Drug Targeting Organ-Specific Strategies

5.3 Renal Delivery Using Macromolecular Carriers: The Low Molecular Weight Protein Approach 135

number of human tumours, having a high over-expression of a membrane-associated folate receptor, in-vitro studies have shown that folic acid derivatization allowed selective delivery to cancer cells in the presence of normal cells.Thus high tumour selectivity was achieved with folate-targeted imaging agents [57], antineoplastic drugs [58,59], protein toxins [60], liposomes [61] and antisense oligonucleotides [62].

Interestingly, after intravenous administration of a radiolabelled folate conjugate (111-In- dium-diethylenetriaminepenta acid (DTPA)-folate) in the rat, the conjugate was rapidly excreted in the urine. Moreover, after intravenous administration to athymic mice with a human tumour cell implant, the radiotracer was not only taken up by the subcutaneous tumour but was also taken up by the kidneys in significant quantities [63], indicating substantial renal selectivity of the folate conjugate. In addition to the kidney, the liver also has a high concentration of the folate-receptor [64].

5.2.3.3 Benefits and Limitations of Folate

To date, the possibility of using folate binding for the purpose of renal drug targeting has not been studied. Since the kidney is not the only organ containing folate-receptors, the physicochemical properties of the conjugate may be important determinants of the success of targeting.

5.3Renal Delivery Using Macromolecular Carriers: The Low Molecular Weight Protein Approach

5.3.1 Introduction

Low molecular weight proteins (LMWP) are freely filtered proteins with a molecular weight of less than 30 000 Dalton and are considered to be suitable as renal-specific drug carriers. The concept is based on four principles:

•The carrier has functional groups allowing drug attachment.

•The LMWP accumulates specifically in the kidney, in particular in the tubular cells through a reabsorption mechanism.

•The physicochemical properties of the LMWP overrule those of the linked drug.

•The drug–LMWP conjugate is stable in the circulation but after arrival in the kidney, the active drug is released in the catabolically-active lysosomes of the proximal tubular cells (Figure 5.7).

As reviewed by Franssen et al. [65], drugs can be directly coupled to LMWPs via the lysine amino group of the protein to form an amide bond. Alternatively, the drug can be coupled to the protein via different spacers such oligopeptides (amide bond), (poly)-alpha-hydroxy acids (ester bond), pH-sensitive cis-aconityl spacers (acid-sensitive amide bond) and SPDP spacers (disulfide bond) (see Chapter 11).The ability of the kidney to release the parent drug from such drug-spacer derivatives and drug–LMWP conjugates by enzymatic or chemical hy-

136 5 Delivery of Drugs and Antisense Oligonunucleotides to the Proximal Tubular Cell

Figure 5.7. Schematic representation of the mechanism by which drug targeting to the proximal tubular cell of the kidney might be achieved using a low molecular weight protein (LMWP) as a carrier.

drolysis of the bond, have been tested in renal cortex homogenates and lysosomal lysates as well as in in vivo studies. It was found that lysosmal proteases can cleave the peptide bond between the carboxylic acid group of a drug and an α-amino group of an amino acid. However, the bond between the carboxylic acid group of the drug and the ε-amino group of lysine could not be cleaved. Since the conjugation of drugs to amino groups of a protein will predominantly occur at the ε-lysine residues and only to a small extent at the N-terminal α-amino group, direct conjugation of a drug via its carboxylic acid group will not result in the quantitative regeneration of the parent compound [66]. Drugs with a terminal carboxyl group, such as naproxen [67], can be released as the parent drug from LMWP conjugates using ester spacers such as L-lactic acid. Increasing spacer length by intercalating a tetra (L-lac- tic acid) moiety between the drug and the protein further increases the rate of drug release, indicating increased accessibility of the bond to the enzymes.

Drugs that have primary amino groups available for conjugation, for instance dopamine and doxorubicin, can in principle be coupled to LMWPs via oligopeptides. In contrast to the carboxypeptidases, the aminopeptidases appear to possess a broader specificity. To allow the release of terminal amino group-containing drugs in the acid environment of the lysosomes without the requirement of enzymes, an acid-sensitive spacer can be used.

Drugs coupled via a disulfide bond like, captopril, are rapidly released from the proteinspacer moiety of the conjugate, enzymatically by β-lyase and/or non-enzymatically by thioldisulfide exchange with endogenous thiols [68].

The different aspects of drug targeting using LMWPs that have been studied to date are discussed below. As an example, we use the data of two conjugates, naproxen–lysozyme and captopril–lysozyme.

5.3 Renal Delivery Using Macromolecular Carriers: The Low Molecular Weight Protein Approach 137

5.3.2 Renal uptake of LMWP Conjugates

5.3.2.1 Renal Uptake of Native LMWPs

Comparison of the kinetic features of different LMWPs revealed that all LMWPs tested so far (such as lysozyme, cytochrome-c and aprotinin) are quickly cleared from the circulation and accumulate rapidly in the kidney [38]. The fractions of the injected LMWP that are reported to be taken up by the kidney vary between 40–80 % of the injected dose. In our studies, using external counting of radioactivity, at least 80 % of the intravenously injected LMWPs was finally taken up by the kidneys, which is in agreement with renal extraction studies [69,70]. However, studies in which the actual amount of LMWP in the kidney was measured directly in the tissue, indicated a lower, but still substantial accumulation of 40% of the injected dose [71,72]. Apart from the kidney, LMWPs do not seem to accumulate elsewhere in the body (Figure 5.8).

From this we concluded that LMWPs are potentially suitable to serve as renal-specific drug carriers: a drug–LMWP conjugate will be rapidly removed from the circulation and the drug can be intra-renally released. Consequently, major distribution to extra-renal tissue and related unwanted effects elsewhere in the body can, in principle, be avoided. It is assumed that secondary redistribution of the generated drug from the kidney is relatively slow so that systemic concentrations remain below the therapeutic window for extra-renal effects.

Figure 5.8. Renal specificity of a radiolabelled LMWP. Gamma-camera imaging after an intravenous injection of a radiolabelled low molecular weight protein (LMWP) in the rat, showing the predominant uptake of the LMWP by the kidneys.

5.3.2.2 Renal Delivery of Naproxen–Lysozyme

Targeting of nonsteroidal anti-inflammatory drugs (NSAIDs) such as naproxen could be of interest for the treatment of proteinuria and tubular defects such as Fanconi syndrome and Bartter’s syndrome [73,74]. Although a conjugate with an ester spacer is preferred to a conjugate with a direct peptide linkage [66,67], we continued our research using naproxen di-

138 5 Delivery of Drugs and Antisense Oligonunucleotides to the Proximal Tubular Cell

rectly conjugated to lysozyme. The synthesis of the conjugate with an ester spacer (naprox- en–L-lactic acid–lysozyme) is cumbersome, but fortunately the catabolite of the conjugate with the direct peptide linkage (naproxen–lysine) appeared to have an inhibitory effect on prostaglandin synthesis in vitro which was equivalent to that of the parent drug [66].

The coupling of 2 moles of naproxen to 1 mole of lysozyme did not affect the renal uptake of lysozyme in the rat: like native lysozyme, the conjugate rapidly accumulated in the kidney [75]. Focusing on the drug moiety of the conjugate, it was shown that conjugation of naproxen to lysozyme distinctly altered the kinetics of the drug. Conjugation to lysozyme resulted in a 70-fold increase in naproxen concentrations in the kidney (Figure 5.9a) [76].

a)

b)

Figure 5.9. The concentration–time course of (a) naproxen and (b) captopril in the kidney after intravenous injection of the parent drug or the drug–lysozyme (LZM) conjugate. Values are given as means + SEM.

5.3.2.3 Renal Delivery of Captopril–Lysozyme

Angiotensin-converting enzyme (ACE) inhibitors such as captopril exert a long-term renoprotective effect. Among other effects, they lower systemic blood pressure and renal plasma flow and effectively reduce urinary protein excretion. Renal delivery of ACE-inhibitors may increase this efficacy and reduce extra-renal side-effects. Renal targeting of an ACE-in- hibitor can also be useful in clarifying the contribution of local ACE inhibition to these renoprotective effects.

A spacer was used to link captopril via a disulfide bond to the LMWP lysozyme. Conjugation of captopril to lysozyme resulted in a 6-fold increase in captopril accumulation in the rat kidney (Figure 5.9b) [77]. This modest enrichment, as compared to that achieved with naproxen–lysozyme, was due to fact that, in contrast to naproxen, free captopril is cleared very efficiently by the kidney itself. Thus, delivery via lysozyme reabsorption only leads to a limited improvement of renal accumulation of captopril.

5.3 Renal Delivery Using Macromolecular Carriers: The Low Molecular Weight Protein Approach 139

Figure 5.10. Accumulation of a radiolabelled LMWP in the lysosomes of the proximal tubular cell. Electron microscope autoradiography of renal proximal tubular cells from a rat injected i.v. with [125I]- tyramine-cellobiose-labelled cytochrome-c, 4 h prior to fixation through the abdominal aorta. An intense lysosomal accumulation of the protein is observed in three dark electron-dense lysosomes . A few grains are seen over the apical endocytic apparatus. Part of the luminal brush border is found in the upper right hand corner. Magnification, x 25 000. Unpublished data from E. I. Christensen, Arhus, Denmark, and M. Haas, Groningen, Netherlands.

5.3.3 Renal Catabolism of LMWP-conjugates

5.3.3.1 Renal Catabolism of Native LMWPs

Morphological (Figure 5.10) and biochemical studies have established that after endocytosis by the proximal tubular cell, LMWPs migrate via endosomes to the proteolytically active lysosomes [78,79]. Within the lysosomes the LMWPs are degraded into small peptides and single amino acids. Whereas the renal uptake rate of various LMWPs appeared to be similar, LMWPs are catabolized with distinct individual differences in their catabolic rate as indicated from the difference in the rate of decline of radioactivity in the kidney (Figure 5.11). The rate of catabolism seemed unrelated to the size or charge of the protein alone [80,81]. Probably multiple structural factors play a role in this process. A crucial factor may be the different endosomal migration times of LMWPs from the tubular lumen to the lysosomes. Whereas cytochrome-c accumulated in the lysosomes within 3 min, lysozyme seemed to migrate for 20 min before the commencement of degradation [72, 82].Also the intrinsic activity of the reabsorbed protein may play a role. For instance, the long renal half-life of aprotinin, an inhibitor of proteolytic enzymes, may be explained by an inhibition of its own degradation, as suggested by Bianchi [71]. These studies suggest that the LMWP method of renal drug targeting results in cell-selective delivery followed by controlled drug release which can be manipulated at various stages of the renal deposition process. The lysosomes are stacked with a variety of proteolytic enzymes in an acidic environment. Programmed drug release from a drug–carrier conjugate may therefore be achieved using peptide, ester or acid-labile bonds

140 5 Delivery of Drugs and Antisense Oligonunucleotides to the Proximal Tubular Cell

Figure 5.11. Time course of clearance from the kidney of radiolabelled LMWPs after intravenous injection. After renal uptake, the radiolabelled protein is gradually catabolized and the radioactive breakdown products released from the kidney, as shown by the decline of renal radioactivity over time.

between the drug and protein carrier. Consequently both the differences in rate of catabolism between LMWPs as well as the rate of hydrolysis of the bond between the drug and carrier may be used to manipulate the rate of drug release in the kidney. The variable migration times of different LMWPs and their conjugates after endocytosis may have consequences for the intracellular concentration profiles. For instance, in order to achieve relatively constant cellular levels of the drug, an LMWP which is only slowly degraded might be preferred as a drug carrier. In contrast, if short-term peak levels of the drug are preferred, treatment with a rapidly processed protein (with a short migration time) may be a more appropriate choice. Certain drugs (e.g. peptides and nucleotides) should be released before entering lysosomes to prevent inactivation by degradative enzymes. For such drugs, a prolonged endosomal migration time combined with simple hydrolysis of the drug–protein linkage in the acidic environment of the endosomes, will be preferred to achieve adequate drug release and prevent an abortive route to the lysosomes.

5.3.3.2 Renal Catabolism of Naproxen–Lysozyme

The coupling of 2 moles of naproxen to 1 mole of lysozyme did not affect the catabolism of lysozyme in rat kidney [66,75]. After delivery to the kidney, naproxen in the form of naprox-

5.3 Renal Delivery Using Macromolecular Carriers: The Low Molecular Weight Protein Approach 141

en–lysine was gradually released from the conjugate. This catabolite was subsequently eliminated from the kidney and after a single injection, drug levels in the renal tissue gradually decreased with a t1/2 of 160 min (Figure 5.9a).

No detectable amounts of naproxen or its lysine conjugates were found in the plasma after administration of the conjugate and it can be inferred that excretion into the urine is the crucial process which determines the elimination rate t1/2. The lack of diffusion into the bloodstream is a favourable property in relation to unwanted extra-renal effects.

5.3.3.3 Renal Catabolism of Captopril–Lysozyme

After renal uptake, captopril was rapidly released from the conjugate as indicated by the rapid decrease in renal captopril levels with time (Figure 5.9b). The difference in renal t1/2 of naproxen and captopril after delivery with lysozyme is likely to be due to an unequal rate of release from the lysozyme conjugates. Whereas naproxen–lysozyme requires a peptidase for cleavage, captopril is released from the conjugate enzymatically by β-lyase and/or non-enzy- maticaly by thiol-disulfide exchange with endogenous thiols. To reduce the rate of capto- pril–lysozyme breakdown, two different cross-linking reagents, SPDP and SMPT, were tested. Although an SMPT link between two proteins is in principle less susceptible to disulfide reduction [83], no difference in degradation rate was found between the SPDP and the SMPT captopril–lysozyme conjugates (Kok et al., unpublished data).

5.3.4 Effects of Targeted Drugs Using an LMWP as Carrier

5.3.4.1 Renal Effects of Naproxen–Lysozyme

Having obtained promising kinetic profiles, the potential renal effects of naproxen–lysozyme in the rat were investigated [84]. Naproxen, as an inhibitor of cyclooxygenase, blocks prostaglandin synthesis. Among other effects, naproxen reduced furosemide-stimulated urinary excretion of prostaglandin E2 (PGE2) as well as the natriuretic and diuretic effects of furosemide. Studies with the conjugate showed that naproxen–lysozyme treatment clearly prevents furosemide-induced excretion of PGE2. This occurred with a dose of naproxen that was not effective in the unconjugated form. Surprisingly, this effect occurred in the absence of a change in natriuretic and diuretic response to furosemide. In this respect the pharmacological effect differed from treatment with a high dose of free naproxen. An explanation for these differences remains to be found. One possibility is that there is a difference in the in- tra-renal kinetics of the NSAID compared with the parent drug. Free naproxen is extensively reabsorbed in the distal tubule of the kidney via which route it may effectively inhibit prostaglandin synthesis in the medullary interstitial cells. On the other hand, naproxen–ly- sine is more hydrophilic and may be unable to reach the sites of prostaglandin synthesis involved in the furosemide-induced excretion of sodium and water. These data shows that renal drug targeting preparations can also be used as a tool to unravel the mechanisms of renal therapeutic effects.

142 5 Delivery of Drugs and Antisense Oligonunucleotides to the Proximal Tubular Cell

5.3.4.2 Renal and Systemic Effects of Captopril–Lysozyme

With regard to the pharmacological effects of the captopril–lysozyme conjugate, the following observations were made (Kok et al., unpublished data). The extent of ACE-inhibition in the plasma and kidney tissue was measured after i.v. administration of captopril–lysozyme and an equimolar dose of free captopril. It was shown that conjugation to lysozyme caused a similar though more sustained inhibition of renal ACE-activity by captopril.The inhibition of plasma ACE-activity was clearly reduced but not entirely prevented by conjugation of captopril to lysozyme. Possibly, the S-S linked drug conjugate is partly degraded in the circulation. It is also possible that after degradation of the conjugate in the kidney, captopril was transported back into the bloodstream. The rapid intracellular release may provide a sufficient driving force for transport across the basolateral membranes.

Captopril–lysozyme did not significantly affect systemic blood pressure whereas an equimolar dose of captopril alone decreased blood pressure significantly. Whereas free captopril (5 mg kg–1) completely prevented an angiotensin-I-induced blood pressure increase, an equimolar amount of captopril–lysozyme did not. However, in line with the direct ACE activity measurements in renal tissue and plasma, in captopril–lysozyme-treated rats the an- giotensin-I-induced blood pressure increase was lower than in untreated rats, suggesting that systemic activity was not fully prevented.

Neither free nor conjugated captopril affected glomerular filtration. Renal plasma flow increased to the same degree after treatment with free or conjugated captopril (1 mg kg–1). Although the complete dose–effect relationship was not studied, we can conclude that conjugation of captopril to lysozyme did not prevent the drug from acting on the renal plasma flow. Whether this effect is determined by intra-renal or systemic ACE-inhibition remains to be investigated.

At present, the synthesis of lysozyme conjugates with ACE-inhibitors other than captopril is under investigation. Some of these ACE-inhibitors may be advantageous for renal delivery. The amount of conjugate required for therapy can be reduced when using an ACE-inhibitor with a higher affinity for ACE (e.g. lisinopril). Furthermore, the stability of the conjugate in plasma may be increased by using an ACE-inhibitor which is conjugated to lysozyme via a linkage that is highly stable in plasma (e.g. lisinopril can in principle be coupled via an acidsensitive spacer).

5.3.5 Renal Disease and LMWP Processing

Proteinuria is one of the most prominent abnormalities found in renal disease and is one of the factors held responsible for the progressive loss of renal function. As a consequence of the glomerular leakage of proteins, the proximal tubular cells are exposed to increasing amounts of protein. This pathological condition can be anticipated to influence the deposition and metabolism of protein-linked drugs. It is likely, in such a situation, that drug–LMWP conjugates will have to compete with the overload of protein for tubular uptake as well as for catabolism. The effect of proteinuria on the renal processing of LMWPs has been examined in a number of studies [85–92]. Collectively, these studies clearly indicate that the effect of proteinuria on renal uptake and degradation of LMWPs depends on the severity and dura-

5.3 Renal Delivery Using Macromolecular Carriers: The Low Molecular Weight Protein Approach 143

tion of the protein leakage. However, it should be noted that tubular reabsorption of LMWPs is only slightly reduced during adriamycin-induced chronic proteinuria [92]. With respect to LMWP catabolism, the data suggest that protein overload will lead to reduced proteolytic degradation. In that case, an acid labile spacer or a disulfide bond should be chosen to guarantee an adequate rate of drug release.

We found a difference in susceptibility to proteinuria between cationic LMWP cy- tochrome-c and neutral LMWP myoglobulin with respect to their catabolism. This may indicate that the effect of proteinuria on LMWP catabolism is determined by the proximal tubular segment in which the LMWPs and the protein overload are processed [88,89,93,94]. We speculate that, through coupling to a specific LMWP, drugs can be delivered specifically to those proximal tubular cells that are predominantly affected by proteinuria.This might be essential for drugs chosen to protect the tubular cell from further damage by proteinuria. In addition, it may be possible to use certain LMWPs as drug carriers to circumvent the protein- uria-affected cells. In that case, treatment of diseases unrelated to proteinuria will not be hindered by the severity of proteinuria.

5.3.6 Renal Delivery of High Doses of LMWPs

The renal cell responsible for the uptake of LMWPs is the proximal tubular cell. LMWPs are relatively freely filtered by the glomerulus and subsequently reabsorbed by the proximal tubular cell by megalin/gp330 receptor-mediated endocytosis [95]. In healthy individuals, the relatively moderate amounts of endogenous LMWPs are completely reabsorbed by the proximal tubular cells. However, for drug targeting purposes, larger doses of LMWP may be required. We compared the urinary loss of intact LMWP after intravenous administration of different doses of LMWP by either single dose injections or by continuous infusions in healthy rats. From these studies, we concluded that after a continuous low-dose infusion the non-reabsorbed fraction is considerably less than that after single high-dose injections. However, infusion could not entirely prevent the loss of intact LMWP into the urine (the loss was 8% of the dose after 100 mg lysozyme kg–1 over 6 h and rose to 33% following 1000 mg lysozyme kg–1 over 6 h).

Cojocel et al. demonstrated clear adverse effects after relatively high doses of lysozyme [96]. We studied these aspects in more detail and concluded that lysozyme should be given in a dose of less than 100 mg–1 kg–1 over 6 h to minimize the negative effects on systemic blood pressure, glomerular filtration and renal blood flow. From these data, it emerged that LMWPs are suitable to serve as drug carriers to the proximal tubular cell of the kidney. However, the conjugate should preferably be administered in low-dose by constant infusion to limit the systemic and renal toxicity and to reduce the urinary loss of the intact conjugate (unpublished data).

5.3.7 Limitations of the LMWP Strategy of Drug Delivery to the Kidney

Among the disadvantages of the LMWP strategy for the treatment of chronic renal disease are the requirement for parenteral administration and the possible immunogenicity of the

144 5 Delivery of Drugs and Antisense Oligonunucleotides to the Proximal Tubular Cell

drug conjugate. With respect to the administration route, the conjugate could possibly be administered subcutaneously or intramuscularly. This is a common administration route for polypeptide drugs such as insulin. If immunogenicity appears to be a serious limitation for chronic treatment, a synthetic polymer may be used as the ‘reabsorptive’ carrier instead [97,98].

For short-term clinical interventions with the aim of protecting the kidney during acute reperfusion or preventing allograft rejection after transplantation, the prerequisite of parenteral administration does not constitute a serious limitation.

5.4 Renal Delivery of Antisense Oligodeoxynucleotides

5.4.1 Introduction

Various macromolecular and pro-drug technologies designed to achieve selective renal drug accumulation and action have been discussed in the previous sections of this chapter. In these approaches, traditional drugs have been modified through coupling to carrier molecules. It is generally accepted that, at least in theory, antisense oligodeoxynucleotides (AS-ODN) offer a new approach for selective treatment [99,100].

In view of the preferential distribution of some AS-ODNs to the kidney the oligonucleotide backbone could even be employed for renal-specific drug delivery because of both their intrinsic activity and the potential of coupling of other agents to them.

Antisense refers to the use of single-stranded synthetic oligonucleotides to inhibit gene expression [99,100].The striking advantage of the antisense approach in comparison to traditional drugs is its potential for specificity. The binding affinity between the oligonucleotide and its target receptor is many orders of magnitude higher compared to that at other binding sites, as a result of the multiple interaction sites that exist on the target receptors [101]. Since affinity is proportional to the number of interactions between a drug and its receptor, the specificity of an AS-ODN depends on its length. The base pairing specificity of an AS-ODN of about 15-17 nucleotides in length appeared to be sufficient to inhibit only one target gene within the entire human genome [99]. For successful inhibition in vivo, the plasma and intracellular stability and the pharmacokinetic profile of the antisense molecule along with the turnover time of the inhibited gene are important determinants.

First, we will briefly review the different aspects that are of importance in the use of antisense for in vivo therapy. Second, we will describe the effects of antisense targeting to the proximal tubule of the kidney that have been obtained so far.

5.4.2 Mechanism of Action of Antisense Oligodeoxynucleotides

AS-ODN are designed to be complementary to the coding (sense) sequence of the mRNA in the cell. After hybridization to target sequences, translational arrest occurs via one of several putative mechanisms. The first mechanism is inhibition of transcription. Secondly, ASODN can prevent the synthesis of fully mature mRNA in the cytosol at the level of splicing,