EUDRAGIT E100 EXHIBITS AN OUTER MEMBRANE-DISORGANIZING EFFECT ON Pseudomonas aeruginosa

ROMERO, VERÓNICA L.; MANZO RUBEN H.; ALOVERO, FABIANA L.

Rosario

Congreso; 2° REUNION INTERNACIONAL DE CIENCIAS FARMACEUTICAS- RICIFA 2012; 2012

UNIVERSIDAD NACIONAL DE ROSARIO

Introduction. It is known that certain polycationic substances such as polymyxin and their derivatives, polylysines and protamine, among others, permeabilize the Gram-negative bacteria outer membrane (OM) making it permeable to solutes that normally are unable to penetrate it. OM is the component of the bacterial envelope responsible for the increased resistance to detergents and hydrophobic antibiotics exhibited by Gram-negative bacteria (1). Substances lacking inherent toxicity yet causing sensitization of bacteria to the action of other potentially inhibitory substances are termed permeabilizers (2). Eudragit E100 is a cationic polymer based on dimethylaminoethyl methacrylate and other neutral methacrylic acid esters. In a previous study, an ofloxacin-containing Eudragit E100 aqueous dispersion (Eu-OFX) showed notorious enhancement in the bactericidal activity of ofloxacin against P. aeruginosa (3). Therefore, we assessed whether the observed effect is attributable to a permeabilizing capacity exhibited by Eudragit E100, since this aspect has not been reported for this polymer, by testing susceptibility to the action of lytic agents and detergents. Materials and methods. An overnight culture of Pseudomonas aeruginosa was suspended in saline and adjusted to an absorbance of 0.3. Cells were incubated for 10 min at 37 °C in the presence of sub-MIC concentration of ofloxacin (32 µg/mL) in Eu-OFX or free solution. Bacterial suspensions treated with drug-free polymer (Eu) were also evaluated. Polymyxin B (100 µg/mL) was used as positive control. Following centrifugation, bacterial cells were re-suspended in saline containing 10 µg/mL lysozyme, 1% Triton X-100 or 0.1 and 1% SDS. After 4 additional minutes of incubation at 37 °C, the optical density at 405 nm was measured. Results were expressed as percentage of the control culture OD405. Low percentages are indicative of strong cell lysis by lytic agents. Results. Exposure of P. aeruginosa to Eu-OFX and free-drug polymer before adding detergent or lysozyme did not produce significant lysis (Table 1). Bacterial cultures were weakly sensitized by Eu-OFX and free-drug polymer to Triton X-100 and lysozyme, but strongly sensitized to SDS. This behavior was similar to that exhibited by polymyxin. In contrast, cultures treated with ofloxacin did not differ from control (Table 1). Conclusions. Fluoroquinolones must enter the cell to become effective. Electrostatic interactions between bacterial surface and Eu-OFX put them in touch quickly. Eu-OFX has a strong OM-permeabilizing activity after brief exposure, causing sensitization to anionic detergents. To our knowledge, this is the first study on the interaction of Eudragit E100® with bacterial cells. Eu-OFX and free-drug polymer behavior are consistent with the action of polymyxin B, known OM-disorganizing polycationic agent. However, our previous studies indicate that free-drug polymer does not kill P. aeruginosa (3). Therefore, the improved bactericidal action exhibited by Eu-OFX could be attributed to the effects on membranes combined with the mechanism of action of the fluoroquinolone. The ability to alter the OM of P. aeruginosa extends the applicability of Eudragit E100 as a vehicle or as an adjuvant or potentiator for fluoroquinolones in topical pharmaceutical preparations. In addition, may be a useful tool to broaden the spectrum of other antibiotics whose clinical use is limited by the impermeability of the bacterial OM. References: 1.Helander IM, Alakomi HL, Latva-Kala K, Koski P. Polyethylenimine is an effective permeabilizer of Gram-negative bacteria. Microbiol 1997; 143:3193-3199. 2.Helander IM, Nurmiaho-Lassila EL, Ahvenainen R, Rhoades J, Roller S. Chitosan disrupts the barrier properties of the outer membrane of Gram-negative bacteria. Int J Food Microbiol 2001; 71:235?244. 3.Romero V, Manzo R, Alovero F. Eudragit E100 potentiates activity of Ofloxacin against Fluoroquinolone-resistant Pseudomonas aeruginosa. Biocell 2009; 33(1):35-36. Table 1. Sensitization of Pseudomonas aeruginosa FQ-R1 to lytic action of lysozyme and detergents Lytic agent Relative turbidity (%) control Eu-OFX Eu(free-drug polymer) Oflo Polymixyn ----- 100±2 94.4±6.8 98±2 100±3 91.5±4.6 Lysozyme 92±3 83±11 82±15 93±5 73±10 SDS 1% 52±4 9±1 17±8 53±5 15±14 SDS 0,1% 100±6 8.6±6.0 16±3 95.5±9.2 14.6±4.7 Triton X-100 88±6 72±1 77±2 92±5 88±6