J. Phys. Chem. B 2009, 113, 14239–14246
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Langmuir Monolayer Study toward Combined Antileishmanian Therapy Involving Amphotericin B and Edelfosine Katarzyna Ha¸c-Wydro,*,† Patrycja Dynarowicz-Ła¸tka,† and Radosław Z˙uk‡ Faculty of Chemistry, Jagiellonian UniVersity, Ingardena 3, 30-060 Krako´w, Poland, and M. Smoluchowski Institute of Physics, Jagiellonian UniVersity, Reymonta 4, 30-059 Kraków, Poland ReceiVed: April 9, 2009; ReVised Manuscript ReceiVed: August 31, 2009
In this work, the results of comparative studies on the effect of lysophospholipid analogue, edelfosine (ED), and its mixtures with the polyene antibiotic, amphotericin B (AmB), on model erythrocyte and parasite membranes are presented. Both compounds are known for their antileishmanian activity; however, the application of AmB is limited by its toxicity, while the treatment with alkyl-lysophospholipids (edelfosine) is expensive in addition to its lower therapeutic activity as compared to the polyene. An additional problem is the emergence of resistance to both groups of drugs. The foregoing facts inspired the investigations toward combined amphotericin B/alkyl-lysophospholipid therapy. In this aspect, the effect of edelfosine on model erythrocyte versus parasite membrane has been verified by the incorporation of the drug into binary sterol/ phospholipid monolayer of the proportion corresponding to the respective cellular membrane. Afterward, amphotericin B/edelfosine mixtures of various proportions (1:9; 1:1, and 9:1) have been added into sterol/ phospholipid model membranes in the concentration of 1, 5, and 10%. The interactions between amphotericin B and edelfosine in mixed monolayers have also been investigated. The obtained results indicate that differences in the organization of erythrocyte versus parasite membranes are of great importance for selectivity of edelfosine toward the parasite membrane. The mixtures of drugs practically do not modify the properties of model erythrocyte membrane; however, their incorporation in the parasite membrane is thermodynamically unfavorable and causes membrane destabilization. The strongest differences in the influence of drug mixtures on erythrocyte versus parasite model membrane occur for 1% content of 1:9 and 1:1 AmB/edelfosine mixture. Moreover, when edelfosine is combined with amphotericin B, a decrease in edelfosine concentration needed to induce a similar effect on parasite membranes as edelfosine alone does is observed. As a consequence of strong interactions between edelfosine and amphotericin B, the decrease of the activity of their mixtures on model membranes was found. Introduction Chemicals of amphiphilic structure, because of their surface activity, find many practical applications. They are used, for example, in the textile industry as coatings for conditioning surfaces (as antistatics, for softening materials or to improve wettability), in the food industry or cosmetics as emulsifiers to stabilize emulsions, and in everyday life as detergents. It is worth mentioning that majority of physiologically active compounds (drug, hormones) in addition to structural components of all living cells (proteins, phospholipids, sterols) have a characteristic of surfactants amphipathic structure. Independently on their application, surfactants occur in mixtures rather than individually and interact with each other. Because of this, surfactant mixtures can show very different properties as compared to their components. In general, three different phenomena can occur, synergy, antagonism, or indifference. Most desirable is synergism, which occurs in a system when a given property (for example, surface tension or critical micelle concentration (CMC)) in the mixture reaches a more favorable value as compared to that attained by either component itself.1 Very wellknown mixtures of enhanced surface-active properties are commercial detergent preparations. The use of surfactants * To whom correspondence should be addressed. E-mail: hac@ chemia.uj.edu.pl. Fax: +48 0-12-634-05-15. Phone: +48 0-12 633-20-79. † Faculty of Chemistry, Jagiellonian University. ‡ M. Smoluchowski Institute of Physics, Jagiellonian University.
differing in the polar group (i.e., one charged and the other noncharged) usually leads to synergism, contrary to mixtures of surfactants differing in the hydrophobic parts, for example, hydrogenated and perfluorinated amphiphiles, which tend to phase separate.2 In analogy to the above-mentioned phenomena occurring in detergent mixtures, the interactions between physiological molecules may alter their properties significantly. A plethora of examples can be found to prove antagonism between drugs or their synergetic interactions.3,4 Both phenomena are of utmost importance in combined therapy. Examples of diseases when a combined therapy can be useful are parasitic infections, for example, visceral leishmaniasis (kala-azar), a tropical protozoal infection transmitted by sandfly bite.5 Conventional treatment with pentavalent antimonials, the cheapest one and in use since the 1930s, is usually not well-tolerated and ineffective in some regions due to the emergence of resistance.6 Amphotericin B (AmB), a macrolide antibiotic well-recognized for its antifungal activity,7 is alternative in treatment of visceral leishmaniasis.8 Although it is known to be one of the most active antileishmanial agents, it has to be administered paranterally and requires long therapy, which is associated with severe toxic side effects. To reduce AmB toxicity, lipid-associated formulations have been developed;8-10 however, they are not frequently applied due to high costs. Leishmanicidal acivity has also been reported for synthetic lysophospholipid (LP) analogues, such as miltefosine11
10.1021/jp9032996 CCC: $40.75 2009 American Chemical Society Published on Web 09/25/2009
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or edelfosine (ED),12 which were first applied as anticancer drugs,13 and because of this are known as antitumor lipids (ATLs). Contrary to AmB, they are not toxic and can be administered orally, although the treatment is quite expensive. One of the major barriers to successful treatment of leishmaniasis is the development of drug resistance, which is not only limited to antimonium drugs6 but it appears also for AmB14 and LPs.15,16 A way to postpone or reduce drug resistance can be administration of both drugs together, that is, AmB and one of the LPs to combat leishmaniasis. Another advantage of such a bitherapy is the possibility of oral treatment for leishmaniasis. Such an idea has already been verified in vitro.17,18 It has been found that miltefosine solubilizes AmB by forming mixed micelles, which provides a new oral delivery system for combined therapy of leishmaniasis. Also, strong interactions between AmB and miltefosine observed in solutions are of great importance for biological activities of both drugs administered together. Combined therapy involving AmB and one of the LPs analogues can also be appropriate in cancer therapy. It is well known that antitumor chemotherapy is usually associated with immunodeficiency that triggers off severe mycoses, and therefore the use of both drugs can simply be the necessity in some cases. The combination of visceral leishmaniasis and coinfections with HIV has also been a major problem.19 Both drugs have been reported to inhibit human immunodeficiency virus,7,13 and therefore their application together can be useful in HIV patients. Because of amphiphatic nature of both amphotericin B and LP analogues, these compounds are active on the level of cellular membrane. Namely, AmB has been reported to bind to membrane sterols thereby disrupting a bilayer by forming channels (transmembrane pores).20,21 Insufficient selectivity of AmB toward mammalian versus microorganisms membranes results in toxicity of amphotericin B to human cells.7,22-24 Moreover, the fact that the resistance of parasites to AmB is related to the modification of the membrane composition, leading to the decrease of membrane ordering,14 and that the antibiotic is able to modify the lipids organization in membrane25 proves that the AmB-membrane interactions are crucial for the activity of that polyene. The affinity to membranes is also characteristic of the lysophospholipid analogues. It has been found that LPs affect the membrane phospholipids proportion (e.g., miltefosine was reported to increase PC and decrease PE content) in parasites membrane26 and decrease cholesterol content in host macrophages membranes.27,28 Moreover, the reduction of the amount of unsaturated fatty acids in membrane is characteristic for parasites resistant to LPs.16 Additionally, both edelfosine and miltefosine are able to modify the lipid rafts organization in membranes.29,30 In view of all the above-mentioned potential applications of combined therapy involving polyene and LP analogues, it is interesting to analyze the interactions between both groups of drugs and to compare the effect of LP analogue and LP analogue/AmB mixture on model membrane properties. Since both amphotericin B and LPs are capable of floating monolayer formation,31-34 it is possible to apply for this kind of investigations the Langmuir monolayer technique, which is used for studying the interactions between membrane components and the influence of membrane active agents on membrane properties and organization.35,36 For our studies, we have selected edelfosine, ED (1-Ooctadecyl-2-O-methyl-rac-glycero-3-phosphocholine) as a representative of LP analogues that has been less frequently studied
Ha¸c-Wydro et al. in the context of its effect on membranes than miltefosine, however, it is more active against some Leishmania species as compared to miltefosine.37,38 Our paper is aimed at gaining insight into the effect of edelfosine on the interactions and properties of model sterol/ phospholipid erythrocyte and parasite membranes and also into the influence of AmB/edelfosine mixtures of various compositions on model membranes organization. The results allow us to verify, how the organization of model membrane is related to edelfosine activity and how the effect of the foregoing drug is modified by the presence of amphotericin B. 2. Experimental Section 2.1. Materials and Measurements. Cholesterol, dipalmitoyl L-R-phosphatidylcholine (DPPC), and amphotericin B (AmB) were products of high purity (>99%) supplied by Sigma. Edelfosine (ED) (g99.1%) was purchased from Biaffin GmbH & Co KG, Germany. Both the phospholipid and edelfosine have been dissolved in chloroform/ethanol 9:1 v/v mixture (p.a. POCh, Poland). The stock solutions of cholesterol have been prepared in chloroform, while amphotericin B was dissolved in N,N-dimethylformamide (p.a. POCh, Poland). From the respective stock solutions, their mixtures of desirable compositions were prepared. Spreading solutions were deposited onto the water subphase with the Hamilton microsyringe, precise to 1.0 µL. After spreading, the monolayers were left to equilibrate for 5 min before the compression was started with the barrier speed of 50 cm2/min. Measurements were performed with the NIMA (UK) Langmuir trough (total area ) 600 cm2) placed on an antivibration table. Surface pressure was measured with the accuracy of (0.1 mN/m using Wilhelmy plate made of filter paper (ashless Whatman Chr1) connected to an electrobalance. The subphase temperature (22 °C) was controlled thermostatically to within 0.1 °C by a circulating water system. The surface pressure (π)-area (A) isotherms were recorded for binary amphotericin B/edelfosine monolayers, for ternary cholesterol/DPPC/edelfosine mixtures, and for quaternary cholesterol/DPPC/amphotericin B/edelfosine mixed systems. To verify the effect of edelfosine and its mixtures with amphotericin B on model erythrocyte and parasite membranes, the cholesterol/ DPPC molar ratio in the studied mixed systems was always constant and reflected the sterols/phospholipids proportion in the respective natural membranes (0.9 and 0.1 for erythrocyte and parasite membranes, respectively).39,40 Cholesterol has been chosen as a dominant sterol in human membranes39 and one of the major sterols in parasites.16,26 Since in both erythrocyte and Leishmanias membranes phosphatidylcholines are a major class of phospholipids, we have used DPPC as representative phospholipids.26,39 Into the mixed cholesterol/DPPC monolayers of the composition mimicking erythrocyte and Leishmania membranes, edelfosine has been incorporated in various concentrations. In further experiments, amphotericn B and edelfosine mixed in proportions 1:9; 1:1, and 9:1 have been added into sterol/phospholipid model membranes. The concentrations of the respective drug mixtures in model membranes were equal to 1, 5, and 10%. 2.2. Data Analysis. From the surface pressure (π)-area (A) isotherms the excess Gibbs energy of mixing values (∆GExc) have been calculated according to eq 1 (for binary mixtures), eq 2 (for ternary mixed systems), and 3 (for quaternary films)
∆GExc ) N
∫0π (A12 - X1A1 - X2A2)dπ
(1)
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∆GExc ) N
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∫0π (A123 - (X1 + X2)A12 - X3A3)dπ (2)
∆GExc ) N
∫0π (A1234 - (X1 + X2)A12 - (X3 + X4)A34)dπ (3)
where N is the Avogadro’s number. A1, A2 and A3 are molecular areas of the respective component in their pure films at a given surface pressure, A12 and A34 are mean molecular areas in the respective cholesterol/DPPC film and in AmB/edelfosine mixture, respectively, A123 is the area per lipid in ternary monolayers, and A1234 is the area per lipid in quaternary monolayers. X1, X2, X3, and X4 are the mol fractions of components 1, 2, 3, 4 in the mixed film. The influence of the investigated drugs on model membranes ordering has been analyzed on the basis of the compression modulus values, calculated according to41
CS-1 ) -A(dπ/dA)
(4)
wherein A is area per molecule at a given surface pressure π. Results 1. The Interactions of Edelfosine with Erythrocyte and Parasite Model Membranes. Figure 1a,b presents the surface pressure-area isotherms for cholesterol/DPPC monolayers mimicking erythrocyte (a) and parasite (b) membranes incorporating edelfosine in different proportions. The isotherm for pure edelfosine monolayer is in a good agreement with the data already published, that is, edelfosine forms very stable mono-
Figure 1. The surface pressure-area (π-A) isotherms of mixed cholesterol/DPPC and edelfosine monolayers mimicking erythrocyte (a) and parasite (b) membranes.
Figure 2. The excess Gibbs energy of mixing (∆GExc) vs composition plots (XED) of mixed cholesterol/DPPC and edelfosine monolayers mimicking erythrocyte (a) and parasite (b) membranes at different constant surface pressures.
layers at the air/water interface of the liquid-expanded character and collapses at surface pressure of about 37 mN/m.34 The sterol/ phospholipid mixture imitating erythrocyte membrane contains higher amount of cholesterol as compared to the parasitemimicking system. This reflects in the shape and position of the foregoing mixtures isotherms. The π-A curve for the erythrocyte model membrane is steeper versus that of the parasite and lifts off at smaller molecular areas. The addition of edelfosine into cholesterol/phospholipid mixture induces a shift of the curves for the respective ternary monolayers toward the isotherm for pure ED film. Only the curves for XED ) 0.9 are located at larger areas than the isotherm for pure edelfosine monolayer. For erythrocyte model membrane system, two collapses appear in the course of the isotherms for monolayers containing higher proportion of edelfosine (XED g 0.5). First collapse is observed at π close to the collapse pressure for pure edelfosine film, while the other one is at the pressure similar to that for the investigated cholesterol/DPPC mixture. This suggests that the monolayer components are miscible in the pressure region below the collapse for edelfosine, and at the first collapse edelfosine molecules are being squeezed out from the mixed film. The excess Gibbs energy of mixing (∆GExc) values (Figure 2) calculated for the investigated monolayers are positive for the parasite model system and negative for erythrocyte mimicking mixtures, except for films rich in edelfosine (XED > 0.5), where the values are slightly positive. The negative values of ∆GExc suggest stronger attractions between molecules in ternary monolayers as compared to those existing in the model membrane and in pure edelfosine monolayer, while the positive values prove that the interactions in ternary mixtures are less attractive (or more repulsive) and the film is thermodynamically less stable as compared to cholesterol/DPPC monolayer. 2. Amphotericin B/Edelfosine Mixed Monolayers. The surface pressure-area isotherm for amphotericin B monolayer
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Figure 3. The surface pressure-area isotherms (a) and the excess Gibbs energy of mixing (∆GExc) vs composition plots (XED) (b) of mixed amphotericin B/edelfosine monolayers.
has a characteristic shape, that is, it exhibits a plateau region separating the liquid-expanded and liquid-condensed regions, which have been attributed to orientational changes of AmB molecules upon compression.31 There are slight differences in the area for the pressure lift-off and plateau surface pressure as compared to the isotherms for AmB published by other authors, which are due to different spreading solvents (3:1(v/v) mixture of DMF and HCl,32 2:1 chloroform/methanol42) or different source of the compound (Bristol-Mayor Squibb lab32 or Sigma42). The π-A isotherms together with ∆GExc versus the composition plots for binary amphotericin B/edelfosine monolayers are presented in Figure 3a,b. The incorporation of edelfosine molecules into AmB film provokes changes in the shape and position of the isotherms for the polyene monolayer. Most pronounced is the effect on AmB plateu region, which gradually vanishes upon increasing of edelfosine content in the mixed monolayer. The negative values of the excess Gibbs energy of mixing suggest stronger attractions (or weaker repulsions) between AmB and edelfosine in the mixed films than in the respective one component monolayers. The strongest interactions occur at low mol fraction of edelfosine (XED ) 0.1). 3. The Effect of Amphotericin B/Edelfosine Mixtures on Erythrocyte and Parasite Model Membranes. To verify the influence of mixtures composed of AmB and edelfosine on erythrocyte and parasite membranes, experiments have been performed in which both drugs mixed in various proportions (AmB to edelfosine 1:9; 1:1, 9:1) have been incorporated into model cholesterol/DPPC membranes. The concentrations of the respective mixtures in monolayers were 1, 5, and 10%. In Figures 4 and 5, the surface pressure-area curves recorded for the investigated films are presented. It is evident that the
Figure 4. The surface pressure-area isotherms of mixed cholesterol/ DPPC monolayers (model erythrocyte membrane) and amphotericin B/edelfosine mixtures of various composition.
addition of edelfosine-AmB mixtures into the model systems is of lower influence on the shape and position of the isotherms as compared to the effect induced by pure edelfosine (Figure 1). As can be observed in Figures 4 and 5 both for erythrocyte and parasite-mimicking membranes the isotherms are localized close to the curve for the respective cholesterol/DPPC monolayers. This may suggest a lower surface activity of edelfosine combined with AmB due to complex formation between both molecules. On the other hand, it should be pointed out that the concentration of drugs in the systems is rather low and therefore its effect on the course of the isotherm is less pronounced. In regards to the shape of the isotherms, only for erythrocyte membranes, containing AmB/edelfosine mixture of 9:1 proportion (Figure 4c), a characteristic plateau region appear. On the basis of the experimentally obtained isotherms, the excess Gibbs energy of mixing values for the studied systems have been calculated at π ) 30 mN/m. This surface pressure value has been chosen because the correlation has been found between the properties of the lipid film imitating membrane leaflets and the properties of a bilayer at surface pressures of 30-35 mN/ m.43 The results of the foregoing calculations for erythrocyte versus parasite membranes are presented in Figure 6a-c (for AmB/edelfosine mixture of 1:9; 1:1, and 9:1 in Figure 6a,b,c, respectively). At first glance, it is evident that the excess Gibbs energy of mixing values for parasite model membrane are
Combined Antileishmanian Therapy Involving AmB and ED
Figure 5. The surface pressure-area isotherms of mixed cholesterol/ DPPC monolayers (model parasite membrane) and amphotericin B/edelfosine mixtures of various composition.
positive, while the values for erythrocyte mimicking monolayers are slightly negative. The exception appears for the erythrocyte membrane containing 1:1 drug mixture in the highest studied proportion (10%). For such a mixture, the ∆GExc value is positive and comparable with that obtained for parasite membrane (Figure 6b). For the 9:1 AmB/edelfosine mixture, ∆GExc values increase with the content of the drugs in the mixed systems, while for the remaining drug mixtures a decrease of the excess Gibbs energy of mixing values can be noted. It should be pointed out that such a drop in ∆GExc values is markedly more pronounced for parasite as compared to the erythrocyte membrane. To analyze the influence of the drugs mixture on the condensation of model membranes, the compression modulus (Cs-1) values have been calculated and the values obtained at π ) 30 mN/m have been presented in Figure 7a,b. As can be observed in Figure 7a,b, the erythrocyte model membrane is more condensed than the parasite one, which is proved by higher values of compression modulus for the former system as compared to the latter. The strongest differences in the effect of the drugs on erythrocyte versus parasite model membrane condensation appear at a lower content of the drug mixture in the monolayer. For all the studied AmB/edelfosine mixtures at their 1% content, the compression modulus values for erythrocyte model membrane practically do not change, while for
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Figure 6. The excess Gibbs energy of mixing (∆GExc) for cholesterol/ DPPC (model erythrocyte or parasite membrane) and amphotericin B/edelfosine mixtures of various proportion vs the concentration of amphotericin B/edelfosine mixture in the system at π ) 30 mN/m.
the parasite membrane drastic increase of CS-1 values is observed. Similar situation appears for 1:9 mixtures at its 5% content in model membranes. For the remaining drug mixtures, a drop of the compression modulus values for the investigated monolayers can be observed. Discussion In this work, the influence of a representative alkyllysophospholipid-edelfosine and its mixtures with amphotericin B of various composition on cholesterol/phospholipid model membranes, mimicking erythrocyte and parasite membranes, has been investigated. Although amphotericin B and edelfosine are the first line drugs for fungal infections and cancer, respectively, however, they both prove also antiparasitic activity against various Leishmania species.6,9,12,14,38,44,45 Among various levels of action of these compounds, their affinity toward membrane should be stressed as a logical consequence of their amphiphatic character. As it has already been mentioned in the Introduction, amphotericin B is used in the treatment of parasitic infections; however, its application is limited by various side effects appearing during therapy and high cost of novel, less toxic formulation of this drug. The alkyl-lysophospholipids (edelfosine, miltefosine, perifosine) are much less toxic than AmB and
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Figure 7. The compression modulus (CS-1) values at π ) 30 mN/m for model erythrocyte (a) or parasite (b) membrane and amphotericin B/edelfosine mixtures of various proportion vs the concentration of amphotericin B/edelfosine mixture in the system.
were found to be exclusive agents against some of Leishmania infections (e.g., canine leishmaniasis).8 On the other hand, as proved in the tests on various parasite species15,37,44 the alkyllysophospholipids are of lower antileishmanian activity than amphotericin B. A promising way to combine the therapeutic properties of both drugs and to reduce the side effects during treatment is simultaneous application of alkyl-lysophospholipids and AmB. The investigations on the properties of polyenes and alkyl-lysophospholipids mixtures prove the increase of in vivo activity of miltefosine combined with AmB, strong interactions between the drugs and complex formation as well as solubilization of AmB aggregates by miltefosine above CMC of the latter antibiotic.17,18,44 The studies presented herein were aimed at verifying the influence of edelfosine on model erythrocyte and parasite membranes and analyzing the effect of AmB and edelfosine mixed in various proportions and incorporated in various concentrations into cholesterol/phospholipid monolayer. The Influence of Edelfosine on Model Erythrocyte and Parasite Membranes. The results obtained for erythrocyte model system prove that in the presence of a low amount of edelfosine the interactions in ternary mixed system are stronger than those between cholesterol and DPPC in binary film as well as between ED-ED in pure edelfosine monolayer. Although the absolute values of ∆GExc are not extremely large (∆GExc ) -900 J/mol at XED ) 0.3, π ) 30 mN/m), the fact that they are negative proves thermodynamic stability of the mixed system. For the parasite model membrane the values of ∆GExc are positive (at π ) 30 mN/m ∆GExc values achieve maximum at ∼600 J/mol), which suggest that the incorporation of edelfosine lowers the lipid-lipid interactions in the parasite membrane and from thermodynamical point of view, is unfavorable for cholesterol/DPPC model system. Differences in the effect of edelfosine on both investigated model membranes result from their different composition. It
Ha¸c-Wydro et al. has been found46 that edelfosine shows significant affinity to cholesterol and strong interactions occur between both molecules in the mixed system. Similar results have been found for miltefosine/sterol mixtures.47 Since the erythrocyte model membrane contains more sterol than that of the parasite, the cholesterol/edelfosine interactions significantly contribute into negative values of the excess Gibbs energy of mixing found for erythrocyte membrane/edelfosine system. Unfavorable effect of edelfosine on the parasite model membrane is connected with the state of the monolayer imitating the membrane system. As it is evident from the calculated compression modulus values (Figure 7), the parasite model membrane is less condensed and thus more accessible for edelfosine molecules than the erythrocyte model system. The foregoing conclusion is based on the results in which stronger affinity of miltefosine toward unsaturated phospholipids has been proved and easier insertion of this drug into less condensed lipid monolayer has been found.16,47 Summarizing, edelfosine molecules induce strongly unfavorable effect on parasite model membrane, which is more fluid than the erythrocyte model system containing higher amount of sterols. The results obtained for ternary cholesterol/DPPC/edelfosine monolayers are of great biological relevance. Both in parasite and erythrocyte membranes the percent of unsaturated fatty acids of the lipids is very similar (49-53%26,39,48). However, erythrocyte membranes contain higher proportion of sterol molecules, which make them less fluid than parasite membranes, in which the sterol/phospholipid proportion is low (cholesterol/phospholipid ratio ) 0.140). These differences in the fluidity of erythrocyte and parasite membranes seem to act as the natural mechanism of selectivity of alkyl-lysophospholipids. This is consistent with the fact that miltefosine molecules do not affect integrity of the erythrocytes membrane.47 Moreover, the membranes of parasites, which are resistant to miltefosine, are of lower content of unsaturated fatty acid (therefore are more ordered) as compared to miltefosine-sensitive Leishmania species.16 Since AmB, unlike edelfosine, has long been known and investigated, many papers have appeared so far, in which the behavior of this antibiotic in model membranes has been discussed. Moreover, in numerous experiments that have been performed, cholesterol/DPPC mixtures (of various proportion of lipids) served as a model of natural membrane for the analysis of the effect of AmB.49-55 In FTIR and DSC studies, it has been found that the incorporation of AmB into binary cholesterol/ DPPC bilayers exerts a perturbing effect on the lipids50 and destabilizes the organization of the phospholipid acyl chains.51 The influence of AmB on motional freedom of acyl chains has been also found in 1H NMR investigations.52 It has been also indicated that cholesterol is the compound perturbing the interactions between the antibiotic and DPPC in model system.51 Moreover, in UV-vis linear dichroism experiments on the behavior of polyenes in lipidic multilayers has been found that the presence of cholesterol in DPPC/AmB system influences the antibiotic orientation and its order degree even in gel phase of DPPC.54 The effect of AmB on cholesterol/DPPC membranes has also been examined in experiments based on AmB penetration into mixed monolayers.55 It has been suggested that the insertion of the drug molecules into cholesterol/DPPC monolayer depends on the composition of model membrane that determines its packing and the interactions between the sterol and phospholipids have been indicated as being crucial for AmB incorporation.
Combined Antileishmanian Therapy Involving AmB and ED The Influence of Edelfosine/Amphotericin B Mixture on Model Erythrocyte and Parasite Membranes. A starting point for the analysis of the effect of the mixtures of the investigated drugs on model membranes are studies on the interactions in edelfosine/AmB mixed monolayers. As proved by the negative values of the excess Gibbs energy of mixing, between the investigated drug molecules strong interactions exist in the mixed systems. These interactions are stronger even than cholesterol-DPPC interactions.56 The foregoing results may suggest that between ED and AmB the complexes are formed with the most stable at XED ) 0.1. The existence of stable complexes has also been found between AmB and DPPC.57 The complex formation between both drugs could be important from the point of view of the effect of AmB/ED mixtures on model membranes systems; the immobilization of drug molecules in the complex may decrease their influence on model membrane. From the comparison of the excess Gibbs energy of mixing values and the compression modulus values obtained for model membranes containing mixtures of drugs, some important conclusions on edelfosine versus edelfosine/AmB effect on erythrocyte and parasite membranes can be drawn. As far as the ∆GExc values are concerned, they are lower (more negative) for the erythrocyte membrane system than for the parasite membrane (one exception is 1:1 drug mixture of it is the highest proportion in the mixed monolayer). Similar differences have been found when edelfosine effect on model membrane was analyzed. However, the values of ∆GExc for erythrocyte membranes containing both edelfosine and amphotericin B are less negative than those for edelfosine-containing model erythrocyte membrane. Since the excess Gibbs energy of mixing values have been calculated in respect to the binary cholesterol/ phospholipid and AmB/edelfosine mixture, the results obtained for erythrocyte model membranes mean that in cholesterol/ DPPC/edelfosine/AmB films the interactions between molecules are only slightly more attractive as compared to those in a model membrane as well as between the drugs in their respective mixtures. The fact that ∆GExc values for lower content of AmB/ edelfosine mixtures (1 and 5% of 1:9, 1:1, and 9:1 mixtures) are rather low (do not exceed -155 J/mol) and that the compression modulus values found for the foregoing mixed systems are similar to those found for binary sterol/phospholipid model membrane allow concluding on rather low effect of the investigated drugs mixtures on model erythrocyte membrane. This may be a consequence of the formation of ED/AmB complexes, which contribute to the drop of the activity of the drugs when they are incorporated together. For parasite model membranes, the obtained results prove that in cholesterol/DPPC/AmB/edelfosine monolayers the interactions between molecules are less attractive (more repulsive) as compared to those in the parasite model membrane and the respective AmB/edelfosine mixture. Positive values of ∆GExc suggest also that the incorporation of both drugs is thermodynamically unfavorable and induces membrane destabilization. Analyzing both ∆GExc and compression modulus values it can be summarized that the strongest effect of the drugs mixture on parasite membrane properties occurs for 1:9 and 1:1 AmB/ edelfosine mixtures at their 1% content in model systems. It should be also pointed out that the foregoing drug mixed systems practically do not affect the properties of model erythrocyte membrane. Therefore, it is evident that for 1% content of 1:9 and 1:1 mixture of edelfosine and amphotericin B the strongest differences between their influences on model erythrocyte versus parasite membranes are observed. Interestingly, the values of ∆GExc obtained for the foregoing mixed monolayers are
J. Phys. Chem. B, Vol. 113, No. 43, 2009 14245 comparable to maximal values obtained for parasite model membrane/edelfosine monolayer (650 versus 600 J/mol for 1% content of 1:9 and 1:1 mixture of edelfosine and amphotericin B and for parasite model membrane/edelfosine at XED ) 0.5). The mol fractions of edelfosine in the parasite membrane/ edelfosine/AmB are 0.009 (for 1:9 drug mixture) and 0.005 (for 1:1 drug mixture). Thus, when edelfosine is combined with amphotericin B, a lower concentration of LP is required to induce similar effect on parasite membranes as exerts edelfosine alone. In this sense the influence of edelfosine on the parasite membrane increases in the presence of a small amount of amphotericin B. It is also interesting that erythrocyte model membranes are practically not affected by the mixtures addition, while the effect on parasite membranes is thermodynamically unfavorable. This can be associated with an easier insertion of AmB/ED complexes into more fluid parasite membrane as compared to the erythrocyte system. For drugs with mixed systems of higher proportion of amphotericin B (AmB/edelfosine ) 9:1), ∆GExc values increase with the content of the drugs in the monolayer both for erythrocyte and parasite model membranes. As it has been found (Figure 3) for the foregoing edelfosine/AmB proportion, the strongest interactions occur and the formed complexes are the most stable; however, the incorporation of the mixture dominated by large polyene molecules may disturb the organization of model membrane. Acknowledgment. K.H.-W. wishes to thank The Foundation for Polish Science for financial support. References and Notes (1) Rosen, M. J. Molecular interaction and synergism in binary mixtures of surfactants. In Phenomena in mixed surfactants systems; Scamehorn, J. F., Ed.; ACS Symposium Series 311; American Chemical Society: Washington DC, 1986. (2) Hoffman, H.; Po¨ssnecker, G. Langmuir 1994, 10, 381. (3) Tallarida, R. J. J. Pharmacol. Exp. Ther. 2001, 298, 865. (4) Barroso, P. A.; Marco, J. D.; Calvopina, M.; Kato, H.; Korenaga, M.; Hashiguchi, Y. J. Antimicrob. Chemother. 2007, 59, 1123. (5) McConville, M. J.; Handman, E. Int. J. Parasitol 2007, 37, 1047. (6) Murray, H. W. Antimicrob. Agents Chemother. 2001, 45, 2185. (7) Zotchev, S. B. Curr. Med. Chem. 2003, 10, 211. (8) Croft, S. L.; Coombs, G. H. Trends Parasitol. 2003, 19, 502. (9) Berman, J. D.; Hanson, W. L.; Capman, W. L.; Aliving, C. R.; Lopez-Berestein, G. Antimicrob. Agents Chemother. 1986, 30, 847. (10) Dupont, B. J. Antimicrob. Chemother. 2002, 49, 31. (11) More, B.; Bhatt, H.; Kukreja, V.; Ainapure, S. S. J. Postgrad. Med. 2003, 49, 101. (12) Azzouz, S.; Maache, M.; Gil Garcia, R.; Osuna, A. Basic Clin. Pharmacol. Toxicol. 2005, 96, 60. (13) Gajate, C.; Mollinedo, F. Curr. Drug Metab. 2002, 3, 491. (14) Mbongo, N.; Loiseau, P. M.; Billion, M. A.; Robert-Gero, M. Antimicrob. Agents Chemother. 1998, 42, 352. (15) Seifert, K.; Matu, S.; Perez-Victoria, F. J.; Castanys, S.; Gamarro, F.; Croft, S. L. Int. J. Antimicrob. Agents 2003, 22, 380. (16) Rakotomanga, M.; Saint-Pierre-Chazalet, M.; Loiseau, P. M. Antimicrob. Agents Chemother. 2005, 49, 2677. (17) Menez, C.; Buyse, M.; Besnard, M.; Farinotti, R.; Loiseau, P. M.; Barratt, G. Antimicrob. Agents Chemother. 2006, 50, 3793. (18) Menez, C.; Legrand, P.; Rosilio, V.; Lesieur, S.; Barratt, G. Mol. Pharmaceutics 2007, 4, 281. (19) Russo, R.; Nigro, L. C.; Minniti, S.; Montineri, A.; Gradoni, L.; Caldeira, L.; Davidson, R. N. J. Infect. 1996, 32, 133. (20) DeKruijff, B.; Demel, R. A. Biochim. Biophys. Acta 1974, 339, 57. (21) Mouri, R.; Konoki, K.; Matsumori, N.; Oishi, T.; Murata, M. Biochemistry 2008, 47, 7807. (22) Odds, F. C.; Brown, A. J. P.; Gow, N. A. R. Trends Microbiol. 2003, 11, 272. (23) Ha¸c-Wydro, K.; Dynarowicz-Ła¸tka, P.; Borowski, E.; Grzybowska, J. J. Colloid Interface Sci. 2005, 287, 476. (24) Falk, R.; Domb, A. J.; Polacheck, J. Antimicrob. Agents Chemother. 1999, 43, 1975.
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