Purpose: This study was conducted to investigate the impact of backbone modifications on the hepatobiliary disposition of oligonucleotides.
Methods: The disposition of backbone-modified antisense oligonucleotides [phosphorothioate (PS) and methylphosphonate (MP)] of the same base-length and sequence (5'-TAC-GCC-AAC-AGC-TCC-3'), complementary to the codon 12 activating mutation of Ki-ras, was investigated in the isolated perfused rat liver. Livers were perfused for 2 hr: perfusate and bile concentrations were analyzed by HPLC. Hepatocellular distribution was examined by measuring the amount of radiolabeled PS oligonucleotide associated with hepatocytes and Kupffer cells. Protein binding of the PS and MP oligonucleotides was determined in rat serum by ultrafiltration.
Results: MP oligonucleotide perfusate concentrations remained constant during the 2-hour perfusion. In contrast, PS oligonucleotide was eliminated slowly by the isolated perfused liver [CI = 1.05 +/- 0.21 mL/min; extraction ratio = 0.06 +/- 0.01]. Uptake of PS oligonucleotide by Kupffer cells appeared to exceed uptake by hepatocytes, based on standard cell separation techniques as well as confocal microscopy. The degree of protein binding in rat serum was greater for the PS oligonucleotide (79.9 +/- 2.2%) than for the MP oligonucleotide (53.0 +/- 4.7%).
Conclusions: Backbone modifications significantly-influence the hepatic clearance of oligonucleotides. Uncharged MP oligonucleotides are not extracted by the isolated perfused rat liver, whereas the charged PS oligonucleotide is processed more readily.