Safety and tolerability of intravenous regadenoson in healthy subjects: A randomized, repeat-dose, placebo-controlled study.

BACKGROUND: Regadenoson is a selective A2A adenosine receptor agonist indicated for radionuclide myocardial perfusion imaging in patients unable to undergo adequate exercise stress. However, the safety, tolerability, and plasma concentrations associated with repeated doses have not previously been assessed. METHOD AND RESULTS: Healthy males and females were randomized to receive intravenous regadenoson [100 µg (3 doses), 200 µg (3 doses), or 400 µg (2 doses)], or placebo (2 or 3 doses; 0.9% sodium chloride); all doses 10 minutes apart. The primary endpoint was vital sign measurements (blood pressure and heart rate). Secondary endpoints included 12-lead electrocardiogram measurements, clinical laboratory evaluations (hematology, chemistry, and urinalysis), and adverse events. Thirty-six subjects were randomized and completed the study. Plasma concentrations of regadenoson increased in a dose-related manner and with successive doses. No consistent effect was observed for systolic blood pressure, although diastolic blood pressure was slightly lower than placebo for all regadenoson groups. Transient, dose-dependent increases in heart rate were observed in all regadenoson groups. There were no serious adverse events; 27 adverse events occurred in 14 regadenoson-treated subjects vs two events in two placebo-treated subjects. CONCLUSION: Repeated doses of regadenoson appeared to be safe and well tolerated in healthy subjects.

ELISA microplate: a viable immunocapture platform over magnetic beads for immunoaffinity-LC-MS/MS quantitation of protein therapeutics?

AIM: Evaluate the performance of ELISA microplates versus commonly used magnetic beads for biological sample cleanup and/or enrichment in immunoaffinity-LC-MS/MS to reduce tedious beads washing procedures and a relatively high assay cost. MATERIALS & METHODS: ELISA microplates were used as immunicapture platform and compared with magnetic beads for sample cleanup for LC-MS/MS quantitation of protein therapeutics. RESULTS: One unmodified and two surface-activated microplates provided comparable linear ranges and sensitivities for a therapeutic protein (mass 78 kDa) using a human serum sample of 100 µl with 1:1 dilution compared with Tosylactivated magnetic beads using 200 µl of human serum without sample dilution. The assays' precision and accuracy were all within acceptable ranges. No nonspecific binding or other selectivity issues were observed. CONCLUSION: The results suggested an ELISA microplate could be a viable immunocapture platform for immunoaffinity-LC-MS/MS quantitation of protein therapeutics.

Guanidinated protein internal standard for immunoaffinity-liquid chromatography/tandem mass spectrometry quantitation of protein therapeutics.

RATIONALE: A protein internal standard (IS) is essential and superior to a peptide IS to achieve reproducible results in the quantitation of protein therapeutics using immunoaffinity-liquid chromatography/tandem mass spectrometry (LC/MS/MS). Guanidination has been used as a protein post-modification technique for more than half a century. A decade ago, the modification was applied to lysine-ending peptides to enhance their MALDI responses and peptide sequencing coverage. However, rarely has tryptic digestion of guanidinated proteins been investigated, likely due to the early conclusion that trypsin did not hydrolyze peptide bonds involving homoarginine in guanidinated proteins. In this study, the opposite was observed. Guanidinated lysine residues of proteins did not hinder the access of trypsin allowing for proteolytic digestion. Based on this observation, a new concept of internal standard, named Guanidinated Protein Internal Standard (GP-IS), was proposed for LC/MS/MS quantitation of protein therapeutics. METHODS: The GP-IS is prepared by treating a portion of the therapeutic protein (analyte) with guanidine to convert arginine residues in the protein into homoarginine residues. After tryptic digestion, the GP-IS produces a series of homoarginine-ending peptides plus another series of arginine-ending peptides. One of the homoarginine-ending peptides, which corresponds to the analyte surrogate (lysine-ending) peptide, was chosen as a peptide internal standard (GP-PIS) for LC/MS/MS quantitation. RESULTS: Using this GP-IS approach, a sensitive and robust immunoaffinity-LC/MS/MS assay was developed and fully validated with a linearity range from 10 to 1000 ng/mL using 200 µL of human serum for the quantitation of an Astellas protein drug in clinical development. CONCLUSIONS: The proposed strategy allows LC/MS/MS to play an ever-increasing role in bioanalytical support for protein therapeutics development because of its capability of completely tracking all variations from the beginning to the end of sample analysis, easier preparation compared to isotope-labeled protein-IS, and greater flexibility for changing to alternate analyte surrogate peptides.

The effect of frequency doubled double pulse Nd:YAG laser fiber proximity to the target stone on transient cavitation and acoustic emission.

PURPOSE: Scant information has been published describing the effect of laser fiber distance from the stone target on the mechanism of calculus fragmentation. Using high speed photography and acoustic emission measurements we characterized the impact of laser fiber proximity on stone comminution. We evaluated the effect of laser fiber distance from the stone target on resultant cavitation bubble formation and shock wave generation. MATERIALS AND METHODS: Stone fragmentation was assessed using a FREDDY (frequency doubled double pulse Nd:YAG) (World of Medicine, Orlando, Florida) laser and a holmium laser. The FREDDY laser was operated using a 420 microm fiber at an output energy of 120 and 160 mJ in single and double pulse settings, and a pulse repetition rate of 1 Hz. The holmium laser was operated using a 200 microm fiber at an output energy of 1 to 3 J and a pulse repetition rate of 1 Hz. The surface of a 1 cm square BegoStone (Bego, Bremen, Germany) attached to an X-Y-Z translational stage was aligned perpendicular to the laser fiber, which was immersed in a Lucite tank filled with water at room temperature. An Imacon 200 high speed camera was used to capture transient cavitation bubbles at a framing rate of up to 1,000,000 frames per second. Acoustic emission signals associated with shock waves generated during the rapid expansion and collapse of the cavitation bubble were measured using a 1 MHz focused ultrasound transducer. RESULTS: At laser fiber distances of 3.0 mm or less cavitation bubbles and shock waves were observed with the FREDDY laser. In contrast to the holmium laser, the bubble size and shock wave intensity of the FREDDY laser was inversely related to the fiber-to-stone distance over the range tested (0.5 to 3.0 mm). CONCLUSIONS: While bubble size was noted to increase with a larger stone-to-fiber distance using the holmium laser, to consistently generate cavitation bubbles and shock waves using the FREDDY laser the laser fiber should be operated within 3.0 mm of the target stone. These findings have significant implications during clinical laser stone fragmentation.

Separation of a BMS drug candidate and acyl glucuronide from seven glucuronide positional isomers in rat plasma via high-performance liquid chromatography with tandem mass spectrometric detection.

A high-performance liquid chromatography/tandem mass spectrometry (LC/MS/MS) method has been developed and validated for the determination of a BMS drug candidate and its acyl glucuronide (1-O-beta glucuronide) in rat plasma. A 50-microL aliquot of each plasma sample was fortified with acetonitrile containing the internal standard to precipitate proteins and extract the analytes of interest. After mixing and centrifugation, the supernatant from each sample was transferred to a 96-well plate and injected into an LC/MS/MS system. Chromatographic separation was achieved isocratically on a Phenomenex Luna C(18), 3 mm x 150 mm, 3 microm column. The mobile phase contained 0.075% formic acid in 70:30 (v/v) acetonitrile/water. Under the optimized chromatographic conditions, the BMS drug candidate and its acyl glucuronide were separated from its seven glucuronide positional isomers within 10 min. Resolution of the parent from all glucuronides and acyl glucuronide from its positional isomers was critical to avoid their interference with quantitation of parent or acyl glucuronide. Detection was by positive ion electrospray MS/MS on a Sciex API 4000. The standard curve, which ranged from 5 to 5000 ng/mL, was fitted to a 1/x(2) weighted quadratic regression model for both the BMS drug candidate and its acyl glucuronide. Whole blood and plasma stability experiments were conducted to establish the sample collection, storage, and processing conditions. The validation results demonstrated that this method was rugged and repeatable. The same methodology has also been used in mouse and human plasma for the determination of the BMS drug candidate and its acyl glucuronide.