High Molar Activity Research Articles

Synthesis of K+ channel radioligand [18F]5-methyl-3-fluoro-4-aminopyridine and PET imaging in mice.

[18F]3-fluoro-4-aminopyridine ([18F]3F4AP) is the first positron emission tomography (PET) radioligand that targets voltage-gated potassium (K+) channels in the brain for imaging demyelination. [18F]3F4AP exhibits high brain penetration, favorable kinetics for PET imaging, and high sensitivity to demyelinating lesions. However, recent studies in awake human subjects indicate lower metabolic stability than in anesthetized animals, resulting in reduced brain uptake. Therefore, there is a need for novel radioligands for K+ channels with suitable pharmacological properties and enhanced metabolic stability. Recent in vitro studies demonstrate that 5-methyl-3-fluoro-4-aminopyridine (5Me3F4AP) exhibits comparable binding affinity to K+ channels, pKa, logD, and membrane permeability as 3F4AP, and a slower enzymatic metabolic rate, suggesting its potential as a K+ channel PET tracer. In this study, we describe the radiochemical synthesis of [18F]5Me3F4AP using an isotope exchange method from the corresponding 3-fluoro-5-methyl-4-nitropyridine N-oxide, followed by a palladium on carbon mediated hydrogenation of the nitro and N-oxide groups. This method yielded [18F]5Me3F4AP with high purity and acceptable molar activity. PET/CT studies using naïve mice demonstrate that [18F]5Me3F4AP effectively crosses the blood-brain barrier and has comparable kinetics to [18F]3F4AP. These findings strongly suggest that [18F]5Me3F4AP is a promising candidate for neuroimaging applications and warrant further studies to investigate its sensitivity to lesions and in vivo metabolic stability.

"[18F]Trifluoroiodomethane- Enabling Photoredox-mediated Radical [18F]Trifluoromethylation for Positron Emission Tomography.

The development of new tracers for positron emission tomography (PET) is highly dependent on the available synthetic tools for their radiosynthesis. Herein, we present theradiosynthesis and application of [18F]trifluoroiodomethane - the first reagent for broad scope radical [18F]trifluoromethylation chemistry in high molar activity. CF218FI can be prepared from [18F]fluoroform with 67±5% AY and >99% RCP. Its synthetic utility is demonstrated by the radiosynthesis of previously unprecedented 18F-labeled a-trifluoromethyl ketones and trifluoromethyl sulfides, important motifs that are present in a range of bioactive compounds. Both protocols are Ru- and photo-mediated and proceed under mild reaction conditions. They show good functional group tolerance evidenced by the respective reaction scopes and make use of easily obtainable starting materials. The products can be isolated in 8.3-11.1 GBq/µmol (starting from ca. 5GBq [18F]fluoride). The applicability to PET tracer synthesis is shown by the radiolabeling of bioactive compounds, such as derivatives of probenecid and febuxostat. In a broader context, this work opens the door to the utilization of radical [18F]trifluoromethylation chemistry for the radiolabeling of PET tracers in high molar activity.

“[18F]Trifluoroiodomethane – Enabling Photoredox‐mediated Radical [18F]Trifluoromethylation for Positron Emission Tomography

The development of new tracers for positron emission tomography (PET) is highly dependent on the available synthetic tools for their radiosynthesis. Herein, we present the radiosynthesis and application of [18F]trifluoroiodomethane – the first reagent for broad scope radical [18F]trifluoromethylation chemistry in high molar activity. CF218FI can be prepared from [18F]fluoroform with 67±5% AY and >99% RCP. Its synthetic utility is demonstrated by the radiosynthesis of previously unprecedented 18F‐labeled a‐trifluoromethyl ketones and trifluoromethyl sulfides, important motifs that are present in a range of bioactive compounds. Both protocols are Ru‐ and photo‐mediated and proceed under mild reaction conditions. They show good functional group tolerance evidenced by the respective reaction scopes and make use of easily obtainable starting materials. The products can be isolated in 8.3‐11.1 GBq/µmol (starting from ca. 5 GBq [18F]fluoride). The applicability to PET tracer synthesis is shown by the radiolabeling of bioactive compounds, such as derivatives of probenecid and febuxostat. In a broader context, this work opens the door to the utilization of radical [18F]trifluoromethylation chemistry for the radiolabeling of PET tracers in high molar activity.

The production and separation of 161Tb with high specific activity at the University of Utah

Targeted radiotherapy (TRT) is an increasingly prominent area of research in nuclear medicine, particularly in the context of treating cancerous tumors. One radionuclide of considerable interest for TRT is terbium-161 (t1/2 = 6.95 days), which undergoes beta emission and shares similar decay properties as 177Lu (FDA-approved as LUTATHERA® and PLUVICTO®). Besides beta emission, 161Tb also emits a significant number of conversion and Auger electrons further enhancing its therapeutic potential. Terbium-161 can be produced using nuclear reactors through an indirect neutron capture reaction, G64160dn,γG64161d→3.66min,β−T65161b, from 160Gd targets. However, a key challenge in utilizing 161Tb for TRT lies in effectively separating target and product materials to attain high specific activity for radiolabeling. Here, we detail the production of no-carrier added 161Tb using low flux research reactors (mean thermal (<0.625 eV) neutron flux: 1.356×1012n∙cm−2∙s−1) like the University of Utah TRIGA Reactor, using enriched 160Gd2O3 targets (1.5 ± 0.3 μCi of 161Tb per mg of 160Gd target per hour of irradiation). We also developed a separation technique based on cation exchange and extraction chromatography, suitable for mCi level irradiations with targets exceeding 200 mg. In a simulated full-scale irradiation, 161Tb was successfully isolated from large mass targets using cation exchange (AG 50W-X8, with 2-hydroxyisobutyric acid at 70 mM, pH 4.75) and extraction chromatography (LN Resin, 0.5–0.75 M HNO3) methods. This resulted in high apparent molar activities of [161Tb]Tb-DOTA (113 ± 3 MBq/nmol), demonstrating high purity 161Tb relevant for potential future preclinical applications.

Electrochemical radiofluorination using a split-bipolar electrode

Electrochemical (radio)fluorination (ECF) is a versatile approach for nucleophilic radiofluorination of electron-rich compounds such as thioether derivatives. However, ECF generally requires high concentrations of supporting salts, which leads to low molar activity (Am) of the final product due to undesired contamination with [19F]F– ions. Here, we demonstrate the first example of radiofluorination using a split bipolar electrode (s-BPE) platform with low concentration of supporting salt. Under optimal reaction conditions, the ECF of methyl (methylthio)acetate (MMTA) using s-BPEs provided [18F]F-MMTA with radiochemical conversion of up to 70 % and high molar activities (28–43 GBq/μmol; 0.74–1.1 Ci/μmol), with approximately 5 mM of tetrabutylammonium perchlorate as supporting electrolyte.

Towards imaging the immune state of cancer by PET: Targeting legumain with 11C-labeled P1-Asn peptidomimetics carrying a cyano-warhead

PurposeM2-type tumor-associated macrophages (TAM) residing in the tumor microenvironment (TME) have been linked to tumor invasiveness, metastasis and poor prognosis. M2 TAMs suppress T cell activation, silencing the recognition of the cancer by the immune system. Targeting TAMs in anti-cancer therapy may support the immune system and immune-checkpoint inhibitor therapies to fight the cancer cells. We aimed to develop a PET tracer for the imaging of M2 TAM infiltration of cancer, using activated legumain as the imaging target. Basic proceduresTwo P1-mimicking inhibitors with a cyano-warhead were labeled with carbon-11 and evaluated in vitro and in vivo with a CT26 tumor mouse model. Target expression and activity were quantified from RT-qPCR and in vitro substrate conversion, respectively. The co-localization of legumain and TAMs was assessed by fluorescence microscopy. The two tracers were evaluated by PET with subsequent biodistribution analysis with the dissected tissues. Parent-to-total radioactivity in plasma was determined at several time points after i.v. tracer injection, using reverse phase radio-UPLC. Main findingsLegumain displayed a target density of 40.7 ± 19.1 pmol per mg total protein in tumor lysate (n = 4) with high substrate conversion and colocalization with M2 macrophages in the tumor periphery. [11C]1 and [11C]2 were synthesized with >95 % radiochemical purity and 12.9–382.2 GBq/μmol molar activity at the end of synthesis. We observed heterogeneous tumor accumulation in in vitro autoradiography and PET for both tracers. However, excess unlabeled 1 or 2 did not compete with tracer accumulation. Both [11C]1 and [11C]2 were rapidly metabolized to a polar radiometabolite in vivo. Principal conclusionsThe legumain tracers [11C]1 and [11C]2, synthesized with high radiochemical purity and molar activity, accumulate in the legumain-positive CT26 tumor in vivo. However, the lack of competition by excess compound questions their specificity. Both tracers are rapidly metabolized in vivo, requiring structural modifications towards more stable tracers for further investigations.

Structure-based discovery of a 4,5-Dihydropyrazole-cored PET ligand for imaging of receptor-interacting serine/threonine-protein kinase 1 (RIPK1) in the brain

Receptor-interacting serine/threonine-protein kinase 1 (RIPK1) regulates programmed cell death and inflammation, contributing to a wide range of human pathologies, including inflammatory disorders, neurodegenerative conditions, and cancer. Despite this, no RIPK1 positron emission tomography (PET) ligand with significant in vivo specificity has been reported to date. In this work, we designed and synthesized a new family of dihydropyrazole-cored ligands suitable for 18F-labeling at the late stage. Among these, WL8 showed a strong binding affinity to RIPK1 (EC50 = 19.9 nM, Kd = 25 nM) and was successfully labeled with 18F in the 6-position of pyridine ring, yielding a high radiochemistry yield of 27.9 % (decay-corrected) and a high molar activity of 18.8–31.2 GBq/μmol. In in vitro autoradiography, [18F]WL8 showed some specific binding in the brain sections of rats and lipopolysaccharide (LPS) model mice. Preliminary PET studies in rat brains revealed that [18F]WL8 could efficiently penetrate the blood-brain barrier and was rapidly washed out. As anticipated, [18F]WL8 exhibited a high initial uptake (brain2min = 4.80 % ID/g) in mouse brains, followed by a rapid washout (brain60min = 0.14 % ID/g), although no clear specific binding to RIPK1 was observed. Moderate in vivo stability was noted for [18F]WL8 in mouse brains with 35.2 % of the parent fraction remaining after 30 min post-administration. Altogether, our work broadens the landscape and offers a new chemotype for RIPK1 PET ligand development.

Validation of the C-X-C chemokine receptor 3 (CXCR3) as a target for PET imaging of T cell activation

PurposeCXCR3 is expressed on activated T cells and plays a crucial role in T-cell recruitment to the tumor microenvironment (TME) during cell-based and immune checkpoint inhibitor (ICI) immunotherapy. This study utilized a 64Cu-labeled NOTA-α-CXCR3 antibody to assess CXCR3 expression in the TME and validate it as a potential T cell activation biomarker in vivo.ProceduresCXCR3+ cells infiltrating MC38 tumors (B57BL/6 mice, untreated and treated with αPD-1/αCTLA-4 ICI) were quantified using fluorescence microscopy and flow cytometry. A commercial anti-mouse CXCR3 antibody (α-CXCR3) was site-specifically conjugated with 2,2,2-(1,4,7-triazacyclononane-1,4,7-triyl)triacetic acid (NOTA) and radiolabeled with 64Cu. Saturation binding of [64Cu]Cu-NOTA-α-CXCR3 was investigated using CHO cells stably transfected with murine CXCR3. Biodistribution and PET imaging studies both at baseline and after 1 to 3 cycles of ICI, respectively, were carried out using different molar activities (10 GBq/µmol to 300 GBq/µmol) of [64Cu]Cu-NOTA-α-CXCR3.ResultsFlow cytometry analysis at baseline confirmed the presence of CXCR3 + T-cells in MC38 tumors, which was significantly increased at day five after ICI (treated 33.8 ± 17.4 vs. control 8.8 ± 6.2 CD3+CXCR3+ cells/mg). These results were qualitatively and quantitatively confirmed by immunofluorescence of tumor cryoslices. In vivo PET imaging of MC38 tumor bearing mice before, during and after ICI using [64Cu]Cu-NOTA-α-CXCR3 (Kd = 3.3 nM) revealed a strong dependence of CXCR3-specificity of tracer accumulation in secondary lymphoid organs on molar activity. At 300 GBq/µmol (1.5 µg of antibody/mouse), a specific signal was observed in lymph nodes (6.33 ± 1.25 control vs. 3.95 ± 1.23%IA/g blocking) and the spleen (6.04 ± 1.02 control vs. 3.84 ± 0.79%IA/g blocking) at 48 h p.i. Spleen-to-liver ratios indicated a time dependent systemic immune response showing a steady increase from 1.08 ± 0.19 (untreated control) to 1.54 ± 0.14 (three ICI cycles).ConclusionsThis study demonstrates the feasibility of in vivo imaging of CXCR3 upregulation under immunotherapy using antibodies. However, high molar activities and low antibody doses are essential for sensitive detection in lymph nodes and spleen. Detecting therapy-induced changes in CXCR3+ T cell numbers in tumors was challenging due to secondary antibody-related effects. Nonetheless, CXCR3 remains a promising target for imaging T cell activation, with anticipated improvements in sensitivity using alternative tracers with high affinities and favorable pharmacokinetics.

C-H Labeling with [18F]Fluoride: An Emerging Methodology in Radiochemistry.

Fluorine-18 is the most routinely employed radioisotope for positron emission tomography, a dynamic nuclear imaging modality. The radiolabeling of C-H bonds is an attractive method for installing fluorine-18 into organic molecules since it can preclude the cumbersome prefunctionalization of requisite precursors. Although electrophilic "F+" reagents (e.g., [18F]F2) are effective for C-H radiolabeling, state-of-the-art methodologies predominantly leverage high molar activity nucleophilic [18F]fluoride sources (e.g., [18F]KF) with substantial (pre)clinical advantages. Reflecting this, multiple nucleophilic C-H radiolabeling techniques of high utility have been disclosed over the past decade. However, the adoption of (pre)clinical C-H radiolabeling has been slow, and PET imaging agents are still routinely prepared via methods that, despite a high level of practicality, are limited in scope (e.g., SNAr, SN2 radiofluorinations). By addressing the drawbacks inherent to these strategies, C-H radiofluorination and radiofluoroalkylation carry the potential to complement and supersede state-of-the-art labeling methods, facilitating the expedited production of PET agents used in disease staging and drug development. In this Outlook, we showcase recent C-H labeling developments with fluorine-18 and discuss the merits, potential, and barriers to adoption in (pre)clinical settings. In addition, we highlight trends, challenges, and directions in this emerging field of study.

Scalability study on [133La]LaCl3 production with a focus on potential clinical applications

BackgroundIn recent years, targeted alpha therapy has gained importance in the clinics, and in particular, the alpha-emitter 225Ac plays a fundamental role in this clinical development. Nevertheless, depending on the chelating system no real diagnostic alternative has been established which shares similar chemical properties with this alpha-emitting radionuclide. In fact, the race to launch a diagnostic radionuclide to form a matched pair with 225Ac is still open, and 133La features attractive radiation properties to claim this place. However, in order to enable its translation into clinical use, upscaling of the production of this PET radionuclide is needed.ResultsA study on optimal irradiation parameters, separation conditions and an exhaustive product characterization was carried out. In this framework, a proton irradiation of 2 h, 60 µA and 18.7 MeV produced 133La activities of up to 10.7 GBq at end of bombardment. In addition, the performance of four different chromatographic resins were tested and two optimized purification methods presented, taking approximately 20 min with a 133La recovery efficiencies of over 98%, decay corrected. High radionuclide purity and apparent molar activity was proved, of over 99.5% and 120 GBq/µmol, respectively, at end of purification. Furthermore, quantitative complexation of PSMA-617 and mcp-M-PSMA were obtained with molar activities up to 80 GBq/µmol. In addition, both 133La-radioconjugates offered high stability in serum, of over (98.5 ± 0.3)% and (99.20 ± 0.08)%, respectively, for up to 24 h. A first dosimetry estimation was also performed and it was calculated that an 133La application for imaging with between 350 and 750 MBq would only have an effective dose of 2.1–4.4 mSv, which is comparable to that of 18F and 68Ga based radiopharmaceuticals.ConclusionsIn this article we present an overarching study on 133La production, from the radiation parameters optimization to a clinical dose estimation. Lanthanum-133 activities in the GBq range could be produced, formulated as [133La]LaCl3 with high quality regarding radiolabeling and radionuclide purity. We believe that increasing the 133La availability will further promote the development of radiopharmaceuticals based on macropa or other chelators suitable for 225Ac.Graphical abstract

Preclinical evaluation of [225Ac]Ac-crown-TATE – An alpha-emitting radiopharmaceutical for neuroendocrine tumors

BackgroundTargeted alpha therapy (TAT) of somatostatin receptor-2 (SSTR2) positive neuroendocrine tumors (NETs) involving Ac-225 ([225Ac]Ac-DOTA-TATE) has previously demonstrated improved therapeutic efficacy over conventional beta particle-emitting peptide receptor radionuclide therapy agents. DOTA-TATE requires harsh radiolabeling conditions for chelation of [225Ac]Ac3+, which can limit the achievable molar activities and thus therapeutic efficacy of such TAT treatments. Macropa-TATE was recently highlighted as a potential alternative to DOTA-TATE, owing to the mild radiolabeling conditions and high affinity toward [225Ac]Ac3+; however, elevated liver and kidney uptake were noted as a major limitation and a suitable imaging radionuclide is yet to be reported, which will be required for patient dosimetry studies and assessment of therapeutic benefit. Previously, [155Tb]Tb-crown-TATE has shown highly effective imaging of NETs in preclinical SPECT/CT studies, with high tumor uptake and low non-target accumulation; these favourable properties and the versatile coordination behavior of the crown chelator may therefore show promise for combination with Ac-225 for TAT. MethodsCrown-TATE was labeled with Ac-225, and radiochemical yield was analyzed as the function of crown-TATE concentration. LogD7.4 was measured as the indication of hydrophilicity. Free [225Ac]Ac3+ release from [225Ac]Ac-crown-TATE in human serum was studied. Biodistribution studies of [225Ac]Ac-crown-TATE in mice bearing AR42J tumors was evaluated at 1, 4, 24, 48, and 120 h, and the absorbed dose to major organs calculated. Therapy-monitoring studies with AR42J tumor bearing mice were undertaken using 30 kBq and 55 kBq doses of [225Ac]Ac-crown-TATE and compared to controls treated with PBS or crown-TATE. Results[225Ac]Ac-crown-TATE was successfully prepared with high molar activity (640 kBq/nmol), and characterized as a moderately hydrophilic radioligand (LogD7.4 = −1.355 ± 0.135). No release of bound Ac-225 was observed over 9 days in human serum. Biodistribution studies of [225Ac]Ac-crown-TATE showed good initial tumor uptake (11.1 ± 1.7% IA/g at 4 h) which was sustained up to 120 h p.i. (6.92 ± 2.03% IA/g). Dosimetry calculations showed the highest absorbed dose was delivered to the tumors. Therapy monitoring studies demonstrated significant (log-rank test, P < 0.005) improved survival in both treatment groups compared to controls. ConclusionsThis preclinical study demonstrated the therapeutic efficacy of [225Ac]Ac-crown-TATE for treatment of NETs, and highlights the potential of using crown chelator for stable chelation of Ac-225 under mild conditions.

[89Zr]ZrCl4 for direct radiolabeling of DOTA-based precursors

IntroductionZirconium-89 (89Zr) is a positron emitter with several advantages over other shorter-lived positron emission tomography (PET) compatible radiometals such as gallium-68 or copper-64. These include practically unlimited availability, extremely low cost, greatly facilitated distribution logistics, positron energy fit for medical PET imaging, and sufficiently long physical half-life to enable PET imaging at later time points for patient-specific dosimetry estimations. Despite these apparent benefits, the reception of 89Zr in the nuclear medicine community has been tepid. The driving factor for the absence of broader adaptation is mostly routed in its final formulation — [89Zr]zirconium oxalate. While serving as a suitable precursor solution for the gold standard chelator deferoxamine (DFO), [89Zr]Zr-oxalate is inaccessible for the most commonly used chelators, such as the macrocyclic DOTA, due to its pre-chelated state.Consequently, pioneering work has been conducted by multiple research groups to create oxalate-free forms of [89Zr]Zr4+, either via chemical conversion of oxalate into other counterion forms or via direct radiochemical isolation of [89Zr]ZrCl4, showing that [89Zr]Zr-DOTA complexes are possible and stable. However, this success was accompanied by challenges, including complex and labor-intensive radiochemical processing and radiolabeling procedures as well as the relatively minuscule conversion rates. Here, we report on the direct production of [89Zr]ZrCl4 avoiding oxalate and metal contaminants to enable efficient radiolabeling of DOTA constructs. MethodsWe based our direct production of [89Zr]ZrCl4 on previously reported methods and further optimized its quality by including an additional iron-removing step using the TK400 Resin. Here, we avoided using oxalic acid and effectively minimized the content of trace metal contaminants. Our two-step purification procedure was automated, and we confirmed excellent radionuclide purity, minimal trace metals content, great reactivity over time, and high specific molar activity. In addition, DOTA-based PSMA-617 and DOTAGA-based PSMA-I&T were radiolabeled to demonstrate the feasibility of direct radiolabeling and to estimate the maximum apparent specific activities. Lastly, the biodistribution of [89Zr]Zr-PSMA-617 was assessed in mice bearing PC3-PIP xenografts, and the results were compared to the previously published data. ResultsA total of 18 batches, ranging from 6.9 to 20 GBq (186 to 541 mCi), were produced. The specific molar activity for [89Zr]ZrCl4 exceeded 0.96 GBq (26 mCi) per nanomole of zirconium. The radionuclidic purity was >99 %, and the trace metals content was in the <1 ppm range. The [89Zr]ZrCl4 remained in its reactive chemical form for at least five days when stored in cyclic olefin polymer (COP) vials. Batches of 11.1 GBq (300 mCi) of [89Zr]Zr-PSMA-617 and 14.4 GBq (390 mCi) of [89Zr]Zr-PSMA-I&T, corresponding to specific activities of 11.1 MBq/μg (0.3 mCi/μg), and 14.4 MBq/μg (0.39 mCi/μg), respectively, were produced. [89Zr]Zr-PSMA-617 animal PET imaging results were in agreement with the previously published data. ConclusionIn this work, we report on a suitable application of TK400 Resin to remove iron during [89Zr]ZrCl4 radiochemical isolation. The breakthrough allows for direct radiolabeling of DOTA-based constructs with [89Zr]ZrCl4, leading to high apparent molar activities and excellent conversion rates.

Improved titanium-44 purification process for establishing a high apparent molar activity titanium-44/scandium-44 generator

44Sc-radiopharmaceuticals are gaining more interest but still lack availability. The proof of principle of a44Ti/44Sc generator, which can produce 44Sc daily, has been established but with some limitations and drawbacks. Despite recent advances, separation of 44Ti from massive quantities of scandium target material is still cumbersome. In this work, the improved radiochemical separation of 44Ti from residual scandium target material was carried out by precipitation of Sc with fluoride ions. Furthermore, two approaches were used to set up a high apparent molar activity small-scale generator. The first method relied on extraction chromatography for fine purification using a DGA resin, followed by loading of the purified 44Ti onto a ZR resin column. In the second method, 44Ti was loaded on the ZR resin directly after the precipitation step. This second method was used to set up a generator of 370 kBq and evaluate by radiolabeling. An apparent molar activity of 2 MBq/nmol was obtained for the radiolabeling with DOTA, the most common and suitable chelate for scandium. This result is comparable with previously published data on 44 m/44Sc.

Application of 99mTc-Labeled WL12 Peptides as a Tumor PD-L1-Targeted SPECT Imaging Agent: Kit Formulation, Preclinical Evaluation, and Study on the Influence of Coligands.

With the development of PD-1/PD-L1 immune checkpoint inhibitor therapy, the ability to monitor PD-L1 expression in the tumor microenvironment is important for guiding therapy. This study was performed to develop a novel radiotracer with optimal pharmacokinetic properties to reflect PD-L1 expression in vivo via single-photon emission computed tomography (SPECT) imaging. [99mTc]Tc-HYNIC-WL12-tricine/M (M = TPPTS, PDA, ISONIC, 4-PSA) complexes with high radiochemical purity (>97%) and suitable molar activity (from 100.5 GBq/μmol to 300 GBq/μmol) were prepared through a kit preparation process. All 99mTc-labeled HYNIC-WL12 radiotracers displayed good in vitro stability for 4 h. The affinity and specificity of the four radiotracers for PD-L1 were demonstrated both in vitro and in vivo. The results of biodistribution studies displayed that the pharmacokinetics of the 99mTc-HYNIC-conjugated radiotracers were significantly influenced by the coligands of the radiotracers. Among them, [99mTc]Tc-HYNIC-WL12-tricine/ISONIC exhibited the optimal pharmacokinetic properties (t1/2α = 8.55 min, t1/2β = 54.05 min), including the fastest clearance in nontarget tissues, highest tumor-to-background contrast (e.g., tumor-to-muscle ratio, tumor-to-blood ratio: 40.42 ± 1.59, 14.72 ± 2.77 at 4 h p.i., respectively), and the lowest estimated radiation absorbed dose, highlighting its potential as a clinical SPECT imaging probe for tumor PD-L1 detection.

Design and radiosynthesis of class-IIa HDAC inhibitor with high molar activity via repositioning the 18F-radiolabel

The design and radiosynthesis of [18F]NT376, a high potency inhibitor of class-IIa histone deacetylases (HDAC) is reported. We utilized a three-step radiochemical approach that led to the radiosynthesis of [18F]NT376 in a good radiochemical yield, (17.0 ± 3%, decay corrected), high radiochemical purity (> 97%) and relatively high molar activity of 185.0 GBq/µmol (> 5.0 Ci/µmol). The repositioning of the 18F-radiolabel into a phenyl ring (18F-Fluoro-aryl) of the class-IIa HDAC inhibitor avoided the shortcomings of the direct radiolabeling of the 5-trifluoromethyl-1,2,4-oxadiazole moiety that was reported by us previously and was associated with low molar activity (0.74–1.51 GBq/µmol, 20–41 mCi/µmol). This radiochemical approach could find a wider application for radiolabeling similar molecules with good radiochemical yield and high molar activity.

Development of a New Positron Emission Tomography Imaging Radioligand Targeting RIPK1 in the Brain and Characterization in Alzheimer's Disease.

Targeting receptor-interacting protein kinase 1 (RIPK1) has emerged as a promising therapeutic stratagem for neurodegenerative disorders, particularly Alzheimer's disease (AD). A positron emission tomography (PET) probe enabling brain RIPK1 imaging can provide a powerful tool to unveil the neuropathology associated with RIPK1. Herein, the development of a new PET radioligand, [11C]CNY-10 is reported, which may enable brain RIPK1 imaging. [11C]CNY-10 is radiosynthesized with a high radiochemical yield (41.8%) and molar activity (305 GBq/µmol). [11C]CNY-10 is characterized by PET imaging in rodents and a non-human primate, demonstrating good brain penetration, binding specificity, and a suitable clearance kinetic profile. It is performed autoradiography of[11C]CNY-10 in human AD and healthy control postmortem brain tissues, which shows strong radiosignal in AD brains higher than healthy controls. Subsequently, it is conducted further characterization of RIPK1 in AD using [11C]CNY-10-based PET studies in combination with immunohistochemistry leveraging the 5xFAD mouse model. It is found that AD mice revealed RIPK1 brain signal significantly higher than WT control mice and that RIPK1 is closely related to amyloid plaques in the brain. The studies enable further translational studies of [11C]CNY-10 for AD and potentially other RIPK1-related human studies.

Development of a Radiolabeled Cyclin-Dependent Kinases 4 and 6 (CDK4/6) Inhibitor for Brain and Cancer PET Imaging.

The synthesis, biochemical evaluation and radiosynthesis of a cyclin-dependent kinases 4 and 6 (CDK4/6) inhibitor and radioligand was performed. NT431, a newly synthesized 4-fluorobenzyl-abemaciclib, exhibited high potency to CDK4/6 and against four cancer cell lines with IC50 similar to that of the parent abemaciclib. We performed a two-step one-pot radiosynthesis to produce [18F]NT431 with good radiochemical yield (9.6 ± 3%, n = 3, decay uncorrected), high radiochemical purity (>95%), and high molar activity (>370 GBq/µmol (>10.0 Ci/µmol). In vitro autoradiography confirmed the specific binding of [18F]NT431 to CDK4/6 in brain tissues. Dynamic PET imaging supports that both [18F]NT431 and the parent abemaciclib crossed the BBB albeit with modest brain uptake. Therefore, we conclude that it is unlikely that NT431 or abemaciclib (FDA approved drug) can accumulate in the brain in sufficient concentrations to be potentially effective against breast cancer brain metastases or brain cancers. However, despite the modest BBB penetration, [18F]NT431 represents an important step towards the development and evaluation of a new generation of CDK4/6 inhibitors with superior BBB penetration for the treatment and visualization of CDK4/6 positive tumors in the CNS. Also, [18F]NT431 may have potential application in peripheral tumors such as breast cancer and other CDK4/6 positive tumors.

Preclinical evaluation of MC1R targeting theranostic pair [155Tb]Tb-crown-αMSH and [161Tb]Tb-crown-αMSH

BackgroundTargeted radionuclide therapy is established as a highly effective strategy for the treatment of metastatic tumors; however, the co-development of suitable imaging companions to therapy remains significant challenge. Theranostic isotopes of terbium (149Tb, 152Tb, 155Tb, 161Tb) have the potential to provide chemically identical radionuclidic pairs, which collectively encompass all modes of nuclear decay relevant to nuclear medicine. Herein, we report the first radiochemistry and preclinical studies involving 155Tb- and 161Tb-labeled crown-αMSH, a small peptide-based bioconjugate suitable for targeting melanoma. Methods155Tb was produced via proton induced spallation of Ta targets using the isotope separation and acceleration facility at TRIUMF with isotope separation on-line (ISAC/ISOL). The radiolabeling characteristics of crown-αMSH with 155Tb and/or 161Tb were evaluated by concentration-dependence radiolabeling studies, and radio-HPLC stability studies. LogD7.4 measurements were obtained for [161Tb]Tb-crown-αMSH. Competitive binding assays were undertaken to determine the inhibition constant for [natTb]Tb-crown-αMSH in B16-F10 cells. Pre-clinical biodistribution and SPECT/CT imaging studies of 155Tb and 161Tb labeled crown-αMSH were undertaken in male C57Bl/6 J mice bearing B16-F10 melanoma tumors to evaluate tumor specific uptake and imaging potential for each radionuclide. ResultsQuantitative radiolabeling of crown-αMSH with [155Tb]Tb3+ and [161Tb]Tb3+ was demonstrated under mild conditions (RT, 10 min) and low chelator concentrations; achieving high molar activities (23–29 MBq/nmol). Radio-HPLC studies showed [161Tb]Tb-crown-αMSH maintains excellent radiochemical purity in human serum, while gradual metabolic degradation is observed in mouse serum. Competitive binding assays showed the high affinity of [natTb]Tb-crown-αMSH toward MC1R. Two different methods for preparation of the [155Tb]Tb-crown-αMSH radiotracer were investigated and the impacts on the biodistribution profile in tumor bearing mice is compared. Preclinical in vivo studies of 155Tb- and 161Tb- labeled crown-αMSH were performed in parallel, in mice bearing B16-F10 tumors; where the biodistribution results showed similar tumor specific uptake (6.06–7.44 %IA/g at 2 h pi) and very low uptake in nontarget organs. These results were further corroborated through a series of single-photon emission computed tomography (SPECT) studies, with [155Tb]Tb-crown-αMSH and [161Tb]Tb-crown-αMSH showing comparable uptake profiles and excellent image contrast. ConclusionsCollectively, our studies highlight the promising characteristics of [155Tb]Tb-crown-αMSH and [161Tb]Tb-crown-αMSH as theranostic pair for nuclear imaging (155Tb) and radionuclide therapy (161Tb).

Preclinical Evaluation of 131I/18F-Labeled Covalent Small-Molecule Inhibitors for STING Status Imaging.

The stimulator of interferon genes (STING) is a vital protein to the immune surveillance of the tumor microenvironment. In this study, we develop novel inhibitor-based radioligands and evaluate their feasibility for noninvasive visualization of STING expression in tumor-bearing mice. Analogous compounds to STING inhibitors C170 and C176 were synthesized and labeled with 131I and 18F to attain [131I]I-NFIP and [18F]F-NFEP, respectively. The radiosynthesis was achieved with high radiochemical purity (>95%) and molar activity (28.56-48.89 GBq/μmol). The affinity and specificity of tracers were assessed through cell uptake and docking experiments, demonstrating that [131I]I-NFIP exhibited high specificity for STING, with a cell-based IC50 value of 7.56 nM. Small-animal PET/SPECT imaging and biodistribution studies in tumor-bearing mice models were performed to verify the tracers' pharmacokinetics and tumor-targeting capabilities (n = 3/group). SPECT imaging demonstrated that [131I]I-NFIP rapidly accumulated in the Panc02 tumor quickly at 30 min post-injection, with a tumor-to-muscle (T/M) ratio of 2.03 ± 0.30. This ratio significantly decreased in the blocking group (1.10 ± 0.14, **P < 0.01, n = 3). Furthermore, tumor uptake and the T/M ratio of [131I]I-NFIP were positively associated with STING expression. In summary, [131I]I-NFIP is the first STING-specific inhibitor-based radioligand offering the potential for visualizing STING status in tumors.

Photoredox Nucleophilic (Radio)fluorination of Alkoxyamines.

Herein, we report a photoredox nucleophilic (radio)fluorination using TEMPO-derived alkoxyamines, a class of substrates accessible in a single step from a diversity of readily available carboxylic acids, halides, alkenes, alcohols, aldehydes, boron reagents, and C-H bonds. This mild and versatile one-electron pathway affords radiolabeled aliphatic fluorides that are typically inaccessible applying conventional nucleophilic substitution technologies due to insufficient reactivity and competitive elimination. Automation of this photoredox process is also demonstrated with a user-friendly and commercially available photoredox flow reactor and radiosynthetic platform, therefore expediting access to labeled aliphatic fluorides in high molar activity (Am) for (pre)clinical evaluation.