Drug Targeting Organ-Specific Strategies

5.4 Renal Delivery of Antisense Oligodeoxynucleotides 147
5.4.5 Cellular Uptake of Antisense Oligonucleotides
Cellular uptake of AS-ODNs is restricted because of their large molecular mass as well as
their polyanionic character. When added directly to cells in culture, only 1–2% of the AS-
ODNs will be cell-associated. Therefore, enhanced AS-ODN uptake is a critical consideration
in developing these agents for therapeutic applications.
The cellular uptake of AS-ODN is an energy-dependent process and takes place in a saturable
and sequence-independent manner [120,121]. The exact mechanism of uptake remains
controversial. From in vitro experiments, some authors have proposed that the uptake
is endocytic and mediated by membrane receptor proteins. The receptor responsible for the
cellular uptake of AS-ODNs was reported to consist of both a 30-kDa protein [122] and an
80-kDa membrane protein [121]. However, other workers have argued that AS-ODN binding
to membrane proteins is relatively non-specific and is mostly charge associated, consistent
with adsorptive endocytosis or fluid-phase pinocytosis [101]. As a result of these conflicting
reports, it is unlikely that in vitro data can be safely extrapolated to what occurs in the
intact organism.
In the kidney, AS-ODNs are filtered and subsequently reabsorbed by the proximal tubular
cells. The AS-ODNs most likely accumulate in the proximal tubular cells via a receptordependent
mechanism [110,123]. This hypothesis supports the apparent saturation of AS-
ODN uptake in the kidney as reflected by a reduction of degree of renal uptake with increasing
AS-ODN dose [110,116,117]. Moreover, Rappaport et al. described the existence of
40 and 97-kDa binding proteins for 18mer phosphorothioates in the renal brush border
membrane [123]. In another study, a protein with a molecular weight of approximately
50 kDa which may serve as a transmembrane channel transporting AS-ODN into the tubular
cell was described [124]. These channels have previously been reported for the uptake of
proteins and phage DNA. The presence of such channels might explain why uptake in the
proximal tubular cells is dependent on the nucleotide length as was demonstrated by Loke
and co-workers [121]. It is noteworthy that scavenger receptors located at the basolateral site
may also be responsible for additional tubular accumulation of AS-ODN [125].
5.4.6 Metabolism and Elimination of Antisense Oligodeoxynucleotides
A prerequisite to acquire an antisense effect is the maintenance of AS-ODN within the target
cells. Several studies have reported that the majority of phosphorothioated AS-ODNs
taken up by the kidney remains intact for several hours [110,113]. In fact, 4 days after administration,
3% of the infused dose was still present in the kidney intactly [110]. Although
several studies have confirmed the presence of intact AS-ODN in the kidney, concomitant
metabolism in the kidney of 20% after 6 h [113], 50% after 48 h [118,126] and 50% after
4 days [114] has also been reported.
In spite of the improved stability to nucleases, achieved through chemical modification,
AS-ODN degradation in plasma still occurs, predominantly from the 3′-terminus. In the liver
and kidney, the major sites of metabolism, AS-ODNs are degraded from the 5′-terminus
as well [127,128].