Patent Application
20080069776
The present disclosure relates to subject matter contained in Japanese Patent Application No. 2006-248849 (filed on Sep. 14, 2006) which is expressly incorporated herein by reference in its entirely.
Examples of the endoscopy according to the present invention include medical endoscopies such as gastrointestinal endoscopy, respiratory endoscopy, vascular endoscopy and peritoneoscopy. Among these, gastrointestinal endoscopy is particularly preferred. According to the present invention, the visible light endoscopes include all of the endoscopes observing under visible light, and also include conventional endoscopes, magnifying endoscopes, and chromoendoscopes observing visible light. Meanwhile, the fluorescent endocopes include endoscopes measuring the fluorescence generated by irradiation of an excitation light, and also include magnifying fluorescent endoscopes. Furthermore, the confocal endoscope refers to an endoscope equipped with a confocal imaging system. In addition, a confocal endoscope conventionally has both a normal observation optical system and a confocal observation optical system.
The histofluorescent stain composition of the present invention contains a compound represented by the Formula (1) described above. In the Formula (1), R1 and R2, which may be identical or different, each represent an alkyl group having 1 to 5 carbon atoms. Examples of the alkyl group include a methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, and the like, and among these, a methyl group and an ethyl group are preferred. Furthermore, it is particularly preferred when both R1 and R2 represent an ethyl group.
X− represents an anion residue, and may be exemplified by HOSO3− (sulfate ion), a halogen ion, or the like. Among these, HOSO3− is particularly preferred.
As a specific preferred example of the compound of Formula (1), Brilliant Green (R1═R2═C2H5, X−═HOSO3−) may be included.
Brilliant Green is widely used as a colorant for cosmetic products. It is a legal colorant that can be used in cosmetic products. The safety of this component has been established. However, it is not fully known that this compound emits fluorescence only when bound to a protein such as albumin, and it is not known at all that the compound exhibits clear fluorescent images of the inner part of a tissue, only when applied to a tissue.
Commercially available products of the Brilliant Green include, for example, Brilliant Green (B4014-25G) from Sigma-Aldrich Company. It is also available under different names such as Pigment Green 1 and Basic Green, and is indicated by C.I. (Color Index Number) 42040.
The content of the compound of Formula (1) in the tissue stain of the present invention is preferably from 0.01 to 70% by weight, more preferably from 0.01 to 50% by weight, and particularly preferably from 0.01 to 20% by weight, from the viewpoints of stainability and the clearness of stained images.
The tissue stain of the present invention can be used in any form, such as liquid, granule, or tablet. In the case of spreading into the gastrointestinal tract or administering submucosally, liquid is preferred, while in the case of orally administering, liquid, granules, tablets and the like are preferred.
The tissue stain of the present invention may have various components blended in according to the form (formulation). For example, a consistency agent, a thickening agent, a surfactant, a sweetening agent, a preservative, a flavoring agent, a pH adjusting agent, water and the like may be blended in.
The pH adjusting agent is an agent adjusting the pH to 5 to 9, and examples thereof include hydrochloric acid, phosphoric acid, citric acid, malic acid, acetic acid and salts thereof, sodium hydroxide, potassium hydroxide, sodium hydrogen carbonate, tetrasodium pyrrolate, and the like. Furthermore, ethanol, water and the like may be added as a solvent. In the case of tablets, components that are known to be used for tablets, such as a binding agent and a disintegrating agent, can be used.
Since the tissue stain of the present invention can stain tissues to be blue in color, the stain is useful as a tissue stain for conventional white light endoscopic observation. The endoscope used herein is a conventional endoscope or a magnifying endoscope, and is useful for endoscopic observation at a magnifying power of 10 to 500 times.
The stainability for tissues varies, from the state of normal tissues to a pre-cancerous state or a state in the presence of tumor. In the case of staining normal tissues, the cellular membrane and cytoplasm of the epithelial cells are stained in the small intestine or the large intestine. On the other hand, in the observation of tumors, features of individual cells include an increase in the nuclear volume, an increase in the amount of nuclear chromatin and hyperchromatism, an increase in the number of nucleoli, abnormality in the number or form of chromosomes, basophilic staining of cells and its association with the proliferation properties of the cells, and the like. Furthermore, in tumor cells, a large number of nuclear chromatins assemble close to the nuclear membrane, thus endoplasmic reticulum being simplified. Moreover, mitochondria become irregular and non-uniform in size, and there is an increased amount of filamentous structures generated in the cells.
The tissue stain composition of the present invention has been found, first of all, to provide good staining of the epithelial cells and the cytoplasm while practically not staining the interstitial space, by observation. Therefore, the morphology of cells in the tissue or the characteristics of cellular arrangement can be detected. The stain composition will be highly useful with an endoscope utilizing the wavelength range which is adequate for the stain composition, since abnormality in tissues and cells is suited for the observation by a confocal imaging system. Furthermore, since the cellular nucleus is not stained, determination can be made more easily in the case of the pre-cancerous state described above, and the like.
Also, the tissue stain composition of the present invention has a property of emitting fluorescence only for bound tissues, and thus necessary information can be obtained even without washing by running a solution. From this point of view, it can also be said that the significance of the information obtained by the stain of the present invention is high.
As indicated by the following Examples, the compound of Formula (1) is characterized in that, as compared to other dyes, the compound does not emit fluorescence in a solution state, but emits fluorescence when spread in the lumen of the body, and that the compound provides clear fluorescence stained images of the inner part of tissues. Therefore, the stain of the present invention is very useful as a histofluorescent stain for endoscopy.
In addition, if the endoscope employing a confocal optical system has both a normal observation optical system and a confocal observation optical system, it is possible to diagnose the surface as well as the inner part of tissues without excising the tissue in the lesion area, by visually observing the lesion area by observation under normal light, and then upon reaching the lesion of question, observing fluorescent stained cross-sectional images of the inner part of the tissue (for example, up to 250 μm) with a confocal endoscope. That is, the morphology of cells or nuclei of a biological tissue can be observed in a live state. As a result, diagnosis of gastrointestinal diseases such as pre-cancerous state, cancer, ulcer, and ulcerative colitis is made possible safely, rapidly and low-invasively, and the degree of precision is remarkably improved.
With regard to such endoscopic observation, the tissue stain of the present invention may be directly spread in the gastrointestinal lumen or administered submucosally, or may be administered orally or intravenously.
Next, the present invention will be described in more detail with reference to Examples, but the present invention is not intended to be limited by these Examples.
Brilliant Green (manufactured by Sigma-Aldrich Company, B4014-25G) was adjusted with distilled water to a concentration of 0.001 mg/mL, and the light absorbance was continuously measured over a wavelength range from 200 to 750 nm using a spectrophotometer (manufactured by Shimadzu Corporation, BioSpec-1600) to determine the wavelength at which the absorption was the maximum. The maximum absorption wavelength of 625 nm thus obtained was irradiated as an excitation wavelength, and the wavelength of the scattered light detected in the perpendicular direction to the light axis of the excitation light was measured using a fluorescence spectrophotometer (manufactured by Shimadzu Corporation, RF-1500), thus to obtain a maximum fluorescence wavelength of 625 nm. The maximum excitation wavelength measured using this fluorescence spectrophotometer was 627 nm. The
As a result, no fluorescence was detected from the stain which was prepared as a solution. The same test was performed using Brilliant Blue FCF, which is widely used as an edible blue dye No. 1, and as a result, Brilliant Blue FCF was found to have a maximum absorption wavelength of 629 nm, a maximum excitation wavelength of 619 nm, and a maximum fluorescence wavelength of 650 nm, showing a clear difference in the fluorescence intensity in the same wavelength region. Brilliant Green was found to emit no fluorescence under a light source in the red wavelength region.
When measured with a fluorescence photometer, Brilliant Green does not show any difference in the maximum absorption wavelength and the maximum fluorescence wavelength. This implies that the compound is not undergoing the process of excitation by an irradiated light and emission of fluorescence.
However, when a biological tissue from the large intestine or the like is stained with Brilliant Green and excited under a certain laser light, observation by means of intense fluorescence can be made.
As a comparison to Brilliant Green, the wavelength pattern of Rhodamine B, which stains the inner part of cells in the lumen of the large intestine or the like in the same manner as Brilliant Green does, was measured. It could be confirmed that Rhodamine B exhibits intense fluorescence even in a solution state, as shown in
Brilliant Green (manufactured by Sigma-Aldrich Company, B4014-25G) was prepared as a solution at 0.1 mg/mL using distilled water. Albumin (from Bovine Serum, Globulin Free; Wako Pure Chemical Industries, Ltd., 013-15104) was prepared as a solution at 2.0 mg/mL using physiological saline. Using a 0.1 M phosphate buffer solution (pH 5) and the 2.0 mg/mL albumin solution prepared above, a liquid mixture of 0.01 mg/mL Brilliant Green and albumin was prepared. The maximum absorption wavelength of Brilliant Green of 625 nm was irradiated as the excitation wavelength, and the wavelength of the scattered light detected in the perpendicular direction to the light axis of the excitation light, was measured using a spectrophotometer (manufactured by Shimadzu Corporation, RF-1500). The maximum fluorescence wavelength was 651 nm.
As a comparison to the liquid mixture of Brilliant Green and albumin, the maximum fluorescence wavelength of an albumin solution was measured with respect to an excitation wavelength of 625 nm, which was shown in
From these, it was determined that although Brilliant Green does not emit fluorescence in the state of an aqueous solution, Brilliant Green emits fluorescence, when bound to albumin, under a light source in the red wavelength region.
Brilliant Green (manufactured by Sigma-Aldrich Company, B4014-25G) was prepared as a solution using distilled water, and the concentration was adjusted to 1.0 mg/mL.
A mouse (ddY, 13 weeks old, male) was subjected to laparotomy under anesthesia, and the large intestine was extracted. Immediately after extraction, the prepared solution of Brilliant Green was applied onto the large intestine, and fluorescence observation of the specimen by a confocal imaging system using a confocal microscope (manufactured by Leica Corp., TCPSP2) was performed. Here, the stainability, penetrability, and fluorescence properties of Brilliant Green were examined.
Observation was performed for the cross-sections at every 5 μm from the luminal surface layer of the flesh specimen of extracted large intestine, to the depth direction (
As can be seen from
Unless otherwise stated, a reference to a compound or component includes the compound or component by itself, as well as in combination with other compounds or components, such as mixtures of compounds.
As used herein, the singular forms “a,” “an,” and “the” include the plural reference unless the context clearly dictates otherwise.
Except where otherwise indicated, all numbers expressing quantities of ingredients, reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about.” Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention, At the very least, and not to be considered as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should be construed in light of the number of significant digits and ordinary rounding conventions.
Additionallly, the recitation of numerical ranges within this specification is considered to be a disclosure of all numerical values and ranges within that range. For example, if a range is from about 1 to about 50, it is deemed to include, for example, 1, 7, 34, 46.1, 23.7, or any other value or range within the range.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2006-248849 | Sep 2006 | JP | national |
