Partitioning of flunitrazepam into model membranes studied by temperature controlled gel filtration chromatography

Abstract

The use of gel filtration chromatography with Sephadex as a separation medium was used in order to study flunitrazepam (FNTZ) partitioning into artificial model membranes consisting of dipalmitoyl‐phosphatidylcholine (dpPC) vesicles, under controlled temperature conditions. In this system two phenomena are taking place simultaneously: the ligand–liposome interaction and the lipid self‐aggregation to form the liposome. The liposome–FNTZ interaction was evidenced by the non‐enantiography of the first derivative of FNTZ elution peak in frontal chromatography through Sephadex G‐75. On the other hand, the presence of FNTZ reduced liposomes mean size and increased their size dispersion as evidenced by molecular filtration through Sephadex G‐200. The dpPC–buffer FNTZ partition coeficient determined in zonal chromatography through Sephadex G‐10 increased about 33% when the temperature rose above the temperature of dpPC transition from the liquid crystalline to the fluid phase. Gel filtration chromatography seems a suitable technique to study lipid liposome–FNTZ interactions at a qualitative level. In addition, this technique has the advantage over other methods of giving the possibility of observing the mutual effects exerted between the drug and the self‐aggregating structure. © 1997 John Wiley & Sons, Ltd.

Flunitrazepam partitioning into natural membranes increases surface curvature and alters cellular morphology

“…natural membranes). Even though, our results suggest that not only in model membranes [9,10] but also in natural membranes (present paper) the non-receptor mediated interaction with FNTZ may induce an increase in the outer monolayer curvature and evaginations. Such curvature changes can be interpreted as a mechanism suitable to relieve the tension generated by drug incorporation into the bilayer.…”

“…Many works have demonstrated that negatively-charged or neutral amphipaths induced echinocytosis by their intercalation into the outer monolayer of the erythrocyte membrane, resulting in net outer monolayer expansion and crenation [16,17] according to the bilayer-couple hypoth-esis of Sheetz and Singer [15]. The ability of benzodiazepines like FNTZ to partition from an aqueous media towards a membrane either artificial [7][8][9][10]25] or natural [5] including erythrocytes [26], was stated using several methodologies like monomolecular layers at the air -water interface, radioligand binding, spectroscopic fluorescence and electron spin resonance. The membrane/buffer partition coefficient for FNTZ had a value around 20 in both types of membranes and although affected by pH, temperature, saline concentration and cholesterol content, it remained within the same order of magnitude [5,25].…”

“…This interaction led to an increase of the relative volume of the polar head groups and to a decrease in the thermodynamic stability of the self-aggregating structure, which was forced towards an increase of its surface curvature. Molecular filtration [10] and electron microscopic [9] studies let us demonstrate that FNTZ is able to diminish the diameter of dipalmitoyl phosphatidylcholine (dpPC) vesicles. It was expected that membranes of more complex composition could show more responsive mechanisms.…”

Flunitrazepam induces geometrical changes at the lipid–water interface

“…The P value for FNTZ is strongly influenced by the physical state of the bilayer membrane, whose changes may be triggered by external parameters like temperature and composition as well as by the characteristics of the surrounding media like pH and ionic strength. Particular interactions may lead to strong local accumulation of FNTZ within the membrane due to a coupling to lateral density fluctuations [17,18].…”

Surface active benzodiazepine-bromo-alkyl conjugate for potential GABAA-receptor purification

“…Due to their lipophilic characteristics, BZDs interact not only with the receptor (specific interaction) but also, through hydrophobic interactions, with the membrane as a whole by a partitioning process (nonspecific interactions, NS) locating mainly at the polar head group region of the biomembranes. [20][21][22][23] Total binding (TB) and nonspecific interactions (NB) are experimentally accessible but specific binding to the receptor (B) has to be calculated as a difference between TB and NB. The latter are irreversible.…”