Optimization of a Nucleophilic Two-Step Radiosynthesis of 6-O-(2-[18F]fluoroethyl)-6-O-desmethyl-diprenorphine ([18F]FE-DPN) for PET Imaging of Brain Opioid Receptors.

We have established a method for nucleophilic one-pot, two-step radiosynthesis of the popular opioid receptor radioligand 6-O-(2-[18F]fluoroethyl)-6-O-desmethyl-diprenorphine ([18F]FE-DPN) from the novel precursor 6-O-(2-tosyloxyethyl)-6-O-desmethyl- 3-O-trityl-diprenorphine (TE-TDDPN), which we designate as the Henriksen precursor. We undertook an optimization of the synthesis conditions, aiming to enhance the accessibility of [18F]FE-DPN for positron emission tomography (PET) studies of µ-opioid receptors. Herein, we report an optimized direct nucleophilic 18F-fluorination and the deprotection conditions for a fully automated radiosynthesis of [18F]FE-DPN on a modified GE Tracerlab FX FE synthesis panel. Starting from 1-1.5 GBq of [18F]fluoride and applying an Oasis Max 1cc cartridge for fluorine-18 trapping with a reduced amount of K2CO3 (5.06 µmol K+ ion), [18F]FE-DPN ([18F]11) was produced with 44.5 ± 10.6 RCY (decay-corrected), high radiochemical purity (>99%), and a molar activity of 32.2 ± 11.8 GBq/µmol in 60-65 min.

Therapeutic Performance Evaluation of 213Bi-Labelled Aminopeptidase N (APN/CD13)-Affine NGR-Motif ([213Bi]Bi-DOTAGA-cKNGRE) in Experimental Tumour Model: A Treasured Tailor for Oncology.

Since NGR-tripeptides (asparagine-glycine-arginine) selectively target neoangiogenesis-associated Aminopeptidase N (APN/CD13) on cancer cells, we aimed to evaluate the in vivo tumour targeting capability of radiolabelled, NGR-containing, ANP/CD13-selective [213Bi]Bi-DOTAGA-cKNGRE in CD13pos. HT1080 fibrosarcoma-bearing severe combined immunodeficient CB17 mice. 10 ± 1 days after cancer cell inoculation, positron emission tomography (PET) was performed applying [68Ga]Ga-DOTAGA-cKNGRE for tumour verification. On the 7th, 8th, 10th and 12th days the treated group of tumourous mice were intraperitoneally administered with 4.68 ± 0.10 MBq [213Bi]Bi-DOTAGA-cKNGRE, while the untreated tumour-bearing animals received 150 µL saline solution. In addition to body weight (BW) and tumour volume measurements, ex vivo biodistribution studies were conducted 30 and 90 min postinjection (pi.). The following quantitative standardised uptake values (SUV) confirmed the detectability of the HT1080 tumours: SUVmean and SUVmax: 0.37 ± 0.09 and 0.86 ± 0.14, respectively. Although no significant difference (p ≤ 0.05) was encountered between the BW of the treated and untreated mice, their tumour volumes measured on the 9th, 10th and 12th days differed significantly (p ≤ 0.01). Relatively higher [213Bi]Bi-DOTAGA-cKNGRE accumulation of the HT1080 neoplasms (%ID/g: 0.80 ± 0.16) compared with the other organs at 90 min time point yields better tumour-to-background ratios. Therefore, the therapeutic application of APN/CD13-affine [213Bi]Bi-DOTAGA- cKNGRE seems to be promising in receptor-positive fibrosarcoma treatment.

Investigation of the Effect on the Albumin Binding Moiety for the Pharmacokinetic Properties of 68Ga-, 205/206Bi-, and 177Lu-Labeled NAPamide-Based Radiopharmaceuticals.

Although radiolabeled alpha-melanocyte stimulating hormone-analogue NAPamide derivatives are valuable melanoma-specific diagnostic probes, their rapid elimination kinetics and high renal uptake may preclude them from being used in clinical settings. We aimed at improving the pharmacokinetics of radiolabeled DOTA-NAPamide compounds by incorporating a 4-(p-iodo-phenyl)-butanoic acid (IPB) into the molecules. Followed by 68Ga-, 205/206Bi-, and 177Lu-labelling, the radiopharmaceuticals ([68Ga]Ga-DOTA-IPB-NAPamide, [205/206Bi]Bi-DOTA-IPB-NAPamide, [177Lu]Lu-DOTA-IPB-NAPamide) were characterized in vitro. To test the imaging behavior of the IPB-containing probes, B16F10 tumor-bearing C57BL/6 mice were subjected to in vivo microPET/microSPECT/CT imaging and ex vivo biodistribution studies. All tracers were stable in vitro, with radiochemical purity exceeding 98%. The use of albumin-binding moiety lengthened the in vivo biological half-life of the IPB-carrying radiopharmaceuticals, resulting in elevated tumor accumulation. Both [68Ga]Ga-DOTA-IPB-NAPamide (5.06 ± 1.08 %ID/g) and [205/206Bi]Bi-DOTA-IPB-NAPamide (4.50 ± 0.98 %ID/g) exhibited higher B16F10 tumor concentrations than their matches without the albumin-binding residue ([68Ga]Ga-DOTA-NAPamide and [205/206Bi]Bi-DOTA-NAPamide: 1.18 ± 0.27 %ID/g and 3.14 ± 0.32; respectively), however; the large amounts of off-target radioactivity do not confirm the benefits of half-life extension for short-lived isotopes. Enhanced [177Lu]Lu-DOTA-IPB-NAPamide tumor uptake even 24 h post-injection proved the advantage of IPB-based prolonged circulation time regarding long-lived radionuclides, although the significant background noise must be addressed in this case as well.

61Cu-Labelled radiodiagnostics of melanoma with NAPamide-targeted radiopharmaceutical.

Malignant melanoma is a major public health problem with an increasing incidence and mortality in the Caucasian population due to its significant metastatic potential. The early detection of this cancer type by imaging techniques like positron emission tomography acts as an important contributor to the long-term survival. Based on literature data, the radio labelled alpha-MSH analog NAPamide molecule is an appropriate diagnostic tool for the detection of melanoma tumors. Inspired by these facts, a new radiotracer, the [61Cu]Cu-KFTG-NAPamide has been synthesized to exploit the beneficial features of the positron emitter 61Cu and the melanoma specificity of the NAPamide molecule. In this work, we report a new member of the CB-15aneN5 ligand family (KFTG) as the chelator for 61Cu(II) complexation. On the basis of the thorough physico-chemical characterization, the rigid [Cu(KFTG)]+ complex exhibits fast complex formation (t1/2 = 155 s at pH 5.0 and 25 °C) and high inertness (t1/2 = 2.0 h in 5.0 M HCl at 50 °C) as well as moderate superoxide dismutase activity (IC50 = 2.3 µM). Furthermore, the [61Cu]Cu-KFTG-NAPamide possesses outstanding features in the diagnostics of B16-F10 melanoma tumors by PET imaging: (T/M(SUVs) (in vivo): appr. 14, %ID/g: 7 ± 1 and T/M (ex vivo): 315 ± 24 at 180 min).

In Vivo evaluation of newly synthesized 213Bi-conjugated alpha-melanocyte stimulating hormone (α-MSH) peptide analogues in melanocortin-1 receptor (MC1-R) positive experimental melanoma model.

Given the rising pervasiveness of melanocortin-1 receptor (MC1-R) positive melanoma malignum (MM) and pertinent metastases, radiolabelled receptor-affine alpha-melanocyte stimulating hormone-analogue (α-MSH analogue) imaging probes would be of crucial importance in timely tumor diagnostic assessment. Herein we aimed at investigating the biodistribution and the MM targeting potential of newly synthesized 213Bi-conjugated MC1-R specific peptide-based radioligands with the establishment of MC1-R overexpressing MM preclinical model. DOTA-conjugated NAP, -HOLD, -FOLD, -and MARSamide were labelled with 213Bi. Ex vivo biodistribution studies were conducted post-administration of 3.81 ± 0.32 MBq [213Bi]Bi-DOTA conjugated deriva-tives into twenty B16-F10 tumor-bearing C57BL/6 J and healthy mice. Organ Level Internal Dose Assessment (OLINDA) and IDAC-Dose were used to calculate translational data-based absorbed radiation dose in human organs. Moderate or low %ID/g uptake of [213Bi]Bi-DOTA conjugated NAP, -HOLD, -and MARSamide and significantly increased [213Bi]Bi-DOTA-FOLDamide accumulation was observed in the thoracic and abdominal organs (p ≤ 0.01). High [213Bi]Bi-DOTA-NAP (%ID/g:3.76 ± 0.96), -and FOLDamide (%ID/g:3.28 ± 0.95) tumor tracer activity confirmed their MC1-R-affinity. The bladder wall received the highest radiation absorbed dose followed by the kidneys (bladder wall: 1.95·10-2 and 8.97·10-2 mSv/MBq; kidneys: 7.47·10-3 vs. 5.88·10-2 mSv/MBq measured by IDAC and OLINDA; respectively) indicating the suitability of the NAPamide derivative for clinical use. These novel [213Bi]Bi-DOTA-linked peptide probes displaying meaningful MC1-R affinity could be promising molecular probes in MM imaging.

Evaluation of the therapeutic efficacy of 213Bi-labelled DOTA-conjugated alpha-melanocyte stimulating hormone peptide analogues in melanocortin-1 receptor positive preclinical melanoma model.

Melanocortin-1 receptor (MC1-R) targeting alpha-melanocyte stimulating hormone-analogue (α-MSH) biomolecules labelled with α-emitting radiometal seem to be valuable in the targeted radionuclide therapy of MC1-R positive melanoma malignum (MM). Herein is reported the anti-tumor in vivo therapeutic evaluation of MC1-R-affine [213Bi]Bi-DOTA-NAPamide and HOLDamide treatment in MC1-R positive B16-F10 melanoma tumor-bearing C57BL/6J mice. On the 6th, 8th and 10th days post tumor cell inoculation; the treated groups of mice were intravenously injected with approximately 5 MBq of both amide derivatives. Beyond body weight and tumor volume assessment, [68Ga]Ga-DOTA-HOLDamide and NAPamide-based PET/MRI scans, and ex vivo biodistribution studies were executed 30,- and 90 min postinjection. In the PET/MRI imaging studies the B16-F10 tumors were clearly visualized with both 68Ga-labelled tracers, however, significantly lower tumor-to-muscle (T/M) ratios were observed by using [68Ga]Ga-DOTA-HOLDamide. After alpha-radiotherapy treatment the tumor size of the control group was larger relative to both treated cohorts, while the smallest tumor volumes were observed in the NAPamide-treated subclass on the 10th day. Relatively higher [213Bi]Bi-DOTA-NAPamide accumulation in the B16-F10 tumors (%ID/g: 2.71 ± 0.15) with discrete background activity led to excellent T/M ratios, particularly 90 min postinjection. Overall, the therapeutic application of receptor selective [213Bi]Bi-DOTA-NAPamide seems to be feasible in MC1-R positive MM management.

Methods for the Determination of Transition Metal Impurities in Cyclotron-Produced Radiometals.

Cyclotron-produced radiometals must be separated from the irradiated target and purified from other metal impurities, which could interfere with the radiolabeling process. We compared different chromatographic and colorimetric methods to determine the amount of transition metals in radioactive samples. Besides commercially available colorimetric tests, 4-(2-pyridylazo)resorcinol and xylenol orange were used as a non-selective metal reagents, forming water-soluble chelates with most of the transition metals immediately. We compared the applicability of pre- and post-column derivatization, as well as colorimetric determination without separation. The studied chromatographic and colorimetric analyses are not suitable to completely replace atomic spectroscopic techniques for the determination of metal contaminants in radioactive samples, but they may play an important role in the development of methods for the purification of radiometals and in their routine quality control.

The Synthesis and Preclinical Investigation of Lactosamine-Based Radiopharmaceuticals for the Detection of Galectin-3-Expressing Melanoma Cells.

Given that galectin-3 (Gal-3) is a ß-galactoside-binding lectin promoting tumor growth and metastatis, it could be a valuable target for the treatment of Gal-3-expressing neoplasms. An aromatic group introduced to the C-3' position of lactosamine increased its affinity for Gal-3. Herein, we aimed at developing a radiopharmaceutical for the detection of Gal-3 positive malignancies. To enhance tumor specificity, a heterodimeric radiotracer capable of binding to both Gal-3 and αvß3 integrin was also synthetized. Arginine-glycine-asparagine (RGD) peptide is the ligand of angiogenesis- and metastasis-associated αvß3 integrin. Following the synthesis of the chelator-conjugated (2-naphthyl)methylated lactosamine, the obtained compound was applied as a precursor for radiolabeling and was conjugated to the RGD peptide by click reaction as well. Both synthetized precursors were radiolabeled with 68Ga, resulting in high labeling yield (>97). The biological studies were carried out using B16F10 melanoma tumor-bearing C57BL6 mice. High tumor accumulation of both labeled lactosamine derivatives­detected by in vivo PET and ex vivo biodistribution studies­indicated their potential for melanoma detection. However, the heterodimer radiotracer showed high hepatic uptake, while low liver accumulation characterized chelator-conjugated lactosamine, resulting in PET images with excellent contrast. Therefore, this novel carbohydrate-based radiotracer is suitable for the highly selective determination of Gal-3-expressing melanoma cells.

Synthesis, Physicochemical, Labeling and In Vivo Characterization of 44Sc-Labeled DO3AM-NI as a Hypoxia-Sensitive PET Probe.

Hypoxia promotes angiogenesis, which is crucial for tumor growth, and induces malignant progression and increases the therapeutic resistance. Positron emission tomography (PET) enables the detection of the hypoxic regions in tumors using 2-nitroimidazole-based radiopharmaceuticals. We describe here a physicochemical study of the Sc(DO3AM-NI) complex, which indicates: (a) relatively slow formation of the Sc(DO3AM-NI) chelate in acidic solution; (b) lower thermodynamic stability than the reference Sc(DOTA); (c) however, it is substantially more inert and consequently can be regarded as an excellent Sc-binder system. In addition, we report a comparison of 44Sc-labeled DO3AM-NI with its known 68Ga-labeled analog as a hypoxia PET probe. The in vivo and ex vivo biodistributions of 44Sc- and 68Ga-labeled DO3AM-NI in healthy and KB tumor-bearing SCID mice were examined 90 and 240 min after intravenous injection. No significant difference was found between the accumulation of 44Sc- and 68Ga-labeled DO3AM-NI in KB tumors. However, a significantly higher accumulation of [68Ga]Ga(DO3AM-NI) was found in liver, spleen, kidney, intestine, lung, heart and brain than for [44Sc]Sc(DO3AM-NI), leading to a lower tumor/background ratio. The tumor-to-muscle (T/M) ratio of [44Sc]Sc(DO3AM-NI) was approximately 10-15-fold higher than that of [68Ga]Ga(DO3AM-NI) at all time points. Thus, [44Sc]Sc(DO3AM-NI) allows the visualization of KB tumors with higher resolution, making it a promising hypoxia-specific PET radiotracer.

Synthesis of Novel, Dual-Targeting 68Ga-NODAGA-LacN-E[c(RGDfK)]2 Glycopeptide as a PET Imaging Agent for Cancer Diagnosis.

Radiolabeled peptides possessing an Arg-Gly-Asp (RGD) motif are widely used radiopharmaceuticals for PET imaging of tumor angiogenesis due to their high affinity and selectivity to αvß3 integrin. This receptor is overexpressed in tumor and tumor endothelial cells in the case of numerous cancer cell lines, therefore, it is an excellent biomarker for cancer diagnosis. The galectin-3 protein is also highly expressed in tumor cells and N-acetyllactosamine is a well-established ligand of this receptor. We have developed a synthetic method to prepare a lactosamine-containing radiotracer, namely 68Ga-NODAGA-LacN-E[c(RGDfK)]2, for cancer diagnosis. First, a lactosamine derivative with azido-propyl aglycone was synthetized. Then, NODAGA-NHS was attached to the amino group of this lactosamine derivative. The obtained compound was conjugated to an E[c(RGDfK)]2 peptide with a strain-promoted click reaction. We have accomplished the radiolabeling of the synthetized NODAGA-LacN-E[c(RGDfK)]2 precursor with a positron-emitting 68Ga isotope (radiochemical yield of >95%). The purification of the labeled compound with solid-phase extraction resulted in a radiochemical purity of >99%. Subsequently, the octanol-water partition coefficient (log P) of the labeled complex was determined to be -2.58. In addition, the in vitro stability of 68Ga-NODAGA-LacN-E[c(RGDfK)]2 was investigated and it was found that it was stable under the examined conditions.

Synthesis of 68Ga-Labeled cNGR-Based Glycopeptides and In Vivo Evaluation by PET Imaging.

Tumor hypoxia induces angiogenesis, which is required for tumor cell survival. The aminopeptidase N receptor (APN/CD13) is an excellent marker of angiogenesis since it is overexpressed in angiogenic blood vessels and in tumor cells. Asparagine-glycine-arginine (NGR) peptide analogs bind selectively to the APN/CD13 recepto, therefore, they are important vector molecules in the development of a PET radiotracer which is capable of detecting APN-rich tumors. To investigate the effect of glycosylation and pegylation on in-vivo efficacy of an NGR-based radiotracer, two 68Ga-labeled radioglycopeptides were synthesized. A lactosamine derivative was applied to glycosylation of the NGR derivative and PEG4 moiety was used for pegylation. The receptor targeting potential and biodistribution of the radiopeptides were evaluated with in vivo PET imaging studies and ex vivo tissue distribution studies using B16-F10 melanoma tumor-bearing mice. According to these studies, all synthesized radiopeptides were capable of detecting APN expression in B16-F10 melanoma tumor. In addition, lower hepatic uptake, higher tumor-to background (T/M) ratio and prolonged circulation time were observed for the novel [68Ga]-10 radiotracer due to pegylation and glycosylation, resulting in more contrasting PET imaging. These in vivo PET imaging results correlated well with the ex vivo tissue distribution data.

Radiosynthesis and Preclinical Investigation of 11 C-Labelled 3-(4,5-Diphenyl-1,3-oxazol-2-yl)propanal Oxime ([11 C]SZV 1287).

The radiosynthesis, as well as the in vivo and ex vivo biodistribution of the 11 C radiolabelled 3-(4,5-diphenyl-1,3-oxazol-2-yl)propanal oxime (6, [11 C]SZV 1287) are reported. SZV 1287 is a novel semicarbazide-sensitive amine oxidase (SSAO) inhibitor and a promising candidate to be a novel analgesic for the treatment of neuropathic pain. Its radiolabelling was developed via a four-step radiosynthesis which started from the reaction of a Grignard reagent with [11 C]CO2 to produce [11 C]oxaprozin (3). In the next step this carboxylic acid 3 was directly reduced to yield the corresponding aldehyde, which was then converted into the oxime. [11 C]SZV 1287 was administered to male NMRI mice. The animals were examined with dynamic PET/MR imaging for 90 minutes. Biodistribution studies were performed at 10, 30, 60 and 120 minutes post injection. The accumulation of the labelled compound was observed in the brain of the animals. The main excretion pathway was found to be through the liver and intestines. These studies provide preliminary information for pharmacokinetic characterization of the SZV 1287.