Naltrexone potentiates anti hiv effect of antiviral durgs 2001 by LDNIM

Drug and Alcohol Dependence 64 (2001) 257– 263 www.elsevier.com/locate/drugalcdep

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: peter137@umn.edu (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

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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.

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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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(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

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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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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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