KR20230054098A - Method and system for evaluating pharmacokinetic and pharmacodynamic distribution of materials using radiotracer in brain animal disease model - Google Patents

Abstract

The present invention relates to a method and system for evaluating pharmacokinetic and pharmacodynamic distribution of materials by using a radioactive isotope in a brain disease animal model. When there is no indication of binding and action in the brain and cardiovascular system for various candidate drugs, which are likely to be used for a combination administration therapy and are able to remedy side effects and weaknesses of an existing Alzheimer's disease (AD) medicine, the present invention may evaluate: what a specific target of a central nervous system (CNS) of a candidate drug material is from among a nerve cell, a neuropathology material (tau, amyloid), a neuroinflammatory cells, or a vascular endothelial cell; whether the candidate drug material has specific binding to a CNS target; and how a binding capacity of the candidate material and pharmacokinetic and pharmacodynamic properties thereof vary depending on changes in a permeability state of blood brain barriers (BBB) of a young normal control mouse, an aging mouse, and an AD transgenic mouse.

Description

Method and system for evaluating pharmacokinetic/pharmacodynamic distribution of substances using radioactive isotopes in brain disease animal models

The present invention relates to a method and system for evaluating the pharmacokinetic/pharmacodynamic distribution of a substance using a radioactive isotope in an animal model of brain disease.

Alzheimer's disease, a leading cause of dementia, currently exceeds 25 million worldwide, and is expected to reach 63 million in 2030 and 114 million in 2050. According to the announcement, the number of dementia patients will increase exponentially in the future, and it is estimated that there will be over 1 million in 2024 and over 2 million in 2041, and the cost of nursing care will also increase rapidly accordingly (Central Dementia Center 2013 Annual Report).

According to the statistics of the Alzheimer's Association (www.alzform.org), the prevalence of dementia is expected to increase by 3 times compared to 2010 by 2050 in the United States, while the prevalence of dementia is expected to increase by about 8 times or more during the same period in Korea, which is more rapid than the United States. implying an economic burden.

In this situation, pharmaceutical companies in each country are developing Alzheimer's treatments competitively. When the top 10 global drug markets are analyzed based on the growth rate by disease, AD treatments rank first, showing an average annual growth rate of more than 34%, and anticancer drugs 18.6%. %, followed by antihypertensive drugs at 18.1% (IMS MIDAS).

In the case of the domestic market, it is 200 billion won, and the size of the US market alone in 2010 is 6.6 billion dollars, and it is expected to reach 13.4 billion dollars in 2017 (GlobalData-PRLog, 2011). Among them, anti-amyloid antibody was recently approved by the US FDA as a treatment for dementia, but ARIA (amyloid related) was detected in brain MRI of a patient who received aducanumab, a representative anti-amyloid antibody. Cerebrovascular side effects such as vasogenic edema and microhemorrhage, which are seen as imaging abnormalities, have been reported.

Against this background, several candidate drugs that can protect the side effects and disadvantages of existing Alzheimer's drugs can be proposed as a combination therapy, but many of these candidate drugs have not been identified for their binding and action in the brain and cerebrovascular system. . Prior to determining the clinical effectiveness of the indication for co-administration of a candidate drug substance to the brain, it will be necessary to verify whether the candidate drug substance is a reliable biomarker targeting the brain or blood vessels.

Specifically, which of the specific targets of the central nervous system (CNS) of the candidate drug substance are neurons, neuropathology (tau, amyloid), neuroinflammatory cells, and vascular endothelial cells, and whether the candidate drug substance is a central nervous system (CNS) target target), and the binding capacity and pharmacodynamic/pharmacokinetic properties of the candidate substance are normal mice (young normal control), aging mouse (aging mouse), and Alzheimer's dementia mouse (AD transgenic mouse) blood-brain barrier (BBB) Only when the evaluation of how the drug changes according to the change in the permeability state of the drug can be used for the diagnosis and treatment of psychiatric disorders, and considering the indications for co-administration of the candidate drug with other drugs targeting the cerebrum, clinical You will be able to make a judgment about the validity of the enemy.

Republic of Korea Patent No. 10-2228460 Republic of Korea Patent Publication No. 10-2011-0046503

Accordingly, the present inventors go beyond the conventional fragmentary histopathological method and radiochemically label the candidate drug substance with [ ⁸⁹ Zr] -DFO or [ ¹²⁵ I] to obtain a positron emission tomography image using a radioactive isotope tracer ( PET) imaging ([ ⁸⁹ Zr]-DFO-labeled PET imaging) and ex-vivo autoradiography, nuclear track emulsion technique, three-dimensional fluorescence microscopy using tissue clearing pretreatment technique By examining the specific binding ability and pharmacokinetic/pharmacodynamic distribution using the latest multi-omics fusion technologies such as volume fluorescence-imaging using tissue clearing, the candidate drug substance is found in the brain or brain. We developed an optimal platform system that comprehensively evaluates whether there are reliable biomarkers targeting blood vessels.

In particular, in order to improve the penetration depth and clearing performance of the antibody when performing tissue clearing, the time of injection of the fluorescently labeled antibody, the tissue sectioning method, and the concentration of the permeation enhancer, for example, triton-X 100 solution, were tested under different conditions. do.

In the present invention, a method for evaluating the pharmacokinetic/pharmacodynamic distribution of a substance in an animal model of brain disease is provided.

The evaluation method includes: 1) labeling a material to be evaluated with a radioactive isotope; 2) injecting the material labeled with the radioactive isotope into an animal model of brain disease; 3) performing positron emission tomography (Positron Emission Tomography) of the animal model brain; 4) cross-sectioning the brain of the animal model and performing autoradiography; 5) Immunoscanning by immersing the brain of the experimental animal in the nuclear track emulsion; and 6) acquiring a 3D fluorescence microscopy image using a tissue clearing preprocessing technique.

The substance is an antibody, peptide, cytokine, immune checkpoint inhibitor, mitogen, growth factor, small RNA, dsRNA (double-stranded RNA), mononuclear blood cell, feeder cell, feeder cell component or It may be any one selected from the group consisting of a replacement factor, a vector comprising one or more polynucleic acids of interest, a chemotherapeutic agent, and a radioactive moiety.

The radioactive isotope may be any one of [ ⁸⁹ Zr] or [ ¹²⁵ I], but is not limited thereto.

In step 2), the material labeled with the radioactive isotope is injected through the tail vein of the animal model.

In step 3), the method may further include measuring standard uptake coefficients of the cerebral cortical sub-region and cerebellum through the PET static image.

In step 3), a step of measuring binding potential of the material by performing compartment analysis through the PET dynamic image may be further included.

In step 4), the distribution of the radioactive isotope-labeled material may be analyzed.

In step 5), immunostaining using a vascular endothelial marker (CD31) and an anti-amyloid antibody or an anti-neurofilament antibody may be further included.

In step 6), tissue clearing pretreatment is performed, and the injection time of the fluorescently labeled antibody, the tissue sectioning method, and the concentration of the permeation enhancer, for example, triton-X 100 solution, can be adjusted to improve the penetration depth and clearing performance of the antibody. The permeation enhancer dissolves cells with a surfactant and promotes the movement of the substance to be evaluated into cells.

In step 6), the fluorescence microscope image may be taken with a macro laser light-sheet illumination imaging system, an ultramicroscope, or a two-photon microscope, but is not limited thereto. no.

The distribution of cells or pathological substances in the brain of the animal model may be analyzed through a 3D fluorescence microscopy image using the tissue clearing preprocessing technique of step 6), but is not limited thereto.

It may be characterized in that it further comprises the step of synthesizing the evaluation results of the steps 3) to 6).

According to another embodiment of the present invention, a system 100 for evaluating the pharmacokinetic/pharmacodynamic distribution of a substance in an animal model of brain disease is provided.

The processor 110 may be implemented as one or more processors and may be configured to process commands of a computer program by performing basic arithmetic, logic, and input/output operations. Commands may be provided to the processor 110 by the storage medium 150 and the communication unit 120 . For example, the processor 110 may be configured to execute commands received according to program codes stored in a recording device such as the storage medium 150 .

The communication unit 120 may provide a function for communicating with external devices (PET image capture device, 3D fluorescence microscope image capture device, etc.) through a network. For example, a request generated according to a program code stored in a recording device such as a storage medium 150 by the processor 110 of the drug efficacy evaluation system 100 is sent to other electronic devices through a network under the control of the communication unit 120. can be conveyed For example, a control signal or command received through the communication unit 120 may be transmitted to the processor 110 or the storage medium 150, and the received image may be stored in the storage medium 150.

The storage medium 150 is a computer-readable recording medium, and may include a random access memory (RAM), a read only memory (ROM), and a permanent mass storage device such as a disk drive. In addition, an operating system and at least one program code may be stored in the storage medium 150 . These software components may be loaded from a computer-readable recording medium separate from the storage medium 150 using a drive mechanism. The separate computer-readable recording medium may include a computer-readable recording medium such as a floppy drive, a disk, a tape, a DVD/CD-ROM drive, and a memory card. In another embodiment, software components may be loaded into the storage medium 150 through the communication unit 120 rather than a computer-readable recording medium. For example, at least one program is stored on the storage medium 150 based on a program (for example, the above-described application) installed by files provided through a network of developers or a file distribution system that distributes installation files of applications. can be loaded into

The input unit 130 may receive an input from a user. The input unit 130 may include an operation panel that receives user input. Specifically, the input unit may include devices capable of receiving various types of user inputs, such as a keyboard, a physical button, a touch screen, a camera, or a microphone. The input unit 130 may receive input of preliminary data necessary for drug efficacy evaluation. The prior data may include, but is not limited to, experimental results for measuring cognitive recovery index, PET images, 3D fluorescence microscopy images, gene expression information, and experimental data for side effect evaluation, etc., based on the drug efficacy evaluation. Any material is possible.

The output unit 140 may display a screen providing information. A user interface generated by the processor may be output. The output unit 140 may output a web page corresponding to a user input. For example, the output unit 140 may include a display panel displaying a screen. Also, the output unit may include a display panel or a speaker.

The storage medium 150 of the system 100 includes a standard uptake coefficient measurement unit 151 for measuring the standard uptake coefficient of the cerebral cortical sub-region and the standard uptake coefficient of the cerebellum through PET static images; a binding capacity measurement unit 152 for measuring binding potential of the material by performing compartment analysis through PET dynamic images; an autoradiography analysis unit 153 that analyzes the distribution of a material labeled with a radioactive isotope through an autoradiography result; a pathological material distribution analysis unit 154 for analyzing a distribution pattern of pathological materials through immunoscanning by immersion in the nuclear track emulsion; It may include a 3D fluorescence microscope image processing unit 155 that analyzes the distribution of cells or pathological substances in the brain of the animal model through 3D fluorescence microscopy images using a tissue clearing preprocessing technique.

The system may further include a final result calculation unit 156 for integrating evaluation results and calculating a final result.

Although the methods and systems of the present invention have been described with reference to specific embodiments, some or all of their components or operations may be implemented using a computer system having a general-purpose hardware architecture.

Among several candidate drugs to be considered for combined administration therapy that can protect the side effects and disadvantages of existing Alzheimer's treatments, the present invention is concerned with the central nervous system (central nervous system) of candidate drug substances when the binding and action in the brain and cerebrovascular system are not known. system, CNS), whether the specific target is nerve cells, neuropathological substances (tau, amyloid), neuroinflammatory cells, and vascular endothelial cells, and whether the candidate drug substance specifically binds to the cerebral target (CNS target), and the candidate How the binding capacity and pharmacodynamic/pharmacokinetic properties of substances change according to changes in the permeability of the blood-brain barrier (BBB) in young normal control, aging mouse, and AD transgenic mouse can be evaluated. The candidate drug substance verified by the system of the present invention can be used as a drug delivery system for diagnosis and treatment of psychiatric disorders, a drug delivery system for dementia antibody treatment with great clinical impact, or as a combination administration agent for synergistic effects.

1 is a diagram schematically showing the neuroprotective effect evaluation method of the present invention.
Figure 2 is a diagram schematically showing a system for the neuroprotective effect evaluation method of the present invention.

Hereinafter, the configuration of the present invention will be described in more detail through examples, but the following examples are intended to aid understanding of the present invention, and the scope of the present invention is not limited thereto.

<Example>

1. Imaging experiments to check the blood brain barrier (BBB) permeability of animal models

1) By injecting [ ¹⁸ F] fluorothymidine (FLT), a radioactive isotope that normally does not pass through the blood-brain barrier, and checking whether it passes through the brain, how was the BBB permeability of AD transgenic mice and normal control mice? Check if it is different.

- Tail vein injection of [ ¹⁸ F]fluorothymidine (FLT) to AD transgenic mice and normal control mice.

- Acquire static PET images for 20 minutes 1 hour after injection.

- Analyze the intake of Brain PET by visual analysis and standard intake factor (SUV) method.

2. Measurement of labeling yield of radioactive isotope labeled material

[ ⁸⁹ Zr] -DFO and [ ¹²⁵ I] are labeled for the material to be checked for BBB permeability, and labeling efficiency is measured. For radioactive isotope labeling, refer to the method of a previous paper [3].

3. To see the pharmacokinetic / pharmacodynamic distribution of the substance in the cerebral blood vessel or nerve tissue area in the in vivo system [ ⁸⁹ Zr]-DFO-labeled PET imaging and in-vivo quantification

AD transgenic mice and normal control mice were anesthetized with isoflurane, placed on a microPET bed, and 0.5 mCi of [ ⁸⁹ Zr]-DFO-labeled material was injected into the tail vein, followed by 90-minute dynamic PET imaging or 60-minute, 24-hour After obtaining static PET images after (1 day), 48 hours (2 days), and 144 hours (6 days), standardized uptake values (SUVs) of cortex subregions were obtained for static images. ) is calculated by normalizing SUVR with cerebellum SUV, or BP _nd (binding potential) is calculated by performing compartment analysis in dynamic images.

4. Ex-vivo autoradiography for precise anatomical localization between blood vessel wall/brain tissue of radioisotope-coupled materials in ex vivo tissue

After MicroPET imaging is completed, the brain is removed and sectioned, followed by autoradiography. For the specific autoradioraphy method, refer to the method implemented in the following paper [4].

5. Ex-vivo nuclear track emulsion for precise anatomical localization between blood vessel wall/brain tissue of radioisotope-coupled materials in ex vivo tissues

AD transgenic mice were injected with [ ¹²⁵ I]-labeled material and The brain is removed and subjected to saline perfusion. Afterwards, the extracted brain tissue is cut into 5 mm thickness at minus 80°C. When a section immunostained with a vascular endothelial marker (CD31) and an anti-amyloid antibody or an anti-neurofilament antibody is immersed in ILFORD K5 emulsion in a dark room, the radiolabeled antibody (otracerlabelled) Antibody sensitized with silver grain on immunostained cross-sections to determine whether antibodies labeled with radioactive isotopes are attached to the vascular endothelium wall, cells of brain parenchyme or amyloid , spatial co-distribution patterns with pathological substances such as tau are also visualized. For the specific protocol, refer to the method described in the following paper [5].

6. 3-dimensional fluorescence imaging of the whole brain using a tissue clearing pre-processing technique for localization and three-dimensional distribution of fluorescently labeled substances in ex vivo tissues

In order to improve the penetration depth and clearing performance of the antibody when performing clearing, the time of injection of the fluorescently labeled antibody, the thickness of tissue sectioning, and the concentration of the triton-X 100 solution are tested under different conditions.

- After fluorescently labeling the substance, inject the fluorescently labeled substance into the jugular vein 2 days before, 1 day before, just before perfusion or during perfusion.

- Lectin for blood vessel staining (Wheat germ agglutinin (WGA) or Lycopersicon esculentum lectin (LEL)) is injected into mice immediately before perfusion.

- After injecting 4% PFA and proceeding with perfusion, extract the cerebrum.

- Section the extracted cerebrum into 1mm, 2mm, and 3mm thickness.

- The tissue is treated with 4% PFA and fixed (fixation). To help antibody penetration, triton-X-100 solution is treated differently at three different concentrations: 0.1%, 0.3%, and 0.5%.

- Treat the tissue clearing solution.

- Treat the permeability solution. Treat with three concentrations of 0.1xPBS, 0.1%, 0.3%, and 0.5% Triton X-100, 20% DMSO, and Serum.

- Process volume immune staining. Volume immunostaining is performed for neuronal axon (chicken neurofilament antibodies) or amyloid (anti-AβAb or thioflavin S).

- Macro laser light-sheet illumination imaging system, UltraMicroscope, two-photon microscopy, or confocal microscopy.

[Reference]

1. Carpenter, Timothy S., et al. "A method to predict blood-brain barrier permeability of drug-like compounds using molecular dynamics simulations." Biophysical journal 107.3 (2014): 630-641.

2. Wolff, Anette, et al. "In vitro blood-brain barrier models—an overview of established models and newmicrofluidic approaches." Journal of pharmaceutical sciences 104.9(2015): 2727-2746.

3. Price, Eric W., et al. " ⁸⁹ Zr-DFO-AMG102 immuno-PET to determine local hepatocyte growth factor protein levels in tumors for enhanced patient selection." Journal of Nuclear Medicine 58.9 (2017): 1386-1394.

4. Aguero, Cinthya, et al. "Autoradiography validation of novel tau PET tracer [F-18]-MK-6240 on human postmortem brain tissue." Actaneuropathologica communications 7.1 (2019): 1-15.

5. Lane, T. "Technical information Ilford nuclear emulsions." HARMANTechnology Ltd., Mobberley, England, UK (2005).

Claims (13)

1) labeling the material to be evaluated with a radioactive isotope;
2) injecting the material labeled with the radioactive isotope into an animal model of brain disease;
3) performing positron emission tomography (Positron Emission Tomography) of the animal model brain;
4) cross-sectioning the brain of the animal model and performing autoradiography;
5) Immunoscanning by immersing the brain of the experimental animal in a nuclear track emulsion; and
6) A method for evaluating the pharmacokinetic/pharmacodynamic distribution of a substance in an animal model of brain disease, comprising acquiring a three-dimensional fluorescence microscopy image using a tissue clearing preprocessing technique.
According to claim 1,
The radioisotope is an evaluation method, characterized in that any one of [ ⁸⁹ Zr] or [ ¹²⁵ I].
According to claim 1,
Evaluation method characterized by injecting the material labeled with the radioactive isotope in step 2) through the tail vein of the animal model.
According to claim 1,
The evaluation method characterized in that it further comprises the step of measuring the standard uptake coefficient of the cerebral cortical sub-region and the standard uptake coefficient of the cerebellum through the PET static image in step 3).
According to claim 1,
The evaluation method characterized in that it further comprises the step of measuring the binding potential of the material by performing compartment analysis through the PET dynamic image in step 3).
According to claim 1,
Evaluation method characterized in that for analyzing the distribution of the radioactive isotope-labeled material in step 4).
According to claim 1,
In step 5), the evaluation method further comprises performing immunostaining using a vascular endothelial marker (CD31) and an anti-amyloid antibody or an anti-neurofilament antibody.
According to claim 1,
In order to improve the penetration depth and clearing performance of the antibody when performing the tissue clearing pretreatment technique in step 6), adjusting the injection time of the fluorescently labeled antibody, the tissue sectioning method, and the concentration of the permeation enhancer, for example, triton-X 100 solution Characterized evaluation method.
According to claim 1,
In step 6), the fluorescence microscope image is taken with a macro laser light-sheet illumination imaging system, an ultramicroscope or a two-photon microscope Evaluation method, characterized in that.
According to claim 1,
Evaluation method, characterized in that for analyzing the distribution of cells or pathological substances in the brain of the animal model through a three-dimensional fluorescence microscope image using the tissue clearing pretreatment technique of step 6).
According to claim 1,
The evaluation method further comprising the step of synthesizing the evaluation results of steps 3) to 6).
A system for evaluating the pharmacokinetic/pharmacodynamic distribution of a substance in an animal model, the system comprising:
1) a standard uptake coefficient measuring unit that measures the standard uptake coefficient of the cerebral cortex subregion and the standard uptake coefficient of the cerebellum through PET static images;
2) a binding capacity measurement unit for measuring binding potential of the material by performing compartment analysis through PET dynamic images;
3) an autoradiography analysis unit for analyzing the distribution of a radioactive isotope-labeled material through an autoradiography result;
4) a pathological substance distribution analysis unit that analyzes the distribution pattern of pathological substances through immunoscanning by immersion in the nuclear track emulsion;
5) A system including a 3D fluorescence microscopy image processing unit that analyzes the distribution of cells or pathological substances in the brain of the animal model through 3D fluorescence microscopy images using a tissue clearing preprocessing technique.
According to claim 12,
The system further comprises a final result calculation unit for integrating the evaluation results and calculating a final result.

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