KR20020000222A - Method for detecting and killing epithelial cancer cells - Google Patents

Abstract

The present invention relates to a diagnostic method for detecting cancerous epithelial cells by selectively marking the mitochondria of cancer cells and delivering a cationic supermicrobial mitochondrial marking agent to the epithelium. Selective lethality of cancerous epithelial cells in the presence of normal cells is achieved by delivering cationic supersomal mitochondrial marking agents to epithelial cancer cells. The lethal may also include the reaction product of the marking agent and the cancer chemotherapeutic drug or in the form of a mixture with the drug.

Description

METHOD FOR DETECTING AND KILLING EPITHELIAL CANCER CELLS

In vivo diagnostic methods of detecting cancerous and precancerous epithelial damage or sarcoma by using dye compositions that selectively "color" abnormal tissues due to dysplasia, hyperplasia, tumorigenesis and other active surface damage are known in the art. Known. This diagnostic method uses a dye that can deliver color to cancerous tissue and can be detected at visible wavelengths or a fluorescent dye that can be detected when delivered to wavelengths outside the visible spectrum by transferring color to a substrate.

For example, methods using fluorescein and fluorescein derivatives are described in Chenz, Chinese Journal of Stomatology ( 27 : 44-47 (1992)) and Filurin (Stomatologiia (Russian) 72 : 44-47 ( 1993) These methods relate to the detection of selectively fluorescent and cancerous / precancerous tissues by application of dyes and then examination under UV light.Another prior art method is cleaning the epithelium with toluidine blue. And then detecting any selectively stained tissue by performing standard visual inspections, which are described in the patent literature (Burkett (US 6,086,852), Tucci (US 5,372,801) and Mashberg (US 4,321,251)). The use of other thiazine dyes and oxazine dyes in a similar manner is described in US Pat. No. 5,882,627 to Pomerantz.

Previously, cancerous tissues were selective because the dyes are retained in relatively large interstitial spaces between cells of cancerous tissues and do not efficiently penetrate the tighter intracellular connections of normal tissues or are selectively retained in such relatively small spaces. There was a theory called "marking".

In contrast to the idea that toluidine blue is selectively retained in relatively large interstitial spaces between cancer cells, cationic dyes such as rhodamine, fluorescein, oxazine and thiazine dyes ( The mechanism of selective staining of epithelial tissues with dyes such as toluidine blue and other cationic parasite marking agents is the selective uptake and selective retention of agents in the mitochondria of cancer cells. This selective uptake and retention of mitochondria is apparently due to the higher electrical potential of the cancerous cells (negative charge on the inside of the membrane) compared to the mitochondria of normal cells. See Chen et al ., Cancder Cells 1 / The Transformed Phenotype, 75-85 (Cold Spring Harbor Laboratory, 1984); Lampidis, et al ., Cancer Research 43 , 716-720 (1983). Indeed, the selective marking of cancer cells by superbiotic cationic dyes and other supergenic cationic marking agents and their retention in the mitochondria of cancer cells is one of the characteristics of cancer cells that appear to be responsible for their rapid cloning growth and metastasis capacity. In other words, the high electrical potential of the mitochondria of cancer cells is a source of cellular energy and a driving force for ATP (adenosine triphosphate) production by the cells.

The present invention relates to a method for detecting epithelial cancer.

In another aspect, the present invention relates to a method for selectively killing epithelial cancer cells.

In a further aspect, the invention relates to a method of detecting epithelial cancer cells in the presence of normal cells and / or selectively killing said cells, wherein the mitochondria of cancer cells are sufficient for sufficient time to identify and / or kill cancer cells. Retains mitochondrial marking agents.

Justice

As used herein, the meanings of the following terms are as follows:

"Cancer" or "cancerous" cells are used in a broad sense, including invasive cancer cells, cancer-in situ cells and severe dysplastic cells.

"Mitochondrial marking agent" means a compound that is selectively absorbed by the mitochondria of living cancer cells and selectively retained within the cancer cells for a time sufficient to identify and / or kill or neutralize them.

By "lethal" a cell is meant to induce cell death, apoptosis or cellular changes that render the cell unable to proliferate and metastasize.

"Additive" means a covalent or non-covalent reaction product of a mitochondrial marking agent and a cancer chemotherapeutic agent.

By "adjuvant" is meant a mitochondrial marking agent that, together with another chemotherapeutic agent, results in enhanced lethality of cancer cells by synergistic or additive effects with other agents.

Summary of the Invention

We have now found a method for the in vivo detection of cancerous epithelial cells by selective marking of mitochondria of cancer cells.

In another aspect, the inventors have discovered a therapeutic method for selectively killing cancer cells in the presence of normal cells.

Our detection method delivers a cationic supermicrobial mitochondrial marking agent to the tissue at the location of the suspected cancerous site on the epithelium (including both normal and cancerous cells), allowing the agent to be absorbed and within the mitochondria of the cancer cells. Optionally retaining. Cancerous cells can then be detected by any suitable method, for example, by instrument or visual inspection under visible or selected invisible light wavelengths.

In a further embodiment, after the marking agent has been absorbed by the mitochondria, the cleaning agent is applied at the location of the suspected cancerous site to enhance the ratio of release of the agent from the mitochondria of normal cells and further increase the selectivity of the diagnostic method. .

According to another important embodiment of the present invention, the present inventors contact the cancerous cells at the locations of the suspected cancerous sites with a cationic parasite mitochondrial marking agent, causing cell death or making the cancer cells substantially incapable of proliferation. It provides a method for selectively killing cancerous epithelial cells, including. The marking agent may be delivered to cancer cells with or without a detergent after each dose in one, continuous or repeated doses.

In a further embodiment of the present invention, the inventors (1) form a reaction product of the cationic superbiotic agent and the chemotherapeutic agent and deliver the reaction product to the cancerous epithelial cells or (2) cancer the cationic supercellular agent. In combination with a chemotherapeutic agent, a method of improving the selectivity or lethality of a cancer chemotherapeutic agent is provided, including the step of enhancing the selectivity or lethality of the chemotherapeutic agent by an additive or synergistic effect, or both.

The above embodiments, other embodiments and additional embodiments of the present invention will be apparent to those skilled in the art, and a more sufficient understanding of the present invention will be provided from the following examples provided to illustrate the present invention and the examples are within the scope of the present invention. It is not provided as an indication of, but the scope of the invention is defined only by the appended claims.

In the practice of the present invention and in the following working examples, cationic superbiotic mitochondrial marking agents include toluidine blue O, alcian blue, malachite green, phenosafranin, acriflavin, pyronin Y, toluylene blue and brilliant green. Dye; And "non-dye" compounds, including peonidine, oxycytiamine, thimonium iodine, elliptinium acetate, and furazolium chloride.

According to a preferred embodiment of the invention, preferred mitochondrial marking agents are dyes of the oxazine and thiazine classes. Thiazine dyes are particularly preferred, including toluidine blue O, azure A, azure B and ring-substituted and N-substituted analogs thereof.

In order to be selectively absorbed and retained by cancer cell mitochondria, the reaction product of the marking agent or marking agent + chemotherapeutic agent must have a molecular weight of about 5,000 or less. In addition, due to the selective marking of various closely related analogs and marked differences in therapeutic activity, the molecular structure of the marking agent significantly affects the ability to selectively mark and / or kill living cancer cells in the presence of normal cells. Seems to exert. These differences in cell marking and lethal capacity are related to the structural properties of the marking agents associated with one or more or all of the mechanisms of action such as the location and type of ring- and N-substituents:

1. The structure of the marking agent molecule, such as the position and nature of the rings and N-substituents on the cationic molecule, affects the utility of the positive charge and the marking agent or their "stacked" group on the membrane or mitochondria of the mitochondria. It interferes with its ability to be attracted by negative charges within it.

2. The structure of the marking agent molecule allows the marking agent to be inserted into the mitochondrial DNA of cancer cells or "stacked" along the outside of the mitochondrial DNA.

3. The structure of the marking agent allows the marking agent or its stacked group to bind to a specific active site of the mitochondria, such as four specific proteins and / or precipitate with cardiolipin on the inner surface of the mitochondrial membrane.

4. The structure of the marking agent affects its reduction potential and its propensity to change to the uncharged "leuco" form.

5. The structure of the marking agent affects its acidity (p K _a ), affecting the ability of the cationic marking agent to deprotonate at physiological pH. Thus, the cationic form of the dye may be attracted to the outer surface of the mitochondrial membrane, and the dye cations on the membrane lose protons and at the same time lose their positive charge, releasing the neutral form of the dye, which more easily penetrates the nonpolar matrix of the membrane. The passage into the inside of the mitochondria can be obtained.

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 evaluated by various methods. One of these is the partition coefficient between the aqueous solution and 1-octanol (ie, log P value) low polar organic solvent. Mechanisms 4 and 5 rely on hydrophobic-lipophilic due to the effect of different solvation of the reactants and products on the reduction potential (oxidized versus reduced form) and p K _a (neutral versus charged form). For example, hydrophobicity interferes with the solvation of protonated tertiary aliphatic amines (R ₃ NH ⁺ ), thereby reducing their acidity compared to secondary amines (R ₂ NH ₂ ⁺ ).

According to a preferred embodiment of the present invention, cationic parasite marking agents having a log P of about -1.0 to about 5 are used.

The following examples are provided to enable those skilled in the art to understand and practice the present invention and to identify preferred embodiments. These examples are for illustrative purposes only and 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 marking agents in living carcinoma cells

Various cationic marking agents at different concentrations at 100, 50, 10 and 1 μg / ml were prepared in RPMI medium containing 20% fetal bovine serum, 1 mM glutamine, hydrocortisone, insulin, transferrin, estradiol, selenium and growth hormone. Prepared.

Carcinoma cells were incubated for 5 minutes at 37 ° C. in a tissue incubator with 5% CO ₂ and 95% relative humidity with respective agents and concentrations and then washed twice via 2 minute incubation with 1% acetic acid. 30 minutes, 1 hour, 2 hours, 4 hours and 8 hours after incubation and washing, cells were collected. Cells were then extracted using 2-butanol and analyzed by spectrophotometry for quantification of marking agents.

The results showed that concentration dependence exists on the accumulation ratio of the marking agent in the mitochondria of both carcinoma cells and normal cells and the selectivity of the release of the marking agent from the cancer cells, but this concentration dependency begins to be less asserted. Saturation concentration for toluidine blue O occurred at concentrations of 10 μg / ml or more. Saturation concentrations of other marking agents were determined similarly. The remaining experiments were performed at the concentrations of 10 μg / ml for toluidine blue O and at the saturation concentrations determined above for other marking agents, unless otherwise noted.

Example 2

Mitochondrial Positioning of Agents in Living Cells

After incubating and washing various cell lines with different cationic marking agents, the mitochondrial positions of the agents were analyzed using confocal high resolution microscopy and phase contrast microscopy.

Live cells were incubated in complete growth medium with 20% fetal bovine serum and growth factors and maintained at 37 ° C. These cells accumulate and retain marking agents in the mitochondria. When these cells are maintained in medium without agonist, the carcinoma cells retain the agent for at least about 1 hour, but normal epithelial cells release the agent in about 15 minutes.

In contrast to live cells, dead cells or cells treated with agents that dissipate mitochondrial membrane translocations lost mitochondrial staining and accumulated agents in the nucleus.

Example 3

Release of agent from mitochondria with dissipation of mitochondrial membrane potential

Epithelial cancer cells were infiltrated using known agents that alter the mitochondrial electrical potential and then treated with cationic parasite mitochondrial marking agents. Such pretreatment agents include azide and cyanide preparations and dinitrophenols.

Epithelial cancer cells were also prestained with various dyes and then post-treated with known agents. Release of the dye from the cells or delivery of the dye to other subcellular zones including the nucleus were analyzed.

Cells pretreated with the agent did not accumulate dye in the mitochondria and mitochondria of prestained cells released the dye upon post-treatment with the agent.

Example 4

In Vitro Transplantation of Squamous Carcinoma

Fresh explants from excised epithelial carcinoma were analyzed for marking agent uptake and retention. After excision, the carcinoma was microdissected from surrounding tissue, cut into 3 mm sections and maintained at 37 ° C. as explant tissue culture. The explants were then incubated with various agents and then extracted for quantification of the agents.

Oral carcinoma (SqCHN) rapidly absorbs and prolongs retention of the agent. The agent began to release from the cells about 1 hour after incubation in a medium without agent. However, agents were released faster when cells were cultured in media that did not contain growth factors, fetal bovine serum and other media additives. Agents also released faster when cells were grown in adverse conditions such as low temperatures.

Example 5

Explant tissue of normal epithelial cells

Surgically obtained cells from normal areas of oral epithelium were cultured as normal epithelial culture. This culture was then incubated with the marking agent for analysis of agent uptake and retention.

Unlike carcinoma cells, normal epithelial cells released agents rapidly from their mitochondria and released much faster from the cells. About 10-15 minutes, most of the agent was released from the mitochondria.

Example 6

Marking Agents-Chemotherapy Additives

In place of the agents of Examples 1-5, the following additions of cationic mitochondrial marking agents and various known chemotherapeutic agents were used, with substantially similar results, but the cancer cell lethality and selectivity of the chemotherapeutic agents were substantially improved.

Marking Agent Chemotherapy

Toluidine Blue O Methotrexate

Rhodamine 123 Nitrogen Mustard

Example 7

Adjuvant Composition

The following combinations of known cancer chemotherapeutic agents and mitochondrial marking agents have shown synergistic or at least additional cancer cell lethality effects:

Chemotherapy Cationic Marking Agent

Texol Toluidine Blue

Taxotere Azur A

Vintrystin Alsian Blue

Selective therapeutic effect

In the following examples, toluidine blue drug was prepared according to the preparation method described in Burkett, US Pat. No. 6,086,852, issued July 11, 2000. The components of the drug were then fractionated by semi-preparative HPLC and obtained analogs identified in the '852 patent as represented by peaks 5, 6, 7 and 8. Compounds represented by peaks 7 and 8 are toluidine blue regioisomers and have ring methyl groups at the -2 position (peak 8) and the -4 position (peak 7). The compound represented by peak 5 is the N-dimethylated derivative of peak 7 and the compound represented by peak 6 is the N-dimethylated derivative of peak 8.

Example 8

Compounds represented by peaks 5, 6, 7, and 8 obtained during fractionation of toluidine blue O were analyzed for their selective toxicity to live oral carcinoma cells (SqCHN) compared to live normal oral epithelial cells. Separate cultures of squamous carcinoma cells and normal epithelial cells were incubated with different dye fractions and then washed with dye free medium. Cells were then incubated in growth medium again and observed over 8 days to determine the size of cell death. Compound of peak 6 resulted in 95% cell death of carcinoma cells compared to only about 20% lethality of normal cells. Compound of peak 8 showed 89% cell death of carcinoma cells, while only causing about 20% lethality of normal cells. Thus, the selective retention of the compounds of peaks 6 and 8 was selectively toxic to carcinoma cells.

The selective introduction of cationic dyes into the mitochondria resulted in the disruption of the mitochondrial electrical potential, a source of cellular energy of cells and the inducer of ATP (adenosine triphosphate) production. The ability of carcinoma cells to divide and metastasize rapidly depends on the availability of these higher energy sources.

However, the effect on the electrical charge does not seem to be just an associated mechanism because the compounds represented by peaks 5 and 7 are cationic dyes and they do not exhibit the same selective toxicity to the carcinoma that peaks 6 and 8 show. Thus, the compounds of peak 6 and peak 8 seem to have other molecular properties that cause their selective toxicity to carcinoma cells.

Example 9

The therapeutic properties of the compounds of peak 6 and peak 8 were determined by additional in vitro tests performed by the method of Example 8, using different isolates of carcinoma cells and normal epithelial cells. Test profiles include other squamous carcinomas of the head and neck, esophagus, lungs, neck and skin, as well as other types of cancers such as adenocarcinomas, lymphomas and sarcomas. In vivo therapeutic treatment of these compounds by performing "delay of tumor growth" and "tumor suppression assay" tests in vivo using animals with tumors, including head and neck and lung carcinomas transplanted into Balb-C mice The benefits were analyzed. These additional in vitro and in vivo tests confirmed the selective toxicity of the compounds of peaks 6 and 8 against a wide variety of cancer cell types.

The present invention has been described in ways that those skilled in the art can understand and practice, and have identified their preferred embodiments.

Claims (10)

A diagnostic method for detecting cancerous epithelial cells in vivo by selectively marking the mitochondria of cancerous epithelial cells, the method comprising delivering a cationic supermicrobial mitochondrial marking agent to the epithelium. A method of selectively killing epithelial cancer cells, the method comprising delivering a cationic supermicrobial mitochondrial marking agent to the epithelial cancer cells. 3. The method of claim 2, wherein the agent is a reaction product of a cationic superbiotic mitochondrial marking agent and a cancer chemotherapeutic drug. The method of claim 2, wherein the agent is delivered to epithelial cancer cells along with another cancer chemotherapeutic drug that selectively kills the cancer cells by a mechanism that is different from the mechanism by which the agent kills cancer cells. 3. The method of claim 1, wherein the cationic superbiotic mitochondrial marking agent is selected to provide a molecular structure that does not interfere with the positive charge attraction of the marking agent by negative charge on the mitochondrial membrane. A method according to claim 1 or 2, characterized in that the cationic superbiotic mitochondrial marking agent is selected to provide a molecular structure that allows binding to a specific site of the mitochondria. 3. The method of claim 1 or 2, wherein the cationic supermicrobial mitochondrial marking agent is selected to provide a structure that is inserted into or stacked along the mitochondrial DNA. 3. The method of claim 1 or 2, wherein the cationic superbiotic mitochondrial marking agent is selected to provide a molecular structure that affects the reduction potential such that it changes to an uncharged leuco form before, during or after entering the mitochondria. Characterized in that the method. 3. The method of claim 1 or 2, wherein the cationic superbiotic mitochondrial marking agent is selected to provide a molecular structure that is deprotonated at physiological pH. 3. The method of claim 1, wherein the cationic superbiotic mitochondrial marking agent has a log p of 0-5. 4.