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WO2001064110A1 - Method for detecting and killing epithelial cancer cells - Google Patents

Method for detecting and killing epithelial cancer cells Download PDF

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Publication number
WO2001064110A1
WO2001064110A1 PCT/US2000/005387 US0005387W WO0164110A1 WO 2001064110 A1 WO2001064110 A1 WO 2001064110A1 US 0005387 W US0005387 W US 0005387W WO 0164110 A1 WO0164110 A1 WO 0164110A1
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WO
WIPO (PCT)
Prior art keywords
cells
agent
marking
cancer cells
agents
Prior art date
Application number
PCT/US2000/005387
Other languages
French (fr)
Inventor
Samuel D. Bernal
Douglas D. Burkett
Ralph E. Green
Original Assignee
Zila, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Zila, Inc. filed Critical Zila, Inc.
Priority to AU2000237154A priority Critical patent/AU2000237154A1/en
Priority to US09/673,991 priority patent/US6649144B1/en
Priority to PCT/US2000/005387 priority patent/WO2001064110A1/en
Priority to CNB018006604A priority patent/CN100544770C/en
Priority to CA002370741A priority patent/CA2370741A1/en
Priority to PCT/US2001/006318 priority patent/WO2001064255A1/en
Priority to BR0104747-7A priority patent/BR0104747A/en
Priority to AU43316/01A priority patent/AU785489B2/en
Priority to MXPA01010886A priority patent/MXPA01010886A/en
Priority to KR1020087020377A priority patent/KR20080080681A/en
Priority to CZ20013861A priority patent/CZ20013861A3/en
Priority to EP01916271A priority patent/EP1214101A4/en
Priority to RU2001132076/15A priority patent/RU2226404C2/en
Priority to IL14614101A priority patent/IL146141A0/en
Priority to KR1020017013731A priority patent/KR100907122B1/en
Priority to JP2001563152A priority patent/JP2003525044A/en
Priority to NZ515202A priority patent/NZ515202A/en
Publication of WO2001064110A1 publication Critical patent/WO2001064110A1/en
Priority to NO20015242A priority patent/NO20015242L/en

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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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • 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
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • This invention relates to methods for detecting epithelial cancer.
  • the invention pertains to methods for selectively killing epithelial cancer cells.
  • 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 retains a mitochondrial marking agent for a time sufficient to permit identification and/or killing such cells.
  • 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 .
  • Adduct means the reaction product of a mitochondrial marking agent and a cancer chemotherapeutic agent.
  • 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.
  • 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.
  • cationic dyes e.g., dyes such as rhodamine, fluoresceins , oxazine and thiazine dyes (including toluidine blue) and other cationic supravital marking agents
  • mitochondria is apparently due to the higher electrical potential (negative charge on the inside of the membrane) of cancerous mitochondrial cells as compared to normal cells. See, e.g., Chen et al . f Cancer Cells 1/The Transformed Phenotype, 75-85 (Cold Spring Harbor Laboratory, 1984); Lampidis, et al . , Cancer Research 43, 716-720 (1983).
  • the selective marking and retention of the mitochondria of cancer cells by 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 ) product of the cells.
  • ATP adenosine triphosphate
  • my method comprises the steps of delivering to tissue in the locus of a suspect cancerous site on the epithelium (which contains both normal and cancerous cells), with a cationic supravital mitochondrial marking agent other than rhodamine, causing said agent to be taken up and selectively retained in the mitochondria of 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.
  • 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 method.
  • I 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 after each dose.
  • I provide a method of improving the selectivity of cancer chemotherapeutic agents comprising the steps of forming an reaction product of a cationic supravital agent and a chemotherapeutic agent and delivering the reaction product to cancerous epithelial cells.
  • cationic supravital mitochondrial marking agents including
  • dyes including toluidine blue 0, alcian blue, malachite green, phenosafranin, acriflavine, pyronine Y, toluylene blue and brilliant green;
  • the marking agent or reaction product of marking agent + chemotherapeutic agent 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.
  • Different concentrations of the various cationic marking agents at 100, 50, 10 and 1 ⁇ g/ml are prepared in RPMI medium complete with 20% fetal calf serum, 1 mM glutamine, hydrocortisone, insulin, transferrin, estradiol, selenium and growth hormone.
  • carcinoma cells are incubated at 37°C in tissue culture incubators with 5% C0 2 and 95% relative humidity, for 5 minutes with each agent and concentration there and then rinsed twice using 2 minute incubations with 1%
  • 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 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 lO ⁇ g/ml and above.
  • the saturation concentrations for the other marking agents are similarly determined.
  • the remaining experiments are conducted with a concentration of lO ⁇ g/ml for toluidine blue O and at the saturation concentrations for the other marking agents so-determined, unless stated otherwise.
  • 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 20% fetal calf serum and growth factors, and maintained at 37°C. These cells accumulate and retain the marking agents in the mitochondria. When these cells are then maintained in a 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.
  • 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.
  • the cells pretreated with these agents did not accumulate dyes in the mitochondria and the mitochondria of the pre-stained cells released the dye upon post- treatment with these agents.
  • 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.
  • 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 substantially improved.

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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

METHOD FOR DETECTING AND KILLING EPITHELIAL 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 retains a mitochondrial marking agent for a time sufficient to permit identification and/or killing such cells.
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 death or cell changes that render a cell incapable of reproduction and metastasizing.
"Adduct" means the reaction product of a mitochondrial marking agent and a cancer chemotherapeutic 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 stained tissue. Such procedures are disclosed, for example in the patents to Tucci et al. (U.S. Patent 5,372,801) and Mashberg (U.S. Patent 4,321,251). Use of certain other thiazine dyes and oxazine dyes in an analogous manner is disclosed in U.S. Patent 5,882,627 o Pomerantz.
Heretofore, it was theorized that such dyes selectively "marked" cancerous tissue because it was retained in the relatively larger interstitial spaces between the cells of cancerous tissue and would not efficiently penetrate the normally tight 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. In turn, this selective staining of and retention in the
mitochondria is apparently due to the higher electrical potential (negative charge on the inside of the membrane) of cancerous mitochondrial cells as compared to normal cells. See, e.g., Chen et al . f 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 and retention of the mitochondria of cancer cells by 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 ) product of the cells.
Summary of the Invention
I have now discovered a method for in-vivo detection of cancerous epithelial cells by selective marking of the mitochondria thereof. My method comprises the steps of delivering to tissue in the locus of a suspect cancerous site on the epithelium (which contains both normal and cancerous cells), with a cationic supravital mitochondrial marking agent other than rhodamine, causing said agent to be taken up and selectively retained in the mitochondria of 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 method.
According to another embodiment of the invention, I 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 after each dose. In a further embodiment of the invention, I provide a method of improving the selectivity of cancer chemotherapeutic agents comprising the steps of forming an reaction product of a cationic supravital agent and a chemotherapeutic agent and delivering the reaction product to cancerous epithelial cells.
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 following working examples, cationic supravital mitochondrial marking agents, including
dyes, including toluidine blue 0, 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.
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.
Example 1
Uptake and Retention of Mitochondrial Marking Agents in Living Carcinoma Cells
Different concentrations of the various cationic marking agents, at 100, 50, 10 and 1 μg/ml are prepared in RPMI medium complete with 20% fetal calf serum, 1 mM glutamine, hydrocortisone, insulin, transferrin, estradiol, selenium and growth hormone.
The carcinoma cells are incubated at 37°C in tissue culture incubators with 5% C02 and 95% relative humidity, for 5 minutes with each agent and concentration there and then rinsed twice using 2 minute incubations with 1%
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 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 lOμg/ml and above. The saturation concentrations for the other marking agents are similarly determined. The remaining experiments are conducted with a concentration of lOμg/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 20% fetal calf serum and growth factors, and maintained at 37°C. These cells accumulate and retain the marking agents in the mitochondria. When these cells are then maintained in a 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 Dissipation 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.
The cells pretreated with these agents did not accumulate dyes in the mitochondria and the mitochondria of the pre-stained cells released the dye upon post- treatment with these agents.
Example 4 Tissue Explants of Sguamous 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.
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 substantially improved.
Marking Agent Chemotherapeutic Agent toluidine blue 0 methotrexate rhodamine 123 nitrogen mustard

Claims

CLAIMS :Having described my 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, I CLAIM:
1. A diagnostic method for in vivo detection of cancerous eptithelial cells by selective marking of the mitochondria thereof, comprising the step of delivering to the epithelium a cationic supravital mitochondrial marking agent.
2. A method for selective killing of epithelial cancer cells comprising the step of delivering to epithelial cancer cells a cationic supravital mitochondrial marking agent.
3. The method of claim 2 in which the agent is the reaction product of a cationic supravital mitochondrial marking agent and a cancer chemotherapeutic drug.
PCT/US2000/005387 2000-02-28 2000-02-28 Method for detecting and killing epithelial cancer cells WO2001064110A1 (en)

Priority Applications (18)

Application Number Priority Date Filing Date Title
AU2000237154A AU2000237154A1 (en) 2000-02-28 2000-02-28 Method for detecting and killing epithelial cancer cells
US09/673,991 US6649144B1 (en) 2000-02-28 2000-02-28 Method for detecting and killing epithelial cancer cells
PCT/US2000/005387 WO2001064110A1 (en) 2000-02-28 2000-02-28 Method for detecting and killing epithelial cancer cells
KR1020087020377A KR20080080681A (en) 2000-02-28 2001-02-27 How to detect and kill epithelial cancer cells
CZ20013861A CZ20013861A3 (en) 2000-02-28 2001-02-27 Use of cationic supravital mitochondrial marker for detection and control of epithelial cancer cells
PCT/US2001/006318 WO2001064255A1 (en) 2000-02-28 2001-02-27 Method for detecting and killing epithelial cancer cells
BR0104747-7A BR0104747A (en) 2000-02-28 2001-02-27 Use of cationic supravital mitochondrial marking agent in the detection and extinction of cancerous epithelial cells
AU43316/01A AU785489B2 (en) 2000-02-28 2001-02-27 Method for detecting and killing epithelial cancer cells
MXPA01010886A MXPA01010886A (en) 2000-02-28 2001-02-27 Method for detecting and killing epithelial cancer cells.
CNB018006604A CN100544770C (en) 2000-02-28 2001-02-27 The method of detection and killing epithelial cancer cells
CA002370741A CA2370741A1 (en) 2000-02-28 2001-02-27 Method for detecting and killing epithelial cancer cells
EP01916271A EP1214101A4 (en) 2000-02-28 2001-02-27 Method for detecting and killing epithelial cancer cells
RU2001132076/15A RU2226404C2 (en) 2000-02-28 2001-02-27 Method for detecting and eliminating epithelial cancer cells
IL14614101A IL146141A0 (en) 2000-02-28 2001-02-27 Method for detecting and killing epithelial cancer cells
KR1020017013731A KR100907122B1 (en) 2000-02-28 2001-02-27 How to detect and kill epithelial cancer cells
JP2001563152A JP2003525044A (en) 2000-02-28 2001-02-27 Methods for detecting and eliminating epithelial cancer cells
NZ515202A NZ515202A (en) 2000-02-28 2001-02-27 Method for detecting and killing epithelial cancer cells
NO20015242A NO20015242L (en) 2000-02-28 2001-10-26 Method for detecting and killing epithelial cancer cells

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JP (1) JP2003525044A (en)
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AU (2) AU2000237154A1 (en)
BR (1) BR0104747A (en)
CA (1) CA2370741A1 (en)
CZ (1) CZ20013861A3 (en)
IL (1) IL146141A0 (en)
MX (1) MXPA01010886A (en)
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EP1214101A1 (en) * 2000-02-28 2002-06-19 Zila, Inc. Method for detecting and killing epithelial cancer cells
EP1294408A1 (en) * 2000-06-30 2003-03-26 Zila, Inc. Rhodamine diagnostic agent and diagnostic methods for detection of epithelial cancer
AU2002367731B2 (en) * 2001-12-14 2008-11-13 Zila Biotechnology, Inc. Stain-directed molecular analysis for cancer prognosis and diagnosis
US7659057B2 (en) * 2000-09-26 2010-02-09 Zila Biotechnology, Inc. Stain-directed molecular analysis for cancer prognosis and diagnosis
EP2446897A1 (en) 2005-01-06 2012-05-02 Novo Nordisk A/S Anti-KIR combination treatments and methods

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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
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
EP1534346A4 (en) * 2002-06-04 2007-07-25 Zila Inc Toluidine blue o drug substance and use thereof for in vivo staining and chemotherapeutic treatment of dysplastic tissues
EP1799097A4 (en) * 2004-09-30 2009-05-06 Zila Biotechnology Inc Light-directed method for detecting and aiding further evaluation of abnormal mucosal tissue
CN102497868A (en) 2009-07-15 2012-06-13 N.V.努特里奇亚 Blend of non-digestible oligosaccharides to stimulate the immune system
CN103185713B (en) * 2011-12-29 2015-12-09 闫文广 For the detection agent composition and method of making the same of epithelial neoplasms cell

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AU2000237154A1 (en) 2001-09-12
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