HEPATIC DISTRIBUTION AND CLEARANCE OF ANTISENSE OLIGONUCLEOTIDES IN THE ISOLATED-PERFUSED RAT-LIVER

Purpose. This study was conducted to investigate the impact of backbon e modifications on the hepatobiliary disposition of oligonucleotides. Methods. The disposition of backbone-modified antisense oligonucleotid es [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 isola ted perfused rat liver. Livers were perfused for 2 hr; perfusate and b ile concentrations were analyzed by HPLC. Hepatocellular distribution was examined by measuring the amount of radiolabeled PS oligonucleotid e associated with hepatocytes and Kupffer cells. Protein binding of th e PS and MP oligonucleotides was determined in rat serum by ultrafiltr ation. Results. MP oligonucleotide perfusate concentrations remained c onstant during the 2-hour perfusion. In contrast, PS oligonucleotide w as eliminated slowly by the isolated perfused liver [Cl = 1.05 +/- 0.2 1 mL/min; extraction ratio = 0.06 +/- 0.01]. Uptake of PS oligonucleot ide by Kupffer cells appeared to exceed uptake by hepatocytes, based o n standard cell separation techniques as well as confocal microscopy. The degree of protein binding in rat serum was greater for the PS olig onucleotide (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 char ged PS oligonucleotide is processed more readily.