Interaction of Clonixin with EPC Liposomes Used As Membrane Models

Interaction of Clonixin with EPC Liposomes Used as Membrane Models

HELENA FERREIRA,1 MARLENE LU´ CIO,1 JOSE´ L.F.C. LIMA,1 CARLA MATOS,2 SALETTE REIS1

1REQUIMTE, Departamento de Quı´mica-Fı´sica, Faculdade de Farma´cia, Universidade do Porto, Rua Anı´bal Cunha, 164, 4050-047 Porto, Portugal 2REQUIMTE, Faculdade Cieˆncias da Sau´de, Universidade Fernando Pessoa, Rua Carlos da Maia, no. 296, 4200-150 Porto, Portugal

Received 15 December 2004; revised 15 February 2005; accepted 16 February 2005 Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jps.20351

ABSTRACT: In this work, an overall analysis of clonixin interaction with liposomes was achieved using different techniques, which allowed the evaluation of the change in different membrane’s characteristics as well as the possible location of the drug in the membrane. Clonixin acidity constants were obtained and the values are 5.5 0.08 and 2.2 0.04. Clonixin partition coefficient (Kp) between liposomes and water was also determined using derivative spectrophotometry, fluorescence quenching, and zeta- potential (z-potential). These three techniques yielded similar results. z-potential measurements were performed and an increase of the membrane negative charge with an increase of drug concentration was observed. Drug location within the bilayer was performed by fluorescence quenching using a set of n-(9-anthroyloxy) fatty acid probes (n ¼ 2, 6, 9, and 12). The fluorescence intensity of all probes was quenched by the drug. This effect is more noticeable for the outer located probe, indicating that the drug is positioning in the external part of the membrane. These same probes were used for steady-state anisotropy measurements to determine the perturbation in membrane structure induced by clonixin. Clonixin increased membrane fluidity in a concentration dependent manner, with the highest perturbation occurring nearby the 2-AS probe, closely located to the bilayer surface. ß 2005 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 94:1277–1287, 2005 Keywords: clonixin; liposomes; drug interaction; light scattering; UV/Vis spectroscopy; fluorescence spectroscopy; partition coefficient; drug location; membrane fluidity

INTRODUCTION phenomena, such as neutrophil function inhibition, oxidative phosphorylation inhibition in Cyclooxygenase (COX), a membrane related mitochondria, signal transduction disruption, enzyme, is the pharmacological target of non- and the consequent interference with intracel- steroidal anti-inflammatory drugs (NSAIDs), lular calcium mobilization and protein kinase C which are therefore commonly used in inflamma- activity alteration, have all been reported by tory diseases treatment. Although low doses of Klein et al.,1 as well as a membrane fluidity NSAIDs inhibit prostaglandins biosynthesis, high alteration, which has been mentioned by several concentrations interfere with processes not de- authors.2–5 Furthermore, NSAIDs have been pendent on these mediators. Membrane related shown to inhibit the cellular proliferation rate, to alter the cell cycle regulation, and to induce apoptosis in cancer cell lines, in a mechanism in- Correspondence to: Salette Reis (Telephone: þ351-222-078- dependent from prostaglandin pathways.1 966. Fax: þ351-222-004-427; E-mail: [email protected].) There is consensual evidence that the lipid Journal of Pharmaceutical Sciences, Vol. 94, 1277–1287 (2005) ß 2005 Wiley-Liss, Inc. and the American Pharmacists Association affinity of the NSAIDs is of major significance

JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 94, NO. 6, JUNE 2005 1277 1278 FERREIRA ET AL. for their toxic and therapeutic actions. Indeed, quenching. Using the z-potential technique, it was depending on their hydrolipophilic character, possible to evaluate the interaction of clonixin NSAIDs can be distributed between the mem- with liposomes by measuring the membrane brane and the aqueous phases. This distribution potential arising from the drug partitioning. In determines their concentration in each phase and fact, biological membranes are charged, due to thereby controls the extents of their penetration ionized components (lipids, glycolipids, glycopro- into the membrane and/or their interactions with teins), and the resulting surface potential plays a phospholipids or other membrane components, critical role in regulatory processes, membrane– such as COX enzymes, which are embedded in membrane interactions, and in their binding capa- the lipid bilayers.6 Thus, for the study of NSAIDs’ city to solutes in solution.13,14 Additionally, to action mechanisms and their side-effects, it is of electrostatic effects, which can affect the confor- great importance to investigate the interactions mation and activity of membrane and membrane- between these drugs and biomembranes. For this bound enzymes,15,16 several cell processes are also purpose, this work was performed using lipo- related to electrostatic or polarization effects on somes of egg yolk phosphatidylcholine (EPC). the cell membrane. In this context, the character- Liposomes are generally accepted to be a suitable ization of the electrostatic membrane properties model for the study of membrane structure and induced by clonixin binding is a fundamental properties, because they are surrounded by a lipid parameter, and it also allows the quantification bilayer structurally similar to the lipidic matrix of clonixin molecules in these membranes. Conse- 6,7 of the cell membranes. Additionally, because of quently, Kp values can be calculated. being constituted by natural lipids, EPC liposomes Fluorescence quenching was also used to mea- can mimic the chemical and structural anisotropic sure clonixin’s coefficient partition. The fluores- environment of cell membranes. EPC liposomes cent n-(9-anthroyloxy)-stearic acids (n-AS, n ¼ 2, also appear to mimic the interfacial character as 6, 9, and 12) are the set of probes most widely well as the ionic, H-bond, dipole–dipole, and used for obtaining information on molecular hydrophobic interactions, which may define parti- aggregates, such as liposomes and natural mem- tioning in real biomembranes.6,7 branes.17–22 For these probes, there is evidence Traditionally, the octanol–water partition coef- that the anthroyloxy fluorophores are located at ficient (Kp) has been used to measure compounds’ a graded series of depths inside a membrane, hydrophobicity, which is correlated to drug activ- depending on its substitution position (n) in the ity. The octanol–water system is a good membrane aliphatic chain.17 Therefore, these probes, due to model when polar group interactions between the their exceptional environmental sensitivity, have solute and the phospholipid bilayer are minimal or been employed to monitor the microenvironment absent. However, since octanol can only model of membranes. These appropriate measurements non-polar interactions,8 better systems are needed allow information about the local membrane for molecules which can establish electrostatic in- structure to be inferred. According to this, besides teractions with polar groups in the membrane. the determination of clonixin’s Kp, fluorescence According to this, the study of clonixin’s partition quenching provides a mean to evaluate the posi- in a liposome/buffer system has been performed. tion and orientation of the drug in the membrane There is a more satisfactory correlation between by a comparative analysis of all probe’s quenching. this parameter and its pharmacological properties Furthermore, the fluorescent probes are capable of since clonixin has proved to be able to establish sensing a ‘‘fluidity’’ gradient through the bilayer electrostatic interactions with polar groups in the leaflet and, therefore, they were used to assess the biomembranes. The drug’s Kp was evaluated by clonixin effect in the lipid membrane fluidity. This derivative spectrophotometry, a technique that was achieved using steady-state anisotropy mea- can be used when a solute’s spectral characteristic surements, since that anisotropy depends upon changes between one media to another. Deriva- the rotational motion of the fluorophore and it is tive spectrophotometry eliminates the intense sensitive to hindrance forces imposed by the background signals that arise from light scattered microenvironment, property that has been widely by lipid vesicles, and it also improves the resolu- used to estimate membrane fluidity.18 Membrane tion of overlapping signals reported by several fluidity assessment gives useful physiologic infor- 9–12 authors. Moreover, the liposome/water Kp was mation as biomembranes need to be in a fluid state also determined by other experimental techni- in order to maintain complete biological function. ques: zeta-potential (z-potential) and fluorescence Indeed, any alteration in membrane fluidity tends

JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 94, NO. 6, JUNE 2005 INTERACTION OF CLONIXIN WITH EPC LIPOSOMES 1279 to change movement and/or orientation of pro- Fluorescence studies were carried out on a teins floating within lipid bilayer, as reported by Perkin-Elmer LS 50B steady-state fluorescence Dave and Witorsch.23 Becceria et al.3 described a spectrometer equipped with a constant-tempera- decrease in lymphocytes membrane fluidity in ture cell holder. All data were recorded at rheumatoid arthritis, and suggested membrane 25.0 0.18C in 1-cm cuvettes with excitation and fluidification as a good indicator of NSAID’s ther- emission slits between 4.0 and 9.0 nm. Excitation apeutic effects. Another important aspect in wavelength was set to 384 nm and emission physiological activity, is the acid–base properties wavelength to 446 nm for 12-AS and 9-AS probes, of drugs. Although little has been published about and to 451 nm for 6-AS and 2-AS ones. Fluo- clonixin’s acid–base chemistry and lipophilicity, rescence intensity data were corrected for absor- this characterization is necessary for thorough bance of the quencher (clonixin) at the excitation understanding of its pharmacokinetic and phar- wavelength.25 macodynamic parameters. z-potential values and size distribution of ex- Finally, clonixin has been chosen for this study truded EPC liposomes, with and without incorpo- because, besides its anti-inflammatory effect, it is rated drug, were determined at pH 7.4 (HEPES usually prescribed as an analgesic. Therefore, it is buffer), at 25.0 0.18C, by quasi-elastic light of great interest to investigate the behavior of scattering analysis using a ZET 5104 cell in a clonixin to compare with others classic NSAIDs, Malvern ZetaSizer 5000, with a 908scattering like nimesulide,24 once that therapeutic and toxic angle. activities can differ in structurally unrelated and homologous NSAIDs. Spectrophotometric Determination of the Acidity Constants EXPERIMENTAL SECTION Clonixin acidity constants were obtained from UV/Vis data in aqueous solution with ionic Reagents and Equipment strength adjusted to 0.1M by addition of NaCl. The anti-inflammatory drug clonixin was gener- The log [Hþ] value was consecutively changed ously supplied by its manufacturer Janssen-Cilag by potentiometric titration of 25.00 mL of an Pharmaceutica (Barcarena, Portugal) and was acidified aqueous solution (with hydrochloric acid used without further purification. The EPC and 30%) of the drug (approximately 30 mM) with ( )-12-(9-anthroyloxy)-stearic acid (12-AS) were NaOH (0.02M), under a nitrogen stream. The purchased from Sigma Chemicals (St. Louis, MO); potentiometric system calibration was performed the other probes, ( )-2-(9-anthroyloxy)-stearic by the Gran method,26 in terms of hydrogen ion acid, ( )-6-(9-anthroyloxy)-stearic acid, and concentration, by titrating solutions of strong acid ( )-9-(9-anthroyloxy)-stearic acid (2-AS, 6-AS, (103M HCl) with strong base (0.02M NaOH). and 9-AS) were purchased from Molecular Probes Absorption spectra were recorded in the system (Eugene, OR). All of these were used as supplied. described in ‘‘Reagents and Equipment.’’ Calcula- All the other chemicals were from Merck (Darm- tions were performed with data obtained from, at stadt, Germany) with pro analysi grade. Solutions least, four independent experiments using the were prepared with double-deionized water (con- program pHab.27 ductivity less than 0.1 ms/cm). The ionic strength of all solutions was adjusted to 0.1M with NaCl. Liposome Preparation and Drug Incorporation The polycarbonate filters with a diameter pore of 100 nm (Nucleopore) were obtained from What- Liposomes were prepared by the thin film hydra- man (Maidstone, England). tion method. According to this method, a known Absorption spectra were recorded at 25.0 amount of EPC was dissolved in chloroform/ 0.18C on a Perkin-Elmer Lambda 45 UV/VIS methanol (9:1). The organic solvent was evapo- spectrophotometer in the range 220–500 nm with rated under a nitrogen stream and the residual 1 nm intervals (a Hitachi U-2000 dual-beam spec- traces of solvent were removed by a further eva- trophotometer, a 220–400 nm range, and 2 nm poration for, at least, 3 h under the same stream. intervals for determination of acidity constants). The resulting dried lipid film was dispersed by the In all the cases, quartz cells were used and the addition of the buffers with different pH values temperature was kept constant by circulating (pH 3.0: 34.6 mM hydrochloric acid, 50 mM glycin; thermostated water in the cell holder. pH 7.4: 10 mM HEPES; pH 10.3: 43.2 mM sodium

JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 94, NO. 6, JUNE 2005 1280 FERREIRA ET AL. hydroxide, 50 mM boric acid; all buffers with buffer), at 25.0 0.18C. Lipid concentration I ¼ 0.1M and for pH 3.0 and 10.3 buffers the final was kept constant at approximately 400 mM. pH was adjusted by addition of strong acid or Clonixin concentrations ranged between 0 and base). These mixtures were then vortexed above 500 mM. The values for viscosity and refrac- their phase transition temperature (room tem- tive index were taken as 0.890 cP and 1.330, perature) to produce multilamellar liposomes respectively.30 (MLVs). MLVs suspensions were then equili- Particle size of the pure vesicles was found to brated for 30 min and extruded ten times through be 133 5 nm (average and standard deviation of polycarbonate filters with a pore diameter of the measurements of six independently prepared 100 nm (Nucleopore)6 at room temperature to suspensions). produce large unilamellar vesicles (LUVs). EPC concentration in vesicle suspensions was deter- Kp Determination by Fluorescence Quenching mined by phosphate analysis using the phospho- molybdate method.28 In fluorescence quenching studies, liposomes pre- In fluorescence studies, the fluorescence probes pared with the 2-AS probe were added to HEPES dissolved in ethanol were gently added to a (pH 7.4, 10 mM, I ¼ 0.1M) buffered solutions of liposome suspension to achieve a final probe to clonixin as described above. The EPC concentra- lipid ratio smaller than 1:100, to prevent changes tion of the liposomes ranged between approxi- in membranes structure. To ensure complete mately 80 and 900 mM and drug concentration incorporation of the probe in the lipid bilayer, the between 0 and 500 mM. The resulting suspensions suspensions were left to stand in the dark for were incubated in the dark for 1 h at room 30 min.29 Subsequent assays of liposome charac- temperature. terization by z-size measurements were made to confirm that no changes occurred in the fluores- Membrane Fluidity and Drug Location Studies cent marked vesicles. After the liposome preparation and/or labeling, Membrane fluidity was estimated by fluorescence the drug samples were prepared by mixing a anisotropy while drug location was determined by known volume of the drug to a suitable aliquot of fluorescence quenching; in both cases all n-AS vesicle suspension in buffer. The correspondent fluorescence probes in LUV suspensions at pH 7.4 reference solutions were identically prepared, in were used. The EPC concentration was approxi- the absence of drug. All suspensions were then mately 500 mM. HEPES (pH 7.4, 10 mM, I ¼ 0.1M) vortexed for 5 min and incubated for 30 min at buffered solutions of clonixin were added to the room temperature. liposomes prepared with the n-AS probes as previously described. Drug concentrations were in the range of 0 to 500 mM. Kp Determination by Derivative Spectrophotometry RESULTS AND DISCUSSION Clonixin Kp was determined in LUVs suspensions at pH 3.0, 7.4, and 10.3. In the derivative Spectrophotometric Determination spectrophotometry studies, a series of buffered of Acidity Constants suspensions containing a fixed concentration of drug (30 mM) and increasing concentrations of Clonixin contains two proton-binding sites (carbo- EPC (in the range 70–1200 mM) were prepared. xylate group and aminopyridinyl moiety) and can The correspondent reference solutions were iden- exist in four protonated forms in solution. Proto- tically prepared in the absence of drug. All nation equilibria suggested are shown in Figure 1, suspensions were then vortexed and incubated in a similar fashion to what has been suggest- in the dark for 30 min at room temperature and ed for a structurally similar molecule, niflumic the absorption spectra were recorded. acid.31 Neutral species are presumed to exist in a negligible percentage, in detriment of the zwitterionic species, and from this assumption, only z-Potential and Size Determinations three protonated states are considered: positive, z-potential values and size distribution of ex- zwitterionic, and negative species. truded EPC liposomes, with and without incorpo- Due to the low solubility of clonixin, acidity rated drug, were determined at pH 7.4 (HEPES constants were determined by spectrophotometry.

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Figure 1. Protonation equilibria of clonixin. Figure 2. Second and third derivative spectra of This method can be used to determine the clonixin clonixin at different concentrations of egg yolk phos- pKa values, since the protonation of one or other phatidylcholine (EPC): (1) 0; (2) 86; (3) 181; (4) 272; (5) functional group by changing the solution pH, 352; (6) 441; (7) 525; (8) 629; (9) 764; (10) 851; (11) 872; results in spectral changes. The pKa1 and pKa2 (12) 958; (13) 1127 mM. values of clonixin obtained were 5.5 0.08 and 2.2 0.04, respectively. These results resemble the ones obtained by Takaćs-Nova´k et al. with The derivative UV spectra of clonixin (Figure 2) 31 niflumic acid and can be used to calculate the pH- show a decrease in absorption intensities in dependent percentages of the species. Therefore, the presence of increasing amounts of EPC. Fur- at blood physiological pH (pH ¼ 7.4), clonixin thermore, the absorption spectra of clonixin in exists predominantly (98.7%) in the negative form, EPC exhibit isosbestic points and a bathochromic increasing this percentage with the pH value. shift in lmax with increasing lipid concentration (Figure 2). These observations suggest that the drug exists in two forms: drug in polar bulk water Kp Determination by 9,11 Derivative Spectrophotometry and in EPC bilayers. Derivative intensities can be related to the Kp of a compound between vesicle suspensions partition coefficient by the following expression: and the aqueous solution is defined as:30 ÂÃ ðÞAbsm Absw Kp½L Vf AT AbsT ¼ Absw þ ð2Þ m 1 þ Kp½L Vf Kp ¼ T ð1Þ ½A w where AbsT, Absm, and Absw are the total, lipid, T where [A ]m is the local drug concentration of A in and aqueous absorbances of the drug, respec- T the lipid phase and [A ]w in the water phase based tively, Kp is the partition coefficient, [L] is the on lipid and water solution volumes, Vm and Vw. lipid concentration, and Vf the lipid molar The concentration of drug partitioned in each volume. For EPC, the mean molecular weight phase can be determined by UV spectrophotome- was considered to be 700 g/mol and Vf to be 0.688 try, by non-linear regression of the data collected L/mol.32 This equation was fitted to the experi- in different EPC concentrations, providing that mental second and third derivative spectrophoto- the solute’s spectral characteristics (e and/or lmax) metric data (DT versus [L]) using a non-linear change when it permeates from the aqueous to the least-squares regression method (Figure 3), at lipid phases. However, it is not usually possible to wavelengths where the scattering is eliminat- obtain Kp using direct spectroscopic methods, ed. The value of Kp obtained in HEPES buffer owing to intense background signals arising from (pH ¼ 7.4) was 660 100. Kp were also determin- lipid vesicles light scattered. Derivative spectro- ed also at pH 3.0 and 10.3, and the results photometry eliminates the effect of background obtained were 530 100 and 870 50, respec- signals and it improves the resolution of over- tively (mean and standard deviation of at least lapping bands.9–12 two independent assays). Results show that,

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the maximum mole lipid:drug ratio that is, the membrane’s loading capacity. The values of smax can be obtained from the plot of s versus the concentration of total drug added to the system, [AT], ﬁtting the binding isotherm:36

T Kb½A y ¼ T ð5Þ 1 þ Kb½A in which y, the degree of saturation, is given by y ¼ s =smax. Knowledge of smax allows the calcu- lation of n: 30

1 smax ¼ ð6Þ ðaA þ nna LÞ Results for potential measurements obtained in Figure 3. Second-derivative spectrophotometric data HEPES buffer (pH 7.4) were obtained at increas- at l ¼ 316 nm for clonixin at different EPC concentrations (0; 86; 181; 272; 352; 441; 525; 629; 764; 851; 872; ing clonixin concentrations, and used to calculate 958; and 1127 mM). The curve represents the best fit to Kp by the mathematical formula described before. Equation 2. The values of smax were determined by fitting the Equation 6 the plot of s versus [AT], as can be observed in Figure 4, which shows an example despite what was observed for other NSAIDs in for this determination at several concentrations previous articles,24,30,33,34 the K values do not p of clonixin. The value of s obtained was 1.20 dramatically change with the change in medium max 0.20103 molecules/A˚ 2 (average and standard pH. Since clonixin shows electrical charged deviations of three independent experiments). groups at any pH value its hydrophilicity is main- The value of was calculated by Equation 6, as- tained and its affinity to the lipid bilayer does not suming a as 60 A˚ 2,37 and as a (molecular surface significantly change. L A area of clonixin) was not determined, one has assumed the same value (60 A˚ 2), because, as described previously, this parameter does not z-Potential Determinations Since clonixin is predominantly in its negative state at pH 7.4, its partition in the liposomes leads to the formation of a charged membrane surface. z-potential values, z, determined in different drug concentrations, can be used to calculate surface potential values (C0); and these allow the determination of surface charge density on the membrane, expressed in number of charged molecules per area unit, s*.30 The determination of Kp from z-potential results can be achieved using Langmuir isotherms:30 ÂÃ s ¼ Kpsmax Ai ð3Þ ÂÃ where, Ai the concentration at the interface can be obtained using the Boltzmann equation.35 The apparent partition coefficient Kp can be trans- 30 formed in Kp by:

½LaA þ n½LaL Kp ¼ Kp ð4Þ ½AmaA þ½LaL Figure 4. Dependence of number of charged molecules per area unit (s*) with concentration of clonixin at where aA and aL represent the molecular area for pH 7.4, in the presence of 400 mM of EPC. The curve the drug and lipid, respectively, and n stands for represents the best ﬁt to Equation 5.

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Table 1. Partition Coefﬁcients (Kp) (Adimensional) for Clonixin in EPC Unilamellar Liposomes (LUV) at pH 7.4, by Derivative Spectrophotometry, Fluorescence Quenching, and z-Potential*

Method Kp Derivative spectrophotometry 660 100 Fluorescence quenching 670 130 z-potential 580 120

*The reported values are the mean of at least two independent measurements; the error that affects each value is the standard deviation. significantly affect the results obtained.30 The value calculated for n was 14.5 2.0. The results obtained show a decrease in Kp Figure 5. Apparent Stern–Volmer constants Kapp values with the increase in drug concentration ð SV Þ (from 580 for 20 mM to 240 for 500 mM), indicating of 2-AS for clonixin in EPC unilamellar liposomes (LUV) obtained for different lipid concentrations ([L]). that the influence of electrostatic effects on Kp values cannot be neglected for higher drug con- liposome suspensions incorporating the fluores- centrations. The value presented in Table 1 is the cent n-AS probes, in which an ester linkage average of the values obtained for clonixin con- attaches the fluorescent anthracene at different centrations lower than 50 mM (the order of positions along the fatty acid chain, is a sensitive magnitude of the drug concentrations used in the method of determining the relative position of other presented techniques). quenching molecules in the lipid bilayer. When n- AS probes are included in the lipid bilayer, the

Kp Determination by Fluorescence Quenching carboxyl terminal group is located at the interfacial region of the membrane and the anthracene Quenching properties provide a means to deter- group is located in precise and known positions mine quantitatively the K of the drugs between p along the membrane depth plane.34 lipid and aqueous phases. For this purpose, the The fluorescence intensity of n-AS probes capacity of clonixin to quench the fluorescence of decreases with an increase of drug concentration. the probe 2-AS was evaluated by determination of app Figure 6 shows this behavior for the 12-AS probe in the apparent Stern–Volmer constant, Ksv ,at app EPC unilamellar liposomes. different lipid concentrations. The Ksv values depend not only on the quencher efficiency but also on its Kp between the aqueous and the lipid phase, since only the quencher molecules in the membrane are responsible for quenching. This dependence can be described by the equation:38

app Kp Ksv ¼ Ksv ð7Þ KpVm þ 1 app The knowledge of Ksv for several lipid concentrations (Figure 5) allows the determination of clonixin Kp and Stern–Volmer constant (KSV), by ﬁtting Equation 7 using a non-linear regression method. The Kp value obtained is included in Table 1.

Drug Location

Measurements of the clonixin quenching efﬁcien- Figure 6. Fluorescence quenching of 12-AS probe in cies, by addition of increasing amounts of drug to EPC unilamellar liposomes (500 mM, pH 7.4) by clonixin.

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The existence of fluidity and polarity gradients effect of molecules on the physical properties of through the plane of the liposomes membrane as phospholipid bilayers. There is considerable evi- mentioned by Thulborn et al.18,39 is reflected in dence indicating that membrane fluidity can be different t0 values found for each probe. However, understood in terms of the rate and amplitude of the effect of the microenvironment differences the anisotropic motion of the phospholipid acyl surrounding the probes is eliminated in bimole- chains. Fluorescent probes are used to report such 38 40 cular constant, kq, and, therefore, the determi- motion. The so-called ‘‘fluiditity gradient’’ can nation of these values provides an useful tool to be investigated using the previously mentioned predict drug location. As the fluorescence lifetime series of n-AS probes. As reported by New,29 this (t0) for all n-(9-anthroyloxy) stearic acid probes provides labeling at a graded series of depths in at pH 7.4 have been reported by Vincent et al.,40 the bilayer. Therefore, fluorescence anisotropy app and from the knowledge of the determined Ksv studies with these probes constitute a relatively values, it is possible to determine an apparent kq easy means of establishing the depth-dependence that is kapp: of fluidity. 38 app The method described by Lakowicz was used Ksv ¼ kapp t0 ð8Þ to measure the steady-state anisotropies (rss), by

The comparison of the kapp values obtained for the equation: each probe as a tool to access drug location is app Ivv GIvh preferable to the comparison of K values, since rss ¼ ð9Þ sv I þ 2GI in the first the effect of lifetime variation within vv vh the probe series is eliminated. The location of the where G is an instrumental correction factor, Ivv anti-inflammatory drug at pH 7.4 was therefore and Ivh are the emission intensities polarized achieved using kapp (Table 2). The observation of vertically and horizontally to the direction of the these values leads to the conclusion that all n-AS polarized light. probes were quenched by clonixin and the relative For a fluorescent molecule dissolved in an quenching efficiencies are in the order 2-AS > 6- isotropic solvent, the anisotropy can be described AS > 9-AS > 12-AS. This suggests that the anti- by the Perrin equation,38 which expresses the inflammatory drug is not deeply buried inside the effect of either lifetime or microviscosity variations lipid bilayer, but is preferably located near the on anisotropy. Under certain specific conditions, in phospholipid headgroups, probably with an elec- which the ‘‘out-of-plane’’ motion of the n-AS probes trostatic binding between the negative drug and in membranes is totally unhindered, the inten- the positive pole of the zwitterionic phophatidyl- sity decays exponentially to zero and the Perrin choline headgroup. This proposed location corro- equation can be used.38,40,41 These statements are borates the observation of clonixin’s structure made on the assumption that clonixin does not (Figure 1), where one can see a strong quenching change the intensity decay of the probe to multi- group (chloride atom), which is responsible for the exponential. quenching of the probes. In addition, clonixin The Perrin equation can be rearranged to yield: shows electrical charged groups and that can ex- plain the noticeable preference for the probes that 0 y þ t0 r ¼ rss ð10Þ lie near the surface of the phospholipids acyl chain. y þ t0

where t and t0 are the ﬂuorescence lifetime of the Membrane Fluidity Studies 0 ﬂuorophore in the absence and presence of the Fluorescence anisotropy variation is a parameter drug, respectively, and y is the rotational correla- that has frequently been used to examine the tion time (directly related to local microviscosity).

app Table 2. Values of Apparent Stern–Volmer Constants ðKSV Þ and Apparent Bimolecular Rate Constants kapp Obtained for Clonixin in Unilamellar Liposomes*

2-AS 6-AS 9-AS 12-AS

app KSV ðÞ=M 8080 300 7970 300 8300 200 8060 300 9 kapp 10 ð=MsÞ 2.59 0.77 2.35 0.56 2.11 0.13 1.56 0.12

*The reported values are the means of two independent measurements; the error is the standard deviation.

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The values of t0 can be easily calculated using the following equation:38 I t 0 ¼ 0 ð11Þ I t0 where I0 and I are the corrected fluorescence intensity of the fluorophores (n-AS probes) in the absence and presence of the drug, respectively. The values of y and t0 have been published for the n-AS probes used.40 Considering, in Equation 10, rss as the value measured for t0 (the value in the absence of quencher) and the published values of y and t0 for n-AS probes,40 a curve is generated with the values of the corrected anisotropy, r0. These r0 values are the variation of anisotropy that should be obtained due to the lifetime changes of the Figure 7. Dependence of the fluidizing effect ratio (%) fluorophore (gives a correction for the influence of for each n-AS probe in EPC unilamellar liposomes the probe itself in membrane microviscosity) and (500 mM, pH 7.4) caused by increasing effective concen- are then compared with the experimental rss trations of clonixin. 0 values. From the difference between rss and r one can assess the real variation of anisotropy caused by the drug without the illusory effect of the headgroup region, increasing the spacing between intrinsic variation due to the decrease of probe lipid molecules. 0 fluorescence lifetime. Once that rssr decreases In this work, besides the quantification of the with increasing clonixin concentration, it is con- interaction of clonixin with EPC liposomes, one cluded that a membrane fluidization happened was able to evaluate the effects of this interaction for the NSAID studied. The fluidizing effect ratio by studying the changes of membrane potential 0 can be calculated by: % fluidizing effect ¼ [(r rss)/ and fluidity. The data obtained for clonixins’ 0 r 0] 100. location and partition are consistent with the z- As clonixin partitions into the membrane and potential studies, all pointing to a preferable some fraction of the quencher remains in the interaction of this drug with the membrane sur- aqueous phase, it is necessary to correct the drug face. In fact, analyzing the dependence of the concentration, by the following equation:38 clonixin concentration on z-potential values, it is evident that clonixin provokes an appreciable Kp½QT decrease of the z-potential of the bilayer. This ½¼Qm ð12Þ Kpam þ ðÞ1 am confirms that this compound is negatively charged at the pH of the studies. Similarly, in previous where [Qm] is the quencher concentration in works, indomethacin and acemetacin proved also membrane and [QT] is the total concentration of to be predominantly in the negative form at the pH quencher added; Kp is the lipid–water Kp and am studied and, consequently, they were able to is the volume fraction of the membrane phase decrease the z-potential of the membrane as (am ¼ Vm/VT; Vm and VT represent the volumes of clonixin did. In spite of this, indomethacin and the membrane and water phases, respectively). acemetacin can reach the inner part of the bilayer Figure 7 shows a plot of % fluidizing effect and this contrasting location may be explained by versus effective drug concentration for each probe the presence of longer hydrocarbonated chains, studied. The graph shows that the fluidizing effect which put the strong quenching group (chloride is practically the same for probes 9-AS, 6-AS, and atom) closely positioned to 12-AS probe.30,34 On 12-AS, and it is more effective for 2-AS. the other hand, another previously studied NSAID As clonixin, from the quenching studies pre- (nimesulide), does not affect the z-potential of the sented, seems to be preferably located near the membrane, since it is almost in a neutral form at membrane surface, this ‘‘fluidizing’’ effect near the the same pH. Therefore, nimesulide can reach the membrane surface can be explained by a free inner part of the bilayer, presenting an inverted volume effect once that clonixin binds to the order of probes’ quenching (12-AS > 9-AS > 6-

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AS > 2-AS) when compared to the results obtained effect on lymphocyte membrane fluidity. Pharma- for clonixin (2-AS > 6-AS > 9-AS > 12-AS). col Res 22:277–285. Both electrostatic and fluidity properties are 5. Simonetti O, Ferretti G, Offidani AM, Gervasi P, fundamental for regulatory mechanisms, such as Curantola G, Bossi G. 1996. Plasma membrane membrane enzyme activity and electric stimulae fluidity of keratinocytes of normal and psoriatic conduction. These in turn, can be directly related skin: A study using fluorescence anisotropy of trimethylammonium diphenylhexatriene (TMA- to inflammatory and analgesic actions of clonixin. DPH). Arch Dermatol Res 288:51–54. In fact, the results show that clonixin can build a 6. Lasic DD. 1993. Liposomes—From Physics to appli- concentration dependent negative charge on the cations. New York: Elsevier. membrane’s surface and it can increase bilayer 7. Mason RP, Rhodes DG, Herbette LG. 1991. Reeval- fluidity substantially, especially on the surface uating equilibrium and kinetic binding parameters where the drug is preferentially located. This for lipophilic drugs based on a structural model for agrees with previous studies performed for other drug interaction with biological membrane. J Med NSAID drugs,24,33,34 where a parallelism between Chem 34:869–877. location and membrane fluidification is also found. 8. Betageri GV, Rogers JA. 1988. The liposome as a However, for the others, NSAIDs studied their distribution model in QSAR studies. Int J of Pharm preferential location and higher membrane fluidi- 46:95–102. 9. Welti R, Mullikin LJ, Yoshimura T, Helmkamp fication effects are observed in the core of the GM. 1984. Partition of amphiphilic molecules bilayer, whereas clonixins’ effects are mainly into phospholipid vesicles and human erythrocyte noticed at the surface. ghosts: Measurements by ultraviolet difference From the overall studies performed one can spectroscopy. J Biochem 23:6086–6091. conclude that anti-inflammatory activity can be 10. Kitamura K, Imayoshi N. 1992. Second-derivative related to membrane effects as described for spectrophotometric determination of the binding clonixin. constant between chlorpromazine and cyclodextrin in aqueous solutions. Ana Sci 8:497–501. 11. Kitamura K, Imayoshi N, Goto T, Shiro H, Mano T, Nakai Y. 1995. Second derivative spectropho- ACKNOWLEDGMENTS tometric determination of partition coefficients of chlorpromazine and promazine between lecithin The authors thank FCT and FEDER for financial bilayer vesicles and water. Anal Chim Acta 304: support through the contract POCTI/FCB/47186/ 101–106. 2002. Some of us, H. F. and M. L., thank FCT for 12. Gu¨ rsoy A, Senyu¨ cel B. 1997. Characterization of the fellowships (BD 6829/01) and (BD 21667/99), ciprofloxacin liposomes: Derivative ultraviolet spec- respectively. trophotometric determinations. J Microencapsul 14:769–776. 13. McLaughlin S. 1989. The electrostatic properties of membranes. Annu Rev Biophys Biophys Chem REFERENCES 18:113–136. 14. Cevc G. 1990. Membrane electrostatics. Biochim 1. Klein T, Ullrich V, Pfeilschifter J, Nu¨ sing R. 1998. Biophys Acta 1131:311–382. On the induction of Cyclooxygenase-2, inducible 15. Russel AJ, Fersht AR. 1987. Rational modifications nitric oxid synthase and soluble phospholipase A2 of enzyme catalysis by engineering surface charge. in rat mesangial cells by a nonsteroidal anti- Nature 328:496–500. inflammatory drug: The role of cyclic AMP. Mol 16. Volwerk JJ, Jost PC, Haas GHDe, Griffith OH. Pharmacol 53:385–391. 1986. Activation of porcine pancreatic phospholi- 2. Knazek RA, Liu SC, Dave JR, Keller RJ. 1981. pase A2 by the presence of negative charges at Indomethacin causes a simultaneous decrease of the lipid–water interface. Biochemistry 25:1726– both prolactin binding and fluidity of mouse liver 1733. membranes. Prostag Med 6:403–411. 17. Blatt E, Sawyer WH. 1985. Depth-dependent fluo- 3. Beccerica E, Piergiacomi G, Cutarola G, Ferretti G. rescent quenching in micelles and membranes. 1989. Effect of antirheumatic drugs on lymphocyte Biochim Biophys Acta 822:43–62. membrane fluidity in rheumatoid arthritis: A 18. Thulborn KR, Tilley LM, Sawyer WH, Treolar FE. fluorescence polarization study. Pharmacology 38: 1979. The use of n-(9-anthroyloxy) fatty acids to 16–22. determine fluidity and polarity gradients in phos- 4. Beccerica E, Ferretti G, Cutarola G, Cervini C. pholipid bilayers. Biochim Biophys Acta 558:166– 1990. Diacethylrhein and rhein: In vivo and in vitro 178.

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