Note: Descriptions are shown in the official language in which they were submitted.
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METHOD FOR DETECTING AND KILLING
EPITHELIAL CANCER~CELLS
Related Application
This application is a continuation-in-part of
copending International Application, PCT/US00/05387,
filed February 28, 2000, entitled "Method of Detecting
and Killing Epitheleal Cancer Cells.
Field of the Invention
This invention relates to methods for detecting
epithelial cancer.
In another respect the invention pertains to methods
for selectively killing epithelial cancer cells.
In a further aspect, the invention concerns methods
for detecting epithelial cancer cells in the presence of
normal cells and/or for selectively killing such cells,
in which the mitochondria of cancer cells retain a
mitochondrial marking agent for a time sufficient to
permit identification and/or killing such cells.
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Definitions
As used herein, the following terms have the
indicated meanings:
"Cancer" or "cancerous" cells are used in the broad
sense, to include invasive cancer cells, cancer-in-
situ cells and severely dysplastic cells.
"Mitochondrial marking agent" means a compound that
is selectively taken up by the mitochondria of
living cancer cells and is selectively retained in
cancer cells for a time sufficient to permit
identification and/or killing or incapacitation
thereof.
"Killing" of cells means either causing cell deathL
apoptosis or cell changes that render a cell
incapable of reproduction and metastasizing.
"Adduct" means the reaction product, either covalent
or noncovalent, of a mitochondrial marking agent and
a cancer chemotherapeutic agent.
"Adjuvant" means a mitochondrial marking agent that,
in combination with another chemotherapeutic agent,
causes improved killing of cancer cells, either
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synergistically or by additive effects with the
other agent.
Background of the Invention
In-vivo diagnostic procedures for detecting
malignant and premalignant epithelial lesions or
carcinomas, employing dye compositions that selectively
"color" tissues that are abnormal due to dysplasia,
hyperplasia, tumorigenesis and other active surface
lesions, are known in the art. These diagnostic methods
employ a dye that imparts color to a cancerous substrate,
which is then detectable under light at visible
wavelengths or a fluorescent dye that imparts color to
the substrate, which is then detectable when illuminated
by light at wavelengths outside the visible spectrum.
For example, procedures employing fluorescein and
fluorescein derivatives are disclosed in Chenz, Chinese
Journal of Stomatology (27:44-47 (1992)) and Filurin
(Stomatologiia (Russian) 72:44-47 (1993)). These
procedures involve application of the dye, followed by
examination under ultraviolet light to detect the
cancerous/precancerous tissue, which is selectively
fluorescent. Another prior art procedure involves
rinsing the epithelium with toluidine blue, followed by
normal visual examination to detect any selectively
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stained tissue. Such procedures are disclosed, for
example in the patents to Burkett (U. S. 6,086,852), Tucci
(U.S. 5,372,801) and Mashberg (U.S. 4,321,251). Use of
other thiazine dyes and oxazine dyes in an analogous
manner is disclosed in U.S. Patent 5,882,627 to
Pomerantz.
Heretofore, it was theorized that such dyes
selectively "marked" cancerous tissue because the dye was
retained in the relatively larger interstitial spaces
between the cells of cancerous tissue and would not
efficiently penetrate the tighter intracellular junctions
of normal tissue or be selectively retained in such
relatively smaller spaces.
Contrary to the belief that toluidine blue
selectively marks cancerous epithelial tissue because it
is selectively retained in the relatively larger
interstitial spaces between cancer cells, the mechanism
of such selective staining of epithelial tissue by
cationic dyes, e.g., dyes such as rhodamine,
fluoresceins, oxazine and thiazine dyes (including
toluidine blue) and other cationic supravital marking
agents, is the selective uptake and selective retention
of the agent in the mitochondria of cancer cells. This
selective mitochondrial uptake and retention is
apparently due to the higher electrical potential
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(negative charge on the inside of the membrane) of
cancerous cells' mitochondria as compared to mitochondria
of normal cells. See, e.g., Chen et al., Cancer Cells
1/The Transformed Phenotype, 75-85 (Cold Spring Harbor
Laboratory, 1984); Lampidis, et al., Cancer Research 43,
716-720 (1983). In fact, the selective marking of cancer
cells by, and retention in the mitochondria of cancer
cells of, supravital cationic dyes and other supravital
cationic marking agents, are related to one of the very
characteristics of cancer cells that appears to be
responsible for their rapid cloning growth and
metastasizing ability, namely, that the higher electrical
potential of the mitochondria of cancer cells is the
source of cellular energy and is the driving force for
ATP (adenosine triphosphate)production by the cells.
Summary of the Invention
We have now discovered a method for in-vivo
detection of cancerous epithelial cells by selective
marking of the mitochondria thereof.
In another respect, we have discovered a therapeutic
method for selectively killing cancer cells in the
presence of normal cells.
Our detection methods comprise the steps of
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delivering a cationic supravital mitochondrial marking
agent to tissue in the locus of a suspect cancerous site
on the epithelium (which contains both normal and
cancerous cells), thus causing said agent to be taken up
and selectively retained in the mitochondria of the
cancer cells. The cancerous cells are then detectable by
any suitable method, for example, instrumental or visual
examination under visible light or under light of
selected invisible wavelengths.
In a further embodiment, after the marking agent is
taken up by the mitochondria, a rinse reagent is applied
to the locus of the suspect cancerous site, thus
enhancing the rate of release of the agent from the
mitochondria of the normal cells and further increasing
the selectivity of the diagnostic methods.
According to another important embodiment of the
invention, we provide a method for selectively killing
cancerous epithelial cells comprising the step of
contacting cancerous cells in the locus of a suspect
cancerous site with a cationic supravital mitochondrial
marking agent, to cause cell death or to render the'
cancer cells substantially incapable of multiplication.
The marking agent can be delivered to the cancer cells in
a single discrete dose, or continuously, or in repeated
discrete doses, with or without employing a rinse reagent
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after each dose.
In a further embodiment of the invention, we provide
a method of improving the selectivity and cancer cell
killing ability of cancer chemotherapeutic agents
comprising the steps of either (1) forming a reaction
product of a cationic supravital agent and a
chemotherapeutic agent and delivering the reaction
product to cancerous epithelial cells or (2) combining
the cationic supravital agent with a cancer
chemotherapeutic agent, to improve the selectivity or
killing ability of the chemotherapeutic agent, either by
additive or synergistic effects, or both.
These, other and further embodiments of the
invention will be apparent to those skilled in the art
and a better understanding of the invention will be
obtained from the following examples which are provided
to illustrate the invention and not as indications of the
scope thereof, which is defined only by the appended
claims.
In the practice of the invention and in the
following working examples, cationic supravital
mitochondrial marking agents, include
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dyes, including toluidine blue O, alcian blue,
malachite green, phenosafranin, acriflavine,
pyronine Y, toluylene blue and brilliant green;
and
"non-dye" compounds, including peonidin,
oxythiamine, tiemonium iodide, elliptinium
acetate and furazolium chloride.
According to the presently preferred embodiment of
the invention, the preferred mitochondrial marking agents
are dyes of the oxazine and thiazine class. The thiazine
dyes are especially preferred, particularly toluidine
blue O, Azure A, Azure B and ring-substitution and N-
substitution analogs thereof.
In order to be selectively absorbed and retained in
cancer cell mitochondria, the marking agent or reaction
product of marking agent + chemotherapeutic agent, must
have a molecular weight of below about 5,000. Further,
because of marked differences in the selective marking
and therapeutic activity of various closely related
analogs, it appears that the molecular structure of the
marking agent significantly affects its ability to
selectively mark and/or kill living cancer cells in the
presence of normal living cells. These differences in
cell marking and killing ability are related to
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structural features, e.g., location and type of ring-
substituents and N-substituents, of the marking agent
molecules that implicate one or more or all of the
following mechanisms of action:
1. The structure of the marking agent molecule,
e.g., position and nature of ring and N-substituents on
the cationic molecule, affects the availability of the
positive charge and hinders the ability of the marking
agent or "stacked" groups of them to be attracted by the
negative charges on the mitochondrial membranes or within
the mitochondria.
2. The structure of the marking agent molecule
permits it to intercalate into or "stack" along the
exterior of mitochondrial DNA of cancer cells.
3. The structure of the marking agent molecule
permits it or stacked groups of them to bind to specific
active sites, e.g., four specific proteins, in the
mitochondria, and/or precipitate with cardiolipins at the
inner surface of the mitochondrial membrane.
4. The structure of the marking agent affects its
reduction potential and its tendency to change to the
uncharged "leuco" form.
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5. The structure of the marking agent affects its
acidity (pKa), and, in turn, the ability of the cationic
marking agent to deprotonate at physiological pH. Thus,
the cationic form of the dye can be attracted to the
outer surface of the mitochondrial membrane, whereupon
the dye cation can lose a proton and concomitantly lose
its positive charge, thereby liberating the neutral form
of the dye, which may more readily penetrate the nonpolar
matrix of the membrane and gain access to the interior of
the mitochondrion.
The intermolecular interactions of mechanisms 1 dye-
membrane), 2 (dye-base pair or dye-dye), and 3 (dye-
protein or dye-lipid) depend on the hydrophobicity-
lipophilicity of the dye, which can be assessed by
various means, one of which is the partition coefficient
between aqueous solution and a low-polarity organic
solvent, such as 1-octanol (i.e., log P values).
Mechanisms 4 and 5 depend on hydrophobicity-
lipophilicity, due to the effect of differential
solvation of reactant and product on reduction potential
(oxidized vs. reduced forms) and pKa (neutral vs. charged
forms). For example, hydrophobicity hampers the
solvation of protonated tertiary aliphatic amines (R3NH+),
thereby decreasing their acidity relative to secondary
amines (RzNH2+) .
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According to the presently preferred embodiment of
the invention, one employs a cationic supravital marking
agent having a log P of from about -1.0 to about 5.
The following examples are presented to enable those
skilled in the art to understand and practice the
invention and to identify the presently preferred
embodiment. These examples are for illustrative purposes
only are not intended to limit the scope of the
invention, which is defined only by the appended claims.
Example 1
Uptake and Retention of Mitochondrial
Markin~qents in Living Carcinoma Cells
Different concentrations of the various cationic
marking agents, at 100, 50, 10 and 1 ~g/ml are prepared
in RPMI medium complete with 20o fetal calf serum, 1 mM
glutamine, hydrocortisone, insulin, transferrin,
estradiol, selenium and growth hormone.
The carcinoma cells are incubated at 37EC in tissue
culture incubators with 5% COZ and 95% relative humidity,
for 5 minutes with each agent and concentration there and
then rinsed twice using 2 minute incubations with 10
acetic acid. After incubation and rinsing, the cells are
harvested, at 30 min., 1 hour, 2 hours, 4 hours and 8
hours. The cells are then extracted with 2-butanol and
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analyzed by spectrophotometry for quantitation of the
marking agent.
The results show that there is a concentration
dependence in the rate of accumulation of marking agent
in the mitochondria of both carcinoma and normal cells
and in the selectivity of release of the marking agent
from cancer cells, but this concentration dependence
starts to become less pronounced. The saturation
concentration for toluidine blue 0 occurs at
concentrations of l0ug/ml and above. The saturation
concentrations for the other marking agents are similarly
determined. The remaining experiments are conducted with
a concentration of l0ug/ml for toluidine blue O and at
the saturation concentrations for the other marking
agents so-determined, unless stated otherwise.
Example 2
Mitochondrial Localization of the Agents in Living Cells
After incubation and rinsing of various cell lines,
using the different cationic marking agents, the
mitochondrial localization of the agents is analyzed
using confocal high resolution microscopy and phase
contrast microscopy.
Living cells, are cultivated in complete growth
medium with 20o fetal calf serum and growth factors, and
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maintained at 37EC. These cells accumulate and retain
the marking agents in the mitochondria. When these cells
are then maintained in an agent-free medium, carcinoma
cells retain the agent for longer than about 1 hour, but
normal epithelial cells release the agent within about
15 minutes.
In contrast to living cells, dead cells or cells
treated with agents that dissipate the mitochondrial
membrane potential lose mitochondrial staining and
accumulate the agents in the nucleus.
Example 3
Release of the Agents from Mitochondria With
Dissir~ation of the Mitochondrial Membrane Potential
Known agents that alter the mitochondrial electrical
potential are used to pretreat epithelial cancer cells,
followed by treatment with the cationic supravital
mitochondrial marking agents. These pretreatment agents
include azide and cyanide preparations and dinitrophenol.
Epithelial cancer cells are also pre-stained with
the various dyes and then are post-treated with these
known agents. The release of the dyes from the cells or
the transfer of the dyes to other subcellular
compartments, including the nucleus is analyzed.
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The cells pretreated with these agents do not
accumulate dyes in the mitochondria and the mitochondria
of the pre-stained cells release the dye upon post-
treatment with these agents.
Example 4
Tissue Explants of Scruamous Carcinomas
Fresh explants of resected epithelial carcinomas are
analyzed for marking agent uptake and retention. After
resection, the carcinomas are microdissected from
surrounding tissue, cut into 3 mm sections and maintained
as explant tissue cultures at 37°C. These explants are
then incubated with the various agents and then extracted
for quantitation of the agent.
Oral carcinoma (SqCHN) have rapid uptake and
prolonged retention of these agents. The agents start to
be released from the cells after about one hour of
cultivation in agent-free medium. However, the agents
are released faster when the cells are incubated in
medium that does not contain growth factors, fetal calf
serum and other medium additives. The agents are also
released faster when the cells are grown in adverse
conditions such as lower temperatures.
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Example 5
Tissue Explants of Normal Epithelial Cells
Cells obtained surgically from normal areas of the
oral epithelium are cultivated as normal epithelial
cultures. These cultures are then incubated with the
marking agents for analysis of the agent uptake and
retention.
Unlike the carcinoma cells, normal epithelial cells
quickly release the agents from their mitochondria and
from the cell much more quickly. By 10-15 minutes, most
of the agent is released from the mitochondria.
Example 6
Marking Agent-Chemotherapeutic Agent Adducts
In place of the agents of Examples 1-5, the
following adducts of cationic mitochondrial marking
agents and various known chemotherapeutic agents are
employed, with substantially similar results, except that
the cancer cell kill rate and selectivity of the
chemotherapeutic agent are substantially improved.
Markina Aaent Chemotherapeutic Aaent
toluidine blue O methotrexate
rhodamine 123 nitrogen mustard
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Example 7
Adjuvant Compositions
The following combinations of known cancer
chemotheraputants with mitochondrial marking agents
exhibit synergistic or at least additive cancer cell
killing effects:
Chemotherapeutant Cationic Marking Aaent
taxol toluidine blue
taxotere azure A
vincristin alcian blue
Selective Therapeutic Effects
In the following examples, a toluidine blue drug
substance is prepared in accordance with the
manufacturing procedures disclose in the U.S. Patent
6,086,852, issued to Burkett on July 11, 2000.
Components of the drug substance are then fractionated
and separated by semi-preparative HPLC, yielding the
analogs identified in the '852 patent as represented by
Peaks 5, 6, 7 and 8. The compounds represented by peaks
7 and 8 are toluidine blue regioisomers, having the ring
methyl group in the -2 position (peak 8) and the -4
position (peak 7). The compound represented by peak 5 is
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the N-demethylated derivative of peak 7 and the compound
represented by peak 6 is the N-demethylated derivative of
peak 8.
Example 8
The compounds represented by peaks 5, 6, 7 and
8, obtained during the fractionation of the toluidine
blue O, are analyzed for their selective toxicity towards
living oral carcinoma cells (SqCHN) compared to living
normal oral epithelial cells. Separate cultures of
squamous carcinoma cells and normal epithelial cells are
incubated with the different dye fractions and then
washed with dye-free medium. The cells are then re-
incubated in growth medium and observed over a period of
8 days to determine the extent of cell killing. The
compound of peak 6 results in 95% cell death of carcinoma
cells, compared to only about 20% killing of normal
cells. The compound of peak 8 shows 89°s cell death of
carcinoma cells whereas it only causes about 20% killing
of normal cells. Thus, the selective retention of the
compounds of peaks 6 and 8 is selectively toxic towards
carcinoma cells.
The selective introduction into the mitochondria of
cationic dyes leads to disruption of the mitochondrial
electrical potential which is the source of cellular
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energy and the driving force of ATP (adenosine
triphosphate) production of the cells. The ability of
carcinoma cells to divide rapidly and to metastasize is
dependent upon the availability of this higher energy
source.
However, effects on electric charge do not appear to
be the only mechanism involved, because the compounds
represented by peak 5 and peak 7 also are cationic dyes
and yet they do not exhibit the same selective toxicity
towards carcinomas that Peak 6 and Peak 8 demonstrate.
Thus, the compounds of peaks 6 and 8 appear to have other
molecular properties that lead to their selective
toxicity towards carcinoma cells.
Example 9
The therapeutic characteristics of the compounds of
peaks 6 and 8 are determined by further in-vitro tests,
conducted in the manner of Example 8, using other
isolates of carcinoma cells and normal epithelial cells.
The testing profile includes other squamous carcinomas of
the head and neck, esophagus, lung, cervix and skin, as
well as other types of cancers, including
adenocarcinomas, lymphomas and sarcomas. In-vivo "delay
of tumor growth" and "tumor regression assay" tests
using tumor-bearing animals, including head and neck and
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lung carcinomas implanted in Balb-C mice, are made to
analyze the in-vivo therapeutic benefit of these
compounds. These further in vitro and in-vivo tests
confirm the selective toxicity of the compounds of peaks
6 and 8 to the wider variety of cancer cell types.
Having described the invention in such manner as to
enable those skilled in the art to understand and
practice it and, having identified the presently
preferred embodiments thereof, WE CLAIM:
