Plasma Protein Binding of Colistin Measured by Ultracentrifugation and Equilibrium Dialysis in Teflon® Cells
Problems in establishing PK/PD: binding to proteins, plastics, etc.
Roger L Nation PhD Monash Institute of Pharmaceutical Sciences Monash University, Melbourne, Australia
ESCMID OnlineECCMID Lecture2015 Library Copenhagen, Denmark 25-28 April 2015 @ by author
PK/PD indices: Link between PK (exposure) & PD (response)
DRUG
Concentration vs time Antibacterial PK PD exposure profile response Toxicity Resistance
Infection HOST BUG Host defense ESCMID Online Lecture Library @ by author PK/PD indices: Link between PK (exposure) & PD (response)
fCmax/MIC PK/PD indices for fAUC/MIC unbound drug
Infection in thigh, lung, etc. fT>MIC
ESCMID Online Lecture Library @ by author PK/PD indices: Link between PK (exposure) & PD (response)
fC /MIC • Dose fractionation studies to max
differentiate among the 3 indices fAUC/MIC • Different endpoints … most
typically CFU count at 24 h Infection in thigh, lung, etc. fT>MIC
Fluoroquinolones against primarily Gram-negative bacilli
Many other examples with different antibiotic classes of identifying relevant PK/PD index ESCMID Online Lectureand targetLibrary values
Craig 2002 @via Ambrose by et al .author CID 2007 PK/PD relationships: How translatable between mice and humans? Fluoroquinolones against infections in humans Forrest et al. JAC 1997; AAC 1993; AAC 1993
Ciprofloxacin 100 Ciprofloxacin positive positive - - 75 AUC/MIC < 125
50
AUC/MIC 125-250 25
AUC/MIC > 250
0 % of patients remaining culture remaining % patients of % of patients remaining culture remaining % patients of 0 2 4 6 8 10 12 14 Days of treatment
Grepafloxacin
ESCMID Online Lecture Library @ by author Craig 2002 Problems in establishing PK/PD: binding to proteins, plastics
Define PD response
Measure of in vitro activity
MIC
Plasma concentration Unbound PK/PD index Hours Define PK Define protein binding Define PK/PD relationship
Problems defining the PK/PD relationship …
… can arise because of ESCMID Online Lecture Librarybinding to labware @ by author PK/PD relationships: Key measurements
Ability to be able to make use of PK/PD relationships for translation to the clinic …
… relies on reliable estimates of:
• MIC
• PK (Cmax, AUC, t1/2) • Plasma protein binding (f)
fCmax/MIC
ESCMID Online Lecture fLibraryAUC/MIC
@ by author fT>MIC MIC: What does it mean?
An in vitro measure of antibacterial activity • Small volume, low bacterial density low CFU • Blunt endpoint: ‘turbid’ versus ‘not turbid’ at a fixed point in time; no CFU count • ‘Not turbid’ ≠ no bacteria • No information on: – time-course of bacterial number in the well (rate and extent of kill) – presence of resistant subpopulations
5 × 104 CFU • Can be influenced by a number of factors e.g. – Broth composition – Inorganic ions 18 h – Binding (adsorption) of antibiotic to the well
CFU ESCMID Online Lecture Library
@Time by author MIC: What does it mean in the context of translation?
• MIC is a number that is a measure of activity under a given set of in vitro conditions • In general, its application in PK/PD indices works well e.g. fluoroquinolones, β lactams • To a large extent, the absolute MIC number doesn’t matter
• Lower MIC estimate (by abolishing binding to the MTT well) means a higher PK/PD target value … … BUT it does not change the exposure needed to achieve a given in vivo response … … NOR does it mean the drug is more ‘potent’ in vivo after a given dose administered
Stasis CFU What IS important is that the same MIC method is used in translation … and that the ESCMID Online LectureMIC is a measure Library of the activity of the antibiotic and that it can be determined reliably @f AUC/MICby author MIC: Binding (adsorption) of antibiotic to plastic
• Binding decreases antibiotic concentration in the bulk solution in well, potentially leading to higher MIC value • Antibiotics affected include: – Oritavancin and dalbavancin – Colistin and polymyxin B
ESCMID Online Lecture Library @ by author Binding of colistin to lab tubes and MIC wells
Karvanen et al. ICAAC 2011 • Colistin binding to lab tubes – Time-dependent (most binding in first 4-8 h) – Concentration-dependent (i.e. saturable)
PP = polypropylene PS = polystyrene
• Extensive, concentration- dependent binding in MIC Spiked colistin Average % of spiked concentration conc. (mg/L) TREK panels … decreased by Without P-80 With P-80 addition of 0.002% of polysorbateESCMID 80 … BUT not Online0.5 Lecture7.5% Library35.7% complete reduction, especially 2.0 46.6% 93.7% at low colistin concentrations @ by author John Turnidge, personal communication Polysorbate 80: What is it and why is it used in micro labs?
• It is an amphiphilic polymer Polysorbate 80 – Hydrophilic head group – Hydrophobic tail
– It is like a ‘soap’ Hydrophilic Hydrophobic
• Non-ionic surface active agent
Polysorbate 80, added in BMD testing to: • Assist dispersion of drug and microbial cells • Enhance reproducibility ESCMID• DecreaseOnline antibiotic bindingLecture to plastic Library @ by authorJones et al. Diagn Microbiol Infect Dis 2007 Polysorbate 80: Surface active agent
Wall of well or tube
Surface of liquid
PolystyreneESCMID Online Lecture Library @ by author Amphiphilic antibiotics
Colistin Surface active Polysorbate 80 Wallace et al. J Phys Chem B 2010
Hydrophilic Hydrophobic
Hydrophobic tail and largely hydrophilic head Dalbavancin Oritavancin group
ESCMID Online Lecture Library @ by author Polysorbate 80: Why does it decrease binding to lab-ware?
Amphiphilic antibiotic Wall of well or tube
Surface of liquid
PolystyreneESCMID Online Lecture Library Inhibition of antibiotic binding may not be complete … extent will depend on the relative@ binding by affinities author of the antibiotic and P-80, and their respective concentrations Polysorbate 80: What concentration to use in MIC measurements? Rennie et al. JCM 2007
Influence of polysorbate 80 concentration on dalbavancin MIC results
P-80 capable of doing more than just decreasing binding of antibiotic to MTT well
Effect
11 strains LHB 4 strains No effect in media ESCMID Online Lecture Library @ by author Polysorbate 80: Use in MIC measurements Oritavancin Arhin et al. AAC 2008
• Oritavancin bound extensively in 96-well plates • Binding was minimised by 0.002% polysorbate 80 or 2% lysed horse blood
• Binding loss was concentration dependent in absence of P-80 • 0.002% P-80 afforded full recovery across the concentration range (in contrast to whatESCMID occurs with colistin) Online Lecture Library @ by author Polysorbate 80: Use in MIC measurements Oritavancin Arhin et al. AAC 2008
• MICs in presence of P-80 substantially lower than in its absence • Fold decrease in MIC in presence of P-80 approximates the fold increase in oritavancin concentration in well via abolition of binding to MTT well
• No substantial effect of P-80 if LHB also present
MIC (mg/L)
ESCMID Online Lecture Library @ by author Polysorbate 80: Use in MIC measurements Colistin and polymyxin B Sader et al. Diagn Microbiol Infect Dis 2012
Colistin • 247 clinical strains – 124 Enterobacteriaceae – 60 Acinetobacter spp. – 63 Pseudomonas aeruginosa
• BMD MICs without and with P-80
• MICs lower with P-80, especially at the low end Polymyxin B
ESCMID Online Lecture Library @ by author Polysorbate 80: Use in MIC measurements Colistin Hindler and Humphries JCM 2013
• 50 clinical strains – 11 Acinetobacter baumannii – 15 Klebsiella pneumoniae – 24 Pseudomonas aeruginosa
• BMD MICs without and with P-80
• MICs mostly lower with P-80 …. but, sometimes the reverse was found
ESCMID Online Lecture Library @ by author Polysorbate 80: Use in MIC measurements Colistin Albur et al. JAC 2014
Examined 146 clinical isolates in 3 replicates: • 2 types of MTTs: non-coated (NMTT) vs. tissue-culture-coated (TCMTT) • With / without P-80 0.002% Colistin MICs for isolates
• HigherESCMID MICs in TCMTT compared Online with NMTT Lecture Library • P-80 lowered MIC estimates in NMTT, but not in TCMTT @ by author Polysorbate 80: Use in MIC measurements Colistin and polymyxin B John Turnidge, personal communication
Study conducted on behalf of CLSI
• MICs of E. coli ATCC 25922 and P. aeruginosa ATCC 27853 • 8 labs, each with 30 replicates of the MIC test • MICs were consistently lowered by P-80
• BUT overall assay variance was not lower with P-80 versus without P-80
Colistin Polymyxin B QC Strain Colistin Polymyxin B + P-80 + P-80 E. coli 0.716 0.693 0.768 0.837 ATCC 25922 P. aeruginosa 0.680 0.660 0.811 0.690 ATCCESCMID 27853 Online Lecture Library @ by author Polysorbate 80: Use in MIC measurements
• Undoubtedly, polysorbate 80 added in BMD testing can decrease the binding of ‘sticky’ antibiotics to the MTT wells … and decrease measured MICs
• Concentration used most commonly is 0.002% • Sounds low … right? • But, 0.002% = 20 mg/L
• Compare with clinically relevant plasma concentrations of:
– Colistin and polymyxin B (~1 - 5 mg/L)
Might P-80 do more than just decrease binding to plastic MTT wells?
e.g. Is there any evidence for antibacterial activity of its own? ESCMIDe.g. Is there any evidenceOnline for a synergistic Lecture effect Library e.g. Is there any other mechanism whereby P-80 might modify MIC results? @ by author Polysorbate 80: Use in MIC measurements Might it do more than decrease binding to plastics?
Any evidence that P-80 can have antibacterial effects in its own right?
• P-80 had MBC of 0.00066% against H. pylori and caused morphological & ultrastructural changes shown by TEM
Swollen cells, altered outer membrane, granular cytoplasm, vesicles
• Also had synergistic effect with metronidazole and clarithromycin ESCMID Online Lecture Library @ by author Figura et al. BMC Microbiol 2012 Polysorbate 80: Use in MIC measurements Might it do more than decrease binding to plastics?
Possible mechanism for P-80 synergistic activity with antibiotics • P-80 is likely to bind to the surface of bacterial cell … aligning the P-80 hydrophobic region of P-80 away from water molecules • Possible effects include permeabilisation of the outer membrane Gram-negative rod … any evidence for this?
• P-80 had no effect on viability of P. aeruginosa … but did increase leakage of cell contents and uptake of a Brown and Richards. J Pharm Pharmacol 1964 fluorescent dye, ANS Brown and Winsley. J Gen Microbiol 1969 & 1971 … indicative of effects on OM
• Contemplate the potential for this to enhanceESCMID permeation of the OnlineOM by Lecture Library antibiotics which are normally excluded from the@ cell or haveby poor author access Polysorbate 80: Use in MIC measurements Might it do more than decrease binding to plastics?
Is there any suggestion of synergy betweem P-80 and polymyxins?
• Report from >40 y ago that P-80, in • The authors may have been correct in concentrations as low as 0.0001%, suggesting synergy acted synergistically with polymyxin B • Can’t tell because they did not measure against P. aeruginosa polymyxin B concentrations in the • There was increased cell leakage, lysis incubation vessel without and with P-80 and death over that seen with polymyxin • Thus, they did not exclude the possibility B alone that the increased effect in the presence • Suggested that the ‘synergy’ was due to of P-80 was simply due to P-80 both agents acting on the outer decreasing binding of polymyxin B to the ESCMIDmembrane Online Lecturevessel and increasing Library its concentration in the incubation @ by author Polysorbate 80: Use in MIC measurements Might it do more than decrease binding to plastics? Turnidge, pers commun
Colistin MIC experiment ‘cooked up’ at Siemens Microscan, Sacramento
Non-stick Mini Muffin Pan Time No P-80 P-80 Compared to targeted colistin concn. 1 hour 93% 95% 24 hour 104% 94% No binding of colistin
Test organism P-80 MIC (mg/L) No 0.5 Results E. coli ATCC 25922 Yes 0.125 confirmed in Australia P. aeruginosa ATCC No 1.0 27853 E. coli ATCC 25922 Yes 0.25 P. aeruginosaESCMID ATCC 27853 Online Lecture Library A. baumannii (2 clinical strains) Colistin MICs lower in the presence of P-80, in the K. pneumoniae@ (2 clinical by strains) author absence of binding to the ‘wells’ synergism Polysorbate 80: Use in MIC measurements Might it do more than decrease binding to plastics?
• In aqueous solution, P-80 can undergo self-association to form micelles • Critical Micelle Concentration (CMC) is 13-15 mg/L (Conc. used in MIC testing is 20 mg/L) • Colistin can also form micelles (Wallace et al. J Phys Chem B 2010)
ESCMID Online Lecture Library @ by author Polysorbate 80: Use in MIC measurements Might it do more than decrease binding to plastics?
Polysorbate 80 Amphiphilic antibiotic Lipophilic antibiotic
Mixed micelle of Antibiotic solubilized P-80 + Antibiotic in P-80 micelle
• Would decrease the ‘free’ concentration of antibiotic ESCMID Online Lecture Library • Might this explain cases where P-80 increases MICs? @ by author Polysorbate 80: Use in MIC measurements Might it do more than decrease binding to plastics? Jones et al. Diag Mic Inf Dis 2007
Tigecycline MIC results tested with (0.002%) and without P-80 for 231 Gram-negative organisms
? “Slight trend” to “Slightly lower higher” MICs MICs
• Might elevated MICs (e.g. tigecycline, colistin under some circumstances, dalbavancin) ESCMIDin presence of P-80 be dueOnline to incorporation Lecture of antibiotic in P-80 micelles?Library @ by author Polysorbate 80: Use in MIC measurements Summary
• Concentration of P-80 (0.002%) seems low, but it is 20 mg/L! • P-80 could influence MIC measurements in a number of ways: 1) Decrease binding of amphiphilic or lipophilic antibiotics to plastics measured MIC Note: P-80 may not totally abolish binding across the relevant antibiotic concentration range 2) Cause morphological / ultrastructural / biochemical effects leading to antibacterial activity or synergistic effect via permeabilisation of the OM or other mechanism measured MIC 3) Decrease ‘free’ concentration of antibiotic due to incorporation of the antibiotic into P-80 micelles measured MIC 4) The net effect of 2 or all 3 of the above • P-80 may push MICs in either direction. Net effect may depend on bacterial species and the antibiotic • It is important to ensure that the activity being measured is due to the antibiotic.
• For colistin and polymyxin B, CLSI / EUCAST have agreed that MIC should be measured by BMD without P-80 P-80 resulted in: –ESCMID Synergy Online Lecture Library – Less than complete blockage of binding to plastic – No improvement@ by in reproducibilityauthor PK/PD relationships: Key measurements
Ability to be able to make use of PK/PD relationships …
… relies on reliable estimates of:
• MIC
• PK (Cmax, AUC, t1/2) • Plasma protein binding (f)
fCmax/MIC
ESCMID Online Lecture fLibraryAUC/MIC
@ by author fT>MIC Drug binding to lab-ware while assaying biological samples
• Will lead to artificially low measured concentrations • Use low-binding tubes • Usually not a problem for samples containing protein (e.g. plasma) • In other cases e.g. – Urine – Dialysate – CSF – Solutions used for spiking calibrators and QC samples add material to decrease binding to test tubes e.g. blank human plasma, albumin, organic solvent ESCMID Online Lecturesuch as acetonitrile Library @ by author PK/PD relationships: Key measurements
Ability to be able to make use of PK/PD relationships …
… relies on reliable estimates of:
• MIC
• PK (Cmax, AUC, t1/2) • Plasma protein binding (f)
fCmax/MIC
ESCMID Online Lecture fLibraryAUC/MIC
@ by author fT>MIC Plasma protein binding methods
Ultrafiltration Equilibrium dialysis Ultracentrifugation Semi-permeable membrane Teflon® cells Plasma Protein-free ‘supernatant’
Plasma Buffer Membrane
Total Unbound conc. conc. Protein-free ultrafiltrate ESCMID Online Lecture Library Better suited to determine protein binding of ‘sticky’ drugs Not@ suitable by authorImportant to minimize binding in protein-free matrix Plasma protein binding of colistin Measured by ultracentrifugation and equilibrium dialysis in Teflon® cells
Methods in good agreement over a wide concentration range Neutropenic infected mice % bound 92.9 ± 3.3% UltraCent 90.4 ± 1.1% Eq Dial (Teflon® cells)
Healthy humans % bound 49.2 ± 3.0% UltraCent
Critically-ill patients (n = 66) % bound 50.8 ± 11.3% UltraCent
Approximately 5-fold difference in ESCMID Online Lectureunbound Library fraction between mice and humans @ by author Colistin against P. aeruginosa in mouse infection models
Thigh infection Lung infection ATCC 27853 ATCC 27853 10 12 A A
10 8
8 6
6 CFU/lung CFU/thigh 10 10 4 CFU per lung per CFU CFU per thigh per CFU Log Log 4 10 10
R2 0.82 R2 0.82 Log
Log 2 2
0 0 0.1 1 10 100 0.1 1 10 100 fAUC/MICfAUC/MIC fAUC/MICfAUC/MIC 10 12 EffectB fAUC/MIC • Similar values for 2 otherB strains Effect fAUC/MIC • Lower response in lung10 Stasis8 9.94 Stasis 34.1 • For an MIC of 1 mg/L and unbound ESCMID Online Lecture8 Library 1-log10 kill 12.4 fraction of ~0.5 as in patients, 1-log10 kill 43.3 6 fAUC/MIC of 12.4 corresponds to a 2-log10 kill 15.8 6 2-log10 kill 51.8 CFU/lung CFU/thigh
plasma colistin C 10 of ~1 mg/L
10 ss,avg 4 @ by author Log Log 4
R2 0.84 R2 0.05 2 2
0 0 0.1 1 10 100 0.1 1 10 100 fAUC/MIC fAUC/MIC Conclusions
• Establishing PK/PD relationships in animal infection models has had a substantial impact on the way in which antibiotics are developed and used clinically • Some antibiotics are amphiphilic or hydrophobic in nature and prone to bind to MTT wells • Polysorbate 80 0.002% has been used in MIC tests to decrease binding to plastics (and to assist in dispersion of bacterial cells and antibiotics) • Important to recognise that polysorbate 80 is physicochemically complex and not always biologically inert – Lack of antibacterial effect in its own right does not mean no potential for synergy – Multiple potential effects in MIC testing (e.g. for colistin, ed binding + synergism) – Effects vary from drug to drug and also from bacterial species to species – Reproducibility is not necessarily improved by adding P-80 • What is needed is an MIC test that provides a reliable measure of activity of the antibiotic • Species differences in plasma protein binding must be considered in translating PK/PD indices for unbound drug • Determination of MIC, PK and plasma protein binding must be undertaken using well validatedESCMID methods Online Lecture Library @ by author
September 22 - 24, San Diego, USA
2nd International Conference on Polymyxins The Polymyxin Jigsaw: More pieces put in place
Reserve the dates today and joinESCMID the polymyxin Online family Lecture after ICAAC/ICC Library 2015! @ by author http://monash.edu/pharm/about/events/polymyxins