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Patent 2113683 Summary

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(12) Patent Application: (11) CA 2113683
(54) English Title: TREATMENT OF PROLIFERATIVE DISORDERS, METASTASES, AND DRUG RESISTANT TUMORS WITH VANADATE COMPOUNDS AND DERIVATIVES OR ANALOGUES THEREOF
(54) French Title: TRAITEMENT DES MALADIES PROLIFERATIVES, METASTASES ET TUMEURS PHARMACORESISTANTES PAR DES COMPOSES VANADATE ET DERIVES OU ANALOGUES DE VANADATE
Status: Dead
Bibliographic Data
(51) International Patent Classification (IPC):
  • A61K 33/24 (2019.01)
  • A61P 35/00 (2006.01)
  • A61P 35/04 (2006.01)
(72) Inventors :
  • CRUZ, TONY (Canada)
(73) Owners :
  • MOUNT SINAI HOSPITAL CORPORATION (Canada)
(71) Applicants :
  • MOUNT SINAI HOSPITAL CORPORATION (Canada)
(74) Agent: BERESKIN & PARR LLP/S.E.N.C.R.L.,S.R.L.
(74) Associate agent:
(45) Issued:
(22) Filed Date: 1994-01-18
(41) Open to Public Inspection: 1995-07-19
Availability of licence: N/A
(25) Language of filing: English

Patent Cooperation Treaty (PCT): No

(30) Application Priority Data: None

Abstracts

English Abstract






The present invention relates to the use of
vanadate compounds or derivatives or analogues of vanadate
compounds as antiproliferative and anti-metastatic agents,
and/or to treat drug resistant tumors in animals; to
compositions containing vanadate compounds adapted for
such use; to methods for the treatment of proliferative
disorders, to methods of reducing the ability of a tumor
to metastasize, and to methods for treating drug resistant
tumors. The invention also relates to methods for testing
for substances which affect cell proliferation.


Claims

Note: Claims are shown in the official language in which they were submitted.


- 26 -

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. A method for the treatment of proliferative
disorders comprising administering an amount of a vanadate
compound, or a derivative or an analogue thereof, which
results in a serum concentration of the vanadate compound,
or derivative or analogue thereof, of at least 5µM.

2. The method as claimed in claim 1 wherein the
intracellular concentration of the vanadate compound is
5µM to 50µM.

3. The method as claimed in claim 1 wherein the
vanadate compound is orthovanadate or vanadyl sulphate.

4. A method for reducing or inhibiting the growth
of a drug resistant tumor comprising administering to the
patient an amount of a vanadate compound, or a derivative
or an analogue thereof effective to reduce or inhibit the
growth of the drug resistant tumor.

5. A method for reducing metastases in a patient
comprising administering an amount of a vanadate compound,
or a derivative or an analogue thereof effective to reduce
metastases.

6. A pharmaceutical composition for use as an
antiproliferative and anti-metastatic agent comprising a
vanadate compound or a derivative or analogue thereof, and
at least one antioxidant, and one or more of a
pharmaceutically acceptable carrier, diluent, or
excipient.

7. A composition as claimed in claim 6, wherein the
vanadate compound is orthovanadate and the antioxidant is

- 27 -
N-acetylcysteine.

8. A method for treating proliferative disorders,
treating drug resistant tumors or reducing metastases
comprising administering an effective amount of a vanadate
compound or a derivative or analogue thereof, and at least
one antioxidant.

9. The method as claimed in claim 8 wherein the
vanadate compound is orthovanadate or vandyl sulphate.

10. The method as claimed in claim 8 or 9 wherein
the antioxidant is N-acetylcysteine, glutathione, Vitamin
E (alpha-tocopherol), Vitamin C (ascorbic acid), beta-
carotene, ergothioneine, zinc, selenium, copper,
manganese, a flavonoid or an estrogen.

11. The method as claimed in claim 9 wherein the N-
acetylcysteine is administered prior to and during the
administration of orthovanadate.

12. The method as claimed in claim 10 wherein the
orthovanadate is administered at a dose which provides a
serum concentration of at least 5µM and the N-
acetylcysteine is administered at a dose which provides a
serum concentration of between 0.5mM to 15mM.

Description

Note: Descriptions are shown in the official language in which they were submitted.


21136~


BP File No. 3153-099/LMK

Title: ~R~A~M~NT OF PROLIFERATIVE DI~oRn~R~
METASTASES, AND DRUG RESISTANT TUMORS WITH VANADATE
COMPOUNDS AND DERIVATIVES OR ANALOGUES ~RRRROF




FIELD OF THE INVENTION
The present invention relates to the use of
vanadate compounds or derivatives or analogues of vanadate
compounds as antiproliferative and anti-metastatic agents,
and/or to treat drug resistant tumors in animals; to
compositions contA i n ing vanadate compounds adapted for
such use; to methods for the treatment of proliferative
disorders, to methods of reducing the ability of a tumor
to metastasize, and to methods for treating drug resistant
tumors. The invention also relates to methods for testing
for substances which affect cell proliferation.

RACRGROUND OF THE INVENTION
Cancer is a global problem which affects an
estimated 5.9 million people worldwide annually. There are
many types of cancer, some of the most common in North
America include breast, lung, colon and lymphatic cancer.
Although chemotherapy has had positive impact on the
survival rate of cancer patients in the last 30 years,
most human cancers are, or become resistant to
chemotherapy. Thus, there is a tremendous need for
anticancer drugs which are more effective and which can
act on drug resistant tumors.
Two important features of cancer cells is their
ability to proliferate abnormally leading to tumor
formation and growth, and to invade other tissues leading
to metastases. It is thought that genetic damage to
specific genes is responsible for the transformation of
cells and the development of cancer in humans. The genetic
damage found in human cancer cells can be divided into two
types. One of these involves the mutation of oncogenes

21136~3
-- 2 --
which results in continuous proto-oncogene activation. The
second involves the mutation of tumor suppressor genes
which results in the loss of their function. Genetic
damage to proto-oncogenes or to tumor suppressor genes
leads to oncogene activation in the absence of stimuli
and to uncontrolled cellular proliferation. Damage has
been found to one or another proto-oncogenes and tumor
suppressor genes with some consistency in a variety of
human malignancies.
Two oncogenic transcription factors, fos and
jun, have been shown to be involved and required for the
induction of genes involved in cellular proliferation and
in particular, in cellular proliferation in many tumor
cell 7ines. Inhibition of the expression of these two
lS genes leads to the inhibition of cellular proliferation.
One of the most life threatening aspects of
cancer is the development of metastases. Generally, most
solid tumors can be removed surgically from the primary
site resulting in a local cure. However, if the cancer
cells have invaded vascular channels and metastasized to
a different organ, then the likelihood of a complete cure
is reduced. Thus, agents which reduce the metastatic
properties of cancer cells would be beneficial for the
treatment of cancer.
The cellular processes thought to play an
important role in metastases include; increased cellular
attachment, tumor cell proteolysis of host tissue, tumor
cell locomotion and colony formation. These processes
occur in a sequential order. First, tumor cells attach to
the basement membrane through their surface receptors of
integrin and non-integrin types to ligands such as
- collagen, laminin and fibronectin in the basement
membrane. After attachment, a localized zone of lysis of
the basement membrane occurs at the point of cell
attachment. The tumor cells produce and secrete
degradative enzymes, such as collagenase and gelatinase,
which degrade the basement membrane and allow the

21136~
_ - 3 -
infiltration and locomotion of tumor cells into the host
organ. There is a positive association between tumor
aggressiveness and the ability of cells to produce a group
of enzymes, matrix metalloproteases, involved in the
invasive process. Inhibition of certain proteases, such as
metalloproteases or serine proteases, have been shown to
prevent invasion and metastasis (Alvarez et al. 1990. J.
Natl. Cancer Inst. 82: 589-595; Schultz et al 1988, Cancer
Res. 48, 5539-5545; and, Wang & Stearns 1988, Cancer Res.
48, 6262-6271)).
Ionic vanadium compounds such as vanadyl or
vanadate salts in combination with thiosulphate or sulfite
compounds have been reported to be useful for treating
malignant tumors, arteriosclerosis and mental syndromes in
the elderly ((U.S. Patent Serial No. 5,045,316 to Kaplan).
Kaplan discloses a daily dose ranging from 0.0043 mg/kg to
0.14 mg/kg of vanadyl or vanadate salts. No mechanism for
the action of vanadate and thiosulphate in the disclosed
treatments is provided by Kaplan.
In the background of the Kaplan patent it is
disclosed that others have reported that vanadium salts
have an antineoplastic effect and dietary vanadyl sulphate
has been reported to inhibit chemically induced mammary
carcinogenesis in rats.
Saxena et al. (Biochem. Pharmacology 45(3): 539-
542, 1993) examined the in vivo effects of vanadate on the
antioxidant status of control and alloxan diabetic rat
livers. Diabetic rats were administered 0.6 mg sodium
orthovanadate/ml in drinking water. It should be noted
that the present inventor has found that oral
administration of orthovanadate to animals at 0.5 mg/ml
results in gastric toxicity (See Example 9 herein).
Antioxidants such as ~-carotene, ~-tocopherol,
vitamin E, vitamin C, and glutathione have been reported
to have anticancer activity (G. Shklar et al. Nutrition
and Cancer, 1993, p.l45). It has also been disclosed that
a mixture of antioxidants (~-carotene, dl-~-tocopherol

_ 4 _ 2113683

acid succinate tvitamin E), vitamin C, and reduced
glutathione) was very effective in preventing
carcinogenesis in an in vivo cancer model and was more
effective than the individual components of the mixture as
cancer chemopreventive agents.
snMMARY OF THE INVENTION
The present inventor has found that the levels
of superoxides or H2O2 in the cell play an important role
in the induction of fos and jun expression. Reducing the
levels of H2O2 by inhibiting its production with diphenyl
iodonium (DPI), or by increasing the levels of
intracellular reducing agents such as N-acetylcysteine and
orthovanadate were shown to completely inhibit fos and jun
expression in response to factors such as IL 1 or
arachidonic acid. Under all of the conditions examined,
inhibition of fos and jun expression results in inhibition
of collagenase expression.
The present inventor also found that
orthovanadate and its analogues are extremely toxic to
proliferating cell lines, at concentrations that are not
toxic to normal nonproliferating cells indicating that
orthovanadate may be useful as a chemotherapeutic agent.
He has also significantly found that orthovanadate acts on
cell lines resistant to conventional drugs such as
colchicine, vinblastine and doxorubicin indicating that
the drug is useful for treatment of drug resistant tumors.
The mechanisms which normally expel chemotherapeutic
agents from cancer cells that are drug resistant do not
recognize the vanadate compounds.
Orthovanadate and analogues thereof were also
shown to suppress tumor growth in an in vivo animal model
(NDAY-D2 model). Doses of at least 0.2 mg/kg were
required to reach concentrations of orthovanadate or
analogues thereof in the serum of the animals to be highly
toxic to cancer cells.
Significant inhibition of tumor growth was
observed when orthovanadate in combination with an anti-


21136~3
-- 5 --
oxidant, N-acetylcysteine, was administered. The action of
orthovanadate and N-acetylcysteine was more effective in
inhibiting tumor growth in vivo than orthovanadate alone.
The present inventor also found that animals
receiving orthovanadate or vanadyl sulphate did not have
detectable levels of metastases.
Accordingly, broadly stated the present
invention relates to a method of modulating fos and jun
expression by regulating concentrations of hydrogen
peroxide.
In accordance with an embodiment of the
invention compounds are used to reduce hydrogen peroxide
and/or superoxides to thus effect a reduction in cell
proliferation. Preferably the compounds are vanadate
compounds, or derivatives or analogues thereof.
The invention also contemplates a pharmaceutical
composition for the treatment of proliferative disorders
comprising an amount of a vanadate compound, or a
derivative or an analogue thereof, effective to reduce
cell proliferation, and one or more of a pharmaceutically
acceptable carrier, diluent, or excipient. In a preferred
embodiment of the invention, the pharmaceutical
composition is used to reduce tumor growth. The invention
further contemplates a method for the treatment of a
proliferative disorder comprising administering an amount
of a vanadate compound, or a derivative or an analogue
thereof, effective to reduce cell proliferation.
The amount of a vanadate compound or derivative
or analogue thereof, effective to reduce cell
proliferation is an amount which results in a serum
concentration of the compound of at least 5~M, preferably
5-50~M, most preferably 10-30~M. Generally a dosage of at
least 0.2 mg/kg, preferably 0.2 mg/kg to 20 mg/Kg will
result in the appropriate serum concentrations in humans
and other mammals.
The invention also relates to a method for
reducing or inhibiting the growth of drug resistant tumors

2113683
-- 6 --
comprising administering an amount of a vanadate compound,
or a derivative or an analogue thereof effective to reduce
or inhibit the growth of drug resistant tumors. T h e
invention further contemplates a method for reducing or
inhibiting metastases comprising administering an amount
of a vanadate compound, or a derivative or an analogue
thereof effective to reduce or inhibit metastases.
The invention also contemplates a composition
comprising a vanadate compound or a derivative or analogue
thereof, and at least one antioxidant, preferably N-
acetylcysteine, which enhances the antiproliferative and
anti-metastatic effects of the vanadate compound and
reduces cell proliferation and metastases. Methods of
treating and preventing proliferative disorders, treating
drug resistant tumors, and reducing metastases using this
composition are also provided.
The invention also relates to methods for
testing a drug for activity in reducing cell
proliferation.
These and other aspects of the present invention
will become evident upon reference to the following
detailed description and attached drawings. In addition,
reference is made herein to various publications, which
are hereby incorporated by reference in their entirety.
BRIFF D~.~RTPTION OF THE DRAWINGS
Further details of the invention are described
below with the help of the examples illustrated in the
accompanying drawings in which:
Figure 1 is a graph showing the FACS analysis of
superoxide production in response to IL 1 and inhibition
of NADPH oxidase by DPI;
Figure 2 is a Northern Blot showing the role of
superoxide production on fos and collagenase expression;
Figure 3 is a Northern blot showing hydrogen
peroxide stimulates fos expression;
Figure 4 is a Northern blot showing that
orthovanadate inhibits fos, jun and collagenase expression;

~ _ 7 _ 21i~6~
Figure 5 is a Northern blot showing N-
acetylcysteine inhibits IL 1 induction of fos and
collagenase expression;
Figure 6 is a graph showing the effect of
orthovanadate on proliferating cells;
Figure 7 is a graph showing that orthovanadate
is toxic to MDAY-D2 and HTB14 cells;
Figure 8 is a graph showing the effect of
different forms of orthovanadate on cell toxicity;
Figure 9 is a bar graph showing that H202
potentiates orthovanadate toxicity;
Figure 10 is a graph showing that orthovanadate
is toxic to cell lines of varying drug resistance;
Figure 11 is a photograph of tumors from
untreated and orthovanadate treated mice;
Figure 12 is a graph showing that orthovanadate
administration suppresses tumor growth in vivo;
Figure 13 is a graph showing the effect of
orthovanadate, vanadyl sulphate and vanadyl hydroperoxide
administration on tumor growth in vivo;
Figure 14 is a graph showing that orthovanadate
and N-acetylcysteine administration completely inhibits
tumor growth, in vivo;
Figure 15 is a photograph showing liver
metastases by NDAY-D2,cells:
Figure 16 are photographs showing the effect of
orthovanadate and vanadyl sulphate on metastases;
Figure 17 is a graph showing a comparison of a
prior art treatment and the orthovanadate/N-acetylcysteine
treatment of the present invention; and
Figure 18 is a Northern blot showing
orthovanadate inhibition of IL 1, PNA and AA induced c-fos
and c-jun expression.
DETATT.~D DESCRIPTION OF THE lNv~N~lION
As hereinbefore mentioned, the present invention
relates to a method of modulating fos and jun expression
by regulating concentrations of hydrogen peroxide.

2113683
- 8 -
Increasing the concentrations of hydrogen peroxide should
result in increased expression of fos and jun and
accordingly an increase in cell proliferation. An increase
in cell proliferation would be useful in the treatment of
conditions involving damaged cells and in particular may
be useful in treating conditions in which degeneration of
tissue occurs such as arthropathy, bone resorption,
inflammatory disease, degenerative disorders of the
central nervous system, and for promoting wound healing.
In accordance with an embodiment of the
invention compounds are used to reduce hydrogen peroxide
and/or superoxides to thus effect a reduction in cell
proliferation. Preferably the compounds are vanadate
compounds, or derivatives or analogues thereof. Suitable
vanadate compounds for use in the present invention are
oxidative forms of vanadate, preferably orthovanadate.
Derivatives of vanadate compounds, preferably
pharmaceutically acceptable salts, esters and complexes of
vanadate compounds including potassium and sodium salts,
and amino acid, carbohydrate and fatty acid complexes, for
example, vanadate complexed with cysteine, dihydroxamate,
and glucuronate may also be used in the present invention.
Suitable analogues may be selected based upon
their functional similarity to vanadate compounds,
including the ability to interact with hydrogen peroxide
to produce hydroxyl radicals or to generally reduce
hydrogen peroxide. Examples of such compounds include
metal ions such as iron, titanium, cobalt, nickel and
chromium complexes, stannum, glutathione, and diphenyl
iodonium. Analogues of vanadate compounds may also be
selected based upon their three dimensional structural
similarity to vanadate compounds. For example, the vanadyl
forms of vanadium may be used in the present invention,
preferably vandyl sulphate.
Most preferably, orthovanadate and vanadyl
sulphate are used in the pharmaceutical compositions,
therapeutic treatments and methods of the present

- 21i~6~3

invention.
Selected derivatives and analogues of vanadate
compounds may be tested for their ability to reduce
hydrogen peroxide, their ability to effect growth of
proliferating cell lines, non-proliferating cell lines,
and drug resistant cell lines, and their ability to
inhibit tumor growth or metastases in animal models
following the methods described herein.
The composition of the invention may contain one
of more antioxidants in combination with a vanadate
compound or analogue or derivative thereof. The
antioxidant(s) are selected based on their ability to
increase the efficacy of the vanadate compounds and reduce
toxicity on normal cells using the methods described
herein. Suitable antioxidants for use in the e~hAncing
composition of the invention include N-acetylcysteine,
glutathione, Vitamin E (alpha-tocopherol), Vitamin C
(ascorbic acid), beta-carotene, ergothioneine, zinc,
selenium, copper, manganese, flavonoids and estrogens, or
derivatives thereof, preferably N-acetylcysteine.
The administration of vanadate compounds or
analogues or derivatives thereof, and optionally one or
more antioxidants, in the forms and modes described herein
reduces hydrogen peroxide to effect a reduction in cell
proliferation, and also reduces metastases of tumors.
Thus, the compositions may be used for the treatment of
proliferative disorders including various forms of cancer
such as leukemias, lymphomas (Hodgkins and non-Hodgkins),
sarcomas, melanomas, adenomas, carcinom~s of solid tissue,
hypoxic tumors, squamous cell carcinomas of the mouth,
throat, larynx, and lung, genitourinary cancers such as
cervical and bladder cancer, hematopoietic cancers, head
and neck cancers, and nervous system cancers, benign
lesions such as papillomas, arthrosclerosis, angiogenesis,
and viral infections, in particular HIV infections. The
compositions of the invention have been shown to be
specifically effective in inhibiting the growth of

211~83
-- 10 --
hematopoietic tumors, human glioma and astrocytoma primary
tumors.
Vanadate compounds or analogues or derivatives
thereof, and optionally one or more antioxidants, in the
compositions described herein may also be used to treat
drug resistant tumors. Examples of drug resistant tumors
are tumors expressing high levels of P-glycoprotein which
is known to confer resistance to multiple anticancer drugs
such as colchicine, vinblastine and doxorubicin, or tumors
expressing the multi-drug resistance protein as described
in R. Deeley et al., Science, 258:1650-1654, 1992.
The compositions of the invention contain
vanadate compounds or derivatives or analogues thereof,
and optionally one or more antioxidants, either alone or
together with other substances. Such pharmaceutical
compositions can be for topical, parenteral (intravenous,
subcutaneous, intramuscular or intramedullary) or local
use. Preferably, a mode of administration is used which
results in a slow continuous release of the active
substances. This may be achieved by intravenous
administration, subcutaneous administration, or using
control release mechanisms such as implants or pumps.
Control release methods generally use control release
polymers and the release of the active ingredient is based
on solubility properties, and the pore size of the
polymers and active ingredients.
In the case of parenteral administration,
solutions, suspensions, emulsions or powders of the
vanadate compound and/or derivative and or analogue
thereof, and optionally antioxidant(s) can be employed,
using one or more pharmaceutically acceptable excipients
or diluents, suitable for the aforesaid uses and with an
osmolarity which is compatible with the physiological
fluids. For local use, those preparations in the form of
creams or ointments for topical use or in the form of
sprays should be considered.
The preparations of the invention can be

2113G$3
.
11
intended for administration to humans and various other
mammals, such as ovines, bovines, equines, swine, canines,
and felines.
The amount of a vanadate compound or derivative
or analogue thereof, effective to reduce cell
proliferation, and/or to reduce metastases or treat drug
resistant tumors is the minimum dose adequate to achieve
a reduction in cell proliferation, reduction or inhibition
of metastases, and/or growth of drug resistant tumors. A
dose which results in a serum concentration of the
compound of at least 5~N, preferably 5-50~M, most
preferably 10-30~M, is required to reduce cell
proliferation and accordingly provide for effective
treatment of proliferative disorders. Generally, a dose of
at least 0.2 mg/kg, preferably 0.2 mg/kg to 20 mg/Kg will
provide an appropriate serum concentration in humans and
other mammals. The above-mentioned doses may be used to
reduce metastases and treat drug resistant tumors. The
selected doses will also depend on individual needs and
the mode of administration.
When the vanadate compound or analogue or
derivative thereof is used in combination with one or more
antioxidants, the doses of the vanadate compound or
analogue or derivative thereof and the antioxidant(s) are
selected so that the vanadate compound and antioxidant(s)
alone would not show a full effect. Generally, the
effective doses of the vanadate compound and the
antioxidant(s) are the minimum doses adequate for enhanced
antiproliferative or anti-metastatic effects. The vanadate
compound and antioxidant(s) may be administered
concurrently, separately, or sequentially.
The vanadate compound and antioxidant may be
prepared and administered as a complex. For example,
vanadate may be complexed with glutathione or N-
acetylcysteine.
In an embodiment of the invention, a dose oforthovanadate compound is administered which provides a

21~83
- 12 -
serum concentration of the compound of at least 5~N,
preferably 5-50~N, most preferably 10-30~N. N-
acetylcysteine is administered prior to, (preferably 20
minutes prior to), and during administration of
orthovanadate, at a dose which provides a serum
concentration of the compound of between 0.5mM to 15.OmM,
preferably 5mN to 12.5 mN. Generally, a dose of between
40.0 mg/kg to 1000 mg/Kg of N-acetylcysteine will provide
an appropriate serum concentration in humans and other
mammals.
The compositions can be prepared by per se known
methods for the preparation of pharmaceutically acceptable
compositions which can be administered to patients, and
such that an effective quantity of the active substance is
combined in a mixture with a pharmaceutically acceptable
vehicle. Suitable vehicles are described, for example, in
Remington's Pharmaceutical Sciences (Remington's
Pharmaceutical Sciences, Mack Publishing Company, Easton,
Pa., USA 1985). On this basis, the pharmaceutical
compositions include, albeit not exclusively, solutions of
the vanadate compounds, derivatives or analogues thereof
in association with one or more pharmaceutically
acceptable vehicles or diluents, and contained in buffered
solutions with a suitable pH and iso-osmotic with the
physiological fluids.
The compositions and treatments are indicated as
therapeutic agents or treatments either alone or in
conjunction with other therapeutic agents-or other forms
of treatment. In particular, the compositions and
treatments described herein may be used to reduce toxicity
of other therapeutic agents. For example the compositions
of the invention may be used in combination with
radiotherapy or chemotherapy, such as multi-drug
chemotherapy for Hodgkins disease or combination
radiotherapy, and chemotherapy for treatment of breast
cancer.
As hereinbefore mentioned the invention also

211~6~
- 13 -
relates to methods for assaying for substances that affect
cell proliferation. The method involves determining the
effect of the substance on the growth of non-proliferating
cells and comparing the effect to that observed for the
substance with proliferating cells. In one embodiment a
substance which is suspected of affecting cell
proliferation is assayed by preparing a non-proliferating
primary cell culture by plating non-proliferating
preferably human or bovine chondrocytes or fat cells, at
high cell density, preferably 2X106 to 4X106 cells /per
well on a six well plate, and preparing a proliferating
cell culture by plating proliferating cells, preferably
chondrocytes at low density preferably 5x105 to lx106 cells
/per well on a six well plate; incubating each of the cell
cultures in media cont~; n ing the substance suspected of
affecting cell proliferation preferably for 1 to 48 huors
at 37C, harvesting cells and quantitating the number of
viable cells, and comparing the number of viable cells in
the proliferating and non-proliferating cell cultures.
The following non-limiting examples are
illustrative of the present invention:
~XAMPLES
Example 1
Identification of signalling mechanisms regulating fos,
jun and collagenase expression.
The sequence of events or second messengers
responsible for the stimulation of fos and jun expression
were investigated.
A. IL 1 induces a transient increase in fos and jun mRNA.
The cytokine interleukin 1 (IL 1) has been used
to identify the intermediate second messengers which
regulate the expression of fos and jun. The reason for
using IL 1 is that it has been shown to stimulate fos and
jun expression, and produce all of the signals required to
induce the expression of matrix metalloproteases. IL 1 was
found to induce a transient increase in fos and jun mRNA
levels which peaks by 30 min to one hour, whereas the

- 14 - 2~3~
appearance of collagenase mRNA is detected by 9 hours and
continues to increase up to 12 hours. This data is
consistent with studies demonstrating that fos and jun
expression is required for collagenase production.
B. IL 1 stimulates the production of reactive oxygen
intermediates
Chondrocytes ( i.e. bovine chondrocytes plated
as described in Kandel R.A. et al. Biochim. Biophys. Acta.
1053, 130-134, 1990) were incubated with
dihydroxyrhodamine for 5 min (DHR) or for 4 hours in the
absence (-IL 1 ) or presence of IL 1 ( + IL 1), or in the
presence of both IL 1 and the NADPH inhibitor, diphenyl
iodonium, ( + IL 1, + DPI). Figure 1 shows that IL 1
stimulates the production of reactive oxygen intermediates
by FACS analysis. The inhibitor of NADPH oxidase, DPI
(diphenyl iodonium), completely inhibits constitutive and
IL 1 induced reactive oxygen intermediates in
chondrocytes. These data indicate that IL 1 stimulates the
production of intracellular superoxides and oxygen
reactive intermediates.
C. Effect of DPI on fos and jun mRNA levels induced by IL
Although IL 1 stimulated superoxide production,
it was not known whether IL 1 induced fos and jun
expression was dependent on the production of superoxides.
In order to elucidate this possibility, the effect of DPI
on fos and jun mRNA levels induced by IL 1 was
investigated. RNA from chondrocytes treated with IL 1 in
the presence and absence of DPI were analyzed by Northern
blot analysis using either fos or collagenase cDNA probes.
The results demonstrated that IL 1 induction of fos and
collagenase is suppressed by DPI, indicating that
superoxide production plays a role in the induction of
these genes (Figure 2). Similar data has been obtained
for IL 1 induced jun expression. Furthermore, inhibition
of fos and jun expression by DPI was sufficient to
suppress IL 1 induced and constitutive collagenase

- 21i3623
-- 15 --
expression. These data indicate that inhibition of
superoxides or H2O2 production prevents the induction of
fos, jun and collagenase expression.
D. Hydrogen peroxide mimics the effect of IL 1 in the
induction of fos expression
Since superoxides are rapidly converted to
hydrogen peroxide in the cell by superoxide dismutase,
whether hydrogen peroxide could mimic the effect of IL 1
in the induction of fos expression was investigated. RNA
was extracted from chondrocytes (Kandel et al. supra)
treated with H2O2 for 30, 60 and 90 minutes and examined by
Northern blot analysis using a fos cDNA probe. As
demonstrated in Figure 3, addition of H2O2 to chondrocytes
also stimulates the expression of fos, suggesting that
this molecule may be a key second messenger in the
induction of the transcription factors, fos and jun.
E. Effect of orthovanadate and N-acetylcysteine on fos
jun and collagenase expression
The effect of orthovanadate and N-acetylcysteine
on fos, jun and collagenase expression were examined.
Bovine articular chondrocytes were isolated and
plated as previously described (Kandel R.A. et al.
Biochim. Biophys. Acta. 1053, 130-134, 1990). In order to
determine the effect of orthovanadate on IL 1 and PMA
(phorbol ester) induced responses, chondrocytes were
incubated with orthovanadate (100 uM) for 2 hours before
stimulation with IL 1 (10 ng/ml) or PMA (100 mg/ml).
Collagenase production was determined by incubating
chondrocytes for 24 hours with IL 1 or PMA and the cell
conditioned medium was assayed for collagenase activity
using an ELISA procedure as described previously (Kandel
et al. supra). PLA2 activity was measured by incorporating
3H-arachidonic acid (3H-AA) into the cells and then
incubating the cells with medium cont~i~ing 1 mg/ml BSA,
either alone or in the presence of IL 1 or PMA, for 10
min. as previously described (Conquer, J.A. 1192, Biochim.
Biophys. Acta. 1134, 1-6). The amount of 3H-AA liberated

21~3683
- 16 -
into the supernatant was determined. To measure PGE2
production, chondrocytes were incubated for 6 hours in
Ham's F12 medium, either alone or with IL 1 or PMA. The
supernatant was analyzed by RIA using an antibody specific
for PGE2 (Dr. S.A. Jones, Mount Sinai Hospital, Toronto,
Can.). In order to ~x~ine the expression of c-fos and c-
jun, chondrocytes were incubated for 1 hour in the
presence of IL-1, PMA or AA (3 ~M). Chondrocytes were
washed in PBS and the total RNA extracted as previously
described (Cruz. et al, 1991, Biochem. J. 277, 327-330).
RNA samples were run on formaldehyde agarose gels and
transferred to nylon membrane for northern analysis using
cDNA probes for c-fos and c-jun.
IL 1 and PMA induced the release of 3H-AA as well
as the production of PGE2 and collagenase by chondrocytes
in monolayer culture. Although orthovanadate (100 uM)
completely inhibited the production of collagenase it did
not inhibit the IL 1 or PMA induced release of 3H-AA or the
production of PGE2. These data would suggest that either
the effect of orthovanadate is occurring downstream from
3H-AA release or that the mechanisms regulating PLA2
activity and PGE2 production are separate from those
regulating collagenase production. The expression of c-
fos and c-jun were stimulated by IL 1, PMA as well as AA
itself in bovine chondrocytes. Orthovanadate completely
inhibited the IL 1, PMA and AA induced c-fos and c-jun
expression, which may be responsible for the inhibition of
collagenase production. These data (See Figures 4 and 18)
suggest that orthovanadate inhibition of collagenase
production may be occurring downstream from the IL 1
induced 3H-AA release by inhibiting c-fos and c-jun
expression in chondrocytes. The data demonstrating that
orthovanadate is a potent inhibitor of fos, jun and
collagenase expression indicates that agents reducing H2O2
levels in cells may serve as potent inhibitors of
expression of fos and jun.
Cells were also incubated as described above

- 17 - 21~3683
with 20 mM N-acetylcysteine for 20 min. and then incubated
with IL 1 for an additional 1 or 12 hours. The RNA was
extracted and examined by Northern blot analysis using
cDNA probes for c-fos and collagenase. N-acetylcysteine
which is converted to GSH intracellularly was also found
to reduce the levels of fos and collagenase expression in
response to IL 1 (Figure 5). Presumably the higher
intracellular levels of GSH reduced HzO2 and superoxide
levels and suppressed the induction of fos and collagenase
expression.
In summary, the results demonstrate that both N-
acetylcysteine and orthovanadate indirectly reduce the
levels of superoxides and H2O2 in cells.
Example 2
Vanadate Comro~ln~.c as potent chemotherapeutic agents in
vitro.
The effect of a class of vanadyl derivatives, on
cellular proliferation in vitro is described below.
A. In vitro effects of Vanadyl Derivatives on normal non-
proliferating and proliferating cells.
As described in example 1,~ orthovanadate
inhibited fos, jun and collagenase expression. If fos and
jun expression are required for cellular proliferation,
then orthovanadate should inhibit chondrocyte
proliferation. In order to compare the effect of
orthovanadate on non-proliferating and proliferating
chondrocytes, chondrocytes were plated at both high cell
density (2x106 to 4X106 cells tper well on a six well
plate) (nonproliferating) and at a lower cell density
(5x105 to lx106 cells /per well on a six well plate)
(proliferating) and then maintained for 48 hours. The
cells were then incubated in media (HAMS F12) contAining
O - 50 ~M Orthovanadate for an additional 48 hours. The
cells were harvested and the number of viable cells
determined.
Figure 6 demonstrates that orthovanadate did not
effect the chondrocytes that were plated at high cell

_ - 18 - 2 113 S~
density but was toxic to cells plated at low cell density.
These data suggest that proliferating cells are sensitive
to orthovanadate, whereas non-proliferating cells are
resistant to orthovanadate toxicity.
B. In vitro effects of orthovanadate on proliferating
tumor cell lines
Fos and jun activity are also required for
cellular proliferation in many tumor cell lines.
Accordingly, the effect of orthovanadate on adherent cells
and cell suspensions were examined. MDAY-D2 (a mouse
lymphoid cell line grown in suspension) and HTB14 cells
(an adherent human primary astrocytoma cell line) were
incubated in media cont~ining 0 - 50 ~M orthovanadate for
48 hours. The cells were harvested and the number of
viable cells determined. Figure 7 demonstrates the effect
of orthovanadate on HTB14 and NDAY-D2 cells.
Orthovanadate treatment resulted in a
concentration dependent increase in cell death. Although
there were slight differences in sensitivity to
orthovanadate between cell types, all cell lines examined
were killed by orthovanadate at concentrations of 5 to 10
times lower than that used in the studies with normal
nonproliferating cells (above). Orthovanadate induced cell
death was observed by 24 hours and complete (over 98~)
within 3 days of continuous treatment. In conclusion,
treatment of cancer cell lines with orthovanadate leads to
cell death at concentrations which had no significant
toxic effects on normal non-proliferating cells.
Example 3
Efficacy of different forms of orthovanadate.
Three different forms of vanadyl compounds were
examined for their effect on viability of cancer cell
lines. MDAY-D2 cells were incubated in media contA i n ing o
- 50 ~M orthovanadate, vanadyl sulphate, or vanadyl
hydroperoxide for 48 hours. The cells were harvested and
the number of viable cells determined. Figure 8
demonstrates the effect of orthovanadate, vanadyl

- 19 2~ ~683
sulphate, and vanadyl hydroperoxide on MDAY-D2 cells. The
results show that all of these agents were equally
effective in killing these cells. Although there were
slight differences in sensitivity, the overall cell death
was similar.
Example 4
Orthovanadate was thought in view of the
investigations described in Examples 1-3, to react with
H2O2 to form hydroxyl radicals which are extremely toxic.
If the orthovanadate induced formation of hydroxyl
radicals is responsible for cell toxicity, then adding
exogenous H2O2 should enhance the effects of orthovanadate.
Accordingly, cells were incubated in media alone or
cont~ining 1 mM H2O2 or 10 ~M orthovanadate or both for 24
hours. The cells were harvested and cell viability
determined. Figure 9 demonstrates the combined effects of
low concentrations of orthovanadate and H2O2 on cell
toxicity. Addition of H2O2 alone had a small effect.
However, addition of H2O2 in combination with orthovanadate
increased cell toxicity significantly in comparison to
orthovanadate alone. The potentiation of cell toxicity by
H2O2 suggests that hydroxyl free radicals generated by
orthovanadate treatment may be responsible for the cell
death.
Example 5
Orthovanadate is toxic to drug resistant cell lines
In many different cancers, tumor cells cannot be
eliminated by the conventional chemotherapeutic agents and
these tumors are designated drug resistant. Although the
mechanisms involved in this process are not well
understood, it is thought that these cancer cells express
a protein which removes the drug from inside-the cell and
reduces its intracellular toxicity. Patients having a drug
resistant tumor have a very poor prognosis. Thus, agents
which would be toxic to drug resistant tumors would be a
valuable chemotherapeutic agent for the treatment of these
patients.

~ - 20 - 2 ~ 1 3 ~ ~ ~
The effect of orthovanadate on three ovarian
cancer cell lines, KB8, KB8-5 and KB85-11, which have
increasing drug resistance, respectively, relative to the
parent cell line, KB3-1 was compared. These drug resistant
cell lines are not killed by several classes of
chemotherapeutic agents such as colchicine, vinblastine
and doxorubicin. In the study, cell lines of increasing
drug resistance (KB8, KB8-5 and KB-85-11) and the parent
cell line, KB3-1, were incubated in media (DMEM)
cont~ining 0-50 ~N orthovanadate for 48 hours. The cells
were harvested and the number of viable cells determined.
As demonstrated in Figure 10, orthovanadate was equally
effective in killing all of the drug resistant cell lines.
Minor differences in sensitivity to orthovanadate was
observed between cell lines, but it was not dependent on
their drug resistance property, and by three days of
orthovanadate administration these differences were not
apparent since most of the cells had died.
In conclusion, the data indicate that
orthovanadate is lethal to drug resistant cell lines and
it may be particularly useful for the treatment of drug
resistant tumors.
Exampl e 6
IN VIVO EFFECTS OF TREATMENT WITH VANADYL COMPOUNDS
In order to examine the ability of vanadyl
compounds to reduce tumor formation, growth and
metastases, a specific animal model which allows
investigation of all of these processes in the same animal
was chosen. This model involves the injection of a
metastatic haematopoietic cell line, NDAY-D2, into mice
subcutaneously. These cells form a tumor at the site of
injection and its size can be easily determined. In
addition, these cells metastasize to the liver and
metastases can be detected histologically after day 17 to
19. This model provides a very sensitive and reproducible
approach to investigate the effect of vanadyl compounds on
tumor growth and metastases.

- 21 - 2113~3
A. Effect of orthovanadate treatment on tumor growth in
vivo
Using the animal model described above, the
effect of subcutaneous administration of orthovanadate on
tumor growth was investigated. A total of 15 mice were
injected subcutaneously with 1 x 105 NDAY-D2 cells on Day
1. On Day 5, small tumors could be observed at the site of
injection. Five mice were injected daily with 50 ~l of
water alone and 10 mice were injected daily with water
contAi ni ng 10 mg/ml orthovanadate. On day 14, the mice
were sacrificed. The tumors were removed from all the
animals, photographed, and weighed. Figure 11 compares
sizes of tumors from two untreated and two orthovanadate
treated mice. The tumors of orthovanadate treated mice
were either undetectable or considerably smaller. Figure
12 demonstrates the size of the tumors for each mouse. In
animals treated with water alone, four mice had tumors
weighing between 1.18 and 1.68 gms. In the orthovanadate
treated mice, 2 mice did not have detectable tumors and
five mice had tumor sizes that were less than 0.16 gms.
B. Efficacy of orthovanadate, vanadyl sulphate and vanadyl
hydroperoxide administration on reducing tumor growth in
vivo
In a separate experiment using the same animal
model, the effect of orthovanadate, vanadyl sulphate and
vanadyl hydroperoxide administration on tumor growth in
vivo was examined. On Day 1, 20 mice were injected with 2
X 105 MDAY-D2 cell subcutaneously. The mice were divided
into four groups of five mice. At day 5, the animals were
injected subcutaneously with 50 ~l of water alone or
cont~ining 10 mg/ml of orthovanadate, 10 mgtml of vanadyl
sulphate, or 10 mg/ml of vanadyl hydroperoxide. This
treatment was continued daily for 16 days. At day 21, the
mice were sacrificed and the tumors dissected and weighed.
One animal died in each of the orthovanadate and vanadyl
sulphate treated groups, and all five died in the vanadyl
hydroperoxide treated group.

`- - 22 - 21~3~3
As demonstrated in Figure 13, the untreated mice
developed tumors which ranged in weights from 2.32 to 4.79
gms. Although the effects of vanadyl sulphate treatment
were quite variable, the treatment reduced tumors size in
all of the animals. The tumors ranged in size from 0.14
gms to 2.18 gms. In the orthovanadate treated group, one
mouse did not have detectable tumors and the re--ining
three mice had tumors which varied in size from 0.15 to
0.38 gms. These data indicate that orthovanadate had the
most efficacy in reducing tumor growth, vanadyl sulphate
was less effective and vanadyl hydroperoxide was too toxic
to evaluate its efficacy.
Example 7
Combination therapy of orthovanadate and N-acetylcysteine
completely inhibited tumor growth and formation
The studies described in the previous examples
indicated that orthovanadate was 80 to 100% effective in
preventing tumor growth in mice. Since N-acetylcysteine
is converted to glutathione in cells, higher levels of
glutathione may not only reduce orthovanadate induced
toxicity but may also reduce tumor formation. Thus,
whether administration of N-acetylcysteine in combination
with orthovanadate was more effective in reducing animal
toxicity and tumor growth in vivo was examined.
Twenty mice were injected subcutaneously with 2
x105 cells on Day 1. At day 4, the mice were divided into
four groups of five mice. Group one (control) received
subcutaneous injections of 50 ~1 of water. Group two
received daily intraperitoneal injections of 50 ~1 of 250
mM N-acetylcysteine. Group three received daily
subcutaneous injections of 50 ~1 of 10 mg/ml of
orthovanadate. Group four received daily intraperitoneal
injections of 50 ~1 of 250 nM N-acetylcysteine and 20
minutes later received 50 ~1 of subcutaneous injection of
~1 of 10 mg/ml of orthovanadate. On day 10 the
treatment was stopped. The animals were sacrificed on Day
13 and analyzed for tumor growth. One orthovanadate

- 23 - 2~ i ~ 6~ 3
treated animal died during the experiment.
Tumors were dissected from control mice and mice
treated with orthovanadate (V04) or N-acetylcysteine
(NAC) or both (NAC/V04). The data shown in Figure 14
represent the weight of each tumor. As demonstrated in
Figure 14, the untreated mice had tumors which weighed
between 0.87 to 1.69 gms. In comparison, N-acetylcysteine
treated mice had tumors which weighed between 0.23 to 1.18
gms, indicating that this agent alone was capable of
reducing tumor growth to some extent. Of the four
orthovanadate treated mice, two had no detectable tumors
and the other two had tumors weighing 0.13 and 0.35 gms.
On the other hand, all five animals receiving
orthovanadate and N-acetylcysteine administration had no
detectable tumors. These experiments clearly indicated
that the combination therapy of orthovanadate and N-
acetylcysteine was the most effective therapy in
inhibiting tumor growth in vivo. Furthermore, N-
acetylcysteine appeared to reduce the slight toxic effects
observed in animals treated with orthovanadate alone.
Example 8
VANADYL COMPOUNDS AS ANTI-METASTATIC AGENTS
Vanadate compounds were found to inhibit
metastatic potential of cancer cells by reducing their
ability to invade other organs. More particularly,
metastases of MDAY-D2 cells was found to occur in the
animal model described in Example 6. Figure 15 shows a
control liver and a liver with metastases. The metastatic
liver was obtained from an animal 24 days following the
administration NDAY-D2 cells. The nodules are quite
numerous and large. In animals sacrificed between 19 and
23 days, the number and size of the nodules were quite
variable from animal to animal, indicating that in order
to ex~rine the anti-metastatic potential of orthovanadate,
animals should be maintained for a minimum of 23 days
following the injection of MDAY-D2 cells.
Preliminary results from histological

- 24 _ 2~36~3
examination of livers obtained following one of the
experiments described above in Example 6 suggested that
orthovanadate and vanadyl sulphate were both effective at
preventing metastases. Livers were removed from animals
treated as described above and prepared for histological
examination. Figure 16 compares liver sections from
untreated (C), orthovanadate (VO)(500~g/day) and vanadyl
sulphate (VS)(500~g/day) treated animals. Nodules are
identified with an arrow. Infiltration of MDAY-D2 cells
and the formation of colonies was observed in the
untreated animals. Animals receiving orthovanadate and
vanadyl sulphate did not have detectable levels of
metastases.
Example 9
Oral administration of orthovanadate at 0.5
mg/ml was found to result in gastric toxicity in
laboratory mice. Furthermore intraperitoneal
administration of high doses of orthovanadate was also
found to be toxic to the animals. However, subcutaneous
injections of up to 500 ~gms orthovanadate is tolerated by
the animals. Slow administration of the orthovanadate
would decrease toxicity and the animals may tolerate
higher doses.
Example 10
Comparison with Raplan U.S. Patent Serial No. 5,045,316
The concentration of vanadate used by Kaplan was
found to be far too low to be effective in inhibiting
tumor growth or metastases. In order to determine whether
Kaplan's optimum conditions were effective, the effect of
the highest concentrations of orthovanadate alone, or
thiosulfate alone, or orthovanadate and thiosulfate
administered together on tumor growth in mice was
investigated. Kaplan reported daily doses ranging from
0.0043 mg/kg to 0.14 mg/kg of vanadyl or vanadate salts
are required for treatment. Assuming an equal
distribution in the body fluids and a water content of
56%, the maximum concentration of orthovanadate in the

- 25 - ~ i 3 6 8 3
serum with these doses at the time of administration is
from .04 ~M to 1.3 ~M.
As demonstrated in Figure 17, no decrease in
tumor growth was observed with any of the agents described
by Kaplan alone, or in combination, at the doses disclosed
by Kaplan. Under the optimum treatment conditions of the
present invention, tumor growth was either not apparent or
less than 80% of control.
From the foregoing, it will be appreciated that,
although specific embodiments of the invention have been
described herein for purposes of illustration, various
modifications may be made without deviating from the
spirit and scope of the invention. Accordingly, the
invention is not limited except as by the appended.

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Administrative Status

Title Date
Forecasted Issue Date Unavailable
(22) Filed 1994-01-18
(41) Open to Public Inspection 1995-07-19
Dead Application 2000-01-18

Abandonment History

Abandonment Date Reason Reinstatement Date
1999-01-18 FAILURE TO PAY APPLICATION MAINTENANCE FEE

Payment History

Fee Type Anniversary Year Due Date Amount Paid Paid Date
Application Fee $0.00 1994-01-18
Registration of a document - section 124 $0.00 1995-05-04
Maintenance Fee - Application - New Act 2 1996-01-18 $100.00 1996-01-09
Maintenance Fee - Application - New Act 3 1997-01-20 $100.00 1997-01-16
Maintenance Fee - Application - New Act 4 1998-01-20 $100.00 1997-12-08
Owners on Record

Note: Records showing the ownership history in alphabetical order.

Current Owners on Record
MOUNT SINAI HOSPITAL CORPORATION
Past Owners on Record
CRUZ, TONY
Past Owners that do not appear in the "Owners on Record" listing will appear in other documentation within the application.
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Office Letter 1994-04-12 1 28
Description 1995-07-19 25 1,211
Claims 1995-07-19 2 65
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Abstract 1995-07-19 1 17
Cover Page 1995-09-15 1 18
Fees 1997-12-08 1 53
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Fees 1996-01-09 1 50