JP2004501982A - Rhodamine diagnostics and diagnostic methods for the detection of epithelial cancer - Google Patents

Abstract

A method for identifying and / or delineating rhodamine dye compositions and cancerous or precancerous epithelial tissue.
[Selection diagram] None

Description

[0001]
(Technical field)
The present invention relates to a novel diagnostic agent for detecting cancerous and precancerous epithelial tissues.
[0002]
(Background technology)
According to another aspect, the present invention relates to a novel method for detecting and / or delineating epithelial cancerous and / or precancerous tissue.
In another aspect, the invention relates to useful diagnostic procedures and agents, particularly for routine diagnosis or treatment of dentists or physicians, such as regular dental or medical examinations and tooth cleaning. In part, it is useful for in vivo screening of patients with potential oral cancer.
[0003]
Yet another aspect of the invention relates to useful treatments and compositions, wherein the invention is more readily available and / or less expensive than the dyes used in prior art procedures, as well as synthetic and And / or use dye staining, which is not complicated to purify.
[0004]
In yet another aspect, the present invention relates to in vivo treatments and compositions with dyes, which, although not in the prior art, can be used by rinsing and / or gargling the entire oral cavity and have a sufficiently low Toxic.
In vivo diagnostic procedures to detect lesions in the oral cavity and precancerous epithelial lesions, such as oral tumors, rely on tissues that have become abnormal due to dysplasia, hyperplasia, tumorigenesis, and other active surface lesions. It uses a dye composition that is selectively retained and is known in the art. For example, procedures using fluorescein and fluorescein derivatives are disclosed in Chenz, (Chinese Journal of Stmatology (27: 44-47 (1992)) and Filurin (Stomatologia (Russian) 72: 44-47 (1993)). The procedure involves visual inspection under ultraviolet light to detect the cancerous / precancerous tissue that applies the dye and then selectively fluoresces.
[0005]
Another prior art procedure involves application in vivo by rinsing with toluidine blue O, followed by routine visual inspection to detect any selectively stained tissue. Such procedures are disclosed, for example, in U.S. Patent No. 5,372,802 to Tucci et al. And in U.S. Patent No. 4,321,251 to Mashberg. Toluidine blue has been used for several decades as a histopathological stain for in vitro use. Through this use, it became known as heterochromatin, which stains DNA- and RNA-rich nuclei from purple to pink. The native deep blue color of toluidine blue 0 changes to purple or pink when the dye binds to nucleic acids and other acidic intracellular macromolecules. Of course, this type of dye relies on dyes gaining access to internal subcellular structures such as the nucleus. Such access is readily obtained only by fixing tissue samples with formaldehyde or other reagents that disrupt cell membranes without destroying general cellular structure.
[0006]
In contrast to the mechanisms involved in in vitro use, staining oral tissue in vivo with Toluidine Blue O is due to its ability to penetrate cell walls and attach to mitochondria, where mitochondria are involved in extracellular matrix. Retains the dye longer than the components.
[0007]
The Mashberg procedure involves applying toluidine blue O as a rinse across the mouth, gargle, and then rinsing with water and acetic acid to remove dyes not retained by the cancerous or precancerous tissue. I do. Preliminary diagnosis by the Mashberg procedure was later confirmed by direct application of the toluidine blue O composition to the suspect site after 10-14 days. Tucci's' 801 patent discloses an improved toluidine blue O composition for use according to the general procedure taught by Mashberg.
[0008]
In vivo procedures, including the use of Lugol solution (iodine) and toluidine blue O, have been described in Papazian, Gastroenterological Clinique et Biologic 9: 16-22 (1985) to detect cancers that coincide with upper aerodigestive cancers in upper aerodigestive cancers. Was suggested to.
[0009]
More recently, U.S. Pat. No. 5,882,627 to Pomerantz disclosed a structurally defined class of oxazine and thiazine dyes that are useful based generally on Mashberg's diagnostic protocol.
[0010]
Bernal et al., Cancer Research 43, 716-720 (1983) disclosed that rhodamine dyes selectively inhibit and kill the growth of carcinoma cells in vitro. Gaboury et al., US Pat. No. 5,773,460, discloses that rhodamine is preferentially retained by many tumor cells, and that certain esters of rhodamine have been photodynamically tested in vitro for certain tumor cell lines. Although proposed to be useful for inhibition, it indicates that systemic toxicity may limit its usefulness in chemotherapy.
[0011]
(Disclosure of the Invention)
Rhodamine, (2- (6-amino-3-imino-3H-xanthen-9-yl) benzoic acid methyl ester and its ionic salts, such as the hydrochloride, are lipophilic cationic dyes of the pyrilium class. This is a method of selectively identifying cancerous and precancerous epithelial tissue by topical application to the epithelium, followed by routine visual experimentation, generally according to the protocol disclosed by the Mashberg '251 patent. And / or effective for depiction.
[0012]
Suitable compositions of rhodamine for applying 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, depending on the buffer system, from about 2.5 to 7.0, preferably 4.0 to 5.0. It is preferable to have a final solution that has a pH in the range of 0 and is substantially isotonic. This can be achieved with an acetic acid-sodium acetate buffer system. Other suitable buffer systems include a mixed acid system such as citric acid-sodium citrate or citric acid-sodium phosphate.
[0013]
The solvent used to provide the liquid rhodamine dye composition of the present invention is an aqueous solvent. According to a preferred embodiment of the present invention, the solvent comprises a pharmaceutically acceptable, ie non-toxic, non-reactive alcohol, such as ethyl alcohol. Such solvents hardly interfere with the mechanism of the dye and do not themselves contribute to reducing the colored form (chromo form) of the dye to the white form (leuco form).
[0014]
The fragrance is stable with respect to other components of the pigment composition and may be added to improve the palatability of the composition when it is used as a "rinse" of the oral cavity .
[0015]
The amount of rhodamine dye in the liquid composition is preferably adjusted to yield a concentration of about 1% by weight of the final composition, although higher concentrations can be used, and lower concentrations may be at least partially It is effective for The inventor prefers to use a dye composition containing about 0.5 to about 3.5% by weight of the rhodamine component.
[0016]
The present invention also contemplates a composition for use in accordance with the methods of the present invention, wherein any leuco form of the dye present in the composition is in a manner similar to that disclosed in the Tucci '801 patent. Oxidized to the chromo form by the inclusion of a pharmaceutically acceptable oxidizing agent.
[0017]
(Example)
The following examples are given to illustrate embodiments of the present invention to those skilled in the art, and are not intended to limit the scope of the invention, which is defined solely by the appended claims.
Example 1 Preparation of a Diagnostic Composition A diagnostic composition is prepared by mixing each of the components shown in the following proportions (% by weight):
Purified water U.S. S. P. 83.85
Glacial acetic acid U.S. S. P. 4.61
Sodium acetate trihydrate U.S. S. P. 2.45
SD18 Ethyl alcohol 7.48
30% hydrogen peroxide, U.S.A. S. P. 0.41
IFF Raspberry IC563457 0.20
Rhodamine 1.00
[0018]
Example 2 Preparation of Rinse Solution A rinse solution is prepared by mixing the following components in the indicated proportions (% by weight):
Purified water U.S. S. P. 98.70
Glacial acetic acid U.S. S. P. 1.00
Sodium benzoate U.S. S. P. 0.10
IFF Raspberry IC563457 0.20
[0019]
Example 3 Clinical Efficacy The clinical efficacy of the compositions of Examples 1 and 2 was determined using the diagnostic protocol disclosed in the '251 patent of Mashberg, toluidine prepared according to the Tucci' 801 patent. Compared to Blue O diagnostic control composition.
[0020]
Patients are first screened for oral pathology using the TBO control composition. After identifying the pathology of the potential cancerous or precancerous condition, all traces of TBO are removed by repeatedly rinsing the putative site with the water and acetate rinse of Example 2.
[0021]
These patients exhibiting oral medical conditions are then considered test subjects for the rhodamine diagnostic composition of Example 1. 2-3 cc of the rhodamine composition is applied by smearing the pathological mucosal surface, followed by a rinse with a rinse mixture and water to remove excess rhodamine composition.
[0022]
Histological examination of the tissue from the area stained by the rhodamine diagnostic composition of Example 1 shows that the rhodamine composition identifies and delineates cancerous and precancerous epithelial tissue at least as much as toluidine blue. Make sure it is valid.
[0023]
(Example 4)
The procedure of Example 3 is repeated, except that the test and control compositions are applied directly to the oral mucosa by rinsing with gargling instead of applying directly to the lesion at the putative site. .
[0024]
We disclose our invention and disclose currently preferred embodiments, so that one skilled in the art can understand and practice the invention, and we claim the following claims.

Claims (3)

A diagnostic method for identifying and delineating cancerous epithelial tissue, comprising the following steps:
(A) applying rhodamine to the epithelium to selectively mark the cancerous tissue; and (b) comparing the color of the proximate tissue with the color of the putative site to presume the cancerous tissue. Visually inspecting the epithelium to identify and delineate the site of the epithelium. In a method for the detection of precancerous epithelial diseases and cancer tumors in vivo, the following sequential steps:
Rinsing the epithelium with a dye staining composition that is selectively retained by the cancerous and precancerous tissue, wherein the staining composition consists essentially of toluidine blue O; Rinsing the epithelium with a rinse composition to remove the staining composition of
And the dyeing composition comprises rhodamine. A biological staining composition for in vivo detection of cancerous and precancerous tissues, comprising: (a) rhodamine;
A composition comprising (b) a pharmaceutically acceptable aqueous solvent, and (c) a pharmaceutically acceptable oxidizing agent for leukorhodamine.