Radiochemical Aspects of Receptor Scintigraphy: labeling with radiometals, optimisation and radiochemical purity

__Abstract__ Radioactieve peptiden zijn stoffen die gebruikt worden voor het in vivo in beeld brengen van kanker. Tumorcellen brengen vaak receptoren in overvloed tot expressie in en op de tumorcellen. Wanneer deze receptoren, aanwezig op de tumorcellen, binden met de radiopeptiden kunnen met behulp van een gammacamera of PET (Positron Mission Tomografie) deze tumoren worden gevisualiseerd. Om met een hoge resolutie te kunnen visualiseren op een gammacamera gaat dit ten koste van de gevoeligheid van een gammacamera. Dit betekent dat er dan meer radioactiviteit nodig is om een scan te maken maar wel met een betere beeldkwaliteit. Een nadeel uit bovenstaande voorbeelden is dat dit in beide gevallen leidt tot een hogere dosis aan straling in oplossing. Deze straling afkomstig van de gebruikte radionucliden zorgen voor radiolyse in de desbetreffende oplossing. Radiolyse is het beschadigen van peptide door hoge dosis aan straling. Door de hogere dosis straling is er een verhoogde kans dat het peptide wordt beschadigd, zelfs voordat het wordt geïnjecteerd. Er is daarom onderzoek gedaan naar een mogelijkheid voor betere bescherming tegen radiolyse van het radiopeptide. De hoeveelheid schade aan radioactief gelabelde peptide door radiolyse wordt uitgedrukt in percentage radiochemische zuiverheid (RCP). RCP is momenteel niet goed gedefinieerd. Er is momenteel een gebrek aan fundamentele kennis voor wat betreft de radiolyse van radiopeptiden. Radiolyse is ook onduidelijk gedefinieerd. Om de invloed van radiolyse te kunnen onderzoeken zijn er in dit proefschrift twee type peptiden als model gebruikt: DOTA- of DTPA-geconjugeerde analogen van somatostatine en bombesine. Deze peptiden worden radioactief gelabeld en worden specifiek gebruikt voor visualisatie van neuro-endocrine, prostaat-, borst-tumoren op een gamma- of PET camera. Zoals beschreven in dit proefschrift zijn peptide receptor scintigraphy (PRS) en peptide receptor radionuclide therapie (PRRT) al meer dan twee decennia succesvol klinisch toegepast. Toch zijn er nog steeds mogelijkheden om deze techniek en de toepassing ervan te verbeteren. De resultaten van de hier beschreven onderzoeken geven meer kennis en inzicht in de verschillende processen van bijvoorbeeld de radiochemische technieken en zorgen uiteindelijk voor een verbetering van de toepassingen. Dit resulteert in een betere beeldvorming en dus in het eerder in beeld brengen van kanker en vooral in een effectieve therapie

Measurement of reaction kinetics of [177Lu]Lu-DOTA-TATE using a microfluidic system

Microfluidic synthesis techniques can offer improvement over batch syntheses which are currently used for radiopharmaceutical production. These improvements are, for example, better mixing of reactants, more efficient energy transfer, less radiolysis, faster reaction optimization, and overall improved reaction control. However, scale-up challenges hinder the routine clinical use, so the main advantage is currently the ability to optimize reactions rapidly and with low reactant consumption. Translating those results to clinical systems could be done based on calculations, if kinetic constants and diffusion coefficients were known. This study describes a microfluidic system with which it was possible to determine the kinetic association rate constants for the formation of [177Lu]Lu-DOTA-TATE under conditions currently used for clinical production. The kinetic rate constants showed a temperature dependence that followed the Arrhenius equation, allowing the determination of Arrhenius parameters for a Lu-DOTA conjugate (A = 1.24 ± 0.05 × 1019 M-1 s-1, EA = 109.5 ± 0.1 × 103 J mol-1) for the first time. The required reaction time for the formation of [177Lu]Lu-DOTA-TATE (99% yield) at 80 °C was 44 s in a microfluidic channel (100 μm). Simulations done with COMSOL Multiphysics® indicated that processing clinical amounts (3 mL reaction solution) in less than 12 min is possible in a micro- or milli-fluidic system, if the diameter of the reaction channel is increased to over 500 μm. These results show that a continuous, microfluidic system can become a viable alternative to the conventional, batch-wise radiolabelling technique

In Vivo Evaluation of Indium-111-Labeled 800CW as a Necrosis-Avid Contrast Agent.

Current clinical measurements for tumor treatment efficiency rely often on changes in tumor volume measured as shrinkage by CT or MRI, which become apparent after multiple lines of treatment and pose a physical and psychological burden on the patient. Detection of therapy-induced cell death in the tumor can be a fast measure for treatment efficiency. However, there are no reliable clinical tools for detection of tumor necrosis. Previously, we studied the necrosis avidity of cyanine-based fluorescent dyes, which suffered long circulation times before tumor necrosis could be imaged due to low hydrophilicity. We now present the application of radiolabeled 800CW, a commercially available cyanine with high hydrophilicity, to image tumor necrosis in a mouse model. We conjugated 800CW to DOTA via a PEG linker, for labeling with single-photon emission-computed tomography isotope indium-111, yielding [ <sup>111</sup> In]In-DOTA-PEG <sub>4</sub> -800CW. We then investigated specific [ <sup>111</sup> In]In-DOTA-PEG <sub>4</sub> -800CW uptake by dead cells in vitro, using both fluorescence and radioactivity as detection modalities. Finally, we investigated [ <sup>111</sup> In]In-DOTA-PEG <sub>4</sub> -800CW uptake into necrotic tumor regions of a 4T1 breast tumor model in mice. We successfully prepared a precursor and developed a reliable procedure for labeling 800CW with indium-111. We detected specific [ <sup>111</sup> In]In-DOTA-PEG <sub>4</sub> -800CW uptake by dead cells, using both fluorescence and radioactivity. Albeit with a tumor uptake of only 0.37%ID/g at 6 h post injection, we were able to image tumor necrosis with a tumor to background ratio of 7:4. Fluorescence and radioactivity in cryosections from the dissected tumors were colocalized with tumor necrosis, confirmed by TUNEL staining. [ <sup>111</sup> In]In-DOTA-PEG <sub>4</sub> -800CW can be used to image tumor necrosis in vitro and in vivo. Further research will elucidate the application of [ <sup>111</sup> In]In-DOTA-PEG <sub>4</sub> -800CW or other radiolabeled hydrophilic cyanines for the detection of necrosis caused by chemotherapy or other anti-cancer therapies. This can provide valuable prognostic information in treatment of solid tumors

Investigation of Factors Determining the Enhanced Permeability and Retention Effect in Subcutaneous Xenografts

Liposomal chemotherapy offers several advantages over conventional therapies, including high intratumoral drug delivery, reduced side effects, prolonged circulation time and the possibility to dose higher. The efficient delivery of liposomal chemotherapeutics relies however on the enhanced permeability and retention (EPR) effect, which refers to the ability of macromolecules to extravasate leaky tumor vessels and accumulate in the tumor tissue. Using a panel of human xenograft tumors, we evaluated the influence of the EPR effect on liposomal distribution in vivo by injection of pegylated liposomes radiolabeled with 111In. Liposomal accumulation in tumors and organs was followed over time by SPECT/CT imaging. We observed that fast growing xenografts, which may be less representative of tumor development in patients, showed higher liposomal accumulation as compared to slow growing xenografts. Additionally, several other parameters determining the EPR effect were evaluated, such as blood and lymphatic vessel density, intratumoral hypoxia, and the presence of macrophages. The investigation of various parameters showed a few correlations. Although hypoxia, proliferation and macrophage presence were associated with tumor growth, no hard conclusions or predictions could be made regarding the EPR effect or liposomal uptake. However liposomal uptake was

Imaging of atherosclerosis, targeting LFA-1 on inflammatory cells with 111In-DANBIRT

Background: 111In-DOTA-butylamino-NorBIRT (DANBIRT) is a novel radioligand which binds to Leukocyte Function-associated Antigen-1 (LFA-1), expressed on inflammatory cells. This study evaluated 111In-DANBIRT for the visualization of atherosclerotic plaque inflammation in mice. Methods and Results: ApoE−/− mice, fed an atherogenic diet up to 20 weeks (n = 10), were imaged by SPECT/CT 3 hours post injection of 111In-DANBIRT (~ 200 pmol, ~ 40 MBq). Focal spots of 111In-DANBIRT were visible in the aortic arch of all animals, with an average Target-to-Background Ratio (TBR) of 1.7 ± 0.5. In vivo imaging results were validated by ex vivo SPECT/CT imaging, with a TBR up to 11.5 (range 2.6 to 11.5). Plaques, identified by Oil Red O lipid-staining on excised arteries, co-localized with 111In-DANBIRT uptake as determined by ex vivo autoradiography. Subsequent histological processing and in vitro autoradiography confirmed 111In-DANBIRT uptake at plaque areas containing CD68 expressing macrophages and LFA-1 expressing inflammatory cells. Ex vivo incubation of a human carotid endarterectomy specimen with 111In-DANBIRT (~ 950 nmol, ~ 190 MBq) for 2 hours showed heterogeneous plaque uptake on SPECT/CT, after which immunohistochemical analysis demonstrated co-localization of 111In-DANBIRT uptake and CD68 and LFA-1 expressing cells. Conclusions: Our results indicate the potential of radiolabeled DANBIRT as a relevant imaging radioligand for non-invasive evaluation of atherosclerotic inflammation

Imaging inflammation in atherosclerotic plaques, targeting SST2 with [111In]In-DOTA-JR11

Background: Imaging Somatostatin Subtype Receptor 2 (SST2) expressing macrophages by [DOTA,Tyr3]-octreotate (DOTATATE) has proven successful for plaque detection. DOTA-JR11 is a SST2 targeting ligand with a five times higher tumor uptake than DOTATATE, and holds promise to improve plaque imaging. The aim of this study was to evaluate the potential of DOTA-JR11 for plaque detection. Methods and Results: Atherosclerotic ApoE−/− mice (n = 22) fed an atherogenic diet were imaged by SPECT/CT two hours post injection of [111In]In-DOTA-JR11 (~ 200 pmol, ~ 50 MBq). In vivo plaque uptake of [111In]In-DOTA-JR11 was visible in all mice, with a target-to-background-ratio (TBR) of 2.23 ± 0.35. Post-mortem scans after thymectomy and ex vivo scans of the arteries after excision of the arteries confirmed plaque uptake of the radioligand with TBRs of 2.46 ± 0.52 and 3.43 ± 1.45 respectively. Oil red O lipid-staining and ex vivo autoradiography of excised arteries showed [111In]In-DOTA-JR11 uptake at plaque locations. Histological processing showed CD68 (macrophages) and SST2 expressing cells in plaques. SPECT/CT, in vitro autoradiography and immunohistochemistry performed on slices of a human carotid endarterectomy sample showed [111In]In-DOTA-JR11 uptake at plaque locations containing CD68 and SST2 expressing cells. Conclusions: The results of this study indicate DOTA-JR1

Influence of tumour size on the efficacy of targeted alpha therapy with 213Bi-[DOTA0,Tyr3]-octreotate

BACKGROUND: Targeted alpha therapy has been postulated to have great potential for the treatment of small clusters of tumour cells as well as small metastases. (213)Bismuth, an α-emitter with a half-life of 46 min, has shown to be effective in preclinical as well as in clinical applications. In this study, we evaluated whether (213)Bi-[DOTA(0), Tyr(3)]-octreotate ((213)Bi-DOTATATE), a (213)Bi-labelled somatostatin analogue with high affinity for somatostatin receptor subtype 2 (SSTR(2)), is suitable for the treatment of larger neuroendocrine tumours overexpressing SSTR(2) in comparison to its effectiveness for smaller tumours. We performed a preclinical targeted radionuclide therapy study with (213)Bi-DOTATATE in animals bearing tumours of different sizes (50 and 200 mm(3)) using two tumour models: H69 (human small cell lung carcinoma) and CA20948 (rat pancreatic tumour). METHODS: Pharmacokinetics was determined for calculation of dosimetry in organs and tumours. H69- or CA20948-xenografted mice with tumour volumes of approximately 120 mm(3) were euthanized at 10, 30, 60 and 120 min post injection of a single dose of (213)Bi-DOTATATE (1.5–4.8 MBq). To investigate the therapeutic efficacy of (213)Bi-DOTATATE, xenografted H69 and CA20948 tumour-bearing mice with tumour sizes of 50 and 200 mm(3) were administered daily with a therapeutic dose of (213)Bi-DOTATATE (0.3 nmol, 2–4 MBq) for three consecutive days. The animals were followed for 90 days after treatment. At day 90, mice were injected with 25 MBq (99m)Tc-DMSA and imaged by SPECT/CT to investigate possible renal dysfunction due to (213)Bi-DOTATATE treatment. RESULTS: Higher tumour uptakes were found in CA20948 tumour-bearing animals compared to those in H69 tumour-bearing mice with the highest tumour uptake of 19.6 ± 6.6 %IA/g in CA20948 tumour-bearing animals, while for H69 tumour-bearing mice, the highest tumour uptake was found to be 9.8 ± 2.4 %IA/g. Nevertheless, as the anti-tumour effect was more pronounced in H69 tumour-bearing mice, the survival rate was higher. Furthermore, in the small tumour groups, no regrowth of tumour was found in two H69 tumour-bearing mice and in one of the CA20948 tumour-bearing mice. No renal dysfunction was observed in (213)Bi-DOTATATE-treated mice after the doses were applied. CONCLUSIONS: (213)Bi-DOTATATE demonstrated a great therapeutic effect in both small and larger tumour lesions. Higher probability for stable disease was found in animals with small tumours. (213)Bi-DOTATATE was effective in different neuroendocrine (H69 and CA20948) tumour models with overexpression of SSTR(2) in mice. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s13550-016-0162-2) contains supplementary material, which is available to authorized users

Semi-automated system for concentrating 68Ga-eluate to obtain high molar and volume concentration of 68Ga-Radiopharmaca for preclinical applications

Introduction: 68Ga-radiopharmaceuticals are common in the field of Nuclear Medicine to visualize receptor-mediated processes. In contrast to straightforward labeling procedures for clinical applications, preclinical in vitro and in vivo applications are hampered for reasons like e.g. volume restriction, activity concentration, molar activity and osmolality. Therefore, we developed a semi-automatic system specifically to overcome these problems. A difficulty appeared unexpectedly, as intrinsic trace metals derived from eluate (Zn, Fe and Cu) are concentrated as well in amounts that influence radiochemical yield and thus lower molar activity. Methods: To purify Gallium-68 and to reduce the high elution volume of a 68Ga-generator, a NaCl-based method using a column containing PS-H+ was implemented in a low volume PEEK system. Influence on reducing osmolality, acidity and the amount of PS-H+ resin (15–50 mg) was investigated. [68Ga]Ga was desorbed from the PS-H+ resin with acidified 2-5 M NaCl (containing 0.05 M of HCl) and 68Ga-activity was collected. DOTA-TATE was used as a peptide model. All buffers and additives used for labeling were mixed with Chelex 100 (~1 g/50 mL) for >144 h and eventually filtered using a 0.22 μm filter (Millipore). Quantification of metals was performed after labeling by HPLC (UV). Results: Gallium-68 activity could be desorbed from PS-H+ cation column with 3 M NaCl, and >60% (120–180 MBq) of [68Ga]Ga was collected in 99% (ITLC), and a radiochemical purity of >95% (HPLC). Conclusion: With the here described concentration system and metal purification technique, a low activity containing 68Ga-generator can be used to label DOTA-peptide in preclinical applicable amounts >60 MBq/nmol (40–60 MBq/0.1 mL) and within 20 min

Therapeutic application of CCK2R-targeting PP-F11: influence of particle range, activity and peptide amount

Background: Targeted radionuclide therapy with high-energy beta-emitters is generally considered suboptimal to cure small tumours (90Y, 177Lu or 213Bi, accounting for the radionuclide specific activities (SAs), the tumour absorbed doses and tumour (radio) biology. Methods: Tumour uptake of 111In-PP-F11 was determined in nude mice bearing CCK2 receptor-transfected A431 xenografts at 1 and 4 h post-injection for escalating peptide masses of 0.03 to 15 nmol/mouse. The absorbed tumour dose was estimated, assuming comparable biodistributions of the 90Y, 177Lu or 213Bi radiolabelled peptides. The linear-quadratic (LQ) model was used to calculate the tumour control probabilities (TCP) as a function of tumour mass and growth. Results: Practically achievable maximum SAs for PP-F11 labelled with 90Y and 177Lu were 400 MBq 90Y/nmol and 120 MBq177Lu/nmol. Both the large elution volume from the 220 MBq 225Ac generator used and reaction kinetics diminished the maximum achieved 213Bi SA in practice: 40 MBq 213Bi/nmol. Tumour uptakes decreased rapidly with increasing peptide amounts, following a logarithmic curve with ED50 = 0.5 nmol. At 0.03 nmol peptide, the (300 mg) tumour dose was 9 Gy after 12 MBq 90Y-PP-F11, and for 111In and 177Lu, this was 1 Gy. A curative dose of 60 Gy could be achieved with a single administration of 111 MBq 90Y labelled to 0.28 nmol PP-F11 or with 4 × 17 MBq 213Bi (0.41 nmol) when its α-radiation relative biological effectiveness (RBE) was assumed to be 3.4. Repeated dosing is preferable to avoid complete tumour receptor saturation. Tumours larger than 200 mg are curable with 90Y-PP-F11; the other radionuclides perform better in smaller tumours. Furthermore, 177Lu is not optimal for curing fast-growing tumours. Conclusions: Receptor saturation, specific radiopharmaceutical activities and absorbed doses in the tumour together favour therapy with the CCK2 receptor-binding peptide PP-F11 labelled with 90Y, despite its longer β-particle range in tissue, certainly for tumours larger than 300 mg. The predicted TCPs are of theoretical nature and need to be compared with the outcome of targeted radionuclide experiments

Maintaining radiochemical purity of [177Lu]Lu-DOTA-PSMA-617 for PRRT by reducing radiolysis

[177Lu]Lu-DOTA-PSMA-617 for PRRT is subject to radiolysis and therefore loses receptor affinity. This will be detrimental for treatment efficacy. In this study optimal quencher(s) (combinations) are determined to maintain radiochemical purity with a downscaled model. Downscaled model in terms of activity, but at similar concentrations. DOTA-PSMA-617 was labeled with [177Lu]LuCl3 with different molar- and volume activities. Either methionine, ethanol or both showed superior effects on the stabilizing radiochemical purity of [177Lu]Lu-DOTA-PSMA-617. As a consequence, radiochemical purity of [177Lu]Lu-DOTA-PSMA-617 could be maintained by the addition of methionine and/or ethanol and downscaled model was proven and complementary