Enhanced anticryptococcal activity of chloroquine in ...

JAC

Journal of Antimicrobial Chemotherapy (2005) 55, 223–228 doi:10.1093/jac/dkh522 Advance Access publication 8 December 2004

Enhanced anticryptococcal activity of chloroquine in phosphatidylserine-containing liposomes in a murine model Masood A. Khan*, Rukhsana Jabeen, T. H. Nasti and Owais Mohammad Interdisciplinary Biotechnology Unit, Aligarh Muslim University, Aligarh-202002, India Received 6 July 2004; returned 17 September 2004; revised 29 October 2004; accepted 1 November 2004

Methods: In the present study, we investigated the antifungal activity of chloroquine entrapped in PS liposomes against Cryptococcus neoformans in the macrophage cell line J 774 and in a murine model. Mice were treated with free as well as liposomal formulations of chloroquine before and after challenging with C. neoformans infection. The anticryptococcal activity of chloroquine was also evaluated in combination with fluconazole in the treatment of systemic murine cryptococcosis. The efficacy of chloroquine treatment was assessed by continued survival as well as by colony forming units (cfu) in liver and brain of treated mice. Results: Chloroquine entrapped in PS liposomes shows increased activity against C. neoformans infection both in in vitro and in vivo studies. Moreover, the antifungal activity of fluconazole increases when used in combination with liposomal chloroquine. Chloroquine in PS liposomes was found to be more effective in comparison with the same dose of free chloroquine or chloroquine entrapped in neutral liposomes. Conclusions: The enhanced anticryptococcal activity of chloroquine in PS liposomes seems to be due to uptake of drug-containing PS liposomes by macrophages. It can be assumed that liposomemediated delivery of chloroquine to macrophages results in an unfavourable (alkaline) environment for the growth of C. neoformans inside macrophages. Keywords: Cryptococcus neoformans, fluconazole, macrophages, therapy

Introduction The AIDS epidemic and advances in the field of medical sciences (including organ transplant development), the use of aggressive chemotherapy and the insertion of intravascular devices account for the dramatic rise in invasive fungal infections over the past decades and their expected rise in coming years.1,2 Fungal pathogens like Candida albicans, Aspergillus fumigatus, Cryptococcus neoformans and Histoplasma capsulatum cause life-threatening infections in HIV-infected patients.3 The current therapy of cryptococcosis is based on the use of polyene and azole groups of antifungal chemotherapeutic agents. The azole antifungal agents, e.g. fluconazole and itraconazole, because of their relative safety and ease of delivery, have subsequently become a critical component of the antifungal armamentarium. Drugs from the polyene class of antifungal agents,

especially amphotericin B, have long been considered the most effective of the systematically administered antifungal agents. Unfortunately, infusion-related toxicities and the frequent association of renal dysfunction with the use of amphotericin B has limited its utility for long duration therapy.4 But in recent years, resistance in pathogenic fungi against azole and polyene antifungal agents has been emerging at an alarming rate.5 To cope with the current trend of antifungal resistance, it has become mandatory to search for safe and broad-spectrum therapeutic agents for prophylaxis and treatment of fungal infections. In previous years, chloroquine has been utilized extensively for treatment of malaria and inflammatory diseases.6,7 Recently, the antifungal activity of chloroquine both in in vitro and in vivo systems has also been documented.8,9 The lipophilic nature of chloroquine makes it easy for it to diffuse freely into membranes in the unprotonated form, but on reaching the intracellular acidic

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*Corresponding author. E-mail: [email protected] ..........................................................................................................................................................................................................................................................................................................................................................................................................................

223 JAC vol.55 no.2 q The British Society for Antimicrobial Chemotherapy 2004; all rights reserved.

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Objectives: The anticryptococcal activity of chloroquine was assessed after incorporation in phosphatidylserine (PS)-containing negatively charged liposomes in a murine model.

M. A. Khan et al.

Materials and methods

separated from liposomal drug by passing the preparation through a Sephadex G-50 column. The liposomal formulation of chloroquine was used in the study.

Estimation of liposome-intercalated chloroquine The intercalation efficiency of chloroquine in the liposomes was estimated spectrophotometrically. A standard curve of chloroquine was plotted at 342 nm, as described previously.22 The amount of drug associated with the liposomes was determined by dissolving the formulation in 1% Triton X-100 and determining the absorbance at 342 nm against a corresponding amount of lipid in a final solution of 1% Triton X-100 as a blank. The amount of chloroquine entrapped in the liposomes was calculated using the standard curve of the drug. The amount of chloroquine associated with the liposomes was found to be 128 ± 14 mg of chloroquine/mmol of lipid P.

Animals Female Swiss mice weighing 20 ± 4 g were used in the study. The animals were given a standard pellet diet (Hindustan Lever Ltd) and water ad libitum. Mice were checked daily for their mortality and moribundity. The techniques used for bleeding, injection as well as sacrifice of animals were approved by the Animal Ethics Committee [Committee for the purpose of control and supervision of Experiments on Animals (CPCSEA), Government of India].

Test strain The strain of C. neoformans (JMCR 102) was obtained from a leukaemia patient of the Jawaharlal Nehru Medical College (JNMC), Aligarh Muslim University, Aligarh. Sabouraud dextrose (SD) agar/ broth was used for growing patient isolates of C. neoformans. The identity of the clinical isolate of C. neoformans was confirmed in the mycology section of the Department of Microbiology, JNMC, Aligarh, India.

Materials All the reagents used in the study were of the highest purity available. Cholesterol was bought from Centron Research Laboratory, Bombay, and used after crystallization with methanol. Fluconazole was procured from Roerig-Pfizer (New York, NY, USA). Chloroquine, PS, MOPS and RPMI 1640 were purchased from Sigma Chemical Co. (St Louis, MO, USA). Egg phosphatidylcholine (egg PC) was isolated and purified according to a previously published procedure.21

Liposomes Chloroquine-containing liposomes were prepared from egg PC, cholesterol and PS (7:2:1), as described previously.22 Briefly, all the ingredients, including chloroquine (drug:lipid, 1:20), were dissolved in a round-bottomed flask in a minimum volume of chloroform/ methanol (1:1, v/v). The solvents were carefully evaporated under reduced pressure to form a thin lipid film on the wall of the flask. The final traces of the solvents were removed by subjecting the flask to vacuum overnight at 48C. Subsequently, the dried lipid film (consisting of egg PC/cholesterol/PS, chloroquine) was hydrated by vortexing with 2.0 mL of 150 mM sterile saline. The suspension of the lipid-drug formulation was sonicated (1 h, 48C) in a bath-type sonicator under N2 atmosphere. The sonicated preparation was centrifuged at 10000 g for 30 min at 48C to remove traces of undispersed lipid. The upper 2/3 volume was taken out and dialysed against normal saline for 24 h at 48C in the dark. Free drug was

Macrophage cell line The murine macrophage cell line J 774 was maintained in minimal essential medium, as described previously.23 J 774 macrophages were mechanically collected with a cell lifter (Costar Italia, Milan, Italy).

Antifungal susceptibility testing The MIC of fluconazole (Roerig-Pfizer) was determined by the broth macrodilution method, according to the guidelines of the National Committee for Clinical Laboratory Standards (NCCLS), document M27-A.24 Stock solution of fluconazole was prepared in water at 10 times the highest concentration tested. Stock solutions were diluted with RPMI 1640 medium (Sigma Chemical Co.) supplemented with L -glutamine, without bicarbonate, buffered to pH 7.4 with 0.165 M MOPS (Sigma Chemical Co.). The final concentration was in the range 0.125– 128 mg/L for fluconazole. Antifungal susceptibility testing was performed in 96-well round-bottomed microtitration plates. Yeast inocula were prepared in sterile water and diluted in RPMI 1640 medium to give a final inoculum concentration of 5 102 – 2.5 103 cells/mL. The plates were incubated at 378C and read after 72 h. MIC was defined as the lowest concentration at which there was 80% inhibition of growth of C. neoformans compared with that in a drug-free control. The MIC of fluconazole for C. neoformans (JMCR 102) was found to be 16 mg/L.

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environment chloroquine becomes protonated and this raises the intravacuolar pH.10 Intracellular pathogens adopt various strategies to survive inside the hostile environment of macrophages. Mycobacterium tuberculosis and Mycobacterium avium modulate the internal environment of phagosomes by selective blocking of vacuolar proton-ATPases.11 Toxoplasma gondii and Legionella pneumophila avoid acidification by inhibiting the fusion of residing vesicles with lysosomes.12,13 The acidic lysosomal pH ( pH 5.0) is required for antimicrobial activity of host proteases. Chloroquine treatment of macrophages inhibits the intracellular multiplication of pathogens. Chloroquine inhibits the proliferation of L. pneumophilia, H. capsulatum and Francisella tularensis by limiting the supply of crucial nutrients required for their growth.14 – 16 It has been demonstrated that chloroquine inhibits the growth of C. neoformans in macrophages by a mechanism not dependent on iron deprivation but by alkalinizing the pH of mononuclear phagocytes.17 Liposomes have been proved to be very useful in the treatment of macrophage-based intracellular infections.18 Phosphatidylserine (PS)-containing negatively charged liposomes were found to target the contents of macrophages via the latter’s specific receptors.19 They increase the therapeutic index of the entrapped drugs by such targeting.20 Bearing in mind this facility of PS-containing liposomes, we utilized them to target chloroquine to sanctuary sites of pathogens in macrophages, which may be effective in the prevention and therapy of C. neoformans infection. The results of the present work show that the use of chloroquine in PS-containing liposomes is effective in the elimination of C. neoformans infection in a murine model.

Role of chloroquine in treatment of C. neoformans infection Preparation of C. neoformans cells for infection

Saline Empty PS liposomes PS liposomal chloroquine Liposomal chloroquine + fluconazole (50 mg/kg) PS liposomal chloroquine + fluconazole (10 mg/kg) PS liposomal chloroquine + fluconazole (20 mg/kg) PS liposomal chloroquine + fluconazole (50 mg/kg) Fluconazole (50 mg/kg)

Stock culture of an encapsulated strain of C. neoformans was maintained on SD agar. Yeast cells were harvested from agar plates into YPD (1% yeast extract, 2% peptone, 5% dextrose) medium at 378C for 36 h. The cells were washed with normal saline at low speed centrifugation (2000 rpm) and diluted to the appropriate concentration in saline prior to use in in vitro as well as in vivo studies.

Antifungal activity of macrophages in presence of chloroquine

Assessment of anticryptococcal activity

Effect of chloroquine against C. neoformans infection in mice The prophylactic and therapeutic role of chloroquine in the elimination of systemic infection of C. neoformans in mice was evaluated. The animals were treated with free as well as liposomal chloroquine at various doses (5, 10 and 20 mg/kg). The dose of 10 mg/kg was found to be most suitable and was used in further experiments. Before 24 h of infection with C. neoformans, each mouse was pretreated with free liposomal chloroquine and PS-liposomal chloroquine via an intraperitoneal route. Each mouse was infected with 5 105 cells of C. neoformans. After 24 h of infection, each mouse was treated with a dose of free as well as liposomal formulations of chloroquine for three consecutive days. Mice (n = 10 in each group) were divided into the following groups: Saline Free chloroquine (10 mg/kg) Liposomal chloroquine (10 mg/kg) PS liposomal chloroquine (5 mg/kg) PS liposomal chloroquine (10 mg/kg) PS liposomal chloroquine (20 mg/kg) The efficacy of various formulations of chloroquine in the different groups was assessed by culturing tissue homogenates of brain and liver of C. neoformans-infected mice, for determination of fungal burden.

Anticryptococcal activity of fluconazole in chloroquine pre-treated mice For prophylactic study, the animals were pre-treated with PS liposomal chloroquine (10 mg/kg) for three consecutive days via an intraperitoneal route. After chloroquine pre-treatment, mice were challenged with C. neoformans infection (7 105 cells/mouse). The treatment with fluconazole was started after 24 h of C. neoformans infection on days 1, 2 and 3 via an intravenous route. The animals were divided into the following groups and each group contained 10 mice.

The role of chloroquine alone or with fluconazole in protection against C. neoformans infection was assessed by survival data and fungal burden in the liver and brain of mice. The animals were observed until day 30 post-infection. For cfu determination, three mice from each group were sacrificed and their brain and liver analysed, as described previously.25 Briefly, weighed portions of the given organ were homogenized in 5 mL of sterile normal saline and an aliquot of the suspension was plated on SD agar plates containing chloramphenicol and gentamicin after appropriate dilution. The plates were incubated for 48 – 72 h at 378C. The numbers of fungal colonies (cfu) were counted and the fungal load in various organs was calculated by multiplying with the dilution factor.

Statistics Analysis of survival of animals was conducted using Kaplan – Meier curve, and various groups were compared by Kruskal – Wallis test. Fungal burden (cfu) in organs was analysed by paired t-test using GraphPad Prism software.

Results Chloroquine in PS liposomes shows increased antifungal activity against C. neoformans in macrophages C. neoformans multiplies inside macrophages, as evidenced by an increase in cfu (12040 ± 1864 to 37886 ± 6470) after 48 h of culture. Treatment of J 774 cells with chloroquine (10 mM) induced a significant inhibition of C. neoformans growth, as compared with the macrophages without drug treatment (37886 ± 6470 to 10250 ± 1280). Results demonstrated that macrophages treated with PS liposomal chloroquine show remarkable reduction (34 ± 8) in cfu, as compared with macrophages treated with free chloroquine or without drug treatment after 48 h of incubation.

Liposomal chloroquine shows increased efficacy against murine cryptococcosis Both free as well as liposomal formulations of chloroquine were administered to mice at various doses (5, 10 and 20 mg/kg) to analyse its protective role against C. neoformans infection. Among the various formulations, PS liposomal chloroquine at a dose of 10 or 20 mg/kg was the most effective in controlling the severity of C. neoformans infection in mice. Animals injected with chloroquine at a dose of 20 mg/kg showed some toxic manifestations (data not shown). Chloroquine at a dose of 10 mg/kg was found to be suitable for in vivo study in terms of both efficacy and toxicity. Free chloroquine at a dose of 10 or 20 mg/kg imparted some protection, but it was inferior to that of PS liposomal chloroquine at equal dose (Table 1).

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J 774 macrophages were seeded in triplicate in 96-well Costar plates with 2 105 cells/well in complete medium supplemented with 5% human serum and incubated at 378C in 5% CO2 for 24 h. The macrophages were then treated with chloroquine formulations at indicated concentrations for 1 h. C. neoformans (1 105 cells/well) was added to the wells containing macrophages and drug and incubated for 2 h. The number of cfu recovered from the lysis of J 774 cells (without drug treatment) after 2 h of phagocytosis was considered to be the initial inoculum (baseline). After 24 and 48 h of incubation, macrophages were lysed with 0.1% Tween 20 and phagocytosed yeasts were recovered and centrifuged. The number of cfu of C. neoformans was determined by dilution and spread plates on SD agar after incubation at 378C for 18 h in triplicate.

M. A. Khan et al. Table 1. Effect of chloroquine (CQ) treatment on establishment of C. neoformans infection in mice No. of cfu/organ on day 4 post-infection Treatment group Saline Free CQ (10 mg/kg) Liposomal CQ (10 mg/kg) PS liposomal CQ (5 mg/kg) PS liposomal CQ (10 mg/kg) PS liposomal CQ (20 mg/kg)

brain

liver

158068 ± 25224 106648 ± 18520 71642 ± 8804 90345 ± 9600 50864 ± 7080 33462 ± 8604

198902 ± 28722 114563 ± 19006 65489 ± 11252 80604 ± 9284 45384 ± 8266 35802 ± 5246

Free chloroquine versus liposomal chloroquine (10 mg/kg), P = 0.0422; free chloroquine versus PS liposomal chloroquine (10 mg/kg), P = 0.0084; liposomal chloroquine versus PS liposomal chloroquine (10 mg/kg), P = 0.0317.

The prophylactic effect of PS liposomal chloroquine was assessed in mice infected with C. neoformans and consequently treated with various doses of fluconazole (10, 20 and 50 mg/kg). The efficacy of fluconazole increased in chloroquine-pre-treated mice in comparison with mice without chloroquine treatment. Mice pre-treated with PS liposomal chloroquine and subsequently treated with fluconazole (50 mg/kg) showed increased _ 80%), followed by those treated with fluconazole at a survival (> dose of 20 mg/kg ( 50%). Fluconazole at lower doses (5 or 10 mg/kg) also showed enhanced anticryptococcal activity in chloroquine pre-treated mice, but it was not significant (Figure 1). Fluconazole treatment at a dose of 50 mg/kg in PS liposomal chloroquine pre-treated mice showed superior efficacy to all other treatment combinations (P < 0.05) except over the mice pre-treated with liposomal chloroquine.

C. neoformans burden in brain and liver The protective role of PS liposomal chloroquine in the elimination of C. neoformans infection from tissues was assessed by quantification of fungal burden in vital organs, i.e. brain and liver. The efficacy of fluconazole also increased with the relative increase in the dose of fluconazole in chloroquine-pre-treated mice. A remarkable reduction in fungal load was observed in the organs of PS liposomal chloroquine-pre-treated animals followed by fluconazole (50 mg/kg) treatment, in comparison with those treated with lower doses of fluconazole (Table 2).

Discussion In the current era of AIDS and iatrogenic immunosuppression, the incidence and prevalence of opportunistic fungal infections have increased dramatically.1 – 3 Although the discovery of new antifungal agents is promising, strategies to enhance the efficacy of currently available drugs may have more immediate impact. The present study was performed to increase the efficacy of fluconazole against C. neoformans infection in mice, by treating them with chloroquine entrapped in PS-containing negatively charged liposomes.

Chloroquine accumulates inside macrophage phagolysosomes by ion trapping, where it exerts potent antifungal activity against H. capsulatum and C. neoformans.8 Iron plays a critical role in the intracellular growth of H. capsulatum within macrophages. Chloroquine inhibits the growth of H. capsulatum by impeding the pH-dependent acquisition of iron either from the transferrin – transferrin receptor complex in the endosome or from the ferritin in the lysosomes.15 On the other hand, the inhibition of C. neoformans growth by chloroquine is independent of iron deprivation but is related to the other phenomenon of pH increase in the subcellular acidic compartments.17 Table 2. Effect of chloroquine (CQ; 10 mg/kg) pre-treatment on the efficacy of fluconazole (Flu) against C. neoformans infection in mice No. of cfu/organ on day 4 post-infection Treatment group brain Saline Empty PS liposomes PS liposomal CQ PS liposomal CQ+Flu (10 mg/kg) PS liposomal CQ+Flu (20 mg/kg) PS liposomal CQ+Flu (50 mg/kg) Liposomal CQ+Flu (50 mg/kg) Flu (50 mg/kg)

liver

168860 ± 23480 189040 ± 27234 138726 ± 26538 145242 ± 31282 89282 ± 10880 85462 ± 16242 60928 ± 8902 56784 ± 8202 47044 ± 9446 39472 ± 6608 7620 ± 1986 4891 ± 1252 25438 ± 4485 16280 ± 2436 58460 ± 8028 69448 ± 12842

Liposomal CQ + Flu 50 mg/kg versus PS liposomal CQ + Flu 50 mg/kg, P = 0.0031; PS liposomal CQ + Flu 20 mg/kg versus PS liposomal CQ + Flu 50 mg/kg, P = 0.0018; PS liposomal CQ + Flu 50 mg/kg versus Flu 50 mg/kg, P = 0.0005.

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Pre-treatment with PS liposomal chloroquine augments the anticryptococcal activity of fluconazole

Figure 1. Prophylactic use of liposomal chloroquine increases the efficacy of fluconazole in mice infected with less-susceptible strains of C. neoformans to fluconazole. Mice were pre-treated with liposomal chloroquine intraperitoneally (10 mg/kg) before infecting them with C. neoformans (7 105 sporespores/animal). After 24 h of infection, the animals were treated with various doses of fluconazole (10, 20, 50 mg/kg) for three consecutive days. Survival was monitored over 30 days after infection. The groups are: filled upright triangles, saline; filled squares, empty PS liposomes; filled circles, PS liposomal chloroquine; open diamonds, PS liposomal chloroquine + fluconazole 10 mg/kg; open squares, PS liposomal chloroquine + fluconazole 20 mg/kg; open inverted triangles, liposomal chloroquine + fluconazole 50 mg/kg; filled diamonds, fluconazole 50 mg/kg; open upright triangles, PS liposomal chloroquine + fluconazole 50 mg/kg. The data are means of three independent experiments.

Role of chloroquine in treatment of C. neoformans infection HIV replication via secretion of proinflammatory cytokines such as tumour necrosis factor (TNF)-a.26 Chloroquine has been found to inhibit the secretion of proinflammatory cytokines such as TNF-a.27 Moreover, chloroquine has been shown to inhibit HIV-1 replication in human peripheral blood lymphocytes.28 Chloroquine treatment might have an extra advantage in blocking secondary infection-induced HIV replication, due to its direct and indirect anticryptococcal effect. Thus, the use of chloroquine for prevention and treatment of fungal infections could result in improved outcome by decreasing morbidity from both the fungal as well as HIV infection.

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PS-containing liposomes have been shown to activate macrophages for enhanced microbicidal activity.19 In the present study, we demonstrated the increased antifungal activity of chloroquine in PS-containing liposomes against C. neoformans both in vitro as well as in vivo. PS liposomal chloroquine was found to impart more protection at lower doses as compared with free chloroquine at higher doses. This is based on the assumption that liposomal chloroquine was taken up by macrophages more efficiently, resulting in the accumulation of a substantial amount of chloroquine in the macrophages in comparison with free chloroquine. C. neoformans shows increased proliferation at pH 5.0 and an increase in the pH of intracellular compartments retards its multiplication.10 Liposome-mediated delivery of chloroquine increases the pH of the acidic compartments of macrophages, creating a long-lasting unfavourable environment for the growth of C. neoformans. Thus macrophages respond with enhanced antifungal activity upon treatment with the same dose of chloroquine in liposomal form. The liposome-mediated chloroquine targeting macrophages also reduces the chances of drug-induced toxic manifestations to other cells even at high concentrations. The isolate of C. neoformans used in the present study did not respond well to fluconazole alone, either in in vitro or in vivo studies. Even at higher doses (20, 50 mg/kg) fluconazole was not significantly effective in the elimination of C. neoformans infection in a murine model. This is in agreement with in vitro fluconazole MIC results (16 mg/L), which clearly shows the dosedependent susceptibility of C. neoformans to fluconazole. The increased therapeutic potential of fluconazole in mice pre-treated with PS liposomal chloroquine can be attributed to the chloroquine-induced unfavourable physiological conditions for the proliferation of C. neoformans inside macrophages. Within acidic lysosomes, chloroquine acquires a net positive charge that prevents it from exiting the phagolysosomes. Chloroquine concentrates within lysosomes up to many fold higher concentration than its extracellular level. This results in alkalization of the acidic constitution of phagosomes. It seems that alkalization of endosomes and lysosomes causes dysfunction of various biochemical reactions crucial for the survival of intracellular pathogens. Thus it can be assumed that higher concentrations of chloroquine inside macrophages may directly kill C. neoformans. It is further supported by the fact that chloro_ 30 mM inhibited cryptococcal growth quine at concentrations > _ 100 mM is fungicidal.10 The mobilization of iron from and at > transferrin and ferritin, two major sources of iron in mononuclear phagocytes, is dependent on an acidic environment. Iron dissociates from transferrin at pH < 6.0 and becomes available to the pathogen.15 Iron availability from ferritin occurs after proteolysis in lysosomes at lower pH. Chloroquine inhibits both these processes by altering the medium inside macrophages and thus restricts the supply of iron to the pathogen. Chloroquine treatment of mice prior to C. neoformans infection increases the efficacy of fluconazole in the treatment of murine cryptococcosis not responding to fluconazole in chloroquine-untreated mice. In combination therapy, chloroquine does not allow the pathogen to proliferate within the cells. At this stage, the administration of fluconazole proves to be more effective in controlling the weakly proliferating fungal cells and eliminates C. neoformans infection from the murine model. Opportunistic fungi and their products have been shown to stimulate HIV replication in latently infected macrophages and lymphocytes.26 C. neoformans exacerbates AIDS by activating

M. A. Khan et al. antibody-bearing liposomes effectively controls chloroquine-resistant Plasmodium berghei infections in mice. Antimicrobial Agents and Chemotherapy 39, 180– 4. 23. Owais, M. & Gupta, C. M. (2002). Liposome mediated cytosolic delivery of macromolecules and its possible role in vaccine development. European Journal of Biochemistry 267, 3946– 56. 24. National Committee for Clinical Laboratory Standards. (1997). Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts: Approved Standard M27-A. NCCLS, Villanova, PA, USA. 25. Khan, M. A., Faisal, S. M., Haque, W. et al. (2002). Immunomodulator tuftsin augments anti-fungal activity of amphotericin B against experimental murine candidiasis. Journal of Drug Targeting 10, 185– 92. 26. Orenstein, J. M., Fox, C. & Wahl, S. M. (1997). Macrophages as a source of HIV during opportunistic infections. Science 276, 1857–61. 27. Weber, S. M. & Levitz, S. M. (2001). Chloroquine antagonizes the proinflammatory cytokine response to opportunistic fungi by alkalizing the fungal phagolysosome. Journal of Infectious Diseases 183, 935–42. 28. Pardridge, W. M., Yang, J. & Diagne, A. (1998). Chloroquine inhibits HIV-1 replication in human peripheral blood lymphocytes. Immunology Letters 64, 45–7.

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environment provides essential intracellular iron required for growth. Infection and Immunity 63, 1478–83. 17. Levitz, S. M., Harrison, S. T., Abdulmoneim, T. et al. (1997). Chloroquine induces human mononuclear phagocytes to inhibit and kill Cryptococcus neoformans by a mechanism independent of iron deprivation. Journal of Clinical Investigation 100, 1640– 6. 18. Bergers, J. J., ten Hagen, T. L., Van Etten, E. W. et al. (1995). Liposomes as delivery systems in the prevention and treatment of infectious diseases. Pharmacy World & Science 17, 1 –11. 19. Gilbreath, M. J., Nacy, C. A., Hoover, D. L. et al. (1985). Macrophage activation for microbicidal activity against Leishmania major: inhibition of lymphokine activation by phosphatidylcholinephosphatidylserine liposomes. Journal of Immunology 134, 3420–5. 20. Tempone, A. G., Perez, D., Rath, S. et al. (2004). Targeting Leishmania (L.) chagasi amastigotes through macrophage scavenger receptors: the use of drugs entrapped in liposomes containing phosphatidylserine. Journal of Antimicrobial Chemotherapy 54, 60– 8. 21. Singleton, W. S., Gray, M. S. & Brown, M. L. (1965). A method for adsorbent fractionation of cottonseed oil for experimental intravenous fat emulsions. Journal of the American Oil Chemists’ Society 42, 53– 6. 22. Owais, M., Varshney, G. C., Choudhury, A. et al. (1995). Chloroquine encapsulated in malaria-infected erythrocyte-specific