JP2016132658A - Diagnosis assistant method for malignant mesothelioma and diagnosis assistant agent - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a medicine and method for increasing efficiency in success of biopsy with thoracoscope for cell diagnosis and tissue diagnosis of malignant mesothelioma.SOLUTION: Excitation light is irradiated onto breast membrane of a target to which a compound represented by formula (I) or salt thereof (5-ALAs) has been administered, fluorescence emitted by excitation onto protoporphyrin induced from the 5-ALAs and accumulated at tumor part is captured in color mode, energy (ER) corresponding to wavelength 636±2 nm and energy (EG) corresponding to wavelength 500±2 nm are calculated, image processing for assisting specification of red fluorescence part is carried out in fluorescence image by utilizing EG value and/or ER value, the red fluorescence part is specified as a part at which biopsy is carried out, and the specified part is subjected to cell diagnosis with the biopsy so as to carry out diagnosis of malignant mesothelioma.SELECTED DRAWING: None

Description

  The present invention relates to a method for assisting diagnosis of malignant mesothelioma and a diagnostic aid for use in such a method, and more particularly, malignant using 5-aminolevulinic acid (5-ALA) or a derivative thereof or a salt thereof. The present invention relates to a method for assisting cytodiagnosis and tissue diagnosis of mesothelioma and a cytodiagnosis and tissue diagnosis auxiliary agent containing 5-ALA for use in such a method.

  Malignant mesothelioma is a tumor caused by asbestos exposure and is characterized by onset after asbestos exposure. Prognosis is poor and early detection is difficult.

  Malignant mesothelioma is caused by exposure to asbestos, so accurate diagnosis is required for workers' certification and asbestos relief. The current definitive diagnosis is cytodiagnosis / histological diagnosis, and confirmation in the central pathological diagnosis is necessary to receive relief measures. In addition, the treatment of malignant mesothelioma requires a large pleural detachment and, in some cases, is highly invasive with the removal of the lung. From this viewpoint, accurate cytodiagnosis and tissue diagnosis are important.

  The current cytodiagnosis and histological diagnosis of malignant mesothelioma involves thoracoscopic biopsy, but in patients who are suspected of having malignant mesothelioma by CT examination etc., the pleura is often already thickened. Often, it is not clear which part should be biopsied. Biopsy will be performed at several thickened locations to perform cytodiagnosis and tissue diagnosis, but there are cases in which malignant mesothelioma cells are not successfully taken, treatment becomes behind, and patients can not receive relief measures Many.

  As a method to solve this, there is an attempt to administer 5-ALA hydrochloride to a patient prior to thoracoscopic biopsy, and to perform accurate biopsy using the fluorescence of protoporphyrin IX accumulated in malignant mesothelioma cells as a guide . This technique, which uses fluorescence of protoporphyrin IX as a guide, has been put to practical use in brain tumors, and has been reported to be effective in bladder cancer, etc., and is also known to be useful in general lung cancer. Unfortunately, malignant mesothelioma is not as successful as other cancers because fluorescence is not observed.

  In addition, narrow-band analysis that irradiates blue light in a narrow wavelength range and observes the distribution of blood vessels that are commonly found in cancer cells and analysis that searches for areas with low autofluorescence are also effective because malignant mesothelioma is scattered in the thickened pleura It cannot be a good guide.

  On the other hand, 5-ALA is known as an intermediate of tetrapyrrole biosynthetic pathway widely present in animals, plants and fungi, and is usually biosynthesized from succinyl CoA and glycine by 5-aminolevulinic acid synthase. Photodynamic diagnosis or photodynamic therapy using 5-ALA has also been developed, and has attracted attention as a diagnostic method / treatment method that is low in invasiveness and maintains QOL. Tumor diagnosis / treatment using 5-ALA or the like Agents and the like have been reported (for example, see Patent Documents 1 to 3). ALA is also known to be useful as an agent for preventing or improving adult diseases, cancer, and male infertility.

International publication WO2011 / 161933 JP 2011-016753 A International Publication WO2009 / 130893

  An object of the present invention is to provide a drug and a technique for increasing the success efficiency of thoracoscopic biopsy for cytodiagnosis and tissue diagnosis of malignant mesothelioma.

  The present inventors add other engineering factors to the administration of 5-ALA used for photodynamic diagnosis of other cancers and the accompanying fluorescence observation of protoporphyrin, and further refine the conditions. Thus, the present invention has been completed by considering various image processing conditions.

That is, the present invention is as follows.
(1) (a) irradiating a pleura of a subject administered with a compound represented by the following formula (I) or a salt thereof (5-ALA) through a thoracoscope; (b) from 5-ALA A step of performing color imaging of fluorescence emitted when protoporphyrin IX (PpIX) induced and accumulated in a tumor site is excited; (c) fluorescence energy corresponding to a wavelength of 636 ± 2 nm by analyzing a color-captured fluorescence image Fluorescence energy (EG) corresponding to (ER) and a wavelength of 500 ± 2 nm is calculated, and image processing that assists the identification of the red fluorescent part by appropriately using the EG value and / or ER value is color imaging fluorescence Malignant with each step of: (d) identifying a red fluorescent part in a color photographed fluorescent image subjected to image processing as a biopsy site; A method to assist cytodiagnosis and tissue diagnosis of mesothelioma.
(In the formula, R ¹ represents a hydrogen atom or an acyl group, and R ² represents a hydrogen atom, a linear or branched alkyl group, a cycloalkyl group, an aryl group, or an aralkyl group).
(2) The method of (1) above, wherein R ¹ and R ² are hydrogen atoms.
(3) Malignant mesothelioma containing the compound represented by the formula (I) or a salt thereof for use in the method of assisting cytodiagnosis / histological diagnosis of malignant mesothelioma according to (1) or (2) above For cell and tissue diagnosis.
(4) The adjuvant for cytodiagnosis / tissue diagnosis of malignant mesothelioma according to (3) above, wherein R ¹ and R ² are hydrogen atoms.
(5) The compound represented by the formula (I) or a salt thereof for preparing an adjuvant for use in the method for assisting cytodiagnosis / histological diagnosis of malignant mesothelioma according to (1) or (2) above Use of.
(6) Use of (5) above, wherein R ¹ and R ² are hydrogen atoms.
(7) (a) irradiating the pleura of a subject administered with the compound represented by the formula (I) or a salt thereof (5-ALA) through a thoracoscope; (b) from the 5-ALA A step of performing color imaging of fluorescence emitted when protoporphyrin IX (PpIX) induced and accumulated in a tumor site is excited; (c) fluorescence energy corresponding to a wavelength of 636 ± 2 nm by analyzing a color-captured fluorescence image Fluorescence energy (EG) corresponding to (ER) and a wavelength of 500 ± 2 nm is calculated, and image processing that assists the identification of the red fluorescent part by appropriately using the EG value and / or ER value is color imaging fluorescence (D) a step of identifying a red fluorescent portion in a color photographed fluorescent image subjected to image processing as a biopsy site; (e) special in step (d) A method for diagnosing malignant mesothelioma, comprising the steps of: performing a cytodiagnosis / histological diagnosis of the determined site by biopsy.

  According to the present invention, the presence and range of a tumor site of malignant mesothelioma can be diagnosed, and a nodule that is difficult to visualize can be diagnosed.

It is a fluorescence screen which shows the optical diagnosis result of the disseminated lesion using 5-ALA. It is a fluorescence screen which shows the result of a malignant pleural mesothelioma (progressive example) biopsy using 5-ALA.

  As a method for assisting cytodiagnosis / histological diagnosis of malignant mesothelioma of the present invention, the compound represented by the formula (I) or a salt thereof (hereinafter, these may be collectively referred to as “5-ALA”). )) Irradiating the pleura of the subject administered with excitation light through a thoracoscope; (b) fluorescence emitted when protoporphyrin IX (PpIX) derived from 5-ALA and accumulated in the tumor site is excited (C) calculating a fluorescence energy (ER) corresponding to a wavelength of 636 ± 2 nm and a fluorescence energy (EG) corresponding to a wavelength of 500 ± 2 nm by analyzing a color-captured fluorescence image; and A step of performing image processing on a color-captured fluorescent image to assist the identification of the red fluorescent portion by appropriately using the EG value and / or ER value; -A method that includes the step of identifying the red fluorescent part in the photographed fluorescent image as a biopsy site; there is no particular limitation, and cancer cells are found in the pleura (thin tissue layer covering the inner side of the rib cage and the lung surface). It is a method to assist cytodiagnosis and histological diagnosis of malignant mesothelioma, which is a common disease.

  Examples of the auxiliary agent for cytodiagnosis / histological diagnosis of malignant mesothelioma of the present invention include 5-ALA for use in the method for assisting cytodiagnosis / histological diagnosis of malignant mesothelioma of the present invention. This invention is a use invention in which the use of 5-ALA's “for use in a method for assisting cytodiagnosis / histological diagnosis of malignant mesothelioma” is limited. Other aspects of the present invention include the use of 5-ALA for preparing an auxiliary agent for cytodiagnosis / histological diagnosis of malignant mesothelioma, and for use in assisting cytodiagnosis / histological diagnosis of malignant mesothelioma. 5-ALA can be mentioned.

  As a method for diagnosing malignant mesothelioma of the present invention, steps (a) to (d) of the method for assisting cytodiagnosis / histological diagnosis of malignant mesothelioma of the present invention are specified in step (d). The method is not particularly limited as long as the method includes the step (e) of performing cytodiagnosis / tissue diagnosis by biopsy, and the cell diagnosis / tissue diagnosis may be performed by collecting cells or tissues from the pleura using a thin needle. A biopsy (biopsy) in which a pathologist looks for signs of cancer under a microscope can be exemplified.

Among the 5-ALAs, 5-ALA or a salt thereof, in which R ¹ and R ^{2 in} the formula (I) are both hydrogen atoms, can be preferably exemplified. 5-ALA is one of the amino acids also called δ-aminolevulinic acid. Further, as the 5-ALA derivative, R ^{1 in} the formula (I) is a hydrogen atom or an acyl group, and R ^{2 in} the formula (I) is a hydrogen atom, a linear or branched alkyl group, a cycloalkyl group, an aryl group. Or compounds other than 5-ALA which are aralkyl groups can be mentioned.

Examples of the acyl group in R ¹ of the formula (I) include linear or branched C 1-8 carbon atoms such as formyl, acetyl, propionyl, butyryl, isobutyryl, valeryl, isovaleryl, pivaloyl, hexanoyl, octanoyl, benzylcarbonyl group and the like. Examples include alkanoyl groups and aroyl groups having 7 to 14 carbon atoms such as benzoyl, 1-naphthoyl and 2-naphthoyl groups.

Examples of the alkyl group in R ² of the formula (I) include a straight chain such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl, hexyl, heptyl, octyl group, etc. A branched alkyl group having 1 to 8 carbon atoms can be exemplified.

The cycloalkyl group in R ² of the formula (I) includes a saturated or partially unsaturated bond such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclododecyl, 1-cyclohexenyl group, etc. Examples thereof include cycloalkyl groups having 3 to 8 carbon atoms.

Examples of the aryl group in R ² of formula (I) include aryl groups having 6 to 14 carbon atoms such as phenyl, naphthyl, anthryl, and phenanthryl groups.

As the aralkyl group in R ² of formula (I), the aryl moiety can be exemplified as the above aryl group, and the alkyl moiety can be exemplified as the above alkyl group. Specifically, benzyl, phenethyl, phenylpropyl, phenyl Examples thereof include aralkyl groups having 7 to 15 carbon atoms such as butyl, benzhydryl, trityl, naphthylmethyl, and naphthylethyl groups.

Examples of the ALA derivative, R ¹ is formyl, acetyl, propionyl, or butyryl group, compound, said R ² is methyl, ethyl, propyl, butyl, compound pentyl group are preferred, the R ¹ and The combination of R ² is formyl and methyl, acetyl and methyl, propionyl and methyl, butyryl and methyl, formyl and ethyl, acetyl and ethyl, propionyl and ethyl, butyryl and ethyl, or R ¹ is hydrogen and R ² is methyl Suitable examples include 5-ALA esters such as ethyl, propyl, butyl, and pentyl groups.

  ALAs only have to act as an active ingredient in the state of 5-ALA of the formula (I) or a derivative thereof in a living body, and various salts, esters, or biosynthetic agents for increasing solubility depending on the administration form. It can be administered as a prodrug (precursor) that is degraded by enzymes in the body. For example, examples of salts of 5-ALA and its derivatives include pharmacologically acceptable acid addition salts, metal salts, ammonium salts, organic amine addition salts, and the like. Examples of acid addition salts include hydrochloride, hydrobromide, hydroiodide, phosphate, nitrate, sulfate, and other inorganic acid salts, formate, acetate, propionate, toluenesulfonic acid Salt, succinate, oxalate, lactate, tartrate, glycolate, methanesulfonate, butyrate, valerate, citrate, fumarate, maleate, malate, etc. Organic acid addition salts can be exemplified. Examples of metal salts include alkali metal salts such as lithium salt, sodium salt and potassium salt, alkaline earth metal salts such as magnesium and calcium salt, and metal salts such as aluminum and zinc. Examples of ammonium salts include ammonium salts and alkylammonium salts such as tetramethylammonium salts. Examples of the organic amine salt include salts such as triethylamine salt, piperidine salt, morpholine salt, and toluidine salt. These salts can also be used as a solution at the time of use.

  Among the above 5-ALAs, desirable ones are various such as 5-ALA and 5-ALA methyl ester, 5-ALA ethyl ester, 5-ALA propyl ester, 5-ALA butyl ester, 5-ALA pentyl ester, etc. These are 5-ALA esters and their hydrochlorides, phosphates, and sulfates, and 5-ALA hydrochloride and 5-ALA phosphate can be particularly preferably exemplified.

  The 5-ALA can be produced by any known method of chemical synthesis, production by microorganisms, and production by enzymes. Moreover, the said 5-ALA may form the hydrate or the solvate, and can use either individually or in combination of 2 or more types as appropriate.

  In the present invention, the administration route of 5-ALA's includes oral administration including sublingual administration, nasal administration, inhalation administration, intravenous administration including infusion, transdermal administration using a cataplasm, suppository, or nasal administration Examples include parenteral administration such as administration by forced enteral nutrition using a gastric tube, nasal intestinal tract, gastric leak tube, or enteral leak tube.

  The dosage form of 5-ALA administered in the present invention can be appropriately determined according to the administration route described above, but injection, nasal drop, instillation, tablet, capsule, fine granule, scattered , Liquid preparations, poultices, suppositories and the like dissolved in liquids, syrups and the like. In addition, for elderly people and infants who have difficulty in swallowing, the form of a disintegrating tablet exhibiting rapid disintegration in the mouth and the form of a liquid suitable for nasogastric tube administration are preferred.

  In order to prepare various dosage forms of 5-ALA administered in the present invention, pharmacologically acceptable carriers, excipients, diluents, additives, disintegrants, binders as necessary. , Coating agents, lubricants, lubricants, lubricants, flavors, sweeteners, solubilizers, solvents, gelling agents, nutrients, etc., specifically water, physiological saline And animal fats and oils, vegetable oils, lactose, starch, gelatin, crystalline cellulose, gum, talc, magnesium stearate, hydroxypropylcellulose, polyalkylene glycol, polyvinyl alcohol, and glycerin. In addition, when preparing these 5-ALA as a dosage form of aqueous solution, in order to prevent decomposition | disassembly of ALAs, it is necessary to pay attention so that aqueous solution may not become alkaline. Removal can also prevent decomposition.

  The dose of 5-ALA can be appropriately changed depending on the age, weight, etc. of the patient, but is 10 mg to 40 mg, preferably 15 mg to 25 mg, more preferably 20 mg per kg of human body weight in terms of 5-ALA. The time from the administration of 5-ALA to the irradiation of excitation light to excite PpIX is preferably a time zone in which sufficient PpIX is accumulated in the tumor tissue, specifically 4 to 8 hours. Is preferred.

  In the method for diagnosing malignant mesothelioma of the present invention, for example, a color fluorescence diagnostic system comprising a thoracoscope, a color fluorescence imager, fluorescence analysis means, image processing means, and biopsy means can be used. The thoracoscope guides and irradiates excitation light to excite PpIX accumulated in the tumor site; the color fluorescence imager is derived from 5-ALA, and accumulates protoporphyrin IX (PpIX) accumulated in the tumor site. ) Is emitted in a color image; the fluorescence analysis means analyzes the color-captured fluorescence image to correspond to a fluorescence energy (ER) corresponding to a wavelength of 636 ± 2 nm and a wavelength of 500 ± 2 nm. Fluorescence energy (EG) is calculated, and the image processing means appropriately uses the EG value and / or ER value to assist the identification of the red fluorescent portion in the color photographing fluorescence image; It has a function of collecting cells and tissues of the red fluorescent part specified in the photographed fluorescent image.

  As a light source of excitation light for exciting PpIX accumulated in the tumor part, the irradiance is strong to enable detection of PpIX even for minutely scattered tumors in the living tissue of the subject, In order to enable accurate automatic identification, a semiconductor laser light source having a small irradiation area is preferable. Specific examples of the excitation light guide unit that guides the excitation light and emits the light from one end to the outside include a thin optical fiber (a thin optical fiber for a light source). As a wavelength oscillated as excitation light emitted from the thin optical fiber for the light source, it is preferable to select a wavelength at which red light having a peak near 636 nm peculiar to PpIX can be observed by exciting the PpIX. It is a violet wavelength close to the ultraviolet light belonging to the absorption peak of PpIX belonging to the Soray band, and specifically 405 ± 5 nm is preferable, and the filter for observing the fluorescence clearly by cutting the excitation light is almost colorless. For this purpose, 405 ± 2 nm is more preferable. As an element used for the light source, a semiconductor mixed crystal such as InGaN can be used, and violet light can be oscillated by changing the compounding ratio of InGaN. Specifically, a compact laser diode having a diameter of about 5.6 mm can be preferably exemplified. A thin optical fiber for measurement can be used to receive fluorescence emitted by PpIX excited by the excitation light, and the thin optical fiber for measurement is integrated with the thin optical fiber for light source, and the received fluorescence is colored. The light is guided to the fluorescence imager.

  EXAMPLES Hereinafter, although an Example demonstrates this invention more concretely, the technical scope of this invention is not limited to these illustrations.

  PpIX accumulation by inserting a thoracoscope into the thoracic cavity of a patient suspected of having malignant mesothelioma who was orally administered 5-ALA 15-20 mg / kg 4 hours before biopsy surgery and irradiating the pleura with excitation light having a wavelength of 405 nm The red fluorescence of the site was observed with a color fluorescence camera. As a result, the presence and extent of the tumor site could be diagnosed, and nodules that were usually difficult to see were also found.

The cases that could be diagnosed are as follows.
7 out of 15 cases of primary lung cancer P1-P2 factor: 5 cases Pleural dissemination example: 2 cases (see FIG. 1)
All 5 non-visualized cases were P0 cases (no pleural infiltration)
-5 cases out of 6 metastatic lung tumors (1 renal cancer, 2 colorectal cancers, 2 breast cancers, 1 uterine cancer). One non-depicted case had a distance from the pleura.
-Two of the two pleural mesothelioma cases were biopsy cases, but clear images were obtained in both cases. (See Figure 2)

  The live cell diagnostic adjuvant and method for malignant mesothelioma of the present invention are useful in the medical field and the like.

Claims (7)

A method for assisting cytodiagnosis and tissue diagnosis of malignant mesothelioma, comprising the following steps (a) to (d):
(A) irradiating excitation light through a thoracoscope on a pleura of a subject to which a compound represented by the following formula (I) or a salt thereof (5-ALA) is administered;
(B) Color imaging of fluorescence emitted when protoporphyrin IX (PpIX) derived from 5-ALA and accumulated in the tumor site is excited;
(C) Analyzing the color fluorescence image to calculate a fluorescence energy (ER) corresponding to a wavelength of 636 ± 2 nm and a fluorescence energy (EG) corresponding to a wavelength of 500 ± 2 nm, and an EG value and / or ER Performing image processing on the color-captured fluorescent image to assist the identification of the red fluorescent part by appropriately using the value;
(D) a step of identifying a red fluorescent portion in a color photographed fluorescent image subjected to image processing as a biopsy site;
(In the formula, R ¹ represents a hydrogen atom or an acyl group, and R ² represents a hydrogen atom, a linear or branched alkyl group, a cycloalkyl group, an aryl group, or an aralkyl group). The method according to claim 1, wherein R ¹ and R ² are hydrogen atoms. A cytodiagnosis / tissue of malignant mesothelioma containing a compound represented by the following formula (I) or a salt thereof for use in the method for assisting cytodiagnosis / histological diagnosis of malignant mesothelioma according to claim 1 or 2 A diagnostic aid.
(In the formula, R ¹ represents a hydrogen atom or an acyl group, and R ² represents a hydrogen atom, a linear or branched alkyl group, a cycloalkyl group, an aryl group, or an aralkyl group). The adjuvant for cytodiagnosis and tissue diagnosis of malignant mesothelioma according to claim 3, wherein R ¹ and R ² are hydrogen atoms. Use of a compound represented by the following formula (I) or a salt thereof for preparing an auxiliary agent for use in the method for assisting cytodiagnosis / histological diagnosis of malignant mesothelioma according to claim 1 or 2.
(In the formula, R ¹ represents a hydrogen atom or an acyl group, and R ² represents a hydrogen atom, a linear or branched alkyl group, a cycloalkyl group, an aryl group, or an aralkyl group). Use according to claim 5, characterized in that R ¹ and R ² are hydrogen atoms. A method for diagnosing malignant mesothelioma comprising the following steps (a) to (e):
(A) irradiating excitation light through a thoracoscope on a pleura of a subject to which a compound represented by the following formula (I) or a salt thereof (5-ALA) is administered;
(B) Color imaging of fluorescence emitted when protoporphyrin IX (PpIX) derived from 5-ALA and accumulated in the tumor site is excited;
(C) The fluorescence image (ER) corresponding to a wavelength of 636 ± 2 nm and the energy (EG) corresponding to a wavelength of 500 ± 2 nm are calculated by analyzing a color photographed fluorescent image, and the EG value and / or ER value are calculated. Performing image processing on the color-captured fluorescent image to assist the identification of the red fluorescent portion by appropriately using
(D) a step of identifying a red fluorescent portion in a color photographed fluorescent image subjected to image processing as a biopsy site;
(E) performing a cytodiagnosis / tissue diagnosis of the site identified in step (d) by biopsy;
(In the formula, R ¹ represents a hydrogen atom or an acyl group, and R ² represents a hydrogen atom, a linear or branched alkyl group, a cycloalkyl group, an aryl group, or an aralkyl group).