Differentiating Oxicam Nonsteroidal Anti-Inflammatory Drugs in

pubs.acs.org/Langmuir © 2009 American Chemical Society

Differentiating Oxicam Nonsteroidal Anti-Inflammatory Drugs in Phosphoglyceride Monolayers Katarzyna Czapla,†,‡ Beata Korchowiec,†,‡ and Ewa Rogalska*,‡ †

Department of Physical Chemistry and Electrochemistry, Faculty of Chemistry, Jagiellonian University, ul. R. Ingardena 3, 30-060 Krakow, Poland and ‡Structure et R eactivit e des Syst emes Mol eculaires Complexes, BP 239, CNRS/Nancy Universit e, 54506 Vandoeuvre-l es-Nancy Cedex, France Received August 16, 2009. Revised Manuscript Received November 23, 2009 Meloxicam, piroxicam, and tenoxicam belong to a highly potent oxicam group of nonsteroidal anti-inflammatory drugs. Whereas the structurally similar oxicams have different pharmacokinetics, treatment efficiency, and adverse effects, their common mechanism of action is the inhibition of a membrane enzyme, cyclooxygenase. Because the prerequisite for accessing the cyclooxygenase by the drugs is interaction with the membrane, the focus of the current study was a comparison of how meloxicam, piroxicam, and tenoxicam interact with lipid monolayers used as models of biological membranes. The monolayers were formed with 1,2-dipalmitoyl-sn-glycero-3-phospho-rac-(1glycerol), 1,2-dipalmitoyl-sn-glycero-3-phospho-L-serine, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine, 1,2-myristoyl-sn-glycero-3-phosphoethanolamine, and 1,2-dilauroylsn-glycero-3-phosphoethanolamine. These systems were examined via surface pressure and surface electrical potential measurements, polarization modulation infrared reflection adsorption spectra, and Brewster angle microscopy. The three oxicams are differentiated in the monolayers; meloxicam shows the highest ability to modify membrane fluidity and surface potential, followed by piroxicam and tenoxicam. The dissimilarity of the biological activity of the oxicams may be linked to different interaction with the membrane, as revealed by the present study.

Nonsteroidal anti-inflammatory drugs (NSAIDs) are at present the most widely used medications in the treatment of inflammation and pain associated with rheumatic diseases.1,2 Among several chemically different classes of NSAIDs,3 antiinflammatory oxicams4 have attracted much pharmacological, therapeutic, and basic research interest. The objective of this research is twofold: first, to gain further insight into the oxicam NSAID-lipid interaction because the therapeutic properties of these molecules are linked to membrane processes and second, to explore the possibility of using lipid layers for differentiating oxicams. NSAIDs are inhibitors of prostaglandin H synthase, more commonly referred to as cyclooxygenase (COX).5 COX is the rate-limiting enzyme for the cellular synthesis of prostaglandins (PGs) and thromboxane A2.6-8 COX exists in two isoforms: the constitutive COX-l, which stimulates the physiological production of PGs, and the inducible COX-2, which is expressed in response to an inflammatory stimulus. Because prostaglandins are important participants in many physiological processes, the

*Corresponding author. E-mail: [email protected]. Tel: þ33 (0)3 83 68 43 45. Fax: þ33 (0)3 83 68 43 22. (1) Ong, C. K. S.; Lirk, P.; Tan, C. H.; Seymour, R. A. Clin. Med. Res. 2007, 5, 19–34. (2) Tsai, R. S.; Carrupt, P. A.; El Tayar, N.; Giroud, Y.; Andrade, P.; Testa, B.; Bree, F.; Tillement, J. P. Helv. Chim. Acta 1993, 76, 842–854. (3) Yazdanian, M.; Briggs, K.; Jankovsky, C.; Hawi, A. Pharm. Res. 2004, 21, 293–299. (4) Lombardino, J. G.; Wiseman, E. H. Med. Res. Rev. 1982, 2, 127–152. (5) Kulkarni, S. K.; Jain, N. K.; Singh, A. Methods Finds Exp. Clin. Pharmacol. 2000, 22, 291–298. (6) Vane, J. R. Nature, New Biol. 1971, 231, 232–235. (7) Mifflin, R. C.; Powell, D. W. Regul. Pept. Lett. 2001, 8, 49–63. (8) Ruan, K.-H.; Deng, H.; Wu, J.; So, S.-P. Arch. Biochem. Biophys. 2005, 435, 372–381.

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new NSAIDs such as meloxicam (MEL) are therefore conceived as selective inhibitors of COX-2.9 It can be noted that COX-1 and COX-2 are monotopic membrane proteins.10,11 The interfacial localization of COX-2 may be important for inhibition by NSAIDs because these compounds preferentially partition in membranes to access the cyclooxygenase active site.12 NSAIDs must pass through the cell membrane and, in addition, either enter the interior of the endoplasmic membrane or pass through the plasma membrane to reach the enzyme.13 Moreover, it is increasingly accepted that cytosolic phospholipase A2 (cPLA2), COX-1/COX-2, and prostacyclin synthase are colocalized in the nuclear envelope and endoplasmic reticulum and are functionally coupled to facilitate the transfer of intermediate metabolites.14,15 The oxicam NSAIDs have different pharmacokinetics,16,17 treatment efficiency,18 and adverse effects.3,18-20 In this situation, (9) Wolfe, M. M.; Lichtenstein, D. R.; Singh, G. N. Engl. J. Med. 1999, 340, 1888–1899. (10) Spencer, A. G.; Woods, J. W.; Arakawa, T.; Singer, I. I.; Smith, W. L. J. Biol. Chem. 1998, 273, 9886–9893. (11) Picot, D.; Loll, P. J.; Garavito, R. M. Nature 1994, 367, 243–249. (12) MirAfzali, Z.; Leipprandt, J. R.; McCracken, J. L.; DeWitt, D. L. J. Biol. Chem. 2006, 281, 28354–28364. (13) Luger, P.; Daneck, K.; Engel, W.; Trummlitz, G.; Wagner, K. Eur. J. Pharm. Sci. 1996, 4, 175–187. (14) Wu, K. K.; Liou, J.-Y. Biochem. Biophys. Res. Commun. 2005, 338, 45–52. (15) Scott, K. F.; Bryant, K. J.; Bidgood, M. J. J. Leukocyte Biol. 1999, 66, 535– 541. (16) Busch, U.; Heinzel, G.; Narjes, H. Eur. J. Clin. Pharmacol. 1995, 48, 269– 272. (17) Olkkola, K. T.; Brunetto, A. V.; Mattila, M. J. Clin. Pharmacokinet. 1994, 26, 107–120. (18) Moser, U.; Waldburger, H.; Schwarz, H. A.; Gobelet, C. A. Scand. J. Rheumatol. Suppl. 1989, 80, 71–80. (19) Avouac, B. Rev. Rhum. Mal. Osteoartic. 1988, 55, 444–450. (20) Jolliet, P.; Simon, N.; Bree, F.; Urien, S.; Pagliara, A.; Carrupt, P.-A.; Testa, B.; Tillement, J.-P. Pharm. Res. 1997, 14, 650–656.

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both a better understanding of the complex, membrane-related processes of COX inhibition and in vitro methods for comparing21 the anti-inflammatory activity and efficiency of different NSAIDs are needed. Valuable insight can be obtained from studies using oxicams and model lipid membranes22 such as small unilamellar vesicles,23 micelles,24 and liposomes.22,25 Indeed, the membrane fusogenic activity,26 lipid antioxidant activity,27 and lipid/water partitioning23 of different oxicams were monitored in phospholipid bilayers. Studies of the interaction with micelles used as membrane models permitted a better understanding of the charge and hydrophobic effect in the action of NSAIDs.28 Studies by Kyrikou et al. using differential scanning calorimetry, Raman spectroscopy, and modeling showed that meloxicam, tenoxicam, piroxicam and lornoxicam are inserted between the polar and hydrophobic regions of lipid bilayers.29 Recently, experiments performed with liposomes allowed the classification of structurally similar oxicams in two groups on the basis of different interactions with the bilayer. It was shown that tenoxicam (TEN) and piroxicam (PIR), known as COX-1 inhibitors, demonstrated a higher partitioning capacity in liposome/water systems, together with a smaller ability to change the membrane fluidity and surface potential in comparison with COX-2 inhibitors lornoxicam and meloxicam.25 Compared to other model membranes, lipid monomolecular films (Langmuir films) are readily amenable to study via a rich variety of experimental techniques.30 The interaction between some drugs and monolayers has already been studied.31-33 To obtain molecular-level information, pure lipid components of cell membranes are used in such studies. In the present work, we show that monomolecular films formed with phosphoglycerides bearing different polar heads and hydrocarbon chains of different lengths can be successfully used both to quantify the impact on the lipid membrane and to differentiate between three structurally closely related oxicam NSAIDs, namely, piroxicam, tenoxicam, and meloxicam, which are molecules generated by isosteric replacement in drug design.2

Experimental Section Materials and Reagents. 1,2-Dipalmitoyl-sn-glycero-3phospho-rac-(1-glycerol) sodium salt (DPPG; P9789, g99%), 1,2-dipalmitoyl-sn-glycero-3-phospho-L-serine sodium salt (DPPS; P1185, g99%), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC; P4329, g99%), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE; P1348, g99%), 1,2-myristoyl-sn-glycero-3-phosphoethanolamine (DMPE; P5693, approximately 99%), 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine (DLPE) (99% pure), (21) Cordero, J. A.; Camacho, M.; Obach, R.; Domenech, J.; Vila, L. Eur. J. Pharm. Biopharm. 2001, 51, 135–142. (22) Ferreira, H.; Lucio, M.; Lima, J. L. F. C.; Cordeiro-da-Silva, A.; Tavares, J.; Reis, S. Anal. Biochem. 2005, 339, 144–149. (23) Chakraborty, H.; Sarkar, M. Biophys. Chem. 2007, 125, 306–313. (24) Chakraborty, H.; Banerjee, R.; Sarkar, M. Biophys. Chem. 2003, 104, 315– 325. (25) Lucio, M.; Ferreira, H.; Lima, J. L. F. C.; Reis, S. Med. Chem. 2006, 2, 447– 456. (26) Chakraborty, H.; Mondal, S.; Sarkar, M. Biophys. Chem. 2008, 137, 28–34. (27) Lucio, M.; Ferreira, H.; Lima, J. L. F. C.; Reis, S. Anal. Chim. Acta 2007, 597, 163–170. (28) Chakraborty, H.; Sarkar, M. Biophys. Chem. 2005, 117, 79–85. (29) Kyrikou, I.; Hadjikakou, S. K.; Kovala-Demertzi, D.; Viras, K.; Mavromoustakos, T. Chem. Phys. Lipids 2004, 132, 157–169. (30) Davies, J. T.; Rideal, E. K. Interfacial Phenomena, 2nd ed; Academic Press: New York, 1963. (31) Corvis, Y.; Barzyk, W.; Brezesinski, G.; Mrabet, N.; Badis, M.; Hecht, S.; Rogalska, E. Langmuir 2006, 22, 7701–7711. (32) Korchowiec, B.; Ben Salem, A.; Corvis, Y.; Regnouf de Vains, J.-B.; Korchowiec, J.; Rogalska, E. J. Phys. Chem. B 2007, 111, 13231–13242. (33) Wieclaw, K.; Korchowiec, B.; Corvis, Y.; Korchowiec, J.; Guermouche, H.; Rogalska, E. Langmuir 2009, 25, 1417–1426.

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tenoxicam (T0909, g99%), and piroxicam (P5654, g98%) were from Sigma-Aldrich, and meloxicam (>99%) was from Boehringer Ingelheim. The saturated lipids were chosen to avoid hydrocarbon chain oxidation. Aqueous solutions of 11 μM TEN, PIR, or MEL were used as subphases in all experiments. This concentration was chosen because it is below the solubility limit of MEL in water; the latter is the least soluble among the three oxicams.2,33-35 Appropriate amounts of NSAIDs were dissolved in 1 L of Milli-Q degassed water, and the solutions were stirred using a magnetic stirrer for 24 h at 20 °C and then filtered through a 0.45 μm PTFE filter. Chloroform and methanol (both ∼99.9% pure) used for preparing phospholipid solutions were from Sigma-Aldrich.

Compression Isotherms and Brewster Angle Microscopy. The surface pressure (Π) and electric surface potential (ΔV) measurements were carried out with a KSV 5000 Langmuir balance (KSV Instruments Ltd., Helsinki, Finland). A Teflon trough (15 cm 58 cm 1 cm) with two hydrophilic Delrin barriers (symmetric compression) was used in compression isotherm experiments. The system was equipped with an electrobalance and a platinum Wilhelmy plate (perimeter 3.94 cm) as a surface pressure sensor. The surface potential was measured using a KSV Spot 1 with a vibrating plate electrode and a steel counter electrode immersed in the subphase. The apparatus was enclosed in a Plexiglas box, and the temperature was kept constant at 20 °C. All solvents used for cleaning the trough and the barriers were of analytical grade. Aqueous subphases for monolayer experiments were prepared with Milli-Q water, which had a surface tension of 72.8 mN m-1 at 20 °C and pH 5.6. Because oxicams are absorbed from the intestinal region, the latter pH value was chosen because it corresponds to the duodenum conditions.3 Monolayers were spread from calibrated solutions (concentration of about 0.5 mg mL-1) of DPPC in chloroform and DPPG, DPPS, DPPE, DLPE, and DMPE in a chloroform/methanol mixture (3:1 v/v) using a microsyringe (Hamilton Co.). After an equilibration time of 20 min, the films were compressed at a rate of 5 mm min-1 barrier-1 by two symmetrically moving barriers. A PC computer and KSV software were used to control the experiments. Each compression isotherm was performed at least three times. The standard deviation was (0.5 A˚2 for the mean molecular area (A), (0.2 mN m-1 for the surface pressure, and (0.005 V for the surface potential measurements. The compression isotherms allowed the determination of the compressibility modulus (CS-1 = -A(∂Π/∂A)T). Collapse parameters ΔVcoll, Πcoll, and Acoll and the parameters corresponding to surface pressures of 10 and 30 mN m-1 were determined directly from the compression isotherms. The morphology of the studied films was visualized using a computer-interfaced KSV 2000 Langmuir balance combined with a Brewster angle microscope (KSV Optrel BAM 300, Helsinki, Finland). The Teflon trough dimensions were 6.5 cm 58 cm 1 cm; other experimental conditions were as described above.

Polarization-Modulation Infrared Reflection-Absorption Spectroscopy. The PM-IRRAS spectra of phospholipid monolayers spread on pure water or on aqueous solutions of drug molecules were registered at 20 °C. The Teflon trough dimensions were 36.5 cm 7.5 cm 0.5 cm; other experimental conditions were as described in the preceding paragraph. PM-IRRAS measurements were performed using a KSV PMI 550 instrument (KSV Instruments Ltd., Helsinki, Finland). The PMI 550 contains a compact Fourier transform IR spectrometer equipped with a polarization-modulation (PM) unit on one arm of a goniometer and an MCT detector on the other arm. The incident angle of the light beam can be freely chosen to be between 40 and 90°; here, the incident angle was 75°. The spectrometer and the PM unit operate at different frequencies, allowing the separation (34) Naidu, N. B.; Chowdary, K. P. R.; Murthy, K. V. R.; Satyanarayana, V.; Hayman, A. R.; Becket, G. J. Pharm. Biomed. Anal. 2004, 35, 75–86. (35) David, V.; Albu, F.; Medvedovici, A. J. Liq. Chromatogr. Relat. Technol. 2004, 27, 965–984.

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Figure 1. Structures of NSAIDs: (A) tenoxicam, (B) piroxicam, and (C) meloxicam. The partition coefficient (log P) values estimated from the drug partitioning between n-octanol and buffer at pH 5.0 are 0.67, 1.57, and 1.91 in A-C, respectively.13.

Figure 2. Compression isotherms of phospholipid monolayers spread in the presence and in the absence of TEN, PIR, or MEL. Results obtained with (A) DPPG, (B) DPPS, (C) DPPC, (D) DPPE, (E) DMPE, and (F) DLPE; Π-A isotherms, solid lines; ΔV-A isotherms, dotted lines; black curves, pure phospholipid film spread on a pure water subphase; red curves, pure phospholipid film spread on the 11 μM TEN aqueous solution subphase; green curves, pure phospholipid film spread on the 11 μM PIR aqueous solution subphase; and blue curves, pure lipid film spread on the 11 μM MEL aqueous solution subphase. Temperature 20 °C; pH 5.6. Langmuir 2010, 26(5), 3485–3492

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Czapla et al. Table 1. Isotherm Parameters at 10 and 30 mN m-1 Π = 10 (mN m-1)

Π = 30 (mN m-1)

A (A˚2)

CS-1 (mN m-1)

ΔV (V)

A (A˚2)

CS-1 (mN m-1)

ΔV (V)

DPPG on water DPPG on 11 μM TEN DPPG on 11 μM PIR DPPG on 11 μM MEL

48 48 51 53

183.4 175.7 119.3 98.7

0.19 0.29 0.34 0.39

44 44 45 46

255.7 225.0 174.1 163.7

0.19 0.30 0.36 0.41

DPPS on water DPPS on 11 μM TEN DPPS on 11 μM PIR DPPS on 11 μM MEL

45 46 49 51

237.6 229.5 123.6 116.1

0.32 0.39 0.41 0.42

42 42 43 45

442.5 359.5 250.9 203.5

0.33 0.41 0.43 0.45

DPPC on water DPPC on 11 μM TEN DPPC on 11 μM PIR DPPC on 11 μM MEL

52 53 57 66

88.4 66.7 49.3 31.8

0.49 0.49 0.51 0.43

46 46 47 49

221.1 205.6 164.7 111.4

0.55 0.55 0.57 0.57

DPPE on water DPPE on 11 μM TEN DPPE on 11 μM PIR DPPE on 11 μM MEL

46 47 49 53

235.0 219.4 162.8 89.7

0.56 0.56 0.56 0.56

43 43 44 46

298.2 277.8 197.0 185.7

0.58 0.58 0.58 0.58

DMPE on water DMPE on 11 μM TEN DMPE on 11 μM PIR DMPE on 11 μM MEL

48 49 52 63

79.1 78.4 50.6 27.2

0.53 0.55 0.53 0.48

43 43 45 46

207.8 205.4 148.7 105.1

0.57 0.59 0.59 0.58

DLPE on water DLPE on 11 μM TEN DLPE on 11 μM PIR DLPE on 11 μM MEL

71 72 77 86

52.8 51.6 47.8 40.8

0.35 0.38 0.37 0.39

54 55 57 61

79.4 76.3 66.0 60.9

0.44 0.47 0.46 0.48

of the two signals at the detector. The PM unit consists of a photoelastic modulator, which is an IR-transparent, ZnSe piezoelectric lens. The incoming light is continuously modulated between s and p polarization at a frequency of 74 kHz. This allows the simultaneous measurement of spectra for the two polarizations, the difference providing surface-specific information and the sum providing the reference spectrum. Because the spectra are measured simultaneously, the effect of water vapor is largely removed. The PM-IRRAS spectra of the film-covered surface, S(f), as well as that of the pure water, S(w), were measured, and the normalized difference ΔS/S = [S(f) - S(w)]/ S(w) is reported. Interferogram scans (6000, 10 scans/s) have been acquired for each spectrum. The peak retardation wavelength for modulation can freely be selected depending on the wavelength region of interest. Half-wave retardations of 1500 and 2900 cm-1 were used to analyze the methylene and carbonyl regions of the spectra, respectively. The spectral range of the device is 800-4000 cm-1, and the resolution is 8 cm-1.

Results and Discussion The three oxicams used in this study are small, rigid molecules (Figure 1) with hydrophobicity increasing in the order of TEN < PIR < MEL.2,35 The Langmuir technique was used to quantify the interaction between the oxicams and model membranes and to explore the possibility of using lipid films to differentiate between structurally closely related molecules. To this end, surface pressure and surface potential measurements as well as PM-IRRAS and BAM experiments were performed. The films were formed with lipids bearing different headgroups, namely, DPPG, DPPS, DPPC, and DPPE, or chains of different lengths, namely, DPPE, DMPE, and DLPE. Surface Potential Measurements. Figure 2 shows compression isotherms of pure phospholipid monolayers spread on a pure water subphase and on 11 μM TEN, PIR, or MEL solutions. The characteristic parameters of the isotherms (Experimental Section) are given in Tables 1 and 2. 3488 DOI: 10.1021/la903052t

Table 2. Isotherm Parameters at Collapse Πcoll (mN m-1)

Acoll (A˚2)

CS-1 (mN m-1)

ΔVcoll (V)


53.1 52.1 50.9 49.1

41 41 41 42

932.6 517.3 366.8 287.0

0.20 0.31 0.37 0.41


51.2 49.8 48.6 48.0

40 41 41 41

1146.2 815.2 343.1 282.9

0.34 0.41 0.44 0.46


56.3 55.7 55.5 54.4

41 41 42 42

287.1 261.3 260.8 221.7

0.57 0.57 0.59 0.59


54.9 54.7 53.1 51.2

40 40 41 41

788.6 653.7 413.3 272.1

0.59 0.59 0.59 0.59


56.4 56.3 54.1 48.7

40 40 41 42

598.6 528.8 472.1 462.8

0.58 0.59 0.59 0.59


54.3 54.1 54.0 52.0

40 40 41 42

301.5 240.1 195.7 155.4

0.54 0.58 0.57 0.59

The effect of the drugs on the surface properties of phospholipid monolayers was clearly observed with the ΔV-A isotherms (Figure 2, dotted lines). Indeed, the ΔV jumps characteristic of the liquid-expanded-gas (LE-G) phase transitions observed in the ΔV-A isotherms of pure phospholipids are less pronounced in Langmuir 2010, 26(5), 3485–3492

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Figure 3. PM-IRRAS spectra of DPPG (A, B), DPPS (C, D), DPPC (E, F), DPPE (G, H), DMPE (I, J), and DLPE (K, L) collected at 30 mN m-1 in the presence or absence of TEN, PIR, or MEL in the system. Black curves indicate a pure phospholipid film spread on the pure water subphase; red curves indicate a pure phospholipid film spread on the 11 μM TEN aqueous solution subphase; green curves indicate a pure phospholipid film spread on the 11 μM PIR aqueous solution subphase; and blue curves indicate a pure phospholipid film spread on the 11 μM MEL aqueous solution subphase. Temperature 20 °C, pH 5.6.

the presence of the drugs in the subphases. The surface potential values of the lipid films spread on the subphase containing NSAIDs are higher than those corresponding to the films spread on pure water. More important changes in the surface potential values were observed with anionic DPPG and DPPS compared to zwitterionic DPPC and DPPE. In the case of phosphatidylethaLangmuir 2010, 26(5), 3485–3492

nolamines, the chain length has no meaningful effect on the surface potential, as shown with DPPE, DMPE, and DLPE. In general, with all phospholipids used, the highest surface potential values are observed in the case of the films spread on the subphases containing MEL (Figure 2, Tables 1 and 2). This result is in accordance with those obtained by Lucio et al.25 It should be DOI: 10.1021/la903052t

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Table 3. Characteristic Vibrational Wavenumbers of Phospholipid Bonds upon Interaction with NSAIDs at Π = 30 mN m-1 νas(CH2) (cm-1)

νs(CH2) (cm-1)

ν(CdO) (cm-1)


2918 2919 2916 2915

2850 2850 2850 2843

1735 1734 1736 1737


2917 2917 2916 2916

2848 2850 2850 2847

1739 1739 1740 1740


2919 2918 2918 2918

2849 2849 2850 2850

1727 1734 1736 1736


2919 2920 2920 2920

2850 2850 2852 2850

1725 1735 1736 1736


2920 2920 2921 2927

2851 2850 2856 2855

1730 1731 1733 1735


2921 2922 2924 2926

2853 2844 2846 2853

1732 1732 1734 1739

noted that the acidity of the phenolic -OH group in PIR and TEN bearing a piridynyl moiety is higher compared to that in MEL.2,36 The higher ΔV values observed in the case of MEL could be linked to its higher lipophilicity compared to that of TEN and PIR;2,33-35 the difference between the lipophilicity of those molecules in nonionic states was revealed recently using modeling.29 Indeed, MEL would penetrate more easily to the lipid films, displace water molecules, and form hydrogen bonds with the phospholipid polar heads, dehydrating and structuring the film. Moreover, the electrostatic interaction with the lipid polar heads may be less repulsive in the case of MEL than with the more polar PIR or TEN. The differentiation between the three oxicams, which is particularly well seen in the case of the negatively charged monolayers, may be linked to this effect. The high ΔV values observed in the presence of the oxicams in the most condensed states of the DPPG and DPPS monolayers may indicate that the drugs are expelled from the hydrocarbon chain region upon compression, as indicated by the Π-A isotherms, but interact with the lipid headgroups. Surface Pressure Measurements. As shown in Figure 2 using Π-A isotherms, the three oxicams are clearly differentiated in the monolayers. Indeed, the Π-A isotherms of the monolayers formed on the subphases containing PIR or MEL are shifted to higher molecular areas compared to pure water, but no meaningful shift can be observed with TEN. This shift is more important for MEL than for PIR (e.g., with DLPE at a surface pressure of 30.0 mN m-1, the isotherm shifts are around 7 and 3 A˚2 with MEL and PIR, respectively). In general, for both MEL and PIR, the shift of the isotherms decreases with the increase in surface pressure (e.g., it is more significant at 10 mN m-1 than at 30 mN m-1 (Table 1) and very low at the collapse point (Table 2)). (36) Jayaselli, J.; Cheemala, J. M. S.; Geetha, R. D. P.; Pal, S. J. Braz. Chem. Soc. 2008, 19, 509–515.

3490 DOI: 10.1021/la903052t

The overall results indicate that MEL and PIR penetrate to the monolayer from the subphase and significantly change the molecular orientation and monolayer ordering, whereas TEN would stay in the subphase and, as indicated by the ΔV-A isotherms, interact with the polar head region of the layer. It can be observed that the negatively charged DPPS and DPPG allow a clear differentiation of the oxicams via the ΔV measurements, which can be easily explained by the interaction of the oxicams with the charged lipid headgroups. In the case of the zwitterionic phosphoethanolamines (PEs), the differentiation is more pronounced using the ΔΠ measurements; the latter is particularly well seen in the shorter-chain DMPE and DLPE (Figure 2A-D). This effect is not surprising because drug penetration from the subphase to the less rigid films formed with the medium-chain lipids is easier. Different isotherm parameters are indicative of the interaction of the oxicams with the monolayers. Indeed, the compressibility modulus values obtained from the Π-A isotherms (Table 1 and 2) are significantly affected by the presence of the drugs in the subphase, in particular, by PIR and MEL. In general, all of the films spread on the subphases containing NSAIDs are more liquidlike than the films spread on pure water, as shown by the corresponding CS-1 values. Likewise, the stability of the monolayers decreases in the presence of drugs in the system, as indicated by the decreasing values of Πcoll (Table 2). However, both CS-1 and Πcoll values indicate that the films containing PIR or MEL are less stable and more liquidlike than those containing TEN. PM-IRRAS: Pure Lipid Monolayers. PM-IRRAS was used to gain more insight into the interaction between NSAIDs and phospholipids.33,37-39 The influence of NSAIDs on the phospholipid monolayers can be monitored via the frequency of the lipid symmetric and antisymmetric methylene group stretching vibrations [(νs(CH2) and νas(CH2)] (around 2850 and 2920 cm-1, respectively) and the CdO stretching band (around 1730 cm-1).40 Indeed, the frequency of the methylene bands is sensitive to the conformation of phospholipid acyl chains.41,42 A shift from 2920 and 2850 cm-1 to lower wavenumbers indicates higher chain ordering in the film, and a shift to higher wavenumbers suggests chain disordering.43 The stretching ν(CdO) band is used to investigate the interfacial region of phospholipids. This frequency is sensitive to hydrogen bonding and provides information about the carbonyl groups’ accessibility to water, and its shift to higher wavenumbers indicates CdO group dehydration.44 The PM-IRRAS spectra of pure phospholipid films formed in the absence and in the presence of NSAIDs were collected at a surface pressure of 30 mN m-1 (Figure 3). The characteristic vibrations of phospholipids, the symmetric and antisymmetric methylene stretching vibrations, and the carbonyl stretching40-45 are clearly visible in the spectra (Figure 3, black curves). (37) Blaudez, D.; Buffeteau, T.; Cornut, J. C.; Desbat, B.; Escafre, N.; Pezolet, M.; Turlet, J. M. Appl. Spectrosc. 1993, 47, 869–874. (38) Blaudez, D.; Buffeteau, T.; Cornut, J. C.; Desbat, B.; Escafre, N.; Pezolet, M.; Turlet, J. M. Thin Solid Films 1994, 242, 146–150. (39) Blaudez, D.; Turlet, J.-M.; Dufourcq, J.; Bard, D.; Buffeteau, T.; Desbat, B. J. Chem. Soc., Faraday Trans. 1996, 92, 525–530. (40) Cornut, I.; Desbat, B.; Turlet, J. M.; Dufourcq, J. Biophys. J. 1996, 70, 305– 312. (41) Dicko, A.; Bourque, H.; Pezolet, M. Chem. Phys. Lipids 1998, 96, 125–139. (42) Pinazo, A.; Wen, X.; Liao, Y.-C.; Prosser, A. J.; Franses, E. I. Langmuir 2002, 18, 8888–8896. (43) Dyck, M.; Kerth, A.; Blume, A.; Loesche, M. J. Phys. Chem. B 2006, 110, 22152–22159. (44) Allouche, M.; Castano, S.; Colin, D.; Desbat, B.; Kerfelec, B. Biochemistry 2007, 46, 15188–15197. (45) Du, X.; Wang, Y. J. Phys. Chem. B 2007, 111, 2347–2356.

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Figure 4. BAM micrographs of DMPE monolayers in the absence and in the presence of TEN, PIR, or MEL. Pure DMPE at 72, 67, and 57 A˚2 (A-C); DMPE on the TEN solution subphase at 73, 68, and 58 A˚2 (D-F); DMPE on the PIR solution subphase at 75, 70, and 60 A˚2 (G-I); DMPE on the MEL solution subphase at 83, 78, and 68 A˚2 (J-L). Scale: the width of the snapshots corresponds to 400 μm.

The νas(CH2) and νs(CH2) bands of the pure, long-chain DPPG, DPPS, DPPC, and DPPE appear at wavenumbers lower than 2920 and 2850 cm-1, respectively (Table 3), which indicates acyl chains with high conformational order.45 It should be noted that in the case of DPPS and DPPG monolayers lower wavenumbers than for DPPC and DPPE were observed, suggesting that the former are more rigid. This is in good agreement with the compression isotherm analysis (Table 1). By the same token, the νas(CH2) and νs(CH2) bands observed for DMPE and DLPE, which appear at slightly higher wavenumbers compared to those for DPPE, indicate chain disordering and a more fluidlike character of the films formed with the PEs bearing shorter chains; this observation is in accordance with the compression isotherm analysis as well (Table 1). The higher values of the ν(CdO) wavenumbers observed in the case of DPPG and DPPS compared to PEs indicate the dehydration of the carbonyl and a decrease in the water accessibility of this moiety in the negatively charged phosphoglycerides. However, the lower values of the ν(CdO) wavenumbers observed in the case of DPPE compared to DMPE and DLPE indicate that a higher number of carbonyl groups are hydrogen bonded in (46) Du, X.; Liang, Y. J. Phys. Chem. B 2000, 104, 10047–10052.

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the former lipid. This effect indicates that hydrogen bond formation is facilitated in rigid monolayers, formed with longer-chain lipids.46-48 PM-IRRAS: Monolayers Formed in the Presence of NSAIDs. In the condensed states of the films, the impact of NSAIDs on the hydrocarbon chains is low. However, the shift of the νas(CH2) and νs(CH2) bands to higher wavenumbers observed with DMPE and DLPE in the presence of MEL and PIR (Figure 3, Table 3) clearly shows the disorganizing effect of these two oxicams. This result is in accordance with the compression isotherm experiments showing that the oxicams penetrate more easily to the more fluid films compared to the films formed with the long-chain phosphoglycerides. The PM-IRRAS results are in accordance with those obtained from the compression experiments and show that the DMPE and DLPE monolayers can be used as probes of the relative hydrophobicity of different molecules. On the basis of the νas(CH2) and νs(CH2) wavenumbers, the hydrophobicity of the three oxicams increases in the order of TEN < PIR < MEL. (47) Leite, V. B. P.; Cavalli, A.; Oliveira, O. N., Jr. Phys. Rev. E: Stat. Phys., Plasmas, Fluids, Relat. Interdiscip. Top. 1998, 57, 6835–6839. (48) Miranda, P. B.; Du, Q.; Shen, Y. R. Chem. Phys. Lett. 1998, 286, 1–8.

DOI: 10.1021/la903052t

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Changes in the lipid headgroup hydration in the presence of the drugs are clearly visible with all lipid monolayers. In general, in the systems containing the drugs, the CdO band is shifted to higher wavenumbers (>1730 cm-1) compared to that for pure lipid monolayers. This shift reflects the dehydratation of carbonyl moieties49 upon interaction with drug molecules. It can be noticed that this effect is more important for MEL than for PIR and more important for PIR than for TEN. Also noteworthy is the fact that with some lipids in the presence of the oxicams a conspicuous signal in the 1715-1720 cm-1 region is observed (e.g., Figure 3D). Although a further experimental check is still necessary, this result may indicate the formation of hydrogen bonds between the lipid carbonyl50 and the electrodonor oxicam groups. It can be concluded that the less polar oxicams embedded in the monolayer would contribute more to the dehydration of the carbonyl groups than would the more polar oxicams present in the water subphase in the polar head region. We propose that for this reason, among the three oxicams studied, MEL has the most pronounced impact both on the hydrocarbon chain organization and on the carbonyl dehydration. Brewster Angle Microscopy. To gain more insight into the structure of phospholipid monolayers upon interaction with NSAIDs, BAM measurements were performed. Because DMPE monolayers form characteristic domains in the liquid expandedliquid condensed (LE-LC) phase-transition region, DMPE was used as a model lipid in these experiments. The BAM images of the pure DMPE monolayers spread on pure water as well as on the TEN, PIR, and MEL solutions are presented in Figure 4. In the first column, the snapshots of the beginning of the LE-LC phase transition are shown, that is, at 72, 73, 75, and 83 A˚2 for DMPE spread on pure water as well as on the TEN, PIR, and MEL solutions. The snapshots shown in the next two columns were taken at molecular areas that were 5 and 15 A˚2 lower, respectively, than the first column, that is, at 67, 68, 70, and 78 A˚2 (second column) and at 57, 58, 60, and 68 A˚2 (third column). It can be seen that the condensed-phase domains of DMPE appearing in the presence of NSAIDs in the subphase are smaller and more numerous. This effect is more pronounced in the case of MEL (Figure 4J-L) than in the systems containing TEN or PIR (Figure 4D-F and Figure 4G-I, respectively). (49) Dziri, L.; Desbat, B.; Leblanc, R. M. J. Am. Chem. Soc. 1999, 121, 9618– 9625. (50) Lewis, R. N.; McElhaney, R. N.; Pohle, W.; Mantsch, H. H. Biophys. J. 1994, 67, 2367–2375.

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Generally, the BAM results suggest the most liquidlike character of the films formed in the presence of MEL, followed by PIR and TEN; this observation is in accordance with the compression isotherm characteristics.

Conclusions The monolayer experiments performed in this study revealed significant differences between the interfacial properties of MEL, PIR, and TEN. The surface pressure and surface potential measurements as well as PM-IRRAS and BAM experiments show that MEL has the most important effect on the lipid films, followed by PIR and TEN. We propose that the least-polar MEL penetrates more easily to the film than do PIR and TEN and the effects observed are proportional to the number of oxicams present in the film. All of the results taken together indicate that the oxicams interact both with the apolar methylene and with the polar carbonyl groups; this leads us to think that the oxicams are localized between the lipid hydrocarbon chains and the polar heads in the monolayer. This proposal is in agreement with the literature.29 It can be expected that the capacity of the NSAIDs to penetrate into the lipid layer is decisive for accessing and inhibiting COX-2 and, consequently, for the therapeutic efficiency of these molecules. From the point of view of analytical purposes, it can be observed that the negatively charged DPPG and DPPS are well suited for differentiating the oxicams via surface potential measurements, whereas the liquid character of the films is important for a differentiation based on the molecular area and PM-IRRAS measurements. We suggest that the insights obtained from the monolayer experiments may be useful for anticipating the chromatographic behavior of structurally similar eluters in reversephase chromatography35 and, in particular, for the rapid evaluation of the pH effect on the properties of labile molecules such as tautomeric PIR used here and other tautomers (e.g., amino acids).51 Acknowledgment. This work was supported by a Hubert Curien partnership (“Polonium”, no. 20077QA) and the Ministry of Science and Higher Education, Poland (project no. 1206/ GDR/2007/03). The technical assistance of Eric Dubs is gratefully acknowledged. We thank Dr. Matthew Fielden for helpful discussions and for revising the English. (51) Headley, A. D.; Starnes, S. D. J. Am. Chem. Soc. 1995, 117, 9309–9313.

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