WO1998001131A1

WO1998001131A1 - Drugs inducing ultrasonic action and apparatus wherein the drugs are used

Info

Publication number: WO1998001131A1
Application number: PCT/JP1997/002285
Authority: WO
Inventors: Kenichi Kawabata; Shinichiro Umemura; Kazuaki Sasaki; Nami Sugita
Original assignee: Hitachi, Ltd.
Priority date: 1996-07-05
Filing date: 1997-07-02
Publication date: 1998-01-15

Abstract

Drugs containing compounds of xanthene dyes or derivatives thereof (including dimers) having xanthene ring(s) and inducing an ultrasonic action of lowering the threshold of acoustic strength causing acoustic cavitation, wherein any of R1 to R8 bonded to carbon atoms of the xanthene dye skeleton is a functional group capable of chemically binding to a halogeno, thiol or amino group (selected from among halogenated acetamide, maleimide, aziridine, isothiocyanate, succinimide and sulfonyl chloride). Because of being able to lower the threshold, these drugs make it possible to safely treat benign or malignant tumors or stones by the irradiation with ultrasonic waves of a low acoustic strenght.

Description

Description: Agent for inducing ultrasonic action and ultrasonic device using the same

The present invention relates to a method for treating acoustic or cavitation using acoustic cavitation, a chemical reaction by ultrasonic waves, generation of bubbles by ultrasonic waves, ultrasonic imaging using bubbles, or sterilization of a liquid. The present invention relates to an agent for inducing an ultrasonic action, which contains a compound having an action for reducing the effect of an ultrasonic wave, and an ultrasonic apparatus using the same. Background art

Techniques for treating tumors, calculi, etc. by irradiating focused ultrasound from outside the body are less invasive than open surgery, lowering physical strength during surgery and quality of life after surgery (Quality of Life). ) Is excellent in principle, and is considered to increase social value in the future. Acoustic cavitation is considered to play an important role in the mechanism by which the therapeutic effect can be obtained by the irradiation of convergent ultrasonic waves, and in the mechanism of promoting the chemical reaction by the irradiation of ultrasonic waves. In the Journal of Acoustical Society of America (J. Acust. Soc. Am., Vol. 89, p. 2740, 1999), acoustic cavitation occurs in the living body and bubbles It is described that when generated, the generated bubbles can produce an ultrasound contrast effect.

Hitherto, as a method for efficiently generating and crushing acoustic cavities, a method using exclusively physical means has been proposed. For example, Japanese Patent Application Laid-Open No. 2-126848 describes a technique of irradiating an ultrasonic wave by changing a sound field at a time interval of 1 ms ec to 100 msec. JP Hei 2 -The technology described in JP-A-26848 focuses on the fact that the ultrasonic irradiation time required to generate acoustic cavitation is 1 msec to 100 msec, and two types of sound fields with different ultrasonic wavefronts are used. The sound field is switched by repeating the cycle of irradiating ultrasonic waves while switching at time intervals of 1 msec to 100 msec, and crushing the hairpin cavitation generated by one sound field by the other sound field. The efficiency of sonochemical action is improved by an order of magnitude at the same ultrasonic power compared to the case without.

In addition, Ultrasonics Sonoch em istryvo 1.3, No. 1, pp. 1-6 (1996) (Kawa bata, K and Umemu ra, S) usually obtains only when there is a reflective object. The technique of the second harmonic superposition method, which obtains an ultrasonic wave having a waveform that is advantageous for generating acoustic cavitation even in a situation where there is no reflector, describes an ultrasonic wave that is advantageous for generating acoustic cavitation. When a multiple frequency of this one frequency component is superimposed on a waveform having one frequency component and used as a sound wave waveform, and a living body is irradiated with ultrasonic waves having multiple center frequencies at the same time, treatment by ultrasonic irradiation is used. It is expected that the effect of ultrasonic irradiation will be enhanced and safety will be improved in the future.

In Japanese Patent Publication No. 6-29196, as a method of chemically enhancing the antitumor cancer effect of ultrasound, a substance such as porphyrin is used as a substance that generates active oxygen by the chemical action of ultrasound. Although the method is described, a substance such as porphyrin has a force that has a function of generating active oxygen secondary by acoustic cavitation generated by ultrasonic waves, and a threshold value for generating acoustic cavitation. Can not cause a decline in

Disclosure of the invention

In the conventional method based on the physical sound field, individual ultrasonic waves are converged, especially when the ultrasonic waves are converged and irradiated to the patient using multiple ultrasonic sources. W

Three

Since it is necessary to reach the target site while maintaining the sound intensity and the relative phase difference between the ultrasonic waves emitted from the source in a constant relationship, it is expected for an acoustically nonuniform object such as a living body. In some cases, a therapeutic effect (effect obtained on a uniform subject) may not always be obtained. If the threshold of the sound intensity at which acoustic cavitation is generated by a chemical method (hereinafter simply referred to as “acoustic cavitation threshold”) can be reduced, the therapeutic effect will be impaired even for non-uniform objects. And treatment by ultrasound is possible. However, we did not keep in mind that the conventionally reported substances that enhance the biological effects of ultrasound have the function of lowering the threshold of acoustic cavitation.

It is known that the threshold of acoustic cavitation changes depending on the interfacial tension and viscosity of a liquid (water in living organisms). Until now, it has not been known that the viscosity of water is changed by relatively low-concentration substances, and it has been practically difficult to change the viscosity of water and reduce the 閎 value of the acoustic cavitation. The addition of a relatively small amount of surfactant can change the interfacial tension, and changing the interfacial tension can change the threshold of acoustic cavitation. Acoustic cavitation in water consists of the process of creating microbubbles that create cavities between water molecules, the process of growing microbubbles, and the process of collapsing the grown bubbles. It is expected that the process of generating the first microbubbles will proceed at a lower acoustic intensity due to the decrease in interfacial tension due to the surfactant, but in actuality, the presence of the surfactant will result in acoustic cavitation. It is known that the threshold is rather high (LA Crum: Nature vol. 278, no. 8, pp. 148-149 (1799)). Decreasing the threshold value of acoustic cavitation may require a high interfacial tension in the collapse of bubbles in the last stage of acoustic cavitation. Focusing solely on surfactant activity or viscosity, acoustic cavities can be created by using low-concentration substances. It was not possible to lower the threshold.

It is an object of the present invention to provide an agent that induces an ultrasonic action (hereinafter referred to as an ultrasonic action-inducing (or attraction) technique, which makes it possible to generate acoustic cavitation with lower acoustic intensity, which was not possible with the prior art approach. In particular, short-lived chemical species such as hydroxyl radicals generated by acoustic cavitation, and secondary generated chemical species caused by these short-lived chemical species. An object of the present invention is to provide an ultrasonic action inducer that does not suppress (prevent) the chemical action of short-lived chemical species in order to perform a chemical treatment based on the drug, and to provide an ultrasonic apparatus using the drug.

The ultrasonic action inducer of the present invention comprises a compound comprising a xanthene dye containing a xanthene ring or a derivative of a xanthene dye, and is an agent for lowering the threshold of acoustic intensity for generating acoustic cavitation. Having two or more halogens bonded to the carbon atom of the skeleton of the xanthene dye, the above derivative being bonded to the carbon atom of the skeleton of the xanthene dye, a halogenated acetate amide group, a maleimide group, an aziridine group, and an isothiocyanate group. Group, a succinimide group, or a sulfonyl chloride group, and this functional group is characterized in that it can be chemically bonded to a thiol group or an amino group. For example, R ₂ shown in Fig. 4 is any functional group of a halogenated acetamido group, maleimide group, aziridine group, isothiocyanate group, succinimid group, and sulfonyl chloride group. . Further, the above derivative has the structure of ONa and COONa in which H of OH and COOH is replaced by Na in FIG. 4, and may be a salt of a xanthene dye.

In addition, a substance in which two molecules of a xanthene dye or a derivative of a xanthene dye containing a xanthene ring are linked by — (CH ₂ ) „-(3 ≤ n (integer) (hereinafter, a xanthene dye or a derivative of a xanthene dye derivative) Dimer), and the threshold of the sound intensity to generate acoustic cavitation. The above derivative has two or more halogens bonded to the carbon atom of the skeleton of the xanthene dye. The derivative has a halogenated acetoamide group bonded to the carbon atom of the skeleton of the xanthene dye. It has a maleimide group, aziridine group, isothiocyanate group, succinimide group, or sulfonyl chloride group, which can be chemically bonded to a thiol group or an amino group. ,, Etc. For example, R ₂ shown in FIG. 10 is any one of a halogenated acetoamide group, a maleimide group, an aziridine group, an isothiocyanate group, a succinimid group, and a sulfonyl chloride group. Further, the above derivative has a structure of ONa in which H of OH is replaced by Na in FIG. 10, and may be a salt of a xanthene dye or a dimer of a derivative of the xanthene dye.

An ultrasonic apparatus using the ultrasonic wave inducer of the present invention includes a compound (including a dimer) of a xanthene dye containing a xanthene ring or a derivative of a xanthene dye, and has a threshold of an acoustic intensity for generating an acoustic cavitation. A means for administering the ultrasonic action inducer to be reduced to a target site of the subject; and a means for superimposing ultrasonic waves having a plurality of center frequencies having an acoustic intensity of 1 O WZ cm ² or less and irradiating the target site. And means for detecting ultrasonic waves from a target site.

An ultrasonic treatment method using the ultrasonic action inducer of the present invention comprises a compound comprising a xanthene dye containing a xanthene ring or a derivative of a xanthene dye (including a dimer), and comprises a threshold of acoustic intensity for generating acoustic cavitation. Administering an ultrasonic action-inducing agent to the target site on the subject, and irradiating the target site with superimposed ultrasonic waves having multiple center frequencies with an acoustic intensity of 1 O WZ cm ² or less And a step of detecting ultrasonic waves from a target site.

It is thought that the surfactant does not promote acoustic cavitation due to the disadvantageous effect of low interfacial tension during acoustic cavitation. You. In addition, surfactants generally form relatively large bubbles, as is prominent in detergents, and it is considered that the large bubbles that have formed adversely affect the formation of acoustic cavitation. In addition, the strong surfactant action of surfactants has the effect of destroying cell membranes of living organisms, causing strong biotoxicity. If surfactants have a substance that has the function of promoting the production of acoustic cavitation. Even so, application to living organisms is difficult. From this point of view, we have a weak interaction with water molecules in organic compounds that have hydrophilicity and lipophilicity (lipophilicity) like surfactants, and do not have strong surfactant activity. A substance that weakens the interaction between water molecules to facilitate the generation of acoustic cavitation and that does not reduce the interfacial tension in general, or a substance that generates bubbles of appropriate size for sound cavitation due to its weak foaming ability. I've been searching. In particular, when the chemical action of acoustic cavitation is used for therapy, a substance that does not inhibit (block) the chemical action of the short-lived species generated by acoustic cavitation is desirable.

Dyes are used to generate short-lived species by light irradiation, and are unlikely to act as scavenging agents for short-lived species. In the search, the evaluation of the effect of acoustic cavitation generation was performed using a method that confirmed the generation of subharmonic waves. This method is an acoustic measurement and can directly measure the movement of the bubble itself compared to other methods. When ultrasonic waves generate short-lived chemical species such as hydroxyl radicals, the subharmonic wave can be measured. The generation must be observed (Ken—ichi Kawabataand Shin—ichiro Umemu ra; The J ourna 1 of Ph ysical Chem istryvol. 100, no. 48, p p. 1 996))).

Ultrasonic waves are applied by an ultrasonic probe (details of the configuration will be described later) that measures the generation of acoustic cavitation in a liquid with the configuration shown in Fig. I. The acoustic signal from the sample was measured to determine whether subharmonics were generated. Figure 2 shows the spectrum of a typical acoustic signal when acoustic cavitation occurs. (Figure 2 shows a 1 MHz solution of Rose Bengal in a 0.1 mM (millimolar) solution. FIG. 4 is a diagram showing a typical spectrum of an acoustic signal obtained when acoustic cavitation is generated in a liquid by simultaneously irradiating ultrasonic waves of ² MHz and 2 MHz at an intensity of 5 W / cm ² for 1 minute. The vertical axis indicates the relative value of the average signal intensity based on 1 × 10 ″ ⁸ (mV) ² ).

1MHz and 2M H2 in a 0.1 mM (mmol) solution of the xanthene dye erythroxin, the thiazine dye methylene blue, the porphyrin dye hematoporphyrin, and the azine dye riboflavin Ultrasonic waves of z were simultaneously irradiated at an intensity of 5 W / cm ² for 1 minute, and a subharmonic signal generated during the irradiation was measured every second. The mean square value of the amplitude of the subharmonic waves in the irradiation time, the subharmonic intensity as relative values relative to the ^{1 X 1 0- 7 (mV)} 2, was used as an indicator of the degree of generation of subharmonics . The higher the signal strength of the subharmonic wave, the more active the acoustic cavitation is, which means that the acoustic cavitation is generated at a lower acoustic intensity.

Figure 3 shows the intensity of subharmonic waves generated when a solution containing various dyes was irradiated with ultrasonic waves. In the control experiment, the intensity of subharmonic waves obtained by irradiating ultrasonic waves to a phosphate buffer solution was measured. Is shown. As is evident from Fig. 3, large subharmonic signals are obtained only in the case of xanthene-based erythrocycin.

In addition, a solution of the dyes classified as xanthene dyes, fluorescein, tetralucorin fluorescein, eosin Y, ellis cincinus, phloxin B, and rose bengal, at a concentration of 0.1 mW [1 MHz and 2 MHz Fig. 6 shows the results of simultaneously irradiating an ultrasonic wave of z with an intensity of 5 WZ cm ² for 1 minute and measuring the subharmonic signal generated during the irradiation every second.

FIG. 4 is a diagram showing the structure of a compound comprising a xanthene dye or a derivative of a xanthene dye containing a xanthene ring used in a test example of the present invention. FIG. 5 is a diagram showing the structure of a compound comprising a derivative of a xanthene dye or a xanthene dye used in a test example of the present invention. Xanthene dyes are also derivatives of phthalic acid and are also called phthalein dyes. All but phenolphthalene contain a xanthene ring in the molecule. In PH 7.4, the xanthene dye has the structure shown in Fig. 4, and a compound having a salt structure of ONa and COONa in which H of OH and COOH is replaced by Na is generally widely used. . Fluorescein (F 1 u 0 rescein), dichlorofluorescein (2 '7' -D ichlorofluorescein), tetrachloro mouth fluorescein (4,5,6,7 — Tetrachlorofluorescein), eosin Ε (osin Y) mouth Shin (E rythrosin), phloxine B (P h 1 oxin B) , rose downy Bengal (Ro sebengal) is, RR _2, R ₃ shown in FIG. _{_{4, R 4, R 5, Re}} , R 7, R 8 Has the structure shown in Fig. 5 as an element. The salt of ONa or COONa in which H of OH and COOH is replaced by Na may be used.

Figure 6 is a diagram showing the intensity of subharmonic waves generated when a solution containing a xanthene dye or a derivative of a xanthene dye is irradiated with ultrasonic waves. The vertical axis represents the intensity of the subharmonic wave at 1 X 1 CT ⁷ (mV) ² Is shown as a relative value based on. As is clear from Fig. 6, the effect of the xanthene dye containing a halogen atom on the acoustic intensity required for the production of acoustic cavitation was reduced, and the halogen atom was added to the skeleton of the carbon atom of the xanthene dye shown in Fig. 4. It has been confirmed that the compound with the compound has the effect of lowering the sound intensity required for the generation of acoustic cavitation, especially for compounds having two or more halogen atoms in the molecule. .

The fact that the threshold force of acoustic cavitation in a liquid in which cells such as cancer cells or red blood cells are suspended, ', is lower than the threshold of acoustic cavitation in water, means that proteins on the cell surface may With the core of cavitation It is thought to be due to Based on the belief that it is more effective to lower the threshold of cavitation in body fluids (water) in living organisms, it is more effective to use cells in body fluids as nuclei of cavitation. We searched for compounds that could be present at a higher rate near the surface.

Chemical substances in which a functional group capable of binding to a thiol group or amino group of an amino acid is chemically bonded to a xanthene dye have high affinity for cell membranes (Cobb, C and Beth, A; Biochemistry 1. 29, No. 36, pp. 8283-8290 (1990)). As a functional group capable of binding to a thiol group or an amino group, halogenated acetamide, maleimide, aziridine, isothiocyanate, succinimide, and sulfonyl chloride are known. For the xanthene dye derivative chemically bonded with a functional group capable of binding to a thiol group or an amino group, the effect of lowering the sound intensity required for generating sound cavitation was investigated. FIG. 7 is a diagram showing the structure of a compound comprising a xanthene dye or a derivative of a xanthene dye having a functional group capable of chemically bonding to a thiol group or an amino group. The structure of these xanthene dye derivatives is as follows: R, R ₂ , R ₃ , R ₄ , R ₅ , R ₆₎ shown in Fig. 4 where R ₇ and R ₈ are the elements or functional groups shown in Fig. 7. . The salt of ONa or COONa in which H of OH and COOH is replaced by Na may be used. R ₂ shown in FIG. 4 is an iodoacetamido group, an isothiocyanate group, a maleimide group, a derivative of a xanthene dye shown in FIG. For acoustic amide (Erythrosin-5-iodoacet amide), erythrosin-5-isothiocyanate, and erythrosin-5-ma1 eimide, acoustic cavitation Lowers the sound intensity required for generation Since interaction with the living body is particularly important for the mechanism, the effect of lowering the threshold of acoustic cavitation was examined using a living body (ddY mouse (os, 5 weeks old)).

Each compound shown in Fig. 7 is administered to ddY mice at a dose of 50 mg / Kg body weight, and the ultrasonic device shown in Fig. 8 is used to measure the production of acoustic cavitation in a living body. (Details will be described later) using 0.5 Μ _{Ζ Ζ} and 1 MHz ultrasonic waves at a sound intensity ratio of 1: 1 to generate subharmonic waves and tissue damage from mouse liver. Was observed.

FIG. 9 is a diagram showing threshold values of acoustic cavitation in mouse liver when a compound comprising a xanthene dye or a derivative of a xanthene dye having a functional group capable of chemically bonding to a thiol group or an amino group is present. In Figure 9, the threshold for acoustic cavitation in the mouse liver was defined as the minimum acoustic intensity that produced both subharmonics and tissue damage. In the control experiment shown in Fig. 9, physiological saline was administered to the mice instead of the acoustic cavitation inducer, and the mice were irradiated with ultrasonic waves. As a result of observing the generation of subharmonic waves and the generation of tissue damage by varying the concentration of each compound solution administered to mice, the functional groups capable of binding to the thiol group were observed for any of the substances shown in Fig. 7. Compared to erythrosine without γ, the 闞 value of acoustic cavitation was reduced to the same or higher level. The same acoustic cavitation threshold value as when erythro-cindoacetamide was administered at a dose of 5 mg ZK g body weight using an erythrocystine without a functional group capable of binding to a thiol group or an amino group. In order to produce a decrease in the concentration, it is necessary to administer a 10-fold dose of 50 mg ZK g body weight, and use a substance having a functional group capable of binding to a thiol group to lower the threshold value of acoustic cavitation at a lower concentration. This has been made possible. The halogenated acetamido group, maleimide group, aziridine group, isothiocyanate group, succinimid group, and sulfonyl chloride group, which are functional groups capable of binding to a thiol group or an amino group, are: the 4th Regardless of the position of R ₂ , R ₃ , R ₄ , R ₅ , R _s , R ₇ , or R ₈ in the carbon atom skeleton of the xanthene dye shown in the figure, a thiol group or is capable of binding with Amino _{_{_{group, R lt R 2, R 3}}} , R 4, R 5, R 6, R 7, even when in any position of the R _a, acoustic needed to generate acoustic Canon bi station It has the effect of reducing strength. Further, a plurality of functional groups capable of bonding to a thiol group or an amino group may be bonded to the carbon atom skeleton of the xanthene dye shown in FIG.

Xanthene dyes and derivatives of xanthene dyes generally have high hydrophilicity and, when administered to a living body, flow out of the living body in a relatively short period of time. When used as an inducer, a protocol that administers to a living body at a relatively high concentration and irradiates the living body with ultrasonic waves in a relatively short time after the administration is effective. In particular, xanthene dyes or derivatives of xanthene dyes are considered to be suitable for treatment of calculi, ultrasonic imaging using bubbles, and the like.

However, when considering the treatment of tumors, it may be difficult to allow highly hydrophilic substances to be present in high concentrations in a part of benign or malignant tumors. Using xanthene dye alone was not enough.

To obtain an acoustic cavitation effect inducer with high fat solubility, we first searched for a substance that was hydrophilic and lipophilic (lipid-soluble) in the same way as xanthene dyes, and had higher lipophilicity than xanthene dyes. Power of searching for dyes Neither hematoporphyrin nor riboflavin shown in Fig. 3 was effective as an acoustic cavitation effect inducer.

Next, we searched for an acoustic cavitation effect inducer in which a hydrophobic group was added to a xanthene dye or its derivative. In order to increase the hydrophobicity of a compound, a method of adding an alkyl group is generally used. Derivatives of xanthene dyes produced by this method have a hydrophobic part and a hydrophilic part in the molecule. Molecules may exist in an aqueous solution or body fluid in the form of micelles, and the physicochemical properties of the molecule in an aqueous solution or body fluid may be a xanthene dye or its derivative. It may be different from the physicochemical properties of the original molecules. In order to change only the hydrophobicity of the xanthene dye or its derivative without altering its original physicochemical properties as much as possible, it is desirable to make it a dimer. A search was made for compounds having a dimeric structure cross-linked by a hydrophobic alkylene group (1 (CH ₂ ) _π —: 3≤n (integer) 0). Compounds having a dimer structure in which two molecules of a xanthene dye or its derivative are cross-linked by one (CH ₂ ) „-(3≤n (integer) ≤20) are described in the literature (Photochem. Ph otobiol). Vol. 47, No. 4, pp. 551-557 (1988)) Figure 10 shows that two of the molecules consisting of a xanthene dye or a derivative of a xanthene dye are — ( ₂ is a diagram showing the structure of a dimer compound formed by bonding with CH ₂ ) _n — — (CH ₂ ) _n — (3≤n (integer) ≤20), whereby two molecules of a xanthene dye or its A compound having a dimeric structure obtained by cross-linking a derivative is administered to mice, and the generation of subharmonic waves and tissue damage from the liver of the mouse is performed using an ultrasonic device shown in Fig. 8 (details will be described later). The production of acoustic cavitation was measured by the method: Rose Bengal, Rose Bengal Dimer (Structure: Fig. 10 And each compound was administered to d dY mice at a dose of 5 OmgZKg body weight, using an ultrasonic device (see details later) with the configuration shown in FIG. Ultrasonic waves of 5 MHz and 1 MHz were simultaneously irradiated at a sound intensity ratio of 1: 1 to observe the generation of subharmonics and the generation of tissue damage from the mouse liver. Fig. 11 shows the threshold value of acoustic cavitation in the mouse liver in the presence of Rose Bengal dimer, In Fig. 11, the threshold value of acoustic cavitation in the mouse liver is: , Subharmonic and tissue damage Is defined as the minimum sound intensity that produces both In the control experiment shown in Fig. 11, mice were injected with physiological saline and irradiated with ultrasound. Administration of the Rose Bengal dimer and administration of Rose Bengal to mice produced acoustic cavitation at approximately the same sound intensity. From these results, it can be seen that a compound having a dimer structure in which two molecules of a xanthene dye or a derivative thereof are crosslinked by one (CH ₂ ) _n- (3 <n (integer) ≤ 20) has high hydrophobicity. It was also found that the acoustic intensity required to produce acoustic cavitation was reduced to the same extent as one molecule of the xanthen dye or its derivative. In FIG. 10, the same effect was obtained when a compound having a salt structure of ONa in which H of OH was replaced with Na was used. Monomers of xanthene dyes or their derivatives are poorly soluble in chloroform, but dimers are readily soluble in chloroform and highly lipophilic. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a diagram showing a configuration of an ultrasonic apparatus for measuring the generation of acoustic cavitation in a liquid in a test example of the present invention.

FIG. 2 shows a typical spectrum of an acoustic signal obtained when acoustic cavitation is generated in a liquid in a test example of the present invention. FIG. 3 shows a test pattern of the present invention. In the example, a diagram showing the intensity of subharmonic waves generated when a solution containing various dyes is irradiated with ultrasonic waves,

FIG. 4 is a diagram showing the structure of a compound comprising a xanthene dye containing a xanthene ring or a derivative of a xanthene dye used in the test example of the present invention, and FIG. 5 is a diagram showing the xanthene dye used in the test example of the present invention. Figure showing the structure of a compound consisting of a dye or a derivative of a xanthene dye.

FIG. 6 is a diagram showing the intensity of subharmonic waves generated when a solution containing a xanthene dye or a derivative of a xanthene dye is irradiated with ultrasonic waves in the test example of the present invention. FIG. 7 is a diagram showing the structure of a compound comprising a xanthene dye or a derivative of a xanthene dye having a functional group capable of chemically bonding to a thiol group or an amino group in a test example of the present invention.

FIG. 8 is a diagram showing a configuration of an ultrasonic device for measuring the generation of acoustic cavitation in a living body in a test example of the present invention.

FIG. 9 shows the results obtained in the test example of the present invention in the mouse liver when a compound comprising a xanthene dye or a derivative of a xanthene dye having a functional group capable of chemically bonding to a thiol group or an amino group is present. Diagram showing the threshold of acoustic cavitation

The first 0 figures In the test examples of the present invention, two molecules consisting of derivatives of xanthene dyes or xanthene dyes one _(CH ₂₎ π - shows the structure of a dimer which is formed by combining the Figure,

Fig. 11 is a graph showing the threshold value of acoustic cavitation in mouse liver in the presence of Rose Bengal and Rose Bengal dimer in the test example of the present invention.

FIG. 12 is a diagram showing a configuration of an ultrasonic apparatus for measuring an antitumor (tumor growth inhibition) effect in a living body in a test example of the present invention.

Fig. 13 is a graph showing the dependence of the intensity of the subharmonic wave generated when a solution containing a xanthene dye is irradiated with ultrasonic waves on the acoustic intensity in the test example of the present invention.

Fig. 14 is a graph showing the dependence of the amount of active oxygen generated on the sound intensity when a solution containing a xanthene dye was irradiated with ultrasonic waves in the test example of the present invention.

FIG. 15 is a graph showing the inhibition rate of tumor growth when mice were administered with Rose Bengal and irradiated with ultrasonic waves in the test example of the present invention.

Fig. 16 is a graph showing the calculus breaking ratio when calculus in a solution containing a xanthene dye was irradiated with ultrasonic waves in the test example of the present invention. Fig. 17 shows the results of the modulation produced when a solution containing a derivative of a xanthene dye having a functional group capable of chemically bonding to a thiol group or an amino group was irradiated with ultrasonic waves in the test example of the present invention. Figure showing the dependence of wave intensity on sound intensity.

Fig. 18 shows the active oxygen content of a solution containing a compound comprising a derivative of a xanthene dye having a functional group capable of chemically bonding to a thiol group or an amino group in a test example of the present invention. Figure showing the dependence of the amount of generated sound on the sound intensity.

FIG. 19 is a graph showing the inhibition rate of tumor growth when mice were administered with erythrochloride acetoacetamide and irradiated with ultrasonic waves in the test example of the present invention. FIG. FIG. 4 is a graph showing stone destruction rates when a stone in a solution containing a compound comprising a derivative of a xanthene dye having a functional group capable of chemically bonding to a thiol group or an amino group is irradiated with ultrasonic waves in a test example; Figure 21 shows that in the test example of the present invention, a solution containing a dimer formed by binding _two molecules of a xanthene dye or a derivative of a xanthene dye by one (CH ₂ ) _π— Fig. 4 shows the dependence of the intensity of the subharmonic generated when sonic waves are irradiated on the acoustic intensity.

FIG. 22 shows a solution containing a dimer formed by bonding _two molecules of a xanthene dye or a derivative of a xanthene dye with — (CH ₂ ) _n — in the test example of the present invention. Diagram showing the dependence of the amount of active oxygen generated on sound intensity when irradiated with ultrasonic waves.

FIG. 23 shows the structure of a dimer formed by bonding _two molecules of a xanthene dye or a derivative of a xanthene dye with — (CH ₂ ) _n — in a test example of the present invention. Figure,

Fig. 24 shows the results obtained when two molecules of a xanthene dye or a derivative of a xanthene dye are linked by — (CH ₂ ) _n — in a test example of the present invention. Of acoustic cavitation in mouse liver Diagram showing the threshold of

FIG. 25 is a graph showing the inhibition rate of tumor growth when mice were administered with Rose Bengal dimer and Eris mouth simple acetate amide dimer in a test example of the present invention and irradiated with ultrasonic waves. is there.

The third diagram, Figure 6, the first 3 diagrams, first 7 view, in the second 1 figure, subharmonic intensity mean square value of the amplitude of subharmonic 1 X 1 0- ⁷ (mV) expressed as a relative value based on ² , and in Fig. 14, Fig. 18, and Fig. 22, the amount of active oxygen generated is the amount that can oxidize iodine ions at a rate of 1 (micromol) in. The relative value is set to 1. BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, in order to explain the present invention in detail, test examples in which the effect of the ultrasonic action inducer of the present invention has been confirmed will be specifically described with reference to the accompanying drawings. However, the present invention is not limited to these test examples. Not something.

Evaluation experiment A: “Measurement of formation of acoustic cavitation in liquid” Fig. 1 shows the results of the test described below, using the ultrasonic action-inducing (attracting) agent of the present invention. FIG. 3 is a diagram illustrating a configuration of an ultrasonic device that measures the generation of acoustic cavitation. As a sample solution 4, a phosphate buffer solution (pH = 7.4) containing an ultrasonic wave inducer at a concentration of 1 × ^10—4 M was used. Fix the bag 3 to the fixture 6 with pinch cocks 5-1 and 5-2, and put it in the water tank 2 filled with deaerated water 1. The waveform generator 14 synthesizes a sine wave having a frequency of 1 MHz and a sine wave having a frequency of 2 MHz, amplifies the resulting signal with an amplifier 13, and inputs the amplified signal to a planar ultrasonic transducer 9 held by a fixture 10. Ultrasonic waves at frequencies of 1 MHz and 2 MHz are simultaneously radiated from the ultrasonic transducer 9 for 1 to 2 minutes. During the ultrasonic irradiation, the sound pinion signal from the sample solution 4 is held by the holder 8. Underwater microphone 7 Measure more. The acoustic signal measured by the underwater microphone 7 is amplified by the amplifier 11 and then input to the spectrum analyzer 12 to extract the signal component of 500 MHz, which is a subharmonic of 1 MHz, every second. From 15, the time average of the square of the subharmonic component is calculated. The time average of the square of the subharmonic component was defined as the subharmonic intensity, which was used as an index of the magnitude of acoustic cavitation generation. The control experiment was performed by replacing the phosphate buffer (PH = 7.4) containing the sonication inducer with a phosphate buffer (pH = = 7.4).

Evaluation experiment B _: “Measurement of the production of acoustic cavitation in a living body” Fig. 8 shows the results of the following test examples in which the ultrasonic action-inducing (attracting) agent of the present invention was used. FIG. 3 is a diagram illustrating a configuration of an ultrasonic device that measures the generation of acoustic cavitation. In anaesthetized d dY mice (5-week-old, female), the ultrasound effect inducer was administered at a dose of 5 mg / Kg body weight (Test Example 7) or a dose of 5 OmgZKg body weight (Test Example 13). After intravenous injection, with the liver 25 exposed outside the body, the mouse 24 was fixed in a water tank 2 filled with degassed water 1, and focused ultrasound at frequencies of 0.5 and 1 MHz was applied at an acoustic intensity ratio. The liver 25 was irradiated at the same time so that the ratio became 1: 1.

Ultrasonic irradiation by the 2nd harmonic superposition method (see literature, Ultrasonics Sonochemistry vol. 3, No. 1, pp. 1-6 (1996) (Kawabata, K and Urnemura, S)) is possible. The converging ultrasonic transducer 26 is composed of piezoelectric elements 28-1, 28-2, ..., 28-N and 0.5MHz ultrasonic waves arranged on a ring for irradiating a 1MHz ultrasonic wave. It consists of piezoelectric elements 29-1, 29-2, ..., 29-N arranged on the irradiation ring. The anesthetized mouse 24 fixed to the fixture 23 is placed in the water tank 2 filled with degassed water 1, and the liver 25 exposed outside the body is fixed at the focal point 27 of the focused ultrasonic transducer 26. Move tool 23. After intravenous injection of an ultrasonic action inducer, ultrasonic waves were conveyed by a convergent ultrasonic transducer 26 for 1 hour. Irradiate for 80 seconds, and visually check for damage to liver 25 during the irradiation time. In addition, the acoustic signal obtained from the liver 25 during ultrasonic irradiation is measured with a submerged microphone 31 to check for the presence of the 250 MHz subharmonic component of 0.5 MHz. The minimum acoustic intensity at which the damage and subharmonic waves of the liver 25 were observed by the irradiation of the ultrasonic wave was set as the acoustic cavitation threshold, and it was determined that the acoustic cavitation was generated and that the biological effect was caused by the action of the acoustic cavitation. In the control experiment, physiological saline was injected intravenously instead of the ultrasonic wave inducer, and irradiated with ultrasonic waves.

The focal point 27 is confirmed using an imaging ultrasonic probe 30 composed of multiple transducers. 32 is a processing circuit for controlling the transmission and reception of ultrasonic waves by the imaging ultrasonic probe 30 and obtaining an image, 34 is a transmission waveform generation circuit for driving the piezoelectric element of the converging ultrasonic transducer, and 33 is an amplifier for amplifying the transmission waveform. Width circuit, 35 is an amplification circuit that amplifies the reception signal of the underwater microphone 31,

Reference numeral 36 denotes a reception waveform processing circuit for calculating a time average of the square of the subharmonic component. Evaluation experiment C: "Measurement of effects that promote sonochemical reactions"

In the following test examples, the effect of the ultrasonic action-inducing (attracting) agent of the present invention to promote the sonochemical reaction was determined by measuring the amount of active oxygen produced, which is the source of the antitumor effect by ultrasonic chemical action. investigated. The amount of generated active oxygen was measured by the oxidation reaction of iodine ions generated by the active oxygen. The progress of the reaction was investigated by spectroscopically measuring the concentration of triiodide ion, an oxidation product of iodine ion. Ultrasonic action inducing agent and phosphoric acid buffer solution containing iodide force Riumu each at a concentration of 1 X 10- ⁴ Micromax and 0. 1 Μ (ρΗ = 7. 4 ) Polyethylene bags (thickness of the present invention 0 03 mm), and were irradiated simultaneously with 1 MHz and ² MHz ultrasonic waves under the conditions of sound intensity of 0 to 1 OWZcm ² and irradiation time of 1 minute. In the control experiment, the phosphate buffer containing an ultrasonic wave inducer (pH 7.4) was added to the phosphate buffer (pH = 7.

4). Evaluation Experiment D: “Measurement of Antitumor (Tumor Growth Inhibition) Effect in Living Body” FIG. 12 shows the results of a test using the ultrasonic action-inducing (attracting) agent of the present invention in the following test examples. FIG. 3 is a diagram showing a configuration of an ultrasonic apparatus for measuring an antitumor (tumor growth inhibition) effect in the inside. The configuration of the ultrasonic device shown in FIG. 12 is the same as the configuration of the ultrasonic device shown in FIG. The measurement of the antitumor effect (tumor growth inhibition test) is performed as follows. Seven-week-old male BAL BZc mice (3 mice per group) were transplanted with C 0 10 n 26 cells subcutaneously in the abdomen, and then administered an ultrasound inducer when the tumor diameter reached approximately 1 cm. It was injected intravenously to give an amount of 5 OmgZkg body weight. The anesthetized mouse 24 fixed to the fixture 23 is placed in the water tank 2 filled with deaerated water 1, and the subcutaneously implanted tumor of about 1 cm in diameter comes to the focal point 27 of the converging ultrasonic transducer 26. Move the fixture 23. After administration by intravenous injection of ultrasonic action inducing agents, converged by the ultrasound transducer 26, in 0. 5 MHz and 1M H sound intensity of each superimposed simultaneously 1.0 WZC m ² ultrasound z, ultrasound 5 Irradiation for 2 s, the acoustic signal obtained from the tumor 37 during the ultrasonic irradiation was measured with an underwater microphone 31 and it was confirmed that the component of 250 MHz, which is a subharmonic of 0.5 MHz, was included. I do. Then, 12 hours after administration of the drug, ultrasonic waves of 0.5 MHz and 1 MHz were superimposed and simultaneously irradiated at an acoustic intensity of 1 OWZ cm ² for 5 minutes each. The weight of 37 was measured, and the tumor growth inhibition rate was calculated by equation (1). The ultrasonic probe 10 for imaging was used to confirm the focal point 27.

Tumor growth inhibition rate (%) = ((mean tumor weight of control group-mean tumor weight of test group) mean tumor weight of control group) X 100 (1) 0 n26 cells were implanted, and then ultrasound was administered without administration of ultrasound, and mice were implanted with C.sub.10n26 cells and then were administered without administration of ultrasound. It consists of a group that performs sound wave irradiation. W

20

In the ultrasonic apparatus shown in FIGS. 8 and 12, the position of the underwater microphone 31 and the position of the imaging ultrasonic probe 30 may be interchanged. When applying the ultrasonic device shown in FIGS. 8 and 12 to the human body, a water jacket may be placed between the front surface of the convergent ultrasonic transducer 26 and the affected area instead of using the water tank 2.

Evaluation experiment E: "Measurement of effects promoting stone destruction"

In the following test examples, the effect of the ultrasonic action-inducing (attracting) agent of the present invention to promote stone destruction by acoustic cavitation was evaluated by a cholesterol dissolution test. Pressurize 1.5 g of cholesterol under the conditions of 400 atm for 1 minute to form a pellet 1 cm in diameter. The pellet is placed in a polyethylene bag (0.03 mm thick) together with phosphate buffer (pH = 7.4) lm 1 containing an ultrasonic wave inducer, and 1 MHz ultrasonic waves are applied for 2 minutes. Irradiated. After irradiation, the sample was allowed to stand for 3 minutes. After removing the supernatant, the sample was dried, the pellet weight was measured, and the calculus breaking ratio was calculated by equation (2). Control experiments were performed by replacing the phosphate buffer (PH = 7.4) containing the sonication inducer with a phosphate buffer (pH = 7.4).

Calculus destruction rate (%) = ((weight of pellet before irradiation-weight of pellet after irradiation) weight of pellet before irradiation) X100 (2) Test Example 1: "Xanthene dye is used in a liquid for acoustic cavitation. Test to confirm the effect of lowering the threshold ”

Figure 13 shows the dependence of the intensity of the subharmonic generated when a solution containing a xanthene dye is irradiated with ultrasonic waves on the acoustic intensity. Figure 13 shows an example of the test results in accordance with Evaluation Experiment A. When an ultrasonic wave inducer such as Phloxine B, Rose Bengal or Erythritis is included, the acoustic intensity is about 2 WZ cm ^2. Above, acoustic cavitation occurs, and as the acoustic intensity increases, the cavitation intensity also increases. In contrast, control experiments in sound depression intensity occurs sound hairpin Kiyabiteshiyon even 1 0 WZc m ² From this, it is clear that the ultrasonic action inducer lowered the acoustic cavitation threshold to 1/5 or less in this test example. In all cases where subharmonics were measured in this test example, visible bubble formation was observed. Therefore, the ultrasonic action-inducing agent of the present invention was effective in generating bubbles by acoustic cavitation. It is clear that

Test Example 2: "Confirmation test of the effect of xanthene dye on accelerating sonochemical reaction"

Figure 14 shows the dependence of the amount of active oxygen generated on the sound intensity when a solution containing a xanthene dye is irradiated with ultrasonic waves. Fig. 14 shows an example of the test results according to the evaluation experiment C. When an ultrasonic action inducer such as Phloxine B, Rose Bengal or Erythritis is included, the sound intensity is about 2 cm ² or more. As a result, active oxygen is generated with increasing acoustic intensity. On the other hand, in the control experiment, no active oxygen was generated even at an acoustic intensity of 1 O WZ cm ² , indicating that the ultrasonic action inducer accelerated the sonochemical reaction in this test example. is there. In addition, since active oxygen has a bactericidal action as well as an antitumor action, it is clear that the ultrasonic action inducer of the present invention is effective for sterilization by ultrasound.

Test example 3: "Confirmation test of rose bengal's antitumor effect (tumor growth inhibition rate)"

Fig. 15 shows the tumor growth inhibition rate when rose bengal was administered to mice and irradiated with ultrasound. Figure 15 shows an example of the test results according to evaluation experiment D, showing the results for the xanthene dye rose bengal. As shown in Fig. 15, the rate of inhibition of tumor growth (inhibition rate) in the case of ultrasonic irradiation was 14.7% in the control experiment, while that in the case of using Rose Bengal was compared. In this case, it was 57.7%, and the antitumor pain effect was about 4 times higher. The effect of rose bengal when ultrasonic irradiation is not performed Although lower than the control experiment, this is considered to be within the error range. The antitumor effect of this test example is obtained by the effect of lowering the threshold of the acoustic cavitation and the effect of accelerating the sonochemical reaction of the ultrasonic action inducer of the present invention. Among xanthene dyes other than Rose Bengal, phloxin B, erythrin syn, tetracro mouth fluorescein, etc., in which halogen is bonded to multiple conjugated carbon atoms, have the same antitumor properties as Rose Bengal. (4) It is expected that an effect can be obtained. In Test Examples 1 and 2, phloxine B and erythrocystin show the same effect as Rose Pengal in lowering the acoustic cavitation threshold and producing active oxygen. Was.

Test Example 4: “Confirmation test of the effect of xanthene dye on accelerating stone destruction” Figure 16 shows the stone destruction rate when stones in a solution containing xanthene dye are irradiated with ultrasonic waves. Fig. 16 shows an example of test results according to evaluation experiment E. As shown in Fig. 16, the phloxin B, rose bengal and erythro-orcin-induced sonication inducers (Macao 1 × 10 " ⁴ ) were compared with the control experiment by 2.4 and 2. It showed a stone destruction rate of 2 to 1.9 times, the highest when using Phloxine B, and all of the ultrasonic action inducers were effective in promoting stone destruction.

Test Example 5: “Confirmation test of the effect of a compound consisting of a derivative of a xanthene dye having a functional group capable of chemically bonding to a thiol group or amino group to reduce the 閎 value of sound cavitation in a liquid”

Figure 17 shows the dependence of the intensity of the subharmonic wave generated when a solution containing a compound consisting of a derivative of a xanthene dye having a functional group capable of chemically bonding to a thiol or amino group is irradiated with ultrasonic waves on the acoustic intensity. FIG. In the first 7 figure shows an example of a test result in accordance with the evaluation experiments A, if containing ultrasonic action inducing agent such as Ellis port Shin Yodoase Bok Ami de is acoustic intensity of about 2 W / cm ² or more Acoustic cavitation has occurred. On the other hand, in the control experiment, even if the sound intensity was 10 WZ cm ² , gagged cavitation was generated. From this, it is clear that the ultrasonic action inducer lowered the acoustic cavitation threshold below 15 in this test example. Also, in all cases where subharmonics were measured in this test example, visible bubble formation was observed, indicating that the ultrasonic action inducer is effective in generating bubbles by acoustic cavitation. It is clear.

Test Example 6: “Confirmation test of the effect of a compound consisting of a derivative of a xanthene dye having a functional group capable of chemically bonding to a thiol group or an amino group to accelerate the sonochemical reaction”

Figure 18 shows the dependence of the amount of active oxygen generated on sound intensity when a solution containing a compound consisting of a derivative of a xanthene dye having a functional group capable of chemically bonding to a thiol group or an amino group is irradiated with ultrasonic waves. FIG. The first 8 figure shows an example of a test result in accordance with the evaluation experiment C, and may include an ultrasound action inducing agent such as Ellis port Shinyo one Doasetoami de is active in acoustic intensity of about 2 W / cm ² or more Oxygen is being produced. In contrast, in the control experiment, no active oxygen was generated even at an acoustic intensity of 1 O WZ cm ² , indicating that the ultrasonic action inducer accelerated the sonochemical reaction in this test example. Is. Since active oxygen has a bactericidal effect as well as an antitumor effect, it is clear that the ultrasonic action inducer is effective for sterilization by ultrasound. Test Example 7: “Confirmation test of the effect of a compound consisting of a xanthene dye or a derivative of a xanthene dye having a functional group capable of chemically bonding to a thiol group or an amino group to lower the threshold of acoustic cavitation in a living body”

FIG. 9 is a diagram showing threshold values of acoustic cavitation in mouse liver when a compound comprising a xanthene dye or a derivative of a xanthene dye having a functional group capable of chemically bonding to a thiol group or an amino group is present. Fig. 9 shows an example of the test results according to the evaluation experiment B. Eris mouth Sin, Eris mouth Sindoacetamide, Eris mouth Sin isothiosinate, The threshold value of acoustic cavitation was lower in all cases of Ellis mouth cinmaremid than in the case where no ultrasonic action inducer was administered. The acoustic cavitation threshold was reduced to about 2.4, 1/12, 1/8 and 1/3 by each ultrasonic action inducer.

Test Example 8: “Confirmation test of antitumor effect (tumor growth inhibition rate) of erythrocyte syndodoctamide”

Fig. 19 is a graph showing the inhibition rate of tumor growth when mice were injected with erythrocyte cindoacetamide and irradiated with ultrasound. Fig. 19 shows an example of the test results obtained in accordance with Evaluation Experiment D, and shows the results for the xanthene dye Eris Mouth Sidodoacetamide. The rate of inhibition of tumor growth (suppression rate) in the case of ultrasonic irradiation was 14.7% in the control experiment, whereas the rate of inhibition in the control experiment was 44.7% when erythrocyte cinnacetamide was used. At 9.7%, an anti-tumor effect of about 3.4 times was obtained.

As shown in Test Examples 6 and 7, the antitumor effect of this test example is obtained by the effect of the sonication inducer on lowering the threshold of the sound tone and the effect of promoting the sonochemical reaction. It is expected that xanthene dyes other than erythrido-syndoacetamide having an affinity for thiol group or amino group will have the same antitumor effect as erythrocyte-scinodacetamide. Mouth synthiothiocyanate and erythrosine maleimide showed almost the same effects as elimination cinnamate acetoamide in lowering the acoustic cavitation threshold and producing active oxygen ₍ Test Example 9: “thiol group or amino acid”). Confirmation of the effect of a compound consisting of a derivative of a xanthene dye having a functional group capable of chemically bonding with a group to accelerate stone destruction ”

Fig. 20 shows an example of the application of ultrasonic waves to a calculus in a solution containing a compound consisting of a derivative of a xanthene dye having a functional group capable of chemically bonding to a thiol group or an amino group. It is a figure which shows the calculus destruction rate at the time of irradiating. Fig. 20 shows an example of test results according to evaluation experiment E, in which xylose dye acetamido, ellis cinthiothiothiocyanate, and ellis cinmareimide were selected as xanthene dye derivatives.

As shown in FIG. 20, Ellis port Shinyo one Doasetoami de, Erisuroshi Ni source thio Xia Natick Bok, Ellis port Shinmareimi de of the ultrasonic action inducing agent (concentration 1 X 10- ⁴ M) is compared to the control experiment , 2. 2. 1. 9, respectively

The stone destruction rate was 1.9 times higher, the highest when using Ellis mouth cinnacetacetamide, and all ultrasonic action inducers were effective in promoting stone destruction.

Test Example 10: “Test for confirming the effect of erythricular cinnamate acetoamide generating bubbles for ultrasound imaging”

We examined the ultrasound contrast effect of the generation of air bubbles by acoustic cavitation in the presence of an ultrasonic action inducer, targeting the bladder of an SD rat. An EUB-450 manufactured by Hitachi, Ltd. was used as the ultrasonic diagnostic apparatus, and an intraoperative fingertip probe (7.5 MHz) was used as the probe. After anesthesia was performed by intravenously injecting an anesthetic into a 20-week-old SD rat, the probe was pressed against the bladder to obtain an ultrasonic B-mode image of the bladder. Strong echo signals were observed only from the periphery of the bladder. Next, a solution prepared by dissolving erythridocindoacetamide in physiological saline to a concentration of 0.5 gZl (liter) was injected intravenously into an SD rattle to a concentration of 5 mg / kg body weight. After 15 minutes, ultrasonic waves of frequencies 0.5 MHz and 1 MHz were simultaneously irradiated at an acoustic intensity of 5 WZcm ² for 5 seconds. During the ultrasound irradiation and for several seconds immediately after the ultrasound irradiation, echo signals from the bladder that were not seen before the ultrasound irradiation were observed.

Test Example 11: “Confirmation of the effect of dimer and its derivative formed by binding two molecules of xanthene dye by — (CH ₂ ) _n — to lower the threshold of acoustic cavitation in liquid test" Fig. 21 shows the components generated when a solution containing a dimer formed by combining _two molecules of a xanthene dye or a derivative of a xanthene dye with one (CH ₂ ) _n — is irradiated with ultrasonic waves. FIG. 6 is a diagram showing the dependence of the harmonic intensity on the acoustic intensity. Figure 23 is a diagram showing the structure of a dimer formed by combining _two molecules of a xanthene dye or a derivative of a xanthene dye with one (CH ₂ ) _n —. shown, shown xanthene dyes or hexa pentene in the dimer of the dye derivative _{_{_{R t, R 2, R 3}}} , R 4> R s, the _{_{_{R 6, R 7, R 8}}} .

Figure 21 shows an example of the test results according to evaluation experiment C. When an ultrasonic wave inducer such as a Rose Bengal dimer is included, the acoustic intensity is about 2 WZ cm ² or more and the acoustic cavitation is not possible. Has occurred. In contrast, control of the acoustic intensity in experiments does not occur acoustic Kiyabiteshiyon even 1 OWZ cm ^2, an ultrasonic action inducing agent in the present test example lowers the acoustic Kiyabiteshi ® emission threshold 1 Z5 below It is clear that there is.

Test Example 12: “Confirmation test of the effect of a dimer and its derivative formed by bonding two molecules of a xanthene dye by — (CH ₂ ) _π— to accelerate the sonochemical reaction”

Fig. 22 shows active oxygen when a solution containing a dimer formed by combining _two molecules consisting of a xanthene dye or a derivative of a xanthene dye by one (CH ₂ ) _n — is irradiated with ultrasonic waves. FIG. 4 is a diagram showing the dependence of the amount of generated sound on the sound intensity. Figure 22 shows an example of the test results according to evaluation experiment C. When an ultrasonic wave inducer such as rose bengal dimer is included, active oxygen is generated at an acoustic intensity of about SWZcm ² or more. I have. In contrast, in the control experiment, no active oxygen was generated even at an acoustic intensity of 1 OWZcm ² . FIG. 23 is a diagram showing the structure of a dimer formed by bonding _two molecules of a xanthene dye or a derivative of a xanthene dye with — (CH ₂ ) _n —, as shown in FIG. Xanthene dye or derivative of xanthene dye R ₂ , R ₃ , R ₄ , R ₅ , R _6l R ₇ , R _{8 in the} _{dimer of} Test Example 13: “Confirmation test of the effect of dimer and its derivative formed by binding two molecules of xanthene dye by one (CH ₂ ) _n — to lower the threshold of acoustic cavitation in living body”

FIG. 24, xanthene dyes or two molecular consisting derivatives of xanthene dyes one _(CH ₂₎ π - when the dimer is formed by bonding by the presence, in the acoustic Kiyabite in mouse liver It is a figure showing a threshold of one shot. Fig. 24 shows an example of the test results according to the evaluation experiment II. Rosebengal dimer, ellis mouth succinoacetamide dimer, eosin isothiosinate dimer, fluorescein male The threshold of acoustic cavitation was lower in all of the mid dimers than in the case where no ultrasonic action inducer was administered. The acoustic cavitation threshold is reduced to about 112, 1/2, 1/8, and 13 by each ultrasonic action inducer. Test Example 14: “One molecule of xanthene dye is one (CH ₂ ). Confirmation test of antitumor effect (tumor growth inhibition rate) of dimer and its derivative formed by binding together

Figure 25 shows the tumor growth inhibition rate when mice were administered with Rose Bengal dimer and Elis succinoacetoamide dimer and irradiated with ultrasound. Fig. 25 shows an example of the test results according to Evaluation Experiment D, and shows the results for dioxbengal dimer and erythrox cinnamate acetamide dimer. The inhibition rate of tumor growth with ultrasound irradiation was 10.1% in the control experiment, whereas it was 70.5% when Rose Bengal dimer was used, and In contrast, when Eris mouth succinoacetamide dimer was used, it was 73.2%, and the antitumor effect was about 7 times higher. The antitumor effect of the rose pengal dimer in this test example is about twice that of the antitumor effect of rose bengal monomer in test example 3. The antitumor effect in this test example was determined from the results of Test Examples 11 and 12. It was confirmed that the ultrasonic action inducer of the present invention was obtained by the effect of lowering the threshold value of acoustic cavitation and the effect of promoting the chemical reaction of sound. It should be noted that a dimer of a xanthene dye having a functional group other than an acetoacetamide group capable of binding to a thiol group or an amino group also has an antitumor effect equivalent to that of an erythrocyte sio-doacetamide dimer. Expected, in fact, in Test Examples 11 and 12, eosin isothiocynate dimer and fluorescein maleide dimer showed lower acoustic cavitation threshold and active oxygen. In the formation, the effect was almost the same as that of the elimination of cinnamate acetamide dimer.

In addition, in the dimer of the xanthene dye or the derivative of the xanthene dye shown in FIGS. 21, 22, 24, and 25, R ₁ is a chlorine-atom-to-substituted dimer ( In Oct ame t hy lenebis—rosebengal, n = 8, the elimination of the erythrocin succin-doacetamide (dimer), which is the odoacet amide group (Dec ame t hy lenebis— ( erythrosin— 5— iodoacet am ide)) and n = 10, eosinisothiocinet (dimer), which is an isothiocyanate group (He xadec ame t hy In lenebis— (eosin—5—isothiocy anate)), n = 16, fluorescein maleide (dimer), which is a maleimide group (aieimide group), is used. leimide)), n = 10

As described above, according to the ultrasonic action inducer of the present invention, the threshold value of sound cavitation can be reduced, and irradiation of ultrasonic waves with low acoustic intensity can benign or malignant tumors and calculi. Treatment can be performed safely. The present invention is summarized as follows. A xanthene dye containing a xanthene ring or It contains a compound consisting of a derivative of a xanthene dye (including a dimer) and is an agent that induces the action of ultrasound that lowers the threshold of sound intensity that produces acoustic cavitation. R i~R ₈ bound to atoms either (FIG. 4 or the 1 0 view), halogen, thiol groups or Amino group capable of chemically bonding functional group (halogenated Asetoami de group, male imide group , Aziridine, isothiocyanate, succinimid, or sulfonyl chloride). The threshold of acoustic intensity for the generation of acoustic cavitation can be lowered, and the treatment of benign or malignant tumors and calculi can be safely performed by irradiating low-intensity ultrasonic waves.

Claims

The scope of the claims

1. A drug that induces an ultrasonic action that lowers the threshold of acoustic intensity that generates acoustic cavitation, comprising a compound comprising a xanthene dye containing a xanthene ring or a derivative of the xanthene dye.

2. The drug according to claim 1, wherein the derivative has two or more halogens bonded to a carbon atom of the skeleton of the xanthene dye.

3. The drug according to claim 1, wherein the derivative has a functional group bonded to a carbon atom of the skeleton of the xanthene dye, and the functional group can be chemically bonded to a thiol group or an amino group. Drugs.

4. The drug according to claim 3, wherein the functional group is a halogenated acetamido group, a maleimide group, an aziridine group, an isothiocyanate group, a succinimid group, or a sulfonyl chloride group. A drug that is either.

5. The drug according to claim 1, wherein the compound has two of the xanthene rings.

6. The drug according to claim 1, wherein the compound comprising two of the xanthene dye or a derivative of the xanthene dye has two, and two of the compounds are one (CH ₂ ) „-(where, Drugs bound by 3≤n (integer) ≤20).

7. The drug according to claim 6, wherein the derivative has two or more halogens bonded to carbon atoms of the skeleton of the xanthene dye.

8. The drug according to claim 6, wherein the derivative has a functional group bonded to a carbon atom of the skeleton of the xanthene dye, and the functional group is capable of chemically bonding to a thiol group or an amino group. Drugs.

9. The drug according to claim 8, wherein the functional group is a halogenated acetate amide group, a maleimide group, an aziridine group, an isothiocyanate group, a succinimid group, or a sulfonyl chloride group. A drug that is any of

10. An agent that induces an ultrasonic action that lowers the threshold of acoustic intensity that generates acoustic cavitation, containing a xanthene dye containing a xanthene ring or a compound that is a derivative of the xanthene dye, is applied to the target site of the subject. a means for administration, and means for irradiating by superimposing the ultrasonic wave at the site of the target with a plurality of center frequencies of the acoustic intensity of 1 OWZcm ² or less, and means for detecting the ultrasonic wave from the site of the target Ultrasound device.

1 1. An agent that contains a xanthene dye containing a xanthene ring or a compound composed of a derivative of the xanthene dye and induces an ultrasonic action that lowers the threshold of acoustic intensity that generates acoustic cavitation is applied to a target site of the subject. Administering, superposing ultrasonic waves having a plurality of center frequencies having an acoustic intensity of 1 OW / cm ² or less, and irradiating the ultrasonic waves to the target site, and detecting ultrasonic waves from the target site. Ultrasound treatment method comprising: