacterized. Without being limited to a specific theory, we
believe this novel PDE activity may be a novel conformation of PDE2
that substantially lacks cAMP-hydrolyzing activity, i.e. it is
cGMP-specific. Classic PDE2 is not cGMP-specific (it also
hydrolyzes cAMP), classic PDE2 is also found in neoplastic cells.
This new PDE and PDE2 are useful in screening pharmaceutical
compounds for desirable anti-neoplastic properties. Basically, in
neoplastic cells when PDE5 and the PDE2 activity (in its novel and
conventional conformations) are inhibited by an anti-neoplastic
PDE5-inhibiting compound, the result is apoptosis. When only PDE5
is inhibited (but not the several forms of PDE2), apoptosis does
not occur.
[0013] In its broadest aspects, this new PDE conformation has
activity characterized by:
[0014] (a) cGMP specificity over cAMP
[0015] (b) positive cooperative kinetic behavior in the presence of
cGMP substrate;
[0016] (c) submicromolar affinity for cGMP; and
[0017] (d) insensitivity to incubation with purified cGMP-dependent
protein kinase
[0018] Other characteristics of this novel PDE include: it has
reduced sensitivity to inhibition by zaprinast and E4021, it can be
separated from classical PDE5 activity by anion-exchange
chromatography, it is not activated by calcium/calmodulin, and it
is insensitive to rolipram, vinpocetine and indolidan.
[0019] Another embodiment of this invention involves evaluating
whether a compound causes an increase in cGMP-dependent protein
kinase G ("PKG") activity and/or a decrease of .beta.-catenin in
neoplastic cells. It has been found that unexpected characteristics
of SAANDs include the elevation of PKG activity and a decrease in
.beta.-catenin in neoplastic cells exposed to a SAAND. We believe
that the elevation of PKG activity is due at least in part by the
increase in cGMP caused by SAANDs inhibition of the appropriate
PDEs, as described above. The other characteristics of SAANDs are
(1) inhibition of PDE5 as reported in the '694 patent above, (2)
inhibition of the novel cGMP-specific PDE conformation, (3)
inhibition of PDE2; (4) the fact that they increase intracellular
cGMP in neoplastic cells, and (5) the fact that they decrease cAMP
levels in some types of neoplastic cells.
[0020] Thus, one embodiment of the novel method of this invention
is evaluating whether a compound causes PKG activity to elevate in
neoplastic cells and whether that compound inhibits PDE5. Another
embodiment of the novel screening method of this invention is
evaluating whether a compound that causes PKG activity to elevate
in neoplastic cells and whether that compound inhibits the novel
cGMP-specific PDE described above and/or PDE2. Still a third
embodiment is evaluating whether a compound causes PKG activity to
elevate in neoplastic cells and whether that compound causes cGMP
to rise in neoplastic cells and/or causes cAMP levels to fall.
Compounds successfully evaluated in such fashions have application
as SAANDs.
[0021] Among other things, this invention relates to novel in vitro
and in vivo methods for selecting compounds for their ability to
treat and prevent neoplasia, especially pre-cancerous lesions,
safely. In particular, the present invention is a method for
selecting compounds that can be used to treat and prevent
neoplasia, including precancerous lesions. The compounds so
identified can have minimal side effects attributable to COX
inhibition and other non-specific interactions associated with
conventional chemotherapeutics. The compounds of interest can be
tested by exposing the novel PDE described above to the compounds,
and if a compound inhibits this novel PDE, the compound is then
further evaluated (e.g., in vitro or in vivo animal or human
testing models or trials) for its anti-neoplastic properties.
[0022] One aspect of this invention, therefore, involves a
screening/selection method to identify a compound effective for
treating neoplasia that includes ascertaining the compound's
inhibition of this novel PDE and/or PDE2 and its inhibition of COX.
Preferably, the screening and selection methods of this invention
further include determining whether the compound inhibits the
growth of tumor cells in vitro or in vivo.
[0023] By selecting compounds in this fashion, potentially
beneficial and improved compounds for treating neoplasia can be
identified more rapidly and with greater precision than possible in
the past for the purposes of developing pharmaceutical compositions
and therapeutically treating neoplasia. Further benefits will be
apparent from the following detailed description.
[0024] This invention also includes pharmaceutical compositions
containing such compounds, as well as therapeutic methods involving
such compounds.
BRIEF DESCRIPTION OF THE DRAWINGS
[0025] FIG. 1 is a graph of the cGMP activities of the cGMP
phosphodiesterases obtained from SW480 neoplastic cells, as assayed
from the eluent from a DEAE-Trisacryl M column.
[0026] FIG. 2 is a graph of cGMP activities of the reloaded cGMP
phosphodiesterases obtained from SW480 neoplastic cells, as assayed
from the eluent from a DEAE-Trisacryl M column.
[0027] FIG. 3 is a graph of the kinetic behavior of the novel PDE
of this invention.
[0028] FIG. 4 illustrates the effect of the sulfide derivative of
sulindac and the sulfone derivative of sulindac (a.k.a. exisulind)
on purified cyclooxygenase activity.
[0029] FIG. 5 illustrates the effects of test compounds B and E on
COX inhibition.
[0030] FIG. 6 illustrates the inhibitory effects of sulindac
sulfide and exisulind on PDE4 and PDE5 purified from cultured tumor
cells.
[0031] FIG. 7 illustrates the effects of sulindac sulfide on cyclic
nucleotide levels in HT-29 cells.
[0032] FIG. 8 illustrates the phosphodiesterase inhibitory activity
of compound B.
[0033] FIG. 9 illustrates the phosphodiesterase inhibitory activity
of compound E.
[0034] FIG. 10 illustrates the effects of sulindac sulfide and
exisulind on apoptosis and necrosis of HT-29 cells.
[0035] FIG. 11 illustrates the effects of sulindac sulfide and
exisulind on HT-29 cell growth inhibition and apoptosis induction
as determined by DNA fragmentation.
[0036] FIG. 12 illustrates the apoptosis-inducing properties of
compound E.
[0037] FIG. 13 illustrates the apoptosis-inducing properties of
compound B.
[0038] FIG. 14 illustrates the effects of sulindac sulfide and
exisulind on tumor cell growth.
[0039] FIG. 15 illustrates the growth inhibitory and
apoptosis-inducing activity of sulindac sulfide and control
(DMSO).
[0040] FIG. 16 illustrates the growth inhibitory activity of
compound E.
[0041] FIG. 17 illustrates the inhibition of pre-malignant,
neoplastic lesions in mouse mammary gland organ culture by sulindac
metabolites.
[0042] FIG. 18A is a SDS protein gel of SW480 cell lysates from
drug-treated cell lysates in the absence of added cGMP, where cells
were treated in culture for 48 hours with DMSO (0.03%, lanes 1 and
2), exisulind (200, 400 and 600 .mu.M; lanes 3, 4, 5) and E4021
(0.1, 1 and 10 .mu.M, lanes 6, 7, 8).
[0043] FIG. 18B is a SDS (X-ray film exposure) gel PKG assay of
SW480 cell lysates from drug-treated cell lysates in the presence
of added cGMP, where cells were treated in culture for 48 hours
with DMSO (0.03%, lanes 1 and 2), exisulind (200, 400 and 600
.mu.M; lanes 3, 4, 5) and E4021 (0.1, 1 and 10 .mu.M, lanes 6, 7,
8).
[0044] FIG. 19 is a bar graph of the results of Western blot
experiments of the effects of exisulind on .beta.-catenin and PKG
levels in neoplastic cells relative to control.
[0045] FIG. 20 is a graph of the cGMP activities of the cGMP
phosphodiesterases obtained from HTB-26 neoplastic cells, as
assayed from the eluent from a DEAE-Trisacryl M column.
[0046] FIG. 21 is a graph of the cGMP activities of the cGMP
phosphodiesterases obtained from HTB-26 neoplastic cells, as
assayed from the eluent from a DEAE-Trisacryl M column with low and
high substrate concentration.
[0047] FIG. 22 is a graph of the cGMP activities of the cGMP
phosphodiesterases obtained from LnCAP neoplastic cells, as assayed
from the eluent from a DEAE-Trisacryl M column
[0048] FIG. 23 is a graph of the cGMP activities of the cGMP
phosphodiesterases obtained from LnCAP neoplastic cells, as assayed
from the eluent from a DEAE-Trisacryl M column with low and high
substrate concentration.
[0049] FIG. 24 is a bar graph illustrating the specificity binding
of the non-catalytic cGMP binding sites of PDE5 for cyclic
nucleotide analogs and selected PDE5 inhibitors.
[0050] FIG. 25 is a graph of the cGMP activities of the cGMP
phosphodiesterases obtained from SW480 neoplastic cells, as assayed
from the eluent from a DEAE-Trisacryl M column using ethylene
glycol in the buffer.
[0051] FIG. 26 is a graph of the cGMP activities of the cGMP
phosphodiesterases obtained from SW480 neoplastic cells grown in
roller bottles, as assayed from the eluent from a DEAE-Trisacryl M
column.
[0052] FIG. 27A shows a time-dependent increase in the amount of
histone-associated fragmented DNA in LNCaP cell cultures following
treatment with 50 .mu.M Compound I.
[0053] FIG. 27B shows the course of treatment of PrEC prostate
cells with Compound I (50 .mu.M) that did not affect DNA
fragmentation for up to 4 days of treatment.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0054] I. The Novel cGMP-Speciflc Phosphodiesterase and PDE2 from
Neoplastic Cells
[0055] A. The Isolation of the Novel PDE Conformation
[0056] The isolated cGMP-specific phosphodiesterase (which appears
to be a novel conformation of PDE2) was first prepared from the
human carcinoma cell line commonly referred to as SW480 available
from the American Tissue Type Collection in Rockville, Md., U.S.A.
SW480 is a human colon cancer cell line that originated from
moderately differentiated epithelial adenocarcinoma. As discussed
below, a similar conformation has also been isolated from
neoplasias of the breast (i.e., HTB-26 cell line) and prostate
(i.e., LNCAP cell line).
[0057] By "isolated" we mean (as is understood in the art) not only
isolated from neoplastic cells, but also made by recombinant
methods (e.g., expressed in a bacterial or other non-human host
vector cell lines). However, we presently believe isolation from
the human neoplastic cell line is preferable since we believe that
the target protein so isolated has a structure (i.e., a
conformation or topography) that is closer to, if not identical
with, one of the native conformations in the neoplastic cell as
possible. This conformation assists in the selection of
anti-neoplastic compounds that will inhibit the target enzyme(s) in
vivo.
[0058] The novel PDE activity was first found in SW480 colon cancer
cell lines. To isolate the novel phosphodiesterase from SW480,
approximately four hundred million SW480 cells were grown to
confluence in and were scraped from 150 cm.sup.2 tissue culture
dishes after two washes with 10 mL cold PBS and pelleted by
centrifugation. The cells were re-suspended in homogenization
buffer (20 mL TMPI-EDTA-Triton pH 7.4: 20 mM Tris-HOAc, 5 mM
MgAC.sub.2, 0.1 mM EDTA, 0.8% Triton-100, 10 .mu.M benzamidine, 10
.mu.M TLCK, 2000 U/mL aprotinin, 2 .mu.M leupeptin, 2 .mu.M
pepstatin A) and homogenized on an ice bath using a polytron
tissumizer (three times, 20 seconds/pulse). The homogenized
material was centrifuged at 105,000 g for 60 minutes at 4.degree.
C. in a Beckman L8 ultracentrifuge, and the supernatant was diluted
with TMPI-EDTA (60 mL) and applied to a 10-milliliter
DEAE-Trisacryl M column pre-equilibrated with TMPI-EDTA buffer. The
loaded column was washed with 60 mL of TM-EDTA, and PDE activities
were eluted with a 120 mL linear gradient of NaOAC (0-0.5 M) in
TM-EDTA, at a flow rate of 0.95 mL/minute, 1.4 mL/fraction. Eighty
fractions were collected and assayed for cGMP hydrolysis
immediately (i.e. within minutes). FIG. 1. shows the column's
elution profile, revealing two initial peaks of cGMP PDE activity,
peaks A and B, which were eluted by 40-50 mM and 70-80 mM NaOAC,
respectively. As explained below, peak A is PDE5, whereas peak B is
a novel cGMP-specific phosphodiesterase activity.
[0059] Cyclic nucleotide PDE activity of each fraction was
determined using the modified two-step radio-isotopic method of
Thompson et al. (Thompson W. J., et al., Adv. Cyclic Nucleotide
Res. 10: 69-92, 1979), as further described below. The reaction was
in 400 .mu.l containing Tris-HCl (40 mM; pH 8.0), MgCl.sub.2 (5
mM), 2-mercaptoethanol (4 mM), bovine serum albumin (30 .mu.g),
cGMP (0.25 .mu.M-5 .mu.M) with constant tritiated substrate
(200,000 cpm). The incubation time was adjusted to give less than
15% hydrolysis. The mixture was incubated at 30.degree. C. followed
by boiling for 45 seconds to stop the reaction. Then, the mixture
was cooled, snake venom (50.mu.g) added, and the mixture was
incubated at 30.degree. C. for 10 minutes. MeOH (1 mL) was added to
stop the reaction, and the mixture was transferred to an
anion-exchange column (Dowex 1-X8, 0.25 mL resin). The eluent was
combined with a second mL of MeOH, applied to the resin, and after
adding 6 mL scintillation fluid, tritium activity was measured
using a Beckman LS 6500 for one minute.
[0060] To fractionate the cGMP hydrolytic activities of peaks A and
B further, fractions 15 to 30 of the original 80 were reloaded onto
the DEAE-Trisacryl M column and eluted with a linear gradient of
NaOAC (0-0.5 M) in TM-EDTA. Fractions were again immediately
assayed for cGMP hydrolysis (using the procedure described above
with 0.2, 2, 5 .mu.M substrate), the results of which are
graphically presented in FIG. 2. One observation about peak B
illustrated in FIG. 2 is that increasing substrate concentration of
cGMP dramatically enhanced activity when contrasted to peak A.
While this observation is consistent with its being a PDE2, the
fact that the enzyme characterized in FIG. 2 is cGMP-specific (see
below) suggests that it has a novel conformation compared to the
classic PDE2 reported in the literature. Peak A activity shows
apparent substrate saturation of high affinity catalytic sites.
[0061] B. The Isolation of Classic PDE2 From SW480
[0062] Two methods were found that allowed "peak B" to be isolated
from SW480 so that the enzyme had the classical PDE2 activity (i.e.
was not cGMP-specific, but was cGMP stimulated). The first method
involved growing the SW480 in 850 cm.sup.2 Corning roller bottles
instead of 150 cm.sup.2 tissue culture flasks. SW480 were grown in
roller bottles at 0.5 rpm with each bottle containing 200 mL of
RPMI 1640, 2 mM glutamine, and 25 mM HEPES. Cells were harvested by
the following procedure. PBS media was warmed to 37.degree. C. for
at least 15 minutes. 200 mL of 5% FBS/RPMI 1640 complete media is
prepared and 5 mL of glutamine were added. 5 mL of
antibiotic/antimycotic were also added.
[0063] 70 mL of the PBS solution was added to 10 mL of
4.times.Pancreatin. The mixture was maintained at room temperature.
The media was removed and the flask was rinsed with 4 mL of PBS
being sure the bottom of the flask was covered. All solution was
removed with a pipet. 4 mL of diluted Pancreatin was added to the
flask, and the flask was swished to cover its bottom. The flask was
incubated at 37.degree. C. for 8-10 minutes. After the incubation,
the flask was quickly checked under an inverted microscope to make
sure all cells were rounded. The flask was hit carefully on its
side several times to help detach cells. 10 mL of cold complete
media were added to the flask to stop the Pancreatin proteolysis.
The solution was swirled over the bottom to collect the cells. The
media was removed using a 25 mL pipet, and the cells placed in 50
mL centrifuge tubes on ice. The tubes were spun at 1000 rpm at
4.degree. C. for 5 minutes in a clinical centrifuge to pellet
cells. The supernatant was poured off and each pellet frozen on
liquid nitrogen for 15 seconds. The harvested cells can be stored
in a -70.degree. C. freezer.
[0064] The PDEs from the harvested SW480 cells were isolated using
a FPLC procedure. A Pharmacia AKTA FPLC was used to control sample
loading and elution on an 18 mL DEAE TrisAcryl M column. About 600
million cells of SW480 were used for the profiles. After
re-suspending cells in homogenization buffer (20 mL
TMPI-EDTA-Triton pH 7.4: 20 mM Tris-HOAc, 5 mM MgAc.sub.2, 0.1 mM
EDTA, 0.8% Triton-100, 10 .mu.M benzamidine, 10 .mu.M TLCK, 2000
U/mL aprotinin, 2 .mu.M leupeptin, 2 .mu.M pepstatin A), samples
were manually homogenized. FPLC buffer A was 8 mM TRIS-acetate, 5
mM Mg acetate, 0.1 mM EDTA, pH 7.5 and buffer B was 8 mM
TRIS-acetate, 5 mM Mg acetate, 0.1 mM EDTA, 1 M Na acetate, pH 7.5.
Supernatants were loaded onto the column at 1 mL per minute,
followed by a wash with 60 mL buffer A at 1 mL per minute. A
gradient was run from 0-15% buffer B in 60 mL, 15-50% buffer B in
60 mL, and 50-100% buffer B in 16 mL. During the gradient, 1.5 mL
fractions were collected.
[0065] The profile obtained was similar (FIG. 26) to the profile
for the novel PDE activity (see, e.g., FIG. 1) obtained above,
except that Peak B isolated in this manner showed cAMP hydrolytic
activity at 0.25 .mu.M substrate that could be activated 2-3 fold
by 5 .mu.M cGMP.
[0066] A second method used to isolate classic PDE2 from SW480 was
done using a non-FPLC DEAE column procedure described above (see
Section IA) with the modification that the buffers contained 30%
ethylene glycol, 10 mM TLCK and 3.6 mM .beta.-mercaptoethanol. The
addition of these reagents to the buffers causes a shift in the
elution profile (see FIG. 25) from low to high sodium acetate so
that peak A moves from 40 to 150 mM, peak B from 75 to 280 mM and
peak C from 200 to 500 mM Na acetate (see FIG. 25). Peak B in FIG.
25 was assayed with 2 .mu.M cAMP substrate and showed a two-fold
activation by 5 .mu.M cGMP (see Figure -Y). The selective PDE2
inhibitor EHNA inhibited 2 .mu.M cGMP PDE activity in this Peak B
with an IC.sub.50 of 1.6 .mu.M and inhibited 2.0 .mu.M cAMP PDE
activity in Peak B with an IC.sub.50 of 3.8 .mu.M (and IC.sub.50 of
2.5 .mu.M with addition of 10 .mu.M rolipram).
[0067] C. cGMP-Specificity of PDE Peak A and the Novel Peak B
Activity
[0068] Each fraction from the DEAE column from Section IA was also
assayed for cGMP-hydrolysis activity (0.25 .mu.M cGMP) in the
presence or absence of Ca.sup.++, or Ca.sup.++-CaM and/or EGTA and
for cAMP (0.25 .mu.M cAMP) hydrolysis activity in the presence or
absence of 5 .mu.M cGMP. Neither PDE peak A and peak B (fractions
5-22; see FIG. 1) hydrolyzed cAMP significantly, establishing that
neither had the activity of a classic cAMP-hydrolyzing family of
PDE (i.e. a PDE 1, 2, 3).
[0069] Ca.sup.++ (with or without calmodulin) failed to activate
either cAMP or cGMP hydrolysis activity of either peak A or B, and
cGMP failed to activate or inhibit cAMP hydrolysis. Such results
establish that peaks A and B constitute cGMP-specific PDE
activities but not classic or previously known PDE1, PDE2, PDE3 or
PDE4 activities.
[0070] For the novel PDE peak B, as discussed below, cyclic GMP
activated the cGMP hydrolytic activity of the enzyme, but did not
activate any cAMP hydrolytic activity (in contrast with the Peak B
from Section IB above). This reveals that the novel PDE peak B--the
novel phosphodiesterase of this invention--is not a cGMP-stimulated
cAMP hydrolysis ("cGS") or among the classic or previously known
PDE2 family activities because the known isoforms of PDE2 hydrolyze
both cGMP and cAMP.
[0071] D. Peak A is a Classic PDE5, but the Novel Peak B--a New
cGMP-Specific PDE--is Not
[0072] To characterize any PDE isoform, kinetic behavior and
substrate preference should be assessed.
[0073] Peak A showed typical "PDE5" characteristics. For example,
the K.sub.m of the enzyme for cGMP was 1.07 .mu.M, and Vmax was
0.16 mmol/min/mg. In addition, as discussed below, zaprinast
(IC.sub.50=1.37 .mu.M) and E4021 (IC.sub.50=3 nM) and sildenafil
inhibited activity of peak A. Further, zaprinast showed inhibition
for cGMP hydrolysis activity of peak A, consistent with results
reported in the literature.
[0074] PDE Peak B from Section IA showed considerably different
kinetic properties as compared to PDE peak A. For example, in
Eadie-Hofstee plots of Peak A, cyclic GMP hydrolysis shows single
line with negative slope with increasing substrate concentrations,
indicative of Michaelis-Menten kinetic behavior. Peak B, however,
shows the novel property for cGMP hydrolysis in the absence of cAMP
of a decreasing (apparent K.sub.m=8.4), then increasing slope
(K.sub.m<1) of Eadie-Hotfstee plots with increasing cGMP
substrate (see, FIG. 3). Thus, this establishes Peak B's
submicromolar affinity for cGMP (i.e., where K.sub.m<1).
[0075] Consistent with the kinetic studies (i.e., FIG. 3) and
positive-cooperative kinetic behavior in the presence of cGMP
substrate, was the increased cGMP hydrolytic activity in the
presence of increasing concentrations of cGMP substrate. This was
discovered by comparing 0.25 .mu.M, 2 .mu.M and 5 .mu.M
concentrations of cGMP in the presence of PDE peak B after a second
DEAE separation to rule out cAMP hydrolysis and to rule out this
new enzyme being a previously identified PDE5. Higher cGMP
concentrations evoked disproportionately greater cGMP hydrolysis
with PDE peak B, as shown in FIG. 2.
[0076] These observations suggest that cGMP binding to the peak B
enzyme causes a conformational change in the enzyme. This confirms
the advantage of using the native enzyme from neoplastic cells, but
this invention is not limited to the native form of the enzyme
having the characteristics set forth above.
[0077] E. Zaprinast- and Sildenafil-Insensitivity of PDE Peak B
Relative to Peak A, and Their Effects on Other PDE Inhibitors
[0078] Different PDE inhibitors were studied using twelve
concentrations of drug from 0.01 to 100 .mu.M and substrate
concentration of 0.25 .mu.M .sup.3H-cGMP. IC.sub.50 values were
calculated with variable slope, sigmoidal curve fits using Prism
2.01 (GraphPad). The results are shown in Table 1. While compounds
E4021 and zaprinast inhibited peak A, (with high affinities)
IC.sub.50 values calculated against the novel PDE activity in peak
B (Section IA) are significantly increased (>50 fold). This
confirms that peak A is a PDE5. These data further illustrate that
the novel PDE activity of this invention is, for all practical
purposes, zaprinast-insensitive and E4021-insensitive.
1TABLE 1 Comparison of PDE Inhibitors Against Peak A and Section IA
Peak B (cGMP Hydrolysis) IC.sub.50 IC.sub.50 Ratio PDE Family Peak
A Peak B (I.sub.C50 Peak A/ Compound Inhibitor (.mu.M) (.mu.M) Peak
B) E4021 5 0.003 8.4 0.0004 Zaprinast 5 1.4 >30 <0.05
Compound E 5 and others 0.38 0.37 1.0 Sulindac 5 and others 50 50
1.0 sulfide Vinpocetine 1 >100 >100 EHNA 2, 5 >100 3.7
Indolidan 3 31 >100 <0.31 Rolipram 4 >100 >100
Sildenafil 5 .0003 >10 <.00003
[0079] By contrast, sulindac sulfide and Compound E and
competitively inhibited both peaks A and B phosphodiesterases at
the same potency (IC.sub.50=0.38 .mu.M for PDE peak A; 0.37 .mu.M
for PDE peak B).
[0080] There is significance for the treatment of neoplasia and the
selection of useful compounds for such treatment in the fact that
peak B (either form of it) is zaprinast-insensitive whereas peaks A
and B are both sensitive to sulindac sulfide and Compound E. We
have tested zaprinast, E4021 and sildenafil to ascertain whether
they induce apoptosis or inhibit the growth of neoplastic cells,
and have done the same for Compound E. As explained below,
zaprinast by itself does not have significant apoptosis-inducing or
growth-inhibiting properties, whereas sulindac sulfide and Compound
E are precisely the opposite. In other words, the ability of a
compound to inhibit both PDE peaks A and B correlates with its
ability to induce apoptosis in neoplastic cells, whereas if a
compound (e.g., zaprinast) has specificity for PDE peak A only,
that compound will not by itself induce apoptosis.
[0081] F. Insensitivity of the Novel PDE Peak B to Incubation with
cGMP-Dependent Protein Kinase G
[0082] Further differences between PDE peak A and the novel peak B
(Section IA) were observed in their respective cGMP-hydrolytic
activities in the presence of varying concentrations of
cGMP-dependent protein kinase G (which phosphorylates typical
PDE5). Specifically, peak A and peak B fractions from Section IA
were incubated with different concentrations of protein kinase G at
30.degree. C. for 30 minutes. Cyclic GMP hydrolysis of both peaks
has assayed after phosphorylation was attempted. Consistent with
previously published information about PDE5, Peak A showed
increasing cGMP hydrolysis activity in response to protein kinase G
incubation, indicating that Peak A was phosphorylated. Peak B was
unchanged, however (i.e., was not phosphorylated and insensitive to
incubation with cGMP-dependent protein kinase G). These data are
consistent with Peak A being an isoform consistent with the known
PDE5 family and Peak B from Section IA being a novel cGMP-specific
PDE activity.
[0083] G. Novel Peak B in Prostate and Breast Cancer Cell Lines
[0084] The novel Peak B was also isolated from two other neoplastic
cell lines, a breast cancer cell line, HTB-26 and a prostate cancer
cell line, LnCAP by a procedure similar to the one above used to
isolate it from SW480. The protocol was modified in several
respects. To provide even greater reproducibility to allow
comparison of different cell lines, a Pharmacia AKTA FPLC was used
to control sample loading and elution on an 18 mL DEAE TrisAcryl M
column. SW840 was run by this same procedure multiple times to
provide a reference of peak B. 200-400 million cells of SW480 were
used for the profiles. 70 million cells of LnCAP were used for a
profile (see FIGS. 22 and 23), and in a separate experiment 32
million cells of HTB-26 were used for a profile (see FIGS. 20 and
21). After re-suspending cells in homogenization buffer, samples
were manually homogenized. FPLC buffer A was 8 mM TRIS-acetate, 5
mM Mg acetate, 0.1 mM EDTA, pH 7.5 and buffer B was 8 mM
TRIS-acetate, 5 mM Mg acetate, 0.1 mM EDTA, 1 M Na acetate, pH 7.5.
Supernatants were loaded onto the column at 1 mL per minute,
followed by a wash with 60 mL buffer A at 1 mL per minute. A
gradient was run from 0-15% buffer B in 60 mL, 15-50% buffer B in
60 mL, and 50-100% buffer B in 16 mL. During the gradient 1.5 mL
fractions were collected. Peaks of cGMP PDE activity eluted around
fraction 65 that was at 400 mM Na acetate (see FIGS. 20-23). This
activity was measured at 0.25 .mu.M cGMP (indicating submicromolar
affinity for cGMP). Rolipram, a PDE4-specific drug, inhibited most
of the cAMP PDE activity (i.e. the cAMP activity was due to PDE4),
indicating that the peak B's cGMP activity were specific for cGMP
over cAMP. All three peak B's (from SW480, HTB-26, and LnCAP) did
not show stimulation with calcium/calmodulin and were resistant to
100 nM E4021, a specific PDE5-specific inhibitor like zaprinast
(see FIGS. 20 and 22). The peak B's also showed a dramatic increase
in activity when substrate was increased from 0.25 .mu.M to 5 .mu.M
cGMP (suggesting positively cooperative kinetics) (see FIGS. 21 and
23). Also, the three peaks show similar inhibition by exisulind and
Compound I, below.
[0085] II. Protein Kinase G and .beta.-Catenin Involvement--in
General
[0086] A series of experiments were performed to ascertain what
effect, if any, an anti-neoplastic cGMP-specific PDE inhibitor such
as exisulind had on cGMP-dependent protein kinase G ("PKG") in
neoplastic cells containing either the adenomatous polyposis coli
gene ("APC gene") defect or a defect in the gene coding for
.beta.-catenin. As explained below, such an inhibitor causes an
elevation in PKG activity in such neoplastic cells. That increase
in activity was not only due to increased activation of PKG in
cells containing either defect, but also to increased expression of
PKG in cells containing the APC defect. In addition, when PKG from
neoplastic cells with either defect is immunoprecipitated, it
precipitates with .beta.-catenin.
[0087] .beta.-catenin has been implicated in a variety of different
cancers because researchers have found high levels of it in
patients with neoplasias containing mutations in the APC
tumor-suppressing gene. People with mutations in this gene at birth
often develop thousands of small tumors in the lining of their
colon. When it functions properly, the APC gene codes for a normal
APC protein that is believed to bind to and regulate
.beta.-catenin. Thus, the discovery that PKG in neoplastic cells
containing either the APC gene defect or the .beta.-catenin defect
is bound to .beta.-catenin indeed strongly implicates PKG in one of
the major cellular pathways that leads to cancer. In addition,
because of the relationship between cGMP-specific inhibition and
PKG elevation upon treatment with SAANDs links cGMP to the
PKG/.beta.-catenin/APC defect in such cells.
[0088] This latter link is further buttressed by the observation
that .beta.-catenin itself is reduced when neoplastic cells
containing the APC defect or the .beta.-catenin defect are exposed
to a SAAND. This reduction in .beta.-catenin is initiated by PKG
itself. PKG phosphorylates .beta.-catenin--which is another novel
observation associated with this invention. The phosphorylation of
.beta.-catenin allows .beta.-catenin to be degraded by
ubiquitin-proteasomal system.
[0089] This phosphorylation of .beta.-catenin by PKG is important
in neoplastic cells because it circumvents the effect of the APC
and .beta.-catenin mutations. The mutated APC protein affects the
binding of the .beta.-catenin bound to the mutant APC protein,
which change in binding has heretofore been thought to prevent the
phosphorylation of .beta.-catenin by GSK-3b kinase. In the case of
mutant .beta.-catenin, an elevation of PKG activity also allows the
mutant .beta.-catenin to be phosphorylated. By elevating PKG
activity in neoplasia with cGMP-PDE inhibition allows for
.beta.-catenin phosphorylation (leading to its degradation) in
neoplastic cells containing either type of mutation.
[0090] In short, these findings not only lead to new pharmaceutical
screening methods to identify further SAAND candidate compounds,
but also buttress the role of cGMP-specific PDE inhibition in
therapeutic approaches to neoplasia. This observation may also
explain the unexpectedly broad range of neoplasias SAANDs can
inhibit since both neoplasia with and without the APC defect can be
treated, as explained above.
[0091] III. Screening Pharmaceutical Compositions Using the
PDEs
[0092] A. In General
[0093] The novel PDE of this invention and PDE2 are useful with or
without PDE5 to identify compounds that can be used to treat or
prevent neoplasms, and that are not characterized by serious side
effects.
[0094] 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 compound is important to the discovery of a
relevant target for pharmaceutical therapy. Screening assays based
on this technology can be combined with other tests to select
compounds that have growth inhibiting and pro-apoptotic
activity.
[0095] 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, several of the present inventors
unexpectedly discovered compounds that selectively induce apoptosis
without substantial COX inhibition also inhibit PDE5. In
particular, and contrary to leading scientific studies, desirable
compounds for treating neoplastic lesions inhibit PDE5 (EC
3.1.4.17). PDE5 is one of at least ten gene families of
phosphodiesterase. PDE5 and the novel PDE of this invention are
unique in that they selectively degrade cyclic GMP and not cAMP,
while the other families of PDE selectively degrade/hydrolyze cAMP
and not cGMP or non-selectively degrade both cGMP and cAMP.
Preferably, desirable compounds used to treat neoplasia do not
substantially inhibit non-selective or cAMP degrading
phosphodiesterase types.
[0096] B. COX Screening
[0097] A preferred embodiment of the present invention involves
determining the cyclooxygenase inhibition activity of a given
compound, and determining the cGMP specific PDE inhibitory activity
of the compound. The test compounds are assessed for their ability
to treat neoplastic lesions either directly 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 irritation is
5-fluoro-2-methyl-1-(p-methylsulfonylbenzylidene)-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 (cGMP-specific
PDEs, such as 1-(3-chloroanilino)-4-phenyphthalazine
("MY5445").
[0098] 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 keratosis), whether the lesions are
clinically identifiable or not.
[0099] As used herein, the terms "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.
[0100] 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; cyclic nucleotide, RIA
represents--radioimmunoassay.
[0101] COX inhibition by a compound 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, but preferred protocols are set forth below.
[0102] Compounds can be evaluated to determine whether they inhibit
the production of prostaglandin E.sub.2 ("PGE.sub.2"), by measuring
PGE.sub.2. Using an enzyme immunoassay (EIA) kit for PGE.sub.2,
such as commercially available from Amersham, Arlington Heights,
Ill. U.S.A. Suitable cells include those that make an abundance of
PG, such as HL-60 cells. HL-60 cells are human promyelocytes that
are differentiated with DMSO into 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 ATCC
(ATCC:CCL240). They can be grown 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 a concentration of
3.times.10.sup.6 cells/mL.
[0103] The differentiated HL-60 cells (3.times.10.sup.6 cells/mL)
are incubated for 15 minutes 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.-6 M) for 15 minutes.
PGE.sub.2 secreted into the external medium is measured as
described above.
[0104] As indicated above, a second method to assess COX inhibition
of a compound is to measure the COX activity in the presence of a
test compound. Two different forms of cyclooxygenase (COX-I and
COX-2) have been reported in the literature to regulate
prostaglandin synthesis. 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.
[0105] 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 minutes 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. After stopping the reaction by adding
thiobarbituric acid and malonaldehyde, enzymatic activity can then
be measured spectrophotometrically at 530 nm.
[0106] Obviously, a compound that exhibits a lower COX-I or COX-2
inhibitory activity in relation to its greater combined PDE5/novel
PDE/PDE2 inhibitory activities may be a desirable compound.
[0107] The amount of COX inhibition is determined by comparing the
activity of the cyclooxygenase in the presence and absence of the
test compound. Residual (i.e., less than about 25%) or no COX
inhibitory activity at a concentration of about 100 .mu.M is
indicative that the compound should be evaluated further for
usefulness for treating neoplasia.
[0108] C. Determining Phosphodiesterase Inhibition Activity
[0109] Compounds can be screened for inhibitory effect on the
activity of the novel phosphodiesterase of this invention using
either the enzyme isolated as described above, a recombinant
version, or using the novel PDE and/or PDE2 together with PDE5.
Alternatively, cyclic nucleotide levels in whole cells are measured
by RIA and compared to untreated and zaprinast-treated cells.
[0110] 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 the PDE 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 isolated PDE of this invention.
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 minutes 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 1 mL Dowex 1-X8 column; and
washed with 1 mL of 100% methanol. The amount of radioactivity in
the breakthrough and the wash from the column is combined and
measured with a scintillation counter. The degree of
phosphodiesterase 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 but with drug solvent).
[0111] Alternatively, the ability of desirable compounds to inhibit
the phosphodiesterases of this invention is reflected by an
increase in cGMP in neoplastic cells exposed to a compound being
screened. The amount of PDE 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. As indicated above, SW-480
contains both PDE5 and the novel PDE of this invention, so when PDE
activity is evaluated in this fashion, a combined cGMP hydrolytic
activity is assayed simultaneously. 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
methyl 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
Physiol., 72:389-395, 1992, which is incorporated herein by
reference).
[0112] In addition, the culture media may be acidified, frozen
(-70.degree. C.) and also analyzed for cGMP and cAMP.
[0113] In addition to observing increases in the content of cGMP in
neoplastic cells caused by desirable compounds, decreases in
content of cAMP have also 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 cGMP-specific
PDE 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 cAMP
content within 24 hours. The intracellular targets of drug actions
are being studied further, but current data support the concept
that the initial rise in CGMP content and the subsequent fall in
cAMP content precede apoptosis in neoplastic cells exposed to
desirable compounds.
[0114] The change in the ratio of the two cyclic nucleotides may be
a more accurate tool for evaluating desirable cGMP-specific
phosphodiesterase inhibition activity of test compounds, rather
than measuring only the absolute value of cGMP, only cGMP-specific
phosphodiesterase inhibition, or only the level of cGMP hydrolysis.
In neoplastic cells not treated with anti-neoplastic compounds, the
ratio of cGMP content/cAMP 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.
[0115] 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.
[0116] 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.
[0117] Verification of the cyclic nucleotide content may be
obtained by determining the turnover or accumulation of cyclic
nucleotides in intact cells. To measure intact cell cAMP,
.sup.3H-adenine pre-labeling is used according to published
procedures (Whalin, M. E., Garrett Jr., R. L., Thompson, W. J., and
Strada, S. J. "Correlation of cell-free brain cyclic nucleotide
phosphodiesterase activities to cyclic AMP decay in intact brain
slices", Sec. Mess. and Phos. Protein Research, 12:311-325, 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
pre-labeling 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).
[0118] The PDE inhibitory activity effect of a compound can also be
determined from a tissue sample. Tissue biopsies from humans or
tissues from anesthesized animals 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 cGMP-specific inhibition
is determined by assaying for the amount of cyclic nucleotides
using RIA procedures as described above.
[0119] The amount of inhibition is determined by comparing the
activity of the novel PDE (or PDE2) in the presence and absence of
the compound. Inhibition of the novel PDE activity (or PDE2) 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 the novel PDE inhibition should be less than 50 .mu.M for
the compound to be further considered for potential use.
[0120] D. Determining Whether a Compound Reduces Tumor Cell
Growth
[0121] In an alternate embodiment, the 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.
[0122] A compound's ability to inhibit tumor cell growth can be
measured using the HT-29 human colon carcinoma cell line obtained
from ATCC. HT-29 cells have previously been characterized as a
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 bovine calf 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.
[0123] 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.
[0124] 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 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.
[0125] E. Determining Whether a Compound Induces Apoptosis
[0126] 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.
[0127] 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.
[0128] Apoptosis 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 by certain chemotherapeutic drugs.
Inappropriate regulation of apoptosis is thought to play an
important role in many pathological conditions including cancer,
AIDS, or Alzheimer's 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.
[0129] Following treatment with a 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).
[0130] 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 re-suspended 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 for morphological features of
apoptosis.
[0131] Apoptosis can also be quantified by measuring an increase in
DNA fragmentation in cells that 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.
[0132] 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-sulfonate]) as
substrate.
[0133] 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., the cytoplasmic
fraction) are transferred into a 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.
[0134] 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.
[0135] Statistically significant increases in 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.
[0136] F. Mammary Gland Organ Culture Model Tests
[0137] Test compounds identified by the above methods can be tested
for antineoplastic activity by their ability to inhibit the
incidence of pre-neoplastic 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 certain NSAIDs,
retinoids, tamoxifen, selenium, and certain natural products, and
is useful for validation of the screening method of the present
invention.
[0138] 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 added to medium 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 pre-malignant
lesions.
[0139] 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 are 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 to glands without lesions. The incidence of mammary lesions
in test compound treated glands is compared with that of the
untreated glands.
[0140] 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 non-regressed structures is also outlined on the digitization
pad and quantitated by the computer.
EXPERIMENTAL RESULTS
[0141] A number of 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:
[0142]
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;
[0143]
B--(Z)-5-Fluoro-2-methyl-1-(3,4,5-trimethoxybenzylidene)-3-acetic
acid;
[0144] C--(Z)-5-Fluoro-2-methyl-1-(p-chlorobenzylidene)-3-acetic
acid;
[0145]
D--rac-(E)-1-(butan-1',4'-olido)-[3',4':1,2]-6-fluoro-2-methyl-3-(p-
-methylsulfonylbenzylidene)-1S-indanyl-N-acetylcysteine;
[0146]
E--(Z)-5-Fluoro-2-methyl-1-(3,4,5-trimethoxybenzylidene)-3-indenyla-
cetamide, N-benzyl;
[0147]
F--(Z)-5-Fluoro-2-methyl-1-(p-methylsulfonylbenzylidene)-3-indenyla-
cetamide, N,N'-dicyclohexyl;
[0148] G--ribo-(E)-1-Triazolo-[2',3':1",
3"]-1-(butan-1',4'-olido)-[3',4':-
1,2]-6-fluoro-2-methyl-3-(p-methylsulfonylbenzylidene)-indan;
and
[0149]
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
[0150] Reference compounds and test compounds were analyzed for
their COX inhibitory activity in accordance with the protocol for
the COX assay, supra. FIG. 4 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.sub.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 were obtained for the COX-2 isoenzyme
(Thompson, et al., Journal of the National Cancer Institute, 87:
1259-1260, 1995).
[0151] FIG. 5 shows the effect of test compounds B and E on COX
inhibition. COX activity was determined as for the compounds shown
in FIG. 4. The data show that neither test compound B and E
significantly inhibit COX-I.
2TABLE 2 Cyclooxygenase inhibitory activity for 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
[0152] In accordance with the protocol, supra, compounds A through
E were evaluated for COX inhibitory activity as reported in Table 2
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
cGMP PDE Inhibition Assay
[0153] Reference compounds and test compounds were analyzed for
their cGMP PDE inhibitory activity in accordance with the protocol
for the assay described supra. FIG. 6 shows the effect of various
concentrations of sulindac sulfide and exisulind on either PDE4 or
cGMP PDE 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 cGMP PDE was 17 .mu.M. The IC.sub.50
value of exisulind for inhibition of PDE4 was 181 .mu.M, and for
inhibition of cGMP PDE was 56 .mu.M. These data show that both
sulindac sulfide and exisulind inhibit phosphodiesterase activity.
Both compounds show selectivity for the cGMP PDE isoenzyme forms
over PDE4 isoforms.
[0154] FIG. 7 shows the effects of sulindac sulfide on either cGMP
or cAMP production as determined in cultured HT-29 cells in
accordance with the assay described, 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 (FIG. 7A). Levels of cAMP were
unaffected by treatment, although a known PDE4 inhibitor, rolipram,
increased cAMP (FIG. 7B). The data demonstrate the pharmacological
significance of inhibiting cGMP PDE, relative to PDE4.
[0155] FIG. 8 shows the effect of the indicated dose of test
compound B on either cGMP PDE or PDE4 isozymes of
phosphodiesterase. The calculated IC.sub.50 value was 18 .mu.M for
cGMP PDE and was 58 .mu.M for PDE4.
[0156] FIG. 9 shows the effect of the indicated dose of test
compound E on either PDE4 or cGMP PDE. The calculated IC.sub.50
value was 0.08 .mu.M for cGMP PDE and greater than 25 .mu.M for
PDE4.
3TABLE 3 cGMP PDE 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
[0157] The above compounds in Table 3 were evaluated for PDE
inhibitory activity, as described in the protocol 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. 8,
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
[0158] Reference compounds and test compounds were analyzed for
their novel PDE inhibitory activity in accordance with the
protocols for the assay, supra. In accordance with those protocols,
FIG. 10 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 show
that both sulindac sulfide and exisulind are capable of causing
apoptotic cell death without inducing necrosis. All data were
collected from the same experiment.
[0159] FIG. 11 shows the effect of sulindac sulfide and exisulind
on tumor growth inhibition and apoptosis induction as determined by
DNA fragmentation. Top figure (11A); growth inhibition (open
symbols, left axis) and DNA fragmentation (closed symbols, right
axis) by exisulind. Bottom figure (11B); 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 were collected from the same
experiment.
[0160] FIG. 12 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.
[0161] FIG. 13 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.
4TABLE 4 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 E4021
<2.0 Zaprinast <2.0 Sildenafil <2.0 EHNA <2.0 Test
compounds A <2.0 B 3.4 C 5.6 D <2.0 E 4.6
[0162] In accordance with the fold induction protocol, supra, the
compounds A through E were tested for apoptosis inducing activity,
as reported in Table 4 above. Compounds B, C and E showed
significant apoptotic inducing activity, greater 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 but did inhibit cGMP-specific
PDE.
[0163] The apoptosis inducing activity for a series of
phosphodiesterase inhibitors was determined. The data are presented
in Table 5 below. HT-29 cell were treated for 6 days with various
inhibitors of phosphodiesterase. Apoptosis and necrosis were
determined morphologically after acridine orange and ethidium
bromide labeling in accordance with the assay described, supra. The
data show that the novel cGMP-specific PDE is useful for screening
compounds that induce apoptosis of HT-29 cells.
5TABLE 5 Apoptosis-Induction 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
[0164] Reference compounds and test compounds were analyzed for
their PDE5 inhibitory activity in accordance with the protocol for
the assay supra. FIG. 14 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 sulindac 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 sulindac sulfide was
approximately 45 .mu.M and 200 .mu.M for the exisulind. The data
show that both sulindac sulfide and exisulind are capable of
inhibiting tumor cell growth.
[0165] FIG. 15 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 (15A top) was measured by counting viable cells after
trypan blue staining. Apoptosis (15B 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.
[0166] FIG. 16 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.
6TABLE 6 Growth-inhibitory activity among a series of compounds %
Inhibition at 100 .mu.M Reference compounds Indomethacin 75 MY5445
88 Sulindac sulfide 88 Exisulind <50 E4021 <50 sildenafil
<50 zaprinast <50 Test compounds A 68 B 77 C 80 D 78 E 62
[0167] In accordance with the screening protocol of section supra,
compounds A through E were tested for growth inhibitory activity,
as reported in Table 6 above. All the test compounds showed
activity exceeding a 100 .mu.M single dose test.
[0168] The growth inhibitory activity for a series of
phosphodiesterase inhibitors was determined. The data are shown in
Table 7 below. HT-29 cells were treated for 6 days with various
inhibitors of phosphodiesterase. Cell growth was determined by the
SRB assay described, supra. The data below taken with those above
show that inhibitors of the novel PDE were effective for inhibiting
tumor cell growth.
7TABLE 7 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 Zaprinast PDE5 >100 .mu.M Sildenafil
PDE5 >100 .mu.M E4021 PDE5 >100 .mu.M
[0169] 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 were determined. The data are shown in Table 8 below.
The IC.sub.50 values were determined by the SRB assay. The data
show the broad effectiveness of these compounds on a broad range of
neoplasias, with effectiveness at comparable dose range. Therefore,
compounds identified and selected by this invention should be
useful for treating multiple forms of neoplasia.
8TABLE 8 Growth Inhibitory Data of Various Cell Lines IC.sub.50
(.mu.M) Cell Type/ Sulindac Tissue specificity sulfide Exisulind
Compound E* HT-29, Colon 60 120 0.10 HCT116, Colon 45 90 MCF7/S,
Breast 30 90 UACC375, Melanoma 50 100 A-427, Lung 90 130 Bronchial
Epithelial Cells 30 90 NRK, Kidney 50 180 (non ras-transformed)
KNRK, Kidney 60 240 (ras transformed) Human Prostate Carcinoma PC3
82 0.90 Colo 205 1.62 DU-145 0.10 HCT-15 0.60 MDA-MB-231 0.08
MDA-MB-435 0.04 *Determined by neutral red assay as described by
Schmid et al., in Proc. AACR Vol 39, p. 195 (1998).
EXAMPLE 5
Activity in Mammary Gland Organ Culture Model
[0170] FIG. 17 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
[0171] To select compounds 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 additional testing and
subsequent human use.
[0172] Qualitative data of various test compounds and the several
protocols are shown in Table 9 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, and presence of
effective cGMP-specific 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.
9TABLE 9 Activity Profile of Various Compounds Mammary Gland COX
PDE Growth Organ Compound Inhibition Inhibition Inhibition
Apoptosis Culture Exisulind - ++ ++ ++ +++ Sulindac ++++ +++ +++
+++ - sulfide MY5445 - +++ +++ +++ + A - - +++ ++ ++ B - +++ +++
+++ ++ D - - ++ - - E - +++ ++++ ++++ ++++ F - - ++ + - G - - +++
++ +++ H - - ++ - - Table 9 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 ++++ Highly active
[0173] Also disclosed is a novel assay for PKG activity, which is
used in the screening methods of this invention, but also has more
general usefulness in assaying for PKG activity for other purposes
(e.g., for studying the role of PKG in normal cellular function).
For explanation purposes, it is useful to describe the PKG assay
first, before describing how PKG activity can be useful in drug
evaluation in ascertaining whether a compound is potentially useful
in the treatment of neoplasia.
[0174] The Novel PKG Assay
[0175] The novel PKG assay of this invention involves binding to a
solid phase plural amino acid sequences, each of which contain at
least the cGMP binding domain and the phosphorylation site of
phosphodiesterase type 5 ("PDE5"). That sequence is known and
described in the literature below. Preferably, the bound PDE5
sequence does not include the catalytic domain of PDE5 as described
below. One way to bind the PDE5 sequences to a solid phase is to
express those sequences as a fusion protein of the PDE5 sequence
and one member of an amino acid binding pair, and chemically link
the other member of that amino acid binding pair to a solid phase
(e.g., beads). One binding pair that can be used is glutathione
S-transferase ("GST") and glutathione ("GSH"), with the GST being
expressed as a fusion protein with the PDE5 sequence described
above, and the GSH bound covalently to the solid phase. In this
fashion, the PDE5 sequence/GST fusion protein can be bound to a
solid phase simply by passing a solution containing the fusion
protein over the solid phase, as described below.
[0176] RT-PCR method is used to obtain the cGB domain of PDE5 with
forward and reverse primers designed from bovine PDE5A cDNA
sequence (McAllister-Lucas L. M. et al, J. Biol. Chem. 268,
22863-22873, 1993) and the selection among PDE 1-10 families.
5'-3', Inc. kits for total RNA followed by oligo (dT) column
purification of mRNA are used with HT-29 cells. Forward primer
(GAA-TTC-TGT-TAG-AAA-AGC-CAC-CAG-AGA-AAT-G, 203-227) and reverse
primer (CTC-GAG-CTC-TCT-TGT-TTC-TTC-CTC-TGC-TG, 1664-1686) are used
to synthesize the 1484 bp fragment coding for the phosphorylation
site and both low and high affinity cGMP binding sites of human
PDE5A (203-1686 bp, cGB-PDE5). The synthesized cGB-PDE5 nucleotide
fragment codes for 494 amino acids with 97% similarity to bovine
PDE5A. It is then cloned into pGEX-5.times.-3
glutathione-S-transferase (GST) fusion vector (Pharmacia Biotech)
with tac promoter, and EcoRI and XhoI cut sites. The fusion vector
is then transfected into E. Coli BL21 (DE3) bacteria (Invitrogen).
The transfected BL21 bacteria is grown to log phase and then IPTG
is added as an inducer. The induction is carried at 20.degree. C.
for 24 hrs. The bacteria are harvested and lysated. The soluble
cell lysate is incubated with GSH conjugated Sepharose 4B
(GSH-Sepharose 4B). The GST-cGB-PDE5 fusion protein can bind to the
GSH-Sepharose beads and the other proteins are washed off from
beads with excessive cold PBS.
[0177] The expressed GST-cGB-PDE5 fusion protein is displayed on
7.5% SDS-PAGE gel as a 85 Kd protein. It is characterized by its
cGMP binding and phosphorylation by protein kinases G and A. It
displays two cGMP binding sites and the K.sub.d is 1.6.+-.0.2
.mu.M, which is close to K.sub.d=1.3 .mu.M of the native bovine
PDE5. The GST-cGB-PDE5 on GSH conjugated sepharose beads can be
phosphorylated in vitro by cGMP-dependent protein kinase and
cAMP-dependent protein kinase A. The K.sub.m of GST-cGB-PDE5
phosphorylation by PKG is 2.7 .mu.M and Vmax is 2.8 .mu.M, while
the K.sub.m of BPDEtide phosphorylation is 68 .mu.M. The
phosphorylation by PKG shows one molecular phosphate incorporated
into one GST-cGB-PDE5 protein ratio.
[0178] To assay a liquid sample believed to contain PKG using the
PDE5-bound solid phase described above, the sample and the solid
phase are mixed with phosphorylation buffer containing
.sup.32P-.gamma.-ATP. The solution is incubated for 30 minutes at
30.degree. C. to allow for phosphorylation of the PDE5 sequence by
PKG to occur, if PKG is present. The solid phase is then separated
from solution (e.g., by centrifugation or filtration) and washed
with phosphate-buffered saline ("PBS") to remove any remaining
solution and to remove any unreacted .sup.32P-.gamma.-ATP.
[0179] The solid phase can then be tested directly (e.g., by liquid
scintillation counter) to ascertain whether .sup.32P is
incorporated. If it does, that indicates that the sample contained
PKG since PKG phosphorylates PDE5. If the PDE5 is bound via fusion
protein, as described above, the PDE5-containing fusion protein can
be eluted from the solid phase with SDS buffer, and the eluent can
be assayed for .sup.32P incorporation. This is particularly
advantageous if there is the possibility that other proteins are
present, since the eluent can be processed (e.g., by gel
separation) to separate various proteins from each other so that
the fusion protein fraction can be assayed for .sup.32P
incorporation. The phosphorylated fusion protein can be eluted from
the solid phase with SDS buffer and further resolved by
electrophoresis. If gel separation is performed, the proteins can
be stained to see the position(s) of the protein, and .sup.32P
phosphorylation of the PDE5 portion of the fusion protein by PKG
can be measured by X-ray film exposure to the gel. If .sup.32P is
made visible on X-ray film, that indicates that PKG was present in
the original sample contained PKG, which phosphorylated the PDE5
portion of the fusion protein eluted from the solid phase.
[0180] Preferably in the assay, one should add to the assay buffer
an excess (e.g., 100 fold) of protein kinase inhibitor ("PKI")
which specifically and potently inhibits protein kinase A ("PKA")
without inhibiting PKG. Inhibiting PKA is desirable since it may
contribute to the phosphorylation of the PKG substrate (e.g.,
PDE5). By adding PKI, any contribution to phosphorylation by PKA
will be eliminated, and any phosphorylation detected is highly
likely to be due to PKG alone.
[0181] A kit can be made for the assay of this invention, which kit
contains the following pre-packaged reagents in separate
containers:
[0182] 1. Cell lysis buffer: 50 mM Tris-HCl, 1% NP-40, 150 mM NaCl,
1 mM EDTA, 1 mM Na.sub.3VO.sub.4, 1 mM NaF, 500 .mu.M IBMX,
proteinase inhibitors.
[0183] 2. Protein kinase G solid phase substrate: recombinant
GST-cGB-PDE5 bound Sepharose 4B (50% slurry).
[0184] 3. 2.times. Phosphorylation buffer: .sup.32P-.gamma.-ATP
(3000 mCi/mmol, 5.about.10 .mu.Ci/assay), 10 mM KH.sub.2PO.sub.4,
10 mM K.sub.2HPO.sub.4, 200 .mu.M ATP, 5 mM MgCl.sub.2.
[0185] 4. PKA Protein Kinase I Inhibitor
[0186] Disposable containers and the like in which to perform the
above reactions can also be provided in the kit.
[0187] From the above, one skilled in the analytical arts will
readily envision various ways to adapt the assay formats described
to still other formats. In short, using at least a portion of PDE5
(or any other protein that can be selectively phosphorylated by
PKG), the presence and relative amount (as compared to a control)
of PKG can be ascertained by evaluating phosphorylation of the
phosphorylatable protein, using a labeled phosphorylation
agent.
[0188] SAANDs Increase PKG Activity in Neoplastic Cells
[0189] Using the PKG assay described above, the following
experiments were performed to establish that SAANDs increase PKG
activity due either to increase in PKG expression or an increase in
cGMP levels (or both) in neoplastic cells treated with a SAAND.
[0190] Test Procedures
[0191] Two different types of PDE inhibitors were evaluated for
their effects on PKG in neoplastic cells. A SAAND, exisulind, was
evaluated since it is anti-neoplastic. Also, a non-SAAND classic
PDE5 inhibitor, E4021, was evaluated to ascertain whether PKG
elevation was simply due to classic PDE5 inhibition, or whether PKG
elevation was involved in the pro-apoptotic effect of SAANDs
inhibition of PDE5 and the novel PDE disclosed in U.S. patent
application Ser. No. 09/173,375 to Liu et al filed Oct. 15,
1998.
[0192] To test the effect of cGMP-specific PDE inhibition on
neoplasia containing the APC mutation, SW480 colon cancer cells
were employed. SW 480 is known to contain the APC mutation. About 5
million SW480 cells in RPMI 5% serum are added to each of 8
dishes:
[0193] 2-10 cm dishes--30 .mu.L DMSO vehicle control (without
drug),
[0194] 3-10 cm dishes--200 .mu.M, 400 .mu.M, 600 .mu.M exisulind in
DMSO, and
[0195] 3-10 cm dishes--E4021; 0.1 .mu.M, 1 .mu.M and 10 .mu.M in
DMSO.
[0196] The dishes are incubated for 48 hrs at 37.degree. C. in 5%
CO.sub.2 incubator.
[0197] The liquid media are aspirated from the dishes (the cells
will attach themselves to the dishes). The attached cells are
washed in each dish with cold PBS, and 200 .mu.L cell lysis buffer
(i.e., 50 mM Tris-HCl, 1% NP-40, 150 mM NaCl, 1 mM EDTA, 1 mM
Na.sub.3VO.sub.4, 1 mM NaF, 500 .mu.M IBMX with proteinase
inhibitors) is added to each dish. Immediately after the cell lysis
buffer is added, the lysed cells are collected by scraping the
cells off each dish. The cell lysate from each dish is transferred
to a microfuge tube, and the microfuge tubes are incubated at
4.degree. C. for 15 minutes while gently agitating the microfuge
tubes to allow the cells to lyse completely. After lysis is
complete, the microfuge tubes are centrifuged full speed (14,000
r.p.m.) for 15 minutes. The supernatant from each microfuge tube is
transferred to a fresh microfuge tube.
[0198] A protein assay is then performed on the contents of each
microfuge tube because the amount of total protein will be greater
in the control than in the drug-treated samples, if the drug
inhibits cell growth. Obviously, if the drug does work, the total
protein in the drug-treated samples should be virtually the same as
control. In the above situation, the control and the E-4021
microfuge tubes needed dilution to normalize them to the high-dose
exisulind-treated samples (the lower dose groups of exisulind had
to be normalized to the highest dose exisulind sample). Thus, after
the protein assays are performed, the total protein concentration
of the various samples must be normalized (e.g., by dilution).
[0199] For each drug concentration and control, two PKG assays are
performed, one with added cGMP, and one without added cGMP, as
described in detail below. The reason for performing these two
different PKG assays is that cGMP specifically activates PKG. When
PKG activity is assayed using the novel PKG assay of this
invention, one cannot ascertain whether any increase the PKG
activity is due to increased cGMP in the cells (that may be caused
by cGMP-specific PDE inhibition) or whether the PKG activity level
is due to an increased expression of PKG protein. By determining
PKG activity in the same sample both with and without added cGMP,
one can ascertain whether the PKG activity increase, if any, is due
to increased PKG expression. Thus, if an anti-neoplastic drug
elevates PKG activity relative to control, one can establish if the
drug-induced increase is due to increased PKG protein expression
(as opposed to activation) in the drug-treated sample if (1) the
drug-treated sample with extra cGMP exhibits greater PKG activity
compared to the control sample with extra cGMP, and (2) the
drug-treated sample without extra cGMP exhibits greater PKG
activity relative to control.
[0200] After, parallel samples with and without added cGMP are
prepared, 50 .mu.L of each cell lysate is added to 20 .mu.L of the
PDE5/GST solid phase substrate slurry described above. For each
control or drug cell lysate sample to be evaluated, the reaction is
started by adding phosphorylation buffer containing 10 .mu.Ci
.sup.32P-.gamma.-ATP solution (200 .mu.M ATP, 4.5 mM MgCl; 5 mM
KH.sub.2PO.sub.4; 5 mM K.sub.2HPO.sub.4;) to each mixture. The
resultant mixtures are incubated at 30.degree. C. for 30 minutes.
The mixtures are then centrifuged to separate the solid phase, and
the supernatant is discarded. The solid phase in each tube is
washed with 700 .mu.L cold PBS. To the solid phase, Laemmli sample
buffer (Bio-Rad) (30 .mu.L) is added. The mixtures are boiled for 5
minutes, and loaded onto 7.5% SDS-PAGE. The gel is run at 150 V for
one hour. The bands obtained are stained with commassie blue to
visualize the 85 Kd GST-PDE5 fusion protein bands, if present. The
gel is dried, and the gel is laid on x-ray film which, if the PDE5
is phosphorylated, the film will show a corresponding darkened
band. The darkness of each band relates to the degree of
phosphorylation.
[0201] As shown in FIGS. 18A and 18B, the SAAND exisulind causes
PKG activity to increase in a dose-dependent manner in both the
samples with added cGMP and without added cGMP relative to the
control samples with and without extra cGMP. This is evidenced by
the darker appearances of the 85 Kd bands in each of the
drug-treated samples. In addition, the SW480 samples treated with
exisulind show a greater PKG phosphorylation activity with added
cGMP in the assay relative to the samples treated with exisulind
alone (i.e. no added cGMP). Thus, the increase in PKG activity in
the drug-treated samples is not due only to the activation of PKG
by the increase in cellular cGMP when the SAAND inhibits
cGMP-specific PDE, the increase in PKG activity in neoplasia
harboring the APC mutation is due to increased PKG expression as
well.
[0202] Also the fact that the E4021-treated SW480 samples do not
exhibit PKG activation relative to control (see FIGS. 18A and 18B)
shows that the increased PKG activation caused by SAANDs in
neoplasia containing the APC mutation is not simply due to
inhibition of classic PDE5.
[0203] As an analytic technique for evaluating PKG activation,
instead of x-ray film exposure as described above, the 85 Kd band
from the SDS page can be evaluated for the degree of
phosphorylation by cutting the band from the gel, and any .sup.32P
incorporated in the removed band can be counted by scintillation
(beta) counter in the .sup.32P window.
[0204] To test the effect of cGMP-specific PDE inhibition on
neoplasia containing the .beta.-catenin mutation, HCT116 colon
cancer cells were employed. HCT116 is known to contain the
.beta.-catenin mutation, but is known not to contain the APC
mutation.
[0205] The same procedure is used to grow the HCT116 cells as is
used in the SW480 procedure described above. In this experiment,
only exisulind and controls were used. The exisulind-treated cells
yielded PKG that was phosphorylated to a greater extent than the
corresponding controls, indicating that PKG activation occurred in
the drug-treated cells that is independent of the APC mutation.
[0206] Thus, for the purposes of the present invention, we refer to
"reducing .beta.-catenin" in the claims to refer to wild type
and/or mutant forms of that protein.
[0207] Confirmation of Increased PKG Expression and Decreased
.beta.-Catenin in SW 480 by Western Blot
[0208] As demonstrated above, SAANDs cause an increase in PKG
expression and an increase in cGMP level, both of which cause an
increase in PKG activity in SAANDs-treated neoplastic cells. This
increase in PKG protein expression was further verified by
relatively quantitative western blot, as described below.
[0209] SW480 cells treated with exisulind as described previously
are harvested from the microfuge tubes by rinsing once with
ice-cold PBS. The cells are lysed by modified RIPA buffer for 15
minutes with agitation. The cell lysate is spun down in a cold
room. The supernatants are transferred to fresh microcentrifuge
tubes immediately after spinning. BioRad DC Protein Assay
(Temecula, Calif.) is performed to determine the protein
concentrations in samples. The samples are normalized for protein
concentration, as described above.
[0210] 50 .mu.g of each sample is loaded to 10% SDS gel. SDS-PAGE
is performed, and the proteins then are transferred to a
nitrocellulose membrane. The blotted nitrocellulose membrane are
blocked in freshly prepared TBST containing 5% nonfat dry milk for
one hour at room temperature with constant agitation.
[0211] A goat-anti-PKG primary antibody is diluted to the
recommended concentration/dilution in fresh TBST/5% nonfat dry
milk. The nitrocellulose membrane is placed in the primary antibody
solution and incubated one hour at room temperature with agitation.
The nitrocellulose membrane is washed three times for ten minutes
each with TBST. The nitrocellulose membrane is incubated in a
solution containing a secondary POD conjugated rabbit anti-goat
antibody for 1 hour at room temperature with agitation. The
nitrocellulose membrane is washed three times for ten minutes each
time with TBST. The detection is performed by using Boehringer
Mannheim BM blue POD substrate.
[0212] As graphically illustrated in FIG. 19, exisulind causes the
drop of .beta.-catenin and the increase of PKG, which data were
obtained by Western blot. SW480 cells were treated with exisulind
or vehicle (0.1% DMSO) for 48 hours. 50 .mu.g supernatant of each
cell lysates were loaded to 10% SDS-gel and blotted to
nitrocellulose membrane, and the membrane was probed with
rabbit-anti-.beta.-catenin and rabbit anti-PKG antibodies.
[0213] SAANDs Reduce .beta.-Catenin Levels in Neoplastic Cells
[0214] This observation was made by culturing SW480 cells with
either 200, 400 or 600 .mu.M exisulind or vehicle (0.1% DMSO). The
cells are harvested 48 hours post treatment and processed for
immunoblotting. Immuno-reactive protein can be detected by Western
blot. Western blot analysis demonstrated that expression of
.beta.-catenin was reduced by 50% in the exisulind-treated cells as
compared to control. These results indicate that .beta.-catenin is
reduced by SAANDs treatment. Together with the results above
establishing PKG activity increases with such treatment and the
results below establishing that .beta.-catenin is phosphorylated by
PKG, these results indicate that the reduction of .beta.-catenin in
neoplastic cells is initiated by activation of PKG. Thus, using PKG
activity in neoplasia as a screening tool to select compounds as
anti-neoplastics is useful.
[0215] The Phosphorylation of .beta.-catenin by PKG
[0216] In vitro, PKG phosphorylates .beta.-catenin. The experiment
that established this involves immunoprecipitating the
.beta.-catenin-containi- ng complex from SW480 cells (not treated
with any drug) in the manner described below under ".beta.-catenin
immunoprecipitation" The immunoprecitated complex, while still
trapped on the solid phase (i.e., beads) is mixed with
.sup.32P-.gamma.-ATP and pure PKG (100 units). Corresponding
controls with out added PKG are prepared.
[0217] The protein is released from the solid phase by SDS buffer,
and the protein-containing mixture is run on a 7.5%SDS-page gel.
The running of the mixture on the gel removes excess
.sup.32P-.gamma.-ATP from the mixture. Any .sup.32P-.gamma.Y-ATP
detected in the 93 Kd .beta.-catenin band, therefore, is due to the
phosphorylation of the .beta.-catenin. Any increase in
.sup.=P-.gamma.-ATP detected in the 93 Kd .beta.-catenin band
treated with extra PKG relative to the control without extra PKG,
is due to the phosphorylation of the .beta.-catenin in the treated
band by the extra PKG.
[0218] The results we obtained were that there was a noticeable
increase in phosphorylation in the band treated with PKG as
compared to the control, which exhibited minimal, virtually
undetectable phosphorylation. This result indicates that
.beta.-catenin can be phosphorylated by PKG.
[0219] The Phosphorylation of Mutant .beta.-catenin by PKG
[0220] The same procedure described in the immediately preceding
section was performed with HCT116 cells, which contain no APC
mutation, but contain a .beta.-catenin mutation. The results of
those experiments also indicate that mutant .beta.-catenin is
phosphorylated by PKG.
[0221] Thus, for the purposes of the present invention, we refer to
the phosphorylation of .beta.-catenin in the claims to refer to the
phosphorylation of wild type and/or mutant forms of that
protein.
[0222] .beta.-Catenin Precipitates With PKG
[0223] Supernatants of both SW480 and HCT116 cell lysates are
prepared in the same way described above in the Western Blot
experiments. The cell lysate are pre-cleared by adding 150 .mu.l of
protein A Sepharose bead slurry (50%) per 500 .mu.g of cell lysate
and incubating at 4.degree. C. for 10 minutes on a tube shaker. The
protein A beads are removed by centrifugation at 14,000.times.g at
4.degree. C. for 10 minutes. The supernatant are transferred to a
fresh centrifuge tube. 10 .mu.g of the rabbit polyclonal
anti-.beta.-catenin antibody (Upstate Biotechnology, Lake Placid,
N.Y.) are added to 500 .mu.g of cell lysate. The cell
lysate/antibody mixture is gently mixed for 2 hours at 4.degree. C.
on a tube shaker. The immunocomplex is captured by adding 150 .mu.l
protein A Sepharose bead slurry (75 .mu.l packed beads) and by
gently rocking the mixture on a tube shaker for overnight at
4.degree. C. The Sepharose beads are collected by pulse
centrifugation (5 seconds in the microcentrifuge at 14,000 rpm).
The supernatant fraction is discarded, and the beads are washed 3
times with 800 .mu.l ice-cold PBS buffer. The Sepharose beads are
resuspended in 150 .mu.l 2.times.sample buffer and mixed gently.
The Sepharose beads are boiled for 5 minutes to dissociate the
immunocomplexes from the beads. The beads are collected by
centrifugation and SDS-PAGE is performed on the supernatant.
[0224] A Western blot is run on the supernatant, and the membrane
is then probed with an rabbit anti .beta.-catenin antibody. Then
the membrane is washed 3 times for 10 minutes each with TBST to
remove excess anti .beta.-catenin antibody. A goat, anti-rabbit
antibody conjugated to horseradish peroxidase is added, followed by
1 hour incubation at room temperature. When that is done, one can
visualize the presence of .beta.-catenin with an HRPO substrate. In
this experiment, we could clearly visualize the presence of
.beta.-catenin.
[0225] To detect PKG on the same membrane, the anti-.beta.-catenin
antibody conjugate is first stripped from the membrane with a 62 mM
tris-HCl buffer (pH 7.6) with 2% SDS and 100 .mu.M
2.beta.-mercaptoethanol in 55.degree. C. water bath for 0.5 hour.
The stripped membrane is then blocked in TBST with 5% non-fat dried
milk for one hour at room temperature while agitating the membrane.
The blocked, stripped membrane is then probed with rabbit
polyclonal anti-PKG antibody (Calbiochem, LaJolla, Calif.), that is
detected with goat, anti-rabbit second antibody conjugated to HRPO.
The presence of PKG on the blot membrane is visualized with an HRPO
substrate. In this experiment, the PKG was, in fact, visualized.
Given that the only proteins on the membrane are those that
immunoprecipitated with .beta.-catenin in the cell supernatants,
this result clearly establishes that PKG was physically linked to
the protein complex containing the .beta.-catenin in the cell
supernatants.
[0226] The same Western blot membrane was also probed after
stripping with anti-GSK3-.beta. antibody to ascertain whether it
also co-precipitated with .beta.-catenin. In that experiment, we
also detected GSK3-.beta. on the membrane, indicating that the
GSK3-.beta. precipitated with the GSK3-.beta. and PKG, suggesting
that the three proteins may be part of the same complex. Since
GSK3-.beta. and .beta.-catenin form part of the APC complex in
normal cells, this that PKG may be part of the same complex, and
may be involved in the phosphorylation of .beta.-catenin as part of
that complex.
[0227] Anti-Neoplastic Pharmaceutical Compositions Containing cGMP
PDE Inhibitors
[0228] As explained above, exisulind is one compound that exhibits
desirable anti-neoplastic properties. Its efficacy and use as an
anti-neoplastic was discovered before it was understood that the
compound acted by inhibiting cGMP-specific PDE activity in
neoplastic cells.
[0229] Among other things, the verification that the selection
process of this invention could be used to select compounds for
human treatment was obtained in human clinical trials in patients
with neoplasias. By understanding after the fact that exisulind was
anti-neoplastic (in vitro), that it had the profile of a desirable
compound meeting the selection criterion of this invention, the
success of the compound in two human clinical trials establishes
that other compounds can be selected meeting the selection
criterion of this invention.
[0230] As indicated above, a number of neoplasias harbor the APC
mutation. Among other things, the verification of the selection
process of this invention was established in human clinical trials
in patients with neoplasia harboring the APC mutation.
[0231] The APC mutation was first discovered in patients with the
hereditary neoplasia, adenomatous polyposis coli ("APC"). The APC
disease is characterized by the appearance in the teen years of
hundreds to thousands of polyps in the colon, and the common
therapy is surgical removal of the colon before the age of 20.
[0232] The first clinical trial involved patients with APC.
[0233] using exisulind. In that study, each patient had already had
his/her colon removed, except for a small section of colon adjacent
the rectum (where the small intestine was attached) to preserve
rectal function. However, such a patient commonly forms polyps in
the small remaining colonic section, which polyps require periodic
removal (e.g., by electrocautery).
[0234] That trial where exisulind was selected was a prevention
trial designed to evaluate the anti-neoplastic characteristics of
the drug by comparing the cumulative number of new polyps formed
over twelve months by the drug and placebo groups. Eligible
patients were those who form between 9 and 44 polyps per year.
Patients were fully ablated (had all polyps removed) at the start
of the study, at the end of 6 months and at the end of 12 months.
The study enrolled thirty-four eligible patients. Based on the
estimated mean number of polyps formed over a year in APC patients
who had historically produced 9 to 44 polyps per year, exisulind
was clinically and statistically significantly better than placebo
in decreasing the rate of polyp formation. Based on the median
number of polyps produced in the first six months of the study,
patients treated with exisulind developed approximately one-third
the number of polyps as patients treated with placebo (median
values 9 polyps/year and 26 polyps/year, respectively; p=0.013).
Based on the median number of polyps produced over the entire 12
months of the study, patients treated with exisulind produced
approximately half the number of polyps as patients treated with
placebo (median values 18 polyps/year and 38 polyps/year,
respectively; p=0.020).
[0235] A separate clinical trial was also performed on male
patients who had prostate cancer, and as a result had their
prostates removed. The study was conducted in patients with
detectable PSA (prostate specific antigen) levels that were rising
following radical prostatectomy, indicating recurrence of prostate
cancer.
[0236] 96 patients were enrolled in the prostate cancer evaluation:
a double-blind, placebo-controlled, multi-center trial involving
exisulind administered to the drug-receiving patients at 500
mg/day. As presented below, the data show a statistically
significant difference in PSA levels between the exisulind-treated
group and the placebo-treated group. PSA levels in the
exisulind-treated group were significantly reduced as compared with
the PSA levels of the placebo-treated group. Although a rising
level of PSA is not itself a disease condition, it is widely
regarded in the medical community as a surrogate marker indicative
of the presence of recurrence of prostate cancer in such men.
[0237] In addition to performing an evaluation based on the
differences in mean PSA levels between the exisulind and placebo
groups as a whole, the interim analysis included subgroup analysis.
The patients in the study were classified into high, intermediate
and low risk groups in terms of their risk of developing metastatic
disease. This classification was performed using the methodology
published in the Journal of the American Medical Association (JAMA
May 5, 1999, pp. 1591-97). To ascertain which study patients fell
into which risk group, medical histories were supplied to a
researcher who was blinded as to whether patients were on drug or
placebo; he assigned study patients to the appropriate risk groups
according to the above referenced published methodology. The
statistical analysis revealed statistically significant differences
in mean PSA levels between exisulind and placebo patients in both
high and intermediate risk groups.
[0238] The data from the prostate study are as follows:
10TABLE 10 Effect of Exisulind On Mean PSA Level In Men
Post-Prostatectomy With Rising PSA Group Placebo Exisulind "p"
value Overall 4.49 2.85 0.0004 High Risk 4.98 2.91 0.0002
Intermediate Risk 6.24 2.95 0.0053
[0239] In these exisulind trials and several others involving the
drug in other indications, safety was evaluated by monitoring
adverse events (AEs), clinical laboratory tests (hematology, serum
chemistry, and urinalysis), vital signs (blood pressure, pulse
rate, respiratory rate, temperature, and weight), physical
examination, and upper endoscopy.
[0240] No outstanding safety issues have been demonstrated in the
clinical trials conducted with exisulind to date in over 400
patients. Exisulind did not demonstrate any blood dyscrasia,
dose-limiting vomiting, or neurological or renal toxicities
associated with convention chemotherapeutics. It also did not cause
any clinically significant changes in vital signs. In fact, in
paired biopsies of polyp and normal colonic tissues in APC
patients, it was found that exisulind increased apoptosis rates in
polyp, but not normal colonic tissues, suggesting minimal effects
on normal tissues.
[0241] At doses above the maximum tolerated dose (MTD=600 mg in
patients with subtotal colectomy; 400 mg in patients with intact
colons; 350 mg in pediatric patients), the only dose-limiting
adverse events found were elevations in liver function tests (LFTs)
that are seen early during treatment. When experienced, LFT
elevations were rapidly reversible, and do not recur when the dose
has been lowered. Other events (e.g., occasional abdominal pain)
were typically short lasting and of mild to moderate intensity, and
did not necessitate discontinuing or lowering of the exisulind
dose.
[0242] In short, these trials demonstrated that exisulind is an
effective, well-tolerated chronic therapy for the clinical
management of neoplasia. Thus, these results illustrate that
selecting an additional compound that inter alia inhibits
cGMP-specific PDE activity (as well as meeting the other selection
criteria of this invention) can result in a therapeutically
effective drug, in vivo.
[0243] A second drug that was also invented before its mechanism of
action was found to involve cGMP inhibition and before it was known
to meet the selection criterion of this invention is
(Z)-5-fluoro-2-methyl-(4-pyridyl-
idene)-3-(N-benzyl)indenylacetamide hydrochloride (Compound I). It
has been demonstrated in in vitro and in vivo evaluations as
anti-neoplastic having activities against a broad range of
neoplasias. It is also safe in animal studies and in a single,
escalating dose human study.
[0244] As one skilled in the art will recognize from the data
presented below, Compound I can safely be given to animals at doses
far beyond the tolerable (and in many cases toxic) doses of
conventional chemotherapeutics or anti-neoplastic NSAIDs. For
example, in an acute toxicity study in rats, single oral doses of
Compound I administered (in a 0.5% carboxy-methylcellulose vehicle)
at doses up to and including 2000 mg/kg resulted in no observable
signs of toxicity. At 4000 mg/kg, body weight gains were slightly
reduced. A single dose of 1000 mg/kg administered intraperitoneally
resulted in reduced body weight gain, with mesenteric adhesions
seen in some animals from this group at necropsy.
[0245] In dogs, the administration of Compound I in capsules at
1000 mg/kg resulted in no signs of toxicity to the single group of
two male and two female dogs. Due to the nature of Compound I
capsules, this dose necessitated the use of at least 13 capsules to
each animal, which was judged to be the maximum number without
subjecting the animals to stress. Therefore, these dogs were
subsequently administered seven consecutive doses of 1000
mg/kg/day. At no time in either dosing phase were any obvious signs
of drug-related effects observed.
[0246] Thus, on a single-dose basis, Compound I is not acutely
toxic. Based on the findings of these studies, the oral LD.sub.50
of Compound I was considered to be greater than 1000 mg/kg in dogs
and 4000 mg/kg in rats, and the intraperitoneal LD.sub.50 was
considered to be greater than 1000 mg/kg in rats.
[0247] A seven-day dose-range finding study in rats, where Compound
I was evaluated by administering it at doses of 0, 50, 500 or 2000
mg/kg/day resulting in no observable signs of toxicity at 50
mg/kg/day. At 500 mg/kg/day, treatment-related effects were limited
to an increase in absolute and relative liver weights in female
rats. At 2000 mg/kg/day, effects included labored breathing and/or
abnormal respiratory sounds, decreased weights gains and food
consumption in male rats, and increased liver weights in female
rats. No hematological or blood chemistry changes nor any
microscopic pathology changes, were seen at any dose level.
[0248] A 28-day study in rats was also carried out at 0, 50, 500
and 2000 mg/kg/day. There were no abnormal clinical observations
attributed to CP-461, and body weight changes, ophthalmoscopic
examinations, hematological and blood chemistry values and
urinalysis examinations were unremarkable. No macroscopic tissue
changes were seen at necropsy. Organ weight data revealed
statistically significant increase in liver weights at 2000
mg/kg/day, and statistically significant increases in thyroid
weights for the 2000 mg/kg/day group. The slight increases at the
lower doses were not statistically significant. Histopathological
evaluation of tissues indicated the presence of traces of
follicular cell hypertrophy, increased numbers of mitotic figures
(suggestive of possible cell proliferation) in the thyroid gland
and mild centrilobular hypertrophy in the liver. These changes were
generally limited to a small number of animals at the 2000
mg/kg/day dose, although one female at 500 mg/kg/day had increased
mitotic figures in the thyroid gland. The findings in the liver may
be indicative of a very mild stimulation of microsomal enzymes,
resulting in increased metabolism of thyroid hormones, which in
turn resulted in thyroid stimulation. Thus, one skilled in the art
will recognize that these effects are extremely minimal compared to
what one would expect at similar doses of conventional
chemotherapeutics or NSAIDs.
[0249] To further establish the safety profile of Compound I, a
study was performed to evaluate whether Compound I-induced
apoptosis of prostate tumor cell lines was comparable to its
effects on prostate epithelial cells derived from normal tissue.
The androgen-sensitive prostate tumor cell line, LNCaP (from ATCC
(Rockville, Md.)) was propogated under standard conditions using
RPMI 160 medium containing 5% fetal calve serum and 2 mM glutamine.
Primary prostate epithelial cell cultures (PrEC) derived from
normal prostate (from Clonetics Inc. (San Diego, Calif.)) were
grown under the same conditions as the tumor cell line except a
serum-free medium optimized for the growth of such cultures was
used (Clonetics Inc). For the experiments, LNCaP or PrEC cells were
seeded in 96 well plates at a density of 10,000 cells per well.
After 24 hours, the cells were treated with either vehicle (0.1%
DMSO) or 50 .mu.M Compound I (free base) solubilized in DMSO. After
various drug treatment times (4, 24, 48, 72, or 99 hours) the cells
were lysed and processed for measurement of histone-associated DNA
as an indicator of apoptotic cell death (see, Piazza et al., Cancer
Research 57: 2452-2459, 1997).
[0250] FIG. 27 shows a time-dependent increase in the amount of
histone-associated fragmented DNA in LNCaP cell cultures following
treatment with 50 .mu.M Compound I(free base). A significant
increase in fragmented DNA was detected after 24 hours of
treatment, and the induction was sustained for up to 4 days of
continuous treatment. By contrast, treatment of PrEC ("normal""
prostate) cells with Compound 1 (50 .mu.M) did not affect DNA
fragmentation for up to 4 days of treatment. These results
demonstrate a selective induction of apoptosis in neoplastic cells,
as opposed to normal cells. This is in marked contrast to
conventional chemotherapeutics that induce apoptosis or necrosis in
rapidly growing normal and neoplastic cells alike.
[0251] Finally as to safety, in a single, escalating dose human
clinical trial, patients, human safety study in which the drug was
taken orally, Compound I produced no significant side effects at
any dose, including doses above the level predicted to be necessary
to produce anti-cancer effects.
[0252] As indicated above, Compound I also exhibits potent
anti-neoplastic properties. The growth inhibition IC.sub.50 value
obtained for Compound I was 0.7 .mu.M in the SW-480 cell line. This
result has been confirmed by evaluating Compound I in rodents using
aberrant crypt foci ("ACF") as an indicator of carcinogenesis (see,
Bird, Cancer Lett. 37: 147-151, 1987). This established rodent
model of azoxymethane ("AOM")-induced carcinogenesis was used to
assess the effects of Compound I (free base and salt) on colon
cancer development in vivo. ACF are precursors to colonic tumors,
and ACF inhibition is predictive of chemo-preventive efficacy.
[0253] In the rats in this experiment, ACF initiation was achieved
by two consecutive weekly injections of the carcinogen. Compound I
was administered one week prior to ACF initiation and for the
duration of the experiment. ACFs were scored after 5 weeks of
treatment. Compound I was administered orally to male Fisher 344
rats in the rat chow. Daily food consumption (mg/kg body weight)
varied over the course of the study, and therefore Compound I dose
was expressed a grams per kg of diet to provide a basis of
comparison between doses. To determine if Compound I had an adverse
effect on growth and/or feeding behavior, body weight was
determined throughout the course of the experiment. The
experimental groups gained less weight than the controls, which was
indicative of bioavailability. However, the weight differences were
less than 10% and not considered to affect ACF formation.
[0254] The free base of Compound I inhibited ACF formation as
measured by a reduction of crypts per colon. The data are
summarized in Table 11. With the exception of the low dose group
(only 0.5 g/kg diet), the differences between treatment and control
groups were substantial, and statistically significant in the case
of the 1.0 and 2.0 g/kg diet group.
11TABLE 11 Inhibition of Aberrant Crypt Foci by Compound I Compound
Mean ACF/colon p (t-test) Dose (g/kg diet) n (+SE) % Control vs.
control Control 10 149 + 9 -- -- 0.5 7 149 .+-. 14 100 0.992 1 10
111 .+-. 9 75 0.008 1.5 10 132 + 4 89 0.101 2.0 10 107 + 15 72
0.029
[0255] Also, Compound I retrospectively met the selection criterion
of this invention, and was one of the compounds used to establish
the validity of this selection criteria. For example, using the
protocols described previously, Compound I has a cGMP-specific PDE
IC.sub.50 value of 0.68 .mu.M utilizing cGMP-specific PDE from HT29
cell extracts. Its COX I inhibition (at 100 .mu.M) was less than
25%.
[0256] As for being pro-apoptotic, Compound I's DNA fragmentation
EC.sub.50 was 15 .mu.M. In addition, the percent apoptosis for
Compound I in SW-480 is shown in Table 12 at various drug
concentrations.
12TABLE 12 Apoptosis Induction of HT-29 Cells of SW-480 Colon
Adenocarcinoma Cells by Compound I as Determined by Morphology
Treatment Dose % Apoptosis Vehicle (0.1% DMSO) -- 1 Compound I 0.35
.mu.M 16 Compound I 0.7 .mu.M 27 Compound I 1.5 .mu.M 88
[0257] Compound I's activity is not confined to activity against
colon cancer cell lines or animal models of colon cancer. It has a
broad range of anti-neoplastic effects in various neoplastic cell
lines. Various types of human cancer cell lines were propagated
under sterile conditions in RPMI 1640 medium with 10% fetal bovine
serum, 2 mM L-glutamine and sodium bicarbonate. To determine growth
inhibitory effects of Compound I, cells were seeded in 96-well
plates at a density of 1000 cells per well. Twenty-four hours after
plating, the cells were dosed with various concentrations of the
free base of Compound I solubilized in DMSO (final concentration
0.1%). The effect of the drug on tumor cell growth was determined
using the neutral red cytotoxicity assay following five days of
continuous treatment. Neutral red is a dye that is selectively
taken up by viable cells by an ATP-dependent transport
mechanism.
[0258] As summarized in Table 13, Compound I (free base) displayed
potent growth inhibitory activity when evaluated against a panel of
cultured human cell lines derived from various tissue origins.
Compound I displayed comparable growth inhibitory effects
regardless of the histogenesis of the tumor from which the cell
lines were derived. The GI.sub.50 value (concentration of drug to
inhibit growth by 50% relative to vehicle control) calculated for
all cell lines was 1-2 .mu.M.
[0259] In addition to the data in the table below, we observed
comparable sensitivity of human leukemia cell lines (CCRF-CEM,
K562, and Molt-4), a myeloma cell line (RPMI8226), a pancreatic
tumor cell line (PAN-1), and an ovarian tumor cell line (OVCAR-3)
to Compound I (HCl salt).
13TABLE 13 Growth Inhibition of Various Human Tumor Cell Lines by
Compound I Cell Line Tumor origin GI.sub.50.mu.M GI.sub.90 .mu.M
Colo 205 Colon 1.6 2.4 HCT-15 Colon 1.7 3.0 HT-29 Colon 2.1 8.0
SW-620 Colon 1.7 2.5 DU145 Prostate 1.6 2.8 PC-3 Prostate 1.7 82.5
NCI-H23 Lung 1.7 2.5 NCI-H322M Lung 2.1 13.2 NCI-H460 Lung 1.9 30.0
NCI-H82 Lung 1.7 5.8 MDA-MB-231 Breast 1.8 77.6 MDA-MB-435 Breast
1.6 2.3 UISO-BCA-1 Breast 1.5 4.7 Molt-4* Leukemia 1.6 ND CCRF-CEM*
Leukemia 1.4 ND K-562* Leukemia 1.8 ND RPMI-8226* Myeloma 1.2 ND
OVCAR* Ovary 1.2 ND PANC-1* Pancreas 2.2 ND *Testing was done with
the free base of the compound unless otherwise indicated with an
asterisk in which case testing was done with the HCl salt.
[0260] Given the animal and human safety characteristics, and the
animal and very broad cell culture efficacy of Compound I, it is
clear that compounds meeting the selection criteria of this
invention (including cGMP-specific PDE inhibition) can are useful
anti-neoplastic therapeutics.
[0261] As to identifying structurally additional cGMP-specific PDE
inhibiting compounds that can be effective therapeutically as
anti-neoplastics, one skilled in the art has a number of useful
model compounds disclosed herein (as well as their analogs
incorporated by reference) that can be used as the bases for
computer modeling of additional compounds having the same
conformations but different chemically. For example, software such
as that sold by Molecular Simulations Inc. release of WebLab.RTM.
ViewerPro.TM. includes molecular visualization and chemical
communication capabilities. Such software includes functionality,
including 3D visualization of known active compounds to validate
sketched or imported chemical structures for accuracy. In addition,
the software allows structures to be superimposed based on
user-defined features, and the user can measure distances, angles,
or dihedrals.
[0262] In this situation, since the structures of other active
compounds are disclosed above, one can apply cluster analysis and
2D and 3D similarity search techniques with such software to
identify potential new additional compounds that can then be
screened and selected according to the selection criteria of this
invention. These software methods rely upon the principle that
compounds, which look alike or have similar properties, are more
likely to have similar activity, which can be confirmed using the
selection criterion of this invention.
[0263] Likewise, when such additional compounds are computer
modeled, many such compounds and variants thereof can be
synthesized using known combinatorial chemistry techniques that are
commonly used by those of ordinary skill in the pharmaceutical
industry. Examples of a few for-hire combinatorial chemistry
services include those offered by New Chemical Entities, Inc. of
Bothell Washington, Protogene Laboratories, inc., of Palo Alto,
Calif., Axys, Inc. of South San Francisco, Calif., Nanosyn, Inc. of
Tucson, Ariz., Trega, Inc. of San Diego, Calif., and RBI, Inc. of
Natick, Mass. There are a number of other for-hire companies. A
number of large pharmaceutical companies have similar, if not
superior, in-house capabilities. In short, one skilled in the art
can readily produce many compounds for screening from which to
select promising compounds for treatment of neoplasia having the
attributes of compounds disclosed herein. To further assist in
identifying compounds that can be screened and then selected using
the criterion of this invention, knowing the binding of selected
anti-neoplastic compounds to PDE5 protein is of interest. By the
procedures discussed below, it was found that preferable, desirable
compounds meeting the selection criteria of this invention bind to
the cGMP catalytic region of PDE5.
[0264] To establish this, a PDE5 sequence that does not include the
catalytic domain was used. One way to produce such a sequence is to
express that sequence as a fusion protein, preferably with
glutiathione S-transferase ("GST"), for reasons that will become
apparent.
[0265] RT-PCR method is used to obtain the cGB domain of PDE5 with
forward and reverse primers designed from bovine PDE5A cDNA
sequence (McAllister-Lucas L. M. et al, J. Biol. Chem. 268,
22863-22873, 1993) and the selection among PDE 1-10 families.
5'-3', Inc. kits for total RNA followed by oligo (dT) column
purification of mRNA are used with HT-29 cells. Forward primer
(GAA-TTC-TGT-TAG-AAA-AGC-CAC-CAG-AGA-AAT-G, 203-227) and reverse
primer (CTC-GAG-CTC-TCT-TGT-TTC-TTC-CTC-TGC-TG, 1664-1686) are used
to synthesize the 1484 bp fragment coding for the phosphorylation
site and both low and high affinity cGMP binding sites of human
PDE5A (203-1686 bp, cGB-PDE5). The synthesized cGB-PDE5 nucleotide
fragment codes for 494 amino acids with 97% similarity to bovine
PDE5A. It is then cloned into pGEX-5X-3 glutathione-S-transferase
(GST) fusion vector (Pharmacia Biotech) with tac promoter, and
EcoRI and XhoI cut sites. The fusion vector is then transfected
into E. Coli BL21 (DE3) bacteria (Invitrogen). The transfected BL21
bacteria is grown to log phase, and then IPTG is added as an
inducer. The induction is carried at 20.degree. C. for 24 hrs. The
bacteria are harvested and lysed. The soluble cell lysate is
incubated with GSH conjugated Sepharose 4B (GSH-Sepharose 4B). The
GST-cGB-PDE5 fusion protein can bind to the GSH-Sepharose beads,
and the other proteins are washed off from beads with excessive
cold PBS.
[0266] The expressed GST-cGB-PDE5 fusion protein is displayed on
7.5% SDS-PAGE gel as an 85 Kd protein. It is characterized by its
cGMP binding and phosphorylation by protein kinases G and A. It
displays two cGMP binding sites, and the K.sub.d is 1.6.+-.0.2
.mu.M, which is close to K.sub.d=1.3 .mu.M of the native bovine
PDE5. The GST-cGB-PDE5 on GSH-conjugated sepharose beads can be
phosphorylated in vitro by cGMP-dependent protein kinase and
cAMP-dependent protein kinase A. The K.sub.m of GST-cGB-PDE5
phosphorylation by PKG is 2.7 .mu.M and Vmax is 2.8 .mu.M, while
the K.sub.m of BPDEtide phosphorylation is 68 .mu.M. The
phosphorylation by PKG shows molecular phosphate incorporated into
GST-cGB-PDE5 protein on a one-to-one ratio.
[0267] A cGMP binding assay for compounds of interest (Francis S.
H. et al, J. Biol. Chem. 255, 620-626, 1980) is done in a total
volume of 100 .mu.L containing 5 mM sodium phosphate buffer
(pH=6.8), 1 mM EDTA, 0.25 mg/mL BSA, H.sup.3-cGMP (2 .mu.M, NEN)
and the GST-cGB-PDE5 fusion protein (30 .mu.g/assay). Each compound
to be tested is added at the same time as .sup.3H-cGMP substrate,
and the mixture is incubated at 22.degree. C. for 1 hour. Then, the
mixture is transferred to Brandel MB-24 cell harvester with GF/B as
the filter membrane followed by 2 washes with 10 mL of cold 5 mM
potassium buffer (pH 6.8). The membranes are then cut out and
transferred to scintillation vials followed by the addition of 1 mL
of H.sub.2O and 6 mL of Ready Safe.TM. liquid scintillation
cocktail to each vial. The vials are counted on a Beckman LS 6500
scintillation counter.
[0268] For calculation, blank samples are prepared by boiling the
binding protein for 5 minutes, and the binding counts are <1%
when compare to unboiled protein. The quenching by filter membrane
or other debris are also calibrated.
[0269] PDE5 inhibitors, sulfide, exisulind, Compound B, Compound E,
E4021 and zaprinast, and cyclic nucleotide analogs, cAMP, cyclic
IMP, 8-bromo-cGMP, cyclic UMP, cyclic CMP, 8-bromo-cAMP,
2'-O-butyl-cGMP and 2'-O-butyl-cAMP are selected to test whether
they could competitively bind to the cGMP binding sites of the
GST-cGB-PDE5 protein. The results were shown in FIG. 24. cGMP
specifically binds GST-cGB-PDE5 protein. Cyclic AMP, cUMP, cCMP,
8-bromo-cAMP, 2'-O-butyl-cAMP and 2'-O-butyl-cGMP did not compete
with cGMP in binding. Cyclic IMP and 8-bromo-cGMP at high
concentration (100 .mu.M) can partially compete with cGMP (2 .mu.M)
binding. None of the PDE5 inhibitors showed any competition with
cGMP in binding of GST-cGB-PDE5. Therefore, they do not bind to the
cGMP binding sites of PDE5.
[0270] However, Compound E does competitively (with cGMP) bind to
PDE 5 (i.e., peak A). (Compound E also competitively (with cGMP)
binds to PDE peak B.). Given that Compound E does not bind to the
cGMP-binding site of PDE5, this the fact that there is competitive
binding between Compound E and cGMP at all means that desirable
compounds such as Compound E bind to the cGMP catalyic site on
PDE5, information that is readily obtainable by one skilled in the
art (with conventional competitive binding experiments) but which
can assist one skilled in the art more readily to model other
compounds. Thus, with the chemical structures of desirable
compounds presented herein and the cGMP binding site information,
one skilled in the art can model, identify and select (using the
selection criteria of this invention) other chemical compounds for
use as therapeutics.
[0271] Compounds selected in accordance with the methodology of
this invention may be formulated into pharmaceutical compositions
as is well understood from the ordinary meaning of the term
"pharmaceutical composition" i.e., a compound (e.g., like the
solids described above) and a pharmaceutically acceptable carrier
for delivery to a patient by oral administration in solid or liquid
form, by IV or IP administration in liquid form, by topical
administration in ointment form, or by rectal or topical
administration in a suppository formulation. Carriers for oral
administration are most preferred.
[0272] As is well known in the art pharmaceutically acceptable
carriers in pharmaceutical compositions for oral administration
include capsules, tablets, pills, powders, troches and granules. In
such solid dosage forms, the carrier can comprise at least one
inert diluent such as sucrose, lactose or starch. Such carriers can
also comprise, as is normal practice, additional substances other
than diluents, e.g., lubricating agents such as magnesium stearate.
In the case of capsules, tablets, troches and pills, the carriers
may also comprise buffering agents. Carriers such as tablets, pills
and granules can be prepared with enteric coatings on the surfaces
of the tablets, pills or granules. Alternatively, the
enterically-coated compound can be pressed into a tablet, pill, or
granule, and the tablet, pill or granules for administration to the
patient. Preferred enteric coatings include those that dissolve or
disintegrate at colonic pH such as shellac or Eudraget S.
[0273] Pharmaceutically acceptable carriers in pharmaceutical
compositions include liquid dosage forms for oral administration,
e.g., pharmaceutically acceptable emulsions, solutions,
suspensions, syrups and elixirs containing inert diluents commonly
used in the art, such as water. Besides such inert diluents,
compositions can also include adjuvants such as wetting agents,
emulsifying and suspending agents, and sweetening, flavoring and
perfuming agents.
[0274] Pharmaceutically acceptable carriers in pharmaceutical
compositions for IV or IP administration include common
pharmaceutical saline solutions.
[0275] Pharmaceutically acceptable carriers in pharmaceutical
compositions for topical administration include DMSO, alcohol or
propylene glycol and the like that can be employed with patches or
other liquid-retaining material to hold the medicament in place on
the skin so that the medicament will not dry out.
[0276] Pharmaceutically acceptable carriers in pharmaceutical
compositions for rectal administration are preferably suppositories
that may contain, in addition to the compounds of this invention
excipients such as cocoa butter or a suppository wax, or gel.
[0277] A pharmaceutically acceptable carrier and compounds of this
invention are formulated into pharmaceutical compositions in unit
dosage forms for administration to a patient. The dosage levels of
active ingredient (i.e., compounds selected in accordance with this
invention) in the unit dosage may be varied so as to obtain an
amount of active ingredient effective to achieve
neoplasia-eliminating activity in accordance with the desired
method of administration (i.e., oral or rectal). The selected
dosage level therefore depends upon the nature of the active
compound administered (e.g., its IC.sub.50, which can be readily
ascertained), the route of administration, the desired duration of
treatment, and other factors. If desired, the unit dosage may be
such that the daily requirement for active compound is in one dose,
or divided among multiple doses for administration, e.g., two to
four times per day. For IV administration, an initial dose for
administration can be ascertained by basing it on the dose that
achieves the IC.sub.50 in the plasma contents of the average adult
male (i.e., about 4 liters). Initial doses of active compound
selected in accordance with this invention can range from 0.5-600
mg.
[0278] The pharmaceutical compositions of this invention are
preferably packaged in a container (e.g., a box or bottle, or both)
with suitable printed material (e.g., a package insert) containing
indications, directions for use, etc.
[0279] 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.
* * * * *