Prophylactic Role of Liposomized Chloroquine Against Murine Cryptococcosis Less Susceptible to Fluconazole Masood A. Khan, 1,2,3 Rukhsana Jabeen, 1 and Owais Mohammad 1 Received June 27, 2004; accepted August 26, 2004 Purpose. The prophylactic role of liposomized chloroquine (lip-CQ) has been assessed against less susceptible Cryptococcus neoformans infection in murine model. Methods. In the current study, we investigated the antifungal activity of lip-CQ against C. neoformans in macrophages cell line (J 774) and murine model. Mice were pretreated with free as well as liposomized formulations of CQ at various doses. The anticryptococcal activity of fluconazole was compared in mice with or without CQ pretreatment. The efficacy of CQ prophylaxis was assessed by survival as well as colony forming units (cfu) in brain and lungs of treated mice. Results. Fluconazole alone was not found significantly effective against C. neoformans in both in vitro and in vivo studies. However, the antifungal activity of fluconazole increases in chloroquine- pretreated mice. Lip-CQ was found to be more effective in compari- son to the same dose of free chloroquine in reducing fungal burden from macrophages in vitro and lungs and brain of C. neoformans infected mice. Conclusions. The enhanced prophylactic activity of lip-CQ seems due to rapid uptake of drug-containing liposomes by macrophages. The liposome-mediated accumulation of CQ in macrophages makes the environment unfavorable (alkaline) for the intracellular multiplica- tion of C. neoformans. Moreover, the increased incidence of multi- drug resistance and diversity of pathogenic microorganisms inhibited or killed by CQ makes it the drug of choice for prophylactic therapy. KEY WORDS: chloroquine; cryptococcosis; fluconazole; liposomes. INTRODUCTION Cryptococcus neoformans is one of the major causes of morbidity and mortality in persons with impaired cell medi- ated immunity, especially those with AIDS or undergoing chemotherapy for organ transplantation and neoplastic dis- eases (1,2). The key point behind the success of C. neofor- mans as a parasite in macrophages is its ability to survive within the acidic environment of phagolysosomes (3). The treatment of fungal diseases such as cryptococcosis is based on the use of polyene and azole groups of antifungal chemotherapeutic agents. Drugs from polyene class of anti- fungal agents, especially amphotericin B, have long been con- sidered the most effective of the systematically administered antifungal agents. Unfortunately, infusion related toxicities and the frequent association of renal dysfunction with the use of Amp B have limited its utility for longer duration (4). 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. Resistance in pathogenic fungi against the less toxic azoles has been reported with rapid rate in recent years (5). To cope with current rate of antifungal resistance, it becomes mandatory to search for safe and broad spectrum therapeutic agents for prophylaxis and treatment of fungal infections. Various pathogens adopt different mechanisms to avoid the low pH of phagolysosomes; for example, Mycobacterium tuberculosis and Mycobacterium avium modulate the internal environment of phagosomes by selective blocking of vacuolar proton-ATPase (6). Toxoplasma gondii and Legionella pneu- mophila avoid acidification by inhibiting the fusion of residing vesicles with lysosomes (7,8). Chloroquine (CQ) has widely been available drug for prophylaxis and therapy of malaria (9). The lipophilic nature of CQ makes it easy to diffuse freely into membrane in the unprotonated form, but on reaching into intracellular acidic environment becomes protonated and thus raises the in- travacuolar pH (9). It inhibits the proliferation of L. pneu- mophilia, H. capsulatum and F. tularensis by limiting avail- ability of crucial nutrients required for the growth of these microbes (10–12). It has been demonstrated that CQ inhibits the growth of C. neoformans in macrophages by a mechanism not dependent on iron deprivation but by alkalinizing the pH of mononuclear phagocytes (13). Liposomes have been proved to be very useful in treat- ment of macrophage-based intracellular infections (14). Pre- viously, we have shown that antigens entrapped in liposomes are avidly taken up by macrophages (14). Keeping in view this property of liposomes, we used them for the targeting of CQ to macrophages, which may prove more effective in prophy- laxis and therapy of C. neoformans infection. They not only reduce the toxicity of free drug but also capable of targeting the substantial part of drug to macrophages. The current study clearly shows that prophylactic use of liposomized chlo- roquine (lip-CQ) is more effective than that of free chloro- quine alone or in mice injected with fluconazole for treatment of C. neoformans infection. MATERIALS AND METHODS All the reagents used in the study were of the highest purity available. Cholesterol was bought from Centron Re- search Laboratory (Bombay, India) and used after crystalli- zation with methanol. Fluconazole (Flu) was procured from Roerig-Pfizer (New York, NY, USA). Chloroquine, morpho- linepropanesulphonic acid (MOPS) and RPMI 1640 were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Egg phosphatidylcholine (egg PC) was isolated and purified according to the published procedure (15). Liposomes Chloroquine-containing liposomes were prepared from egg PC (49 mols) and cholesterol (Chol; 21 mols) as de- scribed earlier (16). Briefly, all the ingredients including chlo- roquine (drug:lipid 1:20) were dissolved in a round-bottomed 1 Interdisciplinary Biotechnology Unit, Aligarh Muslim University, Aligarh 202002, India. 2 Department of Immunology and Medical Microbiology, IUPUI, In- dianpolis, Indiana, USA. 3 To whom correspondence should be addressed. (e-mail: alammasood1@rediffmail.com) Pharmaceutical Research, Vol. 21, No. 12, December 2004 (© 2004) Research Paper 2207 0724-8741/04/1200-2207/0 © 2004 Springer Science+Business Media, Inc.