Modified host defence peptides as beta-lactamase-dependent antibiotic prodrug candidates.

Infections caused by antibiotic resistant bacteria have been listed as top priority diseases by the World Health Organisation (WHO) in terms of their threat to global public health if the need for novel medicines is not met. Host defence peptides (HDPs) are multifunctional molecular effectors of innate immunity, the first line of defence against infection in multicellular organisms. They are characterised by an antimicrobial activity essentially directed at a universal non-protein target (bacterial membrane(s)), complemented by multiple immunomodulatory and anti-inflammatory activities. They are recognised as promising candidates for the development of novel antimicrobial and anti-inflammatory agents, owing to their original and selective mode of action which may circumvent the limitations of classical antibiotics in terms of drug resistance. Despite their potential in medicine, these \u27Nature\u27s antibiotics\u27 have not yet been approved for use as drugs. Their main limitations are a risk of unpredictable side-effects when administered systemically and a lack of stability limiting their efficacy. Several approaches have been proposed to achieve clinical success with host defence peptides, such as conversion into peptidomimetics, liposomal delivery and a prodrug strategy. Prodrugs are pharmacologically inactive molecules metabolised to an active agent. Ideally this metabolic step is catalysed by an enzyme confined to a targeted body site andlor associated with a pathological process. Despite the potential of this approach, no inactive precursors of host defence peptides eligible for disease site specific activation have been reported to date. The present project aimed at developing prodrugs of host defence peptides by conjugating selected candidates to a p-lactam molecule (cephalosporin) as a promoiety. These components are joined by a linker which can release the peptide upon degradation of the cephalosporin by a p-lactamase (elimination promoted-immolative carbamate linker). The technology required to unequivocally and efficiently conjugate a peptide to a cephalosporin has been developed and applied to the successful preparation of P-lactamase-dependent prodrug candidates of host defence peptides, the fmt to be reported for these mediators of innate immunity

Anisamide-targeted cyclodextrin nanoparticles for siRNA delivery to prostate tumours in mice

A hepta-guanidino-β-cyclodextrin (G-CD), its hepta-PEG conjugate (G-CD-PEG), and the corresponding anisamide-terminated PEG conjugate (G-CD-PEG-AA) have been synthesised and compared as delivery vectors for siRNA to prostate cancer cells and tumours in vivo. The G-CD-PEG-AA.siRNA formulations (in which anisamide targets the sigma receptor), but not the non-targeted formulations, induced prostate cell-specific internalisation of siRNA resulting in approximately 80% knockdown in vitro of the reporter gene, luciferase. Following intravenous administration of the anisamide-targeted formulation in a mouse prostate tumour model significant tumour inactivation with corresponding reductions in the level of vascular endothelial growth factor (VEGF) mRNA were achieved, without demonstrating enhanced toxicity. This data imply significant potential for anisamide-conjugated cyclodextrin vectors for targeted delivery of therapeutic siRNAs in the treatment of prostate cancer

A click chemistry route to 2-functionalised PEGylated and cationic beta-cyclodextrins: co-formulation opportunities for siRNA delivery

A new approach to the synthesis of amphiphilic beta-cyclodextrins has used 'click' chemistry to selectively modify the secondary 2-hydroxyl group. The resulting extended polar groups can be either polycationic or neutral PEGylated groups and these two amphiphile classes are compatible in dual cyclodextrin formulations for delivery of siRNA. When used alone with an siRNA, a cationic cyclodextrin was shown to have good transfection properties in cell culture. Co-formulation with a PEGylated cyclodextrin altered the physicochemical properties of nanoparticles formed with siRNA. Improved particle properties included lower surface charges and reduced tendency to aggregate. However, as expected, the transfection efficiency of the cationic vector was lowered by co-formulation with the PEGylated cyclodextrin, requiring future surface modification of particles with targeting ligands for effective siRNA delivery

D6.4 - 3MWth CLC demonstration unit testing

publishedVersio

In vitro investigations of the efficacy of cyclodextrin-siRNA complexes modified with lipid-PEG-octaarginine: towards a formulation strategy for effective neuronal siRNA delivery

Purpose: Development of RNA interference based therapeutics for neurological and neurodegenerative diseases is hindered by a lack of non-viral vectors with suitable properties for systemic administration. Amphiphilic and cationic cyclodextrins (CD) offer potential for neuronal siRNA delivery. Here, we aimed to improve our CD-based siRNA formulation through incorporation of a polyethyleneglycol (PEG) shielding layer and a cell penetrating peptide, octaarginine (R8). Methods: CD.siRNA complexes were modified by addition of an R8-PEG-lipid conjugate. Physical properties including size, charge and stability were assessed. Flow cytometry was used to determine uptake levels in a neuronal cell model. Knockdown of an exogenous gene and an endogenous housekeeping gene were used to assess gene silencing abilities. Results: CD.siRNA complexes modified with R8-PEG-lipid exhibited a lower surface charge and greater stability to a salt-containing environment. Neuronal uptake was increased and significant reductions in the levels of two target genes were achieved with the new formulation. However, the PEG layer was not sufficient to protect against serum-induced aggregation. Conclusions: The R8-PEG-lipid-CD.siRNA formulation displayed enhanced salt-stability due to the PEG component, while the R8 component facilitated transfection of neuronal cells and efficient gene silencing. Further improvements will be investigated in the future in order to optimise stability in serum and enhance neuronal specificity

In Vitro Activities of Synthetic Host Defense Propeptides Processed by Neutrophil Elastase against Cystic Fibrosis Pathogens▿

The antimicrobial and hemolytic activities of a host defense peptide can be controlled by its modification as a propeptide of reduced net charge, which can then be processed by neutrophil elastase, a serine protease involved in chronic airway inflammation and infections associated with cystic fibrosis