Our previous studies showed differences in accumulation and
intracellular activity between ciprofloxacin and moxifloxacin
that may actually result from their differential susceptibility
to efflux by the ciprofloxacin transporter (1
differs from ciprofloxacin by the presence of a C-8 methoxy
group and a bulkier C-7 (octahydropyrrolo (3.4)-pyridinyl versus
piperazinyl for ciprofloxacin) substituent (31
). As previously
), these changes resulted in an increase in hydrophobicity
) for moxifloxacin and –0.79(34
for ciprofloxacin). However, these structural modifications
between moxifloxacin and ciprofloxacin may not account for the
differences in the pK values (from 6.25 to 6.09 (33
these differences are below the experimental errors. Interestingly,
these compounds were much weaker acids than aromatic carboxylic
acids. The reduced acidity may be ascribed to the formation
of an intramolecular hydrogen bond between the carboxyl and
neighboring keto groups in the quinoline ring, resulting in
stabilization of the protonated form of the carboxyl group.
The activity of efflux pumps in general, and those involvedfor ciprofloxacin or moxifloxacin cellular accumulation, inparticular, is closely related to the interaction of the drugwith lipids, regardless of the model proposed for their activity(i.e., flippase or hydrophobic vacuum cleaner). We thereforelooked at the influence of the small changes in chemical structurebetween moxifloxacin and ciprofloxacin on their interactionwith lipids. To address this crucial question, the interactionsbetween model lipid membranes and the two fluoroquinolones wereprobed using a variety of techniques.
Nanoscale investigations by AFM revealed different behaviorsfor ciprofloxacin and moxifloxacin. AFM imaging showed a reductionof the size of the DPPC gel phase domains in presence of fluoroquinolones.The erosion process is greater for moxifloxacin compared tociprofloxacin, and follows an exponential-like function-decreasefor moxifloxacin and a linear-decrease for ciprofloxacin. Consideringboth molecules, the difference in their ability to erode theDPPC gel phase domains might be due to a better insertion ofthe moxifloxacin than the ciprofloxacin into the fluid matrix,and a more marked decrease of the line tension at the boundaryof the DOPC/DPPC phases resulting from a fluidification of theDPPC.
This marked effect of moxifloxacin on lipids as compared tociprofloxacin was confirmed by conformational analysis. In accordancewith experimental data obtained for ciprofloxacin (37) or grepafloxacin(38), the fluoroquinolones are located at the lipid-water interface,near the first carbons of the acyl chains. Both molecules showeda minimum of energy when they are at the phospholipids headgroup/acyl-chainsinterface, and the interaction energy rose markedly when themolecule was forced into the hydrophobic domain. This energyincrease was less marked for moxifloxacin as compared to ciprofloxacin,suggesting a higher affinity of moxifloxacin for lipid phase.
Taken together, our data suggest that ciprofloxacin and moxifloxacininteract in a very different way with lipids. The major challenge,however, is to understand the mechanism, at a molecular level,unraveling the interaction between lipids and fluoroquinolonesand the path of these antibiotic molecules through lipid layers.Previous data reported by Montero et al. (28) showed a shiftof the surface pressure-area isotherms of monolayer toward alower area per molecule in the presence of ciprofloxacin. Weextended these data to one major fluoroquinolone used in clinics,moxifloxacin. To go further in the mechanism involved, we monitoredthe amount of fluoroquinolones in the subphase after the monolayercompression. In agreement with the hypothesis based on a dissolvingeffect in the subphase (39), we did find fluoroquinolones thereinwith a higher proportion of ciprofloxacin as compared to moxifloxacin.Taking into account the amount of fluoroquinolones present insidethe monolayer, we corrected the surface-pressure-area isothermsof the monolayer and again observed a shift toward a lower areaper molecule in the presence of fluoroquinolones. This effectwas more marked with moxifloxacin as compared to ciprofloxacin.The dissolving effect in the aqueous phase is therefore probablyessential, although not fully sufficient, to explain the condensingeffect of fluoroquinolones.
Thus, we investigated a change in the lipid chain conformationand orientation using ATR-FTIR technique (21,40). Indeed, thedrug-induced area condensation of lipids can derive from theacyl ordering attained when trans-gauche isomerization aboutthe carbon-carbon bonds is reduced. The trans conformation isthe most stable and has an estimated energy barrier of 3.5 kcal/molto rotate past the eclipsed configuration to the gauche form.The all-trans conformation allows the chain to be maximallyextended, whereas a gauche bond alters the direction of thechain inducing a kink in the chain. Our results clearly indicatedthat moxifloxacin has a higher ability than ciprofloxacin tomarkedly decrease the number of all-trans conformation. Therelated change in the packing of the acyl chains might allowmoxifloxacin to be located in the pocket created by the presenceof a kink in the acyl chain. In contrast, with ciprofloxacin,the appearance of a kink from the all-trans chain conformationwould be less marked, suggesting a less important change inlipid packing. Interestingly, both condensing effects (lowerarea of mixed monolayer lipids/fluoroquinolones) have also beendescribed when cholesterol was added to fluid-phase phosphatidylcholine(41–43).
The disorder in the lipid chains revealed by the decrease ofall-trans conformations has also been analyzed in terms of orientationand tilt between the molecular axis (the membrane normal) andthe transition dipole moments. In this analysis, the ciprofloxacinshowed an additional cause of disorder, because it modifiesthe orientation of the acyl chain in relation to its higherability to be released in an aqueous phase after monolayer compression.
Differences in the charge distribution of the molecule at thephysiological pH could also explain changes for drug membranelocation and bound hydration shell surrounding the headgroupof membrane lipids, which, in turn, could partly explain themore condensing effect of moxifloxacin as compared to ciprofloxacin.Moreover, the determinations were made in aqueous environment,whereas the condensing effect of moxifloxacin involved the presenceof the drug with a lipidic phase.
All together, we showed that the condensing effect of fluoroquinoloneson lipid layer resulted not only from a dissolving mechanismbut also from an alteration of the intramolecular acyl-chainorder in relation to a reduction in trans-gauche isomerizationabout the carbon-carbon bonds, and change in the average moleculartilt of lipid acyl chain of DPPC. The two fluoroquinolones investigatedshowed difference in their effects. Ciprofloxacin had a lowerability to decrease the all-trans conformation of lipid chainsthan moxifloxacin but showed a higher capacity to affect theorientation of lipid chains and to disorder the membrane. Theseeffects might explain its higher ability to be released fromthe lipid monolayer to aqueous phase and its lower effect onsurface pressure-area isotherms of monolayers. In contrast,moxifloxacin has a lower capacity to induce membrane disorderand does not change the tilt between the molecular axis andthe transition dipole moment. Moxifloxacin has also a highertendency to decrease the number of all-trans conformations withincrease of kink, creating a pocket in which moxifloxacin canbe located. This can explain why the amount of moxifloxacinin the aqueous phase was lower than that found for ciprofloxacinand why the mean molecular area of lipids/fluoroquinolones monolayersafter compression is significantly lower in the presence ofmoxifloxacin as compared to ciprofloxacin.
This model is entirely compatible with the physico-chemicalcharacteristics of the two fluoroquinolones. It suggested thatsmall structural differences among fluoroquinolones (notablyoverall molecular hydrophobicity (Papp = 0.089 vs. 0.031 forciprofloxacin and moxifloxacin, respectively (45)), bulkiness,and/or the internal dynamics of the C-7 substituent, could beimportant for drug lipid interactions and lipid packing. Thediazabicyclonyl-ring at position 7 of moxifloxacin, by aligningthe sn-2 chain, probably contributes to the higher tendencyof this antibiotic to induce a decrease of all-trans configurationas compared to ciprofloxacin. This is in line with data reportedwith n-alkyl-piperazinyl-ciprofloxacin (39).
In conclusion, we provided a comprehensive picture of the interactionof the two major fluoroquinolones ciprofloxacine and moxifloxacinewith lipids, and elucidate fundamental issues such as the relationshipbetween lipid chain conformation and orientation with changesin membrane properties as determined by Langmuir studies andthe ability of drugs to diffuse through membranes. All theseparameters might be related to the activity of membranous proteins.Our work notably showed that an increase in drug lipophilicityand addition of a bulky moiety (moxifloxacin versus ciprofloxacin)produced marked changes in the packing of lipids. This was concomitantwith a lower release of the more lipophilic drug from lipidmonolayer and with a potential inefficient activity of effluxproteins which could be involved in a kind of futile cycle resultingin an increase in cellular accumulation (1). So far, progressin understanding the structure-function relationships of membranesand understanding of the lipid-drug interaction appears to beof crucial importance in understanding the mechanisms involvedin cellular drug accumulation.