European Journal of Clinical Investigation (2001) 31, 248±252
Short Communication
New drug combinations for the treatment of murine AIDS and macrophage protection A. Fraternale, A. Casabianca, A. Tonelli, L. Chiarantini, G. Brandi and M. Magnani University of Urbino, Urbino, Italy
See Commentary on page 190.
Abstract
Background The failure of highly active antiretroviral therapies (HAART) is mainly due to the existence of latent infected reservoirs, such as macrophages and resting CD41 T cells. In this paper, we report the results that we obtained in a murine model of AIDS by alternating the administration of the lympholitic drug 2-Fluoro-ara-AMP (Fludarabine) to eliminate the infected cells, with that of Azidothymidine (AZT) plus reduced glutathione (GSH) encapsulated in erythrocytes, to protect lymphocytes and macrophages not yet infected, respectively. Materials and methods Two groups of infected mice were treated as follows: one group was treated by alternating the administration of Fludarabine and AZT (treatment A), while the other group received the same treatment plus GSH-loaded erythrocytes given with AZT (treatment A 1 L±RBC). Fludarabine was administered intraperitoneally, AZT in the drinking water and GSH was encapsulated in erythrocytes by a procedure of hypotonic dialysis and isotonic resealing. Results The results obtained show that GSH-loaded erythrocytes provide additive effects in all the parameters examined. Conclusions Alternation of a lympholitic drug and antiretroviral drug is effective in reducing the progression of murine AIDS. Addition of a system to protect macrophages provides additive effects in almost all the parameters considered, confirming that combination therapies aimed at protecting different infectable cell compartments are better than treatments protecting mainly lymphocytes. Keywords AZT, drug delivery system, fludarabine, GSH, murine-retrovirus-induced immunodeficiency (MAIDS). Eur J Clin Invest 2001; 31(3): 248±252
Introduction The problems linked to the current anti HIV-1 drug combinations, including drug resistance, toxicity and problems with adherence, prompt researchers to experiment with new therapies. Moreover, the existence of latent infected reservoirs, such as macrophages and resting CD41 T cells, make it impossible to eradicate the virus [1]. Correspondence to: Prof. Mauro Magnani, Istituto di Chimica Biologica `G. Fornaini', UniversitaÁ degli Studi di Urbino, Via Saffi, 2, 61029-Urbino, Italy. Tel.: 1 39 722 305211; Fax: 1 39 722 320188; e-mail:
[email protected] Received 15 August 2000; accepted 27 August 2000 Q 2001 Blackwell Science Ltd
These considerations prompted us to evaluate a new anti-HIV approach, which foresees the use of a lympholitic drug to selectively kill the infected cells, in addition to two antiretroviral drugs to protect both lymphocytes and macrophages. Using a murine model of AIDS, we evaluated the efficacy of alternating the administration of Fludarabine (Schering A.G., Berlin, Germany) and AZT plus GSH-loaded erythrocytes. GSH is an antioxidant molecule that is able to inhibit virus replication [2,3], and intramuscular administration of this drug was found to be effective in delaying murine AIDS [4]. The choice of encapsulating GSH was made on the basis of the evidence that antiretroviral drug-loaded erythrocytes modified to increase macrophage phagocytosis can efficiently protect macrophages against retroviral infections [5].
Treatment of MAIDS by AZT, Fludarabine and GSH
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Fludarabine monophosphate (9-d-arabinofuranosyl-2fluoroadenine-5 0 -phosphate) (Fludarabine; Schering A.G.) is an adenine nucleoside studied extensively in indolent lymphoproliferative diseases [6]. The results reported in this paper show that addition of a system to protect macrophages (GSH-loaded erythrocytes) to a therapeutic approach including lympholitic and antiretroviral drugs [7,8], provides additive effects in almost all the parameters that characterize murine AIDS.
procedure of hypotonic dialysis and isotonic resealing as previously described [9]. GSH was encapsulated at a final concentration of 3´35 1024 ^ 8´7 1025 mmol 1026 erythrocytes. Mice were treated with weekly intraperitoneal injections of 12´3 nmol.
Materials and methods
Flow cytometry analysis
Female C57BL/6 mice were infected by means of two consecutive intraperitoneal injections of LP±BM5 viral mixture at 24 h intervals (1 IU reverse transcriptase, RT).
Whole blood samples were stained with: fluorescein isothiocyanate (FITC)-conjugated antimouse CD4 (L3T4) (Immucor Italia, Milan, Italy) for the detection of CD41 lymphocytes; phycoerythrin (PE)-conjugated antimouse Ly2R (Sigma, Milan, Italy) for the detection of CD8a lymphocytes; FITC-conjugated antimouse CD90´2 (Thy1´2) (Immucor Italia, Milan, Italy) for the detection of T cells; and PE-conjugated antimouse CD45R (B220) for the detection of committed B cell precursors (Immucor Italia). Samples were analyzed by FACS (Becton Dickinson, San Jose, CA, USA).
Fludarabine and AZT Fludarabine was administered intraperitoneally at the concentration of 150 mg kg21 twice a day for 5 days a week. Treatment with Fludarabine was started 30 h after the first viral injection. AZT was administered ad libitum in drinking water at a concentration of 250 mg L21. GSH encapsulation in erythrocytes
Serum immunoglobulin G (IgG) determination Serum IgG levels were determined using an ELISA technique as described [9].
Competitive polymerase chain reaction analysis of LP±BM5 proviral DNA
Murine erythrocytes were loaded with GSH by using a
Total cellular DNA was isolated from lymph nodes of uninfected, infected and infected/treated mice and BM5d
Figure 1 Treatment scheme for LP±BM5-infected C57BL/6 mice, by alternating cycles of Fludarabine, then AZT (treatment
A) or Fludarabine followed by AZT plus GSH-loaded erythrocytes (treatment A 1 L±RBC).
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proviral DNA content was quantified by a competitive PCR assay as described [10].
Lymphocyte proliferative index 3 [H] Thymidine incorporation upon lymphocyte stimulation by phytohemagglutinin (PHA) for T cells and lipopolysaccharide (LPS) for B cells was measured as previously described [11].
Results and discussion In this study the murine model of AIDS was used to evaluate the efficacy of a new combination therapy that included Fludarabine, AZT and GSH-loaded erythrocytes (L±RBC). C57BL/6 mice infected with LP±BM5 were treated as reported in Fig. 1. Two groups of mice were used as controls: one group was infected but not treated and the other was not infected. After the first 6 weeks of treatment and at the end of the 12-week cycle of treatment, the parameters characterizing the disease, such as splenomegaly, lymphadenopathy and hypergammaglobulinemia, were evaluated; moreover, the proviral DNA content in lymph nodes, the peripheral blood lymphocyte phenotypes and the proliferative index for T and B spleen cells were studied.
Figure 2 Inhibition of lymphadenopathy (a), splenomegaly (b) and hypergammaglobulinemia (c) by alternating cycles of Fludarabine, then AZT (treatment A) or Fludarabine followed by AZT plus GSH-loaded erythrocytes (treatment A 1 L±RBC). The parameters were evaluated at the times indicated in the figure and are mean ^ SD of eight animals. Treatment groups are described in Fig. 1. *P , 0´05, A vs. A 1 L±RBC.
Figure 3 Lymphocyte proliferative response to phytohemagglutinin (T cells) and lipopolysaccharide (B cells) observed in LP± BM5-infected C57BL/6 mice, untreated or treated as reported in Fig. 1. Spleen lymphocytes were obtained 12 weeks after infection. All values are mean ^ SD of 3±5 animals. CPM values ^ SD in the controls were 23 400 ^ 3510 (per 106 cells) for T lymphocytes and 49 630 ^ 7400 (per 106 cells) for B lymphocytes. *P , 0´05, **P , 0´01, A vs. A 1 L±RBC.
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Table 1 Inhibition of proviral DNA in the lymph nodes of LP±BM5-infected C57BL/6 mice by alternating cycles of Fludarabine, then AZT (treatment A) or Fludarabine followed by AZT plus GSH-loaded erythrocytes (treatment A 1 L-RBC). Mouse group Infected (I) Proviral DNA copy number I1A Proviral DNA copy number % of inhibition I 1 A 1 L±RBC Proviral DNA copy number % of inhibition
6th week
12th week
92 500 ^ 32 500
290 000 ^ 32 650
59 700 ^ 34 000 39 ^ 32
166 940 ^ 71 054 42 ^ 24
40 462 ^ 33 304 54 ^ 35
85 310 ^ 64 050 71 ^ 22*
BM5d proviral DNA copy number per 50 ng of total cellular DNA was assessed by competitive PCR analysis [8]. Percentages of inhibition refer to infected, non-treated mice. Values are mean ^ SD of five animals for `Infected (I)' and of 10 animals for `I 1 A' and `I 1 A 1 L±RBC' and were obtained at the times indicated in the table. Treatment groups are described in Fig. 1. *P 0´015, A vs. A 1 L±RBC.
The results obtained by the evaluation of splenomegaly, lymphadenopathy and hypergammaglobulinemia show that both treatments were able to reduce these parameters to almost control values (Fig. 2). The quantification of proviral DNA content revealed that treatment A 1 L±RBC was more effective than treatment A in inhibiting this parameter in lymph nodes (Table 1). The data obtained by evaluating the proliferative response of T and B spleen cells to mitogenic stimuli at
12 weeks, show that treatment A 1 L±RBC was more effective than treatment A in restoring the proliferative index of both lymphocyte subpopulations (Fig. 3). Cytofluorimetric analysis of peripheral blood lymphocytes revealed that at 6 weeks both treatments restored all the lymphocyte populations to normal values; at 12 weeks, treatment A 1 L±RBC was found to have given a slight improvement in the percentage of CD45 1 cells with respect to treatment A, while both treatments had the
Figure 4 Phenotyping by flow cytometry of blood lymphocytes from treatment groups described in Fig. 1. Values are mean ^ SD
of duplicate determinations in at least 10 animals and are expressed as percentage of the total lymphocyte population.
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same effect in returning the percentages of CD81 cells and consequently the percentage of Thy 1´21 to normal values (Fig. 4). The data obtained show that treatment A 1 L±RBC gives additive effects to those afforded by the treatment A. These results confirm that combination therapies aimed at protecting different infectable cell compartments (i.e. lymphocytes and macrophages) are better than treatments protecting mainly lymphocytes. Furthermore, addition of alternating cycles of a lympholitic drug to antiviral drugs is safe and worth further studies aimed at depleting endogenous viral reservoirs in the infected host.
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Acknowledgements This work was supported by Ministero della SanitaÁ, Istituto Superiore di SanitaÁ, Progetto AIDS 1998; Progetto Patologia Clinica e Terapia dell'AIDS, azione concertata 1998 and C.N.R. Target Project on Biotechnology. Fludarabine was kindly provided by Schering A.G, Berlin, Germany.
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