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Concentration-dependent Plasma Binding of Colistin:
Impacts of Infection, Neutropenia & Multiple Proteins
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
www.pharm.monash.edu.au/mips
FADDI
Outline
• Background
• Methods
• Results and Discussion
• Conclusions
Outline
• Background
• Methods
• Results and Discussion
• Conclusions
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
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
Colistin
 NH2
() L-Dab
Fatty acid
()L-Dab
L-Thr
() L-Dab
 NH2
 NH2
D-Leu
L-Leu
( ) L-Dab
L-Thr
()L-Dab
()L-Dab
 NH2
 NH2
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
1
R2 = 81%
R2 = 93%
0
-1
-2
-3
-1
-4
-2
1
0.1
1.0
fC
Cmax/MIC
max/MIC
-3
32.8
-4
1.0
10.0 26.3
fAUC/MIC
Bergen et al. submitted
100.0
10.0
R2 = 70%
0
Killing Effect
Killing Effect
1
Killing Effect
0
-1
-2
-3
-4
0
20
40
60
% T>MIC
80
100
Higher plasma binding of polymyxin B in
critically-ill patients
0.5 - 1.5 mg/kg every 12 or 48 h
Plasma concentration (mg/L)
100
Patient 1
Patient 2
Patient 3
Patient 4
Patient 5
Patient 6
Patient 7
Patient 8
10
1
MICs
0.1
0.01
0
120
240
360
480
600
720
Time (min)
Protein binding 78.5 - 92.4% vs 56% in healthy human plasma
Cao et al, JAC 2008
Zavascki et al, CID 2009
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
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/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
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
Unbound fraction of colistin
Plasma binding of colistin: proteins involved
0.8
0.7
Colistin: Low concentration
Colistin: High concentration
0.6
0.5
• Concentration
dependent
• AAG and HSA
0.4
• Healthy human
plasma vs
physiological
concentrations of
HSA/AAG
0.3
0.2
0.1
0.0
Low colistin concentrations: 0.81 - 1.71 mg/L
High colistin concentrations: 5.69 - 11.1 mg/L
Plasma binding of colistin in mice
Unbound fraction (fu)
0.8
Colistin: Low concentration
Colistin: High concentration
0.7
0.6
0.5
0.4
• fu in healthy
mouse plasma is
similar to that in
healthy human
plasma
0.3
0.2
0.1
0.0
Mouse
plasma
Neutropenic Neutropenic
mouse infected mouse
plasma
plasma
Low concentrations:
High concentrations:
1.42 - 1.80 mg/L
12.9 - 14.9 mg/L
• fu in neutropenic
and neutropenic
infected mouse
plasma are lower
Conc-dependent plasma binding of colistin
in mice
Neutropenic infected mouse plasma
Unbound fraction (fu)
0.8
• Colistin
concentration
range
(~0.9 – 30 mg/L)
0.6
0.4
• fu increased ~4-5
fold as plasma
concentration
increased
0.2
0.0
1
10
Colistin (mg/L)
Dudhani et al. A1-576
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
binding of colistin
increased plasma
• 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