MODULATION OF PLASMINOGEN-ACTIVATOR INHIBITOR TYPE-1 BIOSYNTHESIS IN-VITRO AND IN-VIVO WITH OLIGO(NUCLEOSIDE PHOSPHOROTHIOATE)S AND RELATEDCONSTRUCTS

Oligonucleotides with a nucleotide sequence complementary to various r egions of human plasminogen activator inhibitor type-1 (PAI-1) mRNA ha ve been studied as antisense inhibitors of expression of PAI-1 protein in cultured cells [human umbilical vein endothelial cells (HUVEC), hu man aortic smooth muscle cells, human hybrid endothelial cells]. Hexad eca(deoxyribonucleoside phosphorothioate) 13 complementary to a fragme nt of a signal peptide PAI-1 mRNA was found to be most active, giving ca. 70% inhibition of PAI-1 release in a time-and dose-dependent way. The stereo-regular All-S-p and All-R-p diastereomers of 13 were studie d and found to inhibit PAI-1 synthesis in HUVEC in a stereo-dependent manner, with the All-S-p diastereomer considerably more active than th e stereo-random construct and All-R-p isomer. The observed stereo depe ndent activity of oligonucleotide phosphorothioate constructs is presu mably governed by their resistance to nucleases. The corresponding pho sphodiester analogue of 13 was not active unless covalently bound at i ts 5'-end to a lipophilic alcohol residue (menthol, heptadecanol). The observed antisense activity of phosphodiester oligonucleotide bioconj ugates in cultured human hybrid endothelial cells was paralleled by th eir increased stability in human plasma with respect to unconjugated o ligonucleotide. The oligo(deoxyribonucleoside phosphorothioate) comple mentary to the same signal peptide region of rat PAI-1 mRNA was found to reduce the PAI-1 level in blood plasma of rats after intravenous ad ministration into the tail vein. The effect was both time-and dose dep endent. The same oligonucleotide was found to protect against arterial thrombus formation in the rat (lower incidence of venous thrombosis, lower thrombus weight, and increased occlusion time in experimentally induced thrombosis). An anti-PAI-l inhibitory activity has been indepe ndently reported for a 20-mer oligo(2'-O-methyl-ribonucleoside phospho rothioate) complementary to a 3'-untranslated region of human PAI-1 mR NA in cultured HUVEC and human aortic smooth muscle cells. (C) 1997 El sevier Science, Inc.