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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. - PowerPoint PPT Presentation
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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, 2009San Francisco
Concentration-dependent Plasma Binding of Colistin: Impacts of Infection, Neutropenia & Multiple Proteins
Outline
• Background
• Methods
• Results and Discussion
• Conclusions
Outline
• Background
• Methods
• Results and Discussion
• Conclusions
Gram-negative ‘superbugs’: Acinetobacter baumanniiKlebsiella 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, 2009Livermore Ann Med 2003Payne et al. Nat Rev Drug Discov 2007
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
()
L-Thr L-Dab( ) ( )
L-LeuD-LeuL-Dab
L-Dab
()
L-Dab()L-Thr()L-Dab L-DabFatty acid
NH2 NH2
NH2 NH2
NH2
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
Colistin PK/PD index against P. aeruginosa in an in vitro dynamic model
P. aeruginosa ATCC 27853 and PAO1
Bergen et al. submitted
1.0 10.0 100.0-4
-3
-2
-1
0
1
Kill
ing
Effe
ct
1.0 10.0 100.0fAUC/MIC
-4
-3
-2
-1
0
1 R2 = 93%
0 20 40 60 80 100% T>MIC
-4
-3
-2
-1
0
1
Kill
ing
Effe
ct0 20 40 60 80 100
% T>MIC
-4
-3
-2
-1
0
1
Kill
ing
Effe
ct
R2 = 70%
0.1 1.0 10.0Cmax/MIC
-4
-3
-2
-1
0
1
Kill
ing
Effe
ct
0.1 1.0 10.0Cmax/MIC
-4
-3
-2
-1
0
1
Kill
ing
Effe
ct
-4
-3
-2
-1
0
1
Kill
ing
Effe
ct
R2 = 81%
fCmax/MIC
26.3
32.8
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 2008Zavascki et al, CID 2009
MICs
Protein binding 78.5 - 92.4% vs 56% in healthy human plasma
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 proteino 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
Aims
• To investigate the proteins involved in the plasma binding of colistin
• To examine the potential impact of colistin concentration on its plasma binding
Outline
• Background
• Methods
• Results and Discussion
• Conclusions
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 (~107 CFU) was injected into each posterior thigh
o At 6 h, animals were humanely sacrificed & plasma was obtained
Methods
• Purified protein solutions in isotonic phosphate buffer (pH 7.4)o HSA: 22.5 g/L & 45 g/Lo AAG: 0.75 g/L & 3 g/Lo AAG/HSA: 0.75 g/L / 45 g/L; 3 g/L / 45 g/L
• Equilibrium dialysiso 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 ho 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
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
Outline
• Background
• Methods
• Results and Discussion
• Conclusions
Plasma binding of colistin: proteins involved
Unb
ound
frac
tion
of c
olis
tin
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8Colistin: Low concentrationColistin: High concentration • Concentration
dependent
• AAG and HSA
• Healthy human plasma vs physiological concentrations of HSA/AAG
Low colistin concentrations: 0.81 - 1.71 mg/LHigh colistin concentrations: 5.69 - 11.1 mg/L
Plasma binding of colistin in mice
Mouse plasma
Neutropenic mouse plasma
Neutropenic infected mouse
plasma
• fu in healthy mouse plasma is similar to that in healthy human plasma
• fu in neutropenic and neutropenic infected mouse plasma are lower
Low concentrations: 1.42 - 1.80 mg/LHigh concentrations: 12.9 - 14.9 mg/L
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)
• fu increased ~4-5 fold as plasma concentration increased
Outline
• Background
• Methods
• Results and Discussion
• Conclusions
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
Acknowledgements
• FADDI team
• NIH/NIAID R01AI079330 and R01AI070896• Australian National Health & Medical Research Council