Erythrocyte-mediated delivery of a new homodinucleotide active against human immunodeficiency virus and herpes simplex virus

Luigia Rossia, Sonja Serafinia, Loredana Cappellaccib, Emanuela Balestrac, Giorgio Brandid, Giuditta F. Schiavanod, Palmarisa Franchettib, Mario Grifantinib, Carlo-Federico Pernoc,e and Mauro Magnania,*

a Institute of Biochemistry ‘G. Fornaini’ and d Institute of Hygiene, University of Urbino, 61029 Urbino; b Department of Chemical Science, University of Camerino, 62032 Camerino; c Department of Experimental Medicine, University of Rome ‘Tor Vergata’, 00133 Rome; e IRCCS ‘L. Spallanzani’, 00173 Rome, Italy


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Monocyte-derived macrophages (MDMs) play a central role in the pathogenesis of infection by human immunodeficiency virus (HIV-1) and represent one of the main reservoirs of the virus in the body. In addition, MDMs can easily be infected by various herpes viruses, including herpes simplex virus type 1 (HSV-1). We have synthesized a new antiviral agent (Bis-PMEA) that consists of two 9-(2-phosphonylmethoxyethyl)adenine (PMEA) molecules bound by a phosphate bridge. This nucleotide analogue, like the parent compound PMEA, has strong and selective activity against HIV-1 and HSV-1. A drug-targeting system previously developed in our laboratory was used for the selective delivery of these drugs to macrophages. Bis-PMEA and PMEA were encapsulated into autologous erythrocytes by a procedure of hypotonic dialysis and isotonic resealing. Loaded erythrocytes were modified to increase their recognition and phagocytosis by human macrophages. By administering Bis-PMEA-loaded erythrocytes to macrophages, 47% of Bis-PMEA and 28% of PMEA was still present 10 days after phagocytosis; in contrast, only 12% of PMEA was found in macrophages receiving PMEA-loaded erythrocytes. Bis-PMEA-loaded erythrocytes were then added to macrophages infected with HIV-1 and HSV-1 and their antiviral activity evaluated. Remarkable protection was obtained against HIV-1 and HSV-1 infection (95 and 85%, respectively). Therefore, Bis-PMEA acts as an efficient antiviral prodrug that, following selective targeting to macrophages by means of loaded erythrocytes, can protect a refractory cell compartment.


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
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 References
 
In addition to CD4+ T lymphocytes, which are a major target for infection by human immunodeficiency virus type 1 (HIV-1), cells of the mononuclear phagocyte system also play a role as a reservoir for HIV. Indeed, the noncytopathic infection of monocyte-derived macrophages (MDMs) by HIV-1 makes these long-lived cells the major viral reservoirs, with an important role in the pathogenesis of AIDS.14 HIV can persistently infect macrophages in all tissues and organs,5,6 and infected macrophages can fuse with CD4 lymphocytes and thus transfer the virus to these cells.7 Moreover, infected macrophages are able to cause the death of bystander uninfected CD4 lymphocytes8,9 and induce both the chemotaxis and activation of resting T lymphocytes, permitting productive HIV-1 infection.10

MDMs can also easily be infected by various herpes viruses, including herpes simplex virus types 1 and 2 (HSV-1 and HSV-2),11 varicella-zoster virus12 and cytomegalovirus.13 In addition, HSV-1 is able to activate and increase HIV replication,14,15 thereby accelerating the progression of the disease. MDMs coinfected by HIV-1 and HSV-1 and strongly producing both virus progenies have been found in patients.16 Hence, therapeutic strategies are needed that can inhibit replication of both viruses in macrophages.

The adenine analogue 9-(2-phosphonylmethoxyethyl) adenine (PMEA, adefovir) is a promising compound belonging to the class of acyclic nucleoside phosphonates currently being studied (in a prodrug formulation) in HIV-infected patients.17 One of the most interesting features of PMEA is its ability to inhibit both retroviruses (including HIV) and herpes viruses (including HSV-1) in vitro and in vivo in animal models.1823 Thus, the dual antiviral activity of PMEA is an important advantage in the treatment of retrovirus infections, which are frequently complicated by opportunistic herpes virus infections. However, the negative charge of the phosphonate moiety of PMEA significantly impairs its cellular uptake.

Magnani et al.24 have developed a drug-targeting system that allows the administration of antiviral drugs selectively to macrophages. This system was shown to be effective in the protection of macrophages in both murine25 and feline26 models of AIDS. Moreover, this system was recently used by Perno et al.27 to target PMEA to macrophages.

Furthermore, it was shown that it is possible to design and synthesize new antiviral prodrugs that, once in the macrophage, are split into nucleoside analogues that can be converted into the corresponding active phosphorylated drugs by cellular kinases.28,29 In order to increase the selective delivery of PMEA to macrophages, a compound consisting of two molecules of PMEA bound together {di (P1,P2)[2-(adenin-9-yl)ethoxymethyl]phosphonate} (Bis-PMEA) was synthesized and encapsulated into autologous erythrocytes modified to increase their recognition and phagocytosis by human macrophages.

Once inside macrophages, prodrug degradation yielded the pharmacologically active metabolites. The chemical synthesis, metabolism and antiviral activities of the above-mentioned homodimer are reported herein. The results obtained show that the administration of Bis-PMEA-loaded erythrocytes to human macrophages provides an effective in vitro protection against both HIV-1 and HSV-1 infection.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Synthesis of Bis-PMEA and [14C]Bis-PMEA

The homodinucleotide Bis-PMEA (Figure 1Go) was synthesized by reacting the morpholidate derivative of PMEA with PMEA as tributylammonium salt, in anhydrous N,N-dimethylformamide. The radiolabelled dimer [14C]Bis-PMEA was synthesized in a similar way (synthesis will be described in detail elsewhere). PMEA and [adenine-8-14C]PMEA (the latter used for the synthesis of [14C]Bis-PMEA) were kindly provided by Dr Norbert Bischofberger (Gilead Sciences, Foster City, CA, USA).



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Figure 1.  Structure of Bis-PMEA.

 
HPLC analyses of Bis-PMEA and [14C]Bis-PMEA metabolites

Samples were extracted with perchloric acid as in Magnani et al.30 Preliminary experiments (not shown) have shown that Bis-PMEA is stable under these extraction conditions. Neutralized extracts were then used for HPLC determinations. A 5 µm res. elut. 5 C18 90A column (150 x 4.60 mm internal diameter; Varian, Harbor City, CA, USA) protected with a guard column (Pelliguard LC-18, 20 x 4.6 mm internal diameter, 40 µm particles) was used. The mobile phase consisted of two eluents: 25 mM KH2PO4 adjusted to pH 6.0 (buffer A) and buffer A containing 30% (v/v) acetonitrile (buffer B). All buffer solutions, as well as standards and sample solutions, were filtered through a 0.22 µm membrane filter (Millipore, Bedford, MA, USA). The elution conditions were as follows: 5 min at 100% buffer A, up to 100% buffer B over 30 min and hold for 10 min. The gradient was returned to 100% buffer A over 3 min and the initial conditions restored in 2 min. The flow rate was 0.9 mL/min and the detection wavelength was 260 nm. Analyses were performed at room temperature and quantitative measurements were obtained by injection of standards of known concentration. The retention times under the conditions used were 14 min for PMEA and 16 min for Bis-PMEA. A Radiomatic 500TR series flow scintillation analyser (Packard, Meriden, CT, USA) was used to evaluate the radiolabelled compounds.

Bis-PMEA metabolism in erythrocyte lysates

Human red blood cell lysates were obtained as in Rossi et al.29 The haemoglobin (Hb) concentration was 125 mg/mL haemolysate. The haemolysate was incubated for 7 h at 37°C in the presence of 0.16 mM Bis-PMEA and in the absence or presence of 1.0 mM ATP. At times 0, 0.5, 1, 2, 4 and 7 h, 100 µL aliquots were removed and treated for HPLC determinations.

Bis-PMEA and [14C]Bis-PMEA encapsulation in erythrocytes

Bis-PMEA was encapsulated into human erythrocytes by a procedure of hypotonic dialysis, isotonic resealing and reannealing as reported previously24 with some modifications. Briefly, human erythrocytes were washed in 10 mM HEPES (pH 7.4) containing 154 mM NaCl and 5 mM glucose (washing buffer) to remove leucocytes and platelets. Red blood cells (RBC) were resuspended at 70% haematocrit (Ht) in the same washing buffer and were dialysed for 70 min using a tube with a cut-off of 12–14 kDa, against 50 vols of 10 mM NaH2PO4, 10 mM NaHCO3 and 20 mM glucose (pH 7.4) containing 3 mM reduced glutathione and 2 mM ATP. The osmolarity of the buffer was c. 65 mOsm, whereas the erythrocytes reached c. 88 mOsm at the end of the dialysis time. All these procedures were performed at 4°C. After this step, 8 µmol of Bis-PMEA was added to each millilitre of dialysed erythrocytes, which were then incubated for 30 min at room temperature under gentle mixing. Resealing of erythrocytes was obtained by adding 0.1 vol. of 5 mM adenosine, 100 mM inosine, 2 mM ATP, 100 mM MgCl2, 0.194 M NaCl, 1.606 M KCl and 35 mM NaH2PO4 (pH 7.4) per volume of dialysed erythrocytes and incubating the resealed cells at 37°C for 25 min. The resealed cells were washed three times in the washing buffer and either used as they were or processed further to increase their recognition by macrophages. The same procedure was used for encapsulation into human erythrocytes of [14C]PMEA (specific activity 11 198 cpm/nmol) or [14C]Bis-PMEA (specific activity 20 973 cpm/nmol).

Stability of Bis-PMEA in human erythrocytes

The stability of Bis-PMEA in loaded erythrocytes was evaluated by incubation of these cells at 0.8% Ht in RPMI 1640 medium containing 10% fetal calf serum (FCS). At different incubation times, at 37°C in a 5% CO2 atmosphere and under sterile conditions, 5 mL aliquots were processed to determine the concentration of Bis-PMEA and its metabolites. Briefly, Bis-PMEA-loaded RBC were extracted with perchloric acid and analysed by HPLC as described previously, while media were submitted to solid-phase extraction using Isolute TM C18 columns (International Sorben Technology, Mid-Glamorgan, UK) according to the manufacturer's instructions before being analysed by HPLC.

Targeting of Bis-PMEA- and [14C]Bis-PMEA-loaded erythrocytes to macrophages

Targeting of Bis-PMEA-loaded erythrocytes to macrophages was achieved essentially as described in Magnani et al.24 with some modifications. Briefly, suspensions of loaded erythrocytes (10% Ht) in 1.0 mM ZnCl2 were treated with 1.0 mM bis(sulphosuccinimidyl)suberate (BS3) for 15 min at room temperature under gentle mixing and washed once in washing buffer containing 10 mM ethanolamine and once in washing buffer containing 1% (w/v) bovine serum albumin (BSA). These cells were then incubated in autologous plasma for 60 min at 37°C at an Ht of 30% and then washed first in washing buffer containing 2% (w/v) BSA and then in washing buffer without any addition. During the procedure described here, Bis-PMEA is fully retained in RBC, as revealed by HPLC analyses performed before and after RBC membrane modifications (data not shown). Bis-PMEA-loaded erythrocytes were then added to macrophages and their antiviral activity was evaluated (see below). The same procedure was used for targeting of [14C]Bis-PMEA to macrophages.

Stability of [14C]PMEA and [14C]Bis-PMEA in macrophages

[14C]PMEA- and [14C]Bis-PMEA-loaded RBC were modified to increase their recognition by macrophages, as described above. Loaded RBC were added to macrophages at a ratio of 100 RBC per macrophage in 60 mm dishes and phagocytosed for 18 h. Non-ingested RBC were removed by extensive washing (three times) with RPMI 1640 medium, after which the macrophages were incubated at 37°C for 10 days. At 0 and 10 days after phagocytosis, perchloric acid extracts of scraped macrophages were prepared and neutralized with K2CO3. The extracts were concentrated about 10 times in a Speedvac concentrator (Savant, Hicksville, NY, USA) and analysed by HPLC as described. Protein content was determined in the acid insoluble fraction after addition of 100 µL 1.0 M NaOH by Bradford's protein assay (Bio-Rad, Hercules, CA, USA).

Cells and viruses

Peripheral blood mononuclear cells (PBMCs) were obtained from normal seronegative blood donors by separation on Histopaque solution (Sigma, St Louis, MO, USA). Monocytes were separated as described by Rossi et al.29 or by Perno et al.31 After removal of non-adhering cells by repeated washings, monocytes were scraped and collected in a phosphate saline solution. The viability of the cells was >90% as assayed by the trypan blue dye exclusion test. The cells were suspended in RPMI 1640 medium supplemented with 15–20% heat-inactivated FCS and 1% antibiotics (complete medium) and adjusted to a final concentration of 5 x 105 cells/mL. After culture, the monocytes had matured into macrophages as revealed by surface marker analysis, and formed a monolayer (100000 macrophages/well in 1 mL of culture medium). A monocytotropic strain of HIV-1, HTLV-IIIBa-L, and a laboratory-adapted strain of HSV-1, Mc entyre (the latter kindly provided by M. Barbi, Institute of Virology, University of Milan, Italy), were used in experiments of infection with HIV-1 and HSV-1, respectively. Both viruses grew and replicated easily in MDMs.

Assay of anti-HIV-1 activity

For the assay of antiretroviral activity on infected macrophages, RBC loaded with Bis-PMEA (0.9 µmol/mL RBC) were added at a ratio of 500 RBC per macrophage. After 18 h of incubation, non-ingested RBC were removed by extensive washing with culture medium. As a control, macrophage cultures were treated with unloaded RBC, i.e. RBC submitted to the same procedure including transient lysis and subsequent modification to increase macrophage recognition, but without addition of Bis-PMEA.

Human macrophage cultures receiving either Bis-PMEA-loaded or unloaded RBC were then infected for 2 h with HIV-1Ba-L [300 TCID50 (50% tissue culture infective dose)/mL]. After incubation with the virus, cell cultures were extensively washed to remove any residual virus particles. Further controls were performed in each experiment: 1.0 µM Bis-PMEA and 1.0 µM PMEA were given overnight as for RBC.

All cell cultures were maintained for up to 35 days at 37°C and 5% CO2, medium was changed twice a week. No drug was added after infection throughout the experiments. Virus production was assessed in the supernatant, with a commercially available enzyme-linked immunosorbent assay (ELISA) kit able to detect HIV p24gag (Abbott Laboratories, Pomezia, Italy).

Assay of anti-HSV-1 activity

For the assay of antiviral activity on infected macrophages, unloaded RBC and Bis-PMEA-loaded RBC were added and maintained overnight (18 h) before infection at a ratio of 100 RBC per macrophage.

Non-ingested erythrocytes were removed by extensive washing with culture medium. Human macrophage cultures receiving Bis-PMEA-loaded RBC or unloaded RBC were then infected for 2 h with HSV-1 [3 plaque-forming units (pfu)/cell]. After incubation with the virus, cell cultures were extensively washed and fresh medium was added. As control, 1.6 µM Bis-PMEA was added to macrophages overnight as for RBC. The inhibitory effect of the compounds on the replication of HSV-1 was evaluated 48 h after infection by determining the amount of infectious virus in the supernatants by plaque assay in Vero cells. In a parallel experiment, the efficacy of drug-loaded RBC was monitored for 6 days after infection. As control, 1.6 µM Bis-PMEA was added either for the same time as the RBC or during infection and maintained throughout the entire experiment. In all the antiviral assays, loaded and unloaded erythrocytes were used within 24 h of preparation.


    Results
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 Materials and methods
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Metabolism of Bis-PMEA in erythrocyte lysates and intact erythrocytes

To evaluate the metabolism of Bis-PMEA in erythrocyte lysates, the haemolysate was incubated for 7 h at 37°C in the presence of 0.16 mM Bis-PMEA and in the absence or presence of 1.0 mM ATP. The results obtained show that in the absence of ATP, 85% of the homodimer was still present after 7 h of incubation. Moreover, PMEA was never detectable in the presence of 1.0 mM ATP, showing that the prodrug is completely stable for the indicated times when physiological ATP concentrations are present (Figure 2Go). This result is in agreement with the previous observation that an ADP-ribose pyrophosphatase is present in human erythrocytes,32 acts on a number of P1–P2 dinucleotides28,29 and is inhibited by physiological ATP concentration.28,29,32



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Figure 2.  Stability of Bis-PMEA in human erythrocyte lysates. Bis-PMEA (0.16 mM) was incubated with the lysates at 37°C for 7 h in the absence (•) or presence ({blacksquare}) of 1.0 mM ATP ({blacktriangleup}, PMEA formed in the absence of ATP). Aliquots of the incubation mixture were extracted with perchloric acid and analysed by HPLC as described in Materials and methods. The results are the mean ± S.E. obtained from three different experiments.

 
To evaluate the metabolism of Bis-PMEA in intact erythrocytes, human erythrocytes were loaded to a final concentration of 0.5 mM. Increased amounts of drug inside the RBC can be achieved by adding increasing amounts of Bis-PMEA during the dialysis step. The encapsulation of the homodimer by the procedure of hypotonic dialysis, isotonic resealing and reannealing did not give any appreciable alteration in erythrocyte morphology and metabolism (not shown). Bis-PMEA-loaded erythrocytes were incubated for up to 10 days at 37°C in RPMI 1640 medium under sterile conditions. After different incubation times (0, 1, 2, 4, 7 or 10 days) the presence of Bis-PMEA and its metabolites was evaluated. As shown in Figure 3Go, a slow decrease in intracellular Bis-PMEA was observed, reaching as low as 20% of the starting level after 10 days of incubation. The decrease in Bis-PMEA was paralleled by the stoichiometric production of PMEA as revealed by analyses of the culture media. The results for Bis-PMEA in erythrocyte lysates strongly suggest that the homodimer is stable enough in RBC to allow their use as a drug-targeting system.



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Figure 3.  Metabolism of Bis-PMEA in intact erythrocytes. Bis-PMEA was encapsulated into human erythrocytes by a procedure of hypotonic dialysis and isotonic resealing to a final concentration of about 0.5 mM. These cells were then incubated at 37°C under the conditions described in Materials and methods. Values are the mean ± S.E. of three different experiments. Symbols: {blacksquare}, Bis-PMEA in RBC; •, PMEA in RBC; {blacktriangleup}, PMEA outside RBC.

 
[14C]PMEA and [14C]Bis-PMEA metabolism in macrophages

To evaluate the metabolism of [14C]PMEA and [14C]Bis-PMEA in macrophages, human erythrocytes were loaded with [14C]PMEA and [14C]Bis-PMEA to a final concentrations of 3.97 and 0.989 mM, respectively. This difference is partly justified by the lower specific activity of [14C]PMEA (11 198 cpm/nmol) with respect to [14C]Bis-PMEA (20 973 cpm/nmol) and partly by the higher percentage of [14C]PMEA encapsulation into RBC under identical processing conditions (not shown). Loaded RBC were then modified to increase their recognition by macrophages, as described above. The amount of [14C]PMEA and [14C]Bis-PMEA delivered by carrier RBC to macrophages and their stability once inside the target cells were then evaluated.

After 18 h of incubation of [14C]PMEA-loaded RBC with macrophages, 0.63 nmol of PMEA/mg protein were found (time 0). At the same time, macrophages receiving [14C]Bis-PMEA-loaded RBC contained 0.46 nmol/mg protein of Bis-PMEA and 0.32 nmol/mg protein of PMEA. Thus, during the phagocytosis of drug-loaded erythrocytes, some Bis-PMEA has already been converted to PMEA. Ten days after the onset of phagocytosis of the loaded RBC, macrophages receiving [14C]PMEA-loaded RBC still showed 12% of the amount of drug found at time 0. In contrast, 47% of Bis-PMEA and 28% of PMEA were present in macrophages receiving [14C]Bis-PMEA-loaded RBC at day 10. Thus, the intracellular stability of Bis-PMEA is much longer than that of PMEA.

Anti-HIV activity of Bis-PMEA-loaded RBC

To assess the anti-HIV activity of Bis-PMEA-loaded RBC, human macrophages were treated with Bis-PMEA-loaded (c. 0.9 mM inside RBC) or unloaded RBC for 18 h before infection with a macrophage-tropic HIVBa-L strain. Since we have demonstrated previously24 that each macrophage under these experimental conditions phagocytoses one erythrocyte, we calculated that the mean drug concentration in macrophage is in the 0.8–5 µM range for Bis-PMEA. p24 production was determined at 10 days post-infection (Table IGo).


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Table I.  Percentage of virus inhibition (p24) in HIV-infected human macrophages treated with drug-loaded RBC or free drugs
 
The results obtained show that by administering the drug-loaded RBC, 95% inhibition of HIV replication was obtained. Because of this high inhibition efficiency of Bis-PMEA-loaded erythrocytes, we decided to prolong macrophage cultures up to 35 days post-infection. The amount of p24 production by infected macrophages at this time was 40 000 pg/mL, while in macrophages receiving Bis-PMEA-loaded erythrocytes the p24 produced was 2880 pg/mL, with an inhibition of p24 production of c. 93%. It is worth noting that the administration of unloaded RBC was able to inhibit the replication of HIV-1 by c. 30–50%. This effect of unloaded RBC has been described and discussed previously.33 As a control, macrophages were exposed to 1.0 µM Bis-PMEA given as free drug only for 18 h (as for RBC) and then removed. A significant inhibition of HIV replication (60%) was obtained: this result depends on the cleavage of Bis-PMEA to PMEA by serum enzymic activities (data not shown). In addition, the administration of 1.0 µM PMEA (a concentration that is >=40-fold the EC50 of PMEA in HIV-1-infected macrophages) given as free drug for 18 h (as for RBC) was also evaluated. PMEA administration gave a 45% inhibition of p24 production. This efficacy can be easily explained by the long intracellular half-life of the active metabolite of PMEA, PMEApp (c. 18 h).34

Anti-HSV-1 activity of Bis-PMEA-loaded RBC

To evaluate the anti-HSV-1 activity of Bis-PMEA-loaded RBC, human macrophages were treated with Bis-PMEA-loaded (c. 0.9 µmol/mL) or unloaded RBC for 18 h before infection with HSV-1 at 3 pfu/cell. Virus production was assayed 48 h after infection in Vero cells using the plaque assay. The administration of Bis-PMEA-loaded RBC gave an 85% inhibition of HSV-1 replication (Table IIGo). As expected, unloaded RBC were effective in inhibiting HSV-1 replication in human macrophages. Bis-PMEA (1.6 µM; added for the same amount of time as RBC) was used as control and 70% inhibition of HSV-1 replication was observed.


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Table II.  Percentage of virus inhibition in HSV-infected human macrophages by Bis-PMEA-loaded RBC or free drugs
 
In parallel, the anti-HSV-1 efficacy of Bis-PMEA-loaded RBC was evaluated up to 6 days after infection. Their antiviral activity was compared with the efficacy of 1.6 µM free Bis-PMEA given as for RBC (that is, only 18 h before infection) or given during the infection and maintained throughout the entire experiment. The results of one representative experiment are reported in Figure 4Go and show that a single administration of drug-loaded RBC produced a higher or comparable protection against HSV-1 replication than the continuous presence of the drug in the medium. Moreover, it is noteworthy that when Bis-PMEA was administered only for 18 h before infection, as for Bis-PMEA-loaded RBC, its antiviral activity was found to have decayed completely after 2 days of treatment.



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Figure 4.  Percentage of HSV-1 inhibition by Bis-PMEA-loaded RBC or free drug administration to infected macrophages. Bis-PMEA-loaded RBC ({blacksquare}) were added for 18 h before virus challenge and their activity compared with that of Bis-PMEA added as for RBC (•) or added during infection and maintained throughout the entire experiment ({blacktriangleup}). At different times, supernatants were collected and infectivity titres determined using the plaque assay in Vero cells. One of three similar and independent experiments is shown. The mean titre of virus production in the supernatants of infected-untreated culture was 2.15 x 106 ± 3.22 x 105 pfu/mL (mean ± S.E.).

 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Human herpes viruses (HHVs) are distributed worldwide and are among the most frequent causes of viral infections in both immunocompetent and immunocompromised patients. In immunocompetent patients, herpes virus infection is in most cases self-limiting and usually does not require antiviral therapy. In contrast, immunocompromised hosts can develop severe viral disease that can be life-threatening and needs prompt treatment with antiviral agents. In particular, HSV-1/2 infections are common among individuals infected with HIV and the disease is manifested to a much greater extent. Although HSV-1 and HIV-1 have a different principal cellular tropism, there is evidence supporting the possibility that HSV-1 and HIV-1 reciprocally enhance their replication in AIDS patients with non-genital herpes simplex lesions.16 Hence, therapeutic strategies are sorely needed to inhibit the replication of both viruses, especially in macrophages, which are known to be important hosts for both HIV-1 and HSV-1. In this paper, we show that it is possible to protect macrophages against de novo HIV and HSV infections by the administration of a single prodrug consisting of two molecules of PMEA bound together (Bis-PMEA).

PMEA is unusual in that it has both antiretroviral and antiherpesvirus properties and thus may be useful in AIDS patients for the treatment of the opportunistic herpes virus infection and of the retroviral disease. However, the high anionic charge of the phosphonate moiety of PMEA is held responsible for the rather low cellular uptake of PMEA. As regards macrophages, this limitation can be overcome by loading PMEA into RBC and exploiting the natural process of erythrophagocytosis of macrophages following artificial ageing of the drug-loaded erythrocytes, as shown by Perno et al.27 However, we reasoned that Bis-PMEA could be more advantageous than PMEA since, mole per mole, its efficacy is double and it could act as a prodrug for a more sustained delivery. Thus, Bis-PMEA was synthesized and encapsulated into autologous RBC modified to increase their recognition and phagocytosis by human macrophages. The results reported in this paper show that human erythrocytes possess a dinucleotide pyrophosphatase able to cleave the pyrophosphate bridge of Bis-PMEA slowly with subsequent production of stoichiometric amounts of PMEA. This activity is susceptible to ATP inhibition, as shown by the absence of Bis-PMEA degradation in RBC haemolysate supplemented with 1.0 mM ATP. The presence of a dinucleotide pyrophosphatase in human erythrocytes and its susceptibility to ATP inhibition has been described by others.32 When Bis-PMEA was encapsulated into RBC, 50% of the compound was still present inside cells after 5 days of incubation, suggesting that the homodimer is stable enough in RBC to allow their use as a drug-targeting system. Moreover, by administering [14C]Bis- PMEA-loaded RBC to macrophages, 47% of Bis-PMEA and 28% of PMEA were still present 10 days after phagocytosis; in contrast, only 12% of PMEA was found in macrophages receiving [14C]PMEA-loaded RBC. These results strongly suggest the advantage of the dimer form as a prodrug. Thus, once delivered to macrophages, a slow conversion of Bis-PMEA to PMEA occurs, yielding its pharmacologically active metabolite, as demonstrated by the reported antiviral activities. The mechanism hypothesized is as follows: Bis-PMEA is first cleaved into two PMEA monomers by a specific pyrophosphatase, then phosphorylated to PMEA diphosphate (PMEApp), the active antiviral drug. Two different pathways have been suggested for the phosphorylation of PMEA: PMEA can be converted directly to PMEApp by a 5-phosphoribosyl-1-pyrophosphate synthetase35 or, most likely, by AMP kinase, which is able to phosphorylate PMEA in two consecutive steps (PMEA->PMEAp-> PMEApp).

It is worth noting that protection of macrophages against HIV-1 infection is remarkable (95%) upon drug-loaded RBC phagocytosis, while 45% inhibition was obtained after the administration of 1.0 µM PMEA, a concentration that is >=40-fold the EC50 of PMEA in HIV-1-infected macrophages.31 However, it is significant that in these

experiments PMEA was maintained only 18 h before infection (as for RBC) and that this activity can be explained by the long intracellular half-life of PMEApp (about 18 h). Moreover, when 1.0 µM Bis-PMEA was administered always as a free drug for the same amount of time, 60% inhibition of HIV replication was obtained. Since Bis-PMEA does not cross the cellular membranes (data not shown), this anti HIV-1 activity was due to PMEA formed by hydrolysis of Bis-PMEA by means of the serum enzymes present in the RPMI complete culture medium.

When the antiviral activity of Bis-PMEA-loaded RBC against HSV-1 replication was evaluated in macrophages, good protection (85%) from the infection was obtained. As expected, the administration of Bis-PMEA as a free drug for the same time as in RBC was also effective, giving 70% inhibition of HSV-1 replication. However, it is noteworthy that this result was observed only during the first 48 h post- infection, after which time the anti-herpes efficacy of free Bis-PMEA rapidly decayed, while that of the Bis-PMEA-loaded RBC persisted for at least 6 days after infection. Furthermore, the anti-herpes efficacy of a single administration of drug-loaded RBC before infection was comparable to that of Bis-PMEA maintained throughout the entire experiment.

Another point of interest is the surprising finding of a consistent effect of unloaded RBC upon the replication of both HIV and HSV-1 in macrophages (ranging from 30 to 50% depending on the experimental conditions used: donor variability, viral load, etc.). This antiviral status induced by RBC in macrophages may be due to an activation of macrophage functions and/or the production of certain cytokines36 as already discussed.33

The data reported in this paper suggest that Bis-PMEA can be considered a useful prodrug of PMEA that, once inside macrophages, can be slowly converted into PMEA and protect these cells for a longer period of time. Indeed, virus replication was consistently and remarkably inhibited (>90% in HIV-infected monocyte-derived macrophages by RBC-encapsulated Bis-PMEA) even 35 days after virus challenge. This effect is not dramatically greater than that achieved by free PMEA, yet it is noteworthy that it is achieved by a single administration of drug-loaded RBC and is sustained over time. This result resembles that previously published by some of our group with PMEA-loaded RBC.27

In conclusion, the data reported here prove that a new homodinucleotide consisting of two molecules of PMEA bound together (Bis-PMEA), once encapsulated into autologous erythrocytes modified to increase their recognition and phagocytosis, is able to protect macrophages from de novo infection by HIV-1 and HSV-1. Therefore, Bis-PMEA acts as an efficient antiviral prodrug following selective targeting to macrophages by means of loaded erythrocytes.


    Acknowledgments
 
This work was supported by C.N.R. Target Project on Biotechnology and Ministero della Sanità, Istituto Superiore di Sanità (Programma Nazionale di Ricerca sull'AIDS, 1998; Progetto Patologia Clinica e Terapia dell';AIDS, azione concertata 1998), Rome.


    Notes
 
* Correspondence address. Istituto di Chimica Biologica ‘G. Fornaini’, Università degli Studi di Urbino, Via Saffi, 2-61029 Urbino, Italy. Tel: +39-0722-305211; Fax: +39-0722-320188; E-mail: magnani{at}bib.uniurb.it Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Received 12 June 2000; returned 19 September 2000; revised 24 October 2000; accepted 16 March 2001