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

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