Compositions and methods for cancer imaging and cancer radiotherapy - Patents.com

Abstract

Compositions and methods for cancer imaging, cancer detection, and cancer radiotherapy are provided. [Solution] Provided are conjugate compounds comprising a pharmaceutically acceptable marker M and a peptide or pseudopeptide P having up to 30 amino acid residues and capable of binding to the low density lipoprotein receptor (LDLR), and their use in methods for labeling and / or detecting and / or treating cancer cells in a subject by administering the conjugate compound to the subject and analyzing the presence and / or amount of the marker.

Description

[Technical field]

[0001] The present invention relates to compositions and methods for cancer imaging, cancer detection, and cancer radiotherapy. In particular, the present invention relates to conjugate compounds comprising an LDLR targeting peptide and a pharma- ceutically acceptable marker, and methods for labeling, detecting, staging, or treating cancer, particularly cancers that overexpress the Low-Density Lipoprotein Receptor (LDLR). Concerning its use for. [Background technology]

[0002] Cancer remains a significant cause of morbidity and mortality worldwide, with approximately 14 million new cases and 8.2 million cancer-related deaths in 2012, according to the UK and World Health Organization Cancer Surveys.

[0003] Although it is now possible to treat many forms of cancer, some are still difficult to treat. For example, pancreatic cancer is one of the most lethal cancers, with a 75% mortality rate within one year and a 5-year survival rate of around 6%. Approximately 12,000 people are diagnosed with pancreatic cancer each year in France, and around 340,000 worldwide. This cancer is predicted to become the second cause of cancer deaths by 2030. Pancreatic cancer affects both men and women over the age of 50 and is particularly difficult to treat. Similarly, glioblastoma multiforme (GBM), the most common and most aggressive form of malignant primary brain tumor, is a rare but serious disease. In the European Union, Currently, approximately 25,000 new cases are diagnosed each year (www.pubcan.org / cancer). It remains a significant clinical challenge, as current treatments are ineffective. Indeed, 5-year survival is currently around 3%, and despite significant advances in our basic understanding of tumor development, median overall patient survival has increased by only 3.3 months (from 11.3 to 14.6 months) over the past 25 years (Patel MA, et al. 2014 Sep29;6(4):1953-85).

[0004] The majority of cancer deaths could be avoided if diagnosed early. Given that the 5-year survival rate for various cancers varies significantly with the tumor stage at the time of detection, there is a strong need to develop non-invasive imaging tools that can identify tumors as early as possible and identify patients who may benefit from therapeutic intervention. Cancer mortality and morbidity could be significantly improved by the development of effective imaging and therapeutic agents.

[0005] Much effort is being made to develop new methods for early and accurate diagnosis of cancer. Various imaging techniques are used, including positron emission tomography (PET scan) and ), single photon emission computed tomography (SPECT), X-ray computed tomography (CT scan) These include radiological scans, nuclear scans, ultrasound, and magnetic resonance imaging (MRI). Imaging techniques are well developed, but are mostly based on non-specific macroscopic physiological changes in the organ being analyzed. Currently, the most widely used PET tracers in oncology are probes that measure glucose utilization and are recognized tools for cancer diagnosis and staging. 18 However, 18 F-FDG has significant limitations, including moderate uptake in some tumors (e.g., prostate) and high background uptake in certain normal tissues (e.g., brain).

[0006] Selective receptor-targeting peptide-based drugs have attracted considerable attention in molecular imaging of tumor cells that overexpress the corresponding peptide receptors due to their unique properties, such as high affinity and specificity for their targets. Indeed, targeting receptors that are highly expressed in tumors is believed to provide better cancer diagnosis, since the receptor-targeting peptides can distinguish diseased tissues from normal tissues.

[0007] In cancer treatment, many approaches such as chemotherapy, immunotherapy or radiation therapy are used alone or in combination.

[0008] Radiation therapy (also called radiotherapy, x-ray therapy, or irradiation) is based on the use of ionizing radiation to kill cancer cells and shrink tumors. Radiation therapy is used for a variety of tumors to obtain both curative and palliative symptoms. The action of radiation therapy is localized and limited to the treated area. Radiation therapy causes damage or destroys cells in the treated area ("target tissue") by damaging their genetic material, preventing these cells from continuing to grow and divide. Radiation damages both cancer cells and normal cells, but most normal cells are able to recover from the effects of radiation and function properly. The goal of radiation therapy is to damage as many cancer cells as possible while limiting harm to nearby healthy tissue. Ionizing radiation works by damaging the DNA of the cancerous tissue, resulting in cell death.

[0009] There are several types of radiation therapy. -External beam radiotherapy (EBRT), the most common form of radiation therapy External beam radiation therapy (EBRT) is a type of therapy in which the patient sits or lies down on a couch while an external source of ionizing radiation is directed at a specific part of the body. X-rays and electron beams are the most widely used sources of external beam radiation therapy. - Brachytherapy, in which sealed radioactive sources are placed in or adjacent to the area to be treated. - Unsealed source radiation therapy (or unsealed source radionuclide therapy) using solid, liquid, gaseous or other forms of radioactive material (essentially radiopharmaceuticals) called radiopharmaceuticals, which are introduced into the body by various means (mainly injection or ingestion) and localized to a specific location, organ or tissue depending on their properties and route of administration. This includes anything from simple compounds such as sodium iodide, which targets the thyroid gland by trapping iodide ions, to complex biopharmaceuticals such as recombinant antibodies that are attached to a radionuclide and recognize specific antigens on the cell surface. This technology is based on the physical, chemical and biological properties of the radiopharmaceutical to target areas of the body for radiation treatment.

[0010] Radiation therapy has several side effects. The characteristics, severity, and duration of side effects depend on the organ receiving radiation, the treatment itself (type of radiation, dose, fractionation, concomitant chemotherapy), and the condition of the patient. There are acute side effects (nausea, vomiting, intestinal discomfort, bloating, infertility, etc.) and delayed side effects (fibrosis, hair loss, dryness, heart disease, cognitive decline, etc.). Therefore, there is a need to develop targeted radiation therapy that causes fewer side effects.

[0011] Peptide receptor radionuclide therapy (PRRT) is a molecular therapy (also called radioisotope therapy) used to treat certain cancers, such as neuroendocrine cancers. In PRRT, cell-targeting proteins (or peptides) are combined with small amounts of radioactive material or radionuclides to create a special type of radiopharmaceutical called a radiopeptide. When injected into a patient's bloodstream, this radiopeptide travels and binds to specific tumor cells, delivering a high dose of radiation to the cancerous tissue while sparing normal tissue.

[0012] There is a clear unmet medical need in the area of ​​effective imaging and targeting agents. Targeting receptors that are highly expressed in tumors would result in better cancer diagnosis and treatment, as the receptor targeting peptides would distinguish diseased tissue from normal tissue.

[0013] The present application relates to novel compositions and methods for cancer imaging and cancer radiotherapy based on LDLR-targeting agents. The present invention relates to novel compositions and methods for cancer imaging and radiotherapy based on LDLR-targeting agents. We provide LDLR conjugate compounds optimized to correspond to radiotherapeutic agents, and demonstrate that such compounds enable efficient, reliable and selective labeling of LDLR-overexpressing cancer cells, making them most suitable for effective diagnosis or radiotherapy of such cancers. Summary of the Invention [Problem to be solved by the invention]

[0014] The present invention provides novel compositions and methods for effective imaging, diagnosis, detection, or radiotherapy of cancers expressing LDLR. The present invention provides, in particular, conjugated compounds capable of binding to and imaging cancer cells overexpressing LDLR. The present invention allows for the in vivo detection of such cancers and their effective and selective radiotherapy. The present invention shows, in fact, that the conjugated agents of the present invention can distinguish between cancer cells and non-cancerous cells and can be used to deliver sufficient levels of radiation to cancer cells without affecting healthy tissue. [Means for solving the problem]

[0015] Thus, one object of the present invention relates to a conjugate compound of formula (I) for use in a method for labeling and / or detecting cancer cells in a subject by administering said conjugate compound to said subject and analyzing the presence and / or amount of a marker, MP (I), In the formula, M represents a pharma- ceutically acceptable marker; P represents a peptide or pseudopeptide having up to 30 amino acid residues and capable of binding to the LDLR.

[0016] A further object of the present invention is a method for labeling or detecting LDLR-overexpressing cancer in a subject, comprising: i) administering to a subject a compound of formula (I); MP (I), In the formula, M represents a pharma- ceutically acceptable marker; P represents a peptide or pseudopeptide having up to 30 amino acid residues and capable of binding to LDLR; and ii) Analysing the presence and / or amount of markers.

[0017] In certain embodiments, the conjugate compound is a multimeric compound comprising a plurality of monomers of formula (I).

[0018] In a preferred embodiment, M represents a radionuclide or a fluorescent marker and P comprises the amino acid sequence (II), A1-Met-A2-Arg-Leu-Arg-A3-A4 (II), In the sequence, A1 and A4 independently represent cysteine ​​or an analog or isostere thereof, A2 represents proline or an analog or isostere thereof, and A3 represents glycine or an analog or isostere thereof.

[0019] Furthermore, M and P can be linked to each other covalently or through non-covalent interactions, directly or through a spacer, hi certain embodiments, M is linked to P using a prosthetic group or is trapped using a chelating group such as NODAGA, DOTA, DOTA-GA or NOTA, or a functional derivative thereof.

[0020] Another object of the present invention relates to a conjugate compound of formula (III) MCSP (III), In the formula, M represents a pharma- ceutically acceptable radionuclide; C represents a chelator that forms a chelate together with M; S represents a spacer, P represents a peptide or pseudopeptide having up to 30 amino acid residues and capable of binding to the LDLR.

[0021] A further object of the present invention relates to a composition comprising a conjugated compound as described above and its use in medical imaging or radiotherapy.

[0022] A further object of the present invention is a method for treating an LDLR-overexpressing cancer in a subject, comprising administering to the subject a conjugate compound of formula (III): MCSP (III), where M represents a pharma- ceutically acceptable radionuclide suitable for use in radiotherapy; C represents a chelator that forms a chelate together with M; S represents a spacer, P represents a peptide or pseudopeptide having up to 30 amino acid residues and capable of binding to the LDLR.

[0023] The present invention can be used to detect or treat any LDLR-overexpressing cancer, such as pancreatic cancer, adrenal cancer, glioblastoma, prostate cancer, colon cancer, liver cancer, pancreatic cancer, ovarian cancer, lung cancer, or gastric cancer. The present invention can be used in any mammalian subject, such as a human subject. [Brief description of the drawings]

[0024] [Figure 1] Figure 1 is a Western blot showing human and mouse LDLR expression in various cancer cell lines. (A) shows CHO95 (a cell line overexpressing human LDLR fused to eGFP), Hela (cervical cancer), NCI-H295R (adrenal adenocarcinoma), Capan, BxPC3, and Panc1 (pancreatic cancer), MCF-7, and MDA-MB231 (breast cancer). In the right panel, LDLR expression in mouse pancreatic cell line PK4 is compared with mouse normal pancreas. In (B), glioblastoma cell line U87MG (two columns), prostate cancer cell line PC3, and NCI-H295R are shown. [Diagram 2] Figure 2 shows the binding and endocytosis of conjugate A (A), conjugate B (B), conjugate C (C), and conjugate D (D) in CHO cells expressing human LDLR conjugated to GFP. The LDLR-GFP signal is visualized in the second row, and the conjugate signal is visualized directly using A680 fluorescence in the fourth row or using anti-hFc-A594 conjugate secondary antibody in the third row. Cell nuclei were stained with Hoechst blue and the signal is visualized in the first row. Co-labeling of LDLR-GFP and conjugates is visualized as highlighted signal in the fifth row in the merged image. [Diagram 3] In Figure 3, A shows the tissue biodistribution of conjugate A in the pancreas, pancreatic tumor, and kidney (control) of mice implanted with PK4A tumors and injected with conjugate A via the tail vein, and B shows the tissue biodistribution of conjugate C in the pancreas and pancreatic tumor of mice injected with conjugate C. Biodistribution was assessed using quantification by ELISA. [Figure 4] In FIG. 4, A shows whole-body fluorescence imaging of mice implanted with PK4A tumors and analyzed 15, 30, 60, and 120 minutes after intravenous injection of Conjugate C or Conjugate D (control), and B shows ex vivo imaging of pancreatic tumors, healthy pancreas, and liver harvested 4 hours after intravenous injection of Conjugate C or Conjugate D (control). [Diagram 5] 5 shows a reaction scheme for the synthesis of caged-βAla-PEG12-peptide-NH2 compounds. In this scheme, the synthesis of NODAGA-βAla-PEG12-SEQ ID NO:1-NH2 is shown as an example. [Figure 6A-C] 6A-C show PET imaging of mice treated with 18F-FDG (A), 68Ga-CH44 (B), and 68Ga-CH40 (C) 14 days after implantation of NCI-H295R cells. Adrenal tumors are indicated by circles. [Figure 7]Figure 7 shows PET imaging of mice treated with 68Ga-CH44 (A) and 68Ga-CH40 (B) 32 days after implantation of NCI-H295R cells. Adrenal tumors are indicated by circles. [Figure 8] FIG. 8 shows PET imaging of mice administered 68Ga-FG770 (A) and 68Ga-FG769 (B) 37 days after implantation of NCI-H295R cells. [Figure 9A-C] 9A-C show PET imaging of mice treated with 18F-FDG (A), 68Ga-CH44 (B), and 68Ga-CH40 (C) on day 4 after implantation of Pk4a cells. Pancreatic tumors are indicated by circles. [Figure 10A-B] 10A-B show PET imaging of mice treated with 68Ga-CH44 (A) and 68Ga-CH40 (B) 12 days after implantation of Pk4a cells. Pancreatic tumors are indicated by circles. [Figure 11A-E] Figures 11A-E show PET imaging of mice administered 68Ga-CH44 on day 14 (A), 68Ga-CH40 on day 16 (B), 68Ga-CH44 on day 21 (C), 68Ga-CH40 on day 21 (D), and 68Ga-RGD on day 21 (E) after cerebral implantation of U87MG cells. [Figure 12] FIG. 12 shows quantification of 68Ga-CH44, 68Ga-CH40, 68Ga-RGD in animals implanted with U87MG on day 21, expressed as tumor to control ratio. [Figure 13] FIG. 13 shows PET imaging of mice administered 68Ga-CH44 (A) and 68Ga-CH40 (B) 48 days after implantation of NCI-H295R cells. [Figure 14] FIG. 14 shows the structure of compound VHd. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0025] The present invention relates to compositions and methods for cancer imaging and cancer radiotherapy based on LDLR targeting agents.The present invention provides a conjugate compound comprising an LDLR binding moiety and a pharma- ceutically acceptable marker.The present invention further shows that such compounds can effectively label cancer cells in vitro and in vivo, allowing them to be effectively imaged, detected, staged or radiotreated.

[0026] More specifically, the object of the present invention is a method for labeling a cancer or tumor or a cancer-tumor cell using a conjugate compound of formula (I): MP (I), In the formula, M represents a pharma- ceutically acceptable marker; P represents a peptide or pseudopeptide having up to 30 amino acid residues and capable of binding to the low density lipoprotein receptor (LDLR).

[0027] Human LDLR is a transmembrane protein of 839 amino acids that contains three domains: the extracellular domain (1-768), the transmembrane domain (768-790), and the cytoplasmic domain (790-839). The extracellular domain is divided into two subdomains: the LDL-binding domain (1-322) and the extra-LDL-binding domain (322-768). LDLR is reported to be expressed by most nucleated cells, and it is generally accepted that 1000-3000 LDLRs are present on the surface of non-pathological cells. The amount of LDLR in cancer cells can be greatly increased up to more than 100,000. Tumor cells that have been reported to overexpress LDLR include prostate cancer. (Chen et al., 2001, Int. J. Cancer, 91, 41-45), colon cancer (Niendorf et al., 1995, Int. J. Cancer, 61, 461-464), leukemia (Tatidis et al., 2002, Biochem. Pharmacol., 63, 2169-2180), colorectal cancer (Caruso et al., 2001, Anticancer Res., 21, 429-433), breast cancer (Graziani et al., 2002, Gynecol. Oncol., 85, 493-497), glioblastoma brain tumor cells (Malentiska et al., 2000, Cancer Res., 60, 2300-2303; Nikanjam et al., 2007, Int. J.Pharm., 328, 86-94), as well as liver, pancreatic, ovarian, lung, and gastric cancer cells. However, it is not known whether such increased expression may affect the organization and availability of the receptor, particularly the extracellular domain 322-768. Indeed, it has been observed that high expression of the receptor can lead to aggregation or conformational changes. Such aggregation may alter the ability to target drugs to such cells using LDLR targeting agents that bind to the extracellular domain 322-768.

[0028] Interestingly, and despite the ubiquitous expression of LDLR, the conjugates of the present invention can effectively label cells that overexpress LDLR, particularly cancer cells that overexpress LDLR, and allow for effective targeting of such LDLR-expressing cancer cells. Such an effect may be due to the appropriate receptor density, conjugate binding affinity to LDLR, and mechanism of action (non-competitive).

[0029] The present invention demonstrates that the conjugates of the present invention retain the ability to effectively bind LDLR-overexpressing cancer cells.

[0030] The present invention demonstrates that LDLR-overexpressing cells can be targeted with pharma- ceutically acceptable markers by the conjugates of the present invention, suitable for distinguishing LDLR-overexpressing cells (e.g., cancer cells) from cells with normal levels of LDLR (e.g., normal cells).

[0031] The present invention demonstrates that the conjugates of the invention are capable of labeling LDLR-overexpressing cells to sufficient levels, despite variations in LDLR density and possible remodeling.

[0032] Thus, the present invention provides safe and effective labeling agents and radiotherapeutic agents, enabling the design of effective cancer diagnosis and treatment methods.

[0033] Thus, an object of the present invention relates to a conjugate compound of formula (I) for use in a method for labeling and / or detecting cancer cells in a subject by administering said conjugate compound to said subject and analyzing the presence and / or amount of a marker, MP (I), In the formula, M represents a pharma- ceutically acceptable marker; P represents a peptide or pseudopeptide having up to 30 amino acid residues and capable of binding to the LDLR.

[0034] The present invention also relates to a method of labelling, imaging, diagnosing or staging cancer in a subject using the conjugate compound as defined above.

[0035] In certain embodiments, the conjugate compound of the invention further comprises a second targeting group T that binds to a molecule different from the LDLR. An example of such a dual conjugate compound is a compound of formula (IV): Mn-PT-Mm (IV), wherein M and P are as defined above, T represents a second targeting group, n is 1 or 0, and m is 1 or 0. Preferably, m+n=1.

[0036] In a further specific embodiment, the dual conjugate compound has the structure: (MC-)nP-T(-CM)m, wherein M, C, and P are as defined above, T represents a second targeting group, n is 1 or 0, and m is 1 or 0. Preferably, m+n=1.

[0037] T can be any group that binds to a target molecule (or to a cell expressing such a molecule) different from LDLR. Preferably, such a target molecule is a molecule expressed by a cancer cell, such as a pancreatic cancer cell, a prostate cancer cell, an ovarian cancer cell, or an adrenal cancer cell. Examples of such target groups include, but are not limited to, PSMA (prostate specific membrane antigen), neurotensin, somatostatin analogs, and their derivatives.

[0038] [Conjugate] The present invention discloses the use of specific LDLR-targeting conjugates and the design of novel conjugates, which may be monomeric or multimeric structures of formula (I), as described below.

[0039] In a particular embodiment, the conjugate is a compound of formula MP(I) as defined above. Preferably, P comprises the amino acid sequence (II): A1-Met-A2-Arg-Leu-Arg-A3-A4 (II), In the sequence, A1 and A4 independently represent cysteine ​​or an analog or isostere thereof, A2 represents proline or an analog or isostere thereof, and A3 represents glycine or an analog or isostere thereof.

[0040] More specifically, A1 and A4 each independently represent an amino acid residue selected from cysteine ​​(Cys, C) in the D- or L-configuration, or 2-amino-3-mercaptopropanoic acid in the L- or D-configuration and its S-substituted derivatives, S-acetylcysteine ​​or 2-amino-3-(acetylthio)propanoic acid, selenocysteine ​​(Sec, U) or 2-amino-3-(seleno)propanoic acid, cysteinol, 3-mercaptopropanoic acid (Mpa), or penicillamine (Pen).

[0041] In a preferred embodiment, A1 and A4 are each independently a cysteine ​​(Cys), (D)-cys, penicillamine (Pen), and (D)-penicillamine ((D)-Pen).

[0042] A2 is more preferably proline (Pro, P), pyrrolidine or -2-carboxylic acid, homoproline, 2-(2-pyrrolidinyl)ethanoic acid, 3-hydroxyproline (3Hyp), 4-hydroxyproline (4Hyp), 3-methylproline, 3,4-dehydroproline, 3,4-methanoproline, 4-aminoproline, 4-oxoproline, thioproline, thiazolidine-4-carboxylic acid (Thz), 2-oxo Thiazolidine-4-carboxylic acid, indoline-2-carboxylic acid (Idc), pipecolic acid (Pip), piperidine-2-carboxylic acid, nipecoic acid (Nip), piperidine-3-carboxylic acid, A residue selected from 4-oxopipecolic acid, 4-hydroxypipecolic acid, amino-1-cyclohexanecarboxylic acid, or prolinol is shown.

[0043] In a preferred embodiment, A2 is proline, pipecolic acid (Pip), or thiazolyl. and thazin-4-carboxylic acid (Thz).

[0044] A3 is more preferably glycine (Gly, G), 2-aminoethanoic acid, sarcosine (Sar), N-methylglycine (MeGly), N-ethylglycine (EtGly), allylglycine (a 2-aminopent-4-enoic acid, 2-cyclopentylglycine (Cpg), 2-cyclohexylglycine (Chg), 2,2-dipropylglycine (Dpg), 2-(3-indolyl)glycine (IndGly), 2-indanylglycine (Igl), 2-neopentylglycine NptGly, 2-octylglycine (OctGly), 2-propargylglycine (Pra) or 2-aminopent-4-ynoic acid, 2-phenylglycine (Phg), 2-(4-chloro phenylglycine, azaglycine (AzGly), glycinol, or 2-a The residues selected from aminoethanol are shown.

[0045] In a preferred embodiment, A3 is glycine or sarcosine.

[0046] In a preferred embodiment, P is SEQ ID NO:1: (D)-Cys-Met-Thz-Arg-Leu-Arg-Gly-Pen; SEQ ID NO:2: (D)-Cys-Met-Thz-Arg-Leu-Arg-Sar-Pen; SEQ ID NO:3: (D)-Cys-Met-Pip-Arg-Leu-Arg-Sar-Cys; SEQ ID NO: 4: (D)-Cys-Met-Pip-Arg-Leu-Arg-Gly-Pen; SEQ ID NO:5: (D)-Cys-Met-Pip-Arg-Leu-Arg-Sar-Pen; SEQ ID NO: 6: Cys-Met-Pro-Arg-Leu-Arg-Gly-Cys; SEQ ID NO: 7: (D)-Cys-Met-Pro-Arg-Leu-Arg-Gly-Cys; SEQ ID NO: 8: Asp-Ser-Gly-Leu-Cys-Met-Pro-Arg-Leu-Arg-Gly-Cys-Asp-Pro-Arg; SEQ ID NO: 9: (D)-Pen-Met-Thz-Arg-Leu-Arg-Gly-Cys, comprising, essentially consisting of, or consisting of an amino acid sequence selected from any one of SEQ ID NOs: 1 to 9.

[0047] The results obtained by the inventors show that the conjugates of the invention have a higher affinity (K D) (see Example 3). In addition, the conjugates of the present invention accumulate better in pancreatic tumors compared to healthy pancreas (see Example 4). In particular, as shown in the examples, the labeling of cancer tissue can be 10-50 times better than that of non-cancerous tissue. This accumulation level is sufficient to allow a reliable and clear distinction in the images. Furthermore, in the case of use in radiotherapy, it means that the conjugates have a very good specificity for tumor cells compared to healthy cells.

[0048] As mentioned above, the marker M can be any radionuclide or fluorescent marker.

[0049] Fluorescent markers that can be used are, for example, Alexa Fluor 680, CF680, ATTO680, Fluoroprobes682, Cyanine5.5, IRDye680, or Vivotag. In a general manner, all near-infrared (IR) fluorescent markers can be used for imaging, as they allow for minimal tissue autofluorescence. More specifically, Alexa Fluor680 is a bright, photostable near-infrared dye that is similar in spectrum to Cy5.5 dye. Alexa Fluor680 dye is used for stable signal generation in imaging and flow cytometry, is water-soluble, and is pH-insensitive at pH4 to pH10. Alexa Fluo The NHS ester (or succinimidyl ester) of r680 is the most common tool for conjugating this dye to proteins or antibodies. NHS esters of the dyes are conjugated to primary amines (R-NH2) of proteins, amine-modified oligonucleotides, and other amines. The method can be used to label fluorescein-containing molecules.

[0050] In a most preferred embodiment, M is used in medical imaging, e.g., positron emission tomography (PET) imaging, scintigraphy, positron emission tomography-computing. Preferably, M is a radionuclide compatible for use in PET, or single photon emission computed tomography.

[0051] Examples of radionuclides suitable for use in the present invention include: 18 F, 11 C. 15 O. 13 N, 68 Ga, 82 Rb, and 89 Preferred radionuclides for use in imaging may have a short half-life (e.g., 30 minutes to 3 or 4 days) and a low energy emission (e.g., less than a 300 keV gamma-emitting radionuclide). 68 It's Ga. 68 Ga has a short half-life (68 minutes) and is a positron-emitting isotope.

[0052] For use in radiation therapy, the radionuclide is usually a beta-, or high-energy gamma-emitting radionuclide, preferably with a longer half-life (e.g., 1 to 75 days). Preferred radionuclides for use in radiation therapy are: 90 Y, 111 In, 131 I, and 177 In a preferred embodiment, 90 Y, 177 Lu, or 111 In is used.

[0053] The conjugates of the invention can be synthesized by any technique known to those skilled in the art, such as chemical, biological or genetic synthesis. Chemical synthesis involves the addition of one or more peptides, which may include, inter alia, the insertion of natural amino acids, as well as non-natural amino acids (so-called exotic, i.e. non-coded amino acids), such as, for example, D enantiomers and residues with side chains that differ in hydrophobicity and steric hindrance from those of their natural homologues, or methylene (-CH2-) or phosphate (-PO2-) groups, secondary amines (-NH-) or oxygen (-O-) or N-alkyl peptides. Commercially available constructs are available that can introduce peptide sequences containing peptide-like bonds. The peptides or pseudopeptides, or protein portions thereof, can also be obtained from nucleic acid sequences that encode them. These nucleic acid sequences can be DNA or RNA and can be combined with control sequences and / or inserted into biological expression vectors.

[0054] In the conjugate compounds of the invention, the attachment of M to P (and / or P to T) can be by any acceptable means of attachment, taking into account the chemical nature, disorder and number of the agents involved. That is, attachment can be by one or more covalent, ionic, hydrogen, hydrophobic or van der Waals bonds. Also, attachment can be at any site on the peptide or pseudopeptide where a functional group such as -OH, -SH, -CO2H, -NH2, -SO3H, -CN, -N3, -NCS, -PO3H, alkyne, maleimide or succinimide ester is naturally present or introduced. That is, M can be covalently attached (coupled) to the peptide or pseudopeptide either at the N-terminus or C-terminus, at a reactive group on a natural or non-natural amino acid side chain of the peptide sequence, or via a prosthetic group. Similarly, attachment can be at any site on the imaging or radiotherapeutic agent.

[0055] It is preferred that the interaction be strong enough that M does not dissociate from the peptide before it has reached its site of action and that M and the peptide remain bound for the time necessary to perform the imaging procedure or for M to have a therapeutic effect. For this reason, the preferred bonds of the present invention are covalent or ionic bonds. The imaging agent or radiotherapeutic agent is attached to one of its termini. The M can be attached directly to the peptide, either at the N-terminus or C-terminus, or at a side chain of one of the constituent amino acids of the sequence. The imaging or radiotherapeutic agent can also be attached indirectly, by a linker or spacer, either at one of the termini of the peptide, or at a side chain of one of the constituent amino acids of the sequence. In certain embodiments, M is attached to the peptide by entrapment, for example, using a chelator.

[0056] In this regard, in a preferred embodiment, the conjugate compound comprises a chelator C capable of forming a chelate with M. Such a conjugate has an MCP structure. Various chelators can be used. Preferably, C is NODAGA (1,4,7-triazacyclononane-1-glutaric acid-4,7-diacetic acid), DOTA (tetraazacyclododecane-1,4,7,10-tetraacetic acid), NOTA (1,4,7-triazacyclononane-triacetic acid), DOTA -GA (2,2',2''-(10-(2,6-dioxotetrahydro-2H-pyran-3-yl)-1,4,7,10-tetraazacyclododecane-1,4,7triyl)triacetic acid), and functional derivatives thereof. NODAGA and NOTA are preferred for imaging, and DOTA is preferred for radiotherapy.

[0057] The term "functional derivative" means any compound derived from the above-mentioned chelators by replacing one or more of its functional groups (i.e., groups involved in the chelating function) with other functional groups without impairing the chelating function, and / or by adding and / or deleting groups not involved in the chelating function without impairing the chelating function.

[0058] The chelator C can be attached to the peptide P (or T) directly or via a spacer S, which is usually covalently attached to P and C (or T and C). In this regard, in certain embodiments, the conjugate comprises an MCP structure, where M, C and P are as described above, and C is directly attached to the peptide P. In other particular embodiments, the conjugate comprises an MCSP structure, where M, C, S and P are as described above. In further particular embodiments, the conjugate comprises an MCPT, or PTCM, or MCSPT, or PTSCM structure, where M, C, S, P and T are as described above. In further embodiments, P and T are attached via a spacer group.

[0059] S may be alkyl, aryl, PEG, or peptidic and may be selected from bifunctional or polyfunctional agents having functional groups at their termini, such as amines, esters, aldehydes, alkyl or aryl acids, anhydrides, sulfhydryls, or carboxyl groups, cyanogen bromide or chloride, carbonyldiimidazole, succinimide esters, sulfonic acid halides, maleimides, azides, isothiocyanates, groups derived from alkynes, and the like. In certain embodiments, S comprises one or more PEGs, preferably with a beta-alanine at one end. As an example, S is an oligomer of 12 PEGs with a beta-alanine at one end of the chain. In other embodiments, S comprises multiple Gly residues, such as G3 or G4S. In other specific embodiments, S comprises a hydrophilic negatively charged peptide sequence that includes amino acids in the D or L configuration, such as multiple His-Glu repeat units (2 or 3 units), or a hydrophilic neutral sequence that includes amino acids in the D or L configuration, such as Gly-Gly-Gly-Arg-Asp-Asn. In other embodiments, a metabolizable group that is stable in plasma but is specifically cleaved in the kidney by hydrolases in the proximal tubular brush border membrane can be used to reduce retention in the kidney. As an example, a glycyl-lysine bond can be used. That is, S can be any pharmacokinetic modifier.

[0060] In certain embodiments, the present invention relates to a conjugate compound of formula (III): MCSP (III), In the formula, M represents a pharma- ceutically acceptable radionuclide; C represents a chelator that forms a chelate together with M; S represents a spacer, P represents a peptide or pseudopeptide having up to 30 amino acid residues and capable of binding to the low density lipoprotein receptor (LDLR).

[0061] In a particular embodiment, the present invention relates to a conjugate compound as defined above, further comprising a second targeting group T.

[0062] A preferred conjugate compound of the present invention is a compound of formula (III): In the formula, M represents a pharma- ceutically acceptable radionuclide; C is selected from NODAGA, DOTA, NOTA, and DOTA-GA; S represents a spacer comprising a bifunctional drug, PEG or peptide, or S is absent; P represents a peptide or pseudopeptide having up to 30 amino acid residues and capable of binding to the low density lipoprotein receptor (LDLR).

[0063] In the most preferred compounds of the invention, C is NODAGA or DOTA and P comprises an amino acid sequence selected from SEQ ID NOs: 1-9.

[0064] Specific examples of conjugate compounds of the present invention include: 68 Ga-NODAGA-SEQ ID NO:1 68 Ga-NOTA-SEQ ID NO:1 68 Ga-DOTA-SEQ ID NO:1 68 Ga-DOTA-GA-SEQ ID NO:1 18 F-NODAGA-SEQ ID NO:1 18 F-NOTA - SEQ ID NO:1 18 F-DOTA-SEQ ID NO:1 18 F-DOTA-GA-SEQ ID NO:1 90 Y-NODAGA-SEQ ID NO:1 90 Y-NOTA - SEQ ID NO:1 90 Y-DOTA-SEQ ID NO:1 90 Y-DOTA-GA-SEQ ID NO:1 111 In-NODAGA-SEQ ID NO:1 111 In-NOTA - SEQ ID NO:1 111 In-DOTA-SEQ ID NO:1 111 In-DOTA-GA-SEQ ID NO:1 177 Lu-NODAGA-SEQ ID NO:1 177 Lu-NOTA - SEQ ID NO:1 177 Lu-DOTA-SEQ ID NO:1 177 Lu-DOTA-GA - SEQ ID NO:1 68 Ga-NODAGA-SEQ ID NO:2 68 Ga-NOTA-SEQ ID NO:2 68 Ga-DOTA-SEQ ID NO:2 68 Ga-DOTA-GA-SEQ ID NO:2 18 F-NODAGA-SEQ ID NO:2 18 F-NOTA-SEQ ID NO:2 18 F-DOTA-SEQ ID NO:2 18 F-DOTA-GA-SEQ ID NO:2 90 Y-NODAGA-SEQ ID NO:2 90 Y-NOTA-SEQ ID NO:2 90 Y-DOTA-SEQ ID NO:2 90 Y-DOTA-GA-SEQ ID NO:2 111 In-NODAGA-SEQ ID NO:2 111 In-NOTA-SEQ ID NO:2 111 In-DOTA-SEQ ID NO:2 111 In-DOTA-GA-SEQ ID NO:2 177 Lu-NODAGA-SEQ ID NO:2 177 Lu-NOTA-SEQ ID NO:2 177 Lu-DOTA-SEQ ID NO:2 177 Lu-DOTA-GA-SEQ ID NO:2 68 Ga-NODAGA-SEQ ID NO:3 68 Ga-NOTA-SEQ ID NO:3 68 Ga-DOTA-SEQ ID NO:3 68 Ga-DOTA-GA-SEQ ID NO:3 18 F-NODAGA-SEQ ID NO:3 18 F-NOTA - SEQ ID NO:3 18 F-DOTA-SEQ ID NO:3 18 F-DOTA-GA-SEQ ID NO:3 90 Y-NODAGA-SEQ ID NO:3 90 Y-NOTA - SEQ ID NO:3 90 Y-DOTA-SEQ ID NO:3 90 Y-DOTA-GA-SEQ ID NO:3 111 In-NODAGA-SEQ ID NO:3 111 In-NOTA - SEQ ID NO:3 111 In-DOTA-SEQ ID NO:3 111 In-DOTA-GA-SEQ ID NO:3 177 Lu-NODAGA-SEQ ID NO:3 177 Lu-NOTA-SEQ ID NO:3 177 Lu-DOTA-SEQ ID NO:3 177 Lu-DOTA-GA - SEQ ID NO:3 68 Ga-NODAGA-SEQ ID NO:4 68 Ga-NOTA-SEQ ID NO:4 68 Ga-DOTA-SEQ ID NO:4 68 Ga-DOTA-GA-SEQ ID NO:4 18 F-NODAGA-SEQ ID NO:4 18 F-NOTA - SEQ ID NO:4 18 F-DOTA-SEQ ID NO:4 18 F-DOTA-GA-SEQ ID NO:4 90 Y-NODAGA-SEQ ID NO:4 90 Y-NOTA - SEQ ID NO:4 90 Y-DOTA-SEQ ID NO:4 90 Y-DOTA-GA-SEQ ID NO:4 111 In-NODAGA-SEQ ID NO:4 111 In-NOTA - SEQ ID NO:4 111 In-DOTA-SEQ ID NO:4 111 In-DOTA-GA - SEQ ID NO:4 177 Lu-NODAGA-SEQ ID NO:4 177 Lu-NOTA - SEQ ID NO:4 177 Lu-DOTA-SEQ ID NO:4 177 Lu-DOTA-GA - SEQ ID NO:4 68 Ga-NODAGA-SEQ ID NO:5 68 Ga-NOTA-SEQ ID NO:5 68 Ga-DOTA-SEQ ID NO:5 68 Ga-DOTA-GA-SEQ ID NO:5 18 F-NODAGA-SEQ ID NO:5 18 F-NOTA-SEQ ID NO:5 18 F-DOTA-SEQ ID NO:5 18 F-DOTA-GA-SEQ ID NO:5 90 Y-NODAGA-SEQ ID NO:5 90 Y-NOTA - SEQ ID NO:5 90 Y-DOTA-SEQ ID NO:5 90 Y-DOTA-GA-SEQ ID NO:5 111 In-NODAGA-SEQ ID NO:5 111 In-NOTA-SEQ ID NO:5 111 In-DOTA-SEQ ID NO:5 111 In-DOTA-GA-SEQ ID NO:5 177 Lu-NODAGA-SEQ ID NO:5 177 Lu-NOTA - SEQ ID NO:5 177 Lu-DOTA-SEQ ID NO:5 177 Lu-DOTA-GA - SEQ ID NO:5 68 Ga-NODAGA-SEQ ID NO:6 68 Ga-NOTA-SEQ ID NO:6 68 Ga-DOTA-SEQ ID NO:6 68 Ga-DOTA-GA-SEQ ID NO:6 18 F-NODAGA-SEQ ID NO:6 18 F-NOTA - SEQ ID NO:6 18 F-DOTA-SEQ ID NO:6 18 F-DOTA-GA-SEQ ID NO:6 90 Y-NODAGA-SEQ ID NO:6 90 Y-NOTA - SEQ ID NO:6 90 Y-DOTA-SEQ ID NO:6 90 Y-DOTA-GA-SEQ ID NO:6 111 In-NODAGA-SEQ ID NO:6 111 In-NOTA - SEQ ID NO:6 111 In-DOTA-SEQ ID NO:6 111 In-DOTA-GA - SEQ ID NO:6 177 Lu-NODAGA-SEQ ID NO:6 177 Lu-NOTA - SEQ ID NO:6 177 Lu-DOTA-SEQ ID NO:6 177 Lu-DOTA-GA - SEQ ID NO:6 And, 68 Ga-NODAGA-S-SEQ ID NO:1 68 Ga-NOTA-S-SEQ ID NO:1 68 Ga-DOTA-S-SEQ ID NO:1 68 Ga-DOTA-GA-S-SEQ ID NO:1 18 F-NODAGA-S-SEQ ID NO:1 18 F-NOTA-S-SEQ ID NO:1 18 F-DOTA-S-SEQ ID NO:1 18 F-DOTA-GA-S-SEQ ID NO:1 90Y-NODAGA-S-SEQ ID NO:1 90 Y-NOTA-S-SEQ ID NO:1 90 Y-DOTA-S-SEQ ID NO:1 90 Y-DOTA-GA-S-SEQ ID NO:1 111 In-NODAGA-S-SEQ ID NO:1 111 In-NOTA-S-SEQ ID NO:1 111 In-DOTA-S-SEQ ID NO:1 111 In-DOTA-GA-S-SEQ ID NO:1 177 Lu-NODAGA-S-SEQ ID NO:1 177 Lu-NOTA-S-SEQ ID NO:1 177 Lu-DOTA-S-SEQ ID NO:1 177 Lu-DOTA-GA-S-SEQ ID NO:1 68 Ga-NODAGA-S-SEQ ID NO:2 68 Ga-NOTA-S-SEQ ID NO:2 68 Ga-DOTA-S-SEQ ID NO:2 68 Ga-DOTA-GA-S-SEQ ID NO:2 18 F-NODAGA-S-SEQ ID NO:2 18 F-NOTA-S-SEQ ID NO:2 18 F-DOTA-S-SEQ ID NO:2 18 F-DOTA-GA-S-SEQ ID NO:2 90 Y-NODAGA-S-SEQ ID NO:2 90 Y-NOTA-S-SEQ ID NO:2 90 Y-DOTA-S-SEQ ID NO:2 90 Y-DOTA-GA-S-SEQ ID NO:2 111In-NODAGA-S-SEQ ID NO:2 111 In-NOTA-S-SEQ ID NO:2 111 In-DOTA-S-SEQ ID NO:2 111 In-DOTA-GA-S-SEQ ID NO:2 177 Lu-NODAGA-S-SEQ ID NO:2 177 Lu-NOTA-S-SEQ ID NO:2 177 Lu-DOTA-S-SEQ ID NO:2 177 Lu-DOTA-GA-S-SEQ ID NO:2 68 Ga-NODAGA-S-SEQ ID NO:3 68 Ga-NOTA-S-SEQ ID NO:3 68 Ga-DOTA-S-SEQ ID NO:3 68 Ga-DOTA-GA-S-SEQ ID NO:3 18 F-NODAGA-S-SEQ ID NO:3 18 F-NOTA-S-SEQ ID NO:3 18 F-DOTA-S-SEQ ID NO:3 18 F-DOTA-GA-S-SEQ ID NO:3 90 Y-NODAGA-S-SEQ ID NO:3 90 Y-NOTA-S-SEQ ID NO:3 90 Y-DOTA-S-SEQ ID NO:3 90 Y-DOTA-GA-S-SEQ ID NO:3 111 In-NODAGA-S-SEQ ID NO:3 111 In-NOTA-S-SEQ ID NO:3 111 In-DOTA-S-SEQ ID NO:3 111 In-DOTA-GA-S-SEQ ID NO:3 177 Lu-NODAGA-S-SEQ ID NO:3 177 Lu-NOTA-S - SEQ ID NO:3 177 Lu-DOTA-S-SEQ ID NO:3 177 Lu-DOTA-GA-S-SEQ ID NO:3 68 Ga-NODAGA-S-SEQ ID NO:4 68 Ga-NOTA-S-SEQ ID NO:4 68 Ga-DOTA-S-SEQ ID NO:4 68 Ga-DOTA-GA-S-SEQ ID NO:4 18 F-NODAGA-S-SEQ ID NO:4 18 F-NOTA-S-SEQ ID NO:4 18 F-DOTA-S-SEQ ID NO:4 18 F-DOTA-GA-S-SEQ ID NO:4 90 Y-NODAGA-S-SEQ ID NO:4 90 Y-NOTA-S-SEQ ID NO:4 90 Y-DOTA-S-SEQ ID NO:4 90 Y-DOTA-GA-S-SEQ ID NO:4 111 In-NODAGA-S-SEQ ID NO:4 111 In-NOTA-S - SEQ ID NO:4 111 In-DOTA-S-SEQ ID NO:4 111 In-DOTA-GA-S-SEQ ID NO:4 177 Lu-NODAGA-S-SEQ ID NO:4 177 Lu-NOTA-S - SEQ ID NO:4 177 Lu-DOTA-S-SEQ ID NO:4 177 Lu-DOTA-GA-S-SEQ ID NO:4 68 Ga-NODAGA-S-SEQ ID NO:5 68 Ga-NOTA-S-SEQ ID NO:5 68 Ga-DOTA-S-SEQ ID NO:5 68 Ga-DOTA-GA-S-SEQ ID NO:5 18 F-NODAGA-S-SEQ ID NO:5 18 F-NOTA-S-SEQ ID NO:5 18 F-DOTA-S-SEQ ID NO:5 18 F-DOTA-GA-S-SEQ ID NO:5 90 Y-NODAGA-S-SEQ ID NO:5 90 Y-NOTA-S-SEQ ID NO:5 90 Y-DOTA-S-SEQ ID NO:5 90 Y-DOTA-GA-S-SEQ ID NO:5 111 In-NODAGA-S-SEQ ID NO:5 111 In-NOTA-S-SEQ ID NO:5 111 In-DOTA-S-SEQ ID NO:5 111 In-DOTA-GA-S-SEQ ID NO:5 177 Lu-NODAGA-S-SEQ ID NO:5 177 Lu-NOTA-S -SEQ ID NO:5 177 Lu-DOTA-S-SEQ ID NO:5 177 Lu-DOTA-GA-S-SEQ ID NO:5 68 Ga-NODAGA-S-SEQ ID NO:6 68 Ga-NOTA-S-SEQ ID NO:6 68 Ga-DOTA-S-SEQ ID NO:6 68Ga-DOTA-GA-S-SEQ ID NO:6 18 F-NODAGA-S-SEQ ID NO:6 18 F-NOTA-S-SEQ ID NO:6 18 F-DOTA-S-SEQ ID NO:6 18 F-DOTA-GA-S-SEQ ID NO:6 90 Y-NODAGA-S-SEQ ID NO:6 90 Y-NOTA-S-SEQ ID NO:6 90 Y-DOTA-S-SEQ ID NO:6 90 Y-DOTA-GA-S-SEQ ID NO:6 111 In-NODAGA-S-SEQ ID NO:6 111 In-NOTA-S - SEQ ID NO:6 111 In-DOTA-S-SEQ ID NO:6 111 In-DOTA-GA-S-SEQ ID NO:6 177 Lu-NODAGA-S-SEQ ID NO:6 177 Lu-NOTA-S - SEQ ID NO:6 177 Lu-DOTA-S-SEQ ID NO:6 177 Lu-DOTA-GA-S-SEQ ID NO:6 where S is a spacer as defined above.

[0065] In certain embodiments, the conjugate compounds of the invention are in multimeric form, i.e., can contain multiple copies of the P group or the M group, or multiple copies of both. More specifically, the conjugate compounds are MPX i -P j -M kThe polymeric structure may include the structure, where M and P are as described above, X is a cross-linking agent, i is an integer selected from 0 or 1, j is an integer selected from 0, 1, 2, 3, 4, or 5, and k is an integer selected from 0, 1, 2, 3, 4, or 5. In such polymeric structures, when i=0, j and k are also typically 0. Furthermore, k may be equal to or less than j. Also, in such polymeric structures, M may be linked to P as described above (i.e., either directly or using a chelator and / or spacer).

[0066] A specific example of such a multimeric compound is MPXP. j -M k wherein M and P are as defined above, X is a crosslinker, j is 1, and k is 0.

[0067] The crosslinker X can be any chemical crosslinking group compatible with use in the pharmaceutical or veterinary field. The group is preferably biologically and non-toxic. The size of the crosslinker can be adjusted by the skilled artisan. Preferred examples of X groups are polylysine or polyglutamic acid platforms (linear, cyclic or branched blocks) or polyfunctional organic compounds such as PEG(3)-Pentrimer-G1-(NH2+4xN3)*4HCl, which contains one amino and four azide functional groups. In certain embodiments, the crosslinker contains at least two, preferably at least three, reactive functional groups, allowing the attachment of at least two peptides. Examples of reactive functional groups include, for example, amine, acid, thiol, azide, alkyne, carbonyl, or hydrazine. The peptide can be attached to X either directly by covalent bond at the reactive functional group of the drug or via a spacer, which may consist of, for example, glycine or a series of glycines, PEG molecules, or aminohexanoic acid.

[0068] A specific example of such a multimeric compound of the invention is the compound VHd:DOTA-PEG2-E(PEG2-SEQ ID NO:1)-PEG2-E(PEG2-SEQ ID NO:1)-NH2 as shown in FIG.

[0069] The present invention also relates to any one of the above-mentioned conjugate compounds without the marker group M. Such compounds are intermediates in the preparation of the final labeled conjugate compounds. Indeed, as shown in the examples, the conjugates are first prepared and then labeled, usually before administration. Thus, the present invention relates to any one of the above-mentioned conjugate compounds without the marker group M. The present invention also relates to conjugates, particularly C-(S-) n With respect to compounds of structure P, wherein C, S, P and n are linked as defined above, as well as variants thereof and / or multimers thereof further comprising a T group as defined above.

[0070] The present invention also relates to a pharmaceutical composition comprising at least one conjugate compound as defined above. The composition may comprise one or more pharma- ceutically acceptable excipients or diluents.

[0071] The present invention also relates to a diagnostic composition, characterized in that it comprises a diagnostic or medical imaging agent composed of a conjugate compound as defined above.

[0072] [Use in imaging and diagnostics] The conjugates of the invention can be used to label, detect, or diagnose LDLR-expressing cancers in vivo in any mammalian subject, particularly human subjects.

[0073] In this regard, in certain embodiments, M is a radionuclide or fluorescent dye for use in imaging, and the method of labeling and / or detection includes: a) administering a conjugate compound to a subject; b) performing an imaging method; c) analyzing the signal obtained in step b), The presence of the signal indicates the presence of cancer cells and / or the staging of the progression of cancer in the subject.

[0074] The conjugates can be administered by various routes. Preferably, the conjugates are injected by systemic, parenteral or local injection. In particular, the injection can be intravenous, subcutaneous, intramuscular, intraarterial or intratumoral. In a particular embodiment, the conjugate compounds of the present invention are injected intravenously. Such an administration mode allows for proper diffusion of the compound in the body and access to the target tissue. In another embodiment, the administration is performed by intratumoral injection. Such a mode is appropriate when the presence of a tumor is known or suspected, and the method is used, for example, to stage the tumor, evaluate its progression or the effectiveness of a treatment.

[0075] The amount of compound administered can be adjusted by one skilled in the art depending on the purpose (imaging, detection, monitoring) and subject of the method.

[0076] The compounds of the invention can be used in various imaging methods known per se in the art, such as, but not limited to, scintigraphy, positron emission tomography (PET), positron emission tomography-computed tomography (PET-CT), or single photon emission scintigraphy. and computed tomography (SPECT). The preferred method is PET.

[0077] The obtained signal can be a quantitative marker value, an image, a distribution curve, a number of positron annihilation events, etc., depending on the method. In step c), such a signal is analyzed to determine the presence, staging, or progression of cancer in the subject. The term "analysis" refers to any method that allows the determination of whether a signal corresponds to a normal signal. The analysis can involve not only visual but also quantitative analysis. The analysis can include a step of comparing the signal value obtained by imaging with the signal value of known healthy tissue in the same or other subjects. The signal value can also be compared with a reference value.

[0078] A value obtained in step b) that is greater than the reference value or a control from healthy tissue indicates the presence of cancer cells. This can be assessed by comparing the signals obtained after two separate imaging sessions spaced apart.

[0079] The conjugate compounds of the present invention are particularly adapted for labeling and / or detection of cancer cells that overexpress LDLR. In this regard, the term "cells that overexpress LDLR" refers to cells that express at least 10% more LDLR than the standard level of expression. Typically, 1000-3000 LDLRs are present on the surface of "normal" cells. LDLR-overexpressing cells are cells that express at least 20% more LDLR, more specifically at least 50%, 75%, 100%, or 150% more LDLR.

[0080] Specific examples of LDLR-overexpressing cancers that can be detected using the present methods include pancreatic cancer, adrenal cancer, glioblastoma, prostate cancer, colon cancer, liver cancer, pancreatic cancer, ovarian cancer, lung cancer, or gastric cancer.

[0081] [Radiation therapy] In a further embodiment, the present invention also relates to the use of the conjugate compounds of the present invention for treating cancer by radiation therapy.

[0082] The terms "treatment," "treating," "treat," and other similar phrases refer to obtaining a pharmacological and / or physiological effect, such as inhibiting cancer cell growth or promoting cancer cell death.

[0083] In this regard, the method of treatment includes administering to a subject in need of such treatment a conjugate compound as defined above.

[0084] Preferably, the conjugate compound comprises a radionuclide suitable for radiotherapy, such as a beta-, or high energy gamma-emitting radionuclide, preferably with a longer half-life (e.g., 1 day to 75 days). Preferred radionuclides for use in radiotherapy include: 90 Y, 111 In, 131 I, and 177 In a preferred embodiment, 90 Y, 177 Lu, or 111 In is used.

[0085] The conjugate can be administered by various routes. Preferably, the conjugate is injected by systemic, parenteral or local injection. In particular, the injection can be intravenous, subcutaneous, intramuscular, intraarterial or intratumoral. In a particular embodiment, the conjugate compound of the present invention is injected intravenously. Such an administration mode allows for proper diffusion of the compound in the body and delivery to the target tissue. In another embodiment, administration is performed by intratumoral injection.

[0086] The compound must be administered in an amount sufficient to irradiate the tumor, and such an amount can be adjusted by one skilled in the art.

[0087] Treatment can be performed alone or in combination (eg, alternating or concomitantly) with other cancer treatments, such as chemotherapy.

[0088] The method can be used to treat any LDLR-overexpressing cancer, such as pancreatic cancer, adrenal cancer, glioblastoma, prostate cancer, colon cancer, liver cancer, pancreatic cancer, ovarian cancer, lung cancer, or gastric cancer. The method can be used at any stage of cancer development in any mammalian subject, particularly a human subject. Usually, multiple sequential treatment regimens are administered. In therapeutic use, the conjugate compound of the present invention can be in the form of any pharma- ceutically acceptable salt. The term "pharmaceutically acceptable salt" refers, for example and without limitation, to any pharma- ceutically acceptable salt. It refers to pharma- ceutically acceptable base or acid addition salts, hydrates, esters, solvates, precursors, metabolites, or stereoisomers. The term "pharma-ceutically acceptable salts" refers to non-toxic salts that can generally be prepared by reacting a free base with a suitable organic or inorganic acid. These salts retain the biological effectiveness and properties of the free base. Representative examples of such salts include water soluble and water insoluble salts, such as acetate, N-methylglucamine ammonium, amsonate (4,4-diaminostilbene-2,2'-disulfonate), benzenesulfonate, benzoate, bicarbonate, bisulfate, bitartrate, borate, hydrobromide, bromide, butyrate, camsylate, carbonate, hydrochloride, chloride, citrate, clavulanate, dichlorohydrate, diphosphate, edetate, calcium edetate, edisylate, estolate, esylate, fumarate, gluceptate, gluconate, glutamate, glycolylarsanilate, hexafluorophosphate, hexylresorcinate, hydrabamine, hydroxynaphthoate, iodide, isothioate, Salts include, but are not limited to, lactate, lactobionate, laurate, malate, maleate, mandelate, mesylate, methyl bromide, methyl nitrate, methyl sulfate, mucate, napsylate, nitrate, 3-hydroxy-2-naphthoate, oleate, oxalate, palmitate, pamoate (1,1-methylene-bis-2-hydroxy-3-naphthoate, or embonate), pantothenate, phosphate, picrate, polygalacturonate, propionate, p-toluenesulfonate, salicylate, stearate, subacetate, succinate, sulfate, sulfosalicylate, suramate, tannate, tartrate, teoclate, tosylate, triethiodide, trifluoroacetate, and valerate.

[0089] The conjugate compound can also be formulated with any suitable pharmaceutical excipient, carrier or diluent. In this regard, the present invention also relates to a composition comprising a compound as defined above and a pharma-ceutically acceptable carrier or excipient. The pharma-ceutically acceptable carrier can be selected from carriers conventionally used for each administration mode. Depending on the administration mode considered, the compound can be in solid, semi-solid or liquid form. For solid compositions such as tablets, pills, powders or granules loose or contained in gelatin capsules, the active substance can be combined with a) diluents such as lactose, dextrose, sucrose, mannitol, sorbitol, cellulose and / or glycine, b) lubricants such as silica, talc, stearic acid, its magnesium or calcium salts, and / or polyethylene glycol, c) binders such as magnesium and aluminum silicate, starch paste, gelatin, tragacanth gum, methylcellulose, sodium carboxymethylcellulose and / or polyvinylpyrrolidone, d) disintegrants such as starch, agar, alginic acid or its sodium salt, or effervescent mixtures, and / or e) adsorbents, dyes, flavors and sweeteners. Excipients can be, for example, mannitol, lactose, starch, magnesium stearate, sodium saccharin, talc, cellulose, glucose, sucrose, magnesium carbonate and pharmaceutical grade analogues. For semi-solid compositions, e.g., suppositories, the excipients may be, for example, emulsions or oily suspensions, or polyalkylene glycols, e.g., based on polypropylene glycol.Liquid compositions, particularly those for injection or inclusion in soft capsules, may be prepared, for example, by dissolving, dispersing, etc., the active substance in a pharma- ceutically pure solvent, such as water, saline, aqueous dextrose, glycerol, ethanol, oils, and the like.

[0090] The compositions or conjugates of the invention can be administered by any suitable route, for example by parenteral route, such as in the form of a formulation that can be injected via intravenous, infusion, subcutaneous or intramuscular routes, by oral route (or per os), such as in the form of coated or uncoated tablets, gelatin capsules, powders, pellets, suspensions or oral solutions (one such oral dosage form may be with immediate release or with extended or delayed release), by rectal route, such as in the form of suppositories, for example patches, po They can be administered by a topical route, such as in the form of a gel or ointment, in particular by a transdermal route, by an intranasal route, for example in the form of an aerosol and spray, by a lingual route, or by an intraocular route, but this is not limited to these routes.

[0091] The present invention also relates to a method for preparing or producing a conjugate compound as defined above, comprising the step of binding a marker M to a peptide P, preferably using a chelating agent C.

[0092] The present invention also relates to a method of making a pharmaceutical composition comprising the steps of providing a conjugate compound as defined above and formulating said compound with a suitable excipient or diluent.

[0093] Other aspects and advantages of the present invention will become apparent in light of the following examples, which are merely illustrative in nature and are not intended to limit the scope of the present application. EXAMPLES

[0094] Example 1: LDL-receptor (LDLR) expression in mouse and human tissues To assess membrane expression of LDLR in mouse and human cell lines of interest, membrane extracts from human and mouse cancer cell lines were prepared using the ProteoExtract Subcellular Proteome Extraction kit (Calbiochem, LaJolla, CA, USA).

[0095] Membrane extracts were quantified using the BioRad DC Protein assay (Bio-Rad, Hercules, CA, USA) according to the manufacturer's instructions. The cytoplasmic proteins were separated on a 4-12% polyacrylamide gel by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a nitrocellulose membrane (Amersham Biosciences). Then, peroxidase-conjugated donkey anti-goat secondary antibody (Jackson Immunoresearch) was used. The membrane was probed. Finally, the proteins were detected using chemiluminescence.

[0096] As shown in Figure 1A, LDLR expression is enhanced in human cancer cells, including adrenal carcinoma (NCI-H295R), glioblastoma (U87MG), breast cancer (MDA-MB-231), and prostate cancer (PC3). (Modified Chinese hamster ovary cells stably expressing hLDLR fused to GFP (hLDLR-GFP)) are used as a positive control. In human pancreatic cancer cells (Capan, BxPC3 and Panc1), LDLR levels are variable. In other cell types, such as Hela (cervical cancer) or MCF7 breast cancer, LDLR expression levels are significantly lower and barely detectable. LDLR expression is increased in the mouse pancreatic cell line PK4A (derived from a mouse that developed spontaneous tumors in the pancreas) compared to normal pancreatic tissue.

[0097] Example 2: Synthesis of fluorescent LDLR-targeted conjugates, Conjugate A, Conjugate B, Conjugate C, and Conjugate D In the following examples, we disclose the production and use of conjugates A, B, C and D. Conjugates A, B, C and D comprise peptide A (SEQ ID NO:1), peptide B (SEQ ID NO:13), peptide C (SEQ ID NO:6) and peptide D (SEQ ID NO:14), respectively, as shown below. [Table 1]

[0098] [Conjugate C and Conjugate D fusion protein production] Peptide C and peptide D were cloned in fusion with the Fc fragment of IgG1.

[0099] To generate Fc fragments fused to peptide C or peptide D, a plasmid pINFUSE hIgG1-Fc2 (InvivoGen) was used as a template. Plasmid constructs were constructed using the following primers: A megaprimer, designated Primer C or Primer D, was synthesized by PCR using the oligonucleotides: -Forward primer CTTGGCATTATGCACCTCCA (SEQ ID NO: 10) - reverse primer containing the sequence encoding peptide C CTGGCCAGCTAGCACTCAGCAACCGCGAAGACGAGGCATACAAGCACCTTTACCCGGAGACAGGGAG (sequence number 11) - reverse primer containing the sequence encoding peptide D CTGGCCAGCTAGCACTCGCAGGGTCTGCCCAGCATTCTGCAAGCACCTTTACCCGGAGACAGGGAG (SEQ ID NO: 12)

[0100] The product of the PCR reaction was purified, digested with DpnI (an enzyme that digests parental methylated DNA) and amplified using the QuickChange II Site Directed Mutagenesis kit (Agilent) to generate pINFUSE hIgG1- It was used as megaprimer in the second PCR reaction performed with Fc2 plasmid. After transformation of competent bacteria, isolated colonies were obtained, plasmid DNA was prepared, and the cDNA constructs were sequenced in both strands for confirmation. Vectors designated pConjugate-C and pConjugate-D express Fc fragments fused at the C-terminus with peptide C and peptide D after transfection of mammalian cells. The Expi293 expression system (Thermo Fischer) was used for the transient expression of the fusion proteins Conjugate C and Conjugate D in the culture supernatant. 72 hours after transfection, the supernatants were harvested and purified using the Montage antibody protein A PROSEP A kit (Millipore) according to the manufacturer's recommendations.

[0101] [Conjugate A and Conjugate B Protein Synthesis] Conjugation of conjugate A and conjugate B to human IgG1 Fc fragment (Merck Millipore) was carried out in a two-step procedure using the heterobifunctional spacer First, the Fc fragment was reacted with sulfo-SMCC to obtain a reactive protein for thiol moieties, and in the second step, the thiol-functionalized peptide A or B was ligated to the Fc fragment. The antibody was conjugated to a gin-binding Fc-SMCC protein.

[0102] [Synthesis of thiol-functionalized peptides] The amino acid sequences of peptide A and peptide B are cMThzRLRGPen (SEQ ID NO: 1) and cRPLGRMC (SEQ ID NO: 13), respectively, where "Thz" represents thiazolidine and "Pen" represents penicillamine.

[0103] N-α-Fmoc-protected amino acids were chosen for standard orthogonal side chain protection: Fmoc-Cys(Trt)-OH (D or L configuration), Fmoc-Pen(Trt)-OH, Fmoc-Met-OH, Fmoc-Pro-OH, Fmoc-Thz-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Leu-OH, and Fmoc-Gly-OH. All of these were cleaved with piperidine, trifluoroacetic acid (TFA), diisopropylethylamine (DIEA), or ethyl amine (EI). , ethanedithiol (EDT), triisopropylsilane (TIS), benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBop), 1 -[Bis(dimethylamino)methylene]-1H-1,2,3,-triazolo[4,5-β]pyridinium 3-oxide hexafluorophosphate (HATU), along with 2-tritylthio-1-ethylamine hydrochloride (Trt-cysteamine) were purchased from Iris Biotech.

[0104] Dimethylformamide (DMF), propionic anhydride, AcOH, K3[Fe(CN)6 ], ammonium carbonate, and dichloromethane (DCM) were from Sigma-Aldrich. I purchased it.

[0105] Both Pr-peptide AG-CH2-CH2-SH and Pr-peptide BG-CH2-CH2-SH were synthesized by solid-phase peptide synthesis (SPPS) using the Fmoc / tBu method and Fmoc-Gly-Wang resin (100-200 mesh, 1% DVB, 0.7 mmol / g loading) purchased from Iris Biotech. Such a resin allows the synthesis of peptides that are fully deprotected at their side chains and have a free carboxylic acid at the C-terminus. A Gly residue was inserted at the C-terminal position to functionalize while avoiding racemization.

[0106] The cMThzRLRGPen and cRPLGRMC sequences were analyzed using Liberty (CEM) Peptide synthesis was carried out using a microwave synthesizer.

[0107] In the automated synthesis, amino acids were coupled by microwave activation of the acidic functional group of the n+1 amino acid using 4 / 4 / 8 equivalents of aa / DIEA / HATU (relative to resin) in a 0.25 mmol scale synthesis. The coupling time was adjusted to 10 min. Amino acids introduced after a thiazolidine or proline residue require double coupling. Deprotection of the Fmoc group of the new amino acid thus coupled was performed using 20% ​​piperidine in DMF. The last amino acid coupled during peptide elongation was deprotected and N-propionylated using propionic anhydride (2*5 min, Pr2O / DCM:1 / 1).

[0108] Then, the solution was incubated at room temperature (RT) for at least 2 h in a solution containing TFA / TIS / H2O / EDT (94 / 2 / 2 / 2). The resin-bound peptide was cleaved using 100% ethyl acetate. A minimum of 15 mL of cleavage solution was used per gram of resin. The crude peptide was then precipitated using ice-cold ether, centrifuged at 3000 rpm for 8 min, and lyophilized in H2O / 0.1% TFA. A white solid was obtained and used in the cyclization step without further purification.

[0109] Disulfide bridges were obtained by intramolecular cyclization from two appropriately protected Cys or Pen thiol functions in either the L or D configuration. zRLRGPen-G-OH and Pr-cRPLGRMC-G-OH were dissolved in 0.5% AcOH to obtain a final concentration of 0.5 mg / mL. Ammonium carbonate (2 N) was added to the peptide solution to reach a basic pH of approximately 8-9. Then, K3[Fe(CN)6] (0.01 N) was added to the reaction mixture until a bright and constant yellow color was observed. Analytical RP-HP Reactions were monitored by LC. Reactions were typically quantifiable in less than 30 min. The reaction mixture was filtered through a 0.45 μm membrane and purified by preparative RP-HPLC. Fractions with purity greater than 95% were collected and lyophilized to give a pure white powder (final purity >95%). Homogeneity and identity of the pure synthetic peptides were assessed by analytical RP-HPLC. Peptides were fully confirmed for identity by ESI mass spectrometry on an LCQ Fleet (ThermoFisher) used in positive mode.

[0110] i) activation of the first Gly residue with PyBop / DIEA in DMF and reaction with 2-tritylthio-1-ethylamine hydrochloride (Trt-cysteamine), and ii) removal of the cysteamine trityl protection in acidic conditions (DCM / TIS / TFA: 3 / 1 / 1) afforded Pr Thiol functionalization of Pr-peptide BG-OH and Pr-peptide AG-OH was performed at their C-termini using cysteamine.

[0111] [Chemical conjugation of Pr-peptide AG-CH2-CH2-SH and Pr-peptide BG-CH2-CH2-SH to Fc fragment] In the first step, peptide A and peptide B were functionalized with a sulfo-SMCC spacer. Peptide A-SMCC and peptide B-SMCC were reacted with the primary amines of human Fc fragment (AG 714-Millipore) using a molar excess of 25 peptides per Fc. The reaction was incubated in PBS buffer at room temperature (RT) for 1 h. To remove excess peptide, the mixture was purified on a Pierce® dextran desalting column (Pierce). Fc-peptide A and Fc-peptide B conjugate concentrations were measured using an anti-Fc ELISA assay.

[0112] [Chemical synthesis of conjugates A, B, C and D] The fluorophore Alexa680 (A680 excitation: 679 nm; emission: 702 nm) was added to Fc-peptide A To conjugate Fc-peptide B, Fc-peptide C, and Fc-peptide D, the SAIVI Rapid Antibody Labeling Kit (S30045-ThermoFicher) was used. The kits are designed to label antibodies with an optimal degree of labeling (DOL, dye-to-protein ratio) for in vivo imaging applications (a DOL of approximately 2). The DOL of the conjugate (see Table 2) can be measured by absorption spectroscopy using the Beer-Lambert law: absorbance (A) = absorption coefficient (ε) × molar concentration × path length (d). The UV-VIS spectrum of the conjugate solution was measured. By measuring the absorbance at 280 nm (Amax) and 280 nm (protein absorption maximum) (A280), the concentration of bound dye is obtained by c(dye) = Amax / εmax x d, where εmax is the absorption coefficient of the dye at the absorption maximum, and from its absorbance at 280 nm the protein concentration is obtained by c(protein) = Aprot / εprot x d, where εprot is the absorption coefficient of the protein at 280 nm. The DOL is then calculated by DOL = [Amax / εmax] / [Aprot / εprot]. [Table 2] Table 2 shows the DOL of the conjugates. The concentrations of conjugates A, B, C, and D were measured using an anti-Fc ELISA assay.

[0113] Example 3: Affinity of conjugates A, B, C, and D for LDLR (K D Surface plasmon resonance (SPR) measurement of the conjugate binding to h / mL DLR. Doxocytosis properties are stably expressed by CHO cells and cancer cell lines. [Affinity of the conjugate for LDLR (K D )] Recombinant human LDLR (His-tagged) was purchased from SinoBiological (Beijing, China). The interaction of the conjugate with LDLR was examined using a BiacoreT200 (GE Healthcare) and 50 mM HEPES-NaOH ( The test was performed at 25°C using a 100-μM buffer (pH 7.4), 150 mM NaCl, 0.005% Tween-20, and 50 μM EDTA. hLDLR was measured at 30–60 fmol / mm 2The conjugates were immobilized on a NiHC sensor chip (Xantec, Dusseldorf, Germany) at a density of 1000 uL / min. Binding of the conjugates to the LDLR-coated flow cell was corrected for nonspecific binding to the uncoated flow cell. The affinity of the ligands for the LDLR was measured using a single cycle kinetic method. The ligands were diluted in running buffer and injected in increasing concentrations at 30 μl / min for 2 min. The running conditions were similar before the ligand injection. A blank test injection of buffer was performed. Double subtraction sensorgrams were globally fitted to a 1:1 Langmuir binding model in BiacoreT200 Evaluation version 2.0. D are summarized in Table 3 below. [Table 3] Table 3 shows the affinity of conjugates A, B, C, and D for human LDLR.

[0114] [Binding and endocytosis of fluorescent conjugates by LDLR-expressing cells] To assess the ability of conjugates with affinity for h / mLDLR-GFP (conjugates A and C) and control conjugates (conjugates B and D) to be endocytosed by the LDLR, immunocytochemistry experiments were performed involving incubation of all conjugates in live hLDLR-GFP cells for 1 h at 37°C. Afterwards, confocal microscopy analysis was performed.

[0115] In these experiments, with reference to FIG. 2, the LDLR-GFP signal is visualized in column 2, and the conjugate signal is visualized directly using A680 fluorescence in column 4 or using anti-hFc-A594 conjugate secondary antibody in column 3. Cell nuclei are stained with Hoechst blue and the signal can be visualized in column 1. Co-labeling of LDLR-GFP and conjugate can be visualized as highlighted signal in column 5 in the merged image. The results show that conjugates A and C bind and are endocytosed by CHO-hLDLR-GFP cells, whereas control conjugates B and D are not.

[0116] Example 4: ELISA quantification of distribution of LDLR-targeted conjugates injected intravenously into naive or tumor-bearing mice Mouse pancreatic adenocarcinoma PK4A cells were obtained from S. Vasseur (Inserm U1068, Cellular Stress) and have been previously described (Guillaumond F et al., 2013 Proc NatlAcad Sci USA 110(10):3919-3924). Cells were cultured in 100 mL of penicillin (100 U / mL), streptomycin (100 U / mL), and 100 mL of ethanol. Dalbedo supplemented with isocytose (100 μg / mL) and fetal bovine serum (10% Gold Serum, InVitrogen). The cells were cultured in DMEM (DMEM; Invitrogen, Carlsbad, CA) at 37 °C in a 5% CO The cells were used for tumor induction. PK4A tumor xenografts were induced in Hsd at 4 weeks of age. Male athymic nude-Foxn1nu nude mice were purchased from Envigo (Harlan, Indianapolis, IN) and incubated in 150 μL of complete medium. 1 x 10 suspended in the ground 6 Cells were obtained by subcutaneous injection between the shoulders. Tumor size was assessed visually every other day, and animals were used for in vivo imaging experiments. PK4A subcutaneous tumor volumes were approximately 700–1500 mm 3Once tumors reached a tumor size (10–14 days after implantation), 5 nmol of conjugate was injected into the tail vein of tumor-bearing mice. After 2 h for conjugates A and B and 4 h for conjugates C and D, whole blood was collected into heparinized tubes (Sigma Aldrich) and plasma was isolated after centrifugation at 5000 g for 15 min. Mice were then perfused with PBS 1x for 10 min at a rate of 2 ml / min. Organs were removed, weighed, and resuspended in PBS containing protease inhibitor cocktail (Sigma Aldrich). S / 0.1% Triton (Sigma Aldrich). Organ homogenates were After freezing at 80°C for 12 hours and sonication 3x10s, tissue lysates were clarified by centrifugation at 20000g for 15 minutes. Fc concentrations of isolated supernatants were measured by anti-Fc ELISA. Results shown in Figure 3 show 40-fold accumulation of conjugate A and 30-fold accumulation of conjugate C in pancreatic tumor versus pancreas. Kidney accumulation is shown as a control.

[0117] Example 5: Biodistribution of Conjugate C and its scrambled counterpart, Conjugate D, in pancreatic tumors and healthy pancreas. Tumor-bearing mice were intravenously injected with 5 nmol of conjugate C along with conjugate D as a control (n=5). Fluorescence whole-body imaging was monitored at 15, 30, 60, and 120 min after intravenous injection. At the end of the experiment (4 h after intravenous injection of the molecules), pancreas, liver, and tumor tissues were harvested and fluorescence was measured ex vivo. Figure 4 shows that conjugate C significantly accumulates in pancreatic tumors compared to scrambled conjugate D. Moreover, this accumulation was specific to pancreatic tumors, since no accumulation was observed in healthy pancreatic tissues.

[0118] Example 6: Synthesis of NODAGA-SEQ ID NO:1-NH2, DOTA-SEQ ID NO:1-NH2, NODAGA-βAla-PEG12-SEQ ID NO:1-NH2, DOTA-βAla-PEG12-SEQ ID NO:1-NH2, and their respective scrambled controls NODAGA-βAla-PEG12-SEQ ID NO:13-NH2, and DOTA-βAla-PEG12-SEQ ID NO:13NH2 In the following examples, 68 Ga-CH44 is 68 Used as an abbreviation for Ga-NODAGA-βAla-PEG12-SEQ ID NO:1-NH2 、 68 The Ga-FG770 is 68 used as an abbreviation for Ga-DOTA-βAla-PEG12-SEQ ID NO:1-NH2, 68 Ga-CH40 is 68 Used as an abbreviation for Ga-NODAGA-βAla-PEG12-SEQ ID NO:13-NH2 、 68 The Ga-FG769 is 68 Used as an abbreviation for Ga-DOTA-βAla-PEG12-SEQ ID NO:13-NH2. [Table 4] Two types of chelating cages were conjugated to the LDLR-targeting peptide vector. -NODAGA (1,4,7-triazacyclononane-1-glutaric acid-4,7-diacetic acid) allows the synthesis of radiolabeled conjugates for imaging. -DOTA (tetraazacyclododecane-1,4,7,10-tetraacetic acid) and radiation therapy The possibility of radiolabeling was introduced for the preparation of radiolabeled conjugates.

[0119] The macrocycles NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid) and NODAGA are commonly used radiotracers. 68It is the most preferred ligand for gallium chelation. Among these ligands, NODAGA was selected because its chelating cage contains an additional binding moiety compared to NOTA. This ensures that all of the carboxy arms are available to saturate the hexadentate.

[0120] In radiation therapy, 111 indium, 177 Lu, or 90 Yttrium is used in clinical trials. Such radiometals are well suited to the DOTA macrocycle chelator, which forms thermodynamically and kinetically stable complexes with them. In fact, DOTA offers eight donor atoms and a suitable cavity size to form more stable complexes with these radionuclides.

[0121] Derivatives of NODAGA and DOTA were prepared to develop imaging agents and receptor-mediated radiotherapy that can preferentially target LDLR-expressing cancers.

[0122] To evaluate the position and distance of the macrocyclic chelator conjugated to the peptide vector, various constructs were designed and prepared. The chelator was conjugated to the C-terminus, N-terminus, or to the amino side chain of a lysine that was further introduced into the peptide vector sequence. All derivatives consistently bound to the target LDL receptor as assessed by SPR analysis in purified LDLR. Thus, the construct with the chelator at the N-terminus was selected because this synthetic route is more amenable to setup.

[0123] 5 shows a reaction scheme for the synthesis of a cage-linker-peptide-NH2 conjugate. In this scheme, an example of the synthesis of NODAGA-βAla-PEG12-SEQ ID NO:1-NH2 is shown.

[0124] The reagents and conditions for the synthesis reaction are as follows with reference to FIG. Step a: AA (4 equivalents (eq)), HATU (4 eq), DIEA (8 eq), DMF , microwave active, - Step b: Fmoc-PEG12-OH (1.5eq), DIC (4eq), HOBt (2eq), DMF, room temperature (RT), 24 hours, -Step c: Fmoc-(β)Ala-OH(3eq), COMU(3eq), DIEA(8eq), DMF, RT, 3 hours, - Step d: DMF / piperidine (20%) 15 min, TFA / TIS / HO / EDT (94 / 2 / 2 / 2), RT, 2 h, - Step e: AcOH (0.5%), (NH4)2CO3, K3Fe(CN)6, pH = 7-8, [peptide] = 1 mg / mL, Step f: NODAGA-NHS (2 eq), DIEA (10 eq), DMF, RT, 3 0 minutes.

[0125] Analysis and purification methods - Reaction progress and purity were monitored using a Thermo Fisher UltiMate 3000 system equipped with a C18 KinetexTM (5 μm, 150 mm x 4.6 mm). Detection was performed at 214 nm. The elution system consisted of H2O / 0.1% TFA (solution A) and MeCN / 0.1% TFA (solution B). The flow rate was 2 mL / min, and a gradient of 0 to 100% of solution B was performed in 4 min. - The crude product was purified using a Thermo Purification was performed by RP-HPLC on a Fisher UltiMate 3000 system. Detection was performed at 214 nm. The elution system consisted of H2O / 0.1% TFA (solution A) and MeCN / 0.1% TFA (solution B). The flow rate was 20 mL / min.

[0126] [Synthesis of H-SEQ ID NO:1-NH2, H-βAla-PEG12-SEQ ID NO:1-NH2, and H-βAla-PEG12-SEQ ID NO:13-NH2] N-α-Fmoc-protected amino acids were chosen for standard orthogonal side chain protection: Fmoc-Cys(Trt)-OH (D or L configuration), Fmoc-Pen(Trt)-OH, Fmoc-Met-OH, Fmoc-Pro-OH, Fmoc-Thz-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Leu-OH, Fmoc-βALa-OH, and Fmoc-Gly-OH. All of these were cleaved with piperidine, trifluoroacetic acid (TFA), diisopropyl ether, and ethyl acetate (ETA). Iris Biotech Co., Ltd., together with propylethylamine (DIEA), ethanedithiol (EDT), triisopropylsilane (TIS), 1-cyano-2-ethoxy-2-oxoethylideneaminooxy)dimethylamino-morpholino-carbenium hexafluorophosphate (COMU), 1-[bis(dimethylamino)methylene]-1H-1,2,3,-triazolo[4,5-β]pyridinium 3-oxide hexafluorophosphate (HATU), N,N´-diisopropylcarbodiimide (DIC), and 1-hydroxybenzotriazole, Purchased from ch.

[0127] Dimethylformamide (DMF) and dichloromethane (DCM) were purchased from Sigma-Aldrich.

[0128] Fmoc-21-amino-4,7,10,13,16,19-hexaoxaheneicosanoic acid (Fmoc-PEG12-OH) was purchased from PolyPeptide Laboratories. I entered.

[0129] The conjugates H-SEQ ID NO:1-NH2, H-βAla-PEG12-SEQ ID NO:1-NH2, and H-βAla-PEG12-SEQ ID NO:13-NH2 were synthesized by solid-phase peptide synthesis (SPPS) using the Fmoc / tBu method and Fmoc-Rink amide resin (100-200 mesh, 1% DVB, 0.7 mmol / g loading) purchased from Iris Biotech. Such resin allows for complete deprotection at the side chain and C-terminal This allows the synthesis of peptides containing amides.

[0130] Peptide synthesis was carried out on the cMThzRLRGPen (SEQ ID NO: 1) and cRPLGRMC (SEQ ID NO: 13) sequences using a Liberty (CEM) microwave synthesizer, while If so, the linkers Fmoc-βAla-OH and Fmoc-PEG12-OH were manually attached.

[0131] In the automated synthesis, amino acids were coupled by microwave activation of the acidic functional group of the n+1 amino acid using 4 / 4 / 8 equivalents of aa / DIEA / HATU (relative to resin) in a 0.25 mmol scale synthesis. The coupling time was adjusted to 10 min. Amino acids introduced after a thiazolidine or proline residue require double coupling. Deprotection of the Fmoc group of the new amino acid thus coupled was performed using 20% ​​piperidine in DMF. The last amino acid coupled during peptide elongation was deprotected to allow further coupling at the N-terminus. In sequences with a linker introduced between the cage and the peptide vector, Fmoc-PEG12-OH was manually introduced using 1.5 / 4 / 2 equivalents of aa / DIC / HOBt (relative to resin). The reaction was carried out overnight at room temperature. The coupling efficiency could be monitored by the TNBS test. After Fmoc deprotection with piperidine / DMF (20%, 3*5 min), Fmoc-βAla-OH was finally conjugated to the free N-terminus using 3 / 8 / 3 equiv. aa / DIEA / COMU for 3 h at room temperature. The coupling efficiency could be monitored by TNBS test, and Fmoc was removed with 20% piperidine in DMF (3*5 min).

[0132] The resin-bound peptide was then cleaved using a solution containing TFA / TIS / H2O / EDT 94 / 2 / 2 / 2 for at least 2 hours at room temperature (RT). A minimum of 15 mL of cleavage solution was used per gram of resin. The crude peptide was then precipitated using ice-cold ether, centrifuged at 3000 rpm for 8 minutes, and lyophilized in H2O / 0.1% TFA. A white solid was obtained and used in the next step without further purification.

[0133] [Cyclization of conjugates H-SEQ ID NO:1-NH2, H-βAla-PEG12-SEQ ID NO:1-NH2, and H-βAla-PEG12-SEQ ID NO:13-NH2] Disulfide bridges were obtained by intramolecular cyclization from two thiol functionalities on two appropriately protected Cys or Pen in either L or D configuration. AcOH, K3[Fe(CN)6], and ammonium carbonate were purchased from Sigma-Aldrich. Crude conjugates of H-SEQ ID NO:1-NH2, H-βAla-PEG12-SEQ ID NO:1-NH2, and H-βAla-PEG12-SEQ ID NO:13-NH2 were dissolved in AcOH 0.5% to obtain a final concentration of 0.5 mg / mL. Ammonium carbonate (2 N) was added to the peptide solution to reach a basic pH of approximately 8-9. Then, K3[Fe(CN)6] (0.01 N) was added to the reaction mixture until a bright and constant yellow color was observed. Analytical RP-HPL Reactions were monitored by HPLC. Reactions were typically quantifiable in less than 30 min. The reaction mixture was filtered through a 0.45 μm membrane and purified by preparative RP-HPLC. Fractions with purity greater than 95% were collected and lyophilized to give a pure white powder (final purity >95%). Homogeneity and identity of the pure synthetic peptides were assessed by analytical RP-HPLC. Peptides were fully confirmed for identity by ESI mass spectrometry on an LCQ Fleet (ThermoFisher) used in positive mode.

[0134] [NODAGA and DOTA conjugates to H-SEQ ID NO:1-NH2, H-βAla-PEG12-SEQ ID NO:1-NH2, and H-βAla-PEG12-SEQ ID NO:13-NH2] Activated esters of NODAGA mono-N-hydroxysuccinimide (NODAGA-NHS) and DOTA mono-N-hydroxysuccinimide (DOTA-NHS) were used to generate chelators. was conjugated to the peptide.

[0135] NODAGA-NHS and DOTA-NHS were purchased from Chematech (Dijon, France). Obtained from.

[0136] Chelator (4 eq) and DIEA (10 eq) were added to a solution of peptide in DMF (1.2 mM). The reaction mixture was stirred at room temperature. The reaction was assessed to be quantitative by monitoring the reaction by analytical RP-HPLC. The reaction mixture was filtered through a 0.45 μm membrane and purified by preparative RP-HPLC. Fractions with purity greater than 95% were collected and lyophilized to give a pure white powder (final purity >95%). The homogeneity and identity of the pure synthetic peptide was assessed by analytical RP-HPLC. The peptide was fully confirmed for identity by ESI mass spectrometry on an LCQ Fleet (ThermoFisher) used in positive mode.

[0137] Example 7: Affinity of MG04 / VH-DO35, CH44, FG770, CH40, and FG769 for LDLR (K D Surface plasmon resonance (SPR) measurement of [Affinity of the conjugate for LDLR (K D )] Recombinant human LDLR (His-tagged) was purchased from SinoBiological (Beijing, China). The interaction of the conjugate with LDLR was examined using a BiacoreT200 (GE Healthcare) and 50 mM HEPES-NaOH ( The test was performed at 25°C using a 100-μM buffer (pH 7.4), 150 mM NaCl, 0.005% Tween-20, and 50 μM EDTA. hLDLR was measured at 30–60 fmol / mm 2 The conjugates were immobilized on a NiHC sensor chip (Xantec, Dusseldorf, Germany) at a density of 1000 uL / min. Binding of the conjugates to the LDLR-coated flow cell was corrected for nonspecific binding to the uncoated flow cell. The affinity of the ligands for the LDLR was measured using a single cycle kinetic method. The ligands were diluted in running buffer and injected in increasing concentrations at 30 μl / min for 2 min. The running conditions were similar before the ligand injection. A blank test injection of buffer was performed. Double subtraction sensorgrams were globally fitted to a 1:1 Langmuir binding model in BiacoreT200 Evaluation version 2.0. D are summarized in Table 5 below. [Table 5] Table 5 shows the affinity of conjugates CH40, FG769, CH44, FG770, and MG04 for LDLR.

[0138] Example 8: Subcutaneous mouse model of adrenal tumors 68 Ga-CH44 and its scrambled counterpart 68 PET imaging of Ga-CH40> The purpose of this example is to demonstrate the effect of the LDLR-targeting conjugate ( 68 Ga-CH44) and its scramble Control ( 68 The aim of this study was to use PET scans to assess the biodistribution of Ga-CH40 Ta. 18 F-FDG was used as a control.

[0139] 〔material〕 [Radiolabel] CH44 and CH40 68 Radiolabeling was performed using GaCl. 68 Ge / 68 Gallium is generated using a Ga generator (Obninsk). 68 Ga 3+ By form In 200 μL of ammonium acetate buffer (4 M, pH 6), at 25° C. for 15 min. 20 μg of NODAGA-CH44 and 40 μg of NODAGA-CH40 were added to 74–148 MBq (2–4 mCi) of 68 The radiolabeling reaction was carried out by reacting with Ga.

[0140] 68 Ga-CH44 and 68 The specific activities obtained for Ga-CH40 were 12.5 Bq / mmol and 5.5 Bq / mmol, respectively.

[0141] [Tumor implant] According to a protocol approved by the Aix-Marseille Ethics Committee (Comity 14), Animal studies were performed. Four-week-old male BALB / c nude mice were obtained from Charles River Inc. 150 μL of complete culture containing 50% Matrigel (Corning) was used. Mice were implanted subcutaneously in the upper flank with NCI-H295R cells in whole medium. Tumors were 700–1500 mm 3 Mice were used for imaging experiments when the amount contained in

[0142] [Route of administration, dose, and experimental design] Animals were dosed with 10 ± 4 MBq 68 Ga-CH44 or 68 Test substances were administered by a single bolus intravenously at a dose of Ga-CH40.

[0143] Six mice were implanted with NCI-H295R adrenal cancer cells. Fourteen days after implantation, the animals 18 F-FDG, 68 Ga-CH44 and 68 Ga-CH40 was administered intravenously at 24-h intervals. On the 32nd day after implantation, the animals 68 Ga-CH44, and after 24 hours 68 Ga-CH40 was administered intravenously. After each administration, the biodistribution in adrenal cancer xenografts and other tissues was assessed using PET imaging.

[0144] [PET scan] Groups of nude mice (n = 5–7) were treated with 10 ± 4 MBq 68Ga-CH44 was administered intravenously and PET / CT scans were obtained 1 h post-injection (pi). PET and PET / CT studies were performed on a microPET / microCT rodent model scanner (nanoPET / CT, Mediso). Anesthesia was induced with 5% isoflurane and maintained at 1.5%. To improve image quality, For each stationary PET emission scan (energy window: 400-600 keV, Time: 20 min per FOV) and 20 million coincidence events were acquired per mouse. For the dual-modality PET / CT, CT images (35 kVp, 350 ns exposure time, medium zoom) were acquired and analyzed. Anatomical registration and attenuation correction were applied to the corresponding PET scans. Six hours after the first injection, the same group of nude mice received 10 ± 4 MBq of 68 Ga-CH40 was administered and PET / CT scans were obtained 1 hour post-injection (pi). This series of acquisitions constituted the control group.

[0145] 〔result〕 [PET imaging] On day 14 for all three compounds, 68 Ga-CH44 and 68 Image acquisition was performed on Ga-CH40 on the 32nd day. On the 14th day, 18 Imaging of animals injected with F-FDG showed no significant accumulation at the tumor site (Figure 6A).

[0146] On the other hand, on days 14 (Figures 6B and 6C) and 32 (Figures 7A and 7B), the images were not significantly different in most animals. 68 Compared to Ga-CH40 68 Ga-CH4 4 showed significant accumulation.

[0147] [Conclusion] In the experiment, most animals showed 68 We demonstrated clear and selective imaging and labeling of adrenal cancer with Ga-CH44.

[0148] Example 9: Subcutaneous mouse model of adrenal tumors 68 Ga-FG770 and its scrambled counterpart 68 PET imaging of Ga-FG769> The purpose of this example is to demonstrate the effect of the LDLR-targeting conjugate ( 68 Ga-FG770) and its scrambled counterpart ( 68 The aim of this study was to use PET scans to assess the biodistribution of Ga-FG769. The cage used in this study was DOTA, not NODAGA.

[0149] 〔material〕 [Radiolabel] FG770 and FG769 68 Radiolabeling was performed using GaCl. 68 Ge / 68 Gallium is generated using a Ga generator (Obninsk). 68 Ga 3+ shape The solution was dissolved in 100 μL of ammonium acetate buffer (2 M, pH 4.5) at 95°C for 1 h. 20 μg of DOTA-FG770 and 20 μg of DOTA-FG769 were incubated at 74–148 MBq (2–4 mCi) for 5 min. 68 The radiolabeling reaction was carried out by reacting with Ga.

[0150] 68 Ga-FG770 and 68 The specific activities obtained for Ga-FG769 were 6.45 Bq / mmol and 6.16 Bq / mmol, respectively.

[0151] [Tumor implant] According to a protocol approved by the Aix-Marseille Ethics Committee (Comity 14), Animal studies were performed. Four-week-old male BALB / c nude mice were obtained from Charles River Inc. 150 μL of complete culture containing 50% Matrigel (Corning) was used. Mice were implanted subcutaneously in the upper flank with NCI-H295R cells in whole medium. Tumors were 700–1500 mm 3 Mice were used for imaging experiments when the amount contained in

[0152] [Route of administration, dose, and experimental design] Animals were dosed with 10 ± 4 MBq 68 Ga-FG770 or 68 Test substances were administered by a single bolus intravenously at the dose of Ga-FG769.

[0153] Six mice were implanted with NCI-H295R adrenal cancer cells. Thirty-seven days after implantation, the animals 68 Ga-FG770 and 68 Ga-FG769 was administered intravenously at 24-h intervals, respectively. After administration, the biodistribution in adrenal cancer xenografts and other tissues was evaluated using PET imaging.

[0154] [PET scan] A group of nude mice (n=3) received 10±4MBq 68 Ga-FG770 was administered intravenously and PET / CT scans were obtained 1 hour post-injection (pi). PET and PET / CT studies were performed on a microPET / microCT rodent model scanner (nanoPET / CT, Mediso). Anesthesia was induced with 5% isoflurane and maintained at 1.5%. Improving image quality In order to measure the PET emission rate, the During the study, 20 million coincidence events were acquired per mouse over 1 FOV (interval: 20 min). For dual-modality PET / CT, CT images (35 kVp, 350 ns exposure time, medium zoom) were acquired and anatomical registration and attenuation correction were applied to the corresponding PET scans.

[0155] Twenty-four hours after the first injection, the same group of nude mice was treated with 10 ± 4 MBq 68 Ga-FG769 was administered intravenously, and PET / CT scans were obtained at 1 and 4 hours post-injection (pi). Quantitative region of interest (ROI) analysis in PET images was performed using the Intercept This was performed on attenuation- and attenuation-corrected PET images using viewfusion.

[0156] 〔result〕 [PET imaging] 68 Ga-FG770 and 68 Imaging was performed on day 37 for both compounds, Ga-FG769. Imaging images were obtained in most animals. 68 Compared to Ga-FG769 68 The results showed a significant accumulation of Ga-FG770 (FIGS. 8A and 8B, respectively).

[0157] [Conclusion] In the experiment, most animals 68 We demonstrated clear and selective imaging and labeling of adrenal cancer with Ga-FG770.

[0158] Example 10: In a subcutaneous mouse model of pancreatic cancer 68 Ga-CH44 and its scrambled counterpart 68 PET imaging of Ga-CH40> The purpose of this example is to evaluate the activity of the LDLR-targeting conjugate ( 68 Ga-CH44) and its scramble Control ( 68 The aim of this study was to use PET scans to assess the biodistribution of Ga-CH40 Ta. 18 F-FDG was used as a control.

[0159] 〔material〕 [Radiolabel] CH44 and CH4068 Radiolabeling was performed using GaCl. 68 Ge / 68 Gallium is generated using a Ga generator (Obninsk). 68 Ga 3+ By form In 200 μL of ammonium acetate buffer (4 M, pH 6), at 25° C. for 15 min. 20μg of CH44 and 40μg of CH40 were mixed at 74-148MBq (2-4mCi) 6 8 The radiolabeling reaction was carried out by reacting with Ga.

[0160] 68 Ga-CH44 and 68 The specific activities obtained for Ga-CH40 were 12.5 Bq / mmol and 5.5 Bq / mmol, respectively.

[0161] [Tumor implant] According to a protocol approved by the Aix-Marseille Ethics Committee (Comity 14), Animal studies were performed. Four-week-old male BALB / c nude mice were obtained from Charles River Inc. Pk4a cells (1 × 10 6 ) was implanted subcutaneously between the shoulders of mice. 3 Mice were used for imaging experiments when the amount contained in

[0162] [Route of administration, dose, and experimental design] Animals were dosed with 10 ± 4 MBq 68 Ga-CH44 and 68 Test substances were administered by a single bolus intravenously at a dose of Ga-CH40.

[0163] Mice (n = 5–6) were implanted with Pk4a pancreatic cancer cells. Four days after implantation, the animals 18 F-FDG, 68 Ga-CH44 and 68Ga-CH40 was administered intravenously at 24-h intervals. On the 12th day after implantation, the animals 68 Ga-CH44, and after 24 hours 68 Ga-CH40 was administered intravenously.

[0164] After each dose, biodistribution in pancreatic cancer xenografts and other tissues was assessed using PET imaging.

[0165] [PET scan] Groups of nude mice (n = 5–7) were treated with 10 ± 4 MBq 68 Ga-CH44 was administered intravenously and PET / CT scans were obtained 1 h post-injection (pi). PET and PET / CT studies were performed on a microPET / microCT rodent model scanner (nanoPET / CT, Mediso). Anesthesia was induced with 5% isoflurane and maintained at 1.5%. To improve image quality, For each stationary PET emission scan (energy window: 400-600 keV, Time: 20 min per FOV) and 20 million coincidence events were acquired per mouse. For dual-modality PET / CT, CT images (35 kVp, 350 ns exposure time, medium zoom) were acquired and anatomical registration and attenuation correction were applied to the corresponding PET scans. Six hours after the first injection, the same group of nude mice received 10 ± 4 MBq of 68 Ga-CH40 was administered and PET / CT scans were obtained 1 hour post-injection (pi). This series of acquisitions constituted the control group.

[0166] 〔result〕 [PET imaging] 18 F-FDG, 68 Ga-CH44 and 68 On the fourth day, 68 Ga-CH44 and 68Imaging was performed on day 12 for Ga-CH40. Due to the small tumor size, no significant accumulation of the various compounds was observed on day 4 (Figures 9A and 9B, respectively). However, imaging images showed that most animals showed significant accumulation of the various compounds on day 12. 68 Compared to Ga-CH40 68 The results showed a significant accumulation of Ga-CH44 (Figures 10A and 10B, respectively).

[0167] [Conclusion] In the experiment, on the 12th day 68 We demonstrated clear and selective imaging and labeling of pancreatic cancer with Ga-CH44.

[0168] Example 11: In an orthotopic mouse model of glioblastoma 68 Ga-CH44 and its scrambled counterpart 68 PET imaging of Ga-CH40> The purpose of this example is to demonstrate the efficacy of LDLR-targeting conjugates ( 68 Ga-CH44) and its scrambled counterpart Lighting ( 68 The aim of this study was to evaluate the distribution of Ga-CH40 in the body using PET scans. , an integrin-specific marker that has been used in several clinical studies for glioblastoma. 68 A comparison was made on day 21 to Ga-RGD.

[0169] 〔material〕 [Radiolabel] CH44 and CH40 68 Radiolabeling was performed using GaCl. 68 Ge / 68 Gallium is generated using a Ga generator (Obninsk). 68 Ga 3+ By form In 200 μL of ammonium acetate buffer (4 M, pH 6), at 25° C. for 15 min. 20μg of CH44 and 40μg of CH40 were mixed at 74-148MBq (2-4mCi) 6 8 The radiolabeling reaction was carried out by reacting with Ga.

[0170] 68 Ga-CH44 and 68 The specific activities obtained for Ga-CH40 were 12.5 Bq / mmol and 5.5 Bq / mmol, respectively. 68 The specific radioactivity of Ga-RGD was 7.35 Bq / mmol.

[0171] [Tumor implant] According to a protocol approved by the Aix-Marseille Ethics Committee (Comity 14), Animal studies were performed. Six-week-old male BALB / c nude mice were obtained from Charles River Inc. Mice were inoculated with U87MG cells (5 × 10 5 ) were implanted in the striatum of the brain (1 mm anterior, 2 mm lateral to the bregma, 3 mm deep). Mice were used for imaging experiments on days 14 and 21 after injection, with expected volumes of 9 mm 3 and 53mm 3 It was.

[0172] [Route of administration, dose, and experimental design] Animals received 8±4MBq 68 Ga-CH44 and 68 Test substances were administered by a single bolus intravenously at a dose of Ga-CH40.

[0173] Mice (n=6) were implanted with U87MG glioblastoma cells. 14 days after implantation, the animals 68 Ga-CH44 and 68 Ga-CH40 was administered intravenously at 24-h intervals. On the 21st day after implantation, the animals 68 Ga-CH44, 68 Ga-CH40, 68Ga-RGD was administered intravenously at intervals of 24 and 72 hours, respectively.

[0174] After each dose, biodistribution in glioblastoma cancer and other tissues was assessed using PET imaging.

[0175] [PET scan] Nude mice (n=6) were treated with 8±4MBq 68 Ga-CH44 was administered intravenously and PET / CT scans were obtained 1 hour post-injection (pi). PET and PET / CT studies were performed using a microPET / microCT rodent model scanner (nanoPET / CT, Medico-Rad Laboratories, Inc., New York, NY, USA). Anesthesia was induced with 5% isoflurane and maintained at 1.5%. To improve image quality, every stationary PET emission scan was performed (energy window: 400-600 keV, time: 1 A total of 20 million coincidence events were acquired per mouse over a 20-minute period (20 min per FOV). For dual-modality PET / CT, CT images (35 kVp, 350 ns exposure time, medium zoom) were acquired and anatomical registration and attenuation correction were applied to the corresponding PET scans. Six hours after the first injection, the same group of nude mice received 8 ± 4 MBq of CT. 68 Ga-CH40 was administered and PET / CT scans were obtained 1 hour post-injection (pi). This series of acquisitions constituted the control group.

[0176] 〔result〕 [PET imaging] 68 Ga-CH44 and 68 On the 14th day, Ga-CH40 68 Ga-CH44, 68 Ga-CH40, and 68 Imaging was performed on day 21 for Ga-RGD. Imaging images were obtained on day 14 for 4 of 6 mice. 68 The signal of Ga-CH44 is shown. 68Ga-CH40 did not show any significant signal (Figures 11A and 11B, respectively). At day 21, all six mice 68 Ga-CH40 or 68 Significantly stronger than either weak signal of Ga-RGD 68 The tumor / control ratio analysis was performed to quantify the imaging signal. 68 Ga-CH44 68 Ga-CH40 or 68 It was shown that it accumulated significantly more than Ga-RGD (about 4-fold) (FIG. 12).

[0177] [Conclusion] In the experiment, on the 21st day 68 We demonstrate clear and selective imaging and labeling of glioblastoma cancer with Ga-CH44.

[0178] Example 12: Subcutaneous mouse model of adrenal cancer 68 Ga-CH44 and 68 Ga -PET imaging of MG04> The purpose of this example is to determine the efficacy and safety of 100% NODAGA-containing mice after intravenous administration of a xenograft model of adrenal cancer (xenograft model). 68 Ga-CH44), one is D Includes OTA cage and does not have spacer (S) ( 68 Ga-MG04) Targeting LDLR 2 The biodistribution of the two conjugates was assessed and compared using PET scans.

[0179] 〔material〕 [Radiolabel] CH44 and MG04 68 Radiolabeling was performed using GaCl. 68 Ge / 68 Gallium is generated using a Ga generator (Obninsk). 68 Ga 3+ By form In 200 μL of ammonium acetate buffer (4 M, pH 6), at 25° C. for 15 min. , 20 μg CH44 68 The radiolabeling reaction was carried out by reacting 20 μg of MG with Ga in 200 μL of ammonium acetate buffer (pH 4) at 100 °C for 10 min. 04 68 The radiolabeling reaction was carried out by reacting with Ga.

[0180] 68 Ga-CH44 and 68 The specific activities obtained for Ga-MG04 were 331.3±10.5MBq / g and 449.7±148.1MBq / g, respectively. 68 Ga-MG04: 91±16.9%; 68 The rate was 97±1.4% for Ga-CH44.

[0181] [Tumor implant] Animal studies were performed according to protocols approved by the University of Clermont-Auvergne. Four-week-old male BALB / c nude mice were obtained from Charles River Inc. N CI-H295R cells were implanted subcutaneously in the lower neck of mice. Tumors were 200–400 mm 3 Mice were used for imaging experiments when the amount contained in

[0182] [Route of administration, dose, and experimental design] Animals received 21.6 ± 3.6 MBq 68 Ga-CH44 or 150.8±40.3μL 68 Test substances were administered by a single bolus intravenously at the dose of Ga-MG04.

[0183] Six mice were implanted with NCI-H295R adrenal cancer cells. 48 days after implantation, the animals 68 Ga-CH44 and 68Ga-MG04 was administered intravenously at 48-h intervals, respectively. After each administration, the biodistribution of adrenal cancer xenografts and other tissues was evaluated using PET imaging.

[0184] [PET scan] A group of nude mice (n=2) received 21.6±3.6MBq 68 Ga-CH44 was administered intravenously, and PET / CT scans were obtained 1 h postinjection (pi). PET and CT studies were performed using a Dell (eXplore CT120, GE Healthcare). Anesthesia was 5% isopropyl alcohol. The mouse was induced at full run and maintained at 1.5%. To improve image quality, the mouse was induced at full run and maintained at 1.5%. Twenty million coincidence events were acquired per 1000 mSv. For the dual-modality PET / CT, CT images (70 kVp, 32 ns exposure time, medium zoom) were acquired and matched with anatomical registration and attenuation correction. This was applied to PET scans. 68 At 48 hours after Ga-CH44 injection, the same group of nude mice received 21.6 ± 3.6 MBq 68 Ga-MG04 was administered and PET / CT scans were obtained 1 hour post-injection (pi). This series constituted the second group.

[0185] 〔result〕 [PET imaging] 68 Image acquisition was performed on the 48th day for Ga-CH44. 68 Image acquisition was performed for Ga-MG04. On the 48th day (FIG. 13A) and the 50th day (FIG. 13B), the imaging images were 68 Ga-CH44 and 68 Significant accumulation of Ga-MG04 was observed (light gray arrow = tumor, dark gray arrow = kidney). 68Tumor uptake of Ga-MG04 68 73% higher than Ga-CH44. Images are shown at the same threshold (SUVmin=0; SUVmax=1). SUV (Standardized Uptake Value) is

number

[0186] [Conclusion] In the experiment, two molecules 68 Ga-MG04 and 68 We demonstrated clear and selective imaging and labeling of adrenal cancer with Ga-CH44. 68 There is also an advantage in tumor uptake of Ga-MG04 (73%).

Claims

1. A composition for use in the diagnosis or radiotherapy of an LDLR-overexpressing cancer selected from prostate cancer, adrenal cancer, ovarian cancer and glioblastoma, comprising a conjugate compound represented by the formula M-C-P, wherein M represents a pharma- ceutically acceptable radionuclide; C represents a chelator which is DOTA or a functional derivative thereof, forming a chelate with M, said functional derivative of DOTA being a compound derived from DOTA in which the hydroxyl (-OH) groups of DOTA are replaced with one or more amine (-NH 2 ) groups; A composition, wherein P represents a peptide or pseudopeptide having up to 30 amino acid residues, binding to the low density lipoprotein receptor (LDLR), and comprising the amino acid sequence of SEQ ID NO:

1.

2. 2. The composition of claim 1, wherein the compound further comprises a group T selected from an analog of prostate specific membrane antigen (PSMA), neurotensin, and somatostatin.

3. 3. The composition of claim 1 or 2, wherein the compound is a multimer comprising multiple copies of the P group or the M group, or multiple copies of both.

4. The composition of any one of claims 1 to 3, wherein the compound is a multimer comprising multiple monomers of the formula M-C-P.

5. ６８ Ga-DOTA - SEQ ID NO:1, ６８ Ga-DOTA-GA-SEQ ID NO:1, １８ F-DOTA - SEQ ID NO:1, １８ F-DOTA-GA - SEQ ID NO:1, ９０ Y-DOTA - SEQ ID NO:1, ９０ Y-DOTA-GA-SEQ ID NO:1, １１１ In-DOTA - SEQ ID NO:1, １１１ In-DOTA-GA - SEQ ID NO:1, １７７ Lu-DOTA-SEQ ID NO:1, or １７７ The composition according to any one of claims 1 to 4, which is Lu-DOTA-GA-SEQ ID NO:

1.

6. A composition described in any one of claims 1 to 5, which is 68 Ga-DOTA-sequence number 1 or 177 Lu-DOTA-sequence number 1.

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