Differential Effect of Oxicam Non-Steroidal Anti-Inflammatory Drugs on

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Differential Effect of Oxicam Non-Steroidal Anti-Inflammatory Drugs on Membranes and Their Consequence on Membrane Fusion Anupa Majumdar, Debjyoti Kundu, and Munna Sarkar J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.5b03918 • Publication Date (Web): 06 Jul 2015 Downloaded from http://pubs.acs.org on July 7, 2015

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The Journal of Physical Chemistry

Differential Effect of Oxicam Non-Steroidal Anti-Inflammatory Drugs on Membranes and Their Consequence on Membrane Fusion Anupa Majumdar,a Debjyoti Kundub and Munna Sarkara* a

Chemical Sciences Division, Saha Institute of Nuclear Physics, 1/AF, Bidhannagar, Kolkata-

700064, India b

Department of Biochemistry, University of Calcutta, 35, Ballygunge Circular Road, Kolkata -

700 019 , India

_________________________________________________________________ * Corresponding author. Fax: +91-33-23374637. Email:[email protected].

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ABSTRACT: Non-Steroidal Anti-inflammatory drugs (NSAIDs) are the most commonly used analgesic and antipyretics that form an interesting drug group because of their new and alternate functions. The ability of the NSAIDs belonging to the oxicam chemical group, to induce membrane fusion, at low physiologically relevant concentrations, is a new function than has drawn considerable attention. Membrane fusion is dependent on the interplay of physicochemical properties of both drugs and membranes. Here, we have elucidated the effects of different oxicam drugs, Meloxicam, Piroxicam, Tenoxicam, Lornoxicam and Isoxicam on an identical membrane-mimetic system. This highlights only the differential effects of the drugs on drug-membrane interactions, which in turn modulate their role as membrane fusogens. The partitioning behavior and the location of the drugs in dimyristoylphosphatidylcholine vesicles have been studied using second-derivative absorption spectroscopy, fluorescence quenching, steady-state fluorescence anisotropy, and time-resolved fluorescence lifetime measurements. Fusion kinetics has been monitored by fluorescence assays and dynamic light scattering was used to provide a snapshot of the vesicle diameter distribution at different time points. The differential perturbing effect of the drugs on the membrane is dependent both on their partitioning and location. Whereas, partitioning governs the extent of fusion, the location modulates the rates of each step.

Keywords: Membrane fusion; Drug-membrane interaction; Steady state and time resolved fluorescence spectroscopy; Dynamic Light Scattering; Non-Steroidal Anti-Inflammatory drugs; Oxicam


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1. INTRODUCTION Non-Steroidal Anti-inflammatory drugs (NSAIDs) constitute one of the most common classes of drugs available commercially, where they are widely used as effective analgesic, antiinflammatory as well as antipyretic agents. NSAIDs form a diverse group of chemical compounds classified on the basis of different functional groups like the salicylic acid group, the phenylalkanoic acid group, the oxicam group, the anthranillic acid group and finally the coxibs. It is already known that the molecular mechanism behind the established anti-inflammatory action of the NSAIDs lies in their ability to inhibit cyclo-oxygenase enzymes (COX) thereby, inhibiting the conversion of arachidonic acid into prostaglandins (PGs), responsible for inflammation.1-3 Apart from their well established roles, NSAIDs have been considered to be an interesting drug group to look into for new and alternate functions. The new and alternate functions include chemoprevention and chemosuppression against cancer cell lines,4,5 protective effects against neurodegenerative diseases such as Alzheimer’s disease6,7 and Parkinson’s disease.8,9 Some studies suggest that the anti-cancer action of the NSAIDs follows a COX dependant pathway where the over-expression of COX-2 enzyme in cancerous cell lines is inhibited by these NSAIDs. Other findings suggest that NSAIDs have direct anti-proliferative and apoptotic effects in cell lines, irrespective of the levels of expression of the COX enzymes in cells.10 Several proposals to explain the molecular mechanism behind these new functions exist, but a general consensus has not been reached yet. Since COX enzymes reside in membranes and serve as primary targets of these NSAIDs, the drug-membrane interactions play an important role in choosing the structural form of the drugs that will finally be presented to the target. Previously published data from our group suggests that the nature of the membrane mimetic micelle plays a crucial role in choosing a specific 3 ACS Paragon Plus Environment


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prototropic form of the drug for incorporation into the membrane.11 Results on NSAIDmembrane interactions have shown that NSAIDs bring about intricate alterations in the hydrophobicity, permeability, fluidity as well as stability of the membranes,12,13 which in turn have provided valuable insights into how they induce surface injury to the gastrointestinal mucosal membranes. This work has paved ways for development of a novel class of phosphatidylcholine (PC) associated NSAIDs with reduced GI toxicity.14 In vitro experiments on drug-membrane interactions are being extensively carried out by Reis et al. where explanations for the molecular basis of different functions of both new and old drugs are being looked into. It is seen that the initial organization or packing in the membrane model plays a very crucial role in determining the extent and nature of interactions, with the drug.15,16 It is thus evident that drugmembrane interactions basically hold the key to explaining drug action. As a manifestation of such NSAID-membrane interactions, our group has previously shown that Piroxicam (Px) belonging to the oxicam chemical group could permeabilize mitochondrial membranes leading to the release of cytochorome C in the cytosol, which in turn activated the downstream pro-apoptotic caspase pathway.17 This property of piroxicam to permeabilize the mitochondrial membrane was later explained on account of another very interesting property, where three oxicam NSAIDs were found to induce membrane fusion. Two oxicam NSAIDs, meloxicam (Mx) and tenoxicam (Tx) along with Px were found to induce membrane fusion in protein-free membrane mimetic vesicles at very low physiologically relevant concentrations.18,19 Each intermediate step of this membrane fusion process is characterized by specific energy barriers.20 These energy barriers need to be overcome in order to induce membrane fusion and lead the process to completion. For this, aid of external agents called fusogens, is required. Proteins and peptides, the most common class of fusogens, can overcome these energy barriers


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by their own conformational reorganizations.20-24 Even though, oxicam NSAID induced membrane fusion lacks this advantage, the NSAIDs manage to induce and complete the fusion process. The results published by our group indicate that the fusion process is governed by a balanced interplay of the physicochemical properties of both the participating membrane systems as well as the drugs. The modulatory effects of different membrane and drug parameters on oxicam NSAIDs induced membrane fusion have already been established in previous studies.18,19,25,26 Effects of several other lipidic parameters are also in the process of being elucidated, in order to get a complete picture of the mechanistic details associated with NSAID induced membrane fusion process. This study focuses on how the five oxicam NSAIDs, Meloxicam (Mx), Piroxicam (Px), Tenoxicam (Tx), Lornoxicam (Lx) and Isoxicam (Isx), having such small variations in their structures, can modulate their interactions with the membranes, which in turn affects their membrane fusogenic properties. NSAIDs belonging to any particular chemical group are basically generated by the isosteric replacement of certain functional groups.27 This approach involves the replacement of groups or fragments in the first drug from the group with moieties of similar stereo-electronic features to improve the various drug properties e.g. pharmacokinetics and pharmacodynamics. For instance replacement of the benzo ring of piroxicam with a thieno ring yields Tenoxicam (Tx) whereas replacement with a 2-chloro-thieno ring yields Lornoxicam (Lx). Similarly replacement of the pyridine-2-yl ring of piroxicam with 5-methylisoxazol-3-yl and 5-methylthiazol-2-yl rings yields Isoxicam (Isx) and Meloxicam (Mx) respectively (Figure 1).



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Figure 1. Structure of the five oxicam NSAIDs.

Existence of subtle intra-molecular interactions within the molecule intricately affects the overall physicochemical properties of the drugs, imparting each drug a unique character. In this effort we aim to achieve a better understanding of how the differences in the physicochemical behavior of these isosterically substituted oxicam NSAIDs affect their interactions with the membranes, in the context of membrane fusion. A detailed biophysical characterization of the NSAID-membrane interactions has been done in this work, which has then been correlated to the membrane fusion inducing ability of the five oxicam NSAIDs. To highlight the effect of the drugs only, the membrane mimetic system was kept same for all experiments. As a simple membrane mimetic, small unilamellar vesicles (SUVs) made of dimyristoylphosphatidylcholine (DMPC) was used to highlight the role of these NSAIDs on the membrane structure/ morphology, which in turn affects their role as membrane fusogens. It should be mentioned that 6 ACS Paragon Plus Environment

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interaction of four oxicam NSAIDs, Mx, Px, Tx and Lx with egg yolk phosphatidylcholine (EPC) large unilamellar vesicles (LUV), has been previously studied to understand the basis of their COX selectivity,28 but has not been studied in the context of their differential behavior as membrane fusogens. For their experiments they have used EPC LUVs as the model membrane mimetic. The major component of phospholipids extracted from enriched egg yolk is comprised of a mixture of phosphatidylcholines of varying tail lengths and varying degrees of unsaturation,29,

30

as opposed to DMPC SUVs having uniform C14 chain length and no

unsaturation. We have already seen that subtle changes in membrane parameters25,26 profoundly affect membrane properties/dynamics which alters drug membrane interactions, thereby modulating their effects on membrane fusion. Hence, it is expected that in two different lipidic systems, there would be a change in the drug membrane interactions. In this work, different spectroscopic techniques have been employed. Drug-partition coefficients (Kp) has been determined through second derivative absorption spectroscopy. Drug induced membrane fluidization has been monitored using steady-state anisotropy of membrane bound 1, 6 - Diphenyl- 1, 3, 5- Hexatriene (DPH) probe. The relative partitioning of DPH molecules into the bilayer, in presence of the five drugs was studied using fluorescence lifetime studies. Subsequently estimation of drug location in the DMPC SUV systems has been done using both steady-state and nanosecond time-resolved fluorescence quenching studies. Fluorescence based lipid mixing and content mixing assays have been used to monitor the kinetics of the different steps of fusion process of Lx and Isx and compared with that of Px, Mx and Tx. Finally, Dynamic light scattering (DLS) studies have been performed to record the changes in the vesicle size distribution at different time points, encompassing the time frame of the content mixing step, which is the final event in fusion.



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2. EXPERIMENTAL SECTION 2.1 Materials. Dimyristoylphosphatidylcholine (DMPC), piroxicam (Px) (4-hydroxy-2methyl-N-pyridin-2-yl-2H-1,2-benzothiazine-3-carboxamide

1,1-dioxide),

dipicolinic

acid

(DPA), TritonX-100 (ultrapure), isoxicam (Isx) 4-Hydroxy-2-methyl-N-(5-methyl-3-isoxazolyl)2H-1,2-benzothiazine-3-carboxamide 1,1-dioxide, Tenoxicam (Tx) (3E)-3-[hydroxy(pyridin-2ylamino)methylene]-2-methyl-2,3-dihydro-4H-thieno[2,3-e]

[1,2]thiazin-4-one

1,1-dioxide

Terbium chloride(TbCl3), (3-[N-morpholino] propanesulfonic acid) sodium salt (MOPS buffer), 1,6-Diphenyl-1,3,5-hexatriene (DPH), Sephadex G-50 for size-exclusion chromatography were purchased from Sigma-Aldrich, meloxicam (Mx) [4-hydroxy-2-methyl-N-(5-methyl-1,3-thiazol2-yl)-2H-1,2-benzothiazine-3-carboxamide-1,1-dioxide] and Lornoxicam (Lx) (3E)-6-chloro-3[hydroxy(pyridin-2-ylamino)methylene]-2-methyl-2,3-dihydro-4H-thieno[2,3-e][1,2]thiazin-4one

1,1-dioxide

from

LKT

Laboratories,

dihexadecanoyl-sn-glycero-3-phosphoethanolamine,

N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-1,2triammonium

salt

(N-NBD-PE)

and

Lissamine rhodamine B-1,2-dihexadecanoyl-sn-glycero-3-phosphanolamine, triethylammonuim salt

(N-Rh-PE)

from

Invitrogen

Life

Science

Corporation,

and

2-[tris

(hydroxymethyl)methylamine]-1-ethanesulfonic acid (TES buffer), sodium ethylene diamine tetra aceate (EDTA sodium salt) were purchased from SRL (India). Stock solutions of Mx, Px, Tx, Lx and Isx were prepared by dissolving in DMSO. In each case the % (v/v) ratio of DMSO (Merck) in buffer was maintained at 0.3% (v/v) which did not have any significant effect on the integrity of SUV structure or membrane fusion. 2.2 Preparation of Small Unilamellar Vesicles (SUVs) and DPH Labelling. SUVs of DMPC were prepared by the standard method of sonication. DMPC lipid was dissolved in in 2:1 (v/v) chloroform: methanol solvent after which the solution was purged under stream of argon 8 ACS Paragon Plus Environment

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until the solvent was totally removed. The dried lipid film was further dried overnight in a vacuum desiccator at -20 ºC. The dried film was hydrated in 10 mM TES and 100 mM NaCl buffer (pH 7.4) and vortexed thoroughly. This hydrated liposome solution was sonicated for 9 min at equal intervals using dr. Heilscher (Germany) probe sonicator (200 W) until a clear solution was obtained. The samples were then allowed to stand for 40 min at 39 °C to be hydrated completely. The sonicated samples were centrifuged at 10,000 rpm for 15 min to remove titanium particles, introduced as an impurity from the sonicator probe during the process of sonication, and aggregated lipids. For vesicle labelling, appropriate amount of DPH solution (1 mM stock in DMSO) was added to the vesicle solution, maintaining a lipid: probe ratio of 500:1 and incubated for nearly an hour before performing the experiments. The incubation time of an hour was given to ensure complete partitioning of DPH into the bilayer. Incorporation of DPH into membrane bilayer was accompanied by a steady increase in its fluorescence intensity at 428 nm. 2.3

Determination

of

Partition-coefficients

by

Second-Derivative

Absorption

Spectrophotometry. Partition coefficients of the five oxicam NSAIDs, between lipidic and aqueous phase have been determined using second derivative spectrophotometry. The absorption spectra of the five oxicam NSAIDs (30 µM) dissolved in TES buffer (10 mM TES, 100 mM NaCl, pH 7.4), in presence of increasing concentrations of DMPC (0-1500 µM) were recorded, along with the spectra of the corresponding reference solutions prepared in the absence of the drugs, using a JASCO V-650 UV-Vis spectrophotometer. The absorption spectra were measured at 39 °C using quartz cells having 10 mm path length. The corrected spectra have been obtained by subtracting the reference spectra from the sample spectra. The second-derivative spectra have been calculated using Savitzky-Golay method, using the software provided along with the



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JASCO V-650 spectrophotometer. The second-derivative absorbance values are related to the Kp values according to equation 1, as follows:31

D = Dw +

((D l - Dw) × Kp × [L] × γ) ......(1) 1 + (Kp × [L] × γ)

γ is the molar volume, considered as 0.95,32 and [L] is molar concentration of the DMPC SUVs. The Dl and Dw values represent the second derivative absorbance values of drugs in lipidic and aqueous phases respectively, whereas D is the total second derivative absorbance in both the phases. The D values at absorption maxima, have been used to generate D vs. [L] plots. These plots were fitted to the above equation using non-linear least square method to obtain the partition coefficients (Kp) of the five drugs. 2.4 Steady-State Fluorescence Anisotropy Measurements. The fluorescence anisotropy value (r) of hydrophobic membrane probe DPH is measured to monitor the gel-sol phase transition of the lipid bilayer, both in presence and absence of the five oxicam NSAIDs. The ‘r’ values of DPH molecules embedded in the DMPC vesicles (hydrated in 10 mM TES, 100 mM NaCl buffer), have been measured over a range of temperatures starting from 8 ºC to 44 ºC. The drug treated vesicles were incubated for more than an hour to ensure completion of the last step of the fusion process. The anisotropy measurements were carried out in VARIAN Cary Eclipse fluorescence spectrophotometer attached to a constant temperature cell-holder. The excitation and emission wavelengths were set at 365 nm and 428 nm respectively. The anisotropy values were calculated using the following equation:

r=

I VV - G .I VH .......( 2) , where, G = I HV I VV + 2G .I VH I HH

IVV and IVH are the emission intensities of DPH, when the excitation polarizer is vertically oriented and emission polarizer is oriented vertically and horizontally respectively. IHV and IHH 10 ACS Paragon Plus Environment

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are emission intensities of DPH, when excitation polarizer is oriented horizontally with emission polarizer being oriented vertically and horizontally respectively. The plots of ‘r’ vs. temperature were fitted using equation 3,15 to obtain the values of gel-sol phase transition temperature (Tm) and cooperativity of transition (B):

rs = rs 1 + p1T +

rs 2 - rs 1 + p 2T - p1T 1 + 10B((1 / T ) - (1 / Tm ))

.......( 3)

where, T is the absolute temperature (°C), p1 and p2 are the slopes of the linear fits to the anisotropy value against temperature plots before and after the phase transition region respectively and rs1 and rs2 are the corresponding y-intercepts. Non-linear least square fitting of the anisotropy vs. temperature plots were done using Origin 8.0 software. 2.5 Fluorescence Quenching Study. Steady-state fluorescence quenching of membraneincorporated DPH molecules was monitored in the presence of increasing concentration of the drugs Mx, Px, Tx, Lx and Isx. Aliquots of 1 mM DMPC liposomes were prepared and incubated with increasing drug concentrations (0 µM - 120 µM). Before performing any fluorescence measurements, the aliquots were maintained at temperatures above gel-sol phase transition temperature of DMPC, for an hour. This incubation time was given to allow NSAIDs to reach their partition equilibrium between lipidic and aqueous phases. Steady-state fluorescence measurements were measured in HORIBA Jobin Yvon Fluoromax-3 spectrophotometer equipped with a constant temperature cell-holder. Identically prepared aliquots, incubated with varying drug concentrations were used to record corresponding excited state fluorescence lifetimes of DPH molecule. The fluorescence intensity decay measurements were recorded using Jobin Yvon HORIBA picoseconds-resolved time correlated-single-photon counting instrument (TCSPC), having a pulsed diode laser with excitation at 377 nm. Fluorescence emission wavelength was set at 428 nm. All measurements 11 ACS Paragon Plus Environment


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were made using Ludox as a reference standard (τ = 0.00 ns). The pulsing frequency of the diode laser was 1MHz. The instrument response function (IRF) has a full width at half-maximum (fwhm) value of 250 ps and a resolution of 28 ps per channel. The fluorescence decay curves of DPH could be fitted to a bi-exponential decay using following equation:

I(t ) = B 0 + B1 exp[-

t t ] + B 2 exp[- ]........( 4) τ1 τ2

where, B0 is a constant, B1 and B2 are the pre-exponential factors and τ1 and τ2 are the corresponding fluorescence lifetimes. The fitting was done using IBH DAS 6.2 data analysis software. The goodness of fit was determined by the reduced χ2 values and from the plots of the residuals as a function of time. All lifetimes were recorded at room temperature of 30 °C. 2.6 Fusion Measurements. 2.6.1. Lipid Mixing Assay. The lipid mixing step of the fusion process was monitored through a FRET based assay33 where N-NBD-PE and N-Rh-PE were used as donor and acceptor molecules respectively. One set of the vesicles were labeled with 0.8 mol% each of both donor and acceptor probes and mixed with another set of probe-free vesicles at a ratio of 1:9 respectively. A low probe concentration is chosen such that FRET occurs initially in the labelled vesicles. Drug induced fusion between labeled and probe-free vesicles, is accompanied by disruption of FRET, as a result of increase in donor-acceptor distances associated with probe redistribution during lipid mixing. This is manifested as increase in the donor fluorescence (excitation at 460 nm and emission at 530 nm). This increase in the donor fluorescence, in the presence of the oxicam NSAIDs (D/L 0.03), is monitored as a function of time and the associated rate constants of the lipid mixing step are obtained from this kinetic curve. In this work, 10 mM TES, 100 mM NaCl buffer was used for vesicle hydration and the total lipid concentration was


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maintained at 1 mM. The plots obtained from the lipid mixing assay have been expressed as percentage lipid mixing plotted against time. The percentage lipid mixing is given by the following equation 5,19

% Lipid Mixing =

F - F0 × 100........(5) F∞ - F0

where, F= Fluorescence intensity at time t, F0 = initial fluorescence of the labeled liposomes set as 0% fluorescence, and F͚ = the maximum fluorescence set as 100% which was determined by lysing the vesicles with 1%(v/v) TritonX-100 and releasing the probes in aqueous buffer resulting in high dilution. A complete disruption of FRET between N-NBD-PE and N-Rh-PE occurs in the presence of Triton-X, giving the maximum possible donor fluorescence intensity.

2.6.2. Content Mixing Assay. The well established fluorescence based Tb-DPA assay34 has been used to monitor the content mixing step of the fusion process in presence of the oxicam drugs. Pseudo first order reaction condition was maintained by encapsulating 8 mM TbCl3 into one set of vesicles, and 80 mM DPA into another set of vesicles. The free TbCl3 and DPA molecules present in the external buffer were removed by the principle of size-exclusion chromatography, where the two sets of vesicles were passed through two separate Sephadex G50 columns equilibrated with elution buffer (10 mM TES, 100 mM NaCl, 1 mM EDTA). The two sets of probe encapsulated vesicles were mixed in a 1:1 ratio where total lipid concentration was 1mM. In presence of the fusogenic drugs (D/L 0.03), fusion resulted in mixing of internal contents of the vesicles leading to the formation of intensely fluorescent Tb(DPA)3 chelate complex. The increase in the fluorescence intensity of the Tb(DPA)3 complex at 490 nm, on being excited at 275 nm, was monitored as a function of time using Hitachi F-7000 fluorescence spectrophotometer. The content mixing plots were expressed as percentage content mixing against time. To convert the increase in fluorescence intensity to percentage content mixing, the 13 ACS Paragon Plus Environment


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TbCl3 containing vesicles were re-chromatographed using EDTA free elution buffer. The rechromatographed vesicles were then lysed with Triton-X-100 at 1% (v/v) and the corresponding fluorescence intensities measured both in absence and presence of excess amount of DPA defined 0% and 100% fusion limits. The percentage content mixing is then given by the following equation 6,19, 34

% Content Mixing =

(F - F0 ) / F0 (F l - F0l ) / F0l

× 100........(6)

where, F is the fluorescence intensity of the Tb/DPA complex in the presence of drug at time t,

F0 is the fluorescence intensity of the Tb/DPA complex in the presence of drug at t=0, Fl is the fluorescence intensity of the lysed Tb-vesicles in the presence of an adequate amount of DPA, and Fl0 is the fluorescence intensity of the lysed Tb-vesicles. 2.7 Dynamic Light Scattering (DLS). Hydrodynamic number weighted size distributions of DMPC SUVs in absence and presence of the five oxicam NSAIDS have been determined by DLS experiments using the instrument ZetaSizer NanoS of Malvern Instruments; Worcestershire, UK. The instrument operates at 90° scattering angle and 173° detection geometry. All experiments were carried out at 39 °C and parameters like material and dispersant were set according to the nature of the sample used in the experiment. For lipid vesicles, polystyrene latex and 10 mM MOPS were taken as material and dispersant respectively. Results of experiments were interpreted in terms of number percentage with respect to the diameter of the vesicles. Here, the number percentage signifies the relative abundance of different sized vesicles involved in scattering. This was done to better observe the relative changes in the size and distribution of the small vesicles used here, that are not obscured by the presence of a small


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number of larger vesicles or aggregates. The particle scattering intensity is proportional to the square of the molecular weight. Hence, intensity distribution can be somewhat misleading since scattering intensity from only a small amount of aggregates or larger particles can dominate the distribution.

3. RESULTS AND DISCUSSION 3.1 Determination of Partition Coefficients. As has been discussed in the introduction section, characterization of drug-membrane interactions forms the very basis of explaining drug action. The extent of interaction of a drug with lipid bilayer is best quantified by its partitioncoefficient (Kp) between the lipidic and aqueous phases. Large Kp values indicate increased incorporation of the drugs in the membrane bilayer as compared to the aqueous buffer phase irrespective of the location of the drugs within the bilayer. The partition coefficients of the five drugs were determined from second derivative absorption spectroscopy so as to eliminate the scattering of vesicles from their respective absorbance spectra. Representative plots of 2nd derivative absorbance values at absorption maxima vs. increasing molar lipid concentration are given in Figure 2 (a) and (b) for Lx and Isx respectively.



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Figure 2. Plots of second-derivative absorbance values (D) at corresponding λmax against concentration of DMPC vesicles [L] for (a) Lornoxicam and (b) Isoxicam.

The Kp values have been determined by fitting the plots in Figure 2 to equation 1, using a non-linear least squares regression method. The partition coefficient values are given in Table 1. Previously reported octanol/buffer distribution coefficients (log D) at pH 7.4 of the five drugs have been included35 in Table 1 to see whether there is any correlation with the obtained trend in Kp values. Distribution coefficient (log D) of any compound is a measure of the ratio of the concentrations of both ionized and unionized forms of the compound, in octanol and water, at a particular pH and is given by the relation,

log D oc tan ol / water = log

ionized un − ionized [drug ]oc tan ol + [drug ]oc tan ol un − ionized [drug ]ionized water + [drug ]water

..........(7 )


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Table 1: Partition Coefficient Values Obtained for the Drugs Meloxicam, Piroxicam, Tenoxicam, Lornoxicam And Isoxicam between the SUVs of DMPC and Aqueous Phase Using Second Derivative Absorption Spectroscopy

a

Drugs

log Da (Octanol water) (pH 7.4)

Mx

0.09

363 nm

296.4 ± 2.0

Px

-0.05

354 nm

135.0 ± 3.0

Tx

-0.32

368 nm

127.1 ± 2.0

Lx

0.61

377 nm

548.4 ± 46.0

Isx

-0.32

347 nm

205.4 ± 5.6

λmax (at which secondderivative values were plotted)

Partition Coefficient (Kp )

Values have been taken from reference. 35

From Table 1, it was observed that Lx has the highest Kp value followed by Mx, Isx, Px and Tx, having the lowest Kp value. It was observed that the log D value of Lx (pH 7.4) is greater than that of Mx which correlated well with our determined Kp values. However, no such correlation was found to exist between the reported log D and Kp values of Px, Tx and Isx. In fact, even for identical log D values, as in case of Tx and Isx, the Kp values are different. This is because octanol/water mixture is not a good model for membranes. Even though they represent two immiscible phases of different lipophilicity, they cannot mimic the heterogeneous environment of a lipid bilayer. Therefore, octanol/water mixture can neither model the van der Waals interactions, nor the steric effects and the electrostatic environment that a drug would face on entering the lipid bilayer. Thus the differences in the Kp values could arise not only on account of the differences in the log D values but, also on how well the drugs can be accommodated in the bilayer, keeping in mind the steric effects, van der Waals and electrostatic interactions between drugs and the bilayer.36 This is dependent on the structure of the drugs, as 17 ACS Paragon Plus Environment


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well as on their location in the lipidic phase. However, these factors are not only dependent on the drugs but also on the nature of the lipidic phase. This is the reason why Kp values of oxicam NSAIDs in EPC LUVs,28 did not match the present trend observed in case of DMPC SUVs. The effect of structure is best demonstrated by Tx and Isx which have identical log D values but significantly different Kp values (Table 1), with the structure of Isx being favored to enter the lipid bilayer over Tx. Replacement of the pyridine-2-yl ring by 5-methylisoxazol-3-yl as in Isx has been shown to result in the dramatic decrease of basicity of the nitrogen atom of the carboxamide moiety,35 which might favor its entry into the bilayer. It is necessary to find out whether the Kp values could be correlated to the membrane perturbing ability of the drugs. For this, membrane fluidization was measured by monitoring the gel-sol phase transition temperatures (Tm ) of the DMPC bilayer in the presence of these drugs. 3.2 Steady-State Fluorescence Anisotropy Measurements for Monitoring Membrane Fluidity. The phase transition temperature (Tm) and cooperativity (B) values of lipid gel-sol phase transition was estimated by measuring the steady state fluorescence anisotropy (r) of membrane bound probe, DPH, as a function of temperature. There exists a fluidity gradient in the lipid bilayer below and above phase transition temperature with more segmental order existing in the hydrocarbon chain nearer to the head group between C1-C9,15 than in the centre of the bilayer. Hence, the cooperativity B, that measures the size of the cooperative unit that undergoes the gel-sol transition, will be most affected when the drugs are accommodated in the C1-C9 region and will remain unaffected when incorporated in the more disordered central region of the bilayer.


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Figure 3. Steady-state fluorescence anisotropy of DPH, plotted against temperature, in the absence and presence of Lx and Isx (D/L 0.03). The continuous lines are the best fitted curves obtained using equation 3. The anisotropy value gives a measure of the restriction imposed on the mobility of DPH, mainly localized in the tail region of the bilayer. The change in the Tm values of DMPC due to presence of the oxicam NSAIDs gives an idea of the extent of perturbation/fluidization by the drug. Different drugs localize into different regions of the bilayer and results in different changes in both the Tm and cooperativity values. Figure 3 shows the representative plots of DPH steadystate fluorescence anisotropy against increasing temperature, in DMPC bilayers in the presence of Lx and Isx. The temperature range over which phase transition occurs broadens in the presence of drugs intercalating in the C1- C9 region of the lipid bilayer thereby changing the B value. Drugs that get deeper into the bilayer beyond the C9 atom of the bilayer are expected to bring about little or



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no change in the cooperativity parameter (B), which gives a good idea about the approximate location of the drugs.15 The Tm and B values obtained by fitting the above plots to equation 3 are given below in Table 2. From the table it is seen that all the drugs perturb the lipid bilayers to different extents and decrease the Tm values with respect to pure DMPC bilayer. Lx and Mx were found to bring about the maximum change in the values of Tm of DMPC, followed by Px, Isx, and Tx. The four drugs apart from Lx were found to bring about a significant change in the B values. Lx, on the other hand, was found to keep the B value nearly unaltered. This could indicate a comparatively deeper location of Lx in the bilayer core as opposed to the other four drugs, which corroborates with our previous studies. Even though Lx was found to partition to a deeper non-cooperative region in the bilayer, its high partition-coefficient value indicate larger amount of drugs inside the bilayer compared to the other oxicams. This results in enhanced membrane perturbation which is reflected in the maximum change in Tm values. In fact, the decrease in Tm, which is an approximate measure of the fluidization by the drugs, directly correlates to the amount of drugs incorporated in the bilayer as reflected by their Kp values irrespective of their location. Table 2: Gel-Sol Phase Transition Temperature (Tm) and Cooperativity (B) Values Determined From Steady-state Fluorescence Anisotropy Measurements of DPH in DMPC Vesicles Recorded as a Function of Temperature, in the Presence of the Five Oxicam NSAIDs Drugs

(Tm )

Cooperativity(B)

No Druga

24.1 ± 0.0

256.9 ± 1.2

Mxa

23.4± 0.2

134.5 ± 5.1

Pxa Tx Lx Isx

23.8 ± 0.0 23.8 ± 0.1 23.0 ± 0.1 23.5 ± 0.2

170.8 ± 0.7 174.1 ± 0.4 260.1 ± 1.1 157.5 ± 2.6

a

Taken from reference 37. Throughout experiments, pH was maintained at 7.4 and temperature was varied from 8 °C to 44 °C. Each sample was equilibrated for 15-20 min at a particular temperature before recording the fluorescence anisotropy values. 20 ACS Paragon Plus Environment

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We need to establish how individual drugs might affect the relative partitioning of the DPH probe, before presenting our data on quenching of DPH probe by the five drugs to determine their approximate locations in the bilayer, 3.3 Time Resolved Fluorescence of DPH in DMPC Vesicles. Time resolved fluorescence decay profiles of DMPC incorporated DPH alone and in presence of the free drugs, could be best fit to a bi-exponential decay giving a chi square value close to one (Table 3). A short lifetime component τ1 (2.7 – 2.0 ns) and a long lifetime component τ2 (9.3 – 7.7 ns) were obtained in the absence and in the presence of the drugs.37 The short lived component has been attributed to the DPH population located towards the membrane interface and the long lived fluorescence has been attributed to the DPH population buried relatively deeper in the bilayer.38 The short component τ1 is a measure of the disturbances in the membrane-water interface, while the long lived component τ2 is a reflection of changes deeper in the bilayer. For most cases, the short component has been found to have less contribution to the intensity of the decay profile than the long component. Figure 4 shows a representative plot of the time-resolved fluorescence lifetime decay of DPH in the presence of three oxicam drugs Lx, Mx and Isx. The data obtained from bi-exponential fitting of the decay profile is given in Table 3.



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Figure 4: Representative fluorescence decay profiles of DPH in presence of (a) Lx, (b) Mx and (c) Isx at D/L ratio of 0.03).

In the presence of DPH alone, the short lifetime component (τ1 = 2.7 ns) has a 9.7% contribution with the major contribution of 90.3%, coming from the long life time component (τ2 = 9.3 ns). There is a decrease in both τ1 and τ2 in presence of all the five drugs at a constant D/L ratio of 0.03. It is noteworthy that all the five drugs decrease both the lifetime components to different extents. This is indicative of an increase in hydration of the bilayer at both the membrane interface as well as in the interior.39 For the most hydrophilic drugs Tx and Isx, as reflected by their identical log D values (-0.32),35 there is practically no change in the τ1 and τ2 values compared to the control. This indicates that the two hydrophilic drugs cannot perturb the lipid packing enough to allow water penetration. For the most lipophilic Lx, maximum change in the lifetime values compared to the control indicates maximum hydration of the bilayer in its presence. This is consistent with its high Kp and its deepest location into the bilayer compared to the other oxicams, which causes higher perturbation in lipid packing allowing more water penetration. 22 ACS Paragon Plus Environment

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Table 3: Fluorescence Lifetime Studies of DPH Incorporated into DMPC in Presence of Five Oxicam NSAIDs at D/L 0.03 DPH labeled

λem

B1

DMPC in presence of

B2

(ns)

(nm)

No Drug a

τ1

τ2

τ avg

(ns)

(ns)

χ2

9.7

2.7

90.3

9.3

9.1

1.09

12.8

2.2

87.1

8.7

8.4

1.01

9.6

2.1

90.4

9.0

8.9

1.00

Tx

11.4

2.6

88.6

9.2

8.9

1.13

Lx

22.3

2.0

77.5

7.7

7.2

1.04

Isx

10.7

2.6

89.3

9.2

9.0

1.17

Mx Px

a

a

428

a

The values have been quoted from ref 37. Temperature was maintained at 30 °C and pH at 7.4 throughout the experiments. The calculated error lies within ± 5 %. Experiments were repeated at least 3 times and average values are given in the table.

It should be noted that there is a significant increase in the contribution of the short lived component in presence of Lx. This indicates that there is a re-distribution in the probe population resulting in the increase in DPH population at the membrane-water interface (from 9.7% to 22.3%). This is an expected result since high Kp value indicates a larger number of Lx molecules penetrating deeper into the bilayer interior, as concluded from previous sections, causing crowding in the interior. This pushes out the probe molecules and thereby results in an increase in the probe population at the membrane-water interface. These results so far indicate that out of the five oxicams, Lx is incorporated in the deepest portion of the bilayer as evident from the cooperativity value. This resulted in maximum perturbation of the bilayer with enhanced fluidization effect, as reflected by the Tm values. It shows the highest alteration of the lipid packing which is reflected in the increased hydration of the bilayer, as given by the decrease in both the fluorescence lifetimes of DPH. For the other four oxicams, the presence of the drugs does not significantly change the relative partitioning of DPH in the lipid membranes. 23 ACS Paragon Plus Environment


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In the next section, steady-state and time-resolved experiments on quenching of DPH probe by the five drugs, have been further done to determine approximate locations of the drugs in the bilayer. 3.4 Determination of Drug Location using Steady-State and Time-Resolved Fluorescence Quenching of DPH. The relative location of the five drugs within the DMPC bilayer was also determined using fluorescence quenching of membrane bound fluorophore DPH, with the drugs acting as quenchers. As mentioned before, though the DPH molecules distribute themselves into two distinct populations at different regions of the bilayer, the predominant location of the probe lies in the membrane interior, aligned roughly parallel to the acyl chains. Despite the fact that DPH is very hydrophobic, it does not on the average locate very deep inside the bilayer. Rather, it prefers to intercalate in the portions of the lipid acyl chains closer to the membrane surface which are better aligned than regions towards the centre of the bilayer.40 In other words, DPH prefers to pack in the cooperative region of the bilayer,

40

approximately between C1-C915 rather than in the more flexible non-cooperative bilayer interior beyond C9. The intensity of fluorescence emission at 428 nm with excitation at 365 nm, as well as the fluorescence lifetime of DPH, was monitored in the presence of increasing drug concentrations, to get an insight into the nature of the quenching process. From the steady-state as well as time-resolved fluorescence measurements, it was clear that the drugs could effectively quench the fluorescence intensity and decrease the fluorescence lifetime of the DPH fluorophore. If the quenching is in an isotropic medium and is a purely diffusion controlled collisional process, the fluorescence intensity of the fluorophore can be related to the quencher/drug concentration by


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F0 τ = 1 + K SV [Q] = 0 ..........(8) F τ In the above equation 8, F0 and F are the fluorescence intensities of DPH in the absence and presence of the quencher respectively, KSV is the Stern-Volmer quenching constant, τ0 and τ are the average fluorescence lifetimes of DPH in the absence and presence of quencher. [Q] is the total concentration of the quencher. However, in a lipid bilayer, the total quencher concentration is not available to the probe which is inside the bilayer. Hence only that amount of quencher should be considered, that can partition into the membrane phase from solution. This can be calculated from the total drug concentration [Q] and drug partition coefficient (Kp) according to the following equation 41, 42 [Q]m =

K p [Q] 1 + (K p - 1) γL

........(9)

In the above equation, γ is the lipid molar volume which is taken as 0.95 for DMPC32 and L is the molar lipid concentration used in these experiments. Using the above equations τ0/τ and F0/F values of DPH fluorophore molecules are plotted against the respective [Qm] values. A representative plot for Mx is given below in Figure 5a and 5b.



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Figure 5. Representative Stern-Volmer plots of DPH incorporated in DMPC, in the presence of Mx showing (a) τ0/τ vs [Qm] obtained from fluorescence lifetime measurements and (b) F0/F vs [Qm] obtained from steady-state fluorescence intensity measurements.

For all the oxicam drugs, τ0/τ vs. [Qm] plots were found to be linear which represents only the dynamic quenching component, whereas, the F0/F vs. [Qm] plots were found to exhibit a non-linear nature with upward curvature. Despite evidence of different populations of DPH in 26 ACS Paragon Plus Environment

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the lipid bilayer (section 3.3), the upward curvature as opposed to downward curvature of the Stern-Volmer plots suggests that all populations of the DPH in the membrane are accessible to quenchers. The curvature in the steady-state Stern-Volmer plot indicates the presence of a quenching process having both dynamic and static quenching components. The component of static quenching can be attributed either to the formation of a non-fluorescent complex between fluorophore and quencher or to the presence of a quenching ‘sphere of action’ that decreases the fraction of fluorescing DPH molecules. The characteristic fluorescence spectra of DPH did not change in the presence of the quencher and no additional band at longer wavelength was observed. No specific interactions between the drugs and the fluorophore could be identified either in absorption or fluorescence spectra leading us to rule out the possibility of static quenching through ground-state complex formation. Thus the observed positive curvature of the Stern-Volmer plots could be interpreted in terms of the ‘sphere of action’ quenching model. If the quencher is located inside a spherical volume (V) surrounding the fluorophore, at the exact time of excitation of the fluorophore, instantaneous quenching occurs rendering the fluorescence of those particular fluorophore molecules unobservable. To take this additional fluorophorequencher proximity effect into account, the Stern-Volmer equation is modified as given below:

F0 = ( 1 + K D [ Q m ]) e V [ Q ] .......( 10 ) F m

In the above equation, V is the molar ‘sphere of action’ volume. The Stern-Volmer quenching constant (KD) here, corresponds to the dynamic collisional quenching constant. From the slope of the τ0/τ vs. [Qm] plots, KD was calculated. Using these calculated values of KD, the molar ‘sphere of action’ volumes (V) were determined for each of the five oxicam NSAIDs by



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fitting F0/F vs. [Qm] to equation 10.

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The bimolecular quenching rate constant (Kq) was

calculated from the obtained KD values according to the following equation:

Kq =

KD ......( 11 ) τ0

Kq is a measure of the efficiency of quenching. The calculated values of the Stern-Volmer constant as well as the bimolecular quenching constants have been given in Table 4. Table 4. Values of the Stern–Volmer Constant (KD), the Bimolecular Quenching Constant (Kq), the Molar ‘sphere of interaction volume’ (V), Obtained from Quenching Measurements using Steady State and Time Resolved fluorescence of DPH in SUVs of DMPC (1 mM lipid; T = 39 °C at pH 7.4), in Presence of the Five Oxicam NSAIDs NSAID (Quencher)

KD (M-1)

Kq 8 (x 10 M-1s-1 )

V (Molar Sphere of action volume) (M-1)

Meloxicam

14.54 ± 0.56

16.0 ± 0.6

105.31 ± 2.82

Piroxicam

5.24 ± 0.21

5.8 ± 0.2

131.05 ± 2.19

Tenoxicam

2.34 ± 0.11

2.6 ± 0.1

186.56 ± 1.99

Lornoxicam

35.13 ± 2.56

38.6 ± 2.8

76.26 ± 1.00

Isoxicam

4.76 ± 0.16

5.2 ± 0.1

109.76 ± 2.92

As has been mentioned before, most of the DPH molecules prefer to intercalate in the region of the lipid acyl chains closer to the membrane surface which are better aligned than regions towards the centre of the bilayer.40 This also corresponds to the preferred location of the drugs (C1-C9) Mx, Px, Tx and Isx as seen from the changes in their B values given in the previous section. This part of the fatty acyl chains are better aligned and more restricted than the portions of the tails nearer to the centre of the bilayer. Hence, diffusion controlled collisional quenching is expected to be restricted in this region. This is reflected in the relatively lower KD values and the 28 ACS Paragon Plus Environment

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corresponding Kq values of these drugs compared to Lx. The deeper location of Lx towards the more unrestricted region of the tail ends facilitates diffusion controlled collisional quenching, as is reflected in its highest KD and Kq values. On the other hand, the static component, which is reflected by the V values, would be facilitated when the probe and the quencher is trapped in close proximity within a restricted environment as seen in case of Mx, Px, Tx and Isx. This is reflected in the relatively high V values of these drugs compared to Lx. It is evident that each of these drugs partition and get incorporated inside the SUVs with Lx preferring a deeper location when compared to the other four drugs. This is consistent with the Tm and B values. In order to see how the differential partitioning, locations as well as perturbing ability of the drugs are reflected in their ability to induce membrane fusion, we have studied the rates of the intermediate lipid mixing step as well as the final content mixing step. 3.5 Effect on Fusion Kinetics. Fluorescence based fusion assays were used to monitor the rates and extents of lipid mixing, content mixing in presence of the five oxicam drugs, keeping the membrane mimetic system same. The drug concentrations were kept constant (D/L ratio 0.03) in all our experiments. The previous sections showed how only changing the nature of the drugs can lead to differences in their interactions with identical membrane mimetic i.e. DMPC SUVs, as well as the location of the drugs in it. This section will highlight how the differences in the location of the drugs and their interactions with the membrane will manifest in their fusogenic behavior. The representative plots of lipid mixing and content mixing in presence of Lx and Isx are given in Figure 6a and 6b respectively.



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Figure 6: Time courses of, % lipid mixing (a) and % content mixing (b) recorded in the absence and presence of Lx and Isx at D/L 0.03. The solid grey lines are obtained by fitting the plots to a single exponential rate equation given by f = a [1- exp (-kt)]. Control curves recorded at identical conditions in absence of the drugs are also included. This shows that no fusion occurs in absence of the drugs. The rates associated with lipid mixing and content mixing were calculated by fitting the time courses to a single exponential rate equation f = a[1 - exp(-kt)], where “k” is the rate constant and pre-exponential factor “a” is the 30 ACS Paragon Plus Environment

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extent, i.e. the percentage of lipid mixing or content mixing at infinite time. The extent is the percentage population of the vesicles involved in a particular step. The rate constants and extents of the two steps of fusion in presence of the drugs are given in Table 5. Table 5: Rate Constants (k) and Extents (Expressed as %) of Lipid Mixing and Content Mixing in the Presence of Mx, Px, Tx, Lx and Isx Drugs D/L Lipid mixing Lipid mixing Content mixing Content mixing ratio rate constant extent rate constant extent -3 -1 -3 -1 (x 10 s ) (%) (x 10 s ) (%) a a a a Mx 8.16 ± 0.30 39.4 0.85 ± 0.06 8.4a Pxa 6.79 ± 0.40a 35.8a 0.39 ± 0.01a 5.0a 0.03 Tx 10.69 ± 0.45 36.7 0.45 ± 0.05 7.2 Lx 9.32 ± 0.51 44.5 0.82 ± 0.10 14.5 Isx 12.21 ± 0.62 38.5 0.45 ± 0.02 10.0 Temperature was maintained at 39 °C and pH was maintained at 7.4 for all the experiments. a

Values taken from Reference 37.

The extents of both the lipid and content mixing steps correlated well with the Kp values (Table 1). Increase in partitioning (Kp) resulted in increased extent of both the steps. Since extents represent the percentage vesicle population involved in a particular step, it is expected that greater partitioning of the drugs in the vesicles will ensure greater involvement of the vesicles in that step. However, the rates of lipid mixing step do not show such correlation with the partitioning of the drugs in the lipidic phase. It should be mentioned that lipid mixing step involves extensive lipidic rearrangements, requiring different types of lipid tail movements to form the hemifusion stalk intermediate structure. Hindering any movement e.g. tail tilting motion, 43, 44 can inhibit the lipid mixing rates. Both, the amount of drug partitioned in the lipid bilayer and their location will determine how the motions of the lipid tails are inhibited. Hence, unlike the lipid mixing extents the rates are not directly correlated to the Kp values. Hydrophilic Tx and Isx, which localize more 31 ACS Paragon Plus Environment


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towards the head-group region of the bilayer exhibit higher rates of lipid mixing when compared to their more hydrophobic counter parts Mx and Lx. Though all the drugs are responsible for bringing about perturbations in the bilayer to subsequently bring about membrane fusion, there is an underlying difference in the manner in which they bring about the perturbations. The removal of the hydration sheath followed by the opening up of the outer leaflet of lipid bilayer, forms the first step of membrane fusion. Tx and Isx partition into the lipid bilayer and bring about the initial perturbations in the head-group region so as to initiate the first step of fusion, lipid mixing. It might be possible that the drugs localizing with ease near the head-group regions of the bilayer help the outer leaflet of the bilayer to achieve a negative curvature required for promoting the first step of the fusion process. Also, their location is such that they do not restrict the lipid tail movements due to crowding as in case of the hydrophobic Mx and Lx, thereby result in higher lipid mixing rates. Px stands out in its behavior which is its characteristic.11, 45 As mentioned before, the extent of content mixing correlates well with the Kp values. The content mixing rate constants are higher for the more hydrophobic drugs like Mx and Lx that partition deeper in the bilayer, compared to the hydrophilic Tx, Isx and Px. This trend is opposite to what we have seen in case of lipid mixing rate constants. Deeper location of Mx and Lx in the bilayer could perturb the bilayer interior to facilitate the opening of the inner leaflet to form the fusion pore leading to content mixing. Another point to note is that rate of the spontaneous leakage competes with the content mixing more than the lipid mixing step thereby, modulating the rates and extent of the last step of fusion.40 3.6 Dynamic Light Scattering Studies (DLS) of Drug Treated Vesicles Recorded at Different Time Points of the Final Content Mixing Step. Dynamic light scattering (DLS) was used to determine the size distribution of vesicles in presence of different drugs, recorded at


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different time points spanning the content mixing step. In general, two time points were chosen, one during the rise part of the content mixing kinetic curve and the other near the saturation of the same. Figure 7 represents the DLS data for all the oxicam NSAIDs treated vesicles, recorded at these two time points. In each panel the DLS data for pure DMPC vesicles are also included for easy reference. It should be mentioned that the size distribution of DMPC vesicles in absence of the drugs essentially remained unaffected at different time points. As has been pointed out in the experimental section, the DLS data are expressed as number percentage instead of intensity percentage vs. hydrodynamic diameter such that the relative changes in the size and size distribution of these small vesicles is not overshadowed by the presence of a few large vesicles or aggregates. Since scattering intensity is proportional to the square of the molecular weight, the intensity from a small amount of large particles can dominate the distribution. From Figure 7 it is evident that in presence of all the five drugs, there is an increase in the average diameter of the vesicles along with a broadening of the size distribution, for time points taken from the rise part of the content mixing curve. This shows that vesicles with larger average diameters are formed due to fusion, while the broadening reflects presence of mixed population in the different stages of the fusion process. For time points near the saturation, further shift in the average diameter occurs, depending on the fusion kinetics. In some cases, the number percentages are decreased due to the leakage of vesicles leading to rupture of a certain population.



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.

Figure 7. Dynamic light scattering profile expressed in Number % vs diameter (nm) for 100 % DMPC SUVs in the presence of the five oxicam NSAIDs as shown. Temperature was maintained at 39 °C.


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Finally, we must point out that since we have worked with only five drugs, it is difficult to directly correlate the structural features of the drugs with its properties like partitioning in the lipidic phase, fluidization and fusion of the vesicles. To do so, we require a larger set of structurally related drugs. Hence, we can only speculate on what chemical features is essential or most important for the above mentioned properties. In this connection the clear winner is Lx. It partitions maximally in the bilayer, leading to highest perturbing /fluidizing ability, which in turn makes it an effective membrane fusogen as expressed by the highest lipid mixing and content mixing extents.

4. CONCLUSIONS To summarize our findings, a) The Kp values determined from second derivative absorbance spectroscopy, do not correlate fully with the log Doct values (at pH 7.4). This was somewhat expected since octanol/water is not a good model membrane and is unable to model several drug membrane interactions which include steric effects and van der Waals interaction etc. b) Monitoring the gel-sol transition of the lipid bilayer in presence of the drugs, using steady state fluorescence anisotropy of DPH, showed the ability of all the drugs to perturb the bilayer. Measuring the cooperativity of the transition in presence and absence of the drugs allow us to conclude a location between C1-C9 for all the drugs excepting Lx, which is incorporated deeper in the bilayer beyond C9. The same inference has also been made from the fluorescence quenching studies of the DPH probe in presence of the five drugs.



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c) From the data obtained from lifetime measurements of DPH in presence of the drugs in the bilayer matrix, it is seen that all the drugs irrespective of their locations, hydrates the bilayer differentially. Maximum hydration is caused by Lx that partition deep into the hydrophobic core, causing a re-distribution in the probe population between near surface location and deeper location in the bilayer. Thus, these five drugs partition into different locations, cause perturbation in the otherwise strongly packed lipid bilayers, and exhibit different fusogenic behavior, as is evident from the different rate constants and extents associated with the intermediate steps of the fusion process. The rough order of effectiveness of these NSAIDs in inducing membrane fusion in the DMPC vesicles as evident from the extents of content mixing is as follows: Lx> Isx >Mx > Tx> Px. The rate constants do not follow the exact same trend. It is observed that the drugs which localize mainly near the membrane-water interface thereby, perturbing that region, show higher rate constants of lipid mixing involving the outer leaflet of the bilayer, when compared to the more hydrophobic Mx and Lx. On the other hand, Mx and Lx located comparatively deeper in the hydrophobic interior, show higher rate constants of content mixing, due to greater extent of perturbations within the inner hydrophobic tail regions of the bilayer which affects the inner leaflet more, to facilitate pore opening and content mixing. Our DLS data provides us with snapshots of vesicle size distribution at different stages of the final step of NSAID induced membrane fusion. The differential ability of these five oxicam to affect different steps of the fusion process cannot be pinpointed to only on their extent of interaction i.e. partitioning in the bilayer, but location also plays an important role. The membrane fusion itself is a very complex process with the various steps requiring different conditions in the bilayer, to strike the correct structure


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energy balance to overcome the energy barriers governing each step. It is relatively easy to achieve this by altering lipidic parameters, but is more demanding when we try to achieve this only by altering the nature of the drugs generated by isosteric substitution as in case of the oxicam NSAIDs studied here. The fact that each oxicam drug can induce fusion indicates that they can cause the right kind of perturbation in the bilayer, by striking a correct balance between their partitioning and how they incorporate in different regions of the bilayer. Both the partitioning and the location are dependent on the physico-chemical nature of the drugs which differentially affects their ability to act as membrane fusogens.

SUPPORTING INFORMATION: Figure S1, Plots of second-derivative absorbance values (D) at corresponding λmax against concentration of DMPC vesicles [L] for (a) Meloxicam, (b) Piroxicam and (c) Tenoxicam. Figure S2, Representative steady-state fluorescence anisotropy of DPH plotted against temperature, in the presence of Mx, Px and Tx (D/L 0.03). Figure S3, S4, S5, S6, Representative Stern-Volmer plots of DPH incorporated in DMPC vesicles, in the presence of Isx, Tx, Lx amd Px respectively, showing (a) τ0/τ vs [Qm] obtained from fluorescence lifetime measurements and (b) F0/F vs [Qm] obtained from steady-state fluorescence intensity measurements. Figure S7, Time courses of % lipid mixing recorded in the presence of Mx, Px and Tx at D/L 0.03. Figure S8, Time courses of % content mixing recorded in the presence of Mx, Px and Tx at D/L 0.03. This material is available free of charge via the Internet at http://pubs.acs.org. FUNDING This work was financially supported by BARD (Biomolecular Assembly, Recognition and Dynamics) project of Saha Institute of Nuclear Physics, Kolkata, funded by Department of Atomic Energy, Government of India.



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AUTHOR INFORMATION Corresponding Author: Prof. Munna Sarkar *Fax: +91-33-23374637. E-mail: [email protected]. Notes The authors declare no competing financial interest.

ACKNOWLEDGEMENT We acknowledge BARD (Biomolecular Assembly Recognition and Dynamics) project of SINP funded by Department of Atomic Energy, Government of India for financial support. We thank Mr. Ajoy Das of Chemical Sciences Division for his cooperation in handling the TCSPC instrument. A. Majumdar acknowledges the University Grants Commission, India for her Ph.D. fellowship.


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REFERENCES 1. Mitchell, J. A.; Akarasereenont, P.; Thiemermann, C.; Flower, R. J.; Vane, J. R. Selectivity of Nonsteroidal Antiinflammatory Drugs as Inhibitors of Constitutive and Inducible Cyclooxygenase. Proc. Natl. Acad. Sci.1993, 90, 1163-11697. 2. Mitchell, J. A.; Warner, T. D. Cyclo-Oxygenase-2: Pharmacology, Physiology, Biochemistry and Relevance to NSAID Therapy. Br. J. Pharmacol. 1999, 128, 1121-1132. 3. Rao, P.; Knaus, E. E. Evolution of Nonsteroidal Anti-Inflammatory Drugs (NSAIDs): Cyclooxygenase (COX) Inhibition and Beyond. J. Pharm Pharm Sci. 2008, 11, 81s-110s. 4. Keller, J. J.; Giardiello, F. M. Chemoprevention Strategies Using NSAIDs and COX-2 Inhibitors. Cancer Biol. Ther. 2003, 2, 139-148. 5. Ulrich, C. M.; Bigler, J.; Potter, J. D. Non-Steroidal Anti-Inflammatory Drugs for Cancer Prevention: Promise, Perils and Pharmacogenetics. Nat Rev Cancer 2006, 6, 130–140. 6. McGeer, P. L.; McGeer, E. G. NSAIDs and Alzheimer Disease: Epidemiological, Animal Model and Clinical Studies. Neurobiol Aging 2007, 28, 639-647. 7. Wyss-Coray, T.; Rogers, J. Inflammation in Alzheimer Disease—A Brief Review of the Basic Science and Clinical Literature. Cold Spring Harb Perspect Med 2012, 2, 1-23. 8. Esposito, E.; Matteo, V. D.; Benigno, A.; Pierucci, M.; Crescimanno, G.; Giovanni, G. D. Non-Steroidal Anti-Inflammatory Drugs in Parkinson's Disease. Exp. Neurol. 2007, 205, 295312. 9. Moore, A. H.; Bigbee, M. J.; Boynton, G. E.; Wakeham, C. M.; Rosenheim, H. M.; Staral, C. J.; Morrissey, J. L.; Hund, A. K. Non-Steroidal Anti-Inflammatory Drugs in Alzheimer's Disease

and

Parkinson's

Disease:

Reconsidering

the

Role

of

Neuroinflammation.

Pharmaceuticals 2010, 3, 1812-1841.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 40 of 45

10. Chakraborty, H.; Devi, P. G.; Sarkar, M.; Dasgupta, D. New Functions of Old Drugs: Aureolic Acid Group of Anti-Cancer Antibiotics and Non-Steroidal Anti-Inflammatory Drugs. In Recent Advances in Medicinal Chemistry; Rahman, A.-R., Perry, G., Ed.; Bentham Science Publishers: 2014; Vol. 1, pp 3−55. 11. Chakraborty, H.; Banerjee, R.; Sarkar, M. Incorporation of NSAIDs in Micelles: Implication of Structural Switchover in Drug–Membrane Interaction. Biophys. Chem. 2003, 104, 315-325. 12. Lichtenberger, L. M.; Zhou, Y.; Dial E. J.; Raphael, R. M. NSAID Injury to the Gastrointestinal Tract: Evidence that NSAIDs Interact with Phospholipids to Weaken the Hydrophobic Surface Barrier and Induce the Formation of Unstable Pores in Membranes. J.

Pharm. Pharmacol. 2006, 58, 1421-1428. 13. Pereira-Leite, C.; Nunes, C.; Reis, S. Interaction of Nonsteroidal Anti-Inflammatory Drugs with Membranes: in vitro Assessment and Relevance for their Biological Actions. Prog.

Lipid. Res. 2013, 52, 571−584. 14. Lichtenberger, L. M.; Zhou, Y.; Jayaraman, V.; Doyen, J. R.; O’Neil, R. G.; Dial, E. J.; Volk, D. E.; Gorenstein, D. G.; Boggara, M. B.; Krishnamoorti, R. Insight into NSAID-Induced Membrane Alterations, Pathogenesis and Therapeutics: Characterization of Interaction of NSAIDs with Phosphatidylcholine. Biochim Biophys Acta. 2012, 1821, 994-1002. 15. Nunes, C.; Brezesinski, G.; Pereira-Leite, C.; Lima, J. L. F. C.; Reis, S.; Lúcio, M. NSAIDs Interactions with Membranes: A Biophysical Approach. Langmuir 2011, 27, 1084710858. 16. Pinheiro, M.; Silva, A. S.; Pisco, S.; Reis, S. Interaction of Isoniazid with Membrane Models: Implication for Drug Mechanism of Action. Chem. Phys. Lipids 2014, 183, 184-190.


Page 41 of 45

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


17. Chakraborty, H.; Chakraborty, P. K.; Raha, S.; Mandal, P. C.; Sarkar, M. Interaction of Piroxicam with Mitochondrial Membrane and Cytochrome C. Biochim. Biophys. Acta,

Biomembranes 2007, 1768, 1138-1146. 18. Chakraborty, H.; Mondal, S.; Sarkar, M. Membrane fusion: A New Function of Non Steroidal Anti-Inflammatory Drugs. Biophys. Chem. 2008, 137, 28-34. 19. Mondal, S.; Sarkar, M. Non-Steroidal Anti-Inflammatory Drug Induced Membrane Fusion: Concentration and Temperature Effects. J. Phys. Chem. B 2009, 113, 16323-16331. 20. Martens, S.; McMahon, H. T. Mechanisms of Membrane Fusion: Disparate Players and Common Principles. Nat. Rev. Mol. Cell Biol. 2008, 9, 543-556. 21. Weber, T.; Zemelman, B.V.; McNew, J. A.; Westermann, B.; Gmachl, M.; Parlati, F.; Söllner, T. H.; Rothman, J. E. Snarepins: Minimal Machinery for Membrane Fusion. Cell 1998,

92, 759-772. 22. Beniac, D. R.; deVarennes, S. L.; Andonov, A.; He, R.; Booth, T. F. Conformational Reorganization of the SARS Coronavirus Spike Following Receptor Binding: Implications for Membrane Fusion. PLos One 2007, 2, e1082. 23. Fuhrmans, M.; Marrink, S. J. Molecular View of the Role of Fusion Peptides in Promoting Positive Membrane Curvature. J. Am. Chem. Soc. 2012, 134, 1543-1552. 24. Risselada, H. J.; Grubmüller, H. How SNARE Molecules Mediate Membrane Fusion: Recent Insights from Molecular Simulations. Curr. Opin. Struct. Biol. 2012, 22, 187-196. 25. Mondal Roy, S.; Bansode, A. S.; Sarkar, M. Effect of Increase in Orientational Order of Lipid Chains and Head Group Spacing on Non Steroidal Anti-Inflammatory Drug Induced Membrane Fusion. Langmuir 2010, 26, 18967-18975.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 42 of 45

26. Mondal Roy, S.; Sarkar, M. Effect of Lipid Molecule Headgroup Mismatch on Non Steroidal Anti-Inflammatory Drugs Induced Membrane Fusion. Langmuir 2011, 27, 1505415064. 27. Floersheim P.; Pombo-Villar E.; Shapiro, G. Isosterism and Bioisosterism Case Studies with Muscarinic Agonists. Chimia 1992, 46, 323-334. 28. Lúcio, M.; Ferreira, H.; Lima, J. L. F. C.; Reis, S. Interactions between Oxicams and Membrane Bilayers: An Explanation for their Different COX Selectivity. Med Chem. 2006, 2, 447-456. 29. Palacios, L. E.; Wang, T. Egg-Yolk Lipid Fractionation and Lecithin Characterization. J.

Am. Oil Chem. Soc. 2005, 82, 571-578. 30. Walczak, J.; Bocian, S.; Buszewski, B. Two-Dimensional High Performance Liquid Chromatography-Mass Spectrometry for Phosphatidylcholine Analysis in Egg Yolk. Food Anal.

Methods 2015, 8, 661-667. 31. Santos, N. C.; Prieto, M; Castanho, M. A. R. B. Quantifying Molecular Partition into Model Systems of Biomembranes: An Emphasis on Optical Spectroscopic Methods. Biochim.

Biophys. Acta-Biomembr. 2003, 1612, 123-135. 32. Barrantes, F. J.; Antollini, S. S.; Blanton, M. P.; Prieto, M. Topography of Nicotinic Acetylcholine Receptor Membrane-Embedded Domains. J. Biol. Chem. 2000, 275, 37333– 37339. 33. Wasan, E. K.; Harvie, P.; Edwards, K.; Karlsson, G.; Bally, M. B. A Multi-Step Lipid Mixing Assay to Model Structural Changes in Cationic Lipoplexes Used for in vitro Transfection. Biochim. Biophys. Acta-Biomembr. 1999, 1461, 27-46.


Page 43 of 45

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


34. Wilschut, J.; Düzgüneş, N.; Fraley, R.; Papahadjopoulos, D. Studies on the Mechanism of Membrane Fusion: Kinetics of Calcium Ion Induced Fusion of Phosphatidylserine Vesicles Followed by a New Assay for Mixing of Aqueous Vesicle Contents. Biochemistry 1980, 19, 6011-6021. 35. Tsai, R.-S.; Carrupt, P.-A.; Tayar, N. E.; Giroud, Y.; Andrade, P.; Testa, B. Physicochemical and Structural Properties of Non-Steroidal Anti-Inflammatory Oxicams. Helv.

Chim. Acta. 1993, 76, 842-854. 36. Mälkiä, A.; Murtomäki, L.; Urtti, A.; Kontturi, K. Drug Permeation in Biomembranes in vitro and in Silico Prediction and Influence of Physicochemical Properties. Eur. J. Pharm. Sci. 2004, 23, 13-47. 37. Majumdar, A.; Chakraborty, S.; Sarkar, M.

Modulation of Non Steroidal Anti-

Inflammatory Drug Induced Membrane Fusion by Copper Coordination of These Drugs: Anchoring Effect. J. Phys. Chem. B 2014, 118, 13785-13799. 38. Konopásek, I.; Večeř, J.; Strzalka, K.; Amler, E. Short-Lived Fluorescence Component of DPH Reports on Lipid−Water Interface of Biological Membranes. Chem. Phys. Lipids 2004,

130, 135−144. 39. Konopasek, I.; Kvasnicka, P.; Herman, P.; Linnertz, H.; Obsil, T.; Večeř, J.; Svobodova, J.; Strzalka, K.; Mazzanti, L.; Amler, E. The Origin of the Diphenylhexatriene Short Lifetime Component in Membranes and Solvents. Chem. Phys. Lett. 1998, 293, 429−435. 40. Kaiser, R. D.; London, E. Location of Diphenylhexatriene (DPH) and its Derivatives within Membranes: Comparison of Different Fluorescence Quenching Analyses of Membrane Depth. Biochemistry 1998, 37, 8180-8190.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 44 of 45

41. Lakowicz, J. R. Quenching of Fluorescence. In Principles of Fluorescence Spectroscopy; Lakowicz, J. R., Ed.; Springer: New York, U. S. A., 2006; pp. 298-300. 42. Lúcio, M.; Nunes, C; Gaspar, D.; Golebska, K.; Wisniewski, M.; Lima, J. L. F. C; Brezesinski, G.; Reis, S. Effect Of Anti-Inflammatory Drugs in Phosphatidylcholine Membranes: A Fluorescence and Calorimetric Study. Chem. Phys. Lett. 2009, 471, 300-309. 43. Kuzmin, P. I.; Zimmerberg, J.; Chizmadzhev, Y. A.; Cohen, F. S. A Quantitative Model for Membrane Fusion Based on Low-Energy Intermediates. Proc. Natl. Acad. Sci. 2001, 98, 7235-7240. 44. Kozlovsky, Y.; Kozlov, M. M. Stalk Model of Membrane Fusion: Solution of Energy Crisis. Biophys. J. 2002, 82, 882-895. 45. Kundu, S.; Chakraborty, H.; Sarkar, M.; Datta, A. Interaction of Oxicam NSAIDS with Lipid Monolayer: Anomalous Dependence on Drug Concentration. Colloids Surf. B 2009, 70, 157−161.


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Differential Effect of Oxicam Non-Steroidal Anti-Inflammatory Drugs on

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