Antinociceptive effects after intrathecal administration of phosphodiester-, 2'-O-allyl-, and C-5-propyne-modified antisense oligodeoxynucleotides targeting the NMDAR1 subunit in mouse.
In the present study, we have compared the antinociceptive effect of three different types of antisense oligodeoxynucleotides targeting the N-methyl-D-aspartate (NMDA) R1-subunit in mice. The probes were administrated intrathecally three times during a period of 5 days (1, 5 or 25 microg/injection), followed by evaluation using the formalin test. The antinociceptive effect was correlated to in vitro receptor binding in spinal cord sections. The tissue distribution was studied after a single injection of fluorescein-conjugated probes. The phosphodiester probe showed superficial tissue penetration after 30 min and disappeared within 2 h. The probe did, however, significantly reduce both receptor binding in laminae I and II (by 36-44% compared to saline) as well as pain behavior (32% compared to saline), without apparent side effects. The mismatched probe was ineffective at 25 microg, while some reductions in receptor binding and pain behavior were seen after 5 microg. The C-5-propyne-modified phosphorothioate probe showed pronounced tissue penetration and cellular uptake as soon as 30 min after injection which was still detectable after 24 h. Immediately after injection of the highest dose, long-lasting hind-limb paralysis was observed. Receptor binding was reduced but not in a dose-related manner. Pain behavior was significantly reduced by 40% following 25 microg of antisense probe but not after lower doses or 25 microg of mismatched probe. The 2'-O-allyl-modified probe did not significantly reduce receptor binding or pain behavior. Thus, only the phosphodiester probe showed a significant correlation between reduction in pain behavior and receptor binding. These findings demonstrate that antisense technology is associated with specificity problems, but still could provide a valuable tool to study the role of different target proteins in the drug discovery process.
Rydh-Rinder
M
M
AstraZeneca R&D, Södertälje, Sweden. malin.ryidh-rinder@astrazeneca.com
Berge
O G
OG
Hökfelt
T
T
D015763:2-Amino-5-phosphonovalerate / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D000818:Animals; D018691:Excitatory Amino Acid Antagonists / Q000378:metabolism / Q000494:pharmacology; D016650:Fluorescein-5-isothiocyanate; D005456:Fluorescent Dyes; D015316:Genetic Therapy / Q000379:methods*; D007278:Injections, Spinal; D008297:Male; D051379:Mice; D008815:Mice, Inbred Strains; D009619:Nociceptors / Q000187:drug effects / Q000502:physiology*; D020319:Oligodeoxyribonucleotides, Antisense / Q000737:chemistry / Q000493:pharmacokinetics*; D059408:Pain Management; D010147:Pain Measurement; D011869:Radioligand Assay; D016194:Receptors, N-Methyl-D-Aspartate / Q000235:genetics*; D013116:Spinal Cord / Q000378:metabolism; D014316:Tritium
D015763:2-Amino-5-phosphonovalerate; D018691:Excitatory Amino Acid Antagonists; D016650:Fluorescein-5-isothiocyanate; D005456:Fluorescent Dyes; D020319:Oligodeoxyribonucleotides, Antisense; D016194:Receptors, N-Methyl-D-Aspartate; D014316:Tritium; C069052:CGP 39653