Drug Targeting Organ-Specific Strategies

5.4 Renal Delivary of Antisense Oligodeoxynucleotides

145

processing and transport across the nuclear membrane. The third mechanism is inhibition of translation by hybridization of the AS-ODN to the sense sequence and thereby preventing the ribosome from reading the mRNA code. Translation can be inhibited by AS-ODN which bind to important sites for translation such as translation initiation sites, poly(A) signals, and protein-binding regulatory sites. Finally, AS-ODN hybridization to the mRNA initiates specific cleavage of the RNA strand by activated RNase H and this cleavage results in destruction of the coding sequence and inhibition of mRNA translation [101].

5.4.3 Stabilization of Antisense Oligodeoxynucleotides

Phosphodiester AS-ODN are poor candidates for use as therapeutic agents in vivo due to their sensitivity to 3′- and 5′- exo/endonucleases. Because of this, various chemical modifications to the oligonucleotide backbone have been introduced to improve enzymatic stability while preserving their ability to hybridize cognate targets. Most common examples include the phosphorothioated and methylphosphonated analogues which have a sulfur atom and a methyl group, respectively, substituted for a non-bridging oxygen atom (Figure 5.12).

Phosphorothioated AS-ODN retain their negatively charged groups in the phosphodiester backbone and have the ability to induce mRNA degradation via RNase H. However, these compounds have a somewhat lower binding affinity to the target sequence. Moreover, non- sequence-specific activity has been reported for phosphorothioated AS-ODN, probably due to their stronger protein binding capacity [102,103].

Unlike phosphorothioates, methylphosphonated AS-ODN are uncharged compounds with a higher cellular uptake than unmodified AS-ODN. Unfortunately, these compounds appeared to be ineffective in some cell lines. This might be explained by the formation of

Figure 5.12. Chemical structure of antisense oligodeoxynucleotides (AS-ODN). Phosphorothioate and methylphosphonate AS-ODN have a sulfur atom and a methyl group respectively, substituted for a nonbridging oxygen atom to increase stability to nucleases.

146 5 Delivery of Drugs and Antisense Oligonunucleotides to the Proximal Tubular Cell

diastereomers or the inability of methylphosphonates to induce mRNA degradation via RNase H [104].

To avoid the problem of chirality and to improve the potency and limit the non-specific actions of AS-ODN, new compounds are required. Synthesis of new AS-ODNs has further improved their nuclease stability, enhanced of cellular uptake and affinity through modification of the base, sugar and phosphate moieties of the oligonucleotides [105–108].

5.4.4Pharmacokinetic Aspects of Antisense Oligodeoxynucleotides and Renal Distribution

The tissue distribution of AS-ODN after a single intravenous injection has been studied extensively in many species including mouse [109], rat [110], monkey [111] and man [112]. The majority of pharmacokinetic studies have been performed using phosphorothioated ASODNs. In general, the pharmacokinetic profiles of AS-ODNs of varying lengths (up to 20mer) and base compositions are remarkably similar in all species.

In plasma, most of the phosphorothioated AS-ODNs are protein bound [113,114]. Cossum and co-workers revealed that albumin and α2-macroglobulin are responsible for this binding [114]. The protein binding capacity in rat was elevated after administration of doses higher than 15–20 mg kg–1 resulting in a dose-dependent increase in distribution volume and an increase in plasma clearance [115–117].

The rapid elimination from plasma following intravenous administration of phosphorothioated AS-ODN can be explained by a two compartment model in all species, i.e. an initial plasma half-life of less than 1 h [111,113,114] and a slower elimination half-life ranging between 20 and 50 h [111,113].

The kidneys and the liver primarily take up phosphorothioated AS-ODN after parenteral administration, accumulating more than 10% each, while the rest of the organs all accumulate less than 1% of the injected dose [110,111,114,116]. It is noteworthy that renal AS-ODN tissue levels exceed that of any other organ [110,113,114], as confirmed by the tissue to plasma ratios of approximately 85 and 20 for kidney and liver, respectively [113,118].

Autoradiographic studies of the kidney have shown the accumulation of AS-ODN to occur almost exclusively in the proximal tubular cells [110,119]. Oberbauer et al. reported that intravenously injected AS-ODN accumulated in proximal tubular cells, and electron microscopy revealed that AS-ODN did accumulate only in the brush border or lysosomal compartment. This implies that the AS-ODNs were not completely degraded after being taken up by the proximal tubule [110].

In the last 2 years, several AS-ODNs with modified backbone structures and sugar moieties have been developed and these are characterized by a significanty increased stability in plasma [107,108]. Chimeric AS-ODNs, consisting of a mixture of phosphorothioate and methylphosphonate nucleotides, also exhibited increased stability in plasma [106]. It is worth noting that these AS-ODNs also appeared to be more stable in various tissues including the kidney [106,107]. Agrawal et al. [105] and Crooke et al. [108] have shown that changes in the sugar moieties can further improve the tissue distribution of AS-ODNs in favour of the kidney.

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 ASODNs 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 ASODN 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].

148 5 Delivery of Drugs and Antisense Oligonunucleotides to the Proximal Tubular Cell

Elimination of phosphorothioated AS-ODN takes place primarily via the urine. Approximately 30% of the injected dose is found in the urine within 24 h [110,113].Althought in most cases only metabolites of AS-ODN could be demonstrated in the urine [110,118], Agrawal and co-workers described the excretion of intact AS-ODN in the urine after a dose of 30 mg kg–1 [126]. The saturation of plasma protein binding and proximal tubular uptake could explain this observation [114,116].

Excretion via faeces is a minor route of elimination, accounting for less than 10% of the administered dose [113, 126].

5.4.7 Effects of Antisense Targeting to the Proximal Tubule

Noiri et al. used AS-ODN to inhibit production of inducible nitric oxide synthase (iNOS) in an attempt to prevent NO production in an ischaemic kidney. A single intravenous injection of iNOS AS-ODN attenuated acute renal failure and reduced the morphological abnormalities [129].

Oberbauer et al. reported inhibition of a sodium/phosphate (Na/Pi-2) co-transporter by phosphorothioated AS-ODN. A single intravenous injection of the AS-ODN inhibited both the mRNA and the protein for the Na/Pi-2 co-transporter, and consequently suppressed luminal uptake of phosphate by the proximal tubules [130].

Wang et al. injected a Texas-red-labelled phosphorothioated AS-ODN into the dopamine 1A receptor in the rat renal interstitium. Fluorescence was detected after 24 h in both tubular epithelium and intra-renal vasculature. Treatment resulted in a 35% decrease in the dopamine 1A receptor protein, causing a reduction in urinary sodium excretion and urine output [131].

Rat kidneys were perfused ex situ with phosphorothioate intercellular adhesion molecule (ICAM)-1 AS-ODN and exposed to 30 min cold or warm ischaemia. After this time the kidneys were transplanted to syngeneic nephrectomized rats.Treatment with 10 mg antisense reduced the harmful effect of transplantation on renal function [132]

Cheng et al. showed that intravenous infusion of intracellular adhesion molecule (ICAM) AS-ODN markedly reduced ICAM-1 expression, alleviated infiltration of inflammatory cells and accumulation of extracellular matrix in the obstructed kidney of mice with unilateral obstruction of the ureter [133].

Repeated intravenous injection of osteopontin AS-ODN to Goodpasture syndrome rats blocked tubular osteopontin expression, attenuated monocyte infiltration and preserved renal plasma flow. No changes were found in osteopontin mRNA level, glomerular histology or proteinuria. The data suggest that interstitial inflammation as a consequence of glomerular disease can be prevented through a selective inhibition of tubular osteopontin expression u- sing osteopontin AS-ODN [134].

Continuous infusion of transforming growth factor-β (TGF-β) AS-ODN in diabetic mice, decreased kidney TGF-β levels and attenuated the increase in kidney weight, and decreased levels of α1(IV)collagen and fibronectin mRNAs.

The above described studies show that the renal proximal tubular cell is a good target for antisense therapy [135].

5.6 In-Vitro and In-Vino Models for Renal Targeting

149

5.4.8 Benefits and Limitations of Antisense Oligodeoxynucleotides

The introduction of therapy through the delivery of antisense oligodeoxynucleotides holds promise for the treatment of several diseases. It is more specific than conventional drugs while, in contrast with gene targeting, the effect is temporary so that therapy can be terminated when desired. Pharmacokinetic studies have revealed that the proximal tubular cell of the kidney is a suitable target for antisense therapy. Although recent studies have shown antisense oligodeoxynucleotides to be effective in the treatment of renal diseases, antisense targeting is however, a new approach to therapy and all the risks associated with it are not yet known.

5.5 Drugs for Renal Targeting

For the treatment of kidney diseases, several kinds of drugs are currently used. At present, angiotensin-converting enzyme inhibitors are the first choice drugs for the treatment of chronic kidney diseases that are characterized by loss of renal function and proteinuria [136]. These drugs exhibit only moderate side-effects. However, renal targeting of an ACE-in- hibitor may improve the therapy in certain cases. For example, when proteinuria is accompanied by normal blood pressure, hypotension due to ACE-inhibition limits the amount of drug that can be given. Renal inflammation such as glomerulonephritis and tubulointerstitial inflammation are treated with corticosteroids [137]. These drugs have serious side-effects. Renal targeting of these drugs may allow a more aggressive treatment of the inflammation. Also, local suppression of the immune system may be useful to prevent transplant rejection. However, it is as yet unknown whether suppression of the local immune system is sufficient or whether the systemic system should also be suppressed to prevent rejection [138]. Renal tumours are characterized by insensitivity to the common anti-tumour drugs [139]. This is probably due to the unfavourable kinetic profile of these drugs. By renal targeting, an antitumour drug may reach the renal tumour while the extra-renal side-effects will be reduced.

5.6 In-Vitro and In-Vivo Models for Renal Targeting

5.6.1 In-vitro Models

In the isolated perfused kidney model, the artery of the kidney is perfused and urinary samples as well as venous blood samples can be collected to determine the drug concentration.A serious drawback of the model is that isolation and artificial perfusion greatly affect the function of the organ as shown by a dramatic drop in the glomerular filtration rate. Another invitro model is the isolated tubule in which samples can be taken from both the luminal and basolateral sites of the tubule [140,141]. The disadvantage of this technique as well as of the isolated kidney model, is that they require specific equipment and expertise and therefore can only be performed in rather specialized laboratories. Experiments using freshly isolated or cultured cells are more simple to carry out [142,143]. Tubular cells can be grown in a po-

150 5 Delivery of Drugs and Antisense Oligonunucleotides to the Proximal Tubular Cell

larized fashion enabling the addition of drugs and removal of samples from both the luminal and basolateral sites of the cell.

5.6.2 In vivo Models

Obviously, none of the existing animal models of renal diseases are perfect reflections of the human situation. The natural model of progressive loss of renal function is the 5/6 nephrectomy. Drawbacks of this model are the large wound in the remaining kidney and the limited amount of tissue available for analysis. The two-kidney, one-clip Goldblatt model is a good model of renal vascular hypertension. Progressive loss of renal function due to essential hypertension can be studied using the spontaneously hypertensive rat (SHR). Several animal models for diabetic nephropathy exist [144]. Streptozotocin induces diabetes, resulting in a mild proteinuria and tubular dysfunction during the progression of the disease [145]. Also, animal models of spontaneous diabetes have been described [146].The diabetic nephropathy that develops in these models is likely to be a good reflection of the human situation since it is a consequence of the same initial disease.

Several models of toxic nephritis have been developed. After an intravenous injection of adriamycin or puromycin, a chronic nephropathy develops which is characterized by severe proteinuria and glomerular sclerosis [147,148]. The severity of the proteinuria is much higher compared to human proteinuria, while the reduction in the glomerular filtration rate is limited. The progression of proteinuria after puromycin injection occurs in two phases, while adriamycin causes a gradual continuous increase in proteinuria. Overload proteinuria is a model in which bovine albumin is repeatedly injected into rats in large quantities [149]. The proteinuria is less aggressive than in the adriamycin and puromycin models and the model seems to be a better reflection of the human situation. However, this model is more difficult to set up. Several toxic agents, like cyclosporin and cadmium, accumulate in the proximal tubular cell, causing severe tubular damage. These models are a good reflection of tubular damage by toxic agents in humans.

For glomerulonephritis, several immunological models are available [150]. For example, injection of an antibody against thymocytes (anti-Thy 1.1 nephritis) causes a rapid mesangiolysis followed by proliferation [151,152].

In addition to other models [153], tubulointerstitial inflammation and fibrosis can be obtained by ureter obstruction. The inflammation develops very rapidly and is severe. The model is a good reflection of ureter obstruction in humans. However, a serious drawback in using this model for tubular drug delivery studies is the fact that glomerular filtration is absent.

5.7 Concluding Remarks

In this chapter, macromolecular and pro-drug approaches for cell-selective therapeutic intervention in the proximal tubular cell have been described. Using a low-molecular weight protein as a drug carrier, the drug is delivered to the lysosomes of the proximal tubular cell

References 151

via reabsorption from the luminal site. Lysosomal delivery allows drug attachment via an acid-sensitive spacer or via biodegradable peptide or ester moieties. Using the alkylglycoside vector as a drug carrier, the drug is taken up via the basolateral site into the proximal tubular cell. It is as yet unknown to which compartment of the proximal tubular cell the drug is delivered using this carrier, and the subsequent stages such as drug release as well as the kinetics and dynamics during renal diseases remain to be studied. Yet, a basolateral delivery may be advantageous during severe reduction of glomerular filtration and presence of proteinuria. On the other hand, with low-molecular weight proteins a broader range of drugs (with respect to their physicochemical properties) can be delivered to the proximal tubular cells.

Oligonucleotide targeting to the kidney is more feasible than to many other tissues as a result of the glomerular filtration and tubular reabsorption of these poly-anionic agents.The effect is temporary allowing the therapy to be terminated when desired. Up until now, data has only been available on the kinetics and some renal and extra-renal effects of oligonucleotides in healthy animals.

The most relevant studies examining the effects of drug targeting in experimental disease, are yet to come.These studies may provide clues to the role of the proximal tubular cell in the various renal diseases and may determine whether treatment of renal disease can be accomplished by drug targeting to the proximal tubular cell. A further goal of renal targeting is the specific delivery of drugs to the filtration unit of the kidney, the glomerulus, which is also believed to play an important role in the progression of renal disease. Until recently only limited research has been focused on this target in the kidney [76].

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