INTRODUCTION

1. School of Dentistry / Ysgol am Deintyddiaeth CHANGES IN SPUTUM RHEOLOGY FOLLOWING IN VITRO TREATMENT WITH ALGINATE OLIGOMERS Pritchard, Manon F1, 2; Khan, Saira 1; Hill, Katja E1; Hawkins, Karl2; Curtis, Dan2; Doull, Iolo 3; Onsøyen, Edvar 4; Myrvold, Rolf 4; Dessen, Arne 4; Thomas, David W1 1Wound Biology Group, Cardiff University School of Dentistry, Cardiff, UK; 2Centre for Nanohealth, Swansea University, Swansea, UK; 3Respiratory/Cystic Fibrosis Unit, Children's Hospital for Wales, Cardiff, UK; 4AlgiPharma AS, Sandvika, Norway. E-mail: pritchardmf@cardiff.ac.uk INTRODUCTION Progressive lung disease in cystic fibrosis (CF) patients represents a major cause of morbidity and mortality. Bacterial infections in the lungs of CF patients are typified by bacterial overproduction of alginate or extracellular polysaccharide. OligoG is an alginate (ca. 2600 MW) of linear (1-4) linked polymers of β-D-mannuronic acid (M) and α-L-guluronic acid (G),comprised of 90-95% G residues (Fig 1) that has been proven safe in pre-clinical animal and phase I human volunteer studies1. The ability of OligoG to potentiate the activity of antimicrobial/antibiotic therapy and disrupt in vitro biofilms has previously been demonstrated4 (Fig 2). In preliminary rheological studies, we demonstrated that OligoG is able to alter CF sputum viscoelasticityin vitro; potentially aiding the disruption of CF sputum and facilitating its clearance from the diseased lung.  OligoG is currently undergoing Phase IIa human trials2. RESULTS RESULTScontinued Extensional rheology A B C Fig 7. Example of a typical sputum diameter profile highlighting the area of analysis where capillary thinning is dominated by surface tension (circled; Fig 7:A), in the other section drainage due to gravity plays a part. Extensional rheology results of samples divided into control Vs 2% OligoG (Fig 7:B-C) showing the change in extensional viscosity (Pa.s) as the extension rate (s-1) increases. Fig 1. Structure of OligoG showing arrangement of mannuronic acid (M) and guluronic acid (G) monomers. Extensional rheological analysis of CF sputum defined a reproducible model of sputum deformation and filament breakage (circled; Fig 7:A). Further rheometric analysis of sputum samples in this region (n=3) showed marked reductions in extensional viscosity of the 2% OligoG-treated CF sputum (Fig 7:B-C). Videos of the extensional rheometry can be viewed via the QR codes below (Fig 8). Control 2% OligoG 6% OligoG 10% OligoG Longitudinal study A B Fig 2. Confocal laser scanning microscopy of 24 h green fluorescent protein (gfp) labelled P. aeruginosa PAO1 biofilms grown in the presence of 0, 2, 6 and 10% OligoG. A • AIMS & OBJECTIVES B • The specific aims of the studies were to: • Investigate the ability of OligoG to modulate the viscoelastic properties of CF sputum in vitro. • Determine the reproducibility of the effects of OligoG and/or dornase alpha (Pulmozyme®) over time during episodes of acute exacerbation; investigating potential changes in efficacy with disease state. • Develop a reliable, reproducible model system to study and compare the effects of mucolytic agents in vitro. Fig 5. Oscillatory frequency sweep of sputum from longitudinally collected samples (n=10) treated with 0.2% OligoG or 2% OligoG, 100 nM Pulmozyme® (Pz) or a combination showing at 0.16 Hz (A) storage modulus, G’; (B) loss modulus, G’’ of CF sputum (** p>0.01). MATERIALS & METHODS Patient samples: Non-induced CF sputum samples were collected from patients at the Cardiff and Llandough Cystic Fibrosis Unit. A longitudinal study was conducted over 10 weeks (n=10) on samples from a 17 year old patient chronically infected with Pseudomonas aeruginosa. Each sample was treated ex vivo with the following treatment modalities: (1) control with distilled water, (2) 100 nM dornase alpha (rhDNase; Pulmozyme®)3 (3) 0.2% OligoG (4) 2% OligoG (5) Pulmozyme® and 0.2% OligoG (6) Pulmozyme® and 2% OligoG, following the treatment model commonly used for clinical sputum induction3. To further observe effectiveness of 2% OligoG, a further 23 patient sputum samples were collected, treated then subjected to rheological analysis. Samples and test agents were inverted 4 times and incubated for 4 h at 37°C prior to testing. Rheological analysis: The shear rheology of the samples was characterised in terms of linear viscoelastic parameters (the storage and loss moduli, G’ and G’’ respectively) over a frequency range of 0.1-10 Hz at 37°C using a TA-Instruments AR-G2 rheometer. In oscillatory shear, storage (G’) describes the elastic response of the material, whilst loss (G’’) is the viscous response. The longitudinal study (over 10 weeks) demonstrated a statistically significant consistent response to treatment with 2% OligoG (Fig 5). The ability of 2% OligoG to potentiate the changes in both storage and loss modulus was evident, as well as potentiating the response to treatment with Pulmozyme® (p<0.01). Fig 8. Extensional rheology video of CF sputum at 5 s (A) Control (B) 2% OligoG. • CONCLUSIONS • These studies demonstrated the ability of OligoG to effectively: • Modify the rheology of sputum from CF patients in vitro; significantly altering the observed viscoelasticity. • Potentiate the activity of Pulmozyme®, by decreasing the elastic and viscous response of CF sputum under oscillatory shear. • Induce rheological changes in CF sputum over time, during periods of disease exacerbations in an individual patient. • These findings and the ability of OligoG to potentiate the action of antibiotics against multi-drug resistant bacteria4,highlights its potential application in the treatment of biofilm-related chronic infections. The rheological effect of 2% OligoG on CF sputum A decrease in the storage modulus was observed in nearly all sputum samples treated with 2% OligoG compared to the control (Fig 6). This contrasted to treatment with Pulmozyme®, where changes in visco-elasticity were not uniformly observed throughout the study; a positive effect being evident in only 6/10 samples in the longitudinal analyses. This study shows a consistent in vitro effect of OligoG at changing the viscoelasticity of sputum. B A A significant difference in storage modulus (G’) and loss modulus (G’’) between CF sputum in control Vs 2% OligoG (Fig 4) was confirmed (G’ and G’’, p<0.0001). These studies highlight the potential application of OligoG in the treatment of biofilm-related chronic infections. Capillary Break-Up Extensional Rheometry(CaBER) was employed to measure the extensional properties of CF sputum samples (n=3). Extensional viscosity defines the material response to uni-axial deformation.  CaBER experiments were carried out by measuring the mid-filament diameter of the unstable (i.e. thinning) filament formed after a step strain (displacement) event is imposed upon the material. In the final stages of filament break-up, the thinning phenomena is dominated by surface tension effects which permits analysis of the rate-change of the mid-filament diameter to determine the extensional viscosity of the sample (Fig 3). REFERENCES 1. EudraCT number: 2009-009330-33, www.clinicaltrials.gov identifier NCT00970346. 2.EudraCT number: 2010-023090-19, www.clinicaltrials.gov identifier NCT01465529. 3. King et al. (1997) Am J RespirCrit Care Med 156:173-177. 4. Khan et al. (2012) Antimicrob Ag Chemother 56:5134-5141. Fig 6. Timeline graph of 10 sputum samples collected over a period of 10 week, showing change in storage modulus (Pa) at 0.16 Hz when treated with 100 nM Pulmozyme® and 2% OligoG. Fig 4. Shear rheology of control Vs 2% OligoG at 0.16 Hz. (A) storage modulus (G’) and (B) loss modulus (G’’) of CF sputum. ACKNOWLEDGMENTS We gratefully acknowledge the support of Dr Ian Ketchell, Mrs Alison Lynne Hopkins and the patients from the Cardiff and Vale University Health Board's Cystic Fibrosis unit in the provision of samples used in this study. DECLARATION This work was funded by the European Union, Cystic Fibrosis Foundation Therapeutics, Inc., and AlgiPharma AS. TIME Fig 3. Typical stages of capillary break-up. ** **