1. www.pharm.monash.edu.au/mips FADDI RAJESH V. DUDHANI, JIAN LI, ROGER L. NATION Facility for Anti-infective Drug Development & Innovation Drug Delivery, Disposition and Dynamics Monash Institute of Pharmaceutical Sciences ISAP Post-ICAAC Symposium, 15 September, 2009 San Francisco Concentration-dependent Plasma Binding of Colistin: Impacts of Infection, Neutropenia & Multiple Proteins

2. Outline Background Methods Results and Discussion Conclusions

3. Outline Background Methods Results and Discussion Conclusions

4. Gram-negative ‘superbugs’: Acinetobacter baumannii Klebsiella pneumoniae Pseudomonas aeruginosa Virtually NO antibiotics active against G-negatives in next 9 - 11 years Currently, colistin often the only active antibiotic The threat from the PINK corner IDSA 2004, 2006, 2009 Livermore Ann Med 2003 Payne et al. Nat Rev Drug Discov 2007

5. Colistin Colistin (polymyxin E) Antibacterial activity  Narrow spectrum: G-neg bacteria (P. aeruginosa, A. baumannii and K. pneumoniae)  Rapid bactericidal effect: Concentration-dependent  Very modest PAE against P. aeruginosa Currently resistance is low, but emerging Li et al. Lancet Infect Dis 2006

6. Colistin Colistin A: 6-methyloctanoic acid Colistin B: 6-methylheptanoic acid Dab: ,  -Diaminobutyric acid multi-component a weak organic base containing 5 primary amine groups polycation at physiological pH Li et al. Lancet ID 2006

7. Colistin PK/PD index against P. aeruginosa in an in vitro dynamic model P. aeruginosa ATCC 27853 and PAO1 Bergen et al. submitted 1.010.0100.0 -4 -3 -2 0 1 Killing Effect 1.010.0100.0 fAUC/MIC -4 -3 -2 0 1 R 2 = 93% R 2 = 70% R 2 = 81% fC max /MIC 26.3 32.8

8. Higher plasma binding of polymyxin B in critically-ill patients 0.5 - 1.5 mg/kg every 12 or 48 h Cao et al, JAC 2008 Zavascki et al, CID 2009 MICs Protein binding 78.5 - 92.4% vs 56% in healthy human plasma

9. Plasma binding of drugs Crucial to understanding of PK/PD relationship Two plasma proteins commonly involved Human serum albumin (HSA): binds weak organic acids & bases and neutral compounds alpha-1-acid glycoprotein (AAG) o the acute-phase reactant protein o often important for the binding of weak organic basic drugs o plasma concentrations of AAG (~0.75 g/L) are normally much lower than those of HSA (~45 g/L) o concentrations of AAG are increased (~3-5 fold) in a number of stressful conditions, including infection

10. Aims To investigate the proteins involved in the plasma binding of colistin To examine the potential impact of colistin concentration on its plasma binding

11. Outline Background Methods Results and Discussion Conclusions

12. Methods Healthy human plasma (Australian Red Cross) Mouse plasma o Six-week old, female Swiss albino mice (22 - 26 g) were rendered neutropenic by IP cyclophosphamide (150 mg/kg) 4 days and (100 mg/kg) 1 day prior to experimental infection o Neutropenic mice were anesthetized and 50 µL early log-phase P. aeruginosa ATCC 27853 (~10 7 CFU) was injected into each posterior thigh o At 6 h, animals were humanely sacrificed & plasma was obtained

13. Methods Purified protein solutions in isotonic phosphate buffer (pH 7.4) o HSA: 22.5 g/L & 45 g/L o AAG: 0.75 g/L & 3 g/L o AAG/HSA: 0.75 g/L / 45 g/L; 3 g/L / 45 g/L Equilibrium dialysis o Spectra Por 2 ® dialysis membrane (MW cut off 12,000 - 14,000) o Colistin-spiked plasma or solutions of purified protein(s) were dialyzed at 37ºC for 21 h o Initial colistin conc: 3 mg/L (low) & 30 mg/L (high) o In neutropenic infected mouse plasma, initial colistin conc 2.5 – 75 mg/L

14. Methods Colistin concentrations in the protein and buffer solutions were determined using a validated HPLC assay The unbound fraction (f u ) of colistin was calculated from the ratio, at dialysis equilibrium, of concentration in buffer to that in the protein-containing solution

15. Outline Background Methods Results and Discussion Conclusions

16. Plasma binding of colistin: proteins involved Concentration dependent AAG and HSA Healthy human plasma vs physiological concentrations of HSA/AAG Low colistin concentrations: 0.81 - 1.71 mg/L High colistin concentrations: 5.69 - 11.1 mg/L

17. Plasma binding of colistin in mice Mouse plasma Neutropenic mouse plasma Neutropenic infected mouse plasma f u in healthy mouse plasma is similar to that in healthy human plasma f u in neutropenic and neutropenic infected mouse plasma are lower Low concentrations:1.42 - 1.80 mg/L High concentrations:12.9 - 14.9 mg/L

18. Conc-dependent plasma binding of colistin in mice Dudhani et al. A1-576 Neutropenic infected mouse plasma Colistin concentration range (~0.9 – 30 mg/L) f u increased ~4-5 fold as plasma concentration increased

19. Outline Background Methods Results and Discussion Conclusions

20. Both HSA and AAG are important in the binding of colistin in plasma AAG conc increases in infections increased plasma binding of colistin Colistin binding was dependent upon its concentration Similar fAUC/MIC values from in vitro PK/PD model and mouse thigh infection model Assist in defining optimal dosage regimens for colistin Further investigation on colistin binding affinity, capacity to plasma proteins and bacterial cells is warranted

21. Acknowledgements FADDI team NIH/NIAID R01AI079330 and R01AI070896 Australian National Health & Medical Research Council