Monooxorhenium(V) complexes with 222-N2S2 MAMA ligands for bifunctional chelator agents: Syntheses and preliminary in vivo evaluation.

INTRODUCTION: Targeted radiotherapy using the bifunctional chelate approach with 186/188Re(V) is challenging because of the susceptibility of monooxorhenium(V)-based complexes to oxidize in vivo at high dilution. A monoamine-monoamide dithiol (MAMA)-based bifunctional chelating agent was evaluated with both rhenium and technetium to determine its utility for in vivo applications. METHODS: A 222-MAMA chelator, 222-MAMA(N-6-Ahx-OEt) bifunctional chelator, and 222-MAMA(N-6-Ahx-BBN(7-14)NH2) were synthesized, complexed with rhenium, radiolabeled with 99mTc and 186Re (carrier added and no carrier added), and evaluated in initial biological distribution studies. RESULTS: An IC50 value of 2.0±0.7nM for natReO-222-MAMA(N-6-Ahx-BBN(7-14)NH2) compared to [125I]-Tyr4-BBN(NH2) was determined through competitive cell binding assays with PC-3 tumor cells. In vivo evaluation of the no-carrier added 99mTc-222-N2S2(N-6-Ahx-BBN(7-14)NH2) complex showed little gastric uptake and blockable pancreatic uptake in normal mice. CONCLUSIONS: The 186ReO-222-N2S2(N-6-Ahx-BBN(7-14)NH2) complex showed stability in biological media, which indicates that the 222-N2S2 chelator is appropriate for chelating 186/188Re in radiopharmaceuticals involving peptides. Additionally, the in vitro cell studies showed that the ReO-222-N2S2(N-6-Ahx-BBN(7-14)NH2) complex (macroscopically) bound to PC3-tumor cell surface receptors with high affinity. The 99mTc analog was stable in vivo and exhibited pancreatic uptake in mice that was blockable, indicating BB2r targeting.

Synthesis and evaluation of an (18)F-labeled pyrimidine-pyridine amine for targeting CXCR4 receptors in gliomas.

INTRODUCTION: Chemokine receptor-4 (CXCR4, fusin, CD184) is expressed on several tissues involved in immune regulation and is upregulated in many diseases including malignant gliomas. A radiolabeled small molecule that readily crosses the blood-brain barrier can aid in identifying CXCR4-expressing gliomas and monitoring CXCR4-targeted therapy. In the current work, we have synthesized and evaluated an [(18)F]-labeled small molecule based on a pyrimidine-pyridine amine for its ability to target CXCR4. EXPERIMENTAL: The nonradioactive standards and the nitro precursor used in this study were prepared using established methods. An HPLC method was developed to separate the nitro-precursor from the nonradioactive standard and radioactive product. The nitro-precursor was radiolabeled with (18)F under inert, anhydrous conditions using the [(18)F]-kryptofix 2.2.2 complex to form the desired N-(4-(((6-[(18)F]fluoropyridin-2-yl)amino)methyl)benzyl)pyrimidin-2-amine ([(18)F]-3). The purified radiolabeled compound was used in serum stability, partition coefficient, cellular uptake, and in vivo cancer targeting studies. RESULTS: [(18)F]-3 was synthesized in 4-10% decay-corrected yield (to start of synthesis). [(18)F]-3 (tR ≈ 27 min) was separated from the precursor (tR ≈ 30 min) using a pentafluorophenyl column with an isocratic solvent system. [(18)F]-3 displayed acceptable serum stability over 2 h. The amount of [(18)F]-3 bound to the plasma proteins was determined to be > 97%. The partition coefficient (LogD7.4) is 1.4 ± 0.5. Competitive in vitro inhibition indicated 3 does not inhibit uptake of (67)Ga-pentixafor. Cell culture media incubation and ex vivo urine analysis indicate rapid metabolism of [(18)F]-3 into hydrophilic metabolites. Thus, in vitro uptake of [(18)F]-3 in CXCR4 overexpressing U87 cells (U87 CXCR4) and U87 WT indicated no specific binding. In vivo studies in mice bearing U87 CXCR4 and U87 WT tumors on the left and right shoulders were carried out using [(18)F]-3 and (68)Ga-pentixafor on consecutive days. The CXCR4 positive tumor was clearly visualized in the PET study using (68)Ga-pentixafor, but not with [(18)F]-3. CONCLUSIONS: We have successfully synthesized both a radiolabeled analog to previously reported CXCR4-targeting molecules and a nitro precursor. Our in vitro and in vivo studies indicate that [(18)F]-3 is rapidly metabolized and, therefore, does not target CXCR4-expressing tumors. Optimization of the structure to improve the in vivo (and in vitro) stability, binding, and solubility could lead to an appropriate CXCR4-targeted radiodiagnositic molecule.

PET Imaging of Extracellular pH in Tumors with (64)Cu- and (18)F-Labeled pHLIP Peptides: A Structure-Activity Optimization Study.

pH (low) insertion peptides (pHLIP peptides) target acidic extracellular environments in vivo due to pH-dependent cellular membrane insertion. Two variants (Var3 and Var7) and wild-type (WT) pHLIP peptides have shown promise for in vivo imaging of breast cancer. Two positron emitting radionuclides ((64)Cu and (18)F) were used to label the NOTA- and NO2A-derivatized Var3, Var7, and WT peptides for in vivo biodistribution studies in 4T1 orthotopic tumor-bearing BALB/c mice. All of the constructs were radiolabeled with (64)Cu or [(18)F]-AlF in good yield. The in vivo biodistribution of the 12 constructs in 4T1 orthotopic allografted female BALB/c mice indicated that NO2A-cysVar3, radiolabeled with either (18)F (4T1 uptake; 8.9 ± 1.7%ID/g at 4 h p.i.) or (64)Cu (4T1 uptake; 8.2 ± 0.9%ID/g at 4 h p.i. and 19.2 ± 1.8% ID/g at 24 h p.i.), shows the most promise for clinical translation. Additional studies to investigate other tumor models (melanoma, prostate, and brain tumor models) indicated the universality of tumor targeting of these tracers. From this study, future clinical translation will focus on (18)F- or (64)Cu-labeled NO2A-cysVar3.

OPT3 is a component of the iron-signaling network between leaves and roots and misregulation of OPT3 leads to an over-accumulation of cadmium in seeds.

Plants and seeds are the main dietary sources of zinc, iron, manganese, and copper, but are also the main entry point for toxic elements such as cadmium into the food chain. We report here that an Arabidopsis oligopeptide transporter mutant, opt3-2, over-accumulates cadmium (Cd) in seeds and roots but, unexpectedly, under-accumulates Cd in leaves. The cadmium distribution in opt3-2 differs from iron, zinc, and manganese, suggesting a metal-specific mechanism for metal partitioning within the plant. The opt3-2 mutant constitutively up-regulates the Fe/Zn/Cd transporter IRT1 and FRO2 in roots, indicative of an iron-deficiency response. No genetic mutants that impair the shoot-to-root signaling of iron status in leaves have been identified. Interestingly, shoot-specific expression of OPT3 rescues the Cd sensitivity and complements the aberrant expression of IRT1 in opt3-2 roots, suggesting that OPT3 is required to relay the iron status from leaves to roots. OPT3 expression was found in the vasculature with preferential expression in the phloem at the plasma membrane. Using radioisotope experiments, we found that mobilization of Fe from leaves is severely affected in opt3-2, suggesting that Fe mobilization out of leaves is required for proper trace-metal homeostasis. When expressed in yeast, OPT3 does not localize to the plasma membrane, precluding the identification of the OPT3 substrate. Our in planta results show that OPT3 is important for leaf phloem-loading of iron and plays a key role regulating Fe, Zn, and Cd distribution within the plant. Furthermore, ferric chelate reductase activity analyses provide evidence that iron is not the sole signal transferred from leaves to roots in leaf iron status signaling.

Chemical Kinetics Laboratory Discussion Worksheet.

A laboratory discussion worksheet and its answer key provide instructors and students a discussion model to further the students' understanding of chemical kinetics. This discussion worksheet includes a section for students to augment their previous knowledge about chemical kinetics measurements, an initial check on students' understanding of basic concepts, a group participation model where students work on solving complex-conceptual problems, and a conclusion to help students connect this discussion to their laboratory or lecture class. Additionally, the worksheet has a detailed solution to a more advanced problem to help students understand how the concepts they have put together relate to problems they will encounter during later formal assessments.

Method and Basis Set Analysis of Oxorhenium(V) Complexes for Theoretical Calculations.

A variety of method and basis set combinations has been evaluated for monooxorhenium(V) complexes with N, O, P, S, Cl, and Se donor atoms. The geometries and energies obtained are compared to both high-level computations and literature structures. These calculations show that the PBE0 method outperforms the B3LYP method with respect to both structure and energetics. The combination of 6-31G** basis set on the nonmetal atoms and LANL2TZ effective core potential on the rhenium center gives reliable equilibrium structures with minimal computational resources for both model and literature compounds. Single-point energy calculations at the PBE0/LANL2TZ,6-311+G* level of theory are recommended for energetics.