4132 Langmuir 2008, 24, 4132-4139

Binding of Nonsteroidal Anti-inflammatory Drugs to DPPC: Structure and Thermodynamic Aspects

Marlene Lu´cio,*,†,⊥ Frank Bringezu,‡,|,⊥ Salette Reis,† Jose´ L. F. C. Lima,† and Gerald Brezesinski§,⊥

REQUIMTE, Faculdade de Farma´cia, UniVersidade do Porto, Rua Anı´bal Cunha, 4099-030 Porto, Portugal, Institute of Medical Physics and Biophysics, UniVersity of Leipzig, Ha¨rtelstrasse 16, D-04103 Leipzig, Germany, and Max-Planck Institute of Colloids and Interfaces, Research Campus Golm, Am Mu¨hlenberg 1, D-14476 Potsdam, Germany

ReceiVed NoVember 16, 2007. In Final Form: January 8, 2008

The effect of nonsteroidal anti-inflammatory drugs (NSAIDs) on the phase transition and phase properties of 1,2-dipalmitoylphosphatidylcholine (DPPC) has been investigated in both 2D (monolayers at the air/water interface) and 3D (multilayers in lipid/water dispersions) model systems. The 2D membrane models have been characterized by means of pressure-area isotherms and grazing incidence X-ray diffraction (GIXD) measurements. Differential scanning calorimetry (DSC) and simultaneous small- and wide-angle X-ray diffraction have been applied to lipid aqueous dispersions. All NSAIDs studied altered the main transition temperature of the gel to liquid-crystalline phase transition, with the arylacetic acid derivatives (acemetacin and indomethacin) showing the largest effects. A comparison of the results reveals distinct structural features of the membrane models after interaction with the NSAID. All drugs induced perturbations in the lipid liquid-crystalline phase, suggesting a major change in the hydration behavior of DPPC. Again, the largest effects on the structural parameters are found for the arylacetic acid derivatives. The results obtained in the different model systems give indications of the role of the membrane/NSAID interactions that might also be important for NSAID gastric injury.

Introduction serious side effects during medical applications will also depend Cyclooxygenase (COX), also known as prostaglandin synthase on the understanding of the processes initiating and promoting (PGH), is a potent mediator of inflammation.1 Nonsteroidal anti- gastric injury. Such mechanisms are complex, and the cascade inflammatory drugs (NSAIDs) bind to cyclooxygenase, thereby of events leading to mucosal damage must therefore be inhibiting the production of prostaglandins. COX-1 and COX-2 characterized and can also be related to the topical irritancy of are two isoforms of cyclooxygenase.2 COX-1 is a constitutive NSAIDs. Davenport suggested that the normal resistance of the enzyme that produces gastroprotective prostaglandins,3 and gastric mucosa to back diffusion of luminal acid can be disrupted COX-2 is induced by cytokines, mitogens, and endotoxins in by the topical administration of lipid-soluble damaging agents 8 inflammatory cells, which are responsible for the production of such as acetylsalicylic acid or aspirin. Evidence of the direct inflammatory prostaglandins.4 Most of the NSAIDs bind at the superficial damaging effects of other drugs that are members of active sites of both COX-1 and COX-2 with little specificity and the NSAID family was subsequently provided by many inves- lead to side effects such as gastric lesions and renal toxicity. tigators who showed histological, biochemical, and permeability 9-11 The discovery by Vane and colleagues provided new insight changes in the gastric mucosa. However, the “barrier- into both the mechanisms of NSAID therapeutic activities and breaking” activity of the drugs has not been established on a side effects1,2,5-7 such as the development of inhibitors that molecular basis. selectively bind to COX-2, which would be expected to show Despite the complexity of the acid resistance properties of the anti-inflammatory action in vivo with minimal gastric side effects. gastric inner surface, the extracellular lining of surfactant-like However, the sequence of events resulting from cyclooxygenase phospholipids on the surface within the mucus gel layer represents inhibition does not completely explain the overall gastric toxicity an initial line of defense of the stomach and confers hydrophobic, 12 of NSAIDs. The development of novel NSAIDs showing less non-wettable, acid-resistant properties to the mucosa. NSAIDs appear to decrease mucosal hydrophobicity because * Corresponding author. E-mail: [email protected]. Fax: +351-222078961. of their ability to suppress prostaglandin synthesis. In addition, Tel: +351-222078966. they may chemically associate with phospholipids and destabilize † Universidade do Porto. them from the mucus gel layer.13 Such a transition would increase ‡ University of Leipzig. the wettability of the stomach and result in an increase in the § Max-Planck Institute of Colloids and Interfaces. | Current address: Institute of Biotechnology, Martin Luther University, back-diffusion of luminal acid into the mucosa; consequently, Halle-Wittenberg, Kurt-Mothes Strasse 3, 06120 Halle, Germany. the development of erosions must be expected. The aim of the ⊥ These authors have contributed equally. (1) Vane, J. R. Nature 1971, 231, 232-235. (8) Davenport, H. W. Gastroenterology 1964, 46, 245-253. (2) Vane, J. R. Nature 1994, 367, 215-216. (9) Giraud, M. N.; Motta, C.; Romero, J. J.; Bommelaer, G.; Lichtenberger, (3) Mitchell, J. A.; Akarasereenont, P.; Thiemrman, C.; Flower, R.; Vane, J. L. M. Biochem. Pharmacol. 1999, 57, 247-254. R. Proc. Natl. Acad. Sci. U.S.A. 1994, 90, 11693-11697. (10) Morris, G. P.; Wallace, J. L.; Harding, P. L.; Krasse, E. J.; Idle, S. T. Dig. (4) Herschman, H. R. Biochim. Biophys. Acta 1996, 1299, 125-140. Dis. Sci. 1984, 29,6-11. (5) Vane, J. R. Am. J. Med. 1998, 104, 2S-8S. (11) Wallace, J. L. Gastroenterology 1997, 112, 1000-1016. (6) Vane, J. R.; Botting, R. M. Inflammation Res. 1995, 44,1-10. (12) Lichtenberger, L. M.; Graziani, L. A.; Dial, E. J.; Butler, B. D.; Hills, B. (7) Vane, J. R.; Botting, J.; Botting, R. M. ImproVed Nonsteroidal Anti A. Science 1983, 219, 1327-1329. Inflammatory Drugs-COX-2 Enzyme Inhibitors; Kluwer Academic Publishers: (13) Lichtenberger, L. M.; Wang, Z. M.; Romero, J. J.; Ulloa, C.; Perez, J. London, 1996. C.; Giraud, M. N.; Barreto, J. C. Nat. Med. 1995, 1, 154-158.

10.1021/la703584s CCC: $40.75 © 2008 American Chemical Society Published on Web 03/13/2008 Binding of NSAIDs to DPPC Langmuir, Vol. 24, No. 8, 2008 4133

Figure 1. Chemical structures of the NSAIDs investigated: 1-(p-chlorobenzoyl)-5-methoxy-2-methylindole-3-acetic acid carboxymethyl ester (1, acemetacin), 1-(4-chlorobenzoyl)-5-methoxy-2-methyl-3-indoleacetic acid (2, indomethacin), and N-(4-nitro-2-phenoxyphenyl)- methanesulfonamide (3, nimesulide). present study was therefore to investigate the ability of NSAIDs drug adsorption and penetration into the lipid film present at the to bind to or to penetrate into layers of surface-active phos- interface. Structural changes within the DPPC monolayer upon pholipids. The results may elucidate the effects of these drugs adsorption of indomethacin were investigated by synchrotron compromising the integrity of the gastric mucosal barrier. For X-ray experiments at grazing incidence (GIXD). These experi- this purpose, three different NSAIDs (indomethacin, acemetacin, ments were carried out to obtain information on the structure of and nimesulide; Figure 1) were selected, and their influence on the DPPC monolayer interacting with indomethacin. The effect the biophysical properties of 1,2-dipalmitoylphosphatidylcholine of NSAIDs on the lipid organization in 2D monolayers and 3D (DPPC) was studied by different complementary experimental bilayers are investigated and compared. techniques in 2D and 3D model systems. DPPC is a typical phospholipid with regard to its role in Experimental Methods - determining the physical chemical and biological properties of Materials. DPPC, indomethacin, acemetacin, and nimesulide were cellular membranes.14 By dispersing this lipid in water, multi- obtained from Sigma Chemical Co. (St. Louis, MO), and used without lamellar vesicles are spontaneously formed that exhibit many further purification. All other chemicals were purchased from Merck features of the cellular membranes (e.g., the barrier and transport (Darmstadt, Germany). Solutions were prepared with HEPES buffer function or permeability by passive diffusion through the bilayer). (10 mM, 0.1 M NaCl, pH 7.4). Water was purified with a Millipore In aqueous dispersions, DPPC exhibits a rich polymorphism that desktop system, leading to a specific resistance of 18.2 MΩ cm. 19 encompasses crystalline, gel, and liquid-crystalline phases. The Methods. Following the classical thin film hydration method, formation of these structures depends on the environmental the effects of NSAIDs in multilamellar liposomes (MLV) of DPPC were studied by comparing the thermotropic behavior of the conditions (e.g., temperature, pressure, salt conditions, vesicle 14-16 drug homogeneously dissolved with DPPC in an organic solvent preparation, and hydration). According to this, in the current (direct mixing procedure) with that observed by leaving a fixed work differential scanning calorimetry measurements (DSC) were amount of drug in contact with already-prepared vesicles (incubation applied for the thermoanalysis of the phase behavior of the lipid procedure). aqueous dispersions and the effect of the NSAID on the phase- Incubation Procedure. DPPC was dissolved in a chloroform- transition parameters. The results are compared with structural methanoI (9:1 v/v) mixture in a round-bottomed flask. The solvents information from small- and wide-angle X-ray scattering (SAXS/ were removed at 40 °C on a rotary evaporator under a nitrogen WAXS)17 to learn about the influence of the NSAID on the stream until the lipid was dried and distributed as a thin film on the structures of the pure DPPC dispersion. wall of the vessel. The resulting film was kept under vacuum overnight The interaction of the NSAID with lipid bilayers must be to remove the residual solvents. The lipid films were hydrated by considered to be a complex event of adsorption, penetration, and adding 10 mM HEPES buffer to the film and then alternately heated in a water bath at 60 °C and mixed via a vortex mixer at room translocation that involves a change in the lipid hydration and temperature for 5 min. This procedure was repeated three times. molecular orientation of both the lipid head groups and the chains Samples for DSC measurement were then prepared by adding certain forming the hydrophobic membrane core. Using Langmuir amounts of the concentrated stock solutions of the NSAIDs to the monolayers at the air/liquid interface allows one to separate the liposome suspensions in order to obtain the chosen molar fractions adsorption and penetration of the NSAID toward the membrane of the compound. surface from the trans-bilayer events.18 Special interest in using The samples were shaken for1hinawater bath at 60 °C, a phospholipid monolayers for membrane studies arises from the temperature above the DPPC gel to liquid-crystalline phase transition, possibility of varying the physical-chemical parameters at the to allow full hydration of the phospholipid and homogenize the interface, such as molecular density, lateral pressure, surface liposomes, permitting the NSAIDs to reach the partition equilibrium potential, and ionic conditions. In particular, in the study described between the lipid membranes and the aqueous medium. Finally, the samples were aged overnight at 4 °C and again shaken at room here, the option to manipulate the subphase was used to allow temperature for 20 min before the measurement. Direct Mixing Procedure. NSAIDs were mixed with DPPC in (14) Shinitzky, M., Ed. Biomembranes; VCH-Verlagsgesellschaft: Weinheim, - Germany, 1996. organic solvents (9:1 v/v chloroform methanol) in order to obtain (15) Janiak, M. J.; Small, D. M.; Shipley, G. G. Biochemistry 1976, 15. the required molar fraction of the drug. After the dissolution of the (16) New, R. R. C., Ed. Liposomes: A Practical Approach; Oxford University compounds, the preparation of the samples followed the same steps Press: New York, 1990. described in the incubation procedure: lipid film preparation, (17) Jain, M. K. Introduction to Biological Membranes; Wiley and Sons: New York, 1988. (18) Goddard, E. D., Ananthapadmanabha, K. P., Eds. Interactions of (19) Lasic, D. D. Liposomes: From Physics to Applications; Elsevier: New Surfactants with Polymers and Proteins; CRC Press: Boca Raton, 1993. York, 1993. 4134 Langmuir, Vol. 24, No. 8, 2008 Lu´cio et al. rehydration with 10 mM HEPES buffer at a temperature above the for total external reflection. This limits the penetration depth to gel to liquid-crystalline phase transition alternating with vortex approximately 70 Å. The diffracted radiation was detected by a mixing, aging overnight at 4 °C, and shaking at room temperature linear position-sensitive detector (PSD) (OED-100-M, Braun, for 20 min. Garching, Germany) as a function of both the vertical and the DSC measurements were performed using a microcalorimetry horizontal scattering angles. The intensities were corrected for system (MCS DSC, MicroCal Inc., Northampton, MA) as described polarization, effective area, and powder averaging (Lorentz factor). elsewhere.20,21 Samples of NSAID-containing multilamellar vesicle Model peaks taken to be Lorentzian in the in-plane direction and suspensions in buffer (∼1 mg/mL) and the buffer used as reference Gaussian in the out-of-plane direction were fitted to the corrected were degassed before measurement and then were transferred to the intensities. From the Qxy in-plane component of the scattering vector, DSC sample holder. During the measurements, the samples were one gets information about the periodic structure of the monolayer kept under nitrogen overpressure. Each sample was examined in parallel to the water surface. Information about the polar tilt and tilt triplicate to check the reproducibility of the results. The scan rate direction is available from the Qz out-of-plane scattering vector employed was 1 °C/min in the temperature range between 10 and component. The procedure is described in detail in the literature.29 55 °C after an initial isothermal period of 15 min. The obtained data were analyzed using Microcal Origin software. After the calorimetric Results and Discussion scans, aliquots of the scanned samples were used to determine the amount of phospholipid by phosphorus assay.22 DSC was applied to obtain information about the influence For X-ray diffraction measurements, dispersions of DPPC were of NSAIDs on the phase-transition parameters of the pure lipids. prepared in 10 mM HEPES buffer (15-20 wt % of lipid in buffer) Several compounds have been studied and shown to induce by the direct mixing procedure as described above. For the chosen modifications of the phase-transition profile depending on their NSAID/lipid molar ratios, the respective amount of drug was added location in the lipid bilayer. In fact, the presence of an additive from a stock methanol solution before the formation of the thin lipid in phospholipid bilayers affects thermodynamic parameters such films. After the hydration procedure and formation of lipid vesicles, as the main transition and the pretransition temperatures (T , the samples (30 µL) used to fill glass capillaries (Hilgenberg GmbH, m T ), the heat capacity of the samples (c ), and the transition Malsfeld, Germany; wall thickness 0.1 mm), which were flame sealed p p and stored at 4 °C before the measurements. enthalpies (∆H). Compounds entering the lipid bilayers can Small- and wide-angle scattering data where collected using soft change the lipid packing mode and therefore change both Tm and condensed matter beamline A2 at storage ring Doris III of HASYLAB ∆H of the gel to the liquid-crystalline transition.30 (DESY, Hamburg, Germany).23-25 The samples were fixed in a Fully hydrated DPPC bilayers show a characteristic thermo- temperature-controlled sample holder that was heated/cooled between gram consisting of a broader low-enthalpy pretransition at 35.3 °C 15 and 75 °C at a scan rate of 1 °C/min. The data acquisition was and a sharp high-enthalpy main transition at 41.2 °C. Below performed in 10 s exposure increments followed by a 50 s wait time. 35 °C, pure DPPC bilayers form the lamellar gel phase (Lâ’), Additionally, static exposures were taken below and above the main where the acyl chains are in the all-trans conformation and are transition temperature and compared between the heating/cooling tilted from the bilayer normal. Above 41 °C, the lamellar liquid- cycles to check for possible radiation damage. To minimize the X-ray exposure on the sample, a shutter mounted before the sample crystalline LR phase with fluid acyl chains is formed. Between ° was kept closed when no data were acquired. From the peak maximum 35 and 42 C, the phospholipid bilayers form the so-called ripple 15,31 positions, the repeat distances d were obtained after calibration with phase Pâ′. standards: dry rat-tail collagen and p-bromobenzoic acid for the Calorimetric heating curves of DPPC liposomes in HEPES SAXS and the WAXS regions, respectively. The bilayer spacings buffer at pH 7.4 in the absence and presence of different molar were obtained according to fractions of NSAIDs indomethacin, acemetacin, and nimesulide are shown in Figure 2. Thermograms of DPPC in buffer are 1 2 sin θ s ) ) shown in the top row, where the pretransition and main transition d λ hkl temperatures are in good agreement with those previously published.15,32 where s is the scattering vector, 2θ is the scattering angle, hkl are the Miller indices, and λ is the X-ray wavelength (0.15 nm), Depending on the molecular structure and the mole fraction respectively. used, the NSAID studied have different influences on the main The monolayer experiments were performed in aqueous buffer transition temperature Tm (Figure 3) and on the cooperativity of containing 0.1 M NaCl, 10 mM HEPES at pH 7.4, and 1 mM the phase transition (e.g., full width at half-maximum of the indomethacin using a film balance (R&K, Potsdam, Germany) transition peak). Adding nimesulide decreases the Tm values; equipped with a Wilhelmy-type pressure-measuring sensor. The however, at a relatively high molar fraction of 0.4, only a small DPPC monolayers were prepared from 1 mM chloroform solutions reduction of 1.2 °C is found. Arylacetic acid derivatives by spreading the appropriate amount at the air/liquid interface. After acemetacin and indomethacin show a larger shift in the transition 10 min of evaporation time, the monolayer was compressed with 2 -1 -1 - temperatures, and at a molar fraction of 0.4, a reduction of up a velocity of 4 Å molecule min , and the area pressure isotherm ° was recorded. Information about the lipid-chain lattice in the liquid to 5 C (acemetacin) is obtained. These findings suggest that condensed phase was obtained from GIXD experiments using the these compounds show a higher affinity toward the lipid liquid-surface diffractometer on undulator beamline BW1 at HA- membrane and better penetration capabilities. The arylacetic acid SYLAB (DESY, Hamburg, Germany).26-28 The synchrotron beam derivatives are chemically very similar, yet acemetacin differs was adjusted to strike the surface at grazing incidence with an angle from indomethacin because it contains a different residue in of incidence of Ri ) 0.85Rc, where Rc is the critical angle (∼0.13°) (26) Als-Nielsen, J.; Jaquemain, D.; Kjaer, K.; Lahav, M.; Levellier, F.; (20) Branchu, S.; Forbes, R. T.; York, P.; Nyqvist, H. Pharm. Res. 1999, 16, Leiserowitz, L. Phys. Rep. 1994, 246, 251-313. 702-708. (27) Kjaer, K. Physica B 1994, 198, 100-109. (21) Johnson, C. M.; Cooper, A.; Stockley, P. G. Biochemistry 1992, 31, 9717- (28) Bringezu, F.; Brezesinski, G. Colloids Surf., A 2001, 183, 391-401. 9724. (29) Kaganer, V. M.; Mo¨hwald, H.; Dutta, P. ReV. Mod. Phys. 1999, 71, 779- (22) Bartlett, G. R. J. Biol. Chem. 1959, 234, 466-468. 819. (23) Rappolt, M.; Pabst, G.; Rapp, G.; Kriechbaum, M.; Amenitsch, H.; Krenn, (30) Kyrikou, I.; Hadjikakou, S. K.; Kovala-Demertzi, D.; Viras, K.; C.; Bernstoff, S.; Laggner, P. Eur. Biophys. J. 2000, 29, 125-133. Mavromoustakos, T. Chem. Phys. Lipids 2004, 132, 157-169. (24) Rapp, G. Acta Phys. Pol., A 1992, 82, 103-120. (31) Mouritsen, O. G.; Jo¨rgensen, K. Chem. Phys. Lipids 1994, 73,3-25. (25) Rapp, G.; Gabriel, A.; Dosiere, M.; Koch, M. H. J. Nucl. Instrum. Methods (32) Panico, A. M.; Santagati, A.; Cardile, V.; Urso, D.; Gentile, B.; Ronsisvalle, Phys. Res., Sect. A 1995, 357, 178-182. G. Colloid Surf., B 2003, 28,77-81. Binding of NSAIDs to DPPC Langmuir, Vol. 24, No. 8, 2008 4135

Figure 4. Effect of increasing molar fractions of the NSAIDs (b, acemetacin; 9, indomethacin; 2, nimesulide) in DPPC/NSAID mixtures on the Tm values (average of at least three runs). The samples are prepared using the incubation procedure or the direct mixing procedure (filled symbols).

The enthalpy changes (∆H) determined from the peak area remained nearly constant (data not reported because of the negligible variation). This behavior is similar to that observed by the interaction between lipophilic or amphipathic drugs and 35-37 Figure 2. Calorimetric heating curves of DPPC bilayers (top row) DPPC liposomes, as also shown previously and explained containing different molar fractions (xDPPC ) 0.9 - middle row; in terms of a “fluidizing” effect due to the introduction of lipophilic xDPPC ) 0.6 - bottom row) of the NSAIDs studied [(A) acemetacin, drug molecules into the ordered structure of the lipid bilayer. (B) indomethacin, and (C) nimesulide] at pH 7.4. The drug molecules can intercalate between the flexible acyl chains of lipid as interstitial impurities, causing Tm variations without changing ∆H.38 The shift of the Tm values obtained by the direct mixing procedure must be considered to be the maximum effect obtainable if the entire drug was able to reach the vesicle surface, penetrate it, and homogeneously diffuse through the lipid layers. Therefore, this shift is much larger compared with the results observed for the preparation by incubation (Figure 4). Tm shifts resulting from drug incubation are smaller, suggesting that the drug migration through the aqueous medium is initially greatly hindered and that only a small amount that reaches the bilayer surface is able to penetrate the vesicles. Changes in the phase-transition parameters and the complete absence of the pretransition indicate that changes in the structure b of the liquid-crystalline and gel phases of the DPPC dispersion Figure 3. Effect of increasing molar fractions of the NSAIDs ( , must be taken into account. To study the structural effect of acemetacin; 9, indomethacin; 2, nimesulide) in DPPC/NSAID NSAIDs on DPPC bilayers, SAXS/WAXS studies have been mixtures on the Tm values (average of at least three runs). carried out. Figure 5 shows the SAXS region of DPPC in buffer position 11 (Figure 1). This residue, designated the promoiety, and in solutions with 40 mol % of the drugs at 20 °C. The DPPC was added to the original molecule of indomethacin by a multilayers in buffer are well ordered. The peaks show a small metabolically labile chemical linkage. After the metabolic break fwhm, indicating a good correlation between the bilayers (Table of this chemical linkage, acemetacin, which is an inactive drug 1). At 20 °C, the Lâ′ phase has a d value of 6.37 nm, which is (prodrug), is converted to the pharmacologically active substance in good agreement with literature data.39 The addition of 10 mol % indomethacin. The promoiety, when added to a drug, alters the nimesulide does not change the d value in the gel phase but physical properties of the drug to increase water or fat solubility reduces the correlation length between the bilayers drastically or provide site-directed delivery. Accordingly, the larger effect (Table 1). A larger amount of nimesulide (mole fraction of 0.4) on the transition temperature of the lipid observed for acemetacin yields a small increase in the d value to 6.5 nm and a further was indeed due to the presence of the promoiety residue at position decrease in the correlation length. The chain packing is also not 11 (Figure 1), which leads to an increase in both the prodrug significantly influenced by the addition of 10 mol % nimesulide lipid solubility and the size of the molecule. Both are effects that as seen in the WAXS patterns (Figure 6). Two peaks characteristic alter the steric hindrance and electron properties and can thus of an orthorhombic lattice with tilted chains can be resolved at modulate the interaction and/or penetration of drugs into lipid the same positions as for DPPC in buffer (2.36 and 2.41 nm-1). 32 bilayers. The increase in the nimesulide concentration to 40 mol % moves Despite the differences related to the NSAID structures, the the peak positions (2.37 and 2.38 nm-1), which can be explained perturbing effect of all NSAIDs observed in Figure 2 leads to a lowering of the main phase-transition temperature and a (34) Lasonder, E.; Weringa, W. D. Colloid Surf., B 1990, 139, 469-478. broadening of this transition peak as well as to a broadening and (35) Castelli, F.; Puglisi, G.; Giammona, G.; Ventura, C. A. Int. J. Pharm. loss of the pretransition in DPPC bilayers. These observations 1992, 88,1-8. (36) Cater, B. R.; Chapman, D.; Hawes, S. M.; Saville, J. Biochim. Biophys. may be explained by the effect of NSAID molecules incorporated Acta 1974, 363,54-69. into the lipid bilayers, which perturb the alkyl chain ordering of (37) Estep, T. N.; Mountcastle, D. B.; Biltonen, R. L.; Thompson, T. E. 33,34 Biochemistry 1978, 17, 1984-1989. lipids and decrease the cooperativity of the phase transitions. (38) Jo¨rgensen, K.; Ipsen, J. H.; Mouritsen, O. G.; Benett, D.; Zuckhermann, M. J. Biochim. Biophys. Acta 1991, 1062, 227-238. (33) Hwang, S. B.; Shen, T. Y. J. Med. Chem. 1981, 24, 1202-1211. (39) Wiener, M. C.; Sluter, R. M.; Nagle, J. F. Biophys. J. 1989, 55, 315-325. 4136 Langmuir, Vol. 24, No. 8, 2008 Lu´cio et al.

Figure 5. Small-angle X-ray diffraction patterns recorded in static Figure 7. Small-angle X-ray diffraction patterns of DPPC mixed exposures at 20 °C for DPPC (a) and subsequent mixtures with with indomethacin [(a) xDPPC ) 0.9, (b) xDPPC ) 0.6] recorded in ° nimesulide (b), indomethacin (c), and acemethacin (d) (xDPPC ) static exposures at 20 C. 0.6).

Figure 6. Wide-angle X-ray diffraction patterns recorded in static exposures at 20 °C for DPPC (a) and subsequent mixtures with nimesulide (b), indomethacin (c), and acemethacin (d) (x ) Figure 8. Small-angle X-ray diffraction patterns of DPPC (a) and DPPC ) 0.6). subsequent mixtures (xDPPC 0.6) with nimesulide (b), indomethacin (c), and acemethacin (d) recorded in static exposures at 50 °C. Table 1. Bilayer Spacing (d) and Correlation Length (ê)as Calculated from the X-ray Diffraction Analysis for DPPC and the Mixtures Investigated at 20 and 50 °C 20 °C50°C

xDPPC d/(Å) ê/(Å) d/(Å) ê/(Å) DPPC 1.0 63.7 ( 0.5 1084 ( 10 65.8 ( 0.5 1410 ( 10 DPPC + 0.9 63.7 ( 0.5 286 ( 10 68.0 ( 0.5 480 ( 10 nimesulide 0.6 64.9 ( 0.5 185 ( 10 68.2 ( 0.5 476 ( 10 DPPC + 0.9 77.3 ( 0.5 286 ( 10 72.5 ( 0.5 126 ( 10 acemethacin 0.6 74.6 ( 0.5 300 ( 10 72.1 ( 0.5 222 ( 10 DPPC + 0.9 69.7 ( 0.5 467 ( 10 60.6 ( 0.5 706 ( 10 indomethacin 77.0 ( 0.5 368 ( 10 67.6 ( 0.5 236 ( 10 0.6 76.0 ( 0.5 430 ( 10 65.1 ( 0.5 214 ( 10 by a reduced chain tilt that automatically leads to the slightly - increased d value. Figure 9. Pressure area isotherm of DPPC on HEPES buffer (a) and on HEPES buffer containing 1 mM indomethacin (b) at 20 °C. The addition of 10 mol % indomethacin leads to the splitting of the first-order Bragg peak observed in the SAXS region (Figure to a chain cross-sectional area of 0.204 nm2. This value is only 7). One peak is located at a d value of 6.97 nm, and the other slightly larger compared with pure DPPC in buffer (0.203 nm2). reflection has a spacing of 7.7 nm. Obviously, there is a phase Both the correlation between the bilayers and the correlation separation, and the amount of indomethacin is not large enough within one layer is clearly reduced by the presence of in- to reach a homogeneous distribution. If the indomethacin domethacin. concentration is increased to 40 mol %, then one homogeneous The strongest influence on the DPPC structure is observed for phase with a d value of 7.6 nm is formed. The chain packing is acemetacin, with 10 mol % being sufficient to increase the d also drastically influenced because only one Bragg peak has value from 6.37 to 7.46 nm. The increase in the acemetacin been observed, which indicates a hexagonal packing of non- concentration does not change this value remarkably (7.73 nm) tilted chains (Lâ phase). The position of this peak corresponds and does not lead to any phase separation. The bilayer correlation Binding of NSAIDs to DPPC Langmuir, Vol. 24, No. 8, 2008 4137

-1 -1 Figure 10. Diffracted intensities summarized over six Qz intervals (∆Qz ) 0.18 Å , starting from 0.0 Å ) as function of Qxy for DPPC on HEPES buffer containing 1 mM indomethacin at 20 °C and different lateral pressure values (19.5, 25, 30, 40, and 20 mN/m. Schematic representation of the interactions of indomethacin with the monolayers at each lateral pressure assayed.

Table 2. Best-Fit Values of the In-Plane (Qxy) and Out-of-Plane (Qz) Scattering Vector Components, Tilt Angle t, and Cross-Sectional Area of the Chains A0 of DPPC on HEPES Buffer Containing 1 mM Indomethacin at 20 °Ca phase I phase II

-1 -1 -1 -1 pressure π Qxy (Å ) Qxy (Å ) Qxy (Å ) Qxy (Å ) tilt -1 2 -1 -1 -1 2 (mN/m) Qz (Å )A0 (Å ) Qz (Å ) Qz (Å ) Qz (Å ) A0 (Å ) t (deg) 19.5 1.474 21.0 0 25 1.476 20.9 1.344 1.377 1.466 20.3 30.3 0 0.71 0.62 0.08 30 1.480 20.8 1.359 1.394 1.470 20.4 27.9 0 0.65 0.57 0.08 40 1.484 20.7 1.382 1.420 1.478 20.4 24.7 0 0.59 0.49 0.09 Figure 11. Tilt angle of DPPC as function of lateral pressure at 20 1.473 21.0 1.351 1.466 20.3 31.0 20 °C measured on different subphases (b, water; O, PBS or HEPES; 0 0.68 0 2, HEPES containing 1 mM indomethacin). a Phase I is the strongly influenced DPPC phase with upright oriented is again drastically reduced. The increase in the acemetacin molecules, and phase II is the less influenced phase with tilted molecules. concentration reduces the correlation length further. As observed for indomethacin, the chain packing is hexagonal (one Bragg In contrast to the behavior in the gel phase, the addition of 10 peak at 2.39 nm-1) with a cross-sectional area of 0.203 nm2. mol % nimesulide increases the d value slightly to 6.8 nm. Such Compared with DPPC in buffer (Lâ′ phase), the d value in the an increase could be the result of a change in the hydration gel phase (Lâ phase) is increased by 1.1 nm as a result of the behavior of DPPC due to interaction with nimesulide in the interaction with acemetacin. Assuming that the chains are in an disordered liquid-crystalline phase. The ordered gel phase is not all-trans conformation, the change from the tilted (30°)tothe influenced by nimesulide, whereas in the more liquid-like LR non-tilted state would increase the d value by about 0.54 nm. phase, interactions are observed. The remaining 0.56 nm strongly suggests that the head group At 40 mol %, acemetacin as well as indomethacin have very conformation and the hydration must also be changed. Obviously, similar influences on the d values in the gel phase. In both cases, the interaction of acemetacin with the DPPC head groups alters the head group conformation and hydration are changed, allowing their orientation to a more stretched conformation, but such a an upright orientation of the chains. However, pronounced conformational change alone cannot explain the large increase differences between these two systems have been observed in in the d value. Therefore, we conclude that the hydration behavior the LR phase above the main transition. In the case of acemetacin, is affected, leading to a thicker water layer between the lipid chain melting leads to a decrease in the d value by approximately bilayers. 0.4 nm. The melting alone is expected to reduce the bilayer Figure 8 shows the SAXS region in the LR phase above the thickness by 0.8 nm. Therefore, a further increase in the thickness main phase-transition temperature. In all cases, the addition of of the water layer between the lipid bilayers has to be assumed. NSAIDs leads to a pronounced reduction of the bilayer correlation. Such changed hydration behavior can be expected by comparing 4138 Langmuir, Vol. 24, No. 8, 2008 Lu´cio et al. the d values of DPPC in the buffer with those of DPPC in the as well as the cross-sectional areas of the different phases and acemetacin solution. The difference is approximately 0.6 nm. the tilt angles are presented in Table 2. The second phase, which For 10 mol % indomethacin, the splitting of the peaks also occurs appears at 25 mN/m, is an influenced DPPC phase because it in the LR phase. Chain melting leads to a decrease in the d values clearly has a smaller tilt angle that does DPPC on buffer (Figure by approximately 1 nm for both coexisting phases. This is slightly 11). Compression leads to a partial squeezing out of indomethacin, more than expected for the pure melting process. From this which is still interacting with the DPPC head groups. This observation, we conclude that the thickness of the water layer interaction obviously changes the orientation and/or the hydration between the bilayers is decreased. The same conclusion can be of the PC head groups, leading to a smaller area occupied by made for 40 mol % indomethacin, where a reduction of the d these head groups. The decrease in the mismatch of the area value by 1.1 nm is observed. The d value in the LR phase is requirements between the different parts of the DPPC molecules therefore very similar to that observed for DPPC in buffer. allows the reduction of the chain tilt. Therefore, at higher pressures However, there is also the possibility that indomethacin increases two differently influenced DPPC phases coexist. The intensity the fluidity in the chain region, leading to a further decrease in of the broad Bragg peak at zero Qz decreases drastically above the d value. However, indomethacin could increase the thickness the kink in the isotherm (38 mN/m), indicating an almost complete of the water layer between the bilayers, leading to an increase squeezing out of indomethacin and therefore the disappearance in the d value. Both effects can compensate for each other, leading of the nontilted phase. An expansion of the monolayer to 20 to a d value that is similar to that of DPPC in buffer. But the same mN/m increases the intensity of the broad peak at zero Qz, d value might not be an indication of the same structure. indicating a repenetration of indomethacin into the DPPC Figure 9 presents the area-pressure isotherms of DPPC on monolayer. This repenetration process is obviously kinetically buffer and on the 1 mM indomethacin solution measured at 20 °C. hindered and needs a certain time. Therefore, directly after the DPPC on the buffer exhibits the same phase behavior as on monolayer expansion weak peaks belonging to the less influenced water. The lateral pressure starts to increase below a molecular DPPC phase can be seen together with the strong and very broad 2 area of 100 Å . A phase transition from the disordered liquid- peak at zero Qz. expanded (LE) phase to an ordered condensed (LC) phase occurs at around 5 mN/m. On the 1 mM indomethacin solution, the Conclusions isotherm is strongly expanded because of the penetration of The sequence of events resulting from cyclooxygenase indomethacin, and the LE-LC phase transition is shifted to higher inhibition does not totally explain the overall gastric toxicity of lateral pressures around 15 mN/m. Above the phase-transition NSAIDs. Such mechanisms are complex, and the cascade of pressure, the molecular area on the indomethacin solution is still events leading to mucosal damage can also be related to NSAID much larger than on the pure buffer. Further compression leads topical irritancy. The therapeutic and toxic effects of NSAID are to another change in the isotherm slope around 33 mN/m, thus strongly influenced by their lipid affinity. Accordingly, the indicating that most of the penetrated indomethacin is squeezed aim of this article is to report results with regard to the structure out. At pressures above 38 mN/m (kink in the isotherm), the and thermodynamic aspects of the interactions of NSAIDs with molecular area of DPPC on the indomethacin solution is only lipid membranes once these interactions have clinical significance slightly larger than that measured on the pure buffer. in elucidating the effects of NSAIDs on the integrity of the gastric GIXD measurements show a large difference between the mucosal barrier, or they may be useful in the development of DPPC chain lattice on buffer compared with that on the new NSAIDs. indomethacin solution (Figure 10). On the buffer as on water, The NSAIDs interact and penetrate the lipid bilayers and induce DPPC exhibits an oblique chain lattice with strongly tilted variations in the temperature associated with the lipid main phase 40,41 ° ° chains. The tilt angle decreases from 37 at 10 mN/m to 29 transition. The arylacetic acid derivatives (acemetacin and at 40 mN/m (Figure 11). The reason for this large tilt angle is indomethacin) induce greater modifications, probably because the mismatch between the area requirement of the large, strongly of better interaction and penetration into the lipid structure. The hydrated head group and the tightly packed hydrocarbon chains enthalpy changes (∆H) remain nearly constant. The calorimetric 2 with a cross-sectional area of approximately 20.2 Å .Onthe results obtained in the present work agree with the literature,33,34 indomethacin solution, only one very broad Bragg peak is where it was reported that the Tm and cooperativity of the phase observed just above the phase transition (Figure 10). The large transition of DPPC liposomes were decreased by the presence fwhm indicates a very disturbed chain lattice with a small of indomethacin. This behavior is explained in terms of a fluidizing correlation length. The Bragg rod has its maximum at zero Qz, effect due to the introduction of lipophilic drug molecules into indicating an upright orientation of the chains. The cross-sectional the ordered structure of the lipid bilayer. The drug molecules 2 area amounts to 21 Å , which is much larger than for DPPC on intercalate between the flexible acyl chains of lipid as interstitial buffer. Therefore, one can assume that the chains are partially 38 impurities, causing Tm variations without ∆H changing. disordered. The molecular area measured by GIXD is much The NSAIDs studied revealed perturbing effects of the smaller than the area determined from the isotherm (42 compared membrane liquid-crystalline phase, as observed both by SAXS 2 with 74 Å ). This indicates that indomethacin has penetrated into and DSC studies. The observed effects of the NSAIDs are in the DPPC monolayer and occupies a certain space at the interface. agreement with the fluidizing effect of these drugs, which have The penetration leads to a fluidization of the DPPC layer (higher been observed by fluorescence measurements of steady-state phase-transition pressure and larger molecular areas). Further anisotropy.42 compression leads to the appearance of additional Bragg peaks In the lipid gel phase, indomethacin and acemetacin showed around zero Qz and at higher Qz values (Figure 10). The fitting the highest destabilizing effects. Because the model used in the and indexing of these peaks give a chain lattice that is similar current study is made of the same class of phospholipids that line to that of DPPC on buffer (oblique lattice). The peak positions the luminal aspects of the mucus gel layer to provide it with (40) Bringezu, F.; Majerowicz, M.; Wen, S.; Reuther, G.; Tan, K.-T.; Kuhlmann, nonwetting properties, the observed destabilizing effects on J.; Waldmann, H.; Huster, D. Eur. Biophys. J. 2007, 36, 491-498. (41) Dahmen-Levison, U.; Brezesinski, G.; Mohwald, H. Thin Solid Films (42) Lu´cio, M.; Ferreira, H.; Lima, J. L. F. C.; Matos, C.; Castro, B.; Reis, 1998, 327, 616-620. S. Phys. Chem. Chem. Phys. 2004, 6, 1493-1498. Binding of NSAIDs to DPPC Langmuir, Vol. 24, No. 8, 2008 4139 membrane biophysics may be part of the mechanism by which Acknowledgment. This work was supported by the DAAD these NSAIDs attenuate the hydrophobic barrier properties of and CRUP. We are grateful to Dr. Se´rgio Funari and Dr. Kristian the stomach’s mucus phospholipid gel layer with the consequent Kjaer for help at beamlines A2 and BW1. We thank HASYLAB increase in the back diffusion of luminal acid into the mucosa at DESY, Hamburg, Germany, for beam time and support. F.B. and the development of erosions. In comparison, nimesulide, gratefully acknowledges financial support from the DFG which has a less damaging gastrointestinal profile, showed (BR1826/3-2). minimal effects in the lipid gel phase. LA703584S