Structural optimization method to improve the therapeutic performance of radionuclide therapy targeting peptide receptors against cancer.

Abstract

To provide a structural optimization method for improving the therapeutic performance of radionuclide therapy targeting peptide receptors against cancer. [Solution] In a specific embodiment, the present invention provides formula I for cancer treatment: TLX This provides a cancer targeting conjugate containing and a method for using the same. T is an SST2R targeting ligand, L is a linker, X is a chelating agent. In one embodiment, the present invention relates to a novel chelating agent which is 1,4,7,10-tetraazacyclododecane-7-acetamido-1,4,10-triacetic acid.

Description

[Technical Field]

[0001] Statement on federally funded research This invention was developed with government support under R01CA243014 and P50CA174521 granted by the National Institutes of Health. The government has certain rights in this invention.

[0002] Cross-reference of related applications This application claims priority to U.S. Provisional Patent Application No. 62 / 967,497 filed on 29 January 2020, and the entirety of this disclosure is incorporated herein by reference. [Background technology]

[0003] Neuroendocrine neoplasms (NENs) are a heterogeneous group of neoplasms whose incidence has been steadily increasing over the decades (1,2). NENs are generally subdivided into well-differentiated (low to moderate) neuroendocrine neoplasms (NETs) and poorly differentiated (high-grade) neuroendocrine carcinomas (NECs) based on histological, biological, and pathological differences (2,3). In many cases, well-differentiated NETs are less aggressive than poorly differentiated NECs and respond to several targeted therapies (2). The vast majority (>80%) of NENs express somatostatin receptors (3), and among these, somatostatin receptor subtype 2 (SSTR2) is a well-known target of a specific therapy called peptide receptor radionuclide therapy (PRRT). Current developments of SSTR2-targeted PRRT involve the beta particle emitter yttrium-90 ( 90 Y) and Lutetium-177 ( 177 It is based on Lu)(4~8). In particular, 177 Lu labeled DOTA-tyr 3 - Octoreote ( 177 Lu-DOTATATE (Lutathera) is the only radiotherapy approved by the U.S. Food and Drug Administration (FDA) for well-differentiated neoplasms (NETs) (9). The drug has shown therapeutic efficacy by improving patients' tumor response and progression-free survival (PFS) (6-8). However, its effects are limited to partial responses—complete responses are rarely reported.

[0004] An α-particle emitter replaces the conventional β-particle emitter, significantly increasing the radiation dose within cells (up to several hundred times), leading to tumor metastasis (10) due to decay, and similarly resulting in a high relative biological effectiveness (RBE) due to the high linear energy transfer (LET) of α particles (11). Multiple studies have shown that α-particle emitters may be able to treat cancer patients who were resistant to β-particle emitters (12, 13). Lead-212 ( 212 Pb) has a half-life (10.64 h) suitable for clinical application (14) and is an attractive α-particle emitter that well matches the biological half-life (several hours) of peptides in vivo. Also, 212 Pb has a diagnostic pair, lead-203 ( 203 Pb), which can be used for single photon emission computed tomography (SPECT) with photons of 279 keV (81% intensity) (15). 203 The half-life of 212 Pb (51.87 h) is long enough to monitor the body distribution and pharmacokinetics of each patient by continuously imaging up to 4 to 5 half-lives of

[0005] Changes in the peptide structure can significantly alter the binding affinity, pharmacokinetics, and biodistribution of radiolabeled peptides. Therefore, peptide structure changes have the potential to improve the therapeutic outcomes of peptide-based therapies by optimizing these parameters. Approaches to manipulate the maximum performance of peptides for this application include modifying cyclization methods, inserting linkers of appropriate size and composition to connect chelating agents to the peptide backbone, and developing radionuclide-specific chelating agents. Rhenium coordination peptide cyclization (16) and "click" cyclization, as well as further optimization with glycine-glycine (GG) linkers (17), have been evaluated in melanoma models targeting melanocortin receptor subtype 1 (MC1R), suggesting that tumor targeting, pharmacokinetics, and biodistribution can be improved by these approaches. In many other investigations, it has been pointed out that by inserting various linkers (17 - 19) and changing chelating agents (20 - 22), in vivo performance can be improved and radiolabeled peptides can be optimized for optimal tumor targeting.

[0006] In this study, changes in the peptide structure were made by various strategies based on Tyr 3 -octreotide (TOC). A new chelating agent composition, 1,4,7,10-tetraazacyclododecane-7-acetamido-1,4,10-triacetic acid (referred to herein as the Pb-specific chelating agent or PSC), was introduced for Pb isotopes and other 2+-charged radionuclides. The structure was further optimized by adding a polyethylene glycol (PEG) linker between the chelating agent and TOC. DOTATOC, PSCTOC, PSC-PEG2-TOC, and PSC-PEG4-TOC were synthesized by standard Fmoc-based solid-phase peptide synthesis. The performance of each peptide was comprehensively evaluated by radiolabeling efficiency, binding affinity, cell uptake, and biodistribution, and lead compounds were used for 203 SPECT imaging of Pb and 212 Pb therapy / toxicity studies. SUMMARY OF THE INVENTION MEANS FOR SOLVING THE PROBLEM

[0007] As described above, the present invention relates to a novel chelating agent, in one embodiment, which is 1,4,7,10-tetraazacyclododecane-7-acetamido-1,4,10-triacetic acid. The chelating agent is specific to 2+ charged radionuclides, including lead isotopes. The structure includes the chelating agent and Tyr 3 -A polyether linker is included between octreotide (TOC) or other peptides, with polyethylene glycol (PEG) linkers being preferred. The present invention is primarily used to target any cancer expressing somatostatin receptor subtype 2 (SSTR2), including but not limited to neuroendocrine tumors, small cell lung cancer, meningioma, neuroblastoma, medulloblastoma, paraganglioma, and pheochromocytoma. [Modes for carrying out the invention]

[0008] In certain embodiments, the present invention provides a cancer tumor targeting conjugate comprising formula I for cancer treatment. TLX T is an SST2R targeting ligand, L is a linker, X is a chelating agent.

[0009] In certain embodiments, the radiolabeled SST2R-targeting ligand is a peptide, an antibody or antibody fragment, or a small molecule.

[0010] In a particular embodiment, T is Tyr 3 -It is octreotide.

[0011] In certain embodiments, the SST2R-targeting ligand is chemically conjugated to a chelating agent (X) and radiolabeled with a radionuclide used for medical imaging and / or treatment of cancerous tumors.

[0012] In certain embodiments, the radionuclide is Ga-68, In-111, Pb-203, F-18, C-11, Zr-89, Sc-44, Tc-99m, or other medical radionuclides used for imaging.

[0013] In certain embodiments, the radionuclide is Y-90, Pb-212, Bi-212, Bi-213, At-211, Lu-177, Re-188, or other medical radionuclides used in the treatment of cancerous tumors.

[0014] In certain embodiments, L is a chemical linker inserted between a peptide backbone that recognizes the SST2R protein and a chelating agent used to radiolabel the composition using a radionuclide for therapeutic and / or diagnostic imaging, the linker improving the binding and / or internal migration of the composition to cells, improving the retention of the composition in tumors, and improving the clearance of residual composition via other excretion pathways while minimizing radiation exposure to other organs (e.g., kidneys), thereby enabling more precise delivery of radiation to cancerous tissue.

[0015] In certain embodiments, L is a polyether linker containing up to four carbon atoms, consisting of an aliphatic carbon chain that connects the chelating agent to the peptide backbone.

[0016] In certain embodiments, L is PEG n And n is 1 to 4. In certain embodiments, n is 2 or 4.

[0017] In certain embodiments, X is radiolabeled with a radionuclide used in medical imaging and / or treatment of cancerous tumors.

[0018] In certain embodiments, the radionuclide is Ga-68, In-111, Pb-203, Cu-64 or other Cu isotopes, F-18, C-11, Zr-89, Sc-44, Tc-99m, or other medical radionuclides used for imaging.

[0019] In certain embodiments, the radionuclide is Y-90, Pb-212, Cu-67 or other Cu isotopes, Bi-212, Bi-213, At-211, Lu-177, Re-188, or other medical radionuclides used in the treatment of cancerous tumors.

[0020] In certain embodiments, the chelating agent is based on 1,4,7,10-tetraazacyclododecane-7-acetamido-1,4,10-triacetic acid or other chelating agents used to bind radionuclides for imaging or treatment of cancer or other diseases.

[0021] In certain embodiments, the present invention provides a conjugate consisting of PSC-PEG2 / PEG4-TOC.

[0022] In certain embodiments, the drug is administered orally or parenterally.

[0023] In certain embodiments, the drug is administered subcutaneously.

[0024] In certain embodiments, the conjugate is administered orally or parenterally.

[0025] In certain embodiments, the method further comprises administering an anticancer composition.

[0026] In certain embodiments, the conjugate is administered in a single dose.

[0027] In certain embodiments, the conjugate is administered in multiple doses.

[0028] In certain embodiments, the conjugate is administered daily for several days.

[0029] In certain embodiments, the conjugate is administered once a week for one month. In certain embodiments, the conjugate is administered once a week for up to six months.

[0030] In certain embodiments, the conjugate is administered at a dose of 1 mCi for medical imaging.

[0031] In certain embodiments, the conjugate is administered at a dose of up to 10 mCi for medical imaging.

[0032] In certain embodiments, the conjugate is administered at a dose of up to 50 mCi for medical imaging.

[0033] In certain embodiments, the conjugate is administered at a dose of 0.1 mCi for the medical treatment of cancerous tumors.

[0034] In certain embodiments, the conjugate is administered at a dose of up to 1 mCi for the medical treatment of cancerous tumors.

[0035] In certain embodiments, the conjugate is administered in doses up to 10 mCi for the medical treatment of cancerous tumors.

[0036] In certain embodiments, the conjugate is administered at a dose of up to 100 mCi for the medical treatment of cancerous tumors.

[0037] In certain embodiments, the conjugate is administered for more than one month.

[0038] In certain embodiments, the conjugate is administered for more than one year.

[0039] In certain embodiments, the conjugate is administered at a dose of at least 0.05 μg / day.

[0040] In certain embodiments, the present invention provides the use of the above-described conjugate, a) The conjugate is administered simultaneously with one or more anticancer drugs, or b) The conjugate and one or more anticancer drugs are administered sequentially, or c) Administration of one or more anticancer drugs should begin approximately 1 to 10 days before administration of the conjugate, or d) Administration of the conjugate should begin approximately 1 to 10 days before administration of one or more anticancer drugs, or e) Administration of the conjugate and administration of one or more anticancer drugs shall begin on the same day.

[0041] In certain embodiments, the ligand is a peptide.

[0042] In certain embodiments, the peptide is radiolabeled.

[0043] In certain embodiments, the SST2R-targeting ligand is a peptide that binds to somatostatin receptor subtype 2.

[0044] In certain embodiments, the peptide is radiolabeled.

[0045] In certain embodiments, the agent that increases SST2R expression is administered separately, sequentially, or simultaneously with the SST2R-targeting ligand.

[0046] In certain embodiments, the agent that increases SST2R expression is administered approximately 1 day to 6 months prior to the administration of the SST2R-targeting ligand.

[0047] In certain embodiments, the drug is administered orally or parenterally.

[0048] In certain embodiments, the drug is administered subcutaneously.

[0049] In certain embodiments, the SST2R-targeting ligand is administered orally or parenterally.

[0050] In certain embodiments, drug administration is initiated approximately 1 to 10 days before administration of the SST2R-targeted ligand.

[0051] In certain embodiments, the administration of the drug and the administration of the SST2R-targeted ligand are initiated on the same day.

[0052] In certain embodiments, the method further comprises administering an anticancer composition.

[0053] This patent or application document includes at least one drawing drawn in color. Copies of this patent or patent application publication, including the color drawing(s), will be provided by the Patent Office upon request and payment of the necessary fees. [Brief explanation of the drawing]

[0054] [Figure 1][Figure 1A] Structure (A) of the SST2R-targeting ligand DOTATOC. The peptides were synthesized by standard Fmoc-based solid-phase peptide synthesis. They were based on tyr3-octreotide (TOC) and conjugated with DOTA or a novel Pb-specific chelating agent (PSC). For linker-containing peptides, two different sizes of polyethylene glycol (PEG), PEG2 and PEG4, were inserted between the PSC and the peptide backbone. [Figure 1B] Structure (B) of the SST2R-targeting ligand PSCTOC. The peptides were synthesized by standard Fmoc-based solid-phase peptide synthesis. They were based on tyr3-octreotide (TOC) and conjugated with DOTA or a novel Pb-specific chelating agent (PSC). For linker-containing peptides, two different sizes of polyethylene glycol (PEG), PEG2 and PEG4, were inserted between the PSC and the peptide backbone. [Figure 1C] Structure (C) of the SST2R-targeting ligand PSC-PEG2 / PEG4-TOC. The peptides were synthesized by standard Fmoc-based solid-phase peptide synthesis. They were based on tyr3-octreotide (TOC) and conjugated with DOTA or a novel Pb-specific chelating agent (PSC). For linker-equipped peptides, two different sizes of polyethylene glycol (PEG), PEG2 and PEG4, were inserted between the PSC and the peptide backbone. [Figure 2] Good radiolabeling efficiency of PSC-conjugated peptides with the SST2R-targeting ligand DOTATOC, and with 203Pb(A) and 212Pb(B). 18.5 MBq of 203Pb or 14.1 MBq of 212Pb was reacted with 10 nmol of peptide in 0.5 M sodium acetate (NaOAc) buffer (pH=5.4, 1 ml reaction volume). The reaction was carried out at various temperatures (25°C, 50°C, or 85°C) and reaction times (10 min, 20 min, or 30 min) for 203Pb labeling. DOTATOC and PSCTOC were selected for 212Pb labeling, and the reaction was carried out at a constant temperature (85°C) over a longer time (up to 30 minutes). [Figure 3]Competitive inhibition of 125I-tyr3-octreotide (125I-TOC) binding to SSTR2-positive AR42J cells by TOC, DOTATOC, and PSC conjugate peptides. IC50 values: TOC: 3.1±1.1nM, DOTATOC: 11.3±1.3nM, PSC-TOC: 6.2±1.1nM, PSC-PEG2-TOC: 5.3±1.2nM, PSC-PEG4-TOC: 9.4±1.3nM (at least n=6 from at least 3 biological replicates for DOTATOC and PSC-PEG2-TOC, and n=4-6 from 2 biological replicates for TOC, PSC-PEG2-TOC, and PSC-PEG4-TOC). [Figure 4] Cellular uptake of 203Pb-labeled DOTATOC, PSCTOC, and PSC-PEG2-TOC in AR42J cells. 203Pb-labeled peptides, purified by HPLC at 200,000 CPM, were incubated with AR42JSST2R-expressing cells at 37°C for up to 120 minutes. Cellular uptake of each radiotrace was measured. Data are presented as the mean percentage ± SD of cell uptake relative to incubated activity (n=4). [Figure 5] In vivo distribution of 203Pb-labeled SST2R-targeted DOTATOC, PSCTOC, and PSC-PEG2-TOC in thymus-deficient nude mice with AR42J tumors. In vivo distribution of 37 kBq of 203Pb-labeled peptides was observed 1, 3, and 24 hours after intravenous injection (A), showing the percentage of injected volume per g of tissue (%ID / g) and the tumor-to-kidney ratio over time for tumors and liver (B). Data are presented as the mean percentage of injected volume per g of tissue (%ID / g) or the relative mean tumor-to-kidney ratio ± SD (n=3). [Figure 6]These are 203Pb SPECT / CT images of thymus-deficient nu / nu female mice with AR42J tumors. (A) Mice with AR42J tumors were imaged 3 and 24 hours after administration of 11.1 MBq of 203Pb-DOTATOC and 203Pb-PSC-PEG2-TOC. To confirm tumor specificity, 30 nmol of unlabeled peptide was co-injected for blocking imaging. (B) The tumor-to-kidney ratio over time, analyzed from images acquired using Inveon Research Workplace software. (C) Mice were euthanized 30 hours after injection to obtain in vivo distribution. [Figure 7] Stability of PSC-PEG2-TOC in water and human serum. PSC-PEG2-TOC was radiolabeled with 50 MBq (1.34 mCi) of 203Pb, and 9 MBq (0.24 mCi) of purified radiopeptide was added to 3 ml of water or human serum and incubated at 37°C for up to 24 hours. Peptide degradation was monitored at 8 hours and 24 hours using a radio-HPLC system (Agilent 1200 series connected to an IN / USβ-RAM Model 4 radiation detector). [Figure 8] Clinically relevant high specific activity 203Pb radiolabeling of PSC-PEG2-TOC. Radiolabeling was performed at 85°C for 30 minutes with high radioactivity 203Pb using either 90 MBq / nmol of DOTATOC (A), 90 MBq / nmol of PSC-PEG2-TOC (B), or 120 MBq / nmol of PSC-PEG2-TOC (C) as a reference in 0.5 M sodium acetate (NaOAc) buffer (pH=5.4, 1-2 ml reaction volume). [Figure 9] In vivo distribution of 203Pb / 212Pb-labeled PSC-PEG2-TOC in thymic-deficient nude mice with AR42J tumors 3 hours after injection. 74 kBq of 212Pb-PSC-PEG2-TOC (specific activity, 3.7 MBq / nmol) was injected via the tail vein, and its in vivo distribution was obtained 3 hours after injection (n=4). This data was directly compared with previously obtained in vivo distributions of 203Pb-PSC-PEG2-TOC (specific activity, 22.2 MBq / nmol, Figure 5). [Figure 10] Specific tumor binding of radioactive peptides in nude mice with AR42J tumors, indicated by reduced renal accumulation of 203Pb-PSC-PEG2-TOC with co-injection of DL-lysine and tumor blocking with co-injection of excess unlabeled peptide. (A) In vivo distribution of 203Pb-PSC-PEG2-TOC 3 hours after injection in nude mice with AR42J tumors with co-injection of lysine (400 mg / kg), without co-injection of lysine, or with co-injection of unlabeled peptide (for tumor blocking, 10 nmol of PSC-PEG2-TOC). (B) Complete in vivo distribution of 203Pb-PSC-PEG2-TOC 1, 3, 6, and 24 hours after injection with co-injection of DL-lysine (400 mg / kg). Results are expressed as the percentage of injected volume per gram of tissue (%ID / g) ± SD (n=3). [Figure 11] Treatment results of the first 212Pb-PSC-PEG2-TOC therapy experiment in mice with tumors expressing AR42J-SST2R 30 days after treatment. 212Pb-PSC-PEG2-TOC therapy was initiated when the average tumor size reached approximately 150 mm3. 0.37 MBq (10 μCi) and 1.85 MBq (50 μCi) of 212Pb-PSC-PEG2-TOC were co-injected via tail vein with DL-lysine (400 mg / kg) to block renal uptake of radiotherapy drugs. [Figure 12]Dose measurements and toxicity of 212Pb-PSC-PEG2-TOC at an increasing dose (maximum 150 μCi) in CD-1 elite (SOPF) male mice. (A) Body weight change after injection of 212Pb-PSC-PEG2-TOC. After an initial decrease in body weight during the first few days, the body weight of treated mice gradually increased, and the increase in body weight was dose-dependent. (B) In vivo distribution of 212Pb-PSC-PEG2-TOC in CD-1 elite (SOPF) male mice. 212Pb is used for studies including the effects of potential demetallation of 212Pb and redistribution in the bone marrow. (C) Estimated renal dose resulting from an increasing dose of 212Pb-PSC-PEG2-TOC based on its in vivo distribution in CD-1 elite (SOPF) male mice. Organ-level internal dose assessment (OLINDA) V2.1 was used to estimate the dose of mice using a 30 g mouse voxel phantom model. Levels of nephrotoxicity markers resulting from the gradual dose increase of 212Pb-PSC-PEG2-TOC, assessed by urinary neutrophil gelatinase-associated lipocalin (uNGAL, D) on days 1 and 3 after administration, and by blood urea nitrogen (BUN, E) 3 months after administration. (F) Reversible hematological toxicity as indicated by whole blood cell count (CBC) 1 week, 2 weeks, and 4 weeks after administration. [Modes for carrying out the invention]

[0055] The following examples are intended to further illustrate the present invention. They are not intended to limit the invention in any way.

[0056] Materials and methods Peptide synthesis DOTATOC, PSCTOC, PSC-PEG2-TOC, and PSC-PEG4-TOC were synthesized by standard Fmoc-based solid-phase peptide synthesis. The linear peptide D-Phe-Cys-Tyr-D-Trp-Lys-Thr-Cys-Thr(ol) was synthesized on resin at a 100 μmol scale using an automated peptide synthesizer (AAPPTEC Apex396). At the end of the automated synthesis, the N-terminus of the linear peptide was deprotected with 25% piperidine (PIP). For PSC-PEG2-TOC or PSC-PEG4-TOC, a PEG linker (PEG2 or PEG4) was manually added. The peptide resin was suspended in N,N-dimethylformamide (DMF), and 5 equivalents of Fmoc-NH-PEG2 / PEG4-propionic acid (purchased from AAPPTEC), 2-(7-aza-1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HATU), and 1-hydroxybenzotriazole (HOBt), along with 10 equivalents of N,N-diisopropylethylamine (DIPEA), were added and the mixture was allowed to react at 37°C for 2 hours with mixing. Next, the N-terminal Fmoc of the peptide resin was manually deprotected with 25% piperidine (in DMF) with gentle mixing for 10 minutes at 25°C, washed with DMF / dichloromethane (DCM) / methanol, and the above procedure was repeated. Next, the linear peptide with an open N-terminus was resuspended in DMF on the resin, and 5 equivalents of either DOTA-tris(tert-butyl ester), PSC-bis(tert-butyl ester), HATU, and HOBt, along with 10 equivalents of DIPEA, were added and the mixture was allowed to react at 37°C overnight with mixing. The success of each coupling / deprotection step was verified by the Kaiser test, and the process was repeated until successful. Next, the linear peptide was cyclized by iodine oxidation. Iodine (I2, 20 equivalents) was dissolved in 6 ml of DMF and added to the peptide resin, and the reaction was allowed to proceed with trityl deprotection from cysteine, while simultaneously promoting disulfide formation by oxidation over 3 hours.Next, the resin and protecting groups were cleaved from the cyclic peptide by adding 3 mL of a cleavage cocktail (93% trifluoroacetic acid, 3% triisopropylsilane, 4% water) at room temperature for 2 hours, followed by ether precipitation on ice for at least 4 hours. The crude peptide was then purified by semi-preparative high-performance liquid chromatography (HPLC) using a C-18 column (Vydac 10 × 250 mm, 10 μm; Grace, Deerfield, IL). The collected samples were concentrated by rotational evaporation and lyophilized. The purified peptides were characterized by mass spectrometry.

[0057] 203 Pb / 212 Pb radioactive labeling efficiency DOTATOC and PSC conjugate peptides are 203 Pb and 212 Radiation-labeled with Pb. 18.5 MBq 203 Pb or 14.1 MBq 212 PB was reacted with 10 nmol of peptide in 0.5 M sodium acetate (NaOAc) buffer (pH=5.4, 1 ml reaction volume). The reaction proceeded as follows: 203 For Pb labeling, the process was carried out at various temperatures (25°C, 50°C, or 85°C) and reaction times (10 minutes, 20 minutes, or 30 minutes). DOTATOC and PSCTOC were... 212 Selected for Pb labeling, the reaction was carried out over time (maximum 30 minutes) at a constant temperature (85°C). After the reaction, the resulting product was spotted onto pre-dried immediate thin-layer chromatography (iTLC) shards and developed with 10 mM diethylenetriaminepentaacetic acid (DTPA) in 0.1 M NaOAc buffer. The shards were then cut in half, and each part of the iTLC shard (top, free) was separated. 203 Pb / 212 Pb; bottom, peptide labeled 203 Pb / 212 The radioactivity of Pb was measured using a NaI detector, with isotope-specific gamma peaks. 203 Pb, 279 keV; 212 Pb, 239 keV).

[0058] 125I-TOC competitive binding assay TOC, as explained elsewhere (23), uses the conventional chloramine T method to extract iodine-125 ( 125 I) was labeled. 1.0x10 5 AR42J rat pancreatic acinar cells were seeded in 24-well plates coated with poly-D-lysine. After 3 days, the cells were removed from a plate with increased concentration (10 -11 ~10 -6 M) 30,000 CPM in conjugation medium containing TOC, DOTATOC, PSCTOC, PSC-PEG2-TOC, or PSC-PEG4-TOC (RPMI supplemented with 0.2% bovine serum albumin, 1640, 0.3 mM 1,10-phenanthroline) 125 The cells were incubated with I-TOC at 37°C for 2 hours. Next, the cells were washed twice with ice-cold PBS, lysed with 0.5N NaOH, and their radioactivity was measured using a gamma counter. The 50% inhibitory concentration (IC) was then measured. 50 The result was determined using GraphPad Prism V8.0.

[0059] 203 Internal transfer and efflux of Pb-labeled peptides 37MBq 203 Pb was labeled with 10 nmol of DOTATOC, PSCTOC, and PSC-PEG2-TOC, and the labeled peptide was separated from the unlabeled peptide by high-performance liquid chromatography (HPLC) based on the difference in retention times between the labeled and unlabeled peptides using a previously developed separation method (15). Next, the HPLC-separated radioactive peptide was purified using a C-18 cartridge. 2 days prior 2.0 x 10 5 AR42J cells seeded at the specified cell density were purified by HPLC at 200,000 CPM. 203Cells were incubated with Pb-labeled peptides at 37°C for up to 120 minutes. Next, the cells were washed twice with ice-cold PBS, and the membrane-bound radioactive material was washed and collected with 50 mM acidic (pH=4) sodium acetate buffer. The remaining cells were lysed by adding 0.5 N NaOH for 5 minutes. The radioactivity of each portion (membrane-bound and internally transported) was counted using a 310 Cobra II gamma counter (PerkinElmer, Freemont, CA). In the efflux assay, cells were purified by HPLC at 200,000 CPM. 203 Cells were incubated with Pb-labeled peptide at 37°C for 120 minutes. Next, the cells were washed twice with ice-cold PBS and replenished with binding medium. At 60 and 120 minutes, radioactivity was counted for efflux (to medium), membrane binding, and internal distribution (collected using the same method as the internal distribution assay).

[0060] 203 Distribution of Pb-labeled peptides in the body 37kBq 203 Pb-labeled DOTATOC, PSCTOC, and PSC-PEG2-TOC (specific activity: 22.2 MBq / nmol) were injected via the tail vein into female thymus-deficient nu / nu mice with AR42J tumors. The mice were euthanized by cervical dislocation under isoflurane anesthesia at 1, 3, and 24 hours after injection. The target tumors and organs / tissues were collected, and the weight of the collected organs / cells was measured. The radioactivity of the samples was measured using a PerkinElmer 310 Cobra II gamma counter (PerkinElmer, Freemont, CA).

[0061] Dose measurement of tumors and kidneys Particle and Heavy Ion Transport Coding System (PHITS) was used for dosimetry analysis. For renal dosimetry, a DigiMouse voxel phantom model was used, and the model's voxel size was adjusted so that the kidney volume was the same as the average kidney volume (288.7 ± 41.4 mg, 28 mice) obtained from an in vivo distribution study (AR42J, 8-10 weeks). The basic kidney composition and mass density were assumed to be the same as the human reference adult values ​​obtained from the International Commission on Radiological Units and Measurements (ICRU) report 46. For tumor dosimetry, a spherical volume was created based on the average tumor mass (156.9 ± 0.096 mg) of 28 mice. The basic tumor composition (adenoid cystic carcinoma) and mass density (1.04 g / cm³) were used. 3 The data was taken from Maughan et al. 1997 Med Phys 24(8):1241-4 and RM Thomson et al. 2013 Phys. Med. Biol. 58:1123-50. In the Monte Carlo simulation, at least 1 million particles were transported, reducing the statistical uncertainty to less than 1%.

[0062] 203 Pb-DOTATOC and 203 Serial SPECT / CT imaging of Pb-PSC-PEG2-TOC 1.85 GBq (50 mCi, 61.7 MBq / nmol) 203 Pb was labeled with DOTATOC and PSC-PEG2-TOC. Each 11.1 MBq 203 Pb-labeled peptides were injected into mice with AR42J via the tail vein, and images of the mice were taken 3 and 24 hours after injection. Separately, for blocking studies to confirm the tumor specificity of the radioactive tracer, the same radioactivity was used. 203 Pb-PSC-PEG2-TOC was co-injected with 30 nmol of unlabeled PSC-PEG2-TOC. Images were reconstructed and analyzed using Inveon Research Workplace software with the same parameter settings. Standardized uptake values, corrected for body weight (SUVbw), were analyzed to obtain the in vivo distribution in mice 30 hours after administration.

[0063] Water and human serum 203 Stability of Pb-PSC-PEG2-TOC As the identified lead compound, PSC-PEG2-TOC was further evaluated from various aspects. PSC-PEG2-TOC was administered in 50 MBq (1.34 mCi) doses. 203 The radiopeptides were radiolabeled with lead and purified with C-18. 9 MBq (0.24 mCi) of the purified radiopeptide was added to 3 ml of water or human serum and incubated at 37°C for up to 24 hours. After incubation, 203 Serum samples containing Pb-PSC-PEG2-TOC were transferred to an Amicon Ultra centrifugal filter (3K; Millipore) and centrifuged using a Beckman Coulter Avanti J-25I centrifuge. Osmotic samples (serum samples) or samples in water were analyzed using a radioactive HPLC system (Agilent 1200 series connected to an IN / USβ-RAM Model 4 radioactivity detector) to monitor the degree of peptide degradation.

[0064] Clinically relevant high specific activity of PSC-PEG2-TOC 203 Pb radiolabel PSC-PEG2-TOC has a high specific activity of either 90 MBq / nmol or 120 MBq / nmol. 203 The reagents were radiolabeled with lead (Pb). DOTATOC was also labeled at 90 MBq / nmol for reference. The reaction was carried out at 85°C for 30 minutes in 0.5 M sodium acetate (NaOAc) buffer (pH=5.4, 1-2 ml reaction volume). 203 Two μl of the reaction product containing a lead-labeled peptide was spotted onto an iTLC (intensive thin-layer chromatography) strip. The strip was developed using a mobile phase (0.2 M sodium acetate and 20 mM EDTA) and then imaged with a phosphorescent imager (Typhoon FLA7000). The strip was cut in half, and the radioactivity of both sides of the strip was measured. 203 The radioactive labeling efficiency was measured using a NaI detector based on the Pb gamma peak (279 keV).

[0065] In nude mice, AR42J is a leading species.212 Distribution of Pb-PSC-PEG2-TOC in the body 74kBq 212 Pb-PSC-PEG2-TOC (specific activity, 3.7 MBq / nmol) was injected via the tail vein into thymus-deficient nude mice with AR42J, and its distribution in vivo was obtained 3 hours after injection (n=4). This data was obtained previously. 203 The distribution of Pb-PSC-PEG2-TOC in the body was directly compared (specific activity, 22.2 MBq / nmol, Figure 5). The data were as follows: 212 As a substitute for imaging and dose measurement of Pb-PSC-PEG2-TOC, 203 The validity of Pb-PSC-PEG2-TOC was determined.

[0066] In nude mice with AR42J, induced by simultaneous injection of lysine. 203 Distribution of Pb-PSC-PEG2-TOC in the body 37kBq 203 Pb-PSC-PEG2-TOC (specific activity: 22.2 MBq / nmol) was injected via the tail vein into nude mice with AR42J tumors, and we observed whether co-injection of DL-lysine (400 mg / kg, 8 mg / animal) reduced nonspecific renal uptake of the radiotracer, both with and without co-injection. Additionally, 10 nmol of unlabeled peptide (without lysine) and 37 kBq of [unlabeled peptide] were used. 203 A separate group was added for tumor blocking to verify the specificity of tumor targeting by co-injecting Pb-PSC-PEG2-TOC. These mice were then euthanized 3 hours after injection, and their in vivo distribution was evaluated (n=3 for each group). In another experiment, comprehensive in vivo distribution was obtained at 1, 3, 6, and 24 hours after injection with co-injection of DL-lysine, providing complete pharmacokinetic data for further doseometry experiments.

[0067] 212 Pb-PSC-PEG2-TOC therapy 5.0x10 6AR42J rat pancreatic acinar cells were transplanted into the left shoulder of female thymus-deficient nu / nu mice. After 10 days, the average tumor size was approximately 150 mm. 3 When it reaches 274 MBq (7.4 mCi) 212 Pb was reacted with 30 nmol of PSC-PEG2-TOC (9.1 MBq / nmol) at 85°C for 20 minutes in the presence of ascorbic acid (1 mg / ml). After the reaction, the radioactive peptide was purified with C-18 and resuspended in physiological saline containing ascorbic acid (1 mg / ml). 0.37 MBq (10 μCi) and 1.85 MBq (50 μCi) were obtained. 212 Pb-PSC-PEG2-TOC was injected via the tail vein. DL-lysine (400 mg / kg) was administered simultaneously to block the renal uptake of the radiotherapy drug.

[0068] 212 Pb-PSC-PEG2-TOC toxicity test 212 Pb-PSC-PEG2-TOC was administered in escalating doses (0, 0.37, 1.85, 3.33, and 5.55 MBq, or 0, 10, 50, 90, and 150 μCi) to tumor-free CD-1 elite (SOPF) male mice (n=4 in each group). Body weight was measured twice weekly until 3 weeks after injection, and then once weekly thereafter. To assess acute renal tubular toxicity, urine samples were collected (via metabolic case) on days 1 and 3 after administration. Urine samples were centrifuged, and urinary neutrophil gelatinase-associated lipocalin (uNGAL) levels were measured using a mouse NGAL ELISA kit (Kit 042, BIOPORTO Diagnostics) according to the manufacturer's manual. Three months after injection, serum samples were collected by tail vein nicking and sent to IDEXX Laboratories, Inc. for analysis of comprehensive blood chemistry, including blood urea nitrogen (BUN). Further follow-up was performed at 6-7 months for comprehensive blood chemistry testing and renal histopathological analysis. Hematological toxicity was assessed at weeks 1, 2, and 4 after administration by complete blood cell count (CBC) using an automated veterinary blood analyzer (ADVIA120, Siemens Healthineers). Furthermore, 212Pb-PSC-PEG2-TOC in vivo distribution studies were performed at 1 hour, 3 hours, 6 hours, and 24 hours (including bone marrow) to support dosimetry analyses that could correlate with toxicity profiles in vital organs / tissues, including the kidneys and bone marrow. Dose estimation was performed using organ-level internal dose assessment (OLINDA, V2.1) software with a 30g mouse voxel phantom model.

[0069] References 1.Dasari A, Shen C, Halperin D, et al. Trends in the Incidence, Prevalence, and Survival Outcomes in Patients With Neuroendocrine Tumors in the United States.JAMA Oncol. 2017;3:1335-1342. 2.Oronsky B, Ma PC, Morgensztern D, Carter CA. Nothing But NET: A Review of Neuroendocrine Tumors and Carcinomas.Neoplasia. 2017;19:991-1002. 3.Kaemmerer D, Trager T, Hoffmeister M, et al. Inverse expression of somatostatin and CXCR4 chemokine receptors in gastroenteropancreatic neuroendocrine neoplasms of different malignancy.Oncotarget. 2015;6:27566-27579. 4. Imhof A, Brunner P, Marincek N, et al. Response, survival, and long-term toxicity after therapy with the radiolabeled somatostatin analogue [90Y-DOTA]-TOC in metastasized neuroendocrine cancers.J Clin Oncol. 2011;29:2416-2423. 5.Marincek N, Jorg AC, Brunner P, et al. Somatostatin-based radiotherapy with [90Y-DOTA]-TOC in neuroendocrine tumors: long-term outcome of a phase I dose escalation study.J Transl Med. 2013;11:17. 6.Strosberg J, El-Haddad G, Wolin E, et al. Phase 3 Trial of 177Lu-Dotatate for Midgut Neuroendocrine Tumors.New England Journal of Medicine. 2017;376:125-135. 7.Kwekkeboom DJ, de Herder WW, Kam BL, et al. Treatment with the radiolabeled somatostatin analog [177 Lu-DOTA 0,Tyr3]octreotate: toxicity, efficacy, and survival. J Clin Oncol. 2008;26:2124-2130. 8.Brabander T, van der Zwan WA, Teunissen JJM, et al. Long-Term Efficacy, Survival, and Safety of [(177)Lu-DOTA(0),Tyr(3)]octreotate in Patients with Gastroenteropancreatic and Bronchial Neuroendocrine Tumors.Clin Cancer Res. 2017;23:4617-4624. 9.FDA Approves Lutathera for GEP NET Therapy.J Nucl Med. 2018;59:9N. 10.Lee D, Li M, Bednarz B, Schultz MK. Modeling Cell and Tumor-Metastasis Dosimetry with the Particle and Heavy Ion Transport Code System (PHITS) Software for Targeted Alpha-Particle Radionuclide Therapy.Radiat Res. 2018;190:236-247. 11.Sgouros G, Roeske JC, McDevitt MR, et al. MIRD Pamphlet No. 22 (abridged): radiobiology and dosimetry of alpha-particle emitters for targeted radionuclide therapy.J Nucl Med. 2010;51:311-328. 12.Kratochwil C, Bruchertseifer F, Giesel FL, et al. 225Ac-PSMA-617 for PSMA-Targeted alpha-Radiation Therapy of Metastatic Castration-Resistant Prostate Cancer.J Nucl Med. 2016;57:1941-1944. 13.Kratochwil C, Giesel FL, Bruchertseifer F, et al. 213Bi-DOTATOC receptor-targeted alpha-radionuclide therapy induces remission in neuroendocrine tumours refractory to beta radiation: a first-in-human experience.Eur J Nucl Med Mol Imaging. 2014;41:2106-2119. 14.Dos Santos JC, Schafer M, Bauder-Wust U, et al. Development and dosimetry of (203)Pb / (212)Pb-labelled PSMA ligands: bringing “the lead” into PSMA-targeted alpha therapy?Eur J Nucl Med Mol Imaging. 2019;46:1081-1091. 15.Li M, Zhang X, Quinn TP, et al. Automated cassette-based production of high specific activity [(203 / 212)Pb]peptide-based theranostic radiopharmaceuticals for image-guided radionuclide therapy for cancer.Appl Radiat Isot. 2017;127:52-60. 16.Chen J, Cheng Z, Owen NK, et al. Evaluation of an (111)In-DOTA-rhenium cyclized alpha-MSH analog: a novel cyclic-peptide analog with improved tumor-targeting properties.J Nucl Med. 2001;42:1847-1855. 17.Martin ME、Sue O’Dorisio M、Leverich WM、Kloepping KC、Walsh SA、Schultz MK “Click”-cyclized (68)Ga-labeled peptides for molecular imaging and therapy: synthesis and preliminary in vitro and in vivo evaluation in a melanoma model system.Recent Results Cancer Res. 2013;194:149-175. 18.Guo H, Miao Y. Introduction of an 8-aminooctanoic acid linker enhances uptake of 99mTc-labeled lactam bridge-cyclized alpha-MSH peptide in melanoma.J Nucl Med. 2014;55:2057-2063. 19.Schweinsberg C, Maes V, Brans L, et al. Novel glycated [99mTc(CO)3]-labeled bombesin analogues for improved targeting of gastrin-releasing peptide receptor-positive tumors.Bioconjug Chem. 2008;19:2432-2439. 20.Chappell LL, Dadachova E, Milenic DE, Garmestani K, Wu C, Brechbiel MW. Synthesis, characterization, and evaluation of a novel bifunctional chelating agent for the lead isotopes 203Pb and 212Pb.Nucl Med Biol. 2000;27:93-100. 21. Gourni E, Mansi R, Jamous M, et al. N-terminal modifications improve the receptor affinity and pharmacokinetics of radiolabeled peptidic gastrin-releasing peptide receptor antagonists: examples of 68Ga- and 64Cu-labeled peptides for PET imaging.J Nucl Med. 2014;55:1719-1725. 22.Lin M, Welch MJ, Lapi SE. Effects of chelator modifications on (68)Ga-labeled [Tyr (3)]octreotide conjugates.Mol Imaging Biol. 2013;15:606-613. 23.de Blois E, Chan HS, Breeman WA. Iodination and stability of somatostatin analogues: comparison of iodination techniques. A practical overview.Curr Top Med Chem. 2012;12:2668-2676.

[0070] It should be understood that minor changes in dosage and formulation of the compositions and ranges expressed herein may still fall within the scope and spirit of the present invention.

[0071] While the present invention has been described with reference to specific compositions, theories of efficacy, etc., it will be apparent to those skilled in the art that the invention is not intended to be limited by such exemplary embodiments or mechanisms, and that modifications can be made without departing from the scope or spirit of the invention as defined by the appended claims. All such obvious modifications and changes are intended to fall within the scope of the invention as defined by the appended claims. The claims are intended to encompass the claimed components and processes in any order that is effective in achieving the intended purpose therein, unless otherwise indicated in the context.

[0072] The foregoing description is presented for illustrative and explanatory purposes only. It is not intended to be an exhaustive list or to limit the invention to the exact forms disclosed. It is conceivable that other alternative processes and methods obvious to those skilled in the art may be considered to be included in the invention. The foregoing description is merely an example of embodiments. Any other modifications, substitutions, and / or additions may be made, which shall be within the intended spirit and scope of the disclosure. From the foregoing, it may be seen that the exemplary embodiments of the disclosure achieve at least all of the intended purposes. The present invention provides, for example, the following items: (Item 1) Formula I for cancer treatment procedures and medical imaging: TLX A cancer targeting conjugate including, T is a radiolabeled SST2R targeting ligand. L is a linker, X is the conjugate, which is a chelating agent. (Item 2) The aforementioned SST2R targeting ligand is a peptide, the conjugate described in item 1. (Item 3) The SST2R ligand is an antibody, antibody fragment, or small molecule, as described in item 1. (Item 4) The SST2R ligand is radiolabeled with a radionuclide used for medical imaging and / or treatment of cancerous tumors, as described in item 2 or 3. (Item 5) The radioactive nuclide is selected from the group consisting of Ga-68, In-111, Pb-203, F-18, C-11, Zr-89, Sc-44, Tc-99m, Cu-64, and other medical radioactive nuclides used for imaging, as described in item 4. (Item 6) The radionuclide is selected from the group consisting of Y-90, Pb-212, Bi-212, Bi-213, At-211, Lu-177, Re-188, Cu-67 (or other Cu radionuclides), and other medical radionuclides used in the treatment of cancerous tumors, as described in item 4. (Item 7) T is Tyr 3 - Octreotide, the conjugate described in item 1. (Item 8) L is a chemical linker, a conjugate as described in any one of items 1 through 7. (Item 9) L is a polyether linker comprising up to four carbon atoms, consisting of an aliphatic carbon chain connecting the chelating agent to the peptide backbone, as described in any one of items 1 to 7. (Item 10) L is PEG n The conjugate is one of the items 1 through 9, where n is 1 through 4. (Item 11) n is 2 or 4, as described in item 10. (Item 12) X is a chelating agent based on 1,4,7,10-tetraazacyclododecane-7-acetamido-1,4,10-triacetic acid, as described in any one of items 1 to 11. (Item 13) A conjugate that is PSC-PEG2 / PEG4-TOC and is listed in any one of items 1-12. (Item 14) A method of treating cancer, including administering the conjugate described in item 1. (Item 15) The method according to item 14, wherein the conjugate is administered by a method selected from the group consisting of oral, parenteral, and subcutaneous administration. (Item 16) The method according to item 14, wherein the conjugate is administered together with the anticancer composition. (Item 17) The conjugate is administered in a single dose, as described in item 14. (Item 18) The conjugate is administered in multiple doses, as described in item 14. (Item 19) The method described in item 14, wherein the conjugate is administered daily for several days. (Item 20) The conjugate is administered at a dose of up to 150 mCi, as described in item 14.

Claims

1. Formula I for cancer treatment and medical imaging: T-L-X A cancer targeting conjugate including, T is a radiolabeled SST2R targeting ligand, L is a linker, X is the conjugate, which is a chelating agent.

2. The conjugate according to claim 1, wherein the SST2R targeting ligand is a peptide.

3. The conjugate according to claim 1, wherein the SST2R ligand is an antibody, an antibody fragment, or a small molecule.

4. The conjugate according to claim 2 or 3, wherein the SST2R ligand is radiolabeled with a radionuclide used for medical imaging and / or treatment of cancerous tumors.

5. The conjugate according to claim 4, wherein the radionuclide is selected from the group consisting of Ga-68, In-111, Pb-203, F-18, C-11, Zr-89, Sc-44, Tc-99m, Cu-64, and other medical radionuclides used for imaging.

6. The conjugate according to claim 4, wherein the radionuclide is selected from the group consisting of Y-90, Pb-212, Bi-212, Bi-213, At-211, Lu-177, Re-188, Cu-67 (or other Cu radionuclides), and other medical radionuclides used in the treatment of cancerous tumors.

7. T is Tyr ３ - The conjugate according to claim 1, which is an octreotide.

8. L is a chemical linker, the conjugate according to any one of claims 1 to 7.

9. The conjugate according to any one of claims 1 to 7, wherein L is a polyether linker comprising up to four carbon atoms, consisting of an aliphatic carbon chain connecting the chelating agent to a peptide backbone.

10. L stands for PEG ｎ The conjugate according to any one of claims 1 to 9, wherein n is 1 to 4.

11. The conjugate according to claim 10, wherein n is 2 or 4.

12. The conjugate according to any one of claims 1 to 11, wherein X is a chelating agent based on 1,4,7,10-tetraazacyclododecane-7-acetamido-1,4,10-triacetic acid.

13. PSC-PEG ２ / PEG ４ - A conjugate according to any one of claims 1 to 12, which is TOC.

14. A method for treating cancer, comprising administering the conjugate described in claim 1.

15. The aforementioned conjugate is selected from the group consisting of oral, parenteral, and subcutaneous administration. The method according to claim 14, which is administered by [method].

16. The method according to claim 14, wherein the conjugate is administered together with the anticancer composition.

17. The method according to claim 14, wherein the conjugate is administered in a single dose.

18. The method according to claim 14, wherein the conjugate is administered in multiple doses.

19. The method according to claim 14, wherein the conjugate is administered daily for several days.

20. The method according to claim 14, wherein the conjugate is administered at a dose of up to 150 mCi.

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