On-Line HPLC Electrospray Mass Spectrometry of Phosphorothioate Oligonucleotide Metabolites

Gaus, Hans; Owens, Stephen R.; Winniman, Michael; Cooper, Scott; Cummins, Lendell L.

doi:10.1021/ac960557q

Cited by 113 publications

(66 citation statements)

References 14 publications

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“…These data are consistent with the nuclease resistance of 2Ј-MOE-modified nucleotides placed in the 3Ј-and 5Ј-terminal portions of ISIS 301012 (Zhang et al, 1995). There was a notable absence of metabolites ranging from 16 to 19 nucleotides, suggesting little to no exonuclease products that are often seen with unmodified phosphorothioate oligodeoxynucleotides (Temsamani et al, 1994;Cummins et al, 1996;Gaus et al, 1997). The similarity of the excretion metabolite pattern across species suggests similar enzymes are responsible for metabolic cleavage of oligonucleotides.…”

Section: Discussionsupporting

confidence: 61%

Cross-Species Pharmacokinetic Comparison from Mouse to Man of a Second-Generation Antisense Oligonucleotide, ISIS 301012, Targeting Human Apolipoprotein B-100

Yu¹,

Kim²,

Hong³

et al. 2006

Drug Metab Dispos

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221

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ABSTRACT:The pharmacokinetics of a 2-O-(2-methoxyethyl)-modified oligonucleotide, ISIS 301012 [targeting human apolipoprotein B-100 (apoB-100)], was characterized in mouse, rat, monkey, and human. Plasma pharmacokinetics following parental administration was similar across species, exhibiting a rapid distribution phase with t 1/2␣ of several hours and a prolonged elimination phase with t 1/2␤ of days. The prolonged elimination phase represents equilibrium between tissues and circulating drug due to slow elimination from tissues. Absorption was nearly complete following s.c. injection, with bioavailability ranging from 80 to 100% in monkeys. Plasma clearance scaled well across species as a function of body weight alone, and this correlation was improved when corrected for plasma protein binding. In all of the animal models studied, the highest tissue concentrations of ISIS 301012 were observed in kidney and liver. Urinary excretion was less than 3% in monkeys and human in the first 24 h. ISIS 301012 is highly bound to plasma proteins, probably preventing rapid removal by renal filtration. However, following 25 mg/kg s.c. administration in mouse and 5-mg/kg i.v. bolus administration in rat, plasma concentrations of ISIS 301012 exceeded their respective protein binding capacity. Thus, urinary excretion increased to 16% or greater within the first 24 h. Albeit slow, urinary excretion of ISIS 301012 and its shortened metabolites is the ultimate elimination pathway of this compound, as demonstrated by 32% of dose recovered in total excreta by 14 days in a rat mass balance study. The pharmacokinetics of ISIS 301012 in human is predictable from the pharmacokinetics measured in animals. The pharmacokinetic properties of ISIS 301012 provide guidance for clinical development and support infrequent dose administration.

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Section: Discussionsupporting

confidence: 61%

Cross-Species Pharmacokinetic Comparison from Mouse to Man of a Second-Generation Antisense Oligonucleotide, ISIS 301012, Targeting Human Apolipoprotein B-100

Yu¹,

Kim²,

Hong³

et al. 2006

Drug Metab Dispos

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221

160

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“…Herein, we showed that our assay is highly sensitive and specific toward the parent compound and its metabolites, as it detects only the 3V-end intact sequence of G3139 at a single nucleotide resolution, with little or no cross-reactivity with the putative 3V-end metabolites (33,34). In contrast, the assay was not selective toward 5V metabolites; however, 5V-end metabolism has not been considered a major pathway (34 -36).…”

Section: Discussionmentioning

confidence: 99%

Cellular Uptake and Intracellular Levels of the Bcl-2 Antisense G3139 in Cultured Cells and Treated Patients with Acute Myeloid Leukemia

Dai

Chan

Liu

et al. 2005

Clinical Cancer Research

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Purpose: Down-regulation of Bcl-2 by the antisense G3139, currently under clinical evaluations, could restore chemosensitivity in otherwise resistant malignant cells. To date, the mechanism of intracellular accumulation of G3139 following in vivo administration remains to be elucidated.This study aimed to assess whether detectable intracellular concentrations of G3139 are achievable in vivo and how these relate to Bcl-2 down-regulation. Experimental Design: Cellular uptake of G3139 was studied in leukemia myeloid cell lines and blasts collected from treated patients using a newly developed, novel, and highly sensitive ELISAbased assay. Real-time reverse transcription-PCR was used to quantify Bcl-2 mRNA changes in treated cells. Results: The assay was fully validated and showed a limit of quantification of 50 pmol/L.When exposed to 0.33 to10 Amol/L G3139, K562 cells exhibited intracellular concentrations in the range of 2.1 to 11.4 pmol/mg protein. When G3139 was delivered with cationic lipids, a 10-to 25-fold increase of the intracellular concentrations was observed. There was an accumulation of G3139 in the nuclei, and the ratio of nucleus to cytoplasm was increased 7-fold by cationic lipids. Intracellular concentrations of G3139 were correlated with Bcl-2 mRNA down-regulation. Robust intracellular concentrations of G3139 were achieved in vivo in bone marrow (range, 3.4-40.6 pmol/mg protein) and peripheral blood mononuclear cells (range, 0.47-19.4 pmol/mg protein) from acute myeloid leukemia patients treated with G3139. Conclusions:This is the first evidence that measurable intracellular levels of G3139 are achievable in vivo in acute myeloid leukemia patients and that Bcl-2 down-regulation is likely to depend on the achievable intracellular concentrations rather than on plasma concentrations.

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“…These data are consistent with the nuclease resistance of 2Ј-MOE-modified nucleotides placed in the 3Ј-and 5Ј-terminal portions of ISIS 104838 . There was a notable absence of metabolites ranging from 16 to 19 nucleotides in length, suggesting little to no exonuclease products, which are often seen with unmodified phosphorothioate oligodeoxynucleotides (Temsamani et al, 1994;Gaus et al, 1997;.…”

Section: Discussionmentioning

confidence: 99%

PHARMACOKINETICS OF A TUMOR NECROSIS FACTOR-α PHOSPHOROTHIOATE 2′-O-(2-METHOXYETHYL) MODIFIED ANTISENSE OLIGONUCLEOTIDE: COMPARISON ACROSS SPECIES

Geary¹,

Yu²,

Watanabe³

et al. 2003

Drug Metab Dispos

159

128

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This article is available online at http://dmd.aspetjournals.org ABSTRACT:The pharmacokinetics of a 2-O-(2-methoxyethyl)-ribose modified phosphorothioate oligonucleotide, ISIS 104838 (human tumor necrosis factor-␣ antisense), have been characterized in mouse, rat, dog, monkey, and human. Plasma pharmacokinetics after i.v. administration exhibited relatively rapid distribution from plasma to tissues with a distribution half-life estimated from approximately 15 to 45 min in all species. Absorption after s.c. injection was high (80-100%), and absorption after intrajejunal administration in proprietary formulations was as high as 10% bioavailability compared with i.v. administration. Urinary excretion of the parent drug was low, with less than 1% of the administered dose excreted in urine after i.v. infusion in monkeys at clinically relevant doses (<5 mg/ kg). ISIS 104838 is highly bound to plasma proteins, likely preventing renal filtration. However, shortened oligonucleotide metabolites of ISIS 104838 lose their affinity to bind plasma proteins. Thus, excretion of radiolabel (mostly as metabolites) in urine (75%) and feces (5-10%) was nearly complete by 90 days. Elimination of ISIS 104838 from tissue was slow (multiple days) for all species, depending on the tissue or organ. The highest concentrations of ISIS 104838 in tissues were seen in kidney, liver, lymph nodes, bone marrow, and spleen. In general, concentrations of ISIS 104838 were higher in monkey tissues than in rodents at body weight-equivalent doses. Plasma pharmacokinetics scale well across species as a function of body weight alone. This favorable pharmacokinetic profile for ISIS 104838 provides guidance for clinical development and appears to support infrequent and convenient dose administration.

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On-Line HPLC Electrospray Mass Spectrometry of Phosphorothioate Oligonucleotide Metabolites

Cited by 113 publications

References 14 publications

Cross-Species Pharmacokinetic Comparison from Mouse to Man of a Second-Generation Antisense Oligonucleotide, ISIS 301012, Targeting Human Apolipoprotein B-100

Cross-Species Pharmacokinetic Comparison from Mouse to Man of a Second-Generation Antisense Oligonucleotide, ISIS 301012, Targeting Human Apolipoprotein B-100

Cellular Uptake and Intracellular Levels of the Bcl-2 Antisense G3139 in Cultured Cells and Treated Patients with Acute Myeloid Leukemia

PHARMACOKINETICS OF A TUMOR NECROSIS FACTOR-α PHOSPHOROTHIOATE 2′-O-(2-METHOXYETHYL) MODIFIED ANTISENSE OLIGONUCLEOTIDE: COMPARISON ACROSS SPECIES

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