Conjugates and their use as imaging agents

Abstract

The present invention relates to a compound of formula (I) or a pharmaceutically acceptable salt, ester, prodrug or solvate thereof, the use of the compound and a method for preparing the compound, wherein A is -As(OH)2 or an arsenic oxide equivalent group; each of R1, R2, R3 and R4 is independently selected from H, X, OH, NH2, CO, SCN, CH2NH, -NHCOCH3, -NHCOCH2X or NO, and X is a halogen; R5 is -NHCH2COOH, OH or OR6, wherein R6 is C 1‑5 Straight or branched alkyl; Z is a radioactive isotope with a half-life of less than 4 days. The present invention also relates to a diagnostic method using the compound.

Description

Technical Field

[0001] The present invention broadly relates to radiolabeled conjugates according to formula (I) as defined herein and compounds according to formula (II) as defined herein. The present invention further relates to the use of such radiolabeled conjugates in imaging cell death, diagnosis and treatment of disorders associated with cell death, and methods of producing such radiolabeled conjugates. Background Art

[0002] Cell death plays an integral role in cell renewal. Imbalance in cell death, characterized by a significant increase or decrease in cell death relative to cell regeneration, is often associated with disease. For example, excessive cell death is a feature of vascular disease, neurodegenerative diseases, myelodysplastic syndrome, ischemia / reperfusion injury, organ transplant rejection, and neoplastic conditions including tumors and cancer. In particular, cancer is caused by an imbalance between the rate of cell proliferation and survival in a tissue.

[0003] Visualization of cell death therefore has the potential to be a very useful tool for diagnosing and treating a variety of conditions associated with abnormal levels of cell death, as well as for assessing and monitoring cell death, for example, during drug development and testing for tissue toxicity of a given substance. For example, in oncology, successful treatments control cancer cell growth by inhibiting cell proliferation and / or promoting cell death, and the ability to directly image cell death as a means of assessing treatment response is highly desirable, but not available. Currently, imaging used to assess treatment response in oncology (via anatomical techniques such as computed tomography (CT) and magnetic resonance imaging (MRI)) assesses cell death indirectly, either by reduction in tumor size, or by reduction in metabolic activity, most commonly glucose metabolism, via positron emission tomography (PET). While these techniques are widely and routinely used in oncology, they assess cell death indirectly and are therefore subject to false-positive and false-negative results. In addition, these techniques cannot assess cell death in real time and are usually not performed until at least several weeks after the start of treatment (for example, positron emission tomography using 2-fluoro-2-deoxyglucose (FDG PET / CT) is usually not performed until two cycles of chemotherapy - usually 6 to 8 weeks after the start of treatment). In addition, changes in tumor size measured by CT, such as using the Response Evaluation Criteria in Solid Tumours (RECIST1.1), are usually but not always correlated with treatment response. After the start of treatment (usually taking several months), tumor size slowly decreases, and in some cases, despite responding to treatment, it may not decrease at all. Attempts to directly image cell death are limited, such as using derivatives of annexin V and radiolabeled caspase 3 / 7 inhibitors, but these are hampered by complex and expensive production, poor biodistribution, high physiological uptake in particular in the blood and normal tissues / organs (especially the liver and intestine), poor tissue permeability, and the inability to reliably detect tumor cell death after treatment. Given the lack of methods to reliably monitor tumor cell death in response to in vivo treatment, little is known about the kinetics of cell death.

[0004] For these reasons, a method to directly assess tumor cell death in near real time (within a few days of treatment initiation) could be highly beneficial in clinical and oncology research.

[0005] Particularly advantageous imaging agents would allow rapid, continuous imaging commensurate with the time course of cell death, e.g., following administration of a cancer therapy that results in tumor cell death. Such imaging agents would allow visualization of changes in cell death on biologically and clinically relevant time scales. It would therefore be desirable to provide a convenient and sensitive imaging agent that allows noninvasive, effective, and accurate visualization of cell death in a manner and time frame suitable for disease diagnosis and treatment. Summary of the invention

[0006] According to a first aspect, the present disclosure provides a compound of formula (I) or a pharmaceutically acceptable salt, ester, prodrug or solvate thereof,

[0007]

[0008] wherein A is -As(OH)2 or an arsenic oxide equivalent; each of R1, R2, R3 and R4 is independently selected from H, X, OH, NH2, CO, SCN, CH2NH, -NHCOCH3, -NHCOCH2X or NO, and X is a halogen; R5 is -NHCH2COOH, OH or OR6, wherein R6 is C 1-5 Straight or branched chain alkyl; Z is a radioactive isotope with a half-life of less than 4 days. -

[0009] Compounds according to the present disclosure can be used to image cell death in vitro and in vivo. These compounds allow accurate, sensitive and non-invasive detection and measurement of cell death. In particular, such compounds can be used for diagnosis, monitoring and evaluation of the treatment of various diseases and conditions in which cell death is a relevant factor. The radiolabeled compounds of the present invention can be easily synthesized for in vivo use, showing favorable biodistribution, imaging properties and radiation dosimetry, and due to the half-life of the radioisotope used, allow visualization of cell death on a clinically relevant time scale.

[0010] In some embodiments, each of R1, R2, R3 and R4 is H. In some embodiments, R5 is NHCH2COOH. In some embodiments, the compound is a compound of formula (Ia) or a pharmaceutically acceptable salt, ester, prodrug or solvate thereof,

[0011]

[0012] wherein A is as defined above.

[0013] In some embodiments, Z has a half-life of less than 1 day. In some embodiments, Z has a half-life of less than 4 hours. In some embodiments, Z has a half-life of less than 2 hours. In some embodiments, Z is 68 Ga.

[0014] 68 Ga has a half-life of 68 minutes, which means it is particularly useful for visualizing cell death via PET; the use of this short-lived positron-emitting radioisotope allows for frequent, serial and quantitative imaging.

[0015] Particularly preferred compounds are those of formula (I) wherein Z is 68Ga, R1-R4 are H, R5 is -NHCH2COOH, and A is As(OH)2. Such an embodiment provides the advantages described above: easy synthesis, synthesis from readily available and inexpensive starting materials, exhibiting good biodistribution, low uptake by normal organs, favorable imaging properties, favorable radiation dosimetry, and a short half-life suitable for continuous repeated imaging by positron emission tomography.

[0016] The present disclosure provides compounds according to the first aspect for use as imaging agents, for example as imaging agents in positron emission tomography.In a particular embodiment, the present disclosure provides compounds according to the first aspect for use in visualizing cell death.

[0017] According to a second aspect, the present disclosure provides a pharmaceutical composition comprising a compound according to the first aspect and a pharmaceutically acceptable carrier, excipient, diluent, vehicle and / or adjuvant.

[0018] According to a third aspect, the present disclosure provides a compound of formula (II) or a pharmaceutically acceptable salt, ester, prodrug or solvate thereof,

[0019]

[0020] wherein A is -As(OH)2 or an arsenic oxide equivalent; each of R1, R2, R3 and R4 is independently selected from H, X, OH, NH2, CO, SCN, CH2NH, -NHCOCH3, -NHCOCH2X or NO, and X is a halogen; R5 is -NHCH2COOH, OH or OR6, wherein R6 is C 1-5 Straight or branched alkyl. Such compounds can be used to prepare the compounds according to the first aspect above, and can be converted into compounds of formula (I) by radiolabeling with radioactive isotopes.

[0021] In some embodiments, each of R1, R2, R3 and R4 is H. In some embodiments, R5 is NHCH2COOH. In some embodiments, the compound of formula (II) is a compound of formula (IIa) or a pharmaceutically acceptable salt, ester, prodrug or solvate thereof,

[0022]

[0023] wherein A is as defined in formula (II).

[0024] According to a fourth aspect, the present disclosure provides a use of a compound according to the first aspect as an imaging agent. The imaging agent can be used for positron emission tomography. The imaging agent can be used for visualizing cell death.

[0025] According to a fifth aspect, the present disclosure provides a compound of the first aspect for use in therapy. The compound can be used in the treatment of a disorder associated with altered cell death and / or a treatment that causes altered cell death.

[0026] According to a sixth aspect, the present disclosure provides a compound of the first aspect for in vivo diagnosis. The compound can be used for diagnosis of a disorder associated with changes in cell death and / or treatment leading to changes in cell death.

[0027] The compounds used according to the fifth or sixth aspect may be used to treat or diagnose a neoplastic disorder or an autoimmune disorder. The neoplastic disorder may be a tumor. The neoplastic disorder may be cancer.

[0028] According to a seventh aspect, the present disclosure provides a method for diagnosing or treating a disease of a subject or visualizing cell death in a subject, wherein the disease is associated with a change in cell death and / or the treatment of the disease results in a change in cell death, the method comprising administering an effective amount of a compound according to the first aspect. In some embodiments, the disease is a neoplastic disease or an autoimmune disease. The method may further include performing a positron emission tomography scan on the subject after administering the compound according to the first aspect. A plurality of positron emission tomography images may be collected after administering the compound according to the first aspect. In some embodiments, the collection of a plurality of images may allow a more accurate and quantifiable assessment of cell death, because the difference in cell death within a given time period may be determined rather than an absolute value.

[0029] The compound according to the first aspect may be administered intravenously.

[0030] In the method according to the seventh aspect, the neoplastic disorder may be a tumor.The neoplastic disorder may be cancer.

[0031] According to an eighth aspect, the present disclosure provides a method for evaluating a subject's response to a treatment intended to cause a change in the level of cell death, comprising: administering a treatment; administering a compound according to the first aspect; and visualizing cell death. In some embodiments, cell death is visualized by performing positron emission tomography on the subject. In some embodiments, the treatment is chemotherapy, radiation therapy, targeted therapy, or immunotherapy, or a combination thereof.

[0032] According to a ninth aspect, the present invention provides a method for preparing a compound according to the first aspect, wherein Z is 68 Ga, the method comprises 68 Ga is eluted onto a strong cation exchange column; the strong cation exchange column elutes a mixture comprising the compound of formula (II) and a buffer, wherein the pH of the buffer is about 4.5. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Exemplary embodiments of the present disclosure are described herein by way of non-limiting examples only, with reference to the following drawings.

[0034] Figure 1 Shows NODAGA-GSAO and 68 Structure of Ga-NODAGA-GSAO.

[0035] Figure 2 The one used in Example 2 68 Schematic diagram of the Ga-NODAGA-GSAO radiolabeling system

[0036] Figure 3 is the radio-HPLC chromatogram of the final product produced in Example 2.

[0037] Figure 4 is the final product produced in Example 2 after reaction with 2,3-dimercapto-1-propanol (DMP) (with Figure 4 Radio-HPLC chromatogram of the same product).

[0038] Figure 5 Shown in 68 In healthy male rats, 1 and 2 hours after administration of Ga-NODAGA-GSAO 68 Biodistribution of Ga-NODAGA-GSAO (%ID / g).

[0039] Figure 6 The administration of tracer ( 68 a) 1 hour and b) 2 hours after 68 Maximum intensity projection of Ga-NODAGA-GSAO PET CT scan.

[0040] Figure 7 Eight time points after injection for patient 1 are shown. 68 Maximum intensity front projection of Ga-NODAGA-GSAO PET.

[0041] Figure 8 Eight time points after injection for patient 2 are shown. 68 Maximum intensity front projection of Ga-NODAGA-GSAO PET.

[0042] Fig. 9 Eight time points after injection in patient 3 are shown. 68 Maximum intensity front projection of Ga-NODAGA-GSAO PET.

[0043] Fig.10 Eight time points after injection in patient 4 are shown. 68Maximum intensity front projection of Ga-NODAGA-GSAO PET.

[0044] Fig.11 Shows 68 Biodistribution of Ga-NODAGA-GSAO in normal organs of patient 1 over time.

[0045] Fig.12 Shows 68 Biodistribution of Ga NODAGA GSAO in selected normal tissues and tumors of patient 1.

[0046] Fig.13 Shows 68 Biodistribution of Ga NODAGA GSAO in selected normal tissues and tumors of patient 2.

[0047] Fig.14 Shows 68 Biodistribution of Ga NODAGA GSAO in selected normal tissues and tumors of patient 3.

[0048] Fig.15 Shows 68 Biodistribution of Ga NODAGA GSAO in selected normal tissues and tumors of patient 4.

[0049] Fig.16 Shows objects 1-4 68 Biodistribution of Ga NODAGA GSAO in selected normal tissues (mean SUV ± SD).

[0050] Fig.17 Shows objects 1-4 68 Blood pool activity of Ga NODAGA GSAO and 68 Ga NODAGA GSAO is taken up into tumor deposits.

[0051] Fig.18 FDG-PET ( Fig.18 A) and after administration of 205MBq CDI ( 68 CDI-PET (Ga NODAGA GSAO) was performed 60 minutes after Fig.18 B) Maximum intensity front projection image. The tumor was surgically removed, fixed, and adjacent sections were stained for apoptotic cells ( Fig.18 C, brown TUNEL staining, a and b) or morphological staining by hematoxylin and eosin ( Fig.18 C, c and d). DETAILED DESCRIPTION

[0052] 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 the present disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, typical methods and materials are described.

[0053] Throughout the specification, unless the context requires otherwise, the word "comprise" or variations such as "include" or "comprising" will be understood to imply the inclusion of the stated steps or elements or integers, or groups of steps or elements or integers, but not the exclusion of any other steps or elements or integers, or groups of elements or integers. Therefore, in the context of this specification, the term "comprises" means "mainly including but not necessarily only including".

[0054] In the context of this specification, the terms "a" and "an" refer to one or more than one (ie, at least one) of the grammatical object of the article. For example, "an element" refers to one element or more than one element.

[0055] In the context of this specification, the term "about" is understood to refer to a numerical range that a person skilled in the art considers to be equivalent to the stated numerical value in the context of achieving the same function or result.

[0056] In the context of this specification, reference to a numerical range disclosed herein (e.g., 1 to 10) also includes reference to all rational numbers within that range (e.g., 1, 1.1, 2, 3, 3.9, 4, 5, 6, 6.5, 7, 8, 9, and 10) and any range of rational numbers within that range (e.g., 2 to 8, 1.5 to 5.5, and 3.1 to 4.7), and thus, all subranges of all ranges explicitly disclosed herein are hereby explicitly disclosed. These are only examples of what is specifically intended, and all possible combinations of numerical values ​​between the lowest and highest values ​​recited are considered to be explicitly stated in this application in a similar manner.

[0057] As used herein, the term "and / or" means "and" or "or" or both.

[0058] As used herein, the term "subject" refers to any mammal, including but not limited to livestock and other farm animals (e.g., cattle, goats, sheep, horses, pigs, and chickens), performance animals (e.g., racehorses), companion animals (e.g., cats and dogs), laboratory test animals, and humans. Typically the subject is a human.

[0059] As used herein, the terms "treating / treatment / treating", "reduce / reducing", and "prevent / preventing / prevention", etc. refer to any and all applications that remedy or otherwise hinder, delay or reverse the progression of an infection or disease, or at least one symptom of an infection or disease, including reducing the severity of an infection or disease. Thus, the term "treat / treatment / treating" does not necessarily imply that a subject is treated until the infection is completely eliminated or recovered from the disease. Similarly, the term "prevent / preventing / prevention", etc. refers to any and all applications that prevent an infection or disease from occurring or otherwise delay the onset of an infection or disease.

[0060] The term "optionally" is used herein to indicate that a subsequently described feature may or may not exist, or a subsequently described event or circumstance may or may not occur. Thus, this specification will be understood to include and encompass embodiments in which the feature exists and embodiments in which the feature does not exist, and embodiments in which the event or circumstance occurs and embodiments in which the event or circumstance does not occur.

[0061] As used herein, the terms "effective amount" and "effective dose" include within their meaning an amount or dose of a compound that is non-toxic but sufficient to provide the desired effect. The exact amount or dose required will vary from subject to subject, depending on factors such as the species being treated, the age and general condition of the subject, the severity of the condition being treated, the specific compound being administered, and the mode of administration, among others. Thus, it is not possible to specify an exact "effective amount" or "effective dose." However, for any given situation, an appropriate "effective amount" or "effective dose" can be determined by one of ordinary skill in the art using only routine experimentation.

[0062] In the context of this specification, the term "arsenic oxide" refers to the group -As=O. Groups written as -As=O and -As(OH)2 are considered equivalent.

[0063] As used herein, the term "arsenic oxide equivalent" refers to any dithiol-reactive species that exhibits substantially the same affinity for dithiols as -As=O or As(OH)2, and the term includes, for example, groups including transition elements and any trivalent arsenic that hydrolyzes to -As=O or -As(OH)2 when dissolved in an aqueous medium (e.g., cell culture buffers and fluids contained in the treated organism). Typically, arsenic oxide equivalents include dithiol-reactive entities such as As, Ge, Sn, and Sb species. Arsenic oxide equivalents are expected to exhibit the same or substantially the same activity as the corresponding arsenic oxide.

[0064] The term "bifunctional chelator" refers to a chemical moiety comprising a chelating portion capable of binding metals or other ions (e.g., radionuclides) and a chemically reactive functional group for attachment to other chemical entities. In the context of the present application, the term "bifunctional chelator" refers to a related compound prior to chelation with a metal or other ion and / or prior to reaction of the reactive functional group and a related compound once chelated to a metal or other ion and / or attached to another chemical entity via the reactive functional group, the relevant definition being readily apparent from the context. When not chelating a metal or other ion, the bifunctional chelator is suitable for chelating a metal or other ion.

[0065] As used herein, the term "C1-C5-alkyl" and the like refers to a saturated straight or branched hydrocarbon group containing 1 to 3, 1 to 6, or 1 to 12 carbon atoms, respectively. Examples of C1-C5-alkyl include, but are not limited to, methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, pentyl, isopentyl, and neopentyl.

[0066] "Pharmaceutically acceptable salts" refers to those salts which are suitable for use in contact with the tissues of humans and lower animals without excessive toxicity, irritation, allergic response, etc., and with a reasonable benefit / risk ratio, within the scope of sound medical judgment. Unless otherwise specified or understood otherwise from the context, the compounds mentioned herein should be understood to include their pharmaceutically acceptable salts.

[0067] Provided herein are compounds of formula (X) or pharmaceutically acceptable salts, esters, prodrugs or solvates thereof,

[0068]

[0069] wherein A is -As(OH)2 or an arsenic oxide equivalent; each of R1, R2, R3 and R4 is independently selected from H, X, OH, NH2, CO, SCN, CH2NH, -NHCOCH3, -NHCOCH2X or NO, and X is a halogen; R5 is -NHCH2COOH, OH or OR6, wherein R6 is C 1-5 A straight chain or branched alkyl group; Z is a radioactive isotope with a half-life of less than 4 days; and L is a bifunctional chelating agent that chelates Z.

[0070] In some embodiments, the compound of formula (X) is a compound of formula (Xa) or a pharmaceutically acceptable salt, ester, prodrug or solvate thereof:

[0071]

[0072] In a preferred embodiment, Z is 68In a preferred embodiment, L is a radioactive isotope known to chelate with high affinity at room temperature with a half-life of less than 4 days, in particular 68 Bifunctional chelating agent for Ga.

[0073] The present disclosure provides a compound of formula (I) or a pharmaceutically acceptable salt, ester, prodrug or solvate thereof,

[0074]

[0075] wherein A is -As(OH)2 or an arsenic oxide equivalent; each of R1, R2, R3 and R4 is independently selected from H, X, OH, NH2, CO, SCN, CH2NH, -NHCOCH3, -NHCOCH2X or NO, and X is a halogen; R5 is -NHCH2COOH, OH or OR6, wherein R6 is C 1-5 Straight or branched alkyl; Z is a radioactive isotope with a half-life of less than 4 days. In preferred embodiments, each of R1, R2, R3 and R4 is H. In some preferred embodiments, R5 is -NHCH2COOH. In particularly preferred embodiments, the mixture is a compound of formula (Ia) or a pharmaceutically acceptable salt, ester, prodrug or solvate thereof:

[0076]

[0077] In a preferred embodiment, A is an arsenic oxide group As(OH)2.

[0078] In compounds suitable for use in the present invention, the arsenic oxide group (-As(OH)2) can generally be replaced by an arsenic oxide equivalent.

[0079] Such compounds are based on 4-(N-(S-glutathionylacetyl)amino)phenylarsenous acid (GSAO) which has been radiolabeled with a radioisotope using a bifunctional chelator. In a particularly preferred embodiment, the bifunctional chelator is 2,2'-(7-(1-carboxy-4((2,5-dioxopyrrolidin-1-yl)oxy)-4-oxobutyl)-1,4,7-triazolidine-1,4-diyl)diacetic acid (NODAGA), as shown in formula (I) and formula (Ia).

[0080] GSAO is specifically taken up into dead and dying cells. Without wishing to be bound by theory, it is believed that GSAO is retained in the cytoplasmic matrix of dying and dying cells by forming a covalent bond between the As(III) ion and the thiol group of the proximal cysteine ​​residue. GSAO is a trivalent As(III) peptide that has been found to activate the mitochondrial permeability transition pore. GSAO is toxic to proliferating cells and inhibits angiogenesis in vivo (Don AS, Kisker O, Dilda P et al (2003) Apeptide trivalent arsenical inhibits tumor angiogenesis by perturbing mitochondrial function in angiogenic endothelial cells. Cancer Cell 3: 497–509), but is non-toxic to resting endothelial cells in vitro. Conjugation of the γ-glutamyl residues of GSAO to fluorophores or radionuclides, as in the present invention, results in the loss of its anti-angiogenic effect and the acquisition of the ability to recognize dying cells. When the integrity of the plasma membrane is compromised, the GSAO conjugate enters and binds to intracellular proteins, primarily the 90 kDa heat shock protein (Hsp90) (Park D, Don AS, Massamiri T et al (2011) Non-invasive imaging of cell death using an Hsp90 ligand. J Am Chem Soc 133: 2832–2835); this protein is highly abundant in the cytoplasmic matrix, accessible only when the integrity of the cell membrane is compromised during cell death, and is upregulated in many malignancies (Hahn JS. The Hsp90 chaperone machinery: from structure to drug development. BMB Rep. 2009; 42(10): 623-30). The As(III) motif of GSAO cross-links the unpaired thiols of Cys597 and Cys598 of Hsp90 to form a stable cyclic dithioarsenite.

[0081] Therefore, compounds according to the present disclosure that are labeled with a radionuclide moiety can be used to image cell death in vitro and in vivo. In particular, radiolabeled conjugates as described herein can be used to diagnose, treat, and monitor conditions associated with changes in cell death, such as neoplastic diseases or autoimmune diseases, such as tumors or, for example, cancer. The radiolabeled compounds of the present invention provide one or more advantages, can be easily synthesized for in vivo use using readily available and inexpensive materials, show good biodistribution and imaging properties and radiation dosimetry, and are non-invasive imaging and means for measuring cell death. Embodiments of the present disclosure can image treatment responses earlier and in situations that were not possible before (e.g., immediately after treatment begins), and can enable image-guided personalized therapy, which is currently not possible because existing imaging modalities are not accurate or fast enough.

[0082] The use of radioisotopes with a half-life of less than 4 days in the compounds of the present disclosure allows for the assessment of cell death on a practical and clinically relevant time scale. Z can be, for example 11 C. 64 Cu, 13 N. 15 O. {Al 18 F} 2+ , 68 Ga, 89 Zr, 82 Rb or 99 In a preferred embodiment, the half-life of Z is less than 1 day, such as less than 12 hours, such as less than 8 hours, such as less than 6 hours, such as less than 4 hours, or such as less than 2 hours. In a preferred embodiment, the compounds of the present disclosure are suitable for positron emission tomography (PET). In a particularly preferred embodiment, Z is 68 Ga. 68 Ga has a half-life of 68 minutes, which means it is particularly useful for visualizing cell death via PET; the use of this short-lived positron-emitting radioisotope allows imaging on a practical and clinically relevant time scale (i.e., no long wait is required after administration to obtain an image). In addition, in some cases, such a short half-life allows frequent continuous and quantitative imaging. That is, repeated imaging can be performed, allowing accurate changes in cell death to be recorded over a period of time, such as before and after the administration of chemotherapeutic agents or other treatments that induce cell death (e.g., radiotherapy, targeted therapy, or immunotherapy, or a combination thereof). Repeated measurements of the same object allow for a more accurate assessment of cell death compared to single measurements made with reference to standard values ​​or images from different subjects. In contrast, if any changes in cell death are to be visualized, the use of isotopes with longer half-lives will require waiting for weeks between administration and imaging. In some alternative preferred embodiments, Z is {Al18 F} 2+ This radioisotope is particularly advantageous because 18 F is widely available and has a half-life that is short enough (109.7 minutes) to be particularly useful in positron emission tomography, as described above, yet long enough to facilitate the production and distribution of products containing the radioisotope without substantial decay.

[0083] In a particularly preferred embodiment, the compound of formula I is 68 Ga-NODAGA-GSAO (i.e., a compound of formula I, wherein Z is 68 Ga, R1-R4 are H, R5 is -NHCH2COOH, and A is As(OH)2). Such embodiments provide the advantages described above: easy synthesis, synthesis from readily available and inexpensive starting materials, exhibiting good biodistribution, low uptake by normal organs, favorable imaging properties, favorable radiation dosimetry, being non-invasive in use, and having a short half-life suitable for continuous repeated imaging by positron emission tomography and imaging on a clinically relevant and practical time scale.

[0084] According to another aspect, the present disclosure also provides a compound of formula (Y) or a pharmaceutically acceptable salt, ester, prodrug or solvate thereof or a derivative thereof,

[0085]

[0086] wherein A is -As(OH)2 or an arsenic oxide equivalent; each of R1, R2, R3 and R4 is independently selected from H, X, OH, NH2, CO, SCN, CH2NH, -NHCOCH3, -NHCOCH2X or NO, and X is a halogen; R5 is -NHCH2COOH, OH or OR6, wherein R6 is C 1-5 A straight chain or branched chain alkyl group; L is a bifunctional chelating agent.

[0087] In some preferred embodiments, the compound of formula (Y) is a compound of formula (Ya),

[0088]

[0089] The present disclosure provides a compound of formula (Y), which is a compound of formula II or a pharmaceutically acceptable salt, ester, prodrug or solvate thereof,

[0090]

[0091] wherein A is -As(OH)2 or an arsenic oxide equivalent; each of R1, R2, R3 and R4 is independently selected from H, X, OH, NH2, CO, SCN, CH2NH, -NHCOCH3, -NHCOCH2X or NO, and X is a halogen; R5 is -NHCH2COOH, OH or OR6, wherein R6 is C 1-5 Straight chain or branched chain alkyl.

[0092] Compounds of formula (Y) can be used in the synthesis of compounds of formula (X). In particular, compounds of formula (II) can be used to synthesize compounds of formula I by radiolabeling the NODAGA group. This synthesis is schematically represented in Scheme 1 below, using NODAGA-GSAO as the starting material and 68 Ga is exemplified as a radioactive isotope.

[0093]

[0094] Solution 1

[0095] In a preferred embodiment, each of R1, R2, R3 and R4 is H. In a further preferred embodiment, R5 is -NHCH2COOH. In a particularly preferred embodiment, the compound is a compound of formula (IIa) or a pharmaceutically acceptable salt, ester, prodrug or solvate thereof,

[0096]

[0097] wherein A is as defined in formula (II).

[0098] In a preferred embodiment, A is an arsenic oxide group As(OH)2.

[0099] In compounds suitable for use in the present invention, the arsenic oxide group (-As(OH)2) can generally be replaced by an arsenic oxide equivalent.

[0100] The present disclosure provides a method for preparing a compound of formula (I), comprising mixing a radioactive isotope with a half-life of less than 4 days with a compound of formula (II), wherein the compound of formula (I) or formula (II) may be any of those described above. In a preferred embodiment, the mixing is carried out at room temperature, i.e. without heating. ... 68 A method for preparing a compound of formula (I) of Ga, comprising: 68Ga is eluted onto a strong cation exchange column; the strong cation exchange column is eluted with a mixture comprising a compound of formula (II) and a buffer, wherein the pH of the buffer is about 4.5. In some embodiments, the mixing is performed at room temperature, i.e., without heating. In some embodiments, the compounds of formula (I) and formula (II) are compounds of formula (Ia) and formula (IIa), respectively. The present disclosure also provides a method for preparing a compound of formula (X), comprising mixing a radioactive isotope with a half-life of less than 4 days with a compound of formula (Y), wherein the compound of formula (X) or formula (Y) can be any of those described above. In a preferred embodiment, the mixing is performed at room temperature, i.e., without heating. The present disclosure provides a method for preparing Z is 68 A method for preparing a compound of formula (X) of Ga, comprising: 68 Ga is eluted onto a strong cation exchange column; the strong cation exchange column is eluted with a mixture comprising a compound of formula (Y) and a buffer, wherein the pH of the buffer is about 4.5. In some embodiments, the mixing is performed at room temperature, i.e., without heating.

[0101] The present disclosure further provides pharmaceutical compositions and / or therapeutic formulations, ie, compounds of the present disclosure are present together with a pharmaceutically acceptable carrier, excipient, diluent and / or vehicle.

[0102] For medical use, salts of the compounds according to the present disclosure may be used, including pharmaceutically acceptable salts, although other salts may be used in the preparation of the compounds or their pharmaceutically acceptable salts. Pharmaceutically acceptable salts are those salts that are suitable for use in contact with the tissues of humans and lower animals without excessive toxicity, irritation, allergic reaction, etc., and with a reasonable benefit / risk ratio within the scope of sound medical judgment.

[0103] Pharmaceutically acceptable salts are well known in the art.

[0104] For example, suitable pharmaceutically acceptable salts of compounds of the present disclosure can be prepared by mixing a pharmaceutically acceptable acid such as hydrochloric acid, sulfuric acid, methanesulfonic acid, succinic acid, fumaric acid, maleic acid, benzoic acid, phosphoric acid, acetic acid, oxalic acid, carbonic acid, tartaric acid or citric acid with a compound of the present invention. Suitable pharmaceutically acceptable salts of compounds of the present disclosure therefore include acid addition salts.

[0105] For example, SM Berge et al describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 1977, 66: 1-19. The salts can be prepared in situ during the final isolation and purification of the compounds of the present disclosure, or separately by reacting a free base function with a suitable organic acid. Representative acid addition salts include acetate, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrobromide, hydrochloride, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, toluenesulfonate, undecanoate, valerate, and the like. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like, as well as non-toxic ammonium, quaternary ammonium, and amine cations including, but not limited to, ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, and the like.

[0106] The present disclosure also provides prodrugs. In general, prodrugs will be functional derivatives of compounds of the present disclosure that are readily convertible in vivo into the desired (active) compound of the present disclosure, eg, an imaging agent, therapeutic agent, and / or diagnostic agent.

[0107] Typical procedures for the selection and preparation of prodrugs are known to the person skilled in the art and are described, for example, in H. Bundgaard (Ed), Design of Prodrugs, Elsevier, 1985.

[0108] Intermediates and final products can be post-processed and / or purified according to standard methods, for example, using chromatography, distribution methods, (re) crystallization, etc. Compounds, including their salts, can also be obtained in the form of solvates, in particular hydrates. In the context of the present invention, solvates refer to compounds according to the present disclosure in which they form complexes by coordination with solvent molecules in the solid or liquid state. Hydrates are specific forms of solvates coordinated with water. Crystals of compounds of the present invention may, for example, include solvents for crystallization. There may be different crystalline forms.

[0109] The present disclosure also relates to those forms of the process for preparing the compounds according to the present disclosure in which compounds obtainable as intermediates at any stage of the process are used as starting materials and the remaining process steps are carried out, or in which starting materials are formed under the reaction conditions or are used in the form of derivatives, for example in protected form or in the form of salts, or compounds obtainable by the process according to the present invention are produced under the process conditions and further processed in situ.

[0110] Single or multiple administrations of the compound or pharmaceutical composition can be carried out at a dosage level and pattern selected by the treating physician.In any event, the compound or pharmaceutical composition of the present disclosure should provide an amount of the compound sufficient to effectively treat or diagnose a patient or visualize cell death in a subject.

[0111] One skilled in the art will be able to determine by routine experimentation the effective, non-toxic amount of a compound or pharmaceutical composition used in the present invention necessary for detecting cells undergoing cell death and / or treating or preventing the conditions and diseases disclosed herein.

[0112] The compounds of the present disclosure may be administered at a dose of, for example, up to 300 μg, such as up to 250 μg, such as up to 200 μg, such as up to 150 μg, such as up to 100 μg, such as up to 50 μg. In some embodiments, the compounds of the present disclosure are administered at a dose of less than 50 μg, such as 10 to 50 μg.

[0113] Although the compounds of the present disclosure can be administered alone, it is generally preferred that the compounds be administered as pharmaceutical compositions / formulations. In general, pharmaceutical formulations of the compounds of the present disclosure can be prepared according to methods known to those of ordinary skill in the art and thus may include pharmaceutically acceptable carriers, excipients, diluents, vehicles and / or adjuvants.

[0114] The carrier, excipient, diluent, vehicle and adjuvant must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.

[0115] In some embodiments, the pharmaceutical composition of the present disclosure comprises a compound according to the present disclosure and one or more other components selected from ascorbic acid, sodium, phosphate, acetate and chloride. In some embodiments, the pharmaceutical composition comprises all such components. In a preferred form, the pharmaceutical composition of the compound of the present disclosure comprises an effective amount of a compound of the present disclosure and a pharmaceutically acceptable carrier, diluent and / or adjuvant, as shown in Example 3.

[0116] The pharmaceutical compositions of the present disclosure can be administered by standard routes.

[0117] In a particularly preferred embodiment, the compounds or pharmaceutical compositions of the present disclosure are administered intravenously. For administration as injectable solutions or suspensions, non-toxic parenterally acceptable diluents or carriers may include Ringer's solution, isotonic saline, phosphate-buffered saline, ethanol and 1,2-propylene glycol.

[0118] The present disclosure provides compounds and compositions according to the present disclosure for detecting, imaging and / or visualizing cell death. This use can be used for treatment or in vivo diagnosis, or can be used for healthy subjects. For example, compounds according to the present disclosure can be used to visualize cell death in an object by positron emission tomography (PET). When administered intravenously, compounds of the present disclosure will target dying cells and can be visualized by virtue of their radioactive labels, thereby providing information about the level of cell death in different parts of the object. Compounds according to the present disclosure can be used to provide a measure of cell death at a single time point, i.e., by performing a single PET scan. In some embodiments, more than one administration and / or scanning can be performed before and after applying a stimulus that induces cell death (e.g., chemotherapeutic drugs, radiotherapy, targeted therapy or immunotherapy, or a combination thereof) to assess changes in the level of cell death before and after applying the stimulus.

[0119] Visualization of this cell death can be used, for example, to assess the normal tissue toxicity of a substance, environmental condition, or activity (e.g., experimental treatment). Therefore, such compounds can be used for research and drug development. The compound can be used to assess cell death in drug screening in animal models of cancer and other diseases. These compounds can also be used to assess cell death in human tissues, such as during clinical trials, which is beneficial to patient safety, and by providing accurate feedback on the level of cell death at a specific dose, a faster dose escalation regimen may be allowed. This will allow a personalized risk adaptation approach to be adopted during the trial, which is helpful for all patients, especially patients with altered renal or liver function or patients at extreme ages. The compounds disclosed herein are particularly useful in late-stage oncology clinical trials and may help to understand the overall situation and time treatment response, such as drug administration and duration. For example, in the case of a relatively moderate overall response rate, the use of compounds may allow identification of response subgroups, thereby enabling optimization of future research. Such uses of compounds may also help to show regulatory agencies subgroups with significant responses to increase the possibility of potential regulatory and reimbursement approvals.

[0120] Provided herein are compounds according to the present disclosure for use in treatment and in vivo diagnosis, such as by PET imaging. Uses in treatment and in vivo diagnosis can be used for any disease associated with a change in the level of cell death or a disease whose treatment results in a change in the level of cell death. The change in cell death is a change (increase or decrease) in the level of cell death expected in the relevant area of ​​a healthy subject. For example, the compounds of the present disclosure can be used to diagnose neoplastic conditions, such as tumors, such as solid tumors and / or, for example, cancer. For example, a tumor may contain a high level of cell death, and therefore can be visualized using the compounds of the present disclosure. The compounds of the present disclosure can also be used to treat such conditions, by allowing visualization of changes in cell death and its level in response to the administration of treatment, to determine whether the treatment is successful. For example, the successful treatment of such conditions after the administration of a treatment intended to treat a neoplastic condition (such as a tumor, or, for example, cancer) can be determined by visualization of the increased level of cell death at the site of the compounds of the present disclosure. Similarly, the compounds of the present disclosure can be used to diagnose or treat autoimmune conditions in which autoimmunity causes cell death. Examples of autoimmune disorders include, but are not limited to, rheumatoid arthritis, systemic lupus erythematosus (SLE), multiple sclerosis, type 1 diabetes, Crohn's disease, vasculitis, and seronegative arthropathy. The compounds according to the present disclosure can be used to diagnose and / or monitor disorders, and to assess the success of any treatment applied.

[0121] Even where a diagnosis has been made and no treatment has been administered, but the disorder is associated with changes in the level of cell death, the compounds of the present disclosure may be used to monitor the disorder.

[0122] When treating conditions associated with cell death or treating conditions that cause changes in cell death, the compounds of the present disclosure can be used to customize or change the treatment applied, such as the intensity, type or duration of the treatment. Measurement of cell death may indicate whether a particular treatment is effective; when ineffective, an alternative dose or alternative treatment method may be used. When effective, treatment can be continued if necessary, or treatment can be reduced / stopped if necessary. For example, the compounds of the present disclosure can be used to visualize cell death in response to the applied treatment, and the treatment dose is adjusted accordingly based on the level of cell death. For example, it is determined that patients with little or no death of tumor cells after treatment will indicate the need to increase the dose or duration of the current treatment (upgrade), or change to a more intensive or multi-modal treatment to maximize the chance of cure or disease control. On the contrary, accurately assessing the response early in the treatment process will allow the duration or intensity of treatment to be reduced in cancer patients who respond well to avoid treatment-related morbidity and mortality (downgrade) without affecting cure or disease control. Assessment of cell death may lead to the adoption of new treatments, in which case the measurement of cell death after the initial treatment method indicates that the initial method is unsuccessful.

[0123] The present disclosure also provides methods for treating or diagnosing the above-mentioned conditions, or visualizing cell death, or monitoring such conditions by administering the compounds described herein. The present disclosure further provides the use of the compounds described herein in such methods and in the preparation of medicaments for treating such conditions. The present disclosure also provides the use of the compounds described herein as imaging agents (e.g., in PET) and for visualizing cell death.

[0124] The use of the compounds disclosed herein and the treatment or diagnosis methods (e.g., of the above-mentioned conditions) provided herein include administering an effective amount of a compound as described herein to a subject. The method may further include performing PET on the subject after administering a compound as described herein (e.g., immediately after administering a compound as described herein). In alternative embodiments, any suitable imaging method other than PET may be used to image the compounds as described herein.

[0125] In some embodiments, the PET scan is performed after a time interval of at least 10 minutes, such as at least 20 minutes, at least 30 minutes, at least 40 minutes, at least 50 minutes, or at least 1 hour, after the administration of the compound according to the present disclosure. In some embodiments, multiple PET scans can be performed at different times after administration. For example, the compound of the present disclosure can be administered, and the PET scan can be performed immediately after administration and about 30 minutes, about 1 hour, about 2 hours, and about 3 hours after administration. In some embodiments, the method of diagnosis or treatment includes administering a substance that induces cell death in tumor treatment, such as a chemotherapeutic agent, radiotherapy, targeted therapy, or immunotherapy, or a combination thereof, wherein the compound of the present disclosure can be administered to observe the effectiveness of the substance in inducing cell death. For example, a chemotherapeutic agent, radiotherapy, targeted therapy, or immunotherapy, or a combination thereof, can be administered to the subject together with the administration of the compound of the present disclosure, before or after the administration of the compound of the present disclosure. A PET scan can be performed after the administration of the compound to observe the cell death inducing activity of chemotherapy, radiotherapy, targeted therapy, or immunotherapy, or a combination thereof.

[0126] In some embodiments, the administered species (eg, a drug) may have the effect of reducing cell death, and thus visualization of cell death following administration of such species may indicate a reduction in cell death in the target area.

[0127] Certain administered species (e.g., drugs) may take some time to show their effects; therefore, visualization of cell death, such as by PET scanning, may be performed longer after administration of the species, such as 1 day, 3 days, 5 days, 1 week, 2 weeks, or a month after administration; in such cases, a compound according to the present disclosure may be administered prior to the scan in addition to or in lieu of administration of the drug or other species.

[0128] In some specific embodiments, the present disclosure provides a method for evaluating a subject's response to a treatment intended to cause a change in the level of cell death, comprising: administering a treatment; administering a compound according to the present disclosure; and visualizing cell death. In a specific embodiment, cell death is visualized by subjecting the subject to positron emission tomography. In a specific embodiment, the treatment intended to cause a change in the level of cell death is chemotherapy, radiotherapy, targeted therapy, or immunotherapy, or a combination thereof. In a treatment intended to cause an increase in the level of cell death in a specific part of the subject, such as chemotherapy, radiotherapy, targeted therapy, or immunotherapy, or a combination thereof, when a high level of cell death is visualized at the desired location, the assessment will show whether the treatment is successful. In some embodiments, a compound according to the present disclosure is administered before the treatment is administered and / or cell death is also visualized to allow comparison between the levels of cell death before and after the administration of the treatment. In this case, an increase in the level of cell death between the two visualizations may indicate a successful treatment. Conversely, a low level of cell death or a decrease in cell death may indicate that the treatment is unsuccessful or suboptimal. In some alternative embodiments, treatment is directed to reducing the level of cell death in a specific region of a subject; in such embodiments, low levels of cell death or reduced levels of cell death in the region of interest indicate successful treatment, while high levels of cell death or increased cell death indicate unsuccessful or suboptimal treatment.

[0129] In the above methods, the administration of the compounds of the present disclosure and the visualization of cell death can occur, for example, about 1 day, about 2 days, about 3 days, about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, and / or about 6 weeks after the administration of the treatment. In some embodiments, the administration of the compounds of the present disclosure and the visualization of cell death occur within 7 days of the administration of the treatment. In some embodiments, the administration of the compounds of the present disclosure and the visualization of cell death occur at least 4 weeks after the administration of the treatment. In some embodiments, the administration of the compounds of the present disclosure and the visualization of cell death occur more than once after the administration of the treatment. For example, in some embodiments, the administration of the compounds of the present disclosure and the visualization of cell death occur within 7 days and at least 4 weeks after the administration of the treatment.

[0130] In the above methods, visualization of cell death (e.g., by positron emission tomography) can occur after administration of a compound according to the disclosure, e.g., at least 10 minutes, at least 20 minutes, at least 30 minutes, at least 40 minutes, at least 50 minutes, at least 1 hour, or at least 90 minutes. For example, a compound of the disclosure can be administered and visualization can be performed, e.g., immediately after administration of a compound according to the disclosure, or about 30 minutes, about 1 hour, about 90 minutes, about 2 hours, or about 3 hours after administration.

[0131] The present disclosure relates to the above-mentioned methods, compounds according to the present disclosure for use in such methods, uses of compounds according to the present disclosure in such methods, and uses of compounds according to the present disclosure in the preparation of medicaments for use in such methods.

[0132] The present invention in a broad sense may also include, individually or collectively, the components, elements and features mentioned or indicated in the specification of the present application, as well as any or all combinations of any two or more of the components, elements or features, and wherein the specific integers mentioned herein have known equivalents in the art to which the present invention belongs, such known equivalents are deemed to be included herein as if individually set forth.

[0133] Reference in this specification to any previous publication (or information derived therefrom) or any known matter is not and should not be taken as an acknowledgement or approval or any form of suggestion that the previous publication (or information derived therefrom) or known matter forms part of the common general knowledge in the field of endeavor to which this specification relates.

[0134] The present disclosure will now be described with reference to the following specific examples, which should not be construed as limiting the scope of the invention in any way.

[0135] Example

[0136] Example 1

[0137] Synthesis of NODAGA-GSAO

[0138] a) GSAO was prepared using the method described by Park D, Don AS, Massamiri T et al (2011) Non-invasive imaging of cell death using an Hsp90 ligand. J Am Chem Soc 133: 2932-3835; 4-(N-(bromoacetyl)amino)phenylarsonic acid (BRAA) was synthesized from p-arsenic acid and bromoacetyl bromide, and BRAA was reduced to 4-(N-(bromoacetyl)amino)phenylarsonic acid (BRAO). BRAO was coupled with glutathione (GSH) to produce GSAO. GSAO was separated from unreacted BRAO and GSH by C18 chromatography.

[0139] b) Sodium bicarbonate and ultrapure water were purged with nitrogen for 30 minutes before use. Reaction setup and purification were performed under an inert nitrogen atmosphere. GSAO (20.0 mg, 36.5 μmol) obtained in step a) was dissolved in 0.1 N sodium bicarbonate (7.4 mL) at 4°C and stirred for 10 minutes.

[0140] c) NODAGA-NHS (2,2′-(7-(1-carboxy 4((2,5-dioxopyrrolidin-1-yl)oxy)-4-oxobutyl)-1,4,7-triazolidine-1,4-diyl)diacetic acid mono-N-hydroxysuccinimide ester) (34.5 mg, 47.0 μmol) obtained from CheMatech (Dijon, France) was dissolved in anhydrous dimethylformamide (DMF) (1 mL) and added dropwise to the reaction mixture obtained in step b) over 1 hour.

[0141] d) The reaction mixture was stirred for 4 hours, acidified by the addition of 1 M hydrochloric acid (1 mL), flash frozen in liquid nitrogen and freeze-dried.

[0142] NODAGA-GSAO purification

[0143] e) The residue from step d) was redissolved in degassed water (4 mL), filtered (0.45 μm), and purified by reverse phase high performance liquid chromatography (RP-HPLC). A gradient of 2-20% mobile phase B (0.2% trifluoroacetic acid (TFA) in acetonitrile) in mobile phase A (0.2% TFA in ultrapure water) was applied from 0 to 25 minutes. NODAGA-GSAO was eluted at 20.6 minutes. Samples were collected manually and each fraction was immediately purged with nitrogen.

[0144] HPLC was performed in a HPLC system with two LC-20AP pumps, SIL-10AP autosampler, SPD-20A UV / VIS detector and Shimadzu ShimPack GIS-C18 column (150 x 10.0 mm id, 5 μm, 4 mL / min1) (System A). Shimadzu LabSolutions software (Ver. 5.73) was used for data acquisition and processing.

[0145] f) The combined fractions were frozen at -20°C and lyophilized to give 7.3 mg of a white powder (yield 21.6%).

[0146] g) NODAGA-GSAO was dispensed in 54 μg aliquots per 100 μL water and stored at -20°C.

[0147] h) The purity (>95%) of the compound was verified by injecting NODAGA-GSAO solution (5 μL; about 17 mM in water) on a liquid chromatography-mass spectrometer (LC-MS) with mobile phase B (0.1% formic acid (FA) in acetonitrile) in mobile phase A (0.1% FA in mass spectrometry grade water) 2-2-50% at 0-5-45 minutes. NODAGA-GSAO was eluted at 19.4 minutes.

[0148] LC-MS was performed using an Agilent system (Santa Clara, CA, USA) consisting of a 1260 series quaternary pump with built-in degasser, a 1200 series autosampler, a thermostatic column oven, a diode array detector, a fraction collector, a 6120 series single quadrupole mass spectrometer, and an Agilent Zorbax Eclipse XDB-C18 column (150x4.6 mm inner diameter, 5 μm) at 30 °C (System B). The drying gas flow, temperature, and nebulizer were set to 12 L / min, 350 °C, and 35 psi, respectively. Agilent OpenLAB Chromatography Data System (CDS) ChemStation Edition (C.01.05) was used for data acquisition and processing. Electrospray ionization (ESI) was used to analyze aliquots (5 μL) in positive ion mode using a capillary voltage of 3500 V. Nuclear magnetic resonance (NMR) spectra were recorded using a Varian 400-MR NMR spectrometer (Lexington, MA, USA) in 5 mm Pyrex tubes (Wilmad, USA) operating at 399.73 MHz (1H) or 100.51 MHz (13C) at 24 °C using VnmrJ 3.1 software (Agilent Technologies, Santa Clara, CA, USA). 1 H and 13 C) Spectrum. Spectral data are reported in ppm (δ) and referenced to the residual solvent (deuterated dimethyl sulfoxide [DMSO-d6] 2.50 / 39.52 ppm).

[0149] i) The absorbance was measured at 210 and 254 nm and the respective area under the curve (AUC) was used to determine compound purity as a percentage of the total AUC compared to background.

[0150] Example 2

[0151] use 68 Radiolabeling of NODAGA-GSAO with Ga

[0152] a) The body of the BondElute SCX column is cut so that the barbed Luer thread is directly above the column medium when inserted to form a cartridge (hereinafter referred to as an SCX cartridge). The barbed Luer thread should fit securely and firmly into the cut barrel of the BondElute SCX column to form a gas-tight and liquid-tight sealed column.

[0153] b) The SCX cartridge was primed with 1 mL of 5.5 M HCl and then rinsed with 10 mL of water.

[0154] c) Purge the SCX cartridge with air.

[0155] d) An ascorbic acid solution (0.25 M) was obtained by dissolving 44 mg ascorbic acid in 1 mL water (Water Ultrapur, Merck).

[0156] e) Sodium acetate buffer (1.5 M CH3COONa·3H2O, pH 4.5) was prepared by dissolving 10.21 g CH3COONa·3H2O in water (Water Ultrapur, Merck). The pH was adjusted to 4.5 with glacial acetic acid and water was added to a total volume of 50 mL.

[0157] f) A vial of 54 μg of NODAGA-GSAO obtained in Example 1 was thawed and mixed with 100 μL of ascorbic acid solution (used as a free radical scavenger, as GSAO is sensitive to radiolysis and oxidation), 250 μL of sodium acetate buffer and 3.5 mL of water, and the mixture was transferred to a 10 mL vacuum glass reaction vial.

[0158] g) According to the supplier's instructions, 68Ge / 68 Ga was eluted onto the prepared SCX cartridge.

[0159] h) Purge the SCX cartridge with air.

[0160] i) Elute the contents of the SCX cartridge into a reaction vial with 500 μL of NaCl / HCl elution mixture followed by 0.5 mL of air using a B. Braun Sterican needle to minimize leaching of metal ions from the needle. The contents of the reaction vial were briefly mixed and reacted at room temperature for 10 minutes.

[0161] j) Add 3 mL of phosphate buffer to the reaction vial. Remove the contents of the reaction vial using a 10 mL syringe and pass it through a 0.22 μm filter into a new sterile bottle to obtain the final product for injection. No post-purification of the product was performed because 68 Ga-NODAGA-GSAO was not significantly retained on the C-18 cartridge, and a suitable biocompatible post-purification cartridge / solvent system has not yet been identified. 68 Ga-NODAGA-GSAO has high radiochemical purity and specific activity, exceeding 68 Current Release Requirements for Ga Radiopharmaceuticals.

[0162] k) A sterile closed radiolabeling system is used for the above procedures, which is the preferred system for preparation for human use and to minimize the risk of radioactive contamination to operators and the environment. Figure 2 ). This can also be automated using the Radiochemical Synthesis Module.

[0163] 68 Purity of Ga-NODAGA-GSAO

[0164] l) Evaluation of the HPLC system C at 9-9-60% mobile phase B (acetonitrile) in mobile phase A (0.1% TFA in ultrapure water) within 0-6-10 minutes using radioactivity detection 68 The radiochemical purity of Ga-NODAGA-GSAO (about 100 μL sample of the final product obtained in step h above) was more than the sum of all the radiation peaks greater than three times the background. 68 The AUC of the Ga-NODAGA-GSAO peak was used to determine radiochemical purity. The absorbance was also measured at 210 and 280 nm; however, the molar amount was below the limit of reliable absorbance detection and was therefore not used for purity assessment. 68 Ga-NODAGA-GSAO was eluted at a retention time of approximately 3 minutes and 55 seconds, as shown in FIG. Figure 3 The radioactive HPLC chromatogram of the final product is shown below: Region 1 corresponds to 68 Ga, region 2 corresponds to the oxidation products, and region 3 corresponds to 68 Ga-NODAGA-GSAO. Used in final products 68 The release standard of the radiochemical purity of Ga-NODAGA-GSAO is ≥91% (European Pharmacopoeia (2016) 01 / 2013: 2482 Gallium ( 68 Gallium (68Ga) Edotreotide injection correct 8.6. European Pharmacopeia, 9th edition, pages 1150-1152 European Pharmacopeia (2016) 01 / 2013: 2482 Gallium (68Ga) Edotreotide injection correct 8.6. European Pharmacopeia, 9th edition, pages 1150-1152 th edn,pp 1150-1152).

[0165] m) The final product was reacted by reacting 200 μL with 5 μL of DMP / DMSO solution at room temperature for 10 minutes with occasional stirring. 68 Further evaluation of the radiochemical purity of Ga-NODAGA-GSAO. Approximately 100 μL of this mixture was evaluated by HPLC system C at 9-9-60% mobile phase B (acetonitrile) in mobile phase A (0.1% TFA in ultrapure water) using radioactivity detection within 0-6-10 minutes. Approximately 100 μL of this mixture was evaluated by HPLC system C at 9-9-60% mobile phase B (acetonitrile) in mobile phase A (0.1% TFA in ultrapure water) using radioactivity detection within 0-6-10 minutes. The sum of all radiative peaks greater than three times the background was68 The AUC of the Ga-NODAGA-GSAO peak (retention time is about 9 minutes and 30 seconds) should be ≥91%; 68 The benzene-arsenic moiety of Ga-NODAGA-GSAO is combined, which will eliminate the usual 68 The Ga-NODAGA-GSAO peak (retention time about 3 minutes 55 seconds) and a new peak with a retention time of about 9 minutes 30 seconds are generated. This provides specific information about the radiochemical purity of the active GSAO and enables the distinction between 68 Ga-NODAGA-GSAO and other products, such as oxidative degradation products of GSAO. However, this is not accommodated by the release criteria that require minimizing product losses due to degradation. The radio-HPLC chromatogram obtained is shown in Figure 4 Shown: Region 1 corresponds to the unchelated 68 Ga, region 2 corresponds to the oxidation product, and region 3 corresponds to DMP 68 Ga-NODAGA-GSAO. --

[0166] n) Evaluation of colloidal contaminants by instant thin layer chromatography developed in 0.9% NaCl. The colloidal contaminants remain in place, while 68 Ga-NODAGA-GSAO has an Rf > 0.5. The release standard for colloidal contaminants with a total radioactivity Rf ≥ 0.5 is ≥ 90%.

[0167] o) The half-life was determined by performing a minimum of four measurements on a dose calibrator over 10 minutes. The release criterion used was a calculated half-life between 64 and 72 minutes (the half-life needed to be determined to confirm the absence of significant 68Ge breakthrough).

[0168] Sterility and pyrogenicity testing

[0169] p) Sterility and pyrogenicity were initially tested for three series of synthesis in an appropriately accredited laboratory to confirm that the sterility and pyrogenicity of the process complied with the pharmacopoeial guidelines (Ph. European (2016) 01 / 2013: 2482 Gallium ( 68 Ga) Edotreotide injection standard 8.6. European Pharmacopoeia, 9th edition, pages 1150-1152). The preparations were subsequently tested randomly at regular intervals.

[0170] Example 3

[0171] 68 Pharmaceutical formulation of Ga-NODAGA-GSAO

[0172] Compositions containing ingredients in the amounts listed in Table 1 below were prepared.

[0173] Ingredient name quantity unit ascorbic acid 4.4 mg sodium 95 mg Phosphate 109 mg Acetate 22 mg chloride 91 mg <![CDATA[ 68 Ga-NODAGA-GSAO]]> 200 MBq

[0174] Table 1

[0175] Example 4

[0176] 68 Biodistribution of Ga-NODAGA-GSAO

[0177] Biodistribution was studied in 10 healthy male rats aged 6-8 weeks (Lewis, Liverpool Hospital Animal Facility). Five rats were administered 68 Ga-NODAGA-GSAO. Rats were housed individually in cages with water-tight absorbent pads, and 5 rats were killed with a lethal dose of carbon dioxide 1 hour after administration. Blood samples were collected by cardiac puncture immediately after death. Then two of the 5 rats were imaged by PET CT (GE Discovery 710). PET CT scans included CT scans (80 kVp, 20 mA, spiral mode, reconstruction slice thickness 0.625 mm), followed by PET scans (2 beds, 7.5 minutes / bed, 256x256 reconstruction matrix, slice thickness 3.27 mm).

[0178] All rats were then dissected and organs were sampled, weighed and counted in a gamma counter, and cpm values ​​were converted to MBq using known standards. Activity in the remaining carcasses was measured in a dose calibrator.

[0179] The other 5 rats were given 68 Biodistribution studies were conducted two hours after the administration of Ga-NODAGA GSAO.

[0180] The injected activity was corrected by measuring the residual activity left in the syringe after injection in a dose calibrator. To correct for any dose extravasation from the injection site, the tail was harvested and the activity in the tail was subtracted from the administered activity. All calculations were decay corrected using the injection time as reference.

[0181] Biodistribution is expressed as %ID / g and %ID / organ. %Retained activity is the sum of all activity in all individually harvested organs and activity in the rest of the carcass as a percentage of the injected dose. %Recovered activity is the sum of all activity in all individually harvested organs and activity in the rest of the carcass and excreted activity in the impermeable pad as a percentage of the injected dose.

[0182] result

[0183] The mean rat weight was 17 g (range 120 to 229 g, standard deviation 32.2 g). The mean injected activity was 27.3 MBq (range 18.9 to 38.6 MBq, standard deviation 7.4 MBq).

[0184] For the 1 hour biodistribution group, the mean uptake time was 62.6 (range 60 to 65) minutes, and for the 2 hour biodistribution group, the mean uptake time was 122.2 (range 120 to 126) minutes.

[0185] Figure 5 Shown in 68 1 and 2 hours after administration of Ga-NODAGA-GSAO 68 Organ biodistribution of Ga-NODAGA-GSAO in healthy male rats (%ID / g).

[0186] like Figure 5 As shown, 68 The highest concentration of Ga-NODAGA-GSAO is in the kidneys. 68 The organs with the highest Ga-NODAGA-GSAO uptake were the kidney, liver, and small intestine. High kidney and liver uptake are consistent with renal excretion and hepatic metabolism, whereas small intestinal uptake may reflect uptake within dead and dying intestinal epithelial cells.

[0187] At 1 hour, 32.4% (range 24.9 to 38.2%, SD 5.6%) of the injected activity was retained, and at 2 hours, 21.4% (range 11.2 to 32.1%, SD 7.5%) of the injected activity was retained in the animals. The overall mean total recovered activity at 1 hour was 84.9% (range 55.3 to 107.9%, SD 19.0%) of the injected activity, and at 2 hours the total recovered activity was 75.3% (range 50.0 to 120.9%, SD 27.2%) of the injected activity.

[0188] Imaging

[0189] The results shown by PET CT images were consistent with the quantitative biodistribution data. Figure 6 Shown are the results of a) 1 hour and b) 2 hours after tracer administration. 68 Maximum intensity projection of a Ga-NODAGA-GSAO PET CT scan. Images taken 1 hour after tracer administration show high tracer concentrations in the kidneys ( Figure 6 Arrows i) in a) and b) show lower uptake in the liver (arrow ii)). In the mediastinum (arrow iii)), there is residual blood pool activity similar to that in the liver. In images taken 2 hours after administration ( Figure 6 b), The tracer concentration is also high in the kidneys, with lower levels of uptake in the liver. There is no longer visible blood pool activity in the mediastinum. In both sets of images, uptake is present in the small intestine (arrow iv)) and epiphysis (arrow v)), which may be due to specific uptake at sites of high physiological cell death.

[0190] Example 5

[0191] Radiation dosimetry

[0192] The biodistribution data derived above were used to estimate human radiation dosimetry using the method described by Stabin for a standard adult male (Stabin and Siegel 2003). The %ID / g for a given standard male organ was extrapolated from the rat biodistribution data using the following equation:

[0193]

[0194] Monoexponential clearance curves were fitted for each organ and the total remaining tissue using tools in the OLINDA / EXM software. 68 Rapid excretion of GaNODAGAGSAO, all excretion was assumed to be via urine (i.e., urine half-elimination times were calculated using a single exponential fit and were assumed to be 1-% of the total remaining activity at each time point). For the voiding bladder model, it was assumed that the patient would urinate 1 hour after administration.

[0195] The whole body effective dose was estimated to be 2.13E-02 mSv / MBq. Assuming an injection activity of 150 MBq, this results in a whole body effective dose of 3.2 mSv, which is lower than the dose of a diagnostic CT scan of the abdomen and lower than FDG-PET CT. The estimated doses to individual organs in humans are listed in Table 2 below (ULI = upper large intestine, LLI = lower large intestine).

[0196] Table 2

[0197] Target Organs Alpha Beta Photon total ED Cont. Adrenal glands 0.00E000 2.13E-03 1.89E-03 4.02E-03 2.01E-05 brain 0.00E000 8.15E-04 5.73E-04 1.39E-03 6.94E-06 breast 0.00E000 2.13E-03 8.94E-04 3.02E-03 1.51E-04 Gallbladder wall 0.00E000 2.13E-03 2.15E-03 4.28E-03 0.00E000 LLI wall 0.00E000 2.13E-03 5 09E-03 7.23E-03 8.67E-04 Small Intestine 0.00E000 5.18E-03 3 03E-03 8.21E-03 4.10E-05 Stomach wall 0.00E000 2.61E-03 1.61E-03 4.22E-03 5.06E-04 ULI Wall 0.00E000 2.53E-03 2.63E-03 5.16E-03 2.58E-05 Heart wall 0.00E000 3.09E-03 1.55E-03 4.65E-03 0.00E000 kidney 0.00E000 2.27E-02 3.56E-03 2.62E-02 1.31E-04 liver 0.00E000 4.76E-03 2.04E-03 6.80E-03 3.40E-04 lung 0.00E000 5.42E-03 1.38E-03 6.80E-03 8.16E-04 muscle 0.00E000 2.13E-03 2.04E-03 4.18E-03 2.09E-05 Ovaries 0.00E000 2.13E-03 4.89E-03 7.02E-03 1.40E-03 pancreas 0.00E000 3.87E-03 2 03E-03 5.90E-03 2.95E-05 Red pulp 0.00E000 1.49E-03 1.89E-03 3.38E-03 4.05E-04 Osteoblasts 0.00E000 3.31E-03 1.67E-03 4.98E-03 4.98E-05 skin 0.00E000 2.13E-03 1.06E-03 3.19E-03 3.19E-05 spleen 0.00E000 4.05E-03 1.86E-03 5.91E-03 2.95E-05 testis 0.00E000 2.21E-03 3.49E-03 5.69E-03 0.00E000 Thymus 0.00E000 4.48E-03 1.36E-03 5.84E-03 2.92E-05 thyroid 0.00E000 2.13E-03 1.15E-03 3.28E-03 1.64E-04 Bladder wall 0.00E000 2.84E-01 3.92E-02 3.24E-01 1.62E-02 Uterus 0.00E000 2.13E-03 9.42E-03 1.15E-02 5.77E-05 whole body 0.00E000 2.68E-03 1.93E-03 4.61E-03 0.00E000

[0198] discuss

[0199] As shown in the above experiments, 68 GaNODAGAGSAO has superior imaging properties and relatively little interference with the physiological activities of the kidney and liver. In addition, the rapid clearance indicates that imaging between 1 and 2 hours after injection is feasible, making it ideal for use 68 Ga (clinically based on 68 Imaging of somatostatin receptor expression in Ga was performed 45–90 min after injection). 68 GaNODAGAGSAO PET / CT image ( Figure 6 ) is noteworthy for visualization of uptake within the small and large intestine and within the epiphyses of long bones, which may represent uptake in areas of physiologically high cell death. The imaging findings were corroborated by the measured distribution, with uptake at 2 h post-injection being higher than at 1 h post-injection in some other organs (particularly the liver and kidney), suggesting that uptake in the intestine may represent specific binding rather than nonspecific tracer diffusion.

[0200] Estimated human radiation dosimetry was favorable, with an estimated total body effective dose of 0.021 mSv / MBq, which, assuming a standard injected dose of 150 MBq, would provide a total total body effective dose of 3.2 mSv. The dose limiting organ was the bladder wall, with a dose of 0.32 mSv / MBq.

[0201] These combined results show 68 Ga-NODAGA-GSAO could be a promising drug for in vivo imaging of dead and dying cells, and first human studies are warranted.

[0202] Example 6

[0203] Human studies

[0204] The following patients received 200 to 207 MBq 200 MBq 68 Ga-NODAGA-GSAO:

[0205] A 66-year-old male patient with esophageal squamous cell carcinoma

[0206] A 73-year-old woman with metastatic ovarian cancer

[0207] A 66-year-old man with metastatic squamous cell carcinoma of the skin

[0208] An 81-year-old woman presented with invasive ductal breast cancer.

[0209] All subjects tolerated the study well, with no serious adverse events or adverse events, either related or unrelated. There were no significant changes in any clinical, laboratory, or electrocardiographic parameters.

[0210] Biodistribution

[0211] Biodistribution data show that 68 GaNODAGAGSAO is rapidly distributed in the vasculature, with rapid initial clearance followed by a second, slower phase of clearance from the blood pool. Renal uptake and excretion are rapid.

[0212] For patient 1), the percentage of injected dose excreted in urine (%ID) averaged 30% (range 19 to 38%) before 2 hours and 48% (range 21 to 71%) after 3 hours. Figure 7 As shown, the figure shows 68Maximum intensity front projections of Ga-NODAGA-GSAO PET at 8 time points; maximum front projections of FDG PET are shown below for comparison. The location of the tumor is indicated by an arrow at each time point. Low levels of tracer uptake are seen in the remaining organs, which gradually decreases over time (except for the testes and large intestine). There is no significant hepatobiliary excretion. There is little activity in the brain, suggesting that it does not cross the blood-brain barrier to any extent. Imaging results from patients 2-4 are similarly shown in Figure 8 (Patient 2), Fig. 9 (Patient 3), Fig.10 (Patient 4).

[0213] Fig.11 Shows 68 Biodistribution of Ga-NODAGA-GSAO in normal organs over time in Patient 1. In the blood, there was an initial rapid decline in concentration followed by a second, slower phase of clearance. Most organs showed an early peak followed by a gradual decline similar to the second phase of blood elimination, with the exception of the large intestine and testes, which showed an initial increase in concentrations approximately 40 minutes after dosing followed by a slow decline. This may be due to the physiologically higher rate of cell death in these two organs. Note that the bladder wall was assessed separately.

[0214] The biodistribution patterns in organs and tissues were consistent between subjects 1 to 4 (e.g. Fig.16 All of this indicates that 68 Ga NODAGA GSAO is rapidly distributed through the blood pool after injection, with rapid renal uptake and excretion. One hour after injection, renal 68 GaNODAGA GSAO had the highest concentration (4.85±0.70; mean SUV±SD, SUV=standard uptake value), while other tissues and organs 68 Ga NODAGAGSAO levels are relatively low and are cleared over time. 68 Ga NODAGA GSAO had the second highest concentration (3.00 ± 0.62), followed by the blood pool (2.31 ± 0.37) and stomach (2.05 ± 1.34).

[0215] Figures 12 to 15 They are shown separately 68 Biodistribution of Ga NODAGA GSAO in selected normal tissues and tumors of patients 1 to 4. Note that tumor 2 is only available for patients 3 and 4, so Fig.12 and 13 The middle is blank. Fig.16 Biodistribution in selected normal tissues of subjects 1 to 4 is shown (mean SUV±SD).

[0216] Radiation dosimetry

[0217] The whole-body effective dose was estimated by drawing representative spherical volumes of interest within the organ, estimating the %ID / g for each organ, and then calculating the %ID / organ using the organ weights of a standard adult phantom.

[0218] 68 The effective whole-body dose of Ga NODAGA GSAO for subjects 1 to 4 ranged from 2.16 × 10 -2 to 3.38×10 - 2 mSv / MBq, for the regimen used in the first human study, the estimated effective whole-body dose ranged from 13.5 to 15.9 mSv. 68 Detailed organ dosimetry for Ga NODAGA GSAO (Tables 5 to 8). In all cases, the bladder was the dose-limiting organ. For subsequent human studies, fewer time points will be required, thereby reducing the need for low-dose CT, which will reduce the overall radiation dose. This dose level is comparable to many conventional medical imaging procedures that use ionizing radiation, including X-ray computed tomography (CT), SPECT / CT, and PET / CT scans.

[0219] Radiation dose was calculated using Olinda / EXM based on the organ biodistribution described above for subjects 1 to 4. Urinary excretion was modeled based on measurements of activity in collected urine samples and urine volume was measured from images.

[0220] Tables 3 to 6 show the 68 Estimates of individual organ and whole body radiation dosimetry for subjects 1 to 4 of Ga NODAGA GSAO in mSv / MBq (EDE cont. = Effective Dose Equivalent Contribution, ED cont. = Effective Dose Contribution). The estimated whole body dose from one low-dose CT and two ultra-low-dose CTs was 9.2 mSv.

[0221] Table 3 shows the radiation dosimetry estimates for Subject 1. The overall estimated radiation dose for Subject 1 was 14.5 mSv.

[0222] Table 3

[0223]

[0224]

[0225] Table 4 shows the radiation dosimetry estimate for Subject 2. The overall estimated radiation dose for Subject 2 was 13.9 mSv.

[0226] Table 4

[0227] Target Organs Alpha Beta Photon total EDE Cont ED Cont Adrenal glands 0.00E+00 9.53E-03 5.24E-03 1.48E-02 0.00E+00 7.38E-05 brain 0.00E+00 6.40E-04 1.46E-03 2.10E-03 0.00E+00 1.05E-05 breast 0.00E+00 9.48E-03 2.76E-03 1.22E-02 1.84E-03 6.12E-04 Gallbladder wall 0.00E+00 4.69E-03 5.27E-03 9.96E-03 0.00E+00 0.00E+00 LLI wall 0.00E+00 1.37E-02 7.16E-03 2.08E-02 1.25E-03 2.50E-03 Small Intestine 0.00E+00 1.27E-02 5.57E-03 1.83E-02 0.00E+00 9.14E-05 Stomach wall 0.00E+00 1.15E-02 4.97E-03 1.65E-02 0.00E+00 1.98E-03 ULI Wall 0.00E+00 1.19E-02 6.02E-03 1.79E-02 0.00E+00 8.97E-05 Heart wall 0.00E+00 8.27E-03 4.29E-03 1.26E-02 0.00E+00 0.00E+00 kidney 0.00E+00 4.21E-02 7.59E-03 4.97E-02 2.98E-03 2.48E-04 liver 0.00E+00 1.45E-02 5.51E-03 2.00E-02 0.00E+00 1.00E-03 lung 0.00E+00 5.73E-03 3.54E-03 9.27E-03 1.11E-03 1.11E-03 muscle 0.00E+00 6.08E-03 3.83E-03 9.91E-03 0.00E+00 4.95E-05 Ovaries 0.00E+00 4.69E-03 6.95E-03 1.16E-02 2.91E-03 2.33E-03 pancreas 0.00E+00 1.62E-02 5.90E-03 2.21E-02 1.32E-03 1.10E-04 Red pulp 0.00E+00 6.61E-03 4.24E-03 1.08E-02 1.30E-03 1.30E-03 Osteoblasts 0.00E+00 1.14E-02 4.14E-03 1.55E-02 4.65E-04 1.55E-04 skin 0.00E+00 4.69E-03 2.32E-03 7.01E-03 0.00E+00 7.01E-05 spleen 0.00E+00 1.48E-02 5.34E-03 2.02E-02 1.21E-03 1.01E-04 Thymus 0.00E+00 4.69E-03 3.66E-03 8.36E-03 0.00E+00 4.18E-05 thyroid 0.00E+00 1.19E-02 3.24E-03 1.52E-02 4.55E-04 7.59E-04 Bladder wall 0.00E+00 1.84E-01 3.21E-02 2.16E-01 1.30E-02 1.08E-02 Uterus 0.00E+00 4.69E-03 8.98E-03 1.37E-02 0.00E+00 6.84E-05 whole body 0.00E+00 7.76E-03 3.84E-03 1.16E-02 0.00E+00 0.00E+00 Effective dose 2.35E-02

[0228] Table 5 shows the radiation dosimetry estimate for Subject 3. The overall estimated radiation dose for Subject 3 was 13.5 mSv.

[0229] Table 5

[0230] Target Organs EDE Cont. ED Cont. Adrenal glands 0.00E+00 5.97E-03 3.71E-03 9.68E-03 0.00E+00 4.84E-05 brain 0.00E+00 6.13E-04 8.65E-04 1.48E-03 0.00E+00 7.39E-06 breast 0.00E+00 2.02E-03 1.59E-03 3.61E-03 5.41E-04 1.80E-04 Gallbladder wall 0.00E+00 2.02E-03 4.07E-03 6.09E-03 0.00E+00 0.00E+00 LLI wall 0.00E+00 1.44E-02 5.96E-03 2.03E-02 1.22E-03 2.44E-03 Small Intestine 0.00E+00 8.73E-03 4.29E-03 1.30E-02 0.00E+00 6.51E-05 Stomach wall 0.00E+00 4.31E-03 3.12E-03 7.43E-03 0.00E+00 8.91E-04 ULI Wall 0.00E+00 2.02E-03 4.00E-03 6.02E-03 0.00E+00 3.01E-05 Heart wall 0.00E+00 4.65E-03 2.82E-03 7.48E-03 0.00E+00 0.00E+00 kidney 0.00E+00 4.45E-02 6.83E-03 5.13E-02 3.08E-03 2.57E-04 liver 0.00E+00 1.20E-02 4.36E-03 1.64E-02 9.84E-04 8.20E-04 lung 0.00E+00 5.16E-03 2.37E-03 7.53E-03 9.04E-04 9.04E-04 muscle 0.00E+00 5.15E-03 2.83E-03 7.98E-03 0.00E+00 3.99E-05 Ovaries 0.00E+00 2.02E-03 5.52E-03 7.54E-03 0.00E+00 0.00E+00 pancreas 0.00E+00 1.13E-02 4.18E-03 1.54E-02 9.27E-04 7.72E-05 Red pulp 0.00E+00 4.29E-03 2.96E-03 7.25E-03 8.70E-04 8.70E-04 Osteoblasts 0.00E+00 4.95E-03 2.74E-03 7.69E-03 2.31E-04 7.69E-05 skin 0.00E+00 2.02E-03 1.63E-03 3.66E-03 0.00E+00 3.66E-05 spleen 0.00E+00 1.09E-02 3.83E-03 1.47E-02 0.00E+00 7.35E-05 testis 0.00E+00 1.22E-02 4.18E-03 1.64E-02 4.09E-03 3.27E-03 Thymus 0.00E+00 2.02E-03 2.30E-03 4.32E-03 0.00E+00 2.16E-05 thyroid 0.00E+00 8.46E-03 2.38E-03 1.08E-02 3.25E-04 5.42E-04 Bladder wall 0.00E+00 1.91E-01 2.79E-02 2.19E-01 1.31E-02 1.09E-02 Uterus 0.00E+00 2.02E-03 8.36E-03 1.04E-02 0.00E+00 5.19E-05 whole body 0.00E+00 4.94E-03 2.77E-03 7.71E-03 0.00E+00 0.00E+00 Effective dose 2.16E-02

[0231] Table 6 shows the radiation dosimetry estimate for Subject 4. The overall estimated radiation dose for Subject 4 was 15.9 mSv.

[0232] Table 6

[0233]

[0234]

[0235] Tumor uptake

[0236] Fig.17 Shows objects 1-4 68 Ga NODAGA GSAO's blood pool activity and 68 Ga NODAGA GSAO was taken up into tumor deposits (note: in patients 3 and 4, there were two tumor deposits and these were analyzed separately). Although blood pooling and clearance were reproducible, tumor uptake and clearance varied by tumor type.

[0237] In subjects 1 to 4, tumor uptake varied by tumor histology, with high levels of uptake observed in esophageal squamous cell carcinoma (SUVmean 3.8) and metastatic cutaneous squamous cell carcinoma (SUVmean 4.1), and low levels observed in metastatic ovarian cancer (SUVmean 1.9) and breast cancer (SUVmean 1.8). Note that in subjects 3 and 4, there were two tumor deposits, and these were analyzed separately. It is not unexpected that different tumor histologies have different rates of denovo cell death. To confirm this, tumor cell death and serotonin were analyzed in two tumor deposits from patient 3. 68 Histological correlation of tumor uptake of Ga NODAGA GSAO (one in the right axilla with high uptake 68 Ga NODAGA GSAO SUVmean 4.1, and the other in the right upper cervical triangle, its low uptake 68 Ga NODAGAGSAO SUVmean 2.7)( Fig.18 ).

[0238] Dissected tumors were fixed in formalin, embedded in paraffin, and cut into 4 μm thick sections. Adjacent sections were stained for apoptotic cells or morphologically stained by hematoxylin and eosin. For TUNEL staining, sections were deparaffinized in xylene, rehydrated in decreasing concentrations of ethanol, and permeabilized with proteinase K for 20 min at room temperature. Endogenous peroxidase activity was quenched with 3% H2O2 for 5 min. Apoptotic cells were labeled with biotinylated terminal deoxynucleotidyl transferase for 2 h at 37°C in a humidified chamber and then incubated with streptavidin-HRP conjugate for 30 min. HRP-positive cells were visualized using diaminobenzidine and sections were counterstained with methyl green (Sigma). Whole sections were imaged at 10× magnification using PowerMosaic scanning on a Leica DM6000D microscope.

[0239] Fig.18 Figure 2 shows FDG-PET performed 60 minutes after administration of 256 MBq of FDG (fluorodeoxyglucose) in a 66-year-old man (patient 3) with metastatic cutaneous squamous cell carcinoma. Fig.18 A) and after administration of 205MBq CDI ( 68 CDI-PET (Ga NODAGAGSAO) was performed 60 minutes after Fig.18 B) Maximum intensity front projection image. FDG-PET shows two metabolically active lymph node metastases, one in the right axilla and the other in the right upper anterior cervical triangle. These are thought to represent synchronous lymph node metastases from two different cutaneous squamous cell carcinomas that were previously resected. CDI-PET ( 68 Ga NODAGAGSAO) showed strong uptake in the right axillary lymph node metastasis (SUVmean = 4.1) and mild uptake in the right anterior cervical triangle lymph node metastasis (SUVmean = 1.7). The tumor was surgically resected, fixed, and adjacent sections were stained for apoptotic cells ( Fig.18 C, brown TUNEL staining, a and b) or morphological staining by hematoxylin and eosin ( Fig.18 C, c, and d). Arrows in TUNEL staining point to areas of extensive apoptosis.

[0240] Note that those tumors with high uptake had uptake that was twice that of the blood pool and was greater than that in all other organs except the kidney, which is a pathway for excretion. This high level of uptake in certain tumors combined with low activity levels in normal tissues and organs justifies the use of 68 Potential of Ga NODAGA GSAO as an effective imaging agent.

[0241] discuss

[0242] exist 68An interim analysis of the first four patients in the first-in-human study of Ga-NODAGA-GSAO showed it to be safe, well tolerated, and without side effects. Biodistribution and imaging characteristics were favorable, with only low levels of activity in most normal organs. The urinary tract was the only route of excretion. Uptake by dead and dying cells in tumors was observed, and 68 Ga-NODAGA-GSAO tumor uptake variation is consistent with different tumor histologies, and this uptake has been shown histopathologically to correlate with the proportion of dead and dying cells within the tumor. 68 The effective whole-body dose range of Ga NODAGA GSAO is 2.16×10 -2 to 3.38×10 -2 mSv / MBq, for an administration of 200 MBq, the effective whole-body dose is estimated to range from 4.3 to 6.8 mSv. This is comparable to the effective whole-body dose from many other diagnostic radiopharmaceuticals used in PET / CT and SPECT / CT, as well as from other radiological procedures such as X-ray computed tomography (CT).

Claims

1. A compound of formula (I) or a pharmaceutically acceptable salt thereof, Among them, A It is -As(OH)2; Each of R1, R2, R3 and R4 is independently selected from H, X, OH, NH2, CO, SCN, CH2NH, -NHCOCH3, -NHCOCH2X or NO, and X is a halogen; R5 is -NHCH2COOH, OH or OR6, wherein R6 is C 1-5 Straight or branched chain alkyl; Z is 64 Cu, {Al 18 F} 2+ , 68 Ga, 89 Zr, 82 Rb or 99 mTc.

2. The compound according to claim 1, wherein Each of R1, R2, R3 and R4 is H.

3. The compound according to claim 1, wherein R5 is -NHCH2COOH.

4. The compound according to claim 1, which is a compound of formula (Ia) or a pharmaceutically acceptable salt thereof, wherein A and Z are as defined in claim 1.

5. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, having the following structure:

6. The compound according to any one of claims 1 to 4, wherein Z is 68 Ga or {Al 18 F} 2+ .

7. A compound according to any one of claims 1 to 5 for use as an imaging agent.

8. The compound according to claim 7, for use as an imaging agent in positron emission tomography.

9. The compound according to claim 7, for use in visualizing cell death.

10. A pharmaceutical composition comprising the compound according to any one of claims 1 to 9 and a pharmaceutically acceptable excipient.

11. A compound of formula (II) or a pharmaceutically acceptable salt thereof, Among them, A It is -As(OH)2; Each of R1, R2, R3 and R4 is independently selected from H, X, OH, NH2, CO, SCN, CH2NH, -NHCOCH3, -NHCOCH2X or NO, and X is a halogen; R5 is -NHCH2COOH, OH or OR6, wherein R6 is C 1-5 Straight chain or branched chain alkyl.

12. The compound according to claim 11, wherein Each of R1, R2, R3 and R4 is H.

13. The compound according to claim 11, wherein R5 is -NHCH2COOH.

14. The compound according to claim 11 or a pharmaceutically acceptable salt thereof, wherein the compound has the following structure:

15. Use of a compound according to any one of claims 1 to 9 for the preparation of an imaging agent for positron emission tomography.

16. Use of a compound according to any one of claims 1 to 9 for the preparation of an imaging agent for visualizing cell death by positron emission tomography.

17. Use of a compound according to any one of claims 1 to 9 for the preparation of a medicament for the diagnosis of disorders associated with changes in cell death by positron emission tomography.

18. Use of a compound according to any one of claims 1 to 9 for the preparation of a medicament for diagnosing a neoplastic disorder or an autoimmune disorder by positron emission tomography.

19. The use according to claim 18, wherein The neoplastic disorder is a tumor.

20. The use according to claim 18 or 19, wherein The neoplastic disorder is cancer.

21. A method for preparing a compound according to claim 5, comprising 68 Ga is eluted onto a strong cation exchange column; and a mixture comprising the compound according to claim 14 and a buffer is eluted from the strong cation exchange column.

Images