Optimization of elution conditions and comparison of emerging biocompatible columns on the resolving power and detection sensitivity of oligonucleotides by ion-pairing reversed-phase liquid chromatography mass spectrometry.

A generic performance comparison strategy has been developed to evaluate the impact of mobile-phase additives (ion-pairing agent / counter ion systems), distinct stationary phases on resulting resolving power, and MS detectability of oligonucleotides and their critical impurities in gradient IP-RPLC. Stationary-phase considerations included particle type (core-shell vs. fully porous particles), particle diameter, and pore size. Separations were carried out at 60°C to optimize mass transfer (C-term). The incorporation of an active column preheater mitigated thermal mismatches, leading to narrower peaks and overcoming peak splitting. Acetonitrile as organic modifier outweighed methanol in terms of peak-capacity generation and yielded a 30% lower back pressure. Performance screening experiments were conducted varying ion-pairing agents and counter ions, while adjusting gradient span achieved an equivalent effective retention window. Hexafluoromethylisopropanol yielded superior chromatographic resolution, whereas hexafluoroisopropanol yielded significantly higher MS detection sensitivity. The 1.7 µm core-shell particle columns with 100 Å pores provided maximum resolving power for small (15-35 mers) oligonucleotides. Sub-min analysis for 15-35 polyT ladders was achieved operating a 50 mm long column at the kinetic performance limits. High-resolution separations between a 21-mer modified RNA sequence oligonucleotides and its related (shortmer and phosphodiester) impurities and complementary strand were obtained using a coupled column set-up with a total length of 450 mm.

Tissue distribution and depletion kinetics of bortezomib and bortezomib-related radioactivity in male rats after single and repeated intravenous injection of 14 C-bortezomib.

PURPOSE: The body distribution of total radioactivity (TR) and bortezomib was investigated in male Sprague-Dawley rats after single and repeated i.v. (bolus) administration with (14)C-labelled bortezomib (VELCADE) (0.2 mg/kg; 0.28 MBq./kg). METHODS: Bortezomib was dosed on days 1, 4, 8, and 11 (i.e. a clinical dosing cycle) and the animals were sacrificed at selected time points following single and repeated dose administration for the quantification of TR in blood, plasma, and various tissues by liquid scintillation counting following organ dissection or by quantitative whole body autoradiography. In selected tissues, bortezomib levels were determined by LC-MS/MS. RESULTS: In general, plasma TR levels were less than 10% of the corresponding blood concentrations. TR was rapidly and widely distributed to the tissues with only limited penetration into the central nervous system (CNS). In the tissues, highest levels of TR were measured in bortezomib-eliminating organs (liver and kidney), lymphoid tissues, and regions of rapidly dividing cells (e.g. the bone marrow, intestinal mucosa). Low TR concentrations were found in the CNS (tissue-to-blood ratio of approximately 0.05 after repeated dosing). With the exception of the liver, TR consisted almost exclusively of the parent drug. Tissue concentrations of TR and bortezomib increased up to about threefold from the first to the third dose administration, after which they remained constant. CONCLUSION: No undue tissue accumulation of TR and of bortezomib was observed in rats following a full clinical dosing cycle of bortezomib.

A novel radioligand for the ATP-gated ion channel P2X7: [3H] JNJ-54232334.

The ATP-gated ion channel P2X7 has emerged as a potential central nervous system (CNS) drug target based on the hypotheses that pro-inflammatory cytokines such as IL-1ß that are released by microglia, may contribute to the etiology of various disorders of the CNS including depression. In this study, we identified two closely related P2X7 antagonists, JNJ-54232334 and JNJ-54140515, and then tritium labeled the former to produce a new radioligand for P2X7. JNJ-54232334 is a high affinity ligand for the rat P2X7 with a pKi of 9.3±0.1. In rat cortical membranes, [3H] JNJ-54232334 reached saturable binding with equilibrium dissociation (Kd) constant of 4.9±1.3 nM. The compound displayed monophasic association and dissociation kinetics with fast on and off rates. In rat brain sections, specific binding of [3H] JNJ-54232334 was markedly improved compared to the previously described P2X7 radioligand, [3H] A-804598. In P2X7 knockout mouse brain sections, [3H] A-804598 bound to non-P2X7 binding sites in contrast to [3H] JNJ-54232334. In rat or wild type mouse brain sections [3H] JNJ-54232334 bound in a more homogenous and region independent manner. The ubiquitous expression of P2X7 receptors was confirmed with immunohistochemistry in rat brain sections. The partial displacement of [3H] A-804598 binding resulted in the underestimation of the level of ex vivo P2X7 occupancy for JNJ-54140515. Higher levels of P2X7 ex vivo occupancy were measured using [3H] JNJ-54232334 due to less non-specific binding. In summary, we describe [3H] JNJ-54232334 as a novel P2X7 radioligand, with improved properties over [3H] A-804598.