Preproenkephalin targeted antisenses cross the blood-brain barrier to reduce brain methionine enkephalin levels and increase voluntary ethanol drinking.
Antisense potentially can manipulate target gene expression in the brain if it can cross the blood-brain barrier (BBB). We designed three (10mer, 17mer, and 19mer) phosphorothioated antisenses (PS-ODNs) directed against the precursor molecule of methionine enkephalin (Met-Enk), an opiate peptide which suppresses voluntary ethanol drinking. We measured the ability of the antisenses to cross the BBB, accumulate in the brain and CSF, decrease levels of Met-Enk in brain and blood, and affect voluntary ethanol drinking. Each antisense readily crossed the BBB, with 0.07-0.16% of the i.v. dose accumulating per gram of brain. Capillary depletion and CSF sampling each confirmed that the antisenses entered the CNS. Gel electrophoresis of radioactivity recovered from brain and serum showed intact antisense and a higher molecular weight form likely representing antisense bound to protein, but no degradation products. Each antisense molecule and a cocktail of all three reduced Met-Enk levels in brain and serum. Met-Enk levels in the brain were reduced more rapidly and for a longer duration than Met-Enk levels in the serum, indicating a degree of selective targeting to the CNS. Additionally, administration of the cocktail was more effective in reducing Met-Enk levels than any of the individual antisenses. Each antisense increased voluntary ethanol drinking by about 20% and the cocktail increased it by about 80%. Taken together, these results used pharmacokinetic, immunochemical, and behavioral methods to show that PS-ODN antisenses that readily cross the BBB can decrease brain levels of Met-Enk and increase voluntary ethanol drinking.
Banks
William A
WA
Veterans Affairs Medical Center and Division of Geriatrics, Department of Internal Medicine, Saint Louis University School of Medicine, 915N. Grand Blvd, St. Louis, MO 63106, USA. bankswa@slu.edu
Jaeger
Laura B
LB
Urayama
Akihiko
A
Kumar
Vijaya B
VB
Hileman
Stanley M
SM
Gaskin
Frederich S
FS
Llanza
Nancy V
NV
Farr
Susan A
SA
Morley
John E
JE
D000818:Animals; D001692:Biological Transport; D001812:Blood-Brain Barrier; D001921:Brain / Q000378:metabolism*; D004327:Drinking Behavior / Q000502:physiology*; D004744:Enkephalin, Methionine / Q000172:deficiency* / Q000378:metabolism; D004745:Enkephalins / Q000096:biosynthesis / Q000235:genetics*; D000431:Ethanol / Q000008:administration & dosage*; D018390:Gene Targeting; D007275:Injections, Intravenous; D008297:Male; D011498:Protein Precursors / Q000096:biosynthesis / Q000235:genetics*; D016372:RNA, Antisense / Q000235:genetics / Q000378:metabolism*
D004745:Enkephalins; D011498:Protein Precursors; D016372:RNA, Antisense; D000431:Ethanol; D004744:Enkephalin, Methionine; C033660:preproenkephalin