JP2019100945A - Method for imaging protein - Google Patents

Abstract

To provide a method for non-invasively imaging a targeted protein in a sample separately from a non-targeted protein.SOLUTION: The method for imaging protein according to an embodiment includes the steps of: determining an imaging agent (step 1); determining a non-targeted protein having a binding affinity with the imaging agent (step 2); preparing a labelled imaging agent (step 3); preparing a masking agent (step 4); applying the masking agent and the labelled imaging agent to the sample (step 5); and detecting a signal from the label and preparing an image (step 6), the masking agent having a binding affinity with the targeted protein and the non-targeted protein, and the binding affinity of the masking agent with the targeted protein being lower than that of the imaging agent with the targeted protein and the binding affinity of the masking agent with the non-targeted protein being higher than that of the imaging agent with the non-targeted protein.SELECTED DRAWING: Figure 1

Description

  Embodiments of the present invention relate to methods of imaging proteins.

  Mutation of the epidermal growth factor receptor (EGFR) is one of the causes of lung cancer. In patients with EGFR mutation-positive lung cancer, when the molecule-targeted therapeutic agents afatinib and gefitinib are continuously administered for several months to a year and a half, there are cases where the mutant EGFR is further mutated and the therapeutic agent fails. An example of such a further mutation is the T790M mutation in which 790th threonine in exon 20 of the mutant EGFR is mutated to methionine. The T790M mutation accounts for about 50% of resistant mutant EGFR mutations in Japanese patients. Osimertinib is known as a therapeutic agent for the T790M mutation. Although administration of osimerinib has been approved in patients with T790M mutation positive cases, administration to negative cases has not been approved by health insurance. Therefore, a method for detecting T790M mutation with high sensitivity is required. As a method for detecting the T790M mutation, for example, a tissue biopsy for collecting a tissue of a lesion and detecting the T790M mutation, a liquid biopsy for collecting a blood and detecting a gene derived from the T790M mutation, and the like are performed.

  On the other hand, as a method of detecting a disease such as cancer, there is a method of imaging a biomarker such as a protein associated with a disease using an imaging diagnostic apparatus. In imaging, a labeled low molecular weight compound, antibody or the like that binds to a biomarker is administered to a patient, and a signal obtained from the low molecular weight compound or antibody is detected by an imaging diagnostic apparatus. Imaging is an important technology, for example, in the diagnosis or stratification treatment of diseases, and there is a need to develop a method for more accurately detecting a biomarker.

Hirano et al. (2015) Oncotarget, Vol. 6, No. 36, 38789-38803 "In vitro modeling to determine mutation specificity of EGFR tyrosine kinase inhibitors against clinically relevant EGFR mutants in non-small-cell lung cancer"

  The tissue biopsy accurately detects the T790M mutation because the tissue may not be collected from all the lesions, or the tissue may not be collected from all the lesions. I can not do it. As for liquid biopsy, the target T790M mutation-derived gene is not always released into the blood, and even if the gene can be collected, whether it is from the primary lesion or from the metastatic lesion. I can not tell if there is any. In addition, when the test target of these test methods is a patient with advanced cancer, the patient may not tolerate the collection of tissue and blood.

  Imaging overcomes the problems of tissue biopsy and liquid biopsy, as it can detect systemic lesions at once without collecting tissue or blood. However, low molecular weight compounds and antibodies used for imaging in most cases also bind to out-of-test biomarkers having a similar structure to the biomarkers to be tested. Therefore, there is a problem that the test result is false positive and the target biomarker can not be detected accurately.

  The problem to be solved by the present invention is to provide a method for imaging a target protein in a sample in a noninvasive manner in a differentiated manner from non-target proteins.

  The method of imaging a protein according to the embodiment is a method of imaging a target protein in a sample, and includes the following steps. (S1) Determining an imaging agent having binding affinity with a target protein. (S2) Determining a non-target protein having a binding affinity with an imaging agent. (S3) Providing a labeled imaging agent in which the imaging agent is labeled with a labeling substance. (S4) Prepare a masking agent. Here, the masking agent has binding affinity to the target protein and non-target protein, and the binding affinity to the target protein is lower than that of the imaging agent, and the binding affinity to the non-target protein is imaging agent Higher than that of (S5) Administration of a masking agent and a labeled imaging agent to the sample. (S6) Detecting a signal from a labeled substance, and creating an image based on the result of the detection.

It is a flowchart which shows an example of the method of 1st Embodiment. It is a figure explaining the principle of imaging in the method of a 1st embodiment. It is a flowchart which shows an example of the method of 2nd Embodiment. It is a figure explaining the principle of imaging in the method of a 2nd embodiment. It is a figure explaining the principle of imaging in the method of a 2nd embodiment. It is a flowchart which shows an example of the method of 3rd Embodiment. It is a flowchart which shows an example of the method of 4th Embodiment. It is a flowchart which shows an example of the method of 5th Embodiment. It is a figure explaining the principle of the imaging in the method of 5th Embodiment. It is a figure explaining the principle of the imaging in the method of 5th Embodiment.

  Hereinafter, various embodiments will be described with reference to the drawings.

  The method of imaging a protein according to the embodiment is a method of imaging a target protein in a sample.

  The sample is a target on which the examination by the imaging method of the embodiment is to be performed, and may be a target protein that is a target of imaging. The sample is, for example, an animal. An animal is, for example, an organism belonging to mammals, birds, amphibians, reptiles or fish. Mammals belong to, for example, primates such as monkeys and humans, rodents such as mice, rats and guinea pigs, companion animals such as dogs, cats and rabbits, domestic animals such as horses, cows and pigs, or display animals etc. It is a mammal. The sample may be, for example, a tissue or cultured cell derived from the above animal.

  The target protein is a protein to be imaged in the imaging method of the embodiment that may be present in the sample. The target protein is, for example, a protein serving as an index for determining the presence, nature, course, or response to a drug, such as a lesion or lesion of a particular disease, etc., at or near the lesion or lesion of a particular disease. It is a protein specifically expressed. Such target proteins may be, for example, biomarkers. The biomarker may be, for example, a diagnostic marker, a prognostic marker, a pharmacodynamic marker, a monitoring marker or a therapeutic marker. For example, therapeutic markers are targets for molecular targeted therapy of particular diseases.

Specific diseases are, for example, proliferative diseases, inflammatory diseases, autoimmune diseases, mental diseases and the like. The proliferative disorder is, for example, a cancer or a tumor. Examples of cancer or tumor include colorectal cancer, colon cancer, colon cancer, stomach cancer, breast cancer, lung cancer, small cell lung cancer, non-small cell lung cancer, liver cancer, pancreas cancer, thyroid cancer, kidney cancer, brain cancer, cervical cancer, head and neck Head cancer, central nervous system (CNS) cancer, bladder cancer, prostate cancer, testicular cancer, uterine cancer, cervical cancer, ovarian cancer, lymphoma, neuroblastoma, myeloma and / or leukemia and the like.
First Embodiment Hereinafter, a method of imaging a protein according to the first embodiment will be described with reference to FIG. FIG. 1 is a schematic flowchart showing an example of the method of the first embodiment.

  First, in step (S1), an imaging agent is determined. An imaging agent is a substance for imaging a target protein. That is, the imaging agent is a substance having binding affinity to the target protein, and the target protein can be imaged by binding the labeled imaging agent to the target protein.

The term "having binding affinity" for a target protein refers to, for example, being capable of binding to the target protein. Whether it has a binding affinity can be determined, for example, by measuring or calculating an in vitro 50% inhibitory concentration (IC ₅₀ ) value, a dissociation constant or a binding constant, and the like. For example, as the imaging agent, it is preferable to use a substance having a smaller IC ₅₀ value for binding to a target protein or a dissociation constant, and for the binding constant, it is preferable to use a substance having a larger value. That is, it is preferable to use a substance that is more likely to bind to the target protein.

  The imaging agent may be a substance expected to have affinity to the target protein. For example, the imaging agent may be determined according to the structure of the target protein, or a substance having affinity to the target protein may be inferred from actual pharmacological effects or experimental imaging results, and used as the imaging agent. It is also good. Alternatively, a substance known to have binding affinity with a target protein may be used as an imaging agent.

  As an imaging agent, for example, a low molecular weight compound, an antibody, a peptide or a DNA aptamer can be used.

  The low molecular weight compound is, for example, a compound having a molecular weight of about 200 to about 1000 having binding affinity to the target protein. For example, when the target protein is an enzyme, the low molecular weight compound may be an enzyme inhibitor. When the target protein is a receptor, the small molecule compound may be a ligand. Alternatively, the low molecular weight compound may be a compound referred to as “small molecular drug” or “small molecular drug” or the like which is used as a molecular targeted therapeutic drug for a target protein. Such low molecular weight compounds are, for example, tyrosine kinase inhibitors, anaplastic lymphoma kinase inhibitors, Janus kinase inhibitors, poly ADP ribose polymerase (PARP) inhibitors, Raf kinase inhibitors, MEK inhibitors, CDK inhibitors or It may be a proteasome inhibitor or the like.

  An antibody is, for example, an antibody having binding affinity to a target protein. The antibody may be an antibody referred to as "antibody drug" or "antibody drug" or the like which is used as a molecular target therapeutic drug for a target protein. The antibody is, for example, a monoclonal antibody. The antibody may be, for example, a mouse antibody, a chimeric antibody, a humanized antibody or a humanized antibody, or a fragment antibody thereof.

  For example, one imaging agent is selected for one target protein.

  Next, non-target proteins are determined in step (S2).

  Non-target proteins are proteins present in a sample and having binding affinity with an imaging agent. When nontarget proteins are to be imaged with the imaging agent for targeting proteins, the imaging agents are bound because they have binding affinity with the imaging agent, and they are not targeted for imaging that interferes with specific imaging of the target protein. It is a protein. That is, non-target proteins are proteins that can cause false positive results in imaging.

  For example, the non-target protein and the target protein can each have a binding site that can bind to the imaging agent. For example, the non-target protein and the target protein may be a normal protein and a variant thereof. Also, for example, the non-target protein and the target protein may be a mutant protein and a further mutant protein.

  The non-target protein may be a protein predicted to have affinity with the imaging agent. For example, a non-target protein may be determined by structure, or a protein having affinity with an imaging agent may be inferred from actual pharmacological effects or experimental imaging results, and this may be used as a non-target protein. Good. Alternatively, a protein other than a target protein known to have affinity with the imaging agent may be a non-target protein.

  Next, in step (S3), a labeled imaging agent is prepared.

The labeled imaging agent is an imaging agent labeled with a labeling substance. The labeling substance is, for example, a radioactive isotope that emits a positron (positron). Examples of radioactive isotopes that emit positrons include ¹¹ C (carbon 11), ¹³ N (nitrogen 13), ¹⁵ O (oxygen 15), ¹⁸ F (fluorine 18) and the like. The radioactive isotopes that emit positrons are, for example, radioactive isotopes that can be detected by positron emission tomography (PET).

  The labeled imaging agent labeled with a radioactive isotope that emits a positron is, for example, one obtained by replacing any of the elements such as carbon, nitrogen and oxygen contained in the imaging agent with the radioactive isotope. Multiple atoms of the same type of imaging agent may be substituted with the same type of radioactive isotope. Alternatively, any of the above-mentioned radioactive isotopes may be added to any position of the imaging agent which does not affect the binding affinity to the target protein.

Alternatively, the labeling substance may be a compound containing a metal radioactive isotope that emits gamma rays. As such a metal radioactive isotope, ^99m Tc (technetium 99m), ¹¹¹ In (indium 111), ⁶⁷ Ga (gallium 67), ²⁰¹ Tl (thallium 201) or the like can be used. Metal radioactive isotopes that emit gamma rays are, for example, radioactive isotopes that can be detected by Single photon emission computed tomography (SPECT).

The compound containing a metal radioactive isotope is, for example, a complex. The complex has any of the above-mentioned metal radioisotopes as a central metal and has a suitable structure for administration to a sample. For example, preferred ^99m Tc complexes are oxo core complexes, monooxo core complexes or N ₂ S ₂ type complexes, N ₃ S type complexes, N ₄ complexes or dioxo core complexes, etc.

  The labeled imaging agent labeled with a metal radioactive isotope as described above is, for example, one obtained by adding a compound containing any of the above-mentioned metal radioactive isotopes to any position of the imaging agent. When a complex of a metal radioactive isotope is used as a labeling substance, the imaging agent is preferably an antibody.

Alternatively, the labeling substance may be a radioactive isotope of iodine which emits gamma rays. The radioactive isotope of iodine is, for example, ¹²³ I or ¹³¹ I and the like. The labeled imaging agent labeled with such a labeling substance is, for example, one obtained by adding the radioactive isotope of any of the above to any position of the imaging agent.

  In the step (S4), a masking agent is prepared.

  The masking agent is a protein for masking non-target proteins. That is, the masking agent is a substance that binds to non-target proteins in the sample and prevents the labeled imaging agent from binding to non-target proteins. The masking agent is a substance having binding affinity with a non-target protein. The masking agent also has binding affinity with the target protein. And, the binding affinity between the masking agent and the target protein is lower than the binding affinity between the imaging agent and the target protein, and the binding affinity between the masking agent and the non-target protein is the binding between the imaging agent and the non-target protein Less than affinity. Details of this relationship will be described later.

  As a masking agent, for example, a low molecular weight compound, an antibody, a peptide or a DNA aptamer can be used.

  The low molecular weight compound is, for example, a compound having a molecular weight of about 200 to about 1000 having binding affinity to a non-target protein. For example, when the non-target protein is an enzyme, the low molecular weight compound may be an enzyme inhibitor. When the non-target protein is a receptor, the small molecule compound may be a ligand. Alternatively, the low molecular weight compound may be a compound referred to as "small molecular drug" or "small molecular drug" or the like which is used as a molecular target therapeutic drug for non-target proteins. Such low molecular weight compounds are, for example, tyrosine kinase inhibitors, anaplastic lymphoma kinase inhibitors, Janus kinase inhibitors, poly ADP ribose polymerase (PARP) inhibitors, Raf kinase inhibitors, MEK inhibitors, CDK inhibitors or It may be a proteasome inhibitor or the like.

  An antibody is, for example, an antibody having binding affinity to a non-target protein. The antibody may be an antibody referred to as "antibody drug" or "antibody drug" or the like, which is used as a molecular target therapeutic drug for non-target proteins. The antibody is, for example, a monoclonal antibody. The antibody may be, for example, a mouse antibody, a chimeric antibody, a humanized antibody or a humanized antibody, or a fragment antibody thereof.

  The masking agent is, for example, one selected for one non-target protein.

  The imaging agent and the masking agent are different substances. However, the same substance may be used. For example, the imaging agent and the masking agent may both be low molecular weight compounds. Alternatively, the imaging agent and the masking agent may be of different types. For example, the imaging agent may be a low molecular weight compound and the masking agent may be an antibody.

  The relationship between the affinity of the imaging agent and the masking agent to target proteins and non-target proteins is described in detail below. The imaging agent and the masking agent have binding affinity with non-target protein and target protein, respectively. And, the binding affinity between the target protein and the masking agent is lower than the binding affinity between the target protein and the imaging agent, and the binding affinity between the non-target protein and the masking agent is that between the non-target protein and the imaging agent Less than affinity. That is, as shown in Table 1 below, the degree of binding affinity between the target protein and the imaging agent is “A”, the degree of binding affinity between the target protein and the masking agent is “B”, imaging with the non-target protein Assuming that the degree of binding affinity to the agent is "C" and the degree of binding affinity of the non-target protein to the masking agent is "D", the relational expressions A> B and C <D hold.

Also, for example, as the in vitro IC ₅₀ value shows a lower value as the binding affinity is higher, the IC ₅₀ value of the binding to the target protein is smaller in the imaging agent than in the masking agent, and to the non-target protein the IC ₅₀ values for binding of the direction of the masking agent is less than the imaging agent. That is, as shown in Table 2 below, IC and an IC ₅₀ value of the target protein and the imaging agent "a", an IC ₅₀ value of the target protein and the masking agent "b", a non-target protein and imaging agents _Assuming that the ₅₀ value is “c” and the IC ₅₀ value of the non-target protein and the masking agent is “d”, the relational expressions of a <b and c> d hold.

  The labeled imaging agent and the masking agent are preferably provided in a form suitable for administration to a sample in step (S5) described later. For example, the labeled imaging agent and the masking agent are provided in the form contained in one pharmaceutical composition. Alternatively, the labeled imaging agent and the masking agent are provided in the form contained in separate pharmaceutical compositions. The pharmaceutical composition contains, for example, a pharmaceutically acceptable carrier, a diluent, an excipient and the like. The pharmaceutical composition is, for example, in the form of a liquid for intravenous injection.

  A pharmaceutical composition containing a labeled imaging agent labeled with a radioactive isotope that emits positrons is, for example, a preparation for PET. A pharmaceutical composition comprising a labeled imaging agent labeled with a radioactive isotope that emits gamma rays is, for example, a preparation for SPECT.

  The pharmaceutical composition may be obtained commercially, for example, or may be prepared at the place where the imaging method of the embodiment is performed. Since the pharmaceutical composition containing a labeled imaging agent labeled with a radioactive isotope emitting positrons has a short half-life of the radioactive isotope, it is prepared using, for example, a medical cyclotron at the place where the imaging method of the embodiment is performed. It is preferable to do.

  Next, the step (S5) will be described with reference to FIG. First, the masking agent 2 and the labeled imaging agent 3 are administered to the sample 1 ((a) of FIG. 2). The labeled imaging agent 3 is obtained by labeling the imaging agent 4 with the labeling substance 5. The administration can be carried out by, for example, intravenous injection of the pharmaceutical composition prepared in step (S4). When the labeled imaging agent 3 and the masking agent 2 are prepared separately, one may be administered followed by the other sequentially. When a pharmaceutical composition containing both the labeled imaging agent 3 and the masking agent 2 is prepared, it may be administered.

  Since the degree of binding affinity to the administered target protein 6 is higher in the labeled imaging agent 3 than in the masking agent 2, the labeled imaging agent 3 preferentially binds to the target protein 6 (FIG. 2 (b )). Further, since the degree of binding affinity to non-target protein 7 is higher in masking agent 2, masking agent 2 preferentially binds to non-target protein 7 ((b) in FIG. 2). However, since the masking agent 2 also has affinity to the target protein 6, it can also bind to the target protein 6 (arrow 8 in (b) of FIG. 2). In that case, the masking agent 2 dissociates from the target protein 6 when the labeled imaging agent 3 approaches, and the labeled imaging agent 3 binds thereto ((c) in FIG. 2). Moreover, since the labeled imaging agent 3 also has affinity with the non-target protein 7, the labeled imaging agent 3 can also bind to the non-target protein 7 (arrow 9 in (b) of FIG. 2). In that case, the labeled imaging agent 3 dissociates when the masking agent 2 approaches, and the masking agent 2 binds thereto ((c) in FIG. 2). Thereafter, the masking agent 2 and the labeled imaging agent 3 which are not bound to the non-target protein 7 and the target protein 6 are washed away ((d) in FIG. 2). Thus, the labeled imaging agent 3 can bind to the target protein 6 without binding to the non-target protein 7. As a result, the target protein 6 is labeled by the labeling substance 5.

  For example, the difference in affinity between the masking agent 2 and the labeled imaging agent 3 for the target protein 6, ie, “A” and “B” shown in Table 1 or “a” and “b” shown in Table 2 The larger the difference, the lower the possibility that the masking agent 2 will bind to the target protein 6, and the higher the possibility that the masking agent 2 bound to the target protein 6 will replace the labeled imaging agent 3. For example, the value of "a" in Table 2 is preferably one tenth or less of the value of "b". It is more preferable if it is 1/100 or less.

  Also, for example, the differences in the affinity of masking agent 2 and labeled imaging agent 3 for non-target protein 7, ie, "C" and "D" shown in Table 1, or "c" and "d" shown in Table 2 The larger the difference in, the lower the possibility of the labeled imaging agent 3 binding to the non-target protein 7 and the higher the possibility of replacing the imaging agent 3 bound to the non-target protein 7 with the masking agent 2. For example, the value of “d” in Table 2 is preferably one tenth or less of the value of “c”. It is more preferable if it is 1/100 or less.

  Next, in step (S6), a signal from the labeling substance is detected, and an image is created based on the result of the detection.

  When the labeling substance is a radioactive isotope that emits a positron (positron), the signal is emitted in the opposite direction 180 ° resulting from the annihilation of a positron emitted from the radioactive isotope with a nearby electron 2 It is annihilation radiation (gamma ray) of a book. For example, signals can be detected and imaged by co-counting gamma rays emitted in such two directions. That is, by simultaneously detecting two gamma rays with a plurality of gamma ray detectors arranged in a ring around the sample, a radioactive isotope is present on the line connecting the gamma ray detectors in which the gamma rays are detected. Data can be obtained, for example a sinogram. By reconstructing the obtained data, the distribution of radioactive isotopes can be obtained as a tomographic image. For example, detection of such annihilation radiation and creation of an image are PET-CT combining a positron emission tomography (PET) apparatus, a PET apparatus and a computed tomography (CT) apparatus. It can be performed by a nuclear medicine diagnostic apparatus such as an apparatus or a TOF-PET (time-of-flight PET) apparatus.

  When the labeling substance is a radioactive isotope emitting gamma rays, the signal is gamma rays emitted from the radioactive isotope. For example, by detecting such gamma rays with a collimator on the sample side, for example, a gamma ray detector provided with a parallel collimator, the position and power pulse of the gamma ray detector where the gamma ray has arrived and the power pulse is output It is possible to obtain data such as the number of times it has been A planar image can be created from the obtained data. The distribution of radioactive isotopes can be obtained as tomographic images by detecting gamma rays from various directions of a sample with a gamma ray detector, collecting planar images, and reconstructing images with a computer. For example, such gamma ray detection and imaging can be performed by nuclear medicine diagnosis such as single photon emission computed tomography (SPECT) apparatus or SPECT-CT apparatus combining SPECT apparatus and CT apparatus. It can be done using the device.

  The detection of the signal and the creation of the image may be performed on the whole of the sample or on a part of the sample.

  According to the method of imaging a protein described above, it is possible to efficiently prevent the imaging agent from binding to a non-target protein that is not to be imaged. As a result, the imaging result is prevented from being false positive, and the target protein can be detected with high sensitivity. In addition, according to the method of the embodiment, it is possible to non-invasively detect the presence or absence or distribution of the target protein all over the sample at once even in a sample which can not tolerate collection of tissue or blood.

  When the target protein is a protein associated with a specific disease, and the target protein is imaged by the method of the embodiment, for example, it can be diagnosed that the sample suffers from the specific disease. In addition, depending on the position and amount of the target protein imaged, characteristics of the diseased organ such as the presence or absence of metastasis or infiltration, disease characteristics, severity of disease, progress and / or adverse effects on pharmaceuticals or side effects depending on the location or amount of the target protein. It is possible to diagnose etc. Such a diagnosis may be, for example, a companion diagnosis. It is also possible to stratify the sample by its diagnosis. According to the method of the embodiment, such diagnosis does not become false positive, and it is possible to diagnose more accurately.

Second Embodiment A further embodiment of the imaging method of the embodiment is shown in FIG. FIG. 4 is a schematic flowchart showing an example of the method of the second embodiment.

  First, an imaging agent is determined in step (S11), and non-target proteins are determined in step (S12). Steps (S11) and (S12) can be performed, for example, in the same manner as steps (S1) and (S2).

  Next, a labeled imaging agent is prepared in step (S13), and a masking agent is prepared in step (S14). The steps (S13) and (S14) can be performed, for example, in the same manner as the steps (S3) and (S4). In a second embodiment, the labeled imaging agent and the masking agent are provided in separate pharmaceutical composition forms.

  Next, step (S15) and step (S16) will be described with reference to FIG. 4 and FIG.

  In step (S15), masking agent 2 is administered to sample 1 ((a) in FIG. 4). The administration can be performed, for example, by intravenous injection of a pharmaceutical composition containing a masking agent. The method of administration is selected depending on the type of target protein, non-target protein and masking agent.

  The administered masking agent 2 binds to non-target protein 7 (FIG. 4 (b)). For example, it is preferable to leave the sample until the masking agent 2 binds to the non-target protein 7. Since the masking agent 2 also has binding affinity to the target protein 6, it can also bind to the target protein 6 (arrow in (b) of FIG. 4). Thereafter, the masking agent 2 not bound to the non-target protein 7 or the target protein 6 is washed away (FIG. 4 (c)). In this way, the non-target protein 7 is masked by the masking agent 2.

  Next, in step (S16), the labeled imaging agent 3 in which the imaging agent 4 is labeled with the labeling substance 5 is administered to the sample 1 (FIG. 5 (d)). The administration can be performed by, for example, intravenous injection of a pharmaceutical composition containing the labeled imaging agent 3. The method of administration is selected depending on the type of target protein, non-target protein and masking agent.

  The labeled imaging agent 3 thus administered binds to the target protein 6 ((e) in FIG. 5). For example, it is preferable to leave the sample 1 until the labeled imaging agent 3 binds to the target protein 6. When the masking agent 2 is bound to the target protein 6 (arrow in (e) of FIG. 5), the degree of the binding affinity to the target protein 6 is higher in the labeled imaging agent 3. When the labeled imaging agent 3 approaches the masking agent 2 bound to, the masking agent 2 dissociates, and the labeled imaging agent 3 binds thereto ((f) in FIG. 5). Thereafter, the masking agent 2 and the labeled imaging agent 3 not bound to the non-target protein 7 and the target protein 6 are washed away ((g) in FIG. 5). Thus, the labeled imaging agent 3 can bind to the target protein 6 without binding to the non-target protein 7. As a result, the target protein 6 is labeled by the labeling substance 5.

  For example, as described in the first embodiment, the larger the difference in the affinity between the masking agent and the labeled imaging agent for the target protein, the lower the possibility that the masking agent 2 will bind to the target protein 6, and There is a high possibility that the bound masking agent 2 will replace the labeled imaging agent 3.

  Next, in step (S15), a signal from the labeling substance is detected, and an image is created based on the result of the detection. This step can be performed by the same method as the above step (S6).

  According to the method of the embodiment described above, since the labeled imaging agent is administered with the non-target protein masked by the masking agent, the possibility of the labeled imaging agent binding to the non-target protein is reduced. As a result, the target protein can be detected with higher sensitivity.

Third Embodiment In a further embodiment, each of the target protein and the imaging agent may be of multiple types. Such an example will be described with reference to FIG. FIG. 6 is a schematic flowchart showing an example of the method of the third embodiment.

  In the case of this example, the imaging method of the embodiment is a method of imaging the first to nth target proteins in a sample, and includes steps (S21) to (S26) shown in FIG. Here, n is an integer of 2 or more.

  First, in step (S21), first to nth imaging agents having binding affinity with each of the first to nth target proteins are determined. This step is performed, for example, by determining the first to nth imaging agents corresponding to the respective target proteins in the same manner as the above step (S1) for the first to nth target proteins. Can. Assuming that the value of any of 1 to n is t, the affinity of the t-th imaging agent for the t-th target protein is higher than any of the imaging agents other than the t-th target protein for the t-th target protein.

  Next, in step (S22), non-target proteins having binding affinity with the first to n-th imaging agents are determined. In this step, for example, non-target proteins having affinity to the first to n-th imaging agents can be determined by the same method as the above step (S2). The non-target protein may be a protein having an affinity for any of the first to n imaging agents, or a protein having a binding affinity for all of the first to n imaging agents, It is also good.

  Next, in step (S23), first to nth labeled imaging agents are prepared. The 1st to n-th labeling imaging agents are the 1st to n-th imaging agents labeled by the 1st to n-th labeling substances, respectively. The labeling with the first to nth labeling substances of the first to nth imaging agents can be performed, for example, by the same method as the above (S3).

  The first to nth labeling substances are labeling substances having different energies. Thereby, multiple types of target proteins can be distinguished and imaged. However, it is preferable that all of the first to nth labeling substances be radioactive isotopes emitting positrons, or all be radioactive isotopes emitting gamma rays. Thereby the target protein can be imaged using the same device at one time.

  In the step (S24), a masking agent is prepared. The masking agent has binding affinity to the first to nth target proteins and non-target proteins. And, the binding affinity between the masking agent and the first to nth target proteins is lower than any of the respective affinity between the first to nth imaging agent and the first to nth target proteins. In addition, the binding affinity between the masking agent and the non-target protein is higher than any one of the first to nth imaging agents and the non-target protein.

  For example, the relationship between the binding affinity of each substance when using two types of target proteins and one type of non-target protein, and two types of imaging agents and one type of masking agent will be described. For example, as shown in Table 3, the degree of binding affinity between the first target protein, the first imaging agent, the second imaging agent and the masking agent is “E”, “F”, “G”, respectively. The degree of binding affinity between the second target protein and the first imaging agent, the second imaging agent and the masking agent is “H”, “I” and “J”, respectively, and the non-target protein, Assuming that the degrees of binding affinity with the first imaging agent, the second imaging agent and the masking agent are “K”, “L” and “M”, respectively, “E> F, G”, “I> H, The expressions J "and" M> K, L "hold.

  The first to nth labeled imaging agents and masking agents are preferably provided in the form contained in a pharmaceutical composition suitable for administration in step (S25) described later. The first to nth labeled imaging agents may be all contained in separate pharmaceutical compositions, or all may be contained in one pharmaceutical composition, or a plurality of drugs in any combination. It may be included in the composition.

  In step (S25), the first to nth labeled imaging agents and masking agents are administered. When the first to nth labeled imaging agents are contained in separate pharmaceutical compositions, those pharmaceutical compositions may be administered simultaneously or sequentially without intervals. When all the labeled imaging agents are contained in one pharmaceutical composition, the pharmaceutical composition may be administered. For example, a pharmaceutical composition comprising the first to nth labeled imaging agents and a masking agent may be prepared and administered. The administration can be performed, for example, in the same manner as the step (S5). Alternatively, after the masking agent is administered, the first to nth labeled imaging agents may be administered.

  In step (S26), signals from the first to n-th labeling substances are detected, and an image is created based on the detection result. This step can be performed, for example, by detecting the signals from the first to n-th labeling substances and creating an image by the same method as step (S6). In this example, the signals can be distinguished because the energy of the signals from the first to n-th labeling substances are different. A separate image may be created for each of the signals obtained from different labels of energy, or an image may be created that integrates the data obtained with each signal. In the case of integration, it is possible to distinguish and image the first to nth labeling substances by imaging using different colors for each signal.

  According to the method of the third embodiment described above, it is possible to distinguish and image multiple kinds of labeling substances at one time.

Fourth Embodiment In a further embodiment, the non-target protein and the masking agent may each be of multiple types. Such an example will be described with reference to FIG. FIG. 7 is a schematic flowchart showing an example of the method of the fourth embodiment.

  In the case of this example, the imaging method of the embodiment is a method of imaging a target protein in a sample, and includes steps (S31) to (S36) shown in FIG.

  First, in step (S31), an imaging agent having binding affinity to a target protein is determined. This step can be performed, for example, by the same method as the above step (S1).

  Next, in step (S32), first to nth non-target proteins having binding affinity with the imaging agent are determined. This step can be performed, for example, by selecting a plurality of kinds of proteins having a binding affinity with the imaging agent in the same manner as the step (S2).

  In the step (S33), a labeled imaging agent in which the imaging agent is labeled with a labeling substance is prepared. The labeled imaging agent can be prepared, for example, by the same method as the step (S3).

  In the step (S34), first to nth masking agents are prepared. The first to nth masking agents have binding affinity to the target protein and the 1st to nth non-target protein, respectively. That is, assuming that any value of 1 to n is p, the p-th masking agent has binding affinity with the target protein and the corresponding p-th non-target protein. And, the affinity of the p-th masking agent for the p-th non-target protein is higher than any affinity of imaging agents other than the p-th non-target protein for the p-th non-target protein.

  In addition, the binding affinity between the target protein and the first to nth masking agents is lower than the binding affinity between the target protein and the imaging agent, respectively, and the first to nth non-target proteins and the corresponding first The binding affinities with the to n-th masking agents are respectively higher than the affinities of the first to nth non-target proteins and the imaging agent.

  For example, the binding affinity of each substance when one type of target protein and two types of non-target proteins, and one type of imaging agent and two types of masking agent are used will be described. For example, as shown in Table 4, the degree of binding affinity between the target protein and the imaging agent, the first masking agent and the second masking agent is “O”, “P”, “Q”, respectively. Let “R”, “S” and “T” be the degrees of binding affinity between the non-target protein of 1 and the imaging agent, the first masking agent and the second masking agent, respectively, and the second non-target protein and When the degree of binding affinity with the imaging agent, the first masking agent and the second masking agent is “U”, “V”, “W”, respectively, “O> P, Q”, “S> R , T "and" W> U, V "hold.

  The labeled imaging agent and the first to nth masking agents are preferably provided in a form included in a pharmaceutical composition suitable for administration in step (S35) described later. The first to nth masking agents may be all contained in separate pharmaceutical compositions, or all may be contained in one pharmaceutical composition, or a plurality of pharmaceutical compositions in any combination. It may be included in the thing.

  In step (S35), the first to nth masking agents and a labeled imaging agent are administered to the sample. When the first to nth masking agents are contained in separate pharmaceutical compositions, those pharmaceutical compositions may be administered simultaneously or sequentially without intervals. When all the masking agents are contained in one pharmaceutical composition, the pharmaceutical composition may be administered. For example, a pharmaceutical composition comprising a labeled imaging agent and first to nth masking agents may be prepared and administered. This step can be performed, for example, in the same manner as the above step (S5). Alternatively, the labeled imaging agent may be administered after the administration of the first to nth masking agents.

  Next, in step (S36), the signal from the labeling substance is detected, and an image is created based on the result of the detection. This step can be performed, for example, by the same method as the above step (S6).

  According to the fourth embodiment described above, a plurality of types of non-target proteins can be masked simultaneously and efficiently, and the detection accuracy is further improved. Selecting more non-target proteins improves the sensitivity of target protein imaging.

  Alternatively, the target protein and the imaging agent may be of multiple types, and the non-target protein and the masking agent may be of multiple types.

Fifth Embodiment In one embodiment, the target protein is a T790M mutant of mutant EGFR (hereinafter referred to as “mutated EGFR / T790M”), the non-target protein is a mutant EGFR, and the imaging agent is Osimertinib (Osimertinib) and the masking agent is afatinib (Afatinib). Such an example will be described with reference to FIG. FIG. 8 is a schematic flowchart showing an example of the method of the fifth embodiment. The method in this example is a method of imaging mutant EGFR / T790M in a sample, and includes steps (S41) to (S44) shown in FIG.

  First, in the step (S41), labeled osimerinib is prepared. Osimertinib is a compound represented by the following chemical formula.

  Labeled osimertinib is labeled with a labeling substance. The labeling substance is preferably, for example, a radioactive isotope that emits any of the positrons described above. The chemical formula of an example of labeled osimerinib labeled with such a labeling substance is shown below.

In this example, we have shown oximertinib labeled by replacing one carbon with the radioactive isotope ¹¹ C. The position of carbon replaced with a radioactive isotope may not be the position shown in the above chemical formula. Also, multiple carbons may be replaced with ¹¹ C. Also, any nitrogen may be ¹³ N, or any oxygen may be ¹⁵ O.

  In the step (S42), afatinib is prepared. Afatinib is a compound represented by the following chemical formula.

The IC ₅₀ values of the binding of mutant EGFR and mutant EGFR / T 790 M to afatinib and osimerinib are, for example, as shown in Table 5.

  As shown in Table 1, osimerinib and afatinib have binding affinity to mutant EGFR / T790M and mutant EGFR, respectively. And, the binding affinity of afatinib for mutant EGFR / T 790 M is lower than that of osimerinib, and the binding affinity of afatinib for mutant EGFR is higher than that of osimerinib.

  It is preferred that the labeled ocimerinib and afatinib be provided, for example, in a form suitable for being administered to the sample in step (S42). For example, labeled ocimertinib and afatinib are provided in a form contained in one pharmaceutical composition. Alternatively, labeled ocimerinib and afatinib are provided in separate pharmaceutical compositions. The pharmaceutical composition contains, for example, a pharmaceutically acceptable carrier, a diluent, an excipient and the like. The pharmaceutical composition may be, for example, a formulation for PET.

  Next, in step (S43), labeled ocimerinib and afatinib are administered to the sample. For example, although a pharmaceutical composition containing labeled osimertinib and afatinib may be administered, or labeled osimartinib and afatinib separately prepared may be administered sequentially, after administration of afatinib, the labeled osimertinib is administered. Is preferred. Such an example will be described using FIG. 9 and FIG.

  First, afatinib 12 is administered to sample 11 (FIG. 9 (a)). Administration is performed, for example, by intravenous injection. The administered afatinib 12 binds to mutant EGFR13 (FIG. 9 (b)). For example, it is preferable to leave the sample until afatinib 12 binds to mutant EGFR13. Since afatinib 12 also has binding affinity to mutant EGFR / T790M14, it can also bind to mutant EGFR / T790M14 (arrow in (b) of FIG. 9). Thereafter, afatinib 12 not bound to mutant EGFR 13 or mutant EGFR / T 790 M 14 is washed away ((c) in FIG. 9). In this way, the mutant EGFR 13 is masked by afatinib 12.

Next, labeled osimertinib 17 is administered to sample 11 ((d) in FIG. 10). Labeled osimertinib 17 is osimertinib labeled with ¹¹ C16. Administration is performed, for example, by intravenous injection. The labeled osimerinib 17 administered binds to the mutant EGFR 13 (FIG. 10 (e)). When mutant EGFR / T790M14 and afatinib 12 are bound (arrow in (e) of FIG. 10), the afatinib 12 is replaced with labeled osimerinib 17 and the labeled osimerinib 17 binds with mutant EGFR / T790M14 (FIG. (F) of FIG. Thereafter, afatinib 12 and labeled osimerinib 17 not bound to mutant EGFR13 and mutant EGFR / T790M14 are washed away (FIG. 10 (g)). In this way, labeled osimerinib 17 can bind to mutant EGFR / T790M14 without binding to mutant EGFR13. As a result, mutant EGFR / T790M14 is labeled by ¹¹ C16.

  Next, in step (S44), the signal from the labeling substance is detected to create an image. In this example, the signal is two gamma rays generated by pair annihilation of a positron emitted from a radioactive isotope which is a labeling substance and a nearby electron. The detection of gamma rays and the creation of an image can be performed, for example, by the same method as the above step (S6), for example, using a PET apparatus or the like.

  According to the method of the fifth embodiment described above, the mutant EGFR is masked by afatinib, and the binding of osimerinib to the mutant EGFR is prevented. As a result, mutant EGFR / T790M can be distinguished from mutant EGFR and correctly imaged. According to this method, it is possible to non-invasively detect the presence or absence or distribution of the mutant EGFR / T790M in the whole sample even if it is a sample that can not tolerate collection of tissue or blood.

  When mutant EGFR / T790M is imaged by the method of the embodiment, for example, it can be diagnosed that the sample suffers from mutant EGFR / T790M positive lung cancer. In addition, depending on the position and amount of the mutant EGFR / T790M imaged, presence or absence of the mutant EGFR / T790M in the organ where the mutant EGFR / T790M has developed, in the primary focus and in the metastatic focus, the progression and severity of lung cancer, It is possible to diagnose the progress and / or the effect on the drug and the like. According to the method of the embodiment, it is possible to diagnose mutant EGFR / T790M more accurately without false positive diagnosis of mutant EGFR / T790M.

1 ... sample 2 ... masking agent 3 ... labeling imaging agent 4 ... imaging agent 5 ... labeling substance 6 ... target protein 7 ... non-target protein 11 ... sample 12 ... afatinib 13 ... mutant EGFR
14: Mutant EGFR / T 790 M 15: Ocimurtinib 16: ¹¹ C 17: Labeled Ocimertinib

Claims (8)

A method of imaging a target protein in a sample, comprising
(S1) determining an imaging agent having binding affinity to the target protein,
(S2) determining a non-target protein having binding affinity to the imaging agent,
(S3) Providing a labeled imaging agent in which the imaging agent is labeled with a labeling substance,
(S4) A binding affinity to the target protein and the non-target protein, the binding affinity to the target protein is lower than that of the imaging agent, and a binding affinity to the non-target protein is the above Providing a masking agent higher than that of the imaging agent,
(S5) administration of the masking agent and the labeling imaging agent to the sample, and (S6) detecting a signal from the labeling substance, and producing an image based on the detection result How to image.   The method according to claim 1, wherein in the step (S5), the labeled imaging agent is administered after the masking agent is administered.   The method according to claim 1 or 2, wherein the target protein is a biomarker.   The method according to claim 3, wherein the biomarker is a diagnostic marker, a prognostic marker, a pharmacodynamic marker, a monitoring marker or a therapeutic marker.   The method according to any one of claims 1 to 4, wherein the labeling substance is a radioactive isotope that emits positrons. A method for imaging the first to nth target proteins in a sample, wherein n is an integer of 2 or more,
(S21) determining first to nth imaging agents having binding affinity to the first to nth target proteins,
(S22) determining a non-target protein having binding affinity to each of the first to nth imaging agents,
(S23) preparing the first to n-th labeled imaging agents in which the first to n-th imaging agents are labeled with the first to n-th labeling substances;
(S24) The binding affinity with the first to nth target proteins and the non-target protein, and the binding affinity to the first to nth target proteins is respectively the first to nth imaging Providing a masking agent which is lower than that of the agent and whose binding affinity to the non-target protein is higher than that of the first to nth imaging agents,
(S25) administering the masking agent and the first to nth labeled imaging agents to the sample, and (S26) detecting a signal from the first to nth labeled substances, and detecting the result A method of imaging a protein comprising creating an image based on: A method of imaging a target protein in a sample, comprising
(S31) determining an imaging agent having binding affinity to the target protein,
(S32) determining first to nth non-target proteins each having binding affinity with the imaging agent,
(S33) Providing a labeled imaging agent in which the imaging agent is labeled with a labeling substance,
(S34) The protein has binding affinity to the target protein and the first to nth non-target proteins, and the binding affinity to the target protein is lower than that of the imaging agent. providing first to nth masking agents, each of which has a binding affinity to n non-target proteins higher than that of the imaging agent;
(S35) administering the first to nth masking agents and the labeled imaging agent to the sample, and (S36) detecting a signal from the labeled substance, and creating an image based on the result of the detection A method of imaging a protein that involves doing. A method of imaging a T790M mutant of mutant EGFR (hereinafter, referred to as "a mutant EGFR / T790M") in a sample,
(S41) Providing a labeled osimeltinib labeled with osimatinib with a labeling substance,
(S42) preparing afatinib,
(S43) A protein comprising: administering to the sample the afatinib and the labeled osimertinib; and (S44) detecting a signal from the labeled substance and generating an image based on the result of the detection Method.

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