Antiviral Research 47 (2000) 149–158

A new acyclic heterodinucleotide active against HumanImmunodeficiency Virus and Herpes Simplex Virus

Palmarisa Franchetti a,*, Ghassan Abu Sheikha a, Loredana Cappellacci a,Stefano Marchetti a, Mario Grifantini a, Emanuela Balestra b,

Carlo-Federico Perno b,c, Umberto Benatti d, Giorgio Brandi e, Luigia Rossi f,Mauro Magnani f

a Dipartimento di Scienze Chimiche, Uni6ersita di Camerino, 62032 Camerino, Italyb Dipartimento di Medicina Sperimentale e Scienze Biochimiche, Uni6ersita di Roma ‘Tor Vergata’, Roma, Italy

c IRCCS L. Spallanzani, 00173 Roma, Italyd Istituto Policattedra di Chimica Biologica, Uni6ersita di Geno6a, 16132 Geno6a, Italy

e Istituto di Igiene, Uni6ersita di Urbino, 61029 Urbino, Italyf Istituto di Chimica Biologica ‘Giorgio Fornaini’, Uni6ersita di Urbino, 61029 Urbino, Italy

Received 21 May 1999; accepted 25 April 2000

Abstract

The most common therapies against human herpes virus (HSV-1) and human immunodeficiency virus (HIV-1)infectivity are based on the administration of nucleoside analogues. Acyclovir (ACV) is the drug of choice againstHSV-1 infection, while the acyclic nucleoside phosphonate analogue PMPA has shown marked anti-HIV activity ina phase I and II clinical studies. As monocyte-derived macrophages are assumed to be important as reservoirs of bothHSV-1 and HIV-1 infection, new approaches able to inhibit replication of both viruses in macrophages should bewelcome. ACVpPMPA, a new heterodinucleotide consisting of both an antiherpetic and an antiretroviral drug boundby a phosphate bridge, was synthesized and encapsulated into autologous erythrocytes modified to increase theirphagocytosis by human macrophages. ACVpPMPA-loaded erythrocytes provided an effective in vitro protectionagainst both HSV-1 and HIV-1 replication in human macrophages. © 2000 Elsevier Science B.V. All rights reserved.

Keywords: Acyclovir (ACV); Anti-HIV activity; Anti-HSV activity; Human macrophages; (R)PMPA

www.elsevier.com/locate/antiviral

1. Introduction

Human herpes viruses (HSVs) are distributedworldwide and are among the most frequent viralinfections in human immunodeficiency virus type1 (HIV-1) immunocompromised patients. Hence,therapeutic strategies able to inhibit replication of

* Corresponding author. Tel.: +39-0737-402228; fax: +39-0737-637345.

E-mail addresses: frapa@camserv.unicam.it (P. Franchetti),magnani@bib.uniurb.it (M. Magnani).

0166-3542/00/$ - see front matter © 2000 Elsevier Science B.V. All rights reserved.

PII: S 0166 -3542 (00 )00101 -7

P. Franchetti et al. / Anti6iral Research 47 (2000) 149–158150

both viruses are surely welcome (De Clercq,1997). So far, the most common therapeuticstrategies against Human herpes virus type 1(HSV-1) and HIV-1 infectivity have been basedon the administration of nucleoside analoguesalone or in combination with other antiviralagents. However, these antiviral nucleosides haveto be converted to their triphosphorylated deriva-tives by viral and/or cellular kinases (De Clercq,1997). At the cellular level, the main problemsinvolved in the use of such drugs are their limitedphosphorylation in some cells (e.g. antiretroviraldrugs in macrophages) and the cytotoxic sideeffects of the corresponding triphosphates.Acyclic nucleoside phosphonates (ANP), as mim-ics of 5%-monophosphate nucleosides, have provedto be effective inhibitors of different types ofDNA viruses and retroviruses including HIV (DeClercq, 1997, 1998). These compounds enter thecells through an endocytosis-like process and areconverted into diphosphorylated derivatives,which act as alternative substrate to the dNTP/chain terminator in the HIV reverse trancriptase(RT) reaction. Also the 3-hydroxy-2-(phospho-nomethoxy)propyl (HPMP) derivatives, such ascidofovir (HPMPC), the 2-(phospho-nomethoxy)propyl (PMP) derivatives, such asPMPA, and the 2-(phosphonomethoxy)ethyl(PME) derivatives, such as PMEA, in theirdiphosphorylated form (HPMCpp, PMPApp, andPMEApp) interact with the normal substrates forthe viral DNA polymerases. PMPA [9-((R)-2-phosphonomethoxypropyl)adenine] is more afaithful chain terminator of the HIV-1 reversetranscriptase reaction than PMEA (De Clercq,1997 and references cited therein). PMPA exhibitsa slightly higher selectivity index in cell culture,which could be explained by the low affinity ofthe phosphorylated PMPApp form for host DNApolymerases. PMPApp proved to be a less potentinhibitor of each of the cellular polymerases, par-ticularly DNA polymerase g, than PMEApp.Both PMEApp and PMPApp were found to besubstrates for the DNA polymerases a, b and g,and to terminate DNA elongation after their in-corporation into the primer. The lower cytotoxic-ity of PMPA, as compared to PMEA, coulddepend on its less efficient incorporation into

DNA (Cihlar and Chen, 1997). PMPA does notafford activity against herpes viruses, but its activ-ity is selective against a wide range of hepad-naviruses and retroviruses including the humanimmunodeficiency virus (De Clercq, 1997). In par-ticular, PMPA is highly effective in the pre- orpostexposure prophylaxis of simian immunodefi-ciency virus (SIV) infection in rhesus monkeys(Tsai et al., 1997). This acyclic nucleotide is cur-rently undergoing phase III clinical trials for thetreatment of HIV infection in AIDS patients.Acyclovir (ACV) is the drug of choice againstHSV and varicella-zoster virus (VZV) infectionsin immunosuppressed patients.

Cells of macrophage lineage [monocyte/macrophages (M/M)] play a central role in thepathogenesis of infection by HIV and representthe main reservoir of the virus in the body. Inaddition, M/M can be targeted by other viruses,such as HSV-1, which are able to enhance thereplication of HIV (Heng et al., 1994; Perno et al.,1997).

Magnani et al. (1992) have developed a drugtargeting system that allows the selective adminis-tration of antiviral drugs to macrophages. Thissystem has proved to be effective in the protectionof macrophages both in the murine model ofAIDS (Rossi et al., 1993) and feline AIDS (Mag-nani et al., 1994). Furthermore, it was shown thatnew antiviral prodrugs, once in the macrophage,can be split into phosphorylated (and thus acti-vated) nucleoside analogues (Magnani et al.,1996).

Prompted by these considerations, we designedand synthesized ACVpPMPA, a new acyclic het-erodinucleotide consisting of both an antiherpeticand an antiretroviral drug. In ACVpPMPA anacyclic nucleoside (acyclovir) and an acyclic nu-cleoside phosphonate (PMPA) were bound by aphosphate bridge, giving a mixed phosphate-phos-phonate anhydride.

We propose ACVpPMPA as a single moleculeactive against both HSV and HIV replication.ACVpPMPA was incorporated into a carrier suchas the red blood cells (RBCs) for targeted deliverythereby conferring protection on macrophages.These are expected to split the acyclic heterodinu-cleotide symmetrically with the formation of ACV

P. Franchetti et al. / Anti6iral Research 47 (2000) 149–158 151

monophosphate and PMPA, or asymmetricallywith the formation of ACV and PMPAmonophosphate which could then be convertedinto the corresponding active diphosphate formby cellular kinases. In this way, it is possible toincrease the uptake of phosphorylated drugs bycells of the M/M lineage and to overcome the lowphosphorylating capabilities of macrophages. Atthe same time, ACVpPMPA acts as a prodrugthat can be activated by endogenous enzymes toproduce at low concentrations triphosphorylatednucleoside analogues that selectively inhibit theviral reverse transcriptase and/or viral DNApolymerase.

2. Materials and methods

2.1. Reagents

Acyclovir was purchased from Sigma Reagents.(R)PMPA was synthesized as reported by Holy etal. (1995a,b). PMEA was kindly provided byGilead Sciences, Foster City, CA, USA.

2.2. Synthesis

The heterodinucleotide ACVpPMPA was ob-tained by coupling the morpholidate derivative of9-((R)-2-phosphonomethoxypropyl)adenine (1)with the mono n-tri-butylammonium salt of ACVmonophosphate (ACV-MP, 2) as reported inScheme 1. Morpholidate 1 was obtained as N,N %-dicyclohexyl-4-morpholine-carboxamidinium saltby treatment of PMPA with morpholine in thepresence of N,N %-dicyclohexyl-carbodiimide(DCCI) as activating reagent (molar ratio 1/6), inhot aqueous tert-butyl alcohol for 11 h. ACV-MPwas synthesized as described by Yoshikawa et al.(1967), except that after the reaction the mixturewas poured into diethyl ether, and the resultingprecipitate was then separated by centrifugation.The product was then dissolved in water and theaqueous solution was adjusted to pH 7.5 with a 1M NaOH solution; the solvent was removed un-der reduced pressure and dried in vacuo. The finalproduct (100% yield) was identified by mass spec-trometric analysis using an atmospheric pressureionization electrospray ionization (API-ESI)source, which confirmed the expected molecularmass of 305.5 m/z. The tributylammonium salt ofACV-MP was then obtained as described by vanWijk et al. (1992). Reaction of morpholidate 1with ACV-MP (2) (molar ratio 1/1) in anhydrouspyridine for 2 days at room temperature gavecrude ACVpPMPA. The obtained reaction mix-ture was evaporated to dryness and purified bychromatography on silica gel column using 2-propanol/NH4OH/H2O (75:20:5, v/v/v) as eluents.The appropriate fraction was evaporated in vacuoto give a solid residue that was solubilized inaqueous ammonia (30%) and washed severaltimes with diethyl ether. The aqueous fraction wasconcentrated in vacuo to give ACVpPMPA asdiammonium salt. The final product (52% yield)was analyzed by mass spectrometry as describedabove, and showed a mass value of 574.1 m/z,consistent with the expected molecular structure.All measurements were performed in the negativeion mode, and scanning in the 300–800 massrange. The elemental analysis confirmed the struc-ture of ACVpPMPA as diammonium salt (Anal.Calcd. for C17H24N10O9P2

. 2NH3: C 33.56, H 4.97,N 27.62. Found: C 33.38, H 4.71, N 27.40).

Scheme 1. Reaction conditions: (a) morpholine, DCCI, tert-butanol: (b) pyridine.

P. Franchetti et al. / Anti6iral Research 47 (2000) 149–158152

Finally, the structure of ACVpPMPA was alsoconfirmed by nuclear magnetic resonance 1H-NMR spectrum in D2O recorded at 300 MHzwith a Varian VXR-300 spectrometer. The chemi-cal shift values are expressed in d values (parts permillion) relative to tetramethylsilane as an inter-nal standard. The 1H-NMR spectrum showed thepresence of two singlets at d 7.82 (1H, H-2 ofadenine) and at d 8.13 (2H, H-8 of adenine andguanine). Moreover, the 31P-NMR spectrum inD2O, recorded by the above apparatus at 121MHz, revealed the presence of two singlets at d

9.02 (CH2P) and d −10.59 (O–P–O).

2.3. HPLC analysis of ACVpPMPA metabolites

Samples were extracted with perchloric acid(Magnani et al., 1989); neutralized extracts werethen used for HPLC determinations essentially asdescribed by Rossi et al. (1998) with some modifi-cations: the flow rate was 0.9 ml/min and thedetection wavelength was 254 nm. The retentiontimes under the conditions used were 8.5 min forACV, 11.5 min for ACV-MP and 15.4 min forPMPA.

2.4. ACVpPMPA metabolism in erythrocytelysate

Erythrocyte lysate was obtained as described byRossi et al. (1998). The hemolysate (123 mg ofhemoglobin/ml) was incubated at 37°C in thepresence of 0.08 mM ACVpPMPA, in absence orin presence of 1.0 mM ATP. At 0, 0.5, 1.0, 2.0,4.0, and 7.0 h, 100 ml aliquots were removed andprocessed for HPLC determination.

2.5. ACVpPMPA encapsulation in RBCs

Human erythrocytes were loaded withACVpPMPA by a procedure of hypotonic dialy-sis, isotonic resealing, and reannealing, essentiallyas reported by Magnani et al. (1992) with somemodifications: RBCs were dialyzed for 70 min andat the end of the dialysis time 10 mmol ofACVpPMPA were added to each milliliter oferythrocytes. The mixture was then incubated 30min at room temperature before adding 0.1 vol of

the compound solution per volume of dialyzederythrocytes. Cells were washed twice in 10 mMHEPES (pH 7.4) containing 154 mM NaCl and 5mM glucose (buffer A) and used for the experi-ments described below.

2.6. ACVpPMPA metabolism in intacterythrocytes

ACVpPMPA-loaded RBCs were resuspendedat a 0.7% hematocrit in RPMI 1640 mediumcontaining 10% fetal calf serum (FCS) and incu-bated at 37°C, 5% CO2 under sterile condition. Atdifferent incubation times, 5 ml aliquots wereprocessed for the determination of ACVpPMPAand its metabolites by extraction with perchloricacid and analysis by HPLC, as described above.ACVpPMPA metabolites in the incubationmedium were extracted by solid-phase extractionusing Isolute TM C18 columns (International Sor-ben Technology, Mid-Glamorgan, UK) accordingto the manufacturer’s instructions and analyzedby HPLC, as described above.

2.7. Targeting of drug-loaded erythrocytes

ACVpPMPA-loaded RBCs were modified toincrease their recognition by macrophages by aprocedure described in Magnani et al. (1992) andPerno et al. (1997). Briefly, loaded erythrocytesuspensions (10% hematocrit) in 1.0 mM ZnCl2were treated with 1.0 mM bis(sulfosuccin-imidyl)suberate (BS3) for 15 min at room temper-ature and washed once in buffer A containing 10mM ethanolamine, and once in buffer A contain-ing 1% (w/v) bovine serum albumin. Erythrocyteswere then incubated at 37°C in autologous plasmafor 60 min at a hematocrit of 40% and after thistime washed once in buffer A containing 2% (w/v)bovine serum albumin and once in buffer A.Finally, opsonized ACVpPMPA-loaded RBCswere resuspended in buffer A at a hematocrit of20% and used for the targeting to macrophages.

2.8. Cells and 6iruses

Peripheral blood mononuclear cells (PBMCs)were obtained from normal seronegative blood

P. Franchetti et al. / Anti6iral Research 47 (2000) 149–158 153

Table 1Percent virus inhibition (p24) in HIV-infected human macrophages treated with drug-loaded RBCs or free drugsa

% HIV inhibitionTreatments

Days

2014 27 34

0 00 0NoneUnloaded RBCs (1:100) – 31 51 50

28 48– 46Unloaded RBCs (1:500)\99ACVpPMPA-loaded RBCs (1:100) \99 \99 \99

\99 \99ACVpPMPA-loaded RBCs (1:500) \99\990 00 0ACVpPMPA 1.0 mM (overnight)

14PMPA 1.0 mM (overnight) 13 13 142PMEA 1.0 mM (overnight) 18 2 3

64 6878 79ACVpPMPA 1.0 mM (maintained)

a Drugs were added to human macrophages 18 h before infection and maintained overnight until infection, except for 1.0 mM ofACVpPMPA whose efficacy was evaluated also when maintained throughout the entire experiment. When 1.0 mM PMEA and 1.0mM PMPA were maintained throughout the entire experiment, an almost total protection was obtained. All values are the meansof quadruplicate cultures in which variability never exceeded 5%. One-hundred-percent virus production corresponds to 10 000,22 000, 30 000, and 35 000 pg of p24/ml 14, 20, 27, and 34 days after infection, respectively.

donors by separation on Histopaque solution(Sigma, St. Louis, MO). Monocytes were sepa-rated as described by Rossi et al. (1998) or Pernoet al. (1996). After removal of nonadhering cellsby repeated washings, monocytes were scrapedand collected in a phosphate saline solution. Cellviability was greater than 90% as determined bythe trypan blue dye exclusion test. The cells weresuspended in RPMI 1640 medium supplementedwith 15–20% heat-inactivated FCS and 1% an-tibiotics (complete medium), and adjusted to afinal concentration of 5×105 cells/ml. After cul-ture, the monocytes had matured intomacrophages, as revealed by surface marker anal-ysis, and had formed a monolayer. A monocy-totropic strain of HIV-1, HTLV-IIIBa-L and alaboratory-adapted strain of HSV-1, Mc Entyre(the latter kindly provided by M. Barbi, Instituteof Virology, University of Milan, Italy), were usedin experiments of infection with HIV-1 and HSV-1, respectively. Both viruses grew and easily repli-cated in macrophages (Perno et al., 1996).

2.9. Assays of anti-HIV-1 acti6ity

For the assays of antiretroviral activity on in-fected macrophages, RBCs loaded with 1.0 mM

ACVpPMPA were added at a ratio of 100 or 500RBCs per macrophage (Table 1). After 18 h ofincubation, noningested RBCs were removed byextensive washing with culture medium. As a con-trol, macrophages culture were treated with un-loaded RBCs, i.e. RBCs submitted to the sameprocedure including transient lysis and successivemodifications to increase macrophage recognition,but without addition of ACVpPMPA (see below).Human macrophage cultures receiving eitherACVpPMPA loaded or unloaded RBCs were theninfected for 2 h with HIV-1Ba-L [300 CCID50 (50%cell culture infective dose)/ml]. After incubationwith the virus, cell cultures were extensivelywashed to remove any residual virus particles.Further controls were performed in each experi-ment: (1) 1.0 mM ACVpPMPA given overnight(as for RBCs) or maintained throughout the en-tire experiment; (2) 1.0 mM PMEA givenovernight (as for RBCs); (3) 1.0 mM PMPA givenovernight (as for RBCs), and (4) unloaded RBCsprepared and cultured with macrophages exactlyas the loaded ones. Cell cultures were then main-tained at 37°C and 5% CO2 for 5 weeks. Freshmedium was replaced every 5–6 days and virusproduction was assessed in the supernatants, witha commercially available enzyme linked immuno-

P. Franchetti et al. / Anti6iral Research 47 (2000) 149–158154

sorbent assay (ELISA) kit able to detect HIVp24gag.

2.10. Assays for anti-HSV-1 acti6ity

For the assays of antiviral activity on infectedmacrophages, unloaded RBCs and ACVpPMPA-loaded RBCs were added overnight before infec-tion at a ratio of 50 RBCs per macrophage.Noningested erythrocytes were removed by exten-sive washing with culture medium. Humanmacrophage cultures receiving ACVpPMPA-loaded RBCs and unloaded RBCs were then in-fected for 2 h with HSV-1 [3 pfu (plaque-formingunits)/cell]. After incubation with the virus, cellcultures were extensively washed, and freshmedium was added. As a control, 15 mMACVpPMPA was added for the same time as theRBCs. The inhibitory effect of the compounds onthe replication of HSV-1 was evaluated 48 h afterinfection by determining the infectious virus in thesupernatant by plaque assay in Vero cells (Table 2).

In all the antiviral assays, loaded and unloadederythrocytes were used within 24 h of preparation.

3. Results

3.1. Metabolism of ACVpPMPA

To evaluate the metabolism of ACVpPMPA in

Fig. 1. Stability of ACVpPMPA in plasma. ACVpPMPA (0.08mmol/ml plasma) was incubated for 7 h at 37°C. Perchloricacid extracts were prepared at different incubation times andanalyzed by HPLC, as described in Section 2.

human plasma, 0.08 mmol of free drug/ml of plasmawas incubated for 7 h at 37°C. At different incuba-tion times (0, 0.5, 1, 2, 4, and 7 h), aliquots werecollected and processed for HPLC analyses. Theresults obtained (Fig. 1) showed that ACVpPMPAwas almost completely converted to ACV andPMPA already after 1 h of incubation.

When ACVpPMPA was incubated in erythro-cyte lysate in absence of ATP, it was converted toACV-MP and PMPA (Fig. 2); ACV-MP wasrapidly converted to ACV. However, in presence of1 mM ATP, a significant inhibition of this conver-sion was obtained; in this condition 65% ofACVpPMPA was still present after 7 h of incuba-tion at 37°C.

To evaluate the metabolism of ACVpPMPA inintact erythrocytes, human erythrocytes wereloaded with ACVpPMPA to a final concentrationof 0.58 mM. The encapsulation of the heterodimer,by the procedure of hypotonic dialysis, isotonicresealing and reannealing, did not result in anyappreciable alterations in erythrocyte mor-

Table 2Inhibition of HSV-1 replication by ACVpPMPA-loaded RBCsor free drugsa

% HSV inhibitionTreatments

0NoneUnloaded 46

69*ACVpPMPA-loaded RBCs36ACVpPMPA 15 mM (overnight)

a Human macrophages were cultured for 10 days beforetreatment with the drugs. Cells were infected for 2 h with 3pfu/cell of HSV-1. The inhibitory effect of the compounds wasevaluated 48 h after infection by a plaque assay in Vero cells.The values are the mean of three experiments, each performedin duplicate. Standard errors were less than 10%.

* P valueB0.05 versus unloaded (T test). The titer of HSV-1production in the supernatants of infected untreatedmacrophages ranged from 0.5 to 1.0×106 pfu/ml.

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Fig. 2. Stability of ACVpPMPA in human erythrocyte lysates. ACVpPMPA (0.08 mmol/ml) was incubated with the lysates at 37°Cfor 7 h in the absence (A) or presence (B) of 1 mM ATP. Aliquots of the incubation mixture were extracted with perchloric acidand analyzed by HPLC, as described in Section 2.

phology and metabolism (not shown).ACVpPMPA-loaded RBCs were then incubatedup to 10 days at 37°C in RPMI 1640 mediumunder sterile conditions. At different times ofincubation, the presence of ACVpPMPA and itsmetabolites was evaluated (Fig. 3). The resultsobtained show that the intracellular ACVpPMPAdecreases down to about 2% of the starting levelafter 10 days of incubation. The decrease of theheterodimer in the RBCs was paralleled by thestoichiometric production of ACV and PMPAreleased in the culture medium. As suggested bythe results on ACVpPMPA stability in erythro-cyte lysates in the presence of 1 mM ATP (seeabove), the heterodimer is sufficiently stable inRBCs to allow the use of ACVpPMPA in ery-throcytes as a drug targeting system.

3.2. Anti6iral acti6ity of ACVpPMPA-loadedRBCs

To evaluate the anti-HIV activity ofACVpPMPA-loaded RBCs, human macrophageswere treated with ACVpPMPA (approx. 1.0 mM)

inside RBCs or unloaded RBCs. The productionof p24 was determined at different days postinfection (Table 1). As control, the free drugs 1.0mM ACVpPMPA, 1.0 mM PMPA and 1.0 mMPMEA were used. The results show thatACVpPMPA-loaded RBCs were able to com-pletely protect macrophages against HIV-1infection.

To evaluate the anti-HSV-1 activity ofACVpPMPA-loaded RBCs, human macrophageswere treated with ACVpPMPA-loaded RBCs (ap-prox. 1.0 mM inside RBCs). After extensive wash-ing, macrophages were infected for 2 h withHSV-1 (3 pfu/cell). As a control, 15 mMACVpPMPA added for the same time as RBCswas used. The inhibitory effect of the compoundwas evaluated 48 h after infection by a plaqueassay in Vero cells (Table 2). The results obtainedshow that ACVpPMPA-loaded RBCs were ableto inhibit HSV-1 replication by almost 70%,compared to B50% inhibition induced by un-loaded RBCs. Addition of the free drug in themedium reduced HSV-1 production by 36%.

P. Franchetti et al. / Anti6iral Research 47 (2000) 149–158156

It is worth noting that the administration ofunloaded RBC was able to inhibit the replicationof HIV-1 and HSV-1 by about 30–50%. Similarresults were previously reported and discussed byPiedimonte et al. (1992) and Rossi et al. (1998).The inhibitory effects of unloaded RBC dependon the experimental condition used (includingdonor variability, viral load, RBC/macrophagesratio, etc.) and were also observed with latexbeads. This antiviral status may be due to anactivation of macrophage functions and/or theproduction of certain cytokines (Borghi et al.,1995). In any case, the inhibition observed byadministering drug-loaded RBC was always sig-nificantly higher than that observed with un-loaded RBC administration (Table 1 and Table2).

When 1.0 mM ACVpPMPA-loaded RBCswas added to control (non-infected) macrophages,no cytopathic effect was observed (data notshown).

4. Discussion

The development of new therapeutic systemsagainst HIV and HSV infections, based on nu-cleoside analogues and designed to achieve bothantiretroviral and antiherpes activity in specificcell types like monocytes/macrophages, requires acombination of different features. (i) Pharmaco-logically active triphosphorylated nucleoside ana-logues can display unacceptable cell toxicity dueto inhibition of enzyme targets in the host cellother than viral enzymes. Therefore PMPApp andACV-TP and similar compounds cannot be safelyencapsulated as such into RBCs for targeting toinfected cells. (ii) Accordingly, triphosphorylatednucleoside analogues should be formed in thedesired cell type at low concentrations such as toinhibit selectively viral reverse transcriptase and/or viral DNA polymerase; therefore, metaboli-cally suitable prodrugs that can be activated byendogenous enzymes are necessary. (iii) Targetingof such prodrugs to macrophages, where theirbioconversion is required, can be greatly favoredby exploiting the natural property of ery-throphagocytosis. (iv) The selected prodrugsshould be stable enough in the carrier RBCs to bemetabolically converted to the nucleoside triphos-phate analogues only within infectedmacrophages. Furthermore, to favor compliance,single drugs with multiple activities should bepreferred instead of multiple drugs active againsteach pathogen.

All the above requirements seem to be fulfilledby ACVpPMPA loaded into RBCs. Although adinucleotide pyrophosphorylase in human redcells is present, its activity is rather low, probablybecause of its inhibition by ATP, as shown in Fig.2. On the other hand, this heterodinucleotide,while being stable in the loaded red cells, disap-pears in the macrophages within time intervalsthat exceed those required to complete ery-throphagocytosis (data not shown). This propertyis consistent with metabolism of ACVpPMPAlargely occurring in macrophages. ThatACVpPMPA behaves as a prodrug inmacrophages is demonstrated by the anti-HIVand anti-HSV-1 activity of this compound inHIV-1Ba-L- and HSV-1-infected macrophages.

Fig. 3. Metabolism of ACVpPMPA in intact erythrocytes.ACVpPMPA was incapsulated in human erythrocytes by aprocedure of hypotonic dialysis and isotonic resealing to afinal concentration of 0.58 mM. These cells were then incu-bated at 37°C following the conditions described in Section 2.

P. Franchetti et al. / Anti6iral Research 47 (2000) 149–158 157

It is worth noting that protection ofmacrophages from HIV-1 infection is completeupon drug-loaded RBCs phagocytosis and persistsup to the end of the experiment (34 days) at bothRBC/macrophage ratios investigated (Table 1).The same drug given free in solution and main-tained throughout the entire experiment reducedp24 production from 70 to 80%. This limitedactivity is likely due to extracellular conversion ofthe ACVpPMPA dimer to PMPA, which thenenters the macrophages. In fact, ACVpPMPAdiffusion through the cell membrane is unlikely, asshown by the retention of the drug when it isencapsulated into RBCs (i.e. the ACVpPMPAdimer has never been found intact in the RBCculture medium during the stability studies re-ported above). PMPA or PMEA maintained inculture for only 18 h were of very limited efficacy.

The results show that the antiviral effect of thedimer is substantially different in M/M infected byHIV, as compared to M/M infected by HSV-1.These apparently surprising results, can be ex-plained by the different virus lifecycles. Indeed,HIV-RT works only in one step at the beginningof virus replication cycle. After proviral integra-tion, RT is no longer necessary for virus produc-tion. On the contrary, HSV-DNA polymeraseworks during the whole HSV lifecycle, since pro-duction of new viral DNA genome is continuouslyrequired. Thus, it is conceivable that the effect ofthe dimer on HIV-RT can be amplified by thelimited rate of activity of this enzyme in infectedM/M (O’Brien et al., 1994), while, by contrast, theeffect of the same dimer can be reduced by thecontinuous activity of the HSV-DNA polymerase.Further studies are required to achieve final proofof this issue.

Another point of interest is the finding of aconsistent effect of unloaded RBC upon the repli-cation of both HIV-1 and HSV-1 in macrophages(ranging from 30 to 50% depending on the exper-imental condition used) as already discussed (seeSection 3). However, administration ofACVpPMPA-loaded RBC completely inhibitedHIV-1 replication in macrophages.

In conclusion, ACVpPMPA is a new het-erodimer with adequate stability in RBCs andprotective activity in human macrophages against

HIV-l and HSV infections. This dimer maintainsthe high antiretroviral activity of PMPA and at thesame time compensates for the lack of anti-HSVactivity of PMPA. The approach used furthersuggests that nucleoside analogues, if present onlyfor limited periods of time (i.e. 18 h in ourexperiment), are of limited efficacy in protecting themacrophages against HIV-l and HSV infections.Thus, careful pharmacokinetic studies are neededto define the optimal drug dosage able to maintainin vivo therapeutic concentrations for longer peri-ods of time. When encapsulated into the appropri-ate carriers, these nucleoside/nucleotide analoguesare able, upon single pulsed administration, toprovide improved protection.

Acknowledgements

This work was supported by the AIDS Projectof the Italian Ministry of Health, and by C.N.R.Target Project on Biotechnology.

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