Antimicrobial lipid mesophases

Until recently, infectious diseases were thought to be readily controlled. This optimism has led to a fatal complacency in the use of antibiotics, which has resulted in a world-wide increase in pathogenic bacteria that exhibit multi-resistance to conventional antibiotics costing thousands of lives. Therefore, WHO, EU and national health organizations warned that this situation can cause a global health problem, which is further complicated by the fact that a large fraction of hospital-acquired infections are due to antibiotic resistant bacteria. A great concern is also raised by the fact that many of these bacteria, e.g. Staphylococcus aureus, the most common cause of nosocomial wound infections, are becoming resistant to vancomycin, a drug of last resort. This does not only affect people quality of life but also adds health and medical costs (4-5 billion$/year in the US, likely to be in the same order of magnitude in Europe). Accordingly, there is urgent need to develop new drugs to combat antibiotic- resistant bacterial strains. Our research is aimed at developing a new class of antibiotics based on host defense peptides, which are anticipated to be less prone to antibiotic resistance. The main advantage of this class of substances, when considering bacterial resistance, is that they rapidly, within minutes, destroy bacteria. Furthermore, due to the nature of their target, i.e. cell membrane rather than a specific receptor, bacterial resistance is unlikely to occur since substantial modification of the lipid com-position would affect bacterial cell viability. The focus of this project is devoted to the medical application of antimicrobial peptides, because so far their use is limited to topical (transdermal) applications. This is due to the susceptibility of these peptides to rapid enzymatic degradation in the systemic circulation and tissues. One strategy to prevent degradation of peptides, when parenterally administered, will be to encapsulate anti-microbial peptides in taylored liposomes or lipidic mesophases, thereby also lowering toxicity and increasing bio-availability. This approach has not been pursued by other research groups, because of the intrinsic property of antimicrobial peptides to damage the lipid bilayer structure, that does not only form the matrix of membranes but also of liposomes and lipidic mesophases. However, our investigations clearly demonstrated that these peptides discriminate between different lipid subspecies. Based on this knowledge and our longstanding expertise on lipid polymorphism and characterization of supramolecular lipid complexes, stable delivery systems for antimicrobial peptides for medical application should be developped.

Demographic changes such as an ageing population in the Western hemisphere increase the need of antibiotics. This, however, will further enhance the problem of antibacterial drug resistance, which has been ranked as a priority disease (WHO, Nov. 2005). Therefore, it is necessary to develop antimicrobial agents with new mechanisms of action. Antimicrobial peptides are considered as such novel antibiotic agents, because they are less vulnerable to antibiotic resistance mechanisms. The focus of this project was devoted to the medical application of antimicrobial peptides, because so far their use is limited to topical applications owing to the susceptibility of these peptides to rapid enzymatic degradation in blood and tissue. One strategy to prevent degradation of these peptides will be to encapsulate antimicrobial peptides in delivery vehicles (liposomes or other lipidic mesophases), thereby also increasing bio-availability. An additional reason to use liposomes for their delivery is the increased vascular permeability upon bacterial infection leading to the accumulation of liposomes and consequently of the antibiotic agent in the sites of inflammations thereby further improving the treatment of bacterial infections. The goal of this project was the design of lipid based delivery vehicles for antimicrobial peptides that can be used for parenteral application. The concept was to test lipid compositions that yield spontaneously stable unilamellar liposomes in the presence of antimicrobial peptides that enables rapid peptide release. The lipid matrix was formed by lecithin, cholesterol and a negatively charged lipid that is characterized by a cone shaped molecular geometry. The charge was used to control the number of bilayers in a liposome, while the molecular shape controlled the size of the liposome. Indeed, a composition range was found, where these ternary lipid mixtures formed spontaneously unilamellar liposomes of defined size and homogeneity. These liposomes were stable in the presence of peptides over months. Moreover, this procedure has a big advantage as compared to conventional liposomal preparations as no additional technical procedures are necessary for transferring multilamellar structures into unilamellar vesicles. The latter causes low reproducibility in respect of size and encapsulation of drug. Therefore this new method will result in well-defined and reproducible liposomal delivery systems, which in turn will substantially improve therapeutic treatments being of benefit for patients suffering from severe bacterial infection.