RAJESH V. DUDHANI, JIAN LI, ROGER L. NATION

1. RAJESH V. DUDHANI, JIAN LI, ROGER L. NATION Facility for Anti-infective Drug Development & InnovationDrug 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. The threat from the PINK corner 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 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. 1 1 0 0 -1 -1 Killing Effect -2 -2 -3 -3 -4 -4 1.0 1.0 10.0 10.0 100.0 100.0 fAUC/MIC Colistin PK/PD index against P. aeruginosa in an in vitro dynamic model P. aeruginosa ATCC 27853 and PAO1 R2 = 81% R2 = 93% fCmax/MIC R2 = 70% 32.8 26.3 Bergen et al. submitted

8. Higher plasma binding of polymyxin B in critically-ill patients 0.5 - 1.5 mg/kg every 12 or 48 h MICs Protein binding 78.5 - 92.4% vs56% in healthy human plasma Cao et al, JAC 2008 Zavascki et al, CID 2009

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) • the acute-phase reactant protein • often important for the binding of weak organic basic drugs • plasma concentrations of AAG (~0.75 g/L) are normally much lower than those of HSA (~45 g/L) • 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 • 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 • Neutropenic mice were anesthetized and 50 µL early log-phase P. aeruginosa ATCC 27853 (~107 CFU) was injected into each posterior thigh • At 6 h, animals were humanely sacrificed & plasma was obtained

13. Methods • Purified protein solutions in isotonic phosphate buffer (pH 7.4) • HSA: 22.5 g/L & 45 g/L • AAG: 0.75 g/L & 3 g/L • AAG/HSA: 0.75 g/L / 45 g/L; 3 g/L / 45 g/L • Equilibrium dialysis • Spectra Por 2® dialysis membrane (MW cut off 12,000 - 14,000) • Colistin-spiked plasma or solutions of purified protein(s) were dialyzed at 37ºC for 21 h • Initial colistin conc: 3 mg/L (low) & 30 mg/L (high) • 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 (fu) 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 HSA 22.5 g/L Human plasma Low colistin concentrations: 0.81 - 1.71 mg/L High colistin concentrations: 5.69 - 11.1 mg/L AAG 0.75 g/L HSA 45 g/L AAG 3 g/L HSA 45g/L AAG 0.75 g/L HSA 45 g/L AAG 3 g/L

17. Plasma binding of colistin in mice • fu in healthy mouse plasma is similar to that in healthy human plasma • fu in neutropenic and neutropenic infected mouse plasma are lower Mouse plasma Neutropenic mouse plasma Neutropenic infected mouse plasma 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 Neutropenic infected mouse plasma • Colistin concentration range • (~0.9 – 30 mg/L) • fu increased ~4-5 fold as plasma concentration increased Dudhani et al. A1-576

19. Outline • Background • Methods • Results and Discussion • Conclusions

20. Conclusions • 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