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Work Package 2

"Improving pharmacological properties and delivery of available antibiotics or circumvention of resistance mechanisms"

 

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A. Incorporating antimicrobial compounds in nanoparticles. 

Nanomedicines with improved tissue accumulation due to targeting achieved by surface-grafting the nanoparticles might have great potential for treating serious infections. Liposomes (lipid film method), polymeric nanoparticles (e.g. water-in-oil-in-water emulsification), and nanoplexes (ionic gelation) will be characterized by size and surface charge, amount of drug encapsulated, physical and chemical stability at storage conditions as well as upon administration will also be investigated in vitro using tissue homogenates and cell culture models. The transepithelial delivery will be assessed using cell culture models with different phenotypic properties, resembling e.g. the oral epithelium. The cytotoxicity of the drug delivery systems will be assessed and the efficacy of the system tested in well-established experimental animal models.
Contributors:  Hanne Mørck Nielsen, Niels Frimodt-MøllerLotte Jakobsen 

B. Improvement of drug delivery at the target site. 

Complexation or encapsulation may improve the efficacy of antibiotics with limited access to the target site. A novel possibility is to graft the nanoplex or nanoparticle exterior with molecules that aid delivery, membrane-interaction enhancers like specific cell penetrating peptides (CPP) and/or active targeting ligands. Optimally, this would enable the encapsulated or complexed antibiotic to reach it's site of action with fewer disturbances of the normal bacterial flora - perhaps thereby increasing the selective delivery of the antibiotic. A major part of these studies will be aimed at generating a thorough understanding of the mechanisms of interaction with and transport through eukaryotic as well as prokaryotic cell membranes. This will be done by use of model systems consisting of vesicle lipid bilayers, supported bilayers, and cell culture models applying methodologies based on calorimetry, fluorescence, and microscopy. NMR-spectroscopy will be used to elucidate these interactions. The rankings obtained will be correlated to studies of cellular membrane damage by cytotoxicity assay and intracellular trafficking (by confocal laser scanning microscopy). The efficacy of the improved antibiotics will evaluated in well-established experimental animal models.
Contributors: Hanne Mørck Nielsen, Jesper Søborg Bahnsen, Lars Erik Uggerhøj, Reinhard Wimmer, Niels Frimodt-Møller, Lotte Jakobsen

C. Reduction of antibiotic binding to serum proteins. 

The bioavailability of certain antibiotics is severely limited by binding to serum proteins. A novel antimicrobial peptide with high activity against gram-negative bacteria is under evaluation by Novozymes, but the activity is impeded by binding to albumin. Saturation Transfer Difference NMR2 allows an analysis of which parts or functional groups on ligands interact with protein molecules. This technique can be employed on the peptide receptor and a row of serum proteins, in modified form even on natural blood samples and living cells2.These methods will help identify the binding epitope of the ligand (i.e. drug) to its target and to serum proteins. This will allow the design and synthesis of drug variants with reduced binding to serum proteins while maintaining activity.
Contributors: Henrik FranzykPaul Robert HansenLars Erik Uggerhøj, Reinhard WimmerHans-Henrik Kristensen 

D. Inhibition of transport mechanisms. 

One important mechanism of resistance is a change in bacterial membrane transport mechanisms and efflux mechanisms pumping otherwise absorbed antibiotics out of the cell again. We will screen for enhancement of antibiotic activity by simultaneous inhibition of transport and efflux mechanisms with available drugs. We will also study the fate of antibiotics in wounds and the fate of metabolites by NMR based metabonomics with high time-resolution.
Contributors: Lotte Jakobsen, Karen A. Krogfelt, Mette E. Skindersø, Reinhard Wimmer, Lars Erik Uggerhøj, Niels Frimodt-MøllerKlaus Skovbo Jensen 

E. Improvement of peptide penetration over biological membranes. 

Conjugation of antibiotics with cell-penetrating peptides may be used to increase drug concentration in the bacterial cell. We will form hybrid peptides between active antimicrobial species and peptides signalling transport into the bacterial cell.
Contributors: Anders Løbner-OlesenSusanne Kjelstrup 

F.   Designing more effective treatment regimens by human/animal and mathematical modelling of PK/PD. 

Modelling drug activity in vitro and in vivo in individual patients/animals is a crucial component of testing drug activity. Data from in vitro models, validated in in vivo experiments, can be employed to evaluate the use of antibiotics in humans by Monte Carlo simulation and to extrapolate optimum dosages. Furthermore, mathematical modelling is a useful tool to relieve the use of experimental animals and to reduce the time needed for human trials1. Some models for this purpose have already been developed (www.ssi.dk/pkpdsim).
Contributors: Lotte Jakobsen, Niels Frimodt-MøllerKlaus Skovbo Jensen
 

Reference List
 

  1. Frimodt-Moller, N. 2002. How predictive is PK/PD for antibacterial agents? Int.J.Antimicrob.Agents 19:333-339.
  2. Mayer, M. and B. Meyer. 2001. Group epitope mapping by saturation transfer difference NMR to identify segments of a ligand in direct contact with a protein receptor. J.Am.Chem.Soc. 123:6108-6117.

 

Last revised 16 July 2014