BACKGROUND: The cysteine-aspartic acid protease caspase-3 is recognized as the main executioner of apoptosis in cells responding to specific extrinsic and intrinsic stimuli. Caspase-3 represents an interesting biomarker to evaluate treatment response, as many cancer therapies exert their effect by inducing tumour cell death. Previously developed caspase-3 PET tracers were unable to reach routine clinical use due to low tumour uptake or lack of target selectivity, which are two important requirements for effective treatment response evaluation in cancer patients. Therefore, the goal of this study was to develop and preclinically evaluate novel caspase-3-selective activity-based probes (ABPs) for apoptosis imaging.
RESULTS: A library of caspase-3-selective ABPs was developed for tumour apoptosis detection. In a first attempt, the inhibitor Ac-DW3-KE (Ac-3Pal-Asp-βhLeu-Phe-Asp-KE) was 18F-labelled on the N-terminus to generate a radiotracer that was incapable of adequately detecting an increase in apoptosis in vivo. The inability to effectively detect active caspase-3 in vivo was likely attributable to slow binding, as demonstrated with in vitro inhibition kinetics. Hence, a second generation of caspase-3 selective ABPs was developed based on the Ac-ATS010-KE (Ac-3Pal-Asp-Phe(F5)-Phe-Asp-KE) with greatly improved binding kinetics over Ac-DW3-KE. Our probes based on Ac-ATS010-KE were made by modifying the N-terminus with 6 different linkers. All the linker modifications had limited effect on the binding kinetics, target selectivity, and pharmacokinetic profile in healthy mice. In an in vitro apoptosis model, the least hydrophilic tracer [18F]MICA-316 showed an increased uptake in apoptotic cells in comparison to the control group. Finally, [18F]MICA-316 was tested in an in vivo colorectal cancer model, where it showed a limited tumour uptake and was unable to discriminate treated tumours from the untreated group, despite demonstrating that the radiotracer was able to bind caspase-3 in complex mixtures in vitro. In contrast, the phosphatidylethanolamine (PE)-binding radiotracer [99mTc]Tc-duramycin was able to recognize the increased cell death in the disease model, making it the best performing treatment response assessment tracer developed thus far.
CONCLUSIONS: In conclusion, a novel library of caspase-3-binding PET tracers retaining similar binding kinetics as the original inhibitor was developed. The most promising tracer, [18F]MICA-316, showed an increase uptake in an in vitro apoptosis model and was able to selectively bind caspase-3 in apoptotic tumour cells. In order to distinguish therapy-responsive from non-responsive tumours, the next generation of caspase-3-selective ABPs will be developed with higher tumour accumulation and in vivo stability.

The prediction of central nervous system (CNS) active pharmaceuticals and radiopharmaceuticals has experienced a boost by the introduction of computational approaches, like blood-brain barrier (BBB) score or CNS multiparameter optimization values. These rely heavily on calculated pKa values and other physicochemical parameters. Despite the inclusion of various physicochemical parameters in online data banks, pKa values are often missing and published experimental pKa values are limited especially for radiopharmaceuticals. This comparative study investigated the discrepancies between predicted and experimental pKa values and their impact on CNS activity prediction scores. The pKa values of 46 substances, including therapeutic drugs and PET imaging radiopharmaceuticals, were measured by means of potentiometry and spectrophotometry. Experimentally obtained pKa values were compared with in silico predictions (Chemicalize/Marvin). The results demonstrate a considerable discrepancy between experimental and in silico values, with linear regression analysis showing intermediate correlation (R2(Marvin) = 0.88, R2(Chemicalize) = 0.82). This indicates that if one requires an accurate pKa value, it is essential to experimentally assess it. This underscores the importance of experimentally determining pKa values for accurate drug design and optimization. The study\'s data provide a valuable library of reliable experimental pKa values for therapeutic drugs and radiopharmaceuticals, aiding researchers in the field.

Colorectal cancer (CRC) is among the most lethal and prevalent malignancies in the world. Human epidermal growth factor receptor 2 (HER2) is a promising target for the diagnosis and treatment of CRC. In this study, we aimed to design, synthesize and label peptide-based positron emission tomography (PET) tracers targeting HER2-positive CRC, namely [68Ga]Ga-ES-01 and [68Ga]Ga-ES-02. The results show that [68Ga]Ga-ES-01 and [68Ga]Ga-ES-02 possessed hydrophilicity, rapid pharmacokinetic properties and excellent stabilities. [68Ga]Ga-ES-02 demonstrated higher binding affinity (Kd = 24.29 ± 4.95 nM) toward the HER2 in CRC. In HER2-positive HT-29 CRC xenograft mouse model, PET study showed specific tumor uptake after injection of [68Ga]Ga-ES-02 (SUV15min max = 0.87 ± 0.03; SUV30min max = 0.64 ± 0.02). In biodistribution study, the T/M ratios of 68Ga-ES-02 at 30 min after injection reached a maximum of 4.07 ± 0.34. In summary, we successfully synthesized and evaluated two novel peptide-based PET tracers. Our data demonstrate that [68Ga]Ga-ES-01/02 is capable of HER2-positive colorectal cancer, with [68Ga]Ga-ES-02 showing superior imaging effect, enhanced targeting, and increased specificity.

Gallium-68-labeled siderophores as radiotracers have gained interest for the development of in situ infection-specific imaging diagnostics. Here, we report radiolabeling, in vitro screening, and in vivo pharmacokinetics (PK) of gallium-68-labeled schizokinen ([68Ga]Ga-SKN) as a new potential radiotracer for imaging bacterial infections. We radiolabeled SKN with ≥95% radiochemical purity. Our in vitro studies demonstrated its hydrophilic characteristics, neutral pH stability, and short-term stability in human serum and toward transchelation. In vitro uptake of [68Ga]Ga-SKN by Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and S. epidermidis, but no uptake by Candida glabrata, C. albicans, or Aspergillus fumigatus, demonstrated its specificity to bacterial species. Whole-body [68Ga]Ga-SKN positron emission tomography (PET) combined with computerized tomography (CT) in healthy mice showed rapid renal excretion with no or minimal organ uptake. The subsequent ex vivo biodistribution resembled this fast PK with rapid renal excretion with minimal blood retention and no major organ uptake and showed some dissociation of the tracer in the urine after 60 min postinjection. These findings warrant further evaluation of [68Ga]Ga-SKN as a bacteria-specific radiotracer for infection imaging.

Colony-stimulating factor 1 receptor (CSF1R) is a type III receptor tyrosine kinase that is crucial for immune cell activation, survival, proliferation, and differentiation. Its expression significantly increases in macrophages during inflammation, playing a crucial role in regulating inflammation resolution and termination. Consequently, CSF1R has emerged as a critical target for both therapeutic intervention and imaging of inflammatory diseases. Herein, we have developed a radiotracer, 1-[4-((7-(dimethylamino)quinazolin-4-yl)oxy)phenyl]-3-(4-[18F]fluorophenyl)urea ([18F]17), for in vivo positron emission tomography (PET) imaging of CSF1R. Compound 17 exhibits a comparable inhibitory potency against CSF1R as the well-known CSF1R inhibitor PLX647. The radiosynthesis of [18F]17 was successfully performed by radiofluorination of aryltrimethyltin precursor with a yield of approximately 12% at the end of synthesis, maintaining a purity exceeding 98%. In vivo stability and biodistribution studies demonstrate that [18F]17 remains >90% intact at 30 min postinjection, with no defluorination observed even at 60 min postinjection. The PET/CT imaging study in lipopolysaccharide-induced pulmonary inflammation mice indicates that [18F]17 offers a more sensitive characterization of pulmonary inflammation compared to traditional [18F]FDG. Notably, [18F]17 shows a higher discrepancy in uptake ratio between mice with pulmonary inflammation and the sham group. Furthermore, the variations in [18F]17 uptake ratio observed on day 7 and day 14 correspond to lung density changes observed in CT imaging. Moreover, the expression levels of CSF1R on day 7 and day 14 follow a trend similar to the uptake pattern of [18F]17, indicating its potential for accurately characterizing CSF1R expression levels and effectively monitoring the pulmonary inflammation progression. These results strongly suggest that [18F]17 has promising prospects as a CSF1R PET tracer, providing diagnostic opportunities for pulmonary inflammatory diseases.

Neuroinflammation is associated with the pathophysiologies of neurodegenerative and psychiatric disorders. Evaluating neuroinflammation using positron emission tomography (PET) plays an important role in the early diagnosis and determination of proper treatment of brain diseases. To quantify neuroinflammatory responses in vivo, many PET tracers have been developed using translocator proteins, imidazole-2 binding site, cyclooxygenase, monoamine oxidase-B, adenosine, cannabinoid, purinergic P2X7, and CSF-1 receptors as biomarkers. In this review, we introduce the latest developments in PET tracers that can image neuroinflammation, focusing on clinical trials, and further consider their current implications.

Tumour hypoxia is a known microenvironmental culprit for treatment resistance, tumour recurrence and promotion of metastatic spread. Despite the long-known existence of this factor within the tumour milieu, hypoxia is still one of the greatest challenges in cancer management. The transition from invasive and less reliable detection methods to more accurate and non-invasive ways to identify and quantify hypoxia was a long process that eventually led to the promising results showed by functional imaging techniques. Hybrid imaging, such as PET-CT, has the great advantage of combining the structural or anatomical image (offered by CT) with the functional or metabolic one (offered by PET). However, in the context of hypoxia, it is only the PET image taken after appropriate radiotracer administration that would supply hypoxia-specific information. To overcome this limitation, the development of the latest hybrid imaging systems, such as PET-MRI, enables a synergistic approach towards hypoxia imaging, with both methods having the potential to provide functional information on the tumour microenvironment. This study is designed as a systematic review of the literature on the newest developments of PET-MRI for the imaging of hypoxic cells in breast cancer. The analysis includes the affinity of various PET-MRI tracers for hypoxia in this patient group as well as the correlations between PET-specific and MRI-specific parameters, to offer a broader view on the potential for the widespread clinical implementation of this hybrid imaging technique.

Prostate cancer (PCa) is one of the most common tumors in men, with the overexpression of prostate-specific membrane. In this study, we developed four new 68Ga-labeled PSMA-targeting tracers by introducing quinoline, phenylalanine and decanoic acid groups to enhance their lipophilicity, strategically limiting their metabolic pathway through the urinary system. Four radiotracers were synthesized with radiochemical purity >95 %, and exhibited high stability in vivo and in vitro. The inhibition constants (Ki) of SDTWS01-04 to PSMA were in the nanomolar range (<10 nM). Micro PET/CT imaging and biodistribution analysis revealed that 68Ga-SDTWS01 enabled clear tumor visualization in PET images at 1.5 h post-injection, with excellent pharmacokinetic properties. Notably, the kidney uptake of 68Ga-SDTWS01 significantly reduced, with higher tumor-to-kidney ratio (0.36 ± 0.02), tumor-to-muscle ratio (24.31 ± 2.10), compared with 68Ga-PSMA-11 (T/K: 0.15 ± 0.01; T/M: 14.97 ± 1.40), suggesting that 68Ga-SDTWS01 is a promising radiotracer for the diagnosis of PCa. Moreover, SDTWS01 with a chelator DOTA could also label 177Lu and 225Ac, which could be used for the treatment of PCa.

There are several well-described molecular mechanisms that influence cell growth and are related to the development of cancer. Chemokines constitute a fundamental element that is not only involved in local growth but also affects angiogenesis, tumor spread, and metastatic disease. Among them, the C-X-C motif chemokine ligand 12 (CXCL12) and its specific receptor the chemokine C-X-C motif receptor 4 (CXCR4) have been widely studied. The overexpression in cell membranes of CXCR4 has been shown to be associated with the development of different kinds of histological malignancies, such as adenocarcinomas, epidermoid carcinomas, mesenchymal tumors, or neuroendocrine neoplasms (NENs). The molecular synapsis between CXCL12 and CXCR4 leads to the interaction of G proteins and the activation of different intracellular signaling pathways in both gastroenteropancreatic (GEP) and bronchopulmonary (BP) NENs, conferring greater capacity for locoregional aggressiveness, the epithelial-mesenchymal transition (EMT), and the appearance of metastases. Therefore, it has been hypothesized as to how to design tools that target this receptor. The aim of this review is to focus on current knowledge of the relationship between CXCR4 and NENs, with a special emphasis on diagnostic and therapeutic molecular targets.

Here, we describe the characterization of a radioligand selective for GluN2B-containing NMDA receptors, 3-[3H] 1-(azetidin-1-yl)-2-(6-(4-fluoro-3-methyl-phenyl)pyrrolo[3,2-b]pyridin-1-yl)ethanone ([3H]-JNJ- GluN2B-5). In rat cortical membranes, the compound bound to a single site, and the following kinetic parameters were measured; association rate constant Kon = 0.0066 ± 0.0006 min-1 nM-1, dissociation rate constant Koff = 0.0210 ± 0.0001 min-1 indicating calculated KD = Koff/Kon = 3.3 ± 0.4 nM, (mean ± SEM, n = 3). The equilibrium dissociation constant determined from saturation binding experiments in rat cortex was KD of 2.6 ± 0.3 nM (mean ± SEM, n = 3). In contrast to the widely used GluN2B radioligand [3H]-Ro 25-6981, whose affinity Ki for sigma 1 and sigma 2 receptors are 2 and 189 nM, respectively, [3H]-JNJ-GluN2B-5 exhibits no measurable affinity for sigma 1 and sigma 2 receptors (Ki > 10 μM for both) providing distinct selectivity advantages. Anatomical distribution of [3H]-JNJ-GluN2B-5 binding sites in rat, mouse, dog, monkey, and human brain tissue was studied using in vitro autoradiography, which showed high specific binding in the hippocampus and cortex and negligible binding in the cerebellum. Enhanced selectivity for GluN2B-containing receptors translated to a good signal-to-noise ratio in both in vitro radioligand binding and in vitro autoradiography assays. In conclusion, [3H]-JNJ-GluN2B-5 is a high-affinity GluN2B radioligand with excellent signal-to-noise ratio and unprecedented selectivity.