Search

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

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

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.

Introduction: Due to the upregulation of somatostatin receptor subtype-2 (SSTR2) in neuroendocrine tumors, radiolabeled somatostatin analogs have been successfully used so far for diagnostic investigations and peptide receptor radionuclide therapy [1]. In the present work, both the somatostatin agonist (Tyr3)octreotate (TATE) and the somatostatin antagonist JR11 were conjugated with a cyclohexanediaminetriazole (CHDT) chelator for the potential labeling with Al18F, 68Ga or 111In. The radiopharmacological behavior of Al18F and 68Ga labeled CHDT-TATE and CHDT-JR11 were studied in BON-SSTR2 cells [2] in both monolayer (ML) and spheroid (SP) culture. The binding data were determined in static cell culture to have a basis for further assays using a microphysiological system (MPS) with dynamic properties [3]. Methods: Spheroids of BON-SSTR2 cells were created according to a protocol by the vendor (Greiner Bio-One GmbH, Germany). Freshly harvested cells were incubated with magnetic nanoparticles in RPMI medium overnight. After loading, the cells were distributed in a 24-well-plate (3 x 10-4 cells/well) and placed on a plate with corresponding 24-point magnets in order to form SPs in the incubator for up to 5 days [4]. 18F-labeling was performed with Al[18F]F2+ (TR-FLEX Cyclotron, Canada) and 68Ga-labeling with [68Ga]GaCl3 (68Ge/68Ga generator, iThemba Labs, South Africa) under mild conditions (20 min at 40°C). After incubation with the radiolabeled ligands the cells were washed; for internalization purpose the cells were treated with acidic glycine buffer (pH 3.0) to remove the bound molecules from the outer cellular membrane [5]. These samples correspond to the ligand-bound fraction and are measured in a gamma counter alongside the samples with the internalized ligand molecules. Results: Saturation binding analysis of Al18F-labeled CHDT-TATE and CHDT-JR11 revealed very good binding affinities with similar Kd-values toward ML (Al[18F]F-CHDT-TATE 12.4 ± 2.1 nM, Al[18F]F-CHD

Introduction: Radiolabeled prostate-specific membrane antigen (PSMA) ligands have been successfully developed in recent years for diagnosis and therapy of prostate cancer [1]. A potential new PSMA radioligand would, after in vitro characterization, normally be further evaluated in small animals. However, the use of micro-physiological systems (MPS), an organ-on-chip technology in which human cell spheroids and organoids are treated in a circuit under defined conditions [2,3], should lead to a reduction of animal experiments. In the present study, experiments with the novel 64Cu-labeled PSMA antagonist FR52 were performed using 2D or 3D cultures of LNCaP cells in MPS chips. Our aim was to determine binding parameters (Kd, Bmax) in the MPS chip and to compare them with the results of assays in well plates (WP). Methods: Derived from the compound PSMA-617 [4], instead of DOTA a cyclohexanediamine-triazole (CHDT) chelator was conjugated, resulting in the conjugate FR52 (in-house synthesis). FR52 was radiolabeled with 64Cu (molar activity: 14.2 ± 2.2 MBq/nmol). For the assays, 5 x 104 LNCaP cells were placed in 3 wells of an MPS module (à 0.2 cm2), either as monolayer or as spheroids and cultured for 2 to 7 days. The MPS chips include an integrated micropump, driven by a controller, that generates a pulsatile flow of media that runs through the capillaries and keeps the cells alive [5] (Fig. 1). After blocking of adjacent cell samples (PSMA pharmacophore 0.8 mM; 30 min) [64Cu]Cu-FR52 (6 to 200 nM) was added to all samples and pumped through the MPS for 10 min (volume: 0.1 mL/well; 80 bpm; volume flow 6.4 µL/s). After PBS washing and exposing the MPS chips to an imaging plate, the cells were lysed and in vials measured in a gamma counter. Evaluation was processed with the analysis program GraphPad Prism. Results: Saturation assays with [64Cu]Cu-FR52 in MPS revealed similar high binding affinities (Kd) for LNCaP monolayer and spheroids (MPS-monolayer Kd: 33 ± 6 nM; MPS-sph

Heavy alkaline earth metals offer radionuclides which are promising candidates for radiopharmaceutical applications like barium-131 for diagnosis or radium-223/-224 – with similar properties to barium – for targeted alpha-particle therapy. However, there is a lack of suitable chelation agents especially for these metal ions. A series of calix[4]crown-6 derivatives with perfluoroalkylsulfonylcarboxamide functions (RF = CF3, C2F5, i-C3F7, n-C4F9) was synthesized to serve as cage-like chelators for a strong complexation of Ba2+ and Ra2+. These functional ligands are deprotonated even at slightly acidic pH due to the intense electron withdrawing effect of the sulfonamide group. The obtained ligands were easily converted the desired barium complexes as well as into calix-crown compounds containing two sodium ions. DFT calculation methods were used to discover either the binding behavior of the metal ions with the desired ligands as well as the influence of the different donor groups from the chelating moiety of the calixarenes with respect to different pH. Radiolabeling procedures with the radionuclides barium-133 and radium-224 as [133Ba]BaCl2 and [224Ra]Ra(NO3)2 were performed to determine association constant values between 4.1 and 8.2 for the appropriate M2+ complexes using a two-phase extraction procedure. A stability test using physiological Ca2+ solution showed a minor release of approx. 1-7% of the central ions (Ba2+ respectively Ra2+) from the complexes.

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.

Prostate specific membrane antigen (PSMA) is an excellent target for imaging and treatment of prostate carcinoma (PCa). Unfortunately, not all PCa cells express PSMA. Therefore, alternative theranostic targets are required. The membrane protein prostate stem cell antigen (PSCA) is highly overexpressed in most primary prostate carcinoma (PCa) cells and in metastatic and hor-mone refractory tumor cells. Moreover, PSCA expression positively correlates with tumor pro-gression. Therefore, it represents a potential alternative theranostic target suitable for imaging and/or radioimmunotherapy. In order to support this working hypothesis, we conjugated our previously described anti-PSCA monoclonal antibody (mAb) 7F5 with the bifunctional chelator CHX-A″-DTPA and subsequently radiolabeled it with the theranostic radionuclide 177Lu. The re-sulting radiolabeled mAb ([177Lu]Lu-CHX-A″-DTPA-7F5) was characterized both in vitro and in vivo. It showed a high radiochemical purity (>95%) and stability. The labelling did not affect its binding capability. Biodistribution studies showed a high specific tumor uptake compared to most non-targeted tissues in mice bearing PSCA-positive tumors. Accordingly, SPECT/CT images re-vealed a high tumor-to-background ratios from 16 h to 7 days after administration of [177Lu]Lu-CHX-A″-DTPA-7F5. Consequently, [177Lu]Lu-CHX-A″-DTPA-7F5 represents a promising candidate for imaging and in the future also for radioimmunotherapy.

Ziel: macropa-PSMA-Konjugate mit Albuminbinder zur zielgerichteten Alphatherapie mit Actinium-225 wurde innerhalb der Arbeitsgruppe bereits erfolgreich synthetisiert und in vitro und in vivo untersucht. Die dabei eingeführte albuminbindende Einheit bietet die Grundlage zur Erarbeitung eines theranostischen Ansatzes indem, neben der Komplexierung des Alphaemitters Actinium-225, die Bindung von Iod-123 als leicht zugängliches SPECT-Nuklid im gleichen Molekül ermöglicht wird. Vorteilhaft sind die milden Markierungsbedingungen sowie eine passende Halbwertszeit des Iodisotopes (t1/2 =13,2 h), welche die Bildgebung länger zirkulierender Substanzen ermöglicht. Methoden: Die Synthese der peptidomimetischen Ausgangsverbindungen erfolgte angelehnt an die Vorarbeiten durch mehrstufige Peptidkupplungen und die Anbringung des Chelators mittels Cu-katalysierter Click-Reaktion. Anstelle der 4-(p-Iodphenyl)buttersäure wurde der entsprechende Zinnprecursor eingeführt, um die Verbindung durch eine elektrophile, aromatische Substitution mit Iod-123 markieren zu können. Ergebnisse: Durch die erfolgreiche Entwicklung einer Methode zur Iodierung der synthetisierten Zinnprecursoren mit hohen radiochemischen Ausbeuten konnten schließlich die einfach bzw. doppelt PSMA-gebundenen Verbindungen [123I]I-mcp-M-alb-PSMA und [123I]I-mcp-D-alb-PSMA hergestellt werden. Nach der Iodierung der Verbindungen wurde in vitro der Einfluss des 139La-Komplexes als auch der des unkomplexierten Chelators an der PSMA-positiven Zelllinie LNCaP untersucht. Schlussfolgerungen: Die Einführung von Iod-123 in die bereits untersuchten Derivate mit stabilem Iod-127 ergab keine Änderungen der biologischen Ergebnisse. Damit ergibt sich der neue Aspekt des theranostischen Nutzens als hybrides Radiopharmakon mit einer weiteren Markierungsstelle in der Peripherie des eigentlichen Liganden.

The prostate-specific membrane antigen (PSMA) is expressed in approx. 80% of prostate cancer at considerably higher levels in tumor cells compared to healthy tissues. Its upregulation occurs in all stages of the disease. Therefore, PSMA has emerged as an attractive target for molecular imaging and especially targeted radionuclide therapy (endoradiotherapy) of metastatic castration-resistant prostate cancer (mCRPC). Due to its favorable nuclear physical properties, actinium-225 belongs to the clinically applied alpha emitters for therapeutic applications. More than ten clinical trials are displayed for actinium-225-DOTA-based conjugates. A trial using [225Ac]Ac-PSMA-617 as the IMP in men with PSMA-positive prostate cancer is projected to start. Actinium-225 can be chelated with the standard DOTA chelator, but requires rather harsh labeling conditions such as high temperatures (80–95°C) or microwave assistance. Alternatively, with the complexing agent macropa mild labeling conditions, low reaction temperature and high in vivo stability can be realized owing to its perfect characteristics for Ac3+ chelation. Moreover, to further improve the tumor uptake for future targeted alpha therapy, albumin binding units based on 4-iodophenylbutyrate were introduced to increase the tumor uptake. Two 225Ac-radioconjugates with albumin binders were prepared, either containing one or two PSMA-binding units (based on PSMA-617), the second one to raise the binding affinity to the PSMA receptor. The synthetic approach consisted of the functionalization of one or two picolinic side arms with a clickable reactive site for the connection of the vector molecules. The azide-functionalized PSMA derivative, which also contains the albumin binder, was prepared using standard peptide coupling conditions. Cell culture-based studies regarding radioligand affinity and clonogenicity were performed using PSMA-positive LNCaP cells and the biodistribution of the radioconjugates wa

Diazacyclononyne (DACN) derivatives belong to the class of diazamacrocycles containing a strained alkyne bond. Therefore, DACNs are predestinated starting materials to be applied in copper-free strain-promoted click labeling with azide-functionalized bio(macro)molecules. The diazamacrocyclic scaffold is easy to prepare via a double Nicholas reaction. The following functionalization is convenient by the use of functionalized sulfonamides or by the alkylation of the secondary amines provided by the molecule. To make the DACN derivatives suitable for radiolabeling with technetium-99m, two dipicolylamine (DPA) derivatives were prepared allowing the use of the 99mTc-tricarbonyl core. The first DPA-ligand is functionalized with an activated ester to allow convenient labeling of, e.g., amines. The second is based on the DACN macrocycle, allowing the copper-free click labeling. Two PSMA-binding derivatives based on PSMA-617 were chosen for final radiolabeling, one with a terminal amine function the other with an azide function. For the conventional radiolabeling with e.g. primary amines, the DPA chelator was functionalized with a benzoate moiety which was further transferred into the corresponding activated TFP ester using tetrafluorophenol in three steps and an overall yield of 64%. For the click labeling procedure, the functionalized DACN macrocycle was prepared from a sulfonyl-modified diamide (4 steps) which was reacted with butyne-1,3-diol under Nicholas conditions. Finally, the DPA moiety was connected to the DACN via the propylsulfonyl linker. Both derivatives were reacted with (Et4N)2[ReBr3(CO)3] in methanol to obtain both reference complexes (natRe-DPA-TFP ester: 91% yield, natRe-DPA-DACN: 76% yield). [99mTc][Tc(CO)3]+ was prepared according to an established protocol using 99mTc-pertechnetate and K2[H3BCO2] (in-h

Objectives The prostate-specific membrane antigen (PSMA) is expressed in most cases of prostate cancer at considerably higher levels in tumor cells compared to healthy tissues [1-3] and its upregulation occurs in all stage of the disease [4, 5]. Therefore, PSMA has emerged as an attractive target for molecular imaging and especially targeted radionuclide therapy (endoradiotherapy) of metastatic castration-resistant prostate cancer (mCRPC), given the example of [177Lu]Lu-PSMA-617. We recently described the synthesis and in-depth characterization of PSMA radioligands for targeted alpha therapy with actinium-225 [6]. Our present study aimed at the design, synthesis and preclinical evaluation of albumin-binding PSMA ligands in order to optimize their tissue distribution profile and to improve their pharmacokinetic properties. Methods Two novel compounds were prepared by combining a macropa chelator with one or two lysine-ureido-glutamate–based PSMA-binding entities equipped with 4-(p-iodophenyl)butyrate residues. The albumin-binding properties of the 225Ac-labeled conjugates were investigated in vitro using mouse, rat and human serum. The specific interaction of both compounds with human serum albumin was confirmed by microscale thermophoresis. Cell culture-based studies regarding radioligand affinity and clonogenicity were performed on PSMA-positive LNCaP cells. The biodistribution of the radioconjugates was analyzed in LNCaP tumor-bearing mice with ensuing investigation of tissue toxicity by histological examinations. Results Radiolabeling of both PSMA ligands with 225Ac was achieved at up to 5 MBq/nmol with >99% radiochemical purity. In vitro assays confirmed considerable binding of the radioligands to mouse, rat and human serum proteins. Cell binding and survival studies revealed a higher cell binding affinity and an improved cell killing efficiency for the radioconjugate with two PSMA-binding entities compared to the derivative with only one targeting motif. Biodis

Introduction: Potential radiopharmaceuticals are usually evaluated in small animals. Aligned with the 3R principle, animal numbers could be reduced by using “Micro-Physiological Systems (MPS)” which is an organ-on-chip technology. In MPS modules, cultured cells and human organoids can be analysed in a circulatory system under defined conditions [1,2]. In this study, preliminary tests with radiolabeled anti-EGFR antibody cetuximab (C225) were performed using 2D or 3D (spheroid) cultures of A431 cells. The aim was to determine selected pharmacological parameters such as binding affinity of radiolabeled C225 using the MPS. Methods: 104 A431 cells were placed in the 6-well chamber of the MPS module, either as monolayer or as spheroids (0.8 ± 0.3 mm) and cultured for 24 hours. The conjugate NOTA-C225 was radiolabeled with 68Ga or 64Cu (molar activity: 81.1 ± 14.7 MBq/nmol). An integrated micropump, driven by a controller, generates a pulsatile fluid flow through the fluidic channels upon the cells (Fig.1). Medium (total binding) and medium with an EGFR blocking C225 concentration of 2 µM (nonspecific binding) (1mL) was pumped through the MPS for 5 min (80 bpm at a volume flow of 6.4 µL/s). Under saturation conditions, 1.2 to 15 nM of radiolabeled C225 were applied to cells and spheroids. Using the micropump, a total volume of 1 mL of the compound solutions were distributed through the MPS over 15 min. After washing with PBS for 10 min, the MPS modules were exposed to an imaging plate, and bound radiolabeled C225 was determined by phosphor imaging. Evaluation was realised with AIDA /GraphPadPrism. Results/Discussion: We demonstrate that a MPS environment can be employed to determine radioligand binding parameters. The first saturation assays show high binding affinity of the radiolabeled C225 with Kd values in the low nanomolar range (1.7 to 25 nM), being in the range of previous reports [3,4]. Kd of A431 monolayers was similar to that of A431 spheroids. Nonspecific bindi

Objectives: During the last 15 years several PSMA PET ligands for prostate cancer imaging have been developed resulting in the recent approvals of 68Ga-PSMA-11, 18F-PSMA-1007 and 18F-DCFPyL.[1,2] However, 99mTc remains a popular radiometal for SPECT imaging due to its longer half-life and availability from 99Mo/99mTc generators especially in outpatient imaging centers. Due to the widespread availability of SPECT cameras PSMA SPECT ligands have the potential to substantially extend the availability of PSMA-based imaging, but currently no PSMA SPECT ligand is approved. MIP-1404, PSMA-I&S and HYNIC-iPSMA are rare examples of PSMA SPECT tracers in clinical development. Accordingly, the aim of our study was to develop a PSMA ligand for 99mTc SPECT imaging of prostate cancer based on an N2S2 chelator, which enables reliable radiolabeling to form stable 99mTc complexes.[3] Methods: A number of compounds, which contain the PSMA binding motif Glu-urea-Lys, a varying linker and a bisaminothiol (BAT)-type N2S2 chelator, were synthesized. Selected compounds were radiolabeled with 99mTc, followed by assessment of in vitro stability of the formed complexes via radio-HPLC. The specific binding affinity towards PSMA and the internalization of the radioligands were examined in LNCaP cells, supplemented by autoradiographic studies. Tissue distribution and tumor accumulation were evaluated in LNCaP-tumor bearing mice via quantitative SPECT/CT imaging, benchmarked with [99mTc]Tc-PSMA-I&S[4] and [68Ga]Ga-PSMA-11. Results: Among the six 99mTc radioligands synthesized and examined, [99mTc]TcO-ABX474 showed most favorable properties. Radiolabeling of ABX474 (50 µg) was achieved starting from [99mTc]NaTcO4 (0.2-2 GBq) in saline (1.4 mL) using SnCl2 (1 µg in 0.01 M HCl) as reducing agent, in presence of calcium-D-heptagluconate (10 µg) and D-mannitol (1 mg) at pH 5-6. Incubation at r.t. for 20 min followed by heating at 80°C for 20 min afforded the product-containing solution with a radioche

Epidermal growth factor receptors (EGFR) of tyrosine kinase (TK) have shown high expression levels in most cancers and are considered a promising target for cancer diagnosis and therapy. Expanding the investigation for novel targeted radiopharmaceuticals, an EGFR inhibitor such as 4-aminoquinazoline derivatives along with a radionuclide such as technetium-99m (99mTc) could be ideal. Thus, we report herein the synthesis, characterization, and biological evaluation of new “4 + 1” mixed-ligand ReIII- and 99mTcIII-complexes of the general formula [99mTc][Tc(NS3)(CN-R)] bearing tris(2-mercaptoethyl)-amine (NS3) as the tetradentate tripodal ligand and a series of isocyanide derivatives (CN-R) of tyrosine kinase inhibitor (3-bromophenyl)quinazoline-4,6-diamine as the monodentate ligand. The quinazoline isocyanide derivatives 4a-d were prepared in two steps and reacted with the [Re(NS3)PMe2Ph] precursor leading to the final complexes 5a-d in high yield. All compounds were characterized by elemental analysis, IR, and NMR spectroscopies. In vitro studies, for their potency to inhibit the cell growth, using intact A431 cells indicate that the quinazoline derivatives 4a-d and the Re complexes 5a-d significantly inhibit the A431 cell growth. In addition, the EGFR autophosphorylation study of complex 5b shows an IC50 value in the nanomolar range. The corresponding “4 + 1” 99mTc-complexes 6a-d were prepared by employing the [99mTc]TcEDTA intermediate and the appropriate monodentate 4a-d in a two-step synthetic procedure with a radiochemical yield (RCY) from 63 to 77 % and a radiochemical purity (RCP) > 99 % after HPLC purification. Their structures have been established by HPLC comparative studies using the well-characterized Re-complexes 5a-d as reference. All 99mTc-complexes remain stable for at least 6 h, and their logD7.4 values confirmed their anticipated lipophilic character. Biodistribution studies in healthy Swiss albino mice of 99mTc-complexes showed hepatobiliary excreti

Radiopharmaceuticals can be used for targetspecific functional diagnostics, such as PET or SPECT imaging, or radionuclide therapy of diseased tissue, depending on the incorporated radionuclide. Following initial in vitro testing, radiopharmaceutical candidates are usually further characterized in small animals. Since reduction of animal testing is a central precept in preclinical research it would be beneficial to replace at least some of these tests by alternative methods. Using micro-physiological system (MPS) technology, various organ-on-chip models can be created with human cell systems/organoids, which are operated in a circulatory system under defined physiological conditions. Here we present first attempts to introduce MPS for evaluating radiopharmaceuticals using the radiolabeled anti-EGFR antibody cetuximab (C225) as reference compound. In an MPS equipped with six 96-well plate-like microwells in a flow chamber, binding of 64Cu and 68Ga-labeled C225 to cells and spheroids grown from A431 (EGFR-positive) and MDA-MB435S (EGFR-negative) cells was measured and compared to conventional microplates. Specific saturation binding of radiolabeled C225 at increasing concentrations was analyzed using a phosphor imaging system. The affinity of radiolabeled C225 towards A431 spheroids measured in the MPS was in the same range as that of the spheroids in conventional microplates. Within the MPS assays, the results showed a trend towards increased affinity for A431 monolayers compared to the spheroids. The values of binding capacity for radiolabeled C225 on 2D and 3D A431 cell culture models were in the same order of magnitude when measured in MPS or in microplates.

Targeted Alpha Therapy is a research field of highest interest in specialized radionuclide therapy. Over the last decades, several alpha-emitting radionuclides have entered and left research topics towards their clinical translation. Especially, 225Ac provides all necessary physical and chemical properties for a successful clinical application, which has already been shown by [225Ac]Ac PSMA 617. While PSMA 617 carries the DOTA as the complexing agent, the chelator macropa as macrocyclic alternative provides even more beneficial properties regarding labeling and complex stability in vivo. Lanthanum-133 is an excellent positron-emitting diagnostic lanthanide to radiolabel macropa-functionalized therapeutics, since 133La forms a perfect matched theranostic pair of radionuclides with the therapeutic radionuclide actinium-225 which itself can optimally be complexed by macropa as well. 133La was thus produced by cyclotron-based proton irradiation of an enriched 134Ba target. The target (30 mg of [134Ba]BaCO3) was irradiated for 60 min at 22 MeV and 10-15 µA beam current. Irradiation side products in the raw target solution were identified and quantified: 135La (0.4%), 135mBa (0.03%), 133mBa (0.01%) and 133Ba (0.0004%). The subsequent work-up and anion-exchange-based product purification process took approx. 30 min and led to a total amount of (1.2 – 1.8) GBq (decay-corrected to EOB) of 133La, formulated as [133La]LaCl3. After complete decay of 133La, a remainder of ca. 4 kBq of long-lived 133Ba per 100 MBq of 133La was detected and rated as uncritical regarding personal dose and waste management. Subsequent radiolabeling was successfully performed with previously published macropa-derived PSMA inhibitors at a micromolar range (quantitative labeling at 1 µM) and evaluated by radio-TLC and radio-HPLC analyses. The scale up to radioactivity amounts that are needed for clinical application purposes would be easy to achieve by increasing target mass, beam current and/or irradi

Objective: The somatostatin receptor subtype 2 (SSTR2) is a suitable target for the diagnosis and therapy of neuroendocrine tumors using radiolabeled ligands [1]. BON-1S cells have been described to express SSTR2 receptors in vitro [2]. We therefore established 2D and 3D cell culture systems of BON-1S cells. For SSTR2 ligand binding, BON-1S cells cultured as monolayer or spheroids were incubated with an SSTR2 agonist (NODAGA-TATE) or an SSTR2 antagonist (NODAGA-JR11). Both were labeled with either 68Ga- or 64Cu and their respective binding and uptake were determined. Understanding the binding data is a prerequisite for using spheroids of BON-1S cells in a Micro Physiological System (MPS) [3]. Methods: Spheroids of SSTR2 containing BON-1S cells were created as follows: Freshly harvested cells were incubated with magnetic nanoparticles in RPMI medium overnight. After magnetic particle uptake, cells were split into a 24-well-plate (1.5 x 10-4 cells/well). In an incubator, the magnetic particles were moved via a magnetic drive for five days, to allow the formation of spheroids [4]. After incubation with the 68Ga- or 64Cu-labeled radioligands (RCY>95%), the BON-1S cells, either as monolayer or as spheroids, were washed and treated with acidic glycine buffer (pH 3.0) to remove the bound but not yet internalized molecules from the outer cellular membrane. After this washing step, the remaining radioligand molecules are assumed to represent internalized molecules [5]. In the monolayer, internalization was determined after 10 minutes, 1 hour and 3 hours after addition of the radioligand to the respective culture. Results: Saturation analysis of radiolabeled NODAGA-TATE as well as NODAGA-JR11 on BON-S1 monolayer yielded similar Kd values in the low nanomolar range (2.3 to 8.6 nM). Affinity of the respective radiolabeled ligand was much lower on BON-1S spheroids, with Kd values around 30 nM for both 64Cu-labeled ligands and almost 60 nM for the 68Ga-labeled ligands. The maxima

We recognize 131Ba as a SPECT radionuclide, which provides a diagnostic match for the therapeutic alpha-emitting radionuclides 223Ra and 224Ra. The gamma-emitter barium-131 (t½ = 11.5 d) decays with an energy of 123.8 keV (30% intensity) of the first decay via cesium-131 (t½ = 9.7 d) to stable xenon-131, each by electron capture. Our aim was to develop of a straightforward resin-based radiochemical separation to yield 131Ba with high radionuclide purity. Furthermore, the radiolabeling of the complexing agent macropa with [131Ba]Ba2+ using mild labeling conditions was intended. For this purpose, different TLC systems as reaction control were utilized. The radiopharma-cological characterization of 131Ba-labeled macropa in comparison to uncomplexed [131Ba]Ba2+ was carried out in healthy mice, including biodistribution and small animal SPECT/CT experiments.

Currently, targeted alpha therapy is one of the most investigated topics in radiopharmaceutical cancer management. Especially, the alpha emitter 225 Ac provides excellent nuclear properties and is gaining increasing popularity for the treatment of various tumor entities. We herein report on the synthesis of two universal 225Ac-chelators for mild condition radiolabeling and binding sites to conjugate biomolecules via the copper-mediated click chemistry. A convenient radiolabeling procedure was investigated as well as the complex stability proved for both chelators and two PSMA-targeting model radioconjugates. Studies regarding affinity and cell survival were per-formed on LNCaP cells followed by biodistribution studies, which were performed using LNCaP tumor-bearing mice. High efficiency radiolabeling for all conjugates was demonstrated. Cell binding studies revealed a fourfold lower cell affinity for the PSMA conjugate with one targeting vector compared to the conjugate owing two targeting vectors. Additionally, these differences were verified by in vitro cell survival evaluation and biodistribution studies, both showing a higher therapeutic efficiency for the same dose on a cellular leve, a higher tumor up-take (15%ID/g) and a rapid whole body clearance after 24 hours. The synthesized chelators will overcome obstacles of lacking stability and worse labeling needs regarding 225Ac complexation using the DOTA chelator. Moreover, the universal functionalization expands the coverage of these chelators in combination with any sensitive bio(macro)molecule, thus improving treat-ment of any addressable tumor target.

Background and purpose Systemic molecular radiotherapy utilizes internal irradiation by radionuclide-labeled tumor-targeting agents with the potential to destroy (micro-)metastases. However, doses that are applicable in solid tumors do not reach the levels nessecary for tumor control. Thus, the combination of molecular and external radiotherapy is a promising treatment strategy, as enhanced tumor doses can be delivered with and without minor overlapping toxicities. Here, we combined a 90Y-labeled anti-EGFR antibody (Cetuximab) with clinically relevant fractionated radiotherapy in a preclinical trial using head and neck squamous cell carcinoma xenograft tumors. Materials and methods To model 90Y-Cetuximab uptake for treatment schedule optimization, FaDu-bearing mice were injected with near-infrared-labeled-Cetuximab at different time points during radiotherapy with differing doses. Cetuximab uptake was longitudinally followed by in vivo-optical imaging. Tumor control probability experiments with fractionated radiotherapy (30 fx, 6 weeks, 8 dose groups/ arm) in combination with 90Y-Cetuximab were performed to test the curative potential. Results Imaging of near-infrared-labeled-Cetuximab uptake revealed that low to moderate external beam doses can enhance antibody uptake. Using the optimized schedule, combination of molecular and external radiotherapy using 90Y-Cetuximab at a dose that did not result in permanent tumor inactivation in previous experiments, led to substantially increased tumor control compared to radiotherapy alone. Conclusion Our results indicate that combination of radiolabeled therapeutics with clinically relevant fractionated radiotherapy has a remarkable potential to improve curative treatment outcome. Application of some radiation dose prior to injection may improve drug uptake and enable patient stratification and treatment personalization via a corresponding PET-tracer during therapy.

We show the synthesis of an in vivo stable mercury compound with functionality suitable for radiopharmaceuticals. The designed cyclic bisarylmercury was based on the water tolerance of organomercurials, higher bond dissociation energy of Hg-Ph to Hg-S, and the experimental evidence that acyclic structures suffer significant cleavage of one of the Hg-R bonds. The bispidine motif was chosen for its in vivo stability, chemical accessibility, and functionalization properties. Radionuclide production results in 197(m)HgCl2(aq), so the desired mercury compound was formed via a water-tolerant organotin transmetallation. The Hg-bispidine compound showed high chemical stability in tests with an excess of sulphur-containing competitors and high in vivo stability, without any observable protein interaction by human serum assay, and good organ clearance demonstrated by biodistribution and SPECT studies in rats. In particular no retention in the kidneys typical of unstable mercury compounds. The natHg analogue allowed full characterization by NMR and HRMS.