CN100544770C - The method of detection and killing epithelial cancer cells - Google Patents
The method of detection and killing epithelial cancer cells Download PDFInfo
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- CN100544770C CN100544770C CNB018006604A CN01800660A CN100544770C CN 100544770 C CN100544770 C CN 100544770C CN B018006604 A CNB018006604 A CN B018006604A CN 01800660 A CN01800660 A CN 01800660A CN 100544770 C CN100544770 C CN 100544770C
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Classifications
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- A—HUMAN NECESSITIES
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- A61K49/00—Preparations for testing in vivo
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- G—PHYSICS
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- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/001—Preparation for luminescence or biological staining
- A61K49/006—Biological staining of tissues in vivo, e.g. methylene blue or toluidine blue O administered in the buccal area to detect epithelial cancer cells, dyes used for delineating tissues during surgery
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- Oncology (AREA)
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- Molecular Biology (AREA)
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- Immunology (AREA)
- Pathology (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Medicinal Preparation (AREA)
Abstract
A kind of by selected marker cancerous cell mitochondrion, by being delivered to epithelium, cation supravital mitochondrial marking agent detects carcinous epithelial cell diagnostic method.By cation supravital mitochondrial marking agent is delivered to epithelial cancer cells, be implemented in the carcinous epithelial purpose of selective killing under the situation that normal cell exists.This kill agent can also comprise marking agent and cancer chemotherapeutics medicine product or with the mixture of medicine.
Description
Related application
The application is that the part of unsettled International Application PCT/US00/05387 continues, and international application was submitted on February 28th, 2000, and name is called the method for killing epithelial cancer cells " detect and ".
Invention field
The present invention relates to detect epitheliomatous method.
Another aspect of the present invention relates to the method for selective killing epithelial cancer cells.
Aspect further, the present invention relates under the situation that normal cell exists, detect the method for epithelial cancer cells and/or this cell of selective killing, wherein the cancerous cell mitochondrion keeps mitochondrial marking agent in the sufficiently long time, to discern and/or to kill and wound this cell.
Definition
Following term used herein has pointed implication:
" cancer " or " carcinous " cell uses in a broad sense, comprises the cancerous cell of invasion, cancer in situ cell and serious dysplastic cell.
" mitochondrial marking agent " means a kind of chemical compound, and mitochondrion of the cancerous cell that it is lived optionally absorbs, and optionally keeps time enough in cancerous cell, makes identification and/or kill and wound cancerous cell, or makes the cancerous cell incapacitation.
" kill and wound " cell and mean or cause cell death, apoptosis, perhaps cause the change that cell is irreproducible and shift.
" adduct " means the covalently or non-covalently product of mitochondrial marking agent and cancer chemotherapeutic agents.
" adjuvant " means is mitochondrial marking agent, and it combines with other chemotherapeutant, synergistically or by with the addition of other reagent, the ability of killing and wounding cancerous cell is improved.
Background of invention
Use selectivity " dyeing " because of abnormal development, hyperplasia, tumor take place and the dye composite of the abnormal structure that other active surface damage causes, it is known in the art detecting in-vivo diagnostic method pernicious and damage of canceration anterior epithelium cornea or carcinoma.These diagnostic methods use the dyestuff with carcinous substrate staining, and carcinous substrate under visible light is can be detected, or use the fluorescent dye with substrate staining, and when with the rayed of wavelength beyond the visible spectrum, substrate is can be detected.
For example, at Chenz, the method for using fluorescein and fluorescein derivative is disclosed among Chinese stomatology magazine (27:44-47 (1992)) and the Filurin (stomatology (Russia) 72:44-47 (1993)).These methods comprise the use dyestuff, then check under ultraviolet light, organize before detecting cancer/canceration that selectivity sends fluorescence.The method of another kind of prior art comprises with toluidine blue rinsing epithelium, then detects the tissue of any selectively staining by common visual inspection.This method is disclosed, and is for example in Burkett (U.S. 6,086,852), open in the patent of Tucci (U.S. 5,372,801) and Mashberg (U.S. 4,321,251).Use other thiazine dye and oxazine dye open in the United States Patent (USP) 5,882,627 of Pomerantz in a similar fashion.
Up to now, theorize, think that this dye selection " labelling " cancerous tissue is because dyestuff is retained in the relatively large clearance space of cancerous tissue iuntercellular, connect in the cell more closely and can not see through normal structure effectively, or optionally be retained in this less relatively space.
And toluidine blue because of selective retention between cancerous cell in the relatively large gap, thereby the view of selected marker cancer epithelial tissue is opposite, this dye of positive ion, for example the mechanism of rhodamine, fluorescein, oxazines and thiazine dye (comprising toluidine blue) and the super in vivo marker agent of other cation selectively staining epithelial tissue is that marking agent is absorbed and selective retention by selectivity in the cancerous cell mitochondrion.It obviously is owing to compare the mitochondrial high potential of cancerous cell (negative charge on the film inboard) with the normal cell mitochondrion that this optionally mitochondrion absorbs with keeping.Referring to, Chen etc. for example, the phenotype that cancerous cell 1/ changes, 75-85 (cold spring harbor laboratory, 1984); Lampidis etc., cancer research 43,716-720 (1983).In fact, the super live body dye of positive ion is relevant with a specific character of cancerous cell with the reservation in the cancerous cell mitochondrion to the selected marker of cancerous cell with other super live body cation marking agent, it seems that this characteristic be the reason that causes growth of cancerous cell quick clone and transfer ability, promptly, the mitochondrial high potential of cancerous cell is the source of cellular energy, is the driving force that cell produces ATP (adenosine triphosphate).
Summary of the invention
We have now found that and a kind ofly detect the epithelial method of cancer in vivo by selected marker cancer epithelial cell mitochondrion.
On the other hand, we find a kind of under the situation that normal cell exists the Therapeutic Method of selective killing cancerous cell.
Our detection method comprises the following step, cation supravital mitochondrial marking agent is delivered to the tissue in site, the suspicious carcinous position of epithelium (it comprises normal and carcinous cell), thereby causes that described marking agent is absorbed also selective retention in the mitochondrion of cancerous cell.Detect cancerous cell by any suitable method then, for example, under visible light or selected black light, carry out instrument or visual inspection.
In yet another embodiment, marking agent uses rinse reagent in the site at suspicious carcinous position after being absorbed by mitochondrion, thereby improves the speed that marking agent discharges from the normal cell mitochondrion, and then increases the selectivity of diagnostic method.
According to another important embodiment of the present invention, we provide a kind of selective killing cancer epithelial method, comprise the following steps: with cation supravital mitochondrial marking agent at the site at suspicious carcinous position contact cancerous cell, to cause cell death or cancerous cell can not be bred basically.Can single discontinuous dosage, or continuously, or multiple discontinuous dosage is delivered to cancerous cell with marking agent, uses or do not use rinse reagent after each administration.
In another embodiment of the present invention, we provide a kind of method that improves the selectivity of cancer chemotherapeutic agents and kill and wound the cancerous cell ability, comprise the following steps: or (1) forms the product of super in vivo marker agent of cation and chemotherapeutant, and product is delivered to carcinous epithelial cell, perhaps (2) combine the super in vivo marker agent of cation with the cancer chemotherapeutics agent, by add and or synergy, or the two has concurrently, improves the selectivity and the kill capability of chemotherapeutant.
Of the present invention these, other and further embodiment is apparent to one skilled in the art, can understand the present invention better by the following example.Embodiment is used to illustrate the present invention, rather than to the qualification of its scope, scope of the present invention is only limited by claim.
In practice of the present invention and following operation embodiment, cation supravital mitochondrial marking agent comprises
Dyestuff comprises blutene, alcian blue, peacock green, phenosafraine, acriflavine, Pyronine Y, C.I. 49410. and viride nitens;
With
" non--dyestuff " chemical compound comprises peonidin, oxythiamine, tiemonium iodide, elliptinium acetate and furazolium chloride.
The present preferred embodiment according to the present invention, preferred mitochondrial marking agent is oxazines and thiazin dyes.Thiazin dyes is particularly preferred, and blutene especially, reddish black A, reddish black B and ring thereof replace and N-replacement analog.
In order to be absorbed by selectivity and to be retained in the cancerous cell mitochondrion, the product of marking agent or marking agent+chemotherapeutant must have and is lower than about 5,000 molecular weight.In addition because the selected marker of various closely-related analog and the significant difference of therapeutic activity, the molecular structure of marking agent seem appreciable impact its under the situation that normal living cells exists, selected marker and/or kill and wound the ability of cancerous cell alive.The difference of these cell markings and kill capability is relevant with the construction features of marking agent molecule, for example, type and position that the ring-replacement of marking agent molecule and N-replace, it relates to a kind of, multiple or whole in the following mechanism of action:
1. the structure of marking agent molecule, for example, ring and N-replace on the cationic molecule position and character, influence the effectiveness of positive charge and hinder marking agent or its " accumulation " group by on the mitochondrial membrane or the attraction of the negative charge of mitochondrion inside.
2. the structure of marking agent molecule inserts it into the cancerous cell mitochondrial DNA or along the outside " accumulation " of cancerous cell mitochondrial DNA.
3. the structure of marking agent molecule makes itself or its stacked group combine with activity specific site in the mitochondrion, for example with 4 species specificity protein binding, and/or with the cardiolipin of mitochondrial membrane inner surface precipitation.
4. its reduction potential of the structure influence of marking agent and become the trend of uncharged " colourless " form.
5. its acidity of the structure influence of marking agent (pKa), and, the cation marking agent takes off proton under physiological pH ability influenced again.Like this, the dyestuff of cationic form can attracted to the outer surface of mitochondrial membrane, on mitochondrial membrane, dye cations may be lost proton and be followed and lose its positive charge, discharge the dyestuff of neutral form thus, it can more easily see through the non-polar substrate of film, and is entered mitochondrion inside.
Mechanism 1 dyestuff-film), the intermolecular interaction of 2 (dyestuff-base pair or dyestuff-dyestuffs) and 3 (dyestuff-protein or dyestuff-lipids) depends on the hydrophobicity-lipotropy of dyestuff, this character can be assessed by the whole bag of tricks, a kind of method wherein is assessment aqueous solution and low polar organic solvent, as the partition coefficient between the 1-capryl alcohol (that is log P value).Mechanism 4 and 5 depends on hydrophobicity-lipotropy, and this is because reactant and product turn usefulness at the different solvents of reduction potential (oxidised form is to the reduction form) and pKa (neutral form is to charged form).For example, with respect to secondary amine (R
2NH
2 +), hydrophobicity hinders protonated uncle's fatty amine (R
3NH
+) solvation, reduce their acidity thus.
The present preferred embodiment according to the present invention is used from-1.0 to 5 the super in vivo marker agent of cation approximately of log P value.
Provide the following example so that those skilled in the art understands and realizes the present invention, and determine present preferred embodiment.These embodiment only are used for the purpose of explanation, and do not limit the scope of the invention, and scope of the present invention only limits by additional claim.
Embodiment 1
The absorption of mitochondrial marking agent and reservation in the live body cancerous cell
Containing 20% hyclone, the 1mM glutamine, hydrocortisone, insulin, siderophillin, estradiol, in the RPMI culture medium of selenium and growth hormone, the various cation marking agents of preparation 100,50,10 and 1 μ g/ml variable concentrations.
With each marking agent and concentration, has 5%CO
2In tissue culture's calorstat of 95% relative humidity, 37EC cultivated cancerous cell 5 minutes, used twice of the method rinsing of hatching with 1% acetic acid 2 minutes then.After cultivation and the rinsing, at the 30th minute, 1 hour, 2 hours, 4 hours and 8 hours collecting cells.Use 2-butyl alcohol extraction cell then, marking agent is carried out quantitative analysis with spectrophotography.
The result shows that accumulation rate and the marking agent of marking agent in cancerous cell and normal cell mitochondrion has concentration dependent from the selectivity that cancerous cell discharges, but this concentration dependent begins not too obvious.The saturated concentration of blutene appears at 10 μ g/ml and above concentration.Can determine the saturated concentration of other marking agent similarly.Except as otherwise noted, the reservation of blutene experiment is carried out in the concentration of 10 μ g/ml, and the reservation experiment of other marking agent is carried out in the saturated concentration of determining in this way.
Embodiment 2
The mitochondrion location of marking agent in living cells
After cultivation and the various cell lines of rinsing, use different cation marking agents, by using the mitochondrion location of burnt high resolution microscope of copolymerization and the agent of phase contrast microscope evaluation of markers.
In the complete growth medium that comprises 20% hyclone and somatomedin, cultivate living cells, and keep at 37EC.These cells gather in mitochondrion and the retention marker agent.When these cells were grown in the culture medium that does not contain marking agent subsequently, the agent of cancerous cell retention marker surpassed about 1 hour, but normal epithelium cell release mark agent in about 15 minutes.
Compare dead cell and lose mitochondrion dyeing and in nucleus, gather marking agent with the cell that the marking agent of dissipation line mitochondrial membrane potential was handled with living cells.
Embodiment 3
Marking agent discharges from the mitochondrion of mitochondrial membrane potential loss
With the known agent pretreatment epithelial cancer cells that changes the mitochondrion current potential, then handle with cation supravital mitochondrial marking agent.These pretreating reagents comprise azide and cyanide preparation and dinitrophenol,DNP.
Epithelial cancer cells through various dyestuff pre-stainings, carries out post processing with these known agent equally then.Analyze dyestuff to his subcellular fraction at interval, comprise to nuclear transfer from the release of cell or dyestuff.
Can not in mitochondrion, gather dyestuff with the pretreated cell of these reagent, carry out post processing, the mitochondrion released dye of pre-staining cell with these reagent.
Embodiment 4
Squamous cell carcinoma organize explant
The marking agent of analyzing the fresh explant of epithelial cancer of excision absorbs and reservation.After the excision,, be cut into 3mm section from surrounding tissue microdissection carcinoma, and as explantation tissue's culture 37 ℃ of cultivations.These explants and various marking agent are hatched then, extract then to quantize marking agent.
Oral cancer (SqCHN) fast Absorption marking agent and the reservation that prolongs these marking agents.After cultivating about 1 hour in not having the culture medium of marking agent, marking agent begins to discharge from cell.Yet when cell is not containing somatomedin, when cultivating in the culture medium of hyclone and other culture medium additives, marking agent discharges sooner.When cell was grown under unfavorable conditions such as low temperature, marking agent also discharged quickly.
Embodiment 5
Normal epithelium cell organize explant
The cell that obtains from the oral epithelium normal region with surgical method is used as the cultivation of normal epithelial culture.These cultures and marking agent are hatched then, with the absorption and the reservation of evaluation of markers agent.
Different with cancerous cell, normal epithelium cell is from their mitochondrion rapid release marking agent, and discharges sooner from cell.To 10-15 minute, most of marking agents discharged from mitochondrion.
Embodiment 6
Marking agent-chemotherapeutant adduct
Use the marking agent of the adduct alternative embodiment 1-5 of following cation mitochondrial marking agent and various known chemical therapeutic agents, except the selectivity of cancerous cell kill rate and chemotherapeutant improved in fact, the result was similar substantially.
Marking agent
Chemotherapeutant
The blutene methotrexate
The rhodamine chlormethine
Embodiment 7
Adjunvant composition
The following combination of known cancer chemotherapeutics agent and mitochondrial marking agent demonstrate collaborative or add at least and the cancerous cell fragmentation effect:
Chemotherapeutant
The cation marking agent
The paclitaxel toluidine blue
The reddish black A of taxotere
The vincristine alcian blue
The effect of selective therapy
In the following example, according to the United States Patent (USP) 6,086,852 that licensed to Burkett on July 11st, 2000, in disclosed manufacture method prepare the toluidine blue medicine.Use semipreparative HPLC fractional distillation and separate drug component then, production is identified in ' 852 patents, by the analog of peak 5,6,7 and 8 representatives.Chemical compound by peak 7 and 8 representatives is toluidine blue isomery tagma (regioisomers), has cyclohexyl methyl at-2 (peaks 8) and-4 (peaks 7).The chemical compound of peak 5 representatives is the N-demethylation derivatives at peak 7, and the chemical compound of peak 6 representatives is the N-demethylation derivatives at peak 8.
Embodiment 8
Compare with the normal mouth epithelial cells of living, analyze the chemical compound that obtains in the blutene fractional distillation process, the selection toxicity of cancer cell of oral cavity (SqCHN) alive by peak 5,6,7 and 8 representatives.The separation and Culture thing of squamous cancer cell and normal epithelium cell is hatched with different dyestuff fractions, cleans with the culture medium that does not contain dyestuff then.And then in growth medium cultured cell, observe 8 days to determine the degree of cell killing.Compare with about 20% Normocellular killing and wounding only, peak 6 chemical compounds cause the death of 95% cancerous cell.The cancer cell death of peak 8 compound exhibits 89%, and it only causes about 20% Normocellular killing and wounding.Thereby the selective retention of peak 6 and 8 chemical compounds is to have selection toxic to cancerous cell.
Dye of positive ion selectivity enters the interruption that mitochondrion causes the mitochondrion current potential, and this current potential is the source of cellular energy and the driving force that cell produces ATP (adenosine triphosphate).Quick division of cancerous cell and the ability that shifts depend on the effectiveness of this high-energy source.
Yet, unique mechanism that as if effect of electric charge do not related to, because be the dye of positive ion by the chemical compound of peak 5 and peak 7 representatives, they but do not show the selection toxicity to cancer identical with peak 6 and 8 yet.Therefore, it seems that peak 6 and 8 chemical compound cause their selection toxicity to cancerous cell by the molecule performance with other.
Embodiment 9
The treatment characteristic of peak 6 and 8 chemical compounds determines that by further in vivo test the separator of other cancerous cell and normal epithelium cell is used in vivo test, carries out according to the method for embodiment 8.Pilot project comprises other squamous cell carcinomas of head and neck, esophagus, lung, cervix uteri and skin, and the cancer of other type, comprises adenocarcinoma, lymphoma and sarcoma.With taking the in vivo test that the tumor animal carries out " tumor growth retardance " and " tumor regression mensuration ", comprise the carcinoma of implanting Balb-C mice head and neck and lung, to analyze the cylinder therapeutic effect of these chemical compounds.These further external and in vivo tests have confirmed peak 6 and the 8 chemical compounds selection toxicity to more multiple cancerous cell.
So that the mode that those skilled in the art can understand and implement has been described the present invention, and determined the preferred embodiment that the present invention is present.
Claims (2)
1. at-2 toluidine blues with cyclohexyl methyl or-2 the purposes of N-demethylation toluidine blue in the preparation of preparation selective killing squamous cancer cell with cyclohexyl methyl.
2. purposes according to claim 1, wherein said squamous cancer cell are oral squamous carcinoma cell.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| PCT/US2000/005387 WO2001064110A1 (en) | 2000-02-28 | 2000-02-28 | Method for detecting and killing epithelial cancer cells |
| WOPCT/US00/05387 | 2000-02-28 | ||
| USPCT/US00/05387 | 2000-02-28 |
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| CN1365288A CN1365288A (en) | 2002-08-21 |
| CN100544770C true CN100544770C (en) | 2009-09-30 |
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| JP (1) | JP2003525044A (en) |
| KR (2) | KR20080080681A (en) |
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| WO2001064110A1 (en) * | 2000-02-28 | 2001-09-07 | Zila, Inc. | Method for detecting and killing epithelial cancer cells |
| BR0013636A (en) * | 2000-06-30 | 2005-01-11 | Zila Inc | Use of a rhodamine composition for identification and delineation of cancerous epithelial tissue |
| IL149330A0 (en) * | 2000-09-26 | 2002-11-10 | Zila Inc | Method for early prediction of the onset of invasive cancer |
| CA2457407A1 (en) * | 2001-12-14 | 2003-07-17 | Zila, Inc. | Light-directed molecular analysis for cancer prognosis and diagnosis |
| US6929817B2 (en) | 2002-05-14 | 2005-08-16 | National Starch & Chemical Investment Holding Corporation | Slowly digestible starch product |
| US6890571B2 (en) | 2002-05-14 | 2005-05-10 | National Starch And Chemical Investment Holding Corporation | Slowly digestible starch product |
| US7081261B2 (en) * | 2002-05-14 | 2006-07-25 | National Starch And Chemical Investment Holding Corporation | Resistant starch prepared by isoamylase debranching of low amylose starch |
| JP2005535605A (en) * | 2002-06-04 | 2005-11-24 | ジラ バイオテクノロジー・インク | Toluidine blue O drug and its in vivo staining of dysplastic tissue and its use in chemotherapy treatment |
| CA2583427A1 (en) * | 2004-09-30 | 2006-04-20 | Zila Biotechnology, Inc. | Light-directed method for detecting and aiding further evaluation of abnormal mucosal tissue |
| EP1835929B8 (en) | 2005-01-06 | 2016-07-27 | Novo Nordisk A/S | Anti-kir combination treatments and methods |
| CN105510321A (en) * | 2011-12-29 | 2016-04-20 | 闫文广 | Detection agent composition for epithelial tissue tumor cells and preparing method thereof |
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| US4321251A (en) * | 1979-12-19 | 1982-03-23 | The United States Of America As Represented By The Department Of Health And Human Services | Detection of malignant lesions of the oral cavity utilizing toluidine blue rinse |
| US5194373A (en) * | 1985-06-06 | 1993-03-16 | Thomas Jefferson University | Method of determining endothelial cell coverage of a prosthetic surface |
| US4816395A (en) * | 1985-12-19 | 1989-03-28 | Peralta Cancer Research Institute | Method for predicting chemosensitivity of anti-cancer drugs |
| DE69229471T2 (en) * | 1991-10-31 | 1999-11-25 | Zila Inc | BIOLOGICAL DYE COMPOSITION, PROCESS FOR ITS PRODUCTION AND USE FOR MARKING THE OUTLINE OF EPITHELIC CARSINOMAS |
| CA2214829C (en) * | 1996-01-16 | 2002-05-14 | Edwin Pomerantz | Methods and compositions for in-vivo detection of oral cancers and precancerous conditions |
| US6086852A (en) * | 1997-11-13 | 2000-07-11 | Zila, Inc. | In vivo stain composition, process of manufacture, and methods of use to identify dysplastic tissue |
| WO2000038730A2 (en) * | 1998-12-23 | 2000-07-06 | G.D. Searle & Co. | Use of a cyclooxygenase-2 inhibitor and one or more antineoplastic agents for combination therapy in neoplasia |
| WO2001064110A1 (en) * | 2000-02-28 | 2001-09-07 | Zila, Inc. | Method for detecting and killing epithelial cancer cells |
| US6967015B1 (en) * | 2000-07-20 | 2005-11-22 | Zila, Inc. | Diagnostic method for detecting dysplastic epithelial tissue |
| IL149330A0 (en) * | 2000-09-26 | 2002-11-10 | Zila Inc | Method for early prediction of the onset of invasive cancer |
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| NO20015242D0 (en) | 2001-10-26 |
| AU785489B2 (en) | 2007-11-15 |
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| IL146141A0 (en) | 2002-07-25 |
| KR20020000222A (en) | 2002-01-05 |
| NO20015242L (en) | 2001-11-29 |
| EP1214101A1 (en) | 2002-06-19 |
| MXPA01010886A (en) | 2002-05-06 |
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| EP1214101A4 (en) | 2005-04-13 |
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