Quantitative Determination of a Chemically Modified Hammerhead Ribozyme in Blood Plasma Using 96-Well Solid-Phase Extraction Coupled with High-Performance Liquid Chromatography or Capillary Gel Electrophoresis

Development and validation of a sensitive, specific, and rapid hybridization-ELISA assay for determination of concentrations of a ribozyme in biological matrices

“…As polynucleotides like ribozymes and antisense oligonucleotides are developed as therapeutics, the need arises to measure concentrations of these drugs in blood and other biological matrices. Conventional approaches to bioanalytical quantification of polynucleotides have included HPLC, electrophoresis, or capillary gel electrophoresis [1][2][3][4]6]. Overall, these approaches have limited sensitivity (50-100 ng/ml) and are time intensive because they require extraction of the polynucleotide of interest from a biological matrix.…”

“…Following hybridization of ribozyme to target, the complimentary target RNA species is cleaved, resulting in a decrease in target expression. Previous bioanalytical methodology used in the determination of ribozyme or antisense oligonucleotide concentrations required extraction of the drug from the biological matrix followed by chromatography, electrophoresis, or capillary gel electrophoresis for quantitation [1][2][3][4][5][6]. These methods are time-consuming and lack sensitivity; plasma lower-limits of quantitation typically range from 50 to 100 ng/ml.…”

“…Samples were reconstituted with water and TEAA, applied to a SPEC PH 3 ml/15 mg cartridge, and eluted with water/MeOH (1:1). Samples were evaporated, then reconstituted with water and dialyzed on a 0.025 m floating desalting floating membrane filter (Millipore, Billerica MA) for 2 h. Capillary gel electrophoresis of samples was performed as described [2]. This assay had a validated lower limit of quantitation of 105 ng/ml.…”

Chromatographic methods for the determination of therapeutic oligonucleotides

“…Several studies use a proteinase K digestion or methanol precipitation to remove the protein from the biological samples [41,[43][44][45][46]. These approaches avoid issues with the nonspecific and irreversible binding of oligonucleotide to either solid-phase extraction (SPE) cartridges or many containers.…”

Advances of capillary electrophoresis in clinical and forensic analysis (1999-2000)

“…Examples include an inhibitor of angiogenesis that has shown promise in preclinical cancer models, an anti-hepatitis C antiviral drug and a ribozyme against metastatic breast cancer in which the gene HER2 is overexpressed (Ribozyme Pharmaceuticals, Boulder, CO, USA). CGE has successfully been applied to the determination of a chemically modified hammerhead ribozyme in plasma, the assay being based upon serial solid-phase extraction performed on a 96 well plate format with CGE back-end analysis [308]. Furthermore, Prasmickaite et al [309] investigated the intracellular metabolism of a 2'-Omethyl-stabilized ribozyme after uptake by DOTAP transfection or as free ribozyme by CGE and Seavels et al [310] described the use of CGE to study the catalytic activity of a hammerhead ribozyme.…”