JP6395461B2 - Method for producing contrast medium - Google Patents

Description

  The present invention relates to a method for producing a contrast agent.

  In recent years, fluorescent imaging methods and photoacoustic imaging methods have attracted attention as optical imaging methods capable of noninvasive diagnosis.

The fluorescence imaging method is widely used by irradiating a fluorescent dye with light and detecting fluorescence emitted from the dye. The photoacoustic imaging method is a method for obtaining an image of a measurement target by detecting the intensity of the acoustic wave generated by the volume expansion caused by the heat emitted from the molecule to be measured irradiated with light and the generation position of the acoustic wave. . In the fluorescence imaging method and the photoacoustic imaging method, a light absorber is used as a contrast agent for increasing the intensity of fluorescence from the measurement target site and the intensity of acoustic waves. 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. For example, a molecule such as indocyanine green (hereinafter sometimes abbreviated as ICG) can be administered into the body and used as a contrast agent. As light used for contrast, light in the near-infrared wavelength region that has little influence on the human body when irradiated and has high permeability to a living body is preferable. Since ICG absorbs light in the near infrared wavelength region well, it can be suitably used as a contrast agent in optical imaging. In the present specification, ICG refers to a compound represented by the following formula 1. However, the counter ion is not limited to Na ⁺ , and any counter ion such as H ⁺ or K ⁺ can be used.

Here, the contrast agent may bind a ligand to a substance that emits a signal such as fluorescence or acoustic wave in order to easily accumulate in a target site such as a tumor.
Patent Document 1 discloses a contrast agent including a complex composed of a hydrophobic dye having a structure similar to ICG and a protein considered to function as a ligand, as a material related to a diagnostic marker containing a fluorescent functional group. Yes. In Patent Document 1, in order to obtain a complex, a hydrophobic dye (Formula 3 below) and bovine serum-derived albumin (BSA) are bound in an aqueous solution, and then unfiltered by gel filtration using PD-10. The reactive dye is separated and purified.

JP 9-127115 A

  However, the present inventors have found a problem with Patent Document 1. That is, a complex was synthesized using a hydrophobic dye described in Patent Document 1 (formula 3 above) and serum-derived albumin protein as a ligand, and separation with PD-10 was attempted. As a result, the elution volume difference between the unreacted dye and the complex was not observed, and the two could not be separated. This is because the hydrophobic dye aggregates in water during purification and the apparent molecular weight increases, and as in Patent Document 1, the difference in molecular weight between the hydrophobic dye and the complex to which the ligand is bound is utilized. It is thought that it cannot be purified.

  Accordingly, an object of the present invention is to provide a method for producing a contrast agent that can satisfactorily purify a contrast agent containing a complex obtained by reacting a hydrophobic dye and a ligand in water.

The present invention is a method for producing a contrast agent having a complex comprising a hydrophobic dye and a ligand,
Provided is a method for producing a contrast agent, comprising: a reaction step of reacting a hydrophobic dye and a ligand to obtain a complex; and a purification step of adsorbing the unreacted hydrophobic dye to an adsorbent in water.

  By applying the adsorption process using the adsorbent of the present invention, the hydrophobic dye can be purified in water. According to the method for producing a contrast agent according to the present invention, a contrast agent containing a complex obtained by reacting a hydrophobic dye and a ligand is adsorbed with an unreacted hydrophobic dye by using an adsorbent in water, and combined. A contrast medium with high body purity can be obtained.

It is the electrophoresis result which shows the effect of a refinement | purification of a composite_body | complex (cyanine dye-PEG chemical conjugate) and an unreacted cyanine dye. It is a graph which shows the elution pattern of the cyanine dye by the gel filtration chromatography (PD-10) of a composite_body | complex (cyanine dye-PEG chemical conjugate) and an unreacted cyanine dye.

A method for producing a contrast agent having a complex containing a hydrophobic dye and a ligand, comprising a reaction step of reacting the hydrophobic dye and the ligand to obtain a complex, and an unreacted hydrophobic dye in water And a purification step to adsorb to the liquid.
The hydrophobic dye and the ligand may be covalently bonded, or may be non-covalently bonded such as a hydrogen bond or a hydrophobic bond.

  The purification step may be characterized by comprising a step of adsorbing an unreacted hydrophobic dye on the adsorbent and a step of removing the unreacted hydrophobic dye adsorbed on the adsorbent.

  The step of adsorbing the unreacted hydrophobic dye on the adsorbent and the step of removing the unreacted hydrophobic dye adsorbed on the adsorbent are performed simultaneously or before and after.

  In the step of removing the unreacted cyanine dye adsorbed on the adsorbent, removal of the unreacted cyanine dye adsorbed on the adsorbent is selected from at least one means of sedimentation, centrifugation, or filter filtration. Can be characterized.

  Further, the adsorbent preferably has a hydrophobic material for adsorbing the hydrophobic dye. Here, the hydrophobic material is, for example, a material that is highly hydrophobic to such an extent that the dye recovery rate described later in Examples is less than 0.1.

  The adsorbent preferably has at least one selected from the group consisting of a material having an aryl group, a material having an alkyl group having 4 or more carbon atoms, and a material having an acrylic group. This is because these materials are highly hydrophobic.

  Further, the adsorbent preferably has at least one selected from the group consisting of aryl agarose, aryl organic polymer resin, alkyl agarose, alkyl organic dextran and resin having an acrylic group.

  Further, as a specific example of the adsorbent, at least one selected from the group consisting of phenyl agarose, butyl agarose, octyl agarose, a copolymer consisting of allyl dextran and methylene bisacrylamide gel, and a copolymer consisting of ethylene glycol and methacrylate. It is preferable to have a seed.

Further, the problem that the dyes aggregate in water as in the present invention is likely to occur in a dye having a highly hydrophobic structure such as a naphthalene structure. An example of a hydrophobic dye having a naphthalene structure is a cyanine dye. In addition, cyanine dyes include substances having an absorption maximum in the near-infrared wavelength band, and are suitably used as contrast agents. Examples of the cyanine dye include compounds represented by the following general formula (I), more specifically ICG or derivatives thereof.

In the general formula (I), R _{101 to} R ₁₁₂ are each independently a hydrogen atom or a linear or branched alkyl group having 1 to 18 carbon atoms,
In the general formula _{(I), L 11} to _{L 17} is CH or _{CR 15,} independently of one _{another, R 15} is a straight-chain or a branched alkyl group having a carbon number 1 to 18,
In the general formula (I), L _{11 to} L ₁₇ may be bonded to each other to form a 4-membered ring to a 6-membered ring,
In the general formula (I), R _{11 to} R ₁₄ are each independently a hydrogen atom or a linear or branched alkyl group having 1 to 18 carbon atoms,
In the general formula _{(I), A 11} and _{B 11} represents a linear or branched alkylene group having 1 to 18 carbon atoms independently of one another,
In the general formula _{(I), X 11} and _{Y 11} are sulfo groups or linker group each independently, linker group _-NH 2, -COOH, -OH, -SH, epoxy group, a glycidyl group, N - Saku Castanopsis succinimidyl group, N- sulfo succinate CHILLY succinimidyl group, and any one or more including the N- maleimido group.
The compound represented by the above general formula (I) can be chemically bonded to the ligand independently of each other via X ₁₁ and Y ₁₁ . A ligand is a molecule arbitrarily selected from natural or non-natural products.

The manufacturing method of the contrast agent which concerns on one Embodiment of this invention has the following processes.
(A) Reaction step A reaction step in which a hydrophobic dye and a ligand are reacted to obtain a complex.
(B) Purification step Unreacted hydrophobic dye is adsorbed on the adsorbent in water from the solution containing the complex obtained in (a).

  Non-Patent Document 1 (Chemical Physics Volume 220, Issue 3, p. 373-384 (1997)) describes the aggregation property of ICG, which is a hydrophobic dye, in water. ICG shows an absorption maximum in the vicinity of 785 nm in a dispersed state in water, but when aggregated in water, it shows an absorption equal to or higher than that in the vicinity of 700 nm. When ICG is dissolved in an organic solvent such as a surfactant or methanol, absorption near 700 nm is suppressed, suggesting that the dispersibility of ICG is improved in these solutions.

  However, molecules such as proteins that can serve as ligands exhibit irreversible denaturation when exposed to surfactants or organic solvents, and may not be able to perform their original functions. It is preferable to keep it. Moreover, when using as a diagnostic marker, it is assumed that the cyanine compound is administered into blood in the form of an aqueous solution. Therefore, the production method of the present invention that can carry out purification in an aqueous solvent is effective.

(Reaction process)
The reaction step in the present embodiment is not particularly limited as long as a contrast agent having a complex containing a hydrophobic dye and a ligand can be obtained. The hydrophobic dye in the present invention may be a chemical bond having a skeleton in which a hydrophobic dye and a ligand form a covalent bond by a condensation reaction or the like. Further, it may be in the form of a non-covalent complex in which the hydrophobic dye and the ligand have a physical interaction that does not involve a covalent bond as a holding force.

  In the structure in which the hydrophobic dye and the ligand form a covalent bond in the present invention, the hydrophobic dye obtained in the reaction step has an absorption characteristic in the near infrared (described later), and the hydrophobic dye is adsorbed. There is no limitation on the ligand used as long as it does not interact with the material.

  In the configuration of forming a noncovalent complex containing a hydrophobic dye in the present invention, the complex is formed by a physical interaction between the hydrophobic dye and the ligand not via a covalent bond. As a non-covalent complex, for example, an electrostatic interaction, hydrophobic interaction, or a combination of hydrophobic pigments or other materials formed into an aggregate by affinity such as hydrogen bonding Show. Specific examples include liposomes formed from phospholipids such as phosphatidylcholine and cholesterol encapsulating the cyanine dye of the above general formula (I). Other examples include an emulsion formed with a synthetic surfactant molecule such as Tween-20 and the like, in which the hydrophobic dye of the above general formula (I) is encapsulated. That is, there is no limitation on the ligand to be used unless the hydrophobic dye as a raw material interacts with the adsorbent and the hydrophobic dye obtained in the reaction step does not interact with the adsorbent.

  The ligand in the present invention refers to a molecule for binding to a hydrophobic dye, and the ligand is not particularly limited, and can be arbitrarily selected from natural products and non-natural products. Typically, a ligand refers to a substance that specifically binds to a specific receptor. However, in this specification, the ligand generally refers to a substance that can easily reach a target by binding to the ligand. That is, examples include a substance that binds to a specific target such as a tumor and a substance that binds to a tumor marker, such as an antibody. In addition, polymer compounds (synthetic polymers, proteins, saccharides, etc.) that increase the EPR effect by increasing the molecular weight of the complex and easily reach the tumor and accumulate are also examples of ligands. Although the molecular weight of a high molecular compound is not specifically limited, For example, it is 10 k or more. As the ligand, polyethylene glycol and albumin having high biocompatibility are preferable.

  Examples of natural products include cells, antibodies, albumin, collagen, protease, proteins such as peptides and aptamers, amino acids and gene sequences, saccharides such as dextran and glucose, lipids such as phospholipids and cholesterol, and the like. In addition, natural products include those obtained by modifying or designing the materials exemplified above. Specifically, those that have been partially removed by enzymatic treatment, those that have been combined with other materials, and those that have been modified by genetic engineering techniques are also included. In addition, when the cyanine compound is a chemical conjugate, even if the molecule originally contains a substituent for chemical bonding, it is selected from those further introduced with a substituent for chemical bonding. be able to. As an example of the non-natural product, it can be arbitrarily selected from synthetic polymers such as polyethylene glycol and polylactic acid, surfactants, inorganic compounds such as oxides of iron, gold, carbon and the like.

  The reaction process of the present invention is not necessarily carried out in an aqueous solvent system. However, since the next purification step needs to be performed in an aqueous system, if an organic solvent is used, the reaction step may further include a step of removing the organic solvent.

(Purification process)
The purification process in the present embodiment is not particularly limited as long as the unreacted hydrophobic dye can be purified in water using an adsorbent from the reaction solution obtained by reacting the hydrophobic dye with the ligand. Absent.

As an example of the purification step in the present invention, a case where a cyanine compound of the following formula 6 is obtained by a condensation reaction between a cyanine dye of the following formula 4 and a polyethylene glycol (hereinafter sometimes referred to as PEG) derivative of the following formula 5 is used. I will give you. In this reaction system, in addition to the target compound of the following formula 6, the presence of a compound of the following formula 7 as a by-product in the raw materials of the following formula 4, the following formula 5, and the following formula 4 is assumed. Is done. In the exemplified reaction system, an amide bond is formed as a result of the 1: 1 condensation of the carboxyl group of the cyanine dye of the following formula 4 and the terminal amino group of the compound of the following formula 5. Therefore, in general, if a large excess of the compound of the following formula 4 is added to the compound of the following formula 5 in the system, the existence of the compound of the following formula 5 can be ignored in principle. In the purification step, it is necessary to separate the compound of the following formula 4 and the compound of the following formula 7.

  An example of the purification step is purification of ICG (formula 1) encapsulated particles. In this case, ICG molecules that are not encapsulated in the particles are to be purified.

  In the purification step of the present invention, the adsorbent is used to adsorb and remove the raw material hydrophobic dye. A specific example is removal using the interaction between a cyanine dye having a naphthalene structure as represented by the general formula (I) and an adsorbent. In this example, an adsorption phenomenon due to a hydrophobic interaction between a hydrophobic group typified by a naphthalene structure of a cyanine dye skeleton and a hydrophobic group of an adsorbent is used.

  A suitable adsorbent in the present invention is a material having a hydrophobic substituent, and the hydrophobic unit is patched in a silica particle, a hydrophobic polymer (polyacrylamide, polystyrene, polyester, polyamide, polyethylene, etc.), or a water-soluble unit. The polymer present above is preferred.

  On the other hand, when protein is included in the reaction system, such as when protein is used as a ligand, the adsorbent is a polymer in which hydrophobic units are present on the patch in a water-soluble skeleton in order not to denature the protein during the purification process. It is particularly desirable. Further, it is particularly desirable that the difference in specific gravity between the target product and the target product is sufficiently large in order to facilitate separation of the target product and the adsorbent in the purification step.

  Suitable water-soluble skeletons in the present invention include at least one of the group of agarose, dextran, carboxydextran, allyl dextran, polyethylene glycol, polymethyl methacrylate, polyglycolide, pluronic, cellulose, polyalkylcyanoacrylate, polyhydroxybutyrate. It is selected from one or more types of polymers. Further, a suitable hydrophobic unit in the present invention is selected from at least one of the group consisting of butyl group, propyl group, octyl group, phenyl group, allyl group, aryl group, acrylamide group, acrylic group and alkyl group. It is characterized by that.

  From the above, in the present invention, the adsorbent composed of a polymer in which a hydrophobic unit is present on a patch in the water-soluble skeleton is composed of a combination of both alkyl agarose, aryl-agarose, alkyl organic dextran and aryl. It is preferably selected from at least one of the group consisting of organic polymer resins. More preferably, the adsorbent composed of a polymer in which hydrophobic units are present on the patch in the soluble skeleton of the present invention is butyl, phenyl and octyl agarose, ethylene glycol / methacrylate copolymer, allyl dextran / methylene bisacrylamide gel copolymer. Examples thereof include polymers, acrylamide agarose, and polyacrylamide. Further, as an example of such an adsorbent, commercially available gel filtration, ion exchange chromatography, and hydrophobic chromatography resin can be used. Commercially available products include Superose (manufactured by GE Healthcare Japan), Sephacryl (manufactured by GE Healthcare Japan), Sephadex (manufactured by GE Healthcare Japan), Bio-Gels (manufactured by BioRad Laboratories), Fractogel TSK (Merck Millipore) ), Ultragel (manufactured by Paul), Octyl Sepharose (manufactured by GE Healthcare Japan), Butyl Sepharose (manufactured by GE Healthcare Japan), Phenyl Sepharose (manufactured by GE Healthcare Japan), TSKgel Phenyl-5PW (manufactured by Tosoh) , TSKgel Ether-5PW (manufactured by Tosoh), TSKgel Butyl-NPR (manufactured by Tosoh), TOYOPEARL HW (manufactured by Tosoh), etc. Kill.

  The purification step of the present invention can include an adsorption step of an unreacted hydrophobic dye and an adsorbent in the reaction, and a removal step of unreacted hydrophobic dye remaining in the reaction. An adsorption process and a removal process may be performed simultaneously, may be performed before and after, and in that case, the time for performing both processes may partially overlap. Specifically, in the form in which the adsorption step is carried out in a homogeneous system such as stirring with contact between the hydrophobic dye and the adsorbent, the hydrophobic dye and the adsorbent are adsorbed by means such as natural sedimentation, centrifugation or filter processing following the adsorption step. The material and adsorbate are removed (process). Moreover, you may further include the process of wash | cleaning adsorption agent between the adsorption | suction process by an adsorbent, and a removal process. Further, the method may further include a step of recovering the adsorbent by collecting the hydrophobic dye adsorbed using an organic solvent such as methanol after adsorbing the hydrophobic dye on the adsorbent.

  On the other hand, in the embodiment in which purification is performed in a column shape in which an adsorbent is packed in a chromatographic tube or the like, a target hydrophobic dye material can be obtained by collecting the aqueous solution that has passed through the column. Therefore, in such a case, the adsorption step and the removal step are performed simultaneously. As described above, the adsorption process and the removal process may not always be clearly separated, but such a configuration is also included in the present invention.

(Hydrophobic dye)
The cyanine compound represented by the above general formula (I) having a naphthalene structure and a carboxyl group or a salt thereof as an example of a hydrophobic dye in this embodiment will be described.
In addition, the hydrophobic pigment | dye in this embodiment has as a basic skeleton the structure formed by the methine chain | strand and the 5-membered ring containing N couple | bonded with the both ends of this methine chain | strand.

  The hydrophobic dye in the present embodiment is not limited as long as it absorbs light having a wavelength included in the near infrared region of 600 nm to 1300 nm, and more preferably has an absorption characteristic in 700 nm to 900 nm. Further preferred. The hydrophobic dye in this embodiment preferably has a molar extinction coefficient exceeding 100 k (100,000). The hydrophobic dye in the present invention may refer to a substance in general having the above optical characteristics, but is removed from an underwater system in a purification process using an adsorbent, that is, a substance that interacts with an adsorbent. May also be referred to. On the other hand, the hydrophobic dye in the present invention explicitly refers to a substance exhibiting no optical interaction among the substances exhibiting the above optical characteristics. In the present invention, for the convenience of explanation, the hydrophobic dye compound may be referred to as a chemical conjugate of a hydrophobic dye skeleton.

Moreover, in the hydrophobic pigment | dye which concerns on this embodiment, it is more preferable that general formula (I) is the indocyanine green represented by the following formula (i), and its derivative (s).

In the above formula (i), A and B are each independently an integer of 1 to 20.
In the above formula (i), * 1 and * 2 are those in which ligands are chemically bonded independently of each other via a sulfo group or a linker group L. L is _-NH 2, -COOH, -OH, -SH, epoxy group, a glycidyl group, N- fence Castanopsis succinimidyl group, N- sulfo succinate CHILLY succinimidyl group, and N- any one or more of the maleimide group including. The ligand can be arbitrarily selected from natural products or non-natural products.

  The present invention will be described below with reference to examples, but the present invention is not limited to these examples.

[Example 1 to Example 5]
(Evaluation of adsorbent adsorption capacity of cyanine dyes)
In this example, the ability of various adsorbents to capture cyanine dyes was compared. A commercially available gel resin (hereinafter sometimes referred to as a gel) was selected as the adsorbent in this example. Specifically, as the adsorbent, Octyl Sepharose (manufactured by GE Healthcare Japan), which is an agarose in which the octyl group is modified to measure the chain, Sephacryl S-400 (which is a copolymer of allyl dextran and methylene bisacrylamide gel). GE Healthcare Japan), Sephacryl S-500 (GE Healthcare Japan), Sephacryl S-1000 (GE Healthcare Japan), and TOYOPEARL HW-65S, which is a copolymer of ethylene glycol and methacrylate (Manufactured by Tosoh), Sepharose CL4B (manufactured by GE Healthcare Japan) made of agarose as a comparative example, Sephadex G-25 (manufactured by GE Healthcare Japan) made of dextran, Sephadex G-10 (manufactured by GE Healthcare Japan) was used. ICG-Sulfo-OSu (manufactured by Dojindo Laboratories; I254) was used as the cyanine dye. ICG-Sulfo-OSu is a compound having the structure of the above formula 2.

  The gel was taken in each 1 mL tube, 10 mL of 10 mM HEPES solution (pH = 7.1) was added, the gel was precipitated by centrifugation (1000 G, 5 minutes), and the supernatant was removed. This operation was repeated 3 times to obtain an aqueous gel solution in which the gel solvent was replaced with a 10 mM HEPES solution (pH = 7.1). 32 nanomoles of ICG-Sulfo-OSu was added to the gel aqueous solution, and the mixture was rotated and stirred at room temperature for 30 minutes with a rotator (manufactured by ASONE). Thereafter, the gel was precipitated again by centrifugation (1000 G, 5 minutes), and then the supernatant was collected. The same amount of 10 mM HEPES solution (pH = 7.1) as that recovered was added again to wash the gel, and the supernatant was recovered by centrifugation. Thereafter, the liquid volume of the recovered liquid was concentrated to 0.1 mL, the absorbance derived from the dye was measured, and the dye recovery rate was calculated from the absorbance of the administered dye. The low cyanine dye recovery rate calculated here indicates that the cyanine dye is trapped in the gel that has been precipitated by centrifugation, which means that the function (adsorption capacity) as an adsorbent is high. Means high.

In this example, the dye recovery rate was calculated based on the following formula (1).
{(Dye absorbance of centrifugal supernatant) / (Input cyanine dye absorbance)} × 100 Formula (1)

  Table 1 shows the results of calculating the cyanine dye recovery rate by various adsorbents. The control shown in Table 1 is a sample obtained by adding 32 nanomoles of ICG-Sulfo-OSu to 1 mL of 10 mM HEPES solution (pH = 7.1) without using a gel, and rotating and stirring. The dye recovery rate is a relative value when the value of the above formula (1) calculated for the control sample is 100. As a result, the absorbance of the centrifuged supernatant after concentration in all gel materials except Sepharose CL4B, Sephadex G-25, and Sephadex G-10 was below the measurement sensitivity, and the dye recovery rate was 0. That is, it is suggested that the charged cyanine dye was trapped in the gel. On the other hand, with Sepharose CL4B, Sephadex G-25, and Sephadex G-10 used as comparative examples, the absorbance of the centrifuged supernatant after concentration was higher than the measurement sensitivity. As a result, the scavenging ability of the cyanine dye was lower than that of the aforementioned material. Furthermore, the same test as described above was performed using ICG (manufactured by Japan Public Standards Association), which is a compound of the above formula 1, using a gel (Sephaacryl S-400) which showed the properties of the adsorbent this time. As a result, the absorbance of the centrifugal supernatant was less than the sensitivity. Although it has the chemical structure of the above formula 1, the result of the adsorption ability of the adsorbent for the cyanine dye is considered to have the same tendency for ICG.

  A characteristic common to the material showing a high capturing ability for cyanine dyes in this example is that it has a highly hydrophobic functional group. For example, a cyanine dye may be trapped in a material by a hydrophobic interaction that acts between an alkyl chain of Butyl Sepharose or an acrylamide structure that is a crosslinking site of Sephacryl S-400 and a hydrophobic portion of an aromatic ring of a cyanine dye. Conceivable. On the other hand, it is considered that materials such as Sepharose CL4B, Sephadex G-25, and Sephadex G-10 have a weak cyanine dye capturing ability due to a small number of hydrophobic functional groups. It should be noted that Sephadex G-25 suggests that the difference in the interaction between Sephadex G-10 and cyanine dyes is related to the difference in their interaction with water. For example, the absorption amount of the aqueous solvent with respect to 1 g of dry Sephadex is 2.5 g and 1.0 g for Sephadex G-25 and Sephadex G-10, respectively, and comparing them, Sephadex G-10 is relatively more hydrophobic It is suggested. The above results also suggest that the hydrophobicity (hydrophobic interaction) of the material is involved in the cyanine dye retention ability.

[Example 6 to Example 9]
(Purification of cyanine compounds 1)
10 mg of monoamine linear PEG (manufactured by NOF; ME-200EA) was weighed and dissolved in 0.8 mL of 10 mM carbonate buffer (pH 9.6) to obtain a PEG solution. As a cyanine dye, 0.93 mg of ICG-Sulfo-OSu (produced by Dojindo Laboratories; I254) was weighed and dissolved in 0.12 mL of dimethyl sulfoxide to obtain a dye solution. The dye solution was added to the PEG solution, a stir bar was added, and the mixture was stirred with a magnetic stirrer (200 rpm, 2 hours, room temperature) to obtain a reaction solution containing a cyanine dye and a condensate of PEG.

  Take Sephacryl S-400, Sephacryl S-500, Sephacryl S-1000, and TOYOPARRL HW-65S, which were effective adsorbents in the previous example, in 1 mL tubes, and add 10 mM HEPES solution (pH = 7.1). 10 mL was added, the gel was precipitated by centrifugation (1000 G, 5 minutes), and the supernatant was removed. This operation was repeated 3 times to obtain an aqueous gel solution in which the gel solvent was replaced with a 10 mM HEPES solution (pH = 7.1). To this gel aqueous solution, 16 nanomoles of cyanine dye was added from the above reaction solution, and rotated and stirred at room temperature for 30 minutes with a rotator (manufactured by ASONE). Thereafter, the gel was precipitated again by centrifugation (1000 G, 5 minutes), and then the supernatant was collected. The same amount of 10 mM HEPES solution (pH = 7.1) as that recovered was added again to wash the gel, and the supernatant was recovered by centrifugation. Thereafter, the liquid volume of the recovered liquid was concentrated to 0.1 mL, the absorbance derived from the dye was measured, and the dye recovery rate was calculated from the absorbance of the administered dye. The results are shown in Table 2. Although a slight error occurred in the dye recovery rate, the dye recovery rate was approximately 20 to 30% regardless of the type of adsorbent.

  In this example, the amount of the dye added to the adsorbent was not adsorbed to the adsorbent in this example, although it was half the amount of the dye when only the cyanine dye was added in the previous example. A dye component was present. This suggests that components other than the raw material component of the unreacted cyanine dye were not adsorbed.

  The components that were not adsorbed by the above treatment were collected and subjected to electrophoresis evaluation. Electrophoresis was performed by putting 1 pmol of the dye amount out of the collected components into a polyacrylamide gel for electrophoresis. An untreated reaction solution (pigment amount 1 pmol) was also used as a control. The gel after electrophoresis was photographed with a near-infrared fluorescent scanner (manufactured by LI-COR).

  FIG. 1 shows the photographing result of the gel. In the lane (left side) where an unpurified sample not subjected to purification treatment was migrated, fluorescent fractions were confirmed for high molecular weight and low molecular weight, respectively. When molecular weights were determined based on the mobility in the gel, they were confirmed to be a PEG-dye conjugate (cyanine compound) and an unreacted cyanine dye, respectively. On the other hand, strong fluorescence of the PEG-dye conjugate was observed in the lane (right side) in which the sample after column treatment was migrated compared to the fraction of unreacted dye. This result suggests that the colored fraction near the column inlet is unreacted dye, and strongly suggests that the target PEG dye conjugate is separated by capturing the unreacted dye on the gel. As a result. From the above results, it was determined that the method of capturing unreacted dye was effective as a purification method.

[Example 10]
(Purification of cyanine compounds 2)
15 mL of Sephacryl S400 gel (manufactured by GE Healthcare Japan) as an adsorbent was charged into a column container (manufactured by Bio-Rad Laboratories; 732-1010). Then, the solvent in the column was replaced with 10 mM carbonate buffer (pH 9.6) having a volume twice or more the column volume.

  A total amount of a reaction solution, which is a mixture of a cyanine dye and a cyanine compound obtained in the same manner as in the previous example, was added to the above column, and 10 mM carbonate buffer (pH 9.6) was allowed to flow as an eluent. As a result, it was confirmed that the colored fraction was eluted when the elution volume was around 7 mL. On the other hand, a colored fraction was also confirmed near the column inlet. In order to elute the colored fraction in the vicinity of the column inlet, 10 mM carbonate buffer (pH 9.6) of 10 times or more the column volume was further eluted. However, the movement of the colored fraction was not confirmed even by this operation, suggesting that the colored fraction in the vicinity of the inlet was captured by the Sephacryl S400 gel.

  Furthermore, from the result of electrophoresis similar to the previous example, since the amount of unreacted dye was significantly reduced in the eluted fraction, it was judged that the purification method of the present invention was effective.

[Comparative Example 5]
(Evaluation of gel filtration purification of cyanine compounds)
Separation by gel filtration was attempted using the dye material of Example 2. As a gel filtration purification column, a PD-10 column (manufactured by GE Healthcare Japan) was used. The PD-10 column is a column device for gel filtration packed with Sephadex G-25 gel used in Example 1. In this example, an aqueous solution of ICG-Sulfo-OSu and a reaction solution of ICG-Sulfo-OSu and monoamine linear PEG (ME-200EA) were used. 1.3 μmol of the dye material described above is added to the PD-10 column, 0.5 mL of each dye column is sampled, and the dye elution position is measured by measuring the absorbance derived from the dye in each sample. Was observed.

  FIG. 2 shows the elution results of the cyanine dye and the cyanine compound on the PD-10 column. The vertical and horizontal axes of the graph are the relative dye amount and the elution volume, respectively. The relative dye amount is a relative value when the maximum dye elution amount is 1 when the dye alone is passed. As a result, in both cases, colored components were observed at the overlapping position where the elution volume was around 3 mL. That is, it was difficult to separate the cyanine dye, which is a dye alone, and the PEG-dye conjugate, which is a dye compound, with PD-10. The cyanine dye used in this experiment has a molecular weight of about 1,000, and the PEG-dye conjugate has a molecular weight of about 21,000. Considering the molecular weight separation characteristics of PD-10, it is expected that they can be separated, but in reality, the elution positions overlapped, making it difficult to separate them. This is considered to be one of the causes that the elution position was changed because the cyanine dye of this example formed an aggregate in water and the apparent molecular weight was increased. Based on the above, it was judged that it was extremely difficult to purify a dye having a tendency to aggregate in water using the molecular sieving effect.

Claims (10)

A method for producing a contrast agent having a complex containing a compound represented by the following general formula (I) and a ligand,
A reaction step of obtaining the complex by reacting the ligand with said compound,
A purification step of adsorbed by the adsorbent of the unreacted compound in water, was closed,
The purification step has a step of removing the unreacted compound adsorbed on the adsorbent;
The method for producing a contrast agent, wherein in the step of removing the compound, the unreacted compound adsorbed on the adsorbent is removed by at least one of sedimentation, centrifugation, or filter filtration .
(In the general formula (I), R _{101 to} R ₁₁₂ are each independently a hydrogen atom or a linear or branched alkyl group having 1 to 18 carbon atoms;
In the general formula (I), L _{11 to} L ₁₇ are each independently CH or CR ₁₅ , R ₁₅ is a linear or branched alkyl group having 1 to 18 carbon atoms, and the general formula (I ), L _{11 to} L ₁₇ may be bonded to each other to form a 4-membered to 6-membered ring,
In the general formula (I), R _{11 to} R ₁₄ are each independently a hydrogen atom or a linear or branched alkyl group having 1 to 18 carbon atoms,
In the general formula (I), A ₁₁ and B ₁₁ are each independently a linear or branched alkylene group having 1 to 18 carbon atoms,
In the general formula (I), X ₁₁ and Y ₁₁ are each independently a sulfo group or a linker group, and the linker group is —NH ₂ , —COOH, —OH, —SH, an epoxy group, a glycidyl group, N -It contains any one or more of succinimidyloxy group, N-sulfosuccinimidyloxy group, and N-maleimidoalkyl group. ) The method for producing a contrast agent according to claim 1, wherein the adsorbent includes a hydrophobic material. The adsorbent is a material having an aryl group, a material having alkyl group having 4 or more carbon atoms, in claim 1 or 2, characterized in that it comprises at least one selected from the group consisting of a material having an acrylic group The manufacturing method of the contrast agent of description. The adsorbent is characterized by having at least one aryl-agarose, aryl organic polymer resin, alkyl agarose, alkyl organic dextran and resins having an acrylic group is selected from the group consisting of any of claims 1 to 3 2. A method for producing a contrast medium according to claim 1. The adsorbent has at least one selected from the group consisting of a copolymer consisting of phenyl agarose, butyl agarose, octyl agarose, allyl dextran and methylene bisacrylamide gel, and a copolymer consisting of ethylene glycol and methacrylate. The method for producing a contrast agent according to any one of claims 1 to 4 , wherein the method is characterized. A method for producing a contrast agent having a complex containing a compound represented by the following general formula (I) and a ligand,
A reaction step of reacting the compound with a ligand to obtain a complex;
A purification step of adsorbing the unreacted compound on the adsorbent in water,
A method for producing a contrast agent, wherein the adsorbent has at least one selected from the group consisting of aryl agarose, aryl organic polymer resin, alkyl agarose, alkyl organic dextran, and resin having an acrylic group.
(In the above general formula (I), R ₁₀₁ To R ₁₁₂ Are each independently a hydrogen atom, or a linear or branched alkyl group having 1 to 18 carbon atoms,
In the general formula (I), L ₁₁ Thru L ₁₇ Are independently of each other CH or CR ₁₅ And R ₁₅ Is a linear or branched alkyl group having 1 to 18 carbon atoms, and in the general formula (I), L ₁₁ Thru L ₁₇ May be bonded to each other to form a 4-membered to 6-membered ring,
In the above general formula (I), R ₁₁ To R ₁₄ Are each independently a hydrogen atom, or a linear or branched alkyl group having 1 to 18 carbon atoms,
In the above general formula (I), A ₁₁ And B ₁₁ Are each independently a linear or branched alkylene group having 1 to 18 carbon atoms,
In the above general formula (I), X ₁₁ And Y ₁₁ Are each independently a sulfo group or a linker group, and the linker group is -NH ₂ , —COOH, —OH, —SH, an epoxy group, a glycidyl group, an N-succinimidyloxy group, an N-sulfosuccinimidyloxy group, and an N-maleimidoalkyl group. ) A method for producing a contrast agent having a complex containing a compound represented by the following general formula (I) and a ligand,
Reacting the compound with the ligand to obtain a complex;
A purification step of adsorbing the unreacted compound on the adsorbent in water,
The adsorbent has at least one selected from the group consisting of a copolymer consisting of phenyl agarose, butyl agarose, octyl agarose, allyl dextran and methylene bisacrylamide gel, and a copolymer consisting of ethylene glycol and methacrylate. A method for producing a contrast agent.
(In the above general formula (I), R ₁₀₁ To R ₁₁₂ Are each independently a hydrogen atom, or a linear or branched alkyl group having 1 to 18 carbon atoms,
In the general formula (I), L ₁₁ Thru L ₁₇ Are independently of each other CH or CR ₁₅ And R ₁₅ Is a linear or branched alkyl group having 1 to 18 carbon atoms, and in the general formula (I), L ₁₁ Thru L ₁₇ May be bonded to each other to form a 4-membered to 6-membered ring,
In the above general formula (I), R ₁₁ To R ₁₄ Are each independently a hydrogen atom, or a linear or branched alkyl group having 1 to 18 carbon atoms,
In the above general formula (I), A ₁₁ And B ₁₁ Are each independently a linear or branched alkylene group having 1 to 18 carbon atoms,
In the above general formula (I), X ₁₁ And Y ₁₁ Are each independently a sulfo group or a linker group, and the linker group is -NH ₂ , —COOH, —OH, —SH, an epoxy group, a glycidyl group, an N-succinimidyloxy group, an N-sulfosuccinimidyloxy group, and an N-maleimidoalkyl group. ) Method for producing a contrast medium according to any one of claims 1 7, characterized in that said compound is indocyanine green. It said ligand, synthetic polymers, proteins, method for producing contrast agent according to any one of claims 1 8, characterized in that it is selected from at least one of the saccharides. The method for producing a contrast agent according to any one of claims 1 to 9 , wherein the ligand is at least one of polyethylene glycol and albumin.