A nonradioisotope approach to study the in vivo metabolism of phosphorothioate oligonucleotides.
A 25-mer phosphorothioate oligodeoxynucleotide (GEM 91) complementary to the gag gene mRNA of HIV-1 virus was administered intravenously (i.v.) at a dose of 10 mg/kg/day for 8 weeks or 25 mg/kg single dose subcutaneously (SC) to adult Rhesus monkeys. No radioactive markers were used. A capillary gel electrophoresis (CGE) method with UV detection was used to determine the concentration of GEM 91 in plasma and the metabolite profile. The metabolite profile was virtually the same following a single dose of either 10 mg/kg i.v. or 25 mg/kg SC. A different metabolite profile was observed after 4 or 8 weeks of multiple i.v. doses of 10 mg/kg/day. The extract was subjected to matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOFMS) for positive identification. Mass spectrometry confirmed the major metabolic pathway in vivo to be via 3'-end exonuclease activity. The extract was then subjected to a hybridization-assisted ligation reaction in which only 5'-end intact metabolites were labeled. Analysis by CGE with laser-induced fluorescence (LIF) detection allowed each of these metabolites to be quantified with a limit of detection of 1 ppb (ng/ml). MALDI-TOFMS identified components digested from both ends of the DNA. This study demonstrates that the combination of quantitative CGE-LIF and MALDI-TOFMS yields a powerful and unique approach to study the metabolism of phosphorothioate oligonucleotides.
Cohen
A S
AS
Hybridon Inc., Worcester, MA 01605, USA.
Bourque
A J
AJ
Wang
B H
BH
Smisek
D L
DL
Belenky
A
A
D000818:Animals; D004334:Drug Administration Schedule; D015750:Genes, gag / Q000187:drug effects / Q000235:genetics; D015497:HIV-1 / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007275:Injections, Intravenous; D008253:Macaca mulatta; D020319:Oligodeoxyribonucleotides, Antisense; D016376:Oligonucleotides, Antisense / Q000008:administration & dosage* / Q000097:blood / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods; D013873:Thionucleotides / Q000008:administration & dosage* / Q000097:blood / Q000378:metabolism*
D020319:Oligodeoxyribonucleotides, Antisense; D016376:Oligonucleotides, Antisense; D013873:Thionucleotides; C079996:GEM91