JP2011184329A - Compound, particle comprising the compound and contrast agent comprising the particle - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a compound that, when contained in a particle or the like, hardly leaks out from the particle under a condition where water exists around the particle. <P>SOLUTION: The compound is represented by formula (1). X in formula (1) is an integer of at least 4 and at most 20. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a compound, a particle having the compound, and a contrast agent having the particle.

  A photoacoustic tomography (hereinafter abbreviated as PAT) device is known as one of devices for visualizing information inside a living body. In the PAT apparatus, by measuring the intensity and generation position of the photoacoustic signal emitted from the measurement object by irradiating the measurement object with light, the substance distribution inside the measurement object is calculated, A layer image is obtained.

  Here, when indocyanine green is irradiated with light, it is known that indocyanine green emits a photoacoustic signal. In the present specification, indocyanine green refers to a compound represented by the following structure.

  Hereinafter, indocyanine green may be abbreviated as ICG. It is known that when an ICG is irradiated with light, the ICG emits a photoacoustic signal. ICG is hydrophilic.

  In Patent Document 1, light is irradiated to a mouse administered with indocyanine green encapsulated in liposome, and a photoacoustic signal is detected.

  Non-Patent Document 1 describes nanoparticles of polylactic acid glycolic acid copolymer containing indocyanine green.

Special table 2002-508760 gazette

Journal of Photochemistry and Photobiology B: Biology, 74 (2004) 29-38

  The above substances having indocyanine green have the following problems. In Patent Document 1, since indocyanine green is hydrophilic, indocyanine green may leak from the liposome under conditions where water is present around the liposome (for example, in vivo). In Non-Patent Document 1, indocyanine green may leak from the nanoparticles under the condition where water is present around the nanoparticles. When indocyanine green leaks from liposomes or particles, it reacts with water around the particles and decomposes.

  Therefore, for example, when indocyanine green is used as a photoacoustic signal transmitter, there are the following problems. That is, when the liposome or nanoparticle is placed under conditions where water is present around it, such as in a living body, the photoacoustic signal obtained is small when the photoacoustic signal is measured by irradiating light on the liposome or nanoparticle.

  Therefore, an object of the present invention is to provide a compound that, when encapsulated in a particle or the like, hardly leaks from the particle under conditions where water is present around the particle.

  1st this invention is a compound shown by following formula (1).

However, X in Formula (1) is an integer of 4 or more and 20 or less.

  The second aspect of the present invention is a particle comprising a hydrophobic molecule and a hydrophilic molecule, the particle having the hydrophilic molecule on the surface of the particle, and the hydrophobic molecule inside the particle In the method, the particles include the compound represented by the formula (1).

  The third aspect of the present invention is a particle composed of an amphiphilic molecule having a hydrophilic part and a hydrophobic part, the particle having the hydrophilic part on the surface of the particle, and the inside of the particle. In the particle having a hydrophobic site, the particle contains the compound represented by the above formula (1).

  According to the present invention, it is possible to provide a compound that, when encapsulated in a particle, is less likely to leak from the particle under conditions where water is present around the particle.

It is the schematic which shows the structure of the particle | grains concerning the 3rd Embodiment of this invention. It is a graph which shows the time-dependent change of the absorption spectrum of each sample.

  An embodiment of the present invention will be described.

(First embodiment)
The compound according to this embodiment is represented by the following formula (1).

  However, X in said formula (1) is an integer of 4-20. X is preferably 10 or less, and more preferably 8 or less. Hereinafter, the compound represented by the formula (1) may be referred to as indocyanine green tetraalkylammonium salt or ICG tetraalkylammonium salt.

  The compound represented by formula (1) is hydrophobic because it has a hydrophobic alkyl group in the cation moiety in formula (1). Therefore, when the compound represented by the formula (1) is encapsulated in particles having hydrophobic molecules therein, it is difficult to leak from the particles under the condition that water exists around the particles. This compound is easy to handle in a hydrophobic solvent such as an organic solvent.

  When the compound represented by the formula (1) is irradiated with light, the compound emits a photoacoustic signal. That is, it is a photoacoustic signal transmitting substance. Therefore, the compound represented by the formula (1) can be used as a contrast agent for a photoacoustic tomography apparatus.

  In addition, the compound shown by Formula (1) is obtained by carrying out the salt exchange of indocyanine green and the tetraalkyl ammonium halide salt in chloroform, and refine | purifying.

(Second Embodiment)
The particles according to this embodiment are composed of hydrophobic molecules and hydrophilic molecules. And it has a hydrophilic molecule | numerator on the surface of particle | grains, and has a hydrophobic molecule | numerator inside a particle | grain. Furthermore, the particles encapsulate the compound represented by the above formula (1).

  Here, since the compound represented by the formula (1) is hydrophobic, it collects inside the particles where hydrophobic molecules exist. Therefore, the compound represented by the formula (1) is difficult to leak from the particles. As a result, the compound represented by the formula (1) becomes difficult to come into contact with water around the particles and is difficult to decompose.

  Examples of the particles according to this embodiment having the characteristics of having hydrophilic molecules on the surface of the particles and hydrophobic molecules inside the particles include the following.

  First, there may be mentioned particles in which hydrophilic molecules are bonded to the surface of particles made of hydrophobic molecules. In this case, particles made of hydrophobic molecules encapsulate the compound represented by the formula (1).

  Secondly, there are particles composed of a core portion made of a hydrophobic molecule and a shell portion made of a hydrophilic molecule. In this case, the core part which consists of a hydrophobic molecule encloses the compound shown by Formula (1).

  Thirdly, there may be mentioned particles in which hydrophobic molecules are present inside particles made of hydrophilic molecules. In this case, the compound represented by the formula (1) is present inside the particle in which the hydrophobic molecule is present. At this time, the hydrophobic molecules may not form the core part, and may be dispersed in the particles made of hydrophilic molecules.

  In the above description, the hydrophobic molecule may be a hydrophobic polymer, and the hydrophilic molecule may be a hydrophilic polymer.

(Hydrophobic and hydrophilic molecules)
The hydrophobic molecule | numerator which concerns on this embodiment is shown by following formula (2), for example.

  Here, in the above formula (2), n is an integer of 50 or more and 150 or less.

  The hydrophilic molecule according to the present embodiment is represented by the following formula (3), for example.

  Here, in the above formula (3), m is an integer of 2 or more and 1000 or less, and a is an integer of 1 or more and 4 or less. R is any one of a methoxy group, a hydroxyl group, an amino group, a carboxyl group, and a maleimide group.

(Particle size)
The particle size of the particles according to the present embodiment is not particularly limited, but is preferably in the range of 10 nm to 200 nm. This is because when the particle size is 200 nm or less, as will be described later, more particles can be deposited on the tumor site than on the normal site in the living body. Further, when the particle size is 10 nm or more, a sufficient photoacoustic signal is emitted when used as a contrast agent in the photoacoustic tomography apparatus. In addition, the particle diameter here is a particle diameter of the whole particle | grains which consist of a hydrophilic molecule | numerator, a hydrophobic molecule | numerator, and ICG tetraalkylammonium salt. The particle size can be measured by a dynamic light scattering method.

(Method for producing particles)
The particle | grains which concern on this embodiment can be obtained with the manufacturing method which has the following processes, for example.
(I) Step of preparing a solution in which an amphiphilic molecule and a compound represented by formula (1) are dissolved in an organic solvent (ii) The organic solvent is distilled off from the solution obtained in (i), and a dried product is obtained. Step (iii) Step of adding micelles by adding water to the dried product obtained in (ii) and forming micelles As an organic solvent used in step (i) above, for example, dichloromethane, chloroform, Any of toluene, xylene, methanol, or a mixture of two or more of these may be used.
Distillation in the step (ii) is an operation of removing the organic solvent from the solution obtained in (ii) to obtain a dried product. The distillation can be performed by any conventionally known method such as a method using a decompression device such as an evaporator.
In the step (iii), the particle size finally obtained can be adjusted by appropriately selecting the energy of the ultrasonic wave to be irradiated. In the step (iii), the micelle is a granular structure formed by gathering the hydrophobic part at the center and the hydrophilic part on the surface among the amphiphilic molecules. is there.
In addition, you may include processes other than said (i) (ii) (iii).

(Contrast agent for photoacoustic tomography)
The particles according to this embodiment absorb light and emit a photoacoustic signal. In addition, as described above, the compound represented by the formula (1) as the photoacoustic signal transmitting substance existing in the particle is difficult to leak out from the particle, so that the photoacoustic signal emitted from the particle is large. Therefore, it can be used as a contrast agent for PAT.

  The contrast agent according to the present embodiment includes the particles according to the present embodiment and a dispersion medium.

  Here, if the particle size of the particles is 200 nm or less, more particles can be accumulated in the tumor site than in the normal site in the living body by using the EPR (Enhanced Permeability and Retention) effect. As a result, when the particle is administered into the living body and then irradiated with light, the photoacoustic signal emitted from the tumor site is larger than the photoacoustic signal emitted from the normal site. Therefore, the contrast agent according to the present embodiment can specifically detect the tumor site by setting the particle size of the particles to 200 nm or less.

  The dispersion medium is a liquid substance for dispersing the particles according to the present embodiment, and examples thereof include physiological saline and distilled water for injection. In the contrast agent according to the present embodiment, the particles according to the present embodiment may be preliminarily dispersed in the dispersion medium, or the particles according to the present embodiment and the dispersion medium may be used as a kit to be in vivo. Prior to administration, the particles may be used after being dispersed in a dispersion medium.

In consideration of administration and use in a living body, the particles according to the present embodiment preferably have an absorption spectrum peak in the near-infrared wavelength region. This is because it is safe when irradiated with light in the near-infrared wavelength region and has a relatively high permeability to the living body.
Here, the near-infrared wavelength region is a wavelength region from 650 nm to 1000 nm.

  In addition, the contrast agent which concerns on this embodiment may have a pharmacologically acceptable additive other than the particle | grains and dispersion medium which concern on this embodiment as needed.

(Contrast method)
A method for detecting particles according to the present embodiment administered into a living body using a photoacoustic tomography apparatus will be described. The method for detecting particles according to this embodiment includes the following steps.
(A) The step of administering the particles according to the present embodiment into the living body (b) The step of irradiating the living body with light and detecting the photoacoustic signal emitted from the particles according to the present embodiment existing in the living body In the step (b), there are no particular limitations on the device that generates the light that irradiates the living body and the device that detects the photoacoustic signal emitted from the particles.

  The contrast method can also be applied to the contrast agent according to the present embodiment.

(Third embodiment)
This embodiment will be described in detail with reference to FIG. The description of the same points as in the first embodiment will be omitted.

  The particle according to the present embodiment is composed of an amphiphilic molecule 2 having a hydrophilic portion 3 and a hydrophobic portion 4, has a hydrophilic portion on the surface of the particle, and is hydrophobic inside the particle. Has a site. Further, the particle 1 includes a photoacoustic signal transmitting substance 5. The photoacoustic signal transmitting substance 5 is a compound represented by the formula (1).

  Here, since the compound represented by the formula (1) is hydrophobic, it gathers inside the particle in which the hydrophobic portion 4 is present in the amphiphilic molecule 2. Therefore, the photoacoustic signal transmitting substance 5 is difficult to leak from the particles 1. As a result, the photoacoustic signal transmitting substance 5 is less likely to come into contact with water around the particles 1 and is difficult to decompose.

(Amphiphilic molecule)
The amphiphilic molecule according to the present embodiment has a hydrophilic part and a hydrophobic part. The hydrophilic part may contain a hydrophobic molecule, but the whole part is hydrophilic. The hydrophobic part may contain a hydrophilic molecule, but exhibits hydrophobicity throughout the part.

  The amphiphilic molecule in the present embodiment is, for example, an amide bond between a hydrophobic molecule represented by the above formula (2) and a hydrophilic molecule represented by the above formula (3).

  Here, in the above formula (2), n is an integer of 50 or more and 150 or less. In the above formula (3), m is an integer of 2 or more and 1000 or less, and a is an integer of 1 or more and 4 or less. R is any one of a methoxy group, a hydroxyl group, an amino group, a carboxyl group, and a maleimide group.

  The amphiphilic molecule according to the present embodiment is a molecule obtained by polymerizing one of the molecule of the following formula (4) and the molecule of the formula (5), or the formula (4) and the formula (5) are randomly selected. Polymerized molecules or those in which the formula (4) and the formula (5) are alternately polymerized like... AABBAA. Moreover, the molecule | numerator which the molecule | numerator which Formula (4) superposed | polymerized, and the molecule | numerator which superposed | polymerized Formula (5) couple | bonded like * AAAAABBBBB *. However, A shows the structure of Formula (4), B shows A which shows the structure of Formula (5).

  Here, m1 and m2 in the above formulas (4) and (5) are integers of 2 or more and 1000 or less. A1 and a2 are integers of 1 or more and 4 or less. R1 and R2 are any of a methoxy group, a hydroxyl group, an amino group, a carboxyl group, and a maleimide group.

(Particle size)
The particle size of the particles according to the present embodiment is the particle size of the entire particle composed of amphiphilic molecules and ICG tetraalkylammonium salt. The particle size can be measured by a dynamic light scattering method.

  The particle according to the present embodiment may be a particle composed of a liposome and a compound represented by the above formula (1) present in the hydrophobic part of the liposome.

  As mentioned above, although embodiment of this invention was described, this invention is not limited to these.

  Examples of the present invention will be described below.

(Measurement of particle size)
The particle size measurement performed in each example described below was performed using a dynamic light scattering analyzer (manufactured by Otsuka Electronics Co., Ltd., DLS-8000).

(Measurement of photoacoustic signal)
The measurement of the photoacoustic signal performed in each example described below was performed with reference to Japanese Patent Laid-Open No. 06-296612. The measurement conditions are as follows.
Liquid measurement cell: polystyrene cuvette path length: 1 mm
Light source: wavelength tunable titanium sapphire laser LT2211A (manufactured by Lotis TII)
Variable wavelength range: 690 to 1000 nm
Pulse width: about 20ns
Pulsed light energy: 25 mJ (wavelength 800 nm)
Primary excitation laser: Nd / YAG laser LS-2134 (manufactured by Lotis TII)
Primary excitation wavelength 532nm (second harmonic)
Primary excitation pulse width: 16 to 18 ns
Primary excitation pulse energy: 150 mJ
Repetition frequency: 10Hz
Water immersion type ultrasonic probe: V303 (Panametrics-NDT)
Center frequency: 1MHz
Element size: 13mm
Preamplifier: Model 5682 (Olympus)
Amplification degree: +30 dB
Cut-off frequency: 30 MHz
Oscilloscope: DPO 4104 (manufactured by Tektronix)
Laser light detection photodiode: DET10A / M (manufactured by THORLABS)

The measuring method is as follows.
The sample dispersion liquid was filled into a liquid measurement cell, and the laser light irradiation site was immersed in a glass container filled with water for photoacoustic signal measurement. The emission wavelength of the titanium sapphire laser was set to a predetermined wavelength, and laser pulse light was irradiated from one side of the cell to the laser light irradiation site. The water immersion type ultrasonic probe is installed at a position 25 mm from the surface opposite to the laser beam irradiation of the cell, and an electric signal generated by inputting a photoacoustic signal to the probe is used as the preamplifier. Was amplified and input to the oscilloscope. Further, an electric signal was generated by a scattered light photodiode generated when the laser pulse light hits the measurement cell, and was input as a trigger signal to the oscilloscope. Using the average processing function of the oscilloscope, the input voltage waveform obtained by integrating and averaging 128 signals is used as the photoacoustic signal evaluation waveform of the sample dispersion (the difference between the positive and negative peak voltages is the evaluation value of the photoacoustic signal intensity). And).

(Measurement of fading rate)
In the present specification, an index called fading rate is used as the degree of ease of leakage described above. The larger the fading rate, the easier it is to leak, and the smaller it is, the harder it is to leak.

The measurement of the fading rate performed in each example described below was performed as follows.
First, after measuring the absorbance of the maximum absorption wavelength (λmax) at the initial stage (within 30 minutes after the aqueous dispersion of particles was obtained) of each sample (particle aqueous dispersion), the sample was left standing in the dark and left at λmax The change with time in the absorbance was measured. Next, the fading rate of the particles after t days when the initial λmax was A0 and the λmax after t days was At was calculated as fading rate (%) = [(A0−At) / A0] × 100. .

<Example 1>
A molecule represented by the above formula (2) (12.5 mg, a molecular weight of 30000 to 50000, manufactured by Aldrich) and a molecule represented by the above formula (3) (in the formula (3), R is a methoxy group, a Is 94) (94 mg, average molecular weight 10,000, SUNBRIGHT ME-100EA, manufactured by NOF Corporation) was reacted in chloroform (3 ml) to obtain a chloroform solution of amphiphilic molecules.

  Next, ICG (12.5 mg, manufactured by Japan Public Standards Association) and tetraoctylammonium bromide (34 mg, manufactured by Tokyo Chemical Industry) represented by the above structure were subjected to salt exchange in chloroform (50 ml). Thereafter, purification was performed to obtain ICG tetraoctylammonium salt (compound represented by the above formula (1) (X = 8)).

<Example 2>
(Particle A)
The ICG tetraoctylammonium salt (12.5 mg) was added to the chloroform solution (50 ml) of the amphiphilic molecule and dissolved uniformly. Thereafter, chloroform was removed by distillation under reduced pressure using an evaporator to obtain a dried product.

  Next, by adding milli-Q water (20 ml) to this dried product and then irradiating with ultrasonic waves, the aqueous dispersion of particles (particles A) in which particles composed of amphiphilic molecules encapsulate ICG tetraoctylammonium salt. A liquid was obtained. The average particle diameter of the obtained particles A was 50 nm.

  The fading rate of particles A was measured using the aqueous dispersion of particles A obtained above. The results are shown in Table 1 and FIG. Table 2 shows the results of measuring the photoacoustic signal of the aqueous dispersion of particles A obtained above.

<Example 3>
The compound was synthesized in the same manner as in Example 1 except that tetraoctylammonium bromide in Example 1 was replaced with tetraheptylammonium bromide. As a result, ICG tetraheptylammonium salt (compound represented by the above formula (1) (X = 7)) was obtained.

<Example 4>
(Particle B)
Particles were prepared in the same manner as in Example 1 except that the ICG tetraoctyl ammonium salt in Example 2 was replaced with the ICG tetraheptyl ammonium salt. As a result, an aqueous dispersion of particles (particle B) in which particles composed of amphiphilic molecules encapsulate ICG tetraheptylammonium salt was obtained. The average particle diameter of the obtained particles B was 45 nm.

  The fading rate of particles B was measured using the aqueous dispersion of particles B obtained above. The results are shown in Table 1. Table 2 shows the results of measuring the photoacoustic signal of the aqueous dispersion of particles B obtained above.

<Example 5>
The compound was synthesized in the same manner as in Example 1 except that tetraoctylammonium bromide in Example 1 was replaced with tetrahexylammonium bromide. As a result, ICG tetrahexylammonium salt (compound represented by the above formula (1) (X = 6)) was obtained.

<Example 6>
(Particle C)
Particles were prepared in the same manner as in Example 1 except that the ICG tetraoctyl ammonium salt in Example 2 was replaced with the above ICG tetraxyl ammonium salt. As a result, an aqueous dispersion of particles (particle C) in which particles composed of amphiphilic molecules encapsulate ICG tetrahexylammonium salt was obtained. The average particle diameter of the obtained particles C was 50 nm.

  The fading rate of the particles C was measured using the aqueous dispersion of the particles C obtained above. The results are shown in Table 1. Table 2 shows the results of measuring the photoacoustic signal of the aqueous dispersion of particles C obtained above.

<Example 7>
The compound was synthesized in the same manner as in Example 1, except that tetraoctylammonium bromide in Example 1 was replaced with tetrabutylammonium bromide (mg, manufactured). As a result, ICG tetrabutylammonium salt (compound represented by the above formula (1) (X = 4)) was obtained.

<Example 8>
(Particle D)
Particles were prepared in the same manner as in Example 1 except that the ICG tetraoctylammonium salt in Example 2 was replaced with the ICG tetrabutylammonium salt. As a result, an aqueous dispersion of particles (particles D) in which particles composed of amphiphilic molecules encapsulate ICG tetrabutylammonium salt was obtained. The average particle diameter of the obtained particles D was 169 nm.

  The fading rate of the particles D was measured using the aqueous dispersion of the particles D obtained above. The results are shown in Table 1. Table 2 shows the results of measuring the photoacoustic signal of the aqueous dispersion of particles D obtained above.

<Comparative Example 1>
The fading rate was measured using an aqueous solution of ICG represented by the above formula (2) (12.5 mg, manufactured by Japan Public Standards Association). The results are shown in Table 1 and FIG. Table 2 shows the result of measuring the photoacoustic signal.

<Comparative example 2>
(Particle E)
Chloroform was removed from the chloroform solution of the amphiphilic molecules obtained in Example 1 by distillation under reduced pressure to obtain a dried product. Next, after adding an aqueous solution (5.4 mg, 20 ml) of ICG (12.5 mg, manufactured by Japan Standards Association) to the obtained dried product, particles E having ICG are obtained by irradiating with ultrasonic waves. It was. The results of measuring the fading rate of the particles E are shown in Table 1 and FIG. 2C, and the results of measuring the photoacoustic signal of the aqueous dispersion of the particles E are shown in Table 2.

(Evaluation of fading rate)
From the results in Table 1, it was confirmed that the fading rates of the particles A, B, C, and D were smaller than those of the ICG aqueous solution and the particles E. That is, it is considered that the ICG tetraalkylammonium salt is less likely to leak from the particles.
FIGS. 2A, 2B, and 2C are graphs showing changes over time in absorption spectra of an aqueous dispersion of particles A, an aqueous solution of ICG, and an aqueous dispersion of particles E, respectively. However, in FIGS. 2A, 2B, and 2C, the peak value of the absorption spectrum measured immediately after the preparation of the aqueous dispersion of each particle is set to about 2. From this result, it was found that the particle A had a smaller fading rate than the aqueous solution of ICG, the particle E.

(Evaluation of photoacoustic signal)
From the results in Table 2, it was confirmed that the aqueous dispersions of the particles A, B, C, and D can obtain a larger photoacoustic signal than the comparative example. That is, it was found that the particles according to this example can incorporate a large amount of ICG tetraalkylammonium salt.

  As mentioned above, although the examples of the present invention have been described, the present invention is not limited to these examples, and the materials, composition conditions, reaction conditions, etc. are within a range where particles having equivalent functions and effects can be obtained. It can be changed freely.

1 Particle 2 Amphiphilic molecule 3 Hydrophilic site 4 Hydrophobic site 5 Photoacoustic signal transmitter

Claims (5)

A compound represented by the following formula (1).

However, X in Formula (1) is an integer of 4 or more and 20 or less. A particle composed of hydrophobic molecules and hydrophilic molecules,
In the particle having the hydrophilic molecule on the surface of the particle and the hydrophobic molecule inside the particle,
The particle includes the compound according to claim 1. Particles composed of amphiphilic molecules having a hydrophilic part and a hydrophobic part,
In the particle having the hydrophilic part on the surface of the particle and the hydrophobic part inside the particle,
The particle includes the compound according to claim 1.   The particle according to claim 2 or 3, wherein the particle diameter is in a range of 10 nm or more and 200 nm or less.   A contrast agent for photoacoustic tomography, comprising the particles according to any one of claims 2 to 4 and a dispersion medium.