Inhibition of HIV-1 replication in macrophages by red blood cell-mediated delivery of a heterodinucleotide of lamivudine and tenofovir

Article abstract of DOI:10.1080/15257770701508067

Homo- and heterodimers of nucleoside/nucleotide analogues as reverse transcriptase inhibitors are effective on HIV-1-infected human monocyte-derived macrophages (M/M) compared to the single drugs or their combination. Since the combined treatment of lamivudine (3TC) and tenofovir ((R)PMPA) has an antiretroviral efficacy and a synergic effect respect to separate drugs, the heterodinucleotide 3TCpPMPA was synthesized. A single administration of the dimer as free drug or 3TCpPMPA-loaded RBC selectively targeted to M/M was able to almost completely protect macrophages from "de novo" infection. Copyright Taylor & Francis Group, LLC.

Full text of DOI:10.1080/15257770701508067

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Inhibition of HIV-1 Replication in
Macrophages by Red Blood Cell-Mediated
Delivery of a Heterodinucleotide of
Lamivudine and Tenofovir
P. Franchetti a , L. Cappellacci a , R. Petrelli a , P. Vita a , M.
Grifantini a , L. Rossi b , F. Pierigé b , S. Serafini b , M. Magnani b , E.
Balestra c & C.-F. Perno c
a Department of Chemical Sciences , University of Camerino ,
Camerino, Italy
b Institute of Biochemistry “G. Fornaini,” , University of Urbino ,
Urbino, Italy
c Department of Experimental Medicine , University of Rome “Tor
Vergata,” , Rome, Italy
Published online: 10 Dec 2007.
To cite this article: P. Franchetti , L. Cappellacci , R. Petrelli , P. Vita , M. Grifantini , L.
Rossi , F. Pierigé , S. Serafini , M. Magnani , E. Balestra & C.-F. Perno (2007) Inhibition of HIV-1
Replication in Macrophages by Red Blood Cell-Mediated Delivery of a Heterodinucleotide of
Lamivudine and Tenofovir, Nucleosides, Nucleotides and Nucleic Acids, 26:8-9, 953-957, DOI:
10.1080/15257770701508067
To link to this article: http://dx.doi.org/10.1080/15257770701508067
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Nucleosides, Nucleotides, and Nucleic Acids, 26:953–957, 2007
Copyright ꢀ Taylor & Francis Group, LLC
C
ISSN: 1525-7770 print / 1532-2335 online
DOI: 10.1080/15257770701508067
INHIBITION OF HIV-1 REPLICATION IN MACROPHAGES BY RED
BLOOD CELL-MEDIATED DELIVERY OF A HETERODINUCLEOTIDE
OF LAMIVUDINE AND TENOFOVIR
P. Franchetti, L. Cappellacci, R. Petrelli, P. Vita, and M. Grifantini
2
Department of Chemical Sciences, University of Camerino, Camerino, Italy
L. Rossi, F. Pierige´, S. Serafini, and M. Magnani
Institute of Biochemistry “G.
2
Fornaini,” University of Urbino, Urbino, Italy
E. Balestra and C.-F. Perno
of Rome “Tor Vergata,” Rome, Italy
Department of Experimental Medicine, University
2
Homo- and heterodimers of nucleoside/nucleotide analogues as reverse transcriptase inhibitors
2
are effective on HIV-1-infected human monocyte-derived macrophages (M/M) compared to the sin-
gle drugs or their combination. Since the combined treatment of lamivudine (3TC) and teno-
fovir ((R)PMPA) has an antiretroviral efficacy and a synergic effect respect to separate drugs,
the heterodinucleotide 3TCpPMPA was synthesized. A single administration of the dimer as free
drug or 3TCpPMPA-loaded RBC selectively targeted to M/M was able to almost completely protect
macrophages from “de novo” infection.
Keywords Heterodinucleotides; anti-HIV-1 activity; human macrophages
INTRODUCTION
The combination of nucleoside analogues as reverse transcriptase in-
hibitors (NRTIs) with nucleotide analogues or other antiviral drugs resulted
in effective therapy for the treatment of human viral infections. Despite
the clinical benefit associated with the highly active antiretroviral therapy
(HAART), current drugs are not able to eradicate HIV-1 simply due to
the persistence of the virus in cellular reservoirs, predominantly long-lived
memory CD4+ T cells and cells of the macrophage lineage.[1] Indeed, HIV-
1 has been recovered from the peripheral blood monocytes of patients with
maximal viral suppression while on HAART.[2] Viral reservoirs established
This work was supported by grants from AIDS Project of the Italian National Institute of Health and
by FIRB funds. (Red blood cells as drug carriers, RBNE01TBTR.)
Address correspondance to Mario Grifantini, Department of Chemical Sciences, University of
Camerino, 62032 Camerino, Italy. E-mail: mario.grifantini@unicam.it
953

954
P. Franchetti et al.
early during the infection remain unaffected by antiretroviral therapy for a
long time and are able to replenish systemic infection following the failure
or cessation of the treatment.
Since the crucial role of the monocyte-derived macrophages (M/M) as
a HIV-1 reservoir, therapeutic strategy must consider the inhibition of the
viral replication in M/M.[3]
However, since macrophages are resting cells, they phosphorylate (acti-
vate) the antiviral nucleoside analogues at a rate lower than that of periph-
eral blood mononuclear cells.[4] Thus, the delivery of phosphorylated nu-
cleoside analogues directly into infected-macrophages could result in highly
anti-HIV-1 activity.
On this basis, some antiviral double-drugs (homo- and heterodinu-
cleotides) as reverse transcriptase inhibitors and as potential pro-drugs with
efficacy against HIV-1-infected M/M have been reported by us.[5] Herein, we
report on the synthesis of a new heterodinucleotide 3TCpPMPA consisting
of the potent anti-HIV-1 nucleoside and nucleotide analogues lamivudine
(3TC) and tenofovir ((R)PMPA), respectively.
The combination of tenofovir and lamivudine has already been sug-
gested in therapy guidelines for HAART-na¨ıve patients.[6] The efficacy of
the dimer 3TCpPMPA to protect the refractory cell compartment against de
novo HIV infection by simultaneous administration of 3TC and (R)PMPA
in a single molecule has been assessed.
RESULTS AND DISCUSSION
The heterodinucleotide consisting of 3TC and (R)PMPA bound to-
gether by a phosphate bridge was synthesized by coupling the mono n-
tri-butylammonium salt of β-L-3ꢁ-thia-2ꢁ,3ꢁ-dideoxy-cytidine monophosphate
(3TCMP) with (R)-9-(2-phosphonometoxypropyl)adenine as a morpholi-
date derivative (Scheme 1).
At first, the metabolism of 3TCpPMPA in plasma obtained from healthy
seronegative donors was determined. As shown in Figure 1, human plasma
possesses enzymes able to cleave the phosphate bridge of 3TCpPMPA with
the stoichiometric production of PMPA and 3TC (the latter is quickly ob-
tained starting from 3TCMP). The results obtained show that 3TCpPMPA is
stable enough in human plasma (t1/2 = 4 hours) and suggest the possibility
of using 3TCpPMPA as a prodrug of 3TC and PMPA.
The ability of the heterodimer to protect the infected human
macrophages against de novo HIV-1 infection also was determined. Com-
pound was administered to macrophages and maintained all through ex-
periment at a concentration of 1.0 µM and virus production was evaluated
over a period of 35 days. The results show an almost 100% inhibition of viral
replication (Table 1).

Inhibition of HIV-1 Replication
955
SCHEME 1 Reagents and conditions: a) morpholine, dicyclohexylcarbodiimide, t-BuOH/H2O, reflux;
b) N,N-dimethylformamide, 40◦C.
The selective delivery of 3TCpPMPA to macrophages was performed us-
ing a drug targeting system. This system is based on the use of autologous
artificially-aged red blood cells (RBC)7 that allows the direct administration
of nucleoside analogues, already in phosphorylated form, to macrophages.
The heterodinucleotide was encapsulated into autologous erythrocytes by
FIGURE 1 Metabolism of 3TCpPMPA in human plasma. 3TCpPMPA (0.25 mM) was incubated in
human plasma for 24 hours at 37◦C. Perchloric acid extracts were prepared at different incubation
times and analyzed by HPLC, as described above. All values are the mean ( S.D.) of three different
experiments.

956
P. Franchetti et al.
TABLE 1 Percentage of virus production in
HIV-1-infected human macrophages
Virus production (% of infected control)
Time (days)
3TCpPMPA (1.0 µM)
14
21
28
35
0
0
0.3
0.5
Values are expressed in percentage of p24 production
respect to untreated infected macrophages. All values
are the mean ( S.D.) of sextuplicate cultures of three
representative experiments and show the percentage of
inhibiton in p24 produced until 35 days after infection.
One hundred percent virus production corresponds to
23625 2902 pg p24 antigen/mL.
a procedure of hypotonic dialysis, isotonic resealing, and reannealing.[7]
3TCpPMPA-loaded erythrocytes were modified to increase their phagocy-
tosis by human macrophages. Then, the cells were infected by HIV-1Ba−L
and inhibition of HIV-1 replication was assessed by HIV p24gag quantifica-
tion. When 3TCpPMPA was selectively targeted to the macrophagic com-
partment by a single administration of loaded erythrocytes, the protection
of macrophages from de novo infection was nearly complete (99% protec-
tion 3 weeks posttreatment).
In conclusion, the heterodinucleotide 3TCpPMPA could act as an
efficient prodrug that, by the conversion in 3TCMP and PMPA inside
macrophages, is able to protect the refractory cell compartment against de
novo HIV-1 infection for a long time.
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