Labeled Compound Calcium-Sensing Receptor Ligands for Imaging and Methods of Their Use - Patent application

Abstract

The present invention relates to labeled compounds suitable for positron emission tomography (PET) imaging and / or fluorescence imaging, such as near-infrared fluorescence imaging (NIRF). The present invention further relates to methods of using these compounds for performing PET scans, imaging calcium sensing receptor (CSR) positive organs, ablating parathyroid tissue, protecting parathyroid tissue during thyroid surgery, and treating one or more disorders of CSR positive tissue in a subject.

Description

[Technical field]

[0001] Declaration of priority This application claims the benefit of U.S. Provisional Application Serial No. 63 / 220,737, filed July 12, 2021, the entire contents of which are incorporated herein by reference.

[0002] The present invention relates to labeled compounds suitable for positron emission tomography (PET) imaging and / or fluorescence imaging, such as near-infrared fluorescence (NIRF) imaging. The present invention further relates to methods of using these compounds for performing PET scans, imaging calcium sensing receptor (CSR) positive organs, ablating parathyroid tissue, protecting parathyroid tissue during thyroid surgery, and treating one or more disorders of CSR positive tissue in a subject.

[0003] References to U.S. government support This invention was made with government support under Grant No. DK128447 awarded by the National Institutes of Health. The United States Government has certain rights in this invention. [Background technology]

[0004] Primary hyperparathyroidism (PHPT) is a common disorder that causes significant morbidity, affecting more than 100,000 people annually in the United States. The parathyroid gland is a small endocrine gland that normally controls calcium levels in the circulation. PHPT is caused by an endogenous abnormality in the parathyroid gland, which secretes excessive parathyroid hormone (PTH), causing elevated serum calcium levels. Excess hormone also promotes calcium resorption from bone, which over time leads to loss of bone mineral density and may result in osteoporosis. In addition, elevated calcium is excreted by the kidneys, which may cause kidney damage (nephrocalcinosis) and lead to the formation of kidney stones. High circulating calcium also damages blood vessels and predisposes to arteriosclerotic disease. There are also many neuropsychiatric symptoms caused by this disease, such as those mentioned above, including fatigue, depression, and mental "fog."

[0005] Currently, the only treatment for PHPT is surgical removal of one or more affected parathyroid glands. Most people have four parathyroid glands, located adjacent to the larger thyroid gland. Under normal conditions, these parathyroid glands are very small (the size of a lentil, i.e., 3-5 mm) and are not easily visible on current imaging tests. The most common cause of PHPT is the development of a parathyroid adenoma (occurring in approximately 85% of cases), resulting in enlargement of one, two, or three affected glands. In 5-10% of patients with PHPT, all parathyroid glands are affected by hyperplasia. Multiple adenomas or parathyroid hyperplasia are collectively referred to as "multi-gland disease" because pathological analysis cannot reliably distinguish between multiple adenomas and hyperplasia. Accurate detection and identification of the parathyroid glands is important for the management of patients with PHPT. The parathyroid glands are usually located in the neck close to the thyroid gland, but in some cases, due to abnormal migration during embryonic development, they can be located anywhere from the base of the skull to the heart.

[0006] Traditionally, treatment of hyperparathyroidism involves preoperative localization of the parathyroid glands using imaging to direct the surgeon to the abnormal parathyroid gland. The most commonly used modalities are computerized tomography (CT), ultrasound, and the nuclear medicine sestamibi scan. Ultrasound has a sensitivity of about 75% to about 80% and a positive predictive value (PPV) of 90% to 95% when performed in high-volume, experienced centers. Ultrasound is limited by anatomical considerations, as it has a poor ability to detect posteriorly located adenomas and cannot detect ectopic glands. The 99mTc-sestamibi nuclear medicine study uses technetium-labeled sestamibi as a tracer. 99mTc-sestamibi accumulates initially in both the thyroid and parathyroid glands, but is more rapidly washed out of the thyroid gland than the parathyroid gland. Delayed images are obtained after the tracer has washed out of the thyroid gland. While earlier techniques used simple planar scintigraphic imaging, one current technique combines a single photon emission computed tomography scan with a computed tomography scan (SPECT-CT) to improve anatomical resolution. Sestamibi / SPECT-CT has a sensitivity of 80-86% and a positive predictive value of 90-95%. The use of 4D CT (dynamic CT) has also become more common in recent years. The use of 4D CT, or dynamic CT, has also become more common in recent years. Images with enhanced contrast of pre-, early-, and late-phase are obtained. Small lesions showing rapid washout in the thyroid gland area are then examined to identify parathyroid tissue.The overall sensitivity of 4D CT ranges from 62% to 88%, with a PPV ranging from 84% to 90%. Despite the availability of multiple imaging modalities, in approximately 20% of cases the abnormal glands cannot be identified with current techniques. In addition, current imaging studies are poor at detecting "multi-gland disease," and small adenomas, usually less than 1 cm in size, are also often not visualized. These weaknesses are particularly problematic in cases where the initial operation has failed. Reoperations to locate parathyroid glands in an unknown location are difficult and risky, and often unsuccessful. Parathyroid glands may also be found in ectopic locations, often outside the neck. In these cases, imaging is essential to prevent futile exploration of the neck and to guide resection of the malpositioned parathyroid glands.

[0007] Most surgeons rely on visual identification, which requires years of focused experience to do properly. Visual identification of the parathyroid glands is also important in thyroidectomy to prevent inadvertent removal of normal parathyroid glands, which can result in hypoparathyroidism, a highly morbid condition for affected individuals.

[0008] Accurate detection and identification of parathyroid glands is critical for the management of PHPT patients. The present invention overcomes the shortcomings in the art by providing suitable ligands, fluorophores, probes and compositions for the detection of parathyroid tissue and / or calcium-sensing receptor (CSR) positive tissue, and methods for their use during surgery, e.g., pre- and / or intraoperatively to guide exploration during surgery or to preserve normal glands. Summary of the Invention [Means for solving the problem]

[0009] One aspect of the present invention provides a radioisotope-labeled calcium sensing receptor (CSR) ligand comprising an aromatic ring, wherein the radioisotope is directly attached to the aromatic ring at one or more positions of the ring.

[0010] Another aspect of the present invention is 18 A radiolabeled CSR ligand is provided that comprises the formula XX (etelcalcetide hydrochloride) directly attached to F.

[0011] Another aspect of the invention provides labeled CSR ligands suitable for use as positron emission tomography (PET) probes, fluorescence imaging (e.g., NIRF) probes and / or optical probes that comprise a CSR binding moiety, hi some embodiments, the ligand may be an antibody or an antigen-binding fragment thereof.

[0012] Another aspect of the invention provides halogenated fluorophores that contain radioisotopes that can be preferentially taken up in thyroid and / or parathyroid tissue.

[0013] Also provided are PET probes, fluorescent imaging probes (eg, NIRF probes), compositions, and pharmaceutical compositions comprising the ligands and / or fluorophores of the invention.

[0014] Additionally, there is provided a probe and / or composition of the invention for use in imaging, diagnosing, and / or directing treatment of a parathyroid disorder (e.g., primary hyperparathyroidism, secondary hyperparathyroidism, tertiary hyperparathyroidism), a thyroid disorder (e.g., thyroid cancer, goiter, thyroid nodules, Graves' disease), a cardiac disorder (e.g., hypertension), a renal disorder (e.g., nephrocalcinosis, rickets, proteinuria), a reproductive disorder (e.g., infertility, embryonic or fetal development disorder), a lactation disorder (e.g., reduced milk production), a gastrointestinal disorder (e.g., pancreatitis, diabetes, diarrhea, gastrointestinal endocrine disorders), a bone disorder (e.g., osteoporosis), a cancer (e.g., colon cancer), a neurological disorder (e.g., Alzheimer's disease, epilepsy), and / or a pulmonary disorder (e.g., pulmonary hypoplasia, pulmonary hyperplasia).

[0015] A further aspect of the invention provides a method of performing a PET scan on a subject, comprising administering to said subject a ligand, fluorophore, probe and / or composition of the invention.

[0016] Another aspect of the invention provides a method of imaging tissue containing CSR in a subject comprising administering to said subject a ligand, fluorophore, probe and / or composition of the invention.

[0017] Another aspect of the invention provides a method of imaging thyroid and / or parathyroid tissue in a subject comprising administering to said subject a ligand, fluorophore, probe and / or composition of the invention.

[0018] Another aspect of the invention provides a method of simultaneously performing a PET scan and fluorescence imaging (e.g. NIRF imaging) on ​​a subject, comprising administering to the subject a ligand, fluorophore, probe and / or composition of the invention.

[0019] Another aspect of the invention provides a method of identifying parathyroid tissue in a subject, the method comprising performing a PET scan and / or fluorescence imaging (e.g. NIRF imaging) on ​​the subject using a ligand, fluorophore, probe or composition of the invention, wherein the PET scan and / or fluorescence imaging identifies the presence of parathyroid tissue.

[0020] Another aspect of the invention provides a method of removing hyperplastic and / or ectopic parathyroid tissue in a subject, comprising: (a) performing a PET scan and / or fluorescence imaging (e.g., NIRF imaging) on ​​the subject using a ligand, fluorophore, probe and / or composition of the invention, where the PET scan and / or fluorescence imaging identifies the presence of parathyroid tissue; (b) identifying the presence of hyperplastic and / or ectopic parathyroid tissue; and (c) surgically resecting the identified hyperplastic and / or ectopic parathyroid tissue, thereby removing the hyperplastic and / or ectopic parathyroid tissue.

[0021] Another aspect of the invention provides a method of guiding surgery to remove parathyroid tissue in a subject, the method comprising performing a PET scan and / or fluorescence imaging (e.g., NIRF imaging) on ​​the subject using a ligand, fluorophore, probe and / or composition of the invention, wherein the PET scan and / or fluorescence imaging identifies the presence of parathyroid tissue.

[0022] Another aspect of the invention provides a method of guiding surgery to protect parathyroid tissue during thyroid and / or other neck surgery in a subject, the method comprising performing a PET scan and / or fluorescence imaging (e.g., NIRF imaging) on ​​the subject using a ligand, fluorophore, probe and / or composition of the invention, wherein the PET scan and / or fluorescence imaging identifies the presence of parathyroid tissue.

[0023] Another aspect of the invention relates to a method of determining a target site for surgical removal of parathyroid tissue in a subject (e.g., a subject having hyperparathyroidism, or a subject at risk for or suspected of having or developing hyperparathyroidism), the method comprising: (a) performing a PET scan and / or fluorescence imaging (e.g., NIRF imaging) on ​​the subject using a ligand, fluorophore, probe and / or composition of the invention, wherein the PET scan and / or fluorescence imaging identifies the presence of parathyroid tissue; and (b) identifying one or more regions of the subject containing the presence of ectopic and / or hyperplastic parathyroid tissue, wherein the presence of ectopic and / or hyperplastic parathyroid tissue in the one or more regions indicates the one or more regions of the subject as a target site for surgical removal of parathyroid tissue.

[0024] Another aspect of the invention provides a method of treating hyperparathyroidism (e.g., primary hyperparathyroidism, secondary hyperparathyroidism, and / or tertiary hyperthyroidism) in a subject, the method comprising determining the suitability of a subject for surgical removal of parathyroid tissue by performing a PET scan and / or fluorescence imaging (e.g., NIRF imaging) on ​​a subject having hyperparathyroidism, or a subject at risk for or suspected of having or developing hyperparathyroidism, using a ligand, fluorophore, probe or composition of the invention, where the PET scan and / or fluorescence imaging identifies the presence of parathyroid tissue; and treating the hyperparathyroidism based on the results of the PET scan and / or fluorescence imaging.

[0025] Another aspect of the invention provides a method of treating a CSR-positive tissue disorder in a subject, the method comprising: determining the suitability of a subject for such treatment by performing a PET scan and / or fluorescence imaging (e.g., NIRF imaging) on ​​a subject having hyperparathyroidism, or a subject at risk for or suspected of having or developing hyperparathyroidism, using a ligand, fluorophore, probe or composition of the invention, wherein the PET scan and / or fluorescence imaging identifies the presence of CSR-positive tissue; and treating the disorder based on the results of the PET scan and / or fluorescence imaging.

[0026] In some embodiments, the subject may be a pre-operative subject. In some embodiments, the subject may be an intra-operative subject (e.g., the subject is undergoing surgery (e.g., exploratory surgery)).

[0027] These and other aspects of the invention are described in greater detail in the following description of the invention. [Brief description of the drawings]

[0028] [Figure 1] FIG. 1 shows a table and histological images of patient tissue staining for hyperparathyroidism and CSR expression in the parathyroid gland (n=10 for each type of patient specimen). [Diagram 2] Figure 2 shows two schematic diagrams of representative routes for producing synthetic 18F-cinacalcet. Route A (top) shows a representative route for synthesizing 18F-cinacalcet through direct CH radiofluorination. Route B (bottom) shows two synthetic routes (path A and path B) for labeling [18F]-CF3-cinacalcet with CF3 groups. [Diagram 3]Figure 3 shows a schematic of the photoredox radiofluorination that could efficiently introduce 18F into boc-protected cinacalcet (Panel A of Figure 3), which correlates well with standards prepared through the route (Panel B of Figure 3). Panel C of Figure 3 shows a quality control graph of 18F-labeled boc cinacalcet. Unreacted boc cinacalcet could be well separated from the 18F-labeled product. [Figure 4A] Figure 4A shows a graph demonstrating the in vitro stability of 18F-cinacalcet. [Figure 4B] FIG. 4B shows a graph of the in vivo stability of 18F-cinacalcet in non-primates. [Diagram 5] FIG. 5 shows (Panel A of FIG. 5) images of a small animal PET / CT scan demonstrating accumulation of 18F-cinacalcet in the parathyroid region; and (Panel B of FIG. 5) autoradiography and pathological staining of an adjacent slide demonstrating that 18F-cinacalcet is localized to the CSR-positive parathyroid glands instead of the thyroid gland. [Figure 6] FIG. 6 shows two schematic diagrams, where the upper box "A" shows a representative synthetic route for the precursor and standard preparation for T700, and the lower box "B" shows the structures of the T800 dye and several related precursors. [Figure 7] 7 shows a representative route for synthesizing [18F]T700 dye via CH radiofluorination (route "A") and a representative route for synthesizing [18F]T800 dye via deoxy radiofluorination (route "B"). T700 can also be synthesized via deoxy radiofluorination; T800 can also be produced via CH fluorination. [Figure 8]Figure 8 shows (Panel A of Figure 8) images of fluorescence imaging of T700 and T800 dual channel imaging; (Panel B of Figure 8) dual channel imaging showing that T700 was localized in the thyroid gland and T800 was localized in the parathyroid gland; and (Panel C of Figure 8) fluorescence images showing that autofluorescence from mice 1-3 was negligible compared to mouse 3 (injected with T800) imaged in the T800 channel. Mouse 2 was injected with T700 and mouse 1 was injected with saline. [Figure 9] Figure 9 shows 18F-T800 (panel A of Figure 9) and 18F-T700 (panel B of Figure 9) synthesized using the photoredox labeling method. The products correlate well with the cold standard. [Figure 10] Figure 10 shows images of an implanted PHPT model that could be visualized by the T800 (left panel). Image guided surgery showed high contrast between the PHPT and nearby tissue (right panel). [Figure 11] FIG. 11 shows PET images of 18F-cinacalcet PET of the native parathyroid gland of a Rhesus Macaque monkey. [Figure 12] FIG. 12 shows (Panel A of FIG. 12) a schematic diagram of a conventional PET / NIRF probe design (Panel B of FIG. 12) and a 2-in-1 radioactive fluorescent dye PET / NIRF probe design. [Figure 13] FIG. 13 shows a schematic representative route for the chemical synthesis of [19F]F-ZW-cinacalcet and precursors for the photoredox reaction (Panel A of FIG. 13) and the photoredox radiolabeling of Boc-cinacalcet via direct CH fluorination (Panel B of FIG. 13). [Figure 14]Figure 14 shows the results of CSR expression by Western blot in various cell lines (Panel A of Figure 14). The cellular uptake and specific blocking assay of [18F]F-ZW-cinacalcet are shown in Panel B of Figure 14. [Figure 15] Figure 15 shows representative coronal PET images (Figure 15, panel A) and quantitative analysis (Figure 15, panel B) in rats injected with [18F]F-cinacalcet at 0.5, 1, and 2 hours p.i. Arrows indicate the parathyroid glands. [Figure 16] Figure 16 shows representative coronal, sagittal, transverse PET / CT images (Panel A of Figure 16) and 3D volume-rendered PET / CT images (Panel B of Figure 16) in a rat injected with [18F]F-cinacalcet at 0.5 hours. Position lines and filled arrows indicate the parathyroid glands and open arrows indicate the trachea. [Figure 17] Figure 17 shows representative coronary PET images and quantitative analysis in rats injected with [18F]F-cinacalcet for 0 to 60 minutes. The arrow indicates the parathyroid gland. [Figure 18] Figure 18 shows representative transverse dynamic PET images (Figure 18 Panel A), merged PET / CT images (Figure 18 Panel B) and quantitative analysis (Figure 18 Panel C) in rats injected with [18F]F-ZW-cinacalcet from 0 to 60 min. The solid arrow indicates the heart and the open arrow indicates the lungs. [Figure 19] Figure 19 shows the anatomy and excised tissues of local laryngeal and tracheal tissues including thyroid and parathyroid glands (Panel A of Figure 19), matched autoradiography (Panel B of Figure 19) and IHC staining (Panel C of Figure 19) of CSR in rats injected with [18F]F-cinacalcet, and quantitative analysis (Panel D of Figure 19). Circles indicate thyroid including parathyroid glands, and arrows indicate parathyroid glands, **P<0.01. [Figure 20] Figure 20 shows IHC staining of CSR in paraffin embedded tissue sections. Circles indicate the thyroid gland including the parathyroid glands, and arrows indicate the parathyroid glands. [Figure 21] FIG. 21 shows autoradiography of combined IHC and HE staining of CSR in paraffin-embedded tissue sections. [Figure 22] FIG. 22 shows clinical PET / MRI imaging of the parathyroid glands in non-human primates, with arrows indicating the parathyroid glands. [Figure 23] Figure 23 shows HE staining of kidney, liver and heart of mice overdosed with [19F]F-cinacalcet at different time points. The basic structures of glomeruli, renal tubules, hepatic lobular veins and myocardial fibers are intact. Scale bar = 200 μm. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0029] The present invention will now be described hereinafter with reference to the accompanying drawings and examples in which embodiments of the present invention are shown. This description is not intended to be a detailed catalog of all the different ways in which the present invention may be implemented or all the features that may be added to the present invention. For example, features illustrated with respect to one embodiment may be incorporated into other embodiments, and features illustrated with respect to a particular embodiment may be omitted from that embodiment. Thus, the present invention contemplates that in some embodiments of the present invention, any feature or combination of features defined herein may be excluded or omitted. In addition, numerous modifications and additions to the various embodiments suggested herein will be apparent to those skilled in the art in light of this disclosure, without departing from the present invention. Thus, the following description is intended to illustrate some specific embodiments of the present invention, without exhaustively specifying all permutations, combinations and variations thereof.

[0030] 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 invention belongs. The terms used in the description of the present invention in this specification are for the purpose of describing particular embodiments only and are not intended to limit the present invention.

[0031] All publications, patent applications, patents, and other references cited herein are incorporated by reference in their entirety for the teachings relevant to the sentence and / or paragraph in which the reference is set forth.

[0032] Unless the context indicates otherwise, it is specifically intended that the various features of the present invention described herein can be used in any combination.Moreover, the present invention also contemplates that in some embodiments of the present invention, any feature or combination of features described herein can be eliminated or omitted.For example, when the present specification states that a composition comprises component A, component B and component C, it is specifically intended that any of A, B or C, or combinations thereof, can be omitted and removed.

[0033] As used in the detailed description of the invention and the appended claims, the singular forms "a," "an," and "the" are intended to include the plural forms unless the context clearly indicates otherwise. For example, "a" cell means a single cell or multiple cells.

[0034] Also, as used herein, "and / or" refers to and includes any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted as alternatives ("or").

[0035] As used herein, the term "about" when referring to a measurable value, such as an amount or concentration, is meant to include not only the stated value, but also ±10%, ±5%, ±1%, ±0.5%, or even ±0.1% variation of the stated value. For example, "about X" is meant to include X, where X is a measurable value, as well as ±10%, ±5%, ±1%, ±0.5%, or ±0.1% variation of X. Ranges of measurable values ​​provided herein may include any other ranges and / or individual values ​​within the range.

[0036] As used herein, phrases such as "between X and Y" and "between about X and Y" should be interpreted to include X and Y. As used herein, phrases such as "between about X and Y" mean "between about X and about Y", and phrases such as "about X to Y" mean "about X to about Y".

[0037] As used in this specification, the words "comprises" and "comprising" specify the presence of stated features, integers, steps, operations, elements, and / or components, but do not exclude the presence or addition of one or more other features, integers, steps, operations, elements, components and / or groups thereof.

[0038] As used herein, the transitional phrase "consisting essentially of" (and grammatical variations) applied to the compositions of the present invention means that the claims should be interpreted to include the specific materials or steps recited in the claims, and which do not substantially affect one or more of the basic and novel characteristics of the claimed invention. Thus, the word "consisting essentially of" as used in the claims of the present invention is not intended to be interpreted as equivalent to "comprise."

[0039] The term "substantially altered" as applied to a composition means that the therapeutic efficacy of the composition is increased or decreased by at least about 20% or more as compared to the efficacy of a composition consisting of the referenced components.

[0040] As used herein with respect to a property (e.g., structure, function, or other measurable characteristic) of a compound, the terms "substantially retain" and / or "substantially unchanged" refer to maintaining the property "substantially the same" as a comparison object (e.g., a control), where at least about 75%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% of the property (e.g., structure, function, and / or other measurable characteristic) is retained.

[0041] The terms "treat" or "treating" or "treatment" refer to any type of action that imparts a modulating effect, which can be, for example, a beneficial effect, to a subject suffering from a disorder, disease, or condition, including an improvement in the subject's condition (e.g., in one or more symptoms), a delay or reduction in the progression of a condition, and / or a change in clinical parameters, a disease, or condition, etc., as is well known in the art.

[0042] As used herein, the term "therapeutically effective amount" or "effective amount" refers to an amount of a composition, compound or agent of the invention that confers a modulating effect, which can be, for example, a beneficial effect, to a subject suffering from a disorder, disease or condition, including an improvement in the subject's condition (e.g., in one or more symptoms), a delay or alleviation in the progression of a condition, prevention or delay of the onset of a disorder, and / or a change in clinical parameters, disease or condition, etc., as is well known in the art. For example, a therapeutically effective amount or effective amount can refer to an amount of a composition, compound or agent that improves a condition in a subject by at least 5%, e.g., at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100%.

[0043] As used herein, the term "therapeutically effective amount", "effective amount" or "therapeutic amount" is an amount sufficient to provide some improvement or benefit to the subject. Alternatively, a "therapeutically effective amount", "effective amount" or "therapeutic amount" is an amount that will provide some relief, alleviation, reduction or stabilization in at least one clinical symptom in the subject. Those skilled in the art will appreciate that the therapeutic effect need not be complete or curative, so long as some benefit is provided to the subject. The effective amount may vary depending on the age and general condition of the subject, the severity of the condition being treated, the particular agent administered, the duration of treatment, the nature of any concurrent treatment, the pharmacologic carrier used, and similar factors within the knowledge and expertise of the skilled artisan. As appropriate, the effective amount or therapeutic amount in each individual case can be determined by the skilled artisan by reference to the relevant texts and literature and / or by using routine experimentation. (See, e.g., Remington, The Science and Practice of Pharmacy (2006) , 1999-2002). th ed. 2000).

[0044] As used herein, the term "pharmaceutical acceptable" refers to a material that is biologically or otherwise undesirable, i.e., a material that can be administered to an individual in conjunction with a composition of the present invention without causing substantial adverse biological effects or interacting in a deleterious manner with any of the other components of the composition in which it is contained. The material will, of course, be selected to minimize any degradation of the active ingredient and to minimize any side effects in the subject, as will be well known to those of skill in the art (see, e.g., Remington's Pharmaceutical Sciences; 21 st (See, e.g., U.S. Pat. No. 5,333,351, ed. 2005). Exemplary pharma- ceutically acceptable carriers for compositions of the invention include, but are not limited to, sterile pyrogen-free water and sterile pyrogen-free saline.

[0045] The terms "administering" or "administering" a composition of the invention to a subject encompass any route of introducing or delivering a compound to a subject to perform its intended function (e.g., for use in PET and / or fluorescent imaging, e.g., for guiding surgery).

[0046] A "subject" of the present invention may include any animal in need thereof. In some embodiments, the subject may be, for example, a mammal, reptile, bird, amphibian, or fish. Mammalian subjects include, but are not limited to, laboratory animals (e.g., rats, mice, guinea pigs, rabbits, primates, etc.), farm or commercial animals (e.g., cows, pigs, horses, goats, donkeys, sheep, etc.), or livestock (e.g., cats, dogs, ferrets, gerbils, hamsters, etc.). In some embodiments, the mammalian subject may be a primate, or a non-human primate (e.g., chimpanzee, baboon, macaque (e.g., rhesus monkey, cynomolgus monkey, stump-tailed monkey, pig-tailed monkey), monkey (e.g., squirrel monkey, owl monkey, etc.), marmoset, gorilla, etc. In some embodiments, the mammalian subject may be a human.

[0047] A "subject in need" of the methods of the invention can be any subject known or suspected to have a thyroid disorder, and / or a parathyroid disorder, and / or any CSR-expressing tissue disorder, and / or a disease for which imaging and / or surgery may provide a beneficial health effect, or a subject at increased risk of developing said disease.

[0048] A "sample," "biological sample," and / or "ex vivo sample" according to the present invention can be any biological material, e.g., biological fluids, extracts from cells, extracellular matrix isolated from cells, cells (in solution or bound to a solid support), tissues, tissue homogenates, etc., and are well known in the art.

[0049] The terms "amino acid sequence," "polypeptide," "peptide," and "protein" may be used interchangeably to refer to polymers of amino acids of any length. The terms "nucleic acid," "nucleic acid sequence," and "polynucleotide" may be used interchangeably to refer to polymers of nucleotides of any length. As used herein, the terms "nucleotide sequence," "polynucleotide," "nucleic acid sequence," "nucleic acid molecule," and "nucleic acid fragment" may refer to polymers of RNA, DNA, or RNA and DNA that are single- or double-stranded, and that optionally contain synthetic, non-natural and / or modified nucleotide bases.

[0050] As used herein, the term "binding moiety" refers to a portion (e.g., a fragment) of a molecule that binds to another molecule (e.g., a target). For example, as used herein, a "CSR binding moiety" refers to a portion of a molecule (e.g., a ligand, e.g., a fluorophore) that is capable of binding to CSR. The binding moiety may be, for example, isolated from a molecule or compound, synthetically produced de novo, and / or contained within a larger molecule (e.g., a ligand, fluorophore, antibody, etc.).

[0051] As used herein, the term "antigen" refers to a molecule capable of inducing the production of immunoglobulins (e.g., antibodies). Molecules capable of stimulating an antibody and / or immune response may be referred to as antigenic and / or immunogenic, and may be said to have antigenic / immunogenic capabilities. The binding site for an antigen in an antibody may be referred to as an antigen-binding moiety. An antigen-binding moiety may be, for example, isolated from an antibody, synthetically produced de novo, and / or contained within a larger molecule (e.g., an antibody or fragment thereof).

[0052] As used herein, the term "antibody" includes intact immunoglobulin molecules as well as active fragments of the immunoglobulin, such as Fab, F(ab')2 and Fc, that are capable of binding an epitopic determinant (i.e., antigenic determinant) of an antigen. Antibodies that bind to the polypeptides of the present invention are prepared using intact polypeptides and / or fragments that contain small peptides of interest as immunizing antigens. The polypeptides or fragments used to immunize animals can be derived from enzymatic cleavage, recombinant expression, isolation from biological materials, synthesis, etc., and can be conjugated to carrier proteins, if desired. Carriers that are chemically coupled to peptides and proteins for the production of antibodies include, but are not limited to, bovine serum albumin, thyroglobulin, and keyhole limpet hemocyanin. The coupled peptides or proteins are then used to immunize host animals (e.g., mice, rats, goats, sheep, humans, or rabbits). The polypeptide or peptide antigen can also be administered in conjunction with an immunostimulant, as described herein and otherwise known in the art.

[0053] As used herein, the term "antibody" or "antibodies" refers to all types of immunoglobulins, including IgG, IgM, IgA, IgD, and IgE. The antibody may be monoclonal or polyclonal, may be of any species of origin, including mouse, rat, rabbit, horse, goat, sheep, human, and / or may be a chimeric or humanized antibody. See, for example, Walker et al., Molec. Immunol. 26:403-11 (1989). The antibody may be a recombinant monoclonal antibody produced according to the methods disclosed in U.S. Pat. No. 4,474,893 or U.S. Pat. No. 4,816,567. The antibody may also be chemically constructed according to the methods disclosed in U.S. Pat. No. 4,676,980. The antibody may further be a single chain antibody (scFv) or a bispecific antibody.

[0054] Techniques for producing chimeric or humanized antibodies can be used by splicing mouse antibody genes into human antibody genes to obtain molecules with appropriate antigen specificity and biological activity (Morrison et al. 1984. Proc. Natl. Acad. Sci. 81:6851-6855; Neuberger et al. 1984. Nature 312:604-608; Takeda et al. 1985. Nature 314:452-454). Alternatively, techniques described for the production of single chain antibodies can be adapted to produce single chain antibodies specific for the polypeptides and / or fragments and / or epitopes of the invention using methods known in the art. Antibodies with related specificity but different idiotypic composition can be generated by chain shuffling from random combinatorial immunoglobulin libraries (Burton 1991. Proc. Natl. Acad. Sci. 88:11120-3).

[0055] The term "fragment," as used herein with respect to proteins, refers to a polypeptide that is shortened in length relative to a reference polypeptide and that comprises, consists essentially of, and / or is composed of a contiguous amino acid sequence that is identical or nearly identical (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical) to the corresponding portion of the reference polypeptide. Such a polypeptide fragment may, where appropriate, be contained within the larger polypeptide of which it is a component. In some embodiments, the polypeptide fragment comprises, consists essentially of, or consists of at least about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 125, about 150, about 175, about 200, about 225, about 250, about 300, about 350, about 400, about 450, about 500, or more contiguous amino acids. In some embodiments, the polypeptide fragment comprises, consists essentially of, or consists of less than about 2, less than about 3, less than about 4, less than about 5, less than about 6, less than about 7, less than about 8, less than about 9, less than about 10, less than about 11, less than about 12, less than about 13, less than about 14, less than about 15, less than about 20, less than about 25, less than about 30, less than about 35, less than about 40, less than about 45, less than about 50, less than about 55, less than about 60, less than about 65, less than about 70, less than about 75, less than about 80, less than about 85, less than about 90, less than about 95, less than about 100, less than about 125, less than about 150, less than about 175, less than about 200, less than about 225, less than about 250, less than about 300, less than about 350, less than about 400, less than about 450, less than about 500 or less contiguous amino acids.

[0056] The terms "functional fragment" or "active fragment" as used herein with respect to a protein refer to a polypeptide fragment that retains at least about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.5%, or more of at least one biological activity of the full-length polypeptide (e.g., an antigen-binding antibody).

[0057] As used herein, the term "modified" as applied to a polynucleotide or polypeptide sequence refers to a sequence that differs from a wild-type sequence by one or more deletions, additions, substitutions, or any combination thereof. Modified sequences are also referred to as "modified variant(s)."

[0058] As used herein, to "isolate" or "purify" a fragment (or grammatical equivalents) means that the fragment is at least partially separated from at least some of the other components in the starting material.

[0059] Non-limiting examples of antibodies or active antibody fragments include monoclonal antibodies or fragments thereof, chimeric antibodies or fragments thereof, CDR-grafted antibodies or fragments thereof, humanized antibodies or fragments thereof, Fc, Fab, Fab', F(ab')2, Fv, disulfide-linked Fv, single chain antibodies (scFv), single domain antibodies (dAbs), diabodies, multispecific antibodies (e.g., bispecific antibodies) or fragments thereof, anti-idiotypic antibodies or fragments thereof, bifunctional hybrid antibodies or fragments thereof, functionally active epitope-binding antibody fragments, affibodies, nanobodies, and any combination thereof.

[0060] Active antibody fragments included within the scope of the present invention include, for example, Fab, F(ab')2, Fc fragments, and corresponding fragments obtained from antibodies other than IgG. Such fragments can be produced by known techniques. For example, F(ab')2 fragments can be produced by pepsin digestion of the antibody molecule, and Fab fragments can be generated by reducing the disulfide bonds of F(ab')2 fragments. Alternatively, Fab expression libraries can be constructed to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity (Huse et al., (1989) Science 254:1275-1281).

[0061] Monoclonal antibodies can be produced in hybridoma cell lines according to the technique of Kohler and Milstein (Nature 265:495-97 (1975)). For example, a solution containing the appropriate antigen is injected into a mouse, and after a sufficient time, the mouse is sacrificed and spleen cells are obtained. The spleen cells are then immortalized by fusion with myeloma cells or with lymphoma cells, typically in the presence of polyethylene glycol, to produce hybridoma cells. The hybridoma cells are then grown in an appropriate medium and the supernatant is screened for monoclonal antibodies with the desired specificity. Monoclonal Fab fragments can be produced in bacterial cells, such as E. coli, by recombinant techniques known to those skilled in the art. See, for example, W. Huse, (1989) Science 246:1275-81.

[0062] Compounds and Compositions

[0063] Positron emission tomography (PET) is a powerful and rapidly developing technique that is used to quantitatively measure site-specific chemical reactions, their spatial distribution, and metabolic perturbations and biological processes in vivo with high precision and sensitivity.

[0064] Fluorescence imaging has proven to be a valuable tool to increase the sensitivity of intraoperative lesion detection (invasive surgery), despite its limited tissue penetration.

[0065] The present invention provides a novel PET probe and a highly sensitive and specific PET / NIRF imaging technique that can effectively detect the parathyroid gland preoperatively and also guide its subsequent localization during surgery. Without wishing to be bound by theory, this combined technique may significantly reduce surgery time and improve surgical yield.

[0066] One of the problems that the present invention aims to solve is determining the identity of the suspicious tissue and the location of the parathyroid gland (rather than distinguishing PHPT from normal parathyroid glands). In fact, normal parathyroid glands are very small (3-5 mm), which cannot be easily seen with current imaging tests. In PHPT, one or more parathyroid glands become enlarged with autonomy in parathyroid hormone secretion. Normal and normal hyperparathyroidism can be easily distinguished by their difference in size, but the difficult problem is identifying their location. Despite the availability of multiple imaging modalities, in about 20% of cases, the diagnosis is not possible with current techniques, e.g., ultrasound, 99m Current techniques mentioned above, including Tc-sestamibi or 4D CT, are unable to localize the abnormal thyroid gland. This can be a real problem, especially in cases of multigland disease and small adenomas, which may lead to reoperations and fruitless neck exploration. Here, we present a novel PET agent for parathyroid imaging, a CSR-specific radiopharmaceutical [ 18[F]F-ZW-cinacalcet has been developed. This imaging agent is highly sensitive for the parathyroid glands, as demonstrated in both rodents and non-human primates. This imaging approach can guide the surgeon in performing the procedure, especially when the parathyroid glands are not in their normal location. In other situations, a nodule (or nodules) may be seen near the thyroid gland on ultrasound, but its identity as a parathyroid adenoma, as opposed to a lymph node or a focus of ectopic thyroid tissue, may not be immediately apparent. 18 [F]F-ZW-cinacalcet may be useful in identifying the source of suspicious tissue, thereby avoiding unnecessary surgery. 18 The development of [F]F-ZW-cinacalcet is also a successful application of photoredox labeling in PET probe development.

[0067] CSR is a transmembrane G-protein coupled receptor that responds to circulating calcium concentrations. CSR is expressed primarily in the parathyroid gland and kidney, but a variety of tissues express the receptor (Brown, EM and MacLeod, RJ, 2001 Physiol. Rev. 81:239-297). Two calcimimetic drugs that bind to CSR are clinically available: cinacalcet (an orally administered small molecule compound) and etelcalcetide (an intravenously administered synthetic peptide). In addition, antibodies against CSR are also available.

[0068] The synthesis of the compounds of the invention can be carried out by methods known in the art and described herein. In some embodiments, the synthetic route involves radiofluorination (e.g., S NAr radiofluorination, metal-catalyzed radiofluorination, iodonium radiofluorination, laser-induced radiofluorination, arene CH radiolabeling), such as those described in Chen et al. 2019 Science 364(6446):1170-1174, which is incorporated herein by reference. In some embodiments, the synthetic route may include any of the routes described herein, such as those shown in Figures 2, 3, 6, and 7.

[0069] Thus, one aspect of the present invention relates to a radioisotope-labeled calcium sensing receptor (CSR) ligand, wherein the CSR ligand comprises an aromatic ring and the radioisotope is directly attached to the aromatic ring at one or more positions thereof.

[0070] The radioisotope may be any radioisotope label that does not substantially alter the biological activity (e.g., CSR binding) of the ligand. In some embodiments, the radioisotope is 18 F and / or 11 C. In some embodiments, 11 C is 11 C.N., 11 COOH and / or 11 It may also be CH3.

[0071] The radiolabeled CSR ligand of the present invention may contain any type of aromatic ring, including, but not limited to, any type of aromatic ring as described above, including heteroaromatic ring, arene ring, phenyl ring, etc. In some embodiments, the aromatic ring may be an arene ring. In some embodiments, the arene ring may be a naphthalene ring and / or a phenyl ring.

[0072] The radioisotopes of the radiolabeled CSR ligands of the present invention can be coupled to the nucleophilic aromatic S ligand via CH fluorination and / or via nucleophilic aromatic S ligands via CH fluorination. N The ligand may be directly attached to the aromatic ring of the ligand at any position of the ring through any process, including, but not limited to, via the Ar (addition-elimination) mechanism. In some embodiments, the ligand is directly attached to the arene ring at the 1-position, 2-position, 3-position, 4-position, 5-position, etc. 18 F and / or 11 In some embodiments, the ligand may include a C bonded directly to the naphthalene ring at the 1, 2, 3, 4, 5 or other position. 18 F and / or 11 In some embodiments, the ligand may include C attached directly to the naphthalene ring at the 2- and / or 4-position. 18 F and / or 11 In some embodiments, the ligand may include C attached directly to the naphthalene ring at the 2- and / or 4-position. 18 In some embodiments, the ligand may include F attached directly to the naphthalene ring at the 2- and / or 4-position. 11 It may also contain C.

[0073] In some embodiments, the radiolabeled CSR ligand of the invention may comprise Formula II below, where Ar is a substituted aryl and Y is an alkyl.

[0074] [ka]

[0075] Ar may be any aryl, including but not limited to the groups shown below, where X is O, S, NH, or CH2, and R is aryl, alkyl, halo, CF3, NO2, COOMe, OH, OMe, alkene, or alkyne.

[0076] [ka]

[0077] In some embodiments, the radiolabeled CSR ligand of the present invention may comprise Formula III:

[0078] [ka]

[0079] In some embodiments, the radiolabeled CSR ligand of the present invention may comprise Formulae Ia to Ih:

[0080] [ka]

[0081] In some embodiments, the radiolabeled CSR ligand of the invention may comprise Formula I:

[0082] [ka]

[0083] In some embodiments, the Ar group may be linked to the compound by an aliphatic chain linker or a chain that includes a heteroatom.In some embodiments, the linker may be an aliphatic chain.In some embodiments, the linker may further include additional atoms such as, but not limited to, oxygen atoms, sulfur atoms, and nitrogen atoms.

[0084] In some embodiments, the radiolabeled CSR ligand of the invention may comprise any one of Formula VIII, Formula IX, and / or Formula X below.

[0085] [ka]

[0086] In some embodiments, the ligands of the present invention are radiolabeled calcium sensing receptor (CSR) ligands, which may include the CSR ligands described above that contain an aromatic ring, where the ligand does not include Formula XXI below.

[0087] [ka]

[0088] In some embodiments, the radiolabeled CSR ligand of the present invention may include any radiolabeled derivative of the drug NPS-2143 (SB-262470A).Non-limiting examples of the radiolabeled CSR ligand of the present invention derived from the drug NPS-2143 include any one of the following formulas IV, V, VI, and / or VII (NPS-2143 derivatives):

[0089] [ka]

[0090] In some embodiments, the radiolabeled CSR ligand of the invention may comprise any radiolabeled derivative of the drug evocalcet (CAS number 870964-67-3). In some embodiments, the radiolabeled CSR ligand of the invention may comprise the following formula XI, in which the radioisotope may be directly attached to the naphthalene at the 4-position.

[0091] [ka]

[0092] In some embodiments, the radiolabeled CSR ligand of the invention can comprise any one of Formula XII, Formula XIII and / or Formula XIV below.

[0093] [ka]

[0094] In some embodiments, the radiolabeled CSR ligand of the present invention may comprise any radiolabeled derivative of the drug SB-423562 (CAS No. 351490-27-2; Formula XV).

[0095] [ka]

[0096] Non-limiting examples of radiolabeled CSR ligands of the present invention derived from the drug SB-423562 include any one of Formulas XVI, XVII, XVIII and XIX below.

[0097] [ka]

[0098] In some embodiments, the radiolabeled CSR ligand of the invention can include any radiolabeled derivative of the drug etelcalcetide hydrochloride (CAS No. 1334237-71-6; Formula XX). In some embodiments, the radiolabeled CSR ligand of the invention can include any radiolabeled derivative of the drug etelcalcetide hydrochloride (CAS No. 1334237-71-6; Formula XX). 18 It may include the following formula XX (etelcalcetide hydrochloride) directly bonded to F:

[0099] [ka]

[0100] The structures provided herein of labeled cinacalcet, NPS-2143, SB-423562, evocalcet, etelcalcetide hydrochloride, and derivatives thereof are examples of ligands of the invention, but are not intended to be limiting. Other radioisotope labels and other positions of direct attachment of the labels are also contemplated. For example, in some embodiments, etelcalcetide hydrochloride can also be coupled to the ligand, e.g., via chelation, as illustrated in FIG. 12: 64 Cu, 68 Ga and / or 89 It may also contain a Zr label.

[0101] Another aspect of the invention provides labeled calcium sensing receptor (CSR) ligands suitable for use as positron emission tomography (PET) probes, fluorescence imaging (e.g., NIRF) probes and / or optical probes, comprising a labeled calcium sensing receptor (CSR) binding moiety.

[0102] CSR ligands suitable for use as PET, fluorescent, and / or optical probes can be any type of ligand that selectively binds to CSR (eg, contains a CSR binding moiety).

[0103] The label of the labeled CSR ligand of the present invention can be any label that does not substantially alter the biological activity (e.g., CSR binding) of the ligand. In some embodiments, the label can be a fluorescent dye (e.g., a near-infrared (NIR) dye or a NIR-II dye). In some embodiments, the label can be a radioisotope. For example, in some embodiments, the label can be 18 F, 11 C. 68 Ga, 89 Zr, 64 Cu, 87 Y, 124 I, 44 In some embodiments, the label may be, but is not limited to, Sc, etc., or any combination thereof.18 F and / or 11 C. In some embodiments, 11 C is 11 C.N., 11 COOH, and / or 11 It may also be CH3.

[0104] In some embodiments, the ligand may be an antibody or an antigen-binding fragment thereof. For example, in some embodiments, the antibody or antibody fragment may be, but is not limited to, a monoclonal antibody or fragment thereof, a chimeric antibody or fragment thereof, a CDR-grafted antibody or fragment thereof, a humanized antibody or fragment thereof, an Fc, a Fab, a Fab', a F(ab')2, an Fv, a disulfide-linked Fv, a single-chain antibody (scFv), a single-domain antibody (dAb), a diabody, a multispecific antibody (e.g., a bispecific antibody) or fragment thereof, an anti-idiotypic antibody or fragment thereof, a bifunctional hybrid antibody antibody or fragment thereof, a functionally active epitope-binding antibody fragment, an affibody, a nanobody, and any combination thereof.

[0105] In some embodiments, the antibody may be a known antibody having antigen specificity for CSR (e.g., an anti-CSR antibody, also called an anti-CaSR antibody). In some embodiments, the antibody may be, but is not limited to, monoclonal anti-CSR antibody clones 5C10, ADD, 3F12, 611825, EPR24050-59, 6D4, and / or HL1499. In some embodiments, the antibody may be generated de novo.

[0106] Another aspect of the invention provides halogenated fluorophores that contain radioisotopes that are capable of preferential uptake in thyroid and / or parathyroid tissue.

[0107] The radioisotope may be any radioisotope label that does not substantially alter the biological activity of the ligand (e.g., thyroid and / or parathyroid uptake). In some embodiments, the radioisotope is 18 F and / or 11 C. In some embodiments, the radioisotope is 18 It's F.

[0108] In some embodiments, the halogenated fluorophore has the formula XXII: 18 F-T700).

[0109] [ka]

[0110] In some embodiments, the halogenated fluorophore has the formula XXIII: 18 F-T800).

[0111] [ka]

[0112] In some embodiments, the labeled ligands and / or fluorophores of the invention may have a serum stability of at least 70% or more (e.g., at least 70% or more, at least 71% or more, at least 72% or more, at least 73% or more, at least 74% or more, at least 75% or more, at least 76% or more, at least 77% or more, at least 78% or more, at least 79% or more, at least 80% or more, at least 81% or more, at least 82% or more, at least 83% or more, at least 84% or more, at least 85% or more, at least 86% or more, at least 87% or more, at least 88% or more, at least 89% or more, at least 90% or more, at least 91% or more, at least 92% or more, at least 93% or more, at least 94% or more, at least 95% or more, at least 96% or more, at least 97% or more, at least 98% or more, at least 99% or more, or any value or range thereof). For example, in some embodiments, the labeled CSR ligands of the invention (e.g., radioisotope-labeled CSR ligands) and / or halogenated fluorophores of the invention may have a serum stability of at least 70%, at least 85%, or at least 90%. The serum stability of the ligands and / or fluorophores of the invention may be measured by any standard method known in the art, such as the standard methods described above, including but not limited to, by co-incubation with a solution containing serum proteins, for example, by the method described in Qu et al. 2019 Anim.Cells Syst.(Seoul) 23:155-163, which is incorporated herein by reference.

[0113] In some embodiments, the labeled ligands and / or fluorophores of the invention are at least 50% (e.g., at least 50% or more, at least 51% or more, at least 52% or more, at least 53% or more, at least 54% or more, at least 55% or more, at least 56% or more, at least 57% or more, at least 58% or more, at least 59% or more, at least 60% or more, at least 61% or more, at least 62% or more, at least 63% or more, at least 64% or more, at least 65% or more, at least 66% or more, at least 67% or more, at least 68% or more, at least 69% or more, at least 70% or more, at least 71% or more, at least 72% or more, at least 73% or more, at least 74% or more, at least 75% or more, at least 76% or more, at least 77% or more, at least 78% or more, at least 79% or more, at least 80% or more, at least 81% or more, at least 82% or more, at least 83% or more, at least 84% or more, at least 85% or more, at least 86% or more, at least 87% or more, at least 88% or more, at least 89% or more, at least 90% or more, at least 92% or more, at least 93% or more, at least 94% or more, at least 95% or more, at least 96% or more, at least 97% or more, at least 98% or more, at least 99% or more, at least 100% or more, at least 100% or more, at least 100% or more, at least 100% or more or greater, at least 74% or greater, at least 75% or greater, at least 76% or greater, at least 77% or greater, at least 78% or greater, at least 79% or greater, at least 80% or greater, at least 81% or greater, at least 82% or greater, at least 83% or greater, at least 84% or greater, at least 85% or greater, at least 86% or greater, at least 87% or greater, at least 88% or greater, at least 89% or greater, at least 90% or greater, at least 91% or greater, at least 92% or greater, at least 93% or greater, at least 94% or greater, at least 95% or greater, at least 96% or greater, at least 97% or greater, at least 98% or greater, at least 99% or greater, or any value or range thereof. For example, in some embodiments, a labeled CSR ligand of the invention (e.g., a CSR ligand labeled with a radioisotope) and / or a halogenated fluorophore of the invention may have a metabolic stability of at least 50%, at least 65%, at least 70%, at least 80%, at least 85%, or at least 90%. The metabolic stability of the ligands and / or fluorophores of the present invention may be measured by any standard method known in the art, such as any of the standard methods described above, including but not limited to, by HPLC analysis, for example, by the method described in Qu et al. 2019 Anim. Cells Syst. (Seoul) 23:155-163, which is incorporated herein by reference.

[0114] Another aspect of the invention relates to a PET probe which comprises a ligand or fluorophore of the invention.

[0115] Another aspect of the invention relates to an optical probe comprising a ligand or fluorophore of the invention. In some embodiments, the optical probe can be a fluorescent probe. In some embodiments, the probe can be a dual tracer (e.g., an optical probe and a PET probe, e.g., a fluorescent probe and a PET probe).

[0116] Another aspect of the invention relates to a fluorescent imaging probe (e.g., a near-infrared fluorescence (NIRF) probe) comprising a ligand or fluorophore of the invention. In some embodiments, the fluorescent imaging probe of the invention can be a near-infrared fluorescence (NIRF) probe. In some embodiments, the fluorescent imaging probe of the invention can be a conventional (visible light) fluorescent probe (e.g., excited at wavelengths from about 300 nanometers (nm) to about 850 nm). In some embodiments, the fluorescent imaging probe of the invention can be a shortwave infrared (SWIR) fluorescent probe. In some embodiments, the probe can be a dual tracer (e.g., an optical probe and a PET probe, a fluorescent probe and a PET probe, a NIRF probe and a PET probe).

[0117] Another aspect of the invention relates to compositions comprising the ligands, fluorophores, and / or probes of the invention and a pharma- ceutically acceptable carrier.

[0118] In some embodiments, the present invention provides pharmaceutical compositions comprising the ligands, fluorophores and / or probes of the present invention in a pharma- ceutically acceptable carrier, and optionally other medicinal agents, pharmaceutical agents, stabilizers, buffers, carriers, adjuvants, diluents, etc. For injections, the carrier is typically a liquid. For other modes of administration, the carrier may be solid or liquid. For inhalation administration, the carrier is respirable, and is preferably in solid or liquid particulate form.

[0119] "Pharmaceutically acceptable" means not a toxic or otherwise undesirable substance, ie, the substance may be administered to a subject without causing undesired biological effects.

[0120] In some embodiments, compositions of the invention comprising radioisotope-labeled ligands and / or fluorophores of the invention may have a radiopurity of at least 70% or more (e.g., at least 70% or more, at least 71% or more, at least 72% or more, at least 73% or more, at least 74% or more, at least 75% or more, at least 76% or more, at least 77% or more, at least 78% or more, at least 79% or more, at least 80% or more, at least 81% or more, at least 82% or more, at least 83% or more, at least 84% or more, at least 85% or more, at least 86% or more, at least 87% or more, at least 88% or more, at least 89% or more, at least 90% or more, at least 91% or more, at least 92% or more, at least 93% or more, at least 94% or more, at least 95% or more, at least 96% or more, at least 97% or more, at least 98% or more, at least 99% or more, or any value or range thereof). For example, in some embodiments, compositions comprising radioisotope-labeled CSR ligands and / or halogenated fluorophores of the present invention may have a radioactive purity of at least 70% or more, at least 75% or more, at least 80% or more, at least 85% or more, at least 90% or more, at least 95% or more, or at least 98% or more.

[0121] In some embodiments, the ligands, fluorophores, probes and / or compositions of the invention may be for use in imaging, diagnosing and / or directing treatment of disorders. Non-limiting examples of disorders for use in the invention include parathyroid disorders (e.g., primary hyperparathyroidism, secondary hyperparathyroidism, tertiary hyperthyroidism), thyroid disorders (e.g., thyroid cancer, goiter, thyroid nodules, Graves' disease), cardiac disorders (e.g., hypertension), renal disorders (e.g., nephrocalcinosis, rickets, proteinuria), reproductive disorders (e.g., infertility, embryonic or fetal development disorders), lactation disorders (e.g., reduced milk production), gastrointestinal disorders (e.g., pancreatitis, diabetes, diarrhea, gastrointestinal endocrine disorders), bone disorders (e.g., osteoporosis), cancer (e.g., colon cancer), neurological disorders (e.g., Alzheimer's disease, epilepsy), and / or pulmonary disorders (e.g., pulmonary hypoplasia, pulmonary hyperplasia). In some embodiments, the ligands, fluorophores, probes and / or compositions of the invention may be for use in imaging, diagnosing and / or directing treatment of parathyroid disorders. In some embodiments, the ligands, fluorophores, probes and / or compositions of the invention may be for use in imaging, diagnosing and / or directing treatment of thyroid disorders.

[0122] method

[0123] A further aspect of the invention relates to methods of using the compounds of the invention for imaging or therapy.

[0124] One aspect of the invention relates to a method of performing a PET scan on a subject, comprising administering to said subject a ligand, fluorophore, probe and / or composition of the invention.

[0125] Another aspect of the invention relates to a method of imaging tissue containing the calcium sensing receptor (CSR) in a subject, comprising administering to said subject a ligand, fluorophore, probe and / or composition of the invention.

[0126] Another aspect of the invention relates to a method of imaging thyroid and / or parathyroid tissue in a subject comprising administering to said subject a ligand, fluorophore, probe and / or composition of the invention.

[0127] Another aspect of the invention relates to a method of simultaneously performing a PET scan and fluorescence imaging (e.g. NIRF imaging) on ​​a subject, comprising administering to said subject a ligand, fluorophore, probe and / or composition of the invention.

[0128] Another aspect of the invention relates to a method of identifying parathyroid tissue in a subject, the method comprising performing a PET scan and / or fluorescence imaging (e.g. NIRF imaging) on ​​the subject using a ligand, fluorophore, probe or composition of the invention, wherein the PET scan and / or fluorescence imaging identifies the presence of parathyroid tissue.

[0129] Another aspect of the invention provides methods of removing hyperplastic and / or ectopic parathyroid tissue in a subject, comprising: (a) performing a PET scan and / or fluorescence imaging (e.g., NIRF imaging) on ​​the subject using a ligand, fluorophore, probe and / or composition of the invention, wherein the PET scan and / or fluorescence imaging identifies the presence of parathyroid tissue; (b) identifying the presence of hyperplastic and / or ectopic parathyroid tissue; and (c) surgically resecting the identified hyperplastic and / or ectopic parathyroid tissue, thereby removing the hyperplastic and / or ectopic parathyroid tissue.

[0130] Another aspect of the invention provides a method of guiding surgery to remove parathyroid tissue in a subject, the method comprising performing a PET scan and / or fluorescence imaging (e.g., NIRF imaging) on ​​the subject using a ligand, fluorophore, probe and / or composition of the invention, wherein the PET scan and / or fluorescence imaging identifies the presence of parathyroid tissue.

[0131] Another aspect of the invention relates to a method of guiding surgery to protect parathyroid tissue during thyroid and / or other neck surgery in a subject, the method comprising performing a PET scan and / or fluorescence imaging (e.g., NIRF imaging) on ​​the subject using a ligand, fluorophore, probe and / or composition of the invention, wherein the PET scan and / or fluorescence imaging identifies the presence of parathyroid tissue.

[0132] A further aspect of the invention provides a method of determining target areas for surgical removal of parathyroid tissue in a subject, e.g., a subject having hyperparathyroidism, or a subject at risk for or suspected of having or developing hyperparathyroidism, the method comprising: (a) performing a PET scan and / or fluorescence imaging (e.g., NIRF imaging) on ​​the subject using a ligand, fluorophore, probe and / or composition of the invention, wherein the PET scan and / or fluorescence imaging identifies the presence of parathyroid tissue; and (b) identifying one or more regions of the subject containing the presence of ectopic and / or hyperplastic parathyroid tissue, wherein the presence of ectopic and / or hyperplastic parathyroid tissue in the one or more regions indicates the one or more regions as target areas of the subject for surgical removal of parathyroid tissue.

[0133] Another aspect of the invention provides a method of treating hyperparathyroidism (e.g., primary hyperparathyroidism, secondary hyperparathyroidism, and / or tertiary hyperthyroidism) in a subject, the method comprising determining the suitability of a subject for surgical removal of parathyroid tissue by performing a PET scan and / or fluorescence imaging (e.g., NIRF imaging) on ​​a subject having hyperparathyroidism, or a subject at risk for or suspected of having or developing hyperparathyroidism, using a ligand, fluorophore, probe or composition of the invention, where the PET scan and / or fluorescence imaging identifies the presence of parathyroid tissue; and treating the hyperparathyroidism based on the results of the PET scan and / or fluorescence imaging.

[0134] An additional aspect of the invention provides a method of treating a calcium sensing receptor (CSR) positive tissue disorder in a subject, the method comprising: determining the suitability of a subject for such treatment by performing a PET scan and / or fluorescence imaging (e.g., NIRF imaging) on ​​a subject having hyperparathyroidism, or a subject at risk of having or suspected of developing hyperparathyroidism, with a ligand, fluorophore, probe or composition of the invention, wherein the PET scan and / or fluorescence imaging identifies the presence of CSR positive tissue; and treating the disorder based on the results of the PET scan and / or fluorescence imaging.

[0135] Non-limiting examples of disorders relevant to the ligands, fluorophores, probes, compositions and methods of the invention include parathyroid disorders (e.g., primary hyperparathyroidism, secondary hyperparathyroidism, tertiary hyperthyroidism), thyroid disorders (e.g., thyroid cancer, goiter, thyroid nodules, Graves' disease), cardiac disorders (e.g., hypertension), renal disorders (e.g., nephrocalcinosis, rickets, proteinuria), reproductive disorders (e.g., infertility, embryonic or fetal development disorders), lactation disorders (e.g., reduced milk production), gastrointestinal disorders (e.g., pancreatitis, diabetes, diarrhea, gastrointestinal endocrine disorders), bone disorders (e.g., osteoporosis), cancer (e.g., colon cancer), neurological disorders (e.g., Alzheimer's disease, epilepsy), and / or pulmonary disorders (e.g., pulmonary hypoplasia, pulmonary hyperplasia).

[0136] In some embodiments, the subject may have hyperparathyroidism (e.g., primary hyperparathyroidism, secondary hyperparathyroidism, tertiary hyperthyroidism) or may be at risk for or suspected of having or developing hyperparathyroidism (e.g., primary hyperparathyroidism, secondary hyperparathyroidism, tertiary hyperthyroidism). For example, in some embodiments, the subject may have primary hyperparathyroidism or may be at risk for or suspected of having or developing primary hyperparathyroidism. In some embodiments, the subject may have secondary hyperparathyroidism or may be at risk for or suspected of having or developing secondary hyperparathyroidism. In some embodiments, the subject may have tertiary hyperparathyroidism, or may be a subject at risk for tertiary hyperparathyroidism or a subject suspected of having or developing tertiary hyperparathyroidism.

[0137] In some embodiments, the subject may have a thyroid disorder or may be a subject at risk for a thyroid disorder or a subject suspected of having or developing a thyroid disorder, where the thyroid disorder includes, but is not limited to, thyroid cancer, goiter, thyroid nodules, and / or Graves' disease.

[0138] In some embodiments, the subject may be a pre-operative subject.

[0139] In some embodiments, the subject can be an intra-operative subject (e.g., the subject is undergoing surgery (e.g., explorative surgery)).

[0140] In some embodiments, the identified parathyroid tissue may be ectopic parathyroid tissue and / or hyperplastic parathyroid tissue, hi some embodiments, the identified parathyroid tissue may be healthy and / or normal (i.e., non-malignant) parathyroid tissue.

[0141] In some embodiments, the methods of the invention may further comprise quantifying the size of the identified parathyroid tissue in the subject, wherein identified parathyroid tissue that is larger than normal (e.g., by, e.g., 5% or more, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 100% or more, or e.g., 1 fold or more, 2 fold or more, 3 fold or more, 4 fold or more, 5 fold or more, 6 fold or more, 7 fold or more, 8 fold or more, 9 fold or more, 10 fold or more, compared to the subject) identifies abnormal (e.g., malignant, ectopic, hyperplastic and / or adenomatous) parathyroid tissue. Quantification of parathyroid tissue size may be performed via any method known in the art, including but not limited to, via quantification of PET and / or dual tracer probe radiolabel accumulation in the tissue (e.g., as an indirect estimate of size), via visual intensity, and / or via computational methods (e.g., computational methods based on results imaging modalities, including but not limited to, PET, computer tomography (CT), and / or fluorescence imaging).

[0142] In some embodiments, the method of the present invention may further comprise ablating at least a portion of the identified malignant parathyroid tissue. In some embodiments, the method of the present invention may further comprise ablating (all of) the malignant parathyroid tissue, i.e., the entirety of the identified malignant parathyroid tissue. In some embodiments, the method of the present invention may further comprise protecting at least a portion of the identified healthy parathyroid tissue from resection during parathyroid surgery, thyroid surgery, and / or other neck surgery. In some embodiments, the method of the present invention may further comprise protecting (all of) the identified healthy parathyroid tissue, i.e., the entirety of the identified healthy parathyroid tissue, from resection during parathyroid surgery, thyroid surgery, and / or other neck surgery.

[0143] In some embodiments, the methods of the present invention may further comprise scanning the resected thyroid tissue for the presence of parathyroid tissue.

[0144] In some embodiments of the methods of the invention, performing a PET scan on a subject using a ligand, fluorophore, probe or composition of the invention may include administering about 1 to about 15 mCi of the ligand, fluorophore, probe and / or composition, e.g., about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, or about 15 mCi of the ligand, fluorophore, probe and / or composition, or any value or range thereof.

[0145] Exemplary modes of administration include oral, rectal, mucosal, topical, nasal, inhalation (e.g., via aerosol), buccal (e.g., sublingual), vaginal, intrathecal, intraocular, transdermal, intrauterine (or in ovo), parenteral (e.g., intravenous, subcutaneous, intradermal, intramuscular (including administration to skeletal muscle, diaphragm muscle, and / or cardiac muscle), intradermal, intrathoracic, intracerebral, and intraarticular), topical (e.g., both cutaneous and mucosal surfaces (including airway surfaces), as well as transdermal administration), intralymphatic, etc., as well as direct injection into a tissue or organ (e.g., injection into the liver, skeletal muscle, cardiac muscle, diaphragm muscle, or brain). Administration can also be to a tumor (e.g., in or near a tumor or lymph node). In some embodiments, the administration can be via intravenous injection. The most suitable route in any given case will depend on the nature and severity of the condition being imaged and / or treated, and the nature of the particular composition being administered.

[0146] In some embodiments of the methods of the invention, the labeled ligands and / or fluorophores of the invention may have a serum stability of at least 70% or more (e.g., at least 70% or more, at least 71% or more, at least 72% or more, at least 73% or more, at least 74% or more, at least 75% or more, at least 76% or more, at least 77% or more, at least 78% or more, at least 79% or more, at least 80% or more, at least 81% or more, at least 82% or more, at least 83% or more, at least 84% or more, at least 85% or more, at least 86% or more, at least 87% or more, at least 88% or more, at least 89% or more, at least 90% or more, at least 91% or more, at least 92% or more, at least 93% or more, at least 94% or more, at least 95% or more, at least 96% or more, at least 97% or more, at least 98% or more, at least 99% or more, or any value or range thereof) for at least 30 minutes or more (e.g., in vivo after administration). For example, in some embodiments, a labeled ligand of the invention (e.g., a radioisotope-labeled CSR ligand) and / or a halogenated fluorophore of the invention may have a serum stability of at least 70%, at least 85%, or at least 90% for at least 30 minutes, at least 60 minutes, at least 90 minutes, at least 2 hours, or at least 3 hours after administration.

[0147] In some embodiments of the methods of the invention, the labeled ligands and / or fluorophores of the invention are at least 50% (e.g., at least 50% or more, at least 51% or more, at least 52% or more, at least 53% or more, at least 54% or more, at least 55% or more, at least 56% or more, at least 57% or more, at least 58% or more, at least 59% or more, at least 60% or more, at least 61% or more, at least 62% or more, at least 63% or more, at least 64% or more, at least 65% or more, at least 66% or more, at least 67% or more, at least 68% or more, at least 69% or more, at least 70% or more, at least 71% or more, at least 72% or more, at least 73% or more, at least The composition may have a metabolic stability of at least 30 minutes (e.g., in vivo following administration) of 74% or more, at least 75% or more, at least 76% or more, at least 77% or more, at least 78% or more, at least 79% or more, at least 80% or more, at least 81% or more, at least 82% or more, at least 83% or more, at least 84% or more, at least 85% or more, at least 86% or more, at least 87% or more, at least 88% or more, at least 89% or more, at least 90% or more, at least 91% or more, at least 92% or more, at least 93% or more, at least 94% or more, at least 95% or more, at least 96% or more, at least 97% or more, at least 98% or more, at least 99% or more, or any value or range thereof. For example, in some embodiments, a labeled ligand of the invention (e.g., a radioisotope-labeled CSR ligand) and / or a halogenated fluorophore of the invention may have a metabolic stability of at least 50%, at least 65%, at least 70%, at least 80%, at least 85%, or at least 90% for at least 30 minutes, at least 60 minutes, at least 90 minutes, at least 2 hours, or at least 3 hours after administration.

[0148] The present invention will now be described with reference to the following examples. It should be understood that these examples are not intended to limit the scope of the invention claimed, but rather to illustrate specific embodiments. Any variations in the exemplified methods that occur to those skilled in the art are intended to fall within the scope of the present invention.

[0149] Working Example

[0150] The foregoing is illustrative of the present invention, and is not to be construed as limiting the present invention, which is defined by the following claims, with equivalents of the claims to be included therein.

[0151] Example 1: Development of novel PET agents targeting calcification drugs and peptide-based CSR

[0152] In this study, an arene-CH radiolabeling approach (Chen et al. 2019 Science 364:1170-1174) was used to convert a wide range of organic drugs into PET molecular probes under mild and rapid conditions in a relatively simple manner. Several novel aromatic NIRF dyes based on arene-containing drug molecules were synthesized. 18 Described herein is a F-based PET tracer that has been successfully produced for parathyroid detection. 18 F-labeled cinacalcet, a small molecule that targets the calcium-sensing receptor (CSR), and for specific parathyroid and thyroid targeting 18 Contains F-labeled NIRF dye.

[0153] Most imaging techniques (ultrasound, CT, MRI, etc.) are structure-based, i.e., they can show the shape of internal structures. Identification of the structure requires the radiologist's anatomical knowledge. In many cases, a structure can be identified, but its identity remains unknown. Techniques that target molecules or functions specific for a given tissue can be very useful to identify unknown masses. Markers that target tissue-specific molecules can provide information about the identity of the tissue in a way that structural imaging cannot. For example, an ultrasound may show a small nodule near the thyroid gland, but its identity as a parathyroid adenoma may not be immediately apparent, as opposed to a lymph node or a focus of ectopic thyroid tissue. A parathyroid-specific tracer that "lights up" the nodule may reveal its identity. Such an approach is complementary to structural imaging and may be used in combination with other imaging techniques. In fact, a normal parathyroid gland is a 3-5 mm tissue that is difficult to detect using traditional methods. Once one or more of the parathyroid glands become hyperparathyroid, the size grows to about 10 mm or more. Unfortunately, current imaging may identify multiple lesions (some of which may not be related to the parathyroid gland, leading to unnecessary surgery), making it important to determine which lesions are related to the parathyroid gland (for surgical planning). Furthermore, current imaging is poor at detecting small lesions.

[0154] In this study, several examples of PET and PET / NIRF probes were developed based on CSR binding molecules.

[0155] Existing archival clinical specimens were used to test for the presence of CSR using immunohistochemistry (IHC). Normal parathyroid glands can be inadvertently removed during thyroidectomy. Existing clinical specimens were reviewed to determine appropriate tissue blocks. Using standard IHC techniques, tissues were stained for expression of CSR. Expression was scored using an "H" score, which takes into account the intensity of staining (0-3) and the percentage of cells staining at each intensity. For example, if 30% of cells stain 1+, 20% 2+, and 40% 3+, the H score would be 30+40+120=190. The H score ranges from 0-300. In addition, parathyroid adenomas and parathyroid hyperplasias were found in the existing clinical specimens. They were also stained for CSR and compared to normal parathyroid glands. Thirty normal parathyroid glands adjacent to the thyroid gland, 30 parathyroid adenomas, 30 hyperplastic parathyroid glands from patients with primary hyperparathyroidism, and 30 parathyroid glands from patients with secondary (renal) hyperparathyroidism were used in this study. Patients with hyperplasia were identified based on review of the entire clinical record because routine histology cannot reliably distinguish between adenomas and hyperplasia. A 3- to 4-fold difference in staining was expected to be sufficient to distinguish the tissues on imaging.

[0156] Figure 1 shows CSR staining results in normal thyroid, parathyroid, and hyperparathyroid, and demonstrates that CSR expression in hyperparathyroid and parathyroid glands is 6-7 times higher than its expression in nearby thyroid tissue. This difference provides sufficient contrast for PET imaging, and high contrast was observed in the rat and NHP studies described below. These staining results clearly demonstrated that CSR is a valid target for parathyroid imaging.

[0157] Cinacalcet was used as a starting example for developing novel parathyroid targeted PET agents based on calcimimetic drugs. Cinacalcet is a therapeutic agent that binds to CSR with high affinity and selectivity. To successfully convert cinacalcet into an imaging agent, hydrogens on the aromatic ring of cinacalcet were replaced with fluorines, which represents a minimal change in the chemical structure, with the aim of maintaining the CSR binding affinity of the parent compound (Figure 2, top schematic "A"). Conventionally, electron-rich fluorines have been introduced into the aromatic system, which is a key step in the development of imaging agents. 18 It was not easy to introduce F. Recent work on photoredox radiolabeling by the inventors of the present invention provided an innovative method to directly convert the C-H bond to a C-F bond. By replacing H with similarly sized F, we aimed to maintain the binding affinity to CSR, which was confirmed by binding assays. The synthesis of optically pure cinacalcet was achieved by one-step Boc protection. Other 18 F-labeled cinacalcet analogs were also prepared using a similar strategy. In addition to direct CH fluorination, radiofluorination of the CF3 group in cinacalcet was also explored (Figure 2, bottom schematic "B"). Boc-protected cinacalcet was prepared through either direct radiofluorination of FLP or a relatively stable Br intermediate via a frustrated Lewis pair-mediated approach. After one-step deprotection, radioactive molecules with the same chemical structure as the parent cinacalcet agent were obtained. After the precursors were obtained, radiolabeling conditions were explored by varying the light source, temperature, fluoride source, reaction ratio, etc. The isolated yields of the selected agents were greater than 5%.

[0158] Fluorine 18( 18 F) has a relatively long half-life (t 1 / 2=110 min and decays with high efficiency (97%) by positron emission, making it one of the most important radioisotopes in the radiopharmaceutical industry. Aromatic or heteroaromatic systems are common in small molecule drugs and therapeutics, with many aromatic C-H bonds and C(sp 2 )-F bonds are becoming increasingly important, so arena CH to C- 18 Direct conversion to an F bond is ideal. 18 F]TBAF 18 Direct CH[ of aromatics used as a source of fluoride 18 Previous work on [F] fluorination has published a list of model compounds (Chen et al. 2019 Science 364:1170-1174; incorporated herein by reference). In addition to this report, a recent study using a blue laser at ice-cold temperatures has demonstrated that fluorination of 1,2-difluorophenyl fluoride (F) is a promising method for the synthesis of fluorinated 1,2-difluorophenyl fluoride (F) at low temperatures. 18 Deoxy radiofluorination was discovered that allows for the site-specific introduction of F into aromatic systems (Tay et al. 2020 Nature Catalysis, 2020, 3:734-742; incorporated herein by reference).

[0159] In this study, we directly synthesized the arene C–H bond of Boc-protected cinacalcet [ 18 [F] radiofluorination was performed (Figure 3). Photoredox labeling produced the desired product in 19% isolated yield. 18 F-labeled Boc-cinacalcet could be completely separated from Boc-cinacalcet using HPLC separation. The labeling occurred mainly at the 4-position of the naphthalene ring, and the identity was confirmed by co-injection with 4F-Boc-cinacalcet standard. Small radioactive peaks were observed that were likely compounds labeled at other positions, but all of them were 4-[ 18 F]F-Boc cinacalcet could be completely separated. After removing the Boc group, 4-[ 18 F]F-cinacalcet could be obtained with a radiochemical purity of greater than 98% and a molar activity of 2.1 Ci / μmol, which was then injected into normal animals for initial evaluation.

[0160] After obtaining the desired agents targeting CSR, their dissociation constants (Kd) were determined using a saturation assay. Briefly, O cells (positive for CSR expression) were plated at 0.2 × 10 6 Cells / well were seeded and incubated overnight at 37°C, 5% CO2. Then, stock solutions of the radioactive tracer in FBS-free medium were prepared at concentrations ranging from 0.1 nM to 100 μM through the addition of a cold standard. To measure cell binding, increasing concentrations of the radioactive tracer solution were added sequentially into associated wells of one plate, and nonspecific binding of the tracer was assessed in the presence of a large excess of non-radiolabeled compound (500 μM) in another plate. After 1 h of incubation on ice, unbound tracer was removed and gently washed three times with ice-cold PBS, and the cells were harvested with 0.2 N NaOH and radioactivity was measured in a gamma counter. Specific binding was determined by subtracting nonspecific binding from cell binding activity, and Kd values ​​were calculated through the specific binding curves using nonlinear regression curve fits (GraphPad Prism). Desirable agents should have a comparable Kd compared to cinacalcet and etelcalcetide. In addition to Kd values, serum stability and metabolic stability were also performed. The selected agent should have a serum stability of >90% and metabolic stability of >80% at 1 hour post-incubation / injection.

[0161] 18 The in vitro stability of F-cinacalcet was performed and demonstrated a purity of over 90% at 6 hours (Figure 4A). In non-human primate imaging experiments, blood samples were taken from the vein at 1 hour and 3 hours after injection. As shown in Figure 4B, the agent was fairly stable at 1 hour, and significant metabolites were observed at 3 hours. Since PET imaging was completed at 1 hour after injection, 18It was suggested that F-Cinacarect has reasonable stability as a PET drug.

[0162] Normal rats were used as an animal model for initial CSR agent imaging. In contrast to rats, which weigh approximately 10 times more than mice, surgical dissection of the mouse parathyroid gland is technically too challenging. Analysis was performed by individuals with extensive experience in identifying parathyroid glands for pathological, autoradiographic, and microscopic studies. Briefly, animals were anesthetized with isoflurane. Dynamic PET imaging was performed after administration of 1–2 mCi of tracer for the PET probe, and the animals were imaged in a 2-hour dynamic scan. CT scans were also acquired for anatomical registration and attenuation correction. Images were reconstructed and representative dynamic images were created for the study (10-minute epochs). These images were qualitatively scored to assess the visibility of the parathyroid gland relative to background tissue. PET kinetic modeling was also performed to assess the relative kinetics of parathyroid uptake compared to thyroid and other tissues.

[0163] After imaging of each animal, the parathyroid glands were carefully dissected and the uptake in the parathyroid glands was measured, followed by subsequent pathological evaluation to confirm the histology. Autoradiography was performed to examine its distribution in the parathyroid region. Optical imaging guidance during surgery was also performed using the developed CSR PET fluorescence probe. During resection, the surgeon also evaluated the visibility of the lesion with and without the use of fluorescence. Other vital organs, such as the thyroid gland, adjacent tissues, and other vital organs mentioned above, including the major organs, were also dissected to evaluate the relative uptake within these organs. Localization of radioactivity in the thyroid tissue was confirmed by autoradiography combined with pathological staining of adjacent slides.

[0164] 18F-cinacalcet was able to detect the parathyroid glands in rats by targeting CSR. The parathyroid glands are very small (3-5 mm in humans) and are generally below the detection limit of most imaging methods. A novel CSR PET agent 18 F-cinacalcet was injected in rats and small animal PET imaging was performed. As shown in Figure 5, panel A, two hot spots were clearly demonstrated in the parathyroid region of the rat neck. To confirm that the observed signal was derived from the very small parathyroid gland and not from the thyroid or other tissues, the rats were sacrificed and the localization of radioactivity in the gland tissue was evaluated by autoradiography combined with pathological staining of adjacent slides. As shown in Figure 5, panel B, 18 Conclusions: Localization of F-cinacalcet correlated well with CSR-positive parathyroid glands. These results strongly support the approach of using CSR for parathyroid imaging.

[0165] In addition to cinacalcet, etelcalcetide is a calcimimetic drug for the treatment of secondary hyperparathyroidism. This peptide can also be modified to construct PET and PET fluorescence probes.

[0166] Example 2: Targeting the Parathyroid Gland 18 Deployment of F-labeled NIRF dyes

[0167] So far, novel halogenated fluorophores have been discovered for thyroid (T700) and parathyroid (T800) fluorescence imaging without any targeting motif (Wizenty et al. 2020 Molecules 25; Kim et al. 2017 Gland Surg. 6:516-524; Wada et al. 2017 Ann. Thorac. Surg. 103:1132-1141; Hyun et al. 2015 Nat. Med. 21:192-197). Real-time, highly sensitive NIRF imaging allows the parathyroid gland to be differentiated from the thyroid gland and surrounding soft tissues. Clear visualization of the parathyroid gland aids in parathyroid surgery and also minimizes thyroid damage or removal of normal tissue. However, NIR fluorophores still have limited tissue penetration. This can be particularly problematic in detecting parathyroid glands when they are ectopic or in a hidden location, e.g., within the thyroid gland. To address this limitation, the present study used a NIRF dye 19 F element PET isotope 18 F, allowing the parathyroid gland to be detected by PET while maintaining its NIRF properties for image-guided surgery. Since the radioactive compound has the exact same structure as the parent dye, the parathyroid or thyroid homing capability of the dye will not be altered. The data herein are 18 We describe the successful generation of F-labeled T700 and T800, which were demonstrated to be preferentially taken up in the thyroid and parathyroid glands, respectively. These radioactive and fluorescent agents can be used for detection of the parathyroid gland by PET and subsequent image-guided surgery.

[0168] The optical signals of the halogenated cyanine dyes T700 (taken up in the thyroid) and T800 (taken up in the parathyroid) have limited tissue penetration. Standard compounds and asymmetric precursors of T700 and T800 were prepared as shown in FIG. 6. Starting from the asymmetric precursors T700-precursor-H and T800-precursor-H, direct CH radiofluorination gave the symmetric products 18 F-T700 and 18 The CH fluorination of T700-precursor-H and T800-precursor-H can be performed by radiofluorination of multiple arene CH sites, resulting in F-T800 (Figure 7). 18 F-T700 and 18 F-T800 was also synthesized through a newly developed deoxy radiofluorination method (Tay et al. 2020 Nature Catalysis 3:734-742). Briefly, asymmetric precursor T800-precursor-OR was prepared as shown in Figure 6. Radiofluorination was performed at the position bearing the "OR" group. The range of nucleofuge for deoxy radiofluorination includes 4-chlorophenoxyether and other electron-poor aryloxy groups as shown in Figure 6. Selected aryloxy groups for further use were synthesized using the nucleofuge. 18 F-T800 must maintain a quantum yield >5% with excitation >700nm and emission >720nm (ensuring that the labeling process does not adversely affect its optical signal) and maintain serum stability >90% with a 1-hour incubation for in vivo studies.

[0169] [ 18 F]-radiolabelling can be achieved by the direct CH fluorination or deoxyfluorination of stable C-[ 18The in vitro stability of these novel PET / fluorescent dyes was assessed by HPLC at different time points (0.5, 1, 2, 4, and 6 h) after incubation in PBS and bovine serum albumin (BSA). Their resistance (or susceptibility) to radiolysis, especially at high activity concentrations, was also investigated. This information, combined with the obtained fluorescence properties (excitation / emission wavelengths, quantum yield, and photostability) of these fluorinated dyes as described above, allowed the determination of the [F]F bond. 18 It was determined whether the [F]-T700 / T800 dyes exhibited reasonable stability in vitro (>90% after 1 h of incubation, no defluorination). Selected agents that met these criteria were injected into normal mice for in vivo stability evaluation.

[0170] At present, it remains unknown why T800 and T700 are taken up by different glands. However, halogenation and polymethine length appear to be important. Without wishing to be bound by theory, it is hypothesized that the uptake of T700 into the thyroid gland may be the result of an active transport mechanism mediated by the NIS protein. Therefore, I - Blocking studies in the presence of riboflavin will assess whether its uptake can be reduced. Similarly, T800 may be a mineralizing agent, and blocking studies in the presence of calcium ions will assess its blocking effect.

[0171] In vivo studies were performed in normal rats to confirm the localization profile of T700 and T800 dyes and to evaluate the newly created T800 analog. Considering the small size of normal parathyroid glands in rats, fluorescence microscopy and pathology were used to confirm parathyroid uptake and contrast after PET / NIFR imaging. Briefly, imaging agents were injected intravenously (i.v.) and imaged with IVIS and PET / CT at 1 and 4 hours after injection. To determine the dosage of imaging agents, imaging agents were injected in the range of 2 nmol to 100 nmol, and the optimal dosage with high parathyroid signal without inducing too much background signal was selected. After the imaging experiment was completed, the parathyroid glands were removed and the signal-to-noise contrast was further evaluated by fluorescence microscopy, autoradiography (adjacent slides), and pathology. The signal-to-background ratio was calculated using the fluorescence intensity and / or autoradiography signal between the parathyroid gland and the thyroid or adjacent tissues. The agent selected should have a signal to background ratio of >2.

[0172] Both T700 and T800 were synthesized and then scanned with IVIS using dual channel imaging. As shown in panel A of FIG. 8, 675 nm excitation and 720 nm emission channel results in predominant fluorescent signal from T700. In contrast, 745 nm excitation and 780 nm emission channel results in predominant fluorescent signal from T800. Clearly, these parameters can be used in our dual channel imaging. To confirm the specific imaging ability of T700 and T800 in the gland, 0.2 μmol of each agent was injected into Wistar rats, followed by ex vivo imaging. As shown in panel B of FIG. 8, prominent fluorescent signal was observed from T700 signal in parathyroid gland. Ex vivo scanning further confirmed that the T700 signal was located in the thyroid gland and the T800 signal in the parathyroid gland. Clearly, dual channel overlay will help surgeons identify the parathyroid gland in relation to the background thyroid gland and its neighboring tissues.

[0173] Although rat parathyroid glands are much larger than mouse parathyroid glands, ex vivo imaging may still lack the necessary resolution due to the overall small size of the parathyroid glands. The ability to accurately identify the parathyroid glands is an important technique for assessing parathyroid uptake and parathyroid-to-background contrast through fluorescence microscopy or autoradiography in initial screening studies. Autofluorescence from the parathyroid glands was also examined (Figure 8, Panel C). Mice 1, 2, and 3 were injected with saline, T700, and T800 dye, respectively. Tissues containing the parathyroid glands were autofluorescent and imaged in the T700 and T800 channels. As expected, autofluorescence was near background levels at the location of the parathyroid glands.

[0174] The synthesized 18 F]-T800 and [ 18 F]-T700 was confirmed to correlate well with the standard by radio-HPLC (Figure 9). Conventional labeling methods using nitro as a leaving group did not yield the desired product. The key step in the methodology was the addition of a nitro group to the aromatic ring of the cyanine dyes (T700 and T800). 18The first step was to introduce F. This initial success confirmed the feasibility of the method proposed here. Other labeling methods include radio-deoxyfluorination (Neumann et al. 2016 Nature 538:274), fluorination of N-arylsydnones (Narayanam et al. 2017 Angew Chem. Int. Ed. Engl. 56:1306-13010), sulfonium salts (Gendron et al. 2018 J. Am. Chem. Soc. 140:11125-11132) and iodonium salts (Ichiishi et al. 2014 Org. Lett. 16:3224-3227; McCammant et al. 2017 Org. Lett. 19:3939-3942; Rotstein et al. 2014 Nat. Commun. 5:4365), and fluorodemetalation (Lee et al. 2011 Science 334:639-642; Lee et al. 2012 J. Am. Chem. Soc. 134:17456-17458), boronic acids (Mossine et al. 2015 Org. Lett. 17:5780-5783) and esters (Tredwell et al. 2014 Angew Chem. Int. Ed. Engl. 53:7751-7755). There may be concern of high background signal due to the hydrophobicity of the dye. The data herein demonstrated that the contrast of T800 in the parathyroid gland is good compared to the adjacent tissue. In the case of photoredox radiofluorination, there may be multiple potential reaction sites and the ability to be separated from the precursor. In the case of CH fluorination reaction, there may be two or possibly three major products. S N Ar photoredox deoxy radiofluorination leads mainly to one product. The labeling agent can be well separated from the precursor on an F5 column.

[0175] Lead agents were further evaluated in nude mice with transplanted human parathyroid tissue. These experiments confirmed that the developed agents could efficiently target human parathyroid tissue in addition to the murine parathyroid gland.

[0176] To evaluate the newly developed agents in a clinically relevant model, xenografting of human parathyroid tissue was used. Over 15 model mice were established by transplanting human parathyroid glands into nude mice. Using the established T800 as a probe, the transplanted PHPT tissue could be clearly visualized after blood vessels were established at the transplantation site (Figure 10). The transplant was also surgically removed together with the neighboring tissues. Ex vivo imaging demonstrated good contrast between the parathyroid gland and its neighboring tissues.

[0177] Further experiments include conducting a 14-day toxicity study in rodents. A single high dose (>100-fold higher than the proposed human dose for the selected agent) is used for acute toxicity testing. The study is conducted under Good Laboratory Practice (GLP) conditions. Female and male rats are administered a single intravenous (iv) dose of lead agent (>100-fold the imaging dose). The animals are checked twice daily for morbidity / mortality and signs of toxicity. Animals have weight, blood chemistry and hand observation data collected prior to injection and on days 1, 2, 4, 8 and 14. Four groups of animals are studied: Group 1: no fasting control; Group 2: no fasting + lead agent; Group 3: 12-hour fast; and Group 4: 12-hour fast followed by injection of lead agent. On day 15, all remaining animals were euthanized and blood and urine samples were collected for analysis, clinical chemistry, hematology, and coagulation factors. Major organs were collected, weighed, and processed for H&E staining and examination. Statistical analysis among different groups was performed in terms of body weight, organ weight, clinical pathology data, and urinalysis data.

[0178] Example 3: Use of lead agents in non-human primates

[0179] A non-human primate, such as the rhesus monkey, is selected for further characterization.

[0180] 18 A dynamic scan is performed for 2 hours after intravenous (iv) injection of the F-labeled agent. A static scan is also performed focusing on the thyroid and parathyroid glands. The purpose of this experiment is to (1) 18 During the first 2 hours after a brief intravenous (i.v.) infusion of a F-labeled PET agent, 18 To characterize the pharmacokinetics, biodistribution, and metabolic stability of F-labeled PET agents; (2) to estimate dosimetry data from proposed future clinical procedures; (3) to design clinical PET / CT scan protocols, such as those described above including injection doses and scan time points, from which the optimal times for maximal imaging and parathyroid-to-background contrast will be determined.

[0181] In detail, rhesus monkeys (NHPs) are anesthetized with 1.4-4% isoflurane inhalation and maintained with mechanical ventilation. Two venous catheters are applied: one for tracer administration and one for sampling of blood radioactivity concentration. A CT transmission scan is obtained. Next, a CT scan is performed targeting the parathyroid glands. 18F-labeled PET agent (3 to 5 mCi) is administered intravenously (iv) and a 120-minute dynamic PET scan is performed. The scans are performed fasting and non-fasting to compare differences in uptake and contrast. Serial venous blood samples (0.2-0.5 ml) are taken before and 0.5, 5, 30, 60, and 90 minutes after injection (pi) to measure metabolic stability and blood uptake. Temperature, heart rate, ECG, pCO2, pO2, SaO2, and blood pressure are monitored throughout the study. Urine samples for HPLC metabolite analysis are taken at the end of the whole-body scan. PET scan data are analyzed for circulating PET, e.g., by volumetric region of interest (ROI) analysis, extraction of tissue TAC and steady-state SUV; 18 Quantitative analysis of plasma time-activity-curves (TAC) and HPLC data to determine the TAC for the F-PET agent and its metabolites versus time; and the PET scan data as described above, including calculation of cumulative activity for normal organs / tissues, are analyzed.

[0182] Plasma and urine samples were 18 The serum is assayed for F-labeled agents and labeled metabolites. A blood sample is taken and immediately centrifuged at 14,000 rpm for 5 minutes. 100 μL of 50% TFA in PBS is then added to the supernatant serum solution and centrifuged for 5 minutes. The supernatant solution is injected for HPLC analysis. Urine is filtered and then used for HPLC analysis.

[0183] The distribution of the absorbed dose is calculated according to the MIRD method, whereby it is assumed that the integrated radioactivity for each of the source organs is known. 18 Observed feeding organs in which F-PET agents may concentrate include the bladder, kidneys, and liver. 18Using a combination of F-PET scans, attenuation scans, and comparative CT scans, other organs whose anatomical boundaries can be identified are used as additional donor organs for complementation (brain, lower colon, stomach, blood, heart wall, lungs, pancreas, red bone marrow, spleen). 18 Organs in which no F-PET uptake is observed and the boundaries cannot be delineated are treated as background and assigned a residual level of accumulated activity.

[0184] NHPs will be closely monitored for the development of toxicity. Metabolic studies, such as those described above including blood chemistry profile (electrolytes, glucose, calcium, phosphorus, magnesium, bilirubin, albumin, total protein, AST, ALT, ALP), will be performed to assess potential alterations in liver and kidney function.

[0185] Primary hyperparathyroidism is more prevalent in women, with an incidence rate of 66 per 100,000 000 years in women and 25 per 100,000 000 years in men. Therefore, both male and female subjects are used for the preclinical biodistribution and imaging studies described herein. Potential differences between the sexes are compared and calculated in the statistical analysis.

[0186] Newly developed 18 Native parathyroid PET imaging was performed in rhesus monkeys using F-cinacalcet. Importantly, as shown in Figure 11, this CSR agent demonstrated significant uptake within the parathyroid region, indicating that CSR is a valid target for parathyroid imaging. Blood metabolic stability was 18 It was demonstrated that F-cinacalcet has acceptable stability for imaging applications (Figure 4, Panel B).

[0187] Example 4: Development of CSR-targeted PET agents for parathyroid imaging

[0188] BOC-protected Sensipar登録商標 (Cinacalcet, a small molecule compound targeting CSR) 18 The introduction of F (radioactive tag) was efficiently achieved using direct photoredox CH radiofluorination. After simple deprotection, 18 F-labeled cinacalcet ([ 18 The compound, designated [F]F-ZW-cinacalcet, was obtained in 34% yield, and its uptake could be efficiently blocked by a competing drug. The agent also demonstrated fast blood clearance and good plasma stability. 18 [F]F-ZW-cinacalcet PET imaging noninvasively detected the parathyroid glands in mice and rats, and the localization was further confirmed by autoradiography and immunohistochemistry. To further facilitate future clinical applications, PET / MRI scans were performed in non-human primates (NHPs). 18 F]F-ZW-cinacalcet demonstrated significant tissue accumulation in the parathyroid region. 18 We have demonstrated that [F]F-ZW-cinacalcet is safe for future human trials. 18 [F]F-ZW-cinacalcet is a novel imaging agent for the detection of parathyroid glands, which has been demonstrated to be beneficial in the management of patients with primary hyperparathyroidism.

[0189] [ 19[F]Synthesis of F-ZW-cinacalcet and precursors for photoredox reaction. The standard compound of F-ZW-cinacalcet was synthesized based on the scheme shown in panel A of FIG. 13. Briefly, 4-fluoro-1-acetonaphthone was reduced to N-benzyl-1-(4-fluoronaphthalen-1-yl)ethan-1-amine in two steps in 53% yield. After removing the Bn protecting group, conjugation with 3-(trifluoromethyl)hydrocinnamic acid was carried out (87% yield), followed by reduction of the amide bond with NaBH4 (97% yield), which resulted in the desired standard of F-ZW-cinacalcet. A Boc group was also added to F-ZW-cinacalcet to generate standard-Boc-F-ZW-cinacalcet. To synthesize a precursor compatible with photoredox labeling reactions, a Boc protecting group was added to the parent drug cinacalcet (Figure 13, Panel B). Protecting the secondary amine avoided potential oxidation at the nitrogen atom and facilitated direct CH radiofluorination through the photoredox reaction.

[0190] Photoredox radiofluorination. Cinacalcet is a therapeutic agent that binds to CSR with high affinity and selectivity. 18 F can be introduced into the CF3 group of cinacalcet (SP 3 - 18 F bond), and the resulting agent has low stability. There is a need to design new agents based on cinacalcet to improve the stability of the agent with minimal changes to the parent agent's structure. The photoredox labeling method allows the formation of the CF bond through direct CH radiofluorination. This SP in naphthalene 2 -F bond is S-P 3 The compound was more stable than the aryl group, which only replaced one arene-CH bond with an arene-CF bond, and represented minimal changes to the structure of the parent compound.

[0191] [ 18The synthesis of [F]F-ZW-cinacalcet was carried out in two steps by direct CH radiofluorination under photoredox conditions followed by deprotection of the Boc group. 18 Starting from [F]F-TBAF, direct [ 18 F] radiofluorination to give Boc-[ 18 F]F-ZW-cinacalcet was obtained in 42.9±4.3% yield. The labeling occurred mainly at the 4-position of the naphthalene ring, with Boc-[ 19 The identity was confirmed by coinjection with authentic [F]-ZW-cinacalcet. After removal of the solvent, concentrated hydrochloric acid was added and the Boc protecting group was removed by heating at 95°C for 10 min (80% yield). 18 [F]F-ZW-cinacalcet was obtained with a radiochemical purity of >96% and an activity of 4.6 Ci / μmol. An ice bath was used for the labeling reaction, but radiofluorination also proceeds without cooling. Evaporation of the solvent is fast, so special care is required to avoid complete drying of the reaction. The reaction can also be scaled up to produce sufficient agent for non-human primate studies. Due to the hydrophobicity of the drug, 50% EtOH was used in the final step to reduce sterile filter absorption of the drug.

[0192] [ 18 Stability of [F]F-ZW-cinacalcet. The photoredox labeling method was 2 This allows access to a new category of PET agents containing -F bonds. 18 F]F-ZW-cinacalcet has improved stability compared to the CF3 labeling method. 18 [F]F-ZW-cinacalcet was first incubated in PBS (phosphate buffered saline containing 8% EtOH) and aliquots were taken for analysis at different time points. Although a slight peak of hydrophilic impurities was observed after 2 hours of incubation, the purity remained above 95% after 4 hours of incubation. 18The in vivo stability of [F]F-ZW-cinacalcet was examined in non-human primates. Blood samples were taken at 1 and 3 hours after injection. At the 1 hour time point, the majority of the agent remained intact. At the 3 hour time point, hydrophilic metabolites were observed. Metabolites were not determined in this study because imaging was terminated within the first hour.

[0193] Determination of octanol / water partition coefficient (Log P). 18 The Log P of [F]F-ZW-cinacalcet was determined in 1-octanol-water system with a mean Log P = 1.95 ± 0.02. 18 We demonstrated that [F]F-ZW-cinacalcet has lipid solubility.

[0194] Cellular uptake and specific blocking assay. 18 To verify the target specificity of [F]F-ZW-cinacalcet, cellular uptake and blocking experiments were performed. As shown in Figure 14, panel A, Hcc827 (a non-small cell lung cancer cell line) has high CSR expression, which was then selected for in vitro assays. 18 When incubated with [F]F-ZW-cinacalcet, the uptake of radioactivity by Hcc827 cells gradually increased over time (Figure 14, Panel B). To confirm the binding specificity, Hcc827 cells were incubated with [F]F-ZW-cinacalcet. 18 F]F-ZW-cinacalcet, as well as excess cinacalcet, CaCl2, and cold standard [ 19 The cells were co-incubated with [F]F-ZW-cinacalcet (100 μM). 19 F]F-ZW-cinacalcet was detected at 20 min after incubation, 18 F]F-ZW-cinacalcet uptake was blocked 89.9% efficiently. Uptake of over 87% was observed at other time points. 18The uptake of [F]F-ZW-cinacalcet was also efficiently blocked by cinacalcet (71.3±2.9% to 79.0±0.6% reduction) and CaCl2 (71.5±1.2% to 82.9±1.7% reduction). 18 It was shown that F]F-ZW-cinacalcet could be internalized by CSR expressing Hcc827, and the target specificity was confirmed by competitive blocking assay. 18 [F]F-ZW-cinacalcet enabled visualization of CSR expression in vivo through targeted molecular imaging.

[0195] Small animal PET / CT imaging of the parathyroid glands in rodents. Because the parathyroid glands are much smaller, rats were used instead of mice to evaluate the agent. Static PET scans were performed to measure the parathyroid glands in rodents. 18 PET images were acquired in rats at 0.5, 1, and 2 hours after injection of [F]F-ZW-cinacalcet. Representative coronal PET images are shown in Figure 15, panel A. The parathyroid glands could be visualized on the images as early as 0.5 hours with a radiotracer uptake of 0.28 ± 0.16% ID / g and a parathyroid / muscle ratio of 4.9 ± 0.93. Radioactivity in the parathyroid glands decreased over time and returned to background levels at 2 hours (Figure 15, panel B). Uptake in the salivary glands was also observed, which did not decrease over time. Interestingly, high uptake of radioactivity was observed in the rat lungs, which were confirmed by staining to have high CSR expression.

[0196] In vivo 18To better demonstrate the location of [F]F-ZW-cinacalcet, the PET and CT images were merged at the coronal, sagittal, and transverse levels, respectively (Figure 16, Panel A), and a multi-angle 3D volume-rendered PET / CT image was also reconstructed (Figure 16, Panel B). From the spatially oriented PET / CT images, it was easy to identify the parathyroid glands adjacent to the cricoid cartilage and trachea. The radioactive uptake in the parathyroid glands was significantly higher than that in background tissues.

[0197] In rats 18 Dynamic PET images of [F]F-ZW-cinacalcet were acquired for 60 min. Representative PET images and time-activity profiles for the parathyroid gland and muscle are shown in Figure 17. 18 [F]F-ZW-cinacalcet accumulated in the parathyroid gland at the earliest time point and peaked approximately 7 min after injection (0.33 ± 0.05% ID / g), followed by a gradual decline to 0.17 ± 0.01% ID / g at 60 min after injection. 18 Muscle uptake of [F]F-ZW-cinacalcet was significantly lower at all time points (i.e., less than 0.06% ID / g ± 0.01 at 20 min postinjection). These results support the conclusion that the [F]F-ZW-cinacalcet 18 Excellent specificity and tissue kinetics ('fast in' and slow clearance) of [F]F-ZW-cinacalcet were demonstrated.

[0198] Similar to the CSR positive glands, dynamic PET images and merged PET / CT images (panels A and B in Figure 18) show that the lungs showed high levels of radiotracer uptake. According to the regional time-radioactivity curves (Figure 18C), the radiotracer uptake in the lungs showed a "fast in and fast out" trend. The uptake peaked at 2 min post-injection (lungs: 1.00 ± 0.19% ID / g; heart: 0.95 ± 0.14% ID / g) and showed similar kinetics up to 10 min post-injection. Then, the cardiac uptake rapidly declined to 0.15 ± 0.27% ID / g at 60 min post-injection, while the lung uptake remained at a high level of 0.41 ± 0.07% ID / g at 60 min post-injection. In addition, the radiotracer was also observed to be uptaken into the brain, with uptake levels remaining stable at ~0.2% ID / g.

[0199] Autoradiography and pathological examination. 18 To further confirm the parathyroid imaging results from [F]F-ZW-cinacalcet-based PET / CT, ex vivo studies were performed to verify the accuracy of radiotracer targeting by autoradiography and IHC staining. First, local laryngeal and tracheal tissues (Figure 19, panel A) containing the thyroid and parathyroid glands were stained with [F]F-ZW-cinacalcet. 18Sections were immediately prepared from the excised rats injected with [F]F-ZW-cinacalcet and used for autoradiography and IHC staining of CSR, respectively. As shown in Panels B and C of Figure 19, autoradiography was well matched with IHC staining of CSR. The areas with the highest radioactivity uptake in autoradiography were consistent with the IHC stained parathyroid areas. Quantitative analysis of autoradiography and IHC staining was expressed as mean integrated density (Mean IntDen). Radiotracer uptake and CSR expression in parathyroid were significantly higher (P<0.01) than in thyroid background tissue, where the parathyroid / thyroid ratio was 6.60±1.28 in autoradiography and 3.76±1.13 in IHC staining (Panel D of Figure 19). Meanwhile, CSR expression levels were significantly higher than the radiotracer uptake in parathyroid and thyroid (r=0.93, 95% confidence interval 0.46-0.99, R 2 = 0.86) (Panel D of Figure 19).

[0200] To better preserve the structural integrity, some tissues were also fixed in formalin and embedded in paraffin to be sectioned for CSR-IHC staining. The highest expression level of CSR was observed in the parathyroid gland (Figure 20), which was consistent with the results of IHC staining using frozen tissues. In PET imaging, high radioactivity uptake was observed in the lungs. We also performed autoradiography and IHC staining of the lungs (with the heart) and muscle to verify the observations. The results showed that the lungs had extremely high expression of CSR compared to the heart and muscle (Figure 21), and were in good agreement with the data of autoradiography (Figure 21), IHC staining, and H&E staining. This confirmed the [ 18 High uptake of [F]F-cinacalcet was described.

[0201] Clinical PET / MRI imaging of the parathyroid gland in non-human primates. To further facilitate future clinical applications, PET / MRI scans were performed in non-human primates (NHPs) (Figure 22). 18 F]F-ZW-cinacalcet demonstrated clear tissue accumulation in the parathyroid region.

[0202] In vivo testing. 19 An in vivo acute toxicity study of [F]F-cinacalcet was performed using JAX Swiss Outbred mice. Mice were treated with cold standard [ 19 Overdose of [F]F-cinacalcet (57.6 μg, radioactive tracer [ 18 [F]F-cinacalcet (~1000 times the dose) was intravenously injected. Plasma samples were collected and analyzed at various time points. No animal deaths or significant health changes were observed during the study period. Alkaline phosphatase (ALP) and alanine transaminase (ALT) are two important biological indicators associated with liver function. Plasma test results showed no significant changes in ALP and ALT levels in treated mice (Table 1). A small but significant increase in AST was observed 1 hour after injection, which rapidly decreased within 24 hours and returned to normal levels after 2 weeks. It is very common to see a transient increase in blood AST after drug administration, which usually has no real pathological significance. Blood urea nitrogen (BUN) and creatinine are two important indicators for evaluating renal function. 19 In mice injected with [F]F-cinacalcet, no significant changes were observed in the levels of BUN and creatinine compared to vehicle controls. In addition, histopathological staining was performed on kidney, liver and heart tissues harvested at different time points. As shown in Figure 23, 19No obvious pathological changes were observed in mice treated with [F]F-cinacalcet. The basic structures of glomeruli, renal tubules, hepatic central lobular veins, and myocardial fibers remained intact. All these results suggest that the cold standard [ 19 Overdoses of F]F-cinacalcet did not show acute toxicity in mice, and the radioactive probe [ 18 The biological safety of [F]F-cinacalcet was shown to be well within acceptable limits.

[0203] [Table 1]

[0204] ALP: alkaline phosphatase (U / L), ALT: alanine aminotransferase (U / L), AST: aminotransferase (U / L), BUN: blood urea nitrogen (mg / dL), creatinine (mg / dL).

[0205] In summary, cinacalcet, which acts as a mineralizing agent, is commonly used as a therapeutic agent to treat PHPT. Given the high expression of CSR in parathyroid cells, 18 F-labeled cinacalcet was hypothesized to act as a PET agent for parathyroid detection. Previous attempts have been made to generate PET agents based on cinacalcet, but it is believed that the fast metabolism of the previously labeled agents may be the main cause of suboptimal results. In fact, the availability of novel PET agents may be limited due to the lack of efficient and easy labeling methods to modify biologically active small molecules / drugs. Herein, a highly innovative photoredox technique was used, which allows the direct conversion of cinacalcet into a PET agent through arene CH fluorination.

Claims

1. A calcium-sensing receptor (CSR) ligand labeled with a radioisotope, wherein the CSR ligand contains an aromatic ring and the radioisotope is directly bonded to the aromatic ring at one or more positions of the aromatic ring, the CSR ligand labeled with the radioisotope.

2. wherein the radioisotope is 18 F and / or 11 C, and the CSR ligand labeled with the radioisotope according to claim 1.

3. The aforesaid 11 C is 11 CN, 11 COOH and / or 11 CH 3 The CSR ligand labeled with the radioactive isotope according to claim 2, wherein the CSR ligand is as described above.

4. The CSR ligand labeled with the radioisotope according to any one of claims 1 to 3, wherein the aromatic ring is an arene ring.

5. The CSR ligand labeled with the radioisotope according to claim 4, wherein the arene ring is a naphthalene ring and / or a phenyl ring.

6. The ligand is directly bonded to the naphthalene ring at the 2-position and / or 4-position 18 The radiolabeled CSR ligand according to claim 5, comprising F.

7. wherein the ligand is directly bonded to the phenyl ring at the 2- and / or 4-position 18 The radiolabeled CSR ligand according to claim 5, comprising F.

8. The ligand is directly bonded to the naphthalene ring at the 2-position and / or 4-position 11 The radiolabeled CSR ligand according to claim 5, comprising C.

9. wherein the ligand is directly bonded to the phenyl ring at the 2-position and / or 4-position 11 The radiolabeled CSR ligand according to claim 5, comprising C.

10. wherein the ligand is directly bonded to the naphthalene ring at the 4-position 18 The radiolabeled CSR ligand according to claim 6, comprising 18 F.

11. The following formula II: 【Chemical 1】 wherein Ar is a substituted aryl and Y is an alkyl. The CSR ligand labeled with the radioisotope according to any one of claims 1 to 3, comprising the same.

12. Ar is selected from the group consisting of the following: 【Chemical formula 2】 Here, X is O, S, NH or CH 2 and R is aryl, alkyl, halogen atom, CF 3 , NO 2 , COOMe, OH, OMe, alkene or alkyne The CSR ligand labeled with the radioisotope according to claim 11, selected from the group consisting of the same.

13. The following formula III: [Chemical 3] The CSR ligand labeled with the radioisotope according to claim 12, comprising the same.

14. The following: 【Chemical Formula 4】 The CSR ligand labeled with the radioisotope according to any one of claims 1 to 3, selected from the group consisting of the same.

15. The following formula I ( 18 F-sinacalcet): 【Chemical Formula 5】 The CSR ligand labeled with the radioisotope according to claim 12, comprising the same.

16. The following: 【Chemical Formula 6】 The CSR ligand labeled with the radioisotope according to any one of claims 1 to 3, comprising a formula selected from the group consisting of the same.

17. The following: 【Chemical Formula 7】 The CSR ligand labeled with the radioisotope according to any one of claims 1 to 3, comprising a formula selected from the group consisting of the same.

18. The following formula XI (evocalcet): 【Chemical Formula 8】 wherein the radioisotope is directly bonded to naphthalene at the 4-position. The CSR ligand labeled with the radioisotope according to any one of claims 1 to 3, comprising the same.

19. The following: 【Chemical Formula 9】 The CSR ligand labeled with the radioisotope according to claim 18, selected from the group consisting of the same.

20. The following: 【Chemical 10】 The CSR ligand labeled with the radioisotope according to any one of claims 1 to 3, selected from the group consisting of the same.

21. 18 The following formula XX (etelcalcetide hydrochloride) directly linked to F: 【Chemical 11】 A calcium-sensing receptor (CSR) ligand labeled with a radioisotope, which comprises

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