Characterization of Oligonucleotide MetabolismIn Vivovia Liquid Chromatography/Electrospray Tandem Mass Spectrometry with a Quadrupole Ion Trap Mass Spectrometer

Griffey, Richard H.; Greig, Michael J.; Gaus, Hans; Liu, Kenneth; Monteith, David K.; Winniman, Michael; Cummins, Lendell L.

doi:10.1002/(sici)1096-9888(199703)32:3<305::aid-jms482>3.0.co;2-r

Cited by 94 publications

(56 citation statements)

References 8 publications

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“…In addition to the liver, kidneys are another important organ responsible for the degradation of PS-ODN. Significant amounts of chainshortened metabolites were detected in the kidneys, especially in the proximal tubular cells (Griffey et al, 1997;Noll et al, 2005). Metabolism of GTI-2040 and other PS-ODNs was also found in circulation and other organ tissues in addition to liver (Gilar et al, 1997;FIG.…”

Section: Enzyme Kinetics Of An Antisense Drug Gti-2040mentioning

confidence: 96%

“…The underestimation of the in vivo CL int,h1 is likely because of the multiple metabolism sites of PS-ODNs in vivo. In vivo, PS-ODNs extensively distribute to different organs and tissues with the majority of drugs accumulating within the liver and the kidneys (Agrawal et al, 1991;Griffey et al, 1997;Yu et al, 2004;Noll et al, 2005). In the liver, PS-ODNs are not only taken up into the hepatocytes but also rapidly distribute to the nonparenchymal cells, including Kupffer cells and endothelial cells (Nolting et al, 1997;Graham et al, 2001), and only a small amount of drug is excreted into the bile (Lischka et al, 2003).…”

Section: Enzyme Kinetics Of An Antisense Drug Gti-2040mentioning

confidence: 99%

“…GTI-2040 has been shown recently to have promising response and acceptable tolerability in phase I clinical trials as a single agent or in combination with cytarabine for the treatment of advanced solid tumors and acute myeloid leukemia (AML) (Desai et al, 2005;Klisovic et al, 2008). Using a highly specific ion-pair HPLC/tandem mass spectrometry method (Griffey et al, 1997;Gilar and Bouvier, 2000;Dai et al, 2005b), we recently reported the metabolism of GTI-2040 as the sequential nucleotide deletion via the 3Ј exonuclease (Wei et al, 2006). A series of progressively 3Ј chain short-end metabolites were identified in several biological matrices including plasma from AML patients, solutions containing 3Ј exonuclease, and in human liver microsomes.…”

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confidence: 99%

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Enzyme Kinetics of GTI-2040, a Phosphorothioate Oligonucleotide Targeting Ribonucleotide Reductase

Wei

Dai²,

Liu³

et al. 2008

Drug Metabolism and Disposition

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ABSTRACT:Enzyme kinetics of GTI-2040 (5-GGC TAA ATC GCT CCA CCA AG-3), a phosphorothioate ribonucleotide reductase antisense, were investigated for the first time in 3 exonuclease solution and human liver microsomes (HLMs), using the ion-pair high-performance liquid chromatogram method for quantification of the parent drug and two major 3N-1 and 3N-2 metabolites. Enzyme kinetics of GTI-2040 in 3-exonuclease solution were found to be well characterized by the Michaelis-Menten model, using the sum of formation rates of 3N-1 and 3N-2 (ϳtotal metabolism) because of sequential metabolism. In HLMs, a biphasic binding was observed for GTI-2040 with high-and low-affinity constants (K d s) of 0.03 and 3.8 M, respectively. Enzyme kinetics of GTI-2040 in HLMs were found to deviate from Michaelis-Menten kinetics when the total GTI-2040 substrate was used. However, after correction for the unbound fractions, the formation rate of total metabolites could be described by Michaelis-Menten kinetics. Using the free substrate fraction, the K m and V max of GTI-2040 were determined to be 6.33 ؎ 3.2 M and 16.5 ؎ 8.4 nmol/mg/h, respectively. Using these values, in vitro hepatic intrinsic clearance (CL int ) in HLM was estimated to be 2.61 ؎ 0.56 ml/h. The CL int was then used to predict GTI-2040's in vivo intrinsic clearance in humans by a microsomal protein scaling factor, which gave a mean value of 182.7 l/h, representing 24.1% of the observed in vivo mean scaled hepatic intrinsic clearance of 758.7 l/h in patients with acute myeloid leukemia. We concluded that the saturable nonspecific binding of GTI-2040 in HLMs complicated the interpretation of its enzyme kinetics, and scaled intrinsic clearance from HLMs only partially predicted the in vivo intrinsic clearance.Antisense oligonucleotides (ODNs) are short, single-strand DNA molecules designed to hybridize with specific mRNA strands, thereby selectively inhibiting the production of specific gene products (Stein and Cheng, 1993;Dias and Stein, 2002). This approach has been explored to target expression of genes important for malignant transformation and other pathogenetic mechanisms. For a successful therapeutic use of ODN compounds robust in vivo, stability is critical. Previously used unmodified ODNs degraded rapidly in biological fluids by nucleases, which limited their clinical use. More recently, by substituting one of the nonbridge oxygen atoms, phosphorothioate analogs have been synthesized and shown to be more resistant to exonucleases than the unmodified ODNs Aboul-Fadl, 2005). Several phosphorothioate ODNs (PS-ODNs) are currently undergoing clinical evaluation for a number of diseases, including cancer, viral infections, and inflammatory disorders Jansen and Zangemeister-Wittke, 2002;Marcucci et al., 2005). It has been reported that the in vivo pharmacodynamic effects of antisense correlate with the intracellular drug levels and clinical response Dai et al., 2005a;Marcucci et al., 2005). Moreover, it is anticipated that a well defined pharmacokinetics-pharmacodynamics cor...

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Section: Enzyme Kinetics Of An Antisense Drug Gti-2040mentioning

confidence: 96%

Section: Enzyme Kinetics Of An Antisense Drug Gti-2040mentioning

confidence: 99%

mentioning

confidence: 99%

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Enzyme Kinetics of GTI-2040, a Phosphorothioate Oligonucleotide Targeting Ribonucleotide Reductase

Wei

Dai²,

Liu³

et al. 2008

Drug Metabolism and Disposition

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“…MSMS could close this gap as this method was shown to be highly suitable for the characterization of small nucleic acids. For example, the characterization of metabolites of oligonucleotides used as therapeutic agents for the treatment of cancer was only possible via tandem mass spectrometric sequencing [28,29]. However, the deduction of sequence information from an MSMS spectrum is time-consuming, highly technical, lacking strict objectivity due to reliance on human interpretation, and can be performed only in laboratories with extensive experience in MSMS.…”

Section: De Novo Sequencing Strategymentioning

confidence: 99%

Automated de novo sequencing of nucleic acids by liquid chromatography-tandem mass spectrometry

Oberacher

Mayr

Huber

2004

J. Am. Soc. Mass Spectrom.

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We present the first global computer-aided sequencing algorithm for the de novo determination of short nucleic acid sequences. The method compares the fragment ion spectra generated by collision-induced dissociation of multiply charged oligodeoxynucleotide-ions to the m/z values predicted employing established fragmentation pathways from a known reference sequence. The closeness of matching between the measured spectrum and the predicted set of fragment ions is characterized by the fitness, which takes into account the difference between measured and predicted m/z values, the intensity of the fragment ions, the number of fragments assigned, and the number of nucleotide positions not covered by fragment ions in the experimental spectrum. Smaller values for the fitness indicate a closer match between the measured spectrum and predicted m/z values. In order to find the sequence most closely matching the experimental spectrum, starting from a given nucleotide composition all possible oligonucleotide sequences are assembled followed by identification of the correct sequence by the lowest fitness value. Using this concept, sequences of 5-to 12-mer oligodeoxynucleotides were successfully de novo determined. High sequence coverage with fragment ions was essential for obtaining unequivocal sequencing results. Moreover, the collision energy was shown to have an impact on the interpretability of tandem mass spectra by the de novo sequencing algorithm. Experiments revealed that the optimal collision energy should be set to a value just sufficient for complete fragmentation of the precursor ion. T he hyphenation of liquid chromatography (LC) to mass spectrometry (MS) is one of the most powerful methods for the characterization of oligonucleotides today [1]. In this context the combination of ion-pair reversed-phase high-performance liquid chromatography and electrospray ionization mass spectrometry (ICEMS) has emerged as a versatile tool for the analysis of single-and double-stranded nucleic acids ranging in size from a few nucleotides (nt) up to several hundred base pairs (bp) [2][3][4]. Although the occurrence of a sequence variation is predictable on the basis of molecular mass measurements [5][6][7], detailed sequencing information can only be obtained from selective decomposition of oligonucleotides by tandem mass spectrometry (MSMS).The principles of gas-phase collision-induced dissociation (CID) reactions of oligonucleotides have been studied extensively [8 -17] and the resulting product ion spectra are predictable on the basis of known fragmentation pathways. Consequently, MSMS of oligodeoxynucleotides has been successfully applied to characterize or confirm structures [11], to detect chemical modifications [2,10,18], and to identify sequence variations [14, 19 -21]. However, the deduction of sequence information is time-consuming, highly technical, lacking strict objectivity due to reliance on human interpretation, and can be performed only in laboratories with extensive experience in MSMS. Hence, automation of pro...

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“…LC/ MS techniques provide the most accurate and most thorough characterization of oligonucleotides particularly useful for metabolite profiling. Several methods for oligonucleotide quantitation in biological matrixes using LC/MS with solid-phase extraction (SPE) or phenol/chloroform liquid-liquid extraction have been reported in literature (14)(15)(16)(17)(18)(19)(20)(21). Most of the reported assays encountered problems either with MS (e.g.…”

Section: Introductionmentioning

confidence: 99%

Phosphorothioate Oligonucleotide Quantification by μ-Liquid Chromatography-Mass Spectrometry

Erb

Leithner²,

Bernkop‐Schnürch³

et al. 2012

AAPS J

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Abstract. Phosporothioate oligonucleotides represent an important class of therapeutic oligonucleotides, in which none-bridging oxygen atoms of the phosphate groups are replaced by sulfur. These oligonucleotides are designed to treat disease by modulating gene expression of an affected individual. As the development and application of these therapeutical oligonucleotides require analytical support, the development, validation, and application of an assay for the quantitative analysis of a phosporothioate oligonucleotide in rat plasma is described. The method employs ion-pair reversedphase chromatography on a monolithic capillary column with acetonitrile gradients in cyclohexyldimethylammonium acetate for separation and high-resolution tandem mass spectrometry for detection of nucleic acids. Chromatographic parameters (i.e. column temperature, mobile phase composition) as well as mass spectrometric parameters (i.e. spray voltage, gas flow, and capillary position, scan mode) have been optimized for sensitive oligonucleotide quantification. Furthermore, a solid-phase extraction method was developed which enabled processing of 10 μl of plasma. The five-point calibration curve showed linearity over the range of concentrations from 100 to 1,000 nM of the oligonucleotide. The limit of detection was 50 nM. The intra-and inter-day precision and accuracies were always better than 10.2 %. Using this assay, we performed a pharmacokinetic study of the phosporothioate oligonucleotide in rat treated with a single intravenous dose of 0.39 μmol/kg. The assay sensitivity was sufficient to study the early phase elimination of the oligonucleotide. Small amounts of the oligonucleotide were detectable up to 3 h after dosing.

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Characterization of Oligonucleotide MetabolismIn Vivovia Liquid Chromatography/Electrospray Tandem Mass Spectrometry with a Quadrupole Ion Trap Mass Spectrometer

Cited by 94 publications

References 8 publications

Enzyme Kinetics of GTI-2040, a Phosphorothioate Oligonucleotide Targeting Ribonucleotide Reductase

Enzyme Kinetics of GTI-2040, a Phosphorothioate Oligonucleotide Targeting Ribonucleotide Reductase

Automated de novo sequencing of nucleic acids by liquid chromatography-tandem mass spectrometry

Phosphorothioate Oligonucleotide Quantification by μ-Liquid Chromatography-Mass Spectrometry

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