Simple method provides resolution of albumin, lipoprotein, free fraction, and chylomicron to enhance the utility of protein binding assays.

Medicinal chemists have been encouraged in recent years to embrace high speed protein binding assays. These methods employ dialysis membranes in 96-well format or spin filters. Membrane-based methods do not separate lipoprotein binding from albumin binding and introduce interference despite membrane binding controls. Ultracentrifugation methods, in contrast, do not introduce interference if density gradients can be avoided and they resolve lipoprotein from albumin. A new generation of compact, fast ultracentrifuges facilitates the rapid and fully informative separation of plasma into albumin, albumin/fatty acid complex, lipoprotein, protein-free, and chylomicron fractions with no need of salt or sugar density gradients. We present a simple and fast ultracentrifuge method here for two platinum compounds and a taxane that otherwise bound irreversibly to dialysis membranes and which exhibited distinctive lipoprotein binding behaviors. This new generation of ultracentrifugation methods underscores a need to further discuss protein binding assessments as they relate to medicinal chemistry efforts.

Evaluation of high-field asymmetric waveform ion mobility spectrometry coupled to nanoelectrospray ionization for bioanalysis in drug discovery.

The potential of high-field asymmetric waveform ion mobility spectrometry (FAIMS) coupled to nanoelectrospray ionization (nanoESI) as a method to improve sample throughput for bioanalysis in a discovery pharmaceutical setting was explored in this work. The ability of FAIMS to separate gas-phase ions in the millisecond timescale was exploited to eliminate the need for liquid chromatography. Samples were introduced into the FAIMS electrodes/mass spectrometer using offline nanoESI at 20 nL/min and 1.5 kV. Signals were averaged for 30 s after which the next sample could be analyzed. The separation of simple mixtures, e.g., the removal of metabolite and endogenous interferences from parent drug, was demonstrated. Moreover, the application of nanoESI attenuated the ion suppression effects that normally plague conventional electrospray. On average, approximately two-thirds of the neat sample signal intensity was preserved in extracted plasma samples. Standard curves were prepared for several compounds and linearity was obtained over approximately two to three orders of magnitude. This methodology was further tested with the analysis of plasma samples from a mouse pharmacokinetic study. Concentration values determined using nanoESI-FAIMS were comparable to those determined using conventional LC/MS as demonstrated by percent differences of less than 30%. This work demonstrated the proof of concept that the combination of FAIMS and nanospray ionization can be a potentially useful tool to improve the throughput of discovery bioanalysis.

A phase 1 study of the proteasome inhibitor bortezomib in pediatric patients with refractory leukemia: a Children's Oncology Group study.

PURPOSE: A phase 1 study to determine the maximum-tolerated dose, dose-limiting toxicity, pharmacokinetics, and biological effects of bortezomib in children with recurrent/refractory leukemia. EXPERIMENTAL DESIGN: Bortezomib was administered twice weekly for 2 consecutive weeks at either 1.3 or 1.7 mg/m(2) dose followed by a 1-week rest. Bortezomib pharmacokinetics and nuclear factor kappaB (NF-kappaB) binding activity were evaluated during the first treatment cycle. RESULTS: Twelve patients (nine with acute lymphoblastic leukemia, three with acute myelogenous leukemia), median age 11 years (range, 1-18 years), were enrolled between May 2004 and November 2005, of whom seven were not fully evaluable for toxicity due to rapidly progressive disease or uncontrolled infection. Dose-limiting toxicities occurred in two patients at the 1.7 mg/m(2) dose level. One patient experienced grade 3 confusion and the other patient had grade 4 febrile neutropenia associated with grade 4 hypotension and grade 3 creatinine. Pharmacokinetic analysis at 1.3 mg/m(2) revealed a clearance of 11 mL/h/m(2), a central volume of distribution of 6.7 L/m(2), and a terminal half-life of 12.6 h. NF-kappaB activity was examined in five patients and was noted to transiently increase and then decrease 4- to 6-fold by 24 h following bortezomib in two patients. There were no objective clinical responses. CONCLUSIONS: For children with leukemia, the recommended phase 2 dose of bortezomib, administered twice weekly for 2 weeks followed by a 1-week rest, is 1.3 mg/m(2)/dose. Although bortezomib treatment inhibited NF-kappaB activity, bortezomib had little activity as a single agent in this population.

Impact of differential recovery in bioanalysis: the example of bortezomib in whole blood.

A key assumption in pharmaceutical bioanalysis is that spiked standards mimic incurred samples in every analytical aspect. Although deviations from this assumption have been reported in terms of the difference in ion suppression or metabolite interference, the difference of extraction recovery and its impact has been rarely reported and is often characterized as unlikely. In this work, we demonstrated the presence and significance of differential recovery using a real-world example: the assay of bortezomib in whole blood. Recovery differences of up to 10-fold were observed between the spiked standards and the incurred samples when different extraction methods were used. Because of its high impact, it is important that the potential of differential recovery between standards and incurred samples be evaluated during method validation. A simple time course incubation experiment was proposed to screen compounds for potential differential recovery during method validation in heterogeneous matrixes, such as whole blood and tissue. The use of this approach and the interpretation of the results from this experiment were demonstrated using bortezomib in whole blood as an example. The differential recovery of bortezomib is likely to be driven by slow binding to the proteosome present in red blood cells. Spiked samples, however, do not have sufficient time for binding to occur.