HK1237359B - Novel indocyanine compound, synthesis method thereof, purification method thereof, diagnostic composition using indocyanine compound, and device for measuring in vivo kinetics and device for visualizing circulation using diagnostic composition - Google Patents

Abstract

Provided are a novel indocyanine compound by which problems occurring in the existing indocyanine green, such as dissolution in water or physiological saline, can be overcome, a method for synthesizing the same, a method for purifying the same, and a diagnostic composition comprising said novel indocyanine compound. Also provided are a method for evaluating the in vivo kinetics of the novel indocyanine compound, a device for measuring the in vivo kinetics, a method for visualizing the in vivo circulation of a fluid such as blood and a device therefor, each using the aforesaid diagnostic composition. Further, disclosed are a novel indocyanine compound, wherein a hydrophobic moiety of a near infrared fluorescent indocyanine molecule is included in a cavity of a cyclic sugar-chain cyclodextrin to thereby coat the hydrophobic moiety of the indocyanine molecule with glucose, a method for synthesizing the same and a method for purifying the same. Disclosed are a method for capturing a fluorescence image of an organ other than liver via intravenous administration, a method for evaluating the in vivo kinetics of the novel indocyanine compound, a device for measuring the in vivo kinetics thereof, a method for visualizing the in vivo circulation of a fluid such as blood and a device therefor, each using a diagnostic composition comprising the aforesaid novel indocyanine compound.

Description

Indocyanine compound, synthesis and purification method thereof, diagnostic composition, in vivo dynamics measurement device, and circulation visualization device

This patent application is a divisional application of the patent application with application number 201180006735.X, application date January 28, 2011, and invention name "New indocyanine compound, synthesis method and purification method thereof, diagnostic composition using the indocyanine compound, in vivo dynamic measurement device using the diagnostic composition, and circulation visualization device".

Technical Field

The present invention relates to a new indocyanine compound that is a green pigment useful in medical diagnosis, medical surgery, scientific measurement and analysis, printing, note-taking, coating, dyeing, and staining technologies and has the property of emitting near-infrared fluorescence, a method for synthesizing and purifying the same, and a diagnostic composition. Specifically, the invention relates to a cyclic sugar chain cyclodextrin-bound indocyanine compound that is a green pigment and has the property of emitting near-infrared fluorescence, a method for synthesizing and purifying the same, a diagnostic composition using the indocyanine compound, an in vivo dynamics measurement device using the diagnostic composition, and a circulation visualization device.

Background Art

A variety of green pigments, indocyanine compounds that emit near-infrared fluorescence, have been synthesized. These compounds have a wide range of applications in the medical field, for example, as dyes for vitreous surgery, medicinal pigments for liver function tests, medicinal pigments for circulatory function tests, surgical pigments, and near-infrared fluorescent compounds for surgery. In the scientific field, they serve as dyes or compounds for dyeing or fluorescing proteins, sugars, and other substances, and as pigments in printing technology. Among these indocyanine compounds, indocyanine green (hereinafter referred to as "ICG") has been used for nearly 50 years as a drug for liver and circulatory function tests. In recent years, ICG's high light transmittance through biological tissues has been exploited to allow for local administration of ICG to, for example, blood vessels, lymphatic vessels, brain, eyes, stomach, breasts, esophagus, skin, and other sites within the body, thereby allowing for medical procedures and diagnostics by observing its near-infrared fluorescence (Non-Patent Document 1).

Prior art literature

Non-patent literature

Non-patent literature 1: "All about ICG Fluorescence Navigation Surgery", supervised and edited by Mitsuo Kusano, インターメディカ社, (published in November 2008).

Non-Patent Document 2: URL of the Internet homepage described in the instructions for "(Trade Name) Diagnogreen Injection 25 mg" (Daiichi Sankyo Co., Ltd.) (https://www.daiichisankyo.co.jp/med/contents/di/dg2/pi/pdf/pi_dg2_0909.pdf)

Non-patent document 3: R.C. Benson, H.A. Kues, Phys. Med. Biol., 23, 159-163 (1978).

Non-patent document 4: S. Yoneyama, T. Saito, Y. Komatsu, I. Koyama, K. Takahashi, J. Duvoll-Young, IOVS, 37, 1286-1290 (1998).

Non-patent document 5: Y. Ye, W. P. Li, C. J. Anderson, J. Kao, G. V. Nikiforovich, S. Achilefu, J. Am. Chem. Soc., 125, 7766-7767 (2003).

Non-patent literature 6: K. Teranishi and S. Tanabe, ITE Letters on Batteries, New Technologies & Medicine, 1, 53-60 (2000).

Non-patent document 7: MICHAEL O’SHAUGHNESSY et al: 1994 Wiley-Liss, Inc MICROSURGERY 15: 40S412 1994.

Non-patent document 8: RALPH J.P.M et al: 1997 Wiley-Liss, Inc. MICROSURGERY 17: 402-408 1996.

Summary of the Invention

Technical issues

However, the green chromophore (the chemical structure required for the green color) and near-infrared fluorophore (the chemical structure required for near-infrared fluorescence) of ICG are hydrophobic, and sulfonyl groups are attached to the side chain terminals to make them water-soluble. Therefore, existing ICG has the following problems.

In general medical use, ICG preparations are prepared by adding approximately 5 to 10 mL of distilled water to 25 mg of ICG and stirring with vibration to dissolve it. Failure to completely dissolve ICG can cause symptoms such as nausea, retching, fever, and shock (Non-Patent Document 2). Furthermore, because ICG does not dissolve completely, it cannot be initially dissolved in other aqueous solutions such as saline (Non-Patent Document 2).

While ICG is water-soluble due to the sulfonyl groups attached to it, its chemical structure contains many hydrophobic hydrocarbon groups, making it surface-active and prone to adsorption to lipids. Consequently, when injected into tissues such as blood vessels and organs, it can sometimes adhere to non-target tissues by adhering to the injection site, leaking through the injection site, or flowing backward. ICG adhered to tissues is difficult to remove through wiping or aspiration, potentially hindering surgical procedures and medical diagnostics.

ICG has a molecular association property in aqueous solution, one of the reasons for which is its low fluorescence intensity in aqueous solution (Non-Patent Documents 3 and 4).

Furthermore, ICG becomes insoluble over time after being dissolved in water, making it difficult to store it as an aqueous solution for a long period of time. Furthermore, freezing at low temperatures promotes its insolubility.

Furthermore, there is a problem that an ICG preparation containing 5% or less of NaI may cause iodine hypersensitivity in some cases (Non-Patent Document 2).

In addition, ICG rapidly accumulates in the liver and is excreted by the liver when injected intravenously, making fluorescence imaging of other organs such as the kidneys, ureters, bladder, urethra, heart, and lungs difficult.

Furthermore, ICG migrates with the blood when injected intravenously, so its migration to peripheral tissues is minimal, making it difficult to observe its migration to the interstitium.

The present invention aims to resolve the aforementioned problems with existing ICG. Specifically, it aims to provide a new indocyanine compound that is a green pigment and emits near-infrared fluorescence. The compound is characterized by high solubility in water or physiological saline, easy removal from biological tissues, low molecular association in aqueous solution, and high near-infrared fluorescence intensity in aqueous solution, enabling fluorescence imaging of organs other than the liver, such as the kidneys, ureters, bladder, urethra, heart, and lungs. Furthermore, a chemical synthesis method and purification method for the new indocyanine compound with these characteristics are provided. Furthermore, a diagnostic composition containing the new indocyanine compound is also provided. Furthermore, a method and apparatus for visualizing the circulation of blood, lymph, urine, and other water components in a living organism is provided using these diagnostic compositions.

Technical solutions to the problem

The present inventors have conducted intensive research to solve the above problems and have discovered compounds that can emit near-infrared fluorescence. These compounds can be used in surgical procedures and medical diagnoses that utilize the properties of near-infrared fluorescence, thereby solving the above-mentioned problems with ICG.

Specifically, the present invention has been completed by the discovery of a new indocyanine compound characterized by being a green pigment that emits near-infrared fluorescence, having high solubility in water or physiological saline, being easily removed from biological tissues, exhibiting low molecular association in aqueous solutions, and exhibiting high near-infrared fluorescence intensity in aqueous solutions. Furthermore, the present invention has been completed by the discovery of a chemical synthesis method and a purification method for the new indocyanine compound. Furthermore, a diagnostic composition containing the new indocyanine compound is provided. Furthermore, a method for performing fluorescence imaging of organs other than the liver using the diagnostic composition, even during intravenous administration, is provided. Furthermore, a method for evaluating the in vivo dynamics of the new indocyanine compound related to, for example, water balance in a living body and a device for measuring the in vivo dynamics are provided, as well as a method and device for visualizing the circulation of blood, lymph, urine, and other water in a living body.

The first new indocyanine compound of the present invention is a compound in which a cyclic sugar chain cyclodextrin is covalently bound to the green chromophore (chemical structure required for green color) and the near-infrared fluorophore (chemical structure required for emitting near-infrared fluorescence) of ICG. In addition, the second new indocyanine compound of the present invention is characterized in that the naphthyl site belonging to the hydrophobic site of the indocyanine structure is included in the cavity of the cyclodextrin, thereby covering the naphthyl site belonging to the hydrophobic site with a hydrophilic glucose group, so that most areas of the indocyanine molecular structure become three-dimensionally hydrophilic. Therefore, compared with the characteristic of ICG having similar surface activity due to the hydrophilicity of both the hydrophobic site and the sulfonyl group, the compound of the present invention is characterized in that the hydrophobic site in its molecule is covered by cyclodextrin, so it does not have similar surface activity properties. Specifically, the present invention is as follows:

<1> A cyclodextrin-bound indocyanine compound represented by Chemical Formula 1, characterized in that the indocyanine is covalently bound to a cyclic sugar chain cyclodextrin;

[Chemical Formula 1]

(R ₁ , R ₂ , R ₃ , R ₄ , R ₅ , R ₆ , R ₇ , R ₈ , R ₉ , R ₁₀ , R ₁₁ , R ₁₂ , R ₁₃ , R 14 , R ₁₅ , R ₁₆ , R ₁₇ , R ₁₈ , R ₁₉ , R ₂₀ , R _in the formula ₂₁ , R ₂₂ , R _R23 is a hydrogen atom, an alkyl group, an aryl group, a halogen atom, an alkoxy group, an amino group, a carboxyl group, a formyl group, a sulfonyl group, a sulfonic acid group, a phosphate group, an alkoxycarbonyl group, an aryloxycarbonyl group, an alkylcarbonyl group, an arylcarbonyl group, or a heterocycle. Alternatively, it may be a substituent thereof (carboxylic acid, sulfonic acid, phosphoric acid). When the hydrogen ion in the substituent is dissociated, the hydrogen ion may be replaced by a metal ion such as sodium ion, potassium ion, magnesium ion, or calcium ion. The amino group may be selected from primary, secondary, tertiary, and quaternary groups ₍ alkyl groups may be exemplified as substituents bonded to nitrogen). Alternatively, _R8 and _R9 may _be selected _{from CH2} , _{CH2CH2 , CH2CH2CH2} , or _{CH2CH2CH2CH2 . 2.} Alternatively, the hydrogen atoms on these alkyl groups may be replaced by alkyl groups, aryl groups, halogen atoms, alkoxy groups, amino groups, carboxyl groups, formyl groups, sulfonyl groups, sulfonic acid groups, phosphoric acid groups, alkoxycarbonyl groups, aryloxycarbonyl groups, alkylcarbonyl groups, arylcarbonyl groups or heterocyclic groups.)

<2> The cyclodextrin-bound indocyanine compound represented by Chemical Formula 2 is characterized in that at least a portion of the naphthyl group of the indocyanine is included in the cavity of the cyclodextrin.

[Chemical Formula 2]

(R ₁ , R ₂ , R ₃ , R ₄ , R ₅ , R ₆ , R ₇ , R ₈ , R ₉ , R ₁₀ , R ₁₁ , R ₁₂ , R ₁₃ , R 14 , R ₁₅ , R ₁₆ , R ₁₇ , R ₁₈ , R ₁₉ , R ₂₀ , R _in the formula ₂₁ , R ₂₂ , R ₂₃ is a hydrogen atom, an alkyl group, an aryl group, a halogen atom, an alkoxy group, an amino group, a carboxyl group, a formyl group, a sulfonyl group, a sulfonic acid group, a phosphate group, an alkoxycarbonyl group, an aryloxycarbonyl group, an alkylcarbonyl group, an arylcarbonyl group, or a heterocycle. Alternatively, it may be a substituent thereof (carboxylic acid, sulfonic acid, phosphate). When the hydrogen ion in the substituent is dissociated, the hydrogen ion may be replaced by a metal ion such as a sodium ion, potassium ion, magnesium ion, or calcium ion. The amino group is selected from primary, secondary, tertiary, and quaternary groups. Furthermore, R ₈ and R ₉ may be selected from a cyclic structure of CH ₂ , CH ₂ CH ₂ , CH ₂ CH ₂ CH ₂ , or CH ₂ CH ₂ CH ₂ CH ₂ . Alternatively, a functional group in which the hydrogen atom on these alkyl groups is replaced by an alkyl group, an aryl group, a halogen atom, an alkoxy group, an amino group, a carboxyl group, a formyl group, a sulfonyl group, a sulfonic acid group, a phosphate group, an alkoxycarbonyl group, an aryloxycarbonyl group, an alkylcarbonyl group, an arylcarbonyl group, or a heterocycle may be selected.)

<3> The cyclodextrin-bound indocyanine compound represented by Chemical Formula 3 is characterized in that it is the cyclodextrin-bound indocyanine compound represented by Chemical Formula 1 in which an indocyanine and a cyclic sugar chain cyclodextrin are covalently bound via an amide bond.

[Chemical Formula 3]

(R ₁ , R ₂ , R ₃ , R ₄ , R ₅ , R ₆ , R ₇ , R ₈ , R ₉ , R ₁₀ , R ₁₁ , R ₁₂ , R ₁₃ , R 14 , R ₁₅ , R ₁₆ , R ₁₇ , R ₁₈ , R ₁₉ , R ₂₀ , R _in the formula ₂₁ , R ₂₂ , R _R23 is a hydrogen atom, an alkyl group, an aryl group, a halogen atom, an alkoxy group, an amino group, a carboxyl group, a formyl group, a sulfonyl group, a sulfonic acid group, a phosphate group, an alkoxycarbonyl group, an aryloxycarbonyl group, an alkylcarbonyl group, an arylcarbonyl group, or a heterocycle. Alternatively, it may be a substituent thereof (carboxylic acid, sulfonic acid, phosphoric acid), wherein the hydrogen ion in the substituent, when dissociated, may be replaced by a metal ion such as sodium ion, potassium ion, magnesium ion, or calcium ion. The amino group is selected from primary, secondary, tertiary, and _quaternary groups. m and n are integers from 1 to 6. Furthermore, _R8 and _R9 may _{be selected from CH2} , _CH2CH2 , _CH2CH2CH2 , or _{CH2CH2CH2CH2 . 2.} In addition, the functional groups in which the hydrogen atoms on these alkyl groups are replaced by alkyl groups, aryl groups, halogen atoms, alkoxy groups, amino groups, carboxyl groups, formyl groups, sulfonyl groups, sulfonic acid groups, phosphoric acid groups, alkoxycarbonyl groups, aryloxycarbonyl groups, alkylcarbonyl groups, arylcarbonyl groups or heterocyclic rings can also be selected.)

<4> Chemical formula 4 represents a cyclodextrin-bound indocyanine compound, characterized in that it is a cyclodextrin-bound indocyanine compound represented by Chemical formula 2, in which indocyanines and cyclic sugar chain cyclodextrin are covalently bound via an amide bond.

[Chemical Formula 4]

(R ₁ , R ₂ , R ₃ , R ₄ , R ₅ , R ₆ , R ₇ , R ₈ , R ₉ , R ₁₀ , R ₁₁ , R ₁₂ , R ₁₃ , R 14 , R ₁₅ , R ₁₆ , R ₁₇ , R ₁₈ , R ₁₉ , R ₂₀ , R _in the formula ₂₁ , R ₂₂ , R _R23 is a hydrogen atom, an alkyl group, an aryl group, a halogen atom, an alkoxy group, an amino group, a carboxyl group, a formyl group, a sulfonyl group, a sulfonic acid group, a phosphate group, an alkoxycarbonyl group, an aryloxycarbonyl group, an alkylcarbonyl group, an arylcarbonyl group, or a heterocycle. Alternatively, it may be a substituent thereof (carboxylic acid, sulfonic acid, phosphoric acid), wherein the substituent can be replaced by a metal ion such as sodium ion, potassium ion, magnesium ion, or calcium ion when the hydrogen ion dissociates. The amino group is selected from primary, secondary, tertiary, and quaternary groups. _m and n are integers from 1 to 6. Furthermore, _R8 and _R9 may _be selected _{from CH2} , _CH2CH2 , _{CH2CH2CH2 ,} or _{CH2CH2CH2CH2 . 2.} In addition, the functional groups in which the hydrogen atoms on these alkyl groups are replaced by alkyl groups, aryl groups, halogen atoms, alkoxy groups, amino groups, carboxyl groups, formyl groups, sulfonyl groups, sulfonic acid groups, phosphoric acid groups, alkoxycarbonyl groups, aryloxycarbonyl groups, alkylcarbonyl groups, arylcarbonyl groups or heterocyclic rings can also be selected.)

<5> The cyclodextrin-bound indocyanine compound represented by Chemical Formula 5 among the cyclodextrin-bound indocyanine compounds represented by Chemical Formula 3.

[Chemical Formula 5]

(In the formula, m, n, p, and q are integers of 2 to 6. r is an integer of 5 to 7. s is an integer of 0 to 4. R is a hydrogen atom, an alkyl group, an aryl group, a halogen atom, an alkoxy group, an amino group, a carboxyl group, a formyl group, a sulfonyl group, a sulfonic acid group, an alkoxycarbonyl group, an aryloxycarbonyl group, an alkylcarbonyl group, an arylcarbonyl group, or a heterocycle.)

<6> The cyclodextrin-bound indocyanine compound represented by Chemical Formula 6 among the cyclodextrin-bound indocyanine compounds represented by Chemical Formula 4.

[Chemical Formula 6]

(In the formula, m, n, p, and q are integers of 2 to 6. r is an integer of 5 to 7. s is an integer of 0 to 4. R is a hydrogen atom, an alkyl group, an aryl group, a halogen atom, an alkoxy group, an amino group, a carboxyl group, a formyl group, a sulfonyl group, a sulfonic acid group, an alkoxycarbonyl group, an aryloxycarbonyl group, an alkylcarbonyl group, an arylcarbonyl group, or a heterocycle.)

<7> The cyclodextrin-bound indocyanine compound represented by Chemical Formula 7 among the cyclodextrin-bound indocyanine compounds represented by Chemical Formula 3.

[Chemical Formula 7]

(In the formula, m and n are integers of 2 to 6. r is an integer of 5 to 7. s is an integer of 0 to 4. R is a hydrogen atom, an alkyl group, an aryl group, a halogen atom, an alkoxy group, an amino group, a carboxyl group, a formyl group, a sulfonyl group, a sulfonic acid group, an alkoxycarbonyl group, an aryloxycarbonyl group, an alkylcarbonyl group, an arylcarbonyl group, or a heterocycle.)

<8> The cyclodextrin-bound indocyanine compound represented by Chemical Formula 8 among the cyclodextrin-bound indocyanine compounds represented by Chemical Formula 4.

[Chemical Formula 8]

(In the formula, m and n are integers of 2 to 6. r is an integer of 5 to 7. s is an integer of 0 to 4. R is a hydrogen atom, an alkyl group, an aryl group, a halogen atom, an alkoxy group, an amino group, a carboxyl group, a formyl group, a sulfonyl group, a sulfonic acid group, an alkoxycarbonyl group, an aryloxycarbonyl group, an alkylcarbonyl group, an arylcarbonyl group, or a heterocycle.)

<9> The cyclodextrin-bound indocyanine compound represented by Chemical Formula 9 among the cyclodextrin-bound indocyanine compounds represented by Chemical Formula 3.

[Chemical Formula 9]

(In the formula, m and n are integers of 2 to 6. r is an integer of 5 to 7. s is an integer of 0 to 4. R is a hydrogen atom, an alkyl group, an aryl group, a halogen atom, an alkoxy group, an amino group, a carboxyl group, a formyl group, a sulfonyl group, a sulfonic acid group, an alkoxycarbonyl group, an aryloxycarbonyl group, an alkylcarbonyl group, an arylcarbonyl group, or a heterocycle.)

<10> The cyclodextrin-bound indocyanine compound represented by Chemical Formula 10 among the cyclodextrin-bound indocyanine compounds represented by Chemical Formula 4.

[Chemical Formula 10]

(In the formula, m and n are integers of 2 to 6. r is an integer of 5 to 7. s is an integer of 0 to 4. R is a hydrogen atom, an alkyl group, an aryl group, a halogen atom, an alkoxy group, an amino group, a carboxyl group, a formyl group, a sulfonyl group, a sulfonic acid group, an alkoxycarbonyl group, an aryloxycarbonyl group, an alkylcarbonyl group, an arylcarbonyl group, or a heterocycle.)

<11> The cyclodextrin-bound indocyanine compound represented by Chemical Formula 11 among the cyclodextrin-bound indocyanine compounds represented by Chemical Formula 3.

[Chemical Formula 11]

(s is an integer from 0 to 4. R is a hydrogen atom, an alkyl group, an aryl group, a halogen atom, an alkoxy group, an amino group, a carboxyl group, a formyl group, a sulfonyl group, a sulfonic acid group, an alkoxycarbonyl group, an aryloxycarbonyl group, an alkylcarbonyl group, an arylcarbonyl group, or a heterocyclic ring.)

<12> The cyclodextrin-bound indocyanine compound represented by Chemical Formula 12 among the cyclodextrin-bound indocyanine compounds represented by Chemical Formula 4.

[Chemical Formula 12]

(s is an integer from 0 to 4. R is a hydrogen atom, an alkyl group, an aryl group, a halogen atom, an alkoxy group, an amino group, a carboxyl group, a formyl group, a sulfonyl group, a sulfonic acid group, an alkoxycarbonyl group, an aryloxycarbonyl group, an alkylcarbonyl group, an arylcarbonyl group, or a heterocyclic ring.)

<13> The chemical synthesis method of the cyclodextrin-conjugated indocyanine compound described in any one of the above chemical formulas (Chemical Formulas 1, 3, 5, 7, 9, and 11) is characterized by comprising: (1) a step of mixing an indocyanine carboxylic acid compound and an aminocyclodextrin in a solvent; and (2) a step of adding a dehydration condensation agent to carry out a dehydration condensation reaction.

<14> A chemical synthesis method of a cyclodextrin-bound indocyanine compound according to any one of the above chemical formulas (Chemical Formulas 2, 4, 6, 8, 10, 12), characterized in that the cyclodextrin-bound indocyanine compound according to any one of the above chemical formulas (Chemical Formulas 1, 3, 5, 7, 9, 11) is subjected to an inclusion reaction in water.

<15> The cyclodextrin-bound indocyanine compound represented by any one of the above chemical formulae (Chemical Formulae 1 to 12) is purified by column chromatography in which the compound is eluted with a solvent containing HCl.

<16> A diagnostic composition to be used by injection into the body, comprising an aqueous solution containing the cyclodextrin-bound indocyanine compound of any one of the above Chemical Formulas 1 to 12.

<17> The diagnostic composition according to <16>, which contains substantially no iodine.

<18> A device for measuring the dynamics of a cyclodextrin-bound indocyanine compound in vivo, comprising:

an excitation light irradiation unit for irradiating the cyclodextrin-bound indocyanine compound with excitation light, with respect to a part of a living body to which the diagnostic composition according to <16> or <17> is administered;

a fluorescence intensity measuring unit for measuring the intensity of fluorescence emitted by the cyclodextrin-bound indocyanine compound excited by the excitation light irradiation unit; and

The in vivo dynamic calculation unit calculates the temporal change of the time rate of change of the fluorescence intensity from the fluorescence intensity obtained over time from the fluorescence intensity measuring unit, thereby calculating the speed of the cyclodextrin-bound indocyanine compound moving into the interstitial fluid and/or the speed of moving out of the interstitium in a part of the biological body.

<19> A circulation visualization device, comprising:

an excitation light irradiation unit for irradiating the cyclodextrin-bound indocyanine compound with excitation light, with respect to a part of a living body to which the diagnostic composition according to <16> or <17> is administered;

a fluorescence imaging unit for two-dimensionally acquiring the intensity of the fluorescence emitted by the cyclodextrin-bound indocyanine compound excited by the excitation light irradiation unit, thereby obtaining data on the distribution state of the cyclodextrin-bound indocyanine compound in the organism;

a morphological imaging unit that two-dimensionally acquires the intensity of light of wavelengths other than the fluorescence wavelength emitted by the cyclodextrin-bound indocyanine compound, thereby obtaining morphological data of a portion of the biological body; and

The display unit displays the distribution state of the cyclodextrin-bound indocyanine compound in a part of the biological body by superimposing the morphological data obtained by the morphological imaging unit and the distribution state data obtained by the fluorescence imaging unit.

<20> The circulation visualization device according to <19>, wherein the display unit displays a portion of the living body where the distribution amount of the cyclodextrin-bound indocyanine compound is lower than a predetermined reference as a necrotic portion.

<21> The in vivo dynamics measuring device according to <18> above, comprising a swelling progression prediction unit for predicting the degree of swelling of the part of the living body based on the speed of the swelling moving from the part of the living body into and out of the interstitial fluid.

<22> The in vivo dynamic measurement device described in <21> above, wherein the swelling progression prediction unit predicts the degree of swelling progression corresponding to the magnitude of the migration speed into the interstitial fluid after a predetermined time, wherein the predetermined time is the time from the administration of the diagnostic composition to the dispersion of the cyclodextrin-conjugated indocyanine compound in the blood throughout the body.

<23> An in vivo dynamics measurement device, characterized by comprising:

The excitation light irradiation unit irradiates the cyclodextrin-bound indocyanine compound with excitation light at a portion of the living body to which the diagnostic composition according to <16> or <17> is administered and at a control site.

a fluorescence intensity measuring unit for measuring the intensity of fluorescence emitted by the cyclodextrin-bound indocyanine compound excited by the excitation light irradiation unit;

an in vivo dynamic calculation unit for calculating the time-dependent change in the fluorescence intensity obtained from the fluorescence intensity measurement unit over time, thereby calculating the migration speed of the cyclodextrin-bound indocyanine compound into the interstitial fluid and/or the migration speed out of the interstitial fluid in a part of the organism, and

a swelling progression prediction unit for predicting a degree of swelling that will progress in the part of the living body based on a rate of movement of the part of the living body into and out of the interstitial fluid;

wherein the above swelling is carried out in a prediction unit,

determining the relationship between the blood flow in the control site and the blood flow in the portion of the living body based on changes in fluorescence intensity in the portion of the living body and changes in fluorescence intensity in the control site until a predetermined time has passed, wherein the predetermined time is the time from administration of the diagnostic composition to dispersion of the cyclodextrin-bound indocyanine compound into blood throughout the body;

The degree of change in the fluorescence intensity of the part of the biological body after the predetermined time has passed, with the change in the fluorescence intensity of the control part serving as a control, is calculated using this relationship.

The progression of the swelling is predicted based on the calculated degree of change.

<24> An in vivo dynamics measurement device, characterized by comprising:

an excitation light irradiation unit for irradiating the cyclodextrin-bound indocyanine compound with excitation light to a living organ to which the diagnostic composition according to <16> or <17> is administered;

a fluorescence intensity measuring unit for measuring the intensity of fluorescence emitted by the cyclodextrin-bound indocyanine compound excited by the excitation light irradiation unit; and

The in vivo dynamics calculation unit evaluates the in vivo dynamics of the cyclodextrin-bound indocyanine compound in the organ based on the fluorescence intensity acquired over time by the fluorescence intensity measurement unit.

<25> The in vivo dynamics measuring device according to <24>, wherein the organ is any one of a kidney, a ureter, a bladder, and a urethra.

Beneficial effects

The cyclodextrin-bound indocyanine compounds represented by Chemical Formula 1 or Chemical Formula 2 of the present invention provide green pigment compounds that emit near-infrared fluorescence and have the following characteristics: higher solubility in water or physiological saline compared to ICG, easier removal from biological tissues, lower molecular association in aqueous solutions, higher near-infrared fluorescence intensity in aqueous solutions, and the ability to perform fluorescence imaging of organs other than the liver. Furthermore, the cyclodextrin-bound indocyanine compound synthesis method of the present invention provides for the synthesis of useful cyclodextrin-bound indocyanine compounds. Furthermore, the cyclodextrin-bound indocyanine compound purification method of the present invention provides for the purification of useful cyclodextrin-bound indocyanine compounds. Furthermore, the cyclodextrin-bound indocyanine compounds of the present invention exhibit sufficient solubility even without iodine, thus providing diagnostic compositions that do not contain iodine, a cause of iodine allergy. These diagnostic compositions exhibit different in vivo behavior than conventional diagnostic compositions containing only ICG, and their properties can be exploited to provide various beneficial devices.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the results of a test on the adsorption of ICG and the isomeric equilibrium compound (TK1) represented by Chemical Formulas 19 and 20 to human skin. The images show the results of a 1 mM aqueous solution (0.03 mL) of ICG and the isomeric equilibrium compound (TK1) represented by Chemical Formulas 19 and 20 applied to the wrist and ingested immediately (left photo), 5 minutes later and immediately after rinsing with water (center photo), and then immediately after wiping and rinsing with water (right photo). The dots on the right are marked with a red pen.

Figure 2 shows the results of a test on the adsorption of ICG and the isomeric equilibrium compound (TK1) represented by Chemical Formulas 19 and 20 onto cellulose fibers. The images show the results of a cotton swab application of a 1 mM aqueous solution (0.05 mL) of ICG and the isomeric equilibrium compound (TK1) represented by Chemical Formulas 19 and 20, followed by immediate uptake (top photo), and then 3 minutes later, followed by 5 seconds of rinsing with water, followed by immediate uptake (bottom photo).

Figure 3 shows the results of a test on the adsorption of ICG and the isomeric equilibrium compound (TK1) represented by Chemical Formulas 19 and 20 into a biological meat model. The images show a 1 mM aqueous solution (0.05 mL) of ICG and the compound represented by Chemical Formula 20 applied to a 5 mm diameter depression in pork tenderloin immediately after ingestion (left photo), and then rinsed with water for 10 seconds 3 minutes later, followed by immediate ingestion (right photo).

Figure 4 shows the results of a test on the adsorption of ICG and the isomeric equilibrium compound (TK1) represented by Chemical Formulas 19 and 20 onto a biological protein model. The images show a 1 mM aqueous solution (0.05 mL) of ICG and the isomeric equilibrium compound (TK1) represented by Chemical Formulas 19 and 20 applied to a 5 mm diameter depression in chicken breast immediately after ingestion (left photo), and then rinsed with water for 10 seconds 3 minutes later, followed by immediate ingestion (right photo).

Figure 5 shows the test results of the adsorption of ICG and the isomeric equilibrium compound (TK1) represented by Chemical Formulas 19 and 20 onto hydrophobic chemical fibers. The images show a 1 mM aqueous solution (0.05 mL) of ICG and the isomeric equilibrium compound (TK-1) represented by Chemical Formulas 19 and 20 applied to a polypropylene mask, immediately after application (left photo), and then after 20 minutes of rinsing with water for 1 second and immediately after application (right photo).

Figure 6 shows the results of molecular association tests of ICG and the isomerization equilibrium compound (TK1) represented by Chemical Formulas 19 and 20. The left figure shows ICG, and the right figure shows the isomerization equilibrium compound (TK1) represented by Chemical Formulas 19 and 20.

7 is a graph showing the concentration dependency of the fluorescence intensity of ICG, the isomerization equilibrium compound (TK1) represented by Chemical Formulas 19 and 20, and the isomerization equilibrium compound (TK2) represented by Chemical Formulas 15 and 16 in human venous blood.

FIG8 is a graph showing the conditions observed when ICG, the isomerization-equilibrium compound represented by Chemical Formulas 19 and 20 (TK1), and the isomerization-equilibrium compound represented by Chemical Formulas 15 and 16 (TK2) were administered to rats.

FIG9 is a graph showing fluorescence in the peritoneal cavity of rats when ICG, the isomerization-equilibrium compound represented by Chemical Formulas 19 and 20 (TK1), and the isomerization-equilibrium compound represented by Chemical Formulas 15 and 16 (TK2) were administered.

Figure 10 shows images of male Wistar rats (14 weeks old, 300 g) administered 0.075 mL of a 0.1 mM aqueous solution of the isomerized equilibrium compounds (TK2) represented by Chemical Formulas 15 and 16 via the tail vein, followed by imaging using a near-infrared observation system PDE manufactured by Hamamatsu Photonics Co., Ltd. The left image is a black-and-white image using visible light, and the right image is a fluorescent image.

Figure 11 shows near-infrared fluorescence endoscopic images of male Wistar rats (14 weeks old, 300 g) after 0.075 mL of a 0.1 mM aqueous solution of the isomerized equilibrium compounds (TK2) represented by Chemical Formulas 15 and 16 was administered via the tail vein. The left image is a black-and-white image using visible light, and the right image is a fluorescence image.

FIG12 is a graph showing fluorescence at the dorsum of the foot of rats when ICG, the isomerized equilibrium compound (TK1) represented by Chemical Formulas 19 and 20, and the isomerized equilibrium compound (TK2) represented by Chemical Formulas 15 and 16 were administered.

13 is a graph showing the temporal changes in fluorescence intensity at the dorsum of the foot when ICG, the isomerization-equilibrium compound represented by Chemical Formulas 19 and 20 (TK1), and the isomerization-equilibrium compound represented by Chemical Formulas 15 and 16 (TK2) were administered to rats.

14 is a graph showing the temporal changes in fluorescence intensity at the dorsum of the foot when ICG, the isomerization-equilibrium compound represented by Chemical Formulas 19 and 20 (TK1), and the isomerization-equilibrium compound represented by Chemical Formulas 15 and 16 (TK2) were administered to rats.

FIG. 15 is a diagram showing a technique for direct observation of rat blood vessels.

FIG. 16 is a diagram showing a directly observed portion of a rat's blood vessels (spermatozoa cremaster flap).

FIG. 17 is a graph showing the state of fluorescence in blood vessels and surrounding tissues of rats to which ICG was administered.

FIG. 18 is a graph showing fluorescence in blood vessels and surrounding tissues of rats after administration of the isomerization-equilibrated compound (TK1) represented by Chemical Formulas 19 and 20. FIG.

Figure 19 is a graph showing the left paw volume of rats measured immediately after injection (post-injection) and one week after injection (1 week after) for the carrageenan-administered group (edema) and the non-administered group (normal) in the TK1 test. The vertical axis represents volume (mL).

Figure 20 is a graph showing the results of the Von Frey test on the left paw of rats in the TK1 test group, the group that received carrageenan (edema) and the group that did not receive carrageenan (normal), measured immediately after injection and one week after injection. The vertical axis represents the load (g) at which the reaction occurred.

Figure 21 is a graph showing changes in brightness of the left paw surface after TK1 injection immediately after carrageenan injection for a group administered carrageenan (edema) and a group not administered carrageenan (normal). The vertical axis represents brightness (arbitrary unit) and the horizontal axis represents time (seconds).

Figure 22 is a graph showing changes in brightness of the left paw surface one week after carrageenan injection and TK1 injection for a group that received carrageenan (edema) and a group that did not receive carrageenan (normal). The vertical axis represents brightness (arbitrary unit) and the horizontal axis represents time (seconds).

Figure 23 is a graph showing the left paw volume of rats measured immediately after injection (post-injection) and one week after (1 week) for the ICG test group, the group administered carrageenan (edema), and the group not administered (normal). The vertical axis represents volume (mL).

Figure 24 is a graph showing the results of the Von Frey test on the left paw of rats immediately after injection and one week after injection, for groups tested with ICG, those administered carrageenan (edema), and those not administered (normal). The vertical axis represents the load (g) at which the reaction occurred.

Figure 25 is a graph showing the brightness of the left paw surface immediately after ICG injection for a group that received carrageenan (edema) and a group that did not receive carrageenan (normal). The vertical axis represents brightness (arbitrary unit) and the horizontal axis represents time (seconds).

Figure 26 is a graph showing changes in brightness of the left foot surface after ICG injection one week after carrageenan injection in a group that received carrageenan (edema) and a group that did not receive carrageenan (normal). The vertical axis represents brightness (arbitrary units) and the horizontal axis represents time (seconds).

DETAILED DESCRIPTION

The diagnostic composition of the present invention utilizes the cyclodextrin-conjugated indocyanine compound described below as a pigment, making it iodine-free and therefore suitable for diagnostic applications. This diagnostic composition can replace the conventionally used indocyanine green-containing diagnostic compositions. For example, it can be used as a drug for liver function tests or circulatory function tests. Furthermore, by observing the near-infrared fluorescence generated by administration to, for example, blood vessels, lymphatic vessels, brain, eyes, stomach, breast, esophagus, skin, kidneys, ureters, bladder, urethra, lungs, heart, or other internal organs, it can be applied to medical procedures and diagnostics. The pigment contained in the diagnostic composition of the present invention has low binding to the organism, allowing for long-term labeling of desired areas. The diagnostic composition may also contain salts or other additives as isotonic agents, as needed. In addition to pre-prepared aqueous solutions, it can also be used in a form that is dissolved. The diagnostic composition can be administered by injection, intravenous drip, application, or oral administration.

This diagnostic composition is suitable for use in visualizing the circulation of aqueous solutions such as blood, lymph, interstitial water, and urine. By visualizing the circulation of blood and other fluids, for example, it can be used to assess necrosis in the peripheral circulation, evaluate tissue survival after revascularization surgery and transplantation, and diagnose poor blood flow. Furthermore, it can also be used for fundus angiography, which has been previously visualized, cerebral circulation assessment, intraoperative angiography during neurosurgery, sentinel lymph node identification in cancers (such as breast cancer, esophageal cancer, gastric cancer, colorectal cancer, prostate cancer, and skin cancer), evaluation of lymphedema, intraoperative cholangiography, tumor labeling, coronary angiography, and abdominal angiography (hepatic artery, abdominal aorta, digestive tract blood flow, etc.). Fluorescent imaging of the renal excretory system, including the kidneys, ureters, bladder, and urethra, is also possible.

<1. Non-inclusion cyclodextrin-bound indocyanine compounds and their synthesis>

Examples of the non-inclusion type cyclodextrin-bound indocyanine compounds of the present invention include Chemical Formula 1, Chemical Formula 3, Chemical Formula 5, Chemical Formula 7, Chemical Formula 9, and Chemical Formula 11. Their synthesis is accomplished by reacting an indocyanine compound with a cyclodextrin compound in a solution.

In Chemical Formulas 1 and 3, considering the inclusion of a significant portion of the cyclodextrin, _R1 - _R4 and _R13 - _R16 are preferably relatively bulky groups that do not hinder cyclodextrin inclusion. For example, hydrogen or an alkyl or alkoxy group with approximately 1 to 3 carbon atoms. Preferred are hydrogen, methyl, and methoxy groups, with hydrogen being most preferred. For cyclodextrin inclusion, _R5 , _R6 , _R11 , and _R12 are also preferably relatively bulky, but not as bulky as _R1 - _R4 and _R13 - _R16 . For example, hydrogen or an alkyl or alkoxy group with approximately 1 to 6 carbon atoms. _R1 - _R6 and _R11 - _R16 are sites of cyclodextrin inclusion, so even when a hydrophilic functional group is introduced, the entire group is preferably hydrophobic. _R17 , _R18 , _R22 , and _R23 do not significantly affect cyclodextrin inclusion and are not particularly limited as long as they are the substituents listed above. R ₇ , R ₁₀ , R ₁₉ and R ₂₁ are preferably hydrogen from the viewpoint of ease of synthesis. R ₈ , R ₉ and R ₂₀ are not particularly limited as long as they are the substituents described above.

In the case where the non-inclusion type cyclodextrin-bound indocyanine compound of the present invention is covalently bound to the indocyanine compound and the cyclodextrin compound via an amide bond, the synthesis method of the non-inclusion type cyclodextrin-bound indocyanine compound can be achieved by allowing an indocyanine carboxylic acid compound and an aminocyclodextrin compound to undergo a dehydration condensation reaction in a solution.

<2. Inclusion-type cyclodextrin-bound indocyanine compounds>

The cyclodextrin-bound indocyanine compound of the present invention is characterized by being a cyclodextrin-bound indocyanine compound covalently bound to a cyclic sugar chain cyclodextrin represented by Chemical Formula 2, wherein at least a portion of the naphthyl group of the indocyanine is enclosed within the cyclodextrin cavity. Furthermore, as long as the naphthyl group of the indocyanine is enclosed within the cyclodextrin cavity and emits near-infrared fluorescence, the indocyanine group may have a substituent. While there are many types of cyclodextrins, enclosed naphthyl group of the indocyanine is a necessary condition. Examples include α-cyclodextrin, β-cyclodextrin, and γ-cyclodextrin. β-cyclodextrin is preferred. The cyclodextrin may also have a substituent.

In Chemical Formulas 2 and 4, _R1 - _R4 and _R13 - _R16 are preferably small groups that do not hinder cyclodextrin inclusion. Examples include hydrogen, alkyl or alkoxy groups with approximately 1 to 3 carbon atoms. Hydrogen, methyl, and methoxy groups are particularly preferred, with hydrogen being most preferred. For cyclodextrin inclusion, _R5 , _R6 , _R11 , and _R12 , while not as large as _R1 - _R4 and _R13 - _R16 , are also small. Examples include hydrogen, alkyl or alkoxy groups with approximately 1 to 6 carbon atoms. _R1 - _R6 and _R11 - _R16 are sites of cyclodextrin inclusion, so even when introducing hydrophilic functional groups, the entire group is preferably hydrophobic. _R17 , _R18 , _R22 , and _R23 do not significantly affect cyclodextrin inclusion and are not particularly limited as long as they are the substituents listed above. R ₇ , R ₁₀ , R ₁₉ , and R ₂₁ are preferably hydrogen from the perspective of ease of synthesis. R ₈ , R ₉ , and R ₂₀ are not particularly limited as long as they are the substituents described above. The bond between the indocyanine group and the cyclodextrin is not particularly limited as long as it is covalent, and examples thereof include an alkyl bond, an amino bond, an amide bond, a double bond, a triple bond, an ester bond, and an ether bond. However, in chemical synthesis, an amide bond is preferred from the perspective of efficiency.

In order to include at least a portion of the naphthyl groups of the indocyanine in the cyclodextrin cavity, a spacer is preferably used to covalently bond the indocyanine and the cyclic sugar chain cyclodextrin via the spacer. In this case, the extent of inclusion of the naphthyl groups of the indocyanine in the cyclodextrin cavity can be controlled by adjusting the length of the spacer in Chemical Formula 2.

Therefore, the cyclodextrin-bound indocyanine compound formed by covalent bonding of indocyanine and cyclic sugar chain cyclodextrin via a spacer, and the compound characterized by the inclusion of the naphthyl group of indocyanine in the cavity of cyclodextrin, preferably exemplified by Chemical Formula 4. In addition, the substances of Chemical Formula 6, Chemical Formula 8, and Chemical Formula 10 are preferably exemplified. In addition, the substance of Chemical Formula 12 is preferably exemplified. In Chemical Formula 6, m, n, p, and q are preferably m+p and n+q are 5 or more and 7 or less, respectively. From the perspective of easy cyclodextrin inclusion, in all chemical formulas (Chemical Formulas 1 to 10), the number of atoms in the structure (spacer) between the nitrogen atom in the indocyanine structure and the oxygen atom in the cyclodextrin structure is preferably 7 or more and 9 or less.

<3. Synthesis of Inclusion Cyclodextrin-Conjugated Indocyanine Compounds>

The inclusion-type cyclodextrin-bound indocyanine compound of the present invention is as described above, and its synthesis method is to use the non-inclusion-type cyclodextrin-bound indocyanine compound synthesized above as a synthesis precursor and dissolve the compound in an aqueous solution. In the aqueous solution, any substance can be contained as long as it does not hinder inclusion, and the water content is not particularly limited. In addition, the temperature suitable for inclusion is -20°C to 100°C, preferably 0°C to 50°C. In addition, the time required for inclusion is from immediately after addition to the aqueous solution to about 1 month. The inclusion reaction can be in various forms depending on the properties of the non-inclusion-type cyclodextrin-bound indocyanine compound as described above, the temperature of the inclusion reaction, the composition and concentration of the aqueous solution, etc.

Alternatively, the non-inclusion-type cyclodextrin-bound indocyanine compound of the present invention can be converted to an inclusion-type cyclodextrin-bound indocyanine compound by dissolving it in a solvent containing water. While there are no particular restrictions on the water content in the solvent, in principle, inclusion is facilitated when the water content is high, preferably 50% by mass or greater. Furthermore, as long as a substance capable of forming an inclusion-type cyclodextrin-bound indocyanine compound is present in a solvent other than an aqueous solution, inclusion is not necessarily required in an aqueous solution.

In addition, an inclusion-type cyclodextrin-bound indocyanine compound may be formed during the synthesis of a non-inclusion-type cyclodextrin-bound indocyanine compound. In this case, there is no need to conduct the inclusion reaction again.

<4. Synthesis of Non-Inclusion Cyclodextrin-Bound Indocyanine Compounds (Synthesis Example)>

The synthesis of the compound represented by Chemical Formula 11 is given as an example. The indocyanine compound represented by Chemical Formula 11 can be obtained by, for example, adding the compound represented by Chemical Formula 13 synthesized by the method described in Non-Patent Document 5, Chemical Formula 14 synthesized by the method described in Non-Patent Document 6, a water-soluble carbodiimide (WSC: for example, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride) or dicyclohexylcarbodiimide (DCC) as a dehydration condensation agent, and pyridine or N,N-dimethylformamide or an aqueous solution as a solvent, and reacting at -20°C to 60°C for 10 minutes to 100 hours. In addition, in order to activate the reaction, 1-hydroxybenzotriazole (HOBt) can be added as an activator, for example. The amount of the dehydration condensation agent is 2 times the mole of the compound represented by Chemical Formula 13 or more. The solvent used is not limited as long as it can dissolve the reactants and does not hinder the dehydration condensation reaction. There is no limitation on the activator as long as it can activate the dehydration condensation reaction, and there is no limitation on the amount of the activator added as long as it can cause the dehydration condensation reaction to proceed to a desired extent.

[Chemical Formula 13]

[Chemical Formula 14]

<5. Purification of Non-Inclusion Cyclodextrin-Bound Indocyanine Compounds>

The mixture containing the non-inclusion cyclodextrin-bound indocyanine compound synthesized by the above method is dissolved in an acidic aqueous solution and subjected to reverse-phase column chromatography. Elution is performed using, for example, a mixture of acidic water and methanol, a mixture of acidic water and acetonitrile, a mixture of acidic water and ethanol, or a mixture of acidic water and acetone, to isolate and purify the high-purity non-inclusion cyclodextrin-bound indocyanine compound. The acid is not limited as long as it does not decompose the non-inclusion cyclodextrin-bound indocyanine compound, effectively elutes it, and facilitates post-elution handling. Preferred examples include hydrochloric acid, trifluoroacetic acid, acetic acid, sulfuric acid, nitric acid, and formic acid. Hydrochloric acid, trifluoroacetic acid, and acetic acid are preferred. Hydrochloric acid is most preferred. The acid concentration is not limited as long as it does not decompose the non-inclusion cyclodextrin-bound indocyanine compound, effectively elutes it, and facilitates post-elution handling. It is preferably 0.01 mM to 10 mM, and most preferably 0.1 mM to 1 mM. By keeping the concentration within this range, the target compound can be rapidly eluted without decomposing it. The eluted non-inclusion cyclodextrin-bound indocyanine compound can be obtained as a solid by removing the solvent. Freeze-drying can also be used as a solvent removal method.

<6. Synthesis Method (Synthesis Example) and Purification Method (Purification Example) of Inclusion-Type Cyclodextrin-Bound Indocyanine Compounds>

The non-inclusion type cyclodextrin-bound indocyanine compound of Chemical Formula 11 synthesized and purified by the above method is, for example, non-inclusion type in DMSO, but immediately becomes inclusion type upon dissolution in water, as shown in Chemical Formula 12. This phenomenon can be confirmed using ¹ H NMR.

Therefore, as long as the inclusion compound is an aqueous solution, it exists as an inclusion compound and can be used as an inclusion compound. In addition, the inclusion compound in the aqueous solution can be made into a solid by removing the water of the aqueous solution, and a solid of the inclusion compound can also be made.

<7. In vivo dynamics measuring device and circulation visualization device applicable to a living body to which a diagnostic composition has been administered>

In vivo dynamic measurement device

This device is completed based on the fact that the cyclodextrin-bound indocyanine compound of the present invention (hereinafter sometimes appropriately referred to as "compound of the present invention") used in the diagnostic composition of the present invention has different in vivo dynamics such as the degree of interstitial migration from ICG.

That is, the metabolic rate of the compound of the present invention and the rate of migration from the circulatory system, such as blood or lymph, to the interstitium differ from those of ICG. Consequently, the compound of the present invention enters the interstitium at a relatively greater rate than ICG. Therefore, by analyzing the in vivo dynamics of the compound of the present invention, the mechanism of fluid migration (in vivo dynamics) within the body can be accurately evaluated. Here, by maximally maintaining constant measurement conditions (such as the activity state of the organism and ambient temperature), the present device can more accurately measure in vivo dynamics.

One form of abnormal fluid movement in a living body is edema. Here, according to the so-called Stirling hypothesis, the movement of fluid (water) in a living body can be roughly considered to be from arteries to interstitial tissue, and then from interstitial tissue to veins.

The water movement from the artery to the vein can be calculated as (A)-(B)-(C)+(D) based on the pressure difference between the arterial pressure and the interstitial pressure, the water movement outside the circulatory system (A), the water movement into the circulatory system (B) based on the pressure difference between the venous pressure and the interstitial pressure, the water movement from the interstitial fluid to the artery (C) based on the pressure difference between the interstitial fluid osmotic pressure and the arterial blood osmotic pressure, and the water movement from the interstitial fluid to the vein (D) based on the pressure difference between the interstitial fluid osmotic pressure and the venous blood osmotic pressure.

Therefore, when the pressure difference between arterial and venous pressure decreases due to heart failure, or when interstitial fluid osmotic pressure decreases due to nutritional imbalance, the balance of water transfer (A) to (D) is disrupted, causing edema. Furthermore, when there are abnormalities in capillary permeability (abnormalities in the selectivity of material permeation), such as in diabetes, the balance of water transfer (A) to (D) is also disrupted, causing edema. There is no simple method to directly assess abnormal capillary permeability. While the occurrence of edema can be detected by phenomenon, it is currently difficult to determine the specific cause of edema.

Based on the above findings, when abnormal permeability of the capillary wall occurs, the movement of the compound of the present invention can be expected to change compared to cases where edema occurs due to a decrease in the differential pressure between arterial and venous pressure, a decrease in intracellular osmotic pressure, etc. Therefore, when the movement of the compound of the present invention is confirmed to be greater than (or less than) an amount equivalent to water movement, it can be determined that abnormal capillary permeability has occurred. Because it is believed that if no abnormal permeability of the capillary wall occurs, only the movement of the compound of the present invention to an extent equivalent to the change in water movement can be confirmed, it can be determined that an abnormality has occurred in the vascular wall.

In particular, by using two or more compounds of the present invention with different migration rates into the interstitium and measuring the in vivo dynamics of these compounds separately and comparing them, the degree of change in capillary permeability can be evaluated. In other words, by using two or more compounds of the present invention with different permeabilities in the capillary wall and measuring their permeabilities, the occurrence of abnormal vascular wall permeability can be more accurately evaluated. In other words, by using two or more compounds of the present invention with different susceptibilities to abnormal vascular wall permeability and measuring their in vivo dynamics, the factors affecting water migration can be more accurately evaluated. In addition, in addition to evaluating water migration using the compounds of the present invention, water migration can also be measured using conventional methods.

The in vivo dynamics measurement device of the present invention includes an excitation light irradiation unit, a fluorescence intensity measurement unit, an in vivo dynamics calculation unit, and other units. Other units may be selected as needed, such as an in vivo concentration calculation unit that calculates the concentration of a cyclodextrin-bound indocyanine compound based on the fluorescence intensity measured by the fluorescence intensity measurement unit.

This device is a device for measuring at least a portion of a living body to which the diagnostic composition of the present invention has been administered. The dosage of the diagnostic composition is the amount at which fluorescence is observed when the measurement site is irradiated with excitation light. Therefore, the appropriate dosage varies depending on the measurement site. There is no particular limitation on the type of compound of the present invention contained in the diagnostic composition. The compound of the present invention may be used alone or in combination of two or more. In addition, additional diagnostic compositions of the same or different compositions may be added during the measurement process.

The excitation light irradiation unit irradiates the compound of the present invention contained in the administered diagnostic composition with excitation light of a wavelength capable of generating fluorescence. The wavelength of the excitation light irradiated can be limited to an appropriate range. By limiting the wavelength to a minimum possible range, fluorescence and excitation light can be reliably separated. Regarding wavelength limitation, a light source emitting light of an appropriate wavelength can be selected, or a filter can be used to limit the wavelength.

The excitation light irradiation mode is not particularly limited, as long as the generated fluorescence can be measured by the fluorescence intensity measurement unit described later. Examples of excitation light include continuous light, pulsed light, and light with varying intensity. To vary the intensity, the excitation light intensity can be modulated by irradiating the excitation light pulses at predetermined intervals. Preferably, the excitation light intensity is modulated using pulse amplitude modulation.

The excitation light is irradiated to the site to be irradiated through an appropriate optical system. The site to be irradiated is the site where dynamic measurements in the body are to be performed. For example, when evaluating a site where edema occurs, it is preferable to irradiate the site with excitation light directly.

The excitation light irradiation range is not particularly limited. The irradiation range is determined as needed. A narrow irradiation range allows for precise measurement of subdivided in vivo dynamics within the narrow irradiated area. Irradiating a wider range increases the relative amount of the compound of the present invention that fluoresces in response to the excitation light, enabling more precise measurement of fluorescence intensity.

Furthermore, the excitation light irradiation unit is preferably used to irradiate the excitation light while suppressing the influence of ambient light, for example, by irradiating the excitation light in a dark place or by shielding the portion irradiated with the excitation light from external light.

The fluorescence intensity measuring unit measures the fluorescence intensity emitted from the area irradiated with excitation light by the excitation light irradiation unit. The fluorescence intensity is preferably measured by removing light other than fluorescence (ambient light, excitation light, etc.) through a filter that selectively transmits the emitted fluorescence.

When the excitation light irradiation unit uses a unit that irradiates excitation light with modulated intensity, the component that varies according to the modulation can be separated from the measured light intensity and used as the fluorescence intensity. For example, when the excitation light intensity is modulated using pulse amplitude modulation, the fluorescence intensity can be separated by demodulating the light component that varies according to the intensity of the modulated pulse and measuring its intensity. This reduces the impact of ambient light on the fluorescence intensity measurement results.

The in vivo concentration calculation unit is a unit that calculates the in vivo concentration of the compound of the present invention based on the fluorescence intensity measured by the fluorescence intensity measurement unit. The relationship between the fluorescence intensity and the in vivo concentration of the compound of the present invention can be calculated using an appropriate method. For example, a standard curve can be prepared in advance, and the in vivo concentration of the compound of the present invention can be calculated based on the standard curve. In addition, the absolute value of the fluorescence intensity can be directly used as a value related to the in vivo concentration of the compound of the present invention. The in vivo concentration calculation unit calculates the in vivo concentration of the compound of the present invention over time.

The in vivo dynamics calculation unit is a unit that calculates the temporal change of the time rate of change of the in vivo concentration of the compound of the present invention from the in vivo concentration data obtained over time by the in vivo concentration calculation unit. The in vivo dynamics calculation unit calculates the speed of the compound of the present invention moving from the circulatory system to the interstitial tissue and the speed of the compound of the present invention moving from the interstitial tissue to the circulatory system in a part of the body using the temporal change rate of the in vivo concentration obtained. Instead of using the in vivo concentration, the speed of movement into and out of the interstitial tissue can be directly calculated by using the change in fluorescence intensity obtained over time by the fluorescence intensity measurement unit.

It is speculated that there is a high correlation between the movement of the compound of the present invention into (and out of) the interstitium and the movement of water in body fluids. Therefore, by measuring the in vivo behavior of the compound of the present invention, it is possible to evaluate the movement of water in the measurement site. Furthermore, when abnormalities occur in the vascular wall and the permeability of substances is abnormal, the behavior of the compound of the present invention in the body should also change. Therefore, by evaluating the behavior of the compound of the present invention, vascular permeability can be evaluated.

The time rate of change of fluorescence intensity (or concentration in the organism) is obtained by differentiating the time-dependent change in fluorescence intensity (or concentration in the organism) with respect to time, or by calculating the fluorescence intensity (or concentration in the organism) at each specified time and then calculating it as the difference between the fluorescence intensity (or concentration in the organism) before (after) the specified time.

The speed of the compound of the present invention entering the interstitial fluid and the speed of being discharged in a part of the organism can be calculated by the time rate of change of the fluorescence intensity (or biological concentration) obtained. Here, the speed of entering the interstitial fluid and the speed of being discharged can be obtained as relative values by the fluorescence intensity. When more precision is required, it can be calculated according to the concentration of the compound of the present invention in the interstitial and the amount of the interstitial fluid. In addition, the concentration of the compound of the present invention in blood and lymph and its amount are also considered, and it can be calculated more accurately. The concentration of the compound of the present invention in blood, etc. can be measured with good accuracy by actually taking blood.

The following methods can be cited as methods for calculating the concentration in interstitial fluid. The first method is a method for directly approximating the calculated in vivo concentration of the compound of the present invention to the concentration in the interstitial fluid in a part of the organism. As a method related to the first method, the concentration in the interstitial fluid can also be calculated by considering the amount of interstitial fluid. The second method is to calculate the influence ratio of the substance present in the blood vessel on the measured value measured by the fluorescence intensity measurement unit by using other standard substances (ICG, etc.) that do not move to the interstitial on the basis of measuring the concentration of the compound of the present invention in the actual blood, and to calculate the concentration in the interstitial fluid by removing the influence of the concentration of the compound of the present invention in the measured blood.

The time rate of change of the concentration of the compounds of the present invention in interstitial fluid can be calculated by subtracting the speed of movement outside the interstitial fluid from the speed of movement inside the interstitial fluid. Here, it can be assumed that the movement of the compounds of the present invention inside (outside) the interstitial fluid is carried out at a speed related to the water movement in the body fluid, and if the water movement reaches equilibrium, the speed of movement inside the interstitial fluid and the speed of movement outside the interstitial fluid can be considered as constants, therefore, the concentration change (metabolism, discharge, changes caused by movement to tissue, etc.) of the compounds of the present invention in blood and the time rate of change of concentration in interstitial fluid can be used to calculate its movement speed. In addition, even in the case of changes in its movement speed, it is possible to calculate their movement speed by making a model corresponding to this change and applying it.

If the calculated migration speed is outside the range of normal blood vessel wall values, it can be inferred that the blood vessel wall has abnormalities. In addition, as a result of abnormalities in the blood vessel wall, the permeability of the compound of the present invention may be increased or decreased.

It is a well-known fact that acute tissue swelling is caused by various tissue disorders such as acute inflammation, ischemia, and trauma, and is an extremely important tissue reaction in the medical field regardless of the field.

For example, in addition to the simple occurrence of swelling itself being a problem, there are also issues involving all organs, such as the evaluation of intestinal suturing during surgery, the evaluation of the degree of inflammation such as pneumonia, the evaluation of the suitability of organ transplantation, and the evaluation of brain and kidney dysfunction.

However, there is currently no accurate method for predicting the likelihood and extent of tissue swelling, and it can only be estimated empirically based on the degree of tissue damage. Therefore, in most cases, countermeasures can only be discussed after the occurrence of swelling is confirmed, which is a major limitation in treatment.

If the likelihood of swelling can be predicted with high accuracy early on, measures to reduce swelling can be taken early on, minimizing the impact. For example, if the extent of brain swelling that occurs shortly after a cerebral hemorrhage can be accurately predicted, decompression measures and measures to reduce vascular permeability can be implemented early on, minimizing the impact. Similarly, if the impact of ischemic stress can be predicted in advance, secondary complications caused by myocardial infarction and limb injuries can be minimized. Thus, high-precision quantitative prediction technology for swelling has the potential to significantly impact healthcare overall.

Until now, quantification of tissue swelling, including edema, has been qualitatively determined through observation. While quantification has been attempted, it is only applicable in very limited situations and has the disadvantage of being able to evaluate only after swelling has occurred.

To address this issue, the in vivo dynamic measurement device of the present invention calculates the rate of water migration within and outside the interstitium, thereby predicting the progression of swelling. As detailed in the Examples, it is known that ICG does not migrate into the interstitium as swelling progresses. In contrast, the cyclodextrin-conjugated indocyanine compound of the present invention can migrate within and outside the interstitium, and its movement into the interstitium is further promoted by the movement of water as swelling progresses. Therefore, by evaluating the movement of the cyclodextrin-conjugated indocyanine compound of the present invention into the interstitium, the progression of swelling can be predicted.

That is to say, it can be inferred that the degree of swelling that is ultimately formed is affected by the speed of movement of water into and out of the interstitial space, and therefore the degree of final swelling can be evaluated by evaluating the speed of movement of the cyclodextrin-bound indocyanine compound of the present invention, which is related to the speed of movement of water. The speed of movement can be calculated by the concentration of the cyclodextrin-bound indocyanine compound, or directly using the fluorescence intensity. In addition, the speed of movement into and out of the interstitial space can naturally be calculated as an absolute value, or it can be calculated as a relative value (for example, the rate of change of fluorescence intensity can be directly used as a value related to the speed of movement), and the progress of swelling can be predicted based on this value.

The progression of swelling is preferably calculated based on the normal migration rate into the interstitium when there is no swelling. However, the normal migration rate into the interstitium may be unknown. In such cases, a non-swollen site is selected as a control site, and the migration rate of this site is calculated and used as a substitute. Alternatively, the extent of interstitial migration of the cyclodextrin-conjugated indocyanine compound of the present invention can be evaluated by using ICG, which does not migrate within the interstitium, to assess the blood circulation volume and metabolism.

The determination of the extent of movement of the cyclodextrin-bound indocyanine compound of the present invention within the interstitium is carried out after a prescribed time has passed after the diagnostic composition of the present invention is administered into a living body. The prescribed time refers to the time required for the diagnostic composition of the present invention to be distributed in the blood. The reason for using data after the prescribed time for evaluation is that after administration of the diagnostic composition, when it is distributed into the blood after a prescribed time, it is hardly affected by the degree of swelling progression, the concentration in the blood rises rapidly, and the change in fluorescence intensity is not affected by the presence or absence of swelling, and almost no difference is produced. After distribution in the blood, the speed of movement within the interstitium will change according to the presence or absence of swelling progression, so an increase in fluorescence intensity will be selectively confirmed at the site where the movement occurs (i.e., the site where swelling occurs).

As a specific example of a method for estimating the progression of swelling, there is a method in which the blood volume is estimated from body weight, the size of the peak of fluorescence intensity and the time to the peak are calculated from the estimated blood volume; the fluorescence intensity at the time when the edema may change is measured taking into account this time and intensity, and the degree of edema progression and the degree of edema that may occur in the future can be estimated from the results.

Circular Visualization Installation

This device is based on the fact that the cyclodextrin-bound indocyanine compound of the present invention (hereinafter sometimes appropriately referred to as "compound of the present invention") used in the diagnostic composition of the present invention differs from ICG in its in vivo dynamics, such as the degree of migration into the interstitium.

That is, the compound of the present invention easily moves in the interstitial fluid, and by tracking the behavior of the compound of the present invention, the state of blood circulation or lymph circulation can be visualized.

The circulation visualization device of the present invention comprises an excitation light irradiation unit, a fluorescence imaging unit, a morphological imaging unit, and a display unit. This device is a device for measuring at least a portion of a living body to which the diagnostic composition of the present invention is administered. The dosage of the diagnostic composition is the amount in which fluorescence can be observed when the measurement site is irradiated with excitation light. Therefore, the appropriate dosage varies depending on the measurement site. The type of compound of the present invention contained in the diagnostic composition is not particularly limited. The compound of the present invention can be used alone or in combination of two or more. In addition, additional diagnostic compositions having the same or different compositions can be administered during the measurement process.

The excitation light irradiation unit irradiates the compound of the present invention contained in the administered diagnostic composition with excitation light of a wavelength capable of generating fluorescence. The wavelength of the excitation light used for irradiation can be limited to an appropriate range. By limiting the wavelength to a narrow range as much as possible, fluorescence and excitation light can be reliably separated. Wavelength limitation can be achieved by selecting a light source emitting light of an appropriate wavelength or by using a filter to limit the wavelength.

The form of excitation light irradiation is not particularly limited, as long as the generated fluorescence can be measured by the fluorescence imaging unit described below. For example, the excitation light may be continuous light, pulsed light, or light with varying intensity. To vary the intensity, the intensity of the excitation light can be modulated by irradiating the excitation light pulses at predetermined intervals. Preferably, the excitation light is modulated using pulse amplitude modulation.

The excitation light passes through an appropriate optical system and illuminates the desired area. The area to be illuminated is the part of the organism where the circulation is to be visualized, such as the area where tissue necrosis has occurred due to burns, frostbite, inflammation, trauma, infarction, and the like, and its surrounding areas. Necrotic areas of tissue lack blood circulation, and leaving them untreated offers little benefit, so removal is considered. In this case, it is ideal to completely remove only the necrotic area, comparing the necrotic area with the normal area.

Conventionally, identification of necrotic areas has been performed using methods such as angiography using contrast agents or detecting a decrease in body temperature associated with blood circulation. However, angiography has the problem of being inconvenient to use devices such as X-ray irradiation devices, and accurate determination based on body temperature is difficult.

The device of the present invention is used to evaluate necrotic and normal areas based on the presence or absence of body fluid (blood) circulation. Visualizing areas without circulation allows for easy removal. In addition to visualizing necrotic areas, direct observation of blood circulation is also possible, making it easy to identify abnormalities in circulatory function. For example, areas that are not necrotic but have decreased circulatory function due to, for example, infarction can be visualized.

The excitation light irradiation range is determined as needed so as to include the portion to be cyclically visualized.

Furthermore, the excitation light irradiation unit is preferably used to irradiate the excitation light while controlling the influence of ambient light, for example, by irradiating the excitation light in a dark place or by shielding the portion irradiated with the excitation light from external light.

The fluorescence imaging unit two-dimensionally acquires the intensity of fluorescence emitted by the compound of the present invention, which is excited by the excitation light irradiation unit, thereby obtaining data on the distribution status of the compound of the present invention in the organism. In other words, this unit acquires distribution status data representing the distribution of the compound of the present invention in a portion of the organism as two-dimensional image data.

For example, it can be composed of a combination of an appropriate optical system and an imaging element such as a CCD. The resolution of the data obtained two-dimensionally can be set to a necessary value according to the purpose. The determination of fluorescence intensity preferably allows the emitted fluorescence to be selectively transmitted through a filter and removes light (ambient light, excitation light, etc.) other than the fluorescence to be measured.

When using excitation light with modulated intensity as the excitation light irradiation unit, the fluorescence intensity is obtained by separating the components that vary according to the modulation from the measured light intensity. For example, when the excitation light intensity is modulated using pulse amplitude modulation, the fluorescence intensity is separated by demodulating the components of the light that vary according to the modulated pulse intensity and measuring their intensities. This reduces the influence of ambient light on the fluorescence intensity measurement results.

The morphological imaging unit is a unit that two-dimensionally acquires the intensity of light at wavelengths other than the fluorescence wavelength emitted by the compound of the present invention to obtain morphological data of the compound in a portion of a living body. In other words, this unit is a unit that acquires two-dimensional image data of the morphology of a portion of a living body.

The morphological imaging unit can be composed of an appropriate optical system and an imaging element such as a CCD. The resolution of the two-dimensional data can be set according to the desired purpose. In this case, the fluorescence emitted by the excitation light is not measured (or the detection sensitivity is reduced). The morphological imaging unit uses almost all of the aforementioned fluorescence imaging unit and optical system. Finally, a dichroic prism is used on the optical path before it is introduced into the imaging element to guide light with a wavelength corresponding to the fluorescence to the fluorescence imaging unit, and light of other wavelengths can be guided to the morphological imaging unit. By appropriately forming a bidirectional color film, the dichroic prism can reasonably control the wavelength of the separated light.

Furthermore, the fluorescence imaging unit and the morphological imaging unit can share the same camera device. That is, the separation of fluorescence and other light can also be a mathematical separation performed after obtaining their two-dimensional image data. Furthermore, the distribution state data can also be complex image data from the surface of a part of the biological body to the deep layer direction. The optical focus used by the fluorescence imaging unit obtains complex two-dimensional image data in the deep layer direction while moving in the deep layer direction. In addition, as the excitation light irradiation unit, an optical system with a variable focal length is used, and the focus is moved not on the surface of a part of the biological body, but in the deep layer direction, or the excitation light is concentrated and irradiated to a part of the biological body, so that fluorescence can be selectively emitted not only on the surface of the biological body but also in the deep layer inside the biological body, and the circulation of this part can be visualized.

The display unit is a display unit that represents the distribution state of the compound of the present invention in a part of the organism by overlapping the morphological data obtained by the morphological imaging unit and the distribution state data obtained by the fluorescence imaging unit. When the wavelength of the fluorescence is not within the range of visible light, it is preferred to convert the wavelength of the fluorescence into visible light for representation. From the perspective of visualization of the circulation, the representation of the distribution state data takes precedence over the representation of the morphological data. In particular, it is possible to distinguish between the low distribution amount (i.e., fluorescence intensity) of the compound of the present invention and the other parts (whether it is low or not is determined according to the purpose of visualization of the circulation. For example, in the purpose of visualizing the necrotic part, in the part without circulation, i.e., the part where no fluorescence is confirmed) and other parts can be distinguished. For example, the part can be represented as a color different from that of the other parts or a flashing representation. The superposition of the distribution state data and the morphological data can be realized using logic on a computer, etc. The representation of two-dimensional data can be realized by a general display device. The display device is provided between the organism and the measurer, so that the organism can be treated while observing the display device.

＜8. Definitions, etc.＞

In the present invention, "alkyl" refers to a linear or branched alkyl group having 1 to 20 carbon atoms which may have a substituent, for example, a methyl group, ethyl group, propyl group, butyl group, pentyl group, hexyl group, heptyl group, octyl group, nonyl group, decyl group, undecyl group, dodecyl group, tridecyl group, tetradecyl group, pentadecyl group, hexadecyl group, heptadecyl group, octadecyl group, nonadecyl group, eicosyl group or a branched group.

In the present invention, the "alkoxy group" includes, for example, methoxy, ethoxy, propoxy, butoxy, pentyloxy, hexyloxy, methoxyethoxy, methoxypropoxy, ethoxyethoxy, ethoxypropoxy, methoxyethoxyethoxy, etc., in which alkoxy groups having 1 to 20 carbon atoms are bonded in a straight chain or branched form.

In the present invention, the "aryl group" includes aromatic hydrocarbons having 6 to 20 carbon atoms, such as phenyl and naphthyl.

Example

The preferred embodiments of the present invention are described in detail below by way of examples. However, the technical scope of the present invention is not limited to the following embodiments, and various modifications can be made within the scope of the present invention.

<Experiment 1: Synthesis and Purification of Compounds Represented by Chemical Formula 15 and Chemical Formula 16>

A mixture of 0.20 g of the compound represented by Chemical Formula 13, 0.94 g of the compound represented by Chemical Formula 14, 0.18 g of WSC, 0.12 g of HOBt, 4.0 mL of pyridine, and 2.0 mL of N,N-dimethylformamide was stirred at 0°C in the dark for 6 hours. 50 mL of acetone was then added, and the precipitate was filtered under reduced pressure. The precipitate was dissolved in a 0.1% trifluoroacetic acid aqueous solution and subjected to ODS column chromatography. A mixture of water and methanol containing 1 mM hydrochloric acid was used as the eluent to elute the compound represented by Chemical Formula 15. The eluate was concentrated under reduced pressure to obtain 0.65 g of the inclusion-type compound represented by Chemical Formula 16 as a green solid (at the end of the reduced pressure concentration of the eluate, the water content was high, so it naturally became an inclusion-type compound).

[Chemical Formula 15]

[Chemical Formula 16]

Next, the instrumental analysis data for the target compound represented by Chemical Formula 16 are shown. ¹ H NMR (500 MHz, D ₂ O, 26°C, acetone: 2.10 ppm) 1.43 (2H, m), 1.55 (2H, m), 1.99 (6H, s), 2.09 (6H, s), 2.63 (6H, m), 2.80 (6H, m), 2.92 (2H, m), 3.02 (2H, dd, J = 3.7, 9.8 Hz), 3.08 (2H, t, J = 9.2 Hz), 3.2-4.1 (m), 4.19 (2H, t, J = 9.8 Hz), 4.26 (2H, t, J = 9.8 Hz), 4.33 (2H, m), 4.43 (2H, m), 4.71 (2H, d, J = 2.4 Hz) , 4.81 (4H, d, J = 3.7 Hz), 4.91 (2H, d, J = 3.7 Hz), 4.99 (2H, d, J = 3.7 Hz), 5.08 (2H, d, J = 3.7 Hz), 5.13 (2H, d, J = 3.1 Hz), 6.15 (2H, d, J = 13 Hz), 6.52 (2H, t, J = 12 Hz), 7.43 (4H, m), 7.57 (1H, d, J = 12 Hz), 7.57 (2H, d, J = 9.2 Hz), 7.78 (2H, m), 8.06 (3H, m), 8.15 (2H, d, J = 8.5 Hz). ESI-MS m/z calcd for C ₁₃₁ H ₁₉₁ N ₄ O ₇₂ 2972, found 2973 [M] ⁺ .

<Experiment 2: Synthesis and Purification of Compounds Represented by Chemical Formula 19 and Chemical Formula 20>

A mixture of 0.17 g of the compound represented by Chemical Formula 17, 5 mL of methanol, and 0.30 g of t-BuOK was stirred at room temperature for 12 hours. Then, 3 mL of 1M hydrochloric acid and 50 mL of water were added. The precipitate was filtered, washed with water, and then dried under reduced pressure to obtain 0.17 g of the compound represented by Chemical Formula 18.

[Chemical Formula 17]

[Chemical Formula 18]

A mixture of 0.02 g of the compound represented by Chemical Formula 18, 0.081 g of the compound represented by Chemical Formula 14, 0.016 g of WSC, 0.011 g of HOBt, 0.3 mL of pyridine, and 0.2 mL of N,N-dimethylformamide was stirred at 0°C in the dark for 6 hours. 5 mL of acetone was then added, and the precipitate was filtered under reduced pressure, dissolved in a 0.1% trifluoroacetic acid aqueous solution, and subjected to ODS column chromatography. A mixture of water and methanol containing 1 mM hydrochloric acid was used as the eluent to elute the compound represented by Chemical Formula 19. The eluent was concentrated under reduced pressure to obtain 0.045 g of the inclusion-type compound represented by Chemical Formula 20 as a green solid (at the end of the concentration of the eluent under reduced pressure, the water content was high, so it naturally became an inclusion-type compound).

[Chemical Formula 19]

[Chemical Formula 20]

Next, the machine analysis data of the target compound represented by Chemical Formula 20 are shown. ¹ H NMR (500 MHz, D ₂ O,40℃,acetone:2.26ppm)1.54(2H,m),1.68(2H,m),1.98(2H,m),2.19(6H,s),2 .20(2H,m),2.30(6H,s),2.6-2.85(10H,m),2.95(2H,m),3.00(4H,m),3.08 (2H,t,J＝12Hz),3.17(2H,dd,J＝3.7,9.8Hz),3.26(2H,t,J＝9.8Hz),3.35- 4.30(m),4.35(2H,t,J＝9.2Hz),4.50(2H,t,J＝9.2H),4.52(2H,m),4.63(2H ,m),4.87(2H,d,J＝3.7Hz),4.95(d,J＝3.1Hz),4.97(2H,d,J＝3.7Hz),5.08 (2H,d,J＝3.7Hz),5.15(2H,d,J＝4.3Hz),5.25(2H,d,J＝3.7Hz),5.29(2H,d, J＝3.7Hz),6.30(2H,d,J＝14.6Hz),7.58(4H,m),7.73(2H,d,J＝8.5Hz),7.95 (2H,m),8.25(2H,m),8.32(2H,d,J＝14.6Hz),8.35(2H,d,J＝8.5Hz).ESI-MS m/ _z calculated for _{C135H197N4O73 3042} , found 3042 [M] ⁺ .

<Experiment 3: Synthesis and Purification of the Compound Represented by Chemical Formula 21>

A mixture of 0.04 g of the compound represented by Chemical Formula 13, 0.18 g of mono-6-amino-6-deoxy-β-cyclodextrin, 0.05 g of WSC, 0.025 g of HOBt, 0.8 mL of pyridine, and 0.4 mL of N,N-dimethylformamide was stirred at 0°C in the dark for 3 hours. 10 mL of acetone was then added, and the precipitate was filtered under reduced pressure, dissolved in 0.1% trifluoroacetic acid aqueous solution, and subjected to ODS column chromatography. The compound represented by Chemical Formula 21 was eluted using a mixture of water and methanol containing 1 mM hydrochloric acid. The eluate was concentrated under reduced pressure to obtain 0.11 g of the compound represented by Chemical Formula 21 as a green solid.

[Chemical Formula 21]

Next, the instrumental analysis data for the target compound represented by Chemical Formula 21 are shown. ¹ H NMR (500 MHz, D ₂ O, 29°C, acetone: 2.10 ppm) 1.79 (12H, br.), 2.68 (4H, br.), 3.0-4.5 (98H), 4.4 (4H, br.), 4.5-5.3 (14H, br.), 6.18 (2H, br.), 6.46 (2H, br.), 7.3-8.2 (15H). ESI-MS m/z calculated for C ₁₂₅ H ₁₇₉ N ₄ O ₇₀ 2856, found 2856 [M] ⁺ .

<Experiment 4: Synthesis and Purification of the Compound Represented by Chemical Formula 23>

A mixture of 0.02 g of the compound represented by Chemical Formula 22, 0.096 g of mono-6-amino-6-deoxy-β-cyclodextrin, 0.032 g of WSC, 0.5 mL of pyridine, and 0.05 mL of 0.1 M phosphate buffer was stirred in the dark at room temperature for 24 hours. 10 mL of acetone was then added, and the precipitate was filtered under reduced pressure, dissolved in 0.1% trifluoroacetic acid aqueous solution, and subjected to ODS column chromatography. A mixture of water and acetonitrile was used as the eluent to elute the compound represented by Chemical Formula 23. The eluate was concentrated under reduced pressure to yield 0.014 g of the compound represented by Chemical Formula 23 as a green solid.

[Chemical Formula 22]

[Chemical Formula 23]

Next, the instrumental analysis data for the target compound represented by Chemical Formula 21 are shown. ¹ H NMR (500 MHz, D ₂ O, 26°C, acetone: 2.15 ppm) 1.26 (4H, m), 1.5-2.25 (24H, br), 2.7-4.2 (88H), 4.82 (2H, br), 4.90 (8H, br), 4.97 (2H, br), 5.03 (2H, br), 6.11 (2H, br), 6.36 (2H, br), 7.3-8.01 (15H, br). ESI-MS m/z calculated for C ₁₃₁ H ₁₉₁ N ₄ O ₇₀ 2940, found 2940 [M] ⁺ .

<Experiment 5: Synthesis and Purification of the Compound Represented by Chemical Formula 24>

A mixture of 0.20 g of the compound represented by Chemical Formula 22, 0.02 g of 3-amino-3-deoxy-β-cyclodextrin, 0.096 g of WSC, 0.013 g of HOBt, 0.4 mL of pyridine, and 0.2 mL of N,N-dimethylformamide was stirred in the dark at room temperature for 1 hour. Subsequently, 10 mL of acetone was added, and the precipitate was filtered under reduced pressure, dissolved in 0.1% trifluoroacetic acid aqueous solution, and subjected to ODS column chromatography. A mixture of water and methanol was used as the eluent to elute the compound represented by Chemical Formula 24. The eluate was concentrated under reduced pressure to yield 0.013 g of the compound represented by Chemical Formula 24 as a green solid.

[Chemical Formula 24]

Next, the instrumental analysis data for the target compound represented by Chemical Formula 24 are shown. ¹ H NMR (500 MHz, D ₂ O, 29°C, acetone: 2.10 ppm) 1.1-2.5 (28H), 3.0-4.25 (88H), 4.5-5.2 (14H), 7.3-8.02 (15H). ESI-MS m/z calculated for C ₁₃₁ H ₁₉₁ N ₄ O ₇₀ 2940, found 2940 [M] ⁺ .

<Experiment 6: Synthesis and Purification of the Compound Represented by Chemical Formula 25>

A mixture of 0.02 g of the compound represented by Chemical Formula 22, 0.1 g of the compound represented by Chemical Formula 14, 0.032 g of WSC, 0.5 mL of pyridine, and 0.05 mL of 0.1 M phosphate buffer was stirred in the dark at room temperature for 24 hours. 10 mL of acetone was then added, and the precipitate was filtered under reduced pressure, dissolved in 0.1% trifluoroacetic acid aqueous solution, and subjected to ODS column chromatography. A mixture of water and methanol was used as the eluent to elute the compound represented by Chemical Formula 25. The eluate was concentrated under reduced pressure to yield 0.021 g of the compound represented by Chemical Formula 25 as a green solid.

[Chemical Formula 25]

Next, the instrumental analysis data for the target compound represented by Chemical Formula 25 are shown. ¹ H NMR (500 MHz, D ₂ O, 25°C, acetone: 2.10 ppm) 1.0-2.5 (32H), 3.0-4.5 (96H), 4.8-5.2 (14H), 6.09 (2H, br), 6.37 (2H, br), 7.3-8.02 (15H, br). ESI-MS m/z calculated for C ₁₃₇ H ₂₀₃ N ₄ O ₇₂ 3056, found 3056 [M] ⁺ .

<Test 7: Solubility of the Cyclodextrin-Bound Indocyanine Compound of the Present Invention>

Solubility tests were conducted on ICG and the cyclodextrin-conjugated indocyanine compounds of the present invention (Chemical Formulas 16, 20, 21, and 23-25) in water and saline. While the dissolution of powdered ICG from Molecular Probe in water and saline required vigorous agitation for approximately one minute, the cyclodextrin-conjugated indocyanine compounds of the present invention, particularly those represented by Chemical Formulas 16 and 20, dissolved rapidly without the need for agitation.

<Test 8: Adsorption of the Cyclodextrin-Bound Indocyanine Compound of the Present Invention to Human Skin>

ICG and the cyclodextrin-conjugated indocyanine compounds of the present invention (Chemical Formulas 16, 20, 21, 23-25) were tested for their adsorption to human skin. A 1 mM aqueous solution (0.03 mL) of each of ICG and the cyclodextrin-conjugated indocyanine compounds of the present invention (Chemical Formulas 16, 20, 21, 23-25) was dropped onto the wrist, rinsed with water after 5 minutes, and then wiped and rinsed again. The result showed that ICG was not completely washed away by water, while the cyclodextrin-conjugated indocyanine compounds of the present invention, especially the compounds represented by Chemical Formulas 16 and 20, were easily washed away by water, indicating that the adsorption of the cyclodextrin-conjugated indocyanine compounds of the present invention to human skin was significantly lower than that of ICG (Figure 1).

<Test 9: Adsorption of the Cyclodextrin-Bound Indocyanine Compound of the Present Invention to Cellulose Fibers>

ICG and the cyclodextrin-conjugated indocyanine compounds of the present invention (Chemical Formulas 16, 20, 21, 23-25) were tested for their adsorption onto cellulose fibers. A cotton swab (Sanyo Co., Ltd.) was used to apply 0.05 mL of a 1 mM aqueous solution of each of ICG and the cyclodextrin-conjugated indocyanine compounds of the present invention (Chemical Formulas 16, 20, 21, 23-25) to a model cellulose fiber. After 3 minutes, the swab was rinsed in running water (tap water, 1 L/min) for 5 seconds. The results showed that ICG was not completely washed away, while the cyclodextrin-conjugated indocyanine compounds of the present invention, particularly those represented by Chemical Formulas 16 and 20, were easily washed away, indicating that the adsorption of the compounds represented by the cyclodextrin-conjugated indocyanine compounds of the present invention onto cellulose fibers was significantly lower than that of ICG (Figure 2).

<Test 10: Adsorption of the Cyclodextrin-Conjugated Indocyanine Compound of the Present Invention to a Meat Model>

Adsorption of ICG and the cyclodextrin-conjugated indocyanine compounds of the present invention (Chemical Formulas 16, 20, 21, 23-25) onto a biological meat model was tested. Commercially available pork tenderloin was used as the meat model. A 5 mm diameter recess was created in the pork tenderloin. 0.05 mL of a 1 mM aqueous solution of each of ICG and the cyclodextrin-conjugated indocyanine compounds of the present invention (Chemical Formulas 16, 20, 21, 23-25) was applied to the meat model. After 3 minutes, the sample was rinsed in running water (tap water, 1 L/min) for 10 seconds. The results showed that ICG was not completely washed off, while the cyclodextrin-conjugated indocyanine compounds of the present invention, particularly those represented by Chemical Formulas 16 and 20, were easily washed off, demonstrating that the adsorption of the cyclodextrin-conjugated indocyanine compounds of the present invention onto the biological meat model was significantly lower than that of ICG (Figure 3).

<Test 11: Adsorption of the Cyclodextrin-Conjugated Indocyanine Compound of the Present Invention to a Biological Protein Model>

Adsorption of ICG and the cyclodextrin-conjugated indocyanine compounds of the present invention (Chemical Formulas 16, 20, 21, 23-25) onto biological protein models was tested. A 5 mm diameter recess was created in commercially available chicken breast meat as the biological protein model. 0.05 mL of a 1 mM aqueous solution of ICG and the cyclodextrin-conjugated indocyanine compounds of the present invention (Chemical Formulas 16, 20, 21, 23-25) was applied to the breast. After 3 minutes, the samples were rinsed in running water (1 L/min) for 10 seconds. The results showed that ICG was not completely washed away, while the cyclodextrin-conjugated indocyanine compounds of the present invention, particularly those represented by Chemical Formulas 16 and 20, were easily washed away, indicating that the adsorption of the cyclodextrin-conjugated indocyanine compounds of the present invention onto biological protein models was significantly lower than that of ICG (Figure 4).

<Test 12: Adsorption of the Cyclodextrin-Bound Indocyanine Compound of the Present Invention to Hydrophobic Chemical Fibers>

The adsorption of ICG and the cyclodextrin-conjugated indocyanine compounds of the present invention (Chemical Formulas 16, 20, 21, 23-25) onto hydrophobic chemical fibers was tested. A polypropylene mask (Tamagawa Eisai Co., Ltd.) was used as a model hydrophobic chemical fiber. 0.05 mL of a 1 mM aqueous solution of ICG and the cyclodextrin-conjugated indocyanine compounds of the present invention (Chemical Formulas 16, 20, 21, 23-25) was applied to the mask. After 20 minutes (the sample was left to remove any moisture, so it would not adhere to the mask), the mask was rinsed in running water (1 L/min) for one second. The results showed that ICG was not completely washed off, while the cyclodextrin-conjugated indocyanine compounds of the present invention, particularly those represented by Chemical Formulas 16 and 20, were easily washed off, indicating that the adsorption of the cyclodextrin-conjugated indocyanine compounds of the present invention onto hydrophobic chemical fibers was significantly lower than that of ICG (Figure 5).

<Test 13: Molecular Association of Cyclodextrin-Bound Indocyanine Compounds of the Present Invention in Aqueous Solution>

The molecular association properties of ICG and the cyclodextrin-conjugated indocyanine compounds of the present invention (Chemical Formulas 16, 20, 21, 23-25) in aqueous solutions were investigated. Aqueous solutions of ICG and the cyclodextrin-conjugated indocyanine compounds of the present invention (Chemical Formulas 16, 20, 21, 23-25) were prepared at 0.01 mM, 0.025 mM, 0.05 mM, and 0.1 mM, respectively. The solutions were placed in quartz cuvettes with a 1 mm optical path length, and optical absorption spectra were measured at 25°C from 600 nm to 1000 nm. The results showed that ICG exhibited molecular association known as H-aggregation within this concentration range (Figure 6, left). However, the cyclodextrin-conjugated indocyanine compounds of the present invention, particularly those represented by Chemical Formulas 16 and 20, did not exhibit this molecular association within this concentration range (Figure 6, right).

<Test 14: Fluorescence of the Cyclodextrin-Bound Indocyanine Compound of the Present Invention in Aqueous Solution>

The fluorescence properties of ICG and the cyclodextrin-conjugated indocyanine compounds of the present invention (Chemical Formulas 16, 20, 21, 23-25) in aqueous solution were investigated. 0.1 μM aqueous solutions of ICG and the cyclodextrin-conjugated indocyanine compounds of the present invention (Chemical Formulas 16, 20, 21, 23-25) were placed in 1 cm square quartz cuvettes at 25°C. Excitation was performed with 720 nm excitation light (bandpass: 10 nm), and fluorescence spectra were measured (bandpass: 10 nm). Fluorescence efficiency was calculated based on the fluorescence efficiency of ICG, which is 0.13 (in DMSO, 25°C). The results showed that the fluorescence quantum efficiency of ICG was 0.021, while the fluorescence quantum efficiency of the cyclodextrin-conjugated indocyanine compounds of the present invention, particularly the compounds represented by Chemical Formulas 16 and 20, was 0.054 and 0.042, respectively, which are 2.6 times and 2 times that of ICG.

<Test 15: Fluorescence of the Cyclodextrin-Conjugated Indocyanine Compound of the Present Invention in Blood>

The fluorescence of ICG and the cyclodextrin-conjugated indocyanine compounds of the present invention (Chemical Formulas 16, 20, 21, 23-25) in blood was studied. ICG and the cyclodextrin-conjugated indocyanine compounds of the present invention (Chemical Formulas 16, 20, 21, 23-25) were dissolved in human blood to a concentration of 100 μM. The mixture was placed in a triangular quartz cuvette at 25°C and excited with 760 nm excitation light (bandpass: 10 nm), and the surface fluorescence spectrum (bandpass: 10 nm) was measured. The fluorescence intensity at the maximum fluorescence wavelength was 58 (arbitrary units) for ICG. The fluorescence intensities of the cyclodextrin-conjugated indocyanine compounds of the present invention, especially the compounds represented by Chemical Formulas 16 and 20, were 270 (arbitrary units) and 190 (arbitrary units), which are 4.7 times and 3.3 times that of ICG. This is believed to be because the cyclodextrin-conjugated indocyanine compounds of the present invention do not inhibit luminescence in vivo.

<Test 16: Behavior of the Cyclodextrin-Conjugated Indocyanine Compound of the Present Invention in Vivo>

As representative examples, the behaviors of the isomerization-equilibrium compound (TK1) represented by Chemical Formulas 19 and 20 and the isomerization-equilibrium compound (TK2) represented by Chemical Formulas 15 and 16 were evaluated in human blood and rats as biological subjects.

Fluorescence behavior in human blood

The concentration dependence of surface fluorescence intensity of ICG, TK1, and TK2 in human venous blood in triangular cuvettes was evaluated. Specifically, 1.0 mL of human venous blood was added to a triangular quartz cuvette, along with ICG, TK1, and TK2 at specified concentrations. After irradiation with 760 nm (bandpass: 10 nm) excitation light at 25°C, surface fluorescence (bandpass: 10 nm) was measured. The results are shown in Figure 7.

As can be clearly seen from FIG7 , both TK1 and TK2 can emit fluorescence in human venous blood when irradiated with excitation light.

·Study on the feasibility of drug administration in rats

ICG, TK1, and TK2 were prepared as 1 mM aqueous solutions, and 0.1 mL of each solution was injected into the femoral vein of rats after laparotomy. The abdomen was then illuminated with 760 nm excitation light to assess the presence of fluorescence.

The results showed that fluorescence was observed in the internal organs of the rats after the abdominal opening. High accumulation of TK1 and TK2 in the kidneys was observed (as ICG is known to accumulate in the liver). Furthermore, no significant adverse effects on the rats' condition were observed after administration of these drugs, confirming the possibility of relatively safe in vivo administration.

Observation of distribution in rats

Wistar male rats (9 weeks old, weighing 350 g) were anesthetized with ether and then anesthetized by intraperitoneal administration of 0.1 mL of sodium pentobarbital (Nembutal) using a 26G needle. The base of the rat's tail was anesthetized with a rubber band to remove blood, and the tail vein was secured with a 24G indwelling needle, and the terminal line was secured by connecting a three-headed square stopcock and an extension tube. The rat was fixed on a flat table in a supine position (Figure 8). The PDE camera unit manufactured by Hamamatsu Photonics Co., Ltd. was correctly set at a position 16 cm away from the rat's body. However, it was fixed at 20 cm when photographing the whole body. The observation range is the range indicated by the black circle in Figure 8. Thereafter, ICG, TK1, and TK2 at a concentration of 1 mM were administered through the secured terminal line. The dosage was 0.1 mL. The fluorescence in the abdominal cavity when the abdomen was opened 20 minutes after the administration is shown in Figure 9, and the fluorescence of the dorsum of the foot is shown in Figure 12 (image at the time of reaching the maximum brightness).

Figure 9 shows that ICG accumulates primarily in the liver (Figure 9(a)), TK1 primarily in the kidneys (Figure 9(b)), and TK2 also primarily in the kidneys (Figure 9(c)). Near-infrared imaging of internal organs immediately after administration demonstrated clear imaging of the kidneys, ureters, and bladder (Figure 10). Furthermore, near-infrared endoscopy demonstrated the ability to image the ureters (Figure 11).

As can be seen from image 12, fluorescence is clearly observed corresponding to blood flow in the dorsum of the foot. Furthermore, although not shown in the figure, fluorescence was observed after administering ICG, TK1, and TK2 to a rat model of lower limb ischemia in which the right femoral artery was ligated. Since no fluorescence was observed in the ischemic area, the presence or absence of fluorescence can be used to assess the extent of blood flow.

13 and 14 show the temporal changes in fluorescence intensity of the dorsum of the foot. Table 1 shows the time to maximum fluorescence intensity (Imax), the Imax value, and the time to half the Imax value (t _1/2 ).

[Table 1]

Compound
				ICG	0.5min	124	2.3min
TK-1	10min	160	40min
				TK-2	15min	106	55min

As shown in Table 1, Figures 13, and 14, TK1 and TK2 both took longer to reach maximum fluorescence intensity and to reach half-value in time than ICG. This suggests that they can emit fluorescence for a long time in vivo. For example, while ICG's fluorescence intensity drops to its initial value within 10 minutes after administration, TK1 and TK2 exhibit high fluorescence intensity even after one hour.

Observation at the vascular level

According to the method disclosed in non-patent literature 7 and 8 (Figure 15), ICG and TK1 were administered to rats with their blood vessels directly exposed for observation. The situation of the spermatozoa cremaster flap after exposure is shown in Figure 16. As a result, ICG appeared uneven in the blood vessels relative to TK1. In other words, it can be seen that TK1 moved from the blood vessels to the interstitial fluid. In order to conduct a more precise study, microscopic photographs of the blood vessels after administration are shown in Figures 17 (ICG) and 18 (TK1). As can be seen from Figures 17 and 18, when the fluorescence intensity in the blood vessels and the fluorescence intensity in the surrounding tissues are compared, ICG can hardly confirm fluorescence in the interstitial tissue, while TK1 can confirm fluorescence in the interstitial tissue. In addition, although not shown in detail, it is confirmed that the fluorescence intensity in the interstitial tissue increases over time. Therefore, ICG can hardly confirm movement from the blood vessels to the interstitial fluid, but TK1 can confirm movement from the blood vessels to the interstitial fluid.

Evaluation of the correlation between the extent of migration of the cyclodextrin-bound indocyanine compound of the present invention within the interstitium and the magnitude of edema formation

(Evaluation method)

Ten-week-old male Wister rats weighing approximately 350 g were selected as experimental animals (n=4). The rats were anesthetized with isoflurane and injected with 0.1 mL of 0.5% λ carrageenan into the plantar surface of the left hind paw using a 26G needle. Paw edema rapidly developed. Fifteen minutes later, the rats' left paw volume was measured using a rat paw volume meter (MK-101CMP PLETHYSMOMETER, manufactured by Muromachi Machinery Co., Ltd.).

Afterward, isoflurane was turned off and the rat was confirmed to be awake. After the Von Frey test, general anesthesia was re-administered with isoflurane. The rat's tail vein was secured with a 24G indwelling cannula and then secured in a supine position on a horizontal platform. A PDE camera manufactured by Hamamatsu Photonics Co., Ltd. was set up and fixed at a height of 16 cm from the dorsum of the rat's foot.

The PDE camera was used to set the measurement area (ROI) to the central part of the dorsum of the foot. After the measurement began, 0.1 mL of a 1 mmol TK1 aqueous solution was injected through the tail vein, followed by a 5-second flush with 1 mL of saline. Continuous imaging was performed for the first 5 minutes of injection, and the ROI brightness was measured. Thereafter, brightness measurements were taken at 5-minute intervals for 1 minute, continuing until 120 minutes after injection. After the measurement was completed, isoflurane was turned off and the subject returned to consciousness (acute inflammation experiment).

After 7 days, the rats were subjected to Von Frey test, and then the rats were quickly anesthetized with isoflurane. The volume of the rat's left foot was measured using a rat plantar volume meter. After the rat's tail vein was secured with a 24G indwelling needle, it was fixed on a horizontal platform in a supine position. A PDE camera manufactured by Hamamatsu Photonics Co., Ltd. was set up and fixed at a height of 16 cm from the dorsum of the rat's foot. The ROI was set at the center of the dorsum of the foot, and the measurement was started after the light was turned off. 0.1 mL of 1 mmol TK1 aqueous solution was injected from the tail vein and rinsed with 1 mL of normal saline for 5 seconds. Continuous shooting was performed during the first 5 minutes of injection to measure the brightness of the ROI. The brightness was measured for 1 minute at intervals of 5 minutes thereafter, and this continued until 120 minutes after the injection. After the measurement was completed, isoflurane was turned off and the rat returned to consciousness. The same test was performed on the group that was not injected with λ carrageenan.

ICG also implemented the above process.

(result)

The results are shown in Figures 19 to 26. The left paw volume of the rats measured for the group administered with TK1 is shown in Figure 19 (the volume increases toward the top of the vertical axis), the results of the Von Frey test are shown in Figure 20 (the more downward the vertical axis, the more hyperalgesia), the changes in brightness shortly after the injection are shown in Figure 21 (the brightness increases toward the top of the vertical axis), and the changes in brightness one week after the injection of carrageenan are shown in Figure 23 (the brightness increases toward the top of the vertical axis). The left paw volume of the rats measured for the group administered with ICG is shown in Figure 23 (the volume increases toward the top of the vertical axis), the results of the Von Frey test are shown in Figure 24 (the more downward the vertical axis, the more hyperalgesia), the changes in brightness shortly after the injection are shown in Figure 25 (the brightness increases toward the top of the vertical axis), and the changes in brightness one week after the injection of carrageenan are shown in Figure 26 (the brightness increases toward the top of the vertical axis). The values of the vertical axes of all the graphs are arbitrary units.

Significant differences were observed in the patterns of brightness changes within the ROI between ICG and TK1. The brightness rapidly increased shortly after ICG administration, then rapidly decreased, returning to baseline after approximately 10 minutes. In contrast, the initial brightness increase after TK1 administration followed the same trajectory as ICG, with the brightness remaining high thereafter and requiring six hours to return to baseline. Based on these results, we believe that ICG is retained within the blood vessels and does not leak into the interstitium. Therefore, the rapid brightness increase and subsequent rapid decrease are due to passage through the blood vessels distributed within the ROI. In contrast, TK1 exhibits two phases: the initial rapid passage into the blood vessels (hereafter referred to as the "vascular phase") and the subsequent phase in which TK1 fluorescence gradually leaks into the interstitium (hereafter referred to as the "interstitial phase"). The transition from the vascular phase to the interstitial phase corresponds to the specified time.

In the acute inflammation experiment, a significant increase in paw volume was observed in both the ICG and TK1 groups after injection of λ carrageenan, which returned to normal, similar to the tendon side, one week later (Figures 19 and 23). Furthermore, in the Von Frey test, significant hyperalgesia was observed in both the ICG and TK1 groups after injection of λ carrageenan, but returned to normal, similar to the tendon side, one week later (Figures 20 and 24).

Regarding the brightness of the dorsum of the foot, there was no difference between the TK1 group and the control group in the brightness change of the vascular phase, but the brightness in the interstitial phase increased more rapidly than the λ carrageenan-administered group, and maintained a high value throughout the evaluation time (Figure 21). One week after the administration of λ carrageenan and the inflammation subsided, there was no difference in the brightness change of the interstitial phase between the carrageenan-administered side and the tendon side (Figure 22). As the curve in Figure 21 clearly shows, the brightness in the interstitial phase changes in a smooth curve, and the height of the curve depends on the upward rate of change of the interstitial phase. In other words, the change in the value of the interstitial phase can be predicted with high precision based on time series analysis, which is an important basis for the validity of our hypothesis in this work.

No difference in brightness was observed in the ICG group either immediately or one week after injection with lambda carrageenan (Figures 25 and 26). This result indicates that the highly hydrophobic ICG, which is currently used clinically, does not leak fluorescent material outside of blood vessels even in the presence of severe acute inflammation caused by lambda carrageenan.

These results demonstrate that after TK1 administration to a living body, fluorescence intensity at sites predicted for swelling progression can be measured over time during a period corresponding to the interstitial phase. This allows for prediction of subsequent changes in fluorescence intensity, which correlates with the progression of swelling at the site. Therefore, TK1 is suitable for predicting swelling, which is not possible with ICG.

Industrial Application Possibilities

The cyclodextrin-bound indocyanine compounds represented by Chemical Formula 1 or Chemical Formula 2 of the present invention provide green pigment compounds that emit near-infrared fluorescence and have the following characteristics: higher solubility in water or physiological saline compared to ICG, easier removal from biological tissues, lower molecular association in aqueous solutions, higher near-infrared fluorescence intensity in aqueous solutions, and the absence of iodine. Furthermore, the method for synthesizing the cyclodextrin-bound indocyanine compounds of the present invention can provide for the synthesis of useful cyclodextrin-bound indocyanine compounds. Furthermore, the method for purifying the cyclodextrin-bound indocyanine compounds of the present invention can provide for the purification of useful cyclodextrin-bound indocyanine compounds. Furthermore, because the cyclodextrin-bound indocyanine compounds of the present invention exhibit sufficient solubility even without iodine, they can provide diagnostic compositions that are free of iodine, a cause of iodine allergy. These diagnostic compositions exhibit different in vivo behavior than conventional diagnostic compositions containing only ICG, and their properties can be exploited to provide a variety of useful diagnostic methods and diagnostic devices.

Indocyanine compound, synthesis and purification method thereof, diagnostic composition, in vivo dynamics measurement device, and circulation visualization device

This patent application is a divisional application of the patent application with application number 201180006735.X, application date January 28, 2011, and invention name "New indocyanine compound, synthesis method and purification method thereof, diagnostic composition using the indocyanine compound, in vivo dynamic measurement device using the diagnostic composition, and circulation visualization device".

Technical Field

The present invention relates to a new indocyanine compound that is a green pigment useful in medical diagnosis, medical surgery, scientific measurement and analysis, printing, note-taking, coating, dyeing, and staining technologies and has the property of emitting near-infrared fluorescence, a method for synthesizing and purifying the same, and a diagnostic composition. Specifically, the invention relates to a cyclic sugar chain cyclodextrin-bound indocyanine compound that is a green pigment and has the property of emitting near-infrared fluorescence, a method for synthesizing and purifying the same, a diagnostic composition using the indocyanine compound, an in vivo dynamics measurement device using the diagnostic composition, and a circulation visualization device.

Background Art

A variety of green pigments, indocyanine compounds that emit near-infrared fluorescence, have been synthesized. These compounds have a wide range of applications in the medical field, for example, as dyes for vitreous surgery, medicinal pigments for liver function tests, medicinal pigments for circulatory function tests, surgical pigments, and near-infrared fluorescent compounds for surgery. In the scientific field, they serve as dyes or compounds for dyeing or fluorescing proteins, sugars, and other substances, and as pigments in printing technology. Among these indocyanine compounds, indocyanine green (hereinafter referred to as "ICG") has been used for nearly 50 years as a drug for liver and circulatory function tests. In recent years, ICG's high light transmittance through biological tissues has been exploited to allow for local administration of ICG to, for example, blood vessels, lymphatic vessels, brain, eyes, stomach, breasts, esophagus, skin, and other sites within the body, thereby allowing for medical procedures and diagnostics by observing its near-infrared fluorescence (Non-Patent Document 1).

Prior art literature

Non-patent literature

Non-patent literature 1: "All about ICG Fluorescence Navigation Surgery", supervised and edited by Mitsuo Kusano, インターメディカ社, (published in November 2008).

Non-Patent Document 2: URL of the Internet homepage described in the instructions for "(Trade Name) Diagnogreen Injection 25 mg" (Daiichi Sankyo Co., Ltd.) (https://www.daiichisankyo.co.jp/med/contents/di/dg2/pi/pdf/pi_dg2_0909.pdf)

Non-patent document 3: R.C. Benson, H.A. Kues, Phys. Med. Biol., 23, 159-163 (1978).

Non-patent document 4: S. Yoneyama, T. Saito, Y. Komatsu, I. Koyama, K. Takahashi, J. Duvoll-Young, IOVS, 37, 1286-1290 (1998).

Non-patent document 5: Y. Ye, W. P. Li, C. J. Anderson, J. Kao, G. V. Nikiforovich, S. Achilefu, J. Am. Chem. Soc., 125, 7766-7767 (2003).

Non-patent literature 6: K. Teranishi and S. Tanabe, ITE Letters on Batteries, New Technologies & Medicine, 1, 53-60 (2000).

Non-patent document 7: MICHAEL O’SHAUGHNESSY et al: 1994 Wiley-Liss, Inc MICROSURGERY 15: 40S412 1994.

Non-patent document 8: RALPH J.P.M et al: 1997 Wiley-Liss, Inc. MICROSURGERY 17: 402-408 1996.

Summary of the Invention

Technical issues

However, the green chromophore (the chemical structure required for the green color) and near-infrared fluorophore (the chemical structure required for near-infrared fluorescence) of ICG are hydrophobic, and sulfonyl groups are attached to the side chain terminals to make them water-soluble. Therefore, existing ICG has the following problems.

In general medical use, ICG preparations are prepared by adding approximately 5 to 10 mL of distilled water to 25 mg of ICG and stirring with vibration to dissolve it. Failure to completely dissolve ICG can cause symptoms such as nausea, retching, fever, and shock (Non-Patent Document 2). Furthermore, because ICG does not dissolve completely, it cannot be initially dissolved in other aqueous solutions such as saline (Non-Patent Document 2).

While ICG is water-soluble due to the sulfonyl groups attached to it, its chemical structure contains many hydrophobic hydrocarbon groups, making it surface-active and prone to adsorption to lipids. Consequently, when injected into tissues such as blood vessels and organs, it can sometimes adhere to non-target tissues by adhering to the injection site, leaking through the injection site, or flowing backward. ICG adhered to tissues is difficult to remove through wiping or aspiration, potentially hindering surgical procedures and medical diagnostics.

ICG has a molecular association property in aqueous solution, one of the reasons for which is its low fluorescence intensity in aqueous solution (Non-Patent Documents 3 and 4).

Furthermore, ICG becomes insoluble over time after being dissolved in water, making it difficult to store it as an aqueous solution for a long period of time. Furthermore, freezing at low temperatures promotes its insolubility.

Furthermore, there is a problem that an ICG preparation containing 5% or less of NaI may cause iodine hypersensitivity in some cases (Non-Patent Document 2).

In addition, ICG rapidly accumulates in the liver and is excreted by the liver when injected intravenously, making fluorescence imaging of other organs such as the kidneys, ureters, bladder, urethra, heart, and lungs difficult.

Furthermore, ICG migrates with the blood when injected intravenously, so its migration to peripheral tissues is minimal, making it difficult to observe its migration to the interstitium.

The present invention aims to resolve the aforementioned problems with existing ICG. Specifically, it aims to provide a new indocyanine compound that is a green pigment and emits near-infrared fluorescence. The compound is characterized by high solubility in water or physiological saline, easy removal from biological tissues, low molecular association in aqueous solution, and high near-infrared fluorescence intensity in aqueous solution, enabling fluorescence imaging of organs other than the liver, such as the kidneys, ureters, bladder, urethra, heart, and lungs. Furthermore, a chemical synthesis method and purification method for the new indocyanine compound with these characteristics are provided. Furthermore, a diagnostic composition containing the new indocyanine compound is also provided. Furthermore, a method and apparatus for visualizing the circulation of blood, lymph, urine, and other water components in a living organism is provided using these diagnostic compositions.

Technical solutions to the problem

The present inventors have conducted intensive research to solve the above problems and have discovered compounds that can emit near-infrared fluorescence. These compounds can be used in surgical procedures and medical diagnoses that utilize the properties of near-infrared fluorescence, thereby solving the above-mentioned problems with ICG.

Specifically, the present invention has been completed by the discovery of a new indocyanine compound characterized by being a green pigment that emits near-infrared fluorescence, having high solubility in water or physiological saline, being easily removed from biological tissues, exhibiting low molecular association in aqueous solutions, and exhibiting high near-infrared fluorescence intensity in aqueous solutions. Furthermore, the present invention has been completed by the discovery of a chemical synthesis method and a purification method for the new indocyanine compound. Furthermore, a diagnostic composition containing the new indocyanine compound is provided. Furthermore, a method for performing fluorescence imaging of organs other than the liver using the diagnostic composition, even during intravenous administration, is provided. Furthermore, a method for evaluating the in vivo dynamics of the new indocyanine compound related to, for example, water balance in a living body and a device for measuring the in vivo dynamics are provided, as well as a method and device for visualizing the circulation of blood, lymph, urine, and other water in a living body.

The first new indocyanine compound of the present invention is a compound in which a cyclic sugar chain cyclodextrin is covalently bound to the green chromophore (chemical structure required for green color) and the near-infrared fluorophore (chemical structure required for emitting near-infrared fluorescence) of ICG. In addition, the second new indocyanine compound of the present invention is characterized in that the naphthyl site belonging to the hydrophobic site of the indocyanine structure is included in the cavity of the cyclodextrin, thereby covering the naphthyl site belonging to the hydrophobic site with a hydrophilic glucose group, so that most areas of the indocyanine molecular structure become three-dimensionally hydrophilic. Therefore, compared with the characteristic of ICG having similar surface activity due to the hydrophilicity of both the hydrophobic site and the sulfonyl group, the compound of the present invention is characterized in that the hydrophobic site in its molecule is covered by cyclodextrin, so it does not have similar surface activity properties. Specifically, the present invention is as follows:

<1> A cyclodextrin-bound indocyanine compound represented by Chemical Formula 1, characterized in that the indocyanine is covalently bound to a cyclic sugar chain cyclodextrin;

[Chemical Formula 1]

(R ₁ , R ₂ , R ₃ , R ₄ , R ₅ , R ₆ , R ₇ , R ₈ , R ₉ , R ₁₀ , R ₁₁ , R ₁₂ , R ₁₃ , R 14 , R ₁₅ , R ₁₆ , R ₁₇ , R ₁₈ , R ₁₉ , R ₂₀ , R _in the formula ₂₁ , R ₂₂ , R _R23 is a hydrogen atom, an alkyl group, an aryl group, a halogen atom, an alkoxy group, an amino group, a carboxyl group, a formyl group, a sulfonyl group, a sulfonic acid group, a phosphate group, an alkoxycarbonyl group, an aryloxycarbonyl group, an alkylcarbonyl group, an arylcarbonyl group, or a heterocycle. Alternatively, it may be a substituent thereof (carboxylic acid, sulfonic acid, phosphoric acid). When the hydrogen ion in the substituent is dissociated, the hydrogen ion may be replaced by a metal ion such as sodium ion, potassium ion, magnesium ion, or calcium ion. The amino group may be selected from primary, secondary, tertiary, and quaternary groups ₍ alkyl groups may be exemplified as substituents bonded to nitrogen). Alternatively, _R8 and _R9 may _be selected _{from CH2} , _{CH2CH2 , CH2CH2CH2} , or _{CH2CH2CH2CH2 . 2.} Alternatively, the hydrogen atoms on these alkyl groups may be replaced by alkyl groups, aryl groups, halogen atoms, alkoxy groups, amino groups, carboxyl groups, formyl groups, sulfonyl groups, sulfonic acid groups, phosphoric acid groups, alkoxycarbonyl groups, aryloxycarbonyl groups, alkylcarbonyl groups, arylcarbonyl groups or heterocyclic groups.)

<2> The cyclodextrin-bound indocyanine compound represented by Chemical Formula 2 is characterized in that at least a portion of the naphthyl group of the indocyanine is included in the cavity of the cyclodextrin.

[Chemical Formula 2]

(R ₁ , R ₂ , R ₃ , R ₄ , R ₅ , R ₆ , R ₇ , R ₈ , R ₉ , R ₁₀ , R ₁₁ , R ₁₂ , R ₁₃ , R 14 , R ₁₅ , R ₁₆ , R ₁₇ , R ₁₈ , R ₁₉ , R ₂₀ , R _in the formula ₂₁ , R ₂₂ , R ₂₃ is a hydrogen atom, an alkyl group, an aryl group, a halogen atom, an alkoxy group, an amino group, a carboxyl group, a formyl group, a sulfonyl group, a sulfonic acid group, a phosphate group, an alkoxycarbonyl group, an aryloxycarbonyl group, an alkylcarbonyl group, an arylcarbonyl group, or a heterocycle. Alternatively, it may be a substituent thereof (carboxylic acid, sulfonic acid, phosphate). When the hydrogen ion in the substituent is dissociated, the hydrogen ion may be replaced by a metal ion such as a sodium ion, potassium ion, magnesium ion, or calcium ion. The amino group is selected from primary, secondary, tertiary, and quaternary groups. Furthermore, R ₈ and R ₉ may be selected from a cyclic structure of CH ₂ , CH ₂ CH ₂ , CH ₂ CH ₂ CH ₂ , or CH ₂ CH ₂ CH ₂ CH ₂ . Alternatively, a functional group in which the hydrogen atom on these alkyl groups is replaced by an alkyl group, an aryl group, a halogen atom, an alkoxy group, an amino group, a carboxyl group, a formyl group, a sulfonyl group, a sulfonic acid group, a phosphate group, an alkoxycarbonyl group, an aryloxycarbonyl group, an alkylcarbonyl group, an arylcarbonyl group, or a heterocycle may be selected.)

<3> The cyclodextrin-bound indocyanine compound represented by Chemical Formula 3 is characterized in that it is the cyclodextrin-bound indocyanine compound represented by Chemical Formula 1 in which an indocyanine and a cyclic sugar chain cyclodextrin are covalently bound via an amide bond.

[Chemical Formula 3]

(R ₁ , R ₂ , R ₃ , R ₄ , R ₅ , R ₆ , R ₇ , R ₈ , R ₉ , R ₁₀ , R ₁₁ , R ₁₂ , R ₁₃ , R 14 , R ₁₅ , R ₁₆ , R ₁₇ , R ₁₈ , R ₁₉ , R ₂₀ , R _in the formula ₂₁ , R ₂₂ , R _R23 is a hydrogen atom, an alkyl group, an aryl group, a halogen atom, an alkoxy group, an amino group, a carboxyl group, a formyl group, a sulfonyl group, a sulfonic acid group, a phosphate group, an alkoxycarbonyl group, an aryloxycarbonyl group, an alkylcarbonyl group, an arylcarbonyl group, or a heterocycle. Alternatively, it may be a substituent thereof (carboxylic acid, sulfonic acid, phosphoric acid), wherein the hydrogen ion in the substituent, when dissociated, may be replaced by a metal ion such as sodium ion, potassium ion, magnesium ion, or calcium ion. The amino group is selected from primary, secondary, tertiary, and _quaternary groups. m and n are integers from 1 to 6. Furthermore, _R8 and _R9 may _{be selected from CH2} , _CH2CH2 , _CH2CH2CH2 , or _{CH2CH2CH2CH2 . 2.} In addition, the functional groups in which the hydrogen atoms on these alkyl groups are replaced by alkyl groups, aryl groups, halogen atoms, alkoxy groups, amino groups, carboxyl groups, formyl groups, sulfonyl groups, sulfonic acid groups, phosphoric acid groups, alkoxycarbonyl groups, aryloxycarbonyl groups, alkylcarbonyl groups, arylcarbonyl groups or heterocyclic rings can also be selected.)

<4> Chemical formula 4 represents a cyclodextrin-bound indocyanine compound, characterized in that it is a cyclodextrin-bound indocyanine compound represented by Chemical formula 2, in which indocyanines and cyclic sugar chain cyclodextrin are covalently bound via an amide bond.

[Chemical Formula 4]

(R ₁ , R ₂ , R ₃ , R ₄ , R ₅ , R ₆ , R ₇ , R ₈ , R ₉ , R ₁₀ , R ₁₁ , R ₁₂ , R ₁₃ , R 14 , R ₁₅ , R ₁₆ , R ₁₇ , R ₁₈ , R ₁₉ , R ₂₀ , R _in the formula ₂₁ , R ₂₂ , R _R23 is a hydrogen atom, an alkyl group, an aryl group, a halogen atom, an alkoxy group, an amino group, a carboxyl group, a formyl group, a sulfonyl group, a sulfonic acid group, a phosphate group, an alkoxycarbonyl group, an aryloxycarbonyl group, an alkylcarbonyl group, an arylcarbonyl group, or a heterocycle. Alternatively, it may be a substituent thereof (carboxylic acid, sulfonic acid, phosphoric acid), wherein the substituent can be replaced by a metal ion such as sodium ion, potassium ion, magnesium ion, or calcium ion when the hydrogen ion dissociates. The amino group is selected from primary, secondary, tertiary, and quaternary groups. _m and n are integers from 1 to 6. Furthermore, _R8 and _R9 may _be selected _{from CH2} , _CH2CH2 , _{CH2CH2CH2 ,} or _{CH2CH2CH2CH2 . 2.} In addition, the functional groups in which the hydrogen atoms on these alkyl groups are replaced by alkyl groups, aryl groups, halogen atoms, alkoxy groups, amino groups, carboxyl groups, formyl groups, sulfonyl groups, sulfonic acid groups, phosphoric acid groups, alkoxycarbonyl groups, aryloxycarbonyl groups, alkylcarbonyl groups, arylcarbonyl groups or heterocyclic rings can also be selected.)

<5> The cyclodextrin-bound indocyanine compound represented by Chemical Formula 5 among the cyclodextrin-bound indocyanine compounds represented by Chemical Formula 3.

[Chemical Formula 5]

(In the formula, m, n, p, and q are integers of 2 to 6. r is an integer of 5 to 7. s is an integer of 0 to 4. R is a hydrogen atom, an alkyl group, an aryl group, a halogen atom, an alkoxy group, an amino group, a carboxyl group, a formyl group, a sulfonyl group, a sulfonic acid group, an alkoxycarbonyl group, an aryloxycarbonyl group, an alkylcarbonyl group, an arylcarbonyl group, or a heterocycle.)

<6> The cyclodextrin-bound indocyanine compound represented by Chemical Formula 6 among the cyclodextrin-bound indocyanine compounds represented by Chemical Formula 4.

[Chemical Formula 6]

(In the formula, m, n, p, and q are integers of 2 to 6. r is an integer of 5 to 7. s is an integer of 0 to 4. R is a hydrogen atom, an alkyl group, an aryl group, a halogen atom, an alkoxy group, an amino group, a carboxyl group, a formyl group, a sulfonyl group, a sulfonic acid group, an alkoxycarbonyl group, an aryloxycarbonyl group, an alkylcarbonyl group, an arylcarbonyl group, or a heterocycle.)

<7> The cyclodextrin-bound indocyanine compound represented by Chemical Formula 7 among the cyclodextrin-bound indocyanine compounds represented by Chemical Formula 3.

[Chemical Formula 7]

(In the formula, m and n are integers of 2 to 6. r is an integer of 5 to 7. s is an integer of 0 to 4. R is a hydrogen atom, an alkyl group, an aryl group, a halogen atom, an alkoxy group, an amino group, a carboxyl group, a formyl group, a sulfonyl group, a sulfonic acid group, an alkoxycarbonyl group, an aryloxycarbonyl group, an alkylcarbonyl group, an arylcarbonyl group, or a heterocycle.)

<8> The cyclodextrin-bound indocyanine compound represented by Chemical Formula 8 among the cyclodextrin-bound indocyanine compounds represented by Chemical Formula 4.

[Chemical Formula 8]

(In the formula, m and n are integers of 2 to 6. r is an integer of 5 to 7. s is an integer of 0 to 4. R is a hydrogen atom, an alkyl group, an aryl group, a halogen atom, an alkoxy group, an amino group, a carboxyl group, a formyl group, a sulfonyl group, a sulfonic acid group, an alkoxycarbonyl group, an aryloxycarbonyl group, an alkylcarbonyl group, an arylcarbonyl group, or a heterocycle.)

<9> The cyclodextrin-bound indocyanine compound represented by Chemical Formula 9 among the cyclodextrin-bound indocyanine compounds represented by Chemical Formula 3.

[Chemical Formula 9]

(In the formula, m and n are integers of 2 to 6. r is an integer of 5 to 7. s is an integer of 0 to 4. R is a hydrogen atom, an alkyl group, an aryl group, a halogen atom, an alkoxy group, an amino group, a carboxyl group, a formyl group, a sulfonyl group, a sulfonic acid group, an alkoxycarbonyl group, an aryloxycarbonyl group, an alkylcarbonyl group, an arylcarbonyl group, or a heterocycle.)

<10> The cyclodextrin-bound indocyanine compound represented by Chemical Formula 10 among the cyclodextrin-bound indocyanine compounds represented by Chemical Formula 4.

[Chemical Formula 10]

(In the formula, m and n are integers of 2 to 6. r is an integer of 5 to 7. s is an integer of 0 to 4. R is a hydrogen atom, an alkyl group, an aryl group, a halogen atom, an alkoxy group, an amino group, a carboxyl group, a formyl group, a sulfonyl group, a sulfonic acid group, an alkoxycarbonyl group, an aryloxycarbonyl group, an alkylcarbonyl group, an arylcarbonyl group, or a heterocycle.)

<11> The cyclodextrin-bound indocyanine compound represented by Chemical Formula 11 among the cyclodextrin-bound indocyanine compounds represented by Chemical Formula 3.

[Chemical Formula 11]

(s is an integer from 0 to 4. R is a hydrogen atom, an alkyl group, an aryl group, a halogen atom, an alkoxy group, an amino group, a carboxyl group, a formyl group, a sulfonyl group, a sulfonic acid group, an alkoxycarbonyl group, an aryloxycarbonyl group, an alkylcarbonyl group, an arylcarbonyl group, or a heterocyclic ring.)

<12> The cyclodextrin-bound indocyanine compound represented by Chemical Formula 12 among the cyclodextrin-bound indocyanine compounds represented by Chemical Formula 4.

[Chemical Formula 12]

(s is an integer from 0 to 4. R is a hydrogen atom, an alkyl group, an aryl group, a halogen atom, an alkoxy group, an amino group, a carboxyl group, a formyl group, a sulfonyl group, a sulfonic acid group, an alkoxycarbonyl group, an aryloxycarbonyl group, an alkylcarbonyl group, an arylcarbonyl group, or a heterocyclic ring.)

<13> The chemical synthesis method of the cyclodextrin-conjugated indocyanine compound described in any one of the above chemical formulas (Chemical Formulas 1, 3, 5, 7, 9, and 11) is characterized by comprising: (1) a step of mixing an indocyanine carboxylic acid compound and an aminocyclodextrin in a solvent; and (2) a step of adding a dehydration condensation agent to carry out a dehydration condensation reaction.

<14> A chemical synthesis method of a cyclodextrin-bound indocyanine compound according to any one of the above chemical formulas (Chemical Formulas 2, 4, 6, 8, 10, 12), characterized in that the cyclodextrin-bound indocyanine compound according to any one of the above chemical formulas (Chemical Formulas 1, 3, 5, 7, 9, 11) is subjected to an inclusion reaction in water.

<15> The cyclodextrin-bound indocyanine compound represented by any one of the above chemical formulae (Chemical Formulae 1 to 12) is purified by column chromatography in which the compound is eluted with a solvent containing HCl.

<16> A diagnostic composition to be used by injection into the body, comprising an aqueous solution containing the cyclodextrin-bound indocyanine compound of any one of the above Chemical Formulas 1 to 12.

<17> The diagnostic composition according to <16>, which contains substantially no iodine.

<18> A device for measuring the dynamics of a cyclodextrin-bound indocyanine compound in vivo, comprising:

an excitation light irradiation unit for irradiating the cyclodextrin-bound indocyanine compound with excitation light, with respect to a part of a living body to which the diagnostic composition according to <16> or <17> is administered;

a fluorescence intensity measuring unit for measuring the intensity of fluorescence emitted by the cyclodextrin-bound indocyanine compound excited by the excitation light irradiation unit; and

The in vivo dynamic calculation unit calculates the temporal change of the time rate of change of the fluorescence intensity from the fluorescence intensity obtained over time from the fluorescence intensity measuring unit, thereby calculating the speed of the cyclodextrin-bound indocyanine compound moving into the interstitial fluid and/or the speed of moving out of the interstitium in a part of the biological body.

<19> A circulation visualization device, comprising:

an excitation light irradiation unit for irradiating the cyclodextrin-bound indocyanine compound with excitation light, with respect to a part of a living body to which the diagnostic composition according to <16> or <17> is administered;

a fluorescence imaging unit for two-dimensionally acquiring the intensity of the fluorescence emitted by the cyclodextrin-bound indocyanine compound excited by the excitation light irradiation unit, thereby obtaining data on the distribution state of the cyclodextrin-bound indocyanine compound in the organism;

a morphological imaging unit that two-dimensionally acquires the intensity of light of wavelengths other than the fluorescence wavelength emitted by the cyclodextrin-bound indocyanine compound, thereby obtaining morphological data of a portion of the biological body; and

The display unit displays the distribution state of the cyclodextrin-bound indocyanine compound in a part of the biological body by superimposing the morphological data obtained by the morphological imaging unit and the distribution state data obtained by the fluorescence imaging unit.

<20> The circulation visualization device according to <19>, wherein the display unit displays a portion of the living body where the distribution amount of the cyclodextrin-bound indocyanine compound is lower than a predetermined reference as a necrotic portion.

<21> The in vivo dynamics measuring device according to <18> above, comprising a swelling progression prediction unit for predicting the degree of swelling of the part of the living body based on the speed of the swelling moving from the part of the living body into and out of the interstitial fluid.

<22> The in vivo dynamic measurement device described in <21> above, wherein the swelling progression prediction unit predicts the degree of swelling progression corresponding to the magnitude of the migration speed into the interstitial fluid after a predetermined time, wherein the predetermined time is the time from the administration of the diagnostic composition to the dispersion of the cyclodextrin-conjugated indocyanine compound in the blood throughout the body.

<23> An in vivo dynamics measurement device, characterized by comprising:

The excitation light irradiation unit irradiates the cyclodextrin-bound indocyanine compound with excitation light at a portion of the living body to which the diagnostic composition according to <16> or <17> is administered and at a control site.

a fluorescence intensity measuring unit for measuring the intensity of fluorescence emitted by the cyclodextrin-bound indocyanine compound excited by the excitation light irradiation unit;

an in vivo dynamic calculation unit for calculating the time-dependent change in the fluorescence intensity obtained from the fluorescence intensity measurement unit over time, thereby calculating the migration speed of the cyclodextrin-bound indocyanine compound into the interstitial fluid and/or the migration speed out of the interstitial fluid in a part of the organism, and

a swelling progression prediction unit for predicting a degree of swelling that will progress in the part of the living body based on a rate of movement of the part of the living body into and out of the interstitial fluid;

wherein the above swelling is carried out in a prediction unit,

determining the relationship between the blood flow in the control site and the blood flow in the portion of the living body based on changes in fluorescence intensity in the portion of the living body and changes in fluorescence intensity in the control site until a predetermined time has passed, wherein the predetermined time is the time from administration of the diagnostic composition to dispersion of the cyclodextrin-bound indocyanine compound into blood throughout the body;

The degree of change in the fluorescence intensity of the part of the biological body after the predetermined time has passed, with the change in the fluorescence intensity of the control part serving as a control, is calculated using this relationship.

The progression of the swelling is predicted based on the calculated degree of change.

<24> An in vivo dynamics measurement device, characterized by comprising:

an excitation light irradiation unit for irradiating the cyclodextrin-bound indocyanine compound with excitation light to a living organ to which the diagnostic composition according to <16> or <17> is administered;

a fluorescence intensity measuring unit for measuring the intensity of fluorescence emitted by the cyclodextrin-bound indocyanine compound excited by the excitation light irradiation unit; and

The in vivo dynamics calculation unit evaluates the in vivo dynamics of the cyclodextrin-bound indocyanine compound in the organ based on the fluorescence intensity acquired over time by the fluorescence intensity measurement unit.

<25> The in vivo dynamics measuring device according to <24>, wherein the organ is any one of a kidney, a ureter, a bladder, and a urethra.

Beneficial effects

The cyclodextrin-bound indocyanine compounds represented by Chemical Formula 1 or Chemical Formula 2 of the present invention provide green pigment compounds that emit near-infrared fluorescence and have the following characteristics: higher solubility in water or physiological saline compared to ICG, easier removal from biological tissues, lower molecular association in aqueous solutions, higher near-infrared fluorescence intensity in aqueous solutions, and the ability to perform fluorescence imaging of organs other than the liver. Furthermore, the cyclodextrin-bound indocyanine compound synthesis method of the present invention provides for the synthesis of useful cyclodextrin-bound indocyanine compounds. Furthermore, the cyclodextrin-bound indocyanine compound purification method of the present invention provides for the purification of useful cyclodextrin-bound indocyanine compounds. Furthermore, the cyclodextrin-bound indocyanine compounds of the present invention exhibit sufficient solubility even without iodine, thus providing diagnostic compositions that do not contain iodine, a cause of iodine allergy. These diagnostic compositions exhibit different in vivo behavior than conventional diagnostic compositions containing only ICG, and their properties can be exploited to provide various beneficial devices.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the results of a test on the adsorption of ICG and the isomeric equilibrium compound (TK1) represented by Chemical Formulas 19 and 20 to human skin. The images show the results of a 1 mM aqueous solution (0.03 mL) of ICG and the isomeric equilibrium compound (TK1) represented by Chemical Formulas 19 and 20 applied to the wrist and ingested immediately (left photo), 5 minutes later and immediately after rinsing with water (center photo), and then immediately after wiping and rinsing with water (right photo). The dots on the right are marked with a red pen.

Figure 2 shows the results of a test on the adsorption of ICG and the isomeric equilibrium compound (TK1) represented by Chemical Formulas 19 and 20 onto cellulose fibers. The images show the results of a cotton swab application of a 1 mM aqueous solution (0.05 mL) of ICG and the isomeric equilibrium compound (TK1) represented by Chemical Formulas 19 and 20, followed by immediate uptake (top photo), and then 3 minutes later, followed by 5 seconds of rinsing with water, followed by immediate uptake (bottom photo).

Figure 3 shows the results of a test on the adsorption of ICG and the isomeric equilibrium compound (TK1) represented by Chemical Formulas 19 and 20 into a biological meat model. The images show a 1 mM aqueous solution (0.05 mL) of ICG and the compound represented by Chemical Formula 20 applied to a 5 mm diameter depression in pork tenderloin immediately after ingestion (left photo), and then rinsed with water for 10 seconds 3 minutes later, followed by immediate ingestion (right photo).

Figure 4 shows the results of a test on the adsorption of ICG and the isomeric equilibrium compound (TK1) represented by Chemical Formulas 19 and 20 onto a biological protein model. The images show a 1 mM aqueous solution (0.05 mL) of ICG and the isomeric equilibrium compound (TK1) represented by Chemical Formulas 19 and 20 applied to a 5 mm diameter depression in chicken breast immediately after ingestion (left photo), and then rinsed with water for 10 seconds 3 minutes later, followed by immediate ingestion (right photo).

Figure 5 shows the test results of the adsorption of ICG and the isomeric equilibrium compound (TK1) represented by Chemical Formulas 19 and 20 onto hydrophobic chemical fibers. The images show a 1 mM aqueous solution (0.05 mL) of ICG and the isomeric equilibrium compound (TK-1) represented by Chemical Formulas 19 and 20 applied to a polypropylene mask, immediately after application (left photo), and then after 20 minutes of rinsing with water for 1 second and immediately after application (right photo).

Figure 6 shows the results of molecular association tests of ICG and the isomerization equilibrium compound (TK1) represented by Chemical Formulas 19 and 20. The left figure shows ICG, and the right figure shows the isomerization equilibrium compound (TK1) represented by Chemical Formulas 19 and 20.

7 is a graph showing the concentration dependency of the fluorescence intensity of ICG, the isomerization equilibrium compound (TK1) represented by Chemical Formulas 19 and 20, and the isomerization equilibrium compound (TK2) represented by Chemical Formulas 15 and 16 in human venous blood.

FIG8 is a graph showing the conditions observed when ICG, the isomerization-equilibrium compound represented by Chemical Formulas 19 and 20 (TK1), and the isomerization-equilibrium compound represented by Chemical Formulas 15 and 16 (TK2) were administered to rats.

FIG9 is a graph showing fluorescence in the peritoneal cavity of rats when ICG, the isomerization-equilibrium compound represented by Chemical Formulas 19 and 20 (TK1), and the isomerization-equilibrium compound represented by Chemical Formulas 15 and 16 (TK2) were administered.

Figure 10 shows images of male Wistar rats (14 weeks old, 300 g) administered 0.075 mL of a 0.1 mM aqueous solution of the isomerized equilibrium compounds (TK2) represented by Chemical Formulas 15 and 16 via the tail vein, followed by imaging using a near-infrared observation system PDE manufactured by Hamamatsu Photonics Co., Ltd. The left image is a black-and-white image using visible light, and the right image is a fluorescent image.

Figure 11 shows near-infrared fluorescence endoscopic images of male Wistar rats (14 weeks old, 300 g) after 0.075 mL of a 0.1 mM aqueous solution of the isomerized equilibrium compounds (TK2) represented by Chemical Formulas 15 and 16 was administered via the tail vein. The left image is a black-and-white image using visible light, and the right image is a fluorescence image.

FIG12 is a graph showing fluorescence at the dorsum of the foot of rats when ICG, the isomerized equilibrium compound (TK1) represented by Chemical Formulas 19 and 20, and the isomerized equilibrium compound (TK2) represented by Chemical Formulas 15 and 16 were administered.

13 is a graph showing the temporal changes in fluorescence intensity at the dorsum of the foot when ICG, the isomerization-equilibrium compound represented by Chemical Formulas 19 and 20 (TK1), and the isomerization-equilibrium compound represented by Chemical Formulas 15 and 16 (TK2) were administered to rats.

14 is a graph showing the temporal changes in fluorescence intensity at the dorsum of the foot when ICG, the isomerization-equilibrium compound represented by Chemical Formulas 19 and 20 (TK1), and the isomerization-equilibrium compound represented by Chemical Formulas 15 and 16 (TK2) were administered to rats.

FIG. 15 is a diagram showing a technique for direct observation of rat blood vessels.

FIG. 16 is a diagram showing a directly observed portion of a rat's blood vessels (spermatozoa cremaster flap).

FIG. 17 is a graph showing the state of fluorescence in blood vessels and surrounding tissues of rats to which ICG was administered.

FIG. 18 is a graph showing fluorescence in blood vessels and surrounding tissues of rats after administration of the isomerization-equilibrated compound (TK1) represented by Chemical Formulas 19 and 20. FIG.

Figure 19 is a graph showing the left paw volume of rats measured immediately after injection (post-injection) and one week after injection (1 week after) for the carrageenan-administered group (edema) and the non-administered group (normal) in the TK1 test. The vertical axis represents volume (mL).

Figure 20 is a graph showing the results of the Von Frey test on the left paw of rats in the TK1 test group, the group that received carrageenan (edema) and the group that did not receive carrageenan (normal), measured immediately after injection and one week after injection. The vertical axis represents the load (g) at which the reaction occurred.

Figure 21 is a graph showing changes in brightness of the left paw surface after TK1 injection immediately after carrageenan injection for a group administered carrageenan (edema) and a group not administered carrageenan (normal). The vertical axis represents brightness (arbitrary unit) and the horizontal axis represents time (seconds).

Figure 22 is a graph showing changes in brightness of the left paw surface one week after carrageenan injection and TK1 injection for a group that received carrageenan (edema) and a group that did not receive carrageenan (normal). The vertical axis represents brightness (arbitrary unit) and the horizontal axis represents time (seconds).

Figure 23 is a graph showing the left paw volume of rats measured immediately after injection (post-injection) and one week after (1 week) for the ICG test group, the group administered carrageenan (edema), and the group not administered (normal). The vertical axis represents volume (mL).

Figure 24 is a graph showing the results of the Von Frey test on the left paw of rats immediately after injection and one week after injection, for groups tested with ICG, those administered carrageenan (edema), and those not administered (normal). The vertical axis represents the load (g) at which the reaction occurred.

Figure 25 is a graph showing the brightness of the left paw surface immediately after ICG injection for a group that received carrageenan (edema) and a group that did not receive carrageenan (normal). The vertical axis represents brightness (arbitrary unit) and the horizontal axis represents time (seconds).

Figure 26 is a graph showing changes in brightness of the left foot surface after ICG injection one week after carrageenan injection in a group that received carrageenan (edema) and a group that did not receive carrageenan (normal). The vertical axis represents brightness (arbitrary units) and the horizontal axis represents time (seconds).

DETAILED DESCRIPTION

The diagnostic composition of the present invention utilizes the cyclodextrin-conjugated indocyanine compound described below as a pigment, making it iodine-free and therefore suitable for diagnostic applications. This diagnostic composition can replace the conventionally used indocyanine green-containing diagnostic compositions. For example, it can be used as a drug for liver function tests or circulatory function tests. Furthermore, by observing the near-infrared fluorescence generated by administration to, for example, blood vessels, lymphatic vessels, brain, eyes, stomach, breast, esophagus, skin, kidneys, ureters, bladder, urethra, lungs, heart, or other internal organs, it can be applied to medical procedures and diagnostics. The pigment contained in the diagnostic composition of the present invention has low binding to the organism, allowing for long-term labeling of desired areas. The diagnostic composition may also contain salts or other additives as isotonic agents, as needed. In addition to pre-prepared aqueous solutions, it can also be used in a form that is dissolved. The diagnostic composition can be administered by injection, intravenous drip, application, or oral administration.

This diagnostic composition is suitable for use in visualizing the circulation of aqueous solutions such as blood, lymph, interstitial water, and urine. By visualizing the circulation of blood and other fluids, for example, it can be used to assess necrosis in the peripheral circulation, evaluate tissue survival after revascularization surgery and transplantation, and diagnose poor blood flow. Furthermore, it can also be used for fundus angiography, which has been previously visualized, cerebral circulation assessment, intraoperative angiography during neurosurgery, sentinel lymph node identification in cancers (such as breast cancer, esophageal cancer, gastric cancer, colorectal cancer, prostate cancer, and skin cancer), evaluation of lymphedema, intraoperative cholangiography, tumor labeling, coronary angiography, and abdominal angiography (hepatic artery, abdominal aorta, digestive tract blood flow, etc.). Fluorescent imaging of the renal excretory system, including the kidneys, ureters, bladder, and urethra, is also possible.

<1. Non-inclusion cyclodextrin-bound indocyanine compounds and their synthesis>

Examples of the non-inclusion type cyclodextrin-bound indocyanine compounds of the present invention include Chemical Formula 1, Chemical Formula 3, Chemical Formula 5, Chemical Formula 7, Chemical Formula 9, and Chemical Formula 11. Their synthesis is accomplished by reacting an indocyanine compound with a cyclodextrin compound in a solution.

In Chemical Formulas 1 and 3, considering the inclusion of a significant portion of the cyclodextrin, _R1 - _R4 and _R13 - _R16 are preferably relatively bulky groups that do not hinder cyclodextrin inclusion. For example, hydrogen or an alkyl or alkoxy group with approximately 1 to 3 carbon atoms. Preferred are hydrogen, methyl, and methoxy groups, with hydrogen being most preferred. For cyclodextrin inclusion, _R5 , _R6 , _R11 , and _R12 are also preferably relatively bulky, but not as bulky as _R1 - _R4 and _R13 - _R16 . For example, hydrogen or an alkyl or alkoxy group with approximately 1 to 6 carbon atoms. _R1 - _R6 and _R11 - _R16 are sites of cyclodextrin inclusion, so even when a hydrophilic functional group is introduced, the entire group is preferably hydrophobic. _R17 , _R18 , _R22 , and _R23 do not significantly affect cyclodextrin inclusion and are not particularly limited as long as they are the substituents listed above. R ₇ , R ₁₀ , R ₁₉ and R ₂₁ are preferably hydrogen from the viewpoint of ease of synthesis. R ₈ , R ₉ and R ₂₀ are not particularly limited as long as they are the substituents described above.

In the case where the non-inclusion type cyclodextrin-bound indocyanine compound of the present invention is covalently bound to the indocyanine compound and the cyclodextrin compound via an amide bond, the synthesis method of the non-inclusion type cyclodextrin-bound indocyanine compound can be achieved by allowing an indocyanine carboxylic acid compound and an aminocyclodextrin compound to undergo a dehydration condensation reaction in a solution.

<2. Inclusion-type cyclodextrin-bound indocyanine compounds>

The cyclodextrin-bound indocyanine compound of the present invention is characterized by being a cyclodextrin-bound indocyanine compound covalently bound to a cyclic sugar chain cyclodextrin represented by Chemical Formula 2, wherein at least a portion of the naphthyl group of the indocyanine is enclosed within the cyclodextrin cavity. Furthermore, as long as the naphthyl group of the indocyanine is enclosed within the cyclodextrin cavity and emits near-infrared fluorescence, the indocyanine group may have a substituent. While there are many types of cyclodextrins, enclosed naphthyl group of the indocyanine is a necessary condition. Examples include α-cyclodextrin, β-cyclodextrin, and γ-cyclodextrin. β-cyclodextrin is preferred. The cyclodextrin may also have a substituent.

In Chemical Formulas 2 and 4, _R1 - _R4 and _R13 - _R16 are preferably small groups that do not hinder cyclodextrin inclusion. Examples include hydrogen, alkyl or alkoxy groups with approximately 1 to 3 carbon atoms. Hydrogen, methyl, and methoxy groups are particularly preferred, with hydrogen being most preferred. For cyclodextrin inclusion, _R5 , _R6 , _R11 , and _R12 , while not as large as _R1 - _R4 and _R13 - _R16 , are also small. Examples include hydrogen, alkyl or alkoxy groups with approximately 1 to 6 carbon atoms. _R1 - _R6 and _R11 - _R16 are sites of cyclodextrin inclusion, so even when introducing hydrophilic functional groups, the entire group is preferably hydrophobic. _R17 , _R18 , _R22 , and _R23 do not significantly affect cyclodextrin inclusion and are not particularly limited as long as they are the substituents listed above. R ₇ , R ₁₀ , R ₁₉ , and R ₂₁ are preferably hydrogen from the perspective of ease of synthesis. R ₈ , R ₉ , and R ₂₀ are not particularly limited as long as they are the substituents described above. The bond between the indocyanine group and the cyclodextrin is not particularly limited as long as it is covalent, and examples thereof include an alkyl bond, an amino bond, an amide bond, a double bond, a triple bond, an ester bond, and an ether bond. However, in chemical synthesis, an amide bond is preferred from the perspective of efficiency.

In order to include at least a portion of the naphthyl groups of the indocyanine in the cyclodextrin cavity, a spacer is preferably used to covalently bond the indocyanine and the cyclic sugar chain cyclodextrin via the spacer. In this case, the extent of inclusion of the naphthyl groups of the indocyanine in the cyclodextrin cavity can be controlled by adjusting the length of the spacer in Chemical Formula 2.

Therefore, the cyclodextrin-bound indocyanine compound formed by covalent bonding of indocyanine and cyclic sugar chain cyclodextrin via a spacer, and the compound characterized by the inclusion of the naphthyl group of indocyanine in the cavity of cyclodextrin, preferably exemplified by Chemical Formula 4. In addition, the substances of Chemical Formula 6, Chemical Formula 8, and Chemical Formula 10 are preferably exemplified. In addition, the substance of Chemical Formula 12 is preferably exemplified. In Chemical Formula 6, m, n, p, and q are preferably m+p and n+q are 5 or more and 7 or less, respectively. From the perspective of easy cyclodextrin inclusion, in all chemical formulas (Chemical Formulas 1 to 10), the number of atoms in the structure (spacer) between the nitrogen atom in the indocyanine structure and the oxygen atom in the cyclodextrin structure is preferably 7 or more and 9 or less.

<3. Synthesis of Inclusion Cyclodextrin-Conjugated Indocyanine Compounds>

The inclusion-type cyclodextrin-bound indocyanine compound of the present invention is as described above, and its synthesis method is to use the non-inclusion-type cyclodextrin-bound indocyanine compound synthesized above as a synthesis precursor and dissolve the compound in an aqueous solution. In the aqueous solution, any substance can be contained as long as it does not hinder inclusion, and the water content is not particularly limited. In addition, the temperature suitable for inclusion is -20°C to 100°C, preferably 0°C to 50°C. In addition, the time required for inclusion is from immediately after addition to the aqueous solution to about 1 month. The inclusion reaction can be in various forms depending on the properties of the non-inclusion-type cyclodextrin-bound indocyanine compound as described above, the temperature of the inclusion reaction, the composition and concentration of the aqueous solution, etc.

Alternatively, the non-inclusion-type cyclodextrin-bound indocyanine compound of the present invention can be converted to an inclusion-type cyclodextrin-bound indocyanine compound by dissolving it in a solvent containing water. While there are no particular restrictions on the water content in the solvent, in principle, inclusion is facilitated when the water content is high, preferably 50% by mass or greater. Furthermore, as long as a substance capable of forming an inclusion-type cyclodextrin-bound indocyanine compound is present in a solvent other than an aqueous solution, inclusion is not necessarily required in an aqueous solution.

In addition, an inclusion-type cyclodextrin-bound indocyanine compound may be formed during the synthesis of a non-inclusion-type cyclodextrin-bound indocyanine compound. In this case, there is no need to conduct the inclusion reaction again.

<4. Synthesis of Non-Inclusion Cyclodextrin-Bound Indocyanine Compounds (Synthesis Example)>

The synthesis of the compound represented by Chemical Formula 11 is given as an example. The indocyanine compound represented by Chemical Formula 11 can be obtained by, for example, adding the compound represented by Chemical Formula 13 synthesized by the method described in Non-Patent Document 5, Chemical Formula 14 synthesized by the method described in Non-Patent Document 6, a water-soluble carbodiimide (WSC: for example, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride) or dicyclohexylcarbodiimide (DCC) as a dehydration condensation agent, and pyridine or N,N-dimethylformamide or an aqueous solution as a solvent, and reacting at -20°C to 60°C for 10 minutes to 100 hours. In addition, in order to activate the reaction, 1-hydroxybenzotriazole (HOBt) can be added as an activator, for example. The amount of the dehydration condensation agent is 2 times the mole of the compound represented by Chemical Formula 13 or more. The solvent used is not limited as long as it can dissolve the reactants and does not hinder the dehydration condensation reaction. There is no limitation on the activator as long as it can activate the dehydration condensation reaction, and there is no limitation on the amount of the activator added as long as it can cause the dehydration condensation reaction to proceed to a desired extent.

[Chemical Formula 13]

[Chemical Formula 14]

<5. Purification of Non-Inclusion Cyclodextrin-Bound Indocyanine Compounds>

The mixture containing the non-inclusion cyclodextrin-bound indocyanine compound synthesized by the above method is dissolved in an acidic aqueous solution and subjected to reverse-phase column chromatography. Elution is performed using, for example, a mixture of acidic water and methanol, a mixture of acidic water and acetonitrile, a mixture of acidic water and ethanol, or a mixture of acidic water and acetone, to isolate and purify the high-purity non-inclusion cyclodextrin-bound indocyanine compound. The acid is not limited as long as it does not decompose the non-inclusion cyclodextrin-bound indocyanine compound, effectively elutes it, and facilitates post-elution handling. Preferred examples include hydrochloric acid, trifluoroacetic acid, acetic acid, sulfuric acid, nitric acid, and formic acid. Hydrochloric acid, trifluoroacetic acid, and acetic acid are preferred. Hydrochloric acid is most preferred. The acid concentration is not limited as long as it does not decompose the non-inclusion cyclodextrin-bound indocyanine compound, effectively elutes it, and facilitates post-elution handling. It is preferably 0.01 mM to 10 mM, and most preferably 0.1 mM to 1 mM. By keeping the concentration within this range, the target compound can be rapidly eluted without decomposing it. The eluted non-inclusion cyclodextrin-bound indocyanine compound can be obtained as a solid by removing the solvent. Freeze-drying can also be used as a solvent removal method.

<6. Synthesis Method (Synthesis Example) and Purification Method (Purification Example) of Inclusion-Type Cyclodextrin-Bound Indocyanine Compounds>

The non-inclusion type cyclodextrin-bound indocyanine compound of Chemical Formula 11 synthesized and purified by the above method is, for example, non-inclusion type in DMSO, but immediately becomes inclusion type upon dissolution in water, as shown in Chemical Formula 12. This phenomenon can be confirmed using ¹ H NMR.

Therefore, as long as the inclusion compound is an aqueous solution, it exists as an inclusion compound and can be used as an inclusion compound. In addition, the inclusion compound in the aqueous solution can be made into a solid by removing the water of the aqueous solution, and a solid of the inclusion compound can also be made.

<7. In vivo dynamics measuring device and circulation visualization device applicable to a living body to which a diagnostic composition has been administered>

In vivo dynamic measurement device

This device is completed based on the fact that the cyclodextrin-bound indocyanine compound of the present invention (hereinafter sometimes appropriately referred to as "compound of the present invention") used in the diagnostic composition of the present invention has different in vivo dynamics such as the degree of interstitial migration from ICG.

That is, the metabolic rate of the compound of the present invention and the rate of migration from the circulatory system, such as blood or lymph, to the interstitium differ from those of ICG. Consequently, the compound of the present invention enters the interstitium at a relatively greater rate than ICG. Therefore, by analyzing the in vivo dynamics of the compound of the present invention, the mechanism of fluid migration (in vivo dynamics) within the body can be accurately evaluated. Here, by maximally maintaining constant measurement conditions (such as the activity state of the organism and ambient temperature), the present device can more accurately measure in vivo dynamics.

One form of abnormal fluid movement in a living body is edema. Here, according to the so-called Stirling hypothesis, the movement of fluid (water) in a living body can be roughly considered to be from arteries to interstitial tissue, and then from interstitial tissue to veins.

The water movement from the artery to the vein can be calculated as (A)-(B)-(C)+(D) based on the pressure difference between the arterial pressure and the interstitial pressure, the water movement outside the circulatory system (A), the water movement into the circulatory system (B) based on the pressure difference between the venous pressure and the interstitial pressure, the water movement from the interstitial fluid to the artery (C) based on the pressure difference between the interstitial fluid osmotic pressure and the arterial blood osmotic pressure, and the water movement from the interstitial fluid to the vein (D) based on the pressure difference between the interstitial fluid osmotic pressure and the venous blood osmotic pressure.

Therefore, when the pressure difference between arterial and venous pressure decreases due to heart failure, or when interstitial fluid osmotic pressure decreases due to nutritional imbalance, the balance of water transfer (A) to (D) is disrupted, causing edema. Furthermore, when there are abnormalities in capillary permeability (abnormalities in the selectivity of material permeation), such as in diabetes, the balance of water transfer (A) to (D) is also disrupted, causing edema. There is no simple method to directly assess abnormal capillary permeability. While the occurrence of edema can be detected by phenomenon, it is currently difficult to determine the specific cause of edema.

Based on the above findings, when abnormal permeability of the capillary wall occurs, the movement of the compound of the present invention can be expected to change compared to cases where edema occurs due to a decrease in the differential pressure between arterial and venous pressure, a decrease in intracellular osmotic pressure, etc. Therefore, when the movement of the compound of the present invention is confirmed to be greater than (or less than) an amount equivalent to water movement, it can be determined that abnormal capillary permeability has occurred. Because it is believed that if no abnormal permeability of the capillary wall occurs, only the movement of the compound of the present invention to an extent equivalent to the change in water movement can be confirmed, it can be determined that an abnormality has occurred in the vascular wall.

In particular, by using two or more compounds of the present invention with different migration rates into the interstitium and measuring the in vivo dynamics of these compounds separately and comparing them, the degree of change in capillary permeability can be evaluated. In other words, by using two or more compounds of the present invention with different permeabilities in the capillary wall and measuring their permeabilities, the occurrence of abnormal vascular wall permeability can be more accurately evaluated. In other words, by using two or more compounds of the present invention with different susceptibilities to abnormal vascular wall permeability and measuring their in vivo dynamics, the factors affecting water migration can be more accurately evaluated. In addition, in addition to evaluating water migration using the compounds of the present invention, water migration can also be measured using conventional methods.

The in vivo dynamics measurement device of the present invention includes an excitation light irradiation unit, a fluorescence intensity measurement unit, an in vivo dynamics calculation unit, and other units. Other units may be selected as needed, such as an in vivo concentration calculation unit that calculates the concentration of a cyclodextrin-bound indocyanine compound based on the fluorescence intensity measured by the fluorescence intensity measurement unit.

This device is a device for measuring at least a portion of a living body to which the diagnostic composition of the present invention has been administered. The dosage of the diagnostic composition is the amount at which fluorescence is observed when the measurement site is irradiated with excitation light. Therefore, the appropriate dosage varies depending on the measurement site. There is no particular limitation on the type of compound of the present invention contained in the diagnostic composition. The compound of the present invention may be used alone or in combination of two or more. In addition, additional diagnostic compositions of the same or different compositions may be added during the measurement process.

The excitation light irradiation unit irradiates the compound of the present invention contained in the administered diagnostic composition with excitation light of a wavelength capable of generating fluorescence. The wavelength of the excitation light irradiated can be limited to an appropriate range. By limiting the wavelength to a minimum possible range, fluorescence and excitation light can be reliably separated. Regarding wavelength limitation, a light source emitting light of an appropriate wavelength can be selected, or a filter can be used to limit the wavelength.

The excitation light irradiation mode is not particularly limited, as long as the generated fluorescence can be measured by the fluorescence intensity measurement unit described later. Examples of excitation light include continuous light, pulsed light, and light with varying intensity. To vary the intensity, the excitation light intensity can be modulated by irradiating the excitation light pulses at predetermined intervals. Preferably, the excitation light intensity is modulated using pulse amplitude modulation.

The excitation light is irradiated to the site to be irradiated through an appropriate optical system. The site to be irradiated is the site where dynamic measurements in the body are to be performed. For example, when evaluating a site where edema occurs, it is preferable to irradiate the site with excitation light directly.

The excitation light irradiation range is not particularly limited. The irradiation range is determined as needed. A narrow irradiation range allows for precise measurement of subdivided in vivo dynamics within the narrow irradiated area. Irradiating a wider range increases the relative amount of the compound of the present invention that fluoresces in response to the excitation light, enabling more precise measurement of fluorescence intensity.

Furthermore, the excitation light irradiation unit is preferably used to irradiate the excitation light while suppressing the influence of ambient light, for example, by irradiating the excitation light in a dark place or by shielding the portion irradiated with the excitation light from external light.

The fluorescence intensity measuring unit measures the fluorescence intensity emitted from the area irradiated with excitation light by the excitation light irradiation unit. The fluorescence intensity is preferably measured by removing light other than fluorescence (ambient light, excitation light, etc.) through a filter that selectively transmits the emitted fluorescence.

When the excitation light irradiation unit uses a unit that irradiates excitation light with modulated intensity, the component that varies according to the modulation can be separated from the measured light intensity and used as the fluorescence intensity. For example, when the excitation light intensity is modulated using pulse amplitude modulation, the fluorescence intensity can be separated by demodulating the light component that varies according to the intensity of the modulated pulse and measuring its intensity. This reduces the impact of ambient light on the fluorescence intensity measurement results.

The in vivo concentration calculation unit is a unit that calculates the in vivo concentration of the compound of the present invention based on the fluorescence intensity measured by the fluorescence intensity measurement unit. The relationship between the fluorescence intensity and the in vivo concentration of the compound of the present invention can be calculated using an appropriate method. For example, a standard curve can be prepared in advance, and the in vivo concentration of the compound of the present invention can be calculated based on the standard curve. In addition, the absolute value of the fluorescence intensity can be directly used as a value related to the in vivo concentration of the compound of the present invention. The in vivo concentration calculation unit calculates the in vivo concentration of the compound of the present invention over time.

The in vivo dynamics calculation unit is a unit that calculates the temporal change of the time rate of change of the in vivo concentration of the compound of the present invention from the in vivo concentration data obtained over time by the in vivo concentration calculation unit. The in vivo dynamics calculation unit calculates the speed of the compound of the present invention moving from the circulatory system to the interstitial tissue and the speed of the compound of the present invention moving from the interstitial tissue to the circulatory system in a part of the body using the temporal change rate of the in vivo concentration obtained. Instead of using the in vivo concentration, the speed of movement into and out of the interstitial tissue can be directly calculated by using the change in fluorescence intensity obtained over time by the fluorescence intensity measurement unit.

It is speculated that there is a high correlation between the movement of the compound of the present invention into (and out of) the interstitium and the movement of water in body fluids. Therefore, by measuring the in vivo behavior of the compound of the present invention, it is possible to evaluate the movement of water in the measurement site. Furthermore, when abnormalities occur in the vascular wall and the permeability of substances is abnormal, the behavior of the compound of the present invention in the body should also change. Therefore, by evaluating the behavior of the compound of the present invention, vascular permeability can be evaluated.

The time rate of change of fluorescence intensity (or concentration in the organism) is obtained by differentiating the time-dependent change in fluorescence intensity (or concentration in the organism) with respect to time, or by calculating the fluorescence intensity (or concentration in the organism) at each specified time and then calculating it as the difference between the fluorescence intensity (or concentration in the organism) before (after) the specified time.

The speed of the compound of the present invention entering the interstitial fluid and the speed of being discharged in a part of the organism can be calculated by the time rate of change of the fluorescence intensity (or biological concentration) obtained. Here, the speed of entering the interstitial fluid and the speed of being discharged can be obtained as relative values by the fluorescence intensity. When more precision is required, it can be calculated according to the concentration of the compound of the present invention in the interstitial and the amount of the interstitial fluid. In addition, the concentration of the compound of the present invention in blood and lymph and its amount are also considered, and it can be calculated more accurately. The concentration of the compound of the present invention in blood, etc. can be measured with good accuracy by actually taking blood.

The following methods can be cited as methods for calculating the concentration in interstitial fluid. The first method is a method for directly approximating the calculated in vivo concentration of the compound of the present invention to the concentration in the interstitial fluid in a part of the organism. As a method related to the first method, the concentration in the interstitial fluid can also be calculated by considering the amount of interstitial fluid. The second method is to calculate the influence ratio of the substance present in the blood vessel on the measured value measured by the fluorescence intensity measurement unit by using other standard substances (ICG, etc.) that do not move to the interstitial on the basis of measuring the concentration of the compound of the present invention in the actual blood, and to calculate the concentration in the interstitial fluid by removing the influence of the concentration of the compound of the present invention in the measured blood.

The time rate of change of the concentration of the compounds of the present invention in interstitial fluid can be calculated by subtracting the speed of movement outside the interstitial fluid from the speed of movement inside the interstitial fluid. Here, it can be assumed that the movement of the compounds of the present invention inside (outside) the interstitial fluid is carried out at a speed related to the water movement in the body fluid, and if the water movement reaches equilibrium, the speed of movement inside the interstitial fluid and the speed of movement outside the interstitial fluid can be considered as constants, therefore, the concentration change (metabolism, discharge, changes caused by movement to tissue, etc.) of the compounds of the present invention in blood and the time rate of change of concentration in interstitial fluid can be used to calculate its movement speed. In addition, even in the case of changes in its movement speed, it is possible to calculate their movement speed by making a model corresponding to this change and applying it.

If the calculated migration speed is outside the range of normal blood vessel wall values, it can be inferred that the blood vessel wall has abnormalities. In addition, as a result of abnormalities in the blood vessel wall, the permeability of the compound of the present invention may be increased or decreased.

It is a well-known fact that acute tissue swelling is caused by various tissue disorders such as acute inflammation, ischemia, and trauma, and is an extremely important tissue reaction in the medical field regardless of the field.

For example, in addition to the simple occurrence of swelling itself being a problem, there are also issues involving all organs, such as the evaluation of intestinal suturing during surgery, the evaluation of the degree of inflammation such as pneumonia, the evaluation of the suitability of organ transplantation, and the evaluation of brain and kidney dysfunction.

However, there is currently no accurate method for predicting the likelihood and extent of tissue swelling, and it can only be estimated empirically based on the degree of tissue damage. Therefore, in most cases, countermeasures can only be discussed after the occurrence of swelling is confirmed, which is a major limitation in treatment.

If the likelihood of swelling can be predicted with high accuracy early on, measures to reduce swelling can be taken early on, minimizing the impact. For example, if the extent of brain swelling that occurs shortly after a cerebral hemorrhage can be accurately predicted, decompression measures and measures to reduce vascular permeability can be implemented early on, minimizing the impact. Similarly, if the impact of ischemic stress can be predicted in advance, secondary complications caused by myocardial infarction and limb injuries can be minimized. Thus, high-precision quantitative prediction technology for swelling has the potential to significantly impact healthcare overall.

Until now, quantification of tissue swelling, including edema, has been qualitatively determined through observation. While quantification has been attempted, it is only applicable in very limited situations and has the disadvantage of being able to evaluate only after swelling has occurred.

To address this issue, the in vivo dynamic measurement device of the present invention calculates the rate of water migration within and outside the interstitium, thereby predicting the progression of swelling. As detailed in the Examples, it is known that ICG does not migrate into the interstitium as swelling progresses. In contrast, the cyclodextrin-conjugated indocyanine compound of the present invention can migrate within and outside the interstitium, and its movement into the interstitium is further promoted by the movement of water as swelling progresses. Therefore, by evaluating the movement of the cyclodextrin-conjugated indocyanine compound of the present invention into the interstitium, the progression of swelling can be predicted.

That is to say, it can be inferred that the degree of swelling that is ultimately formed is affected by the speed of movement of water into and out of the interstitial space, and therefore the degree of final swelling can be evaluated by evaluating the speed of movement of the cyclodextrin-bound indocyanine compound of the present invention, which is related to the speed of movement of water. The speed of movement can be calculated by the concentration of the cyclodextrin-bound indocyanine compound, or directly using the fluorescence intensity. In addition, the speed of movement into and out of the interstitial space can naturally be calculated as an absolute value, or it can be calculated as a relative value (for example, the rate of change of fluorescence intensity can be directly used as a value related to the speed of movement), and the progress of swelling can be predicted based on this value.

The progression of swelling is preferably calculated based on the normal migration rate into the interstitium when there is no swelling. However, the normal migration rate into the interstitium may be unknown. In such cases, a non-swollen site is selected as a control site, and the migration rate of this site is calculated and used as a substitute. Alternatively, the extent of interstitial migration of the cyclodextrin-conjugated indocyanine compound of the present invention can be evaluated by using ICG, which does not migrate within the interstitium, to assess the blood circulation volume and metabolism.

The determination of the extent of movement of the cyclodextrin-bound indocyanine compound of the present invention within the interstitium is carried out after a prescribed time has passed after the diagnostic composition of the present invention is administered into a living body. The prescribed time refers to the time required for the diagnostic composition of the present invention to be distributed in the blood. The reason for using data after the prescribed time for evaluation is that after administration of the diagnostic composition, when it is distributed into the blood after a prescribed time, it is hardly affected by the degree of swelling progression, the concentration in the blood rises rapidly, and the change in fluorescence intensity is not affected by the presence or absence of swelling, and almost no difference is produced. After distribution in the blood, the speed of movement within the interstitium will change according to the presence or absence of swelling progression, so an increase in fluorescence intensity will be selectively confirmed at the site where the movement occurs (i.e., the site where swelling occurs).

As a specific example of a method for estimating the progression of swelling, there is a method in which the blood volume is estimated from body weight, the size of the peak of fluorescence intensity and the time to the peak are calculated from the estimated blood volume; the fluorescence intensity at the time when the edema may change is measured taking into account this time and intensity, and the degree of edema progression and the degree of edema that may occur in the future can be estimated from the results.

Circular Visualization Installation

This device is based on the fact that the cyclodextrin-bound indocyanine compound of the present invention (hereinafter sometimes appropriately referred to as "compound of the present invention") used in the diagnostic composition of the present invention differs from ICG in its in vivo dynamics, such as the degree of migration into the interstitium.

That is, the compound of the present invention easily moves in the interstitial fluid, and by tracking the behavior of the compound of the present invention, the state of blood circulation or lymph circulation can be visualized.

The circulation visualization device of the present invention comprises an excitation light irradiation unit, a fluorescence imaging unit, a morphological imaging unit, and a display unit. This device is a device for measuring at least a portion of a living body to which the diagnostic composition of the present invention is administered. The dosage of the diagnostic composition is the amount in which fluorescence can be observed when the measurement site is irradiated with excitation light. Therefore, the appropriate dosage varies depending on the measurement site. The type of compound of the present invention contained in the diagnostic composition is not particularly limited. The compound of the present invention can be used alone or in combination of two or more. In addition, additional diagnostic compositions having the same or different compositions can be administered during the measurement process.

The excitation light irradiation unit irradiates the compound of the present invention contained in the administered diagnostic composition with excitation light of a wavelength capable of generating fluorescence. The wavelength of the excitation light used for irradiation can be limited to an appropriate range. By limiting the wavelength to a narrow range as much as possible, fluorescence and excitation light can be reliably separated. Wavelength limitation can be achieved by selecting a light source emitting light of an appropriate wavelength or by using a filter to limit the wavelength.

The form of excitation light irradiation is not particularly limited, as long as the generated fluorescence can be measured by the fluorescence imaging unit described below. For example, the excitation light may be continuous light, pulsed light, or light with varying intensity. To vary the intensity, the intensity of the excitation light can be modulated by irradiating the excitation light pulses at predetermined intervals. Preferably, the excitation light is modulated using pulse amplitude modulation.

The excitation light passes through an appropriate optical system and illuminates the desired area. The area to be illuminated is the part of the organism where the circulation is to be visualized, such as the area where tissue necrosis has occurred due to burns, frostbite, inflammation, trauma, infarction, and the like, and its surrounding areas. Necrotic areas of tissue lack blood circulation, and leaving them untreated offers little benefit, so removal is considered. In this case, it is ideal to completely remove only the necrotic area, comparing the necrotic area with the normal area.

Conventionally, identification of necrotic areas has been performed using methods such as angiography using contrast agents or detecting a decrease in body temperature associated with blood circulation. However, angiography has the problem of being inconvenient to use devices such as X-ray irradiation devices, and accurate determination based on body temperature is difficult.

The device of the present invention is used to evaluate necrotic and normal areas based on the presence or absence of body fluid (blood) circulation. Visualizing areas without circulation allows for easy removal. In addition to visualizing necrotic areas, direct observation of blood circulation is also possible, making it easy to identify abnormalities in circulatory function. For example, areas that are not necrotic but have decreased circulatory function due to, for example, infarction can be visualized.

The excitation light irradiation range is determined as needed so as to include the portion to be cyclically visualized.

Furthermore, the excitation light irradiation unit is preferably used to irradiate the excitation light while controlling the influence of ambient light, for example, by irradiating the excitation light in a dark place or by shielding the portion irradiated with the excitation light from external light.

The fluorescence imaging unit two-dimensionally acquires the intensity of fluorescence emitted by the compound of the present invention, which is excited by the excitation light irradiation unit, thereby obtaining data on the distribution status of the compound of the present invention in the organism. In other words, this unit acquires distribution status data representing the distribution of the compound of the present invention in a portion of the organism as two-dimensional image data.

For example, it can be composed of a combination of an appropriate optical system and an imaging element such as a CCD. The resolution of the data obtained two-dimensionally can be set to a necessary value according to the purpose. The determination of fluorescence intensity preferably allows the emitted fluorescence to be selectively transmitted through a filter and removes light (ambient light, excitation light, etc.) other than the fluorescence to be measured.

When using excitation light with modulated intensity as the excitation light irradiation unit, the fluorescence intensity is obtained by separating the components that vary according to the modulation from the measured light intensity. For example, when the excitation light intensity is modulated using pulse amplitude modulation, the fluorescence intensity is separated by demodulating the components of the light that vary according to the modulated pulse intensity and measuring their intensities. This reduces the influence of ambient light on the fluorescence intensity measurement results.

The morphological imaging unit is a unit that two-dimensionally acquires the intensity of light at wavelengths other than the fluorescence wavelength emitted by the compound of the present invention to obtain morphological data of the compound in a portion of a living body. In other words, this unit is a unit that acquires two-dimensional image data of the morphology of a portion of a living body.

The morphological imaging unit can be composed of an appropriate optical system and an imaging element such as a CCD. The resolution of the two-dimensional data can be set according to the desired purpose. In this case, the fluorescence emitted by the excitation light is not measured (or the detection sensitivity is reduced). The morphological imaging unit uses almost all of the aforementioned fluorescence imaging unit and optical system. Finally, a dichroic prism is used on the optical path before it is introduced into the imaging element to guide light with a wavelength corresponding to the fluorescence to the fluorescence imaging unit, and light of other wavelengths can be guided to the morphological imaging unit. By appropriately forming a bidirectional color film, the dichroic prism can reasonably control the wavelength of the separated light.

Furthermore, the fluorescence imaging unit and the morphological imaging unit can share the same camera device. That is, the separation of fluorescence and other light can also be a mathematical separation performed after obtaining their two-dimensional image data. Furthermore, the distribution state data can also be complex image data from the surface of a part of the biological body to the deep layer direction. The optical focus used by the fluorescence imaging unit obtains complex two-dimensional image data in the deep layer direction while moving in the deep layer direction. In addition, as the excitation light irradiation unit, an optical system with a variable focal length is used, and the focus is moved not on the surface of a part of the biological body, but in the deep layer direction, or the excitation light is concentrated and irradiated to a part of the biological body, so that fluorescence can be selectively emitted not only on the surface of the biological body but also in the deep layer inside the biological body, and the circulation of this part can be visualized.

The display unit is a display unit that represents the distribution state of the compound of the present invention in a part of the organism by overlapping the morphological data obtained by the morphological imaging unit and the distribution state data obtained by the fluorescence imaging unit. When the wavelength of the fluorescence is not within the range of visible light, it is preferred to convert the wavelength of the fluorescence into visible light for representation. From the perspective of visualization of the circulation, the representation of the distribution state data takes precedence over the representation of the morphological data. In particular, it is possible to distinguish between the low distribution amount (i.e., fluorescence intensity) of the compound of the present invention and the other parts (whether it is low or not is determined according to the purpose of visualization of the circulation. For example, in the purpose of visualizing the necrotic part, in the part without circulation, i.e., the part where no fluorescence is confirmed) and other parts can be distinguished. For example, the part can be represented as a color different from that of the other parts or a flashing representation. The superposition of the distribution state data and the morphological data can be realized using logic on a computer, etc. The representation of two-dimensional data can be realized by a general display device. The display device is provided between the organism and the measurer, so that the organism can be treated while observing the display device.

＜8. Definitions, etc.＞

In the present invention, "alkyl" refers to a linear or branched alkyl group having 1 to 20 carbon atoms which may have a substituent, for example, a methyl group, ethyl group, propyl group, butyl group, pentyl group, hexyl group, heptyl group, octyl group, nonyl group, decyl group, undecyl group, dodecyl group, tridecyl group, tetradecyl group, pentadecyl group, hexadecyl group, heptadecyl group, octadecyl group, nonadecyl group, eicosyl group or a branched group.

In the present invention, the "alkoxy group" includes, for example, methoxy, ethoxy, propoxy, butoxy, pentyloxy, hexyloxy, methoxyethoxy, methoxypropoxy, ethoxyethoxy, ethoxypropoxy, methoxyethoxyethoxy, etc., in which alkoxy groups having 1 to 20 carbon atoms are bonded in a straight chain or branched form.

In the present invention, the "aryl group" includes aromatic hydrocarbons having 6 to 20 carbon atoms, such as phenyl and naphthyl.

Example

The preferred embodiments of the present invention are described in detail below by way of examples. However, the technical scope of the present invention is not limited to the following embodiments, and various modifications can be made within the scope of the present invention.

<Experiment 1: Synthesis and Purification of Compounds Represented by Chemical Formula 15 and Chemical Formula 16>

A mixture of 0.20 g of the compound represented by Chemical Formula 13, 0.94 g of the compound represented by Chemical Formula 14, 0.18 g of WSC, 0.12 g of HOBt, 4.0 mL of pyridine, and 2.0 mL of N,N-dimethylformamide was stirred at 0°C in the dark for 6 hours. 50 mL of acetone was then added, and the precipitate was filtered under reduced pressure. The precipitate was dissolved in a 0.1% trifluoroacetic acid aqueous solution and subjected to ODS column chromatography. A mixture of water and methanol containing 1 mM hydrochloric acid was used as the eluent to elute the compound represented by Chemical Formula 15. The eluate was concentrated under reduced pressure to obtain 0.65 g of the inclusion-type compound represented by Chemical Formula 16 as a green solid (at the end of the reduced pressure concentration of the eluate, the water content was high, so it naturally became an inclusion-type compound).

[Chemical Formula 15]

[Chemical Formula 16]

Next, the instrumental analysis data for the target compound represented by Chemical Formula 16 are shown. ¹ H NMR (500 MHz, D ₂ O, 26°C, acetone: 2.10 ppm) 1.43 (2H, m), 1.55 (2H, m), 1.99 (6H, s), 2.09 (6H, s), 2.63 (6H, m), 2.80 (6H, m), 2.92 (2H, m), 3.02 (2H, dd, J = 3.7, 9.8 Hz), 3.08 (2H, t, J = 9.2 Hz), 3.2-4.1 (m), 4.19 (2H, t, J = 9.8 Hz), 4.26 (2H, t, J = 9.8 Hz), 4.33 (2H, m), 4.43 (2H, m), 4.71 (2H, d, J = 2.4 Hz) , 4.81 (4H, d, J = 3.7 Hz), 4.91 (2H, d, J = 3.7 Hz), 4.99 (2H, d, J = 3.7 Hz), 5.08 (2H, d, J = 3.7 Hz), 5.13 (2H, d, J = 3.1 Hz), 6.15 (2H, d, J = 13 Hz), 6.52 (2H, t, J = 12 Hz), 7.43 (4H, m), 7.57 (1H, d, J = 12 Hz), 7.57 (2H, d, J = 9.2 Hz), 7.78 (2H, m), 8.06 (3H, m), 8.15 (2H, d, J = 8.5 Hz). ESI-MS m/z calcd for C ₁₃₁ H ₁₉₁ N ₄ O ₇₂ 2972, found 2973 [M] ⁺ .

<Experiment 2: Synthesis and Purification of Compounds Represented by Chemical Formula 19 and Chemical Formula 20>

A mixture of 0.17 g of the compound represented by Chemical Formula 17, 5 mL of methanol, and 0.30 g of t-BuOK was stirred at room temperature for 12 hours. Then, 3 mL of 1M hydrochloric acid and 50 mL of water were added. The precipitate was filtered, washed with water, and then dried under reduced pressure to obtain 0.17 g of the compound represented by Chemical Formula 18.

[Chemical Formula 17]

[Chemical Formula 18]

A mixture of 0.02 g of the compound represented by Chemical Formula 18, 0.081 g of the compound represented by Chemical Formula 14, 0.016 g of WSC, 0.011 g of HOBt, 0.3 mL of pyridine, and 0.2 mL of N,N-dimethylformamide was stirred at 0°C in the dark for 6 hours. 5 mL of acetone was then added, and the precipitate was filtered under reduced pressure, dissolved in a 0.1% trifluoroacetic acid aqueous solution, and subjected to ODS column chromatography. A mixture of water and methanol containing 1 mM hydrochloric acid was used as the eluent to elute the compound represented by Chemical Formula 19. The eluent was concentrated under reduced pressure to obtain 0.045 g of the inclusion-type compound represented by Chemical Formula 20 as a green solid (at the end of the concentration of the eluent under reduced pressure, the water content was high, so it naturally became an inclusion-type compound).

[Chemical Formula 19]

[Chemical Formula 20]

Next, the machine analysis data of the target compound represented by Chemical Formula 20 are shown. ¹ H NMR (500 MHz, D ₂ O,40℃,acetone:2.26ppm)1.54(2H,m),1.68(2H,m),1.98(2H,m),2.19(6H,s),2 .20(2H,m),2.30(6H,s),2.6-2.85(10H,m),2.95(2H,m),3.00(4H,m),3.08 (2H,t,J＝12Hz),3.17(2H,dd,J＝3.7,9.8Hz),3.26(2H,t,J＝9.8Hz),3.35- 4.30(m),4.35(2H,t,J＝9.2Hz),4.50(2H,t,J＝9.2H),4.52(2H,m),4.63(2H ,m),4.87(2H,d,J＝3.7Hz),4.95(d,J＝3.1Hz),4.97(2H,d,J＝3.7Hz),5.08 (2H,d,J＝3.7Hz),5.15(2H,d,J＝4.3Hz),5.25(2H,d,J＝3.7Hz),5.29(2H,d, J＝3.7Hz),6.30(2H,d,J＝14.6Hz),7.58(4H,m),7.73(2H,d,J＝8.5Hz),7.95 (2H,m),8.25(2H,m),8.32(2H,d,J＝14.6Hz),8.35(2H,d,J＝8.5Hz).ESI-MS m/ _z calculated for _{C135H197N4O73 3042} , found 3042 [M] ⁺ .

<Experiment 3: Synthesis and Purification of the Compound Represented by Chemical Formula 21>

A mixture of 0.04 g of the compound represented by Chemical Formula 13, 0.18 g of mono-6-amino-6-deoxy-β-cyclodextrin, 0.05 g of WSC, 0.025 g of HOBt, 0.8 mL of pyridine, and 0.4 mL of N,N-dimethylformamide was stirred at 0°C in the dark for 3 hours. 10 mL of acetone was then added, and the precipitate was filtered under reduced pressure, dissolved in 0.1% trifluoroacetic acid aqueous solution, and subjected to ODS column chromatography. The compound represented by Chemical Formula 21 was eluted using a mixture of water and methanol containing 1 mM hydrochloric acid. The eluate was concentrated under reduced pressure to obtain 0.11 g of the compound represented by Chemical Formula 21 as a green solid.

[Chemical Formula 21]

Next, the instrumental analysis data for the target compound represented by Chemical Formula 21 are shown. ¹ H NMR (500 MHz, D ₂ O, 29°C, acetone: 2.10 ppm) 1.79 (12H, br.), 2.68 (4H, br.), 3.0-4.5 (98H), 4.4 (4H, br.), 4.5-5.3 (14H, br.), 6.18 (2H, br.), 6.46 (2H, br.), 7.3-8.2 (15H). ESI-MS m/z calculated for C ₁₂₅ H ₁₇₉ N ₄ O ₇₀ 2856, found 2856 [M] ⁺ .

<Experiment 4: Synthesis and Purification of the Compound Represented by Chemical Formula 23>

A mixture of 0.02 g of the compound represented by Chemical Formula 22, 0.096 g of mono-6-amino-6-deoxy-β-cyclodextrin, 0.032 g of WSC, 0.5 mL of pyridine, and 0.05 mL of 0.1 M phosphate buffer was stirred in the dark at room temperature for 24 hours. 10 mL of acetone was then added, and the precipitate was filtered under reduced pressure, dissolved in 0.1% trifluoroacetic acid aqueous solution, and subjected to ODS column chromatography. A mixture of water and acetonitrile was used as the eluent to elute the compound represented by Chemical Formula 23. The eluate was concentrated under reduced pressure to yield 0.014 g of the compound represented by Chemical Formula 23 as a green solid.

[Chemical Formula 22]

[Chemical Formula 23]

Next, the instrumental analysis data for the target compound represented by Chemical Formula 21 are shown. ¹ H NMR (500 MHz, D ₂ O, 26°C, acetone: 2.15 ppm) 1.26 (4H, m), 1.5-2.25 (24H, br), 2.7-4.2 (88H), 4.82 (2H, br), 4.90 (8H, br), 4.97 (2H, br), 5.03 (2H, br), 6.11 (2H, br), 6.36 (2H, br), 7.3-8.01 (15H, br). ESI-MS m/z calculated for C ₁₃₁ H ₁₉₁ N ₄ O ₇₀ 2940, found 2940 [M] ⁺ .

<Experiment 5: Synthesis and Purification of the Compound Represented by Chemical Formula 24>

A mixture of 0.20 g of the compound represented by Chemical Formula 22, 0.02 g of 3-amino-3-deoxy-β-cyclodextrin, 0.096 g of WSC, 0.013 g of HOBt, 0.4 mL of pyridine, and 0.2 mL of N,N-dimethylformamide was stirred in the dark at room temperature for 1 hour. Subsequently, 10 mL of acetone was added, and the precipitate was filtered under reduced pressure, dissolved in 0.1% trifluoroacetic acid aqueous solution, and subjected to ODS column chromatography. A mixture of water and methanol was used as the eluent to elute the compound represented by Chemical Formula 24. The eluate was concentrated under reduced pressure to yield 0.013 g of the compound represented by Chemical Formula 24 as a green solid.

[Chemical Formula 24]

Next, the instrumental analysis data for the target compound represented by Chemical Formula 24 are shown. ¹ H NMR (500 MHz, D ₂ O, 29°C, acetone: 2.10 ppm) 1.1-2.5 (28H), 3.0-4.25 (88H), 4.5-5.2 (14H), 7.3-8.02 (15H). ESI-MS m/z calculated for C ₁₃₁ H ₁₉₁ N ₄ O ₇₀ 2940, found 2940 [M] ⁺ .

<Experiment 6: Synthesis and Purification of the Compound Represented by Chemical Formula 25>

A mixture of 0.02 g of the compound represented by Chemical Formula 22, 0.1 g of the compound represented by Chemical Formula 14, 0.032 g of WSC, 0.5 mL of pyridine, and 0.05 mL of 0.1 M phosphate buffer was stirred in the dark at room temperature for 24 hours. 10 mL of acetone was then added, and the precipitate was filtered under reduced pressure, dissolved in 0.1% trifluoroacetic acid aqueous solution, and subjected to ODS column chromatography. A mixture of water and methanol was used as the eluent to elute the compound represented by Chemical Formula 25. The eluate was concentrated under reduced pressure to yield 0.021 g of the compound represented by Chemical Formula 25 as a green solid.

[Chemical Formula 25]

Next, the instrumental analysis data for the target compound represented by Chemical Formula 25 are shown. ¹ H NMR (500 MHz, D ₂ O, 25°C, acetone: 2.10 ppm) 1.0-2.5 (32H), 3.0-4.5 (96H), 4.8-5.2 (14H), 6.09 (2H, br), 6.37 (2H, br), 7.3-8.02 (15H, br). ESI-MS m/z calculated for C ₁₃₇ H ₂₀₃ N ₄ O ₇₂ 3056, found 3056 [M] ⁺ .

<Test 7: Solubility of the Cyclodextrin-Bound Indocyanine Compound of the Present Invention>

Solubility tests were conducted on ICG and the cyclodextrin-conjugated indocyanine compounds of the present invention (Chemical Formulas 16, 20, 21, and 23-25) in water and saline. While the dissolution of powdered ICG from Molecular Probe in water and saline required vigorous agitation for approximately one minute, the cyclodextrin-conjugated indocyanine compounds of the present invention, particularly those represented by Chemical Formulas 16 and 20, dissolved rapidly without the need for agitation.

<Test 8: Adsorption of the Cyclodextrin-Bound Indocyanine Compound of the Present Invention to Human Skin>

ICG and the cyclodextrin-conjugated indocyanine compounds of the present invention (Chemical Formulas 16, 20, 21, 23-25) were tested for their adsorption to human skin. A 1 mM aqueous solution (0.03 mL) of each of ICG and the cyclodextrin-conjugated indocyanine compounds of the present invention (Chemical Formulas 16, 20, 21, 23-25) was dropped onto the wrist, rinsed with water after 5 minutes, and then wiped and rinsed again. The result showed that ICG was not completely washed away by water, while the cyclodextrin-conjugated indocyanine compounds of the present invention, especially the compounds represented by Chemical Formulas 16 and 20, were easily washed away by water, indicating that the adsorption of the cyclodextrin-conjugated indocyanine compounds of the present invention to human skin was significantly lower than that of ICG (Figure 1).

<Test 9: Adsorption of the Cyclodextrin-Bound Indocyanine Compound of the Present Invention to Cellulose Fibers>

ICG and the cyclodextrin-conjugated indocyanine compounds of the present invention (Chemical Formulas 16, 20, 21, 23-25) were tested for their adsorption onto cellulose fibers. A cotton swab (Sanyo Co., Ltd.) was used to apply 0.05 mL of a 1 mM aqueous solution of each of ICG and the cyclodextrin-conjugated indocyanine compounds of the present invention (Chemical Formulas 16, 20, 21, 23-25) to a model cellulose fiber. After 3 minutes, the swab was rinsed in running water (tap water, 1 L/min) for 5 seconds. The results showed that ICG was not completely washed away, while the cyclodextrin-conjugated indocyanine compounds of the present invention, particularly those represented by Chemical Formulas 16 and 20, were easily washed away, indicating that the adsorption of the compounds represented by the cyclodextrin-conjugated indocyanine compounds of the present invention onto cellulose fibers was significantly lower than that of ICG (Figure 2).

<Test 10: Adsorption of the Cyclodextrin-Conjugated Indocyanine Compound of the Present Invention to a Meat Model>

Adsorption of ICG and the cyclodextrin-conjugated indocyanine compounds of the present invention (Chemical Formulas 16, 20, 21, 23-25) onto a biological meat model was tested. Commercially available pork tenderloin was used as the meat model. A 5 mm diameter recess was created in the pork tenderloin. 0.05 mL of a 1 mM aqueous solution of each of ICG and the cyclodextrin-conjugated indocyanine compounds of the present invention (Chemical Formulas 16, 20, 21, 23-25) was applied to the meat model. After 3 minutes, the sample was rinsed in running water (tap water, 1 L/min) for 10 seconds. The results showed that ICG was not completely washed off, while the cyclodextrin-conjugated indocyanine compounds of the present invention, particularly those represented by Chemical Formulas 16 and 20, were easily washed off, demonstrating that the adsorption of the cyclodextrin-conjugated indocyanine compounds of the present invention onto the biological meat model was significantly lower than that of ICG (Figure 3).

<Test 11: Adsorption of the Cyclodextrin-Conjugated Indocyanine Compound of the Present Invention to a Biological Protein Model>

Adsorption of ICG and the cyclodextrin-conjugated indocyanine compounds of the present invention (Chemical Formulas 16, 20, 21, 23-25) onto biological protein models was tested. A 5 mm diameter recess was created in commercially available chicken breast meat as the biological protein model. 0.05 mL of a 1 mM aqueous solution of ICG and the cyclodextrin-conjugated indocyanine compounds of the present invention (Chemical Formulas 16, 20, 21, 23-25) was applied to the breast. After 3 minutes, the samples were rinsed in running water (1 L/min) for 10 seconds. The results showed that ICG was not completely washed away, while the cyclodextrin-conjugated indocyanine compounds of the present invention, particularly those represented by Chemical Formulas 16 and 20, were easily washed away, indicating that the adsorption of the cyclodextrin-conjugated indocyanine compounds of the present invention onto biological protein models was significantly lower than that of ICG (Figure 4).

<Test 12: Adsorption of the Cyclodextrin-Bound Indocyanine Compound of the Present Invention to Hydrophobic Chemical Fibers>

The adsorption of ICG and the cyclodextrin-conjugated indocyanine compounds of the present invention (Chemical Formulas 16, 20, 21, 23-25) onto hydrophobic chemical fibers was tested. A polypropylene mask (Tamagawa Eisai Co., Ltd.) was used as a model hydrophobic chemical fiber. 0.05 mL of a 1 mM aqueous solution of ICG and the cyclodextrin-conjugated indocyanine compounds of the present invention (Chemical Formulas 16, 20, 21, 23-25) was applied to the mask. After 20 minutes (the sample was left to remove any moisture, so it would not adhere to the mask), the mask was rinsed in running water (1 L/min) for one second. The results showed that ICG was not completely washed off, while the cyclodextrin-conjugated indocyanine compounds of the present invention, particularly those represented by Chemical Formulas 16 and 20, were easily washed off, indicating that the adsorption of the cyclodextrin-conjugated indocyanine compounds of the present invention onto hydrophobic chemical fibers was significantly lower than that of ICG (Figure 5).

<Test 13: Molecular Association of Cyclodextrin-Bound Indocyanine Compounds of the Present Invention in Aqueous Solution>

The molecular association properties of ICG and the cyclodextrin-conjugated indocyanine compounds of the present invention (Chemical Formulas 16, 20, 21, 23-25) in aqueous solutions were investigated. Aqueous solutions of ICG and the cyclodextrin-conjugated indocyanine compounds of the present invention (Chemical Formulas 16, 20, 21, 23-25) were prepared at 0.01 mM, 0.025 mM, 0.05 mM, and 0.1 mM, respectively. The solutions were placed in quartz cuvettes with a 1 mm optical path length, and optical absorption spectra were measured at 25°C from 600 nm to 1000 nm. The results showed that ICG exhibited molecular association known as H-aggregation within this concentration range (Figure 6, left). However, the cyclodextrin-conjugated indocyanine compounds of the present invention, particularly those represented by Chemical Formulas 16 and 20, did not exhibit this molecular association within this concentration range (Figure 6, right).

<Test 14: Fluorescence of the Cyclodextrin-Bound Indocyanine Compound of the Present Invention in Aqueous Solution>

The fluorescence properties of ICG and the cyclodextrin-conjugated indocyanine compounds of the present invention (Chemical Formulas 16, 20, 21, 23-25) in aqueous solution were investigated. 0.1 μM aqueous solutions of ICG and the cyclodextrin-conjugated indocyanine compounds of the present invention (Chemical Formulas 16, 20, 21, 23-25) were placed in 1 cm square quartz cuvettes at 25°C. Excitation was performed with 720 nm excitation light (bandpass: 10 nm), and fluorescence spectra were measured (bandpass: 10 nm). Fluorescence efficiency was calculated based on the fluorescence efficiency of ICG, which is 0.13 (in DMSO, 25°C). The results showed that the fluorescence quantum efficiency of ICG was 0.021, while the fluorescence quantum efficiency of the cyclodextrin-conjugated indocyanine compounds of the present invention, particularly the compounds represented by Chemical Formulas 16 and 20, was 0.054 and 0.042, respectively, which are 2.6 times and 2 times that of ICG.

<Test 15: Fluorescence of the Cyclodextrin-Conjugated Indocyanine Compound of the Present Invention in Blood>

The fluorescence of ICG and the cyclodextrin-conjugated indocyanine compounds of the present invention (Chemical Formulas 16, 20, 21, 23-25) in blood was studied. ICG and the cyclodextrin-conjugated indocyanine compounds of the present invention (Chemical Formulas 16, 20, 21, 23-25) were dissolved in human blood to a concentration of 100 μM. The mixture was placed in a triangular quartz cuvette at 25°C and excited with 760 nm excitation light (bandpass: 10 nm), and the surface fluorescence spectrum (bandpass: 10 nm) was measured. The fluorescence intensity at the maximum fluorescence wavelength was 58 (arbitrary units) for ICG. The fluorescence intensities of the cyclodextrin-conjugated indocyanine compounds of the present invention, especially the compounds represented by Chemical Formulas 16 and 20, were 270 (arbitrary units) and 190 (arbitrary units), which are 4.7 times and 3.3 times that of ICG. This is believed to be because the cyclodextrin-conjugated indocyanine compounds of the present invention do not inhibit luminescence in vivo.

<Test 16: Behavior of the Cyclodextrin-Conjugated Indocyanine Compound of the Present Invention in Vivo>

As representative examples, the behaviors of the isomerization-equilibrium compound (TK1) represented by Chemical Formulas 19 and 20 and the isomerization-equilibrium compound (TK2) represented by Chemical Formulas 15 and 16 were evaluated in human blood and rats as biological subjects.

Fluorescence behavior in human blood

The concentration dependence of surface fluorescence intensity of ICG, TK1, and TK2 in human venous blood in triangular cuvettes was evaluated. Specifically, 1.0 mL of human venous blood was added to a triangular quartz cuvette, along with ICG, TK1, and TK2 at specified concentrations. After irradiation with 760 nm (bandpass: 10 nm) excitation light at 25°C, surface fluorescence (bandpass: 10 nm) was measured. The results are shown in Figure 7.

As can be clearly seen from FIG7 , both TK1 and TK2 can emit fluorescence in human venous blood when irradiated with excitation light.

·Study on the feasibility of drug administration in rats

ICG, TK1, and TK2 were prepared as 1 mM aqueous solutions, and 0.1 mL of each solution was injected into the femoral vein of rats after laparotomy. The abdomen was then illuminated with 760 nm excitation light to assess the presence of fluorescence.

The results showed that fluorescence was observed in the internal organs of the rats after the abdominal opening. High accumulation of TK1 and TK2 in the kidneys was observed (as ICG is known to accumulate in the liver). Furthermore, no significant adverse effects on the rats' condition were observed after administration of these drugs, confirming the possibility of relatively safe in vivo administration.

Observation of distribution in rats

Wistar male rats (9 weeks old, weighing 350 g) were anesthetized with ether and then anesthetized by intraperitoneal administration of 0.1 mL of sodium pentobarbital (Nembutal) using a 26G needle. The base of the rat's tail was anesthetized with a rubber band to remove blood, and the tail vein was secured with a 24G indwelling needle, and the terminal line was secured by connecting a three-headed square stopcock and an extension tube. The rat was fixed on a flat table in a supine position (Figure 8). The PDE camera unit manufactured by Hamamatsu Photonics Co., Ltd. was correctly set at a position 16 cm away from the rat's body. However, it was fixed at 20 cm when photographing the whole body. The observation range is the range indicated by the black circle in Figure 8. Thereafter, ICG, TK1, and TK2 at a concentration of 1 mM were administered through the secured terminal line. The dosage was 0.1 mL. The fluorescence in the abdominal cavity when the abdomen was opened 20 minutes after the administration is shown in Figure 9, and the fluorescence of the dorsum of the foot is shown in Figure 12 (image at the time of reaching the maximum brightness).

Figure 9 shows that ICG accumulates primarily in the liver (Figure 9(a)), TK1 primarily in the kidneys (Figure 9(b)), and TK2 also primarily in the kidneys (Figure 9(c)). Near-infrared imaging of internal organs immediately after administration demonstrated clear imaging of the kidneys, ureters, and bladder (Figure 10). Furthermore, near-infrared endoscopy demonstrated the ability to image the ureters (Figure 11).

As can be seen from image 12, fluorescence is clearly observed corresponding to blood flow in the dorsum of the foot. Furthermore, although not shown in the figure, fluorescence was observed after administering ICG, TK1, and TK2 to a rat model of lower limb ischemia in which the right femoral artery was ligated. Since no fluorescence was observed in the ischemic area, the presence or absence of fluorescence can be used to assess the extent of blood flow.

13 and 14 show the temporal changes in fluorescence intensity of the dorsum of the foot. Table 1 shows the time to maximum fluorescence intensity (Imax), the Imax value, and the time to half the Imax value (t _1/2 ).

[Table 1]

Compound
				ICG	0.5min	124	2.3min
TK-1	10min	160	40min
				TK-2	15min	106	55min

As shown in Table 1, Figures 13, and 14, TK1 and TK2 both took longer to reach maximum fluorescence intensity and to reach half-value in time than ICG. This suggests that they can emit fluorescence for a long time in vivo. For example, while ICG's fluorescence intensity drops to its initial value within 10 minutes after administration, TK1 and TK2 exhibit high fluorescence intensity even after one hour.

Observation at the vascular level

According to the method disclosed in non-patent literature 7 and 8 (Figure 15), ICG and TK1 were administered to rats with their blood vessels directly exposed for observation. The situation of the spermatozoa cremaster flap after exposure is shown in Figure 16. As a result, ICG appeared uneven in the blood vessels relative to TK1. In other words, it can be seen that TK1 moved from the blood vessels to the interstitial fluid. In order to conduct a more precise study, microscopic photographs of the blood vessels after administration are shown in Figures 17 (ICG) and 18 (TK1). As can be seen from Figures 17 and 18, when the fluorescence intensity in the blood vessels and the fluorescence intensity in the surrounding tissues are compared, ICG can hardly confirm fluorescence in the interstitial tissue, while TK1 can confirm fluorescence in the interstitial tissue. In addition, although not shown in detail, it is confirmed that the fluorescence intensity in the interstitial tissue increases over time. Therefore, ICG can hardly confirm movement from the blood vessels to the interstitial fluid, but TK1 can confirm movement from the blood vessels to the interstitial fluid.

Evaluation of the correlation between the extent of migration of the cyclodextrin-bound indocyanine compound of the present invention within the interstitium and the magnitude of edema formation

(Evaluation method)

Ten-week-old male Wister rats weighing approximately 350 g were selected as experimental animals (n=4). The rats were anesthetized with isoflurane and injected with 0.1 mL of 0.5% λ carrageenan into the plantar surface of the left hind paw using a 26G needle. Paw edema rapidly developed. Fifteen minutes later, the rats' left paw volume was measured using a rat paw volume meter (MK-101CMP PLETHYSMOMETER, manufactured by Muromachi Machinery Co., Ltd.).

Afterward, isoflurane was turned off and the rat was confirmed to be awake. After the Von Frey test, general anesthesia was re-administered with isoflurane. The rat's tail vein was secured with a 24G indwelling cannula and then secured in a supine position on a horizontal platform. A PDE camera manufactured by Hamamatsu Photonics Co., Ltd. was set up and fixed at a height of 16 cm from the dorsum of the rat's foot.

The PDE camera was used to set the measurement area (ROI) to the central part of the dorsum of the foot. After the measurement began, 0.1 mL of a 1 mmol TK1 aqueous solution was injected through the tail vein, followed by a 5-second flush with 1 mL of saline. Continuous imaging was performed for the first 5 minutes of injection, and the ROI brightness was measured. Thereafter, brightness measurements were taken at 5-minute intervals for 1 minute, continuing until 120 minutes after injection. After the measurement was completed, isoflurane was turned off and the subject returned to consciousness (acute inflammation experiment).

After 7 days, the rats were subjected to Von Frey test, and then the rats were quickly anesthetized with isoflurane. The volume of the rat's left foot was measured using a rat plantar volume meter. After the rat's tail vein was secured with a 24G indwelling needle, it was fixed on a horizontal platform in a supine position. A PDE camera manufactured by Hamamatsu Photonics Co., Ltd. was set up and fixed at a height of 16 cm from the dorsum of the rat's foot. The ROI was set at the center of the dorsum of the foot, and the measurement was started after the light was turned off. 0.1 mL of 1 mmol TK1 aqueous solution was injected from the tail vein and rinsed with 1 mL of normal saline for 5 seconds. Continuous shooting was performed during the first 5 minutes of injection to measure the brightness of the ROI. The brightness was measured for 1 minute at intervals of 5 minutes thereafter, and this continued until 120 minutes after the injection. After the measurement was completed, isoflurane was turned off and the rat returned to consciousness. The same test was performed on the group that was not injected with λ carrageenan.

ICG also implemented the above process.

(result)

The results are shown in Figures 19 to 26. The left paw volume of the rats measured for the group administered with TK1 is shown in Figure 19 (the volume increases toward the top of the vertical axis), the results of the Von Frey test are shown in Figure 20 (the more downward the vertical axis, the more hyperalgesia), the changes in brightness shortly after the injection are shown in Figure 21 (the brightness increases toward the top of the vertical axis), and the changes in brightness one week after the injection of carrageenan are shown in Figure 23 (the brightness increases toward the top of the vertical axis). The left paw volume of the rats measured for the group administered with ICG is shown in Figure 23 (the volume increases toward the top of the vertical axis), the results of the Von Frey test are shown in Figure 24 (the more downward the vertical axis, the more hyperalgesia), the changes in brightness shortly after the injection are shown in Figure 25 (the brightness increases toward the top of the vertical axis), and the changes in brightness one week after the injection of carrageenan are shown in Figure 26 (the brightness increases toward the top of the vertical axis). The values of the vertical axes of all the graphs are arbitrary units.

Significant differences were observed in the patterns of brightness changes within the ROI between ICG and TK1. The brightness rapidly increased shortly after ICG administration, then rapidly decreased, returning to baseline after approximately 10 minutes. In contrast, the initial brightness increase after TK1 administration followed the same trajectory as ICG, with the brightness remaining high thereafter and requiring six hours to return to baseline. Based on these results, we believe that ICG is retained within the blood vessels and does not leak into the interstitium. Therefore, the rapid brightness increase and subsequent rapid decrease are due to passage through the blood vessels distributed within the ROI. In contrast, TK1 exhibits two phases: the initial rapid passage into the blood vessels (hereafter referred to as the "vascular phase") and the subsequent phase in which TK1 fluorescence gradually leaks into the interstitium (hereafter referred to as the "interstitial phase"). The transition from the vascular phase to the interstitial phase corresponds to the specified time.

In the acute inflammation experiment, a significant increase in paw volume was observed in both the ICG and TK1 groups after injection of λ carrageenan, which returned to normal, similar to the tendon side, one week later (Figures 19 and 23). Furthermore, in the Von Frey test, significant hyperalgesia was observed in both the ICG and TK1 groups after injection of λ carrageenan, but returned to normal, similar to the tendon side, one week later (Figures 20 and 24).

Regarding the brightness of the dorsum of the foot, there was no difference between the TK1 group and the control group in the brightness change of the vascular phase, but the brightness in the interstitial phase increased more rapidly than the λ carrageenan-administered group, and maintained a high value throughout the evaluation time (Figure 21). One week after the administration of λ carrageenan and the inflammation subsided, there was no difference in the brightness change of the interstitial phase between the carrageenan-administered side and the tendon side (Figure 22). As the curve in Figure 21 clearly shows, the brightness in the interstitial phase changes in a smooth curve, and the height of the curve depends on the upward rate of change of the interstitial phase. In other words, the change in the value of the interstitial phase can be predicted with high precision based on time series analysis, which is an important basis for the validity of our hypothesis in this work.

No difference in brightness was observed in the ICG group either immediately or one week after injection with lambda carrageenan (Figures 25 and 26). This result indicates that the highly hydrophobic ICG, which is currently used clinically, does not leak fluorescent material outside of blood vessels even in the presence of severe acute inflammation caused by lambda carrageenan.

These results demonstrate that after TK1 administration to a living body, fluorescence intensity at sites predicted for swelling progression can be measured over time during a period corresponding to the interstitial phase. This allows for prediction of subsequent changes in fluorescence intensity, which correlates with the progression of swelling at the site. Therefore, TK1 is suitable for predicting swelling, which is not possible with ICG.

Industrial Application Possibilities

The cyclodextrin-bound indocyanine compounds represented by Chemical Formula 1 or Chemical Formula 2 of the present invention provide green pigment compounds that emit near-infrared fluorescence and have the following characteristics: higher solubility in water or physiological saline compared to ICG, easier removal from biological tissues, lower molecular association in aqueous solutions, higher near-infrared fluorescence intensity in aqueous solutions, and the absence of iodine. Furthermore, the method for synthesizing the cyclodextrin-bound indocyanine compounds of the present invention can provide for the synthesis of useful cyclodextrin-bound indocyanine compounds. Furthermore, the method for purifying the cyclodextrin-bound indocyanine compounds of the present invention can provide for the purification of useful cyclodextrin-bound indocyanine compounds. Furthermore, because the cyclodextrin-bound indocyanine compounds of the present invention exhibit sufficient solubility even without iodine, they can provide diagnostic compositions that are free of iodine, a cause of iodine allergy. These diagnostic compositions exhibit different in vivo behavior than conventional diagnostic compositions containing only ICG, and their properties can be exploited to provide a variety of useful diagnostic methods and diagnostic devices.

This patent application is a divisional application of the patent application with application number 201180006735.X, application date January 28, 2011, and invention name "New indocyanine compound, synthesis method and purification method thereof, diagnostic composition using the indocyanine compound, in vivo dynamic measurement device using the diagnostic composition, and circulation visualization device".

Technical Field

The present invention relates to a new indocyanine compound that is a green pigment useful in medical diagnosis, medical surgery, scientific measurement and analysis, printing, note-taking, coating, dyeing, and staining technologies and has the property of emitting near-infrared fluorescence, a method for synthesizing and purifying the same, and a diagnostic composition. Specifically, the invention relates to a cyclic sugar chain cyclodextrin-bound indocyanine compound that is a green pigment and has the property of emitting near-infrared fluorescence, a method for synthesizing and purifying the same, a diagnostic composition using the indocyanine compound, an in vivo dynamics measurement device using the diagnostic composition, and a circulation visualization device.

Background Art

A variety of green pigments, indocyanine compounds that emit near-infrared fluorescence, have been synthesized. These compounds have a wide range of applications in the medical field, for example, as dyes for vitreous surgery, medicinal pigments for liver function tests, medicinal pigments for circulatory function tests, surgical pigments, and near-infrared fluorescent compounds for surgery. In the scientific field, they serve as dyes or compounds for dyeing or fluorescing proteins, sugars, and other substances, and as pigments in printing technology. Among these indocyanine compounds, indocyanine green (hereinafter referred to as "ICG") has been used for nearly 50 years as a drug for liver and circulatory function tests. In recent years, ICG's high light transmittance through biological tissues has been exploited to allow for local administration of ICG to, for example, blood vessels, lymphatic vessels, brain, eyes, stomach, breasts, esophagus, skin, and other sites within the body, thereby allowing for medical procedures and diagnostics by observing its near-infrared fluorescence (Non-Patent Document 1).

Prior art literature

Non-patent literature

Non-patent literature 1: "All about ICG Fluorescence Navigation Surgery", supervised and edited by Mitsuo Kusano, インターメディカ社, (published in November 2008).

Non-Patent Document 2: URL of the Internet homepage described in the instructions for "(Trade Name) Diagnogreen Injection 25 mg" (Daiichi Sankyo Co., Ltd.) (https://www.daiichisankyo.co.jp/med/contents/di/dg2/pi/pdf/pi_dg2_0909.pdf)

Non-patent document 3: R.C. Benson, H.A. Kues, Phys. Med. Biol., 23, 159-163 (1978).

Non-patent document 4: S. Yoneyama, T. Saito, Y. Komatsu, I. Koyama, K. Takahashi, J. Duvoll-Young, IOVS, 37, 1286-1290 (1998).

Non-patent document 5: Y. Ye, W. P. Li, C. J. Anderson, J. Kao, G. V. Nikiforovich, S. Achilefu, J. Am. Chem. Soc., 125, 7766-7767 (2003).

Non-patent literature 6: K. Teranishi and S. Tanabe, ITE Letters on Batteries, New Technologies & Medicine, 1, 53-60 (2000).

Non-patent document 7: MICHAEL O’SHAUGHNESSY et al: 1994 Wiley-Liss, Inc MICROSURGERY 15: 40S412 1994.

Non-patent document 8: RALPH J.P.M et al: 1997 Wiley-Liss, Inc. MICROSURGERY 17: 402-408 1996.

Summary of the Invention

Technical issues

However, the green chromophore (the chemical structure required for the green color) and near-infrared fluorophore (the chemical structure required for near-infrared fluorescence) of ICG are hydrophobic, and sulfonyl groups are attached to the side chain terminals to make them water-soluble. Therefore, existing ICG has the following problems.

In general medical use, ICG preparations are prepared by adding approximately 5 to 10 mL of distilled water to 25 mg of ICG and stirring with vibration to dissolve it. Failure to completely dissolve ICG can cause symptoms such as nausea, retching, fever, and shock (Non-Patent Document 2). Furthermore, because ICG does not dissolve completely, it cannot be initially dissolved in other aqueous solutions such as saline (Non-Patent Document 2).

While ICG is water-soluble due to the sulfonyl groups attached to it, its chemical structure contains many hydrophobic hydrocarbon groups, making it surface-active and prone to adsorption to lipids. Consequently, when injected into tissues such as blood vessels and organs, it can sometimes adhere to non-target tissues by adhering to the injection site, leaking through the injection site, or flowing backward. ICG adhered to tissues is difficult to remove through wiping or aspiration, potentially hindering surgical procedures and medical diagnostics.

ICG has a molecular association property in aqueous solution, one of the reasons for which is its low fluorescence intensity in aqueous solution (Non-Patent Documents 3 and 4).

Furthermore, ICG becomes insoluble over time after being dissolved in water, making it difficult to store it as an aqueous solution for a long period of time. Furthermore, freezing at low temperatures promotes its insolubility.

Furthermore, there is a problem that an ICG preparation containing 5% or less of NaI may cause iodine hypersensitivity in some cases (Non-Patent Document 2).

In addition, ICG rapidly accumulates in the liver and is excreted by the liver when injected intravenously, making fluorescence imaging of other organs such as the kidneys, ureters, bladder, urethra, heart, and lungs difficult.

Furthermore, ICG migrates with the blood when injected intravenously, so its migration to peripheral tissues is minimal, making it difficult to observe its migration to the interstitium.

The present invention aims to resolve the aforementioned problems with existing ICG. Specifically, it aims to provide a new indocyanine compound that is a green pigment and emits near-infrared fluorescence. The compound is characterized by high solubility in water or physiological saline, easy removal from biological tissues, low molecular association in aqueous solution, and high near-infrared fluorescence intensity in aqueous solution, enabling fluorescence imaging of organs other than the liver, such as the kidneys, ureters, bladder, urethra, heart, and lungs. Furthermore, a chemical synthesis method and purification method for the new indocyanine compound with these characteristics are provided. Furthermore, a diagnostic composition containing the new indocyanine compound is also provided. Furthermore, a method and apparatus for visualizing the circulation of blood, lymph, urine, and other water components in a living organism is provided using these diagnostic compositions.

Technical solutions to the problem

The present inventors have conducted intensive research to solve the above problems and have discovered compounds that can emit near-infrared fluorescence. These compounds can be used in surgical procedures and medical diagnoses that utilize the properties of near-infrared fluorescence, thereby solving the above-mentioned problems with ICG.

Specifically, the present invention has been completed by the discovery of a new indocyanine compound characterized by being a green pigment that emits near-infrared fluorescence, having high solubility in water or physiological saline, being easily removed from biological tissues, exhibiting low molecular association in aqueous solutions, and exhibiting high near-infrared fluorescence intensity in aqueous solutions. Furthermore, the present invention has been completed by the discovery of a chemical synthesis method and a purification method for the new indocyanine compound. Furthermore, a diagnostic composition containing the new indocyanine compound is provided. Furthermore, a method for performing fluorescence imaging of organs other than the liver using the diagnostic composition, even during intravenous administration, is provided. Furthermore, a method for evaluating the in vivo dynamics of the new indocyanine compound related to, for example, water balance in a living body and a device for measuring the in vivo dynamics are provided, as well as a method and device for visualizing the circulation of blood, lymph, urine, and other water in a living body.

The first new indocyanine compound of the present invention is a compound in which a cyclic sugar chain cyclodextrin is covalently bound to the green chromophore (chemical structure required for green color) and the near-infrared fluorophore (chemical structure required for emitting near-infrared fluorescence) of ICG. In addition, the second new indocyanine compound of the present invention is characterized in that the naphthyl site belonging to the hydrophobic site of the indocyanine structure is included in the cavity of the cyclodextrin, thereby covering the naphthyl site belonging to the hydrophobic site with a hydrophilic glucose group, so that most areas of the indocyanine molecular structure become three-dimensionally hydrophilic. Therefore, compared with the characteristic of ICG having similar surface activity due to the hydrophilicity of both the hydrophobic site and the sulfonyl group, the compound of the present invention is characterized in that the hydrophobic site in its molecule is covered by cyclodextrin, so it does not have similar surface activity properties. Specifically, the present invention is as follows:

<1> A cyclodextrin-bound indocyanine compound represented by Chemical Formula 1, characterized in that the indocyanine is covalently bound to a cyclic sugar chain cyclodextrin;

[Chemical Formula 1]

(R ₁ , R ₂ , R ₃ , R ₄ , R ₅ , R ₆ , R ₇ , R ₈ , R ₉ , R ₁₀ , R ₁₁ , R ₁₂ , R ₁₃ , R 14 , R ₁₅ , R ₁₆ , R ₁₇ , R ₁₈ , R ₁₉ , R ₂₀ , R _in the formula ₂₁ , R ₂₂ , R _R23 is a hydrogen atom, an alkyl group, an aryl group, a halogen atom, an alkoxy group, an amino group, a carboxyl group, a formyl group, a sulfonyl group, a sulfonic acid group, a phosphate group, an alkoxycarbonyl group, an aryloxycarbonyl group, an alkylcarbonyl group, an arylcarbonyl group, or a heterocycle. Alternatively, it may be a substituent thereof (carboxylic acid, sulfonic acid, phosphoric acid). When the hydrogen ion in the substituent is dissociated, the hydrogen ion may be replaced by a metal ion such as sodium ion, potassium ion, magnesium ion, or calcium ion. The amino group may be selected from primary, secondary, tertiary, and quaternary groups ₍ alkyl groups may be exemplified as substituents bonded to nitrogen). Alternatively, _R8 and _R9 may _be selected _{from CH2} , _{CH2CH2 , CH2CH2CH2} , or _{CH2CH2CH2CH2 . 2.} Alternatively, the hydrogen atoms on these alkyl groups may be replaced by alkyl groups, aryl groups, halogen atoms, alkoxy groups, amino groups, carboxyl groups, formyl groups, sulfonyl groups, sulfonic acid groups, phosphoric acid groups, alkoxycarbonyl groups, aryloxycarbonyl groups, alkylcarbonyl groups, arylcarbonyl groups or heterocyclic groups.)

<2> The cyclodextrin-bound indocyanine compound represented by Chemical Formula 2 is characterized in that at least a portion of the naphthyl group of the indocyanine is included in the cavity of the cyclodextrin.

[Chemical Formula 2]

(R ₁ , R ₂ , R ₃ , R ₄ , R ₅ , R ₆ , R ₇ , R ₈ , R ₉ , R ₁₀ , R ₁₁ , R ₁₂ , R ₁₃ , R 14 , R ₁₅ , R ₁₆ , R ₁₇ , R ₁₈ , R ₁₉ , R ₂₀ , R _in the formula ₂₁ , R ₂₂ , R ₂₃ is a hydrogen atom, an alkyl group, an aryl group, a halogen atom, an alkoxy group, an amino group, a carboxyl group, a formyl group, a sulfonyl group, a sulfonic acid group, a phosphate group, an alkoxycarbonyl group, an aryloxycarbonyl group, an alkylcarbonyl group, an arylcarbonyl group, or a heterocycle. Alternatively, it may be a substituent thereof (carboxylic acid, sulfonic acid, phosphate). When the hydrogen ion in the substituent is dissociated, the hydrogen ion may be replaced by a metal ion such as a sodium ion, potassium ion, magnesium ion, or calcium ion. The amino group is selected from primary, secondary, tertiary, and quaternary groups. Furthermore, R ₈ and R ₉ may be selected from a cyclic structure of CH ₂ , CH ₂ CH ₂ , CH ₂ CH ₂ CH ₂ , or CH ₂ CH ₂ CH ₂ CH ₂ . Alternatively, a functional group in which the hydrogen atom on these alkyl groups is replaced by an alkyl group, an aryl group, a halogen atom, an alkoxy group, an amino group, a carboxyl group, a formyl group, a sulfonyl group, a sulfonic acid group, a phosphate group, an alkoxycarbonyl group, an aryloxycarbonyl group, an alkylcarbonyl group, an arylcarbonyl group, or a heterocycle may be selected.)

<3> The cyclodextrin-bound indocyanine compound represented by Chemical Formula 3 is characterized in that it is the cyclodextrin-bound indocyanine compound represented by Chemical Formula 1 in which an indocyanine and a cyclic sugar chain cyclodextrin are covalently bound via an amide bond.

[Chemical Formula 3]

(R ₁ , R ₂ , R ₃ , R ₄ , R ₅ , R ₆ , R ₇ , R ₈ , R ₉ , R ₁₀ , R ₁₁ , R ₁₂ , R ₁₃ , R 14 , R ₁₅ , R ₁₆ , R ₁₇ , R ₁₈ , R ₁₉ , R ₂₀ , R _in the formula ₂₁ , R ₂₂ , R _R23 is a hydrogen atom, an alkyl group, an aryl group, a halogen atom, an alkoxy group, an amino group, a carboxyl group, a formyl group, a sulfonyl group, a sulfonic acid group, a phosphate group, an alkoxycarbonyl group, an aryloxycarbonyl group, an alkylcarbonyl group, an arylcarbonyl group, or a heterocycle. Alternatively, it may be a substituent thereof (carboxylic acid, sulfonic acid, phosphoric acid), wherein the hydrogen ion in the substituent, when dissociated, may be replaced by a metal ion such as sodium ion, potassium ion, magnesium ion, or calcium ion. The amino group is selected from primary, secondary, tertiary, and _quaternary groups. m and n are integers from 1 to 6. Furthermore, _R8 and _R9 may _{be selected from CH2} , _CH2CH2 , _CH2CH2CH2 , or _{CH2CH2CH2CH2 . 2.} In addition, the functional groups in which the hydrogen atoms on these alkyl groups are replaced by alkyl groups, aryl groups, halogen atoms, alkoxy groups, amino groups, carboxyl groups, formyl groups, sulfonyl groups, sulfonic acid groups, phosphoric acid groups, alkoxycarbonyl groups, aryloxycarbonyl groups, alkylcarbonyl groups, arylcarbonyl groups or heterocyclic rings can also be selected.)

<4> Chemical formula 4 represents a cyclodextrin-bound indocyanine compound, characterized in that it is a cyclodextrin-bound indocyanine compound represented by Chemical formula 2, in which indocyanines and cyclic sugar chain cyclodextrin are covalently bound via an amide bond.

[Chemical Formula 4]

(R ₁ , R ₂ , R ₃ , R ₄ , R ₅ , R ₆ , R ₇ , R ₈ , R ₉ , R ₁₀ , R ₁₁ , R ₁₂ , R ₁₃ , R 14 , R ₁₅ , R ₁₆ , R ₁₇ , R ₁₈ , R ₁₉ , R ₂₀ , R _in the formula ₂₁ , R ₂₂ , R _R23 is a hydrogen atom, an alkyl group, an aryl group, a halogen atom, an alkoxy group, an amino group, a carboxyl group, a formyl group, a sulfonyl group, a sulfonic acid group, a phosphate group, an alkoxycarbonyl group, an aryloxycarbonyl group, an alkylcarbonyl group, an arylcarbonyl group, or a heterocycle. Alternatively, it may be a substituent thereof (carboxylic acid, sulfonic acid, phosphoric acid), wherein the substituent can be replaced by a metal ion such as sodium ion, potassium ion, magnesium ion, or calcium ion when the hydrogen ion dissociates. The amino group is selected from primary, secondary, tertiary, and quaternary groups. _m and n are integers from 1 to 6. Furthermore, _R8 and _R9 may _be selected _{from CH2} , _CH2CH2 , _{CH2CH2CH2 ,} or _{CH2CH2CH2CH2 . 2.} In addition, the functional groups in which the hydrogen atoms on these alkyl groups are replaced by alkyl groups, aryl groups, halogen atoms, alkoxy groups, amino groups, carboxyl groups, formyl groups, sulfonyl groups, sulfonic acid groups, phosphoric acid groups, alkoxycarbonyl groups, aryloxycarbonyl groups, alkylcarbonyl groups, arylcarbonyl groups or heterocyclic rings can also be selected.)

<5> The cyclodextrin-bound indocyanine compound represented by Chemical Formula 5 among the cyclodextrin-bound indocyanine compounds represented by Chemical Formula 3.

[Chemical Formula 5]

(In the formula, m, n, p, and q are integers of 2 to 6. r is an integer of 5 to 7. s is an integer of 0 to 4. R is a hydrogen atom, an alkyl group, an aryl group, a halogen atom, an alkoxy group, an amino group, a carboxyl group, a formyl group, a sulfonyl group, a sulfonic acid group, an alkoxycarbonyl group, an aryloxycarbonyl group, an alkylcarbonyl group, an arylcarbonyl group, or a heterocycle.)

<6> The cyclodextrin-bound indocyanine compound represented by Chemical Formula 6 among the cyclodextrin-bound indocyanine compounds represented by Chemical Formula 4.

[Chemical Formula 6]

(In the formula, m, n, p, and q are integers of 2 to 6. r is an integer of 5 to 7. s is an integer of 0 to 4. R is a hydrogen atom, an alkyl group, an aryl group, a halogen atom, an alkoxy group, an amino group, a carboxyl group, a formyl group, a sulfonyl group, a sulfonic acid group, an alkoxycarbonyl group, an aryloxycarbonyl group, an alkylcarbonyl group, an arylcarbonyl group, or a heterocycle.)

<7> The cyclodextrin-bound indocyanine compound represented by Chemical Formula 7 among the cyclodextrin-bound indocyanine compounds represented by Chemical Formula 3.

[Chemical Formula 7]

(In the formula, m and n are integers of 2 to 6. r is an integer of 5 to 7. s is an integer of 0 to 4. R is a hydrogen atom, an alkyl group, an aryl group, a halogen atom, an alkoxy group, an amino group, a carboxyl group, a formyl group, a sulfonyl group, a sulfonic acid group, an alkoxycarbonyl group, an aryloxycarbonyl group, an alkylcarbonyl group, an arylcarbonyl group, or a heterocycle.)

<8> The cyclodextrin-bound indocyanine compound represented by Chemical Formula 8 among the cyclodextrin-bound indocyanine compounds represented by Chemical Formula 4.

[Chemical Formula 8]

(In the formula, m and n are integers of 2 to 6. r is an integer of 5 to 7. s is an integer of 0 to 4. R is a hydrogen atom, an alkyl group, an aryl group, a halogen atom, an alkoxy group, an amino group, a carboxyl group, a formyl group, a sulfonyl group, a sulfonic acid group, an alkoxycarbonyl group, an aryloxycarbonyl group, an alkylcarbonyl group, an arylcarbonyl group, or a heterocycle.)

<9> The cyclodextrin-bound indocyanine compound represented by Chemical Formula 9 among the cyclodextrin-bound indocyanine compounds represented by Chemical Formula 3.

[Chemical Formula 9]

(In the formula, m and n are integers of 2 to 6. r is an integer of 5 to 7. s is an integer of 0 to 4. R is a hydrogen atom, an alkyl group, an aryl group, a halogen atom, an alkoxy group, an amino group, a carboxyl group, a formyl group, a sulfonyl group, a sulfonic acid group, an alkoxycarbonyl group, an aryloxycarbonyl group, an alkylcarbonyl group, an arylcarbonyl group, or a heterocycle.)

<10> The cyclodextrin-bound indocyanine compound represented by Chemical Formula 10 among the cyclodextrin-bound indocyanine compounds represented by Chemical Formula 4.

[Chemical Formula 10]

(In the formula, m and n are integers of 2 to 6. r is an integer of 5 to 7. s is an integer of 0 to 4. R is a hydrogen atom, an alkyl group, an aryl group, a halogen atom, an alkoxy group, an amino group, a carboxyl group, a formyl group, a sulfonyl group, a sulfonic acid group, an alkoxycarbonyl group, an aryloxycarbonyl group, an alkylcarbonyl group, an arylcarbonyl group, or a heterocycle.)

<11> The cyclodextrin-bound indocyanine compound represented by Chemical Formula 11 among the cyclodextrin-bound indocyanine compounds represented by Chemical Formula 3.

[Chemical Formula 11]

(s is an integer from 0 to 4. R is a hydrogen atom, an alkyl group, an aryl group, a halogen atom, an alkoxy group, an amino group, a carboxyl group, a formyl group, a sulfonyl group, a sulfonic acid group, an alkoxycarbonyl group, an aryloxycarbonyl group, an alkylcarbonyl group, an arylcarbonyl group, or a heterocyclic ring.)

<12> The cyclodextrin-bound indocyanine compound represented by Chemical Formula 12 among the cyclodextrin-bound indocyanine compounds represented by Chemical Formula 4.

[Chemical Formula 12]

(s is an integer from 0 to 4. R is a hydrogen atom, an alkyl group, an aryl group, a halogen atom, an alkoxy group, an amino group, a carboxyl group, a formyl group, a sulfonyl group, a sulfonic acid group, an alkoxycarbonyl group, an aryloxycarbonyl group, an alkylcarbonyl group, an arylcarbonyl group, or a heterocyclic ring.)

<13> The chemical synthesis method of the cyclodextrin-conjugated indocyanine compound described in any one of the above chemical formulas (Chemical Formulas 1, 3, 5, 7, 9, and 11) is characterized by comprising: (1) a step of mixing an indocyanine carboxylic acid compound and an aminocyclodextrin in a solvent; and (2) a step of adding a dehydration condensation agent to carry out a dehydration condensation reaction.

<14> A chemical synthesis method of a cyclodextrin-bound indocyanine compound according to any one of the above chemical formulas (Chemical Formulas 2, 4, 6, 8, 10, 12), characterized in that the cyclodextrin-bound indocyanine compound according to any one of the above chemical formulas (Chemical Formulas 1, 3, 5, 7, 9, 11) is subjected to an inclusion reaction in water.

<15> The cyclodextrin-bound indocyanine compound represented by any one of the above chemical formulae (Chemical Formulae 1 to 12) is purified by column chromatography in which the compound is eluted with a solvent containing HCl.

<16> A diagnostic composition to be used by injection into the body, comprising an aqueous solution containing the cyclodextrin-bound indocyanine compound of any one of the above Chemical Formulas 1 to 12.

<17> The diagnostic composition according to <16>, which contains substantially no iodine.

<18> A device for measuring the dynamics of a cyclodextrin-bound indocyanine compound in vivo, comprising:

an excitation light irradiation unit for irradiating the cyclodextrin-bound indocyanine compound with excitation light, with respect to a part of a living body to which the diagnostic composition according to <16> or <17> is administered;

a fluorescence intensity measuring unit for measuring the intensity of fluorescence emitted by the cyclodextrin-bound indocyanine compound excited by the excitation light irradiation unit; and

The in vivo dynamic calculation unit calculates the temporal change of the time rate of change of the fluorescence intensity from the fluorescence intensity obtained over time from the fluorescence intensity measuring unit, thereby calculating the speed of the cyclodextrin-bound indocyanine compound moving into the interstitial fluid and/or the speed of moving out of the interstitium in a part of the biological body.

<19> A circulation visualization device, comprising:

an excitation light irradiation unit for irradiating the cyclodextrin-bound indocyanine compound with excitation light, with respect to a part of a living body to which the diagnostic composition according to <16> or <17> is administered;

a fluorescence imaging unit for two-dimensionally acquiring the intensity of the fluorescence emitted by the cyclodextrin-bound indocyanine compound excited by the excitation light irradiation unit, thereby obtaining data on the distribution state of the cyclodextrin-bound indocyanine compound in the organism;

a morphological imaging unit that two-dimensionally acquires the intensity of light of wavelengths other than the fluorescence wavelength emitted by the cyclodextrin-bound indocyanine compound, thereby obtaining morphological data of a portion of the biological body; and

The display unit displays the distribution state of the cyclodextrin-bound indocyanine compound in a part of the biological body by superimposing the morphological data obtained by the morphological imaging unit and the distribution state data obtained by the fluorescence imaging unit.

<20> The circulation visualization device according to <19>, wherein the display unit displays a portion of the living body where the distribution amount of the cyclodextrin-bound indocyanine compound is lower than a predetermined reference as a necrotic portion.

<21> The in vivo dynamics measuring device according to <18> above, comprising a swelling progression prediction unit for predicting the degree of swelling of the part of the living body based on the speed of the swelling moving from the part of the living body into and out of the interstitial fluid.

<22> The in vivo dynamic measurement device described in <21> above, wherein the swelling progression prediction unit predicts the degree of swelling progression corresponding to the magnitude of the migration speed into the interstitial fluid after a predetermined time, wherein the predetermined time is the time from the administration of the diagnostic composition to the dispersion of the cyclodextrin-conjugated indocyanine compound in the blood throughout the body.

<23> An in vivo dynamics measurement device, characterized by comprising:

The excitation light irradiation unit irradiates the cyclodextrin-bound indocyanine compound with excitation light at a portion of the living body to which the diagnostic composition according to <16> or <17> is administered and at a control site.

a fluorescence intensity measuring unit for measuring the intensity of fluorescence emitted by the cyclodextrin-bound indocyanine compound excited by the excitation light irradiation unit;

an in vivo dynamic calculation unit for calculating the time-dependent change in the fluorescence intensity obtained from the fluorescence intensity measurement unit over time, thereby calculating the migration speed of the cyclodextrin-bound indocyanine compound into the interstitial fluid and/or the migration speed out of the interstitial fluid in a part of the organism, and

a swelling progression prediction unit for predicting a degree of swelling that will progress in the part of the living body based on a rate of movement of the part of the living body into and out of the interstitial fluid;

wherein the above swelling is carried out in a prediction unit,

determining the relationship between the blood flow in the control site and the blood flow in the portion of the living body based on changes in fluorescence intensity in the portion of the living body and changes in fluorescence intensity in the control site until a predetermined time has passed, wherein the predetermined time is the time from administration of the diagnostic composition to dispersion of the cyclodextrin-bound indocyanine compound into blood throughout the body;

The degree of change in the fluorescence intensity of the part of the biological body after the predetermined time has passed, with the change in the fluorescence intensity of the control part serving as a control, is calculated using this relationship.

The progression of the swelling is predicted based on the calculated degree of change.

<24> An in vivo dynamics measurement device, characterized by comprising:

an excitation light irradiation unit for irradiating the cyclodextrin-bound indocyanine compound with excitation light to a living organ to which the diagnostic composition according to <16> or <17> is administered;

a fluorescence intensity measuring unit for measuring the intensity of fluorescence emitted by the cyclodextrin-bound indocyanine compound excited by the excitation light irradiation unit; and

The in vivo dynamics calculation unit evaluates the in vivo dynamics of the cyclodextrin-bound indocyanine compound in the organ based on the fluorescence intensity acquired over time by the fluorescence intensity measurement unit.

<25> The in vivo dynamics measuring device according to <24>, wherein the organ is any one of a kidney, a ureter, a bladder, and a urethra.

Beneficial effects

The cyclodextrin-bound indocyanine compounds represented by Chemical Formula 1 or Chemical Formula 2 of the present invention provide green pigment compounds that emit near-infrared fluorescence and have the following characteristics: higher solubility in water or physiological saline compared to ICG, easier removal from biological tissues, lower molecular association in aqueous solutions, higher near-infrared fluorescence intensity in aqueous solutions, and the ability to perform fluorescence imaging of organs other than the liver. Furthermore, the cyclodextrin-bound indocyanine compound synthesis method of the present invention provides for the synthesis of useful cyclodextrin-bound indocyanine compounds. Furthermore, the cyclodextrin-bound indocyanine compound purification method of the present invention provides for the purification of useful cyclodextrin-bound indocyanine compounds. Furthermore, the cyclodextrin-bound indocyanine compounds of the present invention exhibit sufficient solubility even without iodine, thus providing diagnostic compositions that do not contain iodine, a cause of iodine allergy. These diagnostic compositions exhibit different in vivo behavior than conventional diagnostic compositions containing only ICG, and their properties can be exploited to provide various beneficial devices.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the results of a test on the adsorption of ICG and the isomeric equilibrium compound (TK1) represented by Chemical Formulas 19 and 20 to human skin. The images show the results of a 1 mM aqueous solution (0.03 mL) of ICG and the isomeric equilibrium compound (TK1) represented by Chemical Formulas 19 and 20 applied to the wrist and ingested immediately (left photo), 5 minutes later and immediately after rinsing with water (center photo), and then immediately after wiping and rinsing with water (right photo). The dots on the right are marked with a red pen.

Figure 2 shows the results of a test on the adsorption of ICG and the isomeric equilibrium compound (TK1) represented by Chemical Formulas 19 and 20 onto cellulose fibers. The images show the results of a cotton swab application of a 1 mM aqueous solution (0.05 mL) of ICG and the isomeric equilibrium compound (TK1) represented by Chemical Formulas 19 and 20, followed by immediate uptake (top photo), and then 3 minutes later, followed by 5 seconds of rinsing with water, followed by immediate uptake (bottom photo).

Figure 3 shows the results of a test on the adsorption of ICG and the isomeric equilibrium compound (TK1) represented by Chemical Formulas 19 and 20 into a biological meat model. The images show a 1 mM aqueous solution (0.05 mL) of ICG and the compound represented by Chemical Formula 20 applied to a 5 mm diameter depression in pork tenderloin immediately after ingestion (left photo), and then rinsed with water for 10 seconds 3 minutes later, followed by immediate ingestion (right photo).

Figure 4 shows the results of a test on the adsorption of ICG and the isomeric equilibrium compound (TK1) represented by Chemical Formulas 19 and 20 onto a biological protein model. The images show a 1 mM aqueous solution (0.05 mL) of ICG and the isomeric equilibrium compound (TK1) represented by Chemical Formulas 19 and 20 applied to a 5 mm diameter depression in chicken breast immediately after ingestion (left photo), and then rinsed with water for 10 seconds 3 minutes later, followed by immediate ingestion (right photo).

Figure 5 shows the test results of the adsorption of ICG and the isomeric equilibrium compound (TK1) represented by Chemical Formulas 19 and 20 onto hydrophobic chemical fibers. The images show a 1 mM aqueous solution (0.05 mL) of ICG and the isomeric equilibrium compound (TK-1) represented by Chemical Formulas 19 and 20 applied to a polypropylene mask, immediately after application (left photo), and then after 20 minutes of rinsing with water for 1 second and immediately after application (right photo).

Figure 6 shows the results of molecular association tests of ICG and the isomerization equilibrium compound (TK1) represented by Chemical Formulas 19 and 20. The left figure shows ICG, and the right figure shows the isomerization equilibrium compound (TK1) represented by Chemical Formulas 19 and 20.

7 is a graph showing the concentration dependency of the fluorescence intensity of ICG, the isomerization equilibrium compound (TK1) represented by Chemical Formulas 19 and 20, and the isomerization equilibrium compound (TK2) represented by Chemical Formulas 15 and 16 in human venous blood.

FIG8 is a graph showing the conditions observed when ICG, the isomerization-equilibrium compound represented by Chemical Formulas 19 and 20 (TK1), and the isomerization-equilibrium compound represented by Chemical Formulas 15 and 16 (TK2) were administered to rats.

FIG9 is a graph showing fluorescence in the peritoneal cavity of rats when ICG, the isomerization-equilibrium compound represented by Chemical Formulas 19 and 20 (TK1), and the isomerization-equilibrium compound represented by Chemical Formulas 15 and 16 (TK2) were administered.

Figure 10 shows images of male Wistar rats (14 weeks old, 300 g) administered 0.075 mL of a 0.1 mM aqueous solution of the isomerized equilibrium compounds (TK2) represented by Chemical Formulas 15 and 16 via the tail vein, followed by imaging using a near-infrared observation system PDE manufactured by Hamamatsu Photonics Co., Ltd. The left image is a black-and-white image using visible light, and the right image is a fluorescent image.

Figure 11 shows near-infrared fluorescence endoscopic images of male Wistar rats (14 weeks old, 300 g) after 0.075 mL of a 0.1 mM aqueous solution of the isomerized equilibrium compounds (TK2) represented by Chemical Formulas 15 and 16 was administered via the tail vein. The left image is a black-and-white image using visible light, and the right image is a fluorescence image.

FIG12 is a graph showing fluorescence at the dorsum of the foot of rats when ICG, the isomerized equilibrium compound (TK1) represented by Chemical Formulas 19 and 20, and the isomerized equilibrium compound (TK2) represented by Chemical Formulas 15 and 16 were administered.

13 is a graph showing the temporal changes in fluorescence intensity at the dorsum of the foot when ICG, the isomerization-equilibrium compound represented by Chemical Formulas 19 and 20 (TK1), and the isomerization-equilibrium compound represented by Chemical Formulas 15 and 16 (TK2) were administered to rats.

14 is a graph showing the temporal changes in fluorescence intensity at the dorsum of the foot when ICG, the isomerization-equilibrium compound represented by Chemical Formulas 19 and 20 (TK1), and the isomerization-equilibrium compound represented by Chemical Formulas 15 and 16 (TK2) were administered to rats.

FIG. 15 is a diagram showing a technique for direct observation of rat blood vessels.

FIG. 16 is a diagram showing a directly observed portion of a rat's blood vessels (spermatozoa cremaster flap).

FIG. 17 is a graph showing the state of fluorescence in blood vessels and surrounding tissues of rats to which ICG was administered.

FIG. 18 is a graph showing fluorescence in blood vessels and surrounding tissues of rats after administration of the isomerization-equilibrated compound (TK1) represented by Chemical Formulas 19 and 20. FIG.

Figure 19 is a graph showing the left paw volume of rats measured immediately after injection (post-injection) and one week after injection (1 week after) for the carrageenan-administered group (edema) and the non-administered group (normal) in the TK1 test. The vertical axis represents volume (mL).

Figure 20 is a graph showing the results of the Von Frey test on the left paw of rats in the TK1 test group, the group that received carrageenan (edema) and the group that did not receive carrageenan (normal), measured immediately after injection and one week after injection. The vertical axis represents the load (g) at which the reaction occurred.

Figure 21 is a graph showing changes in brightness of the left paw surface after TK1 injection immediately after carrageenan injection for a group administered carrageenan (edema) and a group not administered carrageenan (normal). The vertical axis represents brightness (arbitrary unit) and the horizontal axis represents time (seconds).

Figure 22 is a graph showing changes in brightness of the left paw surface one week after carrageenan injection and TK1 injection for a group that received carrageenan (edema) and a group that did not receive carrageenan (normal). The vertical axis represents brightness (arbitrary unit) and the horizontal axis represents time (seconds).

Figure 23 is a graph showing the left paw volume of rats measured immediately after injection (post-injection) and one week after (1 week) for the ICG test group, the group administered carrageenan (edema), and the group not administered (normal). The vertical axis represents volume (mL).

Figure 24 is a graph showing the results of the Von Frey test on the left paw of rats immediately after injection and one week after injection, for groups tested with ICG, those administered carrageenan (edema), and those not administered (normal). The vertical axis represents the load (g) at which the reaction occurred.

Figure 25 is a graph showing the brightness of the left paw surface immediately after ICG injection for a group that received carrageenan (edema) and a group that did not receive carrageenan (normal). The vertical axis represents brightness (arbitrary unit) and the horizontal axis represents time (seconds).

Figure 26 is a graph showing changes in brightness of the left foot surface after ICG injection one week after carrageenan injection in a group that received carrageenan (edema) and a group that did not receive carrageenan (normal). The vertical axis represents brightness (arbitrary units) and the horizontal axis represents time (seconds).

DETAILED DESCRIPTION

The diagnostic composition of the present invention utilizes the cyclodextrin-conjugated indocyanine compound described below as a pigment, making it iodine-free and therefore suitable for diagnostic applications. This diagnostic composition can replace the conventionally used indocyanine green-containing diagnostic compositions. For example, it can be used as a drug for liver function tests or circulatory function tests. Furthermore, by observing the near-infrared fluorescence generated by administration to, for example, blood vessels, lymphatic vessels, brain, eyes, stomach, breast, esophagus, skin, kidneys, ureters, bladder, urethra, lungs, heart, or other internal organs, it can be applied to medical procedures and diagnostics. The pigment contained in the diagnostic composition of the present invention has low binding to the organism, allowing for long-term labeling of desired areas. The diagnostic composition may also contain salts or other additives as isotonic agents, as needed. In addition to pre-prepared aqueous solutions, it can also be used in a form that is dissolved. The diagnostic composition can be administered by injection, intravenous drip, application, or oral administration.

This diagnostic composition is suitable for use in visualizing the circulation of aqueous solutions such as blood, lymph, interstitial water, and urine. By visualizing the circulation of blood and other fluids, for example, it can be used to assess necrosis in the peripheral circulation, evaluate tissue survival after revascularization surgery and transplantation, and diagnose poor blood flow. Furthermore, it can also be used for fundus angiography, which has been previously visualized, cerebral circulation assessment, intraoperative angiography during neurosurgery, sentinel lymph node identification in cancers (such as breast cancer, esophageal cancer, gastric cancer, colorectal cancer, prostate cancer, and skin cancer), evaluation of lymphedema, intraoperative cholangiography, tumor labeling, coronary angiography, and abdominal angiography (hepatic artery, abdominal aorta, digestive tract blood flow, etc.). Fluorescent imaging of the renal excretory system, including the kidneys, ureters, bladder, and urethra, is also possible.

<1. Non-inclusion cyclodextrin-bound indocyanine compounds and their synthesis>

Examples of the non-inclusion type cyclodextrin-bound indocyanine compounds of the present invention include Chemical Formula 1, Chemical Formula 3, Chemical Formula 5, Chemical Formula 7, Chemical Formula 9, and Chemical Formula 11. Their synthesis is accomplished by reacting an indocyanine compound with a cyclodextrin compound in a solution.

In Chemical Formulas 1 and 3, considering the inclusion of a significant portion of the cyclodextrin, _R1 - _R4 and _R13 - _R16 are preferably relatively bulky groups that do not hinder cyclodextrin inclusion. For example, hydrogen or an alkyl or alkoxy group with approximately 1 to 3 carbon atoms. Preferred are hydrogen, methyl, and methoxy groups, with hydrogen being most preferred. For cyclodextrin inclusion, _R5 , _R6 , _R11 , and _R12 are also preferably relatively bulky, but not as bulky as _R1 - _R4 and _R13 - _R16 . For example, hydrogen or an alkyl or alkoxy group with approximately 1 to 6 carbon atoms. _R1 - _R6 and _R11 - _R16 are sites of cyclodextrin inclusion, so even when a hydrophilic functional group is introduced, the entire group is preferably hydrophobic. _R17 , _R18 , _R22 , and _R23 do not significantly affect cyclodextrin inclusion and are not particularly limited as long as they are the substituents listed above. R ₇ , R ₁₀ , R ₁₉ and R ₂₁ are preferably hydrogen from the viewpoint of ease of synthesis. R ₈ , R ₉ and R ₂₀ are not particularly limited as long as they are the substituents described above.

In the case where the non-inclusion type cyclodextrin-bound indocyanine compound of the present invention is covalently bound to the indocyanine compound and the cyclodextrin compound via an amide bond, the synthesis method of the non-inclusion type cyclodextrin-bound indocyanine compound can be achieved by allowing an indocyanine carboxylic acid compound and an aminocyclodextrin compound to undergo a dehydration condensation reaction in a solution.

<2. Inclusion-type cyclodextrin-bound indocyanine compounds>

The cyclodextrin-bound indocyanine compound of the present invention is characterized by being a cyclodextrin-bound indocyanine compound covalently bound to a cyclic sugar chain cyclodextrin represented by Chemical Formula 2, wherein at least a portion of the naphthyl group of the indocyanine is enclosed within the cyclodextrin cavity. Furthermore, as long as the naphthyl group of the indocyanine is enclosed within the cyclodextrin cavity and emits near-infrared fluorescence, the indocyanine group may have a substituent. While there are many types of cyclodextrins, enclosed naphthyl group of the indocyanine is a necessary condition. Examples include α-cyclodextrin, β-cyclodextrin, and γ-cyclodextrin. β-cyclodextrin is preferred. The cyclodextrin may also have a substituent.

In Chemical Formulas 2 and 4, _R1 - _R4 and _R13 - _R16 are preferably small groups that do not hinder cyclodextrin inclusion. Examples include hydrogen, alkyl or alkoxy groups with approximately 1 to 3 carbon atoms. Hydrogen, methyl, and methoxy groups are particularly preferred, with hydrogen being most preferred. For cyclodextrin inclusion, _R5 , _R6 , _R11 , and _R12 , while not as large as _R1 - _R4 and _R13 - _R16 , are also small. Examples include hydrogen, alkyl or alkoxy groups with approximately 1 to 6 carbon atoms. _R1 - _R6 and _R11 - _R16 are sites of cyclodextrin inclusion, so even when introducing hydrophilic functional groups, the entire group is preferably hydrophobic. _R17 , _R18 , _R22 , and _R23 do not significantly affect cyclodextrin inclusion and are not particularly limited as long as they are the substituents listed above. R ₇ , R ₁₀ , R ₁₉ , and R ₂₁ are preferably hydrogen from the perspective of ease of synthesis. R ₈ , R ₉ , and R ₂₀ are not particularly limited as long as they are the substituents described above. The bond between the indocyanine group and the cyclodextrin is not particularly limited as long as it is covalent, and examples thereof include an alkyl bond, an amino bond, an amide bond, a double bond, a triple bond, an ester bond, and an ether bond. However, in chemical synthesis, an amide bond is preferred from the perspective of efficiency.

In order to include at least a portion of the naphthyl groups of the indocyanine in the cyclodextrin cavity, a spacer is preferably used to covalently bond the indocyanine and the cyclic sugar chain cyclodextrin via the spacer. In this case, the extent of inclusion of the naphthyl groups of the indocyanine in the cyclodextrin cavity can be controlled by adjusting the length of the spacer in Chemical Formula 2.

Therefore, the cyclodextrin-bound indocyanine compound formed by covalent bonding of indocyanine and cyclic sugar chain cyclodextrin via a spacer, and the compound characterized by the inclusion of the naphthyl group of indocyanine in the cavity of cyclodextrin, preferably exemplified by Chemical Formula 4. In addition, the substances of Chemical Formula 6, Chemical Formula 8, and Chemical Formula 10 are preferably exemplified. In addition, the substance of Chemical Formula 12 is preferably exemplified. In Chemical Formula 6, m, n, p, and q are preferably m+p and n+q are 5 or more and 7 or less, respectively. From the perspective of easy cyclodextrin inclusion, in all chemical formulas (Chemical Formulas 1 to 10), the number of atoms in the structure (spacer) between the nitrogen atom in the indocyanine structure and the oxygen atom in the cyclodextrin structure is preferably 7 or more and 9 or less.

<3. Synthesis of Inclusion Cyclodextrin-Conjugated Indocyanine Compounds>

The inclusion-type cyclodextrin-bound indocyanine compound of the present invention is as described above, and its synthesis method is to use the non-inclusion-type cyclodextrin-bound indocyanine compound synthesized above as a synthesis precursor and dissolve the compound in an aqueous solution. In the aqueous solution, any substance can be contained as long as it does not hinder inclusion, and the water content is not particularly limited. In addition, the temperature suitable for inclusion is -20°C to 100°C, preferably 0°C to 50°C. In addition, the time required for inclusion is from immediately after addition to the aqueous solution to about 1 month. The inclusion reaction can be in various forms depending on the properties of the non-inclusion-type cyclodextrin-bound indocyanine compound as described above, the temperature of the inclusion reaction, the composition and concentration of the aqueous solution, etc.

Alternatively, the non-inclusion-type cyclodextrin-bound indocyanine compound of the present invention can be converted to an inclusion-type cyclodextrin-bound indocyanine compound by dissolving it in a solvent containing water. While there are no particular restrictions on the water content in the solvent, in principle, inclusion is facilitated when the water content is high, preferably 50% by mass or greater. Furthermore, as long as a substance capable of forming an inclusion-type cyclodextrin-bound indocyanine compound is present in a solvent other than an aqueous solution, inclusion is not necessarily required in an aqueous solution.

In addition, an inclusion-type cyclodextrin-bound indocyanine compound may be formed during the synthesis of a non-inclusion-type cyclodextrin-bound indocyanine compound. In this case, there is no need to conduct the inclusion reaction again.

<4. Synthesis of Non-Inclusion Cyclodextrin-Bound Indocyanine Compounds (Synthesis Example)>

The synthesis of the compound represented by Chemical Formula 11 is given as an example. The indocyanine compound represented by Chemical Formula 11 can be obtained by, for example, adding the compound represented by Chemical Formula 13 synthesized by the method described in Non-Patent Document 5, Chemical Formula 14 synthesized by the method described in Non-Patent Document 6, a water-soluble carbodiimide (WSC: for example, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride) or dicyclohexylcarbodiimide (DCC) as a dehydration condensation agent, and pyridine or N,N-dimethylformamide or an aqueous solution as a solvent, and reacting at -20°C to 60°C for 10 minutes to 100 hours. In addition, in order to activate the reaction, 1-hydroxybenzotriazole (HOBt) can be added as an activator, for example. The amount of the dehydration condensation agent is 2 times the mole of the compound represented by Chemical Formula 13 or more. The solvent used is not limited as long as it can dissolve the reactants and does not hinder the dehydration condensation reaction. There is no limitation on the activator as long as it can activate the dehydration condensation reaction, and there is no limitation on the amount of the activator added as long as it can cause the dehydration condensation reaction to proceed to a desired extent.

[Chemical Formula 13]

[Chemical Formula 14]

<5. Purification of Non-Inclusion Cyclodextrin-Bound Indocyanine Compounds>

The mixture containing the non-inclusion cyclodextrin-bound indocyanine compound synthesized by the above method is dissolved in an acidic aqueous solution and subjected to reverse-phase column chromatography. Elution is performed using, for example, a mixture of acidic water and methanol, a mixture of acidic water and acetonitrile, a mixture of acidic water and ethanol, or a mixture of acidic water and acetone, to isolate and purify the high-purity non-inclusion cyclodextrin-bound indocyanine compound. The acid is not limited as long as it does not decompose the non-inclusion cyclodextrin-bound indocyanine compound, effectively elutes it, and facilitates post-elution handling. Preferred examples include hydrochloric acid, trifluoroacetic acid, acetic acid, sulfuric acid, nitric acid, and formic acid. Hydrochloric acid, trifluoroacetic acid, and acetic acid are preferred. Hydrochloric acid is most preferred. The acid concentration is not limited as long as it does not decompose the non-inclusion cyclodextrin-bound indocyanine compound, effectively elutes it, and facilitates post-elution handling. It is preferably 0.01 mM to 10 mM, and most preferably 0.1 mM to 1 mM. By keeping the concentration within this range, the target compound can be rapidly eluted without decomposing it. The eluted non-inclusion cyclodextrin-bound indocyanine compound can be obtained as a solid by removing the solvent. Freeze-drying can also be used as a solvent removal method.

<6. Synthesis Method (Synthesis Example) and Purification Method (Purification Example) of Inclusion-Type Cyclodextrin-Bound Indocyanine Compounds>

The non-inclusion type cyclodextrin-bound indocyanine compound of Chemical Formula 11 synthesized and purified by the above method is, for example, non-inclusion type in DMSO, but immediately becomes inclusion type upon dissolution in water, as shown in Chemical Formula 12. This phenomenon can be confirmed using ¹ H NMR.

Therefore, as long as the inclusion compound is an aqueous solution, it exists as an inclusion compound and can be used as an inclusion compound. In addition, the inclusion compound in the aqueous solution can be made into a solid by removing the water of the aqueous solution, and a solid of the inclusion compound can also be made.

<7. In vivo dynamics measuring device and circulation visualization device applicable to a living body to which a diagnostic composition has been administered>

In vivo dynamic measurement device

This device is completed based on the fact that the cyclodextrin-bound indocyanine compound of the present invention (hereinafter sometimes appropriately referred to as "compound of the present invention") used in the diagnostic composition of the present invention has different in vivo dynamics such as the degree of interstitial migration from ICG.

That is, the metabolic rate of the compound of the present invention and the rate of migration from the circulatory system, such as blood or lymph, to the interstitium differ from those of ICG. Consequently, the compound of the present invention enters the interstitium at a relatively greater rate than ICG. Therefore, by analyzing the in vivo dynamics of the compound of the present invention, the mechanism of fluid migration (in vivo dynamics) within the body can be accurately evaluated. Here, by maximally maintaining constant measurement conditions (such as the activity state of the organism and ambient temperature), the present device can more accurately measure in vivo dynamics.

One form of abnormal fluid movement in a living body is edema. Here, according to the so-called Stirling hypothesis, the movement of fluid (water) in a living body can be roughly considered to be from arteries to interstitial tissue, and then from interstitial tissue to veins.

The water movement from the artery to the vein can be calculated as (A)-(B)-(C)+(D) based on the pressure difference between the arterial pressure and the interstitial pressure, the water movement outside the circulatory system (A), the water movement into the circulatory system (B) based on the pressure difference between the venous pressure and the interstitial pressure, the water movement from the interstitial fluid to the artery (C) based on the pressure difference between the interstitial fluid osmotic pressure and the arterial blood osmotic pressure, and the water movement from the interstitial fluid to the vein (D) based on the pressure difference between the interstitial fluid osmotic pressure and the venous blood osmotic pressure.

Therefore, when the pressure difference between arterial and venous pressure decreases due to heart failure, or when interstitial fluid osmotic pressure decreases due to nutritional imbalance, the balance of water transfer (A) to (D) is disrupted, causing edema. Furthermore, when there are abnormalities in capillary permeability (abnormalities in the selectivity of material permeation), such as in diabetes, the balance of water transfer (A) to (D) is also disrupted, causing edema. There is no simple method to directly assess abnormal capillary permeability. While the occurrence of edema can be detected by phenomenon, it is currently difficult to determine the specific cause of edema.

Based on the above findings, when abnormal permeability of the capillary wall occurs, the movement of the compound of the present invention can be expected to change compared to cases where edema occurs due to a decrease in the differential pressure between arterial and venous pressure, a decrease in intracellular osmotic pressure, etc. Therefore, when the movement of the compound of the present invention is confirmed to be greater than (or less than) an amount equivalent to water movement, it can be determined that abnormal capillary permeability has occurred. Because it is believed that if no abnormal permeability of the capillary wall occurs, only the movement of the compound of the present invention to an extent equivalent to the change in water movement can be confirmed, it can be determined that an abnormality has occurred in the vascular wall.

In particular, by using two or more compounds of the present invention with different migration rates into the interstitium and measuring the in vivo dynamics of these compounds separately and comparing them, the degree of change in capillary permeability can be evaluated. In other words, by using two or more compounds of the present invention with different permeabilities in the capillary wall and measuring their permeabilities, the occurrence of abnormal vascular wall permeability can be more accurately evaluated. In other words, by using two or more compounds of the present invention with different susceptibilities to abnormal vascular wall permeability and measuring their in vivo dynamics, the factors affecting water migration can be more accurately evaluated. In addition, in addition to evaluating water migration using the compounds of the present invention, water migration can also be measured using conventional methods.

The in vivo dynamics measurement device of the present invention includes an excitation light irradiation unit, a fluorescence intensity measurement unit, an in vivo dynamics calculation unit, and other units. Other units may be selected as needed, such as an in vivo concentration calculation unit that calculates the concentration of a cyclodextrin-bound indocyanine compound based on the fluorescence intensity measured by the fluorescence intensity measurement unit.

This device is a device for measuring at least a portion of a living body to which the diagnostic composition of the present invention has been administered. The dosage of the diagnostic composition is the amount at which fluorescence is observed when the measurement site is irradiated with excitation light. Therefore, the appropriate dosage varies depending on the measurement site. There is no particular limitation on the type of compound of the present invention contained in the diagnostic composition. The compound of the present invention may be used alone or in combination of two or more. In addition, additional diagnostic compositions of the same or different compositions may be added during the measurement process.

The excitation light irradiation unit irradiates the compound of the present invention contained in the administered diagnostic composition with excitation light of a wavelength capable of generating fluorescence. The wavelength of the excitation light irradiated can be limited to an appropriate range. By limiting the wavelength to a minimum possible range, fluorescence and excitation light can be reliably separated. Regarding wavelength limitation, a light source emitting light of an appropriate wavelength can be selected, or a filter can be used to limit the wavelength.

The excitation light irradiation mode is not particularly limited, as long as the generated fluorescence can be measured by the fluorescence intensity measurement unit described later. Examples of excitation light include continuous light, pulsed light, and light with varying intensity. To vary the intensity, the excitation light intensity can be modulated by irradiating the excitation light pulses at predetermined intervals. Preferably, the excitation light intensity is modulated using pulse amplitude modulation.

The excitation light is irradiated to the site to be irradiated through an appropriate optical system. The site to be irradiated is the site where dynamic measurements in the body are to be performed. For example, when evaluating a site where edema occurs, it is preferable to irradiate the site with excitation light directly.

The excitation light irradiation range is not particularly limited. The irradiation range is determined as needed. A narrow irradiation range allows for precise measurement of subdivided in vivo dynamics within the narrow irradiated area. Irradiating a wider range increases the relative amount of the compound of the present invention that fluoresces in response to the excitation light, enabling more precise measurement of fluorescence intensity.

Furthermore, the excitation light irradiation unit is preferably used to irradiate the excitation light while suppressing the influence of ambient light, for example, by irradiating the excitation light in a dark place or by shielding the portion irradiated with the excitation light from external light.

The fluorescence intensity measuring unit measures the fluorescence intensity emitted from the area irradiated with excitation light by the excitation light irradiation unit. The fluorescence intensity is preferably measured by removing light other than fluorescence (ambient light, excitation light, etc.) through a filter that selectively transmits the emitted fluorescence.

When the excitation light irradiation unit uses a unit that irradiates excitation light with modulated intensity, the component that varies according to the modulation can be separated from the measured light intensity and used as the fluorescence intensity. For example, when the excitation light intensity is modulated using pulse amplitude modulation, the fluorescence intensity can be separated by demodulating the light component that varies according to the intensity of the modulated pulse and measuring its intensity. This reduces the impact of ambient light on the fluorescence intensity measurement results.

The in vivo concentration calculation unit is a unit that calculates the in vivo concentration of the compound of the present invention based on the fluorescence intensity measured by the fluorescence intensity measurement unit. The relationship between the fluorescence intensity and the in vivo concentration of the compound of the present invention can be calculated using an appropriate method. For example, a standard curve can be prepared in advance, and the in vivo concentration of the compound of the present invention can be calculated based on the standard curve. In addition, the absolute value of the fluorescence intensity can be directly used as a value related to the in vivo concentration of the compound of the present invention. The in vivo concentration calculation unit calculates the in vivo concentration of the compound of the present invention over time.

The in vivo dynamics calculation unit is a unit that calculates the temporal change of the time rate of change of the in vivo concentration of the compound of the present invention from the in vivo concentration data obtained over time by the in vivo concentration calculation unit. The in vivo dynamics calculation unit calculates the speed of the compound of the present invention moving from the circulatory system to the interstitial tissue and the speed of the compound of the present invention moving from the interstitial tissue to the circulatory system in a part of the body using the temporal change rate of the in vivo concentration obtained. Instead of using the in vivo concentration, the speed of movement into and out of the interstitial tissue can be directly calculated by using the change in fluorescence intensity obtained over time by the fluorescence intensity measurement unit.

It is speculated that there is a high correlation between the movement of the compound of the present invention into (and out of) the interstitium and the movement of water in body fluids. Therefore, by measuring the in vivo behavior of the compound of the present invention, it is possible to evaluate the movement of water in the measurement site. Furthermore, when abnormalities occur in the vascular wall and the permeability of substances is abnormal, the behavior of the compound of the present invention in the body should also change. Therefore, by evaluating the behavior of the compound of the present invention, vascular permeability can be evaluated.

The time rate of change of fluorescence intensity (or concentration in the organism) is obtained by differentiating the time-dependent change in fluorescence intensity (or concentration in the organism) with respect to time, or by calculating the fluorescence intensity (or concentration in the organism) at each specified time and then calculating it as the difference between the fluorescence intensity (or concentration in the organism) before (after) the specified time.

The speed of the compound of the present invention entering the interstitial fluid and the speed of being discharged in a part of the organism can be calculated by the time rate of change of the fluorescence intensity (or biological concentration) obtained. Here, the speed of entering the interstitial fluid and the speed of being discharged can be obtained as relative values by the fluorescence intensity. When more precision is required, it can be calculated according to the concentration of the compound of the present invention in the interstitial and the amount of the interstitial fluid. In addition, the concentration of the compound of the present invention in blood and lymph and its amount are also considered, and it can be calculated more accurately. The concentration of the compound of the present invention in blood, etc. can be measured with good accuracy by actually taking blood.

The following methods can be cited as methods for calculating the concentration in interstitial fluid. The first method is a method for directly approximating the calculated in vivo concentration of the compound of the present invention to the concentration in the interstitial fluid in a part of the organism. As a method related to the first method, the concentration in the interstitial fluid can also be calculated by considering the amount of interstitial fluid. The second method is to calculate the influence ratio of the substance present in the blood vessel on the measured value measured by the fluorescence intensity measurement unit by using other standard substances (ICG, etc.) that do not move to the interstitial on the basis of measuring the concentration of the compound of the present invention in the actual blood, and to calculate the concentration in the interstitial fluid by removing the influence of the concentration of the compound of the present invention in the measured blood.

The time rate of change of the concentration of the compounds of the present invention in interstitial fluid can be calculated by subtracting the speed of movement outside the interstitial fluid from the speed of movement inside the interstitial fluid. Here, it can be assumed that the movement of the compounds of the present invention inside (outside) the interstitial fluid is carried out at a speed related to the water movement in the body fluid, and if the water movement reaches equilibrium, the speed of movement inside the interstitial fluid and the speed of movement outside the interstitial fluid can be considered as constants, therefore, the concentration change (metabolism, discharge, changes caused by movement to tissue, etc.) of the compounds of the present invention in blood and the time rate of change of concentration in interstitial fluid can be used to calculate its movement speed. In addition, even in the case of changes in its movement speed, it is possible to calculate their movement speed by making a model corresponding to this change and applying it.

If the calculated migration speed is outside the range of normal blood vessel wall values, it can be inferred that the blood vessel wall has abnormalities. In addition, as a result of abnormalities in the blood vessel wall, the permeability of the compound of the present invention may be increased or decreased.

It is a well-known fact that acute tissue swelling is caused by various tissue disorders such as acute inflammation, ischemia, and trauma, and is an extremely important tissue reaction in the medical field regardless of the field.

For example, in addition to the simple occurrence of swelling itself being a problem, there are also issues involving all organs, such as the evaluation of intestinal suturing during surgery, the evaluation of the degree of inflammation such as pneumonia, the evaluation of the suitability of organ transplantation, and the evaluation of brain and kidney dysfunction.

However, there is currently no accurate method for predicting the likelihood and extent of tissue swelling, and it can only be estimated empirically based on the degree of tissue damage. Therefore, in most cases, countermeasures can only be discussed after the occurrence of swelling is confirmed, which is a major limitation in treatment.

If the likelihood of swelling can be predicted with high accuracy early on, measures to reduce swelling can be taken early on, minimizing the impact. For example, if the extent of brain swelling that occurs shortly after a cerebral hemorrhage can be accurately predicted, decompression measures and measures to reduce vascular permeability can be implemented early on, minimizing the impact. Similarly, if the impact of ischemic stress can be predicted in advance, secondary complications caused by myocardial infarction and limb injuries can be minimized. Thus, high-precision quantitative prediction technology for swelling has the potential to significantly impact healthcare overall.

Until now, quantification of tissue swelling, including edema, has been qualitatively determined through observation. While quantification has been attempted, it is only applicable in very limited situations and has the disadvantage of being able to evaluate only after swelling has occurred.

To address this issue, the in vivo dynamic measurement device of the present invention calculates the rate of water migration within and outside the interstitium, thereby predicting the progression of swelling. As detailed in the Examples, it is known that ICG does not migrate into the interstitium as swelling progresses. In contrast, the cyclodextrin-conjugated indocyanine compound of the present invention can migrate within and outside the interstitium, and its movement into the interstitium is further promoted by the movement of water as swelling progresses. Therefore, by evaluating the movement of the cyclodextrin-conjugated indocyanine compound of the present invention into the interstitium, the progression of swelling can be predicted.

That is to say, it can be inferred that the degree of swelling that is ultimately formed is affected by the speed of movement of water into and out of the interstitial space, and therefore the degree of final swelling can be evaluated by evaluating the speed of movement of the cyclodextrin-bound indocyanine compound of the present invention, which is related to the speed of movement of water. The speed of movement can be calculated by the concentration of the cyclodextrin-bound indocyanine compound, or directly using the fluorescence intensity. In addition, the speed of movement into and out of the interstitial space can naturally be calculated as an absolute value, or it can be calculated as a relative value (for example, the rate of change of fluorescence intensity can be directly used as a value related to the speed of movement), and the progress of swelling can be predicted based on this value.

The progression of swelling is preferably calculated based on the normal migration rate into the interstitium when there is no swelling. However, the normal migration rate into the interstitium may be unknown. In such cases, a non-swollen site is selected as a control site, and the migration rate of this site is calculated and used as a substitute. Alternatively, the extent of interstitial migration of the cyclodextrin-conjugated indocyanine compound of the present invention can be evaluated by using ICG, which does not migrate within the interstitium, to assess the blood circulation volume and metabolism.

The determination of the extent of movement of the cyclodextrin-bound indocyanine compound of the present invention within the interstitium is carried out after a prescribed time has passed after the diagnostic composition of the present invention is administered into a living body. The prescribed time refers to the time required for the diagnostic composition of the present invention to be distributed in the blood. The reason for using data after the prescribed time for evaluation is that after administration of the diagnostic composition, when it is distributed into the blood after a prescribed time, it is hardly affected by the degree of swelling progression, the concentration in the blood rises rapidly, and the change in fluorescence intensity is not affected by the presence or absence of swelling, and almost no difference is produced. After distribution in the blood, the speed of movement within the interstitium will change according to the presence or absence of swelling progression, so an increase in fluorescence intensity will be selectively confirmed at the site where the movement occurs (i.e., the site where swelling occurs).

As a specific example of a method for estimating the progression of swelling, there is a method in which the blood volume is estimated from body weight, the size of the peak of fluorescence intensity and the time to the peak are calculated from the estimated blood volume; the fluorescence intensity at the time when the edema may change is measured taking into account this time and intensity, and the degree of edema progression and the degree of edema that may occur in the future can be estimated from the results.

Circular Visualization Installation

This device is based on the fact that the cyclodextrin-bound indocyanine compound of the present invention (hereinafter sometimes appropriately referred to as "compound of the present invention") used in the diagnostic composition of the present invention differs from ICG in its in vivo dynamics, such as the degree of migration into the interstitium.

That is, the compound of the present invention easily moves in the interstitial fluid, and by tracking the behavior of the compound of the present invention, the state of blood circulation or lymph circulation can be visualized.

The circulation visualization device of the present invention comprises an excitation light irradiation unit, a fluorescence imaging unit, a morphological imaging unit, and a display unit. This device is a device for measuring at least a portion of a living body to which the diagnostic composition of the present invention is administered. The dosage of the diagnostic composition is the amount in which fluorescence can be observed when the measurement site is irradiated with excitation light. Therefore, the appropriate dosage varies depending on the measurement site. The type of compound of the present invention contained in the diagnostic composition is not particularly limited. The compound of the present invention can be used alone or in combination of two or more. In addition, additional diagnostic compositions having the same or different compositions can be administered during the measurement process.

The excitation light irradiation unit irradiates the compound of the present invention contained in the administered diagnostic composition with excitation light of a wavelength capable of generating fluorescence. The wavelength of the excitation light used for irradiation can be limited to an appropriate range. By limiting the wavelength to a narrow range as much as possible, fluorescence and excitation light can be reliably separated. Wavelength limitation can be achieved by selecting a light source emitting light of an appropriate wavelength or by using a filter to limit the wavelength.

The form of excitation light irradiation is not particularly limited, as long as the generated fluorescence can be measured by the fluorescence imaging unit described below. For example, the excitation light may be continuous light, pulsed light, or light with varying intensity. To vary the intensity, the intensity of the excitation light can be modulated by irradiating the excitation light pulses at predetermined intervals. Preferably, the excitation light is modulated using pulse amplitude modulation.

The excitation light passes through an appropriate optical system and illuminates the desired area. The area to be illuminated is the part of the organism where the circulation is to be visualized, such as the area where tissue necrosis has occurred due to burns, frostbite, inflammation, trauma, infarction, and the like, and its surrounding areas. Necrotic areas of tissue lack blood circulation, and leaving them untreated offers little benefit, so removal is considered. In this case, it is ideal to completely remove only the necrotic area, comparing the necrotic area with the normal area.

Conventionally, identification of necrotic areas has been performed using methods such as angiography using contrast agents or detecting a decrease in body temperature associated with blood circulation. However, angiography has the problem of being inconvenient to use devices such as X-ray irradiation devices, and accurate determination based on body temperature is difficult.

The device of the present invention is used to evaluate necrotic and normal areas based on the presence or absence of body fluid (blood) circulation. Visualizing areas without circulation allows for easy removal. In addition to visualizing necrotic areas, direct observation of blood circulation is also possible, making it easy to identify abnormalities in circulatory function. For example, areas that are not necrotic but have decreased circulatory function due to, for example, infarction can be visualized.

The excitation light irradiation range is determined as needed so as to include the portion to be cyclically visualized.

Furthermore, the excitation light irradiation unit is preferably used to irradiate the excitation light while controlling the influence of ambient light, for example, by irradiating the excitation light in a dark place or by shielding the portion irradiated with the excitation light from external light.

The fluorescence imaging unit two-dimensionally acquires the intensity of fluorescence emitted by the compound of the present invention, which is excited by the excitation light irradiation unit, thereby obtaining data on the distribution status of the compound of the present invention in the organism. In other words, this unit acquires distribution status data representing the distribution of the compound of the present invention in a portion of the organism as two-dimensional image data.

For example, it can be composed of a combination of an appropriate optical system and an imaging element such as a CCD. The resolution of the data obtained two-dimensionally can be set to a necessary value according to the purpose. The determination of fluorescence intensity preferably allows the emitted fluorescence to be selectively transmitted through a filter and removes light (ambient light, excitation light, etc.) other than the fluorescence to be measured.

When using excitation light with modulated intensity as the excitation light irradiation unit, the fluorescence intensity is obtained by separating the components that vary according to the modulation from the measured light intensity. For example, when the excitation light intensity is modulated using pulse amplitude modulation, the fluorescence intensity is separated by demodulating the components of the light that vary according to the modulated pulse intensity and measuring their intensities. This reduces the influence of ambient light on the fluorescence intensity measurement results.

The morphological imaging unit is a unit that two-dimensionally acquires the intensity of light at wavelengths other than the fluorescence wavelength emitted by the compound of the present invention to obtain morphological data of the compound in a portion of a living body. In other words, this unit is a unit that acquires two-dimensional image data of the morphology of a portion of a living body.

The morphological imaging unit can be composed of an appropriate optical system and an imaging element such as a CCD. The resolution of the two-dimensional data can be set according to the desired purpose. In this case, the fluorescence emitted by the excitation light is not measured (or the detection sensitivity is reduced). The morphological imaging unit uses almost all of the aforementioned fluorescence imaging unit and optical system. Finally, a dichroic prism is used on the optical path before it is introduced into the imaging element to guide light with a wavelength corresponding to the fluorescence to the fluorescence imaging unit, and light of other wavelengths can be guided to the morphological imaging unit. By appropriately forming a bidirectional color film, the dichroic prism can reasonably control the wavelength of the separated light.

Furthermore, the fluorescence imaging unit and the morphological imaging unit can share the same camera device. That is, the separation of fluorescence and other light can also be a mathematical separation performed after obtaining their two-dimensional image data. Furthermore, the distribution state data can also be complex image data from the surface of a part of the biological body to the deep layer direction. The optical focus used by the fluorescence imaging unit obtains complex two-dimensional image data in the deep layer direction while moving in the deep layer direction. In addition, as the excitation light irradiation unit, an optical system with a variable focal length is used, and the focus is moved not on the surface of a part of the biological body, but in the deep layer direction, or the excitation light is concentrated and irradiated to a part of the biological body, so that fluorescence can be selectively emitted not only on the surface of the biological body but also in the deep layer inside the biological body, and the circulation of this part can be visualized.

The display unit is a display unit that represents the distribution state of the compound of the present invention in a part of the organism by overlapping the morphological data obtained by the morphological imaging unit and the distribution state data obtained by the fluorescence imaging unit. When the wavelength of the fluorescence is not within the range of visible light, it is preferred to convert the wavelength of the fluorescence into visible light for representation. From the perspective of visualization of the circulation, the representation of the distribution state data takes precedence over the representation of the morphological data. In particular, it is possible to distinguish between the low distribution amount (i.e., fluorescence intensity) of the compound of the present invention and the other parts (whether it is low or not is determined according to the purpose of visualization of the circulation. For example, in the purpose of visualizing the necrotic part, in the part without circulation, i.e., the part where no fluorescence is confirmed) and other parts can be distinguished. For example, the part can be represented as a color different from that of the other parts or a flashing representation. The superposition of the distribution state data and the morphological data can be realized using logic on a computer, etc. The representation of two-dimensional data can be realized by a general display device. The display device is provided between the organism and the measurer, so that the organism can be treated while observing the display device.

＜8. Definitions, etc.＞

In the present invention, "alkyl" refers to a linear or branched alkyl group having 1 to 20 carbon atoms which may have a substituent, for example, a methyl group, ethyl group, propyl group, butyl group, pentyl group, hexyl group, heptyl group, octyl group, nonyl group, decyl group, undecyl group, dodecyl group, tridecyl group, tetradecyl group, pentadecyl group, hexadecyl group, heptadecyl group, octadecyl group, nonadecyl group, eicosyl group or a branched group.

In the present invention, the "alkoxy group" includes, for example, methoxy, ethoxy, propoxy, butoxy, pentyloxy, hexyloxy, methoxyethoxy, methoxypropoxy, ethoxyethoxy, ethoxypropoxy, methoxyethoxyethoxy, etc., in which alkoxy groups having 1 to 20 carbon atoms are bonded in a straight chain or branched form.

In the present invention, the "aryl group" includes aromatic hydrocarbons having 6 to 20 carbon atoms, such as phenyl and naphthyl.

Example

The preferred embodiments of the present invention are described in detail below by way of examples. However, the technical scope of the present invention is not limited to the following embodiments, and various modifications can be made within the scope of the present invention.

<Experiment 1: Synthesis and Purification of Compounds Represented by Chemical Formula 15 and Chemical Formula 16>

A mixture of 0.20 g of the compound represented by Chemical Formula 13, 0.94 g of the compound represented by Chemical Formula 14, 0.18 g of WSC, 0.12 g of HOBt, 4.0 mL of pyridine, and 2.0 mL of N,N-dimethylformamide was stirred at 0°C in the dark for 6 hours. 50 mL of acetone was then added, and the precipitate was filtered under reduced pressure. The precipitate was dissolved in a 0.1% trifluoroacetic acid aqueous solution and subjected to ODS column chromatography. A mixture of water and methanol containing 1 mM hydrochloric acid was used as the eluent to elute the compound represented by Chemical Formula 15. The eluate was concentrated under reduced pressure to obtain 0.65 g of the inclusion-type compound represented by Chemical Formula 16 as a green solid (at the end of the reduced pressure concentration of the eluate, the water content was high, so it naturally became an inclusion-type compound).

[Chemical Formula 15]

[Chemical Formula 16]

Next, the instrumental analysis data for the target compound represented by Chemical Formula 16 are shown. ¹ H NMR (500 MHz, D ₂ O, 26°C, acetone: 2.10 ppm) 1.43 (2H, m), 1.55 (2H, m), 1.99 (6H, s), 2.09 (6H, s), 2.63 (6H, m), 2.80 (6H, m), 2.92 (2H, m), 3.02 (2H, dd, J = 3.7, 9.8 Hz), 3.08 (2H, t, J = 9.2 Hz), 3.2-4.1 (m), 4.19 (2H, t, J = 9.8 Hz), 4.26 (2H, t, J = 9.8 Hz), 4.33 (2H, m), 4.43 (2H, m), 4.71 (2H, d, J = 2.4 Hz) , 4.81 (4H, d, J = 3.7 Hz), 4.91 (2H, d, J = 3.7 Hz), 4.99 (2H, d, J = 3.7 Hz), 5.08 (2H, d, J = 3.7 Hz), 5.13 (2H, d, J = 3.1 Hz), 6.15 (2H, d, J = 13 Hz), 6.52 (2H, t, J = 12 Hz), 7.43 (4H, m), 7.57 (1H, d, J = 12 Hz), 7.57 (2H, d, J = 9.2 Hz), 7.78 (2H, m), 8.06 (3H, m), 8.15 (2H, d, J = 8.5 Hz). ESI-MS m/z calcd for C ₁₃₁ H ₁₉₁ N ₄ O ₇₂ 2972, found 2973 [M] ⁺ .

<Experiment 2: Synthesis and Purification of Compounds Represented by Chemical Formula 19 and Chemical Formula 20>

A mixture of 0.17 g of the compound represented by Chemical Formula 17, 5 mL of methanol, and 0.30 g of t-BuOK was stirred at room temperature for 12 hours. Then, 3 mL of 1M hydrochloric acid and 50 mL of water were added. The precipitate was filtered, washed with water, and then dried under reduced pressure to obtain 0.17 g of the compound represented by Chemical Formula 18.

[Chemical Formula 17]

[Chemical Formula 18]

A mixture of 0.02 g of the compound represented by Chemical Formula 18, 0.081 g of the compound represented by Chemical Formula 14, 0.016 g of WSC, 0.011 g of HOBt, 0.3 mL of pyridine, and 0.2 mL of N,N-dimethylformamide was stirred at 0°C in the dark for 6 hours. 5 mL of acetone was then added, and the precipitate was filtered under reduced pressure, dissolved in a 0.1% trifluoroacetic acid aqueous solution, and subjected to ODS column chromatography. A mixture of water and methanol containing 1 mM hydrochloric acid was used as the eluent to elute the compound represented by Chemical Formula 19. The eluent was concentrated under reduced pressure to obtain 0.045 g of the inclusion-type compound represented by Chemical Formula 20 as a green solid (at the end of the concentration of the eluent under reduced pressure, the water content was high, so it naturally became an inclusion-type compound).

[Chemical Formula 19]

[Chemical Formula 20]

Next, the machine analysis data of the target compound represented by Chemical Formula 20 are shown. ¹ H NMR (500 MHz, D ₂ O,40℃,acetone:2.26ppm)1.54(2H,m),1.68(2H,m),1.98(2H,m),2.19(6H,s),2 .20(2H,m),2.30(6H,s),2.6-2.85(10H,m),2.95(2H,m),3.00(4H,m),3.08 (2H,t,J＝12Hz),3.17(2H,dd,J＝3.7,9.8Hz),3.26(2H,t,J＝9.8Hz),3.35- 4.30(m),4.35(2H,t,J＝9.2Hz),4.50(2H,t,J＝9.2H),4.52(2H,m),4.63(2H ,m),4.87(2H,d,J＝3.7Hz),4.95(d,J＝3.1Hz),4.97(2H,d,J＝3.7Hz),5.08 (2H,d,J＝3.7Hz),5.15(2H,d,J＝4.3Hz),5.25(2H,d,J＝3.7Hz),5.29(2H,d, J＝3.7Hz),6.30(2H,d,J＝14.6Hz),7.58(4H,m),7.73(2H,d,J＝8.5Hz),7.95 (2H,m),8.25(2H,m),8.32(2H,d,J＝14.6Hz),8.35(2H,d,J＝8.5Hz).ESI-MS m/ _z calculated for _{C135H197N4O73 3042} , found 3042 [M] ⁺ .

<Experiment 3: Synthesis and Purification of the Compound Represented by Chemical Formula 21>

A mixture of 0.04 g of the compound represented by Chemical Formula 13, 0.18 g of mono-6-amino-6-deoxy-β-cyclodextrin, 0.05 g of WSC, 0.025 g of HOBt, 0.8 mL of pyridine, and 0.4 mL of N,N-dimethylformamide was stirred at 0°C in the dark for 3 hours. 10 mL of acetone was then added, and the precipitate was filtered under reduced pressure, dissolved in 0.1% trifluoroacetic acid aqueous solution, and subjected to ODS column chromatography. The compound represented by Chemical Formula 21 was eluted using a mixture of water and methanol containing 1 mM hydrochloric acid. The eluate was concentrated under reduced pressure to obtain 0.11 g of the compound represented by Chemical Formula 21 as a green solid.

[Chemical Formula 21]

Next, the instrumental analysis data for the target compound represented by Chemical Formula 21 are shown. ¹ H NMR (500 MHz, D ₂ O, 29°C, acetone: 2.10 ppm) 1.79 (12H, br.), 2.68 (4H, br.), 3.0-4.5 (98H), 4.4 (4H, br.), 4.5-5.3 (14H, br.), 6.18 (2H, br.), 6.46 (2H, br.), 7.3-8.2 (15H). ESI-MS m/z calculated for C ₁₂₅ H ₁₇₉ N ₄ O ₇₀ 2856, found 2856 [M] ⁺ .

<Experiment 4: Synthesis and Purification of the Compound Represented by Chemical Formula 23>

A mixture of 0.02 g of the compound represented by Chemical Formula 22, 0.096 g of mono-6-amino-6-deoxy-β-cyclodextrin, 0.032 g of WSC, 0.5 mL of pyridine, and 0.05 mL of 0.1 M phosphate buffer was stirred in the dark at room temperature for 24 hours. 10 mL of acetone was then added, and the precipitate was filtered under reduced pressure, dissolved in 0.1% trifluoroacetic acid aqueous solution, and subjected to ODS column chromatography. A mixture of water and acetonitrile was used as the eluent to elute the compound represented by Chemical Formula 23. The eluate was concentrated under reduced pressure to yield 0.014 g of the compound represented by Chemical Formula 23 as a green solid.

[Chemical Formula 22]

[Chemical Formula 23]

Next, the instrumental analysis data for the target compound represented by Chemical Formula 21 are shown. ¹ H NMR (500 MHz, D ₂ O, 26°C, acetone: 2.15 ppm) 1.26 (4H, m), 1.5-2.25 (24H, br), 2.7-4.2 (88H), 4.82 (2H, br), 4.90 (8H, br), 4.97 (2H, br), 5.03 (2H, br), 6.11 (2H, br), 6.36 (2H, br), 7.3-8.01 (15H, br). ESI-MS m/z calculated for C ₁₃₁ H ₁₉₁ N ₄ O ₇₀ 2940, found 2940 [M] ⁺ .

<Experiment 5: Synthesis and Purification of the Compound Represented by Chemical Formula 24>

A mixture of 0.20 g of the compound represented by Chemical Formula 22, 0.02 g of 3-amino-3-deoxy-β-cyclodextrin, 0.096 g of WSC, 0.013 g of HOBt, 0.4 mL of pyridine, and 0.2 mL of N,N-dimethylformamide was stirred in the dark at room temperature for 1 hour. Subsequently, 10 mL of acetone was added, and the precipitate was filtered under reduced pressure, dissolved in 0.1% trifluoroacetic acid aqueous solution, and subjected to ODS column chromatography. A mixture of water and methanol was used as the eluent to elute the compound represented by Chemical Formula 24. The eluate was concentrated under reduced pressure to yield 0.013 g of the compound represented by Chemical Formula 24 as a green solid.

[Chemical Formula 24]

Next, the instrumental analysis data for the target compound represented by Chemical Formula 24 are shown. ¹ H NMR (500 MHz, D ₂ O, 29°C, acetone: 2.10 ppm) 1.1-2.5 (28H), 3.0-4.25 (88H), 4.5-5.2 (14H), 7.3-8.02 (15H). ESI-MS m/z calculated for C ₁₃₁ H ₁₉₁ N ₄ O ₇₀ 2940, found 2940 [M] ⁺ .

<Experiment 6: Synthesis and Purification of the Compound Represented by Chemical Formula 25>

A mixture of 0.02 g of the compound represented by Chemical Formula 22, 0.1 g of the compound represented by Chemical Formula 14, 0.032 g of WSC, 0.5 mL of pyridine, and 0.05 mL of 0.1 M phosphate buffer was stirred in the dark at room temperature for 24 hours. 10 mL of acetone was then added, and the precipitate was filtered under reduced pressure, dissolved in 0.1% trifluoroacetic acid aqueous solution, and subjected to ODS column chromatography. A mixture of water and methanol was used as the eluent to elute the compound represented by Chemical Formula 25. The eluate was concentrated under reduced pressure to yield 0.021 g of the compound represented by Chemical Formula 25 as a green solid.

[Chemical Formula 25]

Next, the instrumental analysis data for the target compound represented by Chemical Formula 25 are shown. ¹ H NMR (500 MHz, D ₂ O, 25°C, acetone: 2.10 ppm) 1.0-2.5 (32H), 3.0-4.5 (96H), 4.8-5.2 (14H), 6.09 (2H, br), 6.37 (2H, br), 7.3-8.02 (15H, br). ESI-MS m/z calculated for C ₁₃₇ H ₂₀₃ N ₄ O ₇₂ 3056, found 3056 [M] ⁺ .

<Test 7: Solubility of the Cyclodextrin-Bound Indocyanine Compound of the Present Invention>

Solubility tests were conducted on ICG and the cyclodextrin-conjugated indocyanine compounds of the present invention (Chemical Formulas 16, 20, 21, and 23-25) in water and saline. While the dissolution of powdered ICG from Molecular Probe in water and saline required vigorous agitation for approximately one minute, the cyclodextrin-conjugated indocyanine compounds of the present invention, particularly those represented by Chemical Formulas 16 and 20, dissolved rapidly without the need for agitation.

<Test 8: Adsorption of the Cyclodextrin-Bound Indocyanine Compound of the Present Invention to Human Skin>

ICG and the cyclodextrin-conjugated indocyanine compounds of the present invention (Chemical Formulas 16, 20, 21, 23-25) were tested for their adsorption to human skin. A 1 mM aqueous solution (0.03 mL) of each of ICG and the cyclodextrin-conjugated indocyanine compounds of the present invention (Chemical Formulas 16, 20, 21, 23-25) was dropped onto the wrist, rinsed with water after 5 minutes, and then wiped and rinsed again. The result showed that ICG was not completely washed away by water, while the cyclodextrin-conjugated indocyanine compounds of the present invention, especially the compounds represented by Chemical Formulas 16 and 20, were easily washed away by water, indicating that the adsorption of the cyclodextrin-conjugated indocyanine compounds of the present invention to human skin was significantly lower than that of ICG (Figure 1).

<Test 9: Adsorption of the Cyclodextrin-Bound Indocyanine Compound of the Present Invention to Cellulose Fibers>

ICG and the cyclodextrin-conjugated indocyanine compounds of the present invention (Chemical Formulas 16, 20, 21, 23-25) were tested for their adsorption onto cellulose fibers. A cotton swab (Sanyo Co., Ltd.) was used to apply 0.05 mL of a 1 mM aqueous solution of each of ICG and the cyclodextrin-conjugated indocyanine compounds of the present invention (Chemical Formulas 16, 20, 21, 23-25) to a model cellulose fiber. After 3 minutes, the swab was rinsed in running water (tap water, 1 L/min) for 5 seconds. The results showed that ICG was not completely washed away, while the cyclodextrin-conjugated indocyanine compounds of the present invention, particularly those represented by Chemical Formulas 16 and 20, were easily washed away, indicating that the adsorption of the compounds represented by the cyclodextrin-conjugated indocyanine compounds of the present invention onto cellulose fibers was significantly lower than that of ICG (Figure 2).

<Test 10: Adsorption of the Cyclodextrin-Conjugated Indocyanine Compound of the Present Invention to a Meat Model>

Adsorption of ICG and the cyclodextrin-conjugated indocyanine compounds of the present invention (Chemical Formulas 16, 20, 21, 23-25) onto a biological meat model was tested. Commercially available pork tenderloin was used as the meat model. A 5 mm diameter recess was created in the pork tenderloin. 0.05 mL of a 1 mM aqueous solution of each of ICG and the cyclodextrin-conjugated indocyanine compounds of the present invention (Chemical Formulas 16, 20, 21, 23-25) was applied to the meat model. After 3 minutes, the sample was rinsed in running water (tap water, 1 L/min) for 10 seconds. The results showed that ICG was not completely washed off, while the cyclodextrin-conjugated indocyanine compounds of the present invention, particularly those represented by Chemical Formulas 16 and 20, were easily washed off, demonstrating that the adsorption of the cyclodextrin-conjugated indocyanine compounds of the present invention onto the biological meat model was significantly lower than that of ICG (Figure 3).

<Test 11: Adsorption of the Cyclodextrin-Conjugated Indocyanine Compound of the Present Invention to a Biological Protein Model>

Adsorption of ICG and the cyclodextrin-conjugated indocyanine compounds of the present invention (Chemical Formulas 16, 20, 21, 23-25) onto biological protein models was tested. A 5 mm diameter recess was created in commercially available chicken breast meat as the biological protein model. 0.05 mL of a 1 mM aqueous solution of ICG and the cyclodextrin-conjugated indocyanine compounds of the present invention (Chemical Formulas 16, 20, 21, 23-25) was applied to the breast. After 3 minutes, the samples were rinsed in running water (1 L/min) for 10 seconds. The results showed that ICG was not completely washed away, while the cyclodextrin-conjugated indocyanine compounds of the present invention, particularly those represented by Chemical Formulas 16 and 20, were easily washed away, indicating that the adsorption of the cyclodextrin-conjugated indocyanine compounds of the present invention onto biological protein models was significantly lower than that of ICG (Figure 4).

<Test 12: Adsorption of the Cyclodextrin-Bound Indocyanine Compound of the Present Invention to Hydrophobic Chemical Fibers>

The adsorption of ICG and the cyclodextrin-conjugated indocyanine compounds of the present invention (Chemical Formulas 16, 20, 21, 23-25) onto hydrophobic chemical fibers was tested. A polypropylene mask (Tamagawa Eisai Co., Ltd.) was used as a model hydrophobic chemical fiber. 0.05 mL of a 1 mM aqueous solution of ICG and the cyclodextrin-conjugated indocyanine compounds of the present invention (Chemical Formulas 16, 20, 21, 23-25) was applied to the mask. After 20 minutes (the sample was left to remove any moisture, so it would not adhere to the mask), the mask was rinsed in running water (1 L/min) for one second. The results showed that ICG was not completely washed off, while the cyclodextrin-conjugated indocyanine compounds of the present invention, particularly those represented by Chemical Formulas 16 and 20, were easily washed off, indicating that the adsorption of the cyclodextrin-conjugated indocyanine compounds of the present invention onto hydrophobic chemical fibers was significantly lower than that of ICG (Figure 5).

<Test 13: Molecular Association of Cyclodextrin-Bound Indocyanine Compounds of the Present Invention in Aqueous Solution>

The molecular association properties of ICG and the cyclodextrin-conjugated indocyanine compounds of the present invention (Chemical Formulas 16, 20, 21, 23-25) in aqueous solutions were investigated. Aqueous solutions of ICG and the cyclodextrin-conjugated indocyanine compounds of the present invention (Chemical Formulas 16, 20, 21, 23-25) were prepared at 0.01 mM, 0.025 mM, 0.05 mM, and 0.1 mM, respectively. The solutions were placed in quartz cuvettes with a 1 mm optical path length, and optical absorption spectra were measured at 25°C from 600 nm to 1000 nm. The results showed that ICG exhibited molecular association known as H-aggregation within this concentration range (Figure 6, left). However, the cyclodextrin-conjugated indocyanine compounds of the present invention, particularly those represented by Chemical Formulas 16 and 20, did not exhibit this molecular association within this concentration range (Figure 6, right).

<Test 14: Fluorescence of the Cyclodextrin-Bound Indocyanine Compound of the Present Invention in Aqueous Solution>

The fluorescence properties of ICG and the cyclodextrin-conjugated indocyanine compounds of the present invention (Chemical Formulas 16, 20, 21, 23-25) in aqueous solution were investigated. 0.1 μM aqueous solutions of ICG and the cyclodextrin-conjugated indocyanine compounds of the present invention (Chemical Formulas 16, 20, 21, 23-25) were placed in 1 cm square quartz cuvettes at 25°C. Excitation was performed with 720 nm excitation light (bandpass: 10 nm), and fluorescence spectra were measured (bandpass: 10 nm). Fluorescence efficiency was calculated based on the fluorescence efficiency of ICG, which is 0.13 (in DMSO, 25°C). The results showed that the fluorescence quantum efficiency of ICG was 0.021, while the fluorescence quantum efficiency of the cyclodextrin-conjugated indocyanine compounds of the present invention, particularly the compounds represented by Chemical Formulas 16 and 20, was 0.054 and 0.042, respectively, which are 2.6 times and 2 times that of ICG.

<Test 15: Fluorescence of the Cyclodextrin-Conjugated Indocyanine Compound of the Present Invention in Blood>

The fluorescence of ICG and the cyclodextrin-conjugated indocyanine compounds of the present invention (Chemical Formulas 16, 20, 21, 23-25) in blood was studied. ICG and the cyclodextrin-conjugated indocyanine compounds of the present invention (Chemical Formulas 16, 20, 21, 23-25) were dissolved in human blood to a concentration of 100 μM. The mixture was placed in a triangular quartz cuvette at 25°C and excited with 760 nm excitation light (bandpass: 10 nm), and the surface fluorescence spectrum (bandpass: 10 nm) was measured. The fluorescence intensity at the maximum fluorescence wavelength was 58 (arbitrary units) for ICG. The fluorescence intensities of the cyclodextrin-conjugated indocyanine compounds of the present invention, especially the compounds represented by Chemical Formulas 16 and 20, were 270 (arbitrary units) and 190 (arbitrary units), which are 4.7 times and 3.3 times that of ICG. This is believed to be because the cyclodextrin-conjugated indocyanine compounds of the present invention do not inhibit luminescence in vivo.

<Test 16: Behavior of the Cyclodextrin-Conjugated Indocyanine Compound of the Present Invention in Vivo>

As representative examples, the behaviors of the isomerization-equilibrium compound (TK1) represented by Chemical Formulas 19 and 20 and the isomerization-equilibrium compound (TK2) represented by Chemical Formulas 15 and 16 were evaluated in human blood and rats as biological subjects.

Fluorescence behavior in human blood

The concentration dependence of surface fluorescence intensity of ICG, TK1, and TK2 in human venous blood in triangular cuvettes was evaluated. Specifically, 1.0 mL of human venous blood was added to a triangular quartz cuvette, along with ICG, TK1, and TK2 at specified concentrations. After irradiation with 760 nm (bandpass: 10 nm) excitation light at 25°C, surface fluorescence (bandpass: 10 nm) was measured. The results are shown in Figure 7.

As can be clearly seen from FIG7 , both TK1 and TK2 can emit fluorescence in human venous blood when irradiated with excitation light.

·Study on the feasibility of drug administration in rats

ICG, TK1, and TK2 were prepared as 1 mM aqueous solutions, and 0.1 mL of each solution was injected into the femoral vein of rats after laparotomy. The abdomen was then illuminated with 760 nm excitation light to assess the presence of fluorescence.

The results showed that fluorescence was observed in the internal organs of the rats after the abdominal opening. High accumulation of TK1 and TK2 in the kidneys was observed (as ICG is known to accumulate in the liver). Furthermore, no significant adverse effects on the rats' condition were observed after administration of these drugs, confirming the possibility of relatively safe in vivo administration.

Observation of distribution in rats

Wistar male rats (9 weeks old, weighing 350 g) were anesthetized with ether and then anesthetized by intraperitoneal administration of 0.1 mL of sodium pentobarbital (Nembutal) using a 26G needle. The base of the rat's tail was anesthetized with a rubber band to remove blood, and the tail vein was secured with a 24G indwelling needle, and the terminal line was secured by connecting a three-headed square stopcock and an extension tube. The rat was fixed on a flat table in a supine position (Figure 8). The PDE camera unit manufactured by Hamamatsu Photonics Co., Ltd. was correctly set at a position 16 cm away from the rat's body. However, it was fixed at 20 cm when photographing the whole body. The observation range is the range indicated by the black circle in Figure 8. Thereafter, ICG, TK1, and TK2 at a concentration of 1 mM were administered through the secured terminal line. The dosage was 0.1 mL. The fluorescence in the abdominal cavity when the abdomen was opened 20 minutes after the administration is shown in Figure 9, and the fluorescence of the dorsum of the foot is shown in Figure 12 (image at the time of reaching the maximum brightness).

Figure 9 shows that ICG accumulates primarily in the liver (Figure 9(a)), TK1 primarily in the kidneys (Figure 9(b)), and TK2 also primarily in the kidneys (Figure 9(c)). Near-infrared imaging of internal organs immediately after administration demonstrated clear imaging of the kidneys, ureters, and bladder (Figure 10). Furthermore, near-infrared endoscopy demonstrated the ability to image the ureters (Figure 11).

As can be seen from image 12, fluorescence is clearly observed corresponding to blood flow in the dorsum of the foot. Furthermore, although not shown in the figure, fluorescence was observed after administering ICG, TK1, and TK2 to a rat model of lower limb ischemia in which the right femoral artery was ligated. Since no fluorescence was observed in the ischemic area, the presence or absence of fluorescence can be used to assess the extent of blood flow.

13 and 14 show the temporal changes in fluorescence intensity of the dorsum of the foot. Table 1 shows the time to maximum fluorescence intensity (Imax), the Imax value, and the time to half the Imax value (t _1/2 ).

[Table 1]

Compound
				ICG	0.5min	124	2.3min
TK-1	10min	160	40min
				TK-2	15min	106	55min

As shown in Table 1, Figures 13, and 14, TK1 and TK2 both took longer to reach maximum fluorescence intensity and to reach half-value in time than ICG. This suggests that they can emit fluorescence for a long time in vivo. For example, while ICG's fluorescence intensity drops to its initial value within 10 minutes after administration, TK1 and TK2 exhibit high fluorescence intensity even after one hour.

Observation at the vascular level

According to the method disclosed in non-patent literature 7 and 8 (Figure 15), ICG and TK1 were administered to rats with their blood vessels directly exposed for observation. The situation of the spermatozoa cremaster flap after exposure is shown in Figure 16. As a result, ICG appeared uneven in the blood vessels relative to TK1. In other words, it can be seen that TK1 moved from the blood vessels to the interstitial fluid. In order to conduct a more precise study, microscopic photographs of the blood vessels after administration are shown in Figures 17 (ICG) and 18 (TK1). As can be seen from Figures 17 and 18, when the fluorescence intensity in the blood vessels and the fluorescence intensity in the surrounding tissues are compared, ICG can hardly confirm fluorescence in the interstitial tissue, while TK1 can confirm fluorescence in the interstitial tissue. In addition, although not shown in detail, it is confirmed that the fluorescence intensity in the interstitial tissue increases over time. Therefore, ICG can hardly confirm movement from the blood vessels to the interstitial fluid, but TK1 can confirm movement from the blood vessels to the interstitial fluid.

Evaluation of the correlation between the extent of migration of the cyclodextrin-bound indocyanine compound of the present invention within the interstitium and the magnitude of edema formation

(Evaluation method)

Ten-week-old male Wister rats weighing approximately 350 g were selected as experimental animals (n=4). The rats were anesthetized with isoflurane and injected with 0.1 mL of 0.5% λ carrageenan into the plantar surface of the left hind paw using a 26G needle. Paw edema rapidly developed. Fifteen minutes later, the rats' left paw volume was measured using a rat paw volume meter (MK-101CMP PLETHYSMOMETER, manufactured by Muromachi Machinery Co., Ltd.).

Afterward, isoflurane was turned off and the rat was confirmed to be awake. After the Von Frey test, general anesthesia was re-administered with isoflurane. The rat's tail vein was secured with a 24G indwelling cannula and then secured in a supine position on a horizontal platform. A PDE camera manufactured by Hamamatsu Photonics Co., Ltd. was set up and fixed at a height of 16 cm from the dorsum of the rat's foot.

The PDE camera was used to set the measurement area (ROI) to the central part of the dorsum of the foot. After the measurement began, 0.1 mL of a 1 mmol TK1 aqueous solution was injected through the tail vein, followed by a 5-second flush with 1 mL of saline. Continuous imaging was performed for the first 5 minutes of injection, and the ROI brightness was measured. Thereafter, brightness measurements were taken at 5-minute intervals for 1 minute, continuing until 120 minutes after injection. After the measurement was completed, isoflurane was turned off and the subject returned to consciousness (acute inflammation experiment).

After 7 days, the rats were subjected to Von Frey test, and then the rats were quickly anesthetized with isoflurane. The volume of the rat's left foot was measured using a rat plantar volume meter. After the rat's tail vein was secured with a 24G indwelling needle, it was fixed on a horizontal platform in a supine position. A PDE camera manufactured by Hamamatsu Photonics Co., Ltd. was set up and fixed at a height of 16 cm from the dorsum of the rat's foot. The ROI was set at the center of the dorsum of the foot, and the measurement was started after the light was turned off. 0.1 mL of 1 mmol TK1 aqueous solution was injected from the tail vein and rinsed with 1 mL of normal saline for 5 seconds. Continuous shooting was performed during the first 5 minutes of injection to measure the brightness of the ROI. The brightness was measured for 1 minute at intervals of 5 minutes thereafter, and this continued until 120 minutes after the injection. After the measurement was completed, isoflurane was turned off and the rat returned to consciousness. The same test was performed on the group that was not injected with λ carrageenan.

ICG also implemented the above process.

(result)

The results are shown in Figures 19 to 26. The left paw volume of the rats measured for the group administered with TK1 is shown in Figure 19 (the volume increases toward the top of the vertical axis), the results of the Von Frey test are shown in Figure 20 (the more downward the vertical axis, the more hyperalgesia), the changes in brightness shortly after the injection are shown in Figure 21 (the brightness increases toward the top of the vertical axis), and the changes in brightness one week after the injection of carrageenan are shown in Figure 23 (the brightness increases toward the top of the vertical axis). The left paw volume of the rats measured for the group administered with ICG is shown in Figure 23 (the volume increases toward the top of the vertical axis), the results of the Von Frey test are shown in Figure 24 (the more downward the vertical axis, the more hyperalgesia), the changes in brightness shortly after the injection are shown in Figure 25 (the brightness increases toward the top of the vertical axis), and the changes in brightness one week after the injection of carrageenan are shown in Figure 26 (the brightness increases toward the top of the vertical axis). The values of the vertical axes of all the graphs are arbitrary units.

Significant differences were observed in the patterns of brightness changes within the ROI between ICG and TK1. The brightness rapidly increased shortly after ICG administration, then rapidly decreased, returning to baseline after approximately 10 minutes. In contrast, the initial brightness increase after TK1 administration followed the same trajectory as ICG, with the brightness remaining high thereafter and requiring six hours to return to baseline. Based on these results, we believe that ICG is retained within the blood vessels and does not leak into the interstitium. Therefore, the rapid brightness increase and subsequent rapid decrease are due to passage through the blood vessels distributed within the ROI. In contrast, TK1 exhibits two phases: the initial rapid passage into the blood vessels (hereafter referred to as the "vascular phase") and the subsequent phase in which TK1 fluorescence gradually leaks into the interstitium (hereafter referred to as the "interstitial phase"). The transition from the vascular phase to the interstitial phase corresponds to the specified time.

In the acute inflammation experiment, a significant increase in paw volume was observed in both the ICG and TK1 groups after injection of λ carrageenan, which returned to normal, similar to the tendon side, one week later (Figures 19 and 23). Furthermore, in the Von Frey test, significant hyperalgesia was observed in both the ICG and TK1 groups after injection of λ carrageenan, but returned to normal, similar to the tendon side, one week later (Figures 20 and 24).

Regarding the brightness of the dorsum of the foot, there was no difference between the TK1 group and the control group in the brightness change of the vascular phase, but the brightness in the interstitial phase increased more rapidly than the λ carrageenan-administered group, and maintained a high value throughout the evaluation time (Figure 21). One week after the administration of λ carrageenan and the inflammation subsided, there was no difference in the brightness change of the interstitial phase between the carrageenan-administered side and the tendon side (Figure 22). As the curve in Figure 21 clearly shows, the brightness in the interstitial phase changes in a smooth curve, and the height of the curve depends on the upward rate of change of the interstitial phase. In other words, the change in the value of the interstitial phase can be predicted with high precision based on time series analysis, which is an important basis for the validity of our hypothesis in this work.

No difference in brightness was observed in the ICG group either immediately or one week after injection with lambda carrageenan (Figures 25 and 26). This result indicates that the highly hydrophobic ICG, which is currently used clinically, does not leak fluorescent material outside of blood vessels even in the presence of severe acute inflammation caused by lambda carrageenan.

These results demonstrate that after TK1 administration to a living body, fluorescence intensity at sites predicted for swelling progression can be measured over time during a period corresponding to the interstitial phase. This allows for prediction of subsequent changes in fluorescence intensity, which correlates with the progression of swelling at the site. Therefore, TK1 is suitable for predicting swelling, which is not possible with ICG.

Industrial Application Possibilities

The cyclodextrin-bound indocyanine compounds represented by Chemical Formula 1 or Chemical Formula 2 of the present invention provide green pigment compounds that emit near-infrared fluorescence and have the following characteristics: higher solubility in water or physiological saline compared to ICG, easier removal from biological tissues, lower molecular association in aqueous solutions, higher near-infrared fluorescence intensity in aqueous solutions, and the absence of iodine. Furthermore, the method for synthesizing the cyclodextrin-bound indocyanine compounds of the present invention can provide for the synthesis of useful cyclodextrin-bound indocyanine compounds. Furthermore, the method for purifying the cyclodextrin-bound indocyanine compounds of the present invention can provide for the purification of useful cyclodextrin-bound indocyanine compounds. Furthermore, because the cyclodextrin-bound indocyanine compounds of the present invention exhibit sufficient solubility even without iodine, they can provide diagnostic compositions that are free of iodine, a cause of iodine allergy. These diagnostic compositions exhibit different in vivo behavior than conventional diagnostic compositions containing only ICG, and their properties can be exploited to provide a variety of useful diagnostic methods and diagnostic devices.

This patent application is a divisional application of the patent application with application number 201180006735.X, application date January 28, 2011, and invention name "New indocyanine compound, synthesis method and purification method thereof, diagnostic composition using the indocyanine compound, in vivo dynamic measurement device using the diagnostic composition, and circulation visualization device".

Technical Field

The present invention relates to a new indocyanine compound that is a green pigment useful in medical diagnosis, medical surgery, scientific measurement and analysis, printing, note-taking, coating, dyeing, and staining technologies and has the property of emitting near-infrared fluorescence, a method for synthesizing and purifying the same, and a diagnostic composition. Specifically, the invention relates to a cyclic sugar chain cyclodextrin-bound indocyanine compound that is a green pigment and has the property of emitting near-infrared fluorescence, a method for synthesizing and purifying the same, a diagnostic composition using the indocyanine compound, an in vivo dynamics measurement device using the diagnostic composition, and a circulation visualization device.

Background Art

A variety of green pigments, indocyanine compounds that emit near-infrared fluorescence, have been synthesized. These compounds have a wide range of applications in the medical field, for example, as dyes for vitreous surgery, medicinal pigments for liver function tests, medicinal pigments for circulatory function tests, surgical pigments, and near-infrared fluorescent compounds for surgery. In the scientific field, they serve as dyes or compounds for dyeing or fluorescing proteins, sugars, and other substances, and as pigments in printing technology. Among these indocyanine compounds, indocyanine green (hereinafter referred to as "ICG") has been used for nearly 50 years as a drug for liver and circulatory function tests. In recent years, ICG's high light transmittance through biological tissues has been exploited to allow for local administration of ICG to, for example, blood vessels, lymphatic vessels, brain, eyes, stomach, breasts, esophagus, skin, and other sites within the body, thereby allowing for medical procedures and diagnostics by observing its near-infrared fluorescence (Non-Patent Document 1).

Prior art literature

Non-patent literature

Non-patent literature 1: "All about ICG Fluorescence Navigation Surgery", supervised and edited by Mitsuo Kusano, インターメディカ社, (published in November 2008).

Non-Patent Document 2: URL of the Internet homepage described in the instructions for "(Trade Name) Diagnogreen Injection 25 mg" (Daiichi Sankyo Co., Ltd.) (https://www.daiichisankyo.co.jp/med/contents/di/dg2/pi/pdf/pi_dg2_0909.pdf)

Non-patent document 3: R.C. Benson, H.A. Kues, Phys. Med. Biol., 23, 159-163 (1978).

Non-patent document 4: S. Yoneyama, T. Saito, Y. Komatsu, I. Koyama, K. Takahashi, J. Duvoll-Young, IOVS, 37, 1286-1290 (1998).

Non-patent document 5: Y. Ye, W. P. Li, C. J. Anderson, J. Kao, G. V. Nikiforovich, S. Achilefu, J. Am. Chem. Soc., 125, 7766-7767 (2003).

Non-patent literature 6: K. Teranishi and S. Tanabe, ITE Letters on Batteries, New Technologies & Medicine, 1, 53-60 (2000).

Non-patent document 7: MICHAEL O’SHAUGHNESSY et al: 1994 Wiley-Liss, Inc MICROSURGERY 15: 40S412 1994.

Non-patent document 8: RALPH J.P.M et al: 1997 Wiley-Liss, Inc. MICROSURGERY 17: 402-408 1996.

Summary of the Invention

Technical issues

However, the green chromophore (the chemical structure required for the green color) and near-infrared fluorophore (the chemical structure required for near-infrared fluorescence) of ICG are hydrophobic, and sulfonyl groups are attached to the side chain terminals to make them water-soluble. Therefore, existing ICG has the following problems.

In general medical use, ICG preparations are prepared by adding approximately 5 to 10 mL of distilled water to 25 mg of ICG and stirring with vibration to dissolve it. Failure to completely dissolve ICG can cause symptoms such as nausea, retching, fever, and shock (Non-Patent Document 2). Furthermore, because ICG does not dissolve completely, it cannot be initially dissolved in other aqueous solutions such as saline (Non-Patent Document 2).

While ICG is water-soluble due to the sulfonyl groups attached to it, its chemical structure contains many hydrophobic hydrocarbon groups, making it surface-active and prone to adsorption to lipids. Consequently, when injected into tissues such as blood vessels and organs, it can sometimes adhere to non-target tissues by adhering to the injection site, leaking through the injection site, or flowing backward. ICG adhered to tissues is difficult to remove through wiping or aspiration, potentially hindering surgical procedures and medical diagnostics.

ICG has a molecular association property in aqueous solution, one of the reasons for which is its low fluorescence intensity in aqueous solution (Non-Patent Documents 3 and 4).

Furthermore, ICG becomes insoluble over time after being dissolved in water, making it difficult to store it as an aqueous solution for a long period of time. Furthermore, freezing at low temperatures promotes its insolubility.

Furthermore, there is a problem that an ICG preparation containing 5% or less of NaI may cause iodine hypersensitivity in some cases (Non-Patent Document 2).

In addition, ICG rapidly accumulates in the liver and is excreted by the liver when injected intravenously, making fluorescence imaging of other organs such as the kidneys, ureters, bladder, urethra, heart, and lungs difficult.

Furthermore, ICG migrates with the blood when injected intravenously, so its migration to peripheral tissues is minimal, making it difficult to observe its migration to the interstitium.

The present invention aims to resolve the aforementioned problems with existing ICG. Specifically, it aims to provide a new indocyanine compound that is a green pigment and emits near-infrared fluorescence. The compound is characterized by high solubility in water or physiological saline, easy removal from biological tissues, low molecular association in aqueous solution, and high near-infrared fluorescence intensity in aqueous solution, enabling fluorescence imaging of organs other than the liver, such as the kidneys, ureters, bladder, urethra, heart, and lungs. Furthermore, a chemical synthesis method and purification method for the new indocyanine compound with these characteristics are provided. Furthermore, a diagnostic composition containing the new indocyanine compound is also provided. Furthermore, a method and apparatus for visualizing the circulation of blood, lymph, urine, and other water components in a living organism is provided using these diagnostic compositions.

Technical solutions to the problem

The present inventors have conducted intensive research to solve the above problems and have discovered compounds that can emit near-infrared fluorescence. These compounds can be used in surgical procedures and medical diagnoses that utilize the properties of near-infrared fluorescence, thereby solving the above-mentioned problems with ICG.

Specifically, the present invention has been completed by the discovery of a new indocyanine compound characterized by being a green pigment that emits near-infrared fluorescence, having high solubility in water or physiological saline, being easily removed from biological tissues, exhibiting low molecular association in aqueous solutions, and exhibiting high near-infrared fluorescence intensity in aqueous solutions. Furthermore, the present invention has been completed by the discovery of a chemical synthesis method and a purification method for the new indocyanine compound. Furthermore, a diagnostic composition containing the new indocyanine compound is provided. Furthermore, a method for performing fluorescence imaging of organs other than the liver using the diagnostic composition, even during intravenous administration, is provided. Furthermore, a method for evaluating the in vivo dynamics of the new indocyanine compound related to, for example, water balance in a living body and a device for measuring the in vivo dynamics are provided, as well as a method and device for visualizing the circulation of blood, lymph, urine, and other water in a living body.

The first new indocyanine compound of the present invention is a compound in which a cyclic sugar chain cyclodextrin is covalently bound to the green chromophore (chemical structure required for green color) and the near-infrared fluorophore (chemical structure required for emitting near-infrared fluorescence) of ICG. In addition, the second new indocyanine compound of the present invention is characterized in that the naphthyl site belonging to the hydrophobic site of the indocyanine structure is included in the cavity of the cyclodextrin, thereby covering the naphthyl site belonging to the hydrophobic site with a hydrophilic glucose group, so that most areas of the indocyanine molecular structure become three-dimensionally hydrophilic. Therefore, compared with the characteristic of ICG having similar surface activity due to the hydrophilicity of both the hydrophobic site and the sulfonyl group, the compound of the present invention is characterized in that the hydrophobic site in its molecule is covered by cyclodextrin, so it does not have similar surface activity properties. Specifically, the present invention is as follows:

<1> A cyclodextrin-bound indocyanine compound represented by Chemical Formula 1, characterized in that the indocyanine is covalently bound to a cyclic sugar chain cyclodextrin;

[Chemical Formula 1]

(R ₁ , R ₂ , R ₃ , R ₄ , R ₅ , R ₆ , R ₇ , R ₈ , R ₉ , R ₁₀ , R ₁₁ , R ₁₂ , R ₁₃ , R 14 , R ₁₅ , R ₁₆ , R ₁₇ , R ₁₈ , R ₁₉ , R ₂₀ , R _in the formula ₂₁ , R ₂₂ , R _R23 is a hydrogen atom, an alkyl group, an aryl group, a halogen atom, an alkoxy group, an amino group, a carboxyl group, a formyl group, a sulfonyl group, a sulfonic acid group, a phosphate group, an alkoxycarbonyl group, an aryloxycarbonyl group, an alkylcarbonyl group, an arylcarbonyl group, or a heterocycle. Alternatively, it may be a substituent thereof (carboxylic acid, sulfonic acid, phosphoric acid). When the hydrogen ion in the substituent is dissociated, the hydrogen ion may be replaced by a metal ion such as sodium ion, potassium ion, magnesium ion, or calcium ion. The amino group may be selected from primary, secondary, tertiary, and quaternary groups ₍ alkyl groups may be exemplified as substituents bonded to nitrogen). Alternatively, _R8 and _R9 may _be selected _{from CH2} , _{CH2CH2 , CH2CH2CH2} , or _{CH2CH2CH2CH2 . 2.} Alternatively, the hydrogen atoms on these alkyl groups may be replaced by alkyl groups, aryl groups, halogen atoms, alkoxy groups, amino groups, carboxyl groups, formyl groups, sulfonyl groups, sulfonic acid groups, phosphoric acid groups, alkoxycarbonyl groups, aryloxycarbonyl groups, alkylcarbonyl groups, arylcarbonyl groups or heterocyclic groups.)

<2> The cyclodextrin-bound indocyanine compound represented by Chemical Formula 2 is characterized in that at least a portion of the naphthyl group of the indocyanine is included in the cavity of the cyclodextrin.

[Chemical Formula 2]

(R ₁ , R ₂ , R ₃ , R ₄ , R ₅ , R ₆ , R ₇ , R ₈ , R ₉ , R ₁₀ , R ₁₁ , R ₁₂ , R ₁₃ , R 14 , R ₁₅ , R ₁₆ , R ₁₇ , R ₁₈ , R ₁₉ , R ₂₀ , R _in the formula ₂₁ , R ₂₂ , R ₂₃ is a hydrogen atom, an alkyl group, an aryl group, a halogen atom, an alkoxy group, an amino group, a carboxyl group, a formyl group, a sulfonyl group, a sulfonic acid group, a phosphate group, an alkoxycarbonyl group, an aryloxycarbonyl group, an alkylcarbonyl group, an arylcarbonyl group, or a heterocycle. Alternatively, it may be a substituent thereof (carboxylic acid, sulfonic acid, phosphate). When the hydrogen ion in the substituent is dissociated, the hydrogen ion may be replaced by a metal ion such as a sodium ion, potassium ion, magnesium ion, or calcium ion. The amino group is selected from primary, secondary, tertiary, and quaternary groups. Furthermore, R ₈ and R ₉ may be selected from a cyclic structure of CH ₂ , CH ₂ CH ₂ , CH ₂ CH ₂ CH ₂ , or CH ₂ CH ₂ CH ₂ CH ₂ . Alternatively, a functional group in which the hydrogen atom on these alkyl groups is replaced by an alkyl group, an aryl group, a halogen atom, an alkoxy group, an amino group, a carboxyl group, a formyl group, a sulfonyl group, a sulfonic acid group, a phosphate group, an alkoxycarbonyl group, an aryloxycarbonyl group, an alkylcarbonyl group, an arylcarbonyl group, or a heterocycle may be selected.)

<3> The cyclodextrin-bound indocyanine compound represented by Chemical Formula 3 is characterized in that it is the cyclodextrin-bound indocyanine compound represented by Chemical Formula 1 in which an indocyanine and a cyclic sugar chain cyclodextrin are covalently bound via an amide bond.

[Chemical Formula 3]

(R ₁ , R ₂ , R ₃ , R ₄ , R ₅ , R ₆ , R ₇ , R ₈ , R ₉ , R ₁₀ , R ₁₁ , R ₁₂ , R ₁₃ , R 14 , R ₁₅ , R ₁₆ , R ₁₇ , R ₁₈ , R ₁₉ , R ₂₀ , R _in the formula ₂₁ , R ₂₂ , R _R23 is a hydrogen atom, an alkyl group, an aryl group, a halogen atom, an alkoxy group, an amino group, a carboxyl group, a formyl group, a sulfonyl group, a sulfonic acid group, a phosphate group, an alkoxycarbonyl group, an aryloxycarbonyl group, an alkylcarbonyl group, an arylcarbonyl group, or a heterocycle. Alternatively, it may be a substituent thereof (carboxylic acid, sulfonic acid, phosphoric acid), wherein the hydrogen ion in the substituent, when dissociated, may be replaced by a metal ion such as sodium ion, potassium ion, magnesium ion, or calcium ion. The amino group is selected from primary, secondary, tertiary, and _quaternary groups. m and n are integers from 1 to 6. Furthermore, _R8 and _R9 may _{be selected from CH2} , _CH2CH2 , _CH2CH2CH2 , or _{CH2CH2CH2CH2 . 2.} In addition, the functional groups in which the hydrogen atoms on these alkyl groups are replaced by alkyl groups, aryl groups, halogen atoms, alkoxy groups, amino groups, carboxyl groups, formyl groups, sulfonyl groups, sulfonic acid groups, phosphoric acid groups, alkoxycarbonyl groups, aryloxycarbonyl groups, alkylcarbonyl groups, arylcarbonyl groups or heterocyclic rings can also be selected.)

<4> Chemical formula 4 represents a cyclodextrin-bound indocyanine compound, characterized in that it is a cyclodextrin-bound indocyanine compound represented by Chemical formula 2, in which indocyanines and cyclic sugar chain cyclodextrin are covalently bound via an amide bond.

[Chemical Formula 4]

(R ₁ , R ₂ , R ₃ , R ₄ , R ₅ , R ₆ , R ₇ , R ₈ , R ₉ , R ₁₀ , R ₁₁ , R ₁₂ , R ₁₃ , R 14 , R ₁₅ , R ₁₆ , R ₁₇ , R ₁₈ , R ₁₉ , R ₂₀ , R _in the formula ₂₁ , R ₂₂ , R _R23 is a hydrogen atom, an alkyl group, an aryl group, a halogen atom, an alkoxy group, an amino group, a carboxyl group, a formyl group, a sulfonyl group, a sulfonic acid group, a phosphate group, an alkoxycarbonyl group, an aryloxycarbonyl group, an alkylcarbonyl group, an arylcarbonyl group, or a heterocycle. Alternatively, it may be a substituent thereof (carboxylic acid, sulfonic acid, phosphoric acid), wherein the substituent can be replaced by a metal ion such as sodium ion, potassium ion, magnesium ion, or calcium ion when the hydrogen ion dissociates. The amino group is selected from primary, secondary, tertiary, and quaternary groups. _m and n are integers from 1 to 6. Furthermore, _R8 and _R9 may _be selected _{from CH2} , _CH2CH2 , _{CH2CH2CH2 ,} or _{CH2CH2CH2CH2 . 2.} In addition, the functional groups in which the hydrogen atoms on these alkyl groups are replaced by alkyl groups, aryl groups, halogen atoms, alkoxy groups, amino groups, carboxyl groups, formyl groups, sulfonyl groups, sulfonic acid groups, phosphoric acid groups, alkoxycarbonyl groups, aryloxycarbonyl groups, alkylcarbonyl groups, arylcarbonyl groups or heterocyclic rings can also be selected.)

<5> The cyclodextrin-bound indocyanine compound represented by Chemical Formula 5 among the cyclodextrin-bound indocyanine compounds represented by Chemical Formula 3.

[Chemical Formula 5]

(In the formula, m, n, p, and q are integers of 2 to 6. r is an integer of 5 to 7. s is an integer of 0 to 4. R is a hydrogen atom, an alkyl group, an aryl group, a halogen atom, an alkoxy group, an amino group, a carboxyl group, a formyl group, a sulfonyl group, a sulfonic acid group, an alkoxycarbonyl group, an aryloxycarbonyl group, an alkylcarbonyl group, an arylcarbonyl group, or a heterocycle.)

<6> The cyclodextrin-bound indocyanine compound represented by Chemical Formula 6 among the cyclodextrin-bound indocyanine compounds represented by Chemical Formula 4.

[Chemical Formula 6]

(In the formula, m, n, p, and q are integers of 2 to 6. r is an integer of 5 to 7. s is an integer of 0 to 4. R is a hydrogen atom, an alkyl group, an aryl group, a halogen atom, an alkoxy group, an amino group, a carboxyl group, a formyl group, a sulfonyl group, a sulfonic acid group, an alkoxycarbonyl group, an aryloxycarbonyl group, an alkylcarbonyl group, an arylcarbonyl group, or a heterocycle.)

<7> The cyclodextrin-bound indocyanine compound represented by Chemical Formula 7 among the cyclodextrin-bound indocyanine compounds represented by Chemical Formula 3.

[Chemical Formula 7]

(In the formula, m and n are integers of 2 to 6. r is an integer of 5 to 7. s is an integer of 0 to 4. R is a hydrogen atom, an alkyl group, an aryl group, a halogen atom, an alkoxy group, an amino group, a carboxyl group, a formyl group, a sulfonyl group, a sulfonic acid group, an alkoxycarbonyl group, an aryloxycarbonyl group, an alkylcarbonyl group, an arylcarbonyl group, or a heterocycle.)

<8> The cyclodextrin-bound indocyanine compound represented by Chemical Formula 8 among the cyclodextrin-bound indocyanine compounds represented by Chemical Formula 4.

[Chemical Formula 8]

(In the formula, m and n are integers of 2 to 6. r is an integer of 5 to 7. s is an integer of 0 to 4. R is a hydrogen atom, an alkyl group, an aryl group, a halogen atom, an alkoxy group, an amino group, a carboxyl group, a formyl group, a sulfonyl group, a sulfonic acid group, an alkoxycarbonyl group, an aryloxycarbonyl group, an alkylcarbonyl group, an arylcarbonyl group, or a heterocycle.)

<9> The cyclodextrin-bound indocyanine compound represented by Chemical Formula 9 among the cyclodextrin-bound indocyanine compounds represented by Chemical Formula 3.

[Chemical Formula 9]

(In the formula, m and n are integers of 2 to 6. r is an integer of 5 to 7. s is an integer of 0 to 4. R is a hydrogen atom, an alkyl group, an aryl group, a halogen atom, an alkoxy group, an amino group, a carboxyl group, a formyl group, a sulfonyl group, a sulfonic acid group, an alkoxycarbonyl group, an aryloxycarbonyl group, an alkylcarbonyl group, an arylcarbonyl group, or a heterocycle.)

<10> The cyclodextrin-bound indocyanine compound represented by Chemical Formula 10 among the cyclodextrin-bound indocyanine compounds represented by Chemical Formula 4.

[Chemical Formula 10]

(In the formula, m and n are integers of 2 to 6. r is an integer of 5 to 7. s is an integer of 0 to 4. R is a hydrogen atom, an alkyl group, an aryl group, a halogen atom, an alkoxy group, an amino group, a carboxyl group, a formyl group, a sulfonyl group, a sulfonic acid group, an alkoxycarbonyl group, an aryloxycarbonyl group, an alkylcarbonyl group, an arylcarbonyl group, or a heterocycle.)

<11> The cyclodextrin-bound indocyanine compound represented by Chemical Formula 11 among the cyclodextrin-bound indocyanine compounds represented by Chemical Formula 3.

[Chemical Formula 11]

(s is an integer from 0 to 4. R is a hydrogen atom, an alkyl group, an aryl group, a halogen atom, an alkoxy group, an amino group, a carboxyl group, a formyl group, a sulfonyl group, a sulfonic acid group, an alkoxycarbonyl group, an aryloxycarbonyl group, an alkylcarbonyl group, an arylcarbonyl group, or a heterocyclic ring.)

<12> The cyclodextrin-bound indocyanine compound represented by Chemical Formula 12 among the cyclodextrin-bound indocyanine compounds represented by Chemical Formula 4.

[Chemical Formula 12]

(s is an integer from 0 to 4. R is a hydrogen atom, an alkyl group, an aryl group, a halogen atom, an alkoxy group, an amino group, a carboxyl group, a formyl group, a sulfonyl group, a sulfonic acid group, an alkoxycarbonyl group, an aryloxycarbonyl group, an alkylcarbonyl group, an arylcarbonyl group, or a heterocyclic ring.)

<13> The chemical synthesis method of the cyclodextrin-conjugated indocyanine compound described in any one of the above chemical formulas (Chemical Formulas 1, 3, 5, 7, 9, and 11) is characterized by comprising: (1) a step of mixing an indocyanine carboxylic acid compound and an aminocyclodextrin in a solvent; and (2) a step of adding a dehydration condensation agent to carry out a dehydration condensation reaction.

<14> A chemical synthesis method of a cyclodextrin-bound indocyanine compound according to any one of the above chemical formulas (Chemical Formulas 2, 4, 6, 8, 10, 12), characterized in that the cyclodextrin-bound indocyanine compound according to any one of the above chemical formulas (Chemical Formulas 1, 3, 5, 7, 9, 11) is subjected to an inclusion reaction in water.

<15> The cyclodextrin-bound indocyanine compound represented by any one of the above chemical formulae (Chemical Formulae 1 to 12) is purified by column chromatography in which the compound is eluted with a solvent containing HCl.

<16> A diagnostic composition to be used by injection into the body, comprising an aqueous solution containing the cyclodextrin-bound indocyanine compound of any one of the above Chemical Formulas 1 to 12.

<17> The diagnostic composition according to <16>, which contains substantially no iodine.

<18> A device for measuring the dynamics of a cyclodextrin-bound indocyanine compound in vivo, comprising:

an excitation light irradiation unit for irradiating the cyclodextrin-bound indocyanine compound with excitation light, with respect to a part of a living body to which the diagnostic composition according to <16> or <17> is administered;

a fluorescence intensity measuring unit for measuring the intensity of fluorescence emitted by the cyclodextrin-bound indocyanine compound excited by the excitation light irradiation unit; and

The in vivo dynamic calculation unit calculates the temporal change of the time rate of change of the fluorescence intensity from the fluorescence intensity obtained over time from the fluorescence intensity measuring unit, thereby calculating the speed of the cyclodextrin-bound indocyanine compound moving into the interstitial fluid and/or the speed of moving out of the interstitium in a part of the biological body.

<19> A circulation visualization device, comprising:

an excitation light irradiation unit for irradiating the cyclodextrin-bound indocyanine compound with excitation light, with respect to a part of a living body to which the diagnostic composition according to <16> or <17> is administered;

a fluorescence imaging unit for two-dimensionally acquiring the intensity of the fluorescence emitted by the cyclodextrin-bound indocyanine compound excited by the excitation light irradiation unit, thereby obtaining data on the distribution state of the cyclodextrin-bound indocyanine compound in the organism;

a morphological imaging unit that two-dimensionally acquires the intensity of light of wavelengths other than the fluorescence wavelength emitted by the cyclodextrin-bound indocyanine compound, thereby obtaining morphological data of a portion of the biological body; and

The display unit displays the distribution state of the cyclodextrin-bound indocyanine compound in a part of the biological body by superimposing the morphological data obtained by the morphological imaging unit and the distribution state data obtained by the fluorescence imaging unit.

<20> The circulation visualization device according to <19>, wherein the display unit displays a portion of the living body where the distribution amount of the cyclodextrin-bound indocyanine compound is lower than a predetermined reference as a necrotic portion.

<21> The in vivo dynamics measuring device according to <18> above, comprising a swelling progression prediction unit for predicting the degree of swelling of the part of the living body based on the speed of the swelling moving from the part of the living body into and out of the interstitial fluid.

<22> The in vivo dynamic measurement device described in <21> above, wherein the swelling progression prediction unit predicts the degree of swelling progression corresponding to the magnitude of the migration speed into the interstitial fluid after a predetermined time, wherein the predetermined time is the time from the administration of the diagnostic composition to the dispersion of the cyclodextrin-conjugated indocyanine compound in the blood throughout the body.

<23> An in vivo dynamics measurement device, characterized by comprising:

The excitation light irradiation unit irradiates the cyclodextrin-bound indocyanine compound with excitation light at a portion of the living body to which the diagnostic composition according to <16> or <17> is administered and at a control site.

a fluorescence intensity measuring unit for measuring the intensity of fluorescence emitted by the cyclodextrin-bound indocyanine compound excited by the excitation light irradiation unit;

an in vivo dynamic calculation unit for calculating the time-dependent change in the fluorescence intensity obtained from the fluorescence intensity measurement unit over time, thereby calculating the migration speed of the cyclodextrin-bound indocyanine compound into the interstitial fluid and/or the migration speed out of the interstitial fluid in a part of the organism, and

a swelling progression prediction unit for predicting a degree of swelling that will progress in the part of the living body based on a rate of movement of the part of the living body into and out of the interstitial fluid;

wherein the above swelling is carried out in a prediction unit,

determining the relationship between the blood flow in the control site and the blood flow in the portion of the living body based on changes in fluorescence intensity in the portion of the living body and changes in fluorescence intensity in the control site until a predetermined time has passed, wherein the predetermined time is the time from administration of the diagnostic composition to dispersion of the cyclodextrin-bound indocyanine compound into blood throughout the body;

The degree of change in the fluorescence intensity of the part of the biological body after the predetermined time has passed, with the change in the fluorescence intensity of the control part serving as a control, is calculated using this relationship.

The progression of the swelling is predicted based on the calculated degree of change.

<24> An in vivo dynamics measurement device, characterized by comprising:

an excitation light irradiation unit for irradiating the cyclodextrin-bound indocyanine compound with excitation light to a living organ to which the diagnostic composition according to <16> or <17> is administered;

a fluorescence intensity measuring unit for measuring the intensity of fluorescence emitted by the cyclodextrin-bound indocyanine compound excited by the excitation light irradiation unit; and

The in vivo dynamics calculation unit evaluates the in vivo dynamics of the cyclodextrin-bound indocyanine compound in the organ based on the fluorescence intensity acquired over time by the fluorescence intensity measurement unit.

<25> The in vivo dynamics measuring device according to <24>, wherein the organ is any one of a kidney, a ureter, a bladder, and a urethra.

Beneficial effects

The cyclodextrin-bound indocyanine compounds represented by Chemical Formula 1 or Chemical Formula 2 of the present invention provide green pigment compounds that emit near-infrared fluorescence and have the following characteristics: higher solubility in water or physiological saline compared to ICG, easier removal from biological tissues, lower molecular association in aqueous solutions, higher near-infrared fluorescence intensity in aqueous solutions, and the ability to perform fluorescence imaging of organs other than the liver. Furthermore, the cyclodextrin-bound indocyanine compound synthesis method of the present invention provides for the synthesis of useful cyclodextrin-bound indocyanine compounds. Furthermore, the cyclodextrin-bound indocyanine compound purification method of the present invention provides for the purification of useful cyclodextrin-bound indocyanine compounds. Furthermore, the cyclodextrin-bound indocyanine compounds of the present invention exhibit sufficient solubility even without iodine, thus providing diagnostic compositions that do not contain iodine, a cause of iodine allergy. These diagnostic compositions exhibit different in vivo behavior than conventional diagnostic compositions containing only ICG, and their properties can be exploited to provide various beneficial devices.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the results of a test on the adsorption of ICG and the isomeric equilibrium compound (TK1) represented by Chemical Formulas 19 and 20 to human skin. The images show the results of a 1 mM aqueous solution (0.03 mL) of ICG and the isomeric equilibrium compound (TK1) represented by Chemical Formulas 19 and 20 applied to the wrist and ingested immediately (left photo), 5 minutes later and immediately after rinsing with water (center photo), and then immediately after wiping and rinsing with water (right photo). The dots on the right are marked with a red pen.

Figure 2 shows the results of a test on the adsorption of ICG and the isomeric equilibrium compound (TK1) represented by Chemical Formulas 19 and 20 onto cellulose fibers. The images show the results of a cotton swab application of a 1 mM aqueous solution (0.05 mL) of ICG and the isomeric equilibrium compound (TK1) represented by Chemical Formulas 19 and 20, followed by immediate uptake (top photo), and then 3 minutes later, followed by 5 seconds of rinsing with water, followed by immediate uptake (bottom photo).

Figure 3 shows the results of a test on the adsorption of ICG and the isomeric equilibrium compound (TK1) represented by Chemical Formulas 19 and 20 into a biological meat model. The images show a 1 mM aqueous solution (0.05 mL) of ICG and the compound represented by Chemical Formula 20 applied to a 5 mm diameter depression in pork tenderloin immediately after ingestion (left photo), and then rinsed with water for 10 seconds 3 minutes later, followed by immediate ingestion (right photo).

Figure 4 shows the results of a test on the adsorption of ICG and the isomeric equilibrium compound (TK1) represented by Chemical Formulas 19 and 20 onto a biological protein model. The images show a 1 mM aqueous solution (0.05 mL) of ICG and the isomeric equilibrium compound (TK1) represented by Chemical Formulas 19 and 20 applied to a 5 mm diameter depression in chicken breast immediately after ingestion (left photo), and then rinsed with water for 10 seconds 3 minutes later, followed by immediate ingestion (right photo).

Figure 5 shows the test results of the adsorption of ICG and the isomeric equilibrium compound (TK1) represented by Chemical Formulas 19 and 20 onto hydrophobic chemical fibers. The images show a 1 mM aqueous solution (0.05 mL) of ICG and the isomeric equilibrium compound (TK-1) represented by Chemical Formulas 19 and 20 applied to a polypropylene mask, immediately after application (left photo), and then after 20 minutes of rinsing with water for 1 second and immediately after application (right photo).

Figure 6 shows the results of molecular association tests of ICG and the isomerization equilibrium compound (TK1) represented by Chemical Formulas 19 and 20. The left figure shows ICG, and the right figure shows the isomerization equilibrium compound (TK1) represented by Chemical Formulas 19 and 20.

7 is a graph showing the concentration dependency of the fluorescence intensity of ICG, the isomerization equilibrium compound (TK1) represented by Chemical Formulas 19 and 20, and the isomerization equilibrium compound (TK2) represented by Chemical Formulas 15 and 16 in human venous blood.

FIG8 is a graph showing the conditions observed when ICG, the isomerization-equilibrium compound represented by Chemical Formulas 19 and 20 (TK1), and the isomerization-equilibrium compound represented by Chemical Formulas 15 and 16 (TK2) were administered to rats.

FIG9 is a graph showing fluorescence in the peritoneal cavity of rats when ICG, the isomerization-equilibrium compound represented by Chemical Formulas 19 and 20 (TK1), and the isomerization-equilibrium compound represented by Chemical Formulas 15 and 16 (TK2) were administered.

Figure 10 shows images of male Wistar rats (14 weeks old, 300 g) administered 0.075 mL of a 0.1 mM aqueous solution of the isomerized equilibrium compounds (TK2) represented by Chemical Formulas 15 and 16 via the tail vein, followed by imaging using a near-infrared observation system PDE manufactured by Hamamatsu Photonics Co., Ltd. The left image is a black-and-white image using visible light, and the right image is a fluorescent image.

Figure 11 shows near-infrared fluorescence endoscopic images of male Wistar rats (14 weeks old, 300 g) after 0.075 mL of a 0.1 mM aqueous solution of the isomerized equilibrium compounds (TK2) represented by Chemical Formulas 15 and 16 was administered via the tail vein. The left image is a black-and-white image using visible light, and the right image is a fluorescence image.

FIG12 is a graph showing fluorescence at the dorsum of the foot of rats when ICG, the isomerized equilibrium compound (TK1) represented by Chemical Formulas 19 and 20, and the isomerized equilibrium compound (TK2) represented by Chemical Formulas 15 and 16 were administered.

13 is a graph showing the temporal changes in fluorescence intensity at the dorsum of the foot when ICG, the isomerization-equilibrium compound represented by Chemical Formulas 19 and 20 (TK1), and the isomerization-equilibrium compound represented by Chemical Formulas 15 and 16 (TK2) were administered to rats.

14 is a graph showing the temporal changes in fluorescence intensity at the dorsum of the foot when ICG, the isomerization-equilibrium compound represented by Chemical Formulas 19 and 20 (TK1), and the isomerization-equilibrium compound represented by Chemical Formulas 15 and 16 (TK2) were administered to rats.

FIG. 15 is a diagram showing a technique for direct observation of rat blood vessels.

FIG. 16 is a diagram showing a directly observed portion of a rat's blood vessels (spermatozoa cremaster flap).

FIG. 17 is a graph showing the state of fluorescence in blood vessels and surrounding tissues of rats to which ICG was administered.

FIG. 18 is a graph showing fluorescence in blood vessels and surrounding tissues of rats after administration of the isomerization-equilibrated compound (TK1) represented by Chemical Formulas 19 and 20. FIG.

Figure 19 is a graph showing the left paw volume of rats measured immediately after injection (post-injection) and one week after injection (1 week after) for the carrageenan-administered group (edema) and the non-administered group (normal) in the TK1 test. The vertical axis represents volume (mL).

Figure 20 is a graph showing the results of the Von Frey test on the left paw of rats in the TK1 test group, the group that received carrageenan (edema) and the group that did not receive carrageenan (normal), measured immediately after injection and one week after injection. The vertical axis represents the load (g) at which the reaction occurred.

Figure 21 is a graph showing changes in brightness of the left paw surface after TK1 injection immediately after carrageenan injection for a group administered carrageenan (edema) and a group not administered carrageenan (normal). The vertical axis represents brightness (arbitrary unit) and the horizontal axis represents time (seconds).

Figure 22 is a graph showing changes in brightness of the left paw surface one week after carrageenan injection and TK1 injection for a group that received carrageenan (edema) and a group that did not receive carrageenan (normal). The vertical axis represents brightness (arbitrary unit) and the horizontal axis represents time (seconds).

Figure 23 is a graph showing the left paw volume of rats measured immediately after injection (post-injection) and one week after (1 week) for the ICG test group, the group administered carrageenan (edema), and the group not administered (normal). The vertical axis represents volume (mL).

Figure 24 is a graph showing the results of the Von Frey test on the left paw of rats immediately after injection and one week after injection, for groups tested with ICG, those administered carrageenan (edema), and those not administered (normal). The vertical axis represents the load (g) at which the reaction occurred.

Figure 25 is a graph showing the brightness of the left paw surface immediately after ICG injection for a group that received carrageenan (edema) and a group that did not receive carrageenan (normal). The vertical axis represents brightness (arbitrary unit) and the horizontal axis represents time (seconds).

Figure 26 is a graph showing changes in brightness of the left foot surface after ICG injection one week after carrageenan injection in a group that received carrageenan (edema) and a group that did not receive carrageenan (normal). The vertical axis represents brightness (arbitrary units) and the horizontal axis represents time (seconds).

DETAILED DESCRIPTION

The diagnostic composition of the present invention utilizes the cyclodextrin-conjugated indocyanine compound described below as a pigment, making it iodine-free and therefore suitable for diagnostic applications. This diagnostic composition can replace the conventionally used indocyanine green-containing diagnostic compositions. For example, it can be used as a drug for liver function tests or circulatory function tests. Furthermore, by observing the near-infrared fluorescence generated by administration to, for example, blood vessels, lymphatic vessels, brain, eyes, stomach, breast, esophagus, skin, kidneys, ureters, bladder, urethra, lungs, heart, or other internal organs, it can be applied to medical procedures and diagnostics. The pigment contained in the diagnostic composition of the present invention has low binding to the organism, allowing for long-term labeling of desired areas. The diagnostic composition may also contain salts or other additives as isotonic agents, as needed. In addition to pre-prepared aqueous solutions, it can also be used in a form that is dissolved. The diagnostic composition can be administered by injection, intravenous drip, application, or oral administration.

This diagnostic composition is suitable for use in visualizing the circulation of aqueous solutions such as blood, lymph, interstitial water, and urine. By visualizing the circulation of blood and other fluids, for example, it can be used to assess necrosis in the peripheral circulation, evaluate tissue survival after revascularization surgery and transplantation, and diagnose poor blood flow. Furthermore, it can also be used for fundus angiography, which has been previously visualized, cerebral circulation assessment, intraoperative angiography during neurosurgery, sentinel lymph node identification in cancers (such as breast cancer, esophageal cancer, gastric cancer, colorectal cancer, prostate cancer, and skin cancer), evaluation of lymphedema, intraoperative cholangiography, tumor labeling, coronary angiography, and abdominal angiography (hepatic artery, abdominal aorta, digestive tract blood flow, etc.). Fluorescent imaging of the renal excretory system, including the kidneys, ureters, bladder, and urethra, is also possible.

<1. Non-inclusion cyclodextrin-bound indocyanine compounds and their synthesis>

Examples of the non-inclusion type cyclodextrin-bound indocyanine compounds of the present invention include Chemical Formula 1, Chemical Formula 3, Chemical Formula 5, Chemical Formula 7, Chemical Formula 9, and Chemical Formula 11. Their synthesis is accomplished by reacting an indocyanine compound with a cyclodextrin compound in a solution.

In Chemical Formulas 1 and 3, considering the inclusion of a significant portion of the cyclodextrin, _R1 - _R4 and _R13 - _R16 are preferably relatively bulky groups that do not hinder cyclodextrin inclusion. For example, hydrogen or an alkyl or alkoxy group with approximately 1 to 3 carbon atoms. Preferred are hydrogen, methyl, and methoxy groups, with hydrogen being most preferred. For cyclodextrin inclusion, _R5 , _R6 , _R11 , and _R12 are also preferably relatively bulky, but not as bulky as _R1 - _R4 and _R13 - _R16 . For example, hydrogen or an alkyl or alkoxy group with approximately 1 to 6 carbon atoms. _R1 - _R6 and _R11 - _R16 are sites of cyclodextrin inclusion, so even when a hydrophilic functional group is introduced, the entire group is preferably hydrophobic. _R17 , _R18 , _R22 , and _R23 do not significantly affect cyclodextrin inclusion and are not particularly limited as long as they are the substituents listed above. R ₇ , R ₁₀ , R ₁₉ and R ₂₁ are preferably hydrogen from the viewpoint of ease of synthesis. R ₈ , R ₉ and R ₂₀ are not particularly limited as long as they are the substituents described above.

In the case where the non-inclusion type cyclodextrin-bound indocyanine compound of the present invention is covalently bound to the indocyanine compound and the cyclodextrin compound via an amide bond, the synthesis method of the non-inclusion type cyclodextrin-bound indocyanine compound can be achieved by allowing an indocyanine carboxylic acid compound and an aminocyclodextrin compound to undergo a dehydration condensation reaction in a solution.

<2. Inclusion-type cyclodextrin-bound indocyanine compounds>

The cyclodextrin-bound indocyanine compound of the present invention is characterized by being a cyclodextrin-bound indocyanine compound covalently bound to a cyclic sugar chain cyclodextrin represented by Chemical Formula 2, wherein at least a portion of the naphthyl group of the indocyanine is enclosed within the cyclodextrin cavity. Furthermore, as long as the naphthyl group of the indocyanine is enclosed within the cyclodextrin cavity and emits near-infrared fluorescence, the indocyanine group may have a substituent. While there are many types of cyclodextrins, enclosed naphthyl group of the indocyanine is a necessary condition. Examples include α-cyclodextrin, β-cyclodextrin, and γ-cyclodextrin. β-cyclodextrin is preferred. The cyclodextrin may also have a substituent.

In Chemical Formulas 2 and 4, _R1 - _R4 and _R13 - _R16 are preferably small groups that do not hinder cyclodextrin inclusion. Examples include hydrogen, alkyl or alkoxy groups with approximately 1 to 3 carbon atoms. Hydrogen, methyl, and methoxy groups are particularly preferred, with hydrogen being most preferred. For cyclodextrin inclusion, _R5 , _R6 , _R11 , and _R12 , while not as large as _R1 - _R4 and _R13 - _R16 , are also small. Examples include hydrogen, alkyl or alkoxy groups with approximately 1 to 6 carbon atoms. _R1 - _R6 and _R11 - _R16 are sites of cyclodextrin inclusion, so even when introducing hydrophilic functional groups, the entire group is preferably hydrophobic. _R17 , _R18 , _R22 , and _R23 do not significantly affect cyclodextrin inclusion and are not particularly limited as long as they are the substituents listed above. R ₇ , R ₁₀ , R ₁₉ , and R ₂₁ are preferably hydrogen from the perspective of ease of synthesis. R ₈ , R ₉ , and R ₂₀ are not particularly limited as long as they are the substituents described above. The bond between the indocyanine group and the cyclodextrin is not particularly limited as long as it is covalent, and examples thereof include an alkyl bond, an amino bond, an amide bond, a double bond, a triple bond, an ester bond, and an ether bond. However, in chemical synthesis, an amide bond is preferred from the perspective of efficiency.

In order to include at least a portion of the naphthyl groups of the indocyanine in the cyclodextrin cavity, a spacer is preferably used to covalently bond the indocyanine and the cyclic sugar chain cyclodextrin via the spacer. In this case, the extent of inclusion of the naphthyl groups of the indocyanine in the cyclodextrin cavity can be controlled by adjusting the length of the spacer in Chemical Formula 2.

Therefore, the cyclodextrin-bound indocyanine compound formed by covalent bonding of indocyanine and cyclic sugar chain cyclodextrin via a spacer, and the compound characterized by the inclusion of the naphthyl group of indocyanine in the cavity of cyclodextrin, preferably exemplified by Chemical Formula 4. In addition, the substances of Chemical Formula 6, Chemical Formula 8, and Chemical Formula 10 are preferably exemplified. In addition, the substance of Chemical Formula 12 is preferably exemplified. In Chemical Formula 6, m, n, p, and q are preferably m+p and n+q are 5 or more and 7 or less, respectively. From the perspective of easy cyclodextrin inclusion, in all chemical formulas (Chemical Formulas 1 to 10), the number of atoms in the structure (spacer) between the nitrogen atom in the indocyanine structure and the oxygen atom in the cyclodextrin structure is preferably 7 or more and 9 or less.

<3. Synthesis of Inclusion Cyclodextrin-Conjugated Indocyanine Compounds>

The inclusion-type cyclodextrin-bound indocyanine compound of the present invention is as described above, and its synthesis method is to use the non-inclusion-type cyclodextrin-bound indocyanine compound synthesized above as a synthesis precursor and dissolve the compound in an aqueous solution. In the aqueous solution, any substance can be contained as long as it does not hinder inclusion, and the water content is not particularly limited. In addition, the temperature suitable for inclusion is -20°C to 100°C, preferably 0°C to 50°C. In addition, the time required for inclusion is from immediately after addition to the aqueous solution to about 1 month. The inclusion reaction can be in various forms depending on the properties of the non-inclusion-type cyclodextrin-bound indocyanine compound as described above, the temperature of the inclusion reaction, the composition and concentration of the aqueous solution, etc.

Alternatively, the non-inclusion-type cyclodextrin-bound indocyanine compound of the present invention can be converted to an inclusion-type cyclodextrin-bound indocyanine compound by dissolving it in a solvent containing water. While there are no particular restrictions on the water content in the solvent, in principle, inclusion is facilitated when the water content is high, preferably 50% by mass or greater. Furthermore, as long as a substance capable of forming an inclusion-type cyclodextrin-bound indocyanine compound is present in a solvent other than an aqueous solution, inclusion is not necessarily required in an aqueous solution.

In addition, an inclusion-type cyclodextrin-bound indocyanine compound may be formed during the synthesis of a non-inclusion-type cyclodextrin-bound indocyanine compound. In this case, there is no need to conduct the inclusion reaction again.

<4. Synthesis of Non-Inclusion Cyclodextrin-Bound Indocyanine Compounds (Synthesis Example)>

The synthesis of the compound represented by Chemical Formula 11 is given as an example. The indocyanine compound represented by Chemical Formula 11 can be obtained by, for example, adding the compound represented by Chemical Formula 13 synthesized by the method described in Non-Patent Document 5, Chemical Formula 14 synthesized by the method described in Non-Patent Document 6, a water-soluble carbodiimide (WSC: for example, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride) or dicyclohexylcarbodiimide (DCC) as a dehydration condensation agent, and pyridine or N,N-dimethylformamide or an aqueous solution as a solvent, and reacting at -20°C to 60°C for 10 minutes to 100 hours. In addition, in order to activate the reaction, 1-hydroxybenzotriazole (HOBt) can be added as an activator, for example. The amount of the dehydration condensation agent is 2 times the mole of the compound represented by Chemical Formula 13 or more. The solvent used is not limited as long as it can dissolve the reactants and does not hinder the dehydration condensation reaction. There is no limitation on the activator as long as it can activate the dehydration condensation reaction, and there is no limitation on the amount of the activator added as long as it can cause the dehydration condensation reaction to proceed to a desired extent.

[Chemical Formula 13]

[Chemical Formula 14]

<5. Purification of Non-Inclusion Cyclodextrin-Bound Indocyanine Compounds>

The mixture containing the non-inclusion cyclodextrin-bound indocyanine compound synthesized by the above method is dissolved in an acidic aqueous solution and subjected to reverse-phase column chromatography. Elution is performed using, for example, a mixture of acidic water and methanol, a mixture of acidic water and acetonitrile, a mixture of acidic water and ethanol, or a mixture of acidic water and acetone, to isolate and purify the high-purity non-inclusion cyclodextrin-bound indocyanine compound. The acid is not limited as long as it does not decompose the non-inclusion cyclodextrin-bound indocyanine compound, effectively elutes it, and facilitates post-elution handling. Preferred examples include hydrochloric acid, trifluoroacetic acid, acetic acid, sulfuric acid, nitric acid, and formic acid. Hydrochloric acid, trifluoroacetic acid, and acetic acid are preferred. Hydrochloric acid is most preferred. The acid concentration is not limited as long as it does not decompose the non-inclusion cyclodextrin-bound indocyanine compound, effectively elutes it, and facilitates post-elution handling. It is preferably 0.01 mM to 10 mM, and most preferably 0.1 mM to 1 mM. By keeping the concentration within this range, the target compound can be rapidly eluted without decomposing it. The eluted non-inclusion cyclodextrin-bound indocyanine compound can be obtained as a solid by removing the solvent. Freeze-drying can also be used as a solvent removal method.

<6. Synthesis Method (Synthesis Example) and Purification Method (Purification Example) of Inclusion-Type Cyclodextrin-Bound Indocyanine Compounds>

The non-inclusion type cyclodextrin-bound indocyanine compound of Chemical Formula 11 synthesized and purified by the above method is, for example, non-inclusion type in DMSO, but immediately becomes inclusion type upon dissolution in water, as shown in Chemical Formula 12. This phenomenon can be confirmed using ¹ H NMR.

Therefore, as long as the inclusion compound is an aqueous solution, it exists as an inclusion compound and can be used as an inclusion compound. In addition, the inclusion compound in the aqueous solution can be made into a solid by removing the water of the aqueous solution, and a solid of the inclusion compound can also be made.

<7. In vivo dynamics measuring device and circulation visualization device applicable to a living body to which a diagnostic composition has been administered>

In vivo dynamic measurement device

This device is completed based on the fact that the cyclodextrin-bound indocyanine compound of the present invention (hereinafter sometimes appropriately referred to as "compound of the present invention") used in the diagnostic composition of the present invention has different in vivo dynamics such as the degree of interstitial migration from ICG.

That is, the metabolic rate of the compound of the present invention and the rate of migration from the circulatory system, such as blood or lymph, to the interstitium differ from those of ICG. Consequently, the compound of the present invention enters the interstitium at a relatively greater rate than ICG. Therefore, by analyzing the in vivo dynamics of the compound of the present invention, the mechanism of fluid migration (in vivo dynamics) within the body can be accurately evaluated. Here, by maximally maintaining constant measurement conditions (such as the activity state of the organism and ambient temperature), the present device can more accurately measure in vivo dynamics.

One form of abnormal fluid movement in a living body is edema. Here, according to the so-called Stirling hypothesis, the movement of fluid (water) in a living body can be roughly considered to be from arteries to interstitial tissue, and then from interstitial tissue to veins.

The water movement from the artery to the vein can be calculated as (A)-(B)-(C)+(D) based on the pressure difference between the arterial pressure and the interstitial pressure, the water movement outside the circulatory system (A), the water movement into the circulatory system (B) based on the pressure difference between the venous pressure and the interstitial pressure, the water movement from the interstitial fluid to the artery (C) based on the pressure difference between the interstitial fluid osmotic pressure and the arterial blood osmotic pressure, and the water movement from the interstitial fluid to the vein (D) based on the pressure difference between the interstitial fluid osmotic pressure and the venous blood osmotic pressure.

Therefore, when the pressure difference between arterial and venous pressure decreases due to heart failure, or when interstitial fluid osmotic pressure decreases due to nutritional imbalance, the balance of water transfer (A) to (D) is disrupted, causing edema. Furthermore, when there are abnormalities in capillary permeability (abnormalities in the selectivity of material permeation), such as in diabetes, the balance of water transfer (A) to (D) is also disrupted, causing edema. There is no simple method to directly assess abnormal capillary permeability. While the occurrence of edema can be detected by phenomenon, it is currently difficult to determine the specific cause of edema.

Based on the above findings, when abnormal permeability of the capillary wall occurs, the movement of the compound of the present invention can be expected to change compared to cases where edema occurs due to a decrease in the differential pressure between arterial and venous pressure, a decrease in intracellular osmotic pressure, etc. Therefore, when the movement of the compound of the present invention is confirmed to be greater than (or less than) an amount equivalent to water movement, it can be determined that abnormal capillary permeability has occurred. Because it is believed that if no abnormal permeability of the capillary wall occurs, only the movement of the compound of the present invention to an extent equivalent to the change in water movement can be confirmed, it can be determined that an abnormality has occurred in the vascular wall.

In particular, by using two or more compounds of the present invention with different migration rates into the interstitium and measuring the in vivo dynamics of these compounds separately and comparing them, the degree of change in capillary permeability can be evaluated. In other words, by using two or more compounds of the present invention with different permeabilities in the capillary wall and measuring their permeabilities, the occurrence of abnormal vascular wall permeability can be more accurately evaluated. In other words, by using two or more compounds of the present invention with different susceptibilities to abnormal vascular wall permeability and measuring their in vivo dynamics, the factors affecting water migration can be more accurately evaluated. In addition, in addition to evaluating water migration using the compounds of the present invention, water migration can also be measured using conventional methods.

The in vivo dynamics measurement device of the present invention includes an excitation light irradiation unit, a fluorescence intensity measurement unit, an in vivo dynamics calculation unit, and other units. Other units may be selected as needed, such as an in vivo concentration calculation unit that calculates the concentration of a cyclodextrin-bound indocyanine compound based on the fluorescence intensity measured by the fluorescence intensity measurement unit.

This device is a device for measuring at least a portion of a living body to which the diagnostic composition of the present invention has been administered. The dosage of the diagnostic composition is the amount at which fluorescence is observed when the measurement site is irradiated with excitation light. Therefore, the appropriate dosage varies depending on the measurement site. There is no particular limitation on the type of compound of the present invention contained in the diagnostic composition. The compound of the present invention may be used alone or in combination of two or more. In addition, additional diagnostic compositions of the same or different compositions may be added during the measurement process.

The excitation light irradiation unit irradiates the compound of the present invention contained in the administered diagnostic composition with excitation light of a wavelength capable of generating fluorescence. The wavelength of the excitation light irradiated can be limited to an appropriate range. By limiting the wavelength to a minimum possible range, fluorescence and excitation light can be reliably separated. Regarding wavelength limitation, a light source emitting light of an appropriate wavelength can be selected, or a filter can be used to limit the wavelength.

The excitation light irradiation mode is not particularly limited, as long as the generated fluorescence can be measured by the fluorescence intensity measurement unit described later. Examples of excitation light include continuous light, pulsed light, and light with varying intensity. To vary the intensity, the excitation light intensity can be modulated by irradiating the excitation light pulses at predetermined intervals. Preferably, the excitation light intensity is modulated using pulse amplitude modulation.

The excitation light is irradiated to the site to be irradiated through an appropriate optical system. The site to be irradiated is the site where dynamic measurements in the body are to be performed. For example, when evaluating a site where edema occurs, it is preferable to irradiate the site with excitation light directly.

The excitation light irradiation range is not particularly limited. The irradiation range is determined as needed. A narrow irradiation range allows for precise measurement of subdivided in vivo dynamics within the narrow irradiated area. Irradiating a wider range increases the relative amount of the compound of the present invention that fluoresces in response to the excitation light, enabling more precise measurement of fluorescence intensity.

Furthermore, the excitation light irradiation unit is preferably used to irradiate the excitation light while suppressing the influence of ambient light, for example, by irradiating the excitation light in a dark place or by shielding the portion irradiated with the excitation light from external light.

The fluorescence intensity measuring unit measures the fluorescence intensity emitted from the area irradiated with excitation light by the excitation light irradiation unit. The fluorescence intensity is preferably measured by removing light other than fluorescence (ambient light, excitation light, etc.) through a filter that selectively transmits the emitted fluorescence.

When the excitation light irradiation unit uses a unit that irradiates excitation light with modulated intensity, the component that varies according to the modulation can be separated from the measured light intensity and used as the fluorescence intensity. For example, when the excitation light intensity is modulated using pulse amplitude modulation, the fluorescence intensity can be separated by demodulating the light component that varies according to the intensity of the modulated pulse and measuring its intensity. This reduces the impact of ambient light on the fluorescence intensity measurement results.

The in vivo concentration calculation unit is a unit that calculates the in vivo concentration of the compound of the present invention based on the fluorescence intensity measured by the fluorescence intensity measurement unit. The relationship between the fluorescence intensity and the in vivo concentration of the compound of the present invention can be calculated using an appropriate method. For example, a standard curve can be prepared in advance, and the in vivo concentration of the compound of the present invention can be calculated based on the standard curve. In addition, the absolute value of the fluorescence intensity can be directly used as a value related to the in vivo concentration of the compound of the present invention. The in vivo concentration calculation unit calculates the in vivo concentration of the compound of the present invention over time.

The in vivo dynamics calculation unit is a unit that calculates the temporal change of the time rate of change of the in vivo concentration of the compound of the present invention from the in vivo concentration data obtained over time by the in vivo concentration calculation unit. The in vivo dynamics calculation unit calculates the speed of the compound of the present invention moving from the circulatory system to the interstitial tissue and the speed of the compound of the present invention moving from the interstitial tissue to the circulatory system in a part of the body using the temporal change rate of the in vivo concentration obtained. Instead of using the in vivo concentration, the speed of movement into and out of the interstitial tissue can be directly calculated by using the change in fluorescence intensity obtained over time by the fluorescence intensity measurement unit.

It is speculated that there is a high correlation between the movement of the compound of the present invention into (and out of) the interstitium and the movement of water in body fluids. Therefore, by measuring the in vivo behavior of the compound of the present invention, it is possible to evaluate the movement of water in the measurement site. Furthermore, when abnormalities occur in the vascular wall and the permeability of substances is abnormal, the behavior of the compound of the present invention in the body should also change. Therefore, by evaluating the behavior of the compound of the present invention, vascular permeability can be evaluated.

The time rate of change of fluorescence intensity (or concentration in the organism) is obtained by differentiating the time-dependent change in fluorescence intensity (or concentration in the organism) with respect to time, or by calculating the fluorescence intensity (or concentration in the organism) at each specified time and then calculating it as the difference between the fluorescence intensity (or concentration in the organism) before (after) the specified time.

The speed of the compound of the present invention entering the interstitial fluid and the speed of being discharged in a part of the organism can be calculated by the time rate of change of the fluorescence intensity (or biological concentration) obtained. Here, the speed of entering the interstitial fluid and the speed of being discharged can be obtained as relative values by the fluorescence intensity. When more precision is required, it can be calculated according to the concentration of the compound of the present invention in the interstitial and the amount of the interstitial fluid. In addition, the concentration of the compound of the present invention in blood and lymph and its amount are also considered, and it can be calculated more accurately. The concentration of the compound of the present invention in blood, etc. can be measured with good accuracy by actually taking blood.

The following methods can be cited as methods for calculating the concentration in interstitial fluid. The first method is a method for directly approximating the calculated in vivo concentration of the compound of the present invention to the concentration in the interstitial fluid in a part of the organism. As a method related to the first method, the concentration in the interstitial fluid can also be calculated by considering the amount of interstitial fluid. The second method is to calculate the influence ratio of the substance present in the blood vessel on the measured value measured by the fluorescence intensity measurement unit by using other standard substances (ICG, etc.) that do not move to the interstitial on the basis of measuring the concentration of the compound of the present invention in the actual blood, and to calculate the concentration in the interstitial fluid by removing the influence of the concentration of the compound of the present invention in the measured blood.

The time rate of change of the concentration of the compounds of the present invention in interstitial fluid can be calculated by subtracting the speed of movement outside the interstitial fluid from the speed of movement inside the interstitial fluid. Here, it can be assumed that the movement of the compounds of the present invention inside (outside) the interstitial fluid is carried out at a speed related to the water movement in the body fluid, and if the water movement reaches equilibrium, the speed of movement inside the interstitial fluid and the speed of movement outside the interstitial fluid can be considered as constants, therefore, the concentration change (metabolism, discharge, changes caused by movement to tissue, etc.) of the compounds of the present invention in blood and the time rate of change of concentration in interstitial fluid can be used to calculate its movement speed. In addition, even in the case of changes in its movement speed, it is possible to calculate their movement speed by making a model corresponding to this change and applying it.

If the calculated migration speed is outside the range of normal blood vessel wall values, it can be inferred that the blood vessel wall has abnormalities. In addition, as a result of abnormalities in the blood vessel wall, the permeability of the compound of the present invention may be increased or decreased.

It is a well-known fact that acute tissue swelling is caused by various tissue disorders such as acute inflammation, ischemia, and trauma, and is an extremely important tissue reaction in the medical field regardless of the field.

For example, in addition to the simple occurrence of swelling itself being a problem, there are also issues involving all organs, such as the evaluation of intestinal suturing during surgery, the evaluation of the degree of inflammation such as pneumonia, the evaluation of the suitability of organ transplantation, and the evaluation of brain and kidney dysfunction.

However, there is currently no accurate method for predicting the likelihood and extent of tissue swelling, and it can only be estimated empirically based on the degree of tissue damage. Therefore, in most cases, countermeasures can only be discussed after the occurrence of swelling is confirmed, which is a major limitation in treatment.

If the likelihood of swelling can be predicted with high accuracy early on, measures to reduce swelling can be taken early on, minimizing the impact. For example, if the extent of brain swelling that occurs shortly after a cerebral hemorrhage can be accurately predicted, decompression measures and measures to reduce vascular permeability can be implemented early on, minimizing the impact. Similarly, if the impact of ischemic stress can be predicted in advance, secondary complications caused by myocardial infarction and limb injuries can be minimized. Thus, high-precision quantitative prediction technology for swelling has the potential to significantly impact healthcare overall.

Until now, quantification of tissue swelling, including edema, has been qualitatively determined through observation. While quantification has been attempted, it is only applicable in very limited situations and has the disadvantage of being able to evaluate only after swelling has occurred.

To address this issue, the in vivo dynamic measurement device of the present invention calculates the rate of water migration within and outside the interstitium, thereby predicting the progression of swelling. As detailed in the Examples, it is known that ICG does not migrate into the interstitium as swelling progresses. In contrast, the cyclodextrin-conjugated indocyanine compound of the present invention can migrate within and outside the interstitium, and its movement into the interstitium is further promoted by the movement of water as swelling progresses. Therefore, by evaluating the movement of the cyclodextrin-conjugated indocyanine compound of the present invention into the interstitium, the progression of swelling can be predicted.

That is to say, it can be inferred that the degree of swelling that is ultimately formed is affected by the speed of movement of water into and out of the interstitial space, and therefore the degree of final swelling can be evaluated by evaluating the speed of movement of the cyclodextrin-bound indocyanine compound of the present invention, which is related to the speed of movement of water. The speed of movement can be calculated by the concentration of the cyclodextrin-bound indocyanine compound, or directly using the fluorescence intensity. In addition, the speed of movement into and out of the interstitial space can naturally be calculated as an absolute value, or it can be calculated as a relative value (for example, the rate of change of fluorescence intensity can be directly used as a value related to the speed of movement), and the progress of swelling can be predicted based on this value.

The progression of swelling is preferably calculated based on the normal migration rate into the interstitium when there is no swelling. However, the normal migration rate into the interstitium may be unknown. In such cases, a non-swollen site is selected as a control site, and the migration rate of this site is calculated and used as a substitute. Alternatively, the extent of interstitial migration of the cyclodextrin-conjugated indocyanine compound of the present invention can be evaluated by using ICG, which does not migrate within the interstitium, to assess the blood circulation volume and metabolism.

The determination of the extent of movement of the cyclodextrin-bound indocyanine compound of the present invention within the interstitium is carried out after a prescribed time has passed after the diagnostic composition of the present invention is administered into a living body. The prescribed time refers to the time required for the diagnostic composition of the present invention to be distributed in the blood. The reason for using data after the prescribed time for evaluation is that after administration of the diagnostic composition, when it is distributed into the blood after a prescribed time, it is hardly affected by the degree of swelling progression, the concentration in the blood rises rapidly, and the change in fluorescence intensity is not affected by the presence or absence of swelling, and almost no difference is produced. After distribution in the blood, the speed of movement within the interstitium will change according to the presence or absence of swelling progression, so an increase in fluorescence intensity will be selectively confirmed at the site where the movement occurs (i.e., the site where swelling occurs).

As a specific example of a method for estimating the progression of swelling, there is a method in which the blood volume is estimated from body weight, the size of the peak of fluorescence intensity and the time to the peak are calculated from the estimated blood volume; the fluorescence intensity at the time when the edema may change is measured taking into account this time and intensity, and the degree of edema progression and the degree of edema that may occur in the future can be estimated from the results.

Circular Visualization Installation

This device is based on the fact that the cyclodextrin-bound indocyanine compound of the present invention (hereinafter sometimes appropriately referred to as "compound of the present invention") used in the diagnostic composition of the present invention differs from ICG in its in vivo dynamics, such as the degree of migration into the interstitium.

That is, the compound of the present invention easily moves in the interstitial fluid, and by tracking the behavior of the compound of the present invention, the state of blood circulation or lymph circulation can be visualized.

The circulation visualization device of the present invention comprises an excitation light irradiation unit, a fluorescence imaging unit, a morphological imaging unit, and a display unit. This device is a device for measuring at least a portion of a living body to which the diagnostic composition of the present invention is administered. The dosage of the diagnostic composition is the amount in which fluorescence can be observed when the measurement site is irradiated with excitation light. Therefore, the appropriate dosage varies depending on the measurement site. The type of compound of the present invention contained in the diagnostic composition is not particularly limited. The compound of the present invention can be used alone or in combination of two or more. In addition, additional diagnostic compositions having the same or different compositions can be administered during the measurement process.

The excitation light irradiation unit irradiates the compound of the present invention contained in the administered diagnostic composition with excitation light of a wavelength capable of generating fluorescence. The wavelength of the excitation light used for irradiation can be limited to an appropriate range. By limiting the wavelength to a narrow range as much as possible, fluorescence and excitation light can be reliably separated. Wavelength limitation can be achieved by selecting a light source emitting light of an appropriate wavelength or by using a filter to limit the wavelength.

The form of excitation light irradiation is not particularly limited, as long as the generated fluorescence can be measured by the fluorescence imaging unit described below. For example, the excitation light may be continuous light, pulsed light, or light with varying intensity. To vary the intensity, the intensity of the excitation light can be modulated by irradiating the excitation light pulses at predetermined intervals. Preferably, the excitation light is modulated using pulse amplitude modulation.

The excitation light passes through an appropriate optical system and illuminates the desired area. The area to be illuminated is the part of the organism where the circulation is to be visualized, such as the area where tissue necrosis has occurred due to burns, frostbite, inflammation, trauma, infarction, and the like, and its surrounding areas. Necrotic areas of tissue lack blood circulation, and leaving them untreated offers little benefit, so removal is considered. In this case, it is ideal to completely remove only the necrotic area, comparing the necrotic area with the normal area.

Conventionally, identification of necrotic areas has been performed using methods such as angiography using contrast agents or detecting a decrease in body temperature associated with blood circulation. However, angiography has the problem of being inconvenient to use devices such as X-ray irradiation devices, and accurate determination based on body temperature is difficult.

The device of the present invention is used to evaluate necrotic and normal areas based on the presence or absence of body fluid (blood) circulation. Visualizing areas without circulation allows for easy removal. In addition to visualizing necrotic areas, direct observation of blood circulation is also possible, making it easy to identify abnormalities in circulatory function. For example, areas that are not necrotic but have decreased circulatory function due to, for example, infarction can be visualized.

The excitation light irradiation range is determined as needed so as to include the portion to be cyclically visualized.

Furthermore, the excitation light irradiation unit is preferably used to irradiate the excitation light while controlling the influence of ambient light, for example, by irradiating the excitation light in a dark place or by shielding the portion irradiated with the excitation light from external light.

The fluorescence imaging unit two-dimensionally acquires the intensity of fluorescence emitted by the compound of the present invention, which is excited by the excitation light irradiation unit, thereby obtaining data on the distribution status of the compound of the present invention in the organism. In other words, this unit acquires distribution status data representing the distribution of the compound of the present invention in a portion of the organism as two-dimensional image data.

For example, it can be composed of a combination of an appropriate optical system and an imaging element such as a CCD. The resolution of the data obtained two-dimensionally can be set to a necessary value according to the purpose. The determination of fluorescence intensity preferably allows the emitted fluorescence to be selectively transmitted through a filter and removes light (ambient light, excitation light, etc.) other than the fluorescence to be measured.

When using excitation light with modulated intensity as the excitation light irradiation unit, the fluorescence intensity is obtained by separating the components that vary according to the modulation from the measured light intensity. For example, when the excitation light intensity is modulated using pulse amplitude modulation, the fluorescence intensity is separated by demodulating the components of the light that vary according to the modulated pulse intensity and measuring their intensities. This reduces the influence of ambient light on the fluorescence intensity measurement results.

The morphological imaging unit is a unit that two-dimensionally acquires the intensity of light at wavelengths other than the fluorescence wavelength emitted by the compound of the present invention to obtain morphological data of the compound in a portion of a living body. In other words, this unit is a unit that acquires two-dimensional image data of the morphology of a portion of a living body.

The morphological imaging unit can be composed of an appropriate optical system and an imaging element such as a CCD. The resolution of the two-dimensional data can be set according to the desired purpose. In this case, the fluorescence emitted by the excitation light is not measured (or the detection sensitivity is reduced). The morphological imaging unit uses almost all of the aforementioned fluorescence imaging unit and optical system. Finally, a dichroic prism is used on the optical path before it is introduced into the imaging element to guide light with a wavelength corresponding to the fluorescence to the fluorescence imaging unit, and light of other wavelengths can be guided to the morphological imaging unit. By appropriately forming a bidirectional color film, the dichroic prism can reasonably control the wavelength of the separated light.

Furthermore, the fluorescence imaging unit and the morphological imaging unit can share the same camera device. That is, the separation of fluorescence and other light can also be a mathematical separation performed after obtaining their two-dimensional image data. Furthermore, the distribution state data can also be complex image data from the surface of a part of the biological body to the deep layer direction. The optical focus used by the fluorescence imaging unit obtains complex two-dimensional image data in the deep layer direction while moving in the deep layer direction. In addition, as the excitation light irradiation unit, an optical system with a variable focal length is used, and the focus is moved not on the surface of a part of the biological body, but in the deep layer direction, or the excitation light is concentrated and irradiated to a part of the biological body, so that fluorescence can be selectively emitted not only on the surface of the biological body but also in the deep layer inside the biological body, and the circulation of this part can be visualized.

The display unit is a display unit that represents the distribution state of the compound of the present invention in a part of the organism by overlapping the morphological data obtained by the morphological imaging unit and the distribution state data obtained by the fluorescence imaging unit. When the wavelength of the fluorescence is not within the range of visible light, it is preferred to convert the wavelength of the fluorescence into visible light for representation. From the perspective of visualization of the circulation, the representation of the distribution state data takes precedence over the representation of the morphological data. In particular, it is possible to distinguish between the low distribution amount (i.e., fluorescence intensity) of the compound of the present invention and the other parts (whether it is low or not is determined according to the purpose of visualization of the circulation. For example, in the purpose of visualizing the necrotic part, in the part without circulation, i.e., the part where no fluorescence is confirmed) and other parts can be distinguished. For example, the part can be represented as a color different from that of the other parts or a flashing representation. The superposition of the distribution state data and the morphological data can be realized using logic on a computer, etc. The representation of two-dimensional data can be realized by a general display device. The display device is provided between the organism and the measurer, so that the organism can be treated while observing the display device.

＜8. Definitions, etc.＞

In the present invention, "alkyl" refers to a linear or branched alkyl group having 1 to 20 carbon atoms which may have a substituent, for example, a methyl group, ethyl group, propyl group, butyl group, pentyl group, hexyl group, heptyl group, octyl group, nonyl group, decyl group, undecyl group, dodecyl group, tridecyl group, tetradecyl group, pentadecyl group, hexadecyl group, heptadecyl group, octadecyl group, nonadecyl group, eicosyl group or a branched group.

In the present invention, the "alkoxy group" includes, for example, methoxy, ethoxy, propoxy, butoxy, pentyloxy, hexyloxy, methoxyethoxy, methoxypropoxy, ethoxyethoxy, ethoxypropoxy, methoxyethoxyethoxy, etc., in which alkoxy groups having 1 to 20 carbon atoms are bonded in a straight chain or branched form.

In the present invention, the "aryl group" includes aromatic hydrocarbons having 6 to 20 carbon atoms, such as phenyl and naphthyl.

Example

The preferred embodiments of the present invention are described in detail below by way of examples. However, the technical scope of the present invention is not limited to the following embodiments, and various modifications can be made within the scope of the present invention.

<Experiment 1: Synthesis and Purification of Compounds Represented by Chemical Formula 15 and Chemical Formula 16>

A mixture of 0.20 g of the compound represented by Chemical Formula 13, 0.94 g of the compound represented by Chemical Formula 14, 0.18 g of WSC, 0.12 g of HOBt, 4.0 mL of pyridine, and 2.0 mL of N,N-dimethylformamide was stirred at 0°C in the dark for 6 hours. 50 mL of acetone was then added, and the precipitate was filtered under reduced pressure. The precipitate was dissolved in a 0.1% trifluoroacetic acid aqueous solution and subjected to ODS column chromatography. A mixture of water and methanol containing 1 mM hydrochloric acid was used as the eluent to elute the compound represented by Chemical Formula 15. The eluate was concentrated under reduced pressure to obtain 0.65 g of the inclusion-type compound represented by Chemical Formula 16 as a green solid (at the end of the reduced pressure concentration of the eluate, the water content was high, so it naturally became an inclusion-type compound).

[Chemical Formula 15]

[Chemical Formula 16]

Next, the instrumental analysis data for the target compound represented by Chemical Formula 16 are shown. ¹ H NMR (500 MHz, D ₂ O, 26°C, acetone: 2.10 ppm) 1.43 (2H, m), 1.55 (2H, m), 1.99 (6H, s), 2.09 (6H, s), 2.63 (6H, m), 2.80 (6H, m), 2.92 (2H, m), 3.02 (2H, dd, J = 3.7, 9.8 Hz), 3.08 (2H, t, J = 9.2 Hz), 3.2-4.1 (m), 4.19 (2H, t, J = 9.8 Hz), 4.26 (2H, t, J = 9.8 Hz), 4.33 (2H, m), 4.43 (2H, m), 4.71 (2H, d, J = 2.4 Hz) , 4.81 (4H, d, J = 3.7 Hz), 4.91 (2H, d, J = 3.7 Hz), 4.99 (2H, d, J = 3.7 Hz), 5.08 (2H, d, J = 3.7 Hz), 5.13 (2H, d, J = 3.1 Hz), 6.15 (2H, d, J = 13 Hz), 6.52 (2H, t, J = 12 Hz), 7.43 (4H, m), 7.57 (1H, d, J = 12 Hz), 7.57 (2H, d, J = 9.2 Hz), 7.78 (2H, m), 8.06 (3H, m), 8.15 (2H, d, J = 8.5 Hz). ESI-MS m/z calcd for C ₁₃₁ H ₁₉₁ N ₄ O ₇₂ 2972, found 2973 [M] ⁺ .

<Experiment 2: Synthesis and Purification of Compounds Represented by Chemical Formula 19 and Chemical Formula 20>

A mixture of 0.17 g of the compound represented by Chemical Formula 17, 5 mL of methanol, and 0.30 g of t-BuOK was stirred at room temperature for 12 hours. Then, 3 mL of 1M hydrochloric acid and 50 mL of water were added. The precipitate was filtered, washed with water, and then dried under reduced pressure to obtain 0.17 g of the compound represented by Chemical Formula 18.

[Chemical Formula 17]

[Chemical Formula 18]

A mixture of 0.02 g of the compound represented by Chemical Formula 18, 0.081 g of the compound represented by Chemical Formula 14, 0.016 g of WSC, 0.011 g of HOBt, 0.3 mL of pyridine, and 0.2 mL of N,N-dimethylformamide was stirred at 0°C in the dark for 6 hours. 5 mL of acetone was then added, and the precipitate was filtered under reduced pressure, dissolved in a 0.1% trifluoroacetic acid aqueous solution, and subjected to ODS column chromatography. A mixture of water and methanol containing 1 mM hydrochloric acid was used as the eluent to elute the compound represented by Chemical Formula 19. The eluent was concentrated under reduced pressure to obtain 0.045 g of the inclusion-type compound represented by Chemical Formula 20 as a green solid (at the end of the concentration of the eluent under reduced pressure, the water content was high, so it naturally became an inclusion-type compound).

[Chemical Formula 19]

[Chemical Formula 20]

Next, the machine analysis data of the target compound represented by Chemical Formula 20 are shown. ¹ H NMR (500 MHz, D ₂ O,40℃,acetone:2.26ppm)1.54(2H,m),1.68(2H,m),1.98(2H,m),2.19(6H,s),2 .20(2H,m),2.30(6H,s),2.6-2.85(10H,m),2.95(2H,m),3.00(4H,m),3.08 (2H,t,J＝12Hz),3.17(2H,dd,J＝3.7,9.8Hz),3.26(2H,t,J＝9.8Hz),3.35- 4.30(m),4.35(2H,t,J＝9.2Hz),4.50(2H,t,J＝9.2H),4.52(2H,m),4.63(2H ,m),4.87(2H,d,J＝3.7Hz),4.95(d,J＝3.1Hz),4.97(2H,d,J＝3.7Hz),5.08 (2H,d,J＝3.7Hz),5.15(2H,d,J＝4.3Hz),5.25(2H,d,J＝3.7Hz),5.29(2H,d, J＝3.7Hz),6.30(2H,d,J＝14.6Hz),7.58(4H,m),7.73(2H,d,J＝8.5Hz),7.95 (2H,m),8.25(2H,m),8.32(2H,d,J＝14.6Hz),8.35(2H,d,J＝8.5Hz).ESI-MS m/ _z calculated for _{C135H197N4O73 3042} , found 3042 [M] ⁺ .

<Experiment 3: Synthesis and Purification of the Compound Represented by Chemical Formula 21>

A mixture of 0.04 g of the compound represented by Chemical Formula 13, 0.18 g of mono-6-amino-6-deoxy-β-cyclodextrin, 0.05 g of WSC, 0.025 g of HOBt, 0.8 mL of pyridine, and 0.4 mL of N,N-dimethylformamide was stirred at 0°C in the dark for 3 hours. 10 mL of acetone was then added, and the precipitate was filtered under reduced pressure, dissolved in 0.1% trifluoroacetic acid aqueous solution, and subjected to ODS column chromatography. The compound represented by Chemical Formula 21 was eluted using a mixture of water and methanol containing 1 mM hydrochloric acid. The eluate was concentrated under reduced pressure to obtain 0.11 g of the compound represented by Chemical Formula 21 as a green solid.

[Chemical Formula 21]

Next, the instrumental analysis data for the target compound represented by Chemical Formula 21 are shown. ¹ H NMR (500 MHz, D ₂ O, 29°C, acetone: 2.10 ppm) 1.79 (12H, br.), 2.68 (4H, br.), 3.0-4.5 (98H), 4.4 (4H, br.), 4.5-5.3 (14H, br.), 6.18 (2H, br.), 6.46 (2H, br.), 7.3-8.2 (15H). ESI-MS m/z calculated for C ₁₂₅ H ₁₇₉ N ₄ O ₇₀ 2856, found 2856 [M] ⁺ .

<Experiment 4: Synthesis and Purification of the Compound Represented by Chemical Formula 23>

A mixture of 0.02 g of the compound represented by Chemical Formula 22, 0.096 g of mono-6-amino-6-deoxy-β-cyclodextrin, 0.032 g of WSC, 0.5 mL of pyridine, and 0.05 mL of 0.1 M phosphate buffer was stirred in the dark at room temperature for 24 hours. 10 mL of acetone was then added, and the precipitate was filtered under reduced pressure, dissolved in 0.1% trifluoroacetic acid aqueous solution, and subjected to ODS column chromatography. A mixture of water and acetonitrile was used as the eluent to elute the compound represented by Chemical Formula 23. The eluate was concentrated under reduced pressure to yield 0.014 g of the compound represented by Chemical Formula 23 as a green solid.

[Chemical Formula 22]

[Chemical Formula 23]

Next, the instrumental analysis data for the target compound represented by Chemical Formula 21 are shown. ¹ H NMR (500 MHz, D ₂ O, 26°C, acetone: 2.15 ppm) 1.26 (4H, m), 1.5-2.25 (24H, br), 2.7-4.2 (88H), 4.82 (2H, br), 4.90 (8H, br), 4.97 (2H, br), 5.03 (2H, br), 6.11 (2H, br), 6.36 (2H, br), 7.3-8.01 (15H, br). ESI-MS m/z calculated for C ₁₃₁ H ₁₉₁ N ₄ O ₇₀ 2940, found 2940 [M] ⁺ .

<Experiment 5: Synthesis and Purification of the Compound Represented by Chemical Formula 24>

A mixture of 0.20 g of the compound represented by Chemical Formula 22, 0.02 g of 3-amino-3-deoxy-β-cyclodextrin, 0.096 g of WSC, 0.013 g of HOBt, 0.4 mL of pyridine, and 0.2 mL of N,N-dimethylformamide was stirred in the dark at room temperature for 1 hour. Subsequently, 10 mL of acetone was added, and the precipitate was filtered under reduced pressure, dissolved in 0.1% trifluoroacetic acid aqueous solution, and subjected to ODS column chromatography. A mixture of water and methanol was used as the eluent to elute the compound represented by Chemical Formula 24. The eluate was concentrated under reduced pressure to yield 0.013 g of the compound represented by Chemical Formula 24 as a green solid.

[Chemical Formula 24]

Next, the instrumental analysis data for the target compound represented by Chemical Formula 24 are shown. ¹ H NMR (500 MHz, D ₂ O, 29°C, acetone: 2.10 ppm) 1.1-2.5 (28H), 3.0-4.25 (88H), 4.5-5.2 (14H), 7.3-8.02 (15H). ESI-MS m/z calculated for C ₁₃₁ H ₁₉₁ N ₄ O ₇₀ 2940, found 2940 [M] ⁺ .

<Experiment 6: Synthesis and Purification of the Compound Represented by Chemical Formula 25>

A mixture of 0.02 g of the compound represented by Chemical Formula 22, 0.1 g of the compound represented by Chemical Formula 14, 0.032 g of WSC, 0.5 mL of pyridine, and 0.05 mL of 0.1 M phosphate buffer was stirred in the dark at room temperature for 24 hours. 10 mL of acetone was then added, and the precipitate was filtered under reduced pressure, dissolved in 0.1% trifluoroacetic acid aqueous solution, and subjected to ODS column chromatography. A mixture of water and methanol was used as the eluent to elute the compound represented by Chemical Formula 25. The eluate was concentrated under reduced pressure to yield 0.021 g of the compound represented by Chemical Formula 25 as a green solid.

[Chemical Formula 25]

Next, the instrumental analysis data for the target compound represented by Chemical Formula 25 are shown. ¹ H NMR (500 MHz, D ₂ O, 25°C, acetone: 2.10 ppm) 1.0-2.5 (32H), 3.0-4.5 (96H), 4.8-5.2 (14H), 6.09 (2H, br), 6.37 (2H, br), 7.3-8.02 (15H, br). ESI-MS m/z calculated for C ₁₃₇ H ₂₀₃ N ₄ O ₇₂ 3056, found 3056 [M] ⁺ .

<Test 7: Solubility of the Cyclodextrin-Bound Indocyanine Compound of the Present Invention>

Solubility tests were conducted on ICG and the cyclodextrin-conjugated indocyanine compounds of the present invention (Chemical Formulas 16, 20, 21, and 23-25) in water and saline. While the dissolution of powdered ICG from Molecular Probe in water and saline required vigorous agitation for approximately one minute, the cyclodextrin-conjugated indocyanine compounds of the present invention, particularly those represented by Chemical Formulas 16 and 20, dissolved rapidly without the need for agitation.

<Test 8: Adsorption of the Cyclodextrin-Bound Indocyanine Compound of the Present Invention to Human Skin>

ICG and the cyclodextrin-conjugated indocyanine compounds of the present invention (Chemical Formulas 16, 20, 21, 23-25) were tested for their adsorption to human skin. A 1 mM aqueous solution (0.03 mL) of each of ICG and the cyclodextrin-conjugated indocyanine compounds of the present invention (Chemical Formulas 16, 20, 21, 23-25) was dropped onto the wrist, rinsed with water after 5 minutes, and then wiped and rinsed again. The result showed that ICG was not completely washed away by water, while the cyclodextrin-conjugated indocyanine compounds of the present invention, especially the compounds represented by Chemical Formulas 16 and 20, were easily washed away by water, indicating that the adsorption of the cyclodextrin-conjugated indocyanine compounds of the present invention to human skin was significantly lower than that of ICG (Figure 1).

<Test 9: Adsorption of the Cyclodextrin-Bound Indocyanine Compound of the Present Invention to Cellulose Fibers>

ICG and the cyclodextrin-conjugated indocyanine compounds of the present invention (Chemical Formulas 16, 20, 21, 23-25) were tested for their adsorption onto cellulose fibers. A cotton swab (Sanyo Co., Ltd.) was used to apply 0.05 mL of a 1 mM aqueous solution of each of ICG and the cyclodextrin-conjugated indocyanine compounds of the present invention (Chemical Formulas 16, 20, 21, 23-25) to a model cellulose fiber. After 3 minutes, the swab was rinsed in running water (tap water, 1 L/min) for 5 seconds. The results showed that ICG was not completely washed away, while the cyclodextrin-conjugated indocyanine compounds of the present invention, particularly those represented by Chemical Formulas 16 and 20, were easily washed away, indicating that the adsorption of the compounds represented by the cyclodextrin-conjugated indocyanine compounds of the present invention onto cellulose fibers was significantly lower than that of ICG (Figure 2).

<Test 10: Adsorption of the Cyclodextrin-Conjugated Indocyanine Compound of the Present Invention to a Meat Model>

Adsorption of ICG and the cyclodextrin-conjugated indocyanine compounds of the present invention (Chemical Formulas 16, 20, 21, 23-25) onto a biological meat model was tested. Commercially available pork tenderloin was used as the meat model. A 5 mm diameter recess was created in the pork tenderloin. 0.05 mL of a 1 mM aqueous solution of each of ICG and the cyclodextrin-conjugated indocyanine compounds of the present invention (Chemical Formulas 16, 20, 21, 23-25) was applied to the meat model. After 3 minutes, the sample was rinsed in running water (tap water, 1 L/min) for 10 seconds. The results showed that ICG was not completely washed off, while the cyclodextrin-conjugated indocyanine compounds of the present invention, particularly those represented by Chemical Formulas 16 and 20, were easily washed off, demonstrating that the adsorption of the cyclodextrin-conjugated indocyanine compounds of the present invention onto the biological meat model was significantly lower than that of ICG (Figure 3).

<Test 11: Adsorption of the Cyclodextrin-Conjugated Indocyanine Compound of the Present Invention to a Biological Protein Model>

Adsorption of ICG and the cyclodextrin-conjugated indocyanine compounds of the present invention (Chemical Formulas 16, 20, 21, 23-25) onto biological protein models was tested. A 5 mm diameter recess was created in commercially available chicken breast meat as the biological protein model. 0.05 mL of a 1 mM aqueous solution of ICG and the cyclodextrin-conjugated indocyanine compounds of the present invention (Chemical Formulas 16, 20, 21, 23-25) was applied to the breast. After 3 minutes, the samples were rinsed in running water (1 L/min) for 10 seconds. The results showed that ICG was not completely washed away, while the cyclodextrin-conjugated indocyanine compounds of the present invention, particularly those represented by Chemical Formulas 16 and 20, were easily washed away, indicating that the adsorption of the cyclodextrin-conjugated indocyanine compounds of the present invention onto biological protein models was significantly lower than that of ICG (Figure 4).

<Test 12: Adsorption of the Cyclodextrin-Bound Indocyanine Compound of the Present Invention to Hydrophobic Chemical Fibers>

The adsorption of ICG and the cyclodextrin-conjugated indocyanine compounds of the present invention (Chemical Formulas 16, 20, 21, 23-25) onto hydrophobic chemical fibers was tested. A polypropylene mask (Tamagawa Eisai Co., Ltd.) was used as a model hydrophobic chemical fiber. 0.05 mL of a 1 mM aqueous solution of ICG and the cyclodextrin-conjugated indocyanine compounds of the present invention (Chemical Formulas 16, 20, 21, 23-25) was applied to the mask. After 20 minutes (the sample was left to remove any moisture, so it would not adhere to the mask), the mask was rinsed in running water (1 L/min) for one second. The results showed that ICG was not completely washed off, while the cyclodextrin-conjugated indocyanine compounds of the present invention, particularly those represented by Chemical Formulas 16 and 20, were easily washed off, indicating that the adsorption of the cyclodextrin-conjugated indocyanine compounds of the present invention onto hydrophobic chemical fibers was significantly lower than that of ICG (Figure 5).

<Test 13: Molecular Association of Cyclodextrin-Bound Indocyanine Compounds of the Present Invention in Aqueous Solution>

The molecular association properties of ICG and the cyclodextrin-conjugated indocyanine compounds of the present invention (Chemical Formulas 16, 20, 21, 23-25) in aqueous solutions were investigated. Aqueous solutions of ICG and the cyclodextrin-conjugated indocyanine compounds of the present invention (Chemical Formulas 16, 20, 21, 23-25) were prepared at 0.01 mM, 0.025 mM, 0.05 mM, and 0.1 mM, respectively. The solutions were placed in quartz cuvettes with a 1 mm optical path length, and optical absorption spectra were measured at 25°C from 600 nm to 1000 nm. The results showed that ICG exhibited molecular association known as H-aggregation within this concentration range (Figure 6, left). However, the cyclodextrin-conjugated indocyanine compounds of the present invention, particularly those represented by Chemical Formulas 16 and 20, did not exhibit this molecular association within this concentration range (Figure 6, right).

<Test 14: Fluorescence of the Cyclodextrin-Bound Indocyanine Compound of the Present Invention in Aqueous Solution>

The fluorescence properties of ICG and the cyclodextrin-conjugated indocyanine compounds of the present invention (Chemical Formulas 16, 20, 21, 23-25) in aqueous solution were investigated. 0.1 μM aqueous solutions of ICG and the cyclodextrin-conjugated indocyanine compounds of the present invention (Chemical Formulas 16, 20, 21, 23-25) were placed in 1 cm square quartz cuvettes at 25°C. Excitation was performed with 720 nm excitation light (bandpass: 10 nm), and fluorescence spectra were measured (bandpass: 10 nm). Fluorescence efficiency was calculated based on the fluorescence efficiency of ICG, which is 0.13 (in DMSO, 25°C). The results showed that the fluorescence quantum efficiency of ICG was 0.021, while the fluorescence quantum efficiency of the cyclodextrin-conjugated indocyanine compounds of the present invention, particularly the compounds represented by Chemical Formulas 16 and 20, was 0.054 and 0.042, respectively, which are 2.6 times and 2 times that of ICG.

<Test 15: Fluorescence of the Cyclodextrin-Conjugated Indocyanine Compound of the Present Invention in Blood>

The fluorescence of ICG and the cyclodextrin-conjugated indocyanine compounds of the present invention (Chemical Formulas 16, 20, 21, 23-25) in blood was studied. ICG and the cyclodextrin-conjugated indocyanine compounds of the present invention (Chemical Formulas 16, 20, 21, 23-25) were dissolved in human blood to a concentration of 100 μM. The mixture was placed in a triangular quartz cuvette at 25°C and excited with 760 nm excitation light (bandpass: 10 nm), and the surface fluorescence spectrum (bandpass: 10 nm) was measured. The fluorescence intensity at the maximum fluorescence wavelength was 58 (arbitrary units) for ICG. The fluorescence intensities of the cyclodextrin-conjugated indocyanine compounds of the present invention, especially the compounds represented by Chemical Formulas 16 and 20, were 270 (arbitrary units) and 190 (arbitrary units), which are 4.7 times and 3.3 times that of ICG. This is believed to be because the cyclodextrin-conjugated indocyanine compounds of the present invention do not inhibit luminescence in vivo.

<Test 16: Behavior of the Cyclodextrin-Conjugated Indocyanine Compound of the Present Invention in Vivo>

As representative examples, the behaviors of the isomerization-equilibrium compound (TK1) represented by Chemical Formulas 19 and 20 and the isomerization-equilibrium compound (TK2) represented by Chemical Formulas 15 and 16 were evaluated in human blood and rats as biological subjects.

Fluorescence behavior in human blood

The concentration dependence of surface fluorescence intensity of ICG, TK1, and TK2 in human venous blood in triangular cuvettes was evaluated. Specifically, 1.0 mL of human venous blood was added to a triangular quartz cuvette, along with ICG, TK1, and TK2 at specified concentrations. After irradiation with 760 nm (bandpass: 10 nm) excitation light at 25°C, surface fluorescence (bandpass: 10 nm) was measured. The results are shown in Figure 7.

As can be clearly seen from FIG7 , both TK1 and TK2 can emit fluorescence in human venous blood when irradiated with excitation light.

·Study on the feasibility of drug administration in rats

ICG, TK1, and TK2 were prepared as 1 mM aqueous solutions, and 0.1 mL of each solution was injected into the femoral vein of rats after laparotomy. The abdomen was then illuminated with 760 nm excitation light to assess the presence of fluorescence.

The results showed that fluorescence was observed in the internal organs of the rats after the abdominal opening. High accumulation of TK1 and TK2 in the kidneys was observed (as ICG is known to accumulate in the liver). Furthermore, no significant adverse effects on the rats' condition were observed after administration of these drugs, confirming the possibility of relatively safe in vivo administration.

Observation of distribution in rats

Wistar male rats (9 weeks old, weighing 350 g) were anesthetized with ether and then anesthetized by intraperitoneal administration of 0.1 mL of sodium pentobarbital (Nembutal) using a 26G needle. The base of the rat's tail was anesthetized with a rubber band to remove blood, and the tail vein was secured with a 24G indwelling needle, and the terminal line was secured by connecting a three-headed square stopcock and an extension tube. The rat was fixed on a flat table in a supine position (Figure 8). The PDE camera unit manufactured by Hamamatsu Photonics Co., Ltd. was correctly set at a position 16 cm away from the rat's body. However, it was fixed at 20 cm when photographing the whole body. The observation range is the range indicated by the black circle in Figure 8. Thereafter, ICG, TK1, and TK2 at a concentration of 1 mM were administered through the secured terminal line. The dosage was 0.1 mL. The fluorescence in the abdominal cavity when the abdomen was opened 20 minutes after the administration is shown in Figure 9, and the fluorescence of the dorsum of the foot is shown in Figure 12 (image at the time of reaching the maximum brightness).

Figure 9 shows that ICG accumulates primarily in the liver (Figure 9(a)), TK1 primarily in the kidneys (Figure 9(b)), and TK2 also primarily in the kidneys (Figure 9(c)). Near-infrared imaging of internal organs immediately after administration demonstrated clear imaging of the kidneys, ureters, and bladder (Figure 10). Furthermore, near-infrared endoscopy demonstrated the ability to image the ureters (Figure 11).

As can be seen from image 12, fluorescence is clearly observed corresponding to blood flow in the dorsum of the foot. Furthermore, although not shown in the figure, fluorescence was observed after administering ICG, TK1, and TK2 to a rat model of lower limb ischemia in which the right femoral artery was ligated. Since no fluorescence was observed in the ischemic area, the presence or absence of fluorescence can be used to assess the extent of blood flow.

13 and 14 show the temporal changes in fluorescence intensity of the dorsum of the foot. Table 1 shows the time to maximum fluorescence intensity (Imax), the Imax value, and the time to half the Imax value (t _1/2 ).

[Table 1]

Compound
				ICG	0.5min	124	2.3min
TK-1	10min	160	40min
				TK-2	15min	106	55min

As shown in Table 1, Figures 13, and 14, TK1 and TK2 both took longer to reach maximum fluorescence intensity and to reach half-value in time than ICG. This suggests that they can emit fluorescence for a long time in vivo. For example, while ICG's fluorescence intensity drops to its initial value within 10 minutes after administration, TK1 and TK2 exhibit high fluorescence intensity even after one hour.

Observation at the vascular level

According to the method disclosed in non-patent literature 7 and 8 (Figure 15), ICG and TK1 were administered to rats with their blood vessels directly exposed for observation. The situation of the spermatozoa cremaster flap after exposure is shown in Figure 16. As a result, ICG appeared uneven in the blood vessels relative to TK1. In other words, it can be seen that TK1 moved from the blood vessels to the interstitial fluid. In order to conduct a more precise study, microscopic photographs of the blood vessels after administration are shown in Figures 17 (ICG) and 18 (TK1). As can be seen from Figures 17 and 18, when the fluorescence intensity in the blood vessels and the fluorescence intensity in the surrounding tissues are compared, ICG can hardly confirm fluorescence in the interstitial tissue, while TK1 can confirm fluorescence in the interstitial tissue. In addition, although not shown in detail, it is confirmed that the fluorescence intensity in the interstitial tissue increases over time. Therefore, ICG can hardly confirm movement from the blood vessels to the interstitial fluid, but TK1 can confirm movement from the blood vessels to the interstitial fluid.

Evaluation of the correlation between the extent of migration of the cyclodextrin-bound indocyanine compound of the present invention within the interstitium and the magnitude of edema formation

(Evaluation method)

Ten-week-old male Wister rats weighing approximately 350 g were selected as experimental animals (n=4). The rats were anesthetized with isoflurane and injected with 0.1 mL of 0.5% λ carrageenan into the plantar surface of the left hind paw using a 26G needle. Paw edema rapidly developed. Fifteen minutes later, the rats' left paw volume was measured using a rat paw volume meter (MK-101CMP PLETHYSMOMETER, manufactured by Muromachi Machinery Co., Ltd.).

Afterward, isoflurane was turned off and the rat was confirmed to be awake. After the Von Frey test, general anesthesia was re-administered with isoflurane. The rat's tail vein was secured with a 24G indwelling cannula and then secured in a supine position on a horizontal platform. A PDE camera manufactured by Hamamatsu Photonics Co., Ltd. was set up and fixed at a height of 16 cm from the dorsum of the rat's foot.

The PDE camera was used to set the measurement area (ROI) to the central part of the dorsum of the foot. After the measurement began, 0.1 mL of a 1 mmol TK1 aqueous solution was injected through the tail vein, followed by a 5-second flush with 1 mL of saline. Continuous imaging was performed for the first 5 minutes of injection, and the ROI brightness was measured. Thereafter, brightness measurements were taken at 5-minute intervals for 1 minute, continuing until 120 minutes after injection. After the measurement was completed, isoflurane was turned off and the subject returned to consciousness (acute inflammation experiment).

After 7 days, the rats were subjected to Von Frey test, and then the rats were quickly anesthetized with isoflurane. The volume of the rat's left foot was measured using a rat plantar volume meter. After the rat's tail vein was secured with a 24G indwelling needle, it was fixed on a horizontal platform in a supine position. A PDE camera manufactured by Hamamatsu Photonics Co., Ltd. was set up and fixed at a height of 16 cm from the dorsum of the rat's foot. The ROI was set at the center of the dorsum of the foot, and the measurement was started after the light was turned off. 0.1 mL of 1 mmol TK1 aqueous solution was injected from the tail vein and rinsed with 1 mL of normal saline for 5 seconds. Continuous shooting was performed during the first 5 minutes of injection to measure the brightness of the ROI. The brightness was measured for 1 minute at intervals of 5 minutes thereafter, and this continued until 120 minutes after the injection. After the measurement was completed, isoflurane was turned off and the rat returned to consciousness. The same test was performed on the group that was not injected with λ carrageenan.

ICG also implemented the above process.

(result)

The results are shown in Figures 19 to 26. The left paw volume of the rats measured for the group administered with TK1 is shown in Figure 19 (the volume increases toward the top of the vertical axis), the results of the Von Frey test are shown in Figure 20 (the more downward the vertical axis, the more hyperalgesia), the changes in brightness shortly after the injection are shown in Figure 21 (the brightness increases toward the top of the vertical axis), and the changes in brightness one week after the injection of carrageenan are shown in Figure 23 (the brightness increases toward the top of the vertical axis). The left paw volume of the rats measured for the group administered with ICG is shown in Figure 23 (the volume increases toward the top of the vertical axis), the results of the Von Frey test are shown in Figure 24 (the more downward the vertical axis, the more hyperalgesia), the changes in brightness shortly after the injection are shown in Figure 25 (the brightness increases toward the top of the vertical axis), and the changes in brightness one week after the injection of carrageenan are shown in Figure 26 (the brightness increases toward the top of the vertical axis). The values of the vertical axes of all the graphs are arbitrary units.

Significant differences were observed in the patterns of brightness changes within the ROI between ICG and TK1. The brightness rapidly increased shortly after ICG administration, then rapidly decreased, returning to baseline after approximately 10 minutes. In contrast, the initial brightness increase after TK1 administration followed the same trajectory as ICG, with the brightness remaining high thereafter and requiring six hours to return to baseline. Based on these results, we believe that ICG is retained within the blood vessels and does not leak into the interstitium. Therefore, the rapid brightness increase and subsequent rapid decrease are due to passage through the blood vessels distributed within the ROI. In contrast, TK1 exhibits two phases: the initial rapid passage into the blood vessels (hereafter referred to as the "vascular phase") and the subsequent phase in which TK1 fluorescence gradually leaks into the interstitium (hereafter referred to as the "interstitial phase"). The transition from the vascular phase to the interstitial phase corresponds to the specified time.

In the acute inflammation experiment, a significant increase in paw volume was observed in both the ICG and TK1 groups after injection of λ carrageenan, which returned to normal, similar to the tendon side, one week later (Figures 19 and 23). Furthermore, in the Von Frey test, significant hyperalgesia was observed in both the ICG and TK1 groups after injection of λ carrageenan, but returned to normal, similar to the tendon side, one week later (Figures 20 and 24).

Regarding the brightness of the dorsum of the foot, there was no difference between the TK1 group and the control group in the brightness change of the vascular phase, but the brightness in the interstitial phase increased more rapidly than the λ carrageenan-administered group, and maintained a high value throughout the evaluation time (Figure 21). One week after the administration of λ carrageenan and the inflammation subsided, there was no difference in the brightness change of the interstitial phase between the carrageenan-administered side and the tendon side (Figure 22). As the curve in Figure 21 clearly shows, the brightness in the interstitial phase changes in a smooth curve, and the height of the curve depends on the upward rate of change of the interstitial phase. In other words, the change in the value of the interstitial phase can be predicted with high precision based on time series analysis, which is an important basis for the validity of our hypothesis in this work.

No difference in brightness was observed in the ICG group either immediately or one week after injection with lambda carrageenan (Figures 25 and 26). This result indicates that the highly hydrophobic ICG, which is currently used clinically, does not leak fluorescent material outside of blood vessels even in the presence of severe acute inflammation caused by lambda carrageenan.

These results demonstrate that after TK1 administration to a living body, fluorescence intensity at sites predicted for swelling progression can be measured over time during a period corresponding to the interstitial phase. This allows for prediction of subsequent changes in fluorescence intensity, which correlates with the progression of swelling at the site. Therefore, TK1 is suitable for predicting swelling, which is not possible with ICG.

Industrial Application Possibilities

The cyclodextrin-bound indocyanine compounds represented by Chemical Formula 1 or Chemical Formula 2 of the present invention provide green pigment compounds that emit near-infrared fluorescence and have the following characteristics: higher solubility in water or physiological saline compared to ICG, easier removal from biological tissues, lower molecular association in aqueous solutions, higher near-infrared fluorescence intensity in aqueous solutions, and the absence of iodine. Furthermore, the method for synthesizing the cyclodextrin-bound indocyanine compounds of the present invention can provide for the synthesis of useful cyclodextrin-bound indocyanine compounds. Furthermore, the method for purifying the cyclodextrin-bound indocyanine compounds of the present invention can provide for the purification of useful cyclodextrin-bound indocyanine compounds. Furthermore, because the cyclodextrin-bound indocyanine compounds of the present invention exhibit sufficient solubility even without iodine, they can provide diagnostic compositions that are free of iodine, a cause of iodine allergy. These diagnostic compositions exhibit different in vivo behavior than conventional diagnostic compositions containing only ICG, and their properties can be exploited to provide a variety of useful diagnostic methods and diagnostic devices.

Claims (15)

1. A cyclodextrin-bound indocyanine compound, which is represented by the following chemical formula 19 or 20:

[Chemical Formula 19]

In the chemical formula 20, at least a portion of the naphthyl group of indocyanine is encapsulated within the cavities of the cyclodextrin.

2. The cyclodextrin-bound indocyanine compound according to claim 1, wherein the cyclodextrin-bound indocyanine compound is represented by chemical formula 20.

3. A chemical synthesis method for a cyclodextrin-indocyanine compound represented by the following chemical formula 19,

[Chemical Formula 19]

The method includes:

(i) the step of mixing the compound represented by chemical formula 18 and the aminocyclodextrin represented by chemical formula 14 in a solvent; and

(ii) the step of adding a dehydrating condensing agent to the mixture to carry out the dehydration condensation reaction of the mixture.

[Chemical Formula 18]

[Chemical Formula 14]

4. A method for the chemical synthesis of a cyclodextrin-indocyanine compound represented by the following chemical formula 20, wherein at least a portion of the naphthyl group of the indocyanine is encapsulated within the cavity of the cyclodextrin.

[Chemical Formula 20]

The method includes the step of carrying out an inclusion reaction in water of a cyclodextrin represented by the following chemical formula 19 with an indocyanine compound:

[Chemical Formula 19]

5. A method for purifying a cyclodextrin-indocyanine compound represented by the following chemical formula (19), comprising the step of purifying the compound by column chromatography elution with a solvent containing HCl.

[Chemical Formula 19]

6. A diagnostic composition comprising an aqueous solution containing one or more cyclodextrin-bound indocyanine compounds selected from the group consisting of compounds represented by any one of the following chemical formulas (19) or (20), and said diagnostic composition being used by injection into the body.

[Chemical Formula 19]

[Chemical Formula 20]

7. The diagnostic composition according to claim 6 is substantially iodine-free.

8. The use of the cyclodextrin-indocyanine compound according to claim 1 or 2 in the preparation of diagnostic compositions.

9. The application according to claim 8, wherein the diagnostic composition is used for medical surgery or medical diagnosis, wherein near-infrared fluorescence is observed by application of the diagnostic composition to at least one of blood vessels, lymphatic vessels, brain, eye, stomach, breast, esophagus, skin, kidney, ureter, bladder, urethra, lung, and heart.

10. The application according to claim 8, wherein the diagnostic composition is used for visualization of circulation in blood, lymph, interstitial fluid, or urine.

11. The application according to claim 8, wherein the diagnostic composition is used for fluorescence imaging of the renal excretory system including the kidney, ureter, bladder and urethra.

12. The application according to claim 11, wherein the diagnostic composition is used for fluorescence imaging of the ureter.

13. A green solid composed of _{a cyclodextrin} -bound indocyanine compound having an ESI-MSm/z[M] ⁺ value corresponding to the molecular formula _{C135H197N4O73} , and having a chemical structure represented by the following chemical formula ( ₂₀ ):

Furthermore, it is characterized in that at least a portion of the naphthyl group of the indocyanine moiety is encapsulated within the cavity of the cyclodextrin moiety.

14. The green solid according to claim 13, wherein the cyclodextrin-indocyanine compound is prepared by a chemical synthesis method comprising: (i) mixing a compound represented by chemical formula (18) and a compound represented by chemical formula (14) in a solvent; and (ii) adding a dehydrating condensing agent to the mixture to carry out a dehydration condensation reaction of the mixture.

The method may optionally include the step of carrying out an inclusion reaction in water.

15. The green solid according to claim 13, wherein the cyclodextrin-indocyanine compound is purified by column chromatography by eluting the compound with a solvent containing HCl.