Search

A technique to image α-synuclein (αSYN) fibrils in vivo is an unmet scientific and clinical need that would represent a transformative tool in the understanding, diagnosis, and treatment of various neurodegenerative diseases. Several classes of compounds have shown promising results as potential PET tracers, but no candidate has yet exhibited the affinity and selectivity required to reach clinical application. We hypothesized that the application of the rational drug design technique of molecular hybridization to two promising lead scaffolds could enhance the binding to αSYN up to the fulfillment of those requirements. By combining the structures of SIL and MODAG tracers, we developed a library of diarylpyrazoles (DAPs). In vitro evaluation through competition assays against [ 3 H]SIL26 and [ 3 H]MODAG-001 showed the novel hybrid scaffold to have preferential binding affinity for amyloid β (Aβ) over αSYN fibrils. A ring-opening modification on the phenothiazine building block to produce analogs with increased three-dimensional flexibility did not result in an improved αSYN binding but a complete loss of competition, as well as a significant reduction in Aβ affinity. The combination of the phenothiazine and the 3,5-diphenylpyrazole scaffolds into DAP hybrids did not generate an enhanced αSYN PET tracer lead compound. Instead, these efforts identified a scaffold for promising Aβ ligands that may be relevant to the treatment and monitoring of Alzheimer's disease (AD).

Radiolabeling of tetrazines has gained increasing attention due to their important role in pretargeted imaging or therapy. The most commonly used radionuclide in PET imaging is fluorine-18. For this reason, we have recently developed a method which enables the direct aromatic 18 F-fluorination of tetrazines using stannane precursors through copper-mediated fluorinations. Herein, we further optimized this labeling procedure. 3-(3-fluorophenyl)-1,2,4,5-tetrazine was chosen for this purpose because of its high reactivity and respective limited stability during the labeling process. By optimizing parameters such as elution conditions, precursor amount, catalyst, time or temperature, the radiochemical yield (RCY) could be increased by approximately 30%. These conditions were then applied to optimize the RCY of a recently successfully developed and promising pretargeting imaging agent. This agent could be isolated in a decay corrected RCY of 14 ± 3% and Am of 201 ± 30 GBq/µmol in a synthesis time of 70 min. Consequently, the RCY increased by 27%.

Pretargeted nuclear imaging for the diagnosis of various cancers is an emerging and fast developing field. The tetrazine ligation is currently considered the most promising reaction in this respect. Monoclonal antibodies are often the preferred choice as pretargeting vector due to their outstanding targeting properties. In this work, we evaluated the performance of [ 64 Cu]Cu-NOTA-PEG 7 -H-Tz using a setup we previously used for [ 111 In]In-DOTA-PEG 11 -BisPy-Tz, thereby allowing for comparison of the performance of these two promising pretargeting imaging agents. The evaluation included a comparison of the physicochemical properties of the compounds and their performance in an ex vivo blocking assay. Finally, [ 64 Cu]Cu-NOTA-PEG 7 -H-Tz was evaluated in a pretargeted imaging study and compared to [ 111 In]In-DOTA-PEG 11 -BisPy-Tz. Despite minor differences, this study indicated that both evaluated tetrazines are equally suited for pretargeted imaging.

The α7 nicotinic acetylcholine receptor (α7 nAChR) is involved in several cognitive and physiologic processes; its expression levels and patterns change in neurologic and psychiatric diseases, such as schizophrenia and Alzheimer's disease, which makes it a relevant drug target. Development of selective radioligands is important for defining binding properties and occupancy of novel molecules targeting the receptor. We tested the in vitro binding properties of [ 125 I]Iodo-ASEM [(3-(1,4-diazabycyclo[3.2.2]nonan-4-yl)-6-( 125 I-iododibenzo[b,d]thiopentene 5,5-dioxide)] in the mouse, rat and pig brain using autoradiography. The in vivo binding properties of [ 18 F]ASEM were investigated using positron emission tomography (PET) in the pig brain. [ 125 I]Iodo-ASEM showed specific and displaceable high affinity (~1 nM) binding in mouse, rat, and pig brain. Binding pattern overlapped with [ 125 I]α-bungarotoxin, specific binding was absent in α7 nAChR gene-deficient mice and binding was blocked by a range of α7 nAChR orthosteric modulators in an affinity-dependent order in the pig brain. Interestingly, relative to the wild-type, binding in β2 nAChR gene-deficient mice was lower for [ 125 I]Iodo-ASEM (58% ± 2.7%) than [ 125 I]α-bungarotoxin (23% ± 0.2%), potentially indicating different binding properties to heteromeric α7β2 nAChR. [ 18 F]ASEM PET in the pig showed high brain uptake and reversible tracer kinetics with a similar spatial distribution as previously reported for α7 nAChR. Blocking with SSR-180,711 resulted in a significant decrease in [ 18 F]ASEM binding. Our findings indicate that [ 125 I]Iodo-ASEM allows sensitive and selective imaging of α7 nAChR in vitro, with better signal-to-noise ratio than previous tracers. Preliminary data of [ 18 F]ASEM in the pig brain demonstrated principal suitable kinetic properties for in vivo quantification of α7 nAChR, comparable to previously published data.

The bioorthogonal reaction between a tetrazine and strained transcyclooctene (TCO) has garnered success in pretargeted imaging. This reaction was first validated in nuclear imaging using an 111 In-labeled 1,4,7,10tetraazacyclododecane1,4,7,10tetraacetic acid (DOTA)-linked bispyridyl tetrazine (Tz) ([ 111 In]In-DOTA-PEG 11 -Tz) and a TCO functionalized CC49 antibody. Given the initial success of this Tz, it has been paired with TCO functionalized small molecules, diabodies, and affibodies for in vivo pretargeted studies. Furthermore, the single photon emission tomography (SPECT) radionuclide, 111 In, has been replaced with the β-emitter, 177 Lu and α-emitter, 212 Pb, both yielding the opportunity for targeted radiotherapy. Despite use of the 'universal chelator', DOTA, there is yet to be an analogue suitable for positron emission tomography (PET) using a widely available radionuclide. Here, a 68 Ga-labeled variant ([ 68 Ga]Ga-DOTA-PEG 11 -Tz) was developed and evaluated using two different in vivo pretargeting systems (Aln-TCO and TCO-CC49). Small animal imaging and ex vivo biodistribution studies were performed and revealed target specific uptake of [ 68 Ga]Ga-DOTA-PEG 11 -Tz in the bone (3.7 %ID/g, knee) in mice pretreated with Aln-TCO and tumor specific uptake (5.8 %ID/g) with TCO-CC49 in mice bearing LS174 xenografts. Given the results of this study, [ 68 Ga]Ga-DOTA-PEG 11 -Tz can serve as an alternative to [ 111 In]In-DOTA-PEG 11 -Tz., Competing Interests: No conflict of interest has to be reported.