title: Phosphorodiamidate morpholino antisense oligomers inhibit expression of human cytochrome P450 3A4 and alter selected drug metabolism.
abstract: Antisense phosphorodiamidate morpholino oligomers (PMO) inhibit targeted gene expression by preventing ribosomal assembly, thus preventing translation. Inhibition of cytochrome P450 (P450) 3A4 expression was examined in primary human hepatocytes from 11 donors and in Caco-2 cells (stably transfected with CYP3A4 cDNA on an extrachromosomal vector) by evaluating the metabolism of substrate 7-benzyloxy-4-[trifluoromethyl]-coumarin and Western immunoblot analysis. Cellular uptake of PMO was confirmed in both cell systems using fluorescein-labeled PMOs. Three antisense PMO sequences and two control PMO sequences were tested. AVI-4557, a 20-mer PMO with the sequence 5'-CTGGGATGAGAGCCATCACT-3' was selected as the optimal agent. AVI-4557 inhibited expression of CYP3A4 in Caco-2/h3A4 cells by 64% at 24 h following administration of 2.8 microM by an assisted delivery protocol. Inhibition of CYP3A activity was observed in primary human hepatocytes after 24 h exposure to AVI-4557 by an average of 32 +/- 11%. Furthermore, AVI-4557 exposure resulted in a sequence-dependent inhibition of cyclophosphamide-related cytocidal activity and a sequence-dependent induction of paclitaxel-related cytocidal activity in both cell types. Finally, the cytocidal activity of cisplatin was not affected with AVI-4557 treatment in either cell type. These studies indicate AVI-4557 is an effective and specific inhibitor of CYP3A4 expression.
authors.affiliation: Research and Development, AVI BioPharma, Corvallis, Oregon 97333, USA. varora@avibio.com
authors.forename: Vikram
authors.initials: V
authors.lastname: Arora
authors.forename: Melissa L
authors.initials: ML
authors.lastname: Cate
authors.forename: Chandramallika
authors.initials: C
authors.lastname: Ghosh
authors.forename: Patrick L
authors.initials: PL
authors.lastname: Iversen
mesh_terms: D018938:Caco-2 Cells; D002945:Cisplatin / Q000378:metabolism; D003374:Coumarins / Q000378:metabolism; D003520:Cyclophosphamide / Q000378:metabolism; D051544:Cytochrome P-450 CYP3A; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis*; D019793:Fluorescein / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism; D006131:Growth Inhibitors / Q000378:metabolism*; D022781:Hepatocytes / Q000187:drug effects / Q000201:enzymology; D006801:Humans; D009025:Morpholines / Q000737:chemistry / Q000494:pharmacology*; D016376:Oligonucleotides, Antisense / Q000737:chemistry / Q000494:pharmacology*; D017239:Paclitaxel / Q000378:metabolism; D017553:Phosphorus Compounds / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D013379:Substrate Specificity
chemical_list: C419147:7-benzyloxy-4-trifluoromethylcoumarin; D003374:Coumarins; D065607:Cytochrome P-450 Enzyme Inhibitors; D005456:Fluorescent Dyes; D006131:Growth Inhibitors; D009025:Morpholines; D016376:Oligonucleotides, Antisense; D017553:Phosphorus Compounds; D003520:Cyclophosphamide; D003577:Cytochrome P-450 Enzyme System; C436301:phosphorodiamidic acid; C104464:CYP3A protein, human; D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human; D017239:Paclitaxel; D002945:Cisplatin; D019793:Fluorescein