Antimicrobial peptides Drug Processing and Delivery, 2015-05-07 - - PowerPoint PPT Presentation

Formulation of Antimicrobial peptides

Drug Processing and Delivery, 2015-05-07 Uppsala

Helena Bysell SP, Technical Research Institute of Sweden

Grand Challenge

Alexander Fleming, 1945

Antimicrobial resistance

The Review on Antimicrobial Resistance, Jim O’Neill, December 2014

Consumption of antibiotics vs resistant bacteria

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

http://www.ecdc.europa.eu/en/healthtopics/antimicrobial_resistance/esac-net-database/Pages/database.aspx

Methicillin resistant S aureus (MRSA)

Carbapamen resistant Pseudomonas aeruginosa

Average use of antibiotics: Sweden – 7 g/person/year China – 138 g/person/year

Problems: The big pharmaceutical companies have abandoned development of anti-infectives

• low return on big investment

Diagnostics Clinical trials - high costs Regulatory Needs:

• 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

New treatment strategies

Antimicrobial peptides

AMPs in skin

Journal of Investigative Dermatology (2012) 132, 887–895

• 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

Around 2400 AMPs identified today

http://aps.unmc.edu/AP/main.php Critical Reviews in Biotechnology, 2012; 32(2): 143–171

Mechanism of action

• Fast and non-specific mechanism of action
• Bacteria not as prone to develop high level resistance

Nordahl et al J Biol Chem (2005), 280, 34832 Ringstad, Uppsala University thesis, 2009

Efficency and MoA influenced by

• Size
• Conformation
• Net charge
• Charge distribution
• 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 wounds Hakansson et al. Antimicrobial Agents & Chemotherapy 2014;58:2982-2984. Hydrogel Dispersin B (anti-biofilm enzyme), Pluronic F-127 KSL-W Wound infections Gawande PV et al. Current microbiology 2014;68:635- 641. Hydrogel+PLGA Pluronic F-127, PLGA microspheres KSL-W Wound healing Machado et al. BioMed Research International 2013. Multiple emulsion Avocado oil, wheat germ, olive oil, Solagum AX, Span 80, Tween 80 AH-8 Dermal delivery Hoppel et al. Journal of Drug Delivery Science and 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

Malmsten et al, Biomaterials 2009, 30, 5729-36

Sustained release Antimicrobial effect Low toxicity

Formulation of AMPs in nanocarriers - Example

Carriers for protein and peptide drugs Maintain native conformation Limit aggregation

pH-induced release of AMPs from microgels Salt-induced release of AMPs from microgels

Bysell, H et al. (2010) J. Phys. Chemistry B, 114(3),1307-1313 Bysell, H.et al. (2009) Biomacromolecules,10(8):2162-2168

• Temperature
• Specific ions
• Enzymes

Targeted and controlled release Reduced toxicity Increased bioavailability High loading capacity Protection against degradation

FORMAMP Innovative Nanoformulation of Antimicrobial peptides

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

Vision: To reduce the alarming progress of multidrug-resistant bacteria Mission: To develop new sustainable strategies for treatment of infectious diseases

Selected nanocarriers

Selected nanocarriers

WP1 Peptides Peptide drug candidates WP2 Lipidbased nanoformulations LNCs Self-assembly systems

Nanoformulation

WP3 Polymerbased nanoformulations Microgels Dendrimers WP4 Mesoporous silica- based nanoformulations MSNs

Effect studies Formulation in delivery vehicle

WP6 Topical delivery For skin and soft tissue infections, infections in burn wounds Regulatory expertise WP7 Pulmonary delivery For cystic fibrosis and tuberculosis Prototype AMP formulations for clinical testing Clinical expertise WP 5 Effect studies and method development In vitro biological models

• Antibacterial effect
• Immunomodulatory effect
• Effect against biofilms
• Cytotoxicity

In vivo models WP9 Innovation related activities including exploitation WP10 Dissemination & Training WP11-12 Consortium management Regulatory expertise WP8 Process development and preparation for clinical trials

Liquid crystalline phases

LCNPs

Ex 2: Somatostatin Intravenous delivery Incorporation (adsorption and encapsulation) in cubosomes → increased half-lives

Cervin et al Eur J Pharm Sci 2009, 36, 377-385

In vivo skin penetration Ex 3: Cyclosporin-A Topical delivery Incorporation in hexosomes → increased skin delivery, no skin irritation

Lopez et al Phar Res 2006, 23, 1332-1342

In vivo plasma concentration Ex 1: Simvastatin and cyclosporin-A Oral delivery In vivo plasma concentration, single dose Incorporation in cubosomes → increased oral bioavailability and sustained release

Lai et al AAPs PharmSciTech 2009, 10, 960-966 Lai et al Int J Nanomed 2010, 5, 13-23

LCNPs as carriers for peptides

Preparation of LCNPs

Barauskas (2005)

Dispersion of cubic phase: Cubosomes Dispersion of hexagonal phase: Hexosomes

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 (Cryo-TEM) Morphology, structure, particle size Small Angle X-Ray Scattering (SAXS) Structure, phase behavior Ultrafiltration and HPLC analysis Encapsulation efficiency Ellipsometry Adsorption kinetics, Adsorbed mass (“dry” mass), adsorbed layer thickness Quartz crystal microbalance with dissipation (QCM-D) Adsorbed “wet” mass, including contribution from coupled 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

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)
• Mycobacterial killing, intracellular mycobacterial killing, macrophage killing

Cytotoxicity

• MTT assay
• Skin irritation model

• 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

Results highlights

www.formampproject.com

Acknowledgements

FORMAMP team Lukas Boge Lovisa Ringstad Szymon Sollami Delekta David Wennman Martin Andersson and Anand Kumar Rajasekharan, Chalmers University of Technology MAX IV Laboratory is acknowledged for beamtime at beamline I911-SAXS

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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