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Use of Liposome Preparation to Treat Mycobacterial Infections
Immunobiol., vol. 191, pp. 578-583 (1994)
©
1994 by Gustav Fischer Verlag, Stuttgart
Kuzell Institute for Arthritis and Infectious Diseases, Medical Research Institute of San Francisco, Pacific Presbyterian Medical Center, San Francisco, CA, USA
Use of Liposome Preparation to Treat Mycobacterial Infections Lurz E. BERMUDEZ
Abstract Infections caused by organisms of the genus mycobacteria, such as tuberculosis M. avium disseminated infection in AIDS patients and leprosy, are extremely common around the world. Mycobacteria are intracellular organisms that invade and multiply chiefly within phagocytic cells. Antibiotic resistance among mycobacteria is a growing concern. M. tuberculosis resistant to INH and rifampin are increasing in major urban centers of the developed and in the developing world. M. avium is characteristically resistant to most anti-tuberculosis antibiotics. Furthermore, therapy of mycobacterial infections takes a long time and most of the drugs have potential side effects and toxicity. In addition, mycobacteria is found within cells and antimicrobials need to be able to achieve adequate concentration within the compartment where mycobacteria is located. Liposome preparations, containing antibiotics, have a theoretical advantage in being able to deliver high concentrations of antimicrobials into the infected cell. Studies done thus far, in vitro and in vivo, have confirmed this premise, when comparing drug entrapped in liposomes with free drug. This paper summarizes the results obtained using liposome preparations to treat mycobacterial infections.
Introduction Infections caused by organisms of the genus mycobacteria are extremely common around the world. It is estimated that tuberculosis is diagnosed in approximately 8 million people, and is responsible for 3 million deaths every year (1). Disseminated Mycobacterium avium complex (MAC) infection occurs in up to 60 % of patients with AIDS (2) and pulmonary infection with MAC in non-AIDS patients has become more frequent during the last decade (3). Mycobacteria are intracellular organisms, that invade and multiply within chiefly phagocytic cells (4). While M. tuberculosis causes pulmonary infection in most of the infected individuals, MAC usually is associated with pulmonary infection in the HIV-negative popula-
Liposomes for mycobacterial infections . 579
tion and disseminated infection in patients with AIDS. Both bacteria are capable of invading macrophages and monocytes, and survive within the phagocytic cells. M. tuberculosis infect chiefly pulmonary macrophages, while MAC is encountered mainly in reticuloendothelial macrophages. Recent studies have demonstrated that mycobacteria once within macrophages inhibits the fusion of phagosomes with lysosomes (5, 6). Mycobacteria also inhibits the acidification and maturation of the phagosomes, thus living in an intracellular environment with pH in a range of 6.0 to 7.5 (5).
Anti-mycobacterial therapy: current problems and limitations Over the years, tuberculosis has been considered a treatable disease, although it required therapy with three drugs for prolonged periods of time. However, with the surging AIDS epidemic, tuberculosis became a critical public health problem with a number of multiple-resistant strains being isolated in selected cities. In contrast, MAC is known to be resistant to most of anti-tuberculosis antimicrobials available and only recently, through a screening effort in a number of laboratories, effective antibiotics have been discovered (7, 8). The macrolides clarithromycin and azithromycin have been demonstrated to have anti-MAC activity in vitro and in vivo (7-10), and were shown efficacious against MAC disseminated disease in two recent clinical trials (9, 10). However, resistant strains have been isolated after two to three months of single agent therapy in a large percentage of patients. There are a number of factors that need to be taken into account when one considers anti-mycobacterial therapy: (1) the antimicrobial not only needs to be active against extracellular bacteria, but also needs to achieve inhibitory concentration with infected cells. A number of antimicrobials are active against mycobacteria in vitro, but fail to achieve inhibitory concentration within macrophages such as the aminoglycosides. However, it is not known whether infection of macrophages with mycobacteria can alter the mechanisms of transport of antimicrobials into the cell resulting in decreased intracellular concentrations of the drugs. Recent work by BERMUDEZ and colleagues (11) suggests this possibility by showing that human macrophages infected with MAC for longer than three days incorporate significantly less azithromycin (an azalide) than uninfected cells (2). The antimicrobial needs to be active at the pH of the phagosome containing mycobacteria. It is even more relevant for the activity of certain antimicrobials such as aminoglycosides, macrolides, and pyrazinamide that are pH dependent; and (3), ideally, the antimicrobial should be active against latent mycobacteria.
580 . L.E.
BERMUDEZ
Antimicrobials encapsulated in liposomes Synthetic phospholipid bilayer vesicles .assembled into a closed membrane system, called liposomes, are non-toxic and biodegradable and can protect the entrapped drug from enzymatic attack and immune recognition until they reach the target cells. The toxicity of a drug itself can be reduced by this shielding in the liposomes. Upon intravenous injection, liposomes are removed rapidly from circulation by the reticuloendothelial cells into macrophages, where they are slowly degraded. These properties of liposomes make them «ideal» for the therapy of intracellular organisms. Large concentrations of an antimicrobial can be incorporated into the liposomes and delivered to the infected cells. Numerous methods for preparation of various types of liposomes have been described. Liposomes can be basically of two forms. The multi-Iamelar liposomes are made of separated layers and usually are larger than unilamelar liposomes, that can be designed to be very small, measuring between 2 nanometers to a few micrometers in diameter. The clearance of liposomes from the blood is dependent on certain properties of the preparation. In general, liposomes are very positively charged and therefore very efficiently taken out of the blood by cells. More recently, the development of «shealth liposomes» which are neutral preparations or weakly positively charged, made it possible to use liposomes as carriers of drugs that are not taken up by the reticuloendothelial cells. Liposomes have been previously used to treat a number of experimental infections caused by intracellular organisms both in vitro and in vivo, such as infections caused by Salmonella, Brucella, viruses, Candida, and mycobacteria (12, 13), within improved activity when compared with the free drug.
Use of liposome preparations to treat mycobacterial infection The use of liposome preparations for the treatment of mycobacterial infections offers the advantage that high concentrations of active antimicrobials can be delivered to the site of infection. However, administration of a liposome preparation LV. probably will deliver most of the antimicrobial to phagocytic cells in the liver and spleen, and in the particular cases of M. tuberculosis and MAC infection of the lungs, drugs will not reach the bacterial niche. This possibility was well demonstrated in a study done by VLADMIRSKY and colleagues (14) in which streptomycin encapsulated in liposomes was used to treat disseminated infection with M. tuberculosis in mice. The authors found that after 9 days of treatment there was a significant reduction in the number of viable organisms in liver and spleen compared with the reduction in CFU/g obtained in mice treated with free streptomycin. However, the opposite result was obtained in the lungs, where free streptomycin was significantly more efficacious than streptomy-
Liposomes for mycobacterial infections . 581
cin encapsulated in liposomes. The reason for these findings is that the liposome preparation used was not appropriate to treat lung infection. Recent data have shown that liposomes made of glycolipids which are neutral, attain circulation in the blood for longer periods of time than positively charged liposomes and are ingested by lung macrophages. The efficacy of these liposomes has been demonstrated in the treatment of Klebsiella pneumonia in mice. We and others have used liposome preparations to treat experimental disseminated MAC infection in mice (13, 15). Treatment of disseminated MAC infection is challenging due to bacterial resistance or high minimal inhibitory concentration (MIC) of the drug, and ability of the organism to survive intracellularly in macrophages. Aminoglycosides, such as amikacin, have been shown to be modestly effective in vitro and in vivo against MAC. Gentamicin, another antibiotic of the class of aminoglycosides, has MIC 90 of 36 [!g/ml for MAC strains, while the serum level is 2 [!g/ml. Nonetheless, the use of amikacin and gentamicin encapsulated in liposomes demonstrated not only that amikacin and gentamicin liposomes were remarkably active in mice, but also that only 5 doses of amikacin (1 mg/ dose) were sufficient to reduce the load of bacteria in liver and spleen by three orders of magnitude (14). Furthermore, administration of free amikacin was associated with rapid clearance in the urine, but when amikacin was given in liposome form, it was excreted much more slowly. A significant amount of amikacin was still detected in the urine 7 days after administration. These results suggested that liposome preparations will release the entrapped drug very slowly and therefore maintain a sustained concentration of the antibiotic for days after administration, making it possible to inject liposomes probably once a week. More recently, DUZGU N ES and colleagues have used ciprofloxacin encapsulated liposomes to treat MAC infection in mice with success (16). Ciprofloxacin, a quinolone, can concentrate intracellularly, but treatment with liposome-ciprofloxacin showed significantly greater killing of MAC in liver and spleen when compared with free ciprofloxacin. In contrast to aminoglycosides, clarithromycin and ofloxacin are antimicrobials that can achieve high concentrations within macrophages. A recent study by ONYEJI and colleagues (17) showed that liposome-encapsulated ofloxacin and clarithromycin significantly enhanced the activities of the drugs when compared with the antimycobacterial effects of equivalent concentrations of free drugs. Their study also showed that the intracellular concentration of the liposome-entrapped form was markedly higher than the free form of the drugs. This study showed that even with drugs that can concentrate intracellularly , liposome preparations can be associated with an increase in killing of intracellular bacteria. Liposome preparations can deliver high concentrations of antimicrobials into the cell, making it possible for antibiotics with antimycobacterial activity that cannot penetrate into cells, to achieve inhibitory or bactericidal
582 .
L.E.BERMUDEZ
concentrations against mycobacteria, to be used as treatment of mycobacterial diseases. Furthermore, because liposome-entrapped antimicrobials achieve high concentration within phagocytic cells, they may reduce the changes of emergence of resistance. The down side of the use of liposomes for therapy is that first it increases costs, since the patient must be admitted to the hospital; second, we need to learn more about liposome formulations and how to target liposomes to a determined infect cell; and thirdly, we need to reduce the incidence of toxic effects associated with the prolonged use of liposomes, such as renal and lung toxicity. Acknowledgement I thank MONICA F. MAPA for helping to prepare this manuscript.
References 1. BLOOM, B. R. and C. J. L. MURRAY. 1992. Tuberculosis: Commentary on a reemergent killer. Science 257: 1055-1064. 2. NIGHTINGALE, S. D., L. T. BYRD, P. M. SOUTHERN, J. D. JOCHUSCH, S. X. CAL, and B. A. WYNNE. 1992. Disseminated MAC infection: An inevitable complication of HIV infection in children and adults? Incidence of Mycobacterium avium-intracellulare complex bacteremia in human immunodeficiency virus-positive patients. J. Infect. Dis. 165: 1082-1085. 3. PRINCE, D. S., D. D. PETERSOI\i, R. M. STEINER, J. E. GOTTLIEB, R. SCOTT, H. L. ISRAEL, W. G. FIGUEROA, and J. E. FISH. 1989. Infection with Mycobacterium avium complex in patients without predisposing conditions. N. Engl. J. Med. 130: 863-868. 4. CROWLE, A. J., A. Y. TSANG, A. E. VATTER, and M. H. MAY. 1986. Comparison of 15 laboratory and patient-derived strains of Mycobacterium avium for ability to infect and multiply in cultured human macrophages. J. Clin. Microbiol. 24: 812-821. 5. STURGILL-KoSZYCKI, S., P. H. SCHLESINGER, P. CHAKRABORTY, P. L. HADDIX, H. L. COLLINS, A. K. FOK, R. D. ALLEN, S. L. GLUCK, J. HEUSER, and D. G. RUSSELL. 1994. Lack of acidification in Mycobacterium phagosomes produced by exclusion of the vesicular proton-ATPase. Science 263: 678-681. 6. CROWLE, A., R. DAHL, E. Ross, and M. H. MAY. 1991. Evidence that vesicles containing living, virulent Mycobacterium tuberculosis or Mycobacterium avium in cultured human macrophages are not acidic. Inf. Immun. 59: 1823-1827. 7. FERNANDES, P. B., D. J. HARDY, D. McDANIEL, C. W. HANSON, and R. N. SWANSON. 1989. In vitro and in vivo activities of clarithromycin against Mycobacterium avium. Antimicrob. Agents Chemother. 33: 1531-1536. 8. INDERLlED, C. B., P. T. KOLONOSKI, M. Wu, and L. S. YOUNG. 1989. In vitro and in vivo activity of azithromycin (CP 62, 993) against the Mycobacterium avium complex. J. Infect. Dis. 159: 994-997. 9. DAUTZENBERG, B., C. TRUFFOT, S. LEGRIS, M. C. MEYOHAS, H. C. BERLlE, A. MERCAT, S. CHEVRET, and J. GROSSET. 1991. Activity of clarithromycin against Mycobacterium avium infection in patients with the Acquired Immune Deficiency Syndrome. Am. Rev. Respir. Dis. 144: 564-569. 10. YOUNG, L. S., L. WIVIOTT, M. Wu, P. T. KOLONOSKI, R. BOLAN, and C. B. INDERLIED. 1991. Azithromycin reduces Mycobacterium avium complex bacteremia
Liposomes for mycobacterial infections .
583
and relieves the symptoms of disseminated disease in patients with AIDS. Lancet 338: 1107-1109. 11. BERMUDEZ, L. E., C. INDERLlED, and L. S. YOUNG. 1991. Stimulation with cytokines enhances penetration of Azithromycin into human macrophages. Antimicrob. Agents Chemother. 12: 2625-2629. 12. TADAKUMA, T., N. IKEWAKI, T. YASUDA, M. TSUTSUMI, S. SAITO, and S. KAZUHISA. 1985. Treatment of experimental salmonellosis in mice with streptomycin entrapped in liposomes. Antimicrob. Agents Chemother. 1: 28-32. 13. BERMUDEZ, L. E., A. O. YAU-YOUNG, J. P. LIN, J. COOPER, and L. S. YOUNG. 1990. Treatment of disseminated Mycobacterium avium complex infection of beige mice with liposome-encapsulated aminoglycosides. J. Inf. Dis. 161: 1262-1268. 14. VLADIMIRSKY, M. A. and G. A. LADIGNA. 1982. Antibacterial activity of liposomesentrapped streptomycin in mice with M. tuberculosis. Biomedicine 36: 375-377. 15. DUZGUNNES, N., V. PERUMAL, L. KESAVALU, J. A. GOLDSTEIN, R. J. DEBS, and R. J. GANGADHARAM. 1988. Enhanced effect of liposome-encapsulated amikacin on Mycobacterium avium-Mycobacterium intracellulare complex infection in beige mice. Antimicrob. Agents Chemother. 32: 1404-1411. 16. MAJUMDAR, S., D. FLASHER, D. S. FRIEND, P. NASSOS, D. YAjKO, W. K. HADLEY, and N. DUZGUNES. 1992. Efficacies of liposome-encapsulated streptomycin and ciprofloxacin against Mycobacterium avium-M. intracellulare complex infections in human peripheral blood monocyte/macrophages. Antimicrob. Agents Chemother. 36:2808-2815. 17. ONYEjl, C. 0., C. H. NIGHTINGALE, D. P. NICOLAU, and R. QUINTILlANJ. 1994. Efficacies of liposome-encapsulated clarithromycin and ofloxacin against M. avium complex in macrophages. Antimicrob. Agents Chemother. 38: 523-527.
Dr. LUIZ BERMUDEZ, Kuzel! Institute for Arthritis and Infectious Diseases, Medical Research Institute of San Francisco, Pacific Presbyterian Medical Center, 2200 Webster Street, Room 305, San Francisco, CA 9415, USA
©
1994 by Gustav Fischer Verlag, Stuttgart
Kuzell Institute for Arthritis and Infectious Diseases, Medical Research Institute of San Francisco, Pacific Presbyterian Medical Center, San Francisco, CA, USA
Use of Liposome Preparation to Treat Mycobacterial Infections Lurz E. BERMUDEZ
Abstract Infections caused by organisms of the genus mycobacteria, such as tuberculosis M. avium disseminated infection in AIDS patients and leprosy, are extremely common around the world. Mycobacteria are intracellular organisms that invade and multiply chiefly within phagocytic cells. Antibiotic resistance among mycobacteria is a growing concern. M. tuberculosis resistant to INH and rifampin are increasing in major urban centers of the developed and in the developing world. M. avium is characteristically resistant to most anti-tuberculosis antibiotics. Furthermore, therapy of mycobacterial infections takes a long time and most of the drugs have potential side effects and toxicity. In addition, mycobacteria is found within cells and antimicrobials need to be able to achieve adequate concentration within the compartment where mycobacteria is located. Liposome preparations, containing antibiotics, have a theoretical advantage in being able to deliver high concentrations of antimicrobials into the infected cell. Studies done thus far, in vitro and in vivo, have confirmed this premise, when comparing drug entrapped in liposomes with free drug. This paper summarizes the results obtained using liposome preparations to treat mycobacterial infections.
Introduction Infections caused by organisms of the genus mycobacteria are extremely common around the world. It is estimated that tuberculosis is diagnosed in approximately 8 million people, and is responsible for 3 million deaths every year (1). Disseminated Mycobacterium avium complex (MAC) infection occurs in up to 60 % of patients with AIDS (2) and pulmonary infection with MAC in non-AIDS patients has become more frequent during the last decade (3). Mycobacteria are intracellular organisms, that invade and multiply within chiefly phagocytic cells (4). While M. tuberculosis causes pulmonary infection in most of the infected individuals, MAC usually is associated with pulmonary infection in the HIV-negative popula-
Liposomes for mycobacterial infections . 579
tion and disseminated infection in patients with AIDS. Both bacteria are capable of invading macrophages and monocytes, and survive within the phagocytic cells. M. tuberculosis infect chiefly pulmonary macrophages, while MAC is encountered mainly in reticuloendothelial macrophages. Recent studies have demonstrated that mycobacteria once within macrophages inhibits the fusion of phagosomes with lysosomes (5, 6). Mycobacteria also inhibits the acidification and maturation of the phagosomes, thus living in an intracellular environment with pH in a range of 6.0 to 7.5 (5).
Anti-mycobacterial therapy: current problems and limitations Over the years, tuberculosis has been considered a treatable disease, although it required therapy with three drugs for prolonged periods of time. However, with the surging AIDS epidemic, tuberculosis became a critical public health problem with a number of multiple-resistant strains being isolated in selected cities. In contrast, MAC is known to be resistant to most of anti-tuberculosis antimicrobials available and only recently, through a screening effort in a number of laboratories, effective antibiotics have been discovered (7, 8). The macrolides clarithromycin and azithromycin have been demonstrated to have anti-MAC activity in vitro and in vivo (7-10), and were shown efficacious against MAC disseminated disease in two recent clinical trials (9, 10). However, resistant strains have been isolated after two to three months of single agent therapy in a large percentage of patients. There are a number of factors that need to be taken into account when one considers anti-mycobacterial therapy: (1) the antimicrobial not only needs to be active against extracellular bacteria, but also needs to achieve inhibitory concentration with infected cells. A number of antimicrobials are active against mycobacteria in vitro, but fail to achieve inhibitory concentration within macrophages such as the aminoglycosides. However, it is not known whether infection of macrophages with mycobacteria can alter the mechanisms of transport of antimicrobials into the cell resulting in decreased intracellular concentrations of the drugs. Recent work by BERMUDEZ and colleagues (11) suggests this possibility by showing that human macrophages infected with MAC for longer than three days incorporate significantly less azithromycin (an azalide) than uninfected cells (2). The antimicrobial needs to be active at the pH of the phagosome containing mycobacteria. It is even more relevant for the activity of certain antimicrobials such as aminoglycosides, macrolides, and pyrazinamide that are pH dependent; and (3), ideally, the antimicrobial should be active against latent mycobacteria.
580 . L.E.
BERMUDEZ
Antimicrobials encapsulated in liposomes Synthetic phospholipid bilayer vesicles .assembled into a closed membrane system, called liposomes, are non-toxic and biodegradable and can protect the entrapped drug from enzymatic attack and immune recognition until they reach the target cells. The toxicity of a drug itself can be reduced by this shielding in the liposomes. Upon intravenous injection, liposomes are removed rapidly from circulation by the reticuloendothelial cells into macrophages, where they are slowly degraded. These properties of liposomes make them «ideal» for the therapy of intracellular organisms. Large concentrations of an antimicrobial can be incorporated into the liposomes and delivered to the infected cells. Numerous methods for preparation of various types of liposomes have been described. Liposomes can be basically of two forms. The multi-Iamelar liposomes are made of separated layers and usually are larger than unilamelar liposomes, that can be designed to be very small, measuring between 2 nanometers to a few micrometers in diameter. The clearance of liposomes from the blood is dependent on certain properties of the preparation. In general, liposomes are very positively charged and therefore very efficiently taken out of the blood by cells. More recently, the development of «shealth liposomes» which are neutral preparations or weakly positively charged, made it possible to use liposomes as carriers of drugs that are not taken up by the reticuloendothelial cells. Liposomes have been previously used to treat a number of experimental infections caused by intracellular organisms both in vitro and in vivo, such as infections caused by Salmonella, Brucella, viruses, Candida, and mycobacteria (12, 13), within improved activity when compared with the free drug.
Use of liposome preparations to treat mycobacterial infection The use of liposome preparations for the treatment of mycobacterial infections offers the advantage that high concentrations of active antimicrobials can be delivered to the site of infection. However, administration of a liposome preparation LV. probably will deliver most of the antimicrobial to phagocytic cells in the liver and spleen, and in the particular cases of M. tuberculosis and MAC infection of the lungs, drugs will not reach the bacterial niche. This possibility was well demonstrated in a study done by VLADMIRSKY and colleagues (14) in which streptomycin encapsulated in liposomes was used to treat disseminated infection with M. tuberculosis in mice. The authors found that after 9 days of treatment there was a significant reduction in the number of viable organisms in liver and spleen compared with the reduction in CFU/g obtained in mice treated with free streptomycin. However, the opposite result was obtained in the lungs, where free streptomycin was significantly more efficacious than streptomy-
Liposomes for mycobacterial infections . 581
cin encapsulated in liposomes. The reason for these findings is that the liposome preparation used was not appropriate to treat lung infection. Recent data have shown that liposomes made of glycolipids which are neutral, attain circulation in the blood for longer periods of time than positively charged liposomes and are ingested by lung macrophages. The efficacy of these liposomes has been demonstrated in the treatment of Klebsiella pneumonia in mice. We and others have used liposome preparations to treat experimental disseminated MAC infection in mice (13, 15). Treatment of disseminated MAC infection is challenging due to bacterial resistance or high minimal inhibitory concentration (MIC) of the drug, and ability of the organism to survive intracellularly in macrophages. Aminoglycosides, such as amikacin, have been shown to be modestly effective in vitro and in vivo against MAC. Gentamicin, another antibiotic of the class of aminoglycosides, has MIC 90 of 36 [!g/ml for MAC strains, while the serum level is 2 [!g/ml. Nonetheless, the use of amikacin and gentamicin encapsulated in liposomes demonstrated not only that amikacin and gentamicin liposomes were remarkably active in mice, but also that only 5 doses of amikacin (1 mg/ dose) were sufficient to reduce the load of bacteria in liver and spleen by three orders of magnitude (14). Furthermore, administration of free amikacin was associated with rapid clearance in the urine, but when amikacin was given in liposome form, it was excreted much more slowly. A significant amount of amikacin was still detected in the urine 7 days after administration. These results suggested that liposome preparations will release the entrapped drug very slowly and therefore maintain a sustained concentration of the antibiotic for days after administration, making it possible to inject liposomes probably once a week. More recently, DUZGU N ES and colleagues have used ciprofloxacin encapsulated liposomes to treat MAC infection in mice with success (16). Ciprofloxacin, a quinolone, can concentrate intracellularly, but treatment with liposome-ciprofloxacin showed significantly greater killing of MAC in liver and spleen when compared with free ciprofloxacin. In contrast to aminoglycosides, clarithromycin and ofloxacin are antimicrobials that can achieve high concentrations within macrophages. A recent study by ONYEJI and colleagues (17) showed that liposome-encapsulated ofloxacin and clarithromycin significantly enhanced the activities of the drugs when compared with the antimycobacterial effects of equivalent concentrations of free drugs. Their study also showed that the intracellular concentration of the liposome-entrapped form was markedly higher than the free form of the drugs. This study showed that even with drugs that can concentrate intracellularly , liposome preparations can be associated with an increase in killing of intracellular bacteria. Liposome preparations can deliver high concentrations of antimicrobials into the cell, making it possible for antibiotics with antimycobacterial activity that cannot penetrate into cells, to achieve inhibitory or bactericidal
582 .
L.E.BERMUDEZ
concentrations against mycobacteria, to be used as treatment of mycobacterial diseases. Furthermore, because liposome-entrapped antimicrobials achieve high concentration within phagocytic cells, they may reduce the changes of emergence of resistance. The down side of the use of liposomes for therapy is that first it increases costs, since the patient must be admitted to the hospital; second, we need to learn more about liposome formulations and how to target liposomes to a determined infect cell; and thirdly, we need to reduce the incidence of toxic effects associated with the prolonged use of liposomes, such as renal and lung toxicity. Acknowledgement I thank MONICA F. MAPA for helping to prepare this manuscript.
References 1. BLOOM, B. R. and C. J. L. MURRAY. 1992. Tuberculosis: Commentary on a reemergent killer. Science 257: 1055-1064. 2. NIGHTINGALE, S. D., L. T. BYRD, P. M. SOUTHERN, J. D. JOCHUSCH, S. X. CAL, and B. A. WYNNE. 1992. Disseminated MAC infection: An inevitable complication of HIV infection in children and adults? Incidence of Mycobacterium avium-intracellulare complex bacteremia in human immunodeficiency virus-positive patients. J. Infect. Dis. 165: 1082-1085. 3. PRINCE, D. S., D. D. PETERSOI\i, R. M. STEINER, J. E. GOTTLIEB, R. SCOTT, H. L. ISRAEL, W. G. FIGUEROA, and J. E. FISH. 1989. Infection with Mycobacterium avium complex in patients without predisposing conditions. N. Engl. J. Med. 130: 863-868. 4. CROWLE, A. J., A. Y. TSANG, A. E. VATTER, and M. H. MAY. 1986. Comparison of 15 laboratory and patient-derived strains of Mycobacterium avium for ability to infect and multiply in cultured human macrophages. J. Clin. Microbiol. 24: 812-821. 5. STURGILL-KoSZYCKI, S., P. H. SCHLESINGER, P. CHAKRABORTY, P. L. HADDIX, H. L. COLLINS, A. K. FOK, R. D. ALLEN, S. L. GLUCK, J. HEUSER, and D. G. RUSSELL. 1994. Lack of acidification in Mycobacterium phagosomes produced by exclusion of the vesicular proton-ATPase. Science 263: 678-681. 6. CROWLE, A., R. DAHL, E. Ross, and M. H. MAY. 1991. Evidence that vesicles containing living, virulent Mycobacterium tuberculosis or Mycobacterium avium in cultured human macrophages are not acidic. Inf. Immun. 59: 1823-1827. 7. FERNANDES, P. B., D. J. HARDY, D. McDANIEL, C. W. HANSON, and R. N. SWANSON. 1989. In vitro and in vivo activities of clarithromycin against Mycobacterium avium. Antimicrob. Agents Chemother. 33: 1531-1536. 8. INDERLlED, C. B., P. T. KOLONOSKI, M. Wu, and L. S. YOUNG. 1989. In vitro and in vivo activity of azithromycin (CP 62, 993) against the Mycobacterium avium complex. J. Infect. Dis. 159: 994-997. 9. DAUTZENBERG, B., C. TRUFFOT, S. LEGRIS, M. C. MEYOHAS, H. C. BERLlE, A. MERCAT, S. CHEVRET, and J. GROSSET. 1991. Activity of clarithromycin against Mycobacterium avium infection in patients with the Acquired Immune Deficiency Syndrome. Am. Rev. Respir. Dis. 144: 564-569. 10. YOUNG, L. S., L. WIVIOTT, M. Wu, P. T. KOLONOSKI, R. BOLAN, and C. B. INDERLIED. 1991. Azithromycin reduces Mycobacterium avium complex bacteremia
Liposomes for mycobacterial infections .
583
and relieves the symptoms of disseminated disease in patients with AIDS. Lancet 338: 1107-1109. 11. BERMUDEZ, L. E., C. INDERLlED, and L. S. YOUNG. 1991. Stimulation with cytokines enhances penetration of Azithromycin into human macrophages. Antimicrob. Agents Chemother. 12: 2625-2629. 12. TADAKUMA, T., N. IKEWAKI, T. YASUDA, M. TSUTSUMI, S. SAITO, and S. KAZUHISA. 1985. Treatment of experimental salmonellosis in mice with streptomycin entrapped in liposomes. Antimicrob. Agents Chemother. 1: 28-32. 13. BERMUDEZ, L. E., A. O. YAU-YOUNG, J. P. LIN, J. COOPER, and L. S. YOUNG. 1990. Treatment of disseminated Mycobacterium avium complex infection of beige mice with liposome-encapsulated aminoglycosides. J. Inf. Dis. 161: 1262-1268. 14. VLADIMIRSKY, M. A. and G. A. LADIGNA. 1982. Antibacterial activity of liposomesentrapped streptomycin in mice with M. tuberculosis. Biomedicine 36: 375-377. 15. DUZGUNNES, N., V. PERUMAL, L. KESAVALU, J. A. GOLDSTEIN, R. J. DEBS, and R. J. GANGADHARAM. 1988. Enhanced effect of liposome-encapsulated amikacin on Mycobacterium avium-Mycobacterium intracellulare complex infection in beige mice. Antimicrob. Agents Chemother. 32: 1404-1411. 16. MAJUMDAR, S., D. FLASHER, D. S. FRIEND, P. NASSOS, D. YAjKO, W. K. HADLEY, and N. DUZGUNES. 1992. Efficacies of liposome-encapsulated streptomycin and ciprofloxacin against Mycobacterium avium-M. intracellulare complex infections in human peripheral blood monocyte/macrophages. Antimicrob. Agents Chemother. 36:2808-2815. 17. ONYEjl, C. 0., C. H. NIGHTINGALE, D. P. NICOLAU, and R. QUINTILlANJ. 1994. Efficacies of liposome-encapsulated clarithromycin and ofloxacin against M. avium complex in macrophages. Antimicrob. Agents Chemother. 38: 523-527.
Dr. LUIZ BERMUDEZ, Kuzel! Institute for Arthritis and Infectious Diseases, Medical Research Institute of San Francisco, Pacific Presbyterian Medical Center, 2200 Webster Street, Room 305, San Francisco, CA 9415, USA