CA2383697A1 - Rhodamine diagnostic agent and diagnostic methods for detection of epithelial cancer - Google Patents
Rhodamine diagnostic agent and diagnostic methods for detection of epithelial cancer Download PDFInfo
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- CA2383697A1 CA2383697A1 CA002383697A CA2383697A CA2383697A1 CA 2383697 A1 CA2383697 A1 CA 2383697A1 CA 002383697 A CA002383697 A CA 002383697A CA 2383697 A CA2383697 A CA 2383697A CA 2383697 A1 CA2383697 A1 CA 2383697A1
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- rhodamine
- tissue
- cancerous
- epithelium
- diagnostic
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- PYWVYCXTNDRMGF-UHFFFAOYSA-N rhodamine B Chemical compound [Cl-].C=12C=CC(=[N+](CC)CC)C=C2OC2=CC(N(CC)CC)=CC=C2C=1C1=CC=CC=C1C(O)=O PYWVYCXTNDRMGF-UHFFFAOYSA-N 0.000 title claims description 18
- 238000001514 detection method Methods 0.000 title claims description 5
- 238000002405 diagnostic procedure Methods 0.000 title claims description 5
- 201000009030 Carcinoma Diseases 0.000 title claims description 4
- 229940039227 diagnostic agent Drugs 0.000 title description 3
- 239000000032 diagnostic agent Substances 0.000 title description 3
- 239000000203 mixture Substances 0.000 claims abstract description 33
- 238000000034 method Methods 0.000 claims abstract description 17
- 210000000981 epithelium Anatomy 0.000 claims abstract description 12
- 210000001519 tissue Anatomy 0.000 claims description 13
- HNONEKILPDHFOL-UHFFFAOYSA-M tolonium chloride Chemical compound [Cl-].C1=C(C)C(N)=CC2=[S+]C3=CC(N(C)C)=CC=C3N=C21 HNONEKILPDHFOL-UHFFFAOYSA-M 0.000 claims description 11
- 238000001727 in vivo Methods 0.000 claims description 7
- 230000000717 retained effect Effects 0.000 claims description 4
- 239000003125 aqueous solvent Substances 0.000 claims description 2
- 230000009841 epithelial lesion Effects 0.000 claims description 2
- 239000007800 oxidant agent Substances 0.000 claims description 2
- 239000001022 rhodamine dye Substances 0.000 abstract description 4
- 239000000975 dye Substances 0.000 description 17
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 10
- 229950003937 tolonium Drugs 0.000 description 7
- 239000000243 solution Substances 0.000 description 6
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 5
- 229960000583 acetic acid Drugs 0.000 description 4
- 239000002904 solvent Substances 0.000 description 4
- 238000010186 staining Methods 0.000 description 4
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 3
- 235000019441 ethanol Nutrition 0.000 description 3
- 238000000338 in vitro Methods 0.000 description 3
- 230000007170 pathology Effects 0.000 description 3
- 230000000007 visual effect Effects 0.000 description 3
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 2
- 230000001413 cellular effect Effects 0.000 description 2
- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 description 2
- 239000012362 glacial acetic acid Substances 0.000 description 2
- 208000012987 lip and oral cavity carcinoma Diseases 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 210000000214 mouth Anatomy 0.000 description 2
- 231100000252 nontoxic Toxicity 0.000 description 2
- 230000003000 nontoxic effect Effects 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 239000008213 purified water Substances 0.000 description 2
- 150000003839 salts Chemical class 0.000 description 2
- 210000004881 tumor cell Anatomy 0.000 description 2
- BDKLKNJTMLIAFE-UHFFFAOYSA-N 2-(3-fluorophenyl)-1,3-oxazole-4-carbaldehyde Chemical compound FC1=CC=CC(C=2OC=C(C=O)N=2)=C1 BDKLKNJTMLIAFE-UHFFFAOYSA-N 0.000 description 1
- ZCYVEMRRCGMTRW-UHFFFAOYSA-N 7553-56-2 Chemical compound [I] ZCYVEMRRCGMTRW-UHFFFAOYSA-N 0.000 description 1
- 101100309716 Arabidopsis thaliana SD18 gene Proteins 0.000 description 1
- 206010058314 Dysplasia Diseases 0.000 description 1
- 208000000461 Esophageal Neoplasms Diseases 0.000 description 1
- 102000010834 Extracellular Matrix Proteins Human genes 0.000 description 1
- 108010037362 Extracellular Matrix Proteins Proteins 0.000 description 1
- 206010061523 Lip and/or oral cavity cancer Diseases 0.000 description 1
- 208000003445 Mouth Neoplasms Diseases 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 206010030155 Oesophageal carcinoma Diseases 0.000 description 1
- 208000025157 Oral disease Diseases 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 125000002091 cationic group Chemical group 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 210000002421 cell wall Anatomy 0.000 description 1
- 210000003850 cellular structure Anatomy 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000002512 chemotherapy Methods 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- -1 e.g. Chemical compound 0.000 description 1
- 201000004101 esophageal cancer Diseases 0.000 description 1
- 150000002148 esters Chemical class 0.000 description 1
- 230000001747 exhibiting effect Effects 0.000 description 1
- 210000002744 extracellular matrix Anatomy 0.000 description 1
- 238000010562 histological examination Methods 0.000 description 1
- 150000003840 hydrochlorides Chemical class 0.000 description 1
- 206010020718 hyperplasia Diseases 0.000 description 1
- 238000012750 in vivo screening Methods 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 229910052740 iodine Inorganic materials 0.000 description 1
- 239000011630 iodine Substances 0.000 description 1
- 230000003902 lesion Effects 0.000 description 1
- QTWZICCBKBYHDM-UHFFFAOYSA-N leucomethylene blue Chemical compound C1=C(N(C)C)C=C2SC3=CC(N(C)C)=CC=C3NC2=C1 QTWZICCBKBYHDM-UHFFFAOYSA-N 0.000 description 1
- 229920002521 macromolecule Polymers 0.000 description 1
- 210000004379 membrane Anatomy 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 230000003458 metachromatic effect Effects 0.000 description 1
- 210000003470 mitochondria Anatomy 0.000 description 1
- 210000002200 mouth mucosa Anatomy 0.000 description 1
- 150000007523 nucleic acids Chemical group 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 150000004893 oxazines Chemical class 0.000 description 1
- 238000010422 painting Methods 0.000 description 1
- 235000019629 palatability Nutrition 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- 230000000737 periodic effect Effects 0.000 description 1
- MYFATKRONKHHQL-UHFFFAOYSA-N rhodamine 123 Chemical compound [Cl-].COC(=O)C1=CC=CC=C1C1=C2C=CC(=[NH2+])C=C2OC2=CC(N)=CC=C21 MYFATKRONKHHQL-UHFFFAOYSA-N 0.000 description 1
- 235000017281 sodium acetate Nutrition 0.000 description 1
- 239000007974 sodium acetate buffer Substances 0.000 description 1
- 229940087562 sodium acetate trihydrate Drugs 0.000 description 1
- BHZOKUMUHVTPBX-UHFFFAOYSA-M sodium acetic acid acetate Chemical compound [Na+].CC(O)=O.CC([O-])=O BHZOKUMUHVTPBX-UHFFFAOYSA-M 0.000 description 1
- WXMKPNITSTVMEF-UHFFFAOYSA-M sodium benzoate Chemical compound [Na+].[O-]C(=O)C1=CC=CC=C1 WXMKPNITSTVMEF-UHFFFAOYSA-M 0.000 description 1
- 239000004299 sodium benzoate Substances 0.000 description 1
- 235000010234 sodium benzoate Nutrition 0.000 description 1
- 210000004895 subcellular structure Anatomy 0.000 description 1
- 230000001360 synchronised effect Effects 0.000 description 1
- 231100000057 systemic toxicity Toxicity 0.000 description 1
- 239000001016 thiazine dye Substances 0.000 description 1
- 230000000699 topical effect Effects 0.000 description 1
- AURFVNDXGLQSNN-UHFFFAOYSA-K trisodium 2-hydroxypropane-1,2,3-tricarboxylic acid phosphate Chemical compound [Na+].[Na+].[Na+].[O-]P([O-])([O-])=O.OC(=O)CC(O)(C(O)=O)CC(O)=O AURFVNDXGLQSNN-UHFFFAOYSA-K 0.000 description 1
- XPFJYKARVSSRHE-UHFFFAOYSA-K trisodium;2-hydroxypropane-1,2,3-tricarboxylate;2-hydroxypropane-1,2,3-tricarboxylic acid Chemical compound [Na+].[Na+].[Na+].OC(=O)CC(O)(C(O)=O)CC(O)=O.[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O XPFJYKARVSSRHE-UHFFFAOYSA-K 0.000 description 1
Classifications
-
- 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
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/001—Preparation for luminescence or biological staining
- A61K49/0013—Luminescence
- A61K49/0017—Fluorescence in vivo
- A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
- A61K49/0021—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent group being a small organic molecule
- A61K49/0041—Xanthene dyes, used in vivo, e.g. administered to a mice, e.g. rhodamines, rose Bengal
-
- 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
- A61K49/0063—Preparation for luminescence or biological staining characterised by a special physical or galenical form, e.g. emulsions, microspheres
- A61K49/0069—Preparation for luminescence or biological staining characterised by a special physical or galenical form, e.g. emulsions, microspheres the agent being in a particular physical galenical form
- A61K49/0071—Preparation for luminescence or biological staining characterised by a special physical or galenical form, e.g. emulsions, microspheres the agent being in a particular physical galenical form solution, solute
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Veterinary Medicine (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Epidemiology (AREA)
- Physics & Mathematics (AREA)
- Biodiversity & Conservation Biology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Oncology (AREA)
- Optics & Photonics (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
Rhodamine dye compositions and methods for detecting and/or delineating cancerous and precancerous epithelial tissue.
Description
RHODAMINE DIAGNOSTIC AGENT
AND DIAGNOSTIC METHODS FOR DETECTION
OF EPITHELIAL CANCER
This invention relates to a novel diagnostic agent for detection of cancerous and precancerous epithelial tissue.
According to another aspect, the invention pertains to novel methods for detecting and/or delineating cancerous and/or precancerous tissue of the epithelium.
In another respect the invention concerns to such diagnostic procedures and agents useful therein which are especially useful for in vivo screening of patients for possible oral cancer as part of routine dentist's or physician's examinations or procedures, such as periodic dental or physical examinations, dental cleaning, etc.
In yet another aspect the invention relates to such procedures and compositions useful therein, which use dye stains that are more readily available and/or less expensive and less complicated to synthesize and/or purify than the dyes employed in prior art procedures.
AND DIAGNOSTIC METHODS FOR DETECTION
OF EPITHELIAL CANCER
This invention relates to a novel diagnostic agent for detection of cancerous and precancerous epithelial tissue.
According to another aspect, the invention pertains to novel methods for detecting and/or delineating cancerous and/or precancerous tissue of the epithelium.
In another respect the invention concerns to such diagnostic procedures and agents useful therein which are especially useful for in vivo screening of patients for possible oral cancer as part of routine dentist's or physician's examinations or procedures, such as periodic dental or physical examinations, dental cleaning, etc.
In yet another aspect the invention relates to such procedures and compositions useful therein, which use dye stains that are more readily available and/or less expensive and less complicated to synthesize and/or purify than the dyes employed in prior art procedures.
In yet another respect, the invention concerns such in vivo procedures and compositions employing a dye which, despite prior art teachings otherwise, is sufficiently non-toxic that it can be employed by rinsing the entire oral cavity and/or gargling.
In-vivo diagnostic procedures for detecting premalignant epithelial lesions, such as oral lesions and oral carcinomas, employing dye compositions that are selectively retained by tissues rendered abnormal by dysplasia, hyperplasia, tumoriegnesis and other active surface lesions, are known in the art. For example, procedures employing fluorescein or 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 visual examination under ultraviolet light to detect cancerous/precancerous tissue, which is selectively fluorescent.
Another prior art procedure involves in vivo application by rinsing with toluidine blue O, followed by normal visual examination to detect any selectively stained tissue. Such procedures are disclosed, for example, in U.S. Patent 5,372,801 to Tucci, et al, and in U.S. Patent 4,321,251 to Mashberg. Toluidine blue has been used for decades as a histopathological stain for in-vitro use. Through this use it has become known as a metachromatic dye, staining nuclei rich in DNA and RNA a purple to pink color. The inherent deep blue color of toluidine blue 0 is changed to purple or pink when the dye is bound to nucleic acid or other acidic cellular macromolecules. Of course, this type of staining is dependent on the dye gaining access to internal subcellular structures such as the nucleus. Such access is readily obtained only by "fixing" a tissue sample with formaldehyde or other reagent that disrupts the cellular membrane without destroying general cellular structure.
In contrast to the mechanism involved in in-vitro use, the staining of oral tissue in vivo by toluidine blue 0 is due to its ability to penetrate the cell walls and attach to the mitochondria, which retains the dye longer than components of the extracellular matrix.
In-vivo diagnostic procedures for detecting premalignant epithelial lesions, such as oral lesions and oral carcinomas, employing dye compositions that are selectively retained by tissues rendered abnormal by dysplasia, hyperplasia, tumoriegnesis and other active surface lesions, are known in the art. For example, procedures employing fluorescein or 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 visual examination under ultraviolet light to detect cancerous/precancerous tissue, which is selectively fluorescent.
Another prior art procedure involves in vivo application by rinsing with toluidine blue O, followed by normal visual examination to detect any selectively stained tissue. Such procedures are disclosed, for example, in U.S. Patent 5,372,801 to Tucci, et al, and in U.S. Patent 4,321,251 to Mashberg. Toluidine blue has been used for decades as a histopathological stain for in-vitro use. Through this use it has become known as a metachromatic dye, staining nuclei rich in DNA and RNA a purple to pink color. The inherent deep blue color of toluidine blue 0 is changed to purple or pink when the dye is bound to nucleic acid or other acidic cellular macromolecules. Of course, this type of staining is dependent on the dye gaining access to internal subcellular structures such as the nucleus. Such access is readily obtained only by "fixing" a tissue sample with formaldehyde or other reagent that disrupts the cellular membrane without destroying general cellular structure.
In contrast to the mechanism involved in in-vitro use, the staining of oral tissue in vivo by toluidine blue 0 is due to its ability to penetrate the cell walls and attach to the mitochondria, which retains the dye longer than components of the extracellular matrix.
The Mashberg procedure involves application of the toluidine blue 0 solution as a rinse of the entire oral cavity, with gargling, followed by rinses with water and acetic acid to remove dye that is not retained by the cancerous or precancerous tissue. The preliminary diagnosis by the Mashberg procedure is then confirmed by direct application of the toluidine blue 0 composition to the suspect site 10-14 days later. The Tucci '801 patent discloses an improved toluidine blue O composition for use according to the general procedure taught by Mashberg.
Ari in vivo procedure involving use of Lugol's solution (iodine) and toluidine blue 0 was proposed for detecting esophageal cancer synchronous with upper aerodigestive tract cancers in Papazian, Gastroenterologic Clinique et Biologique 9:16-22 (1985).
More recently, Pomerantz U.S. Patent 5,882,627 disclosed a structurally defined class of oxazine and thiazine dyes that are useful in general accordance with the Mashberg diagnostic protocol.
Ari in vivo procedure involving use of Lugol's solution (iodine) and toluidine blue 0 was proposed for detecting esophageal cancer synchronous with upper aerodigestive tract cancers in Papazian, Gastroenterologic Clinique et Biologique 9:16-22 (1985).
More recently, Pomerantz U.S. Patent 5,882,627 disclosed a structurally defined class of oxazine and thiazine dyes that are useful in general accordance with the Mashberg diagnostic protocol.
Bernal et al., Cancer Research 43, 716-720 (1983) disclosed that rhodamine dye selectively inhibits the growth of and kills carcinoma cells in vitro. Gaboury et al., U.S. Patent 5,773,460 discloses that rhodamine is preferentially retained by many tumor cells and proposes that certain esters of rhodamine are useful for vitro photodynamic inhibition of certain tumor cell lines, but indicates that systemic toxicity may limit is usefulness in chemotherapy.
Rhodamine , (2-(6-amino-3-imino-3H-xanthen-9-yl) benzoic acid methyl ester and ionic salts thereof, e.g., hydrochloride salts, is a lipophilic cationic dye of the pyriylium class. It is effective to selectively identify and/or delineate cancerous and precancerous epithelial tissue by topical application to the epithelium, followed by normal visual examination, in general accordance with the protocol disclosed by the Mashberg '251 patent.
Suitable compositions of rhodamine for application of the dye to epithelial tissue are prepared by mixing the dye with a suitable pharmaceutically acceptable solvent. Preferably the pH of the rhodamine solution is adjusted with a suitable pharmaceutically acceptable buffer system to yield a final solution that is substantially isotonic and has a pH in the range of approximately 2.5 to 7.0, preferably 4.0-5Ø This can be accomplished by an acetic acid-sodium acetate buffer system. Other suitable buffer systems include citric acid-sodium citrate or mixed acid salt systems such as citric acid-sodium phosphate and the like.
The solvent used to provide the liquid rhodamine dye compositions of the invention is an aqueous solvent.
According to the presently preferred embodiment of the invention, the solvent included a pharmaceutically acceptable, i.e., non-toxic, non-reactive alcohol, e.g., ethyl alcohol. Such solvents do not appreciably interfere with the staining mechanism and do not themselves contribute to the reduction of chromo forms of the dye to leuco forms.
Flavoring, stable to the other components of the dye composition, may be added to improve the palatability of the composition if it is to be used as an oral "rinse."
The amount of rhodamine dye in the liquid composition is preferably adjusted to yield a concentration of approximately 1~ by weight of the final composition, although higher concentrations can be employed and lower concentrations are at least partially effective. At present, I prefer to employ dye compositions containing from about 0.5 to about 3.5~ by weight of the rhodamine component.
The invention also contemplates compositions for use in accordance with the methods of the invention, in which any leuco form of the dye present in the composition is oxidized to the chromo form, by inclusion of a pharmaceutically acceptable oxidizing agent, in the manner analogous to that disclosed in the Tucci '801 patent.
EXAMPLES
The following examples are presented in order to illustrate practice of the invention to those skilled in the art and not by way of limitation of the scope thereof, which is defined only by the appended claims.
_g_ Example 2 Preparation of Diagnostic Composition A diagnostic composition is prepared by mixing each of the indicated components in the following proportions (% by weight):
Purified Water U.S.P. 83.85 Glacial Acetic Acid U.S.P. 4.61 Sodium Acetate Trihydrate U.S.P. 2.45 SD18 Ethyl Alcohol 7.48 Hydrogen Peroxide 30~, U.S.P. 0.41 IFF Raspberry IC563457 0.20 Rhodamine 1.00 Example 2 Preparation of Rinse Solution A rinse solution is prepared by mixing the following components in the indicated proportions (weight ~):
Purified Water U.S.P. 98.70 Glacial Acetic Acid U.S.P. 1.00 Sodium Benzoate U.S.P. 0.10 IFF Raspberry IC563457 0.20 Example 3 Clinical Effectiveness The clinical effectiveness of the compositions of Examples 1 and 2, is compared to toluidine blue 0 diagnostic control compositions prepared in accordance with the Tucci '801 patent, using the diagnostic protocol disclosed in the Mashberg '251 patent.
Patients are first screened for oral pathology employing the TBO control composition. After identifying potential cancerous or precancerous pathology, all traces of the TBO are removed by repeatedly rinsing the suspect sites with water and the acetic acid rinse of Example 2.
Those patients exhibiting oral pathology are then used as test subjects for the rhodamine diagnostic composition of Example 1. 2-3 cc of the rhodamine composition is applied by painting the pathologic mucosal surface, followed by rinsing with the rinse mixture and water to remove excess rhodamine composition.
Histological examination of tissue from the areas stained by the rhodamine diagnostic composition of Example 1 confirms that the rhodamine composition is at least as effective as toluidine blue O in identifying and delineating cancerous and precancerous epithelial tissue.
Example 4 The procedures of Example 3 are repeated, except that the test and control compositions are applied to the oral mucosa by rinsing, with gargling, instead of by direct application to the locus of the suspect sites.
Equivalent results are obtained.
Having disclosed my invention in such terms as to enable those skilled in the art to understand and practice it, and having disclosed the presently preferred embodiment, I CLAIM:
Rhodamine , (2-(6-amino-3-imino-3H-xanthen-9-yl) benzoic acid methyl ester and ionic salts thereof, e.g., hydrochloride salts, is a lipophilic cationic dye of the pyriylium class. It is effective to selectively identify and/or delineate cancerous and precancerous epithelial tissue by topical application to the epithelium, followed by normal visual examination, in general accordance with the protocol disclosed by the Mashberg '251 patent.
Suitable compositions of rhodamine for application of the dye to epithelial tissue are prepared by mixing the dye with a suitable pharmaceutically acceptable solvent. Preferably the pH of the rhodamine solution is adjusted with a suitable pharmaceutically acceptable buffer system to yield a final solution that is substantially isotonic and has a pH in the range of approximately 2.5 to 7.0, preferably 4.0-5Ø This can be accomplished by an acetic acid-sodium acetate buffer system. Other suitable buffer systems include citric acid-sodium citrate or mixed acid salt systems such as citric acid-sodium phosphate and the like.
The solvent used to provide the liquid rhodamine dye compositions of the invention is an aqueous solvent.
According to the presently preferred embodiment of the invention, the solvent included a pharmaceutically acceptable, i.e., non-toxic, non-reactive alcohol, e.g., ethyl alcohol. Such solvents do not appreciably interfere with the staining mechanism and do not themselves contribute to the reduction of chromo forms of the dye to leuco forms.
Flavoring, stable to the other components of the dye composition, may be added to improve the palatability of the composition if it is to be used as an oral "rinse."
The amount of rhodamine dye in the liquid composition is preferably adjusted to yield a concentration of approximately 1~ by weight of the final composition, although higher concentrations can be employed and lower concentrations are at least partially effective. At present, I prefer to employ dye compositions containing from about 0.5 to about 3.5~ by weight of the rhodamine component.
The invention also contemplates compositions for use in accordance with the methods of the invention, in which any leuco form of the dye present in the composition is oxidized to the chromo form, by inclusion of a pharmaceutically acceptable oxidizing agent, in the manner analogous to that disclosed in the Tucci '801 patent.
EXAMPLES
The following examples are presented in order to illustrate practice of the invention to those skilled in the art and not by way of limitation of the scope thereof, which is defined only by the appended claims.
_g_ Example 2 Preparation of Diagnostic Composition A diagnostic composition is prepared by mixing each of the indicated components in the following proportions (% by weight):
Purified Water U.S.P. 83.85 Glacial Acetic Acid U.S.P. 4.61 Sodium Acetate Trihydrate U.S.P. 2.45 SD18 Ethyl Alcohol 7.48 Hydrogen Peroxide 30~, U.S.P. 0.41 IFF Raspberry IC563457 0.20 Rhodamine 1.00 Example 2 Preparation of Rinse Solution A rinse solution is prepared by mixing the following components in the indicated proportions (weight ~):
Purified Water U.S.P. 98.70 Glacial Acetic Acid U.S.P. 1.00 Sodium Benzoate U.S.P. 0.10 IFF Raspberry IC563457 0.20 Example 3 Clinical Effectiveness The clinical effectiveness of the compositions of Examples 1 and 2, is compared to toluidine blue 0 diagnostic control compositions prepared in accordance with the Tucci '801 patent, using the diagnostic protocol disclosed in the Mashberg '251 patent.
Patients are first screened for oral pathology employing the TBO control composition. After identifying potential cancerous or precancerous pathology, all traces of the TBO are removed by repeatedly rinsing the suspect sites with water and the acetic acid rinse of Example 2.
Those patients exhibiting oral pathology are then used as test subjects for the rhodamine diagnostic composition of Example 1. 2-3 cc of the rhodamine composition is applied by painting the pathologic mucosal surface, followed by rinsing with the rinse mixture and water to remove excess rhodamine composition.
Histological examination of tissue from the areas stained by the rhodamine diagnostic composition of Example 1 confirms that the rhodamine composition is at least as effective as toluidine blue O in identifying and delineating cancerous and precancerous epithelial tissue.
Example 4 The procedures of Example 3 are repeated, except that the test and control compositions are applied to the oral mucosa by rinsing, with gargling, instead of by direct application to the locus of the suspect sites.
Equivalent results are obtained.
Having disclosed my invention in such terms as to enable those skilled in the art to understand and practice it, and having disclosed the presently preferred embodiment, I CLAIM:
Claims (3)
1. A diagnostic method for identifying and delineating cancerous epithelial tissue, comprising:
(a) applying rhodamine to the epithelium, to selectively mark cancerous tissue; and (b) visually examining said epithelium, to identify and delineate suspected cancerous tissue sites, by comparing the color of the suspected sites with the color of the adjacent tissue.
(a) applying rhodamine to the epithelium, to selectively mark cancerous tissue; and (b) visually examining said epithelium, to identify and delineate suspected cancerous tissue sites, by comparing the color of the suspected sites with the color of the adjacent tissue.
2. In a method for in vivo detection of premalignant epithelial lesions and carcinomas, including the steps of sequentially rinsing the epithelium with dye stain composition which is selectively retained by cancerous and precancerous tissue, wherein the stain composition consists essentially of toluidine blue o, and rinsing the epithelium with a rinse composition for removing unretained stain composition, the improvement in which the stain composition comprises rhodamine.
3. A biological stain composition for in vivo detection of cancerous and precancerous tissue, comprising (a) rhodamine, (b) a pharmaceutically acceptable aqueous solvent, and (c) a pharmaceutically acceptable oxidizing agent for leuco rhodamine.
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| PCT/US2000/018126 WO2002002149A1 (en) | 2000-06-30 | 2000-06-30 | Rhodamine diagnostic agent and diagnostic methods for detection of epithelial cancer |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2383697A1 true CA2383697A1 (en) | 2002-01-10 |
Family
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002383697A Abandoned CA2383697A1 (en) | 2000-06-30 | 2000-06-30 | Rhodamine diagnostic agent and diagnostic methods for detection of epithelial cancer |
Country Status (11)
| Country | Link |
|---|---|
| EP (1) | EP1294408A4 (en) |
| JP (1) | JP2004501982A (en) |
| CN (1) | CN1371290A (en) |
| AU (1) | AU784558B2 (en) |
| BR (1) | BR0013636A (en) |
| CA (1) | CA2383697A1 (en) |
| IL (1) | IL148341A0 (en) |
| MX (1) | MXPA02002184A (en) |
| NO (1) | NO20020958L (en) |
| NZ (1) | NZ517445A (en) |
| WO (1) | WO2002002149A1 (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109768151B (en) * | 2019-01-04 | 2020-05-08 | 浙江大学 | Multi-color LED for illumination and display and preparation method thereof |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DK0565668T3 (en) * | 1991-10-31 | 2000-01-31 | Zila Inc | Biological dye composition, method of preparation thereof and for use in epithelial cancer signaling |
| US5618831A (en) * | 1992-11-17 | 1997-04-08 | Fuji Photo Film Co., Ltd. | Composition and method for treating cancer |
| DE69630594T2 (en) * | 1996-01-16 | 2004-09-23 | Zila Inc., Phoenix | METHOD AND MEANS FOR THE IN-VIVO DETECTION OF MOUTH-CREAM AND PRECACCESS STATES |
| AU2000237154A1 (en) * | 2000-02-28 | 2001-09-12 | Zila, Inc. | Method for detecting and killing epithelial cancer cells |
-
2000
- 2000-06-30 NZ NZ517445A patent/NZ517445A/en unknown
- 2000-06-30 MX MXPA02002184A patent/MXPA02002184A/en not_active Application Discontinuation
- 2000-06-30 CN CN00812220.2A patent/CN1371290A/en active Pending
- 2000-06-30 JP JP2002506770A patent/JP2004501982A/en active Pending
- 2000-06-30 IL IL14834100A patent/IL148341A0/en unknown
- 2000-06-30 EP EP00946949A patent/EP1294408A4/en not_active Withdrawn
- 2000-06-30 BR BR0013636-0A patent/BR0013636A/en not_active Application Discontinuation
- 2000-06-30 CA CA002383697A patent/CA2383697A1/en not_active Abandoned
- 2000-06-30 AU AU16752/02A patent/AU784558B2/en not_active Ceased
- 2000-06-30 WO PCT/US2000/018126 patent/WO2002002149A1/en active IP Right Grant
-
2002
- 2002-02-27 NO NO20020958A patent/NO20020958L/en not_active Application Discontinuation
Also Published As
| Publication number | Publication date |
|---|---|
| JP2004501982A (en) | 2004-01-22 |
| AU1675202A (en) | 2002-01-14 |
| NO20020958D0 (en) | 2002-02-27 |
| EP1294408A1 (en) | 2003-03-26 |
| NZ517445A (en) | 2004-03-26 |
| AU784558B2 (en) | 2006-05-04 |
| CN1371290A (en) | 2002-09-25 |
| MXPA02002184A (en) | 2002-09-30 |
| IL148341A0 (en) | 2002-09-12 |
| EP1294408A4 (en) | 2005-01-05 |
| BR0013636A (en) | 2005-01-11 |
| NO20020958L (en) | 2002-04-24 |
| WO2002002149A1 (en) | 2002-01-10 |
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Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| EEER | Examination request | ||
| FZDE | Discontinued |