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Drug and Alcohol Dependence 64 (2001) 257– 263
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Naltrexone potentiates anti-HIV-1 activity of antiretroviral drugs
in CD4+ lymphocyte cultures
Genya Gekker, James R. Lokensgard, Phillip K. Peterson *
Institute for Brain and Immune Disorders, Minneapolis Medical Research Foundation,
Hennepin County Medical Center and the Uni6ersity of Minnesota Medical School, Minneapolis, MN 55404, USA
Received 16 October 1999; received in revised form 30 January 2001; accepted 30 January 2001
Abstract
CD4+ T lymphocytes are the primary cell target for human immunodeficiency virus-1 (HIV-1), and these cells are known to
express opioid receptors. Due to the need for new treatment approaches to HIV-1 infection, we sought to determine whether the
non-selective opioid receptor antagonist naltrexone would affect HIV-1 expression in CD4+ lymphocyte cultures and whether
naltrexone would alter the antiviral properties of zidovudine (AZT) or indinavir. Activated CD4+ lymphocytes were infected with
a monocytotropic or T-cell tropic HIV-1 isolate, and p24 antigen levels were measured in supernatants of drug-treated or
untreated (control) cultures. While naltrexone alone did not affect HIV-1 expression, at a concentration of 10 − 12 –10 − 10 M
naltrexone increased the antiviral activity of AZT and indinavir 2 – 3-fold. Similar findings with a k-opioid receptor (KOR)
selective antagonist supported the possible involvement of KOR in naltrexone’s potentiation of the antiretroviral drugs. The
results of this in vitro study suggest that treatment of alcohol or opiate dependent HIV-1-infected patients with naltrexone is
unlikely to interfere with the activity of antiretroviral drugs. Also, based upon naltrexone’s safety profile and its synergistic activity
in vitro, these findings suggest clinical trials should be considered of naltrexone as an adjunctive therapy of HIV-1 infection.
© 2001 Elsevier Science Ireland Ltd. All rights reserved.
Keywords: Human immunodeficiency virus; Indinavir; Naloxone; Naltrexone; Zidovudine
1. Introduction
The introduction of highly active antiretroviral therapy, i.e. triple combination therapy that includes a
protease inhibitor, has had a dramatic clinical benefit
for many patients infected with human immunodeficiency virus-1 (HIV-1) (The CASCADE Collaboration,
2000; Fauci, 1999). However, a substantial number of
patients have had inadequate responses to current drug
regimens or cannot tolerate their toxic effects. Also,
emergence of HIV-1 strains resistant to available drugs
is a growing problem, and even when highly active
antiretroviral therapy is successful, the virus persists in
sanctuaries unreachable by the drugs or in a latent form
on which drugs have no effect (Fauci, 1999). Thus, an
* Corresponding author. Present address: Department of Medicine,
Hennepin County Medical Center, 701 Park Avenue, Minneapolis,
MN 55415, USA. Tel.: +1-612-3472877; fax: +1-612-9044299.
E-mail address: [email protected] (P.K. Peterson).
ongoing need remains for new treatment approaches to
HIV infection.
The contemporaneous observations made some 20
years ago that CD4+ lymphocytes possess receptors for
opiates (Wybran et al., 1979) and that intravenous drug
users were a major risk group for development of AIDS
(Selik et al., 1984) spawned a large number of studies
that have demonstrated that exogenous opiates, as well
as endogenous opioid peptides, possess immunomodulatory properties (Eisenstein and Hilburger, 1998).
More detailed studies have shown that CD4+
lymphocytes express opioid receptors of the m, k, and d
classes (Sharp et al., 1998). Research in our laboratory,
which has focused primarily on mononuclear phagocytes, has revealed that k-opioid receptor (KOR) agonists can suppress the replication of the monocytotropic
HIV-1SF162 strain in cultures of microglial cells (Chao et
al., 1996) and blood monocyte-derived macrophages
(Chao et al., 2000). In these studies of macrophages, the
antiretroviral activity of the KOR agonist U50,488 was
0376-8716/01/$ - see front matter © 2001 Elsevier Science Ireland Ltd. All rights reserved.
PII: S0376-8716(01)00140-5
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G. Gekker et al. / Drug and Alcohol Dependence 64 (2001) 257–263
blocked by naloxone, a non-selective opioid receptor
antagonist, as well as by the KOR-selective antagonist
nor-binaltorphimine (nor-BNI). In the present study,
we sought to determine whether naloxone or naltrexone, an agent that is widely used in the treatment of
chronic alcoholism, would affect HIV-1 expression in
CD4+ lymphocyte cultures and whether these opiate
antagonists would alter the antiviral activities of the
reverse transcriptase inhibitor, zidovudine (AZT), or
the protease inhibitor, indinavir, in this culture system.
2. Materials and methods
2.1. Reagents
Naloxone, naltrexone, AZT, Dulbecco’s modified Eagle’s medium (DMEM), RPMI 1640, phytohemaglutinin (PHA), L-glutamine, penicillin, and streptomycin
were obtained from Sigma Chemical Co. (St. Louis,
MO). Nor-BNI, a KOR selective antagonist, naltrindole (NTI), a d-opioid receptor (DOR) antagonist,
and b-funaltrexamine (b-FNA), a m-opioid receptor
(MOR) antagonist were kindly provided by P.S. Portoghese (University of Minnesota). All other reagents
were purchased from the indicated sources; fetal bovine
serum (FBS) (Hyclone Laboratories, Logan, UT);
Lymphocyte Separation Medium (ICN Biomedical Inc.,
Aurora, OH); human recombinant interleukin (IL)-2
(Boehringer-Mannheim, Germany).
2.2. Lymphocyte acti6ation and purification
This study was approved by our institutional Human
Subjects Research Committee. Six healthy, HIV-1seronegative laboratory personnel served as donors of
venous blood. After obtaining informed consent, heparinized venous blood was collected, and peripheral
blood mononuclear cells (PBMC) were obtained by
Ficoll-Hypaque
gradient
centrifugation
using
Lymphocyte Separation Medium. PBMC were activated for 3 days with 4 mg/ml PHA in RPMI supplemented with 10% heat-inactivated FBS, 5 U/ml of IL-2,
2 mM-L-glutamine, 100 U/ml penicillin, and 100 mg/ml
streptomycin. Purified CD4+ lymphocytes were then
isolated from the activated PBMC using Dynabeads®
(Dynal, Lake Success, NY), according to directions
supplied by the manufacturer. Briefly, magnetic
polystyrene beads coated with primary monoclonal antibody to CD4 were incubated with activated PBMC
for 45 min at 4°C on an orbital rotator at 1:4 ratio
(cell:bead). The lymphocytes bound to the beads were
separated using a magnet (Dynal MPC) and washed
four times with phosphate-buffered saline (PBS) containing 2% FBS. After isolation, DETACHaBEAD®
was used to remove the isolated CD4+ cells from
Dynabeads® (1 U/100 ml cell suspension was used to
detach positively selected lymphocytes from the magnetic beads using a Dynal-MPC magnet). Isolated
CD4+ lymphocytes were ] 98% pure by FACScan
analysis and were ]98% viable by trypan blue dye
exclusion criteria.
2.3. HIV-1 isolates
The monocytotropic HIV-1SF162 strain was provided
by the NIH AIDS Research and Reference Reagent
Program (National Institute of Allergy and Infectious
Diseases, Rockville, MD). The HIVAT isolate used in
this study was originally recovered from the peripheral
blood of an asymptomatic HIV-1-infected patient and
prepared as previously described (Peterson et al., 1990).
This viral isolate has characteristics most suggestive of
a T-tropic strain (i.e. it replicates readily in the T cell
line H9 and in primary activated CD4+ lymphocytes
but is not expressed in cultures of human microglial
cells, which are primary brain macrophages that are
productively infected by M-tropic but not by T-tropic
HIV-1 strains). HIV-1SF162 or HIV-1AT was added to
purified activated CD4+ lymphocytes at a multiplicity
of infection of 0.02.
2.4. Drug treatment of HIV-1 -infected CD4+
lymphocytes
After 2 h of absorption with HIV-1 at 37°C, CD4+
lymphocytes were washed three times with PBS and
resuspended in culture medium (RPMI 1640, 10% FBS,
5 units/ml of IL-2, penicillin/streptomycin, 2 mg/ml
PHA) containing indicated concentrations of AZT, indinavir, opiate receptor antagonists, or drug combinations. After 4 days post-infection, culture supernatants
were collected in duplicate for HIV-1 p24 antigen (Ag)
assay.
2.5. HIV-1 p24 Ag assay
HIV-1 p24 Ag levels were measured using an enzyme-linked immunoassay (Abbott Laboratories), as
previously described (Chao et al., 1996). A standard
dilution curve derived from known amounts of p24 Ag
was used to quantify the Ag levels in culture supernatants. The sensitivity of this assay is 30 pg/ml.
2.6. Statistical analysis
The effects of various test compounds on HIV-1
replication are expressed as a percentage of p24 Ag
levels in control (untreated) cultures. Where appropriate, data are expressed as means9 S.E.M. of indicated
number of separate experiments using CD4+
lymphocytes from different donors. To compare the
means of two groups, Student’s t-test was used.
G. Gekker et al. / Drug and Alcohol Dependence 64 (2001) 257–263
259
3. Results
3.1. Anti6iral acti6ities of AZT and indina6ir
Before examining the effect of naloxone on HIV-1
expression, concentration-response studies were performed with AZT and indinavir in CD4+ lymphocyte
cultures that were infected with HIV-1AT or with HIV1SF162. While indinavir was considerably more potent
than AZT against HIV-1AT (Fig. 1A), the drugs were
equipotent against HIV-1SF162 (Fig. 1B). Based upon
these results, suboptimal concentrations of AZT (10 − 9
M for both HIV-1AT and HIV-1SF162) and indinavir
(10 − 15 M for HIV-1AT, 10 − 9 M for HIV-1SF162) were
selected for subsequent studies with naloxone or
naltrexone.
Fig. 2. Effect of (A) naloxone and (B) naltrexone on HIV-1AT
expression and on the antiviral activity of AZT. Cultures of HIV-1ATinfected CD4+ lymphocytes were incubated in the absence (control)
or presence of AZT (10 − 9 M), opiate antagonists, or combinations of
opiate antagonists plus AZT, at the indicated concentrations, for 4
days at which time supernatants were collected for measurement of
p24 Ag levels. Data (mean 9S.E.M. of three separate experiments
using CD4+ lymphocytes from three donors) are expressed as percent
of the control p24 Ag values (*) P B0.05 (**), P B0.01 versus AZT
alone.
3.2. Effects of naloxone and naltrexone on AZT
Fig. 1. Antiviral activity of AZT and indinavir versus (A) HIV-1AT
and (B) HIV-1SF162. Cultures of acutely infected CD4+ lymphocytes
were incubated in the absence (control) or presence of drugs, at the
indicated concentrations, for 4 days at which time supernatants were
collected for measurement of p24 Ag levels. Data are expressed as
percent of control p24 Ag values and are representative of three
separate experiments.
Naloxone added alone, at concentrations ranging
from 10 − 14 to 10 − 8 M, to CD4+ lymphocyte cultures
infected with HIVAT had no effect on viral expression
(Fig. 2A). However, when naloxone and a suboptimal
concentration of AZT (10 − 9 M) were added together to
CD4+ lymphocyte cultures, naloxone displayed a bellshaped concentration-response synergistic effect, with
maximal activity at 10 − 10 M naloxone (i.e. AZT’s
antiviral activity was increased 2–3-fold) (Fig. 2A).
Similar results were obtained with naltrexone (Fig. 2B).
When added alone, naltrexone had no effect on HIV1AT expression, but together with AZT, naltrexone
(10 − 10 M) increased the anti-HIV-1 activity of AZT
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G. Gekker et al. / Drug and Alcohol Dependence 64 (2001) 257–263
(10 − 9 M) from 30 to 85%. Because naltrexone was
found to give effects that were comparable to naloxone, naltrexone was used in all subsequent experiments.
To determine whether naltrexone would also potentiate the antiretroviral activity of AZT versus a
monocytotropic strain of HIV-1, a concentration–response study was carried out with CD4+ lymphocytes
infected with HIV-1SF162 (Fig. 3). By itself, naltrexone
again had no effect on viral expression, but when
combined with 10 − 9 M AZT, naltrexone (10 − 10M)
increased the antiviral activity of this reverse transcriptase inhibitor about 2-fold.
3.3. Effects of naltrexone on indina6ir
To ascertain whether the synergistic activity of naltrexone would extend to a protease inhibitor, concentration-response studies of naltrexone were performed
using suboptimal concentrations of indinavir in CD4+
lymphocyte cultures infected with HIV-1AT (Fig. 4A)
or HIV-1SF162 (Fig. 4B). With both viral strains, naltrexone displayed a bell-shaped concentration-response synergistic interaction with indinavir. Maximal
potentiation (2–3-fold) was observed at a naltrexone
concentration of 10 − 12 M.
3.4. Effects of selecti6e opioid receptor antagonists
To determine whether selective opioid receptor antagonists would potentiate the antiviral activity of
Fig. 4. Effect of naltrexone on (A) HIV-1AT and (B) HIV-1SF162
expression and on the antiviral activity of indinavir. Cultures of
acutely HIV-1-infected CD4+ lymphocytes were incubated in the
absence (control) or presence of naltrexone, indinavir (10 − 15 M for
HIV-1AT, 10 − 9M for HIV-1SF162), or naltrexone plus indinavir combinations, at the indicated concentrations, for 4 days at which time
supernatants were collected for measurement of p24 Ag levels. Data
(mean 9S.E.M. of three separate experiments using CD4+
lymphocytes from three donors) are expressed as percent of the
control p24 Ag values. (*) PB0.05, (**) P B0.01 versus indinavir
alone.
Fig. 3. Effect of naltrexone on HIV-1SF162 expression and on the
antiviral activity of AZT. Cultures of HIV-1SF162-infected CD4+
lymphocytes were incubated in the absence (control) or presence of
AZT (10 − 9 M), naltrexone, or a combination of naltrexone plus
AZT, at the indicated concentrations, for 4 days at which time
supernatants were collected for measurement of p24 Ag levels. Data
(mean9 S.E.M. of three separate experiments using CD4+
lymphocytes from three donors) are expressed as percent of the
control value p24 Ag. (*) P B0.05 versus AZT alone.
AZT or indinavir, as had been observed with the
non-selective opioid receptor antagonists naloxone
and naltrexone, varying concentrations of nor-BNI,
NTI, or b-FNA were added to HIV-1AT-infected
CD4+ lymphocytes in the absence or presence of
AZT (10 − 9 M) or indinavir (10 − 15 M). As had been
seen with naloxone and naltrexone, none of the selective opioid receptor antagonists by themselves had an
effect on HIV-1 expression (Fig. 5 and Fig. 6). The
KOR antagonist, nor-BNI, potentiated the antiviral
activity of AZT (Fig. 5A) and indinavir (Fig. 6A) in
a bell-shaped concentration-response manner similar
G. Gekker et al. / Drug and Alcohol Dependence 64 (2001) 257–263
261
to that observed with naloxone and naltrexone. In
contrast to the findings with nor-BNI, the DOR antagonist, NTI, had no effect on the antiviral activity of
AZT (Fig. 5B) or indinavir (Fig. 6B). The MOR antagonist, b-FNA, also had no effect on AZT (Fig. 5C) but
actually inhibited the antiviral activity of indinavir (Fig.
6C). Taken together, these findings suggest that the
potentiating effect of naloxone on AZT and indinavir
may possibly be mediated through an interaction with
KOR.
Fig. 6. Effect of (A) nor-BNI, (B) NTI, and (C) b-FNA on HIV-1AT
expression and on antiviral activity of indinavir. Cultures of HIV1AT-infected CD4+ lymphocytes were incubated in the absence (control) or presence of indinavir (10 − 9 M), opiate receptor antagonists,
or combinations of opiate receptor antagonists plus indinavir, at
indicated concentrations, for 4 days at which time supernatants were
collected for measurement of p24 Ag levels. Data (mean 9 S.E.M. of
three separate experiments using CD4+ lymphocytes from three
donors) are expressed as percent of the control values. (**). PB 0.01
versus indinavir alone.
4. Discussion
Fig. 5. Effect of (A) nor-BNI, (B) NTI, and (C) b-FNA on HIV-1AT
expression and on antiviral activity of AZT. Cultures of HIV-1AT-infected CD4+ lymphocytes were incubated in the absence (control) or
presence of AZT (10 − 9 M), opiate receptor antagonists, or combinations of opiate receptor antagonists plus AZT, at indicated concentrations, for 4 days at which time supernatants were collected for
measurement of p24 Ag levels. Data (mean 9 S.E.M. of three separate experiments using CD4+ lymphocytes from three donors) are
expressed as percent of the control p24 Ag values. (*) P B0.05 versus
AZT alone.
The results of the present study suggest that opiate
antagonists have little or no influence by themselves on
the expression of HIV-1 in CD4+ lymphocytes, the
principal target cell of this virus. This finding is consistent with results of earlier studies on HIV-1 expression
in macrophages (Chao et al., 1996, 2000) and with
more recent findings with HIV-1-infected CD4+
lymphocytes (Peterson et al., 2001). However, the observation that naloxone and naltrexone are capable of
potentiating the antiretroviral activity of both a reverse
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G. Gekker et al. / Drug and Alcohol Dependence 64 (2001) 257–263
transcriptase inhibitor and a protease inhibitor is a
novel finding.
The mechanism of the synergistic activity of naloxone and naltrexone with antiretroviral drugs is
presently unknown. A similar bell-shaped concentration-response effect seen with the KOR antagonist,
nor-BNI, suggests a possible interaction of naloxone
with KOR on CD4+ lymphocytes is involved. The
expression of KOR by T lymphocytes has been characterized both in murine (Alicea et al., 1998; Ignatowski
and Bidlack, 1998) and human (Chuang et al., 1995;
Peterson et al., 2001) cell cultures. Conceivably, an
interaction of naloxone and naltrexone with KOR
could result in increased drug entry into CD4+
lymphocytes, thereby bringing suboptimal intracellular
concentrations to a more fully inhibitory level. Alternatively, activation of KOR could trigger an intracellular
signaling pathway that facilitates the intracellular action of antiretroviral drugs. However, the higher binding affinity for naloxone and naltrexone for MOR
described for brain tissue and the observation that
b-FNA, a MOR selective antagonist, had no effect on
AZT but actually inhibited indinavir’s antiviral activity
underscores the complexity of the interaction of opiate
antagonists with CD4+ lymphocytes and the fact that
the mechanism of action of naloxone and naltrexone in
this cell culture system is unknown.
The phenomenon of synergistic interactions of antimicrobial drugs has long been recognized in the field
of antibacterial therapy. For example, at clinically
achievable concentrations, aminoglycosides have no inhibitory activity against Enterococcus spp., a major
cause of infective endocarditis, but they synergistically
facilitate the killing capacity of penicillin against this
pathogen. Interestingly, recent studies of certain psychotropic drugs suggest that these agents have synergistic antibacterial activity when they are combined with
conventional antibiotics (Munoz-Bellido et al., 2000;
Bettencourt et al., 2000). The results of the present
study suggest that naloxone and naltrexone, drugs used
primally for the purpose of antagonizing opiate interactions in the nervous system, may have synergistic properties when combined with conventional antiviral drugs.
Not surprisingly, naloxone and naltrexone, which are
structurally similar pure opiate receptor antagonists,
were found to have comparable activity in potentiating
the antiviral effects of antiretroviral drugs. Because of
its greater bioavailability after oral administration, naltrexone has been evaluated in clinical trials of opiate
and alcohol dependence (O’Malley et al., 1992; Volpicelli et al., 1992), indications for which it has been
approved by the Food and Drug Administration. Although naltrexone’s mechanism of action in treatment
of alcoholism is incompletely understood, blockade of
endogenous opioid receptors is thought to be involved.
Recent studies suggest that cognitive behavioral ther-
apy and naltrexone may be synergistic in the treatment
of alcohol dependence (Anton et al., 1999). At 50 mg
per day, the dosage used in the treatment of alcohol
dependence, it seems likely that the levels found to be
maximally effective in our in vitro studies of HIV-1-infected CD4+ lymphocytes, i.e. picomolar to nanomolar
range, could be achieved in vivo. It would be of interest
to know whether HIV-1-infected patients who are being
treated concomitantly with antiretroviral drugs and naltrexone for alcoholism have reduced viral loads compared with patients who are not receiving naltrexone.
From a clinical perspective, the results of this in vitro
study are reassuring for they suggest that treatment of
alcoholism in HIV-1-infected patients with naltrexone
would not interfere, and could possibly enhance, the
activity of antiretroviral drugs. Due to its safety profile,
and the urgent need for new therapeutic agents to treat
HIV-1-infected patients who are not responding to or
who cannot tolerate highly active antiretroviral therapy, the results of this study also suggest that clinical
trials should be considered of naltrexone as an adjunctive therapy of this devastating viral infection.
Acknowledgements
This work was supported by US Public Health Service Grants DA04381, DA09924, and T32-DA07239
from the National Institute on Drug Abuse.
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