Synthesis, Radiolabeling, and Characterization of Plasma Protein-Binding Ligands: Potential Tools for Modulation of the Pharmacokinetic Properties of (Radio)Pharmaceuticals

The development of (radio)­pharmaceuticals with favorable pharmacokinetic profiles is crucial for allowing the optimization of the imaging or therapeutic potential and the minimization of undesired side effects. The aim of this study was, therefore, to evaluate and compare three different plasma protein binders (PPB-01, PPB-02, and PPB-03) that are potentially useful in combination with (radio)­pharmaceuticals to enhance their half-life in the blood. The entities were functionalized with a 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) chelator via a l-lysine and β-alanine linker moiety using solid-phase peptide chemistry and labeled with ¹⁷⁷Lu (<i>T</i>_1/2 = 6.65 days), a clinically established radiometal. The binding capacities of these radioligands and ¹⁷⁷Lu–DOTA were evaluated using human plasma and solutions of human serum albumin (HSA), human α₁-acid glycoprotein (α₁-AGP), and human transthyretin (hTTR) by applying an ultrafiltration assay. ¹⁷⁷Lu–DOTA–PPB-01 and ¹⁷⁷Lu–DOTA–PPB-02 bound to a high and moderate extent to human plasma proteins (>90% and ∼70%, respectively), whereas the binding to hTTR was considered negligible (<10%). ¹⁷⁷Lu–DOTA–PPB-03 showed almost complete binding to human plasma proteins (>90%) with a high fraction bound to hTTR (∼50%). Plasma protein binding of the ¹⁷⁷Lu–DOTA complex, which was used as a control, was not observed (<1%). ¹⁷⁷Lu–DOTA–PPB-01 and ¹⁷⁷Lu–DOTA–PPB-02 were both displaced (>80%) from HSA by ibuprofen, specific for Sudlow’s binding site II and coherent with the aromatic structures, and >80% by their respective binding entities. ¹⁷⁷Lu–DOTA–PPB-03 was displaced from hTTR by the site-marker l-thyroxine (>60%) and by its binding entity PPB-03* (>80%). All three radioligands were investigated with regard to the in vivo blood clearance in normal mice. ¹⁷⁷Lu–DOTA–PPB-01 showed the slowest blood clearance (<i>T</i>_1/2,β: >15 h) followed by ¹⁷⁷Lu–DOTA–PPB-03 (<i>T</i>_1/2,β: ∼2.33 h) and ¹⁷⁷Lu–DOTA–PPB-02 (<i>T</i>_1/2,β: ∼1.14 h), which was excreted relatively fast. Our results confirmed the high affinity of the 4-(4-iodophenyl)-butyric acid entity (PPB-01) to plasma proteins, while replacement of the halogen by an ethynyl entity (PPB-02) reduced the plasma protein binding significantly. An attractive approach is the application of the transthyretin binder (PPB-03), which shows high affinity to hTTR. Future studies in our laboratory will be focused on the application of these binding entities in combination with clinically relevant targeting agents for diagnostic and therapeutic purposes in nuclear medicine

Reduced ¹⁸F‑Folate Conjugates as a New Class of PET Tracers for Folate Receptor Imaging

5-Methyltetrahydrofolate (5-MTHF), a reduced folate form, is the biologically active folate involved in many different metabolic processes. To date, there are no studies available in the literature on ¹⁸F-labeled 6<i>S</i>- and 6<i>R</i>-5-MTHF radiotracers for imaging folate receptor (FR)-α-positive tissues. Therefore, the goal of this study was to synthesize four ¹⁸F-labeled 5-MTHF derivatives conjugated at either the α- or γ-carboxylic functionality of glutamate and to assess their suitability for FR-targeting. Organic syntheses of the precursors and the four reference compounds, namely, 6<i>S</i>-α, 6<i>S</i>-γ, 6<i>R</i>-α, and 6<i>R</i>-γ-click-fluoroethyl-5-MTHF, were carried out in low to moderate overall chemical yields. The radiosyntheses of the α- and γ-conjugated ¹⁸F-labeled folate derivatives were accomplished in approximately 100 min, low radiochemical yields (1–7% d.c.) and high molar activities (139–245 GBq/μmol). Radiochemically pure tracers were obtained after the addition of a mixture of antioxidants consisting of sodium ascorbate and l-cysteine. <i>In vitro</i>, all four 5-MTHF conjugates showed similar binding affinities to FR-α (IC₅₀ = 17.7–24.0 nM), whereas folic acid showed a significantly higher binding affinity to the FR-α. Cell uptake and internalization experiments with KB cells demonstrated specific uptake and internalization of the radiofolate conjugates. Metabolite studies in mice revealed high <i>in vivo</i> stability of the radiotracers in mice. Biodistribution and positron emission tomography (PET) imaging studies in FR-positive KB tumor-bearing mice demonstrated that the 6<i>S</i>- and 6<i>R</i>-5-MTHF conjugates exhibited a different accumulation pattern in various organs including the kidneys and the liver, whereas no significant differences in radioactivity accumulation in the kidneys and the liver were found for both the α- and γ-conjugated diastereoisomers. Despite the considerably lower binding affinities of the 5-MTHF derivatives compared to the corresponding folic acid conjugates similar high KB tumor uptake was observed for all the folate conjugates investigated (8–11% IA/g). Based on these results, we conclude that ¹⁸F-labeled 5-MTHF conjugates are a promising new class of radiotracers for targeting FR-positive tumor tissues