Investigation of Tumor Cells and Receptor-Ligand Simulation Models for the Development of PET Imaging Probes Targeting PSMA and GRPR and a Possible Crosstalk between the Two Receptors.

Prostate-specific membrane antigen (PSMA) and gastrin-releasing peptide receptor (GRPR) have both been used in nuclear medicine as targets for molecular imaging and therapy of prostate (PCa) and breast cancer (BCa). Three bioconjugate probes, the PSMA specific: [68Ga]Ga-1, ((HBED-CC)-Ahx-Lys-NH-CO-NH Glu or PSMA-11), the GRPR specific: [68Ga]Ga-2, ((HBED-CC)-4-amino-1-carboxymethyl piperidine-[D-Phe6, Sta13]BN(6-14), a bombesin (BN) analogue), and 3 (the BN analogue: 4-amino-1-carboxymethyl piperidine-[(R)-Phe6, Sta13]BN(6-14) connected with the fluorescent dye, BDP-FL), were synthesized and tested in vitro with PCa and BCa cell lines, more specifically, with PCa cells, PC-3 and LNCaP, with BCa cells, T47D, MDA-MB-231, and with the in-house created PSMA-overexpressing PC-3(PSMA), T47D(PSMA), and MDA-MB-231(PSMA). In addition, biomolecular simulations were conducted on the association of 1 and 2 with PSMA and GRPR. The PSMA overexpression resulted in an increase of cell-bound radioligand [68Ga]Ga-1 (PSMA) for PCa and BCa cells and also of [68Ga]Ga-2 (GRPR), especially in those cell lines already expressing GRPR. The results were confirmed by fluorescence-activated cell sorting with a PE-labeled PSMA-specific antibody and the fluorescence tracer 3. The docking calculations and molecular dynamics simulations showed how 1 enters the PSMA funnel region and how pharmacophore Glu-urea-Lys interacts with the arginine patch, the S1', and S1 subpockets by forming hydrogen and van der Waals bonds. The chelating moiety of 1, that is, HBED-CC, forms additional stabilizing hydrogen bonding and van der Waals interactions in the arene-binding site. Ligand 2 is diving into the GRPR transmembrane (TM) helical cavity, thereby forming hydrogen bonds through its amidated end, water-mediated hydrogen bonds, and π-π interactions. Our results provide valuable information regarding the molecular mechanisms involved in the interactions of 1 and 2 with PSMA and GRPR, which might be useful for the diagnostic imaging and therapy of PCa and BCa.

Human ABC and SLC Transporters: The Culprit Responsible for Unspecific PSMA-617 Uptake?

[177Lu]Lu-PSMA-617 has recently been successfully approved by the FDA, the MHRA, Health Canada and the EMA as Pluvicto®. However, salivary gland (SG) and kidney toxicities account for its main dose-limiting side-effects, while its corresponding uptake and retention mechanisms still remain elusive. Recently, the presence of different ATP-binding cassette (ABC) transporters, such as human breast cancer resistance proteins (BCRP), multidrug resistance proteins (MDR1), multidrug-resistance-related proteins (MRP1, MRP4) and solute cassette (SLC) transporters, such as multidrug and toxin extrusion proteins (MATE1, MATE2-K), organic anion transporters (OAT1, OAT2v1, OAT3, OAT4) and peptide transporters (PEPT2), has been verified at different abundances in human SGs and kidneys. Therefore, our aim was to assess whether [177Lu]Lu-PSMA-617 and [225Ac]Ac-PSMA-617 are substrates of these ABC and SLC transporters. For in vitro studies, the novel isotopologue ([α,ß-3H]Nal)Lu-PSMA-617 was used in cell lines or vesicles expressing the aforementioned human ABC and SLC transporters for inhibition and uptake studies, respectively. The corresponding probe substrates and reference inhibitors were used as controls. Our results indicate that [177Lu]Lu-PSMA-617 and [225Ac]Ac-PSMA-617 are neither inhibitors nor substrates of the examined transporters. Therefore, our results show that human ABC and SLC transporters play no central role in the uptake and retention of [177Lu]Lu-PSMA-617 and [225Ac]Ac-PSMA-617 in the SGs and kidneys nor in the observed toxicities.

Future Treatment Strategies for Cancer Patients Combining Targeted Alpha Therapy with Pillars of Cancer Treatment: External Beam Radiation Therapy, Checkpoint Inhibition Immunotherapy, Cytostatic Chemotherapy, and Brachytherapy.

Cancer is one of the most complex and challenging human diseases, with rising incidences and cancer-related deaths despite improved diagnosis and personalized treatment options. Targeted alpha therapy (TαT) offers an exciting strategy emerging for cancer treatment which has proven effective even in patients with advanced metastatic disease that has become resistant to other treatments. Yet, in many cases, more sophisticated strategies are needed to stall disease progression and overcome resistance to TαT. The combination of two or more therapies which have historically been used as stand-alone treatments is an approach that has been pursued in recent years. This review aims to provide an overview on TαT and the four main pillars of therapeutic strategies in cancer management, namely external beam radiation therapy (EBRT), immunotherapy with checkpoint inhibitors (ICI), cytostatic chemotherapy (CCT), and brachytherapy (BT), and to discuss their potential use in combination with TαT. A brief description of each therapy is followed by a review of known biological aspects and state-of-the-art treatment practices. The emphasis, however, is given to the motivation for combination with TαT as well as the pre-clinical and clinical studies conducted to date.

Synthesis of tritium-labeled Lu-PSMA-617: Alternative tool for biological evaluation of radiometal-based pharmaceuticals.

This project focuses on the generation and evaluation of functional alternatives to radiometal-based pharmaceuticals supporting basic research and the in vitro developmental phase. Employing robust tritium chemistry and non-radioactive metal surrogates in two synthetic and labeling strategies resulted in ([ring-3H]Nal)PSMA-617 and ([α,ß-3H]Nal)PSMA-617. In particular, ([α,ß-3H]Nal)Lu-PSMA-617 exhibited high radiolytic as well as metal-complex stability and was compared to the clinically-established radiopharmaceutical [177Lu]Lu-PSMA-617. The cell-based assays confirmed the applicability of ([α,ß-3H]Nal)Lu-PSMA-617 as a substitute of [177Lu]Lu-PSMA-617 in pre-clinical biological settings.

A New Class of PSMA-617-Based Hybrid Molecules for Preoperative Imaging and Intraoperative Fluorescence Navigation of Prostate Cancer.

The development of PSMA-targeting low-molecular-weight hybrid molecules aims at advancing preoperative imaging and accurate intraoperative fluorescence guidance for improved diagnosis and therapy of prostate cancer. In hybrid probe design, the major challenge is the introduction of a bulky dye to peptidomimetic core structures without affecting tumor-targeting properties and pharmacokinetic profiles. This study developed a novel class of PSMA-targeting hybrid molecules based on the clinically established theranostic agent PSMA-617. The fluorescent dye-bearing candidates of the strategically designed molecule library were evaluated in in vitro assays based on their PSMA-binding affinity and internalization properties to identify the most favorable hybrid molecule composition for the installation of a bulky dye. The library's best candidate was realized with IRDye800CW providing the lead compound. Glu-urea-Lys-2-Nal-Chx-Lys(IRDye800CW)-DOTA (PSMA-927) was investigated in an in vivo proof-of-concept study, with compelling performance in organ distribution studies, PET/MRI and optical imaging, and with a strong PSMA-specific tumor uptake comparable to that of PSMA-617. This study provides valuable insights about the design of PSMA-targeting low-molecular-weight hybrid molecules, which enable further advances in the field of peptidomimetic hybrid molecule development.

Iron-mediated oxidation induces conformational changes within the redox-sensing protein HbpS.

HbpS is an extracellular oligomeric protein, which has been shown to act in concert with the two-component system SenS-SenR during the sensing of redox stress. HbpS can bind and degrade heme under oxidative stress conditions, leading to a free iron ion. The liberated iron is subsequently coordinated on the protein surface. Furthermore, HbpS has been shown to modulate the phosphorylation state of the sensor kinase SenS as, in the absence of oxidative stress conditions, HbpS inhibits SenS autophosphorylation whereas the presence of heme or iron ions and redox-stressing agents enhances it. Using HbpS wild type and mutants as well as different biochemical and biophysical approaches, we show that iron-mediated oxidative stress induces both secondary structure and overall intrinsic conformational changes within HbpS. We demonstrate in addition that HbpS is oxidatively modified, leading to the generation of highly reactive carbonyl groups and tyrosine-tyrosine bonds. Further examination of the crystal structure and subsequent mutational analyses allowed the identification of the tyrosine residue participating in dityrosine formation, which occurs between two monomers within the octomeric assembly. Therefore, it is proposed that oxidative modifications causing structural and conformational changes are responsible for the control of SenS and hence of the HbpS-SenS-SenR signaling cascade.

Rational Linker Design to Accelerate Excretion and Reduce Background Uptake of Peptidomimetic PSMA-Targeting Hybrid Molecules.

The evolution of peptidomimetic hybrid molecules for preoperative imaging and guided surgery targeting the prostate-specific membrane antigen (PSMA) significantly progressed over the past few years, and some approaches are currently being evaluated for further clinical translation. However, accumulation in nonmalignant tissue such as kidney, bladder, spleen, or liver might limit tumor-to-background contrast for precise lesion delineation, particularly in a surgical setting. To overcome these limitations, a rational linker design aims at the development of a second generation of PSMA-11-based hybrid molecules with an enhanced pharmacokinetic profile and improved imaging contrast. Methods: A selection of rationally designed linkers was introduced to the PSMA-targeting hybrid molecule Glu-urea-Lys-HBED-CC-IRDye800CW, resulting in a second-generation peptidomimetic hybrid molecule library. The biologic properties were investigated in cell-based assays. In a preclinical proof-of-concept study with the radionuclide 68Ga, the impact of the modifications was evaluated by determination of specific tumor uptake, pharmacokinetics, and fluorescence imaging in tumor-bearing mice. Results: The modified hybrid molecules carrying various selected linkers revealed high PSMA-specific binding affinity and effective internalization. The highest tumor-to-background contrast of all modifications investigated was identified for the introduction of a histidine- (H) and glutamic acid (E)-containing linker ((HE)3-linker) between the PSMA-binding motif and the chelator. In comparison to the parental core structure, uptake in nonmalignant tissue was significantly reduced to a minimum, as exemplified by an 11-fold reduced spleen uptake from 38.12 ± 14.62 percentage injected dose (%ID)/g to 3.47 ± 1.39 %ID/g (1 h after injection). The specific tumor uptake of this compound (7.59 ± 0.95 %ID/g, 1 h after injection) was detected to be significantly higher than that of the parental tracer PSMA-11. These findings confirmed by PET and fluorescence imaging are accompanied by an enhanced pharmacokinetic profile with accelerated background clearance at early time points after injection. Conclusion: The novel generation of PSMA-targeting hybrid molecules reveals fast elimination, reduced background organ enrichment, and high PSMA-specific tumor uptake meeting the key demands for potent tracers in nuclear medicine and fluorescence-guided surgery. The approach's efficacy in improving the pharmacokinetic profile highlights the strengths of rational linker design as a powerful tool in strategic hybrid-molecule development.

Performance assessment of the ALBIRA II pre-clinical SPECT S102 system for 99mTc imaging.

OBJECTIVE: The performance characteristics of the SPECT sub-system S102 of the ALBIRA II PET/SPECT/CT are analyzed for the 80 mm field of view (FOV) to evaluate the potential in-vivo imaging in rats, based on measurements of the system response for the commonly used Technetium-99 m (99mTc) in small animal imaging. METHODS: The ALBIRA II tri-modal µPET/SPECT/CT pre-clinical system (Bruker BioSpin, Ettlingen, Germany) was used. The SPECT modality is made up of two opposite gamma cameras (Version S102) with Sodium doped Cesium Iodide (CsI(Na)) single continuous crystal detectors coupled to position-sensitive photomultipliers (PSPMTs). Imaging was performed with the NEMA NU-4 image quality phantom (Data Spectrum Corporation, Durham, USA). Measurements were performed with a starting activity concentration of 4.76 MBq/mL 99mTc. An energy window of 20% at 140 keV was selected in this study. The system offers a 20 mm, 40 mm, 60 mm and an 80 mm field of view (FOV) and in this study the 80 mm FOV was used for all the acquisitions. The data were reconstructed with an ordered subset expectation maximization (OSEM) algorithm. Sensitivity, spatial resolution, count rate linearity, convergence of the algorithm and the recovery coefficients (RC) were analyzed. All analyses were performed with PMOD and MATLAB software. RESULTS: The sensitivities measured at the center of the 80 mm FOV with the point source were 23.1 ± 0.3 cps/MBq (single pinhole SPH) and 105.6 ± 5.5 cps/MBq (multi pinhole MPH). The values for the axial, tangential and radial full width at half maximum (FWHM) were 2.51, 2.54, and 2.55 mm with SPH and 2.35, 2.44 and 2.32 mm with MPH, respectively. The corresponding RC values for the 5 mm, 4 mm, 3 mm and 2 mm rods were 0.60 ± 0.28, 0.61 ± 0.24, 0.29 ± 0.11 and 0.20 ± 0.06 with SPH and 0.56 ± 0.20, 0.50 ± 0.18, 0.38 ± 0.09 and 0.23 ± 0.06 with MPH. To obtain quantitative imaging data, the image reconstructions should be performed with 12 iterations. CONCLUSION: The ALBIRA II preclinical SPECT sub-system S102 has a favorable sensitivity and spatial resolution for the 80 mm FOV setting for both the SPH and MPH configurations and is a valuable tool for small animal imaging.

Atomic Nanogenerators in Targeted Alpha Therapies: Curie's Legacy in Modern Cancer Management.

Atomic in vivo nanogenerators such as actinium-225, thorium-227, and radium-223 are of increasing interest and importance in the treatment of patients with metastatic cancer diseases. This is due to their peculiar physical, chemical, and biological characteristics, leading to astonishing responses in otherwise resistant patients. Nevertheless, there are still a few obstacles and hurdles to be overcome that hamper the broader utilization in the clinical setting. Next to the limited supply and relatively high costs, the in vivo complex stability and the fate of the recoiling daughter radionuclides are substantial problems that need to be solved. In radiobiology, the mechanisms underlying treatment efficiency, possible resistance mechanisms, and late side effect occurrence are still far from being understood and need to be unraveled. In this review, the current knowledge on the scientific and clinical background of targeted alpha therapies is summarized. Furthermore, open issues and novel approaches with a focus on the future perspective are discussed. Once these are unraveled, targeted alpha therapies with atomic in vivo nanogenerators can be tailored to suit the needs of each patient when applying careful risk stratification and combination therapies. They have the potential to become one of the major treatment pillars in modern cancer management.

Development of PSMA-1007-Related Series of 18F-Labeled Glu-Ureido-Type PSMA Inhibitors.

In recent years, a number of drugs targeting the prostate-specific membrane antigen (PSMA) have become important tools in the diagnosis and treatment of prostate cancer. In the present work, we report on the synthesis and preclinical evaluation of a series of 18F-labeled PSMA ligands for diagnostic application based on the theragnostic ligand PSMA-617. By applying modifications to the linker structure, insight into the structure-activity relationship could be gained, highlighting the importance of hydrophilicity and stereoselectivity on interaction with PSMA and hence the biodistribution. Selected compounds were co-crystallized with the PSMA protein and analyzed by X-rays with mixed results. Among these, PSMA-1007 (compound 5) showed the best interaction with the PSMA protein. The respective radiotracer [18F]PSMA-1007 was translated into the clinic and is, in the meantime, subject of advanced clinical trials.

Detection of Calcified Aortic Plaques in an Apolipoprotein E Animal Model Using a Human Computed Tomography System for Ultra-High-resolution Imaging: A Feasibility Study.

PURPOSE: The purpose of this study was to prospectively evaluate the feasibility of detecting calcified aortic plaques in apolipoprotein E knockout (ApoE-KO) mice using a state-of-the-art human computed tomography (CT) system. MATERIALS AND METHODS: Eleven ApoE-KO and 9 wild-type mice, all male, were examined in this study. After intraperitoneal injection of 6.6% ketamine all mice underwent 2 ultra-high-resolution CT protocols on a third-generation dual-source CT system at 120 kVp and 130 kVp tube voltage, both performed with a tube current time product of 1300 mAs. Images (0.4 mm) with an increment of 0.2 mm were reconstructed using an iterative reconstruction algorithm. Calcium detectability and scores (Agatston, volume, mass) were determined with a dedicated human calcium scoring software (CaScoring). After the CT examination, a calcium quantification assay of the aortae was performed to determine the aortic calcium content of each mouse. The CT scan time ranged between 40 and 48 seconds. All mice survived the procedure. RESULTS: Calcified plaques could be detected in 8 of 11 ApoE-KO mice. Quantification of calcium levels showed significant differences between those with morphologic calcium plaques detected in CT and those without (3.44±1.6 µg Ca/mg vs. 0.33±0.35 µg Ca/mg; P<0.05). The receiver-operating characteristics analysis revealed a total calcium cut-off value of 0.71 µg Ca/mg for the detection using calcium score algorithms (specificity: 100% and sensitivity: 90%). CONCLUSION: Using a state-of-the-art human CT protocol and an in-human-established calcium scoring system allows for the detection and quantification of calcified aortic plaques in ApoE-KO mice. These results may facilitate preclinical imaging for translational and longitudinal atherosclerotic research studies.

Noncontact recognition of fluorescently labeled objects in deep tissue via a novel optical light beam arrangement.

To date, few optical imaging systems are available in clinical practice to perform noninvasive measurements transcutaneously. Instead, functional imaging is performed using ionizing radiation or intense magnetic fields in most cases. The applicability of fluorescence imaging (e.g., for the detection of fluorescently labeled objects, such as tumors) is limited due to the restricted tissue penetration of light and the required long exposure time. Thus, the development of highly sensitive and easily manageable instruments is necessary to broaden the utility of optical imaging. To advance these developments, an improved fluorescence imaging system was designed in this study that operates on the principle of noncontact laser-induced fluorescence and enables the detection of fluorescence from deeper tissue layers as well as real-time imaging. The high performance of the developed optical laser scanner results from the combination of specific point illumination, an intensified charge-coupled device (ICCD) detector with a novel light trap, and a filtering strategy. The suitability of the laser scanner was demonstrated in two representative applications and an in vivo evaluation. In addition, a comparison with a planar imaging system was performed. The results show that the exposure time with the developed laser scanner can be reduced to a few milliseconds during measurements with a penetration depth of up to 32 mm. Due to these short exposure times, real-time fluorescence imaging can be easily achieved. The ability to measure fluorescence from deep tissue layers enables clinically relevant applications, such as the detection of fluorescently labeled malignant tumors.

PSMA-11-Derived Dual-Labeled PSMA Inhibitors for Preoperative PET Imaging and Precise Fluorescence-Guided Surgery of Prostate Cancer.

Resection of tumors using targeted dual-modality probes combining preoperative imaging with intraoperative guidance is of high clinical relevance and might considerably affect the outcome of prostate cancer therapy. This work aimed at the development of dual-labeled prostate-specific membrane antigen (PSMA) inhibitors derived from the established N,N'-bis[2-hydroxy-5-(carboxyethyl)benzyl]ethylenediamine-N,N'-diacetic acid (HBED-CC)-based PET tracer 68Ga-Glu-urea-Lys(Ahx)-HBED-CC (68Ga-PSMA-11) to allow accurate intraoperative detection of PSMA-positive tumors. Methods: A series of novel PSMA-targeting fluorescent dye conjugates of Glu-urea-Lys-HBED-CC was synthesized, and their biologic properties were determined in cell-based assays and confocal microscopy. As a preclinical proof of concept, specific tumor uptake, pharmacokinetics, and feasibility for intraoperative fluorescence guidance were investigated in tumor-bearing mice and healthy pigs. Results: The designed dual-labeled PSMA inhibitors exhibited high binding affinity and PSMA-specific effective internalization. Conjugation of fluorescein isothiocyanate (10.86 ± 0.94 percentage injected dose [%ID]/g), IRDye800CW (13.66 ± 3.73 %ID/g), and DyLight800 (15.62 ± 5.52 %ID/g) resulted in a significantly increased specific tumor uptake, whereas 68Ga-Glu-urea-Lys-HBED-CC-AlexaFluor488 (9.12 ± 5.47 %ID/g) revealed a tumor uptake similar to that of 68Ga-PSMA-11 (4.89 ± 1.34 %ID/g). The first proof-of-concept studies with the clinically relevant candidate 68Ga-Glu-urea-Lys-HBED-CC-IRDye800CW reinforced a fast, specific enrichment in PSMA-positive tumors, with rapid background clearance. With regard to intraoperative navigation, a specific fluorescence signal was detected in PSMA-expressing tissue. Conclusion: This study demonstrated that PSMA-11-derived dual-labeled dye conjugates are feasible for providing PSMA-specific pre-, intra-, and postoperative detection of prostate cancer lesions and have high potential for future clinical translation.

Investigation of the imaging characteristics of the ALBIRA II small animal PET system for 18F, 68Ga and 64Cu.

AIM: In this study the performance characteristics of the Albira II PET sub-system and the response of the system for the following radionuclides 18F, 68Ga and 64Cu was analyzed. MATERIALS AND METHODS: The Albira II tri-modal system (Bruker BioSpin MRI GmbH, Ettlingen, Germany) is a pre-clinical device for PET, SPECT and CT. The PET sub-system uses single continuous crystal detectors of lutetium yttrium orthosilicate (LYSO). The detector assembly consists of three rings of 8 detector modules. The transaxial field of view (FOV) has a diameter of 80mm and the axial FOV is 148mm. A NEMA NU-4 image quality phantom (Data Spectrum Corporation, Durham, USA) having five rods with diameters of 1, 2, 3, 4 and 5mm and a uniform central region was used. Measurements with 18F, 68Ga and 64Cu were performed in list mode acquisition over 10h. Data were reconstructed using a maximum-likelihood expectation-maximization (MLEM) algorithm with iteration numbers between 5 and 50. System sensitivity, count rate linearity, convergence and recovery coefficients were analyzed. RESULTS: The sensitivities for the entire FOV (non-NEMA method) for 18F, 68Ga and 64Cu were (3.78±0.05)%, (3.97±0.18)% and (3.79±0.37)%, respectively. The sensitivity based on the NEMA protocol using the 22Na point source yielded (5.53±0.06)%. Dead-time corrected true counts were linear for activities ≤7MBq (18F and 68Ga) and ≤17MBq (64Cu) in the phantom. The radial, tangential and axial full widths at half maximum (FWHMs) were 1.52, 1.47 and 1.48mm. Recovery coefficients for the uniform region with a total activity of 8MBq in the phantom were (0.97±0.05), (0.98±0.06), (0.98±0.06) for 18F, 68Ga and 64Cu, respectively. CONCLUSION: The Albira II pre-clinical PET system has an adequate sensitivity range and the system linearity is suitable for the range of activities used for pre-clinical imaging. Overall, the system showed a favorable image quality for pre-clinical applications.

[18F]-Fluorodeoxyglucose-Positron Emission Tomography in Rats with Prolonged Cocaine Self-Administration Suggests Potential Brain Biomarkers for Addictive Behavior.

The DSM5-based dimensional diagnostic approach defines substance use disorders on a continuum from recreational drug use to habitual and ultimately addicted behavior. Biomarkers that are indicative of recreational drug use and addicted behavior are lacking. We performed a translational [18F]-fluorodeoxyglucose-positron emission tomography (FDG-PET) study in the multi-dimensional 0/3crit model of cocaine addiction. Addict-like (3crit) and non-addict-like (0crit) rats, which shared identical life conditions and levels of cocaine self-administration, were acquired for FDG-PET under baseline conditions and following cocaine and yohimbine challenges. Compared to cocaine-naïve control rats, 0crit animals showed higher glucose uptake in the caudate putamen (CPu) and medial prefrontal cortex (mPFC) respect to naïve controls. 3crit animals did not show this adaptive higher glucose utilization, but had lower uptake in several cortical areas. Both cocaine and yohimbine challenges affected glucose uptake in control rats in several brain sites, but not in 0crit and 3crit rats, indicating that impaired glucose mobilization in response to these challenges is not specifically associated with addictive behavior. Compared to 0crit, 3crit rats showed higher reinstatement responses, which were negatively associated with glucose uptake in the ventral tegmental area. Data indicate that cocaine non-addict- and addict-like phenotypes are associated with several potential biomarkers. Specifically, we propose that increased glucose uptake in the CPu and mPFC is a function of controlled drug use, whereas a loss of striatal and prefrontal metabolic activity and reduced uptake in cortical areas are indicative of addictive behavior.

Radiofluorinated N-Octanoyl Dopamine ([18F]F-NOD) as a Tool To Study Tissue Distribution and Elimination of NOD in Vitro and in Vivo.

To mitigate pretransplantation injury in organs of potential donors, N-octanoyl dopamine (NOD) treatment might be considered as it does not affect hemodynamic parameters in braindead (BD) donors. To better assess optimal NOD concentrations for donor treatment, we report on the fast and facile radiofluorination of the NOD-derivative [18F]F-NOD [18F]5 for in vivo assessment of NOD's elimination kinetics by means of PET imaging. [18F]5 was synthesized in reproducibly high radiochemical yields and purity (>98%) as well as high specific activities (>20 GBq/µmol). Stability tests showed no decomposition of [18F]5 over a period of 120 min in rat plasma. In vitro, low cell association was found for [18F]5, indicating no active transport mechanism into cells. In vivo, [18F]5 exhibited a fast blood clearance and a predominant hepatobiliary elimination. As these data suggest that also NOD might be cleared fast, further pharmacokinetic evaluation is warranted.

Inhibition of Rho-Associated Kinase 1/2 Attenuates Tumor Growth in Murine Gastric Cancer.

Gastric cancer (GC) remains a malignant disease with high mortality. Patients are frequently diagnosed in advanced stages where survival prognosis is poor. Thus, there is high medical need to find novel drug targets and treatment strategies. Recently, the comprehensive molecular characterization of GC subtypes revealed mutations in the small GTPase RHOA as a hallmark of diffuse-type GC. RHOA activates RHO-associated protein kinases (ROCK1/2) which regulate cell contractility, migration and growth and thus may play a role in cancer. However, therapeutic benefit of RHO-pathway inhibition in GC has not been shown so far. The ROCK1/2 inhibitor 1-(5-isoquinoline sulfonyl)-homopiperazine (HA-1077, fasudil) is approved for cerebrovascular bleeding in patients. We therefore investigated whether fasudil (i.p., 10 mg/kg per day, 4 times per week, 4 weeks) inhibits tumor growth in a preclinical model of GC. Fasudil evoked cell death in human GC cells and reduced the tumor size in the stomach of CEA424-SV40 TAg transgenic mice. Small animal PET/CT confirmed preclinical efficacy. Mass spectrometry imaging identified a translatable biomarker for mouse GC and suggested rapid but incomplete in situ distribution of the drug to gastric tumor tissue. RHOA expression was increased in the neoplastic murine stomach compared with normal non-malignant gastric tissue, and fasudil reduced (auto) phosphorylation of ROCK2 at THR249 in vivo and in human GC cells in vitro. In sum, our data suggest that RHO-pathway inhibition may constitute a novel strategy for treatment of GC and that enhanced distribution of future ROCK inhibitors into tumor tissue may further improve efficacy.

Opioid receptor activation triggering downregulation of cAMP improves effectiveness of anti-cancer drugs in treatment of glioblastoma.

Glioblastoma are the most frequent and malignant human brain tumors, having a very poor prognosis. The enhanced radio- and chemoresistance of glioblastoma and the glioblastoma stem cells might be the main reason why conventional therapies fail. The second messenger cyclic AMP (cAMP) controls cell proliferation, differentiation, and apoptosis. Downregulation of cAMP sensitizes tumor cells for anti-cancer treatment. Opioid receptor agonists triggering opioid receptors can activate inhibitory Gi proteins, which, in turn, block adenylyl cyclase activity reducing cAMP. In this study, we show that downregulation of cAMP by opioid receptor activation improves the effectiveness of anti-cancer drugs in treatment of glioblastoma. The µ-opioid receptor agonist D,L-methadone sensitizes glioblastoma as well as the untreatable glioblastoma stem cells for doxorubicin-induced apoptosis and activation of apoptosis pathways by reversing deficient caspase activation and deficient downregulation of XIAP and Bcl-xL, playing critical roles in glioblastomas' resistance. Blocking opioid receptors using the opioid receptor antagonist naloxone or increasing intracellular cAMP by 3-isobutyl-1-methylxanthine (IBMX) strongly reduced opioid receptor agonist-induced sensitization for doxorubicin. In addition, the opioid receptor agonist D,L-methadone increased doxorubicin uptake and decreased doxorubicin efflux, whereas doxorubicin increased opioid receptor expression in glioblastomas. Furthermore, opioid receptor activation using D,L-methadone inhibited tumor growth significantly in vivo. Our findings suggest that opioid receptor activation triggering downregulation of cAMP is a promising strategy to inhibit tumor growth and to improve the effectiveness of anti-cancer drugs in treatment of glioblastoma and in killing glioblastoma stem cells.

Targeted alpha-therapy using [Bi-213]anti-CD20 as novel treatment option for radio- and chemoresistant non-Hodgkin lymphoma cells.

Radioimmunotherapy (RIT) is an emerging treatment option for non-Hodgkin lymphoma (NHL) producing higher overall response and complete remission rates compared with unlabelled antibodies. However, the majority of patients treated with conventional or myeloablative doses of radiolabelled antibodies relapse. The development of RIT with alpha-emitters is attractive for a variety of cancers because of the high linear energy transfer (LET) and short path length of alpha-radiation in human tissue, allowing higher tumour cell kill and lower toxicity to healthy tissues. In this study, we investigated the molecular effects of the alpha-emitter Bi-213 labelled to anti-CD20 antibodies ([Bi-213]anti-CD20) on cell cycle and cell death in sensitive and radio-/chemoresistant NHL cells. [Bi-213]anti-CD20 induced apoptosis, activated caspase-3, caspase-2 and caspase-9 and cleaved PARP specifically in CD20-expressing sensitive as well as in chemoresistant, beta-radiation resistant and gamma-radiation resistant NHL cells. CD20 negative cells were not affected by [Bi-213]anti-CD20 and unspecific antibodies labelled with Bi-213 could not kill NHL cells. Breaking radio-/ chemoresistance in NHL cells using [Bi-213]anti-CD20 depends on caspase activation as demonstrated by complete inhibition of [Bi-213]anti-CD20-induced apoptosis with zVAD.fmk, a specific inhibitor of caspases activation. This suggests that deficient activation of caspases was reversed in radioresistant NHL cells using [Bi-213]anti- CD20. Activation of mitochondria, resulting in caspase-9 activation was restored and downregulation of Bcl-x(L) and XIAP, death-inhibiting proteins, was found after [Bi- 213]anti-CD20 treatment in radio-/chemosensitive and radio-/chemoresistant NHL cells. [Bi-213]anti-CD20 seems to be a promising radioimmunoconjugate to improve therapeutic success by breaking radio- and chemoresistance selectively in CD20- expressing NHL cells via re-activating apoptotic pathways through reversing deficient activation of caspases and the mitochondrial pathway and downregulation of XIAP and Bcl-x(L).