Cyclodextrin-Mediated Recruitment and Delivery of Amphotericin B

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Cyclodextrin-Mediated Recruitment and Delivery of Amphotericin B Jia He, Christophe Jean Chipot, Xueguang Shao, and Wensheng Cai J. Phys. Chem. C, Just Accepted Manuscript • DOI: 10.1021/jp3128324 • Publication Date (Web): 10 May 2013 Downloaded from http://pubs.acs.org on May 14, 2013

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The Journal of Physical Chemistry

Cyclodextrin-Mediated Recruitment and Delivery of Amphotericin B Jia He,† Christophe Chipot,‡,§ Xueguang Shao,† Wensheng Cai†,*

College of Chemistry, Nankai University, Tianjin, 300071, P.R. China

Theoretical and Computational Biophysics Group, Beckman Institute for Advanced Science and Technology, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801

Équipe de Dynamique des Assemblages Membranaires, UMR 7565, Université de Lorraine, BP 239, 54506 Vandoeuvre-lès-Nancy cedex, France

* To whom correspondence should be addressed. E-mail: [email protected]. † Nankai University ‡

University of Illinois

§

Université de Lorraine 1

ACS Paragon Plus Environment


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Abstract The clinical use of amphotericin B (AmB), a polyene macrolide antifungal drug, is limited due to its poor bioavailability and pronounced cytotoxicity. Cyclodextrin (CD)-based drug carriers have proven to overcome these shortcomings. In the present contribution, the assembly of AmB with β-CD and γ-CD was investigated systematically using molecular dynamics simulations and free-energy calculations, showing that only the polyene macrolide ring could be included in CDs. The potentials of mean force (PMF) that delineate the process of the macrolide ring entering the cavity of a CD following two possible orientations were determined, revealing distinct inclusion modes for the two CDs. AmB was found to possess two sites within its prolonged macrolide ring where it will bind γ-CD, thereby forming stable complexes – one located at one end of the ring, the other close to the polar head of the drug. Conversely, the macrolide ring cannot enter the cavity of β-CD due to the limited available space. When AmB approaches γ-CD from its primary rim, the AmB:γ-CD complex corresponding to the first binding site was estimated to be energetically favored. Comparison of the free-energy landscapes characterizing the two CDs reveals that γ-CD possesses significantly higher binding affinity to AmB than β-CD, which may explain the experimental observation of their distinct ability to enhance the bioavailability of AmB. Moreover, decomposition of the PMFs into physically meaningful free-energy contributions suggests that van-der-Waals and electrostatic interactions constitute the main driving forces responsible for the formation of the CD inclusion complexes.

Keywords: amphotericin B · cyclodextrin · molecular dynamics simulations · free-energy calculations

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Introduction Amphotericin B (AmB), a popular polyene antifungal drug, has been widely used in the clinical treatment of deep-seated fungal infections. The medical importance of this drug results mainly from its high fungicidal activity, broad antimicrobial spectrum and ability to overcome multidrug resistance.1-2 Nevertheless, it does not meet the criteria required for a good chemotherapeutic agent owing to its toxicity for kidney and low bioavailability.3-4 There are by and large two classes of approaches followed to overcome these shortcomings, namely the design of rational second generation of AmB, or suitable drug delivery systems (DDSs). The discovery of new biologically active derivatives that can be exploited therapeutically to treat disease has, however, stalled, with fewer drugs entering the market every year. In contrast, cyclodextrin (CD)-based DDSs, such as CDs, CD-based copolymers and lipid formulations,5-18 have proven to be a versatile and multifaceted platform for the delivery of AmB. From this point, it is necessary to understand how CDs and AmB recognize and associate.

Delivery of AmB by carriers is evidently an intricate question, which involves a number of steps: i) recruitment of AmB, ii) delivery of AmB in the interfacial environment, iii) release of AmB, iv) association of AmB with sterols in the membrane to form binary complexes, which may subsequently assemble into a barrel-stave channel. CDs have been envisioned to hold great promises as drug carriers. Their function depends intimately on the three-dimensional structure of the CD-drug complexes, while intermolecular interactions strongly affect the conformational preference. On the way towards CD-based AmB delivery systems, a complete understanding of the intermolecular interactions is, therefore, desirable. In this contribution, we focus on the recruitment and the delivery capability of AmB.

On the experimental front, some efforts have been invested to develop new CD-DDSs, which effectively overcome the side effects of AmB. The liposomal AmB formulations with 2-hydroxypropyl-β-CD (HP-β-CD) 3



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and Sulfo butyl ether-β-CD (SBE-β-CD) were found to be significantly less toxic than free AmB or conventional liposomal AmB.5-7 Micelles composed of β-CD-entrapped triblock copolymers with polyrotaxane as a middle block, which varied with the number of β-CD molecules, have also proven to be effective DDSs for the controlled release of AmB and reduction of its toxicity.8-9 On the other hand, experiments show that AmB self-association is responsible for its antibiotic toxicity, while its monomeric form is less toxic.19 Thus, in addition to the controlled release of AmB, formation of AmB-CD complexes may also prevent aggregation of AmB and accordingly reduce its toxicity. The physicochemical properties of AmB-CD complexes have also been investigated.10-18 Formation of AmB:HPγ-CD inclusion complexes was confirmed using different techniques16 and the stability constant and the complexation efficiency of AmB:γ-CD complex and its derivatives in 1:1 and 1:2 stoichiometries were measured through phase-solubility diagram.10 Improvement of aqueous solubility and bioavailability was witnessed in CD-mediated DDSs. However, this phenomenon was not significant for β-CD or its derivatives.16-18 In light of these observations, the following questions need to be addressed. Can AmB assemble with β-CD or its derivatives? If so, why can β-CD or its derivatives not improve significantly the bioavailability of AmB? If not, why can the DDSs based on β-CD or its derivatives be used for reducing toxicity and controlled release of AmB? To answer these questions, a number of key aspects hitherto only partially examined, like the assembly mechanism, the structural stability of the complexes, and driving force responsible for recognition and association of AmB with β-CD and γ-CD ought to be investigated in great detail.

Yet, detailed structural information of the AmB:CD complex is still fragmentary. A number of key issues remain to be addressed, like the driving forces responsible for complexation, or the underlying free-energy change delineating the reaction, which ought to be investigated in greater detail and are admittedly difficult to attain employing conventional experimental methods. Theoretical approaches have proven to constitute an appealing route to gain atomic-level insights into the association mechanism, for example, investigation of 4


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the assembly of drug:CD complexes.20-21 In previous study, theoretical investigation of AmB is mainly focused on the formation of the putative AmB/sterol complexes in a lipid bilayer.22 In the present contribution, molecular dynamics (MD) simulations were used to dissect the assembly of AmB with β-CD and γ-CD at an atomic resolution. The free-energy profiles for AmB:β-CD and AmB:γ-CD assembly following different orientations of the guest with respect to the host were determined employing time-dependent biases acting on collective variables. The preferred assembly modes and the underlying mechanism for the AmB:CD complexes are unveiled here. The present set of results provides a cogent rationalization of the aforementioned experimental observations.

Method Molecular Models. The initial coordinates of AmB and β-CD/γ-CD were based on a crystal structure23 and a previously published work24, respectively. As shown in Scheme 1, due to the “Y-shaped” structure of AmB, there are three potential binding sites, the aminosugar moiety (A), the carboxylic acid moiety (B), and the macrolactone moiety (C), to which the cavity of CD can possibly bind, hence forming different inclusion poses of 1:1 stoichiometry. Two possible orientations, namely the side of the primary or the secondary hydroxyl groups of CD facing AmB, were considered, yielding altogether six possible inclusion poses. Due to spatial mismatch, inclusion of moieties A and B at the primary rim was ruled out. As a result, four remaining possible inclusion poses were investigated. The corresponding initial configurations were thus constructed, as illustrated in Scheme 2. These supramolecular assemblies were subsequently immersed in a periodic box of TIP3P25 water using the VMD package,26 with a margin of at least 8 Å from each edge of the box to any atom of the host:guest complex.

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Scheme 1: Structure of AmBa and CDb

a

Three possible inclusion sites, the aminosugar moiety, the carboxylic acid moiety, and the macrolactone

moiety, are labeled “A”, “B” and “C”. The selected atoms (labeled C1, C2, C3, C4, C5, C6, C7, O8, C9, C10) constitute the reference subset of atoms of AmB. b

β-CD: n=7, γ-CD: n=8.

Molecular Dynamics Simulations. All the atomistic MD simulations described herein were performed using the parallel, scalable MD program NAMD 2.8.27 The carbohydrate solution force field (CSFF) and the multipurpose, macromolecular CHARMM27 force field were used to describe inter- and intramolecular interactions.28 The parameters and charges of the aminosugar and carboxylic acid moiety of AmB were inferred from that of the hexopyranose monosaccharide.29 Chemical bonds involving hydrogen atoms were constrained to their experimental lengths by means of the Shake/Rattle algorithms.30-31 The r-RESPA multiple time-step algorithm was employed to integrate the equations of motion with a time step 2 and 4 fs for short- and long-range interactions, respectively.32 The temperature and the pressure were maintained at 300K and 1 atm, respectively, using Langevin dynamics and the Langevin piston method.33 Long-range electrostatic forces were taken into account by means of the particle mesh Ewald (PME) algorithm.34 A 14-Å cutoff was introduced to truncate van der Waals interactions. Periodic boundary conditions (PBCs) were applied in the three directions of Cartesian space. For each solvated system, the potential energy was minimized using up to 5000 steps of conjugate gradient (CG) with fixed solutes, prior to an additional 6


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geometry optimization of the complete molecular assembly involving an equal number of CG steps. A 2-ns MD trajectory was subsequently generated with weak harmonic restraints - viz. 1.0 kcal/mol-1/Å2 - enforced on the solute. All applied restraints were then removed barring that acting on the longitudinal axis of the macrolactone moiety to maintain it roughly collinear with the z direction to and, thus, avoid spurious interactions of AmB with its images. The ensuing 10-ns MD simulations were carried out to explore the putative binding poses. Visualization and analysis of the MD trajectories were performed with the VMD package.26

Scheme 2: Schematic Representation of the Initial Structure for the Four Possible AmB:CD Inclusion Posesa

a

The aminosugar moiety A (site A), and the carboxylic acid moiety B (site B) inserted in the CD cavity. The

tail of the macrolactone moiety placed in the cavity of CD from its secondary face (site C(I)), from its primary face (site C(II)).

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Free-Energy Calculations. Although AmB can bind to the host CD at three possible locations, our MD simulations show that only the polyene macrolide ring could be included by β-CD or γ-CD. Only the corresponding complexation processes were, therefore, investigated using free-energy calculations. The potentials of mean force (PMF) delineating host:guest association were determined along the model reaction coordinate, ξ, defined as the projection onto the z axis of the distance between the center of mass (COM) of selected atoms of the macrolactone and that of the CD glycosidic oxygen atoms (see Scheme 1 and 3). To obtain such profiles, the adaptive biasing force (ABF) method implemented with the collective variables module of NAMD was utilized.35-40 The center of the CD was maintained at the origin of the coordinate system with the axis of revolution of the central cavity aligned with the z axis. To achieve this goal, the glycosidic oxygen atoms were restrained by means of weak harmonic potentials with a force constant of 1.0 kcal/mol/Å2. The longitudinal axis of the macrolactone moiety of AmB was arbitrarily enforced to remain collinear with the z axis. To increase the efficiency of the calculations, the reaction pathway was broken down into 2-Å wide consecutive windows. Instantaneous values of the force were stored in 0.1-Å wide bins. For each window, a trajectory of at least 5 ns was generated. To eliminate possible non-equilibrium phenomena in the initial configurations 2-ns equilibrium MD simulations were first carried out. The resulting equilibrated molecular assemblies were then used as a starting point for the ABF calculations. The detailed information about studied systems was given in Table S1.

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Scheme 3: Two orientations of AmB Entering the CD Cavity

Results and Discussion Putative Binding Sites of AmB. To investigate the stability and the structural features of the host:guest complexes, 10-ns MD simulations were performed for the different systems depicted in Scheme 2. From the analysis of the trajectories generated, the variations of distances between AmB and CD are gathered in Figure 1. It can be seen that only the inclusion of the polyene macrolide ring into β-CD or γ-CD (sites C(I) and C(II)) is possible, while the other two binding sites, A and B, lead to unstable complexes. Representative snapshots from the trajectories indicate that AmB at locuses A and B, both for β-CD and γ-CD, are quickly displaced from the CD cavity (details are provided in the Supporting Information). Under these premises, AmB is prevented from forming inclusion complexes with CD mainly because of the high hydrophilicity of the charged aminosugar or carboxylic acid moiety.

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Figure 1. Variations of the COM distance, d, between the CD and moiety A (black), moiety B (red), the selected atoms (Scheme 1) of the macrolactone moiety (blue and green), corresponding to the four possible AmB:CD inclusion poses (Scheme 2), respectively.

Free-Energy Profiles. In the light of the above observations, only the inclusion processes wherein the macrolactone moiety of AmB approaches the cavity of CD were explored further using free-energy calculations. Two possible orientations were considered, as depicted in Scheme 3. The PMFs characterizing the entry of the guest molecule in the CD cavity were determined and are gathered in Figure 2. Note that the PMFs were truncated when sampling of interest became insufficient, thus, leading to different ranges of ξ explored for β-CD and γ-CD.

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Figure 2. Free-energy profiles for the inclusion of AmB with (A) β-CD and (B) γ-CD along the model reaction coordinate, ξ, in two orientations. The PMFs were truncated when sampling of interest became insufficient, therefore, leading to different ranges of ξ explored for β-CD and γ-CD.

For AmB:β-CD, the PMFs in both orientations exhibit only one free-energy minimum. The structures of the AmB:β-CD complexes around the minima are depicted in Figure 3, wherein only the substitutes located at the tail of the macrolactone moiety are included in the β-CD cavity. It is apparent that orientation I is energetically favored, which can be ascribed to the larger size of the secondary rim compared to the primary one. Inclusion from orientation II is impossible, as might be inferred from the virtual absence of a minimum in the free-energy landscape.

Figure 3. Snapshots of the inclusion complex of AmB with β-CD near the global minimum of the PMF. (A) ca. ξ= -5 Å, in orientation I, (B) ca. ξ= -6Å, in orientation II. 11



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In the case of AmB:γ-CD, the PMFs obtained for the two orientations have a very similar shape, and both possess two marked local minima. As shown in Figure 4, the molecular structures corresponding to these minima are also very similar for the two orientations. For the structures representative of the first minima (Figures 4(A) and (C)), the tail of the macrolactone moiety lies across the central plane of γ-CD, and its scaffold is included in the cavity at an earlier stage of the insertion process. These features are in agreement with the aforementioned equilibrium MD simulations. The structural arrangements for the second free-energy minima (Figures 4 (B) and (D)) correspond to a deep inclusion of CD with the scaffold of macrolactone moiety, wherein the aminosugar and the carboxylic acid moiety reach neither the primary nor secondary rim of γ-CD and are still exposed to the aqueous environment. Comparing the free-energy profiles, it is interesting to find that the most energetically favored inclusion mode is the association at the first binding site, rather than the deeper one at the second binding site as was initially hypothesized – although the preference is less marked, as will be detailed hereafter.

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Figure 4. Snapshots of the inclusion complex of AmB with γ-CD at the inflection points of the PMF. In orientation I, (A) ca. ξ= 1 Å, near the first minimum, (B) ca. ξ= 12 Å, near the second minimum. In orientation II, (C) ca. ξ= 0 Å, near the first minimum, (D) ca. ξ= 12 Å, near the second minimum.

Examining further the free-energy profiles for the two CDs, it is worth noting that γ-CD possesses significantly higher binding affinity towards AmB than β-CD. This remarkable difference in the propensity of the two species to associate combined with the structural features of the inclusion complexes can rationalize the experimental observation that γ-CD and its derivatives can dramatically enhance the bioavailability of AmB, whereas β-CD does not share this property.16-18 From a theoretical perspective, to obtain an accurate prediction of standard binding free energies, which can be compared with experimental values is by and large a difficult endeavor. During the simulations, geometrical restraints are used to ensure that association can be observed on accessible time scales, which limit the freedom of the drug molecule to move and deform. These restraints effectively reduce configurational entropy, which necessarily impacts the net binding free energy. In their study, Gumbart, 13



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Roux and Chipot,41 demonstrate that the removal of conformational, orientational and positional entropies by means of geometrical restraints should be accounted for to predict with utmost accuracy standard binding free energies. The calculations reported here, however, do not account for the loss of configurational entropy arising from the restraints coupled to the chosen reaction coordinate. This is based on the consideration that the main thrust of the present work is to obtain the relative stability of different binding modes.

Driving Forces Responsible for Assembly. The driving force responsible for the formation of the host:guest complex constitutes a crucial issue in CD inclusion studies. To address this point, physically meaningful free-energy contributions were measured by partitioning the total average force acting along ξ into three main terms, viz. van-der-Waals AmB:CD, electrostatic AmB:CD and AmB-solvent components, and integrating these forces separately. The resulting contributions to the free energy are gathered in Figure 5. Following the above discussion and owing to the low affinity of AmB to β-CD in orientation II, the corresponding decomposition of the PMF is not reported.

As shown in Figure 5A, for AmB:β-CD, moving from the initial location of the guest to the global minimum, ca. -5 Å, both van-der-Waals and electrostatic interactions appear to decrease. This behavior is suggestive that the associated forces drive in a cooperative fashion the formation of the inclusion complex, wherein van-der-Waals contributions are the primary component of the binding free energy. As the AmB moves along the reaction pathway, the free energy increases on account of marked steric hindrances caused by the scaffold of the macrolactone moiety and the limited space available in the β-CD cavity.

For AmB:γ-CD, as shown in Figures 5B and 5C, moving from the initial location of the guest to the second local minimum around 12 Å, in both orientations, van-der-Waals and electrostatic interactions appear to decrease, suggesting that these nonbonded forces constitute the main driving forces responsible for the formation of the γ-CD inclusion complex at the first and the second binding sites, albeit van-der-Waals 14


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interactions are shown to be predominant. The increase of the free energy between the two local minima is primarily caused by AmB-solvent interactions. Past the second minimum, the subsequent free-energy barrier results mainly from an increase of the AmB:γ-CD van-der-Waals contribution. The steric hindrances of the aminosugar and the carboxylic acid moiety eventually prevent AmB from squeezing through the cavity of γ-CD.

To appreciate the effect of the solvent on CD-solvent interactions, the number of hydrogen bonds between AmB and water molecules along the model reaction coordinate was monitored (see the Supporting Information). The interaction of AmB and the solvent steadily decreases in both orientations, which means that the solvation shell of the polar head is disrupted as the latter approaches the torus of CD. Thus, when the CD slides along the prolonged polyene macrolide ring, the AmB-solvent contribution appears to increase steadily.

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Figure 5. Partition of the PMFs into van-der-Waals AmB:CD, electrostatic AmB:CD, and CD-solvent contributions. (A) AmB:β-CD in orientation I, (B) AmB:γ-CD in orientation I, (C) AmB:γ-CD in orientation II.

Structural Analysis of the Inclusion Complexes. To investigate the structural features of the AmB:CD complexes at the minima of the free-energy profiles, additional 10-ns MD simulations were performed bereft of geometric restraints. For the reasons discussed previously, the AmB:β-CD complex in orientation II was not considered. Analysis of the MD trajectories of AmB:β-CD in orientation I shows, by comparison with 16


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the structure in Figure 3A, that the scaffold of the macrolactone moiety is somewhat titled with respect to the principal axis of the CD (see Figure 6A). Most of the hydroxyl groups of the AmB are still exposed to the aqueous environment. Furthermore, the intermolecular hydrogen bonds were analyzed and revealed that one to four hydrogen bonds formed between the macrolactone moiety and the β-CD (see Figure 6B). Based on these structural features, it could be conjectured that, in drug delivery systems designated for AmB, like liposomal drug:CD5-7 or micelles composed of β-CD-entrapped copolymers8-9 , one loaded AmB molecule may recognize and associate with the hydroxyl groups of multiple β-CDs simultaneously through hydrogen-bonding interactions The latter point could rationalize the experimental observation that the loading capacity of AmB increases with the number of entrapped β-CDs.8-9

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Figure 6. (A) Equilibrium structure of the AmB:β-CD complex after 10 ns (up), hydrogen bonds formed between AmB and β-CD (down). (B) Time evolution of the averaged number of hydrogen bonds between the AmB and β-CD obtained from the additional MD simulation of the complex at the minima of the free-energy profiles in orientation I. The hydrogen-bonding criterion is the angle O-H···O>135° and the distance O···O

Cyclodextrin-Mediated Recruitment and Delivery of Amphotericin B

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