Methods and materials for using [18F]-F-AraG in myocardial imaging

Abstract

Embodiments of the present disclosure relate to compositions and methods for performing positron emission tomography (PET), and more particularly, to compositions and methods for use in selected imaging techniques (e.g., studying myocardial physiology). 18 Provided are compositions and methods for the development and use of F-based PET tracers. In particular, as disclosed herein, at the tracer level, [ 18 [F]F-AraG has the potential to be used in many emerging PET methodologies, including those designed for cardiac and / or mitochondrial activity imaging. 18 The use of [F]F-AraG has demonstrated significant benefits over conventional methods in many distinct applications, including the observation of selected physiological phenomena (e.g., myocardial perfusion, myocardial viability, and cardiac inflammation). 18 The method of the present invention provides significant advantages over F-labeled tracers. 18 F]F-AraG, including those designed to monitor patients.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims the benefit of U.S. Provisional Patent Application No. 63 / 057,643, filed on 28 July 2020, title “METHODS AND MATERIALS FOR USING [18F]-F-AraG IN CARDIAC IMAGING” (this application is incorporated herein by reference), which was assigned to a concurrently pending assignee pursuant to Section 119(e) of the U.S. Patent Act. This application relates to a concurrently pending U.S. application filed on 25 February 2021, application number 17 / 185,502, title “Compounds and Methods of Making Compounds” (the contents of which are incorporated herein by reference). [Background technology]

[0002] background Cardiovascular disease (CVD) has remained the leading cause of death in the United States for decades. Furthermore, CVD is the most devastating chronic disease in the U.S. In 2018, stroke and heart failure were the most expensive chronic conditions under the Medicare fee-for-service program.

[0003] This disclosure generally relates to positron emission tomography (PET) and, more specifically, to use in cardiac imaging techniques (e.g., myocardial perfusion studies). 18 This invention relates to compositions and methods for the development and use of F-based PET tracers. 18 The long physical half-life (10⁹ minutes) of fluorine-based tracers enables clinical research without the need for a cyclotron in situ. Furthermore, with modern PET camera technology, quantitative measurements of myocardial radioactivity can be performed with high temporal sampling and good statistical accuracy.

[0004] Cardiac imaging techniques play a central role in the non-invasive diagnosis and risk assessment of cardiovascular disease (CVD), as well as in relevant decisions made by physicians regarding the optimal management of CVD in different individuals. New applications of positron emission tomography (POST) in cardiac imaging are emerging. 18 The development of F-labeled tracers may offer those skilled in the art further opportunities to manage CVD and expand the scope of clinical research. In light of the enormous public health burden of cardiovascular disease, in cardiac imaging technology 18 Further compositions and methods designed to use F-based PET tracers are needed in the art. [Overview of the project] [Means for solving the problem]

[0005] Abstract Compounds used in the methods disclosed herein, 18 F]-F-arabinofuranosylguanine was initially developed as a PET imaging agent for activated T cells. This compound is a derivative of arabinofuranosylguanine (AraG). 18 It is a 1F-labeled analog that can be phosphorylated by two kinases: cytoplasmic deoxycytidine kinase (dCK) and deoxyguanosine kinase (dGK), and can be captured in cells. As discussed below, the inventors have found that at the tracer level, [ 18 We have discovered that [F]F-AraG has the potential to be used in many new PET methodologies (including those designed for cardiac and / or mitochondrial activity imaging). The heart is an organ that requires high energy and is particularly rich in mitochondria. Over the past decade, mitochondrial dysfunction has been recognized as a significant aspect of cardiovascular pathology. As a result, treatments that focus on observing and improving cardiac mitochondrial activity have become a focus of biomedical research.

[0006] The disclosures provided herein are, 18Based in part on the discovery that [¹⁸F]F-AraG is a drug well suited to observe certain physiological phenomena (including, for example, mitochondrial activity in heart cells). In view of this, embodiments of the present invention provide a method for using [¹⁸F]F-AraG in cardiac imaging. In exemplary embodiments of the present invention, the use of [¹⁸F]F-AraG as a PET tracer has been evaluated in healthy volunteers and cancer patients undergoing immunotherapy. In these studies, significant tracer uptake in the heart was observed in both groups. Interestingly, however, the signal observed in the heart wall of healthy volunteers was different from that in cancer patients and patients undergoing immunomodulatory and other treatments. This discovery of different [¹⁸F]F-AraG signal profiles in healthy subjects versus patients treated with therapeutic agents is utilized in the methods disclosed herein (including, for example, methods designed to use the ability of [¹⁸F]F-AraG to image the heart to monitor changes to heart cells caused by drugs or injury). 18 Based in part on the discovery that [¹⁸F]F-AraG is a drug well suited to observe certain physiological phenomena (including, for example, mitochondrial activity in heart cells). In view of this, embodiments of the present invention provide a method for using [¹⁸F]F-AraG in cardiac imaging. In exemplary embodiments of the present invention, the use of [¹⁸F]F-AraG as a PET tracer has been evaluated in healthy volunteers and cancer patients undergoing immunotherapy. In these studies, significant tracer uptake in the heart was observed in both groups. Interestingly, however, the signal observed in the heart wall of healthy volunteers was different from that in cancer patients and patients undergoing immunomodulatory and other treatments. This discovery of different [¹⁸F]F-AraG signal profiles in healthy subjects versus patients treated with therapeutic agents is utilized in the methods disclosed herein (including, for example, methods designed to use the ability of [¹⁸F]F-AraG to image the heart to monitor changes to heart cells caused by drugs or injury). 18 Based in part on the discovery that [¹⁸F]F-AraG is a drug well suited to observe certain physiological phenomena (including, for example, mitochondrial activity in heart cells). In view of this, embodiments of the present invention provide a method for using [¹⁸F]F-AraG in cardiac imaging. In exemplary embodiments of the present invention, the use of [¹⁸F]F-AraG as a PET tracer has been evaluated in healthy volunteers and cancer patients undergoing immunotherapy. In these studies, significant tracer uptake in the heart was observed in both groups. Interestingly, however, the signal observed in the heart wall of healthy volunteers was different from that in cancer patients and patients undergoing immunomodulatory and other treatments. This discovery of different [¹⁸F]F-AraG signal profiles in healthy subjects versus patients treated with therapeutic agents is utilized in the methods disclosed herein (including, for example, methods designed to use the ability of [¹⁸F]F-AraG to image the heart to monitor changes to heart cells caused by drugs or injury). 18 Based in part on the discovery that [¹⁸F]F-AraG is a drug well suited to observe certain physiological phenomena (including, for example, mitochondrial activity in heart cells). In view of this, embodiments of the present invention provide a method for using [¹⁸F]F-AraG in cardiac imaging. In exemplary embodiments of the present invention, the use of [¹⁸F]F-AraG as a PET tracer has been evaluated in healthy volunteers and cancer patients undergoing immunotherapy. In these studies, significant tracer uptake in the heart was observed in both groups. Interestingly, however, the signal observed in the heart wall of healthy volunteers was different from that in cancer patients and patients undergoing immunomodulatory and other treatments. This discovery of different [¹⁸F]F-AraG signal profiles in healthy subjects versus patients treated with therapeutic agents is utilized in the methods disclosed herein (including, for example, methods designed to use the ability of [¹⁸F]F-AraG to image the heart to monitor changes to heart cells caused by drugs or injury). 18 Based in part on the discovery that [¹⁸F]F-AraG is a drug well suited to observe certain physiological phenomena (including, for example, mitochondrial activity in heart cells). In view of this, embodiments of the present invention provide a method for using [¹⁸F]F-AraG in cardiac imaging. In exemplary embodiments of the present invention, the use of [¹⁸F]F-AraG as a PET tracer has been evaluated in healthy volunteers and cancer patients undergoing immunotherapy. In these studies, significant tracer uptake in the heart was observed in both groups. Interestingly, however, the signal observed in the heart wall of healthy volunteers was different from that in cancer patients and patients undergoing immunomodulatory and other treatments. This discovery of different [¹⁸F]F-AraG signal profiles in healthy subjects versus patients treated with therapeutic agents is utilized in the methods disclosed herein (including, for example, methods designed to use the ability of [¹⁸F]F-AraG to image the heart to monitor changes to heart cells caused by drugs or injury).

[0007] The use of [¹⁸F]F-AraG as a PET tracer in the methods disclosed herein offers significant advantages over conventional [¹⁸F] labeled tracers in many separate applications, including the observation of selected physiological phenomena (such as myocardial perfusion, myocardial viability, and cardiac inflammation). Further, as discussed below, the methods of the present invention are designed to use [¹⁸F]F-AraG to observe the physiological responses of patients to various therapeutic agents and include using [¹⁸F]F-AraG as a PET tracer in drug development studies. 18 The use of [¹⁸F]F-AraG as a PET tracer in the methods disclosed herein offers significant advantages over conventional [¹⁸F] labeled tracers in many separate applications, including the observation of selected physiological phenomena (such as myocardial perfusion, myocardial viability, and cardiac inflammation). Further, as discussed below, the methods of the present invention are designed to use [¹⁸F]F-AraG to observe the physiological responses of patients to various therapeutic agents and include using [¹⁸F]F-AraG as a PET tracer in drug development studies. 18 The use of [¹⁸F]F-AraG as a PET tracer in the methods disclosed herein offers significant advantages over conventional [¹⁸F] labeled tracers in many separate applications, including the observation of selected physiological phenomena (such as myocardial perfusion, myocardial viability, and cardiac inflammation). Further, as discussed below, the methods of the present invention are designed to use [¹⁸F]F-AraG to observe the physiological responses of patients to various therapeutic agents and include using [¹⁸F]F-AraG as a PET tracer in drug development studies. 18 The use of [¹⁸F]F-AraG as a PET tracer in the methods disclosed herein offers significant advantages over conventional [¹⁸F] labeled tracers in many separate applications, including the observation of selected physiological phenomena (such as myocardial perfusion, myocardial viability, and cardiac inflammation). Further, as discussed below, the methods of the present invention are designed to use [¹⁸F]F-AraG to observe the physiological responses of patients to various therapeutic agents and include using [¹⁸F]F-AraG as a PET tracer in drug development studies. 18 The use of [¹⁸F]F-AraG as a PET tracer in the methods disclosed herein offers significant advantages over conventional [¹⁸F] labeled tracers in many separate applications, including the observation of selected physiological phenomena (such as myocardial perfusion, myocardial viability, and cardiac inflammation). Further, as discussed below, the methods of the present invention are designed to use [¹⁸F]F-AraG to observe the physiological responses of patients to various therapeutic agents and include using [¹⁸F]F-AraG as a PET tracer in drug development studies.

[0008] The invention disclosed herein has many embodiments. These embodiments are methods of imaging heart cells in a subject, the method comprising the following formula: [ka] A method comprising the steps of: administering a compound having a to a subject, wherein the administration route is selected to allow the compound to be phosphorylated by deoxycytosine kinase and deoxyguanosine kinase present in the cardiac cells of the subject; and then imaging the subject, wherein the detection of the presence of the compound comprises a method by a step of indicating the presence of cardiac cells. In some embodiments, these methods further comprise the step of evaluating one or more parameters of myocardial perfusion in the subject's heart using one or more images of the heart. In some embodiments, these methods further comprise the step of evaluating one or more parameters of myocardial viability in the subject's heart using one or more images of the heart. In some embodiments, these methods further comprise the step of evaluating one or more parameters of inflammation in the subject's heart using one or more images of the heart. In certain embodiments of the present invention, the subject is administered a therapeutic agent, and one or more images of the heart are used to obtain information regarding the effect of the agent on the subject's heart. If necessary, the therapeutic agents used in these methods act on the mitochondria of heart cells.

[0009] Embodiments of the present invention also include a method for imaging selected cell populations (e.g., cardiac cells and leukocytes) in patients suffering from pathological conditions who are being treated with one or more therapeutic agents. Such a method may be used to observe in vivo physiological changes (e.g., cardiotoxicity) resulting from the administration of such therapeutic agents. Typically, such a method involves administering the following formula to a subject being treated with the therapeutic agent: [ka] A step of administering a PET probe compound, wherein the administration route is selected to allow the compound to be phosphorylated by deoxycytosine kinase and deoxyguanosine kinase present in the cells of the subject. These methods further include a step of PET imaging the subject, wherein the detection of the presence of the PET probe compound indicates the presence of cells; and finally, a step of correlating the observed presence of the compound in the cells of the subject with the subject's response to the therapeutic agent. In certain embodiments of these methods, the subject is selected to be a patient diagnosed with cardiovascular disease; and / or the subject is selected to be a patient diagnosed with cancer; and / or the subject is selected to be a patient undergoing treatment for cardiovascular disease or cancer. In illustrative embodiments of the inventions disclosed herein, the therapeutic agent is an anthracycline (e.g., doxorubicin, daunorubicin, epirubicin, idarubicin, etc.) or an immune checkpoint inhibitor (e.g., an immune checkpoint inhibitor selected to affect CTLA-4 or PD-1 / PD-L1 blockade), and one or more images of the heart are used to obtain information regarding physiological phenomena (e.g., cardiotoxicity) resulting from the administration of the anthracycline or the immune checkpoint inhibitor. In illustrative working embodiments, the method includes observing the presence of the PET probe compound in cardiac cells, immune cells and / or lymph nodes (e.g., tumor inflow lymph nodes).

[0010] In a particular embodiment of the present invention, a PET imaging method for imaging cells of a subject responding to the administration of a therapeutic agent comprises the steps of: observing one or more images obtained on a first date; observing one or more images obtained on a second date; and then comparing the images obtained on the first imaging date with the images obtained on the second imaging date in order to observe changes in the patient's physiological state resulting from the administration of the therapeutic agent over time (for example, to distinguish between responders and non-responders to the therapeutic agent). In a particular embodiment of the present invention, the amount of time between the first and second dates includes less than one week. Alternatively, the amount of time between the first and second dates includes at least one week, two weeks, or three weeks, or at least one month, two months, or three months.

[0011] Another embodiment of the present invention is a method for imaging mitochondrial activity in the cells of a subject, wherein the method is given by the following formula: [ka] A method comprising the steps of: administering a compound having to the subject, wherein the administration route is selected to allow the compound to be phosphorylated by deoxycytosine kinase and deoxyguanosine kinase present in the subject's cells; and then imaging the subject, wherein the detection of the presence of the compound includes a step of indicating the presence of mitochondrial activity. In a particular embodiment, the subject is a subject identified as suffering from a mitochondrial deficiency. In another embodiment of the present invention, the method is part of a protocol designed to screen the subject for mitochondrial dysfunction; for example, mitochondrial dysfunction associated with cardiovascular disease, neuropsychiatric disorder, or neurodegenerative disease. If necessary, the above mitochondrial dysfunctions are selected from the group consisting of myocardial perfusion, bipolar disorder, depression, schizophrenia, Alzheimer's disease, Parkinson's disease, Friedreich's ataxia, amyotrophic lateral sclerosis, Huntington's disease, progeria, cardiomyopathy, respiratory chain disorder, mtDNA depletion, myoclonus epilepsy, red ragweed syndrome, encephalomyopathy, lactic acidosis, stroke-like seizures, and optic nerve atrophy.

[0012] Other objects, features, and advantages of the present invention will become apparent to those skilled in the art from the following detailed description. However, it should be understood that the detailed description and specific examples illustrate some embodiments of the present invention, but are illustrated by example and not by limitation. Many changes and modifications within the scope of the present invention can be made without departing from its spirit, and the present invention encompasses all such modifications. [Brief explanation of the drawing]

[0013] Further aspects of this disclosure will be more readily understood by considering the detailed descriptions of its various embodiments set forth below, in conjunction with the accompanying drawings.

[0014] [Figure 1-1A] Figure 1.1A illustrates several synthesis schemes for preparing the compounds of this disclosure. [Figure 1-1B]Figure 1.1B illustrates several synthesis schemes for preparing the compounds of this disclosure. [Figure 1-2] Figure 1.2 illustrates embodiments of R and R'. [Figure 2-1-1] Figure 2.1 illustrates a schematic diagram of the synthesis of the [18F]F-AraG precursor and [18F]F-AraG. [Figure 2-1-2] Figure 2.1 illustrates a schematic diagram of the synthesis of the [18F]F-AraG precursor and [18F]F-AraG. [Figure 2-2] Figure 2.2 illustrates graphs of 5 × 10⁵ CCRF-CEM cells labeled with 18F-AraG (0.6 mCi / ml) at the indicated time and dose. Uptake was measured using a gamma counter. Bars represent the mean ± SEM of the triple determination. [Figure 2-3] Figure 2.3A shows graphs of 5 × 10⁵ CCRF-CEM cells incubated in triplicate with 1 μCi 8H₃-AraG (1 mCi / ml) and gradually increasing doses of cold 2F-AraG or DMSO for either 60 or 120 minutes. The percentage of control uptake was calculated as measured cpm of the sample / cpm of the accumulated 1 μCi 8[H₃]-AraG control × 100. Figure 2.3B shows graphs of 5 × 10⁵ CCRF-CEM, MOLT-4, or RAJI cells (triplicate) labeled with 1 μCi 8-3H-AraG (1 mCi / ml) for 120 minutes in the presence of 1 μM cold 2F-AraG, DMSO, or culture medium. The percentage of control uptake was calculated as shown in Figure 2.3a. [Figure 2-4] Figure 2.4 illustrates a schematic diagram of the metabolism of 2'-deoxyguanosine (dGuo) by T lymphoblasts. In contrast to 2'-dGuo, AraG does not require ribonucleotide reductase activity for integration into DNA and is directly phosphorylated by mitochondrial dGK at low intracellular concentrations. At higher concentrations, it can also be phosphorylated by deoxycytidine kinase and integrated into nuclear DNA (Figure from J Biol Chem. 2008;283:16437-16445). [Figure 3-1]Figure 3.1 illustrates the analytical HPLC profile for simultaneous injection of [18F]F-AraG and a cold F-AraG standard (5% acetonitrile:95% water; 1 mL / min, 254 nm, Phenomenex Gemini C18, 5 μm, 4.6 × 250 mm). [Figure 3-2] Figure 3.2 illustrates a graph showing 5 × 10⁵ CCRF-CEM cells (triple row) exposed to either 3 μCi or 10 μCi of [18F]F-AraG for 60 minutes or 120 minutes. When exposed to 10 μCi, the cells took up approximately twice as much [18F]F-AraG at 60 minutes (p=0.008) and 120 minutes (p=0.001) compared to when exposed to 3 μCi. Error bars represent SEM. [Figure 3-3] Figure 3.3 illustrates a graph showing the incubation of 1 × 10⁶ purified primary T cells stimulated with 100 U / mL IL₂, 50 nM PMA, and 1 μg / mL ionomycin, or unstimulated, with 1 μCi of [18F]F-AraG for 60 minutes. Error bars represent the mean ± SEM of the triple determination (n=4, p=0.14, and 0.003, respectively, by two-sided paired Student's t-test). [Figure 3-4] Figure 3.4 is a schematic diagram of Scheme 1, which describes the synthesis of 2-N-acetyl-6-O-((4-nitrophenyl)ethyl)-9-(3,5-di-O-trityl-2-triphyll-β-D-ribofuranosyl)guanine (2), a precursor of [18F]F-AraG. [Figure 3-5] Figure 3.5 is a schematic diagram of Scheme 2, which describes the synthesis of 2'-deoxy-2'-fluoro-9-β-D-arabinofuranosylguanine 5 (F-AraG). [Figure 3-6] Figure 3.6 is a schematic diagram of Scheme 3, which describes the synthesis of 2'-deoxy-2'-[18F]fluoro-9-β-D-arabinofuranosylguanine 7 ([18F]F-AraG). [Figure 4]Figure 4 illustrates the mechanism for imaging cardiac cells with [18F]F-AraG. [18F]F-AraG is transported to cells via a nucleoside transporter and subsequently [18F]phosphorylated by mitochondrial deoxyguanosine kinase (dGK), and to a lesser extent by cytosol deoxycytidine kinase (dCK). Phosphorylation leads to the capture of [18F]F-AraG, enabling visualization of these cells via PET imaging. [Figure 5] Figure 5 provides data illustrating the kinetic properties of dCK and dGK with respect to [3H]F-AraG. (a) Enzyme reaction kinetics of dGK or dCK with respect to [3H]F-AraG, (b) dGuo-positive control for dGK activity, (c) dCyd-positive control for dCK activity. The line indicates the best fit. The Km of dGK with respect to [3H]F-AraG was found to be 7.27 μM with a Vmax of 23.44 nmol / min / mg, while dCK had a lower affinity for [3H]F-AraG (Km = 50.89 μM, Vmax = 262.5). The inventors observed a high affinity of dCK for dCyd (Km=1.997, Vmax=9.454) and similarly higher affinity (Km=5.083, Vmax=123.2) in a dGuo-positive control for dGK activity. [Figure 6] Figure 6 shows an image of the F-AraG signaling pathway in the heart of a cancer patient. High mitochondrial activity in the heart generates a high F-AraG signaling pathway one hour after tracer injection. [Figure 7] Figure 7 provides data showing the intensity of F-AraG signaling in the heart wall of healthy volunteers and patients receiving immunotherapy (pre-treatment image refers to the image taken 2-3 weeks after a single injection of anti-PD-1 antibody). [Figure 8]Figure 8 provides an illustration illustrating the mechanism of imaging with [18F]F-AraG. [18F]F-AraG is transported into the cell via a nucleoside transporter and subsequently undergoes rate-limiting phosphorylation by mitochondrial deoxyguanosine kinase (dGK). Phosphorylation by dGK leads to capture in mtDNA and potential downstream accumulation, enabling visualization via PET imaging. [Figure 9] Figure 9 provides an illustration illustrating the mechanism of cardiotoxicity and its relevance to imaging with [18F]F-AraG. A. Immune checkpoint inhibitor (ICI) induced inflammation. Briefly, cancer treatments called immune checkpoint inhibitors (ICIs) work by activating the patient's own immune system to fight their cancer. ICI drugs, which disrupt PD-1 and CTLA-4 signaling, interfere with cardioprotective immunosuppressive mechanisms, leading to exaggerated proliferation and activation of immune cells. Excessive inflammatory activity can be visualized by the accumulation of [18F]F-AraG in activated T cells. B. Doxorubicin-induced mitochondrial damage. Doxorubicin readily enters mitochondria and interferes with mtDNA synthesis. Following phosphorylation by dGK, [18F]F-AraG can be incorporated into mtDNA and thus can report the state of its synthesis. [Figure 10] Figure 10 shows maximum intensity projection (MIP) [18F]F-AraG images of a healthy subject (left) and a head and neck cancer patient. In some patients, myocardial uptake was significantly higher than in healthy subjects, indicating abnormal mitochondrial / inflammatory activity. [Figure 11] Figures 11A–11B show data from the evaluation of [3H]F-AraG in human immune cells. Figure 11A. The highest tracer uptake was observed in activated T cells. Macrophages (M1 and M2) and dendritic cells (DCs) also showed accumulation. No significant accumulation was found in B cells, eosinophils, and neutrophils. Figure 11B. Activation of all T cell subtypes resulted in increased tracer uptake, but activated CD8+ cells showed the highest increase. [Figure 12] Figures 12A–12C show data from embodiments of the present invention. Figure 12A. Tumor-infiltrating lymphocytes took up over 80% of tumor-activated [18F]F-AraG, while CD8+ and CD4+ cells acquired the highest percentage of tracers (72%). FACS analysis of isolated lymphocytes showed an activated phenotype (CD44+ CD62L-) in the majority of CD8+ cells (79.9±11.5) and CD4+ cells (88.3±11.7). Figure 12B. [18F]F-AraG signaling in tumors (white circles) and tumor inflow area lymph nodes (red) of mice before and 48 hours after a single anti-PD-1 treatment. Responding mice (R) showed higher [18F]F-AraG signaling in both tumors and tumor inflow area lymph nodes compared to unresponsive mice (NR). Figure 12C. In responders, combined intratumoral and intranodal [18F]F-AraG signaling was significantly higher than in non-responders (2.604±1.083, n=4) (6.587±0.6874, n=4). [Figure 13] Figures 13A–13D show data from embodiments of the present invention. Figure 13A. Paclitaxel / carboplatin treatment (reported to induce immunologically silent death) did not result in a significant change in [18F]F-AraG signal intensity. Figure 13B shows that a dramatic increase in signal intensity was detected after oxaliplatin / cyclophosphamide treatment, which has been shown to induce immunogenic cell death. White circles indicate tumor inflow lymph nodes, and yellow arrows point to the tumor. Figure 13C. The [18F]F-AraG signal detected after oxaliplatin / cyclophosphamide treatment was significantly different from the pre-treatment signal and the signal after paclitaxel-carboplatin treatment. Figure 13D. The ratio of CD8+ (effector) to CD4+FOXP3+ (regulatory) cells in the oxaliplatin / cyclophosphamide group was 27 times higher than in paclitaxel / carboplatin-treated mice. This indicates an immunoactive tumor microenvironment (n=4 per group). [Figure 14]Figures 14A–14D show data from studies of Ctla4+ / - Pdcd1- / - mice exhibiting cardiac immunoinfiltration. Figure 14A. H&E images of Ctla4+ / - Pdcd1- / - mice (left) and humans (right; autopsy sample of myocardium from a patient with complete heart block and ventricular tachycardia after ICI treatment). Figure 14B. Quantification of lymphatic infiltration score and frequency of CD3, CD4, and CD8+ cells as fractions of total nucleated cells from H&E-stained cardiac tissue. Figure 14C. Representative images of CD3, CD4, and CD8 immunohistochemistry (right) stained cardiac tissue sections from female Ctla4+ / - Pdcd1- / - mice. Left panel: IHC; Center panel: Segments with red = positive and blue = negative cells; Right panel: Positive cell density (blue = low, green = intermediate, yellow = high). Heatmap values ​​represent arbitrary density units. Figure 14D. Representative images of cardiac tissue stained with further immunohistochemistry (CD3, F4 / 80+ macrophages and Foxp3+ Tregs) from Ctla4+ / - Pdcd1- / - mice. [Figure 15] Figures 15A–15C outline proposed studies for long-term [18F]F AraG monitoring of cardiotoxicity. Figure 15A. Doxorubicin toxicity. Animals are imaged before and 48 hours after weekly doxorubicin administration. Figure 15B. ICI toxicity. Animals are imaged weekly starting at week 6. Preliminary studies showed that weeks 6–8 were the peak of myocardial immune infiltration. Animals are followed up to week 10. Figure 15C. Dox / immunotherapy toxicity. Mice are treated twice a week for two weeks. [18F]F AraG imaging is performed 48 hours after the second and fourth administrations. Exovivo analysis is performed one day after the final scan. [Figure 16] Figure 16 shows maximum projection (MIP) [18F]F-AraG images of 18FDG and [18F]F-AraG myocardial uptake in head and neck patients. Myocardial uptake of diagnostic 18FDG obtained after fasting was low. Cardiac uptake of [18F]F-AraG in the same patients was higher than in healthy volunteers (see Figure 7). [Figure 17]Figure 17 shows images from a comparison of 18FDG and [18F]F-AraG uptake in rat myocardium. The rats were imaged with a tracer on consecutive days. [18F]F-AraG showed reproducible myocardial uptake, while 18FDG showed variability in myocardial imaging that may interfere with its clinical usefulness. [Figure 18] Figure 18 shows images of myocardial [18F]F-AraG uptake from a healthy female volunteer taken at the indicated time after tracer injection. Cardiac signals were sustained and essentially uniform within the 120-minute imaging timeframe. [Figure 19] Figure 19 shows data on radiomic features extracted from [18F]F-AraG images of mice with tumors treated with anti-CTLA-4 and anti-PD-1 antibodies. Energy, entropy, and homogeneity were found to differ significantly between treated and untreated animals. [Figure 20] Figures 20A–20C show transverse [18F]F AraG PET images of healthy volunteers in Figure 20A and head and neck cancer patients in Figure 20B. Signaling in cancer patients was significantly higher with increasing area of ​​localized elevation (red arrows). Figure 20C provides data showing that [18F]F AraG signaling in the cardiac wall of cancer patients was significantly higher than in healthy subjects, both before and after immunotherapy. Increased signaling before immunotherapy may indicate cardiotoxicity from previous anticancer treatment. [Figure 21] Figure 21 shows data from electrocardiograms and [18F]F AraG images of three head and neck patients. The top patient shows relatively low and uniform [18F]F AraG myocardial uptake and a normal ECG. The other two patients show abnormal ECGs and higher (central) and heterogeneous [18F]F AraG uptake. The data in the figure illustrate an important aspect of the embodiments of the present invention by showing a correlation between tracer uptake and hear abnormalities as shown in the electrocardiogram. [Figure 22]Figure 22 provides maximum projection (MIP) images showing changes in myocardial signaling in a patient with recurrent melanoma before and after immunotherapy infusion. Elevated myocardial uptake in the pre-treatment scan may indicate cardiotoxicity from previous anticancer treatment. A single immunotherapy infusion resulted in dramatically increased myocardial uptake. Increased [18F]F-AraG signaling was also observed in the thyroid and spleen. The data in this figure illustrate a key aspect of the embodiments of the present invention, illustrating how [18F]F AraG can be used to image the effects of ICI on the heart in PET methodology. [Modes for carrying out the invention]

[0015] Detailed explanation Before describing this disclosure in more detail, it should be understood that this disclosure is not limited to the specific embodiments described herein and the embodiments of the invention, and is therefore naturally subject to change. It should also be understood that the terminology used herein is solely for the purpose of describing specific embodiments and is not intended to be limiting. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as that commonly understood by those skilled in the art to which this disclosure belongs. All publications and patents cited herein are incorporated herein by reference as each individual publication or patent is specifically and individually indicated to be incorporated by reference, and are incorporated herein by reference to disclose and describe methods and / or materials, and such publications are cited accordingly. As will be apparent to those skilled in the art upon reading and understanding this disclosure, each of the individual embodiments described and illustrated herein has different components and features, which can be readily separated from or combined with features of any of several other embodiments without departing from the scope or spirit of this disclosure. Any method of description may be in the order of the events being described, or in any other logically possible order.

[0016] Before describing embodiments of this disclosure in detail, it should be understood that, unless otherwise indicated, this disclosure is not limited to and is therefore subject to change, including but not limited to specific materials, reagents, reactants, manufacturing processes, etc. It should also be understood that the terminology used herein is solely for the purpose of describing specific embodiments and is not intended to be limiting. Where logically possible, steps may be performed in a different order, which may be possible in this disclosure.

[0017] Where used herein and in the appended claims, the singular forms “a,” “an,” and “the” should be noted as including the plural unless the context otherwise explicitly states. For example, a reference to “a compound” includes multiple compounds. In this specification and the subsequent claims, many terms are used and defined as having the following meanings unless otherwise clearly intended.

[0018] Each of the applications and patents cited in this context (including during the proceedings of each issued patent; “application cited documents”), each of the documents and references cited in each of those applications and patents, each of the PCT applications and foreign applications that correspond to and / or claim priority from any of these applications and patents, and each of the documents cited or referred to in each of the application cited documents are expressly incorporated herein by reference. Furthermore, any documents or references cited in the text (for example, U.S. Patent Publications No. 20150230762, 20150297760, and 20190054198), any documents or references cited in the preceding list of references before the claims, or within the text itself; and each of these documents or references ("herein cited references"), as well as any documents or references cited in each of the herein cited references (including any manufacturer's specifications, instructions, etc.), are expressly incorporated herein by reference.

[0019] According to this disclosure, the “detectably effective amount” in embodiments of this disclosure is defined as an amount sufficient to produce an acceptable image using an instrument available for clinical use. The detectably effective amount in embodiments of this disclosure may be given in one or more doses. The detectably effective amount in embodiments of this disclosure may vary depending on factors such as the degree of individual sensitivity, the individual’s age, sex, and weight, individual-specific responses, and dosimetry. The detectably effective amount in embodiments of this disclosure may also vary depending on instrument and film-related factors. Optimization of such factors may be within the scope of a person skilled in the art.

[0020] The term “detectable” refers to the ability to detect a signal or presence of an embodiment of the disclosure beyond a background signal. The term “detectable signal” or the phrase “detection of a labeled compound” or “detectable labeled compound” refers to the detection (directly or indirectly) of a labeled compound in a host or sample. Detection of a labeled compound refers to the ability to detect the presence of a labeled compound in a host or sample and distinguish it from other background signals originating from the host or sample. In other words, there is a measurable and statistically significant difference (for example, statistical significance is a difference sufficient to distinguish between the detectable signal and the background (for example, a difference of about 0.1%, 1%, 3%, 5%, 10%, 15%, 20%, 25%, 30%, or 40% or greater)). Standard and / or calibration curves may be used to determine the relative intensity of the detectable signal and / or the background. The detectable signal may be generated from low to high concentrations of labeled compounds. In one embodiment, the detectable signal may be the sum of each of the individual labeled compound signals. In one embodiment, the detectable signal may be generated from sum, integration, or other mathematical processes, formulas, or algorithms. In one embodiment, the sum, integration, or other mathematical processes, formulas, or algorithms may be used to process the detectable signal so that it can be distinguished from background noise, etc.

[0021] As used herein, “agent,” “active agent,” etc., may include the compounds of this disclosure (e.g., labeled compounds). The agents may be contained in a composition or pharmaceutical composition. As used herein, “pharmaceutical composition” refers to a combination of an active agent and a pharmaceutically acceptable carrier. As used herein, “pharmaceutical composition” means a composition suitable for administration to a subject (e.g., a mammal, in particular, a human). Generally, “pharmaceutical compositions” are sterile and preferably free from impurities that could induce an undesirable response in the subject (e.g., the compounds in the pharmaceutical composition are pharmaceutical grade). Pharmaceutical compositions may be designed for administration to a subject or patient in need via many different routes of administration (including oral, intravenous, sphincter, rectal, parenteral, intraperitoneal, intradermal, intra-intramuscular, subcutaneous, and inhalation).

[0022] "Pharmacologically acceptable excipient," "pharmaceutically acceptable diluent," "pharmaceutically acceptable carrier," or "pharmaceutically acceptable adjuvant" generally refers to excipients, diluents, carriers, and / or adjuvants that are generally safe, non-toxic, and not otherwise undesirable biologically, and include excipients, diluents, carriers, and adjuvants acceptable for veterinary and / or human pharmaceutical use. With respect to compositions suitable for human administration, the term "excipient" means, but does not include, those components listed in Remington: The Science and Practice of Pharmacy, Lippincott Williams & Wilkins, 21st edition (2006) (the contents of which are incorporated herein by reference).

[0023] The term “unit dosage form,” as used herein, refers to a physically discontinuous unit suitable for unit administration to human and / or animal subjects (each unit containing a predetermined amount of a compound calculated in sufficient quantity (e.g., host body weight, disease, disease severity, etc.) to produce the desired effect when associated with a pharmaceutically acceptable diluent, carrier, or vehicle). Specifications regarding unit dosage forms depend on the specific compound used, the route and frequency of administration, the effect to be achieved, and the pharmacodynamics associated with each compound in the host.

[0024] The term “effective amount,” as used herein, refers to the amount of an embodiment of the Disclosure (which may be called a labeled compound) administered to a human being, for example, to be used to image cells (e.g., cardiac cells). “Administration” means introducing an embodiment of the Disclosure into a subject. Administration includes, but is not limited to, routes such as intravenous, oral, topical, subcutaneous, intraperitoneal, intra-arterial, inhalation, vaginal, rectal, and nasal, and may also include introduction into cerebrospinal fluid or infusion into body compartments.

[0025] As used herein, the terms “host” or “subject” include humans, mammals (e.g., cats, dogs, horses, etc.), and other living animals. In particular, the host is a human subject. Typical hosts to which embodiments of this disclosure may be administered are mammals, in particular primates, and especially humans. For veterinary applications, a wide variety of subjects are suitable (e.g., livestock (e.g., cattle, sheep, goats, cows, pigs, etc.); poultry (e.g., chickens, ducks, geese, turkeys, etc.); and domesticated animals, in particular pets (e.g., dogs and cats)). For diagnostic and research applications, a wide variety of mammals are suitable subjects (including rodents (e.g., mice, rats, hamsters), rabbits, primates, and pigs (e.g., inbred pigs, etc.)). Furthermore, with regard to in vitro applications (in vitro diagnostic and research applications), the above-mentioned subject fluid and cell samples are suitable for use as "samples," which include, for example, blood, urine, or tissue samples of mammals (especially primates such as humans), or blood, urine, or tissue samples of animals as referred to in relation to veterinary applications.

[0026] The invention disclosed herein has many embodiments. For example, embodiments of the invention include a method for imaging cardiac cells in a subject / patient. Such a method involves the subject being subjected to the following formula: [ka] A step of making a compound having a sugar, wherein the administration route is selected to allow the compound to be phosphorylated by deoxycytosine kinase and deoxyguanosine kinase present in the cardiac cells of the subject; and a step of then imaging the subject, wherein the detection of the presence of the compound includes a step of indicating the presence of cardiac cells. In a particular embodiment of the present invention, the subject is selected to be a patient who has been administered a therapeutic agent or radiotherapy, and the resulting one or more images of the heart are used to obtain information about the effect of the drug or radiotherapy on the heart of the subject. In an illustrative embodiment of the invention disclosed herein, the therapeutic agent is an anthracycline (e.g., doxorubicin) or an immune checkpoint inhibitor, and the resulting one or more images of the heart are used to obtain information about cardiotoxicity resulting from the administration of doxorubicin or the immune checkpoint inhibitor. In a particular embodiment of the present invention, the therapeutic agent is selected to be observed to act on mitochondria in cardiac cells.

[0027] In a particular method of the present invention, the subject is selected to be a patient diagnosed with cardiovascular disease; and / or a patient diagnosed with cancer; and / or a patient undergoing treatment for cardiovascular disease or cancer. Optionally, the subject is a patient treated with at least one therapeutic agent, including an immune checkpoint inhibitor (e.g., an antibody such as pembrolizumab; nivolumab; atezolizumab; avelumab; bevacizumab; and durvalumab) selected to affect CTLA-4 or PD-1 / PD-L1 blockade. In a particular embodiment of the present invention, the method further comprises the steps of observing one or more images of the heart on a first date; observing one or more images of the heart on a second date; and then comparing the images obtained on the first date with the images obtained on the second date to observe changes in the heart over time. In some embodiments of the present invention, the amount of time from the first date to the second date includes less than one week. Or, in other embodiments of the present invention, the amount of time from the first date to the second date includes at least one week, two weeks, or three weeks, or at least one month, two months, or three months. Some embodiments of the present invention include, using one or more images of the heart: the step of evaluating one or more parameters of myocardial perfusion in the subject's heart; and / or the step of evaluating one or more parameters of myocardial viability in the subject's heart; and / or the step of evaluating one or more parameters of inflammation in the subject's heart.

[0028] Embodiments of the present invention also include methods for imaging cells in a subject in response to the administration of a therapeutic agent. Such methods may be used to observe in vivo physiological changes (e.g., cardiotoxicity, drug responsiveness, etc.) resulting from the administration of a therapeutic compound. Typically, such a method involves administering the therapeutic agent to the subject, and then (typically after a period such as at least 48 hours, at least one week, or at least one month) administering the following formula to the subject: [ka] A step of administering a PET probe compound, wherein the administration route is selected to allow the compound to be phosphorylated by deoxycytosine kinase and deoxyguanosine kinase present in the cells of the subject (e.g., cardiac cells, cancer cells, or leukocytes). These methods further include a step of PET imaging the subject, wherein the detection of the presence of the PET probe compound indicates the presence of cells; and a step of finally correlating the observed presence of the compound in the cells of the subject with the subject's response to the therapeutic agent. In some embodiments of the present invention, such a correlation step is performed on a control (e.g., a representative [ in a healthy human). 18 [F]F-AraG PET image profile), obtained from patients before administration of the above drugs, etc. 18This includes comparison to F]F-AraG PET images. In certain working embodiments of such methods disclosed herein, the correlating step includes observing the presence of the PET probe compound in cardiac cells, immune cells and / or lymph nodes (e.g., tumor inflow lymph nodes). In certain embodiments of these methods, the subjects are selected to be patients diagnosed with cardiovascular disease; and / or patients diagnosed with cancer; and / or patients undergoing treatment for cardiovascular disease or cancer. In illustrative embodiments of the inventions disclosed herein, the therapeutic agent is a drug observed to modulate mitochondrial physiological function, an anthracycline (e.g., doxorubicin) or an immune checkpoint inhibitor (e.g., an immune checkpoint inhibitor selected to affect CTLA-4 or PD-1 / PD-L1 blockade), and one or more images of the heart are used to obtain information regarding cardiotoxicity resulting from the administration of doxorubicin or the immune checkpoint inhibitor.

[0029] In a particular embodiment of the present invention, the method for imaging cells in a subject responding to the administration of a therapeutic agent further comprises the steps of: observing one or more images obtained at a first date / time; observing one or more images obtained at a second date / time over time; and then comparing the images obtained at the first date with the images obtained at the second date in order to observe changes in the patient's physiological function resulting from the administration of the therapeutic agent (for example, to distinguish between responders and non-responders to the therapeutic agent). In a particular embodiment of the present invention, the amount of time between the first date and the second date includes less than one week. Alternatively, the amount of time between the first date and the second date includes at least one week, two weeks, or three weeks, or at least one month, two months, or three months.

[0030] A further embodiment of the present invention is a method for imaging mitochondrial activity in subject cells. Typically, these methods are expressed by the following formula: [ka] The method comprises the steps of administering a compound to a subject, wherein the administration route is selected to allow the compound to be phosphorylated by deoxycytosine kinase and deoxyguanosine kinase present in the subject's cells; and subsequently imaging the subject, wherein the detection of the presence of the compound indicates the presence of mitochondrial activity, and consequently, mitochondrial activity in the subject's cells is observed. Typically, in these methods, the subject is a patient selected for having a mitochondrial deficiency. In certain embodiments, the method is used to screen the subject for mitochondrial dysfunction; the mitochondrial dysfunction is a cardiovascular disease, a neuropsychiatric disorder, or a neurodegenerative disease. In some embodiments, the above method is used to screen subjects for mitochondrial dysfunction selected from the group consisting of myocardial perfusion, bipolar disorder, depression, schizophrenia, Alzheimer's disease, Parkinson's disease, Friedreich's ataxia, amyotrophic lateral sclerosis, Huntington's disease, progeria, cardiomyopathy, respiratory chain disorder, mtDNA depletion, myoclonus epilepsy, red ragweed syndrome, encephalomyopathy, lactic acidosis, stroke-like seizures, and optic nerve atrophy.

[0031] In short, in vivo, AraG is metabolized in a specific manner by deoxyguanosine kinase and incorporated into mitochondrial DNA (Figure 2.4). These observations led to the synthesis of a more water-soluble AraG prodrug, 2-amino-6-methoxypurine arabinoside (506U, nelarabine), for potential clinical applications in the treatment of T-cell lymphoblastic diseases. This compound was developed by Glaxo over several years and is now FDA approved for the treatment of relapsed T-cell ALL and T-cell lymphoblastic lymphoma.

[0032] Embodiments of the present disclosure include methods using compounds that can be formed by many processes, such as the [18F]-F-arabinofuranosylguanine compound disclosed in U.S. Patent Application No. 17 / 314,366, filed on May 7, 2021, titled "METHODS AND MATERIALS FOR MAKING PET RADIOTRACERS" (the contents of which are incorporated herein by reference). Such embodiments of the present invention include, for example, the following general formula: [ka] The present invention comprises a [18F]-F-arabinofuranosylguanine compound formed from the composition of a substance containing a compound having; where: PG comprises a protecting group; LG comprises a leaving group. Typically, in such embodiments of the present invention, the nitrogen atom to be bonded to the protecting group is bonded to two protecting groups as represented by "N(PG)2". Alternatively, this nitrogen atom is bonded to a hydrogen atom and one protecting group as represented by "NHPG". In an illustrative embodiment of the present invention, the above composition is precursor 1 or precursor 3: [ka] This includes at least one of the following, where: t Bu contains a tert-butyloxycarbonyl alcohol protecting group; Boc contains a tert-butyloxycarbonylamine protecting group; THP contains a tetrahydropyranyl alcohol protecting group; EOE contains an ethoxyethyl alcohol protecting group; Tf contains a triflate leaving group.

[0033] Embodiments of this disclosure include methods using [18F]-F-arabinofuranosylguanine compounds formed by other processes, such as those disclosed in the U.S. application for the invention “Compounds and Methods of Making Compounds” (filed February 25, 2021, application number 17 / 185,502, the contents of which are incorporated herein by reference). Such methods include compounds such as those shown in Figures 1.1A and 1.1B, having formulas 2, 3, 4, 5, 11, and 12, as well as 2', 3', 4', 5', and 11', and the use of such compounds for imaging, etc.

[0034] Exemplary embodiments for preparing labeled compounds include, in particular, compounds containing the isotope (Ist) and formula 1': [ka] A compound having formula 2' is reacted with this compound to produce formula 2': [ka] A step of forming a compound having; and Deprotection is performed on the compound having the above formula 2', resulting in formula 3: [ka] A step of forming a compound having, where PG is a protecting group, LG is a leaving group, and R is R1, R2, R3, R4, R5, and R6: [ka] A compound having a formula selected from the group consisting of R'1, R'2, R'3, R'4, and R'5: [ka] A compound having a formula selected from the group consisting of, where Ac is an acetyl group and Bz is a benzoyl group, where each of Ac and Bz comprises steps that can be substituted as described herein. Exemplary embodiments of the labeled compound include, in particular, a labeled compound, The above compound is given by formula 3: [ka] The compound contains, where Ist is an isotope, and where R' is R'1, R'2, R'3, R'4, and R'5: [ka] The compound has a formula selected from the group consisting of the following. An illustrative embodiment of the labeled compound is, in particular, formula 2': [ka] The compound comprises a compound having R1, R2, R3, R4, R5, and R6: [ka] A group having a formula selected from the group consisting of the following, where Ac is an acetyl group and Bz is a benzoyl group, where each of Ac and Bz may be replaced as described herein.

[0035] An illustrative embodiment of a method for imaging cardiac cells includes, in particular, the steps of administering the compound of the Disclosure to a subject; and imaging the subject, wherein the detection of the presence of the compound includes the step of indicating the presence of cardiac cells. Another illustrative embodiment of a method for imaging the presence or degree of mitochondrial activity in cells includes, in particular, the steps of administering the compound of the Disclosure to a subject; and imaging the subject, wherein the detection of the presence of the compound includes the step of indicating the presence or degree of mitochondrial activity in the cells of the subject.

[0036] In demonstrating one working embodiment of the present invention, as a PET tracer, [ 18 The use of [F]F-AraG was expressed in healthy volunteers and in cancer patients receiving immunotherapy. In these studies, significant tracer uptake in the heart was observed in both groups. Surprisingly, however, when using this specific PET tracer, the signal observed in the heart wall of healthy volunteers was observed to be different in patients receiving immunotherapy. This unexpected finding was [ 18 [F]F-AraG demonstrates the ability to image changes in cardiac cells caused by drugs such as immunomodulators and chemotherapeutic agents, as well as other damage to the heart, such as damage resulting from radiation exposure. In this context, it can be used to assess mitochondrial activity, [ 18 F]F-AraG may be used in methods to test novel drugs that can improve mitochondrial function and / or to evaluate the efficacy of these types of drugs in improving mitochondrial function. In light of this finding, [ 18 The use of [F]F-AraG is in several distinct applications, including observation of myocardial perfusion, myocardial viability, and cardiac inflammation, compared to existing methods. 18 This addresses the needs in this technology by providing significant advantages over F-labeled tracers. Furthermore, as a PET tracer, [ 18 The use of [F]F-AraG may also be used in cardiac drug development studies and to observe treatment responses to agents that modulate physiological (e.g., mitochondrial) activity (see, e.g., Zinovkin et al., Curr Mol Pharmacol. 2019;12(3):202-214).

[0037] As noted above, embodiments of the present disclosure include compounds such as those shown in Figures 1.1A and 1.1B, having formulas 2, 3, 4, 5, 11, and 12, as well as formulas 2', 4', and 11', and the use of such compounds for imaging, for example. Embodiments of the present disclosure are advantageous because such compounds can be produced in a few simple steps, as described below in detail and in Examples 1-3. In particular, embodiments of the present disclosure provide direct fluorination of guanosine nucleoside precursors, followed by removal of protecting groups. Embodiments of the above method encompass two steps, which occur over a short period of time, and both steps are advantageous over other possible alternative commercial production schemes.

[0038] Embodiments of the above method are shown in schemes A-D and A'-D' in Figures 1.1A and 1.1B. Scheme A is general but uses specific protecting (PG) and leaving (LG) groups, while schemes B-D provide further details. Schemes A'-D' are general in that they do not use specific protecting and leaving groups. It should be noted that reagents may be modified in a manner similar to those described below. More detailed schemes of embodiments of the present disclosure are shown in schemes 1 and 2 of Examples 1 and 2. Generally, embodiments of the above method encompass the step of preparing labeled compounds such as those embodied in formulas 3, 5, and 12. In one embodiment, the above method may encompass the step of reacting a compound containing an isotope (Ist) with a compound having formula 1 in Figure 1.1A to form a compound having formula 2 in Figure 1.1A. The synthesis described in Figures 1.1A and 1.1B is very similar, with the main difference being the specific use of PG and LG in Figure 1.1A. Therefore, the following considerations regarding the synthesis in Figure 1.1A may also apply to the synthesis in Figure 1.1B. Various substitutions related to protecting groups, leaving groups, and reactions described herein may be used in the synthesis described in Figure 1.1B.

[0039] Figure 2.1 illustrates schemes 1 and 2, which relate to specific embodiments of the present disclosure and are described in detail in Example 1. In particular, the above 18 F(FAraG precursor (compound 8) is generated, and then, 18 F(FAraG) is reacted to form a precursor (compound 12). Details of the reaction steps are shown in Figure 2.1, which is similar to that described above with respect to the general synthesis. Figures 3.4 to 3.6 also provide specific details of the synthesis of embodiments of this disclosure, which are described in detail in Example 2.

[0040] Embodiments of this disclosure also encompass methods for imaging cardiac tissue and cells. Generally, embodiments of labeled compounds can be used to image the localization and / or quantity of cardiac cells in a subject (e.g., a living human). The labeled compound may be administered to the subject, and the subject or a portion of the subject may then be imaged using a device such as positron emission tomography (PET) to detect the presence and location within the subject, and / or the amount of the labeled compound present. The presence and / or quantity may be used to detect the presence, location, and / or number / size of cardiac cells, cancer cells, and / or leukocytes in the subject.

[0041] The disclosure may also provide packaged compositions comprising precursors or intermediates (e.g., formulas 1, 1', 2, or 2') to labeled compounds, as well as instructions for preparing and using the labeled compounds (e.g., written instructions for their use). The kits may further include suitable buffers and reagents known in the art for administering embodiments of the disclosure to subjects. [Examples]

[0042] The following examples are provided to give a complete disclosure and explanation of how to prepare and use the present disclosure, and are not intended to limit the scope of what the inventors consider to be such disclosures, nor are they intended to represent all or only experiments in which the following experiments are performed.

[0043] Example 1: Exemplary method for fabricating a PET probe AraG is a nucleoside analog that has shown efficacy in the treatment of T-cell lymphoblastic diseases. It is metabolized in a specific manner by deoxyguanosine kinase and incorporated into mitochondrial DNA. The inventors of this invention have found that, 18 F derivatives were synthesized and used as molecular probes. Those skilled in the art can test uptake and metabolism in cell lines and determine the efficacy of this compound in imaging T lymphoblasts in mouse models and activated T cells in mouse models of acute graft-versus-host disease.

[0044] Previously, the inventors of the present invention have developed a novel 2-deoxyguanosine analog. 18 F derivative, 9-β-D-arabinofuranosylguanine (" 18 F-AraG" or "[ 18 We successfully synthesized "F]F-AraG" and used it as a molecular probe (Figure 2.1). 18 F]AraG cell uptake was evaluated in the leukemia cell line CCRF-CEM, [ 3 This was compared with [H]AraG cell uptake. Figure 2.2 shows the uptake by the CCRF-CEM cell line. 18 This shows the incorporation of [F-AraG] and demonstrates that the above incorporation is dose-dependent. The cold derivative 2F-AraG is 8-[ 3 To determine whether or not it competes with the uptake of [H]-AraG, the inventors used CCRF-CEM, MOLT-4 (leukemia cell line) and Raji (human Burkitt lymphoma cell line) [ 3 We tested the uptake of [H]-Arag and its competition with cold 2F-AraG (Figures 2.3A and 2.3B). Figure 2.3A shows that as the amount of 2F-AraG (1-100 μM) increased, [3 This indicates that a decrease in H]-AraG uptake is induced. Similar results were observed in MOLT4 and Raji cell lines (Figure 2.3B). A competitive assay showed that the cold derivative 2F-Arag exhibits a similar uptake pathway. 3 This demonstrates competition with the uptake of [H]-AraG.

[0045] Early microPET scans in normal nude mice showed that 18 This shows that F-AraG is taken up within the lymph nodes. Figure 2.1 describes an embodiment of a method for preparing the compounds of this disclosure. The above 2'-deoxy-2'-fluoroarabinonucleosides have been reported as antiviral agents (Proc Natl Acad Sci USA, 1992; 89: 2970-2974; Pharmacol 1999; 43: 233-240; J Pharm Chem 1996; 85: 339-344 (each of these is incorporated herein by reference)). The inventors have radiolabeled 8-[ 18 We were investigating 8-[Fluoroguanine derivatives as potential in vivo probes for imaging gene expression using positron emission tomography (PET). Based on a direct radiofluorination reaction, we developed 8-[ 18 We developed a method for preparing fluoroguanine derivatives, and 8-[ 18 [F]fluoroguanosine was synthesized from guanosine (Nuclear Medicine and Biology. 2000; 27(2): 157-162 (this is incorporated herein by reference)). In recent years, 2'-deoxy-2'-[ 18 F]Fluoro-9-β-D-arabinofuranosyladenine ([ 18 The synthesis of [FAA] has been reported (J Label Comp Radiopharm 2003; 46: 805-814 (this is incorporated herein by reference)).

[0046] Example 2: As a PET imaging agent for leukocytes [ 18 F]F-ARAG 9-(β-D-arabinofuranosyl)guanine (AraG) is a guanosine analog that has been shown to be effective in the treatment of T-cell lymphoblastic diseases. To test the possibility of using radiofluorinated AraG as an imaging agent, the inventors have introduced 2'-deoxy-2'-[ 18 F]Fluoro-9-β-D-arabinofuranosylguanine([ 18 We synthesized [F]F-AraG) and investigated its uptake in T cells.

[0047] For this purpose, the inventors have prepared 2-N-acetyl-6-O-((4-nitrophenyl)ethyl)-9-(3',5'-di-O-trityl-2'-O-triphyll-β-D-ribofuranosyl)guanine in DMSO for 45 minutes at 85°C [ 18 [F]KF / K.2.2.2 via direct fluorination 18 F]F-AraG was synthesized in both CCRF-CEM leukemia cell lines (inactivated) and activated primary thymocytes. 18 The uptake of [F]F-AraG was evaluated. The inventors evaluated [ 18 F]F-AraG was successfully prepared. Preliminary cell uptake experiments showed that both CCRF-CEM leukemia cell lines and activated primary thymocytes were [ 18 [F]F-AraG was shown to be incorporated. 18 F]F-AraG was successfully synthesized by direct fluorination of a suitable precursor of guanosine nucleoside. This approach is useful for other important PET probes (e.g., [ 18 F]FEAU, [ 18 F]FMAU and [ 18 It can be used for the synthesis of [F]FBAU). Cell uptake studies have shown that it can be used as a PET imaging agent for T cells. 18 Supports further research to investigate the use of F]F-AraG.

[0048] Scheme 1 is as shown in Figure 3.4, 2-N-acetyl-6-O-((4-nitrophenyl)ethyl)-9-(3,5-di-O-trityl-2-triphyll-β-D-ribofuranosyl)guanine(2), [ 18 The synthesis of the F-AraG precursor is shown. Treatment of 2',5'-di-O-tritylguanosine derivative 1 with CF3SO2Cl / DMAP yielded 2-N-acetyl-6-O-((4-nitrophenyl)ethyl)-9-(3',5'-di-O-trityl-2'-O-triphyll-β-D-ribofuranosyl)guanine (2) in 65% yield. Scheme 2 shows the synthesis of a cold F-AraG standard prepared according to the procedure in the literature (J. Org. Chem. 1992, 57, 7315-7321 (which is incorporated herein by reference)), as shown in Figure 3.5. Derivative 3',5'-di-O-tritylguanosine 1 was converted to 6-O-((4-nitrophenyl)ethyl)-9-(3',5'-di-O-trityl-2'-fluoro-β-D-arabinofuranosyl)guanine 3 using DAST reagent. Deprotection of 3 with DBU yielded 9-(3',5'-di-O-trityl-2'-fluoro-β-D-arabinofuranosyl)guanine (4). Finally, deprotection of 4 with TFA yielded 2'-deoxy-2'-fluoro-9-β-D-arabinofuranosylguanine 5 (F-AraG).

[0049] Radiochemistry: [ 18 The F-labeled guanosine derivative 6 (Scheme 3 as shown in Figure 3.6) was infused in DMSO at 85°C for 45 minutes. 18 Prepared by nucleophilic substitution of triflate in 2 with [F]fluoride ions. HPLC [ 18 Purification of F]6 is performed using deprotonated starting material 2 (AraG) as the final product [ 18 It was necessary to avoid contamination of F]F-AraG 7. 18 F]6 is first hydrolyzed smoothly with a base (0.5M NaOCH3) and then with an acid (1N HCl), 18F]F-AraG 7 was obtained. The radiochemical yield was 7 - 10% (corrected calibration, n = 10). Its specific activity was 0.8 - 1.3 Ci / μmol. The analytical HPLC profile of the co-injection of 7 and cold F-AraG standard is shown in Figure 3.1.

[0050] Cells 18 To evaluate the ability of cells to 18 incorporate F]F-AraG, the CCRF-CEM cell line (acute lymphoblastic T leukemia cells, non-activated) and primary T cells were 18 exposed to F]F-AraG. Figure 3.2 shows the 18 incorporation of F]F-AraG by non-activated CCRF-CEM cells, 18 indicating a dose-dependence with a two-fold (P < XX) increase in F]F-AraG incorporation by cells exposed to 10 μCi compared to cells exposed to 3 μCi. Then, the inventors examined whether activated primary thymocytes derived from normal mouse tissues also 18 accumulate F]F-AraG. Figure 3.3 shows that primary T cells stimulated with 100 U / mL interleukin 2 incorporated 1.4-fold more (p = 0.14) 18 F] F-AraG than unstimulated primary T cells, and primary T cells stimulated with 50 nM PMA and 1 μg / mL ionomycin incorporated 4.7-fold more (p = 0.003) 18 F]F-AraG, showing the data.

[0051] Numerous reports exist on the indirect synthesis of 2'-deoxy-2'-fluoro-9-β-D-arabinofuranosylguanine (F-AraG) in which fluorine is first introduced at the arabino position at C-2, and then the fluorinated sugar reacts with a purine base (Carbohydr. Res. 1975, 42, 233-240, J. Org. Chem. 1985, 50, 3644-3647, and Chem. Pharm. Bull. 1989, 37, 336-339 (each of which is incorporated herein by reference)). However, there is only one report on the direct synthesis of cold F-AraG, in which fluorine is incorporated into the arabino position at C-2 of the sugar by direct fluorination of a guanosine derivative appropriately protected with DAST (J. Org. Chem. 1992, 57, 7315-7321 (which is incorporated herein by reference)). 18 The synthesis of F-labeled F-AraG has not been reported to date. 18 Due to the difficulty in synthesizing [F]-labeled DAST and the long reaction time for DAST-mediated fluorination, 18 [F]DAST method via [ 18 Synthesizing [F]F-AraG is not practical. Compound 2, 18 Prepare the F]F-AraG precursor (as shown in Scheme 1 in Figure 3.4), 1 H and 19 The results were characterized by 1F NMR spectroscopy and high-resolution mass spectrometry. The chemical shift of H2' changed from 4.73 ppm in 1 to 6.03 ppm in 2, due to the electronegativity of the triphyll group at the C2' position. 19 The 1F NMR spectrum showed a singlet state at -75.00 ppm, which is consistent with the chemical shift of sugar triflate. A similar chemical shift trend was observed with respect to the synthesis of adenosine triflate

[19] . To the best of our knowledge, precursor 2 is novel and was first synthesized in our laboratory (a provisional patent application has been filed). Furthermore, for the first time, we have synthesized 2 in DMSO [ 18By directly fluorinating with F]KF / K.2.2.2, at a radiochemical yield of 7 - 10% (decay corrected), with a specific activity of 0.8 - 1.3 Ci / μmol, 18 F]F - AraG (7, Scheme 3 as shown in Figure 3.6) was synthesized. The identity and purity of 7 were confirmed by co - injecting the normal standard compound 5 onto an analytical HPLC column (Figure 3.1).

[0052] In cell culture 18 To evaluate the performance of F]F - AraG 7, the inventors performed several assays. To confirm the ability of cells to 18 take up F]F - AraG, the inventors exposed the CCRF - CEM cell line (acute lymphoblastic T leukemia cells, non - activated) and primary T cells to 18 F]F - AraG. Figure 3.2 shows the uptake of 18 F]F - AraG by CCRF - CEM cells and 18 shows that F]F - AraG uptake is dose - dependent. These data also support that 18 most of the F]F - AraG is taken up by cells within the first hour of exposure. 18 If F]F - AraG (which has an isotope half - life of 110 minutes) is to be finally proven effective as a PET tracer, rapid uptake is necessary. Since the lymphoblastic T cell line was confirmed to 18 take up F]F - AraG, the inventors then investigated whether primary T cells (non - tumorigenic T cells obtained from normal mouse tissues) also 18 take up F]F - AraG. Figure 3.3 shows the data from two independent experiments, which show that non - tumorigenic but activated primary T cells take up 18 F]F - AraG to a recognizable level. The increased uptake of 18 F]F - AraG by activated T cells is relevant as a PET tracer in the detection of graft - versus - host disease (GVHD). 18It may be possible to utilize [¹⁸F]F-AraG. GVHD is a disease mainly caused by T cells. If abnormally activated T cells can be detected by PET, it may facilitate early diagnosis of GVHD in patients without invasive procedures. Since AraG has been reported to induce neurotoxic side effects in some patients at therapeutic serum levels (about 150 μM)

[24] , the inventors, while avoiding potential neurotoxicity in PET patients, 18 In order to optimize [¹⁸F]F-AraG, a dose lower (about 0.5 μM) than the reported therapeutic level of AraG was selected to be utilized in the inventors' assay.

[0053] As considered in this example, 18 [¹⁸F]F-AraG can be synthesized by direct fluorination methods. This approach can currently be used for the synthesis of other important PET tracers that are synthesized by multiple steps and require time-consuming purification processes (e.g., 18 [¹⁸F]FEAU, 18 [¹⁸F]FMAU and 18 [¹⁸F]FBAU) (J. Label. Radiopharm. 2003, 46, 285 - 289 (which is incorporated herein by reference)). Preliminary cellular uptake studies performed in CCRF-CEM cells (non-activated) and activated primary T cells suggest the application of [¹⁸F]F-AraG as a new PET imaging agent for the detection of T cell-origin diseases. 18 [¹⁸F]F-AraG

[0054] Example 3: Use of ¹⁸F-F-ARAG in cardiac imaging Figure 5 shows 3 The kinetic characteristics of dCK and dGK reactions with respect to [³H]F-AraG. (a) 3 Enzyme kinetics of dGK or dCK with respect to [³H]F-AraG. (b) dGuo positive control for dGK activity. (c) dCyd positive control for dCK activity. The lines represent the best fit. 3The Km of dGK for H]F-AraG was found to be 7.27 μM at a Vmax of 23.44 nmol / min / mg, while dCK was [ 3 It showed lower affinity for [H]F-AraG (Km=50.89 μM, Vmax=262.5). The inventors observed high affinity for dCK to dCyd (Km=1.997, Vmax=9.454) and similarly higher affinity for dGuo-positive control for dGK activity (Km=5.083, Vmax=123.2).

[0055] dGK is, 18 Because it has a higher affinity for F]F-AraG, the inventors have found that at the tracer level, [ 18 We considered the possibility that [F]F-AraG can be preferentially phosphorylated by mitochondrial kinases. Mitochondria are organelles responsible for supplying energy to cells by purifying ATP. The heart is a particularly mitochondrial-rich organ due to its high energy requirements. In the last decade, mitochondrial dysfunction has been recognized as a significant aspect of cardiovascular pathology. As a result, treatments focused on cardiac mitochondrial function are rapidly emerging. As a substrate for mitochondrial dGK, [ 18 F]F-AraG was found to be a useful drug for reporting on cardiac mitochondrial activity.

[0056] In an illustrative embodiment of the present invention, as a tracer in the PET methodology, [ 18 The use of F]F-AraG was evaluated in healthy volunteers and cancer patients receiving immunotherapy. Significant cardiac uptake was observed in both groups (Figure 6). Figure 6 shows the cardiac uptake of [ 18 This describes a PET method using F]F-AraG. Our disclosure shows that high mitochondrial activity in the heart is elevated 1 hour after injection of the tracer. 18 This provides clear evidence of the generation of the F]F-AraG signal.

[0057] Interestingly, in this embodiment of the present invention, in the heart wall of a healthy volunteer [ 18 [F]F-AraG signaling was found to be different in patients receiving immunotherapy. In particular, Figure 7 shows the [F]F-AraG signaling in the cardiac wall of healthy volunteers and patients receiving immunotherapy. 18 The intensity of the F]F-AraG signal is shown (the first refers to the pre-treatment image, and the second refers to the image taken 2-3 weeks after a single injection of anti-PD-1 antibody).

[0058] The results provided herein are, 18 F]F-AraG demonstrates the ability to image the heart and report on changes that may be caused by drugs or injury. In light of this, as a PET tracer, [ 18 Using [F]F-AraG, in several distinct applications (including myocardial perfusion, myocardial viability and cardiac inflammation, as well as methods for observing treatment responses to cardiac drug development research and mitochondrial-targeted drugs), existing 18 This could offer significant advantages over F-labeled tracers.

[0059] Example 4: [18F]F-ARAG as an imaging biomarker for early diagnosis and monitoring of cardiotoxicity associated with doxorubicin and immune checkpoint inhibitor therapy This embodiment (a specific predictive aspect) discloses the development of a highly sensitive and specific PET imaging strategy for the early diagnosis of cancer treatment-related cardiotoxicity. The inventors focus on two classes of cancer treatments associated with cardiotoxicity: for example, the administration of immune checkpoint inhibitors (ICIs) (which result in myocardial damage via T cell infiltration and lead to myocarditis), and anthracyclines (e.g., doxorubicin) that cause mitochondrial dysfunction and direct cardiomyocyte death. In this embodiment, the inventors consider the following: 1) [ 18 How F]F-AraG may enable early and pathophysiologically specific identification of cardiotoxicity related to both ICI and doxorubicin treatment;2) 18 FDG and [18 F]F-AraG comparison, and [ 18 Determination of variability in F]F-AraG myocardial uptake; and 3) For example, to perform a retrospective analysis of myocardial uptake in cancer patients, normal physiological [ 18 Determine the range of F-AraG acquisition.

[0060] Life expectancy after cancer diagnosis has improved significantly through earlier diagnosis and more effective cancer treatments. However, the general toxicity of cancer treatments can interfere with treatment, affect survival, and result in debilitating adverse effects and a reduced quality of life. Treatment-related cardiovascular toxicity is a major cause of morbidity and mortality in cancer patients and cancer survivors (1). For many cancer survivors, cardiovascular events, rather than cancer recurrence, represent the primary risk of death in many cancer types (2). Cardiovascular complications can and have been well-documented with older cancer treatments (e.g., anthracyclines) and more selective targeted therapies (e.g., kinase inhibitors) (3,4). Monoclonal antibodies targeting immune checkpoint inhibitors (ICIs), or moduloimmune agents (e.g., programmed death-1 (PD-1), its ligand (PD-L1), or cytotoxic T lymphocyte antigen-4 (CTLA-4)), have yielded impressive clinical outcomes in a fraction of patients with advanced tumors, but a multi-system immune-related adverse effect (including cardiovascular toxicity) has been reported in conjunction with the treatment (5). ICI-related cardiovascular toxicity is not well understood and is largely underreported. Of all immune-related adverse events, myocarditis is the most fatal, with a fatality rate of nearly 50% (6,7). Given the rapidly expanding use of ICIs in cancer treatment, accurate assessment and better understanding of immune-related cardiotoxicity represent a critical, unaddressed clinical challenge for successful patient care and management.

[0061] Due to its non-invasive advantage, cardiac imaging is the most recommended technique for monitoring cardiac function during and after cancer treatment (8,9). Serial measurement of left ventricular ejection fraction (LVEF) by echocardiography is currently the standard technique for detecting cardiotoxicity. However, a decrease in LVEF often appears late in cardiac injury and may indicate irreversible damage. Cardiac MRI allows for the detection of mechanical changes that occur prior to left ventricular dysfunction, but it lacks molecular specificity and sensitivity in ICI-related cardiotoxicity (10).

[0062] The inability of currently used imaging techniques to detect subclinical cardiac involvement has demonstrated a significant impediment to the prevention and better management of cardiac complications in cancer patients and survivors (11). Furthermore, with the advancement of immunotherapy, rapid diagnosis of inflammatory cardiac complications is essential with current diagnostic techniques that are neither highly sensitive nor specific. While high specificity, resolution, and sensitivity make positron emission tomography (PET) the optimal imaging modality for assessing myocardial metabolism and perfusion, its usefulness in the early detection of cancer treatment-related cardiotoxicity has been limited. Nevertheless, PET imaging agents that target early indicators of cardiovascular toxicity offer a powerful, highly specific, and non-invasive tool for detecting subclinical cardiotoxicity.

[0063] In this embodiment, the inventors [ 18 [F]F-AraG, as an imaging biomarker for the early detection and monitoring of cardiotoxicity associated with ICIs and chemotherapy, focuses on PET agents with unique capabilities to assess activated T cell and cardiomyocyte mitochondrial function. 18 F]F-AraG was developed by Namavari et al. to image activated T cells (12). It is made of arabinosylguanosine (AraG) 18It is a 1F-labeled analog of a compound that showed markedly selective accumulation in T cells (13,14), and its prodrug, nelarabine, is used for the treatment of T-cell acute lymphoblastic leukemia and T-cell lymphoblastic lymphoma. 18 F]F-AraG enters T cells via a nucleoside transporter and is captured into the cell primarily through phosphorylation by deoxyguanosine kinase (dGK), an enzyme that is present only in mitochondria and is crucial for supplying nucleotides for mitochondrial DNA synthesis (Figure 8) (15-17). Upon activation, T cells undergo metabolic reprogramming, dramatically increasing both mitochondrial mass and mtDNA (18,19), which is [ 18 [F]F-AraG can be visualized. 18 The ability of [F]F-AraG to image T cell activation and thus provide early indication of an immunotherapy-adapted response in cancer patients is currently being investigated in numerous Phase II studies.

[0064] Myocarditis is the most common and fatal manifestation of ICI-associated cardiovascular toxicity and is thought to be the result of an exaggerated adaptive immune response to antigens shared between cardiomyocytes and tumor cells (Figure 9A) (20). Infiltrating T cells are involved in the pathogenesis of heart failure (21) and have been detected in cases of fatal ICI-myocarditis (7,22). Although not bound by any particular theory or mechanism of action, the inventors believe that [ 18 Since [F]F-AraG can simultaneously detect activated T cell infiltration in tumors and the heart, we believe it will be useful as a tool for the early detection of ICI-related cardiotoxicity.

[0065] As a substrate for mitochondrial dGK (an enzyme in the nucleotide salvage pathway that is responsible for supplying precursors for mtDNA synthesis in a rate-limiting manner) (23), [ 18F]F-AraG is particularly suitable for reporting on mitochondrial status not only in activated T cells but also in cells with a high mitochondrial content (e.g., cardiomyocytes). The cardiotoxicity of doxorubicin (a widely used chemotherapeutic agent) has been thoroughly studied, and many mechanisms have been proposed, all of which focus on dysregulation of mitochondrial function in cardiomyocytes (24). Doxorubicin interacts with mitochondria in multiple ways, affecting membrane potential and leading to oxidation and depletion of mitochondrial DNA (mtDNA) (Figure 9B) (25). Our disclosure relates to the abnormal mitochondrial function in doxorubicin-associated cardiomyopathy, [ 18 [F]F-AraG can visualize this, providing evidence that it enables early detection of doxorubicin-cardiotoxicity.

[0066] The ability to assess cardiotoxicity associated with both ICI and doxorubicin is crucial in the context of increasing the use of combination chemotherapy / immunotherapy approaches that result in enhanced cardiac damage caused by chemotherapy and effector T cell-mediated myocardial injury (26). 18 The ability of [F]F-AraG to assess the mitochondrial state of the cardiomyocyte is demonstrated by myocardial uptake observed in healthy human volunteers. Notably, compared to healthy subjects, some patients receiving immunotherapy showed significantly higher signals in the heart wall. This is because [ 18 [F]F-AraG demonstrates the ability to detect abnormal mitochondria and / or inflammatory activity (Figure 10).

[0067] In this embodiment, the inventors increased the amount of [ 18 The underlying mechanism of F]F-AraG uptake should be considered. The ability to accurately interpret myocardial uptake detected in immunotherapy patients is crucial because it can provide an evaluation of a readily usable clinical tool that simultaneously reports not only the therapeutic response but also the adverse effects of the treatment. 18F]F-AraG may meet the urgent clinical need for non-invasive measurement of mitochondrial function, which could contribute to the development and more successful replacement of mitochondrial-targeted therapies in humans (27). 18 The ability of F]F-AraG PET to detect early cardiac involvement could revolutionize cardio-oncology, a rapidly advancing field focused on balancing therapeutic efficacy and cardiovascular safety, and have a significant impact on patient management. 18 F]F-AraG PET can enable the optimization of treatment, reduction of cardiovascular risk and cardiovascular disease-related morbidity and mortality, and improvement of quality of life in cancer patients and survivors. These benefits to patients, in turn, can reduce the high healthcare and social costs associated with cardiovascular disease.

[0068] The imaging methods currently in use lack molecular specificity to detect the involvement of subclinical cardiac events associated with cancer treatment. The exceptional advantage this offers is that preliminary clinical data ([ 18 This is evident from the data showing that [F]F-AraG PET has great potential to detect indeterminate mitochondrial activity in the heart. In this context, [ 18 F]F-AraG may currently be the only clinically available agent that can be reported for various cancer treatments: chemotherapy, targeted therapy, and damage caused by ICI. Furthermore, its ability to simultaneously report on immune responses in target tissues and off-target toxic effects of immunotherapy is [ 18 This is a characteristic specific to F]F-AraG. This example provides the development of a novel method for evaluating cardiotoxicity associated with ICI- and combination chemotherapy / ICI approaches, for which no diagnostic approach currently exists.

[0069] Embodiment 1. [ 18 [F]F-AraG demonstrates that it enables early identification of cardiotoxicity related to the pathophysiology of mitochondrial damage drugs (e.g., ICIs and doxorubicin). [18 F]F-AraG imaging may correlate with biomarkers of cardiac pathophysiology and may enable early detection of cardiotoxicity.

[0070] Theoretical basis and supporting evidence

[0071] [ 18 F]F-AraG has been comprehensively evaluated in cell culture and in all clinical immune cell-mediated disease models - graft-versus-host disease and rheumatoid arthritis (28,29). 18 F]F-AraG preferentially accumulates in activated CD8+ cells, but some accumulation is also observed in macrophages and dendritic cells (Figure 11). Since T cells play a crucial role in the immune response against tumors, the inventors of this invention have identified [ 18 The focus was on determining the value of F]F-AraG (30,31). From all clinical studies, the effect on activated T cells [ 18 The specificity of F]F-AraG and its usefulness in estimating the response to immunotherapy were confirmed (30). Using a rhabdomyosarcoma model, the inventors determined that more than 80% of the detected intratumor signals originated from tracer accumulation in immune cells (primarily activated CD8+ and CD4+) (Figure 12). Importantly, the [F]F-AraG was detected in colon cancer tumors and in tumor inflow area lymph nodes. 18 The [F]F-AraG signaling could also differentiate between responders and non-responders of anti-PD-1 treatment as early as 48 hours after a single therapeutic dose (Figure 12).

[0072] The discovery of immune responses arising from chemotherapy-induced tumor cell death (also known as immunogenic cell death (32)) has piqued interest in utilizing the immunomodulatory effects of chemotherapy in relation to possible synergistic combinations with immunotherapeutic agents (33,34). Long-term studies of colon cancer models receiving two types of chemotherapy (one known to induce immunogenic cell death, and the other reported to cause immunogenic, silent tumor death) [18 F]F-AraG imaging reveals dramatic differences in the immunomodulatory effects caused by the two chemotherapy regimens mentioned above, and is used to evaluate chemotherapy-induced immunomodulation and the immune activity environment. 18 The usefulness of F]F-AraG was demonstrated (Figure 13) (31).

[0073] Checkpoints (e.g., PD-1 and CTLA-4, which play immunosuppressive roles in antitumor immunity) have also been shown to be crucial players in peripheral immune tolerance to the heart and the prevention of autoimmune myocarditis. ICI drugs that disrupt PD-1 and CTLA-4 signaling can lead to the disruption of peripheral immune tolerance and activation resulting in excessive myocardial T cell infiltration and myocarditis. Those skilled in the art have created both pharmacological and genetic models of ICI-myocarditis by treating mice with anti-CTLA-4 and anti-PD-1 antibodies, as well as by genetically modifying ICI targets. Notably, haploinsufficiency in the CTLA-4 gene (Ctla4+ / -) combined with PD-1 deficiency (Pdcd1- / -) results in myocarditis in approximately 50% of mice that phenotypicly copy ICI-myocarditis in patients (Figure 14)(35). Notably, autoimmunity in Ctla4+ / - Pdcd1- / - mice is limited to the cardiovascular system and characterized by T cell and myeloid infiltration into the myocardium, as observed in humans.

[0074] Although the precise mechanism of doxorubicin-associated cardiac injury is still debated, many studies suggest that mitochondria are a primary target in doxorubicin-induced myocardial injury (25,36,37). Doxorubicin readily enters mitochondria and can form adducts with mtDNA and cardiolipin, inhibiting mitochondrial respiration. Mitochondrial dysfunction appears to be one of the earliest indicators of doxorubicin-associated cardiomyopathy (38). Drugs used to assess cardiac mitochondrial membrane potential, 18 F-Mitophos and 68Preclinical studies with Ga-Galmydar showed reduced cardiac uptake in doxorubicin-treated animals, suggesting the potential for mitochondrial imaging in chemotherapy-induced cardiotoxicity (38,39). Interestingly, doxorubicin treatment in the heart 18 This resulted in increased FDG uptake, suggesting metabolic changes (40).

[0075] [ 18 The demonstrated ability of [F]F-AraG to detect activated T cells and assess mitochondrial function (both playing a central role in the mechanisms of cardiotoxicity) (Figure 9) suggests its potential as a pathophysiologically relevant imaging biomarker for the early detection and monitoring of cardiotoxicity associated with ICI and doxorubicin therapy. Its ability to assess clinically relevant cardiotoxicity associated with both chemotherapeutic and immunotherapeutic agents could be invaluable given the lack of knowledge regarding cardiovascular injury from the rapidly increasing combination chemotherapeutic / immunotherapy approaches.

[0076] One embodiment of the present invention is [ 18 [F]F-AraG can be used to determine the correlation between myocardial uptake and the pathophysiology of cardiotoxicity. 18 To demonstrate that F]F-AraG can enable early and specific imaging of cardiotoxicity, we have [ 18 Using [F]F-AraG, the development of cardiotoxicity caused by ICI, doxorubicin, and doxorubicin / ICI treatment can be monitored, imaging findings can be compared to LVEF measurements, which are the current standard of care, and can be correlated with known markers of cardiac injury.

[0077] Experimental approach Animal models of cardiotoxicity. To investigate dose-dependent doxorubicin cardiotoxicity, mice (n=8 / group, 4 males, 4 females) can be treated with low-dose (4 mg / kg) and high-dose (20 mg / kg) doxorubicin. Control mice (n=8, 4 males, 4 females) can accept vehicle-only injections. For the study of ICI-associated myocarditis, the inventors may use a recently developed preclinical model (35). Ctla4+ / - Pdcd1- / - mice perfectly reproduce the established pathophysiological and clinical features of ICI myocarditis. Ctla4 + / + Pdcd1 - / - Mice can be used as controls. The inventors used eight Ctla4+ / - Pdcd1- / - mice (four males and four females) and eight Ctla4 + / + Pdcd1 - / - A control mouse can be used.

[0078] Furthermore, those skilled in the art have induced a milder form of ICI myocarditis by treating mice with anti-CTLA-4 antibodies and anti-PD-1 antibodies in a specific genetic background. MRL-Fas was treated with anti-CTLA-4 or anti-PD-1 monotherapy or combination therapy twice a week for 8 weeks. lpr Although the mice did not show obvious clinical signs, histological and electron microscopy revealed signs of immune infiltration of the cardiomyocyte and vascular system, as well as signs of endothelial cell damage and sarcomere disruption following combination therapy. The inventors believe that the combination of pharmacological and genetic mice for modeling ICI myocarditis is complementary. Ultimately, as the use of ICIs expands to cancer types in which patients may be exposed to the combination of doxorubicin and ICIs (e.g., triple-negative breast cancer), these mice provide a platform in which the cardiovascular effects of combination therapy can be investigated. To investigate the cardiotoxicity of combined doxorubicin / ICI treatment, the inventors used 16 MRL-Fas mice. lprMice may be used. Eight mice (half male, half female) may be treated with doxorubicin (20 mg / kg) and anti-PD-1 and anti-CTLA-4 antibodies (250 μg). Vehicle-treated MRL-Fas lpr A mouse can be useful as a control device.

[0079] PET and LVEF imaging. 18 F]F-AraG can be generated by conventional methods (for example, at UCSF Radiochemistry Core). For PET / CT, approximately 0.1 ml of [ 18Approximately 200 μCi (7.4 MBq) of [F]F-AraG can be injected via the tail vein and imaged using a dedicated small animal PET / CT (Siemens Inveon). In a subset of mice, the inventors may perform 60 minutes of dynamic PET imaging, followed by CT, at baseline and two post-treatment time points. Electrocardiogram-gated PET may be performed after the last imaging time point to obtain left ventricular ejection fraction (LVEF) information. A timeline for long-term monitoring of cardiotoxicity for all three animal models is shown in Figure 15. Briefly, pharmacological models (doxorubicin and doxorubicin / ICI) can be regularly imaged before and during treatment. A genetic ICI myocarditis model can be imaged three times, once a week, starting at 6 weeks. 60 minutes of list-mode PET data can be reconstructed into dynamic multiframes (12×5, 6×10, 4×30, 6×60, and 10×300 s) using the Expected Value Maximization by Subset with 3D Maximum Posterior Probability (OSEM / MAP) algorithm provided by the scanner manufacturer. Attenuation and scattering corrections to ensure quantitative accuracy can be applied using CT-based attenuation maps and scattering models. ECG-synchronized data with eight cardiac bins can be reconstructed using the same algorithms and corrections. Dynamic multiframe data can be used for two tissue compartment models, and the last 20 minutes of the above data can be used for static data analysis. Rate constants, including the inflow rate constant (Ki), can be derived from the dynamic data, and % injection dose / g (%ID / g) can be derived from the static data. LVEFs can be calculated from the ECG-synchronized data. All of these parameters can be mapped to polar coordinate plots for segmental and segment-specific analysis of the parameters. Image processing can be performed using PMOD / PCARD / PKIN (PMOD Technologies).

[0080] Embodiment 2. 18 F-Fluorodeoxyglucose ( 18 FDG) and [ 18Compare F]F-AraG, [ 18 Determining variability in F]F-AraG myocardial accumulation [ 18 [F]F-AraG may enable glucose-independent assessment of cardiac function and may exhibit low signal variability.

[0081] Theoretical basis and supporting evidence. 18 FDG PET is conventionally used in the diagnosis, staging, and re-staging of tumor patients. The broad applications of FDG have led to ancillary myocardial findings that have stimulated numerous studies aimed at evaluating the usefulness of FDG in cardiac imaging. 18 FDG is now the most widely used and validated PET tracer for assessing myocardial metabolism and viability. It is a radiolabeled analog of glucose. 18 FDG reports on glucose utilization in tissues. To minimize interference from blood glucose and achieve high detection sensitivity in tumors that use glucose as their primary energy source, in tumor patients... 18 The FDG scan is performed under fasting conditions. In a fasting state, the heart 18 FDG uptake is low, reflecting glucose metabolism and increased fatty acids, the heart's primary energy substrate, caused by decreased insulin (Figure 16). Myocardial FDG uptake is dependent on plasma glucose, fatty acid, and insulin levels, but also on diet, activity, and the use of certain medications, resulting in broad spatial and temporal variability in the signaling pathway (42,43). The duration of fasting, investigated as a variable that can be controlled to give a more specific and accurate distinction between myocardial uptake between benign and malignant conditions, does not appear to correlate with myocardial glucose metabolism (43). 18 Achieving good quality FDG images is particularly difficult in diabetic patients (a population with a high rate of cardiovascular complications).

[0082] [ 18The mechanism of [F]F-AraG uptake differs from that of glucose (Figure 8), and consequently, signal variability is predicted to be lower, as it is associated with fasting (Figure 17). Radiolabeled analogs of nucleoside-deoxyarabinoguanosine, 18 The uptake of [F]F-AraG reflects the activity of enzymes in the salvage pathway, which is thought to be the dominant process for maintaining the purine nucleotide pool in cardiomyocytes and lymphoid organs (44,45). Dietary nucleotides are involved in the salvage pathway [ 18 It is a major source of nucleosides that can compete with [F]F-AraG and produce variations in diet-related signaling.

[0083] One embodiment of the present invention is 18 FDG and [ 18 Determine the difference in signal variability between F]F-AraG, and [ 18 The potential causes of [F]F-AraG signal heterogeneity can be better understood. An aspect of the present invention is that in animals exposed to different conditions (fasted or not fasted, anesthetized or active, fed a diet with dietary nucleotides or not) 18 FDG and [ 18 This can be tested by determining the difference between the myocardial signals of F]F-AraG.

[0084] Experimental approach The inventors may investigate the effects of three variables on myocardial uptake: fasting, activity, and diet. For each variable, the inventors may use 16 mice (8 male and 8 female) and divide them into two groups. For the fasting test, one group may be fasted for 8 hours with free access to both feed and water, while the other group may be fasted for 8 hours with access to water. For the activity test, one group of animals may be kept under anesthesia, while the other group may be awakened after tracer injection and before imaging. To investigate the effects of dietary nucleotides, both groups of animals may be supplied with either nucleotide-free feed or feed supplemented with 0.04% nucleotides (a level found to be optimal for immune function) (46).

[0085] PET imaging. All animals were scanned on two consecutive days. 18 FDG and [ 18 Imagery can be performed using F]F-AraG. To assess temporal variability, imaging can be repeated 3 days after the initial scan. PET data can be acquired during a 10-minute static period and reconstructed using 3D OSEM / MAP. Attenuation and scattering corrections to ensure quantitative accuracy can be applied using CT-based attenuation maps and scattering models. %ID / g can be derived from static data. Variability in signal intensity and location can be determined for each tracer and value being compared. The effect of variables on signal intensity can be determined for each tracer. Uptake values ​​can be calculated for the heart, muscle, liver, spleen, kidney, and brain.

[0086] Blood glucose and nucleotide measurements. Glucose levels can be determined in a tail vein blood sample immediately before imaging using a glucose meter. Measurement of blood nucleotides requires a large volume of analyte and can therefore be performed after the final PET scan, following the last intracardiac blood sample (47).

[0087] Image quantification and sample size calculation. The %ID / g between the group with each variable and the control group can be derived from the PET data. The inventors hypothesize that the inventors can determine the dietary variables [ 18 The conclusion is that no significant difference can be observed in the uptake of F]F-AraG. With a sample size of 8 / cohort, and with a significance level α=0.05 and power (1-β)=0.85, a two-sample t-test can be statistically significant if the difference is greater than 30% and the standard deviation is 20%. Therefore, if the difference is smaller than that, there is no significant difference with a power of 0.85, which is what the inventors predicted for this comparative study.

[0088] The inventors predict that the following will be observed between fasting animals and non-fasting animals [ 18 1) There is no / minimal difference in F-AraG signaling, 2) between the anesthetized group and the active group [ 18 There is no / minimal difference in the F-AraG signaling pathway, 3)[ 18 Extent of effect of dietary nucleotides on the F]F-AraG signaling pathway, 4) 18 Compared to FDG, [ 18 Lower volatility in the F]F-AraG signal. This is overall, 18 F]F-AraG 18 It may enable the evaluation of benefits that can be provided beyond FDG, and similarly, benefits unrelated to pathophysiology [ 18 This may indicate a potential cause of variability in the F]F-AraG signaling pathway.

[0089] Embodiment 3. Physiological [ in healthy subjects 18 [F]F-AraG uptake range and increased as an early sign of cardiotoxicity in cancer patients [ 18 Determine the feasibility of using F]F-AraG incorporation. physiological cardiomyocyte 18F-FAraG uptake may be significantly lower than in cancer patients, allowing for the assessment of cardiotoxicity. Post-treatment [18F]F-AraG uptake in cancer patients may be used for early assessment of cardiotoxicity (e.g., ICI or other drugs).

[0090] Theoretical basis and preliminary evidence. 18 For [F]F-AraG to serve as a tool capable of demonstrating cardiotoxicity with high specificity, several conditions must be met: a) physiological uptake in a normal heart should be relatively low and well-defined; b) the signal in a pathological state must be significantly different from the normal cardiac threshold; and c) the signal characteristics (intensity, pattern) should correspond to pathophysiology. One embodiment of the present invention can be used to better understand the differences between physiological and pathological uptake in human subjects by evaluating signal variability in healthy volunteers and performing retrospective analyses of cardiac accumulation in cancer patients.

[0091] [ 18The safety and radiodosimetry of [F]F-AraG were tested in six healthy subjects (3 males and 3 females) during a first-in-human study at UCSF. Myocardial uptake was observed in all six subjects, and the mean SUV in the cardiac wall was 2.9 ± 0.62. The signal persisted throughout the imaging period, indicating tracer capture (Figure 18). The tracer uptake appeared uniform without localized or localized increases. Relatively low uptake and uniform signaling in healthy subjects suggests the possibility of detecting abnormal signal intensity and patterns in pathological conditions; however, the relatively small sample size may not be representative of physiological accumulation in larger populations. To more comprehensively evaluate baseline values, we may, for example, image an additional 30 healthy subjects (15 males and 15 females) and analyze cardiac signals using conventional and radiomic methods. Radiomics (a computational method used to analyze and extract the rich quantitative features (e.g., texture, heterogeneity, and shape) present in images) has been used for the majority of purposes in tumor imaging to improve cancer screening and early detection (48,49). Radiomics has also been used to identify immune contexture features within the tumor microenvironment for a more accurate assessment of immunotherapy responses (50,51). To date, the application of radiomics in cardiac imaging has mainly focused on the analysis of CT and MRI images without the use of molecular biomarkers. Herein, we have developed a method to extract image features with strong biological relevance that can provide a more accurate and pathologically specific assessment of cardiotoxicity, 18 We disclose radiomic analysis of F]F-AraG PET images. In our preliminary preclinical studies, mice treated with anti-PD-1 / CTLA-4 immunotherapy showed significant differences in intratumor signal energy, entropy, and homogeneity compared to untreated mice (Figure 19). Image texture analysis may allow for better characterization and quantification of signal differences observed between healthy volunteers and some cancer patients (Figure 20). 18F]F-AraG is used to evaluate the response to immunotherapy in cancer patients. 18 F]F-AraG is currently being tested in multiple Phase II clinical trials to evaluate its efficacy. To date, more than 40 patients with different types of cancer (head and neck, lung, bladder, squamous cell carcinoma of the breast, melanoma) have been tested. 18 The images were safely created using F]F-AraG. The inventors have used the data collected to date to enable comparative study of findings in a healthy cohort. 18 A comprehensive retrospective analysis of myocardial uptake in F]F-AraG scans can be performed. Our preliminary analysis showed that cancer patients exhibited significantly higher myocardial signaling than healthy subjects both before and after a single infusion of immunotherapy (Figure 20). Higher signaling in the scan before immunotherapy suggests a higher likelihood of cardiotoxic complications from previous anti-cancer treatments (e.g., chemotherapy or radiotherapy). Within a limited patient cohort with accompanying electrocardiograms (ECGs), heterogeneous, stronger myocardial uptake appeared to correlate with abnormal ECG findings (Figure 21). Importantly, in some patients, just a single infusion of immunotherapy resulted in dramatically increased myocardial uptake (Figure 22). Overall, these results suggest the potential of [F]F-AraG as a tool for cardiovascular risk stratification and assessment of early cardiotoxicity. 18 This illustrates the potential of F]F-AraG.

[0092] Experimental approach Clinical trial outline. Trial participants will be at UCSF Nuclear Medicine Clinic [ 18Whole-body PET / CT scans using F]F-AraG may be performed. The study population may consist of 30 healthy subjects (15 males and 15 females, with no known heart disease). To account for age-related differences within each sex group, there may be three age groups: a) 18–40 years, b) 40–65 years, and c) over 65 years. Each age group may have 10 subjects (5 males and 5 females). A careful medical history may be obtained for each subject, with particular emphasis on family history of heart disease and the use of medications and nutritional supplements. All cardiac medications and previous cancer treatments may be recorded and collected for each patient. Subjects may be asked to fast for 6 hours to minimize potential variability from food.

[0093] PET imaging. 18 F]F-AraG can be synthesized for each patient, for each scan base before imaging. 5±0.5mCi[ 18 F]F-AraG can be administered as a bolus intravenous injection. Dynamic imaging can be performed on 2 female and 2 male patients (12 patients in total) from each age group. Dynamic data acquisition can be started immediately after tracer injection and continued for 60 minutes, focusing on the cardiac field. Static images can be obtained 60 minutes after tracer injection in 18 patients (3 males and 3 females from each age group). Non-contrast CT scans (5 mm continuum axial sections) (CTAC) can be obtained for the relevant region using a helical mode with a field of view (FOV) of 38.5 mm / sec, 120 kVp, 40 mA, and a 512 × 512 matrix size. These CTAC images can be fused with PET images and used for attenuation correction and anatomical positioning. Immediately after the CTAC, emission PET scans can be obtained in 3-dimensional Time of Flight (TOF) mode over the same anatomical region. The PET emission scan can be corrected for attenuation using segmented attenuation data from the CTAC scan.

[0094] Image analysis. PET images can be reconstructed using standard iterative algorithms provided by the scanner manufacturer for both dynamic and static imaging data. From dynamic imaging, dynamic multiframes (12×5, 6×10, 4×30, 6×60, and 10×300 s) can be generated for tracer dynamic modeling (two-compartment model). Total data from the last 10 minutes can be generated to show comparable static data. Rate constants, including the inflow rate constant (Ki), can be calculated in the myocardium using PMOD / PCARD / PKIN (PMOD Technologies). Whole-body imaging from static scans is [ 18 This invention aims to show how F]F-AraG can be used for both tumor imaging and myocardial imaging. In this example, the inventors may add a further reconstruction focusing on the same field of view of dynamic imaging, and the following standardized ingestion value measures: SUV maximum, SUV peak, and SUV mean can be calculated with respect to the left ventricle. The American Heart Association (AHA) 17-segment model can be used to quantify these values ​​for static images. Dynamic parameters from dynamic imaging can be correlated with SUV from static images to determine whether static scans provide stable image quantification and whether dynamic scans are not required for practical use.

[0095] Radiomic analysis can be performed using the PyRadiomics library (52), as it is built on non-proprietary software packages (LifeX or 3D-slicer). Before extracting radiomic features, PET images can be normalized to SUV units and resampled to a common voxel size of 1 mm. Radiomic feature vectors can be calculated for each volumetric lesion. A complete list of radiomic features as defined in PyRadiomics can be extracted (53).

[0096] With the embodiments of the present invention, those skilled in the art will see that 1) relatively low, uniform myocardial uptake in healthy subjects, and 2) in healthy subjects [18 [F]F-AraG myocardial uptake is limited to a narrow range, 4) and cancer patients may show significantly higher cardiac uptake than the healthy control population.

[0097] References 1.Curigliano G, Lenihan D, Fradley M, et al. Management of cardiac disease in cancer patients throughout oncological treatment: ESMO consensus recommendations. Ann Oncol. 2020;31:171-190. 2.Carver JR, Shapiro CL, Ng A, et al. American Society of Clinical Oncology clinical evidence review on the ongoing care of adult cancer survivors: cardiac and pulmonary late effects. J Clin Oncol. 2007;25:3991-4008. 3.McGowan JV, Chung R, Maulik A, Piotrowska I, Walker JM, Yellon DM. Anthracycline Chemotherapy and Cardiotoxicity. Cardiovasc Drugs Ther. 2017;31:63-75. 4.Moslehi JJ. Cardiovascular Toxic Effects of Targeted Cancer Therapies. N Engl J Med. 2016;375:1457-1467. 5.Ramos-Casals M, Brahmer JR, Callahan MK, et al. Immune-related adverse events of checkpoint inhibitors. Nat Rev Dis Primers. 2020;6:38. 6.Wang DY, Salem JE, Cohen JV, et al. Fatal Toxic Effects Associated With Immune Checkpoint Inhibitors: A Systematic Review and Meta-analysis. JAMA Oncol. 2018;4:1721-1728. 7.Moslehi JJ, Brinkley DM, Meijers WC. Fulminant Myocarditis: Evolving Diagnosis, Evolving Biology, Evolving Prognosis. J Am Coll Cardiol. 2019;74:312-314. 8.Zamorano JL, Lancellotti P, Rodriguez Munoz D, et al. 2016 ESC Position Paper on cancer treatments and cardiovascular toxicity developed under the auspices of the ESC Committee for Practice Guidelines: The Task Force for cancer treatments and cardiovascular toxicity of the European Society of Cardiology (ESC). Eur J Heart Fail. 2017;19:9-42. 9.Armenian SH, Lacchetti C, Barac A, et al. Prevention and Monitoring of Cardiac Dysfunction in Survivors of Adult Cancers: American Society of Clinical Oncology Clinical Practice Guideline. J Clin Oncol. 2017;35:893-911. 10.Zhang L, Awadalla M, Mahmood SS, et al. Cardiovascular magnetic resonance in immune checkpoint inhibitor-associated myocarditis. Eur Heart J. 2020;41:1733-1743. 11.Steingart RM, Chandrashekhar Y, Marwick TH. Imaging in Cardio-Oncology: Where Are We and Where Should We Be Going? JACC Cardiovasc Imaging. 2018;11:1209-1211. 12.Namavari M, Chang YF, Kusler B, Yaghoubi S, Mitchell BS, Gambhir SS. Synthesis of 2'-deoxy-2'-[18F]fluoro-9-beta-D-arabinofuranosylguanine: a novel agent for imaging T-cell activation with PET. Mol Imaging Biol. 2011;13:812-818. 13.Lambe CU, Averett DR, Paff MT, Reardon JE, Wilson JG, Krenitsky TA. 2-Amino-6-methoxypurine arabinoside: an agent for T-cell malignancies. Cancer Res. 1995;55:3352-3356. 14.Eriksson S, Arner E, Spasokoukotskaja T, et al. Properties and levels of deoxynucleoside kinases in normal and tumor cells; implications for chemotherapy. Adv Enzyme Regul. 1994;34:13-25. 15.Shewach DS, Daddona PE, Ashcraft E, Mitchell BS. Metabolism and selective cytotoxicity of 9-beta-D-arabinofuranosylguanine in human lymphoblasts. Cancer Res. 1985;45:1008-1014. 16.Wang L. Mitochondrial purine and pyrimidine metabolism and beyond. Nucleosides Nucleotides Nucleic Acids. 2016;35:578-594. 17.Kim W, Le TM, Wei L, et al. [18F]CFA as a clinically translatable probe for PET imaging of deoxycytidine kinase activity. Proc Natl Acad Sci U S A. 2016;113:4027-4032. 18.Tan H, Yang K, Li Y, et al. Integrative Proteomics and Phosphoproteomics Profiling Reveals Dynamic Signaling Networks and Bioenergetics Pathways Underlying T Cell Activation. Immunity. 2017;46:488-503. 19.Baixauli F, Acin-Perez R, Villarroya-Beltri C, et al. Mitochondrial Respiration Controls Lysosomal Function during Inflammatory T Cell Responses. Cell Metab. 2015;22:485-498. 20.Khunger A, Battel L, Wadhawan A, More A, Kapoor A, Agrawal N. New Insights into Mechanisms of Immune Checkpoint Inhibitor-Induced Cardiovascular Toxicity. Curr Oncol Rep. 2020;22:65. 21.Blanton RM, Carrillo-Salinas FJ, Alcaide P. T-cell recruitment to the heart: friendly guests or unwelcome visitors? Am J Physiol Heart Circ Physiol. 2019;317:H124-H140. 22.Johnson DB, Balko JM, Compton ML, et al. Fulminant Myocarditis with Combination Immune Checkpoint Blockade. N Engl J Med. 2016;375:1749-1755. 23.Leanza L, Ferraro P, Reichard P, Bianchi V. Metabolic interrelations within guanine deoxynucleotide pools for mitochondrial and nuclear DNA maintenance. J Biol Chem. 2008;283:16437-16445. 24.Varga ZV, Ferdinandy P, Liaudet L, Pacher P. Drug-induced mitochondrial dysfunction and cardiotoxicity. Am J Physiol Heart Circ Physiol. 2015;309:H1453-1467. 25.Wallace KB, Sardao VA, Oliveira PJ. Mitochondrial Determinants of Doxorubicin-Induced Cardiomyopathy. Circ Res. 2020;126:926-941. 26.Xin Yu J, Hodge JP, Oliva C, Neftelinov ST, Hubbard-Lucey VM, Tang J. Trends in clinical development for PD-1 / PD-L1 inhibitors. Nat Rev Drug Discov. 2020;19:163-164. 27.Tian R, Colucci WS, Arany Z, et al. Unlocking the Secrets of Mitochondria in the Cardiovascular System: Path to a Cure in Heart Failure-A Report from the 2018 National Heart, Lung, and Blood Institute Workshop. Circulation. 2019;140:1205-1216. 28.Ronald JA, Kim BS, Gowrishankar G, et al. A PET Imaging Strategy to Visualize Activated T Cells in Acute Graft-versus-Host Disease Elicited by Allogenic Hematopoietic Cell Transplant. Cancer Res. 2017;77:2893-2902. 29.Franc BL, Goth S, MacKenzie J, et al. In Vivo PET Imaging of the Activated Immune Environment in a Small Animal Model of Inflammatory Arthritis. Mol Imaging. 2017;16:1536012117712638. 30.Levi J, Lam T, Goth SR, et al. Imaging of Activated T Cells as an Early Predictor of Immune Response to Anti-PD-1 Therapy. Cancer Res. 2019;79:3455-3465. 31.Levi J, Goth S, Huynh L, Lam T, Huynh TL, Schulte B, Packiasamy JA. (18)F-FAraG PET for CD8 Profiling of Tumors and Assessment of Immunomodulation by Chemotherapy. J Nucl Med. 2020. 32.Casares N, Pequignot MO, Tesniere A, et al. Caspase-dependent immunogenicity of doxorubicin-induced tumor cell death. J Exp Med. 2005;202:1691-1701. 33.Lake RA, Robinson BW. Immunotherapy and chemotherapy--a practical partnership. Nat Rev Cancer. 2005;5:397-405. 34.Galluzzi L, Buque A, Kepp O, Zitvogel L, Kroemer G. Immunological Effects of Conventional Chemotherapy and Targeted Anticancer Agents. Cancer Cell. 2015;28:690-714. 35.Wei SC, Meijers WC, Axelrod ML, et al. A genetic mouse model recapitulates immune checkpoint inhibitor-associated myocarditis and supports a mechanism-based therapeutic intervention. Cancer Discov. 2020. 36.Ashley N, Poulton J. Mitochondrial DNA is a direct target of anti-cancer anthracycline drugs. Biochem Biophys Res Commun. 2009;378:450-455. 37.Jung K, Reszka R. Mitochondria as subcellular targets for clinically useful anthracyclines. Adv Drug Deliv Rev. 2001;49:87-105. 38.Sivapackiam J, Kabra S, Speidel S, Sharma M, Laforest R, Salter A, Rettig MP, Sharma V. 68Ga-Galmydar: A PET imaging tracer for noninvasive detection of Doxorubicin-induced cardiotoxicity. PLoS One. 2019;14:e0215579. 39.McCluskey SP, Haslop A, Coello C, et al. Imaging of Chemotherapy-Induced Acute Cardiotoxicity with (18)F-Labeled Lipophilic Cations. J Nucl Med. 2019;60:1750-1756. 40.Bulten BF, Sollini M, Boni R, Massri K, de Geus-Oei LF, van Laarhoven HWM, Slart R, Erba PA. Cardiac molecular pathways influenced by doxorubicin treatment in mice. Sci Rep. 2019;9:2514. 41.Phillips NR, Sprouse ML, Roby RK. Simultaneous quantification of mitochondrial DNA copy number and deletion ratio: a multiplex real-time PCR assay. Sci Rep. 2014;4:3887. 42.Thut DP, Ahmed R, Kane M, Djekidel M. Variability in myocardial metabolism on serial tumor (18)F-FDG PET / CT scans. Am J Nucl Med Mol Imaging. 2014;4:346-353. 43.Maurer AH, Burshteyn M, Adler LP, Steiner RM. How to differentiate benign versus malignant cardiac and paracardiac 18F FDG uptake at oncologic PET / CT. Radiographics. 2011;31:1287-1305. 44.Radu CG, Shu CJ, Nair-Gill E, Shelly SM, Barrio JR, Satyamurthy N, Phelps ME, Witte ON. Molecular imaging of lymphoid organs and immune activation by positron emission tomography with a new [18F]-labeled 2'-deoxycytidine analog. Nat Med. 2008;14:783-788. 45.Manfredi JP, Holmes EW. Purine salvage pathways in myocardium. Annu Rev Physiol. 1985;47:691-705. 46.Xu M, Zhao M, Yang R, Zhang Z, Li Y, Wang J. Effect of dietary nucleotides on immune function in Balb / C mice. Int Immunopharmacol. 2013;17:50-56. 47.Dudzinska W, Lubkowska A, Dolegowska B, Safranow K, Jakubowska K. Adenine, guanine and pyridine nucleotides in blood during physical exercise and restitution in healthy subjects. Eur J Appl Physiol. 2010;110:1155-1162. 48.Gillies RJ, Kinahan PE, Hricak H. Radiomics: Images Are More than Pictures, They 49.Gillies RJ, Schabath MB. Radiomics Improves Cancer Screening and Early Detection. Cancer Epidemiol Biomarkers Prev. 2020;29:2556-2567. 50.Sun R, Limkin EJ, Vakalopoulou M, et al. A radiomics approach to assess tumour-infiltrating CD8 cells and response to anti-PD-1 or anti-PD-L1 immunotherapy: an imaging biomarker, retrospective multicohort study. Lancet Oncol. 2018;19:1180-1191. 51.Tang C, Hobbs B, Amer A, et al. Development of an Immune-Pathology Informed Radiomics Model for Non-Small Cell Lung Cancer. Sci Rep. 2018;8:1922. 52.van Griethuysen JJM, Fedorov A, Parmar C, et al. Computational Radiomics System to Decode the Radiographic Phenotype. Cancer Res. 2017;77:e104-e107. 53.pyradiomics.readthedocs.io / en / latest / features.html

[0098] It should be noted that ratios, concentrations, quantities, and other measured data may be expressed herein in range form. Since such range forms are used for convenience and brevity, it should be understood that they should also be interpreted in a flexible manner to include not only the numerical values ​​explicitly stated as limits of the range, but also all individual numerical values ​​or subranges that fall within the range as if each numerical value and subrange were explicitly stated. For example, a concentration range of “about 0.1% to about 5%” should be interpreted to include not only the explicitly stated concentrations of about 0.1% by weight to about 5% by weight, but also the individual concentrations (e.g., 1%, 2%, 3%, and 4%) and their subranges within the indicated range (e.g., 0.5%, 1.1%, 2.2%, 3.3%, and 4.4%). In one embodiment, the term “about” may include traditional rounding methods according to the significant figures of the numerical values. Furthermore, the phrase “about 'x' to 'y'” includes “about 'x' to about 'y'.”

[0099] It should be emphasized that the embodiments described above in this disclosure are merely possible examples and are provided only for the purpose of clearly understanding the principles of the disclosure. Many variations and modifications can be made to the embodiments described above in this disclosure without substantially departing from the spirit and principles of the disclosure. All such modifications and variations are intended to be included herein within the scope of the disclosure. In certain embodiments, for example, the following are provided: (Item 1) A method for imaging cardiac cells in a subject, wherein the method is (a) The following equation: [ka] A step of administering a compound having to a subject, wherein the administration route is selected to allow the compound to be phosphorylated by deoxycytosine kinase and deoxyguanosine kinase present in the heart cells of the subject; and (b) A step of imaging the subject, wherein the detection of the presence of the compound corresponds to the presence of heart cells, A method of including. (Item 2) The method according to item 1, wherein the subject is selected to be a patient who has previously been administered a therapeutic agent or radiotherapy, and one or more images of the heart obtained in step (b) are used to obtain information regarding the effect of the agent or radiotherapy on the subject's heart. (Item 3) The method according to item 2, wherein the therapeutic agent is an anthracycline or an immune checkpoint inhibitor, and one or more images of the heart obtained in step (b) are used to obtain information regarding cardiotoxicity resulting from the administration of the anthracycline or the immune checkpoint inhibitor. (Item 4) Using one or more images of the heart obtained in step (b) above, A step of evaluating one or more parameters of myocardial perfusion in the heart of the subject; A step of evaluating one or more parameters of myocardial viability in the heart of the subject; and / or A step of evaluating one or more parameters of inflammation in the heart of the subject, The method described in item 1, which further encompasses the method described in item 1. (Item 5) The method according to item 2, wherein the therapeutic agent is selected from those that have been observed to act on mitochondria in cardiac cells. (Item 6) The subjects will be selected to be patients diagnosed with cardiovascular disease; The subjects will be selected to be patients diagnosed with cancer; The subjects are selected to be patients undergoing treatment for cardiovascular disease or cancer. The method described in item 2. (Item 7) The method according to any one of items 1 to 6, further comprising the steps of: observing one or more images of the heart on a first date; observing one or more images of the heart on a second date; and comparing the images obtained on the first date with the images obtained on the second date in order to observe changes in the heart over time. (Item 8) The method according to item 7, wherein the amount of time between the first date and the second date includes at least one week or at least one month. (Item 9) The method according to item 3, wherein the therapeutic agent comprises at least one immune checkpoint inhibitor selected to affect CTLA-4 or PD-1 / PD-L1 blockade. (Item 10) A method for imaging cells in a subject responding to the administration of a therapeutic agent, wherein the method is: (a) the step of administering the therapeutic agent; (b) The following formula:

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Claims

1. A method for imaging cardiac cell damage in a subject responding to the administration of a therapeutic agent, comprising a composition for monitoring cardiac damage during treatment by imaging the damaged heart, The aforementioned composition has the following formula: 【Chemistry 19】 Contains a compound having The aforementioned method, (a) The step of administering the therapeutic agent; (b) the step of administering the composition to the subject; and (c) A step of imaging the subject, wherein the detection of the presence of the compound corresponds to the presence of the heart cells; and (d) A step of correlating the observed presence of the compound in the heart cells of the subject with the subject's response to the therapeutic agent. It includes, The subjects are selected to be patients diagnosed with cardiovascular disease; The subjects are selected to be patients diagnosed with cancer; or The composition is selected such that the subject is a patient undergoing treatment for cardiovascular disease or cancer.

2. The composition according to claim 1, wherein the correlation step includes observing the presence of the compound in cancer cells and / or lymph nodes.

3. The composition according to claim 1, wherein the therapeutic agent comprises at least one immune checkpoint inhibitor selected to affect CTLA-4 or PD-1 / PD-L1 blockade.

4. The composition according to claim 3, wherein the cancer cells are colon cancer cells.

5. The composition according to claim 1, wherein the method further comprises the steps of observing one or more images on a first date; observing one or more images on a second date; and comparing the images obtained on the first date with the images obtained on the second date in order to observe the subject's response to the administration of the therapeutic agent.

6. The composition according to claim 5, wherein the amount of time from the first date to the second date includes at least one week or at least one month.

7. A method for imaging mitochondrial activity in cardiac cells of a subject with cardiac injury, comprising a composition for monitoring cardiac injury during treatment by imaging mitochondrial activity of the injured heart, wherein the composition has the following formula: 【Chemistry 20】 The method comprises a compound having the following properties: (a) the step of administering the composition to the subject; and (b) a step of imaging the subject, wherein the detection of the presence of the compound corresponds to the presence of mitochondrial activity in the heart cells; and (c) A process for imaging mitochondrial activity. A composition that includes the following:

8. The composition according to claim 7, wherein the subject is selected for having a mitochondrial deficiency.

9. The method is a method for screening the subject for mitochondrial dysfunction; the mitochondrial dysfunction is a cardiovascular disease, a neuropsychiatric disorder, or a neurodegenerative disease, according to claim 7.

10. The composition according to claim 9, wherein the mitochondrial dysfunction is selected from the group consisting of myocardial perfusion, bipolar disorder, depression, schizophrenia, Alzheimer's disease, Parkinson's disease, Friedreich's ataxia, amyotrophic lateral sclerosis, Huntington's disease, progeria, cardiomyopathy, respiratory chain disorder, mtDNA depletion, myoclonus epilepsy, red ragweed syndrome, encephalomyopathy / lactic acidosis / stroke-like seizures, and optic nerve atrophy.

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