JP2000086538A - Contrast medium for mri - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject contrast medium capable of expressing contrasting ability solely in target abnormal cells such as tumor cells and remarkably improving detection sensitivity of the abnormal cells by bringing the contrast medium to contain a complex of a polyionic gadolinium-based contrast medium with an ionic high molecule. SOLUTION: This contrast medium contains a complex of a polyanionic Gd-based contrast medium capable of forming a polyionic complex (e.g. a copolymer, etc., of a metal complex obtained by Gd ion-complexing a cationic high molecule and Gd with a chelating agent and the said chelating agent which is not complexed with Gd ion) and a cationic high molecule, or a complex of a polycationic Gd-based contrast medium [e.g. a binding of a cationic high molecule with a metal complex (Gd-DOTA) expressed by the formula] and an anionic high molecule and expresses solely in the presence of a high polymeric electrolyte in a neutral pH.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a contrast medium for MRI. More specifically, polyanionic gadolinium (G
d) An MRI contrast agent containing a complex of a contrast agent for MRI and a cationic polymer or a complex of a contrast agent for Md MRI and an anionic polymer.

[0002]

2. Description of the Related Art Recent advances in clinical image diagnosis have been remarkable.
X-ray CT (computer tomography), ultrasound imaging, M
Indeed, various imaging diagnostic methods such as RI (magnetic resonance imaging) diagnosis and scintigraphy are used in almost all fields of the whole body. Further, various contrast agents suitable for these diagnostic imaging methods have been developed, and their usefulness has been reported. Especially M
RI diagnostics is a new diagnostic that has recently received considerable attention not only from the field of radiation diagnostics but also from the medical community as a whole. M
Compared with other contrast agents, the contrast agent for RI is superior in the resolution of the concentration in the tissue, and has been confirmed to be highly safe without X-ray exposure, etc., indicating the presence of lesions, normal and pathological sites. It has been pointed out that it is clinically useful for grasping anatomical and functional images of the human. However, its detection ability is still insufficient, but is limited to some diseases and sites, and the development of more sophisticated contrast agents is expected. In particular, 1) at low concentration (low dose), 2)
There is a need for a contrast agent for MRI that can detect target cells such as tumors with high sensitivity and that is 3) non-toxic and 4) can be rapidly excreted from the body. Development of a contrast agent for MRI has been awaited, which is particularly excellent in contrast and which enables imaging with only a tumor or a specific organ without imaging in a region such as a normal tissue where imaging is not necessary.

[0003] Isotope News, July 1998, pp. 7-9, describes a microenvironment-responsive MRI contrast agent in vivo. Gadolinium (Gd) MR
The contrast principle of the contrast agent for I is based on the fact that Gd shortens the longitudinal (T1) relaxation time of water molecules (Lauffer RB, Ch.
em. Rev., 87, 901 (1987)) describes that if the interaction between Gd molecules and water can be controlled in response to microenvironment, on-off of an image signal reflecting the microenvironment becomes possible. . Furthermore, it has been reported that the pH of tumor tissue is lower than that of normal tissue (Vaupel P. et. Al, Cancer Res., 49, 6449).
(1989)) based on cationic polymers and anionic Gd
The change of the T1 relaxation time with the pH change was investigated by combining a system-based contrast agent (Mikawa et al., Proceedings of the Symposium on Polymer Science, 46,
2265, 1997). The complex forms a strong polyion complex (PIC, hereinafter also referred to as a polyion complex) when the amounts of positive and negative charges become almost equal around a neutral pH, and dehydrates the water inside to form a G ion complex.
suppresses the interaction of the d ions with the surrounding water, thus reducing the MR
I contrast ability is not expressed. On the other hand, it is described that the MRI imaging ability can be expressed because a weak acidic pH has an excess of positive charge and cannot form a strong PIC.

[0004]

DISCLOSURE OF THE INVENTION The present invention does not provide imaging at a site where imaging is not required, and expresses the imaging ability only in a target abnormal cell such as a tumor. An object of the present invention is to provide a contrast agent for MRI that significantly improves detection sensitivity.

[0005]

Means for Solving the Problems As a result of intensive studies, the present inventors have noticed that a specific polyelectrolyte is expressed on the surface of abnormal cells such as tumor cells. , The image signal (M
The present invention has been completed by successfully obtaining a contrast agent capable of performing on-off of RI contrast ability). That is, the present invention is as follows. (1) A composite of a polyanionic gadolinium (Gd) -based contrast agent and a cationic polymer or a composite of a polycationic Gd-based contrast agent and an anionic polymer, which can form a polyion complex. MRI that expresses MRI imaging ability only at neutral pH and in the presence of polyelectrolyte
Contrast agent. (2) The polyanionic Gd-based contrast agent is a copolymer of a cationic polymer, a metal complex obtained by Gd ion complexation of Gd with a chelating agent and a chelate agent not Gd ion complexed, wherein the cationic polymer is The contrast agent for MRI according to the above (1), wherein the metal complex or the chelating agent is bound to all of the cations. (3) The cationic polymer copolymerized with the metal complex or the chelating agent is polydiethylaminoethyl methacrylate (PDEAMA), poly L-lysine (PLL), poly L
-Histidine (PLH), polyvinylamine, polyethyleneimine, l, m-ionene, poly (N-alkyl-4-vinylpyridinium chloride) (QPVP), polyvinylbenzyltrimethylammonium chloride (P
VBMA), poly N, N-dimethylaminoethyl acrylate, poly N, N-dimethylaminopropyl acrylamide, poly N, N-dimethyl acrylamide, poly N, N-diethyl acrylamide, polyallylamine,
Poly [N, N- (dimethylamino) ethyl methacrylate], Poly [N, N- (diethylamino) ethyl methacrylate], Poly [N, N- (dipropylamino) ethyl methacrylate], Poly [N, N- (diethyl) (2) The contrast agent for MRI according to the above (2), which is chitosan which is at least one synthetic polymer and / or natural polymer selected from the group consisting of acrylamide] and poly [N, N- (dimethyl) acrylamide]. (4) The metal complex has the formula

[0006]

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Wherein the chelating agent is of the formula

[0008]

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The MR according to the above (2), which is a compound containing
Contrast agent for I. (5) The metal complex is

[0010]

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Wherein the chelating agent is of the formula

[0012]

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The MR according to the above (2), which is a compound containing
Contrast agent for I. (6) The cationic polymer is poly-L-lysine (PLL)
The contrast agent for MRI according to the above (2), (4) or (5), which is: (7) The contrast agent for MRI according to the above (1), wherein the polyanionic Gd-based contrast agent is a polymer contrast agent obtained by polymerizing an anionic metal complex via a spacer molecule. (8) A polymer contrast agent obtained by polymerizing an anionic metal complex via a spacer molecule has a general formula (1)

[0014]

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(Wherein, DTPA represents diethylenetriaminepentaacetic acid, PDA represents 1,3-propanediamine, x1 represents a real number of ₁ to 99, and

[0016]

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Is a DTPA-PDA unit into which Gd has been introduced;
That is

[0018]

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## EQU1 ## Or a complex polymer represented by the general formula (2):

[0020]

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(Where PDA is as described above and DO
TA is 1,4,7,10-tetraazacyclododecane-
N, N ', N'' , N''' - represents a tetra-acetic acid, x ₂ is 1
Represents the real number of ~ 49,

[0022]

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Is a DOTA-PDA unit into which Gd is introduced;
That is

[0024]

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Represents The contrast agent for MRI according to the above (7), which is a complex polymer represented by (7). (9) The cationic polymer is polydiethylaminoethyl methacrylate (PDEAMA), poly L-lysine (P
LL), poly L-histidine (PLH), polyvinylamine, polyethyleneimine, l, m-ionene, poly (N-alkyl-4-vinylpyridinium chloride)
(QPVP), polyvinylbenzyltrimethylammonium chloride (PVBMA), polyN, N-dimethylaminoethylacrylate, polyN, N-dimethylaminopropylacrylamide, polyN, N-dimethylacrylamide, polyN, N-diethylacrylamide, poly Allylamine, poly [N, N- (dimethylamino) ethyl methacrylate], poly [N, N- (diethylamino) ethyl methacrylate], poly [N, N- (dipropylamino) ethyl methacrylate], poly [N, N-
(Diethyl) acrylamide] and poly [N, N-
(Dimethyl) acrylamide], wherein the MRI contrast agent according to the above (1), which is chitosan which is at least one synthetic polymer and / or natural polymer selected from the group consisting of: (10) The polycationic Gd-based contrast agent comprises a cationic polymer and a compound represented by the formula

[0026]

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Wherein the metal complex (Gd-DOTA) is bound to a part of the cations of the cationic polymer, and the metal complex (Gd-DOTA) is bound to a part of the cations of the cationic polymer. The contrast agent for MRI according to the above (1), wherein the part remains unbound. (11) The cationic polymer that binds to the metal complex (Gd-DOTA) is polydiethylaminoethyl methacrylate (PDEAMA), poly L-lysine (PLL), poly L-histidine (PLH), polyvinylamine, polyethyleneimine, l , M-ionene, poly (N-alkyl-4-vinylpyridinium chloride) (QPVP),
Polyvinyl benzyl trimethyl ammonium chloride (PVBMA), poly N, N-dimethylaminoethyl acrylate, poly N, N-dimethylaminopropyl acrylamide, poly N, N-dimethyl acrylamide, poly N, N-diethyl acrylamide, polyallylamine, poly [ N, N- (dimethylamino) ethyl methacrylate], poly [N, N- (diethylamino) ethyl methacrylate], poly [N, N- (dipropylamino)]
Ethyl methacrylate], poly [N, N- (diethyl)
Acrylamide] and at least one selected from the group consisting of poly [N, N- (dimethyl) acrylamide]
The contrast agent for MRI according to the above (10), which is chitosan which is one of a synthetic polymer and / or a natural polymer. (12)
The contrast agent for MRI according to the above (10), wherein the cationic polymer is poly L-lysine (PLL) or chitosan. (13) The anionic polymer is poly-L-glutamic acid, poly-L-aspartic acid, polyacrylic acid (PAA),
Synthetic polymer group consisting of polymethacrylic acid (PMAA), polyvinyl sulfonic acid, polystyrene sulfonic acid (PSS), polystyrene phosphoric acid (PSP) and polyphosphoric acid and sialic Lewis acid, colominic acid, uronic acid, sulfate group and / or phosphoric acid The MRI contrast described in (1) above, which is at least one selected from the group consisting of a natural polymer group consisting of an acidic polysaccharide containing a group, hyaluronic acid, chondroitin, chondroitin sulfate, dermatan sulfate, heparan sulfate, heparin and keratan sulfate. Agent. (14) The MRI contrast agent according to (1), wherein the polymer electrolyte is at least one selected from the group consisting of acidic glycolipids and glucosaminoglucans. (15) The polyion complex comprises (i) poly-L-lysine (PLL) and a formula

[0028]

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A metal complex comprising

[0030]

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The contrast agent for MRI according to the above (1), wherein the contrast agent is a complex of a polyanionic Gd-based contrast agent which is a copolymer with a chelating agent containing: (ii) polydiethylaminoethyl methacrylate (PDEAMA).

Conventionally, two types of contrast agents for MRI, T1-weighted contrast agents and T2-weighted contrast agents, are known. Conventionally, as a T1-weighted contrast agent, gadolinium (Gd), which is a lanthanoid-based metal having a strong effect of shortening the longitudinal relaxation time (T1) of protons, is used as a Gd ion complex with a chelating agent. The T1-weighted contrast agent is a positive contrast agent, and the brightness of the portion where the contrast agent is present increases on the image and shines white. The T2-weighted contrast agent is a contrast agent that shortens the transverse relaxation time (T2) of protons, and is used in which superparamagnetic iron oxide fine particles (magnetite) are colloided with a dextran derivative. The T2-weighted contrast agent is a negative contrast agent, and the brightness of the portion where the contrast agent exists is reduced and appears dark on the image.

A complex of a polyanionic Gd-based contrast agent and a cationic polymer or a complex of a polycationic Gd-based contrast agent and an anionic polymer is usually neutral pH (about pH
In (6) to (8), the positive and negative charges are almost equal, a stable polyion complex is formed, the interaction between Gd ions and surrounding water is suppressed, and the MRI contrast ability is not exhibited. The polyion complex (complex) refers to an assembly of polymer electrolytes having charges of opposite signs.

On the other hand, at the acidic pH (approximately pH 4 to 6), the complex generally increases in positive charge as the polycation is protonated, and decreases in negative as the polyanion shifts to the non-dissociated form. The charge decreases. Therefore, at an acidic pH, the positive charge becomes excessive, a stable polyion complex cannot be formed, and Gd interacts with water to exhibit MRI contrast ability.

At alkaline pH (about pH 8 to 9), the positive charge decreases as the polycation deprotonates, and the negative charge increases as the polyanion shifts to the dissociated form. Therefore, the negative charge becomes excessive, and a stable polyion complex cannot be formed, and Gd and water interact with each other, so that the MRI imaging ability is exhibited.

The polyanionic Gd-based contrast agent used in the present invention is used as a T1-weighted contrast agent.
(A) a copolymer of a cationic polymer, a metal complex obtained by Gd ion complexation of Gd with a chelating agent, and the chelating agent not Gd ion complexed, wherein the cation of the cationic polymer is Examples thereof include a metal complex or a complex to which the chelating agent is bound, and (b) a polymer contrast agent obtained by polymerizing the anionic metal complex or the chelating agent via a spacer molecule. Examples of the cationic polymer include polydiethylaminoethyl methacrylate (hereinafter abbreviated as PDEAMA), poly L-lysine (hereinafter abbreviated as PLL), poly L-histidine (hereinafter abbreviated as PLH), polyvinylamine, polyethyleneimine, l ,
m-ionene, poly (N-alkyl-4-vinylpyridinium chloride) (hereinafter abbreviated as QPVP), polyvinylbenzyltrimethylammonium chloride (hereinafter PV)
BMA), poly N, N-dimethylaminoethyl acrylate, poly N, N-dimethylaminopropyl acrylamide, poly N, N-dimethyl acrylamide, poly N, N-diethyl acrylamide, polyallylamine, poly [N, N- (Dimethylamino) ethyl methacrylate], poly [N, N- (diethylamino) ethyl methacrylate], poly [N, N- (dipropylamino)
Ethyl methacrylate], poly [N, N- (diethyl)
Synthetic polymers such as acrylamide] and poly [N, N- (dimethyl) acrylamide]; and natural polymers such as chitosan. Among them, PLL is preferable.

The chelating agent is represented by the formula

[0038]

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Diethylenetriaminepentaacetic acid (hereinafter abbreviated as DTPA) which is a compound containing

[0040]

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1,4,7,10-tetraazacyclododecane-N, N ′, N ″, N ′ ″-tetraacetic acid (hereinafter abbreviated as DOTA).

The metal complex obtained by Gd ion complexation of Gd with a chelating agent is represented by the following formula:

[0043]

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(Hereinafter abbreviated as Gd-DTPA), and

[0045]

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[Hereinafter, abbreviated as Gd-DOTA]. The metal complex complexed with a Gd ion by a Gd chelating agent and the copolymer of the cationic polymer with the chelating agent not complexed by the Gd chelating agent are the same as the metal complex or the chelating agent for all the cations of the cationic polymer. An agent is bound, and the ratio of the number of the metal complex to the total number of the metal complex and the chelating agent is represented by y%, and the formula: (cationic polymer)-(metal complex) (y%)
Expressed as

The above (cationic polymer)-(metal complex)
The (y%), for example (cationic polymer) - poly _{(Gd-DTPA) (x 3} %) wherein, Gd-DTP
A) are as defined above, x ₃ is a real number of 1 to 99], and (cationic polymer) - poly (Gd-DO
TA) (x _4%) wherein, Gd-DOTA) are as defined above, x ₄ is a is a real number of 1 to 49] include.

The copolymer of the cationic polymer, a metal complex obtained by Gd ion complexation of Gd with a chelating agent, and a chelating agent not Gd ion complexed includes Pd.
LL- poly _{(Gd-DTPA) (x 3} %) ( wherein, PL
L, Gd-DTPA, x ₃ are as defined above) it is preferable.

The polymer contrast agent obtained by polymerizing the anionic metal complex via a spacer molecule has the formula

[0050]

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(Wherein the anionic metal complex is Gd-D
TPA or Gd-DOTA is mentioned, and as the chelating agent, one constituting the anionic metal complex, that is, if the anionic metal complex is Gd-DTPA, D
TPA, which is DOTA if Gd-DOTA, and examples of spacer molecules include amines such as methylene diamine, ethylene diamine, propane diamine, and hexane diamine; and activating compounds such as neocarzinostatin (NCS). , Z are real numbers that vary depending on the chelating agent).

The spacer molecule is preferably propanediamine.

As a polymer contrast agent obtained by polymerizing the anionic metal complex via a spacer molecule, specifically, for example, a compound represented by the following general formula (1):

[0054]

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(Wherein, DTPA represents diethylenetriaminepentaacetic acid, PDA represents 1,3-propanediamine, x1 represents a real number from ₁ to 99, and

[0056]

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Is a DTPA-PDA unit into which Gd has been introduced;
That is

[0058]

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Represents the following. And a complex polymer represented by the general formula (2):

[0060]

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Wherein PDA is as described above and DO
TA is 1,4,7,10-tetraazacyclododecane-
N, N ', N'' , N''' - represents a tetra-acetic acid, x ₂ is 1
Represents the real number of ~ 49,

[0062]

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Is a DOTA-PDA unit into which Gd is introduced,
That is

[0064]

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Represents the following. The complex polymer represented by) is desirable. Particularly preferred is a complex polymer represented by the general formula (I).

The general formula (1) represents that in the complex polymer, x₁
% That DTPA-PDA has Gd introduced.
The complex polymer represented by the general formula (1) is
(Gd-DTPA-PDA) (x₁%).
Similarly, the general formula (2) is expressed as x_TwoGd in% DOTA-PDA
Has been introduced, and poly (Gd-DOTA-
PDA) (x_Two%). x₁And x _TwoNotori
The range to be obtained is as described above.

The anionic metal complex is usually obtained by mixing Gd with a chelating agent in a buffer. A detailed method for preparing poly (Gd-DTPA-PDA) (x ₁ %) will be described later.

A copolymer of the cationic polymer with a metal complex obtained by Gd ion complexation of Gd with a chelating agent and the chelating agent not Gd ion complexed is usually prepared by combining the cationic polymer with a chelating agent. It can be prepared by stirring and mixing in an aqueous solution at room temperature, and then introducing Gd into the chelating portion. For example, the PLL-poly (Gd
-DTPA) (x _3%) (wherein the polymer of x ₃ are as defined above), the European Patent 03316
16 (Example 37), and the amino group of PLL and one of the five carboxyl groups of DTPA are covalently bonded to synthesize PLL-DTPA, followed by Gd
It can be obtained by adding ions.

The polymeric contrast agent obtained by polymerizing the above anionic metal complex via a spacer molecule can be usually prepared by stirring and mixing DTPA or DOTA and the spacer molecule in an aqueous solution at room temperature.

In the present invention, the cationic polymer used for complexing with a polyanionic Gd-based contrast agent to form a polyion complex includes a cationic polymer used for preparing the polyanionic Gd-based contrast agent. Examples are the same as the examples of the conductive polymer, and among them, PDEAMA
And a PLL.

The polycationic Gd-based contrast agent used in the present invention is used as a T1-weighted contrast agent.
A combination of a cationic polymer and a metal complex (Gd-DOTA), wherein a part of the cation of the cationic polymer;
For example, a contrast agent or the like in which the metal complex (Gd-DOTA) is bound to 1 to 99%, preferably 10 to 40%, and the rest of the cation remains unbound.

Examples of the cationic polymer include those similar to those used for preparing the polyanionic Gd-based contrast agent.

As a combination of the cationic polymer as the polycationic Gd-based contrast agent and Gd-DOTA, a polymer in which Gd-DOTA is covalently bonded to a part of PLL cation and a cation of chitosan are used. Gd-DO
Polymers with TA covalently bonded are preferred. In this case,
G covalently bonded to part of the cation part of the cationic polymer
d-DTPA has Gd in all (100%) DOTA sections.
, And DOTA without Gd is not included.

In preparing a polycationic Gd-based contrast agent, the conjugated product of the cationic polymer and the metal complex is usually composed of the cationic polymer and the metal complex (Gd-DOT).
And A) in an aqueous solution at room temperature to 40 ° C.

In the present invention, examples of the anionic polymer used to form a polyionic complex with a polycationic Gd-based contrast agent include poly-L-glutamic acid, poly-L-aspartic acid, and polyacrylic acid. (Hereinafter abbreviated as PAA), polymethacrylic acid (hereinafter abbreviated as PMAA), polyvinyl sulfonic acid, polystyrene sulfonic acid (hereinafter abbreviated as PSS), polystyrene phosphoric acid (hereinafter abbreviated as PS)
P), synthetic polymers such as polyphosphoric acid, sialic Lewis acid, colominic acid, uronic acid, acidic polysaccharides containing a sulfate group and / or a phosphate group, hyaluronic acid, chondroitin, chondroitin sulfate, dermatan sulfate, heparan sulfate And natural polymers such as heparin and keratan sulfate, among which poly-L-glutamic acid and poly-L
-Aspartic acid is preferred.

Normally, in order to obtain excellent contrast performance, MR
It is preferable that the contrast agent for I is polymerized, and the molecular rotation suppresses the molecular rotation speed and the MR excited by a magnetic field.
The energy of the contrast agent for I can be easily transmitted to the surrounding protons, and the proton relaxation time can be shortened, so that the imaging ability can be enhanced. Preferable examples include those having a linear structure in which a greater effect of suppressing molecular motion can be expected. In particular, when a complex is formed via 1,3-propanediamine (PDA) to polymerize, It has an alternating copolymer-like polymer structure and is expected to have high chelate stability. The Gd-based contrast agent can be suitably polymerized by, for example, polymerizing a metal complex used for preparing the Gd-based contrast agent. FIG. 1 shows the process of polymerization.

First, DTPA anhydride and PDA, which are metal chelating agents, were converted to DMF (N, N-dimethylformamide).
DTPA is polymerized by stirring while heating while heating.
With a molecular weight of 5,000 to 3
0000, preferably 20,000 to 30,000 poly-DTPA-PDAs are synthesized. The synthesized product and Gd are mixed in a buffer solution and Gd is introduced into the DTPA site to form a polymerized metal complex, poly (Gd-DTPA-PD).
A) (x ₁ %) is obtained. G to DTPA-PDA
The introduction ratio of d is appropriately selected depending on factors such as the maintenance of water solubility and the state of charge, but is 1 to 99%, preferably 10 to 99%.
Gd is introduced into 30% of the DTPA-PDA section. (Thus, x1 is ₁ to 99, preferably 10 to 30)

In the present invention, the complexing of the polyanionic Gd-based contrast agent with the cationic polymer and the complexing of the polycationic Gd-based contrast agent with the anionic polymer differ depending on the cationic polymer used, but generally, Is, for example, PDE
When AMA is used, the pH is around 5-7, preferably pH
By mixing at room temperature in an aqueous solution near 6.5, P
When LH is used, the pH is around pH 4 to 6, preferably pH 6
When a PLL is used by mixing at room temperature in a nearby aqueous solution, it is performed by mixing at room temperature in an aqueous solution near pH 7 to 9, preferably around pH 8.5.

The ratio of the amount of the polyanionic Gd-based contrast agent to the cationic polymer and the ratio of the amount of the polycationic Gd-based contrast agent to the anionic polymer depend on the kind of the Gd-based contrast agent and the polymer to be used and the conditions for the complexation. Although the charge ratio varies depending on the like, the charge ratio of the polymer to Gd-based
5, more preferably 0.8 to 1.2.

The contrast agent for MRI of the present invention is desirably a poly (Gd-DTPA) (x ₃ %) (x ₃ %) which is a metal complex of a cationic polymer, PLL, and a Gd ion.
₁ is as described above), and a cationic polymer PDEA
A complex with MA, more preferably the complex is x ₃ %
Is 16%, that is, Gd is introduced into the DTPA section of 16%.

Further, it is preferable that a hydrophilic synthetic high-molecular polymer, polysaccharide or the like is bound to the MRI contrast agent of the present invention. The synthetic polymer or polysaccharide is a polyanionic G
It can be bonded by graft copolymerization to a cationic polymer or an anionic polymer (main chain polymer) used to form a complex with a d-based contrast agent or a polycationic Gd-based contrast agent. The binding between the cationic polymer or anionic polymer and the above-mentioned synthetic polymer or polysaccharide differs depending on the cationic polymer or anionic polymer or the synthetic polymer or polysaccharide used. General techniques used for synthesizing copolymers can be used.

[0082] Synthetic high polymers suitable for use in the present invention include polyethylene, polyethylene glycol,
Polyoxyethylene glycol, polyethylene terephthalate, polypropylene, polypropylene glycol,
Polyurethane, polyurethane urea, pluronic acid, pullulon alcohol, polyvinyl polymer, polyvinyl alcohol, polyvinyl chloride, polyvinyl pyrrolidone, nylon, polystyrene, polylactic acid, fluorinated hydrocarbon, fluorinated carbon, polytetrafluoroethylene, polyacrylic acid Examples thereof include esters, polyacrylic acid, polymethacrylic acid, polyacrylamide, and derivatives thereof. In the present invention, the molecular weight is about 1,000 to 1
00,000, preferably about 5,000-50,000
Is used.

The polysaccharides suitable for use in the present invention include arabinan, fructan, fucan, arabinogalactan, galactan, galacturonan, glucan, mannan, xylan, levan, fucoidan, galagenan, galactocarose, pectin, pectin. Acid, amylose, pullulan, glycogen, amylopectin, cellulose, dextran, bustulan, chitin, agarose, keratin, chondroitin, dermatan, hyaluronic acid, alginic acid, xanthan gum, starch, carboxymethylcellulose, hydroxymethylcellulose, hydroxypropylmethylcellulose, methylcellulose, methoxycellulose, Erythrose, threose,
Ribose, arabinose, xylose, lyxose, allose, altrose, glucose, mannose, growth, idose, galactose, talose, erythrulose, ribulose, xylulose, psicose, fructose, sorbose, tagatose, glucuronic acid, gluconic acid, glucaric acid, galacturonic acid , Manronic acid, glucosamine, galactosamine, and neuraminic acid are exemplified, and those having a molecular weight of about 300 to 10,000, preferably about 1,000 to 5,000 are used, but their origin is not particularly limited. .

The graft copolymer is a core-she
11 shows an II structure. In this structure, Gd is encapsulated in a core part having a strong hydrophobic environment, and cannot be contacted with surrounding water molecules, so that imaging is not performed. In addition, since the shell portion has a hydrophilic polymer, water solubility is maintained as a whole. Furthermore, due to its hydrophilicity, interaction such as adsorption of biological components such as proteins in the living body is suppressed, and its incorporation into the reticuloendothelial system can be avoided, thereby prolonging blood retention. Furthermore, target directivity to a specific organ or cell using a specific sugar chain recognition function can be expected. When the graft copolymer is delivered to the target site, the charge balance is lost there, the strong polyion complex cannot be formed, the core-shell structure cannot be maintained, and the surrounding water molecules and Upon contact, the imaging ability is developed.

A composite of a polyanionic Gd-based contrast agent and a cationic polymer constituting the contrast agent for MRI of the present invention;
In addition, a complex of a polycationic Gd-based contrast agent and an anionic polymer usually has almost the same amount of positive and negative charges at neutral pH, forms a strong polyion complex, and its MRI contrast ability is off. However, in the presence of the polyelectrolyte, an imbalance of positive and negative charges is caused, and a part or whole of the polyion is dissociated due to a replacement phenomenon of the polyion forming the polyion complex, thereby causing an interaction between Gd and water. enable. Examples of the polymer electrolyte which causes such a positive / negative charge imbalance include sugar chains and glycoproteins having a negative charge in the polymer, such as sialyl Lewis acid, colominic acid, uronic acid, sulfate group and / or phosphoric acid. Glycolipid containing a group: hyaluronic acid, chondroitin, chondroitin 4-sulfate, chondroitin 6
-Glucosaminoglucans such as sulfuric acid, dermatan sulfate, heparan sulfate, heparin, keratan sulfate, etc., and these substances are abnormal tumor cells, melanoma cells, and other abnormal cells such as inflamed cells. It is known that it is expressed in cells in a state, and therefore, it is possible to perform on-off of the MRI imaging ability targeting a tumor cell or the like.

The contrast agent of the present invention is usually used in the form of a suspension or solution in a solvent such as distilled water for injection, physiological saline or Ringer's solution, and, if necessary, can be pharmacologically acceptable. Additives such as carriers and excipients can be included. The contrast agent can be applied to cells, etc., intravascular (vein, artery) administration, oral administration, rectal administration, vaginal administration,
It can be administered to a living body by intralymphatic administration, intraarticular administration, or the like, and is preferably administered in the form of a solution, emulsion, or suspension. The additive included in the contrast agent of the present invention varies depending on the administration form, administration route, and the like.Specifically, in the case of an injection, a buffer, an antibacterial agent, a stabilizer, a solubilizing agent, Excipients and the like are used alone or in combination. For oral administration (specifically, solutions, syrups, emulsions or suspensions, etc.), coloring agents, preservatives, stabilizers, suspending agents , Emulsifiers, thickeners, sweeteners, fragrances and the like are used alone or in combination. As the various additives, those usually used in the art are used.

The contrast agent of the present invention can be administered and imaged according to a conventional MRI contrast agent. Specific administration methods include intravenous administration and oral administration. More specific dosages can be appropriately increased or decreased depending on the age and body size of the subject to be administered and the site for which imaging is desired, but usually the amount of the Gd-based contrast agent contained therein,
That is, the amount of Gd is selected in the range of 5 to 100 μmol / kg, preferably 10 to 50 μmol / kg.

The contrast agent of the present invention can be suitably used as a contrast agent for various animals besides humans, and its administration form, administration route, dose and the like depend on the weight and condition of the target animal. Select as appropriate.

[0089]

The present invention will be described in more detail with reference to the following Examples and Experimental Examples, which by no means limit the present invention. Example 1: Preparation of polyanionic Gd-based contrast agent-1 (Polymerization of anionic metal complex via spacer molecule)
Polymeric contrast agents) poly (Gd-DTPA-PDA) (Synthesis of x _1%) 1) Poly (Synthesis DMF (N, N-dimethylformamide) DTPAA (diethylenetriaminepentaacetic acid anhydride in 20ml of DTPA-PDA) , Do
jin, 12 mM) was dissolved by heating at 60 ° C. Separately, solutions were prepared by dissolving PDA (1,3-propanediamine, manufactured by Wako, 12 mM) and TEA (triethylamine, manufactured by Wako, 50 mM) in 20 ml of DMF. Both solutions were stirred and mixed at 60 ° C. for 24 hours. The resulting mixture is evaporated to dryness at 80 ° C.
Dissolve in 0 ml of water. The aqueous solution was ethanol precipitated using 100% ethanol. The precipitate was filtered and dried to obtain 5.7 g of crude poly-DTPA-PDA. For further purification, the obtained crude poly-DTPA-PDA was redissolved in 30 ml of water and subjected to ultrafiltration (molecular weight cutoff:
5000d), 0.8 g of purified poly-DTPA-PDA
Obtained. 2) Preparation of poly (Gd-DTPA-PDA) (x ₁ %) 1 ml of 0.1 M gadolinium (Gd) aqueous solution and 1) above
86.2 mg of the poly-DTPA-PDA obtained in 0.1.
Mix in a 1 M phosphate buffer (pH 7.2) at room temperature (G
d: DTPA molar ratio = 1: 2), and G is added to the DTPA site.
d to introduce poly (Gd-DTPA-PDA) (50
%). Similarly, a Gd: DTPA molar ratio of 1:
5 and mixed with poly (Gd-DTPA-PDA) (20
%).

Example 2: PLL (cationic polymer) and
Preparation of Graft Copolymer with Dextran and PLLHCl Salt (manufactured by Peptide Research Laboratories) 100 mg and Dextran (Mw = 2,600, manufactured by Funakoshi) 10
0 mg in 0.1 ml borate buffer (pH 8.5) 15 ml
In the solution, 0.3 M sodium cyanoborohydride was added and reacted at 45 ° C. for 2 days to prepare PLL-g-dextran (dextran graft ratio was 6%).

Example 3 PLL (cationic polymer) and
Preparation of Graft Copolymer with Hyaluronic Acid and PLLHCl Salt (manufactured by Peptide Research Laboratories) 100 mg and Hyaluronic Acid (Mw = 8,000, manufactured by Denki Kagaku Kogyo)
00 mg in 0.1 M borate buffer (pH 8.5) 15 m
in 0.3 M sodium cyanoborohydride,
0.4 M NaCl was added and reacted at 37 ° C. for 2 days.
LL-g-hyaluronic acid (hyaluronic acid graft ratio was 2%) was prepared.

Example 4: Preparation of polyion complex-1 [PDEAMA (cationic polymer) and poly (Gd-D
Of a mixed solution of (TPA-PDA) (20%) (metal complex)
Preparation ] Poly (Gd-DTPA-PDA) obtained in Example 1
(20%) aqueous solution (20 mM Gd / L, 46.24 mg
(Polymer / ml) 4.6 mg of PDEAMA in 100 μl
(Polydiethylaminoethyl methacrylate),
Water was added (each charge is equal at this volume ratio) and made up to 1 ml.

Example 5: Preparation of polyion complex-2 [PLH (cationic polymer) and poly (Gd-DTPA)
-Preparation of mixed solution of (PDA) (20%) (metal complex) ] Poly (Gd-DTPA-PDA) obtained in Example 1
(20%) 14 aqueous solution (20 mM Gd / L, 46.24 m
g polymer / ml) in 100 μl of 4 mg of PLH (poly-
L-Histidine, manufactured by Sigma) was added and mixed (to make each charge equal in this amount ratio), and water was added to 1 ml.

Example 6: Preparation of polyion complex - 3 [PLL-g-dextran (graft-polymerized cation)
Polymer) and poly (Gd-DTPA-PDA) (20
%) (Preparation of mixed solution of (metal complex) ] Poly (Gd-DTPA-PDA) obtained in Example 1
(20%) aqueous solution (20 mM Gd / L, 46.24 mg
(Polymer / ml) PLL obtained in Example 2 in 100 μl
-G-dextran (Mw of dextran = 2,60
0, graft ratio 6%), and mixed (to make each charge equal by this amount ratio), water was added to make 1 ml.

Example 7: Preparation of polyion complex-4 [Poly (Gd-DT) in each pH solution (pH 5 to 9)
PA-PDA) (20%) (metal complex) and PDEAMA
Preparation of Complex of (Cationic Polymer) ] Poly (Gd-DTPA-PDA) prepared in Example 1
(20%) so that the concentration becomes 2 mM Gd in 1 ml.
Added to H5 solution. 4.6 m of PDEAMA in the solution
g was added and each pH (pH 5-
9) and mixed at room temperature for 1 hour (each charge becomes equal at this ratio) to obtain a complex.

Example 8: Preparation of polyion complex-5 [Poly (Gd-DT) in each pH solution (pH 5 to 9)
PA-PDA) (20%) (metal complex) and PLH (click)
Preparation of Complex of On-Polymer)] Poly (Gd-DTPA-PDA) prepared in Example 1
(20%) so that the concentration becomes 2 mM Gd in 1 ml.
Added to H5 solution. 4.5 mg of PLH was added to the solution, adjusted to each pH (pH 5 to 9) with an appropriate amount of 1N NaOH, and mixed at room temperature for 1 hour (each charge becomes equal by this amount ratio) to obtain a complex. Was.

Example 9: Polyanionic Gd-based contrast agent
Synthesis-2 [Preparation of PLL (Gd-DTPA) (16%)] PLL (Gd-DTPA) was obtained from Schering AG (Berlin, Germany). In this embodiment, 16% DT
PLL (Gd-DTP) with Gd introduced into PA
A) (16%) was prepared and used. PLL (Gd-DTP
A) was dialyzed against 0.1 M EDTA solution for 4 days, followed by 1 week against water (MWCO = 3,500) to obtain a polyanion in which Gd ions were dissociated (lyophilized until use). The PLL (Gd-
DTPA) (16%) is shown below (the substance is referred to as [I]).

[0098]

Embedded image

The ratio [Gd] / [DTPA unit] of [I] was confirmed by ICP (inductively coupled plasma) at a measurement wavelength of 342 nm, 247 nm and a high frequency output of 1 KW (ICP emission spectroscopy;
Seiko Electronics Co., Ltd.). As a result, it was 0.16. Next, the number average molecular weight of [I] was determined by GPC (gel permeation c).
hromatography, gel permeation chromatography). GPC was performed using a JASCO880-PU pump system under the conditions of an ultrahydrogel 1000 column (Nippon Water) at a flow rate of 0.8 ml / min (25 ° C.). An aqueous solution containing 0.5 M acetic acid and 0.3 M sodium sulfate was used as a mobile phase. For the detection of polymers, a refractive index detector (830-RI, Jusco) and a polygonal light scattering detector (Dawn-DSP, Wyat)
t Technology). The number average molecular weight of [I] is 5 ×
It was 10 ⁴ .

Example 10: PDEAMA (cationic high
Preparation of poly [2- (dimethylamino) ethyl methacrylate] represented by the following formula (the substance is hereinafter referred to as [II])
In DMF from the corresponding monomer, DEAMA,
In the presence of 2,2′-azobis (2,4-dimethylvaleronitrile), which is a polymerization initiator, at 45 ° C.
It was prepared by radical polymerization over a period of days.

[0101]

Embedded image

After the polymerization, excess acetonitrile was added to the solution with stirring to precipitate the polymer [II]. The solution was treated with a cellulose triacetate membrane (MWCO).
= 20,000, Sartorius) to recover the polymer [II]. The number average molecular weight of [II] was 8.6 × 10 ⁴ .

Experimental Example 1: PLL (Gd-DTPA) (1
6%) and a contrast agent muscle comprising a complex of PDEAMA
And MRI signal intensity in tumor tissue In this experimental example, the in vivo p
H responsiveness was confirmed. (A) Experimental method BAL transplanted with colon 26 adenocarcinoma cells (provided by Shinsuke Nakajima, Asahikawa Medical University) as test animals
B / c nude mice (about 20 g, female, 8 weeks old) were used. 0.15 M of [I] prepared in Example 9 and an equivalent mixture of [I] and [II] prepared in Example 11 were used.
100 μl of a contrast solution prepared with NaCl (pH 7) to a final Gd concentration of 2.0 mM was directly injected into the tumor site and the thigh muscle of the mouse. MRI imaging and MRI signal intensity measurement were performed before injection, immediately after injection,
20 hours after the injection, 1 ml of a disposable syringe (5 mmφ) was filled and 4.7 T animal was filled.
imager (Omega CSI-2, GE-B
M.Ruker Co., Ltd.).
Image is synthesized with T1 and T2−WI (TR / TE = 30)
0/12 ms). The MRI imaging and the administration of each contrast agent were performed under anesthesia. The results are shown in FIG.

(B) Results As is clear from FIG. 2, in the normal muscle part, no change was observed in the MRI signal intensity even after 20 hours from the administration of the contrast agent, and the part was not imaged. Was.
However, the signal intensity of the tumor tissue increased with time, and the tumor tissue was specifically imaged. Such a mechanism of exhibiting the tissue-specific contrast ability is based on the fact that the contrast ability of the contrast agent of the present invention is balanced by the positive and negative charges of the contrast agent by sugar chains such as sialyl Lewis acid expressed on the cell surface of colon 26 cells. This is probably because the polyion complex was dissociated and the imaging ability was turned on.

Experimental Example 2: PLL (Gd-DTPA) (1
6%) and a polyion complex of PDEAMA
When the acidic polysaccharide colominic acid is added at pH 7, the MRI contrast ability (R1 relaxation ability) increases depending on the addition amount.

Experimental Example 3: Poly (Gd-DTPA-PD)
A) In a polyion complex of (20%) and PDEAMA, the mixing ratio of poly (Gd-DTPA-PDA) (20%) and PDEAMA was changed as a charge ratio of-: + (0.5: 1). 1: 1, 1: 1.5, 1:
2) 1: 1 form a stable polyion complex and show little effect of enhancing R1 relaxation ability at neutral pH. 0.5: 1, 1: 1.5 and 1: 2, respectively, cannot form a stable polyion complex due to imbalance of charge of the whole polyion complex, and exhibit an effect of enhancing R1 relaxation ability at neutral pH. . However, if another polymer electrolyte is present, it exhibits a further enhancing effect on the R1 relaxation ability.

Experimental Example 4: PLL (Gd-DTPA) (1
6%) and PDEAMA in a polyion complex,
When the mixing ratio of PLL (Gd-DTPA) (16%) and PDEAMA is changed as a charge ratio of-: + (1: 1, 1: 1.5, 1: 2, 1: 4), 1: 1 Form a stable polyion complex and show little effect of enhancing R1 relaxivity at neutral pH. 1: 1.
Samples having a ratio of 5, 1: 2, and 1: 4 cannot form a stable polyion complex due to the imbalance of charge of the entire polyion complex, and exhibit an effect of enhancing R1 relaxation ability at neutral PH. However, if there are still other polymer electrolytes,
The effect of further enhancing the R1 relaxation ability is shown.

[0108]

The contrast agent of the present invention has a neutral pH (about pH 6).
8), even in the presence of a polyelectrolyte such as expressed on the surface of an abnormal cell, an imbalance of positive and negative charges is caused, and the polyion forming a polyion complex is displaced by one. Part or all of the dissociation causes Gd to interact with the surrounding water, thereby exhibiting MRI contrast ability. The MRI contrast agent of the present invention is a contrast agent capable of on-off switching of MRI contrast ability reflecting a change in a biological environment in which a specific polyelectrolyte is expressed with the appearance of abnormal cells such as tumors. .

[Brief description of the drawings]

FIG. 1. Poly (Gd-DTPA-PDA) (x ₁ %)
FIG. 3 is a chart showing a synthesis process of.

FIG. 2. In tumor tissue and muscle, M
It is a figure which shows the change of the relative signal intensity by administration of a RI contrast agent.

Continued on the front page (72) Inventor Atsushi Maruyama 6-11 Konanodai, Konan-ku, Yokohama-shi, Kanagawa Prefecture Housing 13-105 (72) Inventor Toshihiro Akaike 4-15-23, Shimobotani, Hoya-shi, Tokyo (72) Inventor Miwa Naoto 2-22-16 Matsugaoka, Takatsuki-shi, Osaka F-term (reference) 4C085 HH07 JJ01 KA09 KA28 KB12 KB76 LL18

Claims (15)

[Claims] 1. A complex of a polyanionic gadolinium (Gd) -based contrast agent and a cationic polymer or a complex of a polycationic Gd-based contrast agent and an anionic polymer, which can form a polyion complex. A contrast agent for MRI, which contains and exhibits MRI contrast ability only in the presence of a polymer electrolyte at a neutral pH. 2. The polyanionic Gd-based contrast agent is a copolymer of a cationic polymer and a metal complex obtained by Gd ion complexing Gd with a chelating agent and a chelating agent not Gd ion complexed. The contrast agent for MRI according to claim 1, wherein the metal complex or the chelating agent is bound to all the cations of the polymer. 3. The cationic polymer copolymerized with a metal complex or a chelating agent is polydiethylaminoethyl methacrylate (PDEAMA), poly L-lysine (PLL),
Poly L-histidine (PLH), polyvinylamine, polyethyleneimine, l, m-ionene, poly (N-alkyl-4-vinylpyridinium chloride) (QPV
P), polyvinylbenzyltrimethylammonium chloride (PVBMA), polyN, N-dimethylaminoethyl acrylate, polyN, N-dimethylaminopropylacrylamide, polyN, N-dimethylacrylamide, polyN, N-diethylacrylamide, polyallylamine , Poly [N, N- (dimethylamino) ethyl methacrylate], poly [N, N- (diethylamino) ethyl methacrylate], poly [N, N- (dipropylamino) ethyl methacrylate], poly [N, N- ( The contrast agent for MRI according to claim 2, which is chitosan which is at least one synthetic polymer and / or natural polymer selected from the group consisting of diethyl) acrylamide] and poly [N, N- (dimethyl) acrylamide]. 4. The metal complex represented by the formula: Wherein the chelating agent has the formula: The contrast agent for MRI according to claim 2, which is a compound containing: 5. The method according to claim 1, wherein the metal complex is Wherein the chelating agent has the formula: The contrast agent for MRI according to claim 2, which is a compound containing: 6. The contrast agent for MRI according to claim 2, wherein the cationic polymer is poly L-lysine (PLL). 7. The MRI contrast agent according to claim 1, wherein the polyanionic Gd-based contrast agent is a polymer contrast agent obtained by polymerizing an anionic metal complex via a spacer molecule. 8. A polymer contrast agent obtained by polymerizing an anionic metal complex via a spacer molecule is represented by the general formula (1): (Wherein, DTPA represents diethylenetriaminepentaacetic acid, PDA represents 1,3-propanediamine, x1 represents a real number of ₁ to 99, and the following formula: Is the DTPA-PDA part into which Gd has been introduced, ie, Represents Or a complex polymer represented by the general formula (2): (Where PDA is as described above, DOTA is 1,
4,7,10-tetraazacyclododecane-N, N ',
X ″ represents N ″, N ′ ″-tetraacetic acid, x ₂ represents a real number of 1 to 49, and the following formula: Is the DOTA-PDA part into which Gd has been introduced, ie, Represents The contrast agent for MRI according to claim 7, which is a complex polymer represented by the formula: 9. The cationic polymer may be polydiethylaminoethyl methacrylate (PDEAMA), poly L-lysine (PLL), poly L-histidine (PLH), polyvinylamine, polyethyleneimine, l, m-ionen, poly (N -Alkyl-4-vinylpyridinium chloride) (QPVP), polyvinylbenzyltrimethylammonium chloride (PVBMA), poly N, N-dimethylaminoethyl acrylate, poly N, N-dimethylaminopropyl acrylamide, poly N, N-dimethyl acrylamide, Poly N, N-diethylacrylamide, polyallylamine, poly [N, N- (dimethylamino) ethyl methacrylate], poly [N, N- (diethylamino) ethyl methacrylate], poly [N, N-
(Dipropylamino) ethyl methacrylate], at least one synthetic polymer selected from the group consisting of poly [N, N- (diethyl) acrylamide] and poly [N, N- (dimethyl) acrylamide]. 2. The MR according to claim 1, wherein the molecule is chitosan.
Contrast agent for I. 10. A polycationic Gd-based contrast agent comprising a cationic polymer and a compound represented by the formula: Wherein the metal complex (Gd-DOTA) is bound to a part of the cation of the cationic polymer, and a part of the cation is non-bonded to the metal complex (Gd-DOTA). The contrast agent for MRI according to claim 1, which remains as bonded. 11. The cationic polymer which binds to a metal complex (Gd-DOTA) is polydiethylaminoethyl methacrylate (PDEAMA), poly L-lysine (PL
L), poly L-histidine (PLH), polyvinylamine, polyethyleneimine, l, m-ionene, poly (N-alkyl-4-vinylpyridinium chloride)
(QPVP), polyvinylbenzyltrimethylammonium chloride (PVBMA), polyN, N-dimethylaminoethylacrylate, polyN, N-dimethylaminopropylacrylamide, polyN, N-dimethylacrylamide, polyN, N-diethylacrylamide, poly Allylamine, poly [N, N- (dimethylamino) ethyl methacrylate], poly [N, N- (diethylamino) ethyl methacrylate], poly [N, N- (dipropylamino) ethyl methacrylate], poly [N, N-
(Diethyl) acrylamide] and poly [N, N-
The contrast agent for MRI according to claim 10, which is chitosan which is at least one synthetic polymer and / or natural polymer selected from the group consisting of (dimethyl) acrylamide]. 12. The MR according to claim 10, wherein the cationic polymer is poly L-lysine (PLL) or chitosan.
Contrast agent for I. 13. An anionic polymer comprising poly-L-glutamic acid, poly-L-aspartic acid, polyacrylic acid (P
AA), polymethacrylic acid (PMAA), polyvinyl sulfonic acid, polystyrene sulfonic acid (PSS), polystyrene phosphoric acid (PSP) and polyphosphoric acid and a synthetic polymer group and sialic Lewis acid, colominic acid, uronic acid, sulfate group and / or The MRI according to claim 1, wherein the MRI is at least one selected from the group consisting of an acidic polysaccharide containing a phosphate group, hyaluronic acid, chondroitin, chondroitin sulfate, dermatan sulfate, heparan sulfate, heparin and keratan sulfate. Contrast agent. 14. The MRI contrast agent according to claim 1, wherein the polymer electrolyte is at least one selected from the group consisting of acidic glycolipids and glucosaminoglucans. 15. The polyion complex according to claim 15, wherein (i) poly-L-
Lysine (PLL) and formula And a metal complex comprising G which is a copolymer with a chelating agent containing
2. A complex comprising a d-type contrast agent and (ii) polydiethylaminoethyl methacrylate (PDEAMA).
The contrast agent for MRI of the above.