To clarify the renal disposition characteristics of oligonucleotides at the organ level, the renal handling of model end-capped oligonucleotides, 3'-methoxyethylamine 5'-biotin-decathymidylic acid containing phosphoramidate modifications at 3'- and 5'-terminal internucleoside linkages (T10) and its phosphorothioate (Ts10), were studied in the perfused rat kidney. In a single-pass indicator dilution experiment, venous outflow and urinary excretion patterns and tissue accumulation of radiolabeled oligonucleotides were evaluated under filtering or nonfiltering conditions. No significant binding to bovine serum albumin (BSA) in the perfusate was observed for T10, whereas more than 90% of Ts10 bound to BSA. The steady-state distribution volume of T10 calculated from the venous outflow pattern was larger than that of inulin, which corresponds to the extracellular volume of the kidney, whereas the distribution volume of Ts10 was larger than that of BSA (the intravascular volume). These results suggested their interaction with the vascular wall. Rapid urinary excretion was observed for T10, similar to inulin used as a marker of golmerular filtration rate. On the other hand, urinary excretion of Ts10 was greatly restricted due to its high binding ability (> 90%) to BSA in the perfusate. A significant amount of T10 and Ts10 was accumulated in the kidney (T10, 1.8% of injected dose; Ts10, 1.3%) compared with inulin (0.2%) and BSA (< 0.1%). The accumulation of these oligonucleotides was ascribed to both tubular reabsorption and uptake from the capillary side. In addition, the uptake of T10 from the capillary side was significantly inhibited by simultaneous injection of dextran sulfate, suggesting that the oligonucleotide was taken up as an anionic molecule. These findings will be useful information for the development of delivery systems for antisense oligonucleotides.
The renal disposition characteristics of superoxide dismutase (SOD) and its derivatives, including macromolecular conjugates with polyethylene glycol and carboxymethyl-dextran, a cationized derivative, and glycosylated derivatives with galactose and mannose, were studied in the isolated perfused rat kidney. Renal disposition processes, such as glomerular filtration, tubular reabsorption, and uptake from the capillary side, were quantitatively determined by single-pass indicator dilution experiments under filtering and nonfiltering kidney conditions. Native SOD had a high glomerular filtration rate (40% of that of inulin) and was effectively reabsorbed in the tubules, while no significant uptake was observed from capillary side. Macromolecular conjugates showed restricted glomerular filtration due to an increase in molecular size. Cationization of SOD greatly enhanced its association with the tissue, not only from the luminal side but also from the capillary side, based upon electrostatic interaction. Galactosylated and mannosylated SOD showed reduced tubular reabsorption and increased exposure of the luminal surface to the enzyme. In addition, a small but significant uptake of mannosylated SOD from the capillary side was observed. This uptake was dose-dependent and completely inhibited by mannan, suggesting that mannose receptor-mediated endocytosis existed in the capillary side of the kidney. Thus, we can manipulate the renal disposition profiles of SOD by changing its physicochemical or biological properties through chemical modification.
Therapeutic effect of superoxide dismutase (SOD) and three derivatives: a conjugate with polyethylene glycol (SOD-PEG2), a cationized derivative (cSOD), and a mannosylated derivative (Man-SOD), on acute renal failure induced by ischemia/reperfusion was studied in rats. SOD and derivatives were administered intravenously to the rat after nephrectomy of the right kidney and before and after 60 min occlusion of the left renal artery. At 48 hr after reperfusion, the renal function was evaluated by determining the urinary excretion rate of 14C-inulin injected intravenously. No therapeutic effect on the impaired renal function was shown in the case of low dose SOD (2600 unit/kg) treatment. In contrast, administration of cSOD which was shown to be taken up by the isolated perfused kidney from its capillary side and SOD-PEG2 which maintained high plasma concentration exhibited significant therapeutic effect, as did SOD at ten-fold higher dose (26,000 unit/kg). On the other hand, renal damage was promoted by Man-SOD. Thus, the present study demonstrated that chemical modification may improve the therapeutic effect of SOD on the ischemic acute renal failure and increased SOD concentration in the renal vascular space is an important factor for the improved effect.
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