Synthesis and preliminary in vitro evaluation of DOTA-Tenatumomab conjugates for theranostic applications in tenascin expressing tumors

Tenatumomab is an anti-tenascin murine monoclonal antibody previously used in clinical trials for delivering radionuclides to tumors by both pre-targeting (biotinylated Tenatumomab within PAGRIT) and direct ¹³¹Iodine labeling approaches. Here we present the synthesis and in vitro characterization of three Tenatumomab conjugates to bifunctional chelating agents (NHS-DOTA, NCS-DOTA and NCS-DTPA). Results indicate ST8198AA1 (Tenatumomab-DOTAMA, derived by conjugation of NHS-DOTA), as the most promising candidate in terms of conjugation rate and yield, stability, antigen immunoreactivity and affinity. Labeling efficiency of the different chelators was investigated with a panel of cold metals indicating DOTAMA as the best chelator. Labeling of Tenatumomab-DOTAMA was then optimized with several metals and stability performed confirms suitability of this conjugate for further development. ST8198AA1 represents an improvement of the previous antibody forms because the labeling with radionuclides like ¹⁷⁷Lu or ⁶⁴Cu would allow theranostic applications in patients bearing tenascin expressing tumors.     

Novel inhibitors of advanced glycation endproducts

A number of natural or synthetic compounds as AGE inhibitors have been proposed, discovered or currently being advanced by others and us. We have identified two new classes of aromatic compounds; aryl- (and heterocyclic) ureido and aryl (and heterocyclic) carboxamido phenoxyisobutyric acids, and benzoic acid derivatives and related compounds, as potential inhibitors of glycation and AGE formation. Some of these novel compounds also showed "AGE-breaking" activities in vitro. Current evidence is that chelation of transition metals and/or trapping or indirect inhibition of formation of reactive carbonyl compounds are involved in the mechanisms of action of these novel AGE inhibitors and breakers. Here, we review the inhibitors of glycation and AGE-breakers published to date and present the results of our in vitro and in vivo investigations on a number of these novel AGE inhibitors. These AGE-inhibitors and AGE-breakers may find therapeutic use in the treatment of diseases that AGE formation and accumulation may be responsible for their pathogenesis such as diabetes, Alzheimer's, rheumatoid arthritis, and atherosclerosis.     

Mechanisms of EDDHA effects on the promotion of floral induction in the long-day plant Lemna minor (L.)

EDDHA added in an optimal concentration (20.5 mumol.L-1) to a modified Pirson-Seidel nutrient solution induces flowering in some clones of the species Lemna minor, Lemna gibba and Spirodela polyrrhiza, which in the absence of EDDHA in the same nutrient solution do not flower. By adding EDDHA (20.5 mumol.L-1), floral induction under LD conditions is optimally promoted in the long-day (LD) species Lemna minor. After adding EDDHA to the nutrient solution, before floral induction and during flowering, Zn, Mn and Cu content is significantly increased in plants. Zn-EDDHA (0.86 mumol.L-1), Mn-EDDHA (1.51 mumol.L-1) and Cu-EDDHA (0.12 mumol.L-1), when used individually, greatly promote flowering under LD conditions as compared to flowering in the same nutrient solution with an equivalent quantity of Zn, Mn or Cu in the nonchelate form. If, on the other hand, Zn-EDDHA and Mn-EDDHA are added to the nutrient solution together (instead of Zn and Mn in nonchelate form), their effect on the promotion of flowering is less than in the case of their individual use. This shows that there is antagonism between Zn-EDDHA and Mn-EDDHA that is eliminated by adding EDDHA to the nutrient solution. We obtained the highest percentage of flowering plants (i.e. 74%) if we added EDDHA (20.5 mumol.L-1) to the nutrient solution containing Mn, Zn and Cu in chelate form. 74% of flowering plants actually means that flowering was achieved in all physiologically mature plants. Our results show that EDDHA promotes floral induction in Lemna minor under LD conditions, especially through chelating Zn, Mn and Cu, and, in addition, through eliminating the antagonism between Mn and Zn chelates EDDHA. Zn-EDDHA (0.86 mumol.L-1) also promote floral differentiation, especially cell division of microspore mother cells into dyads and those into microspore tetrads, which can be seen in microphotographs. When investigating possible pathways through which Mn-EDDHA, Zn-EDDHA and Cu-EDDHA promote flowering, we studied the effects of various concentrations of IAA and sucrose added to the nutrient solution as well. The results support the hypothesis that one of the possible pathways in which Mn-EDDHA promotes floral induction is through auxin oxidase, whereas Zn-EDDHA and Cu-EDDHA probably promote it through the enhancement of the photosynthesis and synthesis of sucrose.     

Enzymes and chelating agent in cotton pretreatment

Desized cotton fabric and cotton seed-coat fragments (impurities) have been treated with commercial cellulase (Celluclast 1.5 L), hemicellulase–pectinase (Viscozyme 120 L) and xylanase (Pulpzyme HC) enzymes. Seed-coat fragments hydrolyzed much faster than the cotton fabric itself. This relative difference in hydrolysis rates makes possible a direct enzymatic removal of seed-coat fragments from desized cotton fabric. Addition of chelating agents such as ethylenediamine-tetra-acetic acid (EDTA) markedly enhanced the directed enzyme action. Pretreatments carried out in acidic solution at pH 5 increased the lightness of seed-coat fragments, contrary to the samples treated in neutral medium at pH 7. Alkaline scouring resulted in darker seed-coat fragments except for the samples pretreated with Pulpzyme HC plus EDTA. This effect is similar to that observed in the biobleaching process in pulp and paper industry.     

Preparation of nitrophorin 7(Δ1–3) from Rhodnius prolixus without start–methionine using recombinant expression in Escherichia coli

The heterologous recombinant expression of proteins in Escherichia coli without start–methionine is a common problem. The nitrophorin 7 heme properties and function strongly depend on the accurate N-terminal amino acid sequence. Leading protein expression into the periplasm by fusion with the leader peptide pelB yields functional protein; however, the folded protein sticks to the cell debris. Therefore, the periplasmic fraction was dissolved in guanidinium chloride and folded by a drop-in method. Separation from impurities including residual pelB–nitrophorin 7 required establishing an unconventional chromatographic technique using calcium-loaded Chelating Sepharose as cation exchanger and elution by a linear CaCl₂ gradient.     

Efficient synthesis of new tetradentate ligands with potential applications for 64Cu PET-imaging

We wish to report the synthesis of new tetradentate ligands in less than 3 h in good to excellent yields from a commercially available compound using microwave-assisted technology. First tests of complexation showed a high ability of these ligands to chelate ⁶⁴Cu(II) in very diluted medium. These new systems have the potential to be used for nuclear medicine and particularly for PET-imaging.     

Towards a new attenuating compound: a potentiometric, spectrophotometric and NMR equilibrium study on Fe(III), Al(III) and a new tetradentate mixed bisphosphonate-hydroxypyridinonate ligand

Coordination properties toward Fe(III) and Al(III) of a mixed bisphosphonate-hydroxypyridinonate ligand are presented. Potentiometric, spectrophotometric and NMR results allowed to conclude that Fe(III) and Al(III) coordination takes place on the pyridinone moiety. The high steric hindrance prevents the possibility of simultaneous coordination of both groups to the same metal ion. Quantum mechanical calculations confirm this finding allowing to determine the minimal length of the linker necessary for a stable conformation of complexes in which Fe(III) is coordinated both by pyridinone and bisphosphonate groups.     

Bifunctional 3-hydroxy-4-pyridinone derivatives as potential pharmaceuticals: synthesis, complexation with Fe(III), Al(III) and Ga(III) and in vivo evaluation with 67Ga

A series of extra-functionalized 3-hydroxy-4-pyridinone chelators of hard metal ions, containing different side-chains with peptidomimetic groups, was studied to assess the effect of those groups on the physico-chemical properties, the metal-chelating affinity and the in vivo behaviour of the compounds, in view of their potential pharmaceutical applications. Besides the synthesis of the chelators, the study of their properties in aqueous solution alone and in the presence of M ³⁺ (M = Fe, Ga and Al) was performed by potentiometric/spectroscopic techniques. The octanol/water partition coefficient values of these hydroxypyridinone derivatives cover ca. 3 orders of magnitude (1.1 > log P > −2). They all form very stable tris-chelated M(III) complexes, the pFe and pGa values ranging up to five orders of magnitude. The in vivo studies showed the effect of the ligands on the biodistribution of ⁶⁷Ga citrate and also of ⁶⁷Ga-complexes in mice, in view of the potential use of the ligands or complexes as metal decorporating or as imaging agents, respectively. Although almost all these peptidomimetic hydroxypyridinone derivatives present very rapid clearance rate from most organs, the L-ornithine derivative (H₂L⁹) shows to be superior to the others and as good as Deferiprone as metal decontaminant of Ga. Concerning the ⁶⁷Ga complexes, the benzyl-propylamine (H₂L³) shows considerable bone retention, thus suggesting its potential application as imaging agent.Electronic Supplementary Material Supplementary material is available for this article at An erratum to this article can be found at     

Transient Phytoextraction Agents: Establishing Criteria for the Use of Chelants in Phytoextraction of Recalcitrant Metals

The phytoremediation of recalcitrant metals such as lead and uranium rely on soil amendments to enhance metal availability within the rhizosphere. Because these amendments may persist in soils, agents that not only biodegrade rapidly but also are effective in triggering metal uptake in plants are needed for metals phytoextraction to be considered as an accepted practice. In this study, several biodegradable organic acids and chelating agents were assessed to determine if these amendments can be used in an effective manner, and if their activity and use is consistent with a proposed class of soil amendments for phytoextraction, here termed transient phytoextraction agents (TPAs). A TPA is proposed as an agent that would exhibit both effectiveness in triggering plant accumulation of the targeted metal while minimizing the risk of migration through rapid degradation or inactivation of the soluble complex. Eleven candidate TPAs (acetic acid, ascorbic acid, citric acid, malic acid, oxalic acid, succinic acid, ethylenediaminedisuccinic acid, dicarboxymethylglutamic acid, nitrilotriacetic acid, BayPure® CX 100, and the siderophore desferrioxamine B) were tested in batch studies to evaluate their complexation behavior using contaminated soils, with uranium and lead as the target metals. A growth chamber study was then conducted with Brassica juncea (Indian mustard), Helianthus annuus (sunflower), and Festuca arundinacea (tall fescue) grown in a lead-contaminated soil that was treated with the candidate TPAs to assess phytoextraction effectiveness. For the soils tested, citric acid, oxalic acid, and succinic acid were found to be effective complexing agents for uranium phytoextraction, whereas Baypure® CX 100 and citric acid exhibited effectiveness for lead phytoextraction.     

New tripodal hydroxypyridinone based chelating agents for Fe(III), Al(III) and Ga(III): Synthesis, physico-chemical properties and bioevaluation

Two new tris-hydroxypyridinone based compounds (KEMPPr(3,4-HP)₃ and KEMPBu(3,4-HP)₃) have been developed and studied as strong sequestering agents for iron and the group III of metal ions, aimed as potential pharmacological applications on metal-chelation therapy. Their structure is based on the KEMP acid scaffold to which three 3-hydroxy-4-pyridinone chelating moieties are attached via two different size spacers. After the preparation and characterization of the compounds their physico-chemical properties were studied, in relation with their metal binding affinity and lipophilicity. The KEMPPr(3,4-HP)₃ ligand was also bioassayed to evaluate its in vivo metal sequestering capacity from most organs using an animal model overload with ⁶⁷Ga. These studies showed that, for both in solution and in vivo conditions, the compounds have higher metal chelating efficacy than Deferriprone, the commercially available iron chelator in medical application, thus some perspectives are envisaged as potential pharmaceutical drug candidates for chelating therapy.