Beata Korchowiec

Differentiating Oxicam Nonsteroidal Anti-Inflammatory Drugs in Phosphoglyceride Monolayers

Meloxicam, piroxicam, and tenoxicam belong to a highly potent oxicam group of nonsteroidal anti-inflammatory drugs. Whereas the structurally similar oxicams have different pharmacokinetics, treatment efficiency, and adverse effects, their common mechanism of action is the inhibition of a membrane enzyme, cyclooxygenase. Because the prerequisite for accessing the cyclooxygenase by the drugs is interaction with the membrane, the focus of the current study was a comparison of how meloxicam, piroxicam, and tenoxicam interact with lipid monolayers used as models of biological membranes. The monolayers were formed with 1,2-dipalmitoyl-sn-glycero-3-phospho-rac-(1-glycerol), 1,2-dipalmitoyl-sn-glycero-3-phospho-l-serine, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine, 1,2-myristoyl-sn-glycero-3-phosphoethanolamine, and 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine. These systems were examined via surface pressure and surface electrical potential measurements, polarization modulation infrared reflection adsorption spectra, and Brewster angle microscopy. The three oxicams are differentiated in the monolayers; meloxicam shows the highest ability to modify membrane fluidity and surface potential, followed by piroxicam and tenoxicam. The dissimilarity of the biological activity of the oxicams may be linked to different interaction with the membrane, as revealed by the present study.

Membrane activity of tetra-p-guanidinoethylcalix[4]arene as a possible reason for its antibacterial properties.

Tetra-p-guanidinoethylcalix[4]arene trifluoroacetate salt (CX1) was synthesized recently as an antibacterial agent. It showed to be active in vitro against various Gram-positive and Gram-negative bacteria. To get more insight in the mechanism of the biological activity of this derivative, it was studied upon interactions with model lipid membranes. Langmuir monolayers were formed with zwitterionic 1,2-dimyristoyl-sn-glycero-3-phosphocholine or 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine, and with anionic 1,2-dimyristoyl-sn-glycero-3-phospho-rac-(1-glycerol) and 1,2-dimyristoyl-sn-glycero-3-phospho-L-serine. The two classes of lipids were used, respectively, as model lipids of the eukaryotic and bacterial cell membranes. The monolayers were exposed to CX1 at different concentrations around the minimum inhibitory concentration found for E. coli . The surface pressure-area and surface potential-area compression isotherms, as well as Brewster angle microscopy and polarization-modulation infrared reflection-absorption spectroscopy, were employed to study the monolayers. The results obtained show a higher affinity of CX1 for the anionic lipids, indicating importance of charge-charge interactions. On the basis of a comparative study of the behavior of CX1 and that of p-guanidinoethylphenol trifluoroacetate salt, we propose that interplay of charge-charge and apolar interactions between CX1 and lipids is responsible for the important reorganization of model membranes. This proposal may be helpful in developing new antibacterial calixarene derivatives.

Meloxicam and Meloxicam-β-Cyclodextrin Complex in Model Membranes: Effects on the Properties and Enzymatic Lipolysis of Phospholipid Monolayers in Relation to Anti-inflammatory Activity

Meloxicam, a nonsteroidal anti-inflammatory drug (NSAID), is known as a selective cyclooxygenase-2 inhibitor. Cyclooxygenase-2 is a membrane protein, functionally coupled to an interfacial enzyme, phospholipase A2. Consequently, it may be supposed that the interactions of NSAIDs with lipid membranes play a role in the anti-inflammatory process. In order to investigate the mechanism of this process, Langmuir films formed with 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, 1,2-dilauroyl-sn-glycero-3-phosphocholine, 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine, or 1,2-myristoyl-sn-glycero-3-phosphoethanolamine were exposed to meloxicam and its beta-cyclodextrin inclusion complex. The monolayers were studied by measuring surface pressure, electric surface potential, Brewster angle micrographs, polarization-modulation infrared reflection-absorption spectra, and phospholipase A2 activity; the inclusion complex was studied using molecular modeling. The results obtained show that the monolayers formed in the presence of meloxicam and its complex are expanded and more liquid-like compared to pure lipids. Both compounds modify hydration of the lipid polar heads, orientation of the molecules, morphology of the domains, and the rate of lipolysis catalyzed by phospholipase A2. The latter effect may be involved in the anti-inflammatory activity of meloxicam. Importantly, the effects observed with the meloxicam-beta-cyclodextrin complex are more pronounced compared to those of the free meloxicam. This observation may be relevant for developing new meloxicam preparations with increased bioavailability.

Effects of gemini amphiphilic pseudopeptides on model lipid membranes: A Langmuir monolayer study

Monolayers formed with 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine, 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine, 1,2-dimyristoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] and 1,2-dipalmitoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] at the air/water interface were used as model membranes for studying a potential biological activity of four newly synthesized gemini amphiphilic pseudopeptides (GAPs); some of the GAPs studied showed interesting self-assembly properties. The capacity of GAPs to self-assemble in different environments let us think that these molecules may find biomedical applications in, e.g., drug delivery or transfection. The surface pressure-area and surface potential-area compression isotherms, as well as Brewster angle microscopy and polarization-modulation infrared reflection-absorption spectroscopy were used to study monolayers formed with pure GAPs, pure lipids and lipid/GAPs mixtures. The results obtained show that all four GAPs studied can be incorporated in lipid monolayers. The monolayers containing GAPs are expanded and more liquid-like compared to pure lipids. The overall results indicate that the important changes of the properties induced in the model membranes by GAPs are related to their intrinsic conformational flexibility. This feature of GAPs can be easily adjusted by engineering the structure of the spacer present in the polar head, with the aim to modify lipid membranes in a controlled way.

The Mechanism of Metal Cation Binding in Two Nalidixate Calixarene Conjugates. A Langmuir Film and Molecular Modeling Study

The two new p-tert-butylcalix[4]arene derivatives described here bear one or two nalidixic acid arms linked to the lower calixarene rim via the quinolone carboxylate moiety. These derivatives were synthesized in order to investigate two important features of molecules conceived as potential antibiotics, namely, metal cation complexation and interfacial properties, and the way in which they interrelate. The properties of the calixarene derivatives were studied in monomolecular films spread on pure water and on aqueous subphases containing biologically relevant mono- and divalent metal cations. These systems were examined via surface pressure and surface electrical potential measurements, polarization modulation infrared reflection absorption spectroscopy, and molecular modeling. Molecular modeling shows that important differences exist, first, between the structure and stability of the complexes formed with the two derivatives and, second, between their mono- and dication complexes. Correlating the properties of the monolayers with those of the modeled molecules lets us propose that the derivatives bearing one or two nalidixic pending arms form preferentially inter- and intramolecular complexes, respectively. The results obtained in this study indicate that a possible biological role of the nalidixic arms grafted on the calixarene crown may be revealed upon cation complexation.

Interfacial approach to polyaromatic hydrocarbon toxicity: phosphoglyceride and cholesterol monolayer response to phenantrene, anthracene, pyrene, chrysene, and benzo[a]pyrene.

Interactions of phenantrene, anthracene, pyrene, chrysene, and benzo[a]pyrene (polyaromatic hydrocarbons) with model phospholipid membranes were probed using the Langmuir technique. The lipid monolayers were prepared using 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine, 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol, 1,2-dipalmitoyl-sn-glycero-3-phosphoserine, 1,2-myristoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilauroyl-sn-glycero-3-phosphocholine, and cholesterol. Surface pressure and electrical surface potential were measured on mixed phospholipid/PAH monolayers spread on a pure water subphase. The morphology of the mixed monolayers was followed with Brewster angle microscopy. Polarization-modulation infrared reflection-absorption spectroscopy spectra obtained on DPPE/benzo[a]pyrene showed that the latter interacts with the carbonyl groups of the phospholipid. On the other hand, the activity of phospholipase A2 toward DLPC used as a probe to locate benzo[a]pyrene in the monolayers indicates that the polyaromatic hydrocarbons are not accessible to the enzyme. The results obtained show that all PAHs studied affect the properties of the pure lipid, albeit in different ways. The most notable effects, namely, film fluidization and morphology changes, were observed with benzo[a]pyrene. In contrast, the complexity of mixed lipid monolayers makes the effect of PAHs difficult to detect. It can be assumed that the differences observed between PAHs in monolayers correlate with their toxicity.

Glycolipid-cholesterol monolayers: towards a better understanding of the interaction between the membrane components.

In this work, the interaction between a synthetic analog of archaeal lipids and cholesterol was studied using Langmuir technique. The lipid, β-Mal(3)O(C(16+4))(2), contained phytanyl chains attached via two ether bonds to the sn-2 carbon of the glycerol backbone. The preliminary studies showed that monolayers formed with the pure lipid have a liquid-like character; here, a hypothesis that admixing cholesterol to β-Mal(3)O(C(16+4))(2) could confer a higher rigidity on the films was tested. To check this proposal, two-dimensional miscibility of cholesterol and β-Mal(3)O(C(16+4))(2) in monomolecular films was studied using surface pressure and surface potential measurements, as well as Brewster angle microscopy and polarization-modulation infrared reflection absorption spectroscopy. The stability of the monomolecular films was evaluated based on thermodynamics of mixing of cholesterol and β-Mal(3)O(C(16+4))(2). Atomic level information concerning the orientation of molecules and the degree of hydration of polar headgroups was obtained from molecular dynamics simulations.

Interaction of a β-lactam calixarene derivative with a model eukaryotic membrane affects the activity of PLA2

In this research, the interaction between a membrane phospholipid, 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC), and a p-tert-butylcalix[4]arene derivative bearing 6-aminopenicillanic acid (Calix), conceived as a possible drug carrier, was studied. The Langmuir film balance technique was used to measure surface pressure and electrical surface potential of pure and mixed Calix/DLPC monolayers spread on water at different temperatures. Phospholipase A2 (PLA2) activity was used as well to detect the impact of the calixarene derivative on the monolayer properties. Interaction between the molecules in mixed monolayers has been described quantitatively using thermodynamic functions. Interestingly, low amounts of Calix introduce ordering in the lipid film. This effect may be analogous to that of cholesterol interacting with phospholipids. A lower activity of PLA2 observed with the Calix/DLPC films compared to pure DLPC may be related to structural modifications of the mixed systems.

Enzymatic probing of model lipid membranes: phospholipase A2 activity toward monolayers modified by oxicam NSAIDs.

Three nonsteroidal anti-inflammatory oxicam drugs, namely meloxicam, piroxicam, and tenoxicam, were used to modify the properties of monomolecular films formed with 1,2-dilauroyl-sn-glycero-3-phosphocholine, 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine, or 1,2-dilauroyl-sn-glycero-3-phospho-(1-rac-glycerol). These systems were examined via surface pressure and surface electrical potential measurements, polarization modulation infrared reflection absorption spectroscopy, and Brewster angle microscopy. Moreover, phospholipase A2 activity was used to differentiate between the three drugs. Our results reveal that the oxicams studied modify membrane properties, namely hydration of the lipid polar heads, orientation of the molecules, and morphology of the domains. Phospholipase A2 was shown to be sensitive to the presence of the drugs in the systems studied; the activity of the enzyme correlates with the effect of meloxicam, piroxicam, and tenoxicam on the monolayer properties. The latter indicates that the anti-inflammatory action of oxicams may be related to interference with phospholipase activity in addition to cyclooxygenase inhibition.

Impact of two different saponins on the organization of model lipid membranes

Saponins, naturally occurring plant compounds are known for their biological and pharmacological activity. This activity is strongly related to the amphiphilic character of saponins that allows them to aggregate in aqueous solution and interact with membrane components. In this work, Langmuir monolayer techniques combined with polarization modulation infrared reflection-absorption spectroscopy (PM-IRRAS) and Brewster angle microscopy were used to study the interaction of selected saponins with lipid model membranes. Two structurally different saponins were used: digitonin and a commercial Merck Saponin. Membranes of different composition, namely, cholesterol, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine or 1,2-dipalmitoyl-sn-glycero-3-phospho-rac-(1-glycerol) were formed at the air/water and air/saponin solution interfaces. The saponin-lipid interaction was characterized by changes in surface pressure, surface potential, surface morphology and PM-IRRAS signal. Both saponins interact with model membranes and change the physical state of membranes by perturbing the lipid acyl chain orientation. The changes in membrane fluidity were more significant upon the interaction with Merck Saponin. A higher affinity of saponins for cholesterol than phosphatidylglycerols was observed. Moreover, our results indicate that digitonin interacts strongly with cholesterol and solubilize the cholesterol monolayer at higher surface pressures. It was shown, that digitonin easily penetrate to the cholesterol monolayer and forms a hydrogen bond with the hydroxyl groups. These findings might be useful in further understanding of the saponin action at the membrane interface and of the mechanism of membrane lysis.