Formulation of Antimicrobial peptides Drug Processing and Delivery, 2015-05-07 Uppsala Helena Bysell SP, Technical Research Institute of Sweden
Grand Challenge
Antimicrobial resistance The Review on Antimicrobial Resistance, Jim O’Neill, December 2014 Alexander Fleming, 1945
Consumption of antibiotics vs resistant bacteria Methicillin resistant S aureus (MRSA) Consumption of beta-lactams, penicillins (ATC group J01C) in the community, EU/EEA countries, 2011, expressed as DDD per 1 000 inhabitants and per day Average use of antibiotics: Sweden – 7 g/person/year China – 138 g/person/year Carbapamen resistant Pseudomonas aeruginosa http://www.ecdc.europa.eu/en/healthtopics/antimicrobial_resistance/esac-net-database/Pages/database.aspx
New treatment strategies Problems: The big pharmaceutical companies have abandoned development of anti-infectives - low return on big investment Diagnostics Clinical trials - high costs Needs: Regulatory - New types of antibiotics for treatment of bacterial infections - New types of antibiotics efficient against multidrug resistant bacteria - More strategic use of existing antibiotics - Restrict use of antibiotics - Diagnostic tools
Antimicrobial peptides Around 2400 AMPs identified today http://aps.unmc.edu/AP/main.php • Present in plants, insects , animals and humans • Part of the host defense system • High concentrations in skin, airways, mucosa • Expressed in response to pathogens • Evolutionary well-preserved AMPs in skin Journal of Investigative Dermatology (2012) 132, 887 – 895 Critical Reviews in Biotechnology , 2012; 32(2): 143 – 171
Mechanism of action Nordahl et al J Biol Chem (2005), 280, 34832 • Fast and non-specific mechanism of action • Bacteria not as prone to develop high level resistance Efficency and MoA influenced by - Size - Conformation - Net charge - Charge distribution Ringstad, Uppsala University thesis, 2009 - Hydrophobicity
AMPs in drug delivery AMPs in clinical trials but no products on market Problems with: -Stability (chemical and proteolytic) -Not efficient enough -Toxicity issues -High cost Formulation
Formulation of AMPs Formulation type Components Peptide Target Reference Hydrogel Hydroxypropyl cellulose PLX150 Infections in surgical Hakansson et al. Antimicrobial wounds Agents & Chemotherapy 2014;58:2982-2984. Hydrogel Dispersin B (anti-biofilm KSL-W Wound infections Gawande PV et al. Current enzyme), Pluronic F-127 microbiology 2014;68:635- 641. Hydrogel+PLGA Pluronic F-127, PLGA KSL-W Wound healing Machado et al. BioMed microspheres Research International 2013. Multiple emulsion Avocado oil, wheat AH-8 Dermal delivery Hoppel et al. Journal of Drug germ, olive oil, Solagum Delivery Science and AX, Span 80, Tween 80 Technology 2015;25:16-22. Polymeric wafer Guar gum NP110/NP108 Wound infections O’Driscoll et al. Current microbiology 2013;66:271- 278.
Formulation of AMPs in nanocarriers- Example LL-37 loaded in mesoporous silica to prevent implant-associated infections Sustained release Antimicrobial effect Low toxicity Malmsten et al, Biomaterials 2009, 30, 5729-36
Formulation of AMPs in nanocarriers - Example Carriers for protein and peptide drugs Targeted and controlled release Reduced toxicity Increased bioavailability Maintain native conformation High loading capacity Limit aggregation Protection against degradation pH-induced release of Salt-induced release of AMPs from microgels AMPs from microgels - Temperature - Specific ions - Enzymes Bysell, H.et al. (2009) Biomacromolecules ,10(8):2162-2168 Bysell, H et al. (2010) J. Phys. Chemistry B , 114(3),1307-1313
FORMAMP Innovative Nanoformulation of Antimicrobial peptides Vision: To reduce the alarming progress of multidrug-resistant bacteria Mission: To develop new sustainable strategies for treatment of infectious diseases Facts: Project duration: 2013-2017 Budget: 10.5 MEuro, EU contribution 8 MEuro 16 partners from 5 countries Coordinator: helena.bysell@sp.se www.formampproject.com
Selected nanocarriers
Selected nanocarriers
Nanoformulation WP2 Lipidbased nanoformulations Effect studies LNCs Self-assembly systems Formulation in WP 5 Effect studies and delivery vehicle WP3 Polymerbased method development WP1 Peptides nanoformulations Peptide drug WP6 Topical delivery Microgels In vitro biological models candidates For skin and soft tissue Dendrimers -Antibacterial effect infections, infections in burn -Immunomodulatory effect wounds WP4 Mesoporous silica- -Effect against biofilms based nanoformulations -Cytotoxicity MSNs In vivo models WP7 Pulmonary delivery For cystic fibrosis and tuberculosis Regulatory expertise Clinical expertise Regulatory expertise WP8 Process development and preparation for clinical trials WP9 Innovation related activities including exploitation WP10 Dissemination & Training Prototype AMP WP11-12 Consortium management formulations for clinical testing
Liquid crystalline phases LCNPs
LCNPs as carriers for peptides Ex 3: Cyclosporin-A Ex 1: Simvastatin and cyclosporin-A Ex 2: Somatostatin Topical delivery Oral delivery Intravenous delivery In vivo plasma concentration, single In vivo skin penetration In vivo plasma concentration dose Incorporation in cubosomes → Incorporation (adsorption and Incorporation in hexosomes → increased oral bioavailability and encapsulation) in cubosomes → increased skin delivery, no skin sustained release increased half-lives irritation Lai et al AAPs PharmSciTech 2009, 10, 960-966 Cervin et al Eur J Pharm Sci 2009, 36, 377-385 Lai et al Int J Nanomed 2010, 5, 13-23 Lopez et al Phar Res 2006, 23, 1332-1342
Preparation of LCNPs Dispersion of cubic phase: Cubosomes Dispersion of hexagonal phase: Hexosomes Barauskas (2005)
AMP loading strategies
Key characterization techniques Technique Information Dynamic light scattering (DLS) Particle size, particle size distribution Electrophoretic mobility Surface charge, Zeta potential Transmission Electron Microscopy Morphology, structure, particle size (Cryo-TEM) Small Angle X-Ray Scattering Structure, phase behavior (SAXS) Ultrafiltration and HPLC analysis Encapsulation efficiency Ellipsometry Adsorption kinetics, Adsorbed mass (“dry” mass), adsorbed layer thickness Quartz crystal microbalance with Adsorbed “wet” mass, including contribution from coupled dissipation (QCM-D) water molecules
Phase transitions upon AMP incorporation
AMP loading in LCNPs
In vitro effect studies Antibacterial effect – MIC (minimum inhibitory concentration) / Time-kill -Gram-positive bacteria: Staphylococcus aureus (SA) reference strain, Methicillin-resistant SA (MRSA) -Gram-negative bacteria: Pseudomonas aeruginosa reference strain (PSA ATCC) , Pseudomonas aeruginosa clinical strain, Escherichia coli reference strain , ESBL Escherichia coli , Acinetobacter baumannii reference strain -Mycobacterial killing, intracellular mycobacterial killing, macrophage killing In vitro biofilm models -Developed within FORMAMP for Cystic fibrosis and Burn Wound infections Immunomodulatory effects -Protease sensitivity assay (S. aureus aurelysin, S. aureus V8, P. aeruginosa elastase, Human neutrophil elastase - In vitro inflammation studies (THP-1 cells)- Detection of NF-kB activation -Coagulation analysis (plasma) - determination of prothrombin time (PT) ,activated partial thromboplastin time (aPTT) Cytotoxicity -MTT assay -Skin irritation model
Results highlights -Promising results related to encapsulation efficiency for AMPs in different nanocarrier systems. -MIC analysis show that the antibacterial activity is preserved in 82% of the cases for encapsulated AMPs and also enhanced for 10% of the peptide-carrier combinations. -Preliminary results indicate that AMPs are protected against proteolytic degradation in nanocarriers – A peptide effective against Mycobacterium tuberculosis (both intracellular and extracellular) and harmless to human cells have been identified, synthesized and currently evaluated in vivo (mouse model). www.formampproject.com
Acknowledgements FORMAMP team Lukas Boge Lovisa Ringstad Szymon Sollami Delekta David Wennman MAX IV Laboratory is acknowledged for beamtime at beamline I911-SAXS Martin Andersson and Anand Kumar Rajasekharan, Chalmers University of Technology www.formampproject.com The research in FORMAMP receives funding from the European Union’s Seventh Framework Programme (FP7/2007-2013) under grant agreement n° 604182. http://ec.europa.eu/research
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