Effects of NSAIDs in Membrane Bilayers

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Organic Health Protocol

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T. Mavromoustakos1,1. Kyrikou1, A. Kapou1 and D. Kovala-Demertzi2

'Institute of Organic and Pharmaceutical Chemistry, Vas. Constantinou 48, Athens 11635 2University of Ioannina, Chemistry Department, loannina 45110

Abstract.

The thermal effects of non-steroidal antiinflammatory drugs (NSAIs), aspirin, piroxicam and tenoxicam, in DPPC bilayers without and with cholesterol are studied. All drugs affected the pre- and main transition temperatures of DPPC bilayers without and with cholesterol. Their effects can be summarized to the lowering of the phase transitions and causing an increase of the breadth of the transitions at low concentrations. At higher concentrations their effect is augmented and the pretransition for most of DPPC/NSAID bilayers is abolished. The effect of pH did not appear to modify significantly the thermal effects of these drugs.

Introduction

Aspirin, or acetylsacylic acid, is synonymous for first aid relief of pain, fever, and inflammation. The 100-years old and most popular drug is facing new competition. The unique strength of acetylsacicylic acid is its nonselective behavior and lack of specificity for a single target. It is the portfolio of different properties that makes acetylsacylic acid such a unique drug. The therapeutic effect of acetylsacylic acid is based on a covalent modification of cyclooxygenase and the inhibition of the first step of prostaglandin synthesis. Recently, two different cyclooxygenase isoforms have been characterized COX-1 and COX-2. Inhibition of COX-1 or COX-2 leads to very different pharmacological effects. The COX-1 inhibition is predominantly responsible for anti-thrombotic effects, while antiinflammatory effects are mediated mainly through COX-2. New studies from the last two years revealed that in addition to arthritis and pain, cancer and neurodegenerative diseases like Alzheimer's disease could potentially be treated with COX-2 inhibitors. It has been suggested that the anti-inflammatory and most of the analgetic effects of NSAIDs result from an inhibition of PGs synthesized via COX-2 [1-4].

It was reported in the literature that acetylsalicylic acid (ASA) was 5-10 fold more potent to reduce fever when was intragastrically administered in a lipid-associated state. In another report it was shown that the half-life of ASA was prolonged when the agent was chemically associated with DMPC because of its reducing conversion to salicylic acid [5]. For the above reasons the ASA membrane interactions were studied using fluorescence polarization and NMR spectroscopy. The fluorescence polarization results showed that ASA causes disordering of the membrane bilayers in a concentration depending fashion and lowering of AH. The NMR data showed that ASA was not inserted into membrane bilayers and aromatic rings interacted with membrane bilayers. In addition, the mobility of acetylsalicylic acid was restricted by membrane and the ASA changed membrane viscosity. It was proposed that the association of ASA with membrane might involve both electrostatic and hydrophobic forces. In another study it was shown that aspirin, benzoate and salicylate caused significant increases in membrane conductance [6-8].

These results triggered our research interest to apply DSC and molecular modeling in order to further study the effects of ASA with membrane bilayers and to compare its thermal effects with NSAIDs piroxicam and tenoxicam shown in Figure 1.

Piroxicam, [4-hydroxy-2-methyl-N-pyridin-2-yl)-2H-1,2-benzothiazine-3-

carboxamide-1,1-dioxide], H2pir, is a potent and extensively used non-steroidal antiinflammatory, anti-arthritic drug with a long biological half-life. To date, piroxicam is among the top ten NSAIDs in the market. Tenoxicam [4-hydroxy-2,methyl-N-2-pyridyl-2H-thieno(2,3-e)-l,2-thiazine-3-carboxamide-1,1-dioxide], is a new non-steroidal drug which has anti-inflammatory, analgetic and antipyretic effects: The drug is widely used in the treatment of rheumatic diseases. It is a derivative of oxicam with a thiophene ring replacing the benzene ring in piroxicam. Both piroxicam and tenoxican are the most famous members of this group and are nonselective inhibitors like aspirin We have developed an interest in NSAIDs, the co-ordination chemistry and anti-inflammatory properties of NSAIDs with transition and non-transition metal ions [9,10].

DPPC bilayers were used for these studies because its thermal effects and dynamic properties are well studied. In addition, the incorporation of various drugs in the spontaneously phospholipid bilayers formed after hydration have been studied extensively in an attempt to understand the molecular interactions between drugs with phospholipids. The presence of an additive in membrane bilayers affects the thermodynamic parameters that govern a thermogram such as the maximum of the main phase transition or the pretransition (Tm), the heat capacity of the peaks (Cp) and the line-width (Tmi/2) [8-11]. The nature of the DSC thermograms can be understood if the total intermolecular effects (i.e. interfacial, hydrogen bonds and nonspecific hydrophobic and electrostatic interactions) between the additive and the phospholipid bilayers are considered.

Membrane preparations were formed when the drug was in a neutral and acidic environment (pH=2.5) in order to explore any differential effect of anionic or non- anionic forms of the molecules in membrane bilayers.

The effect of the cholesterol in membrane bilayers containing NSAID was also studied. Cholesterol is a major constituent of membrane bilayers and DPPC/cholesterol bilayers is often more distinctive system to differentiate the thermal properties of drugs. [12].

Aspirin

Piroxicam

Aspirin

Piroxicam

Tenoxicam

Figure 1: Chemical structures ofNSAIDs Results

Differential Scanning Calorimetry: Differential scanning Calorimetry (DSC) is a fast and relatively inexpensive technique that allows the study of the thermotropic properties of the membranes in the absence and presence of bioactive molecules. Therefore, it is used in our laboratory as a diagnostic technique to investigate differential effects that may be caused by the incorporation of additives. When such differential effects are observed, then techniques that offer complementary and more detailed information on the thermal and dynamic properties of membranes, with or without the presence of additives are applied.

Figure 2 shows DSC scans of DPPC and DPPC/NSAID bilayers. The DPPC bilayers exist in the gel phase (Lp ) for temperatures lower than 33 °C, and in the liquid crystalline phase for temperatues higher than 42 °C (IV). In between 33-42 °C the phospholipid bilayers exist in Pp or ripple phase. The obtained DSC scan of fully hydrated DPPC multibilayers shows a pretransition centered at 35 °C and a peak maximum at 41.2 °C. The main phase transition is accompanied by several structural changes in the lipid molecules as well as systematic alterations in the bilayer geometry, but the most prominent feature is the trans-gauche isomerization-taking place in the acyl chain conformation. The average number of gauche conformers indicates the effective fluidity, which depends not only on the temperature, but also on perturbations due to the presence of a drug molecule intercalating between the lipids.

At low concentration of ;t=0.01 (1%-molar ratio) all NSAEDs affect in a similar way the thermal properties of DPPC bilayers, by causing marginal broadening of the pretransition and they cause narrowing of the breadth of the phase transition. The addition of higher concentration of NSAID (x=0.05) augments their thermal effects. In particular, at this concentration aspirin abolishes the pre-transition temperature and causes broadening of the phase transition temperature, effects not observed by piroxicam and tenoxicam. Only piroxicam at this concentration causes significant lowering of the phase transition temperature. At x=0.10 aspirin causes further broadening of the phase transition temperature. Tenoxicam affects only the pretransition by broadening it and piroxicam causes broadening both to the pre- and main transition temperatures. At x=0.20 aspirin does cause a lowering of the phase transition and no further broadening of the phase transition. In contrary, tenoxicam causes a significant broadening and more lowering of the phase transition as well as abolishement of pre-transition. Piroxicam also abolishes the pretransition and causes the most lowering of the phase transition among three preparations. It appears that aspirin causes the most significant effect up tojr=0.10 and lowest at the higher concentration of *=0.20. The drug molecules under study do not appear to affect the enthalpy change {AH) of the main phase transition of DPPC bilayers (see quantitative results on Table 1).

Figure 3 shows DSC scans of the same NSAIDs on DPPC bilayers using identical concentrations and buffer (pH=2.5). Interestingly, similar results have been observed as in aqueous DPPC bilayers for piroxicam and tenoxicam. Aspirin had the most significant effect in all concentrations summarized to:(a) lowering of the phase transition; (b) increase of the breadth of the phase transition and (c) abolish of the pre-transition at lower concentration.

These results show that the effect of pH is only marginal. An exception as is described occurs at x=0.20 with aspirin which causes more significant thermal effects on DPPC bilayers at pH=2.5. This probably reflects that aspirin is more active in membrane bilayers when it is in non-anionic form rather than in anionic form.

Figure 4 shows the effects of NSAIDs in DPPC/cholesterol (x=0.10) bilayers. DPPC/cholesterol bilayers show a phase transition centered at 41.4 °C and no pretransition. The presence of NSAID in this bilayer results in the broadening of the width of phase transition and a slight lowering of the phase transition temperature. The most significant effect in the broadening of the phase transition is observed with aspirin. These results show that cholesterol at low concentrations does not interfere with the way the studied NSAIDs perturb the membrane bilayers. Piroxicam and tenoxicam appear to affect in an almost identical way the DPPC/cholesterol bilayers.

The effects of NSAIDs were also studied in high cholesterol content. DPPC/cholesterol (jc=0.15) shows a considerable broad peak. The presence of the NSAID causes inhomogeneity of the sample, since it causes significant broadeninig and additional peaks (different domains) that probably consist of DPPC/cholesterol, DPPC/NSAID or DPPC/NSAID/cholesterol.

i I i M M M I | i | i | i | i | i I—I i I i I i 1 i 1 i

10 20 30 40 50 60 10 20 30 40 50 60 10 20 30 40 50 60

i I i M M M I | i | i | i | i | i I—I i I i I i 1 i 1 i

10 20 30 40 50 60 10 20 30 40 50 60 10 20 30 40 50 60

Figure 2: DSC scans of NSAIDS when bilayers are formed with the use of water.

Temperature Temperature

Figure 4: DSC scans of NSAIDS when bilayers are formed with the use of water and cholesterol

Temperature Temperature

Figure 4: DSC scans of NSAIDS when bilayers are formed with the use of water and cholesterol

Table 2: Values of Phase Pretransition and Transition Temperatures (Tm), Half-Width Temperature and Enthalpy Changes (AH) of DPPC with or without NSAIDS with the use of water._______

samples

Tml/2

Tm

AH

DPPC alone

0.

50

42.571

41.838

DPPC + aspirin (x=0.01)

0.

50

41.715

41.930

DPPC + aspirin (x=0.05)

0.

50

41.179

41.470

DPPC + aspirin (x=0.10)

0.

60

40.287

40.826

DPPC + aspirin (x=0.20)

0.

75

39.363

39.502

DPPC + tenoxicam (x=0.01)

0.

40

41.729

42.169

DPPC + tenoxicam (x=0.05)

0.

40

41.378

41.340

DPPC + tenoxicam (x=0.10)

0.

50

41.236

41.470

DPPC + tenoxicam (x=0.20)

1.

15

38.285

43.670

DPPC + Piroxicam (x=0.01)

0.

50

41.066

42.502

DPPC + Piroxicam (x=0.05)

0.

60

39.316

42.316

DPPC + Piroxicam (x=0.10)

0.

85

38.985

41.266

DPPC + Piroxicam (x=0.20)

0.

90

36.558

42.128

Table 3: Values of Phase Pretransition and Transition Temperatures (Tm), Half-Width Temperature and Enthalpy Changes (AH) of DPPC with or without NSAIDS with the use of buffer. _

samples

Tml/2

Tm

AH

DPPC alone

0.65

42.20

42.09

DPPC + aspirin (x=0.01)

0. 60

42.94

39.95

DPPC + aspirin (x=0.05)

1.00

41.69

40.60

DPPC + aspirin (x=0.10)

1.35

40.63

41.44

DPPC + aspirin (x=0.20)

1.40

36.53

43.62

DPPC + tenoxicam (x=0.01)

0. 50

42.04

41.63

DPPC + tenoxicam (x=0.05)

0. 50

41.82

41.20

DPPC + tenoxicam (x=0.10)

0. 70

41.24

41.54

DPPC + tenoxicam (x=0.20)

1. 20

38.82

41.00

DPPC + Piroxicam (x=0.01)

0. 55

42.87

40.84

DPPC + Piroxicam (x=0.05)

0.60

41.69

42.31

DPPC + Piroxicam (x=0.10)

0.70

40.85

41.07

DPPC + Piroxicam (x=0.20)

0.90

39.85

40.87

Table 4: Values of Phase Pretransition and Transition Temperatures (Tm), Half-Width Temperature and

Enthalpy Changes (AH) of DPPC / CHOL with or without Compound with the use of water._

samples

DPPC/CHOL (90:10) alone DPPC/CHOL (90:10) + 1 (x=0.10) DPPC/CHOL (90:10) + 2 (x=0.10) DPPC/CHOL (90:10) + 3 (x=0.10) DPPC/CHOL (85:15) alone DPPC/CHOL (85:15) + 1 (x=0.05) DPPC/CHOL (85:15) + 2 (x=0.05) DPPC/CHOL (85:15) + 3 (x=0.05)

Tmi/2

Tm

AH

0.50

41.37

32.28

0.70

40.49

29.30

0.45

40.14

28.15

0.55

40.49

26.25

0.70

41.74

18.33

2.95

40.02

14.50

7.60

32.538

3.61

6.90

32.54

According to the thermograms the three drugs present a similar thermotropic behaviour inside the membrane model. Theoretical calculations confirm that their topography is similar. Each molecule is located near the head-group of the DPPC molecules forming two hydrogen bonds with it. Aspirin is a smaller molecule and is situated lower, towards hydrophobic region. This localization may explain the most perturbing effects in comparison to the other two NSAIDs.

Figure 5: Topography of the nsaids, (a) aspirin, (b) piroxicam, (c) tenoxicam inside the model membrane bilayer

The four oxygen atoms, the pyridyl and imine nitrogens for piroxicam and tenoxicam and the two oxygen atoms for aspirin, exhibit the maximum electron density and negative charge. The highest effective charge and the highest electron density values for the four oxygen atoms and for the pyridyl and imine nitrogen for piroxicam and tenoxicam and for the two oxygen atoms for aspirin show strong electron-donor properties and could rationalize a network of inter-, intra- hydrogen and non-hydrogen or polar bonding [10].

The localization of piroxicam and tenoxicam in the head-group region explains their significant effect on the pre-transition and only marginal effect on the main-transition. The better accommodation of tenoxicam versus piroxicam explains its lowest perturbing effects.

Acknowledgement

We are thankful to GSRT for the funding of this research activity through PENED 99 99ED442. We also thank HELP EPE for the generous gift of piroxicam and tenoxicam.

Piroxicam Chemistry

Figure 5: Topography of the nsaids, (a) aspirin, (b) piroxicam, (c) tenoxicam inside the model membrane bilayer

References

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Part III.

Conformational Analysis of Bioactive Compounds

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10S Press, 2002

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