Methods and Materials for Using [18F]-F-AraG in Myocardial Imaging

Abstract

In cardiac imaging technology 18 Further compositions and methods designed to use F-based PET tracers are provided. Embodiments of the present disclosure provide a method for detecting and measuring myocardial physiology using a hologram, the method comprising: 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 It offers significant advantages over F-labeled tracers.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit under 35 U.S.C. §119(e) of commonly assigned U.S. Provisional Patent Application No. 63 / 057,643, filed July 28, 2020, and entitled "METHODS AND MATERIALS FOR USING [18F]-F-AraG IN CARDIAC IMAGING," which application is incorporated herein by reference. This application is related to a co-pending U.S. application having application serial number 17 / 185,502, filed February 25, 2021, and entitled "Compounds and Methods of Making Compounds," the contents of which are incorporated herein by reference. [Background technology]

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

[0003] The present disclosure relates generally to positron emission tomography (PET), and more particularly to a method for use in cardiac imaging techniques (e.g., myocardial perfusion studies). 18 This document relates to compositions and methods for the development and use of F-based PET tracers. 18 The long physical half-life of F-based tracers (109 min) allows for clinical studies without the need for an on-site cyclotron. Furthermore, with modern PET camera technology, quantitative measurements of myocardial radioactivity concentrations can be performed with a high degree of temporal sampling and good statistical precision.

[0004] Cardiac imaging techniques play a central role in the noninvasive diagnosis and risk assessment of CVD and the associated decisions made by physicians regarding how to optimally manage CVD in different individuals. New techniques useful in cardiac imaging include positron emission tomography (PET). 18 The development of F-labeled tracers may provide those skilled in the art with additional opportunities to manage CVD and further expand the scope of clinical research. Given the enormous public health burden of cardiovascular disease, there is a need for further advances in cardiac imaging techniques. 18 There is a need in the art for additional compositions and methods designed to use F-based PET tracers. Summary of the Invention [Means for solving the problem]

[0005] Abstract The compounds used in the methods disclosed herein, 18 [F]-F-arabinofuranosylguanine was originally developed as a PET imaging agent for activated T cells. This compound is a derivative of arabinofuranosylguanine (AraG). 18 F-labeled analogs, compounds that can be phosphorylated by two kinases: cytoplasmic deoxycytidine kinase (dCK) and deoxyguanosine kinase (dGK) and trapped intracellularly. As discussed below, we have demonstrated that at the tracer level, [ 18 We have discovered that [F]F-AraG has the potential to be used in many emerging PET methodologies, including those designed for cardiac and / or mitochondrial activity imaging. The heart is an organ with high energy requirements and is particularly rich in mitochondria. Over the past decade, mitochondrial dysfunction has been recognized as an important aspect of cardiovascular pathology. Consequently, treatments focused on monitoring and improving cardiac mitochondrial activity are a focus of biomedical research.

[0006] The disclosure provided herein is 18The present invention is based in part on the discovery that [F]F-AraG is a well-suited agent for monitoring certain physiological phenomena, including, for example, mitochondrial activity in cardiac cells. In light of this, embodiments of the present invention provide a method for the detection of [F]F-AraG in cardiac imaging. 18 In an exemplary embodiment of the invention, methods for using [F]F-AraG as a PET tracer are provided. 18 The use of [F]F-AraG was 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 observed in cancer patients and patients undergoing immunomodulatory and other therapies. The different [F]F-AraG uptake in healthy volunteers versus patients treated with therapeutic agents was also observed. 18 This discovery of the [F]F-AraG signal profile is useful in the methods disclosed herein (e.g., to monitor changes to cardiac cells caused by drugs or injury). 18 [F]F-AraG is utilized in methods designed to utilize its ability to image the heart.

[0007]

[0013] as a PET tracer in the methods disclosed herein 18 The use of [F]F-AraG has demonstrated promise over conventional methods in many distinct applications, including the observation of selected physiological phenomena (e.g., myocardial perfusion, myocardial viability, and cardiac inflammation). 18 Further, as discussed below, the method of the present invention provides significant advantages over F-labeled tracers. 18 F]F-AraG was used in studies designed to observe the physiological responses of patients to various therapeutic agents and as a PET tracer in drug development studies. 18 F]F-AraG.

[0008] The invention disclosed herein has many embodiments, including a method of imaging cardiac cells in a subject, the method comprising administering to a subject a compound of the following formula: [ka] and then imaging the subject, wherein detecting the presence of the compound indicates the presence of cardiac cells. In some embodiments, these methods further include assessing 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 include assessing 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 include assessing one or more parameters of inflammation in the subject's heart using one or more images of the heart. In certain embodiments of the 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. Optionally, the therapeutic agent used in these methods acts on the mitochondria of cardiac cells.

[0009] Embodiments of the present invention also include methods for imaging selected cell populations (e.g., cardiac cells and leukocytes) in patients suffering from a pathological condition who are being treated with one or more therapeutic agents. Such methods can be used to observe in vivo physiological changes (e.g., cardiotoxicity) resulting from the administration of such therapeutic agents. Typically, such methods involve administering to a subject receiving treatment with the therapeutic agent a compound of the following formula: [ka] The method further comprises administering a PET probe compound having the formula: wherein the route of administration 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 comprise PET imaging the subject, wherein detecting the presence of the PET probe compound indicates the presence of the cells; and finally, 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 receiving treatment for cardiovascular disease or cancer. In exemplary embodiments of the invention 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 administration of the anthracycline or the immune checkpoint inhibitor. In exemplary working embodiments, the method includes observing the presence of the PET probe compound in cardiac cells, immune cells, and / or in lymph nodes (e.g., tumor-draining lymph nodes).

[0010] In certain embodiments of the present invention, a PET method for imaging cells of a subject responding to administration of a therapeutic agent further comprises 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 to observe changes in the patient's physiological state over time resulting from administration of the therapeutic agent (e.g., to distinguish between responders and non-responders to the therapeutic agent). In certain embodiments of the present invention, the amount of time from the first date to the second date comprises less than one week. Alternatively, the amount of time from the first date to the second date comprises at least one week, two weeks, or three weeks, or at least one month, two months, or three months.

[0011] Another embodiment of the invention is a method of imaging mitochondrial activity in cells of a subject, said method comprising: [ka] The present invention also includes a method comprising administering to the subject a compound having the formula: Optionally, 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 disorders, mtDNA depletion, myoclonic epilepsy, ragged-red fiber syndrome, encephalomyopathy, lactic acidosis, stroke-like episodes, and optic 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. It should be understood, however, that the detailed description and specific examples, while indicating certain embodiments of the present invention, are given by way of illustration and not by way of limitation. Many changes and modifications within the scope of the present invention may be made without departing from the spirit thereof, and the invention includes all such modifications. [Brief explanation of the drawings]

[0013] Further aspects of the present disclosure will be more readily appreciated upon consideration of the following detailed description of various embodiments thereof when taken in conjunction with the accompanying drawings.

[0014] [Figure 1-1A] Figure 1.1A illustrates a number of synthetic schemes for making compounds of the present disclosure. [Figure 1-1B]Figure 1.1B illustrates a number of synthetic schemes for making compounds of the present 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 [18F]F-AraG precursor and [18F]F-AraG. [Figure 2-1-2] Figure 2.1 illustrates a schematic diagram of the synthesis of [18F]F-AraG precursor and [18F]F-AraG. [Figure 2-2] Figure 2.2 depicts a graph of 5 x 105 CCRF-CEM cells labeled with 18F-AraG (0.6 mCi / ml) for the indicated times and doses. Uptake was measured in a gamma counter. Bars represent the mean ± SEM of triplicate determinations. [Figure 2-3] Figure 2.3A illustrates a graph of 5 x 105 CCRF-CEM cells incubated in triplicate with 1 μCi 8H3-AraG (1 mCi / ml) and increasing amounts of cold 2F-AraG or DMSO for either 60 or 120 minutes. Percent control uptake was calculated as measured cpm of sample / accumulated cpm of 1 μCi 8[H3]-AraG control × 100. Figure 2.3B illustrates a graph of 5 x 105 CCRF-CEM, MOLT-4, or RAJI cells (in 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 medium. Percent control uptake was calculated as in Figure 2.3a. [Figure 2-4] Figure 2.4 illustrates a schematic of the metabolism of 2'-deoxyguanosine (dGuo) by T lymphoblasts. In contrast to 2'-dGuo, AraG does not require ribonucleotide reductase activity for incorporation 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 incorporated into nuclear DNA (Figure from J Biol Chem. 2008;283:16437-16445). [Figure 3-1]Figure 3.1 illustrates the analytical HPLC profile of the co-injection of [18F]F-AraG with a cold F-AraG standard (5% acetonitrile:95% water; 1 mL / min, 254 nm, Phenomenex Gemini C18, 5μ, 4.6×250 mm). [Figure 3-2] Figure 3.2 illustrates a graph showing 5 x 10 CCRF-CEM cells (triplicates) exposed to either 3 μCi or 10 μCi of [18F]F-AraG for 60 or 120 minutes. Cells took up approximately twice as much [18F]F-AraG at 60 minutes (p=0.008) and 120 minutes (p=0.001) when exposed to 10 μCi compared to 3 μCi. Error bars represent SEM. [Figure 3-3] Figure 3.3 illustrates a graph showing that 1 x 10 purified primary T cells stimulated or unstimulated with 100 U / mL IL2, 50 nM PMA, and 1 μg / mL ionomycin were incubated with 1 μCi of [F]F-AraG for 60 minutes. Error bars represent the mean ± SEM of triplicate determinations (n ​​= 4, p = 0.14 and 0.003 by two-tailed paired Student's T-test, respectively). [Figure 3-4] Figure 3.4 is a schematic diagram of Scheme 1 describing the synthesis of 2-N-acetyl-6-O-((4-nitrophenyl)ethyl)-9-(3,5-di-O-trityl-2-triphyl-β-D-ribofuranosyl)guanine (2), the [F]F-AraG precursor. [Figure 3-5] Figure 3.5 is a schematic representation of Scheme 2 describing the synthesis of 2'-deoxy-2'-fluoro-9-β-D-arabinofuranosylguanine 5 (F-AraG). [Figure 3-6] Figure 3.6 is a schematic representation of Scheme 3 describing the synthesis of 2'-deoxy-2'-[18F]fluoro-9-β-D-arabinofuranosylguanine 7 ([18F]F-AraG). [Figure 4]Figure 4 shows an illustration of the mechanism for imaging cardiac cells with [F]F-AraG. [F]F-AraG is transported into cells via nucleoside transporters and subsequently [F]phosphorylated by mitochondrial deoxyguanosine kinase (dGK) and, to a lesser extent, cytosolic deoxycytidine kinase (dCK). Phosphorylation results in the capture of [F]F-AraG, allowing visualization of these cells via PET imaging. [Figure 5] Figure 5 provides data showing the kinetic properties of dCK and dGK with [H]F-AraG. (a) Enzyme kinetics of dGK or dCK with [H]F-AraG, (b) dGuo positive control for dGK activity, and (c) dCyd positive control for dCK activity. Lines indicate best fit. The Km of dGK with [H]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 [H]F-AraG (Km = 50.89 μM, Vmax = 262.5). We observed a high affinity of dCK for dCyd (Km=1.997, Vmax=9.454) and a similarly higher affinity in the dGuo positive control for dGK activity (Km=5.083, Vmax=123.2). [Figure 6] Figure 6 shows images of F-AraG signals in the hearts of cancer patients. High mitochondrial activity in the hearts results in high F-AraG signals 1 hour after injection of the tracer. [Figure 7] Figure 7 provides data showing the intensity of the F-AraG signal in the heart wall of healthy volunteers and patients receiving immunotherapy (before refers to pre-treatment images, after refers to images taken 2-3 weeks after a single injection of anti-PD-1 antibody). [Figure 8]Figure 8 provides an illustration showing the mechanism of imaging with [F]F-AraG. [F]F-AraG is transported into cells via nucleoside transporters and subsequently undergoes rate-limiting phosphorylation by mitochondrial deoxyguanosine kinase (dGK). Phosphorylation by dGK leads to trapping in mtDNA and potential downstream accumulation, enabling visualization via PET imaging. [Figure 9] Figure 9 provides an illustration showing the mechanism of cardiotoxicity and its relevance to imaging with [18F]F-AraG. A. Immune checkpoint inhibitor therapy (ICI)-induced inflammation. Briefly, cancer treatments called immune checkpoint inhibitors (ICIs) work by activating a patient's own immune system to fight their cancer. ICI drugs that 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. After phosphorylation by dGK, [18F]F-AraG can be incorporated into mtDNA and thus report on its synthetic status. [Figure 10] Figure 10 provides 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 [H]F-AraG in human immune cells. Figure 11A. 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 subtypes of T cells resulted in an increase in tracer uptake, but activated CD8+ cells showed the highest increase. [Figure 12] Figures 12A-12C show data from an embodiment of the present invention. Figure 12A. Tumor-infiltrating lymphocytes took up more than 80% of tumor-activated [18F]F-AraG, with CD8+ and CD4+ cells acquiring the greatest proportion of the tracer (72%). FACS analysis of isolated lymphocytes revealed 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 signal in the tumor (open circle) and tumor-draining lymph node (red) of a mouse before and 48 hours after a single anti-PD-1 treatment. Responding mice (R) showed higher [18F]F-AraG signal in both the tumor and tumor-draining lymph node compared to non-responding mice (NR). Figure 12C. The combined intratumoral and intranodal [18F]F-AraG signal in responders was significantly higher (6.587±0.6874, n=4) than in non-responders (2.604±1.083, n=4). [Figure 13] Figures 13A-13D show data from an embodiment of the present invention. Figure 13A. Paclitaxel / carboplatin treatment (reported to cause immunologically silent death) did not result in a noticeable change in [18F]F-AraG signal intensity. Figure 13B. A dramatic increase in signal intensity was detected after oxaliplatin / cyclophosphamide treatment, which has been shown to induce immunogenic cell death. Open circles indicate tumor-draining 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 pretreatment 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-fold higher than in paclitaxel / carboplatin-treated mice, indicating an immune-active tumor microenvironment (n=4 per group). [Figure 14]Figures 14A-14D show data from a study of Ctla4+ / - Pdcd1- / - mice exhibiting cardiac immune infiltration. Figure 14A. H&E images of a Ctla4+ / - Pdcd1- / - mouse (left) and a human (right; autopsy sample of myocardium from a patient with complete heart block and ventricular tachycardia after ICI treatment). Figure 14B. Quantification of lymphoid infiltration scores and the frequency of CD3, CD4, and CD8+ cells as a fraction of total nucleated cells from H&E-stained cardiac tissue. Figure 14C. Representative images of cardiac tissue sections stained for CD3, CD4, and CD8 immunohistochemistry (right) from a female Ctla4+ / - Pdcd1- / - mouse. 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 further immunohistochemical (CD3, F4 / 80+ macrophages and Foxp3+ Tregs) stained cardiac tissue from Ctla4+ / − Pdcd1− / − mice. [Figure 15] Figures 15A-15C show an outline of a proposed study of long-term [F]F AraG monitoring of cardiotoxicity. Figure 15A. Doxorubicin toxicity. Animals are imaged before and 48 hours after weekly doxorubicin treatment. Figure 15B. ICI toxicity. Animals are imaged weekly starting at week 6. Preliminary studies have shown peak myocardial immune infiltration between weeks 6 and 8. Animals are followed until week 10. Figure 15C. Dox / immunotherapy toxicity. Mice are treated twice weekly for 2 weeks. [F]F AraG imaging is performed 48 hours after the second and fourth treatments. Ex vivo analysis is performed 1 day after the final scan. [Figure 16] Figure 16 provides maximum intensity projection (MIP) [F]F-AraG images of FDG and [F]F-AraG myocardial uptake in a head and neck patient. Myocardial uptake of diagnostic FDG obtained after fasting was low. [F]F-AraG cardiac uptake in the same patient showed higher uptake than in healthy volunteers (see Figure 7). [Figure 17]Figure 17 shows images from a comparison of FDG and [F]F-AraG uptake in rat myocardium. The rats were imaged with the tracers on consecutive days. [F]F-AraG showed reproducible myocardial uptake, while FDG showed variability that may interfere with its clinical usefulness in myocardial imaging. [Figure 18] Figure 18 shows images of myocardial [F]F-AraG uptake in a healthy female volunteer taken at the indicated time points after injection of the tracer. The cardiac signal was persistent and essentially uniform over the 120-minute imaging time frame. [Figure 19] Figure 19 shows data on radiomics features extracted from [18F]F-AraG images of tumor-bearing mice treated with anti-CTLA-4 and anti-PD-1 antibodies. Energy, entropy, and homogeneity were found to be significantly different between treated and untreated animals. [Figure 20] Figures 20A-20C show transverse [F]F AraG PET images of a healthy volunteer in Figure 20A and a head and neck cancer patient in Figure 20B. The signal in the cancer patient was significantly higher along with the area of ​​focal enhancement (red arrow). Figure 20C provides data showing that the [F]F AraG signal in the heart wall of the cancer patient was significantly higher than in the healthy subject both before and after immunotherapy. The increased signal before immunotherapy may indicate the cardiotoxicity of previous anticancer treatment. [Figure 21] Figure 21 shows data from electrocardiograms and [F]F AraG images of three head and neck patients. The top patient shows relatively low and uniform [F]F AraG myocardial uptake and a normal ECG. The other two patients show abnormal ECGs and higher (middle) and heterogeneous [F]F AraG uptake. The data in the figure illustrate an important aspect of an embodiment of the present invention, as it shows a correlation between tracer uptake and hear abnormalities as shown in the electrocardiogram. [Figure 22]Figure 22 provides maximum intensity projection (MIP) images showing myocardial signal changes in a patient with recurrent melanoma before and after immunotherapy infusion. Higher-than-normal myocardial uptake in pretreatment scans can indicate cardiotoxicity of previous anticancer treatment. Just one immunotherapy infusion resulted in dramatically increased myocardial uptake. Increased [F]F-AraG signal was also observed in the thyroid and spleen. The data in this figure illustrate an important aspect of an embodiment of the present invention, as it demonstrates how [F]F AraG can be used to image the effects of ICIs on the heart in PET methodologies. DETAILED DESCRIPTION OF THE INVENTION

[0015] Detailed Description Before describing the present disclosure in more detail, it should be understood that the present disclosure is not limited to the specific embodiments and embodiments of the present invention described, and thus may, of course, vary. It should also be understood that the terminology used herein is for the purpose of describing specific embodiments only and is not intended to be limiting. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. All publications and patents cited herein are incorporated by reference as if each individual publication or patent was specifically and individually indicated to be incorporated by reference, and are incorporated by reference herein to disclose and describe the methods and / or materials to which the publications are cited. As will be apparent to those skilled in the art upon reading and understanding this disclosure, each of the individual embodiments described and exemplified herein has separate components and features, which can be easily separated from or combined with the features of any of the other embodiments without departing from the scope or spirit of the present disclosure. Any recited method can be carried out in the order of events recited or in any other order which is logically possible.

[0016] Before describing embodiments of the present disclosure in detail, it is to be understood that, unless otherwise indicated, this disclosure is not limited to particular materials, reagents, reactants, manufacturing processes, etc., as such may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting. It is possible in the present disclosure that steps can be carried out in differing order where this is logically possible.

[0017] It must be noted that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include the plural forms unless the context clearly dictates otherwise. Thus, for example, reference to "a compound" includes a plurality of compounds. In this specification and the claims that follow, reference will be made to a number of terms, which shall be defined to have the following meanings unless a contrary intention is apparent.

[0018] Each of the applications and patents cited in this context (including during the prosecution of each issued patent; "application cited documents"), and each of the documents and references cited in each of the applications and patents, and each of the PCT and foreign applications corresponding to and / or claiming 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. Additionally, documents or references cited within the text (e.g., U.S. Patent Application Publication Nos. 20150230762, 20150297760, and 20190054198), in the reference list before the claims, or within the text itself; and each of these documents or references (the "herein cited references"), as well as each document or reference cited within each of the references cited herein (including any manufacturer's specifications, instructions, etc.), are hereby expressly incorporated by reference.

[0019] According to the present disclosure, a "detectably effective amount" of an embodiment of the present disclosure is defined as an amount sufficient to produce an acceptable image using equipment available for clinical use. A detectably effective amount of an embodiment of the present disclosure may be given in one or more administrations. A detectably effective amount of an embodiment of the present disclosure may vary depending on factors such as the individual's degree of susceptibility, the individual's age, sex, and weight, individual-specific responses, dosimetry, etc. A detectably effective amount of an embodiment of the present disclosure may also vary depending on equipment and film-related factors. Optimization of such factors is within the level of one skilled in the art.

[0020] The term "detectable" refers to the ability to detect a signal or presence of an embodiment of the present disclosure above background signals. The term "detectable signal" or phrase "detection of a labeled compound" or "detectable labeled compound" refers to the detection (direct or indirect) 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 (e.g., a statistically significant difference is sufficient difference between the detectable signal and the background to distinguish between the detectable signal and the background (e.g., about a 0.1%, 1%, 3%, 5%, 10%, 15%, 20%, 25%, 30%, or 40% or greater difference between the detectable signal and the background)). Standards and / or calibration curves can be used to determine the relative intensity of the detectable signal and / or the background. The detectable signal can be generated from low to high concentrations of labeled compounds. In one embodiment, the detectable signal may need to be the sum of each of the individual labeled compound signals. In one embodiment, the detectable signal can be generated from a sum, an integral, or other mathematical process, formula, or algorithm. In one embodiment, the sum, integral, or other mathematical process, formula, or algorithm can be used to process the detectable signal so that it can be distinguished from background noise, etc.

[0021] As used herein, "agent," "active agent," and the like can include compounds of the present disclosure (e.g., labeled compounds). The agent can be disposed in a composition or pharmaceutical composition. As used herein, a "pharmaceutical composition" refers to a combination of an active agent and a pharmaceutically acceptable carrier. As used herein, a "pharmaceutical composition" refers to a composition suitable for administration to a subject (e.g., a mammal, particularly a human). Generally, a "pharmaceutical composition" is sterile and preferably free of contaminants that may elicit an undesired response in the subject (e.g., the compounds in the pharmaceutical composition are pharmaceutical grade). Pharmaceutical compositions can be designed for administration to a subject or patient in need thereof via many different routes of administration, including oral, intravenous, buccal, rectal, parenteral, intraperitoneal, intradermal, intraorgan, intramuscular, subcutaneous, inhalation, and the like.

[0022] By "pharmaceutically acceptable excipient," "pharmaceutically acceptable diluent," "pharmaceutically acceptable carrier," or "pharmaceutically acceptable adjuvant" is meant an excipient, diluent, carrier, and / or adjuvant that is generally safe, non-toxic, and not biologically or otherwise undesirable, useful in preparing pharmaceutical compositions, and includes excipients, diluents, carriers, and adjuvants that are acceptable for veterinary use and / or human pharmaceutical use. With respect to compositions suitable for human administration, the term "excipient" refers to any excipient, diluent, carrier, and ... It is meant to include, but not be limited to, those ingredients set forth in 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 physically discrete units suitable for unitary administration for human and / or animal subjects, each unit containing a predetermined amount of compound calculated in an amount sufficient to produce the desired effect (e.g., host weight, disease, disease severity, etc.) in association with a pharmaceutically acceptable diluent, carrier, or vehicle. The specifications for the unit dosage forms depend on the particular compound used, the route and frequency of administration, and the effect to be achieved, as well as 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 present disclosure (which may be considered a labeled compound) administered to a human, which may be used to image cells (e.g., cells of the heart). "Administration" means introducing an embodiment of the present disclosure into a subject. Administration includes, but is not limited to, routes such as intravenous, oral, topical, subcutaneous, intraperitoneal, intraarterial, inhalation, vaginal, rectal, nasal, and may include introduction into the cerebrospinal fluid or infusion into a body compartment.

[0025] As used herein, the term "host" or "subject" includes humans, mammals (e.g., cats, dogs, horses, etc.), and other living animals. In particular, the host is a human subject. Exemplary hosts to which embodiments of the present disclosure can be administered are mammals, particularly primates, 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, particularly 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, for in vitro applications (in vitro diagnostic and research applications), body fluid and cell samples from the above-mentioned subjects are suitable for use as "samples", for example, blood, urine, or tissue samples from mammals (particularly primates such as humans), or blood, urine, or tissue samples from animals referred to in veterinary applications.

[0026] The invention disclosed herein has many embodiments. For example, embodiments of the present invention include a method of imaging cardiac cells in a subject / patient. Such a method comprises administering to the subject a compound of the following formula: [ka] and then imaging the subject, wherein the administration route is selected to allow the compound to be phosphorylated by deoxycytosine kinase and deoxyguanosine kinase present in cardiac cells of the subject; and then imaging the subject, wherein detecting the presence of the compound indicates the presence of cardiac cells. In certain embodiments of the present invention, the subject is selected to be a patient who has been administered a therapeutic agent or radiation therapy, and the one or more images of the heart obtained are used to obtain information about the effect of the agent or radiation therapy on the subject's heart. In exemplary embodiments of the invention disclosed herein, the therapeutic agent is an anthracycline (e.g., doxorubicin) or an immune checkpoint inhibitor, and the one or more images of the heart are used to obtain information about cardiotoxicity resulting from administration of doxorubicin or the immune checkpoint inhibitor. In certain embodiments of the present invention, the therapeutic agent is selected to be one that is observed to act on mitochondria in cardiac cells.

[0027] In certain methods of the present invention, 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.Optionally, the subject is a patient being treated with at least one therapeutic agent, including an immune checkpoint inhibitor selected to affect CTLA-4 or PD-1 / PD-L1 blockade (e.g., antibodies such as pembrolizumab; nivolumab; atezolizumab; avelumab; bevacizumab; and durvalumab).In certain embodiments 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 invention, the amount of time from the first date to the second date comprises less than one week. Alternatively, in other embodiments of the invention, the amount of time from the first date to the second date comprises at least one, two, or three weeks, or at least one, two, or three months. Some embodiments of the invention include using one or more images of the heart to: assess one or more parameters of myocardial perfusion in the subject's heart; and / or assess one or more parameters of myocardial viability in the subject's heart; and / or assess one or more parameters of inflammation in the subject's heart.

[0028] The present invention also encompasses a method for imaging cells in a subject in response to the administration of a therapeutic agent. Such a method can be used to observe physiological changes (such as cardiotoxicity, drug responsiveness, etc.) in vivo resulting from the administration of a therapeutic compound. Typically, such a method comprises 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 to the subject a compound having the following formula: [ka]

[0013] The method further comprises administering a PET probe compound having the formula: 18 F]F-AraG PET image profile), obtained from the patient before administration of the above drugs, etc. 18[F]F-AraG PET images. In certain working embodiments of such methods disclosed herein, the correlating step involves observing the presence of the PET probe compound in cardiac cells, immune cells, and / or lymph nodes (e.g., tumor-draining lymph nodes). 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 exemplary embodiments of the invention disclosed herein, the therapeutic agent is an agent observed to regulate mitochondrial physiology, 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 administration of doxorubicin or the immune checkpoint inhibitor.

[0029] In certain embodiments of the present invention, the method of imaging cells in a subject responsive to administration of a therapeutic agent further comprises 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 to observe changes in patient physiology resulting from administration of the therapeutic agent (e.g., to distinguish between responders and non-responders to the therapeutic agent). In certain embodiments of the present invention, the amount of time from the first date to the second date comprises less than one week. Alternatively, the amount of time from the first date to the second date comprises at least one, two, or three weeks, or at least one, two, or three months.

[0030] Yet another embodiment of the present invention is a method for imaging mitochondrial activity in cells of a subject. Typically, these methods involve imaging mitochondrial activity in cells of a subject using the following formula: [ka] The method includes administering a compound having the formula: 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 cells of the subject; and then imaging the subject, wherein detecting the presence of the compound indicates the presence of mitochondrial activity, thereby observing mitochondrial activity in the cells of the subject. Typically, in these methods, the subject is a patient selected for suffering from 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 disease, or a neurodegenerative disease. In some embodiments, the method is used to screen the subject for a 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 disorders, mtDNA depletion disorders, myoclonic epilepsy, ragged-red fiber syndrome, encephalomyopathy, lactic acidosis, stroke-like episodes, and optic atrophy.

[0031] Briefly, in vivo, AraG is metabolized in a unique 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 application in the treatment of T-lymphoblastic diseases. This compound was developed over several years by Glaxo 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 [F]-F-arabinofuranosylguanine compounds (compounds that can be formed by a number of processes, such as those disclosed in U.S. Patent Application No. 17 / 314,366, filed May 7, 2021, and entitled "METHODS AND MATERIALS FOR MAKING PET RADIOTRACERS," the contents of which are incorporated herein by reference). Such embodiments of the invention include, for example, compounds having the following general formula: [ka] The present invention also includes a [18F]-F-arabinofuranosylguanine compound formed from a composition of matter comprising a compound having the formula: wherein: PG comprises a protecting group; and LG comprises a leaving group. Typically, in such embodiments of the present invention, the nitrogen atom linked to a protecting group is linked to two protecting groups, as represented by "N(PG)2." ​​Alternatively, the nitrogen atom is linked to a hydrogen atom and one protecting group, as represented by "NHPG." In an exemplary embodiment of the present invention, the composition comprises Precursor 1 or Precursor 3: [ka] and wherein: 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; and Tf contains a triflate leaving group.

[0033] Embodiments of the present disclosure encompass methods of using [F]-F-arabinofuranosylguanine compounds formed by other processes, such as those disclosed in U.S. application Ser. No. 17 / 185,502, entitled "Compounds and Methods of Making Compounds," filed February 25, 2021, 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, and 2', 3', 4', 5', and 11', and the use of such compounds for imaging, etc.

[0034] An illustrative embodiment of making a labeled compound involves, among other things, combining a compound containing an isotope (Ist) with a compound of formula 1': [ka] with a compound having formula 2': [ka] forming a compound having the formula: The compound having the above formula 2' is deprotected to give the compound having the formula 3: [ka] wherein PG is a protecting group and LG is a leaving group, and wherein R is selected from R1, R2, R3, R4, R5, and R6: [ka] wherein R' is selected from the group consisting of R'1, R'2, R'3, R'4, and R'5: [ka] wherein Ac is an acetyl group and Bz is a benzoyl group, and wherein each of Ac and Bz can be replaced as described herein. An illustrative embodiment of the labeled compound includes, among others, a labeled compound comprising: The compound has the formula 3: [ka] where Ist is an isotope and where R' is R'1, R'2, R'3, R'4, and R'5: [ka] An illustrative embodiment of the labeled compound is, among others, a compound having a formula selected from the group consisting of: [ka] wherein PG is a protecting group and wherein R is selected from R1, R2, R3, R4, R5, and R6: [ka] wherein Ac is an acetyl group and Bz is a benzoyl group, and wherein each of Ac and Bz can be substituted as described herein.

[0035] An illustrative embodiment of a method for imaging cardiac cells includes, among other things, administering to the subject a compound of the present disclosure; and imaging the subject, wherein detecting the presence of the compound indicates the presence of the cardiac cells. Another illustrative embodiment of a method for imaging the presence or degree of mitochondrial activity in cells includes, among other things, administering to the subject a compound of the present disclosure; and imaging the subject, wherein detecting the presence of the compound indicates the presence or degree of mitochondrial activity in the subject's cells.

[0036] In one working embodiment of the present invention, [ 18 The use of [F]F-AraG was demonstrated in healthy volunteers and in cancer patients undergoing 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 undergoing immunotherapy. This unexpected finding was due to the [F]F-AraG. 18 This study illustrates the ability of [F]F-AraG to image changes to cardiac cells caused by drugs such as immunomodulators, chemotherapeutic agents, etc., as well as other damage to the heart, such as damage resulting from exposure to radiation. In this context, [F]F-AraG may be useful for assessing mitochondrial activity. 18 [F]F-AraG can be used in methods to test new drugs that may improve mitochondrial function and / or evaluate the effectiveness of these types of drugs in improving mitochondrial function. In light of this discovery, [F]F-AraG as a PET tracer 18 The use of [F]F-AraG has demonstrated promise in several distinct applications, including monitoring myocardial perfusion, myocardial viability, and cardiac inflammation. 18 It addresses a need in this technology by offering significant advantages over F-labeled tracers. Furthermore, [ 18 The use of [F]F-AraG can 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 and formulas 2', 4', and 11', and uses of such compounds, for example, for imaging. Embodiments of the present disclosure are advantageous because such compounds can be made in a few simple steps, as described in detail below and in Examples 1-3. In particular, embodiments of the present disclosure provide for the direct fluorination of a guanosine nucleoside precursor, followed by removal of a protecting group. Embodiments of the above method involve two steps, which occur over a short period of time, both of which are advantageous over other possible alternative commercial production schemes.

[0038] Embodiments of the method are shown in Schemes A-D and A'-D' in Figures 1.1A and 1.1B. Scheme A is general but uses a specific protecting group (PG) and leaving group (LG), 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 can be varied in a manner similar to that described below. More detailed schemes of embodiments of the present disclosure are set forth in Schemes 1 and 2 of Examples 1 and 2. In general, embodiments of the method include making labeled compounds such as those embodied in Formulas 3, 5, and 12. In one embodiment, the method can include 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 syntheses described in Figures 1.1A and 1.1B are very similar, with the primary difference being the use of specific PG and LG in Figure 1.1A. Thus, the following discussion of the synthesis in Figure 1.1A may apply to the synthesis in Figure 1.1B. The various substitutions for protecting groups, leaving groups, reactions, etc. described herein may be used in the synthesis described in Figure 1.1B.

[0039] Figure 2.1 depicts Schemes 1 and 2, which relate to specific embodiments of the present disclosure and are described in detail in Example 1. In particular, 18 F(FAraG) precursor (compound 8) is generated, and then 18 The resulting F(FAraG) precursor (compound 12) is reacted to form the F(FAraG) precursor (compound 13). Details regarding the reaction steps are shown in Figure 2.1, which are similar to those described above for the general synthesis. Figures 3.4-3.6 also provide specific details regarding the synthesis of embodiments of the present disclosure, which are described in detail in Example 2.

[0040] Embodiments of the present disclosure also encompass methods for imaging cardiac tissue and cells. Generally, embodiments of labeled compounds can be used to image the location and / or quantity of cardiac cells in a subject (e.g., a living human). The labeled compound can be administered to the subject, and the subject or a portion of the subject can 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 amount can be used to detect the presence, location, and / or number / size of cardiac cells, cancer cells, and / or white blood cells in the subject.

[0041] The present disclosure may also provide packaged compositions containing precursor compounds or intermediates to labeled compounds (e.g., Formulas 1, 1', 2, or 2'), as well as instructions for making and using the labeled compounds (e.g., written instructions for their use). The kits may further include appropriate buffers and reagents known in the art for administering embodiments of the present disclosure to a subject. [Example]

[0042] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present disclosure, and are not intended to limit the scope of what the inventors regard as their disclosure, nor are they intended to represent all or the only experiments performed.

[0043] Example 1: Illustrative method for making a PET probe AraG is a nucleoside analogue that has proven effective in the treatment of T-cell lymphoblastic diseases. It is uniquely metabolized by deoxyguanosine kinase and incorporated into mitochondrial DNA. 18 The F derivative was synthesized and used as a molecular probe. One skilled in the art can test the uptake and metabolism in cell lines and determine the effectiveness 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 present inventors have reported novel 2-deoxyguanosine analogs. 18 F derivative, 9-β-D-arabinofuranosylguanine ( 18 F-AraG" or "[ 18 [F]F-AraG) was successfully synthesized and used as a molecular probe (Figure 2.1). 18 F]AraG cellular uptake was evaluated in the leukemia cell line CCRF-CEM and 3 Figure 2.2 shows the cellular uptake of [H]AraG by the CCRF-CEM cell line. 18 The uptake of 8-[F]AraG is shown and is dose-dependent. 3 To determine whether [H]-AraG competes with the uptake of [H]-AraG by CCRF-CEM, MOLT-4 (a leukemia cell line), and Raji (a human Burkitt's lymphoma cell line), we investigated the uptake of [H]-AraG by CCRF-CEM, MOLT-4 (a leukemia cell line), and Raji (a human Burkitt's lymphoma cell line). 3 H]-AraG uptake and competition with cold 2F-AraG were examined (Figures 2.3A and 2.3B). Figure 2.3A shows that increasing the amount of 2F-AraG (1-100 μM) increased [3 Similar results were observed for MOLT4 and Raji cell lines (Figure 2.3B). Competition assays demonstrated that the cold derivative 2F-Arag exhibits a similar uptake pathway to 8-[H]-AraG. 3 3H]-AraG uptake.

[0045] Initial microPET scans in normal nude mice 18 Figure 2.1 depicts an embodiment of a method for making the compounds of the present disclosure. The 2'-deoxy-2'-fluoro-arabinonucleosides 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 which is incorporated herein by reference). The present inventors have used radiolabeled 8-[ 18 [F]fluoroguanine derivatives have been investigated as potential in vivo probes for imaging gene expression with positron emission tomography (PET). We have developed 8-[F]fluoroguanine derivatives based on a direct radiofluorination reaction. 18 We developed a method for the preparation of 8-[F]fluoroguanine derivatives. 18 F]Fluoroguanosine could be synthesized from guanosine (Nuclear Medicine and Biology. 2000; 27(2): 157-162 (which is incorporated herein by reference). Recently, 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, which is incorporated herein by reference).

[0046] Example 2: [ 18 F]F-ARAG 9-(β-D-arabinofuranosyl)guanine (AraG) is a guanosine analogue that has proven effective in the treatment of T-cell lymphoblastic diseases. To test the feasibility of using radiofluorinated AraG as an imaging agent, we investigated the efficacy of 2'-deoxy-2'-[ 18 F]fluoro-9-β-D-arabinofuranosylguanine ([ 18 [F]F-AraG) and investigated its uptake in T cells.

[0047] For this purpose, we dissolved 2-N-acetyl-6-O-((4-nitrophenyl)ethyl)-9-(3',5'-di-O-trityl-2'-O-triphyl-β-D-ribofuranosyl)guanine in [ 18 via direct fluorination with [F]KF / K.2.2.2 18 [F]F-AraG was synthesized. [F]F-AraG was synthesized in both CCRF-CEM leukemia cell line (non-activated) and activated primary thymocytes. 18 [F]F-AraG incorporation was assessed. We observed [F]F-AraG in 7-10% radiochemical yields (decay-corrected) with specific activities of 0.8-1.3 Ci / μmol. 18 Preliminary cellular uptake experiments showed that both CCRF-CEM leukemia cell line and activated primary thymocytes were able to uptake [F]F-AraG. 18 F]F-AraG uptake was shown. 18 [F]F-AraG was successfully synthesized by direct fluorination of an appropriate precursor of a guanosine nucleoside. This approach is useful for the synthesis of other important PET probes (e.g., [F]F-AraG), which are currently synthesized through multiple steps and require time-consuming purification. 18 F]FEAU,[ 18 F]FMAU and [ 18 Cellular uptake studies have demonstrated the potential of [F]FBAU as a PET imaging agent for T cells. 18 Support further research to explore the use of [F]F-AraG.

[0048] Scheme 1 is a synthesis of 2-N-acetyl-6-O-((4-nitrophenyl)ethyl)-9-(3,5-di-O-trityl-2-triphyl-β-D-ribofuranosyl)guanine (2), [ 18 [F]F-AraG precursor synthesis is shown. Treatment of the 2',5'-di-O-tritylguanosine derivative 1 with CFSOCl / DMAP afforded 2-N-acetyl-6-O-((4-nitrophenyl)ethyl)-9-(3',5'-di-O-trityl-2'-O-triphyl-β-D-ribofuranosyl)guanine (2) in 65% yield. Scheme 2, as shown in Figure 3.5, is a direct copy of a literature procedure (J. Org. The synthesis of cold F-AraG standard was prepared according to Chem. 1992, 57, 7315-7321 (incorporated herein by reference). The 3',5'-di-O-tritylguanosine derivative 1 was converted to 6-O-((4-nitrophenyl)ethyl)-9-(3',5'-di-O-trityl-2'-fluoro-β-D-arabinofuranosyl)guanine 3 with DAST reagent. 3 was deprotected with DBU to give 9-(3',5'-di-O-trityl-2'-fluoro-β-D-arabinofuranosyl)guanine (4). Finally, 4 was deprotected with TFA to give 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 dissolved in [ 18 It was prepared by nucleophilic substitution of the triflate in 2 with [F] fluoride ion. 18 Purification of [F]6 was performed by cleaving the deprotonated starting material 2 (AraG) into the final product [ 18 This was required to avoid contamination with [F]F-AraG 7. 18 F]6 was smoothly hydrolyzed first by base (0.5 M NaOCH3) and then by acid (1 N HCl) to give [ 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 take up 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 uptake of F]F-AraG by non-activated CCRF-CEM cells, 18 indicating a dose-dependence with a 2-fold (P < XX) increase in F]F-AraG uptake by cells exposed to 10 μCi compared to cells exposed to 3 μCi. Next, the inventors investigated 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 take up 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 take up 4.7-fold more (p = 0.003) 18 F]F-AraG, presenting the data.

[0051] There have been many reports on the indirect synthesis of 2'-deoxy-2'-fluoro-9-β-D-arabinofuranosylguanine (F-AraG) in which fluorine is first introduced into the arabino position at C-2 and then the fluorinated sugar is reacted 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 straightforward 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 suitably protected guanosine derivative 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 difficult synthesis of [F]-labeled DAST and the long reaction time of DAST-mediated fluorination, 18 [F] via the DAST method 18 It is not practical to synthesize [F]F-AraG. 18 [F]F-AraG precursor was prepared (Scheme 1 as shown in Figure 3.4), 1 H and 19 They were characterized by F NMR spectroscopy and high-resolution mass spectrometry. The chemical shift of H2′ varied from 4.73 ppm in 1 to 6.03 ppm in 2, due to the electronegativity of the trifyl group at the C2′ position. 19 F NMR showed a singlet at -75.00 ppm, which is consistent with the chemical shift of sugar triflates. Similar chemical shift trends were observed for the synthesis of adenosine triflate

[19] . To the best of our knowledge, precursor 2 is new and has been synthesized for the first time in our laboratory (a provisional patent application has been filed). Also, for the first time, we have reported that 2 was synthesized by the precipitation of [ 18Direct fluorination with [F]KF / K.2.2.2 afforded [F]KF with specific activities of 0.8–1.3 Ci / μmol in 7–10% radiochemical yields (decay-corrected). 18 [F]F-AraG (7, Scheme 3 as shown in Figure 3.6) was synthesized. The identity and purity of 7 was confirmed by co-injection with the authentic standard compound 5 on an analytical HPLC column (Figure 3.1).

[0052] In cell culture [ 18 To evaluate the performance of [F]F-AraG7, we performed several assays. 18 To confirm the ability of [F]F-AraG to incorporate IFN-γ, we cultured the CCRF-CEM cell line (acute lymphoblastic T leukemia cells, non-activated) and primary T cells with [F]F-AraG. 18 Figure 3.2 shows the effect of CCRF-CEM cells on the expression of [F]F-AraG. 18 F]F-AraG incorporation, and [ 18 These data also show that [F]F-AraG uptake is dose-dependent. 18 This confirms that the majority of [F]F-AraG is taken up by cells within the first hour of exposure. 18 Rapid uptake is necessary if [F]F-AraG (which has an isotopic half-life of 110 minutes) is to ultimately prove effective as a PET tracer. 18 Having confirmed that primary T cells (non-tumor-bearing T cells obtained from normal mouse tissues) also incorporate [F]F-AraG, we next investigated whether primary T cells (non-tumor-bearing T cells obtained from normal mouse tissues) also incorporate [F]F-AraG. 18 Figure 3.3 shows data from two independent experiments, demonstrating that non-tumorous but activated primary T cells can incorporate [F]F-AraG to appreciable levels. 18 F]F-AraG uptake by activated T cells. 18 Increased uptake of [F]F-AraG as a PET tracer in the detection of graft-versus-host disease (GVHD) 18This study may enable the use of [F]F-AraG. GVHD is a disease primarily driven by T cells, and the ability to detect abnormally activated T cells by PET may facilitate the 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 (approximately 150 μM)

[24] , we aimed to utilize [F]F-AraG as a tracer for PET while avoiding potential neurotoxicity in patients with PET. 18 To optimize [F]F-AraG, we chose to utilize a dose (approximately 0.5 μM) lower than the reported therapeutic level of AraG in our assays.

[0053] As discussed in this example, 18 [F]F-AraG can be synthesized by a direct fluorination method. This approach is comparable to other important PET tracers (e.g., [F]F-AraG), which are currently synthesized by multiple steps and require time-consuming purification processes. 18 F]FEAU,[ 18 F]FMAU and [ 18 [F]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 support the potential of [F]FBAU as a new PET imaging agent for the detection of diseases of T cell origin. 18 The application of [F]F-AraG is suggested.

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

[0055] dGK is [ 18 Since the α-AraG-binding domain has a higher affinity for [F]F-AraG, we hypothesized that the α-AraG-binding domain has a higher affinity for [F]F-AraG at the tracer level. 18 We considered the possibility that [F]F-AraG could be preferentially phosphorylated by mitochondrial kinases. Mitochondria are organelles responsible for providing energy to cells by producing ATP. The heart is an organ that is particularly rich in mitochondria due to its high energy requirements. Over the past decade, mitochondrial dysfunction has been recognized as an important aspect of cardiovascular pathology. As a result, treatments targeting cardiac mitochondrial function are rapidly emerging. As a substrate for mitochondrial dGK, [F]F-AraG is a mitochondrial kinase. 18 [F]F-AraG has been found to be a useful agent for reporting on cardiac mitochondrial activity.

[0056] In an exemplary embodiment of the present invention, [ 18 The use of [F]F-AraG was evaluated in healthy volunteers and cancer patients undergoing immunotherapy. Significant cardiac uptake was observed in both groups (Figure 6). Figure 6 shows the effect of [F]F-AraG as a tracer in the heart. 18 Our findings demonstrate that high mitochondrial activity in the heart is associated with high [F]F-AraG PET 1 hour after injection of the tracer. 18 We provide evidence that the [F]F-AraG signal clearly occurs.

[0057] Interestingly, in this embodiment of the present invention, [ 18 The [F]F-AraG signal was found to be different from that in patients receiving immunotherapy. In particular, Figure 7 shows the [F]F-AraG signal in the heart wall of healthy volunteers and patients receiving immunotherapy. 18 F]F-AraG signal intensity is shown (before refers to pretreatment images, after refers to images taken 2–3 weeks after a single injection of anti-PD-1 antibody).

[0058] The results provided herein [ 18 In light of this, [F]F-AraG demonstrates its ability to image the heart and report on changes that may be induced by drugs or injury. 18 The use of [F]F-AraG has demonstrated the potential to significantly improve the efficacy of existing methods in several distinct applications, including myocardial perfusion, myocardial viability, and cardiac inflammation, as well as cardiac drug development studies and methods for monitoring treatment responses to mitochondrial-targeted drugs. 18 It may 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 example (a prophetic specific aspect) discloses the development of a highly sensitive and specific PET imaging strategy for the early diagnosis of cancer therapy-related cardiotoxicity. We focus on two classes of cancer therapy associated with cardiotoxicity: for example, administration of immune checkpoint inhibitors (ICIs), which cause myocardial injury via T cell infiltration, resulting in myocarditis, and anthracyclines (e.g., doxorubicin), which cause mitochondrial dysfunction and direct myocardial cell death. In this example, we consider: 1) [ 18 2) how [F]F-AraG may enable early and pathophysiology-specific identification of cardiotoxicity associated with both ICI and doxorubicin treatment; 18 FDG and [18 Comparison of F]F-AraG, and [ 18 3) determining the variability in myocardial uptake of [F]F-AraG; and 3) comparing normal physiological [F]F-AraG in healthy subjects, e.g., to perform retrospective analysis of myocardial uptake in cancer patients. 18 Determine the extent of [F]F-AraG incorporation.

[0060] Life expectancy after a 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 reduced quality of life. Treatment-related cardiovascular toxicity is a major cause of morbidity and mortality in cancer patients and survivors (1). For many cancer survivors, cardiovascular events, rather than cancer recurrence, represent the major risk of death across many cancer types (2). Cardiovascular complications can occur with older cancer treatments (e.g., anthracyclines) and more selective, targeted therapies (e.g., kinase inhibitors), and have been well documented (3, 4). Immune checkpoint inhibitors (ICIs), monoclonal antibodies targeting immune response modulation (e.g., programmed death-1 (PD-1), its ligand (PD-L1), or cytotoxic T lymphocyte antigen-4 (CTLA-4)), have resulted in impressive clinical outcomes in a fraction of patients with advanced tumors; however, multisystem immune-related adverse effects, including cardiovascular toxicity, have been reported associated with treatment (5). ICI-associated cardiovascular toxicity is poorly characterized and is likely largely underreported. Of all immune-related adverse events, myocarditis is the most lethal, with a case-fatality rate approaching 50% (6,7). Given the rapidly expanding application of ICIs in cancer treatment, accurate assessment and better understanding of immune-related cardiotoxicity represent an unmet clinical challenge crucial for successful patient care and management.

[0061] Due to its noninvasive 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, reduced LVEF often appears late in cardiac injury and may indicate irreversible damage. Cardiac MRI allows for the detection of mechanical changes that occur before left ventricular dysfunction, but it lacks the molecular specificity and sensitivity for ICI-associated cardiotoxicity (10).

[0062] The inability of currently used imaging methods to detect subclinical cardiac involvement represents a major obstacle to the prevention and better management of cardiac complications in cancer patients and survivors (11). Furthermore, as immunotherapy progresses, rapid diagnosis of inflammatory cardiac sequelae is essential, as current diagnostic techniques are neither sensitive nor specific. High specificity, resolution, and sensitivity have made positron emission tomography (PET) the gold-standard imaging modality for the assessment of myocardial metabolism and perfusion, yet evaluation of its usefulness in the early detection of cancer therapy-related cardiotoxicity has been limited. Nevertheless, PET imaging agents targeting early indicators of cardiovascular toxicity offer a powerful, highly specific, noninvasive tool for the detection of subclinical cardiotoxicity.

[0063] In this example, we 18 We focus on [F]F-AraG, a PET agent with the unique ability to assess activated T cells and myocardial mitochondrial function as an imaging biomarker for the early detection and monitoring of ICI- and chemotherapy-associated cardiotoxicity. 18 [F]F-AraG was developed by Namavari et al. to image activated T cells (12). It is a nucleotide sequence of arabinosylguanosine (AraG). 18Its F-labeled analog, a compound that showed remarkable selective accumulation in T cells (13, 14), and its prodrug, nelarabine, are used for the treatment of T-cell acute lymphoblastic leukemia and T-cell lymphoblastic lymphoma. 18 [F]F-AraG enters T cells via nucleoside transporters and is captured intracellularly primarily through phosphorylation by deoxyguanosine kinase (dGK), an enzyme found exclusively in mitochondria and crucial for providing 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 associated with [F]F-AraG. 18 [F]F-AraG can be visualized. 18 The ability of [F]F-AraG to image T cell activation and therefore provide an early indication of an adaptive response to immunotherapy in cancer patients is currently being investigated in multiple 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 myocardium and tumor cells (Figure 9A) (20). Infiltrating T cells have been implicated in the pathogenesis of heart failure (21) and have been detected in fatal cases of ICI-myocarditis (7, 22). Without being bound by any particular theory or mechanism of action, the present inventors propose that [ 18 We believe that [F]F-AraG can simultaneously detect activated T cell infiltration in tumors and the heart, and therefore serve as a tool for the early detection of ICI-associated cardiotoxicity.

[0065] As a substrate for mitochondrial dGK, an enzyme in the nucleotide salvage pathway responsible for supplying precursors for mtDNA synthesis in a rate-limiting manner (23), 18[F]F-AraG is uniquely suited to report on mitochondrial status not only in activated T cells but also in mitochondria-rich cells (e.g., myocardium). The cardiotoxicity of doxorubicin (a widely used chemotherapeutic agent) has been thoroughly studied, and numerous proposed mechanisms exist, all of which center on dysregulation of mitochondrial function in the myocardium (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 demonstrates that abnormal mitochondrial function in doxorubicin-associated cardiomyopathy is [F]F-AraG. 18 We provide evidence that doxorubicin-induced cardiotoxicity can be visualized by [F]F-AraG, allowing for early detection of doxorubicin-induced cardiotoxicity.

[0066] The ability to assess cardiotoxicity associated with both ICIs and doxorubicin is crucial in the context of the increasing use of combined chemotherapy / immunotherapy approaches that result in enhanced cardiac damage caused by chemotherapy and by effector T cell-mediated myocardial injury (26). 18 The ability of [F]F-AraG to assess myocardial mitochondrial status is demonstrated by the 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 suggests that [F]F-AraG may be involved in the assessment of myocardial mitochondrial status. 18 F]F-AraG's ability to detect abnormal mitochondrial and / or inflammatory activity (Figure 10).

[0067] In this example, we investigated the increased [ 18 Considering the mechanisms underlying [F]F-AraG uptake, the ability to accurately interpret myocardial uptake detected in patients receiving immunotherapy is crucial, as it may provide a clinically-ready tool for assessing not only treatment response but also concurrently reporting adverse effects of treatment. 18[F]F-AraG may fulfill an urgent clinical need for noninvasive measurements of mitochondrial function, which may aid in the development and more successful translation of mitochondrially targeted therapeutics in humans (27). 18 The ability of [F]F-AraG PET to detect early cardiac involvement could have a profound impact on patient management, with the potential to revolutionize cardio-oncology, a rapidly evolving field focused on balancing treatment efficacy and cardiovascular safety. 18 [F]F-AraG PET may enable 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 may in turn reduce the high health care and societal costs associated with cardiovascular disease.

[0068] Currently used imaging methods lack the molecular specificity to detect subclinical cardiac involvement associated with cancer therapy. This offers exceptional advantages based on preliminary clinical data ([ 18 It is clear from the data that [F]F-AraG PET has great potential to detect variable mitochondrial activity in the heart. 18 [F]F-AraG may currently be the only agent in the clinic that can report on the damage caused by various cancer treatments: chemotherapy, targeted therapy, and ICIs. Furthermore, the ability to simultaneously report on the immune response in target tissues and the off-target toxic effects of immunotherapy is a valuable tool for [F]F-AraG. 18 This example provides the development of a new method for the assessment of cardiotoxicity associated with ICI- and combination chemotherapy / ICI approaches, for which no diagnostic approach currently exists.

[0069] Embodiment 1. 18 We demonstrate that [F]F-AraG allows early identification of cardiotoxicity in relation to the pathophysiology of mitochondrial damaging drugs (e.g., ICI and doxorubicin). [18 [F]F-AraG imaging may correlate with biomarkers of cardiac pathophysiology and may allow early detection of cardiotoxicity.

[0070] Rationale 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). Because T cells play a crucial role in the immune response to tumors, we have focused on [F]F-AraG in immuno-oncology. 18 The focus of this study was to determine the value of [F]F-AraG on activated T cells (30, 31). 18 The specificity of [F]F-AraG and its usefulness in predicting response to immunotherapy have been confirmed (30). Using a rhabdomyosarcoma model, we determined that over 80% of the detected intratumoral signal was derived from tracer accumulation in immune cells (primarily activated CD8+ and CD4+) (Figure 12). Importantly, [F]F-AraG was detected in colon cancer tumors and tumor-draining lymph nodes. 18 [F]F-AraG signals were also able to distinguish between responders and non-responders to anti-PD-1 therapy early in treatment, as little as 48 hours after a single therapeutic dose (Fig. 12).

[0072] The discovery of immune responses resulting from chemotherapy-induced tumor cell death (termed immunogenic cell death (32)) has sparked interest in exploiting the immunomodulatory effects of chemotherapy for 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) have demonstrated that the immune responses 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) are highly effective.18 [F]F-AraG imaging revealed dramatic differences in the immunomodulatory effects caused by the two chemotherapy regimens, providing a useful tool for assessing chemotherapy-induced immunomodulation and immune activation environments. 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 breakdown of peripheral immune tolerance and excessive myocardial T cell infiltration and activation, resulting in myocarditis. Experts have created both pharmacological and genetic models of ICI-myocarditis by treating mice with anti-CTLA-4 and anti-PD-1 antibodies and 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, phenotyping ICI-myocarditis in patients (Figure 14) (35). Notably, autoimmunity in Ctla4+ / - Pdcd1- / - mice is restricted to the cardiovascular system and characterized by T cell and myeloid infiltration into the myocardium, as observed in humans.

[0074] Although the exact mechanism of doxorubicin-related cardiac injury is still under debate, numerous studies have suggested mitochondria as a primary target in doxorubicin-induced myocardial damage (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-related cardiomyopathy (38). Agents that assess cardiac mitochondrial membrane potential, 18 F-Mitophos and 68Preclinical studies with Ga-Galmydar demonstrated reduced cardiac uptake in doxorubicin-treated animals, indicating the potential of mitochondrial imaging in chemotherapy-induced cardiotoxicity (38, 39). Interestingly, doxorubicin treatment significantly reduced cardiac 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 of which play central roles in the mechanisms of cardiotoxicity (Figure 9), indicates its potential to serve as a pathophysiologically relevant imaging biomarker for early detection and monitoring of cardiotoxicity associated with ICI and doxorubicin treatment. The ability to assess clinically relevant cardiotoxicity associated with both chemotherapeutic and immunotherapeutic agents could be invaluable given the lack of knowledge regarding the cardiovascular damage of the rapidly growing combination chemotherapy / immunotherapy approaches.

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

[0077] Experimental Approach Animal model of cardiotoxicity. To investigate dose-dependent doxorubicin cardiotoxicity, mice (n = 8 / group, 4 males and 4 females) can be treated with low (4 mg / kg) and high (20 mg / kg) doses of doxorubicin. Control mice (n = 8, 4 males and 4 females) can receive injections of vehicle only. To study ICI-associated myocarditis, we can use a recently developed preclinical model (35). Ctla4+ / - Pdcd1- / - mice fully recapitulate the well-documented pathophysiological and clinical features of ICI-associated myocarditis. Ctla4 + / + Pdcd1 - / - Mice can be used as controls. We used eight Ctla4+ / - Pdcd1- / - mice (four males and four females) and eight Ctla4 + / + Pdcd1 - / - Control mice may be used.

[0078] Furthermore, researchers have generated a milder form of ICI myocarditis by treating mice with anti-CTLA-4 and anti-PD-1 antibodies in specific genetic backgrounds. MRL-Fas mice treated with anti-CTLA-4 or anti-PD-1 monotherapy, or combination therapy, twice weekly for 8 weeks were treated with CTLA-4 or anti-PD-1 monotherapy. lpr Although the mice did not show obvious clinical signs, histological and electron microscopic examination revealed signs of immune infiltration of the myocardium and vasculature, as well as endothelial cell damage and sarcomere disorganization after combination therapy. We believe that the combination of pharmacological and genetic mice to model ICI myocarditis is complementary. Finally, as the use of ICI expands to cancer types where patients may be exposed to the combination of doxorubicin and ICI (e.g., triple-negative breast cancer), these mice provide a platform in which the cardiovascular effects of combination therapy can be investigated. To examine the cardiotoxicity of combined doxorubicin / ICI treatment, we used 16 MRL-Fas mice. lprMice can be used. Eight mice (half male, half female) can be treated with doxorubicin (20 mg / kg) and anti-PD-1 and anti-CTLA-4 antibodies (250 μg). Vehicle-treated MRL-Fas lpr Mice can serve as controls.

[0079] PET and LVEF imaging. 18 [F]F-AraG can be produced by conventional methods (e.g., at the UCSF Radiochemistry Core). For PET / CT, mice are administered 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, we performed 60 minutes of dynamic PET imaging followed by CT at baseline and two post-treatment time points. Electrocardiogram-gated PET can be performed after the final imaging time point to obtain information on left ventricular ejection fraction (LVEF). 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 imaged before and periodically during treatment. The genetic ICI myocarditis model can be imaged three times, once a week, starting at week 6. Sixty 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 3D OSEM / MAP algorithm provided by the scanner manufacturer. Attenuation and scatter correction to ensure quantitative accuracy can be applied using CT-based attenuation maps and scatter models. ECG-gated 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 data can be used for static data analysis. Rate constants, including the inflow rate constant (Ki), can be derived from the dynamic data, and % injected dose / g (%ID / g) can be derived from the static data. LVEFs can be calculated from the ECG-gated data. All of these parameters can be mapped to polar plots for regional and segmental analysis of the parameters. Image processing can be performed in a PMOD / PCARD / PKIN (PMOD Technologies).

[0080] Embodiment 2. 18 F-fluorodeoxyglucose ( 18 FDG) and [ 18F]F-AraG compared with 18 Determine variability in [F]F-AraG myocardial accumulation [ 18 [F]F-AraG may allow for blood glucose-independent assessment of cardiac function and may exhibit low signal variability.

[0081] Rationale and supporting evidence. 18 FDG PET is routinely used in the diagnosis, staging, and restaging of tumor patients. The widespread use of FDG has resulted in incidental myocardial findings that have stimulated numerous studies aimed at evaluating the utility of FDG in cardiac imaging. 18 FDG is now the most widely used and validated PET tracer for the assessment of 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, FDG is used in tumor patients. 18 FDG scans are performed under fasting conditions. 18 FDG uptake was low, reflecting the increase in glucose metabolism and fatty acids, the main energy substrate for the heart, caused by the decrease in insulin (Figure 16). Myocardial FDG uptake depends on plasma glucose, fatty acid, and insulin levels, but also on diet, activity, and the use of certain drugs, and wide spatial and temporal variability in the signal has been reported (42, 43). The duration of fasting, investigated as a variable that can be controlled to provide more specific and accurate differentiation of myocardial uptake between benign and malignant lesions, does not appear to correlate with myocardial glucose metabolism (43). 18 Good quality FDG images are particularly difficult to achieve in diabetic patients, a population with high rates of cardiovascular comorbidity.

[0082] [ 18The mechanism of [F]F-AraG uptake is different from that of glucose (Figure 8), and consequently signal variability is expected to be lower relative to the fasting state (Figure 17). 18 The uptake of [F]F-AraG reflects the activity of enzymes in the salvage pathway, which is thought to be the predominant process by which purine nucleotide pools are maintained in the myocardium and lymphoid organs (44, 45). Dietary nucleotides are converted into [F]F-AraG in the salvage pathway. 18 F]F-AraG is the major source of nucleosides that can compete with F-AraG and result in variation in meal-related signals.

[0083] One embodiment of the present invention comprises: 18 FDG and [ 18 F]F-AraG to determine the difference in signal variability between 18 Aspects of the present invention may allow for a better understanding of potential sources of [F]F-AraG signal heterogeneity in animals exposed to different conditions (fasted vs. non-fasted, anesthetized vs. active, fed diets with or without dietary nucleotides). 18 FDG and [ 18 This can be tested by determining the difference between the myocardial signals of [F]F-AraG.

[0084] Experimental Approach We can investigate the effects of three variables on myocardial uptake: fasting, activity, and diet. For each variable, we can use 16 mice (8 males and 8 females) and divide them into two groups. For fasting studies, one group can have free access to both food and water, while the other group can be fasted for 8 hours with access to water. For activity studies, one group of animals can be kept under anesthesia, while the other group can be awakened after tracer injection and before imaging. To investigate the effect of dietary nucleotides, both groups of animals can be fed only a nucleotide-free diet and a diet supplemented with 0.04% nucleotides (a level found to be optimal for immune function) (46).

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

[0086] Blood glucose and nucleotide measurements. Glucose levels can be determined in tail vein blood samples immediately prior to imaging using a glucose meter. Because blood nucleotide measurements require large volumes of analyte, they can be measured following a final intracardiac blood draw after the final PET scan (47).

[0087] Image quantification and sample size calculation. The %ID / g between the groups with each variable and the control group can be derived from the PET data. Our hypothesis is that we will not be able to predict the difference due to dietary variables. 18 The conclusion is that no significant difference in [F]F-AraG uptake can be observed. With a sample size of 8 / cohort, a significance level of α=0.05 and a power of (1-β)=0.85, a two-sample t-test can be statistically significant if the difference is greater than 30% with a standard deviation of 20%. Therefore, if the difference is smaller, there will be no significant difference with a power of 0.85, which is what we would expect for an individual comparison study.

[0088] We predict that we will show the following: 1) between fasted and non-fasted animals [ 18 2) no / minimal difference in F]F-AraG signal between the anesthetized and active groups [ 18 F] no / minimal difference in F-AraG signal, 3) [ 18 4) the extent of the effect of dietary nucleotides on the [F]F-AraG signal 18 Compared with FDG, [ 18 Lower variability in the F]F-AraG signal. 18 F]F-AraG 18 It may allow for the evaluation of the benefits that FDG may offer, as well as the benefits that are not related to pathophysiology. 18 This may indicate a potential source of variability in the [F]F-AraG signal.

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

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

[0091] [ 18The safety and radiation dosimetry of [F]F-AraG were tested in six healthy subjects (three males and three females) during a first-in-human study at UCSF. Myocardial uptake was observed in all six subjects, with a mean SUV in the heart wall of 2.9 ± 0.62. The signal persisted throughout the imaging period, indicating tracer capture (Figure 18). The tracer uptake appeared uniform, without focal or localized enhancement. The relatively low density and uniform signal in healthy subjects indicates the possibility of detecting abnormal signal intensity and patterns in pathological conditions, but the relatively small sample size may not be representative of physiological accumulation in a larger population. To more comprehensively assess baseline values, we may, for example, image an additional 30 healthy subjects (15 males and 15 females) and analyze cardiac signals using conventional and radiomics methods. Radiomics, a computational method used to analyze and extract rich quantitative features (e.g., texture, heterogeneity, and shape) present in images, has been largely used in tumor imaging for improved cancer screening and early detection (48, 49). Radiomics has also been used to identify immune contexture features within the tumor microenvironment for more accurate assessment of immunotherapy response (50, 51). To date, the application of radiomics in cardiac imaging has primarily focused on the analysis of CT and MRI images without the use of molecular biomarkers. Here, we demonstrate a novel approach to extract image features with strong biological relevance that may provide a more accurate and pathology-specific assessment of cardiotoxicity. 18 We disclose radiomics analysis of [F]F-AraG PET images. In our preliminary preclinical study, mice treated with anti-PD-1 / CTLA-4 immunotherapy showed significant differences in intratumoral signal energy, entropy, and homogeneity from untreated mice (Figure 19). Texture analysis of the images may allow for better characterization and quantification of the signal differences observed between healthy volunteers and some cancer patients (Figure 20). 18F]F-AraG is a promising candidate for evaluating the response to immunotherapy in cancer patients. 18 [F]F-AraG is currently being tested in multiple Phase II clinical trials to evaluate its usefulness. To date, more than 40 patients with different types of cancer (head and neck, lung, bladder, breast squamous cell carcinoma, and melanoma) have been treated with [F]F-AraG. 18 The present inventors have safely imaged the cerebrospinal fluid (C1F) with [F]F-AraG. To enable comparison of findings in a healthy cohort, we have collected [F]F-AraG. 18 A comprehensive retrospective analysis of myocardial uptake in [F]F-AraG scans can be performed. Our preliminary analysis showed significantly higher myocardial signal in cancer patients than in healthy subjects both before and after a single infusion of immunotherapy (Figure 20). A higher signal in the pre-immunotherapy scan may indicate a cardiotoxic complication of previous anti-cancer treatment (e.g., chemotherapy or radiation therapy). Within a restricted cohort of patients 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 support the use of [F]F-AraG as a tool for cardiovascular risk stratification and assessment of early cardiotoxicity. 18 F]F-AraG.

[0092] Experimental Approach Clinical trial overview. Participants were randomly assigned to receive a 200 mg / kg / day trial at the UCSF Nuclear Medicine Clinic. 18Subjects may undergo a whole-body PET / CT scan with [F]F-AraG. The study population may consist of 30 healthy subjects (15 males and 15 females, with no known cardiac disease). To account for possible age-related differences within each gender 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 special emphasis on family history of cardiac disease and 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 diet.

[0093] PET imaging. 18 [F]F-AraG may be synthesized per patient on a per scan basis prior to imaging. 18 [F]F-AraG may be administered as a bolus intravenous injection. Dynamic imaging may be performed in two female and two male patients from each age group (12 patients total). Dynamic data collection may begin immediately after tracer injection, last for 60 minutes, and focus on the cardiac field. Static images may be acquired in 18 patients (three males and three females from each age group) 60 minutes after tracer injection. Non-contrast CT scans (5 mm continuum axial sections) (CTAC) may be acquired at 38.5 mm / sec with a field of view (FOV) of 38.5 mm / sec in helical mode using 120 kVp, 40 mA, and a 512 × 512 matrix size of the relevant region. The CTAC image may be fused to the PET image and used for attenuation correction and anatomical positioning. Immediately following CTAC, an emission PET scan may be performed over the same anatomical region in a 3-dimensional time-of-flight (3-D time-of-flight) mode. The PET emission scan may be acquired in Time of Flight (TOF) mode, and the PET emission scan may be attenuation corrected using the segmented attenuation data of the CTAC scan.

[0094] Image Analysis. PET images can be reconstructed with standard iterative algorithms provided by the scanner manufacturer for both dynamic and static imaging data. From dynamic imaging, dynamic multiframes (12 x 5, 6 x 10, 4 x 30, 6 x 60, and 10 x 300 s) can be generated for tracer kinetic modeling (two-tissue compartment model). The final 10 minutes of summed data can be generated to represent comparable static data. Rate constants, including the influx rate constant (Ki), can be calculated in the myocardium using PMOD / PCARD / PKIN (PMOD Technologies). Whole-body imaging from static scans can be performed using [ 18 We envision how [F]F-AraG can be used for both tumor imaging and myocardial imaging. For this example, we can add an additional reconstruction focusing on the same field of view for dynamic imaging, and calculate the following standardized uptake value measures for the left ventricle: SUV max, SUV peak, and SUV mean. The American Heart Association (AHA) 17-segment model can be used to quantify these values ​​for static images. Kinetic parameters from dynamic imaging can be correlated with SUV from static images to confirm whether static scans provide stable image quantification and do not require dynamic scans for practical application.

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

[0096] In accordance with embodiments of the present invention, one skilled in the art will be able to determine: 1) relatively low, uniform myocardial uptake in healthy subjects; 2) relatively low, uniform myocardial uptake in healthy subjects;18 4) a narrow range of [F]F-AraG myocardial uptake, which may be significantly higher in cancer patients than in healthy populations.

[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, amounts, and other measured data may be expressed herein in range format. Because such range format is used for convenience and brevity, it should be understood that it should also be interpreted in a flexible manner to include not only the numerical values ​​explicitly recited as range limits, but also all individual numerical values ​​or subranges subsumed within the range as if each numerical value and subrange were explicitly recited. By way of example, a concentration range of "about 0.1% to about 5%" should be interpreted not only to include the explicitly recited concentration of about 0.1% by weight to about 5% by weight, but also to include individual concentrations (e.g., 1%, 2%, 3%, and 4%) and subranges within that recited range (e.g., 0.5%, 1.1%, 2.2%, 3.3%, and 4.4%). In one embodiment, the term "about" may include conventional rounding techniques to reach significant digits of a numerical value. Furthermore, the phrase "about 'x' to 'y'" includes "about 'x' to about 'y'."

[0099] It should be emphasized that the above-described embodiments of the present disclosure are merely possible implementations, set forth merely for a clear understanding of the principles of the present disclosure. Many variations and modifications may be made to the above-described embodiments of the present disclosure without substantially departing from the spirit and principles of the present disclosure. All such modifications and variations are intended to be included herein within the scope of the present disclosure. In certain embodiments, for example, the following are provided: (Item 1) 1. A method of imaging cardiac cells in a subject, the method comprising: (a) the following formula: [ka] administering to the subject a compound having the formula: (b) imaging the subject, wherein detecting the presence of the compound corresponds to the presence of cardiac cells; A method that encompasses (Item 2) Item 10. The method according to item 1, wherein the subject is selected to be a patient who has been administered a therapeutic agent or radiation therapy, and the one or more images of the heart obtained in step (b) are used to obtain information regarding the effect of the agent or radiation therapy on the subject's heart. (Item 3) 3. The method of claim 2, wherein the therapeutic agent is an anthracycline or an immune checkpoint inhibitor, and the one or more images of the heart obtained in step (b) are used to obtain information about cardiotoxicity resulting from administration of the anthracycline or the immune checkpoint inhibitor. (Item 4) using one or more images of the heart obtained in step (b), assessing one or more parameters of myocardial perfusion in the subject's heart; assessing one or more parameters of myocardial viability in the subject's heart; and / or assessing one or more parameters of inflammation in the heart of said subject; The method according to item 1, further comprising: (Item 5) 3. 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 subject is selected to be a patient diagnosed with cardiovascular disease; The subject is selected to be a patient diagnosed with cancer; The subject is selected to be a patient undergoing treatment for cardiovascular disease or cancer. The method described in item 2. (Item 7) 7. The method of 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 to observe changes in the heart over time. (Item 8) 8. The method of claim 7, wherein the amount of time from the first date to the second date comprises at least one week or at least one month. (Item 9) 4. The method of claim 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) 1. A method of imaging cells in a subject in response to administration of a therapeutic agent, the method comprising: (a) administering the therapeutic agent; (b) the following formula: [ka] administering to the subject a compound having the formula: (c) imaging the subject, wherein detecting the presence of the compound corresponds to the presence of the cell; and (d) correlating the observed presence of the compound in the subject's cells with the subject's response to the therapeutic agent; A method that encompasses (Item 11) The subject is selected to be a patient diagnosed with cardiovascular disease; The subject is selected to be a patient diagnosed with cancer; The subject is selected to be a patient undergoing treatment for cardiovascular disease or cancer. Item 11. The method according to item 10. (Item 12) 12. The method of claim 11, wherein the correlating step comprises observing the presence of the compound in cancer cells and / or lymph nodes. (Item 13) 12. The method of claim 11, wherein the therapeutic agent comprises at least one immune checkpoint inhibitor selected to affect CTLA-4 or PD-1 / PD-L1 blockade. (Item 14) Item 14. The method of item 13, wherein the cancer cells are colon cancer cells. (Item 15) Item 12. The method of item 11, further comprising 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 to observe the subject's response to administration of the therapeutic agent. (Item 16) Item 16. The method of item 15, wherein the amount of time from the first date to the second date comprises at least one week or at least one month. (Item 17) 1. A method of imaging mitochondrial activity in cells of a subject, the method comprising: (a) the following formula: [ka] administering to the subject a compound having the formula: (b) imaging the subject, wherein detecting the presence of the compound corresponds to the presence of mitochondrial activity; and (c) imaging mitochondrial activity; A method that encompasses (Item 18) 18. The method of claim 17, wherein the subject is selected as suffering from a mitochondrial defect. (Item 19) 18. The method of claim 17, wherein the method is a method for screening the subject for mitochondrial dysfunction; and the mitochondrial dysfunction is a cardiovascular disease, a neuropsychiatric disease, or a neurodegenerative disease. (Item 20) 20. The method of item 19, 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 disorders, mtDNA depletion disorders, myoclonic epilepsy, ragged-red fiber syndrome, encephalomyopathy, lactic acidosis, stroke-like episodes, and optic atrophy.

Claims

[Claim 1] The invention described in this specification.

Images