Methods and materials for imaging adipose tissue - Patents.com

Abstract

Embodiments of the present disclosure provide compositions and methods for performing positron emission tomography (PET), and more particularly for use in selected imaging techniques, such as the study of selected agents for modulating adipose tissue physiology. 18 Compositions and methods are provided for the development and use of F-based PET tracers. The compounds used in the methods disclosed herein are 18 [F]-F-arabinofuranosylguanine was originally developed as a PET imaging agent for activated T cells.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit under 35 U.S.C. §119(e) of co-pending and commonly assigned U.S. Provisional Patent Application No. 63 / 318,923, filed March 11, 2022, entitled "METHODS AND MATERIALS FOR IMAGING ADIPOSE TISSUE," which is incorporated herein by reference. [Background technology]

[0002] background Brown adipose tissue (BAT) has attracted considerable attention due to its high mitochondrial content, association with cardiometabolic health, and potential therapeutic effects in a range of conditions (from diabetes and obesity to cachexia in cancer) (see, e.g., Becher, T. et al., Brown adipose tissue is associated with cardiometabolic health., Nat Med, 2021, 27(1):p.58-65). Given its potential therapeutic effects associated with modulating BAT activation, processes associated with BAT activation, as well as methods / drugs with the ability to modulate this activity, are under investigation.

[0003] The present disclosure relates generally to positron emission tomography (PET), and more particularly to a method and apparatus for imaging adipose tissue (e.g., brown adipose tissue). 18 The present invention 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 clinical studies without the need for an on-site cyclotron. Furthermore, modern PET camera technology allows quantitative measurements of adipose tissue radioactivity concentrations with rapid sampling and good statistical precision.

[0004] The most commonly used PET imaging agent for imaging brown fat is 2-[F 18 ]Fluoro-2-deoxy-D-glucose (FDG). However, studies have shown that FDG does not image mitochondrial changes (most notably, UCP-1 expression), but rather increases in glucose metabolism in activated brown fat (see, for example, Hankir, MK et al., Dissociation Between Brown Adipose Tissue (18)F-FDG Uptake and Thermogenesis in Uncoupling Protein 1-Deficient Mice., J Nucl Med, 2017, 58(7):p.1100-1103; and Hankir, MK and M. Klingenspor, Brown adipocyte glucose metabolism: a heated subject., EMBO Rep, 2018, 19(9)). Although activated brown fat does indeed exhibit increased glucose metabolism, agents that affect glucose metabolism without brown fat activation will simultaneously result in increases in FDG in brown fat, making FDG a non-specific agent for BAT activation.

[0005] In techniques for adipose tissue imaging, FDG probes do not provide the specificity required to enable one of skill in the art to image BAT activation and / or inactivation in a manner that allows one to evaluate therapeutic agents and other therapeutic interventions. 18 There is a need in the art for additional compositions and methods designed to use F-based PET tracers. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Becher, T. et al., Brown adipose tissue is associated with cardiometabolic health., Nat Med, 2021, 27(1):p.58~65 [Non-Patent Document 2] Hankir, MK et al., Dissociation Between Brown Adipose Tissue (18)F-FDG Uptake and Thermogenesis in Uncoupling Protein 1-Deficient Mice., J Nucl Med, 2017, 58(7):p.1100~1103 [Non-Patent Document 3] Hankir, M.K. and M.Klingenspor, Brown adipocyte glucose metabolism: a heated subject., EMBO Rep, 2018, 19(9) Summary of the Invention [Means for solving the problem]

[0007] overview The compounds used in the methods disclosed herein are 18 [F]-F-arabinofuranosylguanine was originally developed as a PET imaging agent for activated T cells. This compound has the structure of arabinofuranosylguanine (AraG). 18 It is an F-labeled analogue that can be phosphorylated by two cytoplasmic kinases, deoxycytidine kinase (dCK) and deoxyguanosine kinase (dGK), and trapped intracellularly. As discussed below, at the tracer level, 18 The inventors have discovered that [F]F-AraG has the potential to be used in many new PET methodologies, including PET methodologies designed to observe the presence and / or activity of adipose tissue in vivo.

[0008] The disclosure provided herein is 18The present invention is based in part on the discovery that F]F-AraG is a PET imaging agent well suited for observing activation of adipose tissue cells. Embodiments of the invention include, for example, methods of imaging adipose tissue in a subject. Typically, these methods involve the use of a compound of the formula: [ka] The method includes administering to the subject a 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 adipose tissue in the subject; and then imaging the subject by positron emission tomography, wherein detecting the presence of the compound corresponds to the presence of adipose tissue and / or activation of adipose tissue. In some embodiments of the present invention, the adipose tissue is brown adipose tissue. In other embodiments of the present invention, the adipose tissue is bone marrow adipose tissue (e.g., controlled bone marrow tissue).

[0009] In typical embodiments of the present invention, these methods include observing the appearance or level of activated adipose tissue cells. In certain embodiments, adipose tissue is present in a subject diagnosed with a pathological condition (e.g., obesity, diabetes, cachexia, or cancer (e.g., glioblastoma)). In some embodiments of the present invention, the subject is selected to be a patient who has been administered a therapeutic agent and / or undergone a therapeutic intervention (e.g., surgery, radiation, device implantation, etc.), and one or more images of the adipose tissue obtained are used to obtain information regarding the effect of the therapeutic agent or intervention. Although the methods disclosed herein can be used to observe the presence and / or activation of adipose tissue only once, optionally, the method includes observing one or more images of the adipose tissue on a first day; observing one or more images of the adipose tissue on a second day; and comparing the images obtained on the first day with the images obtained on the second day to observe changes in the adipose tissue over time.

[0010] The embodiments of the present invention also include a method of observing the effect of a test agent on the activation of adipose tissue cells (e.g., brown adipose tissue cells, bone marrow adipose tissue cells, etc.). Typically, these methods include combining adipose tissue cells with a test agent; observing whether the activation of adipose tissue cells combined with the test agent changes; and then comparing the activation of adipose tissue cells combined with the test agent to control adipose tissue cells not combined with the test agent, so that the effect of the test agent on the activation of adipose tissue cells is observed. In some embodiments of the present invention, the adipose tissue cells are combined with the test agent in vitro, while in other embodiments, the adipose tissue cells are combined with the test agent in vivo. In certain embodiments of the present invention, the adipose tissue cells are obtained from a subject diagnosed with a pathological condition (e.g., obesity, diabetes, cachexia or cancer).

[0011] 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 some embodiments of the present invention, are given by way of illustration and not by way of limitation. Many changes and modifications that fall within the scope of the present invention may be made without departing from the spirit of the invention, and the invention includes all such modifications.

[0012] Further aspects of the present disclosure will be more readily understood from consideration of the following detailed description of various embodiments of the present disclosure when taken in conjunction with the accompanying drawings. [Brief description of the drawings]

[0013] [Figure 1] FIG. 1 provides a schematic of the mechanism involved in imaging adipose tissue with [18F]F-AraG. [Diagram 2] FIG. 2 provides PET images showing that [18F]F-AraG results in significantly higher accumulation of brown fat (yellow arrows) 23 days after glioblastoma multiforme (gbm) cell implantation (right image) than 14 days (left image). [Diagram 3] Figure 3 provides PET images showing brown fat activation with [18F]F-AraG (upper panel image) and brown fat activation with FDG (lower panel image). [18F]F-AraG shows BAT signal (yellow arrow) only in animals treated with BRL37344 (BAT activator), while FDG shows various degrees of signal in all animals, indicating low specificity. Furthermore, FDG showed increased signal in BAT in mice treated with insulin, which only affects glucose metabolism. The data presented in this figure clearly demonstrate the specificity of [18F]F-AraG and its advantage over FDG for imaging activated BAT. [Figure 4] Figure 4 provides PET images showing that mice exposed to chronic administration of BRL37344 (a BAT activator) exhibit increased [18F]F-AraG accumulation not only in intrascapular BAT but also in the lumbar spine and proximal tibia (yellow arrows). [Diagram 5] Figure 5 provides PET images and data showing that BAT activation is associated with T cell neuroinflammation in multiple sclerosis (Cuprizone-EAE model). Treatment with fingolimod reduces neuroinflammation and results in the absence of BAT activation. [Figure 6] Figure 6 provides PET images showing that simultaneous imaging of activated lymphocytes and adipocytes with [18F]F-AraG reveals concomitant neuroinflammation (A) and BAT activation (B) in patients who recovered from COVID. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] Detailed Description Before the present disclosure is described in more detail, it should be understood that the present disclosure is not limited to the specific embodiments described, and the embodiments of the present invention themselves may naturally vary. It should also be understood that the terms used in this specification are only for the purpose of describing specific embodiments, and are not intended to be limiting. Unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by those of ordinary skill in the art to which this disclosure belongs. All publications and patents cited in this specification are incorporated by reference as if each individual publication or patent was specifically and individually indicated to be incorporated by reference, and all publications and patents cited in this specification are incorporated by reference to disclose and describe the methods and / or materials in which the publications are cited. As will be apparent to those skilled in the art upon reading this disclosure, each of the individual embodiments described and shown in this specification has individual components and features that can be easily separated from or combined with the features of any of the other several embodiments without departing from the scope and spirit of the present disclosure. Any method described may be carried out in the order of events described or in any other order that is logically possible. Unless otherwise indicated, the present disclosure is not limited to particular materials, reagents, reactants, manufacturing processes, etc., which may, as such, 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 also possible in the present disclosure that steps may be carried out in another order, where it is logically possible.

[0015] It must be noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly indicates otherwise. Thus, for example, reference to "a compound" includes a plurality of compounds. Throughout this specification and the appended claims, reference will be made to a number of terms that shall be defined to have the following meanings, unless a contrary intention is apparent.

[0016] Each of the applications and patents cited herein, and each document or reference cited in each of said applications and patents (including each issued patent pending; the "documents cited in the applications"), and each of the PCT and foreign applications or patents that correspond to and / or claim priority from any of these applications and patents, and each of the documents cited or referenced in each of the documents cited in the applications, are hereby expressly incorporated herein by reference. Additionally, documents or references cited in the text, in the list of references preceding the claims, or in the text itself (e.g., U.S. Patent Application Publication Nos. 20150230762, 20150297760, and 20190054198); as well as each of these documents or references ("references cited herein"), and each document or reference cited in each of the references cited herein (including any manufacturer details, instructions, etc.), are hereby expressly incorporated by reference herein.

[0017] According to the present disclosure, the "detectable effective amount" of the embodiments of the present disclosure is defined as an amount sufficient to obtain acceptable images using equipment available for clinical use. The detectable effective amount of the embodiments of the present disclosure may be given in one or multiple doses. The detectable effective amount of the embodiments of the present disclosure may vary according to factors such as the degree of sensitivity of the individual, the age of the individual, the sex of the individual, and the weight of the individual, the idiosyncratic response of the individual, dosimetry, etc. The detectable effective amount of the embodiments of the present disclosure may also vary according to equipment-related factors and film-related factors. Optimization of such factors may be within the level of one skilled in the art.

[0018] The term "detectable" refers to the ability to detect the signal or presence of the embodiment of the present disclosure above background signals. The term "detectable signal" or phrase "detection of labeled compound" or "detectable labeled compound" refers to the detection (direct or indirect) of the labeled compound in a host or sample. Detection of the labeled compound refers to the ability to detect the presence of the labeled compound in a host or sample and distinguish its presence from other background signals originating from the host or sample. In other words, there is a measurable and statistically significant difference between the detectable signal and its background (e.g., a statistically significant difference is a difference sufficient to distinguish between the detectable signal and the background (e.g., a difference of about 0.1%, 1%, 3%, 5%, 10%, 15%, 20%, 25%, 30%, or 40% or more 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 background (e.g., in a method designed to evaluate adipose tissue activation). The detectable signal can be generated from low to high concentrations of the labeled compound. In some embodiments, the detectable signal may need to be the sum of each of the individual labeled compound signals. In some embodiments, the detectable signal can be generated from a sum, an integral, or other mathematical process, formula, or algorithm. In some embodiments, the sum, integral, or other mathematical process, formula, or algorithm can be used to process the detectable signal so that the detectable signal can be distinguished from background noise, etc.

[0019] As used herein, "drug", "active agent", and the like may include a compound of the present disclosure (e.g., a labeled compound). The drug may be disposed in a composition or pharmaceutical composition. As used herein, "pharmaceutical composition" refers to a combination of an active agent and a pharma- ceutical carrier. As used herein, "pharmaceutical composition" refers to a composition suitable for administration to a subject (e.g., a mammal, particularly a human). In general, a "pharmaceutical composition" is sterile and preferably free of contaminants capable of eliciting an undesirable response in a subject (e.g., the compounds in the pharmaceutical composition are pharmaceutical grade). The pharmaceutical composition may be designed for administration to a subject or patient in need thereof by many different routes of administration, including oral, intravenous, buccal, rectal, parenteral, intraperitoneal, intradermal, intracheal, intramuscular, subcutaneous, inhalation, and the like.

[0020] "Pharmaceutically acceptable excipient", "pharmaceutically acceptable diluent", "pharmaceutically acceptable carrier" or "pharmaceutically acceptable adjuvant" means an excipient, diluent, carrier, and / or adjuvant useful in preparing pharmaceutical compositions that are generally safe, non-toxic, and not biologically or otherwise undesirable, and includes excipients, diluents, carriers, and adjuvants acceptable for veterinary and / or human pharmaceutical use. For compositions suitable for human administration, the term "excipient" is meant to include, but is not limited to, ingredients set forth in Remington: The Science and Practice of Pharmacy, Lippincott Williams & Wilkins, 21st Edition (2006), the contents of which are incorporated herein by reference.

[0021] The term "unit dosage form" as used herein refers to physically discrete units suitable as unitary dosages for human and / or animal subjects, each unit containing a predetermined amount of compound calculated (e.g., the body weight of the host, the disease, the severity of the disease, etc.) in an amount sufficient to produce the desired effect in association with a pharma- ceutically 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.

[0022] As used herein, the term "effective amount" refers to an amount of an embodiment of the present disclosure (which may be referred to as a labeled compound) administered that can be used to image cells (e.g., fat cells). "Administering" refers to introducing an embodiment of the present disclosure into a subject. Administration can include, but is not limited to, intravenous, oral, topical, subcutaneous, intraperitoneal, intra-arterial, inhalation, intravaginal, rectal, nasal, introduction into the cerebrospinal fluid, or instillation into a body compartment, and these routes can be used.

[0023] 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. A typical host to which the embodiments of the present disclosure can be administered is a mammal, particularly a primate, especially a human. For veterinary applications, a wide variety of subjects are suitable, such as livestock (e.g., cows, 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 or research applications, a wide variety of mammals are suitable subjects, such as rodents (e.g., mice, rats, hamsters), rabbits, primates, and pigs (e.g., inbred pigs). Furthermore, for in vitro applications (e.g., in vitro diagnostic and in vitro research applications), bodily fluid and cell samples from the subject are suitable for use as "samples", such as blood, urine or tissue samples from mammals (particularly primates, e.g., humans), or from animals referred to in veterinary applications.

[0024] 18 F-AraG (also known as VisAcT) was developed as a PET tracer intended to image activated T cells in vivo. 18 F]F-AraG is a nucleotide analog of arabinofuranosylguanine (AraG), a compound that has been shown to selectively accumulate in T cells. 18 It is an F-labeled analog. Nelarabine, a prodrug of AraG, is approved by the US Food and Drug Administration (FDA) for the treatment of T-cell acute lymphoblastic leukemia and T-cell lymphoblastic lymphoma. 18[F]F-AraG enters T cells via nucleotide transporters and is trapped intracellularly primarily through rate-limiting phosphorylation by deoxyguanosine kinase (dGK), an enzyme found exclusively in mitochondria and critical in providing nucleotides for mitochondrial DNA synthesis. Phosphorylation by dGK leads to trapping and potential downstream accumulation in mtDNA, enabling visualization by PET imaging (Figure 1). 18[F]F-AraG has already shown great promise in assessing T cell involvement in graft-versus-host disease (GVHD), rheumatoid arthritis, cancer and multiple sclerosis (e.g., Ronald, JA 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(11):p.2893-2902; Franc, BL et al., In Vivo PET Imaging of the Activated Immune Environment in a Small Animal Model of Inflammatory Arthritis., Mol Imaging, 2017, 16:p.1536012117712638; Levi, J. et al., Imaging of Activated T Cells as an Early Predictor of Immune Response to Anti-PD-1 Therapy., Cancer Research, 2019, 79(13):p.3455~3465; Levi, J. et al., (18)F-AraG PET for CD8 Profiling of Tumors and Assessment of Immunomodulation by Chemotherapy., J Nucl Med, 2021, 62(6):p.802-807; and Guglielmetti, C. et al., Longitudinal imaging of T-cells and inflammatory demyelination in a preclinical model of multiple sclerosis using (18)F-FAraG PET and MRI., J Nucl Med, 2021).

[0025] In assessing intracranial T cell infiltration in glioblastoma multiforme (GBM) 18During our research into the utility of F-AraG, we found that 18 An unexpected and surprising discovery was that F-AraG can also image and / or evaluate adipose tissue activation (Figure 2). Adipose (or fat) tissue (AT) was once thought to be an inactive tissue that exists primarily to store lipids and was not historically recognized as a critical organ in regulating and maintaining health. With the rise of obesity and more rigorous study, AT is now recognized as a highly complex metabolic organ involved in a number of important physiological functions, including glucose homeostasis and many endocrine capacities. AT dysfunction has been implicated in several disease states, most notably obesity, metabolic syndrome, and type 2 diabetes. Study of AT may provide useful insights in developing strategies to combat these highly prevalent metabolic diseases.

[0026] Adipocytes are highly active endocrine cells that play a central role in global energy homeostasis and are important contributors to some aspects of the immune system. They do so not only by influencing systemic lipid homeostasis, but also through the production and release of numerous adipocyte-specific and adipocyte-enriched hormonal factors, cytokines, and extracellular matrix components (commonly referred to as "adipokines"). Little attention has been paid to the role of adipose tissue in infectious diseases. However, the strong proinflammatory capacity of adipose tissue provides evidence for a key role in the systemic innate immune response. In addition, adipocytes act as an important target for the intracellular parasite Trypanosoma cruzi (the cause of Chagas' disease). In chronic Chagas' disease, adipocytes may represent an important long-term reservoir of the parasite from which recurrences of infection may occur. In other cases (e.g., by certain subtypes of adenovirus), infection with viral particles leads to long-term hyperplasia and hyperproliferation of adipocytes, which associates these adenovirus infections with a high subsequent tendency to become obese. In this context, the study of AT may also provide useful insights in developing strategies to combat these diseases. Moreover, obesity dramatically modifies the adipose tissue microenvironment in many ways, including induction of fibrosis and angiogenesis, increased stem cell mass, and proliferation of pro-inflammatory immune cells. Many of these changes also resemble the shifts observed in the tumor microenvironment, so proximity to adipose tissue may present a hospitable environment for tumor development, providing an important link between adiposity and tumor development.

[0027]

[0013] Embodiments of the present invention include, for example, methods of imaging adipose tissue in a subject. Typically, these methods include imaging adipose tissue comprising a compound of the formula: [ka] The method includes administering a compound having the formula: to a subject, the route of administration being selected to allow the compound to be phosphorylated by deoxycytosine kinase and deoxyguanosine kinase present in adipose tissue in the subject; and then imaging the subject, wherein detecting the presence of the compound corresponds to the presence of adipose tissue. In some embodiments of the present invention, the adipose tissue is brown adipose tissue. In other embodiments of the present invention, the adipose tissue is bone marrow adipose tissue (e.g., controlled bone marrow adipose tissue).

[0028] In a particular embodiment of the invention, the method focuses on imaging activated adipose tissue. In this context, a wide variety of aspects of adipose tissue activation are well known in the art, for example, Fenzi et al., Horm Mol Biol Clin Investig., June 2014;19(1):25-37; van der Vaart et al., Cells., May 6, 2021;10(5):1122; Lim et al., Nat Protoc., March 1, 2012;7(3):606-15; Milton-Laskibar et al., J Physiol Biochem., May 2020;76(2):269-278; Bani Hassan et al., Curr Osteoporos Rep., December 2019;17(6):416-428; Tencerova et al., Best Pract Res Clin Endocrinol Metab., 2021 Jul;35(4); and in U.S. Patent Publication Nos. 20140088487, 20140199278, 20140324130, and 20180202000. In certain embodiments of the invention, the method comprises simultaneously imaging adipose tissue and T cells in a subject, and / or brain infiltration of T cells in a subject; and / or activation of brown adipose tissue and activation of bone marrow adipose tissue (e.g., controlled bone marrow adipose tissue) in a subject.

[0029] In certain embodiments of the invention, these methods include observing the level of activation in adipose tissue associated with a pathology, for example, in a condition in which the subject is a subject diagnosed with a pathological condition (e.g., an infectious disease (e.g., COVID-19 infection), a neuroinflammatory condition, a condition indicating intracerebral lymphocyte activation or infiltration, obesity, diabetes, cachexia, or cancer (e.g., glioblastoma)). In some embodiments of the invention, the subject is selected to be a patient who has been administered a therapeutic agent and / or undergone a therapeutic intervention, and the obtained image or images of adipose tissue are used to obtain information regarding the effect of the therapeutic agent or intervention.

[0030] In certain embodiments of the present invention, the PET method of imaging adipocytes in a subject responding to administration of a therapeutic agent further comprises observing one or more images obtained on a first day; observing one or more images on a second day; and then comparing the images obtained on the first imaging day to the images obtained on the second imaging day to observe the changes in patient physiology resulting from administration of the therapeutic agent over time (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 day to the second day comprises less than one week. Alternatively, the amount of time from the first day to the second day comprises at least one week, two weeks, or three weeks, or at least one month, two months, or three months.

[0031] [ 18 In one exemplary working embodiment designed to show that [F]F-AraG specifically images brown fat activation, we treated mice with BRL37344 and insulin and 18Mice were imaged using both [F]F-AraG and FDG. BRL37344 is a b3 adrenergic receptor agonist known to activate BAT (see, e.g., Peng, XR, et al., Unlock the Thermogenic Potential of Adipose Tissue: Pharmacological Modulation and Implications for Treatment of Diabetes and Obesity., Front Endocrinol (Lausanne), 2015, 6:174), whereas insulin only affects glucose metabolism and not BAT activation.

[0032] The results shown in Figure 3 demonstrate the efficacy of imaging activated BAT. 18 The results clearly demonstrate the specificity of [F]F-AraG and its advantages over FDG. 18 [F]F-AraG showed activation of BAT only with the BAT activator BRL37344, whereas FDG showed increased signal in BAT of mice treated with insulin, which only affects glucose metabolism, and furthermore, FDG also showed signal in BAT in control animals treated with vehicle alone.

[0033] Moreover, and quite unexpectedly but very importantly, chronic administration of BRL37344 (3 days + 1 hour prior to imaging) not only increased BAT in the classical intrascapular region, but also in the lumbar spinal cord and proximal tibia. 18 F]F-AraG signal (Fig. 4 ).

[0034] This finding is of utmost importance because it is in these regions (lumbar spine and proximal tibia) that a specialized type of marrow adipose tissue (MAT) has been found that has properties similar to BAT (e.g., response to cold conditions) (see, e.g., Scheller, EL et al., Region-specific variation in the properties of skeletal adipocytes reveals regulated and constitutive marrow adipose tissues., Nat Commun, 2015, 6:7808).

[0035] Overall, [ 18 The ability of [F]F-AraG to specifically image activation of BAT (and BAT-like MAT) may have a wide variety of applications in both basic science and commerce. For example, embodiments of the invention may be used for drug development of agents that activate or inactivate BAT, and / or to follow treatment regimens for diabetes, obesity, or cachexia. Importantly, [F]F-AraG may be used to specifically image activation of BAT (and BAT-like MAT) activation. 18 [F]F-AraG may serve as an agent that can assess the involvement of activated BAT in GBM and potentially provide a novel therapeutic approach for GBM.

[0036] The embodiment of the present disclosure is 18 [F]-F-arabinofuranosylguanine compounds (compounds that can be formed by a number of processes, such as those disclosed in U.S. Patent Application Serial No. 17 / 314,366, filed May 7, 2021, entitled "METHODS AND MATERIALS FOR MAKING PET RADIOTRACERS," the contents of which are incorporated herein by reference). Such embodiments of the invention include methods of using compounds having the general formula: [ka] A composition comprising a compound having the formula 18F]-F-arabinofuranosylguanine compounds, in which general formula PG comprises a protecting group; LG comprises a leaving group. Typically, in such an embodiment of the invention, the nitrogen atom bound to a protecting group is bound to two protecting groups, as indicated by "N(PG)2". Alternatively, the nitrogen atom is bound to a hydrogen atom and one protecting group, as indicated by "NHPG". In an exemplary embodiment of the invention, the composition comprises Precursor 1 or Precursor 3: [ka] wherein: t Bu comprises a tert-butyloxycarbonyl alcohol protecting group; Boc comprises a tert-butyloxycarbonylamine protecting group; THP comprises a tetrahydropyranyl alcohol protecting group; EOE comprises an ethoxyethyl alcohol protecting group; and Tf comprises a triflate leaving group. Embodiments of the present disclosure may be used to prepare compounds that are capable of forming amines or amines that are capable of forming ... 18 The present invention also includes methods of using the [F]-F-arabinofuranosylguanine compounds.

[0037] An exemplary embodiment of a method for imaging adipocytes includes, among other steps, administering a compound of the present disclosure to a subject; and imaging the subject, where detecting the presence of the compound corresponds to the presence of adipocytes. Another exemplary embodiment of a method for imaging the presence or extent of fat metabolic activation includes, among other steps, administering a compound of the present disclosure to a subject; and imaging the subject, where detecting the presence of the compound corresponds to the presence or extent of adipocyte activation in the subject.

[0038] The embodiments of the present disclosure also include a method of imaging adipose tissue and adipocytes. In general, the embodiments of the labeled compound can be used to image the localization and / or amount of adipocytes in a subject (e.g., a living human). The labeled compound can be administered to the subject, and then the subject or a portion of the subject can be imaged using a device such as positron emission tomography (PET) to detect the presence and location of the labeled compound present in 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 adipocytes, cancer cells, and / or white blood cells in the subject.

[0039] The present disclosure may also provide packaged compositions that include precursor compounds or intermediates to the labeled compounds and instructions on how to make and use the labeled compounds (e.g., instructions describing their use). The kits may further include suitable buffers and reagents known in the art for administering embodiments of the present disclosure to a subject.

[0040] It should be noted that ratios, concentrations, amounts, and other numerical data may be expressed in range format herein. Such range formats are used for convenience and brevity, and therefore such range formats should be understood to be interpreted in a flexible manner to include not only the numerical values ​​expressly recited as the limits of the range, but also all individual numerical values ​​or subranges contained within the range as if each numerical value and subrange were expressly recited. For 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 wt% to about 5 wt%, but also to include individual concentrations (e.g., 1%, 2%, 3%, and 4%) and subranges (e.g., 0.5%, 1.1%, 2.2%, 3.3%, and 4.4%) within the recited range. In some embodiments, the term "about" may include conventional rounding according to significant figures of the numerical value. Furthermore, the phrase "about 'x' to 'y'" includes "about 'x' to about 'y'".

[0041] It should be emphasized that the above-described embodiments of the present disclosure are merely examples of possible implementations and are set forth solely for a clear understanding of the principles of the present disclosure. Many changes 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 changes and modifications are intended to be included herein within the scope of the present disclosure. EXAMPLES

[0042] Example 1: [ 18 [F]F-AraG accumulation is specific to adrenergic stimulation In mice, the regulation of BAT by SNS is driven primarily by β3-adrenergic receptor signaling (Cannon, B. and J. Nedergaard, Brown adipose tissue: function and physiological significance., Physiol Rev, 2004, 84(1):p.277-359; Arch, JR et al., Atypical beta-adrenoceptor on brown adipocytes as target for anti-obesity drugs., Nature, 1984, 309(5964):p.163-5). 18 To examine the accumulation of [F]F-AraG, we treated mice with the well-studied b3-adrenergic receptor agonist BRL37344 (Cawthorne, MA, et al., BRL 35135, a potent and selective atypical beta-adrenoceptor agonist., Am J Clin Nutr, 1992, 55(Suppl 1): p. 252S-257S). Administration of BRL37344 1 hour prior to imaging significantly reduced [F]F-AraG accumulation in intrascapular BAT (iBAT). 18 In iBAT, control animals as well as animals treated with insulin had significantly lower [F]F-AraG accumulation (10.98 ± 1.64%ID / g). 18In comparison, FDG, the most commonly used tracer for imaging BAT activation, showed increased accumulation in iBAT not only with BRL37344 but also in insulin-treated animals. These results are in contrast to FDG, which shows adrenergic-independent uptake in iBAT (Hankir, M. K. and M. Klingenspor, Brown adipocyte glucose metabolism: a heated subject., EMBO Rep, 2018, 19(9)). 18 We provide evidence that [F]F-AraG accumulation represents a process that is specific to adrenergic stimulation.

[0043] Example 2: Long-term adrenergic stimulation increases the expression of 18 Leading to accumulation of [F]F-ARAG Surprisingly, chronic stimulation with BRL37344 not only increased the expression of β-lactamase in iBAT, but also in axillary, lumbar, and tibial and femoral bone marrow. 18 F]F-AraG accumulation.

[0044] Example 3: BAT activation is associated with GBM-induced neuroinflammation Tracking stimulated fat cells 18 The ability of [F]F-AraG allowed the observation of activated iBAT in mice bearing bioluminescently tagged GBM tumors. The activation occurred at different time points and varied in intensity. 18 In one mouse with the highest neuroinflammation evidenced by [F]F-AraG signal, the activation was most dramatic, with iBAT [ 18This resulted in an approximately 9-fold increase in [F]F-AraG signal (14.92%ID / g in OnTx3 vs. 1.70%ID / g in PreTx). In addition to a significant increase in signal in multiple brown fat depots, the mice also showed increased signal in the lumbar spine and proximal femur and tibia bone marrow. See Figure 5.

[0045] Example 4: BAT activation is associated with neuroinflammation in multiple sclerosis Because brain tumors display damage that may lead to stress responses and BAT activation, we further investigated the relationship between BAT activation and inflammation in a mouse model of multiple sclerosis (MS). 18 Assessment of the [F]F-AraG signal demonstrated that BAT activation occurs concomitantly with intracerebral T cell infiltration. Furthermore, treatment with fingolimod, a drug that reduces intracerebral lymphocyte infiltration, resulted in a significant reduction in iBAT signal (see Figure 6). Overall, these results provide evidence of an association between BAT activation and neuroinflammation.

[0046] Example 5: 18 [F]F-ARAG can detect activated brown fat in humans Since neuroinflammation, including T cell involvement, has been associated with the neurological problems experienced by some patients with acute COVID-19 and some patients with long-term COVID-19, we looked for evidence of activated BAT in patients who had recovered from COVID-19. In contrast to healthy volunteers who were not infected with the virus, both neuroinflammation and activated BAT were detected in a small number of patients who had recovered from COVID-19. As shown in Figure 7, [ 18 The location of the [F]F-AraG signal coincided with the cervical and supraclavicular regions, the most commonly observed sites of BAT depots in humans (Figures 7F-H). These results, similar to those observed in mice, suggest that [F]F-AraG signals are consistent with the most commonly observed sites of BAT depots in humans (Figures 7F-H). 18 We show that [F]F-AraG can simultaneously detect both neuroinflammation and BAT activation in humans.

[0047]

change

change

Claims

1. 1. A method of imaging adipose tissue in a subject, the method comprising: (a) Formula: 【Chemistry 5】 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 adipose tissue. A method comprising:

2. the adipose tissue is brown adipose tissue; the adipose tissue is bone marrow adipose tissue; and / or The adipose tissue is activated adipose tissue. The method of claim 1.

3. the method comprising observing the level of cellular activation in the adipose tissue, the level of cellular activation in the adipose tissue being observed by observing the amount of the compound in the adipose tissue. The method of claim 1 or claim 2, further comprising:

4. The method comprises: detecting T cells in the subject; T cell infiltration in the brain of the subject; and / or Brown adipose tissue activation and controlled bone marrow adipose tissue activation in said subject A process for simultaneously imaging The method of claim 1 further comprising:

5. The method of claim 1 , wherein the subject is a subject diagnosed with a pathological condition.

6. 6. The method of claim 5, wherein the pathological condition comprises an infectious disease, a neuroinflammatory condition, a condition exhibiting lymphocyte activation in the brain, obesity, diabetes, cachexia or cancer.

7. The method of claim 6, wherein the pathological condition is COVID-19 infection, glioblastoma, pancreatic cancer or multiple sclerosis.

8. The method of claim 5, wherein the subject is selected to be a patient who has been administered a therapeutic agent and / or undergone a therapeutic intervention, and one or more images of the adipose tissue obtained in step (b) are used to obtain information regarding the effect of the therapeutic agent or intervention.

9. The method includes the steps of: observing one or more images of the adipose tissue on a first day; observing one or more images of the adipose tissue on a second day; and comparing the images obtained on the first day with the images obtained on the second day to observe changes in the adipose tissue over time. The method of claim 8 further comprising:

10. 10. The method of claim 9, wherein the amount of time from the first date to the second date comprises at least one day, at least one week, or at least one month.

11. 1. A method for observing the effect of a test agent on adipose tissue cell activation, the method comprising: combining said adipose tissue cells with said test agent; Observing whether or not there is a change in activation of the adipose tissue cells in combination with the test agent; and comparing the activation of the adipose tissue cells in combination with the test agent to control adipose tissue cells not combined with the test agent, so that the effect of the test agent on the activation of the adipose tissue cells is observed. A method comprising:

12. The method of claim 12, wherein the adipose tissue cells are combined with the test agent in vitro.

13. The method of claim 12, wherein the adipose tissue cells are activated bone marrow adipose tissue cells.

14. The method of claim 12, wherein the adipose tissue cells are obtained from a subject diagnosed with a pathological condition.

15. 15. The method of claim 14, wherein the pathological condition is neuroinflammation, obesity, diabetes, cachexia or cancer.

Images