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Background There are only a handful of true theranostic matched pairs, and in particular the theranostic radiocopper trio 61Cu, 64Cu and 67Cu, for diagnosis and therapy respectively, is a very attractive candidate. In fact, the alternative of two imaging radionuclides with different half-lives is a clear advantage over other theranostic pairs, since it offers a better matching for the tracer biological and radionuclide physical half-lives. Due to the high availability of 64Cu, its translation into the clinic is being successfully carried out, giving the example of the FDA approved radiopharmaceutical Detectnet (copper Cu 64 dotatate injection). However, a shorter-lived PET radionuclide such as 61Cu may as well be beneficial. Results Proton irradiation of enriched 62Ni electrodeposited targets with a compact cyclotron produced the desired radionuclide via the 62Ni(p,2n)61Cu nuclear reaction, leading to 61Cu activities of up to 20 GBq at end of bombardment and 8 GBq at end of purification. Furthermore, two purification methods are compared leading to comparable results regarding separation yield and product purity. Following the radiochemical separation, quality assessment of this product [61Cu]CuCl2 solution proved radionuclidic purities (RNP) over 99.6 % and apparent molar activities (AMA) of 260 GBq/µmol with the 1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid (TETA) chelator, end of purification corrected. Conclusions In the current article a comprehensive novel production method for the PET radionuclide 61Cu is presented, providing an alternative to the most popular production routes. Characterization of the [61Cu]CuCl2 product showed both high RNP as well as high AMA, proving that the produced activity presented high quality regarding radiolabeling up to 9 hours after EOP. Furthermore, production scalability could be easily achieved by increasing the irradiation time.

The use of radionuclides for targeted endoradiotherapy is a rapidly growing field in oncology. In particular, the focus on the biological effects of different radiation qualities is an important factor in understanding and implementing new therapies. Together with the combined approach of imaging and therapy, therapeutic nuclear medicine has made great progress recently. A par-ticular area of research is the use of alpha-emitting radionuclides, which have unique physical properties associated with outstanding advantages, e.g. for single tumor cell targeting. Here, recent results and open questions regarding the production of alpha-emitting isotopes, their chemical combination with carrier molecules and clinical experience from compassionate use reports and clinical trials are discussed.

Cyclic and acyclic ligands containing the hydroxypyridinone (HOPO) moiety as donor group are known as strong coordinating compounds for a wide variety of metal ions. Based on the diaza-crown[18]ether Kryptofix K22, five different tendentate ligands were prepared using 1,2-HOPO, 1,2,3-HOPO and 2,3-Me-HOPO as additional binding moieties. The diaza-crown ether basic skeleton was furnished with two primary amine functions and subsequently reacted with the respective HOPO acids or the HOPO acid chlorides to obtain the desired HOPO derivatives in two synthesis steps after final deprotection. All compounds were evidenced by NMR and MS analyses.

A new Center for Radiopharmaceutical Cancer Research was established at the Helmholtz-Zentrum Dresden-Rossendorf in order to centralize radionuclide production, radiopharmaceutical production and the chemical and biochemical research facilities in 2017. A routine production of several radionuclides was established during the last years. We report about the production methods of metallic and standard PET radionuclides like 11C, 18F, 123I, 64Cu, 67Cu, 67Ga, 131Ba and 131La that are done regularly. In the conclusion we report typical irradiation parameters and achieved saturation yields.

Background: (S)-[18F]FETrp is a promising PET radiotracer for imaging IDO1 activity, one of the main enzymes involved in the tryptophan metabolism that plays a key role in several diseases including cancers. To date, the radiosynthesis of this tryptophan analogue remains highly challenging due to partial racemization occurring during the nucleophilic radiofluorination step. This work aims to develop a short, epimerization-free and efficient automated procedure of (S)-[18F]FETrp from a corresponding enantiopure tosylate precursor. Results: Enantiomerically pure (S)- and (R)-FETrp references as well as tosylate precursors (S)- and (R)-3 were obtained from corresponding N-Boc-(L and D)-tryptophan in 2 and 4 steps, respectively. Manual optimisation of the radiolabelling conditions resulted in >90% radiochemical conversion with more than 99% enantiomeric purity. Based on these results, the (S)-[18F]FETrp radiosynthesis was fully automated on a SynChrom R&D EVOI module to produce the radiotracer in 55.2 ± 7.5% radiochemical yield, 99.9% radiochemical purity, 99.1 ± 0.5% enantiomeric excess, and molar activity of 53.2 ± 9.3 GBq/mol (n = 3). Conclusions: To avoid racemisation and complicated purification processes, currently encountered for the radiosynthesis of (S)-[18F]FETrp, we report herein significant improvements, including a versatile synthesis of enantiomerically pure tosylate precursor and reference compound and a convenient one-pot two-step automated procedure for the radiosynthesis of (S)-[18F]FETrp. This optimised and robust production method could facilitate further investigations of this relevant PET radiotracer for imaging IDO1 activity.

Die Radiumisotope Ra-223/-224 mit ihrem diagnostischen Gammastrahler Ba-131 (SPECT) sind ideale Kandidaten für die zielgerichtete Alphatherapie. Um Ra2+ in Radiokonjugaten zu nutzen, muss dieses jedoch stabil gebunden werden. Der Chelator Macropa (mcp) bildet nach bisherigen Erkenntnissen die stabilsten Komplexe, jedoch sind diese für In-vivo-Anwendungen nicht stabil genug. Daher wurden neuartige makrozyklische Chelatoren auf Basis von macropa entwickelt und zusätzlich eine Anwendung für La, Ac und Pb in Betracht gezogen.

Macropa-PSMA-Konjugate, die zur zielgerichteten Alphatherapie mit Ac-225 dienen, werden mit einem Albuminbinder verknüpft. Neben der Verbesserung des pharmakologischen Verhaltens in vivo bietet die albuminbindende Einheit die Grundlage eines theranostischen Ansatzes. Neben der Komplexierung des Alphaemitters Ac-225 wird zusätzlich die Einführung des SPECT-Nuklids I-123 im gleichen Molekül ermöglicht. Vorteilhaft sind die milden Bedingungen der Radioiodierung sowie eine Halbwertszeit (t1/2 = 13,2 h), welche die Bildgebung länger zirkulierender Substanzen ermöglicht.

This poster highlights the use of the sulfur [18F]fluoride exchange approach to radiolabel a PSMA inhibitor ([18F]2) and test its radiolabeling, in vitro and in vivo properties. • [18F]2 shows good binding kinetics compared to known radiotracers (1 nM) but ~20% defluorination occurs in human serum after 120 min. • [18F]2 binds to LNCaP xenograft sections in a similar pattern as [68Ga]Ga-PSMA-11. • PET imaging experiments show defluorination but also accumulation in LNCaP tumor xenografts (SUVmax = 4.4). • Further work: Radiosynthesis of [18F]4 starting from precursor 3 (in poster)

Macropa-PSMA-Konjugate, die zur zielgerichteten Alphatherapie mit Ac-225 dienen, werden mit einem Albuminbinder verknüpft. Neben der Verbesserung des pharmakologischen Verhaltens in vivo bietet die albuminbindende Einheit die Grundlage eines theranostischen Ansatzes. Neben der Komplexierung des Alphaemitters Ac-225 wird zusätzlich die Einführung des SPECT-Nuklids I-123 im gleichen Molekül ermöglicht. Vorteilhaft sind die milden Bedingungen der Radioiodierung sowie eine Halbwertszeit (t1/2 = 13,2 h), welche die Bildgebung länger zirkulierender Substanzen ermöglicht.

Die Radiumisotope Ra-223/-224 mit ihrem diagnostischen Gammastrahler Ba-131 (SPECT) sind ideale Kandidaten für die zielgerichtete Alphatherapie. Um Ra2+ in Radiokonjugaten zu nutzen, muss dieses jedoch stabil gebunden werden. Der Chelator Macropa (mcp) bildet nach bisherigen Erkenntnissen die stabilsten Komplexe, jedoch sind diese für In-vivo-Anwendungen nicht stabil genug. Daher wurden neuartige makrozyklische Chelatoren auf Basis von macropa entwickelt und zusätzlich eine Anwendung für La, Ac und Pb in Betracht gezogen.

Ziel: Die Herstellung von 6-L-[18F]FDOPA durch elektrophile Radiomarkierung mit [18F]F2 Gas ist eine in Dresden-Rossendorf seit über 20 Jahren praktizierte Methode [1,2]. Mit der Inbetriebnahme eines neuen Zyklotrons TR-Flex und des F2-Gastargets 2018 musste die elektrophile Synthese von [18F]FDOPA angepasst werden.

Ziel: Copper-mediated radiofluorination (CMRF) was a breakthrough of the last decade in the development of non-activated 18F-aryl-bearing radiopharmaceuticals.1 Despite extensive studies and improvements of the radiolabelling conditions, the formation of H-side product 3 and OH-side product 4 still possess a challenge in these Suzuki/Stille type reactions. In our work, we faced similar problems and additionally, the continuous hydrolysis of the boronic ester precursor 1 during the semi-preparative HPLC purification resulted in another impurity 5. In this study, we tried to address and overcome these challenges. Methoden: The CMRF of 1 bearing a non-activating aromatic substituent at para position was optimized by varying the following parameters: solvent (DMA and DMI), reaction time (5 - 20 min), temperature (110 - 130 °C) and molar ratio of precursor 1 to Cu-complex (1:3, 1:4. 2:3, 1:8). Compounds 3 and 4 were synthesized as references for identification of the side-products in the final radiotracer formulation. Various stationary phases (pentafluorophenyl, cyano, phenyl, C18) and mobile phases were tested to separate unwanted side products 3 and 4 by HPLC. Solid phase extraction (SPE) was performed with the C18 plus cartridge before the semi-preparative HPLC purification of [18F]2. Ergebnisse: [18F]2 was achieved with a high radiochemical conversion of 85 % using 2 mg of 1, 10 mg of [Cu(OTf)2(py)4] (molar ratio of 1:4) in n-BuOH/DMI (1:2, v/v) at 110 °C for 5 min. SPE under acidic conditions (pH 2) resulted in around 90 % recovery of [18F]2 compared to only 20 % under neutral conditions. Due to hydrolysis of residual precursor 1 with TFA prior to semi-preparative HPLC purification, the content of 5 in the formulated final product could be reduced from 25 % (without TFA hydrolysis) to 0.5-1 %. The side-products 3 and 4 were successfully separated using a ReproSil C18AQ column (250 x 20 mm) and 48 % THF/ACN (1:1, v/v) buffered with 20 mM ammonium acetate. Compound [

Aim: Cannabinoid receptor 2 (CB2) expression in healthy brain is very low and the upregulation is associated with inflammation, traumatic brain injury, neurodegeneration and cancer[1]. In view of the increasing interest in CB2-targeted therapies, PET offers an attractive strategy to quantify the availability of CB2 in the diseased brain. To achieve this goal, we developed a number of 18F-labelled CB2 ligands and biologically evaluated them in rodents. Here we present a comparative overview of the obtained results. Methods: Structure-activity-relationships-driven target compound identification, organic synthesis and radiofluorination was performed for compounds of the thiazole ([¹⁸F]JHU94620[2] and [¹⁸F]LUZ5[3]), naphthyridin-2-one ([¹⁸F]LU14[4] and [¹⁸F]LU13[5]) and indole ([¹⁸F]RM365) families. The new radioligands were assessed in vitro by binding experiments using CHO(hCB2) cells and rat spleen homogenates and by autoradiography on cryosections of rodent spleen. Furthermore, the radioligands were examined for their metabolic stability and biodistribution by PET. Furthermore, for selected radioligands the binding to highly expressed hCB2 in a rat model overexpressing hCB2(D80N) in the right striatum (AAV-hCB2)[6] was investigated. Results: The low- to subnanomolar hCB2 affinities of the presented radioligands were demonstrated in vitro by Kd values ranging from 0.4 to 2.9 nM with a hCB2 selectivity against hCB1 of >1000-fold. The highest metabolic stability was observed for [18F]RM365 with 55% and 90% and the lowest for [¹⁸F]JHU94620 with 7% and 36% of the initial fraction in plasma and brain 30 min p.i., respectively. The in vivo experiments confirmed the in vitro autoradiographic results, and showed high uptake for [¹⁸F]JHU94620, but low or non-displaceable uptake for [¹⁸F]LU14, [¹⁸F]LUZ5 and [¹⁸F]RM365 in spleen. Contrary to the low uptake of [¹⁸F]LUZ5 in the brain of naïve Wistar rats, target-specific and displaceable uptake for [¹⁸F]LU14 and [¹⁸F]RM365 was de

Ziel/Aim: Der Einsatz von Actinium-225 mit dem DOTA-Konjugat PSMA-617 befindet sich in der klinischen Entwicklung. DOTA ist jedoch nicht ideal, da hohe Temperaturen für die Markierung benötigt werden und der Komplex in vivo nicht stabil genug ist. Ziel ist es, einen besseren, macropa-basierten Chelator zu finden und mit von PSMA-617 abgeleiteten Bindungsvektoren zu verknüpfen. Methodik/Methods: Es wurden 4 verschiedene Konjugate hergestellt, welche sich hinsichtlich der Anzahl der Bindungsmotive sowie ihrer Fähigkeit, an Albumin zu binden, unterscheiden. Die Stabilität der markierten Komplexe wurde über 10 Tage in Puffer und Serum untersucht. Weiterhin wurden Bindungsaffinitäten und Koloniebildungsassays an PSMA-positiven LNCaP-Zellen durchgeführt. Alle Konjugate wurden hinsichtlich ihrer Organverteilung in LNCaP-Tumor-tragenden SCID-Mäusen untersucht. Ergebnisse/Results: Alle 4 Konjugate wurden unter milden Bedingungen radiomarkiert und zeigen bei Zugabe von Gentisinsäure die höchste Stabilität über 10 Tagen. Die Bindungsaffinitäten der Konjugate wurden als KD-Werte bestimmt und liegen zwischen (10-40) nM. Die Untersuchungen zum Zellüberleben zeigen einen therapeutischen Vorteil der bivalenten gegenüber den monovalenten Konjugaten. Alle vier Konjugate erreichten in unterschiedlichen Zeitspannen (bis 5 d p.i.) Tumoranreicherungen von (12-85) %ID/g. Unerwünschte Anreicherungen traten v. a. bei dem dimeren, albuminbindenden Konjugat in Form von Akkumulationen (Leber, Milz, Nieren) auf. Schlussfolgerungen/Conclusions: Die Verwendung von macropa-basierten Konjugaten für die Therapie mit Actinium‑225 oder die zukünftige Bildgebung mit Lanthan‑133 ist vorteilhaft, da die gebildeten Komplexe stabiler sind und damit eine Anreicherung der Aktivität in off-Target-Regionen verringert werden kann. Weiterhin ergibt sich ein Vorteil im Handling, da die Reaktionen schnell und bei Raumtemperatur durchgeführt werden können, was die spätere Umsetzung in eine GMP-gerechte Herstellung

Introduction: The A2A adenosine receptor (A2AAR) is expressed in brain, vasculature and immune cells. According to the alterations of the A2AAR expression in multiple diseases, it is a highly attractive diagnostic and therapeutic target. We developed the A2AAR-specific PET radiotracer [18F]FLUDA and investigated it in healthy mice and piglets, in a rotenone-based mouse model of Parkinson’s disease (RMMPD) and in transgenic mice overexpressing the human A2AAR in heart (TG) [1 3]. Methods: On the basis of a one-pot two-step radiofluorination procedure, a remotely controlled automated radiosynthesis of [18F]FLUDA using the TRACERlab FX2N synthesis module was developed. In vitro autoradiography was performed with cryosections of tissue from animal models. In vivo stability was investigated in mouse by radio-HPLC analyses of blood plasma and brain homogenates. The biodistribution was investigated by dynamic PET/MR studies in healthy mice and piglets under control and blocking conditions (vehicle vs. blocking with 2.5 mg/kg tozadenant and/or 1.0 mg/kg istradefylline) and in both mouse models. The binding potential (BPND) in vivo was calculated using the simplified reference tissue modelling with the cerebellum as reference region. A single dose acute toxicity study was performed in Wistar rats according to the ICH guideline M3(R2). PET-derived radiation dosimetry was estimated in piglets. Results: A reliable and reproducible procedure for the automated production of [18F]FLUDA was successfully established (Fig. 1A) [4]. In vitro autoradiography revealed highly selective binding and high affinity of [18F]FLUDA towards the A2AAR of the three species (KD values 0.7-5.9 nM, Fig. 1B). At 15 min after i.v. injection of [18F]FLUDA in mice, the parent fraction accounted for about 100% in brain and 71% in plasma. PET studies confirmed the specific binding of [18F]FLUDA in vivo to the striatal A2AAR in mice and piglets (BPND=3.9 and 1.3, Fig. 1C). The availability of A2AAR in the P

The cannabinoid receptor 2 (CB2R) is involved in inflammatory processes [1], whereby an increased expression correlates with malignancy in various cancer types like human epidermal growth receptor 2 positive (HER2+) or triple negative breast cancer (TNBC) [2]. Hence, the CB2R is suggested as a pharmacological target, as well as a prognostic biomarker for the stratification and staging of patients [3]. In this study, we evaluated the potential of our novel CB2R-specific radioligand [¹ ⁸F]JHU94620-d8 for the assessment of the CB2R expression in HER2+ breast cancer and TNBC models in vitro. The KD value of [¹ ⁸F]JHU94620-d8 was determined by autoradiography on cryosections of rat and mouse spleen, as well as on rat brains harbouring a local overexpression of the hCB2R (AAV-hCB2R) [4]. The CB2R status was investigated by competitive radioligand binding assays (CRBA) in cell membranes of CHO cells overexpressing the human CB2R (CHOhCB2R), human breast cancer cell lines HCC1954 (HER2+) and MDA-MB-231 (TNBC) with 3.1±0.4 nM [³H]WIN55,212-2 using 10 µM of WIN55,212-2, GW405833 and JHU94620-d8 (each n=3) as agonistic competitors. CB2R expression was validated by immunofluorescence microscopy (IFM). On cryosections of 4T1 tumors the CB2R specific binding of [¹ ⁸F]JHU94620-d8 was investigated by CRBA and the colocalisation of CB2R with Iba1 (macrophages) and CD31 (blood vessels) by IFM. We determined KD values for [¹ ⁸F]JHU94620-d8 of 30 nM in mouse spleen, of 1.0 nM in rat spleen, and of 42 nM in AAV-hCB2R. The cell membrane binding of [³H]WIN55,212-2 was comparable in all used cell lines between 20 ± 1 and 30 ± 11 fmol/106 cells. Competition by JHU94620-d8 decreased the total binding by 57 % (p<0.01) only in CHOhCB2R cells, WIN55,212-2 by 37 % (p=0.01) and 77 % (p<0.01) and GW405833 by 42 % (p<0.01) and 75 % (p<0.01) in HCC1954 and CHOhCB2R cells, respectively, however in MDA MB 231 cells binding was not affected by these compounds (Fig. 1A). The expression of CB2R was confi

Objectives Theranostic concepts and the usage of alpha-particle emitting radionuclides belong to the emerging fields of radiopharmaceutical sciences. Especially and due to its excellent complexation properties, the chelator macropa (mcp)[1,2] was reported to be a suitable complexing agent for 225Ac conjugates[3] and as macrocycle a highly promising starting point for the development of 223/224Ra chelators as well. To follow the theranostic approaches with these alpha emitters, due to their chemical similarities 131Ba[4] for SPECT and 133La[5] for PET are available as diagnostic radionuclide surrogates of radium and actinium, respectively. In our recent study, we aim to establish a new workflow to evaluate and predict the complex stability of new chelating systems by obtaining both protonation constants for respective ligands and association constants for their metal complexes starting with mcp. Methods As a prerequisite for the calculation of stability constants (log(K)), the protonation constants (pKa) of mcp were determined by pH‑dependent 1H NMR studies in the first step. Based on these obtained data, the europium-mcp-complex was examined using time-resolved, laser-induced luminescence spectroscopy (TRLFS) and the log(K) was calculated as reference stability constant. Additionally, Eu-TRLFS was used to determine the Eu-mcp species during titration and pH-dependency of the complexation. The method of isothermal titration calorimetry (ITC) was used to measure the complex stability constants of La-, Ba-mcp-complex, as well as Eu-mcp-complex to value the results in comparison with TRLFS data. The evaluation and calculation of the log(K) values were carried out by parallel factor analysis (PARAFAC). Results Depending on the protonation ability of the functional groups found in mcp (amines and carboxylates), pKa values were obtained as well as the log(K) values for the respective mcp-complexes with Ba, Eu and La and are shown in Table 1. Conclusion By combining 1H-NMR

Ziel: Die Herstellung von 6-L-[18F]FDOPA durch elektrophile Radiomarkierung mit [18F]F2 Gas ist eine in Dresden-Rossendorf seit über 20 Jahren praktizierte Methode [1,2]. Mit der Inbetriebnahme eines neuen Zyklotrons TR-Flex und des F2-Gastargets 2018 musste die elektrophile Synthese von [18F]FDOPA angepasst werden.

Das prostataspezifische Membranantigen (PSMA) ist ein hoch attraktives biologisches Target für die molekulare Bildgebung und für die zielgerichtete Radionuklidtherapie bei kastrationsresistentem Prostatakrebs (mCRPC), da es in allen Phasen der Erkrankung bei den Tumorzellen expremiert ist. Somit bieten hoch affine, PSMA-bindende Biomoleküle eine exzellente Basis zur Entwicklung von Radiokonjugaten für die Therapie mit Alphastrahlern. Aufgrund seiner hervorragenden kernphysikalischen Eigenschaften wird zunehmend Actinium-225 für diese Zwecke in der Nuklearmedizin verwendet. Das dreiwertige Kation Ac3+ muss dazu in einem Chelator hoch stabil komplexiert werden. Um das zu realisieren, wird der Chelator macropa verwendet, welcher eine sehr hohe In-vivo-Stabilität zeigt und Radiomarkierungen unter sehr milden Bedingungen zulässt. Dieser wird mit einer bzw. zwei PSMA-bindenden Einheiten modifiziert, um den 225Ac-Komplex zu den Tumorzellen zu bringen. Zunächst wurden zwei Actinium-225-Radiokonjugate entwickelt und deren pharmakologische Eigenschaften, z.B. die Bindung an Tumorzellen, Anreicherung im Tumor (ca. 10%ID/g für beide Konjugate) und in anderen Organen überprüft. Hier wurde ein ähnliches Verhalten festgestellt, vergleichbar mit dem Standardkonjugat 225Ac-PSMA-617. Die beiden 225Ac-Radiokonjugate wurden weiterentwickelt und mit einem Albumin-Binder (4-Iodphenylbutyrat) versehen. Dadurch wurde eine längere Blutverweilzeit, kombiniert mit einer höheren Aufnahme in den Tumor, erreicht (bis 50%ID/g Monomer, bis 120 %ID/g Dimer), um die Effektivität der Radiotherapie zu erhöhen.

Objectives: Positron Emission Tomography (PET) is a non-invasive molecular imaging technique, which continues to find increasing application and further development for diagnosis of a broad range of disorders and dysfunctions. Since their involvement in pathophysiological processes that lead to cancer, for example, monocarboxylate transporters (MCTs) are of interest for the development of new radioligands for PET, such as [18F]FACH [1, 2] and its deuterated isotopologue [18F]FACH-d6. Both candidates have been already examined previously in preclinical PET studies in healthy mice and piglets, also with regard to their biodistribution and metabolic fate [3]. However, the general finding that in piglets both radioligands showed significant lower metabolic stability than in mice left some important unanswered questions, especially those relevant to potential clinical application. Therefore, this study aimed to further explore the metabolic properties of both radioligands by means of in vitro examinations, which besides discovering species dependency in some respects, might allow predictions for their metabolic stability in humans. Methods: [18F]FACH, [18F]FACH-d6 as well as their corresponding non-radioactive references were incubated with murine (mouse MLM and rat RLM) and human (HLM) liver microsomes and with pig and human S9 fractions, in presence of NADPH, but also other co-factors (e.g. UDPGA). After collection of samples at defined time points, subsequently prepared samples were investigated by radio-HPLC, UV-HPLC or LC-MS/MS. Obtained data allowed calculation of basic metabolic parameters and identification of metabolites formed in vitro. Results: [18F]FACH, incubated in presence of NADPH at 37°C for up to 60 min, showed highest stability in MLM (t1/2= 29913 min), whereas lowest stability was found in RLM (t1/2= 201 min). A moderate stability was found for both HLM (t1/2= 948 min) and pig S9 fraction (t1/2= 522 min). However, in a recent head-to-head comparis

Objectives and Introduction Parkinson’s disease is caused by degeneration of nigro-striatal dopaminergic neurons and denervation of the target neurons in the neostriatum. The resulting disruption of dopaminergic modulation produces an imbalance between antagonistic pathways in the basal ganglia leading to rigidity, tremor, and bradykinesia [1]. One of several treatment options is the so called deep-brain-stimulation (DBS), whereby an electrode is implanted to re-equilibrate the nervous pathways and rescue the pathological imbalance. Though highly effective, DBS is linked to a very complex surgical procedure and can lead to adverse neurological effects [2,3]. The goal of this project is to enable a selective stimulation of striatal dopaminoceptive neurons from outside the brain through polymeric photovoltaic nanoparticles which are transported to the neostriatum using an engineered M13 bacteriophage as nanocarrier. These phages were chosen as a biovector since their filamentous envelop, formed by the major coat protein P8, offers a large surface area which can be modified easily. To monitor its biodistribution in the organism, the engineered bacteriophage is being with [64Cu]CuCl2 enabling PET imaging. Methods: Methods for analysis of the phages by TLC, HPLC and MALDI-TOF MS have been developed. To allow labeling with 64Cu, the bacteriophages were functionalized with 1,4,7‑triazacyclononane,1‑glutaric acid‑4,7-acetic acid (NODA-GA) and the conjugation reaction was analyzed by MALDI-TOF MS. The M13-NODA-GA conjugates were purified using HPLC-SEC. After labeling of the M13-NODA-GA conjugates with inhouse produced [64Cu]CuCl2 and purification by spin filtration, the radiolabeling efficiency was analyzed by radio-TLC and radio-HPLC-SEC. PET imaging was carried out in mice and scans were taken every 15 min up to 1 h after intravenous injection. The mice were sacrificed 70 min post-injection and the radioactivity accumulated in different organs was measured. Results: Conju

L-[11C]Methionine ([11C]Met) is frequently used for the diagnosis of tumours located in brain, head and neck or for tumours induced by the multiple myeloma. The radio synthesis of [11C]Met commonly starts with [11C]CO2 with subsequent transformation to [11C]CH4 followed by transfer into [11C]CH3I which is used for final labelling of the precursor L-homocystein thiolactone hydrochloride (HCTL). [1,2] By performing these steps with the TRACERLab FXC-pro System (GE HC), however, we observed inconstant radiochemical yields and high maintenance efforts especially of the absorber material used within the gas phase iodination. Accordingly, we have searched for optimization of this radio synthesis procedure.

Introduction: Glioma are clinically challenging tumors due to their location and invasiveness, limiting their complete surgical resection. Mutated isocitrate dehydrogenase enzymes (IDH) are frequent alterations - the most common being the IDH1R132H - expressed only in glioma but not in healthy brain, correlating with prognosis. Hence, the evaluation of the IDH mutation status has become essential in clinical patient stratification. Currently, the IDH mutations are determined either directly by invasive biopsies or indirectly by magnetic resonance spectroscopy. Here, we propose a transdisciplinary approach to develop an 18F-labeled ligand to detect the IDH1R132H protein directly and non-invasively by positron emission tomography (PET) imaging. Such an imaging tool could improve the selection of cancer patients who are most likely to benefit from precision medicine. In the present study, we performed the radiofluorination of AG-120 (Ivosidenib), an FDA-approved small molecule inhibitor of mutant-IDH. Methods: The stannyl precursor (HL174) was synthetized according to the published synthesis of AG-120 [1] with minor modifications. The radiosynthesis was performed by copper-mediated radiofluorination of HL174 (Fig. 1) using a TRACERlab FX2 N synthesis module. The inhibitory potency of AG-120 was measured with a diaphorase/resazurin coupled assay using recombinant IDH1R132H or IDH1 (n=2). Internalization of [18F]AG-120 were determined in vitro using U251 human glioblastoma cells stably transfected with IDH1 or IDH1R132H [2]. In vivo metabolism was investigated in healthy CD-1 mice (n=3) by radio-chromatographic analyses of plasma and brain tissue. Dynamic PET (Mediso, nanoScan® PET/CT) imaging studies were performed in nude rats bearing U251-IDH1 (n=2) or U251-IDH1R132H (n=2) glioblastoma. Results: For the first time, diastereomerically pure [18F]AG-120 was prepared by an automated copper-mediated radiolabelling approach without azeotropic drying starting from the stanny

The data concerns the preparation of two new 18F-labeled radiotracers using a ultra-fast radiolabeling method for tumor detection using positron emission tomography (PET) imaging.

UV/Vis of natural Bi(III)- and Pb(II)-L2 and Bi(III)- and Pb(II)-L3; radio-SEC of 133La-L2; radiolabeling kinetics

Radionuclide Theranostics through Radiopharmaceutical Sciences / Radionuklid Theranostik durch Radiopharmazeutische Wissenschaften

The Future of Radionuclide Theranostics through Radiopharmaceutical Sciences

Radionuclide Theranostics through Radiopharmaceutical Sciences (Part 2)

Plenary Lecture: Are applied radiopharmaceutical sciences able to consolidate NextGen radionuclide theranostics?

The applicability of radioligands for targeted endoradionuclide therapy is limited due to radiation-induced deleterious effects to healthy tissues. This applies in particular to the kidneys as primary organs of elimination, which requires dosimetric estimates to justify internal radionuclide therapy. In this context, the targeting of enzymes of the renal brush border membrane by cleavable linkers between target molecule and radiolabel that permit the formation of fast eliminating radionuclide-carrying cleavage fragments gains increasing interest. Herein, we synthesized a small library of ⁶⁴Cu-labeled cleavable linkers and quantified their substrate potentials toward neprilysin (NEP), a highly abundant peptidase at the renal brush border membrane. This allowed for the derivation of structure-activity relationships and selected cleavable linkers were attached to the somatostatin receptor subtype 2 ligand [Tyr³]octreotate. Subsequent radiopharmacological characterization revealed that a substrate-based targeting of NEP in the kidneys with small molecules or peptides entails a certain degree of premature cleavage in the blood circulation by soluble and endothelium-derived NEP. However, for a tissue-specific targeting of NEP in the kidneys, the additional targeting of albumin in the blood by albumin-binding moieties is highlighted.

Octa- and specifically nonadentate ligands with a bispidine scaffold (3,7-diazabicyclo[3.3.1]nonane) are known to be efficiently coordinated to a range of metal ions of interest in radiopharmaceutical chemistry and lead to exceedingly stable and inert complexes. The nonadentate bispidine L2 (with a tridentate bipyridine acetate appended to N3 and a picolinate at N7) has been shown before to be an ideal chelator for 111In3+, 177Lu3+ and 225Ac3+, nuclides of interest for diagnosis and therapy, and a proof-of-principle study with an SSTR2-specific octreotate has shown potential for theranostic applications. We now have extended these studies in two directions. Firstly, we present the ligand derivative L3, where the bipyridine acetate is substituted with terpyridine, a softer donor for metal ions with a preference for more covalency. L3 did not fulfill the hopes because complexation is much less efficient: while for Bi3+ and Pb2+ the ligand is an excellent chelator with similar properties to L2, Lu3+ and La3+ show very slow and inefficient complexation with L3 in contrast to L2, and 225Ac3+ is not fully coordinated, even at an elevated temperature (92% radiochemical yield (RCY) at 80 °C, 60 min, [L3] = 10-4 M). These observations have led to a hypothesis for the complexation pathway that is in line with all experimental data and supported by a preliminary DFT analysis, which is of importance for the design of further optimized bispidine chelators. Secondly, the coordination chemistry of L2 has been extended to Bi3+, La3+ and Pb2+, including solid state and solution structural work, complex stabilities, radiolabeling and radiostability studies. All complexes of this ligand (La3+, Ac3+, Lu3+, Bi3+, In3+, Pb2+), including nuclides for targeted alpha therapy (TAT), single photon emission computed tomography (SPECT), and positron emission tomography (PET) are formed efficiently at physiological conditions, i.e., suitable for the labeling of delicate biological vectors such a

The development of cannabinoid receptor type 2 (CB2R) PET radioligands has been intensively explored due to the pronounced CB2R upregulation in various pathological conditions, such as neuroinflammation and cancer. Herein we report on the enantioselective synthesis of a series of highly affine fluorinated indole-2-carboxamide ligands targeting the CB2R in the brain. Compound RM365 was selected for PET radiotracer development due to its high CB2R affinity (Ki = 2.1 nM) and pronounced selectivity over CB1R (factor >300). A fully automated copper-mediated radiofluorination of [18F]RM365 was established starting from the corresponding aryl boronic acid pinacol ester precursor. Preliminary in vitro evaluation of [18F]RM365 revealed an unprecedentedly high species differences affinity towards CB2R owing a dissociation constant (KD) of 2.32 nM for the human CB2R (hCB2R) and > 10000 nM for the rat CB2R (rCB2R). Metabolism studies in mice revealed high stability of [18F]RM365 with fractions of parent compound of > 90% in the brain and > 54% in the plasma at 30 min p.i.. PET imaging in a rat model of local hCB2R overexpression demonstrate the ability of [18F]RM365 to reach and selectively label the hCB2R in the brain with high signal-to-background ratio. Thus, [18F]RM365 is a very promising PET radioligand for the imaging of upregulated hCB2R expression under pathological conditions with high potential towards clinical application in humans.

The applicability of radioligands for targeted endoradionuclide therapy is limited due to radiation-induced deleterious effects to healthy tissues. This applies in particular to the kidneys as primary organs of elimination, which requires dosimetric estimates to justify internal radionuclide therapy. In this context, the targeting of enzymes of the renal brush border membrane by cleavable linkers between target molecule and radiolabel that permit the formation of fast eliminating radionuclide-carrying cleavage fragments gains increasing interest. Herein, we synthesized a small library of ⁶⁴Cu-labeled cleavable linkers and quantified their substrate potentials toward neprilysin (NEP), a highly abundant peptidase at the renal brush border membrane. This allowed for the derivation of structure-activity relationships and selected cleavable linkers were attached to the somatostatin receptor subtype 2 ligand [Tyr³]octreotate. Subsequent radiopharmacological characterization revealed that a substrate-based targeting of NEP in the kidneys with small molecules or peptides entails a certain degree of premature cleavage in the blood circulation by soluble and endothelium-derived NEP. However, for a tissue-specific targeting of NEP in the kidneys, the additional targeting of albumin in the blood by albumin-binding moieties is highlighted.

Click chemistry, and in particular copper-free click reactions, have gained growing interest for radiolabeling purposes in the field of radiopharmaceutical sciences. [99mTc][Tc(CO)3(H2O)3]+ is an excellent starting complex for radiolabeling of biomolecules under mild conditions. A new chelator, investigated for the copper-free strain-promoted cycloaddition, was synthesized containing the 2,2’-dipicolylamine (DPA) moiety for the 99mTc-tricarbonyl core and compared with a chelator based on activated esters for conventional radiolabeling. For the copper-free click labeling procedure, a DPA containing 4,8-diazacyclononyne moiety was prepared from a sulfonyl-modified diamide (four steps, 64% yield) ensued by the Nicholas reaction with butyne-1,3-diol. The 99mTc-DPA-DACN-complex was prepared with a radiochemical conversion (RCC) of 89% after 30 min. The following SPAAC reaction with an azide-functionalized PSMA molecule was performed within 4 hours to obtain the PSMA (prostate-specific membrane antigen) targeting 99mTc-complex with 79% RCC and without side products. For comparison, a second DPA-chelator based on a tetrafluorophenyl (TFP) ester was prepared (three steps, 64% yield) and was successfully radiolabeled with [[99mTc]Tc(CO)3(H2O)3]+ in 89% RCC after 20 min and >99% radiochemical purity after separation using an RP18 cartridge. The subsequent conjugation of an amine-functionalized PSMA targeting molecule was performed with 23% RCC after 150 min. Two other unknown side products were observed indicating hydrolysis of the TFP ester during the labeling. All nonradioactive Re(CO)3 complexes were synthesized from (Et4N)2[ReBr3(CO)3] (91% yield for the natRe-DPA-TFP ester, 76% yield for the natRe-DPA-DACN) and characterized to confirm the identity of the 99mTc-complexes.

Noninvasive molecular imaging of the PD-1/PD-L1 immune checkpoint is of high clinical relevance for patient stratification and therapy monitoring in cancer patients. Here we report nine small-molecule PD-L1 radiotracers with solubilizing sulfonic acids and a linker–chelator system, designed by molecular docking experiments and synthesized according to a new, convergent synthetic strategy. Binding affinities were determined both in cellular saturation and real-time binding assay (LigandTracer), revealing dissociation constants in the single digit nanomolar range. Incubation in human serum and liver microsomes proved in vitro stability of these compounds. Small animal PET/CT imaging, in mice bearing PD-L1 overexpressing and PD-L1 negative tumors, showed moderate to low uptake. All compounds were cleared primarily through the hepatobiliary excretion route and showed a long circulation time. The latter was attributed to strong blood albumin binding effects, discovered during our binding experiments. Taken together, these compounds are a promising starting point for further development of a new class of PD-L1 targeting radiotracers.

Tracers for bimodal optical imaging and positron emission tomography unite multiple ad-vantages in a single molecule. Their tumor-specific uptake can be visualized after their PET-activation by radiofluorination via PET/CT or PET/MRI allowing for staging or therapy planning, while their non-radioactive moiety additionally facilitates the visualization of malig-nant tissue during intraoperative fluorescence-guided surgery or in histological assessments. The silicon-bridged xanthene core offers the opportunity for radiofluorination with SiFA isotope exchange to obtain a small molecule PET-activatable NIR dye that can be linked to different tar-get vectors. Herein, we demonstrate the PET-activation of a fluorinated silicon pyronine, be-longing to a class of low-molecular weight fluorescence dyes with a large Stokes shift and sol-vent-dependent NIR dye properties, with successful radiochemical conversion. Moreover, a li-brary of unusually functionalized, red-shifted silicon rhodamines was synthesized, which can be easily conjugated by amide bond formation or ‘click-reaction’ approaches.

The potential of designing irreversible alkyne-based inhibitors of cysteine cathepsins by isoelectronic replacement in reversibly acting potent peptide nitriles was explored. The synthesis of the dipeptide alkynes was developed with special emphasis on stereochemically homogenous products obtained in the Gilbert-Seyferth homologation for C-C triple bond formation. 23 dipeptide alkynes and 12 analogous nitriles were synthesized and investigated for their inhibition of cathepsins B, L, S and K. Numerous combinations of residues in the P1 and P2 positions as well as terminal acyl groups, allowed for the derivation of extensive structure-activity relationships, which were rationalized by computational covalent docking for selected examples. The determined inactivation constants of the alkynes at the target enzymes span a range of more than three orders of magnitude (3-10,133 M-1<\sup>s-1<\sup>). Notably, the selectivity profiles of alkynes do not necessarily reflect those of the nitriles. Inhibitory activity at the cellular level was demonstrated for selected compounds.

To follow the design of in vivo stable chelating systems for radiometals, a concise and straightforward method toolbox was developed combining NMR, ITC, and Eu-TRLFS. For this purpose, the macropa chelator was chosen and Lu3+, La3+, Pb2+, Ra2+, and Ba2+ as radiopharmaceutically relevant metal ions. They differ in charge (+2, +3) and coordination properties (main group vs. lanthanides). 1H NMR was used to determine four pKa values (carboxylate functions: 2.40 and 3.13; amine functions: 6.80 and 7.73). Eu-TRLFS was used to validate the exclusive existence of the Mn+:ligand 1:1 complex in the chosen pH range. ITC measurements were accomplished to determine the resulting stability constants of the desired complexes with log K values ranging from 18.5 for the Pb-mcp complex to 7.3 for the Lu-mcp complex. DFT-calculated structures nicely mirror the complexes’ order of stabilities by bonding features. Radiolabeling with macropa using ligand concentrations from 10–3 M to 10–6 M pointed out the complex stability (133La > 131Ba > 177Lu).

This study describes the synthesis, radiofluorination and purification of an anionic amphiphilic teroligomer developed as stabilizer for siRNA-loaded calcium phosphate nanoparticles (CaP-NP). As the stabilizing amphiphile accumulates on nanoparticle surfaces, the fluorine-18 labeled polymer should enable to track the distribution of the CaP-NP in brain tumors after ap-plication by convection-enhanced delivery. At first, an unmodified teroligomer was synthesized with a number average molecular weight of 4550 ± 20 Da by free radical polymerization of a de-fined composition of methoxy-PEG-monomethacrylate, tetradecyl acrylate and maleic anhy-dride. Subsequent derivatization of anhydrides with azido-TEG-amine provided an azido func-tionalized polymer precursor (o14PEGMA-1) for radiofluorination. The 18F-labeling was accom-plished by copper-catalyzed cycloaddition of o14PEGMA-1 with the diethylene glycol - alkyne substituted heteroaromatic prosthetic group [18F]2 which was synthesized with a radiochemical yield (RCY) of about 38% within 60 min using a radiosynthesis module. The 18F-labeled polymer [18F]fluoro-o14PEGMA-2 was obtained after a short reaction time of 2-3 min by using CuSO4/sodium ascorbate at 90 °C. Purification was performed by solid-phase extraction on an anion-exchange cartridge followed by size-exclusion chromatography to obtain [18F]fluoro-o14PEGMA-2 with a high radiochemical purity and an RCY of about 15%.

Pheochromocytomas and paragangliomas (PCCs/PGLs) are catecholamine-producing tumors. In inoperable and metastatic cases, somatostatin type 2 receptor (SSTR2) expression allows for peptide receptor radionuclide therapy with [177Lu]Lu-DOTA-TATE. Insufficient receptor levels, however, limit treatment efficacy. This study evaluates whether the epigenetic drugs valproic acid (VPA) and 5-Aza-2'-deoxycytidine (DAC) modulate SSTR2 levels and sensitivity to [177Lu]Lu-DOTA-TATE in two mouse PCC models (MPC and MTT). Methods: Drug-effects on Sstr2/SSTR2 were investigated in terms of promoter methylation, mRNA and protein levels, and radiotracer binding. Radiotracer uptake was measured in subcutaneous allografts in mice using PET and SPECT imaging. Tumor growth and gene expression (RNAseq) were characterized after drug treatments. Results: DAC alone and in combination with VPA increased SSTR2 levels along with radiotracer uptake in vitro in MPC (high-SSTR2) and MTT cells (low-SSTR2). MTT but not MPC allografts responded to DAC and VPA combination with significantly elevated radiotracer uptake, although activity concentrations remained far below those in MPC tumors. In both models, combination of DAC, VPA and [177Lu]Lu-DOTA-TATE was associated with additive effects on tumor growth and specific transcriptional responses in gene sets involved in cancer and treatment resistance. Effects of epigenetic drugs were unrelated to CpG island methylation of the Sstr2 promoter. Conclusion: This study demonstrates that SSTR2 induction in mouse pheochromocytoma models has some therapeutic benefit that occurs via yet unknown mechanisms. Transcriptional changes in tumor allografts associated with epigenetic treatment and [177Lu]Lu-DOTA-TATE provide first insights into genetic responses of PCCs/PGLs, potentially useful for developing additional strategies to prevent tumor recurrence.

The development of cannabinoid receptor type 2 (CB2R) radioligands for positron emission tomography (PET) imaging was intensively explored. To overcome the low metabolic stability and simultaneously increase the binding affinity of known CB2R radioligands, a carborane moiety was used as a bioisostere. Here we report the synthesis and characterization of carboranebased 1,8-naphthyridinones and thiazoles as novel CB2R ligands. All tested compounds showed low nanomolar CB2R affinity, with (Z)-N-[3-(4-fluorobutyl)-4,5-dimethylthiazole-2(3H)-ylidene]-(1,7-dicarba-closo-dodecaboranyl)-carboxamide (LUZ5) exhibiting the highest affinity (0.8 nM). Compound [18F]LUZ5-d8 was obtained with an automated radiosynthesizer in high radiochemical yield and purity. In vivo evaluation revealed the improved metabolic stability of [18F]LUZ5-d8 compared to that of [18F]JHU94620. PET experiments in rats revealed high uptake in spleen and low uptake in brain. Thus, the introduction of a carborane moiety is an appropriate tool for modifying literature-known CB2R ligands and gaining access to a new class of high-affinity CB2R ligands, while the in vivo pharmacology still needs to be addressed.

PSMA-PET/CT for imaging prostate cancer (PC) has spread worldwide since its clinical introduction in 2011. The majority of experiences have been collected for PSMA-PET-imaging of recurrent PC. Data for primary staging of high-risk PC are highly promising. Meanwhile, a plethora of PSMA-ligands are available for clinical use (e.g. 68Ga-PSMA-11, 68GaPSMA-I&T, 68Ga-PSMA-617, 18F-DCFBC, 18F-DCFPyL, 18F-PSMA-1007, 18F-rhPSMA-7 and 18F-JK-PSMA-7). However, an official approval is available only for 68Ga-PSMA-11 (approved by the US FDA in 2020) and 18F-DCFPyL (approved by the US FDA in 2021). Recommendations for acquisition times vary from 1-2h p.i. It has been shown that for the majority of tumour lesions, the contrast in PSMAPET/CT increases with time. Therefore, additional late imaging can help to clarify unclear findings. PSMA-PET/CT should be performed prior to commencing an androgen deprivation therapy (ADT) since (long term) ADT reduces the visibility of PC lesions. Following injection of PSMA-ligands, hydration and forced diuresis are recommended for PSMA-ligands with primarily excretion via the kidneys in order to increase the visibility of tumour lesions adjacent to the urinary bladder. PSMA-ligands are physiologically taken up in multiple normal organs. For some 18F-labelled PSMA-ligands, presence of unspecific focal bone uptake has been reported. When using these tracers, focal bone uptake without CT-correlate should be interpreted with great caution. Besides prostate cancer, practically all solid tumors express PSMA in their neovasculature thereby taking up PSMA-ligands, although usually at a lower extent compared to PC. Also multiple benign lesions and inflammatory processes (e.g. lymph nodes) take up PSMA-ligands, also usually at lower extent compared to PC.

[18F]Fluoromisonidazole ([18F]FMISO, 1H-1-(3-[18F]fluoro-2-hydroxypropyl)-2-nitroimidazole) is a commonly used radiotracer for imaging hypoxic conditions in cells. Since hypoxia is prevalent in solid tumors, [18F]FMISO is in clinical application for decades to explore oxygen demand in cancer cells and the resulting impact on radiotherapy and chemotherapy. Since the introduction of [18F]FMISO as positron emission tomography (PET) imaging agent in 1986, a variety of radiosynthesis procedures for the production of this hypoxia tracer has been developed. This paper gives a brief overview on [18F]FMISO radiosyntheses published so far from its introduction until now. From a radiopharmaceutical chemist’s perspective, different precursors, radiolabeling approaches and purification methods are discussed as well as used automated radiosynthesizers, including cassette-based and microfluidic systems. In a GMP compliant radiosynthesis using original cassettes for FASTlab we produced [18F]FMISO in 49% radiochemical yield within 48 min with radiochemical purities >99% and molar activities >500 GBq/µmol . In addition, we report an easy and efficient radiosynthesis of [18F]FMISO, based on in-house prepared FASTlab cassettes, providing the radiotracer for research and preclinical purposes in good radiochemical yields (39%), high radiochemical purities (>99%) and high molar activity (>500 GBq/µmol) in a well-priced option.

The understanding of the contribution of the tumour microenvironment to cancer progression and metastasis, in particular the interplay between tumour cells, fibroblasts and the extracellular matrix has grown tremendously over the last years. Lysyl oxidases are increasingly recognised as key players in this context, in addition to their function as drivers of fibrotic diseases. These insights have considerably stimulated drug discovery efforts towards lysyl oxidases as targets over the last decade. This review article summarises the biochemical and structural properties of theses enzymes. Their involvement in tumour progression and metastasis is highlighted from a biochemical point of view, taking into consideration both the extracellular and intracellular action of lysyl oxidases. More recently reported inhibitor compounds are discussed with an emphasis on their discovery, structure-activity relationships and the results of their biological characterisation. Molecular probes developed for imaging of lysyl oxidase activity are reviewed from the perspective of their detection principles, performance and biomedical applications.

L-[11C]Methionine ([11C]Met) is frequently used for the diagnosis of tumours located in brain, head and neck or for tumours induced by the multiple myeloma. The radio synthesis of [11C]Met commonly starts with [11C]CO2 with subsequent transformation to [11C]CH4 followed by transfer into [11C]CH3I which is used for final labelling of the precursor L-homocystein thiolactone hydrochloride (HCTL). [1,2] By performing these steps with the TRACERLab FXC-pro System (GE HC), however, we observed inconstant radiochemical yields and high maintenance efforts especially of the absorber material used within the gas phase iodination. Accordingly, we have searched for optimization of this radio synthesis procedure.

Objectives: Positron Emission Tomography (PET) is a non-invasive molecular imaging technique, which continues to find increasing application and further development for diagnosis of a broad range of disorders and dysfunctions. Since their involvement in pathophysiological processes that lead to cancer, for example, monocarboxylate transporters (MCTs) are of interest for the development of new radioligands for PET, such as [18F]FACH [1, 2] and its deuterated isotopologue [18F]FACH-d6. Both candidates have been already examined previously in preclinical PET studies in healthy mice and piglets, also with regard to their biodistribution and metabolic fate [3]. However, the general finding that in piglets both radioligands showed significant lower metabolic stability than in mice left some important unanswered questions, especially those relevant to potential clinical application. Therefore, this study aimed to further explore the metabolic properties of both radioligands by means of in vitro examinations, which besides discovering species dependency in some respects, might allow predictions for their metabolic stability in humans. Methods: [18F]FACH, [18F]FACH-d6 as well as their corresponding non-radioactive references were incubated with murine (mouse MLM and rat RLM) and human (HLM) liver microsomes and with pig and human S9 fractions, in presence of NADPH, but also other co-factors (e.g. UDPGA). After collection of samples at defined time points, subsequently prepared samples were investigated by radio-HPLC, UV-HPLC or LC-MS/MS. Obtained data allowed calculation of basic metabolic parameters and identification of metabolites formed in vitro. Results: [18F]FACH, incubated in presence of NADPH at 37°C for up to 60 min, showed highest stability in MLM (t1/2= 29913 min), whereas lowest stability was found in RLM (t1/2= 201 min). A moderate stability was found for both HLM (t1/2= 948 min) and pig S9 fraction (t1/2= 522 min). However, in a recent head-to-head comparis

Objectives Theranostic concepts and the usage of alpha-particle emitting radionuclides belong to the emerging fields of radiopharmaceutical sciences. Especially and due to its excellent complexation properties, the chelator macropa (mcp)[1,2] was reported to be a suitable complexing agent for 225Ac conjugates[3] and as macrocycle a highly promising starting point for the development of 223/224Ra chelators as well. To follow the theranostic approaches with these alpha emitters, due to their chemical similarities 131Ba[4] for SPECT and 133La[5] for PET are available as diagnostic radionuclide surrogates of radium and actinium, respectively. In our recent study, we aim to establish a new workflow to evaluate and predict the complex stability of new chelating systems by obtaining both protonation constants for respective ligands and association constants for their metal complexes starting with mcp. Methods As a prerequisite for the calculation of stability constants (log(K)), the protonation constants (pKa) of mcp were determined by pH‑dependent 1H NMR studies in the first step. Based on these obtained data, the europium-mcp-complex was examined using time-resolved, laser-induced luminescence spectroscopy (TRLFS) and the log(K) was calculated as reference stability constant. Additionally, Eu-TRLFS was used to determine the Eu-mcp species during titration and pH-dependency of the complexation. The method of isothermal titration calorimetry (ITC) was used to measure the complex stability constants of La-, Ba-mcp-complex, as well as Eu-mcp-complex to value the results in comparison with TRLFS data. The evaluation and calculation of the log(K) values were carried out by parallel factor analysis (PARAFAC). Results Depending on the protonation ability of the functional groups found in mcp (amines and carboxylates), pKa values were obtained as well as the log(K) values for the respective mcp-complexes with Ba, Eu and La and are shown in Table 1. Conclusion By combining 1H-NMR

Introduction: The A2A adenosine receptor (A2AAR) is expressed in brain, vasculature and immune cells. According to the alterations of the A2AAR expression in multiple diseases, it is a highly attractive diagnostic and therapeutic target. We developed the A2AAR-specific PET radiotracer [18F]FLUDA and investigated it in healthy mice and piglets, in a rotenone-based mouse model of Parkinson’s disease (RMMPD) and in transgenic mice overexpressing the human A2AAR in heart (TG) [1 3]. Methods: On the basis of a one-pot two-step radiofluorination procedure, a remotely controlled automated radiosynthesis of [18F]FLUDA using the TRACERlab FX2N synthesis module was developed. In vitro autoradiography was performed with cryosections of tissue from animal models. In vivo stability was investigated in mouse by radio-HPLC analyses of blood plasma and brain homogenates. The biodistribution was investigated by dynamic PET/MR studies in healthy mice and piglets under control and blocking conditions (vehicle vs. blocking with 2.5 mg/kg tozadenant and/or 1.0 mg/kg istradefylline) and in both mouse models. The binding potential (BPND) in vivo was calculated using the simplified reference tissue modelling with the cerebellum as reference region. A single dose acute toxicity study was performed in Wistar rats according to the ICH guideline M3(R2). PET-derived radiation dosimetry was estimated in piglets. Results: A reliable and reproducible procedure for the automated production of [18F]FLUDA was successfully established (Fig. 1A) [4]. In vitro autoradiography revealed highly selective binding and high affinity of [18F]FLUDA towards the A2AAR of the three species (KD values 0.7-5.9 nM, Fig. 1B). At 15 min after i.v. injection of [18F]FLUDA in mice, the parent fraction accounted for about 100% in brain and 71% in plasma. PET studies confirmed the specific binding of [18F]FLUDA in vivo to the striatal A2AAR in mice and piglets (BPND=3.9 and 1.3, Fig. 1C). The availability of A2AAR in the P

The cannabinoid receptor 2 (CB2R) is involved in inflammatory processes [1], whereby an increased expression correlates with malignancy in various cancer types like human epidermal growth receptor 2 positive (HER2+) or triple negative breast cancer (TNBC) [2]. Hence, the CB2R is suggested as a pharmacological target, as well as a prognostic biomarker for the stratification and staging of patients [3]. In this study, we evaluated the potential of our novel CB2R-specific radioligand [¹ ⁸F]JHU94620-d8 for the assessment of the CB2R expression in HER2+ breast cancer and TNBC models in vitro. The KD value of [¹ ⁸F]JHU94620-d8 was determined by autoradiography on cryosections of rat and mouse spleen, as well as on rat brains harbouring a local overexpression of the hCB2R (AAV-hCB2R) [4]. The CB2R status was investigated by competitive radioligand binding assays (CRBA) in cell membranes of CHO cells overexpressing the human CB2R (CHOhCB2R), human breast cancer cell lines HCC1954 (HER2+) and MDA-MB-231 (TNBC) with 3.1±0.4 nM [³H]WIN55,212-2 using 10 µM of WIN55,212-2, GW405833 and JHU94620-d8 (each n=3) as agonistic competitors. CB2R expression was validated by immunofluorescence microscopy (IFM). On cryosections of 4T1 tumors the CB2R specific binding of [¹ ⁸F]JHU94620-d8 was investigated by CRBA and the colocalisation of CB2R with Iba1 (macrophages) and CD31 (blood vessels) by IFM. We determined KD values for [¹ ⁸F]JHU94620-d8 of 30 nM in mouse spleen, of 1.0 nM in rat spleen, and of 42 nM in AAV-hCB2R. The cell membrane binding of [³H]WIN55,212-2 was comparable in all used cell lines between 20 ± 1 and 30 ± 11 fmol/106 cells. Competition by JHU94620-d8 decreased the total binding by 57 % (p<0.01) only in CHOhCB2R cells, WIN55,212-2 by 37 % (p=0.01) and 77 % (p<0.01) and GW405833 by 42 % (p<0.01) and 75 % (p<0.01) in HCC1954 and CHOhCB2R cells, respectively, however in MDA MB 231 cells binding was not affected by these compounds (Fig. 1A). The expression of CB2R was confi

Introduction: Glioma are clinically challenging tumors due to their location and invasiveness, limiting their complete surgical resection. Mutated isocitrate dehydrogenase enzymes (IDH) are frequent alterations - the most common being the IDH1R132H - expressed only in glioma but not in healthy brain, correlating with prognosis. Hence, the evaluation of the IDH mutation status has become essential in clinical patient stratification. Currently, the IDH mutations are determined either directly by invasive biopsies or indirectly by magnetic resonance spectroscopy. Here, we propose a transdisciplinary approach to develop an 18F-labeled ligand to detect the IDH1R132H protein directly and non-invasively by positron emission tomography (PET) imaging. Such an imaging tool could improve the selection of cancer patients who are most likely to benefit from precision medicine. In the present study, we performed the radiofluorination of AG-120 (Ivosidenib), an FDA-approved small molecule inhibitor of mutant-IDH. Methods: The stannyl precursor (HL174) was synthetized according to the published synthesis of AG-120 [1] with minor modifications. The radiosynthesis was performed by copper-mediated radiofluorination of HL174 (Fig. 1) using a TRACERlab FX2 N synthesis module. The inhibitory potency of AG-120 was measured with a diaphorase/resazurin coupled assay using recombinant IDH1R132H or IDH1 (n=2). Internalization of [18F]AG-120 were determined in vitro using U251 human glioblastoma cells stably transfected with IDH1 or IDH1R132H [2]. In vivo metabolism was investigated in healthy CD-1 mice (n=3) by radio-chromatographic analyses of plasma and brain tissue. Dynamic PET (Mediso, nanoScan® PET/CT) imaging studies were performed in nude rats bearing U251-IDH1 (n=2) or U251-IDH1R132H (n=2) glioblastoma. Results: For the first time, diastereomerically pure [18F]AG-120 was prepared by an automated copper-mediated radiolabelling approach without azeotropic drying starting from the stanny