JP2008017899A - Method of fluorescent diagnosis - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a condition for laser irradiation, etc., necessary for the fluorescent diagnosis. <P>SOLUTION: 1-10 mg/m<SP>2</SP>of talaporfin sodium is administered to a living-body region 107 and accumulated on a tumor as a light-sensitive substance with the affinity to the tumor, and the living-body region 107 is irradiated with laser beams emitted from a semi-conductor laser device 101 at the intensity from 7 to 70 mW/cm<SP>2</SP>to excite the light-sensitive substance. Then, the fluorescence generated from the light-sensitive substance excited with the laser beams is observed, and the region where the tumor is present is determined based on the intensity of the fluorescence. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  In the present invention, a photosensitizer having an affinity for a tumor or other lesion is administered to a living body in advance, and at a stage where the photosensitizer is accumulated in the tumor or other lesion, light having a wavelength matching the absorption wavelength of the photosensitizer is irradiated. The present invention relates to a fluorescence diagnostic method using a photodynamic technique that excites a sensitive substance and captures fluorescence from a tumor or other lesion.

  A semiconductor laser device using a semiconductor laser element is used in various industrial fields because of its small size and light weight. In addition, in other oscillation systems such as a die laser, the laser wavelength distribution is wide, and in a die laser device that oscillates in the visible light wavelength band, the half-value width is more than a dozen nm or more. It is possible to oscillate laser light having a narrow value width of several nm (about 2 nm to 3 nm) and having a uniform wavelength. Further, the semiconductor laser device has a feature that the wavelength of the laser beam can be easily controlled by adjusting the temperature of the semiconductor laser element.

  For example, Patent Document 1 discloses a fluorescence diagnosis / treatment apparatus using a semiconductor laser device having these characteristics. Specifically, Patent Document 1 discloses that a laser beam having a half-value width suitable for an absorption wavelength of a photosensitive material and a treatment purpose is obtained by controlling an oscillation wavelength by adjusting a temperature of a semiconductor laser element. Describes that the photosensitive material can be excited efficiently.

Japanese Patent No. 2596221

However, conventional techniques relating to fluorescence diagnosis including Patent Document 1 do not suggest any conditions such as laser light irradiation conditions and drug concentrations necessary for actually performing fluorescence observation. For example, talaporfin sodium sodium is known as a photosensitive substance. Conventionally, the dose when using talaporfin natonarium as a photosensitive substance has been about 40 mg / m ² . Since talaporfin sodium is expensive, there is a need for dose reduction. However, conventional techniques do not suggest any conditions necessary for fluorescence observation such as laser light irradiation conditions when talaporfin natonarium is used as a photosensitive substance and the dose is reduced.

  In view of the above-described conventional problems, an object of the present invention is to define conditions such as laser light irradiation conditions and drug concentrations necessary for actually performing fluorescence diagnosis.

The present invention relates to a method for diagnosing a tumor in a target region of a subject by fluorescence, wherein the photosensitive substance having affinity for the tumor is 1 mg / m ² or more and 10 mg / m ² or less. by administering Talaporfin Natona helium is integrated into the tumor, by irradiating a laser beam center wavelength to the target area at 5 mW / cm ² or more 70 mW / cm ² or less of the intensity occurs in the following semiconductor laser 660nm or 664nm Fluorescence diagnostic method for exciting the photosensitizer, observing fluorescence emitted from the photosensitizer excited by the laser light, and determining the location of the tumor in the target region based on the intensity of the fluorescence I will provide a.

Although the dose of talaporfin natonarium has been reduced to 1 mg / m ² or more and 10 mg / m ² , the site of the tumor in the target region can be identified based on the intensity of fluorescence. When the intensity of the laser beam exceeds 70 mW / cm ² , the photosensitizer excited by the laser beam generates active oxygen together with the fluorescence, causing damage to the surrounding biological tissue, or modifying its own molecular structure to cause the fluorescence. The photo bleach phenomenon that does not emit the light occurs. Further, when the intensity of the laser beam is less than 7 mW / cm ² , the generated fluorescence becomes weak and observation becomes difficult. Therefore, the intensity of the laser beam is set at 7 mW / cm ² or more 70 mW / cm ² or less.

  When a camera is used for fluorescence observation, if the minimum sensitivity of the camera or the minimum subject illumination is 0.5 lux or more, the tumor site is fluorescent even if the dose of talaporfin natonarium as a photosensitizer is reduced. It can be identified based on the strength of.

  Immediately after administration of the photosensitive substance, there is a high concentration of the photosensitive substance in the blood. Therefore, immediately after the photosensitizer is administered, even if the target area is irradiated with laser light, strong fluorescence is emitted not only from the tumor but also from the blood vessels. Therefore, the location of the tumor in the target area is determined based on the intensity of the fluorescence. It is difficult to do. Specifically, it is difficult to determine the location of the tumor based on the intensity of fluorescence for less than 12 hours after administration of the photosensitive substance. In addition, since the photosensitizer is excreted as time passes after administration of the photosensitizer, the fluorescence from the tumor is weakened and the contrast with the surroundings decreases after a long time after administration of the photosensitizer. This makes it difficult to determine the location of the tumor. Specifically, when 24 hours have passed after administration of the photosensitive substance, it is difficult to determine the site of the tumor based on the intensity of fluorescence. Therefore, it is preferable to observe fluorescence emitted from the photosensitive substance excited by the laser beam within 12 hours to 24 hours after administration of the photosensitive substance to the subject.

  The wavelength of light that excites the photosensitive substance is determined for each substance. Many photosensitive materials have a strong light absorption band near a wavelength of 400 nm. However, excitation light in the vicinity of a wavelength of 400 nm generates natural fluorescence from the living tissue itself and reduces the fluorescence contrast from the photosensitive substance. In addition, the absorption band of hemoglobin contained in blood is in the range of 600 nm to 650 nm or less, and light having a wavelength of 650 nm or less is easily absorbed by hemoglobin, which is inferior in depth to living tissue. Further, when talaporfin natonarium is used as a photosensitive substance, the fluorescence peak from the living body is 672 nm, and it is difficult to separate the laser beam and the fluorescence when the laser beam wavelength is close to this peak wavelength. become. Therefore, since the half-value width of the semiconductor laser light is several nm, it is possible to perform fluorescence observation by exciting the talaporfin sodium sodium at a wavelength of 660 nm or more and 664 nm or less, which is a wavelength below the fluorescence peak, out of the hemoglobin absorption band. preferable.

  According to the present invention, the conditions of fluorescence observation by a photodynamic technique when a semiconductor laser is used as a light source and the dose of talaporfin sodium sodium which is a photosensitive substance is reduced are shown. By identifying and excising or necrotizing, improvement in the therapeutic effect of cancer and the like can be expected.

  FIG. 1 is a block diagram illustrating an example of a medical laser apparatus (fluorescence diagnosis apparatus) used in the fluorescence diagnosis method according to the embodiment. In FIG. 1, reference numeral 101 denotes a semiconductor laser, which has characteristics such that an oscillation wavelength during operation at an operating temperature of 0 ° C. is 664 nm and a half-value width is ± 1 nm. 102 is an optical fiber that guides the laser light oscillated by the semiconductor laser 101, 103 is a collimator lens that collimates the laser light guided by the optical fiber 102, and makes the laser light parallel light, and 104 is parallel light. A wavelength-shaping bandpass filter that removes the fluorescence wavelength component from the spectrum of the laser light. The wavelength 662 nm to wavelength 665 nm has a transmittance of 80% or more, and the wavelength 666 nm or more has a transmittance of less than 1%. Reference numeral 105 denotes an optical lens for uniformly irradiating the biological tissue (target area of the subject) 107, which is an observation area, with the laser light that has passed through the bandpass filter 104, and reference numeral 106 denotes an observation target of the laser light that has passed through the optical lens 105. Is a reflecting mirror for reflecting in the direction of the living tissue 107 to which the photosensitive substance is administered, 108 is a notch filter for removing the wavelength component of the excitation laser light from the fluorescence of the living tissue 107, and 109 is a living body that has passed through the notch filter 108. An objective lens 110 for imaging the fluorescence from the tissue is a CCD camera that captures an image formed by the objective lens 110, and the captured image is displayed on the image display device 111.

  Hereinafter, the fluorescence diagnostic method of the present embodiment will be described.

  First, a photosensitive substance having affinity for a tumor is administered to the subject, and the photosensitive substance is accumulated in the tumor in the living tissue 107.

In this embodiment, chlorin-based talaporfin sodium (trade name Rezaphyrin; manufactured by Meiji Seika Co., Ltd.) is used as a photosensitive substance. Talaporfin sodium has a maximum absorption wavelength of 664 nm and a central wavelength of fluorescence at 672 nm. In the present embodiment, 1 mg / m ² or more and 10 mg / m ² or less of talaporfin sodium are administered to the subject. As described above, in this embodiment, the dose of talaporfin natonarium is significantly reduced as compared with the conventional case (usually about 40 mg / m ² ).

  Next, the photosensitive material is excited by irradiation with laser light generated by the semiconductor laser 101. Then, the fluorescence emitted from the photosensitive substance excited by the laser light is observed, and the location of the tumor in the living tissue 107 is determined based on the intensity of the fluorescence.

  FIG. 2 schematically shows the concentration C1 of the photosensitive substance in the tumor or lesion tissue and the concentration C2 in the normal tissue around the tumor. Immediately after administration of the photosensitive substance, there is a high concentration of the photosensitive substance in the blood. Therefore, immediately after administration of the photosensitive substance, even if the living tissue 107 is irradiated with laser light, strong fluorescence is emitted not only from the tumor but also from the blood vessels. Therefore, based on the intensity of the fluorescence, the site of the tumor in the target region is determined. It is difficult to judge. Specifically, it is difficult to determine the location of the tumor based on the intensity of fluorescence for less than 12 hours after administration of the photosensitive substance. In addition, since the photosensitizer is excreted from the blood as time passes after administration of the photosensitizer, the fluorescence from the tumor becomes weaker after a long time after administration of the photosensitizer, and the fluorescence is detected. At the same time, the contrast with the surrounding area decreases, making it difficult to determine the location of the tumor. Specifically, when 24 hours have passed after administration of the photosensitive substance, it is difficult to determine the site of the tumor based on the intensity of fluorescence. Therefore, laser light irradiation and fluorescence observation are performed within 12 hours to 24 hours after administration of the photosensitive substance to the subject.

  Hereinafter, laser irradiation and fluorescence observation will be described in detail.

  In this embodiment, the semiconductor laser 101 oscillates a laser beam having a center wavelength of 664 nm and a half width of 2 nm (663 nm to 665 nm). The laser light is guided to the collimating lens 103 by the optical fiber 102. The collimating lens 103 collimates the laser light emitted from the optical fiber 102 into parallel light.

  The collimated laser light is incident on the band pass filter 104. FIG. 3 schematically shows the transmittance characteristics of the band-pass filter 104 together with the wavelength characteristics of the excited laser light and fluorescence. 201 is the intensity of the excitation laser beam, 202 is the wavelength characteristic of the transmittance of the bandpass filter 104 that removes part of the wavelength component of the excitation laser beam, and 203 is the fluorescence intensity emitted by the excited photosensitive substance. The wavelength characteristics of each are shown. The band-pass filter 104 cuts the base of the laser light that spreads in terms of wavelength. Specifically, the collimated laser light is attenuated by the band-pass filter 104 to a component having a wavelength of less than 660 nm and a wavelength of 666 nm or more to 1/100 or less.

The laser light transmitted through the band pass filter 104 is irradiated to the living tissue 107 by the optical lens 105 and the reflecting mirror 106. The intensity of the laser beam irradiated to a living tissue 107 is 7 mW / cm ² or more 70 mW / cm ² or less. When the laser light intensity is 15 mW / cm ² or more, the photosensitive substance excited by the laser light generates active oxygen together with the fluorescence, damages the surrounding living tissue, or the molecular structure of the device denatures to emit the fluorescence. The photo bleach phenomenon that does not occur may occur. Therefore, a high power density is not suitable for fluorescence observation. In addition, in the case of less than 7 mW / cm ² , the generated fluorescence becomes weak and observation becomes difficult. Fluorescence having an intensity corresponding to the accumulated concentration of the photosensitive substance is generated from the living tissue 107 to which the photosensitive substance irradiated with the excitation laser light is administered.

  The fluorescence generated from the living tissue 107 and the excitation laser beam reflected by the living tissue 107 are photographed by the CCD camera 110 to which the notch filter 108 is attached and displayed on the image display means 111. Based on the intensity of the fluorescence in the image, the location of the tumor in the living tissue 107 is determined.

  FIG. 4 schematically shows the wavelength characteristics of the notch filter 108 together with laser light and fluorescence. 301 is the wavelength characteristic of the excitation laser light intensity, 302 is the wavelength characteristic of the fluorescence intensity emitted by the photosensitive material excited by the excitation laser light, and 303 is the wavelength characteristic of the transmittance of the notch filter that cuts the excitation laser light. Each is shown. The laser light is cut from the fluorescence by the notch filter 108. Specifically, the region of wavelength 660 nm to wavelength 666 nm of the laser beam 301 and the fluorescence 302 is attenuated to 1/1000 or less (optical density OD3 or more). On the other hand, a fluorescent component having a wavelength longer than 666 nm passes through the notch filter 108 without being attenuated. The intensity of the laser light is already sufficiently weaker than that of fluorescence because the wavelength component of the wavelength less than 660 nm and the wavelength of 666 nm or more is attenuated to 1/100 or less by the bandpass filter 104. Thus, the fluorescence from which the wavelength component of the excitation laser light has been removed is photographed as a fluorescence image by the CCD camera 110 to which the objective lens 109 is attached.

In the present embodiment, as described above, the dose of talaporfin sodium sodium as a photosensitive substance is reduced to 1 mg / m ² or more and 10 mg / m ² or less. The intensity of fluorescence excited by this reduction in dosage tends to be weakened. However, if the minimum sensitivity of the CCD camera 110 or the minimum subject illuminance is 0.5 lux or more, the site where the tumor is present can be reliably identified based on the intensity of fluorescence.

  The CCD camera 110 can be observed with the naked eye. In this case, the objective lens 109 may be removed or replaced as necessary, such as the visual acuity of the observer. A CCD camera with higher sensitivity may be used.

  Table 1 and FIG. 5 below show the relationship between the product of the concentration of the photosensitive substance and the intensity of the excitation laser light and the intensity of the fluorescence. As shown in Table 1 and FIG. 5, the intensity of fluorescence increases as the concentration of the photosensitive substance increases and the intensity of the excitation laser light increases. Further, the concentration of the photosensitive substance and the intensity of the excitation laser beam are in a complementary relationship. For example, when the concentration of the photosensitive substance is reduced to half, the intensity of the fluorescence is maintained almost constant when the intensity of the excitation laser light is increased approximately twice. On the other hand, when the intensity of the excitation laser light is reduced to half, the intensity of the fluorescence is maintained almost constant when the concentration of the photosensitive substance is doubled.

  The fluorescence diagnostic method of the present invention can be used for detection of areas of malignant tumors including gliomas. That is, by capturing fluorescence from tumor cells infiltrating into a normal site, the tumor cells can be excised or necrotized, and an improvement in the therapeutic effect of cancer or the like can be expected.

The block diagram which shows an example of the fluorescence diagnostic apparatus used with the fluorescence diagnostic method concerning embodiment of this invention. The typical graph which shows the elapsed time and density | concentration after administration about a photosensitive substance. The schematic diagram which shows the wavelength distribution of a laser beam and fluorescence, and the wavelength characteristic of a band pass filter. The schematic diagram which shows the wavelength distribution of a laser beam and fluorescence, and the wavelength characteristic of a notch filter. The graph which shows the relationship between the product of the density | concentration of a photosensitive material, the intensity | strength of excitation laser beam, and the fluorescence intensity.

Explanation of symbols

DESCRIPTION OF SYMBOLS 101 Semiconductor laser 102 Optical fiber 103 Collimating lens 104 Band pass filter 105 Optical lens 106 Reflector 107 Biological tissue 108 Notch filter 109 Objective lens 110 CCD camera 201 Wavelength characteristic of excitation laser light intensity 202 Wavelength characteristic of transmittance of the band pass filter 104 203 Wavelength characteristic of fluorescence intensity 301 Wavelength characteristic of excitation laser light intensity 302 Wavelength characteristic of fluorescence intensity 303 Wavelength characteristic of transmittance of notch filter

Claims (3)

A method of diagnosing a tumor in a target area of a subject by fluorescence comprising:
1 mg / m ² or more and 10 mg / m ² or less of talaporfin natonarium is administered to the subject as a photosensitive substance having affinity for the tumor and accumulated in the tumor,
The central wavelength to a target area at 7 mW / cm ² or more 70 mW / cm ² or less of intensity by irradiating a laser beam generated by the following semiconductor laser 660nm or 664nm to excite the light-sensitive material,
Observing the fluorescence emitted by the photosensitive substance excited by the laser light, and determining the location of the tumor in the target region based on the intensity of the fluorescence,
Fluorescence diagnostic method. The fluorescence diagnostic method according to claim 1, wherein a camera is used for observation of the fluorescence, and a minimum subject illuminance of the camera is 0.5 lux or more.   The fluorescence diagnostic method according to claim 1, wherein the fluorescence emitted from the photosensitive substance excited by the laser light is observed within 12 to 24 hours after administration of the photosensitive substance to the subject.

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