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Kinetic fluorescent methods for measuring functional
delivery of membrane active antibiotics
Dr. Scott C. HartselUniversity of Wisconsin-Eau Claire
or
Overview• What is Amphotericin B?• The problem with Amphotericin B/A simple
solution: Hot-Zone!• Applied Photophysics instrumentation to
measure:– Activity of “hot-zone” by fluorescence– Stability/structure of “hot-zone” by CD– Kinetic stability in serum by kinetic diode array
Cholesterol
HO
HO
Ergosterol
O
O
OH OH
OH
OH OH
OH
O
OH
CO2HH
HO O
O
OH NH2
OHMe
Me
Me
Me
Amphotericin B
O
O
OH OH OH
OH
O
OH
CO2HH
HO O
O
OH NH2
OHMe
Me
Me
Me
OH
OH
Nystatin
What is Amphotericin B?
Cholesterol: humans
Ergosterol: fungi
Nystatin
Amphotericin B
Binds strongly to AmB
Binds weakly to AmB
Q:How can you reduce toxicity?
A: Associate with liposomes.
Bolard’s model
•Reducing effective chemical potential of AmB by “tying up” or by macrophage consumption is key.
TOXIC AGGREGATE
A Simple Solution: Hot Zone
• If lowering chemical potential is most important, can we change Amphotericin’s properties without expensive and troublesome lipids?• YES! Heat treating Fungizone (70oC, aqueous, for 20 minutes) creates a new self-associated form. •A superior therapeutic index for Hot-Zone was shown in animal fungal disease models- Francois Gaboriau, Jacques Bolard
•Nickname: Hot-Zone
• We wanted to find out “How and Why?” by asking:
–How has the structural arrangement of AmB changed?–Is the new arrangement stable?–Is the membrane channel forming activity different?–Does heat treatment change interaction with serum components and the immune system?
A Simple Solution: Hot Zone
Hot-Zone-Analysis
Applied PhotophysicsStopped-Flowdiode array and conventional spectrophotometer,spectrofluorimeter, and circular dichroism (CD) spectrometer
Hot-Zone-Absorption Spectra
0
5
10
15
20
25
30
35
40
45
50
300 320 340 360 380 400 420 440
Wavelength, nm
HEAT
CD Spectra (circular dichroism)
Opticallyactivesample
Right and lefthand circularlypolarized light
Polarizedlight beam
Preferentialabsorption ofright handpolarization
CD signal
Absorption
CD Spectra•Self-associated molecules may absorb light as an aggregate by exciton coupling. If the molecules are twisted relative to one another in space they will absorb right and left-handed circularly polarized light differently.
•This gives rise to circular dichroism by the coupled oscillator mechanism. The spectrum will have two equal and opposite bands. The shape and intensity of the CD bands are very sensitive to small changes in the geometry of the molecules.
In phaseOut of phase
CD Spectra• AmB molecules normally have
no CD spectrum in the visible light region, but when self-associated (oligomers)they have intense CD spectra. The dimer is the minimal unit of CD activity.
• This property gives a very sensitive handle on AmB’s supramolecular geometry and changes in that structure.
- +
Hot-Zone-CD Spectra
HEAT
•Circular dichroism: sensitive to small changes in supramolecular structure -
Hot-Zone-CD Spectra•Circular dichroism: sensitive to small changes in supramolecular structure -
Persistence of Hot-Zone-Lyophilization studies show stability
-800
-600
-400
-200
0
200
400
600
800
1000
1200
295 315 335 355 375 395 415
Wavelength
HFZ in Dextrose, before
HFZ in Dextrose, after
• Membranes with 10% ergosterol are fungal models; with cholesterol they are mammalian models.
• With KCl gradient K+ permeation creates a voltage, (K+ selective for AmB).
• H+ equilibrates with
• Pyranine fluorescence responds to pH linearly from ~6.2-7.8. Fluorescence decrease means net cation (K+) selectivity.
Experimental SystemExtruded 1000Å Liposome Membrane Vesicles
High K+ , Cl-Low K+, Cl- iso-osmotic
H+
Amphotericin
2 mM pyranine
K+
H+
Membrane Activity of Hot-Zone
+++
---
- S
O
3
S
O
3
S
O
3
O
-
- -
Fluorescence Decrease
Hot-Zone/Membrane Channel ActivityModel mammalian membranes with
cholesterol
Model fungal membranesWith ergosterol
Hot-Zone has much less activity
Hot-Zone has similar activity !
Change in pH versus Time showing fluorescence detected ion currents on model mammalian membranes comparing Fungizone and Hot-Zone in the presence of 15mg/mL human serum albumin in external buffer (315mM sucrose, 15mM K2HPO4, pH 7.20 at 37C)
-0.9
-0.8
-0.7
-0.6
-0.5
-0.4
-0.3
-0.2
-0.1
0
0.1
0 10 20 30 40 50 60 70 80 90 100
Time (seconds)
Change in pH
15mg/mL HSA in External Buffer 3uM Heat-treated Fungizone 5uM Heat-treated Fungizone 10uM Heat-treated Fungizone
3uM Fungizone 5uM Fungizone 10uM Fungizone 0.3uM Valinomycin
Membrane Activity in the Presence of Serum Components
Hot-Zone-Kinetic Stability (HSA)*Fungizone aggregates are destabilized by serum albumin-500 sec/37C
Hot-zone aggregates are much more stable in the presence of serum albumin
0
500
Hot-Zone-Kinetic Stability (HSA)*
• Extra stability of Hot-Zone probably buys enough time so that AmB aggregates can be safely removed from circulation and monomers subsequently released (like liposomes).
• Fungizone micelles, on the other hand are unstable. The Amphotericin becomes mobile and remains in circulation longer at toxic levels
activeAmB
oligomer
AmBmonomer
Fungizone
AmB channel w/cholesterol or no sterol
AmB/sterol channel w/ergosterol
"Hot-Zone"
Engulfing and slow release from macrophages
A Happy Ending?• The Applied Photophysics system has been used to
measure activity, structure and stability of a new drug delivery system for Amphotericin B
• Hot-Zone is a cheap, easy-to-make and stable formulation of Amphotericin B from Fungizone
• In model membrane and animal systems, Hot-Zone is less toxic and equally effective
• An altered pattern of serum distribution and increased stability may also contribute to lower toxicity
A Happy Ending?
A Happy Ending?
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