JP6398419B2 - Labeling agent containing sorafenib - Google Patents

Description

  The present invention relates to a labeling agent that can be used for measurement of the binding amount of a molecular target drug to a specific biological substance (target molecule) in a tissue, and more particularly, to a labeling agent comprising sorafenib and a label linked thereto.

  Molecular targeting drugs are drugs that treat diseases by elucidating genes and proteins that cause specific diseases and acting on biological substances involved in their abnormal expression and signal transduction at the molecular level. In recent years, some low molecular weight compounds or antibodies have been put to practical use as molecular target drugs for cancer, autoimmune diseases and the like.

  In drug discovery of such molecular target drugs, measuring the amount of drug binding to a specific biological substance (target molecule) is important because it leads to screening of candidate drugs. For this purpose, a label such as a radioisotope, an enzyme that reacts with a specific substrate to develop color, or a fluorophore is linked to a candidate drug, the candidate drug is bound to a target molecule, and bound by a signal derived from the label. A technique for measuring quantities is used.

  For example, Patent Document 1 (International Publication WO2012 / 133347) discloses an immune tissue in which an antibody (trastuzumab or the like) used in an antibody drug is labeled and bound to an antigen (HER2 or the like) targeted by the antibody drug. A staining method is described. Quantum dots and fluorescent substance integrated nanoparticles (organic fluorescent dye integrated silica nanoparticles, etc.) can be used as a label for labeling this antibody, and this immunohistological staining method is an antibody drug. It is also described that the method can be applied to a method for determining the effectiveness of. The use of fluorescent substance-integrated nanoparticles as a label for immunostaining is preferable because quantitative determination can be performed with high accuracy compared to the use of quantum dots or color formers using conventional enzymes.

  On the other hand, low molecular weight compounds instead of antibodies have been used as molecular target drugs for various diseases, and they are used not only as active ingredients of therapeutic drugs but also for assays used to detect target molecules in samples. It is also used when preparing conjugates of For example, Patent Document 2 (Japanese Patent Publication No. 2007-521338) discloses a biological substance such as a binder, a labeling compound (organic fluorescent dye, etc.), and a therapeutic agent (kinase inhibitor, etc.) at the end of a predetermined divalent linker molecule. A compound having a structure in which beneficial components in pharmaceutical or bioanalytical applications are bound is described, and the kinase inhibitor includes a compound having an aromatic ring containing a nitrogen atom (hereinafter referred to as “nitrogen-containing aromatic ring-containing compound”). For example, sorafenib (see compound number VI in the table of paragraph [0070]).

  However, in Patent Document 2, as a site to be bonded to a linker molecule of a compound having an aromatic ring (nitrogen-containing aromatic ring) containing a nitrogen atom, (i) when the nitrogen-containing aromatic ring is located at the end of the compound Selecting the nitrogen atom of the nitrogen-containing aromatic ring or the functional group containing the nitrogen atom directly or indirectly bonded to the nitrogen-containing aromatic ring (amino group, amide group, carbamoyl group, etc.) (Iii) Although an embodiment in which an aromatic ring not containing a heteroatom is located at the terminal of the compound and an aromatic ring carbon atom is selected is disclosed, (iii) When a nitrogen aromatic ring is located, selecting a carbon atom of the nitrogen-containing aromatic ring and its technical significance are not disclosed. For example, for sorafenib, Patent Document 2 only suggests in the general description that a divalent linker molecule is bonded to the nitrogen atom of the carbamoyl group bonded to the nitrogen-containing aromatic ring (pyridyl group) at the end. In addition, in the Examples and the like, the embodiment in which the divalent linker molecule is bound to sorafenib as described above is not specifically disclosed.

  Patent Document 3 (Japanese Patent Publication No. 2012-533579) includes a compound carrying a carboxyl group and a predetermined oligomer composed of an ε-lysine monomer unit as an invention aimed at targeting to the kidney. Furthermore, conjugates in which an active compound may be covalently bound are described, and examples of the active compound include protein kinase inhibitors such as sorafenib. For example, in Example 8 (paragraphs [0181] to [0189]), a conjugate (DOTA-ε-polylysine) containing DOTA, ε-polylysine as the compound carrying the carboxyl group, and a sorafenib derivative as the active compound. -Sorafenib derivatives) are disclosed. In the conjugate obtained by the production method of Example 8, ε-polylysine is a hydroxyethyl group (bonded to the nitrogen atom of the carbamoyl group bonded to the pyridyl group) introduced at the terminal of the sorafenib derivative. Is bound to.

International Publication WO2012 / 133304 Brochure Special table 2007-521338 gazette Special table 2012-533579 gazette

  Many molecular targeting drugs of low molecular weight compounds have an aromatic ring containing a nitrogen atom, such as sorafenib for renal cell cancer and hepatocellular carcinoma. However, in a bioassay relating to such a nitrogen-containing aromatic ring-containing compound, the nitrogen atom of the nitrogen-containing aromatic ring or the nitrogen atom directly or indirectly bonded to the nitrogen-containing aromatic ring obtained by a conventional production method When a labeling agent to which a label is bound via a nitrogen atom of a functional group containing is used, there is a problem that a signal derived from the label is weak. In particular, when fluorescent substance-integrated nanoparticles are used as the label, the fluorescence intensity representing the binding to the target molecule is weak or the number of bright spots is small, making it difficult to evaluate the binding property of the molecular target drug.

  The present invention provides a labeling agent excellent in binding to a target molecule in a bioassay or tissue staining method according to sorafenib or a derivative thereof (sometimes referred to as “sorafenib etc.” in the present specification). Let it be an issue.

  Unlike the conventional cases, the present inventors use a labeling agent to which a labeled body is bound via a carbon atom of a pyrimidine ring such as sorafenib. It has been found that the fluorescence intensity representing the binding to the molecule is increased or the number of bright spots is increased, and the present invention has been completed.

That is, the present invention includes the following inventions.
[Claim 1]
A labeling agent having a structure in which sorafenib or a derivative thereof, which is a molecular target drug, and a label are bonded via a divalent linking group,
A labeling agent, wherein one end of the divalent linking group is bonded to a carbon atom of a pyridine ring of sorafenib or a derivative thereof.
[Section 2]
Item 2. The labeling agent according to Item 1, wherein the label is a fluorescent substance-integrated nanoparticle.
[Section 3]
A bioassay using the labeling agent according to Item 1 or 2.
[Claim 4]
Item 3. A tissue staining method comprising using the labeling agent according to item 1 or 2.

  Since the labeling agent of the present invention has improved binding properties to target molecules such as sorafenib used as a molecular target drug compared to conventional labeling agents, the effect of the molecular target drug (candidate compound) is more accurately evaluated. In addition, it is possible to more accurately quantify the target molecule present in the tissue section (pathological specimen) derived from the patient and estimate the effect of the molecular target drug more accurately.

―Immunostaining agent―
The labeling agent in the present invention is a labeling agent having a structure in which sorafenib or the like as a molecular target drug and a label are bonded via a divalent linking group, and one end of the divalent linking group. Are bonded to a pyridine ring such as sorafenib.

(Molecular targeted drugs)
In the present invention, sorafenib (see the following formula) or a derivative thereof is used as a molecular target drug. Sorafenib derivatives have a functional group introduction, oxidation, reduction, substitution of atoms, etc. to sorafenib that do not significantly change the matrix structure (particularly one end of the divalent linking group is replaced with a carbon atom of the pyridine ring). A variety of compounds that are modified (so as not to prevent binding) and that act as molecular targeting drugs to the same or better extent than sorafenib are included.

(Marker)
The labeled body used in the present invention can be selected from various types of labeled bodies used in the technical field, and is not particularly limited as long as the effects of the present invention are exhibited. Representative labels include radioisotopes, enzymes that develop color by reacting with a specific substrate, phosphors, etc., but they can measure fluorescence intensity or number of bright spots as signals, and are excellent in quantitative performance. A phosphor capable of accurately evaluating the binding property of the molecular target drug to the target molecule is preferable.

  The phosphor can be selected from various known phosphors used in the technical field, and is not particularly limited. Typical phosphors include inorganic semiconductor nanoparticles (also called “quantum dots”), organic fluorescent dyes, and nano-sized aggregates of these phosphors, with each target molecule as a bright spot. Fluorescent substance-integrated nanoparticles, particularly fluorescent dye-containing resin particles, which can emit fluorescence with sufficient intensity to be expressed and can be detected even with a relatively inexpensive low-sensitivity camera, are preferred.

  Note that the “phosphor” in this specification is irradiated with electromagnetic waves (X-rays, ultraviolet rays, or visible rays) having a predetermined wavelength and absorbs energy to excite electrons and return from the excited state to the ground state. A substance that emits surplus energy as electromagnetic waves, that is, a substance that emits "fluorescence" and that can be combined with a "probe". Further, “fluorescence” has a broad meaning and includes phosphorescence having a long emission lifetime in which emission is continued even when irradiation of electromagnetic waves for excitation is stopped, and narrow sense fluorescence having a short emission lifetime.

Inorganic semiconductor nanoparticles Inorganic semiconductor nanoparticles include those containing II-VI group compounds, III-V group compounds, or group IV elements, such as CdSe, CdS, CdTe, ZnSe, ZnS, ZnTe, InP, InN. , InAs, InGaP, GaP, GaAs, Si, Ge, and the like. Also, those having these inorganic semiconductor nanoparticles as a core and a shell formed on the outside thereof, for example, CdSe / ZnS, CdS / ZnS, InP / ZnS, InGaP / ZnS, Si / SiO ₂ , Si / ZnS, Ge Core / shell type inorganic semiconductor nanoparticles such as / GeO ₂ and Ge / ZnS can also be used.

Organic fluorescent dye Examples of organic fluorescent dyes include rhodamine dye molecules, squarylium dye molecules, cyanine dye molecules, aromatic ring dye molecules, oxazine dye molecules, carbopyronine dye molecules, and pyromesene dye molecules. Can do. Alternatively, Alexa Fluor (registered trademark, manufactured by Invitrogen) dye molecule, BODIPY (registered trademark, manufactured by Invitrogen) dye molecule, Cy (registered trademark, manufactured by GE Healthcare) dye molecule, DY dye molecule (registered) Trademark, manufactured by DYOMICS), HiLyte (registered trademark, manufactured by Anaspec), dye molecule, DyLight (registered trademark, manufactured by Thermo Scientific), dye molecule, ATTO (registered trademark, manufactured by ATTO-TEC) Molecules, MFP (registered trademark, manufactured by Mobitec) -based dye molecules, and the like can be used. In addition, the generic name of such a dye molecule is named based on the main structure (skeleton) in the compound or a registered trademark, and the range of fluorescent dyes belonging to each of them must be excessively trial and error by those skilled in the art. It can be grasped appropriately.

・ Fluorescent substance integrated nanoparticles As a representative example of fluorescent substance aggregates, a structure in which particles made of an organic or inorganic substance are used as a base, and a plurality of fluorescent substances are included in and / or adsorbed on the surface thereof. “Fluorescent substance-integrated nanoparticles” which are nano-sized particles having In this case, it is preferable that the base (for example, resin) and the fluorescent substance (for example, organic fluorescent dye) have substituents or sites having charges opposite to each other, and that electrostatic interaction works. .

As the fluorescent substance to be encapsulated in the fluorescent substance integrated nanoparticles, in addition to the inorganic semiconductor nanoparticles and fluorescent dye molecules as described above, for example, Y ₂ O ₃ , Zn ₂ SiO ₄ or the like is used as a base material, and Mn ²⁺ , Eu ³ Examples include “long afterglow phosphors” using ⁺ or the like as an activator.

  Among the matrix forming the phosphor-integrated nanoparticles, the organic substance is a resin generally classified as a thermosetting resin such as melamine resin, urea resin, aniline resin, guanamine resin, phenol resin, xylene resin, furan resin; Resins generally classified as thermoplastic resins such as styrene resin, acrylic resin, acrylonitrile resin, AS resin (acrylonitrile-styrene copolymer), ASA resin (acrylonitrile-styrene-methyl acrylate copolymer); polylactic acid Other resins such as: polysaccharides can be exemplified, and silica (glass) can be exemplified as the inorganic substance.

  The fluorescent substance-integrated nanoparticles can be produced according to a known method (for example, see JP2013-57937A). More specifically, for example, the fluorescent substance-encapsulating silica particles having silica as a base material and encapsulating the fluorescent substance therein are inorganic substances such as inorganic semiconductor nanoparticles, organic fluorescent dyes, and tetraethoxysilane. It can be produced by dropping a solution in which the silica precursor is dissolved into a solution in which ethanol and ammonia are dissolved and hydrolyzing the silica precursor. On the other hand, in the case of fluorescent substance-containing resin particles in which a resin is used as a base and the fluorescent substance is adsorbed on the surface of the resin particles or encapsulated in the resin particles, a resin solution or a dispersion of fine particles is prepared in advance. In addition, it can be prepared by adding a fluorescent substance such as inorganic semiconductor nanoparticles or organic fluorescent dye and stirring the mixture.

  Alternatively, fluorescent substance-containing resin particles can be produced by adding a fluorescent dye to the resin raw material solution and then allowing the polymerization reaction to proceed. For example, when a thermosetting resin such as a melamine resin is used as a base resin, a raw material of the resin (monomer or oligomer or prepolymer, for example, methylol melamine which is a condensate of melamine and formaldehyde), an organic fluorescent dye, Preferably, the organic fluorescent dye-containing resin particles can be prepared by heating a reaction mixture further containing a surfactant and a polymerization reaction accelerator (such as an acid) and advancing the polymerization reaction by an emulsion polymerization method. . In addition, when a thermoplastic resin such as a styrene copolymer is used as a base resin, the raw material of the resin and an organic fluorescent dye (an organic fluorescent dye is bonded in advance as a resin raw material monomer by a covalent bond or the like). The reaction mixture containing a polymerization initiator (benzoyl peroxide, azobisisobutyronitrile, etc.) is heated and the polymerization reaction proceeds by radical polymerization or ionic polymerization. Thus, organic fluorescent dye-containing resin particles can be produced.

  The average particle diameter of the fluorescent substance-integrated nanoparticles (particularly the fluorescent dye-containing resin particles obtained by the above-described production method) is not particularly limited as long as it is a particle diameter suitable for immunostaining of a pathological specimen. Considering the ease of detection and the like, the thickness is usually 10 to 500 nm, preferably 50 to 200 nm. Moreover, the variation coefficient which shows the dispersion | variation in a particle size is 20% or less normally, Preferably it is 5-15%. The fluorescent substance integrated nanoparticles satisfying such conditions can be manufactured by adjusting the manufacturing conditions. For example, when producing fluorescent substance-integrated nanoparticles by emulsion polymerization, the particle size can be adjusted by the amount of surfactant to be added. If the amount of the active agent is relatively large, the particle size tends to be small, and if the amount is relatively small, the particle size tends to be large.

  In addition, the particle diameter of the fluorescent substance-integrated nanoparticles is obtained by taking an electron micrograph using a scanning electron microscope (SEM), measuring the cross-sectional area of the fluorescent substance-integrated nanoparticles, and assuming that the cross-sectional shape is a circle. Further, it can be calculated as the diameter of a circle corresponding to the cross-sectional area. The average particle size and coefficient of variation of a group consisting of a plurality of fluorescent material-integrated nanoparticles are calculated as described above for a sufficient number (for example, 1000) of fluorescent material-integrated nanoparticles, and the average particle size is The coefficient of variation is calculated by the formula: 100 × standard deviation of particle diameter / average particle diameter.

・ Reactive sites (functional groups)
The labeled body naturally has a reactive site capable of binding to a divalent linking group, or such a site needs to be introduced after the labeled body is produced.

  For example, when using an organic phosphor such as a fluorescent substance-integrated nanoparticle based on a resin as a label, the resin constituting the particle itself must have a reactive site from the beginning. For example, it is necessary to introduce a reactive site by surface modification after the formation of particles. Specifically, functional groups such as amino groups can be used for melamine resins, and monomers having functional groups (for example, epoxy groups) in the side chain can be copolymerized for acrylic resins, styrene resins, and the like. Thus, the functional group itself or a functional group converted from the functional group (for example, an amino group generated by reacting aqueous ammonia) can be used, and further, the reaction with the functional group can be performed. Another functional group can also be introduced by using it.

  In addition, when using inorganic phosphors such as fluorescent substance integrated nanoparticles or inorganic semiconductor nanoparticles based on silica as a label, the surface is modified with a silane coupling agent to introduce a desired functional group. can do. For example, if aminopropyltrimethoxysilane is used as a silane coupling agent and reacted with an inorganic phosphor as described above, an amino group can be introduced on the surface.

  The amino group possessed by the label as described above can react with a predetermined functional group possessed by the compound to be a divalent linking group, such as an NHS group, to form a covalent bond. However, the reactive site that can be bonded to the divalent linking group of the label is not limited to an amino group, and may be a predetermined group such as a carboxyl group, a thiol group, an NHS group, a maleimide group, or the like. Various known sites (functional groups) having reactivity with functional groups can be used.

(Divalent linking group)
The divalent linking group for linking the molecular target drug and the label used in the present invention can be selected from various divalent linking groups used in the technical field. It is not particularly limited as long as the effect is achieved.

  In the present invention, one end of the divalent linking group to be bound to the molecular target drug (one end different from the above-described group derived from NHS) is bound to a carbon atom of a pyridine ring such as sorafenib. The method for forming such a divalent linking group is not particularly limited, and a known reaction mode can be applied. For example, a compound having a functional group that reacts with a carbon atom of a pyridine ring to form a covalent bond (first reagent) is reacted with sorafenib or the like, and then the reaction product (intermediate) obtained is reacted with the first reagent. A mode of reacting a compound (second reagent) having a functional group having reactivity with a functional group different from the functional group possessed by a functional group for binding a label such as the aforementioned NHS-derived group Is mentioned.

  A reaction for bonding a divalent linking group to a carbon atom of a pyridine ring such as sorafenib is described in, for example, J. Am. Chem. Soc., 2013, 135 (35), pp 12994-12997. Reaction can be applied. In the reaction, a sodium salt of sulfinic acid having a difluoroalkyl azide group is used as a reaction reagent, and the sulfinic acid group reacts with a carbon atom of a nitrogen-containing aromatic ring to form a covalent bond. In addition, since an azide group reacts with an alkyne (carbon-carbon triple bond) to add a ring, a label having a carbon-carbon triple bond, or a part of a divalent linking group (other than those derived from the reaction reagent) Another reaction reagent having a carbon-carbon triple bond for constituting a portion of (a) can be bound.

  It is preferable to introduce a functional group (a functional group for binding to the labeled body) having reactivity with the functional group of the labeled body in advance at one end of the divalent linking group on the side to be bonded to the labeled body. is there. Examples of such a label binding functional group include a group (NHS group) derived from N-hydroxysuccinimide, which is used as a carboxylic acid activation reagent in organic chemistry and biochemistry. N-hydroxysuccinimide forms an unstable ester bond (active ester) by dehydration condensation with a carboxylic acid, and this active ester reacts with an amine to form an amide bond. Therefore, a label having an amino group at one end of a divalent linking group can be bound by using an NHS group as a label binding functional group.

  Of course, the coupling mode that can be used for coupling to the label at one end of the divalent linking group is not limited to the covalent bond by the reaction between the NHS group and the amino group as described above. As long as the effects of the present invention are exhibited, a wide variety of bonding modes can be used. For example, while introducing biotin into one end of a divalent linking group, the surface of the labeled body is modified with avidin (streptavidin), and these are bound by a biotin-avidin reaction. May be indirectly bound (via a divalent linking group).

-Bioassay-
The bioassay of the present invention is performed using the labeling agent of the present invention, and its embodiment is not particularly limited. For example, there is a bioassay in which the labeling agent of the present invention is added to cultured cells, the labeling agent is bound to the target molecule expressed there, and the signal emitted from the labeling substance is obtained quantitatively. It is possible to evaluate the binding force to the target molecule by comparing the data of the labeling agents using different molecular target drug candidates (for example, sorafenib derivatives), and use it for improving the molecular target drug. Alternatively, the labeling agent of the present invention is added to a specimen such as blood (serum) and bound to a specific molecule targeted by a molecular target drug or a cell expressing it, and the molecule or cell is qualitative. Alternatively, it is possible to use quantitative analysis.

  Such a bioassay is used in the “staining step” and “signal acquisition step” included in the tissue staining method of the present invention described below, and the “staining step for morphology observation” which may be further included as necessary. Accordingly, it can be carried out with appropriate modification in accordance with the subject of cultured cells and specimens instead of tissue sections. Further, if it is not necessary to acquire a photographed image by a digital camera depending on the purpose of the bioassay, it can be modified, for example, to measure fluorescence intensity with a photomultiplier tube (Photomal, PMT).

-Tissue staining method-
The tissue staining method of the present invention is performed using the labeling agent of the present invention, and the embodiment thereof is not particularly limited, and includes, for example, the following steps:
(1) A step of making a tissue section embedded in paraffin suitable for staining (pretreatment step);
(2) a step of staining a target molecule with a labeling agent (staining step);
(3) A step of making the stained tissue section suitable for observation (post-processing step);
(4) As an optional step, a step of staining so that the morphology of cells or the like can be observed in a bright field (morphological observation staining step);
(5) A step of acquiring a signal emitted from the labeled body from the stained tissue section (signal acquisition step).
Specifically, these steps are performed by the following procedure, for example.

(Pretreatment process)
Tissue sections are usually stored in formalin-fixed state and then embedded in paraffin. When staining such a tissue section, a pretreatment step for deparaffinization / hydrophilization of the tissue section is performed in order to enable staining. This pretreatment step may include an activation treatment for bringing the protein into a state suitable for the reaction with the molecular target drug, if necessary.

  The deparaffinization / hydrophilization treatment may be performed, for example, by immersing the tissue section in xylene to remove paraffin, then immersing in ethanol to remove xylene, and further immersing in water to remove ethanol. . These three operations are usually performed at room temperature. Moreover, each immersion time may be about 3 to 30 minutes, and you may replace | exchange each process liquid for a new thing in the middle of immersion as needed.

  In general, the activation treatment is performed by immersing the tissue slice in an activation solution and heating it. As the activation solution, for example, 0.01 M citrate buffer (pH 6.0), 1 mM EDTA solution (pH 8.0), 5% urea, 0.1 M Tris-HCl buffer, and the like can be used. As the heating device, for example, an autoclave, a microwave, a pressure cooker, a water pass, or the like can be used. The heating temperature and time can be, for example, 50 to 130 ° C. and 5 to 30 minutes.

(Dyeing process)
The staining step is a step for staining treatment in which a labeling agent is bound to the target molecule. The staining treatment may be performed, for example, by immersing the tissue section after the pretreatment step in a solution (staining solution) containing a labeling agent. The temperature, time, and other conditions for immersing the tissue section in the staining solution can be appropriately adjusted according to the conventional staining method so that an appropriate signal can be obtained.

  In addition, in the staining step, reference information other than the binding property between the target molecule and the molecular target drug used in the labeling agent is acquired together with the processing for staining the target molecule as described above, if necessary. For this purpose, a process of staining other biological material (reference biological material) may be performed. In that case, two types of staining treatments are performed by immersing the tissue section after the pretreatment step in a solution containing a labeling agent for staining the target molecule and a labeling agent for staining the reference biological material. Can be done in a lump.

(Post-processing process)
The tissue section after the staining step is preferably subjected to treatment such as fixation / dehydration, penetration, and encapsulation so as to be suitable for observation. The immobilization / dehydration treatment may be performed by immersing the tissue section in a fixation treatment solution (crosslinking agent such as formalin, paraformaldehyde, glutaraldehyde, acetone, ethanol, methanol). For clearing, the tissue section after immobilization / dehydration treatment may be immersed in a clearing solution (xylene or the like). The encapsulating process may be performed by immersing the tissue section after the penetration process in the encapsulating liquid. Conditions for performing these treatments, for example, the temperature and immersion time when the tissue section is immersed in a predetermined treatment solution, can be appropriately adjusted according to the conventional staining method so as to obtain an appropriate signal. . If a cover glass is placed on the tissue section after the post-processing step, a specimen having a form suitable for form observation and signal acquisition is obtained.

(Optional process: Dyeing process for morphology observation)
The tissue staining method using the labeling agent of the present invention includes a staining step for morphological observation so that the morphology of cells, tissues, organs, etc. can be observed in a bright field as necessary. Can do. The staining process for morphology observation can be performed according to a conventional method. Regarding morphological observation of tissue sections, cytoplasm, stroma, various fibers, erythrocytes, keratinocytes are stained with red to dark red and / or stained with eosin and / or cell nucleus, lime, cartilage tissue, bacteria, mucus Dyeing using hematoxylin that is stained blue-blue to light blue is standard, and a method of simultaneously performing these two stainings is well known as hematoxylin-eosin staining (HE staining). When the morphological observation dyeing process is included, it may be performed after the dyeing process described above or may be performed before that.

(Signal acquisition process)
The observation / imaging process is a process of acquiring a signal emitted from the labeled body from a tissue section that has undergone the staining process as described above (and the morphological observation staining process performed as necessary). For example, when a preferred phosphor is used as a label in the present invention, a stained tissue section is observed with a fluorescence microscope at a desired magnification while irradiating excitation light corresponding to the phosphor, and the fluorescence microscope is provided. What is necessary is just to image | photograph the dyeing | staining image which can acquire fluorescence intensity or the number of bright spots using a digital camera. In particular, when fluorescent substance integrated nanoparticles preferable as a phosphor are used, it is easy to observe the fluorescent substance integrated nanoparticles that label the molecular target drug bound to the target molecule as a bright spot, not only measuring the fluorescence intensity but also The score can be measured.

  In addition, when the process of staining the reference biological material as described above is performed in the staining process, the signal acquisition process is performed to acquire the signal emitted by the label contained in the labeling agent that stained the target molecule. Also good. In addition, when the morphological observation staining step as described above is performed as an optional step, the tissue section stained for morphological observation is observed in the bright field before or after the signal acquisition step, and a stained image is taken. Processing may be performed.

  The acquired photographed image can be used for quantifying the signal emitted by the labeled body in accordance with the purpose of tissue staining. For example, when a preferred phosphor is used as the label in the present invention, the intensity of fluorescence emitted from the phosphor can be measured and / or the number of fluorescent bright spots can be measured based on image processing.

  An example of software that can be used for image processing is “ImageJ” (open source). By using such image processing software, bright spots with a predetermined wavelength (color) are extracted from the stained image and the sum of the brightness is calculated, or the number of bright spots with a predetermined brightness or higher is measured. Can be performed semi-automatically and quickly.

  The obtained quantitative signal data can be used to improve the molecular target drug by evaluating the binding power to the target molecule by comparing the data of labeling agents using different molecular target drug candidates, for example. is there. In addition, from the data of tissue sections (pathological specimens) derived from patients with a disease targeted by a molecular target drug, the target molecule contained therein is quantified. It is also possible to estimate the effect of the molecular target drug and to obtain useful information for pathological diagnosis, such as presuming that it is high.

[Labeling agent A] Sorafenib-containing labeling agent in which Qdot (registered trademark) 655 is bonded to the 6-position carbon atom of the pyridine ring of sorafenib via a divalent linking group (Synthesis step A-1) Sorafenib ( A sorafenib derivative (5a) in which a divalent linking group was bonded to a carbon atom of the pyridine ring of 1) was synthesized by the following route. The obtained derivative is a mixture of regioisomers (3a), (3b) and (3c), and each compound is separated using liquid chromatography, and the sorafenib derivative (5a) is obtained using regioisomer (3a). Obtained. The route of the synthesis step A-1 is based on the method described in J. Am. Chem. Soc., 2013, 135 (35), pp 12994-12997.

  (Synthesis step A-2) By reacting the sorafenib derivative (5a) with a quantum dot modified with PEG having an amino group at the terminal (Qdot 655 ITK amino (PEG) quantum dots), 6 of the pyridine ring of sorafenib Labeling agent A to which Qdot was linked through a divalent linking group bonded to the carbon atom at the position was obtained.

[Labeling agent B] A sorafenib-containing labeling agent obtained by binding Qdot (registered trademark) 655 to a carbon atom at the 5-position of the pyridine ring of sorafenib via a divalent linking group. The same operation as in Example 1 was carried out except that the sorafenib derivative (5b) was obtained using the regioisomer (3b), via a divalent linking group bonded to the 5-position carbon atom of the pyridine ring of sorafenib. Labeling agent B to which Qdot was linked was obtained.

[Labeling agent C] A sorafenib-containing labeling agent in which a quantum dot: Qdot (registered trademark) 655 was bonded to a carbon atom at the 3-position of the pyridine ring of sorafenib via a divalent linking group was collected using liquid chromatography. The same operation as in Example 1 was performed except that the sorafenib derivative (5c) was obtained using the regioisomer (3c), and a bivalent linking group bonded to the carbon atom at the 3-position of the pyridine ring of sorafenib. Labeling agent C to which Qdot was linked was obtained.

[Labeling agent D] Sorafenib-containing labeling agent in which quantum dot: Qdot (registered trademark) 655 is bonded to the nitrogen atom of the amide group of the pyridine ring of sorafenib via a divalent linking group (Synthesis step D-1) A sorafenib derivative (6) in which a divalent linking group is bonded to a nitrogen atom of an amide group (carbamoyl group) is described in paragraphs [0181] to [0189] of Patent Document 3 (Japanese Patent Publication No. 2012-533579). Synthesized with reference to the method. In the description of Patent Document 3, a desired substance (ε-polylysine) is bound using a sorafenib derivative in which a 2-hydroxyethylcarboxamide group is introduced into a pyridine ring.

  (Synthesis step D-2) Sorafenib derivative (6) is reacted with quantum dots modified with PEG having an amino group at the terminal (Qdot 655 ITK amino (PEG) quantum dots), so that nitrogen of the amide group of sorafenib A labeling agent D to which Qdot was linked through a divalent linking group bonded to an atom was obtained.

[Example 1]
The slide (USBiomax, Inc., T031a) in which spots of hepatocellular carcinoma tissue sections were arranged on the array was deparaffinized and hydrophilized, and then labeling agent A was added onto the tissue. After standing for 2 hours, unreacted labeling agent was removed by washing, and a fluorescent image of the slide was taken using a confocal fluorescence microscope, and the fluorescence intensity was measured.

In this process, a fluorescence microscope “BX-53” (Olympus Co., Ltd.) was used for excitation light irradiation and fluorescence emission observation, and an immunostained image (400 ×) was attached to the fluorescence microscope. A digital camera for microscope “DP73” (Olympus Corporation) was used. Corresponding to the Qdot 655 used as the phosphor, the wavelength of the excitation light to be irradiated is set to 415 to 455 nm using an optical filter for excitation light (Optline Co., Ltd. “QD655-C”), and the fluorescence to be observed The wavelength was set to 648 to 663 nm using an optical filter for fluorescence. The intensity of the excitation light at the time of observation and image photographing with a fluorescence microscope was such that the irradiation energy near the center of the visual field was 30 W / cm ² . The exposure time at the time of image capturing was adjusted to 400 seconds so that the brightness of the image was not saturated.

  The fluorescence intensity was measured by processing a captured image using image processing software “ImageJ” (open source). A region where the luminance of the fluorescence emitted from the phosphor is not less than a predetermined value was extracted, and the sum of the intensities of the fluorescence constituting the region was obtained.

[Example 2]
Except for using the labeling agent B instead of the labeling agent A, the same operation as in Example 1 was performed, and the fluorescence intensity was measured.

[Example 3]
Except that the labeling agent C was used instead of the labeling agent A, the same operation as in Example 1 was performed, and the fluorescence intensity was measured.

[Comparative Example 1]
Except that the labeling agent D was used instead of the labeling agent A, the same operation as in Example 1 was performed, and the fluorescence intensity was measured.

[Example 4]
A slide (USBiomax, Inc., T071) in which spots of renal cell carcinoma tissue sections were arranged on the array was deparaffinized and hydrophilized, and then labeling agent A was added onto the tissue. After standing for 2 hours, unreacted labeling agent was removed by washing, and a fluorescent image of the slide was taken using a confocal fluorescence microscope, and the fluorescence intensity was measured.

[Example 5]
Except that the labeling agent B was used in place of the labeling agent A, the same operation as in Example 4 was performed, and the fluorescence intensity was measured.

[Example 6]
Except that the labeling agent C was used in place of the labeling agent A, the same operation as in Example 4 was performed, and the fluorescence intensity was measured.

[Comparative Example 2]
The fluorescence intensity was measured in the same manner as in Example 4 except that the labeling agent D was used instead of the labeling agent A.

[result]
The results of Examples 1, 2, 3 and Comparative Example 1, and the results of Examples 4, 5, 6 and Comparative Example 2 are shown in the following table. When stained with a labeling agent based on the binding mode of the present invention (labeling agents A, B, C), stained hepatocytes than when using a labeling agent based on the conventional binding mode (labeling agent D) It turns out that the fluorescence intensity of a cancer tissue section and a renal cell cancer tissue section is strong. From this result, Sorafenib used in the conventional labeling agent is likely to interfere with the binding to the target molecule (eg, VEGFR) by Qdot linked as a fluorescent substance, whereas Sorafenib used in the labeling agent of the present invention Is less susceptible to interference by Qdot linked as a phosphor, and is presumed to be able to bind strongly to a target molecule (eg, VEGFR).

[Labeling agent E] A sorafenib-containing labeling agent in which Texas red-integrated melamine resin nanoparticles are bonded to a carbon atom at the 6-position of the pyridine ring of sorafenib via a divalent linking group (Production step 1) Texas red-integrated melamine resin nano Preparation of Particles After 2.5 mg of Texas Red dye molecule “Sulforhodamine 101” (Sigma Aldrich) was dissolved in 22.5 mL of pure water, the solution was stirred for 20 minutes while maintaining the temperature of the solution at 70 ° C. with a hot stirrer. To the solution after stirring, 1.5 g of melamine resin “Nicalak MX-035” (Nippon Carbide Industries, Ltd.) was added, and the mixture was further heated and stirred under the same conditions for 5 minutes. 100 μL of formic acid was added to the stirred solution and stirred for 20 minutes while maintaining the temperature of the solution at 60 ° C., and then the solution was left to cool to room temperature. The cooled solution was dispensed into a plurality of centrifuge tubes and centrifuged at 12,000 rpm for 20 minutes to precipitate Texas red-integrated melamine resin nanoparticles contained in the solution as a mixture. The supernatant was removed, and the precipitated Texas Red integrated melamine resin nanoparticles were washed with ethanol and water to obtain Texas Red integrated melamine resin nanoparticles.

(Preparation process 2) Preparation of terminal amino group PEG-modified Texas red integrated melamine resin nanoparticles Polyethylene glycol having amino groups at both ends and having a number average molecular weight of 10,000 (NOF Corporation, SUNBRIGHT DE-100PA, X- (OCH ₂ CH ₂ ) _n -OX, X: —CH ₂ CH ₂ CH ₂ NH ₂ ) and Texas Red-integrated melamine resin nanoparticles are reacted by heating at 70 ° C. for 1 hour, and terminal amino group PEG-modified Texas Red-integrated melamine resin is reacted. Nanoparticles were obtained.

(Preparation process 3) Preparation of labeling agent E By mixing and reacting the sorafenib derivative (5a) obtained in the synthesis process A-1 of labeling agent A and the terminal amino group PEG-modified Texas Red integrated melamine resin nanoparticles, Labeling agent E was obtained in which Texas Red-integrated melamine resin nanoparticles were linked via a divalent linking group bonded to the 6-position carbon atom of the pyridine ring of sorafenib.

[Example 8]
The same operation as in Example 1 was performed except that the labeling agent B was used instead of the labeling agent A, and the hepatocellular carcinoma tissue section was stained to obtain an observation sample slide. A fluorescence image of the slide was taken using a fluorescence microscope, and the fluorescence intensity (x) was measured. On the other hand, a solution of the labeling agent (Texas Red integrated melamine resin nanoparticles) is diluted with water, and this diluted solution is placed on a glass slide and observed with fluorescence, and the brightest spot with the lowest fluorescence intensity does not change. It was regarded as a molecule (one Texas red-integrated melamine resin nanoparticle), and the fluorescence intensity (y) was measured when only one molecule of the labeling agent was present in one field of view. By dividing the fluorescence intensity (x) by the fluorescence intensity (y) per molecule of the labeling agent thus determined, the number of fluorescent particles contained in the fluorescence image of the observation sample slide was calculated.

[Reference Example 1]
From the fluorescence intensity measured in Example 1, as in Example 8, an attempt was made to calculate the number of fluorescent particles based on the separately determined fluorescence intensity per one particle of the labeling agent. However, because the fluorescence intensity of the labeling agent is low, it is difficult to observe one particle with a fluorescence microscope, the fluorescence intensity per molecule of the labeling agent cannot be obtained, and the number of fluorescent particles cannot be calculated. It was.

[result]
The results of Example 8 and Reference Example 1 are shown in the following table. When fluorescent substance integrated nanoparticles (Texas Red integrated melamine resin nanoparticles) are used as the phosphor, the number of fluorescent particles can also be calculated and more quantitative evaluation is possible. This is preferable in the invention.

Claims (4)

A labeling agent having a structure in which sorafenib or a derivative thereof, which is a molecular target drug, and a label are bonded via a divalent linking group,
A labeling agent, wherein one end of the divalent linking group is bonded to a carbon atom of a pyridine ring of sorafenib or a derivative thereof.   The labeling agent according to claim 1, wherein the label is a fluorescent substance-integrated nanoparticle.   A bioassay using the labeling agent according to claim 1 or 2.   A tissue staining method using the labeling agent according to claim 1 or 2.