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.

A Novel Leu-Enkephalin Prodrug Produces Pain-Relieving and Antidepressant Effects.

Persistent pain is a significant healthcare problem with limited treatment options. The high incidence of comorbid chronic pain and depression significantly reduces life quality and complicates the treatment of both conditions. Antidepressants are less effective for pain and depression than for depression alone and they induce severe side effects. Opioids are highly efficacious analgesics, but rapid development of tolerance, dependence, and debilitating side effects limit their efficacy and safe use. Leucine-enkephalin (Leu-ENK), the endogenous delta opioid receptor agonist, controls pain and mood and produces potent analgesia with reduced adverse effects compared to conventional opioids. High proteolytic instability, however, makes Leu-ENK ineffective after systemic administration and limits its clinical usefulness. KK-103, a Leu-ENK prodrug, was developed to overcome these limitations of Leu-ENK via markedly increased plasma stability in mice. We showed rapid and substantially increased systemic adsorption and blood plasma exposure of KK-103 compared to Leu-ENK. We also observed brain uptake of radiolabeled KK-103 after systemic administration, indicating a central effect of KK-103. We then established KK-103's prolonged antinociceptive efficacy in the ramped hot plate and formalin test. In both models, KK-103 produced a comparable dose to the maximum antinociceptive-effect relationship. The pain-alleviating effect of KK-103 primarily resulted from activating the delta opioid receptor after the likely conversion of KK-103 to Leu-ENK in vivo. Finally, KK-103 produced an antidepressant-like activity comparable to the antidepressant desipramine, but with minimal gastrointestinal inhibition and no incidence of sedation.

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.

Aptamers used for molecular imaging and theranostics - recent developments.

Aptamers are single stranded oligonucleotides that fold into three dimensional structures and are able to recognize a variety of molecular targets. Due to the similarity to antibodies with regards to specificity and affinity and their chemical versatility, aptamers are increasingly used to create targeted probes for in vivo molecular imaging and therapy. Hence, aptamer-based probes have been utilized in practically all major imaging modalities such as nuclear imaging, magnetic resonance imaging, x-ray computed tomography, echography and fluorescence imaging, as well as newer modalities such as surface enhanced Raman spectroscopy. Aside from targeting, aptamers have been used for the creation of sensors that allow the localized detection of cellular markers such as ATP in vivo. This review focuses on in vivo studies of aptamer-based probes for imaging and theranostics since the comprehensive overview by Bouvier-Müller and Ducongé in 2018.

Radiolabeling of a polypeptide polymer for intratumoral delivery of alpha-particle emitter, 225Ac, and beta-particle emitter, 177Lu.

INTRODUCTION: Radiotherapy of cancer requires both alpha- and beta-particle emitting radionuclides, as these radionuclide types are efficient at destroying different types of tumors. Both classes of radionuclides require a vehicle, such as an antibody or a polymer, to be delivered and retained within the tumor. Polyglutamic acid (pGlu) is a polymer that has proven itself effective as a basis of drug-polymer conjugates in the clinic, while its derivatives have been used for pretargeted tumor imaging in a research setup. trans-Cyclooctene (TCO) modified pGlu is suitable for pretargeted imaging or therapy, as well as for intratumoral radionuclide therapy. In all cases, it becomes indirectly radiolabeled via the bioorthogonal click reaction with the tetrazine (Tz) molecule carrying the radionuclide. In this study, we report the radiolabeling of TCO-modified pGlu with either lutetium-177 (177Lu), a beta-particle emitter, or actinium-225 (225Ac), an alpha-particle emitter, using the click reaction between TCO and Tz. METHODS: A panel of Tz derivatives containing a metal ion binding chelator (DOTA or macropa) connected to the Tz moiety directly or through a polyethylene glycol (PEG) linker was synthesized and tested for their ability to chelate 177Lu and 225Ac, and click to pGlu-TCO. Radiolabeled 177Lu-pGlu and 225Ac-pGlu were isolated by size exclusion chromatography. The retention of 177Lu or 225Ac by the obtained conjugates was investigated in vitro in human serum. RESULTS: All DOTA-modified Tzs efficiently chelated 177Lu resulting in average radiochemical conversions (RCC) of >75%. Isolated radiochemical yields (RCY) for 177Lu-pGlu prepared from 177Lu-Tzs ranged from 31% to 55%. TLC analyses detected <5% unchelated 177Lu for all 177Lu-pGlu preparations over six days in human serum. For 225Ac chelation, optimized RCCs ranged from 61 ± 34% to quantitative for DOTA-Tzs and were quantitative for the macropa-modified Tz (>98%). Isolated radiochemical yields (RCY) for 225Ac-pGlu prepared from 225Ac-Tzs ranged from 28% to 51%. For 3 out of 5 225Ac-pGlu conjugates prepared from DOTA-Tzs, the amount of unchelated 225Ac stayed below 10% over six days in human serum, while 225Ac-pGlu prepared from macropa-Tz showed a steady release of up to 37% 225Ac. CONCLUSION: We labeled TCO-modified pGlu polymers with alpha- and beta-emitting radionuclides in acceptable RCYs. All 177Lu-pGlu preparations and some 225Ac-pGlu preparations showed excellent stability in human plasma. Our work shows the potential of pGlu as a vehicle for alpha- and beta-radiotherapy of tumors and demonstrated the usefulness of Tz ligation for indirect radiolabeling.