RU2811734C2 - Radiolabeled progastrin in cancer diagnosis - Google Patents

Abstract

FIELD: pharmaceuticals.

SUBSTANCE: invention relates to a compound or a pharmaceutically acceptable salt thereof for cancer imaging. The proposed compound contains: a progastrin group having the sequence of the following SEQ ID NO: 1, and a chelating moiety, wherein the chelating moiety is a bifunctional chelator selected from the following list: NODAGA (1,4,7-triazacyclononane-1-glutaric acid-4,7-diacetic acid), NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid), p-SCN-Bn-NOTA, p-SCN-Bn-PCTA, p-SCN-Bn-oxo-DO3A, desferrioxamine-p-SCN, DTPA (diethylenetriaminepentaacetic acid) and TETA (1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid). The said chelating group is associated with a radioactive isotope. The progastrin moiety and the chelating moiety are covalently linked. Also the following is proposed: a method of producing the said compound, a method of imaging of one or more cancer cells, organs or tissues in a subject, a method of determining the location of cancer in a subject, a pharmaceutical composition and a kit for imaging one or more cancer cells containing the said compound.

EFFECT: compounds of the invention are capable of binding to cancer cells in vivo, thereby providing imaging of the said cancer, which is particularly useful for identifying the location of the cancer.

12 cl, 4 dwg, 3 tbl

Description

Introduction

Cancer is a multifactorial disease in which a group of cells exhibits uncontrolled growth, invasion in which it invades and destroys neighboring tissues, and sometimes metastasis, or spread to other sites in the body through lymph and blood. These three properties of the malignancy of cancers distinguish them from benign tumors, which do not invade or metastasize.

There are a number of options currently used to treat each type of cancer, including surgery, radiation therapy, chemotherapy, targeted therapy and immunotherapy. Successful cancer therapy targets the primary tumor and any metastases, whether clinically apparent or visible only under a microscope.

It is extremely important for the patient to identify the type of cancer to be treated as early as possible. Cancer that is diagnosed at an early stage is more likely to be successfully treated. If the cancer spreads, effective treatment becomes more difficult and the chances of survival are usually much lower. Therefore, it is imperative to know when to immediately use a heavy and intensive treatment protocol to prevent the spread of aggressive cancer.

Currently, the choice of treatment for solid tumors depends on tumor staging, which is usually performed using the Tumor/Node/Metastasis (TNM) test from the American Joint Committee on Cancer (AJCC). American Joint Committee on Cancer). It is generally believed that although this test and staging system provides valuable information regarding the stage at which a patient's solid tumor was diagnosed, it is inaccurate and insufficient. In particular, it is limited to solid tumors.

Most importantly, TNM analysis cannot identify the earliest stages of tumor progression. These early stages offer the most promising window for therapy. Detecting cancer early in its development makes targeted, effective therapy possible with reduced side effects. It is therefore important to identify patients as early as possible as part of population screening. In this way, cancer can be identified early in the community, making earlier intervention and treatment possible, reducing mortality and suffering from the said disease.

The applicant has recently developed a diagnostic assay based on the detection of progastrin. The selected antibodies were used to perform an ELISA (enzyme-linked immunosorbent assay) analysis to detect progastrin in the blood of patients with different types of cancer and at different stages. The assay, commercialized under the name CancerRead, is particularly effective for detecting different types of cancer, including early stages (WO 2017/114973). Notably, the CancerRead assay demonstrates high sensitivity and specificity for early-stage tumors.

However, even though blood progastrin levels are a reliable biomarker for early stage cancer screening, they do not provide information regarding the primary site of cancer.

Thus, there is a real need for reagents that enable in vivo identification of cancer, so that appropriate therapy can be provided as early as possible.

Description

The present invention relates to a progastrin derivative for imaging cancer in a patient.

In a first aspect, the present invention provides a compound or a pharmaceutically acceptable salt thereof, said compound comprising:

- progastrin grouping, and

- chelating group,

wherein said chelating moiety is possibly associated with a radioactive isotope.

In a preferred embodiment, the progastrin moiety and the chelating moiety are covalently linked. According to this embodiment, the present compound is a conjugate.

The compounds of the invention are particularly useful because they are capable of binding to cancer cells in vivo, thereby enabling imaging of said cancer. This is especially useful for identifying the location of cancer. Notably, radiolabeled progastrin has been used to visualize flow through a variety of technologies, such as single photon emission computed tomography (SPECT) and positron emission tomography (PET).

The term “progastrin” as used herein refers to the mammalian peptide progastrin. Progastrin is formed by cleavage of the first 21 amino acids (signal peptide) from preprogastrin, a 101 amino acid peptide (reference amino acid sequence: AAB19304.1), which is the primary translation product of the gastrin gene. The 80 amino acid progastrin chain is further processed by cleavage and modifying enzymes into several biologically active forms of the hormone gastrin: gastrin 34 (G34) and glycine-extended gastrin 34 (G34-Gly), containing progastrin amino acids 38-71, gastrin 17 (G17) and glycine-extended gastrin 17 (G17-Gly), containing progastrin amino acids 55-71.

In a preferred embodiment, the progastrin derivative is a human progastrin derivative. More specifically, the expression “human progastrin” refers to human progastrin of the sequence SEQ ID No. 1. Human progastrin contains in particular an N-terminal and a C-terminal domain, both of which are not present in the biologically active forms of the gastrin hormone mentioned above. Preferably, the sequence of said N-terminal domain is represented by SEQ ID NO. 2. In another preferred embodiment, the sequence of said C-terminal domain is represented by SEQ ID NO. 3.

Gastrin-producing cells naturally produce progastrin, which matures into gastrin. During digestion, 95% of progastrin is released from the cell as gastrin. A very small amount of progastrin is released as progastrin. Therefore, except during the cleavage period, a healthy person does not have progastrin in his blood.

On the other hand, in pathological conditions progastrin becomes a marker of an early stage. In tumor cells, progastrin does not mature into gastrin and, as a result, is released from the tumor cell. Progastrin can stimulate tumorigenesis (for example, gastric cancer [Burkitt et al., World J Gastroenterol. 15(1): 1-16, 2009, WO 2017/114975], colon cancer [Watson et al., J Cancer. 87(5 ): 567-573, 2002], pancreas [Harris et al., Cancer Res. 64(16): 5624-5631, 2004, WO 2011/083091], ovary [WO 2017/114972], prostate [WO 2018 /178352], esophagus [WO 2017/114976] and lung [WO 2018/178354]) in an autocrine, paracrine or endocrine manner (Dimaline & Varro, J Physiol 592(Pt.14): 2951-2958, 2014), which also serves reason to consider progastrin as a preferable antitumor target in cancers with the expression of these stimulating factors (see, for example, WO 2011/045080, WO 2011/083088, WO 2011/116954, WO 2012/013609, WO 2011/083090, WO 2011/083091, WO 2017/114975, WO 2017/114976, WO 2017/114972, WO 2018/178364). This process does not depend on splitting.

The terms "chelating moiety" or "chelating agent" or "chelator", as used herein, refer to a compound that is capable of chelating any of these radioactive isotopes. The chelating moiety sequesters the corresponding free radioactive isotopes from aqueous solutions, thereby enabling the use of said isotopes for specific biological applications. Preferably, said chelating moiety is a bifunctional chelator. The term "bifunctional chelator" or "bifunctional chelating agent", as used herein, refers to a compound having a metal-binding moiety function and a chemically reactive functional group.

A variety of bifunctional chelators are known in the art. A large number of them are actually commercially available and widely used as PET imaging tools. The structure and physical properties vary among bifunctional chelators. One skilled in the art will select the most appropriate bifunctional chelator for use with the progastrin moiety, taking into account the radioactive isotope used (see, for example, Cutler et al., Chem Rev. 113(2): 858-883, 2013; Price & Orvig, Chem. Soc. Rev. 43(1): 260-290, 2013; Tornesello et al., Molecules 22: E1282, 2017; Brandt et al., J Nucl Med 59(10): 1500-1506, 2018 ; Morais & Ma, Drug Discovery Today: Technologies, 2018, DOI: 10.1016/j.ddtec.2018.10.002).

Examples of bifunctional chelating agents are presented in Table 1.

Thus, the bifunctional chelator is preferably selected from the list of NODAGA, NOTA, DOTA, DOTA-NHS, p-SCN-Bn-NOTA, p-SCN-Bn-PCTA, p-SCN-Bn-oxo-D03A, desferrioxamine-p-SCN , DTPA and THETA.

DOTA, NOTA and NOGADA are widely used bifunctional chelators, especially for ^68Ga labeling. Thus, rapid and quantitative ⁶⁸ Ga radiolabeling of biomolecules can be achieved through the use of well-known chelators such as DOTA, NOTA and NOGADA.

In particular, the chelating agent DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) (or its modified derivatives) has been shown to be an excellent ligand for gallium binding; and DOTA peptides can be quickly and efficiently labeled with ⁶⁸ Ga at high specific activities (Velikyan, Molecules, 20: 12913-12943, 2015). Likewise, diethylenetriaminepentaacetic acid (DTPA) and its derivative are widely used. For example, 1B4M-DTPA, also known as MX-DTPA or thiuxetan, is developed as a component of the chelating agent Zevalin to radiolabel either ¹¹¹ 1p or ⁹⁰ Y (Brechbiel, QJ Nucl Med Mol imaging. 52(2): 166-173, 2008).

NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid) is generally considered to be the gold standard for Ga ³⁺ chelation, with favorable conditions for radiolabeling (RT (room temperature), 30- 60 minutes) and excellent in vivo stability. In fact, it is well known that NOTA and its derivatives form very stable complexes with ⁶⁸ Ga and with ⁶⁴ Cu.

NOTA derivatives, especially NODAGA (1,4,7-triazacyclononane-1-glutaric acid-4,7-diacetic acid), appear to be more suitable for chelating the ⁶⁸ Ga ion than DOTA derivatives. NODAGA is particularly useful for ⁶⁸ Ga and ⁶⁴ Cu labeling due to the high hydrophilicity and in vivo stability of its ⁶⁸ Ga and ⁶⁴ Cu chelates. Clinical studies have demonstrated that radiotracers containing [ ⁶⁸ Ga]NODAGA are well tolerated without drug-related adverse effects in patients (see, e.g., Haubner et al., EurJNucI Med Moi imaging 43:2005-2013, 2016; Kumar et al., J Nucl Med 57(suppl. 2): 1171,2016; Ben Azzouna et al., Endocrine Abstracts 47: OC4, 2016). In fact, [ ⁶⁸ Ga]NODAGA appears to be particularly suitable for in vivo tumor imaging (see, e.g., Oxboel et al., Nucl Med Biol. 41(3):259-267, 2014; Kumar et al. , J Nucl Med 57(suppl. 2): 675, 2016; Kumar et al., J Nucl Med 57(suppl. 2): 1171, 2016; Kumar et al., J Nucl Med 57(suppl. 2): 1298 , 2016; Tornesello et al., Molecules 22: E1282, 2017). NODAGA is available from various suppliers as NODAGA-NHS esters, allowing simple bioconjugation with the amine moiety of progastrin.

Preferably, the chelating agent is selected from DOPA, NOTA and NODAGA. Most preferably, the chelating agent is NODAGA.

The term "radioactive isotope", as used herein, is a version of a chemical element that has an unstable nucleus and emits radiation during its transformation into a stable form. Radioactive isotopes have important applications in medical diagnosis, treatment and research. The radioactive isotope of the present compounds is preferably selected from the list consisting of ⁶⁸ Ga, ⁶⁴ Cu, ⁸⁹ Zr, ^186/188 Re, ⁹⁰ Y, ¹⁷⁷ Lu, ¹⁵³ Sm, ²¹³ Bi, ²²⁵ Ac, ¹¹¹ ln, ^99m Tc, ¹²³ I or ²²³ Ra. These radioactive isotopes are particularly advantageous due to their long half-life and small size, making them particularly suitable for PET/SPECT imaging. More preferably, the radioactive isotope is ⁶⁸ Ga or ⁶⁴ Cu. Even more preferably, said radioactive isotope is ⁶⁸ Ga.

The advantages of ⁶⁸ Ga over other PET radionuclides include mainly its availability from an internal generator, independent of the on-site cyclotron (Shukla & Mittal, J Postgrad Med Edu Res 47(1): 74-76, 2013). Thus, it can be cost-effective and can be produced continuously through a commercially available ⁶⁸ Ge/ ⁶⁸ Ga generator, satisfying the need for proximity of PET centers to cyclotrons required for production of, for example, ¹⁸ F. The method of decay of the radionuclide results in positron imaging -emission tomography (PET) is of high quality and provides accurate quantitative assessment. In addition, the short physical half-life of ⁶⁸ Ga (t _1/2 is 68 min) provides improved dosimetry and repeat imaging, making these agents ideal for clinical use. Notably, this half-life facilitates imaging soon after administration with reduced patient exposure. Small compounds, biological macromolecules, as well as nano- and microparticles, have been successfully labeled with ⁶⁸ Ga, and the resulting agents have demonstrated promising preclinical and clinical imaging capabilities (see, for example, Beylergil et al., Nucl Med Commun. 34(12): 1157 -1165.2013).

Other embodiments of the disclosure include pharmaceutically acceptable salts of the compounds described in any of the above embodiments. As used herein, the term “pharmaceutically acceptable salts” refers to derivatives of the disclosed compounds where the parent compound is modified to form non-toxic acidic or basic salts thereof. Examples of pharmaceutically acceptable salts include inorganic or organic acid salts of basic moieties such as amines; alkaline or organic salts of acidic residues such as, but not limited to, carboxylic acids and the like. Pharmaceutically acceptable salts include conventional non-toxic salts or quaternary ammonium salts of the parent compound formed, for example, from non-toxic inorganic or organic acids. For example, conventional non-toxic acid salts include salts derived from inorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic, orthophosphoric , nitrogen, etc.; and salts derived from organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, pamoic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic, methanesulfonic, sulfanilic, 2- acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethanedisulfonic, oxalic, isethionic, HOOC-(CH ₂ ) _n -COOH, where n is 0-4, etc. Pharmaceutically acceptable salts of the present disclosure can be synthesized from a parent compound that contains a basic or acidic moiety by conventional chemical methods. Typically, such salts can be prepared by reacting the free acid forms of these compounds with a stoichiometric amount of a corresponding base (such as Na, Ca, Mg or K hydroxide, carbonate, bicarbonate or the like) or by reacting the free base forms of these compounds with a stoichiometric amount of the corresponding acid. Such reactions are usually carried out in water or an organic solvent or a mixture of both. Typically non-aqueous media such as ether, ethyl acetate, ethanol, isopropanol or acetonitrile are used when available. Lists of additional suitable salts can be found, for example, in Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, PA, p. 1418 (1985).

In another aspect, the present invention provides a method for preparing a compound of the invention. This method includes the following stages:

a) carrying out the conjugation of the amine-reactive chelating group with the progastrin group; And

b) isolation of the conjugate of progastrin and chelator.

Amine-reactive chelate structures for the radioactive isotope described herein are commercially available, such as, for example, the DOTA-NHS, NOTA-NHS, and NODAGA-NHS esters. Preferably, said amine-reactive chelating moiety is a NODAGA-NHS ester. It is well known to one skilled in the art that NHS esters (N-hydroxysuccinimide esters) will react with primary amines at the N-terminus and side chain of the amino acid lysine (Lys, K) residues of progastrin, and thus are not required detailed description in this document.

Preferably, the method for preparing a compound of the invention further comprises the step of:

c) incubating the conjugate of progastrin and chelator with a complementary radioactive isotope;

which thus leads to the formation of the compound of the invention.

In another aspect, the present invention provides a method for imaging one or more cells, organs, or tissues by exposing the cell to or introducing into the body an effective amount of a compound, wherein the compound comprises a metal isotope suitable for imaging. Imaging can be performed by any suitable technique known to one skilled in the art, especially PET or SPECT.

SPECT and PET are functional imaging techniques used to localize metabolic processes. A radionuclide produced either from a cyclotron or generator is attached to a biologically active molecule, forming a PET radiotracer. The isotopes currently used in SPECT/PET imaging studies are attractive and perhaps better alternatives to ¹⁸ F. ⁶⁸ Ga, ⁶⁴ Cu, ⁸⁹ Zr, ^186/188 Re, ⁹⁰ Y, ¹⁷⁷ Lu, ¹⁵³ Sm, ²¹³ Bi , ²²⁵ Ac or ²²³ Ra are available isotopes that are evaluated for PET imaging due to their light metal properties and ability to bind to chelating agents.

Positron emission tomography (PET) is a nuclear medicine functional imaging technique that provides a three-dimensional image of functional processes in the body. PET is used to localize metabolic processes. A positronically active radionuclide, obtained from either a cyclotron or a generator, is attached to a biologically active molecule to form a PET radiotracer, such as, for example, the compounds described herein. The PET radiotracer is then administered to the patient by injection, oral administration, or inhalation. The system detects pairs of gamma rays indirectly emitted by a radionuclide (tracer) that is injected into the body at the radiotracer. Three-dimensional images of the concentration of the indicator in the body are then constructed through computer analysis. In modern PET-CT scanners (from English computer tomography - computed tomography), three-dimensional visualization is often performed using CT scanning with x-rays, performed on the patient in the same session, on the same device. Immediately after administration of the PET radiotracer, the patient is positioned so that the detectors can detect the incident gamma rays, 2511 keV photons moving in opposite directions, produced when the radionuclide decays, resulting in an annihilation event from a positron, combined with an electron after passing short distance. The detector electronics are synchronized in such a way that the 2 emitted photons are detected on opposite sides and are said to be coincident, and thus must come from the same annihilation event. These matching projections are assigned to the response line and then reconstructed using standard tomography techniques to identify the location of the annihilation event. Using modern "time of flight" information in PET image reconstruction with very fast scintillators, the source of the annihilation event along the response line is identified with improved accuracy.

Radionuclides used in PET scanning are usually isotopes with short half-lives, such as ¹¹ C (about 20 minutes), ¹³ N (about 10 minutes), ¹⁵ 0 (about 2 minutes), ¹⁸ F (about 110 minutes) or ⁸² Rb (approximately 1.27 min). The radioactive isotopes described above, namely the list consisting of ⁶⁸ Ga, ⁶⁴ Cu, ⁸⁹ Zr, ^186/188 Re, ⁹⁰ Y, ¹⁷⁷ Lu, ¹⁵³ Sm, ²¹³ Bi, ²²⁵ Ac or ²²³ Ra, are also widely used in PET. In this regard, as stated above, ⁶⁸ Ga is particularly advantageous due to its half-life of 68 minutes. These radionuclides are incorporated either into compounds commonly used by the body, such as glucose (or glucose analogues), water or ammonium, or into molecules that bind to receptors or other sites, including progastrin. Such labeled compounds are known as radiotracers. PET technology can be used to trace the biological pathway of any compound in a living human (and many other species as well), provided it can be radiolabeled with the PET isotope. In particular, as described below, PET technology can be used to detect cancer in a living person by imaging a radiolabeled probe that specifically binds to cancer cells, such as the compound described herein.

Due to the short half-lives of most positron-active radioactive isotopes, radiotracers have traditionally been produced using a cyclotron in close proximity to PET imaging equipment. The half-life of fluorine-18 is long enough that radioactive tracers labeled with fluorine-18 can be produced on an industrial scale off-site and shipped to imaging centers. On the other hand, ⁶⁸ Ga can be produced in a generator, thus eliminating the need for a cyclotron (Velikyan, Molecules 20: 12913-12943, 2015). In addition, the half-life of gallium-68 is close to that of ¹⁸ F, making this radionuclide especially useful for PET imaging.

Single photon emission computed tomography (SPECT) is a nuclear medicine imaging technique similar to PET. It also uses a radioactive tracer and is based on the detection of gamma rays. Unlike PET, the radioactive tracer used in SPECT emits gamma radiation, which is directly measured.

Embodiments of the invention include a compound of the present invention for use in methods of imaging one or more cells, organs, or tissues involving exposing the cells to, or administering to a subject, an effective amount of an isotope-tagged compound suitable for imaging. In some embodiments, one or more organs or tissues include prostate tissue, kidney tissue, brain tissue, vascular tissue, or tumor tissue. Cells, organs, or tissues may be imaged within the body, or through whole body imaging, or through imaging performed during surgery, or may be removed from the body for imaging.

In another embodiment, the imaging method is suitable for imaging cancer, tumor or neoplasm. As used herein, the term “cancer” refers to or describes a physiological condition in mammals that is typically characterized by unregulated cell proliferation. The terms “cancer” and “cancerous” as used herein are intended to cover all stages of the disease. The term "cancer", as used herein, is any malignant neoplasm that results from unwanted growth, invasion and, under certain conditions, metastasis of damaged cells in the body. Cells that give rise to cancer are genetically disordered and have typically lost their ability to control cell division, cell migration behavior, differentiation status, and/or cell death mechanism. Most cancers produce a tumor, but some cancers do not form hematopoietic tissue, such as leukemia. Cancer usually forms at the primary site, giving rise to primary cancer. Cancer that spreads locally, or to distant parts of the body, is called metastasis.

Thus, “cancer” as used herein can include both benign and malignant tumors. The term "cancer" as used herein can also include both primary and metastatic cancers. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. More specifically, the cancer of the present invention is selected from the group consisting of the following cancers: squamous cell carcinoma (eg, epithelial squamous cell carcinoma), lung cancer, including small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma and squamous cell lung carcinoma, oropharyngeal cancer, cancer nasopharynx, laryngeal cancer, abdominal cancer, esophageal cancer, hepatocellular cancer, stomach cancer, including gastrointestinal cancer and gastrointestinal stromal tumor, pancreatic cancer, glioblastoma, brain cancer, nervous system cancer, cervical cancer, ovarian cancer, liver cancer, bladder cancer, urinary tract cancer, hepatoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, prostate cancer, gallbladder cancer, vulvar cancer, testicular cancer, thyroid cancer, Kaposi's sarcoma, liver carcinoma, anal carcinoma, penile carcinoma, non-melanoma skin cancer, melanoma, cutaneous melanoma, superficial spreading melanoma, lentigo-melanoma, acral lentiginous melanomas, nodular melanomas, multiple myeloma and B-cell lymphoma (including Hodgkin lymphoma; non-Hodgkin's lymphoma, such as, for example, low-grade follicular non-Hodgkin's lymphoma (NHL - from the English non-Hodgkin's lymphoma); small cell lymphocytic (SL - from English small lymphocytic) NHL; moderately differentiated follicular NHL; moderately differentiated diffuse NHL; well-differentiated immunoblastic NHL; well-differentiated lymphoblastic NHL; highly differentiated small cell NHL with an uncleaved nucleus; NHL with a massive defeat; mantle cell lymphoma; AIDS-associated lymphoma; and macroglobules and Waldenström's disease); chronic lymphocytic leukemia (CLL - from the English chronic lymphocytic leukaemia); acute lymphoblastic leukemia (ALL - from the English acute lymphoblastic leukaemia); hairy cell leukemia; chronic myeloblastic leukemia (CML - from the English chronic myeloblasts leukaemia); acute myeloblastic leukemia (AML - from the English acute myeloblasts leukaemia); and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal vascular proliferation associated with phakomatosis, edema (such as proliferation associated with brain tumors), Meigs syndrome, and head and neck cancer , including cancer of the lip and oral cavity, and associated metastases.

In a preferred embodiment, said cancer is lung cancer, lip and oral cavity cancer, oropharyngeal cancer, nasopharyngeal cancer, laryngeal cancer, prostate cancer, esophageal cancer, gallbladder cancer, liver cancer, hepatocellular cancer, stomach cancer, including gastrointestinal cancer and gastrointestinal stromal tumor, pancreatic cancer, Hodgkin's lymphoma, non-Hodgkin's lymphoma, leukemia, multiple myeloma, Kaposi's sarcoma, kidney cancer, bladder cancer, colon cancer, rectal cancer, colorectal cancer, hepatoma, liver carcinoma, carcinoma anal canal, thyroid cancer, non-melanoma skin cancer, skin melanoma, brain cancer, nervous system cancer, testicular cancer, cervical cancer, uterine cancer, endometrial cancer, ovarian cancer or breast cancer.

In a more preferred embodiment, said cancer is esophageal cancer, liver cancer, hepatocellular cancer, gastric cancer, including gastrointestinal cancer and gastrointestinal stromal tumor, pancreatic cancer, Hodgkin's lymphoma, colon cancer, rectal cancer, colorectal cancer colon, hepatoma, liver carcinoma, anal carcinoma, non-melanoma skin cancer, cutaneous melanoma, cervical cancer, uterine cancer, endometrial cancer, ovarian cancer or breast cancer.

The present inventors have discovered that the radiolabeled compounds described herein can be used to study cancer in vitro and in vivo using autoradiography techniques and molecular imaging techniques such as PET or SPECT. The progastrin moiety specifically binds to cancer cells, such that the signal emitted by the radioactive isotope indicates the location of the cancer cells.

According to another aspect, there is provided a method of imaging one or more cancer cells, organs or tissues in a subject determined to be in need thereof, comprising:

a) administering a compound as described herein, or a pharmaceutically acceptable salt thereof, to the specified subject; And

b) identifying said compound by in vivo PET or SPECT imaging.

The compounds of the invention are also useful for diagnosing cancer in a patient. According to this aspect of the invention, there is provided a method for diagnosing cancer in a patient, the method comprising the following steps:

a) administering a compound as described herein, or a pharmaceutically acceptable salt thereof, to the specified subject;

b) identifying said compound by in vivo PET or SPECT imaging; And

c) diagnosing cancer based on identifying stage b).

The progastrin derivatives of the present invention bind only to cancer cells. Any signal detected by PET or SPECT imaging is therefore an indication that cancer cells are present. Thanks to the sensitivity of the present radioactively labeled compounds, it is possible to identify cancer cells in the patient's body and thus diagnose cancer. In addition, the type of cancer can be easily inferred from the location of the primary cancer.

In another aspect, the present invention relates to a method for predicting cancer in a patient, the method comprising the following steps:

a) administering a compound as described herein, or a pharmaceutically acceptable salt thereof, to the specified subject;

b) identifying said compound by in vivo PET or SPECT imaging; And

c) predicting cancer based on identifying stage c).

The term "prognosis", as used herein, means the likelihood of recovery from a disease or a prediction of the likely development or outcome of a disease. For example, the greater the signal detected at stage b), the greater the tumor mass in the patient’s body, the worse the prognosis.

In yet another aspect, the present invention provides a method for determining the location of cancer in a subject in need thereof, comprising:

a) administering a compound as described herein, or a pharmaceutically acceptable salt thereof, to the specified subject; And

b) identifying said compound by in vivo PET or SPECT imaging.

One skilled in the art will immediately appreciate that the invention can also identify the location of cancer at its earliest stages. Notably, the present invention is particularly useful for identifying a cancer site that is too small to otherwise be detected. This is especially advantageous when the only indication that a patient has cancer comes from a biomarker test. For example, an assay including antibodies against progastrin and based on its detection makes it possible to identify the possibility of cancer, even in the absence of any symptom (see, for example, WO 2017/114973).

According to a specific embodiment, a method for detecting the location of cancer in a subject in need thereof includes the following steps:

a) detecting the level of progastrin in a sample of the specified subject;

b) administering a compound as described herein, or a pharmaceutically acceptable salt thereof, to the specified subject; And

c) identifying said compound by in vivo PET or SPECT imaging.

Determination of progastrin concentration in the present method is carried out using any technique known to a person skilled in the field of biochemistry.

Preferably, determining the levels of progastrin in a sample involves contacting said sample with a progastrin-binding molecule and measuring the binding of said progastrin-binding molecule to progastrin.

When expression levels are measured at the protein level, this can generally be done using specific progastrin binding molecules such as, for example, antibodies, in particular using well known technologies such as cell membrane staining, biotinylation or other equivalent techniques followed by immunoprecipitation using specific antibodies, Western blotting, ELISA or ELISPOT (from the English enzyme-linked immunospot method), enzyme-linked immunospot assays (ELISA), radioimmunoassays (RIA - from the English radioimmunoassay), immunohistochemistry (IHC - from English immunohistochemistry), immunofluorescence (IF - from English immunofluorescence), antibody-based microchips or tissue-based microchips in combination with immunohistochemistry. Other suitable techniques include FRET or BRET, single cell microscopy techniques or histochemistry techniques using single or multiple excitation wavelengths and the use of any of the adapted optical techniques such as electrochemical techniques (voltametry and amperometry techniques), atomic force microscopy and radiofrequency techniques e.g. multipolar resonance spectroscopy, confocal and non-confocal, detection of fluorescence, luminescence, chemiluminescence, absorption, reflection, transmittance and birefringence or refractive index (e.g. surface plasmon resonance, ellipsometry, resonant mirror method, waveguide grating coupler method or interferometry), cell ELISA, flow cytometry, radioactive isotope imaging, magnetic resonance imaging, sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) sodium dodecyl sulfate); HPLC (High Performance Liquid Chromatography)-Mass Spectrometry; Liquid chromatography/mass spectrometry (LC-MS/MS)). All of these techniques are well known in the art and do not require further detailed discussion herein. These different techniques can be used to measure progastrin levels.

The method may be particularly selected from the following methods: an immunodetection-based method, a Western blotting-based method, a mass spectrometry-based method, a chromatography-based method, and a flow cytometry-based method. Although any suitable means of performing these assays are included in the invention, methods such as FACS (fluorescence-activated cell sorting), ELISA, RIA, Western blotting and IHC are particularly useful for implementation of the method according to the invention.

It has previously been shown that a subject has cancer if progastrin levels are above 0 pM (see, for example, WO 2017/114973). According to a preferred embodiment, the method includes the following steps:

a) measuring the level of progastrin in a sample of the specified subject;

b) determining that the level of step a) is higher than 0 pM;

c) administering a compound as described herein, or a pharmaceutically acceptable salt thereof, to the specified subject; And

d) detecting said compound by in vivo PET or SPECT imaging.

The term “progastrin-binding molecule” as used herein refers to any molecule that binds progastrin but does not bind gastrin-17 (G17), gastrin-34 (G34), glycine-extended gastrin-17 (G17-Gly), or glycine-extended gastrin-17 (G17-Gly). extended gastrin-34 (G34-Gly). The progastrin binding molecule of the present invention can be any progastrin binding molecule, such as, for example, an antibody molecule or a receptor molecule. Preferably, the progastrin-binding molecule is an anti-progastrin antibody or an antigen-binding fragment thereof. According to a specific embodiment of the method, the level of progastrin is determined by using one or more anti-progastrin antibodies. According to this embodiment, the level of progastrin is determined by bringing one or more anti-progastrin antibodies into contact with a sample of the specified subject.

Said antibody may be a polyclonal or monoclonal antibody. Preferably, the anti-progastrin monoclonal antibody of the present method is any of the anti-hPG monoclonal antibodies disclosed in WO 2017/114973.

The term "biological sample" as used herein also includes a solid tumor sample from a patient to be analyzed when the cancer is a solid tumor. Such a solid tumor sample allows one of ordinary skill in the art to perform any type of biomarker level measurement of the invention. In some cases, the methods of the invention may further include the preliminary step of collecting a solid tumor sample from the patient. The term "solid tumor sample" refers to a sample of tumor tissue. Even in a patient with cancer, the tissue that represents the tumor still contains non-tumor healthy tissue. The “tumor sample” should therefore be limited to tumor tissue collected from the patient. Said “tumor sample” may be a biopsy sample or a sample collected as a result of therapy based on surgical resection.

The biological sample is typically obtained from a eukaryotic organism, most preferably a mammal, or a bird, reptile or fish. Indeed, the “subject” that may be subjected to the method described herein may be any of the mammalian animals, including human, dog, cat, cattle, goat, wild boar, pig, sheep and monkey; or a bird; reptile; or fish. Preferably, the subject is a human; the subject, which is a person, may be known as a "patient".

The phrase “obtaining a biological sample” as used herein means obtaining a biological sample for use in the methods described in this invention. Most often, this will be accomplished by removing a sample of cells from the animal, but may also be accomplished using previously isolated cells (eg, isolated by another person, at a different time, and/or for a different purpose), or by performing the methods of the invention in vivo. Archival samples that have a history of treatment or treatment outcome will be particularly helpful.

This sample can be obtained and, if necessary, prepared in accordance with methods known to one skilled in the art. In particular, it is well known in the art that the sample should be collected from a fasting subject.

Determination of progastrin concentration refers to the determination of the amount of progastrin in a known volume of sample. The progastrin concentration may be expressed relative to a reference sample, for example as a fraction or percentage. Concentration may also be expressed in terms of signal intensity or localization, depending on the method used to determine said concentration. Preferably, the concentration of a compound in a sample is expressed after normalizing the total concentration of related compounds in the sample, for example, the level or concentration of a protein is expressed after normalizing the total concentration of proteins in the sample.

The treatment prescribed to a patient with cancer will depend on the type of cancer. The present invention is particularly advantageous in this regard since the type of cancer can be identified based on the location of said cancer in a patient. Appropriate therapy can be administered to the patient, thereby improving his/her prognosis. The compounds described herein are particularly useful because they enable the visualization and identification of cancer at its earliest stages. It is noteworthy that when their use is combined with the measurement of progastrin levels, as described above, the present compounds provide visualization and identification of cancer even in the absence of any symptom. This is especially useful for identifying the primary site of cancer, since said cancer can be visualized before it has metastasized to distant parts of the patient's body.

According to an aspect of the invention, there is provided a method for identifying the primary site of cancer in a subject in need thereof. The method includes the steps of determining the location of the cancer by the methods described herein and identifying the organ that is affected by the cancer. In one embodiment, the method further comprises in vitro histological examination of a sample of said organ from said patient.

Another aspect of the present invention relates to a composition, generally a pharmaceutical composition, containing a compound as described herein.

The compounds discussed herein may be formulated in various compositions for use in diagnostic or imaging-based therapies. These compositions (eg, pharmaceutical compositions) may be collected as a kit.

Typically, the pharmaceutical composition contains an effective amount (eg, a pharmaceutically effective amount or a detectable effective amount) of a compound described above.

The composition of the disclosure may be formulated as a pharmaceutical composition that contains a compound of the invention and a pharmaceutically acceptable carrier. By "pharmaceutically acceptable carrier" is meant a substance that is not biologically or otherwise undesirable, and that the substance can be administered to a subject without causing any undesirable biological effect or interacting in a detrimental manner with any other components of the pharmaceutical composition in which it is contained. contained. The carrier under natural conditions will be selected to minimize any degradation of the active ingredient and to minimize any harmful side effects in the subject, as will be well known to one of ordinary skill in the art. For a discussion of pharmaceutically acceptable carriers and other components of pharmaceutical compositions, see, for example, Remington's Pharmaceutical Sciences, 18th ed., Mack Publishing Company, 1990. Some suitable pharmaceutical carriers will be obvious to one skilled in the art and include, for example, water (including sterile and/or deionized water), suitable buffers (such as PBS (phosphate buffered saline), saline, cell culture medium (such as DMEM (Dulbecco modified Eagle's medium) )), artificial cerebrospinal fluid, etc.

The pharmaceutical composition or kit of the disclosure may contain other pharmaceutical agents in addition to the compound. The other agent(s) may be administered at any appropriate time during treatment of the patient, either simultaneously or sequentially.

One skilled in the art will appreciate that the specific composition will depend, in part, on the particular agent that is used and the route of administration chosen. Accordingly, there is a wide range of suitable compositions according to the present disclosure.

One skilled in the art will appreciate that a suitable or appropriate composition can be selected, adapted or developed depending on the particular application to be applied. Dosages for the compositions of the disclosure may be presented in unit dosage form. The term "unit dosage form", as used herein, refers to physically discrete units suitable for unit dosage for animal (eg, human) subjects, each unit containing a predetermined amount of the agent of the invention , alone or in combination with other therapeutic agents, calculated in an amount sufficient to obtain the desired effect, together with a pharmaceutically acceptable diluent, carrier or excipient.

One skilled in the art can readily determine the appropriate dosage, regimen, and route of administration for the precise composition to be used to achieve the desired effective amount or effective concentration of agent in an individual patient. The dose of the composition described herein administered to an animal, particularly a human, in the context of the present invention should be sufficient to produce at least a detectable amount of diagnostic response in an individual over a reasonable period of time. The dose used to achieve the desired effect will be determined by a variety of factors, including the activity of the particular agent to be administered, the pharmacodynamics associated with the agent in the host, the severity of the disease state of the infected individuals, other drugs administered to the subject, and the like. Dose Size will also be determined by the presence of any adverse side effects that may accompany the particular agent or composition used. It is generally desirable to keep adverse side effects to a minimum whenever possible. The dose of the biologically active substance will vary; the appropriate quantities for each particular agent will be apparent to a skilled worker.

The pharmaceutical or radiopharmaceutical composition can be administered parenterally, namely by injection, and is most preferably an aqueous solution. Such a composition may optionally contain additional ingredients such as buffers; pharmaceutically acceptable solubilizers (eg, cyclodextrins or surfactants such as Pluronic, Tween or phospholipids); pharmaceutically acceptable stabilizers or antioxidants (such as ascorbic acid, gentisic acid or para-aminobenzoic acid). When a compound described herein is provided as a radiopharmaceutical composition, a method for preparing said compound may further include the steps required to prepare the radiopharmaceutical composition, such as removing the organic solvent, adding a biocompatible buffer, and any optional additional ingredients. For parenteral administration, steps must be included to ensure that the radiopharmaceutical composition is sterile and pyrogen-free. Such steps are well known to those skilled in the art.

According to other embodiments of the disclosure, kits are provided comprising a compound as disclosed herein, or a pharmaceutically acceptable salt thereof. In some embodiments of the disclosure, the kit provides packaged pharmaceutical compositions having a pharmaceutically acceptable carrier and a compound as disclosed herein, or a pharmaceutically acceptable salt thereof. In some embodiments of the disclosure, the packaged pharmaceutical composition will include the reaction precursors necessary to create a compound as disclosed herein, or a pharmaceutically acceptable salt thereof, when combined with a radionuclide. Other packaged pharmaceutical compositions provided by the present disclosure will further contain information containing at least one of the following: instructions for preparing the compounds as disclosed herein, or pharmaceutically acceptable salts thereof, from the supplied precursors, instructions for using the composition for cell imaging, or tissues, in particular, instructions for use of the composition for cancer imaging.

In some embodiments of the disclosure, the present kit contains from about 1 mCi (millicuries) to about 30 mCi of the radiolabeled imaging agent described above, in combination with a pharmaceutically acceptable carrier. The imaging agent and carrier may be provided in solution or lyophilized form. When the imaging agent and kit carrier are in lyophilized form, the kit may optionally contain a sterile and physiologically acceptable reconstitution medium such as water, saline, buffered saline, and the like. The kit may provide a compound described herein document, in solution or in lyophilized form, and these kit components of the disclosure may optionally contain stabilizers such as NaCl, silicate, phosphate buffers, ascorbic acid, gentisic acid and the like. Additional stabilizers of the kit components may be provided in this embodiment, for example, by providing a reducing agent in a form that is resistant to oxidation. The identification and optimization of such stabilizers and methods for stabilization are entirely within the skill of the art.

The term "pharmaceutically acceptable carrier" refers to a biocompatible solution with due regard to sterility, p[Eta], isotonicity, stability, etc., and may include all types of solvents, diluents (including sterile saline, sodium chloride injection, Ringer's solution for injection , dextrose injection, dextrose-sodium chloride injection, lactated Ringer's solution for injection and other aqueous buffer solutions), dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, etc. The pharmaceutically acceptable carrier may also contain stabilizers, preservatives, antioxidants or other additives that are well known to one skilled in the art, or other excipient as known in the art.

Characteristics of the embodiments of the invention will become even more apparent from the following detailed description of the following examples.

Description of graphic materials

Fig. 1: 3rd image of quantitatively assessed ROI (from the English Region of interest - region of interest) on PET/CT. Shown are a tumor (red), liver (blue), kidney (green), heart (blue), muscle (yellow) and brain (pink).

Fig. 2: Sagittal image of a dynamic PET/ST mouse C1S3 at different time points after injection of 68Ga-NODAGA-Progastrin.

Fig. 3: Average biodiversity of ⁶⁸ Ga-NODAGA-Progastrins in each quantified organ over 2 hours PET. Values are expressed as %ID (injected dose)/r plus standard deviation. (A) Kinetics of all quantified regions, (B) Kinetics limited to muscle, brain, and tumor.

Fig. 4: Amount in %ID/r of 68Ga-NODAGA-peptide 1 measured in tumor and muscle (A) and Tumor to Muscle ratio (B) for each mouse after 2 hours of PET/CT data collection.

Examples

Peptide binding

The chelator is prepared at a concentration of 10 mg/ml in 0.2 M sodium bicarbonate at pH 9. Then, 10 equivalents of the chelator are added to an aliquot of progastrin. Conjugation reactions are carried out at 37°C for 2 hours. The final product is purified using AMICON filters. Through these filters, excess unreacted chelator is removed. The inventors obtain a conjugated peptide, which the inventors call NODAGA-Pr gastrin.

Animal model

The T84 colon cancer cell line was cultured in a T75 flask and passaged 4 times after thawing to ensure continued optimal growth rates prior to xenotransplantation in mice. The culture medium used was DMEM-F12 with Glutamax plus 10% fetal bovine serum and 1% antibiotics (Streptomycin, Penicillin). For murine xenografts, cell culture was stopped at 80% confluency and cells were collected in a 1:1 DMEM-F12 solution without serum and Matrigel to a concentration of 1.10 ⁹ cells/100 μl.

Mice were quickly anesthetized with isoflurane and an injection of 100 μl of T84 (1.10 ⁹ cells/100 μl) was performed into the subcutaneous area between the shoulder blades. Animals are placed back into their cages immediately after they are awakened and placed in a room with stable conditions until tumor growth is sufficient for the experiment to be performed.

Radiolabeling and imaging

100 μl of 2 M ammonium acetate solution is added to an aliquot of NODAGA-Progastrin (10 μg solubilized in 50 μl of PBS). Next, 500 μl of gallium-68 eluate, [ ⁶⁸ Ga]GaCl ₃ , from the IRE Elit generator is added to the previously prepared solution. The entire amount is incubated at room temperature for 10 minutes. The final pH is 4.8. The radiochemical purity is greater than 90% (n = 3) and is determined by thin layer chromatography (mobile phase: 0.1 M sodium citrate at pH 5). 2 μl of 10 M sodium hydroxide is added to the final mixture to neutralize the pH. This resulting solution is used as is for biodistribution and PET/CT imaging studies.

Animals are euthanized using gas anesthesia (isoflurane at a concentration of 3% in the case of administration and at a concentration of 1.5-2% in the case of using a mask). A catheter (27G catheter) is inserted into the tail vein. Mice receive an injection of radiotracer in a bolus of 3.5 ± 0.6 MBq for a 2-hour course (Table 2).

All PET/CT imaging is performed using a paporePET/CT® camera (Mediso, Hungary).

Animal images of 3×3 size are obtained. To obtain images of the biodistribution kinetics of NODAGA-Progastrin, 2-hour dynamic PET images (“energy window” 400-600 keV) in combination with a scanner (35 kVp, exposure time/projection 450 ms) are acquired throughout the mouse body (“window” 10 cm). PET data acquisition begins 10 seconds before the start of tracer injection and provides the injection peak. The resulting PET images are then reconstructed through the application of anatomical shift, attenuation correction, and temporal separation. The time divisions are as follows: 10'', 1'', 1', 5', 10', 20', 40', 1 hour, 1 hour 20', 1 hour 20', 1 hour 40' and 2 hours.

Subsequent analysis of 3D PET/CT images was performed using VivoQuant 3.5 software (Invicro, USA). For dynamics, 6 regions of interest (ROIs) are delineated on the scanner, then transferred to PET images for quantification. The organs quantified are liver, kidney, heart, brain, tumor, and muscle (Figure 3). Quantitative results are expressed either as a percentage of the injected dose per gram of tissue (%ID/g)* or as a Tumor/Muscle ratio**.

* %ID/g represents the activity calculated in ROI (MBq)/ (Injected activity (MBq) × Tissue volume (ml)) × 100

** The muscle is considered as a control area for non-specific fixation of the radioactive tracer.

results

Overall, NODAGA-Progastrin biodistribution kinetics were monitored and quantified in a total of 5 mice that developed T84 ectopic tumors from 100 to 600 mm ³ (PET/CT data collection in Table 2).

Mice tumor volumes were measured from CT images (Table 3).

In FIG. 1 illustrates the biological distribution of this tracer during 2 hours of PET imaging in a mouse. The mean %ID/g scoring values of each test area were calculated and presented in FIG. 2.

As expected, the inventors observed high concentrations in the excretory organs liver and kidneys, and much lower levels of activity in muscle or brain, which do not specifically capture the indicator. Even more interesting is that the level of activity in tumor is higher than in muscle in mice with tumor/muscle ratios ranging from 1 to 4 in 5 mice (Figure 3).

Conclusion

The inventors can conclude that in this model there is incorporation of radioactively labeled Progastrin peptide into the tumor.

Claims (25)

1. A compound or a pharmaceutically acceptable salt thereof for cancer imaging, wherein said compound contains: - a progastrin group having the sequence SEQ ID NO: 1, and - chelating group, wherein the chelating group is a bifunctional chelator selected from the following list: NODAGA (1,4,7-triazacyclononane-1-glutaric acid-4,7-diacetic acid), NOTA (1,4,7-triazacyclononane-1,4, 7-triacetic acid), p-SCN-Bn-NOTA, p-SCN-Bn-PCTA, p-SCN-Bn-oxo-DO3A, desferrioxamine-p-SCN, DTPA (diethylenetriaminepentaacetic acid) and TETA (1,4, 8,11-tetraazacyclotetradecane -1,4,8,11-tetraacetic acid); wherein said chelating group is associated with a radioactive isotope and wherein said progastrin group and chelating group are covalently linked. 2. A compound according to claim 1, wherein the bifunctional chelator is NODAGA or NOTA, preferably NODAGA. 3. Connection according to any one of paragraphs. 1 and 2, in which the radioactive isotope is selected from a list consisting of ⁶⁸ Ga, ⁶⁴ Cu, ⁸⁹ Zr, ^186/188 _Re , ⁹⁰ Y, ¹⁷⁷ Lu, ¹⁵³ Sm, ²¹³ Bi, ²²⁵ Ac, ¹¹¹ In, ^99m Tc, ¹²³ I or ²²³ Ra. 4. Connection according to any one of paragraphs. 1-3, in which the radioactive isotope is ⁶⁸ Ga or ⁶⁴ Cu, preferably ⁶⁸ Ga. 5. Method for obtaining a compound according to any one of claims. 1-4, including the following stages: a) carrying out the conjugation of the amine-reactive chelating group with the progastrin group; b) isolation of the conjugate of progastrin and chelator; c) incubation of a conjugate of prograstrin and a chelator with a complementary radioactive isotope; thereby obtaining the compound of the invention. 6. The method of claim 5, wherein the amine-reactive chelating moiety is a NOTA-NHS or NODAGA-NHS ester. 7. Method according to any one of paragraphs. 5 and 6, in which the amine-reactive chelating moiety is a NODAGA-NHS ester. 8. A method of imaging one or more cancer cells, organs or tissues in a subject in need thereof, comprising: a) introducing a compound according to any one of paragraphs. 1-4 or a pharmaceutically acceptable salt thereof to the specified subject; And b) detecting said compound by positron emission tomography (PET) imaging or single photon emission computed tomography (SPECT) imaging in vivo. 9. A method for determining the location of cancer in a subject in need thereof, comprising: a) introducing a compound according to any one of paragraphs. 1-4 or a pharmaceutically acceptable salt thereof to the specified subject; And b) detecting said compound by PET or SPECT imaging in vivo. 10. The method of claim 9, further comprising the preliminary step of determining the level of progastrin, preferably using antibodies to progastrin, in a sample of said subject. 11. Pharmaceutical composition for visualizing one or more cancer cells, containing a compound according to any one of paragraphs. 1-4 and a pharmaceutically acceptable carrier. 12. A kit for imaging one or more cancer cells, containing a compound according to any one of paragraphs. 1-4.