JP2015078150A - Rheumatoid arthritis diagnostic agent - Google Patents

Abstract

The present invention provides a diagnostic agent for rheumatoid arthritis that is highly safe for living bodies and capable of detecting the position of an inflammatory lesion by molecular imaging. A diagnostic agent for rheumatoid arthritis, comprising an amphiphilic block polymer having a hydrophilic block having a sarcosine unit and a hydrophobic block having a lactic acid unit, and nanoparticles containing a labeling agent. The hydrophilic block contains 2 to 300 sarcosine units, and the hydrophobic block contains 5 to 400 lactic acid units. The labeling agent is preferably a fluorescent dye. [Selection] Figure 2

Description

  The present invention relates to a diagnostic agent for rheumatoid arthritis. More specifically, the present invention relates to a diagnostic agent for rheumatoid arthritis containing nanoparticles comprising an amphiphilic block polymer and a labeling agent. More specifically, the present invention relates to a diagnostic agent for rheumatoid arthritis that contains nanoparticles containing an amphiphilic block polymer and a labeling agent, and can detect the position of an inflammatory lesion by molecular imaging based on the labeling agent.

  In the nanoparticle, when the labeling agent is replaced with a drug having an effect of treating rheumatoid arthritis, a drug for treating rheumatoid arthritis is obtained. Further, in the nanoparticles, if the labeling agent has a rheumatoid arthritis therapeutic action, or if both the labeling agent and a drug having a rheumatoid arthritis therapeutic action are included, a diagnostic agent for treating rheumatoid arthritis is obtained.

  Rheumatoid arthritis (RA) is an autoimmune disease in which joints throughout the body are destroyed as it progresses, and it is said that there are approximately 700,000 patients in Japan. As it progresses, there may be significant limitations in daily life due to pain and joint deformation. Arthritis in rheumatoid arthritis is due to inflammation and proliferation of the synovium that covers the joint lumen. In treatment, anti-inflammatory analgesics and anti-rheumatic drugs (methotrexate) are generally used, but depending on the symptoms, sufficient effects may not be obtained.

  The diagnosis of rheumatoid arthritis is performed according to, for example, the American College of Rheumatology (ACR) classification criteria for rheumatoid arthritis, but in early rheumatoid arthritis, the diagnostic sensitivity of the ACR standard is considered to be about 50%.

  Diagnosis of rheumatoid arthritis includes clinical findings, serum rheumatoid factor (autoantibodies against denatured IgG; RF), C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), imaging (simple radiograph, CT, MRI) and the like are used.

  Diagnosing arthritis and other rheumatic diseases in rheumatoid arthritis is often difficult because many symptoms are similar among various diseases. Therefore, a more accurate diagnostic method is required.

  As for the improvement of rheumatoid arthritis diagnosis, for example, JP 2003-57242 A, JP 2005-315772 A, JP 2006-304721 A, JP 2013-21932 A, JP 2013-32358 A, etc. There is a report. These all relate to immunochemical diagnostic techniques.

  JP 2013-515099 A discloses a random copolymer having a zwitterion such as phosphorylcholine and a functional agent (drug, detection agent, imaging agent, labeling agent, diagnostic agent, etc.). Copolymers have been disclosed to be effective for diagnostic and imaging purposes ([0024]). In the publication, the random copolymer is produced by radical polymerization using a monomer that can be linked to a zwitterion-containing monomer such as phosphorylcholine ([0025]), It is disclosed that random copolymers are made from monomers including acrylates, methacrylates, acrylamides, methacrylamides, styrenes, vinyl-pyridines and vinyl-pyrrolidones ([0027]). A zwitterionic component is a compound having both positive and negative charges ([0028]). It is disclosed that the functional agent may contain nanoparticles, and the nanoparticles can be packed with diagnostic and / or therapeutic agents ([0191]). In addition, it is disclosed that the random copolymer is used for imaging by near-infrared rays or MRI ([0258] to [0260]), and it is referred to the treatment of rheumatoid arthritis ([0255]). Each of the above monomers is not sufficiently biocompatible. The publication does not disclose nanoparticles composed of amphiphilic block polymers.

  On the other hand, peptidic nanoparticles with higher biocompatibility are also known. For example, Japanese Patent Application Laid-Open No. 2008-24816 and US Patent Application Publication US2008 / 0019908 disclose peptide-type nanoparticles using an amphiphilic block polymer having polyglutamic acid methyl ester as a hydrophobic block. This publication describes that the particle size of the nanoparticles can be controlled by changing the chain length of the amphiphilic block polymer. It is also shown that the nanoparticles were observed to accumulate in cancer tissue.

  In Chemistry Letters, vol.36, no.10, 2007, p.1220-1221, an amphiphilic block polymer composed of a polylactic acid chain and a polysarcosine chain was synthesized and the amphiphilic block polymer was synthesized. It has been shown that molecular assemblies with a particle size of 20-200 nm have been prepared by self-assembly, which can be used as nanocarriers in DDS.

  International Publication No. 2009/148121 (US Patent Application Publication US2011 / 0104056) and Biomaterials, 2009, Vol. 30, p. In 5156-5160, a linear amphiphilic block polymer in which a hydrophobic block is a polylactic acid chain and a hydrophilic block is a polysarcosine chain is self-assembled in an aqueous solution to form a polymer micelle (lactosome). Is disclosed. Regarding the particle size of the lactosome, paragraph [0127] of WO2009 / 148121 discloses 10 nm to 500 nm, but actually, paragraph [0251] only discloses 30 nm to 130 nm. is there. In addition to high blood retention, lactosomes have been shown to significantly reduce the amount of accumulation in the liver as compared to polymeric micelles that have been developed so far. This lactosome uses a property [Enhanced Permeation and Retention effect (EPR effect)] that nanoparticles with a particle size of several tens to several hundreds of nanometers that are staying in the blood easily collect in the cancer. It can be applied as a nanocarrier for molecular imaging or drug delivery targeting a site.

  Cancer tissue grows faster than normal tissue, and induces new blood vessels in the cancer tissue to acquire oxygen and energy necessary for cell growth. Since this new blood vessel is fragile, it is known that some large molecules leak out of the blood vessel. Furthermore, since the mechanism of substance excretion is not yet developed in cancer tissue, molecules leaking from the blood vessel stay in the cancer tissue to some extent. This phenomenon is known as the EPR effect.

  In WO 2012 / 17685A, a branched amphiphilic block polymer having a branched hydrophilic block containing sarcosine and a hydrophobic block containing polylactic acid is self-assembled in an aqueous solution, Form polymeric micelles (lactosomes) of 10-50 nm.

  WO2012 / 026316 (US Patent Application Publication US2013 / 0149252) and WO2012 / 118136 disclose a switching-type fluorescent nanoparticle probe comprising a lactosome nanoparticle encapsulating a fluorescent dye and the same. A fluorescent molecular imaging method using the above is disclosed.

  International Publication No. 2012/128326 discloses a near red that is composed of a linear amphiphilic block polymer in which a hydrophobic block is a polylactic acid chain and a hydrophilic block is a polysarcosine chain, and causes cytotoxicity by laser irradiation. The use of nanoparticles (lactosomes) containing an outer fluorescent dye for photodynamic therapy is disclosed.

JP 2003-57242 A JP 2005-315772 A JP 2006-304721 A JP 2013-21932 A JP 2013-32358 A JP 2013-515099 A JP 2008-24816 A US Patent Application Publication US2008 / 0019908 WO2009 / 148121 US Patent Application Publication US2011 / 0104056 WO2012 / 176885 WO2012 / 026316 US Patent Application Publication US2013 / 0149252 WO2012 / 118136 WO2012 / 128326

Chemistry Letters, 2007, Vol. 36, No. 10, p. 1220-1221 Biomaterials, 2009, volume 30, p. 5156-5160

  As described above, the current situation is that a diagnostic method for rheumatoid arthritis has not yet been established. In particular, it is more difficult to diagnose early rheumatoid arthritis. Therefore, an accurate diagnosis method for rheumatoid arthritis is required. In particular, there is a need for an accurate diagnostic method that can detect early rheumatoid arthritis.

  An object of the present invention is to provide a diagnostic agent for rheumatoid arthritis, which is highly safe for a living body and can detect the position of an inflammatory lesion by molecular imaging.

  As a result of intensive studies, the present inventors have found that the lactosome nanoparticles encapsulating the labeling agent accumulate at the inflammatory site of rheumatoid arthritis and can detect the location of the inflammatory lesion of rheumatoid arthritis by molecular imaging. It has been found that it can be used as a diagnostic reagent for rheumatoid arthritis.

The present invention includes the following inventions.
(1) A diagnostic agent for rheumatoid arthritis, comprising an amphiphilic block polymer A1 having a hydrophilic block having a sarcosine unit and a hydrophobic block having a lactic acid unit, and nanoparticles containing a labeling agent.

  The “nanoparticles” are basically “molecular assemblies” formed by aggregation of amphiphilic block polymer molecules or self-assembled orientational association. A molecular assembly including the amphiphilic block polymer A1 including a hydrophobic block chain having a lactic acid unit as a basic unit may be referred to as “lactosome”. “Sarcosine” is N-methylglycine.

  The nanoparticles have a molecular assembly (lactosome) as a carrier agent and a labeling agent. As an aspect having a carrier agent, there are the following (5) and (8). In this specification, for the sake of convenience, in any of the following aspects (5) and (8), the labeling agent may be expressed as “encapsulated” in the molecular assembly.

  The “hydrophilicity” of the hydrophilic block chain of the amphiphilic block polymer A1 refers to a property that the hydrophilic block chain is relatively hydrophilic with respect to the hydrophobic block chain having a lactic acid unit. “Hydrophobic” possessed by a hydrophobic block chain refers to a property that the hydrophobic block chain is relatively more hydrophobic than a hydrophilic block chain having a sarcosine unit.

  The labeling agent has a property that enables imaging by detection. Examples of the labeling agent include a substance having a fluorescent group (fluorescent dye), a substance having a radioactive element-containing group, and a substance having a magnetic group.

(2) The rheumatoid arthritis diagnostic agent according to (1), wherein the hydrophilic block contains 2 to 300 sarcosine units. In this specification, the number of structural units, that is, the degree of polymerization is an average degree of polymerization.

(3) The diagnostic agent for rheumatoid arthritis according to (1) or (2) above, wherein the number of lactic acid units contained in the hydrophobic block is 5 to 400.

(4) The diagnostic agent for rheumatoid arthritis according to any one of (1) to (3), wherein the labeling agent is fluorescent dye B.

(5) The fluorescent dye B is a fluorescently labeled polymer B1 having at least five lactic acid units and a fluorescent group, and forms nanoparticles by self-assembling together with the amphiphilic block polymer A1. The rheumatoid arthritis diagnostic agent according to (4) above.

(6) The fluorescent group in the fluorescently labeled polymer B1 has the following formula (I):
Wherein R ₁ is an optionally substituted hydrocarbon group, R ₂ is an optionally substituted divalent hydrocarbon group; R ₃ and R ₃ ′ are hydrogen. Or they are linked together to form a cyclic structure; X is hydrogen or halogen; A ⁻ is an anion, m is 0 or 1; and ring B and ring D are each It is a nitrogen-containing fused aromatic heterocycle which may be the same or different.) The diagnostic agent for rheumatoid arthritis according to (5) above.

(7) The diagnostic agent for rheumatoid arthritis according to (5) or (6) above, wherein the fluorescent group in the fluorescently labeled polymer B1 is a group derived from indocyanine green.

(8) The diagnostic agent for rheumatoid arthritis according to (4), wherein the fluorescent dye B is a fluorescent molecule B2 encapsulated in nanoparticles formed by self-assembly of the amphiphilic block polymer A1.

(9) The near-infrared fluorescent molecule B2 is represented by the following formula (I ′):
(Wherein R ₁ and R ₂ ′ are the same or different, optionally substituted hydrocarbon groups; R ₃ and R ₃ ′ are hydrogen or they are Are linked to each other to form a cyclic structure; X is a halogen; A ⁻ is an anion, m is 0 or 1, and ring B and ring D may be the same or different. The diagnostic agent for rheumatoid arthritis according to (8) above, which is a cyanine compound represented by the following formula: a nitrogen-fused aromatic heterocycle.

(10) The diagnostic agent for rheumatoid arthritis according to (8) or (9), wherein the fluorescent molecule B2 is an indocyanine green molecule.

(11) The diagnostic agent for rheumatoid arthritis according to any one of (1) to (10), wherein the nanoparticles further include a hydrophobic polymer A2 having at least 10 or more lactic acid units.

(12) The diagnostic agent for rheumatoid arthritis according to any one of (1) to (11), wherein the nanoparticles have a particle diameter of 10 to 200 nm. “Particle size” means the particle size that appears most frequently in the particle distribution, that is, the center particle size.

A diagnostic agent for inflammatory synovium of rheumatoid arthritis, comprising an amphiphilic block polymer A1 having a hydrophilic block having a sarcosine unit and a hydrophobic block having a lactic acid unit, and nanoparticles containing a labeling agent.

A diagnostic agent in the range of inflammatory synovium of rheumatoid arthritis, comprising an amphiphilic block polymer A1 having a hydrophilic block having a sarcosine unit and a hydrophobic block having a lactic acid unit, and nanoparticles containing a labeling agent.

A diagnostic agent for the resection range of inflammatory synovium of rheumatoid arthritis, comprising an amphiphilic block polymer A1 having a hydrophilic block having a sarcosine unit and a hydrophobic block having a lactic acid unit, and nanoparticles containing a labeling agent.

  According to the present invention, there is provided a diagnostic agent for rheumatoid arthritis that can detect the position of an inflammatory lesion by molecular imaging based on an encapsulated labeling agent. That is, in the present invention, the nanoparticles are specifically accumulated at the inflammatory site of rheumatoid arthritis due to the EPR effect. Therefore, by detecting the accumulated nanoparticles with a molecular merging device, accurate diagnosis of rheumatoid arthritis, in particular, sliding is performed. An accurate diagnosis of the area of membrane inflammation can be made. This is a great advantage over immunochemical diagnostic procedures.

In Experimental example 2, it is the fluorescence detection image in 3, 6 hours immediately after lactosome administration. The arthritis group is shown at the top and the control group is shown at the bottom. In Experimental example 2, it is a fluorescence detection image in lactosome administration 12, 24, 48 hours. The arthritis group is shown at the top and the control group is shown at the bottom. In Experimental example 2, the result of simultaneous photographing of a real photograph and fluorescence imaging after 24 hours of lactosome administration is shown for both the arthritis group and the control group. FIG. 3A is an actual photograph, and the lower photograph is an enlargement of the ankle joint. FIG. 3B is an image of fluorescence imaging. In Experimental example 2, the fluorescence intensity ratio with respect to the elapsed time from lactosome administration is shown about each group of an arthritis group and a control group. In Experimental example 2, the fluorescence intensity ratio with respect to the elapsed time from lactosome administration is shown about an arthritis group. In Experimental Example 2, the fluorescence intensity in each tissue is shown. FIG. 6A is an actual photograph obtained by excising the skin of the ankle joint, and FIG. 6B is an image of fluorescence imaging corresponding to FIG. FIG. 6C is an actual photograph in which the muscles of the ankle joint are removed after skin resection, and FIG. 6D is an image of fluorescence imaging corresponding to (c). In Experimental Example 2, the fluorescence intensity in each tissue is shown. FIG. 7A is an actual photograph in which the joint capsule and synovium of the ankle joint are excised after muscle resection, FIG. 7B is a partial enlargement of FIG. 7A, and FIG. ) Is an image of fluorescence imaging corresponding to.

  The diagnostic agent for rheumatoid arthritis of the present invention contains an amphiphilic block polymer A1 having a hydrophilic block having a sarcosine unit and a hydrophobic block having a lactic acid unit, and nanoparticles containing a labeling agent. The nanoparticles are molecular assemblies (lactosomes) formed by aggregation of amphiphilic block polymer molecules or by self-assembled orientational association, and the labeling agent is included in the molecular assembly. This will be described below.

[1. Amphiphilic block polymer A1]
The amphiphilic block polymer A1 has a hydrophilic block having a sarcosine unit and a hydrophobic block having a lactic acid unit.

[1-1. Hydrophilic block]
In the present invention, the specific level of the physical property “hydrophilicity” possessed by the hydrophilic block chain is not particularly limited, but at least the entire hydrophilic block chain is hydrophobic with a lactic acid unit described later. A property that is relatively hydrophilic with respect to the functional block chain. Alternatively, it means hydrophilicity to such an extent that an amphiphilic property can be realized as a whole copolymer molecule by forming a copolymer with a hydrophobic block chain. In addition, the hydrophilicity is such that the amphiphilic block polymer can self-assemble in a solvent to form a self-assembly, particularly a particulate self-assembly.

  In the present invention, the amphiphilic block polymer may have a linear structure or a branched structure in the hydrophilic block. In the case of the branched structure, each branch of the hydrophilic block contains sarcosine.

  In the hydrophilic block, the types and ratios of the structural units are appropriately determined by those skilled in the art so that the entire block is hydrophilic as described above. For example, the total number of sarcosine units contained in the hydrophilic block is 2 to 300. Specifically, in the case of a linear type, the total of sarcosine units may be, for example, about 10 to 300, 20 to 200, or about 20 to 100. When the number of structural units exceeds the above range, when the molecular assembly is formed, the formed molecular assembly tends to lack stability. Below the above range, an amphiphilic block polymer is not formed, or formation of a molecular assembly tends to be difficult.

  In the case of the branched type, the total number of sarcosine units contained in all branches may be, for example, 2 to 200, 2 to 100, or 2 to 10. Or the sum total of the sarcosine unit contained in all the some hydrophilic blocks may be 30-200 or 50-100, for example. The average number of sarcosine units per branch can be, for example, 1-60, 1-30, 1-10, or 1-6. That is, each hydrophilic block can be configured to include a sarcosine or polysarcosine chain. When the number of structural units exceeds the above range, when the molecular assembly is formed, the formed molecular assembly tends to lack stability. Below the above range, an amphiphilic block polymer is not formed, or formation of a molecular assembly tends to be difficult.

  In the case of the branched type, the number of branches in the hydrophilic block may be 2 or more, but is preferably 3 or more from the viewpoint of efficiently obtaining particle-shaped micelles when forming a molecular assembly. The upper limit of the number of branches in the hydrophilic block is not particularly limited, but is 27, for example. In particular, in the present invention, in the case of a branched type, the number of branches of the hydrophilic block is preferably 3. The branch structure can be appropriately designed by those skilled in the art.

Sarcosine (i.e. N- methyl glycine) has high water-solubility, also polymers of sarcosine cis than ordinary amide group because of their N-substituted amide - are possible trans isomerization, addition, C ^alpha carbon around Since there is little steric hindrance, it has high flexibility. The use of such a structure as a building block is very useful in that the block has basic characteristics with high hydrophilicity or basic characteristics having both high hydrophilicity and high flexibility.

  Furthermore, it is preferable that the hydrophilic block has a hydrophilic group (for example, represented by a hydroxyl group) at the terminal (that is, the terminal opposite to the linker part).

  In the polysarcosine chain, all sarcosine units may be continuous or non-consecutive, but the molecular design was made so as not to impair the basic characteristics of the polypeptide chain as a whole. It is preferable.

  When the hydrophilic block chain has other structural units other than the sarcosine unit, such a structural unit is not particularly limited, but can be an amino acid (including hydrophilic amino acids and other amino acids). In the present specification, “amino acid” is used in a concept including natural amino acids, unnatural amino acids, and derivatives obtained by modification and / or chemical change thereof. Furthermore, in the present specification, amino acids include α-, β-, and γ-amino acids. Preferably, it is α amino acid. For example, serine, threonine, lysine, aspartic acid, glutamic acid and the like can be mentioned.

  The amphiphilic block polymer A1 may have further groups such as sugar chains and polyethers. In this case, the molecular design is preferably such that the hydrophilic block of the amphiphilic block polymer A1 has a sugar chain or a polyether.

[1-2. Hydrophobic block]
In the present invention, the specific degree of the physical property of “hydrophobic” possessed by the hydrophobic block is not particularly limited, but at least the hydrophobic block is relative to the entire hydrophilic block. In other words, it is a region having a strong hydrophobicity, and it is sufficient if the copolymer molecule is formed so as to be amphiphilic as a whole by forming a copolymer with the hydrophilic block. Alternatively, it is sufficient that the amphiphilic block polymer is hydrophobic enough to allow self-assembly in a solvent to form a self-assembly, preferably a particulate self-assembly.

  The hydrophobic block present in one amphiphilic block polymer may not be branched or may be branched. However, when the hydrophobic block is not branched, the density of the hydrophilic branched shell portion increases with respect to the hydrophobic core portion, so that a stable core / shell molecular assembly having a smaller particle size is formed. It is thought that it is easy to do.

  In the present invention, the hydrophobic block contains a lactic acid unit. The types and ratios of the structural units in the hydrophobic block are appropriately determined by those skilled in the art so that the entire block is hydrophobic as described above. For example, the number of lactic acid units contained in the hydrophobic block is 5 to 400. Specifically, for example, when the hydrophobic block is not branched, the number of lactic acid units may be, for example, 5 to 100, 15 to 60, or 25 to 45. When the hydrophobic block is branched, the total number of lactic acid units contained in all branches may be, for example, 10 to 400, preferably 20 to 200. In this case, the average number of lactic acid units per branch is, for example, 5 to 100, preferably 10 to 100.

  When the number of structural units exceeds the above range, when a molecular assembly is formed, the formed molecular assembly tends to lack stability. If the number of structural units is less than the above range, formation of the molecular assembly tends to be difficult.

  When the hydrophobic block is branched, the number of branches is not particularly limited, but from the viewpoint of efficiently obtaining particle-shaped micelles when forming a molecular assembly, for example, the number of branches in the hydrophilic block may be less than or equal to it can.

Polylactic acid has the following basic characteristics.
Polylactic acid has excellent biocompatibility and stability. For this reason, a molecular assembly obtained from an amphiphilic substance having polylactic acid as a building block is very useful in terms of applicability to a living body, particularly a human body.
In addition, polylactic acid has an excellent biodegradability, so it is rapidly metabolized and has low accumulation in tissues other than cancer tissues in vivo. For this reason, a molecular assembly obtained from an amphiphilic substance having polylactic acid as a building block is very useful in terms of specific accumulation in cancer tissue.
Since polylactic acid is excellent in solubility in a low-boiling solvent, when a molecular assembly is obtained from an amphiphilic substance having such a polylactic acid as a building block, a harmful high-boiling solvent is used. It is possible to avoid use. For this reason, such a molecular assembly is very useful in terms of safety to living bodies.

  In the polylactic acid chain (PLA) constituting the hydrophobic block, all lactic acid units may be continuous or discontinuous. However, the hydrophobic block as a whole has the above basic characteristics. It is preferable that the molecule is designed so as not to be damaged.

  The polylactic acid chain (PLA) constituting the hydrophobic block is a poly L-lactic acid chain (PLLA) composed of L-lactic acid units or a poly D-lactic acid chain composed of D-lactic acid units ( PDLA). Moreover, you may be comprised from both the L-lactic acid unit and the D-lactic acid unit. In this case, the L-lactic acid unit and the D-lactic acid unit may be any of an alternating arrangement, a block arrangement, or a random arrangement.

  When the hydrophobic block chain has other structural units other than the lactic acid unit, the types and ratios of such structural units are not particularly limited as long as the entire block chain is hydrophobic as described above. However, it is preferably a molecule designed so as to have desired properties.

  When the hydrophobic block chain has structural units other than lactic acid units, such structural units can be selected from the group consisting of hydroxy acids other than lactic acid and amino acids (including hydrophobic amino acids and other amino acids). . The hydroxy acid is not particularly limited, and examples thereof include glycolic acid and hydroxyisobutyric acid. Many of the hydrophobic amino acids have an aliphatic side chain, an aromatic side chain, and the like. Natural amino acids include glycine, alanine, valine, leucine, isoleucine, proline, methionine, tyrosine, and tryptophan. Non-natural amino acids include, but are not limited to, amino acid derivatives such as glutamic acid methyl ester, glutamic acid benzyl ester, aspartic acid methyl ester, aspartic acid ethyl ester, and aspartic acid benzyl ester.

[1-3. Synthesis of amphiphilic block polymer A1]
In the present invention, the method for synthesizing the amphiphilic block polymer A1 is not particularly limited, and a known peptide synthesis method, polyester synthesis method, and / or depsipeptide synthesis method can be used.

  Peptide synthesis can be performed, for example, by ring-opening polymerization of N-carboxyamino acid anhydride (amino acid NCA) using a base such as amine as an initiator.

  Polyester synthesis can be performed, for example, by ring-opening polymerization of lactide using a base such as an amine or a metal complex as an initiator. The lactide can be appropriately determined by those skilled in the art in consideration of the desired optical purity of the block chain. For example, L-lactide, D-lactide, DL-lactide, and meso-lactide are appropriately selected, and a person skilled in the art can appropriately determine the amount to be used according to the desired optical purity of the block chain.

  Depsipeptide synthesis includes, for example, a method in which polylactic acid is first synthesized as a hydrophobic block, and then a polypeptide chain that becomes a hydrophilic block is stretched, and a polypeptide chain that becomes a hydrophilic block is synthesized first, And a method of stretching polylactic acid to be a hydrophobic block.

  In the molecular assembly, the chain length of polylactic acid can be adjusted. In the synthesis of the amphiphilic block polymer A1, from the viewpoint of more freely controlling the chain length of the polylactic acid, the polylactic acid, which is a hydrophobic block, is first synthesized, and then the polylactic acid that becomes the hydrophilic block chain is synthesized. It is preferable to carry out a method of extending the peptide chain. In addition, polylactic acid as a hydrophobic block chain in the amphiphilic block polymer A1 can control the degree of polymerization more easily and accurately than polysarcosine as a hydrophilic block chain.

  For the structure and synthesis of the amphiphilic block polymer A1, reference can be made to WO2009 / 148121 (linear type) and WO2012 / 17685 (branched type).

[2. Labeling agent]
The labeling agent only needs to have a property that enables imaging by detection. Examples of the labeling agent include a substance having a fluorescent group (fluorescent dye), a substance having a radioactive element-containing group, and a substance having a magnetic group. Means for detecting these labeling agents are appropriately selected by those skilled in the art. The labeling agent is encapsulated in lactosome nanoparticles as a carrier agent.

[2-1. Labeling group]
The fluorescent group is not particularly limited, and examples thereof include groups derived from cyanine dyes such as fluorescein dyes and indocyanine dyes, rhodamine dyes, and quantum dots. In the present invention, a near-infrared fluorescent group (for example, a group derived from a cyanine dye or quantum dot) may be used.

  In the near-infrared region (700 to 1300 nm), although absorption of each substituent having a hydrogen bond exists, the absorption is relatively small. For this reason, near-infrared light has a characteristic which is easy to permeate | transmit a biological tissue. By utilizing such characteristics of near-infrared light, it is possible to accurately obtain information on the site where the lactosome nanoparticles are accumulated without imposing unnecessary load on the body.

  More specific examples of near-infrared fluorescent groups include groups derived from indocyanine dyes such as ICG (Indocyanine Green), Cy7, DY776, DY750, Alexa790, Alexa750, and the like.

Although it does not specifically limit as a radioactive element containing group, The group derived from sugar, an amino acid, a nucleic acid, etc. labeled with radioactive isotopes, such as ¹⁸ F, is mentioned. One specific example of a method for introducing a radioactive element-containing group is a step of polymerizing lactide using mono-Fmoc (9-fluorenylmethyloxycarbonyl) ethylenediamine, a step of protecting terminal OH with a silyl protecting group, and Fmoc by piperidine treatment. The step of elimination, the step of polymerizing sarcosine-N-carboxyanhydride (SarNCA), the termination of the polymer end, the silyl protecting group is removed, and the sulfonic acid ester (for example, trifluoromethanesulfonic acid ester, paratoluenesulfonic acid) And a method of converting into an ester, etc.) and a step of introducing a radioactive element-containing group. Furthermore, the specific example may be appropriately changed by those skilled in the art.

  The magnetic group is not particularly limited, and examples thereof include those having a magnetic material such as ferrichrome, those found in ferrite nanoparticles, nanomagnetic particles, and the like.

[2-2. Fluorescent dye B]
In the present invention, the labeling agent is preferably a fluorescent dye. The fluorescent dye B will be further described.

  The fluorescent dye B is an element included in the carrier agent. Specifically, it is selected from those having a form in which a near-infrared fluorescent group is bonded to a polymer (near-infrared fluorescent labeling polymer B1) and the near-infrared fluorescent molecule itself (near-infrared fluorescent molecule B2).

[2-2-1. Near-infrared fluorescently labeled polymer B1]
Near-infrared fluorescent labeling polymer B1 has at least the following labeling group and polymer part. The near-infrared fluorescently labeled polymer B1 is an element that can form a molecular assembly by self-assembly with the amphiphilic block polymer A1. Near-infrared fluorescent labeling polymer B1 can be used combining the following 1 type or multiple types.

[2-2-1. Labeling group]
The labeling group is a part that imparts the above-described properties to the near-infrared fluorescent dye B, and specifically, a group derived from a near-infrared fluorescent molecule (near-infrared fluorescent group). For example, groups derived from cyanine-based fluorescent molecules or quantum dots are used. An example of a group derived from a cyanine dye is represented by the following general formula (I).

In the above formula (I), R ₁ is an optionally substituted hydrocarbon group, and R ₂ is an optionally substituted divalent hydrocarbon group.
The hydrocarbon group in R ₁ may be an alkyl group having 1 to 20 carbon atoms, preferably 2 to 5 carbon atoms.
The hydrocarbon group in R ₂ may be an alkylene group having 1 to 20 carbon atoms, preferably 2 to 5 carbon atoms.
The substituents in R ₁ and R ₂ are substituents that may be anionic and can be carboxyl groups, carboxylate groups, metal carboxylate groups, sulfonyl groups, sulfonate groups, metal sulfonate groups, or hydroxyl groups. The metal may be an alkali metal or an alkaline earth metal.

R ₃ and R ₃ ′ are hydrogen or they are connected to each other to form a cyclic structure. R ₃ and R ₃ ′ are linked to each other to form a cyclic structure, thereby allowing the molecular structure of the fluorescent dye to be rigid.

X is hydrogen or halogen. The halogen can be Cl, Br, or I. A ⁻ is an anion, and m is 0 or 1. When m is 0, either _one of R ₁ and R ₂ is an anionic group and takes a betaine structure as a whole molecule. When m is 1, A ⁻ may be a halogen ion such as Cl ⁻ , Br ⁻ or I ⁻ , CIO ₄ ⁻ , BF ₄ ⁻ , PF ₆ ⁻ , SbF ₆ ⁻ , SCN ⁻ or the like.

Ring B and ring D are nitrogen-containing fused aromatic heterocycles which may be the same or different. For example, it may be a nitrogen-containing bicyclic or tricyclic aromatic heterocycle. Preferably, ring B and ring D are the same.
As a preferable embodiment of ring B, the following structures are exemplified.

  As a preferable embodiment of the ring D, the following structures are exemplified.

In the above formula, R ₄ and R ₅ can both be hydrogen. Alternatively, R ₄ and R ₅ can be linked together to form an aryl ring. The aryl ring may be a benzene ring that may be substituted.

  A preferable fluorescent group in the present invention is a group derived from an indocyanine compound represented by the following structural formula (II).

  Specific examples of the fluorescent group in the present invention include ICG group (III), IC7-1 group (IV) and IR820 group (V), wherein ring B and ring D are all nitrogen-containing tricyclic aromatic heterocycles, ring IR783 group (VI) and IR806 group (VII) in which both B and ring D are nitrogen-containing bicyclic aromatic heterocycles, and ring B is a nitrogen-containing tricyclic aromatic heterocycle, and ring C is nitrogen-containing IC7-2 group (VIII) which is a bicyclic aromatic heterocyclic ring is mentioned. The structural formulas of these groups are shown below.

  The ICG group represented by the above formula (III) is excited when irradiated with a near-infrared laser near 810 nm, releases active oxygen and free radicals when returning to a steady state, and further has a cytotoxicity. Produce things.

[2-2-1-2. Polymer part]
The polymer part has 5 or more lactic acid units. For example, it may be one having a lactic acid unit as a main constituent (ie, polylactic acid group), or having a lactic acid unit as a hydrophobic block and further having a hydrophilic block (ie, an amphiphilic block polymer group). It may be. The molecular assembly in the present invention preferably has various properties such that it is excellent in biocompatibility, stability and biodegradability, and the constituent polymer is excellent in solubility in a low boiling point solvent. Therefore, it is preferable that the structure of the polymer part is molecularly designed so as to have these excellent characteristics.

  The polylactic acid group is mainly composed of, for example, 5 to 50, 10 to 50, preferably 15 to 35 lactic acid units. All of the lactic acid units may be continuous or discontinuous. Within the above range, the molecular design is such that the total length of the labeled polymer B1 does not exceed the length of the above-mentioned amphiphilic block polymer A1. Preferably, the molecular design is such that it does not exceed twice the length of the hydrophobic block in the amphiphilic block polymer A1.

  In addition, the structural unit and chain length of the polymer portion of the labeled polymer B1 can be basically determined from the same viewpoint as in the molecular design of the hydrophobic block chain in the above-described amphiphilic block polymer A1. By doing in this way, in a molecular assembly, the effect that it is excellent in affinity with labeled polymer B1 and the hydrophobic block chain of amphiphilic block polymer A1 is also acquired.

  In the labeled polymer B1, the near-infrared fluorescent group can be bonded to the terminal constituent unit of the polymer portion. Further, the labeled polymer B1 can have any component other than the near-infrared fluorescent group and the polymer group that are chemically or biochemically acceptable in terms of molecular design. In this case, the other constituents are included to the extent that the labeled polymer B1 does not exert an influence exceeding the category of “hydrophobicity” defined above as a whole.

  Below, the specific example of labeled polymer B1 in this aspect is shown. In the following formula, n is an integer of 5 to 50. The following compounds have ICG groups as near-infrared fluorescent groups.

  When the labeled polymer B1 is a labeled amphiphilic block polymer, the specific levels of the “hydrophobic” and “hydrophilic” physical properties are the same as those in the molecular design of the above-described amphiphilic block polymer A1. In this case, the structural unit and chain length of the polymer part (namely, amphiphilic block polymer part) of the labeled polymer B1 are basically the same as those in the molecular design of the above-mentioned amphiphilic block polymer A1. Can be determined. By doing in this way, in a molecular assembly, the effect that it is excellent in affinity with labeled polymer B1 and the hydrophobic block chain of amphiphilic block polymer A1 is also acquired.

[2-2-2. Near-infrared fluorescent molecule B2]
The near-infrared fluorescent molecule B2 is an element that can be included in a molecular assembly having the amphiphilic block polymer A1 as a basic element. Near-infrared fluorescent molecule B2 can be used combining the following 1 type or multiple types.

  For example, cyanine fluorescent molecules or quantum dots are used as the near-infrared fluorescent molecule B2. An example of the cyanine fluorescent molecule is represented by the following general formula (I ′).

The structure represented by the above formula (I ′) is the same as the above formula (I) except that the divalent R ₂ in the structure represented by the above formula (I) is replaced with a monovalent R ₂ ′.
In the above formula (I ′), R ₁ and R ₂ ′ may be the same or different and each may be a substituted hydrocarbon group.
The hydrocarbon group in R ₁ and R ₂ ′ may be an alkyl group having 1 to 20 carbon atoms, preferably 2 to 5 carbon atoms. The substituents in R ₁ and R ₂ ′ are substituents that may be anionic and may be carboxyl groups, carboxylate groups, metal carboxylate groups, sulfonyl groups, sulfonate groups, metal sulfonate groups, or hydroxyl groups. . The metal may be an alkali metal or an alkaline earth metal.

R ₃ and R ₃ ′ are hydrogen or they are connected to each other to form a cyclic structure. R ₃ and R ₃ ′ are linked to each other to form a cyclic structure, thereby allowing the molecular structure of the fluorescent dye to be rigid.

X is hydrogen or halogen. The halogen can be Cl, Br, or I. A ⁻ is an anion, and m is 0 or 1. When m is 0, either _one of R ₁ and R ₂ is an anionic group and takes a betaine structure as a whole molecule. When m is 1, A ⁻ may be a halogen ion such as Cl ⁻ , Br ⁻ or I ⁻ , CIO ₄ ⁻ , BF ₄ ⁻ , PF ₆ ⁻ , SbF ₆ ⁻ , SCN ⁻ or the like.

Ring B and ring D are nitrogen-containing fused aromatic heterocycles which may be the same or different. For example, it may be a nitrogen-containing bicyclic or tricyclic aromatic heterocycle. Preferably, ring B and ring D are the same.
As a preferable embodiment of ring B, the following structures are exemplified.

  As a preferable embodiment of the ring D, the following structures are exemplified.

In the above formula, R ₄ and R ₅ can both be hydrogen. Alternatively, R ₄ and R ₅ can be linked together to form an aryl ring. The aryl ring may be a benzene ring that may be substituted.

  A more preferred fluorescent dye in the present invention is an indocyanine compound represented by the following structural formula (II ').

  As specific examples of the fluorescent dye in the present invention, ICG (III ′), IC7-1 (IV ′) and IR820 (V ′), a ring in which both ring B and ring D are nitrogen-containing tricyclic aromatic heterocycles, ring IR783 (VI ′) and IR806 (VII ′), in which both B and ring D are nitrogen-containing bicyclic aromatic heterocycles, and ring B is a nitrogen-containing tricyclic aromatic heterocycle, and ring C is nitrogen-containing A structural formula of IC7-2 (VIII ′) which is a bicyclic aromatic heterocyclic ring is shown below.

  The ICG (indocyanine green) molecule represented by the above formula (III ′) is excited when irradiated with a near-infrared laser near 810 nm, and releases active oxygen and free radicals when returning to a steady state. Furthermore, a degradation product having cytotoxicity is produced.

[2-2-3. Concentration of near-infrared fluorescent dye B]
The concentration of the near-infrared fluorescent dye B may be, for example, 0.1 to 50 mol% or 0.5 to 20 mol% with respect to the total of the polymer constituting the nanoparticles and the near-infrared fluorescent dye B. Moreover, 1 mol%, 5 mol%, or 10 mol% may be sufficient as the minimum of the said range. The upper limit of the above range may be 10 mol%, 5 mol% or 1 mol% on condition that it is larger than the lower limit. In addition, the concentration of other labeling agents may be appropriately selected.

[3. Hydrophobic polymer A2]
In the present invention, the nanoparticles may further contain a hydrophobic polymer A2. The hydrophobic polymer A2 is a hydrophobic polymer having at least 10 or more lactic acid units, and preferably has at least 15 or more lactic acid units. Here, the specific degree of the physical property “hydrophobic” of the hydrophobic polymer A2 is not particularly limited, but at least relative to the hydrophilic block of the amphiphilic polymer A1. Is highly hydrophobic.

In the hydrophobic polymer A2, 10 or more lactic acid units are preferably the main constituent components. However, on the other hand, it is allowed to have other structural units other than lactic acid units. All of the lactic acid units may be continuous or discontinuous.
The structural unit and chain length of the hydrophobic polymer A2 are basically the same as those in the molecular design of the hydrophobic block chain in the above-mentioned amphiphilic block polymer A1 and the polymer part in the near-infrared fluorescently labeled polymer B1. Can be determined. By doing in this way, in a molecular assembly, the effect that it is excellent in affinity with the polymer part in hydrophobic polymer A2 and the hydrophobic block chain of amphiphilic block polymer A1, and / or near-infrared fluorescent labeling polymer B1 Can also be obtained.

  The upper limit of the number of structural units of the hydrophobic polymer A2 is not particularly limited as long as it does not exceed the length of the above-described amphiphilic block polymer A1, but preferably, the hydrophobic block of the amphiphilic block polymer A1 It shall not exceed twice the length. Therefore, the upper limit of the number of structural units of the hydrophobic polymer A2 can be about 200 when the hydrophobic block is not branched, for example. In the present invention, a hydrophobic polymer A2 having a number of structural units of about 10 to 160, preferably about 20 to 100 is often synthesized. When the number of structural units exceeds about 200, when a molecular assembly is formed, the formed molecular assembly tends to lack stability. When the number of structural units is less than 10, the effects described later (hydrophobic core volume increasing effect and particle diameter controlling effect) due to mixing of the hydrophobic polymer A2 become small.

  The blending amount of the hydrophobic polymer A2 can be, for example, an amount such that the use ratio of the amphiphilic block polymer A1 and the hydrophobic polymer A2 is 10: 1 to 1:10 on a molar basis. When the ratio of the hydrophobic polymer A2 exceeds the above range, the molecular assembly itself tends to be difficult to maintain its shape. Moreover, when the ratio of hydrophobic polymer A2 is less than the said range, it exists in the tendency for the below-mentioned effect (hydrophobic core volume increase effect and particle diameter control effect) by mixing hydrophobic polymer A2 to become difficult to be acquired.

[4. Nanoparticle size]
In the present invention, the size of the nanoparticles is, for example, a particle diameter of 10 to 500 nm, or 10 to 200 nm, preferably 20 to 200 nm. Here, the “particle diameter” means a particle diameter having the highest frequency of appearance in the particle distribution, that is, a central particle diameter. Those having a particle size of less than 10 nm are difficult to prepare, and those having a particle size of more than 500 nm may not be preferable as an injection, particularly when administered by injection into a living body. In the present invention, the vascular permeability is enhanced at the inflamed site of rheumatoid arthritis, and the nanoparticles leak out and stay in the extravascular interstitial space due to the EPR effect. It is 20-200 nm, More preferably, it is 20-100 nm, 20-50 nm. With such a particle size, nanoparticles are likely to accumulate at the synovial inflammation site of rheumatoid arthritis, and the position of the inflammatory lesion of rheumatoid arthritis can be detected by molecular imaging.

  In the present invention, the method for measuring the size of the nanoparticles is not particularly limited, and can be appropriately selected by those skilled in the art. Examples thereof include an observation method using a transmission electron microscope (TEM) and a dynamic light scattering (DLS) method. In the DLS method, the migration diffusion coefficient of particles that are in Brownian motion in a solution is measured.

Examples of means for controlling the size of the nanoparticles include controlling the chain length of the amphiphilic block polymer A1. Preferably, the polymerization degree of the hydrophobic block in the amphiphilic block polymer A1 can be adjusted.
In another example, the polymer part of the near-infrared fluorescent labeling polymer B1 as the near-infrared fluorescent dye B and the polymerization degree of the hydrophobic polymer A2 can be adjusted.
In still another example, the amount of the hydrophobic polymer A2 can be controlled. Hydrophobic polymer A2 has the effect of increasing the hydrophobic core of the molecular assembly. In addition, the size of the molecular assembly can be continuously controlled by adjusting the blending amount. Therefore, this means is preferable in that the fine size of the lactosome can be adjusted.

[5. Formation of nanoparticles]
The method for producing the nanoparticle (lactosome) is not particularly limited, and a person skilled in the art appropriately determines the size and characteristics of the desired nanoparticle, the type, nature, content, etc. of the labeling substance (fluorescent dye B etc.) to be supported. You can choose. If necessary, after forming the nanoparticles as described below, the obtained nanoparticles may be subjected to surface modification by a known method. Note that confirmation of the formation of particles may be performed by observation with an electron microscope.

[5-1. Film method]
Since the amphiphilic block polymer A1 and the hydrophobic polymer A2 in the present invention have solubility in a low-boiling solvent, nanoparticles can be prepared using this method.
The film method includes the following steps. That is, a solution containing a polymer constituting a molecular assembly such as amphiphilic block polymer A1 and hydrophobic polymer A2 and a labeling substance (fluorescent dye B or the like) in an organic solvent is prepared in a container (for example, a glass container). A step of removing the organic solvent from the solution to obtain a film containing a polymer and a labeling substance on the inner wall of the container; and adding water or an aqueous solution to the container, and performing ultrasonic treatment as necessary. Converting the film-like substance into a particulate molecular assembly containing a labeling substance to obtain a nanoparticle dispersion. Furthermore, the film method may include a step of subjecting the nanoparticle dispersion to a freeze-drying treatment.

  For a solution containing a polymer that forms a molecular assembly and a labeling substance (fluorescent dye B, etc.) in an organic solvent, the polymer that forms the molecular assembly is stocked in advance in the form of a film. You may prepare by adding the solution containing substance (fluorescent dye B etc.) and melt | dissolving a film.

As the organic solvent used in the film method, a low boiling point solvent is preferably used. The low boiling point solvent in the present invention means a solvent having a boiling point at 1 atm of 100 ° C. or less, preferably 90 ° C. or less. Specific examples include chloroform, diethyl ether, acetonitrile, 2-propanol, ethanol, acetone, dichloromethane, tetrahydrofuran, hexane, and the like.
By using such a low boiling point solvent for dissolving the polymer constituting the molecular assembly and the near-infrared fluorescent dye B, the removal of the solvent becomes very simple. The method for removing the solvent is not particularly limited, and may be appropriately determined by those skilled in the art according to the boiling point of the organic solvent to be used. For example, the solvent may be removed under reduced pressure, or the solvent may be removed by natural drying.

  After the organic solvent is removed, a film containing a polymer constituting the molecular assembly and a labeling substance (such as fluorescent dye B) is formed on the inner wall of the container. Water or an aqueous solution is added to the container to which the film is attached. The water or aqueous solution is not particularly limited, and those skilled in the art may appropriately select a biochemically and pharmaceutically acceptable one. Examples thereof include distilled water for injection, physiological saline, and buffer solution.

  After water or an aqueous solution is added, a heating treatment is performed. A molecular assembly is formed in the process of peeling the film from the inner wall of the container by heating. The heating treatment can be performed, for example, under conditions of 20 to 100 ° C, or 70 to 90 ° C, 1 to 60 minutes, or 5 to 60 minutes. At the end of the heating treatment, a dispersion liquid in which molecular aggregates (nanoparticles) encapsulating a labeling substance (fluorescent dye B or the like) are dispersed in the water or aqueous solution is prepared in a container.

The obtained dispersion can be directly administered to a living body. That is, it does not have to be stored in the state of solventless nanoparticles themselves.
On the other hand, the obtained dispersion may be freeze-dried. A known method can be used as the freeze-drying method without any particular limitation. For example, the nanoparticle dispersion obtained as described above can be frozen with liquid nitrogen and sublimated under reduced pressure. Thereby, a freeze-dried product of nanoparticles can be obtained. That is, the nanoparticles can be stored as a lyophilized product. If necessary, the nanoparticles can be used for use by adding water or an aqueous solution to the lyophilizate to obtain a dispersion of nanoparticles. The water or aqueous solution is not particularly limited, and those skilled in the art may appropriately select a biochemically and pharmaceutically acceptable one. Examples thereof include distilled water for injection, physiological saline, and buffer solution.

  Here, in addition to the nanoparticles formed from the polymer constituting the molecular assembly and the labeling substance (fluorescent dye B, etc.), the dispersion before the lyophilization treatment contributes to the formation of such nanoparticles. Untreated polymers and / or labeling substances (such as fluorescent dye B) can each remain as such. When such a dispersion is subjected to a freeze-drying process, nanoparticles are further formed from the remaining polymer and the labeling substance (fluorescent dye B, etc.) without forming nanoparticles in the course of concentration of the solvent. It becomes possible. Accordingly, it becomes possible to efficiently prepare the nanoparticles.

[5-2. Injection method]
The injection method includes the following steps. In other words, in a container (for example, a test tube), a solution is prepared in which an organic solvent contains a polymer constituting a molecular assembly such as an amphiphilic block polymer A1 and a hydrophobic polymer A2 and a labeling substance (fluorescent dye B, etc.). A step of dispersing the solution in water or an aqueous solution; and a step of removing the organic solvent. Further, in the injection method, a purification treatment step may be appropriately performed before the step of removing the organic solvent.

Examples of the organic solvent used in the injection method include trifluoroethanol, ethanol, 2-propanol, hexafluoroisopropanol, dimethyl sulfoxide, dimethylformamide, and the like.
As water or an aqueous solution, distilled water for injection, physiological saline, buffer solution and the like are used.
As the purification treatment, for example, treatment such as gel filtration chromatography, filtering, and ultracentrifugation can be performed.

  When the molecular assembly obtained in this way is administered into a living body and an organic solvent harmful to the living body is used, it is necessary to strictly remove the organic solvent.

  When preparing a molecular assembly as a vesicle, when preparing an encapsulated type, a substance to be encapsulated is dissolved or suspended in an aqueous solvent such as distilled water for injection, physiological saline, or buffer solution. The solution obtained by dissolving the amphiphilic block polymer A1 and the hydrophobic polymer A2 and / or the functional substance as necessary in the organic solvent is dispersed in the aqueous solution or suspension thus obtained. Good.

[6. Administration of nanoparticles]
As described above, nanoparticles containing the amphiphilic block polymer A1 and the labeling agent are produced. Nanoparticles are used as diagnostic agents for rheumatoid arthritis.

  The living body to which the nanoparticles are administered is not particularly limited, and may be human and non-human animals. Non-human animals are not particularly limited, but mammals other than humans, more specifically, primates, rodents (mouse, rats, etc.), rabbits, dogs, cats, pigs, cows, sheep, horses, etc. Can be mentioned.

The method of administration into the living body is not particularly limited, and can be appropriately determined by those skilled in the art. Therefore, the administration method may be systemic administration or local administration. That is, the molecular probe can be administered by any of injection (needle-type, needle-free), internal use, and external use. The concentration of the nanoparticles in the administration liquid is, for example, 0.5 to 100 mg / ml in terms of the amount in the case of lactosome whose inclusion is ICG-PLLA ₃₀ and the carrier is a molecular assembly of PSar ₇₀ -PLLA ₃₀ , Preferably it may be 0.8-50 mg / ml, more preferably 1-20 mg / ml.

  Vascular permeability is increased at the site of inflammation in rheumatoid arthritis. Due to the EPR (enhanced permeability and retention) effect, the nanoparticles leak out and stay in the extravascular interstitial space. Nanoparticles accumulate at the synovial inflammation site of rheumatoid arthritis. The encapsulated labeling agent (fluorescent dye or the like) is detected by imaging. Thereby, the position of the inflammatory lesion of rheumatoid arthritis can be detected.

  The time from nanoparticle administration to the start of detection can be appropriately determined by those skilled in the art according to the type of labeling agent such as near-infrared fluorescent dye B possessed by the administered nanoparticle and the type of administration target. For example, it can be 3 to 48 hours or 1 to 24 hours after administration. Below the above range, the signal may be too strong and the administration target and other sites (background) may not be clearly separated. Moreover, when it exceeds the said range, a fluorescent probe may be excreted from an administration target.

  For the detection of nanoparticles, a fluorescent imaging device is used when the labeling agent is a fluorescent dye. The fluorescence imaging apparatus is an apparatus for acquiring image data indicating the position of a synovial inflammation site. A fluorescence imaging device detects near-infrared light derived from near-infrared fluorescent dye contained in nanoparticles administered to a subject such as a human body, and observes the position and size of the tissue where the nanoparticles are present. Anything is possible. Therefore, those skilled in the art can appropriately select an apparatus that can visualize near-infrared light derived from a near-infrared fluorescent dye contained in nanoparticles.

  In a preferred embodiment, an apparatus for acquiring image data indicating the position of a synovial inflammation site detects near-infrared fluorescence derived from a near-infrared fluorescent dye contained in a nanoparticle administered to a subject and visualizes it. The image and the surrounding tissue visible light image are extracted simultaneously. Thereby, in a subject such as a human body administered with the nanoparticles of the present invention as a fluorescent contrast agent, a composite image of a visible light image at a synovial inflammation site where nanoparticles are accumulated and an infrared light image from the accumulated nanoparticles Can be obtained. Specifically, an imaging device including a light source, an optical filter, a solid-state imaging device, and a signal processing circuit can be used as follows. As a specific example of the fluorescence imaging apparatus having such means, HyperEye Medical System (manufactured by Mizuho Medical Co., Ltd.) can be cited.

  The light source irradiates the subject with visible light and excitation light of a near-infrared fluorescent dye. Specifically, it may include a visible light emitting unit that emits visible light and an excitation light emitting unit that emits excitation light. The visible light emitting part is, for example, a xenon lamp or a white LED, and the excitation light emitting part is an excitation wavelength of the near-infrared fluorescent dye B included in the nanoparticles (specifically, peaking at 780 nm in the case of ICG). A semiconductor light emitting device such as an LED having a wavelength. Light from the subject is taken into the imaging device via an optical system such as a lens, and enters the solid-state imaging element via an optical filter.

  The optical filter makes the visible light component and the infrared fluorescent component from the subject incident on the solid-state imaging device without being separated. The optical filter independently controls the transparency of visible light, excitation light, and near infrared light. Specifically, visible light and infrared fluorescence are transmitted among light from the subject, and transmission of excitation light is blocked. Further, the optical filter may be capable of individually controlling the emission intensity of each of the visible light emitting unit and the excitation light emitting unit. In such an optical filter, since the transmittance for visible light is set lower than the transmittance for fluorescence, an infrared light image due to weak fluorescence can be clearly displayed on the visible light image. As the optical filter, for example, a thin film formed by laminating metals and dielectrics by a vacuum deposition method is used.

  The solid-state imaging device captures a visible light image and a near-infrared light image based on the transmitted light of the optical filter. Thereby, simultaneous photographing with visible light and near-infrared light becomes possible. Specifically, a mosaic color filter that constitutes a light receiving pixel is arranged on a semiconductor substrate. The color filter is composed of a colored organic material. Depending on the transmission characteristics of each color filter, a light component having sensitivity to each light receiving pixel is determined. In each light receiving pixel, a signal charge corresponding to the visible light component corresponding to the color of the color filter arranged in each pixel of the incident light and a signal charge corresponding to the infrared light component are synthesized and accumulated. . The accumulated signal charge is output to the signal processing circuit.

  The signal processing circuit processes a signal output from the solid-state imaging device. Specifically, an image signal representing a composite image based on a visible light image and a near-infrared light image is generated from a signal read from each light receiving pixel of the image sensor. The generated composite signal is input and displayed on the display device. Specifically, a composite image in which a visible light image of a surgical site and an infrared light image in which the position where the nanoparticles are accumulated is composed of a color tone according to the concentration of the fluorescent dye contained therein is synthesized is monitored. Is displayed.

  In the present invention, since the nanoparticles are specifically accumulated in the inflammatory site of rheumatoid arthritis due to the EPR effect, accurate detection of rheumatoid arthritis, in particular, the range of the synovial inflammatory site is detected by detecting with the above-mentioned fluorescence imaging device. Accurate diagnosis can be performed.

  For labeling agents other than fluorescent dyes, positron emission tomography (PET), single photon emission tomography (SPECT), nuclear magnetic resonance imaging (MRI) Etc. can be used.

  Examples will be shown below to describe the present invention in more detail, but the present invention is not limited to these examples.

[Experimental Example 1: Preparation of ICG lactosome]
Fluorescent dye-encapsulated nanoparticles composed of polysarcosine-polylactic acid amphiphilic block polymer (PSar ₇₀ -PLLA ₃₀ ) and polylactic acid-binding fluorescent compound (ICG-PLLA ₃₀ ) as described below (described as ICG lactosome) ) Was prepared.

(Synthesis of aminated poly-L-lactic acid)
First, aminated poly L-lactic acid (a-PLA) (average polymerization degree 30) was synthesized using L-lactide and N-carbobenzoxy-1,2-diaminoethane hydrochloride (Scheme 1).

  To N-carbobenzoxy-1,2-diaminoethane hydrochloride (310 mg, 1.60 mmol), which is a polymerization initiator, tin octoate (6.91 mg) diffused in toluene (1.0 mL) was added. Toluene was distilled off under reduced pressure, L-lactide (3.45 g, 24 mmol) was added, and a polymerization reaction was performed at 120 ° C. in an Ar atmosphere. After 12 hours, the reaction vessel was air-cooled to room temperature. The obtained yellowish white solid was dissolved in a small amount of chloroform (about 10 mL). Chloroform was added dropwise to cold methanol (100 mL) to obtain a white precipitate. The resulting white precipitate was collected by centrifugation and dried under reduced pressure.

To a solution of the obtained white precipitate (500 mg) in dichloromethane (1 ml) was added 25 v / v% hydrogen bromide / acetic acid (2.0 mL), and the mixture was stirred for 2 hours in the dark and under dry air. After completion of the reaction, the reaction solution was added dropwise to cold methanol (100 mL), and the deposited precipitate was collected by centrifugation. The obtained white precipitate was dissolved in chloroform, washed with a saturated aqueous NaHCO ₃ solution, and dehydrated with anhydrous MgSO ₄ . MgSO ₄ was removed by Celite (R) filtration, followed by vacuum drying to obtain white amorphous powder a-PLA (440 mg).

(Synthesis of polylactic acid-binding fluorescent compound (ICG-PLLA ₃₀ ))
Next, ICG labeling was performed on aminated poly-L-lactic acid (a-PLA) to obtain a polylactic acid-binding fluorescent compound (ICG-PLLA ₃₀ ) (Scheme 2).

A DMF solution containing 1 mg (1.3 eq) of indocyanine green derivative (ICG-sulfo-OSu) was added to a DMF solution containing 1.9 mg (1.0 eq) of a-PLA and stirred at room temperature for about 20 hours. Thereafter, the solvent was distilled off under reduced pressure, and purification was performed with an LH20 column to obtain a compound ICG-PLLA ₃₀ .

(Synthesis of polysarcosine-polylactic acid amphiphilic block polymer (PSar ₇₀ -PLLA ₃₀ ))
A polysarcosine-polylactic acid amphiphilic block polymer (PSar ₇₀ -PLLA ₃₀ ) was synthesized from sarcosine-NCA (Sar-NCA) and aminated poly-L-lactic acid (a-PLA) (Scheme 3).

  Dimethylformamide (DMF) (140 mL) is added to a-PLA (383 mg, 0.17 mmol) and sarcosine-NCA (Sar-NCA) (3.21 g, 27.9 mmol) in an Ar atmosphere, and the mixture is stirred at room temperature for 12 hours. did. After cooling the reaction solution to 0 ° C., glycolic acid (72 mg, 0.95 mmol), O- (benzotriazol-1-yl) -N, N, N ′, N′-tetramethyluronium hexafluorophosphoric acid Salt (HATU) (357 mg, 0.94 mmol) and N, N-diisopropylethylamine (DIEA) (245 μL, 1.4 mmol) were added and reacted at room temperature for 18 hours.

After the DMF was distilled off under reduced pressure using a rotary evaporator, purification was performed using an LH20 column. The fraction in which the peak was detected at UV270 nm was collected and concentrated. The obtained concentrated solution was dropped into diethyl ether at 0 ° C. and reprecipitated to obtain the target product, PSar ₇₀ -PLLA ₃₀ (1.7 g).

(Creation of fluorescent dye-containing nanoparticles)
A chloroform solution (0.2 mM) of polylactic acid-polysarcosine amphiphilic block polymer (PSar ₇₀ -PLLA ₃₀ · 26H ₂ O, MW = 7767) and fluorescent dye ICG-PLLA _{30 as} carrier agents was prepared. For the fluorescent dye ICG-PLLA ₃₀ , a mixed solution of both solutions was prepared in a glass container so that the molar concentration of the fluorescent dye was 20 mol%. Thereafter, lactosomes were prepared according to the film method. The film method was performed as follows. The solvent was distilled off from the mixed solution under reduced pressure to form a film containing a carrier agent and a fluorescent dye on the wall surface of the glass container. Furthermore, water or a buffer solution was added to the glass container in which the film was formed, and after boiling in a water bath at a temperature of 82 ° C. for 20 minutes, the solution was left at room temperature for 30 minutes, filtered through a 0.2 mm filter, and freeze-dried. In this manner, indocyanine green encapsulated nanoparticles (ICG lactosomes) were prepared. When the particle diameter was measured using a dynamic light scattering measurement apparatus (Metavern Instruments, Zetasizer Nano), it was about 30 nm.

[Experimental example 2: Fluorescence imaging using ICG lactosomes in rheumatoid arthritis model mice]
As described below, near-infrared fluorescence imaging using the indocyanine green-encapsulated nanoparticles (ICG lactosomes) in rheumatoid arthritis model mice was performed.

(experimental method)
SKG / Jcl mice (CLEA Japan, Inc.) that spontaneously develop autoimmune arthritis that closely resembled human rheumatoid arthritis immunologically were used. The arthritis (A) group (n = 4) and the control (C) group (n = 4) were divided. Since SKG / Jcl mice are unlikely to spontaneously develop arthritis in the SPF environment, in the arthritis group, mannan 20 mg (Sigma Aldrich M-7504) was intraperitoneally administered at 8 weeks of age and reared for 6 weeks (14 weeks of age). Developed arthritis. At 14 weeks of age, 2.0 mg / mouse of ICG lactosome was administered to both the arthritis group and the control group via the tail vein.

(Arthritis score evaluation)
At 14 weeks of age (before ICG lactosome administration), the arthritis score was evaluated by visual observation of both the arthritis group and the control group. The arthritis score was evaluated for swelling of the fingers, wrist joints, toes, and ankle joints, and the total score of each part was obtained. The arthritis score was 4.73 in the arthritis group and 0 in the control group.

(Pathological evaluation)
Pathological evaluation (safranin O, toluidine blue, HE staining) was performed on both the arthritis group and the control group. In the arthritis group, destruction of articular cartilage (decrease in cartilage proteoglycan and decrease in chondrocytes) and increase in synovium (increase in synovial cells) were observed from the time of 10 weeks of age in comparison with the control group.

  Thus, it was confirmed by the arthritis score evaluation and pathological evaluation that the arthritis group developed autoimmune arthritis.

(Fluorescence imaging)
For both the arthritis group and the control group, immediately after ICG lactosome administration, 3, 6, 12, 24, 48 hours using a fluorescence in vivo imaging device (IVIS Spectrum Xenogen: excitation wavelength: 745 nm, fluorescence wavelength: 840 nm) An image evaluation was performed. FIG. 1 shows images taken immediately after 3, 6 hours, and FIG. 2 shows images taken after 12, 24, 48 hours. 1 and 2, the arthritis group is shown in the upper stage and the control group is shown in the lower stage. In the arthritis group, an increase in fluorescence intensity was observed at the wrist and ankle joints compared to the control group.

  Further, for both the arthritis group and the control group, real photographs and fluorescence imaging were simultaneously taken after 24 hours of lactosome administration. FIG. 3A is an actual photograph, and the lower photograph is an enlargement of the ankle joint. FIG. 3B is an image of fluorescence imaging. From the actual photograph (a), in the arthritis group, swelling of the wrist joint and ankle joint was observed compared to the control group, and in FIG. 3 (b), in the arthritis group, compared with the control group, Increased fluorescence intensity was observed at the wrist and ankle joints.

(Fluorescence intensity ratio)
From the results of the image evaluation, the fluorescence intensity ratio of the region of interest ROI (Region of Interest) was determined as follows.

The maximum width (in the image) of the ankle joint of the control group was about 3.5 mm. Obtain each ROI value [(p / sec) / (μW / cm ² )] before and after fluorescence with a circle of diameter φ: 3.5 mm,
Fluorescence intensity ratio = ROI value after fluorescence / ROI value before fluorescence.

  In FIG. 4, the fluorescence intensity ratio with respect to the elapsed time from lactosome administration is shown about each group of an arthritis group and a control group. Horizontal axis: Elapsed time (hour) since lactosome administration, Vertical axis: fluorescence intensity ratio. At each time, the arthritis (A) group is indicated by the right bar and the control (C) group is indicated by the left bar. There was a significant difference between the arthritis group and the control group by the Mann-Whitney's test. From FIG. 4, in the control group, the fluorescence intensity ratio was small regardless of the elapsed time from the administration of the lactosome. On the other hand, in the arthritis group, the fluorescence intensity ratio increased with the elapsed time from the lactosome administration, and became the largest at 24 hours.

  In FIG. 5, the fluorescence intensity ratio with respect to the elapsed time from lactosome administration is shown about an arthritis group. Horizontal axis: Elapsed time (hour) since lactosome administration, Vertical axis: fluorescence intensity ratio. The same fluorescence intensity ratio as shown in FIG. 4 is shown. There was a significant difference between the 24 hours elapsed from the lactosome administration and the other elapsed times by the Wilcoxon signed rank sum test.

(Fluorescence intensity in each tissue)
In the arthritis group, 24 hours after ICG lactosome administration, the skin, muscle, joint capsule and synovium of the ankle joint were sequentially excised, and the fluorescence intensity was observed.
First, the skin of the ankle joint was excised (FIG. 6 (a)), and the fluorescence intensity was observed (FIG. 6 (b)). That is, it was confirmed that ICG lactosome was not accumulated in the skin.
Next, after the skin was excised, the muscles of the ankle joint were excised (FIG. 6 (c)), and the fluorescence intensity was observed (FIG. 6 (d)), which was the same as when nothing was excised. That is, it was confirmed that ICG lactosome was not accumulated in skin or muscle.
Next, after muscle excision, the joint capsule and synovium of the ankle joint were excised (FIG. 7 (a)), and when the fluorescence intensity was observed (FIG. 7 (c)), almost no fluorescence was observed. That is, it was confirmed that ICG lactosome was accumulated in the joint capsule and synovium.

The above observation results are shown in FIGS. That is, FIG. 6A is an actual photograph obtained by excising the skin of the ankle joint, and FIG. 6B is an image of fluorescence imaging corresponding to FIG.
FIG. 6C is an actual photograph in which the muscles of the ankle joint are removed after skin resection, and FIG. 6D is an image of fluorescence imaging corresponding to (c).
FIG. 7A is an actual photograph in which the joint capsule and synovium of the ankle joint are excised after muscle resection, FIG. 7B is a partial enlargement of FIG. 7A, and FIG. ) Is an image of fluorescence imaging corresponding to.

  From the above results, ICG lactosomes are specifically accumulated at the synovial inflammation site of rheumatoid arthritis and can be detected by fluorescence imaging. Thereby, an accurate diagnosis of rheumatoid arthritis can be performed. It is possible to reduce side effects when taking medication with accurate diagnosis. In particular, an accurate diagnosis of the range of the synovial inflammation site can be performed, which is very effective for specifying the range of synovectomy.

Claims (12)

  A diagnostic agent for rheumatoid arthritis, comprising an amphiphilic block polymer A1 having a hydrophilic block having a sarcosine unit and a hydrophobic block having a lactic acid unit, and nanoparticles containing a labeling agent.   The diagnostic agent for rheumatoid arthritis according to claim 1, wherein the hydrophilic block contains 2 to 300 sarcosine units.   The diagnostic agent for rheumatoid arthritis according to claim 1 or 2, wherein the hydrophobic block contains 5 to 400 lactic acid units.   The diagnostic agent for rheumatoid arthritis according to any one of claims 1 to 3, wherein the labeling agent is a fluorescent dye B.   The fluorescent dye B is a fluorescently labeled polymer B1 having at least 5 lactic acid units and a fluorescent group, and forms nanoparticles by self-assembly with the amphiphilic block polymer A1, The diagnostic agent for rheumatoid arthritis according to claim 4. The fluorescent group in the fluorescently labeled polymer B1 has the following formula (I):
Wherein R ₁ is an optionally substituted hydrocarbon group, R ₂ is an optionally substituted divalent hydrocarbon group; R ₃ and R ₃ ′ are hydrogen. Or they are linked together to form a cyclic structure; X is hydrogen or halogen; A ⁻ is an anion, m is 0 or 1; and ring B and ring D are each 6. The diagnostic agent for rheumatoid arthritis according to claim 5, which is a nitrogen-containing fused aromatic heterocycle which may be the same or different.   The diagnostic agent for rheumatoid arthritis according to claim 5 or 6, wherein the fluorescent group in the fluorescently labeled polymer B1 is a group derived from indocyanine green.   The diagnostic agent for rheumatoid arthritis according to claim 4, wherein the fluorescent dye B is a fluorescent molecule B2 encapsulated in nanoparticles formed by self-assembly of the amphiphilic block polymer A1. The near-infrared fluorescent molecule B2 is represented by the following formula (I ′):
(Wherein R ₁ and R ₂ ′ are the same or different, optionally substituted hydrocarbon groups; R ₃ and R ₃ ′ are hydrogen or they are Are linked to each other to form a cyclic structure; X is a halogen; A ⁻ is an anion, m is 0 or 1, and ring B and ring D may be the same or different. The diagnostic agent for rheumatoid arthritis according to claim 8, which is a cyanine compound represented by a nitrogen-fused aromatic heterocyclic ring.   The diagnostic agent for rheumatoid arthritis according to claim 8 or 9, wherein the fluorescent molecule B2 is an indocyanine green molecule.   The diagnostic agent for rheumatoid arthritis according to any one of claims 1 to 10, wherein the nanoparticles further comprise a hydrophobic polymer A2 having at least 10 or more lactic acid units.   The diagnostic agent for rheumatoid arthritis according to any one of claims 1 to 11, wherein the nanoparticles have a particle diameter of 10 to 200 nm.