U.S. patent application number 11/671823 was filed with the patent office on 2007-10-04 for method of treating cancer using dithiocarbamate derivatives.
This patent application is currently assigned to The University of Utah. Invention is credited to Thomas Preston Kennedy.
Application Number | 20070232692 11/671823 |
Document ID | / |
Family ID | 27102340 |
Filed Date | 2007-10-04 |
United States Patent
Application |
20070232692 |
Kind Code |
A1 |
Kennedy; Thomas Preston |
October 4, 2007 |
Method of Treating Cancer Using Dithiocarbamate Derivatives
Abstract
Dithiocarbamate, particularly tetraethylthiuram disulfide, and
thiocarbamate anions thereof, strongly inhibit the growth of cancer
cells of a variety of cell types. Such inhibitory effect is
enhanced by heavy metal ions such as copper ions, cytokines and
ceruloplasmin. A method is presented for using tetraethylthiuram
disulfide to reduce tumor growth, and to potentiate the effect of
other anticancer agents.
Inventors: |
Kennedy; Thomas Preston;
(Charlotte, NC) |
Correspondence
Address: |
ALSTON & BIRD LLP
BANK OF AMERICA PLAZA
101 SOUTH TRYON STREET, SUITE 4000
CHARLOTTE
NC
28280-4000
US
|
Assignee: |
The University of Utah
|
Family ID: |
27102340 |
Appl. No.: |
11/671823 |
Filed: |
February 6, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10378206 |
Mar 3, 2003 |
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11671823 |
Feb 6, 2007 |
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09735205 |
Dec 12, 2000 |
6548540 |
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10378206 |
Mar 3, 2003 |
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09679932 |
Oct 5, 2000 |
6706759 |
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09735205 |
Dec 12, 2000 |
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09392122 |
Sep 8, 1999 |
6589987 |
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09679932 |
Oct 5, 2000 |
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60099390 |
Sep 8, 1998 |
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Current U.S.
Class: |
514/492 |
Current CPC
Class: |
A61K 31/27 20130101;
A61K 33/04 20130101; A61K 33/245 20130101; Y10S 514/922 20130101;
A61K 45/06 20130101; A61P 29/00 20180101; A61K 33/38 20130101; A61P
35/00 20180101; A61K 33/36 20130101; A61K 2300/00 20130101; A61K
33/38 20130101; A61K 2300/00 20130101; A61K 2300/00 20130101; A61K
2300/00 20130101; A61K 2300/00 20130101; A61K 2300/00 20130101;
A61K 2300/00 20130101; A61K 2300/00 20130101; A61K 2300/00
20130101; A61K 2300/00 20130101; A61K 2300/00 20130101; A61K 33/30
20130101; A61K 33/30 20130101; Y10S 514/825 20130101; A61K 33/32
20130101; A61K 33/24 20130101; A61K 33/34 20130101; A61K 33/245
20130101; A61K 33/26 20130101; A61K 33/36 20130101; A61K 31/27
20130101; A61K 33/24 20130101; A61P 9/10 20180101; A61K 33/32
20130101; A61K 31/325 20130101; A61P 43/00 20180101; A61K 33/26
20130101; Y10S 514/826 20130101; A61P 11/06 20180101; A61P 9/00
20180101; A61K 33/04 20130101; A61K 33/34 20130101; A61K 31/325
20130101 |
Class at
Publication: |
514/492 |
International
Class: |
A61K 31/325 20060101
A61K031/325 |
Claims
1. A method of treating cancer in a patient comprising
administering a therapeutically effective amount of a
dithiocarbamate disulfide, or a dithiocarbamate thiolate anion
thereof, to the patient.
2. The method of claim 1, wherein said administering step comprises
administering a dithiocarbamate disulfide having the formula:
R.sub.1R.sub.2N(S)CS--SC(S)NR.sub.3R.sub.4 wherein R.sub.1,
R.sub.2, R.sub.3, and R.sub.4 are the same or different, and each
is selected from the group consisting of hydrogen, unsubstituted or
substituted alkyl, unsubstituted or substituted alkenyl,
unsubstituted or substituted aryl, unsubstituted or substituted
alkoxy, and unsubstituted or substituted heteroaryl, or either
R.sub.1 and R.sub.2 or R.sub.3 and R.sub.4 and the adjacent N atom
together form an N-heterocyclic ring.
3. The method of claim 2, wherein the dithiocarbamate disulfide is
a disulfide form of a dithiocarbamate selected from the group
consisting of diethyldithiocarbamate, pyrrolodinedithiocarbamate,
N-methyl, N-ethyl dithiocarbamates, hexamethylenedithiocarbamate,
imidazolinedithiocarbamates, dibenzyldithiocarbamate,
dimethylenedithiocarbamate, dipolyldithiocarbamate,
dibutyldithiocarbamate, diamyldithiocarbamate, N-methyl,
N-cyclopropylmethyldithiocarbamate, cyclohexylamyldithiocarbamate,
pentamethylenedithiocarbamate, dihydrxyethyldithiocarbamate, and
N-methylglucosamine dithiocarbamate.
4. The method of claim 1, wherein said administering step comprises
administering a tetraalkyl thiuram disulfide.
5. The method of claim 4, wherein the tetraalkyl thiuram disulfide
is disulfuram.
6. The method of claim 1, wherein said administering step comprises
administering a thiolate anion of a dithiocarbamate disulfide.
7. The method of claim 6, wherein the thiolate anion is
administered in the form of a pharmaceutically acceptable salt.
8. The method of claim 7, wherein the pharmaceutically acceptable
salt is an alkali metal salt.
9. The method of claim 8, wherein the thiolate anion in alkali
metal salt form has the formula: ##STR7## wherein R.sub.2 and
R.sub.3 are the same or different, and each is selected from the
group consisting of hydrogen, unsubstituted or substituted alkyl,
unsubstituted or substituted alkenyl, unsubstituted or substituted
aryl, unsubstituted or substituted alkoxy, and unsubstituted or
substituted heteroaryl; M is an alkali metal; and n is the valence
of the alkali metal.
10. The method of claim 6, wherein the thiolate anion is
diethyldithiocarbamate anion, optionally in the form of an alkali
metal salt.
11. The method of claim 1, further comprising administering a
source of heavy metal ions to the patient in an amount effective to
form a complex with the dithiocarbamate disulfide or
dithiocarbamate thiolate anion thereof.
12. The method of claim 11, wherein the heavy metal is selected
from the group consisting of arsenic, bismuth, cobalt, copper,
chromium, gallium, gold, iron, manganese, nickel, silver, titanium,
vanadium, selenium, and zinc.
13. The method of claim 11, wherein the source of heavy metal ions
is administered in a separate formulation from the dithiocarbamate
disulfide or dithiocarbamate thiolate anion thereof.
14. The method of claim 11, wherein the source of heavy metal ions
is in the form of an aqueous solution of a pharmaceutically
suitable salt.
15. The method of claim 14, wherein the salt form is selected from
the group consisting of a chelate with an organic anion, a sulfate
salt, and a chloride salt.
16. The method of claim 15, wherein the organic anion is selected
from the group consisting of acetate, lactate, glycinate, citrate,
propionate, and gluconate.
17. The method of claim 1, wherein the route of administration is
selected from the group consisting of parenteral, intravenous,
oral, intradermal, subcutaneous, and topical.
18. The method of claim 1, further comprising administering a
therapeutically effective amount of an anticancer agent to the
patient.
19. The method of claim 18, wherein the anticancer agent is a
chemotherapeutic drug.
20. The method of claim 18, wherein the anticancer agent is
selected from the group consisting of busulphan, chlorambucil,
hydroxyurea, ifosfamide, mitomycin, mitotane, chlorambucil,
mechlorethamine, carmustine, lomustine, cisplatin, carmustine,
herceptin, carboplatin, cyclophosphamide, nitrosoureas,
fotemustine, vindescine, etoposide, daunorubicin, adriamycin,
paclitaxel, docetaxel, streptozocin, dactinomycin, doxorubicin,
idarubicin, plicamycin, pentostatin, mitotoxantrone, valrubicin,
cytarabine, fludarabine, floxuridine, clardribine, methotrexate,
mercaptopurine, thioguanine, capecitabine, irinotecan, dacarbazine,
asparaginase, gemcitabine, altretamine, topotecan, procarbazine,
vinorelbine, pegaspargase, vincristine, rituxan, vinblastine,
tretinoin, teniposide, fluorouracil, melphalan, bleomycin,
salicylates, aspirin, piroxicam, ibuprofen, indomethacin, naprosyn,
diclofenac, tolmetin, ketoprofen, nambuetone, oxaprozin,
doxirubicin, nonselective cycclooxygenase inhibitors, and selective
cyclooxygenase-2 (COX-2) inhibitors.
21. A method of treating cancer in a patient comprising:
administering a therapeutically effective amount of a
dithiocarbamate disulfide, or a dithiocarbamate thiolate anion
thereof, to the patient; and administering a source of heavy metal
ions in a separate formulation to the patient in an amount
effective to form a complex with the dithiocarbamate disulfide or
dithiocarbamate thiolate anion thereof, wherein the heavy metal is
selected from the group consisting of arsenic, bismuth, cobalt,
copper, chromium, gallium, gold, iron, manganese, nickel, silver,
titanium, vanadium, selenium, and zinc.
22. The method of claim 21, comprising administering disulfuram and
a source of copper, zinc, gold, or silver.
23. A method of treating cancer in a patient comprising:
administering a therapeutically effective amount of a
dithiocarbamate disulfide, or a dithiocarbamate thiolate anion
thereof, to the patient; administering a source of heavy metal ions
in a separate formulation to the patient in an amount effective to
form a complex with the dithiocarbamate disulfide or
dithiocarbamate thiolate anion thereof; and administering a
therapeutically effective amount of a chemotherapeutic drug to the
patient.
24. The method of claim 23, comprising administering disulfuram, a
source of copper, zinc, gold, or silver, and a chemotherapeutic
drug selected from the group consisting of cisplatin,
cyclophosphamide, etoposide, duanorubicin, adriamycin, paclitaxel,
docetaxel, gemcitabine, fluorouracil, vincristine, vinblastine,
teniposide, and carboplatin.
25. A pharmaceutical composition comprising: a first pharmaceutical
composition comprising a dithiocarbamate disulfide, or a
dithiocarbamate thiolate anion thereof, in a pharmaceutically
acceptable carrier; and a second separate pharmaceutical
composition comprising a source of heavy metal ions in a
pharmaceutically acceptable carrier.
26. The pharmaceutical composition of claim 25, wherein the first
pharmaceutical composition comprises a dithiocarbamate disulfide
having the formula: R.sub.1R.sub.2N(S)CS--SC(S)NR.sub.3R.sub.4
wherein R.sub.1, R.sub.2, R.sub.3, and R.sub.4 are the same or
different, and each is selected from the group consisting of
hydrogen, unsubstituted or substituted alkyl, unsubstituted or
substituted alkenyl, unsubstituted or substituted aryl,
unsubstituted or substituted alkoxy, and unsubstituted or
substituted heteroaryl, or either R.sub.1 and R.sub.2 or R.sub.3
and R.sub.4 and the adjacent N atom together form an N-heterocyclic
ring.
27. The pharmaceutical composition of claim 25, wherein the first
pharmaceutical composition comprises a tetraalkyl thiuram
disulfide.
28. The pharmaceutical composition of claim 27, wherein the
tetraalkyl thiuram disulfide is disulfuram.
29. The pharmaceutical composition of claim 25, wherein the first
pharmaceutical composition comprises a thiolate anion of a
dithiocarbamate disulfide.
30. The pharmaceutical composition of claim 29, wherein the
thiolate anion is in the form of a pharmaceutically acceptable
salt.
31. The pharmaceutical composition of claim 30, wherein the
thiolate anion is in the form of an alkali metal salt.
32. The pharmaceutical composition of claim 31, wherein the
thiolate anion in alkali metal salt form has the formula: ##STR8##
wherein R.sub.2 and R.sub.3 are the same or different, and each is
selected from the group consisting of hydrogen, unsubstituted or
substituted alkyl, unsubstituted or substituted alkenyl,
unsubstituted or substituted aryl, unsubstituted or substituted
alkoxy, and unsubstituted or substituted heteroaryl; M is an alkali
metal; and n is the valence of the alkali metal.
33. The pharmaceutical composition of claim 29, wherein the
thiolate anion is diethyldithiocarbamate anion, optionally in the
form of an alkali metal salt.
34. The pharmaceutical composition of claim 25, wherein the heavy
metal is selected from the group consisting of arsenic, bismuth,
cobalt, copper, chromium, gallium, gold, iron, manganese, nickel,
silver, titanium, vanadium, selenium, and zinc.
35. The pharmaceutical composition of claim 25, wherein the source
of heavy metal ions is in the form of an aqueous solution of a
pharmaceutically suitable salt.
36. The pharmaceutical composition of claim 35, wherein the salt
form is selected from the group consisting of a chelate with an
organic anion, a sulfate salt, and a chloride salt.
37. The pharmaceutical composition of claim 36, wherein the organic
anion is selected from the group consisting of acetate, lactate,
glycinate, citrate, propionate, and gluconate.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application is a continuation of U.S.
application Ser. No. 10/378,206, filed Mar. 3, 2003, which is a
divisional of U.S. application Ser. No. 09/735,205, filed Dec. 12,
2000, now U.S. Pat. No. 6,548,540, which is a continuation-in-part
of U.S. patent application Ser. No. 09/679,932, filed Oct. 5, 2000,
which is a continuation-in-part of U.S. patent application Ser. No.
09/392,122, filed Sep. 8, 1999, which claims priority under 35
U.S.C. .sctn. 119(e) to U.S. Provisional Appl. Ser. No. 60/099,390,
filed Sep. 9, 1998, all of which are hereby incorporated by
reference herein in their entirety.
FIELD OF INVENTION
[0002] This invention generally relates to methods of treating
cancer, and particularly to methods of treating cancer using
dithiocarbamate derivatives.
BACKGROUND OF THE INVENTION
[0003] Cancer, the uncontrolled growth of malignant cells, is a
major health problem of the modern medical era and ranks second
only to heart disease as a cause of death in the United States.
While some malignancies, such as adenocarcinoma of the breast and
lymphomas such as Hodgkin's Disease, respond relatively well to
current chemotherapeutic antineoplastic drug regimens, other
cancers are poorly responsive to chemotherapy, especially non-small
cell lung cancer and pancreatic, prostate and colon cancers. Even
small cell cancer of the lung, initially chemotherapy sensitive,
tends to return after remission, with widespread metastatic spread
leading to death of the patient. Thus, better treatment approaches
are needed for this illness. Also, because almost all currently
available antineoplastic agents have significant toxicities, such
as bone marrow suppression, renal dysfunction, stomatitis,
enteritis and hair loss.
[0004] The end of the twentieth century has seen a more dramatic
increase in the observed incidence of malignant melanoma than for
all other types of tumors. The biology of malignant melanomas
offers an example of the importance of transcription factors for
malignant cell propagation. Malignant melanomas have great
propensity to metastasize and are notoriously resistant to
conventional cancer treatments such as chemotherapy and
.gamma.-irradiation. Development of malignant melanoma in humans
progresses through a multistage process, with transition from
melanocyte to nevi, to radial growth, and subsequently to the
vertical growth, metastatic phenotype of autonomous melanomas,
associated with decreased dependence on growth factors, diminished
anchorage dependence, reduced contact inhibition and increased
radiation and drug resistance.
[0005] Much of the molecular understanding of melanoma progression
has come from studying the response of cultured melanoma cells to
mitogenic stimuli. In culture, melanocyte proliferation and
differentiation are positively regulated by agents that increase
cAMP (See, P. M. Cox, et al., "An ATF/CREB binding motif is
required for aberrant constitutive expression of the MHC class II
Dra promoter and activation by SV40 T-antigen," Nucleic Acids Res.
20:4881-4887 (1992); R. Halaban, et al., "Regulation of tyrosinase
in human melanocytes grown in culture," J. Cell Biol. 97:480-488
(1983); D. Jean, et al., "CREB and its associated proteins act as
survival factors for human melanoma cells," J. Biol. Chem.
273:24884-24890 (1998); P. Klatt, et al., "Nitric oxide inhibits
c-Jun DNA binding by specifically targeted S-glutathionylation," J.
Biol. Chem. 274:15857-15864 (1999); J. M. Lehmann, et. al., "MUC18,
a marker of tumor progression in human melanoma, shows sequence
similarity to the neural cell adhesion molecules of the
immunoglobulin superfamily," Proc. Natl. Acad. Sci. U.S.A.
89:9891-9895 (1989); M. Luca, et al., "Direct correlation between
MUC18 expression and metastatic potential of human melanoma cells,"
Melanoma Res. 3:35-41 (1993); J. P. Richards, et al., "Analysis of
the structural properties of cAMP-responsive element-binding
protein (CREB) and phosphorylated CREB," J. Biol. Chem.
271:13716-13723 (1996); and S. Xie, et al., "Dominant-negative CREB
inhibits tumor growth and metastasis of human melanoma cells,"
Oncogene 15:2069-2075 (1997)), and several cAMP responsive
transcription factors binding to CRE (the consensus motif
5'-TGACGTCA-3', or cAMP response element) play prominent roles in
mediating melanoma growth and metastasis. In MeWo melanoma cells,
the transcription factor CREB (for CRE-binding protein) and its
associated family member ATF-1 promote tumor growth, metastases and
survival through CRE-dependent gene expression. See, D. Jean, et
al., supra. Expression of the dominant negative KCREB construct in
metastatic MeWo melanoma cells decreases their tumorigenicity and
metastatic potential in nude mice. See, S. Xie, et al., "Expression
of MCA/MUC18 by human melanoma cells leads to increased tumor
growth and metastasis," Cancer Res. 57:2295-2303 (1997). The
KCREB-transfected cells display a significant decrease in matrix
metalloproteinase 2 (MPP2, the 72 kDa collagenase type IV) mRNA and
activity, resulting in decreased invasiveness through the basement
membrane, an important component of metastatic potential.
[0006] The cell surface adhesion molecule MCAM/MUC18, which is
involved in metastasis of melanoma (See, J. M. Lehmann, et al.,
supra; M. Luca, et al., supra; S. Xie, et al., supra), is also
down-regulated by KCREB transfection. See, S. Xie, et al., Cancer
Res., supra. In addition, expression of KCREB in MeWo cells renders
them susceptible to thapsigargin-induced apoptosis, suggesting that
CREB and its associated proteins act as survival factors for human
melanoma cells, thereby contributing to the acquisition of the
malignant phenotype. See, D. Jean, et al., supra.
[0007] Melanoma cells aberrantly express the major
histocompatibility complex class II (MHC II) antigens, normally
found only in B-lymphocytes and antigen presenting cells of the
monocyte/macrophage cell line. See, P. M. Cox, et al., "An ATF/CREB
binding motif is required for aberrant constitutive expression of
the MHC class II Dr.alpha. promoter and activation by SV40
T-antigen. Nucleic Acids Res.," 20:4881-4887 (1992). In B.sub.16
melanoma cells this is due to activation of the MHC II DR.alpha.
promoter by constitutive activation of an ATF/CREB motif. CREB
family proteins also bind to the UV-response element (URE,
5'-TGACAACA-3'), and URE binding of the CREB family member ATF2
confers resistance to irradiation and to the chemotherapeutic drugs
cis-platinum, 1-.beta.-D-arabinofuranosylcytosine (araC) or
mitomycin C in MeWo melanoma lines. See, Z. Ronai, et al., "ATF2
confers radiation resistance to human melanoma cells," Oncogene
16:523-531 (1998)). Thus, CREB family transcription factors play
important roles in the malignant potential of this important tumor
type. This has led to the suggestion by others that targeted
molecular disruption of ATF/CREB-mediated transcription might be
therapeutically useful for controlling growth and metastases of
relatively treatment-resistant malignant melanoma. See, D. Jean,
supra, and Z. Ronai, supra.
[0008] The positively charged DNA binding domain of many
transcription factors contains cysteines which can be oxidatively
modified by agents such as hydrogen peroxide or nitric oxide
(NO.sup.-), stimulating repair processes that result in formation
of mixed disulfides between glutathione (GSH) and protein thiols.
See, P. Klatt, et al., supra; and H. Sies, "Glutathione and its
role in cellular functions," Free Rad. Biol. Med. 27:916-921
(1999)). As a consequence of this so-called protein
"S-glutathionylation", the usually positively charged transcription
factor DNA binding domain develops an electronegative charge
imparted by dual carboxylate end groups of GSH. The change in
charge disrupts transcription factor binding to its respective DNA
consensus sequence. See, P. Klatt, et al., supra and H. Sies,
supra. This mechanism has been demonstrated to explain how NO
inhibits c-Jun DNA binding by specifically targeted
S-glutathionylation of cysteines within the DNA binding region, and
a similar mechanism has been suggested for how nitrosative stress
in general might functionally inhibit the activity of Fos,
ATF/CREB, Myb and Rel/NF.kappa.B family transcription factors. See,
P. Klatt, et al., supra.
[0009] The dithiocarbamates comprise a broad class of molecules
giving them the ability to complex metals and react with sulfhydryl
groups and glutathione. After metal-catalyzed conversion to their
corresponding disulfides, dithiocarbamates inhibit cysteine
proteases by forming mixed disulfides with critical protein thiols.
See, C. S. I. Nobel, et al., "Mechanism of dithiocarbamate
inhibition of apoptosis: thiol oxidation by dithiocarbamate
disulfides directly inhibits processing of the caspase-3
proenzyme," Chem. Res. Toxicol. 10:636-643 (1997). CREB contains
three cysteines in the DNA binding region (Cys.sup.300, Cys.sup.310
and Cys.sup.337) which are not essential for DNA binding but might
provide reactive sites for S-glutathionylation. See, S. Orrenius,
et al., "Dithiocarbamates and the redox regulation of cell death,"
Biochem. Soc. Trans. 24:1032-1038 (1996)).
[0010] Recently, dithiocarbamates containing a reduced sulfhydryl
group, e.g., pyrrolidinedithiocarbamate (PDTC) have been shown to
inhibit the proliferation of cultured colorectal cancer cells. See,
Chinery, et al., "Antioxidants enhance the cytotoxicity of
chemotherapeutic agents in colorectal cancer: a p53-independent
induction of p21.sup.WAF1/CIP1 via C/EBP.beta.," Nature Med.
3:1233-1241 (1997); Chinery et al., "Antioxidants reduce
cyclooxygenase-2 expression, prostaglandin production, and
proliferation in colorectal cancer cells." Cancer Res. 58:2323-2327
(1998).
[0011] In addition to their reduced thioacid form, dithiocarbamates
exist in three other forms, e.g., a) the disulfide, a condensed
dimmer of the thioacid, with elimination of reduced sulfhydryl
groups by disulfide bond formation; b) the negatively charged
thiolate anion, generally as the alkali metal salt, such as sodium;
and c) the 1,1-dithiolato complexes of the transition elements, in
which the two adjoining sulfur atoms of the dithiocarbamate are
bound to the same titanium, vanadium, chromium, iron, cobalt,
nickel, copper, silver or gold metal ion. The disulfide, thiolate
anion and transition metal complexes of dithiocarbamates are all
structurally distinct from the reduced form of PDTC used by
Chinery, et al., in that they have no reduced sulfhydryl molecular
moiety and are incapable of functioning as antioxidants by donating
the proton from a reduced sulfhydryl to scavenge electrons of free
radical species. Lacking a reduced sulfhydryl, thiocarbamate
disulfides, thiolate anions and transition metal complexes should,
according to the teachings of Chinery, et al., have no activity as
antiproliferative compounds against cancer, since these three
nonreduced chemical forms of dithiocarbamates are incapable of
functioning as antioxidants.
[0012] In U.S. patent application Ser. No. 09/392,122; filed Sep.
8, 1999, it was reported that the dithiocarbamate disulfide
disulfuram sensitizes tumor cells to cancer chemotherapy and could
be used in conjunction with cancer chemotherapeutic drugs to
increase their effectiveness in treating neoplasms. Recently, this
effect has been explained in work in which disulfuram was shown to
prevent maturation of the P-glycoprotein pump, an ATP-driven 170-kd
efflux pump on the plasma membrane that pumps a variety of
cytotoxic drugs out of cells. See, T. W. Loo, et al., "Blockage of
drug resistance in vitro by disulfuram, a drug used to treat
alcoholism." J. Natl. Cancer Inst. 92:898-902 (2000). This effect
reduces P-glycoprotein-mediated drug resistance in tumor cells and
sensitizes tumor cells to cancer chemotherapy.
[0013] It is therefore an object of the present invention to
provide a method for the treatment of cancer.
[0014] Another object of the present invention is to provide
pharmaceutical compositions for the treatment of cancer.
[0015] It is still another object of the present invention to
provide a relatively less toxic agent available for use alone in
combination with current drugs in order to better treat cancer
patients without risking injury from the therapy itself.
SUMMARY OF THE INVENTION
[0016] The present invention provides a method for treating
established cancer using dithiocarbamate disulfides, or
thiocarbamate anions either alone, or in combination with a heavy
metal ion, and thiocarbamate complexes of heavy metal ions,
[0017] It has been discovered that dithiocarbamate disulfides and
their corresponding thiolate anions alone exhibit potent inhibitory
effects on growth of established tumor cells in the absence of
antioxidant sulfhydryl groups within their structure. Thiocarbamate
disulfides and their corresponding thiolate anions are effective in
inhibiting the growth of established melanomas and non-small cell
lung cancer cells, which are known to be poorly responsive to
currently available neoplastic agents. In addition, it has further
been surprisingly discovered that the antiproliferative and
antineoplastic effect of dithiocarbamate disulfides and their
corresponding thiolate anions on established tumor cells is greatly
potentiated by co-treatment of cancer cells with a transitional
metal salt in a concentration which by itself does not impair
cancer cell growth. The potentiating function of the transition
metal is to facilitate formation of the thiolate anion from the
dithiocarbamate disulfide. Further the tumor cell growth inhibition
effect can be significantly enhanced by the addition of heavy metal
ions such as copper, zinc, gold and silver ion, as examples, or by
administering the thiocarbamate as a heavy metal ion complex.
[0018] The chemical activity of these species is not from
antioxidant action but from stimulating formation of mixed
disulfides between the dithiocarbamate and sulfhydryl moieties of
cysteines located at critical sites on cell proteins, such as the
DNA binding region of transcription factors needed to promote
expression of gene products necessary for malignant cell
proliferation.
[0019] Dithiocarbamates disulfides that are useful in the treatment
of cancer include, but are not limited to, those of the formulas:
R.sub.2R.sub.3N(S)CS--SC(S)NR.sub.2R.sub.3 wherein R.sub.1,
R.sub.2, R.sub.3 and R.sub.4 are the same or different and
represent hydrogen, and unsubstituted or substituted alkyl,
alkenyl, aryl, alkoxy, and heteroaryl groups. It is noted that the
alkyl groups can include cycloalkyl and heterocycloalkyl groups.
R.sub.1, R.sub.2 and the N atom in the formula can together form an
N-heterocyclic ring, which is, e.g., heterocycloalkyl or
heterocycloaryl. Likewise, R.sub.3, R.sub.4 and the N atom in the
formula can together form an N-heterocyclic ring, which is, e.g.,
heterocycloalkyl or heterocycloaryl. Typically R.sub.1 and R.sub.2
are not both hydrogen, and R.sub.3 and R.sub.4 are not both
hydrogen.
[0020] In accordance with another aspect of this invention, a
method for treating established cancer in a patient is provided
comprising administering to the patient a therapeutically effective
amount of a dithiocarbamate disulfide, preferably disulfuram, or
the corresponding diethyldithiocarbamate thiolate metal complex of
the formula: ##STR1## wherein R.sub.2 and R.sub.3 are the same or
different and represent hydrogen, and unsubstituted or substituted
alkyl, akenyl, aryl, alkoxy, and heteroaryl groups; M is a heavy
metal, e.g., arsenic, bismuth, gallium, manganese, selenium, zinc,
titanium, vanadium, chromium, iron, cobalt, nickel, copper, silver
or gold; n is the valence of the metal, and An is an anion selected
from the group consisting of chloride, bromide, iodide, acetate and
low molecular weight organic or inorganic pharmaceutically
acceptable anions.
[0021] In a preferred embodiment, the heavy metal ion is
administered as a complex or chelate with the dithiocarbamate
disulfide or corresponding thiolate anion. Suitable heavy metal
ions include but are not limited to ions of arsenic, bismuth,
cobalt, copper, chromium, gallium, gold, iron, manganese, nickel,
silver, titanium, vanadium, selenium, and zinc.
[0022] In another preferred embodiment, the dithiocarbamate
disulfide or corresponding thiolate anion and the heavy metal ion
are administered in combination with another anticancer agent.
[0023] In addition, the present invention provides a method for
sensitizing cancer cells to chemotherapeutic drugs by the
administration of a dithiocarbamate thiolate anion or a
dithiocarbamate complex with heavy metals in order to effect
inhibition of the tumor cell membrane P-glycoprotein pump which
functions to extrude from cancer cells the anti-neoplastic agents
which are absorbed.
[0024] In yet another embodiment of the present invention, a method
of reducing hypoxic or ischemic damage to the cardiovascular system
of a human is provided using a dithiocarbamate disulfide or
corresponding thiolate anion.
[0025] In accordance with another aspect of the invention, the
present invention provides a pharmaceutical composition that
comprises a pharmaceutically acceptable carrier, and a complex
between a dithiocarbamate and a heavy metal ion. Optionally, the
composition can further contain another anticancer agent.
[0026] The active compounds of this invention can be administered
through a variety of administration routes. For example, they can
be administered orally, intravenously, intradermally,
subcutaneously and topically.
[0027] The present invention is effective for treating various
types of cancer, including but not limited to melanoma, non-small
cell lung cancer, small cell lung cancer, renal cancer, colorectal
cancer, breast cancer, pancreatic cancer, gastric cancer, bladder
cancer, ovarian cancer, uterine cancer, lymphoma, and prostate
cancer. In particular the present invention will be especially
effective in treating melanoma, lung cancer, breast cancer and
prostate cancer. Thus the use of dithiocarbamate disulfides and
thiolate anions in this invention offers a readily available and
easily used treatment for cancers in man and other animals.
BRIEF DESCRIPTION OF THE DRAWINGS
[0028] FIG. 1A shows that M1619 melanoma cells exhibit constitutive
DNA binding activity to the cyclic AMP response element (CRE);
[0029] FIG. 1B shows that the thiocarbamate disulfide disulfuram
and copper inhibit transcription factor binding to CRE;
[0030] FIG. 1C shows that EMSAs performed using nuclear protein
from replicate experiments (n=4) in which near confluent cells were
treated for 8 hr with FBS alone, DMSO vehicle (5 .mu.l/well),
disulfuram (5 .mu.M), CuSO.sub.4 (1.6 .mu.M), or the combination of
disulfuram plus copper;
[0031] FIG. 2 shows the effect of adding disulfuram or disulfuram
plus copper directly to binding reaction on transcription factor to
DNA binding;
[0032] FIG. 3A shows disulfuram and copper reduce expression of the
cell-cycle protein cyclin A;
[0033] FIG. 3B replicates experiments (n=4 each) in which cells
were treated with DMSO vehicle, (5 .mu.l/ml, lanes 1-4), disulfuram
(5 .mu.M, lanes 5-8), (5 .mu.l/ml), CuSO.sub.4 (1.6 .mu.M, lanes
9-12) or the combination of disulfuram and CuSO.sub.4 (lanes
13-16). After 24 hours cells were lysed, immunoblots were performed
to assay for cyclin A;
[0034] FIG. 3C illustrates quantitation of experiments in FIG. 3B
by densitometry;
[0035] FIG. 4A shows that disulfuram inhibits proliferation of
M1619 human melanoma cell lines;
[0036] FIG. 4B illustrates that cell-impermeate Cu.sup.2+ chelator
bathocuproine-disulfonic acid prevents growth inhibition from
disulfuram;
[0037] FIG. 4C shows supplementation of growth medium with copper
enhances the antiproliferative activity of disulfuram;
[0038] FIG. 4D shows that ceruloplasmin can serve as a source of
copper for enhancing the antiproliferative activity of
disulfuram;
[0039] FIG. 5A shows M1619 melanoma cells treated with DMSO
vehicle;
[0040] FIG. 5B shows M1619 melanoma cells treated with 5 .mu.M
disulfuram;
[0041] FIG. 6A shows that disulfuram combined with copper induces
S-phase cell cycle arrest in M1619 melanoma cells and
apopotosis;
[0042] FIG. 6B shows that 5 .mu.M disulfuram combined with copper
induces S-phase cell cycle arrest in M1619 melanoma cells and
apopotosis;
[0043] FIG. 6 C shows that 5 .mu.M disulfuram plus 250 .mu.g/ml
ceruloplasmin (Cerulo) as a source of copper.
[0044] FIG. 7A shows that other metals also protentiate
antiproliferative activity of disulfuram;
[0045] FIG. 7B shows the antiproliferative activity of disulfuram
is enhanced by supplementation of medium with other heavy
metals;
[0046] FIG. 7C shows complexes of disulfuram with gold demonstrate
enhanced antiproliferative activity;
[0047] FIG. 7D shows the antiproliferative activity of the thiolate
sodium diethyldithiocarbamate trihydrate (NaDDC) is reduced by low
concentrations of DTT in the growth medium; and
[0048] FIG. 8 shows the X-ray crystallographic structure of
complexes formed from mixing gold tetrachloride and disulfuram.
DETAILED DESCRIPTION OF THE INVENTION
[0049] The present invention will now be described more fully
hereinafter with reference to the accompanying examples, in which
preferred embodiments of the invention are shown. This invention
may, however, be embodied in many different forms and should not be
construed as limited to the embodiments set forth herein; rather,
these embodiments are provided so that this disclosure will be
thorough and complete, and will fully convey the scope of the
invention to those skilled in the art.
[0050] As used herein, the term "dithiocarbamate disulfides" refers
to compounds having the formula of:
R.sub.1R.sub.2N(S)CS--SC(S)NR.sub.3R.sub.4 wherein R.sub.1,
R.sub.2, R.sub.3 and R.sub.4 are the same or different and
represent hydrogen, and unsubstituted or substituted alkyl,
alkenyl, aryl, alkoxy, and heteroaryl groups. It is noted that the
alkyl groups can include cycloalkyl and heterocycloalkyl groups.
R.sub.1, R.sub.2 and the N atom in the formula can together form an
N-heterocyclic ring, which is, e.g., heterocycloalkyl or
heterocycloaryl. Likewise, R.sub.3, R.sub.4 and the N atom in the
formula can together form an N-heterocyclic ring, which is, e.g.,
heterocycloalkyl or heterocycloaryl. Typically R.sub.1 and R.sub.2
are not both hydrogen, and R.sub.3 and R.sub.4 are not both
hydrogen. Thus, dithiocarbamate disulfide is a disulfide form of
dithiocarbamates that have a reduced sulfhydryl group.
[0051] Many dithiocarbamates are known and synthesized in the art.
Non limiting examples of dithiocarbamates include
diethyldithiocarbamate, pyrrolodinedithiocarbamate, N-methyl,
N-ethyl dithiocarbamates, hexamethylenedithiocarbamate,
imidazolinedithiocarbamates, dibenzyldithiocarbamate,
dimethylenedithiocarbamate, dipolyldithiocarbamate,
dibutyldithiocarbamate, diamyldithiocarbamate, N-methyl,
N-cyclopropylmethyldithiocarbamate, cyclohexylamyldithiocarbamate,
pentamethylenedithiocarbamate, dihydrxyethyldithiocarbamate,
N-methylglucosamine dithiocarbamate, and salts and derivatives
thereof. Typically, a sulfhydryl-containing dithiocarbamate can be
oxidized to form a dithiocarbamate disulfide.
[0052] Sulfhydryl-containing dithocarbamates can be converted to
their corresponding thiolate anions by treatment with an
alkali-metal hydroxide as a proton acceptor, yielding the
structure: ##STR2## wherein R.sub.2 and R.sub.3 are the same or
different and represent hydrogen, and unsubstituted or substituted
alkyl, akenyl, aryl, alkoxy, and heteroaryl groups; M is an alkali
metal selected from the group consisting of from the group
consisting of sodium, potassium, calcium, magnesium, barium, and
lithium; and n is the valence of the alkali metal.
[0053] Finally, the heavy metal complexes of dithocarbamate can be
synthesized either by treatment of the disulfide or the thiolate
anion forms of dithiocarbamates with metal salts, yielding a
variety of useful metal complexes in which the metal forms a
complex with both sulfur atoms: ##STR3## wherein R.sub.2 and
R.sub.3 are the same or different and represent hydrogen, and
unsubstituted or substituted alkyl, akenyl, aryl, alkoxy, and
heteroaryl groups; M is a heavy metal, e.g., arsenic, bismuth,
gallium, manganese, selenium, zinc, titanium, vanadium, chromium,
iron, cobalt, nickel, copper, silver, silver or gold; n is the
valence of the heavy metal; and An is an anion selected from the
group consisting of chloride, bromide, iodide, acetate and low
molecular weight inorganic pharmaceutically acceptable anions.
[0054] Specifically, the preferred gold 1,1-dithio chelates of
dithiocarbamates has the formulae: ##STR4## wherein R.sub.2,
R.sub.3 are ethyl, and An is an anion of low molecular weight.
[0055] Any pharmaceutically acceptable form of dithiocarbamate
disulfides, their corresponding thiolate anions and dithiocarbamate
metal chelates can be used. For example, tetraethylthiuram
disulfide, which is known as disulfuram, is used in one embodiment
of this invention. Disulfuram has the following formula:
R.sub.1R.sub.2N(S)CS--SC(S)NR.sub.3R.sub.4 where R.sub.1, R.sub.2,
R.sub.3 and R.sub.4 are all ethyl. Disulfuram has been used
clinically in the treatment of alcohol abuse, in which disulfuram
inhibits hepatic aldehyde dehydrogenase.
[0056] The thiolate anion derivative of disulfuram is
diethyldithiocarbamate anion, the sodium salt of which has the
following formula: ##STR5## Finally, the heavy metal complex of
diethyldithiocarbamate, exemplified below as the gold (Au III)
1,1-dithiolato complex, is shown: ##STR6## wherein R.sub.2 and
R.sub.3 are ethyl, and An is an anion of small molecular
weight.
[0057] Methods of making dithiocarbamates and their disulfides are
generally known in the art. Exemplary methods are disclosed in,
e.g., Thorn, et al, The Dithiocarbamates and Related Compounds,
Elsevier, New York, 1962; and U.S. Pat. Nos. 5,166,387, 4,144,272,
4,066,697, 1,782,111, and 1,796,977, all of which are incorporated
herein by reference.
[0058] The term "treating cancer" as used herein, specifically
refers to administering therapeutic agents to a patient diagnosed
of cancer, i.e., having established cancer in the patient, to
inhibit the further growth or spread of the malignant cells in the
cancerous tissue/ and/or to cause the death of malignant cells.
[0059] This invention provides a method for treating cancer in a
patient. In accordance with the present invention, it has been
discovered that dithiocarbamate disulfides, their corresponding
thiolate anions, and their heavy metal complexes, such as
disulfuram, the diethyldithiocarbamate anion and
dichloro(ditheylthiocarbamyl)gold (II), respectively, can inhibit
the growth of tumor cells in a heavy metal ion-dependent manner.
Specifically, heavy metal ions such as copper, zinc, gold, and
silver ions significantly enhance the inhibitory effect of
dithiocarbamate disulfides and their thiolate anions on tumor
cells, while depletion of such heavy metal ions prevents growth
inhibition by disulfuram and the diethyldithiocarbamate anion. The
function performed by the metal is to chemically catalyze formation
of or stabilize the thiolate anion form in vivo, so that the
thiolate anion is able to form mixed disulfides with protein
cysteine sulfhydryl groups of cellular proteins.
[0060] In accordance with one aspect of this invention, a method
for treating an established cancer in a patient is provided. A
dithiocarbamate disulfide can be administered to a patient having
established cancer to treat the cancer. Preferably, the thiuram
disulfide administered is a tetra alkyl thiuram disulfide such as
teraethylthiuram disulfide, i.e., disulfuram.
[0061] In another aspect of the invention, the method for treating
cancer in a patient comprises administering to the patient a
therapeutically effective amount of a dithiocarbamate thiolate
anion.
[0062] In another aspect of the invention, the method for treating
cancer in a patient comprises administering to the patient a
therapeutically effective amount of a dithiocarbamate disulfide or
its thiolate anion, and a heavy metal ion.
[0063] Non-limiting examples of heavy metal ions include ions of
arsenic, bismuth, cobalt, copper chromium, gallium, gold iron,
manganese, nickel, silver, titanium, vanadium, selenium and zinc.
Preferably, gold, silver, zinc, selenium, and copper ions are used.
Sources of such heavy metal ions are known to the those skilled in
the art. For example, such ions can be provided in a sulfate salt,
or chloride salt form, or any other pharmaceutically suitable
forms. Preferably, the salt is in a chelated form, complexed with a
pharmaceutically acceptable organic anion such as acetate,
glycinate, gluconate, propionate or lactate so that absorption of
the metal from the gastrointestinal tract is enhanced.
[0064] One or more dithocarbamate disulfide or corresponding
thiolate anions and one or more heavy metal ions can be
administered to the patient. The dithiocarbamate disulfide or
thiolate anion and the heavy metal ion can be administered in
combination or separately. Preferably, they are administered as a
chelating complex. As is known in the art, dithiocarbamates are
excellent chelating agents and can chelate heavy metal ions to form
chelates. Preparation of chelates of dithiocarbamates and heavy
metal ions are known to the ordinary artisan. For example, chelates
of diethyldithiocarbamate and copper, zinc, silver, or gold ions
can be conveniently synthesized by mixing, in suitable solvents,
disulfuram or sodium diethyldithiocarbamate with, e.g., CuSO.sub.4,
ZnCl.sub.2, C.sub.3H.sub.5AgO.sub.3, or HAuCl.sub.4.3H.sub.2O to
allow chelates to be formed. Other dithiocarbamate-heavy metal ion
chelates are disclosed in, e.g., D. Coucouvanis, "The chemistry of
the dithioacid and 1,1-dithiolate complexes," Prog. Inorganic Chem.
11:234-371 (1970); D. Coucouvanis, "The chemistry of the dithioacid
and 1,1-dithiolate complexes, 1968-1977," Prog. Inorganic Chem.
26:302-469 (1978); R. P. Burns, et al., "1,1-dithiolato complexes
of the transition metals," Adv. Inorganic Chem. and Radiochem.
23:211-280 (1980); L. I. Victoriano, et al., "The reaction of
copper (II) chloride and tetralkylhiuram disulfides," J. Coord.
Chem. 35:27-34 (1995); L. I. Victoriano, et al., "Cuprous
dithiocarbamates. Syntheses and reactivity," J. Coord. Chem.
39:231-239 (1996), which are incorporated herein by reference.
[0065] In accordance with another aspect of this invention, a
method for treating cancer in a patient is provided which includes
administering to the patient a therapeutically effect amount of a
dithiocarbamate anion compound and an intracellular heavy metal ion
stimulant, which can enhance the intracellular level of the above
described heavy metal ions in the patient.
[0066] Intracellular heavy metal ion carriers are known. For
example, ceruloplasmin can be administered to the patient to
enhance the intracellular copper level. Other heavy metal ion
carriers known in the art may also be administered in accordance
with this aspect of the invention. The heavy metal ion carriers and
the dithocarbamate disulfide or thiolate anion can be administered
together or separately, and preferably in separate
compositions.
[0067] Ceruloplasmin is a protein naturally produced by the human
body and can be purified from human serum. This 132-kD
glycoprotein, which carries 7 copper atoms complexed over three
43-45 kD domains, is an acute phase reactant and the major
copper-carrying protein in human plasma. See, Halliwell, et al.,
Methods Enzymol. 186:1-85 (1990). When transported into cells, at
least some of the bound cupric ions can be accessible for
complexation with the dithiocarbamate disulfide or thiolate anion
administered to the patient. See, Percival, et al., Am. J. Physiol.
258:3140-3146 (1990). Ceruloplasmin and dithiocarbamate disulfides
or thiolate anions are typically administered in different
compositions. Dithiocarbamate disulfides or thiolate anions can be
administered at about the same time, or at some time apart. For
example, ceruloplasmin can be administered from about five minutes
to about 12 hours before or after dithiocarbamate disulfide or
thiolate anions are administered to the patient.
[0068] In another embodiment, instead of heavy metal ion carriers,
a cytokine is administered to the patient in addition to a
dithiocarbamate disulfide or corresponding thiolate anion. Suitable
cytokines include, e.g., interferon .alpha., interferon .beta.,
interferon .gamma., and interleukin 6 (IL-6). Such cytokines, when
administered to a patient, are capable of inducing an acute phase
response in the body of the patient, thus stimulating elevations of
serum ceruloplasmin in the patient.
[0069] The biochemical and physiological properties of such
cytokines have been studied extensively in the art and are familiar
to skilled artisans. The cytokines can be purified from human or
animal serum. They can also be obtained by genetic engineering
techniques. In addition, commercially available samples of the
above-identified cytokines may also be used in this invention.
Genetically or chemically modified cytokines can also be
administered. For example, it is known that certain peptidic
cytokines have longer circulation time in animals when such
cytokines are conjugated with a water soluble, non-immunogenic
polymer such as polyethylene glycol.
[0070] Typically, the cytokines are administered in a different
composition from the dithiocarbamate disulfide or corresponding
thiolate anion. The cytokines and dithocarbamate disulfide or
thiolate anion can be administered at about the same time, or at
some time apart from each other. For example the cytokines can be
administered from about 5 minutes to about 24 hours before or after
the administration of dithiocarbamate disulfide or thiolate
anion.
[0071] In accordance with another aspect of this invention, the
method of this invention can be used in combination with a
conventional cancer chemotherapy, with the result that the
treatment with dithiocarbamate disulfides or thiolate anions, with
or without heavy metals separately or as dithocarbamate-heavy metal
chelates, will increase the sensitivity of the tumor to
conventional cancer chemotherapy and result in greater
effectiveness of the conventional cancer chemotherapy drug. For
example, the method of this invention can be complemented by a
conventional radiation therapy or chemotherapy. Thus, in one
embodiment of this invention, the method of this invention
comprises administering to a patient a dithiocarbamate disulfide
compound or corresponding thiolate anion and heavy metals, and
another anticancer agent. Treatment by ceruloplasmin or a cytokine,
and a dithiocarbamate disulfide or thiolate anion can also be
conducted along with the treatment with another anticancer agent to
increase the effectiveness of the anticancer agent.
[0072] Any anticancer agents known in the art can be used in this
invention so long as it is pharmaceutically compatible with the
dithiocarbamate disulfide or thiolate anion compound, heavy metal
ion, ceruloplasmin, and/or cytokines used. By "pharmaceutically
compatible" it is intended that the other anticancer agent will not
interact or react with the above composition, directly or
indirectly, in such a way as to adversely affect the effect of the
treatment of cancer, or to cause any significant adverse side
reaction in the patient.
[0073] Exemplary anticancer agents known in the art include
busulphan, chlorambucil, hydroxyurea, ifosfamide, mitomycin,
mitotane, chlorambucil, mechlorethamine, carmustine, lomustine,
cisplatin, carmustine, herceptin, carboplatin, cyclophosphamide,
nitrosoureas, fotemustine, vindescine, etoposide, daunorubicin,
adriamycin, paclitaxel, docetaxel, streptozocin, dactinomycin,
doxorubicin, idarubicin, plicamycin, pentostatin, mitotoxantrone,
valrubicin, cytarabine, fludarabine, floxuridine, clardribine,
methotrexate, mercaptopurine, thioguanine, capecitabine,
irinotecan, dacarbazine, asparaginase, gemcitabine, altretamine,
topotecan, procarbazine, vinorelbine, pegaspargase, vincristine,
rituxan, vinblastine, tretinoin, teniposide, fluorouracil,
melphalan, bleomycin, salicylates, aspirin, piroxicam, ibuprofen,
indomethacin, naprosyn, diclofenac, tolmetin, ketoprofen,
nambuetone, oxaprozin, doxirubicin, nonselective cycclooxygenase
inhibitors such as nonsteroidal anti-inflammatory agents (NSAIDS),
and selective cyclooxygenase-2 (COX-2) inhibitors.
[0074] The anticancer agent used can be administered simultaneously
in the same pharmaceutical preparation with the dithiocarbamate
disulfide or thiolate anion compound, heavy metal compounds or
dithiocarbamate-heavy metal chelates, ceruloplasmin, and/or
cytokines as described above. The anticancer agent can also be
administered at about the same time but by a separate
administration. Alternatively, the anticancer agent can be
administered at a different time from the administration of the
dithiocarbamate disulfide or thiolate anion compound, heavy metal
compounds or dithiocarbamate-heavy metal chelates, ceruloplasmin,
and/or cytokines. Some minor degree of experimentation may be
required to determine the best manner of administration, this being
well within the capability of one skilled in the art once apprised
of the present disclosure.
[0075] The methods of this invention a particularly useful in
treating humans. Also, the methods of this invention are suitable
for treating cancers in animals, especially mammals such as canine,
bovine, porcine, and other animals. The methods are useful for
treating various types of cancer, including but not limited to
melanoma, non-small cell lung cancer, small cell lung cancer, renal
cancer, colorectal cancer, breast cancer, pancreatic cancer,
gastric cancer, bladder cancer, ovarian cancer, uterine cancer,
lymphoma, and prostate cancer. In particular, the present invention
will be especially effective in treating melanoma, lung cancer,
breast cancer, and prostate carcinoma.
[0076] The active compounds of this invention are typically
administered in a pharmaceutically acceptable carrier through any
appropriate routes such as parenteral, intravenous, oral,
intradermal, subcutaneous, or topical administration. The active
compounds of this invention are administered at a therapeutically
effective amount to achieve the desired therapeutic effect without
causing any serious adverse effects in the patient treated.
[0077] The dithiocarbamate disulfide compound disulfuram and its
diethyldithiocarbamate thiolate anion are effective when
administered at amounts within the conventional clinical ranges
determined in the art. Disulfuram approved by the U.S. Food and
Drug administration (Antabuse.RTM.) can be purchased in 250 and 500
mg tablets for oral administration from Wyeth-Ayerst Laboratories
in Philadelphia, Pa. 19101. Typically, it is effective at an amount
of from about 125 to about 1000 mg per day, preferably from 250 to
about 500 mg per day for disulfuram and 100 to 500 mg per day or 5
mg/kg intravenously or 10 mg/kg orally once a week for
diethyldithiocarbamate. However, the dosage can vary with the body
weight of the patient treated. The active ingredient may be
administered at once, or may be divided into a number of smaller
doses to be administered at predetermined intervals of time. The
suitable dosage unit for each administration of disulfuram is,
e.g., from about 50 to about 1000 mg/day, preferably from about 250
to about 500 mg/day. The desirable peak concentration of disulfuram
generally is about 0.05 to about 10 .mu.M, preferably about 0.5 to
about 5 .mu.M, in order to achieve a detectable therapeutic effect.
Similar concentration ranges are desirable for dithiocarbamate
thiolate anions and for dithocarbamate-heavy metal complexes.
[0078] Disulfuram implanted subcutaneously for sustained release
has also been shown to be effective at an amount of 800 to 1600 mg
to achieve a suitable plasma concentration. This can be
accomplished by using aseptic techniques to surgically implant
disulfuram into the subcutaneous space of the anterior abdominal
wall. See, e.g., Wilson, et al., J. Clin. Psych. 45:242-247 (1984).
In addition, sustained release dosage formulations, such as an 80%
poly(glycolic-co-L-lactic acid) and 20% disulfuram, may be used.
The therapeutically effective amount for other dithiocarbamate
disulfide compounds may also be estimated or calculated based on
the above dosage ranges of disulfuram and the molecular weights of
disulfuram and the other dithiocarbamate disulfide compound, or by
other methods known in the art.
[0079] The diethyldithiocarbamate thiolate anion has not been
previously advocated as a cancer chemotherapeutic agent itself, nor
has it been suggested as a treatment to increase the sensitivity of
tumors to cancer chemotherapy drugs. For the treatment of HIV
infection, humans have been treated with doses of 5 mg/kg
intravenous or 10 mg/kg orally, once a week. Minimal side effects
on this dosage regimen include a metallic taste in the mouth,
flatulence and intolerance to alcoholic beverages. An
enteric-coated oral dosage form of diethyldithiocarbamate thiolate
anions to liberate active drug only in the alkaline environment of
the intestine is preferred because of the potential for liberation
of carbon disulfide upon exposure of diethyldithiocarbamate to
hydrochloric acid in the stomach. An oral enteric-coated form of
sodium diethyldithiocarbamate is available in 125 mg tablets as
Imuthiol.RTM. through Institute Merieux, Lyon, France.
[0080] Heavy metal ions can be administered separately as an
aqueous solution in a pharmaceutically suitable salt form. The salt
form is ideally a chelate with an organic anion such as acetate,
lactonate, glycinate, citrate, propionate or gluconate in order to
enhance absorption. However, the heavy metals are preferably
administered in a chelate form in which the ions are complexed with
the dithiocarbamate as a 1,1-dithiolate complex. Thus, the amount
of heavy metal ions to be used advantageously is proportional to
the amount of dithiocarbamate disulfide compound to be administered
based on the molar ratio between a heavy metal ion and the
dithiocarbamate in the chelate. Methods for preparing such chelates
or complexes are known and the preferred methods are disclosed
above and in the examples below.
[0081] The therapeutically effective amount of IL-6 can be from
about 1 to about 100 .mu.g/kg per day, preferably from about 5 to
about 50 .mu.g/kg per day. Interferon .alpha. can be administered
at from about 0.1.times.10.sup.6 to about 10.times.10.sup.6
international units per day, preferably from about 3 to about
8.times.10.sup.6 international units per day, and the
administration frequency can be from about three times per week to
about once per day. Suitable dosage for interferon .beta. can range
from about 1 to about 200 .mu.g per day, preferably from about 10
to about 100 .mu.g per day administered once per week up to once
per day. Interferon .gamma. can be administered at a dosage of from
about 1 to about 1000 .mu.g per day, preferably from about 50 to
about 250 .mu.g per day. Ceruloplasmin may be administered at an
amount of from about 1 to about 100 mg per day, preferably from
about 50 to about 30 mg per day.
[0082] It should be understood that the dosage ranges set forth
above are exemplary only and are not intended to limit the scope of
this invention. The therapeutically effective amount for each
active compound can vary with factors including but not limited to
the activity of the compound used, stability of the active compound
in the patient's body, the severity of the conditions to be
alleviated, the total weight of the patient treated, the route of
administration, the ease of absorption, distribution, and excretion
of the active compound by the body, the age and sensitivity of the
patient to be treated, and the like, as will be apparent to a
skilled artisan. The amount of administration can also be adjusted
as the various factors change over time.
[0083] Advantageously, the active compounds are delivered to the
patient parenterally, i.e., intravenously or intramuscularly. For
parenteral administration, the active compounds can be formulated
into solutions or suspensions, or in lyophilized forms for
conversion into solutions or suspensions before use. Sterile water,
physiological saline, e.g., phosphate buffered saline (PBS) can be
used conveniently as the pharmaceutically acceptable carriers or
diluents. Conventional solvents, surfactants, stabilizers, pH
balancing buffers, anti-bacteria agents, and antioxidants can all
be used in the parenteral formulations, including but not limited
to acetates, citrates or phosphate buffers, sodium chloride,
dextrose, fixed oils, glycerine, polyethylene glycol, propylene
glycol, benzyl alcohol, methyl parabens, ascorbic, acid, sodium
bisulfite, and the like. For parenteral administration, the active
compounds, particularly dithiocarbamate-metal chelates, can be
formulated contained in liposomes so as to enhance absorption and
decrease potential toxicity. The parenteral formulation can be
stored in any conventional containers such as vials, ampoules, and
syringes.
[0084] The active compounds can also be delivered orally in
enclosed gelatin capsules or compressed tablets. Capsules and
tablets can be prepared in any conventional techniques. For
example, the active compounds can be incorporated into a
formulation which includes pharmaceutically acceptable carriers
such as excipients (e.g., starch, lactose), binders (e.g., gelatin,
cellulose, gum), disintegrating agents (e.g., alginate, Primogel,
and corn starch), lubricants (e.g., magnesium stearate, silicon
dioxide), and sweetening or flavoring agents (e.g., glucose,
sucrose, saccharin, methyl salicylate, and peppermint). Various
coatings can also be prepared for the capsules and tablets to
modify the flavors, tastes, colors, and shapes of the capsules and
tablets. In addition, liquid carriers such as fatty oil can also be
included in capsules. For administration of dithiocarbamate
thiolate anions and dithiocarbamate-metal complexes, it is
desirable to administer the compounds as enteric-coated capsules
that are impervious to stomach acid but dissolve in the alkaline
environment of the small intestine, in order to prevent release of
carbon disulfide from dithiocarbamates in the acid environment of
the stomach, and to preserve the integrity of the
dithiocarbamate-metal chelate.
[0085] Other forms of oral formulations such as chewing gum,
suspension, syrup, wafer, elixir, and the like can also be prepared
containing the active compounds used in this invention. Various
modifying agents for flavors, tastes, colors, and shapes of the
special forms can also be included. In addition, for convenient
administration by enteral feeding tube in patients unable to
swallow, the active compounds can be dissolved in an acceptable
lipophilic vegetable oil vehicle, such as olive oil, corn oil, and
safflower oil.
[0086] The active compounds can also be administered topically
through rectal, vaginal, nasal or mucosal applications. Topical
formulations are generally known in the art including creams, gels,
ointments, lotions, powders, pastes, suspensions, sprays, and
aerosols. Typically, topical formulations include one or more
thickening agents, humectants, an/or emollients including but not
limited to xanthan gum, petrolatum, beeswax, or polyethylene
glycol, sorbitol, mineral oil, lanolin, squalene, and the like. A
special form of topical administration is delivery by a transdermal
patch. Methods for preparing transdermal patches are disclosed,
e.g., in Brown, et al., Annual Review of Medicine. 39:221-229
(1988), which is incorporated herein by reference.
[0087] The active compounds can also be delivered by subcutaneous
implantation for sustained release. This may be accomplished by
using aseptic techniques to surgically implant the active compounds
in any suitable formulation into the subcutaneous space of the
anterior abdominal wall. See, e.g., Wilson, et al., J. Clin. Psych.
45:242-247 (1984). Sustained release can be achieved by
incorporating the active ingredients into a special carrier such as
a hydrogel. Typically, a hydrogel is a network of high molecular
weight biocompatible polymers, which can swell in water to form a
gel like material. Hydrogels are generally known in the art. For
example, hydrogels made of polyethylene glycols, or collage, or
poly(glycolic-co-L-lactic acid) are suitable for this invention.
See, e.g., Phillips, et al., J. Pharmceut. Sci. 73:1718-1720
(1984).
[0088] The active compounds can also be conjugated, i.e.,
covalently linked, to a water soluble non-immunogenic high
molecular weight polymer to form a polymer conjugate.
Advantageously, such polymers, e.g., polyethylene glycol, can
impart solubility, stability, and reduced immunogenicity to the
active compounds. As a result, the active compound in the conjugate
when administered to a patient, can have a longer half-life in the
body, and exhibit better efficacy. PEGylated proteins are currently
being used in protein replacement therapies and for other
therapeutic uses. For example, PEGylated adenosine deaminase
(ADAGEN.RTM.) is being used to treat severe combined
immunodeficiency disease (SCIDS). PEGylated L-asparaginase
(ONCAPSPAR.RTM.) is being used to treat acute lymphoblastic
leukemia (ALL).
[0089] Alternatively, other forms of controlled release or
protection including microcapsules and nanocapsules generally known
in the art, and hydrogels described above can all be utilized in
oral, parenteral, topical, and subcutaneous administration of the
active compounds.
[0090] As discussed above, another preferable delivery form is
using liposomes as a carrier. Liposomes are micelles formed from
various lipids such as cholesterol, phospholipids, fatty acids and
derivatives thereof. Active compounds can be enclosed within such
micelles. Methods for preparing liposomal suspensions containing
active ingredients therein are generally known in the art and are
disclosed in, e.g., U.S. Pat. No. 4,522,811, which is incorporated
herein by reference. Several anticancer drugs delivered in the form
of liposomes are known in the art and are commercially available
from Liposome, Inc., of Princeton, N.J. It has been shown that
liposomal delivery can reduce the toxicity of the active compounds,
and increase their stability.
[0091] The active compounds can also be administered in combination
with other active agents that treat or prevent another disease or
symptom in the patient treated. However, it is to be understood
that such other active agents should not interfere with or
adversely affect the effects of the active compounds of this
invention on the cancer being treated. Such other active agents
include but are not limited to antiviral agents, antibiotics,
antifungal agents, anti-inflammation agents, antithrombotic agents,
cardiovascular drugs, cholesterol lowering agents, hypertension
drugs, and the like.
[0092] It is to be understood that individuals placed on
dithiocarbamate disulfide or thiolate anion therapy for their
cancer in any form must be warned against exposure to alcohol in
any form, to avoid the precipitation of nausea and vomiting from
buildup of acetaldehyde in the bloodstream. Subjects therefore must
not only refrain from ingesting alcohol containing beverages, but
should also not ingest over the counter formulations such as cough
syrups containing alcohol or even use rubbing alcohol
topically.
Experimental Procedures
Materials
[0093] Human malignant cell lines were obtained from American Type
Tissue Culture Collection (Rockville, Md.). RPMI medium 1640,
Leibovitz's L-15 medium,
N-2-hydroyethylpiperazine-N'-2-ethanesulfonic acid (HEPES),
antibiotic-antimycotic (10,000 U penicillin, 10,000 .mu.g
streptomycin, and 25 .mu.g amphotericin B/ml), fetal bovine serum
(FBS) and trypsin-ethylenediaminetetraacetic acid (EDTA) solution
were purchased from the GIBCO-BRL division of Life Technologies
(Grand Island, N.Y.). Rabbit polyclonal antibodies against human
Bcl-2, p53, p21.sup.WAF1/Cip1, cyclins A and B1, CREB1, ATF1, ATF2,
c-Jun and Jun B were from Santa Cruz Biotechnology (Santa Cruz,
Calif.). Rabbit polyclonal antibody against c-Fos and A431 cell
lysate standard were from Calbiochem (San Diego, Calif.).
Peroxidase-labeled donkey polyclonal anti-rabbit IgG was from
Amersham Life Sciences (Buckinghamshire, England), and
peroxidase-labeled anti-goat IgG was from Santa Cruz Biotechnology.
Electrophoretic mobility shift assay (EMSA) supplies, including DNA
probes, were purchased from ProMega (Madison, Wis.). Protease
inhibitors were from Boehringer Mannheim (Indianapolis, Ind.). The
diacetate of 2',7'-dichlorofluorescin (DCF-DA) was purchased from
Molecular Probes (Eugene, Oreg.). Pyrrolidinedithiocarbamate
(PDTC), diethyldithiocarbamate, tetraethylthiuram disulfide
(disulfuram), bathocuproinedisulfonic acid (BCPS), metal salts,
nonenzymatic Cell Dissociation Solution.RTM.,
N.omega.-nitro-L-arginine, indomethacin, bovine serum albumin (BSA)
and all other materials were purchased from Sigma Chemical Co. (St.
Louis, Mo.), unless specified.
Culture of Malignant Cell Lines
[0094] Human malignant cell lines were obtained from American Type
Tissue Culture Collection (Rockville, Md.). Melanoma cells lines
CRL 1585 and 1619 were cultured in RPMI 1640 (GIBCO-BRL, Life
Technologies, Grand Island, N.Y.) with 10% FBS and passed with
nonenzymatic Cell Dissociation Solutions (Sigma). The prostate
adenocarcinoma cell line CRL 1435 (PC-3) was also cultured in RPMI
1640 with 10% FBS but passed with 0.05% trypsin and 0.53 mM EDTA.
The squamous lung carcinoma NCI-H520 and the adenosquamous lung
carcinoma NCI-H596 cell lines were grown in RPMI 1640 supplemented
with 10% FBS, 10 mM HEPES and 1.0 mM sodium pyruvate and passed
with trypsin/EDTA. The small cell lung carcinoma NCI-H82 was
cultured as a suspension in RPMI 1640 with 10% FBS. All of the
above were grown in a 37.degree. C. humidified environment
containing 5% CO.sub.2/air. The breast carcinoma cell line
MDA-MB-453 was grown in a 37.degree. C. humidified environment with
free gas exchange with atmospheric air using Leibovitz's L-15
medium with 2 mM L-glutamine and 10% FBS and was passed with
trypsin/EDTA.
Cell Culture Treatments
[0095] Because the disulfide form of dithiocarbamates does not have
a free thiol to act as an antioxidant, most of the experiments were
performed with the tetraethylthiuram disulfide disulfuram. To study
the effect of disulfuram on activation of select genes important
for cellular proliferation, malignant melanoma cells were grown to
confluence on 100.times.15 mm plastic Petri dishes and treated with
5 .mu.M disulfuram or 5 .mu.M disulfuram plus 1.6 .mu.M CuSO.sub.4.
This dose was chosen to approximate the steady state plasma and
tissue concentrations of drug in human subjects on chronic therapy
with this agent. Disulfuram was solubilized in dimethylsulfoxide
(DMSO) so that the final concentration of DMSO was less than
0.3-0.5%. Equal volumes of DMSO were added to control experiments.
Nuclear protein was harvested and electrophoretic mobility gel
shift assays were performed using DNA consensus binding sequence
for the cyclic-AMP responsive element (CRE) as outlined below. To
determine whether disulfuram and metals might directly influence
transcription factor binding, in some experiments, 5 .mu.M
disulfuram and/or CuSO.sub.4 1.6 .mu.M CuSO.sub.4 (final
concentrations) were added to the binding reaction of nuclear
protein obtained from control cells stimulated with 10% FBS alone
in the absence of drugs or metals. In vitro addition of disulfuram
and CuSO.sub.4 to the binding reaction was performed using either
2.5 mM dithiothreitol (DTT) or 3.0 mM GSH as a reducing agent in
the binding buffer.
[0096] The effect of disulfuram (0.15 to 5.0 .mu.M),
diethyldithiocarbamate (DDC, 1.0 .mu.M) or PDTC (0.625 to 5.0
.mu.M) on proliferation of malignant cell lines was studied in
cultures stimulated with 10% FBS. Cell numbers were quantitated
24-72 hours later. In some experiments disulfuram or PDTC were
added immediately after cells were plated. In other experiments,
cells were plated and allowed to grow for 24-72 hours before fresh
media with disulfuram or PDTC was added, and cell numbers were
assayed 24-72 hours later. Synergy was studied between disulfuram
and N,N'-bis(2-chloroethyl-N-nitrosourea (carmustine or BCNU, 1.0
to 1,000 .mu.M) or cisplatin (0.1 to 100 .mu.g/ml) added to medium.
The effect of metals on disulfuram was studied with 0.2 to 10 .mu.M
copper (provided as CuSO.sub.4), zinc (as ZnCl.sub.2), silver (as
silver lactate) or gold (as HAuCl.sub.4.3H.sub.2O) ions added to
growth medium. No pH changes occurred with addition of metal salts
to culture medium. To provide a biologically relevant source of
copper, in some experiments medium was supplemented with human
ceruloplasmin at doses replicating low and high normal adult serum
concentrations (250 and 500 .mu.g/ml).
[0097] Potential redox effects of disulfuram were studied in three
sets of experiments. The importance of cellular glutathione (GSH)
in mediating or modulating thiocarbamate toxicity was studied by
measuring levels of intracellular GSH after treatment with
disulfuram. Disulfuram (5 .mu.M), with or without 1.6 .mu.M
CuSO.sub.4, was added to cells grown to confluence on 100.times.15
mm plastic dishes, and cells were harvested 24 hour later for
measurement of GSH as outlined below. Also, to assess whether a
nonspecific antioxidant effect of disulfuram or PDTC might account
for cellular growth inhibition, we studied the effect of the potent
lipophilic antioxidant probucol (1.0 to 1,000 .mu.M) on
proliferation of malignant cell lines. Finally, the generation of
intracellular oxidants in response to disulfuram (0.625 to 5
.mu.M), copper (0.2 to 1.6 .mu.M CuSO.sub.4) or 1.25 .mu.M
disulfuram plus various concentration of copper was measured
directly.
[0098] To explore the role of cyclooxygenase inhibition on tumor
cell growth, cells were cultured with or without disulfuram in the
presence or absence of the cyclooxygenase-1 (COX1) and
cyclooxygenase-2 (COX2) inhibitors indomethacin (5 .mu.g/ml) or
sodium salicylate (1 mM). To probe whether disulfuram might be
inducing growth retardation by interruption or stimulation of NO
production, proliferation was studied with and without disulfuram
in the presence and absence of the nitric oxide synthese inhibitor
N.omega.-nitro-L-arginine added to growth medium (100 .mu.M).
[0099] Finally, a number of dithiocarbamate effects on cells have
been attributed to increasing the intracellular levels of copper
ions. To further probe the role of copper in mediating cytotoxicity
from disulfuram, cells were cultured with or without addition of
the impermeate Cu.sup.2+ chelator bathocuprioinedisulfonic acid
(BCPS, 100 .mu.M) added to medium to sequester Cu.sup.2+ in the
extracellular compartment. Cells were also treated 12 hours with
various concentration of disulfuram (0.625 to 5.0 .mu.M) and
intracellular copper levels were measured as outlined below.
Electrophoretic Mobility Shift Assays (EMSAs)
[0100] Nuclear protein was isolated and DNA binding reactions were
performed as previously described in detail (See, e.g., R.
Dashtaki, et al., "Dehydroepiandrosterone and analogs inhibit DNA
binding of AP-1 and airway smooth muscle proliferation," J.
Pharmacol. Exper. Ther. 285:876-219 (1998); T. Kennedy, et al.,
"Copper-dependent inflammation and nuclear factor-.kappa.B
activation by particulate air pollution," Am. J. Respir. Cell Mol.
Biol. 19:366-378 (1998)). Monolayers were washed twice in cold DPBS
and equilibrated 10 minutes on ice with 0.7 ml cold cytoplasmic
extraction buffer, CEB (10 mM Tris, pH 7.9, 60 mM KCl, 1 mM EDTA, 1
mM DTT) with protease inhibitors, PI (1 mM Pefabloc, 50 .mu.g/ml
antipain, 1 .mu.g/ml leupeptin, 1 .mu.g/ml pepstatin, 40 .mu.g/ml
bestatin, 3 .mu.g/ml E-64 and 100 .mu.g/ml chymostatin). The
detergent Nonidet P-40 (NP-40) was added to a final concentration
of 0.1% and cells were dislodged with a cell scraper. Nuclei were
pelleted by centrifugation and washed with CEB/PI. Nuclei were then
incubated for 20 minutes on ice in nuclear extraction buffer, NEB
(20 mM Tris, pH 8.0, 400 mM NaCl, 1.5 mM MgCl.sub.2, 1.5 mM EDTA, 1
mM DTT and 25% glycerol) with PI, spun briefly to clear debris and
stored at -80.degree. C. until performance of electrophoretic
mobility shift assays.
[0101] EMSAs were performed using consensus oligonucleotides
(5'-AGAGATTGCCTGACGTCAGAGAGCTAG-3' and
3'-TCTCTAACGGACTGCAGTCTCTCGATC-5') for the cyclic-AMP responsive
element CRE (ProMega, Madison, Wis.), end-labeled by
phosphorylation with [.gamma..sup.32P]-ATP and T4 polynucleotide
kinase. DNA-protein binding reactions were performed with 2 .mu.g
of nuclear protein (as determined by the Pierce method) and
30-80,000 cpm of .sup.32P-end-labelled double-stranded DNA probe in
10 mM Tris-HCl, pH 7.5, 50 mM NaCl, 0.5 mM EDTA, 0.5 mM DTT (except
where indicated), 1 mM MgCl.sub.2, 50 .mu.g/ml poly dI-dC, and 4%
glycerol. All components of the binding reaction with the exception
of labeled probe were combined and incubated at room temperature
for 10 minutes before addition of labeled probe and incubation for
an additional 20 minutes.
[0102] Competition experiments were performed with 10.times.
unlabeled wild-type oligonucleotide sequences for CRE or
NF-.kappa.B (p50, 5'-AGTTGAGGGGACTTTCCCAGGC-3' and
3'-TCAACTCCCCTGAAAGGGTCCG-5'), added before labeled probe.
Supershift experiments were performed by incubating the binding
reaction with 1 .mu.g of supershifting antibody prior to
electrophoresis. Samples were electrophoresed on a 5% nondenaturing
polyacrylamide gel in Tris-glycine-EDTA (TGE, 120 mM glycine and 1
mM EDTA in 25 mM Tris, pH 8.5) buffer. Gels were dried and analyzed
by autoradiography at -80.degree. C. using an image intensifier
screen. Densitometry of bands was performed using Kodak Digital
Science 1D image analysis software (Eastman Kodak, Rochester,
N.Y.).
Measurement of Proliferation in Cell Cultures
[0103] Proliferation of cultured cells was quantitated using a
previously reported colorimetric method based upon metabolic
reduction of the soluble yellow tetrazolium dye
3-[4,5-dimethylthiazol]-2-yl-2,5-diphenyl tetrazolium bromide (MTT)
to its insoluble purple formazan by the action of mitochondrial
succinyl dehydrogenase (See, e.g., S. J. Hirst, et al.,
"Quantifying proliferation of cultured human and rabbit airway
smooth muscle in response to serum and platelet derived growth
factor," Am. J. Respir. Cell Mol. Biol. 7:574-581 (1992); R.
Dashtaki, et al. R., supra; S. S. Brar, et al., "Requirement for
reactive oxygen species in serum-induced and platelet-derived
growth factor-induced growth of airway smooth muscle," J. Biol.
Chem. 274:20017-20026 (1999)). This assay empirically distinguishes
between dead and living cells. For proliferation studies, cells
were seeded into 24-well uncoated plastic plates (Costar) at 50,000
cells per well and cultured with respective media and mitogens.
After 24-96 hours, medium was removed, cells were washed twice with
1 ml of sterile Dulbecco's modified phosphate buffered saline
without Ca.sup.2+ or Mg.sup.2+ (DPBS), the medium was replaced with
1 ml/well fresh medium containing 100 .mu.g/ml MTT, and plates were
incubated an additional hour. MTT-containing medium was removed,
0.5 ml dimethylsulfoxide (DMSO) was added to each well, and the
absorbance of the solubilized purple formazan dye was measured at
540 .mu.m. A total of 4-6 wells were studied at each treatment
condition. Preliminary studies were performed with 50-200 .mu.g/ml
MTT incubated for 15 minutes to 3 hours to determine the optimum
concentration and incubation time at which the rate of conversion
was linear and proportional to the number of cells present. The
absorbence of the MTT formazan reduction product (A.sub.540)
correlated with cell numbers counted by hemocytometer with an
R.sup.2=0.99. In some experiments, the MTT assay and responses to
FBS and inhibitors were also confirmed by performing cell counts on
10 random fields/well of Giemsa-modified Wright's stained
monolayers viewed at 40 power using a 0.01-cm.sup.2 ocular
grid.
Measurement of Cytotoxicity and Apoptosis
[0104] To assess for cytotoxicity, cells were plated at a density
of 50,000 per well on 24 well plates and grown for 24 hours.
Disulfuram was then added. After an additional 36 hours, medium was
removed and replaced with DPBS containing 0.1% trypan blue. Cell
death was assessed by counting the average number of trypan blue
positive cells per 10.times. field in 5 random fields for 4
separate wells.
[0105] To determine whether disulfuram induced apoptosis, cells
grown to confluence on 35 mm Petri dishes or on glass slides were
treated with disulfuram or DMSO as vehicle. Apoptosis was studied
by terminal deoxynucleotidyl transferase (TdT) dependent 3'-OH
fluorescein end-labeling of DNA fragments, using a
Fluorescein-FragEL.TM. DNA fragmentation detection kit (Oncogene
Research Products, Cambridge, Mass.). Apoptosis was also studied by
visually assessing endonuclease dependent DNA fragmentation on
ethidium bromide-stained agarose gels.
DNA Cell Cycle Measurements
[0106] To study the effect of disulfuram on the DNA cell cycle,
cells were grown to confluence in 25 cm.sup.2 plastic flasks and
treated for with 10% FBS plus DMSO vehicle, FBS and DMSO vehicle
plus 250 .mu.g/ml ceruloplasmin as a source of copper, FBS plus 5
.mu.M disulfuram or FBS plus 5 .mu.M disulfuram and 250 .mu.g/ml
ceruloplasmin. After 24 hours cells were trypsinized, washed twice
in cold DPBS with 1 mM EDTA and 1% BSA, fixed 30 minute in ice-cold
70% ethanol, and stained by incubation for 30 minutes at 37.degree.
C. in a 10 .mu.g/ml solution of propidium iodide in DPBS and 1
mg/ml RNase A. DNA cell cycle measurements were made using a
FACStar.sup.PLUS Flow Cytometer (Becton-Dickenson, San Jose,
Calif.).
Immunossay for Proteins
[0107] Cells were lysed and proteins were isolated and quantitated
by immunoassay as previously detailed using 2 .mu.g/ml of primary
rabbit polyclonal antibodies against human bcl-2, p53,
p21.sup.WAF1/Cip1, cyclin A and cyclin B1, and peroxidase-labeled
donkey polyclonal anti-rabbit IgG. Cells were placed on ice, washed
twice with cold DPBS, scraped into 0.5 ml boiling buffer (10%
[vol/vol] glycerol and 2% [wt/vol] sodium dodecyl sulfate [SDS] in
83 mM Tris, pH 6.8) and sheared by four passages through a pipette.
Aliquots were removed for protein determination, using the BCA
protein assay (Pierce). After 10% .beta.-mercaptoethanol and 0.05%
bromophenol blue were added, lysates were boiled for 5 min and
stored at -80.degree. C. until immunoblotting was performed.
Proteins in defrosted samples were separated by SDS-polyacrylamide
gel electrophoresis on 12% polyacrylamide gels (15 .mu.g
protein/lane) and electrotransferred to 0.45 .mu.m Hybond ECL
nitrocellulose membranes (Amersham Life Sciences) using the wet
transblot method in transfer buffer (0.025 M Tris, 0.192 M glycine,
2.6 mM SDS, and 20%[vol/vol] methanol; pH 8.8) at 100 volts for 1
hour. Blots were blocked overnight at 4.degree. C. with blocking
buffer (PBS with 0.1% Tween 20) containing 5% fat-free milk powder
(Carnation, Glendale, Calif.). After rinsing 5 times for 5 minutes
each in PBS containing 0.1% Tween 20, blots were incubated for 1
hour at room temperature with 2.0 .mu.g/ml of primary antibody.
After rinsing again as above, blots were incubated for 1 hr at room
temperature with horseradish peroxidase(HRP)-conjugated secondary
antibody diluted 1:5,000 in blocking buffer. Immunoblots were
rinsed again as above and detected via an enhanced
chemiluminescence method (ECL Western blotting detection system,
Amersham Life Science, Buckinghamshire, England). Autoradiographic
film (X-OMAT AR, Eastman Kodak, Rochester, N.Y.) was exposed to
immunoblots for 10, 30, or 60 seconds to obtain satisfactory
images.
Measurement of Intracellular Copper
[0108] Cells were cultured in 12-well plastic tissue culture plates
at an initial plating density of 50,000 cells/well, grown to
confluence and treated with disulfuram or vehicle DMSO as outlined
above. Media was removed and cells were washed twice with DPBS.
Cells were then scraped into 1.0 ml of 3N HCl/10.0% trichloroacetic
acid and hydrolyzed at 70.degree. C. for 16 hours. The hydrolysate
was centrifuged at 600 gm for 10 minutes to remove debris and
copper was measured in the supernatant using inductively coupled
plasma emission spectroscopy (Model P30, Perkin Elmer, Norwalk,
Conn.) at wavelengths of 325.754 and 224.700 nm. To minimize metal
contamination, plasticware rather than glassware was used in these
experiments, and double-distilled, deionized water was used for all
aqueous media. Results are reported as ng copper/ml of
hydrolysate.
Measurement of Intracellular Generation of Reactive Oxygen
Species
[0109] Generation of reactive oxygen species in response to
disulfuram with or without CuSO.sub.4 was studied using
2',7'-dichlorofluorescin diacetate (DCF-DA, Molecular Probes,
Eugene, Oreg.) and a modification of methods previously reported
(See, J. A. Royall, et al., "Evaluation of 2',7'-dichlorofluorescin
and dihydrorhodamine 123 as fluorescent probes for intracellular
H.sub.2O.sub.2 in cultured endothelial cells," Archiv. Biochem.
Biophys. 302:348-355 (1993)). This method is based upon oxidation
of dichlorofluorescin to 2',7'-dichlorofluorescein by
H.sub.2O.sub.2 in the presence of cellular peroxidases. Cells were
plated in 24 well plastic plates at 50,000 cells per well and grown
to confluence. Media was aspirated from wells and replaced with 100
.mu.l medium containing 10 .mu.M DCF-DA, and plates were incubated
at 37.degree. C. for 30 minutes. The DCF-DA containing media was
aspirated, cells were washed twice with media alone and 100 .mu.l
fresh media was added to wells. With the plate on the fluorescence
micro-plate reader (HTS 7000) cells were stimulated with 25 .mu.l
of media containing 5.times. concentrations of disulfuram and/or
CuSO.sub.4 to provide final concentrations of 0-5.0 .mu.M
disulfuram and/or 0-1.6 .mu.M CuSO.sub.4, respectively. The
relative concentration of dichlorofluoroescein was measured
immediately by monitoring fluorescence at 37.degree. C. using an
excitation wavelength of 485 nm and emission wavelength of 535
nm.
Measurement of Intracellular Glutathione
[0110] Disulfuram (5 .mu.M), with or without 1.6 .mu.M CuSO.sub.4,
was added to cells grown to confluence on 100.times.15 mm plastic
dishes, and cells were harvested 24 hours later for measurement of
GSH using the 5,5'-dithiobis(2-nitrobenzoic acid)-glutathione
reductase recycling assay (See, M. E. Anderson, "Determination of
glutathione and glutathione disulfide in biological samples,"
Methods Enzymol. 113:548-555 (1985)).
Synthesis of Disulfuram-Metal Chelates
[0111] Chelates of disulfuram and a number of metals were
synthesized by vigorous mixing of 150 mg of disulfuram in
chloroform (7.5 mg/ml) with 30 ml of a 5.times. molar excess of
CuSO.sub.4, ZnCl.sub.2, C.sub.3H.sub.5AgO.sub.3 (silver lactate) or
HAuCl.sub.4.3H.sub.2O in double glass distilled deionized water.
The mixture was centrifuged at 1,000 gm for 10 minutes and the
upper aqueous phase was discarded. As the lower chloroform phase
was evaporated, the resulting disulfuram-metal chelates
precipitated.
[0112] In another synthesis, 150 mg of sodium
diethyldithiocarbamate was dissolved in 10 ml of deionized water.
To this was added 250 mg of HAuCl.sub.4.3H.sub.2O. The resulting
precipitate was collected by centrifugation and redisolved in
chloroform. As the chloroform phase was evaporated the resulting
dithiocarbamate-gold chelates were precipitated as crystals.
[0113] These were analyzed to determine their molecular weight,
melting point, solubility, elemental composition and
crystallographic structure.
Statistical Analysis
[0114] Data are expressed as mean values.+-.standard error. The
minimum number of replicates for all measurements was four, unless
indicated. Differences between multiple groups were compared using
one-way analysis of variance. The post-hoc test used was the
Newman-Keuls multiple comparison test. Two-tailed tests of
significance were employed. Significance was assumed at
p<0.05.
EXAMPLE 1
[0115] This example shows dithiocarbamate disulfides inhibit DNA
binding to the cyclic AMP response element.
[0116] M1619 melanoma cells were grown to 60% confluence on
100.times.15 mm plastic Petri dishes, nuclear protein was harvested
and electrophoretic mobility gel shift assays (EMSAs) were
performed using. The results are shown in FIGS. 1A-1C. Treatment of
cells for 6, 12 or 24 hour with the combination of 5 .mu.M
disulfuram and 1.6 .mu.M cupric sulfate substantially interrupts
transcription factor binding to CRE. EMSAs for 2, 6, 12 or 24 hours
of treatment: FBS alone, lanes 1, 5, 9, and 13; FBS+DMSO vehicle,
lanes 2, 6, 10, 14; FBS+disulfuram, lanes 3, 7, 11, 15;
FBS+disulfuram+CuSO.sub.4, lanes 4, 8, 12, 16.
[0117] CRE complexes (I and II) are labeled. Nuclear protein from
proliferating M1619 malignant melanoma cells showed two strong
constitutive bands (I and II) of DNA binding activity in
electrophoretic mobility shift assays with the cyclic AMP response
element (CRE) consensus sequence (FIG. 1A, lane 1). Both bands were
eliminated by addition of 10.times. unlabeled CRE consensus
oligonucleotide to the binding reaction (lane 8). Supershift
experiments demonstrated that the top band II contains the CRE
binding protein activating transcription factor-2 (ATF-2, lane 5),
while the lower complex I contains CREB-1 (lane 2), with ATF-1
(lane 4) as a minor component. Competition experiments shown in
lanes 6-8 demonstrate specificity of the DNA binding reaction: lane
6, FBS (fetal bovine serum) alone; lane 7, FBS with 10.times.
unlabeled CRE probe added to binding reaction; lane 8, FBS with
10.times. unlabeled NF-.kappa.B probe added to binding
reaction.
[0118] As shown in FIG. 1B, disulfuram alone slightly reduced
DNA-binding to CRE, but when combined with treatment of cells with
the transition metal copper, disulfuram eliminated transcription
factor binding to CRE after 6 hours of treatment.
[0119] The upper ATF-2 containing complex proved more sensitive to
inhibition. This is demonstrated in FIG. 1C, which shows
densitometry results performed on the ATF-2 containing upper
complex II experiments is displayed as mean sum intensity of
bands.
[0120] The EMSAs in replicate experiments (n=4) in which near
confluent cells were treated for 8 hours with DMSO vehicle,
disulfuram, copper or the combination of disulfuram plus copper.
The combination of disulfuram plus copper reduced DNA binding of
the upper complex II by half suggests that ATF-2 is extremely
sensitive to inhibition by interactions between thiuramdisulfides
and some metals. At the concentrations employed above, disulfuram
plus copper also inhibited DNA binding of NF-.kappa.B after
treatment for 12 hours and DNA binding of AP-1 after 24 hours (data
not shown), but effects were not as dramatic those on binding to
CRE.
[0121] To determine if inhibition of transcription factor binding
to CRE could be attributed to direct transcription factor
modification by disulfuram and copper, we studied the effect of
adding each agent directly to the binding reaction performed with
nuclear protein from untreated M1619 cells. The results are shown
in FIG. 2. Therein electrophoretic mobility shift assays (EMSAs)
were performed showing that addition of disulfuram plus copper to
the binding reaction reduces DNA binding to CRE. Lane 1, nuclear
protein from fetal bovine serum-stimulated M1619 cells (FBS); lane
2, FBS+DMSO vehicle; lane 3, FBS+disulfuram (5 .mu.M); lane 4,
FBS+1.6 .mu.M CuSO.sub.4; lane 5, FBS+disulfuram+CuSO.sub.4; lane
6, FBS alone; lane 7, FBS+disulfuram; lane 8, FBS+CuSO.sub.4; lane
9, FBS+disulfuram+CuSO.sub.4; lane 10, FBS+disulfuram+CuSO.sub.4.
In lanes 1-5, DTT (2.5 mM) was added to the binding reaction as a
reducing agent, whereas in lane 6-9, GSH (3.0 mM) was used.
Disulfuram alone (lane 3) or disulfuram and copper (lane 5) reduced
transcription factor binding to CRE, but the effect of these agents
was more pronounced when the binding reaction was performed with
GSH (lanes 7 and 9) instead of DTT (lane 3 and 5) as the reducing
agent. Inhibition of binding to CRE by disulfuram and copper in the
presence GSH was reversed by simultaneous addition of the more
potent reducing agent DTT (lane 10).
[0122] The addition of disulfuram alone to the binding reaction
reduced DNA binding to CRE in the upper ATF2 containing complex II
(FIG. 2, lane 3). This effect was magnified when disulfuram was
combined with copper ions (lane 5). These results are consistent
with modest disruption of ATF2 binding to CRE from formation of
mixed disulfides between disulfuram and cysteines in the DNA
binding region, and suggest that copper catalyzes mixed disulfide
generation. However, reduction in CRE binding was much more
pronounced when the binding reaction was performed with GSH instead
of DTT as the reducing agent (FIG. 2, lane 7 for disulfuram, lane 9
for disulfuram plus copper). Inhibition of ATF2 containing complex
II binding to CRE by disulfuram and copper in the presence of GSH
was reversed by simultaneous addition of the potent uncharged
reducing agent DTT (FIG. 2, lane 10).
[0123] These results indicate that GSH, a cellular monothiol found
in mM concentrations within the nuclear compartment might react
with the dithiocarbamate adduct leading to a bulky, negatively
charged GSH-containing mixed disulfide that could more effectively
disrupt DNA binding of ATF2.
EXAMPLE 2
[0124] This example shows that dithiocarbamate disulfides and
copper inhibit cyclin A expression. It is known that heterodimers
of the transcription factors CREB-1 and c-Fos or ATF2 and Jun
family members positively regulate cyclin A expression through
binding to a CRE element in the cyclin A promoter.
[0125] Since disulfuram and copper disrupt transcription factor DNA
binding to CRE, their effect on expression of cyclin A was studied.
FIG. 3A shows disulfuram and copper reduce expression of the
cell-cycle protein cyclin A. M1619 melanoma cells were plated at
equal densities in 60.times.15 mm plastic dishes, grown to 80%
confluence and treated with DMSO vehicle (5 .mu.l/ml), disulfuram
(DS, 5 .mu.M), or the combination of disulfuram and CuSO.sub.4 (1.6
.mu.M). After the indicated times, cells were lysed and protein
extracts were subjected to SDS-polyacrylamide gel electrophoresis
(PAGE) followed by Western blotting using a rabbit polyclonal
antibody (Santa Cruz). Typical experiments are shown for 2, 4, 8,
12, 24 and 36 hours of treatment with disulfuram plus
CuSO.sub.4.
[0126] FIG. 3B replicates experiments (n=4 each) in which cells
were treated with DMSO vehicle, (5 .mu.l/ml, lanes 1-4), disulfuram
(5 .mu.M, lanes 5-8), (5 .mu.l/ml), CuSO.sub.4 (1.6 .mu.M, lanes
9-12) or the combination of disulfuram and CuSO.sub.4 (lanes
13-16). After 24 hours cells were lysed, immunoblots were performed
to assay for cyclin A. In FIG. 3C shows quantitation of experiments
in FIG. 3B by densitometry. Mean sum intensity of bands is
displayed. *p<0.001 compared to all other treatments.
[0127] While disulfuram or copper alone had little effect (FIGS. 3B
and C), treatment with the combination of disulfuram plus copper
progressively decreased cyclin A expression over time (FIG. 3A) and
reduced expression of cyclin A by over two-thirds at 24 hours
(FIGS. 3B and 3C). In contrast, levels of B1 remained unchanged,
and, in the cell lines we studied, disulfuram had no consistent
effect on expression of the cell cycle inhibitor p21.sup.WAF1/CIP1
or the pro- and anti-apoptotic proteins p53 or bcl-2 (data not
shown).
EXAMPLE 3
[0128] This example illustrates that disulfuram is
antiproliferative against melanoma and other tumor cell lines.
Disruption of cyclin A expression should impair cell cycle
progression and cellular proliferation. Therefore, the effect of
disulfuram on M1619 melanoma growth, using concentrations readily
achieved in humans on usual clinical doses was studied. Disulfuram
was a potent inhibitor of growth in vitro for M1619 melanoma (FIG.
4A). FIG. 4A shows that disulfuram inhibits proliferation of M1619
human melanoma cell lines. Cells stimulated with 10% fetal bovine
serum (FBS) were plated at a density of 50,000 cells per well, and
DMSO vehicle (5 .mu.l per ml) or disulfuram (DS) was added to wells
at the indicated concentrations. After 24 hours, proliferation was
quantitated by assessing the cell number-dependent reduction of the
soluble yellow tetrazolium dye
3-[4,5-dimethylthiazol]-2-yl-2,5-diphenyl tetrazolium bromide (MTT)
to its insoluble formazan, measured as the absorbance at 540 nm
(A.sub.540) (6,7). *p<0.01 compared to FBS+DMSO vehicle
control.
[0129] Disulfuram also inhibited growth of a variety of other
malignant cell lines, including M1585 melanoma, prostatic
adenocarcinoma, non-small cell and small cell lung cancer, and
adenocarcinoma of the breast (Table 1). This was true whether
disulfuram was added to culture Each value represents mean.+-.SE
percent inhibition of growth compared to DMSO vehicle treated
control cultures. Cells stimulated with 10% fetal bovine serum
(FBS) were plated at a density of 50,000 cells per well. In some
studies (treatment initially) DMSO vehicle (5 .mu.l per ml) or
disulfuram (DS) was added to wells at the indicated concentrations.
After 48 hours, proliferation was quantitated as described in FIG.
4.
[0130] In other studies (treatment after 24 hours) cells were grown
for 24 hours (M1619, M1585 and H596 lung) or 48 hours (breast).
DMSO vehicle (5 .mu.l per ml) or disulfuram (DS) was added to wells
at the indicated concentrations. After an additional 24 hours
(lung) or 48 hours (breast), proliferation was quantitated as
described in FIG. 4. Percent inhibition of growth was calculated as
100.times.(1.0-A.sub.540 of MTT formazan in disulfuram treated
cells/mean A.sub.540 of MTT formazan in DMSO vehicle treated
cells). In some cell lines, a modest (<10%) but statistically
significant inhibitory effect was observed with DMSO vehicle alone.
Each value represents a mean of at least 4 experiments.
.sup.Ap<0.01 compared to FBS+DMSO vehicle control.
TABLE-US-00001 TABLE 1 DISULFIRAM IS ANTIPROLIFERATIVE FOR
MALIGNANT CELLS Mean Percent Inhibition of Growth Concentration of
Disulfiram (.mu.M) Cell Line 0.625 1.25 2.5 5.0 Treatment initially
Melanoma M1585 100 .+-. 0.sup.A 100 .+-. 0.sup.A 100 .+-. 0.sup.A
100 .+-. 0.sup.A Prostate carcinoma 6 .+-. 6 29 .+-. 5.sup.A 48
.+-. 2.sup.A 86 .+-. 2.sup.A CRL 1435 (PC-3) Squamous lung 76 .+-.
3.sup.A 82 .+-. 4.sup.A 77 .+-. 4.sup.A 78 .+-. 3.sup.A carcinoma
NCI-H520 Adenosquamous lung 47 .+-. 4.sup.A 57 .+-. 4.sup.A 50 .+-.
3.sup.A 50 .+-. 4.sup.A carcinoma NCI-H596 Small cell lung 68 .+-.
3.sup.A 76 .+-. 6.sup.A 76 .+-. 5.sup.A 72 .+-. 3.sup.A carcinoma
NCI-H82 Breast carcinoma 69 .+-. 4.sup.A 94 .+-. 2.sup.A 100 .+-.
0.sup.A 100 .+-. 0.sup.A MDA-MB-453 Treatment after 24 hours
Melanoma M1619 59 .+-. 4.sup.A 35 .+-. 4.sup.A 39 .+-. 3.sup.A 37
.+-. 4.sup.A Melanoma M1585 74 .+-. 4.sup.A 49 .+-. 7.sup.A 41 .+-.
2.sup.A 37 .+-. 6.sup.A Lung carcinoma 30 .+-. 3.sup.A 30 .+-.
3.sup.A 29 .+-. 1.sup.A 34 .+-. 3.sup.A NCI-H596 Breast carcinoma
26 .+-. 5.sup.A 26 .+-. 2.sup.A 39 .+-. 2.sup.A 46 .+-. 4.sup.A
MDA-MB-453
[0131] In FIG. 4B it is shown that the cell-impermeate Cu chelator
bathocuproine-disulfonic acid prevents growth inhibition from
disulfuram. M1619 melanoma cells stimulated and plated as described
in A, and 1.25 .mu.M disulfuram (DS) or DMSO vehicle (5 .mu.l per
ml) was added to wells in the absence or presence of 50 or 100
.mu.M bathocuproine-disulfonic acid (BCPS). After 48 hr
proliferation was quantitated as described. *p<0.001 compared to
FBS+DMSO; +p<0.001 compared to FBS+DS.
[0132] FIG. 4C shows that supplementation of growth medium with
copper enhances the antiproliferative activity of disulfuram. M1619
melanoma cells plated and stimulated as described in FIG. 4A were
grown for 24 hours and supplemented with CuSO.sub.4 or CuSO.sub.4
plus 0.625 .mu.M disulfuram. After an additional 24 hours
proliferation was quantitated. The addition of even 0.2 .mu.M
CuSO.sub.4 to medium converts 0.625 .mu.M disulfuram from a 50%
inhibitory (IC.sub.50) concentration (A) into a 100% inhibitory
(IC.sub.100) concentration of drug. *p<0.001 compared to no
CuSO.sub.4;
[0133] The results shown in FIG. 4D illustrate that ceruloplasmin
can serve as a source of copper for enhancing the antiproliferative
activity of disulfuram. M1619 melanoma cells were plated,
stimulated and grown for 24 hours in the presence or absence of
0.625 .mu.M disulfuram or 5 .mu.l/ml DMSO vehicle in the presence
or absence of human ceruloplasmin (Cerulo) at a concentration
representing the upper level in normal human serum (500 .mu.g/ml).
After 24 hours proliferation was quantitated. *p<0.001 compared
to FBS+DMSO; +p<0.001 compared to FBS+DS.
[0134] Disulfuram induced both necrosis and apoptosis. Treatment of
monolayers with even low doses of disulfuram markedly increased
trypan blue dye uptake (6.+-.2, 8.+-.3.6 and 94.+-.18 trypan blue
positive cells per well, respectively, for untreated, DMSO vehicle
treated or H520 lung adenosquamous carcinoma cells treated with
0.625 .mu.M disulfuram; 12.+-.0.9, 16.5.+-.2.1 and 93.+-.12 trypan
blue positive cells per well, respectively, for untreated,
DMSO-treated or H82 small cell lung cancer cells treated with 0.625
.mu.M disulfuram; p<0.001 compared to untreated or DMSO vehicle
treated controls). Disulfuram also enhanced 3'-OH fluorescein
end-labeling of DNA fragments (FIGS. 5A and 5B) and DNA laddering
on ethidium bromide-stained agarose gels (data not shown).
Consistent with its recently reported effects on P-glycoprotein
mediated drug resistance (See, T. W. Loo, et al., "Blockage of drug
resistance in vitro by disulfuram, a drug used to treat
alcoholism," J. Natl. Cancer Inst. 92:898-902 (2000)), disulfuram
augmented the antiproliferative effect of other antineoplastic
agents on melanoma cells, a tumor notoriously resistant to
chemotherapeutic drugs (Table 2).
[0135] In FIG. 5A, M1619 melanoma cells treated with DMSO vehicle.
In FIG. 5B, M1619 melanoma cells treated with 5 .mu.M disulfuram.
Disulfuram markedly increases 3'-OH fluorescein end-labeling of DNA
fragments. Cells were grown to confluence on 35 mm Petri dishes or
on glass slides and treated for 15 hours with disulfuram or DMSO as
vehicle. Apoptosis was studied by terminal deoxynucleotidyl
transferase (TdT) dependent 3'-OH fluorescein end-labeling of DNA
fragments, using a Fluorescein-FragEL.TM. DNA fragmentation
detection kit (Oncogene Research Products, Cambridge, Mass.).
[0136] Table 2 shows that the combination of disulfuram and
cisplatin or disulfuram and carmustine is significantly more
antiproliferative against M1619 cells than cisplatin or carmustine
alone: TABLE-US-00002 TABLE 2 DISULFIRAM POTENTIATES THE
ANTIPROLIFERATIVE ACTIVITY OF CHEMOTHERAPEUTIC AGENTS A540 of MTT
Formazan A. Cisplatin (ng/ml) DMSO vehicle Disulfiram 2.5 .mu.M 0
1.433 .+-. 0.038 1 1.739 .+-. 0.041 1.369 .+-. 0.033.sup.B 10 1.447
.+-. 0.047 1.221 .+-. 0.028 100 1.372 .+-. 0.052 1.183 .+-.
0.038.sup.A 1,000 1.381 .+-. 0.098 0.921 .+-. 0.027.sup.A B.
Carmustine (.mu.M) DMSO vehicle Disulfiram 0.6 .mu.M 0 0.104 .+-.
0.010 1 0.197 .+-. 0.004 0.042 .+-. 0.003.sup.C 10 0.152 .+-. 0.011
0.025 .+-. 0.002.sup.C 100 0.020 .+-. 0.002 0.030 .+-. 0.023 1,000
0.003 .+-. 0.000 0.004 .+-. 0.000
[0137] In section A M1619 melanoma cells were cultured in 10% FBS
and RPMI 1640 at a density of 50,000 cells/well in 24 well plates.
After 48 hours cisplatin and 2.5 .mu.M disulfuram or DMSO (5 .mu.l
per ml) were added to medium. After an additional 24 hours,
proliferation was quantitated. Each bar represents mean MTT
formazan absorbance in a minimum of 4 experiments. .sup.Ap<0.05
compared to DMSO vehicle; .sup.Bp<0.01 compared to DMSO
vehicle.
[0138] In section B M1619 cells were cultured as above with
addition of carmustine and 0.6 .mu.M disulfuram or DMSO (5 .mu.l
per ml) to medium. After 24 hours, proliferation was quantitated.
Each bar represents mean MTT formazan absorbance in a minimum of 4
experiments. .sup.Cp<0.001 compared to DMSO vehicle.
[0139] Disulfuram was more potent as a growth inhibitor of
neoplastic cell lines than its sulfhydryl-containing relative PDTC.
As an example, the 50% inhibitor concentration (IC.sub.50) against
M1585 melanoma cells was approximately 1.25 .mu.M for PDTC but was
only 0.3 .mu.M for disulfuram. This suggests that the active
antiproliferative construct of thiocarbamates not likely the
reduced thiol-containing monomeric form employed frequently as an
antioxidant.
EXAMPLE 4
[0140] The antiproliferative activity of dithiocarbamate disulfides
depends on complexation with copper. PDTC induces apoptosis in
normal thymocytes that is mediated by complexation of copper from
fetal bovine serum in the medium and subsequent facilitation of
copper transport into cells. Because inhibition of CRE DNA-binding
by disulfuram was shown to be copper dependent in FIGS. 1A-1C and
FIG. 2, the growth inhibition of M1619 cells by disulfuram was
studied to determine whether it was contingent on its ability to
complex with metals present in growth medium. FIG. 4A shows that
disulfuram combined with copper induces S-phase cell cycle arrest
in M1619 melanoma cells and apopotosis. Unsynchronized M1619
melanoma cells were grown in the presence of DMSO vehicle (A), 5
.mu.M disulfuram (B), or 5 .mu.M disulfuram plus 250 .mu.g/ml
ceruloplasmin (Cerulo) as a source of copper (C). Twenty-four hours
later, cells were harvested and flow cytometric analysis was
performed. The proportion of nuclei in each phase of the cell cycle
(brackets) was determined with MODFIT DNA analysis software.
Disulfuram increases the portion of cells in S phase. The
combination of disulfuram and ceruloplasmin further increases the
number of cells in S phase, prevents progression into the G.sub.2-M
cell cycle and induces apoptosis.
[0141] Table 3 below shows that disulfuram greatly enhances
intracellular uptake of copper, while FIG. 4B shows that the
potent, cell impermeate Cu.sup.2+ chelator bathocuproine disulfonic
acid (BCPS) greatly reduces growth inhibition from disulfuram.
Conversely, the antiproliferative activity of disulfuram is greatly
enhanced by supplementation of medium with concentrations of copper
that do not by themselves affect cell growth (FIG. 4C). The copper
transport protein ceruloplasmin, at levels normally present in
human serum, can also serve as a source of copper that can be
complexed to enhance the antiproliferative activity of disulfuram
(FIG. 4D).
[0142] Disulfuram treatment of M1619 melanoma cultures (FIG. 4B)
slightly reduces the number of cells in G.sub.0-G.sub.1 and
increases the portion in S phase of the cell cycle. The addition of
copper from ceruloplasmin to treatment with disulfuram greatly
magnifies these effects. Over two-thirds of cells are in S phase,
none are in G.sub.2-M, and 6% are apoptotic as identified by flow
cytometric cell cycle analysis (FIG. 4C). These studies suggest
that growth inhibition of malignant cell lines by dithiocarbamates
and their disulfides is not only dependent upon interaction with
certain metal ions, but also from complexation with these metal
ions and enhancing their intracellular transport. TABLE-US-00003
TABLE 3 EFFECT OF DISULFIRAM ON INTRACELLULAR COPPER Treatment
Copper (ng/ml) 10% FBS 56 .+-. 7 FBS + DMSO 52 .+-. 4 FBS + 0.625
.mu.M DS 76 .+-. 11 FBS + 1.25 .mu.M DS 102 .+-. 5.sup.A FBS + 2.5
.mu.M DS 160 .+-. 17.sup.A FBS + 5.0 .mu.M DS 195 .+-. 3.sup.B
M1619 melanoma cells were cultured at a density of 50,000
cells/well in 24 well plates in the presence of 10% FBS and grown
to confluence. Disulfuram or DMSO vehicle (5 .mu.l/ml) was added at
the concentrations indicated, and cells were incubated an
additional 6 hours. Supernatant was removed from cells and
monolayers were washed twice with DPBS. Cells were scraped into 1.0
ml 3 N HCL/10% trichloroacetic acid and hydrolyzed at 70.degree. C.
for 16 hours. After centrifugation at 600 g.times.10 min, copper
was measured using inductively coupled plasma emission spectroscopy
at wavelengths of 324.754 and 224.700 nm. Replicates of four are
reported. To minimize metal contamination, plastic ware rather than
glass was used in experiments, and double-distilled, deionized
water was used for all aqueous media. .sup.Ap<0.01 compared to
DMSO control; .sup.Bp<0.001 compared to DMSO control.
EXAMPLE 5
[0143] This example shows dithiocarbate disulfides do not decrease
proliferation through redox mechanisms.
[0144] Disulfuram failed to deplete GSH in M1619 cells (228.+-.18
for FBS alone; 254.+-.7 for DMSO vehicle control; 273.+-.11 nmoles
GSH/.mu.g cell protein for 5 .mu.M disulfuram), and the combination
of 5.0 .mu.M disulfuram and 1.6 .mu.M CuSO.sub.4 even increased
intracellular GSH (293.+-.16 nmoles GSH/.mu.g cell protein;
p<0.05 compared to FBS alone). Likewise, neither disulfuram
(0.625 to 5 .mu.M), CuSO.sub.4 (0.2-1.6 .mu.M) nor the combination
of 1.25 .mu.M disulfuram and 0.2 to 1.6 .mu.M CuSO.sub.4 caused
measurable generation of reactive oxygen species in M1619 cells,
measured using the H.sub.2O.sub.2-sensitive intracellular probe
2',7'-dichlorofluororescin. See, "J. A. Royall, et al., "Evaluation
of 2',7'-dichlorofluorescin and dihydrorhodamine 123 as fluorescent
probes for intracellular H.sub.2O.sub.2 in cultured endothelial
cells," Archiv. Biochem. Biophys. 302:348-355 (1993). The baseline
fluorescence of 1,431.+-.23 units was not increased by any of the
treatments.
[0145] In addition, the potent antioxidant probucol did not
significantly inhibit growth of any of our tumor cell lines (data
not shown). Augmentation of intracellular copper might also
increase levels of the reactive nitrogen species nitric oxide
(NO.sup.-) through Cu.sup.2+-mediated decomposition of
S-nitrosoglutathione and other nitrosothiols (See, D. R. Arnelle,
et al., "Diethyl dithiocarbamate-induced decomposition of
S-nitrosothiols," Nitric Oxide Biol. and Chem. 1:56-64 (1997); M.
P. Gordge, et al., "Copper chelation-induced reduction of the
biological activity of S-nitrosothiols," Brit. J. Pharmacol.
114:1083-1089 (1995); A. C. F. Gorren, et al., "Decomposition of
S-nitrosoglutathione in the presence of copper ions and
glutathione. Archiv. Biochem. Biophys," 330:219-2238 (1996)).
NO.sup.-, in turn, is believed to induce mitochondrial permeability
transition and produce other effects, leading to apoptosis (See, S.
B. Hortelano, et al., "Nitric oxide induces apoptosis via
triggering mitochrondrial permeability transition. FEBS Lett,"
410:373-377 (1997); Y. H. Shen, et al., "Nitric oxide induces and
inhibits apoptosis through different pathways," FEBS Lett.
433:125-131 (1998)).
[0146] While the nitric oxide synthase inhibitor
N.sub..omega.-nitro-L-arginine (LNAME) alone slightly enhanced
cellular growth (23.7.+-.2.3% increase; p<0.01 compared to DMSO
vehicle control), LNAME did not eliminate the antiproliferative
effect of disulfuram (36.8.+-.4.0% inhibition by disulfuram alone
vs 26.7.+-.3.1% inhibition of growth in the presence of disulfuram
plus LNAME; p<0.001 for each compared to DMSO vehicle control
but not significantly different from each other). Finally,
functioning as an antioxidant, PDTC has been postulated to
interfere with growth of colorectal carcinoma in part by reducing
expression of cyclooxygenase-2. See, R. Chinery, Nature Med.,
supra; R. Chinery, Cancer Res, supra. However, cyclooxygenase
inhibitors failed to reduce growth in the cell lines we studied
(data not shown). Thus, taken together, these data suggest that
disulfuram does not appear to inhibit growth by adversely affecting
the cellular redox state.
EXAMPLE 6
[0147] This example illustrates that metals other than copper can
enhance the antiproliferative activity of dithiocarbamate
disulfides. The absorption of copper at both the intestinal and
cellular level is blocked by zinc cations, leading to the use of
zinc acetate as the preferred treatment for Wilson's disease, the
inherited disorder of copper overload.
[0148] High zinc concentrations in culture media affect copper
uptake and transport in differentiated human colon adenocarcinoma
cells therefore it was determined whether zinc supplementation of
medium could inhibit the antiproliferative activity of disulfuram,
which appeared to be copper-dependent. Instead of reducing
activity, zinc chloride also substantially enhanced the
antiproliferative potential of disulfuram (FIG. 7A).
Dithiocarbamates actively complex copper but can chelate other
metals (See, R. P. Burns, et al., "1,1-dithiolato complexes of the
transition elements," Adv. Inorg. Chem. Radiochem. 23:211-280
(1980)), raising the possibility that the activity of disulfuram
might also be enhanced by supplementation with a variety of metal
salts.
[0149] FIGS. 7A-7D show that other metals also protentiate
antiproliferative activity of disulfuram. FIG. 7A shows that zinc
potentiates the antiproliferative activity of disulfuram. M1619
cells were stimulated and plated as in FIG. 4. After 24 hours cells
were treated with indicated concentrations of zinc chloride
(ZnCl.sub.2) in the absence or presence of 0.625 .mu.M disulfuram.
After an additional 24 hr, cell number was quantitated. *p<0.01
compared to no ZnCl.sub.2; +p<0.001 compared to no
ZnCl.sub.2.
[0150] FIG. 7B shows that not only copper and zinc, but also salts
of gold and silver can synergistically enhance the
antiproliferative activity of disulfuram. This further supports the
hypothesis that impairment of cellular proliferation by disulfuram
and possibly other dithiocarbamates and their disulfides is
dependent upon and enhanced catalytically by the presence of heavy
metals. In FIG. 7B the antiproliferative activity of disulfuram is
enhanced by supplementation of medium with other heavy metals.
M1619 cells plated and stimulated as above were treated with FBS
alone, DMSO vehicle (5 .mu.l/ml), disulfuram (DS, 0.15 .mu.M), 5
.mu.M concentrations of metal salts (cupric sulfate, CuSO.sub.4;
silver lactate, C.sub.3H.sub.5AgO.sub.3; gold chloride,
HAuCl.sub.4.3H.sub.2O) or the combination of DS plus metal salts.
After 48 hr cell number was quantitated. *p<0.05 compared to
DMSO; +p<0.001 compared to DS alone.
[0151] In FIG. 7C complexes of disulfuram with gold demonstrate
enhanced antiproliferative activity. M1619 cells plated and
stimulated as above were treated with FBS alone, DMSO vehicle (5
.mu.l/ml), disulfuram (DS, 160 nM) or concentrations of gold
complexed with disulfuram as outlined in Methods (AuDS). After 48
hr cell number was quantitated. *p<0.001 compared to DMSO;
+p<0.001 compared to DS.
EXAMPLE 7
[0152] This example shows thiolate anion formation mediates the
antiproliferative activity of dithiocarbamates and their
disulfides.
[0153] In light of the above findings with metals, chelates of
disulfuram with a number of metal ions, including Cu.sup.2+,
Zn.sup.2+, Ag.sup.1+, or Au.sup.3+ were synthesized. During
generation of disulfuram-metal complexes, chelation of metal ions
from the aqueous phase was suggested by a color change in the
disulfuram-containing chloroform phase (from pale yellow to
brilliant golden orange with complexation of gold ions). All metal
complexes showed increased antiproliferative activity compared to
disulfuram, but the most active compound was formed by the complex
of gold with disulfuram (FIG. 7C), which was antiproliferative at
nM concentrations.
[0154] The x-ray crystallographic structure of this compound
revealed it to be a chelate of gold by the thiolate anion of
diethyldiothiocarbamate, with chlorides occupying the other two
valences of gold (FIG. 8). Complexes were generated as outlined in
Methods. Crystals were mounted on a Nonius Kappa-CCD diffractometer
for evaluation. The crystal diffracted well and a data set was
collected to 27.5.degree. in .theta. using Mo K.alpha. radiation
(.lamda.=0.7173 A). Least-squares refinement on the cell parameters
reveled an orthorhombic P cell with unit cell parameters of
a=11.5167(5), b=7.2472(2), c=12.9350(7) A, and a Volume of
1079.6(1) A.sup.3. Examination of the systematic absences showed
the space group to be Pnma. The structure was solved by direct
methods using SIR92 and revealed the crystal to be
dichloro(diethylthiocarbamyl)gold (II). The structure was confirmed
by the successful solution and refinement of the 83 independent
variables for the 893 reflections (I.gtoreq.3.delta.(I)) to
R-factors of 3.3 and 3.2%, with an ESD of 1.499. The gold complex
is a square planar complex in which the Au and the four coordinated
atoms sit on a mirror at x, 0.25, z. The organic ligand was found
to be disordered with the diethylamine ligand occupying two sites
related to each other through the mirror plane.
[0155] These results suggest that the proximate reactive
dithiocarbamate structure important for promoting cellular mixed
disulfide formation might be the thiolate anion generated from
fully reduced dithiocarbamates or their disulfides by copper and
other metals. To test this hypothesis the ability of the thiolate
sodium diethyldithiocarbamate to inhibit M1619 proliferation alone
or in the presence of a low concentration of DTT added to growth
medium to promote formation of the fully reduced thioacid was
compared. FIG. 7D shows that growth inhibition by the thiolate is
greatly impaired by a concentration of DTT that does not affect
growth of melanoma cells alone. In FIG. 7D the antiproliferative
activity of the thiolate sodium diethyldithiocarbamate trihydrate
(NaDDC) is reduced by low concentrations of DTT in the growth
medium. M1619 cells plated and stimulated above were treated with
FBS alone, NaDDC (1 .mu.M), DTT (100 .mu.M) or NaDDC plus DTT.
After 48 hours cell number was quantitated. *p<0.001 compared to
FBS; +p<0.001 compared to NaDDC alone. Thus, the function of
metals in disrupting transcription factor DNA binding and cell
proliferation may be to promote formation of the dithiocarbamate
anion, the reactive chemical form that condenses into mixed
disulfides with DNA binding region cysteines, with secondary
conjugation to GSH, effecting transcription factor
S-glutathionylation.
[0156] Many modification and other embodiments of the invention
will come to mind to one skilled in the art to which this invention
pertains, having the benefit of the teachings presented in the
descriptions and the associated drawings contained herein.
Therefore, it is to be understood that the invention is not to be
limited to the specific embodiments disclosed and that
modifications and other embodiments are intended to be included
within the scope of the appended claims. Although specific terms
are employed herein, they are used in a generic and descriptive
sense only and not for purposes of limitation.
* * * * *