Evaluation of a 68Ga-Labeled DOTA-Tetrazine as a PET Alternative to 111In-SPECT Pretargeted Imaging.

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 111In-labeled 1,4,7,10tetraazacyclododecane1,4,7,10tetraacetic acid (DOTA)-linked bispyridyl tetrazine (Tz) ([111In]In-DOTA-PEG11-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, 111In, has been replaced with the ß-emitter, 177Lu and α-emitter, 212Pb, 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 68Ga-labeled variant ([68Ga]Ga-DOTA-PEG11-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 [68Ga]Ga-DOTA-PEG11-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, [68Ga]Ga-DOTA-PEG11-Tz can serve as an alternative to [111In]In-DOTA-PEG11-Tz.

Improved radiosynthesis and preliminary in vivo evaluation of the 11C-labeled tetrazine [11C]AE-1 for pretargeted PET imaging.

Pretargeted nuclear imaging based on the ligation between tetrazines and nano-sized targeting agents functionalized with trans-cyclooctene (TCO) has recently been shown to improve both imaging contrast and dosimetry in nuclear imaging of nanomedicines. Herein, we describe the improved radiosynthesis of a 11C-labeled tetrazine ([11C]AE-1) and its preliminary evaluation in both mice and pigs. Pretargeted imaging in mice was carried out using both a new TCO-functionalized polyglutamic acid and a previously reported TCO-functionalized bisphosphonate system as targeting agents. Unfortunately, pretargeted imaging was not successful using these targeting agents in pair with [11C]AE-1. However, brain imaging in pig indicated that the tracer crossed the blood-brain-barrier. Hence, we suggest that this tetrazine scaffold could be used as a starting point for the development of pretargeted brain imaging, which has so far been a challenging task.

Quantitative evaluation of PSMA PET imaging using a realistic anthropomorphic phantom and shell-less radioactive epoxy lesions.

BACKGROUND: Positron emission tomography (PET) with prostate specific membrane antigen (PSMA) have shown superior performance in detecting metastatic prostate cancers. Relative to [18F]fluorodeoxyglucose ([18F]FDG) PET images, PSMA PET images tend to visualize significantly higher-contrast focal lesions. We aim to evaluate segmentation and reconstruction algorithms in this emerging context. Specifically, Bayesian or maximum a posteriori (MAP) image reconstruction, compared to standard ordered subsets expectation maximization (OSEM) reconstruction, has received significant interest for its potential to reach convergence with minimal noise amplifications. However, few phantom studies have evaluated the quantitative accuracy of such reconstructions for high contrast, small lesions (sub-10 mm) that are typically observed in PSMA images. In this study, we cast 3 mm-16-mm spheres using epoxy resin infused with a long half-life positron emitter (sodium-22; 22Na) to simulate prostate cancer metastasis. The anthropomorphic Probe-IQ phantom, which features a liver, bladder, lungs, and ureters, was used to model relevant anatomy. Dynamic PET acquisitions were acquired and images were reconstructed with OSEM (varying subsets and iterations) and BSREM (varying ß parameters), and the effects on lesion quantitation were evaluated. RESULTS: The 22Na lesions were scanned against an aqueous solution containing fluorine-18 (18F) as the background. Regions-of-interest were drawn with MIM Software using 40% fixed threshold (40% FT) and a gradient segmentation algorithm (MIM's PET Edge+). Recovery coefficients (RCs) (max, mean, peak, and newly defined "apex"), metabolic tumour volume (MTV), and total tumour uptake (TTU) were calculated for each sphere. SUVpeak and SUVapex had the most consistent RCs for different lesion-to-background ratios and reconstruction parameters. The gradient-based segmentation algorithm was more accurate than 40% FT for determining MTV and TTU, particularly for lesions [Formula: see text] 6 mm in diameter (R2 = 0.979-0.996 vs. R2 = 0.115-0.527, respectively). CONCLUSION: An anthropomorphic phantom was used to evaluate quantitation for PSMA PET imaging of metastatic prostate cancer lesions. BSREM with ß = 200-400 and OSEM with 2-5 iterations resulted in the most accurate and robust measurements of SUVmean, MTV, and TTU for imaging conditions in 18F-PSMA PET/CT images. SUVapex, a hybrid metric of SUVmax and SUVpeak, was proposed for robust, accurate, and segmentation-free quantitation of lesions for PSMA PET.

Lipophilicity and Click Reactivity Determine the Performance of Bioorthogonal Tetrazine Tools in Pretargeted In Vivo Chemistry.

The development of highly selective and fast biocompatible reactions for ligation and cleavage has paved the way for new diagnostic and therapeutic applications of pretargeted in vivo chemistry. The concept of bioorthogonal pretargeting has attracted considerable interest, in particular for the targeted delivery of radionuclides and drugs. In nuclear medicine, pretargeting can provide increased target-to-background ratios at early time-points compared to traditional approaches. This reduces the radiation burden to healthy tissue and, depending on the selected radionuclide, enables better imaging contrast or higher therapeutic efficiency. Moreover, bioorthogonally triggered cleavage of pretargeted antibody-drug conjugates represents an emerging strategy to achieve controlled release and locally increased drug concentrations. The toolbox of bioorthogonal reactions has significantly expanded in the past decade, with the tetrazine ligation being the fastest and one of the most versatile in vivo chemistries. Progress in the field, however, relies heavily on the development and evaluation of (radio)labeled compounds, preventing the use of compound libraries for systematic studies. The rational design of tetrazine probes and triggers has thus been impeded by the limited understanding of the impact of structural parameters on the in vivo ligation performance. In this work, we describe the development of a pretargeted blocking assay that allows for the investigation of the in vivo fate of a structurally diverse library of 45 unlabeled tetrazines and their capability to reach and react with pretargeted trans-cyclooctene (TCO)-modified antibodies in tumor-bearing mice. This study enabled us to assess the correlation of click reactivity and lipophilicity of tetrazines with their in vivo performance. In particular, high rate constants (>50 000 M-1 s-1) for the reaction with TCO and low calculated logD 7.4 values (below -3) of the tetrazine were identified as strong indicators for successful pretargeting. Radiolabeling gave access to a set of selected 18F-labeled tetrazines, including highly reactive scaffolds, which were used in pretargeted PET imaging studies to confirm the results from the blocking study. These insights thus enable the rational design of tetrazine probes for in vivo application and will thereby assist the clinical translation of bioorthogonal pretargeting.

Imidazole fused phenanthroline (PIP) ligands for the preparation of multimodal Re(I) and 99mTc(I) probes.

A small library of [2 + 1] 99mTc(i) complexes based on phenyl-imidazole-fused phenanthroline (PIP) ligands were synthesized and evaluated as multimodal molecular imaging probes. Using either a two-step or a one-pot synthesis method, 99mTc-PIP complexes containing N-methylimidazole as the monodentate ligand were prepared and isolated in good (54 to 89%) radiochemical yield, with the exception of one derivative bearing a strongly electron-withdrawing substituent. The stability of the [2 + 1] complexes was assessed in saline and in cysteine and histidine challenge studies, showing 6 hours stability, making them suitable for in vivo studies. In parallel, the Re(i) analogues were prepared as reference standards to verify the structure of the 99mTc complexes. The optical properties were consistent with other previously reported [2 + 1] type Re(i) complexes that have been used as cellular dyes and sensors. To facilitate the development of targeted derivatives, a tetrazine-PIP ligand was also synthesized. The 99mTc complex of the tetrazine PIP ligand effectively coupled to compounds containing a trans-cyclooctene (TCO) group including a TCO-albumin derivative, which was prepared as a model targeting molecule. An added benefit of the Re-PIP-Tz construct is that the emission from the metal complex was quenched by the presence of the tetrazine. Following the addition of TCO, there was a 70-fold increase in fluorescence emission, which can in future be leveraged during in vitro studies to reduce background signal.

Trans-Cyclooctene-Functionalized PeptoBrushes with Improved Reaction Kinetics of the Tetrazine Ligation for Pretargeted Nuclear Imaging.

Tumor targeting using agents with slow pharmacokinetics represents a major challenge in nuclear imaging and targeted radionuclide therapy as they most often result in low imaging contrast and high radiation dose to healthy tissue. To address this challenge, we developed a polymer-based targeting agent that can be used for pretargeted imaging and thus separates tumor accumulation from the imaging step in time. The developed targeting agent is based on polypeptide-graft-polypeptoid polymers (PeptoBrushes) functionalized with trans-cyclooctene (TCO). The complementary 111In-labeled imaging agent is a 1,2,4,5-tetrazine derivative, which can react with aforementioned TCO-modified PeptoBrushes in a rapid bioorthogonal ligation. A high degree of TCO loading (up to 30%) was achieved, without altering the physicochemical properties of the polymeric nanoparticle. The highest degree of TCO loading resulted in significantly increased reaction rates (77-fold enhancement) compared to those with small molecule TCO moieties when using lipophilic tetrazines. Based on computer simulations, we hypothesize that this increase is a result of hydrophobic effects and significant rearrangements within the polymer framework, in which hydrophobic patches of TCO moieties are formed. These patches attract lipophilic tetrazines, leading to increased reaction rates in the bioorthogonal ligation. The most reactive system was evaluated as a targeting agent for pretargeted imaging in tumor-bearing mice. After the setup was optimized, sufficient tumor-to-background ratios were achieved as early as 2 h after administration of the tetrazine imaging agent, which further improved at 22 h, enabling clear visualization of CT-26 tumors. These findings show the potential of PeptoBrushes to be used as a pretargeting agent when an optimized dose of polymer is used.

Evaluation of the inverse electron demand Diels-Alder reaction in rats using a scandium-44-labelled tetrazine for pretargeted PET imaging.

BACKGROUND: Pretargeted imaging allows the use of short-lived radionuclides when imaging the accumulation of slow clearing targeting agents such as antibodies. The biotin-(strept)avidin and the bispecific antibody-hapten interactions have been applied in clinical pretargeting studies; unfortunately, these systems led to immunogenic responses in patients. The inverse electron demand Diels-Alder (IEDDA) reaction between a radiolabelled tetrazine (Tz) and a trans-cyclooctene (TCO)-functionalized targeting vector is a promising alternative for clinical pretargeted imaging due to its fast reaction kinetics. This strategy was first applied in nuclear medicine using an 111In-labelled Tz to image TCO-functionalized antibodies in tumour-bearing mice. Since then, the IEDDA has been used extensively in pretargeted nuclear imaging and radiotherapy; however, these studies have only been performed in mice. Herein, we report the 44Sc labelling of a Tz and evaluate it in pretargeted imaging in Wistar rats. RESULTS: 44Sc was obtained from an in house 44Ti/44Sc generator. A 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)-functionalized tetrazine was radiolabelled with 44Sc resulting in radiochemical yields of 85-95%, a radiochemical purity > 99% at an apparent molar activity of 1 GBq/mmol. The 44Sc-labelled Tz maintained stability in solution for up to 24 h. A TCO-functionalized bisphosphonate, which accumulates in skeletal tissue, was used as a targeting vector to evaluate the 44Sc-labelled Tz. Biodistribution data of the 44Sc-labelled Tz showed specific uptake (0.9 ± 0.3% ID/g) in the bones (humerus and femur) of rats pre-treated with the TCO-functionalized bisphosphonate. This uptake was not present in rats not receiving pre-treatment (< 0.03% ID/g). CONCLUSIONS: We have prepared a 44Sc-labelled Tz and used it in pretargeted PET imaging with rats treated with TCO-functionalized bisphosponates. This allowed for the evaluation of the IEDDA reaction in animals larger than a typical mouse. Non-target accumulation was low, and there was a 30-fold higher bone uptake in the pre-treated rats compared to the non-treated controls. Given its convenient half-life and the ability to perform positron emission tomography with a previously studied DOTA-functionalized Tz, scandium-44 (t1/2 = 3.97 h) proved to be a suitable radioisotope for this study.

Pretargeting in nuclear imaging and radionuclide therapy: Improving efficacy of theranostics and nanomedicines.

Pretargeted nuclear imaging and radiotherapy have recently attracted increasing attention for diagnosis and treatment of cancer with nanomedicines. This is because it conceptually offers better imaging contrast and therapeutic efficiency while reducing the dose to radiosensitive tissues compared to conventional strategies. In conventional imaging and radiotherapy, a directly radiolabeled nano-sized vector is administered and allowed to accumulate in the tumor, typically on a timescale of several days. In contrast, pretargeting is based on a two-step approach. First, a tumor-accumulating vector carrying a tag is administered followed by injection of a fast clearing radiolabeled agent that rapidly recognizes the tag of the tumor-bound vector in vivo. Therefore, pretargeting circumvents the use of long-lived radionuclides that is a necessity for sufficient tumor accumulation and target-to-background ratios using conventional approaches. In this review, we give an overview of recent advances in pretargeted imaging strategies. We will critically reflect on the advantages and disadvantages of current state-of-the-art conventional imaging approaches and compare them to pretargeted strategies. We will discuss the pretargeted imaging concept and the involved chemistry. Finally, we will discuss the steps forward in respect to clinical translation, and how pretargeted strategies could be applied to improve state-of-the-art radiotherapeutic approaches.

In Vivo Radionuclide Generators for Diagnostics and Therapy.

In vivo radionuclide generators make complex combinations of physical and chemical properties available for medical diagnostics and therapy. Perhaps the best-known in vivo generator is 212Pb/212Bi, which takes advantage of the extended half-life of 212Pb to execute a targeted delivery of the therapeutic short-lived α-emitter 212Bi. Often, as in the case of 81Rb/81Kr, chemical changes resulting from the transmutation of the parent are relied upon for diagnostic value. In other instances such as with extended alpha decay chains, chemical changes may lead to unwanted consequences. This article reviews some common and not-so-common in vivo generators with the purpose of understanding their value in medicine and medical research. This is currently relevant in light of a recent push for alpha emitters in targeted therapies, which often come with extended decay chains.

Synthesis of Polyester Dendritic Scaffolds for Biomedical Applications.

Using a well-defined poly(2,2-bis(hydroxymethyl)propanoic acid) dendrimer scaffold, a series of G1 to G3 dendrons is functionalized at the periphery with alkynes to enable "Click" functionalization via the copper-catalyzed alkyne-azide cycloaddition (CuAAC). The resulting dendrons are further functionalized at the core with a dipicolylamine (DPA) moiety to enable radiolabeling with 99m Tc for molecular imaging applications. Efficient CuAAC coupling is achieved using an azide-functionalized triethylene glycol monomethyl ether (TEG-N3 ). Removal of copper from the DPA ligand is successfully performed on G1 and G2 dendrimers prior to radiolabeling with 99m Tc. Radiolabeling of the G3 dendrimer is accomplished via transmetallation of the [CuDPA]2+ ligand with 99m Tc, further demonstrating the feasibility of the synthetic strategies in the preparation of dendritic imaging agents. Subsequent attachment of an acyloxymethyl ketone (AOMK) derivative for targeting of cathepsin B is also explored. Despite demonstrating the ability to ligate multiple AOMK ligands, the AOMK-dendrimer conjugates are not able to bind to cathepsin B, which may be attributed to steric hindrance at the dendrimer periphery.

Synthesis and evaluation of radioiodinated acyloxymethyl ketones as activity-based probes for cathepsin B.

Dipeptidyl (acyloxy)methyl ketones (AOMKs) were functionalized with different iodine-containing prosthetic groups to generate a library of candidate cathepsin B probes. Compound 23a, (S)-20-[(S)-2-{[(benzyloxy)carbonyl]amino}-3-phenylpropanamido]-1-(4-iodophenyl)-1,14,21-trioxo-5,8,11-trioxa-2,15-diazadocosan-22-yl 2,4,6-trimethylbenzoate, was identified as a potential lead through in vitro screening, having a Ki = 181 ± 9 nM and demonstrating the ability to effectively label active cathepsin B in vitro. Its less potent analogue 11a, (S)-3-[(S)-2-{[(benzyloxy)carbonyl]amino}-3-phenylpropanamido]-7-[6-(4-iodobenzamido)hexanamido]-2-oxoheptyl 2,4,6-trimethylbenzoate, was also tested as a comparison. Biodistribution studies of the iodine-125-labeled compounds in MDA-MB-231 mouse xenografts exhibited tumor uptake of 0.58% ± 0.06% injected dose per gram (ID/g) for [(125)I]11a and 1.12% ± 0.08% ID/g for [(125)I]23a at 30 min. The tumor-to-blood ratios reached 1.2 for [(125)I]23a and 1.6 for [(125)I]11a after 23 h. The more hydrophilic [(125)I]23a showed an improved clearance profile with a superior tumor-to-muscle ratio of 7.0 compared to 3.4 for [(125)I]11a at 23 h. Iodinated AOMK ligands are suitable in vitro probes for cathepsin B and hold promise as a platform to develop molecular imaging probes.