U.S. patent application number 12/102629 was filed with the patent office on 2008-12-11 for anti-cancer compositions and methods.
This patent application is currently assigned to The Penn State Research Foundation. Invention is credited to Shantu Amin, Dhimant Desai, Gavin P. Robertson, Arati K. Sharma, Arun K. Sharma.
Application Number | 20080306148 12/102629 |
Document ID | / |
Family ID | 39864375 |
Filed Date | 2008-12-11 |
United States Patent
Application |
20080306148 |
Kind Code |
A1 |
Robertson; Gavin P. ; et
al. |
December 11, 2008 |
ANTI-CANCER COMPOSITIONS AND METHODS
Abstract
Anti-cancer compositions and methods are described including one
or more isothiocyanates and/or isoselenocyanates. Methods of
treating a subject are provided according to embodiments of the
present invention which include administering a therapeutically
effective amount of a composition including an isothiocyanate
and/or isoselenocyanate to a subject having a condition
characterized by Akt dysregulation. Administering a therapeutically
effective amount of a composition including an isothiocyanate
and/or isoselenocyanate to a subject detectably increases apoptosis
and/or decreases proliferation of cancer cells, particularly cancer
cells characterized by Akt dysregulation.
Inventors: |
Robertson; Gavin P.;
(Hummelstown, PA) ; Sharma; Arati K.;
(Hummelstown, PA) ; Sharma; Arun K.; (Hummelstown,
PA) ; Amin; Shantu; (Union City, NJ) ; Desai;
Dhimant; (Mechaniscsburg, PA) |
Correspondence
Address: |
GIFFORD, KRASS, SPRINKLE,ANDERSON & CITKOWSKI, P.C
PO BOX 7021
TROY
MI
48007-7021
US
|
Assignee: |
The Penn State Research
Foundation
University Park
PA
|
Family ID: |
39864375 |
Appl. No.: |
12/102629 |
Filed: |
April 14, 2008 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
60911565 |
Apr 13, 2007 |
|
|
|
60959554 |
Jul 13, 2007 |
|
|
|
Current U.S.
Class: |
514/514 ;
435/375; 564/248 |
Current CPC
Class: |
A61P 35/00 20180101;
A61K 45/06 20130101; A61K 9/0014 20130101; A61K 31/095 20130101;
A61K 31/21 20130101; A61K 31/26 20130101; C07C 251/00 20130101;
C07C 391/00 20130101; C07C 331/24 20130101 |
Class at
Publication: |
514/514 ;
564/248; 435/375 |
International
Class: |
A61K 31/21 20060101
A61K031/21; C07C 251/00 20060101 C07C251/00; C12N 5/00 20060101
C12N005/00; A61P 35/00 20060101 A61P035/00 |
Goverment Interests
GOVERNMENT SPONSORSHIP
[0002] The invention was made with government support under
Contract No. HHSN261200566003C awarded by the U.S. Public Health
Service; Contract No. NO2-CB-56603 awarded by the U.S. National
Institutes of Health National Cancer Institute; and Contract No.
CA-127892-01A awarded by the U.S. National Institutes of Health.
The government has certain rights in the invention.
Claims
1. A composition comprising an isoselenocyanate having the
structural formula: R--(CH.sub.2).sub.n--N.dbd.C.dbd.Se, where n is
an integer in the range of 1-8, inclusive, and where R is selected
from the group consisting of: an aromatic group and a non-aromatic
organic group.
2. The composition of claim 1 where the isoselenocyanate has the
structural formula: ##STR00026## where R' is a substituted or
unsubstituted, branched or straight chain, lower alkyl group, and
where n is an integer in the range of 3-8, inclusive.
3. The composition of claim 2 wherein R' is CH.sub.3.
4. The composition of claim 2, where R' is CH.sub.3 and n is 4.
5. A method for synthesis of an isoselenocyanate having the
structural formula: R--(CH.sub.2).sub.n--N.dbd.C.dbd.Se, where n is
an integer in the range of 1-8, inclusive, and where R is selected
from the group consisting of: an unsubstituted aromatic group; an
aromatic group substituted by one or more substituents selected
from the group consisting of: F, Cl, Br, a lower alkyl group, a
lower alkoxy group and a fluorinated lower alkyl group; R'--S(O),
where R' is a lower alkyl group, which may be substituted or
unsubstituted, branched or straight chain; and CH.sub.2.dbd.CH,
comprising: formylation of a starting alkylamine compound having
the structural formula: R--(CH.sub.2).sub.n--NH.sub.2, where n is
an integer in the range of 1-8, inclusive, where R is selected from
the group consisting of: an unsubstituted aromatic group; an
aromatic group substituted by one or more substituents selected
from the group consisting of: F, Cl, Br, a lower alkyl group, a
lower alkoxy group and a fluorinated lower alkyl group; R'--S(O),
where R' is a lower alkyl group, which may be substituted or
unsubstituted, branched or straight chain, to produce a formylated
intermediate having the structural formula:
R--(CH.sub.2).sub.n--NHCHO, where n and R are identical to R and n
of the starting alkylamine; contacting the formylated intermediate
with triphosgene and selenium powder in the presence of
triethylannine.
6. The method of claim 5, wherein the formylated intermediate is
contacted with triphosgene and selenium powder in the presence of
triethylanine and in the presence of a solvent.
7. A pharmaceutical composition, comprising: a compound having the
structural formula: R--(CH.sub.2).sub.n--N.dbd.C.dbd.X, where n is
an integer in the range of 1-8, inclusive, where X is S or Se, and
where R is selected from the group consisting of: an aromatic group
and a non-aromatic organic group.
8. The pharmaceutical composition of claim 7, wherein the compound
is selected from the group consisting of: a phenylalkyl
isothiocyanate, a phenylalkyl isoselenocyanate, and a combination
thereof.
9. The pharmaceutical composition of claim 7, wherein the compound
is an isoselenocyanate having the structural formula selected from
the group consisting of: ##STR00027## where n is 4 or 6; and
##STR00028## where R' is a substituted or unsubstituted, branched
or straight chain, lower alkyl group, and where n is an integer in
the range of 1-8, inclusive; and a pharmaceutically acceptable
carrier.
10. The composition of claim 7, further comprising a
pharmaceutically acceptable carrier.
11. The composition of claim 7, wherein the pharmaceutical
composition is formulated for topical application.
12. A method of treating a subject, comprising: administering a
therapeutically effective amount of a composition comprising a
compound having the structural formula:
R--(CH.sub.2).sub.n--N.dbd.C.dbd.X, where n is an integer in the
range of 1-8, inclusive, where X is S or Se, and where R is
selected from the group consisting of: an aromatic group and a
non-aromatic organic group to a subject in need thereof.
13. The method of claim 12, wherein the composition comprises a
compound having the structural formula:
phenyl-(CH.sub.2).sub.n--N.dbd.C.dbd.X, where n is an integer in
the range of 1-8, inclusive, where X is S or Se.
14. The method of claim 12, wherein the composition comprises an
isoselenocyanate having the structural formula: ##STR00029## where
R' is a substituted or unsubstituted, branched or straight chain,
lower alkyl group, and where n is an integer in the range of 3-8,
inclusive.
15. The method of claim 14 wherein R' is CH.sub.3.
16. The method of claim 12, wherein the composition comprises a
phenylalkyl isoselenocyanate having the structural formula:
##STR00030## where n is 4 or 6.
17. The method of claim 12 wherein the isoselenocyanate is selected
from the group consisting of: an isoselenocyanate glutathione
conjugate; an isoselenocyanate cysteine conjugate; and an
isoselenocyanate N-acetylcysteine conjugate.
18. The method of claim 12 wherein the subject is human.
19. The method of claim 12 wherein the subject has or is at risk of
having cancer.
20. The method of claim 19 wherein the cancer is a melanoma.
21. The method of claim 12 wherein the composition is formulated
for topical application.
22. The method of claim 19 wherein the cancer is characterized by
dysregulation of Akt.
23. The method of claim 19 wherein the cancer is characterized by
dysregulation of Akt3.
24. The method of claim 19, wherein administering the
therapeutically effective amount of the composition to a subject
detectably increases apoptosis and/or decreases proliferation of
cells of the cancer.
25. The method of claim 19, further comprising administration of an
adjunct anti-cancer treatment.
26. A method of modulating Akt dysregulation in a cell, comprising:
contacting the cell with an effective amount of an isoselenocyanate
having the structural formula: R--(CH.sub.2).sub.n--N.dbd.C.dbd.Se,
where n is an integer in the range of 1-8, inclusive, and where R
is selected from the group consisting of: an aromatic group and a
non-aromatic organic group.
27. The method of claim 26 wherein contacting the cell decreases a
component of an Akt signaling pathway selected from the group
consisting of: an Akt1 signaling pathway; an Akt2 signaling
pathway; an Akt3 signaling pathway; and a combination thereof.
Description
REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority from U.S. Provisional
Patent Application Ser. Nos. 60/911,565, filed Apr. 13, 2007 and
60/959,554, filed Jul. 13, 2007, the entire content of both
applications being incorporated herein by reference.
FIELD OF THE INVENTION
[0003] The present invention relates generally to anti-cancer
compositions and methods. In specific embodiments, the present
invention relates to compositions including one or more
isothiocyanates and/or isoselenocyanates, methods for treatment
and/or prevention of patlhological conditions in a subject using
one or more isothiocyanates and/or isoselenocyanates and methods
for synthesis of particular isoselenocyanates.
BACKGROUND OF THE INVENTION
[0004] In spite of recent medical progress, cancer continues to be
one of the most common and deadly diseases. Elucidation of
biochemical pathways involved in development and progression of
various cancers is important to identify potential anti-cancer
treatments as well as to develop agents effective to regulate such
pathways in other aspects of health and disease.
[0005] A particular cancer, melanoma, is the most deadly form of
skin cancer due to its high metastatic potential. Akt3 and
downstream PRAS40 are part of a key signaling cascade activated in
.about.70% of melanomas. Akt3 functions to reduce cellular
apoptosis in early melanomas, thereby promoting development of this
disease. Compositions and methods are required to inhibit the Akt
pathway and inhibit abnormal cell survival and proliferation.
SUMMARY OF THE INVENTION
[0006] Compositions including an isoselenocyanate having the
structural formula R--(CH.sub.2).sub.n--N.dbd.C.dbd.Se, where n is
an integer in the range of 1-8, inclusive, and where R is selected
from the group consisting of: an aromatic group and a non-aromatic
organic group are described herein.
[0007] In embodiments of the present invention, a composition
including an isoselenocyanate having the structural formula:
##STR00001##
where R' is a substituted or unsubstituted, branched or straight
chain, lower alkyl group, and where n is an integer in the range of
3-8, inclusive is provided along with methods of synthesis and use
of such compositions. In particular embodiments, R' is
CH.sub.3.
[0008] Embodiments of compositions of the present invention include
an isoselenocyanate having the structural formula:
##STR00002##
where R' is a substituted or unsubstituted, branched or straight
chain, lower alkyl group, and where n is an integer in the range of
3-8, inclusive. In particular embodiments, R' is CH.sub.3 and n is
4.
[0009] Methods for synthesis of an isoselenocyanate having the
structural formula: R--(CH.sub.2).sub.n--N.dbd.C.dbd.Se, where n is
an integer in the range of 1-8, inclusive, and where R is selected
from the group consisting of: an unsubstituted aromatic group; an
aromatic group substituted by one or more substituents selected
from the group consisting of: F, Cl, Br, a lower alkyl group, a
lower alkoxy group and a fluorinated lower alkyl group; R'--S(O),
where R' is a lower alkyl group, which may be substituted or
unsubstituted, branched or straight chain; and CH.sub.2.dbd.CH, are
provided according to the present invention. Methods of synthesis
of the present invention include formulation of a starting
alkylamine compound having the structural formula:
R--(CH.sub.2).sub.n--NH.sub.2, where n is an integer in the range
of 1-8, inclusive, where R is selected from the group consisting
of: an unsubstituted aromatic group; an aromatic group substituted
by one or more substituents selected from the group consisting of:
F, Cl, Br, a lower alkyl group, a lower alkoxy group and a
fluorinated lower alkyl group; R'--S(O), where R' is a lower alkyl
group, which may be substituted or unsubstituted, branched or
straight chain; and CH.sub.2.dbd.CH, to produce a formylated
intermediate having the structural formula:
R--(CH.sub.3).sub.n--NHCHO, where R and n are identical to R and n
of the starting alkylamine; and contacting the formylated
intermediate with triphosgene and selenium powder in the presence
of triethylamine. In a preferred option, the formylated
intermediate is contacted with triphosgene and selenium powder in
the presence of triethylamine and in the presence of a solvent,
such as dichloromethane.
[0010] Pharmaceutical compositions are provided according to
embodiments of the present invention which include one or more
isothiocyanates and/or isoselenocyanates having the structural
formula R--(CH.sub.2).sub.n--N.dbd.C.dbd.X, where n is an integer
in the range of 1-8, inclusive, where X is S or Se, and where R is
selected from the group consisting of: an aromatic group and a
non-aromatic organic group.
[0011] Pharmaceutical compositions according to embodiments of the
present invention include one or more isothiocyanates and/or
isoselenocyanates having the structural formula
phenyl-(CH.sub.2).sub.n--N.dbd.C.dbd.X, where n is an integer in
the range of 1-8, inclusive, and where X is S or Se.
[0012] Pharmaceutical compositions are provided according to
embodiments of the present invention which include an
isoselenocyanate having the structural formula selected from the
group consisting of:
##STR00003##
where n is 4 or 6;
##STR00004##
, where R' is a substituted or unsubstituted, branched or straight
chain, lower alkyl group, and where n is an integer in the range of
3-8, inclusive; and a pharmaceutically acceptable carrier.
Optionally, the pharmaceutically acceptable carrier is a
particulate carrier. In a further option, the pharmaceutical
composition is formulated for topical application.
[0013] Methods of treating a subject are provided according to the
present invention which include administering an effective amount
of a composition including an isoselenocyanate described herein to
a subject in need thereof. In embodiments of the present invention,
a composition administered to a subject in need thereof includes a
phenylalkyl isoselenocyanate having the structural formula:
##STR00005##
where n is 4 or 6. Optionally, the phenylalkyl moiety is
substituted at an available substitutable site. For example, the
phenylalkyl may be substituted by one or more substituents selected
from the group consisting of: F, Cl, Br, a lower alkyl group, a
lower alkoxy group and a fluorinated lower alkyl group.
[0014] Embodiments of compositions administered to a subject in
need thereof include an isoselenocyanate having the structural
formula:
##STR00006##
where R' is a substituted or unsubstituted, branched or straight
chain, lower alkyl group, and where n is an integer in the range of
3-8, inclusive. Optionally, R' is CH.sub.3. In a further option, R'
is CH.sub.3 and n is 4.
[0015] An isoselenocyanate composition is optionally conjugated to
glutathione, cysteine or N-acetylcysteine to produce an
isoselenocyanate glutathione conjugate; an isoselenocyanate
cysteine conjugate; and an isoselenocyanate N-acetylcysteine
conjugate for administration to a subject in need thereof.
[0016] In embodiments of methods including administration of an
isoselenocyanate to a subject, the subject is human.
[0017] In further embodiments, the subject has or is at risk of
having cancer. In certain embodiments, the subject has cancer or is
at risk for cancer characterized by dysregulation of Akt1, Akt2
and/or Akt3. In further embodiments, the cancer is a melanoma.
[0018] Methods of treating a subject are provided according to
embodiments of the present invention which include administering a
therapeutically effective amount of a composition including an
isothiocyanate and/or isoselenocyanate to a subject in need
thereof, wherein the isothiocyanate or isoselenocyanate has the
structural formula: R--(CH.sub.2).sub.n--N.dbd.C.dbd.X, where n is
an integer in the range of 1-8, inclusive, where X is S or Se, and
where R is selected from the group consisting of: an aromatic group
and a non-aromatic organic group and wherein the subject has a
condition characterized by Akt dysregulation, such as cancer,
pre-neoplastic hyperproliferation, cancer in-situ, neoplasms,
metastasis, tumor or benign growth. In certain embodiments of
methods of treatment of a subject, contacting cells characterized
by Akt dysregulation with a therapeutic amount of an isothiocyanate
and/or isoselenocyanate described herein decreases a component of
an Akt signaling pathway selected from the group consisting of: an
Akt1 signaling pathway; an Akt2 signaling pathway; an Akt3
signaling pathway; and a combination thereof. For example,
contacting the cell with an isothiocyanate and/or isoselenocyanate
decreases a component of an Akt signaling pathway selected from
pAkt1, pAkt2, pAk3, pPRAS40 and a combination thereof. In
embodiments of described methods, treatment of a subject with a
therapeutically effective amount of the composition including an
isothiocyanate and/or isoselenocyanate is substantially without
toxic effect on cells in which Akt is not dysregulated.
[0019] Methods of treating a subject are provided according to
embodiments of the present invention which include administering a
therapeutically effective amount of a composition including an
isothiocyanate and/or isoselenocyanate to a subject in need
thereof, wherein the isothiocyanate or isoselenocyanate has the
structural formula: phenyl-(CH.sub.2).sub.n--N.dbd.C.dbd.X, where n
is an integer in the range of 1-8, inclusive, where X is S or Se,
and wherein the subject has a condition characterized by Akt
dysregulation, such as cancer, pre-neoplastic hyperproliferation,
cancer in-situ, neoplasms, metastasis, tumor or benign growth.
Optionally the phenyl group is substituted.
[0020] Methods according to embodiments of the present invention
include administering a therapeutically effective amount of a
composition including an isoselenocyanate and/or an isothiocyanate
to a subject wherein the administration detectably increases
apoptosis and/or decreases proliferation of cells of the cancer,
pre-neoplastic hyperproliferation, cancer in-situ, neoplasms,
metastasis, tumor or benign growth.
[0021] Optionally, a composition including an isoselenocyanate
and/or isothiocyanate according to embodiments of the present
invention is formulated for topical application, for instance to
treat cancer, pre-neoplastic hyperproliferation, cancer in-situ,
neoplasms, metastasis, tumor or benign growth of the skin.
[0022] Optionally, methods of the present invention additionally
include administration of an adjunct anti-cancer treatment.
[0023] A method of modulating Akt dysregulation in a cell is
provided according to embodiments of the present invention which
includes contacting the cell with an effective amount of an
isothiocyanate or isoselenocyanate having the structural formula:
R--(CH.sub.2).sub.n--N.dbd.C.dbd.X, where n is an integer in the
range of 1-8, inclusive, where X is S or Se, and where R is
selected from the group consisting of: an aromatic group and a
non-aromatic organic group. In certain embodiments of methods of
modulating Akt dysregulation, contacting the cell with an
isoselenocyanate decreases a component of an Akt signaling pathway
selected from the group consisting of: an Akt1 signaling pathway;
an Akt2 signaling pathway; an Akt3 signaling pathway; and a
combination thereof. For example, contacting the cell with an
isoselenocyanate decreases a component of an Akt signaling pathway
selected from pAkt1, pAkt2, pAk3, pPRAS40 and a combination
thereof.
[0024] A method of modulating Akt dysregulation in a cell is
provided according to embodiments of the present invention which
includes contacting the cell with an effective amount of an
isothiocyanate or isoselenocyanate having the structural formula:
phenyl-(CH.sub.2).sub.n--N.dbd.C.dbd.X, where n is an integer in
the range of 1-8, inclusive, and where X is S or Se. Optionally,
the phenyl group is substituted.
[0025] A method of modulating Akt dysregulation in a cell is
provided according to embodiments of the present invention which
includes contacting the cell with an effective amount of an
isoselenocyanate having the structural formula:
R--(CH.sub.2).sub.n--N.dbd.C.dbd.Se, where n is an integer in the
range of 1-8, inclusive, and where R is selected from the group
consisting of: an aromatic group and a non-aromatic organic group.
In certain embodiments of methods of modulating Akt dysregulation,
contacting the cell with an isoselenocyanate decreases a component
of an Akt signaling pathway selected from the group consisting of:
an Akt1 signaling pathway; an Akt signaling pathway; an Akt3
signaling pathway; and a combination thereof. For example,
contacting the cell with an isoselenocyanate decreases a component
of an Akt signaling pathway selected from pAkt1, pAkt2, pAk3,
pPRAS40 and a combination thereof.
BRIEF DESCRIPTION OF THE DRAWINGS
[0026] FIG. 1A is a bar graph showing a comparison of cell
viability following exposure to PBITC or ISC-4, compared to
controls;
[0027] FIG. 1B is a bar graph showing a comparison of cell
viability following exposure to PHITC or ISC-6, compared to
controls;
[0028] FIG. 2A is a bar graph of results of proliferation analysis
of UACC 903 cells treated with DMSO or different concentrations (5
uM, 10 uM, 15 uM) of API-2, PHSC, PBITC or ISC-4 for 24 hours;
[0029] FIG. 2B is a bar graph of results of proliferation analysis
of UACC 903 cells treated with DMSO or different concentrations (5
uM, 10 uM, 15 uM) of API-2, PHSC, PHITC or ISC-6 for 24 hours;
[0030] FIG. 3A is a bar graph showing the effects of treatment of
UACC 903 cells in culture with DMSO or 5 .mu.M, 10 .mu.M or 15
.mu.M of API-2, PHSC, PBITC or ISC-4 for 24 hours on caspase-3/7
activity, an indicator of apoptosis;
[0031] FIG. 3B is a bar graph showing the effects of treatment of
UACC 903 cells in culture with DMSO or 5 .mu.M, 10 .mu.M or 15
.mu.M of API-2, PHSC, PHITC or ISC-6 for 24 hours on caspase-3/7
activity, an indicator of apoptosis;
[0032] FIG. 4A is a bar graph showing results of cell cycle
analysis of UACC 903 cells treated with controls (API-2, PHSC),
PHITC or ISC-4;
[0033] FIG. 4B is a bar graph showing results of cell cycle
analysis of UACC 903 cells treated with controls (API-2, PHSC),
PHITC or ISC-6;
[0034] FIG. 5A is a bar graph showing the effect of isothiocyanates
on tumor development;
[0035] FIG. 5B is a bar graph showing the effect of
isoselenocyanates on tumor development;
[0036] FIG. 6A is a line graph showing chance in tumor size and
body weight (inset) over time in mice treated i. p. with PBITC or
PHITC (0.76 .mu.moles) or ISC-4 or ISC-6 (0.76 .mu.moles,
equivalent to 3 ppm selenium);
[0037] FIG. 6B is a line graph showing change in tumor size and
body weight (inset) over time in mice treated i. p. with PBITC or
PHITC (0.76 .mu.moles) or ISC-4 or ISC-6 (0.76 .mu.moles,
equivalent to 3 ppm selenium);
[0038] FIG. 7A is a bar graph showing results of analysis of
apoptosis following ISC-4 treatment compared to treatment with
PBITC or DMSO, in size and time matched tumors;
[0039] FIG. 7B is a bar graph showing results of analysis of cell
proliferation following ISC-4 treatment compared to treatment with
PBITC or DMSO, in size and time matched tumors;
[0040] FIG. 8 is a bar graph showing results of analysis of levels
of SGOT, SGPT, alkaline phosphatase, glucose and creatinine in
blood collected from animals treated with PBITC, ISC-4 or DMSO
vehicle;
[0041] FIG. 9 is a reproduction of an image of an immunoblot
showing dose dependent decreases in phosphorylated (active) Akt
(S473) and downstream PRAS40 (T246) and a corresponding dose
dependent increase in cleaved PARP, reflective of high levels of
cellular apoptosis in cells treated with ISC-4 or ISC-6;
[0042] FIG. 10A is a reproduction of an image of an immunoblot
showing the effect of ISC-4 on Akt signaling pathway in melanoma
cell line 1205 Lu;
[0043] FIG. 10B is a reproduction of an image of an immunoblot
showing the effect of ISC-4 on Akt signaling pathway in melanoma
cell line WM115;
[0044] FIG. 11 is a bar graph showing quantitation of immunoblot
analysis of tumor protein lysates from animals treated with DMSO,
PBITC or ISC-4 and indicates decreased relative expression of
phosphorylated (active) Akt and downstream PRAS40 of Akt3;
[0045] FIG. 12 is a line graph showing that decreased expression
(activity) of Akt3 reduced the tumor size in animals injected with
Akt3 siRNA treated cells compared to control cells nucleofected
with scrambled siRNA or nucleofection buffer.
[0046] FIG. 13 is a reproduction of an image of an immunoblot
analysis of tumor protein lysates showing reduction in expression
of Akt3 in tumors by siRNA directed against Akt3;
[0047] FIG. 14A is a line graph showing inhibition of tumor
development by 1-isoselenocyanto-4-methylsulfinylbutane (SFN Iso
Se, also interchangeably called ISC-SFN4 herein) compared to
controls.
[0048] FIG. 14B is a line graph showing a lack of significant
changes in body weight in treated animals, indicating that a lack
of toxicity of the administered compounds;
[0049] FIG. 15 is a bar graph showing results of a cell viability
assay measuring inhibitory efficacy of DMSO, ISC-1, ISC-2, ISC-4,
ISC-6, BITC, PEITC, PBITC, or PHITC on UACC 903 cells;
[0050] FIG. 16 is a bar graph showing the effect of DMSO or 5-15
.mu.M of ISC-1, ISC-2, ISC-4, ISC-6, BITC, PEITC, PBITC, or PHITC
on cell proliferation;
[0051] FIG. 17 is a bar graph showing the effect of DMSO or 5-15
.mu.M of ISC-1, ISC-2, ISC-4, ISC-6, BITC, PEITC, PBITC, or PHITC
on apoptosis;
[0052] FIG. 18A is a line graph showing effect of selected
isoselenocyanates on tumor size;
[0053] FIG. 18B is a line graph showing effect of selected
isoselenocyanates on body weight;
[0054] FIG. 18C is a line graph showing effect of selected
isothiocyanates on tumor size; and
[0055] FIG. 18D is a line graph showing effect of selected
isothiocyanates on body weight;
[0056] FIG. 19 is a bar graph showing the effects of topically
applied PBITC or ISC-4 on reconstructed human skin containing GFP
tagged UACC 903 human melanoma cells;
[0057] FIG. 20 is a pair of line graphs showing the effect of
topical ISC4 application on melanoma tumor growth in vivo; and
[0058] FIG. 21 is a pair of line graphs showing the effect of
topical ISC-4 application on melanoma tumor growth in vivo.
DETAILED DESCRIPTION OF THE INVENTION
[0059] Anti-cancer compositions and methods are provided according
to embodiments of the present invention. In certain embodiments,
the present invention relates to compositions including one or more
isothiocyanates and/or isoselenocyanates, methods for treatment
and/or prevention of pathological conditions in a subject using one
or more isothiocyanates and/or isoselenocyanates and methods for
synthesis of particular isoselenocyanates.
[0060] A composition provided according to embodiments of the
present invention includes one or more compounds having the
structural formula: R--(CH.sub.2).sub.n--N.dbd.C.dbd.Se, where n is
an integer in the range of 1-8, inclusive, where R is an aromatic
group or a non-aromatic organic group.
[0061] The term "aromatic" as used herein refers to an optionally
substituted monocyclic or bicyclic hydrocarbon ring system
containing at least one unsaturated aromatic ring. Non-limiting
examples of aromatic groups include phenyl and napthyl.
[0062] In particular embodiments, compositions of the present
invention are phenylalkyl isoselenocyanates having the structural
formula:
##STR00007##
[0063] where n is 1-8.
[0064] R is optionally an aromatic group substituted by one or more
of the following: F, Cl, Br, a lower alkyl group, a lower alkoxy
group or fluorinated lower alkyl group, such as CF.sub.3. In
particular embodiments, R is phenyl group substituted by one or
more of the following: F, Cl, Br, a lower alkyl group, a lower
alkoxy group or fluorinated lower alkyl group, such as
CF.sub.3.
[0065] The term "lower alkoxy" as used herein refers to a straight
chain or branched hydrocarbon group containing from 1-4 carbon
atoms which is appended to the parent molecular moiety through an
oxygen atom. Illustrative examples of lower alkyl groups are
methoxy, ethoxy, propoxy, 2-propoxy, butoxy and tert-butoxy.
[0066] The term "lower alkyl" as used herein refers to a straight
chain or branched hydrocarbon group containing from 1-4 carbon
atoms. Illustrative examples of lower alkyl groups are methyl,
ethyl, iso-propyl, n-propyl, n-butyl, iso-butyl, sec-butyl and
tert-butyl.
[0067] In particular embodiments, a composition of the present
invention includes a sulfoxide composition according to the present
invention having the structural formula:
##STR00008##
where R' is a lower alkyl group, which may be substituted or
unsubstituted, branched or straight chain, and where n is an
integer in the range of 1-8, inclusive.
[0068] In certain embodiments, a composition of the present
invention includes a sulfoxide composition according to the present
invention having the structural formula:
##STR00009##
where R' is a lower alkyl group, which may be substituted or
unsubstituted, branched or straight chain, and where n is an
integer in the range of 3-8, inclusive.
[0069] In still further embodiments, a composition of the present
invention includes a sulfoxide composition according to the present
invention having the structural formula:
##STR00010##
where CH.sub.3 can be substituted or unsubstituted and where n is
an integer in the range of 3-8, inclusive.
[0070] Embodiments of a composition according to the present
invention have the structural formulas:
CH.sub.3--S(O)--(CH.sub.2).sub.3--N.dbd.C.dbd.Se (termed ISC-SFN3);
CH.sub.3--S(O)--(CH.sub.2).sub.4--N.dbd.C.dbd.Se (termed ISC-SFN4
and SFN Iso Se); CH.sub.3--S(O)--(CH.sub.2).sub.5--N.dbd.C.dbd.Se
(termed ISC-SFN5); CH.sub.3--S(O)--(CH.sub.2).sub.6--N.dbd.C.dbd.Se
(termed ISC-SFN6); CH.sub.3--S(O)--(CH.sub.2).sub.7--N.dbd.C.dbd.Se
(termed ISC-SFN7) and
CH.sub.3--S(O)--(CH.sub.2).sub.8--N.dbd.C.dbd.Se (termed ISC-SFN8)
Optionally, CH.sub.3 is substituted or unsubstituted.
[0071] In certain embodiments, R in the structural formula:
R--(CH.sub.2).sub.n--N.dbd.C.dbd.Se, where n is an integer in the
range of 1-8, is a substituted aromatic group. In further
embodiments, R is a phenyl group substituted at one or more
substitutable sites by Cl, Br, F, methyl, methoxy, and/or a
fluorinated lower alkyl group, such as CF.sub.3.
[0072] The non-aromatic organic group optionally includes one or
more heteroatoms such as S, N, O and/or P.
[0073] In further embodiments where R is a non-aromatic organic
group, R is CH.sub.2.dbd.CH. For example, where R is
CH.sub.2.dbd.CH, an isoselenocyanate has the formula:
CH.sub.2.dbd.CH--CH.sub.2--N.dbd.C.dbd.Se.
[0074] In a further option, the group (CH.sub.2).sub.n in the
formula R--(CH--).sub.n--N.dbd.C.dbd.Se, is substituted, For
example, the group (CH.sub.2).sub.n in the formula
R--(CH.sub.2).sub.n--N.dbd.C.dbd.Se, is substituted by one or more
of the following: F, Cl, Br, a lower alkyl group, a lower alkoxy
group or fluorinated lower alkyl group, such as CF.sub.3.
[0075] Compositions including mixtures of two or more
isoselenocyanates are also specifically contemplated and are
considered to be within the scope of the present invention.
[0076] Structures of particular compounds described herein, along
with abbreviations used, are shown below. Particular naturally
occurring and synthetic phenylalkyl isothiocyanate compounds with
increasing chain length in the left column; phenylalkyl
isoselenocyanates of the present invention are shown in the center
column; and phenylhexyl selenocyanate in the right column.
Phenylhexyl selenocyanate (PHSC) is used as a control compound in
particular tests described herein since it is similar to ISC-6 in
structure and contains selenium.
##STR00011##
[0077] Methods for synthesis of an isoselenocyanate are provided
according to embodiments of the present invention.
[0078] A process for synthesis of a compound according to
embodiments of the present invention is shown in Scheme 1.
Treatment of a phenylalkylamine (1) with ethyl formate leads to the
formation of intermediate 2, which on reaction with selenium powder
in the presence of triphosgene and triethylamine, in refluxing
dichloromethane, furnishes the desired isoselenocyanate (3).
##STR00012##
[0079] Methods for synthesis of isothiocyanates and
isoselenocyantes derivatives with a substituted phenyl ring are
also provided by the present invention.
[0080] Substituted phenyl ring, analogs of phenylalkyl
isoselenocyanates are synthesized (Scheme 2a), following
substantially identical methodologies as described in Scheme 1 for
the synthesis of isoselenocyanates. In a similar manner,
substituted phenylalkyl isothiocyanates are synthesized by treating
the corresponding substituted arylalkylamine with thiophosgene and
sodium hydroxide as shown in Scheme 2b. Several combinations of
substitutions of phenyl ring (e.g. 2-, 3- or 4-chloro (Cl), bromo
(Br), fluoro (--F), methyl (CH.sub.3), methoxy (OCH.sub.3) and
trifluoromethyl (--CF.sub.3) substituted arylalkyl isothiocyanates
and isoselenocyanates are synthesized starting from corresponding
appropriately substituted 2-, 3-, and
4-Cl/Br/F/CH.sub.3/OCH.sub.3/CF.sub.3-phenylalkylamines.
##STR00013##
[0081] Methods of synthesis of isosteric selenium analogs of
non-aromatic naturally occurring isothiocyanates are provided by
the present invention. For example, methods of synthesis of
isosteric selenium analogs of sulforaphane i.e.,
1-isoselenocyanato-4-(methylsulfinyl)butane (ISC-SFN4, also called
SFN Iso Se herein), are provided according to embodiments of the
present invention. The synthetic route followed is outlined in
Scheme 3. The key intermediate 1-amino-4-(methylsulfinyl)butane (1
in scheme 3) is synthesized and subjected to a sequence of
reactions as shown in Scheme 3 to obtain the desired ISC-SFN4 (3).
Further analogs, including ISC-SFN3, ISC-SFN6, ISC-SFN7, and
ISC-SFN8 which are isosteric selenium analogs of corresponding
naturally occurring sulfoxide isothiocyanate analogs with varying
alkyl chain length are synthesized using a similar synthetic
strategy.
##STR00014##
[0082] In further embodiments, an allyl isoselenocyanate is
synthesized according to embodiments of the present invention
starting from allylamine as detailed in Scheme 4.
##STR00015##
[0083] Conjugate Compositions
[0084] A compound of the present invention is conjugated to one or
more property-enhancing moieties according to embodiments of the
present invention for modification of one or more characteristics
of the compound. The present invention provides conjugates of
organic isothiocyanates and/or isoselenocyanates, in order to
reduce toxicity, increase solubility and/or increase
bioavailability in particular embodiments of the present invention.
Methods of synthesis of such conjugates are also provided by
embodiments of the presently described invention.
[0085] For example, in particular embodiments, a compound of the
present invention is conjugated to a water solubility-enhancing
moiety, to yield a conjugate which is more water soluble than the
compound. Thus, in particular embodiments, water soluble
isothiocyanate or isoselenocyanate compounds of the present
invention are conjugated to glutathione (GSH), cysteine (Cys) or
N-acetylcysteine (NAC) to yield the corresponding GSH-, Cys-, or
NAC-conjugate.
[0086] An exemplary structure of a glutathione conjugate of a
phenylalkyl isoselenocyanate, where n is an integer in the range of
1-8, inclusive.
##STR00016##
[0087] An exemplary structure of a cysteine conjugate of a
phenylalkyl isoselenocyanate, where n is an integer in the range of
1-8, inclusive:
##STR00017##
[0088] An exemplary structure of an N-acetylcysteine conjugate of a
phenylalkyl isoselenocyanate, where n is an integer in the range of
1-8, inclusive:
##STR00018##
[0089] The NAC-conjugates of organic isoselenocyanates, such as
ISC-4 and ISC-6, are made by reacting corresponding
isoselenocyanate with N-acetylcysteine in aqueous ethanol (50%) at
room temperature under nitrogen atmosphere. The GSH or cysteine
conjugates of isoselenocyanates are synthesized following a similar
procedure.
[0090] Compositions according to embodiments of the present
invention prevent and inhibit cancer cell multiplication and tumor
development and are considered useful as chemotherapeutic and
chemopreventive agents. In addition, isoselenocyanate compositions
according to embodiments of the present invention induce cell death
in cancer cells more effectively than corresponding isothiocyanates
or derivatives thereof. Further, animal studies show significant
reduction in melanoma tumor development by isoselenocyanates of the
present invention at doses three times lower than those of
corresponding isothiocyanates, without significant toxicity.
[0091] Methods and compositions are provided according to the
present invention for treating cancer. Particular cancers treated
using methods and compositions described herein are characterized
by abnormal cell proliferation including, but not limited to,
pre-neoplastic hyperproliferation, cancer in-situ, neoplasms and
metastasis. Methods and compositions of the present invention can
be used for prophylaxis as well as amelioration of signs and/or
symptoms of cancer.
[0092] A therapeutically effective amount of a composition is an
amount which has a beneficial effect in a subject being treated. In
subjects having cancer or at risk for having cancer, such as a
condition characterized by abnormal cell proliferation including,
but not limited to, pre-neoplastic hyperproliferation, cancer
in-situ, neoplasms, metastasis, a tumor, a benign growth or other
condition responsive to an isoselenocyanate composition, a
therapeutically effective amount of a composition is effective to
ameliorate or prevent one or more signs and/or symptoms of the
condition. For example, a therapeutically effective amount of a
composition is effective to detectably increase apoptosis and/or
decrease proliferation of cells of a cancer condition characterized
by abnormal cell proliferation including, but not limited to,
pre-neoplastic hyperproliferation, cancer in-situ, neoplasms,
metastasis, a tumor, a benign growth or other condition responsive
to an isoselenocyanate composition.
[0093] In particular embodiments, cancers treated using methods and
compositions described herein are characterized by Akt
dysregulation.
[0094] Akt, a serine/threonine protein kinase also known as protein
kinase B, has a stimulatory effect on cell cycle progression, cell
proliferation and inhibition of apoptosis. Akt proteins, nucleic
acids and signaling pathway components are described, for instance,
see Testa, J. R. et al., PNAS, 98:10983-10985; Fayard, E. et al.,
J. Cell Sci., 118:5675-5678, 2005; Cheng, J. and S. Nicosia, (2001)
AKT signal transduction pathway in ancogenesis, in Encyclopedic
Reference of Cancer, D. Schwab, Editor. 2001, Springer: Berlin,
Germany, p. 35-7; Datta, S. R., et al. (1999) Cellular survival: a
play in three Akts. Genes Dev, 13(22): 2905-27; Fayard, E. et al.
(2005) J Cell Sci, 118 (Pt 24: 5675-8; Mirza, A. M., Fayard, E. et
al. (2000) 2000. 11 (6: 279-92; Nicholson, K. M. and N. G.
Anderson, (2002) Cell Signal, 2002, 14(5): p. 381-95; Paez, J. and
W. Sellers, (2003) P13K/PTEN/Akt Pathway: A Critical Mediator of
Oncogenic Signaling, in Signal Transduction in Cancer, D. Frankc,
Editor. 2003, Kluwer Academic Publishers: Netherlands; and Testa,
J. R.; P. N. Tsichlis, (2005) Oncogene, 24(50): 7391-3 and other
references listed herein.
[0095] Akt family members, Akt1, Akt2 and Akt3, are activated by
phosphorylation, membrane translocation, increases in gene copy
number and/or loss of a negative regulatory phosphatase, PTEN.
Increased activation of Akt, including increased levels of Akt
and/or increased levels of phosphorylated Akt is an indicator of
Akt dysregulation associated with proliferation and cell survival
in pathogenic conditions, such as cancer.
[0096] Akt3 is active in .about.70% of melanomas. While all three
Akt isoforms are expressed in melanocytes and melanoma cells, Akt3
is the predominantly active family member. Dysregulated Akt3
activity in melanoma cells reduces cellular apoptosis mediated
through caspase-3, thereby promoting melanoma tumor
development.
[0097] Akt dysregulation is determined, for instance, by
measurement of Akt gene copy number, Akt protein or RNA levels
and/or levels of phosphorylated Akt1, in cells known or suspected
to be dysplasic, pre-cancerous, cancerous, metastatic or otherwise
characterized by abnormal cell proliferation compared to normal
cells. Assays for Akt dysregulation include, but are not limited to
immunoassays and nucleic acid assays.
[0098] Methods of treating a subject are provided according to
embodiments of the present invention which include administering a
therapeutically effective amount of a composition including an
isothiocyanate and/or isoselenocyanate to a subject in need
thereof, wherein the isothiocyanate or isoselenocyanate has the
structural formula: R--(CH.sub.2).sub.n--N.dbd.C--X, where n is an
integer in the range of 1-8, inclusive, where X is S or Se, and
where R is selected from the group consisting of: an aromatic group
and a non-aromatic organic group and wherein the subject has a
condition characterized by Akt dysregulation, such as cancer,
pre-neoplastic hyperproliferation, cancer in-situ, neoplasms,
metastasis, tumor or benign growth. In certain embodiments of
methods of treatment of a subject, contacting cells characterized
by Akt dysregulation with a therapeutic amount of an isothiocyanate
and/or isoselenocyanate described herein decreases a component of
an Akt signaling pathway selected from the group consisting of: an
Akt1 signaling pathway; an Akt2 signaling pathway; an Akt3
signaling pathway; and a combination thereof. For example,
contacting the cell with an isothiocyanate and/or isoselenocyanate
decreases a component of an Akt signaling pathway selected from
pAkt1, pAkt2, pAk3, pPRAS40 and a combination thereof. In
embodiments of described methods, treatment of a subject with a
therapeutically effective amount of the composition including an
isothiocyanate and/or isoselenocyanate is substantially without
toxic effect on cells in which Akt is not dysregulated.
[0099] Methods of treating a subject are provided according to
embodiments of the present invention which include adrninistering a
therapeutically effective amount of a composition including an
isothiocyanate and/or isoselenocyanate to a subject in need
thereof, wherein the isothiocyanate or isoselenocyanate has the
structural formula: phenyl-(CH.sub.2).sub.n--N.dbd.C.dbd.X, where n
is an integer in the range of 1-8, inclusive, where X is S or Se,
and wherein the subject has a condition characterized by Akt
dysregulation, such as cancer, pre-neoplastic hyperproliferation,
cancer in-situ, neoplasms, metastasis, tumor or benign growth.
[0100] Methods of treating a subject are provided according to
embodiments of the present invention which include administering a
therapeutically effective amount of a composition including an
isothiocyanate and/or isoselenocyanate to a subject in need
thereof, wherein the isothiocyanate or isoselenocyanate is BITC,
PEITC, PBITC, PHITC, ISC-1, ISC-2, ISC-4 or ISC-6 and wherein the
subject has a condition characterized by Akt dysregulation, such as
cancer, pre-neoplastic hyperproliferation, cancer in-situ,
neoplasms, metastasis, tumor or benign growth.
[0101] Methods of treating a subject are provided according to
embodiments of the present invention which include administering an
effective amount of a composition including an isoselenocyanate to
a subject in need thereof.
[0102] A method of treating a subject is provided according to
embodiments of the present invention which includes administering
to a subject in need thereof a therapeutically effective amount of
a an isoselenocyanate compound having the structural formula;
R--(CH.sub.2).sub.n--N.dbd.C.dbd.Se, where n is an integer in the
range of 1-8, inclusive, where R is an aromatic group or a
non-aromatic organic group.
[0103] In embodiments of the present invention, a method of
treating a subject includes administering an effective amount of an
isoselenocyanate having the structural formula:
##STR00019##
where R' is a substituted or unsubstituted, branched or straight
chain, lower alkyl group, and where n is an integer in the range of
3-8, inclusive.
[0104] In embodiments of the present invention, a method of
treating a subject includes administering an effective amount of an
isoselenocyanate having the structural formula:
##STR00020##
where R' is CH.sub.3 and where n is an integer in the range of 1-8,
inclusive.
[0105] In embodiments of the present invention, a method of
treating a subject includes administering an effective amount of an
isoselenocyanate having the structural formula:
##STR00021##
where R' is CH.sub.3 and n is 4.
[0106] In embodiments of the present invention, a method of
treating a subject, includes administering an effective amount of a
phenylalkyl isoselenocyanate having the structural formula:
##STR00022##
where n is 4 or 6.
[0107] Optionally, an administered isoselenocyanate is an
isoselenocyanate glutathione conjugate; an isoselenocyanate
cysteine conjugate; or an isoselenocyanate N-acetylcysteine
conjugate.
[0108] Optionally, an administered isoselenocyanate is a
pharmaceutically acceptable salt, ester or amide of an
isoselenocyanate described herein.
[0109] Isothiocyanate and/or isoselenocyanate compositions are
provided according to embodiments of the present invention which
inhibit tumor growth by inhibiting an Akt signaling cascade,
particularly an Akt3 signaling cascade, in cells characterized by
Akt dysregulation in certain embodiments.
[0110] Methods including administration of one or more
isothiocyanates and/or isoselenocyanates to a subject in need
thereof are provided according to particular embodiments of the
present invention which have utility, for example, in inhibiting
the Akt signaling cascade and inhibiting cancer cells.
[0111] Inhibitors of the Akt signaling cascade according to
embodiments of the present invention have utility in treatment of
subject having cancer or at risk of having cancer in which Akt
deregulation occurs, such as in melanoma and other cancers
including, but not limited to, cancers of the prostate, breast,
brain, ovary, lung, colon, connective tissues (sarcomas) and soft
tissue.
[0112] Methods of modulating an Akt protein, such as an Akt1, Akt2
and/or an Akt3 protein, in a cell are provided according to
embodiments of the present invention which include contacting the
cell with an effective amount of an isoselenocyanate.
[0113] Pharmaceutical compositions including an isoselenocyanate of
the present invention are also provided according to embodiments of
the present invention.
[0114] A pharmaceutical composition includes an isoselenocyanate of
the present invention and a pharmaceutically acceptable carrier in
particular embodiments of the present invention. The term
"pharmaceutically acceptable carrief" refers to a carrier which is
substantially non-toxic to a subject to which the composition is
administered and which is substantially chenmically inert with
respect to a selenium-containing compound of the present
invention.
[0115] A pharmaceutical composition according to the invention
generally includes about 0.1-99% of an isoselenocyanate of the
present invention. Combinations of isoselenocyanates in a
pharmaceutical composition are also considered within the scope of
the present invention.
[0116] Optionally, a method of treating a subject having cancer or
at risk of having cancer further includes an adjunct anti-cancer
treatment. An adjunct anti-cancer treatment can be administration
of an anti-cancer agent.
[0117] Anti-cancer agents are described, for example, in Goodman et
al., Goodman and Gilman's The Pharmacological Basis of
Therapeutics, 8th Ed., Macmillan Publishing Co., 1990.
[0118] Anti-cancer agents illustratively include acivicin,
aclarubicin, acodazole, acronine, adozelesin, aldesleulcin,
alitretinoin, allopurinol, altretamine, ambomycin, ametantrone,
arifostine, aminoglutethimide, amsacrine, anastrozole, anthramycin,
arsenic trioxide, asparaginase, asperlin, azacitidine, azetepa,
azotomycin, batimastat, benzodepa, bicalutamide, bisantrene,
bisnafide dimesylate, bizelesin, bleomycin, brequinar, bropirimine,
busulfan, cactinomycin, calusterone, capecitabine, caracemide,
carbetimer, carboplatin, carmustine, carubicin, carzelesin,
cedefingol, celecoxib, chlorambucil, cirolemycin, cisplatin,
cladribine, crisnatol mesylate, cyclophosphamide, cytarabine,
dacarbazine, dactinomycin, daunorubicin, decitabine, dexormaplatin,
dezaguanine, dezaguanine mesylate, diaziquone, docetaxel,
doxorubicin, droloxifene, dromostanolone, duazomycin, edatrexate,
eflomithine, elsamitrucin, enloplatin, enpromate, epipropidine,
epirubicin, erbulozole, esorubicin, estramustine, etanidazole,
etoposide, etoprine, fadrozole, fazarabine, fenretinide,
floxuridine, fludarabine, fluorouracil, fluorocitabine, fosquidone,
fostriecin, fulvestrant, gemcitabine, hydroxyurea, idarubicin,
ifosfamide, ilmofosine, interleulcin II (IL-2, including
recombinant interleukin II or rIL2), interferon alfa-2a, interferon
alfa-2b, interferon alfa-n1, interferon alfa-n3, interferon
beta-Ia, interferon gamma-Ib, iproplatin, irinotecan, lanreotide,
letrozole, leuprolide, liarozole, lometrexol, lomustine,
losoxantrone, masoprocol, maytansine, mechlorethamine
hydrochloride, megestrol, melengestrol acetate, melphalan,
menogaril, mercaptopurine, methotrexate, metoprine, meturedepa,
mitindomide, mitocarcin, mitocromin, mitogillin, mitomalcin,
mitomycin, mitosper, mitotane, mitoxantrone, mycophenolic acid,
nelarabine, nocodazole, nogalamycin, ormnaplatin, oxisuran,
paclitaxel, pegaspargase, peliomycin, pentamustine, peplomycin,
perfosfamide, pipobroman, piposulfan, piroxantrone hydrochloride,
plicamycin, plomestane, porfimer, porfiromycin, prednimustine,
procarbazine, puromycin, pyrazofurin, riboprine, rogletimide,
safingol, semustine, simtrazene, sparfosate, sparsomycin,
spirogermanium, spiromustine, spiroplatin, streptonigrin,
streptozocin, sulofenur, talisomycin, tamoxifen, tecogalan,
tegafur, teloxantrone, temoporfin, teniposide, teroxirone,
testolactone, thiamiprine, thioguanine, thiotepa, tiazofurin,
tirapazamine, topotecan, toremifene, trestolone, triciribine,
trimetrexate, triptorelin, tubulozole, uracil mustard, uredepa,
vapreotide, verteporfin, vinblastine, vincristine sulfate,
vindesine, vinepidine, vinglycinate, vinleurosine, vinorelbine,
vinrosidine, vinzolidine, vorozole, zeniplatin, zinostatin,
zoledronate, and zorubicin.
[0119] An adjunct anti-cancer treatment can be a radiation
treatment of a subject or an affected area of a subject's body.
[0120] Pharmaceutical compositions suitable for delivery to a
subject may be prepared in various forms illustratively including
physiologically acceptable sterile aqueous or nonaqueous solutions,
dispersions, suspensions or emulsions, and sterile powders for
reconstitution into sterile injectable solutions or dispersions.
Examples of suitable aqueous and nonaqueous carriers include water,
ethanol, polyols such as propylene glycol, polyethylene glycol,
glycerol, and the like, suitable mixtures thereof; vegetable oils
such as olive oil; and injectable organic esters such as
ethyloleate. Proper fluidity can be maintained, for example, by the
use of a coating such as lecithin, by the maintenance of the
required particle size in the case of dispersions, and by the use
of surfactants, such as sodium lauryl sulfate. Additional
components illustratively including a buffer, a solvent, or a
diluent may be included.
[0121] Such formulations are administered by a suitable route
including parenteral and oral administration. Administration may
include systemic or local injection, and particularly intravenous
injection.
[0122] These compositions may also contain adjuvants such as
preserving, wetting, emulsifying, and dispensing agents. Prevention
of the action of microorganisms can be ensured by various
antibacterial and antifungal agents, for example, parabens,
chlorobutanol, phenol, sorbic acid, and the like. It may also be
desirable to include isotonic agents, for example, sugars, sodium
chloride, and substances similar in nature. Prolonged delivery of
an injectable pharmaceutical form can be brought about by the use
of agents delaying absorption, for example, aluminum monostearate
and gelatin.
[0123] Solid dosage forms for oral administration include capsules,
tablets, pills, powders, and granules. In such solid dosage forms,
selenium-containing compound of the present invention is admixed
with at least one inert customary excipient (or carrier) such as
sodium citrate or dicalcium phosphate or (a) fillers or extenders,
as for example, starches, lactose, sucrose, glucose, mannitol, and
silicic acid, (b) binders, as for example, carboxymethylcellulose,
alignates, gelatin, polyvinylpyrrolidone, sucrose, and acacia, (c)
humectants, as for example, glycerol, (d) disintegrating agents, as
for example, agar-agar, calcium carbonate, plant starches such as
potato or tapioca starch, alginic acid, certain complex silicates,
and sodium carbonate, (e) solution retarders, as for example,
paraffin, (f) absorption accelerators, as for example, quaternary
ammonium compounds, (g) wetting agents, as for example, cetyl
alcohol, glycerol monostearate, and glycols (h) adsorbents, as for
example, kaolin and bentonite, and (i) lubricants, as for example,
talc, calcium stearate, magnesium stearate, solid polyethylene
glycols, sodium lauryl sulfate, or mixtures thereof. In the case of
capsules, tablets, and pills, the dosage forms may also include a
buffering agent.
[0124] Solid compositions of a similar type may also be employed as
fillers in soft and hard-filled gelatin capsules using such
excipients as lactose or milk sugar as well as high molecular
weight polyethyleneglycols, and the like.
[0125] Solid dosage forms such as tablets, dragees, capsules,
pills, and granules can be prepared with coatings and shells, such
as enteric coatings and others well known in the art. They may
contain opacifying agents, and can also be of such composition that
they release the active compound or compounds in a certain part of
the intestinal tract in a delayed manner. Examples of embedding
compositions which can be used are polymeric substances and waxes.
The active compounds can also be in micro-encapsulated form, if
appropriate, with one or more of the above-mentioned
excipients.
[0126] Liquid dosage forms for oral administration include a
pharmaceutically acceptable carrier formulated as an emulsion,
solution, suspension, syrup, or elixir. In addition to the active
compounds, the liquid dosage forms may contain inert diluents
commonly used in the art, such as water or other solvents,
solubilizing agents and emulsifiers, as for example, ethyl alcohol,
isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol,
benzyl benzoate, propyleneglycol, 1,3-butyleneglycol,
dimethylformamide, oils, in particular, cottonseed oil, groundnut
oil, corn germ oil, olive oil, castor oil and sesame oil, glycerol,
tetrahydrofurfuryl alcohol, polyethyleneglycols and fatty acid
esters of sorbitan or mixtures of these substances, and the
like.
[0127] Besides such inert diluents, the composition can also
include adjuvants, such as wetting agents, emulsifying and
suspending agents, sweetening, flavoring, and perfuming agents.
[0128] Suspensions, in addition to an inventive conjugate, may
contain suspending agents, as for example, ethoxylated isostearyl
alcohols, polyoxyethylene sorbitol and sorbitol esters,
microcrystalline cellulose, aluminum metahydroxide, bentonite,
agar-agar or tragacanth, or mixtures of these substances, and the
like.
[0129] In particular embodiments, compositions of the present
invention are formulated for topical application. In further
particular embodiments, compositions of the present invention are
formulated for topical application and are characterized by less
than 10% absorption of an active ingredient in the composition into
the system of an individual treated topically. In still further
particular embodiments, compositions of the present invention are
formulated for topical application and are characterized by less
than 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% absorption of an active
ingredient in the composition into the system of an individual
treated topically. Absorption into the system of an individual can
be measured by any of various methods, particularly assay for the
active ingredient, a metabolite and/or a breakdown product of the
active ingredient in a sample obtained from an individual treated
with the topical formulation. For example, a blood, plasma or serum
sample can be assayed for presence of the active ingredient, a
metabolite of the active ingredient and/or a breakdown product of
the active ingredient.
[0130] A topical formulation can be an ointment, lotion, cream or
gel in particular embodiments. Topical dosage forms such as
ointment, lotion, cream or gel bases are described in Remington:
The Science and Practice of Pharmnacy, 21.sup.st Ed., Lippincott
Williams & Wilkins, 2006, p. 880-882 and p. 886-888; and in
Allen, L. V. et al., Ansel's Pharmaceutical Dosage Forms and Drug
Delivery Systems, 8.sup.th Ed., Lippincott Williams & Wilkins,
2005, p. 277-297.
[0131] Pharmaceutically acceptable carriers and formulation of
pharmaceutical compositions are known in the art, illustratively
including, but not limited to, as described in Remington: The
Science and Practice of Pharmacy, 21.sup.st Ed., Lippincott,
Williams & Wilkins, Philadelphia, Pa., 2006; and Allen, L. V.
et al., Ansel's Pharmaceutical Dosage Forms and Drug Delivery
Systems, 8.sup.th Ed., Lippincott, Williams & Wilkins,
Philadelphia, Pa., 2005.
[0132] The term subject refers to an individual in need of
treatment for a pathological condition, particularly cancer, and
generally includes mammals and birds, such as, but not limited to,
humans, other primates, cats, dogs, cows, horses, rodents, pigs,
sheep, goats and poultry.
[0133] A pharmaceutical composition according to the present
invention is suitable for administration to a subject by a variety
of systemic and/or local routes including, but not limited to,
intravenous, intramuscular, subcutaneous, intraperitoneal, oral,
otic, rectal, vaginal, topical, parenteral, pulmonary, ocular,
nasal, intratumoral and mucosal.
[0134] An inventive composition may be administered acutely or
chronically. For example, a composition as described herein may be
administered as a unitary dose or in multiple doses over a
relatively limited period of time, such as seconds--hours. In a
further embodiment, administration may include multiple doses
administered over a period of days--years, such as for chronic
treatment of cancer.
[0135] With regard to administration of isoselenocyanates to a
mammalian subject, particular exemplary effective dosage ranges
without significant systemic toxicity are described in terms of
amounts of selenium administered via administration of the
isoselenocyanate. Thus, for example, when delivered by a parenteral
route, such as intraperitoneal or intravenous, an exemplary
therapeutically effective dosage of an isoselenocyanate is in the
range of about 1-4 ppm selenium, administered three times per week.
It is noted that the dose range, "about 1-4 ppm selenium" refers to
a dose of "about 1 mg/kg-4 mg/kg of selenium." For example, a dose
of 3 ppm selenium when referring to ISC-4 is equivalent to a dose
of 9.1 mg/kg of ISC-4. Similarly, a dose of 3 ppm selenium when
referring to ISC-6 is equivalent to a dose of 10.17 mg/kg of ISC-6.
In a further example, when delivered topically, an exemplary
therapeutically effective dosage of ISC-4 or ISC-6 is in the range
of about 0.1-1 ppm selenium, administered daily. In a further
example, when delivered orally, an exemplary therapeutically
effective dosage of an isoselenocyanate is in the range of about
1-15 ppm selenium.
[0136] A therapeutically effective amount of a pharmaceutical
composition according to the present invention will vary depending
on the particular pharmaceutical composition used, the severity of
the condition to be treated, the species of the subject, the age
and sex of the subject and the general physical characteristics of
the subject to be treated. One of skill in the art could determine
a therapeutically effective amount in view of these and other
considerations typical in medical practice. In general it is
contemplated that a therapeutically effective amount would be in
the range of about 0.001 mg/kg-100 mg/kg body weight, optionally in
the range of about 0.01-10 mg/kg, and further optionally in the
range of about 0.1-5 mg/kg. Further, dosage may be adjusted
depending on whether treatment is to be acute or continuing.
[0137] Advantageously, anti-cancer compounds according to
embodiments of the present invention are formulated to achieve
lipid-solubility and/or aqueous-solubility.
[0138] In particular embodiments, a pharmaceutically acceptable
carrier is a particulate carrier such as lipid particles including
liposomes, micelles, unilamellar or mulitlamellar vesicles; polymer
particles such as hydrogel particles, polyglycolic acid particles
or polylactic acid particles; inorganic particles such as calcium
phosphate particles such as described in for example U.S. Pat. No.
5,648,097; and inorganic/organic particulate carriers such as
described for example in U.S. Pat. No. 6,630,486.
[0139] A particulate pharmaceutically acceptable carrier can be
selected from among a lipid particle; a polymer particle; an
inorganic particle; and an inorganic/organic particle. A mixture of
particle types can also be included as a particulate
pharmaceutically acceptable carrier.
[0140] A particulate carrier is typically formulated such that
particles have an average particle size in the range of about 1
nm-10 microns. In particular embodiments, a particulate carrier is
formulated such that particles have an average particle size in the
range of about 1 nm-100 nm.
[0141] Embodiments of inventive compositions and methods are
illustrated in the following examples. These examples are provided
for illustrative purposes and are not considered limitations on the
scope of inventive compositions and methods.
EXAMPLES
Example 1
Cell Lines and Culture Conditions
[0142] The human metastatic melanoma cell lines UACC 903 and 1205
Lu; normal human fibroblast cells (FF2441) are maintained in DMEM
(Invitrogen, Carlsbad, Calif.) supplemented with 10% FBS (Hyclone,
Logan, Utah). Vertical growth phase (VGP) melanoma cell line WM115
is maintained in Tu2% medium lacking calcium chloride, supplemented
with 2% heat treated (56.degree. C. for 30 minutes) PBS and
L-glutamine (Mediatech, Handon, Va.) as described in Stahl J M, et
al., Cancer Res 2004; 64:7002-10.
[0143] Colon adenocarcinoma cell line (Caco-2, ATCC No. HTB-37) is
grown either in Advanced DMEM supplemented with 10% heat treated
(56.degree. C. for 30 minutes) FBS and L-glutamine. Fibrosarcoma
(HT-1080; ATCC No. CCL-121), prostate adenocarcinoma (PC-3; ATCC
No. CRL-1435), breast adenocarcinoma cell line (MDA-MB-231; ATCC
No. HB-26), glioblastoma cell line (T98G; ATCC No. CRL-1690) and
human melanoma cell line UACC903 are grown in DMEM supplemented
with 10% FBS.
Example 2
Chemical Synthesis
[0144] Synthetic methods described in Examples 2-12 refer generally
to the following numbered structures:
##STR00023##
[0145] Melting points were recorded on a Fisher-Johns melting point
apparatus and are uncorrected. Unless stated otherwise, proton NMR
spectra were recorded in a Bruker AM 360WB instrument using
CDCl.sub.3 as solvent. The chemical shifts are reported in ppm
downfield from TMS. High-resolution MS (EI) are determined at the
Chemistry Instrumentation Center, State University of New York at
Buffalo, N.Y. Thin-layer chromatography (TLC) is developed on
aluminum-supported, pre-coated silica gel plates (EM Industries,
Gibbstown, N.J.). Column chromatography was conducted on silica gel
(60-200 mesh). Benzyl isothiocyanate (BITC, la), phenylethyl
isothiocyanate (PEITC, 1b), and phenylbutylisothiocyanate (PBITC,
1c) are obtained from commercial sources. Phenylhexylisothiocyanate
(PHITC, 1d) is synthesized as described in Morse, M. A. et al.,
Cancer Res 1991, 51, (7), 1846-50.
Example 3
[0146] A general method for the synthesis of phenylalkylformamides
is described in Elliott, M. C.; Williams, E., Synthesis and
reactions of partially reduced biisoquinolines. Org Biomol Chem
2003, 1 (17), 3038-47. Ethyl formate (120 mmol) was added dropwise
to phenylalkylamine (40 mmol) at room temperature and the resulting
mixture was refluxed for 4-6 h. The excess ethyl formate was
removed under reduced pressure to yield the corresponding
phenylakylformamide as an oil.
[0147] Phenylethylformamide: (AS3.090) .sup.1H NMR (CDCl.sub.3)
.delta. 2.84 (t, 2H, J=6.9 Hz), 3.57 (dt, 21, J=6.9 Hz and 6.6 Hz),
5.68 (br d, 1H, NH), 7.15-7.35 (m, 51), 8.12 (s, 1H, CHO); HRMS (I)
calcd for C.sub.9H.sub.11NO, 149.0835; found, 149.0839.
[0148] Phenylbutylformamide. .sup.1H NMR (CDCl.sub.3) .delta.
1.56-1.62 (m, 2H), 1.66-1.72 (m, 2H), 2.66 (t, 2H, J=6.5 Hz), 3.35
(dt, 2H, J=7.0 and 6.5 Hz), 5.92 (hr s, 1H), 7.18-7.24 (m, 2H),
7.29-7.33 (m, 2H), 8.19 (s, 1H); HRMS (ED calcd for
C.sub.11H.sub.15NO, 177.1148; found, 177.1149.
[0149] Phenylhexylformamide. .sup.1H NMR (CDCl.sub.3) .delta.
1.36-1.41 (m, 4H), 1.52-1.58 (m, 2H), 1.61-1.67 (m, 21), 2.63 (t,
2H, J=7.5 Hz), 3.30 (dt, 211, J=7.0 and 6.5 Hz), 5.58 (br s, 1H),
7.18-7.21 (m, 3H), 7.28-7.31 (m, 2H), 8.19 (s, 1H); HRMS (EI) calcd
for Cl.sub.3H.sub.19NO, 205.1461; found, 205.1462.
Example 4
[0150] Isoselenocyanates are synthesized using a modified method
described in Fernandez-Bolanos, J. G., Lopez, O., Ulgar, V., Maya,
I., and Fuentes, J., Synthesis of O-unprotected glycosyl
selenoureas. A new access to bicyclic sugar isoureas, Tetrahedron
Lett. 2004, 45, 4081-4084. Solid triphosgene is used in a one-pot
dehydration of the formamides in refluxing dichloromethane (Scheme
5).
##STR00024##
[0151] Scheme 5 shows synthesis of compounds 1 and 2; Reagents and
conditions: (a) C.sub.2H.sub.5OCHO, -20.degree. C. to reflux (b)
Et.sub.3N, triphosgene, Se powder, CH.sub.2Cl.sub.2, reflux (c)
CSCl.sub.2, NaOH.
[0152] General experimental procedure for the synthesis of ISC
compounds The synthetic strategy involves the formylation of
phenylalkylamines, followed by treatment with triphosgene and
selenium powder in the presence of triethylamine to furnish the
desired phenylalkyl isoselenocyanates (2) in good yields as
oils.
[0153] The compounds are purified by silica gel column
chromatography and are characterized on the basis of NMR and
high-resolution MS data. (Pure isolated compounds are light yellow
(ISC-1 and ISC-2) to colorless (ISC-4 and ISC-6) which tend to get
a little darker after storing for longer time).
[0154] In a particular example, to a refluxing mixture of the aryl
alkyl formamides (1.5 mmol), triethylamine (6.4 mmol) in
CH.sub.2Cl.sub.2 (5 mL) and 4 .ANG. molecular sieves was added
dropwise a solution of triphosgene (0.8 mmol) in CH.sub.2Cl.sub.2
(2 mL) for a period of 1 hour. After the addition was complete, the
mixture was refluxed for an additional 2.5 hour. Selenium powder
(3.0 mmol) was then added and the resulting mixture was refluxed
for other 6-8 hours. The mixture is cooled, filtered, and the
solvent was evaporated to yield the crude mixture, which is
purified by silica gel column chromatography to afford
isoselenocyanates.
[0155] In a further example, a solution of triphosgene (5.0 mmol)
in CH.sub.2Cl.sub.2 (15 mL) is added over a 1 hour period to a
refluxing mixture of phenylalkylformamides (10.0 mmol),
triethylamine (43.0 mmol) and 4 .ANG. molecular sieves in
CH.sub.2Cl.sub.2) (35 mL). The mixture is then refluxed for an
additional 2.5 hours. Selenium powder (20 mmol) is then added and
resulting mixture refluxed for 6-8 hours. Mixture is cooled,
filtered, and solvent evaporated yielding a crude mixture, which is
purified by silica gel column chromatography generating pure
isoselenocyanates. Isothiocyanates and isoselenocyanates are
>99% pure.
[0156] Phenylalkyl isothiocyanates are obtained commercially (BITC,
PEITC, and PBITC). PHITC is synthesized as described in Morse, M.
A. et al., Cancer Res 1991, 51, (7), 1846-50. Phenylhexylamine (3d)
required for the synthesis of 1d is synthesized by converting
phenylhexyl chloride to the corresponding azide by treatment with
sodium azide in DMF, followed by generation of the 3d by reduction
of azide with lithium aluminium hydride as described in
Gopalakrishnan, G. et al., J. Labelled. Comp. Radiopharma. 1988,
25, (4), 383-393.
Example 5
[0157] Benzyl isoselenocyanate (2a). To a refluxing mixture of the
formamides (1.5 mmol), triethylamine (6.4 mmol) in CH.sub.2Cl.sub.2
(5 mL) and 4 .ANG. molecular sieves was added dropwise a solution
of triphosgene (0.8 mmol) in CH.sub.12Cl.sub.2 (2 mL) for a period
of 1 h. After the addition was complete, the mixture was refluxed
for an additional 2.5 h. Selenium powder (3.0 mmol) was then added
and the resulting mixture was refluxed for other 6 h. The mixture
was cooled, filtered, and the solvent was evaporated to yield the
crude mixture, which was purified by silica gel column
chromatography to afford isoselenocyanates. (AS3.094): viscous oil,
.sup.1H NMR (CDCJ.sub.3) .delta. 4.81 (s, 2H, CH.sub.2), 7.30-7.44
(m, 5H); ARMS (EI) calcd for C.sub.8H.sub.7NSe, 196.9738; found,
196.9741.
Example 6
[0158] Phenylethyl isoselenocyanate (2b). (AS3.091) A mixture of
phenylethylformamide (g, 1.5 mmol), triethylamine (6.4 mmol), 4
.ANG. molecular sieves (g), triphosgene (0.8 mmol), and selenium
powder (3.0 mmol) in CH.sub.2Cl.sub.2 was refluxed and worked up as
mentioned for 2a. The crude residue thus obtained was purified by
silica gel column chromatography (EtOAc/hexanes 2:98) to give 0.96
g (95%) of 2b as an oil. .sup.1H NMR (CDCl.sub.3) .delta. 3.03 (t,
2H, J=6.9 Hz), 3.81 (t, 2H, J=6.9 Hz) 7.20-7.38 (m, 5H); HRMS (EI)
calcd for C.sub.9H.sub.9NSe, 210.9895; found, 210.9892.
Example 7
[0159] Phenylbutyl isoselenocyanate (2c). A mixture of phenylbutyl
formamide (g, 1.5 mmol) triethylamine (6.4 mmol), 4 .ANG. molecular
sieves (g), triphosgene (0.8 mmol), and selenium powder (3.0 mmol)
in CH.sub.2Cl.sub.2 was refluxed and worked up as mentioned for 2a.
The crude residue thus obtained was purified by silica gel column
chromatography (EtOAc/hexanes 2:98) to give 0.96 g (95%) of 2c as
an oil. .sup.1H NMR (CDCl.sub.3) .delta. 1.74-1.76 (m, 4H), 2.66
(t, 2H, J=6.6 Hz), 3.60 (t, 2H, J=5.6 Hz), 7.15-7.32 (m, 5H); HRMS
(EI) calcd for C.sub.11H.sub.13NSe, 239.0208; found, 239.0211.
Example 8
[0160] Phenylhexyl isoselenocyanate (2d). A mixture of phenylbutyl
formamide (g, 1.5 mmol), triethylamine (6.4 mmol), 4 .ANG.
molecular sieves (g), triphosgene (0.8 mmol), and selenium powder
(3.0 mmol) in CH.sub.2Cl.sub.2 was refluxed and worked up as
mentioned for 2a. The crude residue thus obtained was purified by
silica gel column chromatography (EtOAc/hexanes 2:98) to give 0.96
g (95%) of 2d as an oil. .sup.1H NMR (CDCl.sub.3) .delta. 1.37-1.43
(m, 2H), 1.45-1.51 (m, 2H), 1.67 (dt, 2H, J=15.2 and 7.6 Hz), 1.75
(dt, 2H, J=14.9 and 6.7 Hz), 2.64 (t, 2H, J=7.6 Hz), 3.60 (t, 2H,
J=6.7 Hz), 7.19-7.25 (m, 31), 7.32-7.40 (m, 2H); HRMS (EI) calcd
for C.sub.13H.sub.17NSe, 267.0521; found, 267.0529.
Example 9
ISC-4-NAC Conjugate (5)
[0161] Scheme 6 shows the synthesis of NAC conjugate (5) of ISC-4.
The IC.sub.50 of 5 (UACC 903M cells)=17.+-.2.
##STR00025##
Example 10
[0162] 1-isoselenocyanato-4-(methylsulfinyl)butane (ISC-SFN4, also
called SFN Iso Se herein). viscous oil; .sup.1H NMR in CDCl.sub.3:
3.72 (t, 2H, J=6.53 Hz, N--CH.sub.2), 3.10 (t, 2H, J=6.94 Hz,
SO--C.sub.2), 2.97 (s, 3H, S--CH.sub.3),1.98-2.07 (m, 4H,
C--CH.sub.2--CH.sub.2--C).
Example 11
IC.sub.50 Values of ITC and ISC Compounds
[0163] To measure the inhibitory potency (IC.sub.50) of
isothiocyanate and isoselenocyanate derivatives in various cancer
cells, MTS (CellTiter 96 Aqueous Non Radioactive Cell Proliferation
Assay kit, Promega, Madison, Wis.) is used. Cellular viability is
quantified by MIS assay and dose response curves plotted.
5.times.10.sup.3 melanoma cells (UACC 903, 1205 LU or WM115) cells
per well in 100 .mu.L DMEM containing 10% FBS are grown in a
96-well plate for 24 h and then treated with increasing
concentrations of the indicated isothiocyanate or isoselenocyanate
for 24 h. After 24 hours of treatment viable cells are measured by
reading soluble colored formazan product at 490 nm using a
microplate reader. Results of 3 independent experiments are
considered for the determination of IC.sub.50. The IC.sub.50 values
are calculated from the concentration curves using Graph Pad Prism
software and are shown in Table I for cells and compounds
indicated.
TABLE-US-00001 TABLE I IC.sub.50 (.mu.M) 24 h drug treatment Cell
Line BITC PEITC PBITC PHITC ISC-1 ISC-2 ISC-4 ISC-6 UACC 903 15
.+-. 3 12 .+-. 1 15 .+-. 1 15 .+-. 2 16 .+-. 3 12 .+-. 4 12 .+-. 3
10 .+-. 1 1205 Lu >25 24 .+-. 2 19 .+-. 5 11 .+-. 4 >25 18
.+-. 4 11 .+-. 1 7 .+-. 4 WM 115 13 .+-. 1 12 .+-. 1 7 .+-. 1 8
.+-. 1 >25 8 .+-. 1 7 .+-. 2 7 .+-. 2
[0164] Table I shows a comparison of the IC.sub.50 of
isothiocyanates and isoselenocyanates in three independently
derived melanoma cell lines, UACC 903, 1205 Lu and WM115. A general
trend is observed in which increasing carbon chain length and
substitution of selenium for sulfur decreased the IC.sub.50.
Increased potency ranged from 30-70% with increasing chain length
and/or sulfur substituted for selenium. Thus, isothiocyanate
analogs with longer alkyl chain lengths and sulfur substituted for
selenium are better inhibitors of cultured melanoma cells.
Example 12
SAR Study on Cancer Cell Lines
[0165] Certain ITCs (1) and the corresponding ISCs (2) are tested
for their ability to inhibit cell growth in five cancer cell lines
e.g. melanoma, breast, glioblastoma, fibrosarcoma, colon and
prostate cancers. Iin vitro inhibitory efficacy of cancer cell
lines representing different cancer types following treatment with
ITC and ISC is measured using the
3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-
-2H-tetrazolium (MTS) assay (Promega, Madison, Wis.). In brief,
2.5-5.times.10.sup.3 cells per well in 100 .ANG. DMEM containing
10% FBS are grown in a 96-well plate for 24 h and treated with
either control DMSO vehicle or increasing concentrations (2.5-100
.mu.M) of ITC and ISC for 24 hours. At this point cells are treated
individually with either vehicle control DMSO or with increasing
concentrations of ITC or ISC (2.5-100 .mu.M) for 24 h. The
percentages of viable cells compared to control DMSO treated cells
are determined using MTS assay and IC.sub.50 values calculated
using GraphPad Prism version 4.01 (GraphPad software, San Diego,
Calif.). IC.sub.50 value for each compound was determined by at
least three independent experiments and represented with a standard
error (Table II).
[0166] The IC.sub.50 values for compounds 1 and 2 are depicted in
Table II. The values consistently decreased with increasing alkyl
chain length of ITCs in case of MDA-MB-231, T98G, and PC-3 cell
lines; but showed no particular trend in case of HT-1080, Caco-2,
and UACC 903 cells. ISC-1 was least effective in killing cells in
all the cell lines tested compared to its higher alkyl chain
analogs ISC-2 to ISC-6. Among ISC-2, ISC-4 and ISC-6 there was no
particular trend. The values generally decreased with increasing
chain length for all the cancer cell lines tested except breast
cancer cells MDA-MB 231 in case of ISC compounds and sarcoma HT
1080 cells in case of ITCs. Except for ISC-1 and ISC-2 in UACC 903
cells, the ISC derivatives had lower IC.sub.50 values than
corresponding ITCs.
TABLE-US-00002 TABLE II IC.sub.50 (.mu.M) of ITC and ISC
derivatives on different cancer cells Cancer cell lines IC.sub.50
(.mu.M) Breast Glioblastoma Prostate Fibrosarcoma Colon Melanoma
Compounds MDA-MB-231 T-98-G PC-3 HT-1080 Caco-2 UACC 903 1a (BITC)
42 .+-. 3 >100 >50 >50 15 .+-. 2 15 .+-. 3 1b (PEITC) 38
.+-. 6 >100 24 .+-. 2 15 .+-. 1 14 .+-. 2 12 .+-. 1 1c (PBITC)
27 .+-. 2 35 .+-. 1 24 .+-. 2 15 .+-. 1 27 .+-. 2 15 .+-. 1 1d
(PHITC) 24 .+-. 2 26 .+-. 2 17 .+-. 1 29 .+-. 3 49 .+-. 9 15 .+-. 1
2a (ISC-1) 29 .+-. 2 43 .+-. 4 24 .+-. 1 13 .+-. 3 13 .+-. 3 16
.+-. 3 2b (ISC-2) 20 .+-. 3 24 .+-. 1 16 .+-. 1 12 .+-. 3 11 .+-. 1
12 .+-. 4 2c (ISC-4) 21 .+-. 64 27 .+-. 1 19 .+-. 1 11 .+-. 1 12
.+-. 3 12 .+-. 3 2d (ISC-6) 22 .+-. 2 23 .+-. 2 14 .+-. 1 12 .+-. 1
10 .+-. 1 10 .+-. 1 Values are mean .+-. S.E. * Drug treatment for
72 h
[0167] Low values are estimated for isothiocyanates and
isoselenocyanates using ChemDraw 9.0 Ultra and these values are
compared in Table III.
TABLE-US-00003 TABLE III ITCs CLogP ISCs CLogP 1a (BITC) 3.204 2a
(ISC-1) 3.177 1b (PEITC) 3.263 2b (ISC-2) 3.506 1c (PBITC) 4.171 2c
(ISC-4) 4.414 1d (PHITC) 5.229 2d (ISC-6) 5.472 .sup.aLogP was
estimated using ChemDraw 9.0 Ultra
Example 13
[0168] Cell viability of melanoma cells following treatment with
isothiocyanate or an isoselenocyanate is measured using the
3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-
-2H-tetrazolium (MTS) assay (Promega, Madison, Wis.).
[0169] Briefly, 5.times.10.sup.3 melanoma cells (UACC 903, 1205 LU
or WM115) or human fibroblast (FF2441) cells per well in 100 .mu.L
DMEM containing 10% FBS are grown in a 96-well plate for 24 h and
treated with either control DMSO vehicle; phenylhexyl selenocyanate
(PHSC), used as a control compound since it is similar to ISC-6 in
structure and contains selenium; API-2, an Akt inhibitor used for
comparison purposes; or increasing concentrations (2.5-50 .mu.M) of
an isothiocyanate or an isoselenocyanate for 24 h. Cellular
viability compared to control treated cells is measured using the
MTS assay. IC.sub.50 values for each compound in respective cell
lines is determined from three independent experiments using
GraphPad Prism version 4.01 (GraphPad software, San Diego,
Calif.).
[0170] FIG. 1A shows a comparison of cell viability following
exposure to PBITC or ISC-4, compared to controls. FIG. 1B shows a
comparison of cell viability following exposure to PHITC or ISC-6,
compared to controls. Cell viability is determined using MTS assay.
ISC-4 effectively reduces cell viability at concentrations of 10
.mu.M and 15 .mu.M (IC.sub.50=12.+-.3 and 10.+-.1 .mu.M) compared
to controls (API-2, PHSC) or PBITC or PHITC. (IC.sub.50=15.+-.1 and
15.+-.2 .mu.M). The average value represented as the percentage of
control DMSO treated cells. Phenylhexyl selenocyanate (PHSC) is
used as a control compound since it is similar to ISC-6 in
structure and contains selenium. API-2 is an Akt inhibitor used for
comparison purposes. Thus, ISC-4 and ISC-6 are more effective at
inhibiting growth of melanoma cells than sulfur containing PBITC,
PHITC, control PHSC or API-2.
Example 14
[0171] Cellular proliferation in vitro is measured by seeding
5.times.10.sup.3 cells in 96-well plate, followed by treatment for
24 hours with vehicle control DMSO, or 5 .mu.M, 10 .mu.M or 15
.mu.M of APT-2 (a known Akt inhibitor), PHSC, PBITC, PANIC, ISC-4,
or ISC-6. Proliferation rate is measured using a BrdU ELISA kit
(Roche Applied Sciences, Indianapolis, Ind.).
[0172] FIG. 2A shows results of proliferation analysis of UACC 903
cells treated with DMSO or different concentrations (5 uM, 10 uM,
15uM) of API-9, PHSC, PBITC or ISC-4 for 24 hours. FIG. 2B shows
results of proliferation analysis of UACC 903 cells treated with
DMSO or different concentrations (5 uM, 10 uM, 15 uM) of API-2,
PHSC, PHITC or ISC-6 for 24 hours. BrdUrd is used to label cells
for 4-6 hours. At 15 .mu.M concentration, ISC-4 and ISC-6 reduced
UACC 903 cellular proliferative potential by .about.80-90% compared
to controls. Results represent the average of 3 independent
experiments; bars represent SEM. The average value is represented
as the percentage of control DMSO treated cells.
[0173] In contrast to the Akt inhibitor API-2 or the control
6-carbon selenium compound PHSC, increasing concentrations of
PBITC, ISC-4, PHITC or ISC-6 led to decreased proliferative
potential of treated cells. Both ISC-4 and ISC-6 are .about.2-fold
more effective than PBITC or PHITC at inhibiting cellular
proliferation.
Example 15
[0174] Apoptosis rates are measured by seeding 5.times.10.sup.3
cells in 96-well plate, followed by treatment for 24 hours with
vehicle control DMSO; API-2; PHSC; PBITC; PAI.dbd.C; ISC-4; or
ISC-6. Apoptosis rates are measured using an Apo-ONE Homogenous
caspase-3/7 Assay kit (Promega Corporation, Madison, Wis.).
[0175] FIG. 3A shows the effects of treatment of UACC 903 cells in
culture with DMSO or 5 .mu.M, 10 .mu.M or 15 .mu.M of API-2, PHSC,
PBITC or ISC-4 for 24 hours on caspase-3/7 activity, an indicator
of apoptosis. FIG. 3B shows the effects of treatment of UACC 903
cells in culture with DMSO or 5 .mu.M, 10 .mu.M or 15 .mu.M of
API-2, PHSC, PHITC or ISC-6 for 24 hours on caspase-3/7 activity,
an indicator of apoptosis. The isoselenocyanate compounds promote
apoptosis in melanoma cells. Levels of caspase-3/7 activity in
cells exposed to API-2, PHSC, PBITC, PHITC, ISC-4 or ISC-6 are
measured using the Apo-ONE homogeneous caspase-3/7 assay kit. The
graphs in FIGS. 3A and 3B show fold increase in caspase-3/7
activity relative to DMSO vehicle treated cells on the x-axis.
Results represent average of 3 independent experiments. Bars
indicate SEM. In contrast to API-2 and PHSC, increasing
concentrations of PBITC, ISC-4, PHITC and ISC-6 increase cellular
apoptosis of UACC 903 melanoma cells as shown in FIGS. 3A and 3B.
ISC-4 and ISC-6 are .about.2-fold more effective than PBITC at
inducing apoptosis.
Example 16
[0176] Cell cycle analysis is performed by plating
1.5.times.10.sup.6 melanoma cells in 100-mm culture dish. Two days
following plating, cells are treated with DMSO, or 5 .mu.M, 10
.mu.M or 15 .mu.M of API-2, PHSC, PBITC, PHITC, ISC-4, or ISC-6 for
24 hours. Cells are collected by trypsinization and stained using
propidium iodide (13). Trypsinized cells are centrifuged
(500.times.g, for 5 minutes) and treated with 1 mL of propidium
iodide staining solution (100 .mu.g/mL; Sigma, St Louis, Mo.), 20
.mu.g/mL Ribonuclease A (Roche Applied Sciences, Indianapolis,
Ind.) 3 .mu.g/mL Triton X-100 dissolved in 0.1% (W/V) sodium
citrate for 30 minutes at 4.degree. C. Stained cells are analyzed
using the FACScan analyzer (Becton Dickinson, San Jose, Calif.) and
data analyzed using ModFit LT software (Verity Software House,
Topsham, Me.).
[0177] FIG. 4A shows results of cell cycle analysis of UACC 903
cells treated with controls (API-2, PHSC), PBITC or ISC-4, or, DMSO
vehicle control. FIG. 4B shows results of cell cycle analysis of
UACC 903 cells treated with controls (API-2, PHSC), PIIITC or
ISC-6, or, DMSO vehicle control. ISC-4, ISC-6, PBITC or PHITC
treatment increased the sub G0/G1 cell population (an indicator of
apoptosis) and induces G2/M cell cycle arrest in melanoma cells
compared to DMSO or controls API-2 and PHSC. Results represent
average of 2 independent experiments. Enhanced apoptosis induced by
ISC-4 or ISC-6 is confirmed through cell cycle analysis of
asynchronously growing UACC 903 cells. A significant increase is
observed in the sub G0/G1 population in PBITC, ISC-4, PHITC or
ISC-6 treated cells which is indicative of cells undergoing
apoptosis.
[0178] Table IV shows results of cell cycle analysis following
treatment with controls compounds DMSO, API-2 or PHSC or test
compounds PBITC, PHITC, ISC-4 or ISC-6. Treated cells are stained
with propidium iodide and cell cycle analyzed using a FACScan
analyzer such that the proportion of cells in each phase of cell
cycle (G0/G1, St G2/M) is estimated. The G2/M cell population
increases 2-3 fold following 15 .mu.M PBITC, PHITC, ISC-4 or ISC-6
exposure. A significant decrease in G0/G1 cell population occurred
following ITC or ISC treatment. Enhanced apoptosis induced by ISC-4
or ISC-6 is confirmed through cell cycle analysis of asynchronously
growing UACC 903 cells. Analysis of cells in each stage of the cell
cycle showed decreasing numbers of cells in the S- and G1/G0 phase
with a corresponding increase in the G2/M phase.
TABLE-US-00004 TABLE IV UACC 903 - Cell Cycle Analysis Following
Treatment With ISC-4 or ISC-6 and Corresponding Isothiocyanates
Concentration API-2 PHSC PBITC ISC-4 PHITC ISC-6 (.mu.M) DMSO 15 15
5 10 15 5 10 15 5 10 15 5 10 15 subG0/G1 0.4 0.89 2.4 4.9 10.4 20.8
2.9 12.3 22.8 1.4 5.4 12.5 1.5 7.6 15.9 G0/G1 60.8 63.6 61.4 37.4
37.2 42.1 59.4 41.6 35.5 58.7 43.8 40.7 56.8 44.9 41.6 S 27.7 25.6
26.9 27.8 30.9 30.5 24.3 29.0 36.5 28.2 28.7 29.6 31.5 30.5 32.2
G2/M 11.4 10.7 11.5 34.7 31.9 27.4 16.3 29.4 28.0 13.1 27.5 29.7
13.5 24.5 26.1
Example 17
[0179] Isoselenocyanates are effective for inhibiting melanoma
tumor development in preexisting tumors in nude mice.
[0180] Tumor kinetics are measured by subcutaneous injection of
2.5-5.times.10.sup.6 1205 Lu or UACC 903 melanoma cells in 0.2 ml
of DMEM supplemented with 10% FBS above both left and right rib
cages of 4-6 week old female nude mice (Harlan Sprague Dawley,
Indianapolis, Ind.). Six days later when a fully vascularized tumor
has formed, mice are randomly divided into DMSO vehicle control and
experimental groups including 5 mice/group, each mouse having two
tumors. Mice are treated by intraperitoneal (i.p.) injection with
an ITC compound BITC, PEITC, PBITC, or PHITC (2.5 .mu.moles or 0.76
.mu.moles); or treated by i.p injection with an ISC compound
ISC-1-ISC-2, ISC-4 or ISC-6 (0.76 .mu.moles, equivalent to 3 ppm
selenium) thrice per week. (Monday, Wednesday and Friday). Control
mice received an equal volume of the vehicle. Dimensions of the
developing tumors are measured using calipers and the size
estimated in cubic millimeters. Body weight is monitored three
times a week (Monday, Wednesday and Friday).
[0181] FIGS. 5A and 5B show graphs comparing the effect of
isothiocyanates and isoselenocyanates on tumor development using
UACC 903 melanoma cells, a cell line having high Akt3 signaling
activity. Six days following subcutaneous injection of 5 million
UACC 903 melanoma cells, small vascularized palpable tumors are
seen and mice are treated i. p. with 2.5 .mu.moles of an
isothiocyanate or with 0.76 .mu.L moles, equivalent to 3 ppm
selenium, of an isoselenocyanate thrice per week. The bar graphs in
FIGS. 5A and 5B show melanoma tumor volume, as % of vehicle, at day
24. Isoselenocyanates ISC-2, ISC-4 and ISC-6 reduce tumor volume at
doses 3.times. lower than isothiocyanates. ISC, 0.76 .mu.moles vs.
ITC, 2.5 .mu.moles. Thus, isoselenocyanates have increased in vivo
tumor inhibitory effectiveness compared to corresponding
isothiocyanates and effectively reduce melanoma development.
Increasing carbon chain length of isothiocyanates showed less
effective tumor inhibition, FIG. 5A, while increasing carbon chain
length of isoselenocyanates led to greater tumor inhibition, FIG.
5B.
Example 18
Isoselenocyanates Retard Melanoma Tumor Development
[0182] The effect of isothiocyanates and isoselenocyanates on tumor
development is measured in existing tumors formed by subcutaneous
injection of 2.5 or 5 million 1205 Lu or UACC 903 melanoma cells.
Six days following injection of the cells, when small vascularized
palpable tumors are seen, mice are treated i. p. with PBITC or
PHITC (0.76 .mu.moles) or ISC-4 or ISC-6 (0.76 .mu.moles,
equivalent to 3 ppm selenium) thrice per week.
[0183] FIGS. 6A and 6B show graphs of chance in tumor size and body
weight over time in mice treated i. p. with PBRTC or PHITC. (0.76
.mu.moles) or ISC-4 or ISC-6 (0.76 .mu.moles, equivalent to 3 ppm
selenium) thrice per week. Selenium containing analogs ISC-4 or
ISC-6 significantly reduced tumor development compared to DMSO,
PBITC or PHITC controls. While PBITC and PHITC are ineffective at
reducing tumor burden of UACC 903, FIG. 6A, or 1205 Lu, FIG. 6B, at
the administered concentration of 0.76 .mu.moles, administration of
0.76 .mu.moles of ISC-4 or ISC-6 led to significant reductions of
50-60% in tumor size of both cell types. No obvious toxicity is
observed by significant changes in body weight. Data are presented
as mean .+-.SE. Thus, isoselenocyanates (ISC) are effective at
reducing melanoma tumor development at significantly lower
concentrations than corresponding isothiocyanates (ITC).
Example 19
Measurement of Proliferation/Apoptosis Rates in Tumors
[0184] Tumors are established by subcutaneous injection of
2.5-5.times.10.sup.6 1205 Lu or UACC 903 melanoma cells in 0.2 ml
of DMEM supplemented with 10% FBS above both left and light rib
cages of 4-6 week old female nude mice (Harlan Sprague Dawley,
Indianapolis, Ind.). Six days later when a fully vascularized tumor
forms, mice are randomly divided in to DMSO vehicle control and
experimental (BITC, PEITC, PBITC, PHITC, ISC-1-ISC-2, IS C-4 or
ISC-6) groups (5 mice/group; 2 tumors/mouse) and treated i. p. with
ITC compounds (2.5 .mu.moles or 0.76 .mu.moles), ISC compounds
(0.76 .mu.moles, equivalent to 3 ppm selenium) thrice per week.
Monday, Wednesday and Friday). Control mice receive an equal volume
of the vehicle. Dimensions of the developing tumors are measured
using calipers and the size estimated in cubic millimeters. Body
weight is monitored three times a week Monday, Wednesday and
Friday).
[0185] Isoselenocyanates decrease the tumorigenic potential of
melanoma cells by increasing apoptosis.
[0186] FIG. 7A shows that ISC treatment leads to apoptosis in size
and time matched melanoma tumors. Mice bearing tumors matched for
size and time of development are injected i. p. with PBITC (0.76
.mu.moles), ISC-4 (3 ppm equivalent to 0.76 .mu.moles) or DMSO (50
.mu.l) vehicle starting 6 days after subcutaneous injection of
cells and on alternate days thereafter up to day 15. Following
ISC-4 treatment, rates of tumor cell apoptosis and cell
proliferation are compared in size and time matched tumors from
ISC-4 or PBITC treated animals compared to DMSO vehicle. Tumors are
removed from euthanized mice on days 9, 11, 13, and 15 for
measurement of apoptosis and proliferation. Apoptosis and cell
proliferation are measured in formalin-fixed, paraffin-embedded
tumor sections using the TUNEL TMR Red Apoptosis kit from Roche
(Manheim, Germany) or purified mouse anti-human Ki-67 from
PharMingen (San Diego, Calif.), respectively.
[0187] Tumors harvested at day 11 and 13 from mice treated with
ISC-4 showed .about.3-fold (p<0.01; One-way ANOVA) more TUNEL
positive cells compared to control animals treated with DMSO or
PBITC, as shown in FIG. 7A. In contrast, slightly fewer
proliferating tumor cells are observed in ISC-4 treated tumors
compared to PBITC but this difference is not statistically
significant (p>0.05; One-way ANOVA), shown in FIG. 7B.
[0188] A 3-fold increase in number of apoptotic cells is observed
following treatment of UACC 903 tumors with ISC-4 at Day 11 and 13
respectively compared to PBITC or DMSO control. No significant
changes are observed in rate of proliferation. Values shown in
FIGS. 7A and 7B are means from 2 separate experiments with 4-6
fields analyzed from each of 6 tumors per experiment; bars,
.+-.SEM. ISC-4 is more potent than PBITC in inhibiting melanoma
tumor development by increasing apoptosis levels in melanoma
tumors.
Example 20
Toxicity Assessments
[0189] 4-6 weeks old female nude mice (Harlan Sprague Dawley,
Indianapolis, Ind.) are injected i. p. with either control DMSO
vehicle, PBITC or PHITC (0.76 .mu.moles) or ISC-4 or ISC-6 (0.76
.mu.moles equivalent to 3 ppm Se) (n=5), 3 times per week (Monday,
Wednesday and Friday) for 3 weeks. Animals are sacrificed by
CO.sub.2 asphyxiation and blood collected from each animal in
plasma separator tubes with lithium heparin (BD Microtainer, BD,
Franklin Lakes, N.J.) following cardiac puncture and analyzed for
AST (aspartate aminotransferase), ALT, (alanyl aminotransferase),
alkaline phosphatase, glucose and creatinine to ascertain liver,
heart, kidney and pancreas related toxicity. For morphological
examination of blood cells, whole blood is collected in microtainer
tubes containing K.sub.2EDTA (BD Microtainer, BD, Franklin Lakes,
N.J.) and RBC, WBC, lymphocytes, monocytes, eosinophils, platelets,
total hemoglobin and hematocrit percentage analyzed. Blood is also
microscopically examined for segregates, polychromatin bodies, and
smudge cells. A portion of liver, heart, kidney, spleen, intestine
pancreas and adrenal from each animal is formalin fixed and
paraffin-embedded to examine toxicity-related changes in cell or
organ morphology by H&E staining.
[0190] Body weights of isothiocyanate or isoselenocyanate treated
mice compared to the control DMSO vehicle exposed mice showed no
significant differences between groups as shown in the two graph
inserts in FIGS. 6A and 6B.
[0191] Levels of SGOT, SGPT, alkaline phosphatase, glucose and
creatinine are analyzed in the blood collected from the animals
treated with PBITC, ISC-4 or DMSO vehicle. FIG. 8 shows a bar graph
indicating that synthetic isoselenocyanates have negligible
toxicity and treatment with PBITC or ISC-4 did not alter parameters
compared to vehicle DMSO treated animals indicating manageable
toxicity to vital organs with these agents.
[0192] Furthermore, blood parameters (SGOT, SGPT, alkaline
phosphatase, blood urea, glucose and creatinine) indicative of
systemic toxicity did not detect significant liver, kidney or
cardiac related toxicity. Levels of cellular metabolites basal urea
nitrogen (BUN), creative and glucose in animals are also not
significantly different between ISC-4 or PBITC treated and control
animals. Histological examination of hematoxylin and eosin stained
vital organ sections, including the liver reveal that ISC-4
treatment did not significantly change cell morphology or organ
structure. Thus, synthetic selenium containing analog
isothiocyanate treatment had negligible associated systemic
toxicity at the concentrations examined with significant
therapeutic potential. Synthetic isoselenocyanate compounds cause
negligible organ related toxicity following systemic
administration.
Example 21
Statistical Analysis
[0193] Statistical analysis is undertaken using the One-way or
Two-way ANOVA followed by the Tukey's or Bonferroni's post hoc
tests. Results are considered significant at a p-value of
<0.05.
Example 22
Western Blot Analysis of Melanoma Cells Treated with
Isoselenocyanates ISC-4 and ISC-6
[0194] For Western blot analysis, floating, and attached melanoma
cells treated with compounds or control vehicle are harvested by
addition of lyses buffer containing 50 mM HEPES (pH 7.5), 150 mM
NaCl, 10 mM EDTA, 10% glycerol, 1% Triton X-100, 1 mM sodium
orthovanadate, 0.1 mM sodium molybdate, 1 mM phenylmethylsulfonyl
fluoride, 20 .mu.g/ml aprotinin, and 5 .mu.g/ml leupeptin. Whole
cell lysates are centrifuged (>10,000.times.g) for 10 minutes at
4.degree. C. to remove cell debris. Protein concentrations are
quantitated using the BCA assay from Pierce (Rockford, Ill.), and
30 .mu.g of lysate loaded per lane onto NuPAGE Gels from Life
Technologies (Carlsbad, Calif.). Following electrophoresis, samples
are transferred to polyvinylidene difluoride membrane (Pall
Corporation, Pensacola, Fla.). Blots are probed with antibodies
according to each supplier's recommendations: phosphorylated PRAS40
(Thr246) from Invitrogen (Carlsbad, Calif.); Erk2, .alpha.-enolase
and secondary antibodies conjugated with horseradish peroxidase
from Santa Cruz Biotechnology (Santa Cruz, Calif.); and antibodies
to Akt3, phosphorylated-Akt (Ser473), phosphorylated-Erk 1/2
(Thr202/Tyr204) and cleaved PARP from Cell Signaling Technology
(Danvers, MA). Immunoblots are developed using the enhanced
chemiluminescence detection system (Pierce Biotechnology, Rockford,
IL).
[0195] Melanoma cells (UACC 903, 1205 Lu or WM 115) are exposed to
DMSO or increasing concentrations (2.5-15 .mu.M) of
phenylbutylisothiocyanate (PBITC), phenylhexylisothiocyanate
(PHITC), phenylbutylisoselenocyanate (ISC-4) or
phenylhexylisoselenocyanate (ISC-6) for 24 hours. FIG. 9 shows a
representative Western blot analysis for expression/activity of the
Akt signaling pathway and demonstrates dose dependent decrease in
phosphorylated (active) Akt (S473) and downstream PRAS40 (T246) and
a corresponding dose dependent increase in cleaved PARP, reflective
of high levels of cellular apoptosis. A higher level of apoptosis
is observed in cells treated with ISC-4 than in cells treated with
PBITC. Erk2 served as control for equal protein loading.
Isoselenocyanates decrease Akt3 signaling in cultured melanoma
cells and tumors.
[0196] The Western blot in FIG. 9 shows that the isoselenocyanate
compounds ISC-4 and ISC-6 are effective at lower concentrations
compared to the isothiocyanate compounds, completely inhibiting the
pathway at 10 .mu.M compared to corresponding isothiocyanates
requiring .gtoreq.15 .mu.M for similar inhibition. Akt3 pathway
inhibition led to significant apoptosis as measured by the high
levels of cleaved PARP. Higher levels of cleaved PARP are observed
at lower concentrations of ISC-4 and ISC-6 than corresponding
isothiocyanates PBITC or PRUC.
[0197] FIGS. 10A and 10B illustrate Western blots showing the
effect of ISC-4 on Akt signaling pathway in melanoma cell lines
1205 Lu and WM115, respectively. ISC-4 causes significant apoptosis
indicated by elevated cleaved PARP protein levels. Erk-2 served as
a control for equal protein loading.
[0198] Thus, isoselenocyanates ISC-4 and ISC-6 more effectively
inhibit the Akt3 signaling cascade in cultured melanoma cells at
lower concentrations than corresponding sulfur containing
isothiocyanates.
Example 23
Western Blot Analysis of Excised Tumors
[0199] To ascertain the mechanism underlying tumor inhibition,
5.times.10.sup.6 UACC 903 cells are injected into nude mice, 6-days
later mice are treated i. p. with PBITC (0.76 .mu.moles) or PHITC
(0.76 .mu.moles), ISC-4 (0.76 .mu.moles, equivalent to 3 ppm
selenium) or ISC-6 (0.76 .mu.moles, equivalent to 3 ppm selenium)
on alternate days. Size and time matched tumors are harvested at
days 9, 11 and 13 and a small portion of the tumor is flash frozen
in liquid nitrogen, pulverized and lysed in protein lysis buffer
(600-800 .mu.l, 50 mM Tris-HCl, pH 7.5 containing 0.1% Triton
X-100, 1 mM EDTA, 1 mM EGTA, 50 mM sodium fluoride, 10 mM sodium
.beta.-glycerol phosphate, 5 mM sodium pyrophosphate, 1 mM
activated sodium orthovanadate, protease inhibitor cocktail from
Sigma and 0.1% (v/v) 2-mercaptoethanol). Protein concentration is
determined using Bio-Rad protein assay reagent (Bio-Rad
laboratories, Hercules, Calif.) and analyzed by Western blotting to
measure levels of pAkt and downstream pPRAS40 in tumors.
[0200] Western blot analysis of tumors harvested at day 13 from
animals treated with DMSO, PBIT or ISC-4 showed significantly
decreased expression of phosphorylated (active) Akt (p<0.05;
One-way ANOVA) and downstream PRAS40 (p<0.001; One-way ANOVA) in
ISC-4 tumor lysates compared to DMSO control or PBITC treated
tumors. FIG. 11 illustrates a graph of quantitation of Western blot
analysis of tumor protein lysates from animals treated with DMSO,
PBITC or ISC-4 and indicates decreased relative expression of
phosphorylated (active) Akt and downstream PRAS40 of Akt3. Thus,
isoselenocyanates ISC-4 and ISC-6 more effectively inhibit the Akt3
signaling cascade in tumors at lower concentrations than
corresponding sulfur containing isothiocyanates.
Example 24
[0201] Effects of inhibiting Akt3 signaling on melanoma
tumorigenesis are shown using siRNA to inhibit Akt3 protein
expression and thereby inhibit Akt3 activity.
[0202] Duplexed "Stealth".sup.7 siRNA from Invitrogen (Carlsbad,
Calif.) are: AKT3-GGA CUA UCU ACA WEC CGG AAA GAU U (SEQ ID NO. 1)
and scrambled-AAU UCU CCG AAC GUG UCA CGU GAG A (SEQ ID NO. 2).
Nucleofection using Amaxa Nucleofector (Koeln, Germany) is used to
introduce siRNA into UACC 903 cells (Reagent R, program K17). SiRNA
(100 pmoles) against Akt3 or scrambled siRNA are nucleofected into
1.times.10.sup.6 UACC 903 cells, which are then replated in DMEM
supplemented with 10% FBS and allowed to recover for 1.5 days.
Thirty-six hours later 1.times.10.sup.6 UACC 903 cells in 0.2 ml of
DMEM supplemented with 10% FBS are injected subcutaneously into the
left and right flanks of 4 to 6 week old nude mice. Control cells
are nucleofected with scrambled siRNA or nucleofection buffer only.
Dimensions of developing tumors are measured on alternate days
using calipers up to day 17.5. siRNA-mediated inhibition of Akt3
signaling reduced the tumorigenic potential of melanoma cells.
[0203] FIG. 12 is a graph showing that decreased expression
(activity) of Akt3 reduced the tumor size in animals injected with
Akt3 siRNA treated cells compared to control cells nucleofected
with scrambled siRNA or nucleofection buffer. The tumorigenic
potential of melanoma cells is decreased by .about.60%. Thus,
inhibition of Akt3 signaling led to significant melanoma tumor
inhibition.
[0204] FIG. 13 is a Western blot analysis of tumor protein lysates
showing reduction in expression of Akt3, demonstrating effective
knockdown of these proteins in tumors by siRNA directed against
Akt3. .alpha.-enolase served as loading control. A tumor removed
from animals 8 days after introduction of siRNA shows significantly
less Akt3 protein than control tumors into which a scrambled siRNA
had been introduced, demonstrating effective knockdown of Akt3
protein expression using this approach.
Example 25
[0205] Five million UACC903 cells are injected subcutaneously in a
volume of 200 uL at each of two sites in nude mice to establish
tumors. Six days following injection of the cells, mice are divided
into four groups of five mice each. Mice in each group are injected
i.p with 0.76 micromoles of sulforaphane (SFN), 0.76 micromoles
(equivalent to 3 ppm selenium) of
1-isothiocyanto-4-methylselenobutane (SFN Iso Se Me), 0.76
micromoles (equivalent to 3 ppm selenium) of
1-isoselenocyanto-4-methylsulfinylbutane (SEN Iso Se) or DMSO (a
vehicle control).
[0206] FIG. 14A shows inhibition of tumor development by
1-isoselenocyanto-4-methylsulfinylbutane (SEN Iso Se) compared to
controls. No toxicity of the administered compounds is apparent as
indicated by lack of significant changes in body weight, shown in
FIG. 14B.
[0207] IC.sub.50 values for sulforaphane and
1-isoselenocyanto-4-methylsulfinylbutane (SEN Iso Se) are
established in various cell lines. Results are shown in Table
V.
TABLE-US-00005 TABLE V IC-50 values Cell type SFN SFN Iso Se A549
(lung) 73 .+-. 3.6 35 .+-. 1.7 UACC-903 (Melanoma) 40 .+-. 0.89 17
.+-. 0.95 1205LU (Melanoma) >50 39.2 .+-. 2 CaCO2 (Colon
Carcinoma) 48 .+-. 5.9 16 .+-. 0.4 MDA-MB 231 (breast) 57 .+-. 5 20
.+-. 0.46 PC-3 (Prostate) >50 24.8 .+-. 0.58 HT1080 (Soft Tissue
Sarcoma) >50 25.59 .+-. 1.82 IGROV-1 (ovarian carcinoma >50
>50 FF2441 (fibroblast cells) >50 >50
Example 26
[0208] Effect of ISC and ITC compounds on cell viability of
prostate cancer cells LNCaP, PC-3 and DU-145.
[0209] Prostate cell lines, LnCaP, PC-3 and DU-145 are exposed to
different concentrations, 5 micromolar, 10 micromolar or 25
micromolar, of ITCs including BITC, PEITC, PBITC, and PHITC, or
ISCs, including ISC-1, ISC-2, ISC-4, and ISC-4, for 24 hours.
Following incubation, cell viability is assayed using an MTS
assay.
Example 27
[0210] In vitro inhibitory efficacy of DMSO, ISC-1, ISC-2, ISC-4,
ISC-6, BITC, PEITC, PBITC, or PHITC on UACC 903 cells is measured
using the
3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-
-2H-tetrazolium (MTS) assay (Promega, Madison, Wis.). In brief,
2.5-5.times.103 cells per well in 100 microliters DMEM containing
10% FBS are grown in a 96-well plate for 24 h and treated with
either control DMSO vehicle or increasing concentrations (5-15
.mu.M) of the tested isothiocyanate or isoselenocyanate for 24
hours. At this point cells are treated individually with either
vehicle control DMSO or with increasing concentrations of ITC or
ISC (2.5-100 .mu.M) for 24 h. The percentages of viable cells
compared to control DMSO treated cells are determined. FIG. 15
shows results of this assay.
Example 28
[0211] Cellular proliferation rate is measured by seeding
5.times.10.sup.3 human melanoma cell line UACC 903 cells in a
96-well plate, followed by treatment for 24 hours with DMSO or 5-15
.mu.M of ISC-1, ISC-2, ISC-4, ISC-6, BITC, PEITC, PBITC, or PHITC
for 24 hours. Proliferation rates are measured using a BrdU ELISA
kit (Roche Applied Sciences, Indianapolis, Ind.). Results of this
assay are shown in FIG. 16.
Example 29
[0212] Apoptosis rate is measured by seeding 5.times.10.sup.3 human
melanoma cell line UACC 903 cells in a 96-well plate, followed by
treatment for 24 hours with DMSO or 5-15 .mu.M of ISC-1, ISC-2,
ISC-4, ISC-6, BITC, PEITC, PBITC, or PHITC for 24 hours. Apoptosis
rate is measured using an Apo-ONE Homogenous caspase-3/7 Assay kit
(Promega Corporation, Madison, Wis.). Results of this assay are
shown in FIG. 17.
Example 30
Tumorigenicity Assessment
[0213] Tumor kinetics are measured by subcutaneous injection of
5.times.10.sup.6 UACC 903 melanoma cells in 0.2 ml of DMEM
supplemented with 10% FBS above both left and right rib cages of
4-6 week old female nude mice (Harlan Sprague Dawley, Indianapolis,
Ind.). Six days later mice are randomly divided into control (DMSO)
and experimental (ISC-1, ISC-2, ISC-4, ISC-6, BITC, PEITC, PBITC,
PHITC) groups (5 mice/group; 2 tumors/mouse). Six days after
subcutaneous injection of UACC 903 melanoma cells, mice are treated
i. p. with BITC, PEITC, PBITC or PHITC (2.5 .mu.moles), or, ISC-1,
ISC-2, ISC-4 or ISC-6 (0.76 .mu.moles, equivalent to 3 ppm
selenium) thrice per week. (Monday, Wednesday and Friday). Control
mice receive an equal volume of the vehicle, DMSO. The dimensions
of the developing tumors (using calipers) and body weight are
measured three times a week (Monday, Wednesday and Friday) and the
size estimated in cubic millimeters.
[0214] At a dose of 0.76 .mu.moles, ISC-2 to ISC-6 showed about
30-45% reduction in tumor size with the effect increasing with the
increasing alkyl chain length from ISC-2 to ISC-6 (FIG. 18A). ISC-1
failed to show any effect at this concentration. There was no
related toxicity observed at this dose for any of the ISC
derivatives. ITC derivatives are also effective in reducing the
tumor size at a dosage of 2.5 micromoles, .about.3 times higher
than the administered dosage of ISC compounds, shown in FIG. 18C. A
reverse trend of chain length effect is observed, with BITC being
the most effective. FIGS. 18B and 18D shows the body weights of
test compound treated mice treated compared to the control DMSO
vehicle treated mice. No significant difference in weights is
detected between groups, demonstrating negligible toxicity.
Example 31
Western Blotting, Immunoprecipitation, and Kinase Assay for Akt1,
Akt2 and Akt3
[0215] Dysregulation of Akt1, Akt2 and/or Akt3 can be observed in
cells using various well-known techniques
[0216] Western blot procedure for detection of Akt1, Akt2 and/or
Akt3 dysregulation is performed according to standard procedures,
for instance as described herein and in Harlow, E. and Lane, D.,
Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory
Press, 1988; J. D. Pound (Ed.) Immunochemical Protocols, Methods in
Molecular Biology, Humana Press; 2nd ed., 1998; Stahl, J. M., et
al., Cancer Res., 63:2881-2890, 2003; and Stahl, J. M., et al.,
Cancer Res., 64:7002-7010, 2004.
[0217] Immunoprecipitation for detection of Akt1, Akt2 and/or Akt3
dysregulation is performed according to standard procedures, for
instance as described herein and in Harlow, E. and Lane, D.,
Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory
Press, 1988; J. D. Pound (Ed.) Immunochemical Protocols, Methods in
Molecular Biology, Humana Press; 2nd ed., 1998; Stahl, J. M., et
al., Cancer Res., 63:2881-2890, 2003; and Stahl, J. M., et al.,
Cancer Res., 64:7002-7010, 2004. For example, briefly described,
protein is collected from cells after addition of protein lysis
buffer [50 mmol/L Tris-HCl (pH 7.5), 0.1% Triton X-100, 1 mmol/L
EDTA, 1 mmol/L EGTA, 50 mmol/L NaFl, 10 mmol/L sodium
.beta.-glycerol phosphate, 5 mmol/L sodium inorganic pyrophosphate,
1 mmol/L sodium orthovanadate, 0.11% 2-mercaptoethanol, and 0.5%
protease inhibitor mixture (Sigma, St. Louis, Mo.)] to a sample,
such as plates of cells or biopsy material, followed by snap
freezing in liquid nitrogen. Cellular debris is pelleted by
centrifugation (10,000.times.g) of lysates, and protein
concentration is quantitated using the Bio-Rad Protein Assay
(Bio-Rad, Hercules, Calif.). Protein for immunoprecipitation (100
jug) is incubated with 2 .mu.g of Akt1 or Akt2 or 5 .mu.L of Akt3
antibody overnight at 4.degree. C. with constant mixing. A no
antigen negative control is prepared by adding antibody to the
lysis buffer only. Next, 15 .mu.L of equilibrated GammaBind G
Sepharose beads (Amersham Biosciences, Piscataway, N.J.) is added
to each tube and incubated for 2 hours (4.degree. C.) with constant
mixing. Pelleted beads are washed twice with lysis buffer to remove
unbound antibody and protein. Samples are then electrophoresed
under reducing conditions according to the protocol provided by
Invitrogen Life Technologies, Inc. (Carlsbad, Calif.) with the
NuPage Gel System. Western blots of the electrophoresed samples are
probed with an anti-phospho-Akt (Ser-473) antibody and quantitated
by densitometry as described in Stahl, J. M., et al., Cancer Res.,
63:2881-2890, 2003.
[0218] Antibodies for use in immunoassays for Akt1, Akt2 and/or
Akt3 can be obtained commercially, for instance an Akt1 antibody is
available from Cell Signaling Technologies, Beverly, Mass., an Akt2
antibody is available from Santa Cruz Biotechnology, Santa Cruz,
Calif. and an Akt3 antibody is available from Upstate
Biotechnology, Lake Placid, N.Y. An anti-phospho-Akt (Ser-473)
antibodies can also be obtained commercially, e.g. from Cell
Signaling Technologies, Beverly, Mass. Anti-Akt1, Akt2 and/or Akt3
antibodies can also be produced by well-known techniques such as
described, for instance, in Harlow, E. and Lane, D., Antibodies: A
Laboratory Manual, Cold Spring Harbor Laboratory Press, 1988.
[0219] Akt kinase assays are performed according to standard kinase
assay procedures. Briefly described, 15 .mu.L of equilibrated
GammaBind G Sepharose beads are incubated with 2 .mu.g of Akt1 or
Akt2 antibody, or 5 .mu.L of Akt3 antibody in a volume of 350 .mu.L
of lysis buffer at 4.degree. C. with constant mixing for 2 hours.
Microcystin (1 .mu.mol/L) from MP Biomedicals (Irvine, Calif.) is
added to the lysis buffer to ensure complete inactivation of
cellular PP1 and PP2 phosphatases. The antibody/Sepharose complex
is washed twice with 750 .mu.L of lysis buffer and then incubated
with 100 .mu.g of protein in a volume of 350 .mu.L overnight at
4.degree. C. with constant mixing. This complex is washed with 500
.mu.L of lysis buffer (3.times.) and then once with 500 .mu.L of
assay dilution buffer [20 mmol/L 4-morpholinepropanesulfonic acid
(pH 7.2), 25 mmol/L .beta.-glycerol phosphate, 1 mmol/L sodium
orthovanadate, and 1 mmol/L dithiothreitol]. Protein kinase A (PKA)
inhibitor peptide (10 .mu.mol/L) from Santa Cruz Biotechnology
(Santa Cruz, Calif.), 37.5 .mu.mol/L ATP, 17 mmol/L MgCl2, 0.25
.mu.Ci/.mu.L [gamma-.sup.32P]ATP, and 90 .mu.mol/L Akt-specific
substrate Crosstide from Upstate Biotechnology (Lake Placid, N.Y.)
are added to the tubes in assay dilution buffer and incubated at
35.degree. C. for 10 minutes with continuous mixing. Next, 20 .mu.L
of liquid are transferred to phosphocellulose paper, which is
washed three times for 5 minutes with 40 mL of 0.75% phosphoric
acid. After a 5-minute acetone wash, the phosphocellulose was
allowed to dry and transferred to a scintillation vial with 5 mL of
Amersham Biosciences scintillation fluid, and counts per minute
were measured in a Beckman Coulter LS 3801 Liquid Scintillation
System Fullerton, Calif.).
[0220] Detection of Dysregulated Akt in Human Material
[0221] Formalin-fixed paraffin-embedded melanoma specimens are used
for immunohistochemistry to measure phosphorylated Akt. A
phospho-Akt (Ser-473) monoclonal antibody, Cell Signaling
Technologies, is used at a 1:50 titer according to the
manufacturer's recommended protocol. Specificity and intensity of
staining are determined through qualitative comparison with
internal blood vessel endothelium, squamous epithelium, or smooth
muscle controls present in each specimen.
Example 32
[0222] ISC-4 more efficiently inhibits melanoma cell growth
compared to normal human fibroblast cells. 10.times.10.sup.3 normal
human fibroblasts (FF2441) expressing Akt3 at normal levels and
5.times.10.sup.3 metastatic melanoma cells (UACC 903) having
activated Akt3 are plated in 96 well plates in 100 .mu.L DMEM
containing 10% FBS and grown for 24 hours respectively.
Exponentially growing cells are treated with increasing
concentrations (2.5-100 .mu.M) of ISC-4 for 24 hours and IC.sub.50
(.mu.M) values determined.
[0223] Sensitivity of melanoma cells to ISC-4 with elevated Akt3
signaling is compared to fibroblast cells with normal levels of Akt
activity. Three-fold higher drug concentration of ISC-4 (37.5
.mu.M) is required to kill fibroblasts with normal levels of Akt
activity compared to melanoma cells (12.+-.3 .mu.M) with elevated
Akt activity. Thus, cancer cells with constitutively active Akt
signaling are 3-fold more sensitive to ISC-4 than normal cells with
regular Akt activity. These results show that ISC-4 more
effectively kill cells having increased Akt activity than those
having normal levels.
Example 33
[0224] Effect of topical ISC-4 application on melanoma tumor
growth--in vitro (Skin Reconstructs). FIG. 19 is a bar graph
showing the effects of topically applied PBITC or ISC-4 on
reconstructed human skin containing GFP tagged UACC 903 human
melanoma cells.
[0225] Generation of skin containing melanocytic lesions is briefly
described. To create skin in a culture dish, human fibroblasts,
were trypsinized and resuspended in 10% reconstitution buffer, 10%
10.times.DMEM (Mediatech, Herndon, Va.), 2.4 microliters/ml of 10 M
NaOH, and 80% collagen I (BD Discovery Labware Inc., Bedford,
Mass.) at a concentration of 2.5.times.10.sup.5 cells/ml on ice
(Ozbun M A, Meyers C. J Virol 1996, 70: 5437-46.). Mixture was then
aliquoted into 6 or 12 well plates and incubated at 37.degree. C.
for 3 hours. E-media was added to each well to equilibrate the
dermal matrix (Wu Y J, Parker L M, Binder N E, et al., Cell 1982,
31: 693-703.). After two days of growth, keratinocytes and melanoma
cells (WM35-GFP or UACC 903-GFP) were trypsinized and resuspended
at a 1:10 ratio of melanoma cells (nucleofected or untreated) to
keratinocytes in E-media. One milliliter of cell suspension added
to each well on top of the dermal layer. Following two days growth,
reconstructed skin was transferred onto wire grids and fed via
diffusion from E-media below the platforms.
[0226] Reconstructed human skin containing GFP tagged UACC 903
human melanoma cells were treated with 12.5 and 25 .mu.M PBITC or
ISC-4 and the tumor area occupied measured using fluorescence
microscopy.
[0227] The result shows an .about.80% decrease in tumor area
occupied by melanoma cells upon ISC-4 treatment compared to control
DMSO treated or untreated skins. Similar results are observed with
other melanoma cell lines.
Example 34
Effect of Topical ISC-4 Application on Melanoma Tumor Growth--In
Vivo
[0228] Chemopreventive or chemotherapeutic effect of ISC-4 on
cutaneous tumor development is measured by subcutaneous injection
of 1 million UACC 903 cells in 0.2 ml of DMEM-10% FBS above both
the left and right rib cages of 4- to 6-week old female athymic
nude mice using 24 g needles. 24 hours later, animals are treated
daily with ISC-4 (0.063-0.19 .mu.moles equivalent to 0.25-0.75
ppm), PBITC (0.063-0.19 .mu.moles) or vehicle control (acetone) for
3-4 weeks. The dimensions of the developing tumors are measured
alternate days using calipers and the sizes estimated in cubic
millimeters. A minimum of 5 mice per group is used for the topical
treatment.
[0229] FIG. 20 shows a pair of line graphs showing the effect of
topical ISC-4 application on melanoma tumor growth in vivo. Topical
treatment with (0.063-0.19 .mu.moles equivalent to 0.25-0.75 ppm)
ISC-4 leads to decreased tumor size compared to vehicle control (as
shown in the upper graph in FIG. 20) or PBITC (0.063-0.19
.mu.moles) with no systemic toxicity (as shown by body weight
measurements in the lower graph in FIG. 20).
Example 35
Effect of Topical ISC-4 Application on Melanoma Tumor Growth--In
Vivo
[0230] Chemopreventive or chemotherapeutic effect of ISC-4 on
cutaneous tumor development is measured by subcutaneous injection
of 1 million UACC 903 cells in 0.2 ml of DMEM-10% FBS above both
the left and right rib cages of 4- to 6-week old female athymic
nude mice using 24 g needles. 24 hours later, animals were treated
daily with ISC-4 (12.5-50 .mu.M), PBITC (12.5-50 .mu.M), or vehicle
control (acetone) for 3-4 weeks. The dimensions of the developing
tumors are measured alternate days using calipers and the sizes
estimated in cubic millimeters. A minimum of 5 mice per group was
used for the topical treatment.
[0231] Topical treatment with ISC-4 (12.5-50 .mu.M) significantly
reduces melanoma tumor development by .about.50% compared to PBITC
(12.5-50 .mu.M) (as shown in the upper graph in FIG. 21) or vehicle
control with no systemic toxicity (as shown by body weight
measurements in the lower graph in FIG. 21).
REFERENCES
[0232] Serrone L, Hersey P. The chemoresistance of human malignant
melanoma: an update. Melanoma Research 1999; 9:51-8. [0233]
Grossman D, Altieri D C. Drug resistance in melanoma: mechanisms,
apoptosis, and new potential therapeutic targets. Cancer &
Metastasis Reviews 2001; 20:3-11. [0234] Helmbach H, Rossmann E,
Kern M A, Schadendorf D. Drug-resistance in human melanoma.
International Journal of Cancer 2001; 93:617-22 [0235] Markovic S
N, Erickson L A, Rao R D, et al. Malignant melanoma in the 21st
century, part 1: epidemiology, risk factors, screening, prevention,
and diagnosis. Mayo Clin Proc 2007; 82:364-80. [0236] Jemal A,
Thomas A, Murray T, Thun M. Cancer statistics,
2002.[comment][erratum appears in CA Cancer J Clin 2002
March-April; 52(2):119]. Ca: a Cancer Journal for Clinicians 2002;
52:23-47. [0237] Amiri K I, Horton L W, LaFleur B J, Sosman J A,
Richmond A. Augmenting chemosensitivity of malignant melanoma
tumors via proteasome inhibition: implication for bortezomib
(VELCADE, PS-341) as a therapeutic agent for malignant melanoma.
Cancer Res 2004; 64:4912-8. [0238] Gray-Schopfer V, Wellbrock C,
Marais R. Melanoma biology and new targeted therapy. Nature 2007;
445:851-7. [0239] Brazil D P, Hemmings B A. Ten years of protein
kinase B signalling: a hard Akt to follow. Trends Biochem Sci 2001;
26:657-64. [0240] Nicholson K M, Anderson N G. The protein kinase
B/Akt signalling pathway in human malignancy. Cell Signal 2002;
14:381-95. [0241] Stahl J M, Sharma A, Cheung M, et al. Deregulated
Akt3 activity promotes development of malignant melanoma. Cancer
Res 2004; 64:7002-10. [0242] Stahl J M, Cheung M, Sharma A, Trivedi
N R, Shanmugam S, Robertson G P. Loss of PTEN promotes tumor
development in malignant melanoma. Cancer Res 2003; 63:2881-90.
[0243] Madhunapantula S V, Sharma A, Robertson G P. PRAS40
deregulates apoptosis in malignant melanoma. Cancer Res 2007;
67:3626-36. [0244] Keum Y S, Jeong W S, Kong A N. Chemoprevention
by isothiocyanates and their underlying molecular signaling
mechanisms. Mutat Res 2004; 555:191-202, [0245] Zhang Y.
Cancer-preventive isothiocyanates: measurement of human exposure
and mechanism of action. Mutat Res 2004; 555:173-90. [0246] Zhang
Y, Kensler T W, Cho C G, Posner G H, Talalay P. Anticarcinogenic
activities of sulforaphane and structurally related synthetic
norbornyl isothiocyanates. Proc Natl Acad Sci USA 1994; 91:3147-50.
[0247] Hecht S S. Chemoprevention by isothiocyanates. J Cell
Biochem Suppi 1995; 22:195-209. [0248] Zhang Y, Yao S, Li J.
Vegetable-derived isothiocyanates: anti-proliferative activity and
mechanism of action. Proc Nutr Soc 2006; 65:68-75. [0249] Ji Y, Kuo
Y, Morris M E. Pharmacokinetics of dietary phenethyl isothiocyanate
in rats. Pharm Res 2005; 22:1658-66. [0250] El-Bayoumy K, Sinha R,
Pinto J T, Rivlin R S. Cancer chemoprevention by garlic and
garlic-containing sulfur and selenium compounds. J Nutr 2006;
136:864 S-9S. [0251] Miyoshi N, Uchida K, Osawa T, Nakamura Y. A
link between benzyl isothiocyanate-induced cell cycle arrest and
apoptosis: involvement of mitogen-activated protein kinases in the
Bcl-2 phosphorylation. Cancer Res 2004; 64:2134-42. [0252] Chiao
fW, Wu H, Ramaswamy G, et al. Ingestion of an isothiocyanate
metabolite from cruciferous vegetables inhibits growth of human
prostate cancer cell xenografts by apoptosis and cell cycle arrest.
Carcinogenesis 2004; 25: 1403-8. [0253] Reinhold U, Biltz H, Bayer
W, Schmidt K H. Serum selenium levels in patients with malignant
melanoma. Acta Derm Venereol 1989; 69:132-6. [0254] Bandura L,
Drukala J, Wolnicka-Glubisz A, Bjornstedt M, Korohoda W.
Differential effects of selenite and selenate on human melanocytes,
keratinocytes, and melanoma cells. Biochem Cell Biol 2005;
83:196-211. [0255] Brigelius-Flohe R. Selenium compounds and
selenoproteins in cancer. Chem Biodivers 2008; 5:389-95. [0256]
Unni E, Koul D, Yung W K, Sinha R. Se-methylselenocysteine inhibits
phosphatidylinositol 3-kinase activity of mouse mammary epithelial
tumor cells in vitro. Breast Cancer Res 2005; 7:R699-707. [0257] Hu
H, Jiang C, Li G, Lu J. PKB/AKT and ERK regulation of
caspase-mediated apoptosis by methylseleninic acid in LNCaP
prostate cancer cells. Carcinogenesis 2005; 26:1374-81. [0258]
Krishan A. Rapid flow cytofluorometric analysis of mammalian cell
cycle by propidium iodide staining. J Cell Biol 1975; 66:188-93.
[0259] Yang L, Dan H C, Sun M, et al. Akt/protein kinase B
signaling inhibitor-2, a selective small molecule inhibitor of Akt
signaling with antitumor activity in cancer cells overexpressing
Akt. Cancer Res 2004; 64:4394-9. [0260] Feun L G, Blessing J A,
Barrett R J, Hanjani P. A phase II trial of tricyclic nucleoside
phosphate in patients with advanced squamous cell carcinoma of the
cervix. A Gynecologic Oncology Group Study. Am J Clin Oncol 1993;
16:506-8. [0261] Karst A M, Dai D L, Cheng J Q, Li G. Role of p53
up-regulated modulator of apoptosis and phosphorylated Aikt in
melanoma cell growth, apoptosis, and patient survival. Cancer Res
2006; 66:9221-6.
[0262] Any patents or publications mentioned in this specification
are incorporated herein by reference to the same extent as if each
individual publication is specifically and individually indicated
to be incorporated by reference. U.S. Provisional Patent
Application Serial Nos. 60/911,565, filed Apr. 13, 2007 and
60/959,554, filed Jul. 13, 2007, are both incorporated herein by
reference in their entirety.
[0263] The compositions and methods described herein are presently
representative of preferred embodiments, exemplary, and not
intended as limitations on the scope of the invention. Changes
therein and other uses will occur to those skilled in the art. Such
changes and other uses can be made without departing from the scope
of the invention as set forth in the claims.
* * * * *