Effectiveness of [67Cu]Cu-trastuzumab as a theranostic against HER2-positive breast cancer.

PURPOSE: To evaluate the imaging and therapeutic properties (theranostic) of 67Cu-labeled anti-human epidermal growth factor receptor II (HER2) monoclonal antibody trastuzumab against HER2-positive breast cancer (BC). METHODS: We conjugated trastuzumab with p-SCN-Bn-NOTA, 3p-C-NETA-NCS, or p-SCN-Bn-DOTA, and radiolabeled with [67Cu]CuCl2. Immunoconjugate internalization was evaluated in BT-474, JIMT-1 and MCF-7 BC cells. In vitro stability was studied in human serum (HS) and Phosphate Buffered Saline (PBS). Flow cytometry, radioligand binding and immunoreactive fraction assays were carried out. ImmunoSPECT imaging of [67Cu]Cu-NOTA-trastuzumab was done in mice bearing BT-474, JIMT-1 and MCF-7 xenografts. Pharmacokinetic was studied in healthy Balb/c mice while dosimetry was done in both healthy Balb/c and in athymic nude mice bearing JIMT-1 xenograft. The therapeutic effectiveness of [67Cu]Cu-NOTA-trastuzumab was evaluated in mice bearing BT-474 and JIMT-1 xenografts after a single intravenous (i.v.) injection of ~ 16.8 MBq. RESULTS: Pure immunoconjugates and radioimmunoconjugates (> 95%) were obtained. Internalization was HER2 density-dependent with highest internalization observed with NOTA-trastuzumab. After 5 days, in vitro stabilities were 97 ± 1.7%, 31 ± 6.2%, and 28 ± 4% in HS, and 79 ± 3.5%, 94 ± 1.2%, and 86 ± 2.3% in PBS for [67Cu]Cu-NOTA-trastuzumab, [67Cu]Cu-3p-C-NETA-trastuzumab and [67Cu]Cu-DOTA-trastuzumab, respectively. [67Cu]Cu-NOTA-trastuzumab was chosen for further evaluation. BT-474 flow cytometry showed low KD, 8.2 ± 0.2 nM for trastuzumab vs 26.5 ± 1.6 nM for NOTA-trastuzumab. There were 2.9 NOTA molecules per trastuzumab molecule. Radioligand binding assay showed a low KD of 2.1 ± 0.4 nM and immunoreactive fraction of 69.3 ± 0.9. Highest uptake of [67Cu]Cu-NOTA-trastuzumab was observed in JIMT-1 (33.9 ± 5.5% IA/g) and BT-474 (33.1 ± 10.6% IA/g) xenograft at 120 h post injection (p.i.). Effectiveness of the radioimmunoconjugate was also expressed as percent tumor growth inhibition (%TGI). [67Cu]Cu-NOTA-trastuzumab was more effective than trastuzumab against BT-474 xenografts (78% vs 54% TGI after 28 days), and JIMT-1 xenografts (90% vs 23% TGI after 19 days). Mean survival of [67Cu]Cu-NOTA-trastuzumab, trastuzumab and saline treated groups were > 90, 77 and 72 days for BT-474 xenografts, while that of JIMT-1 were 78, 24, and 20 days, respectively. CONCLUSION: [67Cu]Cu-NOTA-trastuzumab is a promising theranostic agent against HER2-positive BC.

Loratadine analogues as MAGL inhibitors.

Compound 12a (JZP-361) acted as a potent and reversible inhibitor of human recombinant MAGL (hMAGL, IC50=46 nM), and was found to have almost 150-fold higher selectivity over human recombinant fatty acid amide hydrolase (hFAAH, IC50=7.24 µM) and 35-fold higher selectivity over human α/ß-hydrolase-6 (hABHD6, IC50=1.79 µM). Additionally, compound 12a retained H1 antagonistic affinity (pA2=6.81) but did not show cannabinoid receptor activity, when tested at concentrations ⩽ 10 µM. Hence, compound 12a represents a novel dual-acting pharmacological tool possessing both MAGL-inhibitory and antihistaminergic activities.

Radiochemical and Biological Evaluation of 3p-C-NETA-ePSMA-16, a Promising PSMA-Targeting Agent for Radiotheranostics.

Bifunctional chelators (BFCs) are a key element in the design of radiopharmaceuticals. By selecting a BFC that efficiently complexes diagnostic and therapeutic radionuclides, a theranostic pair possessing almost similar biodistribution and pharmacokinetic properties can be developed. We have previously reported 3p-C-NETA as a promising theranostic BFC, and the encouraging preclinical outcomes obtained with [18F]AlF-3p-C-NETA-TATE led us to conjugate this chelator to a PSMA-targeting vector for imaging and treatment of prostate cancer. In this study, we synthesized 3p-C-NETA-ePSMA-16 and radiolabeled it with different diagnostic (111In, 18F) and therapeutic (177Lu, 213Bi) radionuclides. 3p-C-NETA-ePSMA-16 showed high affinity to PSMA (IC50 = 4.61 ± 1.33 nM), and [111In]In-3p-C-NETA-ePSMA-16 showed specific cell uptake (1.41 ± 0.20% ID/106 cells) in PSMA expressing LS174T cells. Specific tumor uptake of [111In]In-3p-C-NETA-ePSMA-16 was observed up to 4 h p.i. (1.62 ± 0.55% ID/g at 1 h p.i.; 0.89 ± 0.58% ID/g at 4 h p.i.) in LS174T tumor-bearing mice. Only a faint signal could be seen at 1 h p.i. in the SPECT/CT scans, whereas dynamic PET/CT scans performed after administration of [18F]AlF-3p-C-NETA-ePSMA-16 in PC3-Pip tumor xenografted mice resulted in a better tumor visualization and imaging contrast. Therapy studies with short-lived radionuclides such as 213Bi could further elucidate the therapeutic potential of 3p-C-NETA-ePSMA-16 as a radiotheranostic.

Direct comparison of [18F]AlF-NOTA-JR11 and [18F]AlF-NOTA-octreotide for PET imaging of neuroendocrine tumors: Antagonist versus agonist.

BACKGROUND: [18F]AlF-NOTA-octreotide is an 18F-labeled somatostatin analogue which is a good clinical alternative for 68Ga-labeled somatostatin analogues. However, radiolabeled somatostatin receptor (SSTR) antagonists might outperform agonists regarding imaging sensitivity of neuroendocrine tumors (NETs). No direct comparison between the antagonist [18F]AlF-NOTA-JR11 and the agonist [18F]AlF-NOTA-octreotide as SSTR PET probes is available. Herein, we present the radiosynthesis of [18F]AlF-NOTA-JR11 and compare its NETs imaging properties directly with the established agonist radioligand [18F]AlF-NOTA-octreotide preclinically. METHODS: [18F]AlF-NOTA-JR11 was synthesized in an automated synthesis module. The in vitro binding characteristics (IC50) of [natF]AlF-NOTA-JR11 and [natF]AlF-NOTA-octreotide were evaluated and the in vitro stability of [18F]AlF-NOTA-JR11 was determined in human serum. In vitro cell binding and internalization was performed with [18F]AlF-NOTA-JR11 and [18F]AlF-NOTA-octreotide using SSTR2 expressing cells and the pharmacokinetics were evaluated using µPET/CT in mice bearing BON1.SSTR2 tumor xenografts. RESULTS: Excellent binding affinity for SSTR2 was found for [natF]AlF-NOTA-octreotide (IC50 of 25.7 ± 7.9 nM). However, the IC50 value for [natF]AlF-NOTA-JR11 (290.6 ± 71 nM) was 11-fold higher compared to [natF]AlF-NOTA-octreotide, indicating lower affinity for SSTR2. [18F]AlF-NOTA-JR11 was obtained in a good RCY (50 ± 6 %) but with moderate RCP of 94 ± 1 %. [18F]AlF-NOTA-JR11 demonstrated excellent stability in human serum (>95 % after 240 min). 2.7-fold higher cell binding was observed for [18F]AlF-NOTA-JR11 as compared to [18F]AlF-NOTA-octreotide after 60 min. µPET/CT images demonstrated comparable pharmacokinetics and tumor uptake between [18F]AlF-NOTA-JR11 (SUVmax: 3.7 ± 0.8) and [18F]AlF-NOTA-octreotide (SUVmax: 3.6 ± 0.4). CONCLUSIONS: [18F]AlF-NOTA-JR11 was obtained in good RCY, albeit with a moderate RCP. The cell binding study showed significant higher binding of [18F]AlF-NOTA-JR11 compared to [18F]AlF-NOTA-octreotide, despite the higher IC50 value of AlF-NOTA-JR11. However, pharmacokinetics and in vivo tumor uptake was comparable for both radiotracers. Novel Al18F-labeled derivatives of JR11 with higher SSTR2 affinity should be developed for increased tumor uptake and NET imaging sensitivity.

18F-Labeled Somatostatin Analogs as PET Tracers for the Somatostatin Receptor: Ready for Clinical Use.

Molecular imaging of the somatostatin receptor plays a key role in the clinical management of neuroendocrine tumors. PET imaging with somatostatin analogs (SSAs) labeled with 68Ga or 64Cu is currently the gold standard in clinical practice. However, widespread implementation of 68Ga imaging is often hampered by practical and economic issues related to 68Ge/68Ga generators. 18F offers several advantages to tackle these issues. Recent developments in radiochemistry have allowed a shift from 68Ga toward 18F labeling, leading to promising clinical translations of 18F-labeled SSAs, such as Gluc-Lys-[18F]FP-TOCA, [18F]F-FET-ßAG-TOCA, [18F]AlF-NOTA-octreotide, [18F]SiTATE, and [18F]AlF-NOTA-JR11. This review gives an update of currently available clinical data regarding 18F-labeled SSA tracers and provides justification for the clinical application of this class of tracers.

Gamma counting protocols for the accurate quantification of 225Ac and 213Bi without the need for a secular equilibrium between parent and gamma-emitting daughter.

BACKGROUND: Quantification of actinium-225 through gamma counter measurements, when there is no secular equilibrium between actinium-225 and its gamma emitting daughters bismuth-213 and/or francium-221, can provide valuable information regarding the possible relocation of recoiled daughters such that related radiotoxicity effects can be evaluated. This study proposes a multiple time-point protocol using the bismuth-213 photopeak with measurements before secular equilibrium between actinium-225 and bismuth-213, and a single time-point protocol using both the francium-221 and bismuth-213 photopeak while assuming secular equilibrium between actinium-225 and francium-221 but not between bismuth-213 and actinium-225. RESULTS: Good agreement (i.e. 3% accuracy) was obtained when relying on a multiple time-points measurement of bismuth-213 to quantify both actinium-225 and excess of bismuth-213. Following scatter correction, actinium-225 can be accurately quantified using the francium-221 in a single time-point measurement within 3% of accuracy. The analysis performed on the stability data of [225Ac]Ac-DEPA and [225Ac]Ac-DOTA complexes, before secular equilibrium between bismuth-213 and actinium-225 was formed, revealed considerable amounts of unbound bismuth-213 (i.e. more than 90%) after 24 h of the radiolabeling most likely due to the recoiled daughter effect. CONCLUSION: Both protocols were able to accurately estimate 225Ac-activities provided the francium-221 energy window was corrected for the down scatter of the higher-energy gamma-emissions by bismuth-213. This could prove beneficial to study the recoiled daughter effect and redistribution of free bismuth-213 by monitoring the accumulation or clearance of bismuth-213 in different tissues during biodistribution studies or in patient samples during clinical studies. On the other hand, the single gamma counter measurement protocol, although required a 30 min waiting time, is more time and cost efficient and therefore more appropriate for standardized quality control procedures of 225Ac-labeled radiopharmaceuticals.

3p-C-NETA: A versatile and effective chelator for development of Al18F-labeled and therapeutic radiopharmaceuticals.

Background: Radiolabeled somatostatin analogues (e.g. [68Ga]Ga-DOTATATE and [177Lu]Lu-DOTATATE) have been used to diagnose, monitor, and treat neuroendocrine tumour (NET) patients with great success. [18F]AlF-NOTA-octreotide, a promising 18F-labeled somatostatin analogue and potential alternative for 68Ga-DOTA-peptides, is under clinical evaluation. However, ideally, the same precursor (combination of chelator-linker-vector) can be used for production of both diagnostic and therapeutic radiopharmaceuticals with very similar (e.g. Al18F-method in combination with therapeutic radiometals 213Bi/177Lu) or identical (e.g. complementary Tb-radionuclides) pharmacokinetic properties, allowing for accurate personalised dosimetry estimation and radionuclide therapy of NET patients. In this study we evaluated 3p-C-NETA, as potential theranostic Al18F-chelator and present first results of radiosynthesis and preclinical evaluation of [18F]AlF-3p-C-NETA-TATE. Methods: 3p-C-NETA was synthesized and radiolabeled with diagnostic (68Ga, Al18F) or therapeutic (177Lu, 161Tb, 213Bi, 225Ac and 67Cu) radionuclides at different temperatures (25-95 °C). The in vitro stability of the corresponding radiocomplexes was determined in phosphate-buffered saline (PBS) and human serum. 3p-C-NETA-TATE was synthesized using standard solid/liquid-phase peptide synthesis. [18F]AlF-3p-C-NETA-TATE was synthesized in an automated AllinOne® synthesis module and the in vitro stability of [18F]AlF-3p-C-NETA-TATE was evaluated in formulation buffer, PBS and human serum. [18F]AlF-3p-C-NETA-TATE pharmacokinetics were evaluated using µPET/MRI in healthy rats, with [18F]AlF-NOTA-Octreotide as benchmark. Results: 3p-C-NETA quantitatively sequestered 177Lu, 213Bi and 67Cu at 25 °C while heating was required to bind Al18F, 68Ga, 161Tb and 225Ac efficiently. The [18F]AlF-, [177Lu]Lu- and [161Tb]Tb-3p-C-NETA-complex showed excellent in vitro stability in both PBS and human serum over the study period. In contrast, [67Cu]Cu- and [225Ac]Ac-, [68Ga]Ga-3p-C-NETA were stable in PBS, but not in human serum. [18F]AlF-3p-C-NETA-TATE was obtained in good radiochemical yield and radiochemical purity. [18F]AlF-3p-C-NETA-TATE displayed good in vitro stability for 4 h in all tested conditions. Finally, [18F]AlF-3p-C-NETA-TATE showed excellent pharmacokinetic properties comparable with the results obtained for [18F]AlF-NOTA-Octreotide. Conclusions: 3p-C-NETA is a versatile chelator that can be used for both diagnostic applications (Al18F) and targeted radionuclide therapy (213Bi, 177Lu, 161Tb). It has the potential to be the new theranostic chelator of choice for clinical applications in nuclear medicine.

Bismuth-213 for Targeted Radionuclide Therapy: From Atom to Bedside.

In contrast to external high energy photon or proton therapy, targeted radionuclide therapy (TRNT) is a systemic cancer treatment allowing targeted irradiation of a primary tumor and all its metastases, resulting in less collateral damage to normal tissues. The α-emitting radionuclide bismuth-213 (213Bi) has interesting properties and can be considered as a magic bullet for TRNT. The benefits and drawbacks of targeted alpha therapy with 213Bi are discussed in this review, covering the entire chain from radionuclide production to bedside. First, the radionuclide properties and production of 225Ac and its daughter 213Bi are discussed, followed by the fundamental chemical properties of bismuth. Next, an overview of available acyclic and macrocyclic bifunctional chelators for bismuth and general considerations for designing a 213Bi-radiopharmaceutical are provided. Finally, we provide an overview of preclinical and clinical studies involving 213Bi-radiopharmaceuticals, as well as the future perspectives of this promising cancer treatment option.

Radiolabeling of Human Serum Albumin With Terbium-161 Using Mild Conditions and Evaluation of in vivo Stability.

Targeted radionuclide therapy (TRNT) is a promising approach for cancer therapy. Terbium has four medically interesting isotopes (149Tb, 152Tb, 155Tb and 161Tb) which span the entire radiopharmaceutical space (TRNT, PET and SPECT imaging). Since the same element is used, accessing the various diagnostic or therapeutic properties without changing radiochemical procedures and pharmacokinetic properties is advantageous. The use of (heat-sensitive) biomolecules as vector molecule with high affinity and selectivity for a certain molecular target is promising. However, mild radiolabeling conditions are required to prevent thermal degradation of the biomolecule. Herein, we report the evaluation of potential bifunctional chelators for Tb-labeling of heat-sensitive biomolecules using human serum albumin (HSA) to assess the in vivo stability of the constructs. p-SCN-Bn-CHX-A"-DTPA, p-SCN-Bn-DOTA, p-NCS-Bz-DOTA-GA and p-SCN-3p-C-NETA were conjugated to HSA via a lysine coupling method. All HSA-constructs were labeled with [161Tb]TbCl3 at 40°C with radiochemical yields higher than 98%. The radiolabeled constructs were stable in human serum up to 24 h at 37°C. 161Tb-HSA-constructs were injected in mice to evaluate their in vivo stability. Increasing bone accumulation as a function of time was observed for [161Tb]TbCl3 and [161Tb]Tb-DTPA-CHX-A"-Bn-HSA, while negligible bone uptake was observed with the DOTA, DOTA-GA and NETA variants over a 7-day period. The results indicate that the p-SCN-Bn-DOTA, p-NCS-Bz-DOTA-GA and p-SCN-3p-C-NETA are suitable bifunctional ligands for Tb-based radiopharmaceuticals, allowing for high yield radiolabeling in mild conditions.

Antimalarial N 1,N 3-Dialkyldioxonaphthoimidazoliums: Synthesis, Biological Activity, and Structure-activity Relationships.

Here we report the nanomolar potencies of N 1,N 3-dialkyldioxonaphthoimidazoliums against asexual forms of sensitive and resistant Plasmodium falciparum. Activity was dependent on the presence of the fused quinone-imidazolium entity and lipophilicity imparted by the N1/N3 alkyl residues on the scaffold. Gametocytocidal activity was also detected, with most members active at IC50 < 1 µM. A representative analog with good solubility, limited PAMPA permeability, and microsomal stability demonstrated oral efficacy on a humanized mouse model of P. falciparum.

Optimization of 1,2,5-thiadiazole carbamates as potent and selective ABHD6 inhibitors.

At present, inhibitors of α/ß-hydrolase domain 6 (ABHD6) are viewed as a promising approach to treat inflammation and metabolic disorders. This article describes the development of 1,2,5-thiadiazole carbamates as ABHD6 inhibitors. Altogether, 34 compounds were synthesized, and their inhibitory activity was tested using lysates of HEK293 cells transiently expressing human ABHD6 (hABHD6). Among the compound series, 4-morpholino-1,2,5-thiadiazol-3-yl cyclooctyl(methyl)carbamate (JZP-430) potently and irreversibly inhibited hABHD6 (IC50 =44 nM) and showed ∼230-fold selectivity over fatty acid amide hydrolase (FAAH) and lysosomal acid lipase (LAL), the main off-targets of related compounds. Additionally, activity-based protein profiling indicated that JZP-430 displays good selectivity among the serine hydrolases of the mouse brain membrane proteome. JZP-430 has been identified as a highly selective, irreversible inhibitor of hABHD6, which may provide a novel approach in the treatment of obesity and type II diabetes.