Evaluation of the Tetrakis(3-Hydroxy-4-Pyridinone) Ligand THPN with Zirconium(IV): Thermodynamic Solution Studies, Bifunctionalization, and in Vivo Assessment of Macromolecular 89Zr-THPN-Conjugates.

Zirconium-89 (89Zr) is a suitable radionuclide for positron-emission tomography (PET) of long-circulating targeting vectors such as monoclonal antibodies (mAbs). Due to stability concerns for the most widely used 89Zr-chelating agent desferrioxamine B (DFO) in preclinical studies, alternative 89Zr-chelators are currently being developed. We recently reported on the first tetrakis(3-hydroxy-4-pyridinone) (3,4-HOPO) ligand THPN, which was identified as a promising 89Zr-chelator. In this study, we aimed to further explore this octadentate chelate in vitro and in vivo. The [ZrIV(THPN)] thermodynamic stability was quantified in solution titration studies, which revealed one of the highest formation constants reported for a zirconium chelate (log ßML 50.3(1), pM = 42.8). Solution stabilities with iron(III) were also exceptionally high and can compete with some of the strongest FeIII-chelates. A first bifunctional derivative of the octadentate ligand, p-SCN-Bn-THPN, was then produced in a multistep synthesis. To assess and compare the long-term 89Zr complex stability, bifunctional THPN, as well as the literature chelators p-SCN-Phe-DFO and p-SCN-Phe-DFO*, were conjugated to the high-molecular weight (800 kDa) polymeric carrier hyperbranched polyglycerol (HPG). The functionalized HPGs were radiolabeled with 89ZrIV, and the integrity of the radioconjugates was assessed over several days in vitro and in vivo. While all three radioconjugates remained >95% intact over 5 days in human plasma, the in vivo study in healthy mice revealed higher physiologic stability of the DFO and DFO* radiochelates over bifunctional THPN conjugates. This was evidenced by increased bone uptake of osteophilic 89ZrIV for THPN. This finding contrasts with the exceptionally high thermodynamic stability of the chelate and suggests either a kinetic or metabolic lability, or may stem from coordinative changes due to the covalent conjugation of the 89Zr-THPN radiochelate as suggested by density functional theory (DFT) calculations. These important findings inform the design of next generation 3,4-HOPO chelates with the aim of improving the physiologic stability. This study furthermore demonstrates how HPG can be used as a robust carrier tool to assess and compare the long-term in vivo stability of radiochelates.

H4octox: Versatile Bimodal Octadentate Acyclic Chelating Ligand for Medicinal Inorganic Chemistry.

H4octox, a versatile new octadentate acyclic chelating ligand, has been investigated as an alternative to the acyclic DTPA and the macrocyclic DOTA for trivalent metal ions useful in diagnostic medical imaging or therapeutic applications (Y3+, In3+, La3+, Gd3+, Lu3+). The synthesis of H4octox is straightforward in less steps and thus more economical than those of most previously reported chelators. Complex formation equilibria in the presence of Y3+, In3+, La3+, Gd3+, and Lu3+ revealed fast chelation and high metal-sequestering capacity. Quantitative labeling with 111In3+ was achieved within 15 min at room temperature at ligand concentrations as low as 10-7 M, exactly the properties required for the development of kit-based radiopharmaceuticals. In vitro serum stability studies and in vivo SPECT imaging confirmed excellent complex stability of [111In(octox)]-. Moreover, it is more lipophilic than most of the multidentate carboxylate- or picolinate-based chelators; it therefore shows more liver clearance and provides a complementary choice in the design of metal-based pharmaceuticals and in the tuning of their pharmacokinetic properties. Finally, H4octox showed a large fluorescence enhancement upon complexation with different metals, in particular, with Y3+ and Lu3+, which could be useful for non-radioactive fluorescent stability and cell studies as well as bimodal imaging. Excellent in vitro stability of [Y(octox)]- against transferrin and Fe3+ was confirmed employing this fluorescence.

Development of Novel Monoamine Oxidase-B (MAO-B) Inhibitors with Reduced Blood-Brain Barrier Permeability for the Potential Management of Noncentral Nervous System (CNS) Diseases.

Studies indicate that MAO-B is induced in peripheral inflammatory diseases. To target peripheral tissues using MAO-B inhibitors that do not permeate the blood-brain barrier (BBB) the MAO-B-selective inhibitor deprenyl was remodeled by replacing the terminal acetylene with a CO2H function, and incorporating a para-OCH2Ar motif (compounds 10a-s). Further, in compound 32 the C-2 side chain corresponded to CH2CN. In vitro, 10c, 10j, 10k, and 32 were identified as potent reversible MAO-B inhibitors, and all four compounds were more stable than deprenyl in plasma, liver microsomal, and hepatocyte stability assays. In vivo, they demonstrated greater plasma bioavailability. Assessment of in vitro BBB permeability showed that compound 10k is a P-glycoprotein (P-gp) substrate and 10j displayed mild interaction. Importantly, compounds 10c, 10j, 10k, and 32 displayed significantly reduced BBB permeability after intravenous, subcutaneous, and oral administration. These polar MAO-B inhibitors are pertinent leads for evaluation of efficacy in noncentral nervous system (CNS) inflammatory disease models.

A new tetrapodal 3-hydroxy-4-pyridinone ligand for complexation of 89zirconium for positron emission tomography (PET) imaging.

Zirconium-89 (89Zr) is an ideal radiometal isotope for antibody-based positron emission tomography (immunoPET) as its physical half-life (3.27 days) is a good match with the biological half-life of larger molecular weight targeting molecules, such as antibodies (3-4 days), and its positron emission (BR = 100% EC/ß+, Eß+,avg = 395.5 keV) is suited for high resolution PET imaging. Concerns over the in vivo stability of the most commonly used 89Zr-chelator, desferrioxamine B (DFO), have spurred efforts into the development of alternative 89Zr-chelators that withstand the release of osteophilic 89Zr4+. Herein we report the new chelator 1,3-propanediamine-N,N,N',N'-tetrakis[(2-(aminomethyl)-3-hydroxy-1,6-dimethyl-4(1H)-pyridinone)acetamide] (THPN) based on four 3-hydroxy-4-pyridinone (3,4-HOPO) coordinating groups, as a potentially superior chelator over DFO. THPN has been demonstrated to quantitatively form a monometallic complex with Zr4+ within 10 min at ambient temperature at as low as 10-6 M concentrations of the chelator. The resulting complexes were studied in vitro and in vivo. The 89Zr-THPN complex was stable in serum and outperformed the 89Zr-DFO complex in a direct transchelation challenge. Healthy mice excreted 89Zr-THPN rapidly without signs of demetalation or residual organ uptake. This renders THPN as a promising alternative to DFO and introduces the first octadentate 3,4-HOPO chelator to the field.

Characterization of alendronic- and undecylenic acid coated magnetic nanoparticles for the targeted delivery of rosiglitazone to subcutaneous adipose tissue.

Obesity is a state of positive energy balance where excess white adipose tissue accumulates to the detriment of metabolic health. Improving adipocyte function with systemic administration of thiazolidinediones (TZDs) improves metabolic outcomes in obesity, however TZD use is limited clinically due to undesirable side effects. Here we evaluate magnetic nanoparticles (MNPs) as a tool to target rosiglitazone (Rosi) specifically to adipose tissue. Results show Rosi can be adsorbed to MNPs (Rosi-MNPs) with hydrophobic coatings for which we present binding and release kinetics. Rosi adsorbed to MNPs retained the ability to induce PPARγ target gene expression in cells. Biodistribution analysis of radiolabeled Rosi-MNPs revealed a fat-implanted magnet significantly enhanced localization of Rosi to the targeted adipose tissue when administered by subcutaneous injection to obese mice. We propose MNPs for targeted delivery of anti-diabetic agents to superficially located subcutaneous adipose tissue.