CZ20013861A3 - Use of cationic supravital mitochondrial marker for detection and control of epithelial cancer cells - Google Patents

Abstract

A diagnostic method for detecting cancerous epithelial cells by selectively marking the mitochondria of the cancer cells, by delivering to the epithelium a cationic supravital mitochondrial marking agent. Selective killing of cancerous epithelial cells in the presence of normal cells is effected by delivering a cationic supravital mitochondrial marking agent to epithelial cancer cells. The killing agent can also comprise the reaction product of the marking agent and a cancer chemotherapeutic drug.

Description

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Using a cationic supravital mitochondrial marker to selectively label cancer cell mitochondria, wherein a cationic supravital mirochondrial marker is delivered to the epithelium. The invention also provides the use of a cationic supravital mitochondrial marker for the selective control of epithelial cancer cells by delivering a cationic supravital mitochondrial marker to the epithelium. The killing agent may also comprise a reaction product of the marker and the cancer chemotherapeutic agent, or a mixture of the marker with the chemotherapeutic agent.

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Use of a cationic supravital mitochondrial marker for the detection and control of epithelial cancer cells

Technical field

The invention relates to the use of a cationic supravital mitochondrial marker for the selective labeling of mitochondria that indicates the presence of epithelial cancer.

The invention further relates to the use of a cationic supravital mitochondrial marker for the selective control of epithelial cancer cells.

The invention further relates to the use of a cationic supravital mitochondrial marker for the purpose of detecting epithelial cancer cells in the presence of normal cells and / or selective killing those cells in which the mitochondria of the cancer cells capture the mitochondrial marker for a period sufficient to identify and / or kill them.

BACKGROUND OF THE INVENTION

The following terms, as used herein, have the meanings indicated:

The term cancer or cancer cells is used in its broad sense and includes invasive cancer cells, cancer in situ cells and severe dysplasia cells.

The term mitochondrial marker refers to a compound that is selectively absorbed by mitochondria of living cancer cells and selectively retained in the cancer cells for a period of time sufficient to identify and / or kill or destroy these cancer cells.

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The term cell killing includes either inducing cell death, apoptosis, or cell changes that render the cell incapable of reproducing and forming metastases.

By adduct is meant a reaction product, either covalent or non-covalent, a mitochondrial marker and a cancer chemotherapeutic agent.

The term adjuvant refers to a mitochondrial marker which, in combination with another chemotherapeutic agent, promotes the control of cancer cells, such that the synergistic or additive effects of this marker with said other agent.

In vivo diagnostic procedures for detecting malignant and premalignant epithelial lesions or carcinomas utilize staining agents that selectively stain tissues that are abnormal due to dysplasia, hyperplasia, tumorigenesis, and other active surface lesions known in the art. utilizing a dye that stains the cancer substrate, wherein the stained substrate can be detected in the light at visible wavelengths, or utilizes a fluorescent dye that stains the substrate and the substrate is subsequently detectable under light illumination at wavelengths that lie outside the visible spectrum.

For example, procedures using fluorescein and fluorescein derivatives are described in Chenz, Chinese Journal of Stomatology (27: 44-47) (1992) and Filurin (Stomatologiia (Russia) 72: 44-47 (1993)). These procedures include the application of a dye and subsequent examination in ultraviolet light to detect cancer and / or pre-cancerous tissues that are selectively fluorescent.

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Another known technique involves epithelial flushing with toluidine blue and subsequent examination under normal visible light to detect all selectively stained tissue. Such procedures are described, for example, in patents granted to Burrkett (US 6,086,852), Tuccio (US 5,372,801) and Mashberg (US 4,321,251). The use of other thiazine and oxazine dyes in an analogous manner is described in U.S. Patent 5,882,627 to Pomerantz.

Thus, it is theorized that these dyes selectively label cancerous tissue because they are trapped in relatively larger interstitial spaces between cancer tissue cells and cannot effectively penetrate, or cannot be selectively trapped in, the tightly intracellular junctions of normal tissue.

Contrary to this theory, toluene blue selectively labels cancer epithelial tissue by being selectively trapped in comparatively larger interstitial spaces between cancer cells, the mechanism of selective staining of epithelial tissue with cationic dyes, such as dyes such as rhodamine, fluoresceins, oxazine and thiazine dyes (including toluidine blue) and other cationic supravital markers based on selective uptake and selective retention of this marker in cancer cell mitochondria. This selective mitochondrial uptake and retention is apparently due to the electrical potential (negative charge on the inside of the membrane) of cancer cell mitochondria that is significantly higher than the electrical potential of normal cell mitochondria. See. for example, 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). Ve

01-3422-01-Ma fact is the selective labeling of cancer cells, and retention in cancer cell mitochondria, cationic supravital stains and other cationic supravital markers one property of cancer cells that is associated with is believed to be responsible for rapid cloning and growth ability forming metastases, and in particular it is believed that the higher electrical potential of cancer cell mitochondria is a source of cellular energy and a driving force of these cells for the production of ATP (adenosine triphosphate).

SUMMARY OF THE INVENTION

We have now discovered the use of a cationic supravital mitochondrial marker for the in vivo detection of cancer epithelial cells by selective labeling of the mitochondria of these cells.

The use of a cationic supravital mitochondrial marker for the selective control of cancer cells in the presence of normal cells has also been discovered.

The staining methods of the invention include delivery of a cationic supravital mitochondrial marker to tissue at the epithelial site where cancer (including both normal and cancer cells) is suspected, mitochondrial uptake and capture of the marker in cancer cell mitochondria. The cancer cells are then detected by any suitable method, such as instrumental or visual examination under visible light at wavelengths selected outside the visible spectrum.

In another embodiment of the invention, upon capture of the mitochondrial marker, a rinsing agent is applied to the site of suspected cancer, which will accelerate release of the marker from normal cell mitochondria and thereby increase the selectivity of observable stained cells.

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The invention further provides the use of a cationic supravital mitochondrial marker for the selective control of cancer epithelial cells, which comprises contacting the cancer cells at the site of the anticipated cancer incidence with a cationic supravital mitochondrial marker, the method of which: cell death or substantially prevent the proliferation of these cancer cells. The marker can be delivered to the cancer cells in a single discrete dose, or in a continuous manner or in repeated discrete doses, optionally using a rinsing agent after each dose has been introduced.

The invention also provides the use of a cationic supravital mitochondrial marker with increased selectivity and the ability of cancer chemotherapeutic agents to kill cancer cells, the use comprising (1) forming a reaction product of a cationic supravital agent and a chemotherapeutic agent and delivering the reaction product to cancerous epithelial cells; a cationic supravital agent with a cancer chemotherapeutic agent that results in a synergistic and / or additive increase in the selectivity or ability of the chemotherapeutic agent to kill cancer cells.

These and other embodiments of the invention will become more apparent to those skilled in the art from the following examples. It is to be noted that the following examples are illustrative only and are not intended to limit the scope of the invention as set forth in the appended claims.

The cationic supravital mitochondrial markers used in the practice of the invention and in the following examples include:

Dyes including toluidine blue O, alcian blue, malachite green, phenosafranine, acriflavine, pyronine Y, toluylene blue and brilliant green;

non-dyes, which are compounds including, for example, peonidine, oxythiamine, thiemonium iodide, elliptinium acetate and furazolium chloride.

According to a preferred embodiment of the invention, the preferred mitochondrial dye markers are oxazine and thiazine class dyes. Particularly preferred are thiazine dyes, and in particular toluidine blue O, Azur A, Azur B and their analogs with ring substitution and N-substitution.

In order to selectively absorb and trap the marker in the mitochondria of the cancer cell, the molecular weight of the marker and the marker, respectively, must be determined. The reaction product of the marker and the chemotherapeutic agent is less than about 5000. In addition, due to the difference in labeling with selective labeling and in the therapeutic activity of the various closely related analogues, it is clear that the molecular weight of the marker significantly affects its ability to selectively label and / or kill living cancer cells. in the presence of normal living cells. These differences in cell labeling and killing capacity are related to the structural features of the marker molecules, such as the position and type of ring substituents and N-substituents, which more or less affect the following mechanisms of action:

1. the structure of the marker molecule, such as the position and nature of the ring and N-substituents on the cationic molecule, affects the availability of a positive charge and hinders the marker

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or stacked groups to be attracted by negative charges on the mitochondrial membranes or within the mitochondria.

2. The structure of the marker molecule allows the marker to be inserted into the cancer cell mitochondrial DNA or superimposed on the outer portion of the cancer cell mitochondrial DNA.

3. The structure of the marker molecule allows the marker or stacked groups to bind to specific active sites, for example 4 specific proteins, in mitochondria and / or precipitate with cardiolipins on the inner surface of the mitochondrial membrane.

4. The structure of the marker affects its reduction potential and the tendency to convert to a leuco form without charge.

5. The structure of the marker affects its acidity (pK _a ) and thus the ability of the cationic marker to deprotonate at physiological pH. Thus, the cationic dye may be attracted to the outer surface of the mitochondrial membrane, whereupon the dye cation may lose the proton and at the same time lose its positive charge, thereby becoming a neutral form that readily penetrates the nonpolar membrane matrix and enters the inner portion of the mitochondrion.

The intermolecular interaction of mechanism 1 (dye-membrane), 2 (dye-base pair or dye-dye), and 3 (dye-protein or dye-lipid) depends on the hydrophobicity of lipophilicity, which can be determined in different ways, one of which is the partial coefficient between an aqueous solution and a low polarity organic solvent such as

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for example 1-octanol (for example, log P values). Mechanisms 4 and 5 depend on hydrophobicity-lipophilicity due to the different solvating effect of the reagent and the product on the reducing potential (oxidized vs. reduced form) and pK ₄ (neutral vs. charged form). For example, hydrophobicity prevents the solvation of protonated tertiary aliphatic amines (R ₃ NH ⁺ ), thereby increasing their acidity over secondary amines (R ₂ NH ₂ ⁺ ).

According to a preferred embodiment of the invention, it is suitable to use a cationic supravital marker having a log P value of about -1.0 to 5.

The following examples are intended to enable those skilled in the art to understand and to practice the nature of the invention and to present some particularly preferred embodiments. It should be noted, however, that the following examples are illustrative only and are not intended to limit the scope of the invention as set forth in the appended claims.

DETAILED DESCRIPTION OF THE INVENTION

Example 1

Capture and capture of mitochondrial markers in living cancer cells with estradiol, concentration

Various different cationic markers were prepared in RPMI media enriched with 20% fetal calf serum, 1 mM glutamine, hydrocortisone, insulin, transferrin, selenium and growth hormone, namely concentrations of 100 μρ / γηΐ, 50 μρ / γηΐ, 10 pg / ml and 1 μρ / πιΐ.

AND

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Cancer cells were incubated for 5 minutes with the individual reagents used at defined concentrations at 37 ° C in tissue culture incubators together with 5% CO ₂ and 95% relative humidity, followed by a 2-minute wash with 1% acetic acid. 30 minutes, 1 hour, 2 hours, 4 hours, and 8 hours after incubation and washing, cells were harvested. The cells were then extracted with 2-butanol and analyzed by spectrophotometry to quantify the marker.

The obtained results showed that there is a concentration dependence between the rate of marker accumulation in cancer and normal cell mitochondria and the selectivity of the release of the marker from cancer cells, but this concentration dependence is not very significant. The saturation concentration for toluidine blue O appears to be 10 pg / ml and above. Saturation concentrations for other markers were determined in a similar manner. The remaining experiments were performed, unless otherwise stated, at a saturation concentration of toluidine blue O, i. 10 gg / ml. and at saturation concentrations determined for other markers.

Example 2

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Mitochondrial localization of markers in living cells

After incubation of different cell lines with different cationic markers and washing, the mitochondrial position of the markers was determined using high resolution confocal microscopy and phase contrast microscopy.

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AND

Living cells were cultured in complete growth medium supplemented with 20% fetal calf serum and growth factors and maintained at -37 ° C. These cells accumulated and retained markers in their mitochondria. If these cells were subsequently maintained in the reagent-free marker, then the cancer cells retained the marker for more than about 1 hour, while normal epithelial cells released the marker within 15 minutes.

In contrast to living cells, dead cells or marker-treated cells lose mitochondrial membrane potential, lose the ability to stain mitochondria and accumulate markers in the nucleus.

Example 3

Release of markers from mitochondria associated with loss of mitochondrial membrane potential

Reagents known to alter the mitochondrial electrical potential were used for pretreatment of epithelial cancer cells, followed by treatment with cationic supravital mitochondrial markers. These pretreatment agents included azide and cyanide formulations and dinitrophenols.

Epithelial cancer cells were also pre-stained with various dyes and subsequently treated with known markers. The release of dyes from cells or the transfer of dyes to other subcellular compartments, including the nucleus, is analyzed.

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Cells pretreated with these agents did not accumulate dyes in the mitochondria and mitochondria of the pre-stained cells released the dye after subsequent treatment with these markers.

Example 4

Tissue explants of squamous carcinomas

Fresh explants of epithelial carcinomas were analyzed for marker absorption and retention. After resection, the carcinomas were microscopically separated from the surrounding tissue, sectioned into 3 mm samples and maintained as explanted tissue cultures at 37 ° C. These explants were then incubated with various markers and subsequently extracted to determine the quantitative representation of the marker.

Oral carcinoma (SqCHN) showed rapid absorption and long-term retention of these agents. The reagents began to release from the cells after approximately 1 hour of culture in a marker-free medium. However, when cells were incubated in media that did not contain growth factors, fetal calf serum and other media additives, they released more rapidly. The agents also released more rapidly if the cells were allowed to grow under unfavorable conditions, for example at low temperature.

Example 5

Tissue explants of normal epithelial cells

Cells obtained surgically from normal areas of the oral epithelium were cultured as normal epithelial cultures. These cultures were then incubated with markers in an attempt to determine

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9999 9 Absorption and retention of markers. Unlike cancer cells, normal epithelial cells released markers from their mitochondria and from the cell much faster. Most of the marker was released from mitochondria within 10 to 15 minutes.

Example 6

Marker and chemotherapeutic adducts

Instead of the markers of Examples 1 to 5, the following adducts of cationic mitochondrial markers and various known chemotherapeutic agents were used. Using these adducts, essentially the same results were obtained, with the rate of cancer cell growth and the selectivity of the chemotherapeutic agent much better. ·

Marker Chemotherapeutic agent

Toluidine blue O methotrexate

Rhodamine 123, Mustard Nitrogen

Example 7

Adjuvansy

The following combinations of known cancer chemotherapeutic agents and mitochondrial markers have shown synergistic or at least additive effects in terms of cancer cell control.

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Chemotherapeutic agent

Taxol

Taxotere

Vincristin

Cationic marker toluidine blue azure A alcian blue

Selective therapeutic effects

In the following examples, toluidine blue as an active ingredient was prepared according to the manufacturing procedures described in US Patent 6,086,852, issued to Burkett on July 11, 2000.

The active ingredient components were then fractionated and separated by semi-preparative HPLC to give the analogues defined in the '852 patent which represented peaks 5, 6, 7 and 8. The compounds represented by peaks 7 and 8 are the toluidine blue regioisomers having the -2 position ( peak 8) and at the -4 (peak 7) position a methyl ring. The compound represented by peak 5 is an N-demethylated derivative of peak 7 and the compound represented by peak 6 is an N-demethylated derivative of peak 8.

Example 8

The compounds represented by peaks 6, 7 and 8, obtained during the fractionation of toluidine blue O, were analyzed for their selective toxicity to live oral carcinoma cells (SqCHN) and to live normal oral epithelial cells. Separate cultures of squamous cancer cells and normal epithelial cells were incubated in various dyes, and then washed with dyes with plain medium. The cells were then re-incubated in growth medium for 8 days

01-3422-01-Ma observed to determine the extent of cell death . Compound of peak 6 caused 95% cell death of cancer cells and only about 20% death of normal cells. Compound of peak 8 caused 89% cell death of cancer cells and approximately 20% death of normal cells. Thus, selective retention of compounds of peaks 6 and 8 is selectively toxic to cancer cells.

The selective introduction of cationic dyes into the mitochondria causes disruption of the mitochondrial electrical potential, which is a source of cellular energy and a driving force of cells in the production of ATP (adenosine triphosphate). The ability of cancer cells to divide rapidly and form metastases depends on the availability of this higher energy source.

However, electrical charge interference does not appear to be the only mechanism causing selective cancer toxicity because the compounds represented by peaks 5 and 7 are also cationic dyes and yet do not exhibit the same selective cancer toxicity as those represented by peaks 6 and 8. The compounds of claim 6, wherein the compounds of peaks 6 and 8 have additional molecular properties that lead to selective toxicity to cancer cells.

Example 9 λ

The therapeutic properties of the compound of peaks 6 and 8 were determined by further in vitro assays performed as described in Example 8 using additional cancer cell isolates and normal epithelial cells. The test profile includes other squamous carcinomas of the head and neck, esophagus, lung, cervix and skin, as well as other types of cancer, including adenocarcinomas, limphomas and sarcomas. In an attempt to analyze in vivo • 999

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the therapeutic effect of these compounds was performed in an in vivo tumor growth delay test and a tumor regression test using tumor-bearing animals including Balb-C mice implanted with head, neck and lung carcinomas. These additional in vitro and in vivo tests confirmed the selective toxicity of compounds of peaks 6 and 8 for a broader spectrum of cancer cell types.

Claims (10)

PATENT CLAIMS Use of a cationic supravital mitochondrial marker for selectively labeling mitochondria indicating epithelial cancer cells, wherein a cationic supravital mitochondrial marker is delivered to the epithelium. Use of a cationic supravital mitochondrial marker for the selective control of epithelial cancer cells, wherein a cationic supravital mitochondrial marker is delivered to the epithelium. Use according to claim 2, wherein the marker is a reaction product of a cationic supravital mitochondrial marker and a cancer chemotherapeutic agent. The use of claim 2, wherein the marker is delivered to the epithelial cancer cells together with another cancer chemotherapeutic agent that selectively kills the cancer cells by a mechanism other than that by which the marker cells kill the cells. 01-3422-01-Ma 4 4 44 4 4 • 4 • 4 44 4 4 4 « 4 4 4 4 4 4 4 · 4444 Use according to claim 1 or 2, wherein the cationic supravital mitochondrial marker is selected such that it provides a molecular structure that does not prevent the positive charge of the marker molecule from being negatively charged on the mitochondrial membranes. Use according to claim 1 or 2, wherein the cationic supravital mitochondrial marker is selected such that it provides a molecular structure that allows the marker to bind to a specific site in mitochondria. The use of claim 1 or 2, wherein the cationic supravital mitochondrial marker is selected to provide a molecular structure that is inserted into or superimposed along the mitochondrial DNA. Use according to claim 1 or 2, wherein the cationic supravital mitochondrial marker is selected to provide a molecular structure that affects its reducing potential and allows it to be converted to an uncharged leuco form before, during or after entry into the mitochondria. 4 » 01-3422-01-Ma 14 » 4 · 4 4 4 ♦ 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 W Use according to claim 1 or 2, wherein the cationic supravital mitochondrial marker is selected such that it provides a molecular structure that is deprotonated at physiological pH. Use according to claim 1 or 2, wherein the cationic supravital mitochondrial marker has a log P value of 0 to 5.