WO2019221062A1 - Procédé d'évaluation d'un médicament - Google Patents

Procédé d'évaluation d'un médicament Download PDF

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WO2019221062A1
WO2019221062A1 PCT/JP2019/018948 JP2019018948W WO2019221062A1 WO 2019221062 A1 WO2019221062 A1 WO 2019221062A1 JP 2019018948 W JP2019018948 W JP 2019018948W WO 2019221062 A1 WO2019221062 A1 WO 2019221062A1
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drug
fluorescent
protein
organelle
evaluating
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PCT/JP2019/018948
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English (en)
Japanese (ja)
Inventor
拓司 相宮
古澤　直子
中野　寧
洋 根岸
幸祐 権田
真由美 高野
成史 北村
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コニカミノルタ株式会社
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Priority to JP2020519624A priority Critical patent/JPWO2019221062A1/ja
Publication of WO2019221062A1 publication Critical patent/WO2019221062A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/15—Medicinal preparations ; Physical properties thereof, e.g. dissolubility
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals

Definitions

  • the present invention relates to a drug evaluation method.
  • Cancer is a disease that bisects the cause of death in adults together with vascular diseases such as myocardial infarction and cerebral infarction.
  • vascular diseases such as myocardial infarction and cerebral infarction.
  • the incidence of breast cancer in Japan is lower than in Western countries, but it has been increasing year by year in recent years. In 1998, it surpassed the incidence of gastric cancer and became the first cancer incidence in women. .
  • the annual number of patients with breast cancer exceeds 50,000.
  • the number of breast cancer patients in the world is increasing year by year. According to a 2008 WHO report, breast cancer has the highest incidence among cancers, and the annual number of patients affected is 1.38 million. It accounts for about 23% of women ’s cancer.
  • Organelles are structures that perform various functions in cells, and microtubules, one of which is a fibrous cytoskeleton that exists in eukaryotic cells.
  • a microtubule is composed of two types of proteins called ⁇ -tubulin and ⁇ -tubulin, and the basic unit is a heterodimer in which one molecule is bound to each other.
  • Microtubules are elongated by the addition (polymerization) of this heterodimer and shortened by dissociation.
  • Microtubule elongation and shortening also called microtubule dynamics, has important functions for transporting other various organelles and cell membrane proteins, as well as cell division (mitosis) and cell polarity. It is known to be involved in control.
  • anticancer agents are widely used as anticancer agents because they can inhibit cell growth and cell movement by impairing the structure and control of microtubules.
  • Such drugs include, for example, colchicine and vinca alkaloid anticancer agents (such as vincristine) having an action of suppressing microtubule polymerization, and taxane anticancer having an action of inhibiting microtubule depolymerization.
  • Agents docetaxel, paclitaxel
  • a method for evaluating the drug efficacy of a candidate drug by directly administering the candidate drug screened using cultured cells to a laboratory animal is widely used.
  • a candidate drug is administered to a tumor-bearing model mouse, which is a mouse transplanted with tumor cells, and the efficacy of the candidate drug is evaluated by measuring the size of the tumor after a predetermined time has elapsed.
  • Patent Document 1 a drug that has been shown to have a microtubule inhibitory effect in cell experiments is administered to a model mouse transplanted with breast cancer cells, and the change in tumor volume before and after drug administration is measured.
  • Patent Document 2 discloses a technique for imaging the action of a polypeptide, which is a microtubule elongation inhibitor, using cells expressing CLIP-170, a microtubule-binding protein fused to a fluorescent protein produced by genetic manipulation. Is described.
  • Patent Document 2 directly evaluates the inhibitory action of microtubule elongation by a polypeptide
  • the evaluation is performed using cultured cells instead of mice, and in a more physiological environment. It is not clear whether a similar evaluation can be performed in vivo.
  • the present invention relates to a method for evaluating a drug using a drug that affects the behavior of organelles and a laboratory animal.
  • the present inventors have prepared a specimen from a tissue section collected from a lesioned part of an experimental animal, performed fluorescence imaging, and observed the dynamics of the organelle. It was found that can be evaluated.
  • the present invention provides the following analysis method.
  • a drug evaluation method using an experimental animal having a lesion is A fluorescent protein, A part of transplanted cells expressing a fluorescent protein fused organelle binding protein, which is a protein fused with an organelle binding protein that is a protein that binds to an organelle, Administering to the experimental animal a drug that affects organelle dynamics, A drug evaluation method further comprising the following steps (a) to (c).
  • B A fluorescence imaging step of acquiring a fluorescence image of the specimen.
  • C An information acquisition step of acquiring information based on fluorescent luminescent spots from the image acquired in the imaging step.
  • the information based on the fluorescent bright spot is selected from the number of bright spots of the fluorescent protein, the moving distance and speed of the bright spot of the fluorescent protein, the linearity of the movement, and the distribution and density of the bright spots of the fluorescent protein.
  • the drug evaluation method according to [1] comprising one or more.
  • the drug evaluation method according to any one of [1] to [10], wherein the drug that affects the kinetics of the organelle is a microtubule polymerization inhibitor.
  • a drug evaluation system comprising a fluorescence imaging device and an information processing device
  • the fluorescent photographing device is a device for photographing a fluorescent image
  • the information processing apparatus is Receiving a fluorescence image acquired by the fluorescence imaging device; Obtain information based on the fluorescent luminescent spot from the received fluorescent image, An apparatus for analyzing information based on the fluorescent bright spot, A drug evaluation system for performing the drug evaluation method according to any one of [1] to [16].
  • the drug evaluation system according to [17] further comprising a display device that displays information based on the fluorescent bright spot.
  • the drug evaluation system according to [17] or [18], wherein the information processing apparatus includes an information storage unit that stores information based on the fluorescent bright spot.
  • fluorescence imaging is performed on a specimen prepared from a tissue section collected from a lesioned part of an experimental animal, and the dynamics of organelles are observed, whereby the drug in the lesioned part of the experimental animal is observed. It is possible to evaluate the impact of In addition, since the specimen prepared in the present invention is in a state where the cells remain alive, more detailed information such as the movement distance of the organelle can be acquired.
  • FIG. 1 is a flowchart in the drug evaluation method of the present invention.
  • FIG. 2 is a schematic diagram of a system according to an embodiment of the present invention.
  • FIG. 3 is a flowchart when the medicine evaluation method is performed using the system according to the embodiment of the present invention.
  • the present invention includes a method for evaluating the effect of a drug that affects the organelle dynamics by performing fluorescence imaging using an experimental animal transplanted with cells expressing a fluorescent protein-fused organelle-binding protein.
  • a transplanted lesion part of a cell expressing a fluorescent protein-fused organelle-binding protein which is a protein in which a fluorescent protein and a protein (organelle-binding protein) that binds to an organelle are fused.
  • the experimental animal in the present invention is an experimental animal having a lesioned part, and the lesioned part is a site where cells expressing a fluorescent protein-fused cell organelle binding protein described later are transplanted.
  • the lesion is not particularly limited but is preferably a tumor.
  • the experimental animal is a cancer-bearing animal.
  • the method for transplanting cells expressing the fluorescent protein-fused cell organelle-binding protein is not particularly limited.
  • the cells can be transplanted by subcutaneous injection of a solution in which the cells are suspended.
  • the experimental animal is preferably selected according to the purpose, for example, mouse, rat, rabbit, guinea pig, gerbil, hamster, ferret, dog, minipig, monkey, cow, horse, sheep, etc. Examples include animals that are controlled and have homogeneous genetic requirements. From the viewpoint of establishing an experimental system and facilitating breeding, mice are preferably used. Further, in the present invention, an immunodeficient mouse is preferable because a cell expressing a fluorescent protein-fused cell organelle-binding protein is transplanted. (Fluorescent protein fusion cell organelle binding protein)
  • the fluorescent protein-fused organelle-binding protein of the present invention is a protein in which a fluorescent protein and a protein that binds to an organelle are fused.
  • the structure of the fluorescent protein fusion organelle-binding protein may be selected according to the selected fluorescent protein and organelle-binding protein.
  • GFP is selected as the fluorescent protein
  • EB1 is selected as the organelle-binding protein.
  • the organelle-binding protein is not particularly limited as long as it has a property of binding to an organelle.
  • the organelle-binding protein is preferably a protein that recognizes and binds to the extended end of the microtubule.
  • EB3 end binding protein 3
  • CLIP170 cytoplasmic linker protein 170
  • APC adenomatous polyposis coli
  • STIM1 stromal interaction molecule 1
  • EB1 end binding protein 1
  • the fluorescent protein is not particularly limited as long as it is a fluorescent protein that can be fused and expressed with other proteins. Examples thereof include GFP, RFP, YFP, and the like, and GFP is particularly preferable.
  • the fluorescent protein absorbs energy when irradiated with electromagnetic waves (X-rays, ultraviolet rays, or visible rays) having a predetermined wavelength, and excites electrons to return to the ground state from the excited state. It is a protein that emits (emits) the energy of as “fluorescence”.
  • fluorescence has a broad meaning, and includes phosphorescence that has a relatively long emission lifetime that can continue to emit light even when irradiation of electromagnetic waves for excitation is stopped, and so-called narrow-sense fluorescence that has a relatively short emission lifetime. To do.
  • the cell that expresses the fluorescent protein-fused cell organelle-binding protein is not particularly limited, but is preferably a cultured cell derived from a diseased tissue, and more preferably a cultured cell derived from a tumor tissue. Further, the fluorescent protein-fused organelle-binding protein to be expressed in the cell may be one type or a plurality of types. When multiple types of fluorescent protein-fused organelle-binding proteins are expressed in cells, the fluorescent proteins constituting each fluorescent protein-fused organelle-binding protein are selected from fluorescent proteins that emit fluorescence at different fluorescence wavelengths. It is preferable. In this case, the organelles to which the organelle binding proteins constituting the fluorescent protein fusion organelle binding protein bind may be the same or different.
  • the method for expressing the fluorescent protein-fused organelle-binding protein in the cell is performed by introducing a gene encoding the fluorescent protein-fused organelle-binding protein into the cell.
  • a known method can be used as a method for gene transfer.
  • gene transfer may be performed using a virus particle prepared by a virus vector having a desired gene, or a transfection reagent such as Lipofectamine (registered trademark) may be used.
  • gene transfer may be performed chemically or by a physical method such as electroporation.
  • the step of administering the drug of the present invention is a process of administering a drug that affects the organelle dynamics to the experimental animal.
  • the dosage form, administration route, administration period, number of administrations, etc. of the drug are not particularly limited, and the process of administering the drug may be performed once or multiple times.
  • the drug that affects the organelle dynamics is not particularly limited, but is preferably a drug that affects the microtubule dynamics, more preferably a drug that affects the microtubule polymerization, More preferably, it is a vascular polymerization inhibitor, and specifically, for example, paclitaxel (trade name: paclitaxel, Nippon Kayaku Co., Ltd.), trastuzumab-emtansine (trade name: Kadsaila, Chugai Pharmaceutical Co., Ltd.), brentuximab vedin (product) Name ADCETRIS, Takeda Pharmaceutical Co., Ltd.), Eriblin (trade name Halaven, Eisai Co., Ltd.) and the like.
  • paclitaxel trade name: paclitaxel, Nippon Kayaku Co., Ltd.
  • trastuzumab-emtansine trade name: Kadsaila, Chugai Pharmaceutical Co., Ltd.
  • brentuximab vedin product
  • the amount of the drug is not particularly limited, and an appropriate amount may be administered depending on the type of drug, the weight and age of the experimental animal, the size (volume) of the lesion.
  • the drug is preferably a fluorescently labeled drug labeled with a fluorescent substance.
  • the fluorescent substance can be selected from a group similar to the fluorescent substance used in fluorescent staining described later, such as organic fluorescent dyes, quantum dots, and phosphor integrated nanoparticles described later. It is preferable to select a fluorescent substance that emits fluorescence having a wavelength different from the fluorescence emitted by the fluorescent protein constituting the organelle-binding protein.
  • the method of labeling the drug with a fluorescent substance is not particularly limited, and examples thereof include a method of covalently bonding the drug and the fluorescent substance via a functional group such as an amino group or a hydroxyl group.
  • the drug can be fluorescently labeled using a labeling reagent (kit).
  • sample preparation process In the specimen preparation step in the present invention, a specimen is prepared from a tissue section collected from the lesioned part of the experimental animal.
  • the excised tumor tissue is immersed in a heated gel, and the tumor tissue that has been cooled and solidified is sliced with a scalpel and used as a specimen.
  • the gel is not particularly limited as long as it has a melting temperature higher than room temperature and gels at about room temperature.
  • an agarose gel such as PrimeGel (registered trademark) Agarose or a low melting point acrylamide can be used.
  • the melting temperature is preferably 60 ° C. to 100 ° C., more preferably 65 ° C. to 95 ° C., and still more preferably 65 ° C. to 90 ° C.
  • the solidification temperature is preferably 10 ° C. to 50 ° C., more preferably 20 ° C. to 40 ° C.
  • the concentration of the gel can be appropriately changed depending on the type of gel, environmental conditions such as air temperature, etc. For example, when an agarose gel is used, it is preferably 1% to 2%.
  • the fluorescence emitted from the fluorescent protein by irradiating the sample with excitation light corresponding to the fluorescent protein constituting the fluorescent protein-fused organelle binding protein is obtained as a fluorescent image.
  • Irradiation of these excitation lights can be performed, for example, using a laser light source provided in a fluorescence microscope and an excitation light optical filter that selectively transmits a predetermined wavelength as required.
  • excitation light corresponding to each fluorescent protein is sequentially applied to the specimen sample. By irradiating, the fluorescence emitted from each fluorescent protein can be acquired as a fluorescent image.
  • the excitation light corresponding to the fluorescent substance labeled with the drug and the fluorescent protein constituting the fluorescent protein fusion organelle binding protein The fluorescence emitted from the fluorescent protein and the fluorescent substance by sequentially irradiating the specimen sample with the excitation light can also be acquired as fluorescent images.
  • the sample protein is irradiated with the excitation light corresponding to the fluorescent protein and the excitation light corresponding to the fluorescent substance used for the fluorescent staining in order from the fluorescent protein and the fluorescent substance.
  • the emitted fluorescence can also be acquired as a fluorescence image.
  • the acquisition order of the fluorescent image of the fluorescent protein and the fluorescent image of the fluorescent substance described above is not particularly limited.
  • the first fluorescence imaging step is performed first, and the second is performed next.
  • the fluorescence imaging process, which is performed for the Nth time, may be referred to as the Nth fluorescence imaging process.
  • Fluorescent images can be acquired by, for example, photographing with a digital camera provided in a fluorescence microscope.
  • the fluorescent image may be a still image or a moving image. It is also possible to perform time-lapse photography and acquire a plurality of fluorescent images over time. You may convert the image which carried out the time lapse photography into a moving image using well-known software.
  • a visible light photographing step of obtaining an image by photographing under visible light before or after the fluorescence photographing step may be performed.
  • Information acquisition process In the information acquisition process of the present invention, information based on the fluorescent bright spot is acquired for the image acquired in the fluorescence imaging process.
  • the information based on the fluorescent luminescent spot is preferably derived from the fluorescence emitted by the fluorescent protein constituting the fluorescent protein-fused organelle protein.
  • information derived from the fluorescence emitted by the fluorescent substance used for fluorescent staining for example, the number of bright spots of the fluorescent substance and its localization may be included. .
  • image processing may be performed by any method. For example, when image processing is performed to superimpose images acquired in the first fluorescence imaging step to the Nth fluorescence imaging step, it is possible to observe the mutual positional relationship between the organelles and drugs of the captured cells. it can.
  • image processing is performed using image processing software such as “Imaris” (manufactured by Carl Zeiss) or “ImageJ” (open source), thereby extracting bright spots of a predetermined wavelength (color) from the fluorescent image.
  • image processing software such as “Imaris” (manufactured by Carl Zeiss) or “ImageJ” (open source)
  • the process of calculating the total sum of the brightness, measuring the number of bright spots that exceed the specified brightness, and measuring the movement distance of the bright spots is performed semi-automatically and quickly, so that the desired fluorescence can be obtained. Information based on bright spots can be acquired.
  • the information based on the fluorescent bright spot is not particularly limited, but is selected from the number of bright spots of the fluorescent protein, the moving distance of the bright spot of the fluorescent protein, the moving speed, the linearity of the movement, and the distribution and density of the bright spots of the fluorescent protein. It is preferable to include one or more indexes, and more preferably to include two or more indexes.
  • the linearity of movement can be obtained by quantifying by “linear distance between the starting point and the ending point of the bright spot” ⁇ “moving distance of the bright spot”. The closer the value is to 1, the higher the linearity is.
  • the drug affecting the dynamics of the organelle administered to the experimental animal in the step of administering the drug is a fluorescently labeled drug
  • the number of bright spots derived from the fluorescent substance labeled with the drug is further increased. It is preferable to include.
  • the drug can be evaluated based on the information acquired in the information acquisition process. For example, it can be performed by classifying specimens according to a certain threshold set for arbitrary information, and the rate of change in the number of bright spots is determined to be 50% or more highly effective (Rank A), and 30% to 50%. Less than% can be determined to be moderate (Rank B), and otherwise it can be determined to be less effective (Rank C). In addition, by considering a plurality of pieces of information including information based on fluorescent luminescent spots, more accurate drug evaluation can be performed by making a composite determination.
  • the fluorescence imaging process may optionally be performed again. In this case, a more appropriate image can be taken by changing the fluorescence intensity, the visual field, and the like.
  • the specimen before fluorescence observation may be subjected to fluorescent staining using a fluorescent substance.
  • the target of the fluorescent staining is not particularly limited, but is preferably a drug that affects the organelle dynamics administered to the experimental animal to be evaluated in the present invention.
  • the method of fluorescent staining is not particularly limited, but is preferably fluorescent immunostaining performed using an antibody labeled with a fluorescent substance.
  • the antibody labeled with the fluorescent substance has a configuration of [anti-drug antibody] to [fluorescent substance].
  • the form of the bond represented by “ ⁇ ” is not particularly limited, and examples thereof include a covalent bond, an ionic bond, a hydrogen bond, a coordination bond, physical adsorption, and chemical adsorption. It may be through.
  • [Anti-drug antibody] to [fluorescent substance] can be used as long as [anti-drug antibody] to [fluorescent substance] itself, in which a desired fluorescent substance is bound to a desired antibody, is commercially available. Alternatively, it can be prepared using, for example, a commercially available fluorescent labeling reagent (kit) based on a known technique capable of binding a desired fluorescent substance to a desired antibody (protein).
  • kit fluorescent labeling reagent
  • the fluorescent substance used for the fluorescent staining is not particularly limited, but is preferably a fluorescent substance having a fluorescent wavelength different from the fluorescent wavelength of the fluorescent protein, and phosphor integrated particles are used from the viewpoint of luminance and quantitativeness. Is preferred.
  • the phosphor-integrated particles have a structure in which a plurality of phosphors (for example, fluorescent dyes and semiconductor nanoparticles) are encapsulated and / or adsorbed on the surface of the particles, which are made of organic or inorganic substances.
  • Nano-sized particles having Examples of fluorescent dyes constituting the phosphor-integrated nanoparticles include rhodamine dyes, Cy dyes, AlexaFluro (registered trademark) dyes, BODIPY dyes, squarylium dyes, cyanine dyes, aromatic ring dyes, and oxazine dyes. Dyes, carbopyronine dyes, pyromesene dyes, and the like.
  • Examples of the semiconductor nanoparticles constituting the phosphor-integrated nanoparticles include II-VI group semiconductors, III-V group semiconductors, and IV group semiconductors. Can be mentioned.
  • the phosphor-aggregated particles can be produced according to a known method (for example, see JP2013-57937A).
  • a sample preparation method is a method for preparing a sample for performing the drug evaluation method, wherein cells remain alive from a tissue section collected from a lesion of an experimental animal.
  • This is a method for preparing a specimen that can be photographed with fluorescence.
  • a specimen is prepared by coating a tissue section collected from a lesion of an experimental animal with a melted gel and slicing the tissue section covered with the gel at a solidification temperature lower than room temperature.
  • the gel is not particularly limited as long as it has a melting temperature higher than room temperature and a solidification temperature lower than room temperature.
  • an agarose gel such as PrimeGel (registered trademark) Agarose or a low melting point acrylamide can be used.
  • the melting temperature is preferably 60 ° C. to 100 ° C., more preferably 65 ° C. to 95 ° C., and still more preferably 65 ° C. to 90 ° C.
  • the solidification temperature is preferably 10 ° C. to 50 ° C., more preferably 20 ° C. to 40 ° C.
  • a drug evaluation system is a system for performing the drug evaluation method, and is a drug evaluation system including a fluorescence observation apparatus and an information processing apparatus.
  • the fluorescence observation apparatus is an apparatus that captures a fluorescent image
  • the information processing apparatus receives imaging information acquired by the fluorescence observation apparatus, acquires information based on fluorescent luminescent spots from the received imaging information, This is an apparatus for analyzing information based on fluorescent bright spots.
  • the evaluation system of the present invention preferably further includes an input device that controls the system and a display device that outputs information acquired by the information processing device.
  • the fluorescence imaging apparatus is not particularly limited as long as it is an apparatus capable of capturing a fluorescent image, but is preferably a fluorescence microscope equipped with an imaging apparatus, and further a confocal microscope equipped with an imaging apparatus It is more preferable that The imaging device is not particularly limited as long as it can capture a fluorescent image, but it is preferable that it can capture a multicolor time-lapse image or a moving image.
  • the fluorescence image acquired by the fluorescence imaging apparatus is transmitted to the information processing apparatus.
  • the acquired fluorescent image is preferably converted as a digital image, and may be processed or image processed by a known means.
  • the information processing apparatus includes an information storage unit that stores information based on fluorescent bright spots. For example, at the time of measurement, sample ID information and drug ID information are input from an input device or the like, imaging is performed at a plurality of time points, and data is stored in the information processing device, so that a sample is continuously acquired. Information based on the fluorescent bright spot can be referred to, and the amount of change in the measured value, its ratio, fluctuation, etc. can be observed.
  • the evaluation system has a display device that displays information based on the fluorescent bright spot.
  • the display device includes, for example, a monitor such as a CRT (Cathode Ray Tube) or an LCD (Liquid Crystal Display), and is an image or information processing device photographed by a fluorescence photographing device according to display control of the input device. Display the acquired information.
  • the displayed information may be information that is further organized by an arbitrary method. For example, drug ID (drug name, LOT number, etc.), sample information (mouse ID, cell information transplanted into the mouse, etc.), imaging history, change amount of each information, measurement date / time, captured image, and drug in each sample It is preferable that the determination results and the like are displayed together (see FIG. 2: display device).
  • the program which is one Embodiment of this invention is a program for performing the said chemical
  • a process for causing the fluorescence imaging apparatus to present imaging information to the information processing apparatus a process for causing the information processing apparatus to calculate predetermined information including information based on the fluorescent bright spot from the imaging information received, It is a program for executing a process for calculating a change over time from information calculated at a time point and a process for presenting an evaluation of a drug administered to an experimental animal based on the change.
  • the program may be stored in an information processing apparatus, or other computer-readable recording media such as a magnetic tape (digital data storage (DSS), etc.), a magnetic disk (hard disk drive (HDD), a flexible disk (FD). )), Optical disc (compact disc (CD), digital versatile disc (DVD), Blu-ray disc (BD), etc.), magneto-optical disc (MO), flash memory (SSD (Solid State Drive), memory card, USB memory, etc.)
  • the information processing apparatus or the like stores data necessary for executing the program.
  • the information processing apparatus or the like stores data necessary for executing the program.
  • the information processing apparatus or the like stores data necessary for executing the program.
  • the information processing apparatus or the like stores data necessary for executing the program.
  • the information processing apparatus or the like stores data necessary for executing the program.
  • the information processing apparatus or the like stores data necessary for executing the program.
  • the information processing apparatus or the like stores data necessary for executing the program.
  • the program is stored in an information processing apparatus or
  • the drug evaluation can be performed, for example, by classifying specimens according to a certain threshold set for arbitrary information. For example, regarding the rate of change of the number of bright spots, 50% or more is judged to be highly effective (Rank A), and 30% or more and less than 50% is judged to be moderately effective (Rank B). Judged as low (Rank C). Further, by setting threshold values for a plurality of pieces of information and making a composite determination, more accurate drug evaluation can be performed.
  • Human EB1 cDNA was amplified by reverse transcription polymerase chain reaction (Reverse Transcriptase Polymerase Chain Reaction), and the amplified product was inserted into a pEGFP cloning vector (Clontech) to further amplify EB1-EGFP cDNA.
  • the EB1-EGFP cDNA was excised and inserted into a pLNCX2 retrovirus vector (BD Bioscience) to construct a retrovirus vector into which the EB1-EGFP cDNA was inserted.
  • the constructed retroviral vector is transfected into packaging cells (GP2-293 cells) using Xfect Transfection Reagent (Takara Bio Inc.), cultured for 48 hours, and the recombinant supernatant is recovered by filtering the culture supernatant. did.
  • the human breast cancer cell line KPL-4 was transfected with the EB1-EGFP gene.
  • Transfected cells were transformed into single clones by repeated passages at 37 ° C. and 5% CO 2 with DMEM (Gibco) containing 10% FBS (Gibco) and 400 ⁇ g / ml G418. (Hereinafter, single-cloned cells are referred to as EB1-EGFP-KPL cells).
  • mice 5 ⁇ 7 weeks old female immunodeficient mice (BALB-c nu / nu: Charles River) under general anesthesia with a mixture of 1.5% ketamine and 1.0% xylazine, 2 ⁇ 10 Cancer-bearing mice were prepared by transplanting seven EB1-EGFP-KPL cells subcutaneously in the right hips of the mice. Individuals with tumor diameters of 5 to 10 mm 4 to 5 weeks after transplantation were used for the experiments.
  • 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 particles contained in the solution as a mixture.
  • the supernatant was removed and the precipitated particles were washed with ethanol and water.
  • SEM observation was performed on 1000 of the obtained Texas Red integrated melamine resin particles, and the average particle size was measured. As a result, the average particle size was 80 nm.
  • the antibody was bound to the surface of the Texas red-integrated melamine resin particles thus produced by the following means.
  • the particles subjected to the above surface amination treatment were adjusted to 3 nM using PBS (phosphate buffered saline) containing 2 mM of EDTA (ethylenediaminetetraacetic acid), and the final concentration of this solution was 10 mM.
  • SM (PEG) 12 manufactured by Thermo Scientific, succinimidyl-[(N-maleimipropionamido) -dodecaethyleneglycol] ester was mixed and allowed to react for 1 hour. The mixture was centrifuged at 10,000 G for 20 minutes, the supernatant was removed, PBS containing 2 mM of EDTA was added, the precipitate was dispersed, and centrifuged again. The washing
  • Anti-human IgG antibody (donkey polyclonal antibody; JacksonJImmunoResearch) was treated with N-succinimidyl S-acetylthioacetate (SATA), followed by filtration through a gel filtration column and binding to Texas Red accumulated melamine particles A possible anti-IgG antibody solution was obtained.
  • SATA N-succinimidyl S-acetylthioacetate
  • the Texas Red-integrated melamine particles modified with the maleimide group and the anti-IgG antibody subjected to the thiol group addition treatment were mixed in PBS containing 2 mM of EDTA, reacted at room temperature for 1 hour, and then 10 mM mercaptoethanol. Was added to stop the reaction. After the obtained solution was concentrated with a centrifugal filter, unreacted antibodies and the like were removed using a gel filtration column for purification, and anti-IgG antibody-bound Texas red integrated melamine particles were obtained.
  • Trastuzumab-emtansine 100 ⁇ g (1 mg / mL) (trade name: Kadosaira; Chugai Pharmaceutical Co., Ltd.) was diluted with 100 mM HEPES buffer containing 30% glycerol, and ultrafiltration filter Nanosep 30K (Pall Corporation) was used. Purified.
  • trastuzumab-emtansine 100 ⁇ g (1 mg / mL) with Cy5 ⁇ ⁇ ⁇ NHS ⁇ Ester Mono-reactive dissolved in dimethylsulfoxide (Thermo Fisher) to 8.5 mM for 15 minutes in a 37 °C environment
  • trastuzumab Cy5 was conjugated to the amino group of the lysine residue of emtansine.
  • Cy5-labeled trastuzumab-emtansine was purified according to the procedure described in the kit.
  • Example 1 The tumor-bearing mouse prepared in Preparation Example 1 was anesthetized with 1.5% ketamine + 1% xylazine mixed anesthesia 4-5 weeks after transplantation of GFP-fused EB1-expressing cells, and 1.35 mg with physiological saline.
  • the anticancer drug trastuzumab-emtansine (trade name: Kadsaila; Chugai Pharmaceutical Co., Ltd.) diluted to a concentration of / mL was administered from the tail vein through the tail vein.
  • the same amount of physiological saline (Otsuka raw food injection; Otsuka Pharmaceutical Co., Ltd.) was administered to cancer-bearing mice similarly produced as a control.
  • a culture solution FluoroBrite DMEM Media; Thermo Fisher Scientific
  • the tumor embedded in the gel is sliced with a linear slicer PRO7 (manufactured by Dosaka EM Co., Ltd.) to a thickness of 200 ⁇ m by vibration at 83 to 85 Hz, washed with FluoroBrite (registered trademark) DMEM Media, and 37 ° C. Left in the environment for 1 hour. Thereafter, the tumor slice was placed on a 35 mm glass bottom dish No1.5 containing an appropriate amount of FluoroBrite DMEM Media, and a round cover glass was placed thereon to fix the tumor slice on the dish.
  • the present invention most of the cells remain alive in the tumor section subjected to the above-described treatment, and the living cells are also the object of analysis in the fluorescence observation.
  • Fluorescence is detected using a 488 nm Laser whose output is set to 0.5% as an excitation light source, HV is 75 (au), resolution is 512 ⁇ 512, pixel size is 0.104 ⁇ m / pixel (Zoom 4), Line Average This was done using a resonant vaginal scanner set to 16. Furthermore, Time Lapse photography was performed by continuously photographing for 20 seconds at a scanning speed of 1.07 seconds / 1 frame.
  • the image acquired for 12 cells was analyzed with the image processing software “Imaris”.
  • the number of bright spots observed for each cell during the imaging time (20 seconds) was 2000 per cell in Example 1, and 2710 per cell in the Comparative Example. Of these, 210 per cell in the examples and 400 per cell in the comparative examples were extracted as the number of tracking bright spots.
  • the linear movement distance linear distance of the luminescent spot at the start point and the end point of photographing
  • the moving speed of the luminescent spot and the linearity of the luminescent spot movement were measured, and the average value was calculated.
  • the sum total of the linear movement distances of all bright spots contained in one cell was calculated. The results are shown in Table 1.
  • Example 1 in which trastuzumab-emtansine was administered to a tumor-bearing mouse prepared using KPL-4 cells expressing GFP-fused EB1, the fluorescence emission point was higher than that in Comparative Example 1 in which physiological saline was administered. It was found that the number of points, linear movement distance, and movement speed were greatly reduced. From this, it can be seen that the microtubule polymerization inhibitory action of trastuzumab-emtansine reduces the number of microtubules and also inhibits the movement of microtubules.
  • Example 2 For the tumor-bearing mice prepared in Preparation Example 1, specimen preparation and EB1-EGFP and Cy5 Fluorescence observation was performed by ex vivo imaging.
  • the detection of fluorescence is the same as in Example 1 for EGFP, and for Cy5, the output is set to 4.0% at 640 nm Laser, the excitation light source is HV 80 (au), resolution 512 x 512, pixel This was carried out using a resonant scanner set to a size of 0.104 ⁇ m / pixel (Zoom 4) and Line Average 16.
  • the number of green bright spots (corresponding to GFP) observed for each cell during the imaging time (20 seconds) was 3000 per cell in Example 2 and 5800 per cell in the Comparative Example. Furthermore, a red bright spot (corresponding to Cy5) in each cell was extracted, and their fluorescence intensity was measured. For Cy5, analysis is performed using open software FIJI / ImageJ, and the average signal value of the autofluorescence image is subtracted and normalized per unit area in the region set as the region of interest (ROI). The Cy5 fluorescence intensity of each cell was measured.
  • Example 2 Cells in which green luminescent spots were observed in Example 2 were divided into two groups based on Cy5 fluorescence intensity, and for each group and comparative example, linear movement distances (linear distances of luminescent spots at the start and end points of imaging), luminescent spots The moving speed and the linearity of bright spot movement were measured, and the average value was calculated. Furthermore, the sum total of the linear movement distances of all bright spots contained in one cell was calculated.
  • Example 1 since the moving distance and moving speed of the green fluorescent luminescent spot are greatly reduced, it can be seen that the microtubule polymerization inhibitory action is working. Furthermore, information on the drug that has reached the cell can be obtained simultaneously from the fluorescence of Cy5 labeled with trastuzumab-emtansine. Cells with strong Cy5 fluorescence intensity, that is, cells with a large amount of drug arrival, have a smaller green fluorescent emission distance and movement speed than cells with low Cy5 fluorescence intensity, that is, cells with a small amount of drug arrival, and inhibit microtubule polymerization. It can be seen that the effect is large.
  • Example 3 The cancer-bearing mouse prepared in Preparation Example 1 was administered with a drug (trastuzumab-emtansine) in the same procedure as in Example 1, and a specimen was prepared in the same manner as in Example 1.
  • the prepared specimen was added to the fluorescent staining solution prepared in the culture solution (FluoroBrite DMEM Media; Thermo Fisher Scientific Co., Ltd.) so that the anti-IgG antibody-bound Texas Red-integrated melamine resin particles prepared in Preparation Example 3 were 0.1 nM. Fluorescence staining was performed by immersion and shaking at room temperature for 3 hours. After fluorescent staining, in order to remove excess fluorescent staining solution, it was placed in a tube containing 20 mL of phosphate buffered saline, left at room temperature for 15 minutes, and then immersed in 1 mL of culture solution.
  • the images acquired for 12 cells were analyzed with the image processing software “Imaris” in the same manner as in Example 1.
  • the number of green bright spots (corresponding to GFP) observed at the photographing time (20 seconds) for each cell was 4200 per cell in Example 1, and 6800 per cell in the Comparative Example. Of these, 280 cells per cell were extracted as examples, and 430 cells per cell were extracted as chase points in the comparative example. Further, for each green luminescent spot, the linear movement distance (linear distance of the luminescent spot at the start point and the end point of photographing), the moving speed of the luminescent spot, and the linearity of the luminescent spot movement were measured, and the average value was calculated. Furthermore, the sum total of the linear movement distances of all bright spots contained in one cell was calculated. Further, in addition to the above, the number of red bright spots (corresponding to Texas Red) in each cell was measured, and the average value per cell was calculated. Each result is shown in Table 3.
  • Example 1 since the movement distance and the number of bright spots of the green fluorescent bright spot are greatly reduced as compared with the comparative example, it can be seen that the microtubule polymerization inhibitory action is working. Furthermore, information on the amount of trastuzumab emtansine reaching the cells can be obtained simultaneously from the number of red bright spots indicating Texas Red used for fluorescent staining of trastuzumab emtansine. By staining the microtubule and the drug at the same time in this way, it is possible to understand the relationship between the amount of the drug reaching the cell and the dynamism of the microtubule, and to evaluate the drug more accurately. .

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Abstract

La présente invention concerne un procédé d'évaluation d'un médicament qui influence la dynamique des organites cellulaires, par capture d'une image fluorescente d'organites cellulaires au moyen d'animaux de laboratoire pour le médicament, le procédé comprenant: une étape de préparation d'échantillon dans laquelle un médicament qui influence la dynamique des organites cellulaires est administré à un animal de laboratoire ayant une lésion implantée avec des cellules exprimant une protéine de liaison d'organite cellulaire fusionnée à une protéine fluorescente, et un échantillon est préparé à partir d'une section de tissu obtenue à partir de la lésion de l'animal de laboratoire ; une étape de capture d'image fluorescente pour acquérir une image fluorescente de l'échantillon ; et une étape d'acquisition d'informations pour acquérir des informations à base de points fluorescents à partir de l'image de fluorescence.
PCT/JP2019/018948 2018-05-17 2019-05-13 Procédé d'évaluation d'un médicament WO2019221062A1 (fr)

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WO2022059510A1 (fr) * 2020-09-18 2022-03-24 コニカミノルタ株式会社 Dispositif de traitement d'informations de distribution de médicament, procédé de traitement d'informations de distribution de médicament et programme de traitement d'informations de distribution de médicament
WO2022059509A1 (fr) * 2020-09-18 2022-03-24 コニカミノルタ株式会社 Procédé d'analyse de situation de distribution de médicament et système d'analyse de situation de distribution de médicament
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WO2022059601A1 (fr) * 2020-09-16 2022-03-24 コニカミノルタ株式会社 Procédé de visualisation et procédé d'acquisition d'informations
WO2022059510A1 (fr) * 2020-09-18 2022-03-24 コニカミノルタ株式会社 Dispositif de traitement d'informations de distribution de médicament, procédé de traitement d'informations de distribution de médicament et programme de traitement d'informations de distribution de médicament
WO2022059509A1 (fr) * 2020-09-18 2022-03-24 コニカミノルタ株式会社 Procédé d'analyse de situation de distribution de médicament et système d'analyse de situation de distribution de médicament

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