Elsevier

Analytical Biochemistry

Volume 274, Issue 2, 15 October 1999, Pages 241-248
Analytical Biochemistry

Regular Article
A Nonradioisotope Biomedical Assay for Intact Oligonucleotide and Its Chain-Shortened Metabolites Used for Determination of Exposure and Elimination Half-Life of Antisense Drugs in Tissue

https://doi.org/10.1006/abio.1999.4290Get rights and content

Abstract

Rigorous extraction methods coupled with capillary gel electrophoresis (CGE) provide a basis for a nonradiolabel assay for quantitation of intact antisense drug and its numerous chain-shortened metabolites. As part of the validation of the CGE method, we compared the quantitation of unlabeled ISIS 3521 (ISI 641A) and its chain-shortened metabolites with total radioactivity of [35S]-ISIS 3521. ISIS 3521 was labeled on the fifth nucleotide linkage from the 5′-end with 35S by well-established methods. Multiple tissues collected from rats after administration of [35S]-ISIS 3521 were assayed by both radiolabel (liquid scintillation spectroscopy) and CGE methods. The CGE method provided accurate quantitation of the drug and its metabolites in kidney cortex and liver tissues. The correlation between methods for multiple tissues over time was excellent with 88.5% of the measurements being statistically equivalent. These data suggest that CGE is an accurate means of quantitating oligonucleotide in tissue and that it compares favorably with traditional radiochemical techniques. Clearance half-lives for total measurable oligonucleotides were equivalent to clearance of total radioactivity in both liver and kidney with the longest clearance half-life associated with the kidney.

References (27)

  • N.M. Dean et al.

    J. Biol. Chem.

    (1994)
  • J.A. Phillips et al.

    Biochem. Pharmacol.

    (1997)
  • J. Rappaport et al.

    Kidney Int.

    (1995)
  • J.M. Leeds et al.

    Anal. Biochem.

    (1996)
  • L.L. Cummins et al.

    Bioorg. Med. Chem. Lett.

    (1997)
  • M. Andrade et al.

    Bioorg. Med. Chem. Lett.

    (1994)
  • R. Oberbauer et al.

    Kidney Int.

    (1995)
  • T. Yazaki et al.

    Mol. Pharmacol.

    (1996)
  • Y.-S. Lee et al.

    Mol. Carcinogen.

    (1997)
  • A.F. Dorr

    Proceedings, X111th International Congress of Pharmacology

    (1998)
  • P.A. Cossum et al.

    J. Pharmacol. Exp. Ther.

    (1994)
  • P.A. Cossum et al.

    J. Pharmacol. Exp. Ther.

    (1993)
  • S. Agrawal et al.

    Proc. Natl. Acad. Sci. USA

    (1991)
  • Cited by (30)

    • Elucidation of the Biotransformation Pathways of a Galnac<inf>3</inf>-conjugated Antisense Oligonucleotide in Rats and Monkeys

      2016, Molecular Therapy Nucleic Acids
      Citation Excerpt :

      In light of these observations, and based on internal data, previous animal studies revealed that GalNAc sugars and the trishexylamino (THA) cluster were rapidly cleaved within minutes to hours from GalNAc-conjugated ASOs following subcutaneous (SC) administrations, releasing unconjugated ASOs in liver to exhibit their pharmacological effects. It is well understood that unconjugated ASOs are slowly metabolized to chain-shortened oligonucleotide metabolites by nucleases and eliminated in urine.9 Hence, the distribution, metabolism, and fate of unconjugated ASOs are well established, yet the metabolic pathway of the THA cluster containing GalNAc3 as a targeting moiety has never been reported and is important to help understand the efficacy and safety of the conjugated ASO.

    • Renal uptake and tolerability of a 2'-O-methoxyethyl modified antisense oligonucleotide (ISIS 113715) in monkey

      2012, Toxicology
      Citation Excerpt :

      Representative samples of both kidneys were stained with hematoxylin and eosin and processed from all animals for microscopic examination. Tissue samples were analyzed using a validated capillary gel electrophoresis (CGE) method (Geary et al., 1999). The linear calibration range of the assay was 0.15–20 μM (1.14–151 μg/g for parent compound), with the low end of this range defining the lower limit of quantitation (LLOQ).

    • Biodistribution and metabolism of immunostimulatory oligodeoxynucleotide CPG 7909 in mouse and rat tissues following subcutaneous administration

      2005, Biochemical Pharmacology
      Citation Excerpt :

      Liver and kidney showed a much higher percentage of metabolites than injection site and local lymph nodes, where 3′-exonuclease activity as well as endonuclease activity appeared to be lower. This may be due to different organ distribution of degrading enzymes [29] rather than a reflection of differences in the disposition properties of the individual metabolites. We have no indication for differential distribution pathways for different metabolites.

    View all citing articles on Scopus
    View full text