AGENT DIAGNOSTIC A BASE DE RHODAMINE ET METHODES DE DETECTION DU CANCER EPITHELIAL

RHODAMINE DIAGNOSTIC AGENT AND DIAGNOSTIC METHODS FOR DETECTION OF EPITHELIAL CANCER

Abrégé français

Cette invention concerne des compositions à base de rhodamine et des méthodes permettant de détecter et/ou de délinéer un tissu épithélial cancéreux ou précancéreux.

Abrégé anglais

Rhodamine dye compositions and methods for detecting and/or delineating
cancerous and precancerous epithelial tissue.

Revendications

claims
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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.

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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.

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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.

Description

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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.

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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

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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.

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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.

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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

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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."

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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.

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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 ~):

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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

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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: