Oligonucleotide quantification and metabolite profiling by high-resolution and accurate mass spectrometry.

Aim: Advancements in RNA interference therapeutics have triggered development of improved bioanalytical methods for oligonucleotide metabolite profiling and high-throughput quantification in biological matrices. Results & methodology: HPLC coupled with high-resolution mass spectrometry (LC-HRMS) methods were developed to investigate the metabolism of a REVERSIR™ molecule in vivo. Plasma and tissue samples were extracted using solid-phase extraction followed by LC-HRMS analysis for metabolite profiling and quantification. The method was qualified from 10 to 5000 ng/ml (plasma) and 100 to 50000 ng/g (liver and kidney). In rat liver, intra and interday accuracy ranged from 80.9 to 118.5% and 88.4 to 111.9%, respectively, with acceptable precision (<20% CV). Conclusion: The LC-HRMS method can be applied for metabolite profiling and quantification of oligonucleotides in biological matrices.

In-Vivo Degradation of DNA-Based Therapeutic BC 007 in Humans.

BACKGROUND AND OBJECTIVES: Since there is no clear evidence in the literature to show how non-modified single-stranded DNA (ssDNA) drugs are metabolized in humans, we assessed the metabolism of BC 007, an ssDNA therapeutic, under development as a neutralizer of autoantibodies against G-protein-coupled receptors. In-vitro, investigating its stability in monkey plasma and serum, a successive 3'-exonuclease degradation resulting in several n-x degradation products has been previously reported. Here, we investigated the metabolism of BC 007 in humans after intravenous application to autoantibody-positive healthy subjects, in line with Phase I safety testing. METHODS: 1H-NMR was applied for n-x degradation product search and beta-aminoisobutyric acid (bAIBA) measurement in urine; ultra-performance liquid chromatography-mass spectrometry was also used for the latter. Colorimetric assays were used for quantification of uric acid in serum and urine. RESULTS: Fast degradation prohibited the detection of the intermediate n-x degradation products in urine using 1H-NMR. Instead, NMR revealed a further downstream degradation product, bAIBA, which was also detected in serum shortly after initial application. The purine degradation product, uric acid, confirmed this finding of fast metabolism. CONCLUSION: Fast and full degradation of BC 007, shown by nucleic bases degradation products, is one of the first reports about the fate of a ssDNA product in humans.

Gold-nanoparticle extraction and reversed-electrode-polarity stacking mode combined to enhance capillary electrophoresis sensitivity for conjugated nucleosides and oligonucleotides containing thioether linkers.

We present a capillary electrophoresis method for determining two different C8-conjugated deoxyadenosines, and for oligonucleotides containing them, in which a psoralen or an acridine molecule is bonded to the base via a short alkyl chain containing sulfur ethers at both ends. The sensitivity of the micellar electrokinetic chromatography (MEKC) method was increased by using two preconcentration techniques, micro solid-phase extraction (µSPE) followed by reversed-electrode-polarity stacking mode (REPSM). Variables that affect the efficiency of the extraction in µSPE and preconcentration by REPSM, including the type and volume of extraction nanoparticle, concentration, and injection time, were investigated. Under the optimum conditions, enrichment factors obtained were in the range 360-400. The limits of detection (LODs) at a signal-to-noise ratio of 3 ranged from 2 to 5 nmol L(-1). The relative recoveries of labelled adenosines from water samples were 95-103%. The proposed method provided high enrichment factors and good precision and accuracy with a short analysis time. On the basis of the advantages of simplicity, high selectivity, high sensitivity, and good reproducibility, the proposed method may have great potential for biochemical applications.

Bioanalysis of siRNA and oligonucleotide therapeutics in biological fluids and tissues.

This article summarizes bioanalytical avenues for the determination of siRNA and oligonucleotide therapeutics, with an emphasis on hybridization methods. Aspects of the chemistry and delivery of investigational oligonucleotide therapeutics are considered. The nature of the oligonucleotide under investigation will dictate the best analytical course of action; each method has its advantages and disadvantages, depending upon the oligonucleotide test article and the anticipated toxicokinetic and pharmacokinetic study parameters. Stringent method development and specific validation criteria are essential to attain the best quality results in support of a regulatory filing.

Evidence of oligonucleotides containing 8-hydroxy-2'-deoxyguanosine in human urine.

The product of oxidative damage to DNA, 8-hydroxy-2'-deoxyguanosine (8-OHdG), when detected in urine, is considered to be a global, noninvasive biomarker of in vivo oxidative DNA damage. In this paper we describe a novel approach to confirm the presence of oligonucleotides containing 8-OHdG in human urine. Fractions of urine were prepared by gel-filtration chromatography, and the presence of oligonucleotides was confirmed by ELISA using a monoclonal anti-(single-stranded DNA) antibody. Pools of urine fractions were subsequently prepared according to ELISA reactivity, each containing oligonucleotides with a known range of base numbers. The level of 8-OHdG in each pool was subsequently determined using a commercial ELISA kit. Results confirmed that oligonucleotides containing 8-OHdG are present in urine and, most significantly, oligomers of <30-55 bases were found to be associated with 8-OHdG. This finding strongly supports the involvement of nucleotide excision repair (NER) in the removal of 8-OHdG from the cell. The novel approach adopted in this study was validated using cell culture supernatant obtained from an in vitro model comprising CCRF cells exposed to vitamin C; this model has previously been shown to stimulate removal of 8-OHdG from the cell by an NER-dependent process.

Oligonucleotides in human urine do not contain 8-oxo-7,8-dihydrodeoxyguanosine.

The promutagenic DNA modification 8-oxo-7,8-dihydrodeoxyguanosine is the most frequently used marker for oxidative stress to DNA. The unmodified base and nucleoside and the 8-hydroxylated guanine base and nucleoside are found in urine, the latter used as a global measure of oxidative stress to DNA. Nucleotide excision repair (NER) excises a 27- to 29-mer oligonucleotide with oxidative lesions, and if found in urine, it could be used as a measure of DNA repair in vivo. Enzymatic hydrolysis of human urines followed by HPLC-tandem mass spectrometry was not able to reveal oligonucleotides and/or mononucleotides with the 8-oxo-7,8-dihydrodeoxyguanosine modification. The recovery of a synthetic oligonucleotide with the modification was complete (95% confidence limits: 98-124%). These experiments show that oligonucleotides are excreted into urine, but that 8-oxo-7,8-dihydrodeoxyguanosine is found only as the mononucleoside and is not present in any significant amounts in oligonucleotides. We conclude that oligonucleotides are excreted into urine, and they do not contain oxidized lesions. Either NER products are degraded after excision or NER functions differently in vivo in humans compared with cellular systems.

Pharmacokinetics and distribution of a 33P-labeled anti-human immunodeficiency virus oligonucleotide (AR177) after single- and multiple-dose intravenous administration to rats.

AR177 is a 17-mer oligonucleotide that has anti-human immunodeficiency virus activity in vitro. The disposition of internally labeled 33P-AR177 was studied after the tail vein injection of single and multiple doses (0.7 mg/kg) to rats. After a single dose, the terminal half-life of AR177 in the blood and plasma was 367 and 271 hr, respectively, significantly longer than values reported for other oligonucleotides. Analysis of the AR177 tissue distribution showed that the majority of the dose was distributed to the liver (40%), bone marrow (17%) and renal cortex (15%) at 8 hr after single dosing. Analysis of the AR177 concentrations in tissues showed that the highest concentrations were achieved in the renal cortex (15.0 microg-eq/g), liver (7.4 microg-eq/g), bone marrow (3.9 microg-eq/g), mesenteric lymph node (3.0 microg-eq/g) and spleen (2.4 microg-eq/g) at 8 hr after single dosing. The half-life in these tissues was 9.6, 7.7, 36.8, 10.0 and 30.8 days, respectively. Forty-eight hours after the last of seven i.v. doses given every other day, the concentrations in tissues were as follows: renal cortex, 39.9 microg-eq/g; liver, 33.9 microg-eq/g; bone marrow, 12.7 microg-eq/g; spleen, 9.3 microg-eq/g; mesenteric lymph node, 5.1 microg-eq/g. Twenty-one days after administration of the last dose, tissue concentrations were still high, as follows: renal cortex, 18.6 microg-eq/g; liver, 6.2 microg-eq/g; bone marrow, 12.5 microg-eq/g; mesenteric lymph node, 3.9 microg-eq/g; spleen, 8.1 microg-eq/g. There was low urinary and fecal excretion (urinary excretion of 12.8% and fecal excretion of 6.0% of the total dose over 21 days) after a single dose. Gel filtration and anion-exchange high-performance liquid chromatography and electrophoretic analysis of the radioactivity in tissues indicated that >90% of the radioactivity represented intact AR177 for at least 7 days after drug dosing. These results demonstrate that AR177 has an extended plasma, blood and tissue half-life, is widely distributed and achieves high concentrations in lymphoid and nonlymphoid tissues in rats.

Renal disposition characteristics of oligonucleotides modified at terminal linkages in the perfused rat kidney.

To clarify the renal disposition characteristics of oligonucleotides at the organ level, the renal handling of model end-capped oligonucleotides, 3'-methoxyethylamine 5'-biotin-decathymidylic acid containing phosphoramidate modifications at 3'- and 5'-terminal internucleoside linkages (T10) and its phosphorothioate (Ts10), were studied in the perfused rat kidney. In a single-pass indicator dilution experiment, venous outflow and urinary excretion patterns and tissue accumulation of radiolabeled oligonucleotides were evaluated under filtering or nonfiltering conditions. No significant binding to bovine serum albumin (BSA) in the perfusate was observed for T10, whereas more than 90% of Ts10 bound to BSA. The steady-state distribution volume of T10 calculated from the venous outflow pattern was larger than that of inulin, which corresponds to the extracellular volume of the kidney, whereas the distribution volume of Ts10 was larger than that of BSA (the intravascular volume). These results suggested their interaction with the vascular wall. Rapid urinary excretion was observed for T10, similar to inulin used as a marker of golmerular filtration rate. On the other hand, urinary excretion of Ts10 was greatly restricted due to its high binding ability (> 90%) to BSA in the perfusate. A significant amount of T10 and Ts10 was accumulated in the kidney (T10, 1.8% of injected dose; Ts10, 1.3%) compared with inulin (0.2%) and BSA (< 0.1%). The accumulation of these oligonucleotides was ascribed to both tubular reabsorption and uptake from the capillary side. In addition, the uptake of T10 from the capillary side was significantly inhibited by simultaneous injection of dextran sulfate, suggesting that the oligonucleotide was taken up as an anionic molecule. These findings will be useful information for the development of delivery systems for antisense oligonucleotides.

Quantitative analysis of phosphorothioate oligonucleotides in biological fluids using fast anion-exchange chromatography.

Phosphorothioate oligonucleotides are potentially useful as anti-viral drugs. Classical DNA extraction methods are not as effective on short single-stranded DNA as with longer double-stranced chains. The classical method of phenol-chloroform extraction followed by ethanol precipitation is difficult to quantify, thus monitoring of the pharmacological disposition of these compounds is subject to error. A method has been devised and validated for extraction and analysis of modified oligonucleotides from biological fluids such as urine and serum based on protein kinase digestion and phenol-chloroform extraction. Due to the high native ultraviolet absorbance of the oligomers, detection limits in the low ppb range were obtained without derivatization.

High-performance liquid chromatographic analysis of phosphorothioate analogues of oligodeoxynucleotides in biological fluids.

Phosphorothioate oligodeoxynucleotides (S-ODNs) have potential as anti-viral agents and are being investigated for the chemotherapy of AIDS. A high-performance liquid chromatographic method is described for the analysis, in urine and plasma, of a 28-unit deoxycytidine homopolymer (S-dC28) and a 28-unit S-ODN "antisense" to the rev gene of the human immunodeficiency virus. This method employs ion-pairing HPLC with a polymeric column. Tetrabutylammonium is used as the ion-pairing agent in a mobile phase of acetonitrile in pH 7.0 phosphate buffer. Analysis of the S-ODNs is relatively rapid (20 min) and sensitive (20 nm) and is accomplished by a gradient elution (22.5-30.0% acetonitrile) followed by ultraviolet (266 or 272 nm) absorption detection. This method is likely applicable, with appropriate modifications, to all S-ODNs of similar molecular weight regardless of sequence. The S-ODNs bind very strongly to plasma proteins but are readily prepared for analysis by a phenol extraction procedure. In a preliminary pharmacokinetic study in mice with S-dC28, very rapid elimination of the oligomer from plasma was observed (half-time, 11.6 min). Estimates for the apparent volume of distribution and total body clearance were 3 ml and 0.2 ml/min, respectively. It appears that the majority of the oligomer is eliminated by renal clearance (glomerular filtration), a property likely shared by all S-ODNs of similar molecular mass.