Improved structural optimization methods for the therapeutic and diagnostic performance of peptide receptor targeted radionuclide therapy for cancer

Abstract

The present invention provides, in certain embodiments, a cancer-targeting conjugate comprising Formula I: wherein T is a SST2R-targeting ligand, L is a linker, and X is a chelator, for use in the therapeutic treatment of cancer; and methods of use thereof.

Description

[0001] Statement as to Federally Sponsored Research

[0002] This invention was made with government support under R01 CA243014 and P50 CA 174521 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0003] Cross Reference to Related Applications

[0004] This application claims priority to U.S. Provisional Patent Application Serial No. 62 / 967,497, filed January 29, 2020, the disclosure of which is hereby incorporated by reference in its entirety. BACKGROUND

[0005] Neuroendocrine neoplasms (NENs) are a group of heterogeneous tumors whose incidence has been increasing over decades (1, 2). NENs are generally classified 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). In many cases, well-differentiated NETs are less aggressive than poorly differentiated NECs and they respond to several targeted forms of therapy (2). The majority (>80%) of NENs express somatostatin receptors (3), and among them, somatostatin receptor subtype 2 (SSTR2) is a well-known target for a specific therapy called peptide receptor radionuclide therapy (PRRT). The current development of SSTR2-targeted PRRT is based on the beta particle emitters 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 FDA-approved radiopharmaceutical for the treatment of well-differentiated NETs (9). The drug shows therapeutic benefit by tumor response and increases the progression-free survival (PFS) of patients (6-8). However, the benefit is limited to partial responses and complete responses are rarely reported.

[0006] Alpha particle emitters are an alternative to conventional beta particle emitters that bring significantly higher radiation doses (up to several hundred times) from decay in cells and tumor metastases (10) and higher relative biological effectiveness (RBE) due to the high linear energy transfer (LET) of alpha particles (11). Several studies have shown that alpha particle emitters are promising for the treatment of cancer patients that are difficult to treat with beta particle emitters (12, 13). Lead-212 ( 212Pb) is an attractive alpha particle emitter with a half-life (10.64 h) (14) that is well matched to the biological half-life of the peptide (hours) in vivo. Furthermore, 212 Pb has a diagnostic pair of lead-203 203 Pb), which can be used for single photon emission computed tomography (SPECT) (15) by 279 keV photons (81% intensity). 203 The length of the half-life of Pb (51.87 h) is sufficient to monitor the biodistribution and pharmacokinetics of each patient by serial imaging for up to 4-5 212 The half-life of Pb (51.87 h) is sufficient to monitor the biodistribution and pharmacokinetics of each patient by serial imaging for up to 4-5

[0007] Changes in the structure of the peptide can significantly alter the binding affinity, pharmacokinetics and biodistribution of the radiolabeled peptide. Therefore, changes in the structure of the peptide by improving these parameters can potentially improve the therapeutic outcome of the peptide-based therapy. For this application, the operational approach to the final performance of the peptide includes modifying the cyclization method, inserting a linker of appropriate size and composition that will chelate the chelator to the peptide backbone, and developing radionuclide specific chelators. Further optimization of rhenium coordination peptide cyclization (16) and "click"-cyclization and the use of glycine-glycine (GG) linkers (17) have been evaluated in a melanoma model against the melanocortin receptor subtype 1 (MC1R), demonstrating the potential for improved tumor targeting by the method and pharmacokinetics and biodistribution. Many other studies have shown that by different linker insertion (17-19) and chelator modification (20-22), the radiolabeled peptide can be optimized to achieve optimal tumor targeting with improved in vivo performance.

[0008] In this study, Tyr 3 - Octreotide (TOC) with various strategies to modify the peptide structure. For the Pb isotope and other 2+ charged radionuclides, a new chelator composition, 1,4,7,10-tetraazacyclododecane-7-acetamide-1,4,10-triacetic acid (herein referred to as Pb specific chelator or PSC), was introduced. The structure was further optimized by adding polyethylene glycol (PEG) linkers 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 evaluated comprehensively by radiolabeling efficiency, binding affinity, cellular uptake and biodistribution, and the lead compounds were used 203Pb SPECT imaging and 212 Pb therapy / toxicity studies. SUMMARY

[0009] As mentioned above, the present application relates to a new chelator, which in one embodiment is 1,4,7,10-tetraazacyclododecane-7-acetamide-1,4,10-triacetic acid. The chelator is specific for radionuclides with a 2+ charge, including Pb isotopes. The structure includes a polyether linker, preferably a polyethylene glycol (PEG) linker, between the chelator and Tyr 3 octreotide (TOC) or other peptides. The present application is mainly used for any cancer expressing somatostatin receptor subtype 2 (SSTR2), including but not limited to neuroendocrine tumors, small cell lung cancer, meningioma, neuroblastoma, medulloblastoma, paraganglioma and pheochromacytoma. DETAILED DESCRIPTION

[0010] The present application provides in certain embodiments a cancer targeting conjugate comprising Formula I:

[0011] T-L-X

[0012] wherein T is a SST2R targeting ligand,

[0013] L is a linker, and

[0014] X is a chelator,

[0015] for use in the therapeutic treatment of cancer.

[0016] In certain embodiments, the radiolabeled SST2R targeting ligand is a peptide, or an antibody or antibody fragment, or a small molecule.

[0017] In certain embodiments, T is Tyr 3 octreotide.

[0018] In certain embodiments, the SST2R targeting ligand is radiolabeled with a radionuclide chemically bound to the chelator (X) and used for medical imaging and / or cancerous tumor therapy.

[0019] In certain embodiments, the radionuclide is Ga-68, In-Ill, Pb-203, F-18, C-11, Zr-89, Sc-44, Tc-99m or other medical radionuclides used for imaging.

[0020] 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 for treatment of cancerous tumors.

[0021] In certain embodiments, L is a chemical linker inserted in a position between the peptide backbone that recognizes the SST2R protein and the chelator used for radiolabeling the composition with a radionuclide for therapeutic and / or diagnostic imaging; and the linker improves binding and / or internalization of the composition into cells; improves retention of the composition in tumors; and improves clearance of residual composition through other excretion pathways, thereby delivering radiation more precisely to cancerous tissue while minimizing radiation exposure to other organs (e.g., kidneys).

[0022] In certain embodiments, L is a polyether linker consisting of an aliphatic carbon chain comprising up to 4 carbons that links the chelator to the peptide backbone.

[0023] In certain embodiments, L is PEG n wherein n is 1-4. In certain embodiments, n is 2 or 4.

[0024] In certain embodiments, X is radiolabeled with a radionuclide used for medical imaging and / or treatment of cancerous tumors.

[0025] In certain embodiments, the radionuclide is Ga-68, In-Ill, Pb-203, Cu-64 or other Cu isotopes, F-18, C-ll, Zr-89, Sc-44, Tc-99m, or other medical radionuclides used for imaging.

[0026] 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 for treatment of cancerous tumors.

[0027] In certain embodiments, the chelator is based on 1,4,7,10-tetraazacyclododecane-7- acetamide-1,4,10-triacetic acid or other chelators used for binding radionuclides for diagnostic imaging or treatment of cancer or other diseases.

[0028] The present invention provides, in certain embodiments, a conjugate consisting of PSC-PEG2 / PEG4-TOC.

[0029] In certain embodiments, the agent is administered orally or parenterally.

[0030] In certain embodiments, the agent is administered subcutaneously.

[0031] In certain embodiments, the conjugate is administered orally or parenterally.

[0032] In certain embodiments, the method further comprises administering an anti-cancer composition.

[0033] In certain embodiments, the conjugate is administered in a single dose.

[0034] In certain embodiments, the conjugate is administered in multiple doses.

[0035] In certain embodiments, the conjugate is administered sequentially daily for several days.

[0036] In certain embodiments, the conjugate is administered once a week for 1 month. In certain embodiments, the conjugate is administered once a week for up to 6 months.

[0037] In certain embodiments, the conjugate is administered at a dose of 1 mCi for medical imaging.

[0038] In certain embodiments, the conjugate is administered at a dose of up to 10 mCi for medical imaging.

[0039] In certain embodiments, the conjugate is administered at a dose of up to 50 mCi for medical imaging.

[0040] In certain embodiments, the conjugate is administered at a dose of up to 0.1 mCi for medical treatment of a cancerous tumor.

[0041] In certain embodiments, the conjugate is administered at a dose of up to 1 mCi for medical treatment of a cancerous tumor.

[0042] In certain embodiments, the conjugate is administered at a dose of up to 10 mCi for medical treatment of a cancerous tumor.

[0043] In certain embodiments, the conjugate is administered at a dose of up to 100 mCi for medical treatment of a cancerous tumor.

[0044] In certain embodiments, the conjugate is administered for more than one month.

[0045] In certain embodiments, the conjugate is administered for more than one year.

[0046] In certain embodiments, the conjugate is administered at a dose of at least 0.05 μg / day.

[0047] The present invention provides, in certain embodiments, use of a conjugate as described above, wherein:

[0048] a) the conjugate is administered simultaneously with the one or more anti-cancer agents; or

[0049] b) the conjugate and the one or more anti-cancer agents are administered sequentially; or

[0050] c) administration of the one or more anti-cancer agents begins about 1 day to about 10 days prior to administration of the conjugate; or

[0051] d) administration of the conjugate begins about 1 day to about 10 days prior to administration of the one or more anti-cancer agents; or

[0052] e) administration of the conjugate and administration of the one or more anti-cancer agents begin on the same day.

[0053] In certain embodiments, the ligand is a peptide.

[0054] In certain embodiments, the peptide is radiolabeled.

[0055] In certain embodiments, the ligand that targets SST2R is a peptide that binds to somatostatin receptor subtype 2.

[0056] In certain embodiments, the peptide is radiolabeled.

[0057] In certain embodiments, the agent that increases expression of SST2R is administered separately, sequentially, or simultaneously with the ligand that targets SST2R.

[0058] In certain embodiments, the agent that increases expression of SST2R is administered about one day to about 6 months prior to administration of the ligand that targets SST2R.

[0059] In certain embodiments, the agent is administered orally or parenterally.

[0060] In certain embodiments, the agent is administered subcutaneously.

[0061] In certain embodiments, the ligand that targets SST2R is administered orally or parenterally.

[0062] In certain embodiments, administration of the agent begins about 1 day to about 10 days prior to administration of the ligand that targets SST2R.

[0063] In certain embodiments, administration of the agent and administration of the ligand that targets SST2R begin on the same day.

[0064] In certain embodiments, the method further comprises administering an anti-cancer composition. BRIEF DESCRIPTION OF DRAWINGS

[0065] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0066] Figures 1A-1C Structure of the SST2R targeting ligands DOTATOC Figure 1A , PSCTOC Figure 1B and PSC-PEG2 / PEG4-TOC Figure 1C . Peptides were synthesized by standard Fmoc-based solid phase peptide synthesis. They are based on the tyr 3 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.

[0067] Figures 2A-2B Excellent radiolabeling efficiency of the SST2R targeting ligands DOTATOC and PSC conjugated peptides with 203 Pb(A) and 212 Pb(B). 18.5 MBq of 203 Pb or 14.1 MBq of 212 Pb were reacted with 10 nmol of peptide in 0.5 M sodium acetate (NaOAc) buffer (pH = 5.4, 1 ml reaction volume). For 203 Pb labeling, the reaction was performed at various temperatures (25 °C, 50 °C or 85 °C) and reaction times (10 min, 20 min or 30 min). DOTATOC and PSCTOC were chosen for 212 Pb labeling and the reaction was performed at a fixed temperature (85 °C) and increasing times (up to 30 min).

[0068] Figure 3 Competitive inhibition of the binding of 125 i-tyr 3 octreotide ( 125 i-TOC) to SSTR2 positive AR42J cells by the TOC, DOTATOC and PSC conjugated peptides. IC 50Values, 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 (at least n = 6 from at least three biological replicates of DOTATOC and PSC-TOC; n = 4-6 from two biological replicates of TOC, PSC-PEG2-TOC, and PSC-PEG4-TOC).

[0069] Figure 4 Cellular uptake of Pb-labeled DOTATOC, PSC-TOC, and PSC-PEG2-TOC in AR42J cells. 203 Cellular uptake of Pb-labeled DOTATOC, PSC-TOC, and PSC-PEG2-TOC. 200,000 CPM of HPLC purified 203 Pb-labeled peptides were incubated with AR42J SST2R-expressing cells at 37°C for up to 120 min, and cellular uptake of each radiotracer was measured. Data are shown as the mean percentage ± SD of cellular uptake relative to incubation activity (n = 4).

[0070] Figures 5A-5B . 203 Biodistribution of Pb-labeled SST2R-targeting DOTATOC, PSC-TOC, and PSC-PEG2-TOC in athymic nude mice bearing AR42J tumors. After i.v. injection of 37 kBq of each radiotracer, mice were sacrificed at 1, 3, and 24 h post-injection. 203 Biodistribution of Pb-labeled peptides. After i.v. injection of 37 kBq of each radiotracer, mice were sacrificed at 1, 3, and 24 h post-injection. Biodistribution was observed (A), and the percentage of injected dose per gram of tissue (%ID / g) as a function of time and the tumor-to-kidney ratio (B) are shown for tumor and kidney. Data are shown as the mean percentage of injected dose per gram of tissue (%ID / g) or the relative mean tumor-to-kidney ratio ± SD (n = 3).

[0071] Figures 6A-6C Biodistribution of Pb-labeled SST2R-targeting DOTATOC, PSC-TOC, and PSC-PEG2-TOC in athymic nude mice bearing AR42J tumors. After i.v. injection of 37 kBq of each radiotracer, mice were sacrificed at 1, 3, and 24 h post-injection. 203 Pb SPECT / CT images. (A) Athymic nu / nu female mice bearing AR42J tumors were imaged at 3 h and 24 h post-injection of 11.1 MBq of each radiotracer. Co-injection of 30 nmol of unlabeled peptide was used for blocking imaging to confirm tumor specificity. 203 Pb-DOTATOC and 203 Pb-PSC-PEG2-TOC. Mice bearing AR42J tumors were imaged at 3 h and 24 h post-injection. Co-injection of 30 nmol of unlabeled peptide was used for blocking imaging to confirm tumor specificity. (B) The tumor-to-kidney ratio over time was analyzed from the acquired images using Inveon Research Workstation software. (C) Mice were euthanized at 30 h post-injection and biodistribution was obtained.

[0072] Figure 7Stability of PSC-PEG2-TOC in water and human serum. PSC-PEG2-TOC was radiolabeled with 50 MBq (1.34 mCi) of 203 Pb and 9 MBq (0.24 mCi) of purified radiolabeled 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 h and 24 h by a radio-HPLC system (Agilent 1200 series combined with an IN / US beta-RAM type 4 radio detector).

[0073] Figure 8 Clinical relevant high specific activity of PSC-PEG2-TOC 203 Pb. Radiolabeling was performed at 85°C for 30 min in 0.5 M sodium acetate (NaOAc) buffer (pH = 5.4, 1-2 ml reaction volume) with high activity 203 Pb at 90 MBq / nmol DOTATOC (A), 90 MBq / nmol PSC-PEG2-TOC (B) or 120 MBq / nmol PSC-PEG2-TOC (C) for reference.

[0074] Figure 9 3 h post injection, 203 Pb / 212 Biodistribution of Pb-labeled PSC-PEG2-TOC in athymic nude mice bearing AR42J tumors. 74 kBq of 212 Pb-PSC-PEG2-TOC (specific activity, 3.7 MBq / nmol) was injected via tail vein and biodistribution was obtained 3 h post injection (n = 4). This data was directly compared to the previously obtained 203 Pb-PSC-PEG2-TOC (specific activity, 22.2 MBq / nmol; Fig. 5).

[0075] Figures 10A-10B By co-injection of DL-lysine, 203 The kidney accumulation of pb-PSC-PEG2-TOC was reduced and specific tumor binding of the radiolabeled peptide in AR42J bearing nude mice was demonstrated by co-injection of excess unlabeled peptide and tumor blockade. (A) 3 h post injection with lysine co-injection (400 mg / kg), without lysine co-injection or with unlabeled peptide co-injection (for tumor blockade; 10 nmol PSC-PEG2-TOC), 203Biodistribution of Pb-PSC-PEG2-TOC in nude mice bearing AR42J tumors. (B) In the presence of co-injection of DL-lysine (400 mg / kg), at 1, 3, 6 and 24 h post-injection, 203 Complete biodistribution of Pb-PSC-PEG2-TOC. Results are expressed as percentage of injected dose per gram of tissue (%ID / g) ± S.D. (n=3).

[0076] Figures 11A-11C . Initial tumor weight measurements performed on mice bearing AR42J-SST2R-expressing tumors at 30 days post-treatment 212 Therapeutic results of the Pb-PSC-PEG2-TOC treatment study. 212 Pb-PSC-PEG2-TOC treatment was initiated when the average tumor size became about 150 mm 3 Pb-PSC-PEG2-TOC, by tail vein injection of 0.37 MBq (10 μCi) and 1.85 MBq (50 μCi) 212 Pb-PSC-PEG2-TOC, in the presence of co-injection of DL-lysine (400 mg / kg) to block the uptake of the radiopharmaceutical by the kidneys.

[0077] Figures 12A-12F . Dose escalation (up to 150 μCi) of 212 Dose kinetics and toxicity of Pb-PSC-PEG2-TOC in CD-1 Elite (SOPF) male mice. (A) Body weight changes after injection of 212 Dose kinetics and toxicity of Pb-PSC-PEG2-TOC in CD-1 Elite (SOPF) male mice. (A) Body weight changes after injection of 212 Biodistribution of Pb-PSC-PEG2-TOC in CD-1 Elite (SOPF) male mice. 212 Pb for the study to include 212 Potential demetallation effects and redistribution in the bone marrow of Pb. (C) Estimated kidney doses from the production of increasing doses of 212 Pb-PSC-PEG2-TOC. Organ level internal dose assessment (OLINDA) V2.1 was used for the dose evaluation in mice using a 30 g mouse voxel phantom model. (D-E) Assessment of the kidney damage by urinary neutrophil gelatinase-associated lipocalin (uNGAL; D) at day 1 and day 3 post-administration and blood urea nitrogen (BUN; E) at 3 months post-administration from increasing doses of 212Levels of markers of renal toxicity produced by Pb-PSC-PEG2-TOC. (F) Complete blood counts (CBC) at week 1, week 2, and week 4 post administration indicated reversible hematologic toxicity.

[0078] The following examples are intended to further illustrate the present application. They are not intended to limit the application in any way.

[0079] Materials and Methods

[0080] Peptide synthesis

[0081] DOTATOC, PSCTOC, PSC-PEG2-TOC and PSC-PEG4-TOC were synthesized by standard Fmoc-based solid phase peptide synthesis. Linear peptide D-Phe-Cys-Tyr-D-Trp-Lys-Thr-Cys-Thr(01) was synthesized on resin at 100 pmol scale using an automated peptide synthesizer (AAPPTEC Apex 396) and the N-terminus of the linear peptide was deprotected by 25% piperidine (PIP) at the end of the automated synthesis. For PSC-PEG2-TOC or PSC-PEG4-TOC, the PEG linker (PEG2 or PEG4) was added manually. The peptide-resin was suspended with N,N-dimethylformamide (DMF) and 5 equivalents (equiv.) of Fmoc-NH-PEG2 / PEG4-propionic acid (purchased from AAPPTEC), 2-(7-aza-lH-benzotriazol-l-yl)-l,l,3,3-tetramethyluronium hexafluorophosphate (HATU) and 1-hydroxybenzotriazole (HOBt) and 10 equiv. of N,N-diisopropylethylamine (DIPEA) were added and reacted at 37 °C for 2 h while mixing. The Fmoc on the N-terminus of the peptide-resin was then manually deprotected by 25% piperidine (in DMF) with gentle mixing for 10 min at 25 °C and washed with DMF / dichloromethane (DCM) / methanol and the process was repeated. The linear peptide with an open N-terminus on resin was then re-suspended in DMF and 5 equiv. of DOTA-tris(t-butyl ester) or PSC-bis(t-butyl ester), HATU and HOBt and 10 equiv. of DIPEA were added and reacted overnight at 37 °C while stirring. The success of each coupling / deprotection step was verified by Kaiser test and the process was repeated until success. The linear peptide was then cyclized by iodo-oxidation. Iodine (I2; 20 equiv.) was dissolved in 6 ml DMF and added to the peptide-resin and allowed to react for 3 hours from the trityl deprotection of the cysteines and simultaneously promote disulfide formation by oxidation. The resin and protecting groups were then cleaved from the cyclized peptide by adding 3 mL cleavage cocktail (93% trifluoroacetic acid, 3% triisopropylsilane, 4% water) for 2 h at room temperature followed by ethyl ether precipitation on ice for at least 4 h. The crude peptide was then purified by semi-preparative high-performance liquid chromatography (HPLC) with C-18 column (Vydac 10 x 250 mm, 10 pm; Grace, Deerfield, IL). The collected samples were concentrated by rotary evaporation and lyophilized. The purified peptides were characterized by mass spectrometer.

[0082] 203 Pb / 212 Pb radiolabeling efficiency

[0083] DOTATOC and PSC-conjugated peptides for use 203 Pb and 212 Pb were radiolabeled. 18.5 MBq of 203 Pb or 14.1 MBq of 212 Pb were reacted with 10 nmol of peptide in 0.5 M sodium acetate (NaOAc) buffer (pH = 5.4, 1 ml reaction volume). For 203 Pb labeling, the reaction was performed at various temperatures (25 °C, 50 °C, or 85 °C) and reaction times (10 min, 20 min, or 30 min). DOTATOC and PSCTOC were selected for 212 Pb labeling and the reaction was performed at a fixed temperature (85 °C) and increasing times (up to 30 min). After the reaction, the resulting material was spotted on pre-dried instant thin layer chromatography (iTLC) strips and developed in 0.1 M NaOAc buffer with 10 mM diethylenetriaminepentaacetic acid (DTPA). The strips were then cut in half and the radioactivity of each part (top, no 203 Pb; 212 Pb, 239 keV) was measured using a NaI detector by isotope-specific gamma peaks 203 Pb / 212 Pb; bottom, 203 Pb / 212 Pb-labeled peptides) was measured.

[0084] 125 I-TOC competition binding assay

[0085] TOC was labeled with iodine-125 ( 125 I) by the conventional chloramine T method as described elsewhere (23). 1.0 x 10 5 AR42J rat pancreatic acinar cells were seeded into 24-well plates coated with poly-D-lysine. After 3 days, the cells were incubated with 30,000 CPM of 125 I-TOC in binding medium (RPMI 1640 supplemented with 0.2% bovine serum albumin; 0.3 mM 1,10-phenanthroline) with increasing concentrations (10 -11 to 10 -6 M) of TOC, DOTATOC, PSCTOC, PSC-PEG2-TOC, or PSC-PEG4-TOC at 37 °C for 2 hours. The cells were then washed twice with ice-cold PBS and lysed with 0.5 N NaOH and the radioactivity was measured by a gamma counter. The half maximal inhibitory concentration (IC50) was determined using GraphPad Prism V8.0.50 ).

[0086] 203 Internalization and efflux of Pb-labeled peptides

[0087] AR42J cells were seeded at a density of 37 MBq of 203 Pb were labeled with 10 nmol of DOTATOC, PSCTOC and PSC-PEG2-TOC and the labeled peptides were separated from the unlabeled peptides by a previously developed separation method (15) based on the different retention times of the labeled and unlabeled peptides by high-performance liquid chromatography (HPLC). The HPLC separated radiolabeled peptides were then purified through a C-18 column. AR42J cells seeded at a density of 2.0 x 10 5 AR42J cells seeded at a density of 37 MBq of 203 Pb-labeled peptides were incubated at 37°C for up to 120 min. Cells were then washed twice with ice-cold PBS and membrane-bound radioactivity was washed off and collected by 50 mM acidic (pH = 4) sodium acetate buffer. The remaining cells were lysed by the addition of 0.5 N NaOH for 5 min. The radioactivity of each fraction (membrane-bound and internalized) was counted by a 310 Cobra II gamma counter (PerkinElmer, Freemont, CA). For efflux assays, cells were incubated with 200,000 CPM of HPLC purified 203 Pb-labeled peptides at 37°C for 120 min. Cells were then washed twice with ice-cold PBS and supplemented with binding medium. At 60 min and 120 min, the radioactivity of efflux (into the medium), membrane-bound and internalized (collected by the same way as the internalization assay) was counted.

[0088] 203 Biodistribution of Pb-labeled peptides

[0089] AR42J cells were seeded at a density of 37 kBq of 203 Pb-labeled DOTATOC, PSCTOC and PSC-PEG2-TOC (specific activity: 22.2 MBq / nmol) were injected into female athymic nu / nu mice bearing AR42J tumors via the tail vein. Mice were euthanized by cervical dislocation at 1, 3 and 24 h post-injection under isoflurane anesthesia. Tumors and organs of interest were harvested and the weight of the collected organs was measured. The radioactivity of the samples was measured by a PerkinElmer 310 Cobra II gamma counter (PerkinElmer, Freemont, CA).

[0090] Tumor and kidney dosimetry

[0091] The Particle and Heavy Ion Transport code System (PHITS) was used for dosimetry analysis. For kidney dosimetry, the DigiMouse voxel phantom model was used and the voxel size of the model was adjusted in order to make the kidney volume identical to the average kidney volume from biodistribution studies (carrying AR42J; 8-10 weeks) of female athymic nude mice (288.7+-41.4 mg; 28 mice). The elemental composition and mass density of the kidney were assumed to be identical to the human reference adult values obtained from the International Commission on Radiation Units and measurements (ICRU) report 46. For tumor dosimetry, a spherical volume was constructed based on the average tumor mass of 28 mice (156.9+-0.096 mg). The elemental composition (adenoid cystic carcinoma) and mass density (1.04 g / cm3) of the tumor were assumed to be identical to the values obtained from the ICRU report 46. 3 Adapted from Maughan et al. 1997 Med Phys 24(8): 1241-4 and RM Thomson et al. 2013 Phys. Med. Biol. 58: 1123-50. At least 1 million particles were transported for the Monte Carlo simulation to reduce the statistical uncertainty to less than 1%.

[0092] 203 Tandem SPECT / CT imaging of 203 Pb-DOTATOC versus

[0093] 1.85 GBq (50 mCi; 61.7 MBq / nmol) of 203 Pb-DOTATOC and Pb-PSC-PEG2-TOC were labeled with DOTATOC and PSC-PEG2-TOC. 11.1 MBq of each 203 Pb-labeled peptide was injected into AR42J-bearing mice via the tail vein and mice were imaged 3 h and 24 h post-injection. Separately, the same activity of 203 Pb-PSC-PEG2-TOC was co-injected with 30 nmol of unlabeled PSC-PEG2-TOC for blocking studies to confirm tumor specificity of the radiotracers. Images were reconstructed and analyzed using the Inveon Research Workstation software with the same parameter settings. Analysis was done by body weight corrected standardized uptake value (SUVbw) and biodistribution of mice was obtained 30 h post-administration.

[0094] 203 Stability of Pb-PSC-PEG2-TOC in water and human serum

[0095] As a identified lead compound, PSC-PEG2-TOC was further evaluated in various aspects. PSC-PEG2-TOC was measured using 50 MBq (1.34 mCi). 203 Pb was radiolabeled and purified via 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 h. After incubation, the resulting peptide will have… 203 Serum samples of Pb-PSC-PEG2-TOC were transferred to an Amicon Ultra centrifugal filter (3K; Millipore) and centrifuged using a Beckman Coulter Avanti J-25I centrifuge. Permeate (serum sample) or samples in water were analyzed using a radiometric HPLC system (Agilent 1200 series with IN / US β-RAM 4 radio detector) to monitor peptide degradation.

[0096] Clinically relevant high specific activity of PSC-PEG2-TOC 203 Pb radiolabeling

[0097] PSC-PEG2-TOC was used with high specific activity of 90 MBq / nmol or 120 MBq / nmol. 203 Pb was radiolabeled. DOTATOC was also labeled with 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, 1-2 ml reaction volume). 2 μl of Pb containing... 203 The Pb-labeled peptide reaction product was spotted on an instantaneous thin-layer chromatography (iTLC) band. The sample band was developed in the mobile phase (0.2 M sodium acetate and 20 mM EDTA) and then imaged using a fluorescence imager (Typhoon FLA7000). The band was cut in half and passed through a NaI detector. 203 The radioactivity of the μbγ peak (279keV) on each side of the band is measured to determine the radiolabeling efficiency.

[0098] 212 Pb-PSC-PEG ２ -Biodistribution of TOC in nude mice carrying AR42J

[0099] 74kBq was delivered via the tail vein. 212 μb-PSC-PEG2-TOC (specific activity, 3.7 MBq / nmol) was injected into athymic nude mice carrying AR42J, and biodistribution was obtained 3 h post-injection (n=4). This data was directly compared with previously obtained data. 203The biodistribution of Pb-PSC-PEG2-TOC (specific activity, 22.2 MBq / nmol; Figure 5) was compared. The data determined 203 Pb-PSC-PEG2-TOC as 212 The adequacy of Pb-PSC-PEG2-TOC as an imaging and dosimetric surrogate.

[0100] With lysine co-infusion 203 Biodistribution of Pb-PSC-PEG2-TOC in AR42J-bearing nude mice

[0101] With and without co-injection of DL-lysine (400 mg / kg; 8 mg / animal), 37 kBq of 203 Pb-PSC-PEG2-TOC (specific activity: 22.2 MBq / nmol) was injected into AR42J tumor-bearing nude mice via the tail vein to see if co-injection of lysine could reduce the non-specific kidney uptake of the radiotracer. In addition, by co-injecting 10 nmol of unlabeled peptide (without lysine) with 37 kBq of 203 Pb-PSC-PEG2-TOC, a separate group was added for tumor blockade to verify the specificity of tumor targeting. These mice were then euthanized 3 h post-injection and biodistribution was assessed (n = 3 for each group). In a separate study, with co-injection of DL-lysine, comprehensive biodistribution was obtained at 1, 3, 6, and 24 h post-injection to obtain complete pharmacokinetic data for further dosimetric studies.

[0102] 212 Pb-PSC-PEG2-TOC therapy

[0103] 5.0 x 10 6 AR42J rat pancreatic acinar cells were implanted on the left shoulder of female athymic nu / nu mice. After 10 days, when the average tumor size became about 150 mm 3 , 274 MBq (7.4 mCi) 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 radiolabeled peptide was purified by C-18 and resuspended in saline with ascorbic acid (1 mg / ml). 0.37 MBq (10 μCi) and 1.85 MBq (50 μCi) of 212 pb-PSC-PEG2-TOC were injected via the tail vein. Co-injection of DL-lysine (400 mg / kg) was used to block the kidney uptake of the radiotherapeutic agent.

[0104] 212 Pb-PSC-PEG2-TOC toxicity study

[0105] Increasing doses (0, 0.37, 1.85, 3.33, and 5.55 MBq or 0, 10, 50, 90, and 150 μCi) of 212 Pb-PSC-PEG2-TOC was administered to tumor-free CD-1 Elite (SOPF) male mice (n = 4 for each group). Body weight was measured twice per week until 3 weeks post-injection and once per week thereafter. Urine samples were collected (by metabolic cage) at day 1 and day 3 post-administration to assess acute renal tubular toxicity to the kidneys. Urine samples were centrifuged and levels of urine neutrophil gelatinase-associated lipocalin (uNGAL) were measured using the Mouse NGAL ELISA Kit (kit 042; BIOPORTO Diagnostics) according to the manufacturer’s manual. Three months post-injection, serum samples were collected via tail-vein nick, sent to IDEXX Laboratories, Inc., and analyzed for comprehensive blood chemistry including blood urea nitrogen (BUN). Further follow-up will be conducted at 6-7 months for comprehensive blood chemistry testing and renal histopathology analysis. Hematological toxicity was assessed by complete blood count (CBC) using an automated veterinary hematology analyzer (ADVIA 120, Siemens Healthineers) at week 1, week 2, and week 4 post-administration. In addition, blood samples were collected at 1, 3, 6, and 24 h post-injection for blood chemistry analysis. 212 Pb-PSC-PEG2-TOC biodistribution study (including bone marrow) to support dosimetry analysis related to toxicity profile with key organs / tissues including kidney and bone marrow. Dose estimates were performed using a 30 g mouse voxel phantom model in the Organ Level INternal Dose Assessment (OLINDA, V2.1) software.

[0106] References

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[0130] It is understood that minor dose and formulation modifications can be made to the compositions and ranges expressed herein and still be within the scope and spirit of the application.

[0131] The application has been described with reference to particular illustrative embodiments, effective theory of operation, etc. It will be apparent to one of ordinary skill in the art that the application is not intended to be limited to such illustrative embodiments or mechanisms, and that modifications can be made by persons of ordinary skill in the art without departing from the scope or spirit of the application as defined by the appended claims. It is intended that all such obvious modifications and variations be considered within the scope of the application as defined by the claims appended hereto. The claims are intended to cover components and steps in any order which effectively accomplish the intended goal, unless the order is explicitly dictated by the claim language.

[0132] The foregoing description has been presented for purposes of illustration and description. It is not intended to be an exhaustive list or to limit the application to the precise form disclosed. Other alternative processes and methods which are apparent to those of ordinary skill in the art are considered to be within the scope of the application. The description is merely an example. It is understood that any other modifications, substitutions, and / or additions can be made by one of ordinary skill in the art in the intended spirit and scope of the disclosure. From the foregoing, it will be apparent that the exemplary aspects of the disclosure realize at least all the intended objectives.

Claims

1. A compound having the structure: 。 2. A conjugate comprising a radionuclide chelated to the compound of claim 1.

3. The conjugate of claim 2, wherein the radionuclide is 203 Pb or 212 Pb.

4. The conjugate of claim 3, wherein the radionuclide is 203 Pb.

5. The conjugate of claim 3, wherein the radionuclide is 212 Pb.

6. Use of a conjugate for the manufacture of a medicament for the treatment or imaging of a cancer expressing somatostatin receptor subtype 2 (SSTR2), wherein the conjugate comprises a chelate of a compound having the formula 203 Pb or 212 Pb: ， wherein n is 2.

7. The use of claim 6, wherein the conjugate is used to treat a cancer that expresses SSTR2, and wherein the conjugate comprises 212 Pb.

8. The use of claim 6, wherein the conjugate is used to image a cancer expressing SSTR2, and wherein the conjugate comprises 203 Pb or 212 Pb.

9. The use of any one of claims 6-8, wherein the cancer is selected from neuroendocrine tumors, meningiomas, and medulloblastomas.

10. The use of any one of claims 6-8, wherein the cancer is selected from small cell lung cancer, neuroblastoma, and paraganglioma.

11. The use of any one of claims 6-8, wherein the cancer is pheochromocytoma.

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