Segura‐Sanchez et al. J. Mol. Recognit. 2009 ; 22 :232‐241 DOI :10.1002/jmr.936

Elucidation of the complexation mechanism between (+)-usnic acid and cyclodextrins studied by isothermal titration calorimetry and phase-solubility diagram experiments Freimar SEGURA-SANCHEZa,c, Kawthar BOUCHEMALa*, Geneviève LEBASa, Christine VAUTHIERb, Néréide S. SANTOS-MAGALHAESd, Gilles PONCHELa a. Université Paris Sud-11, UMR CNRS 8612, 92296 Châtenay-Malabry France b. CNRS UMR 8612, Université Paris Sud-11, 92296 Châtenay-Malabry France c. Departamento de Farmacia, Facultad de Química Farmacéutica, Universidad de Antioquia, Medellín, Colombia d. Universidade Federale de Pernambuco (UFPE), Instituto Keizo-Asami, Departamento de Bioquímica, Laboratório de Produtos Naturais, Recife, PE, Brazil In the present work the complexation mechanism between (+)-usnic acid and cyclodextrins (CDs) has been investigated by isothermal titration calorimetry (ITC) and phase-solubility diagrams using pH as a tool for modifying the molecule ionization. ITC experiments have been employed to evaluate the stoichiometry of interaction (N), affinity constants (K) and thermodynamic parameter variation associated with complexation between (+)-usnic acid and α-, β-, HP-β-, SBE-β- and γ-CD. It was shown that (+)-usnic acid did not interact with α-CD and tended to interact more favorably with γ-CD (K=1030 M-1, ∆G=-17.18 kJ.mol-1), than β-CD (K=153 M-1, ∆G=-12.46 kJ.mol-1) forming 1:1 complexes. It was also demonstrated using ITC and solubilization experiments that chemical modifications of the parent β-CD resulted in stronger and more spontaneous interactions (K=281 M-1, ∆G=-13.97 kJ.mol-1, for SBE-β-CD and K=405 M-1, ∆G=-14.87 kJ.mol-1 for HP-β-CD). Analysis of the thermodynamic data suggested that van der Waals forces and hydrogen bonds were responsible for the formation of complexes with a predominance of van der Waals forces. Finally, pH induced modifications of (+)-usnic acid ionization provided important informations relative to the topology of the interaction between (+)-usnic acid molecule and the γ-CD cavity, which were confirmed by molecular modeling. Key words. Isothermal titration calorimetry, complexation thermodynamics, cyclodextrins, (+)-usnic acid, solubilization studies. *Corresponding author: kawthar.bouchemal@u-psud.fr

1. Introduction The antitumour activity of usnic acid -which is the most interesting biological activity of this molecule- was first reported more than two decades ago [Takai et al., 1979]. Among several lichen constituents, (+)-usnic acid enantiomer exhibits a stronger tumours inhibitory activity than that of (-)-enantiomer [Yamamoto et al., 1995]. Nevertheless, practical use of this molecule in anticancer therapy has been rather limited due to its poor solubility in water (less than 0.1 mg.ml-1 at 25°C) [Andrade et al., 2006]. Since solubility improvements represent a serious challenge when foreseeing biomedical applications of (+)-usnic acid, it was the aim of this work to investigate the possibility for (+)-usnic acid to interact with cyclodextrins (CDs) and to determine the thermodynamic characteristics of their complexes.

Complexation of poorly water-soluble drugs with cyclodextrins represents an interesting strategy for increasing their apparent water solubility [Duchêne et al., 1999] and offers further possibilities for their formulation [Loftsson and Duchêne, 2007]. Shaped as a hollow truncated cone, cyclodextrins are cyclic oligosaccharides of D-(+) glucopyranose units obtained from degradation of starch by enzymes produced by Bacillus macerans. The D-(+) glucopyranose units, all in chair conformation, are linked by α-(1,4) glucosidic bonds conferring to cyclodextrins the shape of a truncated cone, with the outer side formed by the secondary 2- and 3-hydroxyl groups and the narrow side by the primary 6-hydroxyl groups. Thanks to this conformation, cyclodextrins have lipophilic inner cavities and hydrophilic outer surfaces. So far, the most probable mode of binding involves the interaction of the less polar region of the guest molecule with the cyclodextrin cavity, while the more polar -and often charged- groups of the guest are exposed to the bulk solvent outside the wider opening of the cavity [Rekharsky and Inoue, 1998; Rekharsky and Inoue, 2006]. Knowledge of the binding constants and the thermodynamic parameters of the interaction are of central importance for understanding the phenomena of molecular recognition of a guest with cyclodextrins. Nowadays, a great deal of efforts has

Segura‐Sanchez et al. J. Mol. Recognit. 2009 ; 22 :232‐241 DOI :10.1002/jmr.936

Indeed, in spite of numerous works describing the effect of the pH medium on the solubilization of (+)-usnic acid by cyclodextrins [Kristmundsdóttir et al., 2002; Kristmundsdóttir et al., 2005], the pH-dependence of the thermodynamic quantities has not been yet defined in the literature. However, since (+)-usnic acid is an ionizable drug presenting three pKa values, the presence of electric charges may play a significant role in drug-cyclodextrin complexation because of ionization modifications of the molecule. Therefore, the ionization degree of (+)-usnic acid was varied by modifying the pH of the medium and these variations were used as a tool for getting further insight to the interaction process. ITC experiments combined with molecular modeling studies allowed to investigate the nature of the interaction forces involved in the complexation process. MATERIALS AND METHODS Reagents (+)-usnic acid (UA), 98% was purchased from Sigma Chemical Company (France), β-CD from Fluka Chemical Company (France), HP-β-CD from Acros Organics (Belgium), α-CD and γ-CD were a gift from Wacker-Chemie (Germany) and SBE-β-CD was a gift from CyDex Pharmaceuticals, Inc (USA). All chemicals were of analytical or reagent grade and were used without further purification. Solutions were prepared by weight using MilliQ® water (Millipore, France). Phosphate buffer solutions 0.05M at pH 7, 7.4 and 8 were prepared according to the methodology described by the US Pharmacopeia 28th edition.

been devoted to understand the thermodynamics of the complex formation between molecules and cyclodextrins [Flamigni, 1993; Inoue et al., 1993; Zia et al., 2000]. This can be achieved by varying different environmental conditions and by using a wide variety of experimental methods including several types of spectroscopic techniques, 1H-NMR, fluorescence, solubilization studies and calorimetry. From the various techniques mentioned above, high sensitivity titration calorimetry is the most modern and sensitive method available at the present time for the determination of thermodynamics of host-guest interactions [Bouchemal, 2008]. ITC helps to determine whether an association process occurs and allows the evaluation of the stoichiometry of the interaction (N), the affinity constant (K), and the enthalpy (∆H) of the interaction from which the entropy (∆S) and the Gibbs free energy of the process (∆G) can be derived [Bouchemal, 2008; Denadai et al., 2007; Fini et al., 2004; Harries et al., 2005; Liu et al., 2008; Sun et al., 2006]. In the work to be presented here, phase-solubility diagrams and ITC experiments were used to investigate (+)-usnic acid solubility enhancements and the thermodynamics of its complexation in aqueous medium by varying the cyclodextrin cavity size (using natural α-, β- and γ-CD), and by using highly water-soluble chemically substituted cyclodextrins [hydroxypropyl-β-CD (HP-β-CD), and (sulfobutylether-β-CD (SBE-β-CD)] (table 1). Because (+)-usnic acid is a lipophilic molecule, which can be partly ionized, interactions with CDs are expected to be rather complex when compared to the case of non ionizable lipophilic molecules. Therefore, the aim of the study was to get further insight into the mechanism of complexation by varying the size of the cyclodextrin cavity, the hydrophilicity of the cyclodextrins and the ionization of the (+)-usnic acid molecule.

Segura‐Sanchez et al. J. Mol. Recognit. 2009 ; 22 :232‐241 DOI :10.1002/jmr.936

Solubilization assays In the aim to investigate the effect of cyclodextrins on the solubility of (+)-usnic acid, an excess of commercial (+)-usnic acid was placed in a glass vial (10 ml) with 2 ml of solubilizing agent that was a solution of α-, β-, HP-β-, SBE-β- or γ-CD in concentrations ranging from 1.5% to 40% w/w (except for α- and β-CD which possess low water solubility: 14.4 and 1.85% w/w respectively). The pH of all these cyclodextrin solutions has been adjusted to 8 using phosphate buffer 0.05M. The effect of (+)-usnic acid ionization on its apparent solubility in a solution of γ-CD 1.5% w/w has been studied by varying the pH of the medium (7, 7.4 and 8). The pH of each solution was adjusted with phosphate buffer 0.05M. In all cases, the vials containing the (+)-usnic acid and a solution of cyclodextrin were kept in a shaker for 108 hours at 25°C, protected from light. This duration was estimated to be long enough for reaching the equilibrium of complexation. Then, the samples were filtered through a 0.22µm membrane filter (Millex, SLAP 0225, Millipore, France) and after appropriate dilution with a suitable buffer solution, (+)-usnic acid concentration in the filtrate was measured spectrophotometrically at 290nm. It was analytically verified that the presence of cyclodextrin did not interfere with the assay. Each experiment was carried out in triplicate. Preparation of (+)-usnic acid solution for ITC experiments For ITC experiments, solutions of (+)-usnic acid were prepared as follows. An excess of commercial (+)-usnic acid (25mg) was placed in a glass vial together with 50ml of phosphate buffer (0.05M) under magnetic stirring during 1 week at a temperature of 37°C. A yellowish suspension was obtained and filtered through a 0.22µm membrane filter (Millex, SLAP 0225, Millipore, France). After appropriate dilution with a suitable buffer solution, (+)-usnic acid concentration in the filtrate was measured spectrophotometrically at 290nm. For all CDs (α-, β-, HP-β-, SBE-β- and γ-CD), the pH was 8 and for γ-CD, additionally pH 7 and 7.4 were used. Isothermal titration calorimetry (ITC) experiments An isothermal calorimeter (ITC) (MicroCal Inc., USA) has been used for determining from a single titration curve simultaneously the enthalpy of the interaction between (+)-usnic acid and cyclodextrins and when appropriate equilibrium constant corresponding to the formation of a complex between those species. The ITC instrument was periodically calibrated either electrically using an internal electric heater, or chemically by measuring the dilution enthalpy of methanol in water. This standard reaction was in excellent agreement (1-2%) with MicroCal constructor data. As a titrant solution, various solutions of cyclodextrins were used, including α-, β-, HP-β-, SBE-β- and γ-CD at molar concentrations of 14.16mM, 13.97mM, 19.76mM, 19.75mM, and 19.69mM, respectively. The pH of these solutions was adjusted to 8. The influence of the pH on the interaction of (+)-usnic acid with γ-CD (19.69mM) has been determined at pH 7, 7.4 and 8. In a typical experiment, a syringe filled with 283µl of a cyclodextrin solution, was used to titrate a solution of (+)-usnic acid into the calorimetric cell accurately thermostated at 298K. Experimental solutions were degassed before each titration. Typically, aliquots of 5µl of titrant (cyclodextrin solution) were delivered over 25s and the corresponding heat flow was recorded as a function of time. Intervals between injections were 120s and agitation speed was 394 rpm. A background titration, consisting in injecting the same

cyclodextrin solution in solely the buffer solution placed in the sample cell, was subtracted from each experimental titration to account for dilution effects. Data consisting in series of heat flows were collected automatically and when appropriate, the interaction process between the two species has been analysed by the mean of either a one-site or two-site binding model proposed in the Windows-based Origin 7 software package supplied by MicroCal. Based on the concentrations of the titrant and the sample, the software used a nonlinear least-squares algorithm (minimization of Chi2) to fit the series of heat flows (enthalpograms) to an equilibrium binding equation, providing best fit values of the stoichiometry, binding constant and change in enthalpy. 2.5. Molecular modeling For a better understanding of the interaction between (+)-usnic acidand γ-cyclodextrin, molecular modeling was used considering the UA molecule in its ionized form. The γ-CD structure was taken from Martin Chaplin web page from London South Bank University [Chaplin, 2008]. The ionized structure of (+)-usnic acid according to table 2 has been drawn and presented to the γ-CD molecule. Further, the dreiding force field was minimized with the software DS ViewerPro 6.0 (Accelrys Software Inc) leading to the more likely supramolecular assembly. Bump monitorization, minimization of the dreiding force field [Mayo et al., 1990] and molecular rendering (solvent accessible surface) were also achieved with DS Viewer Pro 6.0 in the case of these optimized structures.

Segura‐Sanchez et al. J. Mol. Recognit. 2009 ; 22 :232‐241 DOI :10.1002/jmr.936

1.5% w/w. From results presented in figure 2 it was concluded that a limited pH increase from 7 to 8 had a dramatic effect on the solubilization enhancement of the (+)-usnic acid. Indeed, solubility was increased 10 folds (from 0.08, to 0.85 mg.ml-1) when pH was only raised from 7 to 8 using γ-CD. Obviously, this effect was not due to modification of (+)-usnic acid ionization as the effect of a pH increase on (+)-usnic acid solubility was very limited in absence of γ-CD (figure 2). 3.2. ITC experiments A typical ITC titration curve corresponding to the binding interaction of (+)-usnic acid and γ-CD is resented in figure 3.a. Exothermic heat flows which were released after successive injections of 5µL aliquots of cyclodextrins into a solution of (+)-usnic acid were integrated and expressed as a function of the molar ratio between the two reactants. Data were corrected from dilution effects and led to differential binding curves (figures 3.b, 4 and 5). Fitting to different models corresponding to various stoichiometries of interaction were tested. The best fit of the binding curve was obtained when using the standard one-site binding model (1:1) leading to the direct determination of the

Figure 2. Effect of pH on the apparent solubility of (+)-usnic acid in presence of 1.5% (w/w) γ-CD at 298 K (258C) (n=3). Control was treated in similar conditions, except the presence of γ-CD.

Figure 1. (a) Experimental phase solubility diagram of (+)-usnic acid for several types of cyclodextrins at pH 8 (n=3). (b) Typology of phase solubility diagrams according to Higuchi and Connors, 1965 and Challa et al., 2005, representing ‘‘A’’: soluble inclusion complex. ‘‘B’’: formation of insoluble inclusion complex. ‘‘AL’’: linear increase of drug solubility as a function of cyclodextrin concentration. ‘‘AP’’: positively deviated curve. ‘‘AN’’: negatively deviated curve. ‘‘Bs’’: complex with limited solubility. ‘‘BI’’: insoluble complex.

3. Results 3.1. Solubility assays and phase solubility diagrams The influence of the cyclodextrin cavity size (α-, β- and γ-CD) and the hydrophilization of β-CD by chemical substitution (HP-β- and SBE-β-CD) on the apparent solubility of (+)-usnic acid in water has been investigated at pH 8. As shown in figure 1.a, the increase in cyclodextrin concentrations led to the enhancement in apparent solubility of (+)-usnic acid. Experiments using α- and β-CD were hampered by their low water solubility (14.5 and 1.85% w/w, respectively). Solubility enhancement was very poor when using α and β-CD (figure 1.a). The (+)-usnic acid apparent solubilities were 0.12 and 0.15 mg.ml-1 in presence of α and β-CD at concentrations of 10% w/w and 1.5% w/w respectively (Table 3). Highly water-soluble chemically modified β-cyclodextrins (HP-β- and SBE-β-CDs) were used at concentrations up to 40% w/w, leading to considerably higher solubility enhancements (figure 1a). γ-CD had a contrasted effect on (+)-usnic acid apparent solubility since an increase of γ-CD concentration up to 10% w/w led to a maximal apparent solubility of 1.36 mg.ml-1, above which, the solubility of the (+)-usnic acid decreased significantly (figure 1.a). Further, the effect of the pH of the medium on (+)-usnic acid solubilization was investigated using γ-CD at a fixed concentration of

Segura‐Sanchez et al. J. Mol. Recognit. 2009 ; 22 :232‐241 DOI :10.1002/jmr.936

0 5 10 15

-1.8

-1.6

-1.4

-1.2

-1.0

-0.8

-0.6

-0.4

-0.2

0.0

γ-cyclodextrin at pH 8

N 0.994K 1030 M-1 ∆H -8.00 kcal.mol-1

(-33.5 kJ.mol-1)

kcal

.mol

-1 o

f inj

ecta

nt

b

0 50 100 150 200

26

27

28

29

Time (min)

µcal

.s-1

a

Titration curve

Control

Molar RatioFigure 3 Typical ITC data corresponding to the binding interaction of (+)-usnic acid (0.28mM) with γ-CD (19.69mM) in phosphate buffer (0.05M) pH 8 at 298 K (25°C). Left panel (a) shows exothermic heat flows occuring upon successive injection of 5µl aliquots of γ-CD into (+)-usnic acid. The right panel (b) shows integrated heat data as a function of the molar ratio of titrant and titrated molecules, giving a differential binding curve, which was fit to a standard single-site binding model yielding the following parameters N=0.994, K=1030 M-1, and ∆H =-33.5 kJ.mol-1. Control consisted in successive injections of the cyclodextrin solution in solely the buffer solution. Heat flows accounting for dilution effects were further subtracted from each experimental heat flows (b).

stoichiometry of the interaction (N 1:1), binding constant (K) and the enthalpy released upon the interaction between (+)-usnic acid and cyclodextrins (∆H) [Inoue et al., 1993, Bouchemal, 2008]. It is quite instructive to gather in a single table experimental and calculated thermodynamic quantities representing the interaction of (+)-usnic acid with cyclodextrins in various experimental conditions (table 4). From a thermodynamic point of view, the interaction between a guest molecule and a cyclodextrin molecule is stronger and the stability of the complex higher when K values are higher and when ∆H is more exothermic. The interaction is considered to be more spontaneous when ∆G is more negative. Results obtained from ITC experiments concerning the effect of the cyclodextrin cavity size on the thermodynamic characteristics of the inclusion complex presented in figure 4 and table 4 showed that much higher affinities and much stronger interactions were obtained by using γ-CD compared to values obtained with β-CD derivatives. It can be seen from figure 4.d that only heat of dilution was observed after each injection of the α-CD into the (+)-usnic acid solution, meaning that no interactions occured between α-CD and (+)-usnic acid. In a second experiment, the effect of the pH of the medium on the thermodynamics of the interaction between (+)-usnic acid and γ-CD was investigated using ITC. From figure 5, which summarizes the integrated heat profiles and the thermodynamic quantities K, ∆H and ∆G, it can be clearly concluded that a limited increase of the pH of the medium could be responsible for a stronger and more spontaneous interaction. 4. Discussion 4.1. Influence of the cyclodextrin cavity size and size-fit concept It is generally assumed that complexes formed by lipophilic molecules and cyclodextrins result from a direct insertion of at least a

region of the molecule (the “hosted molecule”) into the cyclodextrin cavity. Because naturally occurring α-, β-, and γ-CDs are formed by 6, 7 or 8 glucose units; the number of glucose units determines top to bottom diameters of the cavity (4.7-5.3, 6.0-6.5 and 7.5-8.3Å) and thus its volume (174, 262 and 427Å3 for α-, β-, and γ-CD respectively) (table 1). Therefore, it can be expected that the stability of the complex depends on the fiting of the cavity to the hosted molecule. In the present case, ITC results indicated clearly that the thermodynamic characteristics of the interaction were dramatically affected by the cyclodextrin cavity size. Figure 4 and table 4 show that much higher affinities and much stronger interactions were obtained using γ-CD (K=1030 M-1) compared to β-CD and their derivatives (K varies from 153 to 405 M-1). Further, no interaction could be detected by ITC in the case of α-CD, as can be seen from figure 4.d which shows that thermal events observed after injection of α-CD into (+)-usnic acid solution corresponded solely to the heat of dilution. These data are consistent with the size-fit concept, which predicts the highest complex stabilities for the best size-matched host-guest pairs. Indeed, (+)-usnic acid molecule interacted more favorably with γ- than β-CD because the 7.5-8.3 Å cavity diameter for γ-CD is able to accommodate aromatic groups found in (+)-usnic acid molecule while α-CD cavity is only able to accommodate alkyl chains with a maximum of five to six carbon atoms [Rekharsky et al., 1994; Sanemasa et al., 1990]. As expected, the intensity and the pattern of molecular interactions between (+)-usnic acid and the different type of cyclodextrins had a direct consequence on their solubilization efficiency. When comparing the solubilizing effect of natural cyclodextrins for a fixed concentration of cyclodextrins of 1.5% w/w, the apparent solubility of (+)-usnic acid was the highest with γ-CD when compared to β-CD and almost nil for α-CD (figure 1). Chemical modifications of β-CD resulted in stronger complexation and interaction as shown by higher affinity constants, more negative enthalpies and Gibbs free energies upon the

Segura‐Sanchez et al. J. Mol. Recognit. 2009 ; 22 :232‐241 DOI :10.1002/jmr.936

complexation. The affinity constant with HP-β-CD was higher than with SBE-β-CD. These results could be explained by the fact that the substitution of the hydrophilic edges of the β-CD molecule by hydroxypropyl or sulfobutyl groups led to an extension of the length channel of the β-CD cavity, resulting possibly in an additional masking of the lipophilic regions of the (+)-usnic acid molecule. Interestingly, from a practical point of view, these two chemically substituted cyclodextrins were shown to be excellent solubilizers for many drugs [Irie and Uekama, 1997; Järvinen et al., 1995; Loftsson and Sigurôardóttir, 1994; Rajewski and Stella, 1996; Stella and He, 2008; Stella and Rajewski, 1997]. Not surprisingly, these hydrophilic cyclodextrin derivates were much more effective than natural cyclodextrins for increasing the apparent solubility, because of the formation of highly water soluble complexes with (+)-usnic acid. Despite their relatively low affinity constants when compared to γ-CD, these highly water-soluble complexes would

Figure 4 Typical ITC integrated heat data profiles obtained from the binding interaction of (+)-usnic acid and β-CD (13.97 mM) (a), HP-β-CD (19.76mM) (b) and SBE-β-CD (19.75 mM) (c), expressed as a function of the molar ratio of titrant and titrated molecules. Figure (d) compares the ITC integrated heat data profiles obtained for α-CD, β-CD and γ-CD. Injections of 5µl of cyclodextrin solution we e made in a 0.28 mM (+)-usnic acid solution in phosphate buffer (0.05M) at pH 8. Temperature was fixed at 298 K (25°C).

r

Molar Ratio Molar Ratio

0 2 4 6 8 10 12

-0.35

-0.30

-0.25

-0.20

-0.15

-0.10

-0.05

β-Cyclodextrin

N 0.999K 153 M-1

kcal

.mol

-1 o

f inj

ecta

nta

∆H -4.72 kcal.mol-1 (-19.76 kJ.mol-1 )

Molar Ratio

0 2 4 6 8 10 12

-1.6

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-1.2

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-0.8

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α-CD β-CD γ-CDkc

al.m

ol-1

of i

njec

tant

d

0 5 10 15 20

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1 of i

njec

tant

b

HP-β-Cyclodextrin

N 1-1

kcal

.mol

-

K 405 M ∆H -3.39 kcal.mol-1 (-14.23 kJ.mol-1)

Molar Ratio

0 5 10 15 20

-0.35

-0.30

-0.25

-0.20

-0.15

-0.10

-0.05

SBE-β-Cyclodextrin

N 1K 281 M-1

∆H -5,49 kcal.mol-1

(-23 kJ.mol-1)

kcal

.mol

-1 o

f inj

ecta

nt

c

probably represent interesting candidates for further biological evaluation. The examination of the phase solubility diagrams gave further insight on the complexation process. Higuchi and Connors (1965) categorized phase solubility diagrams into “A” and “B” types; “A” type curves indicate the formation of soluble inclusion complexes while “B” type suggest the formation of inclusion complexes with poor water solubility. “A” type curves are subdivided into “AL” (linear increases of drug solubility as a function of cyclodextrin concentration), “AP” (positively deviating isotherms), and “AN” (negatively deviating isotherms) subtypes. “B” type curves are subdivided into “BS” (complexes of limited solubility) and “BN” (insoluble complexes) (Figure 1.b) [Higuchi and Connors, 1965]. “A” type curves were experimentally obtained when using HP-β-CD and SBE-β-CD (figure 1.a), as was described by Challa and co-workers [Challa et al., 2005]. The HP-β-CD led to “AL” type

Segura‐Sanchez et al. J. Mol. Recognit. 2009 ; 22 :232‐241 DOI :10.1002/jmr.936

0 5 10 15 20 25-1.8

-1.6

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γ-cyclodextrin at pH 7.4

N 1

K 743 M-1 ∆H -7.56 kcal.mol-1 (-31.67 kJ.mol-1)

kcal

.mol

-1 o

f inj

ecta

nt

b

Molar Ratio

0 5 10 15

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0.0

pH 7 pH 7.4 pH 8

Molar Ratio

kcal

.mol

-1 o

f inj

ecta

nt

Figure 5. Effect of pH on typical ITC integrated heat profiles corresponding to the binding interaction of (+)-usnic acid with γ-CD, expressed as a function of the molar ratio of titrant and titrated molecules. Injections of 5µl of a 19.69mM solution of cyclodextrin were made on a 0.28mM (+)-usnic acid solution in phosphate buffer (0.05M) at 298K (25°C) at pH 7 (a), pH 7.4 (b) and pH 8 (c).

0 10 20 30 40 50-1.8

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0.0

γ-cyclodextrin at pH 7

N 1K 747 M-1

∆H -5.32 kcal.mol-1 (-22.27 kJ.mol-1)

Molar Ratio

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.mol

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f inj

ecta

nta

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γ-cyclodextrin at pH 8

N 0.994K 1030 M-1 ∆H -8.00 kcal.mol-1

(-33.5 kJ.mol-1)

Molar Ratio

kcal

.mol

-1 o

f inj

ecta

nt

c

curve indicating a linear increase of drug solubility as a function of cyclodextrin concentration, while a slightly negatively deviating curve was obtained using SBE-β-CD. The α- and β-CD give rise to low (+)-usnic acid solubility increases which reasonably could not be assignated to any type. On the contrary, in the case of γ-CD, an “A” type curve was initially obtained at low concentrations indicating the formation of soluble inclusion complexes. However, at higher concentrations of γ-CD, a “B” type curve was obtained suggesting the formation of poorly water soluble inclusion complexes. It shows unambiguously that high affinity complexes are not necessarily characterized by the highest water solubility, which obviously is of practical

importance when selecting a cyclodextrin derivate for formulation purpose. 4.2. Inclusion thermodynamic analysis For understanding and evaluating the phenomena of molecular interaction of (+)-usnic acid with cyclodextrins, the knowledge of the binding constants and the thermodynamic parameters associated to the association equilibrium is important. In the present case, the formation of a 1:1 complex between cyclodextrins and (+)-usnic acid molecules has been assumed, which is characterized by an affinity constant

Segura‐Sanchez et al. J. Mol. Recognit. 2009 ; 22 :232‐241 DOI :10.1002/jmr.936

K expressed by the following equation:

]][[][

CDUACDUAK •

= (Eq.i)

Where [CD]: concentration of free cyclodextrin, [UA]: concentration of free (+)-usnic acid, [UACD]: concentration of the inclusion complex. From the values of K and ∆H determined from ITC titration curves, the change in Gibbs free energy (∆G) and the entropy of the interaction (∆S) corresponding to the formation of UA•CD complex were calculated from equations (ii) and (iii) :

KRTG ln−=∆ (Eq.ii) GHST ∆−∆=∆ (Eq.iii)

where R is the gas constant (8.314 J.K-1.mol-1) and T is the absolute temperature during the interaction in Kelvin degrees. Complexation thermodynamics have been shown to reflect the nature of the non-covalent interactions occurring between the guest and cyclodextrin molecules [Inoue et al. 1993]. Indeed, many events, including desolvation of water molecules bound to the guest molecule and/or to the cyclodextrin and formation of weak bonds (hydrogen-bonds, hydrophobic interactions), electrostatic bonds, between the guest molecule and the cyclodextrin result in balanced enthalpic and entropic variations. As can be seen from table 4, whatever the conditions, the reactions of complexation of (+)-usnic acid with cyclodextrins were exclusively exothermic phenomena (∆H<0) with negative and defavorable entropic contribution ∆S<0 and mostly enthalpy driven ( STH ∆>∆ ). Large negative entropy changes

usually arise from the significantly reduced translational and conformational freedoms of host and guest upon complexation [Rekharsky and Inoue 2000a.b]. Because large enthalpic gains were observed, it is suggested that the interactions between (+)-usnic acid and cyclodextrins were predominantly mediated by the formation of van der Waals type bonds [Rekharsky and Inoue 2002] whatever the cyclodextrin involved in the complexation and the pH of the medium. These enthalpic gains were compensated by negative T∆S variations. Indeed, a good straight line (R²=0.976) could be obtained when plotting ∆H versus T∆S for the

different reactions of complexation of (+)-usnic acid with cyclodextrins. This confirmed the existence of an enthalpy-entropy compensation effect (figure 6). Because the stability constant of HP-β-CD complex was higher than for SBE-β-CD complex, one could expect a more negative ∆H for HP-β-CD than for SBE-β-CD. On the contrary, a higher value of ∆H was observed for SBE-β-CD, which could be related to the fact that SBE-β-CD is a polyanionic cyclodextrin molecule, due to the substitution of hydroxyls by negatively charged sulfobutyl groups. 4.3. Effect of ionization of (+)-usnic acid on the interaction with γ-CD and proposed structure of the complex Due to the presence of three ionizable functions in the molecule, considerable modifications of the ionization degree of (+)-usnic acid were obtained by varying the pH of the medium in a narrow

-20 -15 -10 -5 0-40

-35

-30

-25

-20

-15

pH 8

HP-β-CD

γ-CD pH 7.4

pH 7

β-CD

∆H (k

J.m

ol-1

)

T∆S (kJ.mol-1)

SBE-β-CD

y = 1.2568x - 12.619R2 = 0.9767

Figure 6. Enthalpy-entropy compensation plot corresponding to inclusion complex formation of (+)-usnic acid (0.28mM) with CDs at 298 K (25°C). The complexation of (+)-UA with various CD cavity size β-CD (13.97mM), and γ-CD (19.69mM) at pH 8 ( ). Complexation of (+)-UA with chemically modified β-CD, SBE-β-CD (19.75mM), HP-β-CD (19.76mM) ( ) at pH 8. Effect of pH changes (7, 7.4 and 8) on γ-CD complexation ( ). See table 4 for the original data.

Segura‐Sanchez et al. J. Mol. Recognit. 2009 ; 22 :232‐241 DOI :10.1002/jmr.936

Figure 7. Optimized structures of inclusion complexes between (+)-usnic acid and γ-CD where (I) and (II) are the two most stable supramolecular assemblies of (+)-usnic acid molecule with γ-CD yielded when using DS ViewerPro 6.0 software. (a), lateral view with secondary face on top), (b) view of secondary face and (c) view of the primary face of the inclusion complexes, respectively.

(II) a b c

(I) a b c

window comprised between 7 and 8 (table 2). In this range of pH, the hydroxyl group in position 7 remained mostly unionized. On the contrary, the raise of the pH progressively induced the ionization of the hydroxyl group in position 9, while the hydroxyl in position 3 was fully ionized in this pH range. The effect of these modifications of ionization were studied in the case of γ-CD at 1.5% w/w, which was the best solubilizing agent for (+)-usnic acid among all tested cyclodextrins at this concentration.

The modification of the thermodynamic characteristics of the interaction, including the increase in the enthalpy of the interaction ∆H and the Gibbs free energy ∆G indicated an increase of the complex stability when the pH was raised from 7 to 8 (table 4). It corresponded to an increase of the apparent solubility of (+)-usnic acid, which was clearly related to the complexation process, as indicated by the very moderate effect of the pH on the water-solubility without the presence of cyclodextrins (figure 2). Clearly, ionization of (+)-usnic acid favored interactions with γ-CD and led to apparent solubility enhancement.

Based on the assumption that uncharged and hydrophobic groups are more likely to interact strongly within the hydrophobic cyclodextrin cavity and that, on the contrary, charged and hydrophilic groups of guest molecules remain in the bulk solution after association with cyclodextrin, attempts were made to propose the most likely structure of the supramolecular assembly resulting from the association of (+)-usnic acid and γ-CD, corresponding to the more stable complex.

Interaction between anionic groups and hydroxyl groups on the exterior of the cyclodextrin through hydrogen bonds has already been reported by Castronuovo [Castronuovo et al., 1997] in the case of α,Ω-dicarboxylic acids and by Hamilton [Hamilton and Chen, 1988a, 1988b] for fenoprofen. Therefore, the negative charge beared in position 3 would contribute to the stability of the complex. Interestingly, structure (II) suggests that not all the volume of the γ-CD cavity is occupied by a molecule of (+)-usnic acid and that interactions would occur only between one of the faces of the (+)-usnic acid molecule and the inner surface of the γ-CD cavity. Indeed, modelization suggests that (+)-usnic acid molecules would adopt a rather planar configuration, favorable to such an interaction. Conclusions The combination of ITC and solubility experiments to molecular modeling was used for investigating the nature of the interactions between (+)-usnic acid and cyclodextrins. ITC led to a straightforward determination of the affinity constant and thermodynamic parameters of the interaction, helping to clarify the mechanism of complexation of this molecule. Obviously, complex formation depended on the cyclodextrin cavity size and was profoundly affected by the ionization state of the (+)-usnic acid molecule. From a practical point of view, solubility enhancements were obtained, which were shown to be considerably dependent on the type of cyclodextrin and the pH of interaction. Acknowledgments Authors would like to thank the Programme AlBan, the European Union Programme of High Level Scholarships for Latin America, scholarship No E06D103256CO for the financial support which enabled Mr Freimar Segura-Sanchez to conduct this study.

Figure 7 (a and b) shows the optimized structure of the complex between (+)–usnic acid and γ-CD. In this model, the un-charged moiety of the (+)-usnic acid molecule interacts with the cyclodextrin cavity through van der Waals forces. The negative charge of (+)-usnic acid molecule located in position 3 would stay outside of the cyclodextrin cavity and tends to form hydrogen bonds with secondary or primary hydroxyl groups of the γ-CD depending on the orientation of the guest molecule in the cyclodextrin cavity (figure 7 (I) or 7 (II), respectively).

Segura‐Sanchez et al. J. Mol. Recognit. 2009 ; 22 :232‐241 DOI :10.1002/jmr.936

References Bouchemal K. 2008. New challenges for pharmaceutical formulations and drug

delivery systems characterization using isothermal titration calorimetry. Drug. Discov. Today. 13(21-22) 960-972. DOI: 10.1016/j.drudis.2008.06.004

Andrade CAS, Santos-Magalhães NS, de Melo CP. 2006. Thermodynamic characterization of the prevailing molecular interactions in mixed floating monolayers of phospholipids and usnic acid. J Colloid Interface Sci. 298(1): 145-53. DOI: 10.1016/j.jcis.2005.11.066

Castronuovo G, Elia V, Velleca F, Viscardi G. 1997. Thermodynamics of the interaction of α-cyclodextrin with α,Ω-dicarboxylic acids in aqueous solutions. A calorimetric study at 25°C. Thermochim. Acta. 292(1-2): 31-37. DOI: 10.1016/S0040-6031(96)03105-X

Challa R, Ahuja A, Ali J, Khar RK. 2005. Cyclodextrins in drug delivery: an updated review. AAPS PharmSciTech. 6(2): E329-E357. DOI: 10.1208/pt060243

Chaplin M. (2008, 22 June, 2008). "Cyclodextrins." In: Water Structure and Science, London South Bank University. From http://www.lsbu.ac.uk/water/cycloh.html.

Denadai AM, Teixeira KI, Santoro MM, Pimenta AM, Cortes ME, Sinisterra RD. 2007. Supramolecular self-assembly of beta-cyclodextrin: an effective carrier of the antimicrobial agent chlorhexidine. Carbohydr. Res. 342(15): 2286-96. DOI: 10.1016/j.carres.2007.05.002

Duchêne D. Wouessidjewe D. and Ponchel G., 1999. Cyclodextrins and carrier systems. J. Controlled Rel., 62, 263-268.

Fini P, Castagnolo M, Catucci L, Cosma P, Agostiano A. 2004. Inclusion complexes of Rose Bengal and cyclodextrins. Thermochim. Acta. 418(1-2): 33-38. DOI: 10.1016/j.tca.2003.11.041

Flamigni L. 1993. Inclusion of fluorescein and halogenated derivatives in α-, β-, and γ-cyclodextrins: a steady-state and picosecond time-resolved study. J. Phys. Chem. 97(38): 9566-9572. DOI: 10.1021/j100140a006

Hamilton JA, Chen L. 1988a. Crystal structure of an inclusion complex of β-cyclodextrin with racemic fenoprofen: direct evidence for chiral recognition. J. Am. Chem. Soc. 110(17): 5833-5841. DOI: 10.1021/ja00225a039

Hamilton JA, Chen L. 1988b. Crystal structures of inclusion complexes of β-cyclodextrin with S-(+)- and (R)-(-)-fenoprofen. J. Am. Chem. Soc. 110(13): 4379-4391. DOI: 10.1021/ja00221a044

Harries D, Rau DC, Parsegian VA. 2005. Solutes probe hydration in specific association of cyclodextrin and adamantane. J. Am. Chem. Soc. 127(7): 2184-90. DOI: 10.1021/ja045541t

Higuchi T, Connors KA. 1965. Phase-solubility techniques. In: Advances in Analytical Chemistry and Instrumentation, Vol. 4, Reilley CN (ed). Interscience: New York; 117-212.

Inoue Y, Hakushi T, Liu Y, Tong L, Shen B, Jin D. 1993. Thermodynamics of molecular recognition by cyclodextrins. 1. Calorimetric titration of inclusion complexation of naphthalenesulfonates with α-, β-, and γ-cyclodextrins: enthalpy-entropy compensation. J. Am. Chem. Soc. 115(2): 475-481. DOI: 10.1021/ja00055a017

Irie T, Uekama K. 1997. Pharmaceutical applications of cyclodextrins. III. Toxicological issues and safety evaluation. Journal of pharmaceutical sciences. 86(2): 147-62. DOI: 10.1021/js960213f

Järvinen T, Järvinen K, Schwarting N, Stella VJ. 1995. beta-cyclodextrin derivatives, SBE4-beta-CD and HP-beta-CD, increase the oral bioavailability of cinnarizine in beagle dogs. J. Pharm. Sci. 84(3): 295-9.

Kristmundsdóttir T, Aradóttir HA, Ingólfsdóttir K, Ôgmundsdóttir HM. 2002. Solubilization of the lichen metabolite (+)-usnic acid for testing in tissue culture. J. Pharm. Pharmacol. 54(11): 1447-52.

Kristmundsdóttir T, Jónsdóttir E, Ôgmundsdóttir HM, Ingólfsdóttir K. 2005. Solubilization of poorly soluble lichen metabolites for biological testing on cell lines. Eur. J. Pharm. Sci. 24(5): 539-43. DOI: 10.1016/j.ejps.2005.01.011

Liu Y, Cao R, Chen Y, He JY. 2008. Effect of β-cyclodextrin charge type on the molecular recognition thermodynamics of reactions with (ferrocenylmethyl)dimethylaminium derivatives. J. Phys. Chem. B. 112(5): 1445-50. DOI: 10.1021/jp0760206

Loftsson T, Duchêne D. 2007. Cyclodextrins and their pharmaceutical applications. Int. J. Pharm. 329(1-2): 1-11. DOI: 10.1016/j.ijpharm.2006.10.044

Loftsson T, Sigurôardóttir AM. 1994. The effect of polyvinylpyrrolidone and hydroxypropyl methylcellulose on HPβCD complexation of hydrocortisone and its permeability through hairless mouse skin. Eur. J. Pharm. Sci. 2(4): 297-301. DOI: 10.1016/0928-0987(94)90013-2

Mayo SL, Olafson BD, Goddard WA. 1990. DREIDING: a generic force field for molecular simulations. J. Phys. Chem. 94(26): 8897-8909. DOI: 10.1021/j100389a010

Rajewski RA, Stella VJ. 1996. Pharmaceutical applications of cyclodextrins. 2. In vivo drug delivery. J. Pharm. Sci. 85(11): 1142-69. DOI: 10.1021/js960075u

Rekharsky MV, Inoue Y. 1998. Complexation Thermodynamics of Cyclodextrins. Chem. Rev. 98(5): 1875-1918. DOI: 10.1021/cr970015o

Rekharsky MV, Inoue Y. 2000a. Chiral recognition thermodynamics of β-cyclodextrin: the thermodynamic origin of enantioselectivity and the enthalpy-entropy compensation effect. J. Am. Chem. Soc. 122(18), 4418-4435. DOI: 10.1021/ja9921118

Rekharsky MV, Inoue Y. 2000b. 1:1 and 1:2 complexation thermodynamics of γ-cyclodextrin with N-carbobenzyloxy aromatic amino acids and ω-phenylalkanoic acids. J. Am. Chem. Soc. 122, 10949-10955. DOI: 10.1021/ja002325l

Rekharsky MV, Inoue Y. 2002. Complexation and chiral recognition thermodynamics of 6-Amino-6-deoxy-β-cyclodextrin with anionic, cationic, and neutral chiral guests: counterbalance between van der Waals and coulombic interactions. J. Am. Chem. Soc. 124(5): 813-826. DOI: 10.1021/ja010889z

Rekharsky MV, Inoue Y. 2006. Microcalorimetry. In: Cyclodextrins and Their Complexes: Chemistry, Analytical Methods, Applications, Dodziuk H (ed). Wiley–VCH Verlag GmbH & Co. KGaA: Weinheim, Germany; 199-230.

Rekharsky MV, Schwarz FP, Tewari YB, Goldberg RN, Tanaka M, Yamashoji Y. 1994. Thermodynamic and NMR Study of the Interactions of Cyclodextrins with Cyclohexane Derivatives. J. Phys. Chem. 98(15): 4098-4103. DOI: 10.1021/j100066a032

Sanemasa I, Osajima T, Deguchi T. 1990. Association of C5-C9 normal alcanes with cyclodextrins in aqueous medium. Bull. Chem. Soc. Jpn. 63(10): 2814-2819.

Stella VJ, He Q. 2008. Cyclodextrins. Toxicol. Pathol. 36(1): 30-42. DOI: 10.1177/0192623307310945

Stella VJ, Rajewski RA. 1997. Cyclodextrins: their future in drug formulation and delivery. Pharmaceutical research. 14(5): 556-67. DOI: 10.1023/A:1012136608249

Sun DZ, Li L, Qiu XM, Liu F, Yin BL. 2006. Isothermal titration calorimetry and 1H NMR studies on host-guest interaction of paeonol and two of its isomers with beta-cyclodextrin. Int. J. Pharm. 316(1-2): 7-13. DOI: 10.1016/j.ijpharm.2006.02.020

Takai M, Uehara Y, Beisler JA. 1979. Usnic acid derivatives as potential antineoplastic agents. J. Med. Chem. 22(11): 1380-4.

Yamamoto Y, Miura Y, Kinoshita Y, Higuchi M, Yamada Y, Murakami A, Ohigashi H, Koshimizu K. 1995. Screening of tissue cultures and thalli of lichens and some of their active constituents for inhibition of tumor promoter-induced Epstein-Barr virus activation. Chem. Pharm. Bull. (Tokyo). 43(8): 1388-90.

Zia V, Rajewski RA, Stella VJ. 2000. Thermodynamics of binding of neutral molecules to sulfobutyl ether β-cyclodextrins (SBE-β-CDs): the effect of total degree of substitution. Pharm. Res. 17(8): 936-41. DOI: 10.1023/A:1007571019908.