Hepatobiliary elimination of bile acid-modified oligodeoxynucleotides in Wistar and TR- rats: evidence for mrp2 as carrier for oligodeoxynucleotides


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Abstract

As therapeutic antisense tools, oligonucleotides (ODNs) must enter cells to bind to their target structures. ODNs distribute in nearly each tissue with relatively high concentrations in kidney and liver from where excretion into urine and bile occurs. To investigate mechanisms involved in hepatic ODN transport, normal mixed backbone phosphodiester/phosphorothioate ODNs (n-ODN) and two different bile acid-conjugated mixed backbone ODNs (1BA-ODN and 2BA-ODN) were applied to two different rat strains, normal Wistar rats and Wistar TR- rats. In normal Wistar rats, concentration-dependent hepatobiliary elimination of the ODNs was observed with a remarkable increase of excretion of the cholic acid BA-ODN conjugates. In contrast to normal Wistar rats, n-ODN excretion into bile by TR- rats, a mutant Wistar rat strain lacking a functional multidrug resistance-associated protein 2 (mrp2) at the canalicular membrane, was strongly diminished, whereas these rats excreted an ODN conjugated with two cholic acid molecules (2BA-ODN) into bile. Concomitant application of substrates transported by mrp2 such as bromosulfophthalein (BSP) or the synthetic chlorogenic acid derivative S 3025 significantly reduced the biliary appearance of normal ODN and 2BA-ODN in Wistar rats and also in TR- rats. To inhibit the expression of cRNA derived from the Na+-dependent taurocholate cotransporting polypeptide (Ntcp), antisense ODNs were constructed which fully retained the antisense properties when coupled with two bile acid molecules. The results indicate that ODNs are secreted via the mrp2 into bile. In the absence of mrp2, further excretory transport systems with affinity for bile acids seem to be relevant for their excretion. The results further indicate that bile acid tagged ODNs are useful tools for liver specific antisense therapy.

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