Predictive value and limitations of preclinical methods in PET-tracer development

Molekulare Bildgebung ist essentiell für die Realisierung einer individuell auf die Bedürfnisse des Patienten zugeschnittenen Therapie (personalisierte Medizin). Die Positronen-Emissions-Tomographie (PET) nutzt die physikalischen Eigenschaften des Positronen-Zerfalls, um die Verteilung eines radioaktiv markierten Arzneimittels (PET-Tracer) in vivo zu verfolgen. PET ermöglicht die nicht-invasive, quantitative und hochsensitive Darstellung molekularer Ereignisse und biologischer Prozesse in Abhängigkeit des eingesetzten PET-Tracers. Auf diese Weise dient PET als molekular-diagnostisches Bildgebungsverfahren, um Krankheiten frühzeitig und präzise zu erkennen. Die ideale PET-Bildgebungsstrategie kombiniert die günstigen Eigenschaften der Zielstruktur mit einem geeigneten physikochemischen und pharmakokinetischen Profil des PET-Tracers sowie einer spezifischen Zielstruktur-Liganden-Wechselwirkung. Eine Herausforderung in der Entwicklung von neuen PET-Tracern ist die genaue Bewertung dieser Eigenschaften, da der translatorische und prädiktive Wert präklinischer Methoden hinsichtlich der Eignung von neu entwickelten PET-Tracern zur Anwendung am Menschen kontrovers diskutiert wird. Die Bildgebungsstrategien im Rahmen dieser Arbeit umfassten den A3-Adenosinrezeptor (A3AR), das 18 kDa Translokator-Protein (TSPO) und den Melanin-konzentrierenden-Hormonrezeptor 1 (MCHR1). [18F]FE@SUPPY, der erste PET-Tracer für den A3AR, erwies sich als lipophile Verbindung und zeigte in einem Mausmodell eine hohe unspezifische Bindung, ungünstige pharmakokinetische Eigenschaften und eine geringe Stabilität. Daher ist [18F]FE@SUPPY in seiner gegebenen Form als PET-Tracer ungeeignet für die A3AR-Bildgebung. Der TSPO PET-Tracer [18F]FEPPA, welcher zur Darstellung der Mikroglia-Aktivierung klinisch angewendet wird, wurde erstmals in vitro bei Darmkrebs untersucht. Vorläufige Tests zeigten vielversprechende Ergebnisse hinsichtlich der Spezifität der Bindung von [18F]FEPPA zu Darmkrebsgewebe und -zellen. Jedoch ist [18F]FEPPA aufgrund des Polymorphismus im TSPO-Gen für die klinische Anwendung nur bedingt geeignet. In einer detaillierten in vitro und in vivo Untersuchung wurden die beiden MCHR1 PET-Tracer [11C]SNAP-7941 und [18F]FE@SNAP hinsichtlich Affinität, Selektivität, metabolischer Stabilität und Qualität der PET-Bildgebungsdaten verglichen. [11C]SNAP-7941 erwies sich als der überlegene PET-Tracer gegenüber [18F]FE@SNAP für eine zukünftige Anwendung am Menschen. Der Fokus einer weiteren Studie lag auf der Evaluierung von [11C]SNAP-7941 für die Bildgebung von braunem Fettgewebe. Neben einer klinischen Anwendung von [11C]SNAP-7941 bei psychischen und neurologischen Störungen wird eine Anwendung bei Stoffwechselerkrankungen immer realistischer.In an era, where personalized medicine is envisioned, molecular imaging is significant to the realization of an individually tailored therapy according to the patient’s needs. Positron emission tomography (PET) uses the favorable physical properties of positron decay to trace the distribution of a radio-labeled pharmaceutical (PET-tracer) in vivo. PET enables a non-invasive, quantitative and highly sensitive visualization of any molecular event or biological process – depending on the employed PET-tracer. In this manner, PET serves as a molecular diagnostic tool that provides an accurate and sensitive detection of diseases in an early stage. The ideal PET imaging strategy combines the favorable characteristics of the target, a suitable physicochemical and pharmacokinetic profile of the PET-tracer and a specific target-ligand-interaction. One key challenge in PET-tracer development is the precise evaluation of these characteristics, as the translational and predictive value of preclinical methods regarding the suitability of newly developed PET-tracers for human application is controversially discussed. PET imaging strategies within the scope of this thesis included the A3 adenosine receptor (A3AR), the 18 kDa translocator protein (TSPO) and the melanin-concentrating hormone receptor 1 (MCHR1). [18F]FE@SUPPY, the first A3AR PET-tracer, evinced as a highly lipophilic compound and showed unspecific binding, unfavorable pharmacokinetics and low in vivo stability in a mouse model. Thus, in its given form, [18F]FE@SUPPY is inadequate to serve as a PET-tracer for A3AR imaging. The well-established TSPO PET-tracer [18F]FEPPA, which is currently in clinical use for microglia activation, was evaluated in vitro in colorectal cancer for the first time. Preliminary testing showed promising results regarding the specificity of [18F]FEPPA binding to colon cancer tissue and cells. However, clinical TSPO PET imaging using [18F]FEPPA is limited due to the polymorphism rs6971. Therefore, this imaging strategy provides only a temporary solution until a PET-tracer is developed, which binds with equal affinity to TSPO in all patients. In a detailed in vitro and in vivo evaluation, the MCHR1 PET-tracers, [11C]SNAP-7941 and [18F]FE@SNAP, were compared regarding affinity, selectivity, metabolic stability and quality of PET imaging data. [11C]SNAP-7941 emerged as the superior tracer compared to [18F]FE@SNAP for future human use. In a further study, special emphasis was placed on the evaluation of [11C]SNAP-7941 for brown adipose tissue imaging, as preliminary data indicated MCHR1 expression. Thus, besides a clinical application of [11C]SNAP-7941 in mental and neurologic disorders, an application in metabolic related diseases is becoming more and more realistic

Experimental Nuclear Medicine Meets Tumor Biology

Personalized treatment of cancer patients demands specific and validated biomarkers for tumor diagnosis and therapy. The development and validation of such require translational preclinical models that recapitulate human diseases as accurately as possible. Moreover, there is a need for convergence of different (pre)clinical disciplines that openly share their knowledge and methodologies. This review sheds light on the differential perception of biomarkers and gives an overview of currently used models in tracer development and approaches for biomarker discovery

Comparison of Radiation Response between 2D and 3D Cell Culture Models of Different Human Cancer Cell Lines

Radiation therapy is one of the most effective tools in cancer therapy. However, success varies individually, necessitating improved understanding of radiobiology. Three-dimensional (3D) tumor spheroids are increasingly gaining attention, being a superior in vitro cancer model compared to 2D cell cultures. This in vitro study aimed at comparing radiation responses in 2D and 3D cell culture models of different human cancer cell lines (PC-3, LNCaP and T-47D) irradiated with varying doses (1, 2, 4, 6, 8 or 20 Gy) of X-ray beams. Radiation response was analyzed by growth analysis, various cell viability assays (e.g., clonogenic assay, resazurin assay) and amount of DNA damage (γH2AX Western Blot). Results showed decreasing cell proliferation with the increase of radiation doses for all cell lines in monolayers and spheroids of LNCaP and T-47D. However, significantly lower radiosensitivity was detected in spheroids, most pronounced in PC-3, evincing radiation resistance of PC-3 spheroids up to 8 Gy and significant growth inhibition only by a dose escalation of 20 Gy. Cell line comparison showed highest radiosensitivity in LNCaP, followed by T-47D and PC-3 in 2D, whereas, in 3D, T-47D showed highest sensitivity. The results substantiate the significant differences in radiobiological response to X-rays between 2D and 3D cell culture models

Experimental Nuclear Medicine Meets Tumor Biology

Personalized treatment of cancer patients demands specific and validated biomarkers for tumor diagnosis and therapy. The development and validation of such require translational preclinical models that recapitulate human diseases as accurately as possible. Moreover, there is a need for convergence of different (pre)clinical disciplines that openly share their knowledge and methodologies. This review sheds light on the differential perception of biomarkers and gives an overview of currently used models in tracer development and approaches for biomarker discovery

Comparison of Radiation Response between 2D and 3D Cell Culture Models of Different Human Cancer Cell Lines

Radiation therapy is one of the most effective tools in cancer therapy. However, success varies individually, necessitating improved understanding of radiobiology. Three-dimensional (3D) tumor spheroids are increasingly gaining attention, being a superior in vitro cancer model compared to 2D cell cultures. This in vitro study aimed at comparing radiation responses in 2D and 3D cell culture models of different human cancer cell lines (PC-3, LNCaP and T-47D) irradiated with varying doses (1, 2, 4, 6, 8 or 20 Gy) of X-ray beams. Radiation response was analyzed by growth analysis, various cell viability assays (e.g., clonogenic assay, resazurin assay) and amount of DNA damage (γH2AX Western Blot). Results showed decreasing cell proliferation with the increase of radiation doses for all cell lines in monolayers and spheroids of LNCaP and T-47D. However, significantly lower radiosensitivity was detected in spheroids, most pronounced in PC-3, evincing radiation resistance of PC-3 spheroids up to 8 Gy and significant growth inhibition only by a dose escalation of 20 Gy. Cell line comparison showed highest radiosensitivity in LNCaP, followed by T-47D and PC-3 in 2D, whereas, in 3D, T-47D showed highest sensitivity. The results substantiate the significant differences in radiobiological response to X-rays between 2D and 3D cell culture models

Sorbitol as a Polar Pharmacological Modifier to Enhance the Hydrophilicity of 99mTc-Tricarbonyl-Based Radiopharmaceuticals

The organometallic technetium-99m tricarbonyl core, [99mTc][Tc(CO)3(H2O)3]+, is a versatile precursor for the development of radiotracers for single photon emission computed tomography (SPECT). A drawback of the 99mTc-tricarbonyl core is its lipophilicity, which can influence the pharmacokinetic properties of the SPECT imaging probe. Addition of polar pharmacological modifiers to 99mTc-tricarbonyl conjugates holds the promise to counteract this effect and provide tumor-targeting radiopharmaceuticals with improved hydrophilicities, e.g., resulting in a favorable fast renal excretion in vivo. We applied the “Click-to-Chelate” strategy for the assembly of a novel 99mTc-tricarbonyl labeled conjugate made of the tumor-targeting, modified bombesin binding sequence [Nle14]BBN(7–14) and the carbohydrate sorbitol as a polar modifier. The 99mTc-radiopeptide was evaluated in vitro with PC-3 cells and in Fox-1nu mice bearing PC-3 xenografts including a direct comparison with a reference conjugate lacking the sorbitol moiety. The glycated 99mTc-tricarbonyl peptide conjugate exhibited an increased hydrophilicity as well as a retained affinity toward the Gastrin releasing peptide receptor and cell internalization properties. However, there was no significant difference in vivo in terms of pharmacokinetic properties. In particular, the rate and route of excretion was unaltered in comparison to the more lipophilic reference compound. This could be attributed to the intrinsic properties of the peptide and/or its metabolites. We report a novel glycated (sorbitol-containing) alkyne substrate for the “Click-to-Chelate” methodology, which is potentially of general applicability for the development of 99mTc-tricarbonyl based radiotracers displaying an enhanced hydrophilicity

CAM-Xenograft Model Provides Preclinical Evidence for the Applicability of [⁶⁸Ga]Ga-Pentixafor in CRC Imaging

Colorectal cancer is one of the leading causes of cancer-related deaths worldwide. Increased expression of CXCR4 has been associated with liver metastasis, disease progression, and shortened survival. Using in vitro cell binding studies and the in ovo model, we aimed to investigate the potential of [68Ga]Ga-Pentixafor, a radiotracer specifically targeting human CXCR4, for CRC imaging. Specific membrane binding and internalisation of [68Ga]Ga-Pentixafor was shown for HT29 cells, but not for HCT116 cells. Accordingly, [68Ga]Ga-Pentixafor accumulated specifically in CAM-xenografts derived from HT29 cells, but not in HCT116 xenografts, as determined by µPET/MRI. The CAM-grown xenografts were histologically characterised, demonstrating vascularisation of the graft, preserved expression of human CXCR4, and viability of the tumour cells within the grafts. In vivo viability was further confirmed by µPET/MRI measurements using 2-[18F]FDG as a surrogate for glucose metabolism. [68Ga]Ga-Pentixafor µPET/MRI scans showed distinct radiotracer accumulation in the chick embryonal heart, liver, and kidneys, whereas 2-[18F]FDG uptake was predominantly found in the kidneys and joints of the chick embryos. Our findings suggest that [68Ga]Ga-Pentixafor is an interesting novel radiotracer for CRC imaging that is worth further investigation. Moreover, this study further supports the suitability of the CAM-xenograft model for the initial preclinical evaluation of targeted radiopharmaceuticals

If It Works, Don’t Touch It? A Cell-Based Approach to Studying 2-[18F]FDG Metabolism

The glucose derivative 2-[18F]fluoro-2-deoxy-D-glucose (2-[18F]FDG) is still the most used radiotracer for positron emission tomography, as it visualizes glucose utilization and energy demand. In general, 2-[18F]FDG is said to be trapped intracellularly as 2-[18F]FDG-6-phosphate, which cannot be further metabolized. However, increasingly, this dogma is being questioned because of publications showing metabolism beyond 2-[18F]FDG-6-phosphate and even postulating 2-[18F]FDG imaging to depend on the enzyme hexose-6-phosphate dehydrogenase in the endoplasmic reticulum. Therefore, we aimed to study 2-[18F]FDG metabolism in the human cancer cell lines HT1080, HT29 and Huh7 applying HPLC. We then compared 2-[18F]FDG metabolism with intracellular tracer accumulation, efflux and the cells’ metabolic state and used a graphical Gaussian model to visualize metabolic patterns. The extent of 2-[18F]FDG metabolism varied considerably, dependent on the cell line, and was significantly enhanced by glucose withdrawal. However, the metabolic pattern was quite conserved. The most important radiometabolites beyond 2-[18F]FDG-6-phosphate were 2-[18F]FDMannose-6-phosphate, 2-[18F]FDG-1,6-bisphosphate and 2-[18F]FD-phosphogluconolactone. Enhanced radiometabolite formation under glucose reduction was accompanied by reduced efflux and mirrored the cells’ metabolic switch as assessed via extracellular lactate levels. We conclude that there can be considerable metabolism beyond 2-[18F]FDG-6-phosphate in cancer cell lines and a comprehensive understanding of 2-[18F]FDG metabolism might help to improve cancer research and tumor diagnosis

SNAPshots of the MCHR1 : a Comparison Between the PET-Tracers [18F]FE@SNAP and [11C]SNAP-7941

Purpose The melanin-concentrating hormone receptor 1 (MCHR1) has become an important pharmacological target, since it may be involved in various diseases, such as diabetes, insulin resistance, and obesity. Hence, a suitable positron emission tomography radiotracer for the in vivo assessment of the MCHR1 pharmacology is imperative. The current paper contrasts the extensive in vitro, in vivo, and ex vivo assessments of the radiotracers [18F]FE@SNAP and [11C]SNAP-7941 and provides comprehensive information about their biological and physicochemical properties. Furthermore, it examines their suitability for first-in-man imaging studies. Procedures Kinetic real-time cell-binding studies with [18F]FE@SNAP and [11C]SNAP-7941 were conducted on adherent Chines hamster ovary (CHO-K1) cells stably expressing the human MCHR1 and MCHR2. Small animal imaging studies on mice and rats were performed under displacement and baseline conditions, as well as after pretreatment with the P-glycoprotein/breast cancer resistant protein inhibitor tariquidar. After the imaging studies, detailed analyses of the ex vivo biodistribution were performed. Ex vivo metabolism was determined in rat blood and brain and analyzed at various time points using a quantitative radio-HPLC assay. Results [11C]SNAP-7941 demonstrates high uptake on CHO-K1-hMCHR1 cells, whereas no uptake was detected for the CHO-K1-hMCHR2 cells. In contrast, [18F]FE@SNAP evinced binding to CHO-K1-hMCHR1 and CHO-K1-hMCHR2 cells. Imaging studies with [18F]FE@SNAP and [11C]SNAP-7941 showed an increased brain uptake after tariquidar pretreatment in mice, as well as in rats, and exhibited a significant difference between the time-activity curves of the baseline and blocking groups. Biodistribution of both tracers demonstrated a decreased uptake after displacement. [11C]SNAP-7941 revealed a high metabolic stability in rats, whereas [18F]FE@SNAP was rapidly metabolized. Conclusions Both radiotracers demonstrate appropriate imaging properties for the MCHR1. However, the pronounced metabolic stability as well as superior selectivity and affinity of [11C]SNAP-7941 underlines the decisive superiority over [18F]FE@SNAP.(VLID)365868