18 F-Fluorination of a supported 2-(aryl-di-tert-butylsilyl)-N-methyl-imidazole for indirect 18 F-labeling of a VH H single-variable domain.

Anchoring an imidazole-di-tert-butyl-arylsilane possessing an azido group to a polystyrene resin provided a heterogeneous precursor that was radiolabeled easily using aqueous [18 F]fluoride. After optimizing the conditions (i.e., using DMSO as solvent and heating at 160°C for 15 min), the desired [18 F]fluorosilane was obtained in 24% radiochemical yield (RCY) and 78% radiochemical purity (RCP) using solid-phase extraction as sole purification. Then, this compound was conjugated by strain-promoted alkyne-azide cycloaddition to a model single-variable domain possessing a cyclooctyne tag, yielding to the desired 18 F-labeled bioconjugate in 2% RCY and >95% RCP after purification by a size exclusion chromatography.

Biosynthesis of [14 C]-11-De-O-Methyltomaymycin, a Precursor of Radiolabelled Antibody Drug Conjugates.

Antibody drug conjugates (ADCs) are one of the most promising technologies to treat cancer as they combine the specificity of an antibody with the high potency of a cytotoxic molecule such as tomaymycin derivatives, which are DNA-interactive antitumor antibiotics previously isolated from bacterial broth. The multistep chemical synthesis of some tomaymycin derivatives is complicated because their structures contain a reactive imine bond. Therefore, we turned to biosynthesis to obtain 14 C radiolabelled tomaymycin derivative to support ADME studies. Following Hurley's work (J. Antibiotics 1977, 30, 349-370; Antimicrob. Agents Chemother. 1979, 15, 42-45; Acc. Chem. Res. 1980, 13, 263-269), the 14 C radiolabel was incorporated efficiently in one step from radiolabelled tyrosine using the strain Streptomyces sp. FH6421. This process has been further optimized by using anthranilic acid as radiolabelled precursor, leading to one of the highest incorporation levels of radiochemical precursors published to date. This biosynthetic strategy is the fastest way to access such radiolabelled precursors.

Investigation of new biomarkers of kidney injury in renal transplant recipients undergoing graft biopsy.

AIM: Urinary and blood kidney biomarkers (BM) remain insufficient for early kidney injury detection. We aimed to compare new kidney BM with histopathological data in kidney allograft recipients. METHODS: Blood and urine samples were collected from consecutive adult patients just before graft biopsy. All kidney samples were classified according to the Banff 2007 classification. The diagnostic performance of 16 new BM was compared to those of urinary proteins, blood urea nitrogen, eGFR, and serum creatinine to identify histopathological groups. RESULTS: Two hundred and twenty-three patients were analyzed. Microalbuminuria and urinary proteins performed well to discriminate glomerular injury from slightly modified renal parenchyma (SMRP). Urinary neutrophil gelatinase-associated lipocalin (NGAL) had the best performance relative to SMRP (AUROC .93) for acute tubular necrosis (ATN) diagnosis. Other BM had a slightly lower AUROC (.89). For the comparison of ATN to acute rejection, several new urinary BM (NGAL, cystatin C, MCP1) and classical BM (eGFR, serum creatinine) gave similar AUROC values (from .80 to .85). Urinary NGAL values in patients with ATN were 10-time higher than those with acute rejection (P=.0004). CONCLUSION: The new BM did not outperform classical BM in the context of renal transplantation. Urinary NGAL may be useful for distinguishing between ATN and acute rejection.

The first radiosynthesis of 2-amino-5-[18F]fluoropyridines via a "minimalist" radiofluorination/palladium-catalyzed amination sequence from anisyl(2-bromopyridinyl)iodonium triflate.

The synthesis of 2-amino-5-[18F]fluoropyridines was achieved in 8-85% yields by palladium-catalyzed reaction of 2-bromo-5-[18F]fluoropyridine with piperidine, dimethylamine, butylamine, methylpiperazine, benzylamine, aniline and 3-aminopyridine. 2-Bromo-5-[18F]fluoropyridine was obtained by radiofluorination of anisyl(2-bromopyridinyl-5)iodonium triflate (88% yield). The radiofluorination step was performed under "minimalist" conditions to guarantee a successful subsequent amination reaction.

Last-step 18F-fluorination of supported 2-(aryl-di-tert-butylsilyl)-N-methyl-imidazole conjugates for applications in positron emission tomography.

Aiming for potential applications in positron emission tomography, fully automated productions of 18F-labelled bioconjugates were achieved using heterogenous precursors obtained by anchoring imidazole-di-tert-butyl-arylsilanes to a polystyrene resin. The reactions were performed using either "batch" or "flow" procedures, avoiding both the time-consuming azeotropic drying and HPLC purifications usually required.

High brain distribution of a new central nervous system drug candidate despite its P-glycoprotein-mediated efflux at the mouse blood-brain barrier.

Efficacy of drugs aimed at treating central nervous system (CNS) disorders rely partly on their ability to cross the cerebral endothelium, also called the blood-brain barrier (BBB), which constitutes the main interface modulating exchanges of compounds between the brain and blood. In this work, we used both, conventional pharmacokinetics (PK) approach and in situ brain perfusion technique to study the blood and brain PK of PKRinh, an inhibitor of the double-stranded RNA-dependent protein kinase (PKR) activation, in mice. PKRinh showed a supra dose-proportional blood exposure that was not observed in the brain, and a brain to blood AUC ratio of unbound drug smaller than 1 at all tested doses. These data suggested the implication of an active efflux at the BBB. Using in situ brain perfusion technique, we showed that PKRinh has a very high brain uptake clearance which saturates with increasing concentrations. Fitting the data to a Michaelis-Menten equation revealed that PKRinh transport through the BBB is composed of a passive unsaturable flux and an active saturable protein-mediated efflux with a km of ≅ 3 µM. We were able to show that the ATP-binding cassette (ABC) transporter P-gp (Abcb1), but not Bcrp (Abcg2), was involved in the brain to blood efflux of PKRinh. At the circulating PKRinh concentrations of this study, the P-gp was not saturated, in accordance with the linear brain PKRinh PK. Finally, PKRinh had high brain uptake clearance (14 µl/g/s) despite it is a good P-gp substrate (P-gp Efflux ratio ≅ 3.6), and reached similar values than the cerebral blood flow reference, diazepam, in P-gp saturation conditions. With its very unique brain transport properties, PKRinh improves our knowledge about P-gp-mediated efflux across the BBB for the development of new CNS directed drugs.

Optimization of liquid chromatography-multiple reaction monitoring cubed mass spectrometry assay for protein quantification: application to aquaporin-2 water channel in human urine.

Aquaporin-2 (AQP2) is a water channel protein located in the kidney collecting ducts that has been studied as a potential biomarker of a wide variety of water handling disorders and that could also be used to monitor lesions in the collecting ducts. Enzyme-linked immunosorbent assay (ELISA), the most commonly used approach for protein assay in biofluids, has a limited potential for biomarker verification due to the restricted possibility to perform multiplex assays, the cost and complexity of assay development for new candidates. Liquid chromatography tandem mass spectrometry (LC-MS/MS), in multiple reaction monitoring (MRM) mode, has been demonstrated as a powerful alternative technique, and applied to multiple protein quantification. An even more specific method, termed MRM cubed (MRM(3)), has recently been developed. This paper focuses on the development of an AQP2 assay in urine by LC-MS/MS, based on the MRM(3) strategy, and the influence of key MRM(3) parameters that enable to increase the method sensitivity by a factor of 10. Linearity is observed within the concentration range 0.5-50ng/mL, intra and inter assay precision ranged from 9 to 35% at the lower limit of quantification (LLOQ), and accuracy from 94 to 114%. This assay could therefore be used in the near future to evaluate human urinary AQP2 as a potential biomarker of kidney collecting duct injury.