CYTOTOXICITY OF PIVOXIL ESTERS OF ANTIVIRAL ACYCLIC NUCLEOSIDE PHOSPHONATES:

Biological effectiveness of antiviral acyclic nucleoside phosphonate adefo vir, 9-[2-(phosphonomethoxy)ethy]ade nine (PMEA) and its more lipophilic (bis)pivaloyloxymethyl ester prodrug adefovir dipivoxil (bis-POM-PMEA) were compared under in vitro conditions in mammalian cell systems. Proliferation of murine splenocytes was inhibited in a concentration-dependent manner, the bis-POM-PMEA being more effective than PMEA. In contrast to PMEA, bis-POM-PMEA inhibited production of nitric oxide (NO) in macrophages activated with interferon-gamma (IFN-gamma) and lipopolysaccharide (LPS). Viability of both splenocytes and macrophages remained uninfluenced by PMEA, whereas pronounced cytocidal effects were exhibited by bis-POM-PMEA. The IC(50)s reached the values of 15 microM and 30 microM in cultures of macrophages and splenocytes, respectively (assayed at the interval of 24 hrs). The effects could partly be mimicked by formaldehyde, a decomposition product of the pivoxil moiety of bis-POM-PMEA. The other possible product, pivalic acid, was ineffective in this respect. The present data are consistent with the view that pivoxil prodrug of PMEA, bis-POM-PMEA possesses enhanced but also broader spectrum of biological effects than the parent compound.


INTRODUCTION
Acyclic nucleoside phosphonate 9-[2-(phosphonomethoxy)ethy]adenine (PMEA; adefovir) is a recognized antiviral agent possessing both antiherpesvirus and antiretroviral activities including anti-HIV effects [1][2][3][4] .The major disadvantage of this class of compounds under therapeutic usage is, however, rather poor intracellular delivery, intestinal permeation and limited oral bioavailability 5,6 .Pharmaceutic formulations which would overcome these obstacles are therefore intensively explored.The negative charge of the PMEA molecule can be reduced through the introduction of the lipophilic (bis)pivaloyloxymethyl (pivoxil) moiety, giving rise to bis-POM-PMEA, i.e. adefovir dipivoxil.The prodrug shows considerably enhanced oral bioavailability in comparison with the parent structure of PMEA (ref. 5,6 , and has been approved by FDA and EMEA for general treatment of hepatitis B (Hepsera ® ).Not only antiviral effectiveness 7 but also more prominent antiarthritogenic activty of bis-POM-PMEA over PMEA 8 have been reported.We have shown recently that PMEA suppresses proliferation of lymphocytes 9 and in contrast to a number of other acyclic nucleoside phosphonates it does not augment production of immune-activated production of nitric oxide by macrophages 10 .The aim of this study was to investigate the influence of PMEA pivoxilation on these cell functions.

Animals
Female mice of the inbred strain C57BL/6, 7-9 wks old, were purchased from Charles River Deutschland (Sulzfeld, Germany).They were kept in transparent plastic cages in groups of eight, and maintained in an Independent Environmental Air Flow Cabinet (ESI Flufrance, Wissous, France).Lighting was set on 0600 to 1800 h, temperature at 22 °C.elsewhere 11,12 .The (bis)pivaloyloxymethyl ester prodrug adefovir dipivoxil (bis-POM-PMEA) was kindly donated by Gilead Sciences (Foster City, CA).Their structure is shown in Fig. 1.Stock solutions were prepared in incomplete phenol red-free RPMI-1640 culture medium containing NaHCO 3 (Sigma-Aldrich, Prague, CR).Working concentrations were prepared by diluting the stock solutions in complete medium RPMI-1640 (see below) and used fresh.

Lymphocytes and lymphocyte proliferation assay
Single-cell suspension of splenocytes was prepared by passing fragmented pooled spleens of mice through a fine nylon sieve.Erythrocytes were removed using the red blood cells lysing buffer (Sigma) containing 0.83 % ammonium chloride in 0.01 M Tris-HCl, pH 7.5.After repeated thorough washing the cells were seeded in triplicate wells of 96-well U-bottom cell culture plates (Nunc, Roskilde, Denmark).The number of cells was 5 × 10 5 /well in final 100 µl.They were cultured for 72 h in the presence or absence of mitogens (ConA, 2.5 µg/ml or LPS, 5 µg/ml) in Heraeus incubator keeping 37 °C, 5 % CO 2 , 100 % relative humidity.
PMEA and bis-POM-PMEA were added immediately after mitogens.Six hours prior to the collection of cells, they were pulsed with 0.5 µCi of methyl-3 H-thymidine.Cells were harvested onto glass microfiber filters using Dynatech Multimash Harvestor 2000.Thymidine incorporation into DNA (cpm) was determined via scintillation counting.

Macrophages and macrophage nitric oxide (NO) production
Mice, sacrificed by cervical dislocation, were injected intraparitoneally with 8 ml sterile saline.Collected and pooled lavage cells were washed, resuspended in culture medium and seeded into 96-well flat-bottom Nunc microplates in 100-µl volumes.Adherent peritoneal cells (macrophages) were isolated by incubating the cells for 2 h at 37 °C, 5% CO 2 , and then thrice vigorously shaking the plate and washing to remove non-adherent cells.Cultures were maintained with or without addition of PMEA and bis-POM-PMEA and in the absence or presence of IFN-γ (5000 pg/ml) plus LPS (1 µg/ml) in Heraeus incubator for 24 h.All experimental variants were set in triplicate.
The concentration of nitrites in supernatants was taken as a measure of NO production 13,14 .It was detected in individual cell-free samples (50 µl) incubated for 10 min at 37 °C with an aliquot of a Griess reagent (1% sulphanilamide/0.1% naphtylendiamine/2.5% H 3 PO 4 ).The absorbance at 540 nm was recorded using a microplate spectrophotometer (Tecan, Austria).A nitrite calibration curve was used to convert absorbance to µM nitrite.

Determination of cell viability
Cell death was analysed using the LDH assay.It is based on the determination of lactate dehydrogenase activity released from the cytosol of damaged cells into the supernatant.The cell supernatants were diluted 1 : 1 and mixed with an aliquot of the LDH kit.After 30-min incubation in the dark at ambient temperature, the reaction was stopped with 2 N HCl.Differences between absorbances at 492 and 690 nm were evaluated.The percentage cytotoxicity of test samples was related to the control samples and to the samples with 100 % dead cells evoked by 1 % Triton, according to the formula: [(exp.value -control value) / (Triton value -control value)] × 100.All control and experimental variants were run in triplicate.

Data analysis
Analysis of variance (ANOVA) with subsequent Dunnett's multiple comparison test, and graphical presentation of data were done using the Prism program (GraphPad Software, San Diego, CA).

Antiproliferative effects of PMEA and bis-POM-PMEA
The following values (means ± SEM) of incorporation of 3 H-thymidine into DNA were found in controls (drugs absent): 1,700 ± 138 cpm for resident, mitogennonstimulated cells, 22,397 ± 1,059 cpm for LPS-stimulated, and 128,473 ± 1,690 cpm for ConA-stimulated cells.Proliferation of nonstimulated and mitogen-stimulated lymphocytes was differentially inhibited by PMEA and bis-POM-PMEA (Fig. 2).Expressed as a percentage of corresponding control values, the inhibitory effects run in parallel in individual experimental groups.The bis-POM-PMEA proved to be significantly more potent (P < 0.01) than PMEA in suppressing proliferation of all nonstimulated (IC 50 s: 2.9 and 20.4 µM, respectively), LPS-stimulated (1.7 and 13.8 µM, respectively), and ConA-treated (0.44 and 1.02 µM, respectively) cells.

Effects of PMEA and bis-POM-PME on nitric oxide (NO) production
Production of NO, triggered primarily in mouse peritoneal macrophages by LPS (10 pg/ml) plus IFN-γ (5000 pg/ml), remained uninfluenced by PMEA (Fig. 3).PMEA was ineffective even in as high concentrations as 500 µM.On the other hand, effects of bis-POM-PMEA on NO production were strongly inhibitory.The dose of Z. Zídek, E. Kmoníčková, A. Holý approximately 5 µM reduced the production of NO by about 50 %.

Cytocidal effects of bis-POM-PMEA
Whereas even high doses (100-500 µM) of PMEA did not influence viability of cells (Fig. 4), bis-POM-PMEA proved to possess remarkable cytocidal activity.The LC 50 , determined at the interval of 24 h, reached the value of 15 µM for macrophages, and 30 µM for splenocytes.The effects were time-dependent, the significant inhibition of viability of both macrophages and lymphocytes being apparent as early as 5-6 h following the addition of bis-POM-PMEA to cells (Fig. 4).

Comparison of biological effects of bis-POM-PMEA with the effects of formaldehyde and pivalic acid
As compared to bis-POM-PMEA, formaldehyde was less potent to inhibit production of NO by macrophages (Fig. 5).Pivalic acid was free of the NO-suppressive effect.Similarly, only formaldehyde, but not pivalic acid, showed moderate cytocidal activity (Fig. 6).

DISCUSSION
Expression of biological potential of PMEA, i.e. 9-[2-(phosphonomethoxy)ethy]adenine (adefovir) is restricted by a relatively high anionic charge of this molecule, brought about by the phosphonate side chain, which

Cytotoxicity of pivoxil esters of antiviral acyclic nucleoside phosphonates: adefovir dipivoxil versus adefovir
This finding prompted us to investigate possible cytocidal potential of bis-POM-PMEA, as well as a profile of biological effects of decomposition products of the pivoxil moiety, i.e. formaldehyde and pivalic acid.Indeed, bis-POM-PMEA was found to remarkably decrease viability of both lymphocytes and macrophages.Similarly, in agreement with other reports 23 , formaldehyde (but not pivalic acid) decreased the cell viability.Furthermore, it inhibited production of NO by macrophages.It may be presumed that enhanced antiproliferative effects of bis-POM-PMEA are due to improved transmembrane permeability, but at least in part they are influenced by formaldehyde-induced dying the cells.Inhibition of NO production seems to be however entirely dependent on formation of formaldehyde, and not on enhanced intracellular concentration of PMEA following administration of the prodrug.It should be noted that formaldehyde (as well as pivalic acid) was applied at concentrations that could theoretically arise if all pivoxil moieties of bis-POM-PMEA molecule are transformed to formaldehyde and pivalic acid.Despite of this, all inhibitory effects of bis-POM-PMEA were more pronounced than those of formaldehyde.Plausible explanation for the discrepancy might be a difference in intracellular accumulation of formaldehyde reached after its direct administration into the cell cultures, or alternatively delivered indirectly by bis-POM-PMEA in the form of pivoxil ancestry.It cannot be excluded that intracellular concentrations of formaldehyde are higher after treatment of cells with bis-POM-PMEA than after addition of exogenous formaldehyde.This question remains to be investigated.
Apart from the inhibitory effects on NO formation and cell viability, formaldehyde has been shown to possess a number of other unwanted effects.It has been sug-  makes it otherwise resistant to metabolism.The pivoxil prodrug, i.e. the (bis)pivaloyloxymethyl ester of PMEA, bis-POM-PMEA (adefovir dipivoxil), overcomes partially this obstacle and has been found to possess higher virostatic efficacy than parent PMEA.Both compounds are transported across the membrane by endocytosis 15 .Once in the cell, bis-POM-PMEA is cleaved gradually to the parent drug 6 , and is subsequently phosphorylated to its mono-and diphosphate (analogues of dADP and dATP).These anabolites are alternative substrates and inhibitors of DNA polymerases 16 and act as chain terminators of the DNA synthesis de novo 11,17 .The cleavage of bis-POM-PMEA and, in particular, the subsequent phosphorylation strongly depend on the cell type and on a variety of enzymes [18][19][20] .We have found that the both drugs inhibit proliferation of murine splenocytes, irrespective of the kind of mitogen stimulation, or without any immune stimulation.The bis-POM-PMEA is several-fold more effective than parent PMEA.As far as the intracellular concentration has been shown to be a crucial factor determining biological efficacy of PMEA 21,22 , it could be presumed that the enhanced effectiveness of bis-POM-PMEA results from the different abilities of PMEA versus bis-POM-PMEA to permeate across cell membranes.
In sharp contrast to a quantitative character of these differences, the influence on production of NO is a novel property and is bound solely to bis-POM-PMEA.Even enormously increased concentrations of PMEA (up to 500 µM) were absolutely devoid of any positive and negative interference with biosynthesis NO.However, the introduction of pivoxil moiety in the molecule of bis-POM-PMEA led to the occurrence of inhibitory effects on NO production Z. Zídek, E. Kmoníčková, A. Holý gested to contribute to allergic respiratory disease 24 , to be a risk factor for initiation of endothelial injury leading to atherosclerosis 25 , and for neurogenic inflammation 26 .Inasmuch as some drugs including antibiotics [27][28][29] and anticancer agents 30,31 are pharmaceutically formulated as pivoxil prodrugs, careful evaluation of their possibly formaldehyde-related side effects and therapeutic implications warrant special consideration.

Fig. 2 .
Fig. 2. Antiproliferative effects of PMEA and bis-POM-PMEA.Mouse lymphocytes were cultured for 72 h in the absence or presence of B-cell mitogen LPS and T-cell mitogen ConA.Six hours prior to the collection of cells, they were pulsed with 0.5 µCi of methyl-3 H-thymidine.Its incorporation into DNA was determined by scintillation counting.Control cpm values are shown in Results.

Fig. 3 .Fig. 4 .
Fig. 3. Effects of PMEA and bis-POM-PMEA on production of NO by macrophages activated with LPS+IFN-γ.Nitrite concentration in supernatants of cells cultured for 24 h was determined by Griess reagent.

Fig. 5 .
Fig. 5. Bis-POM-PMEA-and formaldehyde-induced suppression of NO production in macrophages stimulated with LPS+IFN-γ.Control value for the cells cultivated without the test compounds was 42 ± 2.7 µM nitrite.Formation of NO was inspected after 24-h culture.