JP6648921B2 - Composition having a conjugate of a dye and polyethylene glycol and an additive, and a contrast agent for photoacoustic imaging having the composition - Google Patents

Description

  The present invention relates to a composition having a conjugate of a dye and polyethylene glycol, and an additive that interacts with the conjugate, and a contrast agent for photoacoustic imaging having the composition.

  In recent years, a photoacoustic imaging method has attracted attention as an imaging method capable of non-invasive diagnosis.

  The photoacoustic imaging method is a method of obtaining an image of a measurement target by detecting the intensity of an acoustic wave generated by volume expansion caused by heat emitted from molecules of the measurement target irradiated with light and the generation position of the acoustic wave. . In the photoacoustic imaging method, a dye can be used as a contrast agent for increasing the magnitude of the fluorescence from the measurement target site or the intensity of the acoustic wave.

  Here, Patent Document 1 discloses an example in which a conjugate of a near-infrared dye and a synthetic polyethylene glycol polymer having a molecular weight in the range of 15 to 45 kDa is used as an optical imaging contrast agent.

  On the other hand, Non-Patent Document 1 discloses an example in which a mixture of a conjugate of polyethylene glycol and heparin and a cyclodextrin forms a gel and carries BSA (bovine serum albumin).

JP 2012-520856 A

Biomacromolecules, 2010, 11 (9), pp2204-2221.

  However, the conjugate disclosed in Patent Document 1 with a near-infrared dye and a synthetic polyethylene glycol polymer having a molecular weight in the range of 15 to 45 kDa has high blood retention. For this reason, when used for imaging, it is difficult to distinguish a photoacoustic signal between a photoacoustic imaging contrast agent present in a blood vessel and a photoacoustic imaging contrast agent accumulated in a tumor.

  In addition, a mixture of cyclodextrin and a conjugate of polyethylene glycol and heparin disclosed in Non-Patent Document 1 constitutes a gel, and it is difficult to administer the mixture to the blood.

  Therefore, the present invention uses a composition having a conjugate of a dye and polyethylene glycol, and an additive that interacts with the conjugate, so that the composition has high accumulation in tumors, has little residual in blood, and has low It is intended to provide a contrast agent that can be administered.

  The present invention relates to a composition having a conjugate of a dye and polyethylene glycol, and an additive that interacts with the conjugate, and a contrast agent for photoacoustic imaging having the composition.

  The present invention uses a conjugate of a dye and polyethylene glycol and an additive that interacts with the conjugate, so that the tumor is highly accumulative and has little residual in the blood, so that the tumor depictability is high.

FIG. 1 is a diagram for describing a configuration of a composition according to an embodiment of the present invention.

  Hereinafter, embodiments of the present invention will be described, but the present invention is not limited thereto.

(Configuration of the embodiment)
As shown in FIG. 1, the composition according to this embodiment has a conjugate (A) of a dye and polyethylene glycol, and an additive (B) that interacts with the conjugate.

  After being administered to the blood, aggregates (A) and (B) migrate from the blood to the tumor. Aggregates remaining in the blood during that time are broken down and excreted from the body.

  Therefore, the composition according to the present embodiment has a high ability to accumulate in a tumor and has little residual in the blood, and thus has a high ability to depict a tumor.

(Conjugate of dye and polyethylene glycol)
In the present invention, the conjugate of the dye and polyethylene glycol has an absorption band in a wavelength range of 600 nm to 1300 nm. Having an absorption band means that the light having a wavelength in the range of 600 nm to 1300 nm has a molar extinction coefficient of 10 ⁶ M ⁻¹ cm ⁻¹ or more. In this specification, “polyethylene glycol” includes polyethylene glycol derivatives having a partially substituted structure or the like, and is referred to as polyethylene glycol.

上記式（１）、（５）、（６）、（７）において、Ｒ_３０１乃至Ｒ_３１２は互いに独立に水素原子、ハロゲン原子、−ＰＯ_３Ｔ_３０１、置換または無置換の、フェニル基、チオフェン基、ピリジニル基、または直鎖もしくは分岐の炭素数１乃至１８のアルキル基を表す。
Ｔ_３０１は、水素原子、ナトリウム原子、カリウム原子のいずれかを表す。
Ｒ_３１乃至Ｒ_３４は互いに独立に水素原子、または直鎖もしくは分岐の炭素数１乃至１８のアルキル基を表す。
Ａ_３１、Ｂ_３１、Ｂ_３２は、互いに独立に直鎖もしくは分岐の炭素数１乃至１８のアルキレン基を表す。
Ｌ_３１乃至Ｌ_３７は互いに独立にＣＨ、またはＣＲ_３５であり、Ｌ_３１乃至Ｌ_３７は４員環乃至６員環を形成していてもよい。
Ｒ_３５は、直鎖もしくは分岐の炭素数１乃至１８のアルキル基、ハロゲン原子、置換または無置換のフェニル基、ピリジニル基、ベンジル基、ＳＴ_３０２、または、直鎖もしくは分岐の炭素数１乃至１８のアルキレン基を表す。
Ｔ_３０２は、直鎖もしくは分岐の炭素数１乃至１８のアルキル基、置換または無置換のフェニル基または、直鎖もしくは分岐の炭素数１乃至１８のアルキレン基を表す。
Ｑ_３１は、−ＣＯＮＴ_３１−、−ＮＴ_３１ＣＯ−、−ＮＴ_３１（Ｃ＝Ｏ）ＮＴ_３１−、−ＮＴ_３１（Ｃ＝Ｓ）ＮＴ_３１−、−ＮＴ_３１（Ｃ＝Ｏ）Ｏ−、−Ｏ−、−Ｓ−、−Ｓ（＝Ｏ）_２ＮＴ_３１−、−ＯＰ（＝Ｏ）（ＯＴ_３１）Ｏ−、−Ｓ−Ｓ−、−ＣＴ_３１＝Ｎ−、−ＣＴ_３１＝Ｎ−ＮＨ−、−ＣＴ_３１＝Ｎ−Ｏ−、−ＣＴ_３１＝Ｎ−ＮＨ−Ｏ−、−ＣＯＮＴ_３１−Ｒ_３７−（Ｃ＝Ｏ）Ｏ−、−ＣＯＮＴ_３１−Ｒ_３７−ＣＯＮＴ_３１−、及び下記式（２）、（３）のいずれかを表す。
Ｔ_３１は水素原子、または炭素数１〜５のアルキル基のいずれかを表す。
Ｒ_３７は、−ＣＨ（ＣＯ_２Ｔ_３７）−、−ＣＨ（ＣＨ_２ＣＯ_２Ｔ_３７）−、直鎖もしくは分岐の炭素数１乃至１８のアルキレン基を表す。
Ｔ_３７は水素原子、ナトリウム原子、カリウム原子または炭素数１〜５のアルキル基のいずれかを表す。
Ｒ_３８は、炭素数１乃至１８のアルキル基、水素原子、−ＯＣＨ_３、−ＮＨ_２、−ＯＨ、−ＣＯ_２Ｔ_３８、−Ｓ（＝Ｏ）_２ＯＴ_３８、−Ｐ（＝Ｏ）（ＯＴ_３８）_２、及び−ＯＰ（＝Ｏ）（ＯＴ_３８）_２、のいずれかを表す。
Ｔ_３８は、水素原子、ナトリウム原子、カリウム原子のいずれかを表す。
Ｒ_３９は、水素原子、−ＯＣＨ_３、−ＮＨ_２、−ＯＨ、−Ｓ（＝Ｏ）_２ＯＴ_３９、−ＣＯ_２Ｔ_３９、−Ｐ（＝Ｏ）（ＯＴ_３９）_２、−ＣＯＮＨ−ＣＨ（ＣＯ_２Ｔ_３９）−ＣＨ_２（Ｃ＝Ｏ）ＯＴ_３９、−ＣＯＮＨ−ＣＨ（ＣＯ_２Ｔ_３９）−ＣＨ_２ＣＨ_２（Ｃ＝Ｏ）ＯＴ_３９、及び−ＯＰ（＝Ｏ）（ＯＴ_３９）_２、のいずれかを表す。
Ｔ_３９は、水素原子、ナトリウム原子、カリウム原子のいずれかを表す。
ｑは、１乃至２０の整数である。
ｎは、１乃至２５００の整数である。

上記化学式（１）、（５）、（６）、（７）におけるｎが２以上５００以下であることが好ましく、２以上２５０以下であることが好ましく、２０以上２５０以下であることがさらに好ましい。
また、上記化学式（１）、（５）、（６）、（７）におけるｑが１以上１０以下であることが好ましく、１以上４以下であることがさらに好ましい。
化学式（１）で示される好ましい例として以下の化学式（５０１）、（６０１）をあげることができる。

上記化学式（５０１）、（６０１）中のｊ、ｋは１乃至２５００の整数である。 In the present embodiment, the structure of the conjugate of the dye and polyethylene glycol is represented by the following chemical formulas (1), (5), (6), and (7).

In the above formulas (1), (5), (6) and (7), R _{301 to} R ₃₁₂ are each independently a hydrogen atom, a halogen atom, —PO ₃ T ₃₀₁ , a substituted or unsubstituted phenyl group, thiophene Represents a group, a pyridinyl group, or a linear or branched alkyl group having 1 to 18 carbon atoms.
T ₃₀₁ represents any of a hydrogen atom, a sodium atom, and a potassium atom.
R ₃₁ to R ₃₄ represent each independently a hydrogen atom or a linear or branched alkyl group having 1 to 18 carbon atoms.
A ₃₁ , B ₃₁ , and B ₃₂ independently represent a linear or branched alkylene group having 1 to 18 carbon atoms.
L _{31 to} L ₃₇ are each independently CH or CR ₃₅ , and L _{31 to} L ₃₇ may form a 4- to 6-membered ring.
R ₃₅ represents a linear or branched alkyl group having 1 to 18 carbon atoms, a halogen atom, a substituted or unsubstituted phenyl group, a pyridinyl group, a benzyl group, ST ₃₀₂ , or a linear or branched C 1 to C 18 Represents an alkylene group.
T ₃₀₂ represents a linear or branched alkyl group having 1 to 18 carbon atoms, a substituted or unsubstituted phenyl group, or a linear or branched alkylene group having 1 to 18 carbon atoms.
Q _{31 is, -CONT 31 -, - NT 31} CO -, - NT 31 (C = O) NT 31 -, - NT 31 (C = S) NT 31 -, - NT 31 (C = O) O-, _{-O -, - S -, -} S (= O) 2 NT 31 -, - OP (= O) (OT 31) O -, - S-S -, - CT 31 = N -, - CT 31 = N _{-NH -, - CT 31 = N} -O -, - CT 31 = N-NH-O -, - CONT 31 -R 37 - (C = O) O -, - CONT 31 -R 37 -CONT 31 -, And any of the following formulas (2) and (3).
T ₃₁ represents a hydrogen atom or an alkyl group having 1 to 5 carbon atoms.
R ₃₇ represents —CH (CO ₂ T ₃₇ ) —, —CH (CH ₂ CO ₂ T ₃₇ ) —, or a linear or branched alkylene group having 1 to 18 carbon atoms.
T ₃₇ represents one of hydrogen atom, sodium atom, a potassium atom or an alkyl group having 1 to 5 carbon atoms.
R ₃₈ represents an alkyl group having 1 to 18 carbon atoms, a hydrogen _{atom, -OCH 3, -NH 2, -OH} , -CO 2 T 38, -S (= O) 2 OT 38, -P (= O) ( OT _{38) 2,} and _{-OP (= O) (OT 38} ) 2, represents either.
T ₃₈ denotes either a hydrogen atom, sodium atom, potassium atom.
R ₃₉ is a hydrogen _{atom, -OCH 3, -NH 2, -OH} , -S (= O) 2 OT 39, -CO 2 T 39, -P (= O) (OT 39) 2, -CONH-CH _{(CO 2 T 39) -CH 2} (C = O) OT 39, -CONH-CH (CO 2 T 39) -CH 2 CH 2 (C = O) OT 39, and -OP (= O) _{(OT 39} ) ₂ .
T ₃₉ denotes either a hydrogen atom, sodium atom, potassium atom.
q is an integer of 1 to 20.
n is an integer of 1 to 2500.

In the above chemical formulas (1), (5), (6) and (7), n is preferably 2 or more and 500 or less, more preferably 2 or more and 250 or less, and even more preferably 20 or more and 250 or less. .
Further, q in the above chemical formulas (1), (5), (6) and (7) is preferably 1 or more and 10 or less, more preferably 1 or more and 4 or less.
Preferred examples of the chemical formula (1) include the following chemical formulas (501) and (601).

J and k in the chemical formulas (501) and (601) are integers of 1 to 2500.

(Dye)
The dye of the conjugate of the dye and polyethylene glycol has, as a basic skeleton, a structure in which a methine chain and a 5-membered ring containing N are bonded to both ends of the methine chain.

上記式（１１）、（１５）、（１６）、（１７）において、Ｒ_２０１乃至Ｒ_２１２は互いに独立に水素原子、ハロゲン原子、−ＰＯ_３Ｔ_２０１、ベンゼン環、チオフェン環、ピリジン環、または直鎖もしくは分岐の炭素数１乃至１８のアルキル基を表す。
Ｔ_２０１は、水素原子、ナトリウム原子、カリウム原子のいずれかを表す。
Ｒ_２１乃至Ｒ_２４は互いに独立に水素原子、または直鎖もしくは分岐の炭素数１乃至１８のアルキル基を表す。
Ａ_２１、Ｂ_２１は、互いに独立に直鎖もしくは分岐の炭素数１乃至１８のアルキレン基を表す。
Ｌ_２１乃至Ｌ_２７は互いに独立にＣＨ、またはＣＲ_２５であり、Ｌ_２１乃至Ｌ_２７は４員環乃至６員環を形成していてもよい。
Ｒ_２５は、直鎖もしくは分岐の炭素数１乃至１８のアルキル基、ハロゲン原子、ベンゼン環、ピリジン環、ベンジル基、ＳＴ_２０２、または、直鎖もしくは分岐の炭素数１乃至１８のアルキレン基を表す。
Ｔ_２０２は、直鎖もしくは分岐の炭素数１乃至１８のアルキル基、置換または無置換のフェニル基または、直鎖もしくは分岐の炭素数１乃至１８のアルキレン基を表す。
Ｒ_２８は、水素原子、−ＯＣＨ_３、−ＮＨ_２、−ＯＨ、−ＣＯ_２Ｔ_２８、−Ｓ（＝Ｏ）_２ＯＴ_２８、−Ｐ（＝Ｏ）（ＯＴ_２８）_２、−ＣＯＮＨ−ＣＨ（ＣＯ_２Ｔ_２８）−ＣＨ_２（Ｃ＝Ｏ）ＯＴ_２８、−ＣＯＮＨ−ＣＨ（ＣＯ_２Ｔ_２８）−ＣＨ_２ＣＨ_２（Ｃ＝Ｏ）ＯＴ_２８、及び−ＯＰ（＝Ｏ）（ＯＴ_２８）_２、のいずれかを表す。
Ｔ_２８は、水素原子、ナトリウム原子、カリウム原子のいずれかを表す。
Ｒ_２９は、水素原子、−ＯＣＨ_３、−ＮＨ_２、−ＯＨ、−ＣＯ_２Ｔ_２９、−Ｓ（＝Ｏ）_２ＯＴ_２９、−Ｐ（＝Ｏ）（ＯＴ_２９）_２、−ＣＯＮＨ−ＣＨ（ＣＯ_２Ｔ_２９）−ＣＨ_２（Ｃ＝Ｏ）ＯＴ_２９、−ＣＯＮＨ−ＣＨ（ＣＯ_２Ｔ_２９）−ＣＨ_２ＣＨ_２（Ｃ＝Ｏ）ＯＴ_２９、及び−ＯＰ（＝Ｏ）（ＯＴ_２９）_２、のいずれかを表す。
Ｔ_２９は、水素原子、ナトリウム原子、カリウム原子のいずれかを表す。 The dye in the present embodiment is preferably a compound that can absorb light having a wavelength included in the range of 600 nm to 1300 nm. The dye in this embodiment preferably has a molar extinction coefficient of 10 ⁶ M ⁻¹ cm ⁻¹ or more. Further, the polyethylene glycol part hardly absorbs in the above wavelength range. Therefore, it can be rephrased that the polymer according to the present embodiment preferably has a molar extinction coefficient of 10 ⁶ M ⁻¹ cm ⁻¹ or more for light having any wavelength in the range of 600 nm to 1300 nm.
Preferred examples of the dye in the present embodiment include compounds represented by the following chemical formulas (11), (15), (16), and (17).

The formula (11), in (15), (16), _{(17), R 201} to _{R 212} are each independently a hydrogen atom, a halogen _atom, -PO _{3 T 201,} a benzene ring, a thiophene ring, a pyridine ring or, Represents a linear or branched alkyl group having 1 to 18 carbon atoms.
T ₂₀₁ represents any one of a hydrogen atom, a sodium atom, and a potassium atom.
R _{21 to} R ₂₄ independently represent a hydrogen atom or a linear or branched alkyl group having 1 to 18 carbon atoms.
A ₂₁ and B ₂₁ each independently represent a linear or branched alkylene group having 1 to 18 carbon atoms.
L _{21 to} L ₂₇ are independently CH or CR ₂₅ , and L _{21 to} L ₂₇ may form a 4- to 6-membered ring.
R ₂₅ represents a linear or branched alkyl group having 1 to 18 carbon atoms, a halogen atom, a benzene ring, a pyridine ring, a benzyl group, ST ₂₀₂ , or a linear or branched alkylene group having 1 to 18 carbon atoms. .
T ₂₀₂ represents a linear or branched alkyl group having 1 to 18 carbon atoms, a substituted or unsubstituted phenyl group, or a linear or branched alkylene group having 1 to 18 carbon atoms.
R ₂₈ is a hydrogen _{atom, -OCH 3, -NH 2, -OH} , -CO 2 T 28, -S (= O) 2 OT 28, -P (= O) (OT 28) 2, -CONH-CH _{(CO 2 T 28) -CH 2} (C = O) OT 28, -CONH-CH (CO 2 T 28) -CH 2 CH 2 (C = O) OT 28, and -OP (= O) _{(OT 28} ) ₂ .
T ₂₈ denotes either a hydrogen atom, sodium atom, potassium atom.
R ₂₉ is a hydrogen _{atom, -OCH 3, -NH 2, -OH} , -CO 2 T 29, -S (= O) 2 OT 29, -P (= O) (OT 29) 2, -CONH-CH _{(CO 2 T 29) -CH 2} (C = O) OT 29, -CONH-CH (CO 2 T 29) -CH 2 CH 2 (C = O) OT 29, and -OP (= O) _{(OT 29} ) ₂ .
T ₂₉ denotes either a hydrogen atom, sodium atom, potassium atom.

Further, the compounds represented by the chemical formulas (11), (15), (16) and (17) are represented by the following chemical formulas (101) and (102) (hereinafter, the compounds (101) and (102) In some cases).

(Polyethylene glycol)
As the polyethylene glycol of the conjugate of the dye and the polyethylene glycol, a molecular weight of 100 or more is preferable because of high dispersibility, and a molecular weight of 100,000 or less is preferable because the solution viscosity does not become too large.

  Further, the conjugate of the dye and the polyethylene glycol in the present embodiment may have not only a straight chain but also a branched structure.

  Further, it may have a plurality of amino groups capable of binding to the above dye. This is because the number of dyes bonded per unit of polyethylene glycol can be increased by bonding a plurality of dyes.

上記化学式（２０１）において、Ｄ_１１は、直鎖または分岐の炭素数１〜１８のアルキレン基を表す。
Ｅ_１１は、直鎖または分岐の炭素数１乃至１８のアルキル基、水素原子、−ＯＣＨ_３、−ＮＨ_２、−ＯＨ、−ＣＯ_２Ｔ_３８、−Ｓ（＝Ｏ）_２ＯＴ_３８、−Ｐ（＝Ｏ）（ＯＴ_３８）_２、及び−ＯＰ（＝Ｏ）（ＯＴ_３８）_２、のいずれかを表す。
ｕは、１乃至２０の整数である。
ｖは、１乃至２５００の整数である。 As an example of the polyethylene glycol in the present embodiment, a compound represented by the chemical formula (201) can be given.

In the above chemical formula (201), D ₁₁ represents a linear or branched alkylene group having 1 to 18 carbon atoms.
E ₁₁ is straight or branched alkyl group having 1 to 18 carbon atoms, a hydrogen _{atom, -OCH 3, -NH 2, -OH} , -CO 2 T 38, -S (= O) 2 OT 38, -P _{(= O) (OT 38)} 2, and _{-OP (= O) (OT 38} ) 2, represents either.
u is an integer of 1 to 20.
v is an integer of 1 to 2500.

上記化学式（２０２）においてｔは、１乃至２５００の整数である。
また、上記化学式（２０２）において分子量が１０００〜４００００であることが好ましく、２０００〜３００００であることが好ましく、５０００〜２００００であることがさらに好ましい。 Further, it is more preferable that the compound represented by the chemical formula (201) is represented by the following chemical formula (202) (hereinafter, may be abbreviated as the compound (202)).

In the above chemical formula (202), t is an integer of 1 to 2500.
Further, in the above chemical formula (202), the molecular weight is preferably from 1,000 to 40,000, more preferably from 2,000 to 30,000, and still more preferably from 5,000 to 20,000.

上記化学式（２０４）においてｓは互いに独立に、１乃至２５００の整数である。また、上記化学式（２０４）において分子量が２００００以上６００００以下であることが好ましい。
なお、上記化学式（２０４）においてＸは（ＣＨ_２）_３−ＮＨ_２である。 As other examples, SUNBRIGHT (registered trademark) PA Series (manufactured by NOF) <SUNBRIGHT MEPA-20H, SUNBRIGHT MEPA-50H, SUNBRIGHT MEPA-12T, SUNBRIGHT MEPA-20T, SUNBRIGHT RIGHT REPAGHT RIGHT REPAGHT RIGHT REPAGHT RIGHT REPAG (Registered trademark) EA Series (manufactured by NOF) <SUNBRIGHT ME-050EA, SUNBRIGHT ME-100EA, SUNBRIGHT ME-200EA (compound having a molecular weight of 20,000 in the above chemical formula (202)), SUNBRIGHT ME-300EA, HUNBRIME Methoxyl PEG amine (manufactured by NANOCS) <PG1-AM-35 , PG1-AM-550, PG1-AM-750, PG1-AM-1k, PG1-AM-2k, PG1-AM-5k>, SUNBRIGHT GL2-200PA (made by NOF), SUNBRIGHT GL2-400PA (made by NOF) ), SUNBRIGHT GL2-600PA (made by NOF), SUNBRIGHT GL3-400PA 100U (made by NOF), SUNBRIGHT GL4-600PA (made by NOF), SUNBRIGHT GL4-800PA (made by NOF), SUNBRIGHT PTE2- SUNBRIGHT PTE-400PA (manufactured by NOF, a compound having a molecular weight of 40000 in the following chemical formula (204)), SUNBRIGHT PTE-200PA (manufactured by NOF, a compound having a molecular weight of 20,000 in the following chemical formula (204)), UNBRIGHT HGEO-150PA (day made oil), (NOF) SUNBRIGHT HGEO-400PA, SUNBRIGHT (NOF) PTE2-200MA2, SUNBRIGHT PTE-400MA2 (NOF) can be mentioned.

In the above chemical formula (204), s is an integer of 1 to 2500 independently of each other. Further, in the above chemical formula (204), the molecular weight is preferably from 20,000 to 60,000.
In the above chemical formula (204), X is (CH ₂ ) ₃ —NH ₂ .

(Binding process of dye and polyethylene glycol)
The step of bonding the dye and polyethylene glycol in the present embodiment is performed by the dye represented by the chemical formulas (11), (15), (16), and (17) and the chemical formula (201). The reaction is not particularly limited as long as a conjugate with a compound having a suitable polyethylene glycol can be obtained.

  Examples of the reaction of the conjugate of the dye and the polyethylene glycol include the following. For example, as a reaction between a carboxyl group and an amino group, a method using a condensing agent, a method of forming a salt and performing condensation by a dehydration reaction, a method of using a dehydrating agent, a method of converting a carboxyl group into an acid chloride and reacting with an amino group Any of these can be used.

  As the above-mentioned condensing agent, a carbodiimide-based condensing agent, a phosphoric acid-based condensing agent, and the like can be used.

  Examples of the carbodiimide-based condensing agent include N, N'-dicyclohexylcarbodiimide (DCC) and water-soluble carbodiimide (WSC).

  The amount of the condensing agent to be used is 0.1 mol or more, preferably 1 mol or more with respect to the compounds represented by the chemical formulas (11), (15), (16) and (17). It is. It is also possible to use the condensing agent itself as a reaction solvent.

  The amount of the compound represented by the chemical formulas (11), (15), (16), and (17) used in the reaction step in the present embodiment is determined by the number of amino groups contained in the compound represented by the chemical formula (201). It is preferably in the range of 0.1 to 50.0 moles with respect to More preferably, the molar ratio is in the range of 1.0 to 20.0 times, particularly preferably 1.0 to 10.0 times. This is because the less the compounds represented by the chemical formulas (11), (15), (16) and (17) are used in the reaction step, the less unreacted chemical formulas (11), (15), and This is because the burden of removing the compounds represented by (16) and (17) is small.

(Organic solvent used in the reaction step)
The organic solvent used in the reaction step of the present embodiment combines the compounds represented by the chemical formulas (11), (15), (16), and (17) with the compound represented by the chemical formula (201). There is no particular limitation as long as it is possible.

  Examples of the organic solvent used in the reaction step, hexane, cyclohexane, hydrocarbons such as heptane, acetone, ketones such as methyl ethyl ketone, dimethyl ether, diethyl ether, ethers such as tetrahydrofuran, dichloromethane, chloroform, carbon tetrachloride, Halogenated hydrocarbons such as dichloroethane and trichloroethane, aromatic hydrocarbons such as benzene and toluene, aprotic polar solvents such as N, N-dimethylformamide (hereinafter may be abbreviated as DMF), dimethyl sulfoxide (DMSO) And pyridine derivatives. It is also possible to use a mixture of two or more of these organic solvents.

  Preferably, aprotic polar solvents such as DMF and dimethyl sulfoxide, and halogenated hydrocarbons such as dichloromethane and chloroform are exemplified. This is because the compounds represented by the chemical formulas (11), (15), (16), and (17) have high solubility in these organic solvents, and can be reacted in a state where the compounds are sufficiently dispersed. That's why. The amount of the organic solvent used in the reaction step can be appropriately determined according to the reaction conditions and the like.

  In the reaction step of the present embodiment, the reaction temperature is not particularly limited, but is usually a temperature in the range of 0 ° C. or more and the boiling point of the solvent or less. However, it is desirable to carry out the reaction at an optimum temperature according to the condensing agent used.

  In the reaction step of the present embodiment, the reaction time is, for example, in the range of 1 to 48 hours.

(Purification process)
In the purification step in the present embodiment, the compound having a dye represented by the chemical formula (11), (15), (16), or (17) and a polyethylene glycol represented by the chemical formula (201) is used. The method is not particularly limited as long as the conjugate can be purified.

  Examples of the method for isolating and purifying the conjugate obtained in the above reaction step include normal phase chromatography, size exclusion chromatography, ultrafiltration, and dialysis using an organic solvent.

(Organic solvent used in the purification process)
As the organic solvent used in the purification step in the present embodiment, hexane, cyclohexane, hydrocarbons such as heptane, acetone, ketones such as methyl ethyl ketone, dimethyl ether, diethyl ether, ethers such as tetrahydrofuran, dichloromethane, chloroform, Halogenated hydrocarbons such as carbon tetrachloride, dichloroethane and trichloroethane, aromatic hydrocarbons such as benzene and toluene, aprotic polar solvents such as N, N-dimethylformamide and dimethylsulfoxide, pyridine derivatives, methanol, ethanol, etc. Alcohols. It is also possible to use a mixture of two or more of these solvents.

  Preferable examples include halogenated hydrocarbons such as dichloromethane and chloroform, and alcohols such as methanol and ethanol. This is because when these solvents are used, the dyes represented by the chemical formulas (11), (15), (16), and (17) have good solubility with the conjugate, and the chemical formulas (11), This is because purification can be performed in a state where the dyes represented by (15), (16) and (17) and the conjugate are sufficiently dispersed.

  In addition, it is preferable that the organic solvent used in the purification step and the organic solvent used in the reaction step in the present embodiment are the same.

上記化学式（３０１）中、ｍは、１〜２０の整数から選ばれ、Ｒはそれぞれ独立に上記化学式（４０１）で表わされる構造または水素原子である。
上記化学式（４０１）中、ｎは、１〜２０の整数から選ばれる。 (Additive)
As an example of the additive in the present embodiment, a compound represented by a chemical formula (301) can be given.

In the above chemical formula (301), m is selected from an integer of 1 to 20, and R is each independently a structure represented by the above chemical formula (401) or a hydrogen atom.
In the above chemical formula (401), n is selected from an integer of 1 to 20.

Examples of the additive include α-cyclodextrin (chemical formula (302)), β-cyclodextrin (chemical formula (303)), hydroxypropyl-β-cyclodextrin (chemical formula (304)), γ-cyclodextrin (chemical formula ( 305)). At least one of these may be used, and a plurality of additives may be used.

(Contrast agent for photoacoustic imaging)
In the present embodiment, the contrast agent for photoacoustic imaging has the above composition and a dispersion medium. Examples of the dispersion medium include physiological saline, distilled water for injection, phosphate buffered saline, and aqueous glucose solution.

上記化学式（２０１）において、Ｄ_１１は、直鎖または分岐の炭素数１〜１８のアルキレン基を表す。
Ｅ_１１は、直鎖または分岐の炭素数１乃至１８のアルキル基、水素原子、−ＯＣＨ_３、−ＮＨ_２、−ＯＨ、−ＣＯ_２Ｔ_３８、−Ｓ（＝Ｏ）_２ＯＴ_３８、−Ｐ（＝Ｏ）（ＯＴ_３８）_２、及び−ＯＰ（＝Ｏ）（ＯＴ_３８）_２、のいずれかを表す。
ｕは、１乃至２０の整数である。
ｖは、１乃至２５００の整数である。

上記式（１１）、（１５）、（１６）、（１７）において、Ｒ_２０１乃至Ｒ_２１２は互いに独立に水素原子、ハロゲン原子、−ＰＯ_３Ｔ_２０１、ベンゼン環、チオフェン環、ピリジン環、または直鎖もしくは分岐の炭素数１乃至１８のアルキル基を表す。
Ｔ_２０１は、水素原子、ナトリウム原子、カリウム原子のいずれかを表す。
Ｒ_２１乃至Ｒ_２４は互いに独立に水素原子、または直鎖もしくは分岐の炭素数１乃至１８のアルキル基を表す。
Ａ_２１、Ｂ_２１は、互いに独立に直鎖もしくは分岐の炭素数１乃至１８のアルキレン基を表す。
Ｌ_２１乃至Ｌ_２７は互いに独立にＣＨ、またはＣＲ_２５であり、Ｌ_２１乃至Ｌ_２７は４員環乃至６員環を形成していてもよい。
Ｒ_２５は、直鎖もしくは分岐の炭素数１乃至１８のアルキル基、ハロゲン原子、ベンゼン環、ピリジン環、ベンジル基、ＳＴ_２０２、または、直鎖もしくは分岐の炭素数１乃至１８のアルキレン基を表す。
Ｔ_２０２は、直鎖もしくは分岐の炭素数１乃至１８のアルキル基、置換または無置換のフェニル基または、直鎖もしくは分岐の炭素数１乃至１８のアルキレン基を表す。
Ｒ_２８は、水素原子、−ＯＣＨ_３、−ＮＨ_２、−ＯＨ、−ＣＯ_２Ｔ_２８、−Ｓ（＝Ｏ）_２ＯＴ_２８、−Ｐ（＝Ｏ）（ＯＴ_２８）_２、−ＣＯＮＨ−ＣＨ（ＣＯ_２Ｔ_２８）−ＣＨ_２（Ｃ＝Ｏ）ＯＴ_２８、−ＣＯＮＨ−ＣＨ（ＣＯ_２Ｔ_２８）−ＣＨ_２ＣＨ_２（Ｃ＝Ｏ）ＯＴ_２８、及び−ＯＰ（＝Ｏ）（ＯＴ_２８）_２、のいずれかを表す。
Ｔ_２８は、水素原子、ナトリウム原子、カリウム原子のいずれかを表す。
Ｒ_２９は、水素原子、−ＯＣＨ_３、−ＮＨ_２、−ＯＨ、−ＣＯ_２Ｔ_２９、−Ｓ（＝Ｏ）_２ＯＴ_２９、−Ｐ（＝Ｏ）（ＯＴ_２９）_２、−ＣＯＮＨ−ＣＨ（ＣＯ_２Ｔ_２９）−ＣＨ_２（Ｃ＝Ｏ）ＯＴ_２９、−ＣＯＮＨ−ＣＨ（ＣＯ_２Ｔ_２９）−ＣＨ_２ＣＨ_２（Ｃ＝Ｏ）ＯＴ_２９、及び−ＯＰ（＝Ｏ）（ＯＴ_２９）_２、のいずれかを表す。
Ｔ_２９は、水素原子、ナトリウム原子、カリウム原子のいずれかを表す。

上記化学式（３０１）中、ｍは、１〜２０の整数から選ばれ、Ｒはそれぞれ独立に上記化学式（４０１）で表わされる構造または水素原子である。
上記化学式（４０１）中、ｎは、１〜２０の整数から選ばれる。 (Method of preparing composition)
The method for producing a composition according to the present embodiment comprises bonding polyethylene glycol represented by the following chemical formula (201) with dyes represented by the following chemical formulas (11), (15), (16), and (17). And obtaining a conjugate. The method further comprises a step of mixing the obtained conjugate with an additive represented by the following chemical formula (301).

In the above chemical formula (201), D ₁₁ represents a linear or branched alkylene group having 1 to 18 carbon atoms.
E ₁₁ is straight or branched alkyl group having 1 to 18 carbon atoms, a hydrogen _{atom, -OCH 3, -NH 2, -OH} , -CO 2 T 38, -S (= O) 2 OT 38, -P _{(= O) (OT 38)} 2, and _{-OP (= O) (OT 38} ) 2, represents either.
u is an integer of 1 to 20.
v is an integer of 1 to 2500.

In the above formulas (11), (15), (16), and (17), R _{201 to} R ₂₁₂ each independently represent a hydrogen atom, a halogen atom, —PO ₃ T ₂₀₁ , a benzene ring, a thiophene ring, a pyridine ring, or Represents a linear or branched alkyl group having 1 to 18 carbon atoms.
T ₂₀₁ represents any one of a hydrogen atom, a sodium atom, and a potassium atom.
R _{21 to} R ₂₄ independently represent a hydrogen atom or a linear or branched alkyl group having 1 to 18 carbon atoms.
A ₂₁ and B ₂₁ each independently represent a linear or branched alkylene group having 1 to 18 carbon atoms.
L ₂₁ to _{L 27} is CH or _{CR 25,} independently of one _{another, L 21} to _{L 27} may form a 4-membered ring or 6-membered ring.
R ₂₅ represents a linear or branched alkyl group having 1 to 18 carbon atoms, a halogen atom, a benzene ring, a pyridine ring, a benzyl group, ST ₂₀₂ , or a linear or branched alkylene group having 1 to 18 carbon atoms. .
T ₂₀₂ represents a linear or branched alkyl group having 1 to 18 carbon atoms, a substituted or unsubstituted phenyl group, or a linear or branched alkylene group having 1 to 18 carbon atoms.
R ₂₈ is a hydrogen _{atom, -OCH 3, -NH 2, -OH} , -CO 2 T 28, -S (= O) 2 OT 28, -P (= O) (OT 28) 2, -CONH-CH _{(CO 2 T 28) -CH 2} (C = O) OT 28, -CONH-CH (CO 2 T 28) -CH 2 CH 2 (C = O) OT 28, and -OP (= O) _{(OT 28} ) _2, represents either.
T ₂₈ denotes either a hydrogen atom, sodium atom, potassium atom.
R ₂₉ is a hydrogen _{atom, -OCH 3, -NH 2, -OH} , -CO 2 T 29, -S (= O) 2 OT 29, -P (= O) (OT 29) 2, -CONH-CH _{(CO 2 T 29) -CH 2} (C = O) OT 29, -CONH-CH (CO 2 T 29) -CH 2 CH 2 (C = O) OT 29, and -OP (= O) _{(OT 29} ) ₂ .
T ₂₉ denotes either a hydrogen atom, sodium atom, potassium atom.

In the above chemical formula (301), m is selected from an integer of 1 to 20, and R is each independently a structure represented by the above chemical formula (401) or a hydrogen atom.
In the above chemical formula (401), n is selected from an integer of 1 to 20.

上記化学式（２０２）においてｔは、１乃至２５００の整数である。
また、上記化学式（２０２）において分子量が１０００〜４００００であることが好ましく、５０００〜１００００であることがさらに好ましい。
また、添加剤が、α−シクロデキストリン、β−シクロデキストリン、ヒドロキシプロピル−β−シクロデキストリン、γ−シクロデキストリンのうち少なくともいずれか１つであることが好ましい。
また、上記組成物と分散媒とを混合して光音響イメージング用造影剤を製造してもよい。 In the production method according to the present embodiment, it is preferable that the polyethylene glycol is represented by the following formula (202).

In the above chemical formula (202), t is an integer of 1 to 2500.
Further, in the above chemical formula (202), the molecular weight is preferably from 1,000 to 40,000, more preferably from 5,000 to 10,000.
The additive is preferably at least one of α-cyclodextrin, β-cyclodextrin, hydroxypropyl-β-cyclodextrin, and γ-cyclodextrin.
Further, the above composition and a dispersion medium may be mixed to produce a contrast agent for photoacoustic imaging.

  Hereinafter, the method for producing the composition according to the present embodiment will be described in detail. As a method for preparing the composition of the present embodiment, a known method can be used. For example, a nanoemulsion method, a nanoprecipitation method, a mixing and stirring method using a good solvent, and the like can be given.

  Examples of the solvent used in this preparation method include hydrocarbons such as hexane, cyclohexane and heptane; ketones such as acetone and methyl ethyl ketone; ethers such as diethyl ether and tetrahydrofuran; dichloromethane, chloroform, carbon tetrachloride, dichloroethane, and trichloroethane. Halogenated hydrocarbons such as benzene, aromatic hydrocarbons such as toluene, esters such as ethyl acetate and butyl acetate, aprotic polar solvents such as N, N-dimethylformamide, dimethyl sulfoxide, water, and pyridine Derivatives can be mentioned. These solvents may be used alone or in any combination.

  In the Nanoemulsion method, an emulsion can be prepared by a conventionally known emulsification method. Conventionally known methods include, for example, an intermittent shaking method, a stirring method using a mixer such as a propeller-type stirrer and a turbine-type stirrer, a colloid mill method, a homogenizer method, and an ultrasonic irradiation method. These methods can be used alone or in combination of two or more. The emulsion may be prepared by one-stage emulsification or may be prepared by multi-stage emulsification. However, the emulsification method is not limited to the above method as long as the object of the present invention can be achieved.

  In the Nanoprecipitation method, the composition of the present embodiment can be prepared by a conventionally known method of mixing and stirring an organic solvent dispersion with an aqueous solution, or a method of mixing and stirring an aqueous solution with an organic solvent dispersion. is there.

  In the Nanoemulsion method and the Nanoprecipitation method, it is possible to prepare a conjugate of a dye and polyethylene glycol and an additive by dissolving it in an aqueous solution or an organic solvent dispersion.

  In the mixing and stirring method using a good solvent, a good solvent solution (1) dissolved in a good solvent of a conjugate of a dye and polyethylene glycol and a good solvent solution (2) dissolved in a good solvent of an additive are mixed and stirred. Thereby, the composition of the present embodiment can be prepared.

(Mixing ratio of organic solvent dispersion and aqueous solution)
The weight ratio of the aqueous solution and the organic solvent used in the Nanoemulsion method is not particularly limited as long as an oil-in-water (O / W) emulsion can be formed. Preferably, the weight ratio of the organic solvent to the aqueous solution is in the range of 1: 2 to 1: 1000.

  The weight ratio of the aqueous solution and the organic solvent used in the Nanoprecipitation method is not particularly limited as long as the composition of the present embodiment can be recovered. Preferably, the weight ratio of the organic solvent to the aqueous solution is in the range of 1: 1 to 1: 1000.

  The weight ratio of the good solvent solutions (1) and (2) used in the mixing and stirring method using a good solvent is not particularly limited as long as the composition of the present embodiment can be recovered. Preferably, the weight ratio of the good solvent solutions (1) and (2) used is in the range of 1: 1 to 1: 1000.

(Concentration of pigment and polyethylene glycol in solution, material concentration of additives)
The concentration of the conjugate of the dye and the polyethylene glycol in each solution is not particularly limited as long as they are in a range in which these can be dissolved. A preferable concentration can be 0.0005 to 300 mg / ml.
The concentration of the additive in each solution is not particularly limited as long as it is within a range where these can be dissolved. A preferable concentration can be 0.0005 to 300 mg / ml.

(Organic solvent distillation)
From the prepared composition, the organic solvent can be distilled off, if necessary.
The distillation can be performed by any conventionally known method, and examples thereof include a method of removing by heating, and a method of using a pressure reducing device such as an evaporator.
However, the distillation is not limited to the above method as long as the object of the present invention can be achieved.

(Purification of composition)
The produced composition of the present embodiment can be purified by any conventionally known method. For example, size exclusion chromatography, ultrafiltration, dialysis, and centrifugation can be mentioned.
However, the purification method is not limited to the above method as long as the object of the present invention can be achieved.

(Photoacoustic imaging method)
In one embodiment of the present invention, the composition of the present invention can be used as a contrast agent for photoacoustic imaging. In this specification, photoacoustic imaging is a concept including photoacoustic tomography (tomography). The photoacoustic imaging method using the contrast agent according to the present embodiment includes a step of administering the contrast agent according to the present embodiment to a specimen or a sample obtained from the specimen, and applying a pulsed light to the specimen or the sample obtained from the specimen. Irradiation. And a step of measuring a photoacoustic signal of a substance present in the specimen or a sample obtained from the specimen.

  An example of the photoacoustic imaging method using the contrast agent for acoustic imaging according to the present embodiment is as follows. That is, the contrast agent for acoustic imaging according to the present embodiment is administered to a sample, or added to a sample such as an organ obtained from the sample. Note that the sample refers to humans, laboratory animals, pets, etc., and any other organism without particular limitation, and examples of the sample in or obtained from the sample include organs, tissues, tissue sections, and cells. And cell lysates. After the administration or addition of the substance, the specimen or the like is irradiated with a laser pulse light in a near infrared wavelength range.

  In the photoacoustic imaging method according to the present embodiment, the wavelength of the irradiated light can be selected depending on the laser light source used. In the photoacoustic imaging method according to the present embodiment, in order to efficiently acquire an acoustic signal, near-infrared light having a wavelength of 600 nm to 1300 nm called a “window of a living body” which is less affected by light absorption and diffusion in a living body. It is preferable to irradiate light of the wavelength of the region.

  A photoacoustic signal (acoustic wave) from the contrast agent according to the present embodiment is detected by an acoustic wave detector, for example, a piezoelectric transducer, and is converted into an electric signal. Based on the electric signal obtained from the acoustic wave detector, it is possible to calculate the position and size of the absorber in the sample or the like, or the optical characteristic value distribution such as the molar extinction coefficient. For example, if the contrast agent is detected at or above the reference threshold, it is estimated that the substance is present in the sample, or it can be estimated that the substance is present in the sample obtained from the sample. .

  In the present invention, by suppressing the leakage of the dye, quenching due to the accumulation of the dye is caused, the energy of the irradiated pulse light is prevented from being used for fluorescence emission, and it can be converted into more heat energy. . Therefore, it is possible to more efficiently obtain an acoustic signal.

  Hereinafter, the present invention will be described with reference to Examples to further clarify the features of the present invention. However, the present invention is not limited to these Examples, but includes materials, composition conditions, reaction conditions, and the like. Can be freely changed as long as a composition having the same function and effect can be obtained.

(Photoacoustic characteristics evaluation method)
The measurement of the photoacoustic signal was performed by irradiating the sample with a pulsed laser beam, detecting the photoacoustic signal from the sample using a piezoelectric element, amplifying the sample with a high-speed preamplifier, and acquiring the signal with a digital oscilloscope. The specific conditions are as follows. As a light source, a titanium sapphire laser (manufactured by Lotis) was used. The wavelengths were 750 nm and 780 nm, the energy density was 12 mJ / cm ² , the pulse width was 20 nanoseconds, and the pulse repetition was 10 Hz. Model V303 (manufactured by Panametrics-NDT) was used as the ultrasonic transducer. The center band is 1 MHz, the element size is φ0.5, the measurement distance is 25 mm (Non-focus), and the amplifier is +30 dB (ultrasonic preamplifier Model 5682 manufactured by Olympus). The measurement container was a polystyrene cuvette, the optical path length was 0.1 cm, and the sample volume was about 200 μl. The measurement was performed using a DPO4104 (manufactured by Tektronix) as a trigger: photoacoustic light was detected by a photodiode, and data acquisition was measured 128 times (128 pulses) average.

(Example A1)
105.7 mg of PEG having an amino group (manufactured by NOF CORPORATION, SUNBRIGHT ME-100EA, molecular weight: 10000) and 7.8 mg of the compound (101) as a dye were weighed, and dissolved in 8.0 mL of chloroform. To this solution, 7.3 mg of 4-dimethylaminopyridine (DMAP) (manufactured by Tokyo Chemical Industry Co., Ltd.) was added. To the solution obtained by adding DMAP, 2.8 mg (manufactured by Sigma-Aldrich) of water-soluble carbodiimide 1-Ethyl-3- (3-dimethylaminopropyl) carbodiimide Hydrochloride (hereinafter sometimes abbreviated as WSC) was further added.
The resulting solution was stirred at room temperature for 24 hours.
The obtained reaction solution was separated and purified by dialysis using a dialysis membrane (manufactured by Spectrum Laboratories, molecular weight cut off 3500) and methanol as a solvent.
The solvent of the separated and purified solution was distilled off using an evaporator, and then dried using a vacuum drier to obtain a conjugate (A-1) of the dye and polyethylene glycol.

(Example B1)
2.9 mg of the conjugate (A-1) was dissolved in 2.0 mL of ultrapure water to prepare a conjugate solution (BA-1).
As an additive, 3.2 mg of β-cyclodextrin (manufactured by Tokyo Chemical Industry Co., Ltd.) was dissolved in 2.0 mL of ultrapure water to prepare an additive solution (BA-2).
The conjugate solution (BA-1) was added to the additive solution (BA-2) and stirred at room temperature for 15 minutes to obtain a solution (BA-3).
The solution (BA-3) was freeze-dried to obtain a composition (B-1).

(Example B2)
A composition (B-2) was obtained in the same manner as in Example B1, except that β-cyclodextrin was changed to hydroxypropyl-β-cyclodextrin (manufactured by Tokyo Chemical Industry Co., Ltd.).

(Comparative Example C1)
Except not using (beta) -cyclodextrin, it produced similarly to Example B1 and obtained the composition (C-1).

(Example D1)
2.9 mg of the conjugate (A-1) was dissolved in 1.6 mL of chloroform to prepare a conjugate solution (DA-1).
As an additive, 3.2 mg of β-cyclodextrin was dissolved in 20 mL of ultrapure water to prepare an additive solution (DA-2).
While stirring the additive solution, the conjugate solution was added dropwise to prepare an emulsion preparation solution.
The emulsion preparation liquid was subjected to ultrasonic irradiation using an ultrasonic dispersing apparatus (manufactured by Tommy, UD-200) for an intensity scale of 10 and for 1 minute and 30 seconds to prepare an emulsion.
In order to remove chloroform in the emulsion, the mixture was heated and stirred at 40 ° C. for 4 hours. The recovered solution was filtered with a filter having a pore size of 0.45 μm to obtain a composition (D-1).

(Example D2)
A composition (D-2) was obtained in the same manner as in Example D1, except that β-cyclodextrin was changed to hydroxypropyl-β-cyclodextrin.

(Comparative Example E1)
Except not using (beta) -cyclodextrin, it produced similarly to Example D1 and obtained the composition (E-1).

(Example F1)
738.4 mg of PEG having an amino group (manufactured by NANOCS, PG1-AM-5k, molecular weight: 5000) and 312.0 mg of the above compound (101) as a dye were weighed and dissolved in 20.0 mL of chloroform. To this solution, 48.9 mg of 4-dimethylaminopyridine (DMAP) (manufactured by Tokyo Chemical Industry Co., Ltd.) was added. To the solution obtained by adding DMAP, 76.7 mg (manufactured by Sigma-Aldrich) of water-soluble carbodiimide 1-Ethyl-3- (3-dimethylaminopropyl) carbodiimide Hydrochloride (hereinafter sometimes abbreviated as WSC) was further added.
The resulting solution was stirred at room temperature for 24 hours.
The obtained reaction solution was subjected to separation and purification by dialysis using a dialysis membrane (manufactured by Spectrum Laboratories, molecular weight cut off: 2,000) and methanol as a solvent.
The solvent collected by separation and purification was evaporated using an evaporator, and then dried using a vacuum drier to obtain a conjugate (F-1) of a dye and polyethylene glycol.

(Example F2)
983.5 mg of PEG having an amino group (manufactured by NANOCS, PG1-AM-2k, molecular weight: 2000) and 780.0 mg of the compound (101) as a dye were weighed and dissolved in 20.0 mL of chloroform. To this solution was added 122.2 mg of 4-dimethylaminopyridine (DMAP) (manufactured by Tokyo Chemical Industry Co., Ltd.). To the solution obtained by adding DMAP, 191.7 mg (manufactured by Sigma-Aldrich) of water-soluble carbodiimide 1-Ethyl-3- (3-dimethylaminopropyl) carbodiimide Hydrochloride (hereinafter sometimes abbreviated as WSC) was further added.
The resulting solution was stirred at room temperature for 24 hours.
The obtained reaction solution was separated and purified by dialysis using a dialysis membrane (manufactured by Spectrum Laboratories, molecular weight cut off: 1000) and methanol as a solvent.
After evaporating the solvent from the separated and purified solution using an evaporator, the solution was dried using a vacuum dryer to obtain a conjugate (F-2) of the dye and polyethylene glycol.

(Example F3)
100.0 mg of PEG having an amino group (manufactured by NOF CORPORATION, SUNBRIGHT PTE-200PA, molecular weight: 20,000) and 31.2 mg of the above compound (101) as a dye were weighed, and dissolved in 2.0 mL of chloroform. To this solution, 4.9 mg of 4-dimethylaminopyridine (DMAP) (manufactured by Tokyo Chemical Industry Co., Ltd.) was added. To the solution obtained by adding DMAP, 7.7 mg (manufactured by Sigma-Aldrich) of water-soluble carbodiimide 1-Ethyl-3- (3-dimethylaminopropyl) carbodiimide Hydrochloride (hereinafter sometimes abbreviated as WSC) was further added.
The resulting solution was stirred at room temperature for 24 hours.
The obtained reaction solution was separated and purified by dialysis using a dialysis membrane (manufactured by Spectrum Laboratories, molecular weight cut off: 1000) and methanol as a solvent.
The solvent of the separated and purified solution was distilled off using an evaporator, and then dried using a vacuum dryer to obtain a conjugate (F-3) of the dye and polyethylene glycol.

(Example F4)
50.1 mg of PEG having an amino group (manufactured by NOF CORPORATION, SUNBRIGHT ME-050EA, molecular weight: 5000) and 59.2 mg of the above compound (102) as a dye were weighed, and dissolved in 4.0 mL of chloroform. To this solution, 14.8 mg of 4-dimethylaminopyridine (DMAP) (manufactured by Tokyo Chemical Industry) was added. To the solution obtained by adding DMAP, 22.8 mg (manufactured by Sigma-Aldrich) of water-soluble carbodiimide 1-Ethyl-3- (3-dimethylaminopropyl) carbodiimide Hydrochloride (hereinafter sometimes abbreviated as WSC) was further added.
The resulting solution was stirred at room temperature for 24 hours.
The obtained reaction solution was subjected to separation and purification by dialysis using a dialysis membrane (manufactured by Spectrum Laboratories, molecular weight cut off: 2,000) and methanol as a solvent.
The solvent collected by separation and purification was evaporated using an evaporator, and then dried using a vacuum dryer to obtain a conjugate (F-4) of the dye and polyethylene glycol.

(Example G1)
3.8 mg of the conjugate (F-1) was dissolved in 1.6 mL of chloroform to prepare a conjugate solution (GF-1).
As an additive, 180.0 mg of α-cyclodextrin was dissolved in 20 mL of ultrapure water to prepare an additive solution (GF-2).
While stirring the additive solution, the conjugate solution was added dropwise to prepare an emulsion preparation solution.
The emulsion preparation liquid was subjected to ultrasonic irradiation using an ultrasonic dispersing apparatus (manufactured by Tommy, UD-200) for an intensity scale of 10 and for 1 minute and 30 seconds to prepare an emulsion.
In order to remove chloroform in the emulsion, the mixture was heated and stirred at 40 ° C. for 4 hours.
The recovered solution was filtered with a filter having a pore size of 0.45 micrometers. This solution was freeze-dried to obtain a composition (G-1).

(Example G2)
Except having changed (alpha) -cyclodextrin into (beta) -cyclodextrin, it produced similarly to Example G1 and obtained the composition (G-2).

(Example G3)
A composition (G-3) was obtained in the same manner as in Example G1, except that α-cyclodextrin was changed to hydroxypropyl-β-cyclodextrin.

(Example G4)
Except having changed (alpha) -cyclodextrin into (gamma) -cyclodextrin, it produced similarly to Example G1 and obtained the composition (G-4).

(Comparative Example H1)
A composition (H-1) was obtained in the same manner as in Example G1, except that α-cyclodextrin was not used.

(Example L1)
A composition (L-1) was obtained in the same manner as in Example G1, except that the conjugate (F-1) was changed to the conjugate (F-3).

(Example L2)
A composition (L-2) was obtained in the same manner as in Example L1, except that α-cyclodextrin was changed to β-cyclodextrin.

(Example L3)
A composition (L-3) was obtained in the same manner as in Example L1, except that α-cyclodextrin was changed to hydroxypropyl-β-cyclodextrin.

(Example L4)
A composition (L-4) was obtained in the same manner as in Example L1, except that α-cyclodextrin was changed to γ-cyclodextrin.

(Comparative Example M1)
Except not using a-cyclodextrin, it produced similarly to Example L1 and obtained the composition (M-1).

(Example N1)
A composition (N-1) was obtained in the same manner as in Example G1, except that the conjugate (F-1) was changed to the conjugate (F-4).

(Example N2)
A composition (N-2) was obtained in the same manner as in Example N1, except that α-cyclodextrin was changed to β-cyclodextrin.

(Example N3)
A composition (N-3) was obtained in the same manner as in Example N1, except that α-cyclodextrin was changed to hydroxypropyl-β-cyclodextrin.

(Example N4)
A composition (N-4) was obtained in the same manner as in Example N1, except that α-cyclodextrin was changed to γ-cyclodextrin.

(Comparative Example P1)
A composition (P-1) was obtained in the same manner as in Example N1, except that α-cyclodextrin was not used.

(Example Q1)
A composition (Q-1) was obtained in the same manner as in Example G1, except that the amount of α-cyclodextrin was changed to 360 mg.

(Example Q2)
A composition (Q-2) was obtained in the same manner as in Example G1, except that the amount of α-cyclodextrin used was changed to 90 mg.

(Example Q3)
A composition (Q-3) was obtained in the same manner as in Example G1, except that the amount of α-cyclodextrin was changed to 45 mg.

Table 1 shows the photoacoustic signal intensity (wavelength 780 nm) measurement results of the compositions (B-1), (B-2), and (C-1) obtained above.

(Confirmation of tumor accumulation)
For confirmation of tumor accumulation, female outbred BALB / c Slc-nu / nu mice (6 weeks old at the time of purchase) (Japan SLC, Inc.) were used. The mice were acclimated for one week prior to carcinogenesis using standard diet and bedding in an environment in which they had free access to food and drinking water. Colon 26 (mouse colon cancer cells) was injected subcutaneously into mice. By the time of the experiment, all tumors had settled and the mice weighed 17-22 g. 100 μL (13 nmol as a dye) of the above composition was injected intravenously into the mouse tail of the tumor-bearing mouse.

  Next, the mouse to which the composition was administered was euthanized 24 hours after the administration, and then colon 26 tumor was excised. The tumor tissue was transferred to a plastic tube, and a 1.25-fold amount of a 1% aqueous solution of Triton-X100 with respect to the weight of the tumor tissue was added and homogenized. Then DMSO was added. Using Odyssey (registered trademark) CLx Infrared Imaging System, the fluorescence intensity of the homogenate solution was measured to quantify the amount of dye in the tumor tissue.

Table 2 shows the tumor accumulation rates in the compositions (B-1), (B-2), (C-1), (D-1), (D-2), and (E-1) obtained above. Shows the results of blood concentration.

  For the following compositions, ultrapure water was added in an amount of 0.1 times the amount of the solution before freeze-drying, and used for the tumor accumulation confirmation experiment.

Table 3 shows the compositions (G-1), (G-2), (G-3), (G-4), (H-1), (L-1) and (L-) obtained above. 2) Results of tumor accumulation rate and blood concentration in (L-3), (L-4), (M-1), (Q-1), (Q-2), and (Q-3) Is shown.

  The composition prepared in Example Q1 could not be dispersed when ultrapure water was added in an amount of 0.1 times the amount of the solution before freeze-drying.

  Therefore, the composition according to this example has a favorable ratio between the tumor accumulation rate and the blood concentration, and is suitable as a contrast agent having high tumor delineation ability.

Claims (12)

A compound represented by the chemical formula (1), (5), (6) or (7), and at least one of α-cyclodextrin, β-cyclodextrin, hydroxypropyl-β-cyclodextrin, and γ-cyclodextrin contrast agent having a composition comprising an additive is One.

In the above formulas (1), (5), (6) and (7), R _{301 to} R ₃₁₂ are each independently a hydrogen atom, a halogen atom, —PO ₃ T ₃₀₁ , a substituted or unsubstituted phenyl group, thiophene Represents a group, a pyridinyl group, or a linear or branched alkyl group having 1 to 18 carbon atoms.
T ₃₀₁ represents any of a hydrogen atom, a sodium atom, and a potassium atom.
R ₃₁ to R ₃₄ represent each independently a hydrogen atom or a linear or branched alkyl group having 1 to 18 carbon atoms.
A ₃₁ , B ₃₁ , and B ₃₂ independently represent a linear or branched alkylene group having 1 to 18 carbon atoms.
L _{31 to} L ₃₇ are each independently CH or CR ₃₅ , and L _{31 to} L ₃₇ may form a 4- to 6-membered ring.
R ₃₅ represents a linear or branched alkyl group having 1 to 18 carbon atoms, a halogen atom, a substituted or unsubstituted phenyl group, a pyridinyl group, a benzyl group, ST ₃₀₂ , or a linear or branched C 1 to C 18 Represents an alkylene group.
T ₃₀₂ represents a linear or branched alkyl group having 1 to 18 carbon atoms, a substituted or unsubstituted phenyl group, or a linear or branched alkylene group having 1 to 18 carbon atoms.
Q _{31 is, -CONT 31 -, - NT 31} CO -, - NT 31 (C = O) NT 31 -, - NT 31 (C = S) NT 31 -, - NT 31 (C = O) O-, _{-O -, - S -, -} S (= O) 2 NT 31 -, - OP (= O) (OT 31) O -, - S-S -, - CT 31 = N -, - CT 31 = N _{-NH -, - CT 31 = N} -O -, - CT 31 = N-NH-O -, - CONT 31 -R 37 - (C = O) O -, - CONT 31 -R 37 -CONT 31 -, And any one of the chemical formulas (2) and (3).
T ₃₁ represents a hydrogen atom or an alkyl group having 1 to 5 carbon atoms.
R ₃₇ represents —CH (CO ₂ T ₃₇ ) —, —CH (CH ₂ CO ₂ T ₃₇ ) —, or a linear or branched alkylene group having 1 to 18 carbon atoms.
T ₃₇ represents one of hydrogen atom, sodium atom, a potassium atom or an alkyl group having 1 to 5 carbon atoms.
R ₃₈ represents an alkyl group having 1 to 18 carbon atoms, a hydrogen _{atom, -OCH 3, -NH 2, -OH} , -CO 2 T 38, -S (= O) 2 OT 38, -P (= O) ( OT _{38) 2,} and _{-OP (= O) (OT 38} ) 2, represents either.
T ₃₈ denotes either a hydrogen atom, sodium atom, potassium atom.
R ₃₉ is a hydrogen _{atom, -OCH 3, -NH 2, -OH} , -S (= O) 2 OT 39, -CO 2 T 39, -P (= O) (OT 39) 2, -CONH-CH _{(CO 2 T 39) -CH 2} (C = O) OT 39, -CONH-CH (CO 2 T 39) -CH 2 CH 2 (C = O) OT 39, and -OP (= O) _{(OT 39} ) ₂ .
T ₃₉ denotes either a hydrogen atom, sodium atom, potassium atom.
q is an integer of 1 to 20.
n is an integer of 1 to 2500.

2. The imaging method according to claim 1, wherein the compound represented by the chemical formula (1), (5), (6), or (7) has a structure represented by a chemical formula (501) or (601). 3. Agent .

J and k in the chemical formulas (501) and (601) are integers of 1 to 2500. Formula (1), (5), (6), the contrast agent according to claim 1 or 2, characterized in that n is 2 or more and 500 or less in (7). The compound, in the light of any wavelength in the range of 600nm to 1300 nm, according to any one of claims 1 to 3, wherein the molar absorption coefficient is ¹⁰ 6 ^{M -1 cm} -1 or more Contrast agent . Contrast agent for photoacoustic imaging with a contrast agent, and a dispersion medium according to any one of claims 1 to 4. Bonding polyethylene glycol represented by the following chemical formula (201) with a dye represented by the following chemical formula (11), (15), (16) or (17) to obtain a conjugate; , alpha-cyclodextrin, beta-cyclodextrin, hydroxypropyl -β- cyclodextrin method of γ- cyclodextrin contrast agent characterized by comprising the step of mixing the additive with at least any one .

In Chemical Formula _{(201), D 11} represents a linear or branched alkylene group having 1 to 18 carbon atoms.
E ₁₁ is straight or branched alkyl group having 1 to 18 carbon atoms, a hydrogen _{atom, -OCH 3, -NH 2, -OH} , -CO 2 T 38, -S (= O) 2 OT 38, -P _{(= O) (OT 38)} 2, and _{-OP (= O) (OT 38} ) 2, represents either.
u is an integer of 1 to 20.
v is an integer of 1 to 2500.

In the above formulas (11), (15), (16), and (17), R _{201 to} R ₂₁₂ each independently represent a hydrogen atom, a halogen atom, —PO ₃ T ₂₀₁ , a benzene ring, a thiophene ring, a pyridine ring, or Represents a linear or branched alkyl group having 1 to 18 carbon atoms.
T ₂₀₁ represents any one of a hydrogen atom, a sodium atom, and a potassium atom.
R _{21 to} R ₂₄ independently represent a hydrogen atom or a linear or branched alkyl group having 1 to 18 carbon atoms.
A ₂₁ and B ₂₁ each independently represent a linear or branched alkylene group having 1 to 18 carbon atoms.
L _{21 to} L ₂₇ are independently CH or CR ₂₅ , and L _{21 to} L ₂₇ may form a 4- to 6-membered ring.
R ₂₅ represents a linear or branched alkyl group having 1 to 18 carbon atoms, a halogen atom, a benzene ring, a pyridine ring, a benzyl group, ST ₂₀₂ , or a linear or branched alkylene group having 1 to 18 carbon atoms. .
T ₂₀₂ represents a linear or branched alkyl group having 1 to 18 carbon atoms, a substituted or unsubstituted phenyl group, or a linear or branched alkylene group having 1 to 18 carbon atoms.
R ₂₈ is a hydrogen _{atom, -OCH 3, -NH 2, -OH} , -CO 2 T 28, -S (= O) 2 OT 28, -P (= O) (OT 28) 2, -CONH-CH _{(CO 2 T 28) -CH 2} (C = O) OT 28, -CONH-CH (CO 2 T 28) -CH 2 CH 2 (C = O) OT 28, and -OP (= O) _{(OT 28} ) ₂ .
T ₂₈ denotes either a hydrogen atom, sodium atom, potassium atom.
R ₂₉ is a hydrogen _{atom, -OCH 3, -NH 2, -OH} , -CO 2 T 29, -S (= O) 2 OT 29, -P (= O) (OT 29) 2, -CONH-CH _{(CO 2 T 29) -CH 2} (C = O) OT 29, -CONH-CH (CO 2 T 29) -CH 2 CH 2 (C = O) OT 29, and -OP (= O) _{(OT 29} ) ₂ .
T ₂₉ denotes either a hydrogen atom, sodium atom, potassium atom. The method for producing a contrast agent according to claim 6 , wherein the chemical formula (201) is represented by the following chemical formula (202).

In the above chemical formula (202), t is an integer of 1 to 2500. The method for producing a contrast agent according to claim 7 , wherein the compound represented by the chemical formula (202) has a molecular weight of 1,000 to 40,000. The method for producing a contrast agent according to claim 8 , wherein the compound represented by the chemical formula (202) has a molecular weight of 2,000 to 30,000. The method for producing a contrast agent according to claim 6 , wherein the chemical formula (201) is represented by the following chemical formula (204).

In the above formula (204), s is independently an integer from 1 to 2500, and X is (CH ₂ ) ₃ —NH ₂ . The contrast agent according to any one of claims 6 to 10 , wherein the chemical formulas (11), (15), (16), and (17) are represented by the following chemical formulas (101) or (102). Manufacturing method.

A method for producing a contrast agent for photoacoustic imaging, comprising a step of mixing a contrast medium obtained by the method according to any one of claims 6 to 11 and a dispersion medium.

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