Licofelone-DPPC Interactions: Putting Membrane Lipids on the Radar of Drug Development

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Article Licofelone-DPPC Interactions: Putting Membrane Lipids on the Radar of Drug Development

Catarina Pereira-Leite 1,2 , Daniela Lopes-de-Campos 1, Philippe Fontaine 3 , Iolanda M. Cuccovia 2, Cláudia Nunes 1 and Salette Reis 1,*

1 LAQV, REQUIMTE, Departamento de Ciências Químicas, Faculdade de Farmácia, Universidade do Porto, Rua de Jorge Viterbo Ferreira, 228, 4050-313 Porto, Portugal; [email protected] (C.P.-L.); [email protected] (D.L.-d.-C.); [email protected] (C.N.) 2 Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, Av. Prof. Lineu Prestes, 748, 05508-000 São Paulo, Brazil; [email protected] 3 Synchrotron SOLEIL, L’Orme des Merisiers, Saint Aubin, BP48, 91192 Gif-sur-Yvette, France; [email protected] * Correspondence: [email protected]; Tel.: +351-220-428-672

Academic Editors: Maria Emília De Sousa, Honorina Cidade and Carlos Manuel Afonso Received: 19 December 2018; Accepted: 30 January 2019; Published: 31 January 2019

Abstract: (1) Background: Membrane lipids have been disregarded in drug development throughout the years. Recently, they gained attention in drug design as targets, but they are still disregarded in the latter stages. Thus, this study aims to highlight the relevance of considering membrane lipids in the preclinical phase of drug development. (2) Methods: The interactions of a drug candidate for clinical use (licofelone) with a membrane model system made of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) were evaluated by combining Langmuir isotherms, Brewster angle microscopy (BAM), polarization-modulation infrared reﬂection-absorption spectroscopy (PM-IRRAS), and grazing-incidence X-ray diffraction (GIXD) measurements. (3) Results: Licofelone caused the expansion of the DPPC isotherm without changing the lipid phase transition proﬁle. Moreover, licofelone induced the reduction of DPPC packing density, while increasing the local order of the DPPC acyl chains. (4) Conclusions: The licofelone-induced alterations in the structural organization of phosphatidylcholine monolayers may be related to its pharmacological actions. Thus, the combination of studying drug-membrane interactions with the pharmacological characterization that occurs in the preclinical stage may gather additional information about the mechanisms of action and toxicity of drug candidates. Ultimately, the addition of this innovative step shall improve the success rate of drug development.

Keywords: lipid monolayers; DPPC; licofelone; langmuir isotherms; BAM; PM-IRRAS; GIXD

1. Introduction Drug discovery is primarily based on finding or designing new molecules to target a biomolecule in order to achieve a therapeutic benefit [1]. Proteins are the main biological targets considered in the rational design of novel drugs. However, other biomolecules have been gaining attention in the last decades, with the emergence of DNA and RNA-based gene therapy [2,3] and membrane lipid therapy [4]. Chronologically, lipids were the last biomolecules to be considered as biological targets, for two main reasons: (a) the huge amount of different lipids found in the human body; and (b) powerful lipid profiling techniques, such as high-resolution mass spectrometry, were recently developed [5]. Cell membranes are nowadays considered as a complex, dynamic, and heterogeneous conjunction of lipids, proteins, and carbohydrates, functioning as platforms to control signal transduction.

Molecules 2019, 24, 516; doi:10.3390/molecules24030516 www.mdpi.com/journal/molecules Molecules 2019, 24, 516 2 of 14

In particular, membrane lipids act either as messengers or as regulators of signaling pathways [4].

The adventMolecules 2019 of functional, 24, x lipidomics has been unveiling the specific roles of membrane 2 lipids of 13 on diverse pathophysiological processes. Indeed, alterations in lipid composition and structure are currentlytransduction. related to In diverse particular, pathological membrane conditions, lipids act either including as messengers inflammation, or as regulators cancer, neurodegenerative of signaling disorders,pathways and [4]. metabolic The advent diseases of functional [4,5]. In lipidomics this context, has been membrane unveiling lipid the therapyspecific roles has emergedof membrane with the lipids on diverse pathophysiological processes. Indeed, alterations in lipid composition and structure goal of attaining a therapeutic benefit by designing new compounds able to modify membrane lipid are currently related to diverse pathological conditions, including inflammation, cancer, structures or domains [4]. neurodegenerative disorders, and metabolic diseases [4,5]. In this context, membrane lipid therapy Beyondhas emerged being with considered the goal of for attaining the rational a therapeutic design benefit of novel by designing drugs, membrane new compounds lipids able seem to also to bemodify valuable membrane for the lipid preclinical structures phaseor domains of drug[4]. development, in which the pharmacokinetic, pharmacodynamic,Beyond being and considered toxicity for profiles the rational of the design selected of novel candidate drugs, membrane are fully addressedlipids seem [also1]. Into fact, membranebe valuable model for systems the preclinical are important phase of tools drug to development, predict the in lipophilicity which the pharmacokinetic,of drug candidates, withpharmacodynamic, recognized advantages and toxicity over theprofiles octanol-water of the selected system candidate [6–8], are which fully is addressed one of the [1]. main In fact, aspects influencingmembrane the model pharmacokinetic systems are important properties tools of pharmaceuticalsto predict the lipophilicity [9]. Regarding of drug pharmacodynamics,candidates, with membranerecognized lipids advantages can either overbe the the therapeutic octanol-water target system of drugs [6–8], or which may indirectly is one of regulate the main the aspects activity of influencing the pharmacokinetic properties of pharmaceuticals [9]. Regarding pharmacodynamics, membrane proteins [7,8]. Thus, drug-induced alterations in membrane lipids may result in changes membrane lipids can either be the therapeutic target of drugs or may indirectly regulate the activity in membrane-proteins activity, as already described, for instance, for phospholipase A2 [10]. Finally, of membrane proteins [7,8]. Thus, drug-induced alterations in membrane lipids may result in changes the toxicityin membrane-proteins mechanisms activity, of drugs as already may also described, include for drug-induced instance, for phospholipase modifications A2 [10]. in the Finally, structure of membranethe toxicity lipids, mechanisms as reported of drugs for may commercial also include nonsteroidal drug-induced anti-inflammatory modifications in the drugs structure [7,11 of] and antibioticsmembrane [12]. lipids, as reported for commercial nonsteroidal anti-inflammatory drugs [7,11] and Inantibiotics this context, [12]. this work aims to show the relevance of considering membrane lipids in the preclinical phase of drugIn this development. context, this Forwork that, aims a candidateto show the for relevance clinical use, of considering namely licofelone, membrane was lipids selected, in the and its interactionspreclinical with phase a membrane of drug development. model system, For viz. that, lipid a candidate monolayers, for clinical were evaluated use, namely using licofelone, complementary was experimentalselected, techniques.and its interactions with a membrane model system, viz. lipid monolayers, were evaluated using complementary experimental techniques. Licofelone (Figure1) is a dual cyclooxygenase (COX)/5-lipoxygenase (5-LOX) inhibitor designed Licofelone (Figure 1) is a dual cyclooxygenase (COX)/5-lipoxygenase (5-LOX) inhibitor designed to circumvent the toxic actions of nonsteroidal anti-inflammatory drugs, due to the additional inhibition to circumvent the toxic actions of nonsteroidal anti-inflammatory drugs, due to the additional of 5-LOXinhibition [13]. of Although 5-LOX [13]. the Although released the data released of preclinical data of preclinical and clinical and studiesclinical studies indicate indicate that licofelone that has promisinglicofelone has efficacy promising and tolerabilityefficacy and profilestolerability [13 profiles], this drug [13], this was drug not was marketed not marketed yet, raising yet, raising questions aboutquestions the underlying about the reasons. underlying In this reasons. sense, licofelone In this sense, was licofelonechosen for was this chosen study to for eventually this study unravel to someeventually unknown unravel membrane some unknown lipid-related membrane mechanisms lipid-related of action mechanisms and toxicity, of action which and toxicity, may eventuallywhich contributemay eventually to clarify contribute the delayed to clarify commercialization. the delayed commercialization.

FigureFigure 1. Structural 1. Structural formula formula of of licofelone licofelone andand 1,2-dipalmitoyl-1,2-dipalmitoyl-snsn-glycero-3-phosphocholine-glycero-3-phosphocholine (DPPC). (DPPC).

LipidLipid monolayers monolayers made of made 1,2-dipalmitoyl- of 1,2-dipalmitoyl-sn-glycero-3-phosphocholinesn-glycero-3-phosphocholine (DPPC) (DPPC) at physiological at pH (7.4)physiological were chosen pH (7.4) as membrane were chosen model as membrane system. model Lipid system. monolayers Lipid monolayers are simple are and simple easy-to-make and membrane model systems that mimic the lateral interactions occurring in one leaﬂet of the

Molecules 2019, 24, x 3 of 13 Molecules 2019, 24, 516 3 of 14 easy-to-make membrane model systems that mimic the lateral interactions occurring in one leaflet of the phospholipid bilayer of cell membranes [14]. Moreover, the lipid packing density and the lateral phospholipidpressure of biological bilayer of membranes cell membranes can be [ 14easily]. Moreover, mimicked the by lipidcompressing packing the density lipid andmonolayer the lateral until pressure of biological membranes can be easily mimicked by compressing the lipid monolayer until 30 mN m−1 [15]. DPPC was considered an adequate model lipid, since it is a phosphatidylcholine, the −1 30main mN component m [15]. DPPC of mammalian was considered cell membranes an adequate [16], model and lipid,it forms since monolayers it is a phosphatidylcholine, with defined lipid thephase main transitions component [17,18]. of mammalian cell membranes [16], and it forms monolayers with deﬁned lipid phaseFurthermore, transitions [17 the,18 ].combination of Langmuir troughs with various detection techniques enables the Furthermore, characterization the combinationof lipid monolayers of Langmuir regarding troughs their with phase various transition detection behavior techniques by enablesisotherm themeasurements characterization and Brewster of lipid monolayersangle microscopy regarding (BAM) their [19], phase as well transition as the lipid-lipid behavior and by lipid-drug isotherm measurementsmolecular interactions and Brewster through angle polarization-modulation microscopy (BAM) [19 infrared], as well reflection-absorption as the lipid-lipid and spectroscopy lipid-drug molecular(PM-IRRAS) interactions and grazing-incidence through polarization-modulation X-ray diffraction (GIXD) infrared measurements reﬂection-absorption [14]. In this spectroscopy sense, these (PM-IRRAS)techniques were and grazing-incidence used to evaluate the X-ray molecular diffraction consequences (GIXD) measurements of adding licofelone [14]. In to this the sense, DPPC thesemonolayer. techniques Overall, were usedthe licofelone to evaluate incorporation the molecular in consequences the DPPC monolayer of adding licofelonechanged the to the lipid-lipid DPPC monolayer.interactions, Overall, which the may licofelone ultimately incorporation be on the basis in the of DPPC the pharmacological monolayer changed actions the of lipid-lipid this drug interactions,candidate for which clinical may use. ultimately be on the basis of the pharmacological actions of this drug candidate for clinical use. 2. Results 2. Results

2.1.2.1. Langmuir Langmuir Isotherms Isotherms TheThe surface surface pressure-area pressure-area per per lipid lipid molecule molecule ( π (π-A)-A) isotherms, isotherms, commonly commonly called called Langmuir Langmuir isotherms,isotherms, enable enable studying studying the the phase phase transition transition behavior behavior of of lipid lipid monolayers monolayers upon upon compression, compression, whichwhich is is characteristic characteristic of of each each type type of of lipid lipid [ 20[20].]. In In this this sense, sense, the theπ π-A-A isotherm isotherm of of the the plain plain DPPC DPPC monolayermonolayer was was ﬁrstly firstly characterized characterized to to ensure ensure the the quality quality of of the the data data obtained, obtained, before before evaluating evaluating the the drugdrug effects. effects. TheTheπ π-A-A isotherms isotherms of of DPPC DPPC monolayers monolayers in in the the absence absence and and presence presence of of licofelone licofelone are are shown shown in in FigureFigure2a. 2a. The Theπ π-A-A isotherm isotherm of of plain plain DPPC DPPC is is in in agreement agreement with with the the literature literature [ 17[17,18].,18]. As As expected, expected, itit was was observed observed upon upon compression: compression: a) aa) transition a transition from from the gaseousthe gaseous phase phase to the to liquid-expanded the liquid-expanded (LE) 2 2 phase(LE) phase at ca. at 100 ca. Å1002; b)Å a; b) mixed a mixed LE andLE and liquid-condensed liquid-condensed (LC) (LC) phase phase from from 85 85 to to 60 60 Å 2Å(plateau (plateau 2 2 region);region); andand c) a a LC LC phase phase from from 60 60 Å Åuntil2 until the themonolayer monolayer collapses collapses under under 45 Å 45at a Å surface2 at a pressure surface −1 pressureof ca. 54 ofmN ca. m 54. By mN adding m−1 .licofelone, By adding a concentration-dependent licofelone, a concentration-dependent expansion of the expansion DPPC monolayer of the DPPCwas observed monolayer (Figure was observed2a). This (Figuremeans 2that,a). Thisfor the means same that, surface for thepressure same surfacevalue, the pressure area per value, lipid themolecule area per increased lipid molecule upon licofelone increased addition. upon licofelone It is noteworthy addition. that It is the noteworthy trend of the that licofelone-induced the trend of the licofelone-inducedexpansion of the DPPC expansion monolayer of the DPPC depends monolayer on the surface depends pressure on the surface(Supplementary pressure (Supplementary material, Figure material,S1). The Figure expansion S1). The occurred expansion exponentially occurred exponentially with drug concentration with drug concentration for smaller for values smaller of valuessurface ofpressure, surface pressure, while the while trend the became trend became linear linearas surface as surface pressure pressure increased. increased. This This result result indicates indicates that thatlicofelone licofelone is gradually is gradually squeezed squeezed out out of of the the monolayer monolayer upon upon compression, compression, asas smallersmaller effectseffects were were observedobserved by by increasing increasing surface surface pressure. pressure.

Figure 2. (a) Surface pressure-area per lipid molecule (π-A) isotherms of various DPPC:licofelone Figure 2. (a) Surface pressure-area per lipid molecule (π-A) isotherms of various−1 DPPC:licofelone molar fractions (XDPPC:lico) at pH 7.4; (b) Maximum compressibility modulus (Cs ) vs area per lipid moleculemolar fractions (A) plot (Χ calculatedDPPC:lico) at from pH the7.4; obtained(b) Maximumπ-A isotherms. compressibility modulus (Cs−1) vs area per lipid molecule (A) plot calculated from the obtained π-A isotherms.

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To better assess the effect of adding licofelone to the DPPC monolayer, various parameters were calculated from the obtained π-A isotherms, namely the minimum area per lipid molecule (Amin), −1 −1 the area per lipid molecule at 30 mN m (A30) and the maximum compressibility modulus (Cs ). The results are shown in Table1.A min and A30 were determined by ﬁtting a linear regression to the −1 isotherm under maximum packing conditions (Amin) or around 30 mN m (A30) and extrapolating −1 it to zero surface pressure [21]. Cs describes the proportional relationship between the surface pressure increase and the area per lipid decrease. Hence, it was calculated from the π-A isotherms, as follows [22]: −1 Cs = −A(dπ/dA). (1)

Licofelone induced a more pronounced increase of A30 than Amin (Table1), suggesting, once again, that the drug is partially squeezed out of the monolayer at higher surface pressures. This result is also in line with the πcollapse data (Table1), as no significant alterations were observed for this parameter, confirming the partial exclusion of licofelone from the DPPC monolayer. Concerning the maximum −1 Cs (Figure2b and Table1), no significant differences were observed in the presence of licofelone, meaning that the drug did not markedly modify the elastic properties of the DPPC monolayer.

−1 Table 1. Minimum area per lipid molecule (Amin), area per lipid molecule at 30 mN m (A30), −1 1 maximum compressibility modulus (Cs ) , and surface pressure of collapse (πcollapse) of the lipid monolayer at pH 7.4 as a function of DPPC:licofelone molar fraction (XDPPC:lico). Data are presented as mean ± standard deviation (n = 3).

2 2 −1 −1 −1 XDPPC:lico Amin (Å )A30 (Å ) Cs (mN m ) πcollapse (mN m ) 10:0 56 ± 1 57 ± 1 255 ± 21 54 ± 1 9.5:0.5 56 ± 2 57 ± 2 256 ± 20 54 ± 1 9:1 57 ± 1 59 ± 1 241 ± 20 54 ± 2 8:2 59 ± 2 62 ± 2 232 ± 20 55 ± 1 1 −1 −1 Cs values stand for the mean ± standard deviation of the maximum plateau region of Cs vs A plots.

2.2. Brewster Angle Microscopy The morphological alterations induced upon compression of the DPPC monolayer, in the absence and presence of licofelone, were followed by Brewster angle microscopy (BAM). This technique has the remarkable advantage of enabling the visualization of condensed domains of lipid monolayers without introducing external probes. This is possible once condensed phases display nonzero reﬂectance of a parallel polarized laser beam with the Brewster angle of incidence [19]. The BAM images obtained with the plain DPPC monolayer (Figure3) are in line with the literature [23,24]. By increasing the surface pressure, gray domains with diverse shapes (e.g. dots and bean-shaped domains) appeared on a black matrix (data not shown), and at 10 mN m−1 (Figure3), the domains were in a typical multilobed shape. This result is in agreement with the Langmuir isotherms data (Figure2a), once the appearance of gray domains indicates that the monolayer was transiting from the LE phase to the LC phase [25]. Afterwards, the DPPC domains got closer and closer, displaying smoother contours, until a uniform gray carpet was formed from ca. 20 mN m−1 (Figure3). Molecules 2019, 24, 516 5 of 14 Molecules 2019, 24, x 5 of 13

Figure 3. Brewster angle microscopy (BAM) images of Langmuir monolayers containing various Figure 3. Brewster angle microscopy (BAM) images of Langmuir monolayers containing various DPPC:licofelone molar fractions (10:0, 9.5:0.5, 9:1, and 8:2) at pH 7.4 according to the surface pressure DPPC:licofelone molar fractions (10:0, 9.5:0.5, 9:1, and 8:2) at pH 7.4 according to the surface pressure (10, 20, and 30 mN m−1). Scale bar is the same for all images. (10, 20, and 30 mN m−1). Scale bar is the same for all images. The licofelone-induced alterations in the morphology of the DPPC monolayer were assessed at 10,The 20, licofelone-induced and 30 mN m− 1alterations(Figure3 ).in the At morphology all studied of surface the DPPC pressures, monolayer licofelone were assessed induced at −1 concentration-dependent10, 20, and 30 mN m (Figure modifications. 3). At all Atstudied 10 mN surface m−1, the pressures, gray domains/black licofelone induced matrix concentration- ratio decreased −1 asdependent licofelone modifications. concentration At increased 10 mN m but, nothe modifications gray domains/black were observed matrix ratio in the decreased domains as morphology. licofelone Thisconcentration result suggests increased that but licofelone no modifications may preferentially were observed interact in the with domains the DPPC morphology. molecules This in theresult LE phasesuggests and that may licofelone partially may rescue preferentially DPPC molecules interact in with the morethe DPPC disordered molecules phase, in leadingthe LE phase to smaller and condensedmay partially domains. rescue AtDPPC 20 and molecules 30 mN min− the1, the more licofelone-induced disordered phase, alterations leading onto thesmaller gray condensed condensed −1 carpetdomains. of DPPC At 20 wereand 30 systematically mN m , the assessed licofelone-induced by calculating alterations the average on the histograms gray condensed of the distribution carpet of DPPC were systematically assessed by calculating the average histograms of the distribution of gray of gray values of multiple images using ImageJ 1.52a software (Supplementary material, Figure S2). values of multiple images using ImageJ 1.52a software (Supplementary material, Figure S2). At 20 At 20 mN m−1, various defects in the gray carpet were visible when adding the higher molar fractions mN m−1, various defects in the gray carpet were visible when adding the higher molar fractions of of licofelone (9:1 and 8:2). Indeed, interconnected multilobe-shaped domains were still visible for the licofelone (9:1 and 8:2). Indeed, interconnected multilobe-shaped domains were still visible for the highest molar fraction of drug (Figure3). These observations are in line with the calculated histograms highest molar fraction of drug (Figure 3). These observations are in line with the calculated (Figure S2a), as for the higher molar fractions of drug the number of pixels obtained at smaller gray histograms (Figure S2a), as for the higher molar fractions of drug the number of pixels obtained at values (<150) increased, and these alterations were particularly evident for the 8:2 molar fraction. smaller gray values (<150) increased, and these alterations were particularly evident for the 8:2 molar At 30 mN m−1, less imperfections were observed in the condensed carpet of DPPC upon licofelone fraction. At 30 mN m−1, less imperfections were observed in the condensed carpet of DPPC upon addition. However, darker regions, i.e. less condensed domains, were observed for the 8:2 molar licofelone addition. However, darker regions, i.e. less condensed domains, were observed for the 8:2 fraction (Figure3). The calculated histograms confirmed that less pronounced alterations were induced molar fraction (Figure 3). The calculated histograms confirmed that less pronounced alterations were by licofelone at 30 mN m−1, as symmetric peaks were obtained for all DPPC:licofelone molar fractions induced by licofelone at 30 mN m−1, as symmetric peaks were obtained for all DPPC:licofelone molar (Figure S2b). Moreover, these data suggest that the DPPC carpet may be less condensed upon licofelone fractions (Figure S2b). Moreover, these data suggest that the DPPC carpet may be less condensed incorporation, as the peaks are slightly shifted to lower gray values. upon licofelone incorporation, as the peaks are slightly shifted to lower gray values. 2.3. Polarization-Modulation Infrared Reflection-Absorption Spectroscopy 2.3. Polarization-Modulation Infrared Reflection-Absorption Spectroscopy The interactions of licofelone with the DPPC monolayer were evaluated by polarization-modulation The interactions of licofelone with the DPPC monolayer were evaluated by polarization- infrared reflection-absorption spectroscopy (PM-IRRAS). The drug-induced changes in the modulation infrared reflection-absorption spectroscopy (PM-IRRAS). The drug-induced changes in wavenumbers of the molecular vibration of the phosphate, carbonyl (CO), and methylene (CH2) the wavenumbers of the molecular vibration of the phosphate, carbonyl (CO), and methylene (CH2) groups of DPPC, at the surface pressures of 10 and 30 mN m−1, were analyzed to retrieve information groups of DPPC, at the surface pressures of 10 and 30 mN m−1, were analyzed to retrieve information about the hydration and the conformational order of the lipid monolayer [26]. about the hydration and the conformational order of the lipid monolayer [26].

Molecules 2019, 24, x 6 of 13 Molecules 2019, 24, 516 6 of 14 The wavenumbers of the vibrational bands of the asymmetric phosphate, carbonyl, and symmetricThe wavenumbers and asymmetric of the methylene vibrational groups bands ofof the the asymmetric plain DPPC phosphate, monolayer carbonyl, were in and line symmetric with the −1 literatureand asymmetric [26], namely methylene ca. 1260, groups 1740, of 2854, the plain and DPPC2924 cm monolayer, respectively. were in The line intensity with the of literature the CO [and26], CH2 vibrational bands increased upon compression−1 (Figure 4), because the decrease of the area per namely ca. 1260, 1740, 2854, and 2924 cm , respectively. The intensity of the CO and CH2 vibrational lipidbands molecule increased leads upon to compressionan augment of (Figure the signal4), because arriving the at decreasethe detector, of the as areapreviously per lipid reported molecule for phospholipidleads to an augment monolayers of the [26]. signal arriving at the detector, as previously reported for phospholipid monolayers [26].

Figure 4. Polarization–modulation infrared reﬂection–absorption spectroscopy (PM-IRRAS) spectra in Figurethe molecular 4. Polarization–modulation vibration frequency of infrared the carbonyl reflection–absorption (CO) and the methylene spectroscopy (CH2 )(PM-IRRAS) groups of the spectra DPPC monolayer at pH 7.4 in the absence (X 10:0) and in the presence of licofelone (X 8:2) at in the molecular vibration frequency DPPC:licoof the carbonyl (CO) and the methylene (CH2) DPPC:licogroups of the (a) 10 mN m−1 and (b) 30 mN m−1. Dashed lines are guides to the eye. DPPC monolayer at pH 7.4 in the absence (ΧDPPC:lico 10:0) and in the presence of licofelone (ΧDPPC:lico 8:2) at (a) 10 mN m−1 and (b) 30 mN m−1. Dashed lines are guides to the eye. The effect of the highest molar fraction of licofelone on the PM-IRRAS spectra of the DPPC −1 monolayerThe effect was evaluatedof the highest at 10 molar and 30 fraction mN m of. Thislicofelone molar fractionon the PM-IRRAS was chosen spectra as it caused of the the DPPC most monolayerpronounced was alterations evaluated in at the 10 monolayer and 30 mN (Figures m−1. This2 and molar3), andfraction theresults was chosen are presented as it caused in Figure the most4. pronouncedThe phosphate alterations bands were in the not monolayer plotted as(Figures no alterations 2 and 3), were and the observed results atare both presented surface in pressures. Figure 4. TheOn thephosphate other hand, bands both were the not CO plotted and the as CH no2 alterationsvibrational were bands observed were shifted at both to surface lower wavenumberspressures. On upon licofelone addition. It is noteworthy that the licofelone effects were similar at both surface the other hand, both the CO and the CH2 vibrational bands were shifted to lower wavenumbers upon licofelonepressures studied.addition. This It is resultnoteworthy probably that reﬂects the licofelone the fact effects that the were lipid similar monolayer at both was surface in the pressures LC phase −1 studied.at both 10 This and result 30 mN probably m , irrespective reflects the tofact drug that incorporation the lipid monolayer (Figure was2a). in The the observed LC phase shift at both of the 10 andCO vibrational 30 mN m−1 band, irrespective suggests thatto drug the carbonyl incorporation group (Figure of DPPC 2a). became The observed more hydrated shift of (i.e., the more CO vibrationalH-bonds formation) band suggests in the presencethat the ofcarbonyl licofelone group [21,26 of]. DPPC Moreover, became the decreasemore hydrated of the CH (i.e.,2 vibrational more H- frequencies indicates that licofelone promoted an increment in the conformational order of the DPPC bonds formation) in the presence of licofelone [21,26]. Moreover, the decrease of the CH2 vibrational frequenciesmonolayer, forindicates instance, that by licofelone reducing promoted the number an of incrementgauche rotamers in the conformational in the acyl chains order of DPPC of the and/or DPPC monolayer,increasing the for monolayerinstance, by packing reducing [21 the,26 ].number of gauche rotamers in the acyl chains of DPPC and/or increasing2.4. Grazing-Incidence the monolayer X-Ray packing Diffraction [21,26].

2.4. Grazing-IncidenceGrazing-incidence X-Ray X-ray Diffraction diffraction (GIXD) studies were used to complement the previous results, once they provide information about changes in the structural arrangements and tilt angle variations Grazing-incidence X-ray diffraction (GIXD) studies were used to complement the previous of phospholipids within a monolayer [27,28]. This is possible due to the existence of an evanescent results, once they provide information about changes in the structural arrangements and tilt angle wave that travels along the air/water interface upon an almost completely reﬂection of the incident variations of phospholipids within a monolayer [27,28]. This is possible due to the existence of an beam [27,28]. GIXD measurements were only performed at 30 mN m−1, once similar effects were evanescent wave that travels along the air/water interface upon an almost completely reflection of observed for licofelone at 10 and 30 mN m−1 in PM-IRRAS experiments, and this is the lateral pressure the incident beam [27,28]. GIXD measurements were only performed at 30 mN m−1, once similar of membrane lipids of cell membranes [15]. effects were observed for licofelone at 10 and 30 mN m−1 in PM-IRRAS experiments, and this is the Three Bragg peaks were measured in the diffraction patterns of plain DPPC at pH 7.4 and lateral pressure of membrane lipids of cell membranes [15]. 30 mN m−1 (Figure5a), as previously described [ 12]. Two out of the three Bragg peaks were in-plane Three Bragg peaks were measured in the diffraction patterns of plain DPPC at pH 7.4 and 30 (Figure5b), indicating that DPPC domains with different structural arrangements coexist in the mN m−1 (Figure 5a), as previously described [12]. Two out of the three Bragg peaks were in-plane monolayer. DPPC molecules were organized in a rectangular lattice structure with tilted chains, or in (Figure 5b), indicating that DPPC domains with different structural arrangements coexist in the monolayer. DPPC molecules were organized in a rectangular lattice structure with tilted chains, or

Molecules 2019, 24, 516 7 of 14 Molecules 2019, 24, x 7 of 13 an untiltedin an untilted hexagonal hexagonal lattice. lattice. As the As rectangularthe rectangular and and tilted tilted lattice lattice is usually described described for for the theDPPC DPPC monolayermonolayer prepared prepared in various in various subphases subphases [29 [29,30],,30], domains domains with with hexagonal hexagonal latticelattice were considered considered to be presentto be present in the regular in the regular rectangular rectangular lattice, lattice, and and the the unit unit cell cell of of the the latterlatter was was selected selected for for simplicity. simplicity.

FigureFigure 5. (a )5. Grazing-incidence(a) Grazing-incidence X-ray X-ray diffraction (GIXD) (GIXD) patterns patterns of the of DPPC the DPPCmonolayer monolayer at pH 7.4 atin pH −1 7.4 inthe absence (Χ (XDPPC:licoDPPC:lico 10:0)10:0) and and in the in the presence presence of licofelone of licofelone (ΧDPPC:lico (XDPPC:lico 9:1) at 309:1) mN at 30 m−1 mN. The m . The correspondingcorresponding Qxy-Q-Qz zintensityintensity maps maps are arealso also presented presented for (b for) ΧDPPC:lico (b)X DPPC:lico10:0 and (10:0c) ΧDPPC:lico and ( c9:1.)X DPPC:lico 9:1. From the diffraction patterns, different parameters were determined from the first-order Bragg Frompeaks, the namely diffraction the lattice patterns, repeat distances different (d parameters), the correlation were lengths determined (ξ), and fromthe acyl the chains ﬁrst-order tilt angle Bragg peaks,(θ namely). Traditionally, the lattice the repeat first-order distances peak (indexationsd), the correlation are 11 (out-of-plane) lengths (ξ), and and the 02 acyl(in-plane) chains for tilt the angle (θ). Traditionally,tilted rectangular the ﬁrst-order lattice, and peak 10 (in-plane) indexations for the are untilted 11 (out-of-plane) hexagonal andlattice 02 [28]. (in-plane) From the for Q thexy-Q tiltedz intensity map (Figure 5b), it is possible to conclude that the acyl chains of DPPC were tilted toward rectangular lattice, and 10 (in-plane) for the untilted hexagonal lattice [28]. From the Qxy-Qz intensity the Next Neighbor (NN-tilt), as 11 (out-of-plane) and 02 (in-plane) peaks are present [28]. Thus, the map (Figure5b), it is possible to conclude that the acyl chains of DPPC were tilted toward the Next tilt angle values (θ) were calculated by the following equation: Neighbor (NN-tilt), as 11 (out-of-plane) and 02 (in-plane) peaks are present [28]. Thus, the tilt angle 푄 = 푄 cos(휓∗) tan (휃) values (θ) were calculated by the following equation: (1) where ψ is the azimuth angle, which is zero in the case of NN-tilt. Moreover, the lattice parameters ∗ (푎, b) were calculated as follows: Qz = Qxy cos(ψ ) tan(θ) (1)

푎 = 4휋 2푄 + 2푄 − 2푄 , where ψ is the azimuth angle, which is zero in the case of NN-tilt. Moreover, the lattice parameters(2) (a, b) were calculated as follows: 푏 = 4휋푄 . (3) q 2 2 2 a = 4π/ 2Qxy + 2Qxy − 2Qxy , (2) The results are shown in Table 2, and the1 1obtained 11values for 02the plain DPPC monolayer are in reasonable agreement with the literature [12,31,32].

2 1 b = 4π/Q .2 (3) Table 2. Distances (d) , correlation lengths (ξ), latticexy parameters02 (푎,푏) and acyl-chains tilt angle (θ) determined from the GIXD patterns of the DPPC monolayer at pH 7.4 and at 30 mN m−1, according The results are shown in Table2, and the obtained values for the plain DPPC monolayer are in to lipid:licofelone molar fraction (ΧDPPC:lico). reasonable agreement with the literature [12,31,32]. ΧDPPC:lico 풅ퟏퟏ (Å) 풅ퟎퟐ (Å) 풅ퟏퟎ (Å) 흃ퟏퟏ (nm) 흃ퟎퟐ (nm) 흃ퟏퟎ (nm) 풂 (nm) 풃 (nm) θ (°) Table10:0 2. Distances 4.46 (d ) 1, correlation 4.29 4.14 lengths (ξ), 1.8 lattice parameters 12.6 (a, 9.2b) 2 and 0.522acyl-chains 0.857 tilt angle30 (θ) determined9:1 from 4.53 the GIXD 4.28 patterns of - the DPPC 3.1 monolayer 15.4 at pH 7.4 -and at 30 0.534 mN m−0.8561, according 29 to 1 Standard deviation of lattice repeat distances: ±0.03. 2 Lattice parameters for hexagonal phase: 푎=푏= 0.447 lipid:licofelone molar fraction (XDPPC:lico). nm. ◦ X The diffractiond ¯ (Å) patternd (Å) of DPPCd (Å) monolayersξ ¯ (nm) in theξ presence(nm) ofξ licofelone(nm) showeda (nm) onlyb (nm) two Braggθ ( ) DPPC:lico 02 10 11 02 10 peaks, the 111 (out-of-plane) and the 02 (in-plane) peaks (Figure 5a,c), characteristic of the rectangular 10:0 4.46 4.29 4.14 1.8 12.6 9.2 0.522 0.857 30

9:1 4.53 4.28 - 3.1 15.4 - 0.534 0.856 29 1 Standard deviation of lattice repeat distances: ±0.03. 2 Lattice parameters for hexagonal phase: a = b = 0.447 nm. Molecules 2019, 24, 516 8 of 14

The diffraction pattern of DPPC monolayers in the presence of licofelone showed only two Bragg peaks, the 11 (out-of-plane) and the 02 (in-plane) peaks (Figure5a,c), characteristic of the rectangular lattice structure. Thus, the condensed untilted domains with the smallest lattice repeat distance (d10 = 4.14 Å), i.e. the hexagonal packing, disappeared (Table2). This result may partially justify the expansion of the Langmuir isotherms (Figure2a) toward higher area per lipid. Moreover, the a parameter value of the rectangular lattice structured increased, resulting in higher area per lipid molecule than that obtained with plain DPPC. No signiﬁcant alterations in the θ value was observed upon licofelone addition (Table2), meaning that the orientation of DPPC acyl chains was not inﬂuenced by the drug.

3. Discussion Various experimental techniques were combined to comprehensively characterize the molecular interactions of licofelone with a DPPC monolayer, used as membrane model. First, Langmuir isotherms showed that licofelone caused the expansion of the DPPC monolayer (Figure2a). This effect has been associated with an intercalation of the compound into the phospholipid monolayer [33] and/or an increase of the monolayer fluidity [34]. Since the elastic properties of the monolayer did not varied −1 significantly (Cs values in Table1), the monolayer expansion seems to be essentially caused by the drug intercalation. This hypothesis was further confirmed by the PM-IRRAS data once the conformational order of the DPPC acyl chains increased upon licofelone incorporation (Section 2.3), showing that licofelone did not increase the monolayer fluidity. Despite causing the expansion of the DPPC monolayer, licofelone did not disturb the phase transitions of the DPPC monolayer. The drug only shifted the phase transitions toward higher area per lipid molecule and surface pressures, as revealed by the Langmuir isotherms (Figure2a) and the BAM images (Figure3). Indeed, the typical condensed domains observed in the BAM images of plain DPPC were also detected in the presence of licofelone, without significant morphological alterations. Molecular details about the DPPC-licofelone interactions were retrieved through PM-IRRAS and GIXD experiments. The obtained diffraction patterns revealed that the licofelone-induced expansion of the DPPC isotherm occurs due to the disappearance of highly packed untilted hexagonal domains, as well as the increase of the area per lipid of the tilted rectangular lattice structure. Indeed, the intercalation of licofelone into the DPPC monolayer may disrupt the lipid-lipid interactions, decreasing the DPPC packing density. Lower packing may justify the increase of CO hydration (CO vibrational frequency decreased) upon licofelone addition. Indeed, the CO groups of DPPC have permanent electric dipoles [12], and dipole-dipole interactions with adjacent phospholipids may be disrupted with looser packing, enabling the occurrence of more H-bonds with the aqueous subphase. Despite lowering packing density, the licofelone intercalation also seems to induce a steric constraint on the DPPC acyl chains, which became more ordered (CH2 vibrational frequencies decreased) with reduced trans-gauche isomerizations. To improve clarity, the main alterations induced by licofelone in the DPPC monolayer are illustrated in Figure6. Molecules 2019, 24, x 8 of 13

lattice structure. Thus, the condensed untilted domains with the smallest lattice repeat distance (푑 = 4.14 Å), i.e. the hexagonal packing, disappeared (Table 2). This result may partially justify the expansion of the Langmuir isotherms (Figure 2a) toward higher area per lipid. Moreover, the 푎 parameter value of the rectangular lattice structured increased, resulting in higher area per lipid molecule than that obtained with plain DPPC. No significant alterations in the θ value was observed upon licofelone addition (Table 2), meaning that the orientation of DPPC acyl chains was not influenced by the drug.

3. Discussion Various experimental techniques were combined to comprehensively characterize the molecular interactions of licofelone with a DPPC monolayer, used as membrane model. First, Langmuir isotherms showed that licofelone caused the expansion of the DPPC monolayer (Figure 2a). This effect has been associated with an intercalation of the compound into the phospholipid monolayer [33] and/or an increase of the monolayer fluidity [34]. Since the elastic properties of the monolayer did not varied significantly (Cs−1 values in Table 1), the monolayer expansion seems to be essentially caused by the drug intercalation. This hypothesis was further confirmed by the PM-IRRAS data once the conformational order of the DPPC acyl chains increased upon licofelone incorporation (Section 2.3), showing that licofelone did not increase the monolayer fluidity. Despite causing the expansion of the DPPC monolayer, licofelone did not disturb the phase transitions of the DPPC monolayer. The drug only shifted the phase transitions toward higher area per lipid molecule and surface pressures, as revealed by the Langmuir isotherms (Figure 2a) and the BAM images (Figure 3). Indeed, the typical condensed domains observed in the BAM images of plain DPPC were also detected in the presence of licofelone, without significant morphological alterations. Molecular details about the DPPC-licofelone interactions were retrieved through PM-IRRAS and GIXD experiments. The obtained diffraction patterns revealed that the licofelone-induced expansion of the DPPC isotherm occurs due to the disappearance of highly packed untilted hexagonal domains, as well as the increase of the area per lipid of the tilted rectangular lattice structure. Indeed, the intercalation of licofelone into the DPPC monolayer may disrupt the lipid-lipid interactions, decreasing the DPPC packing density. Lower packing may justify the increase of CO hydration (CO vibrational frequency decreased) upon licofelone addition. Indeed, the CO groups of DPPC have permanent electric dipoles [12], and dipole-dipole interactions with adjacent phospholipids may be disrupted with looser packing, enabling the occurrence of more H-bonds with the aqueous subphase. Despite lowering packing density, the licofelone intercalation also seems to induce a steric constraint on the DPPC acyl chains, which became more ordered (CH2 vibrational frequencies decreased) with Moleculesreduced2019 trans-gauche, 24, 516 isomerizations. To improve clarity, the main alterations induced by licofelone9 of 14 in the DPPC monolayer are illustrated in Figure 6.

Figure 6. Schematic representation of the licofelone-induced alterations in the DPPC monolayer.

The packing density of the DPPC monolayer may also decrease due to the excess of negative charges introduced in the air-water interface due to the licofelone insertion into the DPPC monolayer. Indeed, the bulk pKa of licofelone is 4.8 (according to the MarvinSketch calculator from ChemAxon) and its surface pKa is not expected to increase more than 1 pKa unit according to literature [35–37]. Thus, licofelone has acidic properties (Figure1), being mainly deprotonated at pH 7.4. Since DPPC is a zwitterionic molecule with an anionic phosphate group and a cationic choline group (Figure1), it is conceivable that electrostatic repulsions between the phosphate moiety of DPPC and the carboxylate group of licofelone may be a restriction to highly packed domains of DPPC. This hypothesis also explains the fact that licofelone was partially squeezed out of the monolayer under high surface pressures (Section 2.1), as electrostatic repulsions may drive the expulsion of licofelone from the DPPC monolayer. The structural formula of licofelone (Figure1) also give insights into the putative orientation of the drug in the DPPC monolayer. It is conceivable that licofelone interacts electrostatically with the choline moiety of DPPC, anchoring the drug at the water interface and facing the aromatic rings toward the air, thereby establishing hydrophobic interactions with the DPPC acyl chains. This orientation justifies the steric constraint induced by licofelone in the DPPC acyl chains. Moreover, it is in line with the literature [38–40], regarding the molecular interactions of various anionic anti-inflammatory drugs with phosphatidylcholine molecules. Overall, this study showed the ability of licofelone to induce alterations into the structural organization of DPPC. These actions may be related to the in vivo pharmacological actions of this pharmaceutical. Regarding anti-inflammatory effects, the licofelone-induced changes in the molecular organization of phosphatidylcholines may result in the indirect inhibition of COX and 5-LOX, its therapeutic targets. Despite being structurally and functionally distinct, both COX isoforms (COX-1 and COX-2) are monotopic membrane proteins located in the endoplasmic reticulum and nuclear envelope [41,42], and COX-2 is usually overexpressed in inflammatory conditions [43]. On the other hand, 5-LOX is a cytoplasmatic enzyme that must bind to the nuclear membrane to become activated [44]. Thus, protein-lipid interactions may modulate the activity of both inflammatory enzymes. Indeed, both COX-2 and 5-LOX have preferential affinity for the fluid domains of biological membranes [44,45]. Thus, the drug intercalation and the drug-induced local ordering in the acyl chains region of phosphatidylcholines may cause a reduction in the activity of these enzymes, favoring the therapeutic actions of licofelone. On the other hand, the reduction of the lipid packing density caused by licofelone may result in gastric toxicity, as described for various acidic nonsteroidal anti-inflammatory drugs [7,40]. In fact, defects in the protective phospholipid layers of the gastric Molecules 2019, 24, 516 10 of 14 mucosa may facilitate the penetration of noxious agents, such as protons, digestive enzymes and toxins, prompting the occurrence of gastric damage. In this sense, the combination of in vitro studies on drug-membrane interactions with in vitro and in vivo pharmacological studies in the preclinical phase of drug development shall provide relevant information about the mechanisms of action and toxicity of drug candidates for clinical use. Before the translation from single studies to drug development, different parameters of these in vitro studies must be optimized to better resemble the conditions found by drugs in their in vivo pathway, such as the type of membrane model, the lipid composition, the composition of the aqueous medium, and the drug concentration. Moreover, the development of high-throughput screening methods would be another important step to implement drug-membrane interactions studies in the laborious process of drug development. Despite the long journey ahead, these in vitro studies may be of utmost importance to avoid pitfalls in the latter stages of drug development, increasing the success rate of the process and reducing the corresponding duration and costs.

4. Materials and Methods

4.1. Materials Licofelone was supplied by Cayman Chemical (Ann Arbor, MI, USA). 1,2-Dipalmitoyl-sn-glycero- 3-phosphocholine (DPPC) was purchased from Avanti Polar Lipids, Inc. (Alabaster, AL, USA). Trizma® base was obtained from Sigma-Aldrich Co. (St. Louis, MO, USA) and used to prepare the subphase-Tris-HCl buffer (10 mM, pH 7.4). The subphase was prepared using double-deionized water (κ < 0.1 µS cm−1) and by adjusting the pH with a hydrochloric acid solution (1 M) after Trizma® base dissolution. Chloroform, methanol, and ethanol were supplied by VWR International S.A.S. (Fontenais-sous-Bois, France). The stock solutions of DPPC and licofelone were prepared in chloroform and methanol, respectively.

4.2. Monolayers Preparation A DPPC solution (1 mg mL−1) in chloroform or a DPPC:licofelone solution (9.5:0.5, 9:1, and 8:2 molar fractions) in chloroform:methanol (9:1, v/v) was dropwise spread using a Hamilton syringe (Bonaduz, Switzerland) onto the subphase (Tris HCl buffer, 10 mM, pH 7.4). The lipid or the lipid:drug monolayers were then equilibrated for 15 min to ensure the organic solvents evaporation.

4.3. Langmuir Isotherms Langmuir isotherm measurements were carried out in a Langmuir balance from KSV NIMA (Helsinki, Finland), using a KN-1006 trough with two symmetrical barriers and a strip of ﬁlter paper (Biolin Scientiﬁc, Espoo, Finland) as surface pressure sensor. Surface pressure-area per lipid molecule (π-A) isotherms were acquired by compressing the monolayer at a rate of 5 mm min−1 at 21 (±1) ◦C. Ethanol and double-deionized water (κ < 0.1 µS cm−1) were used to properly clean the trough before and after each measurement. The trough was considered clean when the barriers compression did not cause any increase of the basal surface pressure of the subphase (0 mN m−1).

4.4. Brewster Angle Microscopy Images of the lipid(:drug) monolayers were captured using a Nanoﬁlm_Ultrabam Brewster angle microscope from Accurion GmbH (Goettingen, Germany) coupled with the Langmuir balance described in the previous section. The images with a lateral resolution of 2 µm were taken at 21 (±1) ◦C after setting the surface pressure to 10, 20, or 30 mN m−1. To improve the contrast of BAM images, the brightness was optimized, and the background was corrected using the Accurion Image 1.1.3 software (Accurion GmbH, Goettingen, Germany). Molecules 2019, 24, 516 11 of 14

4.5. Polarization-Modulation Infrared Reflection-Absorption Spectroscopy A PM-IRRAS spectrophotometer from KSV NIMA (Helsinki, Finland) was used for infrared analysis, coupled with a Langmuir balance with a KN-1002 trough with two symmetrical barriers and a strip of filter paper (Biolin Scientific, Espoo, Finland) as surface pressure sensor. PM-IRRAS measurements were performed using an incident angle of 80◦ with respect to the normal of the monolayer surface. After optimizing the interferogram to values higher than 6.5, PM-IRRAS spectra were acquired for 300 s at 21 (±1) ◦C and at 10 and 30 mN m−1. The DPPC spectra in the absence and in the presence of licofelone were corrected with the subphase spectra, recorded previously to the lipid(:drug) spreading.

4.6. Grazing-Incidence X-ray Diffraction GIXD measurements were performed in the SIRIUS beamline at the SOLEIL synchrotron (Gif-sur-Yvette, France). Details about the facility can be found elsewhere [46]. The lipid monolayers were prepared (as described in Section 4.2) in a trough with a helium-flushed sealed chamber. The inert gas was used to reduce the gas scattering and to hamper beam-induced damage to the monolayer. All experiments were performed at 20 (±1) ◦C. GIXD measurements were performed using the following settings, as previously described [12]: 10.5 KeV (λ = 0.118 nm) incident X-ray energy; 0.1 × 2 mm2 (V × H) beam size; and 1.70 mrad incident angle. This angle is below the critical angle of the air-water interface, enabling the refracted wave to be evanescent. Thus, a 5 nm layer underneath the interface was investigated. The scattered intensity was acquired with high resolution (0.03 nm−1 wave vector resolution) using a 1D gas detector fixed on the 2-axis detector arm of the diffractometer, with 2048 channels at 150 mm. The obtained diffraction patterns were displayed as Qxy-Qz intensity maps, which consider the two components of the scattering vector Q, namely Qxy (in-plane component) and Qz (out-of-plane component) [28]. From the vertical integration of the intensity maps along Qz, the position, width and intensity of Bragg peaks were determined to characterize the periodic structure of the monolayer. The Qz component was used to calculate the tilt angle of the lipid acyl chains [47]. The Qxy component, in particular the position of the first-order Bragg peaks, were considered to determine the lattice repeat distances (d = 2π/Qxy)[28]. Moreover, the full-width at half-maximum (w) of the first-order Bragg peaks were considered to calculate the correlation lengths (ξ = 2/w)[12].

Supplementary Materials: The following are available online, Figure S1. Variation of the area per lipid molecule (∆A) according to the licofelone concentration, expressed as DPPC:licofelone molar fraction (10:0, 9.5:0.5, 9:1, 8:2), as a function of surface pressure (π). Figure S2. Average histograms of the distribution of gray values of the BAM images obtained at (a) 20 mN m−1 and (b) 30 mN m−1, according to the DPPC:licofelone molar fraction (10:0, 9.5:0.5, 9:1, 8:2). Author Contributions: Conceptualization, C.P.-L., C.N. and S.R.; Formal analysis, C.P.-L., D.L.-d.-C. and P.F.; Investigation, C.P.-L., C.N. and S.R.; Supervision, I.M.C. and S.R.; Writing–original draft, C.P.-L., D.L.-d.-C. and P.F.; Writing–review and editing, I.M.C., C.N. and S.R. Funding: C.P.-L., D.L.-C. and C.N. were funded by FCT (Fundação para a Ciência e a Tecnologia), POCH (Programa Operacional Capital Humano) and EU (European Union) [grant numbers: SFRH/BD/109621/2015, PD/BD/105957/2014, IF/00293/2015, respectively]. C.P.-L. and I.M.C. were funded by CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico) [grant numbers: 160446/2013-9 and Proc. 301250/2013-8, respectively]. This research was also funded by FAPESP (Fundação de Amparo à Pesquisa do Estado de São Paulo) [Projeto Temático 2013/08166-5] and INCT-FCx. C.P.-L., C.N. and S.R. also thank CALIPSO funded program to perform experiments in SIRIUS beamline at SOLEIL Synchrotron [20130142]; as well as EU [FEDER funds POCI/01/0145/FEDER/007265] and FCT/MEC (Fundação para a Ciência e a Tecnologia and Ministério da Educação e Ciência) [Partnership Agreement PT2020 UID/QUI/50006/2013]. Acknowledgments: The authors thank Manuela Barros and Vânia Dias for administrative and technical support, as well as Ana Catarina Alves and Ana Rute Neves for the help during GIXD data acquisition. Conflicts of Interest: The authors declare no conflict of interest. Molecules 2019, 24, 516 12 of 14

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