JP2014185312A - Compound and contrast medium for photoacoustic imaging having the compound - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a compound high in accumulation property to tumor and large in photoacoustic signal strength generated from the tumor even upon the lapse of a time after administration.SOLUTION: There is provided a compound in which polyethylene glycol and specific cyanine-based compound are bound.

Description

  The present invention relates to a compound and a contrast agent for photoacoustic imaging having the compound.

  A photoacoustic tomography (hereinafter abbreviated as PAT) device is known as one of devices for visualizing information inside a living body. In measurement using a PAT device, the intensity and generation time of a photoacoustic signal emitted from a substance (light absorber) that absorbs light within the object to be measured when the object to be measured is irradiated are measured. An image obtained by calculating the substance distribution inside the measurement body can be obtained.

  Here, any light absorber can be used as long as it absorbs light in a living body and emits an acoustic wave. For example, blood vessels and malignant tumors in the human body can be used as the light absorber. In addition, molecules such as indocyanine green (hereinafter sometimes abbreviated as ICG) can be administered into the body and used as a contrast agent. ICG absorbs light in the near-infrared wavelength region with little influence when irradiated on a human body and high permeability to a living body, and therefore can be suitably used as a contrast agent in a PAT apparatus. In this specification, ICG refers to a compound represented by the following structure.

(1)

However, the counter ion may not be Na ⁺ , and any counter ion such as H ⁺ or K ⁺ can be used.

  However, ICG is known to have a very short half-life in the blood of about several minutes.

  Non-Patent Document 1 reports an example of performing photoacoustic imaging of a rat cerebral blood vessel using a single ICG. According to this report, the photoacoustic signal has dropped to the same level as blood several tens of minutes after the administration of a single ICG in the blood, and the administered substance disappears from the blood immediately after administration. Has been suggested.

  In this way, single ICG disappears from the blood several tens of minutes after being administered into the blood, and therefore, it is considered that the accumulation of the tumor in the tumor after a short time after administration is low.

Optics Letters, 29 (7), 730 (2004)

  Therefore, an object of the present invention is to provide a compound that is highly accumulating in a tumor and has a high intensity of photoacoustic signal emitted from the tumor even when time has elapsed after administration.

The compound according to the present invention is represented by any one of the following formulas (I) to (IV).
^{_{A-L 1 - (CH 2}} ) p- (OCH 2 CH 2) m-O- (CH 2) q-L 2 -A '(I)
^{_{A-L 1 - (CH 2}} ) p- (OCH 2 CH 2) m-O- (CH 2) q-L 2 -M (II)
^{_{N-L 1 - (CH 2}} ) p- (OCH 2 CH 2) m-O- (CH 2) q-L 2 -A (III)
^{_{A-L 1 - (CH 2}} ) p- (OCH 2 CH 2) m-O-R (IV)

In the formula (I), A and A ′ each independently represent any of the following formulas (i) to (vi), and * in the following formulas (i) to (vi) represents L ¹ in the formula (I), It binds to L ^2.

In the formula (I), L ¹ and L ² are each independently —NH—, —O—, —S—, —CO—, or each independently —NH—, —O—, — It is a linker moiety containing one or more of S- and -CO-. p and q are each independently an integer of 1 to 5, and m is an integer of 10 or more and 2500 or less.

  In the formulas (II) and (III), A represents any one of the following formulas (i) to (vi), and * in the following formulas (i) to (vi) represents L1 in the formula (II) or the formula (III) ) To L2.

In formula (II) and formula (III), L ¹ and L ² are each independently —NH—, —O—, —S—, —CO—, or each independently —NH—, It is a linker moiety containing one or more of -O-, -S-, and -CO-. p and q are each independently an integer of 1 to 5, and m is an integer of 10 or more and 2500 or less.

In the formula (II), when L ² is any of —NH—, —O—, and —S—, M is a hydrogen atom, and when L ² is —CO—, M is a hydroxyl group.

In the formula (III), when L ¹ is any of —NH—, —O—, and —S—, N is a hydrogen atom, and when L ¹ is —CO—, N is a hydroxyl group.

In the formula (IV), A represents any one of the following formulas (i) to (vi), and * in the following formulas (i) to (vi) is bonded to L ¹ in the formula (IV).

In the formula (IV), L ¹ is any one of —NH—, —O—, —S—, —CO—, or independently —NH—, —O—, —S—, —CO—. It is a linker part containing any one or more of these. p is an integer of 1 to 5, and m is an integer of 10 or more and 2500 or less. R is a hydrogen atom or a substituted or unsubstituted alkyl group having 1 to 5 carbon atoms.

(I)

(Ii)

(Iii)

(Iv)

(V)

(Vi)

  In the formulas (i) to (vi), Z is a hydrogen atom, a sulfonic acid group, or an indole ring bonded to Z together with a benz [e] indole ring, a benz [f] indole ring, or a benz [g] indole ring. And a hydrogen atom of the cyclic aromatic ring may be substituted with an alkyl group having 1 to 10 carbon atoms, an alkoxy group having 1 to 10 carbon atoms, or a sulfonic acid group.

In formulas (i) to (vi), R ¹ is any one of an alkyl group having 1 to 10 carbon atoms and — (CH ₂ ) _b —SO ₃ ^— (b is an integer of 1 to 10). When R ¹ is an alkyl group, a halogen ion or an organic acid ion may be contained as a counter ion.

R ² and R ³ are each independently a hydrogen atom, an alkyl group having 1 to 10 carbon atoms, an alkoxy group having 1 to 10 carbon atoms, — (CH ₂ ) _b —SO ₃ ^— (b is an integer of 1 to 10) , — (CH ₂ ) _b —SO ₃ X (b is an integer of 1 to 10, and X is sodium, potassium, ammonium, triethylammonium, lysine, or arginine).
In the formulas (i) and (ii), a is an integer of 1 to 10.
In the formulas (i) to (vi), n is 2 or 3.

In the formulas (ii), (iv), and (vi), R ⁴ is an alkyl group having 1 to 10 carbon atoms, — (CH ₂ ) _b —SO ₃ X (b is an integer of 1 to 10, and X is sodium , Potassium, ammonium, triethylammonium, lysine, or arginine).

In the formulas (iii) and (iv), L ³ is a substituted or unsubstituted alkyl group having 1 to 10 carbon atoms, and is substituted with a carbonyl group, an amide group, an ester group, or a piperazyl group in the chain of the alkyl group. May be.

In the formulas (v) and (vi), L ⁴ is a substituted or unsubstituted alkyl group having 1 to 10 carbon atoms, which may be substituted with a carbonyl group, an amide group, or an ester group.

  The compound according to the present invention has a structure in which polyethylene glycol (hereinafter sometimes abbreviated as PEG) and a cyanine compound such as ICG are covalently bonded, so ICG was administered alone to a living body. Compared to the case, the accumulation in the tumor is high, and the intensity of the photoacoustic signal emitted from the tumor is large.

The optical photograph of the tumor transplantation mouse | mouth when photoacoustic imaging was performed in the Example of this invention. The image obtained by the photoacoustic imaging performed in the Example of this invention. The graph of the result obtained by the tumor accumulation property evaluation experiment conducted in the Example of this invention.

An embodiment of the present invention will be described. The compound according to this embodiment has a structure in which a specific cyanine dye is bonded to PEG, and PEG is bonded via a nitrogen atom substituent present in the indole ring of the cyanine dye. The compound according to this embodiment is specifically a compound represented by the following formulas (I) to (IV).
^{_{A-L 1 - (CH 2}} ) p- (OCH 2 CH 2) m-O- (CH 2) q-L 2 -A '(I)
^{_{A-L 1 - (CH 2}} ) p- (OCH 2 CH 2) m-O- (CH 2) q-L 2 -M (II)
^{_{N-L 1 - (CH 2}} ) p- (OCH 2 CH 2) m-O- (CH 2) q-L 2 -A (III)
^{_{A-L 1 - (CH 2}} ) p- (OCH 2 CH 2) m-O-R (IV)

In the formula (I), A and A ′ each independently represent any of the following formulas (i) to (vi), and * in the following formulas (i) to (vi) represents L ¹ in the formula (I), It binds to L ^2.

In the formula (I), L ¹ and L ² are each independently —NH—, —O—, —S—, —CO—, or each independently —NH—, —O—, — It is a linker moiety containing one or more of S- and -CO-. p and q are each independently an integer of 1 to 5, and m is an integer of 10 or more and 2500 or less.

  In the formulas (II) and (III), A represents any one of the following formulas (i) to (vi), and * in the following formulas (i) to (vi) represents L1 in the formula (II) or the formula (III) ) To L2.

In formula (II) and formula (III), L ¹ and L ² are each independently —NH—, —O—, —S—, —CO—, or each independently —NH—, It is a linker moiety containing one or more of -O-, -S-, and -CO-. p and q are each independently an integer of 1 to 5, and m is an integer of 10 or more and 2500 or less.

In the formula (II), when L ² is any of —NH—, —O—, and —S—, M is a hydrogen atom, and when L ² is —CO—, M is a hydroxyl group.

In formula (III), ^{L 1} is -NH -, - O -, - when S- is either, N is a hydrogen atom, when ^{L 1} is -CO-, N represents a hydroxyl group.

In the formula (IV), A represents any one of the following formulas (i) to (vi), and * in the following formulas (i) to (vi) is bonded to L ¹ in the formula (IV).

In the formula (IV), L ¹ is any one of —NH—, —O—, —S—, —CO—, or independently —NH—, —O—, —S—, —CO—. It is a linker part containing any one or more of these. p is an integer of 1 to 5, and m is an integer of 10 or more and 2500 or less. R is a hydrogen atom or a substituted or unsubstituted alkyl group having 1 to 5 carbon atoms.

  In the present specification, * represents a structural formula.

It is synonymous with.

(I)

(Ii)

(Iii)

(Iv)

(V)

(Vi)

  In formulas (i) to (vi), Z is a hydrogen atom, a sulfonic acid group, or a benz [e] indole ring, a benz [f] indole ring, or a benz [g] indole ring together with an indole ring bonded to Z. A cyclic aromatic ring is formed, and the hydrogen atom of the cyclic aromatic ring may be substituted with an alkyl group having 1 to 10 carbon atoms, an alkoxy group having 1 to 10 carbon atoms, or a sulfonic acid group.

In formulas (i) to (vi), R ¹ is any one of an alkyl group having 1 to 10 carbon atoms and — (CH ₂ ) _b —SO ₃ ^— (b is any integer of 1 to 10). .

When R ¹ is an alkyl group, a halogen ion such as a chloride ion, bromide ion or iodide ion, or an organic acid ion such as an acetate ion, a tartrate ion or a succinate ion may be contained as a counter ion. R ² and R ³ are each independently a hydrogen atom, an alkyl group having 1 to 10 carbon atoms, an alkoxy group having 1 to 10 carbon atoms, — (CH ₂ ) _b —SO ₃ ^— (b is any one of 1 to 10 _{integer), - (CH 2) b} -SO 3 X (b is an integer of 1 to 10, X is either sodium, potassium, ammonium, triethyl ammonium, lysine, either arginine) of .
In the formulas (i) and (ii), a is an integer from 1 to 10.
In the formulas (i) to (vi), n is 2 or 3.

In formulas (ii), (iv), and (vi), R ⁴ is independently an alkyl group having 1 to 10 carbon atoms, — (CH ₂ ) _b —SO ₃ X (b is any one of 1 to 10) And X is any of sodium, potassium, ammonium, triethylammonium, lysine and arginine).

In the formulas (iii) and (iv), L ³ is an arbitrary divalent group. For example, it is a substituted or unsubstituted alkyl group having 1 to 10 carbon atoms and may be substituted with a carbonyl group, an amide group, an ester group, a piperazyl group, or the like.

In the formulas (v) and (vi), L ⁴ is an arbitrary divalent group. For example, it is a substituted or unsubstituted alkyl group having 1 to 10 carbon atoms and may be substituted with a carbonyl group, an amide group, an ester group, or the like.

  In the compound according to this embodiment, m in the formulas (I) to (IV) is preferably an integer of 100 or more and 500 or less, and more preferably an integer of 200 or more and 400 or less.

  A and A ′ in the formula (I) may be any one of the formulas (i) to (vi) or a combination of (i) to (vi).

It is preferable that L ¹ and L ² in the formula (I) are both —NH— because the bond between A and L ¹ and A ′ and L ² is strong.

  In addition, -O- is preferable because the compound according to this embodiment is considered to have high dispersibility in an aqueous solution such as serum.

In addition, L ¹ and L ² may each independently be a linker moiety containing any one or more of —NH—, —O—, —S—, and —CO—. Here, the linker site is a site having a function of binding the ethylene glycol chain or alkyl chain in formulas (I) to (IV) to A or A ′. For example, bifunctional compounds such as 1,6-Bismaleimidohexane and Succinimidyl trans-4- (N-Maleimidylmethyl) -Cyclohexane-1-Carboxylate can be mentioned. As another example, an alkyl group having 1 to 10 carbon atoms including a carbonyl group, an amide group, an ester group, a piperazyl group, and the like can be given. In yet another example, a polypeptide having an arbitrary chain length can be used, and the polypeptide may be a capture molecule described below. Polypeptides can form covalent bonds using any of the carbonyl, amino, or thiol groups in the chain. For example, an N-PEG maleimide group and a thiol group can form a maleimide-thiol bond at room temperature under neutral conditions.

  A in formula (i) and formula (ii) is preferably an integer of 2 to 6.

In the formulas (i) to (vi), b in R ¹ , R ² and R ³ is preferably an integer of 2 to 6.

  When a in Formula (i) and Formula (ii) and b in Formula (i) to Formula (vi) are 6 or less, hydrophobicity does not increase, and nonspecific adsorption is difficult in the living body.

  In the present embodiment, the formula (i) is preferably represented by any of the following formulas (i-1) to (i-6).

(I-1)

(I-2)

In formula (i-2), Y ⁻ is any one of halogen ions such as chloride ion, bromide ion and iodide ion, or organic acid ion such as acetate ion, tartrate ion and succinate ion.

(I-3)

(I-4)

(I-5)

(I-6)

  In the present embodiment, the above formula (ii) is preferably represented by any of the following formulas (ii-1) or (ii-2).

(Ii-1)

(Ii-2)

  In the present embodiment, the above formula (iii) is preferably represented by either the following formula (iii-1) or (iii-2).

(Iii-1)

(Iii-2)

  In the present embodiment, the formula (iv) is preferably represented by any of the following formulas (iv-1) or (iv-2).

(Iv-1)

(Iv-2)

Moreover, it is preferable that one or more of the L ¹ and L ² are represented by the following formula (xi).

(Xi)

In the formula (xi), L ⁵ is a polypeptide or a single chain antibody, -NH- is a bond via an amino group of an amino acid in the polypeptide or single chain antibody, -S- is a polypeptide Or it represents the coupling | bonding through the thiol group of the amino acid in a single chain antibody. L ⁶ is an alkyl chain having 1 to 10 carbon atoms including a carbonyl group, an amide group, an ester group, a piperazyl group, and the like. ** binds to the aforementioned *, and *** binds to the alkyl chain side or ethylene glycol chain side of formulas (I) to (IV).

In the formula (xi), L ⁶ is preferably represented by the following formula (xii).
_{- (CH 2) 2 -C (} = O) -NH- (xii)
However, in the formula (xii), the carbon of the ethylene group is bonded to the nitrogen of the maleimide group of the formula (xi), and the nitrogen of —NH— is bonded to *** of the formula (xi). That is, the ethylene group side is bonded to the nitrogen atom of the maleimide group, and the amide group side is bonded to ***.

  The compound according to this embodiment may be further bound with another dye.

  In addition, the compound according to this embodiment may have a capture molecule that specifically binds to a target site.

(Method for preparing compound)
The compound in the present embodiment is prepared by coupling PEG and a specific cyanine-based compound by a known coupling reaction via each functional group. For example, it is bonded to PEG via a nitrogen atom substituent present in the nitrogen-containing heterocycle of a specific cyanine compound. The amino group of the PEG having an amino group is preferably bonded via a sulfosuccinimide ester (sometimes abbreviated as Sulfo-OSu) possessed by a specific cyanine compound. A specific cyanine dye bonded to PEG can be washed and purified by a known purification method such as an ultrafiltration method or a size exclusion column chromatography method. The bond between PEG and a specific cyanine compound may be directly bonded with a functional group such as an amino group of the above PEG and a specific cyanine compound, or various cross-linking materials (crosslinkers). PEG and a specific cyanine compound may be bonded via each other.

  The compound according to the present embodiment is obtained by adding indocyanine green-N-butanoic acid sulfosuccinimide ester (ICG-Sulfo-OSu (registered trademark)) (Dojindo) to PEG having one or more amino groups having a molecular weight of 1000 or more. It is preferable to prepare it by covalent bonding.

(PEG)
PEG used for preparing the compound according to this embodiment is a water-soluble polymer, and has effects such as an increase in serum half-life and a decrease in immunogenicity of the protein. The molecular weight of PEG is preferably in the range of 400 or more and 100,000 or less, and more preferably 20000 or more. If the molecular weight is 20000 or more, it is considered that more PEG can be accumulated at the tumor site than the normal site in the living body due to the EPR (Enhanced Permeability and Retention) effect. Moreover, since the excretion from the kidney is suppressed when the molecular weight is 20000 or more, it can be expected that the retention in blood is longer than that of PEG having a molecular weight of less than 20000. Moreover, since the solution viscosity is so large that the molecular weight of PEG is large, it is preferable that the molecular weight of PEG is 100,000 or less.

  The PEG in the present embodiment has at least one reactive functional group capable of covalently bonding with a specific cyanine compound in one PEG molecule. Here, the reactive functional group can be appropriately selected depending on the functional group of the dye to be bonded. Examples of reactive functional groups include amino groups, hydroxyl groups, thiol groups, carbonyl groups, sulfhydryl groups, epoxy groups, glycidyl groups, N-succinimidyloxy groups, N-sulfosuccinimidyloxy groups, and N-maleimidoalkyls. A group or the like is preferable.

(Specific cyanine compounds)
In the present embodiment, the structure of the specific cyanine compound is represented, for example, by the following formula (V).
BB '(V)

  In the formula (V), B represents the above formula (i) or the above formula (ii).

  In the formula (V), B ′ represents any of the following formulas (vii) to (x).

(Vii)

(Viii)

(Ix)

(X)

  As an example of the above formula (V), a compound represented by the following formula (2) (ICG-Sulfo-OSu (manufactured by Dojindo Laboratories, registered trademark)), a compound represented by the following formula (3), and the following formula (4) are shown. Or a compound represented by the following formula (6), a compound represented by the following formula (6), a compound represented by the following formula (7), or a compound represented by the following formula (8). .

(2)

(3)

(4)

(5)

(6)

(7)

(8)

(Contrast agent for optical imaging)
The contrast agent for optical imaging according to the present embodiment includes the compound according to the present embodiment and a dispersion medium. Further, the contrast agent for optical imaging according to this embodiment may have a pharmacologically acceptable additive such as a vasodilator in addition to the compound according to this embodiment, if necessary.

  Here, the dispersion medium is a liquid substance for dispersing the compound according to the present embodiment, and examples thereof include physiological saline, distilled water for injection, phosphate buffered saline, and aqueous glucose solution. In the contrast agent for optical imaging according to the present embodiment, the compound according to the present embodiment may be preliminarily dispersed in the dispersion medium, or the particles and the dispersion medium according to the present embodiment may be used as a kit. Prior to administration into a living body, the particles may be used after being dispersed in a dispersion medium.

  In this embodiment, optical imaging means imaging (imaging) by irradiating light. That is, by irradiating light other than PEG in the optical imaging contrast agent according to the present embodiment, an acoustic wave, fluorescence, or the like is emitted. Photoacoustic imaging can be performed by detecting the emitted acoustic wave, and fluorescence imaging can be performed by detecting the emitted fluorescence. Photoacoustic imaging is a concept including photoacoustic tomography (tomography).

  When the contrast agent for optical imaging which concerns on this embodiment is used for fluorescence imaging, when used for the contrast agent for fluorescence imaging and photoacoustic imaging, it may be called the contrast agent for photoacoustic imaging.

  The contrast agent for optical imaging according to the present embodiment can be accumulated more in a tumor site than in a normal site in the living body when administered in the living body by using the EPR effect. As a result, after the contrast agent for optical imaging according to the present embodiment is administered into the living body, when the living body is irradiated with light and the acoustic wave from the living body is detected, the acoustic wave emitted from the tumor site is detected as the normal site. It can be made larger than the acoustic wave emitted from.

  Further, the contrast agent for optical imaging according to the present embodiment can also be used for contrasting lymph nodes. Furthermore, it is particularly preferable to use it as a contrast agent for sentinel lymph nodes. This is because the size is larger than that of the dye alone, so that it is more likely to stay in the sentinel lymph node and the accumulation property is expected to be improved.

(Capture molecule)
The capture molecule in the present embodiment is a substance that specifically binds to a target site such as a tumor, a substance that specifically binds to a substance that exists around the target site, and the like, and a chemical substance such as a biomolecule or a pharmaceutical Can be selected arbitrarily. Specific examples include artificial antibodies such as antibodies, antibody fragments, and single chain antibodies, enzymes, biologically active peptides, glycopeptides, sugar chains, lipids, molecular recognition compounds, and the like. These substances can be used alone or in combination. By using the compound according to the present embodiment in which the capture molecule is chemically bonded, specific detection of the target site, tracking of the dynamics, localization, drug efficacy, metabolism, etc. of the target substance can be performed.

(Additive)
The contrast agent for optical imaging according to this embodiment may contain an additive used at the time of lyophilization. Examples of the additive include glucose, lactose, mannitol, polyethylene glycol, glycine, sodium chloride, and sodium hydrogen phosphate. Only one type of additive may be used, or a plurality of types of additives may be used in combination.

(Photoacoustic imaging method)
A method for detecting the compound according to the present embodiment administered in vivo using a photoacoustic imaging apparatus will be described. The method for detecting a compound according to this embodiment includes the following steps (a) and (b). However, the photoacoustic imaging method according to the present embodiment may include steps other than the steps shown below.
(A) A step of irradiating a sample to which a compound according to this embodiment is administered with light in a wavelength region of 600 nm to 1300 nm (b) a step of detecting an acoustic wave generated from the compound present in the sample

  In addition, the compound according to the present embodiment may have a step of reconstructing a spatial photoacoustic signal intensity distribution from the wavelength, phase, time information, and the like of the acoustic wave obtained in (b). Note that three-dimensional image reconstruction can be performed based on the wavelength, phase, and time information of the photoacoustic signal obtained in the step (b). The data obtained by the image reconstruction may take any form as long as the position information of the intensity distribution of the photoacoustic signal can be grasped. For example, the photoacoustic signal intensity may be expressed on a three-dimensional space, or the photoacoustic signal intensity may be expressed on a two-dimensional plane. It is also possible to acquire information with respect to the same observation target by different imaging methods and acquire the positional correspondence between the information and the photoacoustic intensity distribution.

  In the step (a), a specimen to which the compound according to this embodiment is administered by a method such as oral administration or injection can be used.

  In the step (b), there are no particular limitations on the device that generates the light to be irradiated on the specimen and the device that detects the photoacoustic signal emitted from the compound according to the present embodiment.

  The light source for irradiating the specimen with light in the step (b) is not limited as long as the specimen can be irradiated with laser pulse light having at least one wavelength selected from the range of 600 nm to 1300 nm. . Examples of the apparatus for irradiating the laser pulse light include a titanium sapphire laser (LT-2211-PC, manufactured by Lotis), an OPO laser (LT-2214 OPO, manufactured by Lotis), and an alexandrite laser.

  An apparatus for detecting an acoustic wave is not particularly limited, and various apparatuses can be used. For example, a commercially available photoacoustic imaging apparatus (Nexus128, Endra Inc.) can be used.

  The imaging method using the compound according to the present embodiment can image a target site such as a tumor, a lymph node, or a blood vessel through the steps (a) and (b).

  EXAMPLES Hereinafter, although this invention is demonstrated in detail using an Example, this invention is not limited to these Examples. In the following, Mw represents molecular weight. A typical structure of each sample, M5k-ICG, M10k-ICG, M20k-ICG, M30k-ICG, and M40k-ICG prepared in this example is represented by Formula (P1) or Formula (I-1). The molecular weights of polyethylene glycol are 5k, 10k, 20k, 30k, and 40k, respectively. The structure of DE-200PA is represented by the formula (II), and the molecular weight of polyethylene glycol is 20k.

(Example 1) to (Example 4)
(Compound 1 of specific cyanine compound and PEG)
In this example, ICG-Sulfo-OSu (manufactured by Dojindo Laboratories) was used as a specific cyanine compound. ICG-Sulfo-OSu 1 mg (1.25 μmol) was dissolved in 100 μL of DMSO. On the other hand, PEG was weighed in a 1.5 mL plastic tube, and various PEGs were dissolved in 50 mM carbonate buffer (pH 9.0) to adjust the NH ₂ concentration to 0.625 mM. The various PEG derivatives used here are monoamine linear ME-050EA (manufactured by NOF Corporation, Mw5000), monoamine linear ME-100EA (manufactured by NOF Corporation, Mw10000), monoamine linear ME-200EA ( NOF Corporation, Mw 20000), and diamine linear DE-200PA (NOF Corporation, Mw 20000). The above is summarized in Table 1.

The structures of ME-050EA, ME-100EA, and ME-200EA used above are represented by the following formula (p1) and have molecular weights of 5 k, 10 k, and 20 k, respectively.
_{CH 3 O- (CH 2 CH 2} O) n-CH 2 CH 2 NH 2 (p1)
In this specification, k represents 1000, for example, 5k is 5000.

The structure of DE-200PA used above is represented by the following formula (p2) and has a molecular weight of 20k.
_{X- (CH 2 CH 2 O)} n-X (p2)
In the above formula (p2), X is CH ₂ CH ₂ CH ₂ NH ₂
It is represented by

  To a PEG carbonate buffer solution (400 microliters), 20 microliters of an ICG-Sulfo-OSu DMSO solution ([ICG-Sulfo-OSu] = 0.25 micromol) was added. ICG-Sulfo-OSu and PEG were reacted at a reaction ratio of 1. The concentration during the reaction of ICG-Sulfo-OSu was 0.6 mM. After stirring for 24 hours at room temperature under light shielding, the reaction solution was filtered with a 0.22 μm syringe filter to obtain a covalently bonded specific cyanine dye and PEG. These were stored 4 times in the dark.

  Hereinafter, monoamine linear ME-050EA, monoamine linear ME-100EA, monoamine linear ME-200EA, diamine linear DE-200PA, and ICG-Sulfo-OSu covalently bonded to each other are shown in Table 2. Are abbreviated as M5k-ICG, M10k-ICG, M20k-ICG, and D20k-ICG2, respectively. Similarly, ICG-Sulfo-OSu and glycine reacted at a molar ratio of 1: 1 (hereinafter abbreviated as ICG-Gly) were synthesized and used as control samples (Table 2).

  Monoamine linear means that there is one amino group per linear PEG molecule, and diamine linear means that there are two amino groups per linear PEG molecule. To do. The number of amino groups is the same as the number that ICG-Sulfo-OSu can bind.

(Measurement of absorbance)
The absorbance of the solutions of M5k-ICG, M10k-ICG, M20k-ICG, D20k-ICG2, and ICG-Gly was measured. The solution was diluted with 50 mM 4- (2-hydroxyethyl) -1-piperazine etheric acid (HEPES) solution, and the absorbance at 700 nm and 785 nm was measured. The 700 nm absorption is the absorption of the dye in the associated state, while the 785 nm is the absorption of the dye present as a monomer. Therefore, the absorption ratio between 700 nm and 785 nm (abbreviated as 700/785) is an indicator of the association state of the dyes, and it is considered that the larger the value, the higher the ratio of the dyes associated with each other. The absorbance at 785 nm of the reaction solution was calculated by multiplying the absorbance at 785 nm and the dilution rate. The dye concentration of the reaction solution was calculated by dividing the absorbance at 785 nm of the reaction solution by the molar absorption coefficient of the dye, 1.2 × 10 ⁵ (M ⁻¹ xcm ⁻¹ ). Table 3 shows the results of the absorbance measurement.

  ICG-Gly showed a green precipitate during the reaction, while M5k-ICG, M10k-ICG, M20k-ICG, and D20k-ICG2 did not show any precipitate during the reaction and were good in water. It became clear that it showed a good dispersibility. It was shown that the dye alone is unstable in water, but the stability of the dye is improved by covalently bonding with PEG.

(Evaluation of tumor accumulation)
In order to confirm the tumor accumulation property of the compound prepared above, M5k-ICG, M10k-ICG, M20k-ICG, D20k-ICG2 and ICG-Gly were added to the tail vein of tumor transplanted mice transplanted with N87 cell line. Administered. The dose was 13 nmol as the amount of pigment. Mice were euthanized with carbon dioxide gas 24 hours after administration, and N87 tumor tissue was removed. The tumor tissue was transferred to a plastic tube, 1.25 times the amount of 1% Triton X-100 aqueous solution was added to the weight of the tumor tissue, and homogenized using a plastic pestle. Subsequently, dimethyl sulfoxide (DMSO) of 20.25 times the tumor tissue weight was added. The amount of dye in the tumor tissue was quantified by measuring the fluorescence intensity of the homogenate solution in a plastic tube state using IVIS (registered trademark) Imaging System 200 Series (manufactured by Caliper). The rate of dye transfer (% injected dose: abbreviated as% ID) to the tumor tissue relative to the dose (dye 13 nmol) per unit weight of the tumor tissue is shown in Table 4 as the amount of tumor accumulation of the compound (% ID / g). Indicated.

  As a result, the tumor accumulation property of ICG-Gly as a control was 0.6% ID / g, but 1.1, 1. for M5k-ICG, M10k-ICG, M20k-ICG, and D20k-ICG2. The tumor accumulation was increased as the molecular weight was increased to 4, 5.2 and 7.0% ID / g. This seems to be the result of the molecular size increasing with increasing molecular weight and their renal excretion being suppressed. Furthermore, when comparing M20k-ICG and D20k-ICG2 having a molecular weight of 20000, the tumor accumulation was higher in the diamine linear form (compound having dyes attached to both ends of PEG) than in the monoamine linear form. . In diamine linear PEG, there are many dyes per PEG molecule. As a result, the stability of the dye is improved due to the hydrophobic association within the PEG molecular chain or between the molecular chains via the dye, or apparent molecules. It is thought that tumor accumulation increased due to changes in size. Similar to the results obtained in the above (Measurement of Absorbance), it is shown that the tumor accumulation is higher when covalently bound to PEG than the dye alone, and the PEG molecular weight and the form of PEG for tumor accumulation are important parameters. It was shown that.

(Photoacoustic imaging at tumor site)
In order to confirm the tumor imaging ability, M20k-ICG was administered to the tail vein of tumor-transplanted mice transplanted with the N87 cell line in the same manner as in (Tumor accumulation evaluation). The dose was 65 nmol as the amount of pigment. Photoacoustic imaging (irradiation laser wavelength: 785 nm) was performed 5 minutes after administration and 20 hours after administration. FIG. 1 shows an optical photograph of a mouse during photoacoustic imaging, which is an image of a mouse under anesthesia (m in the figure). The FOV in the figure is a tumor part (T in the figure) transplanted subcutaneously in the mouse, and is an observation region (2 cm × 2 cm × 2 cm) in which photoacoustic imaging is performed. FIG. 2 shows the photoacoustic imaging results in the tumor part, and Table 5 shows the photoacoustic intensity ratio in the tumor part when the photoacoustic intensity before administration is 1. In the figure, the white dotted line shows the outline of the mouse, and as is clear from FIG. 2 and Table 5, the intensity of the photoacoustic signal at the tumor part greatly increases immediately after administration (5 minutes later), and light is observed even 20 hours after administration. The acoustic signal was retained, and a photoacoustic signal 2.6 times that before administration was confirmed. From this, it was confirmed that a photoacoustic imaging image of a tumor site was obtained using the compound according to the example of the present invention.

(Preparation of labeled body)
A DMSO solution of ICG-Sulfo-OSu was added to a HEPES solution (pH 7.1) of PEG. ICG-Sulfo-OSu and PEG were reacted at a reaction molar ratio of 3: 1. The concentration of ICG-Sulfo-OSu during the reaction was 0.6 mM. After stirring for 24 hours at room temperature under light shielding, the reaction solution was filtered with a 0.22 μm syringe filter to obtain an aqueous solution in which a specific cyanine dye and PEG were covalently bonded. The near-infrared organic dye bonded to PEG by the reaction can be washed and purified by a known purification method such as an ultrafiltration method or a size exclusion chromatography method.

  In the same manner as in Example 1, a sample prepared by reacting ICG-Sulfo-OSu with glycine (hereinafter abbreviated as ICG-Gly) and an ICG aqueous solution (manufactured by Japan Public Standards Association) as a control sample It was. Using the above samples, the blood retention and tumor accumulation due to the difference in molecular weight and structure were evaluated.

(Example 5) to (Example 10)
The above-mentioned ICG-Sulfo-OSu was used as the near infrared organic dye. ICG-Sulfo-OSu 1 mg (1.25 μmol) was dissolved in 100 μL of DMSO. Meanwhile, PEG was weighed into a 1.5 mL plastic tube. Various PEGs were dissolved in 50 mM carbonate buffer (pH 9.0) to adjust the NH ₂ concentration to 1.25 mM. The various PEGs used here are monoamine linear ME-50EA (manufactured by NOF Corporation, Mw5000), monoamine linear ME-100EA (manufactured by NOF Corporation, Mw10000), monoamine linear ME-200EA (manufactured by Sun Oil company, Mw 20000), monoamine linear ME-300EA (manufactured by NOF Corporation, Mw 30000), monoamine linear ME-400EA (manufactured by NOF Corporation, Mw 40000), diamine linear DE-200PA (NOF Corporation) Manufactured, Mw 20000). A covalent conjugate of a near-infrared organic dye and PEG was prepared in the same procedure as in Example 1 except that the NH ₂ concentration was doubled. Table 6 shows the samples used in this example.

(Photoacoustic measurement on the mouse body surface)
In order to confirm the pharmacokinetics of the compound prepared above, nude mice were administered with M5k-ICG, M10k-ICG, M20k-ICG, M30k-ICG, and photoacoustic measurement of the body surface on the back of the mouse was performed. . The measurement area of this example was selected from an area where the influence of the photoacoustic signal of the main organ of the mouse is considered to be small. Therefore, it is considered that the photoacoustic signal obtained from the back region in this example is close to the dynamics of the compound present in the blood. The measurement was performed before administration, 60 minutes after administration, and 180 minutes after administration, and calculated as relative photoacoustic intensity relative to photoacoustic signal intensity before administration. Table 7 shows the results of summarizing the respective photoacoustic intensities with an irradiation laser wavelength of 797 nm. A compound having a PEG molecular weight of more than 10 k gave a photoacoustic signal more than twice as high as before administration even after 60 minutes. From the above results, it was suggested that a compound to which a PEG having a molecular weight of 10 k or more is bonded is a photoacoustic contrast agent exhibiting a high blood retention compared to Non-Patent Document 1.

(Compound pharmacokinetics)
In order to confirm the blood retention and tumor accumulation of the compound prepared above, M5k-ICG, M10k-ICG, M20k-ICG, M30k-ICG, M40k were used for tumor-transplanted mice transplanted with Colon26 cell line. -ICG, D20k-ICG2 was administered into the tail vein. The dose was 13 nmol as the amount of pigment.

(Blood retention evaluation method)
Blood was collected from mice 24 hours after administration, and blood, 1% Triron X-100 aqueous solution, and DMSO were mixed at a volume ratio of 2: 9: 9 in a plastic tube. Using IVIS (registered trademark) Imaging System 200 Series (manufactured by Caliper), the amount of dye in the blood was quantified by measuring the fluorescence intensity in the state of a plastic tube. Table 8 shows the rate of dye transfer to blood (% injected dose: abbreviated as% ID) relative to the dose (13 nmol of dye) per unit weight of blood as the blood retention (% ID) of the compound. As a result, it was confirmed that the amount of residual blood increased as the molecular weight of PEG increased.

(Tumor accumulation of compounds)
The compound was administered to the tail vein of tumor-transplanted mice transplanted with the Colon26 cell line. The dose was 13 nmol as the amount of pigment. The mouse after blood collection was euthanized with carbon dioxide, and then Colon26 tumor tissue was removed. Table 8 shows the results of the tumor accumulation evaluation of the excised tumor mass by the method described in Example 1. As a result, it was shown that tumor accumulation was improved as the molecular weight of PEG increased.

(Photoacoustic imaging at tumor site)
Similar to the method performed in (evaluation of tumor accumulation) in order to confirm tumor imaging ability, 26 nmol as a pigment amount was administered from the tail vein to a tumor-transplanted mouse transplanted with the Colon26 cell line, and photoacoustic imaging was performed. Carried out. Furthermore, ICG-Sulfo-OSu and glycine reacted at a molar ratio of 1:10 (hereinafter abbreviated as ICG-Gly) were synthesized and used as a control sample. Photoacoustic imaging was performed 1 hour after administration, and was calculated as the relative photoacoustic intensity with respect to the photoacoustic signal intensity of the control ICG-Gly. The irradiation laser wavelength was 797 nm.

  From the results in Tables 8 and 9, the tumor showed higher tumor accumulation than the control sample in tumor photoacoustic imaging, indicating that the molecular weight and structure of PEG are important parameters.

(Example 11) to (Example 12)
(Compound 2 of specific cyanine compound and PEG)
(Preparation of labeled body)
In this example, the compounds described in Formula 7 and Formula 8 were used as specific cyanine compounds. The specific cyanine dye used was prepared by the method described in Bioorganic & Medicinal Chemistry 6 (1998) 2179-2184 (hereinafter referred to as Non-Patent Document 2). 1 mg (1.25 micromol) of dye was dissolved in 100 microliters of DMSO. On the other hand, PEG was weighed and dissolved in a mixed solvent of chloroform and methanol (volume ratio 9: 1) to adjust the NH ₂ concentration to 0.625 mM. The chemical structural formula of the compound produced in Example 11 is as shown in formula (I-2), and the chemical structural formula of the compound produced in Example 12 is as shown in formula (I-3). As the PEG derivative used in this example, monoamine linear ME-200EA (manufactured by NOF Corporation, Mw 20000) was used. In this example, the PEG compound obtained from Formula 7 is described in Formula I-3, and the PEG compound obtained from Formula 8 is that described in Formula I-2.

  The reaction was performed at a reaction molar ratio of 2 between the dye and PEG. The concentration during the reaction of the dye was 0.6 mM. After light-shaking at room temperature for 24 hours under light shielding, the solvent was distilled off and redispersed in 10 mM HEPES buffer. As a result, although the compound described in Formula 7 cannot be dispersed in water by itself, it is clear that it can be dispersed in water by forming a covalent bond with PEG as described in Formula I-3. It became. Subsequently, the reaction solution was filtered with a 0.22 μm syringe filter to obtain a compound in which a specific cyanine dye and PEG were covalently bonded. After production, it was stored 4 times under shading.

(Endokinetic evaluation)
100 microliters (13 nmol as the amount of dye) of PEG compound obtained by intravenous injection of Colon 26 cells subcutaneously into BALB / c Slc-nu / nu mice was intravenously injected. The colon 26 cell mass and blood accumulation 24 hours after administration were evaluated by the method described in Example 12. The results are summarized in Table 10. It was confirmed that a specific cyanine dye acquired dispersibility in water by covalent bonding with PEG, and exhibited tumor accumulation when administered into the body.

(PEG with capture molecule)
(Preparation of single chain antibody hu4D5-8scFv)
Based on the gene sequence (hu4D5-8) of the IgG variable region that binds to HER2, a gene hu4D5-8scFv encoding a single chain antibody (scFv) was prepared. First, cDNA was prepared by linking the VL and VH genes of hu4D5-8 with cDNA encoding peptide (GGGGS) ₃ . A restriction enzyme NcoI was introduced at the 5 ′ end and a restriction enzyme NotI recognition site was introduced at the 3 ′ end. The base sequence is shown below.
SEQ ID NO: 1:
5'- CCATGG GCGGCCGC -3 '
(Restriction enzyme recognition sites are underlined.)

  The gene fragment hu4D5-8scFv was inserted downstream of the T7 / lac promoter of the plasmid pET-22b (+) (Novagen). Specifically, the above cDNA is ligated to pET-22b (+) digested with restriction enzymes NcoI and NotI.

This expression plasmid was transformed into Escherichia coli BL21 (DE3) to obtain a strain for expression. The obtained strain was precultured overnight in 4 ml of LB-Amp medium, and the whole amount was added to 250 ml of 2 × YT medium, and cultured with shaking at 28 ° C. and 120 rpm for 8 hours. Thereafter, IPTG (Isopropyl-β-D (-)-thiogalactopylanoside) was added at a final concentration of 1 mM, and cultured overnight at 28 degrees. The cultured Escherichia coli was centrifuged at 8000 × g for 30 minutes at 4 degrees, and the culture broth of the supernatant was recovered. Ammonium sulfate of 60% by weight of the obtained culture broth was added, and the protein was precipitated by salting out. The salted-out solution was allowed to stand at 4 ° C overnight, and then the precipitate was collected by centrifuging at 8000 × g for 30 minutes at 4 ° C. The resulting precipitate was dissolved in 20 mM Tris · HCl / 500 mM NaCl buffer and dialyzed against 1 L of the same buffer. The protein solution after dialysis was added to a column filled with His · Bind (registered trademark) Resin (Novagen) and purified by metal chelate affinity chromatography via Ni ions. The purified hu4D5-8scFv showed a single band by reducing SDS-PAGE and confirmed that the molecular weight was about 28 kDa. The amino acid sequence of the prepared antibody is shown below. Hereinafter, hu4D5-8scFv is abbreviated as scFv.
SEQ ID NO: 2:
DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSAAALEHHHHHHGGC
(Modification of scFv to PEG)

  The scFv prepared as described above was substituted with a phosphate buffer (2.68 mM KCl / 137 mM NaCl / 1.47 mM KH2PO4 / 1 mM Na2HPO4 / 5 mM EDTA, pH 7.4) containing 5 mM EDTA. Reduction treatment with 2-carboxyethyl) phosphine hydrochloride (TCEP) at 25 ° C. for about 2 hours.

  On the other hand, monomaleimide linear ME-400MA (manufactured by NOF Corporation, MW 40000) was weighed in a 1.5 mL plastic tube. The solution was diluted to 17.9 μM with a phosphate buffer not containing EDTA, mixed with the reduced scFv, and reacted at 25 ° C. for 15 hours or longer. The reaction molar ratio (scFv / PEG) used was 1. Here, “preparation” means added to the reaction system, and “reaction molar ratio of preparation” refers to the molar concentration ratio of scFv and PEG added to the reaction system. Next, a solution of ICG-Sulfo-OSu in DMSO (12.5 mM) and the mixed solution of scFv and PEG were mixed and reacted at 25 degrees for 2 hours. The charging reaction molar ratio (ICG-Sulfo-OSu / scFv) was 3. After filtering this solution (pore size 1.2 μm), scFv that did not bind to PEG was removed by ultrafiltration using a 30 kDa pore size Amicon Ultra-4 (Nihon Millipore), and ICG and scFv were A modified PEG was obtained. This obtained compound is abbreviated as scFv-PEG40.

(Modification of Affibody (registered trademark) to PEG)
A dithiothreitol (DTT) solution was added to an Affibody (registered trademark) solution (manufactured by Affibody) that binds to HER2 to a final concentration of 20 mM, and reduced at 25 ° C. for 2 hours in the dark. Subsequently, DTT was removed from the reaction solution using a PD-10 column (manufactured by GE Healthcare). Next, monomaleimide linear ME-200MA (manufactured by NOF Corporation, MW 20000) or monomaleimide linear ME-400MA (manufactured by NOF Corporation, MW 40000) was weighed into a 1.5 mL plastic tube. The weighed PEG was diluted with a phosphate buffer not containing EDTA to a concentration of 143 μM, mixed with the reduced Affibody (registered trademark), and reacted at 25 ° C. for 15 hours or longer. The charged reaction molar ratio (Affibody (registered trademark) / PEG) was 1. Then, a DMSO solution (12.5 mM) of ICG-Sulfo-OSu and the mixed solution of Affibody (registered trademark) and PEG were mixed and reacted at 25 degrees for 2 hours. The reaction molar ratio of charging (ICG-Sulfo-OSu / Affibody (registered trademark)) was 1. After filtering this solution (pore size 1.2 μm), Affibody (registered trademark) that did not bind to PEG was removed by ultrafiltration using a 10 kDa pore size Amicon Ultra-4 (Nihon Millipore), PEG modified with ICG and Affibody® was obtained. Among the obtained compounds, those using ME-200MA are abbreviated as Af-PEG20, and those using ME-400MA are abbreviated as Af-PEG40.

(Modification of peptide to PEG)
A peptide in which 6 amino acids were added to the carboxyl terminus of the peptide sequence confirmed to be binding to HER2 was synthesized (SEQ ID NO: 3). A cysteine residue present at the carboxyl terminus of this peptide sequence can react with the maleimide group to form a covalent bond.
SEQ ID NO: 3
MARSGLGGKGSC

  Monomaleimide linear ME-200MA (manufactured by NOF Corporation, MW 20000) or monomaleimide linear ME-400MA (manufactured by NOF Corporation, MW 40000) was weighed into a 1.5 mL plastic tube. Weighed PEG was diluted with a phosphate buffer not containing EDTA to 1.57 mM each, mixed with the peptide solution described above, and reacted at 25 ° C. for 15 hours or longer. The reaction molar ratio (peptide / PEG) used was 1. Next, a solution of ICG-Sulfo-OSu in DMSO (12.5 mM) and a mixed solution of the peptide and PEG were mixed and reacted at 25 degrees for 2 hours. The reaction molar ratio of charging (ICG-Sulfo-OSu / peptide) was performed at 1. After filtering this solution (pore size 1.2 μm), peptides that did not bind to PEG were removed by ultrafiltration using a 10 kDa pore size Amicon Ultra-4 (Nippon Millipore), and ICG and peptides were A modified PEG was obtained. Among the obtained compounds, those using ME-200MA are abbreviated as pe-PEG20, and those using ME-400MA are abbreviated as pe-PEG40.

(Evaluation of HER2 binding property of PEG having capture molecule)
The binding property to HER2, which is a target molecule, was evaluated by surface plasmon resonance (SPR) for the PEG having a capture molecule. SPR was measured using Proteon (registered trademark) XPR36 (manufactured by Bio-Rad Laboratories). Recombinant Human ErbB2 / Fc Chimera (manufactured by R & D Systems) was dissolved in acetate buffer (pH 5.0) and immobilized by amine coupling to the carboxyl group on the surface of the GLM sensor chip. The immobilization amount was about 3000 RU (Resonance Unit). Next, PEG having a capture molecule was diluted to various concentrations with phosphate buffer (pH 7.4) containing 0.005% Tween 20, and then injected into the flow cell at a flow rate of 50 microliters / minute. The measurement time was 120 seconds for injection time (binding) and 120 seconds after discontinuation of injection (dissociation). In the binding kinetic analysis experiment, the sensorgram was analyzed using a 1: 1 Langmuir fitting model. The calculated bond dissociation constants (K _D ) are summarized in Table 11. In any sample, binding to HER2 was confirmed, and particularly strong binding was confirmed with scFv-PEG40, Af-PEG20, and Af-PEG40.

(Evaluation of tumor accumulation of PEG with capture molecules)
For evaluation of tumor accumulation, female inbred BALB / c Slc-nu / nu mice (6 weeks old at the time of purchase) (Japan SLC Co., Ltd.) were used. The mice were acclimated in an environment where they could freely consume food and drinking water for one week before the mice were allowed to carry cancer. About 1 week before the imaging experiment, 1 × 10 ⁶ Colon26 mouse colon cancer cells (RIKEN) and 1 × 10 ⁶ HER2 gene artificially introduced into the right shoulder and right thigh of the mice were colon26 mouse colon cancer cells. Were injected subcutaneously into the left shoulder and left thigh of the mouse, respectively. By the time of the experiment, all the tumors had settled and the mice weighed 17-22 g. 200 microliters (13 nmol as ICG) of PEG having a capture molecule or D20k-ICG2 was intravenously injected into a tumor-bearing mouse. After 24 hours from the administration, the mice were euthanized with carbon dioxide gas, and then each cancer tissue was removed. The cancer tissue was transferred to a plastic tube, 1.25 times the amount of 1% Triton X-100 aqueous solution was added to the weight of the cancer tissue, and homogenized using a plastic pestle. Next, 20.25 times DMSO in weight of the cancer tissue was added to prepare a dye extract in the tumor tissue. On the other hand, a cancer tissue was extracted from a tumor-bearing mouse not administered with PEG having a capture molecule. Transfer the cancer tissue to a plastic tube, add 1.25 times the amount of 1% Triton X-100 aqueous solution to the weight of the cancer tissue, and homogenate using a plastic pestle to prepare a Triton X-100 solution of the cancer tissue did. Next, a PEG solution having a known concentration of a capture molecule is diluted to various concentrations with the Triton X-100 solution of the cancer tissue, and 20.25 times the amount of DMSO is added to the diluted solution to obtain a standard solution for calibration. Prepared. Using IVIS (registered trademark) Imaging System 200 Series (XENOGEN), the amount of dye in cancer tissue is quantified by measuring the fluorescence intensity of the dye extract in the tumor tissue and the standard solution for calibration in the state of a plastic tube. did.

  In the tumor accumulation evaluation, blood was collected from the tail vein immediately before euthanizing the mice with carbon dioxide after 24 hours. The collected blood was transferred to a plastic tube, and 4.5% of 1% Triton X-100 aqueous solution was added to the blood volume. Next, DMSO in an amount 4.5 times the volume of blood was added to prepare a blood lysate. Using IVIS (registered trademark) Imaging System 200 Series (XENOGEN), the fluorescence intensity of the blood lysate was measured in the state of a plastic tube. On the other hand, a particle solution having a known concentration was diluted to various concentrations with a 1% Triton X-100 aqueous solution, and the diluted particle solution was mixed with blood collected from the same amount of untreated mice. Subsequently, 1% Triton X-100 aqueous solution was added so that it might become 4.5 times the volume of the blood together with the diluted particle solution. Next, 4.5 times the volume of blood DMSO was added to prepare a blood particle solution for a calibration curve. Similar to the collected blood sample, the fluorescence intensity was measured to prepare a calibration curve. Next, the blood concentration was calculated using the fluorescence intensity of the blood lysate and the prepared calibration curve. The ratio (% ID) of the abundance in blood per dose was calculated by dividing each calculated blood concentration by the total dose.

  The results of tumor accumulation and abundance in blood are summarized in FIG. The amount accumulated in Colon 26 mouse colon cancer cells is shown in white, the amount accumulated in Colon 26 mouse colon cancer cells into which the HER2 gene has been artificially introduced is shown in black, and the ratio of the abundance in the blood per dose is shown in gray.

  All of the PEGs having a capture molecule tended to have a tumor accumulation amount lower than that of D20k-ICG2, but Af-PEG40 was able to confirm inferior tumor accumulation property and abundance in blood. Further, the amount accumulated in the Colon 26 mouse colon cancer cells into which the HER2 gene was artificially introduced was calculated with respect to the amount accumulated in the Colon 26 mouse colon cancer cells. As compared with D20k-ICG2, all of the PEGs having a capture molecule were accumulated in Colon 26 mouse colon cancer cells into which HER2 gene was artificially introduced, and the effect of the HER2 binding property by the capture molecule was confirmed.

Claims (15)

A compound represented by any one of the following formulas (I) to (IV).
^{_{A-L 1 - (CH 2}} ) p- (OCH 2 CH 2) m-O- (CH 2) q-L 2 -A '(I)
^{_{A-L 1 - (CH 2}} ) p- (OCH 2 CH 2) m-O- (CH 2) q-L 2 -M (II)
^{_{N-L 1 - (CH 2}} ) p- (OCH 2 CH 2) m-O- (CH 2) q-L 2 -A (III)
^{_{A-L 1 - (CH 2}} ) p- (OCH 2 CH 2) m-O-R (IV)
In the formula (I), A and A ′ each independently represent any of the following formulas (i) to (vi), and * in the following formulas (i) to (vi) represents L ¹ in the formula (I), It binds to L ^2.
In the formula (I), L ¹ and L ² are each independently any of —NH—, —O—, —S—, —CO—, or —NH—, —O—, —S—, It is a linker moiety containing any one or more of -CO-. p and q are each independently an integer of 1 to 5, and m is an integer of 10 or more and 2500 or less.
In the formulas (II) and (III), A represents any one of the following formulas (i) to (vi), and * in the following formulas (i) to (vi) represents L1 in the formula (II) or the formula (III) ) To L2.
In formula (II) and formula (III), L ¹ and L ² are each independently —NH—, —O—, —S—, —CO—, or each independently —NH—, It is a linker moiety containing one or more of -O-, -S-, and -CO-. p and q are each independently an integer of 1 to 5, and m is an integer of 10 or more and 2500 or less.
In the formula (II), when L ² is any of —NH—, —O—, and —S—, M is a hydrogen atom, and when L ² is —CO—, M is a hydroxyl group.
In the formula (III), when L ¹ is any of —NH—, —O—, and —S—, N is a hydrogen atom, and when L ¹ is —CO—, N is a hydroxyl group.
In the formula (IV), A represents any one of the following formulas (i) to (vi), and * in the following formulas (i) to (vi) is bonded to L ¹ in the formula (IV).
In the formula (IV), L ¹ is any one of —NH—, —O—, —S—, —CO—, or independently —NH—, —O—, —S—, —CO—. It is a linker part containing any one or more of these. p is an integer of 1 to 5, and m is an integer of 10 or more and 2500 or less. R is a hydrogen atom or a substituted or unsubstituted alkyl group having 1 to 5 carbon atoms.

(I)

(Ii)

(Iii)

(Iv)

(V)

(Vi)
In formulas (i) to (vi), Z is a hydrogen atom, a sulfonic acid group, or a benz [e] indole ring, a benz [f] indole ring, or a benz [g] indole ring together with an indole ring bonded to Z. A cyclic aromatic ring is formed, and the hydrogen atom of the cyclic aromatic ring may be substituted with an alkyl group having 1 to 10 carbon atoms, an alkoxy group having 1 to 10 carbon atoms, or a sulfonic acid group.
In formulas (i) to (vi), R ¹ is any one of an alkyl group having 1 to 10 carbon atoms and — (CH ₂ ) _b —SO ₃ ^— (b is any integer of 1 to 10). . When R ¹ is an alkyl group, a halogen ion or an organic acid ion may be contained as a counter ion. R ² and R ³ are each independently a hydrogen atom, an alkyl group having 1 to 10 carbon atoms, an alkoxy group having 1 to 10 carbon atoms, — (CH ₂ ) _b —SO ₃ ^— (b is any one of 1 to 10 _{integer), - (CH 2) b} -SO 3 X (b is an integer of 1 to 10, X is either sodium, potassium, ammonium, triethyl ammonium, lysine, either arginine) of .
In the formulas (i) and (ii), a is an integer from 1 to 10.
In the formulas (i) to (vi), n is 2 or 3. In the formulas (ii), (iv), and (vi), R ⁴ is an alkyl group having 1 to 10 carbon atoms, — (CH ₂ ) _b —SO ₃ X (b is an integer of 1 to 10, and X is sodium , Potassium, ammonium, triethylammonium, lysine, or arginine).
In the formulas (iii) and (iv), L ³ is a substituted or unsubstituted alkyl group having 1 to 10 carbon atoms and may be substituted with a carbonyl group, an amide group, an ester group, or a piperazyl group.
In the formulas (v) and (vi), L ⁴ is a substituted or unsubstituted alkyl group having 1 to 10 carbon atoms, which may be substituted with a carbonyl group, an amide group, or an ester group.   2. The compound according to claim 1, wherein m in the formulas (I) to (IV) is an integer of 100 or more and 500 or less.   The compound according to claim 1 or 2, wherein both A and A 'in the formula (I) are represented by the formula (i).   A in said Formula (IV) is represented by Formula (i), The compound of Claim 1 or 2 characterized by the above-mentioned. The compound according to any one of claims 1 to 4, wherein the formula (i) is represented by any one of the following formulas (i-1) to (i-6).

(I-1)

(I-2)
In formula (i-2), Y ⁻ represents a halogen ion or an organic acid ion.

(I-3)

(I-4)

(I-5)

(I-6)
In the above formulas (i-3) to (i-6), X is any of sodium, potassium, ammonium, triethylammonium, lysine and arginine. The compound according to any one of claims 1 to 4, wherein the formula (ii) is represented by the following formula (ii-1) or (ii-2).

(Ii-1)

(Ii-2)
In the above formulas (ii-1) to (ii-2), X is any of sodium, potassium, ammonium, triethylammonium, lysine, and arginine. The compound according to any one of claims 1 to 4, wherein the formula (iii) is represented by the following formula (iii-1) or (iii-2).

(Iii-1)

(Iii-2) The compound according to any one of claims 1 to 4, wherein the formula (iv) is represented by the following formula (iv-1) or (iv-2).

(Iv-1)

(Iv-2)
In the above formulas (iv-1) to (iv-2), X is any of sodium, potassium, ammonium, triethylammonium, lysine and arginine.   The compound according to any one of claims 1 to 8, further comprising a capture molecule.   The compound of claim 9, wherein the linker moiety is a capture molecule.   The compound according to claim 10, wherein the capture molecule is a polypeptide or a single chain antibody. One or more of the L ¹ and L ² are represented by the following formula (xi), and the compound according to claim 1.

(Xi)
In the formula (xi), L ⁵ is a polypeptide or a single chain antibody, -NH- is a bond via an amino group of an amino acid in the polypeptide or single chain antibody, -S- is a polypeptide Or it represents the coupling | bonding through the thiol group of the amino acid in a single chain antibody. L ⁶ is an alkyl chain having 1 to 10 carbon atoms which may be substituted with any one of a carbonyl group, an amide group, an ester group and a piperazyl group. ** binds to the aforementioned *, and *** binds to the alkyl chain side or ethylene glycol chain side of formulas (I) to (IV). The compound according to claim 12, wherein L ⁶ in the formula (xi) is represented by the following formula (xii).
_{- (CH 2) 2 -C (} = O) -NH- (xii)
In the formula (xii), carbon of the ethylene group is bonded to nitrogen of the maleimide group of the formula (xi), and nitrogen of —NH— is bonded to *** of the formula (xi).   A contrast agent for optical imaging comprising the compound according to any one of claims 1 to 13 and a dispersion medium.   The contrast agent for optical imaging according to claim 14, further comprising an additive.