Erythrocytes as carriers of reduced glutathione (GSH) in the treatment of retroviral infections

A. Fraternale1, A. Casabianca1, L. Rossi1, L. Chiarantini1, G. F. Schiavano2, A. T. Palamara3, E. Garaci4 and M. Magnani1,*

1 Institute of Biological Chemistry ‘Giorgio Fornaini’, Via Saffi, 2; 2 Institute of Hygiene, Via S. Chiara, 27, University of Urbino, 61029 Urbino (PU); 3 Institute of Microbiology, University of Rome ‘La Sapienza’, 00185 Rome; 4 Department of Experimental Medicine and Biochemical Sciences, University of Rome ‘Tor Vergata’, 00133 Rome, Italy

Keywords: antivirals, drug delivery, macrophages, RBCs


    Introduction
 Top
 Introduction
 Selective delivery of antiviral...
 Potential role of reduced...
 Erythrocytes as carriers of...
 Conclusions
 Acknowledgements
 References
 
Macrophages constitute important targets for HIV-1: they serve as virus reservoirs and represent the most important targets in the central nervous system (CNS). Current antiretroviral therapies are of limited efficacy in this cellular compartment, thus every new approach may be of great clinical relevance. In this paper, we show how autologous erythrocytes (RBCs) have been used to deliver reduced glutathione (GSH) selectively to infected macrophages. This approach, in combination with approved antiretroviral drugs, has been used to protect the CNS of mice infected with LP-BM5, a retroviral complex that causes a syndrome known as murine AIDS. Thus, a similar system may be useful in humans and reducing agents such as GSH may become therapeutically relevant when selectively delivered to macrophages.

In HIV-1-infected patients, highly active antiretroviral therapies (HAART) have been used both to reduce viral load in plasma to un-detectable levels, and to increase the number of CD4 cells in the majority of infected individuals. Nonetheless, HIV-1 has not been eradicated by HAART, as demonstrated by virus rebound from latent viral reservoirs, principally CD4 cells and macrophages, after cessation of HAART.1,2 Cells of the macrophage lineage play an important role in HIV-1 pathogenesis. They are the initial HIV-1 cellular targets, and following infection they resist the cytopathic effects of HIV-1. As a consequence, they persist throughout the course of the disease as long-term stable reservoirs of HIV-1 capable of disseminating the virus in other cells and tissues. In fact, infectious virus can be recovered from blood monocytes obtained from patients receiving HAART with no detectable HIV-1 in blood,3 or from macrophages from SHIV-infected monkeys upon lymphocyte depletion.4 Macrophages are also the most important targets for HIV in the CNS, and alterations in neuronal metabolism induced by infected macrophages play a crucial role in the pathogenesis of HIV-related encephalopathy.3 These and other considerations underline the importance of therapeutic approaches able to interfere with HIV infection and replication in macrophages. Among drugs prescribed today, protease inhibitors inhibit HIV replication in chronically infected macrophages (i.e. cells carrying the proviral genome already integrated in the host genome), although at concentrations higher than those effective in de novo infected CD4 lymphocytes. Nucleoside analogues, the first anti-HIV drugs, although very potent in vitro, possess limited penetration in sequestered districts and are poorly phosphorylated in macrophages while non-nucleoside reverse transcriptase inhibitors possess an antiviral activity similar to that found in lymphocytes.5


    Selective delivery of antiviral drugs to macrophages
 Top
 Introduction
 Selective delivery of antiviral...
 Potential role of reduced...
 Erythrocytes as carriers of...
 Conclusions
 Acknowledgements
 References
 
Macrophages have an important role in initial infection and viral propagation. This, together with the poor antiviral activity in macrophages of currently available drugs, has increased efforts dedicated to improving the delivery of antiviral agents to the mononuclear phagocyte system. The task is to overcome pharmacokinetic problems and enhance the activities of drugs in the treatment of HIV infection and AIDS.68 Our group, among others, has developed a cellular drug delivery system based on the use of autologous erythrocytes (RBCs). Hypotonic dialysis is used to encapsulate the antiviral drug into RBCs. The loaded RBCs are then treated with ZnCl2 and BS3, in order to induce irreversible band-3 clustering, and subsequently opsonized by IgG and C3b deposition.9 Thus, macrophages are able to recognize and phagocytose the opsonized erythrocytes.10 Through this mechanism, the drug encapsulated into the erythrocytes is released into the macrophages. Erythrocytes, transformed into carrier cells, present many advantages: they are biodegradable, they circulate for long periods of time (months) and their capacity allows a high percentage of encapsulation. The validity of this approach has been demonstrated widely with various compounds and in several different in vitro and in vivo models.1013


    Potential role of reduced glutathione as an antiviral agent
 Top
 Introduction
 Selective delivery of antiviral...
 Potential role of reduced...
 Erythrocytes as carriers of...
 Conclusions
 Acknowledgements
 References
 
In AIDS patients, chronic inflammation and elevated levels of cytokines seem to be associated with lower levels of GSH: GSH levels decrease rapidly upon infection with HIV and continue to decline as the disease progresses.14 Investigators have shown that agents that increase intracellular levels of GSH inhibit HIV replication.15 The mechanisms of action of GSH are not completely understood. At first, it seemed that the antiviral effect was not directed against viral replication per se, but was essentially related to blocking oxidative stress, typically observed in patients infected by HIV. Oxidative stress is responsible for activation of the cellular transcription factor (NF-{kappa}B), which enhances HIV transcription and replication.16 In fact, NF-{kappa}B is known to be activated in response to various forms of oxidative stress.17 Alternatively, GSH may interfere with the phosphorylation of the I{kappa}B/NF-{kappa}B complex and release of activated NF-{kappa}B, or with the transport of activated NF-{kappa}B into the nucleus (Figure 1a). Successive studies have demonstrated that in vitro HIV-1 infection induces a significant decrease in intracellular GSH in human macrophages, and that both virus production and infectivity can be strongly inhibited by GSH acting in the late stages of viral replication. This causes a selective decrease of specific glycoproteins, such as gp120, which are particularly rich in disulphide bonds (Figure 1b).18 A similar mechanism has been described in GSH-mediated inhibition of different types of viruses.19,20



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Figure 1. Possible mechanisms of action of GSH against HIV. (a) Inhibition of the signal transduction pathway mediated by NF-{kappa}B. (b) Inhibition of HIV protein folding.

 
Other findings revealed that GSH and N-acetylcysteine inhibit the reverse transcriptase (RT) process of HIV-1.21 In vivo, we have demonstrated widely that the administration of GSH at high doses to mice infected with the retroviral complex LP-BM5 is effective in reducing the typical signs of disease and the proviral DNA content in infected organs. Moreover, when GSH is combined with a classical nucleoside inhibitor of RT [i.e. azidothymidine (AZT; Zidovudine)], additional responses can be obtained in all the parameters characterizing murine AIDS.22


    Erythrocytes as carriers of GSH to macrophages
 Top
 Introduction
 Selective delivery of antiviral...
 Potential role of reduced...
 Erythrocytes as carriers of...
 Conclusions
 Acknowledgements
 References
 
On the basis of these considerations, we postulated the use of RBCs to effect a selective delivery of GSH to macrophages. Since HAART is the actual therapy used to combat AIDS, we decided to evaluate the efficacy of GSH-loaded RBCs, first combined with one drug (AZT), and then with two drugs [AZT+2,3-dideoxyinosine (DDI; Didanosine)] used in HAART. The model used to test the efficacy of our approach was again the murine model of AIDS. The first results showed that the combination of AZT and GSH encapsulated in RBCs was more effective than the GSH administered intramuscularly in combination with the same nucleoside analogue. Moreover, when infected mice were treated for 10 weeks with the combination of GSH-loaded RBCs and AZT+DDI, there were additional significant antiviral effects, compared with AZT+DDI therapy alone, in all the parameters analysed. This was particularly evident in the proviral DNA content in the brain and bone marrow where macrophages play a central role. More precisely, ~50% reduction in the proviral DNA content of the brain was obtained through the treatment with AZT+DDI, ~60% when GSH-loaded RBCs were added to AZT monotherapy and ~80% when GSH-loaded RBCs were added to AZT+DDI.13,22 These results are very interesting since, in the age of HAART, the CNS is becoming increasingly important as a sanctuary site for HIV-1, and contradictory results—with regard to penetration and consequently the therapeutic effects of drugs included in HAART—persist.

The importance of this approach is also supported by recent studies demonstrating that the neurodegenerative phenomena observed in HIV-1-associated dementia can be linked to intracellular GSH depletion and loss of mitochondrial function. Hence, restoring both GSH concentration and mitochondrial integrity are possible therapeutic strategies for slowing disease progression and dementia in AIDS patients.23

Moreover, if on the one hand the administration of GSH-loaded RBCs potentiates the antiviral effect of AZT, on the other it can also mitigate the GSH deficiency observed upon AZT treatment.24


    Conclusions
 Top
 Introduction
 Selective delivery of antiviral...
 Potential role of reduced...
 Erythrocytes as carriers of...
 Conclusions
 Acknowledgements
 References
 
The results summarized in this paper suggest that GSH may have an important place in antiviral therapy, and that RBCs are efficient carriers for molecules with antiviral activity both in vitro and in vivo. Since free GSH is oxidized rapidly in biological fluids, RBCs are able to serve as potential carriers avoiding oxidation. In mice infected with retroviral complex LP-BM5, RBCs can be used to effect selective delivery of GSH to macrophages, efficiently protecting those organs where macrophage cells have a pivotal role. The most important result is in the high protection of brain, achieved when GSH-loaded RBCs were added to classical antiretrovirals. This suggests that redox control is an important strategy for oxidative stress-associated disorders, including HIV infection, and that RBCs can be used to enhance delivery of effective antiretroviral agents (i.e. GSH) to the CNS, thus improving therapy for AIDS dementia and related encephalopathies.

Results obtained in the murine model of AIDS have been confirmed in in vitro HIV-1 chronically infected macrophages where one night’s treatment with GSH-loaded RBCs (14 mM GSH inside RBCs) caused a 50%–70% reduction of p24 production. This result is comparable to the inhibition obtained with twice the addition of 20 mM GSH in the medium (data not shown).

In addition to the significant antiviral effect observed, another advantage of the selective delivery of high doses of GSH is the absence of toxic side effects in vitro and in vivo in the mouse model used.

Moreover, it has been demonstrated25 that the intracellular thiol redox status of macrophages influences the ability to generate a Th1- or Th2-type response, and, more precisely, that GSH depletion inhibits Th1-associated cytokine production and/or favours Th2-associated responses; therefore, alterations in immune function because of GSH depletion in macrophages may play a key role in exacerbating HIV and other infectious diseases.25 In our opinion, the specific targeting of GSH-loaded RBCs to macrophages should be useful in restoring immune functions altered by GSH depletion in these cells, and addressing an immune response of wide interest in vaccination studies.

Because of the advantageous properties of RBCs and the availability of a specially designed

apparatus to perform the loading procedure under blood banking conditions,26 we consider that RBCs may be used in a variety of pathological conditions to deliver drugs that do not cross cell membranes and/or are not very stable in biological fluids and/or must be targeted to cells or organs. Furthermore, carrier RBCs can also deliver antisense oligonucleotides to block translation of target mRNA. This may have an application in human cancer gene therapy or peptide delivery.26,27


    Acknowledgements
 Top
 Introduction
 Selective delivery of antiviral...
 Potential role of reduced...
 Erythrocytes as carriers of...
 Conclusions
 Acknowledgements
 References
 
This work was partially supported by grants from the Ministero della Sanità, Istituto Superiore della Sanità, Progetto AIDS Scientific Collaboration Agreement 40D.52; Ministero della Sanità P.F. ex art 12; Ricerca Finalizzata 2000 Convenzione n. ICS 120.5/RF00.123; Progetto FIRB 2001.


    Footnotes
 
* Corresponding author. Tel: +39-0722-305211; Fax: +39-0722-320188; E-mail: magnani{at}uniurb.it Back


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 Top
 Introduction
 Selective delivery of antiviral...
 Potential role of reduced...
 Erythrocytes as carriers of...
 Conclusions
 Acknowledgements
 References
 
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