1. Monash Institute of Pharmaceutical Sciences Sheep as a clinically relevant model to assess the pharmacokinetics of colistin and colistin methanesulfonate after pulmonary and intravenous administration Dr Tri-Hung Nguyen Drug Delivery to the Lung Conference Edinburgh, December 2012

2. 2 Increasing prevalence in hospital environments Product of antimicrobial misuse Sources of MDR lung infections Klebsiella pneumoniae Acinetobacter baumanii Pseudomonas aeruginosa Opportunistic infections Prevalent amongst immunocompromised patients Cystic fibrosis Critically ill Multiple drug resistant Gram negative infections Soon et al. Antimicrob Agents Chemother 2006 05 µm

3. 3 Antimicrobial Development Clatworthy et al., Nat Chem Biol 2007.

4. 4 Antimicrobial Resistance Clatworthy et al., Nat Chem Biol 2007.

5. 5 Declining numbers of new antimicrobial agents Adapted from Spellberg et al. Clin Infect Dis 2011 Antimicrobials with activity against Gram-negatives EMEA 2009

6. 6 Antimicrobial Resistance

7. 7 Colistin (Polymyxin E) Cationic polypeptide antimicrobial First used in the 1950’s and 1960’s

8. 8 Colistin (Polymyxin E) Cationic polypeptide antimicrobial First used in the 1950’s and 1960’s Amphiphilic molecule Hydrophilic peptide ring Peptide Ring

9. 9 Colistin (Polymyxin E) Cationic polypeptide antimicrobial First used in the 1950’s and 1960’s Amphiphilic molecule Hydrophilic peptide ring Hydrophobic tail

10. 10 Colistin (Polymyxin E) Cationic polypeptide antimicrobial First used in the 1950’s and 1960’s Amphiphilic molecule Hydrophilic peptide ring Hydrophobic tail Mode of action Positively charged amino groups Interaction with bacterial cell membrane Positively charged

11. 11 Colistin and colistin methanesulfonate (CMS) Colistin Nephrotoxicity and neurotoxicity on high systemic exposure Superseded by newly developed antibiotics Bronchoconstriction when inhaled

12. 12 Colistin and colistin methansulfonate (CMS) Colistin Nephro- and neurotoxicity on high systemic exposure Superseded by newly developed antibiotics Bronchoconstriction on inhaled use Inactive prodrug Colistin methanesulfonate (CMS) Amines replaced with methylene sulfonic acid moieties

13. 13 Clinical use of colistin and CMS Colistin currently used in clinical practice Inconsistencies with doses administered Need to refine dosing regimens Improve efficacy Prevent resistance Potential toxicity at high dose limits dose excursions

14. 14 Clinical use of colistin and CMS Colistin currently used in clinical practice Inconsistencies with doses administered Need to refine dosing regimens Improve efficacy Prevent resistance Potential toxicity at high dose limits dose excursions Can a pre-clinical model be used to assess targeted delivery of antibiotics and inform clinical practice?

15. 15 In vivo lung models MouseRatGuinea-pigDogSheepHuman Body mass (kg)0.02-0.040.25-0.350.410-154570-80 Nose/mouth breathers Nose Nose/mouth Trachea-bronchial branching pattern Monopodial Dichotomous Irregularly dichotomous Lung mass (g)0.121.5-100250-10001000 Lung volume (mL)0.748.61373628004341 Lung Lobation 5 lobes left lung (1) and right lung (4) Left lung is not divided - 6 lobes left lung (2) and right lung (4) 6 lobes left lung (2) and right lung (4) 5 lobes left lung (2) and right lung (3) Alveoli diameter (μm) 477065126150-300200-400 Tidal volume (ml)0.15-0.180.87-2.08 1.72 – 1.75 11.4 – 16.6 180 – 405400 – 616 Respiratory rate (breaths/min) 16385902315 – 4012

16. 16 Hypothesis Sheep are a suitable preclinical model to assess the localised delivery of an antimicrobial to the lung

17. 17 6 merino sheep Four way randomised crossover study Surgical cannulation Jugular vein:IV administration Carotid artery: Blood collection IV administration 2.5 mg/kg or equivalent Colistin (sulfate salt) CMS (sodium salt) IV infusion at 1 mL/min Blood and broncho-alveolar lavage (BAL) fluid sampled up to 24 hr after administration In-vivo studies

18. 18 Pulmonary administration of colistin and CMS Conducted in conscious sheep Endotracheal tube directed via nostril into the trachea Colistin and CMS solution nebulised over 25 min Blood sampled up to 24 hr after administration BAL fluid collected from lung sections 1, 4 and 24 hr after administration Colistin and CMS plasma analysed via HPLC

19. 19 Data analysis Non-compartmental analysis Population modeling (S-ADAPT and SADAPT-TRAN software/MC-PEM algorithm) Takes into account the true biological variability between sheep Enables prediction of concentration vs. time profiles at other dosage regimens Supports translation from sheep into humans and allometric scaling The PK model can be combined with a pharmacodynamic model to predict antibacterial effects

20. 20 IV colistin pharmacokinetics IV colistin sulfate Fits 3 compartment model Decrease in plasma concentrations over time

21. 21 IV CMS pharmacokinetics IV CMS Fits 3 compartment model Decrease in plasma concentrations over time Similar elimination phase Colistin conversion from CMS Increased concentration over time No colistin/CMS concentrations were detected in epithelial lung fluid (ELF)

22. 22 IV colistin and CMS pharmacokinetics IV colistin sulfate and CMS Fits 3 compartment model Decrease in plasma concentrations over time Similar elimination phase Colistin conversion from CMS Increased concentration over time No colistin/CMS concentrations were detected in epithelial lung fluid (ELF)

23. 23 IV colistin from CMS pharmacokinetics Pharmacokinetic Parameter Colistin from CMS Sheep Colistin from CMS Human Colistin from CMS Rat AUC (mg/L x h)23.9 ± 7.5 t 1/2 (h)9.1 ± 6.1 T max (h)3.1 ± 0.55 C max (mg/L)2.6 ± 0.4

24. 24 IV colistin from CMS pharmacokinetics Pharmacokinetic Parameter Colistin from CMS Sheep Colistin from CMS Humans Colistin from CMS Rats AUC (mg/L x h)23.9 ± 7.5 t 1/2 (h)9.1 ± 6.1 *5.9-14.4 T max (h)3.1 ± 0.55 C max (mg/L)2.6 ± 0.4 ٭Imberti et al. Chest (2010) Garonzik et al. Antimicrob. Agents Chemother. (2011) Plachouras et al. Antimicrob. Agents Chemother. (2009)

25. 25 IV colistin from CMS pharmacokinetics Pharmacokinetic Parameter Colistin from CMS Sheep Colistin from CMS Humans Colistin from CMS Rats AUC (mg/L x h)23.9 ± 7.5 t 1/2 (h)9.1 ± 6.1 *5.9-14.4 # 0.4-1.2 T max (h)3.1 ± 0.55 C max (mg/L)2.6 ± 0.4 ٭Imberti et al. Chest (2010) Garonzik et al. Antimicrob. Agents Chemother. (2011) Plachouras et al. Antimicrob. Agents Chemother. (2009) # Marchand et al. J. Antimicrob. Chemother. (2010) Li et al. Antimicrob Agents Chemother (2003) Li et al. Antimicrob Agents Chemother (2004)

26. 26 Population PK model: IV clearance Pharmacokinetic Parameter Colistin Sheep Colistin Humans Colistin Rats Clearance (L/h)1.3*1.1 # 0.1 * Reed et al. J. Clin Pharmacol. (2002) # Li et al. J. Antimicrob. Chemother. (2003)

27. 27 Local delivery of colistin and CMS to the lung Epithelial lung fluid concentration Colistin and CMS concentration decreased over time Colistin from CMS increased then decreased Conversion from CMS No detectable colistin concentration measured in plasma Consistent with human data < 0.14mg/L* Colistin binding to tissues? * Ratjen et al. J. Antimicrob. Chemother. (2006) Jensen et al. J. Antimicrob. Chemother. (1987)

28. 28 Local delivery of colistin and CMS to the lung Epithelial lung fluid concentration Colistin and CMS concentration decreased over time Colistin from CMS increased then decreased Conversion from CMS No detectable colistin concentration measured in plasma Consistent with human data < 0.14 mg/L* Colistin binding to tissues? * Ratjen et al. J. Antimicrob. Chemother. (2006) Jensen et al. J. Antimicrob. Chemother. (1987) Marchand et al. Antimicrob. Agents Chemother (2010) Systemic plasma concentrations measured in rats

29. 29 Population PK model: Lung delivery PUL colistin dose Colistin in ELF Colistin in lung tissues Colistin clearance (4.6 mL/h) distribution Colistin in absorption compartment absorption

30. 30 Population PK model: Lung delivery PUL CMS dose PUL colistin dose CMS in ELF Colistin in ELF CMS in lung tissues Colistin in lung tissues Metabolism to colistin Colistin clearance (4.6 mL/h) distribution Metabolism to colistin distribution CMS in absorption compartment Colistin in absorption compartment absorption

31. 31 Summary Sheep were examined as a clinically relevant model to assess the PK of the antimicrobial colistin and CMS Decreases in CMS plasma concentrations after IV administration corresponded to an increase in formed colistin No colistin or CMS detected in ELF after IV administration

32. 32 Summary Sheep were examined as a clinically relevant model to assess the PK of the antimicrobial colistin and CMS Decreases in CMS plasma concentrations after IV administration corresponded to an increase in formed colistin No colistin or CMS detected in ELF after IV administration CMS concentrations decreased in ELF over 24 h with increasing colistin concentrations after pulmonary administration No colistin or CMS detected in plasma Correlates to clinical data Population PK modeling indicated that CMS was distributed and metabolised in ELF and lung tissue after pulmonary administration Sheep have been demonstrated to be a clinically relevant model to evaluate the PK of antimicrobials administered to the lung.

33. 33 Acknowledgments Monash Institute of Pharmaceutical Sciences Dr Michelle McIntosh Linh Lieu Biotechnology Research Laboratories (Monash University) Prof Els Meeusen Dr Robert Bishcof Gary Nguyen Centre for Medicine Use and Safety (Monash University) Dr Cornelia Landersdorfer

34. Monash Institute of Pharmaceutical Sciences Sheep as a clinically relevant model to assess the pharmacokinetics of colistin and colistin methansulfonate after pulmonary and intravenous administration Dr Tri-Hung Nguyen Drug Delivery to the Lung Conference Edinburgh, December 2012

35. 35 Population PK model: IV administration IV CMS dose IV colistin dose CMS in plasma Colistin in plasma CMS in slowly equilibrating tissues Colistin in slowly equilibrating tissues Metabolism to colistin colistin elimination distribution Metabolism to colistin distribution Metabolism to colistin CMS in rapidly equilibrating tissues Colistin in rapidly equilibrating tissues CMS elimination distribution

36. 36 Population PK model: IV clearance ParameterColistin from CMSColistin Clearance (L/h)1.320.784 Central volume of distribution (L)0.500.32 Peripheral volume of distribution (Fast equilibrating, L) 7.453.25 Peripheral volume of distribution (Slow equilibrating, L) 92.959.1 Conversion of CMS to colistin in central and fast equilibrating peripheral compartment (h -1 ) 0.05