Note: Descriptions are shown in the official language in which they were submitted.
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METHYLENE BLUE 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
stained tissue. Such procedures are disclosed, for
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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 O 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 O solution as a rinse of the entire oral
cavity, with gargling, followed by rinses with water. and
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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.
An in vivo procedure involving use of Lugol's
solution (iodine) and toluidine blue O 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. However, the Pomerantz
'627 patent expressly excludes methylene blue from this
defined class, asserting that it is too toxic for
application by oral rinse and/or gargling procedures.
Contrary, however, to the disclosure of the
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Pomerantz '627 patent, methylene blue and its ionic
derivatives, in the concentrations and quantities
employed for in vivo tissue staining, is safely employed
as a topically applied selective stain for detecting
and/or delineating epithelial cancerous and precancerous
tissue.
Suitable compositions of methylene blue 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 methylene
blue 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 methylene
blue dye compositions of the invention is an aqueous
solvent. According to the presently preferred embodiment
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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 methylene blue 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 methylene blue 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
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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.
Example 1
Preparation of Diagnostic Composition
A diagnostic composition is prepared by mixing each
of the indicated components in the following proportions
by weight):
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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 300, U.S.P. 0.41
IFF Raspberry IC563457 0.20
Methylene Blue 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 O
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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 methylene blue diagnostic
composition of Example 1. 2-3 cc of the methylene blue
composition is applied by painting the pathologic mucosal
surface, followed by rinsing with the rinse mixture and
water to remove excess methylene blue composition.
Histological examination of tissue from the areas
stained by the methylene blue diagnostic composition of
Example 1 confirms that the methylene blue composition is
at least as effective as toluidine blue 0 in identifying
and delineating cancerous and precancerous epithelial
tissue.
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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:
