A novel approach for developing new antibiotics effective against multidrug resistant bacteria

Development of antibiotic-resistant bacteria is a major and global problem. The lack of new antibiotics limits the treatment and survival of patients suffering from infections caused by multidrug-resistant (MDR) bacteria. The incidence of the so called pandrug resistant bacteria (PDR), and the extensive drug resistant (XDR) Gram-negative bacteria is growing. Currently, only few antibiotic subclasses are available to treat such infections, notably polymyxins and tigecycline and the recent approved Beta-lactam/Beta-lactamase inhibitor combinations, Ceftolozane/ tazobactam and Ceftazidine/avibactam. In November 2015, the emergence of plasmid-mediated polymyxin resistance was for the first time demonstrated. This increases the risk significantly for horizontal gene transfer and rapid dissemination of polymyxin resistance worldwide, which underscores even more the need for new antibiotics effective against Gram-negative organisms. Xellia is the world's leading producer of several generic antibiotics including polymyxin B and colistin (polymyxin E), and have basic knowledge and experience necessary to develop new antibiotics. The overall aim of the project has been to develop at least one new antibiotic to treat infections caused by multidrug-resistant Gram-negative bacteria, included polymyxin resistant pathogens, either a new type of antibacterial molecule or a new polymyxin based compound with increased bioavailability and reduced nephrotoxicity. The project has been a collaboration with Xellia Pharmaceuticals Oslo, SINTEF Materials and Chemistry, Trondheim and Statens Serum Institute in Copenhagen. The Paenibacillus polymyxa bacterium producing polymyxins, and several less described Paenibacillus species, have been selected as sources of potentially valuable new molecules formed by fermentation and that later may be modified by semi-synthetic derivatisation. The genetic potential for antibiotic formation in 5 different Paenibacillus polymyxa strains has been assessed by genome sequencing and annotation. Various secondary metabolites have been expressed by changing the fermentation conditions. New compounds have been isolated from the fermentation broth by fractionation and LC-MS analysis. A significant number of new semi-synthetic derivatives of polymyxins have been prepared. All compounds have been tested for in vitro bioactivity (MIC), and somewhat fewer for in vitro nephrotoxicity. Some selected compounds have been tested for in vivo bioefficacy and nephrotoxicity in mice. There have been several challenges in the project, such as identifying new low abundant bioactive molecules in fermentate with a high degree of complexity seen by thousands of different molecules and with the known polymyxins having high potency/bioactivity often masking activity from the new antibiotics. Furthermore, attempts to reduce the toxicity without losing bioactivity by chemical modification of the polymyxins, or to make new derivatives with higher bioactivity, have also appeared challenging. However, some very promising results have been obtained by effective screening of fermentate and by subsequent chemical modification. A new family of antibiotics was identified in fermentate with activity against MDR Gram-negatives in vitro. In vivo bioefficacy studies are in progress and will continue to some extent until more funding is in place. In summary, an efficient new drug discovery platform has been developed and implemented that include major steps from discovery phase to initial pre-clinical test models. The new platform has proved to identify drug candidates from screening of fermentate resulting in novel lead candidates and several synthetic derivatives with the potential to be further developed to even more efficient antibiotics.

The four year project represents a collaboration between Xellia Pharmaceuticals, SINTEF Materials and Chemistry and Oslo University Hospital or a alternative partner with comparable competence in work with MDR-strains, clinical isolates and evaluation of bioactivity. Its primary objective is to develop at least one new antibiotic effective against multi-drug resistant (MDR) pathogens with an acceptable level of toxicity. The approach taken to reach this goal is to exploit the potential of existing antib iotic producing bacteria to broaden their repertoire of antibiotic substances that are hitherto unknown or not selected for. A novel strategy of combining genome mining, innovative cultivation methods for activation of cryptic genes and rapid, high sensit ive analysis will be employed to induce appearance of these potentially valuable new entities. A broad range of advanced methods will be employed to select, produce, characterize and develop promising lead candidates, from gene sequencing of producer stra ins, targeted genetic modification, classical mutagenesis, miniaturized cultivation with robotic material handling, LCMS metabolic fingerprinting, robotic bioassays using clinical isolates, toxigenic screening with mammalian cell lines, isotope labelling for structure elucidation using NMR and MS, and chemical modification based on structure/activity/toxicology-relationships. Process development for selected candidates and IPR protection will also be part of the project. The socio-economic benefits of a successful project are substantial, and calculations show that introduction of an efficient new antibiotic capable of combating MDR-infections may imply savings in EU alone at an EUR 100 Bn level over a 10 year period post launch (reduced hospital costs a nd productivity gain). Xellia and partners are taking a substantial financial risk by engaging in this project, committing almost NOK 40 mill. over a four year period. Xellia and partners propose a risk sharing with NFR in this major endeavour, and applie s for a financial support of NOK 16 mill. for the same period.