Structural optimization methods to improve the therapeutic performance of peptide receptor-targeted radionuclide therapy for cancer

Abstract

In certain embodiments, the present invention provides compounds of formula I: TLX and methods of using the same, T is an SST2R targeting ligand; L is a linker, X is a chelating agent.

Description

[Technical Field]

[0001] Statement on Federally Sponsored Research This invention was made with government support under R01CA243014 and P50CA174521 awarded by the National Institutes of Health. The government has certain rights in this invention.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 62 / 967,497, filed January 29, 2020, the disclosure of which is incorporated herein by reference in its entirety. [Background technology]

[0003] Neuroendocrine tumors (NENs) are a heterogeneous group of neoplasms, and their incidence has been increasing for several decades (1, 2). NENs are generally subdivided into well-differentiated (low- to intermediate-grade) neuroendocrine tumors (NETs) and poorly differentiated (high-grade) neuroendocrine carcinomas (NECs) based on histological, biological, and pathological differences (2, 3). Well-differentiated NETs are often less aggressive than poorly differentiated NECs and respond to several targeted therapies (2). The majority (>80%) of NENs express somatostatin receptors (3), among which somatostatin receptor subtype 2 (SSTR2) is a well-known target for a specific treatment called peptide receptor radionuclide therapy (PRRT). The current development of SSTR2-targeted PRRT involves the use of a beta particle emitter, yttrium-90 ( 90 Y) and lutetium-177( 177 Lu) (4-8). In particular, 177 Lu labeled DOTA-tyr 3 -Octreotate ( 177 Lu-DOTATATE (Lutathera) is the only radiation therapy approved by the U.S. Food and Drug Administration (FDA) for well-differentiated NETs (9). This drug has demonstrated therapeutic efficacy by facilitating tumor responses and prolonging progression-free survival (PFS) in patients (6-8). However, its efficacy remains limited to partial responses; complete responses are rarely reported.

[0004] Alpha particle emitters are an alternative to conventional beta particle emitters, resulting in significantly increased intracellular radiation doses (up to several hundred times), tumor metastasis through disintegration (10), and a high relative biological effectiveness (RBE) due to the high linear energy transfer (LET) of alpha particles (11). Several studies have shown that alpha particle emitters have the potential to treat cancer patients who have been resistant to beta particle emitters (12, 13). Lead-212 ( 212 Pb) is an attractive α-particle emitter with a half-life (10.64 h) suitable for clinical applications (14), which is well-matched to the biological half-life of peptides in vivo (several hours). 212 Pb contains the diagnostic pair lead-203( 203 Pb), which can be used for single-photon emission computed tomography (SPECT) with 279 keV photons (81% intensity) (15). 203 The half-life of Pb (51.87 h) is 212 Continuous imaging over up to 4–5 half-lives of Pb is long enough to monitor the biodistribution and pharmacokinetics of each patient. (14) Furthermore, theranostic pairs share the same chemistry with respect to radiolabeling and have similar binding affinities and pharmacokinetics when labeled with the same peptide, which is important for accurate dosimetry.

[0005] Changes in peptide structure can significantly alter the binding affinity, pharmacokinetics, and biodistribution of radioactive peptides. Therefore, structural modifications of peptides may improve the therapeutic outcomes of peptidic therapeutics by improving these parameters. Approaches to engineer the maximum performance of peptides for this application include modifying the cyclization method, inserting linkers of appropriate size and composition connecting the chelator to the peptide backbone, and developing radionuclide-specific chelators. Rhenium-coordinated peptide cyclization (16) and "click" cyclization, along with further optimization using a glycine-glycine (GG) linker (17), have been evaluated in a melanoma model targeting the melanocortin receptor subtype 1 (MC1R), suggesting that these approaches may improve tumor targeting, pharmacokinetics, and biodistribution. Numerous other studies have indicated that the insertion of various linkers (17-19) and modification of chelators (20-22) can improve in vivo performance and optimize radioactive peptides for optimal tumor targeting.

[0006] In this study, Tyr 3 Based on octreotide (TOC), peptide structure modification using various strategies was performed. A new chelator composition, 1,4,7,10-tetraazacyclododecane-7-acetamido-1,4,10-triacetic acid (herein referred to as Pb-specific chelator 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 chelator 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, cellular uptake, and biodistribution, and the lead compound was 203 SPECT imaging of Pb and 212 Used in Pb therapy / toxicity studies. Summary of the Invention [Means for solving the problem]

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

[0008] The present invention provides, in certain embodiments, a carcinoma-targeting conjugate comprising Formula I for the therapeutic treatment of cancer: 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, or an antibody or antibody fragment, or a small molecule.

[0010] In certain embodiments, T is Tyr 3 -Octreotide.

[0011] In certain embodiments, the SST2R targeting ligand is chemically conjugated to a chelator (X) and radiolabeled with a radionuclide used in 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 radionuclide used in 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 radionuclide used in the treatment of cancerous tumors.

[0014] In certain embodiments, L is a chemical linker inserted at a position between the peptide backbone that recognizes the SST2R protein and the chelator used to radiolabel the composition with a radionuclide for therapy and / or diagnostic imaging, said linker improving cellular binding and / or internalization of the composition, improving retention of the composition in tumors, and improving clearance of residual composition via other excretion routes while minimizing radiation exposure to other organs (e.g., kidneys), thereby enabling more precise delivery of radiation to cancerous tissues.

[0015] In certain embodiments, L is a polyether linker comprising up to four carbons of an aliphatic carbon chain connecting the chelator 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 radionuclide used in 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 radionuclide 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 the diagnostic 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 agent is administered orally or parenterally.

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

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

[0025] In certain embodiments, the method further comprises administering an anti-cancer 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 consecutive 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 at a dose of 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] The present invention provides in certain embodiments the use of the conjugate described above, a) the conjugate is administered simultaneously with one or more anticancer drugs; or b) the conjugate and the one or more anticancer drugs are administered sequentially; or c) administration of the one or more anticancer agents begins about 1 to about 10 days prior to administration of the conjugate; or d) administration of the conjugate begins about 1 to about 10 days before administration of the one or more anticancer agents; or e) Administration of the conjugate and administration of the one or more anticancer agents 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 expression of SST2R is administered separately, sequentially, or simultaneously with the SST2R targeting ligand.

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

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

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

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

[0050] In certain embodiments, administration of the agent begins about 1 day to about 10 days before administration of the SST2R targeting ligand.

[0051] In certain embodiments, administration of the agent and administration of the SST2R targeting ligand begin on the same day.

[0052] In certain embodiments, the method further comprises administering an anti-cancer composition.

[0053] This patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. [Brief explanation of the drawings]

[0054] [Figure 1][Figure 1A] Structure of the SST2R-targeting ligand DOTATOC (A). 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 chelator (PSC). For peptides with linkers, two different sizes of polyethylene glycol (PEG), PEG2 and PEG4, were inserted between the PSC and the peptide backbone. [Figure 1B] Structure of the SST2R-targeting ligand PSCTOC (B). 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 chelator (PSC). For peptides with linkers, two different sizes of polyethylene glycol (PEG), PEG2 and PEG4, were inserted between the PSC and the peptide backbone. [Figure 1C] Structure of the SST2R-targeting ligand PSC-PEG2 / PEG4-TOC (C). Peptides were synthesized by standard Fmoc-based solid-phase peptide synthesis. They were based on tyr3-octreotide (TOC) and conjugated with DOTA or a new Pb-specific chelator (PSC). For peptides with linkers, 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 203Pb (A) and 212Pb (B). 18.5 MBq of 203Pb or 14.1 MBq of 212Pb were 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, 20, or 30 minutes) for 203Pb labeling. DOTATOC and PSCTOC were selected for 212Pb labeling, and the reaction was carried out at a constant temperature (85 °C) for a period of time (up to 30 minutes). [Figure 3]Competitive inhibition of I-tyr3-octreotide (I-TOC) binding to SSTR2-positive AR42J cells by TOC, DOTATOC, and PSC-conjugated peptides. IC values: TOC: 3.1 ± 1.1 nM, DOTATOC: 11.3 ± 1.3 nM, PSC-TOC: 6.2 ± 1.1 nM, PSC-PEG2-TOC: 5.3 ± 1.2 nM, PSC-PEG4-TOC: 9.4 ± 1.3 nM (n = 6 from at least three biological replicates for DOTATOC and PSC-CTOC, n = 4–6 from two 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. 200,000 cpm of HPLC-purified 203Pb-labeled peptides were incubated with AR42JSST2R-expressing cells at 37°C for up to 120 minutes, and the cellular uptake of each radiotracer was measured. Data are presented as the mean percentage of cellular uptake relative to the incubated activity ± SD (n = 4). [Figure 5] Biodistribution of 203Pb-labeled SST2R-targeted DOTATOC, PSCTOC, and PSC-PEG2-TOC in athymic nude mice bearing AR42J tumors. Biodistribution was observed 1, 3, and 24 hours after intravenous injection of 37 kBq of 203Pb-labeled peptide (A). The percentage of injected dose per gram of tissue (%ID / g) over time for tumor and liver, as well as the tumor-to-kidney ratio, are shown (B). Data are presented as the mean percent injected dose per gram of tissue (%ID / g) or relative mean tumor-to-kidney ratio ± SD (n=3). [Figure 6]203Pb SPECT / CT images of an athymic nu / nu female mouse bearing an AR42J tumor. (A) Mice bearing AR42J tumors were administered 11.1 MBq of 203Pb-DOTATOC and 203Pb-PSC-PEG2-TOC and imaged 3 and 24 hours post-injection. To confirm tumor specificity, 30 nmol of unlabeled peptide was co-injected for blocking imaging. (B) Tumor-to-kidney ratio over time analyzed from images acquired using Inveon Research Workplace software. (C) Mice were euthanized 30 hours post-injection to obtain biodistribution data. [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 radioactive peptide was added to 3 ml of water or human serum and incubated at 37°C for up to 24 h. Peptide degradation was monitored after 8 and 24 h using a radioactive HPLC system (Agilent 1200 series coupled to an IN / US β-RAM Model 4 radiation detector). [Figure 8] Clinically relevant, high-specific-activity Pb ​​radiolabeling of PSC-PEG2-TOC. Radiolabeling was performed with high-activity Pb ​​at 85°C for 30 min with either 90 MBq / nmol DOTATOC (A), 90 MBq / nmol PSC-PEG (B), or 120 MBq / nmol 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] Biodistribution of 203Pb / 212Pb-labeled PSC-PEG2-TOC in athymic nude mice bearing AR42J tumors 3 h after injection. 74 kBq of 212Pb-PSC-PEG2-TOC (specific activity, 3.7 MBq / nmol) was injected via the tail vein, and biodistribution data were obtained 3 h after injection (n=4). This data was directly compared with the previously obtained biodistribution data of 203Pb-PSC-PEG2-TOC (specific activity, 22.2 MBq / nmol, Figure 5). [Figure 10] Specific tumor binding of radioactive peptide in AR42J-bearing nude mice was signaled 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) Biodistribution of 203Pb-PSC-PEG2-TOC at 3 hours post-injection in AR42J tumor-bearing nude mice with co-injection of lysine (400 mg / kg), no co-injection of lysine, or co-injection of unlabeled peptide (10 nmol PSC-PEG2-TOC for tumor blocking). (B) Complete biodistribution of 203Pb-PSC-PEG2-TOC at 1, 3, 6, and 24 hours post-injection with co-injection of DL-lysine (400 mg / kg). Results are percent injected dose per gram of tissue (%ID / g) ± SD (n=3). [Figure 11] Treatment results of the first 212Pb-PSC-PEG2-TOC treatment experiment in mice bearing 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 the tail vein with DL-lysine (400 mg / kg) to block renal uptake of the radiotherapeutic agent. [Figure 12]Dosimetry and toxicity of increasing doses (up to 150 μCi) of 212Pb-PSC-PEG2-TOC 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 over the first few days, treated mice gradually gained weight, and the weight gain was dose-dependent. (B) Biodistribution of 212Pb-PSC-PEG2-TOC in CD-1 elite (SOPF) male mice. 212Pb is used for studies involving the potential demetallization of 212Pb and the effects of redistribution in the bone marrow. (C) Estimated kidney dose resulting from increasing doses of 212Pb-PSC-PEG2-TOC based on biodistribution in CD-1 elite (SOPF) male mice. Organ-level internal dose assessment (OLINDA) v2.1 was used for mouse dose estimation using a 30 g mouse voxel phantom model. Levels of nephrotoxicity markers resulting from increasing doses of 212Pb-PSC-PEG2-TOC assessed by urinary neutrophil gelatinase-associated lipocalin (uNGAL, D) on days 1 and 3 post-dose and by blood urea nitrogen (BUN, E) 3 months post-dose. (F) Reversible hematologic toxicity demonstrated by complete blood count (CBC) at 1, 2, and 4 weeks post-dose. DETAILED DESCRIPTION OF THE INVENTION

[0055] The following examples are intended to further illustrate the present invention, but are not intended to limit it 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, the PEG linker (PEG2 or PEG4) was added manually. The peptide resin was suspended in N,N-dimethylformamide (DMF) and reacted with 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) for 2 hours at 37°C with gentle mixing. The N-terminal Fmoc of the peptide resin was then manually deprotected with 25% piperidine in DMF for 10 minutes at 25°C with gentle mixing, washed with DMF / dichloromethane (DCM) / methanol, and the process was repeated. Next, the linear peptide with an open N-terminus on the resin was resuspended in DMF, and 5 equivalents of either DOTA-tris(tert-butyl ester) or PSC-bis(tert-butyl ester), HATU, and HOBt, as well as 10 equivalents of DIPEA, were added and reacted overnight at 37 °C with mixing. The success of each coupling / deprotection step was verified by Kaiser test, and the process was repeated until successful. Next, the linear peptide was cyclized by iodine oxidation. Iodine (I, 20 equivalents) was dissolved in 6 ml of DMF and added to the peptide resin. The reaction proceeded to promote trityl deprotection from the cysteine ​​and simultaneously promote disulfide formation by oxidation for 3 hours.The resin and protecting groups were then cleaved from the cyclized peptide by adding 3 mL of 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) equipped with a C-18 column (Vydac 10 × 250 mm, 10 μm; Grace, Deerfield, IL). The collected sample was concentrated by rotary evaporation and lyophilized. The purified peptide was characterized by mass spectrometry.

[0057] 203 Pb / 212 Pb radiolabeling efficiency DOTATOC and PSC conjugated peptides 203 Pb and 212 Radiolabeled with Pb. 18.5MBq 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). 203 Pb labeling was carried out at various temperatures (25°C, 50°C, or 85°C) and reaction times (10, 20, or 30 minutes). 212 The Pb-labeling reaction was carried out at a constant temperature (85°C) for a prolonged period (up to 30 min). After the reaction, the resulting material was spotted onto a pre-dried instant thin-layer chromatography (iTLC) strip and developed with 10 mM diethylenetriaminepentaacetic acid (DTPA) in 0.1 M NaOAc buffer. The strip was then cut in half, and each part of the iTLC strip (top, free) was separated. 203 Pb / 212 Pb; bottom, peptide labeled 203 Pb / 212 The radioactivity of Pb was measured by isotope-specific gamma peaks using a NaI detector ( 203 Pb, 279 keV; 212 Pb, 239 keV).

[0058] 125I-TOC competitive binding assay TOC was measured by the conventional chloramine T method using iodine-125( 125 I) labeled with 1.0x10 5 AR42J rat pancreatic acinar cells were plated onto poly-D-lysine-coated 24-well plates. After 3 days, the cells were cultured in increasing concentrations (10 -11 ~10 -6 M) 30,000 CPM in binding medium (RPMI 1640 supplemented with 0.2% bovine serum albumin, 0.3 mM 1,10-phenanthroline) containing TOC, DOTATOC, PSCTOC, PSC-PEG2-TOC, or PSC-PEG4-TOC 125 The cells were incubated with I-TOC for 2 hours at 37°C. Then, the cells were washed twice with ice-cold PBS, lysed with 0.5 N NaOH, and radioactivity was measured in a gamma counter. The 50% inhibitory concentration (IC 50 ) was determined using GraphPad PrismV8.0.

[0059] 203 Internalization and excretion of Pb-labeled peptides 37MBq 203 Pb was labeled with 10 nmol of DOTATOC, PSCTOC, and PSC-PEG2-TOC, and the labeled peptides were separated from the unlabeled peptides by high-performance liquid chromatography (HPLC) based on the difference in retention time between the labeled and unlabeled peptides by a previously developed separation method (15). The HPLC-separated radioactive peptides were then purified on a C-18 cartridge. 2.0x10 5 AR42J cells plated at a cell density of 200,000 CPM were purified by HPLC. 203The cells were incubated with the Pb-labeled peptide at 37°C for up to 120 minutes. The cells were then washed twice with ice-cold PBS, and membrane-bound radioactive material was washed with 50 mM acidic (pH = 4) sodium acetate buffer and collected. The remaining cells were lysed by adding 0.5 N NaOH for 5 minutes. The radioactivity of each fraction (membrane-bound and internalized) was counted using a 310 Cobra II gamma counter (PerkinElmer, Freemont, CA). For efflux assays, the cells were incubated with HPLC-purified PBS at 200,000 CPM. 203 The cells were incubated with Pb-labeled peptide for 120 min at 37°C. Cells were then washed twice with ice-cold PBS and replenished with binding medium. After 60 and 120 min, excreted (into the medium), membrane-bound, and internalized radioactivity (collected in the same manner as in the internalization assay) were counted.

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

[0061] Tumor and kidney dosimetry The Particle and Heavy Ion Transport Code System (PHITS) was used for dosimetry analysis. For kidney dosimetry, a DigiMouse voxel phantom model was used, and the voxel size of the model was adjusted so that the kidney volume was the same as the mean kidney volume (288.7±41.4 mg, 28 mice) of female athymic nude mice obtained from a biodistribution study (8–10 weeks old, bearing AR42J). The basic composition and mass density of the kidney were assumed to be the same as the human reference adult values ​​obtained from the International Commission on Radiation Units and Measurements (ICRU) report. For tumor dosimetry, a spherical volume was created based on the mean tumor mass (156.9±0.096 mg) of 28 mice. The basic composition (adenoid cystic carcinoma) and mass density (1.04 g / cm) of the tumor were calculated. 3 ) was adapted from Maughan et al. 1997 Med Phys 24(8):1241-4 and R.M. Thomson et al. 2013 Phys. Med. Biol. 58:1123-50. Monte Carlo simulations involved transporting at least 1 million particles, reducing the statistical uncertainty to less than 1%.

[0062] 203 Pb-DOTATOC and 203 Sequential 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. 11.1 MBq of each 203 Pb-labeled peptides were injected into AR42J-bearing mice via the tail vein, and the mice were imaged 3 and 24 hours after injection. Separately, for blocking studies to confirm tumor specificity of the radiotracer, 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 the same parameter settings using Inveon Research Workplace software. Standardized uptake values ​​corrected for body weight (SUVbw) were analyzed to obtain the biodistribution in mice 30 hours after administration.

[0063] in water and human serum 203 Stability of Pb-PSC-PEG2-TOC As the identified lead compound, PSC-PEG2-TOC was further evaluated in various aspects. 203 Radiolabeled with Pb and purified with C-18. 9 MBq (0.24 mCi) of purified radioactive peptide was added to 3 ml of water or human serum and incubated at 37°C for up to 24 hours. 203 Serum samples containing Pb-PSC-PEG2-TOC were transferred to Amicon Ultra centrifugal filters (3K; Millipore) and centrifuged in a Beckman Coulter Avanti J-25I centrifuge. The centrifuged permeates (serum samples) or samples in water were analyzed by a radio-HPLC system (Agilent 1200 series coupled to an IN / US β-RAM Model 4 radioactivity detector) to monitor the extent 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 reaction was radiolabeled with Pb. DOTATOC was also labeled at 90 MBq / nmol for reference. The reaction was carried out at 85°C for 30 min in 0.5 M sodium acetate (NaOAc) buffer (pH = 5.4, reaction volume 1–2 ml). 203 Two microliters of the reaction product containing the Pb-labeled peptide was spotted onto an instantaneous thin-layer chromatography (iTLC) strip. The strip was developed with a mobile phase (0.2 M sodium acetate and 20 mM EDTA) and then imaged with a phosphorimager (Typhoon FLA7000). The strip was cut in half, and the radioactivity on both sides of the strip was analyzed. 203 The radiolabeling efficiency was measured by measuring the Pb gamma peak (279 keV) with a NaI detector.

[0065] In nude mice carrying AR42J212 Biodistribution of Pb-PSC-PEG2-TOC 74kBq 212 Pb-PSC-PEG2-TOC (specific activity, 3.7 MBq / nmol) was injected into AR42J-bearing athymic nude mice via the tail vein, and biodistribution data were obtained 3 hours after injection (n=4). 203 The biodistribution of Pb-PSC-PEG2-TOC was directly compared with that of Pb-PSC-PEG2-TOC (specific activity, 22.2 MBq / nmol, Figure 5). 212 As a surrogate for imaging and dosimetry of Pb-PSC-PEG2-TOC, 203 The validity of Pb-PSC-PEG2-TOC was determined.

[0066] in nude mice bearing AR42J by simultaneous injection of ricin 203 Biodistribution of Pb-PSC-PEG2-TOC 37kBq 203 Pb-PSC-PEG2-TOC (specific activity: 22.2 MBq / nmol) was injected via the tail vein into nude mice bearing AR42J tumors, with or without co-injection of DL-lysine (400 mg / kg, 8 mg / animal), to observe whether co-injection of lysine could reduce nonspecific renal uptake of the radiotracer. Also, 10 nmol of unlabeled peptide (without lysine) and 37 kBq of lysine were injected. 203 A separate group was added for tumor blocking to verify the specificity of tumor targeting by co-injection of Pb-PSC-PEG2-TOC. These mice were then euthanized 3 hours after injection to evaluate biodistribution (n = 3 for each group). In a separate experiment, co-injection of DL-lysine was used to obtain comprehensive biodistribution at 1, 3, 6, and 24 hours after injection, providing complete pharmacokinetic data for further dosimetry experiments.

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

[0068] 212 Pb-PSC-PEG2-TOC toxicity test 212 Increasing doses of Pb-PSC-PEG2-TOC (0, 0.37, 1.85, 3.33, and 5.55 MBq, or 0, 10, 50, 90, and 150 μCi) were administered to tumor-free CD-1 elite (SOPF) male mice (n = 4 per group). Body weight was measured twice weekly for 3 weeks after injection, and then weekly thereafter. To assess acute renal tubular toxicity, urine samples were collected (via metabolic bronchoscopy) 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 instructions. Three months after injection, serum samples were collected by tail vein nicking and sent to IDEXX Laboratories, Inc. for comprehensive blood chemistry analysis, including blood urea nitrogen (BUN). Further follow-up was performed 6-7 months later for comprehensive blood chemistry and renal histopathological analysis. Hematologic toxicity was assessed by complete blood count (CBC) using an automated veterinary hematology analyzer (ADVIA120, Siemens Healthineers) at weeks 1, 2, and 4 after administration. 212Pb-PSC-PEG2-TOC biodistribution studies were performed at 1, 3, 6, and 24 hours (including bone marrow) to support dosimetry analysis that could be correlated with toxicity profiles in critical organs / tissues, including kidney and bone marrow. Dose estimation was performed with organ-level internal dose assessment (OLINDA, V2.1) software using a 30 g 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 is understood that minor dosage and formulation variations of the compositions and ranges expressed herein can be made and still fall within the scope and spirit of the invention.

[0071] While the present invention has been described with reference to particular compositions, theories of efficacy, and the like, 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 may be made without departing from the scope or spirit of the invention as defined by the appended claims. All such obvious modifications and variations are intended to be included within the scope of the invention as defined by the appended claims. The claims are intended to cover the claimed elements and steps in any order that is effective to achieve the objectives intended therein, unless the context specifically dictates to the contrary.

[0072] The foregoing description has been presented for purposes of illustration and description. It is not intended to be an exhaustive list or to be limited to the precise form disclosed. It is contemplated that other alternative processes and methods apparent to those skilled in the art are considered to be encompassed by the present invention. The foregoing description is merely exemplary of embodiments. Any other modifications, substitutions, and / or additions may be made that are within the intended spirit and scope of the present disclosure. From the foregoing, it can be seen that the exemplary aspects of the present disclosure achieve at least all of their intended objectives. The present invention provides, for example, the following items. (Item 1) Formula I for the therapeutic treatment of cancer and medical imaging: TLX 1. A carcinoma targeting conjugate comprising: T is a radiolabeled SST2R targeting ligand; L is a linker, The conjugate, wherein X is a chelating agent. (Item 2) 2. The conjugate according to item 1, wherein the SST2R targeting ligand is a peptide. (Item 3) 2. The conjugate according to item 1, wherein the SST2R ligand is an antibody or antibody fragment, or a small molecule. (Item 4) 4. The conjugate according to item 2 or 3, wherein the SST2R ligand is radiolabeled with a radionuclide used in medical imaging and / or treatment of cancerous tumors. (Item 5) 5. The conjugate according to item 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 in imaging. (Item 6) 5. The conjugate according to item 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. (Item 7) T is for Tyr 3 The conjugate according to item 1, which is octreotide. (Item 8) 8. The conjugate according to any one of items 1 to 7, wherein L is a chemical linker. (Item 9) 8. The conjugate according to any one of items 1 to 7, wherein L is a polyether linker comprising up to four carbons of an aliphatic carbon chain connecting the chelator to the peptide backbone. (Item 10) L is PEG n and n is 1 to 4. (Item 11) 11. The conjugate according to item 10, wherein n is 2 or 4. (Item 12) 12. The conjugate according to any one of items 1 to 11, wherein X is a chelating agent based on 1,4,7,10-tetraazacyclododecane-7-acetamido-1,4,10-triacetic acid. (Item 13) PSC-PEG 2 / PEG 4 -TOC. (Item 14) A method for treating cancer, comprising administering the conjugate according to item 1. (Item 15) 15. The method of claim 14, wherein the conjugate is administered by a method selected from the group consisting of orally, parenterally, and subcutaneously. (Item 16) 15. The method of claim 14, wherein the conjugate is administered together with an anti-cancer composition. (Item 17) 15. The method of claim 14, wherein the conjugate is administered in a single dose. (Item 18) 15. The method of claim 14, wherein the conjugate is administered in multiple doses. (Item 19) 15. The method of claim 14, wherein the conjugate is administered daily for several consecutive days. (Item 20) 15. The method of claim 14, wherein the conjugate is administered at a dose of up to 150 mCi.

Claims

1. 1. A composition for the therapeutic treatment of cancer or medical imaging, comprising a compound of formula I: T-L-X and a carcinoma targeting conjugate comprising: T is a radiolabeled SST2R targeting ligand, wherein the SST2R targeting ligand is Tyr ３ -octreotide (TOC), wherein said SST2R ligand is radiolabeled with a Pb-203 radionuclide or a Pb-212 radionuclide; L is a linker, L is PEG n , where n is 2 or 4; The composition, wherein X is a chelating agent, and the chelating agent is 1,4,7,10-tetraazacyclododecane-7-acetamido-1,4,10-triacetic acid (PSC).

2. (a) for imaging cancer, wherein the SST2R ligand is radiolabeled with a Pb-203 radionuclide; or (b) The composition of claim 1, for treating cancer, wherein the SST2R ligand is radiolabeled with a Pb-212 radionuclide.

3. The composition of claim 2 , wherein the composition is administered in conjunction with an anti-cancer composition.

4. The composition of claim 1, wherein the SST2R ligand is radiolabeled with a Pb-203 radionuclide.

5. The composition of claim 1, wherein the SST2R ligand is radiolabeled with a Pb-212 radionuclide.

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