Compositions Comprising a Rapalog and a Radiolabeled Gastrin Analogue, Particularly for Use in the Treatment and / or Diagnosis of CCKB Receptor-Positive Cancers or Tumors - Patent application

Abstract

The present invention relates to compositions comprising (i) rapamycin and / or a rapalog, and (ii) a radiolabeled gastrin analog. The compositions of the present invention can be used to treat and / or diagnose CCKB receptor-positive cancers or tumors, and provide excellent tumor uptake of the radiolabeled gastrin analog, resulting in improved delivery and therapeutic efficacy while preventing and / or reducing cytotoxic side effects.

Description

[Technical field]

[0001] The present invention relates to compositions comprising rapamycin and / or rapalogs and radiolabeled gastrin analogs for the treatment and / or diagnosis of disease. In particular, the present invention relates to compositions for peptide receptor radionuclide therapy (PRRT) applications, which provide superior tumor uptake of radiolabeled gastrin analogs via inhibition of the mammalian target of rapamycin (mTOR), resulting in improved delivery and therapeutic efficacy while preventing and / or reducing cytotoxic side effects.

[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 activation and release of the G protein, subsequently triggering 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 achieve high tumor uptake of the radiolabeled ligand. Therefore, strategies have been explored to increase the uptake of radiopharmaceuticals in tumor or cancer tissues while sparing healthy organs from cytotoxic 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-bound 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] High expression of the cholecystokinin B receptor (CCKBR, sometimes called CCK2R), which belongs to the GPCR family, has been demonstrated in various cancers, including MTC, glioma, colon cancer, ovarian cancer, etc. (Reubi et al. Cancer Res. 1997, 57(7), 1377-1386). Furthermore, the small peptide hormone minigastrin is known to bind with high affinity to CCKBR. Thus, previous studies have proposed the use of radiolabeled gastrin analogues for PRRT, particularly for "theranostic" (therapeutic and diagnostic) applications (Behr et al. Semin Nucl Med. 2002, 32(2), 97-109; Kolenc-Peitl et al. J Med Chem. 2011, 54(8), 2602-2609; Laverman et al. Eur J Nucl Med Mol Imaging. 2011, 38(8), 1410-1416).

[0007] WO 2015 / 067473 177 The present invention discloses a gastrin analog having the formula DOTA-(DGlu)6-Ala-Tyr-Gly-Trp-Nle-Asp-Phe-NH2 (PP-F11N) radiolabeled with Lu. 177 Lu-PP-F11N) is chemically stable (e.g., resistant to proteolysis) and exhibits high tumor uptake as well as low kidney accumulation. 177 It has been shown that Lu-PP-F11N can also accumulate in healthy tissues, including the stomach and large intestine, due to their endogenous expression of CCKBR (Sauter et al. J Nucl Med. 2019, 60(3), 393-399).

[0008] In view of the above, the object of the present invention is to provide a composition (kit-of-parts) for PRRT applications that achieves excellent uptake of radiolabeled gastrin analogues in CCKBR positive cancers or tumors, thus resulting in improved delivery and therapeutic efficacy, while preventing and / or reducing cytotoxic side effects due to accumulation in healthy tissues.

[0009] It is a further object of the present invention to provide compositions (kits of parts) that can be used in methods of treating and / or diagnosing CCKBR positive cancers or tumors.

[0010] Summary of the Invention The present invention provides compositions (kit-of-parts, combination products) that can be used in PRRT applications, particularly in methods of treating and / or diagnosing CCKBR-positive cancers or tumors, such as MTC or glioma. The inventors have found that (pre)treatment of CCKBR-expressing tumor cells with rapamycin and / or a rapalog, such as everolimus (RAD001), results in superior uptake of radiolabeled gastrin analogs in these tumor cells, both in vitro and in vivo, resulting in improved delivery and therapeutic efficacy.

[0011] Furthermore, it has been surprisingly found that excellent in vivo uptake of radiolabeled gastrin analogs occurs specifically in tumor cells but not in healthy tissues such as the gastrointestinal tract that endogenously express CCKBR, thereby preventing and / or reducing side effects that may result from specific or non-specific accumulation of radiolabeled gastrin analogs in healthy tissues.

[0012] Thus, the present invention provides (i) rapamycin and / or a rapalog; (ii) a radiolabeled gastrin analog; The present invention relates to compositions, kits of parts, and combination products comprising:

[0013] The present invention also relates to compositions, kits-of-parts, and combination products as described above for use in methods of treating and / or diagnosing CCKBR positive cancers or tumors, in particular MTC, glioma, gastroenteropancreatic neuroendocrine tumors (GEP-NETs), astrocytoma, gastric cancer, colon cancer, ovarian cancer, breast cancer, and any CCKBR positive cancers and tumors.

[0014] The present invention includes, inter alia, the following embodiments ("items"): 1. (i) rapamycin and / or a rapalog; (ii) a radiolabeled gastrin analog; A composition comprising:

[0015] 2. The composition according to item 1, wherein the rapalog is a compound selected from the group consisting of everolimus (RAD001), temsirolimus (CCI-779), ridaforolimus (AP-23573), and combinations thereof, preferably everolimus (RAD001).

[0016] 3. The radiolabeled gastrin analog has the following formula (1): YS-Axx-Bxx-Cxx-Gly-Trp-Dxx-Asp-Phe-NH2(1) [In the formula, Axx represents an amino acid selected from Glu, D-Glu, Ala, Asp, Gln, Lys, His, Arg, Ser and Asn, preferably Glu or D-Glu, Bxx represents an amino acid selected from Ala, Ser, Val, Cys, Thr and Gly, preferably Ala or Ser, Cxx represents an amino acid selected from Tyr, Ala, Phe, Trp and His, preferably Tyr, Dxx represents an amino acid equivalent to methionine, preferably an amino acid selected from norleucine, 2-amino-5-heptenoic acid, homonorleucine (homoNle), 2-amino-4-methoxybutanoic acid, telluromethionine (Te-Met), selenomethionine (Se-Met) and phenylglycine (Phg), more preferably Nle or Phg; S is a spacer comprising at least one selected from the negative charge-containing amino acids Gln and D-Gln, preferably a tetra- or pentapeptide comprising at least one amino acid selected from Glu, D-Glu, β-glutamic acid (β-Glu), Gln, D-Gln, Asp and D-Asp, more preferably a pentapeptide comprising at least one amino acid selected from Glu, D-Glu, β-Glu, Gln, D-Gln, Asp and D-Asp, Y represents a moiety that contains a radionuclide, e.g., a moiety that chelates a radionuclide or a moiety that covalently binds a radionuclide. 3. The composition according to item 1 or 2, having the following structure:

[0017] 4. The radiolabeled gastrin analog has the following formula (2): Y-DGlu-DGlu-DGlu-DGlu-DGlu-DGlu-Ala-Tyr-Gly-Trp-Dxx-Asp-Phe-NH2(2) wherein Dxx represents an amino acid equivalent to methionine, preferably selected from Nle, 2-amino-5-heptenoic acid, homoNle, 2-amino-4-methoxybutanoic acid, Te-Me, Se-Met and Phg, and Y represents a moiety that contains a radionuclide, e.g., a moiety that chelates a radionuclide, such as a radiometal, or a moiety that covalently binds a radionuclide. 4. The composition according to any one of items 1 to 3, having the following structure:

[0018] 5. The composition according to any one of items 1 to 4, wherein Dxx is Nle and / or Y is 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) or 1,4,7-triazacyclononane,1-glutaric acid-4,7-acetic acid (NODAGA), preferably NODAGA.

[0019] 6. Radioactive nuclides are 18 F, 124 I, 131 I, 86 Y, 90 Y, 177 Lu, 111 In, 188 Re, 64 Cu, 67 Cu, 149 Tb, 161 Tb, 89 Sr, 44 / 43 Sc, 47 Sc and 153 6. The composition according to any one of items 1 to 5, wherein the compound is selected from the group consisting of Sm.

[0020] 7. Radioactive nuclides are 177 Lu, 90 Y and 111 7. The composition according to any one of items 1 to 6, wherein the composition is selected from the group consisting of In.

[0021] 8. Radioactive nuclides 177 8. The composition according to any one of items 1 to 7, wherein Lu.

[0022] 9. The rapalog is everolimus (RAD001) and the radiolabeled gastrin analog is represented by the following formula (3): DOTA-DGlu-DGlu-DGlu-DGlu-DGlu-DGlu-Ala-Tyr-Gly-Trp-Nle-Asp-Phe-NH2(3) [Wherein, DOTA is 177 Chelate Lu] 9. The composition according to any one of items 1 to 8, having the following structure:

[0023] 10. The composition according to any one of items 1 to 9, wherein (i) the rapamycin and / or rapalog and (ii) the radiolabeled gastrin analog are formulated in separate dosage forms and presented together in a co-package or in separate packages, which can be administered simultaneously and / or sequentially, preferably sequentially.

[0024] 11. (i) rapamycin and / or rapalogs, (ii) a radiolabeled gastrin analog; Includes kit of parts.

[0025] 12. The kit-of-parts according to item 11, wherein (i) the rapalog and / or (ii) the radiolabeled gastrin analogue is as defined in any one of items 2 to 9.

[0026] 13. (i) rapamycin and / or rapalogs, (ii) a radiolabeled gastrin analog; Combination products.

[0027] 14. The combination product according to item 13, wherein (i) the rapalog and / or (ii) the radiolabeled gastrin analogue is as defined in any one of items 2 to 9.

[0028] 15. A composition according to any one of items 1 to 10, a kit of parts according to item 11 or 12, or a combination product according to item 13 or 14, for use in treating a CCKB receptor positive cancer or tumor, wherein (i) rapamycin and / or a rapalog is administered together with (ii) a radiolabeled gastrin analogue or (ii) before the radiolabeled gastrin analogue.

[0029] 16. A composition, kit-of-parts or combination product for use according to item 15, wherein (i) the rapamycin and / or rapalog is administered up to 2 months, preferably 1 to 14 days, before (ii) the radiolabeled gastrin analogue.

[0030] 17. A composition, kit-of-parts or combination product for use, wherein (i) rapamycin and / or a rapalog is administered once daily for 1 to 14 consecutive days, preferably 2 to 7 consecutive days, for example 5 consecutive days, before (ii) a radiolabeled gastrin analog is administered.

[0031] 18. The composition, kit of parts or combination product for use according to any one of items 15 to 17, wherein the CCKB receptor positive cancer or tumor is selected from medullary thyroid carcinoma (MTC), glioma, gastroenteropancreatic neuroendocrine tumor (GEP-NET), astrocytoma, gastric cancer, colon cancer, ovarian cancer, breast cancer, and any CCKB receptor positive tumor or cancer.

[0032] 19. A composition, kit-of-parts or combination product for use according to any one of items 15 to 18, wherein the CCKB receptor positive cancer or tumor is MTC.

[0033] 20. A composition, kit-of-parts or combination product for use according to any one of items 15 to 19, wherein (i) rapamycin and / or a rapalog or (ii) a radiolabelled gastrin analog is administered simultaneously with, before or after one or more other therapeutic or therapeutic agents, such as chemotherapeutic agents or immunomodulatory agents.

[0034] 21. A method for treating a CCKB receptor positive cancer or tumor, comprising administering to a patient in need thereof a therapeutically effective amount of (i) rapamycin and / or a rapalog and (ii) a radiolabeled gastrin analogue, comprising the composition according to any one of items 1 to 10, the kit of parts according to item 11 or 12, or the combination product according to item 13 or 14. [Brief description of the drawings]

[0035] [Figure 1] FIG. 1A: Identification of kinase inhibitors that enhance cellular uptake of 177Lu-PP-F11N. (1A) Experimental design: Kinase inhibitor-treated and control untreated A431 / CCKBR cells were subjected to analysis of cellular uptake of 177Lu-PP-F11N as described in the Materials and Methods section below. (1B,C) Volcano plots represent the change in cellular uptake of 177Lu-PP-F11N and proliferation, respectively, shown as log2 [ratio:treated / control]. Dots marked BML-257, SC-514 and rapamycin represent kinase inhibitors that significantly increased the uptake of 177Lu-PP-F11N (P<0.05). [Diagram 2] Figure 2 shows that inhibition of mTORC1 activity enhances internalization of 177Lu-PP-F11N. (2A) Internalization and cell binding activity were measured for control, 100 nM RAD001 and 10 mM RAD001 as shown. Measured 1 hour after incubation with 177Lu-PP-F11N in metformin-treated A431 / CCKBR cells, non-transfected A431 (negative control) and AR42J cells for 20 hours, all experiments were assayed in triplicate, each bar represents the mean ± SD, **p<0.01, **p<0.001, (2B) Mean protein concentration ± SD of lysates used in C, mean protein concentration in control cells was set to 1, (2C) Western blot analysis of whole cell lysates isolated from control cells and 20 hour RAD001- or metformin-treated A431 / CCKBR cells using phosphorylation site-specific S6 antibody, blots were stripped and reprobed with GAPDH antibody for loading control. [Diagram 3]Figure 3 shows that increased CCKBR expression by RAD001 is required to enhance internalization of 177Lu-PP-F11N. WB analysis with anti-CCKBR antibody of glycosylated and deglycosylated (PNGase F treated) lysates from A431 / CCKBR cells after 20 hours of RAD001 treatment (3A) or 48 hours of transfection with luciferase siRNA (control) and CCKBR siRNA (3B) as shown. Blots were stripped and reprobed with GAPDH antibody for loading control, bottom; quantification of CCKBR signal intensity in deglycosylated lysates normalized to GAPDH. CCKBR / GAPDH ratio from untreated or control transfected cells was set to 1. [Figure 4] RAD001 increases CCKBR-dependent 177Lu-PP-F11N tumor uptake in vivo (5 days of treatment). (4A) A431 / CCKBR cells were implanted in immunodeficient (nude) mice and 5 days after implantation, RAD001, metformin and control (PBS) animal groups were dosed daily as indicated by triangles and biodistribution studies were performed the following day. Each bar: biodistribution of 177Lu-PP-F11N analyzed 4 hours after tail vein administration is shown as % of total injected radioactivity per gram of tissue (% injected radioactivity). Bottom row: corresponds to biodistribution after co-injection of blocking peptide. (4B,C) Tumor volumes and mouse weights before (day 1) and after (day 5) treatment in all groups are shown. Each bar represents the mean ± SD. [Diagram 5] Figure 5: RAD001 increases CCKBR-dependent tumor uptake of 177Lu-PP-F11N in vivo (5-day treatment). (5A) SPECT / CT images 2 hours after 5-day 177Lu-PP-F11N injection in metformin-, RAD001- and control (PBS)-treated mice, bottom panel: corresponding radioactive tumor area. (5B) Mean and maximum radioactive concentration of 177Lu-PP-F11N ± SD in radioactive areas of 4 tumors per group. *P<0.05, **P<0.01, ***P<0.001. [Figure 6]Figure 1 shows that RAD001 increases CCKBR-dependent tumor uptake of 177Lu-PP-F11N in vivo (3 days of treatment), (6) A431 / CCKBR cells were implanted into immunodeficient nude mice, RAD001, metformin and control (PBS) animal groups were administered 3 day doses and biodistribution studies were performed the following day, each bar: biodistribution of 177Lu-PP-F11N analyzed 4 hours after tail vein administration shown as % of total injected radioactivity per gram of tissue (% injected radioactivity). [Figure 7] Figure 7 shows that RAD001 increases CCKBR-dependent tumor uptake of 177Lu-PP-F11N in vivo (3-day treatment). (7A) A431 / CCKBR cells were implanted into immunodeficient nude mice, and RAD001, metformin and control (PBS) animal groups were administered 3-day doses and biodistribution studies were performed the following day. Bars: biodistribution of 177Lu-PP-F11N analyzed 4 hours after tail vein administration, shown as % of total injected radioactivity per gram of tissue (% injected radioactivity). (7B) Tumor volume and mouse weight before and after 3-day treatment. Data represent mean ± SD, *P<0.05. [Figure 8] 8A-8B show increased necrosis and decreased numbers of mitotic figures and Ki67 positive cells in tumors treated with RAD001. Paraffin sections prepared from A431 / CCKBR-tumors treated with RAD001, metformin (Met) and PBS (control) were subjected to HE and Ki67 staining as described in Materials and Methods, bars show percentage of necrotic area (8A), number of blood vessels per field (8B) as mean ± SD of tumor group analyzed, right panel; representative HE staining images, arrows in 8B indicate blood vessels, scale bars in 8A are 0.5 mm and in 8B are 20 μm, *P<0.05, **P<0.01, ***P<0.001. [Figure 9]9A-9B show increased necrosis and decreased numbers of mitotic figures and Ki67 positive cells in tumors treated with RAD001. Paraffin sections prepared from A431 / CCKBR-tumors treated with RAD001, metformin (Met) and PBS (control) were subjected to HE and Ki67 staining as described in Materials and Methods, each bar shows the mitotic index (9A) and the percentage of Ki67 positive cells (9B) as the mean ± SD of the tumor group analyzed, right panel; representative HE and Ki67 staining images, arrows in 9A indicate mitotic figures, scale bar in 9AB is 20 μm, *P<0.05, **P<0.01, ***P<0.001. [Figure 10] Figure 10 shows tumor growth inhibition and life span extension in mice treated with RAD001 and 177Lu-PP-F11N, as shown: (10A) Experimental design: Nude mice were implanted with A431 / CCKBR cells, followed by administration of 5 or 10 doses of RAD001 alone or in combination with 60 kBq of 177Lu-PP-F11N, (10B) Tumor growth curves of control and treatment groups. [Figure 11] FIG. 11 shows tumor growth inhibition and life span extension in mice treated with RAD001 and 177Lu-PP-F11N. (FIG. 11) Tumor volumes on days 13, 22 and 25 after the start of treatment, data represent mean ± SD. [Figure 12] FIG. 12 shows tumor growth inhibition and life span extension in mice treated with RAD001 and 177Lu-PP-F11N. Survival rates shown as Kaplan-Meier curves for control and treatment groups.

[0036] Detailed Description of the Invention 1.Definition The term "composition" as used herein shall be understood to refer to a combination of the individual components (i.e., a combination product), i.e., (i) a rapalog and (ii) a radiolabeled gastrin analog, which are kept physically separate from one another but adjacent to one another. Such compositions may be referred to as a "kit of parts." In some embodiments, the term "composition" as used herein encompasses a physical mixture of the individual components, e.g., when (i) a rapamycin and / or a rapalog or (ii) a radiolabeled gastrin analog is used contemporaneously with one or more other therapeutic or therapeutic agents, provided that (i) the rapamycin and / or the rapalog and (ii) the radiolabeled gastrin analog are kept physically separate from one another, e.g., formulated in separate dosage forms.

[0037] The term "rapamycin" (sirolimus, Rapamune®) refers to a macrolide compound, which is known in the art to exhibit immunosuppressive properties by inhibiting the mammalian target of rapamycin (mTOR). Rapamycin has the following chemical structure: [ka] has.

[0038] As used herein, "rapalogs" (for "rapamycin analogs") refer to a class of compounds structurally related to rapamycin, which are known to inhibit the mammalian target of rapamycin complex 1 (mTORC1) by binding to FK binding protein 12 (MacKeigan et al. Neuro-Onc. 2015, 17(12), 1550-1559). Examples of rapalogs include everolimus (RAD001, Afinitor®, Certican®), temsirolimus (CCI-779, Torisel®) and ridaforolimus (AP-23573, MK-8669).

[0039] The inhibitory activity of rapamycin and / or rapalogs against mTORC1 can be determined by measuring the phosphorylation levels of ribosomal protein S6 by Western blot analysis, as further described below.

[0040] 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. The term "gastrin analog" as used herein is meant to encompass all compounds that contain the C-terminal amino acid sequence Gly-Trp-Dxx-Asp-Phe-NH2 (wherein Dxx is Met or an amino acid equivalent to Met), as found in CCKBR-binding endogenous peptide hormones, including gastrin and cholecystokinin (CCK). Gastrin is a linear peptide hormone produced in G cells of the duodenum and the pylorus of the stomach. It is secreted into the bloodstream. The encoded polypeptide is preprogastrin, which is enzymatically cleaved in a post-translational modification to produce progastrin and then various forms of gastrin, including mainly big gastrin (G-34), little gastrin (G-17), and minigastrin (Leu-Glu-Glu-Glu-Glu-Glu-Glu-Ala-Tyr-Gly-Trp-Met-Asp-Phe-NH2), all of which represent "gastrin analogs" within the meaning of the present invention. CCK is a peptide hormone structurally related to gastrin in that both compounds share five C-terminal amino acids, namely Gly-Trp-Met-Asp-Phe-NH2 (wherein Met can be replaced by an amino acid equivalent to Met, such as norleucine). CCK naturally exists in several forms, including, for example, CCK8 (Asp-Tyr-Met-Gly-Trp-Met-Asp-Phe-NH2). A gastrin analogue may, for example, be provided at its N-terminus with a spacer or a moiety capable of chelating a radiometal, such as 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), or a moiety to (covalently) bind a radionuclide, such as 18It can be chemically modified to be covalently attached to F or iodine isotopes. In some embodiments, gastrin analogs can be modified to be covalently attached to imaging moieties for medical use, such as Alexa Fluor® 647, IRDye 680RD or DY-700, or photosensitizers, such as Photofrin, Forscam or Photochlor.

[0041] The expression "gastrin analog" also refers to gastrin, big gastrin, little gastrin, CCK, CCK8 or minigastrin, in particular minigastrin, in which one, two or more amino acid residues are replaced by another natural or non-natural amino acid residue, provided that the resulting "gastrin analog" maintains its pharmacological activity against CCKBR. In some embodiments of the present invention, the aforementioned modifications may, for example, make it possible to achieve improved binding affinity and / or pharmacological activity against CCKBR, improved plasma stability, improved biodistribution, etc. Pharmacological activity in this context means that the gastrin analog retains at least 20%, preferably at least 50%, more preferably at least 80% of the pharmacological (agonist) activity of minigastrin.

[0042] The pharmacological activity of gastrin analogues on CCKBR can be determined by measuring the intracellular increase in calcitonin levels in gastrin analogue-stimulated cells as described by Blaeker et al. (Regulatory Peptides 2004, 118, 111-117).

[0043] In some embodiments, the term "gastrin analog" refers to a compound (peptide) structurally related to the compound having the formula (DGlu)6-Ala-Tyr-Gly-Trp-X-Asp-Phe-NH2, where X represents the amino acid equivalent to methionine. This compound may be modified by adding a spacer or a moiety capable of chelating a radionuclide (e.g., a radioactive metal), such as DOTA, or a moiety that (covalently) binds a radionuclide, such as 18It may be modified to covalently link F or iodine isotopes. When X represents norleucine and the aforementioned compound is modified at its N-terminus by the covalent attachment of DOTA, the compound corresponds to "PP-F11N."

[0044] 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, but α-amino acids, especially α-amino carboxylic acids, are particularly preferred. The term encompasses naturally occurring amino acids as well as synthetic amino acids that are not found in nature.

[0045] As used herein, the term "(D)-amino acid" refers to the (D)-isomer of any naturally occurring or synthetic amino acid. For example, the term "D-Glu" refers to the (D)-isomer of glutamic acid. As used herein, the term "(D)-amino acid" is not meant to encompass non-chiral amino acids such as glycine or other non-chiral amino acids.

[0046] As used herein, the phrase "equivalent amino acid to methionine" refers to a natural or unnatural amino acid that has similar shape and / or electronic properties to methionine. As used herein, the term "equivalent" is meant to encompass amino acids that are essentially equivalent to methionine, such as norleucine (Nle). Examples of amino acids equivalent to methionine include Nle, 2-amino-5-heptenoic acid, homonorleucine (homoNle), 2-amino-4-methoxybutanoic acid, telluromethionine (Te-Met), selenomethionine (Se-Met), and phenylglycine (Phg).

[0047] Unless otherwise specified or dictated otherwise by context, all connections between adjacent amino acid groups are formed by peptide (amide) bonds.

[0048] As used herein, the expression "moiety that chelates or (covalently) binds a radionuclide" refers to a moiety that is (i) capable of donating electrons to a radionuclide, in particular a radiometal, to form a coordination complex therewith, i.e., by forming at least one coordinate covalent bond (dipolar bond) therewith, or (ii) 18 It refers to a moiety (chelator or ligand) that can covalently bind a radionuclide, such as F or iodine isotopes. The chelation mechanism depends on the chelator and / or the radionuclide. For example, DOTA is thought to coordinate radionuclides via a carboxylate group and an amino group (donor group), thereby forming a complex with high stability (Dai et al. Nature Com. 2018, 9, 857).

[0049] The expression "amino acid containing a negative charge" (or "negatively charged amino acid") is used herein to characterize natural or unnatural amino acids whose side chains contain a negatively charged ionizable group (in solution), in particular a carboxylic acid group. Examples of natural or unnatural amino acids containing a negative charge include Glu, D-Glu, β-glutamic acid (β-Glu), Asp, D-Asp, 2-aminoadipic acid, 2-aminopimelic acid, and 2-aminosuberic acid.

[0050] The term "cancer" as used herein refers to a pathological condition in mammalian tissue characterized by abnormal cell growth to form a malignant tumor, which 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. The phrase "CCKBR positive cancer or tumor" as used herein refers to a cancer or tumor characterized by overexpression of CCKBR on the cell surface (Reubi et al. Cancer Res. 1997, 57(7), 1377-1386). Examples of CCKBR positive cancers or tumors include MTC, glioma, gastroenteropancreatic neuroendocrine tumor (GEP-NET), astrocytoma, gastric cancer, colon cancer, ovarian cancer and breast cancer.

[0051] As used herein, the term "internalization" refers to the biological process by which a molecule (e.g., a radiolabeled gastrin analog) is engulfed in a cell membrane and drawn into the cell, such that the molecule (e.g., a radiolabeled gastrin analog) is present within the cell.

[0052] The term "cellular uptake" (of a radiopharmaceutical) refers to the biological process by which a molecule (e.g., a radiolabeled gastrin analog) is internalized and / or bound to a cell membrane, such that the molecule (e.g., a radiolabeled gastrin analog) is able to reside not only within the cell but also at the cell membrane.

[0053] The term "tumor uptake" (of a radiopharmaceutical) refers to the biological process by which a molecule (e.g., a radiolabeled gastrin analog) is taken up by a tumor (cancer) cell. Tumor uptake includes the uptake of a molecule (e.g., a radiolabeled gastrin analog) into tumor cells and / or its retention in the tumor microenvironment. As a result, the molecule (e.g., a radiolabeled gastrin analog) can be present within the tumor (cancer) cell, cell membrane and / or tumor microenvironment.

[0054] The term "co-administration" as used herein refers to the concurrent (simultaneous) administration of both (i) rapamycin and / or rapalog and (ii) radiolabeled gastrin analog, as well as the sequential administration of (i) rapamycin and / or rapalog followed by (ii) radiolabeled gastrin analog.Furthermore, the term "co-administration" is meant to encompass the dosing schedule in which the respective dosages of i) rapamycin and / or rapalog and / or (ii) radiolabeled gastrin analog are changed (increased or decreased) during the course of administration.Preferably, (i) rapamycin and / or rapalog and (ii) radiolabeled gastrin analog are administered according to a sequential pattern in which (i) rapamycin and / or rapalog is administered first for a certain period of time, followed by (ii) radiolabeled gastrin analog.

[0055] 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.

[0056] Hereinafter, in the description of the present invention and in the claims, the use of the terms "containing", "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.

[0057] 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.

[0058] 2. Overview The present invention is based on the discovery that (pre)treatment of CCKBR-expressing tumor cells with rapamycin and / or a rapalog, such as everolimus (RAD001), results in a significant increase in CCKBR protein levels and excellent uptake of radiolabeled gastrin analogues, thereby improving delivery and therapeutic efficacy while preventing and / or reducing cytotoxic side effects due to accumulation in healthy tissue.

[0059] One of the most important goals of efficient PRRT is high tumor uptake of radioactive compounds, which is highly dependent on the expression level and internalization-related activity of the target receptor. The present inventors have surprisingly identified a group of kinase inhibitors that can enhance the uptake of radiolabeled gastrin analogs in CCKBR-positive tumor cells (e.g., A431 / CCKBR cells). In particular, it has been found that (pre)treatment of CCKBR-positive tumor cells with rapalogs (e.g., RAD001) significantly increases CCKBR levels, followed by increased CCKBR-specific internalization of radiolabeled gastrin analogs, resulting in improved delivery and therapeutic efficacy.

[0060] Furthermore, enhanced CCKBR-specific uptake of radiolabeled gastrin analogs was observed in tumor cells but not in healthy organs such as the gastrointestinal tract that endogenously express CCKBR, suggesting that rapalog treatment provides superior tumor cell targeting, thereby preventing and / or reducing potential cytotoxic side effects due to accumulation of radiolabeled gastrin analogs in healthy tissues.

[0061] Without being bound by any theory, it is believed that tumor cells exhibit higher mTORC1 activity compared to healthy tissues, thereby resulting in superior efficacy in tumor cells while preventing and / or reducing its accumulation in healthy tissues.

[0062] 3. Composition The compositions (kit-of-parts, combination products) of the invention are provided in the form of pharmaceutical compositions (formulations) for human or animal use in human and veterinary medicine. Typically, (i) rapamycin and / or rapalogs and (ii) radiolabeled gastrin analogs are presented as separate pharmaceutical compositions for co-administration (kit-of-parts), each respective composition comprising a therapeutically effective amount of (i) rapamycin and / or rapalogs and (ii) radiolabeled gastrin analogs.

[0063] In some embodiments of the invention, the individual components (i) and (ii) are formulated into separate dosage forms, which may be co-packaged or co-presented in separate packages. In some instances, the packaged component (ii) may comprise an unlabeled gastrin analog, which may be labeled with a radionuclide immediately prior to administration, for example, by chelating the radionuclide as further described below.

[0064] The composition (or each part of the kit) can further comprise one or more components selected from carriers, diluents and other excipients. Suitable carriers, diluents and other excipients for use in pharmaceutical compositions are well known in the art and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (Gennaro AR, 1985). Carriers, diluents and / or other excipients can be selected with respect to the intended route of administration and pharmaceutical practice. The composition can comprise any suitable binder, lubricant, suspending agent, coating agent, solubilizer as, or in addition to, the carrier, diluent and / or other excipient.

[0065] The therapeutically effective amount of (i) rapamycin and / or rapalogs and / or (ii) radiolabeled gastrin analogs can be routinely determined by a physician. The specific dose level and dosing frequency for any particular subject / patient may vary and depend on a variety of factors, including the activity of the particular drug compound used, the metabolic stability and duration of action of the compound, the patient's age, weight, general health, sex, diet, mode and time of administration, excretion rate, drug combination, the severity of the particular condition, and the individual being treated. These factors are taken into account by a physician when determining the therapeutically effective dose.

[0066] The rapalog used herein is not particularly limited, as long as it shows mTORC1 inhibitory activity and leads to the enhancement of CCKBR level on cell surface.The level of CCKBR on cell surface can be determined by methods known in the art, for example, by Western blot analysis, as further described below.The mTORC1 inhibitory activity of rapamycin and / or rapalog can be determined by measuring the phosphorylation level of ribosomal protein S6 by Western blot analysis, as further described below.

[0067] According to one embodiment, the rapalog is selected from the group consisting of everolimus (RAD001), temsirolimus (CCI-779), ridaforolimus (AP-23573), and combinations thereof. According to a preferred embodiment, the rapalog is everolimus (RAD001). Some of these compounds are commercially available under the trade names Rapamune® (sirolimus), Afinitor® or Certican® (everolimus), Torisel® (temsirolimus).

[0068] For example, a rapalog such as everolimus (RAD001) can be administered, e.g. orally, in the form of a (dispersible) tablet comprising the rapalog such as everolimus, e.g. as a solid dispersion, at a daily dosage in the range of 0.1 mg to 20 mg, e.g. 0.1 mg to 15 mg, preferably 0.5 mg to 10 mg, e.g. 5 mg.

[0069] The radiolabeled gastrin analog used herein is not particularly limited as long as it binds to CCKBR and exhibits agonist activity thereto. In one embodiment, the gastrin analog is represented by the following formula (1): YS-Axx-Bxx-Cxx-Gly-Trp-Dxx-Asp-Phe-NH2(1) [In the formula, Axx represents an amino acid selected from Glu, D-Glu, Ala, Asp, Gln, Lys, His, Arg, Ser and Asn, preferably Glu or D-Glu, Bxx represents an amino acid selected from Ala, Ser, Val, Cys, Thr and Gly, preferably Ala or Ser, Cxx represents an amino acid selected from Tyr, Ala, Phe, Trp and His, preferably Tyr, Dxx represents an amino acid equivalent to methionine, preferably an amino acid selected from norleucine, 2-amino-5-heptenoic acid, homonorleucine (homoNle), 2-amino-4-methoxybutanoic acid, telluromethionine (Te-Met) and selenomethionine (Se-Met), more preferably Nle; S is negatively charged amino acids, Gln and D-Gln wherein S is a tetra- or pentapeptide comprising at least one amino acid selected from Glu, D-Glu, β-glutamic acid (β-Glu), Gln, D-Gln, Asp and D-Asp, more preferably a pentapeptide comprising at least one amino acid selected from Glu, D-Glu, β-Glu, Gln, D-Gln, Asp and D-Asp; Y represents a moiety that contains a radionuclide, for example a moiety that chelates a radionuclide (eg a radiometal) or a moiety that (covalently) binds a radionuclide.

[0070] In a preferred embodiment, the gastrin analog has the following formula (2): Y-DGlu-DGlu-DGlu-DGlu-DGlu-DGlu-Ala-Tyr-Gly-Trp-X-Asp-Phe-NH2(2) wherein X represents an amino acid equivalent to methionine, such as norleucine (Nle), 2-amino-5-heptenoic acid, homonorleucine (HomoNle), 2-amino-4-methoxybutanoic acid, telluromethionine (Te-Met), selenomethionine (Se-Met) or Phg, and Y represents a moiety that contains a radionuclide, e.g. a moiety that chelates a radionuclide (e.g. a radiometal), e.g. DOTA, NOTA or NODAGA, preferably NODAGA, or a moiety that (covalently) binds a radionuclide.

[0071] In some embodiments, Y (defined in formulas (1) and (2)) comprises a functional group such as a carboxylic acid for covalent attachment to a peptide, i.e., to the N-terminus of a peptide chain or to the side chain of the N-terminal amino acid.

[0072] Examples of moieties (chelators) capable of chelating radionuclides include diethylenetriaminepentaacetic acid (DTPA), deferoxamine (DFO), 1,4,7-triazacyclononane,1-glutaric acid-4,7-acetic acid (NODAGA), 1,4,7,10-tetraazacyclododecane-1-glutaric acid-4,7,10-triacetic acid (DOTAGA), 2,2'-(1,4,7-triazacyclononane-1,4-diyl)diacetic acid (NO2A), 1,4 , 7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), ethylenediaminetetraacetic acid (EDTA), ethylenediaminediacetic acid, triethylenetetraminehexaacetic acid (TTHA), 1,4,8,11-tetraazacyclotetradecane (CYCLAM), 1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid (TETA), 1,4,8 ,11-Tetraazabicyclo[6.6.2]hexadecane-4,11-diacetic acid (CB-TE2A), 2,2',2''-(1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetamide (DO3AM), 1,4,7,10-tetraazacyclododecane-1,7-diacetic acid (DO2A), 1,5,9-triazacyclododecane (TACD), (3a1s,5a1s)-dodecahydro-3a,5a,8a,10a-tetraaza Pyrene (cis-glyoxal-cyclam), 1,4,7-triazacyclononane (TACN), 1,4,7,10-tetraazacyclododecane (cyclen), tri(hydroxypyridinone) (THP), 3-(((4,7-bis((hydroxy(hydroxymethyl)phosphoryl)methyl)-1,4,7-triazonan-1-yl)methyl)(hydroxy)phosphoryl)propanoic acid (NOPO), 3,6,9,15-tetraazabicyclo[9.3.1]Pentadeca-1(15),11,13-triene-3,6,9-triacetic acid (PCTA), 2,2',2'',2''''-(1,4,7,10-tetraazacyclotridecane-1,4,7,10-tetrayl)tetraacetic acid (TRITA), 2,2',2'',2'''-(1,4,7,10-tetraazacyclotridecane-1,4,7,10-tetrayl)tetraacetamide (TRITAM), 2,2',2''-(1,4,7,10-tetraazacyclotridecane-1,4,7-triyl)triacetamide (TRITRAM), trans-N-dimethylcyclam, 2,2',2''-(1,4,7-triazacyl) Chrononane-1,4,7-triyl)triacetamide (NOTAM), oxocyclam, dioxocyclam, 1,7-dioxa-4,10-diazacyclododecane, cross-bridged cyclam (CB-cyclam), triazacyclononanephosphinic acid (TRAP), dipyridoxyl diphosphate (DPDP), meso-tetra-(4-sulfonatophenyl)porphine (TPPS4), ethylene bishydroxyphenylglycine (EHPG), hexamethylenediaminetetraacetic acid, dimethylphosphinomethane (DMPE), methylenediphosphoric acid, dimercaptosuccinic acid (DMPA), and their derivatives.

[0073] In one embodiment, X represents an amino acid equivalent to methionine selected from Nle, 2-amino-4-methoxybutanoic acid, Te-Met, Se-Met, 2-amino-5-heptenoic acid, homoNle and Phg, preferably Nle, and / or Y represents a moiety selected from DOTA, DTPA, NOTA, NODAGA and TETA, preferably DOTA, NOTA or NODAGA, more preferably NODAGA. According to one embodiment, X represents Nle and Y represents DOTA.

[0074] The gastrin analog of formula (1) or (2) shows excellent resistance to degradation by proteases and is stable in systemic circulation.In addition, the gastrin analog of formula (1) or (2) has very low uptake and accumulation in the kidney, and therefore can further reduce side effects caused by non-specific accumulation in healthy tissues (e.g., kidney).

[0075] In a preferred embodiment, the radiolabeled gastrin analog has the following formula (3): DOTA-DGlu-DGlu-DGlu-DGlu-DGlu-DGlu-Ala-Tyr-Gly-Trp-Nle-Asp-Phe-NH2(3)

[0023] In the formula, DOTA is preferably 177 Lu].

[0076] As used herein, a radionuclide may be selected from any naturally occurring or artificially produced atom with excess nuclear energy that emits beta and / or gamma radiation. In one embodiment, a radionuclide is 18 F, 124 I, 131 I, 86 Y, 90 Y, 177 Lu, 111 In, 188 Re, 64 Cu, 67 Cu, 149 Tb, 161 Tb, 89 Sr, 44 / 43 Sc, 47 Sc and 153 Sm.

[0077] According to one preferred embodiment, the radionuclide is 177 Lu, 90 Y and 111 In, more preferably, 177 This is Lu.

[0078] 4. Use of the composition in methods of treating and / or diagnosing disease The compositions (kits of parts) of the invention comprising i) rapamycin and / or a rapalog and (ii) a radiolabeled gastrin analog can be used to treat and / or diagnose disease, in particular to treat and / or diagnose CCKBR positive cancers or tumors. In some embodiments, the treatment can prolong the survival of the subject as compared to the expected survival of the subject in the absence of the treatment.

[0079] The disease treated by the composition (kit-of-parts) can be any neoplastic disease, such as cancer, characterized by expression of CCKBR. Non-limiting examples of CCKBR-positive cancers or tumors include medullary thyroid carcinoma (MTC), glioma, gastroenteropancreatic neuroendocrine tumor (GEP-NET), astrocytoma, gastric cancer, colon cancer, ovarian cancer and breast cancer. An exemplary disease is MTC.

[0080] According to one embodiment, the composition of the present invention is used in a method for treating or diagnosing a CCKBR positive cancer or tumor by co-administering (i) rapamycin and / or rapalog and (ii) radiolabeled gastrin analog to a patient in need of (i) rapamycin and / or rapalog and (ii) radiolabeled gastrin analog. In all aspects of the present invention, one or more rapalogs and one or more radiolabeled gastrin analogs can be co-administered. Co-administration of a rapalog and a radiolabeled gastrin analog includes the concurrent (simultaneous) administration of both (i) rapamycin and / or rapalog and (ii) radiolabeled gastrin analog, as well as the sequential administration of (i) rapamycin and / or rapalog followed by (ii) radiolabeled gastrin analog. When two or more rapalogs are present, they can be administered individually, each by itself, and / or together as a rapamycin / rapalog cocktail. Similarly, when two or more radiolabeled gastrin analogs are present, they can also be administered individually, each by itself, and / or together as a cocktail.

[0081] Without being bound by any theory, it is believed that co-administration allows for sufficient exposure of CCKBR positive tumor cells to the rapalog to inhibit the mTOR pathway and achieve increased CCKBR density on the cell surface prior to exposing the same tumor cells to a radiolabeled gastrin analog, thereby achieving superior targeting of the radiolabeled gastrin analog to the tumor cells. Thus, co-administration includes any method of administering a rapalog in combination with a radiolabeled gastrin analog that achieves this result.

[0082] The rapamycin and / or rapalog and the radiolabeled gastrin analog can be administered in parallel (simultaneously) or independently of one another, for example according to a sequential administration pattern in which (i) the mTOR inhibitor, particularly the rapamycin and / or rapalog, is administered for a predetermined period of time before (ii) the radiolabeled gastrin analog.

[0083] According to one embodiment, (i) rapamycin and / or rapalog is administered simultaneously with (ii) radiolabeled gastrin analog or prior to (ii) radiolabeled gastrin analog. Thus, (i) rapamycin and / or rapalog can be administered on the same day as (ii) radiolabeled gastrin analog, together or within a few hours of each other, but can also be administered up to about two months prior.

[0084] According to a preferred embodiment, (i) rapamycin and / or rapalog is administered first before (ii) radiolabeled gastrin analog.The administration schedule, as well as rapalog and radiolabeled gastrin analog, can be adjusted to patient needs and disease state (progression).The objective pursued by the administration schedule is to administer component (i) to increase CCKBR expression on the surface of CCKBR-positive tumor cells, thereby improving the targeting and uptake of radiolabeled gastrin analog.

[0085] Preferably, the rapamycin and / or rapalog is administered in advance for a period of up to two months, preferably 1 to 14 days, e.g., 2 to 5 consecutive days, such as 3 days, prior to administration of (ii) the radiolabeled gastrin analog. In particular, component (i) can be administered once or several times a day, preferably once a day, for 1 to 14 consecutive days, preferably 2 to 7 consecutive days, e.g., 3 or 5 consecutive days, prior to administration of (ii) the radiolabeled gastrin analog.

[0086] Additionally, co-administration includes administration of one or more doses of the rapalog followed within a few weeks by (ii) two or more doses of the radiolabeled gastrin analog, and thus the rapamycin and / or the rapalog do not need to be re-administered with each subsequent dose of the radiolabeled gastrin analog, but may be administered only once or intermittently during the course of radiolabeled gastrin analog treatment.

[0087] The rapalog may be administered, for example orally, in the form of a (dispersible) tablet comprising the rapalog, for example as a solid dispersion, at a daily dosage in the range of 0.1 mg to 20 mg, for example 0.1 mg to 15 mg, preferably 0.5 mg to 10 mg, for example 5 mg.

[0088] The radiolabeled gastrin analog can be administered once or several times daily.

[0089] In one embodiment, the compositions of the invention can be administered simultaneously with, prior to or subsequent to one or more other therapeutic or therapeutic agents, such as chemotherapeutic agents and / or immunomodulatory agents. Examples of therapeutic or therapeutic agents 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.

[0090] 5. Preparation of Radiolabeled Gastrin Analogues 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.

[0091] 6. Working Example 6.1 List of abbreviations DIEA: Diisopropylethylamine DMEM: Dulbecco's modified Eagle's medium DMF: Dimethylformamide DMSO: Dimethyl sulfoxide DTT: Dithiothreitol ESI: Electrospray ionization FCS: Fetal calf 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 PBS: Phosphate-buffered saline RPMI: Roswell Park Memorial Institute 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 UPLC: Ultra-high performance liquid chromatography

[0092] 6.2 Materials and Methods The following materials and methods were used to evaluate the compositions of the present invention.

[0093] 6.2.1 Cell culture, transfection and treatment The human epidermoid carcinoma A431 stable cell line overexpressing CCKBR, generated as previously described (Aloj et al., J Nucl Med 2004, 45(3), 485-94), was cultured in DMEM, whereas the rat pancreatic acinar cell line AR42J was cultured in RPMI medium supplemented with 10% FCS, 2 mM glutamine and antibiotics (0.1 mg / mL streptomycin, 100 IU penicillin) at 37°C and 5% CO2.

[0094] For CCKBR-specific knockdown, siRNA duplexes against CCKBR or control duplexes against luciferase (Microsynth) were used at a final concentration of 100 nM in Optimem (Gibco): CCKBRseq1 sense RNA 5'-UAUACGAGUAGUAGCACCAdTdT-3', CCKBRseq2 sense RNA 5'-CCGCCAAAGGAUGGAGUACdTdT-3' and control sense RNA 5'-CGUACGCGGAAUACUUCGAdTdT-3'. Cells at 60-80% confluence were transfected with siRNAs using Lipofectamin 3000 according to the user manual. Total protein lysates were subjected to WB analysis.

[0095] Treatment with RAD001 or metformin (both from Selleckchem) diluted in DMSO or water was performed at the indicated time points, and the cells were washed with PBS and used for the analysis described below.

[0096] 6.2.2 Preparation of Gastrin Analogues The gastrin analogs described herein were prepared by standard Fmoc-based SPPS involving 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).

[0097] Coupling reactions for amide bond formation were 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 was carried out with a 20% piperidine solution in DMF. Coupling of the N-terminal labeling moiety was 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).

[0098] The peptide was 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 was precipitated with cold diethyl ether and centrifuged.

[0099] Peptides were 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 × 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 were pooled, concentrated, and lyophilized. Purity was 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 × 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.

[0100] MS analysis was performed in positive and negative modes using an electrospray ionization (ESI) interface.

[0101] 6.2.3 Radiolabeling of Gastrin Analogues The N-terminal DOTA-conjugated gastrin analog PP-F11N (DOTA-(DGlu)6-Ala-Tyr-Gly-Trp-Nle-Asp-Phe) prepared as above and 177 A solution with Lu (available from ITG GmbH) at a nuclide / peptide ratio of 1:30 was prepared in 0.4 M ammonium acetate buffer (pH 5.5), and labeling was carried out at 90° C. for 15 minutes.

[0102] Lutetium incorporation was analyzed by standard HPLC using a C18 column and reached an efficiency of over 95%. Immediately after labeling, appropriate dilutions of radiolabeled gastrin analogs were prepared for targeted radioactivity experiments using a gamma counter.

[0103] 6.2.4 Kinase Inhibitor Library Screen 25,000 cells were seeded per well on an Isoplate 96 TC (PerkinElmer) and treated with 10 μM kinase inhibitors (KIs) for 18 hours (Screen-well® Kinase Inhibitor Library, Enzo Life Sciences AG). The next day, 10,000 cpm of 177 Lu-PP-F11N (prepared as above) was added to each well and the plates were incubated for 2 hours under standard tissue culture conditions. Blocking controls were performed in the presence of 4 μM mini-gastrin LEEEEEAYGWMDF (PSL GmbH).

[0104] After removing the radioactive supernatant, the cells were resuspended in 50 pi of ULTIMA GOLD high flash point LSC cocktail (Sigma) and incubated for 2 hours at room temperature on a horizontal shaker. Radioactivity was measured within 30 seconds per well using a MicroBeta 2450 microplate counter (PerkinElmer). Screens were performed in triplicate.

[0105] 6.2.5 Proliferation assays Pre-treated cells were seeded in 96-well plates as described above, and cell proliferation was analyzed by the CellTiter 96 AQueous non-radioactive cell proliferation assay (Promega) according to the manufacturer's instructions. The absorbance of MTS bioreduced to formazan was measured at 570 nm with a reference of 650 nm using a microplate reader (PerkinElmer). Assays were performed in triplicate.

[0106] 6.2.6 In Vitro Radiolabeled Peptide Internalization Assay 2.5×10 5 Cells were seeded in 6-well plates. After 8 hours, cells were treated with the appropriate inhibitors (as indicated) for 20 hours. The next day, cells were washed with PBS and incubated with 100,000 cpm of 177 Lu-PP-F11N (in DMEM with 0.1% BSA) was incubated in standard TC conditions. For blocking experiments, mini-gastrin (PSL GmbH) was used at a concentration of 4 μM. After 1 or 2 h of incubation, the radioactive medium (together with PBS washes) was collected and the cells were subjected to two washes with glycine buffer (pH=2) for 5 min at room temperature, followed by a lysis step with 1 M NaOH for 15 min at 37°C. All three collected fractions (medium / PBS; glycine washes; lysed cells) were measured in a Cobra II auto gamma counter (Packard); 177 The internalized and membrane-bound fractions of Lu-PP-F11N were expressed as % of total radioactivity. Experiments using blocking peptides with less than 0.1% of total radioactivity (controls for non-specific binding or internalization of glycine-washed or NaOH-lysed cells, respectively) (data not shown) were subtracted from the results obtained. Protein concentrations of NaOH-lysed cell lysates were measured spectrophotometrically using a NanoPhotometer® UV-Vis P-Class (Implen).

[0107] 6.2.7 WB analysis Antibodies against phospho-S6 (D57.2.2F) and GAPDH (14C10) at S235 / S236 were obtained from Cell Signaling Technology, while anti-CCKBR (ab77077) was obtained from Abcam. Cells were 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 were resolved by SDS-PAGE and transferred to PVDF membranes (Millipore) by electroblotting. Membranes were blocked with 5% skim milk in TBST (0.1% Tween 20) for 1 h and incubated with primary antibodies in 2% BSA in TBST overnight followed by incubation with HRP-conjugated secondary antibodies for 2 h. Protein-specific signals were detected by an electron chemiluminescence (ECL) reagent, and the signals were acquired using an ImageQuant RT ECL Imager (GE Healthcare).

[0108] 6.2.8 Deglycosylation Prior to CCKBR detection, protein lysates were subjected to deglycosylation. Briefly, 18 μL of whole cell lysate (approximately 50 μg) was mixed with 2 μL of 10× denaturing buffer (5% SDS, 0.4 M DTT) and incubated at room temperature for 10 min. 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 were added. Finally, 2 μL of PNGase F (Sigma) was added, mixed and collected by centrifugation. The reaction was carried out overnight at 37 °C, followed by WB analysis.

[0109] 6.2.9 Animal testing In this study, we used 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).

[0110] Immunodeficient CD-1 female nude mice (Charles Rivers, Germany) were housed for one week prior to the experiment. Prior to tumor implantation, A431 / CCKBR cells were harvested and 5 million cells in 0.1 mL of sterile phosphate-buffered saline (PBS) containing 0.9% NaCl were subcutaneously injected into mice anesthetized with isoflurane / oxygen inhalation (2 tumors per animal). Five days later, animals were randomly assigned to three experimental groups and tumor size was measured non-invasively with a caliper. RAD001 (3 mg / kg), metformin (200 mg / kg) or PBS (control) were administered daily by intraperitoneal injection for 3 or 5 consecutive days, as indicated. No signs of acute toxicity were observed in any of the groups during the treatment cycle. After treatment, the mice were 177 Lu-PP-F11N was administered into the tail vein (approximately 150 kBq per mouse in a volume of 0.1 mL of sterile PBS) and 4 hours later the mice were humanely euthanized with CO2. Postmortem, tumors and organs were weighed and their radioactivity was measured in a gamma counter. At termination, no significant differences in body or organ weights, overall health, or anatomical structure were observed. All experiments were performed in accordance with Swiss animal protection laws.

[0111] 6.2.10 SPECT / IT Before intravenous injection, 177 Lu-PP-F11N was purified by HPLC, concentrated in a SpeedVac, and diluted in PBS (10MBq in 100μl). 177Tumor uptake of Lu-PP-F11N was monitored by single-photon emission computed tomography (SPECT) combined with X-ray computed tomography (multipinhole small-animal NanoSPECT / CT camera, Mediso Medical Imaging Systems, Inc.) according to the manufacturer's instructions. All mice were sacrificed 2 h after injection and used directly for 5 h SPECT scanning after 10 min CT. Image reconstruction, processing and relative quantification were performed using VivoQuant 3.0 Patch1 software. Otsu's binarization and binding thresholds were applied for selection and analysis of radioactive tumor areas.

[0112] 6.2.11 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).

[0113] 6.2.12 Statistical analysis Analyses of two groups were performed using two-tailed Student's t test, whereas analyses of three or more groups were performed using one-way ANOVA followed by multiple comparison tests using GraphPad Prism 7.00 for Windows. A value of P < 0.05 was considered statistically significant.

[0114] Example 1: In Vitro Internalization of Radiolabeled Gastrin Analogues - Kinase Inhibitor Library Screen To investigate pathways that affect the internalization rate of gastrin analogs, a library of kinase inhibitors (80 compounds) was tested in MTC A431 / CCKBR cells. 177 To measure Lu-PP-F11N uptake, screening was performed according to the procedure described above. An overview of the screening assay is shown in Figure 1A.

[0115] Briefly, cells were subjected to treatment with 10 μM of kinase inhibitor for 18 hours. The next day, 10,000 cpm of 177 Lu-PP-F11N (prepared as above) was added to each well and the plates were incubated for 2 hours under standard tissue culture conditions. After removing the radioactive supernatant, the cells were resuspended and incubated for 2 hours at room temperature on a horizontal shaker. Radioactivity was measured within 30 seconds per well by using a MicroBeta 2450 microplate counter.

[0116] Three inhibitors, namely BML257, SC-514 and rapamycin, 177 We found that Lu-PP-F11N uptake was enhanced by 24–26% compared to untreated controls (Figure 1B). Matching proliferation assays showed no significant changes in cell proliferation for these three inhibitors, suggesting that the enhanced uptake was not due to increased cell numbers after kinase inhibition (Figure 1C). BML-257 and rapamycin are known to target the AKT / mTORC1 pathway.

[0117] BML257 and SC-514 have the following chemical structure: [ka] It is a kinase inhibitor having the formula:

[0118] Example 2: In vitro internalization of radiolabeled gastrin analogues induced by RAD001 and metformin To further investigate interference with AKT / mTORC1 signaling, we investigated the effect of AKT / mTORC1 signaling in MTC A431 / CCKBR cells. 177 Internalization of Lu-PP-F11N was assessed using the well-characterized and clinically approved mTORC1 inhibitors RAD001 (everolimus) and metformin.

[0119] For the assay, cells were treated with metformin or RAD001 for 20 h. 177 The internalization of Lu-PP-F11N was measured according to the procedure described above.

[0120] When A431 / CCKBR cells were treated with metformin and RAD001, 177 Internalization of Lu-PP-F11N was increased by 21.1% and 24.3%, respectively, compared to 16.2% in control cells (ratios [treated / control] of 1.3 and 1.5). Furthermore, treatment of AR42J cells with RAD001 177 Lu-PP-F11N internalization was increased by 10% compared to 5.7% in control cells, with a [RAD001 / control] ratio of 1.7. There was no significant difference in membrane binding activity in RAD001- or metformin-treated cells (Figure 2A). Besides A431 / CCKBR cells treated with RAD001 and metformin, protein levels in AR42J cells treated with RAD001 were reduced to 80%, 79%, and 84%, respectively, compared to controls (Figure 2B).

[0121] The effects of RAD001 and metformin treatment were examined by WB analysis, which showed a lack of mTORC1-regulated phosphorylation of ribosomal protein S6 at Ser235 / 236 in protein lysates obtained from treated cells ( Fig. 2C ).

[0122] Example 3. RAD001-induced CCKBR expression and in vitro 177 Internalization of Lu-PP-F11N The expression of CCKBR in A431 / CCKBR cells was evaluated in cells treated with RAD001, and the results are shown in Figure 3A.

[0123] Treatment with 50 nM or 100 nM RAD001 was found to increase CCKBR expression (approximately 2.2-fold), suggesting that increased internalization of radiolabeled gastrin analog in RAD001-treated cells was due to elevated CCKBR expression. Detection of endogenous CCKBR was confirmed by RNA interference, whereby transfection of double siRNA against CCKBR reduced CCKBR expression levels to 55% compared to control cells transfected with double siRNA against the luciferase gene (Figure 3B).

[0124] Example 4: In vivo in MTC tumors treated with RAD001 177 CCKBR-specific internalization of Lu-PP-F11N (5-day treatment) To examine the effects of RAD001 and metformin in vivo, A431 / CCKBR cells were implanted subcutaneously into immunodeficient nude mice, and 5 days after tumor formation, mice were subjected to treatment with RAD001, metformin, or PBS (control) as described above.

[0125] Biodistribution analysis showed that in animals treated with RAD001 177 A statistically significant increase in tumor uptake of Lu-PPF-11N was observed (P=0.0057), with a ratio of [RAD001 / control] of 1.79, whereas metformin treatment 177 It only modestly increased Lu-PP-F11N internalization, with a [metformin / control] ratio of 1.14, which did not reach statistical significance (Figure 4A).

[0126] Furthermore, in healthy organs 177 The biodistribution of Lu-PP-F11N was not significantly different among all groups. Coadministration of the radiolabeled gastrin analog with the unlabeled blocking peptide minigastrin (a competitor of CCKBR binding) reduced the biodistribution of Lu-PP-F11N in all three groups.177 Tumor uptake of Lu-PP-F11N was significantly reduced, demonstrating the specific binding of the radiolabeled gastrin analog to CCKBR ( Fig. 4A , bottom panel).

[0127] As shown in Figures 4B and 4C, RAD001 / 177 Treatment with Lu-PP-F11N significantly reduced tumor volume, while the overall mouse weight was unaffected. In contrast, metformin / 177 Treatment with Lu-PP-F11N did not affect tumor volume or mouse body weight.

[0128] To further explore whether the observed increase in tumor uptake was influenced by differences in tumor burden between RAD001 and control groups, statistical analysis was performed on size-matched tumor groups. Tumors treated with doses ranging from 50 to 150 mg or 50 to 180 mg of RAD001 showed no significant differences in tumor burden. 177 A statistically significant increase in the uptake of Lu-PP-F11N was found, suggesting that RAD001 treatment increased tumor uptake of radiolabeled minigastrin in a tumor burden-independent manner (data not shown).

[0129] Further SPECT / CT imaging demonstrated that after 5 days of treatment with RAD001, in nude mice bearing A431 / CCKBR tumors, compared with controls 177 Increased tumor uptake of Lu-PP-F11N was confirmed (Figure 5A). Relative quantification of the acquired images showed that the mean and maximum concentrations of radiolabeled minigastrin were significantly higher (P<0.001) in tumors treated with RAD001 compared to controls (Figure 5B). The mean and maximum concentration ratios [RAD001 / control] reached 3.11 and 3.17, respectively, whereas metformin treatment showed no significant changes in the mean and maximum concentration ratios [metformin / control] at 1.03 and 1.17, respectively.

[0130] Example 5: In vivo analysis of CCKBR tumors treated with RAD001 177CCKBR-specific internalization of Lu-PP-F11N (3-day treatment) Fewer than five doses of RAD001 177 To examine whether Lu-PP-F11N tumor uptake could be increased, the same A431 / CCKBR xenograft mouse model was subjected to treatment with RAD001 (three doses), metformin or PBS (control) for three days.

[0131] Biodistribution analysis showed that in animals treated with RAD001 177 There was a statistically significant increase in tumor uptake of Lu-PP-F11N (P=0.0016) with a [RAD001 / control] ratio of 1.56 (Figure 6), whereas co-injection with a blocking peptide (minigastrin) showed CCKBR-specific uptake. 177 Three doses of metformin significantly reduced Lu-PP-F11N tumor uptake. 177 There was no significant effect on the uptake of Lu-PP-F11N (Fig. 7A).

[0132] In healthy organs 177 The biodistribution of Lu-PP-F11N was not significantly different among all groups. Three daily doses of RAD001 significantly reduced tumor volume without affecting the overall mouse weight, whereas metformin treatment had no effect on tumor volume or mouse weight (Figure 7B).

[0133] Example 6: In vivo necrosis, mitotic figures and Ki67 positive cell counts in CCKBR tumors treated with RAD001 Paraffin tumor sections prepared from nude mice bearing A431 / CCKBR tumors treated with five daily doses of RAD001, metformin, and PBS (control) were subjected to immunohistochemistry. Five tumors were analyzed in the RAD001 and PBS groups, and four tumors in the metformin group.

[0134] As shown in Figure 8A, treatment with RAD001 increased necrosis (10.6 ± 6.4%) compared to PBS-treated tumors (1.4 ± 1.2%), whereas metformin showed no significant difference compared to the control. Analysis of the number of blood vessels showed no statistical difference between the analyzed groups, suggesting that the observed increase in in vivo uptake of radiolabeled minigastrin was due to an increase in CCKBR expression in tumors treated with RAD001 (Figure 8B). A decrease in the mitotic index (0.75 ± 0.36%) and the number of Ki67-positive cells (61 ± 8.6%) was detected in tumors treated with RAD001 compared to control tumors showing a mitotic index of 1.08 ± 0.2% and Ki67-positive cells of 80 ± 3.1% (Figures 9A and 9B).

[0135] Metformin treatment did not cause significant changes in mitotic numbers or the number of Ki67-positive cells compared to controls.

[0136] Example 7: RAD001 and IFN-γ in A431 / CCKBR tumor-bearing nude mice 177 Evaluation of therapeutic efficacy against combined treatment with Lu-PP-F11N To evaluate the therapeutic efficacy of the combination treatment, tumor growth and median survival time of immunodeficient A431 / CCKBR tumor-bearing nude mice were compared with those of RAD001 alone or in combination with RADIOTHERAPY, as shown in FIG. 10A. 177 It was examined in combination with Lu-PP-F11N after 5 or 10 doses.

[0137] animal treatment research For animal studies, the human epidermoid carcinoma A431 cell line overexpressing CCKBR was previously generated and kindly provided by Dr. Luigi Aloj. A431 / CCKBR cells were cultured at 37°C and 5% CO2 in DMEM supplemented with 10% FCS, 2 mM glutamine and antibiotics (0.1 mg / mL streptomycin, 100 IU penicillin). Prior to tumor implantation, 5 × 10 A431 / CCKBR cells were cultured in 0.1 mL of phosphate-buffered saline (PBS) containing 0.9% NaCl. 6were injected subcutaneously into CD-1 female nude mice (Charles Rivers, Germany) anesthetized with isoflurane / oxygen inhalation. Six days later, animals were randomly assigned to experimental groups and tumor size was measured non-invasively with a caliper. RAD001 (3 mg / kg), or PBS (control), was administered by daily intraperitoneal injection for 5 or 10 days as indicated. HPLC-purified 60 MBq of RAD001 in 100 μL of PBS was 177 Lu-PPF11N was injected intravenously, while the control group was injected with 100 μL of PBS. The tumor diameter and mouse body weight were recorded daily. Tumor volume was calculated using the formula V = (W × L) / 2. When the tumor volume was 1.5 cm 3 Nude mice were sacrificed when the total number of mice reached 100. Furthermore, mice present in all groups that were unwell (n = 2 in total) or had ulcerated tumors (n = 7 in total) were sacrificed early. These mice were excluded from the analysis. All experiments were performed in accordance with the Swiss animal protection law.

[0138] In this study, GraphPad Prism 7.00 for Windows was used for all statistical analyses. One-way analysis of variance combined with the Benjamini, Krieger and Yekutieli two-stage linear step-up procedure for multiple comparison testing was used for all treatment groups. Log-rank (Mantel-Cox) tests were performed to compare differences in survival curves between 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.

[0139] result RAD001 and 177 Both single treatments of Lu-PP-F11N as well as their combination significantly inhibited tumor growth (Figure 10B). On day 13, when all mice were still present in all groups, the mean tumor volume in the control group was 0.97 cm 3 reached. 177 Lu-PP-F11N, 5, and 10 doses of RAD001, and 177The mean tumor size of mice treated with 5 or 10 doses of RAD001 in combination with Lu-PP-F11N was significantly reduced (P<0.05) compared with the control group, to 0.63, 0.31, 0.11 and 0.15 or 0.08 cm, respectively. 3 (Figure 11). 177 Tumor growth in mice treated with Lu-PP-F11N and 177 This was significantly reduced compared with Lu-PP-F11N monotherapy.

[0140] On day 22, 5 or 10 doses of RAD001 177 The mean tumor size of mice treated with Lu-PP-F11N in parallel was significantly reduced compared to the groups receiving 5 or 10 doses of RAD001 alone. 177 The average tumor size of mice treated in parallel with Lu-PP-F11N was significantly reduced compared to monotherapy with 10 doses of RAD001. As shown in the Kaplan-Meier curves in Figure 12, all treatments increased life span compared to the control group.

[0141] The median survival time in the control group was 19.5 days, whereas 177 Lu-PP-F11N, 5 or 10 doses of RAD001, 177 The median survival time of mice treated with 5 or 10 doses of RAD001 in combination with Lu-PP-F11N was extended to 28, 27, 32, 36 and 43 days, respectively, as shown in Table 1 below.

[0142] [Table 1]

[0143] During the treatment, there was a constant increase in body weight, with no significant difference between the control and treated mouse groups (data not shown).

Claims

1. For use in the treatment of CCKB receptor positive cancers or tumors, (i) rapamycin and / or a rapalog; (ii) a radiolabeled gastrin analog; A composition comprising: the rapalog is a compound selected from the group consisting of everolimus (RAD001), temsirolimus (CCI-779), ridaforolimus (AP-23573), and combinations thereof; and The radiolabeled gastrin analog has the following formula (2): Y-DGlu-DGlu-DGlu-DGlu-DGlu-Ala-Tyr-Gly-Trp-Dxx-Asp-Phe-NH ２ (2) wherein Dxx represents an amino acid selected from Nle, 2-amino-5-heptenoic acid, homoNle, 2-amino-4-methoxybutanoic acid, Te-Me, Se-Met and Phg, and Y represents a moiety containing a radionuclide. A composition comprising:

2. 2. The composition of claim 1, wherein Dxx is Nle and / or Y is 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) or 1,4,7-triazacyclononane,1-glutaric acid-4,7-acetic acid (NODAGA).

3. The radionuclide is １８ F. １２４ I, １３１ I, ８６ Y. ９０ Y. １７７ Lu, １１１ In, １８８ Re, ６４ Cu, ６７ Cu, １４９ Tb, １６１ Tb, ８９ Sr, ４４／４３ Sc, ４７ Sc and １５３ The composition of claim 1 or 2, wherein the metal is selected from the group consisting of Sm.

4. The radionuclide is １７７ Lu, ９０ Y and １１１ The composition of any one of claims 1 to 3, wherein said In is selected from the group consisting of In.

5. The radionuclide １７７ The composition of any one of claims 1 to 4, wherein Lu is

6. The rapalog is everolimus (RAD001) and the radiolabeled gastrin analog is represented by the following formula (3): DOTA-DGlu-DGlu-DGlu-DGlu-DGlu-Ala-Tyr-Gly-Trp-Nle-Asp-Phe-NH ２ (3) [Wherein DOTA is １７７ Chelates Lu The composition of any one of claims 1 to 5, having the formula:

7. The composition of any one of claims 1 to 6, wherein (i) rapamycin and / or rapalog and (ii) radiolabeled gastrin analog are formulated in separate dosage forms that can be administered simultaneously and / or sequentially and are presented together in a co-package or in separate packages.

8. (i) rapamycin and / or a rapalog; (ii) a radiolabeled gastrin analog; A kit of parts comprising: A kit-of-parts for use in the treatment of CCKB receptor positive cancers or tumors, wherein said (i) rapalog and / or said (ii) radiolabeled gastrin analogue are as defined in any of claims 1 to 7.

9. (i) rapamycin and / or a rapalog; (ii) a radiolabeled gastrin analog; A combination product comprising:

8. A combination product for use in the treatment of CCKB receptor positive cancers or tumors, wherein said (i) rapalog and / or said (ii) radiolabeled gastrin analogue are as defined in any of claims 1 to 7.

10. A kit-of-parts as described in claim 8, or a combination product as described in claim 9, wherein (i) rapamycin and / or a rapalog is administered together with (ii) radiolabeled gastrin analogue or prior to (ii) radiolabeled gastrin analogue.

11. 11. The kit-of-parts or combination product for use according to claim 10, wherein said (i) rapamycin and / or rapalog is administered up to 2 months prior to said (ii) radiolabeled gastrin analog.

12. 11. The kit-of-parts or combination product for use according to claim 10, wherein said (i) rapamycin and / or rapalog is administered once daily for 1 to 14 consecutive days before said (ii) radiolabeled gastrin analog is administered.

13. The kit-of-parts or combination product for use according to any one of claims 10 to 12, wherein the CCKB receptor positive cancer or tumor is selected from medullary thyroid carcinoma (MTC), glioma, gastroenteropancreatic neuroendocrine tumor (GEP-NET), astrocytoma, gastric cancer, colon cancer, ovarian cancer, breast cancer, and any CCKB receptor positive tumor or cancer.

14. The kit-of-parts or combination product for use according to any one of claims 10 to 13, wherein the CCKB receptor positive cancer or tumor is MTC.

15. A kit-of-parts or combination product for use according to any one of claims 10 to 14, wherein said (i) rapamycin and / or rapalog or said (ii) radiolabeled gastrin analog is administered simultaneously with, before or after one or more other therapeutic or therapeutic agents, such as chemotherapeutic agents or immunomodulatory agents.

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