Therapy of leishmaniasis: Superior efficacies of liposomeencapsulated

Transcription

1 Proc. Nati. Acad. Sci. USA Vol. 75, No. 6, pp , June 1978 Medical Sciences Therapy of leishmaniasis: Superior efficacies of liposomeencapsulated drugs (antimonial compounds/phospholipids/model membranes/parasites) CARL R. ALVING*, EDGAR A. STECKt, WILLIE L. CHAPMAN, JR.t, VIRGINIA B. WAITS*, LARRY D. HENDRICKSt, GLENN M. SWARTZ, JR.t, AND WILLIAM L. HANSONt * Department of Immunology, and t Division of Medicinal Chemistry, Walter Reed Army Institute of Research, Washington, D.C. 12; and * Departments of Pathology and Parasitology, College of Veterinary Medicine, The University of Georgia, Athens, Georgia 362 Communicated by Roscoe. Brady, April 3, 1978 ABSTRACT Liposomes containing antimonial compounds trapped in the aqueous phase were tested in the treatment of experimental leishmaniasis. The rationale of this approach was based on the hypothesis that the liposomes and the parasite are taken up by the same cell, the reticuloendothelial cell, and we present electron microscopic evidence that supports this hypothesis. Suppression of leishmaniasis was quantified by determining the total number of parasites per liver from impression smears. When two antimonials, meglumine antimoniate and sodium stibogluconate, were encapsulated within lipo somes, each was more than 7 times more active compared to either of the free (unencapsulated) drugs. After infection, if untreated, all of the hamsters eventually would die from the disease. Liposomeencapsulated meglumine antimoniate was about 3364 times more effective in causing a drop in the death rate than was the free antimonial. The efficacy of treatment was influenced by the lipid composition and charge of the liposomes. For example, positively charged liposomes containing egg phosphatidylcholine were much less effective than negatively charged ones. In contrast, positively and negatively charged sphingomyelin liposomes were equally effective. Liposomes containing phosphatidylserine (which were negatively charged, but also had a much higher charge density) were among the lesseffective preparations. Among those tested, the most consistently efficacious liposomes contained highly saturated longchain phospholipids (eg., dipalmitoyl phosphatidylcholine), cholesterol, and a negative charge. We conclude that liposomes may be useful as carriers of drugs to treat infectious diseases involving the reticuloendothelial system. The toxicities of antimony are very similar to those of arsenic. Encapsulation of antimonial drugs and reduction of the dose required for effective therapy should minimize such systemic toxicities as acute cardiomyopathy and toxic nephritis. Synthetic phospholipid bilayer vesicles, also called liposomes, have been used as models of cell membranes (13) and as carriers for a variety of therapeutic agents (47). Upon intravenous injection, liposomes are removed rapidly by the reticuloendothelial cells and are at least partly localized in lysosomes where they are slowly degraded (811). Because of the latter properties, liposomes have been proposed as carriers of agents to treat diseases of the reticuloendothelial system (reviewed in refs. 47). Despite their potential, liposomes have not been developed for widespread use in humans, nor have they been developed for use in any infectious disease. Among the most important diseases of humans are those caused by intracellular parasites of reticuloendothelial cells; such diseases afflict tens of millions of people. Leishmania, a hemoflagellate protozoan, causes leishmaniasis. The disease has a prevalence of at least 12 million parasitic infections in humans, and it represents an immense public health problem in the The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C solely to indicate this fact Middle East, Africa, India, China, Central and South America, and other tropical and subtropical areas throughout the world (1214). The parasite, which resides chronically in reticuloendothelial cells, causes visceral. (kala azar), mucocutaneous, and cutaneous manifestations (1214). In humans, the untreated visceral disease is usually fatal, and all forms may be severe (or fatal), lingering, and recurrent, despite treatment with antimonial compounds, the drugs of choice (1214). The antimonials are limited in effectiveness because of severe systemic toxicities similar to those of arsenicals (15). In a preliminary report we showed that liposomes containing a trapped antimonial compound were at least 35 times as effective as the drug alone in suppressing experimental leishmaniasis in hamsters (16). In the present paper we describe the liposomal membrane factors that influence the antileishmanial efficacy of encapsulated antimonial drugs, and we present morphologic evidence that shows that liposomes travel to the intracellular site of the parasite. MATERIALS AND METHODS Lipids were purchased from the following sources: dimyristoyl, dipalmitoyl, and distearoyl phosphatidylcholine (DMPC, DPPC, and DSPC) (Sigma Chemical Co., St. Louis, MO); egg phosphatidylcholine (PC) and beef sphingomyelin (SM) (Pierce Chemical Co., Rockville, IL); beef phosphatidylserine (PS) (Applied Science Laboratories, State College, PA); cholesterol (Chol) (Calbiochem, La Jolla, CA); dicetyl phosphate (DCP) and stearylamine (SA) (K and K Laboratories, Plainview, NY). Meglumine antimoniate (Glucantime) was purchased from Rhodia, Inc., New York, NY. Sodium stibogluconate (Pentostam) powder was generously supplied as a gift by R. A. Neal, Wellcome Foundation Ltd., Beckenham, Kent, England. Suppressive effects of drugs on leishmanial infections were tested by a modification (16, 17) of a method described by Stauber and associates (18). In brief, golden hamsters (57 g) were injected intracardially with 17 amastigotes of the Khartoum strain of Leishmania donovani obtained from spleens of donor hamsters. At either 17 or 24 days postinoculation of parasites (as indicated for each experiment), test drugs were administered intracardially under "blind" experimental conditions for 4 consecutive days. Thirtytwo days after the start of treatment the surviving animals were sacrificed, and the total numbers of parasites per liver were determined from impression smears (17, 18). Percent of parasite suppression at each dosage level was calculated by comparison of each animal to the average of a control group of 11 infected, untreated animals Abbreviations: DMPC, DPPC, DSPC, and egg PC, dimyristoyl, dipalmitoyl, distearoyl, and egg phosphatidylcholine; SM, sphingomyelin; PS, phosphatidylserine; Chol, cholesterol; DCP, dicetyl phosphate; SA, stearylamine.

2 296 Medical Sciences: Alving et al. Proc. Natl. Acad. Sci. USA 75 (1978) a 71A i 6_, A A CL5 ~4 3A 1A FIG. 1. Suppression of Leishmania by liposomeencapsulated antimonials. Liposomes consisted of DMPC/CholJDCP (molar ratio, 2/1.5/.22). Treatment of the hamsters (11 animals for each point) was begun 17 days after infection, and onefourth of the indicated doses were injected intracardially daily for 4 days. The surviving animals were sacrificed 32 days after start of therapy. Control groups also were injected with equivalent volumes of liposomes swollen in.15 M NaCl., Liposomes containing meglumine antimoniate;, liposomes containing sodium stibogluconate; *, meglumine antimoniate alone;,, sodium stibogluconate alone. that had been injected intracardially with corresponding volumes of isotonic saline. The liposomes (multilamellar) were swollen, by using a Vortex mixer, in either.15 M NaCI or.3 M antimonial drug such that the phosphatide was 1 mm with respect to the final suspension. They were washed three times by dilution in 1 vol of.154 M NaCl and centrifugation at, X g for 1 min at 22. The final pellet was suspended with sufficient.15 M NaCl so that the phosphatide was approximately 18 mm with respect to the aqueous suspension. An aliquot (.4 ml) was shaken with 2.1 ml of water and 2.5 ml of chloroform to disrupt the liposomes; the chloroform was washed twice with 2.5 ml of water; and the combined aqueous phases were sent to analytical laboratories (either to Galbraith Laboratories Inc., Knoxville, TN, or to Stanford Research Institute, Menlo Park, CA, or both) for quantitative antimony analysis. The washed liposomes trapped ca 211% of the antimonial drug present in the original swelling solution. Liver tissue was fixed for electron microscopy in 3% (vol/vol) glutaraldehyde/.1% (wt/vol) cacodylate buffer, ph 7.3, for 3 hr and washed overnight in buffer. It was then postfixed in 1% (wt/vol) osmium tetroxide in the same buffer for 1 hr, dehydrated in an acetone series, imbedded in Spurr's medium, and stained with uranyl acetate lead citrate. RESULTS Comparison of LiposomeEncapsulated and Nonencapsulated Antimonials. Two different antimonials, meglumine antimoniate and sodium stibogluconate, were incorporated into liposomes and tested for relative antileishmanial activities. As shown in Fig. 1, the two liposomeencapsulated drugs had approximately equivalent effectiveness. Based on the data of Fig. 1, under the conditions used the amount of antimonial required for 5% suppresion of leishmanial infection was more than 7 times greater for each of the free drugs than for either of two liposomeencapsulated drugs. For 9% suppression, approximately 6 times as much free, compared to liposomeencapsulated, meglumine antimonate was required. Liposomes alone (control) provided no antileishmanial activity. Influence of Liposomal Membrane Composition on Suppression of Leishmania. AMthough all liposomes that contained c 6 a ~~~~~~~~~~~ A FIG. 2. Influence of liposomal phospholipid type and liposomal charge on suppression. The animals were infected and treated as described in the legend of Fig. 1, except that therapy was begun 24 days after infection. Liposomes contained phospholipid (SM, egg PC, or PS), Chol, and DCP (where present) or SA (where present) in molar ratios of 2/1.5/.22., SM/Chol/DCP. *, SM/Chol/SA;, egg PC/Chol/DCP; o, egg PC/Chol/SA; A, PS/Chol; A, meglumine antimoniate alone. trapped antimonial were superior in potency to the drug alone, certain lipid combinations were better than others. Fig. 2 compares liposomes prepared with phospholipids from natural sources (beef SM, beef PS, or egg PC). The liposomes also had either a negative charge due to the presence of DCP or PS or a positive charge due to the presence of SA. From the data of Fig. 2 it is evident that negatively charged PScontaining or positively charged egg PCcontaining liposomes were less potent than either negatively charged egg PC, negatively charged SM, or positively charged SMcontaining liposomes. The surface charge density on the liposomes containing PS was much greater than that on liposomes containing DCP or SA, and this might have adversely influenced the relative effectiveness of PScontaining liposomes. Previous studies have shown that SMcontaining liposomes have greater inherent stability than do many other liposomes (3), and this also might help to explain the greater effectiveness of SM or of synthetic saturated phospholipids compared to PS. Different stabilities probably do not explain the greater potency of negatively charged, compared to positively charged, egg PCcontaining liposomes. In the presence of normal hamster serum, negatively and positively charged egg PCcontaining liposomes had approximately equal stability (unpublished data). Fig. 3 demonstrates that in a 24day infection, negatively charged, positively charged, or neutral liposomes prepared with DSPC or DMPC and containing meglumine antimoniate all gave similar leishmanial suppression results. Because of the differences between negatively and positively charged egg PCcontaining liposomes (Fig. 2), the effect of the concentrations of charged liposomal lipids on leishmanial suppression was tested in more detail using DMPC in a 17day infection (Fig. 4). Positively charged liposomes containing meglumine anti

3 Medical Sciences: Alving et al. Proc. Natl. Acad. Sci. USA 75 (1978) 2961 c 6 CL. 5 ar C FIG. 3. Influence of synthetic phospholipid fatty acyl chain length and liposomal charge on suppression. The animals were infected and treated as described in the legend of Fig. 1, except that therapy was begun 24 days after infection. The liposomes contained the indicated lipids in molar ratios of 2/1.5/.22., DSPC/Chol/DCP; *, DSPC/Chol/SA; *, DMPC/Chol/SA; v, DMPC/Chol. moniate were slightly, but consistently, less active in Leishmania suppression than were negatively charged ones. Effects of Antimonial Therapy on Death Rates. Intracardiac injection of saline alone resulted in a high (1525%) mortality among infected hamsters within 1 week (Fig. 5). Few animals died in the 2nd or 3rd week, but about 44% died in the 4th and 5th weeks, presumably due to leishmaniasis. Such late mortality did not occur with uninfected animals. Treatment with meglumine antimoniate alone at levels of mg/kg FIG. 4. Influence of liposomal charge concentration on suppression. The animals were infected and treated as described in the legend of Fig. 1. DMPC/Chol/DCP:, 2/1.5/.22; A, 2/1.5/.88. DMPC/Chol/SA:, 2/1.5/.22; 3 2/1.5/.44; a, 2/1.5/.88. resulted in a higher mortality in the 3rd week, but did not greatly reduce overall mortality due to leishmaniasis. An obvious decline in death rate in the 3rd5th weeks after treatment occurred only after injection of 832 mg of the drug per kg (Fig. 5). At the highest dose level a slight increase in mortality was observed in the 1st week, possibly due to acute toxicity from the drug. 3 ( 1 _.1 c._ 1 a 1 a O_ 3 Meglumine antimoniate injected, mg/kg 1 _ Time after starting treatment, wk FIG. 5. Effect of free antimonial (unencapsulated) drug on death rate. The animals were infected and treated as described in the legend of Fig. 1. The initial numbers of animals were as follows: mg/kg (isotonic saline injected), 88; 1352 mg/kg, 22; 8 mg/kg, 44; 416 mg/kg, 55; 832 mg/kg,

4 2962 Medical Sciences: Alving et al. Proc. Natl. Acad. Sci. USA 75 (1978) ' E c 1 m.1 c 1 Meglumine antimoniate injected, mg/kg e1,,,:;. ;.j:,~ Time after starting treatment, wk FIG. 6. Death rates among animals treated with liposomcencapsulated meglumine antimoniate. The animals were infected and treated with liposomes as described in the legend to Fig. 1. The initial numbers of animals were as follows: mg/kg (liposomes contained isotonic saline), 66;.31.3 mg/kg, 66; mg/kg, 55; 13 mg/kg, 44; 48 mg/kg, 33. Deaths due to leishmaniasis in the 4th and 5th weeks of infection occurred more often in those animals receiving liposomes containig saline (Fig. 6) than in animals that received saline alone (Fig. 5). Injection of liposomes containing meglumine antimoniate reduced the death rates in the 4th and 5th weeks, even at the lowest doses of drug. Mortality due to leishmaniasis was virtually eliminated by a dose of 2.5 mg of liposomeencapsulated drug per kg. Thus, when compared with the amount of free (unencapsulated) antimonial drug required for an obvious effect (832 mg/kg) (Fig. 5), liposomeencapsulated drug at a similar level of activity ( mg/kg) had an efficacy that was times greater in prevention of Leishmaniaassociated mortality. These estimates of relative potency based on mortality rates compare favorably with those derived from Fig. 1 based on suppression of Leishmania infection (see above). Increased mortality in the 1st week due to liposomeencapsulated drug was not observed below 4 mg/kg (Fig. 6). Electron Microscopy. Electron micrographs of liver and spleen showed cells, presumably reticuloendothelial cells, that contained parasites adjacent to liposomal vesicles (Fig. 7). The multilamellar structures of the liposomes often were not well preserved, as reported by others (911, 19), but in some cases individual lamellae, as shown in Fig. 7, were sharply defined. DISCUSSION This study demonstrates that, under the conditions used, two liposomeencapsulated antimonial drugs were each more than.2 pm FIG. 7. Electron micrograph of hamster liver showing the detailed lamellar structure of a liposome (L) adjacent to an intracellular parasitic Leishmania (P). The liposomes, containing encapsulated drug, were injected into a donor hamster at about 35 days following leishmanial infection. The hamster was sacrificed 6 hr later. 7 times more effective as an antileishmanial agent than was the corresponding antimonial drug alone. Antimonial compounds, particularly meglumine antimoniate and sodium stibogluconate, are the drugs of choice in leishmaniasis, and the latter substance, which is an investigational new drug, is the only one available in the United States for treating all forms of leishmaniasis. In case of failure with the antimonials, amphotericin B often is used as a last resort (1214). The major difficulty with these forms of chemotherapy is the high sytemic toxicity, particularly to the heart, kidney, and liver (1214). Antimony belongs to the same periodic group as arsenic, and acute and chronic poisoning is similar to that caused by arsenic (15). Although pentavalent antimonials, such as meglumine antimoniate and sodium stibogluconate, are a great improvement over trivalent compounds, the pentavalents, particularly at large or prolonged dosage or in malnourished individuals, still are very toxic (12). Based on our data, toxicity of antimonials should be minimized after incorporation into liposomes because less than.15% of an ordinary therapeutic dose can be used for equivalent therapy. Our data on the increase in acute mortality due to the largest doses of drug in the first week of therapy (Figs. 5 and 6) suggest that the therapeutic index, defined by the ratio toxic dose/effective dose, may be much greater for the liposomeencapsulated drug than for the unencapsulated drug. Although we do not have definitive evidence at present, we anticipate that encapsulation of the drug in liposomes and rapid uptake of liposomes by reticuloendothelial cells will minimize adverse systemic antimonial effects, especially those due to acute cardiomyopathy and toxic nephritis. Rnecentilastiesthae sugiedstated that certain lifosomes by

5 Medical Sciences: Alving et al. themselves may have adverse pharmacologic effects. Generally these effects were produced by brain phospholipids, especially PS (, 21) or after injection of liposomes into brain (22). In the present study, liposomes containing brain PS were among the least effective ones, whereas those containing brain SM were among the more effective (Fig. 2). Although we do not have direct evidence for toxicity of liposomes per se, the mortality due to leishmaniasis in the 4th and 5th weeks following treatment was greater among animals injected with liposomes containing saline than it was among animals injected with saline alone (Figs. 5 and 6). The reason for the increased leishmaniasis mortality with liposomes alone is unknown, but it does raise the possibility that liposometherapy might aggravate certain concomitant pathologic conditions of the reticuloendothelial system other than that for which treatment is being given. Various studies have indicated that the tissue distribution of liposomes can be altered by changing the lipid composition and charge of the membrane (5, 6). We have found by empirical testing that the most effective lipid combinations for treating experimental leishmaniasis in hamsters are those that contain cholesterol and synthetic highly saturated phospholipids. Liposomes that are neutral or negatively charged are often more effective than positively charged ones. The data shown in Fig. 1 demonstrate that, under the conditions employed, unencapsulated meglumine antimoniate was more active than sodium stibogluconate against leishmaniasis. This study also demonstrates that, when encapsulated within liposomes, each of these drugs had greatly increased efficacies compared to either of the unencapsulated compounds. After this work was completed, two other laboratories reported use of liposomes for treatment of experimental visceral leishmaniasis in mice (23, 24). This work was supported in part by United States Army Medical Research and Development Contract DAMD17C Bangham, A. D. (1968) Prog. Biophys. Mol. Biol. 18, Papahadjopoulos, D. (1973) in Biological Horizons in Surface Science, eds Prince, L. M. & Sears, D. F. (Academic, New York), pp Alving, C. R. (1977) in The Antigens, ed. Sela, M. (Academic, New York), Vol. IV, pp Proc. Natl. Acad. Sci. USA 75 (1978) Gregoriadis, G. (1976) N. Engl. J. Med. 295, 7471, Tyrrell, D. A., Heath, T. D., Colley, C. M. & Ryman, B. E. (1976) Biochim. Blophys. Acta 457, Poste, G., Papahadjopoulos, D. & Vail, W. J. (1976) in Methods in Cell Biology, ed. Prescott, D. M. (Academic, New York), Vol. 14, pp Fendler, J. H. & Romero, A. (1977) Life Sci., Gregoriadis, G. & Ryman, B. E. (1972) Biochem. J. 129, Segal, A. W., Wills, E. J., Richmond, J. E., Slavin, G., Black, C. D. V. & Gregoriadis, G. (1974) Br. J. Exp. Pathol. 55, Rahman, Y.E. & Wright, B. J. (1975) J. Cell Biol. 65, de Barsy, T., Devos, P. & Van Hoof, F. (1976) Lab. Invest. 34, Steck, E. A. (1974) in Progress in Drug Research, ed. Jucker, E. (Birkhauser Verlag, Basel, Switzerland), Vol. 18, pp Biagi, F. (1976) in Tropical Medicine, eds. Hunter, G. W., III, Swartzwelder, J. C. & Clyde, D. F. (W. B. Saunders Co., Philadelphia, PA), 5th Ed., pp Mahmoud, A. A. F. & Warren, K. S. (1977) J. Infect. Dis. 136, Harvey, S. C. (1975) in The Pharmacological Basis of Therapeutics, eds. Goodman, L. S. & Gilman, A. (Macmillan, New York), 5th Ed., pp Alving, C. R., Steck, E. A., Hanson, W. L., Loizeaux, P. S., Chapman, W. L., Jr., & Waits, V. B. (1978) Life Sd. 22, Hanson, W. L., Chapman, W. L., Jr. & Kinnamon, K. E. (1977) Int. J. Parasitol. 7, Stauber, L. A., Franchino, E. M. & Grun, J. (1958) J. Protozool. 5, MattenbergerKreber, L., Auderset, G., Schneider, M., Louis Broillet, A., Strolin Benedetti, M. & Malno8, A. (1976) Experlentia 32, Bruni, A., Toffano, G., Leon, A. & Boarato, E. (1976) Nature, Masturzo, P., Gallamini, A., Murialdo, G., Nizzo, M. C. & Toffano, G. (1977) N. Engl. J. Med. 297, Adams, D. H., Joyce, G., Richardson, V. J., Ryman, B. E. & Wisniewski, H. M. (1977) J. Neurol. Sci. 31, Black, C. D. V., Watson, G. J. & Ward, R. J. (1977) Trans. R. Soc. Trop. Med. Hyg. 71, New, R. R. C., Chance, M. L., Thomas, S. C. & Peters, W. (1978) Nature 272,5556.