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(The FASEB Journal. 2000;14:1784-1792.)
© 2000 FASEB

Novel hepatotrophic prodrugs of the antiviral nucleoside 9-(2-phosphonylmethoxyethyl)adenine with improved pharmacokinetics and antiviral activity

E. A. L. BIESSEN*1, A. R. P. M. VALENTIJN{dagger}, R. L. A. DE VRUEH*, E. VAN DE BILT*, L. A. J. M. SLIEDREGT*, P. PRINCE*, M. K. BIJSTERBOSCH*, J. H. VAN BOOM{dagger}, G. A. VAN DER MAREL{dagger}, P. J. ABRAHAMS{ddagger} and T. J. C. VAN BERKEL*

* Division of Biopharmaceutics, LACDR,
{dagger} Department of Bio-Organic Chemistry, LIC, Leiden University; and
{ddagger} Department of Radiation Genetics and Chemical Mutagenesis, Leiden University Medical Center, Leiden, The Netherlands

1Correspondence: Division of Biopharmaceutics, Leiden/Amsterdam Center for Drug Research, Sylvius Laboratories, Wassenaarseweg 72, Leiden, The Netherlands. E-mail: biessen{at}lacdr.leidenuniv.nl


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The device of new hepatotrophic prodrugs of the antiviral nucleoside 9-(2-phosphonylmethoxyethyl)adenine (PMEA) with specificity for the asialoglycoprotein receptor on parenchymal liver cells is described. PMEA was conjugated to bi- and trivalent cluster glycosides (K(GN)2 and K2(GN)3, respectively) with nanomolar affinity for the asialoglycoprotein receptor. The liver uptake of the PMEA prodrugs was more than 10-fold higher than that of the parent drug (52±6% and 62±3% vs. 4.8±0.7% of the injected dose for PMEA) and could be attributed for 90% to parenchymal cells. Accumulation of the PMEA prodrugs in extrahepatic tissue (e.g., kidney, skin) was substantially reduced. The ratio of parenchymal liver cell-to-kidney uptake—a measure of the prodrugs therapeutic window—was increased from 0.058 ± 0.01 for PMEA to 1.86 ± 0.57 for K(GN)2-PMEA and even 2.69 ± 0.24 for K2(GN)3-PMEA. Apparently both glycosides have a similar capacity to redirect (antiviral) drugs to the liver. After cellular uptake, both PMEA prodrugs were converted into the parent drug, PMEA, during acidification of the lysosomal milieu (t1/2{approx}100 min), and the released PMEA was rapidly translocated into the cytosol. The antiviral activity of the prodrugs in vitro was dramatically enhanced as compared to the parent drug (5- and 52-fold for K(GN)2-PMEA and K2(GN)3-PMEA, respectively). Given the 15-fold enhanced liver uptake of the prodrugs, we anticipate that the potency in vivo will be similarly increased. We conclude that PMEA prodrugs have been developed with greatly improved pharmacokinetics and therapeutic activity against viral infections that implicate the liver parenchyma (e.g., HBV). In addition, the significance of the above prodrug concept also extends to drugs that intervene in other liver disorders such as cholestasis and dyslipidemia.—Biessen, E. A. L., Valentijn, A. R. P. M., de Vrueh, R. L. A., van de Bilt, E., Sliedregt, L A. J. M., Prince, P., Bijsterbosch, M. K., van Boom, J. H., van der Marel, G. A., Abrahams, P. J., van Berkel, T. J. C. Novel hepatotrophic prodrugs of the antiviral nucleoside 9-(2-phosphonylmethoxyethyl)adenine with improved pharmacokinetics and antiviral activity.


Key Words: asialoglycoprotein • HBV • HSV-1 • drug targeting • liver


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
WORLDWIDE, CHRONIC HEPATITIS B virus (HBV) infection is one of the most prominent infections. The most frequently applied therapies based on interferon alpha and liver transplantation are only partially effective. Interferon alpha results in a complete seroconversion in only one-third of the treated patients and is accompanied by serious dose-dependent side effects, whereas the success rate of the latter is limited due to recurrent HBV infection (1 2 3) . This has led to the device of drugs with a more specific antiviral activity (4) . Promising results were recently reported for nucleoside analogs like lamivudine and famciclovir, which combine a high antiviral efficacy with a low toxicity (5 , 6) . The emergence of resistant viral strains, however, indicates that a broader panel of antiviral therapeutics is imperative (7 8 9) . Accordingly, a new generation of potent anti-HBV drugs was developed: the acyclic nucleoside phosphonates (4 , 10 , 11) . The therapeutic potential of these phosphonates is considerably reduced due to extensive renal clearance and low hepatic uptake (12 13 14) . High doses are therefore required for antiviral activity, which narrows the therapeutic window (10) and may lead to nephrotoxicity (13 , 14) .

The biological fate of these phosphonates may be considerably improved by site-specific delivery. Various carriers have been suggested for the delivery of anti-HBV drugs to the liver parenchyma, including modified (neo)lipoproteins (15 , 16) , (neo)glycoproteins (17 18 19) , and polymers (20 21 22 23) . Specific delivery to the aimed target could be realized by providing the carrier with substrates for the remnant and the asialoglycoprotein receptor, which are specifically located on parenchymal liver cells to mediate the uptake and lysosomal processing of chylomicron remnant (24) and galactose-terminated glycoproteins, respectively (25) . Fiume and co-workers were the first to demonstrate that the biodistribution of antiviral nucleosides like 9-beta-D-arabinofuranosyladenine 5'-monophosphate (ara-AMP) could be markedly improved by conjugation to lactosylated human serum albumin (17 18 19) . The albumin conjugates were generally tolerated quite well by mice and HBV-infected patients even after repeated injection, although occasionally immune responses were observed (17 , 19) . The carrier-conjugated ara-AMP efficiently reduced viremia without causing neurotoxic side effects at doses of 35–50 mg carrier/kg·day (18) . Lactosylated poly-L-lysine may be even a better carrier for drug delivery to the liver parenchyma as it can be administered intramuscularly (i.m.), synthesized in a chemically controlled fashion, and can handle a heavy drug load (21 , 22) . Uptake of poly-L-lysine immobilized nucleosides by liver parenchyma could be markedly enhanced to 70–80% of the injected dose (21) , leading to a three- to sixfold enhancement of the antiviral activity after i.m. injection into Woodchuck hepatitis virus-infected woodchucks (22) . As compared to the above macromolecular drug carriers, hepatotrophic prodrugs in which the antiviral drug is coupled to a small synthetic ligand for the asialoglycoprotein receptor may offer additional advantages. The prodrugs are chemically well defined, less immunogenic, do not require intricate pharmaceutical formulation protocols, and are tailored for large-scale production.

In the present study, we illustrate the potential of the prodrug approach for the antiviral nucleoside phosphonate 9-(2-phosphonylmethoxyethyl)adenine (PMEA). To this end, PMEA was coupled to di- and trivalent cluster glycosides with nanomolar affinity for the asialoglycoprotein receptor (26 27 28 29) to yield a hepatotrophic prodrug. It is shown that the biodistribution profile of the PMEA prodrugs is substantially improved, leading to a strongly enhanced antiviral activity as compared to the parent drug.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents
PMEA was synthesized as described previously by Holy et al. (30) . [Adenine-2,8-3H]PMEA (0.33 Ci/mmol) was purchased from Moravek Biochemicals (Brea, Calif.). Ethylenediamino-([3H]-)PMEA was synthesized as described (31) . The synthesis and purification of K(GN)2, K2(GN)3, ([3H])PMEA-K(GN)2, and ([3H])PMEA-K2(GN)3 are described in detail elsewhere (29 ; A. R. P. M. Valentijn et al., unpublished results). PMEA mono- and diphosphate were kindly provided by Dr J. Balzarini (Rega Institute, Leuven, Belgium). Na[125I] (carrier free in NaOH) was obtained from Amersham Int. (Bucks, U.K.). Collagenase (types I and IV from Clostridium perfrigans), bovine serum albumin (type V), and agarose-bound neuraminidase (from Clostridium perfrigans, type IV-A) were purchased from Sigma (St. Louis, Mo.). Dulbecco’s modified Eagle medium was obtained from Flow Laboratories (Irvine, Scotland, U.K.). Ketamine (100 mg/ml; HCl salt) was from Eurovet (Bladel, The Netherlands). Hypnorm (0.315 mg/ml of fentanyl citrate and 10 mg/ml of fluanisone) and Thalamonal (0.05 mg/ml of fentanyl citrate and 2.5 mg/ml of droperidol) were from Janssen-Cilag Ltd. (Saunderton, England). Digitonin was from Fluka Chemie (Zwijndrecht, The Netherlands). Human orosomucoid was isolated and subsequently desialylated enzymatically (21) . The resulting asialoorosomucoid (ASOR) was radiolabeled with carrier-free 125I by the ICl method as described by Bilheimer et al. (32) . All other reagents were of analytical grade.

Determination of the acid-induced PMEA release from the PMEA prodrugs
PMEA-K(GN)2 and PMEA-K2(GN)3 (0.1 µmol) (for chemical structure, see Fig. 1 ) were incubated at 37°C in 1 ml of freshly isolated rat plasma [diluted 1:1 with phosphate-buffered saline (PBS), 10 mM NaPi, 150 mM NaCl, pH 7.4), or 50 mM sodium acetate buffer (pH 4.7). At the indicated times, samples of 50 µl were taken. The plasma samples were lyophilized and the residues resuspended in 200 µl of ice-cold MeOH/H2O (70:30 v/v). The suspensions were centrifuged for 5 min at 14,000 rpm, the supernatants were lyophilized, and the residues were dissolved in buffer A (50 mM NH4HCO3; pH 7.5). Subsequently, all samples were analyzed for the presence of PMEA glycoconjugates and released PMEA by reversed-phase high-performance liquid chromatography (HPLC) on a Macherey-Nagel Nucleosil C8 column (5 µm; 250x4.6 mm) using a linear elution gradient from 100% buffer A to 100% CH3CN at a flow of 1 ml/min. UV detection (260 nm) was performed using a Jasco UV-975 detector (Tokyo, Japan). Data were processed using Borwin chromatography software (JMBS Developments, Le Fontanil, France). PMEA, PMEA-K(GN)2, PMEA-K2(GN)3, and ethylenediamino-PMEA eluted at 0%, 33%, 38%, and 40% CH3CN, respectively.



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  		Figure 1. Chemical structures of the PMEA prodrugs.

Experimental animals
Male Wistar rats, weighing ~250 g, were used (Broekman Instituut BV, Someren, The Netherlands). The animals received humane care and were handled in compliance with the guidelines issued by the Dutch authorities. Animals were anesthetized prior to the experiments by subcutaneous injection of a mixture, containing ketamine·HCl, fentanyl citrate, droperidol, and fluanisone (75, 0.04, 1.1, and 0.75 mg/kg, respectively).

Competition of 125I-ASOR binding to parenchymal liver cells
Male Wistar rats of ~250 g were anesthetized as described above and the parenchymal liver cells were isolated after perfusion of the liver with collagenase (type IV, 0.05%) at 37°C, by the method of Seglen, modified as described previously (33) . The viability of the cells as determined by trypan blue exclusion was >90%, while the purity was >95%. Competition studies of [125I]-ASOR binding to isolated parenchymal liver cells by K(GN)2, K2(GN)3, PMEA-K(GN)2, and PMEA-K2(GN)3 were performed as described previously (34) . The binding data were analyzed according to a single site model using a computerized nonlinear fitting program (GraphPad Sofware, Inc., San Diego, Calif.) to calculate the Ki values. For the herpes simplex virus type 1 (HSV-1) replication studies, data obtained from two or three experiments of eight data points in fivefold [per PMEA(prodrug)] were combined and analyzed by nonlinear regression according to a sigmoidal dose-response model. Hence, SD of the pEC50 values indicate goodness-of-fit and accuracy and cannot be used for statistical analysis as such.

Determination of the serum decay and liver association of [3H]PMEA (prodrugs)
Male Wistar rats were anesthetized as described above. The abdomen was opened and [3H]PMEA, [3H]PMEA-K(GN)2, or [3H]PMEA-K2(GN)3 was injected into the vena cava inferior at the renal vein bifurcation at a dose of 170 nmol/kg body weight dissolved in 0.5 ml PBS. If indicated, rats received an injection of asialofetuin at 1 min prior to administration of the radiolabel. At the indicated times blood samples of 0.2–0.3 ml were taken from the vena cava inferior and liver lobules were excised. The blood samples were centrifuged for 2 min at 20,000 g and the radioactivity in the supernatants was counted. At the end of the experiment, the remainder of the liver and some other tissues were removed. Tissue samples were counted for radioactivity after complete combustion using a Packard Tri-Carb 306 sample oxidizer (Packard, Downers Grove, Ill.). The amount of liver tissue tied off successively did not exceed 15% of the total liver mass. Radioactivities in liver and other tissues were corrected for radioactivity in serum present in the tissue at the time of sampling (35) .

Determination of the distribution of [3H]PMEA (prodrugs) over liver cells
Male Wistar rats were anesthetized and injected with [3H]PMEA, [3H]PMEA-K(GN)2, or [3H]PMEA-K2(GN)3 at a dose of 170 nmol/kg body weight (in 0.5 ml PBS) as described above. After 10 min, the liver was cannulated, perfused at 8°C with collagenase (type I, 0.02%), and parenchymal, Kupffer, and endothelial cells were isolated from the liver as described in detail earlier (36) . Samples of cell suspensions were counted for radioactivity after complete combustion using a Packard Tri-Carb 306 sample oxidizer and the protein content of the cell fractions was determined. The contributions of the various cell types to the total liver uptake were calculated as described previously (36) .

Intracellular fate of [3H]PMEA and [3H]PMEA-K(GN)2 in parenchymal liver cells
Parenchymal liver cells (2.106 cells/ml), isolated by the method of Seglen as described above, were incubated for 6 h with [3H]PMEA-K(GN)2 (100 nM) or [3H]PMEA (1 µM) at 37°C. At the indicated times, aliquots of 1 ml were taken, mixed with an equal volume of MOPS/digitonin buffer (17 mM MOPS, pH 7.4, containing 250 mM sucrose, 2.5 mM EDTA, 400 U/l Trasylol, and 0.006% digitonin), and incubated for 20 min at 4°C to lyse the cellular membranes. The incubation mixtures were centrifuged (5 min; 3000 rpm), the supernatants were lyophilized, and the residues were resuspended in 200 µl of ice-cold MeOH/H2O (70:30, v/v). After centrifugation (5 min; 14,000 rpm), the supernatants were lyophilized; the residues were resuspended in 125 µl PBS and stored at -80°C until further analysis. The sample content of PMEA (prodrugs) and derived metabolites was determined by HPLC analysis on a Partisil 10 SAX column (250x4.6 mm) using a 20 min elution gradient from 5 mM to 700 mM potassium phosphate (pH 5.5) at a flow of 1 ml/min. Under these conditions, ethylenediamino-PMEA, PMEA, PMEA monophosphate, PMEA-K(GN)2, and PMEA diphosphate eluted at 9.2 min, 11.2 min, 14.1 min, 16.2 min, and 23.1 min, respectively. The recoveries of PMEA, PMEA-K(GN)2, PMEA monophosphate, and PMEA diphosphate for the whole extraction and chromatographic analysis were 75–83%.

Inhibition of HSV-1 replication in HepG2 cells by PMEA (prodrugs)
Wild-type HSV-1, Glasgow strain 17 syn+, was grown as described previously (37) . To minimize interexperimental variation, the same virus stock was used for all experiments. In brief, the HSV-1 plaque assay was performed as follows. Confluent monolayer cultures of HepG2 cells (seeded at a density of 1.2 106 cells/25 cm2 petri dish and grown to confluency for 4–5 days) were incubated for 4 h at 37°C with PMEA (prodrugs) in RPMI+10% fetal calf serum (FCS). After thorough washing with PBS, the cultures were infected with HSV-1 (2.107 pfu in PBS+1% FCS) and incubated at room temperature for 1.5 h. After incubation, the cells were rinsed with PBS to remove virus. Overlay medium containing 1% agar was added to the HepG2s and the monolayer cultures were incubated for 2 days at 37°C. After 2 days, an HSV-1-induced cytopathogenic effect could be observed. Cell cultures were stained by adding a second overlay of agar containing 0.00125% (w/w) neutral red and the plaques were counted on days 4 and 5.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
In vitro analysis of the PMEA prodrugs
[3H]PMEA was coupled to two lysine-based cluster galactosides, the bivalent K(GN)2 (Kd=24 nM) and the trivalent K2(GN)3 (Kd=5.2 nM), as described previously (29 , 31) . In short, ethylenediamino-[3H]PMEA was synthesized and reacted with the pentafluorophenyl esters of K(GN)2 and K2(GN)3 to furnish the desired prodrugs, designated [3H]PMEA-K(GN)2 and [3H]PMEA-K2(GN)3 (Fig. 1) .

To assess the effect of conjugation of PMEA to K(GN)2 and K2(GN)3 on the affinity of the galactosides for the asialoglycoprotein receptor, we performed competition studies of [125I]-asialoorosomucoid ([125I]-ASOR) binding to isolated parenchymal liver cells. The PMEA prodrugs were able to inhibit [125I]-ASOR binding in a competitive fashion and with high affinity (Fig. 2 ). The inhibition constants of PMEA-K(GN)2 and PMEA-K2(GN)3, as calculated from the competition curves, amounted 46 nM (pKi= 7.33±0.11) and 3.5 nM (pKi= 8.46±0.08), respectively, which is close to that of the cognate ligands K(GN)2 and K2(GN)3 (24 and 5 nM, respectively; 29 ).



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  		Figure 2. Competition studies of [125I]-ASOR binding to parenchymal liver cells by PMEA-K(GN)2 (•) and PMEA-K2(GN)3 ({circ}). Freshly isolated rat parenchymal liver cells were incubated for 2 h at 4°C with a fixed concentration of [125I]-ASOR (5.5 nM) in the presence of PMEA-K(GN)2 or PMEA-K2(GN)3. Specific binding of [125I]-ASOR is defined as the difference between total binding and nonspecific binding (determined in the presence of 100 mM GalNAc), and is plotted as % of the control (without inhibitor).

In vivo fate of [3H]PMEA (prodrugs)
Rats were intravenously injected with [3H]PMEA or [3H]PMEA prodrugs at a therapeutic dose of 170 nmol/kg body weight, and both the liver-associated radioactivity and the radioactivity in serum were monitored. [3H]PMEA, [3H]PMEA-K(GN)2, and [3H]PMEA-K2(GN)3 were rapidly cleared with serum half-lives of 1.1 ± 0.1, 1.1 ± 0.1, and 1.2 ± 0.2 min, respectively. At 40 min after injection, [3H]PMEA was mainly recovered in the kidneys (45±6%), skin (20±2%), muscle (4.5±0.7%), liver (4.2±0.3%), and serum (1.8±0.8%). Uptake of [3H]PMEA by the other organs was less than 1% of the injected dose (Fig. 3A ). Apart from the kidneys (and to a lesser extent the skin), the specific recovery per gram wet tissue of [3H]PMEA in the other tissues was relatively low. Coupling of PMEA to K(GN)2 markedly altered the tissue distribution of the drug (Fig. 3A ). Kidney uptake of [3H]PMEA was decreased threefold to 14 ± 3% after glycoconjugation. In addition, dermal accumulation of [3H]PMEA-K(GN)2 was significantly reduced to 10 ± 2% of the injected dose, whereas the recovery in muscle and serum levels did not differ from that of free [3H]PMEA. A similar change in tissue distribution was observed after coupling of PMEA to K2(GN)3. A more detailed study of the kinetics of the hepatic uptake of [3H]PMEA and [3H]PMEA prodrugs showed that the liver association of the drug was enhanced by 12- to 15-fold after glycoconjugation to K(GN)2 and K2(GN)3 (51±6 and 62±3% of the dose, respectively) (Fig. 3B ). Preinjection of rats with asialofetuin—a specific substrate of the asialoglycoprotein receptor (38) —reduced the liver uptake of [3H]PMEA-K(GN)2 and [3H]PMEA-K2(GN)3 by 79% and 65%, respectively. Involvement of the asialoglycoprotein receptor in the increased hepatic uptake of the prodrugs was further substantiated by analysis of the contribution of the liver cell types to hepatic uptake. Rats were injected with the radiolabeled (pro)drugs; at 10 min after injection the parenchymal, Kupffer, and endothelial liver cells were isolated (Fig. 4 ). Liver uptake of [3H]PMEA could be attributed to Kupffer cells (24±4%) and parenchymal liver cells (57±3%). By contrast, liver uptake of [3H]PMEA-K(GN)2 and [3H]PMEA-K2(GN)3 could be ascribed almost exclusively to parenchymal liver cells (87.3±0.7 and 87.2±0.3% of the total hepatic uptake, respectively).



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  		Figure 3. Biodistribution of PMEA (prodrugs). A) Major recovery sites of PMEA (open bars), PMEA-K(GN)2 (hatched bars), and PMEA-K2(GN)3 (filled bars). Rats were injected with [3H]PMEA and [3H]PMEA prodrugs at a dose of 170 nmol/kg. At 40 min after injection of the radiolabel, rats were killed, organs were excised, weighed, and the tissue-associated radioactivities were determined and corrected for entrapped serum. Values represent means ± SE of 3–4 rats. B) Kinetics of the liver uptake of PMEA (prodrugs): effect of asialofetuin treatment. [3H]PMEA ({blacktriangleup}), [3H]PMEA-K(GN)2 (•, {circ}), or [3H]PMEA-K2(GN)3 ({blacksquare}, {square}) were i.v. injected into rats at a dose of 170 nmol/kg without (•, {blacksquare}) or with ({circ}, {square}) a preinjection of asialofetuin (50 mg/kg). At the indicated times, radioactivities in the liver were determined. Values are means ± SE of 3 animals.



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  		Figure 4. Contribution of the various cell types in liver to the hepatic uptake of PMEA (prodrugs). Rats were injected with [3H]PMEA (open bars), [3H]PMEA-K(GN)2 (filled bars), or [3H]PMEA-K2(GN)3 (hatched bars) at a dose of 170 nmol/kg. At 10 min after injection, the liver was perfused and the parenchymal (PC), Kupffer (KC), and endothelial cells (EC) were isolated. The association of radioactivity to each cell type was determined. Values are means ± SE of three rats and are expressed as % of the total injected dose.

Intracellular fate of [3H]PMEA and [3H]PMEA-K(GN)2 in parenchymal liver cells
The question emerges as to whether the enhanced cellular uptake of the PMEA prodrugs by the asialoglycoprotein receptor also leads to an improved availability of the parent drug in the cytosol. To facilitate the release of PMEA from the prodrug and enable its transfer into the cytosol, PMEA was conjugated to the cluster glycosides via an acid-labile phosphonamidate bond. Both PMEA prodrugs were rapidly hydrolyzed during incubation at pH 4.7, the ambient pH in the lysosomal compartment (39) (Fig. 5A ), but remained stable during incubation in freshly isolated plasma (<10% release after 5 h). The half-lives of PMEA-K(GN)2 and PMEA-K2(GN)3 at pH 4.7, as calculated from the hydrolysis curves, were very comparable and amounted to 113 ± 4 and 101 ± 4 min, respectively. With the rapid acid-induced hydrolysis a first requisite is met toward effective targeting of PMEA. The next issue involves the translocation of the parent drug from the lysosomes to the cytosol. Parenchymal liver cells were incubated with [3H]PMEA (1 µM) or [3H]-K(GN)2 (100 nM); the cellular membranes (but not the endosomal/lysosomal membranes) were permeabilized by digitonin treatment, thereby releasing their cytosolic content. Under these conditions, the total recovery of lactate dehydrogenase, a cytosolic enzyme, in the supernatant was 98%, whereas that of acid phosphatase, a lysosomal enzyme, was ~10%. Analysis of the supernatants for the presence of [3H]PMEA and metabolites revealed that the cytosols of PMEA-K(GN)2-treated cells contained much higher concentrations of PMEA than that of PMEA-treated HepG2s (Fig. 5B ), even though extracellular concentration of PMEA-K(GN)2 was 10-fold lower than that of PMEA. Half-maximal PMEA levels in the cytosol were attained within 30 min of incubation with PMEA and PMEA-K(GN)2, whereas the intracellular PMEA levels leveled off after 2 h of incubation.



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  		Figure 5. Intracellular fate of the PMEA (prodrugs). A) Acid-induced hydrolysis: PMEA-K(GN)2 ({circ}, •) or PMEA-K2(GN)3 (0.1 µmol) ({square}, {blacksquare}) were incubated at 37°C in sodium acetate (50 mM, pH 4.7); at the indicated time points, 50 µl samples were taken. The samples were subsequently analyzed for the presence of the PMEA prodrugs (•, •) and PMEA ({circ}, {square}) by reversed-phase chromatography using a C8 column. B) Recovery of PMEA (prodrugs) in the cytosol of parenchymal liver cells. Parenchymal liver cells (2.106 cells/ml) were incubated for up to 5 h at 37°C with [3H]PMEA (1 µM, {circ}) or [3H]PMEA-K(GN)2 (100 nM; (•). At the indicated time points, cells were treated for 20 min at 4°C with 0.006% digitonin to permeabilize the cellular membranes. After centrifugation, the supernatants were analyzed for PMEA (metabolite) content on a Partisal Sax (10 µ) column, the integrals of the assigned PMEA peak were calculated and plotted against the incubation time. A typical elution profile (t=5 h) is given in the insert, at which the elution volume of PMEA is indicated by an arrow.

Antiviral activity of the PMEA prodrugs
Finally, we assessed whether the improved extra- and intracellular kinetics of the PMEA prodrugs is accompanied by an increased therapeutic activity. We have set up an HSV-1 infection assay to monitor the antiviral activity of PMEA in HepG2 cells. As PMEA is a broad spectrum antiviral agent that is equally potent against HSV-1 and HBV infection (40) , this assay yields a reliable estimate of the anti-HBV activity of PMEA in parenchymal liver cells. PMEA fully inhibited HSV-1 replication at 75 µM (Fig. 6A ). The dose-response curve obeyed first-order Michaelis-Menten kinetics. The concentration giving 50% inhibition of HSV-1 replication (EC50), as calculated from the dose-response curves (pEC50=5.61±0.13; EC50=2.5 µM), corresponded well with previously reported EC50 values for inhibition of HBV infection of HepG2 cells (1.2 µM) (41) . The PMEA prodrugs displayed a considerably higher antiviral activity. The pEC50 value of PMEA-K(GN)2 amounted 6.30 ± 0.06 (EC50=0.5 µM), while that of PMEA-K2(GN)3 was almost two log units higher (pEC50=7.33±0.35; EC50=0.047 µM). In the presence of excess N-acetyl galactosamine (50 mM), the dose-effect curve of PMEA-K2(GN)3 shifted to coincide with that of underivatized PMEA (pEC50=6.09±0.09; EC50=0.8 µM) (Fig. 6B ). This indicated that the stimulated antiviral activity of the prodrug could be reversed almost completely by blocking asialoglycoprotein receptor-mediated uptake of the prodrug.



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  		Figure 6. Inhibition of PMEA (prodrugs) on HSV-1-infected HepG2 cells. A) Dose-effect study of the PMEA (prodrugs). HepG2 cells were incubated for 4 h at 37°C with PMEA (0.75–75 µM, {circ}), PMEA-K(GN)2 (0.075–7.5 µM, {blacksquare}), or PMEA-K2(GN)3 (0.0075–2.5 µM, •). Infection and plaque assays were performed as described in Materials and Methods. The number of plaques is normalized for the number of plaques of an untreated control (100–150/dish) and represents means ± SD (n=5). B) Effect of N-acetyl galactosamine on the antiviral activity of PMEA-K2(GN)3. HepG2 cells were incubated for 4 h at 37°C with PMEA-K2(GN)3 (7.5–2,500 nM) in the presence ({square}) or absence of N-acetyl galactosamine (50 mM, {blacksquare}), subsequently infected with HSV-1, and processed as described above.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We have investigated the potential of tissue-specific prodrugs. According to this concept, a drug is coupled directly to a synthetic high affinity tag for a tissue-specific receptor to enhance the concentration of the prodrug in the target tissue and to reduce its toxicity in nontarget tissue. Compared with conventional, often macromolecular drug carriers (15 16 17 18 19 20 21 22 23) , the tissue-specific prodrug concept may offer the following advantages. The prodrug is chemically well defined, probably nonimmunogenic, does not require an intricate pharmaceutical formulation protocol, and is tailored for large-scale production. In this study, we synthesized a prodrug of the antiviral nucleoside PMEA that is selective for the liver parenchyma, the primary site of HBV infection. As target receptor, we chose the asialoglycoprotein receptor on liver parenchyma (25) . This receptor is coupled to a very efficient internalization machinery and therefore was exploited earlier to enhance drug uptake by parenchymal liver cells (16 17 18 19 20 21 22 23) . The hepatic asialoglycoprotein receptor is still present in patients with acute hepatitis, chronic hepatitis, and liver fibrosis, although the intrahepatic distribution may be slightly different (42 , 43) . A ligand library of bi- and trivalent cluster glycosides was screened for high-affinity ligands for the asialoglycoprotein receptor (29) . From this library, K(GN)2 and K2(GN)3 were selected on the basis of their affinity (5 and 24 nM, respectively) and the presence of a functional group fit for coupling to a drug. The use of 5 and 24 nM tags allowed us to assess the minimal affinity required for successful prodrug targeting. Competition studies showed that conjugation of PMEA to the glycoside ligands via an acid-labile phosphonamidate bond did not affect receptor recognition.

In concert with previous studies, PMEA was rapidly cleared from the bloodstream (12) . The major sites of elimination were the kidney, skin, and muscle. Apart from the extensive renal accumulation, PMEA distributed nonspecifically over the total body fluid. The biodistribution pattern of the prodrugs was markedly different from that of PMEA; the liver appeared to be the major recovery site for PMEA-K(GN)2 and PMEA-K2(GN)3. In addition, renal and dermal uptake was significantly reduced, which implies that the use of hepatotrophic prodrugs may also aid to reduce the side effects of nucleoside phosphonates such as nephrotoxicity and dermal lesion (10) . The enhanced hepatic uptake of the PMEA prodrugs can be ascribed almost completely to parenchymal liver cells. Preinjection of asialofetuin reduced the liver uptake of the PMEA prodrugs to base levels, establishing that the hepatic accumulation of the prodrugs was mediated by the asialoglycoprotein receptor. Accordingly, the ratio of liver parenchyma-to-kidney uptake of PMEA-K(GN)2 (1.86±0.57) and PMEA-K2(GN)3 (2.69±0.24) was much higher that that of PMEA (0.06±0.01). Preliminary studies show that after injection of [3H]PMEA-K(GN)2 into the vena penis, hepatic radioactivity is sustained at a fairly high level for up to 24 h (31.4% of the injected dose; data not shown), whereas the intrahepatic concentration of free [3H]PMEA at this time point is already reduced to 0.42% of the injected dose. Therefore, we assume that the rate of PMEA redistribution after hepatic uptake of the prodrugs will probably be slower than that of renal excretion, so that the kidney-to-liver ratio is maintained at a fairly high level.

The targeting efficacy of the K(GN)2 and K2(GN)3 tags were in the same order of magnitude despite the 10-fold higher affinity of the latter. Apparently, a 24 nM tag is sufficient for preventing untimely renal excretion of PMEA and redirecting the low molecular weight drug to the aimed target. A second conclusion may be that the targeting efficacy of both glycoconjugates is similar to that of conventional drug carriers such as lactosaminated poly-L-lysine (21 , 22) , neoHDL (16) , or human serum albumin (17 18 19 20) .

Our incubation studies demonstrate that PMEA-K(GN)2 and PMEA-K2(GN)3 remain stable in plasma, but are rapidly hydrolyzed in an acidic environment (pH 4.7) owing to the presence of an acid-labile phosphonamidate bond. This indicates that the PMEA prodrugs are rapidly hydrolyzed during lysosomal processing to furnish the parent drug PMEA. The rate of PMEA release during acid-catalyzed hydrolysis was found to be higher than the release rate of adenine arabinoside 5'-monophosphate from arabinogalactan (~0.2%/h) (23) . The adenine arabinoside 5'-monophosphate drug, however, was coupled to the carrier via a phosphoramidate bond, which is less acid-sensitive than a phosphonamidate bond (44) . The high stability of the prodrug at neutral pH and the rapid hydrolysis at acidic pH make phosphonamidate-based prodrugs very suitable for the prodrug approach. A rapid release of the parent drug PMEA within the target tissue will aid to build up therapeutic levels of PMEA biphosphate, the active metabolite whose formation depends on the rate and extent of prodrug influx, hydrolysis, and secretion by parenchymal liver cells as well as on the rate of metabolic inactivation of PMEA (diphosphonate) or prodrug.

The liberated PMEA is efficiently transferred to the cytosol after uptake and lysosomal processing. After incubation of parenchymal liver cells with PMEA-K(GN)2, the cytosol contains almost 10-fold more PMEA than after incubation with the parent drug PMEA (0.27 vs. 3.0 µM). The ratio of cytosolic to extracellular PMEA amounted to 0.27 after incubation with PMEA and ~30 for PMEA-K(GN)2. Clearly, the prodrug is accumulated intracellularly via an active transport process. Moreover, these findings suggest that PMEA is able to pass the lysosomal membrane once it is released from the prodrug. Although specific PMEA transporters have been identified (45 , 46) , these transporters were localized mainly on the cellular membrane. In view of the high stability of the phosphonate group of PMEA against phosphatase, 5'-nucleotidase, or acid-induced hydrolysis (47) , we anticipate that extensive dephosphorylation of PMEA and subsequent phosphorylation will play only a limited role in the translocation to the cytosol. More likely, transfer to the cytosol proceeds through passive diffusion of fully protonated PMEA, which represents ~0.2% of the total lysosomal PMEA at pH 4.7. Once in the cytosol (pH 7.4), the translocated PMEA is rapidly deprotonated to restore the existing concentration gradient between lysosomes and cytosol.

The enhanced cytosolic concentration of PMEA after incubation of parenchymal liver cells with the prodrugs is accompanied by a markedly increased antiviral activity. The prodrugs display a 5- to 52-fold lower EC50 than PMEA, even though the ligand recognition by the asialoglycoprotein receptor on our HepG2 strain is suboptimal. In fact, the affinity of ASOR for the asialoglycoprotein receptor on HepG2 cells was ~10-fold lower than for the parenchymal liver cell receptor (116±34 nM vs. 8.9±2.8 nM; data not shown). When we take this into account, the observed EC50 values of the prodrugs closely reflect the affinities of the cognate glycoside ligands. As the antiviral activity of PMEA-K2(GN)3 could be inhibited for >95% by blocking asialoglycoprotein receptor-mediated uptake with N-acetyl galactosamine, it is evident that functional receptors are required for the PMEA prodrugs to exert an antiviral activity.

PMEA is a broad-spectrum antiviral agent that inhibits the replication of both hepadna- and herpetoviruses by acting as a viral DNA polymerase-mediated 3' chain terminator (40 , 48) . Indeed, the reported anti-HBV activity of PMEA in HepG2–2.2.15 cells and the anti HSV-1 activity in our HepG2 cells were essentially similar (1.2 µM and 2.5 µM, respectively) (41) , while Midoux et al. found similar values for inhibition of HSV-1 infection of macrophages by PMEA (49) . Accordingly, the HSV-1 infection assay provides a reliable estimate of the anti-HBV activity of PMEA in liver cells. The high antiviral potency, the favorable biodistribution profile, and the prolonged antiviral activity make PMEA-K2(GN)3 a promising antiviral agent to be implemented in currently available anti-HBV therapies. Under in vivo conditions, the antiviral activity of K(GN)2 and K2(GN)3-tagged PMEA may reflect the liver parenchyma uptake of both prodrugs (44.5 and 54.1% of the injected dose, respectively). As PMEA-K(GN)2 contrasts favorably with PMEA-K2(GN)3 in terms of drug- to-prodrug mass ratio, the former may be an even more promising hepatotrophic prodrug.

In conclusion, the device of a new hepatotrophic prodrug of PMEA with improved antiviral activity is described. Uptake of the prodrug by the liver parenchyma is increased by 20-fold after conjugation to the high-affinity cluster glycosides K(GN)2 and K2(GN)3. A 24 nM tag appeared to suffice for redirecting drugs to the liver and avoiding untimely renal excretion of the drug. On internalization, the prodrug is rapidly hydrolyzed to afford the parent drug, which in turn is translocated to the cytosol. The improved biodistribution profile of PMEA prodrugs is accompanied by a similarly increased antiviral activity. Moreover, the presented prodrug concept is also suitable for drugs that intervene in other liver disorders including cholestasis, and dyslipidemia and hepatitis C infection.


   ACKNOWLEDGMENTS
 
We would like to acknowledge Dr. Jan Balzarini (Leuven, Belgium) for his generous gift of mono- and dephosphorylated 9-(2-phosphonylmethoxyethyl)adenine. This study was supported by grant no. 349-3363 from the Foundation of Technical Sciences (S.T.W.) and grant no. 902–21-150 from the Dutch Organization for Scientific Research (N.W.O.).

Received for publication October 7, 1999. Revision received March 1, 2000.
   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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