Periodic Report Summary 2 - FORMAMP (Innovative Nanoformulation of Antimicrobial Peptides to Treat Bacterial Infectious Diseases)
Project Context and Objectives:
The long-term objective of the FORMAMP project is to significantly change the treatment strategies for infectious diseases and to reduce the evolution of multi-resistant bacteria. The project aims to develop new and innovative formulation strategies, based on the combination of nanotechnology-based delivery systems and antimicrobial peptides (AMPs), for the treatment of infectious diseases caused by bacteria such as P. aeruginosa (Pseudomonas aeruginosa), MRSA (Methicillin-resistant Staphylococcus aureus), and MTB (Mycobacterium tuberculosis). Clinical indications that are addressed include skin and soft tissue infections, infections in burn wounds, and infections in the lung such as cystic fibrosis and tuberculosis. A number of these indications are associated with the formation of multispecies biofilms, drastically impairing the function of available treatments. An important objective of the FORMAMP project is therefore to develop treatment strategies that also takes into account the problems associated with biofilms for clinical indications were this is of importance.
The objectives of the project are divided into two sections. The first part covers screening and evaluation of a number of different nanostructured systems, i.e. liquid crystalline nanoparticles, lipidic nanocapsules, microgels, dendrimers, and mesoporous silica nanoparticles for encapsulation or functionalization of a number of clinical AMP candidates. The second stage involves development of functional drug delivery systems, i.e topical sprays, gels and aerosols, based on the most promising nanoformulations developed during the screening phase. Important aspects in both stages are to increase the stability and control delivery of the AMPs. For cystic fibrosis and infections in burn wounds, the formulations will be specifically designed to target and breaking down the biofilms associated with these conditions.
To summarize, the aim of the project is to i) evaluate and further develop a number of well-characterized nanostructured materials with excellent properties for peptide delivery applications, and ii) develop functional prototype formulations based on the most promising nanostructured materials for AMP delivery that can be used to treat specific infections at their site of action.
By such strategic formulation setup, the stability of AMPs in the formulations and after administration can be increased, the peptides can be targeted to their site of action and the efficiency of the AMPs can be significantly increased. In addition, by developing formulations that can be applied locally, i.e. directly on the infected site (when no systemic administration is required), the dose can be minimized and unwanted side-effects significantly reduced. A low dose is also highly relevant in order to decrease the progress of multi-resistant bacteria.
Importantly also, in parallel with the research activities described above, strategies for preparation for process development and manufacture as well as clinical trials and regulatory aspects are covered within the project.
Project Results:
Different nanocarrier systems have been evaluated for their potential to increase the stability and improve the delivery of AMPs. These include lipid-based nanocarriers, polymer-based nanocarriers as well as mesoporous silica particles. The focus for the nanoformulation groups have been to develop the different carrier materials for the purpose of AMP delivery and to investigate the interaction of AMPs with the different carrier systems, focusing on optimizing loading and release. Extensive characterization studies have been performed in order to get a better understanding for the physicochemical characteristics and to better design the particles for drug delivery applications. Results show that sufficiently high loading capacities and physical stability can be obtained for the nanocarrier systems by tuning the interactions between the nanocarriers and the peptides.
Several rounds of in vitro effect studies of AMP-loaded nanocarriers have been performed. These include in vitro MIC (minimum inhibitory concentration) and time-kill on several Gram-positive and Gram-negative bacterial strains, including resistant strains. Results show that the antibacterial effect is preserved in the absolute majority of the cases compared to free AMPs and more interestingly also enhanced for some systems. In parallel, results also show that peptide-loaded nanocarriers can enter the macrophages and deliver the peptides by slow release. The proteolytic stability of the nanoformulated AMPs was assessed in an enzyme-sensitivity assay, with results showing that lipid nanoformulations and mesoporous silica particles may protect sensitive AMPs from proteolytic degradation. Cytotoxicity of the AMPs and the nanoformulated AMPs was evaluated in vitro and the majority of the tested formulations were well tolerated, displaying no or very low cytotoxicity in this model.
Based on the results described above, a selection of AMP-nanocarrier to prioritize in further formulation studies, namely development of functional delivery systems for topical or pulmonary administration have been selected.
The work with topical delivery systems has focused on incorporating the nanocarriers into gels and creams and optimizing these formulations depending on the properties of the nanocarriers and the peptides, respectively, and also the target indication. The most promising nanocarriers for topical delivery have been identified and the peptide effect when encapsulated in these systems has been evaluated. Also, several stability studies (have been initiated in order to evaluate the effect of pH, temperature and type of delivery system on the peptide storage stability. In parallel, a number penetrations enhancers for use in formulations for biofilm degradation have been evaluated by use of different biofilm models. During the second reporting period, ex vivo studies analyzing the antibacterial effect of topical skin formulations have been performed, showing good results and enabling a refined selection of formulations for upcoming in vivo studies.
Development work on pulmonary delivery systems of nanocarriers has focused on nebulization and powder formulation by freeze drying and spray drying where it has been shown that nebulization and powder formulation is possible for the systems evaluated within the project. Focus has been on development of an AMP formulation targeting tuberculosis and here dose-finding studies have been initiated in an infected mice model showing promising results for peptide-loaded nanocarriers.
In addition, ex vivo studies have been performed, with respect to analyzing the safety of inhaled nanoformulations, showing good results and enabling a refined selection of formulations for upcoming in vivo studies.
The work package related to preparation for process development and clinical trials have mainly focused on assessment of excipients and production protocol used for the different nanoformulations with a regulatory perspective as well as scale-up possibilities. In addition, a Cost-of-Goods analysis for the different systems has been performed. A review of the available results within FORMAMP has been carried out with respect to selection of systems to include in the preparatory CMC work.
The 6 first manuscripts with results generated in the FORMAMP project have been published in peer-reviews journals and 2 patent drafts have been generated to date. The visibility of FORMAMP in social media has increased with establishment of a Twitter account and several popular science articles in newspapers have been published.
Potential Impact:
The development of new sustainable treatment strategies for bacterial infectious diseases is of great importance to overcome one of the major challenges to our global health, antimicrobial resistance. The results from the FORMAMP project will be of vital importance for the improvement of the health of European citizens and worldwide. Today over 700 000 people worldwide die each year due to resistant bacteria and the cost for society is tremendous reaching over 1.5 billion euro per year, only in Europe. Therefore development of effective therapies will improve the quality of life as well as societal and economic conditions in Europe. The development of new antibiotics is now in dramatic decline relative to needs against infections due to multidrug resistant bacteria. A major advantage of the therapies suggested in the FORMAMP project is that AMPs are much less prone to induce high-level resistance in bacteria due to their non-specific and fast action, thus the currently ever increasing problems with spreading of resistance could be vastly diminished. In the same time, this research will also provide the ability to find new treatments that have less impact on environment, animal flora and humans, which should represent a significant benefit to community.
While there has been considerable activities regarding AMP identification and optimization, and while promising effects have been observed in vitro, but also in various animal models and early clinical trials, no AMP has yet reached the market. Anotable factor behind this is the limited efforts placed on formulation of AMPs and delivery systems. In analogy to low molecular-weight drugs as well as various biomacromolecular drugs, modern nanotechnology-based delivery system may offer substantial functional advantages for AMPs. These advantages include controlling of drug release rate, reduction of chemical and biological degradation, improved bioavailability, and reduced toxicity. Considerable research in placed in both such delivery systems and in AMP identification separately, but there is a complete lack of rational AMP delivery system design in both academic research and industrial AMP therapeutics development. Thus, combining the two, i.e. using modern nanotechnology-based delivery systems for formulation of AMPs, has strong potential to enable this much needed class of new antibiotics to reach the market.
The concept of the FORMAMP project is novel sustainable therapies based on different nanostructured materials for the treatment of infectious bacterial diseases in Europe and worldwide. Due to increasing global trade and travel the spread and prevention of drug-resistant bacterial infections are a global health concern. Through the research and development within the project, this novel therapy will be brought closer to clinical testing at a relatively high speed. This will be enabled by the unique combination of actors and their specific expertise in the FORMAMP consortium. When facing a threatening and challenging problem like multidrug-resistant bacteria and development of future therapies for infectious diseases, an extensive, strongly interdisciplinary collaboration, like the one represented by the consortium in the FORMAMP project, is a prerequisite.
Nanostructured carrier systems in addition to local administration may facilitate the achievement of a higher concentration of an AMP in a given tissue than could otherwise be achieved by e.g. intravenous administration. A higher tissue concentration will translate into faster killing of the bacteria and potentially lower doses of the AMP and thus reduced cost and risk of side effects which will of benefit both to the society as well as to the individual citizens suffering from infectious diseases. The nanotechnologies also enable the AMP to be formulated as topical and inhalable formulation facilitating administration outside a hospital setting.
List of Websites:
www.formampproject.com
The long-term objective of the FORMAMP project is to significantly change the treatment strategies for infectious diseases and to reduce the evolution of multi-resistant bacteria. The project aims to develop new and innovative formulation strategies, based on the combination of nanotechnology-based delivery systems and antimicrobial peptides (AMPs), for the treatment of infectious diseases caused by bacteria such as P. aeruginosa (Pseudomonas aeruginosa), MRSA (Methicillin-resistant Staphylococcus aureus), and MTB (Mycobacterium tuberculosis). Clinical indications that are addressed include skin and soft tissue infections, infections in burn wounds, and infections in the lung such as cystic fibrosis and tuberculosis. A number of these indications are associated with the formation of multispecies biofilms, drastically impairing the function of available treatments. An important objective of the FORMAMP project is therefore to develop treatment strategies that also takes into account the problems associated with biofilms for clinical indications were this is of importance.
The objectives of the project are divided into two sections. The first part covers screening and evaluation of a number of different nanostructured systems, i.e. liquid crystalline nanoparticles, lipidic nanocapsules, microgels, dendrimers, and mesoporous silica nanoparticles for encapsulation or functionalization of a number of clinical AMP candidates. The second stage involves development of functional drug delivery systems, i.e topical sprays, gels and aerosols, based on the most promising nanoformulations developed during the screening phase. Important aspects in both stages are to increase the stability and control delivery of the AMPs. For cystic fibrosis and infections in burn wounds, the formulations will be specifically designed to target and breaking down the biofilms associated with these conditions.
To summarize, the aim of the project is to i) evaluate and further develop a number of well-characterized nanostructured materials with excellent properties for peptide delivery applications, and ii) develop functional prototype formulations based on the most promising nanostructured materials for AMP delivery that can be used to treat specific infections at their site of action.
By such strategic formulation setup, the stability of AMPs in the formulations and after administration can be increased, the peptides can be targeted to their site of action and the efficiency of the AMPs can be significantly increased. In addition, by developing formulations that can be applied locally, i.e. directly on the infected site (when no systemic administration is required), the dose can be minimized and unwanted side-effects significantly reduced. A low dose is also highly relevant in order to decrease the progress of multi-resistant bacteria.
Importantly also, in parallel with the research activities described above, strategies for preparation for process development and manufacture as well as clinical trials and regulatory aspects are covered within the project.
Project Results:
Different nanocarrier systems have been evaluated for their potential to increase the stability and improve the delivery of AMPs. These include lipid-based nanocarriers, polymer-based nanocarriers as well as mesoporous silica particles. The focus for the nanoformulation groups have been to develop the different carrier materials for the purpose of AMP delivery and to investigate the interaction of AMPs with the different carrier systems, focusing on optimizing loading and release. Extensive characterization studies have been performed in order to get a better understanding for the physicochemical characteristics and to better design the particles for drug delivery applications. Results show that sufficiently high loading capacities and physical stability can be obtained for the nanocarrier systems by tuning the interactions between the nanocarriers and the peptides.
Several rounds of in vitro effect studies of AMP-loaded nanocarriers have been performed. These include in vitro MIC (minimum inhibitory concentration) and time-kill on several Gram-positive and Gram-negative bacterial strains, including resistant strains. Results show that the antibacterial effect is preserved in the absolute majority of the cases compared to free AMPs and more interestingly also enhanced for some systems. In parallel, results also show that peptide-loaded nanocarriers can enter the macrophages and deliver the peptides by slow release. The proteolytic stability of the nanoformulated AMPs was assessed in an enzyme-sensitivity assay, with results showing that lipid nanoformulations and mesoporous silica particles may protect sensitive AMPs from proteolytic degradation. Cytotoxicity of the AMPs and the nanoformulated AMPs was evaluated in vitro and the majority of the tested formulations were well tolerated, displaying no or very low cytotoxicity in this model.
Based on the results described above, a selection of AMP-nanocarrier to prioritize in further formulation studies, namely development of functional delivery systems for topical or pulmonary administration have been selected.
The work with topical delivery systems has focused on incorporating the nanocarriers into gels and creams and optimizing these formulations depending on the properties of the nanocarriers and the peptides, respectively, and also the target indication. The most promising nanocarriers for topical delivery have been identified and the peptide effect when encapsulated in these systems has been evaluated. Also, several stability studies (have been initiated in order to evaluate the effect of pH, temperature and type of delivery system on the peptide storage stability. In parallel, a number penetrations enhancers for use in formulations for biofilm degradation have been evaluated by use of different biofilm models. During the second reporting period, ex vivo studies analyzing the antibacterial effect of topical skin formulations have been performed, showing good results and enabling a refined selection of formulations for upcoming in vivo studies.
Development work on pulmonary delivery systems of nanocarriers has focused on nebulization and powder formulation by freeze drying and spray drying where it has been shown that nebulization and powder formulation is possible for the systems evaluated within the project. Focus has been on development of an AMP formulation targeting tuberculosis and here dose-finding studies have been initiated in an infected mice model showing promising results for peptide-loaded nanocarriers.
In addition, ex vivo studies have been performed, with respect to analyzing the safety of inhaled nanoformulations, showing good results and enabling a refined selection of formulations for upcoming in vivo studies.
The work package related to preparation for process development and clinical trials have mainly focused on assessment of excipients and production protocol used for the different nanoformulations with a regulatory perspective as well as scale-up possibilities. In addition, a Cost-of-Goods analysis for the different systems has been performed. A review of the available results within FORMAMP has been carried out with respect to selection of systems to include in the preparatory CMC work.
The 6 first manuscripts with results generated in the FORMAMP project have been published in peer-reviews journals and 2 patent drafts have been generated to date. The visibility of FORMAMP in social media has increased with establishment of a Twitter account and several popular science articles in newspapers have been published.
Potential Impact:
The development of new sustainable treatment strategies for bacterial infectious diseases is of great importance to overcome one of the major challenges to our global health, antimicrobial resistance. The results from the FORMAMP project will be of vital importance for the improvement of the health of European citizens and worldwide. Today over 700 000 people worldwide die each year due to resistant bacteria and the cost for society is tremendous reaching over 1.5 billion euro per year, only in Europe. Therefore development of effective therapies will improve the quality of life as well as societal and economic conditions in Europe. The development of new antibiotics is now in dramatic decline relative to needs against infections due to multidrug resistant bacteria. A major advantage of the therapies suggested in the FORMAMP project is that AMPs are much less prone to induce high-level resistance in bacteria due to their non-specific and fast action, thus the currently ever increasing problems with spreading of resistance could be vastly diminished. In the same time, this research will also provide the ability to find new treatments that have less impact on environment, animal flora and humans, which should represent a significant benefit to community.
While there has been considerable activities regarding AMP identification and optimization, and while promising effects have been observed in vitro, but also in various animal models and early clinical trials, no AMP has yet reached the market. Anotable factor behind this is the limited efforts placed on formulation of AMPs and delivery systems. In analogy to low molecular-weight drugs as well as various biomacromolecular drugs, modern nanotechnology-based delivery system may offer substantial functional advantages for AMPs. These advantages include controlling of drug release rate, reduction of chemical and biological degradation, improved bioavailability, and reduced toxicity. Considerable research in placed in both such delivery systems and in AMP identification separately, but there is a complete lack of rational AMP delivery system design in both academic research and industrial AMP therapeutics development. Thus, combining the two, i.e. using modern nanotechnology-based delivery systems for formulation of AMPs, has strong potential to enable this much needed class of new antibiotics to reach the market.
The concept of the FORMAMP project is novel sustainable therapies based on different nanostructured materials for the treatment of infectious bacterial diseases in Europe and worldwide. Due to increasing global trade and travel the spread and prevention of drug-resistant bacterial infections are a global health concern. Through the research and development within the project, this novel therapy will be brought closer to clinical testing at a relatively high speed. This will be enabled by the unique combination of actors and their specific expertise in the FORMAMP consortium. When facing a threatening and challenging problem like multidrug-resistant bacteria and development of future therapies for infectious diseases, an extensive, strongly interdisciplinary collaboration, like the one represented by the consortium in the FORMAMP project, is a prerequisite.
Nanostructured carrier systems in addition to local administration may facilitate the achievement of a higher concentration of an AMP in a given tissue than could otherwise be achieved by e.g. intravenous administration. A higher tissue concentration will translate into faster killing of the bacteria and potentially lower doses of the AMP and thus reduced cost and risk of side effects which will of benefit both to the society as well as to the individual citizens suffering from infectious diseases. The nanotechnologies also enable the AMP to be formulated as topical and inhalable formulation facilitating administration outside a hospital setting.
List of Websites:
www.formampproject.com