WO2015170573A1 - Lymph node imaging agent - Google Patents

Abstract

The purpose of the present invention is to provide a lymph node imaging agent which enables the imaging of a lymph node even when used in a small amount and which can be administered in a simple manner for the imaging. An anionic branched polymer which can be used as a lymph node imaging agent, has at least one labeling substance bound thereto, and has a molecule diameter of 5 to 20 nm.

Description

Lymph node imaging agent

The present invention relates to a lymph node imaging agent.

The sentinel lymph node (primary lymph node) is the lymph node that first passes when cancer cells metastasize, and detection of the lymph node is very important in detecting and diagnosing metastatic cancer. Is done.

Non-Patent Document 1 discloses a method for detecting a lymph node using radiolabeled liposomes. Such liposomes are anionic liposomes having a molecular size of about 100 nm, and are known to be phagocytosed by macrophages present in the lymph nodes when they are administered subcutaneously, and as a result, accumulate in the lymph nodes.

Non-Patent Document 2 discloses that poly L-lysine dendrimers having a benzyl sulfate group on the surface and a molecular diameter of about 2.8 nm accumulate in lymph nodes by subcutaneous administration and tail vein administration. .

Non-Patent Document 3 discloses a method for detecting a lymph node by administering 20 to 40 nm quantum dots into the tail vein.

Non-Patent Document 4 discloses a lymph node imaging method using a radiolabeled polyamidoamine amine dendrimer, and discloses that G4 to G8 dendrimers are suitable for imaging. As an administration method, a method of directly administering such a dendrimer to the mammary gland is disclosed.

Patent Document 1 discloses a complex of a dendrimer prepared using polyamidoimine and polyamidoamine and alginic acid or gamma polyglutamic acid having a molecular diameter of about 24 nm and a zeta potential of about −49 mV. A method of imaging a sentinel lymph node by administering Foot pad is disclosed.

International publication 2011/105520 pamphlet

As described above, various methods have been developed so far for imaging the sentinel lymph node. In order to image the sentinel lymph node, it is not realistic to administer a contrast agent directly to the lymphatic vessel because the lymph node has a minute and complicated structure, and contrasting to the lymph node via the stroma It is preferred to deliver the agent.

However, the known administration methods for imaging lymph nodes are difficult in terms of administration methods such as administration to the mammary gland and subcutaneous administration, or methods requiring a large amount of administration. Has drawbacks.

In view of such a current situation, an object of the present invention is to provide a lymph node imaging agent capable of imaging a lymph node with a small amount and a simple administration method.

As a result of intensive studies to solve these problems, the present inventors have found that imaging of lymph nodes can be performed with a small dose by using an anionic branched polymer having a specific molecular diameter. It was.

The present invention has been completed based on such knowledge, and includes the inventions of the following broad aspects.

Item 1. An anionic branched polymer having at least one labeling substance and having a molecular diameter of 5 to 20 nm, which is used as a lymph node imaging agent.

Item 2. The anionic branched polymer according to Item 1, wherein the zeta potential is −10 mV or less.

Item 3. The anionic branched polymer according to Item 1 or 2, which has at least one kind of anionic group.

Item 4. The anionic branched polymer according to Item 3, wherein the anionic group is at least one selected from the group consisting of a carboxyl group, a sulfone group, and a phenol group.

Item 5. The anionic branched polymer according to Item 3, wherein the anionic group is a carboxyl group.

Item 6 In any one of Items 1 to 5, wherein the labeling substance is at least one selected from the group consisting of a radioisotope, a low molecular organic dye, a fluorescent dye, and a gadolinium coordination chelating agent. The anionic branched polymer described.

Item 7. The anionic branched polymer according to any one of Items 1 to 6, which is used as a lymph node imaging agent for intradermal administration.

Item 8. The anionic branched polymer according to any one of Items 1 to 7, wherein the anionic branched polymer is a graft polymer, a star polymer, a hyperbranched polymer, a dendron, a dendrimer, a dendron polymer, or a dendrigraft polymer. .

Item 9: A lymph node imaging agent comprising the anionic branched polymer according to any one of Items 1 to 8.

Item 10. A method for imaging a lymph node, comprising a step of administering the anionic branched polymer according to any one of Items 1 to 9.

The anionic branched polymer according to the present invention can be suitably used as an imaging agent for lymph nodes.

By using the anionic branched polymer according to the present invention as an imaging agent for lymph nodes, it is possible to image the lymph nodes by a small amount and a simple administration method, for example, intradermal administration.

NMR spectra and partial structures of various dendrimers prepared in this example. NMR spectra of various dendrimers prepared in this example. NMR spectra of various dendrimers prepared in this example. NMR spectra of various dendrimers prepared in this example. The figure (sentinel lymph node) which shows distribution after administering the various dendrimers produced in the present Example. The figure (liver) which shows distribution after administering various dendrimers produced in a present Example. The figure (spleen) which shows distribution after administering various dendrimers produced in the present Example. The figure which shows distribution after administering the various dendrimers produced in a present Example (kidney). The figure (administration site | part) which shows distribution after administering the various dendrimers produced in the present Example. The figure (blood) which shows distribution after administering the various dendrimers produced in the present Example. The figure (lung) which shows distribution after administering various dendrimers produced in a present Example. The figure (heart) which shows distribution after administering various dendrimers produced in the present Example. The figure (muscle) which shows distribution after administering the various dendrimers produced in the present Example. SPECT / CT images taken after administration of various dendrimers prepared in this example. SPECT / CT images taken after administration of various dendrimers prepared in this example.

<Anionic branched polymer>
The anionic branched polymer according to the present invention is used as a lymph node imaging agent. The anionic branched polymer according to the present invention has at least one labeling substance and has a molecular diameter of 5 to 20 nm.

Since the branched polymer according to the present invention is 5 nm or more, it exhibits the effect of being easily accumulated in the lymph node without diffusing from the administration site. Moreover, since it is 20 nm or less, the effect which is hard to be recognized by macrophages etc. is exhibited.

The branched polymer according to the present invention is preferably about 5 to 15 nm. Since it is 15 nm or less, it exerts an effect that it is more difficult to be recognized by macrophages.

The average molecular diameter is a molecular diameter measured based on the dynamic light scattering method (DLS) method, as shown in the following examples, and the measured value obtained based on the principle of DLS is expressed in a polydisperse system. It is the average particle size of the smallest distribution in the volume-based particle size distribution obtained by analysis by a certain Marquardt method. The measuring instrument used for the dynamic light scattering method is ELSZ-DN2 (Otsuka Electronics) or its equivalent.

A branched polymer is a polymer having a branched structure. Broadly speaking, a branched polymer in which a molecular chain extending from a branch point existing in the main chain or the central point is a single chain, such as a graft polymer and a star polymer. A molecular chain extending from a branch point existing in the main chain or the central point, such as a hyperbranched polymer, a dendron, a dendritic polymer (dendrimer), a dendron polymer, etc. There are branched polymers in which the number of terminals increases as the distance from the starting point increases.

In addition, a dendrigraft polymer in which a branch polymer is further connected in a graft polymer form to the branch polymer of the graft polymer is also included in the branched polymer according to the present invention.

Dendrimers have regular branches in the molecular chain extending from the center, can be synthesized step by step (also called generations), are perfectly aligned in shape and size, are regular and symmetrical It is a branched polymer rich in properties. On the other hand, hyperbranched polymers and dendron polymers are branched polymers that do not have regularity, symmetry, and the like as in dendrimers.

The dendron polymer is a branched polymer in which a dendron that is a partial structure of a dendrimer is bonded to a side chain of a main chain polymer.

Since the branched polymer according to the present invention has a plurality of branched chains as described above, it is common in that it can have a plurality of anionic groups. Therefore, any of the branched polymers can exhibit the effects of the present invention.

The branched polymer according to the present invention is not particularly limited as long as it is the above-described branched polymer. For example, it is preferably a dendrimer.
The branched polymer according to the present invention has an anionic property, and due to the anionic property, when such a polymer is administered to a living body, it exhibits an effect of being accumulated in a lymph node.

The fact that the branched polymer according to the present invention is anionic can be determined based on the zeta potential of the branched polymer measured using sodium chloride as a solvent.

The specific zeta potential of the branched polymer according to the present invention is not particularly limited. The zeta potential is usually about −10 mV or less, preferably about −20 mV or less.

Such an anionic property of the branched polymer according to the present invention can be imparted depending on the anionic group of the branched polymer, and it is preferable that the branched polymer has an anionic group on the surface group. More preferably, it has an anionic group at the terminal of the branched polymer. Such an anionic group is preferably covalently bonded to the branched polymer.

The ratio of the anionic group when the branched polymer according to the present invention has an anionic property on the surface group is usually 100% or less and about 50% or more with respect to the entire surface group of the branched polymer. Preferably about 60% or more, more preferably about 70% or more, more preferably about 80% or more, more preferably about 85% or more, more preferably about 90% or more, more preferably about 95% or more, 98 % Or more is most preferable.

Specific types of anionic groups are not particularly limited, and examples thereof include a carboxyl group, a sulfone group, and a phenol group. These branched polymers according to the present invention may contain only one type of anionic group or two or more types. Among the anionic groups described above, a carboxyl group is particularly preferable.

The anionic branched polymer according to the present invention has at least one labeling substance. These labeling substances are not particularly limited. For example, radioisotopes such as ¹¹ C, ¹³ N, ¹⁵ O, and ¹⁸ F compounds (for PET), radioisotopes such as ¹¹¹ In compounds (for SPECT), indigo Low molecular organic dyes such as carmine or patent blue V; fluorescent dyes such as indocyanine green, fluorescein, rhodamine, Alexa Fluor dye; and contrast agents for nuclear magnetic resonance imaging such as gadolinium chelating agents.

The anionic branched polymer according to the present invention can be produced by a known method. In order to impart anionicity to the branched polymer, an anionic group may be bound to the branched polymer using a known method. Such a bonding mode is not particularly limited, and for example, covalent bonding is preferable.

The labeling substance to be bound to the branched polymer can also be imparted using a known method, and the labeling substance can be bound to the branched polymer through another compound such as a chelating agent. Such a binding mode is not particularly limited, and can be appropriately determined in view of, for example, the physical properties of the branched polymer and the physical properties of the labeling substance.

The anionic branched polymer according to the present invention is used as a lymph node imaging agent. The specific method of use can be the same as described in <Lymph node imaging agent according to the present invention> described later.

<Lymph node imaging agent according to the present invention>
The lymph node imaging agent according to the present invention includes the above-described anionic branched polymer according to the present invention. The content of the anionic branched polymer is not particularly limited. The specific content is usually about 0.001 to 100 parts by weight with respect to 100 parts by weight of the lymph node imaging agent, for example. That is, the anionic branched polymer according to the present invention itself can be used as the lymph node imaging agent according to the present invention.

The dosage form of the lymph node imaging agent according to the present invention is not particularly limited. As such a dosage form, for example, in view of the dosage, administration method and the like as described below, a desired dosage form can be obtained together with various conventionally known pharmaceutically acceptable carriers.

Specific dosage forms include injections such as liquids, implantable injections, and sustained injections; dialysis agents such as peritoneal dialysis agents and hemodialysis agents.

These dosage forms can be manufactured based on known documents such as the 16th revised Japanese Pharmacopoeia Manual.

In addition to the above-mentioned anionic branched polymer, the lymph node imaging agent according to the present invention includes a pH adjuster; a preservative; an isotonic agent; an antiseptic; a solvent such as water for injection, sesame oil, soybean oil, corn oil, olive oil; Stabilizers such as inert gases (nitrogen, carbon dioxide, etc.), chelating agents (EDTA, thioglycolic acid, etc.), reducing substances (sodium bisulfite, L-ascorbic acid, etc.); paraoxybenzoic acid esters, chlorobutanol, benzyl alcohol Preservatives such as phenol, benzalkonium chloride; binding solubilizers (sodium iodide, sodium benzoate, sodium nicotinate, etc.), solvent-soluble solubilizers (alcohol, propylene glycol, liquid macrogol, etc.), etc. Solubilizing agents; local anesthetics such as procaine hydrochloride and benzyl alcohol; glucose, sorbi Such as citric acid, acetic acid, sodium phosphate, and those for injection; carmellose sodium, PVP, aluminum monostearate, these for injection, etc. A suspending agent or the like can be contained.

The dosage of the lymph node imaging agent according to the present invention is not particularly limited. The dosage form, administration method, site of lymph node imaging, purpose, sex of individual to be administered, age, weight, imaging technique, It can be set as appropriate in view of the type of labeling substance. Such a dose is usually about 50 to 300 μL in terms of volume.

The administration method of the lymph node imaging agent of the present invention is not particularly limited, and examples thereof include intravenous administration, subcutaneous administration, and intradermal administration. Of these, intradermal administration is preferred.

The apparatus capable of performing imaging using the lymph node imaging agent according to the present invention can be appropriately selected according to the type of labeling substance contained in the above-mentioned anionic branched polymer, and is not particularly limited. Examples of such an apparatus usually include a nuclear medicine examination apparatus, an MRI apparatus, a fluorescence detection apparatus, and the like. A specific imaging method can be performed by appropriately changing the conditions in accordance with the manual of the above apparatus.

<Method for Imaging and Imaging Lymph Nodes According to the Present Invention>
The method for imaging a lymph node according to the present invention includes a step of administering the above-mentioned anionic branched polymer.

The anionic branched polymer, the dose, the administration method, the equipment used for imaging, and the like should be as described in the above <Anionic branched polymer according to the present invention> or <Lymph node imaging agent according to the present invention>. it can.

<Example>
Examples for explaining the present invention in more detail are shown below. However, it goes without saying that the present invention is not limited to the following examples.

Production Example 1: Preparation of chelated PAMAM dendrimer Polyamidoamine (PAMAM) dendrimers having various sizes were modified with p-SCN-Bn-DTPA (2- (4-isothiocyanobenezyl) -diethylethylenepentaetic acid) as a chelating agent.

PAMAM dendrimers used was obtained from ALDRICH, PAMAM dendrimer, ethylenediamine core, generation 2.0 solution (412406;. Hereinafter, call this in this embodiment the _{G2-NH 2), PAMAM dendrimer} , ethylenediamine core, generation 4 0.0 solution (41449; hereinafter referred to as G4-NH _{2 in} this example), PAMAM dendrimer, ethylene core, generation 6.0 solution (536717; hereinafter referred to as G6-NH in this example) ₂ and referred to.), and PAMAM dendrimer, ethylenediamine c re, generation 8.0 solution (536741; . Hereinafter, the it in this example as G8-NH ₂₎ is, p-SCN-Bn-DTPA was obtained from MACROCYCLICS (B-305).

The solvent was distilled off under reduced pressure, p-SCN-Bn-DTPA was added to the various PAMAM dendrimers dried in vacuo and freeze-dried, and the mixture was stirred in 125 mM NaHCO ₃ buffer at 37 ° C. for 24 hours.

Wherein the addition amount of p-SCN-Bn over DTPA is, G2-NH ₂ 10 equivalents, G4-NH ₂ and G6-NH ₂ 7 equivalents and G8-NH _2, is 17 equivalents.

Then the resulting solution G2-NH ₂ is MwCO1000 dialysis _{membrane, G4-NH 2, G6-} NH 2, and G8-NH ₂ was purified using the ultrafiltration membrane MwCO3000, and then lyophilized.

The amount of p-SCN-Bn-DTPA binding was estimated from a calibration curve of p-SCN-Bn-DTPA by measuring a UV spectrum.

Production Example 2: Preparation of acetylated / carboxylated PAMAM dendrimer The chelated PAMAM dendrimer having various sizes described above was further subjected to acetylation or carboxylation.

(Acetylation)
G2-NH ₂ , G4-NH ₂ , G6-NH ₂ and G8-NH ₂ modified with p-SCN-Bn-DTPA were dissolved in 4 ml of NaHCO ₃ buffer (125 mM). While maintaining pH 9-10, acetic anhydride and 4N NaOHaq were added in portions (10 μl acetic anhydride; 50 μl 4N NaOH) alternately (× 20 times; room temperature, 1 hour).

The mixture was then stirred overnight and purified (G2-NH ₂ was purified using a MwCO1000 dialysis membrane, G4-NH ₂ , G6-NH ₂ , and G8-NH ₂ were purified using a MwCO3000 ultrafiltration membrane. And then lyophilized.

The reaction rate was identified using a fluorescamine color reaction. In addition, the reaction was confirmed using ¹ H NMR measurement (FIGS. 1 to 4). It was revealed that an acetylation peak was present at 1.95 ppm.

Various samples in which G2-NH ₂ , G4-NH ₂ , G6-NH ₂ , and G8-NH ₂ are acetylated are referred to as G2-Ac, G4-Ac, G6-Ac, and G8-Ac in the following examples, respectively. Call it.

(Carboxylation)
G2-NH ₂ , G4-NH ₂ , G6-NH ₂ and G8-NH ₂ modified with p-SCN-Bn-DTPA were dissolved in 4 ml of NaHCO ₃ buffer (125 mM). While maintaining pH 9-10, succinic anhydride and 1N NaOHaq were added in portions (room temperature, 1 hour).

The mixture was then stirred overnight and purified (G2-NH ₂ was purified using a MwCO1000 dialysis membrane, G4-NH ₂ , G6-NH ₂ , and G8-NH ₂ were purified using a MwCO3000 ultrafiltration membrane. And then lyophilized.

The reaction rate was identified using a fluorescamine color reaction. In addition, the reaction was confirmed using ¹ H NMR measurement (FIGS. 1 to 4). It was revealed that a carboxyl group peak was present at 2.4 ppm.

Various samples obtained by acetylating G2-NH ₂ , G4-NH ₂ , G6-NH ₂ , and G8-NH ₂ were used as G2-COOH, G4-COOH, G6-COOH, and G8-COOH, respectively, in the following examples. Call it.

The efficiency of the acetylation and carboxylation was calculated by subtracting the amount of unreacted amino groups remaining in the various PAMAM dendrimers obtained with fluorescamine. The results are shown in Table 1.

The physical properties of various PAMAM dendrimers produced were measured. Various dendrimers were dissolved in PBS to a concentration of 10 mg / mL, the particle size was measured using ELSZ-DN2 (Otsuka Electronics) based on the dynamic light scattering (DLS) method, and the zeta potential was ELSZ- It measured using DN2 (Otsuka Electronics). The smallest distribution in the volume-based particle size distribution obtained by analyzing the measured values by the Marquardt method which is a polydisperse system was defined as the particle size. The results are shown in Table 2.

The zeta potential was measured using PBS as a solvent, and as a result, a result that the zeta potential was on the negative side as a whole was obtained. The zeta potential measured for unchelated G4-NH ₂ PAMAM dendrimer with 10 mM NaCl was +2.82 mV, whereas the zeta potential measured with PBS was -15.63 mV.

* “Modification number” indicates the number of modifications by p-SCN-Bn-DTPA per molecule of various dendrimers.

Production Example 3: Preparation of radiolabeled PAMAM dendrimer The above 12 kinds of PAMAM dendrimers (20 nmol) were dissolved in 1.2 mL of ammonium acetate (0.15 M), and ¹¹¹ InCl ₃ (22.2 MBq to 37 MBq) was added. And allowed to react at room temperature for 1 hour.

Thereafter, the reaction product was purified using a PD-10 column (solvent: PBS). Then, it refine | purified using the ultrafiltration membrane of MwCO3000, and the solvent was substituted by the physiological saline from PBS.

Example 1: Biodistribution experiment The 12 types of radiolabeled PAMAM dendrimers prepared in Production Example 3 were each adjusted to 0.37 MBq (0.15 to 0.37 MBq / DTAnmol) / 100 μL, and the total amount was Slc. : SD rat (male, 5 weeks old, 120-140 g) was intradermally administered to the back of the foot, and the administration site was massaged for 30 seconds (n = 4).

Rats were euthanized at 1, 6 and 24 hours after administration, respectively, and the administration site and various organs (popliteal lymph nodes, blood, spleen, kidney, liver, heart, lung, and muscle) were taken out, their weight and radio Activity was measured. The results are shown in FIGS.

It was found that the uptake into the lymph node was significantly higher in G4 and higher anionic ends (G4-COOH, G6-COOH, and G8-COOH).

It was found that the uptake of anionic end (-COOH) was also high in the reticuloendothelial system liver and spleen. As described later, neutral (-Ac) is considered to have high uptake in the liver and spleen due to high uptake in blood.

It was found that some of G2 and G4 are excreted from the kidney because they are small molecules.

It is clear that the retention at the administration site is higher in the order of cationic (—NH ₂ ), anionic (—COOH), and neutral (—Ac), and higher in the order of G8, G6, G4, and G2. It became.

It was found that uptake of neutral G6 and G8 (G6-Ac and G8-Ac) was high in blood. Lung, heart, and muscle were found to show uptake similar to blood.

Most of the cationic (—NH ₂ ) remained at the site of administration, and the anionic (—COOH) was taken up by the lymph nodes, liver, and spleen. Since neutral G8-Ac and G6-Ac are difficult to interact with various organs, uptake is considered to be high.

Example 2: Among the samples prepared in SPECT Imaging Production Example 3, 7.4 MBq each of 6 types of G8-COOH, G6-COOH, G6-NH ₂ , G6-Ac, G4-COOH, and G2-COOH were used. / 100 μL, and the entire amount was intradermally administered to the back of the foot of Slc: SD rats (male, 5 weeks old, 120-140 g), and the administration site was massaged for 30 seconds (n = 1).

24 hours after administration, SPECT imaging was performed using FX Pre-clinical platform (Gamma Medica Ideas Inc) (60 sec / projection; 64 projection). The captured images are shown in FIGS. The site surrounded by a circle in the figure is an administration site, the region surrounded by a square is the primary lymph node, and the region surrounded by a triangle is the secondary lymph node.

It was found that G8-COOH and G6-COOH were able to image not only the sentinel lymph node (primary lymph node) but also the secondary lymph node. With G4-COOH, the sentinel lymph node could be imaged, but with G2-COOH, it was found that the molecular weight was too small to diffuse. It was found that G6-NH ₂ stays considerably at the administration site and that G6-Ac diffuses from the administration site.

From the above, it is considered that G6-COOH or G8-COOH is suitable for imaging of sentinel lymph nodes.

Claims (10)

1. An anionic branched polymer having at least one labeling substance and having a molecular diameter of 5 to 20 nm, which is used as a lymph node imaging agent.
2. The anionic branched polymer according to claim 1, having a zeta potential of -10 mV or less.
3. The anionic branched polymer according to claim 1 or 2, which has at least one kind of anionic group.
4. The anionic branched polymer according to claim 3, wherein the anionic group is at least one selected from the group consisting of a carboxyl group, a sulfone group, and a phenol group.
5. The anionic branched polymer according to claim 3, wherein the anionic group is a carboxyl group.
6. The anion according to any one of claims 1 to 5, wherein the labeling substance is at least one selected from the group consisting of a radioisotope, a low molecular organic dye, a fluorescent dye, and a gadolinium coordination chelating agent. Branched polymer.
7. The anionic branched polymer according to any one of claims 1 to 6, which is used as a lymph node imaging agent for intradermal administration.
8. The anionic branched polymer according to any one of claims 1 to 7, wherein the anionic branched polymer is a graft polymer, a star polymer, a hyperbranched polymer, a dendron, a dendrimer, a dendron polymer, or a dendrigraft polymer.
9. A lymph node imaging agent comprising the anionic branched polymer according to any one of claims 1 to 8.
10. A method for imaging a lymph node, comprising a step of administering the anionic branched polymer according to any one of claims 1 to 9.

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