Development of Peptide-Based Probes for Molecular Imaging of the Postsynaptic Density in the Brain.

The postsynaptic density (PSD) comprises numerous scaffolding proteins, receptors, and signaling molecules that coordinate synaptic transmission in the brain. Postsynaptic density protein 95 (PSD-95) is a master scaffold protein within the PSD and one of its most abundant proteins and therefore constitutes a very attractive biomarker of PSD function and its pathological changes. Here, we exploit a high-affinity inhibitor of PSD-95, AVLX-144, as a template for developing probes for molecular imaging of the PSD. AVLX-144-based probes were labeled with the radioisotopes fluorine-18 and tritium, as well as a fluorescent tag. Tracer binding showed saturable, displaceable, and uneven distribution in rat brain slices, proving effective in quantitative autoradiography and cell imaging studies. Notably, we observed diminished tracer binding in human post-mortem Parkinson's disease (PD) brain slices, suggesting postsynaptic impairment in PD. We thus offer a suite of translational probes for visualizing and understanding PSD-related pathologies.

Indium-111 radiolabelling of a brain-penetrant Aß antibody for SPECT imaging.

Background: The development of bispecific antibodies that can traverse the blood-brain barrier has paved the way for brain-directed immunotherapy and when radiolabelled, immunoPET imaging. The objective of this study was to investigate how indium-111 (111In) radiolabelling with compatible chelators affects the brain delivery and peripheral biodistribution of the bispecific antibody RmAb158-scFv8D3, which binds to amyloid-beta (Aß) and the transferrin receptor (TfR), in Aß pathology-expressing tg-ArcSwe mice and aged-matched wild-type control mice. Methods: Bispecific RmAb158-scFv8D3 (biAb) was radiolabelled with 111In using CHX-A"-DTPA, DOTA, or DOTA-tetrazine (DOTA-Tz). Affinity toward TfR and Aß, as well as stability, was investigated in vitro. Mice were then intravenously administered with the three different radiolabelled biAb variants, and blood samples were collected for monitoring pharmacokinetics. Brain concentration was quantified after 2 and 72 h, and organ-specific retention was measured at 72 h by gamma counting. A subset of mice also underwent whole-body Single-photon emission computed tomography (SPECT) scanning at 72 h after injection. Following post-mortem isolation, the brains of tg-ArcSwe and WT mice were sectioned, and the spatial distribution of biAb was further investigated with autoradiography. Results: All three [111In]biAb variants displayed similar blood pharmacokinetics and brain uptake at 2 h after administration. Radiolabelling did not compromise affinity, and all variants showed good stability, especially the DOTA-Tz variant. Whole-body SPECT scanning indicated high liver, spleen, and bone accumulation of all [111In]biAb variants. Subsequent ex vivo measurement of organ retention confirmed SPECT data, with retention in the spleen, liver, and bone - with very high bone marrow retention. Ex vivo gamma measurement of brain tissue, isolated at 72 h post-injection, and ex vivo autoradiography showed that WT mice, despite the absence of Aß, exhibited comparable brain concentrations of [111In]biAb as those found in the tg-ArcSwe brain. Conclusions: The successful 111In-labelling of biAb with retained binding to TfR and Aß, and retained ability to enter the brain, demonstrated that 111In can be used to generate radioligands for brain imaging. A high degree of [111In]biAb in bone marrow and intracellular accumulation in brain tissue indicated some off-target interactions or potential interaction with intrabrain TfR resulting in a relatively high non-specific background signal.

211 At-Astatination of Arenes Using Trimethylgermanyl Precursors.

A set of 211At-astatoarenes were synthesized from corresponding trimethylgermyl arenes with radiochemical conversions (RCCs) of up to 90%. Both electron rich and electron poor substrates were successfully radiolabeled at room temperature (RT) using relatively low precursor amounts (0.15 µmol/ 0.02 mL solvent (7.5 mM)). Ready access to ortho-, para- and meta- astatinated arenes was achievable. Optimized reaction conditions were successfully applied to label a poly (ADP-ribose) polymerase (PARP) inhibitor with a RCC of approx. 50%. We believe that trimethylgermanyl derivatives are a viable addition to the astatination precursor toolbox and facilitate astatination of arenes. The developed labeling method should easily be applicable for productions under good manufacturing practice (GMP).

Development of a 99mTc-labeled tetrazine for pretargeted SPECT imaging using an alendronic acid-based bone targeting model.

Pretargeting, which is the separation of target accumulation and the administration of a secondary imaging agent into two sequential steps, offers the potential to improve image contrast and reduce radiation burden for nuclear imaging. In recent years, the tetrazine ligation has emerged as a promising approach to facilitate covalent pretargeted imaging due to its unprecedented kinetics and bioorthogonality. Pretargeted bone imaging with TCO-modified alendronic acid (Aln-TCO) is an attractive model that allows the evaluation of tetrazines in healthy animals without the need for complex disease models or targeting regimens. Recent structure-activity relationship studies of tetrazines evaluated important parameters for the design of potent tetrazine-radiotracers for pretargeted imaging. However, limited information is available for 99mTc-labeled tetrazines. In this study, four tetrazines intended for labeling with fac-[99mTc(OH2)3 (CO)3]+ were synthesized and evaluated using an Aln-TCO mouse model. 3,6-bis(2-pyridyl)-1,2,4,5-Tz without additional linker showed higher pretargeted bone uptake and less background activity compared to the same scaffold with a PEG8 linker or 3-phenyl-1,2,4,5-Tz-based compounds. Additionally, improved bone/blood ratios were observed in pretargeted animals compared to animals receiving directly labeled Aln-TCO. The results of this study implicate 3,6-bis(2-pyridyl)-1,2,4,5-Tz as a promising scaffold for potential 99mTc-labeled tetrazines.

Investigation of imaging the somatostatin receptor by opening the blood-brain barrier with melittin - A feasibility study using positron emission tomography and [64Cu]Cu-DOTATATE.

DOTATATE is a somatostatin peptide analog used in the clinic to detect somatostatin receptors which are highly expressed on neuroendocrine tumors. Somatostatin receptors are found naturally in the intestines, pancreas, lungs, and brain (mainly cortex). In vivo measurement of the somatostatin receptors in the cortex has been challenging because available tracers cannot cross the blood-brain barrier (BBB) due to their intrinsic polarity. A peptide called melittin, a main component of honeybee venom, has been shown to disrupt plasma membranes and increase the permeability of biological membranes. In this study, we assessed the feasibility of using melittin to facilitate the passage of [64Cu]Cu-DOTATATE through the BBB and its binding to somatostatin receptors in the cortex. Evaluation included in vitro autoradiography on Long Evans rat brains to estimate the binding affinity of [64Cu]Cu-DOTATATE to the somatostatin receptors in the cortex and an in vivo evaluation of [64Cu]Cu-DOTATATE binding in NMRI mice after injection of melittin. This study found an in vitro Bmax = 89 ± 4 nM and KD = 4.5 ± 0.6 nM in the cortex, resulting in a theoretical binding potential (BP) calculated as Bmax/KD ≈ 20, which is believed suitable for in vivo brain PET imaging. However, the in vivo results showed no significant difference between the control and melittin injected mice, indicating that the honeybee venom failed to open the BBB. Additional experiments, potentially involving faster injection rates are required to verify that melittin can increase brain uptake of non-BBB permeable PET tracers. Furthermore, an evaluation of whether a venom with a narrow therapeutic range can be used for clinical purposes needs to be considered.

Synthesis and evaluation of fluorine-18 labelled tetrazines as pre-targeting imaging agents for PET.

BACKGROUND: The brain is a challenging target for antibody-based positron emission tomography (immunoPET) imaging due to the restricted access of antibody-based ligands through the blood-brain barrier (BBB). To overcome this physiological obstacle, we have previously developed bispecific antibody ligands that pass through the BBB via receptor-mediated transcytosis. While these radiolabelled ligands have high affinity and specificity, their long residence time in the blood and brain, typical for large molecules, poses another challenge for PET imaging. A viable solution could be a two-step pre-targeting approach which involves the administration of a tagged antibody that accumulates at the target site in the brain and then clears from the blood, followed by administration of a small radiolabelled molecule with fast kinetics. This radiolabelled molecule can couple to the tagged antibody and thereby make the antibody localisation visible by PET imaging. The in vivo linkage can be achieved by using the inverse electron demand Diels-Alder reaction (IEDDA), with trans-cyclooctene (TCO) and tetrazine groups participating as reactants. In this study, two novel 18F-labelled tetrazines were synthesized and evaluated for their potential use as pre-targeting imaging agents, i.e., for their ability to rapidly enter the brain and, if unbound, to be efficiently cleared with minimal background retention. RESULTS: The two compounds, a methyl tetrazine [18F]MeTz and an H-tetrazine [18F]HTz were radiolabelled using a two-step procedure via [18F]F-Py-TFP synthesized on solid support followed by amidation with amine-bearing tetrazines, resulting in radiochemical yields of 24% and 22%, respectively, and a radiochemical purity of > 96%. In vivo PET imaging was performed to assess their suitability for in vivo pre-targeting. Time-activity curves from PET-scans showed [18F]MeTz to be the more pharmacokinetically suitable agent, given its fast and homogenous distribution in the brain and rapid clearance. However, in terms of rection kinetics, H-tetrazines are advantageous, exhibiting faster reaction rates in IEDDA reactions with dienophiles like trans-cyclooctenes, making [18F]HTz potentially more beneficial for pre-targeting applications. CONCLUSION: This study demonstrates a significant potential of [18F]MeTz and [18F]HTz as agents for pre-targeted PET brain imaging due to their efficient brain uptake, swift clearance and appropriate chemical stability.

Development and Preclinical Evaluation of [211At]PSAt-3-Ga: An Inhibitor for Targeted α-Therapy of Prostate Cancer.

The application of prostate-specific membrane antigen (PSMA)-targeted α-therapy is a promising alternative to ß--particle-based treatments. 211At is among the potential α-emitters that are favorable for this concept. Herein, 211At-based PSMA radiopharmaceuticals were designed, developed, and evaluated. Methods: To identify a 211At-labeled lead, a surrogate strategy was applied. Because astatine does not exist as a stable nuclide, it is commonly replaced with iodine to mimic the pharmacokinetic behavior of the corresponding 211At-labeled compounds. To facilitate the process of structural design, iodine-based candidates were radiolabeled with the PET radionuclide 68Ga to study their preliminary in vitro and in vivo properties before the desired 211At-labeled lead compound was formed. The most promising candidate from this evaluation was chosen to be 211At-labeled and tested in biodistribution studies. Results: All 68Ga-labeled surrogates displayed affinities in the nanomolar range and specific internalization in PSMA-positive LNCaP cells. PET imaging of these compounds identified [68Ga]PSGa-3 as the lead compound. Subsequently, [211At]PSAt-3-Ga was synthesized in a radiochemical yield of 35% and showed tumor uptake of 19 ± 8 percentage injected dose per gram of tissue (%ID/g) at 1 h after injection and 7.6 ± 2.9 %ID/g after 24 h. Uptake in off-target tissues such as the thyroid (2.0 ± 1.1 %ID/g), spleen (3.0 ± 0.6 %ID/g), or stomach (2.0 ± 0.4 %ID/g) was low, indicating low in vivo deastatination of [211At]PSAt-3-Ga. Conclusion: The reported findings support the use of iodine-based and 68Ga-labeled variants as a convenient strategy for developing astatinated compounds and confirm [211At]PSAt-3 as a promising radiopharmaceutical for targeted α-therapy.

Evaluation of F-537-Tetrazine in a model for brain pretargeting imaging. Comparison to N-(3-[18F] fluoro-5-(1,2,4,5-tetrazin-3-yl)benzyl)propan-1-amine.

Brain pretargeted nuclear imaging for the diagnosis of various neurodegenerative diseases is a quickly developing field. The tetrazine ligation is currently the most explored approach to achieve this goal due to its remarkable properties. In this work, we evaluated the performance of F-537-Tetrazine, previously developed by Biogen, and N-(3-[18F]fluoro-5-(1,2,4,5-tetrazin-3-yl)benzyl)propan-1-amine, previously developed in our group, thereby allowing for the direct comparison of these two imaging probes. The evaluation included synthesis, radiolabeling and a comparison of the physicochemical properties of the compounds. Furthermore, their performance was evaluated by in vitro and in vivo pretargeting models. This study indicated that N-(3-[18F] fluoro-5-(1,2,4,5-tetrazin-3-yl)benzyl)propan-1-amine might be more suited for brain pretargeted imaging.

A facile synthesis of precursor for the σ-1 receptor PET radioligand [18 F]FTC-146 and its radiofluorination.

The σ-1 receptor is a non-opioid transmembrane protein involved in various human pathologies including neurodegenerative diseases, inflammation, and cancer. The previously published ligand [18 F]FTC-146 is among the most promising tools for σ-1 molecular imaging by positron emission tomography (PET), with a potential for application in clinical diagnostics and research. However, the published six- or four-step synthesis of the tosyl ester precursor for its radiosynthesis is complicated and time-consuming. Herein, we present a simple one-step precursor synthesis followed by a one-step fluorine-18 labeling procedure that streamlines the preparation of [18 F]FTC-146. Instead of a tosyl-based precursor, we developed a one-step synthesis of the precursor analog AM-16 containing a chloride leaving group for the SN 2 reaction with 18 F-fluoride. 18 F-fluorination of AM-16 led to a moderate decay-corrected radiochemical yield (RCY = 7.5%) with molar activity (Am ) of 45.9 GBq/µmol. Further optimization of this procedure should enable routine radiopharmaceutical production of this promising PET tracer.

Repeated low doses of psilocybin increase resilience to stress, lower compulsive actions, and strengthen cortical connections to the paraventricular thalamic nucleus in rats.

Psilocybin (a classic serotonergic psychedelic drug) has received appraisal for use in psychedelic-assisted therapy of several psychiatric disorders. A less explored topic concerns the use of repeated low doses of psychedelics, at a dose that is well below the psychedelic dose used in psychedelic-assisted therapy and often referred to as microdosing. Psilocybin microdose users frequently report increases in mental health, yet such reports are often highly biased and vulnerable to placebo effects. Here we establish and validate a psilocybin microdose-like regimen in rats with repeated low doses of psilocybin administration at a dose derived from occupancy at rat brain 5-HT2A receptors in vivo. The rats tolerated the repeated low doses of psilocybin well and did not manifest signs of anhedonia, anxiety, or altered locomotor activity. There were no deficits in pre-pulse inhibition of the startle reflex, nor did the treatment downregulate or desensitize the 5-HT2A receptors. However, the repeated low doses of psilocybin imparted resilience against the stress of multiple subcutaneous injections, and reduced the frequency of self-grooming, a proxy for human compulsive actions, while also increasing 5-HT7 receptor expression and synaptic density in the paraventricular nucleus of the thalamus. These results establish a well-validated regimen for further experiments probing the effects of repeated low doses of psilocybin. Results further substantiate anecdotal reports of the benefits of psilocybin microdosing as a therapeutic intervention, while pointing to a possible physiological mechanism.

A pilot study of cerebral metabolism and serotonin 5-HT2A receptor occupancy in rats treated with the psychedelic tryptamine DMT in conjunction with the MAO inhibitor harmine.

Rationale: The psychedelic effects of the traditional Amazonian botanical decoction known as ayahuasca are often attributed to agonism at brain serotonin 5-HT2A receptors by N,N-dimethyltryptamine (DMT). To reduce first pass metabolism of oral DMT, ayahuasca preparations additionally contain reversible monoamine oxidase A (MAO-A) inhibitors, namely ß-carboline alkaloids such as harmine. However, there is lacking biochemical evidence to substantiate this pharmacokinetic potentiation of DMT in brain via systemic MAO-A inhibition. Objectives: We measured the pharmacokinetic profile of harmine and/or DMT in rat brain, and tested for pharmacodynamic effects on brain glucose metabolism and DMT occupancy at brain serotonin 5-HT2A receptors. Methods: We first measured brain concentrations of harmine and DMT after treatment with harmine and/or DMT at low sub-cutaneous doses (1 mg/kg each) or harmine plus DMT at moderate doses (3 mg/kg each). In the same groups of rats, we also measured ex vivo the effects of these treatments on the availability of serotonin 5-HT2A receptors in frontal cortex. Finally, we explored effects of DMT and/or harmine (1 mg/kg each) on brain glucose metabolism with [18F]FDG-PET. Results: Results confirmed that co-administration of harmine inhibited the formation of the DMT metabolite indole-3-acetic acid (3-IAA) in brain, while correspondingly increasing the cerebral availability of DMT. However, we were unable to detect any significant occupancy by DMT at 5-HT2A receptors measured ex vivo, despite brain DMT concentrations as high as 11.3 µM. We did not observe significant effects of low dose DMT and/or harmine on cerebral [18F]FDG-PET uptake. Conclusion: These preliminary results call for further experiments to establish the dose-dependent effects of harmine/DMT on serotonin receptor occupancy and cerebral metabolism.

In Vitro and In Vivo Evaluation of Pellotine: A Hypnotic Lophophora Alkaloid.

Quality of life is often reduced in patients with sleep-wake disorders. Insomnia is commonly treated with benzodiazepines, despite their well-known side effects. Pellotine (1), a Lophophora alkaloid, has been reported to have short-acting sleep-inducing properties in humans. In this study, we set out to evaluate various in vitro and in vivo properties of 1. We demonstrate that 1 undergoes slow metabolism; e.g. in mouse liver microsomes 65% remained, and in human liver microsomes virtually no metabolism was observed after 4 h. In mouse liver microsomes, two phase I metabolites were identified: 7-desmethylpellotine and pellotine-N-oxide. In mice, the two diastereomers of pellotine-O-glucuronide were additionally identified as phase II metabolites. Furthermore, we demonstrated by DESI-MSI that 1 readily enters the central nervous system of rodents. Furthermore, radioligand-displacement assays showed that 1 is selective for the serotonergic system and in particular the serotonin (5-HT)1D, 5-HT6, and 5-HT7 receptors, where it binds with affinities in the nanomolar range (117, 170, and 394 nM, respectively). Additionally, 1 was functionally characterized at 5-HT6 and 5-HT7, where it was found to be an agonist at the former (EC50 = 94 nM, Emax = 32%) and an inverse agonist at the latter (EC50 = 291 nM, Emax = -98.6). Finally, we demonstrated that 1 dose-dependently decreases locomotion in mice, inhibits REM sleep, and promotes sleep fragmentation. Thus, we suggest that pellotine itself, and not an active metabolite, is responsible for the hypnotic effects and that these effects are possibly mediated through modulation of serotonergic receptors.

Next generation fibroblast activation protein (FAP) targeting PET tracers - The tetrazine ligation allows an easy and convenient way to 18F-labeled (4-quinolinoyl)glycyl-2-cyanopyrrolidines.

Small-molecular fibroblast activation protein inhibitor (FAPI)-based tracer have been shown to be promising Positron Emission Tomography (PET) 68Ga-labeled radiopharmaceuticals to image a variety of tumors including pancreatic, breast, and colorectal cancers, among others. In this study, we developed a novel 18F-labeled FAPI derivative. [18F]6 was labeled using a synthon approach based on the tetrazine ligation. It showed subnanomolar affinity for the FAP protein and a good selectivity profile against known off-target proteases. Small animal PET studies revealed high tumor uptake and good target-to-background ratios. [18F]6 was excreted via the liver. Overall, [18F]6 showed promising characteristics to be used as a PET tracer and could serve as a lead for further development of halogen-based theranostic FAPI radiopharmaceuticals.

A Fluorescent Probe as a Lead Compound for a Selective α-Synuclein PET Tracer: Development of a Library of 2-Styrylbenzothiazoles and Biological Evaluation of [18F]PFSB and [18F]MFSB.

A method to detect and quantify aggregated α-synuclein (αSYN) fibrils in vivo would drastically impact the current understanding of multiple neurodegenerative diseases, revolutionizing their diagnosis and treatment. Several efforts have produced promising scaffolds, but a notable challenge has hampered the establishment of a clinically successful αSYN positron emission tomography (PET) tracer: the requirement of high selectivity over the other misfolded proteins amyloid ß (Aß) and tau. By designing and screening a library of 2-styrylbenzothiazoles based on the selective fluorescent probe RB1, this study aimed at developing a selective αSYN PET tracer. [3H]PiB competition binding assays identified PFSB (Ki = 25.4 ± 2.3 nM) and its less lipophilic analogue MFSB, which exhibited enhanced affinity to αSYN (Ki = 10.3 ± 4.7 nM) and preserved selectivity over Aß. The two lead compounds were labeled with fluorine-18 and evaluated using in vitro autoradiography on human brain slices, where they demonstrated up to 4-fold increased specific binding in MSA cases compared to the corresponding control, reasonably reflecting selective binding to αSYN pathology. In vivo PET imaging showed [18F]MFSB successfully crosses the blood-brain barrier (BBB) and is taken up in the brain (SUV = 1.79 ± 0.02). Although its pharmacokinetic profile raises the need for additional structural optimization, [18F]MFSB represents a critical step forward in the development of a successful αSYN PET tracer by overcoming the major challenge of αSYN/Aß selectivity.

Development of 11 C-labeled CRANAD-102 for positron emission tomography imaging of soluble Aß-species.

CRANAD-102, a selective near-infrared fluorescent tracer targeting soluble amyloid-ß (Aß) species, has recently attracted attention due to its potential to be used as a diagnostic tool for early stages of Alzheimer's disease (AD). Development of a positron emission tomography (PET) tracer based on CRANAD-102 could as such allow to noninvasively study soluble and protofibrillar species of Aß in humans. These soluble and protofibrillar species are thought to be responsible to cause AD. Within this work, we successfully 11 C-labeled CRANAD-102 via a Suzuki-Miyaura reaction in a RCС of 48 ± 9%, with a RCP of >96% and a molar activity (Am ) of 25 ± 7 GBq/µmol. Future studies have to be conducted to evaluate if [11 C]CRANAD-102 can be used to detect soluble protofibrils in vivo and if [11 C]CRANAD-102 can be used to detect AD earlier as possible with current diagnostics.

A nanobody recognizes a unique conserved epitope and potently neutralizes SARS-CoV-2 omicron variants.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) Omicron variant sub-lineages spread rapidly worldwide, mostly due to their immune-evasive properties. This has put a significant part of the population at risk for severe disease and underscores the need for effective anti-SARS-CoV-2 agents against emergent strains in vulnerable patients. Camelid nanobodies are attractive therapeutic candidates due to their high stability, ease of large-scale production, and potential for delivery via inhalation. Here, we characterize the receptor binding domain (RBD)-specific nanobody W25 and show superior neutralization activity toward Omicron sub-lineages in comparison to all other SARS-CoV2 variants. Structure analysis of W25 in complex with the SARS-CoV2 spike glycoprotein shows that W25 engages an RBD epitope not covered by any of the antibodies previously approved for emergency use. In vivo evaluation of W25 prophylactic and therapeutic treatments across multiple SARS-CoV-2 variant infection models, together with W25 biodistribution analysis in mice, demonstrates favorable pre-clinical properties. Together, these data endorse W25 for further clinical development.

The Structural Combination of SIL and MODAG Scaffolds Fails to Enhance Binding to α-Synuclein but Reveals Promising Affinity to Amyloid ß.

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 [3H]SIL26 and [3H]MODAG-001 showed the novel hybrid scaffold to have preferential binding affinity for amyloid ß (Aß) 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 Aß 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 Aß ligands that may be relevant to the treatment and monitoring of Alzheimer's disease (AD).

Clearing and Masking Agents in Pretargeting Strategies.

'Pretargeting' led to increased target-to-background ratios of nanomedicines in short timeframes. However, clearing or masking agents are needed to reach the full potential of pretargeted approaches. This review gives an overview of clearing and masking agents employed in pretargeting strategies in both preclinical and clinical settings and discusses how these agents work.

Quantitative Evaluation of a Multimodal Aptamer-Targeted Long-Circulating Polymer for Tumor Targeting.

Aptamers are promising targeting agents for imaging and therapy of numerous diseases, including cancer. However, a significant shortcoming of aptamers is their poor stability and fast excretion, limiting their application in vivo. Common strategies to overcome these challenges is to chemically modify aptamers in order to increase their stability and/or to apply formulation technologies such as conjugating them to polymers or nanocarriers in order to increase their circulation half-life. This is expected to result in improved cellular uptake or retention to passively targeted nanomedicines. Herein, we report a modular conjugation strategy based on click chemistry between functionalized tetrazines and trans-cyclooctene (TCO), for the modification of high molecular weight hyperbranched polyglycerol (HPG) with sgc8 aptamer, fluorescent dyes, and 111In. Our data indicate strong affinity of sgc8 against a range of solid tumor-derived cell lines that have previously not been tested with this aptamer. Nevertheless, nonspecific uptake of scrambled ssDNA-functionalized HPG in cells highlights inherent challenges of aptamer-targeted probes that remain to be solved for clinical translation. We validate HPG-sgc8 as a nontoxic nanoprobe with high affinity against MDA-MB-468 breast and A431 lung cancer cells and show significantly increased plasma stability compared to free sgc8. In vivo quantitative SPECT/CT imaging indicates EPR-mediated tumor uptake of HPG-sgc8 and nontargeted or scrambled ssDNA-conjugated HPG but no statistically significant difference between these formulations in terms of total tumor uptake or retention. Our study emphasizes the need for stringent controls and quantification in the evaluation of aptamer-targeted probes. For this purpose, our versatile synthesis strategy provides a simple approach for the design and evaluation of long-circulating aptamer-conjugated nanoformulations.

Pretargeted imaging beyond the blood-brain barrier.

Pretargeting is a powerful nuclear imaging strategy to achieve enhanced imaging contrast for nanomedicines and reduce the radiation burden to healthy tissue. Pretargeting is based on bioorthogonal chemistry. The most attractive reaction for this purpose is currently the tetrazine ligation, which occurs between trans-cyclooctene (TCO) tags and tetrazines (Tzs). Pretargeted imaging beyond the blood-brain barrier (BBB) is challenging and has not been reported thus far. In this study, we developed Tz imaging agents that are capable of ligating in vivo to targets beyond the BBB. We chose to develop 18F-labeled Tzs as they can be applied to positron emission tomography (PET) - the most powerful molecular imaging technology. Fluorine-18 is an ideal radionuclide for PET due to its almost ideal decay properties. As a non-metal radionuclide, fluorine-18 also allows for development of Tzs with physicochemical properties enabling passive brain diffusion. To develop these imaging agents, we applied a rational drug design approach. This approach was based on estimated and experimentally determined parameters such as the BBB score, pretargeted autoradiography contrast, in vivo brain influx and washout as well as on peripheral metabolism profiles. From 18 initially developed structures, five Tzs were selected to be tested for their in vivo click performance. Whereas all selected structures clicked in vivo to TCO-polymer deposited into the brain, [18F]18 displayed the most favorable characteristics with respect to brain pretargeting. [18F]18 is our lead compound for future pretargeted neuroimaging studies based on BBB-penetrant monoclonal antibodies. Pretargeting beyond the BBB will allow us to image targets in the brain that are currently not imageable, such as soluble oligomers of neurodegeneration biomarker proteins. Imaging of such currently non-imageable targets will allow early diagnosis and personalized treatment monitoring. This in turn will accelerate drug development and greatly benefit patient care.