Alpha-radiolabelled gastrin analogs and their use in methods of treating CCKB receptor positive diseases - Patents.com

Abstract

The present invention relates to alpha-radiolabeled gastrin analogs and their use in peptide receptor radionuclide therapy (PRRT) applications. In particular, the present invention relates to alpha-radiolabeled gastrin analogs that exhibit excellent biodistribution and therapeutic efficacy while preventing and / or reducing toxicity to healthy tissue. The present invention also relates to the use of alpha-radiolabeled gastrin analogs in methods for treating CCKB receptor-positive diseases.

Description

[Technical field]

[0001] The present invention relates to alpha-radiolabeled gastrin analogs and their use in peptide receptor radionuclide therapy (PRRT) applications.In particular, the present invention relates to alpha-radiolabeled gastrin analogs, such as gastrin analogs labeled with actinium-255, that exhibit excellent biodistribution and therapeutic effects while preventing and / or reducing toxicity to healthy tissue.The present invention also relates to the use of alpha-radiolabeled gastrin analogs in methods for treating CCKB receptor-positive diseases, such as tumors or cancer.

[0002] 2. Background of the Invention G protein-coupled receptors (GPCRs) constitute a superfamily of membrane proteins that function to transmit chemical signals across cell membranes. Binding of a ligand to a GPCR induces a conformational change in the GPCR that allows it to activate and release the G protein, which then triggers a signaling pathway.

[0003] Overexpression of G protein-coupled receptors (GPCRs) that selectively bind peptide ligands allows the development of peptide receptor radionuclide therapy (PRRT) for human cancers (Lappano et al. Nat Rev Drug Discov. 2011, 10(1), 47-60). One of the most important goals of PRRT is to increase the uptake of radiolabeled ligands into target cells (e.g., cancer cells) and thus result in radiation-induced DNA damage and cell death. Therefore, strategies have been explored to increase the uptake of radiopharmaceuticals into target cells while sparing the surrounding healthy tissues from side effects.

[0004] GPCRs targeted by agonist ligand-based therapeutics undergo a conformational change resulting in the exchange of GDP for GTP on the G protein alpha subunit (Ga). Subsequent dissociation of the Gα and Gβγ subunits from the receptor activates various kinase signaling pathways including protein kinase A and C (PKA; PKC) as well as phosphoinositide 3 kinase (PI3K) and mitogen-activated protein kinase (MAPK) (O'Hayre et al. Curr Opin Cell Biol. 2014, 27, 126-135). Activated GPCRs then undergo desensitization via an arrestin-mediated internalization process, whereby they can be transported to lysosomes for degradation or to endosomes for recycling back to the cell surface (Rajagopal et al. Cell Signal. 2018, 41, 9-16). This internalization process allows the delivery of ligand-conjugated radionuclides to target cells, e.g., cancer cells.

[0005] Medullary thyroid carcinoma (MTC) is a neuroendocrine tumor derived from calcitonin-producing C cells. Accounting for 3-5% of all thyroid cancers, MTC is a relatively rare cancer (Hadoux et al. Lancet Diabetes Endocrinol. 2016, 4(1), 64-71). Unfortunately, responses to conventional chemotherapy (usually doxorubicin alone or in combination with cisplatin) are only transient, limiting its usefulness to a minority of patients. Furthermore, MTC cells do not accumulate iodine and therefore do not respond to radioactive iodine treatment (Verburg et al. Methods. 201, 55(3), 230- 237). Currently, MTC accounts for 14% of thyroid cancer-related deaths, suggesting the need for better treatments, especially in patients with metastatic disease (Roman et al. Cancer. 2006, 107(9), 2134-2142).

[0006] Small cell lung cancer (SCLC) is an aggressive cancer that most commonly occurs in the lung. It usually presents as a large, rapidly expanding lesion that originates from the centrally located tracheobronchial airways and invades the mediastinum. One-third of patients diagnosed with localized stage SCLC have been cured with chemoradiotherapy, with a cure rate of about 25%. However, both advanced stage and recurrent SCLC are often considered incurable, and available treatments, such as chemotherapy, are usually administered with palliative intent. The prognosis for patients with recurrent SCLC remains poor, with a median overall survival of about 6 months (Travis et al. J Thorac Oncol. 2015, 10(9), 1243- 1260).

[0007] Extrapulmonary small cell carcinoma (EPSCC) refers to small cell carcinomas that arise outside the lung. They most commonly appear in the digestive and urinary systems. EPSCC is a rare neoplasm that accounts for 2.5%-5.0% of all small cell carcinomas and 0.1%-0.4% of all cancers. EPSCC has an aggressive natural history characterized by rapid local progression, early widespread metastasis, and recurrence after treatment. The prognosis for patients diagnosed with EPSCC is relatively poor despite chemotherapy, with a median survival of 3-27 months and an overall 5-year survival rate of approximately 13% (Nakazawa et al. Oncol Lett. 2012, 4(4), 617-620).

[0008] High expression of cholecystokinin B receptor (CCKBR, sometimes also called CCK2R), which belongs to the GPCR family, has been verified in various cancers, including MTC, glioma, SCLC, colon cancer, ovarian cancer, etc. (Reubi et al. Cancer Res. 1997, 57(7), 1377-1386). Furthermore, the small peptide hormone "mini-gastrin" is known to bind with high affinity to CCKBR. Thus, previous studies have proposed the use of radiolabeled gastrin analogs for PRRT, especially for "theranostics" (therapeutic and diagnostic) applications.

[0009] Behr et al.90 Y, 153 Sm), Auger electron sources (e.g. 111 In, 67 Ga) and an alpha-emitting source (e.g., 213 Bi, 225 have proposed the use of mini-gastrin analogs containing a chelating moiety (DTPA) that is highly stable towards a variety of radionuclides, including CCKBR (Ac). However, radiolabeled gastrin analogs, regardless of the radionuclide used, have been found to tend to accumulate in healthy tissues that endogenously express CCKBR, particularly the stomach. This accumulation can lead to harmful side effects (toxicity) in healthy tissues, such as hemorrhagic gastritis, and may therefore limit the dose of radiation that can be administered to a subject in need of treatment (J Nucl Med. 2001, 42(5):68P; Semin Nucl Med. 2002, 32(2), 97-109).

[0010] WO 2015 / 067473 177 Lu (hereinafter referred to as “ 177have described a gastrin analogue of formula DOTA-(DGlu)6-Ala-Tyr-Gly-Trp-Nle-Asp-Phe-NH2 (PP-F11N) labeled with 1,2-difluorophenyl ether (DGlu)6-Ala-Tyr-Gly-Trp-Nle-Asp-Phe-NH2 (PP-F11N) (referred to as "Lu-PP-F11N"), which shows good tumor uptake as well as low accumulation in the kidney, i.e. a good "tumor to kidney ratio". However, recent studies have found that this compound may accumulate in healthy tissues (i.e. in the stomach) due to their endogenous CCKBR expression, thereby limiting radiation dose and / or resulting in deleterious side effects (Sauter et al. J Nucl Med. 2019, 60(3), 393-399; Rottenburger et al. J Nucl Med. 2020, 61(4), 520-526). Furthermore, it has been shown that lutetium-177 may damage surrounding healthy cells due to its long tissue penetration range (cross-fire effect) and may show limited efficacy due to insufficient DNA damage, especially in the treatment of advanced (metastatic) stage disease such as small neoplasms and / or (micro)metastases.

[0011] In view of the above, the object of the present invention is to provide a compound for PRRT use that has excellent biodistribution and therapeutic effect, and also has low toxicity to healthy tissue.Furthermore, the object of the present invention is to provide a compound that can be used in the method for treating CCKB receptor positive diseases, especially CCKB receptor positive diseases in advanced stages.

[0012] Summary of the Invention The present invention provides compounds, i.e., alpha radiolabeled gastrin analogs, that have excellent biodistribution and therapeutic effects, and also have low (or no) toxicity to healthy tissues.In particular, the inventors have found that by labeling certain gastrin analogs with alpha radionuclides, it is possible to achieve excellent biodistribution and therapeutic effects, while preventing and / or reducing harmful side effects due to accumulation of the radioactive compound in healthy tissues that endogenously express CCKBR (e.g., in the stomach) and irradiation of healthy tissues surrounding the target cells.These findings are particularly surprising, since alpha radionuclide-labeled gastrin compounds are associated with increased toxicity in healthy tissues, particularly in the stomach, due to their high endogenous CCKBR expression.

[0013] Therefore, the present invention relates to a compound represented by the following formula (1): Y-(DGlu)6-Ala-Tyr-Gly-Trp-Axx-Asp-Phe-NH2(1) [In the formula, Axx represents an amino acid equivalent to methionine; Y represents a moiety that chelates an alpha radionuclide. The present invention relates to an alpha radiolabeled gastrin analog having the formula:

[0014] The compound of the present invention can be used for PRRT.Therefore, the present invention also relates to alpha radiolabeled gastrin analogue for use in the method of treating CCKBR positive disease, particularly GC, PADC, SCLC, EPSCC, MTC, glioma, GEP-NET, colon cancer, ovarian cancer, breast cancer and any CCKB receptor positive disease.

[0015] The present invention particularly includes the following embodiments (hereinafter referred to as "items"): 1. The following formula (1): Y-(DGlu)6-Ala-Tyr-Gly-Trp-Axx-Asp-Phe-NH2(1) [In the formula, Axx represents an amino acid equivalent to methionine; Y represents a moiety that chelates an alpha radionuclide. An alpha radiolabeled gastrin analog having the formula:

[0016] 2. The alpha radiolabeled gastrin analogue according to item 1, wherein Axx represents an amino acid selected from the group consisting of isoleucine (Ile), norleucine (Nle), 2-amino-5-heptenoic acid, homonorleucine (HomoNIe), homocysteine ​​(HomoCys), 2-amino-4-methoxybutanoic acid, telluromethionine (Te-Met), selenomethionine (Se-Met) and phenylglycine (Phg), preferably Nle.

[0017] 3. Y is 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), 1,4,7,10,13,16-hexaazacyclohexadecane-1,4,7,10,13,16-hexaacetic acid (HEHA), 2-(4-isothiocyanatobenzyl)-1,4,7,10,13,16-hexaazacyclohexadecane-1,4,7,10,13,16-hexaacetic acid (HEHA-NCS), [6,6'-({9-hydroxy-1,5-bis(methoxycarbonyl)-2,4-di(pyridin-2-yl)-3,7-diazabicyclo[3.3.1]nonane-3,7-diyl}bis(methylene)dipicolinic acid] (H2Bispa 2 3. An alpha radiolabeled gastrin analogue according to item 1 or 2, which is a moiety derived from N,N'-bis[(6-carboxy-2-pyridyl)methyl]-4,13-diaza-18-crown-6 (Macropa), 6-[[16-[(6-carboxypyridin-2-yl)methyl]-1,4,10,13-tetraoxa-7,16-diazacyclooctadec-7-yl]methyl]-4-isothiocyanatopyridine-2-carboxylic acid (Macropa-NCS), bis(phenyliminodiacetate)-diazacrown ether (Macropid) or t-Bu-calix[4]arenetetracarboxylic acid, preferably from DOTA or HEHA, more preferably from DOTA.

[0018] 4. Alpha radionuclides are212 Bi, 213 Bi, 225 Ac, 225 Fm, 211 At, 223 Ra, 149 Tb, 212 Pb, 226 Th and 227 Th, preferably 212 Bi, 213 Bi, 225 Ac, 225 Fm, 149 Tb, 212 Pb, 226 Th and 227 From Th, more preferably 213 Bi, 225 Ac and 149 4. An alpha radiolabeled gastrin analog according to any one of items 1 to 3, selected from: Tb.

[0019] 5. Alpha radionuclides 225 5. An alpha radiolabeled gastrin analog according to any one of items 1 to 4, wherein said alpha radiolabeled gastrin analog is Ac.

[0020] 6. The alpha radioactive nuclide is (i) or (ii) of the following: (i) a linear energy transfer (LET) of 20 keV / μm to 190 keV / μm, preferably 30 keV / μm to 160 keV / μm, more preferably 50 keV / μm to 150 keV / μm, in particular about 80 keV / μm; and (ii) a tissue penetration range (TPR) of 20 μm to 150 μm, preferably 30 μm to 120 μm, more preferably 40 μm to 100 μm; 6. An alpha-radiolabeled gastrin analog according to any one of items 1 to 5, which satisfies at least one of the following:

[0021] 7. The following formula (2): Y-(DGlu)6-Ala-Tyr-Gly-Trp-Nle-Asp-Phe-NH2(2) [In the formula, Y is 225 represents a moiety derived from DOTA that chelates Ac] 7. An alpha radiolabeled gastrin analog according to any one of items 1 to 6, wherein

[0022] 8. An alpha-radiolabeled gastrin analog for use in a method of treating one or more CCKB receptor positive diseases comprising administering to a human subject diagnosed with a CCKB receptor positive disease a therapeutically effective dose of an alpha-radiolabeled gastrin analog, An alpha-radiolabelled gastrin analog, wherein the alpha-radiolabelled gastrin analog is as defined in any one of items 1 to 7.

[0023] 9. An alpha radiolabeled gastrin analog having the formula (2): Y-(DGlu)6-Ala-Tyr-Gly-Trp-Nle-Asp-Phe-NH2(2) [In the formula, Y is 225 represents a moiety derived from DOTA that chelates Ac] 9. An alpha radiolabeled gastrin analog for use according to item 8, represented by the formula:

[0024] 10. The alpha radiolabeled gastrin analog for use according to item 8 or 9, wherein the CCKB receptor positive disease is selected from gastric cancer (GC), pancreatic adenocarcinoma (PADC), small cell lung cancer (SCLC), extrapulmonary small cell carcinoma (EPSCC), medullary thyroid carcinoma (MTC), glioma, gastroenteropancreatic neuroendocrine tumor (GEP-NET), colon cancer, ovarian cancer, breast cancer, and any CCKB receptor positive cancer or tumor.

[0025] 11. The alpha radiolabeled gastrin analogue for use according to any one of items 8 to 10, wherein the CCKB receptor positive disease is selected from SCLC, EPSCC and MTC, preferably from SCLC and MTC, more preferably MTC.

[0026] 12. The alpha radiolabeled gastrin analogue for use according to any one of items 8 to 11, wherein the therapeutically effective dose administered to a human subject is 10 to 10,000 kBq / kg, preferably 30 to 1000 kBq / kg, more preferably 50 to 200 kBq / kg.

[0027] 13. A method for treating a CCKB receptor positive disease comprising administering a therapeutically effective dose of an alpha-radiolabeled gastrin analog to a human subject in need thereof, A method for the treatment of CCKB receptor positive diseases, wherein the alpha radiolabeled gastrin analogue is as defined in any one of items 1 to 7, preferably as defined in item 7. [Brief description of the drawings]

[0028] [Figure 1] FIG. 1 shows the internalization of 177Lu-PP-F11N and 225Ac-PP-F11N in A431 / CCKBR cells. The internalization and cell binding activities were measured after 2 hours of incubation with 177Lu-PP-F11N or 225Ac-PP-F11N, all experiments were assayed in triplicate, and each bar represents the mean value ± SD. [Diagram 2] FIG. 1 shows the biodistribution of 225Ac-PP-F11N in xenografted nude mice. Radioactivity uptake in tumor, kidney, stomach, liver, bone and blood is shown as percentage of total injected radioactivity per gram of tissue (%iA(injected radioactivity) / g) at 1, 4, 24, 48 hours and 7 days after administration of 225Ac-PP-F11N. Each bar represents the mean ± SD, n=4 per time point. [Diagram 3] FIG. 1 shows the biodistribution of 225Ac-PP-F11N and 177Lu-PP-F11N in xenografted nude mice. Comparative analysis of radioactivity uptake in indicated organs 4 hours after injection of 225Ac-PP-F11N (black bars) and 177Lu-PP-F11N (white bars). Each bar represents the mean ± SD, n=4, *P<0.01, **P<0.01, ***P<0.001. [Figure 4] Figure 1 shows tumor growth inhibition and life span extension in mice treated with 225Ac-PP-F11N. After tumor implantation, PBS or 30, 45, 60, 90 and 120 kBq of purified 225Ac-PP-F11N were administered as indicated to nude mice bearing A431 / CCKBR tumors (n=9 excluded from control and 60 kBq groups: n=18). Tumor growth curves in different treatment groups. Values ​​are expressed as mean ± SD. [Diagram 5] FIG. 1 shows histological analysis of kidney and stomach sections after treatment. Representative images of HE staining of organs isolated from control, 60 and 120 kBq 225Ac-PP-F11N-treated A431 / CCKBR xenografted nude mice. [Figure 6] FIG. 1 shows SPECT / CT images after treatment with 45 kBq or 60 kBq of 225Ac-PP-F11N. SPECT-CT images of 111In-PP-F11N from two mice in which no tumors were detected after treatment with 45 kBq (left) and 60 kBq (right) of 225Ac-PP-F11N. [Figure 7] FIG. 1 shows the quality control of 225Ac-PP-F11N. Samples from fractions collected during HPLC purification were eluted onto a thin layer chromatography (TLC) plate (0.1 M succinic acid in water / acetonitrile, 40 / 60, v / v), elution was stopped, and 30 min after elution a phosphor screen (MultiSensitive; available from PerkinElmer) was exposed to the TLC plate (exposure time: 2 min) and then scanned at a resolution of 300 dpi using a Cyclone Plus Storage Phosphor System (PerkinElmer) to measure the activity distribution, which showed (A) gamma emission mainly due to unbound radionuclides (221Fr, 213, Bi, 209Tl), and (B) a phosphor screen (MultiSensitive; PerkinElmer) was exposed to the same TLC plate 24 h after elution (exposure time: 2 min) and scanned to measure the activity distribution, which showed only daughter nuclides derived from α-decayed 225Ac.

[0029] Detailed Description of the Invention 1.Definition The term "gastrin analog" as used herein refers to a class of compounds (peptides) that are structurally related to the endogenous peptide hormone "gastrin" and can bind to CCKBR. In particular, the term "gastrin analog" defines a compound that includes a C-terminal pentapeptide Gly-Trp-Dxx-Asp-Phe-NH2, where Dxx is the amino acid equivalent to methionine, and is similar to the C-terminal amino acid sequence (Leu-(Glu)5-Glu-Glu-Glu-Glu-Ala-Tyr-Gly-Trp-Met-Asp-Phe-NH2) found in CCKBR-binding endogenous peptide hormones such as gastrin, cholecystokinin (CCK) and minigastrin. Gastrin analogs can be chemically modified to allow covalent attachment of chelating moieties for radiometals, such as 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA).

[0030] The term "amino acid" as used herein refers to a compound that contains or is derived from at least one amino group and at least one acidic group, preferably a carboxyl group. The distance between the amino group and the acidic group is not particularly limited. α-, β-, and γ-amino acids are suitable, with α-amino acids, especially α-amino carboxylic acids, being particularly preferred. The term encompasses naturally occurring amino acids as well as synthetic amino acids not found in nature. Unless otherwise specified or otherwise dictated by context, all connections between adjacent amino acid groups are formed by peptide (backbone amide) bonds. The peptides described herein are listed from left to right in the conventional amino → carboxy direction.

[0031] As used herein, the phrase "amino acid equivalent to methionine" (or "methionine bioisostere") refers to a natural or unnatural amino acid that has similar shape and / or electronic properties to methionine. As a result, a compound in which a methionine residue is replaced with an amino acid equivalent to methionine will exhibit similar properties, e.g., binding affinity, agonist activity, etc., as the original compound containing methionine. Here, the term "equivalent" is meant to encompass all amino acids that are essentially equivalent to methionine in that the backbone and / or side chain of said amino acid are defined to mimic methionine, e.g., sterically, electronically, etc. Examples of amino acids equivalent to methionine include isoleucine (Ile), norleucine (Nle), homocysteine ​​(HomoCys), 2-amino-5-heptenoic acid, homonorleucine (HomoNIe), 2-amino-4-methoxybutanoic acid, telluromethionine (Te-Met), selenomethionine (Se-Met), and phenylglycine (Phg), etc. In some embodiments, the term "amino acid equivalent to methionine" refers to an amino acid residue that, when substituted for methionine in the amino acid sequence of a mini-gastrin, produces a gastrin analog that retains at least 20%, preferably at least 50%, more preferably at least 80% of the pharmacological (agonist) activity of the mini-gastrin against CCKBR. Pharmacological activity can be determined by measuring the intracellular increase in calcitonin levels in gastrin analog-stimulated cells, as described by Blaeker et al. (Regulatory Peptides 2004, 118, 111-117).

[0032] As used herein, the expression "moiety that constitutes an alpha radionuclide" (or "moiety that chelates or (covalently) binds an alpha radionuclide") means (i) a moiety that is capable of donating electrons to an alpha radionuclide to form a coordination complex therewith, i.e., by forming at least one coordinate covalent bond (dipole bond) therewith; or (ii) 211 At and 223It refers to a moiety (chelator or ligand) that can covalently bind an alpha radionuclide, such as Ra. The chelation mechanism depends on the chelator and / or the radionuclide. For example, DOTA can bind a radionuclide, e.g., via a carboxylate group and an amino group (donor group), e.g. 225 It is believed that it coordinates Ac, thereby forming a highly stable complex (Dai et al. Nature Com. 2018, 9, 857). 225 Non-limiting examples of moieties capable of chelating alpha radionuclides, such as Ac, include DOTA, 1,4,7,10,13,16-hexaazacyclohexadecane-1,4,7,10,13,16-hexaacetic acid (HEHA), 2-(4-isothiocyanatobenzyl)-1,4,7,10,13,16-hexaazacyclohexadecane-1,4,7,10,13,16-hexaacetic acid (HEHA-NCS), [6,6'-({9-hydroxy-1,5-bis(methoxycarbonyl)-2,4-di(pyridin-2-yl)-3,7-diazabicyclo[3.3.1]nonane-3,7-diyl}bis(methylene)dipicolinic acid] (H2Bispa 2 ), N,N'-bis[(6-carboxy-2-pyridyl)methyl]-4,13-diaza-18-crown-6 (Macropa), 6-[[16-[(6-carboxypyridin-2-yl)methyl]-1,4,10,13-tetraoxa-7,16-diazacyclooctadec-7-yl]methyl]-4-isothiocyanatopyridine-2-carboxylic acid (Macropa-NCS), bis(phenyliminodiacetate)-diazacrown ether (Macropid), and t-Bu-calix[4]arenetetracarboxylic acid.

[0033] The expression "moiety derived from a compound" as used herein refers to a moiety (Y) attached to an adjacent moiety, which moiety (Y) differs from the molecule from which it is derived only by the structural element responsible for the bond to the adjacent moiety. This can include a covalent bond formed by an existing functional group, or a covalent bond newly introduced for this purpose and an adjacent functional group. For example, the expression "moiety derived from 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)" refers to a moiety derived from the peptide * -(DGlu)6-Ala-Tyr-Gly-Trp-Axx-Asp-Phe-NH2 is covalently bound to the N-terminus via the carboxyl group to form a peptide (amide) bond. * indicates a covalent bond to the DOTA moiety).

[0034] The term "alpha-emitting nuclide" (or "alpha particle-emitting nuclide", "alpha emitter", "alpha") in this context means an unstable element (radionuclide) that undergoes radioactive decay by emitting alpha particles. Non-limiting examples of alpha-emitting nuclides include: 212 Bi, 213 Bi, 225 Ac, 225 Fm, 211 At, 223 Ra, 149 Tb, 212 Pb, 226 Th and 227 Examples include Th.

[0035] As used herein, the term "cancer" refers to a pathological condition characterized by the abnormal growth of cells in mammalian tissue to form malignant tumors that may invade or spread to other tissues or parts of the body to form "secondary" tumors known as metastases. A tumor contains one or more cancer cells.

[0036] As used herein, the term "internalization" refers to the biological process by which a molecule, such as an alpha-radiolabeled gastrin analog, is engulfed in a cell membrane and drawn into the cell, such that the molecule, such as an alpha-radiolabeled gastrin analog, is present intracellularly.

[0037] The term "cellular uptake" refers to a biological process in which a molecule is internalized and / or bound to a cell membrane. As a result, the molecule can be present not only inside the cell but also on the cell membrane. In a similar manner, the term "tumor uptake" (or "tumor cell uptake") refers to a biological process in which a molecule, such as a radiolabeled gastrin analog, is taken up by a tumor (cancer) cell. As a result, the molecule, such as a radiolabeled gastrin analog, can be present inside the tumor (cancer) cell and / or on the cell membrane.

[0038] The expression "CCKB receptor positive disease" as used herein refers to a disease, such as a tumor or cancer, characterized by (over)expression of CCKBR on the cell surface (Reubi et al. Cancer Res. 1997, 57(7), 1377-1386; Dufresne et al. Physiol Rev 2006, 86, 805-847). (Over)expression of CCKBR, in particular expression on the cell surface, can be determined by techniques known in the art, such as immunohistochemistry or Western blot analysis, as further described below. Non-limiting examples of CCKBR positive diseases include stomach cancer (GC), pancreatic adenocarcinoma (PADC), small cell lung cancer (SCLC), extrapulmonary small cell carcinoma (EPSCC), medullary thyroid carcinoma (MTC), glioma, gastroenteropancreatic neuroendocrine tumor (GEP-NET), colon cancer, ovarian cancer, breast cancer. In the context of the present disclosure, the expression "CCKB receptor positive disease" is meant to include disease caused by primary tumor metastasis and spread to other parts of the body, i.e. advanced stage disease. For example, if CCKB receptor (positive) disease (cancer) occurs in the colon and metastasizes to other parts of the body, the cancer cells found in this other part of the body are (CCKBR positive) colon cancer cells.

[0039] As used herein, the expression "human subject diagnosed with CCKBR positive disease" refers to a human subject with a positive diagnosis for at least one of the aforementioned CCKBR positive diseases, for example, a positive diagnosis of MTC or SCLC. In this context, the diagnosis and status of the disease can be determined by a physician based on established screening guidelines, such as those available from the American Cancer Society (ACS). For example, "positive diagnosis" can mean that the subject has a histological and / or cytological disease status, and optionally one or more of the following: (1) radiographically documented disease progression or recurrence after at least one systemic treatment regimen; (2) at least one non-irradiated extracranial measurable target lesion documented according to Response Evaluation Criteria in Solid Tumors 1.1 (RECIST 1.1) after the last anticancer therapy; (3) at least one tumor lesion according to the Positron Emission Response Evaluation Criteria in Solid Tumors 1.1 (PERCIST 1.0); and (4) Eastern Cooperative Oncology Group (ECOG) score of 0-2.

[0040] The expression "effective dose" (or "effective amount") as used herein refers to the total amount of radioactivity (in becquerels / kilogram) administered to a subject (patient) to treat a disease, e.g., the dose required to reduce or stop the proliferation of cancer cells and / or reduce the number of proliferating cancer cells. If the course of treatment includes one or more (administration) cycles, the effective dose refers to the total amount of radioactivity administered to the subject over the entire course, i.e., over all cycles. The term "cycle" in this context refers to the period during which a compound is administered to a subject (treatment time) and then the patient is allowed to rest (rest time) before entering another cycle. The treatment can include one or more cycles, e.g., up to 10 cycles. A series of cycles is usually referred to as a "course" and can last for several months, e.g., 3 to 6 months, depending on the length of each cycle. The effective dose can be determined by a physician based on dosimetry. The effective dose and frequency of administration for any particular subject / patient may vary and will depend on a variety of factors, including the patient's age, weight, overall health, sex, diet, mode and time of administration, excretion rate, severity of the disease, and the individual being treated. These factors will be taken into account by the physician when determining the effective dose.

[0041] When "preferred" embodiments / features are mentioned in this specification, combinations of these "preferred" embodiments / features shall also be deemed to be disclosed, insofar as such combinations of "preferred" embodiments / features make technical sense.

[0042] Hereinafter, in the description of the present invention and in the claims, the use of the terms "including" and "comprising" shall be understood as meaning that in addition to the elements mentioned, additional unmentioned elements may be present. However, these terms shall also be understood to disclose the term "consisting of" as a more limited embodiment, so that additional unmentioned elements may not be present, as long as it is technically meaningful.

[0043] Unless the context dictates otherwise and / or an alternative meaning is expressly provided herein, all terms are intended to have their generally accepted meaning in the art as reflected by the IUPAC Gold Book (status as of November 1, 2017), or The Dictionary of Chemistry, Oxford, 6th Ed.

[0044] 2. Overview The present invention relates to the coupling of certain gastrin analogs (i.e., gastrin analogs of formula (1)) with alpha radionuclides, particularly 225 This is based on the discovery that labeling with Ac results in superior uptake into target cells, e.g., cancer cells, resulting in superior biodistribution and therapeutic efficacy, while preventing and / or reducing toxicity (harmful side effects) to healthy tissues, i.e., tissues that endogenously express CCKBR and / or tissues surrounding the target cells. These results are particularly surprising since the use of alpha-labeled compounds has been associated with cytotoxic side effects, e.g., radiation nephropathy, hemorrhagic gastritis, etc., likely due to the high energy of the emitted alpha particles.

[0045] The compound of the present invention can be used in the method of treating CCKBR positive disease, particularly GC, PADC, SCLC, EPSCC, MTC, glioma, GEP-NET, colon cancer, ovarian cancer, breast cancer, and any CCKB receptor positive disease.In addition, it is expected that the compound of the present invention is particularly suitable for treating CCKBR positive disease that has reached the advanced (metastatic) stage, for example, metastatic MTC or metastatic SCLC.

[0046] 3. Alpha-radiolabelled Gastrin Analogues The present invention relates to alpha radionuclides, i.e. 225The present invention relates to Ac-labeled gastrin analogues (compounds).The compounds of the present invention show excellent in vivo biodistribution and therapeutic effect on target cells expressing CCKBR, such as cancer cells.In addition, the compounds of the present invention show low toxicity (or no toxicity) on tissues that endogenously express CCKBR and / or tissues surrounding target cells.

[0047] Without being bound by any theory, it is believed that the use of gastrin analogs of formula (1) allows for excellent targeting and uptake in cells expressing CCKBR, resulting in excellent biodistribution, while labeling with alpha radionuclides allows for excellent therapeutic effects (i.e., high levels of DNA damage in target cells) due to the high energy of the emitted alpha particles. Furthermore, the inventors have surprisingly observed that the use of alpha radiolabeled gastrin analogs does not result in harmful side effects (toxicity) in healthy tissues (e.g., in the stomach) and / or in the surroundings of the target cells that endogenously express CCKBR. These results suggest that the compounds of the present invention show low levels of accumulation in healthy tissues, and also significantly reduce the undesirable emission of alpha radionuclides to surrounding tissues due to the short penetration range of the alpha radionuclides (i.e., reduced crossfire effect). This is because 177 This is in contrast to other radionuclides, such as Lu, which can cause damage to surrounding healthy tissue due to their long penetration range (crossfire effect).

[0048] The alpha radiolabeled gastrin analogs of the present invention have the following formula (1): Y-(DGlu)6-Ala-Tyr-Gly-Trp-Axx-Asp-Phe-NH2(1) [In the formula, where Axx represents an amino acid equivalent to methionine, and Y represents a moiety that chelates an alpha radionuclide.

[0049] In one embodiment, Axx represents an amino acid equivalent to methionine selected from the group consisting of isoleucine (Ile), norleucine (Nle), 2-amino-5-heptenoic acid, homonorleucine (HomoNIe), homocysteine ​​(HomoCys), 2-amino-4-methoxybutanoic acid, telluromethionine (Te-Met), selenomethionine (Se-Met) and phenylglycine (Phg). Preferably, Axx is Nle.

[0050] In one embodiment, Y is selected from the group consisting of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), 1,4,7,10,13,16-hexaazacyclohexadecane-1,4,7,10,13,16-hexaacetic acid (HEHA), 2-(4-isothiocyanatobenzyl)-1,4,7,10,13,16-hexaazacyclohexadecane-1,4,7,10,13,16-hexaacetic acid (HEHA-NCS), [6,6'-({9-hydroxy-1,5-bis(methoxycarbonyl)-2,4-di(pyridin-2-yl)-3,7-diazabicyclo[3.3.1]nonane-3,7-diyl}bis(methylene)dipicolinic acid] (H2Bispa 2 ), N,N'-bis[(6-carboxy-2-pyridyl)methyl]-4,13-diaza-18-crown-6 (Macropa), 6-[[16-[(6-carboxypyridin-2-yl)methyl]-1,4,10,13-tetraoxa-7,16-diazacyclooctadec-7-yl]methyl]-4-isothiocyanatopyridine-2-carboxylic acid (Macropa-NCS), bis(phenyliminodiacetate)-diazacrown ether (Macropid) or t-Bu-calix[4]arenetetracarboxylic acid. Preferably, Y represents a moiety derived from DOTA or HEHA. More preferably, Y is DOTA.

[0051] In a preferred embodiment, Axx is Nle and Y is DOTA. Hereinafter, this compound is referred to as "PP-F11N."

[0052] In one embodiment, the alpha radionuclide is 212 Bi, 213 Bi, 225 Ac, 225 Fm, 211 At, 223 Ra, 149 Tb, 212 Pb, 226 Th and 227 Preferably, the alpha radionuclide is selected from 212 Bi, 213 Bi, 225 Ac, 225 Fm, 149 Tb, 212 Pb, 226 Th and 227 More preferably, the alpha radionuclide is selected from 213 Bi, 225 Ac and 149 Tb, and most preferably the alpha radionuclide is selected from 225 It is Ac.

[0053] In one embodiment, the alpha radionuclide is one of the following (i) and (ii): (i) a linear energy transfer (LET) of 20 keV / μm to 190 keV / μm, preferably 30 keV / μm to 160 keV / μm, more preferably 50 keV / μm to 150 keV / μm, in particular about 80 keV / μm; and (ii) a tissue penetration range (TPR) of 20 μm to 150 μm, preferably 30 μm to 120 μm, more preferably 40 μm to 100 μm; At least one of the above conditions, and preferably both of them, is satisfied.

[0054] In a more preferred embodiment, the alpha radiolabeled gastrin analog has the following formula (2): Y-(DGlu)6-Ala-Tyr-Gly-Trp-Nle-Asp-Phe-NH2(2) [In the formula, Y is 225 represents a moiety derived from DOTA that chelates Ac. Hereinafter, this compound will be referred to as " 225It is written as "Ac-PP-F11N".

[0055] The alpha radiolabeled gastrin analogs can be provided in the form of pharmaceutical compositions for use in human medicine. Such compositions typically contain a therapeutically effective dose of the alpha radiolabeled gastrin analogs of the invention and one or more other components, such as carriers, diluents, and the like. In one embodiment, the pharmaceutical composition can contain an mTOR inhibitor, in particular rapamycin and / or a rapalog. In another embodiment, the pharmaceutical composition does not contain an mTOR inhibitor, in particular rapamycin and / or a rapalog. The term "mTOR inhibitor" in this context refers to a compound that inhibits the mammalian target of rapamycin (mTOR). The term "rapamycin" (sirolimus) refers to a macrolide compound known in the art to exhibit immunosuppressive properties by inhibiting the mammalian target of rapamycin (mTOR), while the term "rapalog" (short for "rapamycin-analog") refers to a class of compounds structurally related to rapamycin that are known to inhibit the mammalian target of rapamycin of complex 1 (mTORC1) by binding to FK binding protein 12. Examples of rapalogs include everolimus (RAD001), temsirolimus (CCI-779) and ridaforolimus (AP-23573, MK-8669).

[0056] 4. Use of the Compounds in Methods for Treating Disease The compounds of the present invention can be used in the method of treating one or more CCKBR positive diseases.Treatment can be therapeutic and / or prophylactic treatment, the purpose of which is to prevent, reduce or stop the progression of CCKBR positive diseases through targeted destruction of tumor cells.In some embodiments, treatment can prolong the survival time of patients compared with the expected survival time if not treated.

[0057] The method for treating CCKBR positive disease comprises administering a therapeutically effective dose of an alpha radiolabeled gastrin analog (or a pharmaceutical composition comprising the same) to a human subject diagnosed with one or more CCKBR positive diseases. The alpha radiolabeled gastrin analog administered to the subject is a compound represented by formula (1) above. Preferably, the compound administered to the subject is PP-F11N labeled with an alpha radionuclide.

[0058] In a preferred embodiment, the alpha radiolabeled gastrin analog administered to the subject is 225 It is Ac-PP-F11N.

[0059] The CCKB receptor (positive) disease to be treated is not particularly limited, provided that target cell is characterized by the expression of CCKBR (CCKBR positive).In one embodiment, the CCKB receptor positive disease to be treated is selected from gastric (stomach) cancer (GC), pancreatic adenocarcinoma (PADC), small cell lung cancer (SCLC), extrapulmonary small cell carcinoma (EPSCC), medullary thyroid carcinoma (MTC), glioma (e.g. astrocytoma), gastroenteropancreatic neuroendocrine tumor (GEP-NET), colon cancer, ovarian cancer, breast cancer, and any CCKB receptor positive cancer or tumor.

[0060] In one embodiment, the CCKB receptor positive disease to be treated is selected from SCLC, EPSCC and MTC.Preferably, the CCKB receptor positive disease to be treated is SCLC and / or MTC.More preferably, the CCKB receptor positive disease is MTC.

[0061] Furthermore, the compounds of the present invention, e.g. 225Ac-PP-F11N is expected to show excellent therapeutic effect when administered to subjects diagnosed with at least one of the above-mentioned diseases that have reached advanced (metastatic) stage, because the alpha particles emitted can cause high levels of DNA double-strand breaks in disseminated cancer cells.This is in contrast to other radionuclides such as lutetium-177, which may be ineffective in treating disseminated disease, possibly due to activation of DNA repair mechanisms and radioresistance.Therefore, the disease to be treated can be one or more selected from any CCKB receptor positive disease in advanced stage, preferably the above-mentioned CCKB receptor positive disease in advanced stage, particularly metastatic MTC or metastatic SCLC.

[0062] The observed therapeutic effect may be a reduction in the number of cancer cells, a reduction in tumor size, an inhibition or delay in the infiltration of cancer cells into peripheral organs, an inhibition of tumor growth, and / or an alleviation of one or more symptoms associated with CCKBR-positive disease.

[0063] Therapeutic effective dose can be routinely determined by a physician.The dose level and frequency of administration for any particular subject / patient can vary and depend on a variety of factors, including the activity of the compound used, the metabolic stability and duration of action of the compound, the age, weight, overall health, sex, diet, mode and time of administration, excretion rate, drug combination, the severity of specific symptoms, and the individual being treated.These factors are taken into account by a physician when determining therapeutic effective dose.

[0064] In one embodiment, the therapeutically effective dose of the alpha-radiolabelled gastrin analog administered to the patient is between 10 and 40,000 kBq / kg. Preferably, the therapeutically effective dose is between 30 and 1,000 kBq / kg, more preferably between 50 and 200 kBq / kg. The alpha-radiolabelled gastrin analog can be administered once or several times a day.

[0065] The molecule can be administered to the patient once or over a series of treatments, i.e., over one or more administration cycles. In particular, the compound can be administered to the subject once or twice per 2-10 week cycle, preferably once per 4-8 week cycle, more preferably once per 6 week cycle or once per 8 week cycle. When the compound is administered twice per cycle, the therapeutically effective dose is divided into two half-doses that are administered separately over the course of the cycle. The number of cycles can be from 1 to up to 10 cycles, preferably from 2 to 8 cycles, more preferably from 4 to 6 cycles.

[0066] In one embodiment, the compound is administered to a subject according to one of the following administration patterns: (A) once every 6 week cycle for at least two 6 week cycles, preferably once every 6 week cycle for three 6 week cycles; or (B) once every 8 week cycle for at least two 8 week cycles, preferably once every 8 week cycle for three 8 week cycles.

[0067] In one embodiment, a patient diagnosed with one or more CCKBR positive diseases meets one or more of the following (a)-(h) prior to administration of the compound: (a) serum creatinine ≤1.5 × ULN or estimated glomerular filtration rate (eGFR) >50 mL / min based on the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) equation; (b) blood absolute neutrophil count (ANC) ≥1500 cells / μL; (c) platelet count ≥100,000 cells / μL; (d) blood hemoglobin ≥9 g / dL; (e) AST or ALT below 3 × ULN; (f) serum albumin >20 g / L; (g) total bilirubin less than or equal to 2.5 mg / dL; (h) alkaline phosphatase below 5 × ULN.

[0068] In one embodiment, the method may include a step of determining whether the subject satisfies one or more of (a)-(h) above.

[0069] In one embodiment, the compound is administered to the subject by injection, particularly by intravenous injection. In this regard, the compound can be provided as a solution in a pharma- ceutically acceptable injectable carrier, such as an aqueous carrier (e.g., water or 0.9% sodium chloride). An effective dose can be administered in a volume of 1-200 mL, preferably 5-50 mL, for example about 10 mL. The infusion rate can be 35-60 mL / h, for example about 50 mL / h.

[0070] According to one embodiment, the method comprises the following steps: (α) preparing an injectable solution of the compound by dissolving the compound in a pharma- ceutically acceptable injectable carrier to obtain an injectable solution of the compound; (β) administering to the patient the injectable aqueous solution of the compound obtained from step (α), preferably at an infusion rate of 35-60 mL / h, e.g., 50 mL / h, over an infusion period of 20-60 minutes, e.g., 30-45 minutes. Includes.

[0071] According to a further embodiment, the compound is administered simultaneously with, before and / or after one or more other therapeutic agents or treatments, such as a chemotherapeutic agent, an immunomodulatory agent, a proton pump inhibitor (PPI) or a histamine H2 receptor antagonist.

[0072] In one embodiment, the compound (or a pharmaceutical composition comprising it) is administered in combination with (i.e., simultaneously with, before and / or after) rapamycin and / or a rapalog. In a further embodiment, the compound is administered in combination with (i.e., simultaneously with, before and / or after) everolimus. In yet another embodiment, the compound is not administered in combination with (i.e., simultaneously with, before and / or after) rapamycin and / or a rapalog.

[0073] In one embodiment, the compounds of the present invention can be administered simultaneously with, before or after one or more other therapeutic agents or treatments, such as chemotherapeutic agents and / or immunomodulatory agents. Examples of therapeutic agents or treatments that can be used include antitumor agents, such as alkylating agents, alkaloids or kinase inhibitors, immunomodulatory agents, and pharma- ceutically acceptable salts and derivatives thereof.

[0074] 5. Preparation of Alpha-radiolabelled Gastrin Analogue The following provides a method for preparing radiolabeled gastrin analogues.Gastrin analogues can be synthesized by relying on standard Fmoc-based solid phase peptide synthesis (SPPS), including on-resin peptide coupling and convergent strategy.The general strategies and methodologies that can be used to prepare and radiolabel the gastrin analogues of the present invention are well known to those skilled in the art and are further described below.

[0075] In some embodiments, the present disclosure provides: 225 The present invention provides a method for purifying and measuring the purity of compounds labeled with an alpha radionuclide, such as Ac. 225 Purification and quality control of Ac-labeled compounds can be particularly challenging because alpha radionuclides are short-range sources that usually exhibit α decay but in some cases no γ emission. Thus, compounds labeled with alpha radionuclides may not be (directly) detectable by conventional autoradiography systems. Furthermore, their low concentrations in purified solutions (typically in the picomolar range) may preclude detection of alpha radiolabeled compounds by standard detection methods, e.g., ultraviolet absorption spectrometry.

[0076] Therefore, the present inventors have developed a method for purifying and measuring the purity of alpha-radiolabeled compounds. 225 Ac-labeled DOTA-(DGlu)6-Ala-Tyr-Gly-Trp-Nle-Asp-Phe-NH2 ( 225Ac-PP-F11N) is described below as an exemplary compound.

[0077] (1) A compound containing a chelating moiety (e.g., PP-F11N) 225 Label with Ac (e.g., in solution as described below) 225 The labeled solution is purified by chromatography (HPLC) and the eluate fractions are collected.

[0078] (2) The fractions are sampled and individually developed (eluted) on a solid support (e.g., on a TLC plate) containing a stationary phase using an appropriate solvent system (e.g., 0.1 M succinic acid in water / acetonitrile, 40 / 60, v / v), after which the (unbound) gamma-emitting nuclides present in the labeling solution decay, 225 Daughter nuclides derived from Ac ( 225 The mixture is allowed to stand for a predetermined period of time (eg, 6 to 24 hours) until only Ac (produced by α-decay of Ac) is present.

[0079] (3) Measure the activity distribution on the plate and determine the fractions 225 The purity of the Ac-labeled compound can be determined.

[0080] The activity distribution on the TLC can be measured using a Cyclone® Plus Storage phosphor system and a MultiSensitive or Super Resolution storage phosphor screen (available from PerkinElmer, Switzerland), which may be appropriately calibrated. To perform the measurement, the phosphor screen (preliminarily erased with a white light box) is illuminated onto the TLC plate, placed in a Cyclone® Plus radioactive imaging system, and scanned. The exposure time (phosphor screen → TLC plate) can be 1-60 min, preferably 2-10 min, more preferably 2 min. The measurement of the purity / quantity of the compounds in the collected fractions can be performed using OptiQuant® software.

[0081] The above methods allow for the sensitive detection and quantification of alpha-radiolabelled compounds.

[0082] 6. Working Example 6.1 List of abbreviations DIEA: Diisopropylethylamine DMEM: Dulbecco's modified Eagle's medium DMF: Dimethylformamide DTT: Dithiothreitol ESI: Electrospray ionization FBS: Fetal bovine serum HATU: 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate HBTU: 3-[bis(dimethylamino)methyliumyl]-3H-benzotriazole-1-oxide hexafluorophosphate HPLC: High-performance liquid chromatography IU: International Unit MS: Mass spectrometry PBS: Phosphate-buffered saline SQD: Single quadrupole detector SPECT: Single-photon emission computed tomography SPPS: Solid Phase Peptide Synthesis TBST: Tris-buffered saline with Tween 20 TFA: Trifluoroacetic acid TIS: Triisopropylsilane TLC: Thin Layer Chromatography UPLC: Ultra-high performance liquid chromatography

[0083] 6.2 Materials and Methods The following materials and methods can be used to prepare and evaluate the compounds of the invention.

[0084] 6.2.1 Cell culture, transfection and treatment CCKBR-overexpressing human epidermoid carcinoma A431 stable cell line generated as previously described (Aloj et al., J Nucl Med 2004, 45(3), 485-94) can be cultured in DMEM supplemented with 10% FBS (Bio Concept, Switzerland), 2 mM glutamine and antibiotics (0.1 mg / mL streptomycin, 100 IU penicillin) at 37°C in a humidified incubator containing 5% CO2.

[0085] 6.2.2 Preparation of Gastrin Analogues The gastrin analogs described herein can be prepared by standard Fmoc-based SPPS, including on-resin peptide coupling and convergent strategies using an Activo-P-11 automated peptide synthesizer (Activotec) and Rink Amide resin (loading: 0.60 mmol / g; Novabiochem).

[0086] The coupling reaction for amide bond formation is carried out at room temperature for 30 min using 3 equivalents of Fmoc amino acid activated with HBTU (2.9 equivalents) in the presence of DIEA (6 equivalents). Fmoc deprotection is carried out with a 20% piperidine solution in DMF. The coupling of the N-terminal labeling moiety is carried out at room temperature for 30 min using 3 equivalents of DOTA tris-t-Bu ester (Novabiochem) activated with HATU (2.9 equivalents) in the presence of DIEA (6 equivalents).

[0087] The peptide is cleaved from the resin with simultaneous side-chain deprotection by treatment with TFA / TIS / water (95 / 2.5 / 2.5, v / v / v) for 60 min. After concentration of the cleavage mixture, the crude peptide is precipitated with cold diethyl ether and centrifuged.

[0088] Peptides can be purified on a Waters Autopurification HPLC system coupled to an SQD mass spectrometer equipped with an XSelect Peptide CSH C18 OBD Prep column (130 Å, 5 μm, 19 mm x 150 mm) using solvent systems A (0.1% TFA in water) and B (0.1% TFA in acetonitrile) with a 20-60% gradient of B over 30 min at a flow rate of 25 mL / min. Appropriate fractions are associated, concentrated, and lyophilized. Purity is determined on a Waters Acquity UPLC system coupled to an SQD mass spectrometer equipped with a CSH C18 column (130 Å, 1.7 μm, 2.1 mm x 50 mm) using solvent systems A (0.1% TFA in water) and B (0.1% TFA in acetonitrile) with a 5-85% gradient of B over 5 min at a flow rate of 0.6 mL / min.

[0089] MS analysis can be performed in positive and negative modes using an electrospray ionization (ESI) interface.

[0090] 6.2.3 Radiolabeling of Gastrin Analogues (a) To prepare the gastrin analog radiolabeled with the alpha radionuclide, i.e., actinium-225, a stock solution of the N-terminally DOTA-conjugated gastrin analog DOTA-(DGlu)6-Ala-Tyr-Gly-Trp-Nle-Asp-Phe-NH2 (PP-F11N; prepared as described in section 6.2.1 above) in metal-free water is prepared and stored at -20°C (concentration: 0.58 mM).

[0091] 6 μL 225Ac solution (6 MBq in 0.1 M HCl; available from ITG GmbH, Germany), 100 μL of PP-F11N stock solution (0.58 mmol), 120 μL of ammonium acetate (0.4 M, pH 5.5) and 21 μL of ascorbic acid (100 mg / mL freshly prepared in sodium acetate buffer; total pH = 5-5.5) were heated at 90 °C for 15 min in a clean, sterile Eppendorf® tube (1.5 mL). 2 μL of 0.5 mM EDTA in metal-free water was then added to complex any free metal in the reaction mixture.

[0092] The reaction batch can be cleaned up using a Merck Hitachi LaChrom 2D HPLC system equipped with an autosampler, a radiation monitor (RM-19, EBERLINE Instrument Corporation, Santa Fe, NM), a UV detector (Pharmacia LKB-UV-M II), a 515 HPLC pump and an L-7100 pump connected to a Dr. Maisch reversed-phase C-18 Stability BS-C23 column (150×4.6 mm) (5 μm, 150×4.6 mm, Xterra™, MS, Waters, USA). The mobile phase consists of metal-free water (A) and 0.1% TFA (Sigma-Aldrich, USA) in acetonitrile (VWR Chemicals, USA; HPLC-grade) (B). After sample injection, solvent A (68%) in solvent B is applied at a flow rate of 3 mL / min for 5 min to load all the sample onto the column. A gradient of solvent A (50-10%) in solvent B was then applied over 25 min at a flow rate of 1 mL / min. 225 Ac-PP-F11N and unlabeled PP-F11N are separated. 225 The elution time of Ac-labeled PP-F11N is longer than that of unlabeled PP-F11N after the UV peak of unlabeled PP-F11N. Add 250 μL of 100 mg / mL fresh sodium acetate buffer to a clean, sterile Eppendorf tube. 225 The Ac-PP-F11N fraction can be collected.

[0093] Obtained 225 The purity of the Ac-PP-F11N fraction can be measured by developing a sample on a TLC plate (TLC plate coated with RP-18 modified silica gel 60, 5×10 cm; Merck) using 0.1 M succinic acid in water / acetonitrile 40 / 60 (v / v) as the eluent. The TLC plate is left to stand for 24 hours and then placed in a cassette containing a MultiSensitive Storage phosphor screen (available from PerkinElmer). After 2 minutes, the phosphor screen is placed in a Cyclone®Plus Storage phosphor system and the activity distribution is measured.

[0094] The collected fractions used in the following experiments 225 The purity of the Ac-PP-F11N compound was confirmed to be 97% (Figure 7).

[0095] (b) Gastrin analog radiolabeled with Indium-111 ( 225 To prepare the Ac-PP-F11N treated SPECT images, 23.4 nmol of PP-F11N and 120 MBq of 111 In (69.5 pmol, available from ITG GmbH, Germany) was prepared in 92 μL of 0.4 M ammonium acetate buffer (pH 5.5) and labeling was carried out for 20 min at 95° C. with the addition of 66 μL of 0.5 M ascorbic acid. 2 μL of 0.5 mM EDTA in metal-free water was then added to complex any free metal in the reaction mixture.

[0096] Indium incorporation can be analyzed by standard HPLC using a C18 column, reaching an efficiency of over 95%. Directly after labeling, appropriate dilutions of radiolabeled gastrin analogs for targeted radiation experiments are prepared using a gamma counter.

[0097] (c) To prepare the gastrin analog radiolabeled with lutetium-177 (used in the comparative experiments), PP-F11N was used in a nuclide / peptide ratio of 1:30.177 A solution of Lu (available from ITG GmbH, Germany) was prepared in 0.4 M ammonium acetate buffer (pH 5.5) and labeling was carried out at 90° C. for 15 min.

[0098] Lutetium uptake can be analyzed by standard HPLC using a C18 column, reaching an efficiency of over 95%. Directly after labeling, appropriate dilutions of radiolabeled gastrin analogs for targeted radiation experiments are prepared using a gamma counter.

[0099] 6.2.4 In Vitro Radiolabeled Peptide Internalization Assay For internalization assays, 1 × 10 6 Cells / well were cultured overnight in a 6-well plate. The next day, the cells were washed with PBS and incubated with 160 Bq purified 225 Ac-PP-F11N (or 100.000cpm 177 Lu-PP-F11N) in DMEM containing 0.1% BSA under standard tissue culture conditions for 2 h. For blocking experiments, 4 μM LEEEEEAYGWMDF peptide was used.

[0100] After incubation, the supernatant is collected (together with 2x PBS washes). The cells are then incubated twice with 0.05M ice-cold glycine buffer (pH=2) for 5 min, and then lysed with 1M NaOH for 15 min at 37° C. All three collected fractions (supernatant / PBS; glycine solution; lysed cells) can be measured in a Packard Cobra II Auto-Gamma counter (PerkinElmer, Switzerland). 225 The internalized and membrane-bound fractions of Ac-PP-F11N are shown as percentages of the total activity. Nonspecific membrane binding (glycine fraction) or internalization (NaOH-lysed cells) from experiments with blocking peptides were subtracted from the results obtained.

[0101] 6.2.5 Proliferation assays In the cell proliferation assay, 4 × 10 3Cells / well were seeded in a 96-well plate. The next day, 225 Ac-PP-F11N (0.01–316.23 kBq / mL) was added to A431 / CCKBR cells. After 2 h of incubation, unbound 225 The medium containing Ac-PP-F11N is removed and the cells are incubated with fresh medium for an additional 24 h.

[0102] Cell proliferation can be analyzed using the CellTiter 96 AQueous non-radioactive cell proliferation kit (Promega AG, Switzerland) according to the manufacturer's instructions. The absorbance of the formazan product is measured at 570 nm with a reference of 650 nm using a microplate reader (PerkinElmer, Switzerland). The absorbance of control (untreated) cells corresponds to 100% cell viability. 225 The activity level of Ac-PP-F11N was calculated and expressed as the half-maximal effective activity level (EA) in cell killing. 50 The assay was performed in triplicate.

[0103] 6.2.6WB analysis The expression level of CCKBR can be determined by Western blot analysis as follows:

[0104] Antibody against CCKBR (ab77077) is available from Abcam (UK). Cells are homogenized in lysis buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% Triton X, 1 mM sodium orthovanadate, 1 mM NaF and 0.1% SDS supplemented with protease inhibitor cocktail (Roche). Aliquots of 50 μg protein extracts are resolved by SDS-PAGE and transferred to PVDF membranes (Millipore) by electroblotting. Membranes are blocked with 5% skim milk in TBST (0.1% Tween 20) for 1 h, incubated with primary antibodies overnight in 2% BSA in TBST, followed by incubation with HRP-conjugated secondary antibodies for 2 h. Protein-specific signals can be detected by chemiluminescence (ECL) and signals can be acquired using an ImageQuant RT ECL Imager (GE Healthcare).

[0105] Prior to CCKBR detection, protein lysates are subjected to deglycosylation. Briefly, 18 μL of whole cell lysate (approximately 50 μg) are mixed with 2 μL of 10× denaturing buffer (5% SDS, 0.4 M DTT) and incubated for 10 min at room temperature. Then, 4 μL of 10× Glycobuffer (0.5 M sodium phosphate, pH 7.5), 4 μL of 10% Tween-20, and 10 μL of water are added. Finally, 2 μL of PNGase F (Sigma) is added, mixed, and collected by centrifugation. The reaction is carried out overnight at 37 °C and subjected to WB analysis.

[0106] 6.2.7 Animal testing In this study, we use a human A431 / CCKBR xenograft mouse model. Importantly, immunodeficient mice bearing A431-CCK2R xenografts have been previously used for preclinical evaluation of the pharmacokinetics, biodistribution, dosimetry or toxicity of radiolabeled mini-gastrin analogs required for regulatory approval of phase 1 clinical trials in medullary thyroid carcinoma (MTC) patients (Maina et al., Eur J Pharm Sci. 2016;91:236-42).

[0107] For biodistribution studies, 2 × 10 cells in 0.1 mL of sterile phosphate-buffered saline (PBS) were 6 A431 / CCKBR cells are injected subcutaneously (sc) into the left and right flanks (2 tumors per animal) of anesthetized nude mice (immunodeficient CD-1 female nude mice; Charles Rivers, Germany). 12–14 days after implantation, tumors grow to approximately 0.1–0.2 cm 3 Nude mice bearing A431 / CCKBR tumors were treated with 38 kBq of HPLC-purified 225 Ac-PP-F11N (0.08 pmol) or 150 kBq HPLC purification 177 Lu-PP-F11N (0.21 pmol) was administered intravenously. Mice were sacrificed 1, 4, 24, 48 h and 7 days after administration of the radiolabeled peptide, and the weights of tumors and organs dissected after mortem were measured, and their activities were measured using a gamma counter (Packard Cobra II Auto Gamma, PerkinElmer, Switzerland).

[0108] In the treatment study, nude mice were treated with 5 × 10 6 Inject 100 A431 / CCKBR cells subcutaneously into the left shoulder. 5-7 days after tumor implantation, approximately 0.1-0.2 cm 3 Nude mice bearing A431 / CCKBR tumors were randomly assigned to groups and were incubated with HPLC-purified 100 μL of PBS. 225 Ac-PP-F11N is administered intravenously at 30, 45, 60, 90, and 120 kBq. The control group is injected with 100 μL of PBS. The tumor diameter and the weight of the mice are recorded every day during the working day. The tumor volume is calculated according to the formula V = (W 2 The tumor volume was calculated using the formula (Faustino-Rocha et al. Lab Anim. 2013, 42(6), 217-224). Nude mice were treated with a tumor volume of 1.5 cm 3 Data are from two sets of experiments including control, 30, 45, 60 kBq groups and control, 60, 90, 120 kBq treatment groups.

[0109] All experiments are performed in accordance with Swiss animal protection laws.

[0110] 6.2.8 SPECT / CT For SPECT / CT experiments, HPLC-purified radiolabeled 111 In-PP-F11N was concentrated in a SpeedVac, diluted to 13 MBq per 100 μL of PBS, and administered intravenously to nude mice (13 MBq / 100 μL per mouse). 225 in A431 / CCKBR tumor-bearing nude mice after treatment with Ac-DOTA-PP-F11N 111 Imaging of In-PP-F11N is performed by single photon emission computed tomography (SPECT) combined with X-ray computed tomography (multipinhole small-animal Nano SPECT / CT camera, Mediso Medical Imaging Systems). All mice are sacrificed 2 hours after injection and directly used for 7.5 min CT followed by 45 min SPECT scan. Image reconstruction and processing are performed by using VivoQuant 3.0 Patch 1 software.

[0111] 6.2.9 Immunochemistry Paraffin sections of formalin-fixed A431 / CCKBR tumors were subjected to deparaffinization. Rehydrated slides were pretreated with 10 mM citrate buffer, pH 6.0, at 98°C for 60 min, followed by incubation with 4% nonfat milk in PBS for 90 min. Avidin / biotin blocker (Invitrogen) treatment and detection used the ABC method according to the manufacturer's instructions. For monoclonal antibodies against Ki67 (Thermo Scientific, SP6), signals were recorded using an automated instrument reagent system (Discovery XT, Ventana Medical System Inc.) according to the user manual. Images of sections counterstained with hematoxylin were taken (Nikon, YTHM) and analyzed using ImageAccess Enterprise7 and ImageJ software (Schneider et al. Nat Methods 2012, 9(7), 671-675).

[0112] 6.2.10 Bioinformatics and Statistical Analyses All statistical analyses were performed using GraphPad Prism 7.00 for Windows. Two-tailed Student's t-tests with heteroscedasticity were used for the two groups in the biodistribution studies, whereas the control group and all 225 One-way analysis of variance combined with Dunnett's multiple comparison test was used to compare the Ac-PP-F11N-treated groups. Log-rank (Mantel-Cox) test and Gehan-Breslow-Wilcoxon test were performed to compare the different survival curves of the treatment and control groups. The end point was defined as death in the survival curves. A value of P<0.05 was considered statistically significant. Results are reported as the mean ± standard deviation of at least three independent replicates.

[0113] Example 1: 225 In vitro internalization and cytotoxic effects of Ac-PP-F11N To evaluate the affinity and specificity of the alpha radiolabeled gastrin analogs of the present invention for CCKBR, purified 225Ac-PP-F11N (prepared as described above) was used to perform the in vitro internalization assay described above. 225 The internalization rate of Ac-PP-F11N reached 45%, and the membrane binding activity was 1% of the total activity. The results are shown in Figure 1. In the presence of a blocking peptide (LEEEEEAYGWMDF), the internalization rate was suppressed to 1.4%, 225 It was demonstrated that Ac-PP-F11N was specifically taken up into cells by CCKBR. 177 The experiment was repeated using Lu-PP-F11N (prepared as above). 225 Ac-PP-F11N and 177 Both compounds, Lu-PP-F11N, were found to exhibit similar internalization rates in vitro.

[0114] To analyze the cytotoxic effects, the cell proliferation assay described above was performed. 50 Calculate the value, 225 It reached 6.2±1.1 kBq / mL 24 hours after Ac-PP-F11N treatment (data not shown). The maximum cytotoxic effect (0% cell viability) was reached at 100 kBq / mL.

[0115] These results demonstrate that the compounds of the present invention exhibit CCKBR-specific cellular uptake (internalization rate 45%) and potent cytotoxic effects.

[0116] Example 2: 225 In vivo biodistribution of Ac-PP-F11N The in vivo biodistribution of the compounds of the present invention is 225 Ac-PP-F11N was used to evaluate the biodistribution studies described above.

[0117] Biodistribution studies were performed in nude mice bearing A431 / CCKBR tumors at 1, 4, 24, 48 hours and 7 days after radiopharmaceutical application. 225High tumor uptake of Ac-PP-F11N was observed at 1 and 4 h post-injection, reaching 13% and 11.2% / g (%iA (injected radioactivity) / g) of injected activity, respectively (Figure 2). After 24, 48 and 7 days, tumor uptake decreased as expected, to 7.2%, 5.7% and 4.5%iA / g, respectively. Analysis of healthy organs showed 1.5%iA / g in the stomach at 1 h p.i., due to endogenous expression of CCKBR. Gastric activity was similar within 24 h (1.3% and 1.2%iA / g at 4 and 24 h), but decreased to 0.8% and 0.3%iA / g at 48 and 7 days post-injection. A 50% decrease in radiolabeled gastrin accumulation from 8.1% to 4.2%iA / g was observed in the kidneys during the first 4 h. Furthermore, activity levels decreased from 3.8% to 1.2% 48 hours and one week after injection. In the other organs analyzed, activity levels reached a maximum of 0.38% to 0.02% iA / g at one hour after injection and decreased from 0.15% to 0.01% at one week.

[0118] moreover, 225 Ac-PP-F11N is a lutetium-177 labeled mini-gastrin analog ( 177 Lu-PP-F11N) at 4 hours after injection (Figure 3). The tumor uptake was almost the same, but 225 Ac-PP-F11N is 177 It showed moderately high nonspecific uptake in liver and bone compared with Lu-PP-F11N, reaching <2% iA / g at all assay time points.

[0119] The compounds of the present invention exhibited excellent biodistribution properties, i.e., excellent tumor-kidney and tumor-stomach ratios at the time points analyzed, and showed a significant increase in tumor tissue 225 Specific accumulation of Ac-PP-F11N and CCKBR in organs endogenously expressing CCKBR 225 It was found that the mice showed low accumulation of Ac-PP-F11N. Furthermore, no significant differences in body or organ weights, overall health status, or anatomical structure were observed.

[0120] Example 3: 225 In vivo therapeutic effect of Ac-PP-F11N To evaluate the therapeutic efficacy of the compounds of the present invention, 225 The above-mentioned therapeutic test was carried out using Ac-PP-F11N. 225 Tumor growth and median survival time were evaluated in immunodeficient A431 / CCKBR tumor-bearing nude mice after administration of five different doses of Ac-PP-F11N.

[0121] As shown in Figure 4, 225 Treatment with Ac-PP-F11N significantly inhibited tumor growth in a dose-dependent manner. On day 11, when all mice were still present in all groups, the mean tumor volume in the control group was 0.78 cm 3 reached 30, 45, 60, 90 and 120 kBq. 225 The mean tumor sizes of Ac-PP-F11N-treated mice were 0.54 (P = 0.0142), 0.31 (P < 0.001), 0.12 (P < 0.001), 0.11 (P < 0.001) and 0.07 cm, respectively. 3 In the control group, the tumor volume was 1.5 cm on the 11th day after PBS injection (P<0.001). 3 The first mouse was euthanized because it exceeded the endpoint of 30, 45, 60, 90, and 120 kBq. 225 In the Ac-PP-F11N-treated group, initial tumors grew to 1.5 cm on days 12, 13, 21, 33, and 44, respectively. 3 During treatment, there was no weight loss or other signs of cytotoxicity, and weight gain was observed in all groups. A moderate increase in weight was observed in the control group, presumably due to more rapid tumor growth.

[0122] moreover, 225 Treatment with Ac-PP-F11N extended life span in a dose-dependent manner. The mean survival time in the control group was 17 days, whereas the mean survival time in the control group was 17 days. 225 The mean survival time of Ac-PP-F11N-treated mice was extended to 22, 27, 34, 44 and 58 days, respectively. The results are shown in Table 1 below.

[0123] [Table 1]

[0124] ( * ) P values ​​were obtained by comparing differences in survival curves between treated and control groups using the Log-rank (Mantel-Cox) test.

[0125] From the above results, it has become clear that the compound of the present invention exhibits excellent therapeutic effects and is capable of preventing harmful side effects caused by the accumulation of the compound in healthy tissues.

[0126] Example 4: Histopathological analysis and PET / SPECT imaging For healthy organs 225 To further analyze the side effects (toxicity) of Ac-PP-F11N, the control and 225 Kidneys and stomachs isolated from Ac-PP-F11N-treated mice were stained with hematoxylin and eosin (HE) at the late stage of treatment. Organs from the control group were isolated 11–26 days after PBS injection, whereas 60 kBq and 120 kBq 225 From the Ac-PP-F11N treated group, the cells were isolated between 34 and 49 days after treatment. 225 There was no difference from the Ac-PP-F11N treatment group (FIG. 5), indicating that there were no signs of acute radiation toxicity during alpha-ray therapy in the stomach and kidneys.

[0127] As shown in Figure 6, 45 kBq (left photo) and 60 kBq (right photo) 225 In tumor-free mice treated with Ac-PP-F11N 111 SPECT / CT images of In-PP-F11N show no detectable tumors, with radioactive signals only detected in excretory organs such as the kidneys and bladder.

[0128] These results demonstrate not only the excellent therapeutic effect achieved by the compounds of the present invention, but also the low toxicity to healthy tissues, especially to the stomach. The lack of toxicity to the stomach is surprising, since the use of alpha-radiolabeled gastrin compounds has previously been associated with cytotoxic side effects, such as hemorrhagic gastritis (Semin Nucl Med. 2002, 32(2), 97-109). Thus, the compounds of the present invention can be effectively used in methods for treating CCKBR-positive diseases, especially CCKBR-positive cancers such as MTC.

Claims

1. The following formula (1): Y-(DGlu) ６ -Ala-Tyr-Gly-Trp-Axx-Asp-Phe-NH ２ (1) [In the formula, Axx represents norleucine (Nle), Y is, ２２５ and represents a moiety derived from DOTA that chelates Ac, and the moiety derived from DOTA represents a DOTA molecule that is covalently bonded to the N-terminus of DGlu via the carboxyl group of DOTA to form a peptide (amide) bond. An alpha radiolabeled gastrin analog having the formula:

2. The 225 Ac has the following (i) and (ii): (i) a linear energy transfer (LET) of 20 keV / μm to 190 keV / μm; and (ii) Tissue penetration range (TPR) of 20 μm to 150 μm The alpha-radiolabeled gastrin analog of claim 1 , which satisfies at least one of the following:

3. 1. An alpha-radiolabelled gastrin analog for use in a method of treating one or more CCKB receptor positive diseases comprising administering to a human subject diagnosed with a CCKB receptor positive disease a therapeutically effective dose of an alpha-radiolabelled gastrin analog, 3. An alpha-radiolabelled gastrin analog, wherein said alpha-radiolabelled gastrin analog is as defined in claim 1 or 2.

4. The alpha radiolabeled gastrin analog for use according to claim 3, wherein the CCKB receptor positive disease is selected from gastric cancer (GC), pancreatic adenocarcinoma (PADC), small cell lung cancer (SCLC), extrapulmonary small cell carcinoma (EPSCC), medullary thyroid carcinoma (MTC), glioma, gastroenteropancreatic neuroendocrine tumors (GEP-NET), colon cancer, ovarian cancer, breast cancer, and any CCKB receptor positive cancer or tumor.

5. The alpha radiolabeled gastrin analogue for use according to claim 3 or 4, wherein the CCKB receptor positive disease is selected from SCLC, EPSCC and MTC.

6. The alpha-radiolabelled gastrin analogue for use according to any one of claims 3 to 5, wherein the therapeutically effective dose administered to a human subject is between 10 and 40,000 kBq / kg.

7. A pharmaceutical composition for treating a CCKB receptor positive disease comprising an alpha radiolabeled gastrin analog, 3. A pharmaceutical composition, wherein said alpha-radiolabelled gastrin analog is as defined in claim 1 or 2.

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